Nanoparticulate Drug Delivery Systems

DRUGS AND THE PHARMACEUTICA SCIENCES

A Series o! Te"t#oo$s an% Monograp&s

E"ecutive E%itor

'ames S(ar#ric$
P&armaceuTec&) Inc* Pine&urst) Nort& Carolina

A%visory +oar%
arry * Augs#urger
University o! Marylan% +altimore) Marylan%

Harry G* +rittain
Center !or P&armaceutical P&ysics Mil!or%) Ne( 'ersey

'enni!er +* Dressman
'o&ann ,ol!gang Goet&e University -ran$!urt) Germany

Ant&ony '* Hic$ey

University o! Nort& Carolina Sc&ool o! P&armacy C&apel Hill) Nort& Carolina

'e!!rey A* Hug&es
University o! -lori%a College o! P&armacy Gainesville) -lori%a

A.a/ Hussain
San%o/ Princeton) Ne( 'ersey

Trevor M* 'ones
T&e Association o! t&e +ritis& P&armaceutical In%ustry on%on) Unite% 0ing%om

Hans E* 'unginger
ei%en1Amster%am Center !or Drug Researc& ei%en) T&e Net&erlan%s

2incent H* * ee
University o! Sout&ern Cali!ornia os Angeles) Cali!ornia

Step&en G* Sc&ulman
University o! -lori%a Gainesville) -lori%a

'erome P* S$elly
Ale"an%ria) 2irginia

Eli/a#et& M* Topp
University o! 0ansas Sc&ool o! P&armacy a(rence) 0ansas

Geo!!rey T* Tuc$er
University o! S&e!!iel% Royal Hallams&ire Hospital S&e!!iel%) Unite% 0ing%om

Peter 3or$
University o! +ra%!or% Sc&ool o! P&armacy +ra%!or%) Unite% 0ing%om

4* P&armaco$inetics) Milo Gi#al%i an% Donal% Perrier 5* Goo% Manu!acturing Practices !or P&armaceuticals6 A Plan !or Total 7uality Control) Si%ney H* ,illig) Murray M* Tuc$erman) an% ,illiam S* Hitc&ings I2 8* Microencapsulation) e%ite% #y '* R* Ni"on 9* Drug Meta#olism6 C&emical an% +ioc&emical Aspects) +ernar% Testa an% Peter 'enner :* Ne( Drugs6 Discovery an% Development) e%ite% #y Alan A* Ru#in ;* Sustaine% an% Controlle% Release Drug Delivery Systems) e%ite% #y 'osep& R* Ro#inson <* Mo%ern P&armaceutics) e%ite% #y Gil#ert S* +an$er an% C&ristop&er T* R&o%es =* Prescription Drugs in S&ort Supply6 Case Histories) Mic&ael A* Sc&(art/ >* Activate% C&arcoal6 Anti%otal an% ?t&er Me%ical Uses) Davi% ?* Cooney 4@* Concepts in Drug Meta#olism Ain t(o partsB) e%ite% #y Peter 'enner an% +ernar% Testa 44* P&armaceutical Analysis6 Mo%ern Met&o%s Ain t(o partsB) e%ite% #y 'ames ,* Munson 45* Tec&niCues o! Solu#ili/ation o! Drugs) e%ite% #y Samuel H* 3al$o(s$y 48* ?rp&an Drugs) e%ite% #y -re% E* 0arc& 49* Novel Drug Delivery Systems6 -un%amentals) Developmental Concepts) +iome%ical Assessments) 3ie ,* C&ien 4:* P&armaco$inetics6 Secon% E%ition) Revise% an% E"pan%e%) Milo Gi#al%i an% Donal% Perrier 4;* Goo% Manu!acturing Practices !or P&armaceuticals6 A Plan !or Total 7uality Control) Secon% E%ition) Revise% an% E"pan%e%) Si%ney H* ,illig) Murray M* Tuc$erman) an% ,illiam S* Hitc&ings I2 4<* -ormulation o! 2eterinary Dosage -orms) e%ite% #y 'ac$ +lo%inger 4=* Dermatological -ormulations6 Percutaneous A#sorption) +rian ,* +arry 4>* T&e Clinical Researc& Process in t&e P&armaceutical In%ustry) e%ite% #y Gary M* Matoren 5@* Microencapsulation an% Relate% Drug Processes) Patric$ +* Deasy 54* Drugs an% Nutrients6 T&e Interactive E!!ects) e%ite% #y Dap&ne A* Roe an% T* Colin Camp#ell 55* +iotec&nology o! In%ustrial Anti#iotics) Eric$ '* 2an%amme 58* P&armaceutical Process 2ali%ation) e%ite% #y +ernar% T* o!tus an% Ro#ert A* Nas& 59* Anticancer an% Inter!eron Agents6 Synt&esis an% Properties) e%ite% #y Rap&ael M* ?tten#rite an% George +* +utler 5:* P&armaceutical Statistics6 Practical an% Clinical Applications) San!or% +olton

5;* Drug Dynamics !or Analytical) Clinical) an% +iological C&emists) +en.amin '* Gu%/ino(ic/) +urro(s T* 3oun$in) 'r*) an% Mic&ael '* Gu%/ino(ic/ 5<* Mo%ern Analysis o! Anti#iotics) e%ite% #y A%.oran As/alos 5=* Solu#ility an% Relate% Properties) 0ennet& C* 'ames 5>* Controlle% Drug Delivery6 -un%amentals an% Applications) Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y 'osep& R* Ro#inson an% 2incent H* ee 8@* Ne( Drug Approval Process6 Clinical an% Regulatory Management) e%ite% #y Ric&ar% A* Guarino 84* Trans%ermal Controlle% Systemic Me%ications) e%ite% #y 3ie ,* C&ien 85* Drug Delivery Devices6 -un%amentals an% Applications) e%ite% #y Praveen Tyle 88* P&armaco$inetics6 Regulatory D In%ustrial D Aca%emic Perspectives) e%ite% #y Peter G* ,elling an% -rancis * S* Tse 89* Clinical Drug Trials an% Tri#ulations) e%ite% #y Allen E* Cato 8:* Trans%ermal Drug Delivery6 Developmental Issues an% Researc& Initiatives) e%ite% #y 'onat&an Ha%gra!t an% Ric&ar% H* Guy 8;* ACueous Polymeric Coatings !or P&armaceutical Dosage -orms) e%ite% #y 'ames ,* McGinity 8<* P&armaceutical Pelleti/ation Tec&nology) e%ite% #y Isaac G&e#reE Sellassie 8=* Goo% a#oratory Practice Regulations) e%ite% #y Allen -* Hirsc& 8>* Nasal Systemic Drug Delivery) 3ie ,* C&ien) 0ennet& S* E* Su) an% S&yiE-eu C&ang 9@* Mo%ern P&armaceutics6 Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y Gil#ert S* +an$er an% C&ristop&er T* R&o%es 94* Speciali/e% Drug Delivery Systems6 Manu!acturing an% Pro%uction Tec&nology) e%ite% #y Praveen Tyle 95* Topical Drug Delivery -ormulations) e%ite% #y Davi% ,* ?s#orne an% Anton H* Amann 98* Drug Sta#ility6 Principles an% Practices) 'ens T* Carstensen 99* P&armaceutical Statistics6 Practical an% Clinical Applications) Secon% E%ition) Revise% an% E"pan%e%) San!or% +olton 9:* +io%egra%a#le Polymers as Drug Delivery Systems) e%ite% #y Mar$ C&asin an% Ro#ert anger 9;* Preclinical Drug Disposition6 A a#oratory Han%#oo$) -rancis * S* Tse an% 'ames '* 'a!!e 9<* HP C in t&e P&armaceutical In%ustry) e%ite% #y Go%(in ,* -ong an% Stanley 0* am 9=* P&armaceutical +ioeCuivalence) e%ite% #y Peter G* ,elling) -rancis * S* Tse) an% S&ri$ant 2* Ding&e

9>* P&armaceutical Dissolution Testing) Umes& 2* +ana$ar :@* Novel Drug Delivery Systems6 Secon% E%ition) Revise% an% E"pan%e%) 3ie ,* C&ien :4* Managing t&e Clinical Drug Development Process) Davi% M* Cocc&etto an% Ronal% 2* Nar%i :5* Goo% Manu!acturing Practices !or P&armaceuticals6 A Plan !or Total 7uality Control) T&ir% E%ition) e%ite% #y Si%ney H* ,illig an% 'ames R* Sto$er :8* Pro%rugs6 Topical an% ?cular Drug Delivery) e%ite% #y 0ennet& +* Sloan :9* P&armaceutical In&alation Aerosol Tec&nology) e%ite% #y Ant&ony '* Hic$ey ::* Ra%iop&armaceuticals6 C&emistry an% P&armacology) e%ite% #y A%rian D* Nunn :;* Ne( Drug Approval Process6 Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y Ric&ar% A* Guarino :<* P&armaceutical Process 2ali%ation6 Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y Ira R* +erry an% Ro#ert A* Nas& :=* ?p&t&almic Drug Delivery Systems) e%ite% #y As&im 0* Mitra :>* P&armaceutical S$in Penetration En&ancement) e%ite% #y 0ennet& A* ,alters an% 'onat&an Ha%gra!t ;@* Colonic Drug A#sorption an% Meta#olism) e%ite% #y Peter R* +iec$ ;4* P&armaceutical Particulate Carriers6 T&erapeutic Applications) e%ite% #y Alain Rollan% ;5* Drug Permeation En&ancement6 T&eory an% Applications) e%ite% #y Dean S* Hsie& ;8* Glycopepti%e Anti#iotics) e%ite% #y Rama$ris&nan Nagara.an ;9* Ac&ieving Sterility in Me%ical an% P&armaceutical Pro%ucts) Nigel A* Halls ;:* Multiparticulate ?ral Drug Delivery) e%ite% #y Isaac G&e#reESellassie ;;* Colloi%al Drug Delivery Systems) e%ite% #y 'Frg 0reuter ;<* P&armaco$inetics6 Regulatory D In%ustrial D Aca%emic Perspectives) Secon% E%ition) e%ite% #y Peter G* ,elling an% -rancis * S* Tse ;=* Drug Sta#ility6 Principles an% Practices) Secon% E%ition) Revise% an% E"pan%e%) 'ens T* Carstensen ;>* Goo% a#oratory Practice Regulations6 Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y San%y ,ein#erg <@* P&ysical C&aracteri/ation o! P&armaceutical Soli%s) e%ite% #y Harry G* +rittain <4* P&armaceutical Po(%er Compaction Tec&nology) e%ite% #y GFran Al%er#orn an% C&rister NystrFm <5* Mo%ern P&armaceutics6 T&ir% E%ition) Revise% an% E"pan%e%) e%ite% #y Gil#ert S* +an$er an% C&ristop&er T* R&o%es <8* Microencapsulation6 Met&o%s an% In%ustrial Applications) e%ite% #y Simon +enita

<9* ?ral Mucosal Drug Delivery) e%ite% #y Mic&ael '* Rat&#one <:* Clinical Researc& in P&armaceutical Development) e%ite% #y +arry +lei%t an% Mic&ael Montagne <;* T&e Drug Development Process6 Increasing E!!iciency an% Cost E!!ectiveness) e%ite% #y Peter G* ,elling) ouis asagna) an% Umes& 2* +ana$ar <<* Microparticulate Systems !or t&e Delivery o! Proteins an% 2accines) e%ite% #y Sma%ar Co&en an% Ho(ar% +ernstein <=* Goo% Manu!acturing Practices !or P&armaceuticals6 A Plan !or Total 7uality Control) -ourt& E%ition) Revise% an% E"pan%e%) Si%ney H* ,illig an% 'ames R* Sto$er <>* ACueous Polymeric Coatings !or P&armaceutical Dosage -orms6 Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y 'ames ,* McGinity =@* P&armaceutical Statistics6 Practical an% Clinical Applications) T&ir% E%ition) San!or% +olton =4* Han%#oo$ o! P&armaceutical Granulation Tec&nology) e%ite% #y Dilip M* Pari$& =5* +iotec&nology o! Anti#iotics6 Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y ,illiam R* Stro&l =8* Mec&anisms o! Trans%ermal Drug Delivery) e%ite% #y Russell ?* Potts an% Ric&ar% H* Guy =9* P&armaceutical En/ymes) e%ite% #y Al#ert au(ers an% Simon Sc&arpG =:* Development o! +iop&armaceutical Parenteral Dosage -orms) e%ite% #y 'o&n A* +ontempo =;* P&armaceutical Pro.ect Management) e%ite% #y Tony 0enne%y =<* Drug Pro%ucts !or Clinical Trials6 An International Gui%e to -ormulation D Pro%uction D 7uality Control) e%ite% #y Donal% C* Mon$&ouse an% C&ristop&er T* R&o%es ==* Development an% -ormulation o! 2eterinary Dosage -orms6 Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y Gregory E* Har%ee an% '* Desmon% +aggot =>* ReceptorE+ase% Drug Design) e%ite% #y Paul e!! >@* Automation an% 2ali%ation o! In!ormation in P&armaceutical Processing) e%ite% #y 'osep& -* %eSpaut/ >4* Dermal A#sorption an% To"icity Assessment) e%ite% #y Mic&ael S* Ro#erts an% 0ennet& A* ,alters >5* P&armaceutical E"perimental Design) Garet& A* e(is) Di%ier Mat&ieu) an% Roger P&anETanE uu >8* Preparing !or -DA PreEApproval Inspections) e%ite% #y Martin D* Hynes III >9* P&armaceutical E"cipients6 C&aracteri/ation #y IR) Raman) an% NMR Spectroscopy) Davi% E* +ugay an% ,* Paul -in%lay >:* Polymorp&ism in P&armaceutical Soli%s) e%ite% #y Harry G* +rittain

>;* -ree/eEDrying1 yop&ili/ation o! P&armaceutical an% +iological Pro%ucts) e%ite% #y ouis Rey an% 'oan C* May ><* Percutaneous A#sorption6 DrugsHCosmeticsHMec&anismsHMet&o%ology) T&ir% E%ition) Revise% an% E"pan%e%) e%ite% #y Ro#ert * +ronaug& an% Ho(ar% I* Mai#ac& >=* +ioa%&esive Drug Delivery Systems6 -un%amentals) Novel Approac&es) an% Development) e%ite% #y E%it& Mat&io(it/) Donal% E* C&ic$ering III) an% ClausEMic&ael e&r >>* Protein -ormulation an% Delivery) e%ite% #y Eugene '* McNally 4@@* Ne( Drug Approval Process6 T&ir% E%ition) T&e Glo#al C&allenge) e%ite% #y Ric&ar% A* Guarino 4@4* Pepti%e an% Protein Drug Analysis) e%ite% #y Ronal% E* Rei% 4@5* Transport Processes in P&armaceutical Systems) e%ite% #y Gor%on * Ami%on) Ping I* ee) an% Eli/a#et& M* Topp 4@8* E"cipient To"icity an% Sa!ety) e%ite% #y Myra * ,einer an% ois A* 0ot$os$ie 4@9* T&e Clinical Au%it in P&armaceutical Development) e%ite% #y Mic&ael R* Hamrell 4@:* P&armaceutical Emulsions an% Suspensions) e%ite% #y -rancoise Niellou% an% Gil#erte MartiEMestres 4@;* ?ral Drug A#sorption6 Pre%iction an% Assessment) e%ite% #y 'enni!er +* Dressman an% Hans ennernIs 4@<* Drug Sta#ility6 Principles an% Practices) T&ir% E%ition) Revise% an% E"pan%e%) e%ite% #y 'ens T* Carstensen an% C* T* R&o%es 4@=* Containment in t&e P&armaceutical In%ustry) e%ite% #y 'ames P* ,oo% 4@>* Goo% Manu!acturing Practices !or P&armaceuticals6 A Plan !or Total 7uality Control !rom Manu!acturer to Consumer) -i!t& E%ition) Revise% an% E"pan%e%) Si%ney H* ,illig 44@* A%vance% P&armaceutical Soli%s) 'ens T* Carstensen 444* En%oto"ins6 Pyrogens) A Testing) an% Depyrogenation) Secon% E%ition) Revise% an% E"pan%e%) 0evin * ,illiams 445* P&armaceutical Process Engineering) Ant&ony '* Hic$ey an% Davi% Gan%erton 448* P&armacogenomics) e%ite% #y ,erner 0alo() Urs A* Meyer an% Rac&el -* Tyn%ale 449* Han%#oo$ o! Drug Screening) e%ite% #y Rama$ris&na Seet&ala an% Pra#&avat&i +* -ernan%es 44:* Drug Targeting Tec&nology6 P&ysical D C&emical D +iological Met&o%s) e%ite% #y Hans Sc&reier 44;* DrugHDrug Interactions) e%ite% #y A* Davi% Ro%rigues 44<* Han%#oo$ o! P&armaceutical Analysis) e%ite% #y ena ?&annesian an% Ant&ony '* Streeter 44=* P&armaceutical Process ScaleEUp) e%ite% #y Mic&ael evin

44>* Dermatological an% Trans%ermal -ormulations) e%ite% #y 0ennet& A* ,alters 45@* Clinical Drug Trials an% Tri#ulations6 Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y Allen Cato) yn%a Sutton) an% Allen Cato III 454* Mo%ern P&armaceutics6 -ourt& E%ition) Revise% an% E"pan%e%) e%ite% #y Gil#ert S* +an$er an% C&ristop&er T* R&o%es 455* Sur!actants an% Polymers in Drug Delivery) Martin Malmsten 458* Trans%ermal Drug Delivery6 Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y Ric&ar% H* Guy an% 'onat&an Ha%gra!t 459* Goo% a#oratory Practice Regulations6 Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y San%y ,ein#erg 45:* Parenteral 7uality Control6 Sterility) Pyrogen) Particulate) an% Pac$age Integrity Testing6 T&ir% E%ition) Revise% an% E"pan%e%) Mic&ael '* A$ers) Daniel S* arrimore) an% Dana Morton Gua//o 45;* Mo%i!ie%ERelease Drug Delivery Tec&nology) e%ite% #y Mic&ael '* Rat&#one) 'onat&an Ha%gra!t) an% Mic&ael S* Ro#erts 45<* Simulation !or Designing Clinical Trials6 A P&armaco$ineticE P&armaco%ynamic Mo%eling Perspective) e%ite% #y Hui C* 0im$o an% Step&en +* Du!!ull 45=* A!!inity Capillary Electrop&oresis in P&armaceutics an% +iop&armaE ceutics) e%ite% #y Rein&ar% H* H* Neu#ert an% HansEHermann RJttinger 45>* P&armaceutical Process 2ali%ation6 An International T&ir% E%ition) Revise% an% E"pan%e%) e%ite% #y Ro#ert A* Nas& an% Al!re% H* ,ac&ter 48@* ?p&t&almic Drug Delivery Systems6 Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y As&im 0* Mitra 484* P&armaceutical Gene Delivery Systems) e%ite% #y Alain Rollan% an% Sean M* Sullivan 485* +iomar$ers in Clinical Drug Development) e%ite% #y 'o&n C* +loom an% Ro#ert A* Dean 488* P&armaceutical E"trusion Tec&nology) e%ite% #y Isaac G&e#reESellassie an% C&arles Martin 489* P&armaceutical In&alation Aerosol Tec&nology6 Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y Ant&ony '* Hic$ey 48:* P&armaceutical Statistics6 Practical an% Clinical Applications) -ourt& E%ition) San!or% +olton an% C&arles +on 48;* Compliance Han%#oo$ !or P&armaceuticals) Me%ical Devices) an% +iologics) e%ite% #y Carmen Me%ina 48<* -ree/eEDrying1 yop&ili/ation o! P&armaceutical an% +iological Pro%ucts6 Secon% E%ition) Revise% an% E"pan%e%) e%ite% #y ouis Rey an% 'oan C* May 48=* Supercritical -lui% Tec&nology !or Drug Pro%uct Development) e%ite% #y Peter 3or$) U%ay +* 0ompella) an% +oris 3* S&e$unov 48>* Ne( Drug Approval Process6 -ourt& E%ition) Accelerating Glo#al Registrations) e%ite% #y Ric&ar% A* Guarino

49@* Micro#ial Contamination Control in Parenteral Manu!acturing) e%ite% #y 0evin * ,illiams 494* Ne( Drug Development6 Regulatory Para%igms !or Clinical P&armacology an% +iop&armaceutics) e%ite% #y C&an%ra&as G* Sa&a.(alla 495* Micro#ial Contamination Control in t&e P&armaceutical In%ustry) e%ite% #y uis 'imene/ 498* Generic Drug Pro%uct Development6 Soli% ?ral Dosage -orms) e%ite% #y eon S&argel an% I//y 0an!er 499* Intro%uction to t&e P&armaceutical Regulatory Process) e%ite% #y Ira R* +erry 49:* Drug Delivery to t&e ?ral Cavity6 Molecules to Mar$et) e%ite% #y Tapas& 0* G&os& an% ,illiam R* P!ister 49;* Goo% Design Practices !or GMP P&armaceutical -acilities) e%ite% #y An%re( Signore an% Terry 'aco#s 49<* Drug Pro%ucts !or Clinical Trials) Secon% E%ition) e%ite% #y Donal% Mon$&ouse) C&arles Carney) an% 'im Clar$ 49=* Polymeric Drug Delivery Systems) e%ite% #y Glen S* 0(on 49>* In.ecta#le Disperse% Systems6 -ormulation) Processing) an% Per!ormance) e%ite% #y Diane '* +urgess 4:@* a#oratory Au%iting !or 7uality an% Regulatory Compliance) Donal% Singer) RalucaEIoana Ste!an) an% 'aco#us van Sta%en 4:4* Active P&armaceutical Ingre%ients6 Development) Manu!acturing) an% Regulation) e%ite% #y Stanley Nusim 4:5* Preclinical Drug Development) e%ite% #y Mar$ C* Rogge an% Davi% R* Ta!t 4:8* P&armaceutical Stress Testing6 Pre%icting Drug Degra%ation) e%ite% #y Steven ,* +aertsc&i 4:9* Han%#oo$ o! P&armaceutical Granulation Tec&nology6 Secon% E%ition) e%ite% #y Dilip M* Pari$& 4::* Percutaneous A#sorption6 DrugsHCosmeticsHMec&anismsHMet&o%ology) -ourt& E%ition) e%ite% #y Ro#ert * +ronaug& an% Ho(ar% I* Mai#ac& 4:;* P&armacogenomics6 Secon% E%ition) e%ite% #y ,erner 0alo() Urs A* Meyer an% Rac&el -* Tyn%ale 4:<* P&armaceutical Process ScaleEUp) Secon% E%ition) e%ite% #y Mic&ael evin 4:=* Microencapsulation6 Met&o%s an% In%ustrial Applications) Secon% E%ition) e%ite% #y Simon +enita 4:>* Nanoparticle Tec&nology !or Drug Delivery) e%ite% #y Ram +* Gupta an% U%ay +* 0ompella 4;@* Spectroscopy o! P&armaceutical Soli%s) e%ite% #y Harry G* +rittain 4;4* Dose ?ptimi/ation in Drug Development) e%ite% #y Ra.es& 0ris&na

4;5* Her#al SupplementsEDrug Interactions6 Scienti!ic an% Regulatory Perspectives) e%ite% #y 3* ,* -rancis am) S&ie(EMei Huang) an% Step&en D* Hall 4;8* P&armaceutical P&otosta#ility an% Sta#ili/ation Tec&nology) e%ite% #y 'osep& T* Piec&oc$i an% 0arl T&oma 4;9* Environmental Monitoring !or Cleanrooms an% Controlle% Environments) e%ite% #y Anne Marie Di"on 4;:* P&armaceutical Pro%uct Development6 In 2itroEIn 2ivo Correlation) e%ite% #y Da$s&ina Murt&y C&ilu$uri) Ganga%&ar Sun$ara) an% Davi% 3oung 4;;* Nanoparticulate Drug Delivery Systems) e%ite% #y Deepa$ T&assu) Mic&el Deleers) an% 3as&(ant Pat&a$ 4;<* En%oto"ins6 Pyrogens) A Testing an% Depyrogenation) T&ir% E%ition) e%ite% #y 0evin * ,illiams 4;=* Goo% a#oratory Practice Regulations) -ourt& E%ition) e%ite% #y San%y ,ein#erg 4;>* Goo% Manu!acturing Practices !or P&armaceuticals) Si"t& E%ition) e%ite% #y 'osep& D* Nally

Nanoparticulate Drug Delivery Systems

e%ite% #y

Deepa$ T&assu
UC+ P&arma) Inc* Roc&ester) Ne( 3or$) U*S*A*

Mic&el Deleers
UC+ P&arma) C&emin %u -oriest +raine lKAlleu%) +elgium

3as&(ant Pat&a$
UC+ Manu!acturing) Inc* Roc&ester) Ne( 3or$) U*S*A*

Ne( 3or$ on%on

In!orma Healt&care USA) Inc* 5<@ Ma%ison Avenue Ne( 3or$) N3 4@@4; L 5@@< #y In!orma Healt&care USA) Inc* In!orma Healt&care is an In!orma #usiness No claim to original U*S* Government (or$s Printe% in t&e Unite% States o! America on aci%E!ree paper 4@ > = < ; : 9 8 5 4 International Stan%ar% +oo$ Num#erE4@6 @E=9>8E>@<8E< AHar%coverB International Stan%ar% +oo$ Num#erE486 ><=E@E=9>8E>@<8E> AHar%coverB T&is #oo$ contains in!ormation o#taine% !rom aut&entic an% &ig&ly regar%e% sources* Reprinte% material is Cuote% (it& permission) an% sources are in%icate%* A (i%e variety o! re!erences are liste%* Reasona#le e!!orts &ave #een ma%e to pu#lis& relia#le %ata an% in!ormation) #ut t&e aut&or an% t&e pu#lis&er cannot assume responsi#ility !or t&e vali%ity o! all materials or !or t&e conseCuences o! t&eir use* No part o! t&is #oo$ may #e reprinte%) repro%uce%) transmitte%) or utili/e% in any !orm #y any electronic) mec&anical) or ot&er means) no( $no(n or &erea!ter invente%) inclu%ing p&otocopying) micro!ilming) an% recor%ing) or in any in!ormation storage or retrieval system) (it&out (ritten permission !rom t&e pu#lis&ers* -or permission to p&otocopy or use material electronically !rom t&is (or$) please access (((*copyrig&t* com A&ttp611(((*copyrig&t*com1B or contact t&e Copyrig&t Clearance Center) Inc* ACCCB 555 Rose(oo% Drive) Danvers) MA @4>58) ><=E<:@E=9@@* CCC is a notE!orEpro!it organi/ation t&at provi%es licenses an% registration !or a variety o! users* -or organi/ations t&at &ave #een grante% a p&otocopy license #y t&e CCC) a separate system o! payment &as #een arrange%* Tra%emar$ Notice6 Pro%uct or corporate names may #e tra%emar$s or registere% tra%emar$s) an% are use% only !or i%enti!ication an% e"planation (it&out intent to in!ringe* i#rary o! Congress CatalogingEinEPu#lication Data Nanoparticulate %rug %elivery systems 1 e%ite% #y Deepa$ T&assu) Mic&el Deleers) 3as&(ant Pat&a$* p* M cm* EE ADrugs an% t&e p&armaceutical sciences M v* 4;;B Inclu%es #i#liograp&ical re!erences an% in%e"* IS+NE486 ><=E@E=9>8E>@<8E> Aal$* paperB IS+NE4@6 @E=9>8E>@<8E< Aal$* paperB 4* Drug %elivery systems* 5* Nanoparticles* I* T&assu) Deepa$* II* Deleers) Mic&el* III* Pat&a$) 3as&(ant* I2* Series* NDN M6 4* Drug Delivery SystemsEEmet&o%s* 5* Nanostructures* 8* Drug Carriers* ,4 DR=>8+ v*4;; 5@@< 1 ,+ 89@ N4=9 5@@<O

RS4>>*:*N8; 5@@< ;4:P*;EE%c55

5@@;@:49;4

2isit t&e In!orma ,e# site at (((*in!orma*com an% t&e In!orma Healt&care ,e# site at (((*in!orma&ealt&care*com

-ore(or%

T&e use o! molecular or macromolecular entities an% superstructures %erive% t&ereo! !or t&e %elivery o! %rugs &as a long &istory* Anti#o%ies) !or instance) (ere suggeste% early last century as a means to %irect anticancer %rugs to tumor cells in t&e #o%y e"pressing t&e correspon%ing antigen* T&eir use in t&e !orm o! monoclonals is no( at t&e !ore!ront o! targete% t&erapy* -ollo(ing a%vances in t&e %iscovery o! cell receptors) receptorE#in%ing macromolecules (ere a%%e% to t&e armamentarium o! systems !or t&e targeting o! %rugs* Parallel to t&ese %evelopments &as #een) since t&e early 4><@s) t&e e"ploitation o! liposomes as a %elivery system !or %rugs an% vaccines* T&ese superstructures) !orme% spontaneously !rom amp&ipat&ic lipi% molecules) toget&er (it& a %iverse collection o! ot&er promising superstructures %erive% !rom a &uge variety o! natural an% synt&etic monomeric or polymeric units) &ave evolve% to sop&isticate% versions t&roug& t&e incorporation onto t&eir sur!ace o! macromolecules t&at contri#ute to optimal p&armaco$inetics o! actives an% t&eir %elivery to (&ere t&ey are nee%e%* An ever increasing num#er o! %rugE an% vaccineE %elivery systems are #eing teste% clinically) (it& many alrea%y mar$ete%* Recently) %rugE%elivery systems &ave #een re%iscovere% as t&e #iological %imenE sion o! nanotec&nology* A lea%ing article in a prestigious scienti!ic .ournal tells us t&at Q#iologists are em#racing nanotec&nologyRt&e engineering an% manipulation o! entities in t&e 4 to 4@@ nm rangeRan% are e"ploiting its potential to %evelop ne( t&erapeutics an% %iagnostics*S ,&at else is ne(T) you mig&t sayU Nonet&eless) t&e pre!i" nano A!rom t&e Gree$ (or% !or %(ar! B is a use!ul one #ecause it &elps %e!ine %rugE%elivery systems o! a certain si/e range* Re!lecting t&is tren% o! si/e %e!inition) Nanoparticulate Drug Delivery Systems is a (ort&y attempt to #ring toget&er a (i%e range o! %rugE%elivery systems !or t&e %elivery Atargete% or ot&er(iseB) t&roug& a variety o! routes o! a%ministration) o! %rugs) %iagnostics) an% vaccines in t&e treatment or prevention o! %isease) no( encapsulate% in t&e term Qnanome%icine*S Importantly) t&e #oo$ inclu%es a (ealt& o! t&e latest a%vances in t&e tec&nology o! nanoparticulates) inclu%ing electrospinning) !ormation o! microcrystals) pro%uction o! liCui% crystalline p&ases) an%) last #ut not least) t&e tec&nology o! metallic nanoparticles* T&e e%itors) Deepa$ T&assu) Mic&el Deleers) an% 3as&(ant Pat&a$) are to #e complimente% !or #ot& t&eir .u%icial selection o! nanosystems an% c&oice o! t&e international panel o! contri#utors*

Gregory Gregoria%is T&e Sc&ool o! P&armacy University o! on%on on%on) U*0*

iii

Pre!ace

-or many %eca%es) t&e interest in mo%i!ying %rugE%elivery systems &as #een a prominent t&rust o! p&armaceutical researc&* In recent years) %ue to tremen%ous e"pansion in t&e %i!!erent scienti!ic %omains an% s$ill sets) t&e scope &as #een (i%E ene% to incorporate many !aculties in t&e %rugE%elivery researc& covering p&ysics) polymer sciences) electrical engineering) #ioelectronics) genetics) #iotec&nology) an% molecular p&armaceutics* P&armaceutical in%ustry researc& culture is !acing an uncertain !uture* Hig&er clinical %evelopment cost couple% (it& %eclining %rugE%iscovery process an% lo(er clinical success rates is %ecreasing t&e !lo( o! ne( c&emical entities in t&e researc& an% %evelopment pipeline* Due to t&e a%vent o! analytical tec&niCues an% capa#ilities to measure t&e particle si/es in nanometer ranges) particulate %rugE%elivery systems researc& an% %evelopment &as #een moving !rom t&e microE to t&e nanosi/e scale* Signi!icant researc& interests are geare% to(ar%s utili/ing t&e tec&niCues (&ere t&e particles can #e re%uce% almost to nanometer ranges) t&us re%ucing t&e %ose an% reactive nature o! t&e molecule* T&is can %eliver t&e %rug at t&e targete% sites* T&e #oo$ presente% &ere(it& is an attempt to %escri#e t&e researc& e!!orts #eing %one in t&is %irection #y t&e glo#al scienti!ic community* Nanoparticulate %rugE%elivery systems are a c&allenging area) an% t&ere are pulsating c&anges &apE pening almost every %ay* T&is is an attempt to cover t&e recent tren%s an% emerging tec&nologies in t&e area o! nanoparticulate %rugE%elivery systems* T&e !irst c&apter covers a complete overvie( o! t&e nanoparticulate %rugE %elivery system) covering (i%e applications an% evaluation o! t&e nanoparticulate %rugE%elivery system in various !iel%s* C&apter 5 encompasses !ormulations o! nanosuspensions !or parenteral %elivery* T&e t&ir% c&apter covers t&e polymerE #ase% nanoparticulate %rugE%elivery systems* C&apters 9 to ; !ocus on nano!i#ers) nanocrystals) an% lipi%E#ase% nanoparticulate %rugE%elivery systems) respectively* C&apters < to 4@ %iscuss t&e engineering aspects an% %i!!erent tec&niCues use% !or nanoparticulate %rugE%elivery systems) inclu%ing nanoengineering) aerosol !lo( reactor) supercoole% smectic nanoparticles) an% metallic nanoparticles) respectively* C&apters 44 an% 45 !ocus on #iological reCuirements an% t&e role o! nano#iotec&nolE ogy in t&e %evelopment o! nanome%icines* C&apters 48 to 54 e"tensively cover t&e applications o! nanoparticulate %rugE%elivery systems) inclu%ing lipi% nanopartiE cles !or %ermal applicationsM gene carriers !or restenosisM ocular) central nervous system) gastrointestinal applicationsM a%.uvant !or vaccine %evelopmentM an% transE %ermal systems* It is our &ope t&at t&is multiaut&ore% #oo$ on nanoparticulate %rugE%elivery systems (ill assist an% enric& t&e rea%ers in un%erstan%ing t&e %iverse types o! nanoparticulate %rugE%elivery systems availa#le or un%er %evelopment) as (ell as &ig&lig&t t&eir applications in t&e !uture %evelopment o! nanome%icines* T&is #oo$ is eCually relevant to aca%emic) in%ustrial) as (ell as scientists (or$ing in p&armaE ceutical %rug %elivery (orl%(i%e* T&e te"t is planne% in suc& a (ay t&at eac&

vi

Pre!ace

c&apter represents an in%epen%ent area o! researc& an% can #e easily !ollo(e% (it&E out re!erring to ot&er c&apters* ,e (oul% li$e to e"press our sincere t&an$s to Tony +en!onte !or t&e !igures in C&apters 4 an% 48 an% to in%a Glat&er !or rea%ing t&e manuscript an% suggesting corrections an% punctuation* Special t&an$s to our e%itors) Stevan Volo) 3vonne Honigs#erg) an% S&erri Ni/iole$) (&o &elpe% us to get t&roug& t&e pro.ect success!ully* ast) #ut not least) (e (oul% li$e to e"press our sincere gratitu%e to all t&e aut&ors (&o &ave ta$en time !rom t&eir #usy sc&e%ules to #e part o! t&is pro.ect an% (ritten (on%er!ul c&apters t&at a%%e% #ot& t&e %ept& an% value to t&is #oo$*

Deepa$ T&assu Mic&el Deleers 3as&(ant Pat&a$

Contents

-ore(or%Gregory Gregoria%is * * * * iii Pre!ace * * * * v Contri#utors * * * * i" 4* Nanoparticulate DrugEDelivery Systems6 An ?vervie( 4 Deepa$ T&assu) 3as&(ant Pat&a$) an% Mic&el Deleers 5* Nanosuspensions !or Parenteral Delivery 88 +arrett E* Ra#ino( 8* Nanoparticles Prepare% Using Natural an% Synt&etic Polymers :4 Su%&ir S* C&a$ravart&i) Dennis H* Ro#inson) an% Sin.an De 9* Nano!i#erE+ase% Drug Delivery ;4 Matt&e( D* +ur$e an% Dmitry u/&ans$y :* Drug NanocrystalsRT&e Universal -ormulation Approac& !or Poorly Solu#le Drugs <4 'an MFsc&(it/er an% Rainer H* MJller ;* ipi%E+ase% Nanoparticulate Drug Delivery Systems => 'un ,u) Wiao#in V&ao) an% Ro#ert '* ee <* Nanoengineering o! Drug Delivery Systems As&(at& 'ayagopal an% 2* Prasa% S&astri >>

=* Aerosol -lo( Reactor Met&o% !or t&e Synt&esis o! Multicomponent Drug NanoE an% Microparticles 444 'anne Raula) Hannele Eeri$Iinen) Anna I&%e) an% Es$o I* 0auppinen >* Supercoole% Smectic Nanoparticles 45> Hei$e +un.es an% 'u%it& 0untsc&e 4@* +iological an% Engineering Consi%erations !or Developing TumorETargeting Metallic Nanoparticle DrugEDelivery Systems 494 Giulio -* Paciotti an% a(rence Tamar$in 44* +iological ReCuirements !or Nanot&erapeutic Applications 4:> 'osep& -* C&iang 45* Role o! Nano#iotec&nology in t&e Development o! Nanome%icine 4<8 0* 0* 'ain

vii

viii 48* P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems 4=: 3as&(ant Pat&a$) Deepa$ T&assu) an% Mic&el Deleers 49* ipi% Nanoparticles ASoli% ipi% Nanoparticles an% Nanostructure% ipi% CarriersB !or Cosmetic) Dermal) an% Trans%ermal Applications 548 Eliana +* Souto an% Rainer H* MJller 4:* NanoECarriers o! Drugs an% Genes !or t&e Treatment o! Restenosis 58: Einat Co&enESela) 2ictoria Ela/ar) Hila EpsteinE+aras&) an% Gers&on Golom# 4;* ?cular Applications o! Nanoparticulate DrugEDelivery Systems 5<4 Annic$ u%(ig 4<* Nanoparticulate Systems !or Central Nervous System Drug Delivery 5=4 'eanEC&ristop&e ?livier an% Manuela Pereira %e ?liveira 4=* Nanoparticles !or Gene Delivery6 -ormulation C&aracteristics 5>4 'aspreet 0* 2asir an% 2ino% a#&aset(ar 4>* Gastrointestinal Applications o! Nanoparticulate DrugEDelivery Systems 8@: Maria Rosa Gasco 5@* Nanoparticles as A%.uvantE2ectors !or 2accination 84< Socorro Espuelas) Carlos Gama/o) MarXa 'osG +lancoEPrieto) an% 'uan M* Irac&e 54* Trans%ermal Applications o! Nanoparticulates 85< 'ong(on S&im In%e" * * * * 88>

Contents

Contri#utors

MarXa 'osG +lancoEPrieto Department o! P&armacy an% P&armaceutical Tec&nology) University o! Navarra) Pamplona) Spain Hei$e +un.es Department o! P&armaceutical Tec&nology) Institute o! P&armacy) -rie%ric& Sc&iller University 'ena) 'ena) Germany Matt&e( D* +ur$e Department o! P&armaceutical Development) Gla"oSmit&0line) Researc& Triangle Par$) Nort& Carolina) U*S*A* Su%&ir S* C&a$ravart&i Department o! P&armaceutical Sciences) University o! Ne#ras$a Me%ical Center) ?ma&a) Ne#ras$a) U*S*A* 'osep& -* C&iang Department o! C&emistry an% +ioc&emistry) State University o! Ne( 3or$ at ?neonta) ?neonta) Ne( 3or$) U*S*A*) an% Department o! C&emistry) Tsing&ua University) +ei.ing) C&ina Einat Co&enESela Department o! P&armaceutics) Sc&ool o! P&armacy) T&e He#re( University o! 'erusalem) 'erusalem) Israel Sin.an De Researc& an% Development) Perrigo Company) Allegan) Mic&igan) U*S*A*

Mic&el Deleers Glo#al P&armaceutical Tec&nology an% Analytical Development AGPTADB) UC+) +raine lPAlleu%) +elgium Hannele Eeri$Iinen P&armaceutical Pro%uct Development) ?rion Corporation ?rion P&arma) Espoo) -inlan% 2ictoria Ela/ar Department o! P&armaceutics) Sc&ool o! P&armacy) T&e He#re( University o! 'erusalem) 'erusalem) Israel Hila EpsteinE+aras& Department o! P&armaceutics) Sc&ool o! P&armacy) T&e He#re( University o! 'erusalem) 'erusalem) Israel Socorro Espuelas Department o! P&armacy an% P&armaceutical Tec&nology) University o! Navarra) Pamplona) Spain Carlos Gama/o Department o! Micro#iology) University o! Navarra) Pamplona) Spain Maria Rosa Gasco Nanovector srl) Torino) Italy

Gers&on Golom# Department o! P&armaceutics) Sc&ool o! P&armacy) T&e He#re( University o! 'erusalem) 'erusalem) Israel 'uan M* Irac&e Department o! P&armacy an% P&armaceutical Tec&nology) University o! Navarra) Pamplona) Spain 0* 0* 'ain 'ain P&arma+iotec&) +asel) S(it/erlan% As&(at& 'ayagopal +iomaterials) Drug Delivery) an% Tissue Engineering a#oratory) Department o! +iome%ical Engineering) 2an%er#ilt University) Nas&ville) Tennessee) U*S*A* Es$o I* 0auppinen NanoMaterials Group) a#oratory o! P&ysics an% Center !or Ne( Materials) Helsin$i University o! Tec&nology) an% 2TT +iotec&nology) Helsin$i) -inlan% i"

" 'u%it& 0untsc&e Department o! P&armaceutical Tec&nology) Institute o! P&armacy) -rie%ric& Sc&iller University 'ena) 'ena) Germany 2ino% a#&aset(ar Department o! P&armaceutical Sciences) University o! Ne#ras$a Me%ical Center) ?ma&a) Ne#ras$a) U*S*A* Anna I&%e NanoMaterials Group) a#oratory o! P&ysics an% Center !or Ne( Materials) Helsin$i University o! Tec&nology) Helsin$i) -inlan% Ro#ert '* ee Division o! P&armaceutics) College o! P&armacy) NCI Compre&ensive Cancer Center) NS- Nanoscale Science an% Engineering Center !or A!!or%a#le Nanoengineering o! Polymeric +iome%ical Devices) T&e ?&io State University) Colum#us) ?&io) U*S*A* Annic$ u%(ig Department o! P&armaceutical Sciences) University o! Ant(erp) Ant(erp) +elgium Dmitry u/&ans$y Department o! Corporate Tec&nology) Donal%son Company) Inc*) Minneapolis) Minnesota) U*S*A* Rainer H* MJller Department o! P&armaceutical Tec&nology) +iotec&nology) an% 7uality Management) -reie UniversitIt +erlin) +erlin) Germany 'an MFsc&(it/er Department o! P&armaceutical Tec&nology) +iotec&nology) an% 7uality Management) -reie UniversitIt +erlin) +erlin) Germany 'eanEC&ristop&e ?livier P&armacologie %es MG%icaments AntiEIn!ectieu") -aculty o! Me%icine an% P&armacy) an% INSERM) ERI @58) Poitiers) -rance Giulio -* Paciotti CytImmune Sciences) Inc*) Roc$ville) Marylan%) U*S*A* 3as&(ant Pat&a$ UC+ Manu!acturing) Inc*) Roc&ester) Ne( 3or$) U*S*A* Manuela Pereira %e ?liveira P&armacologie %es MG%icaments AntiEIn!ectieu") -aculty o! Me%icine an% P&armacy) an% INSERM) ERI @58) Poitiers) -rance 'anne Raula NanoMaterials Group) a#oratory o! P&ysics an% Center !or Ne( Materials) Helsin$i University o! Tec&nology) Helsin$i) -inlan% +arrett E* Ra#ino( +a"ter Healt&care Corporation) Roun% a$e) Illinois) U*S*A*

Contri#utors

Dennis H* Ro#inson Department o! P&armaceutical Sciences) University o! Ne#ras$a Me%ical Center) ?ma&a) Ne#ras$a) U*S*A* 2* Prasa% S&astri +iomaterials) Drug Delivery) an% Tissue Engineering a#oratory) Department o! +iome%ical Engineering) 2an%er#ilt University) Nas&ville) Tennessee) U*S*A* 'ong(on S&im Nanotec&nology Researc& Team) S$in Researc& Institute) RYD Center) Amorpaci!ic Corporation) 0younggi) Sout& 0orea Eliana +* Souto Department o! P&armaceutical Tec&nology) +iotec&nology) an% 7uality Management) -reie UniversitIt +erlin) +erlin) Germany a(rence Tamar$in CytImmune Sciences) Inc*) Roc$ville) Marylan%) U*S*A* Deepa$ T&assu UC+ P&arma) Inc*) Roc&ester) Ne( 3or$) U*S*A*

'aspreet 0* 2asir Department o! P&armaceutical Sciences) University o! Ne#ras$a Me%ical Center) ?ma&a) Ne#ras$a) U*S*A* 'un ,u Division o! P&armaceutics) College o! P&armacy) T&e ?&io State University) Colum#us) ?&io) U*S*A* Wiao#in V&ao Division o! P&armaceutics) College o! P&armacy) T&e ?&io State University) Colum#us) ?&io) U*S*A*

Nanoparticulate DrugEDelivery Systems6 An ?vervie(

Deepa$ T&assu
UC+ P&arma) Inc*) Roc&ester) Ne( 3or$) U*S*A*

3as&(ant Pat&a$
UC+ Manu!acturing) Inc*) Roc&ester) Ne( 3or$) U*S*A*

Mic&el Deleers
Glo#al P&armaceutical Tec&nology an% Analytical Development AGPTADB) UC+) +raine lPAlleu%) +elgium

INTR?DUCTI?N Nanotec&nology an% nanoscience are (i%ely seen as &aving a great potential to #ring #ene!its to many areas o! researc& an% applications* It is attracting increasing investE ments !rom governments an% private sector #usinesses in many parts o! t&e (orl%* Concurrently) t&e application o! nanoscience is raising ne( c&allenges in t&e sa!ety) regulatory) an% et&ical %omains t&at (ill reCuire e"tensive %e#ates on all levels* T&e pre!i" nano is %erive% !rom t&e Gree$ (or% %(ar!* ?ne nanometer AnmB is eCual to oneE#illiont& o! a meter) t&at is) 4@ Z> m* T&e term Qnanotec&nologyS (as !irst use% in 4><9) (&en Norio Taniguc&i) a scientist at t&e University o! To$yo) 'apan) re!erre% to materials in nanometers* T&e si/e range t&at &ol%s so muc& interest is typiE cally !rom 4@@ nm %o(n to t&e atomic level appro"imately @*5 nm) #ecause in t&is range materials can &ave %i!!erent an% en&ance% properties compare% (it& t&e same material at a larger si/e* -igure 4 s&o(s t&e nanometer in conte"t A4B* Nanotec&nologies &ave #een use% to create tiny !eatures on computer c&ips !or t&e last 5: years* T&e natural (orl% also contains many e"amples o! nanoscale structures) !rom mil$ Aa nanoscale colloi%B to t&e sop&isticate% nanosi/e% an% nanostructure% proteins t&at control a range o! #iological activities) suc& as !le"ing muscles) releasing energy) an% repairing cells* Nanoparticles ANPsB occur naturally an% &ave #een in e"istence !or t&ousan%s o! years as pro%ucts o! com#ustion an% coo$ing o! !oo%* Nanomaterials %i!!er signi!icantly !rom ot&er materials %ue to t&e !ollo(ing t(o ma.or principal !actors6 t&e increase% sur!ace area an% Cuantum e!!ects* T&ese !actors can en&ance properties suc& as reactivity) strengt&) electrical c&aracteristics) an% in vivo #e&avior* As t&e particle si/e %ecreases) a greater proportion o! atoms are !oun% at t&e sur!ace compare% to insi%e* -or e"ample) a particle si/e o! 8@ nm &as :[ o! its atoms on t&e sur!ace) at 4@ nm 5@[) an% at 8 nm :@[ o! t&e atoms are on sur!ace A4B* T&us) an NP &as a muc& greater sur!ace area per unit mass compare% (it& larger particles) lea%ing to greater reactivity* In tan%em (it& sur!ace area e!!ects) Cuantum e!!ects can #egin to %ominate t&e properties o! matter as si/e is re%uce% to t&e nanosE cale* T&ese can a!!ect t&e optical) electrical) an% magnetic #e&avior o! materials* T&eir in vivo #e&avior can #e !rom increase% a#sorption to &ig& to"icity o! nanomaterials*

METH?DS ?- MEASUREMENTS AND CHARACTERIVATI?N ?- NAN?MATERIA S Nanometrology is t&e science o! measurements at t&e nanoscale) an% its application un%erlies all t&e nanoscience an% nanotec&nology* T&e a#ility to measure an%
4

T&assu et al*

-IGURE 4 ASee color insert*B engt& scale s&o(ing t&e nanometer in conte"t* T&e lengt& scale o! interest !or nanoscience an% nanotec&E nologies is !rom 4@@ nm %o(n to t&e atomic scale appro"imately @*5 nm* Source6 -rom Re!* 4*

Nanoparticulate DrugEDelivery Systems

c&aracteri/e materials) as (ell as %etermine t&eir s&ape) si/e) an% p&ysical proE perties at t&e nanoscale is vital !or nanomaterials an% %evices* T&ese nee% to #e pro%uce% to a &ig& %egree o! accuracy an% relia#ility) to reali/e t&e applications o! nanotec&nologies* Nanometrology inclu%es lengt& an%1or si/e A(&ere %imenE sions are typically in nanometersB as (ell as measurement o! !orce) mass) electrical) an% ot&er properties* -our commonly use% tec&niCues are6 transmission electron microscopy ATEMB) scanning electron microscopy ASEMB) scanning pro#e tec&niCues Nscanning pro#e microscopy ASPMBO) an% optical t(ee/ers AsingleE#eam gra%ient trapB*

Transmission Electron Microscopy TEM is use% to investigate t&e internal structure o! microE an% nanostructures* It (or$s #y passing electrons t&roug& t&e samples an% t&en using magnetic lenses to !ocus t&e image o! t&e structure* TEM can reveal t&e !inest %etails o! t&e internal structure) in some cases t&e in%ivi%ual atoms* TEM (it& &ig&Eresolution transmission electron microscopy is t&e important tool !or t&e stu%y o! NP*

Scanning Electron Microscopy SEM uses t&e #asic tec&nology %evelope% !or TEM) #ut t&e #eam o! electrons is !ocuse% to a %iameter spot o! appro"imately 4 nm on t&e sur!ace o! t&e specimen an% scanne% repetitively across t&e sur!ace* It reveals t&at t&e sur!ace topograp&y o! t&e sample (it& t&e #est spatial resolution currently ac&ieve% is on t&e or%er o! 4 nm*

Scanning Pro#e Tec&niCues AScanning Pro#e MicroscopyB SPM uses t&e interaction #et(een a s&arp tip an% a sur!ace to o#tain t&e image* T&e s&arp tip is &el% very close to t&e sur!ace to #e e"amine% an% is scanne% #ac$ an% !ort&* As t&e tip is scanne% across t&e sample) t&e %isplacement o! t&e en% o! t&e cantilever is measure%) using a laser #eam* T&is can image insulating materials simply #ecause t&e signal correspon%s to t&e !orce #et(een t&e tip an% t&e sample) (&ic& re!lects t&e topograp&y #eing scanne%* T&e scanning tunneling microscope #roug&t a No#le pri/e !or p&ysics to Ger% +innig an% Heinric& in 4>=;* T&e atomic !orce microscope uses t&is SPM tec&niCue) (&ic& re!lects t&e sur!ace topograp&y o! t&e samples*

?ptical T(ee/ers ASingleE+eam Gra%ient TrapB ?ptical t(ee/ers use a single laser #eam A!ocuse% #y a &ig&ECuality microscope o#.ectiveB to a spot on t&e specimen plane* T&e ra%iation pressure an% gra%ient !orces !rom t&e spot create an optical trap) (&ic& &ol%s a particle at its center* Small interatomic !orces an% %isplacements can #e measure% #y t&is tec&niCue* Samples t&at can #e analy/e% range !rom single atoms to micrometerEsi/e% sp&eres to stran%s o! DNA an% living cells* Numerous traps can #e use% simultaneously (it& ot&er optical tec&niCues) suc& as scalpels) (&ic& can cut t&e particle #eing stu%ie%* 2arious analytical tec&niCues utili/e% in nanometrology are enumerate% in Ta#le 4*

MANU-ACTURE ?- NAN?MATERIA S T&ere are a (i%e variety o! tec&niCues t&at are capa#le o! creating nanostructures (it& various %egrees o! Cuality) spee%) an% cost* T&ese manu!acturing approac&es

9
TA+ E 4 Analytical Tec&niCues Use% !or C&aracteri/ation o! Nanoparticles Name o! t&e tec&niCue aser %i!!raction P&oton correlation spectroscopy ,i%eEangle WEray scattering Di!!erential scanning colorimetry Proton nuclear magnetic resonance spectroscopy Electron spin resonance Electron transmission microscopy Se%imentation velocity analysis an% EM D S an% cryoETEM D S an% TEM -lo( cytometry an% E ISA met&o% -luorometry -luorescence an% TEM Re!erence A5B

T&assu et al*

A8B A9B A:B A;B A<B A=B A>B

A##reviations6 D S) %ynamic lig&t scatteringM E ISA) en/ymeElin$e% immunosor#ent assayM EM) electron microscopyM NP) nanoparticleM TEM) transmission electron microscopy*

!all un%er t(o categories6 #ottomEup an% topE%o(n* -igure 5 illustrates t&e types o! materials an% pro%ucts (&ic& can #e manu!acture% using t&ese t(o approac&es A4B* +ottomEUp Manu!acturing +ottomEup manu!acturing involves t&e #uil%ing o! nanostructures atom #y atom or molecule #y molecule* T&is can #e %one in t&ree (ays6 c&emical synt&esis) sel!E assem#ly) an% positional assem#ly* C&emical synt&esis is a met&o% o! pro%ucing ra( materials) suc& as molecules or particles) (&ic& can t&en #e use% eit&er %irectly in pro%ucts in t&eir #ul$E%isor%ere% !orm or as t&e #uil%ing #loc$s o! more a%vance% or%ere% materials* -igure 8 repreE sents t&e generic processes t&at are involve% in t&e pro%uction o! NPs A4B6 4* Sel!Eassem#ly is a pro%uction tec&niCue in (&ic& atoms or molecules arrange t&emselves into or%ere% nanoscale structures #y p&ysical or c&emical interactions (it&in t&e smaller units* T&e !ormation o! salt crystals an% sno(!la$es (it& t&eir intricate structure are e"amples o! t&e sel!Eassem#ly process* Alt&oug& sel!E assem#ly occurs in nature) in in%ustry it is relatively ne( an% not a (ellE esta#lis&e% process A4B*

-IGURE 5 T&e use o! #ottomEup an% topE%o(n tec&niCues in manu!acturing nanoparticles* A##reviation6 MEMS) microelectromec&anical system* Source6 -rom Re!* 4*

Nanoparticulate DrugEDelivery Systems

-IGURE 8 T&e generic processes t&at are involve% in t&e pro%uction o! nanoparticles* Source6 -rom Re!* 4*

5* In positional assem#ly) atoms) molecules) or clusters are %eli#erately manipulate% an% positione% one #y one* Tec&niCues suc& as SPM !or (or$ on sur!aces or optical t(ee/ers in !ree space are use% !or t&is* Positional assem#ly is e"tremely la#orious an% rarely use% as an in%ustrial process*

TopEDo(n Manu!acturing TopE%o(n manu!acturing involves starting (it& a larger piece o! material) an% etc&ing) milling) or mac&ining a nanostructure !rom it #y removing material* TopE %o(n met&o%s o!!er relia#ility an% %evice comple"ity* T&ese are &ig&er in energy usage an% pro%uce more (aste t&an t&e #ottomEup met&o%s* Alt&oug& t&e nanotec&nologies &ave #een use% #y in%ustries !or many %eca%es Asemicon%uctor an% c&emical in%ustryB) it is still very muc& at in!ancy stage* In recent years) t&e tools use% to c&aracteri/e materials ATa#le 4B &ave le% to #etter un%erstan%ing o! t&e #e&avior an% properties o! matter on a very small scale* Increase% $no(le%ge o! t&e relations&ip #et(een t&e structure an% properties o! nanomaterials &as ena#le% t&e pro%uction o! materials an% %evices (it& &ig&er per!ormance an% increase% !unctionality* At t&e same time) t&ere are uncertainties (&ic& nee% to #e a%%resse% a#out t&e %irection t&at nanotec&nology (ill ta$e) an% a#out t&e &a/ar%s to &umans an% t&e environment t&at are presente% #y certain aspects o! t&is tec&nology A4@B* T&ere are several goo% reports an% revie(s (&ic& cover t&e pro%uction an% c&aracteri/ation o! NPs an% nanoparticulate %rugE%elivery systems ANPDDSsB* 2en$ates(arlu an% Man.unat& A44B &ave %iscusse% t&e preparation an% c&aracteriE /ation o! clo/apine NPs* T&ey use% &ot &omogeni/ation an% later ultrasonication met&o% to !ormulate soli%Hlipi% nanoparticles AS NsB incorporating clo/apine* Dingler an% Go&la A45B &ave %iscusse% t&e pro%uction o! S N an% scaling up stu%ies an% Gasco A48B &as patente% a met&o% !or pro%ucing S N* Me&nert an% Ma%er A49B &ave (ritten an e"cellent revie( a#out t&e S N pro%uction an% c&aracteri/ation* Many revie(s are reporte% #y Muller et al* A4:B an% ot&ers A5H:)4;H4=B* Rigal%ie et al* A4>B &ave s&o(n t&e &ig&E&y%rostaticEpressure tec&niCue to preserve an% sterili/e t&e sp&erulites) an NPDDS* Several papers an% patents are reporte% #y our group A5@H5;B an% Ro%rigue/ et al* %escri#e% a &ig&Epressure emulsi!ication an% &omogeni/ation process !or NPDDS preparation A4<B* Micro!lui%ics is #eing e"plore% !or applications in NPDDS* It is #ase% on instruments t&at are capa#le o! trans!erring small volumes o! liCui%s ranging !rom microliters to nanoliters* Micro!lui%ic Qla#EonEt&eEc&ipS tec&nology reCuires an un%erstan%ing o! t&e !orces t&at control !lui% movement an% reaction con%itions an% #rings t&e potential #ene!its o! miniaturi/ation) integration) an% automation*

T&assu et al*

Manu!acturing suc& c&ips com#ines met&o%s !rom microc&ip in%ustry (it& e"pertise in !lui% mec&anics) #ioc&emistry) an% &ar%(are engineering to create miniature integrate% #ioc&emicalEprocessing systems* A micro!lui%ics plat!orm provi%es #etter Cuality %ata an% allo(s s&orter assay %evelopment times* ?(ing to t&e %irect measurement at nanoscale an% t&e &ig&ECuality %ata generate% #y micro!lui%ics) t&is tec&nology plat!orm is !in%ing a place in %rug %iscovery as (ell as NPDDS A5<H84B*

DRUGEDE I2ER3 S3STEMS An i%eal %rugE%elivery system possesses t(o elements6 t&e a#ility to target an% to control t&e %rug release* Targeting (ill ensure &ig& e!!iciency o! t&e %rug an% re%uce t&e si%e e!!ects) especially (&en %ealing (it& %rugs t&at are presume% to $ill cancer cells #ut can also $ill &ealt&y cells (&en %elivere% to t&em* T&e re%uction or prevenE tion o! si%e e!!ects can also #e ac&ieve% #y controlle% release* NPDDS provi%e a #etter penetration o! t&e particles insi%e t&e #o%y as t&eir si/e allo(s %elivery via intravenous in.ection or ot&er routes* T&e nanoscale si/e o! t&ese particulate sysE tems also minimi/es t&e irritant reactions at t&e in.ection site* Early attempts to %irect treatment to a speci!ic set o! cells involve% attac&ing ra%ioactive su#stances to anti#o%ies speci!ic to mar$ers %isplaye% on t&e sur!ace o! cancer cells* Anti#o%ies are t&e #o%yPs means o! %etecting an% !lagging t&e presence o! !oreign su#stances* Anti#o%ies speci!ic to certain proteins can #e mass pro%uce% in la#oratories) ironiE cally using t&e cancer cells* T&ese approac&es &ave yiel%e% some goo% results) an% NPDDSs are %emonstrating lot o! potential in t&is area*

ipi%E+ase% Colloi%al Nano%rugEDelivery Systems ipi% nanocapsules are su#micron particles ma%e o! an oily liCui% core surroun%e% #y a soli% or semisoli% s&ell* NPDDSs (ere primarily %evelope% to com#ine t&e colloi%al sta#ility o! soli% particle suspensions in #iological !lui%s an% t&e solu#iE li/ing properties o! liCui%s A85)88B* S Ns (ere invente% at t&e #eginning o! 4>>@s an% are pro%uce% eit&er #y &ig&Epressure &omogeni/ation or #y microemulsion tec&niCue A89B* S Ns consist o! soli% matri" an% can #e %escri#e% as parenteral emulsions in (&ic& t&e liCui%Hlipi% oil is replace% #y a soli%Hlipi%* ?(ing to t&eir soli% particle matri") t&ey can protect incorporate% ingre%ients against c&emical %egra%ation A8:B an% allo( mo%i!ication o! release o! t&e active compoun%s A8;B* Homogeni/ation !ollo(e% #y ultrasonication (as use% !or t&e pro%uction o! clo/apineEloa%e% S Ns A44B* Colloi%al %rug carriers o!!er a num#er o! potential a%vantages as %elivery systems) suc& as #etter #ioavaila#ility !or poorly solu#le %rugs* ?t&er a%vantages o! t&ese S Ns inclu%e6 use o! p&ysiological lipi%s) t&e avoi%ance o! organic solvents in t&e preparation process) a (i%e potential application spectrum Aoral) %ermal) an% intravenousB) &ig&Epressure &omogeni/ation as an esta#lis&e% pro%uction met&o% A(&ic& allo(s largeEscale pro%uctionB) improve% #ioavaila#ility) protection o! sensiE tive %rug molecules !rom t&e environment A(ater an% lig&tB) an% a controlle% release c&aracteristic A49B* Common %isa%vantages o! S Ns inclu%e6 particle gro(t&) unpreE %icta#le gelation ten%ency) une"pecte% %ynamics o! polymorp&ic transitions) an% in&erently lo( incorporation capa#ilities %ue to crystalline structure o! t&e S N A49B* T&e $ey parameters in c&aracteri/ing t&e S N inclu%e6 concentration) nanocapE sule si/e an% s&ape) t&ic$ness) an% s&ell composition) %e!ining t&e !ree/eE%rying

Nanoparticulate DrugEDelivery Systems

<

-IGURE 9 Propose% topology !or lipi% nanocapsules !ree/eE%rie% in t&e presence o! tre&alose* Source6 -rom Re!* 8<*

con%itions suc& as cryoprotectant) pressure) an% temperature cycle* Some o! t&e !acE tors !or t&e !ormulation o! t&e lipi% NP are6 t&e %rug payloa% %epen%s on t&e oil content) t&e evolution o! t&e &y%rop&ilicHlipop&ilic #alance o! solutol HS4: is t&e %riving !orce o! S N !ormation A-ig* 9B) an% t&e S N %iameter %epen%s on #ot& t&e -oil1- solutol an% t&e solutol HS4:1 ipoi% S 4@@ ratios A8<B* +esi%es nanoemulsions) nanosuspensions A8=B) mi"e% micelles an% liposomes) meltEemulsi!ie% NPE#ase% lipi%s) an% soli%s at room temperature &ave #een %evelope% A4:B* T&e lo( incorpoE ration capa#ilities (ere overcome #y using liCui%Hlipi% nanostructure% carriers A8>B* Several e"cellent revie(s an% papers on t&e S N are reporte% A49)8=)9@H99B*

Recent Tren%s in Soli%H ipi% Nanoparticle Researc& Recently) a lipi%E#ase% solventE!ree !ormulation process &as #een %evelope% to prepare lipi% nanocapsules in t&e nanometer range A85)9:B* T&is process ta$es a%vantage o! t&e variation o! t&e &y%rop&ilicHlipop&ilic #alance o! an et&o"ylate% &y%rop&ilic sur!actant as a !unction o! t&e temperature) lea%ing to an inversion p&ase* In t&e !irst step) several temperature cycles applie% aroun% t&e inversionE p&ase temperature lea% to %roplet si/e %ecrease an% &omogeni/ation* In a secon% step) !ast cooling lea%s to t&e crystalli/ation o! t&e lecit&in Aintro%uce% in t&e !ormulation #ot& as lipop&ilic cosur!actant an% constituting material o! t&e rigi% s&ellB) (&ic& lea%s to t&e !ormation o! a sta#le lipi% nanocapsule suspension* T&is suspension can #e !ree/eE%rie% an% resuspen%e% in an aCueous me%ium e"tempoE raneously* T&e !ree/eE%rying can alter t&e topology o! t&e NPsM &ence (&ile %oing so) t&e structure o! t&e NPs nee%s to #e preserve% A8<B*

T&assu et al*

'ores et al* A5B &ave stu%ie% p&ysicoc&emical investigations o! S N an% oilE loa%e% S N using nuclear magnetic resonance an% electron spin resonance* T&ey &ave investigate% various tec&niCues to evaluate an% c&aracteri/e t&em using p&oton correlation spectroscopy* aser %i!!raction (as use% !or particle si/e %eterE mination) an% !iel% !lo( !ractionation (it& multiangle lig&t scattering %etection (as use% to separate t&e particles %ue to t&eir Sto$es ra%ius* It &elpe% in un%erstan%ing t&e topograp&y o! t&e particles* CryoETEM (as use% to stu%y t&e ultrastructure o! t&e NPs* S Ns &ave #een s&o(n to con%ense DNA into nanometric colloi%al particles capa#le o! trans!ecting mammalian cells in vitro A9;B* Compare% (it& stan%ar% DNA carriers suc& as cationic lipi%s or cationic polymers) S N o!!ers several tec&E nological a%vantages suc& as a relative ease o! pro%uction (it&out organic solvents) t&e possi#ility o! largeEscale pro%uction (it& Cuali!ie% pro%uction lines) goo% storE age sta#ilities) possi#ility o! steam sterili/ation) an% lyop&ili/ation A9<B* In a stu%y #y Ru%olp& et al* A9<B) a %iametric tyrosine aminotrans!erase ATATB pepti%e %erive% !rom t&e arginineEric& moti! o! t&e HI2E4 TAT protein t&at !unctions as nuclear locali/ation seCuence an% as a protein trans%uction %omain coul% #e use% to su#stantially en&ance gene trans!er e!!iciency o! S NE#ase% vectors) lea%ing to gene e"pression levels even &ig&er t&an o#serve% !or polyet&ylenimine APEIB gene vectors* T&is mig&t allo( aerosol application o! !ragile gene %elivery systems to lungs in t&e !uture stu%ies* T&e common groun% o! cationic liposome nanoemulsions A9=B an% S Ns !or trans!ection is t&e nee% !or cationic lipi%s to !acilitate %eo"yri#onucleic aci% ADNAB #in%ing* In liposome !ormulations) t&ese lipi%s are arrange% as #ilayers aroun% an aCueous core* Interaction (it& DNA initiates structural rearrangements into %i!!erent structures %epen%ing on t&e $in% o! lipi%) lipi%1DNA ratio) incu#ation me%ia) an% time A8=B* Ta#att et al* A9>B &ave s&o(n eCuipotency o! S Ns an% liposome !ormuE late% !rom t&e cationic lipi%s in in vitro DNA trans!ection e!!iciency* A stu%y #y 0ogure et al* A:@B %emonstrate% t&e %evelopment o! a multiE !unctional envelopeEtype nano%evice !or a geneE%elivery system* T&is containe% mem#raneEpermea#le pepti%e R= (it& less cytoto"icity* T&is system can incorporate various !unctional %evices suc& as a speci!ic ligan% to a speci!ic cell) intracellular sorting %evices t&at permit en%osomal escape) an% nuclear locali/ation* T&is lipi%E #ase% %evice can #e a use!ul tool !or gene %elivery !or gene t&erapy an% #ioc&emical researc& A-ig* :6 sc&ematic steps !or nano%evicesB* ee et al* A:4B reporte% an increase% sta#ility an% controlle% release o! lovastatin #y microencapsulating t&e %rugEloa%e% lipi% NPs* Several stu%ies &ave s&o(n t&e application o! S N !ormulation !or t&e %elivery o! paclita"el an% its proE%rug !or cancer treatment A:5B* Hou et al* A:8B &ave %escri#e% t&e mo%i!ie% &ig& s&ear &omogeni/ation an% ultrasoun% tec&niCues to pro%uce S Ns* Mo%el %rug mi!epristone (as incorporate% in S Ns) an% t&e mean particle si/e (as !oun% to #e 4@; nm* T&e %rug entrapment e!!iciency (as more t&an =<[ an% s&o(e% relatively long sta#ility) as t&e lea$ages (ere small* ?l#ric& et al* A:9B %escri#e% t&e potential %elivery o! &y%rop&ilic antitE rypanosomal %rug %imina/ine %iaceturate to t&e #rain o! in!ecte% mice !ormulating t&e lipi% %rug con.ugate NP #y com#ination o! stearic an% oleic aci%s* An e"cellent (or$ on an in vivo evaluation o! to#ramycin S Ns an% t&eir %uo%enal a%ministraE tion is %escri#e% #y Cavalli et al* A::B) an% is !urt&er %iscusse% in t&e !ollo(ing c&apters in %etail* ,illiams et al* A:;B &ave stu%ie% lipi%E#ase% NP !ormulation o! SN8=) a campE tot&ecin analog use% as antineoplastic %rug* T&ey s&o(e% improve% %rug loa%ing

Nanoparticulate DrugEDelivery Systems

>

-IGURE : T&ree steps involve% in constructing t&e multi!unctional envelopeEtype nano%evice* Source6 -rom Re!* :@*

an% goo% lactone sta#ility in t&e presence o! &uman serum al#umin AHSAB* T&e NPDDS s&o(e% prolonge% circulation in murine #loo% an% #etter e!!icacy against a resistant mo%el o! &uman colon carcinoma in nu%e mice* It (as also %emonstrate% t&at t&e #loo% &al!Eli!e o! SN8= (as greatly prolonge% #y incorporation in NPs*

Nanoparticulate Polymeric Micelles as Drug Carriers Polymeric micelles &ave attracte% muc& attention in %rug %elivery) partly #ecause o! t&eir a#ility to solu#ili/e &y%rop&o#ic molecules) t&eir small particle si/e) goo% t&ermo%ynamic solution sta#ility) e"ten%e% release o! various %rugs) an% prevenE tion o! rapi% clearance #y t&e reticuloen%ot&elial system ARESB A:<B* Critical micelle concentration ACMCB) similar to lo(EmolecularE(eig&t sur!actants) is t&e $ey c&arE acteri/ation parameter !or polymeric micelles* CMC is t&e concentration at (&ic& t&e amp&ip&ilic polymers in aCueous solution #egin to !orm micelles (&ile coe"istE ing in t&e eCuili#rium (it& t&e in%ivi%ual polymer c&ains or unimers* At CMC or slig&tly a#ove t&e CMC) t&e micelles !orm loose aggregates an% contain some (ater in t&e core A:=B* ,it& !urt&er increases in amp&ip&ilic polymer concentration) t&e unimer to micelle eCuili#rium s&i!ts to(ar%s micelle !ormation* T&e micellar strucE ture t&en #ecomes more compresse% an% sta#le) (&ereas resi%ual solvent is e"clu%e% !rom t&e core) an% t&e micelle si/e is re%uce%* T&e lo(er CMC values correlate to more sta#le micelles* T&is concept is especially important !rom t&e p&armacological point o! vie() as upon %ilution (it& a large volume o! t&e #loo%) micelles (it& &ig& CMC values may %issociate into unimers an% t&eir content may precipitate out) (&ereas t&e micelles (it& lo( CMC are more li$ely to remain t&e same* T&us) to %evelop improve% %rugE%elivery systems) amp&ip&ilic molecules t&at are a#le to !orm more sta#le micelles (it& lo(er CMC values are appropriate can%i%ates* A !ascinatE ing stu%y reporte% #y D.or%.evic et al* A:>B utili/e% scorpionEli$e amp&ip&ilic macromolecules* T&ey use% in%omet&acin as a mo%el %rug !or t&e stu%y an% reporte% t&is met&o% as convenient !or %rug %elivery (&ile minimi/ing %rug to"icity an% ma"imi/ing t&e %rug e!!ectiveness* Generally) t&e amp&ip&ilic core1s&ell structure o! polymeric micelles is !orme% !rom #loc$ copolymers) (&ic& are &y%rop&o#ic polymer c&ains lin$e% to &y%rop&ilic

4@

T&assu et al*

polymer c&ains A;@B* Association o! t&e &y%rop&o#ic portions o! t&e #loc$ copolyE mers creates t&e inner micelle core %ue to t&eir co&esive interactions (it& eac& ot&er in aCueous me%ia Ai*e*) &y%rop&o#ic interactionsB) (&ereas t&e outer &y%rop&ilic portions surroun% t&e inner &y%rop&o#ic core as a &y%rate% s&ell A;4B* Polymeric micelles are sel!Eassem#lies o! #loc$ copolymers in aCueous me%ia* Many a%vantages &ave #een %emonstrate% (it& t&eir uniCue core s&ell arc&itecture* Hy%rop&ilic s&ells !rom t&e aCueous e"terior segregate t&e &y%rop&o#ic cores* Hy%rop&o#ic %rugs can #e solu#ili/e% into t&e &y%rop&o#ic core structures o! polyE meric micelles at concentrations muc& &ig&er t&an t&eir intrinsic (ater solu#ility* Polymeric micelles are $no(n to &ave &ig& %rugEloa%ing capacity) &ig& (ater soluE #ility) an% appropriate si/e !or long circulation in t&e #loo% A;5B* T&e &y%rop&ilic s&ell surroun%ing t&e micellar core can protect un%esira#le p&enomena suc& as intermicellar aggregation) or precipitation) protein a%sorption) an% cell a%&esion* T&e c&emical composition o! t&e polymeric micelles can #e tailorEma%e to &ave %esira#le p&ysicoc&emical properties !or %rug solu#ili/ation A;8B* T&e &y%rop&o#ic %rug is incorporate% into t&e &y%rop&o#ic core #y interactions suc& as metalHligan% coor%ination #on%ing an% electrostatic interaction* T&e e"tent o! %rug solu#ility %epen%s on t&e compati#ility #et(een t&e %rug an% t&e micelle core A;9B* ?ne o! t&e limitations o! %rugEloa%e% polymeric micelles is lo( sta#ility in aCueous solution) an% t&e sta#ility #ecomes even lo(er as t&e %rugEloa%ing content increases A;:B* 2arious types o! %rugs can #e loa%e% into t&e &y%rop&o#ic core o! polymeric micelles #y c&emical con.ugation or p&ysical entrapolymeric micelles sent utili/ing various interactions suc& as &y%rop&o#ic interactions) or ionic interactions) or &y%roE gen #on%ing* -urt&ermore) t&e &y%rop&o#ic core serves as a reservoir !rom (&ic& t&e %rug is release% slo(ly over an e"ten%e% perio% o! time* T&e &y%rop&o#ic inner core is solu#ili/e% #y t&e &y%rop&ilic s&ell) (&ic& prevents t&e inactivation o! t&e coreEencapsulate% %rug molecules #y %ecreasing t&e contact (it& t&e inactivating species in t&e aCueous A#loo%B p&ase* As t&e outer &y%rop&ilic part o! t&e polymeric micelles interacts (it& #iocomponents suc& as cells an% proteins) it a!!ects t&eir p&armaco$inetics an% %isposition) as (ell as t&eir sur!ace properties A;;B*

Polymeric Micelles an% Solu#ili/ation o! Drugs Solu#ili/ation o! %rugs is a comple" mec&anism t&at involves %i!!erent parameters) !or e"ample) &y%rop&o#icity) molecular volume) crystallinity) !le"i#ility) c&arge) an% inter!acial tension against (ater* T&e lac$ o! (ater solu#ility &ampers t&e use o! many potent p&armaceuticals* Polymeric micelles are sel!Eassem#le% nanocarriers (it& versatile properties t&at can #e engineere% to solu#ili/e) target) an% release &y%rop&o#ic %rugs in a controlle% release !as&ion* Un!ortunately) t&eir largeEscale use is limite% #y t&e incorporation met&o%s availa#le* T&is poses a pro#lem (&en sterile %osage !orms are !ormulate%* Polymeric micelles present a core s&ell arc&iE tecture t&at results !rom t&e sel!Eassem#ly o! t&e amp&ip&ilic #loc$ polymers in a selective solvent a#ove a t&res&ol% concentration re!erre% to as critical association concentration A;<B* T&eir structure is suc& t&at t&e core provi%es an isolate% cargo space (&ere &y%rop&o#ic %rugs can partition* T&is is o! great signi!icance as many potent p&armaceuticals are &ig&ly &y%rop&o#ic #y nature* T&e nanometric si/e o! polymeric micelles varies !rom 4@ to 4@@ nm an% t&e !le"i#le &ig&ly &y%rate% corona minimi/es nonselective scavenging an% rapi% clearance #y t&e monolayer p&agoE cyte system* T&ese %rug carriers can e"travasate an% accumulate passively in regions presenting lea$y vasculatures suc& as tumors) in!lame% an% in!racte% tissues A;=B*

Nanoparticulate DrugEDelivery Systems

44

-IGURE ; Sc&eme o! t&e !ree/eE%rying proce%ure !or (aterEsolu#le amp&ip&ilic nanocarriers* Source6 -rom Re!* 99*

Recently) polymeric micelles &ave also #een s&o(n to %istri#ute to %e!ine% cytoE plasmic organelles A;>B an% increasing e!!orts are no( %irecte% at targeting t&e su#cellular components A99B* A simple met&o% to &ave &ig&er %rug loa%ing in t&e amp&ip&ilic nanocarriers polymeric micelles (as %evelope% #y -ournier et al* A99B* -igure ; s&o(s t&e sc&ematic pro%uction o! polymeric micelles an% its !ree/eE%rying proce%ure* Polymeric Micelles an% Reticuloen%ot&elial System Polymeric micelles provi%e an attractive c&aracteristic in t&at t&ey can avoi% upta$e o! t&e %rugs #y RES in vivo an% &ence t&ese can circulate in t&e #loo% !or a longer time* T&is a%vantage comes !rom t&e structure o! a micelle) t&e &y%rop&ilic portions o! t&e amp&ip&ilic #loc$ copolymer !orm t&e outer s&ell an% are e"pose% to #o%y !lui%) an% &ence t&e micelles can #e protecte% !rom p&agocytic cells an% plasma proteins in #loo%* Anot&er important #iological a%vantage o! polymeric micelles is t&e EPR>Een&ance% permea#ility an% retention e!!ect or passive targeting* As a result) polymeric micelles can slo(ly accumulate in malignant or in!lame% tissues %ue to t&e elevate% levels o! vascular permea#ility !actors in suc& cells A<@B* Polymeric micelles seem to #e i%eal carriers !or poorly (aterEsolu#le %rugs #ecause o! t&eir

45

T&assu et al*

%istinct a%vantages suc& as &ig& solu#ility) long circulation o! %rug in #loo%) perE meation o! an anticancer %rug #y t&e EPR e!!ect A<4B) an% simple sterili/ation* T&ey &ave t(o ma.or %isa%vantages6 p&ysical insta#ility upon %ilution limits t&eir p&armaceutical application an% (aterEsolu#le %rugs cannot #e in t&e micelles*

Recent Tren%s in Polymeric Micelles Researc& -rancis et al* A<5B &ave stu%ie% t&e polysacc&ari%eE#ase% polymeric micelles !or t&e %elivery o! cyclosporine A* T&ey %emonstrate% t&at coupling o! &y%rop&o#ic groups to (aterEsolu#le polysacc&ari%es signi!icantly promotes t&e solu#ili/ing po(er o! eit&er %e"tran or &y%ro"ypropyl cellulose AHPCB to(ar% cyclosporine A* T&e #ioa%E &esive properties o! HPC en&ance t&e association o! polymeric micelles to(ar% cacoE5 cell monolayers an% !acilitate internali/ation o! t&e polymer an% t&e transport o! t&e %rug* T&e polysacc&ari%eE#ase% polymeric micelles o!!er uniCue opportuniE ties !or t&e oral %elivery o! lipop&ilic %rugs* Similar stu%ies !or cyclosporine &ave #een reporte% using S N A<8B) polycaprolactone NPDDS A<9B) polyElactic aci% polyE et&ylene glycol NPs A<:B) an% c&itosan %erivatives A<;B* A ne( mo%ality o! %rug targeting tumors is #ase% on %rug encapsulation in polymeric micelles !ollo(e% #y a locali/e% triggering o! t&e %rug intracellular upta$e in%uce% #y ultrasoun%) (&ic& is !ocuse% into t&e tumor volume A<<B* A rationale #e&in% t&is approac& is t&at %rug encapsulation in polymeric micelles %ecreases a systemic concentration o! !ree %rug) %iminis&es intracellular %rug upta$e #y normal cells) an% provi%es passive %rug targeting o! tumors via en&ance% penetration an% retention e!!ect as a result o! a#normal permea#ility o! tumor #loo% vessels A<=B* Drugs targeting tumors re%uce un(ante% %rug interactions (it& &ealt&y tissues A<>B* ,it& micelle accumulation in t&e tumor interstitium) an e!!ective intracellular %rug upta$e #y t&e tumor cells s&oul% #e ensure%) ma$ing it possi#le !or ultrasonic irra%iation to #e use% A<<B* T&e in vitro an% in vivo e"periments &ave suggeste% t&at polymeric micelles can #e %egra%e% into unimers un%er t&e action o! ultrasoun%) (&ic& may provi%e an a%%itional a%vantage o! in vivo sensiti/ation o! multi%rugE resistant cells A=@B* It is suggeste% t&at t&is tec&niCue can #e use!ul in treatment o! ovarian carcinoma tumors o! small si/eM &ence early %etection is necessary !or tumor treatment*

P? 3MERE+ASED NAN?PARTICU ATE DRUGEDE I2ER3 S3STEMS Several polymers an% nonlipi% materials &ave #een evaluate% as carriers !or %rugs in t&e nanoparticulate !orms* T&ese materials &ave s&o(n %i!!erent properties an% a%vantages (&en !ormulate% as %rugE%elivery systems* A #rie! %escription o! eac& o! t&e polymeric systems !ollo(s*

Hy%rogelE+ase% Nanoparticulate DrugEDelivery Systems A progressively increasing interest &as #een pai% to sel!Eassem#le% &y%rogel NPs !rom &y%rop&o#i/e% (aterEsolu#le polymers %ue to t&eir potential #iome%ical an% p&armaceutical applications A=4B* T&e NPs &ave s&o(n various structural an% r&eological !eatures in aCueous solutions %epen%ing on t&e structure o! t&e parent (aterEsolu#le polymer) con.ugate% &y%rop&o#ic moiety or groups) an% t&e %egree o! su#stitution* T&e !ormation o! sel!Eassem#le% NPs is t&eori/e% #y a !reeEenergyE minimi/e% structure) s&aring a common !eature o! assem#ly o! polymeric micelles* Ho(ever) t&ere e"ists a %i!!erence in t&e interior structure #et(een NPs an%

Nanoparticulate DrugEDelivery Systems

48

polymeric micelles !orme% !rom amp&ip&ilic #loc$ copolymers* T&e interior o! polymeric NPs consists o! %isperse% multiple &y%rop&o#ic islan% %omains in a &y%rop&ilic sea %omain %ue to t&e ran%om association o! &y%rop&o#ic moieties con.ugate% to solu#le macromolecules* Polymeric micelles provi%e one inner core o! &y%rop&o#ic segments (it& a &y%rop&ilic s&ell A=5)=8B* T&e NPs !orme% !rom polyE mers containing moiety s(itc&ing its &y%rop&ilicity #y e"ternal stimuli is e"pecte% to e"&i#it stimuli responsive sur!ace property plus macroscopic &y%rogel #ul$ property* T&ese properties mig&t lea% to t&e accumulation o! t&e NPs at a %isease site an% t&e c&ange o! %rugErelease #e&avior !rom slo(EtoE!ast %rug release* An interesting stu%y using pullulan acetate1sul!onami%e con.ugates in sel!Eassem#le% NPs responsive to pH c&ange (as reporte% #y Na et al* A=4B* Amp&ip&ilic #loc$ copolymers are (i%ely stu%ie% as potential NPDDSs as t&ey are capa#le o! !orming aggregates in aCueous solutions A=9)=:B* T&ese aggreE gates are comprise% o! a &y%rop&ilic s&ell an% &y%rop&o#ic core* T&ey are goo% ve&icles !or %elivering &y%rop&o#ic %rugs #ecause t&e %rugs are protecte% !rom possi#le %egra%ation #y en/ymes* C&anging t&e composition o! &y%rop&o#ic an% &y%rop&ilic #loc$s on t&e polymer c&ains can vary t&e morp&ology o! NPs proE %uce% !rom amp&ip&ilic #loc$ copolymers* 2arious !orms o! morp&ologies suc& as sp&ere) vesicles) ro%s) lamellas) tu#es) large compoun% micelles) an% large comE poun% vesicles &ave #een reporte%* Some o! t&ese structures are goo% can%i%ates !or %rugE%elivery applications A=;B* Compare% (it& normal s&ell micelles) vesicles (it& a &y%rop&ilic core an% &y%rop&o#ic layers are #etter !or %rug %elivery* In t&e clinical stu%ies) it &as #een s&o(n t&at vesicles improve t&e treatment e!!icacy o! anticancer %rugs suc& as %o"oru#icin %ue to en&ance% permea#ility an% retention properties A=<B* T&e #loc$ copolymers comprise% o! commercial pluronic systems an% #io%egra%a#le polyAlactic aci%B are very goo% carriers !or %rug %elivery an% controlle% release applications A==B* V&ang et al* A=>B synt&esi/e% tri#loc$ copolymers o! polyAcaprolactoneE coElacti%eBH#EpolyAcaprolactoneEcoElacti%eB APC AHPEGHPC AB #y ringEopening copolymeri/ation o! caprolactone an% lacti%e in t&e presence o! polyet&ylene glycol* T&ey entrappe% an anticancer %rug) a camptot&ecin %erivative #y nanoprecipitation tec&niCue* T&e in vitro an% in vivo evaluation o! t&is NPDDS s&o(e% a potential !or use (it& poorly solu#le anticancer %rugs* T&ey %emonstrate% t&at t&e %rug release !rom t&ese systems can #e controlle% #y controlling t&e particle si/e) as t&ey !oun% t&e larger t&e NPs si/e) t&e lo(er (as t&e %rug release* T&e #o%y %istri#ution o! t&ese NPs s&o(e% t&at t&e #loo% concentration can #e maintaine% !or a longer time) an% t&e tissue #o%y %istri#ution (as a!!ecte% #y t&e particle si/e A>@B* Several ot&er groups &ave s&o(n t&e application o! t&e tri#loc$ copolymers !or NPDDSs A>4H>8B* 3oo an% Par$ A>9B &ave s&o(n !olate receptorEtargete% P GAHPEG micelles entrapE ping a &ig& loa%ing amount o! %o"oru#icin) s&o(ing #etter upta$e o! t&e %rug* T&e in vitro an% in vivo stu%ies &ave s&o(n t&e accumulation o! t&e %rug in t&e tumor cells in a siteEspeci!ic manner* An e"cellent revie( on #loc$ copolymer micelles !or %rug %elivery) %esign) c&aracteri/ation) an% #iological signi!icance is (ritten #y 0atao$a et al* A;@B* Anot&er revie( on applications o! polyAet&ylene o"i%eB #loc$ copolymerHpolyAamino aci%sB micelles is pu#lis&e% #y avasani!ar et al* A>:B* 2rie/ema et al* A>;H>=B &ave reporte% some interesting met&o%s to pro%uce #loc$ copolymers* NPs #ase% on &y%rogels are #eing %evelope% !or t&e %elivery o! macroE molecules) an% some o! t&e can%i%ates o! &y%rogel utili/e% !or t&is purpose are enumerate% in Ta#le 5* Many polymeric carriers (ere reporte% use!ul in t&e

49
TA+ E 5 Hy%rogel Matrices +ase% on natural materials Collagen Gelatin Starc& Alginates Synt&etic polymers PolyAnEvinyl pyrroli%ineB PolyAvinyl alco&olB PolyAp&osp&a/enesB PolyNet&ylene o"i%eE#EpolyApropylene o"i%eBO copolymers PE?HPP?HPAA gra!t copolymer P AGBA1PE?1PlAGAB copolymers P2AEgEP GA gra!t polymers PEGTHP+T copolymers ApolyactiveB MAHoligolacti%eHPE?Holigolacti%eHMA

T&assu et al*

Responsive polymers Met&acrylates PolyANEisopropylacrylami%eB CST PE?HPP?HPE? Pluronics

C&itosans De"trans

P GAHPE?HP GA A CSTB

Source6 -rom Re!* 4@9*

!ormulation o! NPDDSs) especially in t&e treatment o! cancer) !or e"ample) polyA5 et&ylE5Eo"a/olineB #loc$EpolyEcaprolactone A>>B) polyal$yl cyanoacrylate polymers A4@@B) P GA NPs A4@4B) polysacc&ari%e %ecorate% polyiso#utyl cyanoacrylate NPs A4@5B) an% serum al#umin NPs A4@8B* Den%rimerE+ase% DrugEDelivery Systems T&reeE%imensional treeEli$e #ranc&e% macromolecules possess some !ascinating c&aracteristics6 a (ellE%e!ine% structure) a very narro( molecular (eig&t %istri#ution) a t&reeE%imensional structure tune% #y %en%rimer generation an% %en%ron structure) an% !le"i#ility !or tailore% !unctional groups (it& &ig& %ensity on t&e perip&ery A;;B* Stu%ies o! #iome%ical application o! %en%rimers are #ecoming more an% more attractive especially in t&e !iel% o! nonviral gene vector an% NPDDS A4@:)4@;B* P&oto%ynamic t&erapy o! cancer involves t&e systemic a%ministration o! p&otosensiti/ers to soli% tumor tissues an% local illumination (it& lig&t o! a speci!ic (avelengt&) lea%ing to p&otoc&emical %estruction o! cancer cells via generation o! singlet o"ygen or supero"i%e !rom molecular o"ygen* Suita#le carriers an% %elivery o! p&otosensiti/ers s&oul% &ave a simple #ut e!!ective strategy to reali/e &ig& selectivity) &ig& p&oto%ynamic e!!icacy) an% &ave less si%e e!!ects* It is a c&allenge to !ormulate t&e p&otosensiti/ers* V&ang et al* A4@<B reporte% t&e use o! %en%rimer polymeric micelles !or t&e %elivery o! p&otosensiti/ers success!ully*

Calcium Car#onate Nanoparticles Ueno et al* A4@=B &ave reporte% t&e incorporation o! &y%rop&ilic %rugs an% #ioactive proteins into soli% calcium car#onate NPs* T&e si/e o! t&e NPs (as controlle% #y mi"ing spee% an% (as aroun% 4@: to 45= nm* T&ese CaC? 8 NPs (ere sta#le an% sustaine% t&e release o! t&e %rug #etamet&asone p&osp&ate*

Proticles6 ProtamineE+ase% Nanoparticulate Drug Carriers Protamine is a nonantigenic an% virtually nonto"ic pepti%e !rom t&e sperm) t&e compoun% %erive% !rom salmon) t&e most (i%ely use% source) an% &as a molecular mass aroun% :@@@ g1mol* It can #e use% as a carrier system !or %elivery o! DNA or oligonucleoti%es an% it is #eing use% as t&e cationic component* Several groups &ave %escri#e% t&e applications o! proticles as %rugEcarrier systems A4@>H445B* In most stu%ies) t&e pepti%e (as employe% toget&er (it& relatively large %ou#leEstran%e%

Nanoparticulate DrugEDelivery Systems

4:

DNA in a t(oEstep proce%ure* In t&e !irst step) it is con%ense% (it& DNA into a comE pact particle an% su#seCuently t&e comple" (as incorporate% into protamine or suitE a#le cationic liposomes* In some cases) trans!erring (as also use%* T&e term QproticlesS (as use% to represent oligonucleoti%es1protamine NPs #y Dinaure et al* A>:B* 2ogel et al* A4@>B s&o(e% inclusion o! HSA in t&e proticles le% to %ramatic sta#ility o! t&e particles* T&ey reporte% many a%vantages o! t&is system suc& as6 t&e proticle pro%uction #y sel!Eassem#ly is simple an% rat&er rapi%* T&e e"cipients use% are nonantigenic an% &ave very lo( to"icity an% are (ell accepte% in p&armaceuE tics* T&e particles are relatively sta#le in (ater an% cell culture me%ium* T&ey s&o( an increase% upta$e #y a variety o! cells) as compare% to na$e% oligonucleoti%es) an% a!ter cellular upta$e) t&e oligo%eo"yri#onucleoti%e A?DNB1protamine NPs rea%ily release t&e active agent* T&ey reporte% t(o %istinct %isa%vantages6 !irst) t&ey imme%iately s&o( massive aggregation an% precipitation (&en pro%uce% or trans!erre% into solutions containing salts at p&ysiological ionic strengt& or even at concentrations in t&e range o! mmol1l) an% secon%ly) t&ose containing t&e more sta#le Ap&osp&orot&ioates PT?sB instea% o! ?DNs A%iestersB %o not release t&eir nucleic aci% a!ter particle upta$e #y cells A4@>B*

C&itosanE+ase% Nanoparticulate DrugEDelivery System C&itosan) a polycationic polymer) comprising %Eglucosamine an% NEacetylE%E glucosamine lin$e% #y #EA4)9BEglycosi%ic #on%s) &as #een e"tensively researc&e% !or NPDDSs !or %elivering anticancer %rugs) genes) an% vaccines A448H44;B* In t&ese applications) it is important to assess t&e e!!ectiveness o! upta$e o! t&e carrier an% associate% %rug cargo into t&e target cells* C&itosan) #eing a natural polymer) is #ioE compati#le* C&itosan NPs (ere also evaluate% !or ocular applications* T&e cationic polysacc&ari%e c&itosan s&o(e% e"cellent properties suc& as #io%egra%a#ility) nonE to"icity) #iocompati#ility) an% mucoa%&esiveness) (&ic& are %esira#le !or t&e ocular %elivery systems* An interesting stu%y #y Campos et al* A44<B %emonstrate% t&at C&itosan NPs (ere a#le to interact an% remain associate% to t&e ocular mucosa !or an e"ten%e% perio% o! time) t&us promising carriers !or en&ancing an% controlling t&e release o! %rugs to t&e ocular sur!ace* A revie( pu#lis&e% #y He.a/i et al* A4@8B %iscusses various aspects o! c&itosanE%elivery systems covering t&e availa#ility) p&ysicoc&emical) an% #iological properties o! c&itosan* T&e revie( covere% various applications o! c&itosanE%elivery systems !or colonEspeci!ic %elivery) as a#sorption en&ancers) an% !or GI tract %elivery systems inclu%ing t&e NPDDS* Par$ et al* A44>B &ave assesse% t&e application o! sel!Eaggregates !orme% #y mo%i!ie% glycol c&itosan as a carrier !or pepti%e %rugs* T&ey e"&i#ite% compara#le #iological activity to parenteral pepti%es* -luorescein isot&iocyanate A-ITCBEla#ele% pepti%es (ere release% !rom t&e sel!Eaggregates in a sustaine% manner !or appro"imately a %ay* A report using c&itosan alginate com#ination nanosp&eres s&o(e% t&e utility o! t&ese nanosp&eres !or %rugE%elivery systems !ormulation A45@B* Sel!Eassem#le% NPs containing &y%roE p&o#ically mo%i!ie% c&itosan !or gene %elivery (as reporte% #y 3oo et al* A454B* T&ey %emonstrate% t&at mo%i!ie% glycol c&itosan NPs compose% o! &y%rop&o#i/e% DNA en&ance% t&e trans!ection e!!iciencies in vitro as (ell as in vivo* 0umar et al* A455B s&o(e% t&e application o! c&itosanE#ase% NPs in treating allergic ast&ma*

Silicone NanoporeEMem#raneE+ase% DrugEDelivery System TopE%o(n micro!a#rication tec&niCues &ave #een use% to create nanopore mem#ranes consisting o! arrays o! parallel rectangular c&annels) (&ic& range !rom < to :@ nm*

4;

T&assu et al*

T&e original met&o% (as pioneere% #y C&u et al* A458B an% consisting o! t(o #asic steps) sur!ace micromac&ining o! nanoc&annels into a t&in !ilm on t&e top o! t&e silicon (a!er an% !orming t&e nanopore mem#rane #y etc&ing a(ay t&e #ul$ o! t&e silicone (a!er un%erneat& t&e t&inE!ilm structure* T&e e"perimental an% mat&ematE ical results &ave s&o(n t&at t&e %evices out!itte% (it& silicone nanopore mem#ranes can regulate t&e %rugE%elivery $inetics o! a (i%e range o! %rugs* Moreover) t&e mec&anism o! release is attri#uta#le to a novel constraine% %i!!usion mec&anism provi%e% #y t&e precise geometry o! t&e nanopore mem#rane itsel!) an% no moving parts suc& as pistons are reCuire%* T&e %rugs can li$ely #e loa%e% into t&e %evice reservoir in a range o! p&ysical states) inclu%ing solutions) crystalline) or microE ni/e% suspensions* -le"i#ility (it& respect to t&e p&ysical !orm o! encapsulate% %rugs provi%es options to su#stantially increase t&e loa%e% %ose an% %uration o! t&e t&erapy) as (ell as promoting approac&es to increase sta#ility o! proteins) (&ic& are intrinsically unsta#le in an aCueous solution at #o%y temperature A459H45;B*

Polyester Polysacc&ari%e Nanoparticles NPs can #e prepare% !rom pre!orme% copolymers #y met&o%s suc& as emulsi!icationE solvent evaporation) nanoprecipitation) or salting out) all o! (&ic& reCuire %issoluE tion o! polymers in organic solvents* emarc&an% et al* A45<B reporte% a stu%y using inter!acial migrationEsolvent evaporation met&o% lea%ing to NP !ormation using a novel !amily o! amp&ip&ilic copolymers #ase% on De"tran gra!te% (it& polycapE rolactone si%e c&ains* T&ey reporte% t&at t&ese materials (ere !oun% to #e a#le to sel!Eorgani/e an% precipitate in t&e presence o! mi"tures o! (ater an% et&yl acetate* Et&yl acetate in a (ater emulsion (as sta#le an% pro%uce% t&e #est NPs*

Al#umin an% Gelatin Nanosp&eres Since t&e !irst reports on t&e preparation o! uni!ormly si/e% al#umin microsp&eres in t&e early 4><@s) t&ese #io%egra%a#le) #iocompati#le particles &ave !oun% various applications* Initially conceive% as a %iagnostic tool) al#umin particles &ave #een utili/e% as %rugEcarrier systems A45=B* More t&an 4@@ t&erapeutic an% %iagnostic agents (ere incorporate% into al#umin particles an% &ave #een investigate% !or intravenous) intramuscular) intraEarterial) an% intraEarticular a%ministration* Al#umin particles are (ell suite% !or %rug targeting an% %rug %elivery #ecause o! t&eir lac$ o! to"icity an% antigenicity* Compare% (it& ot&er colloi%al carrier systems suc& as liposomes) al#umin nanosp&eres &ave #etter sta#ility) s&el! li!e) controlla#le %rugE release properties) an% &ig&er loa%ing properties !or &y%rop&ilic molecules %ue to %rugE#in%ing properties o! native al#umin* Al#umin particles can #e o#taine% #y many met&o%s* In a stu%y #y Muller et al* A45=B) t&ey optimi/e% t&e manu!acturing tec&niCues o! al#umin nanosp&eres (it& average %iameter o! 5@@ nm* T&ey stu%ie% t&e e!!ect o! !ive %i!!erent process varia#les on particle si/e) poly%ispersity) an% yiel%) to optimi/e t&e preparation tec&niCue to reac& su#E5@@Enm particles* An interesting novel %rugE%elivery system !or improve% allEtrans retinoic aci% AatRAB t&erapy !or e"ternal treatments o! p&otoE%amage% s$in (as %evelope%* T&e researc& team prepare% inorganicEcoate% atRA NPs using #oun%aryEorgani/e% reacE tion %roplets* T&e inter!acial properties o! organic arc&itecture in atRA micelles (ere use% to template t&e nucleation o! inorganic materials* ,&en a%ministere%) t&ey !oun% a #oost in t&e pro%uction o! &yaluronan among t&e intercellular spaces o! t&e #asal an% spinous cell layers o! t&e epi%ermis* NanoEatRA tec&nology !or atRA t&erapy coul% not only e!!iciently regulate $eratinocyte cell proli!eration an% %i!!erentiation)

Nanoparticulate DrugEDelivery Systems

4<

#ut also mar$e%ly pro%uce t&e a%%itional #ene!it* Human s$in severely in.ure% #y c&ronic ultraviolet irra%iation may #e completely repaire% %ue to t&e accelerate% turnover o! s$in tissue) (&ic& is in%uce% #y nanoEatRA A45>H484B* Anti#o%yEmo%i!ie% gelatin NPs &ave #een reporte% to #e a carrier system !or targeting t&e speci!ic TElymp&ocytes #y +alt&asar et al* A485B* Gelatin NPs (ere !orme% #y t(oEstep %esolvation process* T&ey s&o(e% t&e utility o! t&is system !or targeting t&e lymp&ocytes*

Polymeric Nanocapsules as Drug Carriers T&ese (ere !irst prepare% #y solu#ili/ation o! t&e outer s&ell material in an organic solvent A488B* Interesting #iop&armaceutical per!ormances o! %rugs encapsulate% in polymeric NPs &ave #een reporte% !or t&e oral A489B) t&e parenteral A48:)48;B) an% t&e ocular routes A48<B* Ho(ever) t&e in%ustrial constraints o! solvent &an%ling) limite% scale) an% particular e!!orts nee%e% to %ecrease resi%ual solvent %o(n to !e( parts per million in%uce% &ig& manu!acturing costs* A clear aCueous nano%ispersion o! porpo!ol) a lipop&ilic anest&etic agent) (as %evelope% #y C&en et al* A48=B) (&ic& possesse% p&ysical an% c&emical sta#ility* It &a% #etter re% #loo% cell compati#ility an% improve% micro#ial resistance compare% (it& t&e mar$ete% pro%uct %iprivan t&at is an oilE#ase% emulsion* T&ey s&o(e% t&e ne( nano%ispersion using a com#ination o! polo"amer) PEG 9@@) polysor#ate =@) propylE ene glycol) an% citric aci% $no(n as TPI 548 - A48=B* An interesting stu%y o! t&e in vitro %egra%ation o! polymer polyE%lElactic aci% APD AB) polyE%lElacticEcoEglycolic aci% AP GAB) an% polyet&yleneEo"i%eE#ase% NPs s&o(e% t&at it too$ t(o years to %egra%e t&e PD AE#ase% NPs) (&ereas it too$ 4@ (ee$s to %egra%e P GA NPs A48>B* PolyAmet&yl vinyl et&erEcoEmaleic an&y%ri%eB AP2A1MAB is a #io%egra%a#le polyEan&y%ri%e (i%ely use% !or %eveloping polymeric micelles as NPDDSs (&ic& possess #ioa%&esive as (ell as mucoa%&esive properties* Ar#os et al* A49@B reporte% t&e application o! t&ese in t&e !ormulation o! NPDDSs an% s&o(e% t&at t&e #ioa%E &esive properties o! t&ese NPs appear to mo%ulate gastrointestinal transit pro!iles* An interesting stu%y (as reporte% #y uu et al* A494B on %eveloping polymeric micelles o! nanostructure% DNA %elivery sca!!ol% #y electrospinning o! P GA Apolylacti%eEcoEglycoli%eB an% P AHPEG ApolyA%lElacti%eBEpolyAet&ylene glycolBB* T&ey s&o(e% t&at t&e release o! plasmi% DNA (as sustaine% over 5@ %ays) an% t&e DNA release% (as structurally intact an% capa#le o! cell trans!ection an% #ioactivE ity* It (as t&e !irst success!ul %emonstration o! plasmi% DNA incorporation into a polymer sca!!ol% using electrospinning* ?t&er groups &ave also use% t&e nanosi/e% sca!!ol% !or %rug %elivery success!ully !or vascular en%ot&elial gro(t& !actor A495B) !or osteotropic !actors A498B) an% !or plasmi% DNA A499B* A &y%rotropic polymer system using N)NE%iet&yl nicotinami%e (as reporte% to #e use!ul !or t&e NPDDS o! t&e poorly (aterEsolu#le %rug paclita"el* T&e micelles range% #et(een 8@ an% :@ nm an% coul% #e easily re%issolve% in an aCueous system* T&ey %emonstrate% &ig&er loa%ing capacity an% p&ysical sta#ility compare% (it& ot&er polymeric micelles A;;B* Son et al* A48@B &ave s&o(n t&e accumulation o! %o"oru#icinEloa%e% glycol c&itosan nanoaggregates in tumor cells #y en&ance% an% permeation e!!ects* Several ot&er stu%ies also reporte% t&e accumulation o! %rugs in tumor cells in vivo using NPDDSs A49;)49<B* Anee% A49=B &as (ritten an e"cellent overvie( o! t&e current %rugE%elivery systems use% !or cancer gene t&erapy) covering #ot& t&e viral an% nonviral vectors !or carrying t&e t&erapeutic genes*

4=

T&assu et al*

Polystyrene Nanosp&eres T&ese mono%isperse polystyrene microparticles) also calle% late" microsp&eres) are use% in a (i%e range o! immuno%iagnostic assays) as si/e stan%ar%s !or cali#ration o! eCuipolymeric micellesent) in cell #iology applications) an% so on* T&e p&ysical a%sorption o! t&e polystyrene particles is use% to #in% ligan%s to t&e sur!ace o! t&e particles* Sa$uma et al* A49>B stu%ie% t&e mucoa%&esion o! polystyrene NPs &aving sur!ace &y%rop&ilic polymeric c&ains in t&e GI tract in rats* T&ey reporte% t&at t&e mucoEa%&esion o! polyANEisopropylacrylami%eB NPs strongly en&ance% t&e a#sorpE tion o! salmon calcitonin* Anot&er interesting stu%y s&o(ing applications o! polystyE rene NPs (as reporte% #y Haya$a(a et al* A4:@B* To esta#lis& an e!!ective tool !or t&e prevention o! HI2E4 transmission) lectinEimmo#ili/e% polystyrene NPs (ere synt&eE si/e% an% e"amine% !or t&eir HI2E4 capture activity* It s&o(e% t&at (&en concanavaE lin A (as immo#ili/e% on t&e sur!ace o! polystyrene NPs Amean %iameters o! 9@@ nmB (it& polyAmet&acrylic aci%B #ranc&es an% incu#ate% (it& HI2E4 suspension at room temperature !or ;@ minutes) t&e NPs ac&ieve% \8*8log an% a 5*5log re%uction o! viral in!ectivity in HI2E4 suspension at a concentration o! 5 an% @*: mg1ml) respectively) %emonstrating t&e potential o! t&is tec&niCue !or prevention o! viral transmission A4:@B* Similar stu%ies (ere also reporte% #y A$as&i et al* A4:4B* ?ga(ara et al* A4:5B (rote a revie( on &epatic %isposition o! polystyrene NPs an% t&e implications !or rational %esign o! particulate %rug carriers* T&e clearance o! colloi%al particles !rom t&e #loo% circulation occurs #y p&agocytosis an%1or en%ot&elial cells) mainly in t&e liver) spleen) an% #one marro(* T&e relative %istri#ution o! t&e in.ecte% particles in t&ese organs is $no(n to %epen% on various !actors suc& as t&e si/e an% t&e sur!ace properties o! t&e particles) an% t&e type o! serum proteins a%sor#e% onto t&e sur!ace o! t&e particles* T&e #asic principles #e&in% t&eir %istri#ution c&aracteristics into t&e RES) &o(ever) remain unclear A4:5B* An interesting stu%y reporte% #y ?ga(ara et al* A4:8B s&o(e% t&at precoating (it& serum al#umin &as re%uce% t&e receptorE me%iate% &epatic %isposition o! polystyrene nanosp&eres* T&is tec&niCue can #e use% to prevent rapi% clearance #y t&e mononuclear p&agocyte system in vivo*

S?ME C?MMERCIA 3 A2AI A+ E NAN?PARTIC ES Melamine Nanosp&eres T&e melamine Apolymet&ylenemelamineB nanosp&eres an% microsp&eres are ma%e !rom crossElin$e% melamine an% &ave some a%vantages %epen%ing on t&e applicaE tion compare% (it& polystyrene particles* T&ey &ave a &ig&er %ensity A4*:4 g1cm 8B) are very sta#le) can #e store% in%e!initely) can #e resuspen%e% in (ater) %o not s(ell or s&rin$ in most organic solvents) an% are &eat resistant up to 8@@] C* T&ese monoE %isperse AC2 4H5[B melamine microparticles are &y%rop&ilic an% can #e suspen%e% in (ater an% t&eir re!ractive in%e" is 4*;=* T&e sur!ace o! plain melamine microparE ticles is terminate% (it& met&ylol groups) (&ic& coul% #e rea%ily !unctionali/e% in t&e %esire% manner A4:9B*

Plain Polymet&yl Met&acrylate an% +io%egra%a#le Polylacti%e Nanosp&eres Plain polymet&yl met&acrylate particles are availa#le as 4@[ suspension) (&en &ig&er concentrations !or pro%uction are necessary A4:9B* Polylacti%e AP AB is a #ioE %egra%a#le t&ermoplastic %erive% !rom lactic aci%* It resem#les clear polystyrene) provi%es goo% est&etics Agloss an% clarityB) #ut it is sti!! an% #rittle an% nee%s mo%iE !ications !or most practical applications Ai*e*) plastici/ers increase its !le"i#ilityB*

Nanoparticulate DrugEDelivery Systems

4>

T&ese particles are ma%e !rom P A (it& a %ensity o! 4*@5 g1cm 8* T&ey are supplie% as 4[ aCueous solution A4@ mg1mlB an% are sta#le at a neutral pH !or at least t&ree mont&s* Degra%ation starts t&roug& #asic or aci%ic pH or en/ymatic &y%rolysis*

Magnetic Plain De"tran Nanosp&eres T&e super paramagnetic NPs on t&e #asis o! %e"tran (it& a si/e o! 5:@ nm &ave a magnetite content o! >@[* A permanent magnet can easily separate :@ an% 4@@ nm particles !rom 48@ an% 5:@ nm particles* Suc& smaller si/es can only #e separate% #y a Q&ig& gra%ient magnetic !iel%S %evice*

Gol% Nanosp&eres Gol% particles are o! &ig&est Cuality an% can #e use% in t&e pro%uction o! %iagnostic tests as (ell as con.ugation stu%ies o! proteins an% anti#o%ies* T&e particles &ave a very narro( si/e %istri#ution AC2 #et(een :[ an% 4:[ %epen%ing on si/eB an% are availa#le !rom 5 to 5:@ nm* T&e num#er o! particles1ml is given in t&e pro%uct1 or%ering ta#le* T&e solutions are sta#ili/e% (it& HAuCl 9* Gol% an% silver colloi%s or sols are availa#le in a num#er o! %i!!erent si/es* T&ere are 49 %i!!erent gol% colloi% si/es an% are o!!ere% in !our pac$ing si/es* T&e pro%ucts are #est store% at room temperature) alt&oug& storage at 9]C is an option* Ho(ever) temperatures too close to !ree/ing (ill %esta#ili/e t&e sol) causing aggregation an% pro%uct loss A4:9B*

Silver Nanosp&eres Silver nanosp&eres are o! &ig&est Cuality an% can #e use% in t&e pro%uction o! %iagE nostic tests as (ell as con.ugation stu%ies o! proteins an% anti#o%ies* T&e particles &ave a very narro( si/e %istri#ution AC2 #et(een 4@[ an% 5@[ %epen%ing on si/eB an% are availa#le !rom 5 to 5:@ nm*

Silica Nanosp&eres T&ese monoE%isperse silica particles (it& a %ensity o! 5*@g1cm 8 are simple to %ispense an% to separate* Alt&oug& polystyrene particles A% ^ 4*@9B are %i!!icult to separate #y centri!ugation un%er a si/e o! :@@ nm) silica particles %o set %o(n easily an% are easy to resuspen%* T&e silica particles are sta#le in (ater an% organic solvents) pro%uce% un%er a ne( %ying met&o%* Silica particles are easy to !unctionE ali/e an% availa#le as !luorescent particles* T&ey are use!ul !or coupling o! DNA) oligonucleoti%es) oligopepti%es) proteins) lectins) an% anti#o%ies* T&e silica particles are also availa#le (it& %i!!erent !unctional groups as HNH 5) an% HC??H) al#umin) protein A) epo"y) NHS) NTA) an% EDTA A4:9B* i et al* A4::B &ave prepare% an% c&aracteri/e% porous &ollo( silica NPs !or controlle% release applications* T&ey reporte% a novel met&o% !or preparing &ollo( silica nanosp&eres (it& a porous s&ell structure via t&e solHgel route an% using inorganic calcium car#onate NPs as a template (it& 4@@Enm %iameter an% a (all t&ic$ness 4@ nm o! t&e nanosp&eres* Several !actors (ere !oun% to a!!ect t&e %rug release rate !rom t&e nanosp&eres A4::B*

Alumina Nanosp&eres Alumina nanosp&eres an% microsp&eres &ave #een use% in various applications #ecause o! t&eir si/e uni!ormity an% &ig& %egree o! sp&erical particles Aas a result) &ig& !lo(a#ility an% &ig& pac$ing %ensityB* Properties o! alumina) suc& as &ig&

5@

T&assu et al*

t&ermal con%uctivity) &eat resistance) &ar%ness) an% so on) are use!ul in !ormulation* T&ese particles are o!ten use% as !illers !or t&ermal con%uctivity s&eets* Alumina nanosp&eres are availa#le (it& plain) amino) an% car#o"yl !unctional groups* T&e alumina microsp&eres an% nanosp&eres can #e prepare% (it& many !unctional groups suc& as al#umin) protein A) epo"y) NHS) NTA) EDTA) an% many ot&ers* T&ey are use!ul !or coupling o! DNA) oligonucleoti%es) oligopepti%es) proteins) lectins) an% anti#o%ies*

Car#on Nanotu#es In t&e last 4: years) it &as #een an e"citing time !or t&e !iel% o! car#on nanomaterials* T&e %iscoveries o! !ullerenes an% car#on nanotu#es &ave attracte% t&e attention o! many researc&ers all over t&e (orl% A4:;B* Ii.ima A4:<B !irst %iscovere% t&ese in 4>>4* T&ey are no( commercially availa#le an% can #e manu!acture% on large scales A4:=B* T&ere are several companies (&o manu!acture t&e car#on nanotu#es an% gra%ually t&eir prices are re%ucing* T&ey &ave a great potential in t&e %rugE%elivery systems an% many ot&er applications A4:>B*

DI2ERSE AND EMERGING TRENDS IN NAN?TECHN? ?G3 APP ICATI?NS Nanotec&nology is ma$ing a great impact in many areas an% some o! t&e ma.or areas are as !ollo(s* +iological Analysis an% Discovery T&e #asic science #e&in% i%enti!ying t&e presence o! a particular gene or protein &as #een %eveloping !or some time* T&e intro%uction o! nano!lui%ics o!!ers an e"cellent opportunity to (or$ (it& smaller amounts o! material* It can #e use% to segregate proteins an% nucleic aci%s ADNA an% RNAB #ase% on si/e an% s&ape* Nanomem#ranes o!!er a great potential in t&is area A4;@B* T&ere are several groups (or$ing on t&e i%ea o! passing a single DNA or RNA t&rea% t&roug& a nanosi/e% pore) !orcing it to straig&ten out an% pass t&roug& a #ase part o! !un%amental co%ing element o! nucleic aci% A4;4)4;5B* C&anging electrical gra%ients on eit&er si%e o! t&e structure containing t&e pore) or Cuantum tunneling current across t&e pore) coul% #e use% to i%enti!y t&e particular #ase t&at is passing t&roug&* T&e a#ility to seCuence a (&ole genome) t&e sum total o! genes in an organism in a matter o! &ours) &as #een proE pose% as a potential application o! t&is approac&* T&e impact o! t&ese tec&niCues in !ormulation o! %rugE%elivery systems an% t&eir t&erapeutic applications (ill #e (ort& (atc&ing %uring t&e coming !e( years*

Nanoparticles Tagging Anot&er #oon to analysis (ill li$ely come !rom t&e attac&ing o! NPs to molecules o! interest* NPs small enoug& to #e&ave as Cuantum %ots can #e ma%e to emit lig&t at varying !reCuencies* T&ese attac&e% molecules can #e spectroscopically measure% an% (ill allo( many %i!!erent molecules to #e measure% !rom t&e same single sample* Anot&er similar approac& relies on getting t&e molecules to #in% nano(ires t&at &ave stripes on t&em similar to #ar co%es* T&ese %etection tec&nologies are getting closer to commerciali/ation* T&ese can also !urt&er #e applica#le in t&e %evelopment o! %rugE%elivery systems A4;8B*

Nanoparticulate DrugEDelivery Systems

54

Nanostructure% Materials Nanostructure% materials couple% (it& liCui% crystals an% c&emical receptors o!!er t&e possi#ility o! c&eap) porta#le #io%etectors t&at mig&t) !or instance) #e (orn as a #a%ge* Suc& a #a%ge coul% c&ange color in t&e presence o! a variety o! c&emicals an% (oul% &ave applications in &a/ar%ous environments A4;8B* T&ese can also #e e"plore% as NPDDSs*

SingleEMolecule Detection T&ere are met&o%s to %etect a single p&oton* I! one can ma$e a nanostructure suc& as a Cuantum %ot (&ic& (ill emit a p&oton in t&e presence o! a particular molecule) one can ma$e a %evice t&at can %etect a single molecule o! a su#stance* 2arious groups are (or$ing in t&is %irection* T&e $ey commercial aspects o! t&e use o! microE an% nanotec&nology in #io%etection relate to porta#ility) cost) an% sensitivity A4;9B*

Protective Nanoparticles Against Pat&ogens NPs are %isruptive to #acteria an% viruses simply #y virtue o! t&eir p&ysical nature* T&is &as le% to i%eas o! lacing !a#rics in &ospitals (it& suc& NPs or nanoparticulate creams t&at can #e sprea% on t&e #o%y or sprays t&at can #e in&ale%) protecting against various pat&ogens A4;8B*

Nanotu#es an% Cellular Manipulation Nanotu#es an% cellular manipulation &ol% a great promise o! #eing use!ul in #ioE logical applications an% as NPDDSs* T&ere are tu#es small enoug& to suc$ out a nucleus !rom a cell an% place it into anot&er* T&is is t&e tec&niCue #e&in% cloning #ut nanotu#es are !iner still an% o!!er t&e potential o! ma$ing pro#es an% %elivery mec&anisms t&at can #e even more precise A4;:)4;;B*

Nanoengineere% Prost&etics T&ere are several %evices (&ic& can #e plugge% into parts o! central nervous sysE tems ACNSsB) %esigne% !or processing visual or au%itory in!ormation* Nanotec&nology is c&anging all t&at6 t&ere is a potential !or replacing a !e( o! our organs !or seeing) &earing) an% touc&) alt&oug& connecting to CNS an% avoi%ing classical pro#lems o! re.ection (ill #e a c&allenge* Synergies #et(een our application areas) materials (it& greatly improve% strengt& an% %esigner sur!ace properties o!!er potential !or use in all manner o! implants) !rom arti!icial &earts to &ip implants* All t&ese (ill &ave e"cellent potential in NPDDSs in t&e near !uture A4;8B*

T&iomer Nanoparticles T&iolate% polymers %esignate% as t&iomers are t&iol si%eEc&ainE#earing polymers* ?(ing to t&e intro%uction o! t&ese !unctional groups) t&iomers e"&i#it comparatively stronger mucoa%&esive) co&esive) en/yme in&i#itory) an% permeationEen&ancing properties* T&ey &ave e"cellent potential in t&e !ormulation o! t&e NPDDSs* Several attempts are going on %eveloping NPDDSs !or insulin an% calcitonin using t&iomer NPs* A nasal %elivery system !or &uman gro(t& &ormone) a pulmonary %elivery system !or vasoactive intestinal pepti%e) a noninvasive %elivery system !or t&e 0 H antigen) an% nasal %elivery systems !or amyloi%E#in%ing pepti%es are #eing generE ate% an% researc&e% using t&iomer polymeric NPs A4:9)4;<H4;>B*

55

T&assu et al*

Nanostructure% Monolit&s -unctional nanostructure% monolit&s are #eing %evelope% an% optimi/e% !or t&e analysis o! protein an% pepti%es #y nanoEHP CHMS1MS* RingEopening metat&esis polymeri/ation AR?MPB is employe% to %evelop nanostructure% separation me%ia* R?MP is a living polymeri/ation tec&niCue capa#le o! controlling polymeri/ation at a molecular level* T&ese s&o( a #etter per!ormance o! protein an% pepti%e analysis using nanocoupling tec&niCues* Deman% !or nanoseparation p&ases is increasing &eavily as nanocoupling tec&niCues) particularly nanoEHP CEMS) are #ecoming more important*

Anti#o%yECoate% Nanosp&eres T&e @*< to @*> _m goat antimouse IgGEcoate% polystyrene particles are prepare% #y using passive a%sorptionM t&e coate% particles are sta#le !or several years un%er proper storage con%itions* Anti#o%yEcoate% polystyrene particles &ave anti#o%y contents 49 _g1mg soli%) @*5 _g1cm 5) 4*: ` 4@9) IgG1particle) an% #in%ing capacity to IgGE-ITC 9 _g1mg* T&ese can #e use% !or targete% %elivery o! %rugs A4<@B*

Nanocrystallites An interesting application o! nanotec&nology (as reporte% #y 3in et al* A4<4B (&ere t&ey use% pluronic in sprayE%rying to pro%uce t&e nanocrystallites o! t&e %rug nee%E ing improve% #ioavaila#ility or improve% solu#ility* T&ey utili/e% Pluronic -45< an% %evelope% several com#inations o! %rug (it& pluronic) an% sprayE%rie% t&ese com#inations using t&e organic solvent*

Nano&y#ri%s A novel met&o% o! %elivering nonionic poorly (aterEsolu#le %rugs suc& as camptot&ecin (as %evelope% an% reporte% #y Tyner et al* A4<5B* Camptot&ecin (as !irst incorporate% into micelles %erive% !rom negatively c&arge% sur!actants* T&e negatively c&arge% micelles (ere t&en encapsulate% in NPs o! magnesiumH aluminum &y%ro"i%es #y an ionEe"c&ange process* T&e resulting structures (ere terme% nano&y#ri%s* T&ey release% camptot&ecin rapi%ly (it& complete release (it&in 4@ minutes at #ot& 9*= an% <*5 pH* T&e encapsulation process allo(e% almost t&ree times t&e %rug loa%ing an% &as e"cellent potential !or %rugE%elivery applicaE tions* T&is comple" (as s&o(n to provi%e similar cytoto"ic c&aracteristics to na$e% %rug #ut t&e nano&y#ri%s can #e a%ministere% in a %oseEcontrolle% !as&ion %ue to goo% %ispersion o! t&e comple"es in (ater* T&e a#ility to attac& targeting #iologiE cally active molecules to t&e outsi%e sur!ace o! t&e nano&y#ri%s as (ell as t&e potenE tial controlle% release properties o! t&e comple"es in%icate t&at t&ese &y#ri%s can #e use% !or speci!ic %elivery o! poorly solu#le nonionic %rugs A4<5B* Anot&er interesting stu%y !rom t&e same group (as reporte% !or t&e applicaE tion o! t&e nano&y#ri%s as a nonviral vector !or gene %elivery* T&e nano&y#ri%s (ere synt&esi/e% #y t&e intercalation o! a !ull gene an% promoter enco%ing green !luorescent protein #et(een layers o! an inorganic &ost* T&e nano&y#ri%s (ere %elivere% to > Glioma cells) 'EG8 C&oriocarcinoma placental cells) an% car%iac myocytes* All cells (ere a#le to internali/e an% tolerate t&e nano&y#ri%s an% e"presse% t&e gene (it& some cell lines &aving up to >@[ trans!ection e!!iciency A4<8B* T&e nano&y#ri%s mimic $ey !eatures o! viral %elivery systems* Unli$e ot&er reporte% inorganic vectors) t&e &ost encapsulates t&e DNA molecule #et(een t&e inorganic

Nanoparticulate DrugEDelivery Systems

58

layers) protecting it !rom premature recognition or %egra%ation* In a%%ition) t&e presence o! &y%ro"yl group on t&e &ost sur!ace provi%es t&e means to lin$ #iologiE cally active molecules to t&e e"terior sur!ace o! t&e nano&y#ri%s) suggesting t&e possi#ility o! targeting t&e %rugE%elivery systems* ?t&er a%vantages o! nano&y#ri% systems inclu%e less !oreign DNA an% t&e a#ility to %eliver multiple genes to cells* In a%%ition) use o! t&e nano&y#ri%s is e"pecte% to increase control o! t&e e"pression level o! target genes #y regulating t&e amount o! DNA intro%uce% into t&e cells* Suc& %ose control is %i!!icult to ac&ieve in viral vectors* Preparation o! viral conE structs can #e timeEconsuming an% te%ious* As t&e nano&y#ri%s are applica#le to a (i%e range o! genes) t&ey may #e prepare% t&roug& a simple oneEstep process) t&us greatly s&ortening t&e amount o! time nee%e% to pro%uce a !unctional vector A4<8B*

Nanocontainer Tec&nology Nanotec&nology promises ne( avenues to me%ical %iagnosis) treatment) an% %rug %elivery* In t&is respect) t&ere are some special in.ecta#le nanove&icles t&at are proE gramma#le to(ar%s speci!ic targets an% are a#le to eva%e t&e immune %e!ense an% are versatile enoug& to #e suite% as carriers o! comple" !unctionality* +iotinE !unctionali/e% ApolyEA5Emet&ylo"a/olineBE#EpolyA%imet&yl silo"aneBE#EpolyA5Emet&yl o"a/olineB tri#loc$ copolymers (ere sel!Eassem#le% to !orm nanocontainers) an% #iotinylate% targeting ligan%s (ere attac&e% #y using Streptavi%in as a coupling agent* Speci!ically) !luorescenceEla#ele% nanocontainers (ere targete% against t&e scavenger receptor A4 !rom macrop&ages) an important cell in &uman %isease* In &uman an% transgenic cell lines an% in mi"e% cultures) receptor #in%ing o! t&ese generic carriers (as !ollo(e% #y vascular upta$e* o( nonspeci!ic #in%ing supE porte% t&e stealt& properties o! t&e carrier (&ile t&e cytoto"icity (as a#sent* T&ese versatile carriers appear to #e promising !or %iagnostic an% t&erapeutic %rugE %elivery purposes A4<9B*

Electrospun Nano!i#ers as DrugEDelivery Systems Electrostatic spinning is a versatile tec&niCue applie% to various microE an% nano!a#rication areas using numerous polymers* T&e tec&niCue involves .etting a liCui% stream o! a %rug1polymer solution to a potential #et(een : an% 8@ $2M !i#ers at su#micrometer %iameters can #e !orme% (&en electrical !orces overcome t&e sur!ace tension o! t&e %rug1polymer solution at t&e air inter!ace Aterme% a Taylor coneB suc& t&at a .et !orms A4<:B* As t&e .et accelerating t&roug& t&e electric !iel%) t(o possi#le outcomes &ave #een &ypot&esi/e%6 AiB ra%ial !orces #ecome increasingly important leaning to splaying o! t&e solution stream or AiiB a continuE ous single !ilament is generate% #ase% on #en%ing insta#ility A4<;B* As t&e solvent evaporates) !i#er can #e collecte% on t&e screen to give a non(oven !a#ric or colE lecte% on a spinning man%rill* T&e !i#er %iameter is a !unction o! solution sur!ace tension) %ielectric constant o! polymer solution) !ee%ing rate) an% electrostatic !iel%* T&e nature o! t&e polymer can also %irect t&e use o! t&e electrospun !i#ers) (it& (aterEsolu#le polymers giving rise to imme%iate release %osage !orms an% (ater insolu#le giving sustaine% release systems* A report #y 2errec$ et al* A4<<B &as use% t&ese nano!i#ers !or t&e release o! itracona/ole* T&e !i#er %iameter range% !rom :@@ nm to a !e( micrometers* T&ey s&o(e% t&at complete release o! t&e poorly solu#le %rug can #e ac&ieve% an% t&e rate o! %rug release can #e tailore%) s&o(ing applications o! t&ese nano!i#ers !or (oun% &ealing) #uccal) an% topical applications A4<=B*

59

T&assu et al*

-UTURE DIRECTI?NS T&e ma.ority o! commercial NP applications in me%icine are geare% to(ar%s %rug %elivery* In #iosciences) NPs are replacing organic %yes in applications t&at reCuire &ig& p&otosta#ility as (ell as &ig& multiple"ing capa#ilities* T&ere are some ne( %evelopments in %irecting an% controlling t&e !unctions o! nanopro#es) !or e"amE ple) %riving magnetic NPs to t&e tumor an% t&en ma$ing t&em eit&er release t&e %rug loa% or .ust &eating t&em in or%er to %estroy t&e surroun%ing tissue* T&e ma.or tren% in !urt&er %evelopment o! nanomaterials is to ma$e t&em multi!unctional an% controlla#le #y e"ternal signals or #y local environment) t&us essentially turning t&em into nano%evices A4;9B*

RE-ERENCES
4* T&e Royal Society* Nanoscience an% nanotec&nologies6 opportunities an% uncertainties* on%on6 Royal Society) 5@@969* 5* 'ores 0) Me&nert ,) Ma%er 0* P&ysicoc&emical investigations on soli%Hlipi% nanopartiE cles an% on oil loa%e% soli%Hlipi% nanoparticles6 a nuclear magnetic resonance an% elecE tron spin resonance stu%y* P&arm Res 5@@8M 5@645<9* 8* Maestrell -) Mura P) Alonso M'* -ormulation an% c&aracteri/ation o! triclosan su#Emicron emulsions an% nanocapsules* ' Microencapsul 5@@9M 546=:<* 9* oc&mann D) 2ogel 2) ,eyermann ') et al* P&ysicoc&emical c&aracteri/ation o! protamineEp&osp&orot&ioate nanoparticles* ' Microencapsul 5@@9M 546;5:* :* Ge/e A) Putau" ' ) C&oisnar% ) 'e&an P) ,ouessi%.e(e D* ong term s&el! sta#ility o! amp&ip&ilic +Ecyclo%e"trin nanosp&eres suspensions monitore% #y %ynamic lig&t scatE tering an% cryoEtransmission electron microscopy* ' Microencapsul 5@@9M 546;@<* ;* ,ang W) Dai ') C&en V) et al* +ioavaila#ility an% p&armaco$inetics o! cyclosporine AE loa%e% pH sensitive nanoparticles !or oral a%ministration* ' Control Release 5@@9M ><6954* <* 3oo HS) Par$ TG* +io%egra%a#le nanoparticles containing proteinH!atty aci% comple"es !or oral %elivery o! salmon calcitonin* ' P&arm Sci 5@@9M >869==* =* Mo 3) im 3* Mec&anistic stu%y o! t&e upta$e o! (&eat germ agglutininEcon.ugate% P GA nanoparticles #y A:9> cells* ' P&arm Sci 5@@9M >865@* >* Alp&an%ary HP) A#ou#a$er M) 'aillar% D) Couvreur P) 2aut&ier C* 2isuali/ation o! insuE lin loa%e% nanocapsules6 in vitro an% in vivo stu%ies a!ter oral a%ministration to rats* P&arm Res 5@@8M 5@64@<4* 4@* Hoet P) Ho&l!el% I+) Salata ?2* NanoparticlesR$no(n an% un$no(n &ealt& ris$s* ' Nano#iotec&nol 5@@9M 5645* 44* 2en$ates(arlu 2) Man.unat& 0* Preparation) c&aracteri/ation an% in vitro release $inetics o! clo/apine soli%Hlipi% nanoparticles* ' Control Release 5@@9M >:6;5<* 45* Dingler A) Go&la S* Pro%uction o! soli%Hlipi% nanoparticles AS NB6 scaling up !easi#ilities* ' Microencapsul 5@@5M 4>644* 48* Gasco MR* Met&o% !or pro%ucing soli%Hlipi% microsp&eres &aving narro( si/e %istri#uE tion* US Patent :)5:@)58;) 4>>8* 49* Me&nert ,) Ma%er 0* Soli%Hlipi% nanocapsules pro%uction) c&aracteri/ation an% appliE cations* A%v Drug Deliv Rev 5@@4M 9<64;:* 4:* Muller RH) Ma%er 0) Go&la S* Soli%Hlipi% nanoparticles AS NB !or controlle% %rug %eliveryHa revie( o! t&e state o! t&e art* Eur ' P&arm +iop&arm 5@@@M :@64;4* 4;* +ec$ RCR) Po&lman AR) Guterres SS* NanoparticlesEcoate% microparticles6 preparation an% c&aracteri/ation* ' Microencapsul 5@@9M 5469>>* 4<* Ro%rigue/ SG) Allemann E) -essi H) Doel$er E* P&ysicoc&emical parameters associate% (it& nanoparticles !ormation in t&e salting out) emulsi!icationE%i!!usion an% nanopreE cipitation met&o%s* P&arm Res 5@@9M 546495=* 4=* iao W) ,ie%mann TS* Measurement o! processE%epen%ent material properties o! p&armaceutical soli%s #y nanoin%entation* ' P&arm Sci 5@@9M >96<>*

Nanoparticulate DrugEDelivery Systems

5:

4>* Rigal%ie 3) argeteau A) emagnen G) et al* E!!ects o! &ig& &y%rostatic pressure on several sensitive t&erapeutic molecules an% a so!t nanoE%isperse% %rug %elivery system* P&arm Res 5@@8M 5@65@8;* 5@* -ru&ling ') Penasse ,) +rassine C) et al* Intracellular penetration o! liposomes containing a (ater insolu#le antimitotic %rug in 454@ cells* Eur ' Cancer 4>=@M 4;649@>* 54* aurent G) a%uron C) Ruyssc&aert 'M) Deleers M* In&i#ition o! cationic liposomes o! 8HEt&ymi%ine incorporation into DNA o! 454@ cells* Res Commun C&em Pat&ol P&armacol 4>=4M 846:4:* 55* HecC ') Deleers M) -anara D) 2ranc$" H) Amig&i 0* Preparation an% c&aracteri/ation o! nanocrystals !or solu#ility an% %issolution rate en&ancement o! Ni!e%ipine* Int ' P&arm 5@@:M 5>>64;<H4<<* 58* HecC ') Deleers M) -anara D) et al* Preparation an% in vitro1in vivo evaluation o! nanoE si/e% crystals !or %issolution rate en&ancement o! UC+E8:99@E8) a &ig&lyE%ose% poorly (ater solu#le (ea$ #ase* Eur ' P&arm +iop&arm 5@@;M ;968;@H8;=* 59* 2ranc$" H) Demoustier M) Deleers M* P&armaceutical composition comprising nanoE capsules* US Patent @::@@559) Marc& 4>) 4>>;* 5:* -anara D) Grognet P) +er(aer M) Deleers M* Use o! p&armaceutical compositions capaE #le o! #eing gelle% in Perio%ontology* US Patent ;9<4><@) ,? @>>:;<5;) EP @4@<8949) ?cto#er 5>) 5@@5* 5;* -anara D) +er(aer M) Deleers M* P&armaceutical compositions capa#le o! #eing gelle%* US Patent ;9;9>=<) ,?@>>:;<5:) EP@4@<894:) ?cto#er 4:) 5@@5* 5<* a2an DA) McGuire T) anger R* Small scale systems !or in vivo %rug %elivery* Nat +iotec&nol 5@@8M 54644=9* 5=* Salt/man ,M) ?l#ric&t , * +uil%ing %rug %elivery into tissue engineering* Nat Rev 5@@5M 4464<<* 5>* Viaie +) +al%i A) ei M) Gu 3) Siegel RA* Har% an% so!t micromac&ining !or #iomems6 revie( o! tec&niCues an% e"amples o! applications in micro!lui%ics an% %rug %elivery* A%v Drug Deliv Rev 5@@9M :;649:* 8@* Su 3C) in * A (ater po(ere% micro%rug %elivery system* ' Electroc&em Syst 5@@9M 486<:* 84* McAllister D2) Allen MG) Prausnit/ MR* Micro!a#ricate% micronee%les !or gene an% %rug %elivery* Annu Rev +iome% Eng 5@@@M 565=>* 85* Heurtault +) Saulnier P) +enoit 'P) Proust 'E) Pec& +) Ric&ar% '* ipi% nanocapsules) preparation) met&o% an% use as a me%icine* Patent No* ,@@41;985=) 5@@@* 88* Heurtault +) Saulnier P) Pec& +) +enoit 'P) Proust 'E* Inter!acial sta#ility o! lipi% nanocapE sules* Colloi%s Sur! + +iointer!aces 5@@8M 8@655:* 89* Muller RH) Dingler T) Sc&neppe T) Go&la S* In6 ,ise D) e%* Han%#oo$ o! P&armaceutical Controlle% Release Tec&nology* Ne( 3or$6 Marcel De$$er) 5@@@68:>* 8:* 'enning 2) 0orting MS) Go&la S* 2itamin A loa%e% soli% lipi% nanoparticles !or topical use6 %rug release properties* ' Control Release 5@@@M ;;644:* 8;* Mu&len AV) Sc&(ar/ C) Me&nert ,* Soli%Hlipi% nanoparticles AS NsB !or controlle% %rug %eliverH%rug release an% release mec&anism* Eur ' P&arm +iop&arm 4>>=M 9:649>* 8<* Dulieu C) +a/ile D* In!luence o! lipi% nanocapsules composition on t&eir aptness to !ree/e %rying* P&arm Res 5@@:M 5565=:* 8=* Muller RH) 'aco#s C) 0ayser ?* Nanosuspensions as particulate %rug !ormulations in t&erapy rational !or %evelopment an% (&at (e can e"pect !or t&e !uture* A%v Drug Deliv Rev 5@@4M 9<68* 8>* 'ores 0) Me&nert ,) Drec&esler M) +un.es H) 'o&ann C) Ma%%er 0* Investigations on t&e structure o! soli%Hlipi% nanoparticles AS NB an% oil loa%e% soli% lipi% nanoparticles #y p&oton correlation spectroscopy) !iel% !lo( !ractionation an% transmission electron microscopy* ' Control Release 5@@9M >:654<* 9@* Muller RH) Ra%t$e SA) ,issing SA* Soli%Hlipi% nanoparticles AS NB an% nanostructure% lipi% carriers AN CB in cosmetic an% %ermatological preparations* A%v Drug Deliv Rev 5@@5M :96484* 94* Muller RH) Ra%t$e M) ,issing SA* Nanostructure% lipi% matrices !or improve% microE encapsulation o! %rugs* Int ' P&arm 5@@5M 5956454* 95* C&esnoy S) Huang * Structure an% !unction o! lipi% DNA comple"es !or gene %elivery* Annu Rev +iop&ys Struct 5@@@M 5>65<*

5;
98* Mer%an T) 0opece$ ') 0issel T* Prospects !or cationic polymers in gene an% oligonucleE oti%es t&erapy against cancer* A%v Drug Deliv Rev 5@@4M 9<64;:* 99* -ournier E) Du!resne MH) Smit& DC) Ranger M) erou" 'C* A novel step %rug loa%ing proce%ure !or (ater solu#le amp&ip&ilic nanocarriers* P&arm Res 5@@9M 546>;5* 9:* Heurtault +) Saulnier P) Pec& +) Proust 'E) +enoit 'P* A novel p&ase inversion #ase% process !or t&e preparation o! lipi% nanocarriers* P&arm Res 5@@5M 4>6=<:* 9;* Ta#att 0) Sameti M) ?l#ric& C) Muller RH) e&r CM* E!!ect o! cationic lipi% an% matri" lipi% composition on soli%Hlipi% nanoparticles me%iate% gene trans!er* Eur ' P&arm +iop&arm 5@@9M :<64::* 9<* Ru%olp& C) Sc&illinger U) ?rti/ A) Ta#att 0) Plan$ C) Muller RH) Rosenec$er '* Application o! novel soli%Hlipi% AS NB gene vector !ormulations #ase% on a %imeric HI2E4 TAT pepE ti%e in vitro an% in vivo* P&arm Res 5@@9M 5464;;5* 9=* iu -) 3ang ') Huang ) iu D* Ne( cationic lipi% !ormulation !or gene trans!er* P&arm Res 4>>;M 4864=:;* 9>* Ta#att 0) 0neuer C) Sameti M) et al* Trans!ection (it& %i!!erent colloi%al systems6 comE parison o! soli%Hlipi% nanoparticles an% liposomes* ' Control Release 5@@9M ><6854* :@* 0ogure 0) Moriguc&i R) Sasa$i 0) Ueno M) -uta$i S) Haras&ima H* Development o! a nonviral multi!unctional envelopEtype nano%evice #y a novel lipi% !ilm &y%ration met&o%* ' Control Release 5@@9M >=684<* :4* ee 0E) C&o SH) ee H+) 'eong S3) 3u$ SH* Microencapsulation o! lipi% nanoparticles containing lipop&ilic %rug* ' Microencapsul 5@@8M 5@69=>* :5* Stevens P') Se$i%o M) ee R'* A !olate receptor targete% lipi% nanoparticles !ormulation !or a lipop&ilic paclita"el pro%rug* P&arm Res 5@@9M 54654:8* :8* Hou DV) Wie CS) Huang 0') V&u CH* T&e pro%uction an% c&aracteristics o! soli%Hlipi% nanoparticles AS NsB* +iomaterials 5@@8M 5964<=4* :9* ?l#ric& C) Gessner A) Sc&ro%er ,) 0ayser ?) Muller RH* ipi%H%rug con.ugate nanoE particles o! t&e &y%rop&ilic %rug %imina/eneEcytoto"icity testing an% mouse serum a#sorption* ' Control Release 5@@9M >;695:* ::* Cavalli R) +argoni A) Po%io 2) Muntoni E) Vara GP) Gasco MR* Duo%enal a%ministration o! soli%Hlipi% nanoparticles loa%e% (it& %i!!erent percentages o! to#ramycin* ' P&arm Sci 5@@8M >564@=:* :;* ,illiams ') ans%o(n R) S(eit/er R) Romano(s$i M) a+ell R) Ramas(ami R) Unger E* Nanoparticle %rug %elivery system !or intravenous %elivery o! topoisomerase in&i#itors* ' Control Release 5@@8M >464;<* :<* 0a#anov A2) +atra$ova E2) Ala$&ov 23* Pluronic #loc$ copolymers as novel polymer t&erapeutics !or %rug an% gene %elivery* ' Control Release 5@@5M =564=>* :=* Torc&ilin 2P* Structure an% %esign o! polymeric sur!actantE#ase% %rug %elivery systems* ' Control Release 5@@4M 58648<* :>* D.or%.evic ') +arc& M) U&ric& E* Polymeric micelles #ase% on amp&ip&ilic scorpion li$e macromolecules6 novel carriers !or (ater insolu#le %rugs* P&arm Res 5@@:M 55659* ;@* 0atao$a 0) Hara%a A) Nagasa$i 3* +loc$ copolymer micelles !or %rug %elivery6 %esign) c&aracteri/ation an% #iological signi!icance* A%v Drug Del Rev 5@@5M :96448* ;4* 0a$i/a(a 3) 0ata$oa 0* +loc$ copolymer micelles !or %elivery o! genes an% relate% comE poun%s* A%v Drug Del Rev 5@@5M :965@8* ;5* Rosler A) 2an%ermeulen GM) 0lo$ HA* A%vance% %rug %elivery %evices via sel! assemE #ly o! amp&ip&ilic #loc$ copolymers* A%v Drug Del Rev 5@@4M :86>:* ;8* Hu& 0M) ee SC) C&o 3,) ee ') 'eong 'H) Par$ 0* Hy%rotropic polymer micelle system !or %elivery o! paclita"el* ' Control Release 5@@:M 4@46:>* ;9* 0(on GS) 0ata$oa 0* +loc$ copolymers micelles as long circulating %rug ve&icles* A%v Drug Del Rev 4>>:M 4;65>:* ;:* +urt HM) V&ang W) Tolei$is P) Em#ree ) Hunter , * Development o! copolymers o! polyA%)l lacti%eB an% met&o"ypolyet&ylene glycol as micellar carriers !or paclita"el* Colloi%s Sur! + +iointer!aces 4>>>M 4;64;4* ;;* -is&er M) 2ogtle -* Den%rimers6 !rom %esign to applicationRa progress report* Ange( C&em Int E% Engl 4>>>M 8=6==:* ;<* Du!resne MH) -ournier -) 'ones MC) Ranger M) erou" 'C* +loc$ copolymers micellesH engineering versatile carriers !or %rugs an% #iomacromolecules* In6 Gurny R) e%*

T&assu et al*

Nanoparticulate DrugEDelivery Systems

5<

;=*

;>* <@* <4* <5*

<8* <9*

<:*

<;*

<<* <=*

<>*

=@* =4*

=5*

=8*

=9* =:* =;* =<* ==* =>*

C&allenges in Drug Delivery !or t&e Ne( Millennium* Saint Priest6 +ulletin Tec&niCue Gatte!osseP>;) 5@@864@8* 0(on G) Su(a S) 3o$oyama T) ?$ano T) Sa$urai 3) 0ata$oa 0* En&ance% tumor accumulation an% prolonge% circulation times o! micelles !orming polyAet&ylene o"i%eE aspartateB #loc$ copolymerHa%riamycin con.ugates* ' Control Release 4>>9M 5>64<* Savic R) uo ) Eisen#erg A) Maysinger D* Micellar nanoEcontainers %istri#ute to %e!ine% cytoplasmic organelles* Science 5@@8M 8@@6;4:* Mae%a H) ,u ') Sa(a T) Matsumura 3) Hori 0* Tumor vascular permea#ility an% EPR e!!ect in macromolecular t&erapeutics6 a revie(* ' Control Release 5@@@M ;:65<4* +rigger I) Du#ernet C) Couvreur P* Nanoparticles in cancer t&erapy an% %iagnosis* A%v Drug Deliv Rev 5@@5M :96;84* -rancis M-) Cristea M) 3ang 3) ,inni$ -M* Engineering polysacc&ari%eE#ase% polymeric micelles to en&ance permea#ility o! cyclosporine A across cacoE5 cells* P&arm Res 5@@:M 5565@>* Uga/io E) Cavalli R) Gasco MR* Incorporation o! cyclosporine A in soli%Hlipi% nanoE particles AS NB* Int ' P&arm 5@@5M 5946894* 2arela MC) Gu/man M) Molpeceres ') A#erturas MDR) Puyol DR) Puyol MR* Cyclosporine loa%e% polycaprolactone nanoparticles6 immunosuppression an% nep&roto"icity in rats* Eur ' P&arm Sci 5@@4M 4569<4* Gre! R) 7uellec P) Sanc&e/ A) Calvo P) Dellac&erie E) Alonso M'* Development an% c&aracteri/ation o! CyEA loa%e% polylacticHpolyet&ylene glycol PEG micro an% nanoE particles6 comparison (it& conventional P A particulate carriers* Eur ' P&arm +iop&arm 5@@4M :46444* Campos AMD) Sanc&e/ A) Alonso M'* C&itosan nanoparticles6 a ne( ve&icle !or t&e improvement o! t&e %elivery o! %rugs to t&e ocular sur!aces) application to cyclosporine A* Int ' P&arm 5@@4M 55964:>* Gao VG) -ain HD) Rapoport N* Controlle% an% targete% tumor c&emot&erapy #y micellarE encapsulate% %rug an% ultrasoun%* ' Control Release 5@@:M 4@565@8* ,u ') A$ai$e T) Hayas&i%a 0) ?$amoto T) ?$uyama A) Mae%a H* En&ance% vascular permea#ility in soli% tumor involving pero"ynitrite an% matri" metalloproteinases* 'pn ' Cancer Res 5@@4M >5698>* Torc&elin 2P) u$yanov AN) Gao A) Papa&a%.opoulos +* Immunomicelles6 targete% p&armaceutical carriers !or poorly solu#le %rugs* Proc Natl Aca% Sci USA 5@@8M 4@@6;@8>* 0a#anov A2) +atra$ova E2) Ala$&ov 23* An essential relations&ip #et(een ATP %epletion an% c&emosensiti/ing activity o! Pluronic #loc$ copolymers* ' Control Release 5@@8M >46<:* Na 0) ee 0H) +ae 3H* pH sensitivity an% pH %epen%ent interior structural c&ange o! sel! assem#le% &y%rogel nanoparticles o! pullulan acetate1oligoEsul!onami%e con.ugate* ' Control Release 5@@9M ><6:48* Cammas S) Su/u$i 0) Sone C) Sa$urai 3) 0atao$a 0) ?$ano T* T&ermo responsive polymer nanoparticles (it& a core s&ell micelle structure as site speci!ic %rug carriers* ' Control Release 4>><M 9=64:<* C&ung 'E) 3o$oyama M) 3amato M) Aoyagi T) Sa$urai 3) ?$ano T* T&ermo responsive %rug %elivery !rom polymeric micelles constructe% using #loc$ copolymers o! poly ANEisopropylacrylami%eB an% polyA#utylmet&acrylateB* ' Control Release 4>>>M ;5644:* Panyam ') a#&aset(ar 2* +io%egra%a#le nanoparticles !or %rug an% gene %elivery to cells an% tissues* A%v Drug Deliv Rev 5@@8M ::685>* 2aut&ier C) Du#ernet C) -atal E) Alp&an%ary HP) Couvreur P* PolyAal$ylcyanoacrylatesB as #io%egra%a#le materials !or #iome%ical applications* A%v Drug Deliv Rev 5@@8M ::6:4>* Disc&er DE) Eisen#erg A* Polymeric vesicles* Science 5@@5M 5><6>;<* Harrington 0') e(ans$i CR) Ste(art 'S,* iposomes as ve&icles !or targete% t&erapy o! cancer* Part 5* Clinical %evelopment* Clin ?ncol 5@@@M 4564;* Wiong W3) Tam 0C) Gan H* Release $inetics o! &y%rop&o#ic an% &y%rop&ilic mo%el %rugs !rom Pluronic -45<1polyAlactic aci%B nanoparticles* ' Control Release 5@@:M 4@86<8* V&ang ) Hu 3) 'iang W) 3ang C) u ,) 3ang 3H* Camptot&ecin %erivativeEloa%e% polyAcaprolactoneEcoElacti%eBE#EPEGE#EpolyAcaprolactoneEcBElacti%e nanoparticles an% t&eir #ioE%istri#ution in mice* ' Control Release 5@@9M >;648:*

5=
>@* Mog&imi SM) Hunter AC) Murray 'C* ong circulating an% target speci!ic nanopartiE cles6 t&eory to practice* P&armacol Rev 5@@4M :865=8* >4* S&en%erova A) +ur$e TG) Sc&(en%eman SP* Sta#ili/ation o! 4@E&y%ro"y camptot&ecin in polyAlacti%eEcoEglycoli%eB microsp&ere %elivery ve&icles* P&arm Res 4>><M 49649@;* >5* Hu 3) 'iang W) Ding 3) et al* Preparation an% %rug release #e&aviors o! nimo%ipineE loa%e% polyAcaprolactoneBEpolyEAet&ylene o"i%eB polylacti%e amp&ip&ilic copolymer nanoparticles* +iomaterials 5@@8M 59658>:* >8* Riley T) Stolni$ S) Heal% CR) et al* P&ysicoc&emical evaluation o! nanoparticles assemE #le% !rom polyAlactic aci%B polyAet&ylene glycolB AP AHPEGB #loc$ copolymers as %rug %elivery ve&icles* angmuir 5@@4M 4<684;=* >9* 3oo HS) Par$ TG* -olate receptor targete% #io%egra%a#le polymeric %o"oru#icin micelles* ' Control Release 5@@9M >;65<8* >:* avasani!ar A) Samuel ') 0(on GS* PolyAet&ylene o"i%eB #loc$ polyAlEamino aci%B micelles !or %rug %elivery* A%v Drug Deliv Rev 5@@5M :964;>* >;* 2rie/ema DM) Hoog#oom ') 2elonia 0) et al* 2esicles an% polymeri/e% vesicles !rom t&iE op&ene containing ro% coil #loc$ copolymers* Nolte Ange( C&em Int E% 5@@8M 956<<5* ><* 2rie/ema DM) 0ros A) Gel%er RD) Cornelissen '' M) Ro(an AE) Roelan% 'M* Electro!orme% giant vesicles !rom t&iop&ene containing ro% coil %i#loc$ copolymers* Macromolecules 5@@9M 8<69<8;* >=* 2rie/ema DM) Araganoes MC) Elemans HAA,) Cornelissen '' M) Ro(an AE) Roelan% 'M* Sel! assem#le% nanoreactors* C&em Rev 5@@:M 4@:649::* >>* ee SC) 0im C) 0(on IC) C&ung H) 'eong S3* Polymeric micelles o! polyA5Eet&ylE5E o"a/olineBE#loc$HpolyAcaprolactoneB copolymer as a carrier !or paclita"el* ' Control Release 5@@8M =>698<* 4@@* 2aut&ier C) Du#ernet C) C&auvierre C) +rigger I) Couvreur P* Drug %elivery to resistant tumors6 t&e potential o! polyAal$yl cyanoacrylateB nanoparticles* ' Control Release 5@@8M >864:4* 4@4* Mu ) -eng SS* A novel controlle% release !ormulation !or t&e anticancer %rug paclita"el ATa"olB6 P GA nanoparticles containing vitamin E TPGS* ' Control Release 5@@8M =;688* 4@5* C&auvierre C) a#arre D) Couvreur P) 2aut&ier C* Novel polysacc&ari%e %ecorate% polyAiso#utyl cyanoacrylateB nanoparticles* P&arm Res 5@@8M 5@64<=;* 4@8* ,artlic$ H) Sc&mitt SS) Stre#&ar%t 0) 0reuter ') anger 0* Tumor cell %elivery o! antisense oligonucleoti%es #y &uman serum al#umin nanoparticles* ' Control Release 5@@9M >;69=8* 4@9* Crommelin D'A) Strom G) 'is$ot ,) Stene$es R) Mastro#attista E) Hennin$ ,E* Nanotec&nological approac&es !or t&e %elivery o! macromolecules* ' Control Release 5@@8M =<6=4* 4@:* I&re HR) Gagne ) -rec&et 'M') S/o$a -C* Polyester %en%ritic systems !or %rug %elivery applications6 in vitro an% in vivo evaluations* +iocon.ug C&em 5@@5M 4869:8* 4@;* iu M') 0ono 0) -rec&et R* ,ater solu#le %en%ritic unimolecular micelles) t&eir potenE tial as %rug %elivery agents* ' Control Release 5@@@M ;:6454* 4@<* V&ang GD) Hara%a A) Nis&iyama N) et al* Polyion comple" micelles entrapping catiE onic %en%rimer porp&yrin6 e!!ective p&otosensiti/er !or p&oto%ynamic t&erapy o! cancer* ' Control Release 5@@8M >86494* 4@=* Ueno 3) -utaga(a H) Ta$agi 3) Ueno A) Mi/us&ima 3* Drug incorporating calcium carE #onate nanoparticles !or a ne( %elivery system* ' Control Release 5@@:M 4@86>8* 4@>* 2ogel 2) oc&mann D) ,eyermann ') et al* ?ligonucleoti%eEprotamineEal#umin nanoE particles6 preparation) p&ysical properties an% intracellular %istri#ution* ' Control Release 5@@:M 4@86>>* 44@* Dinaure N) oc&mann D) Demir&an I) et al* Intracellular trac$ing o! protamine1 antisense oligonucleoti%es nanoparticles an% t&eir in&i#itory e!!ect on HI2E4 transactiE vation* ' Control Release 5@@9M >;69><* 444* oc&mann D) 2ogel 2) ,eyermann ') et al* P&ysicoc&emical c&aracteri/ation o! proE tamine p&osp&orot&ioate nanoparticles* ' Microencapsul 5@@9M 546;98* 445* An%o T) 3amasa$i M) Su/u$i 0* Protamines6 Isolation) C&aracteri/ation) Structure an% -unction* +erlin6 Springer) 4><8* 448* Huang M) 0&or E) im 3* Upta$e an% cytoto"icity o! C&itosan molecules an% nanoE particles6 e!!ects o! molecular (eig&t an% %egree on %eacetylation* P&arm Res 5@@9M 546899*

T&assu et al*

Nanoparticulate DrugEDelivery Systems

5>

449* 44:*

44;* 44<*

44=* 44>* 45@* 454*

455*

458* 459* 45:* 45;*

45<* 45=* 45>*

48@*

484* 485*

488* 489* 48:*

48;*

'anes 0A) -resneau MP) Mara/uela A) -a#ra A) Alonso M'* C&itosan nanoparticles as %elivery systems !or %o"oru#icin* ' Control Release 5@@4M <865::* Ma VS) im 3* Upta$e o! C&itosan an% associate% insulin in t&e cacoE5 call monolayers6 a comparison #et(een C&itosan molecules an% C&itosan nanoparticles* P&arm Res 5@@5M 4>649==* Illum ) Gill I') Hinc&cli!!e M) -is&er AN) Davis SS* C&itosan as a novel nasal %elivery system !or vaccines* A%v Drug Deliv Rev 5@@4M :46=4* Campos AM) Die#ol% 3) Carval&o E ) Sanc&e/ A) Alonso M'* C&itosan nanoparticles as ne( ocular %rug %elivery systems6 in vitro sta#ility) in vivo !ate an% cellular to"icity* P&arm Res 5@@9M 546=@8* He.a/i R) Ami.i M* C&itosan #ase% gastrointestinal %elivery systems* ' Control Release 5@@8M =>64:4* Par$ 'H) 0(on S) Nam '?) et al* Sel! assem#le% nanoparticles #ase% on glycol C&itosan #earing : +Ec&olanic aci% !or RGD pepti%e %elivery* ' Control Release 5@@:M >:6:<>* De S) Ro#inson D* Polymer relations&ips %uring preparation o! C&itosanEalginate an% polyElElysineEalginate nanosp&eres* ' Control Release 5@@8M =>64@4* 3oo HS) ee 'E) C&ung H) 0(on IC) 'eong S3* Sel! assem#le% nanoparticles containing &y%rop&o#ically mo%i!ie% glycol C&itosan !or gene %elivery* ' Control Release 5@@:M 4@8658:* 0umar M) 0ong W) +e&era A0) Hellermann GR) oc$ey R-) Mo&apatra SS* C&itosan I-NEcEpDNA nanoparticles ACINB t&erapy !or allergic ast&ma* Gene 2accines T&er 5@@4M 468* C&u ,H) C&in R) Huen T) -errari M* Silicone mem#rane nanoE!ilters !rom sacri!icial o"i%e removal* ' Micro Electroc&em Syst 4>>>M =689* Martin -) ,alc/a$ R) +oiars$i A) et al* Tailoring (i%t& o! micro!a#ricate% nanoc&annels to solute si/e can #e use% to control %i!!usion $inetics* ' Control Release 5@@:M 4@56458* Desai TA* Micro!a#rication tec&nology !or pancreatic cell encapsulation* E"pert ?pin +iol T&erap 5@@5M 56;88* Desai TA) Hans!or% D) -errari M* C&aracteri/ation o! micromac&ine% silicone mem#ranes !or immunoisolation an% #ioseparation applications* ' Mem#r Sci 4>>>M 4:>6554* emarc&an% C) Couvreur P) +esnar% M) Costantini D) Gre! R* Novel polyesterHpolysacE c&ari%e nanoparticles* P&arm Res 5@@8M 5@645=9* Muller +G) euen#erger H) 0issel T* Al#umin nanosp&eres as carriers !or passive %rug targeting6 an optimi/e% manu!acturing tec&niCue* P&arm Res 4>>;M 48685* 3amaguc&i 3) Nagasa(a T) Na$amura N) et al* Success!ul treatment o! p&oto%amage% s$in o! nano scale atRA particles using novel trans%ermal %elivery* ' Control Release 5@@:M 4@965>* im S') 0im C0* -ormulation parameters %etermining t&e p&ysicoc&emical c&aracterisE tics o! soli%Hlipi% nanoparticles loa%e% (it& all transEretinoic aci%* Int ' P&arm 5@@5M 598648:* im S') ee M0) 0im C0* Altere% c&emical an% #iological activities o! all trans incorpoE rate% in soli%Hlipi% nanoparticles po(%ers* ' Control Release 5@@9M 4@@6:8* +alt&asar S) Mic&aelis 0) Dinauer N) +riesen 2H) 0reuter ') anger 0* Preparation an% c&aracteri/ation o! anti#o%y mo%i!ie% gelatin nanoparticles as carrier system !or upta$e in lymp&ocytes* +iomaterials 5@@:M 5;65<58* Couvreur P) +arratt G) -attal E) egran% P) 2aut&ier C* Nanocapsule tec&nology6 a revie(* Crit Rev T&er Drug Carrier Syst 5@@5M 4>6>>* Dalencon -) Am.au% 3) a!!orgue C) Derouin -) -essi H* AtovaCuone an% ri!a#utine loa%e% nanocapsules6 !ormulation stu%ies* Int ' P&arm 4>>=M 4:8645<* Hu#ert +) At$inson ') Guerret M) Ho!!man M) Devissaguet 'P) Maincent P* T&e preparaE tion an% acute anti&ypertensive e!!ects o! a nanoparticular !orm o! %aro%ipine) a %i&yE %ropyri%ine calcium entry #loc$er* P&arm Res 4>>4M =6<89* +arrat G) Puisieu" -) 3u ,P) -ouc&er C) -essi H) Devissaguet 'P* Anti metaEstatic activE ity o! MDPElEalanyl c&olesterol incorporate% into various types o! nanocapsules* Int ' ImmunoEP&armacol 4>>9M 4;69:<* Calvo P) Alonso M') 2ila'ato ' ) Ro#inson 'R* Improve% ocular #ioavaila#ility o! In%omet&acin #y novel ocular %rug carriers* ' P&arm P&armacol 4>>;M 9=6449<*

48<*

8@
48=* C&en H) V&ang V) Almarsson ?) Marier '-) +er$ovit/ D) Gar%ner CR* A novel lipi% !ree nano%ispersion !ormulation o! propo!ol an% its c&aracteri/ation* P&arm Res 5@@:M 5568:;* 48>* V(eers M T) Eng#ers GHM) Gri.pma D,) -ei.en '* 2itro %egra%ation o! nanoparticles prepare% !rom polymers #ase% on %lElacti%e glycoli%e an% polyAet&ylene o"i%eB* ' Control Release 5@@9M 4@@689<* 49@* Ar#os P) Campenero MA) Arangoa MA) Rene%o M') Irac&e 'M* In!luence o! t&e sur!ace c&aracteristics o! P2M1MA nanoparticles on t&eir #ioa%&esive properties* ' Control Release 5@@8M =>64>* 494* uu 30) 0im 0) Hsiao +S) C&u +) Ha%.iargurou M* Development o! a nanostructure% DNA %elivery sca!!ol% via electroEspinning o! P GA an% P AHPEG #loc$ copolymers* ' Control Release 5@@8M =>6894* 495* Murp&y , ) Peters MC) 0o&n DH) Mooney D'* Sustaine% release o! vascular en%ot&eE lial gro(t& !actor !rom minerali/e% polyAlacti%eEcoEglycoli%eB sca!!ol%s !or tissue engiE neering* +iomaterials 5@@@M 5965:54* 498* ,&ang 0) Gol%stic$ T0) Healy 0E* A #io%egra%a#le polymer sca!!ol% !or %elivery o! osteotropic !actors* +iomaterials 5@@@M 5965:9:* 499* Pere/ C) Sanc&e/ A) Putnam D) Ting D) anger R) Alonso M'* PolyAlactic aci%BH polyAet&ylene glycolB nanoparticles as ne( carriers !or t&e %elivery o! plasmi% DNA* ' Control Release 5@@4M <:6>:5* 49:* Son 3') 'ang 'S) C&o 3,) et al* +io%istri#ution an% antitumor e!!icacy o! %o"oru#icin loa%e% glycol C&itosan nanoEaggregates #y EPR e!!ect* ' Control Release 5@@8M >4648:* 49;* Nigave$ar SS) Sung 3) lanes M) et al* 8H %en%rimer nanoparticles organ1tumor %istri#ution* P&arm Res 5@@9M 5469<;* 49<* +alog& P) Nigave$ar SS) Coo$ AC) Minc ) 0&an M0* Development o! %en%rimer gol% ra%ioactive nanoEcomposites to treat cancer microvasculature* P&arma C&em 5@@8M 56>9* 49=* Anee% AE* An overvie( o! current %elivery systems in cancer gene t&erapy* ' Control Release 5@@9M >964* 49>* Sa$uma S) Su%o R) Su/u$i N) 0i$uc&i H) A$as&i M) Hayas&i M* Mucoa%&esion o! polystyrene nanoparticles &aving sur!ace &y%rop&ilic polymeric c&ains in t&e gastroinE testinal tract* Int ' P&arm 4>>>M 4<<64;4* 4:@* Haya$a(a T) 0a(amura M) ?$amoto M) +a#a M) et al* Concanavalin AEimmo#ili/e% polystyrene nanosp&eres capture HI2E4 virions an% gp45@6 potential approac& to(ar%s prevention o! viral transmission* ' Me% 2irol 4>>=M :;685<* 4:4* A$as&i M) Nii$a(a T) Seri/a(a T) Haya$a(a T) +a#a M* Capture o! HI2E4 gp45@ an% virions #y lectin immo#ili/e% polystyrene* +iocon.ug C&em 4>>=M >6:@* 4:5* ?ga(ara 0I) Higa$i 0) 0imura T* Ma.or %eterminants in &epatic %isposition o! polystyE rene nanosp&eres6 implication !or rational %esign o! particulate %rug carriers* Crit Rev T&er Drug Carrier Syst 5@@5M 4>69:* 4:8* ?ga(ara 0I) -urumoto 0) Nagayama S) et al* Precoating (it& serum al#umin re%uces receptorEme%iate% &epatic %isposition o! polystyrene nanosp&eres6 implications !or rational %esign o! nanoparticles* ' Control Release 5@@9M 4@@69:4* 4:9* +ern$opESc&nurc& A) 0ast CE) Guggi D* Permeation en&ancing polymers in oral %elivery o! &y%rop&ilic macromolecules6 t&iomer1GSH systems* ' Control Release 5@@8M >86>:* 4::* i VV) ,en W) S&ao ) C&en '-* -a#rication o! porous &ollo( silica nanoparticles an% t&eir applications in %rug release control* ' Control Release 5@@9M >=659:* 4:;* V&ou ?) S&imo%a H) Gao +) ?& S) -leming ) 3ue G* Materials science o! car#on nanoE tu#es6 !a#rication) integration an% properties o! macroscopic structures o! car#on nanoE tu#es* Acc C&em Res 5@@5M 8:64@9:* 4:<* Ii.ima S* Helical microtu#ules o! grap&itic car#on* Nature 4>>4M 8:96:;* 4:=* Maurin G) Stepane$ I) +ernier P) Colomer '-) Nagy '+) Henn -* Segmente% an% opene% multi(alle% car#on nanotu#es* Car#on 5@@4M 8>645<8* 4:>* Nanotec&nology No(* Nanotu#es survey) April 5@@8* 4;@* Nam 'M) T&a"ton CC) Mir$in CA* Nanoparticles #ase% #io #ar co%es !or t&e ultrasensiE tive %etection o! proteins* Science 5@@8M 8@464==9* 4;4* Ma&ta# R) Rogers 'P) Murp&y C'* Protein si/e% Cuantum %ot luminescence can %istinE guis& #et(een Qstraig&t)S Q#entS an% Q$in$e%S oligonucleoti%es* ' Am C&em Soc 4>>:M 44<6>@>>*

T&assu et al*

Nanoparticulate DrugEDelivery Systems

84

4;5* Cao 3C) 'in R) Nam 'M) T&a"ton CS) Mir$in CA* Raman %ye la#ele% nanoparticles pro#es !or proteins* ' Am C&em Soc 5@@8M 45:649;<;* 4;8* CMP Scienti!ica* Nanotec&6 T&e tiny revolution* copyrig&te%) 5@@4* 4;9* Salata ?2* Applications o! nanoparticles in #iology an% me%icine* ' Nano#iotec&nol 5@@9M 568* 4;:* Pan$&urst 7A) Connolly ') 'ones S0) Do#son '* Applications o! magnetic nanoparticles in #iome%icine* ' P&ys D Appl P&ys 5@@8M 8;6R4;<* 4;;* Reic& DH) Tanase M) Hultgren A) +auer A) C&en CS) Meyer G'* +iological applications o! multi!unctional magnetic nano(ires* ' Appl P&ys 5@@8M >86<5<:* 4;<* 0ast CE) Guggi D) angot& N) +ern$opESc&nurc& A* Development an% in vivo evaluaE tion o! an oral %elivery system !or lo( molecular (eig&t &eparin #ase% on t&iolate% polycar#op&il* P&arm Res 5@@8M 5@6>84* 4;=* +ern$opESc&nurc& A) Ho!!er MH) 0rum 0* T&iomers !or oral %elivery o! &y%rop&ilic macromolecular %rugs* E"pert ?pin Drug Deliv 5@@9M 46=<* 4;>* eitner 2M) Guggi D) 0raulan% AH) +ern$opESc&nurc& A* Nasal %elivery o! &uman gro(t& &ormone6 in vitro an% in vivo evaluation o! a t&iomer1GSH microparticulate %elivery system* ' Control Release 5@@9M 4@@6=<* 4<@* Commercially availa#le nanoparticles an% nanosp&eres !or me%ical applications* (((* microsp&eresEnanosp&eres*com* 4<4* 3in SW) -ranc&ini M) C&en ') et al* +ioavaila#ility en&ancement o! a C?WE5 in&i#itor +MSE89<@<@) !rom a nanocrystalline %ispersion prepare% #y spray %rying* ' P&arm Sci 5@@:M >964:>=* 4<5* Tyner 0M) Sc&i!!man SR) Giannelis EP* Nano&y#ri%s as %elivery ve&icles !or campE tot&ecin* ' Control Release 5@@9M >:6:@4* 4<8* Tyner 0M) Ro#erson MS) +erg&orn 0A) et al* Intercalation) %elivery an% e"pression o! t&e gene enco%ing green !luorescence protein utili/ing nano&y#ri%s* ' Control Release 5@@9M 4@@68>>* 4<9* +ro/ P) +enito SM) Sa( C ) et al* Cell targeting #y a generic receptor targete% polymer nanocontainer plat!orm* ' Control Release 5@@:M 4@569<:* 4<:* Rene$er DH) C&un I* Nanometer %iameter o! polymer) pro%uce% #y electrospinning* Nanotec&nology 4>>;M <654;* 4<;* 0at& DS) Ro#inson 0,) Atta(ia MA) 0o -,) aurencin CT* +ioresor#a#le nano!i#er #ase% systems !or (oun% &ealing6 optimi/ation o! !a#rication parameters* Transactions o! t&e 5=t& Annual Meeting !or t&e Society o! +iomaterials) 5@@56498* 4<<* 2errec$ G) C&un I) Peeters ') Rosen#latt ') +re(ster ME* Preparation an% c&aracteri/aE tion o! nano!i#ers containing amorp&ous %rug %ispersions generate% #y electrostatic spinning* P&arm Res 5@@8M 5@6=4@* 4:>* 2errec$ G) C&un I) Rosen#latt ') et al* Incorporation o! %rugs in an amorp&ous state into electrospun nano!i#ers compose% o! a (ater insolu#le nonE#io%egra%a#le polymer* ' Control Release 5@@8M >5689>*

Nanosuspensions !or Parenteral Delivery

+arrett E* Ra#ino(
+a"ter Healt&care Corporation) Roun% a$e) Illinois) U*S*A*

HIST?RICA INTR?DUCTI?N Nee% T&e nee% !or nanosuspensions as a %osage !orm (as recogni/e% as a means to a%minister t&erapeutic Cuantities o! (aterEinsolu#le %osage !orms A4B* T&e conE tinuing nee% !or suc& a tool (as rein!orce% (it& t&e upta$e o! &ig&Et&roug&put receptorE#ase% screening assays employe% #y t&e p&armaceutical in%ustry %uring t&e 4>>@s* T&is tec&niCue searc&es !or %rugs t&at e"&i#it strong #in%ing to &y%roE p&o#ic target receptor poc$ets an% t&ere!ore pro%uces %rug lea%s t&at ten% to #e poorly (ater solu#le A5B* It (as not only t&e $in% o! molecule t&at c&ange%) #ut also t&e vast num#ers o! suc& %rug lea%s t&at emerge% !rom %iscovery e!!orts) t&at compelle% a solution to t&e resulting !ormulation conun%rum* Typically) t&ese in vitro assays (ere con%ucte% in %imet&yl sul!o"i%e) to o#viate pro#lems o! (ater insolu#ility %uring t&e preliminary %iscovery p&ase* ,it& progression to animal stu%ies) &o(ever) nonto"ic ve&icles (ere soug&t t&at (oul% permit assessment o! t&e to"icity o! t&e %rug can%i%ate itsel!) (it&out inter!ering e!!ects %ue to t&e ve&icle A8)9B* Nanosuspensions commen%e% t&emselves as suita#le can%i%ates #ecause6 AiB solvents (ere not nee%e%) AiiB small particulate si/e permitte% intravenous %elivery) an% AiiiB t&e soli% crystal p&ase permitte% &ig& loa%ing) (&ic& permitte% t&e animal stu%ies to #e con%ucte% at many multiples o! t&e inten%e% %ose in man*

Pre%ecessor Tec&nology ECually important as t&e %eman% o! applications (as t&e rea%y availa#ility o! #uil%ing #loc$s !or implementation o! t&e ne( tec&nology* Homogeni/ers &a% #een in use since t&e late nineteent& century !or &omogeni/ation o! mil$) an% more recently in t&e latter &al! o! t&e t(entiet& century !or commercial pro%uction o! intravenous lipi% emulsions* T&ere!ore) suc& issues o! cleana#ility) sterili/a#ility) noncontamination o! !lui% processing streams (it& &y%raulics) an% so on) &a% #een long resolve%* It remaine% !or more e!!icient valve %esigns to #e %evelope%) transE mitting more o! t&e energy to t&e particle A:B* Similarly) milling eCuipment !or parE ticulates) as) !or e"ample) t&e paint in%ustry) (as availa#le an% amena#le to t&e c&anges necessary !or p&armaceutical use* T&is (as accomplis&e% #y %eveloping crossElin$e% polystyrene grin%ing me%ia t&at (as smaller an% relatively nonconE taminating) resulting in smaller %rug particles A;B* Precipitation an% crystalli/ation tec&niCues (ere muc& ol%er) #ut signi!icant c&emical engineering un%erstan%ing an% optimi/ation occurre% %uring t&e last century A<B* Supercritical !lui% processing A=B (as commercially %evelope% relatively recently* Su#stantial un%erstan%ing o! t&e role o! sur!actants) #ot& !or !ormulation sta#ili/ation A>B an% !or impacting p&armaE co$inetics A4@B) #ecame clari!ie%* Here) lessons (ere learne% !rom earlier %evelope% %rugE%elivery plat!orms) liposomes an% emulsions) regar%ing prolonging circulation times o! particulate %osage !orms*

88

89

Ra#ino(

Com#ination o! Component Tec&nologies T&e component tec&nologies %escri#e% a#ove (ere o!ten com#ine% to improve per!ormance an% overcome in%ivi%ual %e!iciencies) in t&e %evelopment o! nanoE suspensions* T&us) nanosuspensions (ere lyop&ili/e% !or greater sta#ility A44B) !orE mulate% (it& solu#le molecular analogs A45B) applie% to liCui% %rugs to !orm emulsions A48B) an% com#ine% in matri" pellet !ormulations A49B an% multivesicular lipi% systems A4:B* To re%uce particle si/e to t&e nanometer range) supercritical !lui% tec&niCues (ere com#ine% (it& an ultrasonic vi#rating sur!ace to en&ance mi"ing an% atomi/e t&e .et into nano%roplets A4;B) or spraye% into Cuenc&ing sur!actant solutions A4<B* Ta"ol nanosuspensions (ere prepare% #y emulsion templating) t&at is %issolving %rug into a volatile solvent) &omogeni/ing in an aCueous solution conE taining suita#le sta#ili/ers) an% evaporating solvent to recover nanosuspensions A4=B* -inally) rapi% precipitation an% &omogeni/ation (ere synergistically com#ine% to o#tain sta#le nanosuspensions A4>B* Homogeni/ation crac$s crystals along t&eir %e!ect planes) in%uce% #y rapi% precipitation) to re%uce t&eir si/e !urt&er* A%%itionally) t&e mec&anical s&oc$ o! &omogeni/ation o!ten in%uces conversion o! t&e unsta#le) initially !orme% polymorp& resulting !rom precipitation) to t&e more t&ermoE %ynamically sta#le polymorp&*

-?RMU ATI?N APPR?ACHES AND MANU-ACTURING METH?DS Strategy T&e preparation o! sta#le nanosuspensions must recogni/e t&e t&ermo%ynamic !orces at (or$* -or a given mass o! %rug su#stance) as particle si/e is re%uce%) sur!ace area is increase%* In an aCueous me%ium) t&is signi!icantly increases t&e sur!ace !ree energy o! t&e %rug system* Strong) an% t&ere!ore sta#le #on%s (it&in t&e %rug crysE tal lattice) on t&e one &an%) an% intermolecular &y%rogen #on%s o! (ater molecules) on t&e ot&er &an%) are %isrupte%* Instea%) t&ey are replace% #y a large inter!acial area o! &y%rop&ilic (ater molecules in pro"imity (it& a &y%rop&o#ic %rug sur!ace* Suc& a system (ill ten% to re%uce t&e energetically un!avora#le area #y particle gro(t& an% #y aggregation* ?st(al% ripening represents one mec&anism #y (&ic& t&is may occur* +y t&e ?st(al%H-reun%lic& eCuation) smaller particles &ave a &ig&er sur!ace energy t&an larger particles A5@B* T&is lea%s to greater %issolution o! smaller particles (it& conseCuent increasing si/e o! larger particles* As a result) t&e %istriE #ution o! t&e suspension s&i!ts to increasing particle si/e* To a%%ress t&is as (ell as irreversi#le agglomeration) !ormulation strategy is %esigne% to sta#ili/e particle si/e over time* Utili/ation o! sur!actant e"cipients is an essential part o! !ormulation strategy* T&e particular sur!actants are selecte% to #e compati#le (it& #ot& p&ases) an% so interpose t&emselves #et(een t&e &y%rop&o#ic %rug sur!ace on one si%e an% t&e aCueous me%ia on t&e ot&er* T&e e!!ectiveness o! t&eir interaction is measure% #y t&e re%uction in sur!ace !ree energy* An ionically c&arge% sur!actant (ill provi%e electrostatic repulsion o! neig&#oring particles) (&ic& (ill ten% to in&i#it aggregaE tion* Ho(ever) i! t&e particles overcome t&is longErange r Z5 repulsion A(&ere r is t&e interparticulate %istanceB) as #y a%%ition o! a salt to increase %ielectric s&iel%ing) t&ey may still approac& eac& ot&er too closely* T&ey may t&us #e su#.ect to t&e s&ortE acting Ar Z;B) #ut muc& stronger) attractive on%on %ispersion !orces resulting !rom instantaneous %ipole polari/ation interactions A54B* Un%er t&is circumstance) t&e particles (ill aggregate* To !orestall t&is li$ely possi#ility) a secon% type o! sur!acE tant) %esigne% to prevent too close contact #y nonelectrostatic means) is also use% in

Nanosuspensions !or Parenteral Delivery

8:

com#ination (it& an ionic sur!actant* T&is may #e a neutral) #loc$ copolymer type o! molecule &aving &y%rop&o#ic an% &y%rop&ilic %omains) !or e"ample) polo"amE ers* As particles #earing suc& sur!actants approac& eac& ot&er) t&e movement o! t&eir polymeric c&ains #ecomes constraine%) entailing a loss o! entropy* T&is steric repulsion t&ere!ore is use% synergistically (it& ionic repulsion to minimi/e aggregation over time*

Precipitation Precipitation is accomplis&e% #y %issolving t&e %rug in a suita#le solvent an% t&en mi"ing (it& a nonsolvent) e!!ecting supersaturation (it& resultant precipitation* T&is is a t(oEpart process6 initially) soli%Ep&ase nuclei must !orm !rom molecules in solution) an% su#seCuently crystals (ill gro(* Con%itions suc& as c&oice o! solvent) temperature) or%er) an% spee% o! a%%ition are c&osen to ac&ieve %i!!erent goals* To prepare %rug crystals as small as possi#le) rapi% creation o! many nuclei is optiE mi/e% (&ile minimi/ing su#seCuent gro(t&* To accomplis& t&is) t&e %rug solution may #e a%%e% rapi%ly to t&e nonsolvent) e!!ecting nearly imme%iate supersaturaE tion an% gro(t& o! many nuclei A55B* +ecause o! t&e prepon%erance o! nonsolvent relative to %rug solution) gro(t& is limite%* T&is or%er o! a%%ition is opposite to (&at is typically accomplis&e% in p&armaceutical tec&nology) (&ere t&e goal is to ac&ieve large) pure crystals #y slo(er gro(t&* Despite attempts at optimi/ation) it is !oun% t&at precipitation alone is una#le to generate suita#le particles* T&e particle si/e is usually too large !or in.ecta#le applications) an% t&e %istri#ution ten%s to #e nonuni!orm* -urt&ermore) #ecause o! t&e spee% o! !ormation) suc& suspensions are typically not t&ermo%ynamically sta#le an% ten% to evolve over time* T&e metasta#le %istri#ution may ten% to gro( in si/e i! crystalline) or evolve to a crystalline !orm i! !orme% initially in t&e amorE p&ous state* I! crystalline) rapi%ly !orme% precipitates also ten% to &ave many lattice %e!ects) an% nee%leEli$e morp&ology*

Homogeni/ation Piston gap &omogeni/ation &a% #een use% !or comminution o! liCui%Ep&ase systems) suc& as emulsions an% liposomes* Haynes A4B !irst %escri#e% t&e use o! t&e tec&niCue !or si/e re%uction o! soli% crystalline %rugs in t&e presence o! sur!actant* T&e tec&E niCue currently involves passage o! a %rug suspension un%er &ig& pressure A:@@@H :@)@@@ psiB t&roug& a varia#ly a%.uste%) narro( gap* As t&e velocity o! t&e !lui% must increase t&roug& t&e stricture) t&e pressure %rops in accor%ance (it& +ernoulliPs principle* T&e pressure %rop causes (ater vapor #u##les to !orm in t&e gap) #ut t&ese su#seCuently collapse as t&e !lui% enters a &ig&erEpressure area* T&e rapi% pressure collapse causes cavitations) (&ic& provi%es t&e energy to crac$ t&e partiE cles* T&e greatest si/e re%uction occurs in t&e !irst !e( passes o! t&e suspension t&roug& t&e gap) (it& %iminis&ing #ene!it t&erea!ter* T&is may result !rom initial crac$ing o! t&e crystals along %e!ect planes) (&ic& #ecome e"&auste% as t&e crystals #ecome smaller* It is o#serve% t&at increasing pressure usually %ecreases t&e ultiE mate particulate si/e* Si/e re%uction o! t&e suspension) as #y .et milling) an% preE (etting o! t&e slurry (it& sur!actant are &elp!ul ancillary tec&niCues t&at can #e use% A58B* T&e mean si/e o! t&e particles attaina#le (it& t&is approac& is typically 8@@ nm to 5 _m* Suc& suspensions may #e terminally sterili/e% #y autoclaving i! t&e %rug (it&stan%s &eat* I! t&e %rug is &eatEsensitive) aseptic pro%uction starting !rom sterile ra( materials is possi#le*

8;

Ra#ino(

Com#ine% Precipitation1Homogeni/ation Precipitation may #e com#ine% (it& &omogeni/ation to overcome %isa%vantages associate% (it& eit&er tec&niCue alone* HeatEsensitive %rugs may #e %issolve% in solvent) (&ic& is t&en aseptically !iltere%* Microprecipitation %o(nstream o! t&e !ilter (ill provi%e sterile crystals (&ic& may t&en #e passe% t&roug& a &omogeni/er* It is !oun% t&at com#ining #ot& tec&niCues pro%uces particles t&at are signi!icantly smaller t&an can #e attaine% #y eit&er tec&niCue alone* -urt&ermore) (it& precipiE tation alone) it o!ten &appens t&at t&e initially o#taine% precipitate proves to #e unsta#le) un%ergoing !urt&er c&ange* T&is may involve an amorp&ous to crystalline conversion or simply crystal gro(t& over time* It is !oun%) &o(ever) t&at su#seCuent &omogeni/ation arrests t&is gro(t& #y provi%ing t&e energy necessary to overcome t&e activation #arrier #et(een t&e initially !orme% precipitate an% t&e t&ermoE %ynamically sta#le material*

IN 2ITR? AND IN 2I2? PREDICTI?N ?- STA+I IT3 AND PHARMAC?0INETICS -ormulation Selection Rational %ecisionEma$ing !or !ormulation %evelopment o! a poorly (aterEsolu#le compoun% procee%s seCuentially #y %etermining t&e most appropriate met&o% !or t&e compoun%* I! t&e simpler approac&es o! pH a%.ustment) salt preparation) use o! cosolvents) or use o! lipi%s prove ine!!ective) t&en nanosuspensions may #e consi%E ere%* T&is approac& is reserve% !or t&e most %i!!icult compoun%s (&ic& &ave &ig& crystal energy A&ig& melting temperatureB A59B) in a%%ition to &ig& log P) as (ell as a reCuirement !or &ig& loa%ing* -or t&ese compoun%s) success!ul !ormulation (it& nanosuspensions can #e e"pecte%) provi%e% t&e appropriate sur!actant pac$age is utili/e%) an% sterili/ation issues can #e a%%resse%*

Molecular Determinants o! Particle Si/e Prior to em#ar$ing upon a speci!ic nanosuspension program) t&e strategy !or sucE cess!ul !ormulation can #e %etermine% #y evaluation o! t&e molecular structure (it& regar% to -igure 4* T&e log o! t&e (ater solu#ility Ameasure% or calculate%)

Nucleation rate

og A-ree Energy) slo( ErgsB

Insolu#le Asta#leB1 crystalli/er Amany

%e!ects1smaller particle si/eB

Too solu#le1 slo( crystalli/er

Amany %e!ects1smaller particle si/eB

Insolu#le Asta#leB1 !ast crystalli/er A!e(

%e!ects1larger particle si/eB
?st(al% ripening
og ASolu#ility) MB

Too solu#le1 !ast crystalli/er A!e(

%e!ects1larger particle si/eB

-IGURE 4 Planning !ormulation strategy o! nanosuspensions #ase% on molecular !le"i#ility an% solu#ility* Suita#le can%i%ates are insolu#le an% sta#le to gro(t& via ?st(al% ripening* A%%itionally) t&e nucleation rate is a measure o! t&e %e!ect num#er o! t&e crystals) (&ic& %etermines !ria#ility an% a#ility to !orm smaller particles*

Nanosuspensions !or Parenteral Delivery

8<

(it& regar% only to t&e structure) (it&out nee% !or melting pointB is plotte% along t&e &ori/ontal a"is* Too &ig& a (ater solu#ility (ill lea% to ?st(al% ripening Aincrease o! t&e particle si/e o! t&e populationB* Along t&e vertical a"is is plotte% entropy) a measure o! t&e %egrees o! !ree%om o! t&e molecule) as an in%icator o! t&e %isor%er li$ely to #e in t&e crystal* Crystal %e!ects are an important consi%eration #ot& (&ere nanosuspensions are manu!acture% #y gro(t&) !or e"ample) crystalliE /ation) as (ell as #y attrition) #y milling) or &omogeni/ation* In t&e case o! crystalliE /ation processes) it is t&e smaller) more rigi% molecules) (it& less entropy to lose) t&at !it more rea%ily) an% t&ere!ore rapi%ly) into t&e gro(ing crystal lattice A54B* T&is coul% lea% to a too rapi% crystalli/ation rate) resulting in particles t&at are e"cesE sively large* -or attrition processes) on t&e ot&er &an%) it is $no(n t&at impact !orce crac$s crystals along t&eir %e!ect planes A5:H5=BM smaller particles (ill result (&ere t&ere are many %e!ects* T&e molecular gasEp&ase entropy) as calculate% #y vi#raE tional mo%e analysis o! t&e structure) is in%icative o! t&e %e!ect rate an% is plotte% along t&e vertical a"is* -or molecules t&at are pre%icte% to #e too solu#le Agreater t&an several &un%re% ppm solu#ilityB) ot&er !ormulation approac&es may #e more appropriate* I! nanosuspensions must #e use%) t&en lyop&ili/ation s&oul% #e conE si%ere% !or sta#ility* Particular sur!actant pac$ages an% more attention to processing con%itions s&oul% #e anticipate% !or molecules t&at crystalli/e Cuic$ly) to ensure success!ul !ormulation A5>B*

In 2itro Dissolution It is also a%vantageous to #e a#le to pre%ict t&e in vivo p&armaco$inetic #e&avior o! t&e suspension* T&is can #e %one e"perimentally a!ter t&e !ormulation &as #een ma%e #y in.ecting a #olus o! t&e nanosuspension into a solution an% plotting t&e resulting lig&t transmittance* P&ysical persistence o! particles (ill %ecrease transE mittance) in%icating slo( solu#ility rate* +esi%es t&e e"treme e"amples o! comE poun%s t&at %o not %issolve at all) an% t&ose t&at %issolve virtually instantaneously) are t&ose (&ose %issolution rate is %epen%ent upon particle si/e or in!usion rate* A plasmaEsimulating solvent may #etter appro"imate t&e in vivo case* It is !oun% t&at t&e %issolution rate can also #e calculate% a priori #y $no(ing only t&e sur!acE tants use% in t&e !ormulation an% t&e structure o! t&e molecule) #y utili/ing t&e Sto$esHEinstein an% ?st(al%H-reun%lic& eCuations A5>)8@B* T&us) #e!ore un%erta$E ing %evelopment) t&e in vivo p&armaco$inetics can #e estimate% to %etermine t&e suita#ility o! t&e approac&*

P&armaco$inetic Pro!iles Possi#le p&armaco$inetic pro!iles o! in.ecta#le %rugs are s&o(n in -igure 5* +ase% upon in vitro %issolution) a !ast or slo(E%issolving nanosuspension may #e in%iE cate%* A !ast %issolving !ormulation (ill yiel% a p&armaco$inetic pro!ile essentially i%entical to an a priori solution !ormulation Aprepare% (it&) e*g*) &ig& solvent level or e"treme pHB o! t&e %rug A84)85B* As a result) t&e tissue %istri#ution (ill also #e eCuivalent) as in t&e case !or !lur#ipro!en A88B* In contrast) t&e nanoparticles o! a slo(E%issolving !ormulation may #e p&agocyti/e% !irst #y t&e !i"e% macrop&ages o! t&e liver an% spleen A89B* As t&ey are su#seCuently %issolve% (it&in t&e cell) t&e %rug (ill #e slo(ly release%* T&e resulting I2 %epot e!!ect (ill yiel% a relatively lo( Cma") an apparent %ip in t&e plasma curve (it&in appro"imately 8@ minutes o! in.ecE tion) !ollo(e% #y a %istinct rise in t&e plasma concentration) pea$ing at aroun% si" &ours !or t&e rat) !ollo(e% #y a prolonge% tailEo!! lasting over one &un%re% &ours A-ig* 5AB*

8=

Ra#ino(

P06 Rat vs* Dog @*= Drug Concentration Solution1Drug Drug Aag1m B @*; @*9 @*5 @ @ 4@ 5@ 8@ 9@ Time A&B :@ ;@ <@ =@ Rat Dog

Nanosuspension1Drug Nanosuspension1 Drug Meta#olite Solution1Drug Meta#olite

AAB

5@

9@

;@=@4@@ Time A&oursB

45@

49@

A+B

Drug Concentration Aug1m B ,&ole #loo%

Drug Concentration Ag1m B

Nanoparticles

Microparticles

Plasma

ACB

Time A&B

ADB

Time A&B

-IGURE 5 Sc&ematic in vivo p&armaco$inetic AP0B pro!iles o! nanosuspensions* AAB Comparison o! p&armaco$inetic pro!iles !or slo(E%issolving nanosuspension versus solution !ormulations !or #ot& parent compoun% an% meta#olite* A+B Comparison o! P0 pro!ile o! slo(E%issolving nanosuspension !or rat versus %og* ACB Comparison o! P0 pro!ile o! slo(E%issolving nanosuspension in plasma versus (&ole #loo%* ADB Comparison o! plasma P0 pro!iles o! nanoparticles versus microparticles) a%ministere% su#cutaneously*

T&e &al!Eli!e is noticea#ly longer in t&e %og A-ig* 5+B* ,&ere meta#olites are also trac$e%) it is o#serve% t&at t&eir gro(t& is %elaye%) an% t&eir elimination is proE longe% A-ig* 5AB* In comparison (it& microparticulate suspensions a%ministere% su#cutaneously) nanosuspensions may %emonstrate a &ig&er C ma" an% AUC A-ig* 5DB* T&is occurs #ecause %issolution o! in.ecte% particles in a %epot site o!ten constitutes t&e rateE%etermining step !or migration into t&e #loo% compartment* As particle si/e is re%uce%) sur!ace area is increase%) (&ic& increases %issolution rate* ?!ten) appreE cia#ly &ig&er %rug concentrations appear in a (&ole #loo% assay as compare% (it& plasma A-ig* 5CB) assuming t&at t&e %ensity o! t&e nanosuspension is su!!iciently greater t&an t&at o! plasma* T&is may suggest re% cell #in%ing) #ut coul% also #e in%icative o! upta$e #y circulating macrop&ages*

SA-ET3 ?- IN'ECTA+ E NAN?SUSPENSI?NS T&e speci!ication o! sa!e levels o! nanoparticulate suspensions t&at may #e a%minisE tere% intravenously may #e %e!ine% rationally in vie( o! a (i%e %ata#ase t&at &as #y no( #een accumulate%* To place t&is into perspective) 9@ years ago concerns (ere e"presse% a#out particulate matter in intravenous solutions) (&ere e"treme e"amples (it& %ire conseCuences (ere e"aggerate% !or t&at relatively ne( %osage !orm A8:B* Sa!ety o! in.ecte% particulates (ill #e consi%ere% !rom t(o perspectives6 AiB potential vascular occlusion as a !unction o! t&e si/e) num#er) an% composition o! t&e particles an% AiiB monocyte p&agocytic system response*

Nanosuspensions !or Parenteral Delivery

8>

In 2ivo Distri#ution as a -unction o! Particle Si/e P&armaco$inetics o! organ %istri#ution is %epen%ent on particle si/e an% rate o! in!usion* Nonmeta#oli/a#le particles larger t&an < _m are trappe% in t&e pulmoE nary vasculature !or e"tensive perio%s o! time* In t&e lung) alveolar macrop&ages provi%e a mec&anism !or passing particles less t&an 45 _m A8;B t&roug& t&e capillary (alls permitting e"cretion into t&e sputum out o! t&e lung* T&e e"tensive collateral circulation o! t&e pulmonary vasculature appears to mitigate t&e potential #loc$age o! capillaries #y particles) (it& anticipate% re%uction o! #loo% !lo() i! t&e particle loa% is $ept su!!iciently lo( A8<B* Ho(ever) t&ere is evi%ence o! capillary occlusion in t&e lungs o! recipients o! trans!usion o! un!iltere% #loo%) (&ic& can contain particles o! 5@ to :@@ _m in si/e* T&is e!!ect (as eliminate% (it& t&e use o! Dacron b (ool %ept& !ilters o! 9@ to =@ _m* T&is suggests #ot& an approac& to %eal (it& &ig& particulate #ur%ens o! t&erapeutic nanosuspension %osage !orms) as (ell as accepta#ility o! particles c 9@ _m !rom suc& pro%ucts A8<B* I! not %issolve% initially) particles smaller t&an < _m escape !rom t&eir initial lung seCuestration rat&er Cuic$ly A8=B) (it&in minutes) an% un%ergo p&agocytosis #y t&e !i"e% macrop&age cells o! t&e liver an% spleen A8>)9@B* T&is is a normal #e&avE ior o! t&ese cells (&en presente% (it& micro#es an% !oreign material o! si/e less t&an a#out = _m* In several rat stu%ies) no evi%ence o! an in!lammatory reaction (as !oun%* Histologically) a lo( inci%ence o! !ocal myocar%ial %egeneration (as !oun% (it& 4@ an% 9@ _m particles* Apparently sa!e levels o! = ` 4@ ; particles1$g o! si/e @*9 to 4@ _m or 9 ` 4@: particles1$g o! particles o! si/e 9@ _m coul% #e a%ministere%* +y (ay o! comparison) ?ptisond AAmers&am Healt&B is an approve% al#umin microsp&ere suspension !or ec&ocar%iograp&ic imaging* Alt&oug& t&e resi%ence time in t&e #o%y is very s&ort #ecause o! t&e ultrasoun%Ein%uce% %isruption o! t&e particles) t&ere is a loa% o! smaller particles resulting !rom %isintegration o! t&e priE mary particles t&at must #e cleare% #y t&e monocyte p&agocytic system AMPSB* T&e particle si/e mean %iameter is 5*@ to 9*: _m) (it& >8[ less t&an 4@ _m) #ut (it& a range e"ten%ing up(ar% to 85 _m* T&e concentration is : ` 4@ = to = ` 4@= microE sp&eres1m ) (it& a ma"imal recommen%e% %ose o! =*< m per contrast stu%y A94B* T&e ma"imum num#er o! particles t&at can #e in.ecte% !or t&is approve% pro%uct is t&ere!ore < ` 4@>) or >*> ` 4@< $gZ4* A%%itionally) macroaggregate% al#umin in.ection is an approve% pro%uct) routinely use% in %iagnostic imaging* T&e aggregate% particles are !orme% #y %enaE turing &uman al#umin in a &eating an% aggregation process* Eac& vial contains !our to eig&t million particles* +y lig&t microscopy) more t&an >@[ o! t&e particles are #et(een 4@ an% <@ _m) (&ereas t&e typical average si/e is 5@ to 9@ _mM none is greater t&an 4:@ _m* T&e suggeste% range o! particle num#ers !or a single in.ection is 5@@)@@@ to <@@)@@@ (it& t&e recommen%e% num#er #eing appro"imately 8:@)@@@ A95B* +y (ay o! comparison) t&e USP c<==\ microscopic test !or particulate matter in smallEvolume parenteral intravenous solutions permits 8@@ particles \5: _m* T&ere!ore) con!ormance o! I2 %rug nanosuspensions to t&e limits containe% (it&in t&e USP c<==\ stan%ar% (ill ensure signi!icant sa!ety !actors relative to t&e current practice o! pulmonary per!usion o! ra%iograp&ic particulate in.ections* T&e a#ove analysis a%%resses t&e &ig& si/e en% o! t&e particle si/e %istri#ution o! a nanosuspension %rug !ormulation* T&e mean value is !ar smaller) #elo( 4 _m* Ta#le 4 summari/es ma"imal I2 levels o! tolerate% %oses) reporte% in t&e literature*

9@
TA+ E 4 Ma"imal evels o! In.ecte% Particles (it& ?utcomes Particle si/e A_mB 4*8 @*:H4*4< @*9)9)4@ 8*9 8*< 8*9 5*@H9*: @*9 Protocol Aparticle %ose1$gB +olus) ; ` 4@> +olus) 4*; ` 4@45 Rats) #olus) = ` 4@; Dogs) #olus) 4 ` 4@4@ Dogs) repet* #olus) 5*9 ` 4@= Dogs) 5 min #olus) =*> ` 4@< Humans) #olus) >*> ` 4@< Rats) #olus) 5*: ` 4@45 ?utcome P0 stu%y A98B P0 stu%y A9:B ,ell tolerate% A8>B ,ell tolerate% A9=B ,ell tolerate% A9@B ,ell tolerate% A8;B ?ptison) approve% pro%uct A94B ,ell tolerate% A'erome Gass) +a"ter Healt&care) personal communicationB ,ell tolerate% A'erome Gass) +a"ter Healt&care) personal communicationB

Ra#ino(

@*9

Dogs) in!usion) 4*8 ` 4@45

Response o! t&e Monocyte P&agocytic System T&e ma.ority o! t&e animal stu%ies per!orme% in t&e literature involve% inert) nonE meta#oli/a#le polystyrene) crossElin$e% styrene %ivinylE#en/ene) or late" microE sp&eres* A meta#oli/a#le %rug nanoparticulate (ill #e processe% t&roug& t&e p&agolysosomes o! t&e macrop&ages muc& !aster t&an (ill inert particles A:4B* T&is poses muc& less #ur%en on t&e macrop&ages an% ena#les t&em to cycle !aster* I! t&e reticuloen%ot&elial system #ecomes overloa%e% #y p&agocytic activity) t&en reticuE loen%ot&elial #loc$age coul% occur A:5B) #ut only i! t&e p&agocytic overloa% is continue% an% &eavy A:8):9B) #ecause t&ese cells can %igest all #io%egra%a#le su#stances A::B* Clinically) a%ministration o! liposomal %o"oru#icin Aa cytoto"ic agent an% macrop&age targeterB %i% not result in more !reCuent opportunistic in!ecE tions in patients (it& AIDSErelate% 0aposiPs sarcoma compare% to patients treate% (it& com#inations o! %o"oru#icin) #leomycin) an% vincristine A:;B* T&is pro#a#ly results !rom t&e compensatory increase in macrop&age num#ers an% activity (&en su#.ecte% to &ig& p&agocytic loa%s A:<B*

APP ICATI?NS ?- PARENTERA -?RMU ATI?NS Regional Anest&etics Pursuing a local or regional anest&etic agent !or postEop or c&ronic pain) +oe%e$er et al* A:=B investigate% t&e anest&etic e!!ect o! a lecit&inEcoate% tetracaineEHI nanoE suspension* It (as pro%uce% #y sonication) &aving =@[ o! t&e particle si/e %istri#uE tion #et(een 4@@ an% :@@ nm A#y Coulter counterB an% no particles \: _m* A#out @*8 cm8 o! sample (as in!iltrate% su#cutaneously in ratsP tails) an% local anest&esia (as %etecte% (it& a &emostat* Animals serve% as t&eir o(n controls #y testing #ot& pro"imally an% %istally to t&e in.ection site* Rats in.ecte% (it& 4@[ lecit&inEcoate% tetracaine nanocrystals s&o(e% a tail #loc$ %istal to t&e in.ection site lasting 98*9 e 4*5 &ours An ^ >B) (&ereas t&ose in.ecte% (it& 4[ tetracaine solution &a% a mean tail #loc$ time o! =*: e 4*= &ours An ^ :B* +ot& t&ese groups regaine% a positive response to tail clamping %istal to t&e in.ection site) t&us ruling out nerve in.ury as t&e mec&aE nism o! local anest&esia* T&ose rats receiving 4@[ tetracaine solution eit&er %ie% (it&in 4@ minutes or %evelope% (et gangrene o! t&e tail* Negative controls o! leciE t&in mem#ranes (it&out %rug an% :[ %e"trose s&o(e% no anest&etic e!!ect* No evi%ence o! gross local tissue %amage or systemic to"icity (as o#serve% (it& t&e nanosuspensions* Comparison o! in!lammation scores) #ase% on neutrop&ilic accumulation) reveale% no statistically signi!icant %i!!erence in t&e level o! tissue

Nanosuspensions !or Parenteral Delivery

94

in!lammation !or t&e 4@[ nanosuspension versus 4[ solution* All agents reac&e% t&e nonin.ecte% control level o! in!lammation #y 49 %ays A:>B* T&e nanosuspension t&us provi%e% sustaine% release (it&out pea$s in %elivery o! (&at (oul% ot&er(ise #e a to"ic concentration o! t&e %rug* +ecause o! t&e potential !or neuroto"icity !rom t&e nanosuspension %epot i! place% pro"imal to nerves) a neuroto"icity stu%y (as con%ucte%* E"posure o! t&e rat sciatic nerve) !ollo(e% #y e"tra !ascicular a%ministration o! t&e test an% control artiE cles to t&e !ascia surroun%ing t&e nerve) an% reclosure o! t&e (oun% (as per!orme%* At <5 &ours) t&e sciatic nerves (ere e"cise%) !i"e%) staine%) an% score% microscopiE cally !or e%ema* T&ere (as no statistical %i!!erence #et(een t&e 4@[ tetracaine nanosuspension) t&e 4[ tetracaine solution (&ic& is use% clinically) or :[ %e"trose* All o! t&e agents) &o(ever) cause% statistically signi!icant) alt&oug& minimal) neural e%ema (&en compare% to nonin.ecte% nerve A;@B* Intra%ermal to"icity o! a lecit&inEcoate% suspension o! %e/ocine) prepare% similarly to t&at !or tetracaine a#ove) (as evaluate% in a rat mo%el* It (as compare% (it& t&at !or Dalganb) a commercial solution !ormulation o! t&e analgesic) consisting o! 4*:[ %e/ocine in a 8@1<@ propylene glycol1(ater mi"ture A;4B* T(enty rats (ere stu%ie% in eac& o! t&e !our groups) receiving @*@: m ID in.ections onto t&e s&ave% mi%#ac$* S$in reactions (ere evaluate% !or <5 &ours using t&e !ollo(ing criteria6 %iscoloration @ to 5M #lanc&ing @ to 5M ulceration @ to 5M esc&ar !ormation @ to 5) (it& @ #eing no e!!ect an% 5 t&e most severe* A cytoto"ic in%e" (as calculate% #y multiE plying t&e total score a#ove #y t&e area o! in.ury* T&e in%e" !or 5:@ _g microcrystal %e/ocine at ;*@ e @*< (as signi!icantly A p c @*@4B less t&an t&at !or 5:@ _g Dalgan in.ection) (&ic& &a% an in%e" o! 59*5 e 4*9* Negative control groups o! lecit&in memE #ranes an% 4@[ %e"trose &a% still lo(er cytoto"icity in%e"es at 4*= e @*8 an% @*; e @*5) respectively* Despite &aving less cytoto"icity) t&e microcrystal %e/ocine !ormulation resulte% in a signi!icantly A p c @*@4B e"ten%e% plateau o! analgesia o! 889 e 4;*> minutes versus 9= e <*: minutes !or Dalgan* T&us) t&e intensity an% %uration o! analgesia (ere prolonge% (&ile ren%ering t&e %rug more tissueEcompati#le*

Intravenous Malignant Hypert&ermia P&osp&olipi%Ecoate% nanosuspensions o! %antrolene an% so%ium %antrolene (ere stu%ie% !or treatment o! malignant &ypert&ermia A;5B* T&e currently availa#le %osage !orm) Dantriumb Intravenous) is a lyop&ili/e% !ormulation (&ic& must #e reconstitute% slo(ly to pro%uce a @*88 mg1m solution) t&us reCuiring a large a%ministration volume o! a#out ;@@ m ) at an al$aline pH o! >*:) as (ell as inclu%E ing mannitol A;8B* T&e a%ministrationErelate% issues are particularly onerous in t&e setting o! an intraoperative emergency maneuver* To increase loa%ing o! t&is poorly (aterEsolu#le %rug in a rea%ily reconstitute% !ormat) eit&er %antrolene or its so%ium salt (as coate% (it& egg p&osp&olipi% prepare% #y &omogeni/ation using a microE !lui%i/er* -ollo(ing lyop&ili/ation) rapi% reconstituta#ility (it&in one minute to pro%uce 4@[ to 4:[ suspensions (as ena#le% !or #ot& t&e %antrolene nanosusE pension ANSEDB an% t&e so%ium %antrolene nanosuspension ANSENaDB* Particle si/es o! 8@@ to =@@ nm !or NSENaD an% :@@ to =@@ nm !or NSED (ere measure%* Dissolution o! #ot& nanosuspensions (as rapi%* A !ormal comparison !or NSENaD yiel%e% p&armaco$inetics eCuivalent to t&at !or t&e solution !ormulation o! t&e %rug* A strainEgauge trans%ucer o! !orelim# a%%uction measure% t&e %oseH response curves* T&ese (ere also compara#le) an% are summari/e% as t&e e!!ective

95

Ra#ino(

%ose necessary to pro%uce :@[ an% >:[ o! t&e plateau response) as (ell as t&e magE nitu%e o! t&e plateau response* Similar values (ere also !oun% in normal s(ine* Alt&oug& t&e NSED !ormulation gave a slig&tly &ig&er ED :@ t(itc& %epression t&an Dantrium in malignant &ypert&ermiaEsuscepti#le s(ine A4*@ e @*5 vs* @*; e @*4 mg1$gB) t&e more important ED>: (as statistically eCuivalent A8*: e @*9 vs* 5*< e @*:B* ED >: is more signi!icant #ecause t&e %rug is %ose% to nearEplateau e!!ect* T&e nanosuspenE sions success!ully treate% or prevente% malignant &ypert&ermia in s(ine mo%els* ,it& NSENaD) 5*: mg1$g I2 #olus in s(ine (as o#serve% to cause pulmonary artery APAPB &ypertension as (ell as systemic &ypotension* T&is (as similar to t&e response seen (it& t&e in.ection o! un%issolve% %antrolene po(%er* T&e systemic &ypotension (as eliminate% (it& a%%ition o! a ; _m !ilter) an% t&e PAP increase (as signi!icantly re%uce% #ut not eliminate%* NSED) on t&e ot&er &an%) pro%uce% only minimal PAP increase (&en in.ecte% at t&e same rate (it& a 5 _m !ilter* It (as susE pecte% t&at postin!usion aggregation o! particles (as responsi#le !or t&ese e!!ects) 4>[ o! t&e NSENaD #eing o#serve% in aggregates o! greater t&an 8 _m %iameter) !ollo(ing 5@@` %ilution* In contrast) no aggregation (as seen !or NSED !ollo(ing %ilution* Earlier) it &a% #een %emonstrate% t&at agglomeration o! in.ecte% particles coul% sometimes #e seen a!ter !ast A4 m 1: secB #ut not a!ter slo( A4 m 1minB in.ecE tion o! nanoparticles) essentially lea%ing to plug !lo( A;9);:B* A%%itional e"planaE tion (as provi%e% #y ,ar% an% 3al$o(s$i A;;B (&o !oun% t&at plug !lo( occurre% (&ere t&e in.ection rate matc&e% t&e #loo% !lo( rate* I! one in.ecte% eit&er slo(er or !aster t&an t&is rate) !aster %issipation o! t&e #olus (oul% occur* T&e sa!er strategy is slo(er in!usion A;;B* In %ogs) rapi% I2 #olus up to 4@ mg1$g o! un!iltere% NSED coul% #e a%ministere% (it& no PAP c&ange) suggesting t&at s(ine may #e a more sensitive mo%el t&an t&e %og*

Anti!ungal Preclinical Stu%ies (it& DrugESuscepti#le -ungal Strain ,&en !ormulate% as a nanosuspension) t&e anti!ungal agent itracona/ole coul% #e I2 %ose% to t&e rat (it& no mortality at nearly 4@ times t&e D :@ o! t&e commercially availa#le &y%ro"ypropylEfEcyclo%e"trinEsolu#ili/e% %rug) Sporano" b A 'anssen P&armaceutical Pro%ucts) *P*B* T&is may occur #ecause o! re%uce% C ma") an% t&ereE !ore re%uce% Cma"Ein%uce% to"icity) inasmuc& as t&e slo(ly %issolving itracona/ole particles are initially seCuestere% #y t&e MPS* Su#seCuently) t&e %rug is release% over prolonge% t&erapeutic) yet apparently) su#to"ic levels* T&is sa!ety pro!ile permitte% a muc& greater %ose to #e a%ministere% to rats an% %ogs* In Can%i%a al#icansEc&allenge% immunoEsuppresse% rat mo%els) t&e greater %ose resulte% in &ig&er %rug levels at t&e site o! in!ection in t&e $i%ney (&ic& signi!icantly re%uce% colony counts in t&at organ* In !act) colony counts o! /ero (ere !oun% (it& su!!iciently &ig& %oses o! t&e nanosusE pension* Treatment (it& Sporano"b resulte% in a %ecrease in t&e colonyE!orming units) #ut t&ey %i% not %ecline to /ero* T&ese results are consistent (it& t&e (or$ o! An%es A;<B) (&o !oun% t&at !or %rugs o! t&e a/ole class) t&e AUC1MIC ratio is t&e critical p&armaco$inetic1p&armaco%ynamic parameter associate% (it& treatment e!!icacy* Here) AUC is t&e area un%er t&e %rug plasma curve an% MIC is t&e minimum in&i#iE tory concentration* E!!icacy o! a/oles is in%epen%ent o! pea$ concentration*

Preclinical Stu%ies (it& DrugEResistant -ungal Strain Animal results suggest t&at &ig&er %osing) ac&ieva#le (it& nanosuspensions) may cause a reconsi%eration o! t&e in vitroHin vivo correlation o! anti!ungal suscepti#ility

Nanosuspensions !or Parenteral Delivery

98

testing* Current gui%elines !or interpretive #rea$points o! MIC !or mucosal Can%i%a in!ections are6 suscepti#le g@*45: _g1m M suscepti#le) %epen%ent upon %ose ASEDDB6 @*5: to @*: _g1m M an% resistant h4*@ _g1m A;=B* T&at is to say) t&e !ungal in!ection s&oul% #e clinically treata#le i! t&e results o! t&e suscepti#ility testing in%icate an MIC g @*45: _g1m M an% itracona/ole s&oul% not #e clinically e!!ective i! t&e MIC h 4*@ _g1m * +et(een @*5: an% @*: _g1m ) t&e organism may #e consi%ere% susE cepti#le) %epen%ent upon %ose ASEDDB* It is clear) &o(ever) t&at t&ese gui%elines (ere !ormulate% (it& assumptions ma%e a#out t&e %ose o! itracona/ole t&at can #e a%minE istere%) %epen%ent upon current approve% %rug la#eling* I! greater %osing #ecomes clinically ac&ieva#le t&roug& nanosuspensions) t&en t&e interpretive #rea$points o! MIC) correspon%ing to (&at is consi%ere% treata#le) may #e revise% up(ar%* Preliminary animal results suggest t&at e!!icacy to(ar% !ungal strains) convenE tionally consi%ere% resistant to itracona/ole) can #e %emonstrate% !or itracona/ole nanosuspensions* T&us) a pre%nisolone immunoEcompromise% rat mo%el (as c&alE lenge% (it& a Can%i%a strain consi%ere% resistant to itracona/ole #y t&e a#ove gui%eE lines AMIC ^ 4; _g1m B* Survival o! t&e ma.ority o! t&e nanosuspensionEtreate% animals (as o#serve% #y t&e en% o! t&e 4@E%ay e"periment) (&ereas all o! t&e Sporano"bEtreate% animals &a% %ie% A;>B*

Clinical Stu%y T&e p&armaco$inetics o! itracona/ole nanosuspension &as #een stu%ie% clinically in allergenic &ematopoietic stem cell transplant recipients over a 49E%ay intravenous course o! treatment A<@B* ?n %ays !ive an% si" prior to t&e transplant) 5@@ mg o! nanosuspension (as given I2 every 45 &ours !ollo(e% #y 5@@ mg every 59 &ours !or t&e ne"t 45 %ays* Stea%y state (as not reac&e%) an% t&e t&erapeutic level o! :@@ _g1 (as maintaine% in !ive o! t&e si" cases !or at least nine %ays a!ter treatment cessaE tion* T&e p&armaco$inetic parameters !oun% are compare% in Ta#le 5 (it& t&ose o! t&e commercial Sporano"b solution) as liste% in t&e P&ysiciansP Des$ Re!erence A<4B an% ot&er literature* It is seen t&at in comparison (it& t&e solution !ormulation Sporano" b) t&e nanocrystal suspension occupie% a larger volume o! %istri#ution an% (as cleare% more slo(ly to give a longer &al!Eli!e an% larger area un%er t&e plasma concentraE tion curve !or t&e !irst 59 &ours) AUC 59* T&is p&armaco$inetic #e&avior is consistent (it& seCuestration in t&e MPS %epot) causing re%uce% clearance an% increase% volume o! %istri#ution* Su#seCuent release results in greatly prolonge% %elivery !or t&e nanosuspension) as s&o(n #y &al!Eli!e an% more e!!icient utili/ation o! t&e %rug) as mani!este% #y &ig&er AUC* T&e clinical results con!irme% t&e animal %ata an% suggest t&at t&e %aily %osing esta#lis&e% !or Sporano"b may #e re%uce% to a !reCuency commensurate (it& t&e prolonge% &al!Eli!e o! t&e nanosuspension* T&e maintenance o! t&erapeutic

TA+ E 5 Comparison o! Clinical P&armaco$inetics +et(een Itracona/ole Nanosuspension an% Sporano"b Itracona/ole nanosuspension 2ss A B Cl A 1&rB ti A&rB AUC59 A_g &r1 B 4;<< e =5< 8*8: e 4*= 89; e 55: terminal :4::= e 4@;8: Sporano" <>; e 4=: 55*> e :*< 8:*9 e 5>*9 mean an% 8@ terminal A<5B 8@;@: e =>;4

99

Ra#ino(

levels !or nine %ays !ollo(ing termination o! treatment is especially interesting) an% suggests utility (&en patients migrate !rom intravenous to oral %osing) !ollo(ing &ospital %isc&arge) !or e"ample* Having a relia#le internal %epot o! %rug (oul% #e use!ul %uring t&e transition process to ensure continuity o! %elivere% %ose* -urt&er clinical stu%ies (oul% #e nee%e% to con!irm t&is point* Anticancer6 Paclita"el C&aracteri/ation an% Manu!acture A#ra"aned !or In.ecta#le Suspension is an approve%) commerciali/e% nanosuspenE sion !ormulation* As s&o(n #y transmission electron microscopy) it consists o! a core o! paclita"el) surroun%e% #y an al#umin s&ell (it& an overall mean si/e o! 48@ nm) spanning a range o! a#out 4@@ to 5@@ nm A<8B* It is manu!acture% #y a%%ing a met&ylene c&lori%e solution o! paclita"el to an aCueous solution o! &uman serum al#umin) using lo(Espee% &omogeni/ation* T&e al#umin migrates to t&e aCueousH solvent inter!ace o! t&e emulsion t&at is create%* Application o! &ig&Epressure &omogeni/ation re%uces t&e particle si/e an% crossElin$s t&e al#umin coating via t&e %isul!i%e #on%s) t&us sta#ili/ing t&e particle* T&e met&ylene c&lori%e is t&en volatili/e%) leaving an aCueous suspension o! nanoparticles* T&e particles consist o! an amorp&ous paclita"el core coate% #y a 5:Enm t&ic$ s&ell o! al#umin (it& #oun% paclita"el* T&e particle si/e is su!!iciently small so as to #e sterileE!iltere% A4=B* Importantly) A#ra"ane is !ormulate% (it&out Cremop&or E ) (&ic& &as #een assoE ciate% (it& a &ost o! pro#lems inclu%ing sensitivity reactions) t&e nee% to in!use over prolonge% perio%s o! time) leac&ing o! plastici/er !rom in!usion sets) an% so on*

P&armaco$inetics Clinical p&armaco$inetic stu%ies !or A#ra"ane &ave %e!ine% parameters relative to Ta"olb summari/e% in Ta#le 8 A<9)<:B* T&e s&orter 8@Eminute %uration o! in!usion o! A#ra"ane un%erstan%a#ly results in &ig&er plasma C ma" t&an t&at !or Ta"olb a%minE istere% (it& a t&reeE&our in!usion* T&e nearEeCuivalence o! &al!Elives) alt&oug& not o#serve% earlier !or mice) re!lects a similar terminal elimination rate o! %rug !rom t&e tissue compartment* I#ra&im et al* &ave attri#ute% t&e re%uce% AUC o! t&e nanoE suspension to possi#ly several !actors* T&e !irst is !aster partitioning o! t&e nanosusE pension paclita"el out o! t&e vascular compartment) in comparison (it& Ta"ol b* It is $no(n t&at Cremop&or micelles re%uce t&e !ree paclita"el plasma !raction availa#le !or cellular partitioning) t&us re%ucing tissue %istri#ution !rom t&e central #loo% compartment* Not #eing !ormulate% (it& Cremop&or) A#ra"ane mig&t #e e"pecte% to e"it t&e circulation !aster* T&e secon% reason involves increase% en%ot&elial tranE scytosis via al#uminEme%iate% gp;@ receptor upta$e o! t&e particles A<;B*

Clinical Trial A#ra"ane an% Ta"olb (ere stu%ie% &ea% to &ea% in a P&ase III trial involving 9;@ #reast cancer patients* Ta"olb (as a%ministere% using t&e stan%ar% protocol o!
TA+ E 8 Comparison o! Clinical P&armaco$inetics +et(een A#ra"ane Paclita"el Nanosuspension an% Ta"olb Drug1%ose A#ra"ane A48: mg1m51 8@ minB Ta"ol A48: mg1m518 &rB Cma" Ang1m B ;4@@ 54<@ AUCA@HjB Ang &r1m B ;95< <>:5 ti A&rB 4: 48 Cl A 1&r1m5B 54 4=

Nanosuspensions !or Parenteral Delivery

9:

4<: mg1m5 #y 8E&our in!usion) inclu%ing preme%ication (it& steroi% an% anti&istaE mines to in&i#it Cremop&orErelate% &ypersensitivity* In contrast) A#ra"ane (as given at 5;@ mg1m5 over a s&orter %uration 8@ minutes) (it&out preme%ication or GECS- support* Despite t&e more aggressive protocol !or A#ra"ane) t&e to"icity (as no (orse6 t&ere (ere no &ypersensitivity reactionsM neutropenia %ecrease%M (&ereas neuropat&y increase% some(&at* T&is is signi!icant given t&e correlation esta#lis&e% #et(een t&e %uration o! plasma paclita"el concentrations e"cee%ing @*4 _mol1 (it& %ecline o! a#solute (&ite #loo% cell count A<9)<:)<<B* In t&is trial) A#ra"ane also pro%uce% a &ig&er tumor response rate versus paclita"el A88[ vs* 4>[B an% a longer time to tumor progression A54*> ($ vs* 4;*4 ($B A<=B* T&e improve% e!!icacy o! t&e nanosuspension may #e surprising in vie( o! t&e purporte% #ene!it o! Cremop&or E in in&i#iting t&e PEglycoprotein e!!lu" pump) t&ere#y en&ancing %rug level in tumor cells A<>B* T&e allege% #ene!it o! t&is is pro#a#ly overrate%) inasmuc& as Cremop&or is retaine% in t&e central #loo% comE partment) an% t&ere!ore %oes not enter t&e tumor tissue A=@B* T&ere are ancillary #ene!its to not !ormulating (it& Cremop&or* As corticosteroi%s %o not &ave to #e ta$en as preme%ication) t&ere is t&e possi#ility !or com#ining paclita"el (it& I E5 or inter!eron !or treatment o! metastatic melanoma) renal cell carcinoma) an% so on* T&e current Cremop&orEcontaining !ormulation cannot #e use% #ecause steroi%s) use% !or preme%ication) lyse lymp&o$ine activate% $iller A A0B cells) t&us mitigatE ing t&e #ene!its o! t&e cyto$ines A4=B*

Intrat&ecal Delivery Regional %elivery o! (aterEinsolu#le %rugs o!!ers t&e possi#ility o! increasing local t&erapeutic concentrations (&ile %ecreasing systemic si%e e!!ects* In early (or$) epi%ural in.ection o! a 4@[ #utam#en suspension inten%e% !or c&ronic cancer pain (as (ell tolerate% in %ogs an% &umans A99)=4B* As a treatment mo%ality !or intracE ta#le #rain tumors) t&e tec&niCue o! %irect in.ection into t&e ventricles o! t&e #rain is $no(n as convective en&ance% %elivery* In a%%ition to simply placing t&e %rug (it&in t&e central nervous system) t&is pressure gra%ient microin!usion o! %rug overcomes %i!!usion #arriers associate% (it& &ig& intratumoral interstitial pressures an% %isor%ere% tumor vasculature* As a result) %rug is more rapi%ly %istri#ute% t&roug&out t&e target volume A9;B* As an e"ample o! t&is application) intrat&ecal %elivery o! nanosuspension #usul!an to a mouse mo%el o! neoplastic meningitis le% to a signi!icant increase in survival A9<)9>B* T&e p&armaco$inetics (as %etermine% in patients a!!licte% (it& neoplastic meningitis) (&o receive% t&e %rug #ot& #y an ?mmaya reservoir !or intraventricular %elivery an% via lum#ar puncture* T&e %rug (as (ell tolerate% an% resulte% in %elaye% progression o! %isease A:@B*

C?NC USI?NS Gro(t& in t&e applications o! nanosuspension tec&nology &as occurre% in response to t&e voluminous num#er o! (aterEinsolu#le %rug can%i%ates t&at &ave emerge% !rom %iscovery programs* T&e in&erent &ig& loa%ing o! t&is %osage !orm %istinE guis&es it !rom liposomes) emulsions) cyclo%e"trins) an% polymeric nanoparticles) permitting %osing in animal to"icity stu%ies at t&e reCuire% multiples o! anticipate% &uman e"posure* Parenteral applications !or su#cutaneous) intramuscular) intraE %ermal) intravenous) epi%ural) an% intrat&ecal %elivery &ave #een stu%ie% in animals) (it& en&ance% e!!icacy* At t&e same time) t&e sa!ety pro!ile &as #een o#serve% to #e

9;

Ra#ino(

improve% in many cases (&en compare% to conventional solution !orms o! t&e %rugs* T&is occurs %ue to %eletion o! no"ious e"cipients) c&ange in t&e p&armaco$inetic pro!ile) or regional %elivery) t&us minimi/ing systemic to"icity* T&ese t&erapeutic an% sa!ety #ene!its &ave #een %emonstrate% !or several %rugs !or %i!!erent %isease in%ications in clinical trials* ?n t&e #asis o! t&e success!ul clinical applications) it is anticipate% t&at gro(t& o! t&is !ormulation tool (ill accelerate*

RE-ERENCES
4* Haynes DH* P&osp&olipi%Ecoate% microcrystals6 in.ecta#le !ormulations o! (aterE insolu#le %rugs* US Patent No* :)@>4)4==) 4>>5* 5* ipins$i CA) om#ar%o -) Dominy +,) -eeney P'* E"perimental an% computational approac&es to estimate solu#ility an% permea#ility in %rug %iscovery an% %evelopment settings* A%v Drug Deliv Rev 4>><M 5868* 8* ,einer M) +ernstein I * A%verse Reactions to Drug -ormulation Agents* Ne( 3or$6 Marcel De$$er) 4>=>* 9* Ga% SC* 2e&icles an% e"cipients* In6 Ga% SC) e%* Drug Sa!ety Evaluation* Ne( 3or$6 ,iley) 5@@5 Ac&apter 48*=B* :* Pan%ol!e ,D* Development o! t&e ne( Gaulin MicroEGapd &omogeni/ing valve* ' Dairy Sci 4>=5M ;:65@8:* ;* Meris$oE iversi%ge E) iversi%ge GG) Cooper E* Nanosi/ing6 a !ormulation approac& !or poorlyE(aterEsolu#le compoun%s* Eur ' P&arm Sci 5@@8M 4=6448* <* Myerson AS) Gin%e R* Crystals) crystal gro(t&) an% nucleation* In6 Myerson AS) e%* Han%#oo$ o! In%ustrial Crystalli/ation* +oston6 +utter(ort&EHeinemann) 4>>5* =* 0nutson + ) De#ene%etti PG) Tom ',* Preparation o! microparticulates using supercritiE cal !lui%s* In6 Co&en S) +ernstein H) e%s* Microparticulate Systems !or t&e Delivery o! Proteins an% 2accines* Ne( 3or$6 Marcel De$$er) 4>>;6=>H45;* >* Holm#erg 0) 'onsson +) 0ron#erg +) in%man +) e%s* Sur!actants an% Polymers in ACueous Solution* 5n% e%* Susse"6 ,iley) 5@@8* 4@* Stolni$ S) Illum ) Davis SS* ong circulating microparticulate %rug carriers* A%v Drug Del Rev 4>>:M 4;64>:* 44* Peters 0) eit/$e S) Die%eric&s 'E* Preparation o! a clo!a/imine nanosuspension !or intraE venous use an% evaluation o! its t&erapeutic e!!icacy in murine Myco#acterium avium in!ection* ' Antimicro# C&emot&er 5@@@M 9:6<<* 45* Illig 0') ,ol! G ) +acon ER) et al* A nonsur!actant (etting agent !or t&e preparation o! very small nanoparticle suspensions o! an io%inate% contrast agent* P&arm Tec& 4>>>M >5H4@9* 48* Haynes D* Micro%roplets o! (aterEinsolu#le %rugs an% in.ecta#le !ormulations o! conE taining same* US Patent No* 9)<5:)995) 4>==* 49* 2ergote G') 2ervate C) 2an Driessc&e I) et al* An oral controlle% release matri" pellet !orE mulation containing nanocrystalline $etopro!en* Int ' P&arm 5@@4M 54>6=4* 4:* Solis R) +elare%. S) Rogue 2) et al* Encapsulation o! nanosuspensions into Depo!oam b particles* A#stract k :494* In6 5=t& International Symposium on Controlle% Release o! +ioactive Materials) San Diego) CA) 'une 58H5<) 5@@4* 4;* C&attopa%&yay P) Gupta R+* Pro%uction o! anti#iotic nanoparticles using supercritical C?5 as antisolvent (it& en&ance% mass trans!er* In% Eng C&em Res 5@@4M 9@68:8@* 4<* Pace G,) 2ac&on MG) Mis&ra A0) et al* Process to generate su#micron particles o! (aterEinsolu#le compoun%s* US Patent No* ;)4<<)4@8) 5@@4* 4=* Desai NP) Tao C) 3ang A) et al* Protein sta#ili/e% p&armacologically active agents) met&E o%s !or t&e preparation t&ereo! an% met&o%s !or t&e use t&ereo!* US Patent No* ;)<9>)=;=) 5@@9* 4>* 0ipp 'E) ,ong 'CT) Doty M') Re##ec$ C * Microprecipitation met&o% !or preparing su#micron suspensions* US Patent No* ;);@<)<=9 +5) 5@@8* 5@* 0ipp 'E* P&armaceutical nanotec&nology revie(6 t&e role o! soli% nanoparticle tec&nolE ogy in t&e parenteral %elivery o! poorly (aterEsolu#le %rugs* Int ' P&arm 5@@9M 5=964@> Ac&apters 4; an% 4<B*

Nanosuspensions !or Parenteral Delivery

9<

54* Moore ,'* P&ysical C&emistry* 8r% e%* Engle(oo% Cli!!s) N'6 PrenticeEHall) 4>;5* 55* Estrin '* Precipitation processes* In6 Myerson AS) e%* Han%#oo$ o! In%ustrial Crystalli/ation* +oston6 +utter(ort&s1Heinemann) 4>>86484H49>* 58* Muller RH) +enita S) +o&m +) e%s* Emulsions an% Nanosuspensions !or t&e -ormulation o! Poorly Solu#le Drugs* Stuttgart6 Me%p&arm Scienti!ic Pu#lis&ers) 4>><* 59* 3al$o(s$y SH* Tec&niCues o! Solu#ili/ation o! Drugs* Ne( 3or$6 Marcel De$$er) 4>=464H49* 5:* Huttenrauc& R* -un%amentals o! p&armaceutics* Acta P&arm Tec&nol 4>==M 8964* 5;* Grant D) 3or$ P* Entropy o! processing6 a ne( Cuantity !or comparing t&e soli% state %isor%er o! p&armaceutical materials* Int ' P&arm 4>=;M 8@64;4* 5<* Du%%u SP) Grant D* T&e use o! t&ermal analysis in t&e assessment o! crystal %isruption* T&ermoc&im Acta 4>>:M 59=6484* 5=* Gri!!it& AA* P&il Trans Roy Soc on%on) Ser A 4>54M 55464;8* 5>* 0ipp '* +a"ter Healt&care Corp*) unpu#lis&e% %ata) 5@@9* 8@* ee R,) S&a( 'M) McS&ane ') ,oo% R,* Particle si/e re%uction* In6 iu R) e%* ,aterE Insolu#le Drug -ormulation* Denver6 Interp&arm 5@@@69::H9>5* 84* 2iernstein H) Stump! C* Similar central actions o! intravenous met&o&e"itone suspension an% solution in t&e ra##it* ' P&arm P&armacol 4>>5M 996;;* 85* Clement MA) Pug& ,) Pari$& I* Tissue %istri#ution an% plasma clearance o! a novel microcrystalEencapsulate% !lur#ipro!en !ormulation* P&armacologist 4>>5M 8965@9* 88* Pace SN) Pace G,) Pari$& I) Mis&ra A0* Novel in.ecta#le !ormulations o! insolu#le %rugs* P&arm Tec& 4>>> Marc&M 44;* 89* Mog&imi SM) Hunter AC) Murray 'C* ongEcirculating an% targetEspeci!ic nanoparticles6 t&eory to practice* P&armacol Rev 5@@4M :865=8* 8:* Garvan 'M) Gunner +,* T&e &arm!ul e!!ects o! particles in intravenous !lui%s* Me% ' Aust 4>;9M 564* 8;* 0an$e M) Simmons GH) ,eiss DI) et al* Clearance o! 94Ce la#elle% microsp&eres !rom #loo% an% %istri#ution in speci!ic organs !ollo(ing intravenous an% intraarterial a%minE istration in #eagle %ogs* ' P&arm Sci 4><8M ;6:@=* 8<* +ar#er TA* Patient issues relate% to particulate matter* In6 +ar#er TA) e%* P&armaceutical Particulate Matter) Analysis an% Control* +u!!alo Grove) I 6 Interp&arm Press) 4>>8 Ac&apter WIIB* 8=* Singer 'M* ' Res Soc 4>;>M ;6:;4* 8>* Gesler RM) Garvin P') 0lamer +) et al* T&e #iological e!!ects o! polystyrene late" particles a%ministere% intravenously to ratsRa colla#orative stu%y* +ull Parent Drug Assoc 4><8M 5<64@4* 9@* Sc&roe%er HG* Distri#ution o! ra%iola#ele% su#visi#le microsp&eres a!ter intravenous a%ministration to #eagle %ogs* ' P&arm Sci 4><=M ;<6:@=* 94* ?ptison Pro%uct Description) Amers&am Healt&* 95* Dra"Image MAA 0it !or t&e preparation o! Tec&netium Tc >>m Al#umin Aggregate% In.ection* Pro%uct insert) -e#ruary 5@@5* 98* ,il$ins D') Meyers PA* +r ' E"p Pat&ol 4>;;M 9<6:;=* 99* S&ulman M* Treatment o! cancer pain (it& epi%ural #utylEamino #en/oate suspension* Reg Anest& 4>=<M 4564* 9:* Sc&oen#erg MD) Gilman PA) Muma( 2R) Moore RD* T&e p&agocytosis o! uni!orm polyE styrene late" particles AP PB #y t&e reticuloen%ot&elial system ARESB in t&e ra##it* +rit ' E"ptl Pat& 4>;4M 9569=;* 9;* Hall ,A) Rustam/a%e& E) As&er A * ConvectionEen&ance% %elivery in clinical trials* Neurosurg -ocus 5@@8M 4964* 9<* Grossman SA) 0ra#a$ M'* eptomeningeal carcinomatosis* Cancer Treatment Rev 4>>>M 5:64@8* 9=* Slac$ 'D* Acute &emo%ynamic e!!ects an% #loo% pool $inetics o! polystyrene microE sp&eres !ollo(ing intravenous a%ministration* ' P&arm Sci 4>=4M <@6;;@* 9>* Arc&er GE et al* Intrat&ecal #usul!an treatment o! &uman neoplastic meningitis in at&ymic nu%e rats* ' NeuroE?ncol 4>>>M 996588* :@* 7uinn 'A) Glant/ M) Petros ,) et al* P&ase I trial o! intrat&ecal Sparta.ect #usul!an !or patients (it& neoplastic meningitis* A#stract No* 84=* In6 Eig&t& ASC? Annual Meeting) ?rlan%o) - ) May 4=H54) 5@@5*

9=
:4* 0reuter '* Nanoparticles* In6 0reuter ') e%* Colloi%al Drug Delivery Systems* Ne( 3or$6 Marcel De$$er) 4>>9 6 54>H895) c&* :* :5* Daemen T) Ho!ste%e G) Ten 0ate MT) et al* iposomal %o"oru#icinEin%uce% to"icity6 %epletion an% impairment o! p&agocytic activity o! liver macrop&ages* Int ' Cancer 4>>:M ;46<4;* :8* 2anEtten E,M* A%ministration o! liposomal agents an% #loo% clearance capacity o! t&e mononuclear p&agocyte system* Antimicro# Agents C&emot&er 4>>=M 9564;<<* :9* +a$$erE,ou%en#erg IA'M* A%ministration o! liposomal agents an% t&e p&agocytic !uncE tion o! t&e mononuclear p&agocytic system* Int ' P&arm 4>>=M 4;56:* ::* Pes$o '* P&ysiological conseCuences o! in.ecte% particulates* In6 0napp 'V) +ar#er TA) ie#erman A) e%s* iCui%E an% Sur!aceE+orne Particle Measurement Han%#oo$* Ne( 3or$6 Marcel De$$er) 4>>;* :;* +ogner 'R* iposomal %o"oru#icin in t&e treatment o! a%vance% AIDSErelate% 0aposi sarcoma* ' AcCuir Immune De!ic Syn%r 4>>9M <69;8* :<* Daemen T) Regts ') Meesters M) et al* To"icity o! %o"oru#icin entrappe% (it&in longE circulating liposomes* ' Control Release 4>><M 9964* :=* +oe%e$er +H) o.es$i E,) 0line MD) et al* UltraElong %uration local anest&esia pro%uce% #y in.ection o! lecit&inEcoate% tetracaine microcrystals* ' Clin P&armacol 4>>9M 896;>>* :>* 0line MD) +oe%e$er +H) Matti" ME) et al* Intra%ermal to"icity o! lecit&inEcoate% microE crystalline tetracaine* Anest&esiology 4>>5M <<6A=@@* ;@* +oe%e$er +H) 0line MD) Matti" ME) et al* Perip&eral neuroto"icity o! lecit&inEcoate% tetE racaine microcrystals* Anest&esiology 4>>8M <>6A=5:* ;4* DeMeo R) Haynes D) +i$&a/i G* Comparing e!!icacy an% intra%ermal to"icity o! lecit&inE coate% microcrystal %e/ocine* Anest&esiology 4>>8M <>6A=;:* ;5* 0aran SM) o.es$i E,) Haynes DH) et al* Intravenous lecit&inEcoate% microcrystals o! %antrolene are e!!ective in t&e treatment o! malignant &ypert&ermia6 an investigation in rats) %ogs) an% s(ine* Anest& Analg 4>>;M =56<>;* ;8* o.es$i E,) 0aran SM) +oe%e$er +H) et al* ecit&inEcoate% %antrolene so%ium microE crystals vs* %antrolene so%ium6 %oseHresponse to muscular t(itc& in s(ine* Anest&esiology 4>>8M <>6A98=* ;9* 0reuter ') Tau#er U) Illi 2* Distri#ution an% elimination o! polyAmet&ylE5EC49Emet&acrylateB nanoparticle ra%ioactivity a!ter in.ection in rats an% mice* ' P&arm Sci 4><>M ;=64998* ;:* +orc&ar% G) 0reuter '* T&e role o! serum complement on t&e organ %istri#ution o! intraE venously a%ministere% polyAmet&yl met&acrylateB nanoparticles6 e!!ects o! preEcoating (it& plasma an% (it& serum complement* P&arm Res 4>>;M 4864@::* ;;* ,ar% G) 3al$o(s$y S* Stu%ies in p&le#itis* ' Parenter Sci Tec&nol 4>>8M 9<64;4* ;<* An%es D* In vivo p&armaco%ynamics o! anti!ungal %rugs in treatment o! can%i%iasis* Antimicro# Agents C&emot&er 5@@8M 9<644<>* ;=* Re" 'H) P!aller MA) Galgiani 'N) et al* Development o! interpretive #rea$points !or anti!ungal suscepti#ility testing6 conceptual !rame(or$ an% analysis o! in vitroHin vivo correlation %ata !or !lucona/ole) itracona/ole) an% Can%i%a in!ections* Clin In!ect Dis 4>><M 59658:* ;>* Ra#ino( +E) ,&ite RD) Glosson ') et al* En&ance% e!!icacy o! NAN?EDGE itracona/ole nanosuspension in an immunosuppresse% rat mo%el in!ecte% (it& an itracona/oleE resistant C* al#icans strain* A#stract kR;4=9* In6 Amer* Assn* o! P&arm* Sci* Ann* Meeting) Salt a$e City) Uta&) ?cto#er 5;H8@) 5@@8* <@* Donnelly 'P) Mouton ',) +li.levens NMA) et al* P&armaco$inetics o! a 49 %ay course o! itracona/ole nanocrystals given intravenously to allogeneic &aematopoietic stem cell transplant AHCSTB recipients* A#stract kAE85* In6 94st Interscience Con!erence on Antimicro#ial Agents an% C&emot&erapy) C&icago) I ) Decem#er 4;H4>) 5@@4* <4* SP?RAN?Wb AItracona/oleB In.ection* Approve% la#eling* In6 PDR := e% P&ysiciansP Des$ Re!erenceb* Montvale) N'6 T&omson PDR) 5@@964<<5* <5* ,illems ) van %er Geest R) %e +eule 0* Itracona/ole oral solution an% intravenous !orE mulations6 a revie( o! p&armaco$inetics an% p&armaco%ynamics* ' Clin P&arm T&erap 5@@4M 5;64:>* <8* +lum ' ) +everi%ge R) Ro#ert N) et al* A+IE@@< Nanoparticle paclita"el6 %emonstration o! antiEtumor activity in ta"aneEre!ractory metastatic #reast cancer* A#stract ;9* Proc Am Soc Clin ?ncol 5@@8*

Ra#ino(

Nanosuspensions !or Parenteral Delivery

9>

<9* I#ra&im N0) Desai N) eg&a S) et al* P&ase I an% p&armaco$inetic stu%y o! A+IE@@<) a cremop&orE!ree) proteinEsta#ili/e%) nanoparticle !ormulation o! paclita"el* Clin Cancer Res 5@@5M =64@8=* <:* Ta"olb Apaclita"elB In.ection* Direction insert* Princeton) N'6 +ristolEMyers SCui## ?ncology 5@@8* <;* Desai N) Trieu 2) 3ao R) et al* Increase% en%ot&elial transcytosis o! nanoparticle al#uminE #oun% paclita"el AA+IE@@<B #y en%ot&elial gp;@ receptors6 a pat&(ay in&i#ite% #y Ta"ol* Poster A? @;;* In6 5<t& San Antonio +reast Cancer Symposium) San Antonio) TW) Decem#er 5@@9* <<* Hui/ing MT) 0eung AC-) Rosing H) et al* P&armaco$inetics o! paclita"el an% meta#olites in a ran%omi/e% comparative stu%y in platinumEpretreate% ovarian cancer patients* ' Clin ?ncol 4>>8M 446545<* <=* ee D* Current trials o! a nanoparticle al#uminE#oun% ta"ane !ormulation in metastatic #reast cancer* Clin +reast Cancer 5@@9 -e#M 8>4* <>* ,oo%coc$ DM) 'e!!erson S) insenmeyer ME) et al* Reversal o! t&e multi%rug resistance p&enotype (it& cremop&or E ) a common ve&icle !or (aterEinsolu#le vitamins an% %rugs* Cancer Res 4>>@M :@694>>* =@* Sparre#oom A) 2er(ei. ') 2an %er +urg ME ) et al* Disposition o! Cremop&or E in &umans limits t&e potential !or mo%ulation o! t&e multi%rug resistance p&enotype in vivo* Clin Cancer Res 4>>=M 964>8<* =4* S&ulman M* E!!ect o! epi%ural an% su#arac&noi% in.ections o! a 4@[ #utam#en suspension* Reg Anest& 4>>@M 4:6495*

Nanoparticles Prepare% Using Natural an% Synt&etic Polymers

Su%&ir S* C&a$ravart&i an% Dennis H* Ro#inson
Department o! P&armaceutical Sciences) University o! Ne#ras$a Me%ical Center) ?ma&a) Ne#ras$a) U*S*A*

Sin.an De
Researc& an% Development) Perrigo Company) Allegan) Mic&igan) U*S*A*

INTR?DUCTI?N I%eally) #iologically active agents s&oul% #e encapsulate% (it&in nanoparticles using polymers (it& (ellE%e!ine% p&ysical an% c&emical properties* T&ese polymers protect t&e active ingre%ient an%) a!ter local or systemic a%ministration) potentially target an% t&en release t&e %rug in a controlle%) pre%icta#le manner* T&e use o! polymeric nanoparticle !or %rug %elivery is a strategy t&at aims to optimi/e t&eraE peutic e!!ects (&ile minimi/ing a%verse e!!ects* T&e purpose o! t&is c&apter is to &ig&lig&t t&e ma.or !actors relate% to t&e use o! nanoparticles !a#ricate% !rom synt&etic or natural polymeric materials t&at &ave #een use% in %rug %elivery an% imaging* A compre&ensive revie( o! t&is area o! researc& is #eyon% t&e scope o! t&is c&apter an% &ence t&e rea%ers are re!erre% to ot&er revie(s in t&e areas !or a%%iE tional in!ormation A4H8B*

P? 3MERIC CARRIERS USED T? PREPARE NAN?PARTIC ES Polymers use% in controlle% %rug %elivery) inclu%ing nanoparticles) may #e classiE !ie% as eit&er AiB natural an% synt&etic) or AiiB #io%egra%a#le an% non#io%egra%a#le* E"amples o! naturally occurring #io%egra%a#le an% #iocompati#le polymers use% to prepare nanoparticles inclu%e6 cellulose) gelatin) pullulan) c&itosan) alginate) an% glia%in* T&e c&aracteristics an% per!ormance) particularly in vivo) o! nanoparticles prepare% using natural polymers may #e less pre%icta#le as t&ese polymers may vary (i%ely in c&emical composition an% &ence) p&ysical properties* In a%%ition) natural polymers are o!ten mil%ly immunogenic* Conversely) it is possi#le to synE t&esi/e polymers (it& precise c&emical composition) resulting in &ig&ly pre%icta#le p&ysical properties suc& as solu#ility) permea#ility) an% rates o! #io%egra%ation* As a result) synt&etic polymers are also more easily %esigne% !or speci!ic applicaE tions) suc& as controlle% rates o! %issolution) permea#ility) %egra%ation) an% erosion) as (ell as !or targeting* E"amples o! synt&etic #io%egra%a#le polymers use% to preE pare nanoparticles inclu%e6 polylacti%e AP AB) polyEAlacti%eEcoEglycoli%eB AP GAB) polyan&y%ri%es) polyElEcaprolactone) an% polyp&osp&a/ene* +io%egra%a#ility an% #iocompati#ility are important properties o! polymeric materials t&at are to #e in.ecte% or implante% into t&e #o%y* Non#io%egra%a#le polymeric nanoparticles may #e use% !or controlle% %rug %elivery an% also in t&e complimentary !iel% o! %iagnostic imaging* E"amples o! non#io%egra%a#le) synt&etic polymers use% in %rug %elivery inclu%e polymet&yl met&acrylate (&ile polystyrene particles &ave #een use% as %iagnostic agents*

:4

:5

C&a$ravart&i et al*

Natural +io%egra%a#le Polymers Use% to Prepare Nanoparticles Alginates Alginates are linear) un#ranc&e% polysacc&ari%es compose% o! ran%om c&ains o! guluronic an% mannuronic aci%s A9B* In aCueous me%ia) t&e so%ium ions !rom salts o! t&ese anionic) &eteropolymers e"c&ange (it& %ivalent cations) suc& as calcium) to !orm (aterEinsolu#le gels A:B* +ecause o! t&e !avora#le con%itions %uring manu!acE ture) alginates are i%eal carriers !or oligonucleoti%es A;B) pepti%es A<B) proteins A<B) (aterEsolu#le %rugs) or %rugs t&at %egra%e in organic solvents* Alginates are nonE immunogenic an% availa#le in a (i%e range o! molecular (eig&ts as c&aracteri/e% #y t&eir in&erent viscosity* Alginate nanoparticles are prepare% #y e"tru%ing an aCueous so%ium alginate solution t&roug& a narro(E#ore nee%le into an aCueous solution o! a cationic agent) suc& as calcium ions) c&itosan) or polyElElysine* T&ese cations crossElin$ t&e guluronic an% mannuronic aci%s to !orm an eggE#o" structure t&at !orms t&e core o! t&e gel matri"* In vivo) t&erapeutic agents are release% (&en t&e matri" re%issolves %ue to t&e reversi#le e"c&ange o! %ivalent cations (it& monE ovalent ions) especially so%ium present in p&ysiological !lui%* A %isa%vantage o! t&e use o! alginates is t&at t&is reversi#le ion e"c&ange may result in t&e rapi% release o! t&e t&erapeutic agent* Ho(ever) an e"ample o! t&e use o! alginate nanoparticles to sustain anti#acterial %rug levels a#ove t&e minimum in&i#itory concentration in t&e liver) lungs) an% spleen a!ter pulmonary a%ministration (as %emonstrate% using isonia/i%) ri!ampicin) an% pyra/inami%e A=B* ?ne met&o% to prolong release !rom alginate particles is to coat t&em (it& a cationic polymer) !or e"ample) polyElElysine or c&itosan* In t&is application) t&e mass ratio o! alginate to cationic polymer is critical in terms o! release c&aracteristics an% particle si/e A>B*

C&itosan C&itosan is a natural polymer o#taine% #y %eacetylation o! c&itin) a component o! cra# s&ells* It is a cationic polysacc&ari%e compose% o! linear fA4)9BElin$e% %Eglucosamine* T&e various met&o%s use% to prepare c&itosanE#ase% nanoparticles an% t&eir applications &ave #een e"tensively revie(e% A4@B* C&itosan can entrap %rugs #y numerous mec&anisms inclu%ing c&emical crossElin$ing) ionic crossElin$ing) an% ionic comple"ation A44B*

Gelatin Gelatin is a natural) #io%egra%a#le protein o#taine% #y aci%E or #aseEcataly/e% &y%rolysis o! collagen* It is a &eterogenous mi"ture o! singleE or multiEstran%e% polypepti%es compose% pre%ominantly o! glycine) proline) an% &y%ro"yproline resi%ues an% is %egra%e% in vivo to amino aci%s* Gelatin nanoparticles are prepare% #y a t(oEstep) %esolvation process A45B* +rie!ly) t&is coacervation proce%ure involves t&e a%%ition o! eit&er anot&er more (aterEsolu#le polymer or) a (aterEmisci#le nonsolvent !or gelatin) to an aCueous gelatin solution a#ove its gel temperature o! a#out 9@]C* T&e concentrate% gelatin liCui% particles are isolate% an% &ar%ene% #y c&emical crossElin$ing (it& glutaral%e&y%e* Alternately) t&ese particles can #e preE pare% using a simple o1( emulsion or (1o1( microemulsion met&o%* Gelatin nanoparticles &ave #een use% to %eliver paclita"el) met&otre"ate) %o"oru#icin) DNA) %ou#leEstran%e% oligonucleoti%es) an% genes* PEGylation o! t&e particles signi!icantly en&ances t&eir circulation time in t&e #loo% stream A48B an% increases t&eir upta$e into cells #y en%ocytosis* Anti#o%yEmo%i!ie% gelatin nanoparticles &ave #een use% !or targete% upta$e #y lymp&ocytes A49B*

Nanoparticles Prepare% Using Natural an% Synt&etic Polymers

:8

Pullulan Similar to %e"tran an% cellulose) t&e glucans in pullulan are (aterEsolu#le) linear polysacc&ari%es t&at consist o! t&ree mE4)9Elin$e% glucose molecules polymeri/e% #y mE4); lin$ages on t&e terminal glucose A4:B* Pullulan is a !ermentation pro%uct o! t&e yeast Aureo#asi%ium pullulans* ,&en ma%e &y%rop&o#ic #y acetylation) t&ese polymers (ill sel!Eassociate to !orm nanoparticles (it& a &y%rop&o#ic core t&at (ill encapsulate &y%rop&o#ic %rugs* Pullulan nanoparticles &ave #een prepare% #y %ialysis o! an organic solution against (ater* In one met&o%) a reverse micellar solution o! t&e anionic sur!actant) Aerosol ?T) in nE&e"ane (as prepare% an% an aCueous solution o! t&e %rug an% pullulan a%%e% A4;B* T&e nanoparticles are sta#iE li/e% #y crossElin$ing (it& glutaral%e&y%e* T&ese %elivery systems &ave #een use% in %elivering cytoto"ic %rugs) genes) an% as pHEsensitive %elivery systems*

Glia%in Glia%in is a glycoprotein t&at) as a component o! gluten) is e"tracte% !rom glutenE ric& !oo% suc& as (&eat !lour* T&ey are slig&tly &y%rop&o#ic an% polar* +ioactive molecules o! varia#le polarity can #e encapsulate% into glia%in nanoparticles* Glia%in nanoparticles can #e prepare% #y a %esolvation met&o% t&at e"ploits t&e insolu#ility o! t&is polymer in (ater A4<B* +rie!ly) glia%in nanoparticles are precipiE tate% (&en an et&anolic solution o! glia%in is poure% into an aCueous solution* Glia%in nanoparticles &ave #een use% to %eliver transEretinoic aci%) mEtocop&erol) an% vitamin E* ectins &ave #een con.ugate% to t&e sur!ace o! glia%in nanoparticles to target t&e colon an% treat Helico#acter pylori in!ections A4<B*

Synt&etic +io%egra%a#le Polymers Use% to Prepare Nanoparticles Polylacti%e an% Polylacti%eEcoEGlycoli%e T&e &y%rop&o#ic P A may #e use% alone or copolymeri/e% (it& polyEglycolic aci% to !orm a range o! P GA o! (i%ely varying polymeric ratios an% &ence p&ysicoE c&emical properties* T&ese -DAEapprove% polymers &ave #een (i%ely use% in %rug %elivery inclu%ing nanoparticles* P A an% P GA polymers %egra%e #y ran%om #ul$ &y%rolysis t&at is cataly/e% in aci%ic me%ia* T&e met&o%s use% to prepare P A an% P GA nanoparticles as (ell as t&eir range o! applications) p&ysical properties) #iological !ate) an% targeta#ility &ave #een compre&ensively revie(e% A5B*

Polyan&y%ri%es Polyan&y%ri%es are #io%egra%a#le polymers (it& a &y%rop&o#ic #ac$#one an% a &y%rolytically la#ile an&y%ri%e lin$age* T&ey are synt&esi/e% #y ringEopening polymeri/ation an% %egra%e #y sur!ace &y%rolysis A8B* T&e application o! polyanE &y%ri%es &as #een limite% to !ilm an% microsp&ere !ormulation !or sustaine% release o! a %rug or protein at t&e site*

PolyEnECaprolactones Met&o%s use% to prepare nanoparticles using polyE lEcaprolatones &ave #een previE ously revie(e% A4=B an% inclu%e emulsion polymeri/ation) solvent %isplacement) %ialysis) an% inter!acial polymer %eposition* T&ese semicrystalline polymers are c&emically sta#le) possess a lo( glass transition temperature) an% %egra%e slo(ly* Hence) t&ey &ave t&e potential !or longEterm %rug %elivery* PolyE lEcaprolactone nanoparticles &ave #een use% as ve&icles to %eliver a (i%e range o! %rugs inclu%ing tamo"i!en) retinoic aci%) an% griseo!ulvin*

:9

C&a$ravart&i et al*

Polyal$ylECyanoacrylates Polyal$ylEcyanoacrylate APACAB nanoparticles are prepare% #y t&e conventional emulsionEevaporation tec&niCue* In a%%ition to sustaining %rug release) PACA nanoparticles &ave t&e a#ility to overcome multi%rug resistance at #ot& t&e cellular an% su#cellular levels A4>B* T&e potential !or targete% %elivery o! PACA nanoparticles to cells &as #een %emonstrate% #y con.ugation o! polysacc&ari%es to t&e sur!ace A5@B*

Non#io%egra%a#le Polymers Use% to Prepare Nanoparticles Polymet&acrylate APMAB an% polymet&yl met&acrylate APMMAB &ave #een (i%ely use% in a variety o! p&armaceutical an% me%ical applications* Speci!ically) PMMA Eu%ragitb nanoparticles can #e prepare% #y nanoprecipitation met&o% A54B t&at involves a%%ing &y%roalco&olic solution o! t&e polymer to an organic solvent* Incorporation o! polyEacrylic aci% into nanoparticles increase% t&e trans!ection e!!iE ciency o! DNA* T&e si%e c&ain o! PMMA can #e mo%i!ie% to ma$e t&ese polymers possess pHE%epen%ent solu#ility an% &as #een use% to prepare pHEsensitive nanoparticles to increase t&e oral #ioavaila#ility*

T&ermosensitive Nanoparticles Polyvinyl caprolactone nanoparticles e"ist in s(ollen state (&en %isperse% in (ater at room temperature* Ho(ever) t&ey s&rin$ upon increasing temperature a#ove t&e volume p&ase transition temperature) e"pelling (ater A55B* PolyANEisopropyl acrylE ami%eB ANIPAAMB &as #een e"tensively use% to prepare t&ermosensitive &y%rogelE #ase% nanoparticles* T&is polymer &as a lo( critical solution temperature o! 85]C to 89]C) a#ove (&ic& it s&rin$s to release t&e %rug*

Soli%H ipi% Nanoparticles Alt&oug& not polymers) puri!ie% triglyceri%es an% (a"es t&at are soli% at am#ient an% #o%y temperatures can also #e use% as nanoparticulate carriers* T&ese may #e prepare% #y various met&o%s inclu%ing &ig&Espee% &omogeni/ation) microemulE sions) emulsionEsolvent evaporation) an% ultrasonication A4B* It is possi#le to prepare lipi% nanoparticles (it& &ig& %rug loa%ing) particularly (it& lipop&ilic %rugs t&at sustain release %ue to t&eir &y%rop&o#icity an% lo( %rug %i!!usivity in t&e matri"*

CHARACTERIVATI?N ?- NAN?PARTIC ES P&ysical Properties o! Polymers T&e !ollo(ing p&ysicoc&emical properties o! polymers greatly in!luence t&e properE ties) met&o% o! preparation) an% per!ormance o! nanoparticles* Molecular ,eig&t T&e molecular (eig&t o! a polymer in!luences p&ysical properties suc& as glass transition temperature) viscosity in solutions) solu#ility) crystallinity) %egra%ation rate) an% mec&anical strengt&* In general) polymers (it& a lo(er molecular (eig&t e"&i#it a lo(er viscosity an% tensile strengt&) an% %egra%e more rapi%ly* Hence) selection o! a polymer (it& an appropriate molecular (eig&t is important !or t&e inten%e% application* -or e"ample) i! a polymer %egra%es #y aci%Ecataly/e%) #ul$ &y%rolysis) a lo(EmolecularE(eig&t polymer (ill %egra%e !aster %ue to autocatalyE sis #y t&e greater proportion o! oligomers !orme%* Ine!!icient polymeric synt&esis

Nanoparticles Prepare% Using Natural an% Synt&etic Polymers

::

may !orm polymers (it& &ig& poly%ispersity t&at %egra%e more rapi%ly t&an &omoE polymers o! similar molecular (eig&t* Degree o! Crystallinity T&e mec&anical properties o! polymers can #e altere% #y t&e %egree o! crystallinity* -or e"ample) #ecause o! t&e uni!orm arrangement o! its c&ains (it&in t&e lattice structure) a crystalline polymer (ill %egra%e slo(ly t&an an amorp&ous !orm* Ho(ever) pure crystalline polymers are #rittle an% usually less suite% to %rugE %elivery applications* -urt&er) amorp&ous polymers possess poor mec&anical toug&ness* T&ere!ore) t&e polymers use% in %rug %elivery are usually a mi"ture o! crystalline an% amorp&ous !orms*

Hy%rop&o#icity -actors t&at in!luence t&e &y%rop&o#icity o! t&e polymer inclu%e molecular (eig&t) aCueous solu#ility o! t&e monomers) an% t&e %egree o! #ranc&ing* Alt&oug& increase in molecular (eig&t increases t&e &y%rop&o#icity) an increase in t&e %egree o! #ranc&ing results in a more (aterEsolu#le polymer* Hence) nanoparticles prepare% using a &y%rop&o#ic polymer e"&i#it %ecrease% (ater penetration an% (etta#ility) resulting in relatively slo(er %rug release an% polymer %egra%ation times t&an similar &y%rop&ilic !orms* Ho(ever) t&e incorporation o! a &y%rop&ilic polymer or a%%itives into nanoparticles prepare% using &y%rop&o#ic polymers (ill !orm pores in aCueous me%ia to increase t&e rate o! polymer %egra%ation an% erosion as (ell as %rug release* It is important to reali/e t&at t&e met&o% use% to prepare nanoparticles (ill #e in!luence% #y t&e relative &y%rop&ilicity an% &y%rop&o#icity o! #ot& t&e polymer an% t&e %rug*

Copolymer Ratio T&e c&oice o! a polymer an% t&e met&o% o! polymeri/ation %irectly a!!ect t&e type o! copolymer as (ell as its molecular (eig&t) crystallinity) an% &y%rop&o#icity* In general) copolymers use% to prepare nanoparticles typically contain #ot& &y%roE p&o#ic an% &y%rop&ilic segments (&ic& !acilitate greater !le"i#ility in preparation an% more pre%icta#le p&ysical properties suc& as release c&aracteristics* +io%egra%a#ility an% +iocompati#ility A #io%egra%a#le polymer must %egra%e into p&ysiologically inert pro%ucts t&at are eliminate% #y t&e #o%y* -or e"ample) P GA polymers &y%roly/e to !orm lactic an% glycolic aci%s t&at are !urt&er %egra%e% into normal constituents o! t&e #o%y* +iocompati#le polymers are %e!ine% as t&ose t&at %o not elicit immune reacE tion or in!lammation) are sta#le !or t&e %uration o! action) an% are completely meta#oli/e% in t&e #o%y* Most #io%egra%a#le polymers use% in %rug %elivery are speci!ically inten%e% !or parenteral a%ministration) (&ic& is not a prereCuisite !or oral %elivery*

Solu#ility T&e solu#ility spectrum o! a polymer in!luences t&e met&o% o! preparation an% in vivo per!ormance o! t&e nanoparticles* In general) i! #ot& t&e %rug an% t&e polymer are solu#le in organic solvents) a simple o1( emulsion tec&niCue or p&ase separation can #e use% to prepare t&e nanoparticles* -or protein an% pepti%e %rugs) a mil%er aCueous environment is pre!era#le an% t&ere!ore) t&e c&oice o! t&e polymer is critiE cal an% a multiple emulsion tec&niCue may #e use%*

:;

C&a$ravart&i et al*

DrugHPolymer Interactions C&emical an% p&ysical interactions t&at occur %epen% on t&e c&emical nature o! #ot& t&e polymer an% t&e %rug as is (ell %ocumente% in t&e literature* DrugHpolyE mer interactions are c&ie!ly %etermine% #y c&arge) solu#ility) an% &y%rop&o#icity an% may greatly alter t&e properties o! t&e polymer) !or e"ample) glass transition temperature an% %egree o! crystallinity) as (ell as t&e properties o! t&e nanopartiE cles) !or e"ample) release c&aracteristics* T&e interactions t&at occur #et(een a %rug an% a polymer can #e i%enti!ie%) an% in some cases Cuanti!ie%) using %i!!erential scanning calorimetry ADSCB) t&ermogravimetric analysis ATGAB) soli%Estate 48C NMR) -ourier trans!orm in!rare% spectroscopy) -lory Huggins interaction parameters) an% comparison o! total or partial solu#ility parameters A58B* -or e"ample) isot&ermal calorimetry &as #een use% to %etermine t&e #in%ing a!!inity #et(een alginates an% c&itosan as (ell as poly lElysine A>B* Di!!erences in t&e solu#ility o! t&e %rug in t&e polymer results in t&e microp&ase separation o! t&e %rug (it&in t&e matri" A59B* 7uanti!ying t&e #in%ing a!!inity using isot&ermal calorimetry can i%enti!y an elecE trostatic interaction #et(een t&e polymer an% t&e %rugs*

Preparation o! Polymeric Nanoparticles T&e main met&o%s use% to prepare polymeric nanoparticles inclu%e emulsionE solvent evaporation) p&ase separation) an% use o! supercritical !lui% tec&nology* T&ese met&o%s (ill not #e %escri#e% &ere as t&ey &ave #een e"tensively revie(e% in t&e literature A4)5)9);H49)4;)4<H55)59)5:B*

Properties o! Nanoparticles t&at A!!ect +iological Per!ormance Route o! A%ministration Nanoparticles &ave #een a%ministere% #y t&e !ollo(ing routes6 oral) intravenous) su#cutaneous) intrat&ecal) intraocular) an% pulmonary* ,&en selecting a route to a%minister nanoparticles) it is important to consi%er t&e sta#ility o! t&e %rug in #ioE logical !lui%s) as (ell as anatomical an% p&ysiological c&aracteristics o! t&e route o! a%ministration an% at t&e target site* ?ral %elivery o! nanoparticles may signi!icantly increase t&e #ioavaila#ility o! poorly solu#le %rugs* Ho(ever) a!ter intravenous a%ministration) t&e #ioavaila#ility o! %rugs may %ecrease as macrop&ages (ill interE nali/e &y%rop&o#ic unmo%i!ie% nanoparticles* T&is pro#lem may #e circumvente% #y coating t&e sur!ace o! nanoparticles (it& &y%rop&ilic polyet&ylene glycol) (&ic& con!ers Qstealt&S properties to t&ese particles* It is important to note t&at) a!ter pulE monary a%ministration) nanoparticles may %rain into t&e lymp&atic system an%) as (it& all ne#uli/ers an% aerosols) t&e site o! %eposition o! t&e particles (ill %epen% on t&e type o! t&e %evice use%* Alt&oug& not (i%ely employe%) intrat&ecal %elivery o! nanoparticles may sustain t&e release o! t&e encapsulate% %rug*

Particle Si/e an% Particle Si/e Distri#ution T&e particle si/e an% particle si/e %istri#ution are critical !actors in t&e per!ormance o! nanoparticles) as #atc&es (it& (i%e particle si/e %istri#ution s&o( signi!icant variations in %rug loa%ing) %rug release) #ioavaila#ility) an% e!!icacy* Particle si/e an% particle si/e %istri#ution can #e %etermine% using lig&t scattering tec&niCues an% #y scanning or transmission electron microscopy A-ig* 4B* -ormulation o! nanoparticles (it& a narro( si/e %istri#ution (ill #e a c&allenge i! emulsion cannot #e pro%uce% (it& a narro( %roplet si/e %istri#ution* As nanoparticles are internalE i/e% into cells #y en%ocytosis) an increase in particle si/e (ill %ecrease upta$e an%

Nanoparticles Prepare% Using Natural an% Synt&etic Polymers

:<

-IGURE 4 Scanning electron microscope image o! polyEAlacti%eE coEglycoli%eB nanoparticles*

potentially) #ioavaila#ility o! t&e %rug* T&e e"tent o! en%ocytosis is %epen%ent on t&e type o! t&e target cell* Veta Potential T&e c&arge on t&e sur!ace o! t&e nanosp&eres (ill in!luence t&eir %istri#ution in t&e #o%y an% e"tent o! upta$e into t&e cells* +ecause cell mem#ranes are negatively c&arge%) t&ere is greater electrostatic a!!inity !or positively c&arge% nanoparticles* T&ere!ore) t&e sur!ace o! cationic or neutral nanoparticles may #e mo%i!ie% to con!er a positive c&arge to en&ance e!!icacy* -or e"ample) positively c&arge% tripalmitin nanoparticles containing etoposi%e prolonge% resi%ence time in t&e #loo%) proE %uce% &ig&er #loo% concentrations) %ecrease% clearance #y t&e liver) an% increase% %istri#ution into t&e #rain an% #one A5;B*

Drug oa%ing an% oa%ing E!!iciency Alt&oug& %rug loa%ing e"presses t&e percent (eig&t o! active ingre%ient encapE sulate% to t&e (eig&t o! nanoparticles) %rug loa%ing e!!iciency is t&e ratio o! t&e e"perimentally %etermine% percentage o! %rug content compare% (it& actual) or t&eoretical mass o! %rug use% !or t&e preparation o! t&e nanoparticles* T&e loa%ing e!!iciency %epen%s on t&e polymerH%rug com#ination an% t&e met&o% use%* Hy%rop&o#ic polymers encapsulate larger amounts o! &y%rop&o#ic %rugs) (&ereas &y%rop&ilic polymers entrap greater amounts o! more &y%rop&ilic %rugs* Several !ormulation parameters) suc& as emulsi!ier type) (eig&t ratio o! polymer to %rug) an% organic to aCueous p&ase ratio) (ill in!luence t&e e"tent o! %rug loa%ing*

Dissolution Pro!ile T&e in vitro release o! %rugs !rom nanoparticles may appro"imate t&e %rug release pro!ile insi%e t&e #o%y alt&oug& t&e rate is usually !aster in vivo %ue to t&e presence o! en/ymes an% sur!actants in #iological !lui%s* An in vitro %issolution me%ium mimics t&e pH an% salt concentrations in t&e #o%y* Particularly !or &y%rop&o#ic %rugs) it is critical %uring %issolution testing t&at sin$ con%itions are maintaine% an% pH an% salt concentrations o! #iological !lui% are appro"imate%* I! solu#ility o! t&e %rug in t&e me%ia is a limitation) it may #e necessary to a%% sur!actants to t&e %issolution me%ia* T&e release %ata must #e evaluate% using (ellE$no(n eCuations

:=

C&a$ravart&i et al*

to %etermine parameters suc& as t&e mec&anismAsB o! release) or%er o! release) %isE solution rate constant) e"tent o! release !rom t&e nanoparticles) an% %uration* Sur!ace Mo%i!ication T&e sur!ace o! nanoparticles may #e mo%i!ie% to con.ugate targeting ligan%s or alter #io%istri#ution* Coating o! nanoparticles (it& &y%rop&ilic polymers suc& as polyet&ylene glycol) &eparin) or %e"tran) protects t&em !rom #eing engul!e% #y t&e macrop&ages or $up!!er cells) t&ere#y increasing t&eir circulation time an% en&ancing %rug #ioavaila#ility* Sur!ace mo%i!ication o! nanoparticles also involves alteration o! t&e sur!ace c&arge as %iscusse% previously*

+iocompati#ility Tissue response to polymeric particles occurs in t&ree p&ases* In p&ase I) mil% in!lamE mation is o#serve% (it& monocytes) lymp&ocytes) an% leucocytes surroun%ing t&e particle* In P&ase II) monocytes %i!!erentiate into macrop&ages) (&ic& !urt&er %i!!erentiate into giant #o%y cells an% engul! t&e particles* In some cases) !i#rous tissue may #e !orme%* P&ase III involves !urt&er %egra%ation o! t&e particles* T&e type o! immune response elicite% #y t&e nanoparticles (ill %epen% on t&e route o! a%ministration) t&e type o! t&erapeutic agent encapsulate%) an% t&e c&oice o! t&e polymer* +ecause o! %ynamic !lo() minimal immune responses are generally o#serve% a!ter intravenous a%ministration*

IN 2ITR? CE CU TURE Internali/ation o! Nanoparticles into Cells T&e %i!!erent mec&anisms #y (&ic& t&e nanoparticles are ta$en up #y t&e cells inclu%e en%ocytosis) pinocytosis) !lui%Ep&ase %i!!usion) carrierEme%iate%) an% !acilitate% transport* Alt&oug& en%ocytosis an% carrierEme%iate% transport are ATPE%epen%ent) !acilitate% transport an% %i!!usion are energyEin%epen%ent* Nanoparticles un%ergo en%olysosomal escape (&ic& increases accumulation o! particles insi%e t&e cell A5<B* ReceptorEme%iate% transport involves t&e internali/ation o! nanoparticles t&roug& speci!ic cell sur!ace receptors on t&e cell mem#rane) an% is e"ploite% in t&e %esign o! nanoparticles to speci!ically target t&ese receptors A-ig* 5B* Nanoparticles are

Nanosp&ere #oun% to receptor igan% con.ugate% to nanosp&ere Receptor me%iate% en%ocytosis o! nanoE sp&eres Recycling o! receptor

NUC EUS

Internali/ation o! nanosp&ere

-IGURE 5 ASee color insert*B Sc&ematic %iagram o! speci!ic receptor targeting o! nanoE sp&eres using ligan%s*

Nanoparticles Prepare% Using Natural an% Synt&etic Polymers

:>

primarily internali/e% #y en%ocytosis* ?nce t&e particles are internali/e%) t&ey tranE scytose across cells %eeper into t&e tissue* Recently) it (as %emonstrate% t&at t&e %rug content in t&e cells increase% (it& particle si/e) suggesting t&at in a%%ition to particle internali/ation) %i!!usion o! !ree %rug release% outsi%e t&e cells may also play a role in en&ancing t&e total %rug content o! t&e cells A5=B*

Protection !rom E!!lu" Pumps TransEmem#rane pumps) suc& as pEglycoprotein an% multi%rugEresistanceEassociate% protein) are present on t&e apical si%e o! t&e cells an% actively e!!lu" !oreign su#E stances) inclu%ing %rugs) out o! t&e cell) signi!icantly re%ucing t&e %rug upta$e* Nanoparticles can protect t&e encapsulate% %rug !rom t&ese e!!lu" pumps*

Targete% Delivery Some limitations o! %rugs %elivere% using nanoparticulate ve&icles inclu%e nonE speci!ic upta$e into cells) accumulation o! particles in nonspeci!ic regions) an% ina#ility to %i!!erentiate #et(een %isease% an% normal tissues* Targete% %elivery o! nanoparticles increases t&e e"tent o! %rug upta$e at t&e site o! action (&ile potenE tially re%ucing a%verse e!!ects* Targete% %elivery is #ase% on6 AiB p&ysiological or e"ternally in%uce% p&enomena ApH an% t&ermoEsensitive nanoparticles) ultraE soun%Etriggere% nanoparticles) magnetic nanoparticlesBM AiiB cellE or tissueEspeci!ic targeting Ananoparticles targeting trans!errin) !olate) epi%ermal gro(t& !actorBM an% AiiiB permea#ilityEen&ancing targets Nnanoparticles targeting transEactivating tranE scriptional !actor ATATB pepti%e) integrinO* Targete% %elivery o! nanoparticles can signi!icantly improve t&e t&erapeutic e!!icacy an% sa!ety o! %rugs*

RE-ERENCES
4* ,issing SA) 0ayser ?) Muller RH* Soli%Hlipi% nanoparticles !or parenteral %rug %elivery* A%v Drug Deliv Rev 5@@9M :;645:<H45<5* 5* +ala I) Hari&aran S) 0umar MN* P GA nanoparticles in %rug %elivery6 t&e state o! t&e art* Crit Rev T&er Drug Carrier Syst 5@@9M 5468=<H955* 8* 0umar N) anger RS) Dom# A'* Polyan&y%ri%es6 an overvie(* A%v Drug Deliv Rev 5@@5M :96==>H>4@* 9* Tonnesen HH) 0arlsen '* Alginate in %rug %elivery systems* Drug Dev In% P&arm 5@@5M 5=6;54H;8@* :* Ra.aonarivony M) 2aut&ier C) Couarra/e G) Puisieu" -) Couvreur P* Development o! a ne( %rug carrier ma%e !rom alginate* ' P&arm Sci 4>>8M =56>45H>4<* ;* Gon/ale/ -erreiro M) Tillman ) Har%ee G) +o%meier R* C&aracteri/ation o! alginate1 polyElElysine particles as antisense oligonucleoti%e carriers* Int ' P&arm 5@@5M 58>69<H:>* <* ,ee S) Gom#ot/ ,R* Protein release !rom alginate matrices* A%v Drug Deliv Rev 4>>=M 8465;<H5=:* =* Va&oor A) S&arma S) 0&uller G0* In&ala#le alginate nanoparticles as antitu#ercular %rug carriers against e"perimental tu#erculosis* Int ' Antimicro# Agents 5@@:M 5;65>=H8@8* >* De S) Ro#inson D* Polymer relations&ips %uring preparation o! c&itosanEalginate an% polyElElysineEalginate nanosp&eres* ' Control Release 5@@8M =>64@4H445* 4@* Agni&otri SA) Malli$ar.una NN) Amina#&avi TM* Recent a%vances on c&itosanE#ase% microE an% nanoparticles in %rug %elivery* ' Control Release 5@@9M 4@@6:H5=* 44* Pra#a&aran M) Mano '-* C&itosanE#ase% particles as controlle% %rug %elivery systems* Drug Deliv 5@@:M 45694H:<* 45* Coester C') anger 0) van +riesen H) 0reuter '* Gelatin nanoparticles #y t(o step %esolvationR a ne( preparation met&o%) sur!ace mo%i!ications an% cell upta$e* ' Microencapsul 5@@@M 4<64=<H4>8*

;@

C&a$ravart&i et al*

48* 0aul G) Ami.i M* +io%istri#ution an% targeting potential o! polyAet&ylene glycolBE mo%i!ie% gelatin nanoparticles in su#cutaneous murine tumor mo%el* ' Drug Target 5@@9M 456:=:H:>4* 49* +alt&asar S) Mic&aelis 0) Dinauer N) von +riesen H) 0reuter ') anger 0* Preparation an% c&aracterisation o! anti#o%y mo%i!ie% gelatin nanoparticles as %rug carrier system !or upta$e in lymp&ocytes* +iomaterials 5@@:M 5;65<58H5<85* 4:* ,ol! +,) Garle# 0A) C&oe 3S) Hump&rey PM) Ma$i 0C* Pullulan is a slo(ly %igeste% car#o&y%rate in &umans* ' Nutr 5@@8M 48864@:4H4@::* 4;* Gupta M) Gupta A0* Hy%rogel pullulan nanoparticles encapsulating p+UD acV plasE mi% as an e!!icient gene %elivery carrier* ' Control Release 5@@9M >>64:<H4;;* 4<* Umama&es&(ari R+) 'ain N0* Receptor me%iate% targeting o! lectin con.ugate% glia%in nanoparticles in t&e treatment o! Helico#acter pylori* ' Drug Target 5@@8M 44694:H958M %iscussion 958 H 949* 4=* Sin&a 2R) +ansal 0) 0aus&i$ R) 0umria R) Tre&an A* PolyEepsilonEcaprolactone microE sp&eres an% nanosp&eres6 an overvie(* Int ' P&arm 5@@9M 5<=64H58* 4>* 2aut&ier C) Du#ernet C) C&auvierre C) +rigger I) Couvreur P* Drug %elivery to resistant tumors6 t&e potential o! polyAal$yl cyanoacrylateB nanoparticles* ' Control Release 5@@8M >864:4H4;@* 5@* C&auvierre C) a#arre D) Couvreur P) 2aut&ier C* Novel polysacc&ari%eE%ecorate% polyAiso#utyl oyanoacrylateB nanoparticles* P&arm Res 5@@8M 5@64<=;H4<>8* 54* U#ric& N) Sc&mi%t C) +o%meier R) Ho!!man M) Maincent P* ?ral evaluation in ra##its o! cyclosporinEloa%e% Eu%ragit RS or R nanoparticles* Int ' P&arm 5@@:M 5==64;>H4<:* 55* 2i&ola H) au$$anen A) Hirvonen ') Ten&u H* +in%ing an% release o! %rugs into an% !rom t&ermosensitive polyANEvinyl caprolactamB nanoparticles* Eur ' P&arm Sci 5@@5M 4;6;>H<9* 58* iu ') Wiao 3) Allen C* PolymerH%rug compati#ility6 a gui%e to t&e %evelopment o! %elivE ery systems !or t&e anticancer agent) ellipticine* ' P&arm Sci 5@@9M >86485H498* 59* S&en E) Pi/sc/e$ R) D/ia%ul +) Narasim&an +* Microp&ase separation in #ioero%i#le copolymers !or %rug %elivery* +iomaterials 5@@4M 5565@4H54@* 5:* 3i 3M) 3ang T3) Pan ,M* Preparation an% %istri#ution o! :E!luorouracil A45:B I so%ium alginateE#ovine serum al#umin nanoparticles* ,orl% ' Gastroenterol 4>>>M :6:<H;@* 5;* Re%%y H) S&arma R0) C&uttani 0) Mis&ra A0) Murt&y RR* Etoposi%eEincorporate% tripalmitin nanoparticles (it& %i!!erent sur!ace c&arge6 !ormulation) c&aracteri/ation) ra%iola#eling) an% #io%istri#ution stu%ies* AAPS ' 5@@9M ;6e58* 5<* Panyam ') V&ou ,V) Pra#&a S) Sa&oo S0) a#&aset(ar 2* Rapi% en%oElysosomal escape o! polyA%lElacti%eEcoEglycoli%eB nanoparticles6 implications !or %rug an% gene %elivery* -ASE+ ' 5@@5M 4;6454<H455;* 5=* De S) Miller D,) Ro#inson DH* E!!ect o! particle si/e o! nanosp&eres an% microsp&eres on t&e cellularEassociation an% cytoto"icity o! paclita"el in 9T4 cells* P&arm Res 5@@:M 556<;;H<<:*

Nano!i#erE+ase% Drug Delivery

Matt&e( D* +ur$e
Department o! P&armaceutical Development) Gla"oSmit&0line) Researc& Triangle Par$) Nort& Carolina) U*S*A*

Dmitry u/&ans$y
Department o! Corporate Tec&nology) Donal%son Company) Inc*) Minneapolis) Minnesota) U*S*A*

INTR?DUCTI?N Electrospinning is a process t&at (as originally %evelope% in t&e early 4>8@s) #ut %i% not receive muc& attention until recent %eca%es* Most li$ely t&e increase% interE est is %ue to t&e re!ocusing o! more researc& groups on nanotec&nology* Alt&oug& electrospinning &as e"iste% !or a signi!icant perio% o! time an% is relatively easy to e"ecute) t&e p&ysics o! electrospinning nano!i#ers is only un%erstoo% to a limite% e"tent* A typical electrospinning process involves %issolving t&e %rug o! interest an% a polymer in an appropriate solvent* T&e solution is t&en place% in a syringe) an% a &ig& voltage is applie%* A small amount o! t&e polymer solution is %ra(n out o! t&e syringe) !orming a Taylor cone* Increasing t&e applie% voltage !urt&er results in t&e initiation o! a c&arge% !lui% .et) (&ic& !ollo(s a c&aotic tra.ectory o! stretc&ing an% #en%ing until it reac&es t&e groun%e% target* A sta#le .et is !orme% (&en t&e c&arge is increase% a#ove a critical voltage) an% t&ere is a #alance #et(een t&e sur!ace tension o! t&e !lui% an% t&e repulsive nature o! t&e c&arge %istri#ution on t&e sur!ace o! t&e !lui%* T&e presence o! molecular entanglements in t&e polymer solution prevents t&e .et !rom #rea$ing into %roplets AelectrosprayingB) an% (&en com#ine% (it& t&e electrical !orces results in a (&ipEli$e motion o! t&e .et) $no(n as #en%ing insta#ility* T&is process typically results in t&e %ra(ing o! a virtually en%less !i#er (it& a nanometerEsi/e% to micrometerEsi/e% %iameter* T&e !inal pro%E uct is a t&reeE%imensional non(oven mat o! entangle% nano!i#ers (it& a &ig& sur!aceEareaEtoEvolume ratio A4B* Scanning electron microscopy ASEMB is a typical met&o% to evaluate t&e nano!i#ers pro%uce% t&roug& electrospinning) as s&o(n in -igure 4* Electrospun nano!i#ers &ave a very large sur!aceEareaEtoEvolume ratio) as large as 4@@@ times t&at o! a micro!i#er* T&is p&ysical property &as generate% a signi!icant amount o! interest in t&e #iome%ical an% p&armaceutical in%ustries particularly !or %rug %elivery o! poorly solu#le %rug su#stances* In t&e p&armaE ceutical in%ustry) recent tren%s in %rug %iscovery &ave le% to t&e %evelopment o! a signi!icant num#er o! &ig&ly potent compoun%s (it& e"tremely lo( solu#ility) t&us reCuiring a%vance% met&o%s to create e!!icacious p&armaceutical pro%ucts t&at overcome t&e solu#ility issues o! t&e %rug* Currently) t&e p&armaceutical in%ustry uses met&o%s suc& as milling to re%uce t&e particle si/e o! a %rug su#stance) #ut t&is is a &ig&Eenergy met&o% (&ic& can lea% to sta#ility issues an% o!ten cannot pro%uce truly nanosi/e% %rug particles* In conventional %ry milling) t&e limit o! !ineness is reac&e% in t&e region o! 4@@ _m* ,et grin%ing or (et #ea% milling pro%uces !urt&er re%uction in t&e particle si/e) #ut o!ten not #elo( t&e micrometer range*

;4

;5

+ur$e an% u/&ans$y

-IGURE 4 Sample SEMs o! various types o! GRAS polymer nano!i#ers pro%uce% #y electrospinE ning* AAB Cellulose acetate) A+B P2Ac) ACB polyet&ylene o"i%e) an% ADB 0olli%onb SR A+AS- AG) u%(igs&a!en) GermanyB* A##reviations6 GRAS) generally regar%e% as sa!eM P2Ac) polyvinylaceE tateM SEM) scanning electron microscopy*

T&e utility o! t&e electrospinning process !or p&armaceutical pro%ucts is t&e singleEstep creation o! nanosi/e% %rug particles (it& a lo(Eenergy process* T&e selection o! t&e polymer also controls t&e %rugErelease properties* T&ere!ore) immeE %iateErelease nano!i#ers can #e create% #y (aterEsolu#le polymers) entericErelease nano!i#ers can #e create% #y enteric polymers suc& as met&acrylic aci% copolymers) an% sustaine%Erelease nano!i#ers can #e create% #y polylactic aci% or polyvinyl aceE tate polymers* Alt&oug& t&e %iameter o! electrospun !i#ers is o!ten c&aracteri/e% using SEM as proo! t&at t&ey are nanosi/e%) it is important to note t&at t&e si/e o! t&e %rug particles em#e%%e% in t&e nanosi/e% !i#er is signi!icantly smaller t&an t&e %iameter o! t&e !i#ers t&emselves* -urt&er utility may #e !oun% (it& electrospinning #y em#e%%ing it at t&e en% o! t&e c&emical synt&esis o! t&e %rug su#stance) (&ic& (oul% streamline t&e transE !er o! material !rom c&emical %evelopment to p&armaceutical %evelopment* T&e last step in t&e c&emical synt&esis o! a %rug is usually a puri!ication1precipitation step to create a %rug po(%er* T&en t&e po(%er is trans!erre% to p&armaceutical %evelopment teams) (&ic& o!ten mill t&e po(%er to a particular particle si/e an% !urt&er granulate1process t&e material into a ta#let* T&is po(%er &an%ling an% proE cessing) (&ic& o!ten reCuires sa!ety controls) can #e avoi%e% i! t&e last step in t&e

Nano!i#erE+ase% Drug Delivery

;8

c&emical synt&esis is trans!orme% into a step (&ere t&e %rug an% a pre!erre% polymer are in an appropriate solution !or electrospinning* T&en t&e %rug1polymer solution can #e %irectly electrospun into a !inal pro%uct) t&us creating a seamless process !rom c&emical synt&esis t&roug& creation o! an appropriate !inal p&armaceutical pro%uct* T&is (oul% eliminate po(%er &an%ling o! t&e %rug su#stance) an% possi#ly re%uce variations in t&e !inal particle si/e o! t&e %rug su#stance %uring manu!acturing*

DISS? UTI?N ENHANCEMENT -?R IMMEDIATEERE EASE D?SAGE -?RMS P&armaceutical ta#lets or capsules (&ic& imme%iately release t&eir %rug cargo (&en orally ingeste% are t&e most common %osage !orm in t&e p&armaceutical in%ustry* Creating a pro%uct (&ic& per!orms t&is tas$ is c&allenging (it& lo(Esolu#ility %rugs* Inappropriate !ormulation o! lo(Esolu#ility %rugs can result in slo( or limE ite% %rug %issolution in t&e gastrointestinal AGIB !lui%s an% su#seCuently minimal %rug #eing a#sor#e% into systemic circulation* As mentione% a#ove) currently a common tec&niCue to overcome t&is lo( #ioavaila#ility issue is to re%uce t&e particle si/e o! t&e %rug su#stance to increase t&e %issolution o! t&e %rug into t&e GI !lui%s* T&e particle si/e o! t&e %rug is %irectly relate% to t&e rate t&at it %issolves* T&e e!!ect o! particle si/e on t&e %rug %issolution process can #e mat&ematically %escri#e% #y t&e Nernst an% +runner %i!!usion layer mo%el A5B6

%7D ^SACsHCgB %t& (&ere 7 is t&e amount o! t&e %rug %issolve%) t t&e time) D t&e %i!!usion coe!!icient o! t&e %rug in t&e solu#ili/ing !lui%s o! t&e GI tract) S t&e e!!ective sur!ace area o! t&e %rug particles) & t&e t&ic$ness o! a stationary layer o! solvent aroun% t&e %rug partiE cles) Cs t&e saturation solu#ility o! t&e %rug in t&e stationary layer &) an% Cg t&e concentration o! t&e %rug in t&e #ul$ !lui%s o! t&e GI tract* +ase% on t&e %rug %issolution eCuation %escri#e% in t&e previous paragrap&) it is clearly evi%ent t&at t&e sur!ace area o! t&e %rug particle is a $ey p&ysicoc&emiE cal property (&ic& can #e use% to increase t&e rate o! %rug %issolution* T&e particle si/e o! t&e %rug su#stance is o!ten use% as a surrogate mar$er !or sur!ace areaM t&ereE !ore) a %ecrease in t&e particle si/e (ill increase t&e sur!ace area an% t&us increase %rug %issolution* As mentione% earlier) t&e current particle si/e re%uction tec&noloE gies o!ten reac& a limit aroun% 4 micron* In contrast) #y !irst intent electrospinning pro%uces nano!i#ers less t&an 4 micron) t&us electrospinning presents a mec&anism to allo( !urt&er re%uction o! t&e particle si/e #eyon% t&e current tec&nologies* T&e electrospun nano!i#ers are pre!era#ly a &omogeneous mi"ture o! polymer an% %rug) t&us t&e particle si/e o! t&e %rug s&oul% #e signi!icantly less t&an t&e %iameter o! t&e nano!i#er* Alt&oug& t&e e"act particle si/e o! t&e %rug in t&e nano!i#er remains c&allenging to Cuanti!y) t&e %issolution rate can provi%e in%irect evi%ence t&at signi!icant particle si/e re%uction &as occurre%* Re%uction o! t&e particle si/e o! t&e %rug su#stance t&roug& electrospinning (it& a rapi%ly %issolving polymer causes t&e %rug %issolution rate to #e very rapi%) as s&o(n in -igure 5* Anot&er a%vantage o! electrospinning as a %issolutionEen&ancement tool is t&e a#ility to control t&e morp&ology o! t&e %rug su#stance A8)9B* +y selecting a polymer suc& as &y%ro"ypropylmet&ylcellulose acetate succinate AHPMCEASB) (&ic& &as an amorp&ous c&aracter) one can create an amorp&ous %rug su#stance

;9

+ur$e an% u/&ans$y

-IGURE 5 AAB Dissolution o! a mo%el lo(Esolu#ility %rug !rom polyet&ylene o"i%e electrospun !i#ers in a USP 5 %issolution apparatus* A+B Scanning electron microscopy image o! t&e nano!i#ers*

t&roug& electrospinning* Amorp&ous %rug su#stances are at a &ig&er energy state) t&ere!ore) in general &ave &ig&er solu#ility an% &ig&er %issolution rate compare% (it& crystalline materials* T&us) t&e creation o! an amorp&ous %rug su#stance represents anot&er tec&niCue !or %issolution en&ancement* Amorp&ous %rugs are generally less sta#le p&ysically an% c&emically t&an correspon%ing crystalline materials) so appropriate sta#ility assessment nee%s to #e per!orme% A:B* Alt&oug& sta#ility issues can limit t&e use o! amorp&ous %rug su#stances) t&ere are several marE $ete% oral pro%ucts suc& as 2ancocinb A2irop&arma) USAB) Plen%il ERb AAstraVeneca t%*) U0B) Cesametb AEli illy an% Co*) USAB) an% Certicanb ANovartis AG) +asel) S(it/erlan%B t&at contain amorp&ous %rug su#stances* In a%%ition to increasing t&e solu#ility an% %issolution #y creating an amorE p&ous %rug su#stance (it& HPMCEAS) it &as also #een s&o(n to re%uce in vivo preE cipitation o! t&e %rug #y maintaining t&e %rug as a supersaturate% solution in t&e GI tract* SprayE%rying (it& t&e %rug su#stance an% HPMCEAS &as #een per!orme% to create amorp&ous particles to increase t&e %rug e"posure !or rapi% screening o! %rugs in preclinical mo%els A;B* Electrospinning coul% #e use% in a similar !as&ion an% it &as t(o a%%itional #ene!its6 !irst) t&e a#ility to create smallerE%iameter !i#ers t&an t&e typical particle si/e o! sprayE%rie% material) an% secon%) process collects t&e pro%uct onto a groun%e% sur!ace) (&ic& results in very &ig& e!!iciencies A>>[oB an% simpli!ie% recovery* An e"ample o! t&e use o! electrospinning to create amorp&ous materials !or %issolution en&ancement (as per!orme% #y 2errec$ et al* A<B* In t&is case) t&e researc&ers use% &y%ro"ypropylmet&ylcellulose as t&e electrospinning polymer) an% itracona/ole as t&e mo%el %rug* ?n t&e #asis o! %i!!erential scanning calorimetry) t&ey generate% %ata supporting t&e conclusion t&at itracona/ole (as in t&e amorE p&ous !orm) an% per!orme% %issolution stu%ies to evaluate t&e rate o! release* T&ese %ata also &ig&lig&te% t&e !act t&at #y optimi/ing t&e electrospinning con%itions to pro%uce !i#ers (it& a %iameter o! 8@@ to :@@ nm versus 4 to 9 _m) t&e %issolution rate can #e !urt&er increase% A<B*

SUSTAINEDERE EASE E ECTR?SPUN -I+ERS T&ere is signi!icant interest in t&e use o! electrospinning !or %issolution en&anceE ment o! poorly (aterEsolu#le %rugsM &o(ever) t&e ma.ority o! t&e literature on

Nano!i#erE+ase% Drug Delivery

;:

-IGURE 8 AAB Dissolution o! a mo%el lo(Esolu#ility %rug !rom polyvinylacetate electrospun !i#ers in a USP 5 %issolution apparatus* A+B Scanning electron microscopy image o! t&e nano!i#ers*

electrospinning o! p&armaceutical or #iome%ical pro%ucts ten%s to #e !or (oun% %ressings or ot&er pro%ucts (&ic& &ave sustaine% release o! %rug su#stance* T&is reveals t&e large range o! %rugErelease pro!iles (&ic& can #e o#taine% #y care!ul selection o! t&e polymer* -or release o! a %rug su#stance !or multiple %ays to mont&s) a polymer in t&e #io%egra%a#le !amily) suc& as polyglycoli%e) polylactic aci%) or polycaprolactone) can #e use%* A copolymer o! lEcaprolactone an% et&yl et&ylene p&osp&ate (as use% to sustain t&e release o! &uman fEnerve gro(t& !actor !or at least t&ree mont&s A=B* Ho(ever) selection o! t&e polymer nee%s to #e care!ully screene% #ecause t&e polymer %rug compati#ility &as #een s&o(n to play a critical role in t&e %istri#ution o! %rug (it&in t&e !i#er A>B* Polymers (&ic& &y%rate or s(ell #ut are insolu#le can also #e use% to create sustaine%Erelease nano!i#ers* 2errec$ et al* A4@B electrospun segmente% polyureE t&ane itracona/ole !i#ers to pro%uce a sustaine% release o! t&e %rug su#stance* T&is type o! release can #e ac&ieve% t&roug& t&e use o! generally regar%e% as sa!e AGRASB polymers !or oral %rug %elivery suc& as polyvinylacetate AP2AcB nano!i#ers* An e"ample o! %rug release o! a poorly (aterEsolu#le %rug su#stance !rom electrospun !i#ers o! P2Ac is s&o(n in -igure 8* As s&o(n #y t&e e"amples a#ove) various polymerE#ase% met&o%s to control %rug release suc& as enteric polymers) #io%egra%a#le polymers) or even polysacc&aE ri%e can #e utili/e% (it& electrospinning* T&e intrinsic properties o! t&e polymer !orm t&e #asis !or t&e type o! %rug release t&at occurs !rom t&e nano!i#ers* Alt&oug& proo! o! concept &as #een ac&ieve% (it& electrospun nano!i#ers !or %issolution en&ancement an% controlle% %rug release) t&e use o! electrospinning in t&e p&armaE ceutical in%ustry is still in its in!ancy* More a%vances suc& as !unctionali/e% nano!i#ers an% nanotu#e structures t&roug& coEa"ial electrospinning are li$ely to occur A44)45B*

ARGEESCA E MANU-ACTURING Alt&oug& electrospinning is still at an early stage in t&e p&armaceutical in%ustry) commercial use o! nano!i#ers pro%uce% #y electrospinning in ot&er in%ustries &as alrea%y #een esta#lis&e%* Donal%son CompanyPs nano!i#er !ilter me%ia pro%uction &as increase% (ell #eyon% 4@)@@@ m51%ay %uring t&e last 5@ years* An SEM e"amE ple o! t&e !ilter utili/ation o! a nano!i#er (e# layer on t&e sur!ace o! a spun#on% non(oven is s&o(n in -igure 9*

;;

+ur$e an% u/&ans$y

-IGURE 9 E"amples o! nano!i#er composites electrospun #y Donal%son* Nano!i#ers on t&e sur!ace o! AAB !ilter paper an% A+B spun#on% non(oven*

+y utili/ing many o! t&e current manu!acturing practices use% in t&e !iltration in%ustry) t&e largeEscale manu!acturing o! electrospun nano!i#ers !or p&armaceutical applications can progress at a rapi% rate) inclu%ing t&e a%option an% implementaE tion o! appropriate Cuality control tools !or nano!i#er pro%uction suc& as automate% !i#er si/ing an% mec&anical integrity testing A48B*

Automate% -i#er Si/ing -ull process control o! t&e nano!i#er pro%uction reCuires measurement an% control o! !i#er si/e) !i#er si/e %istri#ution) an% Cuantity o! !i#ers* T&ere!ore) t&ere is a nee% !or a tool t&at can %irectly measure mean !i#er si/e an% !i#er si/e %istri#ution* Routine sampling an% measurement o! t&ese properties are ac&ieve% using an SEM an% a %evelope% analysis met&o%ology* Measuring nano!i#er %iameters usually consists o! manually comparing t&e %iameter o! !i#ers in a p&otomicrograp& to a $no(n scale* T&e process is very timeEconsuming) an% operator consistency an% !atigue can re%uce t&e accuracy* Automating t&e activity o! si/ing !i#ers is a natural solution to t&e pro#lem* -or %rugE%elivery applications) !i#er %iameter an% !i#er %iameter %istri#ution (ill #e important parameters to measure* It is e"pecte% t&at !i#er %iameter is one o! t&e varia#les t&at can #e controlle% to tune t&e rate o! !i#er %issolution an% control %rug release Ai*e*) a #igger !i#er &as lo(er sur!ace area an% (oul% lea% to a %elaye% release compare% (it& a smaller !i#erB* A proprietary algorit&m &as #een %evelope% at Donal%son Company to overcome t&e limitations o! commercially availa#le so!tE (are* In t&e automate% !i#er si/ing process) t&e SEM image is !irst croppe% to a %esire% si/e) an% un(ante% %etails are eliminate%* T&e cali#ration #ar !rom t&e SEM image is use% to set t&e num#er o! pi"els per micrometer* Ne"t) t&e program conE verts t&e image to #lac$ an% (&ite using a gray scale !unction* T&e #lac$ Anon!i#erB areas are sorte% accor%ing to si/e* Starting (it& t&e largest #lac$ area) a straig&t line is %e!ine% on t&e #or%er pi"els an% a %iameter is %ra(n !rom one pi"el across t&e (&ite area at >@]* T&e %iameter %ra(ing stops (&en anot&er #lac$ pi"el is interE secte%* T&e process is repeate% aroun% t&e #lac$ s&ape at an interval o! appro"iE mately every !ive pi"els) an% t&en aroun% eac& #lac$ area in or%er o! si/e A-ig* :B* All !i#er %iameter lengt&s are recor%e% an% a running &istogram is generate% A-ig* ;B* T&e process t&at use% to ta$e &ours o! painsta$ing measurements !or an operator can no( #e complete% in secon%s* C&aracteri/ation o! !i#er si/e) !i#er si/e %istri#ution) an% Cuantity o! !i#ers is critical to ensure a Cuality pro%uct* In a%%ition)

Nano!i#erE+ase% Drug Delivery

;<

-IGURE : Nano!i#er scanning electron microscopic p&otograp& (it& !i#er %iameter measurement lines s&o(n*

t&e mec&anical integrity o! a nano!i#er structure can #e important !or sustaine%E release %rugE%elivery applications suc& as (oun% %ressings) tissue engineering) an% regenerative me%icine* 0no(le%ge o! t&e strainHstress c&aracteristics is imporE tant in un%erstan%ing t&e per!ormance o! nano!i#er composites un%er %ynamic stress suc& as t&e gastric compression an% peristaltic action o! t&e GI tract* ?(ing to t&e small si/e o! !i#ers an% e"tremely lo( (eig&t o! t&e layer) tra%itional measureE ment met&o%s %o not give use!ul results* It is anticipate% t&at t&e mec&anical integE rity o! nano!i#ers s&oul% #e engineere%) measure%) an% controlle%*

Mec&anical Integrity Testing T&e D +en%ing Tester (as %evelope% to stu%y t&e strain properties an% !ailure mec&anisms o! nano!i#ers applie% to t&e sur!ace o! anot&er material* -irst) a sample

-IGURE ; ASee color insert*B Results o! automate% !i#er si/e analysis*

;=

+ur$e an% u/&ans$y

-IGURE < AAB Specimen #e!ore %e!ormation6 4) sampleM 5) .a(sM 8) cylin%er* A+B Specimen a!ter %e!ormation*

is secure% in t&e tester an% positione% in t&e optical microscope* A motor t&en #en%s an% e"ten%s t&e sample aroun% a cylin%er (it& $no(n %iameter* T&e strain con%ition appro"imates plane strain #ecause t&e sample is t&in compare% (it& t&e relatively large ra%ius o! t&e cylin%er* T&us) %i!!erences in t&e strain con%ition #et(een t&e upper an% lo(er sur!aces o! t&e sample are minimal* An area o! appro"imately ; mm ` 9 mm A9 mm in t&e %irection o! stretc&B is o#serve% an% can #e varie%* An angle o! %e!ormation) m) is measure% using a scale on t&e apparatus* T&e #asic sc&ematic representation o! t&e tester is s&o(n in -igure <A an% +* T&e general vie( o! t&e tester is s&o(n in -igure =* T&e strain can #e calculate% !rom t&e measure% angle6 9pmR 8;@ @

e^

(&ere l is t&e calculate% strain) m t&e measure% angle) R t&e cylin%er %iameter) an% @ t&e initial lengt& o! t&e specimen* A camera mounte% on t&e microscope sen%s a %ynamic image to a monitor t&at is use% to o#serve t&e sample t&roug&out t&e test* T&e !irst sign o! relative

-IGURE = Dmitry u/&ans$y #en%ing tester*

Nano!i#erE+ase% Drug Delivery

;>

movement #et(een t&e components o! t&e composite structure is an in%icator o! critical strain* An operator recor%s t&e relative movements an% angles o! t&e !irst %estruction in t&e nano!i#er layer an% !ull %estruction o! nano!i#er layer* Comparison o! t&e angles !or %i!!erent composites gives a measure o! integrity o! t&e material*

C?NC USI?N Electrospun nano!i#ers &ave s&o(n utility in a range o! p&armaceutical an% me%iE cal applications !or imme%iate an% controlle% %rug release) (&ic& (ill increase t&e nee% !or !uture process re!inement an% largeEscale manu!acturing capa#ilities to convert t&ese novel concepts into commercial pro%ucts* Electrospinning represents a nanotec&nology t&at is stea%ily maturing an% its use (ill gro( as companies implement t&is tec&nology as a plat!orm %rugE%elivery tec&niCue*

RE-ERENCES
4* Mc0ee MG) ,il$es G ) Col#y RH) ong TE* Correlations o! solution r&eology (it& electrospun !i#er !ormation o! linear an% #ranc&e% polyesters* Macromolecules 5@@9M 8<64<;@* 5* Hoener +A) +enet V* -actors in!luencing %rug a#sorption an% %rug availa#ility* In6 +an$er GS) R&o%es CT) e%s* Mo%ern P&armaceutics* 9t& e%* Ne( 3or$6 Marcel De$$er) 5@@5 Ac&apter 9B* 8* Ignatious -) +al%oni 'M* Electrospun p&armaceutical compositions* 5@@4M ,? @41:9;;< A4* 9* Ignatious -) Sun * Electrospun amorp&ous p&armaceutical compositions* 5@@94M ,? 5@@91@498@9* :* 3u * Amorp&ous p&armaceutical soli%s6 preparation) c&aracteri/ation an% sta#ili/ation* A%v Drug Del Rev 5@@4M 9=65<* ;* S&an$er RM* DrugHpolymer systems !or t&e supersaturation o! GI luminal !lui%* In6 Presente% at AAPS Annual Meeting) +altimore) MD) Novem#er <H49) 5@@9* <* 2errec$ G) C&un I) Peeters ') Rosen#latt ') +re(ster ME* Preparation an% c&aracteri/ation o! nano!i#ers containing amorp&ous %rug %ispersion generate% #y electrostatic spinE ning* P&arm Res 5@@8M 5@6=4@* =* C&e( S3) ,en ') 3im E0-) eong 0,* Sustaine% release o! proteins !rom electrospun #io%egra%a#le !i#ers* +iomacromolecules 5@@:M ;65@4<* >* V&eng ') 3ang ) iang 7) et al* In!luence o! t&e %rug compati#ility (it& polymer solution on t&e release $inetics o! electrospun !i#er !ormulation* ' Control Release 5@@:M 4@:698* 4@* 2errec$ G) C&un I) Rosen#latt ') et al* Incorporation o! %rugs in an amorp&ous state into electrospun nano!i#ers compose% o! a (aterEinsolu#le) non#io%egra%a#le polymer* ' Control Release 5@@8M >5689>* 44* Casper C ) 3amaguc&i N) 0iic$ 0 ) Ra#olt '-* -unctionali/ing electrospun !i#ers (it& #iologically relevant macromolecules* +iomacromolecules 5@@:M ;64>>=* 45* Huang VEM) V&ang 3EV* MicroEstructures an% mec&anical per!ormance o! coEa"ial nano!i#ers (it& %rug an% protein cores an% polycaprolactone s&ells* Gao%eng Wue"iao Hua"ue Wue#ao 5@@:M 5;6>;=* 48* u/&ans$y D* 7uality control in manu!acturing o! electrospun nano!i#er composites* In6 Presente% at International Non(ovens Tec&nical Con!erence) +altimore) MD) Septem#er 4:H4=) 5@@8*

Drug NanocrystalsRT&e Universal -ormulation Approac& !or Poorly Solu#le Drugs

'an MFsc&(it/er an% Rainer H* MJller
Department o! P&armaceutical Tec&nology) +iotec&nology) an% 7uality Management) -reie UniversitIt +erlin) +erlin) Germany

INTR?DUCTI?N During t&e last t(o %eca%es) many mo%ern tec&nologies &ave #een esta#lis&e% in t&e p&armaceutical researc& an% %evelopment area* T&e automation o! t&e %rug %iscovery process #y tec&nologies suc& as &ig&Et&roug&put screening) com#inatoE rial c&emistry) an% computerEai%e% %rug %esign is lea%ing to a vast num#er o! %rug can%i%ates possessing a very goo% e!!icacy* Un!ortunately) many o! t&ese %rug canE %i%ates are e"&i#iting poor aCueous solu#ility* ong #e!ore one o! t&ese compoun%s can reac& t&e mar$et) it nee%s to #e !ormulate% !or t&e p&armacological activity tests an% !or t&e preclinical stu%ies* T&e great c&allenge !or t&e p&armaceutical %evelopment is to create ne( !ormulation approac&es an% %rugE%elivery systems to overcome solu#ility pro#lems o! t&ese %rug can%i%ates (&ic& are also o!ten assoE ciate% (it& poor oral #ioavaila#ility A4)5B* T&e %issolution velocity Alo( solu#ility in general is correlate% (it& lo( %issolution velocity) la( #y NoyesH,&itneyB an% intestinal permea#ility are $ey %eterminants !or t&e #ioavaila#ility) particularly !or perorally a%ministere% %rugs* To evaluate an% c&aracteri/e p&armaceutical compoun%s (it& respect to t&eir aCueous solu#ility an% intestinal permea#ility) a #iop&armaceutics classi!ication system &as #een %evelope% A8)9B* T&e system %ivi%es t&e %rug compoun%s into !our classes* Poorly solu#le compoun%s can #elong to eit&er class II or class I2* Class I2 means t&at t&e %rug s&o(s simultaneously poor solu#ility an% lo( permea#ility* A solu#ility en&ancement cannot necessarily solve t&e #ioavaila#ility pro#lems o! class I2 %rugs in any case* Drug can%i%ates !or a success!ul improvement o! t&eir #ioavaila#ility #y a solu#ili/ation tec&niCue #elong to class II (&ic& means t&at t&eir #ioavaila#ility is only limite% #y t&eir poor aCueous solu#ility1%issolution velocity* T&e term Qsolu#ili/ation tec&niCuesS in t&e present conte"t means tec&noE logies (&ic& increase t&e %issolution velocity %c1%t an%Ri%eallyRalso t&e saturation solu#ility cs* T&ere are many conventional approac&es !or t&e solu#ili/ation o! poorly solu#le %rugs* Salt !ormation an% pH a%.ustment are t&e !irst attempts i! t&e molecule is ioni/a#le) #ecause in general t&e ioni/e% species &as a &ig&er aCueous solu#ility compare% to t&e neutral one* I! t&is approac& !ails) o!ten cosolvents suc& as propylene glycol) are use%) especially !or parenteral or liCui% oral %osage !orms* Systemic to"icity or pain on in.ection are typical %ra(#ac$s associate% (it& cosolE vents A:B* ?t&er systems contain a large amount o! sur!actants to solu#ili/e %rugs #y an increase% (etting o! t&e &y%rop&o#ic compoun%* Ho(ever) t&e e"ten%e% use o! sur!actants can also cause si%e e!!ects* A typical e"ample is t&e &ypersensitivity reaction cause% #y t&e Cremop&or E b in Ta"olb A;B* Anot&er e"ample o! sur!actants

<4

<5

MFsc&(it/er an% MJller

is mi"e% micelles) !or e"ample) 2aliumb MM A<B* In case o! lipop&ilic %rugs) a lo(E melting point emulsi!ication system suc& as microemulsions) sel!Eemulsi!ying DDSs) or sel!Emicroemulsi!ying DDSs) coul% #e use%* T&e %ra(#ac$s o! t&ese systems inclu%e #atc&EtoE#atc& varia#ility) c&emical insta#ilities) an% &ig& sur!acE tant concentration* Anot&er approac& is t&e use o! liposomes to incorporate &y%rop&o#ic %rugs in p&osp&oElipi% #ilayers o! uniE or multilamellar vesicles* ?ne e"ample is t&e %rug Amp&otericin +) (&ic& is mar$ete% as liposomal !ormulation Am#isome b* A speci!ic approac& is t&e !ormation o! inclusion comple"es) !or e"ample) (it& cyclo%e"trins* Cyclo%e"trines are cyclic oligomers o! %e"trose or %e"trose %erivatiE ves) (&ic& can !orm a reversi#le) noncovalent association (it& poorly solu#le %rugs to solu#ili/e t&em* Especially) t&e more (ater solu#le an% less to"ic %erivatives) suc& as sul!o#utylet&erEfEcyclo%e"trin ACaptisold) CyDe") Inc*B an% &y%ro"yE propylEfEcyclo%e"trin AHPEfEcyclo%e"trinB) are use% in %i!!erent p&armaceutical !ormulations A=B* Sporano"b #y 'o&nson an% 'o&nson1'anssen AItracona/ole1HPE fEcyclo%e"trineB an% Vel%o"b #y P!i/er AViprasi%one1S+EEfEcyclo%e"trineM CaptisolbB are e"amples o! mar$ete% pro%ucts* In or%er to #uil% t&is comple") it is in general reCuire% t&at t&e %rug molecule !its into t&e cyclo%e"trin cavity* -or t&at reason) t&is promising speci!ic approac& can #e use% only !or a limite% num#er o! %rugs* Anot&er %ra(#ac$ o! t&is tec&nology is t&e &ig& e"cipient level o! t&e resulting pro%uct* To sum up) t&e prementione% tec&nologies are success!ully applie% !or a num#er o! %rugs* T&e mar$ete% pro%ucts prove t&eir acceptance an% applica#ility* T&e success o! a tec&nology can #e rate% in t(o areas6

4* time #et(een %eveloping t&e tec&nology an% !irst mar$et pro%ucts an% 5* num#er o! pro%ucts on t&e mar$et in total Aor more precisely) num#er o! pro%ucts launc&e% per yearB* +ase% on t&ese success criteria) t&e per!ormance o! most speci!ic !ormulation approac&es appears rat&er poor* iposomesRre%iscovere% #y +ing&am in 4>;=R nee%e% a#out 5@ years to come to mar$et* T&e num#er o! pro%ucts is relatively lo() %e!initely %istinctly #e&in% e"pectations* Similar is t&e situation !or cyclo%e"trines) currently moving !or(ar% (it& t&e ne() #etter tolerate% %erivatives* T&ese tec&noE logies are commonly use% as primary strategies* Ho(ever) t&ey are more or less speci!ic approac&es !or t&e solu#ili/ation o! a certain %rug can%i%ate* T&ey can #e use% only in compliance (it& certain reCuirements %etermine% #y t&e %rug an% route o! a%ministrationM t&ere is no universal !ormulation approac&* Muc& smarter are nonspeci!ic !ormulation approac&es applica#le to almost any %rug molecule Aapart !rom a !e( e"ceptionsB* Particle si/e re%uction &as #een a nonspeci!ic !ormulation approac& !or many years* T&e microni/ation o! %rugs is applie% to increase t&eir sur!ace area* Increasing t&e sur!ace area (ill proportionally increase t&e rate o! %issolution an% t&e rate o! %i!!usion Aa#sorptionB* Microni/ation means trans!er o! relatively coarse %rug po(%er to micrometer crystals using colE loi% mills or .et mills* T&e mean %iameter o! suc& microni/e% %rug po(%ers is in t&e range o! appro"imately 5 to : _m) correspon%ing to a si/e %istri#ution o! appro"iE mately @*4 to 5@ _m A>B* ?(ing to t&eir particle si/e %istri#ution) suc& !ormulations in general cannot #e use% !or intravenous AI2B in.ections* Microni/ation cannot improve t&e saturation solu#ility o! a %rug su#stance* In cases o! practically insoE lu#le p&armaceutical compoun%s or compoun%s o! very lo( solu#ility) t&e e!!ect o! microni/ation on t&e #ioavaila#lity is not su!!icient* -or t&at reason) t&e ne"t

Drug Nanocrystals

<8

conseCuent step (as to go %o(n one !urt&er %imension in si/e) (&ic& means to re%uce t&e particle si/e in t&e nanometer range* Since t&e 4>=@s) (&en %rug nanoparticles (ere pro%uce% #y ist an% Suc$er A4@B via precipitation) various tec&niCues !or t&e pro%uction o! %rug nanocrystals &ave #een %evelope%* T&is c&apter (ill give an overvie( o! t&e #ene!icial !eatures o! %rug nanocrystals) (ill %iscuss t&e most important pro%uction tec&niCues) an% point out some reasons (&y %rug nanocrystals can #e seen as a universal !orE mulation approac& !or poorly solu#le %rugs*

DE-INITI?N Drug nanocrystals are pure soli% %rug particles (it& a mean %iameter #elo( 4@@@ nm* A nanosuspension consists o! %rug nanocrystals) sta#ili/ing agents suc& as sur!actants an%1or polymeric sta#ili/ers) an% a liCui% %ispersion me%ium* T&e %isE persion me%ia can #e (ater) aCueous solutions) or nonaCueous me%ia* T&e term Q%rug nanocrystalsS implies a crystalline state o! t&e %iscrete particles) #ut %epen%E ing on t&e pro%uction met&o% t&ey can also #e partially or completely amorp&ous* Drug nanocrystals &ave to #e %istinguis&e% !rom polymeric nanoparticles) (&ic& consist o! a polymeric matri" an% an incorporate% %rug* Drug nanocrystals %o not consist o! any matri" material*

PH3SIC?CHEMICA PR?PERTIES ?- DRUG NAN?CR3STA S T&e increase% saturation solu#ility an% t&e accelerate% %issolution velocity are t&e most important %i!!erentiating !eatures o! %rug nanocrystals* In general) t&e saturation solu#ility AcsB is %e!ine% as a %rugEspeci!ic constant %epen%ing only on t&e solvent an% t&e temperature* T&is %e!inition is only vali% !or %rug particles (it& a minimum particle si/e in t&e micrometer range* A particle si/e re%uction %o(n to t&e nanometer range can increase t&e %rug solu#ility* T&e saturation solu#ility o! soli% particles %epen%s on t&eir particle ra%ius an% t&eir lattice structure accor%ing to t&e ?st(al%H-reunlic& eCuation an% t&e 0elvin eCuation6 S 5pq 5 Mq ln ^^ S@ rRT r rRT (&ere S is t&e %rug solu#ility at temperature T) S @ t&e solu#ility i! r ^ j) M t&e molecular (eig&t o! t&e compoun%) p t&e molar volume) q t&e inter!acial sur!ace tension) an% r t&e %ensity o! t&e compoun%* -rom t&e ?st(al%H-reun%lic& eCuation) it can #e conclu%e% t&at a %rug s&o(s &ig&er solu#ility i! t&e particle ra%ius is %ecrease%* T&is e!!ect is not su#stantial !or larger particles #ut (ill #e more pronounce% !or particles #elo( 4 to 5 _m) espeE cially (ell un%er 5@@ nm* Anot&er important !actor in!luencing t&e solu#ility is t&e crystalline structure o! t&e %rug* T&e &ig&er t&e soli% %ensity an% t&e melting point are) t&e lo(er t&e solu#ility in general is* In contrast) a polymorp& !orm (it& a lo(er pac$aging s&o(s a &ig&er molar volume an% lo(er soli% %ensity A44B* T&e 0elvin eCuation can also #e use% to %escri#e t&e correlation o! increase% saturation solu#ility #y %ecrease% particle si/e* T&e 0elvin eCuation %escri#es t&e vapor pressure as a !unction o! t&e curvature o! liCui% %roplets in a gas p&ase* T&e vapor pressure increases (it& increasing curvature A%ecreasing particle si/eB*

A4B

<9

MFsc&(it/er an% MJller

T&is can #e trans!erre% to soli% %rug particles in a liCui% me%ium6 t&e %issolution pressure increases (it& %ecreasing particle si/e A45B* %c" DA ^A cs Z c " B %t& (&ere %c"1%t is t&e %issolution velocity) D t&e %i!!usion coe!!icient) A t&e sur!ace o! t&e %rug particle) & t&e t&ic$ness o! %i!!usional layer) c s t&e saturation solu#ility o! t&e %rug) an% c" t&e concentration in surroun%ing liCui% at time "* T&e increase% %issolution velocity is t&e c&aracteristic !eature o! %rug nanoE crystals* T&e NoyesH,&itney eCuation NECuation A5BO %escri#es no( an increase in %issolution velocity is proportional to an increase in sur!ace area* -or e"ample) (&en moving !rom a sp&erical :@ _m particle to microni/e% : _m particles) t&e total sur!ace area enlarges #y a !actor o! 4@) moving to :@@ nm nanocrystals #y a !actor o! 4@@* T&e %ecrease in t&e %i!!usional %istance & is an a%%itional !actor accelerating t&e %issolution velocity* Accor%ing to t&e Pran%tl eCuation NECuation A8BO) t&e %i!!usional %istance & is re%uce% (it& increasing curvature o! ultra!ine particles* Toget&er (it& t&e increase% saturation solu#ility o! ultra!ine particles) t&e concentration gra%ient in t&e NoyesH,&itney eCuation is signi!icantly increase%* -or t&at reason) nanoniE /ation can %istinctly increase t&e %issolution velocity o! poorly solu#le %rugs6 A5B

41 5 &H ^ $ 41 8 2 (&ere &H is t&e &y%ro%ynamic #oun%ary layer t&ic$ness) 2 t&e relative velocity o! t&e !lo(ing liCui% against a !lat sur!ace) $ a constant) an% t&e lengt& o! t&e sur!ace in t&e %irection o! !lo(* Drug nanocrystals can #e use% !or a c&emical sta#ili/ation o! c&emically la#ile %rugs* T&e %rug paclita"el can #e preserve% !rom %egra%ation (&en it is !ormulate% as a nanosuspension A48)49B* T&e same result (as !oun% !or t&e c&emically la#ile %rug omepra/ole* ,&en !ormulate% as a nanosuspension) t&e sta#ility (as %istinctly increase% in comparison to t&e aCueous solution A4:B* T&e increase% sta#ility can #e e"plaine% #y a s&iel% e!!ect o! t&e sur!actants an% t&e %rug protection #y a monoE layer ma%e o! %egra%e% %rug molecules (&ic& re%uce t&e accessi#ility !or %estrucE tive agents A4;B*

A8B

P?TENTIA C INICA AD2ANTAGES ?- DRUG NAN?CR3STA S Application !or ?ral Delivery T&e oral route is t&e most important an% pre!erre% route o! a%ministration* T&e !ormulation o! %rug nanocrystals can impressively improve t&e #ioavaila#ility o! perorally a%ministere% poorly solu#le %rugs* In 4>>:) iversi%ge an% Cun%y A4<B reporte% an increase in #ioavaila#ility !or t&e %rug Dana/ol !rom :*4 e 4*>[ !or t&e conventional suspension to =5*8 e 4@*4[ !or t&e nanosuspension* T&e increase% %issolution velocity an% saturation solu#ility lea% to !ast an% complete %rug %issolution) an important prereCuisite !or %rug a#sorption* ,&enever a rapi% onset o! a poorly solu#le %rug is %esire%) t&e !ormulation o! %rug nanocrystals can #e #ene!icial) !or e"ample) in case o! analgesics* T&e analgesic napro"en) !ormulate% as a nanosuspension) &as s&o(n a re%uce% t ma" #ut simultaE neously appro"imately t&ree!ol% increase% AUC in comparison to a normal suspenE sion ANaprosynbB A4=B*

Drug Nanocrystals

<:

+esi%es t&e !aster onset o! action) t&e napro"en nanosuspension &as also s&o(n a re%uce% gastric irritancy A4>)5@B* I! a#sorption (in%o(s limit t&e %rug a#sorption or #y !oo% e!!ects) %rug nanocrystals &ave a%vantages in comparison to conventioE nal suspensions* ,u et al* &ave reporte% re%uce% !e%E!aste% ratio an% an improve% #ioavaila#ility !or nanocrystalline aprepitant AM0E@=;>B) t&e active ingre%ient in Emen%b) in #eagle %ogs* Anot&er important a%vantage o! %rug nanocrystals is t&eir a%&esiveness an% t&e increase% resi%ence time) (&ic& can positively in!luence t&e #ioavaila#ility* T&e mucoa%&esiveness can #e raise% #y t&e use o! mucoa%&esive polymers in t&e %ispersion me%ium A54)55B* A%%itionally t&e utili/e% mucoa%&esive polymers can prevent t&e %rug !rom %egra%ation* T&e re%uce% particle si/e can #e also e"ploite% !or improve% %rug targeting) as reporte% !or in!lammatory tissues A58B or t&e lymp&atic %rug upta$e A59B*

Parenteral A%ministration o! Drug Nanocrystals T&e parenteral application o! poorly solu#le %rugs) particularly intravenous AI2B a%ministration o! practically insolu#le compoun%s) using cosolvents) sur!actants) liposomes) or cylco%e"trines) is o!ten associate% (it& large in.ection volumes or to"ic si%e e!!ects* CarrierE!ree nanosuspensions ena#le potential &ig&er loa%ing capacity compare% to ot&er parenteral application systems* Using nanosuspensiE ons) t&e application volume can #e %istinctly re%uce% compare% to solutions A4:B* To !ul!il t&e %istinctly &ig&er regulatory &ur%les) t&e %rug nanocrystals nee% to #e pro%uce% in an aseptic process* Alternatively) nanosuspensions can #e sterili/e% #y autoclaving A5:B or alternatively #y gamma irra%iation as (ell as sterile !iltration A5;B* ,&en a %rug is a%ministere% as a nanosuspension) t&e rapi% %issolution o! t&e nanocrystals (ill mimic t&e plasma concentration pro!ile o! a solution* Drug nanoE suspensions can #e !ormulate% (it& accepte% sur!actants an% polymeric sta#ili/ers !or I2 in.ection* In contrast) solutions o! poorly solu#le %rugs reCuire t&e use o! cosolvents an%1or &ig& sur!actant contents Ae*g*) C&remop&or E in Ta"ol bB) (&ic& can cause un%esire% si%e e!!ects A;B* Comparing t&e to"icity o! Ta"ol b (it& a pacliE ta"el nanosuspension) t&e latter &as s&o(n a %istinctly re%uce% to"icity* T&e nanosuspension (as muc& #etter tolerate%) resulting in an appro"imately %ou#le% D:@ value A5<B* T&e same e!!ect o! increase% tolerate% %ose (as !oun% !or t&e anti!ungal %rug itracona/ole* Itracona/ole is mar$ete% as Sporano" I2 b #y 'anssen P&armaceutica Pro%ucts) *P*) an inclusion comple" o! itracona/ole an% 5E&y%roE "ypropylEfEcyclo%e"trine AHPEfECDB* T&e pro%uct e"&i#its a signi!icant acute to"iE city a#ove 4@ mg1$g an% an D:@ value lo(er t&an 9@ mg1$g (&en a%ministere% as a #olus in t&e cau%al vein o! rats* In contrast) a 4[ nanosuspension o! itracoE na/ole coul% #e a%ministere% up to 85@ mg1$g (it&out animal mortality* +esi%es t&e %ecrease% acute to"icity) t&e nanosuspension &as also s&o(n a prolonge% e!!ect) (&ere#y t&e a%ministration intervals coul% #e e"ten%e% almost t&ree times in comE parison to t&e %aily a%ministration o! Sporano" I2 b A44B* Comparing a clo!a/imine nanosuspension (it& a liposomal !ormulation) #ot& are similarly e!!ective in t&e treatment o! arti!icially in%uce% Myco#acterium avium in!ections* T&e targeting to t&e reticuloen%ot&elial system) t&e lung) liver) an% spleen (as compara#le to t&e liposomal !ormulation A5=B* -urt&ermore) a special targeting can #e ac&ieve% #y a sur!ace mo%i!ication using t&e concept o! Q%i!!erential protein a%sorption*S A sur!ace mo%i!ication o! %rug nanocrystals (it& t&e sur!actant T(een =@ lea%s to a pre!erential a%sorption o! apolipoprotein E* T&is protein a%sorption ena#les a targete% %elivery

<;

MFsc&(it/er an% MJller

o! %rug nanocrystals to t&e #rain* AtovaCuone %rug nanocrystals mo%i!ie% (it& T(een =@ &ave s&o(n an e"cellent e!!icacy in t&e treatment o! To"oplasmosis A5>B* Drug Nanocrystals !or Pulmonary Drug Delivery Delivery o! (aterEinsolu#le %rugs to t&e respiratory tract is very important !or t&e local or systemic treatment o! %iseases* Many important %rugs !or pulmonary %eliE very s&o( poor solu#ility simultaneously in (ater an% nonaCueous me%ia) !or e"ample) important corticosteroi%s suc& as #u%esoni%e or #eclomet&asone %ipropioE nate* In t&e past) most o! t&ese %rugs (ere a%ministere% as aerosols) #ut in compliance (it& t&e Montreal Protocol o! 4>=< t&e use o! c&loro!luorocar#on AC-CB must #e avoE i%e%* T&ere!ore) alternatives suc& as %ry po(%er in&alers or metere% %ose in&alers (it&out C-C AMDIB (ere %evelope%* T&ese systems are !ille% (it& microni/e% %rug po(%ers pro%uce% #y .etEmilling* T&e mean particle si/e in t&e lo(er micrometer range A8H5: _mB results in a signi!icant orop&aryngeal %eposition o! larger particles lea%ing to increase% occurrence o! can%i%asis* A%%itionally) t&e oral %eposition o! t&e %rug lea%s to gastrointestinal AGIB %rug a#sorption !ollo(e% #y systemic si%e e!!ects A8@B* Nanosuspensions can #e success!ully applie% to overcome t&ese pro#lems* T&e ne#uli/ation o! nanosuspensions generates aerosol %roplets o! t&e pre!erre% si/e loa%e% (it& a large amount o! %rug nanocrystals* Using ne#uli/e% nanosuspensions) t&e respira#le !raction is %istinctly increase% in comparison to conventional MDIs A8@B* T&e smaller t&e particle si/e o! t&e %rug nanocrystals) t&e &ig&er t&e %rug loa%ing o! t&e aerosol %roplets A84)85B* T&ere!ore) t&e reCuire% ne#uli/ation time is %istinctly re%uce% A88B* +esi%es t&is) %rug nanocrystals s&o( an increase% mucoa%&esiveness) lea%ing to a prolonge% resi%ence time at t&e mucosal sur!ace o! t&e lung*

?t&er A%ministration Routes Dermal nanosuspensions are mainly o! interest i! conventional !ormulation approaE c&es !ail* T&e use o! %rug nanocrystals lea%s to an increase% concentration gra%ient #et(een t&e !ormulation an% t&e s$in* T&e increase% saturation solu#ility lea%s to Qsupersaturate%S !ormulations) en&ancing t&e %rug a#sorption t&roug& t&e s$in* T&is e!!ect can !urt&er #e en&ance% #y t&e use o! positively c&arge% polymers as sta#ili/ers !or t&e %rug nanocrystals* T&e opposite c&arge lea%s to an increase% a!!inity o! t&e %rug nanocrystals to t&e negatively c&arge% stratum corneum Aunpu#lis&e% %ataB* T&e ocular %elivery o! nanoparticles) inclu%ing %rug nanocrystals) is also o! &ig& interest* T&e %evelopment o! suc& colloi%al %elivery systems !or op&t&almic use aims at %ropa#le %osage !orms (it& a &ig& %rug loa%ing an% a longElasting %rug action* T&e a%&esiveness o! t&e small nanoparticles) (&ic& can #e !urt&er increase% #y t&e use o! mucoa%&esive polymers) lea%s to a more consistent %osing* +lurre% vision can #e re%uce% #y t&e use o! su#micronEsi/e% %rug particles A89)8:B*

PARTIC E SIVE REDUCTI?N TECHNI7UES Nanoparticles Pro%uce% #y Me%ia Milling Processes T&e use o! me%ia mills !or t&e pro%uction o! ultra!ine particles is very common* -rom t&e !irst &al! o! t&e t(entiet& century) #all mills (ere $no(n !or t&e pro%uction o! ultra!ine particle suspensions A8;B* In 4>>4) iversi%ge et al* A8<B &ave a%apte% t&is tec&niCue !or t&e pro%uction o! sur!aceEmo%i!ie% %rug nanoparticles* In or%er to pro%uce nanocrystalline %ispersions #y t&e NanoCrystals b tec&nology) a milling c&am#er is c&arge% (it& milling me%ia) %ispersion me%ium Anormally (aterB) staE #ili/er) an% t&e %rug* T&e %rug particles are re%uce% in si/e #y s&ear !orces an%

Drug Nanocrystals

<<

!orces o! impaction generate% #y a movement o! t&e milling me%ia* Small milling pearls or larger milling #alls are use% as milling me%ia* ,it& a re%uction in t&e si/e o! grin%ing me%ia in a me%ia mill) t&e num#er o! contact points is increase% e"poE nentially) resulting in improve% grin%ing an% %ispersing action Ai*e*) lea%ing to smaller particlesB* T&e pearls or #alls consist o! ceramics AceriumE or yttriumEsta#ili/e% /ircoE nium %io"i%eB) stainless steel) glass) or &ig&ly crossElin$e% polystyrene resinEcoate% #ea%s* A pro#lem associate% (it& t&e pearl milling tec&nology is t&e erosion !rom t&e milling material %uring t&e milling process* +uc&mann et al* A8=B reporte% t&e !ormaE tion o! glass microparticles (&en using glass as milling material* In or%er to re%uce t&e Cuantity o! impurities cause% #y an erosion o! t&e milling me%ia) t&e milling #ea%s (ere coate% (it& &ig&ly crossElin$e% polystyrene resin A8>B* A perpetual pro#lem is t&e a%&erence o! pro%uct to t&e large inner sur!ace area o! t&e milling system* T&e inner sur!ace area is ma%e up o! t&e sur!ace area o! t&e c&am#er an% o! all milling #ea%s toget&er* Even in recirculation systems) t&is pro%uct a%&erence causes a pro%uct loss* ?! course) t&is un%esira#le %rug loss can #e an issue in very e"pensive %rugs) espeE cially (&en very small Cuantities o! ne( c&emical entities ANCEsB are processe%* In general) t&ere are t(o milling principles6 eit&er t&e complete container is move% in a comple" movement lea%ing conseCuently to movement o! t&e milling material or t&e milling me%ium is move% #y an agitator* Assume t&at <;[ o! t&e milling c&am#er volume (ill #e !ille% (it& milling material Alarger #atc&es are %i!!iE cult to pro%uce in a #atc& mo%eB* In a 4@@@E mill) t&is correspon%s to <;@E milling material) #ase% on t&e apparent %ensity o! /ircon o"i%e pearls #eing 8*;> $g1 (&ic& correspon%s to 5*= tons o! milling material* -or t&at reason) agitator #ea% mills in recirculation mo%e are pre!erre% !or t&e pro%uction o! larger #atc&es* -igure 4 s&o(s a crossEsection o! an agitator #ea% mill in a &ori/ontal arrangement* T&e suspension is pumpe% vertically t&roug& t&e #ea% mill* In t&is case) t&e pro%uct is separate% !rom t&e milling me%ia #y a %ynamic separator gap* In ot&er cases) separator cartri%ges are use%* T&e recirculation mo%e prolongs t&e reCuire% milling time) #ecause t&e resi%ence time o! t&e %rug particles un%er impaction o! s&ear !orces is re%uce%* T&e milling time %epen%s on many !actors) suc& as &ar%ness o! t&e %rug) sur!actant content) temperature) viscosity o! t&e %ispersion me%ium) speci!ic energy input) an% si/e o! t&e milling me%ia* Milling perio%s !rom 8@ minutes up to several %ays are reporte% A4=B* Milling eCuipment is no( availa#le !rom t&e la#scale A94B to t&e large pro%uction scale* +esi%es ot&er !actors) t&is is an important prereCuisite !or t&e commercial pro%uction o! nanocrystalline %rugs* In 5@@@)

-IGURE 4 Agitator #ea% mill (it& %ynamic separator gap Acontinuous mo%eB6 4) pro%uct inletM 5) pro%uct outletM 8) cooling .ac$etM 9) milling pearlsM :) rotor (it& grin%ing %is$sM ;) %ynamic separator gap* Source6 -rom Re!* 9@*

<=

MFsc&(it/er an% MJller

Rapamuneb (as launc&e% #y ,yet& as t&e !irst pro%uct containing sirolimus NanoCrystalsb* T&e coate% Rapamuneb ta#lets are more convenient an% s&o( a 5<[ increase% #ioavaila#ility compare% to t&e Rapamune b solution A95B* T&is is an e"ample to compare t(o !ormulation strategies* T&e oral solution s&o(s t&e principE les o! cosolvents an% sur!actants) (&ereas t&e ta#lets s&o(s t&e nice per!ormance o! a particle si/e re%uction tec&niCue* Emen% b is t&e secon% pro%uct incorporating t&e NanoCrystalb tec&nology* It (as intro%uce% to t&e mar$et in 5@@8 #y Merc$* Emen%b is a capsule containing pellets o! nanocrystalline aprepitant) sucrose) microE crystalline cellulose) &yprolose) an% so%ium %o%ecylsul!ate A98B* T&e t&ir% pro%uct is TriCor) a nanocrystalline !eno!i#rate ta#let mar$ete% in 5@@9 #y A#ott* Megaace ES) an oral suspension containing megestrol acetate !or t&e treatment o! HI2EassoE ciate% anore"ia an% cac&e"ia) (as launc&e% as a !ourt& pro%uct late in t&e mi%%le o! t&e year 5@@:* To sum up) t&e (et me%ia milling is a via#le particle si/e re%uction tec&noE logy* T&e per!ormance &as #een proven #y !our -DAEapprove% pro%ucts* +ut ot&er nanoni/ation tec&nologies also can #ene!it !rom t&e success o! t&e NanoCrystal b tec&nology #y t&e attraction o! t&e si/e re%uction principle in general*

Precipitation Met&o%s T&e classical precipitation process) $no(n as Qvia &umi%a paratum)S is actually a very ol% p&armaceutical proce%ure* ater) t&is #asic i%ea (as applie% !or t&e pro%uction o! nanocrystalline %rug particles A99B* T&e !irst application o! t&e preciE pitation tec&niCue (as %evelope% #y ist an% Suc$er A4@B* It is $no(n as &y%rosol tec&nology) an% t&e IP is o(ne% #y San%o/ Ano( NovartisB* A poor (aterEsolu#le %rug is %issolve% in an organic me%ium) (&ic& is (aterEmisci#le* A pouring o! t&is solution into a nonsolvent) suc& as (ater) (ill cause a precipitation o! !inely %isperse% %rug nanocrystals* As simple as t&e particle !ormation process is) t&e preservation o! t&e nanocrystalline particle si/e is %i!!icult* T&e !ine particles ten% to gro( up) %riven #y a p&enomenon calle% Q?st(al% ripening*S T&is is a process (&ere small particles are %issolve% in !avor o! larger particles* Suc$er A9:B suggeste% imme%iate lyop&ili/ation to preserve t&e particle si/e* T&e crystalline state o! t&e particles o#taine% #y t&e precipitation process can #e controlle%* Depen%ing on t&e employe% met&o%) amorp&ous %rug nanoparticles can also #e generate% A9;B* +etaEcarotene is %issolve% in a (aterEmisci#le organic solvent toget&er (it& %igesti#le oil* T&is solution is a%mi"e% to an aCueous solution o! a protective colloi% AgelatineB causing a precipitation o! amorp&ous nanoparticuE late #etaEcarotene* A!ter an annealing step an% sprayE%rying) a sta#le amorp&ous pro%uct can #e o#taine%* T&is NanoMorp& b tec&nology) invente% #y Au(eter et al*) is use% #y t&e company SoliCs* Anot&er approac& to preserve t&e si/e o! t&e precipitate% nanocrystals is t&e use o! polymeric gro(t& in&i#itors) (&ic& are pre!era#ly solu#le in t&e aCueous p&ase* T&e increase% viscosity o! t&e aCueous p&ase can re%uce particle gro(ing* T&e resulting suspension is su#seCuently sprayE%rie% to o#tain a %ry po(%er (it& a relatively &ig& %rug loa%ing A9<B* Using t&is tec&niCue) a tremen%ous increase in %issolution rate A!rom 9[ to >8[ (it&in 5@ minutesB (as s&o(n !or a poor (aterE solu#le %rug ECUE@4 A9=B* Alt&oug& t&e !easi#ility o! preparing %rug nanocrystals #y precipitation &as #een s&o(n #y many groups) no commercial %rug pro%uct using t&is tec&nology &as entere% t&e mar$et* To use t&e prementione% met&o%s) it is reCuire% t&at t&e %rug is solu#le in at least one (aterEmisci#le solvent* T&is is

Drug Nanocrystals

<>

o!ten not t&e case !or NCEs* Many %rugs are simultaneously poorly solu#le in aCueous an% nonaCueous me%ia* Even i! t&ere is a suita#le solvent availa#le) it is %i!!icult to remove t&is solvent completely* Solvent resi%ues can #e potential ris$ !actors !or %rug alteration an% to"ic si%e e!!ects* In a%%ition) in cases o! amorp&ous %rug nanoparticles) it is seen as very critical to preserve t&e amorp&ous c&aracter t&roug&out t&e s&el! li!e o! a pro%uct* Recrystalli/ation (oul% impair t&e oral #ioavaila#ility* T&is e!!ect is less critical in !oo% pro%ucts #ecause o! less strict regulatory reCuirements t&at allo( more tolerance*

Nanoparticles Pro%uce% #y Hig&EPressure Homogeni/ation Hig&Epressure &omogeni/ation is anot&er universal approac& to re%uce t&e particle si/e o! poorly solu#le compoun%s* Consi%ering t&e &omogeni/ation eCuipment an% t&e &omogeni/ation con%itions) it &as to #e %ivi%e% #et(een t&ree tec&nologies* Micro!lui%i/er Tec&nology AIDDEPTM Tec&nologyB Particles can #e generate% #y a &ig& s&ear process using .etEstream &omogeni/ers) suc& as Micro!lui%i/ers AMicro!lui%i/er b) Micro!lui%ics) Inc*B* A !rontal collision o! t(o !lui% streams un%er pressures up to 4<@@ #ar lea%s to particle collision) s&ear !orces) an% also cavitation !orces* To preserve t&e particle si/e) sta#ili/ation (it& p&osE p&olipi%s or ot&er sur!actants an% sta#ili/ers is reCuire%* A ma.or %isa%vantage o! t&is process is t&e reCuire% pro%uction time* In many cases) :@ to 4@@ timeEconsuming passes are necessary !or a su!!icient particle si/e re%uction A9>):@B* S$yeP&arma Cana%a) Inc* Apreviously RTP) Inc*B applies t&is principle !or its IDDEPd tec&nology to pro%uce su#micron particles o! poorly solu#le %rugs A:4B*

PistonEGap Homogeni/ation in ,ater ADissocu#es b Tec&nologyB Drug nanocrystals can also #e pro%uce% #y &ig&Epressure &omogeni/ation using piston gap &omogeni/ers* Depen%ing on t&e &omogeni/ation temperature an% t&e %ispersion me%ia) t&ere is a %i!!erence #et(een t&e Dissocu#es b tec&nology an% t&e Nanopureb tec&nology* T&e Dissocu#esb tec&nology (as %evelope% #y MJller et al* in 4>>9 A:5B an% later acCuire% #y S$yeP&arma P C* It involves t&e pro%uction o! nanosuspensions in (ater at room temperature* T&e tra%e name Dissocu#es b alrea%y in%icates t&e improve% %issolution #e&avior an% t&e cu#ic s&ape o! t&e resulting %rug nanocrystals A:8B* A %rug po(%er is %isperse% in an aCueous sur!acE tant solution* T&e resulting macrosuspension is su#seCuently !orce% #y a piston t&roug& a tiny &omogeni/ation gap applying pressures up to 9@@@ #ar* Depen%ing on t&e viscosity o! t&e suspension an% t&e &omogeni/ation presE sure) t&e (i%t& o! t&e &omogeni/ation gap is in t&e range o! : to 5@ _m A:9B* -igure 5 s&o(s a crossEsection t&roug& a pistonEgap &omogeni/er* Accor%ing to +ernoulliPs la() t&e resulting &ig& streaming velocity o! t&e suspension causes an increase in %ynamic pressure t&at is compensate% #y a re%uction in static pressure #elo( t&e vapor pressure o! t&e aCueous p&ase* T&e (ater starts #oiling an% t&e !ormation o! gas #u##les occurs* T&ese gas #u##les collapse imme%iately (&en t&e liCui% leaves t&e &omogeni/ation gap) resulting in cavitationEcause% s&oc$ (aves* T&e enormous po(er o! t&ese s&oc$ (aves) tur#ulent !lo() an% s&ear !orces #rea$ t&e %rug particles A::B* T&e %etaile% illustration s&o(s t&e principle o! %iminution mec&anisms in t&e &omogeni/ation gap A-ig* 8B* At t&e #eginning) t&e particles are #ro$en at crystal imper!ectionsM (it& continuing t&e &omogeni/ation process) t&e num#er o!

=@

MFsc&(it/er an% MJller

-IGURE 5 CrossEsection o! a pistonEgap &omogeni/er* A macrosuspension is !orce% t&roug& a very tiny &omogeni/ation gap in or%er to pro%uce %rug nanocrystals* Source6 -rom Re!* 9@*

imper!ections %ecreases an% almost per!ect small crystals (ill remain* T&e reCuire% num#er o! cycles is mainly in!luence% #y t&e &ar%ness o! t&e %rug) t&e !inesse o! t&e starting material an% t&e reCuirements o! t&e application route or t&e !inal %osage !orm) respectively* In general) 4@ to 5@ &omogeni/ation cycles are su!!icient to o#tain a unimo%al si/e %istri#ution in t&e nanometer range A:;H:=B* T&e use o! (ater as %ispersion me%ium is associate% (it& some %isa%vanE tages* Hy%rolysis o! (aterEsensitive %rugs can occur) as (ell as pro#lems %uring %rying steps* In cases o! t&ermola#ile %rugs or %rugs possessing a lo( melting point) a complete (ater removal reCuires relatively e"pensive tec&niCues) suc& as lyop&ili/ation* -or t&ese reasons) t&e Dissocu#es b tec&nology is particularly suiE ta#le i! t&e resulting nanosuspension is %irectly use% (it&out mo%i!ications) suc& as %rying steps*

-IGURE 8 Diminution principles %uring &ig&Epressure &omogeni/ation A%etaile% illustration o! a &omogeni/aE tion gap in crossEsectionB6 4) implosion area AcavitationBM 5) #oiling area an% crystal collisionM 8) s&ear !orcesM 9) tur#ulent !lo(* Source6 -rom Re!* 9@*

Drug Nanocrystals

=4

Nanopureb Tec&nology In 4>>>) MJller et al* A:>B !oun% t&at a similar e!!ective particle %iminution can also #e o#taine% in nonaCueous or (aterEre%uce% me%ia* T&e proprietary tec&niCue is $no(n as Nanopureb tec&nology %evelope% an% o(ne% #y P&armaSol Gm#H1+erlin* +y using %ispersion me%ia (it& a lo(Evapor pressure an% per!orming t&e &omogeniE /ation process at lo( temperatures Ae*g*) @]CB) t&e cavitation in t&e &omogeni/ation gap is %istinctly re%uce% or %oes not e"ist at all* It coul% #e s&o(n t&at even in t&e a#sence o! cavitation) a su!!icient particle si/e %iminution (as o#taine%* T&e tur#uE lent !lo( an% s&ear !orces %uring t&e &omogeni/ation process are strong enoug& to #rea$ t&e %rug particles an% to pro%uce %rug nanocrystals* T&e &ig&Epressure &omoE geni/ation in nonaCueous or (aterEre%uce% me%ia is particularly #ene!icial i! t&e nanosuspension &as to #e trans!erre% into a tra%itional !inal %osage !orm* +y re%ucing t&e (ater content in t&e %ispersion me%ium) t&e reCuire% energy is minimi/e% !or %rying steps) suc& as sprayE%rying) !lui%i/e% #e% %rying) or upon suspension layE ering onto sugar sp&eres* T&e evaporation processes can #e per!orme% un%er mil%er con%itions) (&ic& is #ene!icial !or temperatureEsensitive %rugs* Pro%uction o! nanoE suspensions at @]C or even #elo( can prevent temperature la#ile %rugs !rom %egra%ation A4:B* I! t&e &ig&Epressure &omogeni/ation is carrie% out completely in nonaCueous me%ia) even (aterEsensitive %rugs can #e processe% (it&out &y%rolysis* Nanosuspensions pro%uce% in liCui% polyet&ylene glycol APEGB or &otEmelte% PEGs can #e %irectly !ille% into gelatine or &y%ro"y propyl met&yl cellulose AHPMCB capsuE les A;@B* Depen%ing on t&e reCuirements o! t&e !inal !ormulation) t&e (ater content can #e varie% !rom (aterE!ree to isotonic con%itions !or intravenous suspensions* Irrespective o! t&e employe% tec&niCue ADissocu#es b or NanopurebB) t&e pro%uction o! %rug nanocrystals #y &ig&Epressure &omogeni/ation is a pro%uctionE !rien%ly process* Homogeni/ation is a lo(Ecost processM approve% pro%uction lines are alrea%y in use !or t&e pro%uction o! p&armaceutical pro%ucts) suc& as emulsions !or parenteral nutrition A45B* T&e process can #e easily trans!erre% !rom t&e la#scale to t&e large pro%uction scale* Hig&Epressure &omogeni/ation can #e per!orme% starting !rom @*: m AAvestin Emulsi-le"E+8) Avestin) Inc*) Cana%aB up to large #atc& si/es o! 5@@@ 1&r ARannie 44=) AP2 &omogenisers) Denmar$B A5;);4B* ,&en pro%ucing nanosuspenions #y &ig&Epressure &omogeni/ation even at &ar% &omogeni/ation con%itions) only a noncritical pro%uct contamination #elo( 4 ppm (as o#serve% A;5B* Suspensions (it& %rug concentration up to 8@[ an% more can #e easily processe% #y &ig&Epressure &omogeni/ation A;8B*

Com#ination Tec&nologies Nanoe%geb Tec&nology +a"terPs NAN?EDGEb process relies on t&e com#ination o! a microprecipitation tec&niCue (it& a su#seCuent annealing step #y applying &ig& s&ear an%1or t&ermal energy A;9B* A !ine suspension is !orme% #y a%%ing an organic solution o! t&e (aterE insolu#le %rug to an antisolvent) !or e"ample) aCueous sur!actant solution* Depen%ing on t&e precipitation con%itions) eit&er small amorp&ous or crystalline %rug particles in t&e nanometer range or !ria#le nee%leEli$e crystals in t&e micromeE ter range are !orme%* ConseCuently) t&e !ollo(ing &ig&Eenergy input can &ave t(o e!!ects on t&e pre!orme% particles* Small amorp&ous or crystalline %rug particles (ill #e preserve% in si/e #y an annealing step (it&out c&anging t&e mean %iameter* It coul% #e s&o(n t&at t&e ten%ency to crystal gro(t& can #e re%uce% #y energy input a!ter t&e precipitation step* In case long !ria#le nee%leEli$e crystals are o#taiE ne%) t&ey (ill #e re%uce% in si/e #y t&e &ig&Eenergy input using &ig&Epressure

=5

MFsc&(it/er an% MJller

&omogeni/ers* Accor%ing to t&e patent A;9B) particle si/es in t&e range o! 9@@ to 5@@@ nm can #e o#taine%* T&e organic solvent utili/e% &as to #e care!ully remove% !rom t&e !inal nanosuspension (it&out c&anging t&e particle si/e o! t&e %rug nanoE crystals* ?t&er(ise) crystal gro(t& can #e promote% #y an increase% solu#ility o! t&e %rug* Any content o! t&e organic solvent %issolve% in t&e aCueous p&ase can act as a Qcosolvent)S lea%ing to an increase% ten%ency to ?st(al% ripening* Also) to"ic e!!ects can #e cause% #y potential solvent resi%ues) especially i! t&e nanosuspension is t&e !inal pro%uct* -or t&ese reasons) t&e NAN?EDGE b process is particularly suiE ta#le !or %rugs t&at are solu#le in nonaCueous me%ia possessing lo( to"icity) suc& as NEmet&ylE5Epyrroli%inone*

Nanopureb WP Tec&nology Consi%ering t&e commonly use% particle si/e re%uction tec&niCues) t&e pro%uction o! nanosuspensions is in most cases associate% (it& a &ig&Eenergy input A&ig&E spee% me%ia mills) &ig&Epressure &omogeni/ationB an% a relatively long perio% #et(een t&e %rug synt&esis an% t&e !inal pro%uct* A microni/e% %rug material Asi/e 4@H4@@ _mB is recommen%e% !or milling an% &ig&Epressure &omogeni/ation procesE ses A8<)::B* T&ere!ore) t&e %rug o!ten &as to #e .etEmille% #e!ore t&e nanoni/ation* Impurities cause% #y an a#rasion !rom t&e milling material or solvent resi%ues !rom t&e precipitation process are un%esira#le* T&ey can causes si%e e!!ects especially i! t&e %rug is a%ministere% !or t&e treatment o! c&ronic %iseases* T&e minimal ac&ieE va#le particle si/e is signi!icantly %etermine% #y t&e &ar%ness o! t&e %rug* In cases o! very &ar% %rugs) an increasing energy input A#y e"ten%ing t&e milling time or t&e num#er o! &omogeni/ation cyclesB (ill not lea% to a smaller particle si/e* In general) t&e smallest ac&ieva#le si/e o! nanocrystals is aroun% 5@@ nmM only un%er special con%itions can a#out 4@@ nm #e pro%uce%* Ho(ever) especially crystals #elo( 4@@ nm (oul% s&o( an e"tremely !ast %issolution an% simultaneously a great increase in saturation solu#ility A44B* T&ere!ore) suc& particles are o! &ig& commerE cial interest* In 5@@:) MFsc&(it/er A;:B %evelope% a ne( com#ination met&o% !or t&e pro%uction o! %rug nanosuspensions) (&ic& is no( o(ne% #y P&armaSol Gm#H* T&e Nanopureb WP tec&nologogy Aprocess variant6 H95B ena#les e"tension o! t&e per!ormance o! t&e Nanopureb tec&nology to very &ar% an% crystalline materials* Mo%i!ication o! t&e starting material #y an evaporation process #e!ore t&e su#seE Cuently per!orme% &ig&Epressure &omogeni/ation can signi!icantly re%uce t&e num#er o! &omogeni/ation cycles A;;B* ?(ing to t&e reason t&at t&e solvent (ill #e remove% completely #e!ore &omogeni/ation #y t&e evaporation process) various solvents can #e use% !or t&e mo%i!ication process (it&out restrictions %ue to to"icity reasons* A%%itionally) e"cipients can #e a%%e% to t&e %rug solution to increase t&e num#er o! crystal imper!ections upon %rying* -igure 9 ma$es clear t&e e!!ectiveness o! t&e ne( com#ination met&o% in comparison to t&e classical &ig&Epressure &omogE eni/ation* T&e application o! t&e H95 tec&nology lea%s to %istinctly re%uce% particle si/e #y a simultaneous re%uction o! t&e reCuire% num#er on &omogeni/ation cycles* T&is (ill conseCuently re%uce t&e pro%uction costs an% t&e (ear on t&e &omogeniE /ation eCuipment* H>; is anot&er tec&nology #elonging to t&e Nanopure b WP plat!orm* T&e tec&E nology) %evelope% in 5@@: #y MFsc&(it/er an% em$e A;<B) is a mo%i!ie% process #ase% on &ig&Epressure &omogeni/ation* +y using t&is un%isclose% tec&nology) it (as s&o(n t&at %rug nanocrystals #elo( 4@@ nm can #e pro%uce% #y &ig&Epressure &omogeni/ation*

Drug Nanocrystals

=8

-IGURE 9 Comparison o! t&e ne( &omogeni/ation tec&nology H95 Ale!t B (it& t&e conventional &omogeni/ation in (ater Arig&t B) per!orme% in pistonE gap &omogeni/er) in!luence o! cycle num#ers) results represent t&e laser %i!!ractometry %iameters AvolumeE (eig&te%) Coulter s 58@) +ec$manE Coulter) GermanyB* Source 6 -rom Re!* 9@*

H>; results in a translucent nanosuspension in comparison to anot&er nanoE suspension pro%uce% using t&e Dissocu#esb tec&nology* -igure : s&o(s t(o nanoE suspensions* T&e le!t nanosuspension (as pro%uce% applying t&e conventional &ig&Epressure &omogeni/ation in (aterM t&e rig&t nanosuspension (as pro%uce% #y using t&e H>; tec&nology* Alt&oug& #ot& !ormulations are #eing compose% similarly) t&e rig&t !ormulation is translucent %ue to t&e signi!icantly re%uce% particle si/e* T&e particle si/e analysis also provi%es t&e evi%ence o! t&e per!ormance o! t&e H>; met&o%* Almost >>[ o! t&e particles (ere smaller t&an 4@@ nm Nlaser %i!!ractometry A DB D>>[) volume si/e %istri#ution) S 58@) +ec$manECoulter) Germany unpuE #lis&e% %ata (it& permission !rom Re!* 9@O* A very small particle si/e an% a narro( range o! particle si/e %istri#ution can #e o#taine% #y using t&e H>; tec&nology*

?t&er Tec&niCues !or t&e Pro%uction o! Drug Nanocrystals Milling) &ig&Epressure &omogeni/ation) an% precipitation are t&e main met&o%s employe% !or t&e pro%uction o! %rug nanocrystals* T&e importance !or improvement o! t&e #ioavaila#ility o! poorly solu#le %rugs #y t&e pro%uction o! %rug nanocrystals is (i%ely accepte%* T&e intensive researc& !or ne( tec&nologies le% to many ot&er

-IGURE : ASee color insert*B T(o nanosuspensions compose% similarly) pro%uce% #y &ig&Epressure &omogeE ni/ation) conventional met&o% Ale!t B versus translucent nanosuspension Aparticle si/e (ell #elo( 4@@ nmB resulting !rom H>; tec&nology* T&e re% laser #eam is re!lecte% #y t&e tiny nanocrystals* Source 6 -rom Re!* 9@*

=9

MFsc&(it/er an% MJller

approac&es !or t&e pro%uction o! %rug nanocrystals* Even nonp&armaceutical comE panies) suc& as Do( C&emical) are entering t&e mar$et o! poorly solu#le %rugs (it& solu#ilityEen&ancing tec&nologies* SprayE!ree/ing into liCui% A;=B an% evaporative precipitation into aCueous solutions A;>B are e"amples !or suc& ne( tec&nologies*

-INA -?RMU ATI?NS -?R DRUG NAN?CR3STA S In or%er to s&o( t&eir a%vantages in vivo) t&e %rug nanocrystals nee% to #e trans!erre% into t&e rig&t %osage !orm* Nanosuspensions can #e %irectly use% as oral suspensions to overcome t&e %i!!iculties o! s(allo(ing ta#lets #y pe%iatric or geriatric patients* ?ne e"ample is Megace b A+ristol Meyers SCui##B) an oral suspenE sion o! megestrol acetate) use% !or t&e treatment o! HI2Eassociate% anore"ia an% cac&e"ia* T&e application o! t&ese nanosuspensions can improve t&e solu#ility o! t&e %rug an% t&e %issolution rateM a%%itionally) suspensions can #e applie% !or reaE sons o! tasteEmas$ing* Nanosuspensions can also #e use% %irectly !or parenteral %rug a%ministration* Alt&oug& nanosuspensions &ave s&o(n a su!!icient longEterm sta#ility (it&out ?st(al% ripening) !or intravenous pro%ucts a lyop&ili/ation step is recommen%e% in or%er to avoi% aggregation or ca$ing o! settle% %rug nanocrystals* T&e lyop&ili/e% pro%uct can #e easily reconstitute% #e!ore use #y a%%ing isotonic (ater) aCueous glucose solution) or ot&er reconstitution me%ia A5<)<@B* ,it&out Cuestion) #ot& t&e patients an% t&e mar$eting e"perts pre!er t&e oral a%ministration o! tra%itional %osage !orms* Hence) to enter t&e p&armaceutical mar$et success!ully in most cases %rug nanocrystals &ave to #e !ormulate% as tra%itional proE %ucts) suc& as ta#lets or capsules* A per!ect soli% %osage !orm s&oul% preserve t&e in vivo per!ormance o! %rug nanocrystals* ,&en reac&ing t&e target part o! t&e GI tract) t&e %osage !orm s&oul% release t&e %rug nanocrystals as a !ine) nonaggregate% suspenE sion* ?t&er(ise) sel!Eagglomeration or aggregation can impair t&e %rug release A<4B* Using nanosuspensions as granulation !lui% !or a !urt&er ta#let pro%uction is a very simple approac&* T&e nanosuspension is a%mi"e% to #in%ers an% ot&er e"ciE pients) an% t&e granules are !inely compresse% to ta#lets* T&is %osage !orm is limiE te% in t&e ma"imum ac&ieva#le %rug content* A ma"imum %rug content o! a#out :@[ or less is suggeste% in or%er to ensure a complete %isintegration into a !inely %isperse% suspension A<5B* Nanosuspensions can also #e use% !or t&e pro%uction o! matri" pellets A-ig* ;B or as layering %ispersions in a !lui%i/e% #e% process* A!ter t&e pellet preparation) t&e cores can #e coate% (it& several polymers in or%er to mo%i!y t&e release pro!ile o! t&e !inal !ormulation A<8H<:B* A very smart !ormulation approac& is t&e Nanopure b tec&nology* Nanocrystals pro%uce% in nonaCueous me%ia) suc& as liCui% PEG or oils Ae*g*) MiglyolB) can #e %irectly !ille% into gelatine or HPMC capsules* T&e pro%uction o! %rug nanocrystals in melte% PEGs is a ne( strategy !or t&e pro%uction o! !inal %osage !orms contaiE ning %rug nanocrystals* A!ter per!orming t&e &ig&Epressure &omogeni/ation in melte% PEG at a#out ;@]C) t&e mi"ture can #e soli%i!ie%* T&e resulting matri") !i"ing t&e %rug nanocrystals in separate% state) can #e compresse% to ta#lets or %irectly !ille% into capsules A<;B* SprayE%rying o! t&e nanosuspensions is anot&er costEe!!ective approac& to trans!er nanosuspensions into %ry pro%ucts* T&e %rug nanocrystals can %irectly #e pro%uce% #y &ig&Epressure &omogeni/ation in aCueous solutions o! (aterEsolu#le matri" materials) !or e"ample) polymers Npolyvinylpyrroli%one) polyvinylalco&ol or longEc&aine% PEG) sugars Asacc&arose) lactoseB or sugar alco&ols Amannitol) sor#itolBO* A!ter(ar%s) t&e resulting nanosuspension can #e sprayE%rie% un%er appropriate

Drug Nanocrystals

=:

-IGURE ; SEM p&otograp& o! uncoate% matri" core containing %rug nanocrystals6 Ale!t B overvie( Amagni!ication ;@`B) Arig&t B %etaile% magni!ication A4@@@`B s&o(ing %rug nanocrystals com#ine% (it& t&e #in%er material*

con%itions* T&e %ry po(%er) compose% o! %rug nanocrystals em#e%%e% in a (aterE solu#le matri") can #e !ille% in &ar% gelatine capsules !or oral a%ministration A<<B* Anot&er attractive approac& using t&e sprayE%rying principle is %escri#e% as Q%irect compress tec&nologyS A<=B* actose an% ot&er matri"E!orming materials) suc& as microni/e% polymer po(%ers or lipi%s) are a%mi"e% to t&e priorEpro%uce% nanosuspension* T&e resulting suspension is trans!erre% into a %rugHmatri"HcomE poun% #y sprayE%rying* Su#seCuently) t&e !reeE!lo(a#le po(%er can #e use% !or %irect compression o! !ast %issolving or prolonge% release ta#lets* Alternatively) t&e po(%er can also #e !ille% into &ar% gelatine capsules*

C?NC USI?N Poor aCueous solu#ility is clearly recogni/e% #y t&e p&armaceutical in%ustry as a ma.or pro#lem* T&e use o! %rug nanocrystals is a universal !ormulation approac& to increase t&e t&erapeutic per!ormance o! t&ese %rugs in any route o! a%ministration* Almost any %rug can #e re%uce% in si/e to t&e nanometer range* ?(ing to t&eir great !ormulation versatility %rug nanocrystals are no longer only t&e last c&ance rescue !or a !e( %rugs* Many insolu#le %rug can%i%ates are in clinical trials !ormuE late% as %rug nanocrystals Aat present a#out 4@B* 2arious nanoni/ation tec&niCues are availa#le* Pro%uction !acilities are avaiE la#le to pro%uce tons o! nanosuspensions* Currently) attention is turne% to improving t&e %iminution per!ormance to pro%uce %rug nanocrystals (ell #elo( 4@@ nm) also in cases o! very &ar% %rugs* -irst approac&es (ere alrea%y success!ul* Ne( tec&nologies are in %evelopment to pro%uce !inal %osage !orms (it& &ig&er %rug loa%ings) #etter re%ispersa#ility at t&eir site o! action) an% an improve% %rug targeting*

RE-ERENCES
4* Meris$oE iversi%ge E* Nanocrystals6 resolving p&armaceutical !ormulation issues assoE ciate% (it& poorly (aterEsolu#le compoun%s in particles* ?rlan%o6 Marcel De$$er) 5@@5* 5* ipins$i C* Poor aCueous solu#ilityRan in%ustry (i%e pro#lem in %rug %elivery* Am P&arm Rev 5@@5M :6=5H=:* 8* -DA* ,aiver o! in vivo #ioavaila#ility an% #ioeCuivalence stu%ies !or imme%iateErelease soli% oral %osage !orms #ase% on a #iop&armaceutics classi!ication system* -DA) 5@@@*

=;

MFsc&(it/er an% MJller

9* 0an!er I* Report on t&e international (or$s&op on t&e #iop&armaceutics classi!ication system A+CSB6 scienti!ic an% regulatory aspects in practice* ' P&arm P&arm Sci 5@@5M :A4B64H9* :* Gupta P0) Patel 'P) Ha&n 0R* Evaluation o! pain an% irritation !ollo(ing local a%miniE stration o! parenteral !ormulations using t&e rat pa( lic$ mo%el* ' P&arm Sci Tec&nol 4>>9M 9=A8B64:>H4;;* ;* Singla A0) Garg A) Aggar(al D* Paclita"el an% its !ormulations* Int ' P&arm 5@@5M 58:A4H5B64<>H4>5* <* Mattila MA) Suistomaa M* Intravenous preme%ication (it& %ia/epam6 a comparison #et(een t(o ve&icles* Anaest&esia 4>=9M 8>A>B6=<>H==5* =* Ue$ama 0* Design an% evaluation o! cyclo%e"trinE#ase% %rug !ormulation* C&em P&arm +ull ATo$yoB 5@@9M :5A=B6>@@H>4:* >* MJller RH) Peters 0) +ec$er R) 0russ +* Nanosuspensions6 a novel !ormulation !or t&e i*v* a%ministration o! poorly solu#le %rugs* -irst ,orl% Meeting APGI1AP2) +u%apest) 4>>:* 4@* ist M) Suc$er H* Hy%rosols o! p&armacologically active agents an% t&eir p&armaceutical compositions comprising t&em* US :)8=>)8=5) 4>>4* 44* 0ipp 'E* T&e role o! soli% nanoparticle tec&nology in t&e parenteral %elivery o! poorly (aterEsolu#le %rugs* Int ' P&arm 5@@9M 5=9A4H5B64@>H455* 45* MJller RH) +F&m +H * Nanosuspensions* In6 MJller RH) +enita S) +F&m +) e%s* Emulsions an% Nanosuspensions !or t&e -ormulation o! Poorly Solu#le Drugs* Stuttgart6 Me%p&arm) 4>>=649>H4<9* 48* Troester -* Cremop&orE!ree aCueous paclita"el nanosuspensionRpro%uction an% c&emiE cal sta#ility* Controlle% Release Society 84st annual meeting) Honolulu) HI) 5@@9* 49* iversi%ge E) ,ei * Sta#ili/ation o! c&emical compoun%s using nanoparticulate !ormuE lations* US 5@@4M>:5@85 5@@4@>49* CAN 48=659885<) US 5@@8@:9@95 A4) 5@@8* 4:* MFsc&(it/er ') Ac&leitner G) Pomper H) Muller RH* Development o! an intravenously in.ecta#le c&emically sta#le aCueous omepra/ole !ormulation using nanosuspension tec&nology* Eur ' P&arm +iop&arm 5@@9M :=A8B6;4:H;4>* 4;* MJller RH) 0ec$ CM* C&allenges an% solutions !or t&e %elivery o! #iotec& %rugsRa revie( o! %rug nanocrystal tec&nology an% lipi% nanoparticles* ' +iotec&nol 5@@9M 448A4H8B64:4H4<@* 4<* iversi%ge GG) Cun%y 0C* Particle si/e re%uction !or improvement o! oral #ioavaila#iE lity o! &y%rop&o#ic %rugs* I A#solute oral #ioavaila#ility o! nanocrystalline %ana/ol in #eagle %ogs* Int ' P&arm 4>>:M 45:6>4H><* 4=* Meris$oE iversi%ge E) iversi%ge GG) Cooper ER* Nanosi/ing6 a !ormulation approac& !or poorly (aterEsolu#le compoun%s* Eur ' P&arm Sci 5@@8M 4=A5B6448H45@* 4>* iversi%ge GG) Con/entino) P&il* Drug particle si/e re%uction !or %ecreasing gastric irritancy an% en&ancing a#sorption o! napro"en in rats* Int ' P&arm 4>>:M 45:68@>H848* 5@* Eic$&o!! ,M) Engers DA) Mueller 0R* Nanoparticulate NSAID compositions) Application* US >:E8=:;49) ::4=<8=) 4>>;* 54* 'aco#s C) 0ayser ?) MJller RH* Pro%uction an% c&aracterisation o! mucoa%&esive nanosuspensions !or t&e !ormulation o! #upravaCuone* Int ' P&arm 5@@4M 549A4H5B68H<* 55* MJller RH) 'aco#s C* +uparvaCuone mucoa%&esive nanosuspension6 preparation) optimisation an% longEterm sta#ility* Int ' P&arm 5@@5M 58<A4H5B64:4H4;4* 58* amprec&t A) U#ric& N) 3amamoto H) et al* +io%egra%a#le nanoparticles !or targete% %rug %elivery in treatment o! in!lammatory #o(el %isease* ' P&armacol E"p T&er 5@@4M 5>>A5B6<<:H<=4* 59* Hussain N) 'aitley 2) -lorence AT* Recent a%vances in t&e un%erstan%ing o! upta$e o! micropE articulates across t&e gastrointestinal lymp&atics* A%v Drug Deliv Rev 5@@4M :@A4H5B64@<H495* 5:* Na GC) Stevens 'r ') 3uan +?) Ra.agopalan N* P&ysical sta#ility o! et&yl %iatri/oate nanocrystalline suspension in steam sterili/ation* P&arm Res 4>>>M 4;A9B6:;>H:<9* 5;* MJller RH) 'aco#s C) 0ayser ?* Nanosuspensions !or t&e !ormulation o! poorly solu#le %rugs* In6 Niellou% -) MartiEMestres G) e%s* P&armaceutical Emulsions an% Suspensions* Ne( 3or$6 Marcel De$$er) 5@@@68=8H9@<* 5<* +F&m +* Pro%uction an% c&aracterisation o! nanosuspensions as novel %elivery system !or %rugs (it& lo( #ioavaila#ility* In6 P&armaceutical Tec&nology* +erlin6 -ree University o! +erlin) 4>>>* 5=* Peters 0 et al* Preparation o! a clo!a/imine nanosuspension !or intravenous use an% evaE luation o! its t&erapeutic e!!icacy in murine Myco#acterium avium in!ection* ' Antimicro# C&emot&er 5@@@M 9:A4B6<<H=8*

Drug Nanocrystals

=<

5>* Sc&oler N et al* AtovaCuone nanosuspensions s&o( e"cellent t&erapeutic e!!ect in a ne( murine mo%el o! reactivate% to"oplasmosis* Antimicro# Agents C&emot&er 5@@4M 9:A;B64<<4H4<<>* 8@* ?stran%er 0D) +osc& H,) +on%an/a DM* An in vitro assessment o! a NanoCrystal #eclomet&asone %ipropionate colloi%al %ispersion via ultrasonic ne#uli/ation* Eur ' P&arm +iop&arm 4>>>M 9=A8B6 5@<H54:* 84* 'aco#s C) MJller RH* Pro%uction an% c&aracteri/ation o! a #u%esoni%e nanosuspension !or pulmonary a%ministration* P&arm Res 5@@5M 4>A5B64=>H4>9* 85* ,ie%mann TS) DeCastro ) ,oo% R,* Ne#uli/ation o! NanoCrystals6 pro%uction o! a respira#le soli%EinEliCui%EinEair colloi%al %ispersion* P&arm Res 4>><M 49A4B6445H44;* 88* 0ra!t ,0 et al* T&e p&armaco$inetics o! ne#uli/e% nanocrystal #u%esoni%e suspension in &ealt&y volunteers* ' Clin P&armacol 5@@9M 99A4B6;<H<5* 89* Mainar%es RM et al* Colloi%al carriers !or op&t&almic %rug %elivery* Curr Drug Targets 5@@:M ;A8B68;8H8<4* 8:* 0eipert S* ?p&talmi$a6 eta#lierte Ar/nei!ormen un% neue 0on/epte* In6 MJller RH) Hil%e#ran% GE) e%s* P&arma/eutisc&e Tec&nologie6 Mo%erne Ar/nei!ormen* Stuttgart6 ,issensc&a!tlic&e 2erlagsgesellsc&a!t m#H) 4>>=6<<H>=* 8;* Pa&l MH* Ver$leinerungstec&ni$* Pra"is(issen 2er!a&renstec&ni$* 0Fln6 Ts2 R&einlan% Gm#H) 4>>4* 8<* iversi%ge GG) Cun%y 0C) +is&op '-) C/e$ai DA* Sur!ace Mo%i!ie% Drug Nanoparticles* USA) 4>>5* 8=* +uc&mann S) -isc&li ,) T&iel -P) Ale" R* ACueous microsuspension) an alternative intraE venous !ormulation !or animal stu%ies* In6 95n% annual congress o! t&e International Association !or P&armaceutical Tec&nology AAP2B) Main/) 4>>;* 8>* +runo 'AD) +rian D* Gusto( Evan) Illig) 0at&leen ') Ra.agopalan) Nats) Sarpot%ar* Met&o% o! grin%ing p&armaceutical su#stances* US :):4=)4=<) 4>>5* 9@* MFsc&(it/er '* P&armaceutical Tec&nology* P&*D* T&esis* +erlin) -ree University o! +erlin* In preparation* 94* Cunning&am ' ) Elaine) Cooper) Eugene R) iversi%ge) Gary G* Milling microgram Cuantities o! nanoparticulate can%i%ate compoun%s* US 5@@9@4<8;>;) 5@@9* 95* ,yet& Researc& Drug In!ormation) Rapamune ASirolumusB ?ral Solutions an% Ta#lets* Company Communications) 5@@9* 98* Merc$) Drug In!ormation Emen%* 5@@9* 99* 2iolanto MR* Met&o% !or ma$ing uni!ormly si/e% particles !rom (aterEinsolu#le organic compoun%s* US 9)=5;);=>) 4>=>* 9:* Suc$er H* Hy%rosole) eine Alternative !Jr %ie parenterale An(en%ung von sc&(er (asE serlFslic&en ,ir$sto!!en* In6 MJller RH) Hil%e#ran% GE) e%s* P&arma/eutisc&e Tec&nologie6 Mo%erne Ar/nei!ormen* Stuttgart6 ,issensc&a!tlic&e 2erlagsgesellsc&a!t m#H) 4>>=68=8H8>4* 9;* Au(eter H) +o&n H* u%%ec$e E* Sta#le) aCueous %ispersions an% sta#le) (aterE%isperE si#le %ry po(%ers o! "ant&op&ylls) t&eir pro%uction an% t&eir use) Application* ,? 5@@@E EP89;<) 5@@@@;;;;:) 5@@@* 9<* Mueller +,) Rasenac$) Nor#ert* Met&o% !or t&e pro%uction an% t&e use o! microparticles an% nanoEparticles #y constructive micronisation* ,? @@5@@8@=@@89A8) 5@@8* 9=* Rasenac$ N) Harten&auer H) MJller +,* Microcrystals !or %issolution rate en&ancement o! poorly (aterEsolu#le %rugs* Int ' P&arm 5@@8M 5:9A5B648<H49:* 9>* Pari$& IS) Ulagara.* Composition an% met&o% o! preparing microparticles o! (aterE insolu#le su#stances* >8>;>>><) 4>><* :@* Dearn AR* AtovaCuone p&armaceutical compositions* US) ><959=) ;)@4=)@=@) 5@@@* :4* Haynes DH* P&osp&olipi%Ecoate% microcrystals6 in.ecta#le !ormulations o! (aterE insolu#le %rugs* US :)@>4)4=<) 4>>5* :5* MJller RH+) Ro#ert) 0russ) +ern%) Peters) 0atrin* P&arma/eutisc&e Nanosuspensionen /ur Ar/neisto!!appli$ation als Systeme mit er&F&ter SIttigungslFslic&$eit un% Fsungsgesc&(in%ig$eit* :8* MJller RH'C0 ?* DissoCu#esRa novel !ormulation !or poorly solu#le an% poorly #ioavaila#le %rugs) In6 Ro#erts M'R'HMS) e%* Mo%i!ie%ERelease Drug Delivery Tec&nology* Ne( 3or$6 Marcel De$$er) 5@@8648:H49>* :9* MJller RH) MFsc&(it/er ') +us&ra# -N* Manu!acturing o! nanoparticles #y milling an% &omogenisation tec&niCues* In6 0ompella G) e%* Nanoparticle Tec&nology !or Drug Delivery* Ne( 3or$6 Marcel De$$er* Su#mitte%*

==

MFsc&(it/er an% MJller

::* MJller RH) 'aco#s C) 0ayser ?* Nanosuspensions as particulate %rug !ormulations in t&erapy6 rationale !or %evelopment an% (&at (e can e"pect !or t&e !uture* A%v Drug Deliv Rev 5@@4M 9<A4B68H4>* :;* Grau M') 0ayser ?) MJller RH* Nanosuspensions o! poorly solu#le %rugsRrepro%uci#iE lity o! small scale pro%uction* Int ' P&arm 5@@@M 4>;A5B64::H4:>* :<* MJller RH) Peters 0* Nanosuspensions !or t&e !ormulation o! poorly solu#le %rugs* I* Preparation #y a si/eEre%uction tec&niCue* Int ' P&arm 4>>=M 4;@A5B655>H58<* :=* MJller RH) +F&m +H ) Grau M'* NanosuspensionsRa !ormulation approac& !or poorly solu#le an% poorly #ioavaila#le %rugs* In6 ,ise D ) e%* Han%#oo$ o! P&armaceutical Controlle% Release* Ne( 3or$6 Marcel De$$er) 5@@@689:H8:<* :>* MJller RH) 0rause 0) Ma%er 0* Met&o% !or controlle% pro%uction o! ultra!ine microparE ticles an% nanoparticles) Application* ,? 5@@@EEP;:8:) 5@@4@@8;<@) 5@@4* ;@* +us&ra# -N) MJller RH* Drug nanocrystals6 Amp&otericin +Econtaining capsules !or oral %elivery* P&ila%elp&ia6 AAPS) 5@@9* ;4* ie%t$e S et al* In!luence o! &ig& pressure &omogenisation eCuipment on nano%ispersions c&aracteristics* Int ' P&arm 5@@@M 4>;A5B64=8H4=:* ;5* 0rause 0P et al* Heavy metal contamination o! nanosuspensions pro%uce% #y &ig&E pressure &omogenisation* Int ' P&arm 5@@@M 4>;A5B64;>H4<5* ;8* 0rause 0P) MJller RH* Pro%uction an% c&aracterisation o! &ig&ly concentrate% nanosusE pensions #y &ig& pressure &omogenisation* Int ' P&arm 5@@4M 549A4H5B654H59* ;9* 0ipp 'E,) 'osep& C&ung Ta$) Doty) Mar$ ') Re##ec$) C&ristine * Microprecipitation met&o% !or preparing su#micron suspensions* US ;)=;>);4<) 5@@4* ;:* MFsc&(it/er* Met&o% !or t&e pro%uction o! ultraE!ine su#micron suspensions* DE 4@ 5@@: @44 <=;*9) 5@@:* ;;* MJller RH) MFsc&(it/er '* ?ral %rug nanocrystal !omulations #ase% on nonEaCueous1 (aterEre%uce% &omogeni/ation tec&nology* In6 Annual meeting o! t&e Controlle% Release Society) Miami) 5@@:* ;<* MFsc&(it/er 'P) em$e A* Met&o% !or t&e gentle pro%uction o! ultra!ine particle suspenE sions) Germany* DE 4@ 5@@: @4< <<<*=) 5@@:* ;=* Hu ') 'o&nston 0P) ,illiams R? III* Spray !ree/ing into liCui% AS- B particle engineering tec&nology to en&ance %issolution o! poorly (ater solu#le %rugs6 organic solvent versus organic1aCueous coEsolvent systems* Eur ' P&arm Sci 5@@8M 5@A8B65>:H8@8* ;>* C&en W et al* Preparation o! cyclosporine as nanoparticles #y evaporative precipitation into aCueous solution* Int ' P&arm 5@@5M 595A4H5B68H49* <@* Grau M* Investigation o! %issolution velocity) saturation solu#ility an% sta#ility o! ultra!ine %rug suspensions* Dissertation* P&arma/eutisc&e Tec&nologie* +erlin6 -ree University o! +erlin) 5@@@* <4* Ste(art P') V&ao -3* Un%erstan%ing agglomeration o! in%omet&acin %uring t&e %issoluE tion o! micronise% in%omet&acin mi"tures t&roug& %issolution an% %eEagglomeration mo%eling approac&es* Eur ' P&arm +iop&arm 5@@:M :>A5B684:H858* <5* 0ir$o! N* Creation an% c&aracteri/ation o! Nanoparticles* In6 85n% annual meeting an% e"position o! t&e Controlle% Release Society) Miami) 5@@:* <8* MFsc&(it/er ') MJller RH* -rom t&e %rug nanocrystal to t&e !inal mucoa%&esive oral %osage !orm) 5@@9* <9* MFsc&(it/er ') MJller RH* Spray coate% pellets as carrier system !or mucoa%&esive %rug nanocrystals* Eur ' P&arm +iop&arm* Su#mitte%* <:* MFsc&(it/er ') MJller RH* Controlle% %rug %elivery system !or oral application o! %rug nanocrystals* In6 5@@9 AAPS annual meeting an% e"position) +altimore) MD) 5@@9* <;* +us&ra# N-) MJller RH* Nanocrystals o! poorly solu#le %rugs !or oral a%ministration* Ne(Drugs 5@@8M :65@H55* <<* +us&ra# -N) MJller RH* Drug nanocrystals !or oral %eliveryRcompoun%s #y spray %rying* P&ila%elp&ia6 AAPS) 5@@9* <=* MJller RH* Preparation o! a matri" materialRe"cipient compoun% containing a %rug* ,? 4>><EEP;=>8) ,? >=5::>@) 4>>=*

ipi%E+ase% Nanoparticulate Drug Delivery Systems

'un ,u an% Wiao#in V&ao
Division o! P&armaceutics) College o! P&armacy) T&e ?&io State University) Colum#us) ?&io) U*S*A*

Ro#ert '* ee
Division o! P&armaceutics) College o! P&armacy) NCI Compre&ensive Cancer Center) NS- Nanoscale Science Engineering Center !or A!!or%a#le Nanoengineering o! Polymeric +iome%ical Devices) T&e ?&io State University) Colum#us) ?&io) U*S*A*

INTR?DUCTI?N P&osp&olipi%s) upon &y%ration) spontaneously !orm #ilayer mem#rane vesicles AliposomesB or may act as sur!actants in !orming microE or nanoemulsions or soli%Hlipi% nanoparticles* P&osp&olipi%s) triglyceri%es) an% c&olesterol are t&e main ingre%ients o! liposomes an% lipi% nanoparticles* T&ey are natural components o! #iological mem#ranes an% lipoproteins an% are) t&ere!ore) presume% to #e &ig&ly #iocompati#le A4B* Drugs an% cellEtargeting ligan%s can #e incorporate% into t&ese structures #y encapsulation A!or &y%rop&ilic moleculesB) lipi%Ep&ase solu#ili/ation A!or lipop&ilic moleculesB) con.ugation to a lipi% anc&or Aas a lipi%E%erivati/e% proE %rugB) or electrostatic comple"ation A!or polyEanionic molecules suc& as nucleic aci%sB) %epen%ing on t&eir speci!ic p&ysicoc&emical properties* iposomes an% lipi% nanoparticles smaller t&an 8@@ nm are potentially suita#le !or systemic a%ministraE tion* In t&is c&apter) various aspects o! t&ese nanoparticulate systems are %iscusse% in t&e conte"t o! %rug %elivery*

IP?S?MES iposomes are p&osp&olipi% #ilayers (it& an entrappe% aCueous volume* ?n t&e #asis o! t&e num#er o! layers AlamellarityB an% %iameter) liposomes are classi!ie% into multilamellar vesicles AM 2s) %iameter \5@@ nmB) large unilamellar vesicles A%iameter 4@@H9@@ nmB) an% small unilamellar vesicles A%iameter c4@@ nmB* ?n t&e #asis o! sur!ace c&arge A/eta potentialB) t&ey are classi!ie% into cationic) neutral) an% anionic liposomes* In a%%ition) liposomes can #e %eli#erately engineere% to possess uniCue properties) suc& as long systemic circulation time) target cell speci!icity) pH an% re%uctive environmental sensitivity) an% temperature sensitivity* T&ese are ac&ieve% #y selecting t&e appropriate lipi% composition an% sur!ace mo%i!ication !or t&e liposomes*

METH?DS ?- IP?S?ME PREPARATI?N AND CHARACTERIVATI?N iposomes !orm spontaneously (&en p&osp&olipi%s are &y%rate%* A%%itional steps are o!ten necessary to mo%i!y t&e si/e %istri#ution an% lamellarity o! liposomes* Several met&o%s &ave #een esta#lis&e% !or liposome preparation #ase% on t&e scale
=>

>@

,u et al*

o! t&e preparation an% ot&er consi%erations) suc& as %rug encapsulation e!!iciency* T&e !irst step in liposome preparation is to %issolve lipi% ingre%ients in a suita#le solventM t&is is t&en !ollo(e% #y lipi% &y%ration an% particle si/e re%uction* -or e"ample) lipi%s are !irst %issolve% in c&loro!orm1met&anol an% %rie% into a t&in !ilm on a rotary evaporator an% are t&en &y%rate% in an aCueous #u!!er a#ove t&e p&aseEtransition temperature A5B* T&is process (ill result in t&e !ormation o! &eteroE geneous M 2s* T&e lamellarity o! t&e M 2s can #e re%uce% #y repeate% cycles o! !ree/ing an% t&a(ing* I! p&osp&olipi%s are %issolve% in a (aterEmisci#le solvent suc& as et&anol an% rapi%ly %ilute% into an aCueous #u!!er) liposomes (it& relative small particle si/es can #e generate% %irectly* T&is is t&en !ollo(e% #y removal o! t&e solvent !rom t&e liposome preparation an%1or !urt&er si/eEmo%i!ying steps A8B* Particle si/e can t&en #e re%uce% #y a num#er o! met&o%s) inclu%ing sonication) e"trusion) an% &omogeni/ation* Sonication) typically using an ultrasonic pro#e sonicator) can re%uce liposome particle si/e #y in%ucing cavitation A9B* E"trusion is per!orme% using a &ig&Epressure !ilter unit) suc& as t&e ipe"d e"tru%er #y Nort&ern ipi%s) Inc*) containing a trac$Eetc&e% polycar#onate mem#rane at a temperature a#ove t&e p&aseEtransition temperature o! t&e liposomal #ilayer A:B* T&e polycar#onE ate mem#rane &as straig&t t&roug& cylin%rical pores o! precise %iameters an% can (it&stan% pressure o! 8@@@ psi (it& proper support* iposomes e"tru%e% t&roug& t&e polycar#onate mem#ranes typically &ave narro( particle si/e %istri#ution* argeE scale pro%uction o! liposomes is possi#le using t&e e"trusion met&o% employing &ig&Esur!aceEarea e"trusion !ilter units* Anot&er potentially scala#le met&o% !or liposome particle si/e re%uction is &ig&Epressure &omogeni/ation usually at pressures a#ove :@@@ psi A5B* T&is met&o% can #e use% !or continuous pro%uction o! liposomes or nanoparticles at large scale* In t&e la#scale) liposomes can #e synt&esi/e% #y %eterE gent %ialysis in (&ic& lipi%s are !irst solu#ili/e% in a %ialy/a#le %etergent) suc& as octylglucosi%e an% t&en %ialy/e% against a #u!!er A;B* Alternatively) reverse%Ep&ase evaporation met&o% may #e use% to !orm a (aterEinEoil emulsion in a volatile organic solvent !ollo(e% #y p&ase inversion upon solvent removal* T&is met&o% (as %esigne% to ma"imi/e entrapment e!!iciency o! (aterEsolu#le agents A<B* Met&o%s suita#le !or %rug loa%ing into liposomes %epen% on t&e properties o! t&e %rug* ipop&ilic %rugs can #e co%issolve% (it& t&e lipi%s %uring liposome preE paration* Hy%rop&ilic %rugs can #e passively entrappe% into liposomes %uring liposome !ormation* Alternatively) %rugs can #e incorporate% into liposomes #y a pH gra%ientE%riven remote loa%ing proce%ure* -or e"ample) an in(ar%E%irecte% pH gra%ient coul% #e esta#lis&e% #y entrapment o! a lo( pH #u!!er Ae*g*) pH 9 so%ium citrateB or #y entrapping ammonium sul!ate !ollo(e% #y e"ternal #u!!er e"c&ange resulting in re%uction o! intraliposomal pH* A%%ition o! a (ea$ly #asic %rug) suc& as %o"oru#icin an% vincristine) results in nearECuantitative loa%ing o! t&e %rug into t&e liposomes %ue to intraliposomal protonation o! t&e %rug molecules an% comE ple"ation (it& entrappe% counterion A=)>B* -or polyelectrolytes suc& as DNA) loa%ing into liposomes can #e ac&ieve% #y electrostatic comple"ation (it& incorporation o! cationic lipi%s into t&e liposome composition A4@)44B* Structure o! DNA comple"es o! cationic liposomes may not #e similar to t&e structure o! typical liposomes an% is &ig&ly %epen%ent on lipi% &ea% group structure) cationicEtoEanionic c&arge ratio) an% $inetics o! comple" !ormation A45B* iposomes can #e puri!ie% #y a num#er o! met&o%s* At t&e la#scale) liposomes can #e puri!ie% #ase% on t&eir si/e #y &ig&Espee% centri!ugation) si/e e"clusion c&romatograp&y) or %ialysis A8B* At a larger scale) liposomes can #e puri!ie% #y tangential !lo( %ia!iltration A48B* iposomes can also #e lyop&ili/e% in t&e presence

ipi%E+ase% Nanoparticulate Drug Delivery Systems

>4

o! a cryoprotectant) typically a %isacc&ari%e suc& as sucrose) lactose) or tre&alose) (&ic& can prevent vesicle !usion an% particle si/e increase) alt&oug& signi!icant lea$age o! aCueous content may occur upon re&y%ration o! t&e liposomes A49B* A liposomal !ormulation can #e c&aracteri/e% #y a num#er o! esta#lis&e% met&E o%s* -irst) particle si/e %istri#ution can #e measure% #y %ynamic lig&t scattering) #y cryoE or negativeEstaining electron microscopy) or #y atomic !orce microscopy* Sur!ace electrical property o! liposomes can #e measure% #y /eta potential measurement* ?t&er use!ul analyses inclu%e colloi%al sta#ility an% rate o! %rug release in storage an% in plasma #y %ialysis) $inetics o! upta$e) an% internali/ation o! !luorescence la#ele% lipoE somes in culture% cells #y !luorescence microscopy an% !lo( cytometry* Cytoto"icity o! %rugEcarrying an% empty liposomes can #e stu%ie% in tissue culture* -urt&ermore) plasma clearance $inetics) tissue #io%istri#ution) to"icity) an% t&erapeutic e!!icacy o! %rugEcarrying liposomes can #e assesse% in appropriate animal mo%els*

IP?S?MES AS DRUG CARRIERS T&e application o! liposomes as a %rugE%elivery system &as #ecome more popular over t&e last %eca%es) #ecause o! t&eir #iocompati#ility an% versatility in carrying sysE temically a%ministere% %rugs suc& as c&emot&erapeutics an% anti#iotics (it& narro( t&erapeutic (in%o(s* A variety o! t&erapeutic agents &ave #een incorporate% into liposomes* Several &ave reac&e% clinical use* T&ese inclu%e liposomal %o"oru#icin A4:B ADo"ildB) %aunoru#icin A4;B ADauno"omedB) amp&otericin + A4<B AAmp&otecd) Am#isomed) an% A#elcetdB) cytara#ine A4=B ADepocytedB) an% vertepor!in A4>B A2isu%ynedB* Numerous liposomal !ormulations are in clinical trial) inclu%ing t&ose !or vincristine) allEtrans retinoic aci%) topotecan) an% cationic liposomeE#ase% t&erapeutic gene trans!er vectors* Many more are in preclinical evaluation inclu%ing liposomal !ormulations o! c&emot&erapeutics) neutron capture agents) oligonuE cleoti%es) plasmi% DNA) p&otosensiti/ers) anti#iotics) an% vaccines A5@B* +esi%es potential use in systemic gene %elivery) cationic liposomes are routinely use% as trans!ection reagents !or plasmi% DNA an% oligonucleoti%es in t&e la#oratory* iposomal %elivery o! anticancer %rugs &as #een s&o(n to greatly e"ten% t&eir systemic circulation time) re%uce to"icity #y lo(ering plasma !ree %rug conE centration) an% !acilitate pre!erential locali/ation o! %rugs in soli% tumors #ase% on increase% en%ot&elial permea#ility an% re%uce% lymp&atic %rainage) or en&ance% permea#ility an% retention AEPRB e!!ect A54H58B* -or e"ample) liposomal entrapment o! %o"oru#icin greatly re%uces its %oseElimiting car%ioto"icity* Clearance o! %rugs in a liposomal !ormulation is me%iate% #y p&agocytic cells o! t&e reticuloen%ot&elial system ARESB) primarily locate% in liver an% spleen* iposomal entrapment can protect %rugs suc& as nucleic aci%s !rom rapi% meta#olism #y plasma en/ymes A59B* In a%%ition) liposomal %elivery o! %rugs appears to me%iate #ypassing o! P glycoprotein APgpBErelate% multi%rug resistance in tumor cells* iposomes also present a plat!orm !or %elivery o! %rug com#inations* -or e"ample) ,ang et al* A5:B coencapsulate% %o"oru#icin an% verapamil Aa Pgp in&i#itorB into liposomes an% stu%ie% t&eir in vitro cytoto"icity* T&e result %emonstrate% e!!ective reversal o! mulE ti%rug resistance in %o"oru#icinEresistant cell lines*

-?RMU ATI?N STRATEGIES -?R IP?S?MES ipi% Composition !or Increase% Sta#ility In 2ivo As %rug permea#ility over t&e lipi% #ilayer is re%uce% in t&e QgelS state compare% to Q!lui%S state) sta#ility o! liposomal entrapment can #e ma"imi/e% #y selecting

>5

,u et al*

&ig&Ep&aseEtransition p&osp&olipi%s) !or e"ample) p&osp&ati%ylc&olines APCsB (it& long an% saturate% !atty acyl c&ains) suc& as %istearoyl PC an% &y%rogenate% soy PC) (&ic& remain in a gel state at p&ysiological temperature* A%%ition o! 8@ to :@ mol[ o! c&olesterol can !urt&er improve sta#ility o! t&e lipi% #ilayers #y !illing in gaps #et(een PC molecules* Having a tig&t #ilayer also re%uces insertion o! plasma proteins an% re%uces RES clearance o! t&e liposomes*

Sterically Sta#ili/e% iposomes RES clearance o! liposomes is me%iate% #y a%sorption o! plasma proteins to t&e #ilayer sur!ace* Incorporation o! 8 to 4@ mol[ o! polyet&ylene glycol APEGBE con.ugate% lipi%) suc& as monomet&o"yEPEG Amolecular (eig&t 5@@@BH%istearoyl p&osp&ati%ylet&anolamine AmPEG5@@@HDSPEB &as #een s&o(n to greatly e"ten% t&e circulation time o! liposomes #y provi%ing steric &in%rance on t&e #ilayer sur!ace A54)55B* PEGylate% liposomes e"&i#it circulation &al!Eli!e o! up to t(o %ays comE pare% to several &ours !or nonEPEGylate% liposomes* T&e prolongation in mean resi%ence time o! PEGylate% liposomes is %ue to slo(er clearance o! t&ese lipoE somes #y RES organs A5;B* T&is can) in a%%ition) increase EPR e!!ectEme%iate% tumor locali/ation an% antitumor t&erapeutic e!!icacy*

pHESensitive iposomes Alt&oug& &ig& sta#ility o! liposomes prior to reac&ing t&e cellular target is generally %esira#le) e!!icient release o! liposomal %rug in t&e target tissue an%1or cell is essenE tial !or its t&erapeutic activity* Environmentally sensitive liposomes are %esigne% to ta$e a%vantage o! t&e %i!!erences in t&e microenvironment o! a soli% tumor or insi%e t&e cell an% to un%ergo %esta#ili/ation on reac&ing t&eir target* pHEsensitive lipoE somes are %esigne% to %esta#ili/e at mil%ly aci%ic pH !oun% in soli% tumors an% in en%osomal compartments* T&ese are typically compose% o! %ioleoyl p&osp&ati%ylE et&anolamine AD?PEB) (&ic& &as a coneEs&ape% geometry t&at !avors transition !rom #ilayer to HII p&ase) an% a (ea$ly aci%ic amp&ip&ile) suc& as oleic aci% or c&olesteryl &emisuccinate) (&ic& sta#ili/es t&e #ilayer structure at neutral pH #ut not at mil%ly aci%ic pH A5<H5>B* pHEsensitive liposomes &ave #een s&o(n to #e muc& more e!!ective in !acilitating en%osomal release o! mem#raneEimpermea#le %rugs !rom internali/e% liposomes in cells* ?t&er non#ilayerE!avoring lipi%s) suc& as oleyl alco&ol an% %iolein) are also e!!ective in !orming pHEsensitive liposomes A8@)84B* Alternatively) a pHEsensitive oligopepti%e t&at un%ergoes coil to mE&eli" con!ormational transition) suc& as glutamic aci%EalanineEleucineEalanine AGA AB) in!luen/a !usion pepti%es) an% pHEresponsive polymer polyE5Eet&ylEacrylic aci% lin$e% to a lipi% anc&or) can also me%iate intracellular %isruption o! t&e en%osomal mem#rane A85H89B*

-usogenic an% En%osomolytic iposomes T&ese liposomes can #e constructe% #y reconstitution o! envelope proteins o! viruses into liposomes or encapsulation o! &emolysins !rom #acteria (it& varying %egrees o! pH %epen%ence A8:B* In a%%ition to pH) t&e re%ucing an% en/ymatic environment insi%e t&e en%osomal compartment can #e utili/e% to trigger t&e cleavage o! %isulE !i%e or en/ymeEsensitive lin$er t&at may #e incorporate% into a #ilayerEsta#ili/ing lipi% A8;)8<B* -or e"ample) gelonin) a type I plant to"in) (as coencapsulate% insi%e pHEsensitive liposomes (it& listeriolysin ? A ?B) t&e poreE!orming protein t&at me%iates escape o! t&e intracellular pat&ogen listeria monocytogens !rom t&e

ipi%E+ase% Nanoparticulate Drug Delivery Systems

>8

en%osome into t&e cytosol A8=B* Suc& a strategy resulte% in a %ramatic improvement on t&e cytoto"icity o! encapsulate% gelonin against t&e murine +4; melanoma cell line) over !ree gelonin or gelonin encapsulate% in nonE ?Econtaining pHEsensitive liposomes* In anot&er stu%y) Mastro#attista et al* A8:B &ave %emonstrate% t&at coencapsulation o! a pHE%epen%ent !usogenic pepti%e A%iIN-E<B an% %ip&t&eria to"in A c&ain ADTAB in nonEpHEsensitive immunoliposomes promotes e!!icient cytoE solic %elivery o! DTA*

TemperatureESensitive iposomes ocal release o! liposomal %rug can also #e triggere% #y &ypert&ermia #y a%opting a #ilayer composition (it& p&aseEtransition temperature slig&tly a#ove 8<]C) suc& as %ipalmitoyl p&osp&ati%ylc&oline or con.ugation to a t&ermosensitive polymer A8>)9@B* TemperatureEsensitive liposomes t&at s&o( p&ase transition at 9@]C can #e synt&esi/e% #y incorporating lipi%Econ.ugate% copolymers o! NEisopropylacrylE ami%e an% NEacryloylpyrroli%ine A94B* A t&ermosensitive liposome !ormulation entrapping %o"oru#icin AT&ermoDo"dB is currently un%ergoing clinical evaluation*

Cationic iposomes T&ese liposomes can !orm electrostatic comple"es (it& plasmi% DNA an% !acilitate gene trans!er A95B* A (i%e variety o! cationic lipi%s &ave #een synt&esi/e% inclu%ing t&ose (it& a monovalent &ea%group) suc& as 4)5E%ioleoylE8Etrimet&ylammoniumE propane) NEN5)8EA%ioleylo"yBpropylOEN)N)NEtrimet&ylammonium c&lori%e) an% 8EfENNEANP)NPE%imet&ylaminoet&ylBcar#amoylOEc&olesterol) an% t&ose (it& a multiE valent &ea%group) suc& as 5)8E%ioleylo"yENEN5AspermineEcar#o"ami%oBet&ylOEN)NE %imet&ylE4Epropanaminium tri!luoroacetate* D?PE is o!ten use% as a &elper lipi% in t&ese liposomes to en&ance t&eir !usogenicity A98B* Cationic liposomes (it& multivalent cationic lipi%s !orm particles (it& con%ense% structure (it& plasmi% DNA) (&ereas t&ose (it& monovalent cationic lipi%s &ave #een s&o(n to !orm e"ten%e% spag&ettiEli$e structures* Alt&oug& cationic liposomes e"&i#it e!!icient gene trans!er activity in tissue culture an% are currently commonly use% reagents !or in vitro trans!ection) only lo(Elevel trans!ection in select tissues) typically t&e lung) can #e o#taine% upon systemic a%ministration o! cationic liposome1DNA comE ple"es in murine mo%els A99)9:B* T&is mig&t #e %ue to t&e trapping o! t&e cationic comple"es in t&e capillary #loo% vessels in t&e lung) (&ic& is t&e !irstEpass organ encountere% #y intravenously a%ministere% liposome comple"es* In a%%ition to plasmi% DNA) cationic liposomes &ave #een use% in t&e %elivery o! antisense oligoE %eo"yri#onucleoti%es A?DNsB an% siRNA into cells* iposomes (it& a (ea$ly #asic cationic lipi%) suc& as 4)5E%ioleylE8E%imet&ylammoniumEpropane) can e!!iciently incorporate plasmi% DNA or ?DNs at aci%ic pH) (&ere t&e lipi%s are largely catE ionic an% return to a mostly neutral /eta potential at pH <*9) (&ere t&e lipi%s are mostly %eprotonate% A9;B* T&ese liposomes &ave longer circulation time t&an cationic liposomes carrying permanently c&arge% cationic lipi%s an% may #e use!ul !or systemic %elivery o! DNA to soli% tumors* Cationic liposome comple"es (it& plasmi% DNA) ?DNs) or siRNA can also activate t&e innate immune system an% may play a role in immunot&erapy A9<B* In a%%ition to nucleic aci% %elivery) cationic liposomes carrying c&emot&erapy agent paclita"el &ave #een s&o(n to pre!erenE tially target tumor en%ot&elium) suggesting a possi#le role !or t&ese liposomes in tumorEtargete% %rug %elivery* Sc&mittESo%y et al* A9=B s&o(e% t&at cationic

>9

,u et al*

liposomal paclita"el e"&i#its &ig& selectivity !or tumor en%ot&elium an% is &ig&ly e!!icacious in tumor gro(t& in&i#ition*

Targete% iposomes iposomes can #e targete% to speci!ic cell populations via incorporation o! a targetE ing moiety* T&e targeting ligan% can consist o! a lipi%Eanc&ore% anti#o%y or anti#o%y !ragment) trans!errin) !olate) or car#o&y%rate* Immunoliposomes are synt&esi/e% #y con.ugation o! t&e liposome to an anti#o%y Ne*g*) antiEHER5 A9>B) antitrans!errin receptor A:@):4B) antiECD5@ A:5B) an% antiECD4> A:8BO or an anti#o%y !ragment suc& as -a# A:9B an% sc-v A::B* HER5 is a receptor tyrosine $inase) a pro%uct o! t&e HER5 AcEer#+5B protoEoncogene) (&ic& &as #een s&o(n to play an important role in t&e %evelopment an% progression o! #reast an% ot&er cancers* Par$ et al* A:;):<B reporte% t&at antiEHER5 immunoliposomes) (it& encapsulate% %o"oru#icin) %isplaye% signi!icantly en&ance% t&erapeutic e!!icacy in !our %i!!erent #reast cancer "enogra!t mo%els (&en compare% to nontargete% liposomes) !ree %rug) or !ree anti#o%y* -or targeting PEGylate% liposomes) it is &elp!ul to incorporate a long PEGE#ase% lin$er #et(een t&e targeting ligan% an% t&e lipi% anc&or* Incorporation o! t&e targeting moiety can #e accomplis&e% %uring liposome !ormation #y %etergent %ialysis) or postliposome !ormation #y con.ugation to reactive lipi%s or postinsertion o! ligan%s !rom micelles o! lipi%E%erivati/e% anti#o%ies A:=):>B* T&e last met&o% seems &ig&ly promising !or !uture clinical %evelopment* In a%%ition to anti#o%ies) ot&er targeting moieties suc& as trans!errin A:4);@B an% !olic aci% A;4H;8B &ave #een !reCuently evaluate% in targeting liposomes to tumor cells t&at overe"press t&e trans!errin or t&e !olate receptor* Targete% liposomes are speci!ically ta$en up #y target cells an% &ave #een s&o(n to #e &ig&ly e!!icient in %rug %elivery an% to #ypass multi%rug resistance in cell lines A;9B* Tumor locali/ation o! targete% liposomes o!ten %oes not greatly e"cee% t&at o! nontargete% liposomes #ecause #io%istri#ution o! liposomes is %ictate% #y vascular permea#ility an% t&e associate% EPR e!!ect* -urt&ermore) t&ere is concern t&at liposomes) %ue to t&eir si/e) cannot penetrate into soli% tumors) (&ic& typically &ave &ig& interstitial pressure* Nevert&eless) targete% liposomes suc& as antiEHER5 immunoliposomes an% !olate receptorEtargete% liposomes &ave s&o(n improve% antitumor e!!icacy in murine mo%els over nontargete% control liposomes A:4);8B* eu$emias) (&ic& &ave greater accessi#ility !rom circulation) are also potentially goo% targets !or targete% liposomes) as suggeste% #y recent reports* T&e a%vantage o! using immunoliposome !or MA#E#ase% targete% t&erapy in leu$eE mia e"ists in6 AiB liposomes containing &ig& payloa% o! cytoto"ic agents &ave unreE stricte% access to malignant cells an% AiiB application o! a c&emot&erapeutic agent t&at &as alrea%y s&o(n clinical e!!icacy can potentially #ring synergistic e!!ect (it& t&erapeutic MA#) #ase% on a %i!!erent $illing mec&anism* Pan et al* A;:B stu%ie% t&e t&erapeutic e!!icacy o! !olate receptorEtargete% liposomes in com#ination (it& upregulation o! -REf e"pression in an ascitic "enogra!t mo%el o! acute myelogenous leu$emia using allEtrans retinoic aci%* In vivo antitumor activity o! !olate receptorE targete% liposomal %aunoru#icin in t&e 454@E'- ascitic murine leu$emia mo%el &as also #een reporte% #y Pan an% ee A;;B* T&e result s&o(e% t&at !olate receptorE targete% liposomes coul% e!!ectively target receptorEpositive leu$emia cells in vivo*

ipi% Nanoparticles ipi% nanoparticles are nanoscale sp&erical particles compose% o! lipi%s (it& a lipi%ic core* T&ese are suita#le !or %elivery o! lipop&ilic t&erapeutic agents* T&e

ipi%E+ase% Nanoparticulate Drug Delivery Systems

>:

molecule o! interest can #e !ormulate% to lipi% nanoparticle matri" t&roug& lipi%E p&ase %issolution* T&ey &ave consi%era#le utility as controlle% %elivery system !or %rugs an% vaccines* ipi% nanoparticles can #e synt&esi/e% #y com#ining an oil p&ase Ae*g*) trioleinB (it& p&osp&olipi%s as emulsi!iers* T&e oily core can #e use% to incorporate lipi%Esolu#le %rugs suc& as paclita"el A;<);=B) &ematoporp&yrin A;>B) an% lipi%Econ.ugate% pro%rugs A<@B* T&ey can #e synt&esi/e% #y similar met&o%s as t&ose use% in liposomes) suc& as &ig&Epressure &omogeni/ation* i$e liposomes) t&ese particles are cleare% #y RES) locali/e% in tumors via EPR e!!ect) an% can #e ma%e long circulating #y incorporation o! PEGylate% lipi%*

C?NC USI?NS ipi%E#ase% nanoparticulates are versatile %rug carriers (it& signi!icant potential !or clinical applications* Tec&nological a%vances suc& as intro%uction o! remote loa%ing met&o%s) PEGylate% liposomes) an% targete% liposomes provi%e% a%%itional a%vanE tages* In a%%ition to mo%ulating to"icity) p&armaco$inetics) an% #io%istri#ution) liposomal %elivery &as s&o(n promise as a mec&anism to overcome multi%rug resisE tance* -urt&ermore) liposomes are promising %elivery ve&icles !or novel t&erapeutic agents suc& as siRNA an% %rugs t&at lac$ aCueous solu#ility* Particularly promising !or !uture %evelopment are targete% liposomes) (&ic& &ave yet to #e t&oroug&ly evaluate% in clinical stu%ies* Given current tren%s) lipi%E#ase% nanoparticulates are li$ely to &ave an e"pan%ing role in %rug %elivery in t&e clinical setting*

RE-ERENCES
4* Allen TM* iposomal %rug !ormulations6 rationale !or %evelopment an% (&at (e can e"pect !or t&e !uture* Drugs 4>>=M :;6<9<* 5* C&ater.ee S) +aner.ee D0* Preparation) isolation) an% c&aracteri/ation o! liposomes conE taining natural an% synt&etic lipi%s* In6 +asu SC) +asu M) e%s* iposome Met&o%s an% Protocols* Toto(a6 Humana Press) 5@@5 Ac&apter 4B* 8* Martin -'* P&armaceutical manu!acturing o! liposomes* In6 Tyle P) e%* Speciali/e% Drug Delivery System Manu!acturing an% Pro%uction Tec&nology* Ne( 3or$6 Marcel De$$er) 4>>@ Ac&apter ;B* 9* Huang H* Stu%ies on p&osp&ati%ylc&oline vesicles6 !ormulation an% p&ysical c&aracterE istics* +ioc&emistry 4>;>M =6899* :* Hope M') +ally M+) ,e## G) Cullis PR* Pro%uction o! large unilamellar vesicles #y a rapi% e"trusion proce%ure c&aracteri/ation o! si/e %istri#ution) trappe% volume) an% a#ility to maintain a mem#rane potential* +ioc&im +iop&ys Acta 4>=:M ::6=45* ;* Hauser H) Grains N* Spontaneous vesiculation o! p&osp&olipi%s6 a simple an% Cuic$ met&o% o! !orming unilamellar vesicles* Proc Natl Aca% Sci USA 4>=5M <>64;=8* <* S/o$a -) Papa&a%.opoulos D* Proce%ure !or preparation o! liposomes (it& large internal aCueous space an% &ig& capture #y reverseEp&ase evaporation* Proc Natl Aca% Sci USA 4><=M <:694>9* =* +olotin EM) Co&en R) +ar 0) et al* Ammonium sul!ate gra%ients !or e!!icient an% sta#le remote loa%ing o! amp&ipat&ic (ea$ #ases into liposomes an% ligan%oliposomes* ' iposome Res 4>>9M 969::* >* +oman N ) Masin D) Mayer D) Cullis PR) +ally M+* iposomal vincristine (&ic& e"&i#E its increase% %rug retention an% increase% circulation longevity cures mice #earing P8== tumors* Cancer Res 4>>9M :965=8@* 4@* Gao W) Huang * Cationic liposomeEme%iate% gene trans!er* Gene T&er 4>>:M 56<4@* 44* Meyer ?) 0irpotin D) Hong 0) Stern#erg +) Par$ ',) ,oo%le MC* Cationic liposomes coate% (it& polyet&ylene glycol as carrier !or oligonucleoti%es* ' +iol C&em 4>>=M 5<864:;54*

>;
45* ,ro#ell I) Collins D* -usion o! cationic liposomes (it& mammalian cells occurs a!ter en%ocytosis* +ioc&im +iop&ys Acta 4>>:M 458:65>;* 48* Martin -') Morano '0* US Patent 9)<:5)95:) 4>==* 49* T&omas DM) Nancy +* yop&ili/ation o! liposomes* In6 An%re( S') e%* iposomes Rational Design* Ne( 3or$6 Marcel De$$er) 4>>>65;4* 4:* Tar%i PG) +oman N ) Cullis PR* iposomal %o"oru#icin* ' Drug Target 4>>;M 9645>* 4;* +ellott R) Auvrignon A) e#lanc T) et al* P&armaco$inetics o! liposomal %aunoru#icin ADaunoWomeB %uring a p&ase I to II stu%y in c&il%ren (it& relapse% acute lymp&o#lastic leu$aemia* Cancer C&emot&er P&armacol 5@@4M 9<64:* 4<* Gregoria%is G) -lorence AT* iposomes in %rug %elivery6 clinical) %iagnostic an% op&t&alE mic potential* Drugs 4>>8M 9:64:* 4=* Hama%a A) 0a(aguc&i T) Na$ano M* Clinical p&armaco$inetics o! cytara#ine !ormulations* Clin P&armaco$inet 5@@5M 946<@:* 4>* 0onan 3N) Gurny R) Allemann E* State o! art in t&e %elivery o! p&otosensiti/ers !or p&oto%ynamic t&erapy* ' P&otoc&em P&oto#iol + 5@@5M ;;6=>* 5@* -lorence AT* iposomes as p&armaceuticals* In6 An%re( S') e%* iposomes Rational Design6 Intro%uction* Ne( 3or$6 Marcel De$$er) 4>>>* 54* Ga#i/on A) Papa&a%.opoulos D* iposome !ormulations (it& prolonge% circulation time in #loo% an% en&ance% upta$e #y tumors* Proc Natl Aca% Sci USA 4>==M =:6;>9>* 55* 0li#anov A ) Maruyama 0) Torc&ilin 2P) Huang * Amp&ipat&ic polyet&yleneglycols e!!ectively prolong t&e circulation time o! liposomes* -E+S ett 4>>@M 5;=658:* 58* Uster PS) Allen TM) Daniel +E) Men%e/ C') Ne(man MS) V&u GV* Insertion o! polyAet&ylene glycolB %erivati/e% p&osp&olipi%s into preE!orme% liposomes results in prolonge% in vivo circulation time* -E+S ett 4>>;M 8=;6598* 59* ee R') ,ang S) Tur$ M') o( PS* T&e e!!ects o! pH an% intraliposomal #u!!er strengt& on t&e rate o! liposome content release an% intracellular %rug %elivery* +iosci Rep 4>>=M 4=6;>* 5:* ,ang ') Go& +) u , ) et al* In vitro cytoto"icity o! stealt& liposomes coEencapsulating %o"oru#icin an% verapamil on %o"oru#icinEresistant tumor cells* +iol P&arm +ull 5@@:M 5=6=55* 5;* +ra%ley A') Devine D2) Ansell SM) 'an/en ') +roo$s DE* In&i#ition o! liposomeEin%uce% complement activation #y incorporate% polyAet&ylene glycolBElipi%s* Arc& +ioc&em +iop&ys 4>>=M 8:<64=:* 5<* Simtes S) Slepus&$in 2) Du/gunes N) Pe%roso %e ima MC* ?n t&e mec&anisms o! interE nali/ation an% intracellular %elivery me%iate% #y pHEsensitive liposomes* +ioc&im +iop&ys Acta 5@@4M 4:4:658* 5=* Slepus&$in 2A) Simoes S) Da/in P) et al* Sterically sta#ili/e% pHEsensitive liposomes6 intracellular %elivery o! aCueous contents an% prolonge% circulation in vivo* ' +iol C&em 4>><M 5<5658=5* 5>* DJ/gJnes N) Strau#inger RM) +al%(in PA) -rien% DS) Papa&a%.opoulos D* ProtonE in%uce% !usion o! oleic aci%Ep&osp&ati%ylet&anolamine liposomes* +ioc&emistry 4>=:M 5968@>4* 8@* Guo ,) Gosselin MA) ee R'* C&aracteri/ation o! a novel %ioleinE#ase% PDII vector !or gene %elivery* ' Control Release 5@@5M =86454* 84* Su%imac$ '') Guo ,) T.ar$s ,) ee R'* A novel pHEsensitive liposome !ormulation conE taining oleyl alco&ol* +ioc&im +iop&ys Acta 5@@5M 4:9;684* 85* C&ung 'C) Gross D') T&omas ' ) Tirrell DA) ?psa&lE?ng R* pHEsensitive) cationEselecE tive c&annels !orme% #y a simple synt&etic polyelectrolyte in arti!icial #ilayer memE #ranes* Macromolecules 4>>;M 5>69;8;* 88* C&en T) C&oi S) Einstein S) 0lippenstein MA) Sc&errer P) Cullis PR* ProtonEin%uce% perE mea#ility an% !usion o! large unilamellar vesicles #y covalently con.ugate% polyA5Eet&ylE acrylic aci%B* ' iposome Res 4>>>M >68=<* 89* i ,') Nicol -) S/o$a -C 'r* GA A6 a %esigne% synt&etic pHEresponsive amp&ipaE t&ic pepti%e (it& applications in %rug an% gene %elivery* A%v Drug Deliv Rev 5@@9M :;6>;<* 8:* Mastro#attista E) 0oning GA) van +loois ) -ilipe AS) 'is$oot ,) Storm G* -unctional c&aracteri/ation o! an en%osomeE%isruptive pepti%e an% its application in cytosolic %elivery o! immunoliposomeEentrappe% proteins* ' +iol C&em 5@@5M 5<<65<48:*

,u et al*

ipi%E+ase% Nanoparticulate Drug Delivery Systems

><

8;* Simoes S) Slepus&$in 2) Gaspar R) %e ima MC) Du/gunes N* Gene %elivery #y negative c&arge% ternary comple"es o! DNA) cationic liposomes) an% trans!errin or !usigenic pepti%es* Gene T&er 4>>=M :6>::* 8<* Aronso&n AI) Hug&es 'A* Nuclear locali/ation signal pepti%es en&ance cationic liposomeE me%iate% gene t&erapy* ' Drug Target 4>>=M :64;8* 8=* Provo%a C') Stier EM) ee 0D* Tumor cell $illing ena#le% #y listeriolysin ?EliposomeE me%iate% %elivery o! t&e protein to"in gelonin* ' +iol C&em 5@@8M 5<=68:4@5* 8>* Nee%&am D) De(&irst M,* T&e %evelopment an% testing o! a ne( temperatureEsensitive %rug %elivery system !or t&e treatment o! soli% tumors* A%v Drug Deliv Rev 5@@4M :865=:* 9@* 0ong G) De(&irst M,* Revie( &ypert&ermia an% liposomes* Int ' Hypert&er 4>>>M 4:689:* 94* 0ono 0) Na$ai R) Morimoto 0) Ta$agis&i T* T&ermosensitive polymerEmo%i!ie% liposomes t&at release contents aroun% p&ysiological temperature* +ioc&im +iop&ys Acta 4>>>M 494;658>* 95* +rig&am 0 ) Meyric$ +) C&ristman +) Magnuson M) 0ing G) +erry C 'r* In vivo transE !ection o! murine lungs (it& a !unctioning pro$aryotic gene using a liposome ve&icle* Am ' Me% Sci 4>=>M 5>=65<=* 98* San%rine A ) %e ei. -) Hoe$stra D) Molema G* In vivo c&aracteristics o! cationic liposomes as %elivery vectors !or gene t&erapy* P&arm Res 5@@5M 4>64::>* 99* V&u N) iggitt D) iu 3) De#s R* Systemic gene e"pression a!ter intravenous DNA %elivery into a%ult mice* Science 4>>8M 5>=65<=* 9:* Mc ean ',) -o" EA) +alu$ P) et al* ?rganEspeci!ic en%ot&elial cell upta$e o! cationic liposomeHDNA comple"es in mice* Am ' P&ysiol 4>><M 5<86H8=<HH99@* 9;* Re%%y 'A) Dean D) 0enne%y MD) o( PS* ?ptimi/ation o! !olateEcon.ugate% liposomal vectors !or !olateEreceptorEme%iate% gene t&erapy* ' P&arm Sci 4>>>M ==64445* 9<* Siou% M) Sorensen DR* Cationic liposomeEme%iate% %elivery o! siRNAs in a%ult mice* +ioc&em +iop&ys Res Commun 5@@8M 8456455@* 9=* Sc&mittESo%y M) Striet& S) 0rasnici S) Sauer +* Neovascular targeting t&erapy6 paclita"el encapsulate% in cationic liposomes improves antitumoral e!!icacy* Clin Cancer Res 5@@8M >6588:* 9>* +aselga ') Norton ) Al#anell ') 0im 3M) Men%elso&n '* Recom#inant &umani/e% antiEHER5 anti#o%y AHerceptinB en&ances t&e antitumor activity o! paclita"el an% %o"oru#icin against HER51neu overe"pressing &uman #reast cancer "enoE gra!ts* ' Cancer Res 4>>=M :=65=5:* :@* i H) 7ian V* Trans!errin1trans!errin receptorEme%iate% %rug %elivery* Me% Res Rev 5@@5M 55655:* :4* Is&i%a ?) Maruyama 0) Tana&as&i H) I(atsuru M* iposomes #earing polyet&yleneglyE colEcouple% trans!errin (it& intracellular targeting property to t&e soli% tumors in vivo* P&arm Res 5@@4M 4=64@95* :5* Sapra P) Allen TM* Internali/ing anti#o%ies are necessary !or improve% t&erapeutic e!!iE cacy o! anti#o%yEtargete% liposomal %rugs* Cancer Res 5@@5M ;56<4>@* :8* Is&i%a T) 0irc&meier M') Moase EH) Valips$y S) Allen TM* Targete% %elivery an% trigE gere% release o! liposomal %o"oru#icin en&ances cytoto"icity against &uman + lymp&oma cells* +ioc&im +iop&ys Acta 5@@4M 4:4:6499* :9* Pastorino -) +rignole C) Marimpietri D) et al* Do"oru#icinEloa%e% -a#u !ragments o! antiE%isialogangliosi%e immunoliposomes selectively in&i#it t&e gro(t& an% %issemination o! &uman neuro#lastoma in nu%e mice* Cancer Res 5@@8M ;86=;* ::* Marty C) ?%ermatt +) Sc&ott H) et al* Cytoto"ic targeting o! -> teratocarcinoma tumours (it& antiEEDE+ !i#ronectin sc-v anti#o%y mo%i!ie% liposomes* +r ' Cancer 5@@5M =<64@;* :;* Par$ ',) Hong 0) 0irpotin D+) Col#ern G* AntiEHER5 immunoliposomes6 en&ance% e!!icacy attri#uta#le to targete% %elivery* Clin Cancer Res 5@@5M =644<5* :<* Par$ ',) 0irpotin D+) Hong 0) et al* Tumor targeting using antiE&er5 immunolipoE somes* ' Control Release 5@@4M <96>:* :=* Sc&i!!elers RM) 0oning GA) ten Hagen T ) et al* AntiEtumor e!!icacy o! tumor vascuE latureEtargete% liposomal %o"oru#icin* ' Control Release 5@@8M >4644:*

>=
:>* Hoo% 'D) +e%nars$i) M-) Ricar%o G) et al* Tumor regression #y targete% gene %elivery to t&e neovasculature* Science 5@@5M 5>;659@9* ;@* Iinuma H) Maruyama 0) ?$inaga 0) et al* Intracellular targeting t&erapy o! cisplatinE encapsulate% trans!errinEpolyet&ylene glycol liposome on peritoneal %issemination o! gastric cancer* Int ' Cancer 5@@5M >>648@* ;4* Su%imac$ ') ee R'* Drug targeting via t&e !olate receptor* A%v Drug Deliv Rev 5@@@M 94649<* ;5* V&ao W) ee R'* TumorEselective targete% %elivery o! genes an% antisense oligo%eo"yriE #onucleoti%es via t&e !olate receptor* A%v Drug Deliv Rev 5@@9M :;644>8* ;8* Pan W7) ,ang H) ee R'* Antitumor activity o! !olate receptorEtargete% liposomal %o"oE ru#icin in a 0+ oral carcinoma murine "enogra!t mo%el* P&arm Res 5@@8M 5@694<* ;9* Goren D) Horo(it/ AT) T/emac& D) Tars&is& M) Valips$y S) Ga#i/on A* Nuclear %elivery o! %o"oru#icin via !olateEtargete% liposomes (it& #ypass o! multi%rugE resistance e!!lu" pump* Clin Cancer Res 5@@@M ;64>9>* ;:* Pan W7) V&eng W) S&i G) ,ang H) Ratnam M) ee R'* A strategy !or t&e treatment o! acute myelogenous leu$emia #ase% on !olate receptor typeEf targete% liposomal %o"oruE #icin com#ine% (it& receptor in%uction using allEtransEretinoic aci%* +loo% 5@@5M 4@@6:>9* ;;* Pan W7) ee R'* In vivo antitumor activity o! !olate receptorEtargete% liposomal %aunoE ru#icin in a murine leu$emia mo%el* Anticancer Res 5@@:M 5:6898* ;<* Mo 3) im * Nanoparticles o! polyAlacti%eB1vitamin E TPGS copolymer !or cancer c&emot&erapy6 synt&esis) !ormulation) c&aracteri/ation an% in vitro %rug release* ' Control Release 5@@:M 4@<68@* ;=* 3e& T0) u V) ,ient.es MG) Au ' ES* -ormulating paclita"el in nanoparticles alters its %isposition* P&arm Res 5@@:M 556=;<* ;>* Stevens P') Se$i%o M) ee R'* Synt&esis an% evaluation o! a &ematoporp&yrin %erivative in a !olate receptorEtargete% soli%Hlipi% nanoparticle !ormulation* Anticancer Res 5@@9M 5964;4* <@* Stevens P') Se$i%o M) ee R'* A !olate receptorEtargete% lipi% nanoparticle !ormulation !or a lipop&ilic paclita"el pro%rug* P&arm Res 5@@9M 54654:8*

,u et al*

<

Nanoengineering o! Drug Delivery Systems

As&(at& 'ayagopal an% 2* Prasa% S&astri
+iomaterials) Drug Delivery) an% Tissue Engineering a#oratory) Department o! +iome%ical Engineering) 2an%er#ilt University) Nas&ville) Tennessee) U*S*A*

INTR?DUCTI?N C&allenges in %rug %elivery inclu%e t&e attainment o! tuna#le release pro!iles) %rug solu#ility an% structural sta#ility) #iocompati#ility) an% t&e con!inement o! t&eraE peutic action to %isease% sites* Increase% attention &as #een %irecte% to(ar% t&e nanoscale manipulation or nanoengineering o! %rugE%elivery systems ADDSsB !or con!erring uniCue properties on t&e %rug or %rug ve&icle o!ten not ac&ieva#le #y eit&er conventionalE Ae*g*) !reeE!ormB or microscaleE%rug carriers* Nanoengineering may #e ac&ieve% at various levels %uring !ormulation o! a %rug (it&in a carrier* E"amples inclu%e mo%ulation o! t&e %rug environment at t&e molecular level) alterE ation o! t&e p&ysicoc&emical c&aracteristics o! t&e %rug Ae*g*) solu#ility an% strucE tural integrityB) control o! %rugE%i!!usive mo#ility) an% mo%i!ication o! t&e #ul$ an% sur!ace c&emistry o! t&e %rug nanocarrier* T&ese approac&es &ave potential in t&e ac&ievement o! !avora#le t&erapeutic en%points) suc& as en&ance% %rug e!!icacy !or prolonge% intervals) minimal %rug to"icity) re%uce% %osage an% cost #ur%en) an% improve% patient compliance* In t&is c&apter) (e %iscuss recent signi!icant a%vances in DDSs in (&ic& nanoengineering is t&e ena#ling tec&nology !or suc& o#.ectives) (it& an emp&asis on t&e application o! nanoengineering principles to t&e %esign o! controlle%E an% triggere%Erelease DDSs) en&ancement o! %rug activity an% sta#ility) an% smart tissueEtargete% t&erapies* Speci!ic attention is given to t&e uniCue e!!ects o! nanoscale mo%i!ications on critical %rugE%elivery parameters* ,e evaluate t&e promise o! suc& approac&es in %rugE%elivery tec&nology) an% %iscuss !uture consi%E erations relevant to t&eir clinical implementation*

C?NTR?

EDE AND TRIGGEREDERE EASE S3STEMS

To %evelop suita#le t&erapies !or a %iverse range o! %iseases) a &ost o! natural an%1 or synt&etic #iomaterials &ave #een selecte% #ase% on t&eir p&ysicoc&emical c&aracE teristics (&ic& ena#le t&e %evelopment o! sustaine%E an% triggere%Erelease DDSs* Sustaine%Erelease systems are a%ministere% !or t&e maintenance o! %rug concentraE tion (it&in optimal t&erapeutic (in%o(s over e"ten%e% perio%s ranging !rom &ours to mont&s* In ot&er cases) !or instance (&ere instant %rug activity is reCuire%) DDSs can #e tune% !or pulsatile release in response to varying p&ysiological stimuli or e"ternal triggers) suc& as aci%ic pH an% acoustic pulses* Nanoscale mo%ulation o! %rugEtransport c&aracteristics) (it& or (it&out entrapment (it&in a nanocarrier) can #e ac&ieve% #y !ormulation (it& novel %rugEcomple"ation agents !or en&anceE ment o! #ot& controlle%E an% triggere%Erelease applications*

Polymeric Drug Delivery Systems !or Sustaine% an% Triggere% Release T&e engineering o! %rug carriers to #ear one or more polymeric agents) (it& eac& #loc$ &aving a speci!ic role in t&e solu#ility an% %egra%ative suscepti#ility o!
>>

4@@

'ayagopal an% S&astri

t&e resulting ve&icle) allo(s !or nanoscale %esign o! controlle%Erelease DDSs* +io%egra%a#le polymerE#ase% DDSs !eature la#ile groups) t&e presence o! (&ic& can #e tune% to control t&e e"tent o! %egra%ation an% &ence %rug release over time* T&e (ellEc&aracteri/e% an% #iocompati#le polyA%)lElacti%eEcoEglycoli%eB AP GAB %rug carriers are an e"ample o! a system (&ic& un%ergoes #ul$ erosion #y &y%rolysis) ena#ling its utili/ation in controlle%Erelease applications* T&e P GA #loc$ copolyE mer is compose% o! polyAlactic aci%B an% polyAglycolic aci%B) t&e !ormer o! (&ic& is slo(er to %egra%e %ue to increase% crystallinity an% steric &in%rance to scission #y (ater* +ot& are sa!ely eliminate% #y t&e #o%y* 2arying t&e relative composition o! t&e t(o #loc$s on a P GA nanoparticle sur!ace provi%es a means o! tailoring %rugE release rates via mo%ulation o! t&e &y%rop&ilicHlipop&ilic #alance* P GA can also #e incorporate% into nanoparticulate DDSs !or sustaine% intracellular %rug %elivery* T&is is a #ene!icial conseCuence o! t(o events6 !irst) t&e nonspeci!ic en%ocytic internali/ation o! P GA nanoparticles into su#cellular compartments an% secon%) t&e escape o! P GA nanoparticles into t&e cytoplasm ma%e possi#le #y sur!ace cationi/ation (it&in aci%ic en%olysosomal compartments A4B* T&ese !eatures sugE gest t&e versatility o! P GA nanoparticles as intracellular sustaine%Erelease %epots !or gene or %rug %elivery) (&ic& #ypass previous c&allenges suc& as nucleic aci% %egra%ation in t&e cytoplasm) or %rug e!!lu" pumps (&ic& can #e a #arrier to passive %rug %i!!usion* A more recent DDS utili/ing polyesterE#ase% &y%rolytic %egra%ation &as #een reporte% #y A&me% an% Disc&er A5B* In t&is application) an amp&ipat&ic polymersome compose% o! a (aterEsolu#le polyet&ylene glycol APEGB an% &y%rolytically la#ile polylactic aci% or polyAcaprolactoneB #loc$ is synt&esi/e% #y sel!Eassem#ly an% can #e utili/e% to encapsulate &y%rop&o#ic or &y%rop&ilic t&erapeutics) or imaging agents* Release is tuna#le !rom &ours to (ee$s #y a%.ustE ment o! t&e &y%rop&ilicHlipop&ilic #alance) (&ic& controls &y%rolyticE%epen%ent poration o! t&e mem#rane* Alt&oug& encapsulation e!!iciencies are similar to (ellEc&aracteri/e% %rugE%elivery carriers suc& as liposomes) polymersomes e"&i#it en&ance% mec&anical strengt& %ue to increase% mem#rane t&ic$ness an% e"ten%e% circulation &al!Eli!e) suggesting t&e utility o! t&is carrier !or applications reCuiring prolonge% circulation in vivo (it& a resistance to %esta#ili/ation mec&anisms* In a !un%amentally %i!!erent approac&) t&erapeutics can #e %irectly incorporate% into a polymer #ac$#one Atra%e name PolymerDrug) Polymeri" Corp*B A8B* A%.ustment o! t&e p&ysical properties o! t&e resulting polymer o!!ers a p&armaco$inetic tuning mec&anism) an% #io%egra%ation pro%ucts are inert* T&is tec&nology o!!ers a plat!orm !or t&e lo(Eris$ incorporation o! t&erapeutics (it& $no(n sa!ety pro!iles t&at can #e implemente% in implanta#le or %egra%a#le DDSs* Polymeric DDSs &ave also #een applie% as a% &oc triggere%Erelease systems* ?ne route to t&is goal is t&e #len%ing o! polymers to create polymer c&ain composiE tions (&ic& are more suscepti#le to rapi% %egra%ation* +y t&is approac&) sustaine%E release polymers can #e mo%i!ie% !or rapi% release o! encapsulate% contents (&en nee%e%* -or e"ample) alt&oug& P GA is a (ellEc&aracteri/e% copolymer !or #ioE %egra%a#le sustaine%Erelease systems) its intracellular release pro!ile may not #e rapi% or e!!icient enoug& !or some geneE%elivery applications* ittle et al* a%%resse% t&is c&allenge #y co#len%ing P GA (it& a pHEsensitive polyA fEamino esterB (&ic& is insolu#le at p&ysiologic pH) (it& a solu#ility transition occurring (it&in t&e aci%ic pH range o! lysosomes A9B* Ta$ing a%vantage o! t&e rapi% solu#ility o! t&e polymer #elo( pH ;*:) polyA fEamino esterB #len%s (it& P GA in microparticleE containing genetic vaccines rapi%ly release% t&eir contents (it&in %en%ritic cells A:B) an% (ere also note% to &ave &ig&er plasmi% DNAEloa%ing e!!iciencies A;B* T&e same

Nanoengineering o! Drug Delivery Systems

4@4

polymer (as also incorporate% (it&in polyAet&ylene o"i%eB APE?BEengineere% nanoparticles !or t&e en&ance%) pHEselective %elivery o! Ta"ol (it&in tumors A<B* In anot&er triggere%Erelease strategy) micelles consisting o! a &y%rop&ilic PE? an% a &y%rop&o#ic polypropylene o"i%e APP?B in a tri#loc$ copolymer !ormulation APE?HPP?HPE?B (ere o#serve% to rapi%ly release %o"oru#icin an% ru#o"yl upon nanoparticle pertur#ation in%uce% #y ultrasonic cavitation A=B* In a%%ition) upon removal o! t&e ultrasonic pulse) release% %rug (as o#serve% to #ecome reencapsuE late% (it&in micelles) an% intracellular presence o! 4@[ Pluronic micelles e"pe%ite% t&e sealing o! ultrasoun%Ein%uce% cell mem#rane poration) resulting in minimal %rug e!!lu" A>)4@B* Toget&er) t&ese %ata in%icate t&at Pluronic micelles can tig&tly #ear t&eir cargoes Ain t&is case) %ue to PP?H&y%rop&o#ic interactions (it& %rugB (it& a spatially an% temporally controlle% acousticErelease mec&anism* Ta$ing a%vantage o! reencapsulation upon removal o! acoustic pulses) t&erapeutic action can #e con!ine% solely (it&in t&e sonop&oretic range) an% %ue to t&e micelleEin%uce% mem#rane QsealingS e!!ect) (&ic& increases intracellular %rug retention) a lo(er %osage may #e use%* T&ese !in%ings rein!orce t&e concept t&at nanoscale %elegation o! polymer #loc$ roles in %rug release) solu#ility o! t&e nanocarrier) an% interactions (it& t&e surroun%ing environment an% e"ternal stimuli can #e &arnesse% !or %iverse an% po(er!ul %rugE%elivery approac&es*

ipi%E+ase% Drug Delivery Systems !or Sustaine% an% Triggere% Release Controlle%Erelease DDSs &ave also #een !ormulate% using t&e uniCue properties con!erre% #y lipi%E#ase% materials* T&e !lui%ity o! lipi% %omains on t&e nanoscale is a parameter (&ic&) (&en tune%) allo(s !or a mec&anism controlling (ater in!lu"* Palm oil) a #iocompati#le vegeta#le oil) (as use% as a &y%rop&o#ic e"cipient along (it& t&e p&osp&olipi% %ipalmitoyl p&osp&ati%ylc&oline !or entrapment an% conE trolle% release o! t&e &ig&ly &y%rop&ilic %rug ter#utaline sul!ate !or pulmonary a%ministration A44B* Hy%rogenation o! palm oil e"cipient (as utili/e% to tig&tly pac$ &y%rop&o#ic %omains toget&er !or a tig&ter microsp&ere #arrier* Degra%ation o! t&e sprayE%rie% &y%rop&o#ic coat resulte% in t&e sustaine% release o! %rug nanoparticles (it&out #urst e!!ects o#serve% (it& !reeE%rug nanoparticles* T&is approac& may serve as a potential &ig&Ecompliance met&o% o! %elivering %rugs o! varying solu#ility t&roug& t&e pulmonary route !eature% #y noninvasive circulaE tory access as sprayE%rie% microsp&eres (it& nearEoptimal aero%ynamic %iameters) as suggeste% #y E%(ar%s et al* A45B !or improve% %eepElung %eposition (it& re%uce% clearance #y alveolar macrop&ages* Using a .u%icious selection o! components (it& appropriate p&ysicoc&emical c&aracteristics) lipi%E#ase% DDSs can also #e !ormulate% !or instant triggerE#ase% release applications* +y altering t&e p&ase transition o! t&e lipi% c&ains !rom closely pac$e% arrangements AcrystallineB to loose) %isor%ere% !lui% c&ains) i%eally at an ac&ieva#le) clinically relevant temperature) %i!!usion o! %rug !rom internal lipi% nanocarrier compartments can #e ac&ieve%* Suc& p&ase transitions can occur (it&in s&arp) pre%icta#le temperature ranges on t&e or%er o! a !e( %egrees celsius) an% are primarily a !unction o! &y%rocar#on lengt&) e"tent o! saturation) c&arge) an% t&e presence o! ot&er structures (&ic& a!!ect lipi% pac$ing) suc& as c&olesterol or %ou#le #on%s* In lo(Etemperature t&ermosensitive liposomes A TS sB) !or e"ample) t&e gel p&ase rapi%ly transitions to t&e liCui% crystalline p&ase at 95]C upon mi"ture !ormulation o! a conventional p&osp&olipi% com#ination (it& lysolecit&in) at (&ic&

4@5

'ayagopal an% S&astri

temperature e"cessive e!!lu" o! %rug is o#serve%) (it& !ull release o! %o"oru#icin (it&in 5@ secon%s A48B* T&e rapi% release o! &ig& Cuantities o! %rug !rom TS s &as important conseCuences on triggere% c&emot&erapeutic applications* Pea$ intratuE moral %o"oru#icin concentrations upon &ypert&ermia o! TS s (ere measure% to #e 8@E!ol% &ig&er t&an !reeE%rug an% t(o!ol% &ig&er t&an previous t&ermosensitive liposome %esigns* ManganeseEgui%e% loa%ing o! TS s (it& %o"oru#icin &as #een carrie% out to ac&ieve similar loa%ing concentrations as nontemperatureEsensitive liposomes A49B) suggesting t&at &ypert&ermiaE#ase% c&emot&erapy may #e a via#le clinical option to conventional liposomes !or %elivering more to"ic payloa%s to t&e tumor space* Given t&e current clinical usage o! liposomal !ormulations suc& as Do"il) similar liposomal !ormulations triggere% #y &ypert&ermia may #e rea%ily accepte% i! t&ermal %osimetry protocols can #e stan%ar%i/e%* Novel nanoengineerE ing approac&es involving t&e incorporation o! transitionEtemperatureElo(ering components as (ell as metallic %rugEloa%ing sc&emes to ac&ieve &ig& encapsulaE tion e!!iciencies are &elp!ul in !acilitating t&e implementation o! liposomes (it& en&ance% trigger !unctionality in t&e clinic*

Mo%ulation o! Drug Environment an% P&ysicoc&emical Properties !or Sustaine% an% Triggere% Release In many cases) nanoscale mo%ulation o! t&e actual %rug structure is nee%e% to en&ance its solu#ility or ot&er(ise increase its e!!iciency o! transport* In alternative a%ministration routes) suc& as t&e trans%ermal pat&(ay) t&is type o! mo%i!ication is gaining increase% attention* T&e #ene!its o! trans%ermal a%ministration are several !ol%6 AiB !irstEpass liver meta#olism associate% (it& oral an% systemic t&erapy is avoi%e%) AiiB t&ere is generally increase% patient compliance Ae*g*) (earing a patc& vs* repeate% in.ectionsB) an% AiiiB a%verse reactions associate% (it& conventional a%ministrations o! %rug may #e avoi%e% in t&is !orm* Ho(ever) t&e s$in &as lo( permea#ility to most t&erapeutics) especially &ig&EmolecularE(eig&t compoun%s) an% t&us is not #y itsel! an e!!icient a%ministration site !or most t&erapies* Several approac&es &ave #een reporte% to a%%ress t&is c&allenge) an% inclu%e p&ysical s$in #arrier pertur#ation an%1or t&e promotion o! %rug penetration using ultrasoun% Asonop&oresisB) electroporation) %rug molecule electrop&oresis Aiontop&oresisB) an% c&emically assiste% cotransport A4:B* T&e latter involves t&e nanoscale mo%ulation o! %rugE%i!!usive mo#ility #y t&e transient in%uction o! s$in permea#ility) an%1or t&e improvement o! %rug portioning (it&in t&e s$in* 0no(n permeation en&ancers inclu%e !atty aci%s) et&anol) an% ot&er s$inEpermea#le solvents A4:B* ee et al* A4;)4<B reporte% t&e synergistic e!!ect o! nEmet&yl pyrroli%one an% oleic aci% in t&e en&anceE ment o! &y%rop&o#ic an% &y%rop&ilic %rug !lu" A-ig* 4B* NEMet&yl pyrroli%one is per!ectly misci#le (it& (ater !or comi"ing in aCueE ous trans%ermal !ormulations) an% is t&oug&t to e"ert its e!!ects #y promotion o! &y%rogen #on%ing (it& t&e t&erapeutic) as (ell as t&e transient %isruption o! lipi% #ilayers) ena#ling %rug partitioning in t&e stratum corneum !or en&ance% !lu" A4;)4<B* Given en&ancements in %rug transport o#serve% (it& p&ysical pertur#aE tion met&o%s) t&e com#ination o! c&emical permeation en&ancers (it& ultrasoun%) a (i%ely availa#le tec&nology) is li$ely to &ave a signi!icant impact on t&e transport o! a (i%e spectrum o! %rugs !or sustaine% release* T&e alteration o! %rug solu#ility is o!ten critical to its t&erapeutic e!!icacy* A tec&niCue o! mo%ulating %rug solu#ility an% t&us its partitioning %ynamics is supramolecular comple"ation* In t&is approac&) t&e t&erapeutic is com#ine% (it& a

Nanoengineering o! Drug Delivery Systems

4@8

-IGURE 4 Top6 -lu" en&ancement o! &y%rop&ilic HCl salt %rugs !rom 464 H5?1nEmet&yl pyrrolE i%one cosolvent t&roug& strippe% &uman ca%aver s$in* +ottom6 Transport o! li%ocaine an% nEmet&yl pyrroli%one !rom H5?1nEmet&yl pyrroli%one 4[ oleic aci% systems* A##reviation6 NMP) NEmet&yl pyrroli%one* Source6 -rom Re!* 4<*

sta#ili/ing agent to !orm an inclusion comple") (it& t&e overall structure t&en in&erE iting t&e p&ysicoc&emical properties o! t&e comple"ing agent* Cyclo%e"trins ACDsB) cyclic polysugars) &ave #een $no(n to per!orm t&is tas$ very e!!iciently in oralE %osage !ormulations) ena#ling po(er!ul sustaine%Erelease applications (&ic& are not ac&ieva#le (it& t&e !reeE%rug alone* -or e"ample) t&e poorly (aterEsolu#le antiE micro#ial agent c&lor&e"i%ine (as comple"e% (it& fECDs o! varying lipop&ilicity A4=B* T&e CDH%rug comple" (as loa%e% into P GAEimplanta#le DDSs) creating a concentration gra%ient* En&ance% solu#ility con!erre% #y t&e comple" allo(e% !or improve% %rug %i!!usivity) (&ereas t&e lipop&ilicity o! t&e %rugHCD inclusion comple" coul% #e altere% to mo%ulate sustaine%Erelease $inetics* -urt&ermore) t&e antimicro#ial activity o! t&e c&lor&e"i%ine (as en&ance% #y comple"ation (it& CD) t&roug& a plausi#le mec&anism t&at involves en&ancement o! t&e interaction o! t&e %rug (it& t&e glycocaly" Apolysacc&ari%e coatB on t&e #acteria AUS Patent ;);>>):@:B* T&us) t&e %irect alteration o! %rug p&ysicoc&emical properties is a po(er!ul approac& in e"ten%ing t&e scope o! %rugs t&at can #e a%ministere% #y DDSs (it& &ig& e!!icacy*

ENHANCEMENT ?- DRUG STRUCTURA STA+I IT3 AND DURATI?N ?- ACTI2IT3 Several %iverse approac&es &ave #een reporte% !or t&e preservation o! %rug structural integrity an% en&ancement o! t&erapeutic activity* T&erapeutics suc& as proteins an% pepti%es are especially prone to in vivo %egra%ation an% clearance) (&ic& are #arriers to t&eir #ioavaila#ility an% %uration o! action* ?t&er t&erapies may #e poorly solu#le) or %egra%e (it&in &ars& nanocarrier environments over time in circulation or in storage prior to a%ministration* Approac&es to a%%ress t&ese c&allenges on t&e nanoscale inclu%e t&e maintenance o! controlle% %rug microenvironments (it&in

4@9

'ayagopal an% S&astri

t&e ve&icle) polymeric sur!ace engineering to mo%ulate %rug interactions (it& t&e surroun%ing environment) an% t&e %evelopment processing tec&niCues (&ic& utili/e suita#le temperatures) solvents) an%1or comple"ation agents (&ic& con!er sta#ility upon la#ile %rugs) t&us en&ancing t&eir e!!icacy an% applications*

-ormulation Processing Strategies !or En&ance% Drug Sta#ility an% Activity Many promising t&erapeutics are limite% in clinical applications #y poor solu#ility* Suc& %rugs also &ave lo( %issolution rates (&ic& &in%er #ioavaila#ility even in cases o! rapi% %rug upta$e* Previous approac&es !ocuse% on increasing %issolution rates #y increasing t&e sur!ace area o! t&e %rug po(%er using milling o! t&e %rug to create microparticles* Ho(ever) !or lo(Esolu#ility %rugs) t&is is o!ten not a su!!icient sur!ace area en&ancement to promote a%eCuate %issolution* Nanoengineering o! t&e %rug particle !ormulation itsel! &as #een per!orme% to improve %rug solu#ility an% %issolution* -or e"ample) a Qnanoni/ationS process utili/ing &ig&Epressure &omoE geni/ation tec&niCues (as %evelope% #y 0ec$ an% Muller A4>B) (&ic& avoi%s un%esira#le e!!ects associate% (it& solvent precipitation an% pearl milling Atra%e name6 DissoCu#esB* T&e %ecrease in particle si/e an% %i!!usion %istance a!!or%e% #y DissoCu#es provi%es !or an en&ance% %rug %issolution rate an% solu#ility* T&e latter is strictly a conseCuence o! t&e !act t&at t&e %rug is less t&an 4 _m in %iameter Aa#ove (&ic& si/e) temperature an% solvent c&aracteristics are primary %eterminants o! particle solu#ilityB) an% increase% curvature o! t&e %rug particle increases its %issolution pressure) (&ic& t&us en&ances solu#ility* ?(ing to t&e &omogeneity o! t&e particles (it&in t&e nanosuspension) (&ic& may inclu%e electrostatic an% steric sta#ili/ing agents) potentially %egra%ative processes suc& as ?st(al% ripening are not o#serve%) provi%ing %rug sta#ility in storage* To attain suita#le intracarrier environments !or %rug sta#ility (it&in DDSs suc& as P GA nanoparticles) antaci% salts &ave #een intro%uce% %uring encapE sulation to neutrali/e t&e aci%ic internal microclimate) re%ucing protein aggregation an% %egra%ation A5@B* Sacc&ari%es suc& as tre&alose &ave #een use% as a cryoproE tectant in %rugs encapsulate% (it&in soli%Hlipi% nanoparticles A54B* T&e CDs previously %iscusse% are $no(n to protect la#ile %rugs against &y%rolysis) o"i%aE tion) an% p&oto%egra%ation A55B* T&e utility o! CD comple"ation !or t&e protection o! a t&ermally la#ile %rug) r&o%ium AIIB citrate) (as reporte% #y Sinisterra et al* A58B) &ig&lig&ting t&e potential o! CD comple"ation to sta#ili/e %rugs !rom &ig&E temperature compression an% in.ection mol%ing processes use% to manu!acture polymeric DDSs A-ig* 5B* Hy%rop&ilic CDs (it& &y%rop&o#ic cavities) suc& as HP fCD) can serve as nanoscale s&iel%s (&ic& protect &y%rop&o#ic resi%ues on proteins) !or re%uce% %enaE turation an% aggregation) an% re%uce un(ante% polymerH%rug interactions (it&in t&e carrier* T&ese are e"amples o! nanoscaleE%rugEsta#ili/ation strategies (&ic& can #e utili/e% to en&ance solu#ility an% structural con!ormation o! %rugs !or incorporation into nanoscale DDSs) or simply !or improve% transport parameters o! t&e %rug itsel!*

Polymeric Strategies !or Prolongation o! Drug Action Polymeric sur!ace !unctionali/ation o! DDSs is no( commonly use% to mo%ulate t&e interaction o! DDSs (it& t&e surroun%ing environment) !or en&ance% %rug activity t&roug& en&ance% &al!Eli!e in t&e #o%y) an% re%uce% #io%egra%ation* PEG is (ell $no(n !or its a#ility to %iminis& carrier clearance #y t&e reticuloen%ot&elial

Nanoengineering o! Drug Delivery Systems

4@:

-IGURE 5 AAB T&ermogravimetric curves o! &y%ro"ypropylEfEcyclo%e"trin AHPfCDB ARB) R&AIIB citrate AEv EvB) an% association comple" #et(een R&AIIB citrate an% HPfCD AE E EB* A+B Di!!erential t&ermogravimetric ADTGB curves o! HPfCD ARB) R&AIIB citrate AEv EvB) an% association comple" #et(een R&AIIB citrate an% HPfCD AE E EB* T&e t&ermogravimetric an% DTG curves o! t&e R&AIIB citrateHHPfCD comple" in%icate only one t&ermal transition at &ig&er temperature) aroun% 84@]C) (&ic& (as accompanie% #y an =@[ mass loss* T&is %ata suggests t&at HPfCD comple"ation (it& R&AIIB citrate results in en&ance% t&ermal sta#ility* Source6 -rom Re!* 58*

system an% in&i#it protein sur!ace a%sorption* T&is is a conseCuence o! t&e e"clusion volume e"erte% #y t&e !reely mo#ile polymer c&ain o! t&e carrier sur!ace* T&e #ioE compati#le PEG imparts (ater solu#ility to t&e con.ugate% APEGylate%B t&erapeutic* T&is strategy &as #een recently utili/e% !or t&e protection o! t&erapeutic proteins) as rein!orce% #y t&e increasing num#er o! PEGylate% %rugs in t&e clinic) suc& as PEGAS3S Ainter!eron al!aE5#) Roc&eB an% E"u#era Ain&ale% insulin) P!i/erB* Strategies !or utili/ing PEGylation !or !uture proteinE#ase% t&erapies are %epen%ent on t&e %evelopment o! PEGylation c&emistries (&ic& con!er t&e a%vantages o! PEG (it&E out signi!icant losses in protein !unctionality* T&is goal is #eing ac&ieve% (it& t&eraE peutic monoclonal anti#o%ies* Ran%om PEGylation o! protein amine groups using PEG couple% (it& NHS esters (as implicate% in t&e loss o! monoclonal anti#o%yE #in%ing a!!inities in several cases* Ho(ever) attac&ment o! PEG to -a#u !ragments using cysteine crossElin$ing in t&e &inge region Ai*e*) t&roug& t&iolEmaleimi%e

4@;

'ayagopal an% S&astri

c&emistryB re%uce% or eliminate% most a!!inity pro#lems (&ile con!erring en&ance% circulation &al!Eli!e A59B* To e"pan% t&is i%ea) con.ugation o! #ranc&e% an% linear #i!unctional PEGs to singleEc&ain anti#o%ies Asc-vsB (it& an engineere% unpaire% cysteine to create multivalent con.ugates (as also carrie% out (it& success A5:)5;B* Alt&oug& t&e pro#lem is more %i!!icult !or proteins ot&er t&an anti#o%ies (&ic& &ave t&e uniCue #ene!it o! unpaire% cysteines not nee%e% !or #iological activity) siteE speci!ic PEGylation &as also #een reporte% using en/ymatic or siteE%irecte% mutaE genetic approac&es A5<)5=B* T&e tec&nology !or siteEspeci!ic PEGylate t&erapeutic proteins is an area o! active researc& an% t&at is li$ely to e"pan% t&e li#rary o! availa#le proteinE#ase% t&erapies*

TARGETED THERAPIES Several approac&es !or t&e speci!ic targeting o! t&erapeutics to %isease sites &ave #een %evelope%) !or t&e purposes o! ma"imi/ing t&e e!!ective %ose %elivere% to t&e site (&ile lo(ering t&e total %ose nee%e%) an% sparing &ealt&y tissue !rom potential a%verse %rug e!!ects via con!inement o! t&erapeutic activity* Generally) t&ese strategies involve optimi/ation o! geometry o! t&e nanocarrier to optimi/e tissue upta$e o! DDSs) a generally passive route) an% t&e #iocon.ugation o! ligan%s suc& as pepti%es to t&e nanocarrier sur!ace to promote interactions (it& %isease% tissue) an active targeting mec&anism*

Strategies !or Passive Tissue Targeting T&e geometry an% mec&anical properties o! a carrier &as a pro!oun% in!luence on its t&erapeutic e!!icacy in t&at t&e a#ility !or DDSs to target tissue can #e %epen%ent on t&em* -or e"ample) sp&erical an% in!le"i#le carriers &ave #een suggeste% to &ave re%uce% transport in tumor interstitial mo%els compare% to !le"i#le an% e"ten%e% structures) an% %ue to tumor vessel pore si/e restrictions) e"travasation o! nanosp&eres in cancerous tissue may #e a &ig&ly si/eE%epen%ent event A5>B* Gastrointestinal mucosa &as si/eE%epen%ent upta$e p&enomena a!!ecting oral %osage o! #io%egra%E a#le nanoparticles A8@)84B* It !ollo(s t&at t&e p&ysical c&aracteristics o! DDSs must #e optimi/e% !or t&e intrinsic properties o! t&e speci!ic targeting site* T&e lea$y vasculature o! tumors &as #een e"ploite% #y t&e (ellE$no(n en&ance% permea#ility an% retention e!!ect) (&ere#y longEcirculating PEGylate% Qstealt&S liposomes are capa#le o! accuE mulating in t&e tumor space %ue to a lac$ o! rapi% clearance con!erre% #y PEG) comE #ine% (it& t&e accessi#ility o! t&e tumor pores to passive liposomal upta$e*

Strategies !or Actively Targete% T&erapies T&e #iocon.ugation o! ligan%s) suc& as monoclonal anti#o%ies) proteins) or pepti%es to t&e nanocarrier sur!ace) &as #een e"ploite% on many nanoparticulate DDSs !or t&e purpose o! concentrating t&erapeutic action to speci!ic sites* Nonspeci!ic pepti%eE #ase% internali/ation systems) suc& as t&ose #ase% on cellEpenetrating pepti%es) &ave #een s&o(n to #e e!!icient in preliminary in vitro stu%ies) an% may internali/e in an energyEin%epen%ent mec&anism %epen%ing on t&e cargo involve% A85)88B) #ut t&eir use !or in vivo targeting may not #e clinically relevant %ue to t&e a#ility o! t&ese pepti%es to nonspeci!ically target many cell types irrespective o! pat&ology* Cancerous cells &ave #een targete% #y nanoparticles to(ar% uniCue sur!ace antigens in&erent to t&e tumor type Ae*g*) HER5 in #reast cancerB A89B or ra%iation area A8:B* ?t&er targets e"ploite% #y nanoparticulate systems (it& promising t&erapeutic outcomes inclu%e

Nanoengineering o! Drug Delivery Systems

4@<

!olate receptors A8;B an% cellEa%&esion molecules A8<H8>B) #ot& prominent e"amples o! ligan%Eme%iate% internali/ation processes t&at are clinically relevant in a num#er o! pat&ologies* Strategies !or Drug Delivery System Transport Across Tig&t En%ot&elial 'unctions Tig&t en%ot&elial #arriers) suc& as t&e #loo%H#rain #arrier A+++B an% #loo%Hretinal #arrier) pose signi!icant c&allenges to t&e transport o! t&erapies) most o! (&ic& traverse suc& #arriers only un%er e"treme pat&ological con%itions* Met&o%s to traverse t&ese tig&t .unctions (oul% ena#le t&e clinical usage o! multiple t&erapies an% imaging mo%alities using contrast agents* Nanoparticulate %elivery across t&e +++ !or t&e transport &as #een ac&ieve% (it& numerous approac&es* Transport o! ligan%Ecoate% PEGylate% polylacti%eHP GA nanoparticles across t&e +++ &as #een %emonstrate% A9@B* A novel strategy (as recently reporte% !or P GA nanoparticle transport across t&e +++ (it& t&e sur!ace engineering o! pepti%es similar to synt&etic opioi%s A94B* ?ur la#oratory is currently investigating t&e potential o! !unctionali/e% soli%Hlipi% nanoparticles to transport magnetic resonance contrast agents) proteins) an% !luorescent pro#es across t&e +++* T&us) one can retain t&e %esira#le properties o! nanocarriers (it& t&e a%%e% !eature o! +++ penetration) !or t&e %elivery o! t&eraE peutic or %iagnostic agents (&ic& are not lipi%Esolu#le enoug& or %o not meet t&e p&ysical criteria to normally cross t&e +++*

-UTURE DIRECTI?NS T&e potential o! nanoengineering strategies in t&e %evelopment o! controlle%E an% triggere%Erelease DDSs) preservation o! %rug activity) an% %iseaseEspeci!ic targeting &as #een presente%* Many o! t&ese stu%ies are preliminary in nature) an% various systems reCuire !urt&er to"icity an% e!!icacy %ata to !acilitate t&eir transition to cliniE cal trials* Ho(ever) t&e synt&etic an% natural #asic units (&ic& !orm t&e #asis o! t&ese strategies) suc& as t&e t&erapeutics) P GA) an% soli% lipi%s) &ave entere% t&ese p&ases* T&e success o! t&ese initial nano%elivery systems (ill us&er in t&e clinical application o! com#inatorial) integrative strategies (&ic& constitute QsmartS %elivery systems) suc& as t&e multimo%al Qnanocell)S a P GA) PEG) an% p&osp&olipi%E#ase% nanoparticle (&ic& %elivers #ot& antiangiogenic an% c&emot&erapeutic agents (it& temporally controlle% $inetics A95B* ?t&er consi%erations) suc& as t&e a#ility to scaleEup la#oratory tec&niCues !or mass pro%uction o! DDSs an% t&e a#ility to upgra%e e"isting) approve% systems (it& step(ise !unctional a%%itions) (ill also e"pe%ite t&eir clinical usage* Nanoengineering o!!ers t&e uniCue a#ility to manipuE late t&e smallest interactions at t&e most !un%amental scales) (&ic& is certain to provi%e a series o! ma.or a%vancements in %rug %elivery tec&nology*

AC0N?, EDGMENTS T&is c&apter (as ma%e possi#le #y generous !un%ing !rom t&e 2an%er#ilt University Institute !or Integrative +iosystems Researc& an% E%ucation A2II+REB) an% t&e 2an%er#ilt 2ision Researc& Center Training Grant ANEI T85 E3?<48:B* RE-ERENCES
4* Panyam ') a#&aset(ar 2* +io%egra%a#le nanoparticles !or %rug an% gene %elivery to cells an% tissue* A%v Drug Deliv Rev 5@@8M ::A8B685>H89<*

4@=

'ayagopal an% S&astri

5* A&me% -) Disc&er DE* Sel!Eporating polymersomes o! PEGHP A an% PEGHPC 6 &y%rolyE sisEtriggere% controlle% release vesicles* ' Control Release 5@@9M >;A4B68<H:8* 8* Sc&melt/er RC) Sc&malen#erg 0E) U&ric& E* Synt&esis an% cytoto"icity o! salicylateE #ase% polyAan&y%ri%e estersB* +iomacromolecules 5@@:M ;A4B68:>H8;<* 9* ynn DM) Ami.i MM) anger R* pHEresponsive polymer microsp&eres6 rapi% release o! encapsulate% material (it&in t&e range o! intracellular pH* Ange( C&em Int E% Engl 5@@4M 9@A>B64<@<H4<4@* :* ittle SR) ynn DM) Ge 7) et al* PolyE#eta amino esterEcontaining microparticles en&ance t&e activity o! nonviral genetic vaccines* Proc Natl Aca% Sci USA 5@@9M 4@4A5;B6 >:89H>:8>* ;* ittle SR) ynn DM) Puram S2) anger R* -ormulation an% c&aracteri/ation o! polyA#eta amino esterB microparticles !or genetic vaccine %elivery* ' Control Release 5@@:M 4@<A8B6 99>H9;5* <* Potineni A) ynn DM) anger R) Ami.i M* PolyAet&ylene o"i%eBEmo%i!ie% polyA#etaEamino esterB nanoparticles as a pHEsensitive #io%egra%a#le system !or paclita"el %elivery* ' Control Release 5@@8M =;A5H8B6558H589* =* Husseini GA) Myrup GD) Pitt ,G) C&ristensen DA) Rapoport N3* -actors a!!ecting acoustically triggere% release o! %rugs !rom polymeric micelles* ' Control Release 5@@@M ;>A4B698H:5* >* Marin A) Muniru//aman M) Rapoport N* Mec&anism o! t&e ultrasonic activation o! micellar %rug %elivery* ' Control Release 5@@4M <:A4H5B6;>H=4* 4@* Rapoport N) Marin A) uo 3) Prest(ic& GD) Muniru//aman MD* Intracellular upta$e an% tra!!ic$ing o! Pluronic micelles in %rugEsensitive an% MDR cells6 e!!ect on t&e intracE ellular %rug locali/ation* ' P&arm Sci 5@@5M >4A4B64:<H4<@* 44* Coo$ R?) Pannu R0) 0ella(ay I,* Novel sustaine% release microsp&eres !or pulmonary %rug %elivery* ' Control Release 5@@:M 4@9A4B6<>H>@* 45* E%(ar%s DA) +enE'e#ria A) anger R* Recent a%vances in pulmonary %rug %elivery using large) porous in&ale% particles* ' Appl P&ysiol 4>>=M =:A5B68<>H8=:* 48* C&en 7) Tong S) De(&irst M,) 3uan -* Targeting tumor microvessels using %o"oru#icin encapsulate% in a novel t&ermosensitive liposome* Mol Cancer T&er 5@@9M 8A4@B6 4844H484<* 49* C&iu GN) A#ra&am SA) Ic$enstein M) et al* Encapsulation o! %o"oru#icin into t&ermoE sensitive liposomes via comple"ation (it& t&e transition metal manganese* ' Control Release 5@@:M 4@9A5B6 5<4H5==* 4:* Mitragotri S) anger R) 0ost '* En&ancement o! trans%ermal transport using ultrasoun% in com#ination (it& ot&er en&ancers* Han%#oo$ o! P&armaceutical Controlle%ERelease Tec&nology* Ne( 3or$6 Marcel De$$er) 5@@@* 4;* ee P') anger R) S&astri 2P* Novel microemulsion en&ancer !ormulation !or simultaneE ous trans%ermal %elivery o! &y%rop&ilic an% &y%rop&o#ic %rugs* P&arm Res 5@@8M 5@A5B65;9H5;>* 4<* ee P') anger R) S&astri 2P* Role o! nEmet&yl pyrroli%one in t&e en&ancement o! aCueE ous p&ase trans%ermal transport* ' P&arm Sci 5@@:M >9A9B6>45H>4<* 4=* 3ue IC) Po!! ') Cortes ME) et al* A novel polymeric c&lor&e"i%ine %elivery %evice !or t&e treatment o! perio%ontal %isease* +iomaterials 5@@9M 5:A4<B68<98H8<:@* 4>* 0ec$ CM) Muller RH* Drug nanocrystals o! poorly solu#le %rugs pro%uce% #y &ig& pressure &omogenisation* Eur ' P&arm +iop&arm 5@@;M ;568H4;* 5@* V&u G) Mallery SR) Sc&(en%eman SP* Sta#ili/ation o! proteins encapsulate% in in.ecta#le polyAlacti%eEcoEglycoli%eB* Nat +iotec&nol 5@@@M 4=A4B6:5H:<* 54* Heiati H) Ta(as&i R) P&illips NC* Drug retention an% sta#ility o! soli% lipi% nanopartiE cles containing a/i%ot&ymi%ine palmitate a!ter autoclaving) storage an% lyop&ili/ation* ' Microencapsul 4>>=M 4:A5B64<8H4=9* 55* o!tsson T) +re(ster ME* P&armaceutical applications o! cyclo%e"trins 46 %rug solu#iliE /ation an% sta#ili/ation* ' P&arm Sci 4>>;M =:A4@B64@4<H4@5:* 58* Sinisterra RD) S&astri 2P) Na..ar R) anger R* Encapsulation an% release o! r&o%iumAIIB citrate an% its association comple" (it& &y%ro"ypropylE#etaEcyclo%e"trin !rom #io%eE gra%a#le polymer microsp&eres* ' P&arm Sci 4>>>M ==A:B6:<9H:<;* 59* C&apman AP) Antoni( P) Spitali M) ,est S) Step&ens S) 0ing D'* T&erapeutic anti#o%y !ragments (it& prolonge% in vivo &al!Elives* Nat +iotec&nol 4>>>M 4<A=B6<=@H<=8*

Nanoengineering o! Drug Delivery Systems

4@>

5:* Al#rec&t H) +ur$e PA) Natara.an A) et al* Pro%uction o! solu#le Sc-vs (it& CEterminalE !ree t&iol !or siteEspeci!ic con.ugation or sta#le %imeric Sc-vs on %eman%* +iocon.ug C&em 5@@9M 4:A4B64;H5;* 5;* Natara.an A) Wiong C3) Al#rec&t H) DeNar%o G ) DeNar%o S'* C&aracteri/ation o! siteE speci!ic Sc-v PEGylation !or tumorEtargeting p&armaceuticals* +iocon.ug C&em 5@@:M 4;A4B6448H454* 5<* Ca/alis CS) Haller CA) SeaseECargo ) C&ai$o! E * CEterminal siteEspeci!ic PEGylation o! a truncate% t&rom#omo%ulin mutant (it& retention o! !ull #ioactivity* +iocon.ug C&em 5@@9M 4:A:B64@@:H4@@>* 5=* Sato H* En/ymatic proce%ure !or siteEspeci!ic pegylation o! proteins* A%v Drug Deliv Rev 5@@5M :9A9B69=<H:@9* 5>* Stro& M) Vimmer 'P) Du%a DG) et al* 7uantum %ots spectrally %istinguis& multiple speE cies (it&in t&e tumor milieu in vivo* Nat Me% 5@@:M 44A;B6;<=H;=5* 8@* Desai MP) a#&aset(ar 2) Ami%on G ) evy R'* Gastrointestinal upta$e o! #io%egra%E a#le microparticles6 e!!ect o! particle si/e* P&arm Res 4>>;M 48A45B64=8=H4=9:* 84* Desai MP) a#&aset(ar 2) ,alter E) evy R') Ami%on G * T&e mec&anism o! upta$e o! #io%egra%a#le microparticles in CacoE5 cells is si/e %epen%ent* P&arm Res 4>><M 49A44B64:;=H4:<8* 85* Hall#rin$ M) -loren A) lmCuist A) Pooga M) +art!ai T) angel U* Cargo %elivery $inetics o! cellEpenetrating pepti%es* +ioc&im +iop&ys Acta 5@@:M 4:A5B64@4H4@>* 88* Pa%ari 0) Saali$ P) Hansen M) et al* Cell trans%uction pat&(ays o! transportans* +iocon.ug C&em 5@@:M 4;A;B648>>H494@* 89* 0irpotin D) Par$ ',) Hong 0) et al* Sterically sta#ili/e% antiEHER5 immunoliposomes6 %esign an% targeting to &uman #reast cancer cells in vitro* +ioc&emistry 4>><M 8;A4B6;;H<:* 8:* Halla&an D) Geng ) 7u S) et al* IntegrinEme%iate% targeting o! %rug %elivery to irra%iE ate% tumor #loo% vessels* Cancer Cell 5@@8M 8A4B6;8H<9* 8;* Hilgen#rin$ AR) o( PS* -olate receptorEme%iate% %rug targeting6 !rom t&erapeutics to %iagnostics* ' P&arm Sci 5@@:M >9A4@B6548:H549;* 8<* 0elly 0A) Allport 'R) Tsour$as A) S&in%eEPatil 2R) 'osep&son ) ,eissle%er R* Detection o! vascular a%&esion moleculeE4 e"pression using a novel multimo%al nanoparticle* Circ Res 5@@:M >;A8B685<H88;* 8=* Muro S) Ga.e(s$i C) 0oval M) Mu/y$antov 2R* ICAME4 recycling in en%ot&elial cells6 a novel pat&(ay !or sustaine% intracellular %elivery an% prolonge% e!!ects o! %rugs* +loo% 5@@:M 4@:A5B6;:@H;:=* 8>* Muro S) 0oval M) Mu/y$antov 2* En%ot&elial en%ocytic pat&(ays6 gates !or vascular %rug %elivery* Curr 2asc P&armacol 5@@9M 5A8B65=4H5>>* 9@* ?livier 'C* Drug transport to #rain (it& targete% nanoparticles* NeuroR" 5@@:M 5A4B64@=H44>* 94* Costantino ) Gan%ol!i -) Tosi G) Rivasi -) 2an%elli MA) -orni -* Pepti%eE%erivati/e% #ioE %egra%a#le nanoparticles a#le to cross t&e #loo%H#rain #arrier* ' Control Release 5@@:M 4@=A4B6=9H>;* 95* Sengupta S) Eavarone D) Capila I) et al* Temporal targeting o! tumour cells an% neovasE culature (it& a nanoscale %elivery system* Nature 5@@:M 98;A<@:@B6:;=H:<5*

Aerosol -lo( Reactor Met&o% !or t&e Synt&esis o! Multicomponent Drug NanoE an% Microparticles
'anne Raula
NanoMaterials Group) a#oratory o! P&ysics an% Center !or Ne( Materials) Helsin$i University o! Tec&nology) Helsin$i) -inlan%

Hannele Eeri$Iinen
P&armaceutical Pro%uct Development) ?rion Corporation ?rion P&arma) Espoo) -inlan%

Anna I&%e
NanoMaterials Group) a#oratory o! P&ysics an% Center !or Ne( Materials) Helsin$i University o! Tec&nology) Helsin$i) -inlan%

Es$o I* 0auppinen
NanoMaterials Group) a#oratory o! P&ysics an% Center !or Ne( Materials) Helsin$i University o! Tec&nology) an% 2TT +iotec&nology) Helsin$i) -inlan%

INTR?DUCTI?N NanoE an% microparticle %rug carriers &ave potential applications !or a%ministraE tion o! t&erapeutic molecules A4)5B* Targete% %rug %elivery can #e ac&ieve% #y small particles %ue to t&eir ten%ency to accumulate in targete% areas o! t&e #o%y* Moreover) t&e solu#ility o! material !rom t&e nanoscale o#.ects is nota#ly en&ance% %ue to t&e increase% sur!aceEtoEvolume ratio o! small particles* As (ell) t&e systemic si%e e!!ects in %rug targeting) !or e"ample) into a cancerous tumor) can #e minimi/e% #y t&e %ecrease a particle si/e A8B* Su#Emicron) soli%Estate %rug particles t&at ten% to #e unsta#le !or many %rug molecules can #e sta#ili/e% #y speci!ic polymers* In t&ese composite particles) t&e polymer may act) in a%%ition to sta#ili/er) as !unctional mateE rial t&at controls t&e release an% %i!!usion o! a %rug %epen%ing on t&e environmental con%itions suc& as pH) temperature) ionic strengt&) &umi%ity) an% so on A9):B* Several met&o%s &ave #een stu%ie% !or t&e preparation o! t&e %rug nanoE an% microparticles* A common liCui% route met&o% to prepare t&e %rug nanoE an% microparticles is t&e use o! an emulsion* Pro#a#ly) t&e most use% met&o% is an oilE inE(ater emulsion consisting o! t(o immisci#le solvents suc& as c&loro!orm an% (ater A;B* As t&e %rug particles s&o( a large ten%ency to(ar%s agglomeration an% gro(t&) sur!actants &ave to #e a%%e% to sta#ili/e t&e %roplets an% particles* ?t&er relate% met&o%s inclu%e saltingEout A<B) nanoprecipitation Ainter!acial precipitationB A=B) p&ase separation A>B) an% evaporative precipitation into aCueous solution A4@B* In general) t&e pro%uction o! particles inclu%ing t(o or more %rug molecules is %i!!icult* Si/eEre%uction tec&niCues) suc& as (et milling an% &ig&Epressure &omogeniE /ation) &ave also #een use% to prepare nanoE an% microparticles A44B* Hig&Es&ear

444

445

Raula et al*

!orces an% t&us &ig& energies use% in milling processes can create uncontrolla#le c&anges in t&e pro%uct) suc& as c&emical %egra%ation) c&anges in sur!ace energetics) an% %amage in crystal structure* -urt&ermore) long processing times increase t&e ris$ o! micro#iological contamination* Sur!actants are nee%e% to re%uce particle agglomeration an% sintering %uring (et milling* Multicomponent particles (it& controlle% morp&ology an% sur!ace c&aracteristics Acomposition an% morp&ologyB cannot #e pro%uce%* Small particles &ave also #een prepare% using supercritical !lui%s eit&er as solvents or antisolvents !or t&e %rug an% t&e polymer A45B* T&e preparation o! uni!orm multicomponent particles consisting o! a %rug an% a polymer &as #een s&o(n to #e %i!!icult %ue to %i!!erent crystalli/ation an% precipitation $inetics o! t&e %rug an% t&e polymer molecules an% %ue to partitioning o! t&e %rug into superE critical !lui%* SprayE%rying &as #een (i%ely use% !or t&e pro%uction o! micrometerEsi/e% particles A48B* SprayE%rying involves t&e conversion o! a solution %roplet into a %ry particle #y evaporation o! t&e solvent in a oneEstep process* +ot& (aterEsolu#le an% Einsolu#le compoun%s can #e prepare%* T&us) t&e recovery o! t&e %rug in t&e partiE cles is almost Cuantitative* Also temperatureEla#ile compoun%s suc& as proteins an% en/ymes &ave #een success!ully sprayE%rie%* T&e solvent properties an% t&e sprayE%rying varia#les can control t&e particle properties) especially morp&ology* Multicomponent particles (it& sp&erical morp&ology can #e pro%uce%* Ho(ever) particles are typically amorp&ous) an% sur!ace structure as (ell composition cannot #e controlle% in %etail* T&is c&apter presents t&e novel aerosol !lo( reactor met&o% !or t&e synt&esis o! singleE an% multicomponent nanoparticles as (ell as nanostructure%) micronE si/e% particles A49H54B* ,e %emonstrate t&e pro%uction o! po(%ers ma%e !rom %i!!erent %rug an%1or e"cipient materials* +esi%es t&e %rug molecules) t&e polyE meric %rug nanoparticles containe% met&acrylic polymers) (&ic& are p&armaceutiE cally accepta#le A55)58B* It (as o#serve% t&at t&e solu#ility o! a %rug (it&in t&e polymer matri" %epen%s not only on t&e amount o! t&e %rug) #ut also on t&e polymer itsel!* T&e %rug %issolution !orm o! t&e nanoparticles) interactions #et(een t&e %rug an% t&e polymer) an% %rug crystallinity (it&in t&e polymer particles (ill #e %iscusse%* T&is c&apter also %iscusses t&e !ormation o! t&e polymeric nanoE particles !rom several solvents* T&e stu%ie% su#.ects (ere polymer solu#ility in a solvent me%ium an% t&e in!luence o! solvent vapor pressure* C&emical an% p&ysical sta#ility o! %ry po(%ers in storage is crucial* Amorp&ous materials ten% to crystalli/e in time) t&us #uil%ing #ri%ges #et(een in%ivi%ual particles* T&is in turn a!!ects t&e po(%er !lo(a#ility an% &an%ling* A part o! t&is (or$ presents t&e attempts to crystalli/e t&e %rug (it&in t&e aerosol reactor in %i!!erent con%itions* T&is c&apter %iscusses t&e reactor con%itions a!!ecting t&e %rug crystalli/ation* A%%ing leucine %erivatives to %rug particles &as #een s&o(n to re%uce t&e a%&esion #et(een particles A59)5:B* -urt&ermore) a mEamino aci% lEleucine is a surE !aceEactive compoun% in aCueous solutions* It is also important t&at lEleucine is generally regar%e% as sa!e material !or t&e &uman #o%y* T&is c&apter %iscusses t&e preparation o! t&e composite po(%ers using lEleucine to increase t&e sta#ility o! po(%ers* Main materials (ere so%ium c&lori%e) (&ic& is a representative inorganic material t&at crystalli/es (it& ease) an% lactose t&at !orms amorp&ous particles* T&e incorporation o! lEleucine mo%i!ies particle sur!ace structure an% c&anges) !or instance) sp&erical particles to (rin$le% ones %epen%ing on t&e lEleucine content*

Aerosol -lo( Reactor Met&o%

448

Moreover) lEleucine &as #een s&o(n to rein!orce t&e structure o! particles (&ere material is in a ru##ery state* AER?S? - ?, REACT?R METH?D In t&e aerosol !lo( reactor met&o%) t&e solution containing soluteAsB is atomi/e% to pro%uce %roplets t&at are trans!erre% (it& t&e ai% o! a carrier gas to a &eate% !lo( reactor Apart I in -ig* 4B* T&e inert carrier gas is eit&er %ry or saturate% (it& a solvent* In t&e latter) t&e %roplets remain (et in t&e reactor until t&e aerosol is &eate% Apart II

III* P? 3MERIC DRUG NAN?PARTIC ES AB an% CB matri"Etype +B an% DB coreEs&ell

%ru g mole cule s %ru g crystals polymer c&ains

TAam#ientB

C?

ECTI?N

DI UTI?N

HEATING V?NE

II* PARTIC E -?RMATI?N HEATING E %rying V?NE

E sel!Eassem#ling AcoreEs&ellB E molecular arrangement Acrystal gro(t&B E morp&ology trans!ormation

Temperature increases

Dry or solvent saturate% carrier gas mi"e% (it& %roplets

Starting solution6
%rug) polymer) an%1or ot&er e"cipients %issolve% in solvent

I* DR?P ET GENERATI?N E .et ne#uli/er E ultrasonic ne#uli/er E electrospray

-IGURE 4 Sc&ematical presentation o! t&e novel aerosol !lo( reactor met&o% !or t&e pro%uction o! nano an% micronsi/e% %rug po(%ers*

449

Raula et al*

in -ig* 4B* T&e !ast %rying promotes t&e !ormation o! amorp&ous particles* Allo(ing time !or %roplet %rying provi%es also time !or molecular arrangements suc& as crysE tal gro(t& to occur* 2arying !lo( rate an% t&e resi%ence time) %rying rate o! particles an% accor%ingly particle morp&ology can #e manipulate% in t&e process* Do(nstream !rom t&e &eate% section) t&e %ry aerosol is %ilute% (it& %ry inert gas to avoi% solvent con%ensation onto %ry particles* Polymeric %rug nanoparticles o! several types) t&at is) matri" an% core s&ell as s&o(n in part III in -igure 4 &ave #een pro%uce%* Moreover) t&is aerosol met&o% pro%uces %ry partocles %irectly (it&out a nee% !or !urt&er puri!ication) an% no a%%itives are reCuire%* In t&e !ollo(ing) (e %escri#e t&e pro%uction o! nanoE an% micronEsi/e% %ry particles containing t&e %rugs) polymers) an% some mo%el materials*

EWPERIMENTA Materials Drug materials #eclomet&asone %ipropionate A+DPM Sicor S*p*A*) ItalyB) $etopro!en A5EA8E#en/oylp&enylB propionic aci%B ASigma) U*S*A*B) napro"en NASBE5EA;Emet&o"yE5E nap&t&ylB propionic aci%O ASigma) U*S*A*B) an% acetyl salicylic aci% AASAM SigmaEAl%ric&) GermanyB (ere use% as receive%* Re!erence materials lactose monoE &y%rate Aprovi%e% #y ?rion P&arma) -inlan%B) so%ium c&lori%e) NaCl A '*T* +a$er) Hollan%B) an% e"cipient material lEleucine A-lu$a) S(it/erlan%B (ere use% as receive%* P&armaceutically accepte% met&acrylic polymers Eu%ragit b 4@@ ARF&m P&arma) GermanyB) Eu%ragitb E4@@ ARF&m P&arma) GermanyB) an% Eu%ragitb RS ARF&m P&arma) GermanyB (ere use% as receive%* Solvents et&anol A>>*;[) Al$o ?y.) -inlan%B) tetra&y%ro!uran ATH-M '*T* +a$ers) U*S*A*B) an% toluene A '*T* +a$ers) U*S*A*B (ere use% as receive%* ,ater (as puri!ie% #y ionEe"c&ange AMilliporeB) an% (as measure% to &ave pH ;*

Precursor Solutions -or pure polymer solutions) t&e polymer (as %issolve% in eit&er TH- or et&anol* In a case o! solvent mi"tures) (ater or TH- (as a%%e% into polymer solution (&ile stirring t&e mi"ture* T&e volume ratios o! t&e solvents (ere @*4) 4*@) an% >*@* T&e total concentration o! t&e polymer varie% #et(een @*5 an% 4*: g1 * T&e eCuili#rium concentrations !or Eu%ragitb 4@@ in toluene A@*@:= g1 B an% in (ater A@*@5> g1 B (ere %etermine% a!ter !iltration an% solvent evaporation* T&e %rugHpolymer solutions (ere prepare% #y separately %issolving t&e polyE mer an% t&e %rug in et&anol using a magnetic stirrer an% t&en mi"ing t&e solutions in respective amounts* T&e total concentration o! a %rug an% an e"cipient varie% #et(een @*5: an% 5*@ g1 * +DP solutions : an% 5: g1 (ere prepare% #y %issolving t&e %rug in et&anol at room temperature* A saturate% +DP solution (as prepare% #y stirring t&e solution !or 4 &our until no !urt&er %issolution o! +DP (as o#serve%* ACueous lEleucine) lactose) an% NaCl precursor solutions (ere prepare% #y separately %issolving in (ater an% stirring) an% t&en t&e solutions (ere com#ine% in respective amounts* ASA (as %issolve% in et&anol) an% t&e solution (as comE #ine% (it& t&e aCueous lEleucine solution* T&e total solution concentration varie% !rom @*5: to 8@*@ g1 *

Particle Pro%uction #y t&e Aerosol -lo( Reactor Met&o% T&e setEup o! t&e aerosol !lo( reactor is presente% in -igure 5* All t&e e"perimental con%itions !or t&e preparation o! nanoE an% microparticles are given in Ta#le 4* T&e

Aerosol -lo( Reactor Met&o%

44:

2ac* 5: 1min C&arger

E"&aust p

E PI impactor
2ac* >*; 1min

+ PI impactor
Computer

2ac* @*8 1min N5) 8*@ 1min Electrostatic SEM1TEM sampler T p Porous tu#e aerosol %iluter PreE impactor

Dilution) N5 p

?ptional &eater

DMA
0rE=:

-ilter

2ac* 8*@ 1min

CPC Heater T&ermal insulation an%1or &eating tape Critical ori!ice

p T

2ac* @*8 1min

Computer

Pressure meter Temperature meter 2alve

Reactor tu#e6 Stainless steel Inner %iameter 8@ mm Heate% lengt& =@@ mm

Collison
N5 p

Pyrosol

N5 p -res&E"cess starting solution solution Solution Precursor !ee%ingsolution container

-IGURE 5 E"perimental setEup use% in t&e preparation o! nano an% microparticles* N5 ^ clean) %ry pressuri/e% nitrogen* A##reviations6 2ac*) vacuumM 1min) liters per minuteM 0rE=:) aerosol neutrali/er using =:0r fEsourceM + PI) #ernerEtype lo( pressure impactorM CPC) con%ensation particle counterM DMA) %i!!erential mo#ility analy/erM E PI) electrical lo(Epressure impactorM SEM) scanning electron microscopeM TEM) transmission electron microscope*

solutions to pro%uce nanoparticles (ere atomi/e% using a CollisonEtype air .et atomi/er ATSI 8@<;) TSI) Inc*) Particle Instruments) St* Paul) U*S*A*B* T&e atomi/er pro%uces a lognormal %roplet si/e %istri#ution (it& a geometric num#er mean %roplet si/e o! appro"imately 8@@ nm an% a geometric stan%ar% %eviation #et(een 4*; an% 5*@* T&e microparticle %roplets (ere generate% (it& an ultrasonic ne#uli/er AR+I Pyrosol <>@4) Meylan) -ranceB* T&e %roplets (ere carrie% in %ry nitrogen gas into a &eate% reactor (&ere aerosol !lo( (as laminar* Depen%ing on t&e !lo( rate) t&e resi%ence time !or %roplet1particles in t&e reactor can #e mo%i!ie%* T&e temperE ature o! t&e stainless steel reactor (as controlle% (it& !our separate &eaters* Do(nstream !rom t&e &eate% section o! t&e reactor) t&e aerosol (as %ilute% #y %ry nitrogen in a porous %iluter to avoi% solvent con%ensation onto particle sur!ace as (ell as particle %eposition to reactor (alls via t&ermop&oresis* A!ter !ully mi"ing t&e reactor an% %ilution gas !lo(s) a sample o! particles (as collecte% #y

44;
TA+ E 4 Summary o! t&e Con%itions !or t&e Pro%uction o! Nan?E an% Micronsi/e% Dry Po(%ers Polymeric %rug nanoparticles Collisona +DP) $etopro!en) napro"en Eu%ragitb 4@@) E4@@ an% RS4@@ ,ater) et&anol) TH-) toluene @*5H4*: +DPComposite Eleucine microparticlesmicroparticles Pyrosol# +DP Et&anol :H:@ 4*:H5*@ @*@>H4*; :@H4:@ 4;H54 4@H<: :@H4@@

Raula et al*

Type o! po(%er Droplet generation Materials Solvents Precursor solution concentration Ag1 B Carrier gas !lo( rate A 1minB Precursor solution consumption Am 1minB Reactor (all temperature A]CB Resi%ence time in reactor AsB -lo( rate in %ilution A 1minB Dilution gas temperature A]CB
aCollisonEtype air .et

Pyrosol# NaCl) lactose) acetyl salicylic aci%) Eleucine ,ater 4*@H5<*: 4*9 @*5=H@*9: 5@H4@@ 4>H59 <; 5@

4*:H8*: @*5H@*9 9@H5@@ ;H54 5: 5@

atomi/er*

ne#uli/er* #Ultrasonic

A##reviations6 +DP) #eclomet&asone %ipropionateM TH-) tetra&y%ro!uran*

a pointEtoEplane electrostatic precipitator AInTo" Pro%ucts) Al#uCuerCue) U*S*A*B onto eit&er a plain or car#onEcoate% copper transmission electron microscope ATEMB gri% AAgar Scienti!ic t%*) Esse") U*0*B* argeECuantity collections (ere con%ucte% #y a +ernerEtype lo(Epressure impactor A+ PIB A5;B or a smallEscale cyclone A5<B* Particle si/e an% si/e %istri#ution (ere measure% %irectly !rom t&e gas p&ase %o(nE stream !rom t&e %ilution an% mi"ing processes* Computer !lui% %ynamics calculaE tions o! t&e reactor tu#e (ere per!orme% !or %i!!erent !lo( rates g 8*: 1min (it& temperatures covering t&e temperature range use% in t&e e"periments* T&e calculaE tions s&o(e% t&at in t&e &eate% /one a !ully %evelope% laminar !lo( (as ac&ieve%) an% t&e (all temperature (as reac&e% !or all t&e particles A5=B*

Instrumentation an% C&aracteri/ation T&e particle si/e Ageometric num#er mean %iameter) GNMDB an% poly%ispersity Ageometric stan%ar% %eviation) GSDB o! t&e nanoparticles in t&e gas p&ase) t&at is) at t&e reactor outlet) (ere %etermine% (it& a TSI scanning mo#ility particle si/er eCuippe% (it& a %i!!erential mo#ility analy/er ADMA) mo%el 8@=4B ATSI) Inc*) Particle Instruments) St* Paul) U*S*A*B an% a con%ensation particle counter ACPC) mo%el 8@5<B ATSI) Inc*) Particle Instruments) St* Paul) U*S*A*B* T&e average values o! GNMD an% GSD (ere %etermine% !rom t&ree to si" measurements* T&e ma"imum stan%ar% errors (ere :[ an% @*:[ !or GNMD an% GSD) respectively* T&e GNMD an% GSD o! t&e pro%uce% microparticles (ere %etermine% (it& an electrical lo(Epressure impactor AE PIM De$ati t%*) Tampere) -inlan%B A5>)8@B* ?ile% porous collection stages ADe$ati t%*) Tampere) -inlan%B (ere use% to avoi% t&e #ouncing o! t&e particles !rom upper stages to lo(er ones* T&e GNMD an% GSD o! t&e particles (ere calculate% using eCuations GNMD ^ e"pAx An i ln DiB1NB an% GSD ^ e"pAAxni Aln Di H ln GNMDB5B1AN H 4BB415) respectively* Here ni is t&e num#er o!

Aerosol -lo( Reactor Met&o%

44<

particles in t&e it& group) D i t&e aero%ynamic %iameter o! t&e it& group) an% N t&e total num#er o! particles) t&at is) xni* Mass me%ian aero%ynamic %iameter AMMADB an% GSD o! t&e %isperse% parE ticles (ere %etermine% (it& t&e + PI an% using eCuations MMAD ^ e"pAx Am i ln DiB1M an% GSD ^ e"pAAx Ami D8i AlnDi H lnMMADB5BB1Ax AmiD8iB H4BB415) respecE tively* Here mi is t&e mass !raction o! t&e particles on t&e collection stage an% M t&e sum o! mass !ractions an% is unity* In all t&e e"periments) t&e %ensity o! t&e particle is assume% to #e 4 g1cm8* T&e morp&ology o! t&e particles (as analy/e% (it& a !iel% emission scanning electron microscope ASEMM eo DSM>=5 Gemini) E? Electron Microscopy) Inc*) ?#er$oc&en) GermanyB* Internal structure o! t&e particles (as stu%ie% using a TEM ATEMM P&ilips CM5@@ -EG) -EI Company) Ein%&oven) T&e Net&erlan%sB* Some o! t&e samples (ere coate% (it& platinum sputtering in or%er to sta#ili/e t&e particle un%er electron #eam an% to en&ance image contrast* T&e crystallinity o! nanoparticles (as stu%ie% using WEray %i!!raction AWRDM P&ilips P, 4<4@) Ein%&oven an% Almelo) T&e Net&erlan%sB (it& Cu 0m ra%iation* Di!!raction angles A5yB Agoniometer P, 4=5@B use% in t&e recor%ing o! t&e WRD patterns (ere 8] to 9@]* T&e crystallinity o! in%ivi%ual microparticles (as investiE gate% (it& TEM as (ell* T&e t&ermal properties o! t&e nanoparticles (ere stu%ie% using a %i!!erential scanning calorimeter ADSCM Mettler Tole%o DSC =55e) Mettler Tole%o AG) Grei!ensee) S(it/erlan%B (&ere t&e samples (ere &eate% !rom 5:]C to 8@@]C or !rom Z:@]C to 5@@]C using a &eating rate o! 4@]C1min* A nitrogen purge o! :@ m 1min (as use% in t&e oven*

RESU TS AND DISCUSSI?N Polymer Nanoparticles T&is section %iscusses t&e control o! t&e polymer Eu%ragit b 4@@ nanoparticle morp&ology (&en pro%uce% !rom %i!!erent solvent me%ia A54B* T&e morp&ology o! nanoparticles %epen%s on #ot& t&e %roplet %rying rate an% t&e polymer solu#ility in solvent me%ium* 2apor pressure o! t&e solvent is essential #ecause it mainly %eterE mines t&e solvent evaporation rate* 2apor pressures !or TH-) et&anol) toluene) an% (ater at 5:]C are 54*;) <*=<) 8*<>) an% 8*4< $Pa) respectively* Also) a concept o! solvent Cuality !or a polymer is note(ort&y A84B* T&e solu#ility o! Eu%ragit b 4@@ in use% solvents (as e"perimentally o#serve%) an% !oun% to %ecrease in t&e !ollo(ing or%er6 et&anol \ TH- \ toluene \ (ater* T&e !irst t(o solvents are goo% solvents !or Eu%ragit b 4@@* T&e si/e o! t&e %ry Eu%ragitb 4@@ particles !rom et&anol an% TH- solutions increase%) respectively) !rom <: to 48@ an% ;: to >: nm (it&in t&e concentration range !rom @*5 to 4*: g1 * Accor%ingly) t&e GSD o! t&e particles) t&at is) t&e sprea% o! t&e si/e %istri#ution o! t&e particles !rom TH- increase% !rom 4*> to 5*5 #ut t&at !rom et&anol remaine% t&e same) z4*>* ,it& a%%e% (ater) t&e solvent Cuality !or t&e polymer (orsE ens) t&at is) t&e polymer coil s&rin$s in t&e solution prior to atomi/ation* As a result) t&e si/e o! %ry nanoparticles prepare% !rom et&anol1(ater ^ 464 solution %ecrease% appro"imately to t(oEt&ir%s o! t&e particles !rom pure et&anol* Moreover) t&e si/es !rom TH-1(ater ^ 464 solutions at @*5 an% 4*@ g1 %ecrease%) respectively) to !ourE !i!t&s an% t(oEt&ir%s o! t&e particles !rom pure TH-* Polymer particles !rom toluene an% (ater (ere small (it& t&e particle si/e aroun% 8: nm an% t&e GSD less t&an 4*>* -igure 8 s&o(s e"emplary SEM images o! t&e particles prepare% !rom %i!!erent solvent me%ia* T&e !igure summari/es t&e !actors in!luencing t&e particle !ormaE tion* T&ree main routes are %iscusse%6 AAB NonE&ollo( soli% particles can #e o#taine%

44=

Raula et al*

%rug an%1or polymer %issolve% in %roplet Toluene) sat1!ilt

C
Cp{

A + #

Et&anol) @*5 g1

4@@ nm

5@@ nm

TH-6H5?^46>) @*5 g1

Et&anol) 4*@ g1

! % e
5@@ nm

c
TH-) @*: g1
:@@ nm

TH- 4*@ g1

TH-6H5?^>64) @*5 g1

5@@ nm :@@ nm

:@@ nm

-IGURE 8 Sc&eme %escri#es t&e in!luence o! t&e e"perimental parameters on t&e particle !ormation an% resulting particle morp&ology* A %roplet containing %rug an%1or polymer %ries in t&e reactor mainly in t&ree (ays6 A) soli%i!ying) +) !ilm !ormation) an% C) early precipitation* Along t&e soli% arro( is t&e solvent vapor pressure) solvent evaporation rate ) an% polymer concentration increase* Along t&e %as&e% arro( is t&e solvent Cuality !or t&e polymer AEu%ragit b 4@@B %ecreases*

(&en solventAsB evaporates slo(ly* T&e particles are o!ten sp&erical* A+B I! solvent evaporation is !ast) t&e soluteAsB !orms a crust or !ilm on t&e %roplet sur!ace* I! t&e crust is impermea#le to solvent) t&e particle (ill !orm a &ollo( interior %ue to presE sure #uil%Eup on !urt&er solvent evaporation) (&ic& e"pan%s t&e particle A85H8;B* Hollo( sp&erical particles can also collapse to raisinE or lensEs&ape% particles ASEM images c an% % in -ig* 8B A89)8<H8>B* ACB Polymer #eing close to its solu#ility limit in solvent prior to atomi/ation precipitates at very early stages o! solvent evaporation* -or clari!ication) t&e solvent evaporation rate an% polymer concentration increase along a curve% soli% arro(* ,&en !ollo(ing t&e curve% %as&e% arro() t&e solvent Cuality !or t&e polymer (orsens* At some point o! %roplet %rying) t&e polymer {reac&es its solu#ility limit AC pB an% precipitates Aimage g in -ig* 8B* T&ese particles &ave o!ten very irregular s&ape* In #et(een t&ese t(o e"tremes) t&e particles may &ave very unusual morp&ologies) suc& as #listere% Aimage ! in -ig* 8B an% (rin$le% Aimage e in -ig* 8B structures A54B*

Polymeric Drug Nanoparticles T&is section %iscusses t&e pro%uction o! polymeric %rug nanoparticles (&ere t&e %rug an% t&e polymer !orm a soli% solution A4=)4>B* T&e morp&ological sta#ility o! t&e nanoparticles) t&at is) (&et&er t&e state o! matter o! t&e %rug (ill c&ange %uring storage) %epen%s on t&e polymer use% an% (ill #e %iscusse%* Ta#le 5 lists t&e e"periE mental con%itions as (ell as t&e p&ysical properties o! some o! t&e polymeric %rug nanoparticles* T&e solvent (as et&anol in every solution* Nanoparticles prepare%

Aerosol -lo( Reactor Met&o%

44>

TA+ E 5 Summary o! t&e Con%itions an% P&ysical C&aracteristics o! Some o! t&e Drug Nanoparticles Pro%uce% Drug material 0etopro!en @[ 0etopro!en 4@[ 0etopro!en 5:[ 0etopro!en 88[ 0etopro!en :@[ 0etopro!en ;<[ 0etopro!en 88[ 0etopro!en 88[ Napro"en :@[ Napro"en :@[ Napro"en :@[ Napro"en :@[ Napro"en :@[ Eu%ragitb 4@@ 4@@[ 4@@ >@[ 4@@ <:[ 4@@ ;<[ 4@@ :@[ 4@@ 88[ E4@@ ;<[ RS ;<[ 4@@ :@[ 4@@ :@[ 4@@ :@[ 4@@ :@[ 4@@ :@[ C%rug Ag1 B 5 5 5 5 5 5 5 5 4 5*: : 4@ 5: Treactor A]CB =@ =@ =@ =@ =@ =@ =@ =@ =@ =@ =@ =@ =@ GNMD AnmB 45@ 45@ 44; 445 4@= 4@< 4@; >; >9 459 48; 4;@ 5@4 GSD 4)= 4)= 4)= 4)> 4)> 4)> 4)< 4)< 4)= 4)= 4)= 4)< 4);

Drug concentration in precursor solution ACDRUGB an% reactor (all temperature ATREACT?RB (&en pro%ucing %rugE polymer composite nanoparticles are s&o(n* Also geometric num#er mean %iameter an% geometric stan%ar% %eviation o! t&e particle si/e %istri#ution as %etermine% (it& t&e %i!!erential electrical mo#ility met&o% are s&o(n* A##reviations6 GNMD) geometric num#er mean %iameterM GSD) geometric stan%ar% %eviation*

(it& various proportions o! $etopro!en an% Eu%ragit b 4@@ at t&e reactor (all temperature o! =@]C s&o(e% a %ecreasing particle si/e as a !unction o! t&e amount o! $etopro!en* ,it& t&e same amount) 88[ A(1(B o! $etopro!en #ut a %i!!erent polymer) t&e particle si/e %ecrease% in or%er Eu%ragit b 4@@ \ Eu%ragitb E4@@ \ Eu%ragitb RS* T&e e!!ect o! solution concentration on particle si/e is also s&o(n in Ta#le 5* T&e amorp&ous !orm o! t&e %rug &as a ten%ency to spontaneously convert to a crystalline !orm A9@)94B* T&e structural sta#ili/ation) &o(ever) can #e ac&ieve% #y incorporating t&e %rug into t&e polymer matri"* PolymerE%rug nanoparticles (ere prepare% !rom t&e %rug materials +DP A4=B) napro"en A4>B) an% $etopro!en A4>B* -or napro"en an% $etopro!en) it (as o#serve% t&at t&ere is a limit to &o( muc& %rug can #e incorporate% in an amorp&ous !orm into nanoparticles* ,&en t&e amount o! t&e %rug in t&e nanoparticles (as !or $etopro!en g 88[ A(1(B an% !or napro"en g 4@[) t&e nanosp&eres (ere amorp&ous) prove% #y WRD) DSC) an% TEM* No crystalE linity or grain #oun%aries (ere !oun%M instea%) t&e nanoparticles (ere smoot& (it& a uni!orm interior* T&e !ormation o! an amorp&ous) polymerH%rug structure &as previously #een o#serve% !or sprayE%rie% particles containing met&acrylic polyE mers an% $etopro!en %rug A95)98B* ,&en t&e amount o! t&e %rug in t&e nanoparticles (as increase% to :@[ A(1(B $etopro!en an% 5:[ napro"en) an en%ot&ermic transiE tion correspon%ing to melting o! t&e %rug crystals (as %etecte% (it& DSC A-ig* 9B* Moreover) in t&e WRD analyses) t&e #roa% #ac$groun% %i!!raction pattern o! t&e amorp&ous structure #ecame overlappe% #y pea$s correspon%ing to t&e %i!!raction !rom t&e %rug crystal lattice* Ho(ever) t&e %rug molecules on t&e particle sur!ace crystalli/e%) t&us !orming large crystalline #ri%ges #et(een neig&#oring nanoparticles) t&at is) p&aseEtransitionEin%uce% sintering o! t&e nanoparticles occurre%* Molecular si/e (as assume% to a!!ect %rug crystalli/ation %uring particle !orE mation* +DP AM( :54 g1molB is a larger molecule t&an $etopro!en A5:9 g1molB or napro"en A58@ g1molB molecules* T&e molecular mo#ility o! +DP (as e"pecte% to #e slo( %ue to its large si/e an% &ig& glass transition temperature AT g ;;]CB A99)9:B*

45@

Raula et al*

-IGURE 9 DSC t&ermograms o! t&e Eu%ragitb 4@@ nanoparticles (it& %rugs #eclomet&asone %ipropionate A+DPB) $etopro!en) an% napro"en Amar$e% in t&e !iguresB* T&e content o! +DP AaB 4@@[ A(1(B) A#B =@[) AcB ;@[) A%B :@[) AeB 9@[) A!B 5@[) an% AgB @[* T&e content o! $etopro!en AaB 4@@[) A#B :@[) AcB 88[) A%B 5:[) AeB 4@[) A!B @[* T&e content o! napro"en AaB 4@@[) A#B :@[) AcB 88[) A%B 5:[) AeB 4@[) A!B @[* A##reviations6 cr) crystalli/ationM m) meltingM gt) glass transition*

T&e glass transition temperature) Tg) (as calculate% using t&e relation Tg z @*< Tm (&ere temperatures (ere in 0elvin A9;)9<B) a melting temperature) T m) measure% #y DSC A4=B* T&us) t&e amorp&ous state o! pure +DP nanoparticles) t&at is) no a%%e% polymer) is $inetically preserve% even t&oug& it is not t&ermo%ynamically sta#le* T&e nanoparticles containing +DP #ot& (it& an% (it&out polymer s&o(e% single) separate nanoparticles* Crystalli/ation o! +DP in t&e nanoparticles coul% #e in%uce% #y &eating) (&ic& can #e seen as an e"ot&erm in DSC measurements A-ig* 9B* In t&e amorp&ous structure) t&e %rug is solu#ili/e% #y t&e polymer) an% t&e t(o components !orm an amorp&ous soli% solution A9=B* T&e interactions #et(een t&e %rug an% t&e polymer molecules %etermine t&e solu#ility o! t&ese materials in eac& ot&er A9>B* ,&en t&e %rugHpolymer molecular interactions are compara#le to t&e %rugH%rug an% t&e polymerHpolymer interactions) t&e %rug is (ell solu#ili/e% #y t&e polymer an% large amounts o! %rug can #e incorporate% in t&e polymer matri" (it&out %rug crystalli/ation A:@B* Ho(ever) (&en t&e %rugHpolymer interactions are (ea$er t&an t&e %rugH%rug or t&e polymerHpolymer interactions) t&e %rug an% t&e polymer &ave a pre!erence to interact (it& t&e molecules o! t&eir o(n $in%) lea%ing to a potential !or %rug crystalli/ation* T&e amount o! %rug #elo( t&e soluE #ility limit o! t&e %rug in t&e polymer can #e solu#ili/e% #y t&e polymer matri" A:4):5B* ?n increasing t&e amount o! t&e %rug over t&is limit) t&e %rug is no longer solu#le in t&e amorp&ous polymer) #ut can !orm separate crystallites A:8B* An analE ogy to t&e solu#ility o! t&e %rug in t&e polymer can #e !oun% !rom plastici/ation) (&ere !le"i#le small molecules are mi"e% (it& t&e polymer to lo(er t&e T g A:8H::B* Also in plastici/ation) it is essential t&at t&e plastici/ing material !orms a uni!orm mi"ture (it& t&e polymer) an% is solu#ili/e% #y t&e polymer* ConseCuently) !rom t&is point o! vie() $etopro!en (as a more e!!ective plastici/ing agent !or t&e Eu%ragitb 4@@ polymer t&an napro"en A:8B* T&us) t&e c&oice o! t&e polymer is important !or controlling t&e amorp&ousHcrystalline transition* T&e nanoparticles (ere store% at t&ree %i!!erent con%itions to stu%y t&e in!luence o! temperature an% relative &umi%ity on t&e morp&ology o! particles* T&e nanoparticles containing 88[

Aerosol -lo( Reactor Met&o%

454

A(1(B o! $etopro!en an% ;<[ o! Eu%ragitb 4@@ polymer (ere store% !or t&ree mont&s in a re!rigerator) at 5:]C an% @[ o! relative &umi%ity) an% at 5:]C an% <:[ o! relative &umi%ity Asat* NaClB* A!ter storage !or t&ree mont&s) no c&anges in t&e morp&ology o! t&e nanoparticles (ere o#serve%) t&at is) still sp&eres (ere o#serve%* As (ell) no crystalli/ation o! t&e %rug (it&in t&e nanoparticle occurre%* To conE clu%e) t&ese nanoparticles (ere p&ysically sta#le in all t&e con%itionsM &o(ever) c&emical sta#ility (as not stu%ie% %uring t&is perio% o! time*

Nanostructure% Drug Microparticles Drug Crystalli/ation in t&e Reactor Amorp&ous materials ten% to crystalli/e over storage causing pro#lems in insta#ilE ity #ut also in po(%er &an%ling* T&ere!ore) t&e crystalline !orm o! t&e %rug is pre!erre%* T&e e"perimental con%itions to crystalli/e #eclomet&asone %ipropionate particles (&ile pro%ucing t&em (ill #e %iscusse% in t&is section A:;B* T(o solutions) : an% 5: g1 ) (ere atomi/e% in t&e &eate% reactor at %i!!erent temperatures* Ta#le 8 summari/es t&e p&ysical c&aracteristics o! t&e particles* T&e saturate% precursor solutions prior to atomi/ation inclu%e% crystal see%s t&at (ere e"pecte% to in%uce t&e particle crystalli/ation* T&e si/es o! t&e po(%ers (ere in a respira#le si/e range A4H: _mB an% t&e si/e %istri#utions (ere relatively narro( Ai*e*) GSD c5*4B* T&e main issue o! t&is (or$ (as to e"plore t&e reactor con%itions a!!ecting t&e particle crystalli/ation (&ile pro%ucing t&em* Ta#le 8 also lists t&e state o! matter Acrystalline an%1or amorp&ousB in t&e !inal po(%ers* o( solution concentration as (ell as lo( reactor temperature resulte% in smoot&Esur!ace% sp&eres t&at (ere amorp&ous* Ho(ever) t&e increase in reactor temperature seeme% to promote t&e crystalli/ation process in t&e reactor* Appearance o! roug&ness on t&e particle sur!ace (as in%icative o! crystals A-ig* :AB) an% it (as) in !act) prove% #y t&e WRD analysis* -or comparison) !ully amorp&ous) smoot&Esur!ace% +DP po(%er is s&o(n in -igure :+* T&e evaporation o! et&anol !rom t&e %roplets is $inetically !aster t&an t&e organi/ation o! t&e %rug molecules to crystals* T&ere!ore) t&e +DP po(%ers (ere o!ten amorp&ous* Ho(ever) t&e %i!!usion o! t&e +DP molecules in t&e amorp&ous soli% state %epen%s on Tg an% t&e overall rate o! crystalli/ation* T&e latter is at its ma"imum at t&e mi%Etemperature #et(een T g an% Tm A94B* As t&e Tg an% Tm (ere)

TA+ E 8 Summary o! t&e Pro%uction Con%itions an% P&ysical C&aracteristics o! t&e +eclomet&asone Dipropionate Po(%ers Prepare% !rom Et&anolic Solutions C+DP Ag1 B :a 5:a 5:# 5:a Saturate%# Treactor A]CB 4:@ :@ 4@@ 4:@ 4@@ MMAD A_mB 4*9 5*: 4*< 5*5 5*4 GSD 4*= 5*@ 5*@ 5*@ 5*4 State o! matterc Amorp&ous Amorp&ous Some crystallites Crystalline Crystalline core

T&e concentration o! +DP in precursor solution AC+DPB an% reactor (all temperature AT reactorB (&en proE %ucing %rug microparticles are s&o(n* Also mass me%ian aero%ynamic %iameter an% geometric stan%E ar% %eviation o! t&e particle si/e %istri#ution as %etermine% (it& t&e +ernerEtype lo( pressure impactor are s&o(n* a-lo( rate 4*: 1min t&roug& pyrosol* #-lo( rate 4@ 1min t&roug& pyrosol) t&en %ivi%e% into : tu#es (&ere 5 1min* cDirectly !rom t&e pro%uction* A##reviations6 +DP) #eclomet&asone %ipropionateM GSD) geometric stan%ar% %eviationM MMAD) mass me%ian aero%ynamic %iameter*

455

Raula et al*

-IGURE : SEM images o! t&e #eclomet&asone %ipropionate po(%ers in %i!!erent state o! matter6 AAB roug& an% crystalline an% A+B smoot& an% amorp&ous*

respectively) ;;]C Asee t&e relation a#oveB an% 545]C) t&e ma"imum crystalli/aE tion rate (as e"pecte% to #e aroun% 48>]C* T&is e"plains t&e signi!icance o! reactor temperature !or t&e particle crystalli/ation* T&ermal treatment accelerate% crystal gro(t& in t&e sites o! crystal nucleation* T&e slurry solution (it& crystal nuclei (as e"pecte% to promote &eterogeneous nucleation an% %irecting t&e crystal gro(t& A:<B* T&e particles pro%uce% at 4@@]C (ere sp&eres (it& smoot& particle sur!ace t&at is a strong in%ication o! amorp&ous state* Ho(ever) t&e WRD analysis s&o(e% t&at t&e po(%er (as crystalline* T&e particles containe% crystalline core o! t&e original crystal see%s* T&e crystals (ere covere% #y amorp&ous %rug material* Postannealing o! t&e collecte% amorp&ous particles at various temperatures initiate% an%1or accelerate% crystalli/ation* T&e crystalli/ation un!ortunately e"ten%e% #et(een neig&#oring particles !orming crystalline #ri%ges #et(een t&em* As po(%ers ten% to agglomerate an% !orm clusters) t&e crystal #ri%ge !ormation (as inevita#le* Ho(ever) t&e agglomerates can #e #ro$en easily to in%ivi%ual particles #y mil% milling*

Coating o! Particles #y a Pepti%e T&e morp&ology as (ell as sta#ility o! t&e particles can #e mo%i!ie% (it& a sur!aceE active pepti%e suc& as lEleucine A:=B* T&is is analog to t&e sta#ili/ation o! %rug nanoparticles #y polymers as %iscusse% earlier* T&e materials c&osen !or t&e prepaE ration o! t&e composite lEleucine po(%ers &ave %i!!erent p&ysical an% c&emical properties* NaCl crystalli/es (it& ease) (&ereas large lactose molecules are not a#le to organi/e %uring t&e %roplet %rying an% t&e po(%ers are amorp&ous in t&e collecE tion* Moreover) using t&e relation T g @*< Tm) t&e Tg o! ASA (as estimate% to #e rat&er lo() aroun% 48]C* Accor%ingly) pure ASA microparticles s&oul% &ave poor p&ysical sta#ility (&en collecte%* eucine %erivatives are sur!aceEactive materials in aCueous solutions* +esi%es t&e sur!ace tension measurements) t&e sur!ace activity o! a compoun% can #e %eterE mine% #y sur!ace accumulation parameters t&at re!lect t&e %egree o! &y%rop&o#icE ity o! t&e compoun%* T&is is calculate% #y t&e eCuation ZA|}1|cB) (&ere a c&ange in sur!ace tension |} is measure% as a !unction o! solution concentration |c A:>B* ?n t&e !lat sur!ace) t&e ZA|}1|cB values !or glycine) SE) lE) %iE) an% triEleucine are Z@*> A} values !rom Re!* :>B) 9@ A} values !rom Re!* :>B) 5@ A} values !rom Re!* ;@B) =<@ A} values !rom Re!* 59B) an% 4949 A} values !rom Re!* 59B) respectively* T&e accumulaE tion o! t&e sur!aceEactive molecules on %roplet sur!ace is most pro#a#ly en&ance%

Aerosol -lo( Reactor Met&o%

458

-IGURE ; SEM image Ale!t B o! t&e pure Eleucine particles prepare% !rom aCueous 4: g1 solution* T&e sc&eme on t&e rig&t presents t&e sel!Eassem#ling o! Eleucine molecules on t&e sur!ace o! t&e %roplet at t&e inter!ace o! (ater an% air*

%ue to a &ig& sur!aceEtoEvolume ratio o! t&e %roplet* Pure lEleucine particles &a% sp&erical #ut strongly (rin$le% structure Asee an SEM image in -ig* ;B* lE eucine molecules gat&ere% at t&e airH(ater inter!ace on t&e %roplet t&us !orm a !ilm* T&is !ilm increasingly prevents t&e evaporative removal o! (ater as it t&ic$ens* A %rying particle e"pan%s (&ile (ater molecules penetrate t&roug& t&e lEleucine !ilm an% a!ter complete removal o! (ater t&e !ilm collapses to give (rin$le% morp&ology Asee t&e sc&eme in -ig* ;B* During t&e sur!ace organi/ation) t&e lEleucine molecules crystalli/e to some e"tent giving partly polycrystalline structure Asee %i!!raction patterns in t&e inset o! -ig* ;B* Ta#le 9 summari/es t&e p&ysical c&aracteristics o! t&e pro%uce% composite po(%ers* T&e si/e o! saline composite particles increase% slig&tly (it& t&e a%%e% lEleucine an% t&e sprea% o! si/e %istri#ution %i% not vary) (it& GSD #eing aroun% 5*:* ,it& t&e a%%e% lEleucine !rom @ to <*: g1 ) &o(ever) t&e si/e o! lactose particles %ecrease% nota#ly !rom @*> to #elo( @*: _m) (&ereas t&e si/e %istri#ution #roa%ene% an% GSD increase% !rom 4*> to 8*=*

TA+ E 9 Summary o! t&e Pro%uction Con%itions an% P&ysical C&aracteristics o! t&e Eleucine Composite Po(%ers Material Eleucine NaCl 5@ g1 NaCl 5@ g1 NaCl 5@ g1 actose 5@ g1 actose 5@ g1 actose 5@ g1 ASA 4: g1 C
Eleucine Ag1

GNMD A_mB @*;< @*:9 @*;9 @*;; @*>4 @*:5 @*:4 @*98

GSD 5*4 5*< 5*; 5*: 4*> 8*: 8*= 4*>

State o! matter Partly polycrystallinea Crystallinea)# Crystalline# Crystalline# Amorp&ousa)# Amorp&ous# Amorp&ous# Amorp&ous#

4: @ 5*: <*: @ 5*: <*: <*:

T&e concentrations o! Eleucine in aCueous precursor solutions AC EleucineB (&en pro%ucing %rug microparticles at t&e reactor (all temperature o! 4@@ ]C are s&o(n* Also geometric num#er mean %iameter an% geometric stan%ar% %eviation o! t&e particle si/e %istri#ution as %etermine% (it& t&e electrical lo( pressure impactor AE PIB are s&o(n* aTEM* #WRD* A##reviations6 GNMD) geometric num#er mean %iameterM GSD) geometric stan%ar% %eviation*

459

Raula et al*

-IGURE < SEM images o! AAB NaCl) A+B NaCl1 Eleucine) ACB lactose) an% ADB lactose1 Eleucine parE ticles* In t&e precursor solutions t&e concentration o! NaCl an% lactose (as 5@ g1 ) an% t&at o! EleuE cine (as <*: g1 *

T&e morp&ology o! t&e composite particles (as controlle% #y t&e amount o! lEleucine* T&e sur!ace o! lactose particles c&ange% !rom smoot& to (rin$le%) (&ereas cu#ic crystalline salt particles c&ange% !rom sp&eroi%al to lea!y ones (it& increasing lEleucine content A-ig* <AHDB* T&e WRD stu%ies s&o(e% t&at t&e pro%uce% po(%ers (ere amorp&ous e"cept saline composite particles t&at &ave t&e strong %i!!raction pea$s o! NaCl at 84*<] an% 9:*:] A5yB* T&e structure o! t&e NaCl composite particles (as stu%ie% #y t&e evaporative removal o! lEleucine in t&e oven at 5@@]C !or 4@ minutes ATsu# o! lEleucine is z49:H49=]CB A;4B* A!ter t&e t&ermal treatment) t&e remaining particle (as a salt cu#e (it& &oles on t&e sur!ace* T&is s&o(e% t&at t&e lEleucine molecules !orme% t&e s&ell aroun% t&e salt core) partly mi"ing (it& salt core* T&e structure o! lactose composite po(%er coul% not stu%ie% #ecause t&e Tg o! lactose mono&y%rate Az4@4]CB (as lo(er t&an t&e temperature !or lEleucine removal* T&e materials t&at &ave Tg #elo( t&e collection temperature an% %o not crysE talli/e in t&e reactor cannot #e collecte% as compact in%ivi%ual particles* A#ove T g) t&e material is in ru##ery state) an% is so!t an% even !lui%ly* Pure ASA particles pro%uce% !rom an et&anolic solution A4: g1 B (ere !oun% !lat an% crystallineRa roug& particle sur!ace is in%icative o! crystallinityRon t&e collector sur!ace* T&ey !latten %uring collection (it& t&e !ormation o! ru##ery !ilm t&at su#seCuently crysE talli/es a!ter collection* T&us) lEleucine &as #een use% to sta#ili/e t&e structure o! t&e ASA particles* T&e aCueous lEleucine solution (as mi"e% (it& t&e et&anolic ASA solution an% t&en %roplets (ere pro%uce%* At lo( lEleucine concentration A5*: g1 B) some !lat particles (ere still o#serve% A-ig* =AB #ut at t&e concentration o! <*: g1 all t&e particles (ere sp&erical an% compact (it& porous structure A-ig* =+B*

Aerosol -lo( Reactor Met&o%

45:

-IGURE = SEM images o! acetyl salicylic aci% composite particles prepare% !rom aCueous et&anol solution* T&e concentration o! ASA (as 4: g1 an% t&at o! Eleucine (as AAB 5*: g1 an% A+B <*: g1 in et&anol1(ater solution* -lat an% crystalline particles (&ere Eleucine content is lo( A5*: g1 B are circle% in t&e image*

T&e p&ysical sta#ility) t&at is) morp&ological c&anges o! t&e po(%ers (ere e"amine% in t(o con%itions6 at 5:]C an% @[ relative &umi%ity) an% at 5:]C an% 99[ relative &umi%ity Asat* 05C?8B !or t&ree mont&s* A!ter t&ree mont&s) pure lEleucine as (ell as all saline particles s&o(e% no c&anges in morp&ology* Ho(ever) pure lactose po(%er s&o(e% nec$ !ormation #et(een particles even at @[ relative &umi%ity #ut t&is %i% not occur (it& lEleucineEcontaining lactose particles* T&e sur!ace o! lactose particles (as &ar%ene% #y an lEleucine layer an% suc& #ri%ges #et(een neig&#oring particles (ere not o#serve%*

C?NC USI?NS T&e aerosol !lo( reactor met&o% is a novel met&o% to pro%uce %rug particles !rom atomi/e% solutions* T&e reactor can #e mo%i!ie% to pro%uce particles o! %i!!erent si/es) t&at is) nanoE or microparticles* T&is met&o% &as #een use% to prepare sp&erical an% amorp&ous matri"Etype %rugHpolymer nanoparticles as (ell as micrometerEsi/e% %ry po(%ers !or %ry po(%er in&alation* E"perimental con%iE tions in!luence% %rastically t&e particle !ormation an% morp&ology* Solvent vapor pressure as (ell as t&e crust !ormation on t&e %roplet sur!ace mainly %etermine% t&e resulting morp&ology o! t&e polymer nanoparticles* Moreover) t&e solute solu#ility in t&e starting solution also a!!ecte% t&e particle s&ape* T&e state o! %rug in t&e polymer nanoparticles %epen%s on t&e %rug molecule itsel!) t&at is) si/e an% c&emical structure* ,it& increasing si/e o! t&e molecule) t&e %i!!usion (it&in t&e polymer matri" %ecrease%* As it (as s&o(n) large %rug molecule +DP (as amorE p&ous in all particles) (&ereas smaller molecules $etopro!en an% napro"en (ere amorp&ous to some e"tent* As t&e num#er o! t&ese latter molecules increase%) t&ey crystalli/e% an% t&e nanoparticles coalesce%* T&e %i!!erence in t&e interaction #et(een t&e %rug an% t&e polymer (as o#serve% as t&e c&ange o! t&e glass transition temperature o! t&e particles* T&e %rug crystalli/ation %epen%e% on t&e t&ermal &istory e"perience% in t&e reactor* T&e molecular mo#ility increase% (it& increasing reactor temperature t&at in some cases pro%uce% !ully or partly crystalE line %rug particles* Particle morp&ology an% sur!ace properties o! t&e microparticles can #e mo%i!ie% #y t&e pepti%e lEleucine* ?(ing to t&e sur!ace activity o! t&e pepti%e in

45;

Raula et al*

aCueous solutions) it is also possi#le to sta#ili/e t&e structure o! t&e %rug particles t&at are ot&er(ise structurally unsta#le* AC0N?, EDGMENT
-inancial support !rom t&e Aca%emy o! -inlan% is grate!ully ac$no(le%ge%*

RE-ERENCES
4* E%elstein AS) Cammarata RC* Nanomaterials6 Synt&esis) Properties an% Applications* +ristol6 ', Arro(smit& t%*) 4>>;64* 5* Ravi 0umar MN2* Nano an% microparticles as controlle% %rug %elivery %evices* ' P&arm P&armaceut Sci 5@@@M 86589) an% re!erences t&erein* 8* +rigger I) Du#ernet C) Couvreur P* Nanoparticles in cancer t&erapy an% %iagnosis* A%v Drug Deliv Rev 5@@5M :96;84* 9* Galaev I3u) Mattiasson +* T&ermoreactive (aterEsolu#le polymers) nonionic sur!actants) an% &y%rogels as reagents in #iotec&nology* En/yme Micro# Tec&nol 4>>8M 4:68:9* :* 3u$ SH) +ae 3H* P&aseEtransition polymers !or %rug %elivery* Crit Rev T&er Drug Carrier Syst 4>>>M4;68=:* ;* AllGmann E) Curny R) Doel$er E* DrugEloa%e% nanoparticlesRpreparation met&o%s an% %rug targeting issues* Eur ' P&arm +iop&arm 4>>8M 8>64<8* <* AllGmann E) Doel$er E) Curny R* Drug loa%e% polyAlactic aci%B nanoparticles pro%uce% #y a reversi#le saltingEout process6 puri!ication o! an in.ecta#le %osage !orm* Eur ' P&arm +iop&arm 4>>8M 8>648* =* -essi H) Puisieu" -) Devissaguet 'P) Ammoury N) +enita S* Nanocapsule !ormation #y inter!acial polymer %eposition !ollo(ing solvent %isplacement* Int ' P&arm 4>=>M ::6R4* >* Ni(a T) Ta$euc&i H) Hino T) No&ara M) 0a(as&ima 3* +io%egra%a#le su#micron carriE ers !or pepti%e %rugs6 preparation o! %)lElacti%e1glycoli%e copolymer AP GAB nanoE sp&eres (it& na!arelin acetate #y a novel emulsionEp&ase separation met&o% in an oil system* Int ' P&arm 4>>:M 45469:* 4@* C&en W) 3oung T') Sar$ari M) ,illiams R?) 'o&nston 0P* Preparation o! cyclosporine A nanoparticles #y evaporative precipitation into aCueous solution* Int ' P&arm 5@@5M 59568* 44* iversi%ge GG) Cun%y 0C* Particle si/e re%uction !or improvement o! oral #ioavaila#ilE ity o! &y%rop&o#ic %rugs* I* A#solute oral #ioavaila#ility o! nanocrystalline %ana/ol in #eagle %ogs* Int ' P&arm4>>:M 45:6>4* 45* +o%meier R* Polymeric microsp&eres prepare% #y spraying into compresse% car#on %io"i%e* P&arm Res 4>>:M 4564544* 48* +roa%&ea% ') Pouan S0E) R&o%es CT* T&e spray %rying o! p&armaceuticals* Drug Dev In% P&arm 4>>5M 4=644;>* 49* ,atana#e ,) 0auppinen EI) A&onen P) +ro(n DP) Muttonen E* Com#ination %rugs !or t&e treatment o! ast&ma* Patent Application -I 5@@@554;) 5@@@* 4:* ,atana#e ,) 0auppinen EI) A&onen P) +ro(n DP) Muttonen E* Com#ination particles* Patent Application -I 5@@@554:) 5@@@* 4;* ,atana#e ,) A&onen P) 0auppinen EI) et al* In&alation particles* Patent Application ,? ,orl% IP? @49>5;8) 5@@4* 4<* Eeri$Iinen H) ,atana#e ,) 0auppinen EI) A&onen PP* Aerosol !lo( reactor met&o% !or synt&esis o! %rug nanoparticles* Eur ' P&arm +iop&arm 5@@8M ::68:<* 4=* Eeri$Iinen H) 0auppinen EI* Preparation o! polymeric nanoparticles containing corticosteroi%s #y a novel aerosol !lo( reactor met&o%* Int ' P&arm 5@@8M 5;86;>* 4>* Eeri$Iinen H) 0auppinen EI) 0ansi$as '* Polymeric %rug nanoparticles prepare% #y an aerosol !lo( reactor met&o%* P&arm Res 5@@9M 54648;* 5@* 0auppinen EI) Eeri$Iinen H) +ro(n DP) Raula ') Hua '* Nanoparticles an% a met&o% !or t&e preparation o! nanoparticles* Patent -I5@@844=8) 5@@8* 54* Raula ') Eeri$Iinen H) 0auppinen EI* In!luence o! t&e solvent composition on t&e morp&ology o! polymer %rug composite nanoparticles* Int ' P&arm 5@@8M 5=9648* 55* S&u$la A'* Polymet&acrylates* In6 ,a%e A) ,eller P') e%s* Han%#oo$ o! P&armaceutical E"cipients* ,as&ington) DC6 P&armaceutical Press) 4>>968;5H8;;*

Aerosol -lo( Reactor Met&o%

45<

58* Dittgen M) Durrani M) e&mann 0* Acrylic polymers6 a revie( o! p&armaceutical applications* STP P&arma Sci 4>><M <69@8* 59* ec&ugaE+allesteros D) 0uo MEC* Dry po(%er compositions &aving improve% %ispersivity* ,? ,orl% IP? @4185499) 5@@4* 5:* C&e( N30) C&an HE0* E!!ect o! &umi%ity on t&e %ispersion o! %ry po(%ers* In6 Dal#y RN) +yron PR) -arr S') Peart ') e%s* Respiratory Drug Delivery 2II* Raleig&6 Serentec Press) 5@@@6;4:* 5;* Hillamo R) 0auppinen EI* ?n t&e per!ormance o! t&e +erner lo( pressure impactor* Aerosol Sci Tec&nol 4>>4M 49688* 5<* V&u 3) ee 0,* E"perimental stu%y on small cyclones operating at &ig& !lo( rates* ' Aerosol Sci 4>>>M 8@648@8* 5=* +ro(n DP* Development o! a t&reeE%imensional couple% !lo() species an% aerosol mo%el6 applications to particle %eposition in gas tur#ines an% aerosol !ormation an% gro(t& in .et engine e"&austs* P&*D* T&esis* University o! Cincinnati) Cincinnati) USA) 4>>;* 5>* 0es$inen ') Pietarinen 0) e&timI$i M* Electrical lo( pressure impactor* ' Aerosol Sci 4>>5M 5868:8* 8@* Moisio M* Real time si/e %istri#ution measurement o! com#ustion aerosols* P&*D* T&esis* Tampere University o! Tec&nology) Tampere) -inlan%) 4>>>* 84* Gros#erg A3u) 0&o$&lov AR* Giant Molecules) Here) T&ere) an% Every(&ere* USA6 Aca%emic Press) 4>><M 44<64@>H45:* 85* C&e S) Sa$urai ?) S&ino/a$i 0) Mi/utani N* Particle structure control t&roug& intrapartiE cle reactions #y spray pyrolysis* ' Aerosol Sci 4>>=M 5>65<4* 88* V&ou WD) V&ang SC) Hue#ner ,) ?(n#y PD) Hongc&en Gu* E!!ect o! t&e solvent on t&e particle morp&ology o! spray %rie% PMMA* ' Mater Sci 5@@4M 8;68<:>* 89* Esposito E) Roncarati R) Cortesi R) Cervellati -) Nastru//i C* Pro%uction o! Eu%ragit microparticles #y sprayE%rying tec&niCue6 in!luence o! e"perimental parameters on morE p&ological an% %imensional c&aracteristics* P&arm Dev Tec&nol 5@@@M :65;<* 8:* ,ang -E') ,ang CEH* Sustaine% release o! etani%a/ole !rom spray %rie% microsp&eres prepare% #y nonE&alogenate% solvents* ' Control Release 5@@5M =465;8* 8;* Gurav A) 0o%as T) Pluym T) Wiong 3* Aerosol processing o! materials* Aerosol Sci Tec&nol 4>>8M 4>6944* 8<* Maa 3E-) Nguyen PEAT) Hsu S,* SprayE%rying o! airHliCui% inter!ace sensitive recomE #inant &uman gro(t& &ormone* ' P&arm Sci 4>>=M =<64:5* 8=* eong 0H* Morp&ological control o! particles generate% !rom t&e evaporation o! soluE tion %roplets6 e"periment* ' Aerosol Sci 4>=<M 4=6:5:* 8>* Giunc&e%i P) Torre M ) Maggi ) Conti +) Conte U* Cellulose acetate trimetillate et&ylcelE lulose #len%s !or nonEsteroi%al antiEin!lammatory %rug ANSAIDB microsp&eres* ' Microencapsul 4>>;M 486=>* 9@* 3u * Amorp&ous p&armaceutical soli%s6 preparation) c&aracteri/ation an% sta#ili/ation* A%v Drug Deliv Rev 5@@4M 9=65<* 94* Hancoc$ +C) Vogra!i G* C&aracteristics an% signi!icance o! t&e amorp&ous state in p&armaceutical systems* ' P&arm Sci 4>><M =;64* 95* Palmieri G-) +onacucina G) Di Martino P) Martelli S* GastroEresistant microsp&eres containing $etopro!en* ' Microencapsul 5@@5M 4>6444* 98* Palmieri G-) Elisei I) Di Martino P) Martelli S* -ormulation o! microparticulate systems !or mo%i!ie% release containing $etopro!en*In6 4>t& P&armaceutical Tec&nology Con!erence) Italy) 5@@@* 99* Morimoto 3) 0o$u#o T) Sugi#ayas&i 0* Di!!usion o! %rugs in acrylicEtype pressureEsensiE tive a%&esive matri"* II* In!luence o! interaction* ' Control Release 4>>5M 4=6448* 9:* Saleem M) As!our AE-A) De 0ee D* Di!!usion o! organic penetrants t&roug& lo( %ensity polyet&ylene A DPEB !ilms6 e!!ect o! si/e an% s&ape o! t&e penetrant molecules* ' Appl Polym Sci 4>=>M 8<6;4<* 9;* +yrn S) P!ei!!er RR) Ganey M) Hoi#erg C) Pooc&i$ian G* P&armaceutical soli%s6 a strateE gic approac& to regulatory consi%erations* P&arm Res 4>>:M 456>9:* 9<* Hancoc$ +C) Vogra!i G* T&e relations&ip #et(een t&e glass transition temperature an% t&e (ater content o! amorp&ous p&armaceutical soli%s* P&arm Res 4>>9M 4469<4* 9=* euner C) Dressman '* Improving %rug solu#ility !or oral %elivery using soli% %isperE sions* Eur ' P&arm +iop&arm 5@@@M :@69<*

45=
9>* Du#ernet C* T&ermoanalysis o! microsp&eres* T&ermoc&im Acta 4>>:M 59=65:>* :@* -or% ' * T&e use o! t&ermal analysis in t&e stu%y o! soli% %ispersions* Drug Dev In% P&arm 4>=<M 4864<94* :4* 'enCuin MR) McGinity ',* C&aracteri/ation o! acrylic resin matri" !ilms an% mec&aE nisms o! %rugHpolymer interactions* Int ' P&arm 4>=<M 4@4658* :5* Du#ernet C) Roulan% 'C) +enoit 'P* I#upro!enEloa%e% et&ylcellulose microsp&eres6 analysis o! t&e matri" structure #y t&ermal analysis* ' P&arm Sci 4>>4M =@64@5>* :8* ,u C) McGinity ',* NonEtra%itional plastici/ation o! polymeric !ilms* Int ' P&arm 4>>>M 4<<64:* :9* 'enCuin MR) ie#o(it/ SM) Sara#ia RE) McGinity ',* P&ysical an% c&emical !actors in!luencing t&e release o! %rugs !rom acrylic resin !ilms* ' P&arm Sci 4>>@M <>6=44* ::* in SE3) C&en 0ES) RunEC&u * ?rganic esters o! plastici/ers a!!ecting t&e (ater a#sorpE tion) a%&esive property) glass transition temperature an% plastici/er permanence o! Eu%ragit acrylic !ilms* ' Control Release 5@@@M ;=6898* :;* I&%e A) Raula ') 0auppinen EI) ,atana#e ,) A&onen PP) +ro(n DP* Aerosol synt&esis o! in&alation particles via a %ropletEtoEparticle met&o%* Part Sci Tec&nol 5@@:M 596<4* :<* Ro%rXgue/EHorne%o N) Murp&y D* Signi!icance o! controlling crystalli/ation mec&aE nisms an% $inetics in p&armaceutical systems* ' P&arm Sci 4>>>M ==6;:4* :=* Raula ') 0ur$ela 'A) +ro(n DP) 0auppinen EI* Stu%y o! t&e %ispersion #e&avior o! EleuE cine containing microparticles synt&esi/e% (it& an aerosol !lo( reactor met&o%* Po(%er Tec&nol 5@@;* :>* ,eiss#uc& I) -rolo( -) A%%a%i ) a&av M) eisero(it/ * ?riente% crystalli/ation as a tool !or %etecting or%ere% aggregates o! (aterEsolu#le &y%rop&o#ic mEamino aci%s at t&e airHsolution inter!ace* ' Am C&em Soc 4>>@M 4456<<4=* ;@* Matu#ayasi N) Miyamoto H) Nami&ira ') 3ano 0) Tana$a T* T&ermo%ynamic Cuantities o! sur!ace !ormation o! aCueous electrolyte solutions*2* ACueous solutions o! alip&atic amino aci%s* ' Colloi% Inter!ace Sci 5@@5M 5:@6984* ;4* Svec H') Cly%e DD* 2apour pressures o! some amino aci%s* ' C&em Eng Data 4>;:M 4@64:4*

Raula et al*

>

Supercoole% Smectic Nanoparticles

Hei$e +un.es an% 'u%it& 0untsc&e
Department o! P&armaceutical Tec&nology) Institute o! P&armacy) -rie%ric& Sc&iller University 'ena) 'ena) Germany

INTR?DUCTI?N Among t&e %i!!erent types o! nanoparticulate %rug carrier systems) lipi% nanoparE ticles are particularly promising (it& regar% to p&ysiological compati#ility as t&ey can #e prepare% pre%ominantly or even e"clusively on t&e #asis o! p&ysiological compoun%s* Colloi%al lipi% emulsions an% soli% lipi% nanoparticles are t&e classical e"amples o! matri"Etype lipi% nanoparticles) (&ic& are mainly un%er investigation as carriers !or poorly (aterEsolu#le an% lipop&ilic %rugs* T&ese t(o types o! nano%ispersions eac& &ave t&eir o(n a%vantages an% %isa%vantages (it& regar% to t&eir use as %rug carrier systems* -or e"ample) colloi%al lipi% emulsions o! natural an% semisynt&etic oils &ave a soli% #asis o! tec&nological $no(E&o( an% a (ellE %ocumente% recor% o! p&ysiological compati#ility arising !rom t&eir %eca%eElong use in parenteral nutrition A4B* Also t&eir interactions (it& %rugs &ave #een c&aracteri/e%) an% some %rugEloa%e% !ormulations are commercially availa#le A4H8B* T&e %rug incorE poration capacity o! t&e liCui% %roplets is comparatively &ig& in comparison (it& soli% lipi% nanoparticles an% can #e mo%i!ie% #y variation o! t&e oil compoun%* ?n t&e ot&er &an%) t&e liCui% nature o! t&e emulsion %roplets may lea% to %isa%vantages* In cases o! insu!!icient sta#ili/ation) t&e %roplets may gro( #y coalescenceM incorpoE rate% %rug molecules &ave a &ig& mo#ility an% can easily migrate into t&e sur!actant layer or lea$ into t&e aCueous p&ase* Soli% lipi% nanoparticles (it& t&eir rigi% core (ere %evelope% to overcome t&ese limitations A9):B* It &as) &o(ever) turne% out t&at t&e usually crystalline nature o! t&e soli% particle core counteracts incorporation o! larger amounts o! %rugs so t&at t&e %rug carrier capacity o! t&ese particles is !reE Cuently rat&er lo( A;B* Moreover) t&e p&ysical #e&avior o! t&e systems is o!ten Cuite comple" Ae*g*) (it& regar% to crystalli/ation) polymorp&ic transitions) an% interpartiE cle interactionsB an% certain propose% a%vantages o! soli% lipi% nanoparticles) suc& as t&e a#ility to provi%e #etter control o! %rug release) still remain to #e proven* In searc& o! an alternative lipi% carrier system) (&ic& coul% com#ine t&e a%vantages o! lipi% emulsions an% soli% lipi% nanoparticles) (e (ere aiming to preE pare carrier particles (it& a less or%ere% state t&an soli% lipi% nanoparticles in or%er to ac&ieve a &ig&er incorporation capacity !or !oreign su#stances*T&e particles s&oul%) &o(ever) still re%uce t&e mo#ility o! incorporate% %rugs an% sur!ace active agents as muc& as possi#le* ,e !ocuse% our interest on liCui% crystalline p&ases Aalso calle% mesop&asesB) (&ic& com#ine a certain mo#ility on t&e molecular level (it& an o!ten rat&er &ig& macroscopic viscosity* -ormation o! liCui% crystalline p&ases can #e in%uce% #y a%%ition o! solvents Alyotropic mesop&asesB or #y temperature At&ermoE tropic mesop&asesB* ,&ereas lyotropic mesomorp&ism is !reCuently encountere% an% also utili/e% in t&e area o! p&armaceutics A<)=B) t&ermotropic mesop&ases &ave not yet receive% muc& attention in t&is !iel%) alt&oug& t&ermotropic mesomorp&ism &as #een %escri#e% !or some %rug su#stances A>B* T&e solventE!ree c&aracter o! lipop&ilic t&ermotropic mesop&ases ma$es t&em similar to t&e %isperse% p&ase o!

45>

48@

+un.es an% 0untsc&e

colloi%al lipi% emulsions an% suspensions) an% t&us promising to #e e"plore% as potential matri" materials in colloi%al %rug %elivery systems* THERM?TR?PIC MES?PHASES ,&ile lyotropic mesop&ases are !orme% #y amp&ip&ilic molecules in t&e presence o! a suita#le solvent) t&ermotropic mesomorp&ism is a speci!ic property o! certain su#stances (it& strongly anisometric molecular s&ape t&at %oes not reCuire any a%%itives an% occurs in %epen%ence on temperature A4@B* T&ermotropic mesop&ases are classi!ie% into t(o main groups6 calamitic A!orme% #y molecules (it& ro%Eli$e s&ape) -ig* 4B an% %iscotic Amolecules (it& %iscEli$e s&apeB mesop&ases* T(o main calamitic mesop&ases) t&e smectic an% t&e nematic p&ases) can #e %istinguis&e%* ,&en a su#stance !orms a smectic as (ell as a nematic p&ase) t&e smectic p&ase al(ays e"ists at lo(er temperatures* In t&e smectic p&ase) t&e molecules are aligne% si%e #y si%e !orming a layere% structure* Di!!erent mo%i!ications o! t&e smectic p&ase &ave #een %escri#e%) !or e"ample) t&e molecules can #e arrange% perpen%icular Asmectic A p&aseB or tilte% Asmectic C p&aseB (it& respect to t&e smectic layers* Smectic A p&ases are typical !or saturate% c&olesterol esters suc& as c&olesteryl myristate ACMB an% palmitate ACPBM !or c&olesteryl oleate AC?B) a smectic C p&ase is assume% A45B* In t&e nematic p&ase) t&e molecules are arrange% nearly in parallel #ut not in speci!ic layers* T&e c&olesteric p&ase) (&ic& is c&aracteristic o! c&olesterol esters) can #e regar%e% as a t(iste% nematic p&ase* In%ivi%ual nematic molecular layers are t(iste% against eac& ot&er in a certain angle !orming a &elical structure* T&e %istance #et(een t&e molecular layers (it& t&e same orientation Apitc&B %epen%s on temperature an% is o!ten in t&e range o! t&e (avelengt& o! visi#le lig&t lea%ing to c&aracteristic color e!!ects* T&ermotropic mesop&ases are #roa%ly applie% in t&e tec&nical !iel%) !or e"amE ple) !or liCui% crystal %isplays) an%) conseCuently) most $no(n t&ermotropic mesoE gens &ave #een %evelope% !or tec&nical applications A48B* C&olesterol !atty aci% esters as a class o! p&ysiological su#stances are) &o(ever) also capa#le o! !orming t&ermotropic mesop&ases A45B* As t&eir smectic p&ase &as a &ig& viscosity) it appeare% promising to provi%e t&e %esire% c&aracteristics !or t&e %evelopment o! t&e novel type o! lipi% nanoparticles*

CH? ESTER3 M3RISTATE NAN?PARTIC ES6 GENERA PR?PERTIES AND PHASE +EHA2I?R -or !irst investigations on t&e preparation o! smectic nanoparticles) t&e p&ysioE logical ester CM (as use% as mo%el matri" lipi% %ue to its !ully reversi#le an%

-IGURE 4 Structures o! t&erE motropic calamitic mesop&ases* Source6 A%apte% !rom Re!* 44*

Supercoole% Smectic Nanoparticles

484

-IGURE 5 ATopB Di!!erential scanning caloE rimetry &eating an% cooling curves A:]C1minB o! CM in #ul$ an% in colloi%al %ispersion A:[ CM) 8*5[ S4@@) @*=[ SGCB* T&e samples (ere &eate% to t&e isotropic melt Acurve not s&o(n !or t&e #ul$ materialB) coole% %o(n #elo( t&e crystalli/ation temperature) an% &eate% again AsmEc&6 smecticHc&olesteric) c&Ei6 c&olestericH isotropic p&ase transitionB* A+ottomB SmallE an% (i%eEangle WEray %i!!raction patterns o! CM in #ul$ an% in colloi%al %ispersion A:[ CM) 8*5[ S4@@) @*=[ SGCB* +ul$6 crystalline po(%er AAB at 5@]C an% smectic mesop&ase A+B at ;@]C A!orme% upon cooling t&e isotropic meltB* Dispersions6 store% at 9]C ACB an% at 58]C ADB measure% at 5@]C* T&e grap&s o! t&e #ul$ material an% t&e %ispersions are not on t&e same linear intensity scale As ^ 41% ^ 5 sin ~1 (&ere 5~ is t&e scattering angle an% is t&e (avelengt&) eCual to @*4: nmB* A##reviations6 CM) c&olesteryl myristateM SGC) so%ium glycoE c&olateM S4@@) puri!ie% soy #ean lecit&in ipoi% S4@@ A ipoi% 0G) DE u%(igs&a!enB*

(ellEc&aracteri/e% p&ase #e&avior A44)49B* Crystalline CM melts aroun% <5]C into a smectic p&ase) trans!orms into t&e c&olesteric p&ase aroun% <>]C) an% !inally melts into an isotropic liCui% aroun% =9]C A-ig* 5B* Upon cooling t&e melt) t&e liCui% crystalline p&ase transitions are o#serve% at a#out t&e same temperatures as upon &eating* Crystalli/ation occurs #et(een a#out 9@]C an% 8@]C* T&us) t&e smectic p&ase o! #ul$ CM %oes not e"ist un%er p&armaceutically relevant con%itions suc& as #o%y an% room temperature* -or colloi%al particles o! CM) t&e same p&ase transiE tions are) in principle) o#serve% as !or t&e #ul$ material e"cept !or t&e %istinctly &ig&er supercooling o! t&e smectic p&ase upon cooling A-ig* 5B* T&e !act t&at CM crystalli/ation starts only #elo( 5@]C in t&e colloi%al state is t&e #asis !or t&e prepaE ration o! smectic nanoparticles* ,&en t&e &ot %ispersions are only coole% to room temperature an% store% un%er t&is con%ition) t&eir smectic state can #e retaine% !or many mont&s A44)49B* Upon &eating o! a sta#le smectic CM %ispersion) only t&e liCui% crystalline p&ase transitions are o#serve%* T&ese transitions are very small an%) !or t&e colloi%al %ispersions) #roa% an% not (ell separate%) ma$ing t&eir CuanE ti!ication %i!!icult* As an a%%itional !eature to c&aracteri/e t&e smectic state o! t&e nanoparticles) t&e very s&arp smallEangle WEray re!lection arising !rom t&e layere% structure o! t&e smectic p&ase can #e use% A-ig* 5B* T&is smectic re!lection is #roa%er an% less intensive !or t&e nanoparticles) #ut its position is compara#le (it& t&at o! t&e #ul$ material (&en measure% un%er compara#le con%itions* As t&e repeating unit AspacingB o! t&e smectic p&ase increases (it& %ecreasing temperature A4:B) a larger spacing o! t&e strongly supercoole% smectic nanoparticles is measure% at 5@]C A44B* In t&e (i%eEangle range) only %i!!use WEray scattering is o#serve% #ecause o! t&e lac$ o! molecular or%er* -or t&e crystalline material At&e nanoparticles can #e crystalli/e% #y storage at) e*g*) 9]CB) c&aracteristic (i%eEangle re!lections are visi#le

485

+un.es an% 0untsc&e

#esi%e smallEangle re!lections o! !irst an% &ig&er or%ers* -or t&e crystalline nanoparE ticles) t&e re!lections are less s&arp) #ut t&eir positions are compara#le (it& t&ose o! t&e #ul$ lipi%* PREPARATI?N METH?DS -?R SMECTIC NAN?PARTIC ES ACueous %ispersions o! smectic c&olesterol ester nanoparticles can #e prepare% #y &ig&Epressure &omogeni/ation o! a &ot preEemulsion Ao#taine%) e*g*) #y ultraEturra" vorte"ingB at temperatures a#ove t&e melting point o! t&e respective matri" lipi% in t&e presence o! emulsi!iers* T&e process) $no(n as &ig&Epressure meltE&omogeni/ation) is also commonly use% !or t&e preparation o! soli% lipi% nanoparticles A9):B an%) in principle) similar to t&e preparation o! colloi%al !at emulsions A8)4;B* A!ter &omoE geni/ation Ausing a micro!lui%i/er or pistonEgap &omogeni/erB) t&e &ot nanoparticle %ispersions are usually !iltere% A@*5 or : _mB* Hig&Epressure meltE&omogeni/ation yiel%s %ispersions o! smectic nanoparticles (it& mean particle si/es #et(een 4@@ an% 5@@ nm) avoi%ing t&e use o! organic solvents A44)49B* T&e &ig& temperature %uring t&e process o! meltE&omogeni/ation may lea% to a partial %egra%ation o! t&ermally sensitive compoun%s an% %rugs* To avoi% t&is pro#lem an% !or t&e preparation o! %ispersions (it& smaller particles) a mo%i!icaE tion o! t&e soEcalle% emulsi!ication solventEevaporation met&o% A4<B can #e use%* T&e lipi% an% lipop&ilic sta#ili/ers are %issolve% in an organic solvent) (&ic& is not misci#le (it& (ater) !or e"ample) cyclo&e"ane* A cru%e preEemulsion (it& t&e aCueous p&ase Acontaining &y%rop&ilic sur!actantsB is prepare% #y ultraEturra" vorte"ing an% t&e %ispersion is &ig& pressure &omogeni/e%) !or e"ample) using a micro!lui%i/er* T&e (&ole process is carrie% out at room or slig&tly lo(er temperature* T&e organic solvent is remove% un%er re%uce% pressure an% at slig&tly elevate% temperature* T&e %ispersions are !iltere% an% &eate% a#ove t&e melting temperature o! t&e matri" lipi% !or a#out 4@ minutes to ensure t&e smectic state o! t&e nanopartiE cles* ,it& t&is met&o%) %ispersions (it& mean %iameters %istinctly #elo( 4@@ nm can #e prepare% A44B* Resi%ual organic solvent in t&e %ispersions may) &o(ever) #e a %isa%vantage o! t&is proce%ure A4=)4>B* Particle si/e is a crucial point in t&e %evelopment o! %ispersions t&at are sta#le upon storage (it& regar% to t&e metasta#le smectic state o! t&e matri" material) as &as #een s&o(n !or CM %ispersions sta#ili/e% on t&e #asis o! p&osp&olipi%s* Dispersions o! smaller particles are more sta#le against recrystalli/ation upon storE age t&an %ispersions (it& larger particle si/es A49B* Moreover) t&e storage temperaE ture &as to #e a%.uste% to t&e %ispersion properties6 I! t&e %ispersions are store% too close to t&e recrystalli/ation temperature o! t&e nanoparticles) t&e matri" lipi% (ill crystalli/e %uring storage* -or a%ministration o! nanoparticles via t&e parenteral or ocular route) sterility o! t&e %ispersions is an important issue* -irst stu%ies in%icate t&at %ispersions o! supercoole% smectic nanoparticles can #e sterili/e% #y autoclaving at 454]C (it&out increase in particle si/e Asta#ili/ation (it& p&osp&olipi%1#ile salt mi"turesB or (it& only a small increase in particle si/e Nsta#ili/ation (it& polo"amer 4== or polo"amE ine ATetronic >@=) +AS-) DE u%(igs&a!enBO* SmallEsi/e% %ispersions can also #e !ilE tere% t&roug& @*5E_m !ilters as outline% a#ove*

IN- UENCE ?- THE STA+I IVER S3STEM Colloi%al %ispersions are t&ermo%ynamically unsta#le systems #ecause o! t&eir &ig& inter!acial energy* -or t&e preparation an% sta#ili/ation o! colloi%al lipi% particles)

Supercoole% Smectic Nanoparticles

488

sur!ace active agents) (&ic& accumulate in inter!aces an% re%uce t&e inter!acial energy) are reCuire%* Depen%ing on t&e properties o! t&e sur!ace active agent) sta#iE li/ation occurs #y electrostatic Ac&arge% sur!actantsB or steric Ae*g*) amp&ip&ilic polymersB sta#ili/ation or a com#ination o! #ot&* Sta#ili/ers may also in!luence t&e p&ase #e&avior o! %isperse% lipi%s) li$e t&eir crystalli/ation an% polymorp&ic #e&avior A5@H55B* -urt&ermore) t&e sta#ili/er system %etermines t&e sur!ace properE ties in!luencing t&e !ate o! t&e nanoparticles in vivo A58)59B* -or t&e sta#ili/ation o! smectic CM nanoparticles) puri!ie% p&osp&olipi%s Asoy#ean an% eggEyol$ p&osE p&olipi%sB alone or in com#ination (it& t&e #ile salt so%ium glycoc&olate) so%ium glycoc&olate alone) so%ium oleate) %i!!erent polymers Npolo"amer) polo"amine) partially acetylate% polyvinyl alco&ol AP2ABO) T(een =@) a sucrose ester mainly containing sucrose monolaurate) so%ium caseinate) an% gelatin polysuccinate &ave #een teste% so !ar* ,it& e"ception o! t&e sugar ester) all sta#ili/ers le% to sta#le colloi%al %ispersions a!ter meltE&omogeni/ation (it& respect to macroscopic appearE ance an% particle si/e upon longEterm storage* T&e smectic state o! t&ese nanopartiE cles coul% clearly #e i%enti!ie% #y t&e c&aracteristic smallEangle WEray re!lection (it&out in!luence o! t&e sta#ili/er system on t&e position o! t&e re!lection* T&e sta#ili/er system %oes) &o(ever) strongly in!luence t&e p&ase #e&avior o! CM nanoparticles) particularly t&e crystalli/ation process A5:B* ?n t&e #asis o! t&e crystalli/ation pattern an% t&e recrystalli/ation ten%ency upon storage) t(o groups o! sta#ili/ers can #e %istinguis&e%6

Sta#ili/ers in%ucing a multiple crystalli/ation event %epen%ing on t&e t&ermal &istory o! t&e sample an% a &ig& recrystalli/ation ten%ency upon storage Ap&osp&olipi%s) so%ium oleate) sucrose monolaurateB* Sta#ili/ers in%ucing a monomo%al crystalli/ation event mostly in%epen%ent o! t&e t&ermal &istory o! t&e sample an% a lo( recrystalli/ation ten%ency upon storage Aso%ium glycoc&olate) synt&etic polymers) gelatin polysuccinate) so%ium caseinateB*

T&e common !eature o! sta#ili/ers resulting in smectic nanoparticles (it& a multiple crystalli/ation pattern) an% a comparatively &ig& recrystalli/ation ten%ency upon storage is t&e presence o! an acyl c&ain in t&e molecule* All correspon%ing %ispersions s&o( a very comple" crystalli/ation #e&avior A-ig* 8B) (&ic& is pro#a#ly cause% #y particles o! %i!!erent s&apes Asp&erical an% cylin%ricalB present in t&e %ispersions as o#serve% in electron microscopy Asee #elo(B A49B* In contrast) smectic nanoparticles sta#ili/e% (it& polymers an% so%ium glycoc&olate alone &ave a %isE tinctly lo(er crystalli/ation ten%ency upon storage %espite t&eir relatively &ig& crystalli/ation temperature A-ig* 8B* Electron microscopic investigations o! t&ese %ispersions in%icate a more &omogeneous particle structure Asee #elo(B* T&e %isE persion sta#ili/e% (it& T(een =@ Aa sta#ili/er (&ic& also contains an acyl c&ainB cannot #e clearly classi!ie% into one o! t&e groups mentione% a#ove* Alt&oug& store% smectic nanoparticles sta#ili/e% (it& T(een =@ %isplay only one crystalli/ation event upon cooling in %i!!erential scanning calorimetry ADSCB an% t&e recrystalli/aE tion ten%ency o! t&ese smectic nanoparticles is lo() !res&ly melte% nanoparticles give a #imo%al crystalli/ation pattern upon cooling*

IN- UENCE ?- THE MATRIW C?MP?SITI?N CM nanoparticles are e"cellent mo%el systems !or stu%ying t&e in!luence o! paraE meters suc& as particle si/e an% sta#ili/er system on storage sta#ility an% p&ase #e&avior* T&eir comparatively &ig& crystalli/ation temperature) (&ic& is o#serve%

489

+un.es an% 0untsc&e

-IGURE 8 Crystalli/ation onset temperatures A%i!!erential scanning calorimetry) cooling o! t&ermally untreate% samples) :]C1minB AtopB an% amount o! recrystalli/e% matri" lipi% a!ter storage A#ottomB !or nine mont&s Aeig&t mont&s !or samples mar$e% (it& an asteris$B in %epenE %ence on t&e sta#ili/er system* All %ispersions contain :[ CM an%) (it& e"ception o! SGC A5[B) 9[ sta#ili/ers* A##reviations6 CM) c&olesteryl myristateM E=@) puri!ie% egg yol$ lecit&in ipoi% E=@M P2A) polyvinyl alco&olM S4@@) puri!ie% soyE #ean lecit&in ipoi% S4@@M SGC) so%ium glycoE c&olateM sucrose M ) sucrose monolaurate*

even in optimi/e% systems) preclu%es) &o(ever) t&eir %evelopment into a ro#ust %rug %elivery system !or practical use* Suc& a system s&oul% %isplay a &ig& sta#ility against recrystalli/ation upon storage) pre!era#ly also at temperatures %o(n to a#out @]C (&ic& can occur %uring transport an% may #e reCuire% %uring storage to protect sensitive %rugs or e"cipients) !or e"ample) p&osp&olipi%s) !rom %egra%aE tion* T&ere!ore) c&olesteryl nonanoate ACNB) an ester (it& nine car#on atoms in t&e acyl c&ain) an% C?) containing an unsaturate% C4= c&ain) (ere teste% as alternative matri" materials to increase t&e crystalli/ation sta#ility o! smectic nanoparticles A5;B* +ot& esters %o not crystalli/e in t&e colloi%al state) neit&er %uring cooling to Z4@]C nor upon storage Aeven not at re!rigerator temperaturesB* Pure C? is) &o(ever) unsuita#le as matri" lipi%) #ecause its liCui% crystalline to isotropic p&ase transition is very close to #o%y temperature so t&at C? nanoparticles (oul% lose t&eir smectic state upon a%ministration* T&e use o! CN seems to #e very promising !rom t&e p&ysicoc&emical point o! vie() #ut its nonp&ysiological nature reCuires some sa!ety issues to #e resolve% prior to its use as %rug carrier* +ot& esters can also #e use% as a%%itives to longerEc&aine% c&olesterol esters suc& as CM or CP) (&ic& &as an even &ig&er crystalli/ation ten%ency t&an CM) !or t&e preparation o! smectic nanoparticles #eing more sta#le against recrystalli/ation* T&e a%mi"ture o! alrea%y relatively small amounts o! t&ese esters to CM A4@[) 5@[) 9@[ CN) 5@[ C? relate% to t&e (&ole matri" lipi%B or CP A9@[) :@[ CN) :@[ C? relate% to t&e (&ole matri" lipi%B lea%s to smectic nanoparticles (&ic& are sta#le against recrystalli/ation over at least 4= mont&s at 58]C alt&oug& t&e crystalli/ation temperature is only slig&tly %ecrease% particularly !or t&e CME#ase% %ispersions

Supercoole% Smectic Nanoparticles

48:

-IGURE 9 Particle si/e Np&oton correlation spectroscopy APCSBO an% crystalli/ation onset temperE atures o! !res&ly molten samples A%i!!erential scanning calorimetry) :]C1minB in %epen%ence on t&e matri" composition Ale!tB an% sta#ili/er system Arig&t B* -or t&e %ispersion (it& a CM1CNEmatri" sta#ili/e% (it& polyvinyl alco&ol) only t&e very #eginning o! t&e crystalli/ation event (as recor%e% so t&at t&e onset temperature Ac@]CB coul% not #e e"actly %etermine%* A##reviations6 CM) c&olesteryl myristateM CN) c&olesteryl nonanoateM C?) c&olesteryl oleateM CP) c&olesteryl palmitateM P2A) polyE vinyl alco&olM SGC) so%ium glycoc&olateM S4@@) puri!ie% soy #ean lecit&in ipoi% S4@@*

A-ig* 9B* In contrast) t&e amount o! recrystalli/e% matri" lipi% (as =[ an% >9[ !or t&e %ispersions (it& a pure CM or CP matri") respectively* A%mi"ture o! &ig&er amounts o! CN A;@[ o! t&e matri"B to CM smectic nanoparticles resulte% in %isperE sions (&ic& coul% #e store% at 9]C (it&out nanoparticle recrystalli/ation* T&e crysE talli/ation temperatureEsuppressing e!!ect o! CN a%mi"ture can #e !urt&er increase% #y t&e use o! polymeric sta#ili/ers suc& as P2A an% polo"amer in %ispersions (it& a mi"e% c&olesterol ester matri" A-ig* 9B*

INC?RP?RATI?N ?- M?DE DRUGS I#upro!en) micona/ole) etomi%ate) an% progesterone (ere use% as poorly (aterE solu#le mo%el %rugs !or t&e preparation o! %rugEloa%e% smectic nanoparticles A44B* T&e %ispersions containing :[ CM as matri" lipi% an% a p&osp&olipi%1#ile salt mi"E ture A8*51@*=[B as sta#ili/ers (ere prepare% #y &ig&Epressure meltE&omogeni/ation* T&e lo(er melting %rugs i#upro!en) micona/ole) an% etomi%ate coul% #e incorpoE rate% in an amount o! 4@[ relative to t&e lipi% matri" #y %issolving t&e %rug in t&e c&olesterol ester melt* Hig&er %rug loa%s &ave not #een investigate% yet #ut a %rug loa% o! 4@[ is alrea%y &ig&er t&an ac&ieve% !or soli% lipi% nanoparticles (it& micona/ole an% i#upro!en A5<B* Progesterone) (&ic& melts a#ove 4@@]C) coul% #e %issolve% only at a concentration o! 4[ in t&e CM melt (it&in an appropriate time Ac8@ minutesB* Drug incorporation into t&e %ispersions %i% not in!luence t&e occurrence an% position o! t&e smallEangle WEray re!lection c&aracteristic o! t&e smectic nanoparticle state* Particularly t&e liCui% crystalline p&ase transitions (ere) &o(ever) s&i!te% to lo(er temperatures in DSC) in%icating an interaction o! t&e %rug molecules (it& t&e

48;

+un.es an% 0untsc&e

-IGURE : Crystalli/ation onset temE peratures A%i!!erential scanning caloriE metry) :]C1min) !res&ly molten samples) main crystalli/ation eventB o! %rugEloa%e% %ispersions in comparison to t&ose o! a correspon%ing %rugE!ree %ispersion (it& compara#le particle si/e in %epen%ence on storage time*

smectic p&ase* T&e %ecrease o! t&e liCui% crystalline p&ase transition temperatures (as %i!!erent an% least pronounce% in t&e %ispersion (it& 4[ progesterone* E"cept !or t&e %ispersion loa%e% (it& progesterone) t&e crystalli/ation temperature (as also %ecrease% in t&e presence o! %rug A-ig* :B) in%icating t&at %rug incorporation may &ave a #ene!icial e!!ect on t&e recrystalli/ation ten%ency* T&e %rugEloa%e% %ispersions (ere sta#le upon storage !or at least 45 mont&s (it& respect to particle si/e) macroscopic appearance) an% recrystalli/ation o! matri" material* ?nly in t&e progesteroneEloa%e% %ispersion (as a small amount o! recrysE talli/e% matri" lipi% A9[B %etecte% a!ter 45 mont&s o! storage* -irst preliminary stu%ies point to a rapi% release o! at least a ma.or !raction o! i#upro!en an% etomi%ate !rom t&e smectic nanoparticles into t&e aCueous p&ase* Suc& #e&avior (oul% lea% to carrierEin%epen%ent %rug %istri#ution a!ter I2 a%minE istration as is also o!ten o#serve% !or lipi% emulsions A8B* T&e use o! more lipop&ilic pro%rugs Ae*g*) estersB or ionEpairs is e"pecte% to en&ance %rug retention a!ter a%ministration an% mig&t also allo( loa%ing (it& comparatively &y%rop&ilic compoun%s A5=H84B*

U TRASTRUCTURE ?- CH? ESTER3 M3RISTATE NAN?PARTIC ES Di!!erent electron microscopic tec&niCues can #e use% !or t&e investigation o! t&e ultrastructure o! colloi%al lipi% particles* In cryoelectron microscopy) a t&in !ilm o! rapi%ly !ro/en %ispersion is vie(e% %irectly) an% a goo% impression o! t&e s&ape o! t&e pro.ecte% particles is o#taine%* In contrast) !ree/eE!racturing) (&ere t&e !ro/en sample is !racture%) an% t&e !racture plane s&a%o(e% (it& a t&in &eavy metal layer lea%s to images t&at yiel% in!ormation also a#out t&e inner structure o! t&e particles* Electron microscopic images in%icate a mostly nonsp&erical) nearly cylin%riE cal s&ape o! t&e CM nanoparticles* T&e particle s&ape is strongly in!luence% #y t&e sta#ili/er system ranging !rom nearly e"actly cylin%rical particles to Qpaving stoneE li$eS ones A-ig* ;B* In %ispersions sta#ili/e% on t&e #asis o! p&osp&olipi%s an% (it& so%ium oleate) an a%%itional particle !raction (as o#serve% in cryoEpreparations (&ic& (as very unsta#le in t&e electron #eam* An onionEli$e internal structure %etecte% !or some particles in !ree/eE!racture% specimen o! p&osp&olipi%Esta#ili/e% %ispersions points to a sp&erical s&ape o! t&ese particles* ConseCuently) t&ese %isE persions seem to contain t(o %i!!erent types o! smectic nanoparticlesRcylin%rical an% sp&erical ones* ?(ing to t&e layere% structure o! t&e smectic p&ase) a cylin%riE cal particle s&ape s&oul% #e energetically more !avora#le t&an a sp&erical one an%)

Supercoole% Smectic Nanoparticles

48<

-IGURE ; Cryoelectron micrograp&s o! selecte% %ispersions containing :[ CM* AA an% +B Dispersion sta#ili/e% (it& t&e p&osp&olipi%1#ile salt system A8*51@*=[B* A+B T&e !raction o! insta#le) Q#u##lingS particles is clearly visi#le* ACB Dispersion sta#ili/e% (it& 9[ so%ium oleate* ADB Dispersion sta#ili/e% (it& 9[ polyvinyl alco&ol* AEB Dispersion sta#ili/e% (it& 9[ polo"amer 4==* A-B DisperE sion sta#ili/e% (it& 9[ T(een =@* Magni!ications illustrate t&e %epen%ence o! t&e particle s&ape on t&e sta#ili/er system* T&e !ree/eE!racture micrograp& s&o(s an image o! a CM particle (it& an onionEli$e inner structure A8*5[ S4@@) #ar ^ 49@ nmB* A sc&ematic mo%el representation o! t&e structure o! cylin%rical an% sp&erical CM nanoparticles is also given* A##reviations6 CM) c&olesteryl myristateM S4@@) puri!ie% soy #ean lecit&in ipoi% S4@@M SGC) so%ium glycoc&olate*

t&ere!ore) t&e increase% sensitivity o! t&e sp&erical particles to(ar% t&e electron #eam mig&t #e cause% #y a &ig&er energy level o! t&ese particles* A cylin%rical s&ape &as also #een o#serve% in cryoelectron microscopic investigations o! native lo(E %ensity lipoprotein A D B particles A85)88B (&ic& contain a core o! c&olesterol esters t&at s&oul% #e in t&e smectic p&ase un%er t&e con%itions use% !or sample preparation at room temperature A89)8:B*

48=

+un.es an% 0untsc&e

+esi%es smectic nanoparticles) ot&er colloi%al structures suc& as micelles) mi"e% micelles) an% vesicles can #e present in t&e %ispersions %epen%ing on t&e sel!E association c&aracteristics o! t&e sta#ili/ers use%* Particularly in t&e %ispersion staE #ili/e% (it& so%ium oleate) a variety o! %i!!erent structures (as o#serve% in agreement (it& literature %ata on c&olesterol esterE!ree samples A8;B A-ig* ;B* T&e presence o! suc& ot&er colloi%al structures is generally not %esira#le as t&ey may also solu#ili/e lipop&ilic %rugs an% coul% in%uce re%istri#ution processes o! incorporate% %rugs upon storage* Moreover) t&ey may lea% to a%%itional p&ysiological e!!ects* -or e"ample) liposomes !orme% #y an e"cess o! p&osp&olipi%s use% !or t&e sta#ili/ation o! colloi%al !at emulsions !or parenteral nutrition A8<B in!luence !at meta#olism an% can lea% to a %ecrease% %egra%ation o! t&e !at emulsion particles A8=B*

SUMMAR3 AND C?NC USI?N Supercoole% smectic c&olesterol ester nanoparticles are a promising ne( carrier system !or t&e %elivery o! lipop&ilic %rugs* T&ey can #e prepare% #y %i!!erent met&E o%s) !or e"ample) (it& t&e esta#lis&e% &ig&Epressure meltE&omogeni/ation tec&E niCue* T&eir sta#ility against recrystalli/ation %epen%s on t&e matri" composition) emulsi!ier system) an% on t&e particle si/e* I! properly %esigne% (it& respect to composition) preparative) an% storage parameters) t&ey are sta#le on storage in liCui% %ispersion (it& regar% to particle si/e an% liCui% crystalline state o! t&e matri" material !or p&armaceutically relevant perio%s o! time* T&e &ig& viscosity o! t&e liCui% crystalline matri" is e"pecte% to counteract coalescence processes* ipop&ilic %rugs can #e loa%e% into t&e nanoparticles as s&o(n a#ove !or i#upro!en) etomiE %ate) an% micona/ole (&ic& can #e incorporate% at consi%era#le concentration* Alt&oug& #asic $no(le%ge &as alrea%y #een collecte% on t&e c&aracteristics o! t&is ne( type o! particles) t&ey are still in an early stage o! %evelopment as %rug %elivery systems* -uture activities (ill !ocus on t&e !urt&er optimi/ation o! t&eir composiE tion) in particular (it& respect to sta#ility against recrystalli/ation) %rug incorporaE tion) an% retention as (ell as t&eir sur!ace c&aracteristics* T&e lipi%ic nature an% small particle si/e o! supercoole% smectic nanoparticles o!!er several interesting possi#ilities (it& virtually all (ays o! a%ministration Ae*g*) parenteral) ocular) %ermal) or peroralB* In !uture) correspon%ing interactions (it& #iological systems (ill &ave to #e stu%ie% in %etail* -irst cytoto"icity stu%ies &ave alrea%y reveale% promising results* Concerning parenteral) in particular intravenous a%ministration) t&e in!luence o! t&e ne( type o! matri" structure on t&e p&armacoE $inetics an% #io%istri#ution reCuires intensive investigation* As supercoole% smecE tic nanoparticles s&o( similarities in composition an% structure (it& t&e proteinE!ree core o! D s A85)88B) t&ey may &ave interesting potential as easily accessi#le arti!icial constructs (it& similar properties) !or e"ample) !or %rug targeting to certain tumors or t&e #rain via t&e D receptor A8>H94B*

RE-ERENCES
4* yons RT) Carter EG* ipi% emulsions !or intravenous nutrition an% %rug %elivery* In6 Gunstone -D) Pa%ley -+) e%s* ipi% Tec&nologies an% Applications* Ne( 3or$6 Marcel De$$er) 4>><6:8:H::;* 5* ,as&ington C* Sta#ility o! lipi% emulsions !or %rug %elivery* A%v Drug Deliv Rev 4>>;M 5@6484*

Supercoole% Smectic Nanoparticles

48>

8* 0lang S) +enita S* Design an% evaluation o! su#micron emulsions as colloi%al %rug carriers !or intravenous a%ministration* In6 +enita S) e%* Su#micron Emulsions in Drug Targeting an% Delivery* Amster%am6 Har(oo% Aca%emic Pu#lis&ers) 4>>=644>H4:5* 9* Me&nert ,) MI%er 0* Soli% lipi% nanoparticles6 pro%uction) c&aracteri/ation an% appliE cations* A%v Drug Deliv Rev 5@@4M 9<64;:* :* +un.es H) Sie$mann +* Manu!acture) c&aracteri/ation an% applications o! soli% lipi% nanoparticles as %rug %elivery systems* In6 +enita S) e%* Microencapsulation* 5n% e%* Ne( 3or$6 Marcel De$$er) 5@@;6548H5;=* ;* ,estesen 0) +un.es H) 0oc& MH'* P&ysicoc&emical c&aracteri/ation o! lipi% nanoE particles an% evaluation o! t&eir %rug loa%ing capacity an% sustaine% release potential* ' Control Release 4>><M 9=6558* <* Malmsten M* iCui% crystalline p&ases* In6 Malmsten M) e%* Sur!actants an% Polymers in Drug Delivery* Ne( 3or$6 Marcel De$$er) 5@@56:4H=;* =* MJllerEGoymann CC* iCui% crystals in %rug %elivery* In6 S(ar#ric$ ') +oylan 'C) e%s* Encyclope%ia o! P&armaceutical Tec&nology* Ne( 3or$6 Marcel De$$er) 5@@56=89H=:8* >* +un.es H) Ra%es T* T&ermotropic liCui% crystalline %rugs* ' P&arm P&armacol 5@@:M :<6=@<* 4@* 0el$er H) Hat/ R* Han%#oo$ o! iCui% Crystals* ,ein&eim6 2erlag C&emie) 4>=@* 44* 0untsc&e ') ,estesen 0) Drec&sler M) 0oc& MH') +un.es H* Supercoole% smectic nanoE particles6 a potential novel carrier system !or poorly (ater solu#le %rugs* P&arm Res 5@@9M 5464=89* 45* Gins#urg GS) At$inson D) Small DM* P&ysical properties o! c&olesteryl esters* Prog ipi% Res 4>=9M 58648:* 48* Ma%&usu%ana N2* Recent a%vances in t&ermotropic liCui% crystals* Curr Sci 5@@4M =@64@4=* 49* 0untsc&e ') 0oc& MH') Drec&sler M) +un.es H* Crystalli/ation #e&avior o! supercoole% smectic c&olesteryl myristate nanoparticles containing p&osp&olipi%s as sta#ili/ers* Colloi% Sur! + 5@@:M 9965:* 4:* ,en%or!! 'H) Price -P* T&e structure o! mesop&ases o! c&olesteryl esters* Mol Cryst iC Cryst 4><8M 59645>* 4;* +oc$ T) 0leine#u%%e P) MJller +,* Manu!acture o! emulsions #y means o! &ig&Epressure &omogeni/ation6 in!luence o! &omogeni/ation parameters) oils an% sur!actants* In6 MJller RH) +enita S) +F&m +) e%s* Emulsions an% Nanosuspensions !or t&e -ormulation o! Poorly Solu#le Drugs* Stuttgart6 Me%p&arm Scienti!ic Pu#l*) 4>>=65@4H58;* 4<* S.FstrFm +) 0ron#erg +) Carl!ors '* A met&o% !or t&e preparation o! su#micron particles o! sparingly (ater solu#le %rugs #y precipitation in o1(Eemulsions* I* In!luence o! emulsi!ication an% sur!actant concentration* ' P&arm Sci 4>>8M =56:<>* 4=* S.FstrFm +) ,estesen 0) +ergenst&l +* Preparation o! su#micron %rug particles in lecit&inEsta#ili/e% o1( emulsions* II* C&aracteri/ation o! c&olesteryl acetate particles* Int ' P&arm 4>>8M >96=>* 4>* S.FstrFm +) 0aplun A) Talmon 3) Ca#ane +* Structures o! nanoparticles prepare% !rom oilEinE(ater emulsions* P&arm Res 4>>:M 4568>* 5@* Garti N) 3ano '* T&e roles o! emulsi!iers in !at crystalli/ation* In6 Garti N) Sato 0) e%s* Crystalli/ation Processes in -ats an% ipi% Systems* Ne( 3or$6 Marcel De$$er) 5@@46544H5:@* 54* Ueno S) Hama%a 3) Sato 0* Controlling polymorp&ic crystalli/ation o! nEal$ane crystals in emulsion %roplets t&roug& inter!acial &eterogeneous nucleation* Cryst Gro(t& Des 5@@8M 86>8:* 55* +un.es H) 0oc& MH'* Saturate% p&osp&olipi%s promote crystalli/ation #ut slo( %o(n polymorp&ic transitions in triglyceri%e nanoparticles* ' Control Release 5@@:M 4@<655>* 58* Illum ) ,est P) ,as&ington C) Davis SS* T&e e!!ect o! sta#ili/ing agents on t&e organ %istri#ution o! lipi% emulsions* Int ' P&arm 4>=>M :9694* 59* iu -) iu D* ongEcirculating emulsions AoilEinE(aterB as carriers !or lipop&ilic %rugs* P&arm Res 4>>:M 4564@;@* 5:* 0untsc&e ') ,estesen 0) +un.es H* Supercoole% smectic nanoparticlesRin!luence o! t&e sta#ili/er system on p&ysicoc&emical properties* Proc Int Symp Control Release +ioact Mater 5@@8M 8@64>>*

49@

+un.es an% 0untsc&e

5;* 0untsc&e ') ,estesen 0) +un.es H* Supercoole% smectic nanoparticlesRin!luence o! t&e matri" composition on t&e p&ase #e&avior* Proc Int Symp Control Release +ioact Mater 5@@8M 8@64=5* 5<* +un.es H* In!luence o! %i!!erent !actors on t&e structure an% properties o! nanoparticles !rom soli% triglyceri%es* P&*D* T&esis* University o! 'ena) 4>>=* 5=* Cavalli R) Caputo ?) Gasco MR* Soli% liposp&eres o! %o"oru#icin an% i%aru#icin* Int ' P&arm 4>>8M =>6R>* 5>* Murt&a ' ) An%o H3* Synt&esis o! t&e c&olesteryl ester pro%rugs c&olesteryl i#upro!en an% c&olesteryl !lu!enamate an% t&eir !ormulation into p&osp&olipi% microemulsions* ' P&arm Sci 4>>9M =864555* 8@* Meyer 'D) Manning MC* Hy%rop&o#ic ion pairing6 altering t&e solu#ility properties o! #iomolecules* P&arm Res 4>>=M 4:64==* 84* 2ersluis A') Rump ET) Rensen PCN) van +er$el T'C) +i.ster#osc& M0* Synt&esis o! a lipop&ilic %aunoru#icin %erivate an% its incorporation into lipi%ic carriers %evelope% !or D receptorEme%iate% tumor t&erapy* P&arm Res 4>>=M 4:6:84* 85* van Ant(erpen R) Gil$ey 'C* CryoEelectron microscopy reveals &uman lo( %ensity lipoprotein su#structure* ' ipi% Res 4>>9M 8:65558* 88* van Ant(erpen R) C&en GC) Pullinger CR) et al* CryoEelectron microscopy o! lo( %ensity lipoprotein an% reconstitute% %iscoi%al &ig& %ensity lipoprotein6 imaging o! t&e apolipoE protein moiety* ' ipi% Res 4>><M 8=6;:>* 89* Dec$el#aum R') S&ipley GG) Small DM* Structure an% interactions o! lipi%s in &uman plasma lo( %ensity lipoproteins* ' +iol C&em 4><<M 5:56<99* 8:* 0roon PA* T&e or%erH%isor%er transition o! t&e core c&olesteryl esters o! &uman plasma lo( %ensity lipoprotein6 a proton nuclear magnetic resonance stu%y* ' +iol C&em 4>=4M 5:;6:885* 8;* E%(ar%s 0) Silvan%er M) 0arlsson G* Aggregate structure in %ilute aCueous %ispersions o! oleic aci%1so%ium oleate an% oleic aci%1so%ium oleate1egg p&osp&ati%ylc&oline* angmuir 4>>:M 446595>* 8<* Roten#erg M) Ru#in M) +or A) Meyu&as D) Talmon 3) ic&ten#erg D* P&ysicoEc&emical c&aracteri/ation o! intralipi% emulsions* +ioc&im +iop&ys Acta 4>>4M 4@=;65;:* 8=* +ac& AC) -GrG/ou ') -rey A* P&osp&olipi%Eric& particles in commercial parenteral !at emulsions6 an overvie(* Prog ipi% Res 4>>;M 8:6488* 8>* -irestone RA* o(E%ensity lipoprotein as a ve&icle !or targeting antitumor compoun%s to cancer cells* +iocon.ugate C&em 4>>9M :64@:* 9@* De&ouc$ +) -enart ) De&ouc$ MP) Pierce A) Torpier G) Cecc&elli R* A ne( !unction !or t&e D receptor6 transcytosis o! D across t&e #loo%H#rain #arrier* ' Cell +iol 4>><M 48=6=<<* 94* 0reuter '* Nanoparticulate systems !or #rain %elivery o! %rugs* A%v Drug Deliv Rev 5@@4M 9<6;:*

4@

+iological an% Engineering Consi%erations !or Developing TumorETargeting Metallic Nanoparticle DrugEDelivery Systems
Giulio -* Paciotti an% a(rence Tamar$in
CytImmune Sciences) Inc*) Roc$ville) Marylan%) U*S*A*

INTR?DUCTI?N Currently) !irstEline treatment o! resecta#le soli% tumors most commonly involves surgery !ollo(e% #y a regimen o! c&emot&erapy an%1or ra%iation* Un!ortunately) t&is strategy o!ten !ails #ecause o! recurrent or metastatic %isease* To c&ange t&is para%igm) ne( cancer t&erapies must %eliver multi!unctional t&erapeutics capa#le o! %estroying t&e &eterogeneous population o! tumor cells present (it&in soli% tumors* Targeting cancer t&erapeutics to soli% tumors is !acilitate% #y particleE%elivery systems capa#le o! escaping p&agocytic clearance #y t&e reticuloen%ot&elial system ARESB A4H8B* Un%er i%eal con%itions) suc& %elivery systems pre!erentially e"travasate t&e tumor vasculature an% accumulate (it&in t&e tumor microenvironment A9):B* A%%itionally) t&ese nanot&erapeutics may #e engineere% to contain tumorEtargeting ligan%s t&at #in% to speci!ic cells (it&in soli% tumors to anc&or t&e nanoparticle (it&in t&e soli% tumor* +y %esign) particleE%elivery systems capa#le o! seCuestering cancer %rugs solely (it&in a tumor may also re%uce t&e accumulation o! t&e %rugs in &ealt&y organs A4H:B* ConseCuently) t&ese %elivery systems may increase t&e relative e!!icacy or sa!ety o! cancer t&erapies) an% t&us serve to increase t&eir t&erapeutic in%e"* In recent years) t&e !iel% o! nanoparticleE#ase% %rug %elivery &as #een reinvigoE rate% #y a convergence o! nanotec&nology an% me%icine A;H>B* In essence) t&e #len%E ing o! t&ese !iel%s is lea%ing to t&e generation o! innovative synt&etic vectors (it& t&e potential o! ac&ieving t&e long soug&t a!ter goal o! tumorEtargete% %rug %elivery6 getE ting t&e active agentAsB solely (&ere t&ey are nee%e%) t&e soli% tumor* -urt&ermore) t&e versatility o! t&ese nanoparticle systems may !or t&e !irst time lea% to t&e %evelopE ment o! multi!unctional nanot&erapeutics A-ig* 4B t&at %etect an% attac$ t&e &eterogeE neous population o! tumor cells present in a soli% tumor* As s&o(n in -igure 4) t&ese putative vectors may consist o! an immuneEavoi%ance moiety Aa Qstealt&S moietyB) a tumorEtargeting moti!) multiple t&erapeutics) an% a %iagnostic1sensing component) all o! (&ic& are %elivere% on a single nanoparticle no larger t&an :@ nm* ?ver t&e past 5@ years) t&e !iel% o! nanoparticleE#ase% %rug %elivery !ocuse% on t(o c&emically %istinct colloi%al particles6 liposomes an% #io%egra%a#le polyE mers A4@H49B* +ot& %elivery systems encapsulate1entrap t&e active %rug (it&in t&eir structures an% release t&e active agent as t&e particle lyses) in t&e case o! liposomes) or %isintegrates as %escri#e% !or #io%egra%a#le polymers* A recent ne(comer to t&is !iel% is t&e metallic nanoparticle* i$e t&eir organic counterparts) t&e metallic partiE cles &ave a long &istory o! use in #iologyM &o(ever) only recently &ave t&ey #een !ormulate% into tumorEtargeting vectors* T&is c&apter is %ivi%e% into t&ree parts* T&e Q+iological Consi%erationsS section !ocuses on t&e #iological #arriers !acing metallicEnanoparticleE#ase%) tumorEtargete%

494

495
T&erapeutic 4

Paciotti an% Tamar$in

Stealt& Moiety

T&erapeutic 5

Diagnostic1 Sensing Component

Tumor Targeting Moti!

T&erapeutic 8

-IGURE 4 Sc&ematic representaE tion o! a multi!unctional nanoparticle t&erapeutic*

%rug %elivery) an% (ill center on t&e %elivery o! protein an% smallEmoleculeE#ase% t&erapies* Some o! t&ese #arriers are naturally present in &ealt&y portions o! t&e #o%y) (&ereas ot&ers are esta#lis&e% %uring tumor gro(t& an% progression* T&e section QMetallic Nanoparticle DrugEDelivery SystemsS (ill %escri#e t&ree e"amE ples o! metallicEnanoparticleE#ase%) tumorEtargete% %rug %elivery an% t&e speci!ic means use% to overcome t&ese #arriers* Speci!ically) an overvie( o! magneticEiron an% colloi%al gol%E#ase% nanoparticle %rugE%elivery systems (ill #e %iscusse%* T&e !inal section (ill !ocus on t&e %esign an% manu!acturing o! multi!unctional metallic nanoparticles t&at target t&e %elivery o! multiple cancer t&erapies (it& a single metallic nanoparticle*

+I? ?GICA C?NSIDERATI?NS Clearance #y t&e Reticuloen%ot&elial System T&e painsta$ing lessons learne% !rom t&e liposomalE an% polymerE#ase% %rugE %elivery systems must #e consi%ere% in t&e !ormulation o! t&e metallic nanopartiE cles* T&ere is ample prece%ent suggesting t&at imme%iately a!ter t&eir e"posure to t&e circulation) unprotecte% metallic nanoparticles (ill most li$ely un%ergo opsinE i/ation in t&e #loo% an% clearance #y t&e RES* ?psini/ation is t&e process #y (&ic& #loo%E#orne proteins) $no(n as opsins) #in% to t&e metallic nanoparticle sur!ace* ?psonic proteins inclu%e immunoglo#ulins) complement proteins C4 an% C8) apolipoproteins) von ,ille#ran% !actor) t&rom#ospon%in) !i#ronectin) an% mannoseE #in%ing protein A4:H4>B* ?nce #oun% to t&e sur!ace o! t&e particles) opsins act as ligan%s t&at are recogni/e%) #oun%) an% internali/e% #y a class o! macrop&ages $no(n as 0up!!er cells* 0up!!er cells are speciali/e% macrop&ages t&at resi%e in t&e liver an% represent t&e primary cellular component o! t&e scavenging system o! t&e #o%y) $no(n as t&e RES* Un%er p&ysiologic con%itions) t&e RES rapi%ly an% very e!!iciently clears opsini/e% particulates) suc& as #acteria an% colloi%al particles) !rom t&e #o%y* -or e"ample) in mice intravenously in.ecte% (it& unprotecte% colloi%al gol% nanopartiE cles Ai*e*) colloi%al gol% nanoparticles t&at contain only %rug #oun% to t&eir sur!aceB) (e o#serve% t&at >@[ to >:[ o! t&ese gol% nanoparticles are cleare% !rom t&e circuE lation (it&in : to 4@ minutes a!ter in.ection* T&ese o#servations are consistent (it& t&e &istorical #io%istri#ution %ata o! unprotecte% liposomes* Early (or$ in t&e !iel% o! particleE#ase% %rug %elivery %emonstrate% t&at RES clearance (as satura#le) #ecause pretreating animals (it& &ig& %oses o! %rug or place#o nanoparticle vectors Ai*e*) vectors lac$ing t&e active pro%uct ingre%ientB

+iological an% Engineering Consi%erations

498

e"cee%e% t&e p&agocytic capacity o! t&e RES A5@H59B* ConseCuently) nanoparticles not trappe% in t&e liver or spleen (ere availa#le to %eliver %rugs to soli% tumors* A more practical approac& to a%%ress RES upta$e an% clearance is t&e mo%i!ication o! t&e nanoparticle sur!ace to inclu%e &y%rop&ilic #loc$ers suc& as polyet&ylene glycol APEGB as (ell as #loc$ copolymers o! t&e tetronic an% pluronic !amilies o! sur!actants* Gra!ting1#in%ing t&ese &y%rop&ilic moieties onto t&e sur!ace o! t&e colloi%al nanoparticles ma%e t&em Qinvisi#leS to t&e RES* Typically) suc& sterically sta#ili/e% nanoparticles e"&i#it prolonge% circulatory &al!Eli!e an% as %iscusse% #elo( suc& nanoparticle !ormulations may passively accumulate in soli% tumors t&roug& e"travasation o! t&e lea$y vasculature t&at !ee% t&em A5:H5=B* T&e nature an% con!ormation o! potential stealt& molecules in!luence t&e a#ility o! t&e immune system to %etect an% clear nanoparticles* As an e"ample) Mog&imi A5>B %emonstrate% t&at t&e concentration an% con!ormation o! one suc& polymer) polo"amer 9@<) signi!icantly altere% t&e #io%istri#ution o! polystyrene nanoparticles a!ter su#cutaneous in.ection* ,&en t&e et&ylene o"i%e AET?WB tails o! t&e polo"amer polymer (ere gra!te% at relatively lo( concentrations) t&e molecule assume% a !lat or mus&roomEli$e con!iguration on t&e sur!ace o! t&e particles) (&ereas at &ig&er polymer concentrations t&e closely pac$e% ET?W tails assume% a #rus&Eli$e con!ormation on t&e sur!ace o! t&e particles* T&e particles (it& t&e mus&roomEli$e con!iguration (ere rea%ily pic$e% up #y t&e macrop&ages present in t&e primary or secon%ary lymp& no%es) (&ereas t&ose carrying t&e #rus&Eli$e con!iguration eva%e% t&e lymp& no%es) travele% t&roug& t&e lymp&atic system an% reentere% t&e circulation* In our la#oratory) (e also o#serve% t&at t&e manner #y (&ic& a stealt& molecule #in%s to %rugEcoate% colloi%al gol% nanoparticles in!luences t&eir #io%isE tri#ution A8@B* As %escri#e% a#ove) >:[ o! various !ormulations o! %rugEsta#ili/e%) mono%isperse% colloi%al gol% nanoparticles Nnanoparticles #oun% (it& a saturating amount o! t&e protein t&erapeutic) tumor necrosis !actor ATN-B alp&aO) (ere cleare% #y t&e RES (it&in : to 4@ minutes o! intravenous in.ection* T&e !ate o! t&ese mono%isperse% nanoparticles (as evi%ent upon animal necropsy as #ot& t&e livers an% spleens (ere #lac$ (it& aggregate% colloi%al gol% precipitates* Several #loc$ copolymers inclu%ing pluronic) tetronic) car#o(a") an% monot&iolate% !orms o! PEG APEGETHI? B (ere teste% !or t&eir a#ility to #loc$ RES upta$e an% clearance* Alt&oug& all !our classes o! polymers #oun% an% sta#ili/e% t&e colloi%al gol% nanoparticles in a test tu#e) only PEGETHI? prevente% RES upta$e an% clearance (&en in.ecte% intravenously* Unli$e t&e polo"amerEsta#ili/e% nanoparticles) (e #elieve t&at t&e pluronicE) tetronicE) an% car#o(a"E#ase% polymers (ere una#le to #in% to t&e sur!ace o! t&e colloi%al gol% nanoparticle* Ho(ever) t&e presence o! t&e single sul!&y%ryl group on t&e PEG molecule APEGETHI? B allo(e% it to #in% %irectly to t&e sur!ace o! t&e particle) intersperse% #et(een t&e protein t&erapeutic* In t&is case) (e #elieve t&at t&e molecules o! PEGETHI? assume% a #rus&Eli$e con!iguraE tion on t&e sur!ace o! t&e nanoparticles) an% t&e #iological response Ai*e*) avoi%ing immune recognitionB (as similar to t&at seen in t&e e"ample a#ove* Sur!ace c&aracteristics inclu%ing si/e) sur!ace contour1!acets) an% c&arge may also regulate (&ic& opsins #in% an% t&e e"tent to (&ic& t&ey a%&ere to t&e sur!ace o! t&e particles* Particle si/e can a!!ect particle clearance #y t(o mec&anisms A84H89B* -irst) larger particles more e!!iciently activate t&e complement pat&(ay !or clearance #y t&e RES* Secon%) in t&e re% pulp o! t&e spleen) t&e interen%ot&elial slits present on t&e en%ot&elial cells o! t&e venous sinusoi%s may !ilter particles) merely #ase% on si/e Alarger t&an 5@@ nmB* T&e latter e!!ect seems more pro#lematic !or t&e

499

Paciotti an% Tamar$in

earlier !ormulations o! liposomes rat&er t&an metallic nanoparticles as metalE#ase% nanoparticles range in si/e !rom : to 9@ nm* Also) as %escri#e% #elo() particle si/e may in!luence t&e a#ility o! nanoparticles to e!!ectively %eliver %rugs to t&e tumor interstitium* Sur!ace c&arge must also #e consi%ere% in !ormulating metallic nanoparticles #ecause t&e overall sur!ace c&arge may %etermine t&e %egree to (&ic& particles are cleare% #y t&e RES) as (ell as t&eir a#ility to interact (it& en%ot&elial cells t&at !orm t&e tumor neovasculature* -or e"ample) t&e RES rea%ily clears anionic lipi% particles) (&ereas neutral lipi% particles are not rea%ily cleare% A4=)88H8:B* Cationic liposomes containing an RESEavoi%ance system Ai*e*) PEGB pre!erentially #in% to t&e sur!ace o! tumor en%ot&elia A8;)8<B* T&e process o! particle opsini/ation an% RES clearance is an active process #y (&ic& particleE#oun% opsins are recogni/e%) #oun%) an% internali/e% #y t&e macroE p&ages present in t&e liver an% spleen* ?vercoming t&is #iological #arrier is .ust t&e !irst step in tumorEtargete% %rug %elivery* So) even (&en RES clearance is a%eCuately a%%resse%) t&e p&ysical #arriers esta#lis&e% %uring soli% tumor !ormation an% proE gression must ne"t #e a%%resse% !or t&e %evelopment o! a success!ul tumorEtargete% metallic %rug %elivery vector*

Tumor Angiogenesis an% 2asculari/ation In 4><4) -ol$man A8=B lai% out &is &ypot&esis %escri#ing t&e angiogenic mo%el o! tumor gro(t& an% metastases* A central component o! t&is &ypot&esis is t&at soli% tumors) in response to p&ysiologic stresses suc& as &ypo"ia) secrete !actors t&at cause ne( #loo% vessels to sprout an% gro( !rom e"isting #loo% vessels to(ar% t&e tumor* Eventually t&ese angiogenic #loo% vessels #ecome ine"ora#ly ent(ine% in t&e soli% tumor mass* Suc& angiogenesis is reCuire% to ensure continue% tumor gro(t& an% progression) an% t&is process is currently t&e !ocus o! many novel cancer t&erapies* Angiogenesis) t&e process #y (&ic& ne( #loo% vessels sprout !rom e"isting normal #loo% vessels) occurs in a variety o! %isease states inclu%ing cancer an% in!lammation as (ell as %uring normal p&ysiological events suc& as t&e t&ic$ening o! t&e uterine (all) !ollicle %evelopment) an% (oun% &ealing* During (oun% &ealing) !or e"ample) angiogenesis an% clot !ormation are &ig&ly interconnecte% processes t&at are regulate% #y platelets an% solu#le #loo% proteins A8>B* Upon in.ury) tissue !actor is release% an% in%uces clot !ormation t&roug& t&e (ellEc&aracteri/e% casca%e o! clotting !actors t&at ultimately lea% to t&e !ormation o! a clot #y t&e t&rom#inEme%iate% conversion o! !i#rinogen to !i#rin* T&rom#in also activates platelets to release a variety o! potent proEangiogenic !actors inclu%ing various !orms o! vascular en%ot&elial gro(t& !actors A) +) an% C A2EG- A) +) an% C) respectivelyB) !i#ro#last gro(t& !actorE5) an% angiopoietinE5* -inally) t&rom#in stimulates en%ot&elial cells to secrete a variety o! en/ymes responsi#le !or t&e %egra%ation o! t&e #asement mem#rane causing t&e release o! 2EG- !rom t&ese compartments A8>)9@B* Platelets may also #e recruite% an% activate% to secrete t&ese proEangiogenic !actors #y interaction (it& collagen or in%irectly t&roug& interaction (it& von ,ille#ran% !actor* T&ese proEangiogenic !actors signal t&e surroun%ing vascular en%ot&elial cells to gro( an% migrate t&roug& t&e !i#rin clot A94B* To $eep t&e angiogenic process in c&ec$) platelets also secrete antiangiogenic !actors suc& as t&rom#ospon%inE4) plasminogen) an% TG-fE4) (&ic& serve to #loc$ angiogenesis) an% in%uce t&e e"pression o! en/ymes t&at &elp #rea$ %o(n t&e clot* Tig&t control

+iological an% Engineering Consi%erations

49:

#et(een t&e proE an% antiangiogenic signals ensures t&at as t&e process o! tissue repair an% remo%eling is complete%) angiogenesis is terminate%* ,it&in soli% tumors) &o(ever) t&e &omeostatic mec&anism !or controlling ne( #loo% vessel !ormation is a#errant) o!ten !avoring angiogenesis an% continue% tumor gro(t& an% metastasis* -or e"ample) t&e constitutive e"pression o! tissue !actor #y tumor an% supporting stromal cells may in%uce t&e clotting mec&anism #y circulatE ing coagulating !actors) suc& as !actor 2IIa) to ultimately cause plateletEme%iate% coagulation an% release o! 2EG- A95)98B* In%ee% t&ere is a reasona#le prece%ent in t&e literature s&o(ing a potential lin$ #et(een t&e state o! coagulation an% tumor progression A99)9:B* Gasic et al* A9;B %emonstrate% t&is potential lin$ #y s&o(ing t&at anti#o%ies (&ic& #loc$e% platelet !unction in&i#ite% t&e !ormation o! pulmoE nary metastases* More recently) Cramerer et al* A9<B &ig&lig&te% a mo%el in (&ic& circulating tumor cells use secrete% tissue !actor as a means o! in%ucing t&e clotting mec&anism to arrest t&eir migration !rom t&e normal vasculature) to traverse t&e en%ot&elial cell #arrier) an% ultimately to !orm secon%ary metastases* A ne(ly i%enti!ie% an% more pro#lematic cell type may provi%e soli% tumors (it& not only angiogenic support) #ut also (it& strong protection against t&e a#ility o! t&e immune system to in%uce an antitumor response* TumorEassociate% macroE p&ages ATAMsB are monocyteE%erive% macrop&ages t&at migrate !rom t&e vasculaE ture to regions o! &ypo"ia in soli% tumors* Unli$e (il%Etype macrop&ages t&at are present in normal tissues an% in (ellEo"ygenate% areas o! tumors) TAMs e"&i#it altere% p&enotypes* -or e"ample) TAMs secrete immunosuppressive cyto$ines) suc& as I E4@) TG-f) an% prostaglan%ins A9=H:@B* -inally) TAMs resi%ing in t&e &ypo"ic centers o! tumors are not e!!ective at antigen presentation A:4B* T&us) TAMs may in%irectly support tumor gro(t& #y #lunting t&e a#ility o! t&e immune system to mount an e!!ective antitumor response* In !act) once trappe% in t&e &ypo"ic regions o! tumors) TAMs may actually #egin to support tumor gro(t&* In response to t&e lo( o"ygen tension present in t&e &ypo"ic regions o! soli% tumors) TAMs upregulate t&e e"pression o! &ypo"iaE in%uci#le !actors one an% t(o AHI-E4 an% HI-E5) respectivelyB A:5H:9B* Tumor cells also e"press t&ese !actors in response to &ypo"ia A::B* T&e HI- !amily o! proteins are transcription !actors t&at translocate to t&e nucleus) #in% speci!ic DNA regions $no(n as &ypo"iaEresponse elements to stimulate gene e"pression) an% ultimately lea% to 2EG- secretion #y t&e macrop&ages an% tumor cells A:5)::):;B*

2ascular En%ot&elial Gro(t& -actor 2EG- (as i%enti!ie% in t&e late 4><@s !or its a#ility to in%uce vascular permea#ility o! venules an% small veins present in t&e postcapillary ple"us* 2EG-Ein%uce% permea#ility is me%iate% #y t&e !ormation o! vesicular vacuolar organelles A22?sB A:<H:>B* 22?s are a series o! interconnecte% vesicles an% vacuoles t&at span t&e entire %istance o! en%ot&elial cells !rom t&e lumenal to t&e a#lumenal si%e o! t&e en%ot&elial cells* In t&e a#sence o! 2EG-) access o! macromolecules to t&e 22?s is limite% #y t&e presence o! a mem#raneEcovere% stomata (&ic& is connecte% to t&e plasma mem#rane o! t&e en%ot&elial cell* In response to 2EG- stimulation) t&e stomata open to allo( passage o! tracer elements) plasma) an% plasma proteins !rom t&e lumenal si%e o! t&e en%ot&elial cell) t&roug& t&e 22?s) to t&e e"travascular space o! tissues* A%%itional structures suc& as intercellular openings an% intracellular &oles may also increase t&e permea#ility o! tumor #loo% vessels* -or e"ample) Has&i/ume

49;

Paciotti an% Tamar$in

et al* A;@B %escri#e% roun% or oval openings #et(een t&e en%ot&elial #loo% vessel cells supporting t&e gro(t& o! MCaEI2 tumors* T&ese openings are muc& larger t&an t&e !enestrations A:@H=@ nmB A;@B reporte% !or tumor #loo% vessel en%ot&elium an% range% !rom @*8 to 9*< _m (it& an average o! 4*< _m* In a%%ition) transcellular &oles ranging !rom @*5 to @*> _m (ere also reporte%* Alt&oug& a%%itional stu%y is nee%e%) t&e presence o! suc& e"traE!enestral openings suggests a potential mec&aE nism #y (&ic& nanoparticleE%elivery systems passively accumulate in soli% tumors rat&er t&an ot&er &ealt&y !iltering organs suc& as t&e liver) an organ in (&ic& !enesE trae ranging !rom :@ to 8@@ nm &ave #een %escri#e%* T&e increase in vascular permea#ility an% t&e %eposition o! a !i#rin clot are among t&e responses attri#ute% to 2EG-Eme%iate% angiogenesis* ,it&in t&e !i#rin clot) 2EG-Eme%iate% angiogenesis starts (it& t&e generation o! a mot&er vessel) a vessel c&aracteri/e% #y poor coverage (it& #ot& a #asement mem#rane an% periE cytes* Su#seCuently) t&e !ormation o! ne( #loo% vessels may occur #y one o! t&ree mec&anisms* -irst) t&e mot&er vessel may un%ergo sprouting) en%ot&elial cell migration) an% proli!eration* Secon%) t&e mot&er vessel may un%ergo #ri%ging in (&ic& translumenal #ri%ges) !orme% #et(een t&e en%ot&elial cells) serve to %ivi%e t&e lumen o! t&e mot&er vessels into smaller c&annels* Ultimately) t&ese multic&anE nel vessels give rise to ne( %aug&ter vessels* -inally) %uring intussusceptive vessel !ormation) t&e mot&er vessel invaginates to give rise to t(o vessels*

C&aracteri/ation o! t&e Angiogenic Tumor +loo% 2essels During normal p&ysiologic con%itions) t&e neonatal vasculature is un%er t&e tig&t control o! t(o processes6 vasculogenesis an% angiogenesis* 2asculogenesis) t&e %e novo generation o! #loo% vessels) is initiate% !rom a precursor stem cell or angioE #last) (&ereas %uring angiogenesis t&ese ne(ly !orme% vessels are instructe% to un%ergo #ranc&ing an% sprouting #y signals inclu%ing 2EG-* T&ese !ragile) ne(ly !orme% #loo% vessels are rein!orce% #y a cast o! supporting cellular Amural cellsB an% structural elements inclu%ing pericytes) smoot& muscle cells) an% t&e e"tracelE lular matri") a process (&ic& is regulate% an% coor%inate% #y e"tracellular signals inclu%ing PDG-) EDG4) an% S4P4 A;4B* -inally) %uring em#ryonic %evelopment) t&e vasculature is tailore% an% remo%ele% to !it t&e nee%s o! t&e organ it supplies #y t&e locali/e% e"pression o! c&emoattractants an% repellents suc& as ep&rins an% neuropilins A;5H;9B* ?verall) t&e process results in t&e !ormation o! &ig&ly or%ere% vascular #e%s comprise% o! arteries) arterioles) capillaries) venules) an% veins* In tumors) &o(ever) t&e organi/ation o! angiogenic #loo% vessels resem#les &ap&a/ar% arrays o! sprouting #loo% vessels) tumor cells) an% pericyteEli$e cells (&ic& collectively !orm a vessel t&at o!tentimes lac$s an organi/e% e"tracellular matri"* T&e &yperpermea#ility o! t&ese #loo% vessels) a process (&ic& is in part me%iate% #y 2EG-) allo(s t&e e"travasation o! colloi%al particles suc& as colloi%al car#on an% colloi%al gol%) as (ell as tracer molecules inclu%ing ra%ioisotopes) !luorescently la#ele% %e"trans) an% al#umin A:=);:H;<B* In certain instances) eryt&rocytes cross t&e vascular #e% an% pool to !orm vascular la$es o! #loo% cells A;@B* Recently) t&e tumor cells t&emselves (ere reporte% to !orm t&e soEcalle% Qtumor #loo% vessels*S C&ang et al* A;=B %escri#e% t&e presence o! QmosaicS #loo% vessels in a murine colon tumor mo%el* T&ese mosaic #loo% vessels consiste% o! #ot& tumor an% en%ot&elial cells (&ic& (ere %istinguis&e% #y !luorescence* In t&ese stu%ies) C&ang implante% S4<9T murine colon carcinoma tumor cells) (&ic& (ere sta#ly trans!ecte% to e"press green !luorescence protein AG-PB) into SCID mice*

+iological an% Engineering Consi%erations

49<

,it& tumor !ormation) t&e intracellular e"pression o! G-P (as use% to i%enti!y t&e tumor cells) (&ereas Cy: an% r&o%amineEcon.ugate% antiECD8414@: systems (ere use% to i%enti!y t&e en%ot&elial cells* T&ey report t&at nearly 4:[ o! t&e vessels e"amine% %i% not s&o( any en%ot&elial cell staining) #ut %i% s&o( t&e green !luoE rescence o! t&e implante% tumor cell line) creating t&e lumen o! t&e Qtumor #loo% vessel*S In contrast to t&e %ata reporte% #y Mons$y et al* A;>B Asee laterB) (&o reporte% t&at t&e permea#ility o! angiogenic #loo% vessels (as in!luence% #y t&e location o! tumor cell implantation) t&e location o! tumor cell implantation in t&e stu%ies con%ucte% #y C&ang et al* %i% not a!!ect t&e presence or percentage o! t&e mosaic cells* T&ese mosaic #loo% vessels are t&oug&t to represent one mec&anism #y (&ic& s&e%%ing tumor cells enter t&e circulation in !ormation o! metastases* -urt&ermore) given t&at mosaic cells) similar to t&ose %escri#e% a#ove) (ere also i%enti!ie% in #iopsies o! colon cancer patients A;=B &as signi!icant implications !or angiogenicEtargete% cancer t&erapies) #ecause t&e main target !or t&ese t&erapies) t&e ne(ly !orme% #loo% vessels) may not #e present in all cases*

Tumor Angiogenesis an% Interstitial -lui% Pressure -or some time no( t&e in&erent lea$iness o! t&e tumor vasculature &as #een a target !or %eveloping nanoparticleE#ase%) tumorEtargete% %elivery vectors* +ecause o! t&eir si/e) t&ese particleE#ase% t&erapeutics may passively e"travasate t&e lea$y tumor vasculature to accumulate (it&in soli% tumors* TumorH&ost interactions are also s&o(n to control t&e %egree o! en%ot&elial cell lea$iness* Mons$y et al* A;>B %emonstrate% t&at t&e %egree o! tumor #loo% vessel porosity is %epen%ent not only on t&e tumor type) #ut also on its location* -or e"ample) t&e in&erent vascular perE mea#ility o! &uman #reast cancer tumors (as %epen%ent on t&e site o! implantation* Alt&oug& tumors implante% in t&e cranial (all (ere &ig&ly angiogenic) (&en compare% (it& t&e same tumors implante% in mammary !at pa%) t&ey (ere also less permea#le to !luorescently la#ele% in%icators* Nevert&eless) t&e !ormation o! an intratumor clot) in t&e presence o! continE ue% angiogenesis) an% a lateE!orming lymp&atic system com#ine to increase t&e interstitial !lui% pressure AI-PB o! soli% tumors* T&is &ypertensive state represents a ma.or o#stacle !or t&e penetration o! macromolecularE or nanoparticleE#ase% t&erapies to soli% tumors* To e!!ectively overcome t&is #arrier) nanoparticleE#ase% %elivery systems must contain elements t&at #rea$ %o(n t&is interstitial pressure gra%ient* Descri#e% #elo( are t(o classes o! t&erapeutic compoun%s s&o(n to %isrupt t&e I-P gra%ient*

Antiangiogenic T&erapies ?ne clear c&oice !or re%ucing t&e I-P in soli% tumors is to #loc$ 2EG- signaling* Preclinical an% clinical stu%ies (it& monoclonal anti#o%ies %esigne% to #loc$ t&e interaction o! 2EG- (it& its receptor s&o( t&at t&e anti#o%y not only in&i#its t&e !ormation o! ne( #loo% vessels) #ut also causes t&e regression o! alrea%y esta#E lis&e% vessels) an o#servation (&ic& is consistent (it& t&e role t&at 2EG- serves as a survival !actor !or ne(ly !orme% #loo% vessels A<@)<4B* More interestingly) treatments (it& suc& monoclonal anti#o%ies cause an apparent reorgani/ation an% restructuring o! t&e #loo% vessels (it&in soli% tumors* T&is process) terme% normali/ation A<@)<5B) involves a pruning o! e"isting #loo% vessels) improve% covering o! t&e normali/ing #loo% vessels (it& pericytes) an% reesta#lis&ment o! #asement mem#rane* ?verall) normali/ation results in improve%

49=

Paciotti an% Tamar$in

#loo% !lo( in soli% tumors) %ecreases t&e I-P o! tumors Aan o#servation (&ic& &as also #een %emonstrate% in colon cancer patientsB) an% esta#lis&es a &y%rostatic presE sure gra%ient across t&e vascular (all* T&e com#ination o! t&ese events improves t&e accumulation an% e!!icacy o! secon%ary t&erapies) suc& as c&emot&erapy* T&ere is) &o(ever) one caveat associate% (it& t&e antiE2EG-Ein%uce% normalE i/ation o! t&e tumor vasculature an% its propose% synergy (it& c&emot&erapy* Apparently) t&ere is a perio% o! time) $no(n as t&e Qnormali/ation (in%o(S A<4B) in (&ic& synergy o! antiE2EG- t&erapy (it& ra%iation or c&emical t&erapies is optimal* T&is perio% o! time is temporally !lan$e% Ai*e*) prior to an% imme%iately !ollo(ing t&e normali/ation (in%o(B #y t(o time perio%s in (&ic& t&e com#ination is less e!!ective* Not surprisingly) prior to normali/ation) (&en soli% tumors are supporte% #y t&eir torturous1lea$y circulation vasculature) t&e synergy o! antiE2EG- treatE ment an% c&emot&erapy proves ine!!ective* During normali/ation) t&e %rop in I-P couple% (it& t&e reesta#lis&ment o! a vascular pressure gra%ient improves t&e e!!icacy o! secon%ary t&erapies* -inally) as t&e antiE2EG- t&erapy causes t&e %eat& o! t&e tumor neovasculature) t&e e!!icacy o! antiE2EG- an% ra%io or c&emot&erapies once again %ecreases* T&ese %ata) &o(ever) suggest t&at in or%er to ma"imi/e t&e synergy #et(een antiE2EG- an% c&emot&erapy) t&e normali/ation (in%o( in cancer patients must #e clearly i%enti!ie% to ensure ma"imal synergy #et(een t&e t(o t&erapies*

Cyto$ineEMe%iate% Re%uction o! Interstitial -lui% Pressure TN- (as initially %escri#e% as a serum !actor isolate% !rom t&e #loo% o! en%oto"inE treate% mice) (&ic& (as capa#le o! in%ucing &emorr&agic necrosis o! soli% tumors* Nevert&eless) in t&e 4>=@s &opes o! using TN- as a cancer t&erapy (ere nearly %as&e% #y t&e li!eEt&reatening to"icities it in%uce% A<5B) a !ate s&are% #y many cytoE $ines s&o(ing promise in preclinical tumor mo%els* T&e ma.or to"icity o#serve% %uring t&e systemic a%ministration o! TN- is severe &ypotension* 3et t&e pioneering (or$s o! ienar% an% e.uene A<8)<9B %emonstrate% vast improvements in t&e t&erapeutic in%e" o! TN- #y limiting its %elivery to soli% tumors* -or TN-) t&e isolate% lim# protocol AI PB %emonstrate% t&at t&e com#inaE tion o! surgically locali/e% %elivery o! t&e cyto$ine (it& regionally a%ministere% c&emot&erapies in%uce% sustaina#le antitumor responses in patients !ailing tra%iE tional stan%ar%s o! care A<5H<9B* +y locali/ing t&e %elivery o! TN- to soli% tumors) ienar% an% e.uene &arnesse% TN- to in%uce at least one o! t&e possi#le mec&aE nisms #y (&ic& t&e cyto$ine in%uces an antitumor response* -or e"ample) TN- may %irectly cause apoptosis o! various tumor cell types A<:)<;B) activate an% %rive immuneE#ase% antitumor responses A<<B) an% in%uce vascular lea$ in tumor #loo% vessels A<=)<>B) lea%ing to a %estruction o! t&e I-P gra%ient in soli% tumors A=@)=4B* T&e clinical o#servations ma%e on t&e timing1or%er o! TN- an% c&emot&erE apy treatment is consistent (it& a re%uction in t&e I-P* In or%er to ac&ieve ma"imal antitumor responses) TN- treatment eit&er prece%e% or (as given simultaneously (it& c&emot&erapy* Reversing t&e or%er) t&at is) giving t&e c&emot&erapy !irst !ollo(e% #y TN- treatment) pro%uce% less t&an optimal results* In e!!ect) I P (it& TN- serve% to sensiti/e t&e tumors to c&emot&erapy) an% in preclinical mo%els) t&is o#servation correlate% (it& improve% upta$e o! c&emot&erapeutic agents #y t&e soli% tumors A=5B* In t&e !ollo(ing section) (e (ill %escri#e our recent e!!orts to simulate t&e I P using colloi%al gol%E#ase% nanot&erapeutics t&at seCuester not only TN-) #ut also a c&emot&erapeutic Ai*e*) paclita"elB in murine tumors*

+iological an% Engineering Consi%erations

49>

+arriers ,it&in t&e Tumor Interstitium6 Intratumor +arriers In or%er to ac&ieve e!!ective %elivery o! antineoplastic agents to soli% tumors) nanoparticle %elivery systems must e!!ectively eva%e immune %etection #y t&e RES) !in% an% e"travasate t&e lea$y tumor vasculature) an% simultaneously possess met&o%s !or re%ucing t&e I-P* 3et) even (&en t&ese prereCuisites are met) t&e intraE tumor microenvironment presents one a%%itional o#stacle t&at may t&(art e!!ective %rug %elivery to t&e tumor interstitium* T&ese nanot&erapeutics must overcome t&e spatial #arrier esta#lis&e% #y t&e e"tracellular matri" o! soli% tumors* T&is viscous matri" consists o! a !i#rous mes& o! proteoglycanEassem#le% collagen !i#ers in (&ic& stromal an% tumor cells are suspen%e%* T&e orientation o! collagen type I !i#ers present in t&e e"tracellular matri" may retar% t&e tra!!ic$ing an% penetration o! large macromolecules suc& as IgG) IgM) an% &ig&EmolecularE(eig&t %e"tran an% nanoparticleE%elivery systems t&roug& t&e tumor interstitium* T&e main o#stacle pose% #y collagen is t&e spatial orientation o! t&e in%ivi%ual collagen !i#rils an% collagen #un%les* Depen%ing on t&e tumor mo%el) t&e small space #et(een in%ivi%ual collagen !i#ers may &in%er t&e moveE ment o! particles larger t&an 9@ nm) (&ereas t&e collagen #un%les may retar% t&e tra!!ic$ing o! particles t&at are <: to 48@ nm in %iameter* Interestingly) in murine tumor mo%els) t&e relative contri#ution o! t&e collagen mes& appears to #e conE trolle% #y tumorH&ost interactions #ecause t&e %ensity o! collagen an% t&e comple"E ity o! t&e mes& are %epen%ent on t&e site o! tumor implantation A=8B*

Active vs* Passive Tumor Targeting Passive e"travasation t&roug& t&e lea$y tumor vasculature is t&e primary mec&aE nism #y (&ic& nanoparticle vectors gain access to t&e tumor interstitium* Upon e"travasation) t&ese nanot&erapeutics rely on %i!!usion) rat&er t&an convection) as t&e primary mec&anism !or %elivering %rugs to cancer cells* In e!!ect) t&e passive accumulation o! t&e nanot&erapeutics is %esigne% to limit t&e #io%istri#ution o! t&e t&erapeutics* To !urt&er improve t&e %elivery o! t&e t&erapeutic payloa%) t&e nanoparticles may #e engineere% to contain tumorEtargeting ligan%s t&at gui%e t&e %elivery o! t&ese vectors to tumor) en%ot&elial) an% potentially stromal cells* Suc& targeting ligan%s also assist in t&e upta$e o! t&e nanot&erapeutics #y tumor cells #y receptorE me%iate% mec&anisms inclu%ing receptorEme%iate% en%ocytosis an% potolysis* E"amples o! tumorEtargeting ligan%s inclu%e oligosacc&ari%es) !olic aci%) EG-) an% TN-) as (ell as anti#o%yE%irecte% cell sur!ace receptors A=9H=>B*

Tumor Mo%els ?ne !inal c&allenge in %eveloping nanoparticle t&erapeutics lies in t&e e"trapolation o! preclinical !in%ings) o!ten con%ucte% in ro%ent tumor mo%els) to t&e clinical setE ting* Typically) a!ter s&o(ing promise in suc& preclinical stu%ies) can%i%ate %rug t&erapies are teste% in -DAEgui%e% G P to"icology stu%ies) an% in%ee% t&ese stu%E ies are reCuisite to ensure patient sa!ety* 3et) testing suc& t&erapeutics in patients (it& naturally occurring cancer is t&e critical test* In a%vance o! !ormal clinical trials) treating companion animals Ai*e*) petsB (it& inopera#le1unresponsive tumors on a compassionate use #asis may provi%e a%%itional insig&t to %ata o#taine% !rom animal tumor mo%els* It is estimate% t&at in t&e U*S*A* alone t&ere are 48@ million cats an% %ogs) o! (&ic& 4@[ to 5:[ present (it& a variety o! naturally occurring soli% tumors A>@B* ,e #elieve t&at suc& terminally ill animals may serve as sentinels

4:@

Paciotti an% Tamar$in

!or t&e &uman con%ition an% may provi%e valua#le in!ormation regar%ing %rug per!ormance in &uman cancer patients* META IC NAN?PARTIC E DRUGEDE I2ER3 S3STEMS

Alt&oug& metallic nanoparticles &ave a long &istory o! use in #iology) t&eir applicaE tion to tumorEtargete% %rug %elivery &as only recently #een %escri#e%* -or e"ample) colloi%al gol% nanoparticles &ave #een use% as %iagnostic in%icators an% t&erapeuE tics since t&e early 4>:@s* Similarly) alt&oug& t&e magnetic properties o! iron nanoparticles &ave ma%e t&em a valua#le tool !or magnetic resonance imaging AMRIB) t&eir use in magneticEtargete% %rug %elivery to soli% tumors &as only recently #een %escri#e%* In t&e !ollo(ing sections) (e %escri#e t&e c&aracteri/ation o! t&ese nanoparticulates) to"icologic consi%erations) an% !ormulation into tumorEtargete% %rugE%elivery vectors*

Primer on t&e Use o! Colloi%al Gol% an% Iron Nanoparticles in +iotec&nology Gol% Nanoparticles In 4=:<) Mic&ael -ara%ay A>4B (&en generating multicolore% solutions #y reacting gol% c&lori%e (it& so%ium citrate) &e coul% not &ave appreciate% t&e !act t&at &e (as laying t&e !oun%ation !or t&e !iel% o! nanotec&nology* Un#e$no(nst to &im (&at &e actually %escri#e% (as t&e synt&esis o! colloi%al gol% nanoparticles t&at range% !rom 45 to 4@@ nm in %iameter* Since t&at time gol% nanoparticles &ave #een use% to meet a variety o! nee%s in science an% me%icine* In t&e 4>:@s) t&e %iscovery t&at t&e particles #oun% protein #iologics (it&out altering t&eir activity pave% t&e (ay !or t&eir use in &an%E&el% immuno%iagnostics an% in &istopat&ology A>5B* ?! particular importance to t&e current %iscussion (as t&e longEstan%ing use o! colloi%al gol% in t&e treatment o! r&eumatoi% art&ritis as (ell as t&e use o! ra%ioactive gol% nanoparE ticles to treat liver cancer Asee laterB* More recently) gol% nanoparticles &ave #een assem#le% into sca!!ol%s !or use in DNA %iagnostics an% #iosensors A>8B*

Iron Nanoparticles Iron nanoparticles &ave #een use% in #ot& %iagnostics an% t&erapeutics agents) (it& speci!ic applications as contrasting agents !or MRI an% magnetically targete% %rug %elivery* T(o types o! iron o"i%e nanoparticles &ave #een use% as imaging agents6 superparamagnetic iron o"i%e ASPI?B an% ultraEsmall superparamagnetic iron o"i%e AUSPI?B nanoparticles A>9B* Typically) t&ese nanoparticles are coate% (it& a variety o! sta#ili/ing agents inclu%ing %e"tran) al#umin) starc&) or silicones* T&e ma.or %i!!erE ence #et(een SIP?s an% USPI?s relates to t&eir si/e an% circulatory &al!Eli!e* +ot& particles may #e use% as contrasting agents to image t&e gastrointestinal tract) liver) spleen) an% lymp& no%es) alt&oug& t&e USPI?s may #e use% to %emonstrate #loo% pooling in %iseases suc& as #rain an% myocar%ial isc&emia*

Sa!ety o! Metallic Nanoparticle A%ministration in Man Unli$e #io%egra%a#le particles suc& as liposomes an% polymericE#ase% nanopartiE cles) metallic nanoparticles are relative ne(comers to t&e !iel% an% t&us t&e availE a#le to"icology %ata !or eac& nanoparticle system are limite%* -rom a &istorical perspective) t&e Cuestion o! colloi%al gol% to"icity may #e gleane% !rom t&e use o! ra%ioactive colloi%al gol% nanoparticles as a cancer t&erapy in t&e late 4>:@s*

+iological an% Engineering Consi%erations

4:4

Speci!ically) ra%ioactive colloi%al gol% nanoparticles (ere ma%e !rom Au 4>= an% use% !or t&e treatment o! liver cancer an% sarcoma A>:)>;B* Sa!ety %ata reveale% t&at t&e o#serve% to"icities o! t&ese intravenously a%ministere% ra%ioactive nanopartiE cles (ere %ue to ra%iation e"posure* No %emonstra#le to"icities (ere note% !rom t&e particles t&emselves* In ot&er reports) gol% (as also reporte% to #e inert an% #iologically compati#le A><B* Data !rom our G P to"icology stu%y on t&e sa!ety o! %rug sta#ili/e% colloi%al gol% nanoparticles agree (it& t&e &istorical %ata APaciotti et al*) unpu#lis&e% o#servationsB* i$e gol%) ironE#ase% nanoparticles are also #ioE logically inert %rug carriers Ale"iou A>=B an% u#&e A>>B s&o(e% t&at %e"tranEcoate% magnetite particles e"&i#it no %iscerna#le to"icity Ai*e*) D :@B in mice an% rats A>=)>>B* -urt&ermore) clinical stu%ies on t&e use o! epiru#icin %emonstrate% t&at !erro!lui%s (ere (ell tolerate%) (it& to"icity #eing limite% to t&e active agent) rat&er t&an t&e iron nanoparticles t&emselves A>>)4@@B*

Metallic NanoparticleE+in%ing C&emistry In&erent in t&e %evelopment o! metallic nanoparticle %rug vectors is an un%erstan%E ing o! t&e #in%ing mec&anisms involve% in a%sor#ing proteins an% ot&er %rugs to t&e sur!ace o! metallic nanoparticles* -or colloi%al gol%) proteins an% ot&er %rugs #in% to t&e sur!ace o! t&e colloi%al gol% particle #y one o! t&ree mec&anisms* T(o o! t&ese mec&anisms) ionic an% &y%rop&o#ic #in%ing) are relatively (ea$ interactions t&at o!ten result in t&e generation o! poorECuality vectors* T&e t&ir% met&o% involves t&e !ormation o! a %ative1coor%inate covalent #on% #et(een !ree sul!&y%ryl1t&iols o! t&e #iomolecule an% t&e gol% atoms present on t&e sur!ace o! t&e particles* Dative #on%s are very sta#le) possessing t&e energy eCuivalence o! a covalent #on%) an% are only %isrupte% #y strong re%ucing agents suc& as %it&iot&reitol or mercaptoE et&anol A8@)4@4B* A%%itionally) (e recently generate% !unctionali/e% colloi%al gol% nanoparticles containing various sur!ace groups t&at are use!ul !or covalent %rug immo#ili/ation* T&ese !unctional groups) (&ic& inclu%e NH 5) C??H) SH) an% ?H) may #e use% to lin$ %rugs t&roug& (ellE%escri#e% crossElin$ing c&emistries suc& as NHSE#ase% ester !ormation an% EDCE#ase% coupling ASilin et al*) unpu#lis&e% o#servationsB* -or ironE#ase% particles) %rug #in%ing primarily occurs t&roug& in%irect couE pling o! t&e %rug to t&e polymer coat surroun%ing t&e particle* -or e"ample) Allport an% ,eissle%er A4@5B attac&e% t&e HI2 tat pepti%e to t&e sur!ace o! %e"tranEcoate% iron nanoparticles* Alternatively) 'ain et al* A4@8B use% a %ou#le coating o! oleic aci% an% pluronic -45< aroun% t&e central iron nanoparticle to allo( %o"oru#icin to parE tition in t&is &y%rop&o#ic layer (&ile relying upon t&e pluronic coating to increase t&e &y%rop&ilic nature o! t&e particle Asee earlierB* ?ne uniCue aspect o! magnetic particles is t&at t&e particles t&emselves represent a novel t&erapeutic* -or e"ample) t&roug& magnetic e"citation) t&e particles #ecome &eate% an% &ave #een s&o(n to #e use!ul in t&ermal a#lation o! cancer cells Asee laterB*

Preclinical an% Clinical Stu%ies on Gol% an% Iron Nanoparticle Drug Delivery Gol% Nanoparticles C3TE;@>4 is a multivalent %rug t&at is assem#le% on 5;Enm particles o! colloi%al gol% an% %esigne% to actively seCuester recom#inant &uman TN- alp&a (it&in soli% tumors A8@B* T&e %rug is manu!acture% #y covalently lin$ing molecules o! TN- an% t&iolE%erivati/e% polyet&ylene glycol APEGETHI? B onto t&e sur!ace o! t&e colloi%al

4:5

Paciotti an% Tamar$in

gol% nanoparticles A8@B* -ollo(ing intravenous a%ministration) C3TE;@>4 rapi%ly accumulates in MCE8= colon carcinoma tumors an% s&o(s little to no accumulation in t&e livers) spleens Ai*e*) t&e RESB) or ot&er &ealt&y organs o! t&e animals* T&e tumor accumulation (as evi%ence% #y a mar$e% c&ange in t&e color o! t&e tumor as it acCuire% t&e #rig&t re%1purple color o! t&e colloi%al gol% nanoparticles) an% (as coinci%ent (it& t&e active an% tumorEspeci!ic seCuestration o! TN-* -inally) C3TE ;@>4 (as less to"ic an% more e!!ective in re%ucing tumor #ur%en t&an native TN#ecause ma"imal antitumor responses (ere ac&ieve% at lo(er %oses o! t&e %rug* ?ur e"perimental %ata suggest t&at t&e in&erent lea$iness o! t&e tumor neovasculaE ture !acilitates t&e passive e"travasation o! C3TE;@>4 into t&e soli% tumor) limiting t&e #io%istri#ution o! TN- an% avoi%ing &ealt&y organs an% tissues* ?nce insi%e t&e tumor) eac& molecule o! TN- #oun% to t&e sur!ace o! PEGylate% colloi%al gol% nanoparticles may serve one o! t&e t(o !unctions* -irst) as e"pecte% !rom TN-Ps $no(n #iological action) C3TE;@>4 serves as t&e anticancer t&erapeuticM secon% an% more importantly) TN- serves as a tumorEtargeting ligan%* T&e a#ove stu%ies (ere con%ucte% in t&e TN-Esensitive colon carcinoma mo%el) MCE8=) an% t&us t&e o#serve% antitumor e!!ect may &ave #een %ue to a %irect action1#in%ing o! TN- on t&ese tumor cells* Ho(ever) in &uman TN-Einsensitive +4;1-4@ melanoma tumors) C3TE;@>4 e"&i#ite% %i!!erential p&armaco%ynamics* Alt&oug& C3TE;@>4 cause% similar accumulation o! TN- in +4;1-4@ tumors) only transient in&i#ition o! tumor gro(t& (as o#serve%* T&ese %ata com#ine% (it& t&e $no(n cellular &eterogeneity o! soli% tumors support t&e %evelopment o! multi!unctional metallic nanot&eraE peutics t&at attac$ soli% tumors on multiple levels* An e"ample o! suc& a vector is presente% #elo(*

Iron Nanoparticles T&e %elivery o! t&e epiru#icinEcon.ugate% iron particles (as %one #y intravenous in.ection o! t&e nanoparticles into a vein) (&ic& (as locate% contralateral to t&e tumor* At t&e same time) a magnetic !iel%) ranging !rom @*: to @*= T) (as esta#lis&e% an% maintaine% !or 9: minutes aroun% t&e site o! t&e tumor* Preclinical %ata in ro%ent mo%els %emonstrate% t&at not only coul% t&is strategy concentrate t&e nanoparticle %rug vectors (it&in t&e soli% tumor) #ut also signi!icantly improve t&e antitumor e!!icacy o! epiru#icin treatment* In clinical trials) t&ese stu%ies s&o( similar !in%ings6 t&e magnetic particles (ere %etecte% in :@[ o! t&e patients treate%) (&ic& correlate% (it& t&e tumor areas s&o(ing t&e coloring o! t&e iron nanopartiE cles* T&ese %ata (ere !urt&er con!irme% #y &istological e"amination o! tumor tissue* Data !rom preclinical stu%ies reveale% t&at t&e particles (ere cleare% #y t&e RES a!ter removal o! t&e magnetic !iel%* ?t&er &ealt&y !iltering organs suc& as t&e lung an% t&e $i%ney %i% not s&o( t&e presence o! t&e nanoparticles A>=H4@4B*

Metallic Nanoparticle !or t&e T&ermal A#lation o! Tumors In 4>:<) Gilc&rist et al* A4@9B propose% t&e use o! t&ermal t&erapy as a treatment !or soli% tumors* Data supporting suc& an approac& (as later provi%e% #y 'or%an et al* A4@:B an% Neilsen et al* A4@;B) s&o(ing t&at tumor cells (ere sensitive to temperaE tures a#ove 94]C* T&e means o! treating soli% tumors (it& &eat &as a%vance% !rom t&e use o! ultrasonic an% micro(aves to t&e use o! magnetic nanoparticles t&at &eat in response to alternating magnetic !iel%s* T&e %egree o! &eating an% in turn t&e strengt& o! treatment is %etermine% #y t&e intensity o! t&e magnetic !iel% an% t&e si/e o! t&e o#.ect #eing magneti/e%* +a#incova et al* A4@<B %emonstrate% t&at t&e use o! t&is approac& !avore% t&e %estruction o! neoplastic cells over &ealt&y cells*

+iological an% Engineering Consi%erations

4:8

T(o approac&es &ave #een trie% to improve t&e e!!icacy o! t&ermal a#lation t&erapy* T&e !irst mo%i!ication involves coupling o! t&e t&ermal t&erapy (it& more convenE tional t&erapies suc& as c&emoE or ra%iot&erapy* T&e secon% met&o% increases t&e ma"imal treatment temperature !rom 9: to 9< to ::]C* Nevert&eless) at t&ese &ig& temperatures normal cells are a!!ecte%) a si%e e!!ect (&ic& may #e overcome #y %irect intratumor in.ection o! t&e particles Asee Re!* >8 !or revie(B* Hirsc& et al* A4@=B recently %escri#e% a secon% nanoparticle approac& !or t&e t&ermal t&erapy o! soli% tumors* T&ey %escri#e t&e synt&esis o! a gol%1silica nanos&ell comprise% o! an aminate% silica core particle (&ic& (as stu%%e% (it& ultraEsmall A4H8 nmB gol% particles* T&ese small gol% nanoparticles are #oun% to t&e sur!ace o! t&e silica core t&roug& t&eir interaction (it& t&e amine group* T&e ultraE small gol% particles (ere su#seCuently use% as nucleation sites upon (&ic& a%%iE tional gol% (as re%uce% to !orm t&e encase% gol% nanos&ell* T&is process allo(e% !or t&e synt&esis o! gol%Ecovere% particles (it& a tuna#le plasmon #an% in t&e nearE in!rare% region* E"posing t&ese particles to a lig&t source Ai*e*) %io%e laserB (it& a (avelengt& o! =5@ nm causes t&e electrons present on t&e gol% sur!ace Ai*e*) plasE monsB to #ecome e"cite%) resulting in particle &eating* Interstitial in.ection o! t&ese particles near t&e tumor !ollo(e% #y laser lig&t e"citation cause% a signi!icant re%ucE tion o! transmissi#le venerea tumors AT2TB tumors gro(ing in SCID mice*

Multi!unctional Nanot&erapeutics ?ur previous %iscussion &as centere% on t&e use o! metallic nanoparticles to target %elivery o! singular t&erapies) (&et&er c&emical) #iological) or p&ysical Ae*g*) &eatB) to soli% tumors* Nevert&eless) in recent years) it &as #ecome increasingly clear t&at soli% tumors may #e vie(e% as uniCue organs t&at are constantly a%apting to t&eir c&anging environment #y altering t&eir p&enotype* As previously %iscusse%) soli% tumors represent a collage o! cells) (&ic& !or t&e most part (or$ in concert to ensure continue% tumor gro(t& an% metastasis* It seems unli$ely t&at any given single agent) regar%less o! its a#ility to per!orm in e"perimental tumor mo%els) (ill yiel% t&e e"acting results in cancer patients* T&us) one o! t&e !inal c&allenges t&at metallic nanoparticle %rugE%elivery sysE tems !ace is to attac$ t&e &eterogeneous population o! tumor cells present (it&in a soli% tumor #y %elivering multiple t&erapeutics on a single nanoparticle* Com#inational cancer treatments &ave #een a &allmar$ o! recent clinical strategies to treat cancer* ?ne e"ample) previously %iscusse%) t&e isolate% lim# per!usion) %emonstrates %ramatic e!!icacy o! t&e com#ination o! TN- an% c&emot&erapy to treat re!ractory cancer patients A<5H<9B* More recently) a similar com#ination) Avastin an% :E-U) &as %emonstrate% signi!icant #ene!it in colorectal cancer patients A<@)<4B* ?ur o(n e"perience (it& C3TE;@>4 also supports t&e %evelopment o! multi!uncE tional metallic nanot&erapies* ,e o#serve% t&at alt&oug& C3TE;@>4 seCuestere% TN(it&in +4;1-4@ tumors) it pro%uce% only marginal antitumor e!!ects in t&is mo%el* Metallic nanoparticles may %eliver t&ese com#ination t&erapies eit&er spaE tially) on t&e same particle) or temporally) over t&e course o! t&e %isease* ?ur initial e!!orts to %evelop suc& a t&erapeutic centere% on %eveloping a colloi%al gol% nanoparticle vector t&at simulates t&e I P on a single nanoparticle* Recall t&at I P involves t&e surgical isolation o! t&e #loo% supply o! a soli% tumor to allo( !or t&e regional %elivery o! TN- an% a c&emot&erapeutic) avoi%ing systemic to"icities* To accomplis& t&is goal reCuire% t&e #in%ing o! a tumorEtargeting ligan%) a c&emot&erapeutic) an% an RESEavoi%ing molecule onto t&e same 5:Enm particle o! colloi%al gol%* Speci!ically) t&is nanoparticle) terme% C3TE54@@4) uses TN- as t&e

4:9

Paciotti an% Tamar$in

tumorEtargeting ligan%) a t&iolate% paclita"el analog as t&e t&erapeutic) an% PEGE THI? as an RESEavoi%ance molecule* To %ate) (e &ave o#serve% t&at C3TE54@@4 %elivers signi!icantly more TN- an% paclita"el to soli% tumors gro(ing in mice) (&en compare% to native TN-1paclita"el* -urt&ermore) (e o#serve% t&at unli$e C3TE;@>4) (&ic& only cause% transient tumor in&i#ition) C3TE54@@4 in%uce% sigE ni!icant antitumor responses in t&is TN-Einsensitive tumor mo%el APaciotti et al*) unpu#lis&e% o#servationsB*

SUMMAR3 Depicte% in -igure 4 is a simpli!ie% #lueprint !or %eveloping multi!unctional metallic nanot&erapeutics* ?n t&e #asis o! t&e current $no(le%ge o! t&e #iology o! tumor neovasculari/ation as #rie!ly summari/e% a#ove) t&ere is a rational strategy !or using metallic particles to #ot& target t&e tumor an% to %eliver (ellE$no(n) potent) #ut potentially to"ic cancer t&erapies to a #roa% spectrum o! soli% tumors* A%%itionally) as ne( t&erapies are %iscovere%) t&e versatility o! t&e metallic nanoparE ticle sur!ace may allo( !or t&e rapi% %evelopment an% evaluation o! ne( vectors in #ot& preclinical an% clinical settings* ,it& t&e %evelopment o! t&e !irst generation o! metallic nanoparticle cancer t&erapies almost rea%y !or clinical trials) t&e potential promise o! t&ese ne( cancer t&erapeutics may #e reali/e% in years not %eca%es*

RE-ERENCES
4* Papisov MI* T&eoretical consi%erations o! RESEavoi%ing liposomes6 molecular mec&anisms an% c&emistry o! liposome interactions* A%v Drug Deliv Rev 4>>=M 85644>H48=* 5* Mog&imi SM) Patel HM* SerumEme%iate% recognition o! liposomes #y p&agocytic cells o! t&e reticuloen%ot&elial systemRt&e concept o! tissue speci!icity* A%v Drug Deliv Rev 4>>=M 8569:H;@* 8* ,oo%le MC* Controlling liposome #loo% clearance #y sur!ace gra!te% polymers* A%v Drug Deliv Rev 4>>=M 85648>H4:5* 9* Na!ayasu A) Uc&iyama 0) 0i(a%a H* T&e si/e o! liposomes6 a !actor) (&ic& a!!ects t&eir targeting e!!iciency to tumors an% t&erapeutic activity o! liposomal antitumor %rugs* A%v Drug Deliv Rev 4>>>M 9@6<:H=<* :* Maruyama 0) Is&i%a ?) Ta$i/a(a T) Mori#e 0* Possi#ility o! active targeting to tumor tissues (it& liposomes* A%v Drug Deliv Rev 4>>>M 9@6=>H4@5* ;* Salata ?2* Applications o! nanoparticles in #iology an% me%icine* '* Nanotec&nol 5@@9M 568H=* <* Mog&imi SM) Hunter AC) Murray 'C* Nanome%icine6 current status an% !uture prosE pects* -ASE+ ' 5@@:M 4>6844H88@* =* Mog&imi SM) Hunter AC) Murray 'C* ongEcirculating an% targetEspeci!ic nanoparticles6 t&eory to practice* P&armacol Rev 5@@4M :865=8H84=* >* -errari M* Cancer nanotec&nology6 opportunities an% c&allenges* Nat Rev Cancer 5@@:M :64;4H4<4* 4@* MJller RH) MI%er 0) Go&la S* Soli%Hlipi% nanoparticles AS NB !or controlle% %rug %eliveryRa revie( o! t&e state o! t&e art* Eur ' P&arm +iop&arm 5@@@M :@64;4H4<<* 44* 'ain R) S&a& NH) Malic$ A,) R&o%es CT* Controlle% %rug %elivery #y #io%egra%a#le polyAesterB %evices6 %i!!erent preparative approac&es* Drug Dev In%ust P&arm 4>>=M 596<@8H<5<* 45* Ra!!erty DE) El!a$i MG) Montgomery PC* Preparation an% c&aracteri/ation o! a #io%eE gra%a#le microparticle antigen1cyto$ine %elivery system* 2accine 4>>;M 496:85H:8=* 48* ?ga(a 3* In.ecta#le microcapsules prepare% (it& #io%egra%a#le polyAaE&y%ro"yB aci%s !or prolonge% release o! %rugs* ' +iomater Sci Polym E% 4>><M =68>4H9@>* 49* Maruyama 0) Ta$i/a(a T) Ta$a&as&i N) et al* Targeting e!!iciency o! PEGEimmunoliE posomeEcon.ugate% anti#o%ies at PEG terminals* A%v Drug Deliv Rev 4>>=M 59658:H595*

+iological an% Engineering Consi%erations

4::

4:* A#solom D* ?psonins an% %ysopsonins6 an overvie(* Met&o%s En/ymol 4>=;M 48565=4H84=* 4;* Patel HM* Serum opsins an% liposomes6 t&eir interaction an% opsonop&agocytosis* Crit Rev T&er Drug Carrier Syst 4>>5M >68>H>@* 4<* Serra M2) Mannu -) Mater A) Turrini -) Arese P* En&ance% IgG an% complementEin%eE pen%ent p&agocytosis o! sul!ati%eEenric&e% &uman eryt&rocytes #y &uman monocytes* -E+S ett 4>>5M 8446;<H<@* 4=* C&onn A) Cullis PR) Devine D2* T&e role o! sur!ace c&arge in t&e activation o! t&e classic an% alternative pat&(ays o! complement activation #y liposomes* ' Immunol 4>>4M 49;69589H9594* 4>* Mog&imi SM) Patel HM* Altere% tissue speci!ic opsonic activities an% opsonop&agocytosis o! liposomes in tumor #earing rats* +ioc&em +iop&ys Acta 4>>;M 44<>64:<H4;:* 5@* A#ra RM) +os(ort& ME) Hunt CA* iposome %isposition in vivo6 e!!ect o! preE%osing (it& liposomes* Res Commun C&em Pat&ol P&armacol 4>=@M 5>689>H8;@* 54* Sou&ami R ) Patel HM) Ryman +E* T&e e!!ect o! reticuloen%ot&elial #loc$a%e on t&e #loo% clearance an% tissue %istri#ution o! liposomes* +ioc&em +iop&ys Acta 4>=4M ;<968:9H8<4* 55* Mog&imi SM) Davis SS* Innovations in avoi%ing particle clearance !rom t&e #loo% #y 0up!!er cells6 cause !or re!lection* Crit Rev T&er Drug Carrier Syst 4>>9M 44684H:>* 58* +ergCvist ) Sun%#erg R) Ry%en S) Stran% SE* T&e Qcritical colloi% %oseS in stu%ies o! reticuloen%ot&elial !unction* ' Nucl Me% 4>=<M 5=64959H495>* 59* +ra%!iel% ',* A ne( loo$ at reticuloen%ot&elial #loc$a%e* +r ' E"p Pat&ol 4>=@M ;46;4<H;58* 5:* +a#an D-) Seymour ,* Control o! tumour vascular permea#ility* A%v Drug Deliv Rev 4>>=M 8964@>H44>* 5;* Clelan% ' * Protein %elivery !rom #io%egra%a#le microsp&eres* In6 San%ers) Hen%ren) e%s* Protein Delivery6 P&ysical Systems* Ne( 3or$6 Plenum Press) 4>><64H98* 5<* Illum ) Davis SS) Muller RH) Ma$ E) ,est P* T&e organ %istri#ution an% circulation time o! intravenously in.ecte% colloi%al carriers sterically sta#ili/e% (it& a #loc$ copolymerR polo"amine >@=* i!e Sci 4>=<M 9@68;<H8<9* 5=* Re%&ea% HM) Davis SS) Illum * Drug %elivery in polyAlacti%eEcoEglycoli%eB nanopartiE cles sur!ace mo%i!ie% (it& polo"amer 9@< an% polo"amine >@=6 in vitro c&aracterisation an% in vivo evaluation* ' Control Release 5@@4M <@68:8H8;8* 5>* Mog&imi SM* Mo%ulation o! lymp&atic %istri#ution o! su#cutaneously in.ecte% polo"E amer 9@<Ecoate% nanosp&eres6 t&e e!!ect o! et&ylene o"i%e c&ain con!iguration* -E+S ett 5@@8M 594H599* 8@* Paciotti G-) Myer ) ,einreic& D) et al* Colloi%al gol%6 a novel nanoparticle vector !or tumor %irecte% %rug %elivery* Drug Deliv 5@@9M 4464;>H4=8* 84* C&en T) ,eiss * T&e role o! t&e sinus (all in t&e passage o! eryt&rocytes t&roug& t&e spleen* +loo% 4><8M 946:5>H:<8* 85* Mog&imi SM) Porter C'H) Muir IS) Illum ) Davis SS* NonEp&agocytic upta$e o! intraveE nously in.ecte% microsp&eres in t&e rat spleen6 in!luence o! particle si/e an% &y%rop&ilic coating* +ioc&em +iop&ys Res Commun 4>>4M 4<<6=;4H=;;* 88* Senior ') Gregoria%is G* Is &al!Eli!e o! circulating small unilamellar liposomes %etermine% #y c&anges in t&eir permea#ility* -E+S ett 4>=5M 49:64@>H449* 89* 2ola$anis 'E) ,irt/ 0* Interaction o! CEreactive protein (it& arti!icial p&osp&ati%ylc&oE line #ilayer* Nature A on%B 4><>M 5=4644:H4:<* 8:* Devine D2) +ra%ley A'* T&e complement system in liposome clearance6 can complement %eposition #e in&i#ite%T A%v Drug Deliv Rev 4>>=M 8564>H8>* 8;* T&urston G) Mc ean ',) Ri/en M) et al* Cationic liposomes target angiogenic en%ot&elial cells in tumors an% c&ronic in!lammation in mice* ' Clin Invest 4>>=M 4@4649@4H4948* 8<* Camp#ell R+) -u$uruma D) +ro(n E+) Ma//ola M) I/umi 3) 'ain R0* Cationic c&arge %etermines t&e %istri#ution o! liposomes #et(een t&e vascular an% e"travascular comE partment* Can Res 5@@5M ;46;=84H;=8;* 8=* -ol$man '* Tumor angiogenesis6 t&erapeutic implications* N Engl ' Me% 4><4M 5=:644=5H44=;* 8>* Daly ME) Mar$is A) Ree% M) e(is CE* Homeostatic regulators o! tumor angiogenesis6 a source o! antiangiogenic agents !or cancer treatmentT ' Natl Cancer Inst 5@@8M >:64;;@H4;<8*

4:;

Paciotti an% Tamar$in

9@* Heems$er$ ',) +evers EM) in%&out T* Platelet activation an% #loo% coagulation* T&rom# Haemost 5@@5M ==64=;H4>8* 94* Carmeliet P* Mec&anisms o! angiogenesis an% arteriogenesis* Natl Me% 5@@@M ;68=>H8>:* 95* Pine%o HM) 2er&eul HM) DPAmato R') -ol$man '* Involvement o! platelets in tumor angiogenesisT ancet 4>>=M 8:564<<:H4<<<* 98* C&en ') +ier&aus A) Sc&ie$o!er S) et al* Tissue !actorRa receptor involve% in t&e control o! cellular properties) inclu%ing angiogenesis* T&rom# Haemost 5@@4M =;6889H89:* 99* 0a$$ar A0) emoine NR) Scully M-) Te##utt S) ,illiamson RC* Tissue !actor e"pression correlates (it& &istological gra%e in &uman pancreatic cancer* +r ' Surg 4>>:M =5644@4H44@9* 9:* Hama%a 0) 0uratsu ') Saita& 3) Ta$es&ima H) Nis&i T) Us&io 3* E"pression o! tissue !actor correlates (it& gra%e o! malignancy in &uman glioma* Cancer 4>>;M <<64=<<H4==8* 9;* Gasic G') Gasic T+) Ste(art CC* AntiEmetastatic e!!ects associate% (it& platelet re%ucE tion* PNAS AUSAB 4>;=M ;469;H:5* 9<* Cramerer E) 7a/i AA) Duong D) Cornelissen I) A%vincula R) Coug&lin SR* Platelets) proteaseEactivate% receptors) an% !i#rinogen in &ematogenous metastasis* +loo% 5@@9M 4@968><H9@4* 9=* Mantovani A) So//ani S) ocati M) Allavena P) Sica* Macrop&age polari/ation6 tumorE associate% macrop&ages as a para%igm !or polari/e% M5 mononulcear p&agocytes* Tren%s Immunol 5@@5M 586:9>H:::* 9>* Elgert 0D) Alleva DG) Mullins D,* Tumor in%uce% immune %ys!unction6 t&e macroE p&age connection* ' eu$oc +iol 4>>=M ;965<;:H5>@* :@* ,atson GA) -u 3W) ope/ DM* Splenic macrop&ages !rom tumorE#earing mice coE e"pressing MACE4 an% MACE5 antigens e"ert immunoregulatory !unctions via t(o %isE tinct pat&(ays* ' eu$oc +iol 4>>4M 9>645;H48=* :4* ,atson GA) ope/ DM* A#errant antigen presentation #y macrop&ages !rom tumorE #earing mice is involve% in t&e %o(nregulation o! t&eir TEcell responses* ' Immunol 4>>:M 4::6459H489* :5* +ur$e +) Tang N) Cor$e 0P) et al* E"pression o! HI-E4a #y &uman macrop&ages6 implicaE tions !or t&e use o! &ypo"iaEregulate% cancer t&erapy* ' Pat&ol 5@@5M 4>;65@9H545* :8* Mur%oc& C) Giannou%is A) e(is CE* Mec&anism o! regulating t&e recruitment o! macE rop&ages into &ypo"ic areas o! tumors an% ot&er isc&emic tissues* +loo% 5@@9M 4@965559H5589* :9* +ur$e +) Giannou%is A) Cor$e 0P) et al* Hypo"iaEin%uce% gene e"pression in &uman macrop&ages6 implications !or isc&emic tissues an% &ypo"ia regulate% gene t&erapy* Am ' Pat&ol 5@@8M 4;864588H4598* ::* 0os&i$a(a N) Iyo/umi A) Gassmann M) Ta$enaga 0* Constitutive upregulation o! &ypo"iaEin%uci#le !actorE4 alp&a mRNA occurring in &ig&ly metastatic carcinoma cells lea%s to vascular en%ot&elial gro(t& !actor overe"pression un%er &ypo"ic e"posure* ?ncogene 5@@8M 556;<4<H;<59* :;* 0u(ai T) Tana$a 03) Tana$a S) et al* E"pression o! &ypo"iaEin%uci#le !actorE4 alp&a is associate% (it& tumor vasculari/ation in &uman colorectal carcinoma* Int ' Cancer 5@@8M 4@:64<;H4=4* :<* 0o&n S) Nagy 'A) Dvora$ H-) et al* Pat&(ays o! macromolecular tracer transport across venules an% small veins6 structural #asis !or t&e &yperpermea#ility o! tumor #loo% vesE sels* a# Invest 4>>5M ;<6:>;H;@<* :=* Dvora$ AM) 0o&n S) Morgan ES) et al* T&e vesiculoEvacuolar organelle A22?B6 a %istinct en%ot&elial cell structure t&at provi%es a transcellular pat&(ay !or macromolecular e"travasation* ' eu$oc +iol 4>>;M :>64@@H44:* :>* -eng D) Nagy ') Hipp ') et al* 2esiculoEvacuolar organelles an% t&e regulation o! venule permea#ility to macromolecules #y vascular permea#ility !actor) &istamine) an% seroE tonin* ' E"p Me% 4>>;M 4=864>=4H4>=;* ;@* Has&i/ume H) +alu$ P) Mori$a(a S) et al* ?penings #et(een %e!ective en%ot&elial cells e"plain tumor vessel lea$iness* Am ' Pat&ol 5@@@M 4:;648;8H48=@* ;4* 'ain R0* Molecular regulation o! vessel maturation* Nat Me% 5@@8M >6;=:H;>8* ;5* Mu$ouyama 3S) S&in D) +ritsc& S) Ta#iguc&i M) An%erson D'* Sensory nerves %etermine t&e pattern o! arterial %i!!erentiation an% #loo% vessel #ranc&ing in t&e s$in* Cell 5@@5M 4@>6;>8H<@:*

+iological an% Engineering Consi%erations

4:<

;8* Neu!el% G) Co&en T) S&raga N) ange T) 0essler ?) Her/og 3* T&e neuropilins6 multiE !unctional semap&orin an% 2EG- receptors t&at mo%ulate a"on gui%ance an% angiogenE esis* Tren%s Car%iovasc Me% 5@@5M 45648H4>* ;9* Eic&mann A) e No#le -) Autiero M) Carmeliet P* Gui%ance o! vascular an% neural net(or$ !ormation* Curr ?pin Neuro#iol 5@@:M 4:64@=H44:* ;:* Nagy 'A) Masse EM) Her/#erg 0T) et al* Pat&ogenesis o! ascites tumor gro(t&6 vascular permea#ility !actor) vascular &yperpermea#ility) an% ascites !lui% accumulation* Cancer Res 4>>:M ::68;@H8;=* ;;* ic&ten#el% HC) 3uan -) Mic&el CC) 'ain R0* Per!usion o! single tumor microvessels6 application to vascular permea#ility measurement* Microcirculation 4>>;M 8689>H8:<* ;<* Dvora$ H-) Nagy 'A) -eng D) +ro(n -) Dvora$ AM* 2ascular permea#ility !actor vasE cular en%ot&elial gro(t& !actor an% t&e signi!icance o! microvascular &yperpermea#ility in angiogenesis* Curr Top Micro#iol Immunol 4>>>M 58<6><H485* ;=* C&ang 3S) %iTomaso E) McDonal% DM) 'ones R) 'ain R0* Mosaic #loo% vessels in tumors6 !reCuency o! cancer cells in contact (it& !lo(ing #loo%* PNAS 5@@@M ><649;@=H49;48* ;>* Mons$y , ) Carreira CM) Tsu/u$i 3) Go&ongi T) -u$umara 3) 'ain R0* Role o! &ost microenvironment in angiogenesis an% microvascular !unctions in &uman #reast cancer "enogra!ts6 mammary !at pa% versus cranial tumors* Clin Cancer Res 5@@5M =64@@=H4@48* <@* Tong RT) 3ves +ouc&er 3) Sergey 2) et al* 2ascular normali/ation #y vascular en%ot&elial gro(t& !actor receptor 5 #loc$a%e in%uces a pressure gra%ient across t&e vasculature an% improves %rug penetration in tumors* Cancer Res 5@@9M ;968<84H8<8;* <4* 'ain R0* Normali/ation o! tumor vasculature6 an emerging concept in antiangiogenic t&erapy* Science 5@@:M 8@<6:=H;5* <5* Ale"an%er HR) +artlett D ) i#utti S0) -ra$er D ) Moser T) Rosen#erg SA* Isolate% &epatic per!usion (it& tumor necrosis !actor an% melp&alan !or unresecta#le cancers con!ine% to t&e liver* ' Clin ?ncol 4>>=M 4;649<>H49=>* <8* e.eune -'* Hig& %ose recom#inant tumour necrosis !actor ArTN-aB a%ministere% #y isoE lation per!usion !or a%vance% tumors o! t&e lim#s6 a mo%el !or #ioc&emot&erapy o! cancer* Eur ' Cancer 4>>:M 8464@@>* <9* ienar% D) e.eune -) E(alen$o I* In transit metastases o! malignant melanoma treate% #y &ig& %ose rTN- alp&a in com#ination (it& inter!eronEgamma an% melp&alan in isolation per!usion* ,orl% ' Surg 4>>5M 4;6589* <:* Cars(ell EA) ?l% ') 0assel R ) Green S) -iore N) ,illiamson +* An en%oto"in in%uce% serum !actor t&at causes necrosis o! tumors* Proc Natl Aca% Sci USA 4><:M <568;;;* <;* Helson ) Green S) Cars(ell EA) ?l% '* E!!ect o! tumor necrosis !actor on culture% &uman melanoma cells* Nature 4><:M 5:=6<84* <<* Asami T) Iami M) Tana$a 3* In vivo antiEtumor mec&anism o! natural &uman tumor necrosis !actor involving a TEcell me%iate% immunologic route* 'pn ' Cancer Res 4>=>M =@644;4* <=* Na(rot& PP) Stern DM* Mo%ulation o! en%ot&elial cell &omeostatic properties #y tumor necrosis !actor* ' E"p Me% 4>=;M 4;86<9@* <>* +rett ') Gerlac& H) Na(rot& P) Stein#erg S) Go%man G) Stern D* Tumor necrosis !actor1 cac&ectin increases permea#ility o! en%ot&elial cell monolayers #y a mec&anism involving regulatory G proteins* ' E"p Me% 4>=>M 4;=6;8<* =@* 0ristensen CA) No/ue M) +ouc&er 3) 'ain R0* Re%uction o! interstitial !lui% pressure a!ter TN-Ealp&a treatment o! t&ree &uman melanoma "enogra!ts* +r ' Cancer 4>>;M <96:88* =4* Ne%re#o T) +erg A) Ree% R0* E!!ect o! tumor necrosis !actorEm) I E4f) an% I E; on interE stitial !lui% pressure in rat s$in* Heart Circ P&ysiol 4>>>M 9;6H4=:<* =5* van %er 2een AH) %e,ilt 'H,) Eggermont AMM) van Tiel ST) Seyn&aeve A +) ten Hagen T M* TN-Em augments intratumoral concentrations o! %o"oru#icin in TN-Em #ase% isoE late% lim# per!usion in rat sarcoma mo%els) an% en&ances antiEtumor e!!ects* +r ' Cancer 5@@@M =56><8H>=@* =8* Pluen A) +ouc&er 3) Ramanu.an S) et al* Role o! tumorH&ost interactions in interstitial %i!!usion o! macromolecules6 cranial vs* su#cutaneous tumors* PNAS 5@@4M >=69;5=H9;88* =9* Cristiano R') Rot& 'A* Epi%ermal gro(t&E!actor me%iate% DNA %elivery into lungEcancer cells via t&e epi%ermal gro(t& !actor receptor* Cancer Gene T&er 4>>;M 869H4@*

4:=

Paciotti an% Tamar$in

=:* Gottsc&al$ S) Cristiano R') Smit& C) ,oo S C* -olateEme%iate% receptor me%iate% DNA %elivery into tumor cellsRpostsomal %isruption results in en&ance% geneEe"pression* Gene T&er 4>>9M 464=:H4>4* =;* Sing& M* Trans!errin as a targeting ligan% !or liposomes an% anticancer %rugs* Curr P&arm Des 4>>>M :6998H9:4* =<* Curnis -) Sacc&i A) Corti A* Improving c&emot&erapeutic %rug penetration in tumors #y vascular targeting an% #arrier alteration* ' Clin Invest 5@@5M 44@69<:H9=5* ==* Tu!!in G) ,aelti E) Hu(yler ') Hammer C) Marti HP* Immunoliposomes targeting mesangial cells6 a promising strategy !or speci!ic %rug %elivery to t&e $i%ney* ' Am Soc Nep&rol 5@@:M 4;688>:H88@:* =>* Sac&%eva MS* Drug targeting systems !or cancer c&emot&erapy* E"pert ?pin Invest Drug 4>>=M <64=9>H4=;9* >@* Mar$eting survey* Ro#ert H* Smit& Sc&ool o! +usiness) University o! Marylan% College Par$) April 5@@9* >4* -ara%ay M* E"perimental relations o! gol% Aan% ot&er metalsB to lig&t* P&ilos Trans R Soc on%on 4=:<M 49649:* >5* C&an%ler ') Ro#inson N) ,&iting 0* Han%ling !alse signals in gol%E#ase% tests* I2D Tec&nol 5@@4M <689* >8* Mir$in CA) etsinger R ) Mucic RC) Stor&o!! ''* A DNA #ase% met&o% !or rationally assem#ling nanoparticles into macroscopic materials* Nature 4>>;M 8=56;@<* >9* +erry CC) Curtis ASG* -unctionalisation o! magnetic nanoparticles !or applications in #iome%icine* ' P&ys Appl P&ys 5@@8M 8;6R4>=HR5@;* >:* Ru#in P) evitt SH* T&e response o! %isseminate% reticulum cell sarcoma to t&e intravenous in.ection o! colloi%al ra%ioactive gol%* ' Nucl Me% 4>;9M :6:=4* >;* Root S,) An%re(s GA) 0nieseley RM) Tyor MP* T&e %istri#ution an% ra%iation e!!ects o! intravenously a%ministere% colloi%al Au 4>= in man* Cancer 4>:9M <6=:;* ><* Tang ) iu ) El(ing H+* Complement activation an% in!lammation triggere% #y mo%el #iomaterial sur!aces* ' +iome% Mater Res 4>>=M 946888H89@* >=* Ale"iou C) Arnol% ,) 0lein R') et al* ocoregional cancer treatment (it& magnetic targeting* Can Res 5@@4M ;@6;;94H;;9=* >>* u##e AS) Ale"iou C) +ergemann C* Clinical applications o! magnetic %rug targeting* ' Surg Res 5@@4M >:65@@H5@;* 4@@* u##e AS) Ale"iou C) Reiss H) et al* Clinical e"periences (it& magnetic %rug targeting6 a p&ase I stu%y (it& 9uEepi%o"oru#i%in in 49 patients (it& a%vance% soli% tumors* Cancer Res 4>>;M :;69;=;H9;>8* 4@4* Hermanson GT* +iocon.ugate Tec&niCues* Aca%emic Press) 4>>;6:>9H:><* 4@5* Allport 'R) ,eissle%er* In vivo imaging o! gene an% cell t&erapies* E"p Hematol 5@@4M 5>6458<H459;* 4@8* 'ain T0) Morales MA) Sa&oo S0) eslieEPelec$y D ) a#&aset(ar* Iron o"i%e nanopartiE cles !or sustaine% %elivery o! anticancer agents* Mol P&arm 5@@:M 564>9H5@:* 4@9* Gilc&rist R0) Me%al R) S&orey ,D) Hanselman RC) Parrot 'C) Taylor C+* Selective in%uctive &eating o! lymp& no%es* Ann Surg 4>:<M 49;6:>;H;@;* 4@:* 'or%an A) ,ust P) Sc&ol/ R) et al* Cellular upta$e o! magnetic !lui%s particles an% t&eir e!!ects on &uman a%enocarcinoma cells e"pose% to AC magnetic !iel%s in vitro* Int ' Hypert&ermia 4>>;M 456<@:H<55* 4@;* Neilsen ?S) Horsman M) ?vergaar% '* A !uture !or &ypert&ermia in cancer treatmentT Eur ' Cancer 5@@4M 8<64:=<H4:=>* 4@<* +a#incova M) Sourivong P) es/c/yns$a D) +a#inec P* +loo%Especi!ic (&oleE#o%y elecE tromagnetic &ypert&ermia* Me% Hypot&eses 5@@@M ::69:>H9;@* 4@=* Hirsc& R) Sta!!or% R') +an$son 'A) et al* Nanos&ellEme%iate% near in!raEre% t&ermal t&erapy o! tumors un%er magnetic resonance gui%e* PNAS 5@@8M 4@@648:9>H48::9*

44

+iological ReCuirements !or Nanot&erapeutic Applications

'osep& -* C&iang
Department o! C&emistry an% +ioc&emistry) State University o! Ne( 3or$ at ?neonta) ?neonta) Ne( 3or$) U*S*A*) an% Department o! C&emistry) Tsing&ua University) +ei.ing) C&ina

INTR?DUCTI?N In or%er to %iscuss t&e #iological reCuirements !or nanot&erapeutic application) a #rie! %iscussion o! cellular an% tissue structure is necessary to provi%e some #asic #ac$E groun% !or rea%ers* Un%erstan%ing t&e structure an% t&e !unction o! living cells an% t&eir interactions (it& t&eir environments is essential to un%erstan% t&e Q#iological reCuirementS an% t&e interactions o! nanot&erapeutic %evices (it& living systems* Cells are alive an% are t&e smallest units t&at e"&i#it t&e property o! li!e* T&ey can #e culture% in vitro an% coul% %ie #y t&emselves* T&e structure o! cells is a very comple" an% (ellEorgani/e% system* Cells per!orm many !unctions in living t&ings* T&e !ollo(ing are !unctions o! cells6 4* 5* 8* 9* sel!Ereplications an% regulations) ta$ing energy !rom t&e meta#olites) carrying out mec&anical an% translocation (or$s) an% pro%ucing meta#olic !unctions an% c&emical reactions using en/ymes*

T&ere are t(o classes o! cells6 pro$aryotic an% eu$aryotic* T&ey &ave %i!!erent si/es an% %i!!erent internal structures* T&e !ormer) suc& as #acteria) is simpler) an% t&e latter) suc& as !ungi) plants) an% animals) is more comple" in structure* T&e contents o! a cell are calle% Qprotoplasm*S T&ey can #e !urt&er %ivi%e% into cytoplasm Aall t&e protoplasm e"cept t&e contents o! t&e nucleusB an% nucleoplasm Aall o! t&e materials) plasma an% DNA) etc* (it&in t&e nucleusB*

STRUCTURE ?- CE

Every cell &as t&ree ma.or components6 plasma AcellB mem#rane) cytoplasm) an% nucleus A-ig* 4B* Plasma ACellB Mem#rane Plasma mem#rane is a semipermea#le outer #oun%ary o! cells* It is a #ilayer c&ain o! molecules Ap&osp&olipi%sB* T&e mem#rane &as a &y%rop&o#ic tail pointing in(ar%) an% a &y%rop&ilic &ea% (&ic& is in contact (it& t&e surroun%ing environE ments* C&olesterol is also a component o! t&e mem#rane in t&e &y%rop&o#ic part o! t&e mem#rane* Plasma mem#rane controls t&e transport o! nutrients) (ater) an% ions) suc& as Nao) 0o) Ca5o) Mg5o) an% so on*

Cytoplasm All parts are (it&in t&e cell mem#rane* T&e !lui%s contain salts) sugars) lipi%s) vitamins) nucleoti%es) amino aci%s) RNA) an% proteins* Cell meta#olism) replication)
4:>

4;@

C&iang

-IGURE 4 ASee color insert*B Structure o! a typical cell*

an% gro(t& all occur (it&in t&e cytoplasm* Cytosol is a part o! cytoplasm) (&ic& re!ers to a proteinEric& environment* Cytos$eleton is t&e net(or$ !i#ers o! proteins) (&ic& maintain t&e s&ape o! t&e cell* Ri#osomes are t&e sites !or protein synt&esis an% consist o! ri#osomal RNA ArRNAB an% structural proteins* Mitoc&on%ria) lysosomes) pero"isomes) an% c&loroplast Ain plants onlyB are also locate% in t&e cytoplasm* Pero"isomes are mem#raneE#oun% vesicles containing en/ymes to reguE late &y%rogen pero"i%e in t&e cells* En%oplasmic reticulum is t&e interconnecte% mem#rane !unction !or protein synt&esis an% transport* Roug& en%oplasmic reticuE lum ARoug& ERB is connecte% to t&e nuclear envelop* T&e smoot& ER is involve% in transport an% ot&er cell !unctions* T&e Golgi #o%y !unctions as an intracellular tool !or sorting ne( proteins ma%e !rom Roug& ER* ysosomes are use% in p&agocytosis in (&ic& !oreign materials are #roug&t into t&e cell !or #rea$%o(n o! t&e materials* Mitoc&on%ria) Aorganelles in glo#ular s&apeB &ave t(o mem#ranes* T&ey serve as sites o! energy release an% a%enosine trip&osp&ate AATPB !ormation* ?rganisms in storage areas use vacuoles) singleEmem#rane organelles*

Nucleus T&e nucleus contains c&romatin) DNAHc&romosomes) nucleoli) an% nuclear proE teins* T&e nucleolus is t&e site !or rRNA gene transcriptions* T&e nuclear envelope is use% to separate cytoplasm an% nucleus* It is a structural !rame !or t&e nucleus* T&e nuclear mem#rane acts as a #arrier an% prevents !ree passage o! molecules #et(een nucleus an% cytoplasm* Nuclear pore comple"es regulate t&e e"c&ange o! molecules #et(een nucleus an% cytoplasm* Nuclear mem#ranes are associate% (it& Roug& ER* Ri#osomes are #oun%e% to Roug& ER*

THE CHEMICA C?MP?SITI?NS ?- CE

Cells are ma%e o! (ater) organic an% inorganic ions) an% organic molecules* ,ater plays an important role in cells* It is a polar molecule (it& a %ipole moment o! 4*>9 Da* ,ater &as t&e capa#ility to !orm &y%rogen #on%ing %ue to t&e polarity

+iological ReCuirements !or Nanot&erapeutic Applications

4;4

property) an% also interacts (it& cations an% anions* Many organic molecules are nonpolar an% are (aterEinsolu#le in an aCueous environment* T&e !ormer is calle% Q&y%rop&ilic)S (&ereas t&e latter is calle% Q&y%rop&o#ic*S Inorganic ions in cells containing so%ium ion ANaoB) potassium ion A0oB) calcium ion ACa5oB) magnesium 5ion AMg5oB) mono&y%rop&osp&ate ion AHP? 9 B) c&lori%e ion AClZB) an% #icar#onate ion AHC?8B play critical roles in cell !unctions* T&e organic molecules in cells can #e classi!ie% as small organic molecules an% macromolecules* ipi%s #elong to t&e small organic molecules category) (&ereas car#o&y%rates At&e polysacc&ari%esB) proteins) an% nucleic aci%s #elong to t&e macromolecules category*

Small ?rganic Molecules ipi%s ipi%s inclu%e triacylglycerol) p&osp&oglyceri%e) glycolipi%) steroi%) (a") terpene) an% prostaglan%in an% are nonpolar molecules (&ic& are solu#le in organic solE vents* T&e simplest lipi%s are !atty aci%s consisting o! long &y%rocar#on c&ains o! 4; to 4= car#ons (it& a car#o"ylic group AHC??B* T&e ot&er en% contains nonpolar CRH #on%s (&ic& (ill not interact (it& (ater* T&ere are t(o types o! !atty aci%s6 saturate% an% unsaturate%* Unsaturate% !atty aci%s &ave one or more %ou#le CRRC #on%s* P&osp&olipi%s are t&e principal components o! cell mem#ranes* T(o !atty aci%s an% a p&osp&ate are com#ine% (it& glycerol to !orm p&osp&oglyceri%es* Triacylglycerols) or !ats) &ave t&ree !atty aci%s #oun% to a glycerol molecule* Energy sources are store% in !ats* P&osp&olipi%s are amp&ipat&ic molecules) one en% is (aterEsolu#le an% t&e ot&er en% is (aterEinsolu#le* Anot&er type o! p&osp&oE lipi%s is sp&ingomelin) (&ic& is t&e only nonglycerol p&osp&olipi% in t&e cell mem#rane*

Macromolecules Car#o&y%rates Simple sugars an% polysacc&ari%es are car#o&y%rates* Simple sugars) suc& as glucose) serve as ma.or nutrients !or cells* T&e c&emical reactions o! car#o&y%rates (ill provi%e energy !or cells an% also provi%e sources !or t&e synt&esis o! ot&er cellular pro%ucts* Polysacc&ari%es are also use% !or protein transport to an% associaE tion (it& ot&er parts o! t&e cellular system* Simple sugar or monosacc&ari%e &as t&e e"perimental !ormula ACH5?Bn* Glucose) one o! t&e simple sugars in (&ic& (it& n ^ ;) is t&e simplest sugar) ACH5?B;) or C;H45?;* ?ligosacc&ari%es are !orme% #y con%ensation o! several simple sugars) (&ereas polysacc&ari%es are !orme% (it& a large num#ers o! simple sugars* Glycogen is a polysacc&ari%e in animals* ?ligosacc&ari%es an% polysacc&ari%es are also use% !or c&emical interaction #et(een cells*

Nucleic Aci%s Nucleic aci%s are genetic in!ormation materials in cells* DNA A%eo"yri#onucleic aci%B is t&e genetic material in t&e nucleus* RNA Ari#onucleic aci%B is responsi#le !or cellular activities in t&e cell* T&ere are several types o! RNAs* Messenger RNA AmRNAB serves as a template !or protein synt&esis* It carries genetic in!ormation to ri#osomes !rom DNA* Trans!er RNA AtRNAB an% ri#osomal RNA are also use% in protein synt&esis* Polymeri/ation o! nucleoti%es) a nucleic aci% #ase attac&e% to a sugar) an% p&osE p&ate !orms DNA an% RNA* ATP) a type o! nucleoti%e) is a c&emical energy source

4;5

C&iang

in cells* A %etaile% %escription o! DNA) RNA) an% relate% su#.ects can #e !oun% in any cell an% molecular #iology te"t* Nucleic aci%s act in many cellular processes* T&ey can pro%uce t&eir replications) %irect protein synt&esis) an% carry out in!ormaE tional trans!er* Proteins Proteins are #iopolymers consisting o! many %i!!erent amino aci%s* Amino aci%s &ave a general structural !ormulas o! RECAHBANH 8BoAC??B* Con%ensation o! t(o amino aci%s #et(een t&e car#o"yl group o! one an% t&e amino group o! t&e ot&er !orms a pepti%e #on%* T&e si%e c&ains are eit&er straig&t car#on c&ains or car#on rings* T&ere are appro"imately 5@ suc& amino aci%s* T&e c&aracteristics o! proteins are #ase% on t&e properties o! t&e amino aci%s* Some amino aci%s &ave nonpolar si%e c&ains) suc& as glycine) alanine) valine) leucine) isoleucine) proline) cysteine) met&ionine) p&enylalanine) an% tryptop&an* T&us) t&e si%e c&ain o! t&ese amino aci%s (ill not interact (it& (ater or polar molecules* Amino aci%s serine) t&reonine) tyrosine) asparagine) an% glutamine &ave polar si%e c&ains o! eit&er ?H group) or ami%e group A?RRCHNH5B* T&ey are &y%rop&ilic an% (ill !orm &y%rogen #on%ing (it& (ater* ysine an% arginine) (it& c&arge% groups in t&e si%e c&ain) are &y%rop&ilic an% (ill interact (it& (ater* Histi%ine) acting as a positive c&arge% or neutral amino aci% #ase% on t&e pH value) &as t&e a#ility to pro%uce H o !or en/yE matic catalysis* +ot& aspartic aci% an% glutamic aci% &ave a car#o"yl group at t&e en% o! t&e si%e c&ain* T&ey are very &y%rop&ilic* In !orming proteins) t&is en% is locate% on t&e protein sur!ace* Amino aci% lin$e% #y pepti%e #on%s is calle% proE tein* T&e properties o! proteins are #ase% on t&e seCuence o! amino aci%s* T&e propE erties o! proteins also %epen% on t&e con!ormation o! t&e proteins* Many proteins per!orm en/yme catalytic reactions (it&in t&e cells* A catalyst can eit&er increase or slo( %o(n t&e reaction rate (it&out consuming t&e catalyst* Suc& a catalyst is calle% an Qen/ymeS in #iological reaction systems* T&e catalytic action o! en/ymes usually increases t&e rate o! reaction* A #asic $no(le%ge o! c&emical $inetics is essential to un%erstan%ing any en/ymatic process*

DE2E ?PMENT ?- NAN?TECHN? ?G3 Nanotec&nology &a% a !ast pace o! %evelopment !or t&e past %eca%e* It &as #een use% to rearrange molecules so t&at every atom can #e place% in t&e most e!!icient (ay* Anot&er term !or suc& arrangement is Qmolecular nanotec&nologyS or molecE ular manu!acturing* T&us) it can #e use% to construct s&apes) mac&ines) an% pro%E ucts at t&e atomic level* Nanotec&nology can also #e %e!ine% as t&e application o! science t&at %eals (it& materials in 4@@Enm si/e) #ut it is not a miniaturi/ation* At t&is range) materials pro%uce% (ill e"&i#it ne( properties (e are loo$ing !or* In t&is range) t&e sur!ace area increases %rastically) (&ic& (ill e"&i#it ne( c&emical an% p&ysical properties* Nanotec&nology also provi%es applications in energy storage) energy pro%uction) agriculture) air pollution) nanoelectronics) an% &ealt&E care* In t&e &ealt&care application) use o! nanotec&nology as tools to manipulate #iomolecules to regulate li!e an% %eat&) illness) an% &ealt& (ill #e t&e main goal to ac&ieve* ?ne o! t&e e"amples is t&e use o! molecular %iagnostics) (&ic& (ill ena#le selection o! t&e most e!!icient treatment !or eac& in%ivi%ual* Nanoparticles are t&oug&t to &ave potential as novel intravascular pro#es !or %iagnostics Ae*g*) imagE ingB an% t&erapeutic purposes Ae*g*) %rug %eliveryB* T&e critical reCuirement is t&e

+iological ReCuirements !or Nanot&erapeutic Applications

4;8

a#ility to target speci!ic tissues an% cell types an% escape !rom #iological particuE late !ilters* T&is is t&e soEcalle% Qreticuloen%ot&elial system*S T&e a%vantage o! nanoEtec&nology over microsystems is t&at nanot&erapeutics &as &ig&er intracelluE lar upta$es) allo(ing %rug release in %i!!erent cellular compartments suc& as cytoE plasm an% nucleus* It can also #e con.ugate% (it& a ligan% to !avor a targete% t&erapeutic approac&* Practically) %rug loa% is a science #ase% on si/e an% structure o! %evice* E"amples are t&e use o! nanoparticles as controlle% %rug %elivery !or cancer treatment* A com#ination o! microE an% nanoparticles can also #e use% !or %rugE%elivery systems) suc& as micro1nanosp&eres) micro1nanocapsules) an% liposomes* Suc& com#inations (ill %i!!er in structure an% #iop&armaceutical proE perties !or %i!!erent t&erapeutic uses* iposomes %iscovere% #y +aug&man A4B are t&e smallest arti!icial vesicles o! sp&erical s&ape t&at can #e pro%uce% !rom natural unto"ic p&osp&olipi%s an% c&olesterols* iposomes can #e use% as %rug carriers an% loa%e% (it& a variety o! molecules) suc& as small %rug molecules) proteins) nucleoti%es) plasmi%s) an% carriers !or lipop&ilicEantitumor %rug NEocta%ecylE4E fE ara#ino!uranosyl cystine A5B) a/i%ot&ymi%ine) an% %i%eo"ycyti%ine* T&ere are ot&er %rugE%elivery system approac&es in a%%ition to liposomes) suc& as polymer microE capsules) microsp&eres) polymer con.ugates) an% nanoparticles as mentione% preE viously* T&e most !un%amental reCuirement !or nanot&erapeutic %evices is to %eliver any %rug at t&e rig&t time an% in t&e target (&ere it is nee%e% an% at t&e level t&at is reCuire%* Development o! %rugE%elivery systems is also aiming at t&e t&erapeutic an% to"icological properties o! e"isting c&emot&erapies* Nanoporous mem#ranes (it& < to > nm pores o!!er si/eE#ase% e"clusion an% controlle% %i!!uE sion o! molecule %rugs* Common tec&nology !or %rug a%ministration an% %evices inclu%e6 su#cutaneous) implants) surgical) oral) intravenous) an% possi#le pulmoE nary in&alation) (&ereas t&e c&aracteristics o! common routes o! %rug a%ministraE tion are s&o(n in Ta#le 4* Researc& an% tec&nology %evelopment at nanoscale provi%es a !un%aE mental un%erstan%ing o! materials at nanoscale in or%er to create %evices an% systems in me%icineHnanome%icine* Recently) a pro!essional organi/ation QAmerican Aca%emy o! Nanome%icine AAANMBS (as esta#lis&e% in +altimore) Marylan%) on August 4:) 5@@:* AANM is %evote% to t&e stu%y o! nanome%icine an% nanoE t&erapeutic %evices*

-A+RICATI?N ?- NAN?DE2ICES It is (ort&y to revie( an% intro%uce t&e t(o approac&es in !a#rication o! nano%eE vices an% any nanoparticles at t&is point* T&e !irst one is t&e topE%o(n approac& an% t&e secon% one is calle% #ottomEup* T&e topE%o(n approac& involves t&e re%ucE tion o! materials !rom #ul$ si/e to microE an% to nanoscale* -or a t&reeE%imensional case) i! t(o %imensions are $ept in macroscale) #ut t&e t&ir% one is re%uce% to nanoscale) t&e structure is $no(n as Cuantum (ell* I! one %imension is $ept in macE roscale an% t&e ot&er t(o are re%uce% to nanoscale) t&is is calle% nano(ire* I! all t&ree %imensions are re%uce% to nanoscale) it is re!erre% to as Cuantum %ot* T&e topE%o(n approac& #egins (it& a re%uction o! macroscale su#stance to nanoscale pro%uct* it&ograp&ic tec&niCue as use% in microc&ip !a#rication is an e"ample* T&e stan%ar% process is liste% #elo(* An nEtype silicon (a!er %ope% (it& pEtype impurities can #e use% to create t&e %rain an% source o! a transistor* A step(ise illustration is s&o(n as !ollo(s A-ig* 5B*

4;9
TA+ E 4 C&aracteristics o! Common Routes o! Drug A%ministration Route Intravenous A#sorption pattern A#sorption circumvente% Potentially imme%iE ate e!!ects Special utility 2alua#le !or emergency use Permits titration o! %osage Suita#le !or large volumes an% !or irritating su#stances) i! %ilute% Suita#le !or some insolu#le suspensions an% !or implantation o! soli% pellets

C&iang

imitations an% precautions Increase% ris$ o! a%verse e!!ects Must in.ect solutions slo(ly) as a rule Not suita#le !or oily solutions or insolu#le su#stances Not suita#le !or large volumes

Su#cutaneous

Prompt) !rom aCueous solution

Intramuscular

Slo( an% sustaine%) !rom repository preparations Prompt) !rom aCueous solution

Possi#le sloug& !rom irritating su#stances Suita#le !or mo%erate volumes oily ve&icles) an% some irritating su#stances Preclu%e% %uring anticoE agulant me%ication

Slo( an% sustaine%) !rom repository preparations ?ral ingestion 2aria#leM %epen%s upon many !actors Most convenient) sa!e) an% economical

May inter!ere (it& interpreE tation o! certain %iagnosE tic tests Ae*g*) creatine p&osp&o$inaseB ReCuires patient cooperaE tion* A#sorption potenE tially erratic an% incomplete !or %rugs t&at are poorly solu#le an% a#sor#e% slo(ly

4* A silicon (a!er is cut !rom monocrystal silicon A4 4 4B %irection* 5* A layer o! silicon o"i%e is %eposite% on nEsilicon* 8* A p&otosensitive emulsion is %ope% on Si? 5* T&e etc&e% area is remove% #y c&emical process* 9* A layer o! Si?5 is !orme% #y o"i%ation process on top o! t&e silicon* A pEtype o! impurities is use% to cover t&e area to #e metali/e%* :* ?"i%ation is processe% again* ;* Removal o! t&e secon% o"i%e is per!orme%* <* ?"i%ation again on t&e ne(ly gro(n areas* =* Removal o! t&e t&ir% layer again* >* T&e !inal metalli/ation (ill pro%uce t&e !inal transistor*

T&ere are many #ottomEup processes in use !or !a#rication o! nanomaterials* T&e !ollo(ing are .ust a !e( an% inclu%e solHgel process) c&emical vapor %eposition AC2DB) laser pyrolysis) molecular con%ensation) &y%rot&ermal process) an% many ot&er ne(ly %eveloping tec&niCues* A particular met&o% %evelope% #y Wu A8B use% (aterEinEoil microemulsion an% &y%rot&ermal microemulsion to prepare C%S nanocrystal* T&e group also prepare% Ni? nanoring an% mono%isperse% monoE clinic /irconia AmEVr?5B via &y%rot&ermal met&o% in et&anolH(ater system A9B* A ne( tool6 QDipEpen nanolit&ograp&yS (as %evelope% #y Mir$in an% co(or$ers A:B* T&is ne( met&o% can #e use% !or %irectE(rite scanning pro#eE#ase% lit&ograp&y

+iological ReCuirements !or Nanot&erapeutic Applications

4;:

NEType Silicon

N A5B T&e !irst o"i%ation %eposit t&ic$ Si?5 layer

A4B Su#strate material E Silicon

P N A8B -irst o"i%e removal #y etc&ing N

A9B Doping is per!orme% #y t&ermal %eposit

P N A:B Secon% t&ic$ o"i%ation

P N A;B Secon% o"i%e removal

P N A<B Gate o"i%ation

P N

A=B T&ir% o"i%e removal H e"poses source an% %rain

A>B -inal metalli/ation resulte% in !inis&e% transistor

A4@B Tunnels (it& crossover

-IGURE 5 Stan%ar% met&o% !or !a#rication o! pEc&annel metal o"i%e semicon%uctor !iel% e!!ect transistor AM?S-ETB*

(&ic& uses an atomic !orce microscopy AA-MB tip to %eliver c&emical reagents on a target su#strate* A-M is a scanning tec&niCue to pro%uce &ig& resolution 8ED image o! less t&an 4 nN sample sur!ace* It can #e use% to measure t&e !orce #et(een an A-M tip an% t&e sample sur!ace Acon%uctor or insulatorB* T&e small !orces are measure% #y measuring t&e motion o! a very !le"i#le cantilever #eam &aving an e"tra small mass*

4;;

C&iang

In A-M) t&e !orces #et(een t&e tip an% sample are %etecte% rat&er t&an tunneling current* It is a goo% tool to pattern many organic molecules A; H44B) organic A45H49B an% #iological A4:)4;B polymers) an% colloi%al particles A4<H4>B to metal ions A5@H55B* T&e solHgel met&o% is use% to pro%uce colloi%al nanoparticles !rom liCui% p&ase) especially !or o"i%e nanoparticles A58H5:B* It is a &y%rolysis an% con%ensation o! metal al$o"i%es process* In &y%rolysis) precipitation !rom solution !orms insoluE #le &y%ro"i%es* T&e &y%ro"i%es are t&en converte% to o"i%es #y %e&y%ration* C2D met&o% &as #een use% to pro%uce singleE(alle% car#on nanotu#es eit&er at raise% temperature At&ermal C2DB) or (it& plasmaEen&ance% c&emical vapor %eposition APEC2DB* Atomic or molecular con%ensation is use% !or pro%ucing metallic nanoparticles* A ra( material is &eate% in a vacuum to vapori/e t&e material* Rapi% coating o! t&e vaporEp&ase metal in no#le gas results in t&e !ormation o! metal nanoparticles* ?"i%e nanoparticles (ill !orm #y mo%i!ying t&is met&o% using o"ygen instea% o! no#le gas* Nanoparticles o! many materials) suc& as organic) #iological molecules) metals) an% inorganic o"i%es) can #e pro%uce% #y c&emical sel!Eassem#ly tec&niCue* T&is tec&niCue can #e use% !or attac&ing molecules to a speci!ic sur!ace o! a su#strate* Many researc& groups use sel!Eassem#ly as a met&o%* ?ne o! t&em is to !a#ricate al$ylsilo"anes on silicone %io"i%e A5;B* Anot&er e"ample is al$anet&iolates on gol% A5<B* Nanotec&nology can #e use% !or t&erapeutics %ue to t&e compati#le si/es o! proteins an% nanoscale particles* -or e"ample) &emoglo#in &as a si/e o! 9*: ` < nm 5 %imensionM lipoprotein) 5@ nm5 in sp&erical s&apeM mEglo#ulin) 9*8 ` 5; nm5M an% !i#rinogen) 9 ` <; nm5* T&ese are in t&e nanometer range*

NAN?THERAPEUTIC DE2ICES T&e !ollo(ing is a list o! systems in microE an% nanot&erapeutic %elivery %evices6 4*oral %rug %elivery 5*in.ectionE#ase% %rug %elivery 8*trans%ermal %rug %elivery 9*#one marro( in!usion :*organEsystemEspeci!ic %rug %elivery a* pulmonary %rug %elivery #* nasal %elivery to central nervous system c* car%iovascular system AC2B %* genitoEurinary tract e* gastrointestinal tract !* ocular %rug %elivery ;* controlle% release system <* novel pac$aging an% !ormulation a* !ast %issolving system #* c&e(a#le ta#lets c* solu#ility en&ancement =* target %rug %elivery a* polymer an% collagen system #* particleE#ase% system iB t&erapeutic monoclonal anti#o%ies iiB liposomes iiiB microparticles

+iological ReCuirements !or Nanot&erapeutic Applications

4;<

c* mo%i!ie% #loo% cells %* nanoparticles e* viralEassiste% intracellular gene %elivery) an% !* nonviral intracellular gene %elivery >* implant %rugE%elivery system Some o! t&e a#ove %elivery systems are o#vious !rom t&e title* Some %o nee% cerE tain e"planations an% e"amples* T&e targete% %rug %elivery is .ust one o! t&e cases* PolyAaminoB amine %en%rimers A5=)5>B are one o! t&e polymer systems* +loc$ copolymer &as potential to encapsulating large num#ers o! guest molecules (it&in t&e cavity !or t&erapeutic applications* Nanoparticles engineere% (it& speci!ic #in%ing po(er can #e suspen%e% in #o%y !lui%s or even in.ecte% into t&e circulation system* Nanoparticles can #e use% #ot& Cuantitatively an% Cualitatively !or in vivo %etection o! tumor cells* At t&e present time) most nanoE t&erapeutic %evices an% %rugE%elivery systems are concentrate% on oncology !or %etection an% curing tumor cells* In particular) nanot&erapeutic systems can reac& tumor cells muc& easier t&an ot&er cells* Tumor tissue is usually %i!!erE ent !rom normal tissue* T&is p&enomenon provi%es t&e soEcalle% Qen&ance% permea#ility an% retention AEPRB e!!ectS A8@B* Antitumor agent %elivery (it& EPR (ill #e more e!!ective* T&e application o! nanoparticles to C2s &as t(o purposes6 %etection an% targete% %rug %elivery* Recently) a report #y Corie o$ in t&e May 5@@: Issue o! t&e Tec&nology Revie( %escri#e% a ne( %iagnostic tool calle% Qmeta#olomicsS (&ic& can #e use% to %etect %iseases at an earlier stage an% can cure t&e %iseases as (ell* T&e principles involve% in meta#olomics are t&e analysis o! a large num#er o! small molecules) suc& as sugar an% !ats to reveal a#normal #e&aviors* T&is (ill lea% to meta#olic !ingerprints to give an earlier an% more accurate %iagnosis !or %iseases* Implanta#le %evices consisting o! seale% arrays o! reservoirs can #e use% !or in vivo an% in vitro %rug analysis an% %elivery* T&ese %evices are !ille% (it& c&emicals t&at can #e release% on %eman% an% can c&ec$ t&e e!!icacy o! %rug release% over a long perio% o! time* Implanta#le #rain pro#es &ave #een stu%ie% !or neural activities* T&e results can #e use% in t&e treatment o! various mental an% ot&er #rain %isor%ers* Anot&er e"ample is t&e nanoro#ots in.ecte% into t&e &uman #o%y to (or$ in patientsP #loo%streams* In !uture) me%ical nano%evices can routinely #e implante% or even in.ecte% into t&e #loo%stream to monitor (ellness an% to participate in t&e repair o! t&e system t&at %eviates !rom t&e normal state* Implanta#le sensors coul% #e use% in t&e %etection o! glucose !or %ia#etes* Regulation o! glucose meta#olism in t&e &uman #o%y #y insulin can #e ac&ieve% #y t&e %esign %escri#e% #y Gouc& A84B* T&e %evice can #e programme% to (arn o! &ypoglycermia Alo( #loo% glucoseB or &yperglycermia A&ig& #loo% glucoseB* Suc& in%ication can gui%e %ia#etics to a%.ust insulin in.ection or even coul% #e couple% to an implante% pump to %eliver insulin correctly* T&e principle o! operation is #ase% on glucose o"i%ase) (&ic& cataly/es t&e reaction6

Glucose o ?5 o 5?

gluconic aci% o H ?

?"ygen is use% !or t&e reaction* T&e unreacte% o"ygen (ill #e %etecte% in t&e !orm o! electric current* T&us) t&e amount can #e cali#rate% in relation to t&e current o! a system (it&out t&e presence o! t&e en/yme) t&e o"i%ase* Anot&er #iosensor %evelE ope% recently is to measure t&e level o! H 5?5 using Pt electro%e A85B* ?ne more

4;=

C&iang

met&o% mentione% &ere is t&e measurement o! glucose concentration !or %ia#etes #y %irect electroc&emistry o! glucose o"i%ase a#sor#e% on a colloi%al gol%Emo%i!ie% car#on paste electro%e A88B* Castillo et al* A89B &ave measure% glucose concentration #ase% on en/ymeE#ase% amperometric #iosensor* T&e searc& !or nanot&erapeutic applications is mainly %ue to t&e limitation o! current t&erapeutics) especially c&emot&erapies an% ra%iot&erapies* T&ese t&eraE peutics &ave a &ig& %egree o! to"icity* T&ey also pro%uce many invasive si%e e!!ectsM not only %o t&ey give un!avora#le t&erapeutic e!!ects) #ut t&ey also cause %amages %uring a%ministrations o! suc& %rugs or ra%iation treatments* T&ere are many a%vantages o! nanot&erapeutic systems over t&e tra%itional %rug a%ministration* -or any %rug %elivery) t&e penetrations o! strong aci%s1#ases %epen% on t&e permeE a#ility o! cell mem#rane* Penetration o! %rug (it& nano%evices into mitoc&on%ria !ollo(s t&e same principle as cell mem#rane* Also pH value #et(een intracellular an% e"tracellular !lui%s is small ApH o! <*@H<*9B* T&e gra%ient o! %rug across t&e plasma mem#rane is small* In general) (ea$ #ases are slig&tly concentrate% insi%e cells) (&ereas (ea$ aci%s are less concentrate% insi%e cells* o(ering pH value) or increasing t&e concentration o! Ho ion o! t&e e"tracellular !lui%s) (ill cause an increase in t&e intracellular concentration*

RE7UIREMENTS -?R NAN?THERAPEUTIC APP ICATI?NS 4* Transit an% penetration properties o! nanoscale particles6 Nanoparticles o! si/e less t&an 5@ nm can transit t&roug& #loo% vessel (all* T&ey can also penetrate #loo%H#rain #arrier or stomac& epit&elium* 5* Interaction6 t&e nanoscale si/e o! nanoparticles can interact (it& #iomolecules on t&e cell sur!aces) #ut (ill not c&ange t&e #iological properties o! t&e molecules* T&ey also &ave t&e a#ility to interact (it& receptors) nucleic aci%s) an% proteins at t&e molecular scale* 8* Intracellular imaging6 Nanoparticles) suc& as Cuantum %ots) can #e use% !or intracellular imaging as t&ey can accommo%ate large num#ers o! atoms) or molecules o! imaging agent) suc& as ga%olinium) to increase t&e sensitivity !or %etection* 9* Sur!ace c&emistry6 mo%i!ying or altering t&e sur!ace o! nanoparticles to !orm covalency (it& c&emical an% #iological species results in t&e increase o! t&e solu#ility an% #iocompati#ility* -or e"ample) attac&ing a &y%rop&ilic polymer to t&e nanoparticle sur!ace (ill increase &y%ration* As a#sorption %epen%s on solu#ility) it eventually increases t&e a#sorption* T&ey can also #e use% to encapsule insolu#le compoun%s* :* Coating an% uncoating6 nanoparticles coate% (it& &y%rop&ilic polymers &ave longer &al!Eli!eM uncoate% nanoparticles use% in intravenous in.ection can #e cleare% !rom #loo%stream #y reticuloen%ot&elial system* ;* Ionic attac&ments6 Attac&ing t&e sur!ace (it& ionic cation or anion species can in!luence t&e #iocompati#ility o! nanoparticles an% t&e a#ility to traverse #iological #arriers* An e"ample is %en%rimers (it& amine) a cationic group on t&e sur!ace (as more cytoto"ic t&an car#o"ylicEterminate% %en%rimers* <* iposomes use% as %rugE%elivery ve&icles* T&ey (ere %evelope% in early 4>;@s* iposomes are vesicles enclose% #y lipi% #ilayer !rom sel!Eassem#ly process o! amp&ip&ilic molecules* T&ey &ave #een use% to stu%y mem#ranes) also as vesicles !or controlle% %rug %elivery) catalysis) nonviral gene %elivery) an% %iagnostic %evices* T&e %isa%vantage o! liposomes application is %ue to t&e

+iological ReCuirements !or Nanot&erapeutic Applications

4;>

TA+ E 5 Applications o! Nanoparticles in T&erapeutics Nanoparticle Descriptions Re!erences A8;)8<B A8=B A8>)9@B A94B

Nanosp&eres Drug is uni!ormly %isperse% Nanocapsules Drug is enclose% #y polymer mem#rane in a vesicular system Micelles Amp&ip&ilic #loo%Ecopolymer t&at can sel!Eassociate in aCueous solution CeramicNanosp&ere o! 4<35?8H4>Al5?8H;9Si?5 Amol[B composition) 5@H 8@ _m %iameter !or targete% ra%iot&erapy o! liver cancernanoparticles Anonra%ioactive =>3 can #e activate% #y neutron #om#ar%ment to >@3) a _Eemitter At415 ^ ;9*4 &rB iposomesArti!icial sp&erical vesicles !rom natural p&osp&olipi%s an% c&olesterol Den%rimersMicromolecules consisting o! a num#er o! #ranc&es aroun% t&e inner core TNT systemDevelope% #y Triton +ioSystem (it& t&e U*S* Army a# attac$ing cancer in t&ree steps6 Ai B patient receiving a single in!usion containing trillions o! magnetic #iosp&ere) #oun% to vanti#o%iesM Aii B t&e #iosp&ere (ill see$ an% attac& to cancer cells in #loo%E streamM Aiii B s(itc& on t&e magnetic !iel% in t&e cancer region (ill cause t&e #iosp&eres to &eat up to $ill t&e cancer cells in minutes

A98B A99B A9:B

A##reviation6 TNT) targete% nano t&erapeutics*

TA+ E 8 Some Nanoparticles Use% in +iological Researc& Nanoparticle Den%rimers Application Re!erences A99)9;B A9<B A9=B A9>):@B A9>B A:4B A:5H:9B A::B A:;B A:<H:>B A:>B A;@B A;4H;8B A;9H;;B A;<B A;=);>B
A##reviation6 C I?) cross lin$e% iron o"i%e*

Targeting o! cancer cells) %rug %elivery) imaging) #oron neutron capture t&erapy Ceramic nanoparticlePassive targeting o! cancer cells ipi%Eencapsulate% per!luorocarE Passive targeting o! cancer cells #on nanoemulsions Magnetic nanoparticlesSpeci!ic targeting o! cancer cells) tissue imaging HHRHEtargete% silicaEcoate%Speci!ic targeting o! cancer cells lipi% micelles T&iamineEtargete% nanoparticles Directe% trans!er across CacoE5 cells iposomesSpeci!ic targeting o! cancer cells) gene t&erapy) %rug %elivery NanoparticleEaptamerTargeting o! prostate cancer cells #iocon.ugate Antiangiogenesis t&erapyAntiE-l$ anti#o%yEcoate% >@3 nanoparticles Gol% nanos&ellsTissue imaging) t&ermal a#lative cancer t&erapy AntiEHER5 anti#o%yEtargete%+reast cancer t&erapy gol%1silicon nanoparticles C I? paramagneticImaging o! migrating cells nanoparticles 7uantum %otsTissue imaging SiliconE#ase% nano(iresRealEtime %etection an% titration o! anti#o%ies) virus %etection) c&ipE#ase% #iosensors Car#on nanotu#esElectronic #iosensors Trans!ersomesNoninvasive vaccine %elivery) %rug %elivery

4<@

C&iang

#iological insta#ility) so t&ey &ave a s&ort li!etime* T&is &as #een improve% !rom researc& on t&e synt&esis o! t&e polymeric nanocontainers* =* Targete% %rug %elivery o! nanoparticles can #e ac&ieve% #y #in%ing monoclonal anti#o%ies on t&e sur!aces o! t&e targete% cells* 2arious nanoparticles &ave #een trie% in imaging tumors in animal an% &uman trials* Samples are s&o(n in Ta#le 5 !rom McNeil A8:B* Stu%ies o! #iological e!!ects o! c&emiE cal an% me%ical %evices on immunological) in!lammatory) an% proli!erate e!!ects o! nanomaterials) an% t&e interactions o! nanomaterials as applie% in me%ical %evices an% to"icological ris$s &ave #een carrie% out at various la#oratories ATa#le 8B*

RE-ERENCES
4* +ang&man AD* Di!!usion o! univalent ions across t&e lamellae o! s(ollen p&osp&olipi%s* ' Mol +iol 4>;:M 48658=* 5* Si#ylle 0) 0ollerE ucae M) Sc&ott H) Sc&(en%ener RA* Interactions (it& &uman #loo% in vitro an% p&armaco$inetic properties in mice o! liposomal N9Eocta%ecylE4EfEDEara#inoE !uranosylcytosine) a ne( anticancer %rug* '* P&arm E"p T&er4>><M 5=5A8B64:<5* 8* Wu S* iCui%EP&ase Synt&esis an% Mo%i!ication o! Colloi%al Particles) M* S* T&esis* Tsing&ua University) 5@@:* 9* Sun WiaoEming* SolutionE+ase% Synt&esis) C&aracteri/ation an% Property Investigation o! o(EDimensional -unctional Nanomaterials) P&*D* T&esis* Tsing&ua University) 5@@:* :* Piner RD) V&u ') Wu -) Hong SH) Mir$in CA* DipEpen nanolit&ograp&y* Science 4>>>M 5=86;;4* ;* Hong SH) V&u ') Mir$in CA* Multiple in$ nanolit&ograp&y to(ar% a multiple pen nanoEplotter) <* A nanoEplotter (it& #ot& parallel an% serial (riting capa#ilities* Science 4>>>M 5=;6:58* <* Hong SH) Mir$in CA* A nanoHplotter (it& #ot& parallel an% serial (riting capa#ilities* Science 5@@@M 5==64=@=* =* ,ein#erger DA) Hong) SH) Mir$in CA) ,essels +,) Higgins T+* Com#inatorial generation an% analysis o! nanometerEan% micrometerEscale silicon !eatures via %ipEpen nanolit&ograp&y an% (et c&emical etc&ing* A%v Mater 5@@@M 4564;@@* >* V&ang H) i V) Mir$in CA* DipEpen nanolit&ograp&yE#ase% met&o%ology !or preparing arrays o! nanostructures !unctionali/e% (it& oligonucleoti%es* A%v Mater 5@@5M 49649<5* 4@* V&ang H) C&ung S,) Mir$in CA* -a#rication o! su#E:@Enm soli% state nanostructures) #ase% on %ipEpen nanolit&ograp&y* Nano ett 5@@8M 8698* 44* Ivanisevic A) McCum#er 02) Mir$in CA* SiteE%irecte% e"c&ange stu%ies (it& com#inaE torial li#raries o! nanostructures* ' Am C&em Soc 5@@5M 459644>><* 45* Maynor +,) -ilocamo S-) Grinsta!! M,) iu '* NanoE%irect (riting o! polymer nanosE tructures6 polyAt&iop&eneBnano(ires on semicon%ucting an% insulating sur!aces* ' Am C&em Soc 5@@5M 4596:55* 48* im 'H) Mir$in CA* Electrostatically %riven %ipEpen nanolit&ograp&y o! con%ucting polymers* A%v Mater 5@@5M 49649<9* 49* Noy A) Miller AE) 0lare 'E) ,ee$s + ) ,oo%s +,) De3oreo ''* -a#rication o! luminesE cent nanostructures an% polymer nano(ires using %ipEpen nanolit&ograp&y* Nano ett 5@@5M 564@>* 4:* ,ilson D ) Martin R) Hong S) CroninEGolom# M) Mir$in CA) 0aplan D * Nanopatterning collagen #y %ipEpen nanolit&ograp&y* Proc Natl Aca% Sci USA 5@@4M >=648;;@* 4;* Demers M) Ginger DS) Par$ S') i V) C&ung S,) Mir$in CA* Direct patterning o! mo%iE !ie% oligonucleoti%e on metals an% insulators #y %ipEpens* Science 5@@5M 5>;64=8;* 4<* +en Ali +) ?n%arcu&u T) +rust M) 'oac&im C* A-M tip nanoprinting o! gol% nanoclusE ters* angmuir 5@@5M 4=6=<5* 4=* iao 'H) Huang ) Gu N* -a#rication o! nanoparticle pattern t&roug& atomic !orce tipE in%uce% %eposition on mo%i!ie% siliconsur!aces* C&in P&ys ett 5@@5M 4>6489*

+iological ReCuirements !or Nanot&erapeutic Applications

4<4

4>* Garno 'C) 3ang 33) Amro NA) Cruc&onEDupeyrat S) C&en S) iu G3* Precise positioning o! nanoparticles on sur!aces using scanning pro#e lit&ograp&y* Nano ett 5@@8M 868=>* 5@* i 3) Maynor +,) iu '* Electroc&emical A-M Q%ipEpenS nanolit&ograp&y* ' Am C&em Soc 5@@4M 458654@:* 54* Maynor +,) i 3) iu '* QIn$S !or A-M Q%ip penS nanolit&ograp&y* angmuir 5@@4M4<65:<:* 55* Porter A 'r) C&oi HC) Sc&melt/er 'M) Ri##e AE) Elliott CC) +uria$ 'M* Electroless nanoparticle !ilm %eposition compati#le (it& p&otolit&ograp&y) microcontact printing) an% %ipEpen nanolit&ograp&y patterning tec&nologies* Nano ett 5@@5M 5648;=* 58* Ito& H) Utampanya S) Star$ '2) 0la#un%e 0') Sc&lup 'R* Nanoscale metal o"i%e particles as c&emical reagent* intrinsic e!!ects o! particle si/e on &y%ro"yl content an% on reactivE ity an% aci%1#ase properties o! ultra!ine magnesium o"i%e* C&em Mater 4>>8M :6<4* 59* Pal$ar 2R* SolHgel %erive% nanostructure qEalumina porous sp&eres as an a%sor#ent in liCui% c&romatograp&y* Nanostruct Mater 4>>>M 44A8B68;>* 5:* Interrante 2) Hamp%enESmit& M') e%s* C&emistry o! A%vance% Materials6 An ?vervie(* Ne( 3or$6 ,ileyE2CH) 4>>=* 5;* ,asserman SR) Tao 3T) ,&ite&ea%es GM* Structure an% reactivity o! al$ylsilo"ane monE olayers !orme% #y reaction o! al$yltric&lorosilanes on silicon su#strates* angmuir 4>=>M :64@<9* 5<* +ain CD) Evall ') ,&itesi%es GM* -ormation o! monolayers #y t&e coa%sorption o! t&iols on gol%6 variation in t&e &ea% group) tail group) an% solvent* ' Am C&em Soc 4>=>M 4446<4::* 5=* Ha($er C') -rec&et 'M'* Preparation o! polymers (it& controlle% molecular arc&itecE ture) a ne( convergent approac& to %en%rite macromolecules* ' Am C&em Soc 4>>@M 4456<;8=* 5>* Tomalia DA* Den%rimer molecules* Sci Am 4>>:M May6;5* 8@* Duncan R* Drug targeting6 (&ere are (e no( an% (&ere are (e goingT ' Drug Targeting 4>><M :A4B64* 84* Gouc& D* In6 -ung 3C) e%* Intro%uction to +ioengineering* Singapore6 ,orl% Scienti!ic) 5@@4* 85* 0im ) Parris NA) Potts R?) Tierney M'* +iosensor) iontop&oretic sampling system) an% met&o%s o! use t&ereo!* Unite% States Patent ;)<8;)<<<) 5@@9* 88* iu +) 'u '* Reagentless glucose #iosensor #ase% on %irect electron trans!er o! glucose o"i%ase immo#ili/e% on colloi%al gol% mo%i!ie% car#on paste electro%e* +iosens +ioelectron 5@@8M 4>A8B64<<8* 89* Castillo ') Gaspar S) Sou$&arev 2) Domeanu A) Rya#ov A) Csregi E* +iosensors !or li!e Cuality %esign %evelopment an% applications* Sens Actuators + 5@@9M 4@564<>* 8:* McNeil SE* Nanotec&nology !or t&e #iologist* ' eu$oc +iol 5@@:M <=* 8;* Sant&i 0) D&anara. SA) 'osep& 2) Ponnusan$ar S) Sures& +* A stu%y on t&e preparation an% antiEtumor e!!icacy o! #ovine serum al#umin nanosp&eres containing :E!luorouracil* Drug Dev In% P&arm 5@@5M 5=644<4* 8<* ,als& S) Ru#enstein R) Veitlin P) eong 0,* T&erapeutic nanosp&eres* US Patent No* ;)5@<)4>:) 4>>=* 8=* 2elinova M') Sta!!&orst R,HM) Mul%er ,'M) et al* Preparation an% sta#ility o! lipi%E coate% nanocapsules o! cisplatin6 anionic p&osp&olipi% speci!icity* +ioc&im +iop&ys Acta 5@@9M 4;;8648:* 8>* 0(on GS* Polymeric micelles !or %elivery o! poorly (aterEsolu#le compoun%s* Crit Rev T&erap Drug Carrier 5@@86 5@A:B* 9@* E&r&ar%t G') Day DE* T&erapeutic use o! >@3 microsp&eres* Int ' Ra% Appl Instrum + 4>=<M 49A8B6588* 94* 0o$u#o T* In6 +enENissan +) S&er D) ,als& ,) e%s* +ioceramics* 2ol* 4:* Ueti$onEVuric&6 Trans Tec& Pu#lications) 5@@86:58* 95* I$enaga M) ?&ura 0) 3amamura T) 0oyoura 3) ?$a M) 0o#u$o T* ocali/e% &ypert&erE mic treatment o! e"perimental #one tumors (it& !erromagnetic ceramics* ' ?rt&op Res 4>>8M 446=9>* 98* Geng ) ?sus$y 0) 0on.eti S) Halla&an -A* Ra%iationEgui%e% %rug %elivery to tumor #loo% vessels results in improve% tumor gro(t& %elay* ' Control Release 5@@9M >>68;>* 99* 7uintana A) Rac/$a E) Pie&ler) et al* Design an% !unction o! %en%rimerE#ase% &erapeutic nano%evice targete% to tumor cells t&roug& t&e !olate receptor* P&arm Res 5@@5M 4>6484@* 9:* Triton +ioSystem* (((*siliconiron*com1maga/ine1coverstory1in%e"*s&tme*

4<5
9;* +a$er 'R) 'r* T&e synt&esis an% testing o! antiEcancer t&erapeutic %evices* +iome% Micro%ev 5@@4M 86;4* 9<* Roy I) ?&ulc&ans$yy T3) Pu%avar HE) et al* CeramicE#ase% Nanoparticles entrapping (ater solu#le p&otosensiti/ing antiEcancer %rugs* ' Am C&em Soc 5@@8M 45:6<=;@* 9=* an/a GM) ,inter P) Carut&ers S) et al* Novel paramagnetic contrast agents !or molecuE lar imaging an% targete% %rug %elivery* P&arm +iotec&nol 5@@9M :69>:* 9>* +ergey E') ,ang WP) 0re#s ' et al* DC magnetic !iel% in%uce% magnetocytolysis o! cancer cells targete% #y 44ER44 magnetic nanoparticless in vitro* +iome% Micro%ev 5@@5M 965>8* :@* 'ira$ D) 0ri/ ') Heryne$ 2) et al* MRI o! transplante% pancreatic islets* Magn Reson Me% 5@@9M :56455=* :4* Russ(llE'ones G') Art&ur ) ,al$er H* 2itamin +45Eme%icate% transport o! nanopartiE cless across CacoE5 cells* Int P&arm 4>>>M 4<>659<* :5* Du#ey P0) Mis&ra 2) 'ain S) Ma&or S) 2yas SP* iposomes mo%i!ie% (it& cyclic RGD pepti%e !or tumor targeting* ' Drug Target 5@@9M 4565:<* :8* Res/$a RC) 'aco#s A) 2oges '* iposomes me%iate% suici%e gene t&erapy in &umans* Met&o%s En/ymol 5@@:M 8>465@@* :9* Ten Hagen T * iposomal cyto$ines in t&e treatment o! in!ectious %isease an% cancer* Met&o%s En/ymol 5@@:M 8>4645:* ::* -aro$&/a% ?G) 'on S) 0&a%em&osseini A) Tran TN) avan DA) anger R* NanoparticleE aptamer #iocon.ugates6 a ne( approac& !or targeting prostate cancer Cells* Cancer Res 5@@9M ;96<;;=* :;* i ) ,arc&o( CA) Dant&i SN) et al* A novel antiangiogenesis t&erapy using an integrin antagonist or antiE!l$ anti#o%yEcoate% >@3Ela#ele% nanoparticle* Int ' Ra%iat ?ncol +iol P&ys 5@@9M :=6454:* :<* So$olov A) Aaron ') Hsu +) et al* ?ptical system !or in vivo molecular imaging o! Cancer* Tec&nol Cancer Res Treat 5@@8M 569>4* :=* ,ang G) Huang T) Murray R,) Menar% ) Nu//o RG* NearEIR luminescence o! monolE ayerEprotecte% metal clusters* ' Am C&em Soc 5@@:M 45<6=45* :>* Hirsc& R) Sta!!or% R') +an$$son 'A) et al* Nanos&elEme%iate% nearEin!rare% t&ermal t&erapy o! tumors un%er magnetic resonance gui%ance* Proc Natl Aca% Sci USA 5@@8M 4@@648:9>* ;@* 0irsc&er M-) Allport 'R) Graves EE) et al* In vivo &ig& resolution t&reeE%imensional imaging o! antigen speci!ic cytoto"ic TElymp&ocyte tra!!ic$ing to tumor* Cancer Res 5@@8M ;86;=8=* ;4* 2oura E+) 'ais(al '0) Mattoussi H) Simon SM* Trac$ing metastatic tumor cell e"travasaE tion (it& Cuantum nanocrystals an% !luorescence emissionEscanning microscopy Nat Me% 5@@9M 4@6>>8* ;5* ,u W) +ruc&e/ MP* a#eling cellular targets (it& semicon%uctor Cuantum %ot con.uE gates* Met&o%s Cell +iol 5@@9M <:64<4* ;8* Hirsc& R) 'aco#son '+) ee A) Halas N') ,est ' * A (&ole #loo% immunoassay using gol% nanos&ells* Anal C&em 5@@8M <:658<<* ;9* Cui 3) ,ei 7) Par$ H) ie#er CM* Nano(ire nanosensors !or &ig&ly sensitive an% selecE tive %etection o! #iological an% c&emical species* Science 5@@4M 5>8645=>* ;:* +unimovic& 3 ) Ge G) +everly 0C ) Ries RS) Hoo% ) Heat& 'R* Electroc&emically proE gramme% spatially selective #io!unction/li/ation o! silicon (ires* angmuir 5@@9M 5@64@;8@* ;;* Patols$y -) V&eng G) Hay%en ?) a$a%amyali M) V&uang W) ie#er CM* Electrical %etecE tion o! single viruses* Proc Natl Aca% Sci USA 5@@9M 4@4649@4<* ;<* C&en R' ) +angsaruntip S) Drouvala$is 0A) et al* NonEcovalent !unctionali/ation o! car#on nanotu#es !or &ig&ly speci!ic electronic #iosensors* Proc Natl Aca% Sci USA 5@@8M 4@@69>=9* ;=* Gupta PN) Mis&ra 2) Ra(at A) et al* NonEinvasive vaccine %elivery in trans!ersomes noisome) an% liposomes6 a comparative stu%y* Int ' P&arm 5@@:M 5>86<8* ;>* Simoes S ) Dega%o T ) opes RM) et al* Developments in t&e rat a%.uvanr art&ritis mo%el an% its use in t&erapeutic evaluation o! novel nonEinvasive treatment #y S?D in trans!ersomes* ' Control Release 5@@:M 4@8694>*

C&iang

45

Role o! Nano#iotec&nology in t&e Development o! Nanome%icine

0* 0* 'ain
'ain P&arma+iotec&) +asel) S(it/erlan%

INTR?DUCTI?N Given t&e in&erent nanoscale !unctional components o! living cells) it (as inevita#le t&at nanotec&nology (oul% #e applie% in #iotec&nology giving rise to t&e term Qnano#iotec&nology)S (&ic& (ill #e use% in t&is c&apter an% in%icates #iotec&nolE ogy as t&e main !iel%* A less recogni/e% an% less !reCuently use% term) almost synonymously) is Q#ionanotec&nology)S (&ic& implies application in li!e sciences o! nanotec&nology as t&e main %iscipline* An upEtoE%ate %escription o! nano#ioE tec&nologies an% t&eir applications in &ealt&care are given in a special report on t&is topic A4B* T&e topic o! t&is #oo$ is t&e nanoparticulate %rugE%elivery systems* T&is c&apter (ill provi%e an integrate% overvie( o! application o! nano#iotec&nologyE #ase% molecular %iagnostics) %rug %iscovery) an% %rug %elivery in t&e %evelopment o! nanome%icine (it& t&e relations&ips as s&o(n in -igure 4*

R? E ?- NAN?+I?TECHN? ?G3 IN M? ECU AR DIAGN?STICS Nanomolecular %iagnostics is t&e use o! nano#iotec&nology in molecular %iagnostics an% can #e terme% Qnano%iagnosticsS A5B* In contrast to %rug %elivery (&ic& uses mainly nanoparticles) nano%iagnostics uses #ot& particulate an% nonparticulate tec&nologies) (&ic& are %escri#e% in %etail in a #oo$ on t&is topic A8B* Some e"amples o! t&e use o! nanoparticles !or molecular %iagnosis are given &ere an% %escri#e% &ere as t&ey can #e com#ine% (it& %rug %elivery an% t&erapeutics*

Nanoparticles !or Molecular Diagnostics Nanoparticles t&at are commonly use% !or %iagnostics are

gol% particles magnetic an% supramagnetic nanoparticles Cuantum %ot A7DB tec&nology nanoparticle pro#es DNAHprotein an% nanoparticle con.ugates

Gol% Nanoparticles +its o! DNA an% RamanEactive %yes can #e attac&e% to gol% particles no larger t&an 48 nm in %iameter* T&e gol% nanoparticles assem#le onto a sensor sur!ace only in t&e presence o! a complementary target* I! a patterne% sensor sur!ace o! multiple DNA stran%s is use%) t&e tec&niCue can %etect millions o! %i!!erent DNA seCuences simultaneously* NanoparticleE#ase% DNA %etection systems are 4@ times more sensitive an% 4@@)@@@ times more speci!ic t&an current genomic %etection systems* ClearRea%b ANanosp&ere) Inc*B) a nanoparticle tec&nology) ena#les microarrayE#ase%
4<8

4<9

'ain

+I?TECHN? ?G3

NAN?TECHN? ?G3

DRUG DISC?2ER3

I-E SCIENCES

NAN?+I?TECHN? ?G3 DRUG DE I2ER3 NAN?ARRA3S

PHARMACEUTICA DE2E ?PMENT

NAN?MEDICINE

M? ECU AR DIAGN?STICS

L 'ain P&arma+iotec&

-IGURE 4 Integration o! nano#iotec&nologies !or t&e %evelopment o! nanome%icine*

multiple" single nucleoti%e protein ASNPB genotyping o! &uman genomic DNA (it&out t&e nee% !or target ampli!ication A9B* T&is %irect SNP genotyping met&o% reCuires no en/ymes an% relies on t&e &ig& sensitivity o! t&e gol% nanoparticle pro#es* A QspotEan%Erea%S colorimetric %etection met&o% !or i%enti!ying nucleic aci% seCuences is #ase% on t&e %istanceE%epen%ent optical properties o! gol% nanoE particles (it&out t&e nee% !or conventional signal or target ampli!ication A:B*

7uantum Dots T&ere is consi%era#le interest in t&e use o! 7Ds as inorganic !luorop&ores) o(ing to t&e !act t&at t&ey o!!er signi!icant a%vantages over conventionally use% !luorescent mar$ers* -or e"ample) 7Ds &ave !airly #roa% e"citation spectra Z !rom ultraviolet to re% Z t&at can #e tune% %epen%ing on t&eir si/e an% composition* Potential applicaE tions o! 7Ds in molecular %iagnostics are

cancer genotyping (&ole #loo% assays multiple"e% %iagnostics DNA mapping immunoassays an% anti#o%y tagging %etection o! pat&ogenic microorganisms

Magnetic Nanoparticles Magnetic nanoparticles are a po(er!ul an% versatile %iagnostic tool in #iology an% me%icine* It is possi#le to incorporate su!!icient amounts o! superparamagnetic iron o"i%e nanoparticles into cells) ena#ling t&eir %etection in vivo using magnetic resoE nance imaging AMRIB A;B* +oun% to a suita#le anti#o%y) magnetic nanoparticles are use% to la#el speci!ic molecules) structures) or microorganisms*

Role o! Nano#iotec&nology in t&e Development o! Nanome%icine

4<:

R? E ?- NAN?+I?TECHN? ?G3 IN DRUG DISC?2ER3 T&e postgenomic era is revolutioni/ing t&e %rugE%iscovery process* T&e ne( c&alE lenges in t&e i%enti!ication o! t&erapeutic targets reCuire e!!icient an% costEe!!ective tools* a#elE!ree %etection systems use proteins or ligan%s couple% to materials t&e p&ysical properties o! (&ic& are measura#ly mo%i!ie% !ollo(ing speci!ic interactions* Among t&e la#elE!ree systems currently availa#le) t&e use o! metal nanoparticles o!!ers en&ance% t&roug&put an% !le"i#ility !or realEtime monitoring o! #iomolecular recogE nition at a reasona#le cost* Some nanotec&nologies (ill accelerate target i%enti!icaE tion) (&ereas ot&ers (ill evolve into t&erapeutics* T&is is closely relate% to %rug %elivery) anot&er important p&armaceutical aspect o! nano#iotec&nology*

Nanoparticles !or Drug Discovery Nanocrystals A7DsB an% ot&er nanoparticles Agol% colloi%s) magnetic nanoparticles) nano#arco%es) nano#o%ies) %en%rimers) !ullerenes) an% nanos&ellsB &ave receive% consi%era#le attention recently (it& t&eir uniCue properties !or potential use in %rug %iscovery* Use o! Gol% Nanoparticles !or Drug Discovery Gol% nanoparticles can emit lig&t so strongly t&at it is rea%ily possi#le to o#serve a single nanoparticle at laser intensities lo(er t&an t&ose commonly use% !or multiE p&oton a#sorptionEin%uce% luminescence A<B* Moreover) gol% nanoparticles %o not #lin$ or #urn out) even a!ter &ours o! o#servation* T&ese o#servations suggest t&at metal nanoparticles are a via#le alternative to !luorop&ores or semicon%uctor nanoE particles !or #iological la#eling an% imaging* ?t&er a%vantages o! t&e tec&niCue are t&at t&e gol% nanoparticles can #e prepare% easily) &ave very lo( to"icity) an% can rea%ily #e attac&e% to molecules o! #iological interest* In a%%ition) t&e laser lig&t use% to visuali/e t&e particles is a (avelengt& t&at causes only minimal %amage to most #iological tissues* T&is tec&nology coul% ena#le trac$ing o! a single molecule o! a %rug in a cell or ot&er #iological samples*

Use o! 7uantum Dots !or Drug Discovery T&e use o! 7Ds !or %rug %iscovery &as #een e"plore% e"tensively* +ot& a%vantages an% %ra(#ac$s &ave #een investigate% A=B* A%vantages o! t&e use o! 7Ds !or %rug %iscovery are as !ollo(s6

En&ance% optical properties as compare% (it& organic %yes* 7Ds o!!er great imaging results t&at coul% not #e ac&ieve% #y organic %yes* Multiple lea%s can #e teste% on cell culture simultaneously* Similarly) t&e a#sorption o! several %rug molecules can #e stu%ie% simultaneously !or a longer perio% o! time* Using t&e sur!ace !unctionali/ation properties o! 7Ds) targeting capa#ilities can #e a%%e% as (ell* Due to t&e inorganic nature o! 7Ds) t&eir interaction (it& t&eir imme%iate environment at in vivo states can #e minimal compare% (it& t&eir organic counterparts* Dra(#ac$s o! 7Ds !or %rug %iscovery are as !ollo(s6

Si/e variation %uring t&e synt&esis o! single color %ots is 5[ to 9[ an% !or applications suc& as capillary electrop&oresis or gel electrop&oresis) it coul%

4<;

'ain

create !alse results* T&ere!ore) 7D synt&esis tec&niCues nee% to &ave improve% Cuality control (it& respect to si/e %istri#ution #e!ore t&ey can #e seriously utili/e% in %rugE%iscovery researc&* -or a#sorption) %istri#ution) meta#olism an% e"cretion AADMEB purposes) #lue 7Ds A%iameter o! 8*< nmB are t&e smallest class o! t&e 7D !amily #ut t&ey are consi%era#ly larger t&an organic %yes* Hence) t&e use o! 7Ds !or t&is purpose mig&t not #e %esira#le in special cases* Similarly) t&e num#er o! !unctional groups attac&e% to an organic %ye is usually 4) or it can #e controlle% very precisely* T&e transport o! a large volume A%ue to multiple attac&ments o! %rug molecules to a single 7DB across t&e mem#rane (ill #e more %i!!icult t&an a single molecule itsel!* T&e Q#lin$ingS c&aracteristics o! 7Ds (&en t&ey are e"cite% (it& &ig&Eintensity lig&t coul% #e a limiting !actor !or !ast scan systems suc& as !lo( cytometry*

?t&er Nanotec&nologies !or Drug Discovery None o! t&e nanoparticles availa#le is i%eal !or all reCuirements o! %rug %iscovery* T&e c&oice may %epen% on t&e nee%s* Nano%evices suc& as nano#iosensors an% nano#ioc&ips are #eing use% to improve %rug %iscovery an% %evelopment* Nanoscale assays can signi!icantly contri#ute to costEsaving in screening campaigns* In a%%iE tion) some nanosu#stances may #e potential %rugs o! t&e !uture* T&ese inclu%e %en%rimers) !ullerenes) an% nano#o%ies A>B* Den%rimers are a novel class o! t&reeE%imensional nanoscale) coreEs&ell strucE tures t&at can #e precisely synt&esi/e% !or a (i%e range o! applications* T&ey are most use!ul in %rug %elivery an% can also #e use% !or t&e %evelopment o! ne( p&arE maceuticals (it& novel activities* Polyvalent %en%rimers interact simultaneously (it& multiple %rug targets* T&ey can #e %evelope% into novelEtargete% cancer t&eraE peutics A4@B* A $ey attri#ute o! t&e !ullerene molecules is t&eir numerous points o! attac&ment) allo(ing !or precise gra!ting o! active c&emical groups in t&reeE%imensional orientaE tions* T&is attri#ute) t&e &allmar$ o! rational %rug %esign) allo(s !or positional control in matc&ing !ullerene compoun%s to #iological targets* In concert (it& ot&er attri#utes) namely t&e si/e o! t&e !ullerene molecules) t&eir re%o" potential an% t&eir relative inertness in #iological systems) it is possi#le to tailor reCuisite p&armaco$inetic c&arE acteristics to !ullereneE#ase% compoun%s an% optimi/e t&eir t&erapeutic e!!ect A44B* Nano#o%ies are t&e smallest availa#le intact antigenE#in%ing !ragments &ar#oring t&e !ull antigenE#in%ing capacity o! t&e naturally occurring &eavyEc&ain anti#o%ies* Nano#o%ies &ave t&e potential o! a ne( generation o! anti#o%yE#ase% t&erapeutics as (ell as %iagnostics !or %iseases suc& as cancer A45B*

R? E ?- NAN?+I?TECHN? ?G3E+ASED DRUG DE I2ER3 IN DE2E ?PMENT ?- NAN?MEDICINE Several nanoparticleE#ase% tec&nologies !or %rug %elivery are %escri#e% in various c&apters o! t&is #oo$* T&is section (ill #rie!ly %escri#e t&e relevance o! t&ese tec&E nologies !or practical &uman t&erapeutics* An important clinical aspect o! nanoparticleE#ase% t&erapeutics is targete% %rug %elivery* ,&en nanoparticles are use% in t&e treatment o! cancer) t&eir po(erE !ul targeting a#ility an% potential !or large cytoto"ic payloa% %ramatically en&ance t&e e!!icacy o! conventional p&armaceuticals as (ell as novel t&erapeutic approac&es) suc& as gene t&erapy) ra%ioimmunot&erapy) an% p&oto%ynamic t&erapy*

Role o! Nano#iotec&nology in t&e Development o! Nanome%icine

4<<

T&ere are particular a%vantages o! %rug %elivery !or t&e treatment o! various %iseases #y nanoscale %evices* T&ere are several reCuirements !or %eveloping a %evice small enoug& to e!!iciently leave t&e vasculature an% enter cells to per!orm multiple) smart tas$s* Ho(ever) t&e ma.or reCuirement involves si/e* 2ascular pores limit egress o! t&erapeutics to materials less t&an appro"imately :@ nm in %iameter) an% cells (ill not internali/e materials muc& greater t&an 4@@ nm* As a result) t&e only currently availa#le tec&nology t&at !ul!ills t&ese criteria consists o! synt&etic nano%evices* T&ese are %esigne%) synt&etic materials (it& structures less t&an 4@@ nm in si/e* Unli$e !ictional mec&anical nanomac&ines) #ase% on %evices t&at &ave #een Qs&run$enS to nanometer %imensions) several true nanomolecular structures &ave no( #een synt&esi/e% an% applie% to %rug %elivery) gene trans!er) antimicro#ial t&erapeutics) an% immuno%iagnostics* Nanoparticles are important !or %elivering %rugs intravenously so t&at t&ey can pass sa!ely t&roug& t&e #o%yPs smallest #loo% vessels) !or increasing t&e sur!ace area o! a %rug so t&at it (ill %issolve more rapi%ly) an% !or %elivering %rugs via in&alation* Porosity is important !or entrapping gases in nanoparticles) !or controlE ling t&e release rate o! t&e %rug) an% !or targeting %rugs to speci!ic regions* ?(ing to t&eir small si/e) lipi% nanocapsules mig&t #e promising !or an in.ecta#le as (ell as !or an oral %rugE%elivery system) provi%ing su!!icient %rug solu#ility to avoi% em#oli/ation in #loo% a!ter intravenous in.ection as (ell as a positive e!!ect o! %rug a#sorption a!ter oral a%ministration* A %rugE%elivery system !or intravenous a%minE istration o! i#upro!en &as #een %evelope% (&ic& e"&i#its sustaine%Erelease properE ties #y eit&er oral or intravenous route an% may #e use!ul !or t&e treatment o! postoperative pain*

NAN?MEDICINE +esi%es nanoparticles) various nanotec&nologies an% ot&er nanomaterials t&at are currently un%er investigation in me%ical researc& an% %iagnostics (ill soon !in% a practical application in practice o! me%icine* Nano#iotec&nologies are #eing use% to create an% stu%y mo%els o! &uman %isease) particularly immune %isor%ers* Intro%uction o! nano#iotec&nologies in me%icine (ill not create a separate #ranc& o! me%icine #ut simply implies improvement o! %iagnosis as (ell as t&erapy an% can #e re!erre% to as nanome%icine* T&is #roa% term covers various t&erapeutic areas inclu%ing treatments t&at may reCuire surgical intervention* Applications o! nano#iotec&nology in me%icine are s&o(n in Ta#le 4*

Clinical Nano%iagnostics Application o! nanotec&nology in molecular %iagnostics (ill &ave a tremen%ous impact on t&e practice o! me%icine* +iosensor systems #ase% on nanotec&nology coul% %etect emerging %isease in t&e #o%y at a stage t&at may #e cura#le* T&is is e"tremely important in management o! in!ections an% cancer* Some o! t&e #o%y !unctions an% responses to treatment (ill #e monitore% (it&out cum#ersome la#oE ratory eCuipment* Some e"amples are a ra%iotransmitter small enoug& to put into a cell an% acoustical %evices to measure an% recor% t&e noise a &eart ma$es* Nano%iagnostics (ill also #e integrate% (it& nanot&erapeutics*

Nanoen%oscopy En%oscopic microcapsules t&at can #e ingeste% an% precisely positione% are #eing %evelope%* A control system (ill ena#le t&e capsule to attac& to t&e %igestive tract

4<=
TA+ E 4 Nanome%icine in t&e T(entyE-irst Century Nano%iagnostics Molecular %iagnostics Nanoen%oscopy Nanoimaging Nanotec&nologyE#ase% %rugs Drugs (it& improve% met&o%s o! %elivery Regenerative me%icine Tissue engineering (it& nanotec&nology Transplantation me%icine E"osomes !rom %onor %en%ritic cells !or %rugE!ree organ transplants Nanoro#otic treatments 2ascular surgery #y nanoro#ots intro%uce% into t&e vascular system Nanoro#ots !or %etection an% %estruction o! cancer Implants +ioimplanta#le sensors t&at #ri%ge t&e gap #et(een electronic an% neurological circuitry Dura#le re.ectionEresistant arti!icial tissues an% organs Implantations o! nanocoate% stents in coronary arteries to elute %rugs an% to prevent reocclusion Implantation o! nanopumps !or %rug %elivery Minimally invasive surgery using cat&eters Miniaturi/e% nanosensors implante% in cat&eters to provi%e realEtime %ata to surgeons Nanosurgery #y integration o! nanoparticles an% e"ternal energy

'ain

Source 6 -rom Re!* 4*

an% move (it&in it* Precisely positione% microcapsules (oul% allo( p&ysicians to vie( any part o! t&e insi%e lining o! t&e %igestive tract in %etail) resulting in more e!!icient) accurate) an% less invasive %iagnoses* In a%%ition) t&ese capsules coul% #e mo%i!ie% to inclu%e treatment mec&anisms as (ell) suc& as t&e release o! a %rug or c&emical near a %isease% area* PillCamd capsule AGiven Imaging t%*) 3oCneam) IsraelB) an en%oscope to visuali/e small intestine a#normalities) (as approve% in 5@@4* ?t&er companies are no( pro%ucing ingesti#le capsules !or t&is purpose* T&e patient ingests t&e capsule) (&ic& contains a tiny camera) an% intestinal peristalsis propels t&e capsule !or appro"imately eig&t &ours* During t&is time) t&e camera snaps t&e pictures an% images t&at are transmitte% to a %ata recor%er (orn #y t&e patient* T&e p&ysicians can revie( t&e images later on to ma$e t&e %iagnosis) #ut some a#normalities may #e misse% as t&is met&o% &as only a :@[ success rate in %etection o! %iseases* Controlling t&e positioning an% movement on a nanoscale (ill greatly improve t&e accuracy o! t&is met&o%* Similar nanoro#ots are un%er %evelopment !or ot&er parts o! t&e #o%y*

Nano#iotec&nology !or Developing StemECellE+ase% T&erapies StemEcellE#ase% t&erapies are one o! t&e most promising areas o! %evelopment in &uman t&erapeutics* Nano#iotec&nology can #e applie% to %elivery o! gene t&erapy using genetiEcally mo%i!ie% stem cells an% !urt&er applie% in trac$ing stem cells intro%uce% into t&e &uman #o%y* Nano!i#rous sca!!ol%s are #eing %evelope% !or stem cells to mimic t&e nanoE meterEscale !i#ers normally !oun% in t&at matri" A48B* T&ey are #eing use% to gro( stem cells %erive% !rom a%ipose tissue* T&ey can conceiva#ly #e use% !or tissue repair*

Role o! Nano#iotec&nology in t&e Development o! Nanome%icine

4<>

Application o! Nano#iotec&nology in 2arious T&erapeutic Areas Nano#iotec&nology &as #een applie% in almost every area o! &uman &ealt&care* Some e"amples are given o! applications in important t&erapeutic areas6 cancer) neurological %isor%ers) car%iovascular %iseases) an% in!ections* ?ncology Nanoparticles can %eliver c&emot&erapy %rugs %irectly to tumor cells an% t&en give o!! a signal a!ter t&e cells are %estroye%* Drugs %elivere% t&is (ay are several times more potent t&an stan%ar% t&erapies* Com#ination o! gol% nanoparticles !ollo(e% #y WEray treatment re%uces t&e si/e o! t&e tumors) or completely era%icates t&em in mice A49B* T&e tec&niCue (or$s #ecause gol%) (&ic& strongly a#sor#s W rays) selecE tively accumulates in tumors* T&is increases t&e amount o! energy t&at is %eposite% in t&e tumor compare% (it& near#y normal tissue* Gol% nanoparticle ra%iot&erapy !or patients is un%er consi%eration !or clinical trials* Nanos&ells may #e com#ine% (it& targeting proteins an% use% to a#late target cells* T&is proce%ure can result in t&e %estruction o! soli% tumors or possi#ly metasE tases not ot&er(ise o#serva#le #y t&e oncologist* In a%%ition) nanos&ells can #e utili/e% to re%uce angiogenesis present in cancer* E"periments in animals) in vitro an% in tissue %emonstrate t&at speci!ic cells Ae*g*) cancer cellsB can #e targete% an% %estroye% #y an amount o! in!rare% lig&t t&at is ot&er(ise not &arm!ul to surroun%E ing tissue* T&is proce%ure may #e per!orme% using an e"ternal Aoutsi%e t&e #o%yB in!rare% laser* Prior researc& &as in%icate% t&e a#ility to %eliver t&e appropriate levels o! in!rare% lig&t at %ept&s o! up to 4: cm) %epen%ing upon t&e tissue* P&otot&ermal tumor a#lation in mice &as #een ac&ieve% #y using nearEin!rare%E a#sor#ing nanoparticles A4:B* Nanos&ells ena#le a seamless integration o! cancer %etection an% t&erapy* It is (it&in t&e realm o! possi#ility to use molecular tools to %esign a miniature %evice t&at can #e intro%uce% in t&e #o%y) locate an% i%enti!y cancer cells) an% !inally %estroy t&em* T&e %evice (oul% &ave a #iosensor to i%enti!y cancer cells an% a supply o! anticancer su#stance t&at coul% #e release% on encountering cancer cells* A small computer coul% #e incorporate% to program an% integrate t&e com#ination o! %iagnosis an% t&erapy an% provi%e t&e possi#ility to monitor t&e in vivo activities #y an e"ternal %evice* As t&ere is no universal anticancer agent) t&e computer proE gram coul% matc& t&e type o! cancer to t&e most appropriate agent* Suc& a %evice coul% #e implante% as a prop&ylactic measure in persons (&o %o not &ave any o#viE ous mani!estations o! cancer* It (oul% circulate !reely an% coul% %etect an% treat cancer at t&e earliest stage* Suc& a %evice coul% #e reprogramme% t&roug& remote control an% ena#le c&ange o! strategy i! t&e lesion encountere% is ot&er t&an cancer*

Disor%ers o! t&e Central Nervous System Applie% nano#iotec&nology aime% at t&e regeneration an% neuroprotection o! t&e central nervous system ACNSB (ill signi!icantly #ene!it !rom #asic nanotec&nology researc& con%ucte% in parallel (it& a%vances in cell #iology) neurop&ysiology) an% neuropat&ology A4;B* 7D tec&nology is use% to gat&er in!ormation a#out &o( t&e CNS environment #ecomes in&ospita#le to neuronal regeneration !ollo(ing in.ury or %egenerative events #y stu%ying t&e process o! reactive gliosis* Glial cells) &ouseE $eeping cells !or neurons) &ave t&eir o(n communication mec&anisms t&at can #e triggere% to #ecome reactive !ollo(ing in.ury* 7Ds) (it& a%%e% #ioactive molecules) mig&t spur gro(t& o! neurites in a (ay t&at provi%es a me%ium t&at (ill encourage t&is gro(t& in a %irecte% (ay*

4=@

'ain

Nanoparticles can #e use% as an ai% to neurosurgery* Iron o"i%e nanoparticles can outline not only #rain tumors un%er MRI) #ut also ot&er lesions in t&e #rain t&at may ot&er(ise &ave gone unnotice% A4<B* Car%iovascular Diseases T&e %iagnosis an% treatment o! unsta#le plaCue is an area in (&ic& nanotec&nology coul% &ave an imme%iate impact* Nanopro#es can #e targete% to plaCue compoE nents !or noninvasive %etection o! patients at ris$* Targete% nanoparticles) multiE !unctional macromolecules) or nanotec&nologyE#ase% %evices coul% %eliver t&erapy to a speci!ic site) locali/e% %rug release #eing ac&ieve% eit&er passively A#y pro"imE ity aloneB or actively At&roug& supply o! energy as ultrasoun%) nearEin!rare%) or magnetic !iel%B* Targete% nanoparticles or %evices coul% also sta#ili/e vulnera#le plaCue #y removing material) !or e"ample) o"i%i/e% lo(E%ensity lipoproteins* Devices a#le to attac& to unsta#le plaCues an% (arn patients an% emergency me%iE cal services o! plaCue rupture (oul% !acilitate timely me%ical intervention* Restenosis a!ter percutaneous coronary intervention continues to #e a serious pro#lem in clinical car%iology* Recent a%vances in nanoparticle tec&nology &ave ena#le% t&e %elivery o! N0>44) an antiproli!erative %rug) selectively to t&e #alloonE in.ure% artery !or a longer time A4=B* N0>44 is a coreEs&ell nanoparticle o! polyet&ylene glycolE#ase% #loc$ copolymer encapsulating %o"oru#icin* It accumulates in vascular lesions (it& increase% permea#ility* In a #alloon in.ury mo%el o! t&e rat caroti% artery) intravenous a%ministration o! N0>44 signi!icantly in&i#ite% t&e neointimal !ormation* T&e e!!ect o! N0>44 (as %ue to in&i#ition o! vascular smoot& muscle proli!eration #ut not to en&ancement o! apoptosis or in&i#ition o! in!lammatory cell recruitment* N0>44 (as (ell tolerate% (it&out any a%verse systemic e!!ects* T&ese results suggest t&at nanoparticle tec&nology is a promising an% sa!e approac& to target vascular lesions (it& increase% permea#ility !or t&e prevention o! restenosis a!ter #alloon in.ury* A novel approac& to prevention o! restenosis involves incorporation o! nitric o"i%e AN?BEeluting nano!i#ers into stents !or antit&rom#ogenic action* N? &as vasoE %ilating action as (ell) (&ic& may #e #ene!icial in isc&emic &eart %isease*

In!ections An important role o! nanotec&nology in t&e management o! in!ections is use o! !orE mulations (&ic& improve t&e action o! $no(n #acterici%al agents* T&e #acterici%al properties o! some agents are mani!est only in nanoparticulate !orm* Certain !ormuE lations o! nanoscale po(%ers possess antimicro#ial properties* T&ese !ormulations are ma%e o! simple) nonto"ic metal o"i%es suc& as magnesium o"i%e AMg?B an% calcium o"i%e ACa?) limeB in nanocrystalline !orm) carrying active !orms o! &aloE gens) !or e"ample) Mg?*Cl5 an% Mg?*+r5* ,&en t&ese ultra!ine po(%ers contact vegetative cells o! Esc&eric&ia coli) +acillus cereus) or +acillus glo#igii) over >@[ are $ille% (it&in a !e( minutes* i$e(ise) spore !orms o! t&e +acillus species are %econE taminate% (it&in several &ours* Dry contact (it& o!lato"ins an% contact (it& MS5 #acteriop&age Asurrogate o! &uman enterovirusB in (ater also cause %econtaminaE tion in minutes* A nanopo(%er o! Mg? can scour contaminate% rooms o! ant&ra" spores A4>B* Unli$e anti#acterial gases an% !oams (&ic& are messy) corrosive) an% ruin electrical eCuipment) t&e po(%er can #e spraye% into rooms an% s(ept or vacuume% up* T&e c&emical spec$s attract oppositely c&arge% spores* T&e particles t&en cut open an% c&emically #rea$ %o(n t&e sporesP toug& outer s&ell* Silver po(%er (it& particle si/e ranging !rom :@ to 4@@ nm &as a &omogeE neous %istri#ution o! nanoparticles in t&e material an% antiin!ective properties*

Role o! Nano#iotec&nology in t&e Development o! Nanome%icine

4=4

Silver nanoparticles &ave #een incorporate% in commercial preparations !or (oun% care to prevent in!ection* A simple molecule !rom a &y%rocar#on an% an ammonium compoun% &as #een use% to pro%uce a uniCue nanotu#e structure (it& antimicro#ial capa#ility A5@B* T&e Cuaternary ammonium compoun% is $no(n !or its a#ility to %isrupt cell mem#ranes an% causes cell %eat&) (&ereas t&e &y%rocar#on %iacetylene can c&ange colors (&en appropriately !ormulate%M t&e resulting molecule (oul% &ave t&e %esire% properties o! #ot& a #iosensor an% a #ioci%e* Antimicro#ial nanoemulsions) containing (ater an% soy#ean oil (it& uniE !ormly si/e% %roplets in t&e 5@@ to 9@@ nm range) can %estroy micro#es e!!ectively (it&out to"icity or &arm!ul resi%ual e!!ects A54B* T&e nanoparticles !use (it& t&e mem#rane o! t&e micro#e an% t&e sur!actant %isrupts t&e mem#rane) $illing t&e micro#e* T&e classes o! micro#es era%icate% are viruses Ae*g*) HI2) &erpesB) #acteria Ae*g*) E* coli) SalmonellaB) spores Ae*g*) ant&ra"B) an% !ungi Ae*g*) Can%i%a al#icans) +yssoc&lamys !ulvaB*

R? E ?- NAN?+I?TECHN? ?G3 IN THE DE2E ?PMENT ?- PERS?NA IVED MEDICINE Personali/e% me%icine simply means t&e prescription o! speci!ic t&erapeutics #est suite% !or an in%ivi%ual* It is usually #ase% on p&armacogenetic) p&armacogenomic) an% p&armacoproteomic in!ormation) #ut ot&er in%ivi%ual variations in patients are also ta$en into consi%eration A55)58B* Personali/e% me%icine is #eginning to #e recogni/e% an% is e"pecte% to #ecome a part o! me%ical practice (it&in t&e ne"t %eca%e* Molecular %iagnostics is an important component o! personali/e% me%iE cine* Improvement o! %iagnostics #y nanotec&nology &as a positive impact on personali/e% me%icine* Nanotec&nology &as potential a%vantages in applications in pointEo!Ecare %iagnosis) !or e"ample) on patientPs #e%si%e or t&e outpatient clinic) sel!E%iagnostics !or use in t&e &ome) an% integration o! %iagnostics (it& t&erapeuE tics* All o! t&ese (ill !acilitate t&e %evelopment o! personali/e% me%icines* Cancer is a goo% e"ample o! a%vantages o! personali/e% management* In cases o! cancer) t&e variation in #e&avior o! cancer o! t&e same &istological type !rom one patient to anot&er is also ta$en into consi%eration* Personali/ation o! cancer t&erapies is #ase% on a #etter un%erstan%ing o! t&e %isease at t&e molecular level) an% nanotec&nology (ill play an important role in t&is area A59B*

C?NC UDING REMAR0S AND -UTURE PR?SPECTS Disease an% ot&er %istur#ances o! !unction are cause% largely #y %amage at t&e molecular an% cellular level) #ut current surgical tools are large an% cru%e* Even a !ine scalpel is a (eapon more suite% to tear an% in.ure t&an &eal an% cure* It (oul% ma$e more sense to operate at t&e cell level to correct t&e cause o! %isease) rat&er t&an c&op o!! large lesions as a result o! t&e %istur#ances at cell level* Nanotec&nology (ill ena#le construction o! computerEcontrolle% molecular tools t&at are muc& smaller t&an a &uman cell an% #uilt (it& t&e accuracy an% preciE sion o! %rug molecules* Suc& tools (ill #e use% !or interventions in a re!ine% an% controlle% manner at t&e cellular an% molecular levels* T&ey coul% remove o#strucE tions in t&e circulatory system) $ill cancer cells) or ta$e over t&e !unction o! su#celE lular organelles* Instea% o! transplanting arti!icial &earts) a surgeon o! t&e !uture (oul% #e transplanting arti!icial mitoc&on%rion*

4=5

'ain

Nanotec&nology (ill also provi%e %evices to e"amine tissue in minute %etail* +iosensors t&at are smaller t&an a cell (oul% give us an insi%e loo$ at cellular !uncE tion* Tissues coul% #e analy/e% %o(n to t&e molecular level) giving a completely %etaile% Qsnaps&otS o! cellular) su#cellular) an% molecular activities* Suc& a %etaile% %iagnosis (oul% gui%e t&e appropriate treatment* An increasing use o! nano#iotec&nology #y t&e p&armaceutical an% #iotec&E nology in%ustries is anticipate%* Nanotec&nology (ill #e applie% at all stages o! %rug %evelopment Z !rom !ormulations !or optimal %elivery to %iagnostic applicaE tions in clinical trials* It is e"pecte% t&at (it&in t&e ne"t !e( years) (e (ill &ave a #etter un%erstan%E ing o! &o( to coat or c&emically alter nanoparticles to re%uce t&eir to"icity to t&e #o%y) (&ic& (ill allo( us to #roa%en t&eir use !or %isease %iagnosis an% !or %rug %elivery* +iome%ical applications are li$ely to #e some o! t&e earliest* T&e !irst clinical trials are anticipate% !or cancer t&erapy*

RE-ERENCES
4* 'ain 00* Nano#iotec&nology6 applications) mar$ets) an% companies* +asel6 'ain P&arma +iotec& Pu#lications) 5@@<* 5* 'ain 00* Nano%iagnostics6 application o! nanotec&nology in molecular %iagnostics* E"pert Rev Mol Diagn 5@@8M 964:8H4;4* 8* 'ain 00* Nano#iotec&nology in Molecular Diagnostics* Nor(ic&) U0) Nor(ic&6 Hori/on Scienti!ic Press) 5@@;* 9* +ao 3P) Hu#er M) ,ei T-) et al* SNP i%enti!ication in unampli!ie% &uman genomic DNA (it& gol% nanoparticle pro#es* Nucleic Aci%s Res 5@@:M 886e4:* :* Stor&o!! '') ucas AD) Garimella 2) +ao 3P) MJller UR* Homogeneous %etection o! unamE pli!ie% genomic DNA seCuences #ase% on colorimetric scatter o! gol% nanoparticle pro#es* Nat +iotec&nol 5@@9M 556 ==8H==<* ;* +ulte ',) Ar#a# AS) Douglas T) et al* Preparation o! magnetically la#ele% cells !or cell trac$ing #y magnetic resonance imaging* Met&o%s En/ymol 5@@9M 8=;65<:H5>>* <* -arrer RA) +utter!iel% - ) C&en 2,) -our$as 'T* Hig&ly e!!icient multip&otonEa#sorptionE in%uce% luminescence !rom gol% nanoparticles* Nano ett 5@@:M :6448>H4495* =* ?/$an M* 7uantum %ots an% ot&er nanoparticles6 (&at can t&ey o!!er to %rug %iscoveryT Drug Discov To%ay 5@@9M >64@;:H4@<4* >* 'ain 00* T&e role o! nano#iotec&nology in %rug %iscovery* Drug Discov To%ay 5@@:M 4@6498:H4995* 4@* 0u$o(s$aE atallo '-) Can%i%o 0A) Cao V) et al* Nanoparticle targeting o! anticancer %rug improves t&erapeutic response in animal mo%el o! &uman epit&elial cancer* Cancer Res 5@@:M ;:6:84<H:859* 44* ,ilson SR* Nanome%icine6 !ullerene an% car#on nanotu#e #iology* In6 ?sa(a E) e%* Perspectives in -ullerene Nanotec&nology* 0lu(er Aca%emic Pu#lis&ers) 5@@5* 45* Revets H) De +aetselier P) Muyl%ermans S* Nano#o%ies as novel agents !or cancer t&erE apy* E"pert ?pin +iol T&er 5@@:M :6444H459* 48* 0ang W) Wie 3) 0niss DA* A%ipose tissue mo%el using t&reeE%imensional cultivation o! prea%ipocytes see%e% onto !i#rous polymer sca!!ol%s* Tissue Eng 5@@:M 4469:=H9;=* 49* Hain!el% ') Slat$in DN) Smilo(it/ HM* T&e use o! gol% nanoparticles to en&ance ra%ioE t&erapy in mice* P&ys Me% +iol 5@@9M 9>6N8@>HN84:* 4:* ?PNeal DP) Hirsc& R) Halas N') et al* P&otoEt&ermal tumor a#lation in mice using near in!rare%Ea#sor#ing nanoparticles* Cancer ett 5@@9M 5@>64<4H4<;* 4;* Silva GA) C/eisler C) Niece 0 ) et al* Selective %i!!erentiation o! neural progenitor cells #y &ig&Eepitope %ensity nano!i#ers* Science 5@@9M 8@8648:5H48::* 4<* Neu(elt EA) 2arallyay P) +ago AG) et al* Imaging o! iron o"i%e nanoparticles #y MR an% lig&t microscopy in patients (it& malignant #rain tumours* Neuropat&ology an% Applie% Neuro#iology 5@@9M 8@69:;H9<4*

Role o! Nano#iotec&nology in t&e Development o! Nanome%icine

4=8

4=* U(ato$u T) S&imo$a(a H) A#e 0) et al* Application o! nanoparticle tec&nology !or t&e prevention o! restenosis a!ter #alloon in.ury in rats* Circ Res 5@@8M >56e;5He;>* 4>* Stoimenov P0 et al* Metal o"i%e nanoparticles as #acterici%al agents* angmuir 5@@5M 4=6;;<>H;;>;* 5@* ee S+) 0oepsel R) Stol/ D+) et al* Sel!Eassem#ly o! #ioci%al nanotu#es !rom a singleE c&ain %iacetylene amine salt* ' Am C&em Soc 5@@9M 45;6489@@H489@:* 54* Hamou%a T) Myc A) Donovan +) et al* A novel sur!actant nanoemulsion (it& a uniCue nonEirritant topical antimicro#ial activity against #acteria) envelope% viruses an% !ungi* Micro#iological Researc& 5@@4M 4:;6 4H<* 55* 'ain 00* Personalise% me%icine* Curr ?pinion Mol T&er 5@@5M 96:9=H::=* 58* 'ain 00* Personali/e% Me%icine6 Scienti!ic an% Y Commercial Aspects* +asel6 'ain P&arma +iotec& Pu#lications) 5@@<* 59* 'ain 00* Role o! nano#iotec&nology in %eveloping personali/e% me%icine !or cancer* TCRT 5@@:M 96;9:H;:@*

48

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

3as&(ant Pat&a$
UC+ Manu!acturing) Inc*) Roc&ester) Ne( 3or$) U*S*A*

Deepa$ T&assu
UC+ P&arma) Inc*) Roc&ester) Ne( 3or$) U*S*A*

Mic&el Deleers
Glo#al P&armaceutical Tec&nology an% Analytical Development AGPTADB) UC+) +raine lPAlleu%) +elgium

INTR?DUCTI?N T&e success!ul intro%uction o! t&e !irst %rugE%elivery system ADDSB #roug&t a#out tremen%ous interest in t&e researc& an% application o! t&ese systems) especially !or t&e entry o! %rugs in t&e systemic circulation o! t&e #o%y* T&e goal o! any DDS is to provi%e a t&erapeutic amount o! %rug to t&e proper site in t&e #o%y (&ile ac&ieving it rapi%ly an% maintaining %esire% %rug concentrations in t&e #o%y circulation* Most %rugs are %elivere% to patients using a systemic approac&) t&e #elie! #eing t&at i! you !loo% t&e #o%y (it& enoug& active compoun%s) t&ere (ill #e a %esire% t&eraE peutic e!!ect on t&e #o%y* T&e DDSs are suppose% to %eliver t&e %rug at a rate %icE tate% #y t&e nee%s o! t&e #o%y over a speci!ie% perio% o! t&e treatment* T&e i%eali/e% o#.ective !or t&e DDS points to t(o ma.or aspects) namely spatial placement an% temporal %elivery o! t&e %rug* Spatial placement relates to targeting a %rug to a speci!ic organ or tissue* Temporal %elivery re!ers to controlling t&e rate o! %elivery to t&e target tissues A4B* Hun%re%s o! %rugE%elivery pro%ucts &ave #een intro%uce% in t&e mar$et an% many are no( in %i!!erent stages o! %evelopment* ately) in t&e !iel% o! p&armaceutical researc&) a plet&ora o! %rugE%elivery groups an% companies &ave emerge%) partly as a result o! surging interest in generic %rug %evelopment an% continue% tec&nological a%vances* T&e %rugE%elivery tec&nolE ogy can en&ance t&e t&erapeutic as (ell as t&e commercial value o! t&e &ealt&care pro%ucts* It ta$es into consi%eration t&e carrier) t&e route) an% t&e target) an% %evelops a strategy o! processes or %evices %esigne% to increase t&e t&erapeutic e!!icacy o! t&e %rug an% in many cases re%uce t&e si%e e!!ects o! t&e %rug* T&e %rugE%elivery sector &as evolve% !rom #eing simply a part o! t&e p&armaE ceutical pro%uction process to a %riving !orce !or innovation an% pro!its* In%ustry an% commercial interests in %rugE%elivery continue to #uil% stea%ily* T&e #ene!its to t&e patients can #e seen in improve% compliance an% me%ical outcomes* T&e p&armaceuE tical in%ustry is a#le to e"ten% t&e patent protection t&roug& novel DDSs an% #ring ne( t&erapies to t&e mar$et* U*S* %eman%s !or DDSs (ill gro( nearly >[ annually to more t&an =5 #illion #y 5@@<* Gro(t& opportunities e"ten% into all t&erapeutic classes o! p&armaceuticals6 respiratory) central nervous system ACNSB) an% car%iovascular agents (ill remain t&e top t&ree applications #ase% on t&e special !ormulating nee%s o! me%icines !or con%itions suc& as ast&ma) art&ritis) an% &ypertension* T&e ot&er areas suc& as anticancer %rugs) &ormones) an% vaccines (ill also !ollo( t&e trac$*

4=:

4=;

Pat&a$ et al*

NAN?PARTICU ATE DRUGEDE I2ER3 S3STEMS Nanoparticulate %rugE%elivery systems ANPDDSsB are #eing e"plore% !or t&e purpose o! solving t&e c&allenges o! %rug %elivery* Coming in many s&apes an% si/es) most carriers are less t&an 4@@ nm in %iameter* NPDDSs provi%e met&o%s !or targeting an% releasing t&erapeutic compoun%s in very %e!ine% regions* T&ese ve&iE cles &ave t&e potential to eliminate or at least ameliorate many pro#lems associate% (it& %rug %istri#ution* As many %rugs &ave a &y%rop&o#ic component) t&ey o!ten su!!er !rom pro#lems o! precipitation in &ig& concentration) an% t&ere are many e"amples o! to"icity issues (it& e"cipients %esigne% to prevent %rug aggregation* To com#at t&ese issues) many NPDDSs provi%e #ot& &y%rop&o#ic an% &y%rop&ilic environments) (&ic& !acilitate %rug solu#ility* Alternatively) many %rugs su!!er !rom rapi% #rea$%o(n an%1or clearance in vivo* Encapsulating t&e %rugs in a protective environment) NPDDSs increase t&eir #ioavaila#ility) t&ere#y allo(ing t&e clinicians to prescri#e lo(er %oses* ,it& recent a%vances in polymer an% sur!ace con.ugation tec&niCues as (ell as micro!a#rication met&o%s) per&aps t&e greatest !ocus in %rugE%elivery tec&nology is in t&e %esign an% applications o! NPDDSs* Ranging !rom simple metalHceramic core structure to comple" lipi%Hpolymer matrices) t&ese su#micron !ormulations A5B are #eing !unctionali/e% in numerous (ays to act as t&erapeutic ve&icles !or a variety o! con%itions A-ig* 4B* NPDDSs can #e %e!ine% as t&e DDSs (&ere nanotec&nology is use% to %eliver t&e %rug at nanoscale* +elo( 4@@ nm) materials e"&i#it %i!!erent) more %esira#le p&ysical) c&emical) an% #iological properties* Given t&e enormity an% imme%iacy o! t&e unmet nee%s o! t&erapeutic areas suc& as CNS %isor%ers) t&is can lea% to %rugs t&at can e"ten% li!e an% save untimely %eat&s A5B*

A%vantages o! Nanoparticulate DrugEDelivery Systems T&e nanoparticles ANPsB may o!!er some a%vantages suc& as protection o! %rugs against %egra%ation) targeting t&e %rugs to speci!ic sites o! action) organ or tissues)

-IGURE 4 ASee color insert*B Di!!erent types o! nanoparticulate structures*

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

4=<

an% %elivery o! #iological molecules suc& as proteins) pepti%es) an% oligonucleoti%es* A num#er o! %i!!erent strategies &ave #een propose% in or%er to mo%i!y t&e p&ysicoE c&emical c&aracteristics o! t&e NPs) an% t&us t&eir interactions (it&in t&e #iological systems* -or e"ample) it is possi#le to c&ange t&e c&emical nature o! t&e polymeric matri" o! t&e NPs an% t&ere#y alter certain #iological p&enomena suc& as #iorecogniE tion) #io%istri#ution) #ioa%&esion) #iocompati#ility) an%1or #io%egra%ation* Some polymeric materials use% !or t&is purpose are gelatin) c&itosan ACSB) so%ium alginate) polyAal$ylBcynoacrylates) polyAlactic aci%B AP AB) polyAlacticEcoEglycolic aci%B AP GAB) polyAet&ylene glycolEcoEAlacticEglycolic aci%B) polyAcaprolactoneB) an% polymet&yl met&acrylate A8H:B* Anot&er approac& to mo%i!y t&e #iological response is #ase% on t&e incorporation o! suita#le a%.uvants in t&e NPs) li$e proteins suc& as al#umin) invasins) an% lectins) an% polymers suc& as polo"amers an% polo"amines A;B* Di!!erent manu!acturing met&o%s can also ena#le mo%i!ications o! t&e p&ysicoE c&emical c&aracteristics o! NPs suc& as si/e) s&ape) structure) morp&ology) te"ture) an% composition A8B*

Manu!acturing Tec&niCues !or Nanoparticulate DrugEDelivery Systems Conventionally) t(o groups o! manu!acturing tec&niCues &ave #een reporte% !or pro%ucing NPs* T&e !irst involves polymeri/ation o! t&e monomers) (&ereas t&e secon% one is #ase% on %ispersion o! t&e per!orme% polymers* T&e saltingEout A<B) emulsi!icationE%i!!usion A=B) an% nanoprecipitation A>B can #e cite% as typical e"amE ples o! t&e secon% met&o%* NP is a collective term use% to %escri#e t&e nanosp&eres an% nanocapsules ANCsB* T&e %i!!erence #et(een t&ese !orms lies in t&e morp&ology an% t&e arc&itecture* NCs are compose% o! a liCui% core Agenerally an oilB surroun%e% #y polymeric mem#rane) (&ereas nanosp&eres are !orme% #y a %ense polymeric matri" A4@B* NCs are p&armaceutically attractive %ue to t&eir oilE#ase% central caviE ties) (&ic& allo( a &ig& encapsulation level !or lipop&ilic su#stances) ena#ling improve% %rug %elivery* It is possi#le to avoi% %rug precipitation %uring preparation an% su#seCuent sta#ility pro#lems cause% #y t&e presence o! t&e %rug on t&e sur!ace o! t&e NPs* T(o tec&niCues are (i%ely use% to prepare a #io%egra%a#le NC*

Inter!acial Polymeri/ation o! Al$ylcyanoacrylate Monomers In t&is process) t&e cyanoacrylate monomer an% t&e lipop&ilic %rug are %issolve% in a mi"ture o! oil an% et&anol* T&is organic solution is t&en a%%e% slo(ly to (ater or a #u!!er solution ApH 8H>B containing sur!actants suc& as polo"amers or p&osp&olipE i%s* NCs are !orme% spontaneously #y anionic polymeri/ation o! t&e cyanoacrylate in t&e oily p&ase a!ter contact (it& &y%ro"yl ions (&ic& act as initiators*

Inter!acial Deposition o! Per!orme% Polymers In t&is process) t&e lipop&ilic %rug) oil polymer) an% optionally p&osp&olipi%s are %issolve% in a (aterEmisci#le solvent Ae*g*) acetoneB* T&is solution is t&en poure% (&ile stirring into an aCueous solution containing a nonionic sur!actant Ae*g*) polo"amer 4==B* NCs are instantly !orme% #y t&e !ast %i!!usion o! solvent into (ater) (&ic& provo$es t&e spontaneous emulsi!ication o! t&e oily solution in t&e !orm o! nano%roplets (&ere t&e %issolve% polymer (ill !orm a !ilm aroun% t&e %roplets t&at contain t&e %rug A44B* T&e met&o% o! inter!acial polymeri/ation is not i%eal !or t&ree reasons6 AiB t&e pro#a#le presence o! t&e resi%ual) AiiB potentially to"ic monomers or oligomers) an%

4==

Pat&a$ et al*

t&e possi#ility o! crossEreaction (it& t&e %rug) an% AiiiB t&e %i!!iculty in pre%icting t&e molecular (eig&t o! t&e resulting polymer A45B* T&e principal %ra(#ac$ o! t&is met&o% is t&e polymer aggregation t&at is !reCuently o#serve% (&en (or$ing (it& &ig& polymer concentration or lo( organic solvent1(ater ratio* Guerrero et al* A48B &ave %escri#e% a ne( process #ase% on an emulsi!icationE%i!!usion tec&niCue) overE coming t&ese %ra(#ac$s* T&is stu%y %emonstrate% t&at t&e emulsi!icationE%i!!usion tec&niCue represents a via#le alternative !or preparing #io%egra%a#le NCs starting !rom per!orme% polymers* It is simple an% versatile) an% permits &ig& e!!iciency o! entrapment o! lipop&ilic %rugs A48B* It is important to note t&at a %eeper un%erstan%ing o! t&e p&ysicoc&emical p&enomena involve% %uring t&e NPs !ormation is also necessary* Speci!ically) t&e relations&ip #et(een p&ysicoc&emical parameters an% t&eir Cuantitative e!!ects on NP !eatures coul% #e an invalua#le tool in t&e controlle% engineering o! particles* 0no(le%ge o! t&ese !un%amental relations&ips (oul% allo( NPs to #e %esigne% (it& %e!ine% si/e an% sur!ace c&aracteristics !or %elivery to speci!ic cells or organs (it&out reCuiring e"&austive e"perimental proce%ures* Ro%rigue/ et al* A8B stu%ie% t&e in!luence o! certain p&ysicoc&emical properties o! t&e aCueous an% organic p&ases use% %uring NP preparation an% t&e e!!ects on t&e c&aracteristics o! NPs pro%uce% #y saltingEout) emulsi!icationE%i!!usion) an% nanoprecipitation met&o%s) an% conclu%e% t&at t&e mean si/e o! t&e NPs coul% #e narro(e%) using %i!!erent met&o%s* -or e"ample) saltingEout o!!ere% NP mean si/e range #et(een 458 an% <4@ nm* Emulsi!ication met&o% gave 44@ to <4: nm mean si/e) (&ereas nanoprecipiE tation gave a very narro( si/e range %istri#ution o! 49< to 59: nm A8B* T&e (aterHsolvent interaction an% %i!!usion motion o! t&e solvent play an important role in e"plaining t&e variation o! t&e NP si/e %uring NP preparation #y t&e nanoprecipitation met&o%* Common %isa%vantages o! soli%Hlipi% nanoparticles AS NsB inclu%e6 particle gro(ing) unpre%icta#le gelation ten%ency) une"pecte% %ynamics o! polymorp&ic transitions) an% in&erent lo( incorporation rates resulting !rom t&e crystalline structure o! t&e soli% lipi%s A49B*

NAN?PARTICU ATE DRUGEDE I2ER3 S3STEM APP ICATI?NS NPDDSs &ave #een utili/e% !or t&eir t&erapeutic applications over a (i%e range) !rom cancer treatments to some over t&e counter A?TCB preparations* Many %rugs &ave #een use% as mo%el %rugs !or speci!ic spatial an% temporal applications* Ta#le 4 enumerates various %rugs (&ic& &ave #een use% in NPDDSs* In t&e p&armaceutical !ormulations) NPDDSs can #e use% (it& a%vantages* Ta#le 5 s&o(s applications o! NPDDSs to a%%ress various !ormulations issues*

Nanoparticulate DrugEDelivery Systems !or Proteins an% Pepti%es arge num#ers o! ne( t&erapeutic proteins an% pepti%es are #eing %iscovere%) t&us protein %rugE%elivery tec&nologies are o! ever increasing importance* Tra%itionally) t&e protein is %elivere% parenterally via solutions t&at are in.ecte% su#cutaneously) intramuscularly) an% intravenously* Alt&oug& suc& in.ections #eneE !it !rom &ig& #ioavaila#ility) t&ey !ail to provi%e sustaine% plasma concentrations an% su!!er !rom poor patient compliance %ue to t&e reCuire% !reCuency o! in.ecE tions* NPDDSs are %esigne% to provi%e t&e %rug release over an e"ten%e% perio% o! time) t&ere#y minimi/ing t&e nee% !or !reCuent in.ections* T&ese can #e use% !or systemic or oral %elivery) an% t&e #io%egra%a#le nature o! t&e nanoparticulate materials alleviates t&e nee% !or surgical removal*

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

4=>

TA+ E 4 Drugs Use% !or Nanoparticulate Drug Delivery Systems Name o! t&e %rug Aclacinomycin A%riamycin Anti!ungal %rugs AtovaCuone +etamet&asone +i!ona/ole +rimoni%ine +u%esoni%e Camptot&ecin Cep&alosporin Cisplatin Clotrima/ole Clo#etasol Clo/apine Cur%lan %erivative6 anticancer %rugs +ECyclo%e"trin Cyclosporine Cyclosporine Cyclosporine Cyclop&osp&ami%e Diclo!enac Carrier P+CA NPs P+CA NPs Su#microni/e% emulsion S N CaCo8 NPs + Cyclo%e"trin NPs Polyacrylic NPs Polylactic aci% NPs nanosuspension S N Nanocon.ugates Polymeric micelles + Cyclo%e"trin NPs S N S N S N Nanosp&ere S N Stearic aci% NPs HPMCP P+CA NCs Inorganic microparticles Polylacti%e Polylacti%e Caprolactone ipi%E#ase% emulsion S N P+CA NPs Supercritical car#on %io"i%eRP GA ipi%E#ase% ipi% %rug con.ugate ipi%E#ase% NPs Colloi%al NPs Nanosuspension ipi%E#ase% emulsion Met&acrylate polymers S N S N S N P glycoli%e polymer Polycaprolactone an% Eu%ragit S 4@@ P+CA NPs Polysor#ate =@Ecoate% P+CA NPs Colloi%al carriers Magnetic NPs S N nanocrystals ipi%E#ase% %elivery S N Cetyl alco&ol1polysor#ate NPs Gelatin NCs P GA S N P GA P GA NC S N Re!erences A4:B A4;B A4<B A4=B A4>B A5@B A54B A55B A58B A59B A5:B A5@B A5;B A5<B A5=B A5>B A8@B A84B A85B A88B A89B A8:B A8;B A8<B A8:)8=B A8;)8>B A8<B A8=B A8>B A9@B A94B A95B A98B A99B A9:B A9;B A9<B A9=B A9>B A:@B A:4B A:5B A95B A:8B A:9H:;B A:<B A:=B A:>B A;@B A;4B A:9B A;5B A;8B A;9B

Dana/ol Daro%ipine Delargin De"amet&asone Dimina/ine Dimina/ene%iaceturate Ga%olinium :E-luouracil -lur#ipro!en Halo!antrine Heparin Hy%rocortisone I%aru#icin In%omet&acin Isonia/i% 0etopro!en 0ytorp&in operami%e Met&otre"ate Mito"antrone Ni!e%ipine ?nta/olost Paclita"el

P&enot&ia/ine Pilocarpine Pra/iCuantel Pre%nisolone AContinue% B

4>@
TA+ E 4 Drugs Use% !or Nanoparticulate Drug Delivery Systems AContinue%B Name o! t&e %rug Porpo!ol Progesterone Protamine p&osp&orot&ioate Pyra/inami%e Retinal Ri!a#utine Ri!amycin Tamo"i!en Tara/epi%e T&iamine To#ramycin Tretinoin Triclosan Tu#ocurarine U#i%ecarone UC+E8:99@E8 2incristine 2itamin A Want&one Carrier ipi%E!ree NC S N NP comple"es P glycoli%e S N S N P glycoli%e Polycaprolactone NPs Cyclo%e"trin ipi% S N S N Su#micron emulsion an% NCs Polysor#ate =@Ecoate% P+CA NPs S N Nanocrystals Colloi%al carriers S N P GA

Pat&a$ et al*

Re!erences A;:B A9:B A;;B A9=B A;<B A4=B A9=B A;=B A;>B A<@B A<4B A<5B A<8B A<9B A<:B A<;B A95B A<<B A<=B

A##reviations6 HPMCP) &y%ro"ypropyl met&yl cellulose p&t&alateM NC) nanocapsulesM NPs) nanoparticlesM P+CAM poly#utylcyno acrylateM P GA) polyElacticEcoEglycolic aci%M S N) soli%Hlipi% nanoparticles*

+io%egra%a#le nanoparticulate %elivery !or protein must satis!y several tec&E nical reCuirements* Among t&ese6 t&e proteins s&oul% #e encapsulate% (it& a &ig& loa%ing e!!iciency) an% remain sta#le t&roug&out t&e manu!acturing process an% t&e course o! t&eir inten%e% %osing perio%* NPs nee% to #e less t&an 45: _m in %iameter an% !orm a !ree !lo(ing po(%er so t&at t&ey can #e resuspen%e% in an in.ecta#le ve&icle an% passe% t&roug& a nee%le* T&e release pro!ile o! t&e %rug nee%s to #e

TA+ E 5 Nanoparticulate Drug Delivery Systems6 -ormulation Applications A%%ressing t&e %rugE%elivery pro#lems Solving t&e issues relate% to solu#ility ?vercoming t&e poor #ioavaila#ility o! t&e %rugs Issues (it& !e%1!aste% varia#ility P&armaco$inetic varia#ility -in%ing solutions (it& nanoparticulate %rugs Tec&nology a%vances Re%uction in particle si/e o! t&e poorly (aterEsolu#le %rugs Increase% active agent sur!ace area +ene!its !or !aster %issolution Greater #ioavaila#ility Smaller %rug %oses Diminis&e% to"icity Decrease% %osing varia#ility P&armaco%ynamic !actors6 applica#le to pepti%es an% ot&er %rugs T&ese can #e !ormulate% as receptorEspeci!ic T&ese can #e more resistant to unspeci!ic %egra%ation T&ey can %eliver t&e %rug in encapsulate% !orm to %elay t&e %egra%ation) set a %epot !orm !or prolonge% signaling) an% increase t&e treatment e!!icacy as compare% to su#stitution o! t&e natural !orm o! pepti%e

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

4>4

repro%uci#le an% t&erapeutically e!!ective an% pose no p&armacological or to"icological ris$s %ue to rapi% early release or #urst* Macromolecules suc& as proteins an% DNA play an increasingly important role in our arsenal o! t&erapeutic agents* Delivery o! t&ese molecules to t&eir site o! action at t&e %esire% rate is a c&allenge #ecause t&eir transport t&roug& comE partmental #arriers) !or e"ample) en%ot&elium an% epit&elium in t&e #o%y) is ine!!icient an%1or t&ey are rea%ily meta#oli/e%* -or controlle% release or siteE speci!ic %elivery o! suc& macromolecules) %elivery systems are reCuire% (&ic& nee% to #e more sop&isticate% t&an our present %ay strategies* T&ese systems must #e customEma%e) ta$ing into account #ot& molecular si/e an% speci!ic c&aracteristics o! t&ese molecules* ?ne &as to #uil% a plat!orm o! %i!!erent %elivE ery strategies t&at use input !rom tec&nical) p&armaceutical) an% #iome%ical %isciplines to meet t&ese c&allenges* T&e %evelopment o! appropriate DDSs !or ne( macromolecules coming out o! t&e #iotec& in%ustry is a meaning!ul c&allenge to p&armaceutical scientists* Proteins) pepti%es) oligonucleoti%es) an% genes are very unsta#le compoun%s t&at nee% to #e protecte% !rom %egra%ation in t&e #iological environment* T&eir e!!icacy is &ig&ly limite% #y t&eir ina#ility to cross #iological #arriers an% to reac& t&e target sites* T&ey are vulnera#le to &ars& con%itions in t&e gastrointestinal tract) lea%ing to c&emical an% en/ymatic %egra%ation* T&e !uture o! t&ese molecules solely %epen%s on t&e %elivery systems an% appropriate carriers* T&ere are t&ree possi#le pat&(ays !or protein an% pepti%e %rug a#sorption t&roug& t&e GI tract* T&e !irst is via t&e MEcells o! PayerPs patc&es) t&e secon% via a transcellular route involving enterocytes) an% t&e t&ir% via paracellular avenues t&roug& tig&t .unctions A;)<>B* T&e nanosystems are provi%ing a via#le alternative !or t&ese %rugs suc& as liposomes) polymeric micelles) an% NPDDSs* ?ne o! t&e crucial an% pervasive trou#les in &uman t&erapy is to ac&ieve a #alance #et(een to"icity an% t&erapeutic e!!ect o! t&e %rugs* T&ere!ore) t&e siteEspeci!ic %elivery coul% re%uce suc& si%e e!!ects at nontarget sites an% increase t&e e!!icacy* Ro%rigues et al* A=@B &ave reporte% an interesting (or$ on lectin nanocarrier con.ugate* T&ey use% %e"tran1polyAeEcaprolactoneB polyester polymers an% con.ugate% (it& t&ree %i!!erE ent proteins) lectins !rom leaves o! +au&inia monan%ra an% ens culinaris) an% #ovine serum al#umin A+SAB* T&e NPs &aving a si/e aroun% 5@@ nm coul% #e use% !or %elivering proteins A=@B* A polypepti%e &ormone consisting o! 85 amino aci%s plays a crucial role in #ot& #one remo%eling an% calcium &omeostasis* 3oo an% Par$ A=4B !ormulate% salmon calcitonin AsCTB into #io%egra%a#le P GA NPs using sCT oleate comple"es* T&e sCT oleate comple"es (ere prepare% #y &y%rop&o#ic ion pairing* SCT NPs (ere rea%ily ta$en up #y CacoE5 cells an% sCT (as transporte% across t&e CacoE5 monolayer in vitro* In vivo e"periments s&o(e% sCT (as orally a#sor#e%* 2ranc$" et al* A=5B also reporte% similar results (&ere t&ey use% an NC !ormulation (it& &y%rop&ilic core !or %elivering salmon calcitonin in rats* A stu%y #y Alp&an%ary et al* A=8B &as s&o(n t&e crossing o! insulin t&roug& t&e intestinal epit&elial #arrier to t&e #loo% compartment (&ere it (as a#sor#e% #y porE tions o! t&e MEcellE!ree epit&elium* T&e insulin (as incorporate% in #io%egra%a#le poly Aal$ylcynoacrylateB NCs A=8B* An e"cellent revie( %iscusses t&e strategies o! en&ancing t&e immunostimulaE tory e!!ects o! CpG oligonucleoti%es an% outlines t&e latest %evelopment in t&e application o! liposomes an% NPDDSs !or t&e %elivery o! oligonucleoti%es (it& an e"tensive literature survey A=9B* eac& et al* A=:B %emonstrate% t&at e"cipientE!ree

4>5

Pat&a$ et al*

protein NPs prepare% #y sprayE!ree/ing into liCui% tec&nology A=;B can #e %isperse% into P GA an% P A microparticles an% t&e #urst e!!ect can #e prevente%* T&e uni!orm encapsulation o! t&e sta#le proteins at &ig& loa%ing (as ac&ieve% (it& minimal #urst e!!ect* NPs #ase% on &y%rogels are #eing %evelope% !or t&e %elivery o! macromolecules* An interesting mec&anistic stu%y reporte% #y Mo an% im A=<B e"&i#ite% upta$e o! (&eat germ agglutininEcon.ugate% NPs #y A:9> cells* In t&is stu%y) t&ey prepare% t&e P GA NPs #y solvent %i!!usion met&o% A==B an% later sur!ace mo%i!ie% (it& (&eat germ agglutinin t&roug& a t(oEstep car#o%iimi%e met&o%* Cellular upta$e (as stu%ie% using con!luent A:9> cells as an in vitro mo%el o! t&e type II alveolar epit&elial cells* Upta$e o! t&e ,GAEcon.ugate% P GA NPs (as compare% to t&at o! NPs similarly mo%i!ie% (it& t&e #ovine serum al#umin A+SAB to %emonstrate t&e speci!icity o! t&e sur!ace ,GA in en&ancing t&e cellular upta$e o! t&e NPs* T&e mec&anism o! upta$e (as stu%ie% #y per!orming t&e upta$e e"periments un%er several in&i#iting con%itions* T&ey conclu%e% t&at t&e gra!ting o! ,GA on P GA NPs &as increase% t&e upta$e #y !ive to eig&t times) &ence t&is met&o% can #e e"ploite% !or t&e intracellular %elivery o! t&erapeutic an% %iagnostic agents A=<B* Targete% %elivery o! proteins an% DNA reCuires a carrier system in su#miE cron si/e or nanosi/e* T&is carrier nee%s to #e target site Acell or tissueBEspeci!ic* ?!ten) t&e actual target site location is intracellular) an% t&e %elivery o! t&e carrier payloa% at t&is intracellular target site is a prereCuisite !or t&erapeutic success* -or e"ample) plasmi% DNA nee%s to #e %elivere% insi%e t&e nucleus o! t&e target cell #e!ore t&e cell can e"press t&e %esire% t&erapeutic protein* A very goo% e"ample o! t&is system is t&e immunoliposomes) (&ere liposomes carrying t&e %rug (it& monoclonal anti#o%ies or monoclonal anti#o%y !ragments are covalently attac&e% to t&e #ilayer !or targeting purposes* A selection o! monoclonal anti#o%ies) (it& in%uce% en%ocytic upta$e) can lea% to t&e entrance o! immunoliposomes A-ig* 5B into tumor cells A=9)=>B* Anot&er application o! liposomeE%epen%ent %rug is %ip&t&eria to"in A ADTAB c&ain* iposome %epen%ent means t&at t&e %rug as suc& cannot reac& its target site o! action insi%e t&e cell (it&out a carrier) as it cannot pass t&e cytoplasmic mem#rane

-IGURE 5 Immunoliposomes6 t&e use o! nanotec&nology to #uil% a carrier system !or siteEspeci!ic %elivery o! a protein* T&e antiEEG-R anti#o%y permits en%ocytosis &elping t&e immunoliposomes to enter t&e target cells a!ter cell #in%ing* T&e pepti%e %i IN-E< in%uces t&e release o! liposomeE entrappe% %ip&t&eria to"in A c&ain !rom immunoliposomes* A##reviation6 EG-R) epi%ermal gro(t& !actor receptor* Source6 -rom Re!* =>*

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

4>8

(it&out &elp Aa carrierB* Suc& a %rug (ill s&o( neit&er t&e %esire% nor t&e un%eE sire% p&armacological e!!ects* Dip&t&eria to"in) a protein consisting o! an A c&ain couple% (it& a + c&ain) can rea%ily enter cells t&roug& t&e transporter + c&ain* Upon entering t&e cell) t&e A c&ain causes t&e cell $ill (it& e"ceptional e!!iciency #y #loc$ing ri#osomal activity* T&us DTA Alac$ing + c&ainB alone nee%s a cellE speci!ic transport system) t&at is) a system t&at transports it into t&e %esire% target cells) !or e"ample) tumor cells A>@B* T&e %esign o! a customEma%e carrier at t&e nanometer level is a targete% %elivery system !or plasmi% DNA to e!!iciently trans!er only to target cells* T&e catE ionic polymer pDMAEMA ApolyEA5BEA%imet&ylaminoBet&yl met&acrylateB con%enses plasmi% DNA e!!ectively into 4@@ nm NPs Apolyple"esB* In vitro trans!ection is very e!!icient #ut in vivo (as ine!!ective* Polyple"es (ere su#seCuently coate% (it& lipi%s yiel%ing lipopolyple"es) (&ic& %emonstrate% target cellEspeci!ic #in%ing c&aracterisE tics) an% (ere a#le to trans!ect t&e cells (it& &ar%ly any cell to"icity A>4B*

?cular Applications o! Nanoparticulate DrugEDelivery System Topical op&t&almic %rugs &ave generally poor a#sorption in t&e eye %ue to t&e corE neaPs lo( permea#ility to %rugs an% noncorneal !actors suc& as rapi% tear turnover) nasolacrimal %rainage) an% systemic a#sorption* ?ne o! t&e ma.or pro#lems in ocular %elivery is provi%ing an% maintaining an a%eCuate concentration o! t&e t&erE apeutic agent in t&e precorneal area* Topical %rop a%ministration o! op&t&almic %rugs in aCueous solutions results in e"tensive %rug loss %ue to tear !lui% an% eyeli% %ynamics A54)>5)>8B* Most noninvasive approac&es !or en&ancing ocular %rug a#sorption involve t&e use o! pro%rugs) t&e use o! viscosity agents %esigne% to proE long t&e %rug resi%ence time) an% colloi%al systems A>9)>:B* Polymeric NPs are attractive colloi%al systems #ecause t&ey %emonstrate increase% sta#ility an% &ave a longer elimination &al!Eli!e in tear !lui% Aup to 5@ minB) t&an %o conventional %rugs applie% topically to t&e eye) (&ic& &ave &al!Elives o! .ust one to t&ree minutes* NPDDSs &ave #een evaluate% !or ocular applications to en&ance a#sorption o! t&eraEpeutic %rugs) improve #ioavaila#ility) re%uce systemic si%e e!!ects) an% sustain intraocular %rug levels A>;B* NPDDSs &ave s&o(n potential in t&e treatment o! e"ternal eye %iseases A><B* P GA &as #een evaluate% an% prove% to #e a very use!ul #io%egra%a#le polymer !or NPDDS !ormulation %ue to its me%ical use) #iocompati#ility) an% sa!ety A>=B* 7a%%oumi et al* A>:B &ave stu%ie% t&e c&aracterisE tics an% mec&anism o! upta$e o! P GAE#ase% NPDDSs !or op&t&almic application* T&ey suggeste% t&at P GAE#ase% NPDDSs coul% #e use% !or t&e en&ancement o! %rug a#sorption in t&e eye an% t&e controlle% release o! proteins an% %rugs* Some reports in ocular applications are very novel approac&es involving periocular routes !or retinal %rug %elivery o! Celeco"i# an% Al%ose re%uctose in&i#itors A>>H4@4B* Salgueiro et al* A88B %emonstrate% op&t&almic application o! cyclop&osp&ami%eE loa%e% poly#utylcyanoacrylate AP+CAB nanosp&ere as an immunosuppressive agent* T&e morp&ometrical properties suc& as average particle si/e an% poly%isparity in%e" o! t&ese DDSs are a%eCuate !or op&t&almic application (it&out in%uce% corneal or con.unctival irritation A88B*

Nanoparticulate DrugEDelivery Systems !or Pulmonary Treatment Pulmonary %rug %elivery !or #ot& systemic an% local treatments &as many a%vE antages over ot&er %elivery routes #ecause t&e lungs &ave a large sur!ace area A98H4@5 m5B) t&in a#sorption #arrier) an% lo( en/ymatic activity* In a%%ition) t&e

4>9

Pat&a$ et al*

alveoli o! t&e lungs &ave a slo(er mucociliary clearance t&an t&e air(ays) an% t&e lung epit&elia are t&inner an% more permea#le* T&ere is a potential !or possi#le sysE temic a#sorption o! t&e pepti%es an% proteins t&roug& t&e alveolar region o! t&e lungs* Several stu%ies &ave e"&i#ite% t&e a#sorption o! &ig&EmolecularE(eig&t %rugs suc& as insulin) &eparin) an% GCS- Arecom#inant &uman granulocyte colonyE stimulating !actorB t&roug& pulmonary DDSs A4@5H4@9)4@9aB* As t&ese pepti%es &ave a s&ort li!e) t&e %evelopment o! %elivery systems (it& sustaine% p&armacological action (oul% #e very use!ul* Innovative) noninvasive) in&ala#le DDSs !or pepti%es are #eing e"plore% !or lung %isease t&erapy (it& t&e vasoactive intestinal pepti%e A2IPB #eing use% in t&e treatment o! severe lung %iseases* ?(ing to its $no(n antiin!lammatory an% vaso%iE lative properties) it &as #een %emonstrate% to possess a &ig& t&erapeutic potential !or ot&er lung %iseases) (&ic& are common in in%ustriali/e% countries* 2IP un!ortuE nately reveals a variety o! #i!unctions me%iate% #y at least t(o %i!!erent receptors on t&e cell sur!ace* T&e (ays t&oug&t to %eal (it& t&is situation are to %evelop a receptorE speci!ic system to ma$e it more speci!ic in !unction* T&ese nee% to #e protecte% #y licensing) an% allo( !or superior treatment compare% to natural 2IP* It can #e ac&ieve% #y %esigning) engineering) an% pro%uction o! analog pepti%es) (&ic& (ill #e very use!ul* +y mo%i!ying t&e pepti%e) 2IP in t&e amino aci% seCuence #ecome more receptorEselective an% more resistant to unspeci!ic %egra%ation* It can %eliver t&e ne( pepti%es in nanoencapsulate% !orm ANPDDSB to %elay t&e %egra%ation) an% to set in %epot !orm !or prolonge% signaling) increasing t&e treatment e!!icacy as compare% to su#stitution o! t&e natural !orm o! t&e pepti%e* An enormous %iversity o! t&erapeutic agents is currently a%ministere% to t&e patients via aerosol in&alation) an% t&e num#er o! potential %rug can%i%ates !or pulmonary application increase %aily* T&e ma.or areas o! researc& an% t&erapeutic applications are ast&ma A4@:B) cystic !i#rosis A4@;B) lung cancer A4@<B) tu#erculosis A4@=H4@>B) pulmonary &ypertension A44@B) an% %ia#etes A444B* Nanostructure% %rug %elivery an% targeting systems are tools to overcome t&e limitations o! lung %elivE ery #y sta#ili/ing an% protecting t&e release in t&e #ronc&i an% ma$e lung t&erapy t&roug& in&alation possi#le an% e!!ective* Some o! t&e polymers suc& as P GA) protamine) t&iomer) an% lipi%E#ase% particles can #e loa%e% #y 2IP or ne( %esigne% analogs* T&e parameters) (&ic& nee% to #e teste%) (ill #e improve% #y in vitro en/ymatic sta#ility) in vitro longEterm %rug release) an% retar%ing properties an% #ioavaila#ility o! t&e carrier* InsulinEloa%e% P+CA NPs (ere stu%ie% #y V&ang et al* A445BM t&ey %emonE strate% t&at t&e pulmonary a%ministration o! t&ese NPs coul% signi!icantly prolong t&e &ypoglycemic e!!ect o! insulin* It (as reporte% t&at t&e #ioavaila#ility o! insulin NPs (as relatively &ig&er t&an t&at o! solution (&en a%ministere% #y pulmonary route to normal rats) #ut (&en NPs (ere a%ministere% su#cutaneously t&e #ioavailE a#ility (as comparatively lo(er compare% to solution a%ministere% t&e same (ay A445B* Anot&er stu%y using P GA NPs to %eliver insulin #y ne#uli/ation also s&o(e% t&e use!ulness o! NPDDSs !or insulin A448B* An interesting stu%y (as reporte% #y iu et al* A449)44:B incorporating estra%iol an% colloi%al gol% NPs in P GA NPs to #e use% as a mo%el !or t&e pulmonary DDSs* T&ey propose% t&at large porous NPs can #e use% as %elivery systems !or t&e pulmonary tract* Several issues complicating t&e %evelopment o! aerosol !ormulation inclu%e6 compoun% loss %uring in&alation) %osing %i!!iculties) en/ymatic %egra%ation (it&in t&e lungs) an% t&e &ig& cost o! pro%uction* NanoparticulateEcontrolle% release DDS &as t&e potential to overcome many o! t&ese pro#lems* Suc& !ormulation may #e incorporate% in aerosol !orm) remaining sta#le against !orces o! %egra%ation %uring

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

4>:

aerosoli/ation* It can target a speci!ic site or cell population in t&e lung) protect t&e %rugEaggressive elements in t&e pulmonary tract) an% release t&e compoun% in a pre%etermine% manner concurrently* It can #e inert to t&e surroun%ing tissues an% contains no irritant or to"ic a%%itives an% %egra%es (&en applica#le (it&in an accepta#le perio% o! time) pro%ucing no to"ic #ypro%ucts A44;B* Polymeric nanoparticulate systems s&o( promise in !ul!illing t&e stringent reCuirements o! t&e pulmonary DDSs* An interesting stu%y is reporte% #y Dailey et al* A44;B using s&ortEc&ain P GA gra!te% onto an amineEsu#stitute% polyAvinyl alco&olB #ac$#one A8E%iet&yl aminoE4Epropylamine A=[BHpolyAvinyl alco&olBEgra!te% polyAlacti%eEcoEglycoli%eB ADEAPAHP2A EgEP GAB polymer* T&is polymer &as amp&ip&ilic properties an% is &ig&ly suite% !or t&e pulmonary %elivery system* It (as also reporte% t&at #y a%%ing varying amounts o! polyanion suc& as car#o"ymet&yl cellulose) %e"tran sul!ate) or even DNA to t&e polymer ring %uring NPs !ormation) NPs o! varia#le p&ysicoc&emical properties coul% #e generate% ena#le* T&is can &ig& loa%ing o! various %rugs along (it& greater sta#ility* Ho(ever) t&ese polymer %erivatives (ere !oun% to %egra%e !rom 59 &ours to (it&in a (ee$* Some relate% stu%ies &ave %escri#e% t&ese aspects in %etail A44<)449B* NPs may #e very e!!ective DDSs !or various pulmonary t&erapeutic sc&emes* T&e stu%y #y Dailey et al* A44=B investigate% t&e e!!ect o! ne#uli/ation tec&nology an% NP c&aracteristics on t&e !eatures o! aerosol generation* T&ey conclu%e% t&at #io%eE gra%a#le NPs containe% in t&e suspensions %i% not a!!ect t&e aerosol %roplet si/e in a clinically relevant mannerM &o(ever) #ot& NP c&aracteristics an% t&e tec&niCue o! aerosoli/ation in!luence NP aggregation occurring %uring t&e aerosoli/ation A44=B* 2ila et al* A44>B &ave s&o(n t&at polyet&ylene glycol APEGB coating o! t&e P A NPs increase% t&e a#sorption o! %rug in nasal mucosa* Pan%ey et al* A9>B %emonstrate% t&e application o! NPDDSs !or t&e treatment o! e"perimental tu#erculosis using polyA%)lElacti%eEcoEglycoli%eB as a polymer* T&ey use% an in&ala#le system using t&e NPs an% t&ree antiET+ %rugs Ri!ampicin) Isonia/i%) an% Pyra/inami%e A9>B*

Nanoparticulate DrugEDelivery Systems !or Central Nervous System T&e entry o! a %rug molecule into t&e #rain is limite% #y one o! t&e most c&allenging #arriers) t&e #loo%H#rain #arrier A+++B* T&e +++ consists o! a continuous layer o! en%ot&elial cells .oine% toget&er #y tig&t .unctions AVonulae occlu%ensB) (&ic& severely restrict paracellular transport across t&e #arrier* T&e +++ allo(s passive %i!!usion o! small lipi%Esolu#le molecules) (&ereas &y%rop&ilic su#stances or molecules (it& &ig& molecular (eig&t &ave minimal passive permeation* T&e mec&anism o! permea#ility regulation inclu%es macrovascular en%ot&elial tig&t .unctions) en/ymatic regulation) an% active #rain e!!lu"* Transport across +++ is a%%itionally regulate% #y a num#er o! transporters inclu%ing very e!!ective e!!lu" transporters suc& as multi%rug resistanceEassociate% protein or pEglycoprotein* Several strategies &ave #een trie% to cross t&e +++M one alternative strategy is to use %rugEcarrier systems suc& as liposomes) anti#o%ies) an% NPs A45@B* Numerous stu%ies &ave s&o(n t&e applications o! NPs !or #rain targeting A:4):5)454)455B* He"apepti%e Dalargin) a eu encep&alin analogue (it& no +++ permea#ility a%sor#e% to t&e sur!ace o! P+CA NPs) cause% central analgesia a!ter I2 a%ministraE tion A458B* ?t&er %rugs Tu#ocurarine A<5B) Do"oru#icin A459B) 0ytorp&in A:4B) an% operami%e A:5B (ere also use% as mo%el %rugs !or t&ese purposes* +rain upta$e o! NPs in t&ese stu%ies (as suggeste% #ase% on t&e !act t&at t&e %rugs a%sor#e% to P+CA NPs cause% a resultant p&armacologic e!!ect in t&e CNS A:4)<5)458B* +rain

4>;

Pat&a$ et al*

%istri#ution o! %rugs %elivere% on t&e sur!ace o! NPs (as also con!irme% #y CuantiE !ication o! t&e %rug in t&e #rain tissue itsel! A459B* Stu%ies &ave also s&o(n t&e intact presence o! NPs in #rain cells in vivo A45:B* 0o/iara et al* A45@B trie% to Cuanti!y t&e presence o! t&e NPs in #rain in situ an% stu%ie% t&e impact on +++ parameters A45;B* Anot&er stu%y !rom t&e same la#oratories s&o(e% t&e e!!ectiveness o! using microE emulsions as precursors to engineer NPs* T&e a%vantages (it& t&e microemulsions (ere simplistic pro%uction o! t&e NPs appro"imately 4@@ nm in %iameter) possi#le incorporation o! &y%rop&o#ic %rugs in oil %roplets) an% inclusion o! siteEspeci!ic ligan%s* T&ey also reporte% t&e $inetic mo%eling o! #rain upta$e) an% t&eir %ata suggeste% t&e pro#a#le mec&anism o! #rain entry A<@B*

Nanoparticulate DrugEDelivery Systems !or En/ymes Many scientists attempte% application o! nanocarriers !or %elivery o! t&erapeutic en/ymes A45<)45=B* A catalaseE%elivery system seems to #e an i%eal testing mo%el !or evaluating t&e $ey aspects o! en/yme %elivery #y nanocarriers* T&ere is a !ascinatE ing stu%y %escri#ing t&e loa%ing an% protection o! an active en/yme catalase into a polyEnanocarrier compose% o! %i#loc$ PEGHP GA copolymers A45<B* It s&o(e% nanocarriers in t&e si/e range o! 5@@ to :@@ nm protecting at least 5:[ o! t&e loa%e% proteases* -igure 8 %emonstrates t&e met&o% o! !ormulation o! P GAHP A NPs

-IGURE 8 -ormulation o! P GAHPEG nanoparticles6 AAB sc&eme (it& t&e %ou#le emulsion synE t&esis proce%ure an% A+B #y sonication in DCM1acetone mi"ture* ACB T&e loa%ing e!!iciency (as %etermine% #y tracing t&e amount o! ra%iola#elle% catalase containe% insi%e eit&er t&e micro particles Ai*e*) pellet o#taine% a!ter centri!uging !or 4: minutes at 4@@@ W gB or nanoparticles !ractions A5n% pellet o#taine% #y centri!uging !or 8@ minutes at 55)@@@ W gB* oa%ing e!!iciency (as greatly en&ance% Aeig&t!ol%B (&en a !ree/e t&a( cycle Agrey #arB (as inclu%e% in t&e primary emulsion step* T&e %ata in t&is !igure is presente% as M e S*E*M* An ^ 8B* Source6 -rom Re!* 45<*

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

4><

carrying t&e en/ymes A45<B* Nanocapsulation &elps to lengt&en t&e t&erapeutic (in%o( #y %esigning #io%egra%a#le polymeric nanocarriers) (&ic& protect encapE sulate% catalase !rom lysosomal proteolysis) t&us prolonging t&e %uration o! t&e %esire% e!!ects* T&ey &ypot&esi/e% t&at t&e polyEnanocarriers !ormation) si/ing) an% loa%ing s&oul% a potential #asis !or a more general !rame(or$ !or t&e !ormulation o! NPDDSs) especially !or en/ymes* Many en/ymes using small su#strates %i!!usE ing t&roug& polymer s&ells suc& as sugars) amino aci%s) an% glutat&ione may #e amena#le to loa%ing into protecting polyEnanocarriers* T&e stu%y recommen%e% testing o! t&e %elivery ve&icles in cell culture an% animal stu%ies) as a ne( strategy !or a prolonge% protection against vascular o"i%ative stress A45<B* ,eissen#oc$ et al* A45>B s&o(e% t&e application o! (&eat germ agglutinin to en&ance t&e a#sorption o! P GA NPs* T&e (&eat germ agglutinin &as cytoEa%&esive an% cytoEinvasive properties* T&is process o! sur!ace engineering t&e P GA NPs #y (&eat germ agglutinin promises &ig& versatility o! application in t&e searc& !or #iorecognitive ligan%s en&ancing t&e cytoEa%&esion) cytoEinvasion) an% pro#a#ly transcellular transport o! colloi%al carriers a!ter peroral a%ministration A45>B* Gre! et al* A48@B an% oc&ner et al* A484B reporte% novel sur!ace engineering o! t&e NPDDS*

Proticles6 Protamine Nanoparticles as DrugEDelivery Systems Proticles are novel NPs compose% o! protamine) a pepti%e use% in many p&armaE ceutical !ormulations (it& DNA) proteins suc& as al#umin an% ot&er t&erapeutiE cally active su#stances* 'ung&ans et al* A485B reporte% t&e use o! proticles as %elivery systems !or oligonucleoti%es* T&e sta#ility o! t&e particles an% t&e oligonucleoti%es #oun% to t&e proticles (as e"amine% in !etal cal! serum an% cell culture me%ium* Proticles signi!icantly %ecrease% cellular gro(t& in a cell proli!eration assay using oligonucleoti%es against t&e cEmyc protoEoncogene* Proticles can also #e use% !or %iagnostics* NPs can #in% large amounts o! su#stances on t&eir sur!ace #y a%sorpE tion) an% so t&ey can #e use% as an a#sor#er* Proticles (it& ligan%Especi!ic as amyloi%E#eta can #in% t&e neuroto"ic amyloi%E#eta protein* T&e neuroEprotective e!!ects o! t&is %elivery system may !in% a novel t&erapy !or Al/&eimerPs %isease* Amyloi%E#in%ing pepti%e %epot system is aime% at %eveloping t&erapy !or Al/&eimerPs %isease* Amyloi%E#in%ing pepti%es are su#stances (&ic& can %isinteE grate amyloi% plaCues* T&ese are &i%%en in t&e interior o! proticles to cross t&e +++ an% !or slo( controlle% release to ac&ieve a &ig& concentration o! amyloi%E#in%ing pepti%es in t&e #rain over a long perio%* T&is can #e !urt&er stu%ie% using t&e MRI (it& ga%olinium as a contrast su#stance* 2IPs) (&ic& are use% in t&e treatment o! severe lung %iseases) can #e pac$age% in proticles an% can #e use% to create a pulmonary %epot !or t&e treatment !or 45 to 59 &ours* Antigens are #oun% to t&e sur!ace o! proticles !or e!!icient composition o! t&e proticles t&at mig&t !urt&er #oost t&e immune response* Some recent reports s&o( t&e application o! proticles as DDSs A488H48:B*

Mucoa%&esive Nanoparticulate DrugEDelivery Systems Mucosal sur!aces are t&e most common an% convenient routes !or %elivering %rugs to t&e #o%y* Ho(ever) macromolecular %rugs suc& as pepti%es an% proteins are una#le to overcome t&e mucosal #arrier an% are %egra%e% #e!ore reac&ing t&e #loo%stream* NPDDSs s&o( a promising strategy !or %elivering %rugs t&roug& mucosa* Polysacc&ari%e C&itosan ACSB is mucoa%&esive an% CS NPs) CSEcoate% oil nano%roplets AnanocapsulesB) an% CSEcoate% lipi% NPs &ave s&o(n interesting

4>=

Pat&a$ et al*

possi#ilities !or t&is purpose A48;B* CSEcoate% systems &ave e"&i#ite% an important capacity to en&ance t&e intestinal a#sorption o! t&e pepti%e salmon calcitonin an% in vivo) a longElasting %ecrease in t&e calcemia levels o#serve% in rats A48<B* Ta$euc&i et al* A48=B &ave (ritten a revie( on mucoa%&esive NPDDSs !or pepti%e %rugs* T&ey %iscusse% t&e preparation an% met&o%s !or evaluation o! mucoE a%&esive nanoparticulate systems* Mucoa%&esive properties (ere con!erre% on t&e systems #y coating (it& mucoa%&esive polymers suc& as CS an% Car#apol* T&e !easiE #ility o! t&is sur!ace a%&esion (as con!irme% #y measuring t&e /eta potential* T&ey suggeste% t&at t&ese %elivery systems coul% #e use% !or %elivery o! pepti%es #y oral an% pulmonary a%ministration* Nanoprecipitation tec&niCues using P GA an% P A polymers (ere !oun% to #e use!ul !or nanoparticulate %elivery o! proteins an% &ave s&o(n more versatility an% !le"i#ility in t&e !ormulation !or protein %elivery A48>B* Ta$euc&i et al* A49@B %escri#e% mucoa%&esive P GA nanosp&eres prepare% #y sur!ace mo%i!ication (it& CS !or oral pepti%e %elivery* CSEmo%i!ie% nanosp&eres (ere applie% to improve t&e pulmonary %elivery o! pepti%es #y ne#uli/ation* T&e particle average %iameter o! ;:@ nm o! t&e aCueous %ispersion o! t&e nanosp&eres (as an important !actor to enclose t&e particles in t&e aerosoli/e% aCueous %roplets pro%uce% (it& t&e ne#uli/er* T&e elimination rate o! CSEmo%i!ie% nanosp&eres !rom t&e lungs (as %ecrease% signi!icantly %ue to t&eir mucoa%&esive property a!ter pulmonary a%ministration compare% to t&at o! t&e unmo%i!ie% nanosp&eres) an% as a result t&e p&armacological action (as signi!icantly prolonge%* It is also con!irme% t&at t&e CS on t&e sur!ace o! t&e nanosp&eres en&ance% t&e a#sorption #y opening t&e intercellular tig&t .unctions in t&e lung epit&elium A4@9B*

Nanoparticulate Drug Delivery in Cancer Treatment Several NPDDSs are reporte% !or t&e application in cancer t&erapy) trans!erring conE .ugate% paclita"elEloa%e% NPs A494B) nanovaccines A495B) a%riamycinEloa%e% NPs !or &epatoma treatment A498B) magnetic P+CA nanosp&eres (it& aclacinomycin A in gastric cancer A4:B) nearEin!rare% a#sorption nanosp&eres A499B) polypropylenimine %en%rimer NPs !or oligonucleoti%es A49:B) lyticEpepti%eE#oun% magnetite NPs !or #reast cancer treatment A49;B) ceramicE#ase% NPs entrapping (aterEinsolu#le p&otoE sensiti/ing anticancer %rugs A49<B) an% polyAepsilonEcaprolactoneB NPs !or t&e %elivery o! Tamo"i!en !or #reast cancer treatment A49=)49>B* 3oo an% Par$ A4:@B &ave reporte% a stu%y o! !olate receptor targete% anticanE cer t&erapy using Do"oru#icinEPEG !olate nanocon.ugates* Do"oru#icin an% !olate (ere) respectively) con.ugate% to alp&a an% omega terminal o! t&e PEG c&ain* T&e con.ugates assiste% !ormation o! Do"oru#icin nanoaggregates (it& average si/e o! 5@@ nm in %iameter (&en com#ine% (it& an e"cess amount o! %epronate% Do"oru#icin in an aCueous p&ase* In vivo stu%ies &ave s&o(n signi!icant re%uction in t&e tumor volume in a &uman tumor "enogra!t nu%e mouse mo%el* Controlle% release o! paclita"el t&roug& su#microemulsion (it& particle si/e o! 9: to 5<@ nm (as evaluate% in vitro an% in vivo !or t&eir antitumor activity #y 0ang et al* A4:4B* T&ey use% P GA polymer !or !ormulating sel!Eemulsi!ying DDSs an% s&o(e% t&e e!!ectiveness o! t&e system*

Nanoparticles in t&e Treatment o! 2ascular T&rom#osis T&e !ormation o! #loo% clots in t&e circulatory system is associate% (it& a range o! serious me%ical con%itions) inclu%ing &eart attac$s) pulmonary em#olisms) stro$es) an% %eep vein t&rom#osis* T&e main component o! t&e clot is t&e insolu#le protein

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

4>>

!i#rin* Treatment o! vascular t&rom#osis involves t&e use o! t&rom#olytic %rugs t&at #rea$ up t&e !i#rin) allo(ing t&e clot to %isperse* +iocompati#le NPs are use% to %evelop suc& %elivery systems) (&ic& can carry t&e t&rom#olytic %rugs* C&ellini A4:5B e"plaine% t&at t&e t&rom#olytic %rugs are po(er!ul agents) (it& serious si%e e!!ects li$e causing &emorr&age i! t&ey are given systemically* Ho(ever) orally t&ey are less e!!icient* I! t&ey can #e incorporate% in NPs) t&ey can #e %elivere% %irectly to t&e speci!ic site) using less %rug materials) an% t&e treatment (ill #e costEe!!ective (it& less si%e e!!ects* T&e %rug (ill #e release% !rom t&e NPs #y %i!!usion) %egra%aE tion) or erosion* A sustaine% release NP !ormulation may #e more &elp!ul A4:5B*

Nanoparticulate DrugEDelivery Systems !or Gene T&erapy Intracellular gene %elivery involves c&anging t&e e"pression o! genes in or%er to prevent) cure) or treat a %isor%er or a %isease* T&ere!ore) t&is treatment met&o% alters t&e e"pression o! a gene an% corrects a %e!ective gene t&at may #e t&e cause o! a %isease or a %isor%er* Nonviral vectors !or gene t&erapy &ave in&erent a%vantages o! sa!ety an% !le"i#ility over viral vectors) alt&oug& t&ey are less e!!icient* Serious issues o! integration (it& t&e &ost genome to permanently alter its genetic structure) sel!Ereplication capa#ility) recom#ination potential) an% t&e possi#ility o! compleE ment activation AimmunogenicityB o! t&e ot&er(ise trans!ection e!!icient viral vectors limit t&eir use !or gene %elivery* In t&e last %eca%e) t&e !ocus o! %evelopment is on nonviral geneE%elivery systems* Speci!ic c&aracteristics t&at must #e inclu%e% in nonviral vectors inclu%e small si/e an% sta#ility against aggregation in #loo%) serum) an% e"tracellular !lui%) t&e a#ility to #e e!!iciently internali/e% #y t&e target cells an% t&e a#ility to %isassem#le an% release t&e payloa% into t&e cell nucleus once internali/e%* Gene t&erapy &as attracte% consi%era#le interest !or t&e treatment o! li!eE t&reatening %iseases* Several viral an% nonviral vectors Atransporting %evicesB are un%er investigation A4:8)4:9B* ?ne o! t&e common %isa%vantages o! #ot& types is rapi% clearance !rom t&e #loo% circulation to !irstEpass organs suc& as liver an% lungs* A !reCuently applie% strategy to circumvent t&is rapi% elimination is to coat t&e outer sur!ace o! t&e comple"es (it& &y%rop&ilic unc&arge% polymers #y (&ic& t&e positive c&arge o! t&ese lipo1polyple"es is s&iel%e% A4::H4:<B* Several polymers suc& as polyElElysine) CS A4:=B polyami%oamine %en%rimers A4:>B) polyet&ylenimine APEIB A4;@B) polyA9 amino#utylElEglycolic aci%B A4;4B) polyElEglutamic aci% A4;5B) polyAfEamino estersB A4;8B) polyA5A%imet&ylaminoBet&yl met&acrylateB) plasmi%E lipi% particles &ave #een use% !or t&is purpose (it& PEG coating A4;4)4;5)4;9B* -un&o!! et al* A4;:B &ave reporte% t&e use o! PEGEs&iel%e% %ou#leElayere% micelles !or gene %elivery* V&ang et al* A4;;B reporte% galactosylate% ternary DNA1polyE p&osp&orami%ate NPs as &epatocyteEtargete% gene carriers* V&ao et al* A4;<H4<@B (rote an e"cellent revie( on polyp&osp&oesters in %rug an% gene %elivery* A report %escri#e% t&e application o! P GA6polo"amer an% P GA6polo"amine #len% NPs as ne( carriers !or t&e gene %elivery6 plasmi% DNA A4<4B* T&ey %evelope% several !orE mulations an% !oun% t&at all NP !ormulations provi%e% continuous an% controlle% release o! t&e plasmi% (it& minimal #urst e!!ect* In a%%ition) t&e release rate an% %uration (ere %epen%ent on t&e composition o! t&e particle matri"* Va&r et al* A<5B &ave create% a layerE#yElayer step(ise sel!Eassem#ly o! t&e polyelectrolytes polyAallylamine &y%roc&lori%eB* PolyAstyrenesul!onateB (as use% to create a macromolecular nanos&ell aroun% %rug NPs Aappro"imately 4:@ nm in %iameterB* De"amet&asone (as c&osen as a mo%el %rug* T&e polymeric nanos&ell on t&e

5@@

Pat&a$ et al*

sur!ace o! t&e %rug NPs provi%es a template upon (&ic& sur!ace mo%i!ications can #e ma%e to create stealt& or targete% DDSs A4<5B* 0aul et al* A4<8B in t&eir stu%y use% PEGEmo%i!ie% gelatin NPs as longEcirculating intracellular %elivery systems (it& a mean particle si/e o! 8@@ nm* T&ey coul% e!!iE ciently encapsulate &y%rop&ilic macromolecules inclu%ing plasmi% DNA* T&ese particles (ere internali/e% #y tumor cells an% (ere !oun% near t&e nucleus a!ter 45 &ours* T&e PEGylate% gelatin NPs (ere also very e!!icient in e"pressing GEP* Drug loa%ing an% %rugErelease rates !rom NPs are important parameters !or t&e !ormulation o! NPDDSs to optimi/e t&e t&erapeutic e!!icacy o! t&e encapsulate% %rug A4<9H4<=B* Panyam et al* A4<=B s&o(e% t&at soli%Estate solu#ility o! t&e %rug in polymer coul% #e an important %eterminant t&at coul% in!luence t&e %rug loa%ing in NPs as (ell as release c&aracteristics o! t&e encapsulate% %rug* In a recent stu%y #y 0aul et al*) plasE mi% DNA (as !ormulate% (it& gelatin an% PEGylate% gelatin (it& average %iameter o! 5@@ nm !or targete% systemic %elivery to soli% tumors* T&e results s&o(e% ;4[ trans!ection e!!iciency) (&ic& (as attri#ute% to a #iocompati#le) #io%egra%a#le longEcirculating carrier system A4<>B* Den%rimers are emerging as a ne( generation o! nonviral vectors !or gene %elivery #ecause o! t(o %istinctive !eatures6 t&eir structures can #e controlle% an% t&eir c&emistry can #e a%apte% !or various reCuirements suc& as %rug or gene %elivE ery A4=@)4=4B* T&ey also #elong to t&e polyamine group o! nonviral vectors) (&ic& inclu%es polyElElysine) polyet&ylenimine) an% polyami%oamine* T&e versatility o! t&ese vectors &as #een e"ploite% to attac& various ligan%s suc& as trans!errin) sugars) an% anti#o%ies !or receptorEtargeting %en%rimers* A ne( !amily o! composite %enE %rimers (it& lipi%ic amino aci%s (as synt&esi/e% #y +ayle et al* an% use% !or transE porting DNA #ot& singleE an% %ou#leEstran%e% as (ell as RNA A4=5B* Polyami%oamine %en%rimer APAMAMB represents one o! t&e most e!!icient polymeric gene carriers* V&ang et al* A4=8B &ave s&o(n t&eir utility !or gene %elivery* Wia et al* A4<<B report a novel DDS recently #y preparing mono%isperse% NPs consisting o! interpenetrating polymer net(or$s AIPNsB o! polyacrylic aci% APAAcB an% isopropylacrylami%e #y a see% an% !ee% met&o%* T&e aCueous %ispersion o! IPN NPs (as !oun% to #e a uniCue NPDDS %ue to its a#rupt inverse t&ermoreversi#le gelation at aroun% 88]C* T&e IPN an% %rugs (ere t&oroug&ly mi"e% as an aCueous solution at room temperature an% !orme% a %rugE%elivery gel at #o%y temperature* T&e %rugE%elivery mo%el (as !oun% very use!ul #ecause suc& a %ispersion an% %rug (as mi"e% (it&out c&emical reaction an% t&e liCui% can #e in.ecte% into a #o%y to !orm in situ a gelle% %rug %epot to release t&e %rug slo(ly A4<<B* +enita et al* A4=9B %escri#e% t&e application o! P GAHPEI NPs !or gene %elivery to pulmonary epit&elium* Di(an et al* A4=:B &ave s&o(n t&at antigen %elivery in #io%egra%a#le NPs can !acilitate in%uction o! strong TEcell response) particularly o! t&e TH4 type) at an e"tremely lo( %ose o! CPG oligonucleoti%es* Suc& re%uction in %ose (oul% #e a%vantageous !or minimi/ing t&e potential si%e e!!ects o! t&ese novel a%.uvants A4=:B* R&aese et al* A4=;B reporte% NPs consisting o! DNA) &uman serum al#umin) an% polyet&ylenimine to #e a carrier !or t&e nonviral gene %elivery* T&ey coul% ac&ieve optimum trans!ection e!!iciency) an% %isplaye% a lo( cytoto"icity (&en teste% in cell culture* T&ey recommen%e% t&ese carriers !or %elivering DNA !or I2 a%ministration* An interesting strategy !or t&e treatment o! various vascular %iseases uses poly Amet&yli%ene malonate 5*4*5B NPs (&ic& is a #iocompati#le polymer t&at en&ances t&e periEa%ventitial a%enoviral gene %elivery A4=<B*

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

5@4

Gene t&erapy strategies &ave #een propose% !or a vast an% %iverse range o! %isor%ers !or (&ic& currently availa#le treatments are %eeme% unsatis!actory* E!!ective %elivery o! genes into cells &as #een consi%ere% a ma.or &ur%le in ac&ieving success!ul gene t&erapy* A num#er o! %elivery systems #ase% on viral A4==B or nonviE ral vectors A4=>)4>@B &ave #een %evise%M none o! t&em &ave proven to #e completely satis!actory* 2iral vectors can only intro%uce genes) not ot&er macromolecules suc& as siRNA or antisense nucleoti%e* -urt&ermore) si%e reactions suc& as &ost immune response an% insertional mutagenesis lea%ing to %eat&) carcinogenesis) or germline alternations &ave le% to serious concerns a#out t&e use o! viruses as gene trans!er vectors A4>4H4>9B* Several analytical tec&niCues (ere reporte% to #e use!ul !or t&e c&aracteri/ation an% esta#lis&ing t&e structure1!unction relations&ip o! polyami%oE amine1DNA %en%rimers as nanoparticulate %rug1geneE%elivery systems* +raun et al* A4>:B use% %ynamic an% electrop&oretic lig&t scattering tec&niCue !or particle si/e) an% p&ase analysis lig&t scattering !or /eta potential* Et&ium #romi%e %isplacement assay &as #een utili/e% !or %etermining t&e interaction #et(een t&e %en%rimer an% DNA) an% e"tent o! gene upta$e* Circular %ic&roism spectroscopy (as use% !or c&arE acteri/ing &elical structure o! DNA (it&in %en%rimer DNA comple"es* -ourier trans!orm in!rare% spectroscopy (as use% as a complimentary tec&niCue to !urt&er investigate t&e secon%ary structure o! DNA component comple"es* Isot&ermal titraE tion calorimetry (as employe% to investigate t&e t&ermo%ynamics o! #in%ing o! %en%rimers an% DNA comple"es* Di!!erential scanning calorimetry (as applie% to evaluate t&e t&ermal sta#ility o! DNA1%en%rimer comple"es A4>:B* Vaitsev et al* A>;B use% a strategy #ase% on t&e !ormation o! polyelectrolyte NPs an% later %eposition o! negatively c&arge% polyelectrolytes onto a DNA core* T&ey s&o(e% t&at t&ese negatively c&arge% particles e"&i#ite% colloi%al sta#ility an% &ig& trans!ection e!!iciency in an in vivo mo%el A4>;B* T&e mec&anistic pat&(ays !or gene e"pression are limite% #y at least !ive ma.or #arriers6 in vivo sta#ility) cell entry) en%osome escape) cytosolic transport) an% nuclear entry* T&e nuclear mem#rane restricts t&e transport o! t&e plasmi% DNA an% t&e e!!iciency o! t&e DNA trans!er !rom t&e cytoplasm to t&e nucleus &as #een estimate% to #e a#out 4@ Z9* In or%er to o#tain t&e &ig& gene e"pression) t&e genes intro%uce% into t&e cells must #e re%uce% to a compact si/e (&ic& can pass t&roug& nuclear pores* T&e collapsing o! t&e DNA into NPs o! re%uce% negative or increase% positive c&arges Ai*e*) DNA con%ensationB &as receive% consi%era#le interest %ue to its #iological importance in DNA pac$aging in virus &ea%s A4><B* Gene %elivery usually ta$es a%vantage o! t&e en%ocytic pat&(ay o! t&e cell* T&e cells continually ingest a part o! t&eir plasma mem#rane via en%ocytosis to !orm en%ocytic vesicles* T&e cells can ta$e up solvent an% solute #y t&e en%ocytosis activity later* En%ocytic vesicles incorporating DNAE#earing particles are transE !erre% to en%osomes an% t&en to lysomes) (&ere li#eration o! DNA some&o( ta$es place* Ho(ever) t&e plasma mem#rane is %irectly lin$e% to an% !unctionally integrate% (it& t&e un%erlying cytos$eletonM t&e en%ocytosis at t&e mem#rane (oul% reCuire t&e rearrangement o! actin an% tu#ulin* Nonviral vectors &ol% several a%vantages over mo%i!ie% virus employe% !or gene %elivery) in terms o! improve% an% pre%icta#le sa!ety pro!ile) a &ig& DNAEcarrying capacity) increase% versatility) t&e ease o! largeEscale pro%uction) an% Cuality control* Nevert&eless) t&eir e!!iciency lags #e&in% t&at o! viral systems* T&ose nonviral vectors e!!icient in trans!ection are o!ten to"ic #ecause o! t&eir nonE %egra%a#le property A4=>B* T&e gene trans!er e!!iciency o! nonto"ic vectors) !or e"ample) #io%egra%a#le cationic polymers) is o!ten not satis!actory A4>=B* i et al*

5@5

Pat&a$ et al*

&ave reporte% t&e synt&esis) c&aracteri/ation o! polyA%)lElacti%eEcoE9E&y%ro"yElE porlineB polymer !or t&e purpose o! gene %elivery) an% t&ey stu%ie% %egra%ation) cytoto"icity) as (ell as pDNA release $inetics an% sustaine% gene e"pression o! t&is polymerE#ase% system* T&ey s&o(e% t&e use!ulness o! t&ese polymers (it& multiple a%vantages A4>@B* Gupta an% Gupta A4>4B &ave s&o(n t&e application o! t&e Pullulan) a (aterE solu#le) neutral linear polysacc&ari%e !or gene %elivery* T&ey s&o(e% t&at t&ese NPs &a% &ig& trans!ection potential) coul% release DNA e!!iciently) an% (ere sta#le against %egra%ation #y DNAse* As speci!ic ligan%s can #e #oun% to t&e NP sur!ace) t&ese particles o!!er t&e possi#ility !or a%%itional targeting strategies* 0a#anov et al* A4>>B suggeste% an interesting approac& using polymer genomics in t&eir recent pu#lication* Pluronic) t&e AE+EA amp&ip&ilic #loc$ copolymers o! polyAet&ylene o"i%eB) can upregulate t&e e"pression o! selecte% genes in cells an% alter t&e genetic response to antineoplastic agents in cancer* T&ey reporte% t&at t&ese #loc$ copolymers alone as (ell as in com#ination (it& polyet&ylenimine can upregulate t&e e"pression o! t&e reporter genes in sta#ly trans!ecte% cells* T&is un%erscores t&e a#ility o! selecte% synt&etic polymers to en&ance transgene e"pression t&roug& a mec&anism t&at augments improve% DNA %elivery into cell* Pluronic is genetically #enign (&en com#ine% (it& an antineoplastic agent Do"oru#icin* It %rastically alters p&armacogenomic responses to t&is agent an% prevents t&e %evelopment o! multiE %rug resistance in #reast cancer cells* T&ey propose% t&e nee% !or a t&oroug& assessment o! p&armacogenomic e!!ects o! polymer t&erapeutics to ma"imi/e t&e clinical outcomes an% un%erstan% t&e p&armacological an% to"icological e!!ects o! polymerE#ase% %rugs an% %elivery systems A4>>B* Ta#att et al* A5@@B compare% t&e cationic S N an% liposomes !or gene trans!er as a carrier* T&ey !oun% t&at DNA #in%ing %i!!ere% only marginally in t&ese t(o systems* T&ey conclu%e% t&at cationic lipi% composition seems to #e more %ominant !or t&e in vitro trans!ection per!ormance t&an t&e $in% o! arrange% colloi%al strucE ture* Hence) cationic S N e"ten%s t&e range o! &ig&ly potent nonviral trans!ection agents #y one (it& !avora#le an% %istinct tec&nological properties A5@@B* ,issing et al* A5@4B pu#lis&e% a revie( (&ic& %escri#es t&e use o! NPs #ase% on soli% lipi% !or t&e parenteral application o! %rugs* Di!!erent types o! NPs #ase% on soli% lipi%s suc& as S Ns) nanostructure% lipi% carriers) an% lipi% %rug nanocon.ugates are intro%uce% an% structural %i!!erences are pointe% out* Di!!erent pro%uction met&o%s inclu%ing t&e suita#ility !or largeEscale pro%uction are %escri#e% along (it& t&e sta#ility issues) an% %rugEincorporation mec&anisms into t&e particles are %iscusse% in %etail* T&e #iological activity o! parenterally applie% S Ns an% #iop&armaceutical aspects suc& as p&armaco$inetic pro!iles as (ell as to"icity aspects are revie(e% A5@4B*

NAN?PARTICU ATE S3STEMS6 0N?,N AND UN0N?,N RIS0S An e"cellent revie( (as recently pu#lis&e% #y Hoet et al* A5@5B (it& an e"tensive literature survey in (&ic& t&ey suggeste% t&at t&e particles in t&e nanosi/e range coul% certainly enter into t&e &uman #o%y t&roug& lungs) gastrointestinal system) mucosa) an% t&e s$in* It is possi#le t&at some particles penetrate %eep in t&e %ermis an% gra%ually may #e ta$en up #y t&e #o%y* T&e c&ances o! penetration %epen% on t&e si/e an% sur!ace properties o! t&e particles an% also point o! contact* T&e %istriE #ution o! particles in t&e #o%y systems is also a !unction o! t&e sur!ace c&aracterisE tics o! t&e NPs* T&ere mig&t #e a critical si/e involve% #eyon% (&ic& t&e movement o! t&e particles mig&t #e restricte%* T&e p&armaco$inetic #e&avior o! %i!!erent types

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

5@8

o! t&e nanosystems reCuires a %etaile% investigation an% a %ata#ase o! &ealt& ris$s associate% (it& %i!!erent NPs nee%s to #e create%* T&e increase% ris$ o! car%iopulE monary %isease reCuires speci!ic measures to #e ta$en !or every ne(ly %evelope% nanoparticulate pro%uct* T&ere is no universal NP to !it all t&e casesM eac& NP system nee%s to #e treate% in%ivi%ually (&en a &ealt& ris$ is e"pecte%* T&e tests currently use% to veri!y sa!ety o! materials s&oul% #e applica#le to i%enti!y &a/ar%ous NPs) an% more stringent an% e!!icient testing proce%ures are nee%e% to evaluate t&e nanoparticulate systems) especially (&en use% as a !oo% component or as DDSs A44>)5@8H5@;B* A stu%y reporte% #y +ilati et al* A5@<B %iscusse% t&e processing an% !ormulation issues relate% to P GA proteinEloa%e% NPs prepare% #y %ou#leE emulsion met&o%* T&ey evaluate% t&e e!!ect o! some typical !ormulation !actors an% processing con%itions on t&e mean si/e an% t&e %rug entrapment e!!iciency o! P GA NPs* T&ey !oun% t&at t&e parameters t&at generally increase t&e entrapment e!!iciency are AiB &ig& molecular (eig&t o! t&e polymer) AiiB t&e presence o! uncappe% car#o"ylic en% groups (&en P GA is use%) AiiiB t&e use o! met&ylene c&lori%e instea% o! et&yl acetate) an% AivB an increase% nominal %rug loa%ing* An interesting stu%y regar%ing t&e GI upta$e an% transport o! S N to t&e lymp&atic system (as reporte% #y +argoni et al* A5@=B* T&ey s&o(e% t&at particle si/e is a critical %etermiE nant o! t&e !ate o! NPs a%ministere% orallyM larger particles may #e retaine% !or longer time in PeyerPs patc&es) (&ereas smaller particles are transporte% to t&e t&oracic %uct* Anot&er stu%y #y Passirani et al* A5@>B reporte% t&at NPs #earing &epE arin or %e"tran covalently #oun% to polymet&ylmet&acrylate (as !oun% to #e in t&e circulation !or a long time* T&e potent capacity !or opsoni/ation o! t&e polymet&ylE met&acrylate core (as &i%%en #y t&e protective e!!ect o! eit&er polysacc&ari%e* In t&e case o! &eparin NPs) t&e stealt& e!!ect (as pro#a#ly increase% #y its in&i#iting properties against complement activation* Silveira et al* A54@B reporte% a ne( type o! NPs (&ere t&ey use% polyiso#utylcyanoacrylate an% cyclo%e"trin com#ination as polymeric carrier* ?(ing to t&e presence o! many lipop&ilic sites #elonging to t&e cyclo%e"trins (&ic& (ere !irmly anc&ore% to t&e structure o! t&e NPs) t&ese types o! carriers (ere very use!ul to en&ance an% increase t&e loa%ing o! t&e lipop&ilic %rugs (it& pro#a#le less si%e e!!ects* +ut) t&e ris$s involve% nee% to #e veri!ie% an% esta#lis&e% #y appropriate sensitive met&o%s to ensure t&e sa!ety o! t&e NPDDS*

RE-ERENCES
4* Gennaro AR) e%* RemingtonPs6 T&e science an% practice o! p&armacy* 5@t& e%* USA6 ippincott ,illiams an% ,il$ins) 5@@@6>@8* 5* ,illis RC* Goo% t&ings in small pac$ages* Mo%ern Drug Discov 5@@9M <64* 8* Ro%rigue/ SG) Allemann E) -essi H) Doel$er E* P&ysicoc&emical parameters associate% (it& nanoparticles !ormation in t&e salting out) emulsi!icationH%i!!usion an% nanopreE cipitation met&o%s* P&arm Res 5@@9M 546495=* 9* Couvreur P) +arratt G) -attal E) egran% P) 2aut&ier C* nanocapsule tec&nology6 a revie(* Crit Rev T&er Drug Carrier Syst 5@@5M 4>6>>* :* Hans M ) o(man AM* +io%egra%a#le nanoparticles !or %rug %elivery an% targeting* Curr ?pin Soli% State Mater Sci 5@@5M ;684>* ;* -lorence AT* T&e oral a#sorption o! micro an% nano particulates6 neit&er e"ceptional nor unusual* P&arm Res 4>><M 4965:>* <* +in%sc&ae%ler C) Gurny R) Doel$er E* Process !or preparing a po(%er o! (ater insolu#le polymer (&ic& can #e re%isperse% in a liCui% p&ase) t&e resulting po(%er an% utili/ation t&ereo!* Patent ,? ==1@=@44) 4>==* =* erou" 'C) Allemann E) Doel$er E) Gurny R* Ne( approac& !or t&e preparation o! nanoparE ticles #y an emulsi!icationH%i!!usion met&o%* Eur ' P&arm +iop&arm 4>>:M 94649*

5@9
>* -essi H) Puisieu" -) Devissaguet 'P) Ammoury N) +enita S* Nanocapsule !ormation #y inter!acial polymer %eposition !ollo(ing solvent %isplacement* Int ' P&arm 4>=>M ::6R4* 4@* Puglisi G) -resta M) Giammona G) 2entura CA* In!luence o! t&e preparation con%itions on polyet&yl cyanoacrylate nanocapsule !ormation* Int ' P&arm 4>>:M 45:65=8* 44* 0reuter '* Nanoparticles* In6 0reuter ') e%* Colloi%al Drug Delivery Systems* Ne( 3or$6 Marcel De$$er) 4>>9654>* 45* Guterres SS) -essi H) +arratt G) Devissaguet 'P) Puisieu" -* PolyA%)lElacti%eB nanocapE sules containing %iclo!enac* I* -ormulation an% sta#ility stu%ies* Int ' P&arm 4>>:M 4486:<* 48* Guerrero D7) Allmann E) Doel$er E) -essi H* Preparation an% c&aracteri/ation o! nanoE capsules !rom per!orme% polymers #y a ne( process #ase% on emulsi!icationE%i!!usion tec&niCue* P&arm Res 4>>=M 4:64@:;* 49* Me&nert ,) Ma%er 0* Soli%Hlipi% nanoparticles6 pro%uction) c&aracteri/ation an% appliE cations* A%v Drug Deliv Rev 5@@4M 9@64;:* 4:* Gao H) ,ang '3) S&en WV) Deng 3H) V&ang ,* Preparation o! magnetic poly#utylcyanoE acrylate nanosp&eres encapsulate% (it& aclacinomycin A an% its e!!ect on gastric tumor* ,orl% ' Gastroenterol 5@@9M 4@65@4@* 4;* C&en 'H) ing R) 3ao 7) et al* En&ance% antitumor e!!icacy on &epatoma #earing rats (it& a%riamycin loa%e% nanoparticles a%ministere% into &epatic artery* ,orl% ' Gastroenterol 5@@9M 4@64>=>* 4<* Piemi MP3) 0orner D) +enita S) Marty 'P* Positively an% negatively c&arge% su#micron emulsions !or en&ance% topical %elivery o! anti!ungal %rugs* ' Control Release 4>>>M :=64<<* 4=* Dalencon -) Am.au% 3) a!!orgue C) Derouin -) -essi H* AtovaCuone an% Ri!a#utine loa%e% nanocapsules6 !ormulation stu%ies* Int ' P&arm 4>>=M 4:8645<* 4>* Ueno 3) -utaga(a H) Ta$agi 3) Ueno A) Mi/us&ima 3* Drug incorporating calcium carE #onate nanoparticles !or a ne( %elivery system* ' Control Release 5@@:M 4@86>8* 5@* Memisoglu E) +oc&ot A) ?/alp M) Sen M) Duc&ene D) Hincal AA* Direct !ormation o! nanosp&eres !rom amp&ip&ilic #eta cyclo%e"trin inclusion comple"es* P&arm Res 5@@8M 5@644<* 54* De T0) Ro%man D') Holm +A) Prasa% PN) +ergey E'* +rimoni%ine !ormulation in polyE acrylic aci% nanoparticles !or op&t&almic %elivery* ' Microencapsul 5@@8M 5@68;4* 55* 'aco#s C) Muller RH* Pro%uction an% c&aracteri/ation o! a +u%esoni%e nanosuspension !or pulmonary a%ministration* P&arm Res 5@@5M 4>64=>* 58* 3ang S) V&u ') u 3) iang +) 3ang C* +o%y %istri#ution o! camptot&ecin soli%Hlipi% nanoparticles a!ter oral a%ministration* P&arm Res 4>>>M 4;6<:4* 59* Corte/ R2) +ac$mann N) Senter PD) et al* E!!icient cancer t&erapy (it& a nano#o%y #ase% con.ugate* Cancer Res 5@@9M 4:65=:8* 5:* Ca#ral H) Nis&iyama N) ?$a/a$i S) 0oyama H) 0atao$a 0* Preparation an% #iological properties o! %ic&loroA4)5E%iaminocyclo&e"aneB platinum AIIB ADACHPtB loa%e% polyE meric micelles* ' Control Release 5@@:M 4@46558* 5;* Hu -7) 3uan H) V&ang HH) !ang M* Preparation o! soli%Hlipi% nanoparticles (it& Clo#etasol propionate #y a novel solvent %i!!usion met&o% in aCueous system an% p&ysE icoc&emical c&aracteri/ation* Int ' P&arm 5@@5M 58>6454* 5<* 2en$ates(arlu 2) Man.unat& 0* Preparation) c&aracteri/ation an% in vitro release $inetE ics o! Clo/apine soli%Hlipi% nanoparticles* ' Control Release 5@@9M >:6;5<* 5=* 0un N) 0eunEHong P) Sung ,0) 3ou H+* Sel! assem#le% &y%rogel nanoparticles !rom cur%lan %erivatives6 c&aracteri/ation) anticancer %rug release an% interaction (it& a &epatoma cell line AHep G5B* ' Control Release 5@@@M ;>655:* 5>* Ge/e A) Putau" ' ) C&oisnar% ) 'e&an P) ,ouessi%.e(e D* ong term s&el! sta#ility o! amp&ip&ilic +Ecyclo%e"trin nanosp&eres suspensions monitore% #y %ynamic lig&t scatE tering an% cryoEtransmission electron microscopy* ' Microencapsul 5@@9M 546;@<* 8@* ?l#ric& C) 0ayser ?) Muller RH* ipase %egra%ation o! Dynasan 449 an% 44; soli%Hlipi% nanoparticlesRe!!ect o! sur!actants) storage time an% crystallinity* Int ' P&arm 5@@5M 58<644>* 84* V&ang 7) 3ie G) i 3) 3ang 7) Nagai T* Stu%ies on t&e cyclosporine AEloa%e% stearic aci% nanoparticles* Int ' P&arm 5@@@M 5@@64:8*

Pat&a$ et al*

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

5@:

85* ,ang W) Dai ') C&en V) et al* +ioavaila#ility an% p&armaco$inetics o! cyclosporine AEloa%e% pH sensitive nanoparticles !or oral a%ministration* ' Control Release 5@@9M ><6954* 88* Salgueiro A) Egea MA) Espina M) 2alls ?) Garcia M * Sta#ility an% ocular tolerance o! cyclop&osp&ami%e loa%e% nanosp&eres* ' Microencapsul 5@@9M 546548* 89* +ec$ RCR) Po&lman AR) Guterres SS* NanoparticlesEcoate% microparticles6 preparation an% c&aracteri/ation* ' Microencapsul 5@@9M 5469>>* 8:* Porter C'H) 0au$onen AM) +oy% +') E%(ar%s GA) C&arman ,N* Suscepti#ility to lipase me%iate% %igestion re%uces t&e oral #ioavaila#ility o! Dana/ol a!ter a%ministration as a me%ium c&ain lipi% #ase% microemulsion !ormulation* P&arm Res 5@@9M 54649@:* 8;* Hu#ert +) At$inson ') Guerret M) Ho!!man M) Devissaguet 'P) Maincent P* T&e prepaE ration an% acute anti&ypertensive e!!ects o! a nanoparticle !orm o! Daro%ipine) a %i&y%ropyri%ine calcium entry #loc$er* P&arm Res 4>>4M =6<89* 8<* Sc&roe%er U) Sommer!iel% P) Sa#el +A* E!!icacy o! oral Delargin loa%e% nanoparticles %elivery across #loo%H#rain #arrier* Pepti%es 4>>=M 4>6<<<* 8=* T&ote A') Gupta R+* -ormation o! nanoparticles o! a &y%rop&ilic %rug using superE critical car#on %io"i%e an% microencapsulation !or sustaine% release) nanome%icine6 nanotec&nology* +iol Me% 5@@:M 46=:* 8>* ?l#ric& C) Gessner A) Sc&ro%er ,) 0ayser ?) Muller RH* ipi%H%rug con.ugate nanoparticles o! t&e &y%rop&ilic %rug %imina/eneEcytoto"icity testing an% mouse serum a#sorption* ' Control Release 5@@9M >;695:* 9@* ?l#ric& C) Gessner A) 0ayser ?) Muller RH* ipi% %rug con.ugate nanoparticles as novel carrier system !or t&e &y%rop&ilic antitrypanosomal %rug %imina/ene%iaceturate* ' Drug Targeting 5@@5M 4@68=<* 94* ?ye(umi M?) 3o$el RA) 'ay M) Coa$ley T) Mumper R'* Comparison o! cell upta$e) #ioE%istri#ution an% tumor retention o! !olate coate% an% PEG coate% Ga%olinium nanoparticles in tumor #earing mice* ' Control Release 5@@9M >:6;48* 95* Moutar%ier 2) Tosini -) 2lieg&e P) Cara ) Delpero 'R) Clerc T* Colloi%al anticancer %rugs #ioavaila#ility in oral a%ministration* Int ' P&arm 5@@8M 5;@658* 98* Castelli -) Messina C) Sarpietro MG) Pignatello R) Puglisi G* -lur#ipro!en release !rom Eu%ragit RS an% R aCueous nanosuspensions6 a $inetic stu%y #y DSC an% %ialysis e"periments* AAPS P&arm Sci Tec& 5@@5M 86>* 99* 0&oo SM) Hum#erstone A') Porter C'H) E%(ar%s GA) C&apman ,N* -ormulation %esign an% #ioavaila#ility assessment o! lipi%ic sel! emulsi!ying !ormulations o! Halo!antrine* Int ' P&arm 4>>=M =<64;9* 9:* Du!resne MH) erou" 'C* Stu%y o! t&e micelli/ation #e&avior o! %i!!erent or%er amino #loc$ copolymers (it& &eparin* P&arm Res 5@@9M 5464;@* 9;* Cavalli R) Peira E) Caputo ?) Gasco MR* Soli%Hlipi% nanoparticles as carriers o! &y%rocortisone an% progesterone comple"es (it& #eta cyclo%e"trins* Int ' P&arm 4>>>M 4=56:>* 9<* Vara GP) +argoni A) Cavelli R) -un%aro A) 2ig&etto D) Gasco MR* P&armaco$inetics an% tissue %istri#ution o! I%aru#icin loa%e% soli%Hlipi% nanoparticles a!ter %uo%enal a%minE istration to rats* ' P&arm Sci 5@@5M >464859* 9=* Calvo P) Alonso M') 2ila'ato ' ) Ro#inson 'R* Improve% ocular #ioavaila#ility o! In%omet&acin #y novel ocular %rug carriers* ' P&arm P&armacol 4>>;M 9=6449<* 9>* Pan%ey R) S&arma A) Va&oor A) S&arma S) 0&uller G0) Prasa% +* PolyA%)lElacti%eEcoE glycoli%eB nanoparticles #ase% in&ala#le sustaine% %rug %elivery system !or e"perimental tu#erculosis* ' Antimicro# C&emot&er 5@@8M 4@64@>8* :@* Palmiri G-) +onacucina G) Martino DP) Martelli S* Gastroresistant microsp&eres containE ing 0etopro!en* ' Microencapsul 5@@5M 4>6444* :4* Sc&ro%er U) Sommer!iel% P) Urlic& S) Sa#le +* Nanoparticle tec&nology !or %elivery o! %rugs across t&e #loo%H#rain #arrier* ' P&arm Sci 4>>=M <=648@:* :5* Alyauti%in RN) Petrov 2E) anger 0) +ert&ol% A) 0&ar$ievic& DA) 0reuter '* Delivery o! operami%e across t&e #loo%H#rain #arrier (it& polysor#ate =@ coate% nanoparticles* P&arm Res 4>><M 49685:* :8* Ale"iou C) 'urgons R) Sc&mi% R') et al* Magnetic %rug targeting #io%istri#ution o! t&e magnetic carrier an% t&e c&emot&erapeutic agent Mito"antrone a!ter locoEregional cancer treatment* ' Drug Target 5@@8M 44648>*

5@;
:9* Cavalli R) Caputo ?) Carlotti ME) Trotta M) Scarnecc&ia C) Gasco MR* Stu%y #y W ray %i!!raction an% %i!!erential scanning calorimetry o! t(o mo%el %rugs) P&enot&ia/ine an% Ni!e%ipine) incorporate% into lipi% nanoparticles* Eur ' P&arm +iop&arm 4>>:M 94685>* ::* HecC ') Deleers M) -anara D) 2ranc$" H) Amig&i 0* Preparation an% c&aracteri/ation o! nanocrystals !or solu#ility an% %issolution rate en&ancement o! ni!e%ipine* Int ' P&arm 5@@:M 5>>64;<* :;* HecC ') Nollevau" G) Deleers M) et al* In vitro transport stu%ies o! ni!e%ipine nanopartiE cles across cacoE5 HT 5>E:M54 culture an% cocultures* Eur ' P&arm +iop&arm* In press* :<* Hauss D') -ogal SE) -icorilli '2) et al* ipi% #ase% %elivery systems !or improving t&e #ioavaila#ility an% lymp&atic transport o! a poorly (ater solu#le T+9 in&i#itor* ' P&arm Sci 4>>=M =<64;9* :=* C&en D+) 3ang TV) u , ) V&ang 7* In vitro an% in vivo stu%y o! t(o types o! long cirE culating soli%Hlipi% nanoparticles containing Paclita"el* C&em P&arm +ull 5@@4M 9>64999* :>* 0o/iara 'M) oc$man PR) Allen DD) Mumper R'* Paclita"el nanoparticles !or t&e potenE tial treatment o! #rain tumors* ' Control release 5@@9M >>65:>* ;@* 3e& T0) u V) ,oent.es MG) Au ' S* -ormulating Paclita"el in nanoparticles alters its %isposition* P&arm Res 5@@:M 556=;<* ;4* De S) Miller D,) Ro#inson DH* E!!ect o! particle si/e o! nanosp&eres an% microsp&eres on t&e cellular association an% cytoto"icity o! Paclita"el in 9T4 cells* P&arm Res 5@@:M 556<;;* ;5* 3onc&eva 0) 2an%ervoort ') u%(ig A* In!luence o! process parameters o! &ig& pressure emulsi!ication met&o% on t&e properties o! Pilocarpine loa%e% nanoparticles* ' Microencapsul 5@@8M 5@699>* ;8* Mainer%es RM) Evangelista RC* Pra/iCuantel loa%e% P GA nanoparticles6 preparation an% c&aracteri/ation* ' Microencapsul 5@@:M 55648* ;9* Mu&len AV) Me&enert ,* Drug release an% release mec&anisms o! pre%nisolone loa%e% soli%Hlipi% nanoparticles* P&arma/ie 4>>=M :86::5* ;:* C&en H) V&ang V) Almarsson ?) Marier '-) +er$ovit/ D) Gar%ner CR* A novel lipi% !ree nano%ispersion !ormulation o! Propo!ol an% its c&aracteri/ation* P&arm Res 5@@:M 5568:;* ;;* oc&mann D) 2ogel 2) ,eyermann ') et al* P&ysicoc&emical c&aracteri/ation o! protE amineEp&osp&orot&ioate nanoparticles* ' Microencapsul 5@@9M 546;5:* ;<* Muller RH) Do#ruc$i R) Ra%oms$a A* Soli%Hlipi% nanoparticles as a ne( !ormulation !or Retinal* Acta Pol P&arm Drug Res 4>>>M :;644<* ;=* S&enoy D+) Ami.i MM* PolyAet&ylene o"i%eBEmo%i!ie% polyAvarepsilonEcaprolactoneB nanoparticles !or targete% %elivery o! Tamo"i!en in #reast cancer* Int ' P&arm 5@@:M 5>865;4* ;>* 'aco#s C) 0ayser ?) Muller RH* Nanosuspensions as a ne( approac& !or t&e !ormulation !or t&e poorly solu#le %rug Tara/epi%e* Int ' P&arm 5@@@M 4>;64;4* <@* oc$man PR) ?ye(umi M?) 0o/ira 'M) Ro%er 0E) Mumper R') Allen DD* +rain upta$e o! t&iamine coate% nanoparticles* ' Control Release 5@@8M >865<4* <4* +argoni A) Cavalli R) Vara GP) -un%aro A) Caputo ?) Gasco MR* Transmucosal transport o! To#ramycin incorporate% in soli%Hlipi% nanoparticles a!ter %uo%enal a%ministration to rats* Part II* Tissue %istri#ution* P&armacol Res 5@@4M 9869><* <5* Maria M) C&iara S) Donatella 2) Giuseppe ) Anna M-* Niosomes as carriers !or Trentinoin6 preparation an% properties* Int ' P&arm 5@@5M 589658<* <8* Maestrell -) Mura P) Alonso M'* -ormulation an% c&aracteri/ation o! Triclosan su#E micron emulsions an% nanocapsules* ' Microencapsul 5@@9M 546=:<* <9* Alyauti%in RN) Te/i$ov E+) Ramage P) 0&ar$evic& DA) +egly D') 0reuter '* Signi!icant entry o! Tu#ocurarine into t&e #rain o! rats #y a%sorption to polysor#ate =@ coate% polyA#utyl cyanoacrylateB nanoparticles6 an in situ #rain per!usion stu%ies* ' Microencapsul 4>>=M 4:6;<* <:* +un.es H) Drec&sler M) 0oc& MH') ,estesen 0* Incorporation o! t&e mo%el %rug U#i%ecarone in to soli%Hlipi% particles* P&arm Res 5@@4M 4=65=<* <;* HecC ') Deleers M) -anara D) et al* Preparation an% in vitro1in vivo evaluation o! nanoE si/e% crystals !or %issolution rate en&ancement o! UC+E8:99@E8) a &ig&ly %ose% poorly (ater solu#le (ea$ #ase* Eur ' P&arm +iop&arm* 5@@;M ;968;@*

Pat&a$ et al*

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

5@<

<<* 'ennings 2) 0orting MS) Go&la S* 2itamin A loa%e% soli%Hlipi% nanoparticles carrier system !or topical use6 %rug release properties* ' Control Release 5@@@M ;;644:* <=* Tei"eira M) Alonso M') Pinto MM) +ar#osa CM* Development an% c&aracteri/ation o! P GA nanosp&eres an% nanocapsules containing Want&one an% 8Emet&o"y "ant&one* Eur ' P&arm +iop&arm 5@@:M :>69>4* <>* Delie -* Evaluation o! nano an% micro particles upta$e #y t&e gastrointestinal tract* A%v Drug Deliv Rev 4>>=M 896554* =@* Ro%rigues 'S) Magal&aes NSS) Coel&o C++) Couvreur P) Ponc&el G) Gre! R* Novel core ApolyesterBEs&ell Apolysacc&ari%eB nanoparticles6 protein loa%ing an% sur!ace mo%i!icaE tion (it& lectins* ' Control Release 5@@8M >564@8* =4* 3oo HS) Par$ TG* +io%egra%a#le nanoparticles containing proteinH!atty aci% comple"es !or oral %elivery o! salmon calcitonin* ' P&arm Sci 5@@9M >869==* =5* 2ranc$" H) Demoustier M) Deleers) M* Ne( nanocapsule !ormulation (it& &y%rop&ilic core6 Application to t&e oral a%ministration o! salmon calcitonin in rats* Eur ' P&arm +iop&arm 4>>;M 95689;* =8* Alp&an%ary HP) A#ou#a$er M) 'aillar% D) Couvreur P) 2aut&ier C* 2isuali/ation o! insulin loa%e% nanocapsules6 in vitro an% in vivo stu%ies a!ter oral a%ministration to rats* P&arm Res 5@@8M 5@64@<4* =9* Mut(iri G0) Nic&ani A0) +a#iu$ S) +a#iu$ A* Strategies !or en&ancing t&e immunosE timulatory e!!ects o! CpG oligo%eo"ynucleoti%es* ' Control Release 5@@9M ><64* =:* eac& ,T) Simpson DT) 2al TN) et al* Uni!orm encapsulation o! sta#le protein nanoE particles pro%uce% #y spray !ree/ing !or t&e re%uction o! #urst release* ' P&arm Sci 5@@:M >96:;* =;* 3u V) Rogers T ) Hu ') 'o&ston 0P) ,illiams R?* Preparation an% c&aracteri/ation o! microparticles containing pepti%e pro%uce% #y a novel process6 spray !ree/ing into liCui%* Eur ' P&arm +iop&arm 5@@5M :96554* =<* Mo 3) im 3* Mec&anistic stu%y o! t&e upta$e o! (&eat germ agglutininEcon.ugate% P GA nanoparticles #y A:9> cells* ' P&arm Sci 5@@9M >865@* ==* Mura$ami H) 0o#ayas&i M) Ta$euc&i H) 0a(as&ima 3* Preparation o! polyA%)lElacti%eE coEglycoli%eB nanoparticles #y mo%i!ie% spontaneous emulsi!ication solvent %i!!usion met&o%* Int ' P&arm 4>>>M 4=<6498* =>* Crommelin D'A) Storm G) 'is$oot ,) Stene$es R) Mastro#attista E) Hennin$ ,E* Nanotec&nological approac&es !or t&e %elivery o! macromolecules* ' Control Release 5@@8M =<6=4* >@* Nassan%er U0) Steeren#erg PA) Poppe H) et al* In vivo targeting o! ?2ET 8 immunoliE posomes to ovarian carcinoma ascetic cells A?2CARE8B in ant&ymic nu%e mice* Cancer Res 4>>5M :56;9;* >4* Mastro#attista E) 0apel RHG) Eggen&ui/en MH) et al* ipi% coate% polyple"es !or targete% gene %elivery to ovarian carcinoma cells* Cancer Gene T&er 5@@4M =69@:* >5* De T0) Ho!!man AS* A reverse microemulsion polymeri/ation met&o% !or preparation o! #ioa%&esive polyacrylic aci% nanoparticles !or mucosal %rug %elivery6 loa%ing an% release o! timolol maleate* Arti! Cells +loo% Su#st Immo#il +iotec& 5@@4M 5>684* >8* De T0) Ho!!man AS* An op&t&almic !ormulation o! #eta a%renoreceptor antagonist) levE o#eta"olol) using polyacrylic aci% nanoparticles as carriers) loa%ing an% release stu%ies* ' +ioactive Compati#le Polym 5@@4M 4;65@* >9* +ourlais C ) Acar ) Via H) Sa%o PA) Nee%&am T) everge R* ?p&t&almic %rug %elivery systems6 recent a%vances* Prog Retin Eye Res 4>>=M 4<688* >:* 7a%%oumi MG) Ue%a H) 3ang ') Dav%a ') a#&aset(ar 2) ee 2H * T&e c&aracteristics an% mec&anisms o! upta$e o! P GA nanoparticles in ra##it con.unctival epit&elial cell layers* P&arm Res 5@@9M 546;94* >;* Vimmer A) C&etoni P) Saettone M) Ver#e H) 0reuter '* Evaluation o! pilocarpine loa%e% al#umin nanoparticles as controlle% %rug %elivery systems !or t&e eye* II* Coa%ministration (it& #ioa%&esive an% viscous polymers* ' Control Release 4>>:M 88684* ><* Diepol% R) 0reuter ') et al* Comparison o! %i!!erent mo%els !or t&e testing o! pilocarpine eye %rops using conventional eye %rops an% a novel %epot nanoparticles !ormulation* Grae!es Arc& Clin E"p ?p&t&almol 4>=>M 55<64==* >=* Gil%ing D0) Ree% AM* +io%egra%a#le polymers !or use in surgery6 polyglycolic1poly lactic aci% &omo an% copolymers* Polymers 4><>M 5@649:>*

5@=
>>* Rag&ava S) Hammon% M) 0ompella U+* Periocular routes !or retinal %rug %elivery* E"p ?pin Drug Deliv 5@@9M 46>>* 4@@* Ayalasomaya.ula SP) 0ompella U+* Retinal %elivery o! celeco"i# is severalE!ol% &ig&er !ollo(ing su#con.unctival a%ministration compare% to systemic a%ministration* P&arm Res 5@@9M 5464<><* 4@4* Sun$ara G) Ayalasomaya.ula SP) C&eru$u RS) 2ennerstrom ' ) De Ruiter ') 0ompella U+* Systemic an% ocular p&armaco$inetics o! NE9E#en/oylaminop&enylsul!onylglyE cine A+APSGB) a novel al%ose re%uctase in&i#itor* ' P&arm P&armacol 5@@9M :;68:4* 4@5* Contreras G) Morcol T) +ell S') Hic$ey A'* Evaluation o! novel particles as pulmonary %elivery systems !or insulin in rats* AAPS P&arm Sci 5@@8M :6E>* 4@8* 3amamoto A* Improvement o! transmucosal a#sorption o! #iologically active pepti%e %rugs* 3a$uga$u Vass&i 5@@4M 4546>5>* 4@9* 3amamoto H) 0uno 3) Sugimoto S) Ta$euc&i H) 0a(as&ima 3* Sur!ace mo%i!ie% P GA nanosp&eres (it& c&itosan improve% pulmonary %elivery o! calcitonin #y mucoa%&esion an% opening o! t&e intercellular tig&t .unctions* ' Control Release 5@@:M 4@568<8* 4@9a* Courrier HM) +ut/ N) 2an%amme T-* Pulmonary %rug %elivery systems6 recent %evelopments an% prospects* Crit Rev T&er Drug Carrier Syst 5@@5M 4>695:* 4@:* Pa&l A) S/elenyi I* Ast&ma t&erapy in t&e ne( millennium* In!lamm Res 5@@5M :465<8* 4@;* Contreras G) Hic$ey A'* P&armaceutical an% #iotec&nological aerosols !or cystic !i#rosis in t&erapy* A%v Drug Deliv Rev 5@@5M :9649>4* 4@<* S&arma S) ,&ite D) Imon%i AR) Plac$e ME) 2ail DM) 0ris MG* Development o! in&aE lational agents !or oncological use* ' Clin ?ncol 5@@4M 4>64=8>* 4@=* Suare/ S) ?PHara P) 0a/antseva M) et al* Air(ays %elivery o! ri!ampicin microparticles !or t&e treatment o! tu#erculosis* ' Antimicro# C&emot&er 5@@4M 9=6984* 4@>* S&arma R) Sa"ena D) D(ive%i A0) Misra A* In&ala#le microparticles containing %rug com#inations to target alveolar macrop&ages !or treatment o! pulmonary tu#erculosis* P&arm Res 5@@4M 4=649@:* 44@* V(issler +* In&ale% vaso%ilators* Anaest&esist 5@@5M :46;@8* 444* ?(ens DR* Ne( &ori/onsEalternative routes !or insulin t&erapy* Nat Rev Drug Discov 5@@5M 46:5>* 445* V&ang 7) S&en V) Nagai T* Prolonge% &ypoglycemic e!!ect o! insulin loa%e% poly#uE tylcyanoacrylate nanoparticles a!ter pulmonary a%ministration to normal rats* Int ' P&arm 5@@4M 54=6<:* 448* 0a(as&ima 3) 3amamoto H) Ta$eguc&i H) -u.io$a S) Hino T* Pulmonary %elivery o! insulin (it& ne#uli/e% %)lElacti%e1glycoli%e copolymer) nanosp&eres to prolong &ypoglycemic e!!ect* ' Control Release 4>>>M ;565<>* 449* Tsapis N) +ennett D) 'ac$son +) ,eit/ DA) E%(ar%s DA* Tro.an particles6 large porous carriers o! nanoparticles !or %rug %elivery* Proc Natl Aca% Sci USA 5@@5M >>645@@4* 44:* iu 3) Tsapis N) E%(ar%s DA* Investigating sustaine%Erelease nanoparticles !or pulmonary %rug %elivery* Cam#ri%ge) MA6 Harvar% University Press) 5@@8* 44;* Dailey A) 0leemann E) ,ittmar M) et al* Sur!actant !ree #io%egra%a#le nanoparticles !or aerosol t&erapy #ase% on t&e #ranc&e% polyesters) DEAPAEP2A EgEP GA* P&arm Res 5@@8M 5@65@44* 44<* 'ung T) 0amm ,) +reiten#ac& A) 0le#e G) 0issel T* oa%ing o! tetanus to"oi% to #ioE %egra%a#le nanoparticles !rom #ranc&e% poly Asul!o#utylEpolyvinyl alco&olBEgEAlacE ti%eEc@Eglycoli%eB nanoparticles #y protein a%sorption6 a mec&anistic stu%y* P&arm Res 5@@5M 4>644@:* 44=* Dailey A) Sc&me&l T) Gessler T) et al* Ne#uli/ation o! #io%egra%a#le nanoparticles6 impact o! ne#uli/er tec&nology an% nanoparticles c&aracteristics on aerosol !eatures* ' Control Release 5@@8M =;6484* 44>* 2ila A) Gill H) McCallion ?) Alonso M'* Transport o! P AHPEG particles across t&e nasal mucosa6 e!!ect o! particle si/e an% PEG coating %ensity* ' Control Release 5@@9M >=6584* 45@* 0o/iara 'M) oc$man PR) Alen DD) Mumper R'* In situ #loo%H#rain #arrier transport o! nanoparticles* P&arm Res 5@@8M 5@64<<5* 454* oc$man PR) Mumper R') 0&an MA) Allen DD* Nanoparticle tec&nology !or %rug %elivery across t&e #loo%H#rain #arrier* Drug Dev In% P&arm 5@@5M 5=64*

Pat&a$ et al*

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

5@>

455* 0reuter '* Nanoparticulate systems !or #rain %elivery o! %rugs* A%v Drug Deliv Rev 5@@4M 9<6;:* 458* Sc&ro%er U) Sa#le +A* Nanoparticles6 a %rug carrier system to pass t&e #loo%H#rain #arrier) permit central analgesic e!!ects o! i*v* %alargin in.ections* +rain Res 4>>;M <4@6454* 459* Gulyaev A) Gelperina SE) S$i%an IN) Antorpove AS) 0ivman G3) 0reuter '* Signi!icant transport o! Do"oru#icin into t&e #rain (it& polysor#ate =@ coate% nanoparticles* P&arm Res 4>>>M 4;64:;9* 45:* 0reuter ') Alyaut%in RN) 0&ar$evic& DA) Ivanov AA* Passage o! pepti%es t&roug& t&e #loo%H#rain #arrier (it& colloi%al polymer particles AnanoparticlesB* +rain Res 4>>:M ;<964<4* 45;* oc$man PR) 0o/iara ') Ro%er 0E) Allen DD* In vivo an% in vitro assessment o! #aseline #loo%H#rain #arrier parameters in t&e presence o! novel nanoparticles* P&arm Res 5@@8M 5@6<@:* 45<* D/iu#la TD) 0arim A) Mu/y$antov 2R* Polymer nanocarriers protecting active en/yme cargo against proteolysis* ' Control release 5@@:M 4@5695<* 45=* Allen TM) Cullis PR* Drug %elivery systems6 entering t&e mainstream* Science 5@@9M 8@864=4=* 45>* ,eissen#oc$ A) ,irt& M) Ga#or -* ,GA gra!te% P GA nanosp&eres preparation an% association (it& CacoE5 single cells* ' Control Release 5@@9M >>68=8* 48@* Gre! R) Couvreur P) +arratt G) Mysia$ine E* Sur!ace engineere% nanoparticles !or multiple ligan% #in%ing* +iomaterials 5@@8M 5969:5>* 484* oc&ner N) Pittner -) ,irt& M) Ga#or -* ,&eat germ agglutinin #in%s to t&e epi%ermal gro(t& !actor o! arti!icial CacoE5 mem#ranes as %etecte% #y silver nanoparticles en&ance% !luorescence* P&arm Res 5@@8M 5@6=88* 485* 'ung&ans M) 0reuter ') Vimmer A* Antisense %elivery using protamineHoligonucleoti%e particles* Nucleic Aci% Res 5@@@M 5=6e9:* 488* 2ogel 2) oc&mann D) ,eyermann ') et al* ?ilgonucleoti%eEprotamine al#umin nanoparticles) preparation) p&ysical properties an% its %istri#ution* ' Control Release 5@@9M 4@86>>* 489* Mayer G) 2ogel 2) ,eyermann ') et al* ?ligonucleoti%eHprotamineHal#umin nanopartiE cles6 protamine sul!ate causes %rastic si/e re%uction* ' Control Release 5@@:M 4@;64=4* 48:* ,eyermann ') oc&mann D) Vimmer A* Comparison o! antisense oligonucleoti%es %rug %elivery systems* ' Control Release 5@@9M 4@@6944* 48;* 2ila A) Sanc&e/ A) To#io M) Calvo P) Alonso M'* Design o! #io%egra%a#le particles !or protein %elivery* ' Control Release 5@@5M <=64:* 48<* Prego C) Garcia M) Torres D) Alonso M'* Transmucosal macromolecular %rug %elivery* ' Control Release 5@@:M 4@464:4* 48=* Ta$euc&i H) 3amamoto H) 0a(as&ima 3* Mucoa%&esive nanoparticulate systems !or pepti%e %rug %elivery* ' Control Release 5@@9M >=64* 48>* +ilati U) Allemann E) Doel$er E* Development o! a nanoprecipitation met&o% inten%e% !or t&e entrapment o! &y%rop&ilic %rugs into nanoparticles* Eur ' P&arm 5@@:M 596;<* 49@* Ta$euc&i H) 3amamoto H) 0a(as&ima 3* Mucoa%&esive nanoparticulate systems !or pepti%e %rug %elivery* A%v Drug Deliv Rev 5@@4M 9<68>* 494* Sa&oo S0) Ma ,) a#&aset(ar 2* E!!icacy o! trans!erring con.ugate% paclita"el loa%e% nanoparticles in a murine mo%el o! prostate cancer* Int ' Cancer 5@@9M 445688:* 495* -i!is T) Gamvrellis A) CrimeenEir(in +) et al* Si/e %epen%ent immunogenicity6 t&erapeuE tic an% protective properties o! nano vaccines against tumors* ' Immunol 5@@9M 4<86849=* 498* C&en 'H) ,ang ) ing R) et al* +o%y %istri#ution o! nanoparticles containing a%riamyE cin in.ecte% into t&e &epatic artery o! &epatoma #earing mice* Dig Dis Sci 5@@9M 9>644<@* 499* ?PNeal DP) Hirsc& R) Halas N') Payne 'D) ,est ' * P&oto t&ermal tumor a#lation in mice using near in!ra re% a#sor#ing nanoparticles* Cancer ett 5@@9M 58659@;* 49:* Sant&a$umaran M) T&omas T) T&omas T'* En&ance% cellular upta$e o! a triple"E!orming oligonucleoti%es #y nanoparticles !ormation in t&e presence o! polypropylenimine %en%rimers* Nucleic Aci% Res 5@@9M 4:654@5* 49;* 0umar CS) eusc&ner C) Doomes EE) Henry ) 'u#an M) Hormes '* E!!icacy o! lytic pepti%e #oun% magnetite nanoparticles in %estroying #reast cancer cells* ' Nanosci Nanotec&nol 5@@9M 9659:*

54@
49<* Roy I) ?&ulc&ans$yy T3) Pu%avar HE) et al* Ceramic #ase% nanoparticles entrapping (ater solu#le p&otosensiti/ing anticancer %rugs a novel %rug carrier system !or p&oto%ynamic t&erapy* ' Am C&em Soc 5@@8M 45:6<=;@* 49=* C&a(la 'S) Ami.i MM* Cellular upta$e an% concentrations o! tamo"i!en upon a%minisE tration in polyAepsilonEcaprolactoneB nanoparticles* AAPS P&arm Sci 5@@8M :6E8* 49>* C&a(la 'S) Ami.i MM* +io%egra%a#le polyAepsilonEcaprolactoneB nanoparticles !or tumor targete% %elivery o! Tamo"i!en* Int ' P&arm 5@@:M 59>645<* 4:@* 3oo HS) Par$ TG* -olate receptor targete% %elivery o! Do"oru#icin nano aggregates sta#ili/e% #y Do"oru#icin PEGE!olate con.ugate* ' Control Release 5@@9M 4@@659<* 4:4* 0ang +0) C&on S0) 0im SH) et al* Controlle% release o! paclita"el !rom microemulsion containing P GA an% evaluation o! anti tumor activity in vitro an% in vivo* Int ' P&arm 5@@9M 5=;649<* 4:5* C&ellini E* Nanotec&nologies an% nanosciences) $no(le%geE#ase% multi!unctional materials an% ne( pro%uction processes an% %evices) TAT 3S pro.ect* Italy6 University o! Pisa) 5@@8* 4:8* ,agner E* Strategies to improve DNA polyple"es !or in vivo gene trans!er6 (ill arti!icial viruses #e t&e ans(erT P&arm Res 5@@9M 546=* 4:9* Pic&on C) Gonsalves C) Mi%ou" P* Histi%ine ric& pepti%es an% polymers !or nucleic aci%s %elivery* A%v Drug Deliv Rev 5@@4M :86<:* 4::* 2er#aan -') ?usorren C) Snel C') et al* Steric sta#ili/ation o! polyA5A%imet&yl aminoB et&yl met&acrylateB #ase% polyple"es me%iates prolonge% circulation an% tumor targeting in mice* ' Gen Me% 5@@9M ;6;9* 4:;* ?gris M) ,al$er G) +lessing T) 0irc&esis R) ,olsc&e$ M) ,agner E* Tumor targete% gene t&erapy6 Strategies !or t&e preparation o! ligan% polyet&ylene glycolEpolyet&ylenE imine1DNA comple"es* ' Control Release 5@@8M >464<8* 4:<* ?upic$y D) ?gris M) Ho(ar% PR) Das& 0) Ul#ric& 0) Seymore ,* Importance o! lateral an% steric sta#ili/ation o! polyelectrolyte gene %elivery vectors !or e"ten%e% systemic circulation* Mol T&er 5@@5M :69;8* 4:=* +orc&ar% G* C&itosan !or gene %elivery* A%v Drug Deliv Rev 5@@4M :5649:* 4:>* ?&sa$i M) ?$u%a T) ,a%a A* In vitro gene trans!ection using %en%ritic polyAlElysineB* +io Con.ug C&em 5@@5M 486:4@* 4;@* -orrest M ) 0oer#er 'T) Pac$ D,* A %egra%a#le polyet&ylenimine %erivative (it& lo( to"icity !or &ig&ly e!!icient gene %elivery* +iocon.ug C&em 5@@8M 496>89* 4;4* im 3+) Han S?) 0ong HU* +io%egra%a#le polyester) polyAalp&aEA9Eamino#utylBElE glycolicEaci%B as a nonto"ic gene carrier* P&arm Res 5@@@M 4<6=44* 4;5* De$ie ) Tonc&eva 2) Du#ruel P* PolyElEglutamic aci% %erivatives as vectors !or gene t&erapy* ' Control release 5@@@M ;:64=<* 4;8* A$inc A) An%erson DG) ynn DM* Synt&esis o! polyEA#etaEamino estersB optimi/e% !or &ig&ly e!!ective gene %elivery* ' Am C&em Soc 5@@@M 45564@<;4* 4;9* +ettinger T) Remy 'S) Er#ac&er P* Si/e re%uction o! galactosylate% PEI1DNA comple"es improves lectin me%iate% gene trans!er into &epatocytes* +iocon.ug C&em 4>>>M 4@6::=* 4;:* -un&o!! AM) Monge S) Teeu(en R) 0oning GA) Nieu(en#roc$ NMES) Crommelin D'A) Ha%%leton DM) Henni$ ,E) van Nostrum C-* PEG s&iel%e% polymeric %ou#le layere% micelles !or gene %elivery* ' Control Release 5@@:M 4@56<44* 4;;* V&ang W7) ,ang W ) V&ang PC) et al* Galactosylate% ternary DNA1polyp&osp&oramiE %ate nanoparticles me%iate &ig& gene trans!ection e!!iciency in &epatocytes* ' Controlle% Release 5@@:M 4@56<9>* 4;<* V&ao V) ,ang ') Mao H7* Polyp&osp&oesters in %rug an% gene %elivery* A%v Drug Deliv Rev 5@@8M ::69=8* 4;=* i 3) ,ang ') i CG* CNS gene trans!er me%iate% #y a novel controlle% release system #ase% on DNA comple"es o! %egra%a#le polycation PPEEEA6 a comparison (it& polyet&ylenimine1DNA comple"es* Gene T&er 5@@9M 4464@>* 4;>* ,ang ') Gao S') V&ang PC* Polyp&osp&orami%ate gene carriers6 E!!ect o! c&arge group on gene trans!er e!!iciency* Gene T&er 5@@9M 4464@@4* 4<@* ,ang ') Vg&ang PC) u H-* Ne( polyp&osp&orami%ate (it& a spermi%ine si%e c&ain as a gene carrier* ' Control Release 5@@5M =864:<*

Pat&a$ et al*

P&armaceutical Applications o! Nanoparticulate DrugEDelivery Systems

544

4<4* Csa#a N) Caamano P) Sanc&e/ A) Domingue/ -) Alonso M'* P GA6 Polo"amer an% P GAM polo"amine #len% nanoparticles) ne( carriers !or gene %elivery* +iomacro Mol 5@@:M 4@65<4* 4<5* Va&r AS) %e2illers M) Pis&$o M2* Encapsulation o! %rug nanoparticles in sel! assem#le% macromolecular nanoEs&ells* angmuir 5@@:M 5469@8* 4<8* 0aul G) Parsons C ) Ami.i M* PolyAet&ylene glycolB mo%i!ie% gelatin nanoparticles !or intracellular %elivery* P&arm Eng 5@@8M 5864* 4<9* Panyam ') a#&aset(ar 2* +io%egra%a#le nanoparticles !or %rug an% gene %elivery to cells an% tissues* A%v Drug Deliv Rev 5@@8M ::685>* 4<:* Panyam ') a#&aset(ar 2* Sustaine% cytoplasmic %elivery o! %rugs (it& intracellular receptors using #io%egra%a#le nanoparticles* Mol P&arm 5@@9M 46<<* 4<;* Mog&imi SM) Hunter AC) Murray 'C* ong circulating an% target speci!ic nanopartiE cles) t&eory an% practice* P&armacol Rev 5@@4M :865=8* 4<<* Wia W) Hu V) MarCue/ M* P&ysically #on%e% nanoparticles net(or$s6 a novel %rug %elivery system* ' Control Release 5@@:M 4@8654* 4<=* Panyam ') ,illiams D) Das& A) Pelec$y D ) a#&aset(ar 2* Soli% state solu#ility in!luE ences encapsulation an% release o! &y%rop&o#ic %rugs !rom P GA1P A nanoparticles* ' P&arm Sci 5@@9M >864=@9* 4<>* 0aul G) Ami.i M* Tumor targete% gene %elivery using poly Aet&ylene glycolB mo%i!ie% gelatin nanoparticles6 in vitro an% in vivo stu%ies* P&arm Res 5@@:M 556>:4* 4=@* Es!an% R) Tomalia DA* PolyAami%oamineB PAMAM %en%rimers6 !rom #iomimetic to %rug %elivery an% #iome%ical applications* Drug Discov To%ay 5@@4M ;695<* 4=4* Tot& I) Salt&ivel T) ,il%erspin A-) et al* Novel cationic lipi%ic pepti%e %en%rimer vectors6 in vitro gene %elivery* STP P&arm Sci 4>>>M >6>8* 4=5* +ayele H0) Salt&ivel T) ?PDonnell M) et al* 2ersatile pepti%e %en%rimers !or nucleic aci% %elivery* ' P&arm Sci 5@@:M >9699;* 4=8* V&ang W7) ,ang W ) Huang S,) et al* In vitro gene %elivery using polyami%oamine %en%rimers (it& a trimesyl core* +iomacromolecules 5@@:M ;6894* 4=9* +enita +M) Rome.in S) 'ungingger HE) +orc&ar% G* P GAHPEI nanoparticles !or gene %elivery to pulmonary epit&elium* Eur ' P&arm +iop&arm 5@@9M =:64* 4=:* Di(an M) Elamanc&illi P) Cao M) Samuel '* Dose sparing o! CpG oligo%eo"ynucleoti%e vaccine a%.uvants #y nanoparticles %elivery* Curr Drug Deliv 5@@9M 469@:* 4=;* R&aese S) +riesen H2) ,aigmann HR) 0reuter ') anger 0* Human serum al#uminEpolE yet&ylenimine nanoparticles !or gene %elivery* ' Control Release 5@@8M >564>>* 4=<* 7uiang +) Segev A) +eliar% I) Nili N) Strauss +H) Se!ton M2* PolyAmet&yli%ene malonate 5*4*5B nanoparticles6 a #iocompati#le polymer t&at en&ances peri a%ventitial a%enoviral gene %elivery* ' Control Release 5@@9M >=699<* 4==* ee HC) 0im S) 0im 0) S&in H) 3oon '* Remission in mo%els o! type 4 %ia#etes #y gene t&erapy using a single c&ain insulin analogue* Nature 5@@@M 9@=69=8* 4=>* Go%#ey ,T) ,u 00) Mi$os AG* Trac$ing t&e intracellular pat& o! polyAet&ylenimineB1 DNA comple"es !or gene %elivery* Proc Natl Aca% Sci USA 4>>>M >;6:4<<* 4>@* i V) Huang * Sustaine% %elivery an% e"pression o! plasmi% DNA #ase% on #io%egra%E a#le polyester) polyA%)lElacti%eEcoE9E&y%ro"yElEprolineB* ' Control Release 5@@9M >=698<* 4>4* Gupta M) Gupta A0* Hy%rogel pullulan nanoparticles encapsulating p+UD acV plasE mi% as an e!!icient gene %elivery carrier* ' Control Release 5@@9M >>64:<* 4>5* Mars&all E* Gene t&erapy %eat& prompts revie( o! a%enovirus vector* Science 5@@@M 5=;65599* 4>8* +oyce N* Trial &alte% a!ter gene s&o(s up in semen* Nature 5@@4M 9496;<<* 4>9* C&ec$ E* Gene t&erapy) a tragic set#ac$* Nature 5@@5M 95@644;* 4>:* +raun CS) 2etro 'A) Tomalia DA) 0oe GS) Mi%%augg& CR* Structure1!unction relationE s&ips o! polyami%oamine1DNA %en%rimers as gene %elivery ve&icles* ' P&arm Sci 5@@:M >96958* 4>;* Vaitsev S) Cartier R) 2y#orov ?) et al* Polyelectrolyte nanoparticles me%iate vascular gene %elivery* P&arm Res 5@@9M 5464;:;* 4><* Montigny ,') Houc&ens CR) Illenye S) et al* Con%ensation #y DNA looping !acilities trans!er o! large DNA molecules into mammalian cells* Nucleic Aci% Res 5@@4M 5>64>=5*

545
4>=* im 3) 0im C) 0im 0) Par$ '* Development o! a sa!e gene %elivery system using #io%eE gra%a#le polymer) polyA9Eamino#utylBElEglycolic aci%B* ' Am C&em Soc 5@@@M 4556;:59* 4>>* 0a#anov A2) +atra$ova E2) Sria%i#&atla S) 3ang V) 0elly D ) Ala$ov 23* Polymer genomics6 S&i!ting t&e gene an% %rug %elivery para%igms* ' Control Release 5@@:M 4@465:>* 5@@* Ta#att 0) 0neuer C) Sameti M) et al* Trans!ection (it& %i!!erent colloi%al systems6 comparison o! soli%Hlipi% nanoparticles an% liposomes* ' Control release 5@@9M ><6854* 5@4* ,issing SA) 0ayser ?) Muller RH* Soli%Hlipi% nanoparticles !or parenteral %rug %elivery* Drug Deliv Rev 5@@9M :;645:<* 5@5* Hoet P) Ho&l!el% I+) Salata ?2* NanoparticlesE$no(n an% un$no(n &ealt& ris$s* ' Nano#iotec&nol 5@@9M 5645* 5@8* Service R-* Nanomaterials s&o( signs o! to"icity* Science 5@@8M 8@@6598* 5@9* +ro(n 'S) Veman 0 ) +ennett ,D* Ultra!ine particle %eposition an% clearance in t&e &ealt&y an% o#structe% lung* Am ' Respir Crit Care Me% 5@@5M 4;;6459@* 5@:* 0reilgaar% M* In!luence o! microemulsions on cutaneous %rug %elivery* A%v Drug Deliv Rev 5@@5M :96S<<* 5@;* Dav%a ') a#&aset(ar 2* C&aracteri/ation o! nanoparticles upta$e #y t&e en%ot&elial cells* Int ' P&arm 5@@5M 5886:4* 5@<* +ilati U) Allemann E) Doel$er E* PoluA%El lacti%eEcoEglycoli%eB protein loa%e% nanoparticles prepare% #y t&e %ou#le emulsion met&o%Eprocessing an% !ormulation issues !or en&ance% entrapment e!!iciency* ' Microencapsul 5@@:M 5565@:* 5@=* +argoni A) Cavalli R) Caputo ?) -un%aro A) Gasco MR) Vara GP* Soli%Hlipi% nanopartiE cles in lymp& an% plasma a!ter %uo%enal a%ministration to rats* P&arm Res 4>>=M 4:6<9:* 5@>* Passirani C) +arratt G) Devissaguet 'P) a#arre D* ong circulating nanoparticles #earing &eparin or %e"tran covalently #oun% to poly Amet&yl met&acrylateB* P&arm Res 4>>=M 4:64@9;* 54@* Silveira AM) Ponc&el G) Puisieu" -) Duc&ene D* Com#ine% poly Aiso#utylcyanoacrE ylateB an% cyclo%e"trins nanoparticles !or en&ancing t&e encapsulation o! lipop&ilic %rugs* P&arm Res 4>>=M 4:64@:4*

Pat&a$ et al*

49

ipi% Nanoparticles ASoli% ipi% Nanoparticles an% Nanostructure% ipi% CarriersB !or Cosmetic) Dermal) an% Trans%ermal Applications
Eliana +* Souto an% Rainer H* MJller
Department o! P&armaceutical Tec&nology) +iotec&nology) an% 7uality Management) -reie UniversitIt +erlin) +erlin) Germany

INTR?DUCTI?N T&e s$in) toget&er (it& t&e mucosal tissues o! t&e %igestive) respiratory) an% urogenital tracts) !orms t&e !rontier o! t&e #o%y) separating t&e e"ternal environE ment !rom internal organs* T&e epi%ermis !orms t&e #arrier #et(een t&ose Q(orl%s)S t&at is) #et(een t&e most varie% con%itions o! temperature an% &umi%ity o! t&e e"ternal environment) in contrast to t&e very sta#le internal environment o! t&e living tissues an% #o%y !lui%s* T&us) epi%ermis #e&aves as t&e protective organ s&o(ing !unctions o! p&ysical protection o! t&e #o%y) sensation) an% temperature control) as (ell as p&ysical #arrier against t&e e"c&ange o! c&emical su#stances into an% out o! t&e &uman organism* T&ese %ays) t&e p&armaceutical in%ustry supplies t&e costumer (it& many p&ysicoc&emically %i!!erent %osage !orms inten%e% !or s$in application) ranging !rom po(%ers to liCui%s) as (ell as semisoli% !ormulations* ,&en %eveloping suc& !ormulations) t&e p&armaceutical tec&nologists must &ave in min% many concerns) suc& as sta#ility) compati#ility) an% costumer accepta#ility o! t&e ve&icle an% active ingre%ient) as (ell t&e #ioavaila#ility o! t&is latter in cases o! %ermal an% transE %ermal treatment A4B* It is a !act t&at preparations !or topical) %ermal) an% trans%ermal use &ave t&e uniCue !eature t&at t&eir p&ysical properties are almost as important as any p&arE macologically active ingre%ient t&at t&ey contain* T&e composition o! t&e ve&icle an% active compoun%s %eserves special emp&asis) particularly #ecause t&e intimate contact (it& t&e s$in is al(ays accompanie% #y possi#le ris$s o! a%verse reactions* T&e treatment o! s$in lesions is o!ten con%ucte% conservatively) mainly inten%e% to soot&e t&e s$in %uring t&e progress o! natural tissue repair* T&e principles o! !ormuE lation closely resem#le t&ose applica#le to cosmetic pro%ucts an% in #ot& cases a goo% %eal o! caution is necessary to ensure t&e success o! t&e t&erapy* Concerning %ermatological an% trans%ermal t&erapy) t&e precise Cuantity o! active ingre%ient (&ic& is necessary to ac&ieve a given response cannot #e pre%icte% (it& certainty* T&is lac$ o! precision is largely %ue to varia#ility o! s$in penetration #y t&e active ingre%ient) (&ic& is relate% to t&e t&ic$ness o! t&e epi%ermis an% its $eratin layer) as (ell as to mec&anical removal o! t&e applie% !ormulation i! t&e a!!ecte% area is not covere% #y a %ressing* To overcome many o! t&ese a#oveEmentione% %ra(#ac$s) attempts &ave #een ma%e to intro%uce lipi% nanoparticles into t&e cosmetic an% p&armaceutical !iel%s*

548

549

Souto an% MJller

Soli% lipi% nanoparticles AS NsB A5B an% nanostructure% lipi% carriers AN CB A8B are novel colloi%al %elivery systems (it& many cosmetic an% %ermatological !eatures) suc& as a%&esive properties (&en applie% to t&e s$in A9B* T&ese properties #ring many ot&er a%vantages suc& as occlusion an% s$in &y%ration) a#sorptionEincreasing e!!ects) active penetration en&ancement) an% controlle%Erelease properties A9):B* S N an% N C systems %i!!er #ecause S Ns (ere %evelope% #y e"c&anging t&e liCui% lipi% AoilB o! oilEinE(ater Ao1(B emulsions #y a soli% lipi% A;B) (&ic& can #ring many a%vantages in comparison to a liCui% core A<B* T&e concept o! N C (as %evelope% #y nanostructuring t&e lipi% matri") in or%er to give more !le"i#ility !or mo%ulation o! %rug release) increasing t&e %rug loa%ing an% preventing its lea$age* T&is approac& (as accomplis&e% #y mi"ing soli% lipi%s (it& liCui% lipi%s AN C conceptB) instea% o! &ig&ly puri!ie% lipi%s (it& relatively similar molecules AS N conceptB* T&e result is a less or%ere% lipi% matri" (it& many imper!ections) (&ic& can accommo%ate a &ig&er amount o! %rug A:)=H4@B* T&e mi"ture o#taine% (it& soli% an% liCui% lipi%s nee%s to #e soli% at least at 9@]C to ma$e sure t&at it %oes not melt at room an% #o%y temperature i! use% !or %rug %elivery* A%vantages o! using lipi%s as carrier systems !or s$in a%ministration are relate% to t&eir p&ysiological an% (ellEtolerate% nature) (&ic& re%uces t&e ris$ o! to"icological pro#lems an% local irritancy A:);)44B* A range o! very %i!!erent lipi%s (it& generally recogni/e% as sa!e status &as #een use% to pro%uce S Ns an% N Cs) ranging !rom &ig&ly puri!ie% lipi%s) !or e"ample) tristearin A45B in S Ns) to mi"tures o! monoE) %iE) an% triacylglycerols in N Cs) inclu%ing monoaci% an% polyaci% acylglycerols* ,&en trans!orming t&e #ul$ lipi% into a nanoparticulate !orm Ai*e*) S Ns or N CsB) a melting point %epression is) in general) o#serve%* T&is melting point %epression is %escri#e% in t&e T&omson eCuation (&ic& itsel! is %erive% !rom t&e 0elvin eCuation A48B* An a%%itional melting point %epression occurs (&en a !oreign compoun% Aactive ingre%ient or sur!actant moleculeB is %issolve% in t&e lipi% matri") !or e"ample) sur!actant (ill partition !rom t&e (ater p&ase to t&e lipi% p&ase) an% activeEloa%e% lipi% nanoparticles s&o( a melting point %epression (&en a molecularE%isperse% active ingre%ient is present* ,&en comparing t&e lipi% materials as #ul$ an% as nanoparticulate systems) melting an% recrystalli/ation temperatures can #e %i!!erent) especially in c&emically poly%isperse% lipi%s* ,&en pro%ucing lipi% nanoparticles) t&e %i!!erence can #e #et(een 4@]C to 5@]C or even more* -or e"ample) lipi%s) suc& as trilaurin AC 45B) (&ic& &as an original melting point o! 98]C to 9<]C) s&o( a very pronounce% superE cooling (&en !ormulate% as lipi% nanoparticles* In suc& compoun%s) crystalli/ation (ill not ta$e place anymore at room temperature) an% it (ill only &appen i! coole% %o(n to !ree/ing temperatures A49B* ipi% molecules s&o( %i!!erent t&reeE%imensional structures) t&at is) polymorp&ic !orms6 unsta#le m) metasta#le fu) an% as t&e most sta#le t&e f mo%i!icaE tion ATa#le 4B* In cases o! mi"tures o! acylglycerols) polymorp&ic transitions occur !rom fu to fi an% t&en to f) (&ic& means t&at anot&er interme%iate polymorp& !orm is present* Most #ul$ lipi%s are o#taine% in f mo%i!ication or at least pre%ominantly in f mo%iE !ication* In many cases) t&e pro%uction o! lipi% nanoparticles lea%s to a c&ange in t&e relative !raction o! t&e polymorp&ic !orms* Depen%ing on t&e c&emical nature o! t&e lipi% an% on t&e parameters esta#lis&e% !or t&e pro%uction o! lipi% nanoparticles) %i!!erent !ractions o! m an% fu mo%i!ication (ill #e o#taine%* T&is can lea% again to a re%uction o! t&e melting point) or more precisely c&ange in !orm an% s&i!t o! t&e

ipi% Nanoparticles !or Cosmetic) Dermal) an% Trans%ermal Applications

54:

TA+ E 4 T&reeEDimensional Structure o! t&e Crystal ?r%er in t&e T&ree Main Polymorp&s !rom Monoaci% Triacylglycerols
m mo%i!ication fu mo%i!ication

f mo%i!ication

Crystal system Su#cell

He"agonal ?rt&or&om#ic

Monoclinical ?rt&or&om#ic

Triclinical Triclinical

Source6 -rom Re!s* 4:) 4;*

melting pea$* In contrast) it mig&t #e t&at t&e create% polymorp&ic !orms are not longEterm sta#le) t&at is) t&ere is a gra%ual trans!ormation to more sta#le mo%i!icaE tions) (&ic& means increasing t&e content o! fu1fi an% !inally f* T&is p&enomenon is not %esire% an% t&e c&anges in lipi% structure can cause %rug e"pulsion %uring storage an% c&anges in t&e release pro!ile o! t&e incorporate% active molecules* To avoi% suc& un%esire% e!!ects) lipi% mi"tures s&oul% #e c&osen (&ic& trans!orm relaE tively !ast to more sta#le mo%i!ications %irectly a!ter particle pro%uction Ai*e*) (it&in one to t&ree %aysB* It is also per!ectly accepta#le or it can even #e planne% to trigger %rug release) (&en t&e generate% !ractions o! t&e %i!!erent polymorp&ic !orms remain unc&ange% %uring storage an% %o not trans!orm #ac$ to t&e f mo%i!ication o! t&e #ul$ lipi%* Drug e"pulsion !rom lipi% matrices is a (ellE$no(n p&enomenon !rom suppositories A4<B* Concerning t&e %i!!erent possi#ilities !or &o( t&e active ingre%iE ents can #e incorporate% (it&in t&e lipi% nanoparticle matri") one can mention6 AiB t&e replacement o! &ost molecules in t&e lattice #y a guest molecule or AiiB its incorE poration in #et(een t&e &ost molecules* Ho(ever) !or t&is latter &ypot&esis t&e guest molecule nee%s to &ave a si/e less t&an 5@[ o! t&e &ost molecule* Active moleE cules can #e locali/e% in #et(een t&e lipi% lamellae (&ic& t&en results in an increase o! t&e lattice spacing % Ai*e*) interatomic %istance %e!ine% #y t&e +raggPs eCuationM see Re!* 4=B* T&ere is also t&e possi#ility t&at t&e active ingre%ient is present in t&e !orm o! amorp&ous clusters) mainly locali/e% in t&e imper!ections o! t&e crystal* In general) t&e accommo%ation o! t&e active molecules is improve% (it& t&e increase o! t&e num#er o! imper!ections in t&e lipi% crystals A4>B* Active loa%ing can #e increase% #y using rat&er cru%e lipi% mi"tures) suc& as t&e ones use% in cosmetic pro%ucts* An even !urt&er improvement o! active loa%ing can #e ac&ieve% #y

54;

Souto an% MJller

controlle% nanostructuring o! t&e lipi% matri") t&at is) creating as many imper!ecE tions as possi#le* Depen%ing on t&e nature o! t&e lipi%s use% !or #len%ing an% t&e lipi% matri") %i!!erent types o! N Cs (ill #e o#taine%* T&e crystalline nature o! t&e lipi% matri" is) t&ere!ore) t&e %ominant !actor %etermining t&e active loa%ing) t&e release pro!ile pattern) an% also t&e longEterm sta#ility o! S Ns an% N Cs regar%ing t&e release an% su#seCuent #ioavaila#ility o! t&e active ingre%ient* T&us) t&e crystalline status nee%s to #e closely monitore% (&en %eveloping a S N or N C !ormulation*

HIST?RICA +AC0GR?UND AND PR?DUCTI?N ?- S? ID IPID NAN?PARTIC ES AND NAN?STRUCTURED IPID CARRIERS T&e !irst lipi% particles (ere pro%uce% #y t&e researc& group o! Speiser) t&e !at&er o! t&e nanoparticles) in Vuric& A5@B A '* Paris Matc&B* Hig&Espee% stirring (as applie% !or t&e pro%uction o! an o1( emulsion #et(een a melte% lipi% p&ase an% a &ot aCueous sur!actant solution* T&e o#taine% emulsion (as t&en coole% an% t&e inner lipi% p&ase !orme% soli% particles* Ho(ever) in general) t&e use o! stirring tec&niCues &as some %ra(#ac$s) suc& as t&e relatively #roa% si/e %istri#ution o! particles an% t&e !act t&at relatively &ig& concentrations o! sur!actant molecules are usually reCuire% to o#tain a mean particle %iameter in t&e nanometer range* T&e pro%uct %evelope% #y Speiser (as calle% Qlipi% nanopelletsS an% (as inten%e% !or oral %elivery* T&e patent o#taine% #y Speiser (as not !ollo(e% up #y t&e o(ner Rentsc&ler) an% patent protection no longer e"ists in a num#er o! countries* A similar process (as %evelope% an% patente% #y Dom#) (&o prepare% parE ticle %ispersions applying a sonication proce%ure* T&e pro%uct %evelope% #y Dom# (as calle% Qliposp&eresS A54B) an% t&ey also !oun% no #roa% application in p&armaceutical pro%ucts* In 4>>4) t&e patent application o! t&e !irst generation o! lipi% nanoparticlesR S NsR(as su#mitte% %escri#ing t&e nanoparticle pro%uction #y &ig&Epressure &omogeni/ation AHPHB A55B an% also via microemulsion tec&niCue A58B* HPH is a tec&niCue #roa%ly use% in %i!!erent researc& areas) an% it is also esta#lis&e% in p&armaceutical pro%uction) !or e"ample) !or t&e pro%uction o! emulE sions !or parenteral nutrition AIntralipi% b) ipo!un%inb) ipovenoesbB A59H5;B* Using t&is tec&niCue) t&e pro#lems o! ot&er nanoparticles Apolymeric nanoparticlesB) suc& as t&e lac$ o! scaling up an% largeEscale pro%uction lines) &as #een overcome* In a%%ition) HPH lea%s to a pro%uct #eing relatively &omogeneous in si/e) t&at is) possessing a &ig&er p&ysical sta#ility o! t&e aCueous %ispersionM in general) poly%isperse %ispersions s&o( a greater ten%ency to aggregate or coalesce* -urt&ermore) it is a simple an% very costEe!!ective pro%uction tec&niCue* T&ere are #asically t(o %i!!erent pro%uction met&o%s6 t&e &ot an% t&e col% HPH tec&niCues* -or t&e &ot HPH tec&niCue) t&e lipi% is melte% at appro"imately :]C to 4@]C a#ove its melting point) t&e active ingre%ient is %issolve% or !inely %isperse% in t&e melt an% t&en t&e activeEingre%ient containing lipi% melt is %isperse% #y stirring in a &ot sur!actant solution* T&e o#taine% preemulsion is &omogeni/e% applying a pressure #et(een 5@@ an% :@@ #ar an% t(o to t&ree &omogeni/ation cycles* A!ter t&e &omogeni/ation) a &ot nanoemulsion is o#taine%M cooling lea%s to recrystalli/ation o! t&e lipi% an% !ormation o! lipi% nanoparticles* -or t&e col% HPH tec&niCue) t&e lipi% melt containing t&e active ingre%ient is coole%) an% a!ter soli%i!ication is groun% using a mortar mill* T&e o#taine% lipi% microparticles are !urt&er %isperse% in a col% aCueous sur!actant solution* T&e

ipi% Nanoparticles !or Cosmetic) Dermal) an% Trans%ermal Applications

54<

resulte% presuspension is &omogeni/e% in t&e soli% state at or #elo( room temperaE ture #y cooling t&e &ig&Epressure &omogeni/er* T&e s&eer !orces an% cavitation !orces in t&e &omogeni/er are strong enoug& to #rea$ t&e microparticles %irectly into lipi% nanoparticles* -or t&e pro%uction o! lipi% nanoparticles via t&e microemulsion tec&niCue) t&e lipi% is melte%) t&e sur!actant) cosur!actant) an% (ater are a%%e% in suc& conE centrations t&at a microemulsion results A5<B* T&e si/e o! t&e microemulsion region in t&e p&ase %iagram is a !unction o! temperature) t&at is) t&e microemulsion can #e converte% to a %i!!erent system (&en) !or e"ample) re%ucing t&e temperature* T&ere!ore) !or particle pro%uction) t&e microemulsion nee%s to #e $ept at t&e elevate% temperature %uring t&e process* T&e &ot microemulsion is %ilute% into col% (ater) lea%ing to a Q#rea$ingS o! t&e microemulsion an% su#seCuent !ormaE tion o! an ultra!ine nanoemulsion* T&e %ilution (it& (ater an% t&e re%uction o! temperature narro(ing t&e microemulsion region are t&e reasons !or #rea$ing o! t&e microemulsion A5=B* ?ne %isa%vantage o! t&is proce%ure is t&e %ilution o! t&e particle suspension #y (ater) o#taining concentrations usually #elo( 4[ o! particle content* ,&en processing to a !inal %osage !orm) a very large amount o! (ater nee%s to #e remove%* ?t&er approac&es !or t&e pro%uction o! lipi% nanoparticles &ave #een a%apte% !rom polymeric nanoparticle pro%uction proce%ures) !or e"ample) t&e solvent emulsi!icationEevaporation met&o% %escri#e% #y S.FstrFm an% +ergenst&l A5>B) t&e solvent %isplacement met&o% %escri#e% #y -essi et al* A8@B) an% t&e emulsi!icaE tionE%i!!usion tec&niCue patente% #y 7uintanarEGuerrero et al* in 4>>> A84B* T&e novel p&aseEinversionE#ase% tec&niCue &as #een %escri#e% #y Heurtault et al* A85)88B !or t&e pro%uction o! S Ns* T&e solvent emulsi!icationEevaporation is a met&o% analogous to t&e pro%ucE tion o! polymeric nanoparticles an% microparticles #y solvent evaporation in o1( emulsions* T&e lipi% is %issolve% in an organic solvent (&ic& s&o(s no misci#ility (it& (ater Ne*g*) cyclo&e"ane A5>)89B) c&loro!orm A89B) or met&ylene c&lori%e A8:)8;BO* T&e organic solution is %isperse% in aCueous sur!actant p&ase) t&e solvent is t&en remove% #y evaporation A89B* It can #e applie% !or t&e incorporation o! &y%rop&ilic molecules suc& as pepti%es an% proteins) (&ic& must #e previously %issolve% into a (ater p&ase preparing in t&is case a (aterEinEoilEinE(ater A(1o1(B emulsion A8;B* In t&e solvent %isplacement met&o%) an organic solvent (&ic& is misci#le (it& (ater is use%* In t&is case) t&e lipi% material is previously %issolve% in a semipolar (aterEmisci#le solvent) suc& as et&anol) acetone) or met&anol A8<H9@B* In t&e emulsi!icationE%i!!usion tec&niCue) #en/yl alco&ol A94B or tetra&y%ro!uE ran A95B) (&ic& is previously saturate% (it& (ater) is use% to ensure t&e initial t&ermo%ynamic eCuili#rium #et(een t&e t(o liCui%s A(ater an% solventB* T&en) t&e (aterEsaturate% organic solvent is use% to %issolve t&e lipi%) an% a!ter t&is an o1( emulsion is prepare%* ?(ing to t&e saturation o! t&e organic solvent (it& (ater) no solvent %i!!uses !rom t&e %roplets into t&e e"ternal (ater p&ase !rom t&e o1( emulsion* Removal o! solvent !rom t&e %roplets an% particle !ormation is ac&ieve% #y a%%ing a%%itional (ater to t&e emulsion an% e"tracting t&e solvent A94B* A very interesting approac& #ase% on t&e p&ysics #e&in% it is t&e p&aseE inversionE#ase% tec&niCue) (&ic& is a t(oEstep met&o%* -irst) all components are place% on a magnetic stirrer using a temperature program !rom room temperature to) !or e"ample) =:]C* T&is is !ollo(e% #y progressive cooling to ;@]C* T&ree temperature cycles A=:H;@H=:H;@H=:]CB are applie% to reac& t&e inversion process %e!ine% #y temperature range* In step 5) an irreversi#le s&oc$ is in%uce% #y %ilution

54=

Souto an% MJller

(it& col% (ater* T&is !ast coolingH%iluting process lea%s to t&e !ormation o! sta#le nanoparticles A98B* M?RPH? ?G3 AND STRUCTURE ?- S? ID IPID NAN?PARTIC ES AND NAN?STRUCTURED IPID CARRIERS Di!!erent mo%els &ave #een %escri#e% in t&e literature !or &o( active molecules can #e incorporate% into S Ns an% N Cs A9B* -or eac& o! t&e carriers) t&ree #asic types are %escri#e% A-ig* 4B* T&e type o! S Ns %epen%s on t&e c&emical nature o! t&e active ingre%ient an% lipi%) t&e solu#ility o! actives in t&e melte% lipi%) nature an% concentration o! sur!actants) type o! pro%uction A&ot vs* col% HPHB) an% t&e pro%uction temperature* T&ere!ore) t&ree incorporation mo%els &ave #een propose% A9:B6 4* S N Type I or &omogeneous matri" mo%el) 5* S N Type II or %rugEenric&e% s&ell mo%el) an% 8* S N Type III or %rugEenric&e% core mo%el* T&e S N Type I or &omogeneous matri" mo%el is %erive% !rom a soli% solution o! lipi% an% active ingre%ient* A soli% solution can #e o#taine% (&en S Ns are pro%uce% #y t&e col% &omogeni/ation met&o%* A lipi% #len% can #e pro%uce% containing t&e active in a molecularly %isperse% !orm* A!ter soli%i!ication o! t&is #len%) it is groun% in its soli% state t&us avoi%ing or minimi/ing t&e enric&ment o! active molecules in %i!!erent parts o! t&e lipi% nanoparticle* T&e S N Type II or %rugEenric&e% s&ell mo%el is ac&ieve% (&en S Ns are pro%uce% via t&e &ot HPH tec&niCue) an% t&e active ingre%ient concentration in t&e melte% lipi% is lo(* During t&e cooling process o! t&e &ot o1( nanoemulsion) t&e lipi% (ill precipitate !irst) lea%ing to a stea%ily increasing concentration o! active molecules in t&e remaining lipi% melt (it& increasing !raction o! lipi% soli%iE !ie%* An activeE!ree lipi% core is !orme%M (&en t&e active reac&es its saturation solu#ility in t&e remaining melt) an outer s&ell (ill soli%i!y containing #ot& active

-IGURE 4 ASee color insert*B +asic types o! soli% lipi% nanoparticles an% nanostructure% lipi% carriers* A##reviations6 N C) nanostructure% lipi% carrierM S N) soli% lipi% nanoparticle* Source6 -rom Re!* 99*

ipi% Nanoparticles !or Cosmetic) Dermal) an% Trans%ermal Applications

54>

an% lipi%* T&e enric&ment in t&e outer area o! t&e particles causes #urst release* T&e percentage o! active ingre%ient locali/e% in t&e outer s&ell can #e a%.uste% in a controlle% (ay #y altering t&e pro%uction parameters* A typical e"ample o! an activeEenric&e% s&ell mo%el is t&e incorporation o! coen/yme 74@ A9;)9<B* T&e S N Type III or %rugEenric&e% core mo%el can ta$e place (&en t&e active ingre%ient concentration in t&e lipi% melt is &ig& an% at or relatively close to its saturation solu#ility* Cooling %o(n o! t&e &ot oil %roplets (ill in most cases re%uce t&e solu#ility o! t&e active in t&e meltM (&en t&e saturation solu#ility is e"cee%e%) active molecules precipitate lea%ing to t&e !ormation o! a %rugEenric&e% core* Regar%ing t&e mo%els %escri#e% !or N Cs) -igure 4 also s&o(s t&ree %i!!erent structures6

4* N C Type I or imper!ect crystal mo%el) 5* N C Type II or amorp&ous mo%el) an% 8* N C Type III or multiple mo%el* N C type I is %e!ine% as t&e imper!ect crystal mo%el) #ecause once in its matri" t&ere are many imper!ections (&ic& are a#le to accommo%ate t&e active molecules* T&is mo%el is o#taine% (&en mi"ing soli% lipi%s (it& small amounts o! liCui% lipi%s AoilsB* ?(ing to t&e %i!!erent c&ain lengt&s o! t&e !atty aci%s an% t&e mi"ture o! monoE) %iE) an% triacylglycerols) t&e matri" o! N Cs is not a#le to !orm a &ig&ly or%ere% structure A:B* N C type II is calle% t&e amorp&ous mo%el #ecause it is create% (&en mi"ing special lipi%s (&ic& %o not recrystalli/e anymore a!ter &omogeni/ation an% cooling) suc& as &y%ro"yoctacosanyl&y%ro"ystearate an% isopropylmyristate* T&ese lipi%s are a#le to create soli% particles o! amorp&ous lipi% structure) (&ic& can avoi% t&e occurrence o! crystalli/ation) minimi/ing %rug e"pulsion) #ecause t&e matri" is maintaine% in t&e polymorp&ic m !orm* N C type III is %escri#e% as t&e multiple mo%el* T&is mo%el &as #een %evelope% to improve t&e loa%ing capacity o! several %rugs) suc& as t&e ones (&ose solu#ility in liCui% lipi%s is &ig&er t&an in soli% lipi%s A9=)9>B* T&is type is %erive% !rom (1o1( emulsions) (&ic& consist o! an oilEinE!atEinE(ater %ispersion* 2ery small oil nanocompartments are create% insi%e t&e soli% lipi% matri" o! t&e nanoE particles generate% #y a p&aseEseparation process A:B* T&is mo%el is o#taine% (&en mi"ing soli% lipi%s (it& liCui% lipi%s AoilsB in suc& a ratio t&at t&e solu#ility o! t&e oil molecules in t&e soli% lipi% is e"cee%e%* T&e melte% lipi% an% t&e &ot oil are #len%e%M t&us) t&e t(o lipi%s must s&o( a misci#ility gap at t&e use% concentraE tions) at appro"imatel 9@]C* A &ot o1( nanoemulsion is pro%uce% at a &ig&er temperature Aappro"* =@]CB) t&en t&e lipi% %roplets are coole%* ,&en reac&ing t&e misci#ility gap) t&e oil precipitates !orming tiny oil %roplets in t&e melte% soli% lipi%* Su#seCuent soli%i!ication o! t&e soli% lipi% as soli% nanoparticle matri" lea%s to !i"ation o! t&e oily nanocompartments*

DE2E ?PMENT ?- C?SMETIC AND TRANSDERMA PR?DUCTS +ASED ?N S? ID IPID NAN?PARTIC ES AND NAN?STRUCTURED IPID CARRIERS T&e scienti!ic literature reports several approac&es !or t&e %evelopment o! pro%ucts #ase% on S Ns an% N Cs A9):B*

55@

Souto an% MJller

Incorporation o! Soli% ipi% Nanoparticles an% Nanostructure% ipi% Carriers into Semisoli% Preparations Similar to liposomes) polymeric nanoparticles) an% microsponge systems) aCueous S N or N C %ispersions can #e a%%e% to semisoli% preparations suc& as lotions) creams) an% &y%rogels A:@):4B* T&e a%vantage o! t&is proce%ure is t&e association o! a (ellEesta#lis&e% topical !ormulation (it& several attractive a%vantages o! t&e lipi% nanoparticles in t&e same !inal pro%uct* T&e incorporation o! S Ns or N Cs into a cream consists o! t&e a%%ition o! t&e lipi% nanoparticles as a &ig&ly concentrate% %ispersion) t&at is) (it& :@[ soli% ApartiE cleB content to a !res&ly prepare% o1( cream or %uring t&e pro%uction o! suc& cream A9>):5B* In t&e !irst case) a part o! t&e (ater in t&e cream !ormulation is replace% #y a &ig&ly concentrate% S N or N C %ispersion) an% a!ter t&at t&e pro%uction process is run normally* T&e lipi% nanoparticles are su!!iciently sta#ili/e% to avoi% t&eir coalesE cence (it& t&e inner oil %roplets o! t&e emulsion* I! t&e pro%uction process o! t&e emulsion is per!orme% at a temperature &ig&er t&an t&e melting point o! t&e lipi% nanoparticles) t&ese latter (ill melt #ut (ill recrystalli/e %uring t&e cooling at t&e en% o! t&e process* T&e secon% approac& is more elegant) #ut in t&is case t&e cream is pro%uce% as usual) &o(ever (it& a re%uce% (ater content in or%er to compensate !or t&e (ater a%%e% (it& t&e aCueous S N or N C %ispersion* A!ter t&e pro%uction o! t&e cream) t&e concentrate% S N or N C %ispersion is a%%e% #y stirring at room temperature* T&is process avoi%s t&e melting o! t&e nanoparticles) avoi%ing t&ere!ore un%esire% c&anges (it&in t&e internal particle structure* I! t&e aim is t&e a%%ition o! S Ns or N Cs to &y%rogels) t&e proce%ure is even simpler* T&e &y%rogels can #e previously prepare%) an% a!ter t&at S N or N C %isperE sion is %ilute% (it&in t&e semisoli% !ormulation A:@B* Alternatively) a concentrate% S N or N C %ispersion is a%%e% #e!ore t&e gelation process* Ho(ever) it s&oul% #e note% t&at electrolytes !reCuently use% to .elli!y synt&etic polymers can %esta#ili/e t&e lipi% nanoparticles* T&e sur!ace electrical c&arge A/eta potentialB is re%uce% lea%ing to aggregation o! suspen%e% particles* T&is p&enomenon s&oul% #e consi%ere% (&en) !or e"ample) a%%ing electrolytes in t&e !orm o! so%ium &y%ro"i%e !or t&e preparation o! car#omer gels* T&e type o! neutrali/ing agent a!!ects t&e aggregation o! S Ns an% N Cs) an% it can #e avoi%e% or minimi/e% (&en using Tristan b an% Neutrolb TE as neutrali/ing agents A9>B* As soon as t&e gel is !orme%) aggregation (ill not occur anymore #ecause S Ns an% N Cs (ill #e p&ysically sta#ili/e% an% entrappe% in t&e gel net(or$* In general) S N an% N C %ispersions) (&ic& are unsta#le #ecause o! su#optimal sur!actant com#ination) can #e !urt&er sta#ili/e% a!ter t&eir incorporation into &y%rogels* T&is opens t&e opportunity to use sta#ili/ers) (&ic& are not t&at e!!icient in provi%ing a longEterm sta#ility o! t&e aCueous %ispersion) #ut are regulaE tory accepte%* T&e S N an% N C %ispersions nee% to #e sta#le only !or t&e !e( &ours until t&ey are incorporate% into t&e semisoli% !ormulation) (&ere t&ey (ill #e sta#ili/e% #y t&e gel net(or$*

Pro%uction o! Gels +ase% on Soli% ipi% Nanoparticles an% Nanostructure% ipi% Carriers T&e pro%uction o! gels #ase% on S Ns an% N Cs consists o! topical !ormulations &aving only lipi% nanoparticles in t&eir composition* A lipi% nanoparticle %isperE sion is previously prepare%) an% t&en t&e gelling agent is a%%e%) pre!erentially nonelectrolyte agents) suc& as cellulose %erivatives A9>B or ot&er natural gums A:8B* -or many gel preparations) an amount o! 9[ to 4@[ o! lipi% nanoparticles is

ipi% Nanoparticles !or Cosmetic) Dermal) an% Trans%ermal Applications

554

su!!icient* To ma$e pro%uction more pro!ita#le) a concentrate% particle suspension Ae*g*) 9@[H:@[B can #e prepare% an% t&en %ilute% to t&e %esire% !inal concentration* T&is approac& &as certain a%vantages) suc& as t&e release o! actives #eing controlle% only #y t&e lipi% nanoparticle matri"* T&e %esire% properties can #e a%.uste% in a very controlle% (ay #y attenuating t&e lipi% nanoparticle !eatures*

Pro%uction o! Creams +ase% on Soli% ipi% Nanoparticles an% Nanostructure% ipi% Carriers T&e pro%uction o! creams #ase% on S Ns an% N Cs consists o! t&e pro%uction o! aCueous %ispersions o! lipi% nanoparticles in a &ig& concentration) up to :@[ or ;@[* ,&en applying &ig&er lipi% concentrations) ointments (it& a #icontinuous structure (ill #e !orme%) t&at is) t&e &omogeni/ation process lea%s to (ellE%e!ine% particles an% not to an ointmentEli$e system* It is $no(n t&at t&e viscosity o! t&e preparations increases (it& t&e lipi% conE centration A:4B) t&at is) at a#out 9@[ to 9:[ t&e !ormulations are creamEli$e) a#ove :@[ t&ey #ecome pasteEli$e) an% (&en increase% to a soli% content o! =@[ or >@[ t&e !ormulations are soli% an% can #e cut (it& a $ni!e* CreamEli$e an% pasteEli$e !ormulations are suita#le !or %ermal application o! actives) (&ereas t&e soli%Eli$e systems are usually o! &ig& interest !or oral a%ministration o! lipi% particles to e"ploit t&e a#sorptionEen&ancing e!!ect o! lipi%s A:9B* -or t&e pro%uction o! suc& !ormulations) a &ig&ly concentrate% stoc$ suspension A:@[H;@[ lipi% nanopartiE clesB is !irst prepare% #y HPH* T&en) a%%itional melte% lipi% is a%%e% step(ise an% %isperse% again) #uil%ing a concentration o! up to =@[ or >@[ o! soli% content* In t&e melte% state) t&e o#taine% pro%uct is still liCui% an% it &as a relatively lo( visE cosity* A!ter cooling %o(n) t&e system #ecomes creamEli$e) pasteEli$e) or soli%Eli$e*

-EATURES ?- S? ID IPID NAN?PARTIC ES AND NAN?STRUCTURED IPID CARRIERS AND S0IN E--ECTS P&ysical Sta#ility in ACueous Dispersions an% in Creams P&ysical sta#ility o! t&e aCueous S N an% N C %ispersions) t&at is) t&e a#sence o! particle aggregation an% creaming) is a prereCuisite !or t&e !ormulation o! cosmetic an% p&armaceutical pro%ucts #ase% on S Ns an% N Cs* ?(ing to t&eir small si/e in t&e nanometer range) lipi% nanoparticles are naturally sta#le) an% creaming o! aCueous %ispersions or nanoparticle se%imentation mig&t not occur* T&e particles are $ept in suspension #y t&e +ro(nian motion o! t&e (ater molecules* ?(ing to t&eir sur!ace electrical c&arge) t&e particles are sta#ili/e% #y electrostatics repulsion) an% t&e p&ysical sta#ility is even !urt&er en&ance% (&en sterically sta#ili/ing polymers) suc& as T(een =@ or polo"amer) are use% as sur!actants in t&e !ormulaE tion* T&e &ig& lipi% nanoparticle sta#ility as aCueous %ispersion &as #een reporte% !or more t&an t&ree years A::B* -urt&ermore) i! necessary S N an% N C can #e incorporate% in creams) gels) lotions) or #o%y mil$s) to increase t&eir p&ysical sta#ility as reporte% a#ove* Apart !rom aggregation an% creaming) anot&er type o! insta#ility coul% #e Cuestione%) t&at is) t&e %issolution o! lipi% nanoparticles in t&e liCui% oil o! t&e a#oveEmentione% semisoli% !ormulations* In contrast to liposomes) S Ns an% N Cs &ave t&e a%vantage t&at t&e sta#ility can #e proven Cuantitatively #y %i!!erential scanning calorimetry ADSCB* In t&e DSC measurements) t&e melting o! t&e nanoparE ticles an% t&e melting energy in 'oule per gram can #e %etermine%* Comparing t&e melting energy on t&e %ay o! nanoparticles incorporation into t&e cream (it& t&e

555

Souto an% MJller

melting energy o#taine% a!ter a certain storage time) (ill allo( t&e calculation o! t&e total percentage o! nanoparticles still present in t&e !ormulation* T&is is not possi#le to per!orm (it& liposomes* It is easy to prove t&e e"istence o! liposomes in a proE %uct a!ter certain storage time #y electron microscopy) &o(ever t&e Cuanti!ication o! t&eir num#er is not possi#le or it is e"tremely %i!!icult to per!orm Ain t&is case using) e*g*) Cuantitative electron microscopy analysisB* T&is is a ma.or o#stacle !or intro%ucing a liposomal cosmetic !ormulation into t&e mar$et in 'apan* Ho(ever) t&is is not a ma.or pro#lem (it& S Ns an% N Cs #ecause t&e e"act amount a!ter a certain storage time can #e easily analy/e% Cuantitatively #y DSC A:;B*

oa%ing Capacity) Entrapment E!!iciency) an% Controlle%ERelease Properties Important parameters to evaluate t&e suita#ility o! a carrier system are t&e loa%ing capacity an% t&e entrapment e!!iciency !or t&e active ingre%ients* A !ull range o! mo%el active ingre%ients &as #een incorporate% into S Ns an% N Cs* An up%ate% list &as #een recently provi%e% #y our group A:<B* oa%ing capacity o! 4@[ to 5@[ (as o#taine% !or tetracaine an% etomi%ate (it& p&ysical sta#ility o! t&e particles remaining A:=B Aplease note t&at t&e loa%ing capacity is calculate% in percent o! t&e lipi% massB* 2itamins A an% E an% t&eir %erivatives &ave #een incorE porate% into S Ns up to 5:[ A:;B* In t&is stu%y) t&e incorporation (as limite% #y t&e !act t&at &ig&er percentages %i% not lea% to any more soli% particles* Ho(ever) i! t&ese active ingre%ients are #len%e% (it& even &ig&er melting lipi%s) t&en t&e loa%ing capacity can #e !urt&er increase%* In case o! !ull misci#ility o! t&e active ingre%ient (it& t&e lipi%) loa%ing capacities o! :@[ an% more can #e ac&ieve%* T&e loa%ing capacity can #e 4@@[ in cases (&ere t&e active ingre%ient is lipoE p&ilic an% soli% itsel!* T&e entrapment e!!iciency is t&e percentage o! active ingre%ient (&ic& is entrappe% insi%e t&e lipi% particles* -or lipop&ilic active ingre%ients) t&e entrapment e!!iciencies are typically #et(een >@[ an% >=[* T&e lo(est values o#serve% (ere appro"imately =@[) !or e"ample) tetracaine A:>B an% clotrima/ole A;@B* -or &y%roE p&ilic compoun%s) t&e loa%ing capacity an% t&e entrapment e!!iciency are o#viously lo(er* 2alues o! a#out :@[ &ave #een o#taine% !or t&e e"tremely &y%rop&ilic mo%el compoun% Iotrolan) an WEray contrast agent A;4B* Iotrolan (as use% as a mo%el comE poun% #ecause o! its e"tremely &ig& &y%rop&ilicityM t(o parts o! Iotrolan %issolve in only one part o! (ater* Mo%el %rugs !or pepti%es an% proteins Ae*g*) lyso/ymeB &ave also #een incorporate%* It coul% #e proven t&at lyso/yme remaine% c&emically intact a!ter incorporation into t&e S N an% (as still active A;5B* ,it& &y%rop&ilic pepti%es) loa%ing capacities up to 5@[ &ave #een ac&ieve% !or prolonge% release !rom t&e particles in vivo in t&e animal mo%el* T&e release o! incorporate% ingre%ients !rom t&e lipi% nanoparticles can #e mo%ulate% accor%ing to t&e nee%s !rom very !ast to very slo(* -igure 5 compares t&e release o! clotrima/ole o#taine% !rom S Ns an% N Cs using -ran/ %i!!usion cells A;@B* In N C !ormulations) lipi% nanoparticles &ave a liCui% core) an% clotrima/ole is incorporate% in t&e oil less tig&tly in comparison to t&e soli% lipi% matri" o! S N !ormulations* In t&e latter) %rug molecules are incorporate% into t&e crystalline matri" an% t&eir %i!!usional mo#ility is %ecrease% A;@B* In general) %i!!usion t&roug& t&e carrier is t&e main mec&anism o! controlle% release as %escri#e% #y -ic$Ps la( o! %i!!usion A:B* Ho(ever) %rug %i!!usion

ipi% Nanoparticles !or Cosmetic) Dermal) an% Trans%ermal Applications

558

8@ 5: Drug 5@ release% N[O 4: 4@ : @ @ : 4@4: Time N&O 5@

S N N C

5:

-IGURE 5 Clotrima/ole release pro!iles !rom tripalmitinE#ase% soli% lipi% nanoparticles an% nano structure% lipi% carrier* A##reviations6 N C) nanostructure% lipi% carrierM S N) soli% lipi% nanoparticle* Source6 -rom Re!* ;@*

coe!!icient cannot #e consi%ere% constant) #ut it is %epen%ent upon %rug concenE tration* ?(ing to a large %rug loa%ing) t&e %egree o! %i!!usion can #e %ecrease%* T&ere are too many molecules trying to %i!!use an% t&ey limit t&eir o(n permeE ation A&in%ering e!!ectsB* Also) t&e previously %escri#e% incorporation mo%els !or S Ns A%rugEenric&e% core or s&ellB un%erline t&ese !in%ings* Drug loa%ing is very important (it& regar% to release c&aracteristics A;B* Generally) t&e increase o! %rug loa%ing lea%s to an acceleration o! t&e %rug release* Ho(ever) in particular cases) increasing t&e %rug loa%ing may slo( %o(n t&e release) (&ic& can #e e"plaine% #y possi#le %rug crystalli/ation insi%e t&e nanoparticles* -igure 8 s&o(s t&e re%istri#ution e!!ect occurring %uring t&e S N pro%uction #y t&e &ot HPH tec&niCue A;B* Accor%ing to c&aracteristics) suc& as %rug solu#ility an% its partitioning coe!!icient) %ispersing t&e %rugEcontaining lipi% melt in a &ot aCueous sur!actant solution (ill lea% to %istri#ution o! %rug into t&e aCueous p&ase* I! t&e aCueous p&ase contains a &ig&er sur!actant concentration) in most cases t&is lea%s to a #etter solu#ility o! t&e lipop&ilic %rug in t&e (ater p&ase Ae*g*) #y solu#iliE /ationB an% t&us more pronounce% %istri#ution o! %rug to t&e (ater p&ase* T&e solu#ility o! t&e active ingre%ient in t&e (ater p&ase can #e !urt&er increase% #y c&oosing &ig&er pro%uction temperatures) t&at is) in t&is case more active (ill partition to t&e (ater p&ase A;B* T&e opposite e!!ect (ill occur (&en t&e o#taine% &ot nanoemulsion is in t&e cooling process) t&at is) t&e solu#ility o! t&e active in t&e aCueous p&ase %ecreases* ipi% starts to precipitate !orming a lipi% core (it& a lo(er active concentration t&an in t&e original %rugEcontaining lipi% melt* -urt&er cooling lea%s to !urt&er re%uction o! t&e active solu#ility in t&e (ater p&ase an% re%istri#ution #ac$ to t&e lipi% p&ase) &o(ever %ue to t&e !ormaE tion o! t&e soli% lipi% core) only t&e outer s&ell is accessi#le !or t&e active* In t&is case) most o! t&e active (ill #e release% in t&e !orm o! a #urst* T&e e"tent o! t&e #urst release can #e) t&ere!ore) mo%i!ie% #y controlling t&e amount o! active in t&e outer s&ell o! t&e o#taine% particles* T&is p&enomenon &as #een o#serve% as a

559

Souto an% MJller

-IGURE 8 Partitioning e!!ect o! %rug %uring pro%uction o! lipi% nanoparticles #y &ig&Epressure &omogeni/ation tec&niCue* Source6 -rom Re!* ;*

!unction o! pro%uction temperature an% sur!actant concentration* +urst release can also #e avoi%e% applying t&e col% HPH process A;8);9B* C&emical Protection o! Incorporate% Su#stances It is $no(n t&at sta#ili/ation o! c&emically la#ile actives against %egra%ation Ae*g*) &y%rolysis an% o"i%ationB can #e ac&ieve% using a soli% matri" suc& as t&e one o! polymeric particles A;:B* T&is is also vali% !or t&e lipi% nanoparticles* T&e sta#ility o! retinol an% coen/yme 74@ coul% #e en&ance% (&en incorporate% into lipi% nanoparE ticles ma%e !rom a mi"ture o! Compritol b=== AT? an% Miglyolb=45 A;;);<B* 0etocona/ole coul% also #e protecte% to a relative e"tent using t&e same lipi% A;=B* ?(ing to t&e !lui% c&aracter o! carriers suc& as liposomes an% emulsions) t&e active ingre%ients (ill partition #et(een t&e liCui% oil an% (ater p&ases A:;B* Permanent e"c&ange o! molecules #et(een t&ese t(o p&ases (ill %ecompose t&e active molecules in t&e (ater p&ase) an% simultaneously non%egra%e% compoun% (ill partition !rom t&e oil to t&e (ater p&ase A#ase% on partitioning coe!!icient a!ter NernstB* T&e partitioning ratio #et(een non%egra%e% an% %egra%e% active (ill #e maintaine% A-ig* 9) upperB* In comparison to a soli% matri" AS N or N CB) t&e active (ill #e in t&is case !i"e% insi%e t&e lipi% particles* E"c&ange #et(een t&e inner an% outer p&ases (ill not &appen or it (ill occur very slo(ly A-ig* 9) lo(erB* T&ese o#serE vations emp&asi/e t&e protection an% c&emical sta#ili/ation e!!ects o! incorporate% actives into S Ns an% N Cs A;>B*

?cclusive E!!ects an% S$in Hy%ration ?(ing to t&e small particle si/e o! S Ns an% N Cs) t&ese carriers s&o( a%&esive properties A:5)<@B) properties t&at &ave also #een o#serve% (&en using liposomes* ,&en in contact (it& t&e s$in) lipi% nanoparticles create a t&in !ilm (it& very narro(

ipi% Nanoparticles !or Cosmetic) Dermal) an% Trans%ermal Applications

55:

-IGURE 9 C&emical protection o! la#ile actives in emulsion %roplets versus lipi% nanoparticles* -ree partitioning o! non%egra%e% an% %egra%e% active molecules in t&e emulsion system AupperB) #ut &in%ere% #y t&e soli% state o! t&e lipi% nanoparticles Alo(erB* Source6 -rom Re!* ;;*

interspaces #et(een t&e particles* T&e !ormation o! t&is !ilm can #e sense% (&en applying an S N or N C !ormulation onto t&e s$in* Even relatively small amounts o! lipi% nanoparticles in a cream Ae*g*) :[B can create t&is !ilm A:;B* -igure : s&o(s t&at t&e relatively &ig& occlusivity is a special !eature o! lipi% nanoparticles A-ig* :) lo(erB) (&en compare% (it& lipi% microparticles A-ig* :) upperB o! i%entical lipi% content* T&e rat&er lo( occlusivity o! microparticles is attri#ute% to t&e large spaces #et(een t&e micrometer carriers still allo(ing t&e evaporation o! (ater* T&e small spaces AcapillariesB #et(een t&e nanoparticles are &y%ro%ynamically un!avora#le an% limit (ater loss Asmall %iameter o! pores) &ig&er resistance to (ater vapor !lo(B* T&is !ilm &in%ers (ater evaporation) (&ic& means t&at an occlusive e!!ect lea%s to increase% s$in &y%ration* T&is property mig&t #e very interesting !or s$in protection %uring t&e %ay) (&en applying place#o S Ns or N Cs* T&e s$in constantly comes into contact (it&

55;

Souto an% MJller

-IGURE : Mec&anism o! t&e occlusion e!!ect %epen%ing on t&e particle si/e* A soli% lipi% microE particle %ispersion A%iameter 4 _m) upperB in comparison to an aCueous soli% lipi% nanoparticles or nanostructure% lipi% carrier %ispersion A%iameter 58@ nm) lo(erB* Source6 -rom Re!* ;;*

a (i%e variety o! c&emicals (&ic& are normally present in t&e atmosp&ere* T&ese inclu%e (ater an% %issolve% mineral salts present as !ree electrolytes toget&er (it& t&e atmosp&eric gases* S$inPs permea#ility to t&ese %i!!erent su#stances varies (i%ely* ?cclusion o! t&e s$in lea%s to increase% &y%ration an% su#seCuently to t&e smoot&ing o! (rin$lesRan e!!ect utili/e% in many cosmetic pro%ucts* ?n t&e #asis o! t&is property) S NE an% N CEcontaining pro%ucts are e"pecte% to &ave also antiE aging e!!ects) especially (&en preparing t&em (it& s$inEcaring active ingre%ients) suc& as several vitamins an% cerami%es* Cerami%es can #e #len%e% (it& &ig&er melting lipi%s lea%ing to soli% nanoparticles) an% (&en applie% t&ey (ill promote restoration o! t&e %amage% protective lipi% layer o! t&e s$in A9B*

Penetration En&ancement o! Incorporate% Su#stances ?(ing to occlusion properties o! lipi% nanoparticles an% su#seCuent increase% s$in &y%ration) t&ese carriers can improve t&e penetration o! t&e incorporate% actives into t&e s$in* T&e penetrationEen&ancement e!!ect o! lipi% nanoparticles &as #een teste% (it& tocop&erol an% tocop&erol acetate* ipi% nanoparticles loa%e% (it& t&ose actives &ave #een applie% to t&e s$in an% t&e penetration compare% to alco&olic solution o! t&e same compoun%s #y applying a stripping test Acumulative penetration as a !unction o! t&e num#er o! stripsB* Penetration o#serve% (it& lipi% nanoparticle !ormulations (as t(ice as &ig& as o#taine% (it& t&e re!erence% alco&olic solution* In a%%ition) t&ese %ata also &ave s&o(n t&at active ingre%ients incorporate% into lipi% nanoparticles (ere o#viously release% !rom t&e carriers (&en applie% to t&e s$in A;;B*

ipi% Nanoparticles !or Cosmetic) Dermal) an% Trans%ermal Applications

55<

S? ID IPID NAN?PARTIC ES AND NAN?STRUCTURED IPID CARRIERS AS 2EHIC ES -?R C?SMETIC) DERMA ) AND TRANSDERMA ACTI2ES T&e intro%uction o! lipi% particles into p&armaceutical tec&nology is reporte% to #e very ol%) an% recently attempts &ave #een ma%e to intro%uce lipi% nanoparticles into t&e mar$et #y cosmetic an% p&armaceutical companies* T&e !irst pro%uct is alrea%y on t&e mar$et an% it &as #een intro%uce% #y t&e company 3amanouc&i Ae*g*) Nano#asebB in Polan%) covere% #y t&e 3amanouc&i patent A<4B* T&is pro%uct contains activeE!ree lipi% nanoparticles an% it is inten%e% !or cosmetic purposes* Even t&oug& cosmetic !ormulations %o not necessarily &ave any p&ysiological !unction) t&ey resem#le topical applications !or %ermatological use in many aspects* -or e"ample) t&e purpose o! a lipstic$ is essentially %ecorative) #ut its !ormulation em#o%ies a num#er o! e"cipients commonly use% in p&armaceutical pro%ucts* Having consi%ere% t&e (ays in (&ic& t&erapeutic applications are !ormulate%) it is only necessary to e"amine t&e mo%i!ications o! t&ese tec&niCues employe% in t&e cosmetic !iel%) (it& special re!erence to !ragrance) !eel) !res&ness) appearance) an% s$in persisE tence* -actors suc& as personal taste an% !as&ion also &ave a strong in!luence (&en people purc&ase t&e pro%uct* T&us) in or%er to %evelop a success!ul pro%uct) t&e !ormulation must e"&i#it #ot& !unctional attri#utes an% est&etic appeal* +esi%es lipstic$s) !acial ma$eup pro%ucts can also #e %evelope% t&at &ave lipi% nanoparticles* T&ey serve to improve t&e uni!ormity o! coloring) a%% ne( color) %isguise #lemis&es) an% impart a smoot& matt !inis&* T&e !ace e"poses t&e greatest single area o! t&e #o%y sur!ace to climatic in!luence an% in vie( o! t&e ma.or est&etic signi!icance o! its appearance) t&ere is a real nee% !or protection* T&e relative t&inness o! t&e !acial epi%ermis a%%s !urt&er importance to suc& protection* Also cleansing creams) (&ic& are oilEmisci#le preparations use!ul to remove ma$eup resi%ues (it&out t&e energetic %egreasing t&at may result !rom using soap an% (ater) can #e !ormulate% (it& S Ns an% N Cs* T&e aCueous p&ase o! t&e %ispersion &as sur!acE tant properties to remove t&e ma$eup (&ile t&e lipi% nanoparticles a%&ere to t&e s$in protecting it* ?t&er e"amples are !ace po(%ers Alipi% nanoparticles can #e sprayE%rie%B !or a #asis coloring or mas$ing to cover minor %e!ects) lipstic$s) &air %ressings) nail preparations) %epilatories) an% s&aving ai%s* Accor%ing to mar$et stu%ies) an important !actor !or a cosmetic to #e purc&ase% is not only t&at t&e pac$aging must &ave an est&etic appeal) #ut so must t&e appearE ance o! t&e !ormulation itsel!* ,&ite pro%ucts are pre!erre% #y t&e consumer) lipi% nanoparticles can #e use% to (ea$en) !or e"ample) yello(is& actives Acoen/yme 74@) vitamin CB* T&is (&itening e!!ect is o! special interest !or actives (&ic& are %egra%e% (it& %egra%ation pro%ucts possessing a color) even i! t&ey %o not in!luence t&e pro%uct Cuality A9B* ,&itening an% lig&tening properties o! lipi% nanoparticles are currently #eing e"plore% A<4H<9B* Deo%orants an% antiperspirants can also &ave lipi% nanoparticles particularly i! !ormulate% (it& antiseptics) !or e"ample) &e"ac&lorop&ene or tric&lorop&ane) (&ic& appear to increase t&e permea#ility o! t&e s(eat %uct an% t&us re%uce t&e amount o! t&e secretion reac&ing t&e sur!ace o! t&e s$in* Aging o! t&e s$in &as #een attri#ute% to a c&ange in t&e circulating se" &ormones) #ut alterations o! t&e collagen an% elastic tissues) (&ic& are accelerate% #y c&ronic U2 e"position) is pro#a#ly more signi!icant* It is) t&ere!ore) li$ely t&at U2Ea#sor#ing cosmetics s&oul% &ave a truly protective role in t&is conte"t* Molecular U2 #loc$ers &ave #een incorporate% into lipi% nanoparticles A<:H=@B*

55=

Souto an% MJller

A si%e e!!ect o! t&ese sunscreens is penetration into t&e s$in lea%ing to s$in irritaE tion or even allergic reactions A=4B* Particulate U2 #loc$ers !reCuently use% are titanium %io"i%e particles (&ic& &ave also #een loa%e% into N Cs A=5B* T&ere is also an ongoing controversial %iscussion (&et&er an% to (&at e"tent titanium %io"i%e particles penetrate into t&e s$in A=8)=9B (&ere t&ey can potentially interact (it& t&e immune system A=:B* A!ter incorporation o! molecular U2 #loc$ers into lipi% nanoparticles) a synergistic e!!ect o! t&e U2 scattering cause% #y t&e S N t&emselves an% t&e U2 a#sor#ance o! t&e molecular sunscreen (as o#serve% A=;B* T&is opens t&e Cuestion o! re%ucing t&e concentration o! t&e molecular sunscreen) an% simultaneously its potential si%e e!!ects) (&ile also maintaining t&e U2EprotecE tive level* Apart !rom re%uction o! si%e e!!ects) t&is result is o! commercial interest !or e"pensive U2 #loc$ers* In a%%ition) it (as !oun% t&at penetration o! molecular sunscreens into t&e s$in (as re%uce% (&en comparing S Ns to a tra%itional o1( emulsion system o! similar composition A<=B* T&e only element common to nearly all cosmetic pro%ucts is t&e employment o! a per!ume to impart a pleasing !ragrance* ipi% nanoparticles can also #e use% !or prolonge% release o! per!umes) !ragrances) an% insect repellents) in comparison to o1( emulsions an% Eau %e Toilettes A=9B* T&e release can #e slo(e% %o(n #y incorporating t&e per!ume in a soli% matri" instea% in a liCui% lipi% particle Aoil %ropE letB A<:B* T&is is very interesting i! t&e aim is to create a once a %ay application (it& continuing scent* -un%amentally) per!umes are %erive% !rom t&e essential oils o! #otanical origin an% most !ormulae still rely to a consi%era#le e"tent in t&e use o! natural oils* T&us) N Cs are suita#le to %eliver oily !ragrances %ue to t&e presence o! an inner liCui% core in t&eir structure* Alt&oug& oily !ragrances are Cuite e"pensive) using a prolonge% release system suc& as N C) lo(er amounts o! oil (ill #e nee%e%M in a%%ition) t&ey can #e use% in soaps an% less e"pensive pro%ucts* Prolonge% release (as also o#serve% !or insect repellents) suc& as lemon oil A<:)==H>@B* Dermal an% trans%ermal !ormulations may also #e compoun%e% as !reeE !lo(ing soli%s Apo(%ersB) semisoli%s) or liCui%s* T&e %ecision to employ a particular %osage !orm is mainly %epen%ent on consi%erations o! !unctional suita#ility* T&e e!!ectiveness o! #ot& topical an% trans%ermal systems is relate% to t&e e"tent o! percutaneous a#sorption o! t&e %rug* T&us) t&ere is a ten%ency to vie( t&ese systems as #eing closely relate% in terms o! !unctionality* Ho(ever) (&en systems suc& as S Ns an% N Cs are place% on t&e s$in to %eliver t&e incorporate% %rugs) t&ese can act6 AiB in t&e local tissues imme%iately #eneat& t&e application site) AiiB in t&e %eep regions in t&e vicinity o! t&e application site) an% AiiiB in t&e systemic circulation* ?! course) (&en moving %o(n t&is progression) t&e %rugE%elivery c&allenge #ecomes increasingly %i!!icult* To ac&ieve systemic circulation) %rug release over long periE o%s must #e provi%e%* T&e main !eature o! lipi% nanoparticles inten%e% !or %ermal an% trans%ermal %elivery o! %rugs is t&eir controlle%Erelease properties A4@)>4B* T&e release pro!ile accomplis&e% #y lipi% nanoparticles is %epen%ent on t&eir structure A;@B* As %iscusse% previously) %epen%ing on t&e pro%uction met&o% A&ot vs* col% &omogeni/ationB) t&e composition o! t&e !ormulation Ai*e*) sur!actant) lipi%sB) t&e solu#ili/ing properties o! t&e matri" !or t&e %rug nanoparticles (it& a %i!!erent structure (ill #e o#taine% A:);8);9B* Depen%ing on t&e matri" structure) t&e release pro!iles (ill vary !rom very !ast) me%ium) or e"tremely prolonge% release A;@B* T&e %egree o! t&e initial #urst release) !or e"ample) %esire% !or topical applications) coul% #e e"plaine% #y t&e %issolution properties o! t&e %rugs %uring t&e pro%uction process #eing a !unction o! lipi%) sur!actant) an% pro%uction temperature* T&e un%erstan%ing o! t&is mec&anism allo(s t&e controlle% pro%uction o! lipi% nanoparticles (it& a %e!ine% initial %ose*

ipi% Nanoparticles !or Cosmetic) Dermal) an% Trans%ermal Applications

55>

To reac& p&armacologically a%eCuate systemic levels in trans%ermal t&erapy) a su!!icient amount o! %rug nee%s to cross t&e s$in !rom t&e application site to t&e circulation* Conventional trans%ermal systems are ointments an% a%&esive systems o! precisely %e!ine% si/e) !or (&ic& i%eally t&ere (oul% #e no local accumulation* Ho(ever) in t&ose cases t&e %rug molecules are !orce% to cross t&roug& a small %i!E !usional (in%o( %e!ine% #y t&e contact area o! t&e trans%ermal system* ConseCuently) irritation an%1or sensiti/ing e!!ects un%erlying t&is system mig&t #e o#serve%* To overcome suc& inconvenience) t&e use o! lipi% nanoparticles mig&t #e #ene!icial particularly %ue to t&e !act t&at t&ey are compose% o! p&ysiological an% (ellE accepte% lipi%s* -urt&ermore) s$in lipi%s A!atty aci%s) cerami%esB can also !igure in t&e lipi% nanoparticle composition* In a%%ition to c&emical sta#ili/ation o! incorporate% %rugs A;=B) %rug penetration can #e improve% #y occlusion an% !urt&er &y%ration e!!ects*

C?NC UDING REMAR0S T&ere is no %ou#t t&at lipi% nanoparticles com#ine a%vantages o! ot&er carrier sysE tems) suc& as emulsions) liposomes) an% polymeric nanoparticles* Consi%ering t&eir special properties) S Ns an% N Cs (ill !in% applications in cosmetic pro%ucts #ut also in p&armaceutical !ormulations) t&at is) !or %ermal an% trans%ermal %elivery* ?ver t&e last %eca%e) %ermal an% trans%ermal %elivery o! %rugs &as #ecome one o! t&e most promising areas o! p&armaceutical researc& an% %ue to intensive e!!orts) t&ousan% o! patents &ave #een !ile% %ealing (it& ne( concepts an% ne( tec&nologies* Apart !rom t&eir special !eatures) ma.or a%vantages o! S Ns an% N Cs are %e!initely t&e possi#ility !or largeEscale pro%uction) t&e costEe!!ective pro%uction met&o%) an% t&e relatively lo( cost o! e"cipients an% ot&er components* ?(ing to t&e (orl%(i%e patent protection) t&e pro%uct e"clusivity can #e guarantee% giving no a%vantage to competitors* Registere% tra%e names (orl%(i%e o!!er t&e possi#ilE ity !or sale using a pro%uct name optimi/e% consi%ering mar$eting aspects* P&armaceutical an% cosmetic preparations are ma%e in a (i%e range o! #atc& si/es an% t&e scale o! operations strongly in!luences t&e !inal Cuality o! t&e o#taine% pro%uct* ,&erever t&ose !ormulations are prepare%) ra( materials &ave to #e store%) t&e ingre%ients an% t&e !inal pro%uct &ave to #e trans!erre% !rom one point to anot&er* T&ey &ave to #e transporte% an% processe% in contact (it& various constructional materials an% !inally store% in #ul$ #e!ore trans!er to !inal containE ers* ,&en %ealing (it& aCueous S N an% N C %ispersions) t&ese nee% to #e p&ysiE coc&emically sta#le to avoi% !ailure o! t&e scale o! pro%uction* argeEscale pro%uction is possi#le !or S Ns an% N Cs) (&ic& means t&at not only t&e eCuipment is availa#le) #ut also t&e o#taine% #atc&es are p&ysicoc&emically sta#le A=5)>5B*

RE-ERENCES
4* Ha%gra!t '* Passive en&ancement strategies in topical an% trans%ermal %rug %elivery* Int ' P&arm 4>>>M 4=964H;* 5* MJller RH) uc$s 'ES* A/neisto!!trIger aus !esten ipi%teilc&enH!este ipi% Nanosp&Iren AS NB) Germany* European Patent @;@:9><) 4>>;* 8* MJller RH) MI%er 0) ippac&er A) 'enning 2* -estE!lJssig A&al#!esteB ipi%parti$el un% 2er!a&ren /ur Herstellung &oc&$on/entrierter ipi%parti$el%ispersionen* PCT applicaE tion PCT1EP@@1@9:;:) 4>>=* 9* MJller RH) Me&nert ,) Souto E+* Soli% lipi% nanoparticles AS NB an% nanostructure% lipi% carriers AN CB !or %ermal %elivery* In6 +ronaug& ) e%* Percutaneous A#sorption* Ne( 3or$6 Marcel De$$er) 5@@:6<4>H<8=*

58@

Souto an% MJller

:* MJller RH) Ra%t$e M) ,issing SA* Soli% lipi% nanoparticles AS NB an% nanostructure% lipi% carriers AN CB in cosmetic an% %ermatological preparations* A%v Drug Deliv Rev 5@@5M :96S484HS4::* ;* MJller RH) MI%er 0) Go&la S* Soli% lipi% nanoparticles AS NB !or controlle% %rug %eliveryRa revie( o! t&e state o! art* Eur ' P&arm +iop&arm 5@@@M :@64;4H4<<* <* Sie$mann +) ,estesen 0* Su#micron lipi% suspensions Asoli% lipi% nanoparticlesB versus lipi% nanoemulsions6 similarities an% %i!!erences* In6 +enita S) e%* Su#micron Emulsions in Drug Targeting an% Delivery* Amster%am6 Har(oo% Aca%emic Pu#lis&ers) 4>>=65@:H54=* =* MJller RH) Ra%t$e M) ,issing SA* Nanostructure% lipi% matrices !or improve% microencapsulation o! %rugs* Int ' P&arm 5@@5M 5956454H45=* >* MJller RH) Ra%t$e M) ,issing SA* Soli% lipi% nanoparticles an% nanostructure% lipi% carriers* In6 Nal(a HS) e%* Encyclope%ia o! Nanoscience an% Nanotec&nology* os Angeles) Cali!ornia6 American Scienti!ic Pu#lis&ers) 5@@9698H:;* 4@* MJller RH) ,issing SA* ipopearls !or topical %elivery o! active compoun%s an% conE trolle% release* In6 Rat&#one M') Ha%gra!t ') Ro#erts MS) e%s* Mo%i!ie%ERelease Drug Delivery Systems* Ne( 3or$6 Marcel De$$er) 5@@86:<4H:=<* 44* Me&nert ,) MI%er 0* Soli% lipi% nanoparticlesRpro%uction) c&aracteri/ation an% appliE cations* A%v Drug Deliv Rev 5@@4M 9<64;:H4>;* 45* +un.es H) ,estesen 0) 0oc& MH'* Crystalli/ation ten%ency an% polymorp&ic transitions in triglyceri%e nanoparticles* Int ' P&arm 4>>;M 45>64:>H4<8* 48* Hunter R'* -oun%ations o! Colloi%al Science* ?"!or%6 ?"!or% University Press) 4>=;* 49* ,estesen 0) +un.es H* Do nanoparticles prepare% !rom lipi%s soli% at room temperature al(ays possess a soli% lipi% matri"T Int ' P&arm 4>>:M 44:645>H484* 4:* Hagemann ',* T&ermal #e&aviour an% polymorp&ism o! acylglyceri%es* In6 Garti N) Sato 0) e%s* Crystalli/ation an% Polymorp&ism o! -ats an% -atty Aci%s* Ne( 3or$6 Marcel De$$er) 4>==6>H>;* 4;* HernCvist * Crystal structures o! !ats an% !atty aci%s* In6 Garti N) Sato 0) e%s* Crystalli/ation an% Polymorp&ism o! -ats an% -atty Aci%s* Ne( 3or$6 Marcel De$$er) 4>==6><H48<* 4<* MJller +,* Suppositorien* Stuttgart6 ,issensc&a!tlic&e 2erlagsgesellsc&a!t m#H) 4>=;* 4=* 0risc&ner H* Ein!J&rung in %ie RFntgen!einstru$turanalyse* 2ol* 9* Au!lage6 2ie(eg 2erlag +raunsc&(eig) 4>>@* 4>* ,estesen 0) +un.es H) 0oc& MH'* P&ysicoc&emical c&aracteri/ation o! lipi% nanopartiE cles an% evaluation o! t&eir %rug loa%ing capacity an% sustaine% release potential* ' Control Release 4>><M 9=6558H58;* 5@* Speiser P* ipi%nanopellets als TrIgersystem !Jr Ar/neimittel /ur peroralen An(en%ung* European Patent EP @4;<=5:) 4>>@* 54* Dom# A'* iposp&eres !or controlle% %elivery o! su#stances* USP Patent USS 4===8<) 4>>8* 55* MJller RH) uc$s 'S* Ar/neisto!!trIger aus !esten ipi%teilc&en) -este ipi%nanosp&Iren AS NB) 4>>4* 58* Gasco MR* Met&o% !or pro%ucing soli% lipi% microsp&eres &aving a narro( si/e %istriE #ution* US Patent :)5:@)58;) 4>>8* 59* Hall#erg D) Holm I) ?#el A ) Sc&u#ert& ?) ,retlin% A* -at emulsion !or complete intravenous nutrition* Postgra% Me% 4>;<M 956A49>HA4:5* 5:* ,retlin% A* Development o! !at emulsions* ' Parenter Enteral Nutr 4>=4M :658@H58:* 5;* ,retlin% A* Recollections o! pioneers in nutrition6 lan%mar$s in t&e %evelopment o! parenteral nutrition* ' Am Coll Nutr 4>>5M 4468;;H8<8* 5<* Cavalli R) Caputo ?) Marengo E) Pattarino -) Gasco MR* T&e e!!ect o! t&e components o! microemulsions on #ot& si/e an% crystalline structure o! soli% lipi% nanoparticles AS NB containing a series o! mo%el molecules* Die P&arma/ie 4>>=M :868>5H8>;* 5=* Cavalli R) Marengo E) Ro%rigue/ ) Gasco MR* E!!ects o! some e"perimental !actors on t&e pro%uction process o! soli% lipi% nanoparticles* Eur ' P&arm +iop&arm 4>>;M 98644@H44:* 5>* S.FstrFm +) +ergenst&l +* Preparation o! su#micron %rug particles in lecit&inEsta#ili/e% o1( emulsions* I* Mo%el stu%ies o! t&e precipitation o! c&olesteryl acetate* Int ' P&arm 4>>56 ==6:8H;5*

ipi% Nanoparticles !or Cosmetic) Dermal) an% Trans%ermal Applications

584

8@* -essi C) Devissaguet 'EP) Puisieu" -) T&ies C* Process !or t&e preparation o! %ispersi#le colloi%al systems o! a su#stance in t&e !orm o! nanoparticles* US Patent :)44=):5=) 4>>5* 84* 7uintanarEGuerrero D) -essi H) AllGman E) Doel$er E* Pseu%olate" preparation using a novel emulsionE%i!!usion process involving %irect %isplacement o! partiallyE(aterE misci#le solvents #y %istillation* Int ' P&arm 4>>>M 4==64::H4;9* 85* Heurtault +) Saulnier P) Pec& +) Proust 'EE) Ric&ar% ') +enoit 'EP* Nanocapsules lipi%iCues) procG%G %e prGparation et utilisation comme mG%icament) -rance) 5@@@* 88* Heurtault +) Saulnier P) Pec& +) Proust 'EE) +enoit 'EP* A novel p&ase inversionE#ase% process !or t&e preparation o! lipi% nanocarriers* P&arm Res 5@@5M 4>6=<:H==@* 89* Sie$mann +) ,estesen 0* Investigations on soli% lipi% nanoparticles prepare% #y precipiE tation in o1( emulsions* Eur ' P&arm +iop&arm 4>>;M 9864@9H4@>* 8:* Reit&meier H) Herrmann ') GFp!eric& A* Development an% c&aracteri/ation o! lipi% microparticles as a %rug carrier !or somatostatin* Int ' P&arm 5@@4M 54=6488H498* 8;* GarcXaE-uentes M) Torres D) Alonso M'* Design o! lipi% nanoparticles !or t&e oral %elivery o! &y%rop&ilic macromolecules* Colloi% Sur! + 5@@5M 5<64:>H4;=* 8<* Sc&u#ert MA) MJllerEGoymann CC* Solvent in.ection as a ne( approac& !or manu!acE turing lipi% nanoparticlesRevaluation o! t&e met&o% an% process parameters* Eur ' P&arm +iop&arm 5@@8M ::645:H484* 8=* Du#es A) ParrotE ope/ H) A#%el(a&e% ,) et al* Scanning electron microscopy an% atomic !orce microscopy imaging o! soli% lipi% nanoparticles %erive% !rom amp&ip&ilic cyclo%e"trins* Eur ' P&arm +iop&arm 5@@8M ::65<>H5=5* 8>* Hu -7) 3uan H) V&ang HH) -ang M* Preparation o! soli% lipi% nanoparticles (it& clo#etasol propionate #y a novel solvent %i!!usion met&o% in aCueous system an% p&ysiE coc&emical c&aracteri/ation* Int ' P&arm 5@@5M 58>6454H45=* 9@* Hu -7) Hong 3) 3uan H* Preparation an% c&aracteri/ation o! soli% lipi% nanoparticles containing pepti%e* Int ' P&arm 5@@9M 5<865>H8:* 94* Trotta M) De#ernar%i -) Caputo ?* Preparation o! soli% lipi% nanoparticles #y a solvent emulsi!icationE%i!!usion tec&niCue* Int ' P&arm 5@@8M 5:<64:8H4;@* 95* S&a&gal%ian P) Gual#ert ') Assa 0) Coleman A,* A stu%y o! t&e !ree/eE%rying con%itions o! cali"arene #ase% soli% lipi% nanoparticles* Eur ' P&arm +iop&arm 5@@8M ::64=4H4=9* 98* Heurtault +) Saulnier P) Pec& +) et al* T&e in!luence o! lipi% nanocapsule composition on t&eir si/e %istri#ution* Eur ' P&arm Sci 5@@8M 4=6::H;4* 99* Souto E+* S N an% N C !or topical %elivery o! anti!ungals* P&*D* T&esis* +erlin6 -ree University o! +erlin) 5@@:* 9:* Me&nert ,) /ur MJ&len A) Dingler A) ,ey&ers H) MJller RH* Soli% lipi% nanoparticles AS NBRein neuartiger ,ir$sto!!ECarrier !Jr 0osmeti$a un% P&arma/euti$a* II* ,ir$sto!!E In$orporation) -reiset/ung un% Sterili/ier#ar$eit* P&arm In% 4>><M :>6:44H:49* 9;* /ur MJ&len A* -este ipi%HNanoparti$el mit prolongierter ,ir$sto!!li#eration6 Herstellung) ang/eitsa#ilitIt) C&ara$terisierung) -reiset/ungsver&alten un% Mec&anismen* P&*D* T&esis* +erlin6 -reie UniversitIt +erlin) 4>>;* 9<* u$o(s$i G) ,erner U* Investigation o! sur!ace an% %rug release o! soli% lipi% nanoparE ticles loa%e% (it& acyclovir* Int Symp Control Release +ioact Mater 4>>=M 5:695:H95=* 9=* 'enning 2* -este ipi%ENanoparti$el AS NB als TrIgersystem !Jr %ie %ermale Applil$ation von Retinol* P&*D* T&esis* +erlin6 -reie UniversitIt +erlin) 4>>>* 9>* 'enning 2) Sc&I!erE0orting M) Go&la S* 2itamin AEloa%e% soli% lipi% nanoparticles !or topical use6 %rug release properties* ' Control Release 5@@@M ;;644:H45;* :@* Souto E+) ,issing SA) +ar#osa CM) MJller RH* Evaluation o! t&e p&ysical sta#ility o! S N an% N C #e!ore an% a!ter incorporation into &y%rogel !ormulations* Eur ' P&arm +iop&arm 5@@9M :=6=8H>@* :4* Souto E+) MJller RH* R&eological an% in vitro release #e&aviour o! clotrima/oleEcontaining aCueous S N %ispersions an% commercial creams* Die P&arma/ie* :5* ,issing SA) ippac&er A) MJller RH* Investigations on t&e occlusive properties o! soli% lipi% nanoparticles AS NB* ' Cosmet Sci 5@@4M :56848H859* :8* Souto E+* S N an% N C as %rug carriers o! clotrima/ole !or topical !ormulations* Master T&esis* ?porto6 ?porto University) 5@@8* :9* MJller RH) 0ec$ CM* C&allenges an% solutions !or t&e %elivery o! #iotec& %rugsRa revie( o! %rug nanocrystal tec&nology an% lipi% nanoparticles* ' +iotec&nol 5@@9M 44864:4H4<@*

585

Souto an% MJller

::* MJller RH) Dingler A) ,ey&ers H) /ur MJ&len A) Me&nert ,* Soli% lipi% nanoparticlesH ein neuartiger ,ir$sto!!ECarrier !Jr 0osmeti$a un% P&arma/euti$a* 8* Mitteilung6 ang/eitsta#ilitIt) Ge!rierE un% SprJ&troc$nung) To"i/itIt) An(en%ung in 0osmeti$a un% P&arma/euti$a* P&arm In% 4>><M :>6;49H;4>* :;* Dingler A) Hil%e#ran% G) Nie&us H) MJller RH* Cosmetic antiEaging !ormulation #ase% on vitamin EEloa%e% soli% lipi% nanoparticles* Proc Int Symp Control Release +ioact Mater 4>>=M 5:6988H989* :<* Souto E+) MJller RH* ipi% nanoparticles AS N an% N CB !or %rug %elivery* In6 Dom# A') Ta#ata 3) Ravi 0umar MN2) -ar#er S) e%s* Nanoparticles !or P&armaceutical Applications* os Angeles) Cali!ornia6 American Scienti!ic Pu#lis&ers) 5@@<64@8H455* :=* MJller RH) Me&nert ,) uc$s 'ES) et al* Soli% lipi% nanoparticles AS NBRan alternative colloi%al carrier system !or controlle% %rug %elivery* Eur ' P&arm +iop&arm 4>>:M 946;5H;>* :>* Sc&(ar/ C) Me&nert ,* Soli% lipi% nanoparticles AS NB !or controlle% %rug %elivery* II* Drug incorporation an% p&ysicoc&emical c&aracteri/ation* ' Microencapsul 4>>>M 4;65@:H548* ;@* Souto E+) ,issing SA) +ar#osa CM) MJller RH* Development o! a controlle% release !orE mulation #ase% on S N an% N C !or topical clotrima/ole %elivery* Int ' P&arm 5@@9M 5<=6<4H<<* ;4* ,ey&ers H* -este ipi% Nanoparti$el AS NB !Jr %ie ge(e#sspe/i!isc&e Ar/neisto!!appli$ation) Herstellung) C&ara$terisierung o#er!lIc&enmo%i!i/ierter -ormulierungen* P&*D* T&esis* +erlin6 -reie UniversitIt +erlin) 4>>:* ;5* Almei%a A') Runge S) MJller RH* Pepti%eEloa%e% soli% lipi% nanoparticles AS NB6 in!luE ence o! pro%uction parameters* Int ' P&arm 4>><M 49>65::H5;:* ;8* /ur MJ&len A) Me&nert ,* Drug release an% release mec&anism o! pre%nisolone loa%e% soli% lipi% nanoparticles* Die P&arma/ie 4>>=M :86::5H:::* ;9* /ur MJ&len A) Sc&(ar/ C) Me&nert ,* Soli% lipi% nanoparticles AS NB !or controlle% %rug %eliveryR%rug release an% release mec&anism* Eur ' P&arm +iop&arm 4>>=M 9:649>H4::* ;:* Sa$uma S) Su/u$i N) Su%o R) Hi(atari 0) 0is&i%a A) A$as&i M* ?ptimi/e% c&emical structure o! nanoparticles as carriers !or oral %elivery o! salmon calcitonin* Int ' P&arm 5@@5M 58>64=:H4>:* ;;* Dingler A* -este ipi%ENanopartic$el als $olloi%ale ,ir$sto!!trIgersysteme /ur %erE malen Appli$ation* P&*D* T&esis* +erlin6 -reie UniversitIt +erlin) 4>>=* ;<* MJller RH) Dingler A* -este ipi%ENanoparti$el A ipopearlsdB als neuartiger Carrier !Jr $osmetisc&e un% %ermatologisc&e ,ir$sto!!e* P&arm Veit Dermo 4>>=M 9>644H4:* ;=* Souto E+) MJller RH* S N an% N C !or topical %elivery o! $etocona/ole* ' Microencapsul 5@@:M 556:@4H:4@* ;>* Dingler A) u$o(s$i G) P!legel P) MJller RH) Go&la S* Pro%uction an% c&aracteri/ation o! ipopearls !or cosmetics* Proc Int Symp Control Release +ioact Mater 4>><M 5:6>8:H>8;* <@* ,issing SA) MJller RH* T&e in!luence o! t&e crystallinity o! lipi% nanoparticles on t&eir occlusive properties* Int ' P&arm 5@@5M 59568<<H8<>* <4* %e 2ringer T* Topical preparation containing a suspension o! soli% lipi% particles* European Patent >45@@;;9) 4>>5* <5* Teeranac&ai%ee$ul 2) Souto E+) MJller RH) 'unyaprasert 2+* E!!ect o! sur!actant on t&e p&ysical an% c&emical sta#ility o! ascor#yl palmitateEloa%e% N C system* In6 AAPS annual meeting an% e"position) Nas&ville) U*S*A*) Novem#er 5@@:6;H4@* <8* Teeranac&ai%ee$ul 2) Souto E+) 'unyaprasert 2+) MJller RH* ongEterm p&ysical sta#ilE ity o! 74@Eloa%e% N C at %i!!erent storage con%itions* In6 AAPS annual meeting an% e"position) Nas&ville) U*S*A*) Novem#er 5@@:6;H4@* <9* Souto E+) Teeranac&ai%ee$ul 2) 'unyaprasert 2+) MJller RH* Encapsulation o! nicotinE ami%e into nanostructure% lipi% carriers* In6 4:t& international symposium on microenE capsulation) Parma) Italy) Septem#er 5@@:64=H54* <:* ,issing SA* S N als innovatives -ormulierungs$on/ept !Jr p!legen%e un% protective %ermale Vu#ereitungen* P&*D* T&esis* +erlin6 -reie UniversitIt +erlin) 5@@5* <;* ,issing SA) MJller RH* T&e %evelopment o! an improve% carrier system !or sunscreens !ormulations #ase% on crystalline lipi% nanoparticles* Int ' P&arm 5@@5M 59568<8H8<:*

ipi% Nanoparticles !or Cosmetic) Dermal) an% Trans%ermal Applications

588

<<* ,issing SA) MJller RH* Soli% lipi% nanoparticles as carrier !or sunscreens6 in vitro release an% in vivo s$in penetration* ' Control Release 5@@5M =4655:H588* <=* ,issing SA) MJller RH* In vitro an% in vivo s$in permeation o! sunscreens !rom soli% lipi% nanoparticles AS NdB) supercoole% melts an% emulsions* In6 Procee%ings o! t&e !ourt& (orl% meeting APGI1AP2) -lorence) 5@@56448:H448;* <>* ,issing SA) MJller RH* A novel sunscreen system #ase% on tocop&erol acetate incorporate% into soli% lipi% nanoparticles* Int ' Cosmet Sci 5@@4M 586588H598* =@* ,issing SA) MJller RH* Soli% lipi% nanoparticles AS NB as sunscreens6 a%vantages over conventional emulsion #ase% systems* Proc Int Symp Control Release +ioact Mater 5@@4M 5=6:55H:58* =4* Mariani E) Neu&o!! C) +argagna A) et al* Synt&esis) in vitro percutaneous a#sorption an% p&ototo"icity o! ne( #en/yli%ene %erivatives o! 4)8)8Etrimet&ylE5Eo"a#icycloA5*5*5B octanE;Eone as potential U2 sunscreens* Int ' P&arm 4>>=M 4;46;:H<8* =5* Saupe A* P&arma/eutisc&E$osmetisc&e An(en%ungen Nanostru$turierter ipi%carrier AN CB6 ic&tsc&ut/ un% P!lege* P&*D* T&esis* +erlin6 -reie UniversitIt +erlin) 5@@9* =8* +ennat C* ic&tsc&ut/ mit Mi$ropigmentenH+eitrag /ur p&ysi$oc&emisc&en C&ara$terisierung)galenisc&enSta#ilisierungun%Untersuc&ung%es Penetrationsver&altens von mi$ro!einen Titan%io"i% un% Vin$%io"i%* P&*D* T&esis* +raunsc&(eig6 TU +raunsc&(eig) 4>>>* =9* +ennat C) MJllerEGo(mann CC* S$in penetration an% sta#ili/ation o! !ormulations containing micro!ine titanium %io"i%e as p&ysical U2 !ilter* Int ' Cosmet Sci 5@@@M 5565<4H5=8* =:* Hage%ornE e(e$e U) ippol% +C* Accumulation o! sunscreens an% ot&er compoun%s in $eratinous su#strates* Eur ' P&arm +iop&arm 4>>=M 9;654:H554* =;* MJller RH) ,issing SA) MI%er 0* Sunscreens containing U2 ra%iation re!lecting o! a#sor#ing agents) protecting against &arm!ul U2 ra%iation an% rein!orcing t&e natural s$in #arrier* PCT Int Appl ,? @41@8;:5) 5@@4* =<* Hommoss A) Souto E+) MJller RH* Assessment o! t&e release pro!iles o! a per!ume incorporate% into N C %ispersions in comparison to re!erence nanoemulsions* In6 AAPS annual meeting an% e"position) Nas&ville) U*S*A*) Novem#er 5@@:6;H4@* ==* 3a/i$si/EIscan 3) ,issing SA) MJller RH) He$imoglu S* Di!!erent pro%uction met&o%s !or soli% lipi% nanoparticles AS NB containing t&e insect repellent DEET* In6 Procee%ings o! t&e !ourt& (orl% meeting APGI1AP2) -lorence) 5@@56<=>H<>@* =>* 3a/i$si/EIscan 3) He$imoglu S) Sargon M-) 0as S) Hincal AA* In vitro release an% s$in permeation o! DEET incorporate% soli% lipi% nanoparticles in various ve&icles* In6 Procee%ings o! t&e !ourt& (orl% meeting APGI1AP2) -lorence) 5@@5644=8H44=9* >@* 3a/i$si/EIscan 3) ,issing SA) He$imoglu S) MJller RH* Development o! a novel carrier system !or vitamin 0 using soli% lipi% nanoparticles AS NdB* In6 Procee%ings o! t&e !ourt& (orl% meeting APGI1AP2) -lorence) 5@@56<=<H<==* >4* MJller RH) ippac&er A) Go&la S* Soli% lipi% nanoparticles AS N B as carrier system !or t&e controlle% release o! %rugs* In6 ,ise D) e%* Han%#oo$ o! P&armaceutical Controlle% Release Tec&nology* Ne( 3or$6 Marcel De$$er) 5@@@68<<H8>4* >5* Sc&neppe T* Ent(ic$lung un% 7uali!i/ierung einer Pilotanlage /ur GMPE un% 7ME gerec&ten Herstellung von !esten ipi%ENanopartic$eln* P&*D* T&esis* +erlin6 -reie UniversitIt +erlin) 4>>=*

4:

NanoECarriers o! Drugs an% Genes !or t&e Treatment o! Restenosis

Einat Co&enESela) 2ictoria Ela/ar ) Hila EpsteinE+aras&) an% Gers&on Golom#
Department o! P&armaceutics) Sc&ool o! P&armacy) T&e He#re( University o! 'erusalem) 'erusalem) Israel

INTR?DUCTI?N Restenosis Percutaneous coronary interventions APCIsB are (i%ely employe% !or t&e revascuE lari/ation o! arteries o#structe% #y an at&erosclerotic plaCue in patients (it& symptomatic coronary artery %isease) (&ic& usually presents as angina or myocarE %ial in!arction A4B* T&e PCI proce%ures inclu%e #alloon %ilation) en%oluminal stenting) e"cisional at&erectomy) intravascular #rac&yt&erapy) an% laser a#lation* T&e successE !ul treatment o! stenotic coronary arteries #y PCI is limite% #y t&e occurrences o! restenosis) (&ic& continues to #e a serious complication A5H;B* Restenosis is c&aracE teri/e% #y t&ree stages o! response to t&e vessel (all in.ury6 AiB acute elastic recoil) AiiB negative remo%eling) an% AiiiB neointimal proli!eration A<B* Coronary artery stenting !ollo(ing #alloon angioplasty signi!icantly %ecrease% restenosis rate #y solving t&e pro#lems o! elastic recoil an% vessel negative remo%eling A:)=H4@B) &o(ever) inEstent restenosis) %ue to neointimal proli!eration) remains t&e ma.or limiting !actor o! PCI* An in!lammatory &ealing response is triggere% #y t&e mec&anical %amage to t&e arterial (all A44H49B* At !irst) platelets are activate% an% attac&e% aroun% t&e site o! in.ury) !ollo(e% #y a%&esion o! in!lammatory cells* Cyto$ines an% gro(t& !actors are secrete% #y a%.acent platelets) macrop&ages) an%1or en%ot&elial cells) inclu%E ing plateletE%erive% gro(t& !actor APDG-B) !i#ro#last gro(t& !actor A-G-B) an% interleu$inE4f AI E4fB A4:B) lea%ing to proli!eration an% migration o! smoot& muscle cells ASMCsB to(ar%s t&e arterial lumen) (it& su#seCuent secretion o! a#un%ant e"tracellular matri" !orming neointima an% narro(ing t&e artery* T&e multi!actorial pat&ogenesis o! restenosis allo(s several options (&ere p&armacological agents mig&t #e applie% to prevent t&e %isease process A4;)4<B* A large num#er o! clinical trials &ave investigate% various systemic %rug t&erapies in an attempt to re%uce restenosis* P&armacological t&erapies can #e %ivi%e% into categories #ase% on mec&anisms o! action6 prevention o! t&rom#us !ormation) prevention o! vascular recoil an% remo%eling) an% prevention o! in!lammation an% cell proli!eration A4;B* None o! t&e &uman trials %emonstrate% any #ene!icial e!!ect on t&e inci%ence o! restenosis* Systemic p&armacological approac&es &ave !aile% in &umans %ue to ina#ility to ac&ieve t&e reCuire% %ose at t&e site o! in.ury (it&out causing systemic si%e e!!ects A4=B* As a response to t&e !ailure o! t&e systemic a%minE istration) t&e concept o! local %rug %elivery emerge%) o!!ering t&e a%vantage o! &ig& local concentration an% minimi/ing systemic si%e e!!ects %ue to t&e relatively lo(er systemic concentrations* Until recently) locally %elivere% %rugs &ave #een unsuccess!ul in &umans %ue to rapi% (as&out o! t&e %rug A4>B) in%icating t&e nee%

ECual contri#ution to t&is (or$*

58:

58;

Co&enESela et al*

!or a controlle% a%ministration o! t&e %rug !or an a%eCuate perio% o! time* T&e most success!ul approac& to %ate is %rugEeluting stents) %elivering me%ication %irectly to t&e site o! vascular in.ury !rom polymeric coate% stents A5@H59B* T&e !irst approve% an% commercially availa#le %rugEeluting stent (as t&e Cyp&er ACor%is) a 'o&nson Y 'o&nson CompanyM Ne( +runs(ic$) Ne( 'ersey) U*S*A*B) containing Rapamycin ASirolimusB) a naturally occurring macroli%e anti#iotic an% a potent immunosupE pressive agent A5:)5;B* Ta"us A+oston Scienti!ic CorporationM Natic$) Massac&usetts) U*S*A*B (as t&e secon% approve% %rugEeluting stent containing Paclita"el APW B) a microtu#uleEsta#ili/ing agent (it& potent antiproli!erative activity A5<)5=B* Ho(ever) %espite t&eir clinical success) t&e longEterm e!!icacy an% sa!ety o! t&ese t(o %rugEeluting stents are yet to #e con!irme%*

NanoECarriers NanoEcarrier systems) suc& as liposomes) oilEinE(ater emulsions) an% polymeric nanoparticles ANPB) &ave #een e"tensively investigate% over t&e last !e( %eca%es as a (ay to mo%i!y t&e #io%istri#ution o! %rugs A5>H8;B* An encapsulate% %rug !ollo(s t&e %istri#ution o! t&e carrier) rat&er t&an %epen%ing on t&e %rugPs p&ysicoc&emical properties an% molecular structure* T&e t&erapeutic e!!icacy o! p&armacological agents is %epen%ent on t&eir #io%istri#ution) as (ell as t&eir elimination route an% $ineticsM t&us) nanoEcarriers can #e use% to improve t&e t&erapeutic in%e" o! %rugs* NanoEcarriers &ave attractive #iological properties) since carrierEincorporate% p&arE maceuticals are protecte% !rom t&e inactivation e!!ect o! e"ternal con%itions) yet %o not cause un%esira#le si%e reactions A8<H8>B* In a%%ition) nanoEcarriers may #e utiE li/e% to ac&ieve targete% t&erapiesM targeting to a %esira#le site may lea% to a more e!!ective t&erapeutic %rug action an% prevent si%e e!!ects A9@B*

iposomes iposomes are microscopic p&osp&olipi% vesicles (it& a #ilayere% mem#rane structure an% &ave receive% a lot o! attention %uring t&e past 8@ years as p&armaE ceutical carriers o! great potential A94)95B* iposomes are !orme% #y t&e sel!EassemE #ly o! p&osp&olipi% molecules in an aCueous environment* iposomes are use% as #iocompati#le carriers o! %rugs) pepti%es) proteins) plasmi% DNA) an% antisense oligonucleoti%es or ri#o/ymes !or p&armaceutical) cosmetic) an% #ioc&emical purposes A95B* T&e enormous versatility in particle si/e A98)99B an% in t&e p&ysical parameters o! t&e lipi%s a!!or%s an attractive potential !or constructing tailorEma%e ve&icles !or a (i%e range o! applications A9:H;8B* iposomes can #e %ivi%e% into t&ree su#groups6 classical AconventionalB liposomes t&at are s&ort circulating A;9BM long circulating liposomes) typically mem#raneEmo%i!ie% (it& polyet&yleneglycol APEGB A8<)8>);:H;=BM an% immunoliposomes) liposomes (it& sur!aceEattac&e% ligan%s capa#le o! recogni/ing an% #in%ing to cells o! interest A;>)<@B*

Nanoparticles NP are soli% colloi%al particles in t&e nanoEsi/e range an%) (&en !ormulate% !rom #iocompati#le polymers) t&ey can #e use% as %rug carriers !or t&erapeutic applicaE tions* Depen%ing on t&eir %esign) NP can #e %ivi%e% into t(o ma.or su#groups6 nanosp&eres ANSB an% nanocapsules ANCB* T&e NS are compose% o! a matri" structure) an% NC are c&aracteri/e% #y a polymeric rateElimiting mem#rane A<4B* T&e %rug may #e c&emically #oun% to t&e particleE!orming polymer) a%sor#e% on t&e NP sur!ace) or entrappe% in t&e NP A<5)<8B*

NanoECarriers o! Drugs an% Genes

58<

T&e NP can #e prepare% #y met&o%s involving eit&er polymeri/ation o! %isperse% monomers or %ispersion o! pre!orme% polymers* T&e use o! pre!orme% polymers is pre!era#le) since t&ey are (ell c&aracteri/e% an% contain no resi%ual monomers or polymeri/ation reagents A<9B* T&e c&oice o! t&e polymer %epen%s upon t&e t&erapeutic application o! t&e system) %esire% %rug release) an% #ioE compati#ility* T&e NP may #e !a#ricate% using #iopolymers Agelatin) al#umin) casein) polysacc&ari%es) lectins) etc*B an% synt&etic polymers Apolycianoacrylates) polyesters) polyan&y%ri%e) etc*B* T&e #iopolymers are not capa#le o! provi%ing protracte% release $inetics !or small %rug molecules) t&us limiting possi#le applicaE tions to %elivery o! #iological macromolecules or %rugs !or (&ic& imme%iate action is %esira#le* T&e polymers most commonly use% in t&is !iel% o! researc& are t&e synt&etic alip&atic #io%egra%a#le polyesters) polylacti%e AP AB) polyglycoli%e AP GB) an% t&eir coEpolymer AP GAB* T&e P GA polymers &ave #een use% (i%ely as #iomaterials !or me%ical applications over t&e last 8@ years an% are regar%e% as #iocompati#le an% nonto"ic A88B*

NanoECarriers in t&e Treatment o! Restenosis In %esigning a %elivery system) it is important to consi%er t&e route o! a%ministraE tion Alocal or systemicB an% me%ication %ose) as (ell as mec&anism an% %uration o! t&e %esire% %rug action* iposomes an% NP as %rug carriers can #e utili/e% !or t&e treatment o! restenosis* T&e nanocarriers can #e %esigne% to &ave varying %egra%ation an% %rug release pro!iles accor%ing to speci!ic reCuirements A<8B) as (ell as tissue an% cell locali/ation properties) allo(ing control o! t&eir p&armacoE logical action selectivity* ?ptimal %rug %elivery reCuires pre!erential locali/ation to t&e site o! in.ury) (&ile maintaining a reservoir o! %rug su!!icient !or %esire% activity %uration) in or%er to avoi% upta$e into nonin.ure% segments o! t&e artery an% nonspeci!ic cells A<:)<;B* Several mo%i!ications o! liposomes (ere propose% to cause selective cell targeting !or t&e car%iovascular system A<<B* Mo%i!ications promoting targeting to t&e in.ure% artery can #e %ivi%e% accor%ing to t&e speci!ic ligan%s an% receptors on cells involve%6 AiB GPII#EIIIa receptor on activate% platelets A<=B) AiiB tissue !actor ATPB on vascular en%ot&elial cells) AiiiB upregulate% PDG- receptor !or SMCs migration1proli!eration) an% AivB EE an%1or PEselectine #ot& on en%ot&elial cells an% platelets A<>)=@B* Speci!ic targeting !or activate% cells causes re%uction o! si%e e!!ects in t&e nonactivate% nonin.ure% site %ue to lo(er or no e"pression on resting vascular cells A=4B*

?CA DRUG DE I2ER3 IN RESTEN?SIS It is accepte% t&at t&e ultimate antirestenotic t&erapy (ill utili/e t&e concept o! local %rug %elivery) consi%ering t&e regional nature o! t&e restenotic process* T&is &ypot&esis is strongly supporte% #y t&e virtual !ailure o! systemically a%ministrate% p&armacological agents to control restenosis A4<)=5B) on one &an%) an% t&e success o! %rugEeluting stents A5<)5=B) on t&e ot&er* ocal %elivery o! %rugs %irecte% against particular steps in t&e pat&ogenesis o! restenosis en&ances e!!icacy #y increasing %rug concentration to t&erapeutic levels in t&e imme%iate vicinity o! vascular in.ury (it& minimal or t&e a#sence o! systemic si%e e!!ects A=8H>@B* In a%%ition) t&erapeutic agents (it& s&ort &al!Eli!e or c&emically unsta#le agents are %elivere% %irectly to t&eir site o! target (it& minimal loss o! t&erapeutic activity prior to t&eir upta$e* -urt&ermore) t&e incorporation o! t&e %rug into a carrier system allo(s controlling its release) t&us !itting t&e %rug release to t&e pat&ology %eman%s A<8B*

58=

Co&enESela et al*

iposomes !or local %rug %elivery o!!er t&e a%vantages o! e"tensive upta$e #y a variety o! cells an% speci!ic locali/ation in t&e cells A98)99B* +y controlling liposome si/e) one can control t&e %elivery A9=)>4H>9B* Small vesicles Ac4:@ nm %iameterB #in% to cell sur!ace receptors an% are transporte% #y receptorEme%iate% en%ocytosis* A!ter internali/ation) t&e vesicle !uses (it& lysosomes) (&ic& in%uce t&e #rea$E %o(n o! t&e lipi%s an% release o! t&eir contents A>:H4@:B* arge particles A\4:@ nm %iameterB) on t&e ot&er &an%) are ta$en up principally #y p&agocytosis) (&ic& is usually limite% to p&agocytic cells #ut can #e in%uce% in many ot&er cell types (it& appropriate ligan%s A4@;B* In #ot& cases) t&e liposome coul% eit&er #e %egra%e% in t&e lo( pH environment) or it coul% !use %irectly (it& t&e en%osomal or lysosomal mem#rane* -urt&ermore) t&e incorporation o! %rug (it&in liposomal vector allo(s com#ination o! rapi% #urst release an%) at t&e same time) sustaine% release #y simple controlling o! t&e liposomal !ormulation* In local %rug %elivery) t&e ultrasmall si/e o! NP is an important a%vantage) since it ena#les &ig&er arterial upta$e #ecause o! t&e apparent ease o! access to t&e arterial (all A4@<B* -is&#ein et al* A4@=B &ave s&o(n t&at r&o%amine + containing polymeric NP (as trace% in rat caroti% arteries 59 &ours a!ter intraluminal %elivery) (&ereas no !luorescent %ye (as present in t&e arterial segments treate% (it& an eCual amount o! r&o%amine + in solution A-ig* 4B) in%icating t&at t&e NP (ere a#le to prevent t&e rapi% elimination o! t&e !ree %rug* -urt&ermore) NP o!!er t&e possiE #ility o! intraEarterial %elivery via porous cat&eters t&at coul% #e inclu%e% as a !inal step in interventional car%iac cat&eteri/ation A4@>H444B*

-IGURE 4 ASee color insert*B Con!ocal images o! #alloonEin.ure% rat caroti% arterE ies a!ter intaluminal %elivery o! r&o%amine solution an% r&o%amineEcontaining nanoE particles* T&e arteries (ere &arveste% >@ minutes AA an% DB) eig&t &ours A+ an% EB) an% one %ay AC an% -B a!ter %elivery* A##reviation6 NP) nanoparticles* Source6 -rom Re!* 4@=*

NanoECarriers o! Drugs an% Genes

58>

Gene T&erapy !or t&e Treatment o! Restenosis T&e local nature o! t&e restenosis p&enomenon ma$es it an attractive target !or gene t&erapy) given t&at t&e tissue reaction t&at %evelops is %irectly accessi#le #y t&e intervention itsel! A445B* T&e success o! gene t&erapy in t&e prevention o! restenosis %epen%s on t&e i%enti!ication o! appropriate molecular targets) a suita#le vector system c&osen !or e!!icient vessel (all targeting) an% met&o%s !or vascular gene %elivery (it&out pro%ucing un%ue %amage or %istal tissue isc&emia A448B* T&e appropriate genetic mo%i!ication) per!orme% locally at t&e time o! angioplasty) coul% in%uce a longEterm #ene!it in potency #y !un%amentally re%irecting t&e &ealing response at its roots A449B* Several molecular species &ave #een implicate% in t&e stimulation o! vascular smoot& muscle cells A2SMCsB to initiate t&e intimal &yperplasic process A44:)44;B6 PDG-) #asic !i#ro#last gro(t& !actor A#-G-B) trans!orming gro(t& !actor #eta ATG-E fB) angiotensin II) an% activator proteinE4 AAPE4B A44<H45@B* Anot&er strategy is !ocuse% on t&e cell cycle mac&inery o! cells involve% in proli!eration an% neointima !ormation A454)455B* T&e 2SMC proli!eration %epen%s on t&e increase% e"pression o! certain cell cycle regulatory proteins at critical points along t&e cell cycle) inclu%ing proli!erating cell nuclear antigen APCNAB) transcripE tion !actor E5-) nuclear !actorE+) cell %ivision cycle 5 Ac%c5B $inase) t&e cyclins) cyclinE%epen%ent $inase Ac%$B) an% nuclear protooncogenes AcEmyc an% cEmy#B) molecules t&at appear to #e critical in t&e mitogenic signaling events A458H45=B* It &as #een &ypot&esi/e% t&at #y #loc$ing t&e gene e"pression or !unction o! one or more o! t&ese proteins) one coul% prevent t&e progression o! 2SMC t&roug& t&e cell cycle an% in&i#it neointimal gro(t&* T&us) %i!!erent regulatory molecules can #e speci!ic targets !or gene t&erapy A5)45>B* Gene t&erapy can #e %e!ine% as a trans!er o! nucleic aci%s to t&e somatic cells o! an in%ivi%ual (it& a resulting t&erapeutic e!!ect) suc& as synt&esis o! missing or %e!ective proteins Aintro%uction o! intact gene seCuencesB or e"pression #loc$a%e o! %iseaseErelate% genes Aantisense tec&nology) %ecoy oligonucleoti%es) an% ri#o/ymesB A48@H488B* Somatic gene t&erapy &as emerge% as a promising approac& !or t&e prevention o! restenosis A489B* T&e aim o! antisenseEs&ort %ou#leEstran%e% DNA or RNA oligonucleoti%es strategy is to in&i#it protein synt&esis #y selectively #loc$ing t&e initiation o! translation or increasing t&e mRNA %egra%ation #y RNase H A484)485)48:B* Natural p&osp&o%iester antisense oligomers are suscepti#le to rapi% %egra%ation #y nucleases* To overcome t&is pro#lem) a num#er o! c&emically mo%i!ie% nuclease resistant analogs) suc& as p&osp&orotioate% oligo%eo"ynucleE oti%es APTE?DNsB an% c&imeric oligomers Amorp&olino an% pepti%e nucleic aci%sB &ave #een %esigne% A48;H48=B* Some success in altering intimal &yperplasia in restenosis &as #een ac&ieve% using antisense oligonucleoti%es in cell culture an% in animal mo%els A48>)49@B* Treatment (it& PTE?DNs against cEmyc) using a polymerE #ase% %elivery system) resulte% in only partial in&i#ition o! 2SMC gro(t& in vitro an% !aile% to re%uce overall intimal &yperplasia in rat arterial in.ury mo%el A494B* Intro%uction o! cEmy# PTE?DNs) incorporate% in t&e same carrier system) using t&e same animal mo%el) (as !oun% to #e e!!ective in vivo A495B* Ho(ever) in !urt&er stu%ies) t&e results (ere not con!irme%* Trans!ection o! cisEelement %ou#leEstran%e% oligo%eo"ynucleoti%es A%ecoyB &as #een reporte% as a po(er!ul tool in a ne( class o! antigene strategies !or gene t&erapy* Synt&etic %ou#leEstran%e% DNA (it& &ig& a!!inity !or a target transcription !actor may #e intro%uce% into target cells as a Q%ecoyS cis element to #in% t&e transcriptional !actor* Trans!ection o! suc& Q%ecoyS oligonucleoti%es results in

59@

Co&enESela et al*

attenuation o! aut&entic cisHtrans interaction) lea%ing to t&e removal o! t&e transE !actors !rom t&e en%ogenous cisEelement (it& su#seCuent mo%ulation o! gene e"presE sion A498B* Several stu%ies &ave provi%e% evi%ence o! t&e in vivo application o! t&is molecular approac& as a t&erapeutic strategy against car%iovascular %isease A488B* A ma.or pro#lem encountere% (it& gene t&erapy an%) particularly) t&e t&eraE peutic use o! oligonucleoti%es) is t&e lo( cellular permea#ility %ue to t&eir large molecular si/e an% &ig& c&arge %ensity an% lysosomal %egra%ation* T&e upta$e o! !oreign genetic material Ana$e% DNA) RNA) or oligonucleoti%eB #y mammalian cells is an ine!!icient process A44=)499B* To improve t&eir cellular %elivery) several vector systems (ere %evelope% A49:)49;B* 2iruses are among t&e most precise an% sta#le gene vectors* Retroviral) a%enoviral) an% a%enoEassociate% vectors &ave t&e a%vantage o! e"ploiting t&e natural mec&anisms o! t&e #o%yPs cell receptors) t&us increasing t&eir e!!icacy an% lea%ing to a sta#le an% longEterm e"pression o! t&e genetic material in t&e cells (&ere t&ey &ave #een inserte% A49<)49=B* Ho(ever) en%ogenous virus recom#ination) oncogenic e!!ects) immunogenicity) an% un$no(n longEterm e!!ects lea% to serious limitations regar%ing t&eir use in gene t&erapy A49:)49>H4:8B* Moreover) t&e recent severe to"icity an% si%e e!!ects o! acute TEcell leu$emia in some patients t&at emerge% in a recent clinical trial o! retroviral vector in WElin$e% severe com#ine% immuno%e!iciency &ig&lig&ts t&e importance o! nonviral gene %elivery A49>)4:9B*

iposomal Gene Delivery Nonviral trans!ection systems) suc& as cationic liposomes) &emagglutinating virus o! 'apan AH2'BEliposomes) an% #io%egra%a#le polymeric NP) are generally pre!erre% over viruses #ecause t&ey are nonimmunogenic) relatively easy to assem#le) !orm sta#le comple"es (it& plasmi% DNA) provi%e protection o! t&e plasmi% DNA !rom %egraE %ative nucleases) an% are amena#le to scaleEup !or in%ustrial pro%uction A4::)4:;B* T&eir %ra(#ac$ is t&at t&ey are less e!!icient t&an t&e viral vectors A448)4:<B*

Cationic iposomes Comple"ing recom#inant DNA (it& cationic liposomes consisting o! cationic an% neutral lipi%s) also $no(n as cationic lipople"es) are relatively e!!icient in %elivering DNA into cells A4:=B) &o(ever) t&e comple" can #e inactivate% in t&e presence o! serum) an% t&ere &ave #een reports o! insta#ility upon storage A4:>B* In a%%ition) cytoto"icity o! cationic liposomes remains a concern A4;@)4;4B* T&e %egree o! success!ul gene trans!er is &ig&ly %epen%ent on t&e cationic lipi% type) liposomal !ormulation) an% cell type A4;5H4;:B* Cationic vectors #in% to negatively c&arge% DNA) resulting in a con%ensation reaction an% t&e !ormation o! sta#le comple"es in t&e nano range* -usion (it& t&e cell mem#rane or en%ocytosis allo(s incorporation o! DNA into t&e cell A4;;B* Recent reports suggest t&at ne( liposomal !ormulations) in%ivi%ually optimi/e% !or t&e targete% tissue) (it& #etter protocols an%1or continE uously a%ministere% ApolyEBEcationic liposomes may su#stantially increase trans!ecE tion e!!iciencies A4;<H4;>B* Trans!ection results &ave #een %i!!erent in various cell types an% cell linesM t&us) eac& reagent apparently &as celltypeEspeci!ic an% a%%iE tional speciesEspeci!ic c&aracteristics A4<@B* Ne(er lipi%s are #eing %evelope% in or%er to en&ance vascular gene trans!er (&ile minimi/ing to"icity* Ne( compoun%s) suc& as AeBENEA8EaminopropylBEN)NE%imet&ylE5)8E#isA%o%ecylo"yBE4Epropanaminium #romi%e AGAPED RIEB) an% %ioleoylp&osp&ati%ylet&anolamine AD?PEB) &ave #een s&o(n to increase t&e vascular %elivery o! plasmi% DNA #y 4:E!ol% compare% (it& previous cationic liposomes A4<4B) screene% nonviral !ormulations trans!ecting

NanoECarriers o! Drugs an% Genes

594

primary 2SMCs in vitro (it& %i!!erent concentrations an% com#inations o! nonviral vectors as (ell as varying DNA1vector ratios an% a%.uvants* Upta$e e!!iciencies range% !rom @*4[ to 5@[ o! trans!ecte% 2SMC #y c&anging liposomal parameters* Use o! cationic liposomes A ipo!ectamineB !or intro%uction o! PTE?DNs) tarE geting &uman PCNA mRNA) in vitro in!luence% t&e oligonucleoti%e upta$e in &uman 2SMC an% &uman at&erosclerotic plaCue) %i% not en&ance antisense e!!ect) #ut increase% t&e magnitu%e o! speci!ic 2SMC in&i#ition compare% to t&e na$e% seCuences A4<5B* Nuclear !actorE+ AN-E+B %ecoy oligonucleoti%es trans!ecte% #y cationic liposome met&o% A ipo!ectamine an% T!W:@B in&i#ite% tumor necrosis !actorEm ATN-EmBEin%uce% e"pression o! interleu$inE; AI E;B an% intracellular a%&esion moleculeE4 AICAME4B in mouse en%ot&elial cells A4<8B an% proli!eration o! ra##it 2SMC A4<9B in vitro* 4)8EDioleoylo"yE5EANEN:OEcar#amoylEspermineBE propane AD?CSPERB) a recently %evelope% polycationic spermin (it& no a%%ition o! neutral lipi%) &as s&o(n t&e #est e!!iciency (it& less to"icity* In vivo stu%ies) comparing 4)8E%ioleoylo"yE5EA;Ecar#o"yEspermylBEpropylami%e AD?SPERB an% ipo!ectamine AD?SPA1D?PE 864 ,1,B con%ucte% in t&e pig #alloon angioplasty mo%el using plasmi%s co%ing !or t&e anti#acterial pepti%e cecropin A) s&o(e% greater re%uction in neointima !ormation using D?SPER cationic liposomes in comE parison to ipo!ectamine as nonviral vector A4<:B* T&e mec&anism o! en&ancement o! gene trans!ection rate using D?CSPER is not (ell un%erstoo%* Nonviral vectors !acilitate gene trans!er in %i!!erent (ays) even in t&e same cell type) %ue to comple" e"tracellular an% intracellular events involve% %uring t&e trans!ection process A4<;)4<<B* Mem#rane #in%ing) internali/ation) en%osomal release) uncoating) an% nuclear translocation constitute #asic cellular events %uring nonviral gene trans!er an% may #e involve% in %i!!erent %egrees %epen%ing on t&e vector use%* Com#ination o! anionic liposomes1DNA an% calcium ions &as #een suggeste% #y Patil et al* A4<=B to ena#le e!!icient trans!ection (it& sa!er pro!ile* Increase% trans!ection o! nonviral vector can #e ac&ieve% in vitro as (ell as in vivo #y controlling liposomal !ormulaE tion an% trans!er con%itions A4<:)4<<)4<>B*

-usigenic iposome Hemagglutinating 2irus o! 'apanE iposomes A2irosomB T&e H2' or Sen%ai virus is a mem#er o! t&e murine paramy"ovirus !amily) conE taining a singleEstran%e% RNA virus genome (it& an envelope A4;:)4=@B* T&e H2'E envelope contains t(o glycoproteins6 HN A&emagglutinating neuramini%aseB an% - A!usion proteinB* T&ese H2'Eenvelope proteins are involve% in cell !usion* T&e H2' virus is an envelope% large particle ranging !rom 8@@ to ;@@ nm in %iameter* T&e viral particle) (&ic& is negatively c&arge% an% attac&e% to sialic aci% At&e H2' receptorB) !uses (it& cell mem#rane) an% releases its genome into cytoplasm %irectly) rat&er via t&e en%ocytosis A4=4B* T&e H2'Eliposome gene trans!er tec&nology (as %evelope% in late 4>=@s Ae*g*) Re!* 4=5B an% early 4>>@s Ae*g*) Re!s* 4=8 an% 4>5B to intro%uce nucleic aci%s) oligo%eo"ynucleoti%e A?DNB) an% protein (it& &ig& e!!iciency* T&e molecules inclu%e% in H2'Eliposomes are %elivere% %irectly into various types o! mammalian cells #y means o! t&e virus cell !usigenic c&aracter o! H2'* T&e H2'Eliposomes are constructe% #y a com#ination o! inactivate% viral particles an% cationic liposomes to pro%uce a nonviral gene trans!er system A4;:)4=5)4=8B* T&e H2'Eliposome nucleic aci%s or ?DN are more e!!icient an% sa!er t&an ot&er nonviral vectors A4=9)4=:B* Moreover) ?DN %elivere% #y H2'Eliposomes s&o(e% rapi% accumulation in t&e nucleus) (&ic& persiste% up to t(o (ee$s* In t&e secon% generation) H2'Eliposomes

595

Co&enESela et al*

(ere mo%i!ie% !rom cationic to anionic* Anionic H2'Eliposomes s&o(e% a :E to 4@E!ol% &ig&er gene e"pression in several cell types A4=;B* T&e H2'Eliposomes &ave lo( immunogenic an% lo( pat&ogenic pro!ile Asa!ety e"periments (ere con%ucte% in mon$eys t&at %emonstrate% t&e sa!ety) !easi#ility) an% t&erapeutic potential o! t&e H2'Eliposome vector !or &umansB A458)4=<B* T&e H2'EliposomesP versatility o!!ere% a (i%e range o! %i!!erent molecules an% &ig& trans!ection e!!iciency into a variety o! cells #ot& in vitro an% in vivo A44<)488)499)4=;H4>9B* Intact gene seCuences (ere trans!erre% using H2'Eliposomes !or prevention o! e"perimental restenosis* Plasmi%s) containing cDNA o! en%ogenous neointimal gro(t& in&i#itors) suc& as nitric o"i%e synt&ase AN?SB) prostacyclin synt&ase APGISB) tissue !actor pat&(ay in&i#itor AT-PIB) (ere %elivere% to t&e vessel (all in vivo #y means o! H2'Eliposomes A4=;)4==)4=>B* Trans!ection o! N?S in&i#ite% neointima !ormation #y <@[) 49 %ays a!ter #alloon in.ury o! rat caroti% artery A4==B* Intro%uction o! cDNA enco%ing &uman PGIS into en%ot&eliumE%enu%e% rat caroti% arteries resulte% in strong PGIS e"pression in neointimal cells at %ay < an% signi!icant in&i#iE tion o! vascular lesion !ormation generate% at %ay 49 a!ter #alloon in.ury AneointiE mal1me%ial area6 4*5 e @*9B) in comparison to ve&icle control group A4*< e @*:M P c @*@4B A4=>B* ?ne (ee$ a!ter angioplasty an% T-PI gene %elivery to ra##it iliac artery) T-PI protein (as %etecte% in neointima an% me%ia o! t&e vessel (all* At !our (ee$s) t&e minimal luminal %iameter (as signi!icantly greater AP c @*@4B an% t&e mean percentage o! stenosis (as signi!icantly lo(er A8< e 4= vs* =8 e 4=[) P c @*@4B in t&e T-PIEtrans!ecte% group t&an in t&e ot&er t&ree control groups A4=;B* T&e H2'Eliposomes (ere utili/e% to en&ance t&e e!!iciency o! cellular upta$e an% t&e sta#ility o! most commonly use% p&osp&orot&ioate% antisense oligonucleE oti%es) (&ile minimi/ing t&eir nonspeci!ic to"icity in several in vitro an% in vivo stu%ies A458)4>@H4>5B* Cyclin +4 an% c%c5 $inase antisense oligonucleoti%es) transE !erre% into in.ure% rat caroti% arteries #y H2'Eliposomes) (ere locali/e% primarily in cell nuclei an% cause% partial) #ut speci!ic an% signi!icant re%uction o! intimal &yperplasia up to eig&t (ee$s a!ter trans!ection) (&ereas cyclin +41c%c5 antisense com#ination completely a#olis&e% neointima !ormation A458B* Trans!ection o! antiE sense angiotensinEconverting en/yme AACEB oligonucleoti%es resulte% in in&i#ition o! neointimal gro(t& #y :@[ (it&out e!!ect on #loo% pressure) &eart rate) an% serum ACE activity A4>8B* Sustaine% in&i#ition o! neointima gro(t& (as pro%uce% #y a single a%ministration o! H2'Eliposomes containing antisense c%$5 $inase A;:[ neointima in&i#itionB or com#inations o! c%$51c%c5 A=:[B an% c%c51PCNA A:@H ;@[B oligonucleoti%es A4>4)4>5B* Trans!ection o! %ecoy ?DNs targeting transcripE tion !actor E5- using H2'Eliposomes in&i#ite% t&e in%uction o! cEmyc) c%c5) an% PCNA e"pression) as (ell as 2SMC proli!eration in vitro an% mar$e%ly suppresse% neointima !ormation compare% (it& controlEtreate% vessels in t&e mo%el o! rat caroti% in.ury Aneointima1me%ia ratio !or E5-Etrans!ecte% segments6 @*5>4 e @*@;4 vs* untrans!ecte% ones6 4*44< e @*48=B A4>9B* H2'Eliposomal trans!er o! APE4 %ecoy ?DN resulte% in signi!icantly %ecrease% 2SMC gro(t& AP c @*@:B an% migration AP c @*@4B in cell culture an% attenuation o! intimal &yperplasia in rat vessel (all in vivo* Pretreatment o! rat caroti% artery (it& APE4 %ecoy ?DN #e!ore #alloon in.ury (as more e!!ective t&an postEtreatment AP c @*@4B in in&i#iting neointimal !ormation A44<)4>8)4>9B*

Polymeric NP !or Gene Delivery +io%egra%a#le NP also present a potentially a%vantageous %rug %elivery system to trans!er genes !or restenosis prevention) %ue to t&eir nonviral nature an% t&eir

NanoECarriers o! Drugs an% Genes

598

a#ility to pro%uce local activity o! %erive% gene pro%ucts A4>:B* Antisense ?DNs utili/e% to in&i#it t&e !unction o! gro(t& regulatory or cellEcycle genes AcEmy#) cEmyc) PCNA) c%c5) an% c%$5B o! SMCs &ave #een s&o(n to %ecrease intimal t&ic$ening in e"perimental restenosis A48>)49@)495)4>4)4>;)4><B* Nevert&eless) !ully p&osp&orot&ioate% antisense ?DNs analogs (ere teste%) (&ic& %emonstrate &ig& a!!inity !or various cellular proteins) resulting in nonspeci!ic e!!ects* In a%%ition) &ig& concentrations o! p&osp&orot&ioate% ?DNs in&i#it DNAEpolymerases an% RNase H) (&ic& impairs t&eir e!!ectiveness as antisense agents A484B* Co&en et al* A4>=B synt&esi/e% a novel partially p&osp&orot&ioate% antisense ?DN seCuence !or t&e purpose o! minimi/ing t&e nonspeci!ic e!!ects o! t&e !ully p&osp&orot&ioE ate% ?DNs* T&e ?DN (as %esigne% to %o(nregulate PDG-REf) (&ic& plays a pivotal role in t&e en&ancement o! SMCs migration a!ter #alloon angioplasty A4>>B* T&e antisense (as incorporate% into a P GAE#ase% nanopaticulate system) an% t&e antirestenotic e!!icacy (as e"amine% in t&e rat caroti% artery mo%el A-ig* 5AB* T&e purpose o! t&e encapsulation in NP (as to ena#le t&e intracellular %elivery o! t&e ?DN) (&ic& e"&i#it lo( cellular permea#ility %ue to its large molecular (eig&t an% &ig& c&arge %ensity* T&e %ou#le emulsionHsolvent evaporation met&o% A5@@B (as employe% !or t&e preparation o! t&e P GAEantisense NP) yiel%ing NP (it& a sp&erical s&ape) &omogeneous si/e %istri#ution Az8@@ nmB) &ig& encapsulation e!!iciency A=4[B) an% sustaine% release o! PDG-REf antisense !or over a mont&* In or%er to ac&ieve &ig& encapsulation) calcium ions (ere a%%e% to t&e !ormulation) t&at minimi/e% t&e escape o! t&e negatively c&arge% ?DNs to t&e e"terior p&ase A-ig* 5+B) similar to plasmi% DNA encapsulation as (as previously reporte% #y t&e same group A-ig* 5CB A5@4B* Partially p&osp&orot&ioate% antisense seCuences (ere !oun% to #e more speci!ic t&an t&e !ully p&osp&orot&ioate% analogs in vitro* T&e in vivo e!!iciency (as e"amine% #y locally a%ministrating treatment an% controls to rat caroti% arteries) imme%iately a!ter #alloon in.ury* T&e rats (ere sacri!ice% a!ter 49 %ays* Scram#le% ?DN seCuence Ana$e% an% encapsulate%B %i% not s&o( any e!!ect) (&ic& in%icates t&at t&e antirestenotic e!!ect (as seCuenceEspeci!ic* A signi!icant antirestE enotic e!!ect o! t&e na$e% antisense seCuence an% t&e antisenseEloa%e% NP (as o#serve% in comparison to #lan$ NP t&at &a% no %eleterious e!!ect on t&e arteries) (&ile t&e na$e% antisense s&o(e% a strong tren% to(ar%s a greater e!!ect t&an t&e nanoparticulate antisense) alt&oug& t&is tren% %i% not reac& statistical signi!icance* It (as suggeste% t&at t&e %ose o! t&e ?DN %elivere% !rom t&e NP (as mar$e%ly lo(er in comparison to t&e na$e% antisense) since t&e release o! t&e antisense !rom t&e NP (as very slo(* It (as also propose% t&at t&e upta$e into t&e artery (as in !avor o! t&e %issolve% material rat&er t&an t&e suspen%e% NP* It can #e conclu%e% t&at (&ile a more speci!ic an% e!!ective antisense seCuence (as %esigne%) t&e !ormuE lation s&oul% #e !urt&er improve% to meet t&e speci!ic reCuirements o! t&e %isease* T&e !ailures o! &uman clinical stu%ies A48@)5@5H5@9B suggest t&at t&e promise o! gene t&erapy in restenosis (as not !ul!ille%*

ocal Drug Delivery o! Drugs Tyrp&ostins Tyrp&ostins are a !amily o! structurally relate% %rugs t&at possess in&i#itory activity against receptorE#oun% an% cytoplasmic protein tyrosine $inases APT0B A5@:)5@;B* Derivati/ation o! t&e #asic structure o! t&ese compoun%s gives rise to in&i#itors

599

Co&enESela et al*

-IGURE 5 AAB ASee color insert*B In&i#ition o! neointimal !ormation 49 %ays a!ter #alloon in.ury to rat caroti% artery an% intraluminal instillations (it& 5@ _M A4 nmoleB antisense or scram#le% partially p&osE p&orot&ioate% oligo%eo"ynucleoti%es APTE?DNsB encapsulate% in P GA nanoparticles Atotal o! :@ _ suspensionB* P&otomicrograp&s o! representative &istological sections* 2erc&oe!!Ps elastin stain) magni!ication `45*:* A+B ASee color insert*B -luorescence micrograp&s o! #lan$ an% PTE?DNE loa%e% P GA nanoparticles* T&e PTE?DNs (ere covalently la#ele% (it& -ITC prior encapsulation* Note !luorescence signal in over >@[ o! t&e particles* ACB Scanning electron micrograp&s o! #lan$ A4B an% plasmi% DNA loa%e% A5B P GA nanoparticles) prepare% #y %ou#le emulsionHsolvent evapoE ration met&o%* Gol% coating A5@@ secon%sB) magni!ication `:0) 8@ $2) #ar ^ 4@ _M* A##reviations6 A) a%ventitiaM ) l(nenM M) me%iaM N) neointimaM NP) nanoparticlesM SCENP) scram#le% NPM ASENP) antisense NP* Source6 -rom Re!s* 4>= an% 5@4*

NanoECarriers o! Drugs an% Genes

59:

e"&i#iting a &ig& %egree o! speci!icity to PT0 o! a given type) suc& as PDG-REf A5@<)5@=B* T&e antirestenotic e!!icacy o! locally %elivere% in&i#itors AGE45>: an% AG E5@98) !ormulate% in P AE#ase% NP) &as recently #een evaluate% A4@=)44@)5@>B* T&ese t(o tyrp&ostins are lo( M,) aromatic compoun%s) (&ereas AG E5@98 &as some solu#ility in (ater %ue to t&e presence o! a polar moiety in its structure an% AGE45>: is practically insolu#le in (ater* Tyrp&ostin AGE45>: %emonstrate% SMCEspeci!ic in&i#itory e!!ect on cell gro(t& in vitro an% e" vivo A444)54@B via in&iE #ition o! PDG-REfEautop&osp&orilation) (&ic& supports t&e suggeste% mec&anism o! action* T&e AG E5@98 is a novel tyrp&ostin c&aracteri/e% #y a &ig&er a!!inity to t&e receptor an% increase% in&i#itory potency A5@<B* T&e tyrp&ostins (ere success!ully entrappe% in t&e P AE#ase% NP #y t&e nanoprecipitation met&o% A544B) (&ic& involves a spontaneous gra%ientE%riven %i!!usion o! (ater misci#le organic solvents into continuous aCueous p&ase A44@)545)548B* T&e release o! #ot& tyrp&ostins (as stu%ie% using t&e e"ternal sin$ met&o%* T&e AG E5@98 e"&i#ite% a more rapi% release in comparison to AGE45>:) apparently %ue to partial a%sorption o! t&e !ormer on t&e NP A44@)549B* Rapi% %rug release associate% (it& su#stantial %rug %istri#ution onto NP sur!ace &as previously #een reporte% !or ot&er semiEpolar compoun%s !ormulate% in NP #y nanoprecipitation A54:B* Selecte% NP !ormulations o! AGE45>: an% AG E5@98 (ere evaluate% in animal mo%els o! restenosis A4@=)44@B* Neointimal !ormation (as signi!icantly re%uce% #y locally %elivere% >@ nm NP o! #ot& AGE45>: an% AG E5@98 in comparison to control animals) #ut not #y 4;@ nm NP at t&e same %rug %oses A4@=)44@B* T&e insigE ni!icant e!!ect o! t&e 4;@ nm particles mig&t #e associate% (it& t&e less e!!icient ingress in t&e arterial tissue A4@=B an% possi#ly (it& re%uce% upta$e #y SMCs A54;B* T&e small NP (ere c&aracteri/e% #y a rapi% elimination in t&e !irst >@ minutes !ollo(e% #y a slo(er late elimination A4@=B* T&e early !aster elimination o! t&e smaller particles (as pro#a#ly cause% #y t&e easier migration to t&e a%ventitia) !acilitating t&eir elimination t&roug& t&e vasa vasorum* T&eir re%uce% late (as&out (as pro#a#ly %ue to t&eir #etter penetration into %eep arterial structures an% creaE tion o! a %rug %epot t&at is relatively inaccessi#le to leac&ing #y #loo% !lo(* T&e in!erior antirestenotic e!!ect o! t&e large particles is in goo% accor% (it& t&eir lo( tissue %rug level at a%vance% time points* T&e NP o! AG E5@98 e"&i#ite% &ig&er antirestenotic e!!icacy compare% to AGE45>: loa%e% NP o! compara#le si/e an% molar %rug loa%ing) (&ic& may #e attri#ute% to t&e lo(er potency o! t&e secon% molecule* T&e pig mo%el is consi%ere% to #e more relevant !or t&e pat&op&ysiology o! &uman restenosis A54<B* -urt&er e"amination o! t&e antirestenotic e!!ect o! NP !ormulations o! t&e t(o tyrp&ostins (as per!orme% in t&e pig mo%el) an% a signi!iE cant antirestenotic e!!ect (as o#serve% in comparison (it& unloa%e% NP o! t&e same si/e A44@B*

De"amet&asone De"amet&asone ADWMB is a glucocorticosteroi% %rug t&at (as !oun% to signi!iE cantly re%uce neointimal proli!eration in t&e rat caroti% artery mo%el) !ollo(ing peria%ventitial %elivery A54=B* T&ere are several possi#le mec&anisms !or t&e antirestenotic e!!ect o! glucocorticosteroi%s) inclu%ing in&i#ition o! PDG- pro%ucE tion A54>B) I E4f transcription A55@B Aa cyto$ine t&at stimulates SMCs proli!erationB) an% re%uction o! t&e in!lammatory response A554B* In lig&t o! t&ese results) Gu/man et al* &ave %eci%e% to e"amine t&e antirestenotic e!!icacy o! locally %elivere% DWM P GAE#ase% NP A4@>B*

59;

Co&enESela et al*

T&e result o! t&e in vivo e!!icacy stu%y in t&e rat caroti% artery mo%el %emonE strate% a signi!icant %ecrease in t&e intima1me%ia ratio (it& DWM NP) in compariE son to #lan$ NP an% DWM NP in.ecte% intraperitoneally AIPB A4@>)555B* T&e DWM levels in t&e arterial (all (ere %etecta#le up to t(o (ee$s !ollo(ing intraEarterial a%ministration o! DWM NP) (&ile no %etecta#le DWM levels (ere o#serve% #eyon% t&ree &ours a!ter IP a%ministration) in%icating t&e success!ul locali/e% %elivery o! t&e NP A4@>)555B* T&e antirestenotic e!!ect o! DWM %elivere% in NP (as compare% to silicone perivasular %elivery* T&e e!!icacy o! t&e silicone implant (as !oun% to #e &ig&er #y more t&an t(oE!ol%) in spite o! t&e &ig&er %rug %ose %elivere% into t&e artery #y t&e NP A4@>)54=)555B* It (as &ypot&esi/e% t&at t&e in!erior e!!ect o! t&e NP (as %ue to t&eir less e!!icient arterial upta$e an% s&orter resi%ence time in t&e artery) in%icating t&e nee% !or !ormulation improvement*

5EAminoc&romone UE=;=>8 AU=;B T&e UE=; is an antiproli!erative cytostatic %rug t&at (as !oun% to &ave an antic&emoE tactic an% antimutagenic activity on SMCs in vitro A558B* A signi!icant antirestenotic e!!ect in #alloonEin.ure% rat caroti% arteries (as ac&ieve% #y systemically in.ecte% UE =;* Ho(ever) %ue to rapi% plasma clearance an% poor oral availa#ility) t&e I2 %oses reCuire% to ac&ieve t&e t&erapeutic e!!ect (ere very &ig& an% prolonge% A558B* Hump&rey et al* A559B e"amine% t&e in&i#itory e!!ect o! P GAE#ase% UE=; NP on neointimal proli!eration in porcine coronary arteries su#.ecte% to #alloon in.ury* T&e emulsi!ication solvent evaporation met&o% (as employe% to !ormulate NP o! 44@ e 9@ nm* T&e release (as #ip&asic an% relatively prolonge%M over 9@[ o! t&e loa%e% %rug (as release% over t&e !irst 59 &ours) !ollo(e% #y a slo( p&ase (it& e"ponentially %ecreasing release rate over t&e ne"t t(o (ee$s* It (as !oun% t&at > mg o! UE=; encapsulate% (it&in ;@ mg o! P GA NP) locally a%ministere% #y %irect intramural in!usion via t&e Dispatc& b cat&eter) signi!icantly re%uces neointimal &yperplasia %evelopment in severely #alloonEin.ure% porcine coronary arteries* T&e antiproli!erative e!!ect o! locally %elivere% nanoparticulate UE=; (as limite% primarily to t&ose arteries t&at &a% e"tensive IE 1me%ial ruptures* No e!!ect (as o#serve% in mo%erately in.ure% vessels) (&ic& (as pro#a#ly %ue to insu!!icient retention o! active %rug in t&ese vascular sites*

T&e E!!ect o! -ormulation C&aracteristics an% Delivery Con%itions on Arterial Upta$e In or%er to optimi/e t&e intraEarterial locali/ation o! t&erapeutic agents using t&e NP nanoEcarrier system) several groups e"amine% t&e per!ormance o! NP as a !unction o! !ormulation c&aracteristics as (ell as %elivery con%itions* Particle si/e (as !oun% to #e an important %eterminant o! t&e arterial upta$e* Song et al* A55:B reporte% on a t&reeE!ol% lo(er arterial upta$e o! large P GAE#ase% NP A5;; nmB in comparison to smallEsi/e% NP A4@@ nmB in t&e e" vivo %og !emoral mo%el* ,este%t A55;B %emonstrate% a si/eE%epen%ent NP penetration into an intact ra##it vessel (all6 (&ile smaller NP A4@@H5@@ nmB (ere %eposite% in t&e inner regions o! t&e vessel) larger NP A:49 nmB accumulate% primarily at t&e luminal sur!ace o! t&e aorta* It (as %emonstrate% #y t&e aut&ors t&at t&e resi%ence properties o! tyrp&ostinsE loa%e% NP in rat caroti% arteries (ere in!luence% #y t&eir si/e* T&e smaller NP A>@ nmB s&o(e% #etter ingress into t&e arterial tissue t&an t&e large NP A4;@ nmB) as in%icate% #y a 8*9E!ol% &ig&er initial %rug concentration in t&e tissue) more t&an eig&tE!ol% &ig&er amount o! NP remaining associate% (it& t&e artery !ollo(ing t&e

NanoECarriers o! Drugs an% Genes

59<

rapi% NP (as&out p&ase an% &ig&er local %rug levels maintaine% over 49 %ays !ollo(ing a%ministration A4@=B* -urt&ermore) t&e >@ nm NP) #ut not t&e 4;@ nm NP) signi!icantly re%uce% neointimal &yperplasia in t&e rat caroti% mo%el) esta#lis&ing !or t&e !irst time a correlation #et(een NP si/e an% antirestenotic e!!icacy A4@=)44@B* Drug loa%ing in t&e NP carrier (as also !oun% to #e a signi!icant !actor in t&e arterial upta$e o! NP* It (as %emonstrate% t&at &ig&er UE=; loa%ing re%uce% t&e arterial UE=; levels in #ot& e" vivo an% in vivo stu%ies A<;B) (&ic& can #e attri#ute% to !aster release $inetics o! t&e %rug !rom t&e !ormulation containing t&e &ig&er %rug loa%ing* An increase in t&e NP concentration in t&e in!usion solution increase% t&e arterial UE=; levels up to a certain NP concentration #eyon% (&ic& t&e arterial upta$e o! t&e NP (as limite%) pro#a#ly %ue to c&arge neutrali/ation A<;B* T&e same group also investigate% t&e in!luence o! sur!ace mo%i!ications o! P GAE#ase% NP containing UE=; on t&eir arterial upta$e A<;)55<B* In general) cationic sur!ace mo%iE !ying agents %emonstrate% increase% arterial %rug levels* T&e NP sur!ace) mo%i!ie% (it& a cationic compoun% %i%o%ecyl%imet&ylammonium #romi%e ADMA+B) %emE onstrate% <E to 4@E!ol% greater UE=; arterial levels in comparison to unmo%i!ie% NP in %i!!erent e" vivo an% in vivo stu%ies* T&e mec&anism responsi#le !or t&e en&ance% arterial upta$e (as assume% to involve an alteration in t&e NP sur!ace c&arge A<;B* T&e a!orementione% %ata con!irm t&at NP properties) suc& as si/e an% sur!ace c&emistry) play a critical role in t&eir arterial upta$e an% %epositionM &o(ever) t&eir success!ul application also %epen%s on t&e %elivery con%itions) as (ell as on possiE #le encountere% anatomic #arriers* In particular) !or NP %elivere% to t&e artery #y in!usion t&roug& a #alloon cat&eter %elivery system) it (as reporte% t&at t&e in!uE sion %uration an% pressure in!luence t&e NP arterial locali/ation* Repeate% s&ort in!usions o! NP suspension (ere !oun% to #e t(oE!ol% more e!!ective in terms o! %rug arterial levels t&an a single prolonge% in!usion) (&ic& (as not signi!icantly %i!!erent in e!!icacy !rom a s&ort single in!usion A55<B* In a %i!!erent stu%y) an en&ancement o! particle %eposition at t&e %elivery site (as o#serve% as a conseE Cuence o! an in!usion pressure increase% !rom 5 to 9 atm* Recently) it (as also reporte% t&at t&e locali/ation o! t&e NP in a coronary artery o! a pig (as in!luence% #y t&e cat&eter type A55=B* T&e e!!iciency o! arterial locali/ation o! NP (as evaluate% in t&e porcine coronary mo%el using t(o cat&eters %i!!ering in t&eir (or$ principle* In t&e Dispac&b cat&eter) t&e %rug is in!use% into small close% c&am#ers !orme% (&en t&e #alloon is in!late% an% can %i!!use into t&e arterial (all un%er pressure* T&e in!usion using t&is cat&eter can #e prolonge%) since t&e #loo% continues to !lo(* T&e secon% cat&eter (as t&e In!iltrator b in (&ic& t&e microEin.ectorEports on t&e sur!ace o! t&e #alloon penetrate t&e arterial (all upon in!lation o! t&e #alloon an% allo( intramural %elivery* T&is cat&eter cannot #e use% !or prolonge% in!usion since it #loc$s t&e #loo% !lo( completely* T&e Dispac& b (as !oun% to #e more e!!icient t&an t&e In!iltratorbM it (as suggeste% t&at t&at t&e microEin.ectorEports may &ave !aile% to penetrate t&e intimal layer* Possi#le anatomic #arriers to any particle transE portation t&roug& t&e (all tissue are t&e intact internal an% e"ternal elastic lamina* Moreover) t&e presence o! an at&erosclerotic plaCue on t&e inner luminal sur!ace strongly in!luences t&e particle penetration into t&e arterial (all A55;B* T&is o#servaE tion points to at an a%%itional aspect o! local NP %elivery strategies in restenosis t&at s&oul% #e ta$en un%er consi%eration* In summary) (&ile NP present a promising %rug carrier system !or arterial local %rug %elivery) NP !ormulation properties) as (ell as t&eir %elivery protocol) s&oul% #e care!ully optimi/e% to ac&ieve t&e antirestenotic e!!ect* T&e lac$ o! clinical trials suggests t&at t&e promise !rom t&e stu%ies (as not !ul!ille%*

59=

Co&enESela et al*

S3STEMIC DRUG DE I2ER3 T&e !ailure o! t&e numerous systemic treatment attempts to prevent arterial restE enosis in &umans raises %ou#ts regar%ing t&e !easi#ility o! t&e systemic approac&* Ho(ever) an e!!icient systemic treatment coul% potentially a%%ress pro#lems in&erent in t&e local treatment strategy) regar%less o! its initial success rate* As oppose% to local t&erapy) a systemic %rug %elivery system may allo( t&e treatment o! multiple lesions (it& a single in.ection an% provi%e an option !or convenient a%ministration o! repeat %oses !or t&e treatment optimi/ation) as (ell as !le"i#ility in c&oosing t&e type an% num#er o! stents to #e %eploye% !or varying lesion lengt&s) artery si/es) an% anatomic locations* Numerous stu%ies esta#lis&e% t&e potential o! systemic treatment (it& lipoE somal an% nanoparticulate !ormulations A>:H><B* +y controlling t&eir !ormulation c&aracteristics an% composition) t&ese nanoEcarriers can present a &ig&ly e!!icient %rug %evice (it& speci!ic release pro!ile an% %e!inite target sites A8=)8>);9)>4)>5)>9) 55>H585B* In a%%ition) %ue to t&eir small si/e an% #iocompati#le components) lipoE somes an% NP are sa!e !or parenteral a%ministration* -urt&ermore) liposomes an% NP can #e sur!aceEmo%i!ie% (it& tissueEspeci!ic ligan% to ac&ieve arterial locali/aE tion) !ollo(ing systemic a%ministrationM &o(ever) to %ate) no signi!icant %evelopE ments &ave #een reporte% !or targete% %elivery systems in vivo* Systemic nanoEcarriers t&erapy !or restenosis inclu%es systemic a%ministraE tion inten%e% !or arterial locali/ation) attaine% eit&er passively A#y controlling t&e !ormulation propertiesB or actively A#y attac&ing t&e ligan% on t&e nanoEcarrier sur!aceB) an% systemic a%ministration aime% at a systemic target*

Systemic Delivery !or Arterial ocali/ation Do"oru#icin Do"oru#icin is a %rug clinically use% !or t&e treatment o! cancerM it is o!ten a%minisE tere% as a liposomal !ormulation pegylate% or in ultrasmall nanosi/e in or%er to re%uce its upta$e #y t&e mononuclear p&agocyte system AmpsB an% re%uce t&e %rug to"icity A588B* Do"oru#icin %amages DNA #y intercalation o! t&e ant&racycline portion) metal ion c&elation) or #y generation o! !ree ra%icals* It &as also #een s&o(n to in&i#it DNA topoisomerase II) (&ic& is critical to DNA !unction* T&e cytoto"ic activity o! t&e %rug is not cellEcycle speci!ic* N0>44 are coreEs&ell NP !orme% #y PEGE#ase% #loc$ copolymer encapsulating %o"oru#icin A589B* ,&en N0>44 compoE nents are %issolve% in t&e aCueous p&ase) t&ey !orm sta#le NP Apolymeric micellesB (it& an average %iameter o! 9@ nm* N0>44 NP (ere !oun% to selectively penetrate t&roug& tumor vessel (alls) &aving en&ance% permea#ility A589B* En&ance% vascuE lar permea#ility t&at (as !irst recogni/e% in tumor tissues A58:B (as later on also o#serve% in in!lammatory an% in!ecte% tissues A58;H58=B* U(ato$u et al* A58>B &ypot&esi/e% t&at #alloonEin.ure% coronary arteries also &ave en&ance% permea#ilE ity an% suggeste% t&at t&ey coul% #e a goo% target !or N0>44* T&ey con!irme% t&at #alloon in.ury causes a mar$e% an% sustaine% increase in t&e vascular permea#ility !or at least seven %ays using Evans +lue staining* T&e N0>44 accumulate% in t&e vascular lesion) (&ere permea#ility (as increase%M t&e tissue concentrations o! %o"E oru#icin (ere up to !ourE!ol% &ig&er t&ree &ours !ollo(ing I2 in.ection o! N0>44 in comparison to !ree %o"oru#icin* o( %rug levels (ere o#serve% in t&e unin.ure% contralateral arteries use% as a control* T&e accumulation (as attri#ute% to t(o !actors6 t&e si/e o! t&e NP) (&ic& (as a%eCuate !or en&ance% accumulation) an% t&e NP negative sur!ace c&arge) (&ic& create% an attraction o! t&e !ormulation to

NanoECarriers o! Drugs an% Genes

59>

t&e positively c&arge% luminal sur!ace o! t&e in.ure% #loo% vessel* T&e I2 in.ecte% N0>44 imme%iately an% t&ree an% si" %ays a!ter t&e #alloon in.ury) #ut not %o"oruE #icin alone) signi!icantly in&i#ite% restenosis in t&e rat caroti% artery !our (ee$s a!ter t&e in.ury in #ot& singleE an% %ou#leEin.ury mo%els* It (as %emonstrate% #y immunostaining t&at t&e antirestenotic e!!icacy o! %o"oru#icin NP (as ac&ieve% #y t&e in&i#ition o! SMCs proli!eration) rat&er t&an %ue to en&ancement o! apoptosis or in&i#ition o! in!lammatory cell recruitment* T&e RNA protection assay %emonE strate% t&at t&e e"pression o! several cyto$ines (as in&i#ite% #y N0>44) #ut not t&at o! apoptosisErelate% molecules* Ho(ever) even in lig&t o! t&e encouraging results using t&e N0>44 particles) t&e clinical potential o! t&is !ormulation is limite% %ue to t&e &ig& to"icity o! %o"oru#icin*

Paclita"el T&e PW is a potent antiproli!erative %rug clinically use% to treat cancer* T&e PW promotes polymeri/ation o! t&e mE an% fEunits o! tu#ulin an% causes a#normal sta#ili/ation o! microtu#ules reCuire% !or G5 transition into MEp&ase A59@)594B* In lo( %oses) t&e structural c&anges in t&e cytos$eleton result in a nearly complete in&i#ition o! gro(t& an% proli!eration an% migration o! SMCs !or a long perio%* T&e most common %elivery o! PW !or t&e prevention o! restenosis is local via %rugEelutE ing stents A5=B* 0olo%gie et al* A595B c&ose to e"amine t&e !easi#ility o! systemic a%ministration o! nanopariculate PW !or t&e treatment o! restenosis* T&e rationale (as t&at systemic %elivery o! PW (oul% provi%e a more uni!orm stente% arterial segment e"posure to t&e %rug) treatment o! multiple lesions) an% rea%ily a%.uste% target %ose* Nevert&eless) it s&oul% #e note% t&at t&e NP (ere a%ministrate% locally to t&e iliac #i!urcation* A systemic I2 in!usion (as only employe% !or t&e repeat %ose at 5= %ays* T&e NP use% (as a novel al#uminEsta#ili/e% 48@ nm NP !ormulaE tion o! PW AnPW B A598B* Using al#umin as t&e sta#ili/er solve% t&e &ypersensitivity reactions A%espite prop&yla"isB an% long in!usion rates o! 8 to 59 &ours associate% (it& t&e !ormer nPW containing Cremop&or E as t&e sur!actant A599B* Doses o! nPW (ere a%ministrate% as a 4@Eminute intraEarterial in!usion t&roug& a #alloon cat&eter at t&e iliac #i!urcation to NV (&ite ra##its) imme%iately a!ter #ilateral iliac artery stent implantation* T&e nPW re%uce% neointimal gro(t& at 5= %ays proE %uce% in a %oseHresponse manner) accompanie% #y incomplete &ealing* T&e e!!icacy (as not sustaine% a!ter >@ %ays !ollo(ing a single %ose o! nPW o! t&e &ig&est %ose teste% A: mg1$gB* Ho(ever) a secon% 8*: mg1$g %ose a%ministrate% intravenously 5= %ays a!ter stenting le% to persistent re%uction o! neointimal !ormation at >@ %ays (it& almost complete &ealing* It (as suggeste% t&at t&e novel PW !ormulation may o!!er an antirestenotic t&erapy (it& re%uce% to"icity t&at may overcome some o! t&e limitations o! t&e %rugEcoate% stents* Ho(ever) t&ere are no proven a%vantages over t&e clinically use%) &ig&ly success!ul) Ta"us) %rugEeluting stent*

Systemic Delivery !or a Systemic Target +etter insig&ts an% un%erstan%ing o! t&e involvement o! innate immunity as a vital !actor in t&e progression o! restenosis allo(e% t&e evolution o! a ne( systemic t&erapy) %irecte% against t&e immune cells* Innate immunity triggers t&e &ealing response t&at lea%s to neointimal !ormation A59:H59<B* Monocytes1macrop&ages play a $ey role in t&e in!lammation casca%e) resulting in restenosis o! #loo% vessels A59=)59>B* Emerging e"perimental an% clinical %ata in%icate t&at t&e in!lammatory cell num#er (it&in t&e vessel (all is a po(er!ul pre%ictor o! t&e e"tent o! cellular

5:@

Co&enESela et al*

proli!eration an% intimal t&ic$ening A5:@)5:4B* Monocytes1macrop&ages comprise up to ;@[ o! neointimal cells a!ter stentEin%uce% arterial in.ury in ra##it) porcine) an% non&uman primate mo%els an% in &uman autopsy specimens A5:4B* It (as recently s&o(n #y -u$u%a et al* A5:5B t&at t&e circulating monocytes count in &uman patients increase% an% reac&e% its pea$ t(o %ays a!ter stent implantation) an% t&at t&e ma"imum monocytes count a!ter stent implantation s&o(e% signi!icant positive correlation (it& inEstent neointimal volume at si" mont&s !ollo(Eup in patients a!ter stent implantations* Macrop&ages #elong to t&e MPS* T&ey originate !rom pluripotent stem cells o! #one marro( t&at are precursors !or all &ematopoietic cells) t&at is) lymp&ocytes) eryt&rocytes) osteoclasts) neutrop&ils) an% mononuclear p&agocytes A5:8B* A!ter certain %i!!erentiation steps in t&e #one marro() t&e committe% stem cells give rise to monocytes) (&ic& move to t&e circulation) migrate to %istinct tissue compartE ments) an% %i!!erentiate into macrop&ages* Macrop&ages are an essential part o! t&e immune systemM in normal stea%y state in t&e #o%y) t&ey !orm a constant population (it& a #alance #et(een rene(al an% cell %eat&* In in!lammation) t&e %istri#ution an% %evelopment o! macrop&ages c&anges signi!icantly A5:9H5:;B* -ollo(ing a stimulus suc& as in!lammation) t&e amount o! circulating monocytes an% t&eir migration to t&e site o! in!lammation increases signi!icantly* It (as &ypot&esi/e% #y t&e aut&ors t&at systemic inactivation o! monocytes an% macrop&ages may lea% to attenuation o! neointimal !ormation* In or%er to target t&erapeutic agents to monocytes an% macrop&ages) t&e aut&ors use% liposomes* iposomes are rea%ily ta$en up #y cells o! t&e MPS A!ormerly $no(n as RESB) macrop&ages) in particular) an% to some e"tent) neutrop&ils) #y t&e process o! p&agocytosis* CellEspeci!ic %elivery system !or monocytes an% macrop&ages %epletion coul% #e #ene!icial !or t&e attenuation o! t&e restenotic processes) (&ile causing minimal to"icity to nonEp&agocytic cells A59:B* Monocyte %epletion (as previously ac&ieve% #y a%ministration o! ot&er particulates) silica) as#estors A5:<B) an% carrageenan A5:=B* T&ese met&o%s resulte% in partial %epletion as (ell as un(ante% e!!ects on nonEp&agocytic cells* Rooi.en A5:>B (as t&e !irst to use a liposomal !ormulation containing a #isp&osp&onate A+PB in or%er to %eplete systemic monocytes* iposomes !or t&e treatment o! restenosis via monocytes1macrop&ages %epletion s&oul% possess optimal si/e %istri#ution) since liposomes si/e control circulation time* T&e general tren% !or liposomes o! similar composition is t&at increasing si/e translates into more rapi% upta$e #y MPs A>4)>9B) in particular #y circulation monocytes* iposomes sur!ace c&arge is also an important !actorM c&arge% liposomes are cleare% more rapi%ly t&an neutral) an% negatively c&arge% liposomes are elimiE nate% !aster t&an positively c&arge% liposomes A;8)55>)5;@B* T&e e!!ect o! lipi%s composition) c&oosing lipi%s (it& speci!ic transition p&ase) is an a%%itional imporE tant !actor t&at is intricately interrelate% (it& t&e e!!ect o! c&arge* Circulation time is %epen%ent on mem#rane pac$ing an% permea#ility consi%erations) as t&e incluE sion o! &ig&Ep&ase transition lipi%s (ill increase circulation li!etime* T&e presence o! c&olesterol in liposomes pro#a#ly &as one o! t&e most important roles in t&e maintenance o! mem#rane #ilayer sta#ility an%) conseCuently) long circulation time in vivo A5;4B* In t&e a#sence o! c&olesterol) liposomes are %esta#ili/e% #y &ig&E%enE sity lipoprotein AHD B particles an%) upon release) t&eir components can #e rea%ily eliminate% !rom t&e circulation A5;5)5;8B* iposomes (it& c&olesterol %isplay negative correlation #et(een clearance an% sta#ility in plasma A5;5B* Designing an optimal liposomal !ormulation !or t&e treatment o! restenosis reCuires consi%eration o! all

NanoECarriers o! Drugs an% Genes

5:4

t&ese !actors toget&er* Negatively c&arge% liposomes at t&e range o! 5@@ nm are pre!era#le %ue to t&e compromise #et(een &ig& p&agocytic capacity #y monocytes an% lo(er systemic to"icity* In or%er to ac&ieve optimal release pro!ile an% plasma sta#ility) incorporation o! c&olesterol is essential* Conventional liposomes) negaE tively c&arge% (it& an average si/e o! 5@ nm containing c&olesterol) (ere utili/e% to ac&ieve #etter an% improve% p&agocytosis capacity an%) at t&e same time) suita#le release pattern an% plasma sta#ility* T&us) t&e MPS cells) mainly monocytes) are t&e target site !or t&e systemic treatment o! restenosis*

+isp&osp&onates T&e +Ps) #oneEsee$ing agents) are a !amily o! %rugs t&at in&i#it #one resorption via osteoclast inactivation an% are use% clinically in several calciumErelate% %isor%ers) suc& as tumor osteolysis an% osteoporosis* T&e +Ps are &y%rop&ilicEc&arge% moleE cules t&at %o not penetrate cells A5;9)5;:B* A!ter a%ministration to patients or animals) t&ey accumulate mainly in #one tissue) an% are cleare% rapi%ly !rom t&e circulation into t&e urine A5;9H5;;B* T&e in vivo e!!ect on #one #y +Ps is me%iate% #y t&e p&agoE cytosis o! t&e #oneEa%sor#e% +P #y osteoclasts* ?steoclasts an% macrop&ages s&are a common &ematopoietic progenitor cell in t&e #one marro(* iposomes) a particulate %osage !orm) can #e use% to en&ance t&e intracellular %elivery o! +Ps into p&agocytic cells) suc& as) monocytes1macrop&ages) in cell culture an% in animals A5:>)5;<B* Negatively c&arge% liposomes (ere prepare% #y t&in lipi% !ilm &y%ration* T&e liposomes (ere compose% o! %istearoylp&osp&ati%ylc&oline ADSPCB) p&osE p&ati%ylglycerol ADSPGB) an% c&olesterol ACH? B at a ratio o! 86564) respectively A-ig* 8AB* Anionic liposomes are nonto"ic) an%) a!ter p&agocytosis #y monocytes1 macrop&ages) t&e lipi% #ilayers o! t&e liposomes are %isrupte% un%er t&e in!luence o! t&e lysosomal p&osp&olipases in t&e macrop&age* T&e %rug) (&ic& is %issolve% in t&e aCueous compartments) is release% into t&e cell* -urt&ermore) a !ree +P) release% #y lea$age !rom liposomes or release% !rom %ea% macrop&ages) (ill not enter cells in amounts t&at are a#le to %istur# t&eir meta#olism* T&is approac&) name% t&e liposomeEme%iate% macrop&age Qsuici%eS tec&niCue) (as intensively use% to elimiE nate macrop&ages !rom %i!!erent compartments o! t&e #o%y in animals to stu%y t&e role o! macrop&ages in pat&ological an% immunological con%itions A5;=B* In macrop&ages) cell line ARA,5;9B an% &uman monocytes Aprimary cultureB) &ig&ly en%ocytotic cells) it (as !oun% t&at encapsulation o! +Ps in liposomes en&ances t&eir in&i#itory activity 5@E to 4@@@E!ol% compare% (it& !ree %rug A-ig* 8+B A5;>H5<5B* T&e SMCs an% en%ot&elial cells AECsB are insensitive to t&e liposomal %rug %elivery A59:B* En&ance% p&agocytosis into cells may #e ac&ieve% #y negative an% positive c&arge o! t&e liposomes A9:):>);8)55>)5;<B* Positively c&arge% lipi%s are not approve% #y t&e -DA !or clinical use* T&e sur!ace c&arge %ensity o! t&e liposomal #isp&osp&onates A +PsB &as #een optimi/e% !or minimal lea$age an% e!!ective intracellular %elivery o! encapsulate% %rugs A5;@)5<4B* Activate% macrop&ages secrete cyto$ines an% gro(t& !actors) lea%ing to en&ance% in!lammation) an% in%uce SMC proli!eration an% migration to(ar%s t&e arterial lumen) !orming neointima an% narro(ing t&e artery A5<8H5=@B* T&e +Ps &ave numerous #ioc&emical e!!ects on cellular meta#olism) ranging !rom t&e in&i#iE tion o! general cell meta#olism to mo%ulation o! cyto$ine secretion A5=4B* Nonamino +Ps as clo%ronate an% eti%ronate &ave antiEin!lammatory properties A5=5H5=9B* T&ey re%uce cyto$ine secretion upon stimuli* In contrast to t&e nonEaminoE+Ps) clo%roE nate an% eti%ronate) t&e aminoEcontaining +Ps) alen%ronate an% pami%ronate at a %ose t&at %oes not $ill t&e cells) s&o(e% proin!lammatory properties on macrop&age

5:5

Co&enESela et al*

-IGURE 8 AAB CryoETEM microscopy o! DSPC6DSPG6CH? liposomes o#taine% #y t&in !ilm &y%ration met&o%) an% e"tru%e% t&roug& @*5 _m polycar#onate mem#ranes* A+B E!!ect o! liposomal !ormulations6 #isp&osp&onates A+PsB Aalen%ronate an% clo%ronateB) empty an% !ree %rugs on RA, 5;9 cell survival* Curves represent percent o! cell in&i#ition (it& %i!!erent +P concentrations* Cell count in #u!!er only (as %etermine% to #e 4@@[ An ^ 8B* ACB ASee color insert*B o(E an% &ig&Epo(er p&otomicrograp&s o! &yperc&olesterolemic ra##it iliac artery stents at 5= %ays A2er&oe!! stainingB o! control AI an% IIB an% o! animals treate% (it& A 8mg1$g AIII an% I2B an% ;mg1$g A2 an% 2IB* ADB +ar grap& s&o(ing re%uction in intimal area an% increase% luminal area in treate% animals* AEB +ar grap& s&o(ing re%uction in stenosis A[B in AEtreate% animals An ^ 4; arteries1group) {P c @*@:B* A##reviation6 A) liposomal alen%ronate* Source 6 -rom Re!s* 5;> an% 5>5*

NanoECarriers o! Drugs an% Genes

5:8

!unctions #y in%ucing t&e secretion o! cyto$ines !rom macrop&ages A5=:B* It &as #een s&o(n t&at +Ps also %iminis& secretion o! reactive o"ygen species #y &uman neutrop&ils) polymorp&onuclear leu$ocytes) an% macrop&ages in vitro A5=;H5==B* Encapsulation o! +Ps in liposomal !ormulations en&ance% t&eir potency* iposomal clo%ronate A CB (as more t&an ten times more potent an in&i#itor o! cyto$ine secretion !rom RA, 5;9 t&an t&e !ree %rug A5=>B* T&e PDG-E++ is a strong c&emoattractant !or vascular SMCs involve% in neointima !ormation secon%ary to vascular in.ury A5>@)5>4B* In vivo stu%ies conE %ucte% in t&e aut&orsP la#oratory reveale% t&at PDG-fR activation Ai*e*) tyrosine p&osp&orylationB is mar$e%ly increase% to 48:[ o! #aseline levels in #alloonEin.ure% arteries o! untreate% rats) (&ereas it (as #arely %etecta#le in CEtreate% rats Ai*e*) #elo( #aseline activityB A5;>B* Macrop&ages are a ric& source o! gro(t& !actors an% cyto$ines) (&ic& !acilitates SMCs migration to t&e in.ure% vessel A59=B* Suppression o! t&ese me%iators activation in treate% animals Arat an% ra##itB correspon%s to a su#stantial re%uction o! PDG-E++ protein levels in t&e lesion) (&ic& can e"plain t&e re%uce% SMC migration an% neointimal !ormation in treate% animals* T&e aut&orsP %ata are in con.unction (it& re%uction in arterial an% #loo% cyto$ines) I E4f) TN-m) N-+ an% MMPE5 activity) !ollo(ing in.ury A59:B* T&e systemic inactivation results in re%uce% e"pression o! local in!lammatory me%iators) lea%ing to re%uce% activaE tion an% proli!eration o! SMC an% %ecrease% neointimal !ormation* It (as reporte% #y t&e aut&ors t&at +Ps %eplete #loo% monocytes A-ACSB) tissue macrop&ages) an% total ,+C ACoulterB count in t&e ra##it #alloon angioplasty mo%el A(it& or (it&out stentingB A59:)5;>B* Systemic a%ministration o! +P re%uce% circulating monocytes as (ell as tissue in!iltration an% accumulation o! macroE p&ages* T&e ,+C count increase% slig&tly 9= &ours a!ter surgery) (it& no signi!iE cant %i!!erence #et(een controls an% t&e +P groups A5;>B* Monocyte num#er at 59 an% 9= &ours a!ter #alloon in.ury an% stenting (as signi!icantly lo(er in +PsE treate% animals* +loo% monocytes %epletion an% elevating ,+C (as partial an% transient) lasting si" %ays a!ter I2 in.ection o! +Ps* SiteEspeci!ic re%uction o! macE rop&ages num#ers (as o#serve% at t&e in.ure% arterial site in +PsEtreate% ra##its) t&ree an% si" %ays a!ter in.ury A59:B* -urt&ermore) t&e num#er o! tissue macroE p&ages in liver an% spleen (ere re%uce% #y +Ps at ; %ays a!ter treatment* iposomes loa%e% (it& t&e !luorescent mar$er) R&o%amine) (it& or (it&out a +P (ere utili/e% to support t&e notion t&at +Ps e"ert t&eir e!!ects systemically A59:)5;>B* Depletion o! systemic monocytes t&at carry t&e liposomes to t&e in.ure% site s&oul% result in re%uce% arterial upta$e o! liposomes* In%ee%) !luorescentEla#ele% +Ps signi!icantly re%uce% t&e !luorescent signal in t&e in.ure% arterial (all) (&ereas massive !luorescent (as %etecte% (it&out t&e +P A59:B* T&e +Ps (ere in.ecte% at %i!!erent %osage regimens to rats an% &yperc&olesE terolemic ra##its to e"amine t&e t&erapeutic e!!ect* T&e rat caroti% artery in.ure% #y a #alloon cat&eter &as #een (i%ely use% as a mo%el o! angioplasty* T&e rat mo%el is a Qproli!erationS mo%el (it&out !oam cells* T&is !orm o! in.ury causes imme%iate coagulation an% t&rom#osis casca%e in (&ic& platelets a%&ere) sprea%) an% %egranulate on t&e %enu%e% sur!ace o! t&e artery) an%) appro"imately 59 &ours later) SMC #egin to proli!erate* T&e +Ps) clo%ronate an% alen%ronate) (ere in.ecte% to male sa#ra rats) 4: mg1$g an% 8 mg1$g) respectively* Mar$e% neointimal !ormation an% %ecrease% luminal area (ere o#serve% in control animals* T&e C an% liposomal alen%ronate A AB suppresse% intimal gro(t& (&en a%ministere% I2 on %ay Z4 an% o;* T&e N1M ratios (ere re%uce% #y ;@[ an% ;>[ !or C an% A) respectively A59:)5;>)5>5B*

5:9

Co&enESela et al*

T&e &yperc&olesterolemic ra##it mo%el simulates t&e &uman mo%el) since !oam cells are involve%* iposomal clo%ronate an% alen%ronate (ere in.ecte% one %ay #e!ore #alloon angioplasty an% si" %ays a!ter A4: mg1$g an% 4*: mg1$g) respecE tivelyB) resulting in signi!icant attenuation o! intimal &yperplasia an% luminal stenosis 5= %ays a!ter surgery A59:)5;>B* Stenosis (as re%uce% !rom <: e =[ in t&e control6 empty liposomes) saline an% !ree +P) to 94 e =[ (it& C treatment an% ;= e :[ (it& A A8 mg1$gB treatment* Stenting proce%ure in t&e Iliac artery resulte% in a#un%ant concentric neointimal !ormation compose% o! SMC an% !oam cells) (it& #ot& intraluminal an% out(ar% neointimal gro(t&* uminal stenosis 5= %ays a!ter stenting (as := e 44[* T&e A A8 mg1$gB signi!icantly re%uce% neointimal !ormaE tion compare% (it& control groups* No signi!icant %i!!erence (as o#serve% #et(een animals treate% (it& 8 or ; mg1$g A or 4: mg1$g C A-ig* 8CB* Di!!erent %osage regiments (ere e"amine%M multiple %oses o! C A4: mg1$gB or A A4*: mg1$gB one %ay #e!ore #alloon angioplasty an% si" %ays a!ter (ere !oun% to &ave t&e same e!!ect as one %ose at %ay Z4* C&anging t&e time o! in.ection !rom Z4 to one in.ection si" %ays a!ter surgery &a% no e!!ect* Treatment o! A) a single %ose) at t&e time o! in.ury reCuire% %ose a%.ustment) elevation o! %ose !rom 4*: to 8 mg1 $g A5;>B* A %rug potency e!!ect relations&ip o! re%ucing restenosis (as esta#lis&e% #y t&e aut&ors) alen%ronate \ pami%ronate \ ISAE48E4 \ clo%ronate A5;;)5>8B* Nonamino +Ps as clo%ronate are several or%ers o! magnitu%e less potent t&an t&e amino +P) alen%ronate) in in&i#iting osteoclasts an% conseCuently #oneErelate% %isor%ers) suc& as tumor osteolysis an% osteoporosis A5;:B* Consistent e!!ect (as esta#lis&e% (it& monocytes1macrop&age in&i#ition A59:B* Having esta#lis&e% t&e antirestenotic e!!ect o! +Ps encapsulate% in liposomes) t&e aut&ors !urt&er investigate% t&e use o! polymeric NP !or t&e same purpose) in or%er to e"amine (&et&er a polymeric nanoparticulate !ormulation containing +Ps coul% e"ert a compara#le e!!ect on +Ps* Similar to liposomes) a success!ul targeting to monocytes1macrop&ages reCuires $no(le%ge o! t&e particle c&aracteristics t&at promote t&eir p&agocytosis* T&e p&ysicoc&emical properties o! NP) suc& as si/e an% sur!ace c&emistry) also play an important role in opsonin a%sorption an% t&e elimiE nation o! NP !rom t&e circulation A5>9)5>:B* P&agocytosis may #e increase% (it& increasing sur!ace c&arge o! t&e particle) particularly (&en t&e c&arge is negative A5>;B* In a%%ition) largerEsi/e% particles are more incline% to #e p&agocytose% t&an smaller ones A5><B* Encapsulation o! &y%rop&ilic agents) suc& as +Ps) in a nanoEcarE rier #ase% on a lipop&ilic polymer is c&allenging %ue to t&e agentsP a!!inity !or t&e e"ternal aCueous p&ase A9@)55:)5>=)5>>B* T&e encapsulation e!!iciency o! &y%rop&ilic +Ps in NP may #e optimi/e% #y t&e in situ !ormation o! t&eir salts (it& lo( solu#ilE ity in (ater) suc& as poorly (ater solu#le comple" (it& calcium* T&e %ou#le emulE sionHsolvent evaporation met&o% A5@@B (as use% !or t&e NP preparation) an% t&eir antirestenotic e!!ect (as e"amine% in animal mo%els* Unli$e ot&er NP !ormulations e"amine% !or restenosis t&erapy releasing t&e t&erapeutic agent in a slo( release manner) t&e release rate o! t&e +Ps (as %esigne% to #e rapi% insi%e t&e p&agocytic cell) protecting t&e %rug in circulation !or a su!!icient perio% till p&agocytosis* T&e NP o! an amino#isp&osp&onate AISAB (ere !oun% to release ==[ o! t&e %rug (it&in 5@ minutes an% t&e release (as complete% (it&in t(o &ours* T&e ISA NP (it& encapsulation yiel% o! ;>[ an% average si/e o! 8>5 nm) #ot& su#cutaneously an% intravenously in.ecte% one %ay #e!ore #alloon angioplasty A4: mg1$gB) resulte% in signi!icant attenuation o! intimal &yperplasia an% stenosis at 49 %ays in t&e rat caroti% in.ury mo%el compare% (it& control animals treate% (it& !ree ISA) carrier solution or #lan$ NP* Alen%ronate NP (it& encapsulation yiel% o! ::*4[ an%

NanoECarriers o! Drugs an% Genes

5::

average si/e o! 558 nm) su#cutaneously a%ministere% A4*: mg1$gB one %ay #e!ore an% si" %ays a!ter #alloon angioplasty) resulte% in a re%uction o! intimal &yperplaE sia an% stenosis at 5= %ays in t&e ra##it mo%el* T&e e!!ect o! +Ps encapsulate% in nanoEcarriers on SMCs proli!eration is in%iE rectly me%iate% #y t&e in&i#itory e!!ect on monocytes1macrop&ages* +y systemic a%ministration o! +Ps encapsulate% in nanoEcarriers) systemic monocytes an% tissue macrop&ages %epletion is ac&ieve%) re%ucing t&e num#er o! monocytes1macroE p&ages availa#le) an% t&ere#y re%ucing t&eir accumulation in t&e arterial (all an% t&e su#seCuent contri#ution to SMCs migration an% proli!eration* T&is approac&) #iologic targeting) is !un%amentally %i!!erent t&an any ot&er treatment mo%ality in restenosis* ,&ile ot&er nanoEcarrier !ormulations) even i! a%ministrate% systemiE cally) are aime% to e!!ect locally in&i#iting local events relate% (it& t&e restenotic course o! action) t&e encapsulate% +Ps present a systemic t&erapy to a systemic process) regar%less o! t&e proce%ure an% t&e %evices use%* I! e!!ective in a clinical setting) it may #e an easily a%ministere%) costEe!!ective mo%ality t&at allo(s !le"i#iE lity in c&oosing t&e type an% num#er o! stents to #e %eploye% an% may serve as an a%.unct t&erapy in &ig&Eris$ patients*

SUMMAR3 To %ate) local %rug %elivery is consi%ere% to #e t&e most !avora#le treatment !or restenosis) (it& %rugEeluting stents #eing t&e lea%ing approac& in clinical practiceM &o(ever) t&eir longEterm e!!icacy an% to"icity s&oul% #e !urt&er e"amine%* In a%%iE tion) t&e antirestenotic e!!ectiveness in &ig&Eris$ groups) as (ell as in cases o! small vessels) long lesions) an% ostial or #i!urcation lesions is yet to #e esta#lis&e%* Anot&er strategy !or t&e prevention o! restenosis #y t&e local route investiE gate% over t&e last !e( %eca%es is nanoEcarriers) inclu%ing liposomes an% NP* T&ese carriers o!!er a potentially improve% %elivery system) since t&ey incorporate t&e a%vantages o! local %rug %elivery an%) in a%%ition) ena#le targeting to speci!ic cells* iposomes an% polymeric NP (ere !oun% to #e &ig&ly e!!icient in t&e local %elivery o! #ot& p&armaceutical agents an% gene pro%ucts* In t&e !iel% o! gene t&erapy !or restenosis) numerous liposomal !ormulations (ere e"amine%* T&e !ormulations (ere mo%i!ie% #y eit&er sur!ace c&arge or nonimmunogenic viral vectors !or t&e en&ancement o! t&eir penetration t&roug& t&e arterial (all as (ell as t&eir incorpoE ration into speci!ic cells an% speci!ic intracellular locali/ation* Ho(ever) t&us !ar) all t&e approac&es in gene t&erapy !or t&e prevention o! restenosis !aile% to pro%uce satis!actory results in clinical trails* T&e use o! NP !or gene t&erapy in restenosis is in its early stages* T&e systemic approac& !or restenosis prevention A(it& a !ree %rug in solutionB !aile% to pro%uce satis!actory results in clinical trials* T&is is #ecause t&erapeutic %rug levels in t&e in.ure% artery (ere not ac&ieve% !ollo(ing systemic a%ministration* T&e strategy o! most systemic e"perimental t&erapies !or restenosis is to ac&ieve arterial locali/ation !ollo(ing t&e systemic %elivery o! a p&armaceutical agent* T&e single uniCue approac& o! systemic a%ministration aime% at a systemic target) terme% Q#iological targeting)S is t&e +PsEencapsulate% nanoEcarriers* T&is latter approac& (as %evelope% in vie( o! t&e para%igm c&ange t&at restenosis is a systemic %isease mani!este% in local &yperplasia) (&ic& calls !or a systemic interE vention* Encapsulate% +Ps are targete% to %eplete circulating monocytes) (&ic& are converte% into macrop&ages at t&e site o! in.ury) an% play an important role in t&e course o! restenosis* Suc& targeting reCuires speci!ic !ormulation properties)

5:;

Co&enESela et al*

inclu%ing relatively larger particle si/e an% &ig& sur!ace c&arge* +ot& liposomes an% NP (ere !oun% to #e e!!ective carriers !or targeting o! +P to monocytes) s&o(ing &ig&ly promising results as an antirestenosic t&erapy in rat an% ra##it restenosis mo%els* NanoEcarriers (it& t&eir %iverse si/e) &ig& %rug entrapment capacity) t&e a#ility to control t&eir c&aracteristics) an% %rug release pro!ile present an a%vantaE geous %elivery system !or t&erapeutic agents in restenosis) suita#le !or #ot& local an% systemic a%ministration* T&e systemic approac&) i! a%%resse% properly) coul% provi%e signi!icant a%vantages over local %rug %elivery*

RE-ERENCES
4* Garas SM) Hu#er P) Scott NA* ?vervie( o! t&erapies !or prevention o! restenosis a!ter coronary interventions* P&armacol T&er 5@@4M >5A5H8B64;:H4<=* 5* +ennett MR) ?PSullivan M* Mec&anisms o! angioplasty an% stent restenosis6 implications !or %esign o! rational t&erapy) P&armacol T&er 5@@4M >4A5B649>H4;;* 8* +ittl 'A* A%vances in coronary angioplasty* N Engl ' Me% 4>>;M 88:A4<B645>@H48@5* 9* C&orny M) -is&#ein I) Golom# G) Drug %elivery systems !or t&e treatment o! restenosis) Crit Rev T&er Drug 5@@@M 4<A8B659>H5=9* :* -isc&man D ) eon M+) +aim DS) et al* A ran%omi/e% comparison o! coronaryEstent placement an% #alloon angioplasty in t&e treatment o! coronaryEartery %isease* N Engl ' Me% 4>>9M 884A=B69>;H:@4* ;* i##y P) Gan/ P* Restenosis revisite%Rne( targets) ne( t&erapies* N Engl ' Me% 4>><M 88<A;B694=H94>* <* Mint/ GS) Popma '') Pic&ar% AD) et al* Arterial remo%eling a!ter coronary angioplastyHA serial intravascular ultrasoun% stu%y* Circulation 4>>;M >9A4B68:H98* =* Ho!!mann R) Mint/ GS) Dussaillant GR) et al* Patterns an% mec&anisms o! inEstent restenosisRa serial intravascular ultrasoun% stu%y* Circulation 4>>;M >9A;B6459<H45:9* >* o(e HC) ?esterle SN) 0&ac&igian M* Coronary inEstent restenosis6 current status an% !uture strategies* ' Am Coll Car%iol 5@@5M 8>A5B64=8H4>8* 4@* Mu%ra H) Regar E) 0lauss 2) et al* Serial !ollo(Eup a!ter optimi/e% ultrasoun%Egui%e% %eployment o! Palma/ESc&at/ stentsRinEstent neointimal proli!eration (it&out signi!iE cant re!erence segment response* Circulation 4>><M >:A5B68;8H8<@* 44* +rasen 'H) 0ivela A) Roser 0) et al* Angiogenesis) vascular en%ot&elial gro(t& !actor an% plateletE%erive% gro(t& !actorE++ e"pression) iron %eposition) an% o"i%ationEspeci!ic epitopes in stente% &uman coronary arteries* Arterioscl T&rom 2as 5@@4M 54A44B64<5@H4<5;* 45* -ar# A) Sangiorgi G) Carter A') et al* Pat&ology o! acute an% c&ronic coronary stenting in &umans* Circulation 4>>>M >>A4B699H:5* 48* Gre(e PH) Dene$e T) Mac&raoui A) +armeyer ') Muller 0M* Acute an% c&ronic tissue response to coronary stent implantation6pat&ologic !in%ings in &uman specimen* ' Am Coll Car%iol 5@@@M 8:A4B64:<H4;8* 49* 0orno(s$i R) Hong M0) Tio -?) +ram(ell ?) ,u HS) eon M+* Instent restenosis6 conE tri#utions o! in!lammatory responses an% arterial in.ury to neointimal &yperplasia* ' Am Coll Car%iol 4>>=M 84A4B6559H58@* 4:* +ayesEGenis A) Camp#ell 'H) Carlson P') Holmes DR) Sc&(art/) RS* Macrop&ages) myo!iE #ro#lasts an% neointimal &yperplasia a!ter coronary artery in.ury an% repair* At&erosclerosis 5@@5M 4;8A4B6=>H>=* 4;* Herrman 'PR) Hermans ,RM) 2os ') Serruys P,* P&armacological approac&es to t&e prevention o! restenosis !ollo(ing angioplastyRt&e searc& !or t&e &olyEgrail*4* Drugs 4>>8M 9;A4B64=H:5* 4<* an%/#erg +R) -ris&man ,H) erric$ 0* Pat&op&ysiology an% p&armacological approac&es !or prevention o! coronary artery restenosis !ollo(ing coronary artery #alloon angioplasty an% relate% proce%ures* Prog Car%iovasc Dis 4>><M 8>A9B68;4H8>=* 4=* -a"on DP* Systemic %rug t&erapy !or restenosisRQDe.a vu all over again)S Circulation 5@@5M 4@;A4=B655>;H55>=*

NanoECarriers o! Drugs an% Genes

5:<

4>* 2ersaci -) Gaspar%one A) Tomai -) et al* Immunosuppressive t&erapy !or t&e prevention o! restenosis a!ter coronary artery stent implantation AIMPRESS stu%yB* ' Am Coll Car%iol 5@@5M 8>A:B64:aH4:a* 5@* Colom#o A) Dr/e(iec$i ') +anning A) et al* Ran%omi/e% stu%y to assess t&e e!!ectiveness o! slo(E an% mo%erateErelease polymerE#ase% paclita"elEeluting stents !or coronary artery lesions* Circulation 5@@8M 4@=A<B6<==H<>9* 54* Gru#e E) Sil#er S) Hauptmann 0E) et al* Si"E an% t(elveEmont& results !rom a ran%E omi/e%) %ou#leE#lin% trial on a slo(Erelease paclita"elEeluting stent !or %e novo coronary lesions* Circulation 5@@8M 4@<A4B68=H95* 55* Morice M) Serruys P,) Sousa 'E) et al* A ran%omi/e% comparison o! a sirolimusEeluting stent (it& a stan%ar% stent !or coronary revasculari/ation* Ne( Engl ' Me% 5@@5M 89;A58B64<<8H4<=@* 58* Moses ',) eon M+) Popma '') et al* SirolimusEeluting stents versus stan%ar% stents in patients (it& stenosis in a native coronary artery* Ne( Engl ' Me% 5@@8M 89>A49B6484:H4858* 59* Sc&o!er ') Sc&luter M) Gers&lic$ AH) et al* SirolimusEeluting stents !or treatment o! patients (it& long at&erosclerotic lesions in small coronary arteries6 %ou#leE#lin%) ranE %omise% controlle% trial AEESIRIUSB* ancet 5@@8M 8;5A>8>@B64@>8H4@>>* 5:* Mar" S?) 'ayaraman T) Go ?) Mar$s AR* RapamycinE-0+P in&i#its cell cycle regulators o! proli!eration in vascular smoot& muscle cells* Circ Res 4>>:M <;A8B6945H94<* 5;* Poon M) Mar" S?) Gallo R) +a%imon '') Tau#man M+) Mar$s AR* Rapamycin in&i#its vascular smoot& muscle cell migration* ' Clin Invest 4>>;M >=A4@B655<<H55=8* 5<* Par$ S') S&im ,H) Ho DS) et al* A paclita"elEeluting stent !or t&e prevention o! coronary restenosis* N Engl ' Me% 5@@8M 89=A4;B64:8<H4:9:* 5=* Stone G,) Ellis SG) Co" DA) et al* A polymerE#ase%) paclita"elEeluting stent in patients (it& coronary artery %isease* N Engl ' Me% 5@@9M 8:@A8B6554H584* 5>* A&san - ) Rivas IP) 0&an MA) Suare/ AIT* Targeting to macrop&ages6 role o! p&ysicoc&emiE cal properties o! particulate carriersEliposomes an% microsp&eresRon t&e p&agocytosis #y macrop&ages* ' Control Release 5@@5M <>A4H8B65>H9@* 8@* Allen TM) Moase EH* T&erapeutic opportunities !or targete% liposomal %rug %elivery* A%v Drug Deliv Rev 4>>;M 54644<H488* 84* 0reuter '* Drug Targeting (it& nanoparticles* Eur ' Drug Meta# P& 4>>9M 4>A8B65:8H5:;* 85* erou" 'C) Co/ens R) Roesel ' ) et al* P&armaco$inetics o! a novel HI2E4 protease in&i#iE tor incorporate% into #io%egra%a#le or enteric nanoparticles !ollo(ing intravenous an% oral a%ministration to mice* ' P&arm Sci 4>>:M =9A45B648=<H48>4* 88* Mog&imi SM) Hunter AC) Murray 'C) ongEcirculating an% targetEspeci!ic nanoparticles6 t&eory to practice* P&armacol Rev 5@@4M :8A5B65=8H84=* 89* Nis&io$a 3) 3os&ino H* ymp&atic targeting (it& nanoparticulate system* A%v Drug Deliv Rev 5@@4M 9<A4B6::H;9* 8:* Torc&ilin 2P* Drug targeting* Eur ' P&arm Sci 5@@@M 44Asuppl 5B6S=4HS>4* 8;* 3o$oyama M) ?$ano T* Targeta#le %rug carriers6 present status an% a !uture perspective* A%v Drug Deliver Rev 4>>;M 546<<H=@* 8<* Ga#i/on A) C&emla M) T/emac& D) Horo(it/ AT) Goren D* iposome longevity an% sta#ility in circulation6 e!!ects on t&e in vivo %elivery to tumors an% t&erapeutic e!!icacy o! encapsulate% ant&racyclines* ' Drug Target 4>>;M 8A:B68>4H8>=* 8=* Ga#i/on A) C&isin R) Amselem S) et al* P&armaco$inetic an% imaging stu%ies in patients receiving a !ormulation o! liposomeEassociate% a%riamycin* +r ' Cancer 4>>4M ;9A;B6445:H4485* 8>* Ga#i/on A) Papa&a%.opoulos D* iposome !ormulations (it& prolonge% circulation time in #loo% an% en&ance% upta$e #y tumors* Proc Natl Aca% Sci USA 4>==M =:A4=B6;>9>H;>:8* 9@* Goven%er T) Stolni$ S) Garnett MC) Illum ) Davis SS* P GA nanoparticles prepare% #y nanoprecipitation6 %rug loa%ing an% release stu%ies o! a (ater solu#le %rug* ' Control Release 4>>>M :<A5B64<4H4=:* 94* Torc&ilin 2P* iposomes as targeta#le %rug carriers* Crit Rev T&er Drug Carrier Syst 4>=:M 5A4B6;:H44:* 95* Torc&ilin 2P* Recent a%vances (it& liposomes as p&armaceutical carriers* Nat Rev Drug Discov 5@@:M 9A5B649:H4;@*

5:=

Co&enESela et al*

98* S/o$a -) ?lson -) Heat& T) 2ail ,) May&e( E) Papa&a%.opoulos D* Preparation o! unilaE mellar liposomes o! interme%iate si/e A@*4H@*5 mumolB #y a com#ination o! reverse p&ase evaporation an% e"trusion t&roug& polycar#onate mem#ranes* +ioc&im +iop&ys Acta 4>=@M ;@4A8B6::>H:<4* 99* ?lson -) Hunt CA) S/o$a -C) 2ail ,') Papa&a%.opoulos D* Preparation o! liposomes o! %e!ine% si/e %istri#ution #y e"trusion t&roug& polycar#onate mem#ranes* +ioc&im +iop&ys Acta 4><>M ::<A4B6>H58* 9:* 0oso#e T) Moriyama E) To$uo$a 3) 0a(as&ima N* Si/e an% sur!ace c&arge e!!ect o! :Eaminolevulinic aci%Econtaining liposomes on p&oto%ynamic t&erapy !or cultivate% cancer cells* Drug Dev In% P&arm 5@@:M 84A<B6;58H;5>* 9;* V&u ') 3an -) Guo V) Marc&ant RE* Sur!ace mo%i!ication o! liposomes #y sacc&ari%es6 vesicle si/e an% sta#ility o! lactosyl liposomes stu%ie% #y p&oton correlation spectrosE copy* ' Colloi% Inter!ace Sci 5@@:M 5=>A5B6:95H::@* 9<* Nomura SM) Mi/utani 3) 0urita 0) ,atana#e A) A$iyos&i 0) C&anges in t&e morp&ology o! cellEsi/e liposomes in t&e presence o! c&olesterol6 !ormation o! neuronEli$e tu#es an% liposome net(or$s* +ioc&im +iop&ys Acta 5@@:M 4;;>A5B64;9H4;>* 9=* Nagayasu A) Uc&iyama 0) 0i(a%a H* T&e si/e o! liposomes6 a !actor (&ic& a!!ects t&eir targeting e!!iciency to tumors an% t&erapeutic activity o! liposomal antitumor %rugs* A%v Drug Deliv Rev 4>>>M 9@A4H5B6<:H=<* 9>* Is&i%a ?) Maruyama 0) Sasa$i 0) I(atsuru M* Si/eE%epen%ent e"travasation an% interstiE tial locali/ation o! polyet&yleneglycol liposomes in soli% tumorE#earing mice* Int ' P&arm 4>>>M 4>@A4B69>H:;* :@* +a.oria R) Contractor S-* E!!ect o! t&e si/e o! liposomes on t&e trans!er an% upta$e o! car#o"y!luorescein #y t&e per!use% &uman term placenta* ' P&arm P&armacol 4>><M 9>A<B6;<:H;=4* :4* Nagayasu A) S&imoo$a T) 0i(a%a H* E!!ect o! vesicle si/e on in vivo release o! %aunoruE #icin !rom &y%rogenate% egg p&osp&ati%ylc&olineE#ase% liposomes into #loo% circulaE tion* +iol P&arm +ull 4>>:M 4=A<B64@5@H4@58* :5* Nagayasu A) S&imoo$a T) 0inouc&i 3) Uc&iyama 0) Ta$eic&i 3) 0i(a%a H* E!!ects o! !luE i%ity an% vesicle si/e on antitumor activity an% myelosuppressive activity o! liposomes loa%e% (it& %aunoru#icin* +iol P&arm +ull 4>>9M 4<A<B6>8:H>8>* :8* iu D) Mori A) Huang * Role o! liposome si/e an% RES #loc$a%e in controlling #io%istriE #ution an% tumor upta$e o! GM4Econtaining liposomes* +ioc&im +iop&ys Acta 4>>5M 44@9A4B6>:H4@4* :9* 'ones MN) Nic&olas AR* T&e e!!ect o! #loo% serum on t&e si/e an% sta#ility o! p&osp&oliE pi% liposomes* +ioc&im +iop&ys Acta 4>>4M 4@;:A5B649:H4:5* ::* +ar/a M) Stuart M) S/o$a -) 'r* E!!ect o! si/e an% lipi% composition on t&e p&armaco$inetE ics o! intravitreal liposomes* Invest ?p&t&almol 2is Sci 4>=<M 5=A:B6=>8H>@@* :;* Sato 3) 0i(a%a H) 0ato 3* E!!ects o! %ose an% vesicle si/e on t&e p&armaco$inetics o! liposomes* C&em P&arm +ull ATo$yoB 4>=;M 89A4@B69599H95:5* :<* Ric&ar%s R ) Ha##ersett RC) Sc&er I) et al* In!luence o! vesicle si/e on complementE %epen%ent immune %amage to liposomes* +ioc&im +iop&ys Acta 4>=;M =::A5B6558H58@* :=* Magin R ) Hunter 'M) Niesman MR) +ar$ GA* E!!ect o! vesicle si/e on t&e clearance) %istri#ution) an% tumor upta$e o! temperatureEsensitive liposomes* Cancer Drug Deliv 4>=;M 8A9B6558H58<* :>* Sc&(en%ener RA) agoc$i PA) Ra&man 3E* T&e e!!ects o! c&arge an% si/e on t&e interaction o! unilamellar liposomes (it& macrop&ages* +ioc&im +iop&ys Acta 4>=9M <<5A4B6>8H4@4* ;@* Margolis +) Ney!a$& AA) 'r* T&e %istri#ution o! soli% liposomes o! %i!!erent si/e on t&e sur!ace o! epit&elial cells* Eur ' Cell +iol 4>=8M 84A5B65:;H5;5* ;4* 3ager P) S&eri%an 'P) Peticolas , ) C&anges in si/e an% s&ape o! liposomes un%ergoing c&ain melting transitions as stu%ie% #y optical microscopy* +ioc&im +iop&ys Acta 4>=5M ;>8A5B69=:H9>4* ;5* Ra&man 3E) Cerny EA) Patel 0R) au EH) ,rig&t +'* Di!!erential upta$e o! liposomes varying in si/e an% lipi% composition #y parenc&ymal an% $up!!er cells o! mouse liver* i!e Sci 4>=5M 84A4>B65@;4H5@<4* ;8* 'uliano R ) Stamp D* T&e e!!ect o! particle si/e an% c&arge on t&e clearance rates o! lipoE somes an% liposome encapsulate% %rugs* +ioc&em +iop&ys Res Commun 4><:M ;8A8B6;:4H;:=*

NanoECarriers o! Drugs an% Genes

5:>

;9* asic DD* Novel applications o! liposomes* Tren%s +iotec&nol 4>>=M 4;A<B68@<H854* ;:* asic DD) Martin -') Ga#i/on A) Huang S0) Papa&a%.opoulos D* Sterically sta#ili/e% liposomes6 a &ypot&esis on t&e molecular origin o! t&e e"ten%e% circulation times* +ioc&im +iop&ys Acta 4>>4M 4@<@A4B64=<H4>5* ;;* asic DD) 2allner '') ,or$ing P0* Sterically sta#ili/e% liposomes in cancer t&erapy an% gene %elivery* Curr ?pin Mol T&er 4>>>M 4A5B64<<H4=:* ;<* Papa&a%.opoulos D) Allen TM) Ga#i/on A) et al* Sterically sta#ili/e% liposomes6 improveE ments in p&armaco$inetics an% antitumor t&erapeutic e!!icacy* Proc Natl Aca% Sci USA 4>>4M ==A59B644)9;@H44)9;9* ;=* ,oo%le MC) asic DD* Sterically sta#ili/e% liposomes* +ioc&im +iop&ys Acta 4>>5M 4448A5B64<4H4>>* ;>* Hoso$a(a S) Taga(a T) Ni$i H) Hira$a(a 3) No&ga 0) Nagai$e 0* E!!icacy o! immunoliE posomes on cancer mo%els in a cellEsur!aceEantigenE%ensityE%epen%ent manner* +r ' Cancer 5@@8M =>A=B64:9:H4::4* <@* Huang A) 0ennel S') Huang * Interactions o! immunoliposomes (it& target cells* ' +iol C&em 4>=8M 5:=A55B649)@89H49)@9@* <4* Allemann E) Gurny R) Doel$er E* DrugEloa%e% nanoparticlesRpreparation met&o%s an% %rug targeting issues* Eur ' P&arm +iop&arm 4>>8M 8>A:B64<8H4>4* <5* 0reuter '* NanoparticleE#ase% %rug %elivery systems* ' Control Release 4>>4M 4;A4H5B64;>H4<;* <8* a#&aset(ar 2) Song CW) evy R'* Nanoparticle %rug %elivery system !or restenosis* A%v Drug Deliver Rev 4>><M 59A4B6;8H=:* <9* Soppimat& 0S) Amina#&avi TM) 0ul$arni AR) Ru%/ins$i ,E* +io%egra%a#le polymeric nanoparticles as %rug %elivery %evices* ' Control Release 5@@4M <@A4H5B64H5@* <:* a#&aset(ar 2* evy R'* Implants !or siteEspeci!ic %rug %elivery* ' Appl +iomater 4>>4M 5A8B6544H545* <;* a#&aset(ar 2) Song C) Hump&rey ,) S&e#us$i R) evy R'* Arterial upta$e o! #io%eE gra%a#le nanoparticles6 e!!ect o! sur!ace mo%i!ications* ' P&arm Sci 4>>=M =<A4@B6455>H4589* <<* estini +') Sagnella SM) Wu V) et al* Sur!ace mo%i!ication o! liposomes !or selective cell targeting in car%iovascular %rug %elivery* ' Control Release 5@@5M <=A4H8B658:H59<* <=* G&a!!ari S) 0ereia$es D') inco!! AM) et al* Platelet glycoprotein II#1IIIa receptor #loc$a%e (it& a#ci"ima# re%uces isc&emic complications in patients un%ergoing %irectional coronary at&erectomy* EPI ?G Investigators* Evaluation o! PTCA to improve longEterm outcome #y c<E8 GP II#1IIIa receptor #loc$a%e* Am ' Car%iol 4>>=M =5A4B6<H45* <>* Everts M) 0oning GA) 0o$ R') et al* In vitro cellular &an%ling an% in vivo targeting o! EE selectin%irecte% immunocon.ugates an% immunoliposomes use% !or %rug %elivery to in!lame% en%ot&elium* P&arm Res 5@@8M 5@A4B6;9H<5* =@* Hu(yler ') Cerletti A) -ric$er G) E#erle AN) Dre(e '* +yEpassing o! Pglycoprotein using immunoliposomes* ' Drug Target 5@@5M 4@A4B6<8H<>* =4* E%elman ER) 0arnovs$y M'* Contrasting e!!ects o! t&e intermittent an% continuous a%ministration o! &eparin in e"perimental restenosis* Circulation 4>>9M =>A5B6<<@H<<;* =5* e!$ovits ') Topol E'* P&armacological approac&es !or t&e prevention o! restenosis a!ter percutaneous coronary intervention* Prog Car%iovasc Dis 4>><M 9@A5B6494H4:=* =8* Ito& T) Nonogi H) Miya/a$i S) et al* ocal %elivery o! argatro#an !or t&e prevention o! restenosis a!ter coronary #alloon angioplasty6 a prospective ran%omi/e% pilot stu%y* Circ ' 5@@9M ;=A<B6;4:H55* =9* 0avanag& CA) Roc&ev 3A) Gallag&er ,M) Da(son 0A) 0eenan A0* ocal %rug %elivery in restenosis in.ury6 t&ermoresponsive coEpolymers as potential %rug %elivery systems* P&armacol T&er 5@@9M 4@5A4B64H4:* =:* Tepe G) Du%a SH) 0alino(s$i M) et al* ocal intraEarterial %rug %elivery !or prevention o! restenosis6 comparison o! t&e e!!iciency o! %elivery o! %i!!erent ra%iop&armaceuticals t&roug& a porous cat&eter* Invest Ra%iol 5@@4M 8;A:B659:H59>* =;* Ettenson DS) E%elman ER* ocal %rug %elivery6 an emerging approac& in t&e treatment o! restenosis* 2asc Me% 5@@@M :A5B6><H4@5* =<* +onan R* ocal %rug %elivery !or t&e treatment o! t&rom#us an% restenosis* ' Invasive Car%iol 4>>;M =A=B68>>H9@5*

5;@

Co&enESela et al*

==* +rieger D* Topol E* ocal %rug %elivery systems an% prevention o! restenosis* Car%iovasc Res 4>><M 8:A8B69@:H948* =>* Gra%usEPi/lo I) ,ilens$y R ) Marc& 0 ) et al* ocal %elivery o! #io%egra%a#le microE particles containing colc&icine or a colc&icine analogue6 e!!ects on restenosis an% impliE cations !or cat&eterE#ase% %rug %elivery* ' Am Coll Car%iol 4>>:M 5;A;B64:9>H4::<* >@* inco!! AM) Topol E') Ellis SG* ocal %rug %elivery !or t&e prevention o! restenosis* -act) !ancy) an% !uture* Circulation 4>>9M >@A9B65@<@H5@=9* >4* Allen TM) Austin GA) C&onn A) in ) ee 0C* Upta$e o! liposomes #y culture% mouse #one marro( macrop&ages6 in!luence o! liposome composition an% si/e* +ioc&im +iop&ys Acta 4>>4M 4@;4A4B6:;H;9* >5* Allen TM) Hansen C) Rutle%ge '* iposomes (it& prolonge% circulation times6 !actors a!!ecting upta$e #y reticuloen%ot&elial an% ot&er tissues* +ioc&im +iop&ys Acta 4>=>M >=4A4B65<H8:* >8* Allen TM) Me&ra T* Recent a%vances in sustaine% release o! antineoplastic %rugs using liposomes (&ic& avoi% upta$e into t&e reticuloen%ot&elial system* Proc ,est P&armacol Soc 4>=>M 856444H449* >9* Allen TM) C&onn A* arge unilamellar liposomes (it& lo( upta$e into t&e reticuloenE %ot&elial system* -E+S ett 4>=<M 558A4B695H9;* >:* Mic&aelis M) Vimmer A) Han%.ou N) Cinatl ') Cinatl ') 'r* Increase% systemic e!!icacy o! ap&i%icolin encapsulate% in liposomes* ?ncol Rep 5@@:M 48A4B64:<H4;@* >;* Asrani S) DPAnna S) Al$anE?nyu$sel H) ,ang ,) Goo%man D) Veimer R* Systemic to"icology an% laser sa!ety o! laser targete% angiograp&y (it& &eat sensitive liposomes* ' ?cul P&armacol T&er 4>>:M 44A9B6:<:H:=9* ><* 7i WR) Maitani 3) Nagai T* Rates o! systemic %egra%ation an% reticuloen%ot&elial system upta$e o! calcein in t&e %ipalmitoylp&osp&ati%ylc&oline liposomes (it& soy#eanE %erive% sterols in mice* P&arm Res 4>>:M 45A4B69>H:5* >=* Vu(alaE'agiello '* En%ocytosis me%iate% #y receptorsR!unction an% participation in oral %rug %elivery* Postepy Hig Me% Dos( 5@@8M :<A8B65<:H5>4* >>* Muro S) 0oval M) Mu/y$antov 2* En%ot&elial en%ocytic pat&(ays6 gates !or vascular %rug %elivery* Curr 2asc P&armacol 5@@9M 5A8B65=4H5>>* 4@@* +at&ori G) Cervena$ ) 0ara%i I* CaveolaeRan alternative en%ocytotic pat&(ay !or targete% %rug %elivery* Crit Rev T&er Drug Carrier Syst 5@@9M 54A5B6;<H>:* 4@4* Re.man ') ?#erle 2) Vu&orn IS) Hoe$stra D* Si/eE%epen%ent internali/ation o! particles via t&e pat&(ays o! clat&rinE an% caveolaeEme%iate% en%ocytosis) +ioc&em ' 5@@9M 8<<APt 4B64:>H4;>* 4@5* Ruc$ert P) +ates SR) -is&er A+* Role o! clat&rinE an% actinE%epen%ent en%ocytotic pat&E (ays in lung p&osp&olipi% upta$e* Am ' P&ysiol ung Cell Mol P&ysiol 5@@8M 5=9A;B6 >=4H >=>* 4@8* Ta$ei 0) Slepnev 2I) Hauc$e 2) De Camilli P* -unctional partners&ip #et(een amp&ip&ysin an% %ynamin in clat&rinEme%iate% en%ocytosis* Nat Cell +iol 4>>>M 4A4B688H8>* 4@9* Perry DG) Daug&erty G ) Martin ,') II* Clat&rinEcoate% pitEassociate% proteins are reCuire% !or alveolar macrop&age p&agocytosis* ' Immunol 4>>>M 4;5A4B68=@H8=;* 4@:* +ec$ 0A) C&ang M) +ro%s$y -M) 0een 'H* Clat&rin assem#ly protein APE5 in%uces aggregation o! mem#rane vesicles6 a possi#le role !or APE5 in en%osome !ormation* ' Cell +iol 4>>5M 44>A9B6<=<H<>;* 4@;* 3amas&ita C) Sone S) ?gura T) 0i(a%a H) Potential value o! cetylmannosi%emo%i!ie% liposomes as carriers o! macrop&age activators to &uman #loo% monocytes* 'pn ' Cancer Res 4>>4M =5A:B6:;>H:<;* 4@<* Eccleston DS) inco!! AM* Cat&eterE#ase% %rug %elivery !or restenosis* A%v Drug Deliver Rev 4>><M 59A4B684H98* 4@=* -is&#ein I) C&orny M) +anai S) et al* -ormulation an% %elivery mo%e a!!ect %isposition an% activity o! tyrp&ostinEloa%e% nanoparticles in t&e rat caroti% mo%el* Arterioscler T&rom# 2asc +iol 5@@4M 54A>B64989H498>* 4@>* Gu/man A) a#&aset(ar 2) Song CW) et al* ocal intraluminal in!usion o! #io%egra%aE #le polymeric nanoparticlesRA novel approac& !or prolonge% %rug %elivery a!ter #alE loon angioplasty* Circulation 4>>;M >9A;B64994H499=*

NanoECarriers o! Drugs an% Genes

5;4

44@*

444*

445* 448* 449* 44:* 44;* 44<*

44=* 44>*

45@*

454* 455*

458*

459* 45:*

45;*

45<*

45=*

45>* 48@* 484* 485*

+anai S) C&orny M) Gert/ SD) et al* ocally %elivere% nanoencapsulate% tyrp&ostin AAG E5@98B re%uces neointima !ormation in #alloonEin.ure% rat caroti% an% stente% porcine coronary arteries) +iomaterials 5@@:M 5;A9B69:4H9;4* +anai S) ,ol! 3) Golom# G) et al* PDG-Ereceptor tyrosine $inase #loc$er AG45>: selecE tively attenuates smoot& muscle cell gro(t& in vitro an% re%uces neointimal !ormation a!ter #alloon angioplasty in s(ine* Circulation 4>>=M ><A4>B64>;@H4>;>* Caplice N) Simari R* Gene trans!er !or coronary restenosis* Curr Interv Car%iol Rep 4>>>M 4A5B64:<H4;9* E&san A) Mann M'* Antisense an% gene t&erapy to prevent restenosis* 2asc Me% 5@@@M :A5B64@8H449* De%ieu '-) Ma&!ou%i A) e Rou" A) +ranellec D* 2ectors !or gene t&erapy o! car%iovasE cular %isease* Curr Car%iol Rep 5@@@M 5A4B68>H9<* Pauletto P) Sartore S) Pessina AC* Smoot&EmuscleEcell proli!eration an% %i!!erentiation in neointima !ormation an% vascular restenosis* Clin Sci A on%B 4>>9M =<A:B69;<H9<>* -in$el T* Epstein E* Gene t&erapy !or vascular %isease* -ASE+ ' 4>>:M >6=98H=:4* A&n 'D) Moris&ita R) 0ane%a 3) et al* In&i#itory e!!ects o! novel APE4 %ecoy oligo%eo"yE nucleoti%es on vascular smoot& muscle cell proli!eration in vitro an% neointimal !ormation in vivo* Circ Res 5@@5M >@A45B6485:H4885* 0aiser S) To#ore$ M* iposomeEme%iate% &ig&Ee!!iciency trans!ection o! &uman en%ot&elial cells* ' 2asc Res 5@@4M 8=A5B6488H498* Tomita N) A/uma H) 0ane%a 3) ?gi&ara T) Moris&ita R* Gene t&erapy (it& transcripE tion !actor %ecoy oligonucleoti%es as a potential treatment !or car%iovascular %iseases* Curr Drug Targets 5@@8M 9A9B688>H89;* 0ume M) 0omori 0) Matsumoto T) et al* A%ministration o! a %ecoy against t&e activator proteinE4 #in%ing site suppresses neointimal t&ic$ening in ra##it #alloonEin.ure% arteries* Circulation 5@@5M 4@:A4@B6455;H4585* 0ullo I') Simari RD) Sc&(art/ RS) 2ascular gene trans!er6 !rom #enc& to #e%si%e* Arterioscler T&rom# 2asc +iol 4>>>M 4>A5B64>;H5@<* i 'M) +roo$s G* Cell cycle regulatory molecules Acyclins) cyclinE%epen%ent $inases an% cyclinE%epen%ent $inase in&i#itorsB an% t&e car%iovascular systemM potential targets !or t&erapyT Eur Heart ' 4>>>M 5@A;B69@;H95@* Moris&ita R) Gi##ons GH) 0ane%a 3) ?gi&ara T) D/au 2'* P&armaco$inetics o! antiE sense oligo%eo"yri#onucleoti%es AcyclinE+E4 an% C%cE5 $inaseB in t&e vessel (all inE vivoRen&ance% t&erapeutic utility !or restenosis #y Hv.Eliposome %elivery* Gene 4>>9M 49>A4B648H4>* Clo(es A,) Sc&(art/ SM* Signi!icance o! Cuiescent smoot& muscle migration in t&e in.ure% rat caroti% artery* Circ Res 4>=:M :;A4B648>H49:* -ingerle ') 'o&nson R) Clo(es A,) Ma.es$y M,) Rei%y MA* Role o! platelets in smoot& muscle cell proli!eration an% migration a!ter vascular in.ury in rat caroti% artery* Proc Natl Aca% Sci USA 4>=>M =;A54B6=945H=94;* Clo(es A,) Clo(es MM) Au 3P) Rei%y MA) +elin D* Smoot& muscle cells e"press uro$inase %uring mitogenesis an% tissueEtype plasminogen activator %uring migration in in.ure% rat caroti% artery* Circ Res 4>>@M ;<A4B6;4H;<* C&an A0) 0almes A) Ha($ins S) Daum G) Clo(es A,* +loc$a%e o! t&e epi%ermal gro(t& !actor receptor %ecreases intimal &yperplasia in #alloonEin.ure% rat caroti% artery* ' 2asc Surg 5@@8M 8<A8B6;99H;9>* S&i 3) Hutc&inson HG) Hall D') Vale(s$i A* Do(nEregulation o! cEmyc e"pression #y antisense oligonucleoti%es in&i#its proli!eration o! &uman smoot&Emuscle cells* Circulation 4>>8M ==A8B644>@H44>:* Iso#e M) Moris&ita R) Su/u$i '* Ne( approac&es to treat car%iovascular %iseases targetE ing N-$+* ' Mol Cell Car%io 5@@9M 8<A4B684;H84;* Cava//anaECalvo M) T&ras&er A) Mavilio -* T&e !uture o! gene t&erapy) Nature 5@@9M 95<A;><<B6<<>H<=4* Stein CA* T&e e"perimental use o! antisense oligonucleoti%es6 a gui%e !or t&e perple"e%* ' Clin Invest 5@@4M 4@=A:B6;94H;99* am#ert G) -attal E) Couvreur P* Nanoparticulate systems !or t&e %elivery o! antisense oligonucleoti%es* A%v Drug Deliv Rev 5@@4M 9<A4B6>>H445*

5;5

Co&enESela et al*

488* Moris&ita R) Ao$i M) 0ane%a 3* Decoy oligo%eo"ynucleoti%es as novel car%iovascular %rugs !or car%iovascular %isease* Ann N3 Aca% Sci 5@@4M >9<) 5>9H8@4M %iscussion 8@4H8@5* 489* Gottsc&al$ U) C&an S* Somatic gene t&erapy6 present situation an% !uture perspective* Drug Res 4>>=M 9=64444H445@* 48:* Des&pan%e D) Ro.anasa$ul 3* Antisense oligonucleoti%e t&erapeutics6 a class o! its o(n* P&armaceutical Ne(s 4>>;M 864:H4=* 48;* Santiago -S) 0&ac&igian M* Nucleic aci% #ase% strategies as potential t&erapeutic tools6 mec&anistic consi%erations an% implications to restenosis* ' Mol Me% 5@@4M <>A45B6;>:H<@;* 48<* Taylor M-* Emerging antisense tec&nologies !or gene !unctionali/ation an% %rug %elivery* DDT 5@@4M ;6S><HS4@4* 48=* Scanlon 0') ?&ta 3) Is&i%a H) et al* ?ligonucleoti%eEme%iate% mo%ulation o! mammaE lian gene e"pression* -ASE+ ' 4>>:M >A48B645==H45>;* 48>* Sirois MG) Simons M) E%elman ER* Antisense oligonucleoti%e in&i#ition o! PDG-RE #eta receptor su#unit e"pression %irects suppression o! intimal t&ic$ening* Circulation 4>><M >:A8B6;;>H;<;* 49@* 2illa AE) Gu/man A) Poptic E') et al* E!!ects o! antisense cEmy# oligonucleoti%es on vascular smoot& muscle cell proli!eration an% response to vessel (all in.ury* Circ Res 4>>:M <;A9B6:@:H:48* 494* E%elman ER) Simons M) Sirois MG) Rosen#erg RD* CEmyc in vasculoproli!erative %isease* Circ Res 4>>:M <;A5B64<;H4=5* 495* Simons M) E%elman ER) De0eyser ' ) anger R) Rosen#erg RD* Antisense cEmy# oligoE nucleoti%es in&i#it intimal arterial smoot& muscle cell accumulation in vivo* Nature 4>>5M 8:>A;8>@B6;<H<@* 498* Moris&ita R) Higa$i ') Tomita N) ?gi&ara T* Application o! transcription !actor Q%ecoyS strategy as means o! gene t&erapy an% stu%y o! gene e"pression in car%iovascular %isE ease* Circ Res 4>>=M =5A4@B64@58H4@5=* 499* Mc0ay M'* Ga#alla MA* Gene trans!er t&erapy in vascular %iseases* Car%iovasc Drug Rev 5@@4M 4>A8B659:H5;5* 49:* Ma&ato RI) Ta$a$ura 3) Has&i%a M) Development o! targete% %elivery systems !or nucleic aci% %rugs* ' Drug Target 4>><M 9A;B688<H8:<* 49;* Tomita N) Moris&ita R* Antisense oligonucleoti%es as a po(er!ul molecular strategy !or gene t&erapy in car%iovascular %iseases* Curr P&arm Des 5@@9M 4@A<B6<><H=@8* 49<* Na#el E) Plaut/ GE) Na#el G) SiteEspeci!ic gene e"pression in vivo #y %irect gene transE !er into t&e arterial (all* Science 4>>@M 59>645=:H45==* 49=* ,ilson ') +iriny ) Salomon R* Implantation o! vascular gra!t line% (it& genetically mo%i!ie% en%ot&elial cells* Science 4>=>M 59964899H489;* 49>* T(om#ly R* -or gene t&erapy) no(ECuanti!ie% ris$s are %eeme% trou#ling* ' Natl Cancer Inst 5@@8M >:A49B64@85H4@88* 4:@* Smit& AE* 2iral vectors in gene t&erapy* Annu Rev Micro#iol 4>>:M 9>6=@<H=8=* 4:4* Miller DP) A%am M) Miller A* Gene trans!er #y retrovirus vectors occurs only in cells t&at are actively replicating at t&e time o! in!ection* Mol Cell +iol 4>>@M 4@6958>H9595* 4:5* 3ang 3) Nunes -) +erencsi 0) Cellular immunity to viral antiEgene limits E4 %elete% a%enoviruses !or gene t&erapy* Proc Natl Aca% Sci USA 4>>9M >4A4@B699@<H9944* 4:8* Ne(man 0) Dunn P) ?(ens '* A%enovirusEme%iate% gene trans!er into normal ra##it arteries results in prolong vascular cell activation) in!lammation) an% neointimal &yperE plasia* ' Clin Invest 4>>:M >;65>::H5>;:* 4:9* Mar(ic$ C) -DA &alts gene t&erapy trials a!ter leu$aemia case in -rance* +M' 5@@8M 85;A<8=5B64=4* 4::* +ro(n MD) Sc&at/lein AG) Uc&eg#u I-* Gene %elivery (it& synt&etic Anon viralB carriers* Int ' P&arm 5@@4M 55>A4H5B64H54* 4:;* V&%anov RI) Po%o#e% ?2) 2lassov 22* Cationic lipi%EDNA comple"eslipople"esE!or gene trans!er an% t&erapy* +ioelectroc&emistry 5@@5M :=A4B6:8H;9* 4:<* 0&urana R) Martin '-) Vac&ary I* Gene t&erapy !or car%iovascular %isease6 a case !or cautious optimism) Hypertension 5@@4M 8=A:B6454@H454;* 4:=* Clar$ P) Hers& E* Cationic lipi% me%iate% gene trans!er6 current concepts* Curr ?pin Mol T&er 4>>>M 464:=H4<;*

NanoECarriers o! Drugs an% Genes

5;8

4:>* Mars&all ') Nietups$i '+) ee ER) et al* Cationic lipi% structure an% !ormulation consi%E erations !or optimal gene trans!ection o! t&e lung) ' Drug Target 5@@@M <A;B69:8H9;>* 4;@* -ilion MC) P&illips NC* To"icity an% immunomo%ulatory activity o! liposomal vectors !ormulate% (it& cationic lipi%s to(ar% immune e!!ector cells* +ioc&im +iop&ys Acta 4>><M 485>A5B689:H8:;* 4;4* Do$$a S) Tole%o D) S&i W) Castranova 2) Ro.anasa$ul 3* ?"ygen ra%icalme%iate% pulmonary to"icity in%uce% #y some cationic liposomes* P&arm Res 5@@@M 4<A:B6:54H:5:* 4;5* -ilion MC) P&illips NC* AntiEin!lammatory activity o! cationic lipi%s* +r ' P&armacol 4>><M 455A8B6::4H::<* 4;8* Du/gunes N) Gol%stein 'A) -rien% DS) -elgner P * -usion o! liposomes containing a novel cationic lipi%) NEN5)8EA%ioleylo"yBpropylOEN)N)NEtrimet&ylammonium6 in%uction #y multivalent anions an% asymmetric !usion (it& aci%ic p&osp&olipi% vesicles* +ioc&emistry 4>=>M 5=A58B6>4<>H>4=9* 4;9* Camp#ell R+) -u$umura D) +ro(n E+) et al* Cationic c&arge %etermines t&e %istri#uE tion o! liposomes #et(een t&e vascular an% e"travascular compartments o! tumors* Cancer Res 5@@5M ;5A58B6;=84H;=8;* 4;:* D/au 2') Mann M') Moris&ita R) 0ane%a 3* -usigenic viral liposome !or gene t&erapy in car%iovascular %iseases* Proc Natl Aca% Sci USA 4>>;M >8A54B644954H4495:* 4;;* -elgner 'H) 0umar R) Sri%&ar CN) et al* En&ance% gene %elivery an% mec&anism stu%E ies (it& a novel series o! cationic lipi% !ormulations* ' +iol C&em 4>>9M 5;>A9B65::@H5:;4* 4;<* -elgner P ) Ga%e$ TR) Holm M) et al* ipo!ection6 a &ig&ly e!!icient) lipi%Eme%iate% DNAEtrans!ection proce%ure* Proc Natl Aca% Sci USA 4>=<M =9A54B6<948H<94<* 4;=* -elgner P ) Tsai 3') Su$&u ) et al* Improve% cationic lipi% !ormulations !or in vivo gene t&erapy* Ann N3 Aca% Sci 4>>:M <<5M 45;H48>* 4;>* 0ic&ler A) Vauner ,) ?gris M) ,agner E* In!luence o! t&e DNA comple"ation me%ium on t&e tras!ection e!!iciency o! lipospermine1DNA particles* Gene T&er 4>>=M :6=::H=;@* 4<@* Grot& D) 0eil# ?) e&mana C) Sc&nei%er M) Ru%o M* Preparation an% c&aracterisation o! a ne( liposperamine !or gene %elivery into various cells* Int ' P&arm 4>>=M 4;5) 498H4:<* 4<4* Armeanu S) Pelise$ ') 0raus/ E) et al* ?ptimi/ation o! nonviral gene trans!er o! vascular smoot& muscle cells in vitro an% in vivo* Mol T&er 5@@@M 4A9B68;;H8<:* 4<5* Pic$ering 'G) Isner 'M) -or% CM) et al* Processing o! c&imeric antisense oligonucleE oti%es #y &uman vascular smoot& muscle cells an% &uman at&erosclerotic plaCue* Implications !or antisense t&erapy o! restenosis a!ter angioplasty* Circulation 4>>;M >8A9B6<<5H<=@* 4<8* Tomita N) Moris&ita R) Tomita S) et al* Transcription !actor %ecoy !or nuclear !actorE $appa+ in&i#its tumor necrosis !actorEalp&aEin%uce% e"pression o! interleu$inE; an% intracellular a%&esion moleculeE4 in en%ot&elial cells* ' Hypertens 4>>=M 4;A<B6>>8H4@@@* 4<9* 0alino(s$i M) 2ie&o!er 0) Hamann C) et al* ocal a%ministration o! N-E$appa + %ecoy oligonucleoti%es to prevent restenosis a!ter #alloon angioplasty6 an e"perimental stu%y in Ne( Vealan% (&ite ra##its* Car%iovasc Intervent Ra%iol 5@@:M 5=A8B6884H88<* 4<:* Ni$ol S) Pelise$ ') Engelmann MG) Rollan% PH) Armeanu S* Prevention o! restenosis using t&e gene !or cecropin comple"e% (it& D?CSPER liposomes un%er optimi/e% con%itions* Int ' Angiol 5@@@M >A5B6=<H>9* 4<;* iu 3) -ong S) De#s R'* Cationic liposomeEme%iate% gene %elivery in vivo* Met&o%s En/ymol 5@@8M 8<86:8;H::@* 4<<* iu 3) iggitt D) V&ong ,) Tu G) Gaensler 0) De#s R* Cationic liposomeme%iate% intraE venous gene %elivery* ' +iol C&em 4>>:M 5<@A95B659)=;9H59)=<@* 4<=* Patil SD) R&o%es DG) +urgess D'* Anionic liposomal %elivery system !or DNA trans!ecE tion* AAPS ' 5@@9M ;A9B6e5>* 4<>* Miller AD* T&e pro#lem (it& cationic liposome1micelleE#ase% nonEviral vector systems !or gene t&erapy* Curr Me% C&em 5@@8M 4@A49B644>:H4544* 4=@* +it/er M) Armeanu S) auer UM) Neu#ert ,'* Sen%ai virus vectors as an emerging negativeEstran% RNA viral vector system* ' Gene Me% 5@@8M :A<B6:98H::8*

5;9

Co&enESela et al*

4=4* Na$anis&i M) Uc&i%a T) Suga(a H) Is&iura M) ?$a%a 3) E!!icient intro%uction o! contents o! liposomes into cells using H2'* E"p Cell Res 4>=:M 4:>68>>H9@>* 4=5* 0ene%a 3) I(ai 0) Uc&i%a T* Intro%uction an% e"pression o! t&e &uman insulin gene in a%ult rat liver* ' +iol C&em 4>=>M 4=;645>H489* 4=8* Tomita N) Higa$i ') Moris&ita R) 0ato H) Mi$ami H) 0ene%a 3* Direct in vivo gene intro%uction into rat $i%ney* +ioc&em +iop&ys Res Commun 4>>5M 4=;645>H489* 4=9* 0ene%a 3) I(ai 0) Uc&i%a T* Increase% e"pression o! DNA cointro%uce% (it& nucear protein in a%ult rat liver* Science 4>=>M 598) 8<:H8<=* 4=:* 0ene%a 3) Sae$i 3) Moris&ita R* Gene t&erapy using H2'Eliposomes6 #est o! #ot& (orl%sT Mol Me% To%ay 4>>>M :) 5>=H8@8* 4=;* 3in W) 3utani C) I$e%a 3) et al* Tissue !actor pat&(ay in&i#itor gene %elivery using H2'E A2E liposomes mar$e%ly re%uces restenosis in at&erosclerotic arteries* Car%iovasc Res 5@@5M :;A8B69:9H9;8* 4=<* Moris&ita R) Gi##ons GH) 0ane%a 3) ?gi&ara T) D/au 2'* Noval an% e!!ective gene trans!er tec&niCue !or stu%y o! vascular renin angiotensin system* ' Clin Invest 4>>8aM >465:=@H5:=:* 4==* 2D eyen HE) Gi##ons GH) Moris&ita R) et al* Gene t&erap&y in&i#iting neointimal vascular lesion6 in vivo trans!er o! en%ot&elial cell nitric o"i%e synt&ase gene* Proc Natl Aca% USA 4>>:M >5) 448<H4494* 4=>* To%a$a T) 3o$oyama C) 3anamoto H) et al* Gene trans!er o! &uman prostacyclin synE t&ase prevents neointimal !ormation a!ter caroti% #alloon in.ury in rats* Stro$e 4>>>M 8@A5B694>H95;* 4>@* Ao$i M) Moris&ita R) Matsus&ita H) et al* In&i#ition o! t&e p:8 tumor suppressor gene results in gro(t& o! &uman aortic vascular smoot& muscle cells* Potential role o! p:8 in regulation o! vascular smoot& muscle cell gro(t&* Hypertension 4>>>M 89A5B64>5H5@@* 4>4* Moris&ita R) Gi##ons GH) Ellison 0E) et al* Intimal &yperplasia a!ter vascular in.ury is in&i#ite% #y antisense c%$ 5 $inase oligonucleoti%es* ' Clin Invest 4>>9M >8A9B649:=H49;9* 4>5* Moris&ita R) Gi##ons GH) Ellison 0E) et al* Single intraluminal %elivery o! antisense c%c5 $inase an% proli!eratingEcell nuclear antigen oligonucleoti%es results in c&ronic in&i#ition o! neointimal &yperplasia* Proc Natl Aca% Sci USA 4>>8M >@A4=B6=9<9H=9<=* 4>8* Moris&ita R) Gi##ons GH) Tomita N) et al* Antisense oligo%eo"ynucleoti%e in&i#ition o! vascular angiotensinEconverting en/yme e"pression attenuates neointimal !ormation6 evi%ence !or tissue angiotensinEconverting en/yme !unction* Arterioscler T&rom# 2asc +iol 5@@@M 5@A9B6>4:H>55* 4>9* Moris&ita R) Gi##ons GH) Horiuc&i 0E) et al* A gene t&erapy strategy using a transcripE tion !actor %ecoy o! t&e E5- #inging site in&i#its smoot& muscle proli!eration in vivo* Proc Natl Aca% USA 4>>:M >56:=::H:=:>* 4>:* a#&aset(ar 2) C&en +R) Muller D,M) et al* GeneE#ase% t&erapies !or restenosis* A%v Drug Deliver Rev 4>><M 59A4B64@>H45@* 4>;* Iaccarino G) Smit&(ic$ A) e!$o(it/ R') 0oc& ,'* Targeting GA#eta gammaB signaling in arterial vascular smoot& muscle proli!eration6 a novel strategy to limit restenosis) Proc Nat Aca% Sci USA 4>>>M >;A<B68>9:H8>:@* 4><* In%ol!i C) Avve%imento E2) Rapacciuolo A) et al* In&i#ition o! cellular ras prevents smoot&Emuscle cellEproli!eration a!ter vascular in.ury inEvivo* Nat Me% 4>>:M 4A;B6:94H:9:* 4>=* Co&enESac$s H) Na.a.re& 3) Tc&ai$ovs$i 2) et al* Novel PDG- #eta R antisense encapsulate% in polymeric nanosp&eres !or t&e treatment o! restenosis* Gene T&er 5@@5M >A58B64;@<H4;4;* 4>>* Hug&es AD) Clunn G-) Re!son ') DemoliouEMason C* PlateletE%erive% gro(t& !actor APDG-B6 actions an% mec&anisms in vascular smoot& muscle) Gen P&armacol 4>>;M 5<A<B64@<>H4@=>* 5@@* Vam#au" M-) +onneau" -) Gre! R) et al* In!luence o! e"perimental parameters on t&e c&aracteristics o! polyAlactic aci%B nanoparticles prepare% #y a %ou#le emulsion met&o%* ' Control Release 4>>=M :@A4H8B684H9@* 5@4* Co&en H) evy R') Gao ') et al* Sustaine% %elivery an% e"pression o! DNA encapsulate% in polymeric nanoparticles* Gene T&er 5@@@M <A55B64=>;H4>@:* 5@5* Moris&ita R* Recent progress in car%iovascular gene t&erapyM Emerging to real %rugT Pre!ace* Curr Gene T&er 5@@9M 9A5B6iHiA4B*

NanoECarriers o! Drugs an% Genes

5;:

5@8* Moris&ita R) Perspective in progress o! car%iovascular gene t&erapy* ' P&armacol Sci 5@@9M >:A4B64H=* 5@9* Moris&ita R) Recent progress in gene t&erapy !or car%iovascular %isease* Circ ' 5@@5M ;;A45B64@<<H4@=;* 5@:* evit/$i A) Tyrp&ostins6 tyrosine $inase #loc$ers as novel antiproli!erative agents an% %issectors o! signal trans%uction* -ASE+ ' 4>>5M ;A49B685<:H85=5* 5@;* evit/$i A) Ga/it A* Tyrosine $inase in&i#ition6 an approac& to %rug %evelopment* Science 4>>:M 5;<A:5@:B64<=5H4<==* 5@<* Ga/it A) 3ee 0) Uec$er A) et al* Tricyclic Cuino"alines as potent $inase in&i#itors o! PDG-R $inase) -lt8 an% 0it* +ioorg Me% C&em 5@@8M 44A>B65@@<H5@4=* 5@=* 0ovalen$o M) Ronnstran% ) Hel%in CH) et al* P&osp&orylation siteEspeci!ic in&i#ition o! plateletE%erive% gro(t& !actor #etaEreceptor autop&osp&orylation #y t&e receptorE #loc$ing tyrp&ostin AG45>;* +ioc&emistry 4>><M 8;A54B6;5;@H;5;>* 5@>* -is&#ein I) C&orny M) Ra#inovic& ) +anai S) Gati I) Golom# G* Nanoparticulate %elivE ery system o! a tyrp&ostin !or t&e treatment o! restenosis* ' Control Release 5@@@M ;:A4H5B6554H55>* 54@* -is&#ein I) ,alten#erger ') +anai S) et al* ocal %elivery o! plateletE%erive% gro(t& !actor receptorEspeci!ic tyrp&ostin in&i#its neointimal !ormation in rats* Arterioscler T&rom# 2asc +iol 5@@@M 5@A8B6;;<H;<;* 544* -essi H) Puisieu" -) Devissaguet 'P) Ammoury N) +enita S* Nanocapsule !ormation #y inter!acial polymer %eposition !ollo(ing solvent %isplacement* Int ' P&arm 4>=>M ::A4B) R4HR9* 545* 7uintanarEGuerrero D) Allemann E) -essi H) Doel$er E* Preparation tec&niCues an% mec&anisms o! !ormation o! #io%egra%a#le nanoparticles !rom pre!orme% polymers* Drug Dev In% P&arm 4>>=M 59A45B64448H445=* 548* C&orny M) -is&#ein I) Danen#erg HD) Golom# G* ipop&ilic %rug loa%e% nanosp&eres prepare% #y nanoprecipitation6 e!!ect o! !ormulation varia#les on si/e) %rug recovery an% release $inetics* ' Control Release 5@@5M =8A8B68=>H9@@* 549* C&orny M) -is&#ein I) Danen#erg HD) Golom# G* Stu%y o! t&e %rug release mec&anism !rom tyrp&ostin AGE45>:Eloa%e% nanosp&eres #y in situ an% e"ternal sin$ met&o%s* ' Control Release 5@@5M =8A8B69@4H949* 54:* +rigger I) C&amina%e P) Marsau% 2) et al* Tamo"i!en encapsulation (it&in polyet&ylene glycolEcoate% nanosp&eres* A ne( antiestrogen !ormulation* Int ' P&arm 5@@4M 549A4H5B68<H95* 54;* Pra#&a S) V&ou ,V) Panyam ') a#&aset(ar 2* Si/eE%epen%ency o! nanoparticleEme%iE ate% gene trans!ection6 stu%ies (it& !ractionate% nanoparticles* Int ' P&arm 5@@5M 599A4H5B64@:H44:* 54<* Sc&(art/ RS* Neointima an% arterial in.ury6 %ogs) rats) pigs) an% more* a# Invest 4>>9M <4A;B6<=>H<>4* 54=* 2illa AE) Gu/man A) C&en ,) Golom# G) evy R') Topol E'* ocal %elivery o! %e"amet&asone !or prevention o! neointimal proli!eration in a rat mo%el o! #alloon angioplasty* ' Clin Invest 4>>9M >8A8B64598H459>* 54>* +er$ +C) Rao GN* Angiotensin IIEin%uce% vascular smoot& muscle cell &ypertrop&y6 PDG- AEc&ain me%iates t&e increase in cell si/e* ' Cell P&ysiol 4>>8M 4:9A5B68;=H8=@* 55@* ee S,) Tsou AP) C&an H) et al* Glucocorticoi%s selectively in&i#it t&e transcription o! t&e interEleu$in 4 #eta gene an% %ecrease t&e sta#ility o! interleu$in 4 #eta mRNA* Proc Natl Aca% Sci USA 4>==M =:A9B645@9H45@=* 554* 0ling D) -ingerle ') Harlan 'M* In&i#ition o! leu$ocyte e"travasation (it& a monoclonal anti#o%y to CD4= %uring !ormation o! e"perimental intimal t&ic$ening in ra##it caroti% arteries* Arterioscler T&rom# 4>>5M 45A>B6>><H4@@<* 555* Gu/man A) a#&aset(ar 2) Song C) 'ang 3) inco!! AM) evy R* Single intraluminal in!usion o! #io%egra%a#le polymeric nanoparticles matri"e% (it& %e"amet&asone %ecreases neointimal !ormation a!ter vascular in.ury* Circulation 4>>:M >5A=B6 48>9H48>9* 558* Eric$son A) +onin PD) ,is&$a DG) et al* In vitro an% in vivo in&i#ition o! rat vascular smoot& muscle cell migration an% proli!eration #y a 5Eaminoc&romone UE=;>=8* ' P&armacol E"p T&er 4>>9M 5<4A4B694:H954* 559* Hump&rey ,R) Eric$son A) Simmons CA) et al* T&e e!!ect o! intramural %elivery o! polymeric nanoparticles loa%e% (it& t&e antiproli!erative 5Eaminoc&romone UE=;>=8

5;;

Co&enESela et al*

55:*

55;*

55<*

55=*

55>* 58@*

584* 585* 588* 589* 58:*

58;* 58<* 58=*

58>* 59@* 594* 595*

598*

599* 59:*

on neointimal &yperplasia %evelopment in #alloonEin.ure% porcine coronary arteries* A%v Drug Deliver Rev 4>><M 59A4B6=<H4@=* Song CW) a#&aset(ar 2) Murp&y H) et al* -ormulation an% c&aracteri/ation o! #io%eE gra%a#le nanoparticles !or intravascular local %rug %elivery) ' Control Release 4>><M 98A5H8B64><H545* ,este%t U) +ar#uETu%oran ) Sc&aper A0) 0alino(s$i M) Al!$e H) 0issel T* Deposition o! nanoparticles in t&e arterial vessel #y porous #alloon cat&eters6 locali/ation #y con!oE cal laser scanning microscopy an% transmission electron microscopy* AAPS P&armSci 5@@5M 9A9B6E94* Song C) a#&aset(ar 2) Cui W) Un%er(oo% T) evy R'* Arterial upta$e o! #io%egra%a#le nanoparticles !or intravascular local %rug %elivery6 results (it& an acute %og mo%el* ' Control Release 4>>=M :9A5B65@4H544* Panyam ') o! ') ?P eary E) a#&aset(ar 2* E!!iciency o! %ispatc& an% in!iltrator car%iac in!usion cat&eters in arterial locali/ation o! nanoparticles in a porcine coronary mo%el o! restenosis* ' Drug Target 5@@5M 4@A;B6:4:H:58* Ga#i/on A* Papa&a%.opoulos D* T&e role o! sur!ace c&arge an% &y%rop&ilic groups on liposome clearance in vivo* +ioc&im +iop&ys Acta 4>>5M 44@8A4B6>9H4@@* Ga#i/on A) Price DC) Hu#erty ') +resalier RS) Papa&a%.opoulos D* E!!ect o! liposome composition an% ot&er !actors on t&e targeting o! liposomes to e"perimental tumors6 #io%istri#ution an% imaging stu%ies* Cancer Res 4>>@M :@A4>B6;8<4H;8<=* asic DD* Colloi% c&emistry* iposomes (it&in liposomes* Nature 4>><M 8=<A;;5=B6 5;H5<* asic DD) Papa&a%.opoulos D* iposomes revisite%* Science 4>>:M 5;<A:5@5B645<:H45<;* Ga#i/on A) S&mee%a H) +aren&ol/ 3* P&armaco$inetics o! pegylate% liposomal %o"oruE #icinHrevie( o! animal an% &uman stu%ies* Clin P&armaco$inet 5@@8M 95A:B694>H98;* Na$anis&i T) -u$us&ima S) ?$amoto 0) et al* Development o! t&e polymer micelle carrier system !or %o"oru#icin* ' Control Release 5@@4M <9A4H8B65>:H8@5* Mae%a H* T&e en&ance% permea#ility an% retention AEPRB e!!ect in tumor vasculature6 t&e $ey role o! tumorEselective macromolecular %rug targeting* A%v En/yme Regul 5@@4M 9464=>H5@<* Mae%a H* Role o! micro#ial proteases in pat&ogenesis* Micro#iol Immunol 4>>;M 9@A4@B6 ;=:H;>>* Mae%a H) ,u ') ?$amoto T) Maruo 0) A$ai$e T* 0alli$reinE$inin in in!ection an% cancer* Immunop&armacology 4>>>M 98A5H8B644:H45=* Molla A) 3amamoto T) A$ai$e T) Miyos&i S) Mae%a H* Activation o! HagemanE!actor an% pre$alli$rein an% generation o! $inin #y various micro#ial proteinases* ' +iol C&em 4>=>M 5;9A4=B64@:=>H4@:>9* U(ato$u T) S&imo$a(a H) A#e 0) et al* Application o! nanoparticle tec&nology !or t&e prevention o! restenosis a!ter #alloon in.ury in rats) Circ Res 5@@8M >5A<B6e;5He;>* Ro(ins$y E0) Done&o(er RC* DrugEt&erapyHPaclita"el ATa"olB* Ne( Engl ' Me% 4>>:M 885A4:B64@@9H4@49* Sc&i!! P+) Hor(it/ S+* Ta"ol sta#ili/es microtu#ules in mouse !i#ro#last cells* Proc Natl Aca% Sci USA* 4>=@M <<A8B64:;4H4:;:* 0olo%gie -D) 'o&n M) 0&urana C) et al* Sustaine% re%uction o! inEstent neointimal gro(t& (it& t&e use o! a novel systemic nanoparticle paclita"el* Circulation 5@@5M 4@;A4@B644>:H44>=* I#ra&im N0) Desai N) eg&a S) et al* P&ase I an% p&armaco$inetic stu%y o! A+IE@@<) a cremop&orE!ree) proteinEsta#ili/e%) nanoparticle !ormulation o! paclita"el* Clin Cancer Res 5@@5M =A:B64@8=H4@99* ,eiss R+) Done&o(er RC) ,ierni$ PH) et al* Hypersensitivity Reactions !rom Ta"ol* ' Clin ?ncol 4>>@M =A<B645;8H45;=* Danen#erg HD) -is&#ein I) Gao ') et al* Macrop&age %epletion #y clo%ronateEcontaining liposomes re%uces neointimal !ormation a!ter #alloon in.ury in rats an% ra##its* Circulation 5@@5M 4@;A:B6:>>H;@:* -el%man ') Aguirre ) Viol M) et al* Interleu$inE4@ in&i#its intimal &yperplasia a!ter angioplasty or stent implantation in &yperc&olesterolemic ra##its* Circulation 5@@@M 4@4A=B6>@=H>4;*

59;*

NanoECarriers o! Drugs an% Genes

5;<

59<* Rogers C) E%elman ER) Simon DI) A mA# to t&e #eta5Eleu$ocyte integrin MacE4 ACD44#1 CD4=B re%uces intimal t&ic$ening a!ter angioplasty or stent implantation in ra##its* P Natl Aca% Sci USA 4>>=M >:A4<B64@)489H4@)48>* 59=* Ross R* Mec&anisms o! %iseaseHat&erosclerosisRan in!lammatory %isease) N Engl ' Me% 4>>>M 89@A5B644:H45;* 59>* Rogers C) ,elt -G) 0arnovs$y M') E%elman ER* Monocyte recruitment an% neointimal &yperplasia in ra##its* Couple% in&i#itory e!!ects o! &eparin* Arterioscler T&rom# 2asc +iol 4>>;M 4;A4@B64845H484=* 5:@* Tana$a H) Su$&ova G0) S(anson S') et al* Sustaine% activation o! vascular cells an% leu$ocytes in t&e ra##it aorta a!ter #alloon in.ury* Circulation 4>>8M ==A9 Pt 4B64<==H4=@8* 5:4* ,elt -) Tso C) E%elman E) et al* eu$ocyte recruitment an% e"pression o! c&emo$ines !ollo(ing %i!!erent !orms o! vascular in.ury) 2asc Me% 5@@8M =64H<* 5:5* -u$u%a D) S&ima%a 0) Tana$a A) 0a(ara#ayas&i T) 3os&iyama M) 3os&i$a(a '* Circulating monocytes an% inEstent neointima a!ter coronary stent implantation* ' Am Coll Car%iol 5@@9M 98A4B64=H58* 5:8* van -urt& R* ?rigin an% turnover o! monocytes an% macrop&ages* Curr Top Pat&ol 4>=>M <>645:H4:@* 5:9* 2an -urt& R) P&agocytic cells6 %evelopment an% %istri#ution o! mononuclear p&agoE cytes in normal stea%y state an% in!lammation* In6 In!lammation6 +asic Principles an% Clinical Correlates* Sny%er R) e%* Ne( 3or$6 Raven Press) t%) 4>==65=4H5>:* 5::* Pa$ianat&an DR) E"tracellularEmatri" proteins an% leu$ocyte !unction* ' eu$oc +iol 4>>:M :<A:B6;>>H<@5* 5:;* Carlos TM) Harlan 'M* eu$ocyteEen%ot&elial a%&esion molecules* +loo% 4>>9M =9A<B65@;=H54@4* 5:<* 0agan E) Hartmann DP* Elimination o! macrop&ages (it& silica an% as#estos* Met&o%s En/ymol 4>=9M 4@=685:H88:* 5:=* S&e$ PN) u$ovic& S* T&e role o! macrop&ages in promoting t&e anti#o%y response me%iate% #y liposomeEassociate% protein antigens* Immunol ett 4>=5M :A;B68@:H8@>* 5:>* van Rooi.en N) van Nieu(megen R* Elimination o! p&agocytic cells in t&e spleen a!ter intravenous in.ection o! liposomeEencapsulate% %ic&loromet&ylene %ip&osp&onate* An en/ymeE&istoc&emical stu%y* Cell Tissue Res 4>=9M 58=A5B68::H8:=* 5;@* Caselles T) 2illalian ') -rernan%e/ '* In!luence o! liposome c&arge an% composition on t&eir interaction (it& &uman #loo% serum proteins* Mol Cell +ioc&em 4>>8M 45@644>H45;* 5;4* Gregoria%is G) Davis C* Sta#ility o! liposome in vivo an% in vitro is promote% #y t&eir c&olesterol content an% t&e presence o! #loo% cells* +ioc&em +iop&ys Res Commun 4><>M =>645=<H45>8* 5;5* Senior ') Gregoria%is G* Sta#ility o! small unilamellar liposomes in serum an% clearance !rom t&e circulation6 t&e e!!ect o! t&e p&osp&olipi% an% c&olesterol components* i!e Sci 4>=5M 8@65458H548;* 5;8* Damen ') Di.$stra ') Regts ') Sc&erp&o! G* E!!ect o! lipoproteinE!ree plasma on t&e interE action o! &uman plasma &ig& %ensity lipoprotein (it& egg yol$ p&osp&ati%ylc&olin liposomes* +ioc&em +iop&ys Acta 4>=@M ;5@6>@H>>* 5;9* -leisc& H) +isp&osp&onatesHP&armacology an% use in t&e treatment o! tumorEin%uce% &ypercalcemic an% metastatic #oneE%isease* Drugs 4>>4M 95A;B6>4>H>99* 5;:* -leisc& H* +isp&osp&onates6 mec&anisms o! action* En%ocr Rev 4>>=M 4>A4B6=@H4@@* 5;;* Ro%an) GA* Mec&anisms o! action o! #isp&osp&onates* Annu Rev P&armacol To"icol 4>>=M 8=68<:H8==* 5;<* Mon$$onen ') 2al.a$$a R) Ha$asalo M) Urtti A* T&e e!!ects o! liposome sur!aceEc&arge an% si/e on t&e intracellular %elivery o! clo%ronate an% gallium inEvitro* Int ' P&arm 4>>9M 4@<A8B64=>H4><* 5;=* 2an Rooi.en N* T&e liposomeEme%iate% macrop&age suici%eP tec&niCue* ' Immunol Met&o%s 4>=>M 459A4B64H;* 5;>* Danen#erg HD) -is&#ein I) Epstein H) et al* Systemic %epletion o! macrop&ages #y liposomal #isp&osp&onates re%uces neointimal !ormation !ollo(ing #alloonEin.ury in t&e rat caroti% artery* ' Car%iovasc P&armacol 5@@8M 95A:B6;<4H;<>*

5;=

Co&enESela et al*

5<@* Mon$$onen ') Heat& TD* T&e e!!ects o! liposomeEencapsulate% an% !ree clo%ronate on t&e gro(t& o! macrop&ageEli$e cells inEvitroRt&e role o! calcium an% iron* Calci! Tissue Int 4>>8M :8A5B648>H49;* 5<4* Mon$$onen ') iu$$onen ') Tas$inen M) Heat& TD) Urtti A* Stu%ies on liposome !ormuE lations !or intraarticular %elivery o! clo%ronate* ' Controlle% Rel 4>>:M 8:A5H8B649:H4:9* 5<5* Selan%er 0S) Mon$$onen ') 0ar&u$orpi E0) Har$onen P) Hannuniemi R) 2aananen H0* C&aracteristics o! clo%ronateEin%uce% apoptosis in osteoclasts an% macrop&ages* Mol P&armacol 4>>;M :@A:B6445<H448=* 5<8* Diacovo TG) Rot& S') +uccola 'M) +ainton D-) Springer TA* Neutrop&il rolling) arrest) an% transmigration across activate%) sur!aceEa%&erent platelets via seCuential action o! PEselectin an% t&e #eta 5Eintegrin CD44#1CD4=* +loo% 4>>;M ==A4B649;H4:<* 5<9* arsen E) Celi A) Gil#ert GE) et al* PADGEM protein6 a receptor t&at me%iates t&e interE action o! activate% platelets (it& neutrop&ils an% monocytes* Cell 4>=>M :>A5B68@:H845* 5<:* Ham#urger SA) Mcever RP* GmpE49@ me%iates a%&esion o! stimulate% platelets to neuE trop&ils* +loo% 4>>@M <:A8B6::@H::9* 5<;* McEver RP) Cummings RD* Role o! PSG E4 #in%ing to selectins in leu$ocyte recruitE ment* ' Clin Invest 4>><M 4@@A44B6S><HS4@8* 5<<* Simon DI) C&en V) Wu H) et al* Platelet glycoprotein i#alp&a is a counterreceptor !or t&e leu$ocyte integrin MacE4 ACD44#1CD4=B) ' E"p Me% 5@@@M 4>5A5B64>8H5@9* 5<=* Diacovo TG) De!ougerolles AR) +ainton D-) Springer TA) A !unctional integrin ligan% on t&e sur!ace o! plateletsHIntercellularEa%&esion moleculeE5* ' Clin Invest 4>>9M >9A8B64598H45:4* 5<>* ,e#er C) Springer) TA* Neutrop&il accumulation on activate%) sur!aceEa%&erent plateE lets in !lo( is me%iate% #y interaction o! MacE4 (it& !i#rinogen #oun% to alp&a II# #eta 8 an% stimulate% #y plateletEactivating !actor* ' Clin Invest 4>><M 4@@A=B65@=:H5@>8* 5=@* i##y P) S&c(art/ D) +rogi E) Tana$a H) Clinton S* A casca%e mo%el !or restenosis* A special case o! at&erosclerosis progression* Circulation =;A;SB) III9<HIII:5) 4>>5* 5=4* -ast D0) -eli" R) Do(se C) Neuman ,-) -leisc& H* T&e e!!ects o! %ip&osp&onates on t&e gro(t& an% glycolysis o! connectiveEtissue cells in culture* +ioc&em ' 4><=M 4<5A4B6><H4@<* 5=5* -eli" R) +ette" 'D) -leisc& H* E!!ect o! %ip&osp&onates on t&e synt&esis o! prostaglan%E ins in culture% calvaria cells* Calci! Tissue Int 4>=4M 88A:B6:9>H::5* 5=8* ?&ya 0) 3ama%a S) -eli" R) -leisc& H* E!!ect o! +isp&osp&onates on prostaglan%in synt&esis #y rat #oneEcells an% mouse calvaria in culture* Clin Sci 4>=:M ;>A9B69@8H944* 5=9* Giuliani N) Pe%ra//oni M) Passeri G) Girasole G* +isp&osp&onates in&i#it I E; pro%uction #y &uman osteo#lasticEli$e cells* Scan% ' R&eumatol 4>>=M 5<A4B68=H94* 5=:* Ma$$onen N) Salminen A) Rogers M') et al* Contrasting e!!ects o! alen%ronate an% clo%rE onate on RA, 5;9 macrop&ages6 t&e role o! a #isp&osp&onate meta#olite* Eur ' P&arm Sci 4>>>M =A5B64@>H44=* 5=;* Hyvonen PM) 0o(oli$ M') Human neutrop&il priming6 c&emiluminescence mo%i!ie% #y &y%ro"yapatite an% t&ree #isp&osp&onates in vitro* ' Clin a# Immunol 4>>8M 9@A5B6;>H<;* 5=<* Serretti R) Core P) Muti S) Sala!!i -* In!luence o! %ic&loromet&ylene %ip&osp&onate on reactive o"ygen species pro%uction #y &uman neutrop&ils* R&eumatol Int 4>>8M 48A9B648:H48=* 5==* Mian M) +enetti D) Aloisi R) Rosini S) -anto//i R* E!!ects o! #isp&osp&onate %erivatives on macrop&age !unction* P&armacology 4>>9M 9>A:B688;H895* 5=>* Mon$$onen ') Pennanen N) apin.o$i S) Urtti A* Clo%ronate ADic&loromet&ylene +isp&osp&onateB in&i#its PSEstimulate% IlE; an% TN- pro%uction #y Ra(E5;9 cells* i!e Sciences 4>>9M :9A49B6Pl55>HPl589* 5>@* Ma.es$y M,) Rei%y MA) +o(enEPope D-) Hart CE) ,ilco" 'N) Sc&(art/ SM* PDGligan% an% receptor gene e"pression %uring repair o! arterial in.ury* ' Cell +iol 4>>@M 444A: Pt 4B6549>H54:=* 5>4* ,alten#erger '* Mo%ulation o! gro(t& !actor action6 Implications !or t&e treatment o! car%iovascular %iseases* Circulation 4>><M >;A44B69@=8H9@>9* 5>5* Danen#erg HD) Golom# G) Groot&uis A) et al* iposomal alen%ronate in&i#its systemic innate immunity an% re%uces inEstent neointimal &yperplasia in ra##its* Circulation 5@@8M 4@=A55B65<>=H5=@9*

NanoECarriers o! Drugs an% Genes

5;>

5>8* Co&en H) Al!eriev IS) Mon$$onen ') et al* Synt&esis an% preclinical p&armacology o! 5E A5Eaminopyrimi%inioB et&yli%eneE4)4E#isp&osp&onic aci% #etaine AISAE48E4BEa novel #isp&osp&onate* P&arm Res 4>>>M 4;A>B648>>H49@;* 5>9* ?ga(ara 0) 3os&i%a M) 0u#o ') et al* Mec&anisms o! &epatic %isposition o! polystyrene microsp&eres in rats6 E!!ects o! serum %epen% on t&e si/es o! microsp&eres* ' Control Release 4>>>M ;4A8B6594H5:@* 5>:* ?ga(ara 0) 3os&i%a M) Ta$a$ura 3) Has&i%a M) Higa$i 0) 0imura T* Interaction o! polystyrene microsp&eres (it& liver cells6 roles o! mem#rane receptors an% serum proE teins* +#aEGen Su#.ects 4>>>M 49<5A4H5B64;:H4<5* 5>;* Roser M) -isc&er D) 0issel T* Sur!aceEmo%i!ie% #io%egra%a#le al#umin nanoE an% microsp&eres* II6 e!!ect o! sur!ace c&arges on in vitro p&agocytosis an% #io%istri#ution in rats* Eur ' P&arm +iop&arm 4>>=M 9;A8B65::H5;8* 5><* Ta#ata 3) I$a%a 3* E!!ect o! sur!ace c&arges on in vitro p&agocytosis an% #io%istri#ution in rats* +iomaterials 4>>=M >A9B68:;H8;5* 5>=* Ni(a T) Ta$euc&i H) Hino T) 0unou N) 0a(as&ima 3* Preparations o! #io%egra%a#le nanosp&eres o! (aterEsolu#le an% insolu#le %rugs (it& D) Elacti%e glycoli%e copolyE mer #y a novel spontaneous emulsi!ication solvent %i!!usion met&o%) an% t&e %rug release #e&avior* ' Control Release 4>>8M 5:A4H5B6=>H>=* 5>>* Ni(a T) Ta$euc&i H) Hino T) 0unou N) 0a(as&ima 3* InEvitro %rugErelease #e&avior o! D) Elacti%e1glycoli%e copolymer APlgaB nanosp&eres (it& na!arelin acetate prepare% #y a novel spontaneous emulsi!ication solvent %i!!usion met&o%* ' P&arm Sci 4>>9M =8A:B6<5<H<85*

4;

?cular Applications o! Nanoparticulate DrugEDelivery Systems

Annic$ u%(ig
Department o! P&armaceutical Sciences) University o! Ant(erp) Ant(erp) +elgium

INTR?DUCTI?N T&e a#sorption o! topically applie% op&t&almic %rugs is very poor #ecause o! e!!icient mec&anisms protecting t&e eye !rom &arm!ul materials an% agents* T&ese protective mec&anisms) suc& as re!le" #lin$ing) lac&rymation) tear turnover) an% %rainage) result in t&e rapi% removal o! !oreign su#stances !rom t&e eye sur!ace* Moreover) t&e very tig&t epit&elium o! t&e cornea also compromises t&e permeE ation o! %rug molecules* ConseCuently) t&e contact time o! conventional eye %rops is only a#out !ive minutes) an% typically less t&an :[ o! t&e applie% %ose penetrates passively across t&e cornea an% reac&es t&e intraocular tissues A4B* T&us) !reCuent instillations o! eye %rops are necessary to maintain t&erapeutic %rug level in t&e tear !ilm or at t&e site o! action* Ho(ever) t&e !reCuent use o! &ig&ly concentrate% %rug solutions may in%uce to"ic si%e e!!ects a!ter a#sorption via t&e #loo% vessels in t&e con.unctival stroma or nasal mucosa into systemic circulation* Moreover) cellular %amage at t&e ocular sur!ace coul% occur A5H9B* T&e rationale !or t&e %evelopment o! various particulate systems !or sustaine% %rug %elivery is #ase% on possi#le entrapment o! t&e particles in t&e ocular mucus layer covering t&e eye sur!ace) an% t&e interaction o! #ioa%&esive polymer c&ains (it& mucins* T&is (ill increase t&e precorneal resi%ence time o! t&e %rug) allo(ing !or an e"tension o! t&e a#sorption time* -urt&ermore) #y controlling %rug release an% en&ancing %rug a#sorption) t&is coul% also improve corneal %rug penetration* ?ne attractive !eature o! utili/ing nanoparticles !or ocular %rug %elivery is t&at it is in a liCui% %osage !orm (&ic& can #e easily a%ministere% #y patients A9H=B* Anot&er signi!icant c&allenge is to %eliver t&erapeutic %oses o! %rugs to treat %iseases a!!ecting t&e posterior segment o! t&e eye* It is %i!!icult to %eliver %rugs to t&e posterior segment #y topical application #ecause o! t&e %i!!usional %istance an% t&e counterE%irectional intraocular convection !rom t&e ciliary #o%y to Sc&lemmPs canal* T&e most logical (ay is to %eliver %rugs #y intraocular in.ections t&us #ypassE ing anatomical an% p&ysiological #arriers* Ho(ever) %rugs o! s&ort &al!Elives Ae*g*) anti#iotics) antiviral %rugsB (oul% reCuire repeate% in.ections) (&ic& coul% increase t&e ris$ o! retinal %etac&ment or &emorr&age* T&ere!ore) #io%egra%a#le nanopartiE cles (ere %evelope% !or intraocular a%ministration in or%er to o#tain a controlle%) sustaine% %rug release an% t&us re%ucing t&e num#er o! in.ections reCuire% A>B* T&e c&oice o! t&e polymer !or preparing particulate systems (ill %epen% on t&e p&ysicoc&emical properties o! t&e %rug* T&e goal is to ac&ieve &ig& %rug loa%E ing in or%er to minimi/e t&e reCuire% volume to #e instille% into t&e eye* Corneal upta$e an% transport o! nanoparticles s&oul% #e !acilitate% #y small particle si/e A4@@H5@@ nmB A=B*

5<4

5<5

u%(ig

DrugErelease $inetics are regulate% #y t&e composition an% preparation proceE %ure o! t&e particles) t&e molecular (eig&t an% %egra%ation o! t&e polymers) an% t&e p&ysicoc&emical properties o! t&e entrappe% %rug molecule A9);H=B* As e"cellent revie(s on t&e use o! nanoparticles in ocular %rug %elivery &ave #een pu#lis&e% previously) t&is c&apter (ill mainly !ocus on t&e latest relevant researc& reporte% in literature A:H>B*

P? 3MERS ?- NATURA ?RIGIN As t&ey are #iocompati#le) polymers o! natural origin suc& as proteins an% polysacE c&ari%es &ave #een investigate% !or use in t&e pro%uction o! microE an% nanopartiE cles* A %enaturation process in%uce% #y eit&er &eating or cooling an% su#seCuent c&emical crossElin$ing proce%ures to create a %enser particle matri" prepare protein nanoparticles* Anot&er preparation met&o% is #ase% on %esolvation o! t&e macroE molecules) (&ic& lea%s to precipitation or t&e !ormation o! a coacervate p&ase* A crossElin$ing agent Ae*g*) glutaral%e&y%eB is a%%e% to &ar%en t&e native particles A:);)4@B* ?(ing to t&e presence o! c&arge% groups) protein nanoparticles can #e use% as a matri" in (&ic& t&e %rug molecules may #e p&ysically entrappe% or covalently lin$e% A44B* Some e"amples o! particles !or ocular purpose reporte% in literature are summari/e% in Ta#le 4* 2arious in vivo stu%ies in ra##its reporte% t&at a prolonge% e!!ect o! %rugs Apilocarpine) piro"icamB incorporate% in al#umin particles (as o#serve% (&en compare% to commercial preparations or aCueous an% viscous solutions A9):)44B*

TA+ E 4 Types o! ?cular Particulate Dosage -orms Use% in Animal Stu%ies ,&ic& ,ere Prepare% !rom Proteins an% Polysacc&ari%es Polymer Al#umin Al#umin Drug ?#servations Re!erences A45B A48B

C&itosan C&itosan C&itosan PE? insert Pectin

Starc& acetate

Carrageenan gelatin

Piro"icam Increase% #ioavaila#ility compare% to commercial eye %rops GanciclovirA!ter intravitreal in.ection in rats) t&e resi%ence time is prolonge% A5 (ee$sBM no in!lammationM goo% tolerance Cyclosporin A T&erapeutic concentrations in cornea an% con.unctiva %uring at least 9= &rs -luorescentHig& amounts o! nanoparticles into corneal la#elan% con.unctival epit&elia ?!lo"acinCma" in aCueous &umor increase% compare% to plain PE? inserts Piro"icam5*:E!ol% &ig&er #ioavaila#ility in aCueous &umor compare% to commercial eye %rops RA!ter 8 &rs incu#ation) =[ o! culture% retinal epit&elial cells too$ up microsp&eres Timolol:*;E an% 5*:E!ol% &ig&er #ioavaila#ility in aCueous &umor compare% to commercial eye %rops an% in situ gelling system) respectively

A49B A4:B A4;B A4<B

A4=B

A4>B

Note 6 Unless as in%icate%) in vivo tests (ere per!orme% on ra##its* A##reviation6 PE?) polyAet&ylene o"i%eB*

?cular Applications o! Nanoparticulate DrugEDelivery Systems

5<8

Ho(ever) in one stu%y) topical application o! &y%rocortisoneEloa%e% al#umin particles in ra##its resulte% in a lo(er tissue concentration o! t&e %rug as compare% to t&e application o! a simple %rug solution A:B* T&is result is possi#ly %ue to t&e strong #in%ing o! t&e %rug to t&e particles* T&e retention o! nanoparticles (as !oun% to #e &ig&er in in!lame% tissue as compare% (it& t&e normal tissue A:B* Ganciclovir) t&e most (i%ely use% antiviral %rug !or t&e treatment o! cytomegalovirus retinitis) (as !ormulate% in nanoparticles !or intravitreal a%minisE tration in rats* A prolonge% resi%ence in t&e vitreous cavity At(o (ee$sB s&o(e% no evi%ence o! in!lammatory reaction in t&e retinal tissue an% also %i% not a!!ect t&e vision A48B* T&e types o! polysacc&ari%es (&ic& &ave #een investigate% !or t&e pro%uction o! ocular particulates are c&itosan) pectin) an% carrageenan* C&itosan) a %eacetylate% c&itin) is #io%egra%a#le) #iocompati#le) an% nonto"ic A<)=B* T&e polycationic c&itosan (as investigate% as an op&t&almic ve&icle #ecause o! its pro#a#le superior mucoa%&esiveness %ue to its a#ility to pro%uce molecular attractive !orces #y electrostatic interactions (it& t&e negative c&arges o! t&e mucus* Numerous stu%ies in%icate% an increase in t&e precorneal retention time o! c&itosan solutions A<B* Moreover) c&itosan appears to en&ance t&e permea#ility o! t&e cornea transiently %ue to t&e interaction (it& tig&t .unction structures A<B* -or t&e preparation o! c&itosan nanoparticles) a num#er o! tec&niCues are employe%* T&ese inclu%e covalent c&emical crossElin$ing) ionic crossElin$ing Ae*g*) ionotropic gelation (it& tripolyp&osp&ateB) or %esolvation* C&itosan microsp&eres &ave #een prepare% #y (aterEinEoil evaporation an% sprayE%rying* +y a%.usting t&e molecular (eig&t an% t&e %eacetylation %egree o! t&e polymer use%) one coul% ac&ieve t&e %egra%ation an% release rate reCuire% !or a speci!ic %rug A<B* In one stu%y) t&e animals treate% (it& cyclosporine AEloa%e% nanoparticles (ere !oun% to s&o( signi!icantly &ig&er corneal an% con.unctival %rug levels t&an t&ose treate% (it& a suspension o! cyclosporin A in a c&itosan aCueous solution or in (ater A5H;E!ol% increaseB A49B* In a recent stu%y in ra##its) De Campos an% co(or$E ers s&o(e% t&at t&e amounts o! !luorescent nanoparticles in cornea an% con.unctiva (ere signi!icantly &ig&er t&an t&ose !or a control solution* T&ese amounts (ere !airly constant !or up to 59 &ours* A &ig&er retention o! c&itosan nanoparticles in t&e con.unctiva as compare% to t&at in t&e cornea (as o#serve%* Con!ocal microscopy stu%ies suggest t&at nanoparticles penetrate into t&e corneal an% con.unctival epit&elia #y a paracellular1transcellular pat&(ay (&ic& is %i!!erent !rom t&e pat&E (ay use% #y t&e polyAal$ylcyanoacrylateB APACAB an% polyAepsilonEcaprolactonB APEC B nanoparticles A<)4:B* To improve t&e release $inetics o! o!lo"acin) c&itosan microsp&eres loa%e% (it& o!lo"acin (ere also incorporate% in polyAet&ylene o"i%eB APE?B inserts* T&e in vivo results in ra##its %i% not %emonstrate a #iop&armaceutical improvement compare% to plain PE? inserts* Ho(ever) a signi!icant increase in t&e C ma" value in t&e aCueous &umor (as o#serve% (&ic& coul% partly #e %ue to t&e permea#ilityE en&ancing e!!ect o! c&itosan across t&e cornea A4;B* Pectin nanoparticles loa%e% (it& !luorescein s&o(e% an increase in t&e precorneal retention time !rom @*: to 5*: &ours) (&en compare% (it& a !luorescein solution* In vivo tests (it& piro"icamEloa%e% pectin particles in%icate% a 5*:E!ol% increase in t&e amount o! %rug concentration in t&e aCueous &umor as compare% to a commercial eye %rop solution A4<B* Tuovinen et al* A4=B reporte% t&e !irst stu%y on en/ymeEsensitive micropartiE cles ma%e o! t&e natural #io%egra%a#le potato starc& acetate !or retinal targeting*

5<9

u%(ig

A!ter a t&reeE&our incu#ation) a#out =[ o! t&e cells o! a retinal pigment epit&elium ARPEB culture too$ up microparticles* T&e via#ility o! culture% RPE cells (as at least =5[ a!ter 59E&our incu#ation (it& t&e microparticles* T&e %egra%ation o! potato starc& acetate in RPE cells is cataly/e% #y esterases an% amylases* Consi%ering t&e lo( to"icity) it seems t&at t&ese microparticles are suita#le !or %rug %elivery to t&e posterior segment o! t&e eye A4=B* Carrageenans are a group o! (aterEsolu#le sul!ate% galactans e"tracte% !rom #ro(n sea(ee%* Microsp&eres containing gelatin) lam#%a carrageenan) an% timolol (ere prepare% #y sprayE%rying* T&e %i!!erent ratios o! carrageenan an% gelatin prove% to #e use!ul in mo%ulating t&e %rugErelease pro!iles an% mucoa%&esive properties* A!ter a%ministration o! a microsp&ere A:@1:@ mi"tureB suspension) t&e #ioavaila#ility AAUC valuesB o! timolol in t&e aCueous &umor in ra##its (ere :*; an% 5*: times &ig&er in comparison (it& commercial eye %rops an% in situ gelling system) respectively A4>B*

ACR3 ATES PolyAal$ylcyanoacrylatesB In t&e past) t(o #io%egra%a#le an% #iocompati#le polymers) PACAs an% polyAal$y lmet&acrylatesB) (ere Cuite popular !or use in t&e preparation o! %rug carriers o! particle si/e range !rom 5@@ to :@@ nm* T&e use o! t&ese polymers is #ase% on t&eir mucoa%&esive or #ioa%&esive properties* T&e %i!!erence #et(een t&e t(o polymers lies mainly in t&eir %egra%ation rate6 polymers (it& a longer si%e c&ain are %egra%e% more slo(ly) resulting in a slo(er release o! t&e %rug incorporate% in PACA particles A9 H;B* T&e %rug is eit&er incorporate% in PACA particles %uring t&e polymeri/ation process or a%sor#e% to t&e nanoparticle sur!ace a!ter t&e particle is !orme%* T&e ultiE mate particle si/e o! t&e particles an% t&e %egree o! %rug loa%ing are %epen%ent on several preparation parameters suc& as t&e pH o! t&e preparation me%ium an% t&e type o! sta#ili/er or sur!actant use% A9H;)=B* PACA nanosp&eres (ere s&o(n to a%&ere to t&e eye sur!ace) an% (ere ta$en up #y a transcellular pat&(ay in t&e outer cell layers o! con.unctiva an% cornea* T&is coul% #e e"plaine% #y eit&er en%ocytosis or lysis o! t&e cell (all in%uce% #y particle %egra%ation pro%ucts* Alt&oug& t&e concentration o! t&e %rug in ocular tissues (as s&o(n to #e &ig&er in in!lame% tissues) a con%ition (&ere t&e permea#ility o! t&e cell mem#rane is increase%) no !ull penetration across t&e cornea into t&e anterior c&am#er (as o#serve% A:B* Many stu%ies con!irme% t&at t&e use o! nanoparticles (it& t&ese polymers can improve t&e clinical e!!ects o! a t&erapeutic agent Ai*e*) in glaucoma t&erapy or anti#iotic t&erapyB an% also minimi/es its si%e e!!ects A9 H;B* Sustaine% %rug release !rom t&e polymer matri" an% prolonge% t&erapeutic e!!ect (ere o#serve%) e"cept in t&e situation (&ere t&e %rug &a% a &ig& a!!inity !or t&e polymer* T&e increase% #ioE logical response (as attri#ute% to improve% ocular penetration an% #ioa%&esion* T&e precorneal resi%ence time o! PACA nanoparticles coul% !urt&er #e increase% #y incorporating t&e particles into a polyet&ylene glycol APEGB gel or coating t&e partiE cles (it& PEG A5@)54B* AcyclovirEloa%e%) PEGEcoate% polyet&ylE5Ecyanoacrylate s&o(e% a 5:E!ol% increase in t&e %rug level in aCueous &umor (&en compare% (it& an aCueous solution o! t&e !ree %rug* T&is result is pro#a#ly %ue to a longer interacE tion o! t&e nanoparticles (it& t&e corneal epit&elium an% t&e penetrationEen&ancing e!!ect o! PEG A54B*

?cular Applications o! Nanoparticulate DrugEDelivery Systems

5<:

Acrylate Derivatives Polyacrylic aci% APAAB an% car#omers are use% in t&e preparation o! viscous eye %rops) arti!icial tears) &y%rogels) an% inserts* T&e mucoa%&esive properties o! t&ese polymers are mainly %ue to &y%rogen #on%ing an% interpenetration o! t&e polymer c&ains an% t&e mucus layer at t&e eye sur!ace A9B* T&e &y%ration state an% pH o! microsp&eres in t&e lacrimal !lui% (ere s&o(n to #e a !actor a!!ecting t&e resi%ence time o! t&e microsp&eres in t&e eye* In one stu%y) ra%iola#ele% PAA microsp&eres ma%e (it& Car#opol b >@< crossElin$e% (it& maltose (as applie% eit&er in %ry !orm or in a pre&y%rate% !orm* T&e clearE ance rate o! t&e microsp&eres applie% in t&e %ry !orm (as !oun% to #e &ig&er t&an t&e pre&y%rate% !orm pro#a#ly %ue to incomplete &y%ration in t&e lac&rymal !lui%* -urt&ermore) t&e clearance o! &y%rate% microsp&eres at pH <*9 is &ig&er t&an at pH :*@ #ecause t&e presence o! protonate% car#o"yl groups at pH :*@ in PAA permits en&ance% #ioa%&esion t&roug& &y%rogen #on% !ormation (it& mucins ATa#le 5B A55B* In a recent stu%y) De et al* A5=B %emonstrate% t&e #iocompati#ility an% a%&esive properties o! PAA nanoparticles on &uman corneal epit&elial cells* A controlle% release o! t&e partly ionically entrappe% #rimoni%ine (as o#taine% %ue to t&e cationEe"c&ange properties o! t&e polyanionic PAA matri" o! t&e nanoparticles A5=B* A ma.or pro#lem o! PACA nanoparticles is t&e lo( loa%ing capacity !or &y%rop&ilic %rugs* To improve %rug loa%ing) attempts are ma%e to increase t&e &y%rop&ilicity o! t&e particle sur!ace #y copolymeri/ation o! met&ylmet&acrylate AMMAB an% sul!opropylmet&acrylate ASPMB* +oun% %rug molecules are release% !rom t&e carrier #y competitive replacement #y ot&er ions* T&ese copolymer nanoparticles loa%e% (it& t&e muscarinic agonists arecai%ine propargyl ester AAPEB an% ASBEAoBacecli%ine (ere evaluate% in ra##its* A t(o!ol% increase in %rug a#sorpE tion (as o#taine% (&en t&e nanoparticles (ere instille% toget&er (it& #ioa%&esive &yaluronan A58B* 2arious cationic acrylic copolymers (ere also e"amine% to prepare nanoparticles (it& a positive c&arge in or%er to !acilitate an e!!ective a%&esion o! t&e %elivery system to t&e ocular sur!ace A=B* Pignatello et al* A59H5;B !ormulate% nanoparticles (it&

TA+ E 5 ?vervie( o! In 2ivo Stu%ies Carrie% ?ut in Ra##its Using Nanoparticles Prepare% (it& Acrylate Derivatives Type o! polymer Car#opol >@< Drug
444In%ium

?#servations -aster elimination o! microsp&eres in %ry !orm t&an (&en instille% as a %ispersion Com#ination (it& mucoa%&esive polymers increase% #ioavaila#ility #y t(o!ol% Hig&er %rug levels in t&e aCueous &umor compare% to aCueous solution %ue to sustaine% release an% increase% precorneal retention Eig&t!ol% longer %ecrease o! I?P a!ter a%ministration o! crossElin$e% particles compare% to eye %rops

Re!erences A55B

Copolymers MMAHSPM Eu%ragit R 4@@ an% RS4@@

Propargyl ester AAPEB ASBEAoBacecli%ine I#upro!enM -lur#ipro!en

A58B

A59H5;B

PNIPAAm

Epinep&rine

A5<B

A##reviations6 APE) arecai%ine propargyl esterM I?P) intraocular pressureM MMA) met&ylmet&acrylateM PNIPAAm) polyENEisopropylacrylami%eM SPM) sul!opropylmet&acrylate*

5<;

u%(ig

Eu%ragitb R an% RS using a mo%i!ie% Cuasiemulsion solvent %i!!usion tec&niCue an% solvent evaporation met&o%* Using t&ese nanoparticles) t&ey %emonstrate% t&at susE taine% release an% increase% a#sorption o! t&e incorporate% nonsteroi%al) antiin!lamE matory %rugs (ere ac&ieve%* -urt&ermore) no in!lammation or %iscom!ort (as o#serve% in t&e ra##itPs eye) suggesting t&at t&e nanoparticles (ere (ell tolerate% A59B* Hsiue et al* A5<B investigate% t&e use o! a t&ermosensitive polymer) polyENE isopropylacrylami%e APNIPAAmB) in controlle%Erelease %elivery systems !or glaucoma t&erapy* CrossElin$e% PNIPAAm nanoparticles containing epinep&rine (ere a%ministrate% to ra##its) an% its e!!ect on t&e intraocular pressure AI?PB (as monitore%* T&e results in%icate% t&at a %ecrease% pressure response (&ic& laste% eig&t times longer t&an t&at o! conventional eye %rops (as o#serve%*

P? 3AEPSI ?NECAPR? ACT?NB Anot&er #iocompati#le an% #io%egra%a#le polymer is PEC * It is slig&tly more &y%rop&o#ic (&en compare% (it& PACA A;)5>B* PEC nanoparticles an% nanoE capsules !or op&t&almic use can #e prepare% #y solvent e"traction or solvent evaporation met&o% A;B* Upon instillation) t&e PEC particles !orm aggregates an% resi%e in t&e culE%eEsac) gra%ually releasing t&e %rug* T&is &ypot&esis (as put !or(ar% to e"plain t&e muc& more pronounce% re%uction in I?P in glaucomatous ra##its a!ter a%ministration o! PEC nanoparticles (&en compare% to PACA an% P GA microsp&eres A9H;B* PEC nanoparticles also seem to #e a#le to penetrate t&e outer corneal cell layers) #ut contrary to PACA) no cellular %amage (as o#serve%* T&is suggests t&at an en%ocytic mec&anism may #e involve%* T&e penetration o! t&e particles is !oun% to #e si/eE%epen%ent6 nanoparticles #ut not microsp&eres (ere !oun% in t&e corneal cells A9H;B* Numerous in vivo stu%ies %emonstrate% t&at en&ance% corneal a#sorption an% prolonge% t&erapeutic e!!ects o! %rugs are possi#le (&en t&e %rugs are incorpoE rate% in PEC nanoparticles* T&e %rugs teste% (ere6 #eta"olol) metipranolol) carteolol) cyclosporin A) an% in%omet&acin* -urt&ermore) nanocapsules seem to %isplay a #etter e!!ect t&an nanosp&eres) pro#a#ly #ecause t&e %rug entrappe% (as in t&e unioni/e% !orm in t&e oily core o! t&e %elivery system an% coul% %i!!use more easily to t&e cornea A9H;)=B* A strategy %esigne% to en&ance t&e interaction (it& t&e mucus layer in t&e eye &as #een investigate% #y Calvo et al* A8@B (&o coate% PEC particles (it& cationic #ioa%&esive polymers* Compare% (it& noncoate% particles) t&e corneal an% aCueous &umor in%omet&acin concentrations (ere %ou#le% !or c&itosanEcoate% nanocapsules* Ho(ever) coating (it& polylysine %i% not seem to improve t&e #ioavaila#ility o! t&e teste% %rug* T&e rise in #ioavaila#ility (as attri#ute% to t&e structural similarity #et(een c&itosan an% mucin) rat&er t&an to its positive c&arge A8@B* C&itosanEcoate% PEC particles e"&i#it an important interaction (it& t&e mucus layer) #ut t&e penetraE tion in t&e corneal epit&elium is lo(er (&en compare% (it& t&at o! t&e uncoate% particles A<B* De Campos et al* A84B compare% t&e e!!ect o! coating PEC nanocapsules (it& PEG versus c&itosan on t&e interaction o! PEC nanocapsules (it& t&e ocular mucosa* T&e in vivo stu%y s&o(e% t&at t&e nanoparticles entere% t&e corneal epiE t&elium #y a transcellular pat&(ay* T&e penetration rate an% %ept& (ere %epen%ent on t&e coating composition* PEG coating en&ance% t&e passage o! t&e PEC particles

?cular Applications o! Nanoparticulate DrugEDelivery Systems

5<<

nanocapsules across t&e (&ole epit&elium) (&ereas t&e c&itosan coating !avore% t&e retention in t&e super!icial layers o! t&e epit&elium A84B* ConseCuently) t&e %esign o! colloi%al carriers (it& %i!!erent ocular %istri#ution seems to #e possi#le* P? 3AD) E ACTIC ACIDB AND P? 3AD) E ACTIDEEC?EG 3C? IDEB As polyA%)lElactic aci%B AP AB an% polyA%)lElacti%eEcoEglycoli%eB AP GAB are -DAEapprove% pro%ucts Aa%%itivesB !or parenteral use) microsp&eres an% nanoparE ticles ma%e o! t&ese polymers (ere investigate% primarily !or t&e controlle% release o! %rugs a!ter intravitreal or su#con.unctival in.ection rat&er t&an !or topical application A9):)>)5>B* P A is a synt&etic) #iocompati#le) an% #io%egra%a#le polymer an% P GA a copolymer o! polyAlactic aci%B an% polyAglycolic aci%B* T&e %egra%ation rate %epen%s on t&e molecular (eig&t) con!ormation) an% polymer composition A>)5>)85B* T&e %rug is release% out o! t&e sp&eres #y %i!!usion an% #y &y%rolysis o! t&e P A1P GA matri"* In an attempt to optimi/e t&e mucoa%&esive properties) P A can #e copolymeri/e% (it& ot&er polymers Ae*g*) PEC or PEGB) so t&at more appropriate QtailorEma%eS polymers can #e %evelope% A88B* Several met&o%s &ave #een propose% !or t&e preparation o! t&e P A1P GA particles* Ho(ever) t&e most popular tec&niCue is t&e emulsi!ication solvent evaporation met&o% A>)85B* T&e upta$e o! P GA nanoparticles in con.unctival epit&elium is %epen%ent on t&e particle si/e6 4@@ nm particles e"&i#ite% t&e &ig&est upta$e as compare% to larger particles A=@@ nm an% 4@ _mB* T&e satura#le particle upta$e occurs via a%sorptiveEme%iate% en%ocytosis (&ic& is in%epen%ent !rom clat&rinE an% caveolinE4Eme%iate% pat&(ays A=)89B* Some e"amples o! P A1P GA particles !or ocular purpose reporte% in literature are summari/e% in Ta#le 8* Giannavola et al* c&ange% t&e sur!ace properties o! P A nanoparticles loa%e% (it& acyclovir #y incorporating PEGylate% 4)5E%istearoylE8Ep&osp&ati%ylet&anolamine ADSPEEPEGB into t&e polymer instea% o! coating t&e e"ternal sur!ace as in t&e case o! PACA nanoparticles A55)8:B* A!ter

TA+ E 8 Evaluation o! PolyAD) E actic Aci%B an% PolyA D) E acti%eECoEGlycoli%eB Particles !or ?p&t&almic Use in Animal Stu%ies Drug Acyclovir Application Instillation ?#servations A 45*;E!ol% increase o! aCueous &umor AUC !or PEGEcoate% nanoparticles compare% (it& %rug suspension T&erapeutic scleral levels %uring < %ays o( ocular to"icity an% no irritation A t(o!ol% increase o! aCueous &umor AUC (it& respect to %rug solution an% sustaine% release DoseE%epen%ent angiogenic response Re!erences A8:B

:E-luorouracil 2ancomycin

Con.unctival implantation Instillation

A8;B A8<B

r&2EG+u%esoni%e

Su#retinal an% intravitreal in rats Su#con.unctival in.ection in rats

A8=B Sustaine% %rug level in retina an% ot&er ocular tissues %uring 49 %ays A8>B

Note6 Unless ot&er(ise in%icate%) all in vivo tests (ere per!orme% on ra##its* A##reviation6 AUC) area un%er t&e curve*

5<=

u%(ig

a%ministration o! t&e nanoparticle suspension in t&e ra##itPs eye) t&e AUC @H; values in t&e aCueous &umor (ere !oun% to #e signi!icantly greater !or t&e PEGE coate% P A nanosp&eres t&an !or t&e uncoate% particles* A %rop o! 9=[ in AUC value (as o#serve% (&en PEGEcoate% particles (ere a%ministrate% to t&e eye a!ter removal o! t&e mucus layer* T&is %ecrease (as attri#ute% to t&e a#sence o! PEGHmucin interactions* T&us) %i!!erences in #ioavaila#ility o! t&e %rugs coul% #e correlate% (it& t&e %i!!erent interactions #et(een t&e nanoparticlePs sur!ace an% t&e corneal epit&elium* Hig&er an% prolonge% vancomycin concentration (as s&o(n in t&e aCueous &umor #y incorporating t&e %rug in P GA microsp&eres* Ho(ever) increasing t&e viscosity o! t&e microsp&ere suspension #y a%%ing a viscosi!ying agent A&y%ro"yE propyl celluloseB %i% not seem to improve any !urt&er %rug a#sorption) pro#a#ly #ecause o! t&e rapi% ocular clearance o! t&e &ig&ly &y%rop&ilic %rug* No irritation or ocular %iscom!ort (as o#serve% in ra##its A8<B* Microsp&eres &ave #een investigate% !or use in %elivering many op&t&almic %rugs !or intravitreal or su#con.unctival in.ections* T&is inclu%es retinoic aci%) a%riamycin) :E!luorouracil) %e"amet&asone) cyclosporin A) an% ganciclovir A>)88B* It &as #een s&o(n t&at intracellular %rug %elivery to retinal epit&elial cells may #e possi#le #y coating t&e P A microsp&eres (it& gelatin) pro#a#ly via p&agocytosis A>B* Recent stu%ies %emonstrate% t&at P A1P GA microsp&eres may #e use!ul in sustaine% or even e"ten%e% slo( %rug %elivery targete% to t&e posterior segment o! t&e eye !or t&e treatment o! c&ronic %iseases or gene t&erapy A8=)8>B* As t&e integrity an% activity o! proteins an% oligonucleoti%es is preserve% (&en encapsulate% (it&in P A) t&e nanoparticles (ere evaluate% !or %elivering t&ese molecules to t&e retina A>B* A!ter intravitreal in.ection in rats) transretinal movement o! t&e nanoparticles (it& a pre!erential locali/ation in t&e retinal epit&elial cells (as o#serve%* A mil% transient in!lammatory reaction a!ter in.ection (as reporte%* T&e presence o! nanoparticles (it&in t&e retinal epit&elium cells coul% #e %etecte% even a!ter !our mont&s o! a single in.ection* T&is suggests t&at a stea%y an% continuous %elivery o! %rugs coul% #e ac&ieve% A9@B* Ho(ever) inter!erence (it& visual acuity %ue to !loating o! nanoparticles in t&e vitreous cavity coul% #e a %ra(E #ac$* +e!ore clinical implementation o! t&e nanoparticles) t&e possi#le e!!ects o! t&e nanoparticles on t&e retinal !unction an% t&e vision &ave to #e investigate% A>)9@B*

IPIDS Instea% o! using macromolecules) several lipi%s (ere propose% !or use to prepare nanoparticles* Soli%Hlipi% nanoparticles AS NsB consist o! a #iocompati#le lipi% core an% an amp&ip&ilic sur!actant as an outer s&ell* T&e a%vantages o! S Ns are6 scala#le manu!acturing process using &ot or col% &ig&Epressure &omogeni/ation) easy mo%ulation o! t&e %rugErelease pro!ile) no organic solvents) t&e (i%e range o! lipi%1sur!actant com#inations) an% t&e &ig& %rug payloa% A94B* Cavalli et al* A95B evaluate% t&e use o! S Ns as carriers !or to#ramycin* Compare% to commercial eye %rops) t&e to#ramycinEloa%e% S Ns pro%uce% a signi!icantly &ig&er #ioavaila#ility6 a 4*:E!ol% increase in Cma" an% !our!ol% increase in AUC* T&e S N %ispersion (as (ell tolerate%) (it& no evi%ence o! ocular irritation A95B* T&e longer retention o#serve% !or S Ns on t&e corneal sur!ace an% in t&e culE%eEsac is pro#a#ly relate% to t&eir relatively small si/e* T&e nanoparticles are presume% to #e entrappe% an% retaine% in t&e mucus layer*

?cular Applications o! Nanoparticulate DrugEDelivery Systems

5<>

C?NC USI?NS T&e in vivo stu%ies t&at (ere use% to evaluate t&e use o! nanoparticles !or ocular %rug %elivery (ere primarily per!orme% on animals* -rom t&e results to %ate) one coul% conclu%e t&at t&e nanoparticles s&oul% possess #ioE or mucoa%&esive proE perties in or%er to ac&ieve a long precorneal retention time an% to improve %rug a#sorption* T&e intraocular use o! #io%egra%a#le) slo(Ereleasing nanoparticles loo$s very promising !or %rug %elivery targete% to t&e tissues o! t&e posterior segment in or%er to treat c&ronic %iseases or !or gene t&erapy*

RE-ERENCES
4* ee 2H ) Ro#inson 'R* Revie(6 topical ocular %rug %elivery6 recent %evelopments an% !uture c&allenges* ' ?cul P&armacol 4>=;M 56;<* 5* Salminen * Revie(6 systemic a#sorption o! topically applie% ocular %rugs in &umans* ' ?cul P&armacol 4>>@M ;6598* 8* +au%ouin C* Si%e e!!ects o! antiglaucomatous %rugs on t&e ocular sur!ace* Curr ?pin ?p&t&almol 4>>;M <6=@* 9* e +ourlais C et al* ?p&t&almic %rug %elivery systemsRrecent a%vances* Prog Retinal Eye Res 4>>=M 4<688* :* Vimmer A) 0reuter '* Microsp&eres an% nanoparticles use% in ocular %elivery systems* A%v Drug Deliv Rev 4>>:M 4;6;4* ;* 0reuter '* Nanoparticles* In6 Colloi%al Drug Delivery Systems* Ne( 3or$6 Marcel De$$er) 4>>9 AC&apter :B* <* Alonso M') Sanc&e/ A* T&e potential o! c&itosan in ocular %rug %elivery* ' P&arm P&armacol 5@@8M ::649:4* =* Ra#inovic&EGuilatt et al* Cationic vectors in ocular %rug %elivery* ' Drug Targeting 5@@9M 456;58* >* 3asu$a(a T et al* Drug %elivery systems !or vitreoretinal %iseases* Progr Retin Eye Res 5@@9M 5865:8* 4@* Ars&a%y R* Al#umin microsp&eres an% microcapsules6 met&o%ology o! manu!acturing tec&niCues* ' Control Release 4>>@M 496444* 44* Mero%io M et al* GanciclovirEloa%e% al#umin nanoparticles6 c&aracteri/ation an% in vitro release properties* Eur ' P&arm Sci 5@@4M 4565:4* 45* Giunc&e%i P et al* Al#umin microsp&eres !or ocular %elivery o! piro"icam* P&arm P&armacol Commun 5@@@M ;649>* 48* Mero%io M et al* ?cular %isposition o! ganciclovirEloa%e% al#umin nanoparticles a!ter intravitreal in.ection in rats* +iomaterials 5@@5M 5864:=<* 49* De Campos AM) Sanc&e/ A) Alonso M'* C&itosan nanoparticles6 a ne( ve&icle !or t&e improvement o! t&e %elivery o! %rugs to t&e ocular sur!ace* Application to cyclosporin A* Int ' P&arm 5@@4M 55964:>* 4:* De Campos AM et al* C&itosan nanoparticles as ne( ocular %rug %elivery systems6 in vitro sta#ility) in vivo !ate) an% cellular to"icity* P&arm Res 5@@9M 546=@8* 4;* Di Colo G et al* E!!ect o! c&itosan on in vitro release an% ocular %elivery o! o!lo"acin !rom ero%i#le inserts #ase% on polyAet&ylene o"i%eB* Int ' P&arm 5@@5M 59=644:* 4<* Giunc&e%i P et al* Pectin microsp&eres as op&t&almic carriers !or piro"icam6 evaluation in vitro an% in vivo in al#ino ra##its* Eur ' P&arm Sci 4>>>M >64* 4=* Tuovinen et al* Starc& acetate microparticles !or %rug %elivery into retinal pigment epiE t&eliumRin vitro stu%y* ' Control Release 5@@9M >=69@<* 4>* +on!eroni MC et al* CarrageenanEgelatin mucoa%&esive systems !or ionEe"c&ange% #ase% op&t&almic %elivery6 in vitro an% preliminary in vivo stu%ies* Eur ' P&arm +iop&arm 5@@9M :<69;:* 5@* Desai SD) +lanc&ar% '* Pluronic -45<E#ase% ocular %elivery systems containing #io%eE gra%a#le polyiso#utylcyanoacrylate nanocapsules o! pilocarpine* ' Drug Deliv Target T&er Agents 5@@5M <65@4*

5=@
54* -resta M et al* ?cular tolera#ility an% in vivo #ioavaila#ility o! polyAet&ylene glycolB APEGBEcoate% polyet&ylE5Ecyanoacrylate nanosp&eresEencapsulate% acyclovir* ' P&arm Sci 5@@4M >@65==* 55* Durrani AM) -arr S') 0ella(ay I,* Precorneal clearance o! mucoa%&esive microsp&eres !rom t&e ra##it eye* ' P&arm P&armacol 4>>:M 9<6:=4* 58* anger 0 et al* Met&ylmet&acrylate sul!opropylmet&acrylate copolymer nanoparticles !or %rug %elivery* Part III* Evaluation as %rug %elivery system !or op&t&almic applications* Int ' P&arm 4>><M 4:=654>* 59* Pignatello R) +ucolo C) Puglisi G* ?cular tolera#ility o! Eu%ragit RS 4@@ b an% R 4@@b nanosuspensions as carriers !or op&t&almic controlle% %rug %elivery* ' P&arm Sci 5@@5M >465;8;* 5:* Pignatello R et al* Eu%ragit RS 4@@b nanosuspensions !or op&t&almic controlle% %elivery o! i#upro!en* Eur ' P&arm 5@@5M 4;6:8* 5;* Pignatello R et al* -lur#ipro!enEloa%e% acrylate polymer nanosuspensions !or op&t&alE mic application* +iomaterials 5@@5M 586859<* 5<* Hsiue GEH et al* Preparation o! controlle% release op&t&almic %rops) !or glaucoma t&erapy using t&ermosensitive polyENEisopropylacrylami%e* +iomaterials 5@@5M 5869:<* 5=* De T0 et al* Polycar#o"ylic aci% nanoparticles !or op&t&almic %rug %elivery6 an e" vivo evaluation (it& &uman cornea* ' Microencapsul 5@@9M 546=94* 5>* Des&pan%e AA) Heller ') Gurny R* +ioero%i#le polymers !or ocular %elivery* Crit Rev T&er Drug Carrier Syst 4>>=M 4:68=4* 8@* Calvo P) 2ilaE'ato ' ) Alonso M'* Evaluation o! cationic polymerEcoate% nanocapsules as ocular %rug carriers* Int ' P&arm 4>><M 4:8694* 84* De Campos AM et al* T&e e!!ect o! a PEG versus a c&itosan coating on t&e interaction o! %rug colloi%al carriers (it& t&e ocular mucosa* Eur ' P&arm Sci 5@@8M 5@6<8* 85* +ala I) Hari&aran S) 0umar MN2R* P GA nanoparticles in %rug %elivery6 t&e state o! t&e art* Crit Rev T&erap Drug Carrier Syst 5@@9M 5468=<* 88* Sint/el M+ et al* +iomaterials in op&t&almic %rug %elivery* Eur ' P&arm +iop&arm 4>>;M 9568:=* 89* 7ua%%oumi MG et al* T&e c&aracteristics an% mec&anisms o! P GA nanoparticles in ra##it con.unctival epit&elial cell layers* P&arm Res 5@@9M 546;94* 8:* Giannavola C et al* In!luence o! preparations on acyclovirEloa%e% polyE%)lElactic aci% nanosp&eres an% e!!ect o! PEG coating on ocular %rug #ioavaila#ility* P&arm Res 5@@8M 5@6:=9* 8;* C&iang CEH et al* In vitro an% in vivo evaluation o! an ocular %elivery system o! :E !luorouracil microsp&eres* ' ?cular P&armacol T&erap 5@@4M 4<6:9:* 8<* Gavini E et al* P GA microsp&eres !or ocular %elivery o! a pepti%e %rug) vancomycin using emulsi!ication1sprayE%rying as preparation met&o%6 in vitro1in vivo stu%ies* Eur ' P&arm +iop&arm 5@@9M :<65@<* 8=* Clelan% ' et al* Development o! polyEA%)lElacti%eEcoglycoli%eB microsp&ere !ormulations containing recom#inant &uman vascular gro(t& !actor to promote local angiogenesis* ' Control Release 5@@4M <5648* 8>* 0ompella U+) +an%i N) Ayalasomaya.ula SP* Su#con.unctival nanoE an% microparticles sustain retinal %elivery o! #u%esoni%e) a corticosteroi% capa#le o! in&i#iting 2EGe"pression* Invest ?p&t&almol 2is Sci 5@@8M 99644>5* 9@* +ourges 'E et al* ?cular %rug %elivery targeting t&e retina an% retinal epit&elium using polylacti%e nanoparticles* Invest ?p&t&almol 2is Sci 5@@8M 9968:;5* 94* Me&nert ,) MI%er 0* Soli%Hlipi% nanoparticles pro%uction) c&aracteri/ation an% applications* A%v Drug Deliv Rev 5@@4M 9<64;:* 95* Cavalli R et al* Soli%Hlipi% nanoparticles AS NB as ocular %elivery systems !or to#ramyE cin* Int ' P&arm 5@@5M 58=6594*

u%(ig

4<

Nanoparticulate Systems !or Central Nervous System Drug Delivery

'eanEC&ristop&e ?livier an% Manuela Pereira %e ?liveira
P&armacologie %es MG%icaments AntiEIn!ectieu") -aculty o! Me%icine an% P&armacy) an% INSERM) ERI @58) Poitiers) -rance

INTR?DUCTI?N T&e central nervous system ACNSB is isolate% !rom t&e (&ole #o%y #y t&e #loo%H #rain #arrier A+++B (&ic& creates a strictly controlle% e"tracellular !lui% environment protecting t&e #rain parenc&yma !rom t&e #loo% composition variation an% !rom #loo%E#orne potentially CNSEto"ic compoun%s* T&is tig&t p&ysiological #arrier limits %rastically %rug %i!!usion to(ar%s t&e #rain parenE c&yma an% is consi%ere% as t&e #ottlenec$ in #rain %rug %evelopment an% as an important limiting !actor !or t&e !uture applications o! #iotec&nologyE%erive% neurot&erapeutics A4B* T&e +++ can #e circumvente% #y intraventricular or intraE cere#ral a%ministration* T&ese invasive an% ris$y tec&niCues are limite% to t&e treatments o! restraine% #rain areas %ue to t&e poor tissue %i!!usion o! in.ecte% materials !rom t&e a%ministration sites* Postsurgical a%ministrations are t&e usual a%ministration routes !or relatively large %rugE%elivery systems ADDSsB) suc& as microsp&eres an% (a!ers) an% are presently limite% in clinical practice to t&e a%.uvant treatment o! #rain tumors a!ter surgical resection A5)8B* T&e noninvasive access !rom t&e #loo% compartment via t&e +++ is t&e most convenient an% sa!est (ay to treat t&e entire #rain space* T&ere are in%ee% aroun% 9@@ miles o! #loo% capillaries in t&e average 48@@Eg &uman #rain) (&ic& constitute a large inter!ace o! appro"imately 5@ m5 sur!ace area A4:@ cm51gB !or #loo%EtoE#rain e"c&anges* Nanoparticulate DDSs AnanoEDDSsB) mostly liposomes an% nanoparticles) &ave #een investigate% !or t&e #rain %elivery o! t&erapeutic agents (&ic& poorly %i!E !use t&roug& t&e +++* ?(ing to t&eir nanometric si/e) t&ese nanocontainers !reely circulate in #loo% capillaries an% can #e conveniently a%ministere% #y t&e intraveE nous route* Ta#le 4 summari/es t&e i%eal properties t&at nanoEDDS s&oul% possess !or #rain %rug %elivery* T&e transit (it&in t&e #loo% compartment reCuires t&e nanoEDDS to escape !rom t&e mononuclear p&agocyte system AMPSB* ?nce (it&in t&e #rain vasculature) nanoEDDSs &ave to trigger t&eir translocation t&roug& t&e +++ to %eliver t&eir content in t&e #rain* T&is c&apter revie(s t&e various paramE eters t&at s&oul% #e consi%ere% (&en %esigning nanoEDDSs !or #rain %elivery an% t&e present ac&ievements in t&e !iel%*

THE + ??DH+RAIN +ARRIER AND THE NEED -?R +RAINESPECI-IC NAN?EDRUG DE I2ER3 S3STEMS Normal +loo%H+rain +arrier A scienti!ic consensus locates t&e +++ at t&e en%ot&elia o! #rain capillaries (&ic& are in close relations&ip (it& t&e surroun%ing pericytes) actrocytes) neurons) an% glial cells A-ig* 4B* To summari/e) t&e +++ results !rom t&e uniCue properties o!
5=4

5=5

?livier an% Pereira %e ?liveira

TA+ E 4 I%eal Properties o! NanoEDrug Delivery Systems !or Drug Delivery Across t&e +loo%H+rain +arrier General !or %rug %elivery Nonto"ic) #io%egra%a#le) an% #iocompati#le Amena#le to small molecules) pepti%es) proteins) or nucleic aci%s Agenes) antisense %rugsB Minimal nanoEDDS e"cipientEin%uce% %rug alteration Ac&emical %egra%ation1alteration) protein %enaturationB Mo%ulation o! %rugErelease pro!iles Scala#le an% costEe!!ective manu!acturing process General !or !ree circulation in #loo% No capillary !iltration6 particle %iameter c4 _m P&ysical sta#ility in #loo% Ano aggregation) no %issolution) no interaction (it& #loo% cellsB Avoi%ance o! t&e MPS) prolonge% #loo% circulation time Particular !or #rain %elivery Noninvasive %elivery !rom t&e #loo% circulation into t&e #rain parenc&yma via t&e +++ (it&out in%ucing +++ alteration) t&ere!ore #y a transcytotic pat&(ay Particle %iameter c4@@ nm to !it t&e loa%ing capacity o! transcytotic vesicles E"traE or intracellular %rug %elivery to su#set o! #rain cells or #rain tumor cells

A##reviations6 +++) #loo%H#rain #arrierM DDS) %rug %elivery systemM MPS) mononuclear p&agocyte system*

en%ot&elial cells A4)9B6 AiB t&e tig&t .unctions t&at connect a%.acent en%ot&elial cells an% p&ysically restrict solute !lu" #et(een t&e #loo% an% t&e #rain (it& t&e conseE Cuence t&at passive %i!!usion to(ar%s #rain is limite% to small lipop&ilic compoun%s Aoptimal log Po1( is 4H8B o! molecular (eig&ts #elo( 9@@ to :@@ Da) AiiB an ela#orate system o! transport proteins t&at allo(s t&e selective in!lu" transport o! &y%rop&ilic solutes Agenerally #y carrierEme%iate% transport or CMTB an% macromolecules A#y receptorEme%iate% transcytosis or RMTB necessary !or CNS maintenance an% e"tracellular !lui% &omeostasis) an% t&at re.ect potentially CNSEto"ic compoun%s or meta#olites N#y active e!!lu" transport AAETB proteinsO) #ot& in!lu" an% e!!lu" transport proteins #eing t&e cause o! t&e o#serve% %eviations !rom t&e precept o! lipop&ilicityE#ase% solute penetra#ility into t&e #rain) AiiiB a meta#olic #arrier (&ic& serves as a #iotrans!ormation an% %eto"i!ication system) an% AivB a negligi#le pinocyE totic activity* T&e &ig& transen%ot&elial electrical resistance value A 5@@@ cm 5B measure% across t&e #rain capillary en%ot&elium is in%icative o! t&e very lo( ionic permea#ility* As a conseCuence) +++ accounts !or t&e restricte% CNS access o! more t&an >=[ o! all smallEmolecule %rugs an% o! 4@@[ o! largeEmolecule %rugs A9B*

+loo%H+rain +arrier Alteration in Pat&ology In various #rain pat&ologies) inclu%ing #rain trauma) stro$e) septic encep&alopat&y) or neuro%egenerative %iseases) or in meta#olic %isor%ers suc& as %ia#etes mellitus) t&e alterations o! t&e +++ permea#ility result in lea$age o! normally restraine% plasma components an% contri#ute to neuroin!lammation an% neuronal %amage A:B* It is still controversial (&et&er t&e +++ permea#ility increase results !rom t&e opening o! passage(ays t&roug& #rain en%ot&elial cells) %e!ine% as vesiculoEtu#uE lar systems or vesiculoEvacuolar organelles A;B) or !rom tig&t .unction %egra%ation A:B* ittle is $no(n on t&e %egree o! +++ permea#ili/ation in in!lammatory #rain pat&ologies) #ut its actual impact on nanoEDDS passive %i!!usion is li$ely to #e lo(* In a rat mo%el o! cere#ral isc&emia an% reper!usion) polystyrene nanoparticles A5@ nm in %iameterB (ere s&o(n to e"travasate into t&e #rain interstitial !lui% A<B* In t&e case o! larger nanoEDDSs suc& as liposomes) t&e upta$e #y in!iltrating

Nanoparticulate Systems !or Central Nervous System Drug Delivery

5=8

astrocyte !oot process

Pinocytotic vesicles

en%ot&elial cell
mitoc&on%ria

nucleus

#loo%
M

4
AET

A
o

o o

o o o o o o

5
CMT

EE oE o EoE o EEooEo o
o AMT

o o o

o o o

RMT o

C
3 3

3 3333

3 3 3 3

EE
o

RMT
3 3 3

E
AMT

9
o

RMT

3 3 3 3

t.

3 3

e"tracellular matri" pericyte

a"onal en%ing

-IGURE 4 Sc&ematic %iagram o! t&e neurovascular association !orming t&e +++ toget&er (it& t&e molecular transport pat&(ays across t&e +++ A4H9B an% propose% transcytotic #rainE%elivery mec&E anisms !or nanoEDDSs AAHDB* +rain en%ot&elial cells are c&aracteri/e% (it& tig&t .unctions At.B t&at loc$ t&e aCueous paracellular pat&(ays) negligi#le pinocytotic activity) &ig& transport) an% meta#olic activity* T&ey are in close relations&ip (it& pericytes) astrocyte !oot processes) a"onal en%ings) an% microglial cells Anot represente%B* Passive %i!!usion across t&e en%ot&elial cells is t&e passage(ay o! small lipop&ilic molecules AM( #elo( 9@@H:@@ DaB A4B* Restraine% #y tig&t .unctions an% t&e plasma mem#ranes) &y%rop&ilic compoun%s reCuire speciali/e% transport to reac& #rain6 CMT sysE tems !or small molecules A5B) RMT !or macromolecules A8B) or a#sorptiveEme%iate% transcytosis AAMTB !or cationic molecules A9B* Compoun%s traversing en%ot&elial cells may #e re.ecte% #ac$ into t&e #loo% compartment #y AET systems as intact molecules or as meta#olites AMB* Propose% tranE scytotic pat&(ays !or nanoEDDSs are AMT triggere% #y positively c&arge% sur!aces AAB or RMT trigE gere% #y natural su#strate ligan%s suc& as trans!errin A+B or pepti%omimetic anti#o%ies %irecte% against e"o!acial epitopes o! trans!errin or insulin receptors ACB* T&e &ypot&etic Q%i!!erential protein a%sorption mec&anismS accor%ing to (&ic& t&e nanoEDDS sur!ace (oul% a%sor#) !rom #loo%) natuE ral su#strates o! +++ receptors Ae*g*) apolipoproteins + or E) su#strates lo(E%ensity lipoprotein recepE torsB an% t&en un%ergo en%ocytosis1transcytosis is also represente% ADB* A##reviations6 AET) Active e!!lu" transportM AMT) a#sorptiveEme%iate transcytosisM +++) #loo%H#rain #arrierM CMT) carrierEme%iate% transportM DDSs) %rug %elivery systemsM RMT) receptorEme%iate% transcytosis*

macrop&ages is pro#a#ly t&e mec&anism !or t&eir increase% %istri#ution into in!lammatory #rain tissues A=B* In t&e case o! #rain tumors) t&e +++ integrity is locally compromise% #y t&e a#sence o! tig&t .unctions) allo(ing !or tumor core penetration an% retention o! %rugs) macromolecules) or nanoEDDS ot&er(ise e"clu%e% !rom normal #rain A>B* T&is p&enomenon is $no(n as t&e en&ance% permea#ility an% retention AEPRB e!!ect o! tumors* Generally) gro(ing tumor margins an% a%.acent normal tissue remain

5=9

?livier an% Pereira %e ?liveira

unreac&a#le* In e"perimental rat #rain tumors) t&e vascular pore cuto!! si/e %eterE mine% (it& longEcirculating liposomes or microsp&eres range% !rom 4@@ to ::@ nm) %epen%ing on t&e tumor cell line A4@B* +y optimi/ing t&e EPR e!!ect or #y %ecreasing perip&eral %istri#ution o! antitumor agents) nanoEDDS !ormulations (ere generally more e!!icient t&an solutions* In rats) %o"oru#icinEloa%e% nanoparticles or liposomes increase% %elivery to e"perimental #rain tumors A44)45B) lea%ing to &ig&er e!!iciency A45B* Do"oru#icin !ormulate% as pegylate% liposomes Amar$ete% un%er Do"il b or Caely"b tra%emar$sB s&o(e%) &o(ever) a mo%erately increase% e!!iciency against &ig&Egra%e gliomas in clinical practice) compare% to t&e solution A48B* Using con!ocal imaging) MR imaging) an% &istological e"amination o! e"perimental rat #rain tumors) Strau#inger et al* A49B s&o(e% t&at intravenously a%ministere% liposomes line% tumor capillaries or #loo% vessels an% poorly sprea% (it&in tumors* Un%er repeate% a%ministrations o! Do"ilb liposomes) e"tensive regions o! &emorr&age (it&in t&e tumor tissue occurre%) suggesting a %estruction o! tumor vasculature or un%erlying tumor cells* T&e opening o! t&e tumor stroma (oul% permit su#seCuent %oses o! liposomes to %i!!use more %eeply into tumors an% !inally e"ert t&eir cytoto"ic e!!ect on margins* Upta$e e!!iciency an% intracellular %elivery may #e improve% (it& tumorEspeci!ic nanoEDDSs) #ut t&e tumor cells t&at in!iltrate t&e normal #rain parenc&yma remain protecte% #y intact +++* T&e com#ination o! nanoEDDSs a#le to cross +++ (it& tumorEspeci!ic nanoEDDSs may #e a means to optimi/e antitumor t&erapy*

Can%i%ate Drugs !or +rainETargete% Delivery (it& NanoEDrug Delivery Systems Generally) smallEmolecule %rugs can #e c&emically %esigne% or mo%i!ie% Ae*g*) #y pro%rug synt&esisB to #e a%eCuately lipop&ilic !or passive %i!!usion t&roug& t&e +++ an% %o not nee% DDSs !or #rain %elivery* Ho(ever) %rug lipi%i/ation is not al(ays applica#le or e!!ective) especially (&en %rugs are particular c&emical entities t&at cannot #e mo%i!ie% (it&out losing t&eir p&armacological activities an%1or are su#strates o! e!!lu" transport proteins present at t&e +++) suc& as t&e PEglycoprotein* -urt&ermore) %rug lipi%i/ation generally results in increase% meta#olism an% perip&E eral %istri#ution) (&ic& necessitates &ig&er %oses) potentially at t&e cost o! more !reCuent a%verse reactions* In suc& cases) or (&en small %rug molecules un%ergo meta#oli/ation in #rain en%ot&elial cells) nanoEDDS !ormulations s&oul% #e consi%E ere% as a means !or improving #rain %elivery* Anot&er group o! molecules t&at necessitates nanoEDDS !ormulations are t&e ne( largeEmolecule t&erapeutics potenE tially e!!icient to treat CNS6 pepti%es) proteins) suc& as neurotrop&ic !actors) antisense %rugs) or genes Aplasmi%sB* ?(ing to t&eir poor sta#ility in #iological !lui%s) rapi% en/ymatic %egra%ation) un!avora#le p&armaco$inetic properties) an% lac$ o! %i!!uE sion to(ar%s t&e CNS) t&ey coul% #e a%vantageously !ormulate% in #rainEtargete% protective nanocontainers* Compare% to conventional %rugs) t&ey possess a &ig& intrinsic p&armacological activity* T&e small %ose nee%e% !or t&erapeutic e!!iciency (oul% easily !it t&e loa%ing capacity o! nanoEDDSs an% avoi% t&e a%ministration o! large amount o! potentially to"ic nanoEDDS e"cipients* T&e c&oice #et(een lipoE somes or nanoparticles (ill %epen% on !ormulation aspects) release $inetics) an% sta#ility upon storage* Proteins are generally unsta#le in t&e presence o! organic solvent or soli% inter!aces* It may) t&ere!ore) #e c&allenging to maintain t&eir #iologiE cal activities upon entrapment (it&in nanoparticles t&at nee% solvents Apolymeric NPB or &eating steps Alipi% NPB !or preparation* -ormulation a%%itives may improve t&eir sta#ility A4:B) or liposome !ormulations may #e more appropriate* Some %rugs

Nanoparticulate Systems !or Central Nervous System Drug Delivery

5=:

(ill reCuire release into t&e #rain interstitial !lui% to #e e!!ective* ?t&ers) suc& as plasmi% genes or antisense oligonucleoti%es) nee% to #e %elivere% intracellularly !or e!!ectiveness) (&ic& means t&at a!ter crossing t&e +++ nanoEDDS s&oul% #e also a#le to cross plasma mem#ranes o! targete% #rain cells*

NAN?EDRUG DE I2ER3 S3STEMS -?R N?NIN2ASI2E DRUG +RAIN DE I2ER3 +asic Principles Accor%ing to t&e p&armaco$inetic rule) t&e percent o! in.ecte% %ose o! a %rug t&at is %elivere% per gram #rain A[ID1gB is %irectly proportional to t&e +++ permea#ilityH sur!ace area APSB pro%uct an% t&e area un%er t&e plasma concentration curve AAUC) [ID min _ Z4B6 [ID1g ^ PS ` AUC A4B* T&is rule also stan%s !or nanoEDDS #rain %elivery* -or optimal e!!iciency) nanoEDDSs a%ministere% I2 s&oul% remain in t&e #loo% compartment) avoi%ing useless perip&eral %istri#ution) in or%er to reac& t&e #rain vasculature an% s&oul% possess an appropriate +++ PS in or%er to %eliver t&eir content #eyon% t&e +++ into t&e #rain parenc&yma* ?(ing to t&eir si/e) nanoE DDSs cannot cross t&e en%ot&elium o! #rain capillaries #y passive %i!!usion t&roug& normal +++* Alt&oug& e!!ective !or promoting liposome %elivery into t&e #rain parenc&yma A4;)4<B an% e"plore% !or improving antitumor %rug %i!!usion into #rain tumors A4=B) increasing +++ permea#ility using &yperosmolar solutions or t&e selecE tive +5 #ra%y$inin Cereport Ala#ra%imil or RMPE<B s&oul% #e limite% to s&ortEterm t&erapy) as t&is promotes t&e nonEselective entry o! #loo%E#orne compoun%s an% may result in sei/ures an% permanent neuropat&ological c&anges* ,it& preserving t&e +++ integrity) t&e %elivery o! nanoEDDSs into t&e #rain parenc&yma implies t&at nanoEDDSs s&oul% not only recogni/e speci!ic sites at t&e +++) #ut also trigger t&eir o(n trans!er across t&e en%ot&elial cells* In%ee% intravenously a%ministere% pegylate% immunoliposomes %irecte% against #rain astrocytes %isplaye% longE circulating properties Aelimination &al!Elives o! =H4: &oursB) #ut (ere una#le to cross t&e +++ an% misse% t&eir targets A4>B* ?n t&e #asis o! an alrea%y long researc&) #ot& steps are #eing rationally appre&en%e% an% $ey parameters are (ell %e!ine% !or liposomes* Ho(ever) %espite real ac&ievements) optimi/ation is still nee%e% in t&e case o! nanoparticles A5@B*

Circulating in t&e +loo% Compartment I! not especially %esigne% to escape !rom t&e MPS upta$e) intravenously a%minisE tere% nanoEDDSs are rapi%ly cleare% !rom t&e #loo% stream A#loo% &al!Elives are generally aroun% t(o to t&ree minutesB an% mostly accumulate in liver an% spleen* It is generally a%mitte% t&at opsoni/ation) t&e !irst step !or MPS recognition) is !avore% #y &y%rop&o#ic sur!aces) (&ic& promote protein a%sorption) an% negative sur!aces) (&ic& are activators o! t&e complement system A54B* In contrast) &y%roE p&ilic coating sterically sta#ili/es nanoEDDSs an% re%uces opsoni/ation an% MPS upta$e* T&ese rat&er simplistic vie(s s&oul%) &o(ever) #e tempere%) as recent evi%ence s&o(e% t&at sterically sta#ili/e% particles (ere e!!iciently opsoni/e% an% activate% t&e complement system) (&ile $eeping t&eir stealt& properties A55B* ,&atever t&e mec&anism may #e) t&e incorporation o! polyet&ylene glycol APEGB %erivati/e% lipi%s into t&e p&osp&olipi%s #ilayer o! liposomes resulte% in longE circulating liposomes) generally re!erre% to as sterically sta#ili/e% liposomes) (it& apparent terminal &al!Elives up to >@ &ours in &umans A58B* ongEcirculating nanoparE ticles are &o(ever still elusive* P&armaco$inetic stu%ies are o!ten contra%ictory) i! not

5=;

?livier an% Pereira %e ?liveira

lac$ing* In t&e case o! pegylate% polyAlacti%eB or polyAlacti%eEcoEglycoli%eB nanoparE ticles) t&e upEtoE%ate most stu%ie% polymeric nanoEDDSs) sur!ace coating (it& PEG molecular (eig&ts o! :@@@ or over) (ere s&o(n to #e e!!icient at re%ucing opsoni/aE tion) #ut #loo% &al!Elives (ere !oun% to #e very varia#le ranging !rom less t&an one to si" &ours A5@B* T&e varia#ility in %ensityErelate% PEG con!ormation may e"plain t&e rapi% clearance o! a signi!icant !raction o! intravenously in.ecte% QlongE circulatingS nanoparticles #y t&e MPS A55B* Contrary to liposomes) t&e variety in nanoparticle core nature Apolymers or lipi%sB t&at c&aracteri/es t&e researc& on nanoparticles resulte% in t&e %ispersion o! researc& e!!ort (&ic& (as pro#a#ly at t&e origin o! t&e limite% ac&ievements in t&e longEcirculating nanoparticle %esign*

NanoEDrug Delivery System Transport T&roug& +rain En%ot&elial Cells T&e reasone% approac&es presently un%er investigation !or triggering nanoEDDS translocation across +++ are #ase% on AMT or RMT A-ig* 4B* Une"pecte%ly) several researc& teams !oun% t&at sur!actantEcoate% nanoparticles %elivere% t&eir contents to t&e #rain* As t&ey (ere not #ase% on t&eoretical concept) t&ese met&o%s (ere re!erre% to as QempiricalS approac&es* A#sorptiveEMe%iate% Transcytosis Cationi/e% proteins Aal#umin) immunoglo#ulinsB &ave #een %emonstrate% to un%ergo a#sorptiveEme%iate% en%ocytosis AAMEB t&roug& t&e +++ in vivo A59B* AME is triggere% #y an electrostatic interaction #et(een t&e positively c&arge% protein an% t&e negatively c&arge% plasma mem#ranes* It is t&ere!ore not #rainE speci!ic) an% cationi/e% proteins also accumulate in liver) $i%ney) an%1or lung tissues* Ho(ever) t&e relatively &ig& capacity o! AME s&oul% #e !avora#le to #rain %elivery A5:B* Preservation o! appropriate p&armaco$inetic pro!iles %epen%s on t&e %egree o! cationi/ation A59B* As an application o! suc& a concept) cationic #ovine serum al#uminEcon.ugate% pegylate% nanoEDDSs Nliposomes A5;B an% pegylate% polylacti%e nanoparticles A5<BO (ere s&o(n to un%ergo en%ocytosis in in vitro +++ mo%el* It is still to #e %emonstrate% (&et&er t&ese cationic nanoEDDSs (ill possess appropriate p&armaco$inetic pro!iles #y t&e intravenous a%ministration route*

ReceptorEMe%iate% Transcytosis ?(ing to a &ig& stereospeci!icity !or t&eir su#strate) CMT reCuires %rugs (it& molecular structures mimic$ing t&e en%ogenous nutrient an% is not a realistic option !or nanoEDDS #rain %elivery A4)5:B* ?nly RMT may #e use% !or transcytotic %elivery o! nanoEDDSs t&roug& t&e +++* ?(ing to t&e u#iCuity o! RMT) relative #rain speciE !icity may #e ac&ieve% #y targeting receptors t&at are overe"presse% at t&e luminal si%e o! #rain en%ot&elial cells compare% to ot&er organs) !or e"ample) t&e receptors o! trans!errin) insulin) insulinEli$e gro(t& !actor AIG-B) or lo(E%ensity lipoproteins A D B A9B* NanoEDDSs s&oul% #e less t&an 4@@ nm in %iameter to !it t&e loa%ing capacity o! t&ese transport systems an% s&oul% &ave on t&eir sur!ace receptorE recogni/ing ligan% Anatural su#strate or anti#o%yB capa#le o! triggering transcytoE sis* Trans!errin ligan% (as investigate% !or liposome #rain targeting (it& promising results A5=B) #ut an apolipoprotein EE%erive% pepti%e !aile% to trigger liposome en%ocytosis via D receptor A5>B* Anti#o%ies %irecte% against e"o!acial receptor epitopes t&at %o not inter!ere (it& t&e natural ligan%E#in%ing sites s&oul% #e preE !erre% in or%er to avoi% potential competition #et(een targete% nanoEDDSs an% en%ogenous natural ligan% Atrans!errin) apolipoproteinsB) p&armacological activity

Nanoparticulate Systems !or Central Nervous System Drug Delivery

5=<

AinsulinB) or lin$age to plasmaE#in%ing proteins AIG-B A9B* T&e most a%vance% (or$ #ase% on t&is QTro.an &orseS concept &as #een carrie% out #y Par%ri%ge an% co(or$E ers (it& pegylate% immunoliposomes A9B* T&e receptorEspeci!ic targeting ligan%s locate% at t&e tip o! 4[ to 5[ o! t&e PEG 5@@@ stran%s are pepti%omimetic monoclonal anti#o%ies a#le to trigger t&e activation o! trans!errin or insulin receptors* A!ter intravenous a%ministration) t&ese immunoliposomes %elivere% t&eir content AsmallE %rug molecules) plasmi%sB into t&e #rain parenc&yma (it&out %amaging t&e +++ A8@H88B* ?(ing to t&e presence o! trans!errin or insulin receptors on t&e neuronal plasma mem#rane) plasmi%Eloa%e% immunoliposomes permitte% neuronal nuclear %elivery) resulting in gene e"pression in t&e entire #rain space A84H88B* As RMT receptors are also a#un%ant in some perip&eral tissues) ectopic e"pression in non#rain organs (as eliminate% using a #rainEspeci!ic gene promoter A88B* In a ;E&y%ro"y%opamine rat mo%el o! Par$insonPs %isease) t&e selective gene e"pression in nigrostriatal neurons resulte% in t&e restoration o! tyrosine &y%ro"ylase activity an% in reversal o! motor impairment A88B* As immunoliposomes %eliver t&eir conE tent Cuic$ly) as s&o(n #y t&e relatively s&ortElasting plasmi% e"pression in #rain A88B) t&ey (oul% reCuire mont&ly a%ministrations to sustain a p&armacological e!!ect A84B* T&is potential inconvenience !or t&e treatment o! c&ronic #rain %isor%ers may #e correcte% using pegylate% polyAlacti%eB immunonanoparticles (it& sustaine%Erelease properties A5@)89B*

Empirical Approac&es 2arious (or$s reporte% on t&e #ene!icial e!!ect o! sur!actantEcoate% lipi% or polyE meric nanoparticles !or t&e #rain %elivery o! %rugs* No #rainEtargeting ligan% is present on nanoparticle sur!ace an% t&e various mec&anisms un%erlying increase% #rain upta$e o! entrappe% compoun%s remain &ypot&etic* Soli%H ipi% Nanoparticles Soli%Hlipi% nanoparticles are #asically constitute% o! a liCui% or soli%Hlipi% core AliCui% glyceri%e or (a"B surroun%e% #y a soli% s&ell ma%e o! an association o! several anionic sur!actants Apolysor#ates) PEG !atty aci% ester) PEG !atty alco&ol et&er) polo"amersB an%1or ionic sur!actants Alecit&in) etc*B an% %o not AgenerallyB necessitate organic solvents !or t&eir preparation A8:B* E"perimental %emonstration o! S N Qstealt&nessS is scarce an% more o!ten #ase% on t&e presuppose% antiopE sonic e!!ect o! PEGylate% sur!actant rat&er t&an on e"perimental evi%ence A8;B* Several e"perimental (or$s s&o(e% t&at sur!actantEcoate% soli%Hlipi% nanopartiE cles coul% increase t&e #rain upta$e o! loa%e% %rugs t&at poorly %i!!use into t&e #rain (&en a%ministere% as solutions* A pro%rug o! %ioctanoyl!luoro%eo"yuri%ine &a% a signi!icantly &ig&er #rain %istri#ution (&en a%ministere% as pluronic -;=Ecoate% soli%Hlipi% nanoparticle !ormulations compare% to t&e solution A8<B* T&e aut&ors propose% as #rain upta$e mec&anisms eit&er an increase in gra%ient concentration #et(een #loo% an% #rain resulting !rom a &ig&er plasma AUC Acompare% to t&e %rug solutionB) or an en%ocytosis #y en%ot&elial cells* In ot&er stu%ies) intravenously a%ministere% to rats or ra##its) %o"oru#icin Qstealt&S S N coate% (it& PEG5@@@ stearate also increase% #rain upta$e compare% to t&e commerE cial solution A8;)8=B* In an in situ rat #rain per!usion mo%el) paclita"el #rain upta$e (as increase% (&en !ormulate% in (a" nanoparticles inclu%ing polysor#ate ;@ an% +ri. <= as sur!actants) compare% to t&e control solution A8>B* Control nanoparticles (ere s&o(n to un%ergo a signi!icant #rain upta$e A9@B (it&out altering +++ permeE a#ility A94)95B* Une"pecte%ly) nanoparticle translocation t&roug& t&e +++ (as not

5==

?livier an% Pereira %e ?liveira

retaine% as t&e mec&anism !or increase% paclita"el #rain upta$e) an% t&e aut&ors !avore% a mo%ulation o! paclita"el e!!lu" #y PEglycoprotein transporter A8>B* Poly#utylcyanoacrylate Nanoparticles A%sor#e% onto polysor#ateEcoate% poly#utylcyanoacrylate AP+CAB nanoparticles a%ministere% intravenously) compoun%s (it& poor #rain %i!!usion A%o"oru#icin) loperami%e) tu#ocurarine) t&e &e"apepti%e %alarginB (ere success!ully %elivere% to t&e #rain (&ere t&ey in%uce% p&armacological e!!ects A!or revie() see Re!* 98B* As t&ese nanoparticles %o not possess targeting ligan%s on t&eir sur!ace) t&e conE cept o! Q%i!!erential protein a%sorptionS (as propose% #y MJller A99B* Accor%ing to t&is concept) some #loo% proteins pre!erentially a%sor# onto nanoparticle surE !aces %epen%ing on t&eir p&ysicoc&emical properties* Apolipoproteins + an% E (oul% #e t&e #rainEtargeting proteins t&at a%sor# onto polysor#ateEcoate% nanoparticles an% permit nanoparticle en%ocytotic %elivery into t&e #rain vessel en%ot&elia via D receptors A9:B* T&is mec&anism may also account !or polysorE #ate =@Ecoate% polyAlacti%eB nanoparticle interaction (it& #rain microvessels A9;B* Ho(ever) it (as s&o(n t&at in rats treate% (it& polysor#ate =@Ecoate% P+CA nanoparticles Apolysor#ate =@6 5: mg1$g) nanoparticles6 :@ mg1$gB inulin spaces increase% #y 4@[ Anonsigni!icantB a!ter 4@ minutes an% #y >>[ Asigni!icantB a!ter 9: minutes compare% to controls A9<B* A nanoparticleEin%uce% nonspeci!ic +++ permea#ili/ation) resulting !rom t&e synergistic e!!ect o! nanoparticle to"icity an% &ig& polysor#ate =@ concentrations) (as propose% as an alternative mec&anism to t&e #rain translocation o! nanoparticles across t&e +++ A9=B*

C?NC USI?N Tec&nology no( e"ists !or %esigning sa!e #rainEtargete% longEcirculating nanoE DDSs* T&e PEGylate% immunoliposomes #ase% on t&e concept o! molecular QTro.an &orseS t&at targets t&e transcytotic +++ receptors an% !erries t&e liposomes t&oug& t&e +++ permitte% t&e e!!icient noninvasive) nonviral %elivery o! plasmi% genes into t&e #rain parenc&yma cells* T&ey constitute t&e present most convincing %emonE stration o! success!ul #rain %elivery o! comple" macromolecules #y nanoEDDSs !rom t&e #loo% circulation t&roug& t&e +++* T&e empirical use o! simpler polymeric or lipi% nanoparticles also resulte% in promising results* T&e mec&anisms un%erlyE ing t&eir still &ypot&etic translocation t&roug& t&e +++ nee% !urt&er investigations !or vali%ation* T&us) %espite t&e comple"ity o! t&e issue) t&ere &ave #een real ac&ievements in nanoEDDS #rain %elivery) #ut t&e (ay is still long !rom #enc& results to clinical applications*

RE-ERENCES
4* Par%ri%ge ,M* T&e #loo%H#rain #arrier6 #ottlenec$ in #rain %rug %evelopment* NeuroR" 5@@:M 568* 5* +enoit 'P) -aisant N) 2enierE'ulienne MC) Menei P* Development o! microsp&eres !or neurological %isor%ers6 !rom #asics to clinical applications* ' Control Release 5@@@M ;:65=:* 8* Guerin C) ?livi A) ,eingart 'D) a(son HC) +rem H* Recent a%vances in #rain tumor t&erapy6 local intracere#ral %rug %elivery #y polymers* Invest Ne( Drugs 5@@9M 5565<* 9* Par%ri%ge ,M* +rain Drug Targeting6 T&e -uture o! +rain Drug Development* Cam#ri%ge University Press) 5@@4* :* Petty MA) o EH* 'unctional comple"es o! t&e #loo%H#rain #arrier6 permea#ility c&anges in neuroin!lammation* Prog Neuro#iol 5@@5M ;=6844*

Nanoparticulate Systems !or Central Nervous System Drug Delivery

5=>

;* ossins$y AS) S&ivers RR* Structural pat&(ays !or macromolecular an% cellular transE port across t&e #loo%H#rain #arrier %uring in!lammatory con%itions* Histol Histopat&ol 5@@9M 4>6:8:* <* 3ang CS) C&ang CH) Tsai P') C&en ,3) Tseng -G) o ,* NanoparticleE#ase% in vivo investigation on #loo%H#rain #arrier permea#ility !ollo(ing isc&emia an% reper!usion* Anal C&em 5@@9M <;699;:* =* Rousseau 2) Deni/ot +) e 'eune '') 'allet P* Early %etection o! liposome #rain locali/ation in rat e"perimental allergic encep&alomyelitis* E"p +rain Res 4>>>M 45:A8B65::H5;9* >* Papa%opoulos MC) Saa%oun S) +in%er D0) Manley GT) 0ris&na S) 2er$man AS* Molecular mec&anisms o! #rain tumor e%ema* Neuroscience 5@@9M 45>A9B64@44H4@5@* 4@* Ho##s S0) Mons$y , ) 3uan -) et al* Regulation o! transport pat&(ays in tumor vessels6 role o! tumor type an% microenvironment* Proc Natl Aca% Sci USA 4>>=M >:A=B69;@<H9;45* 44* +rigger I) Mori/et ') Au#ert G) et al* PolyAet&ylene glycolBEcoate% &e"a%ecylcyanoacrylate nanosp&eres %isplay a com#ine% e!!ect !or #rain tumor targeting* ' P&armacol E"p T&er 5@@5M 8@8A8B6>5=H>8;* 45* Steiniger SC) 0reuter ') 0&alans$y AS) et al* C&emot&erapy o! glio#lastoma in rats using %o"oru#icinEloa%e% nanoparticles* Int ' Cancer 5@@9M 4@>6<:>* 48* Hau P) -a#el 0) +aumgart U) et al* Pegylate% liposomal %o"oru#icinEe!!icacy in patients (it& recurrent &ig&Egra%e glioma* Cancer 5@@9M 4@@A;B644>>H45@<* 49* Strau#inger RM) Arnol% RD) V&ou R) Ma/urc&u$ R) Slac$ 'E* Antivascular an% antitumor activities o! liposomeEassociate% %rugs* Anticancer Res 5@@9M 59A5AB68><H9@9* 4:* Sc&(en%eman SP* Recent a%vances in t&e sta#ili/ation o! proteins encapsulate% in in.ectE a#le P GA %elivery systems* Crit Rev T&er Drug Carrier Syst 5@@5M 4>6<8* 4;* Sa$amoto A) I%o T* iposome targeting to rat #rain6 e!!ect o! osmotic opening o! t&e #loo%H#rain #arrier* +rain Res 4>>8M 5;M ;5>A4B64<4H4<:* 4<* Wie 3) 3e ) V&ang W) et al* Transport o! nerve gro(t& !actor encapsulate% into liposomes across t&e #loo%H#rain #arrier6 in vitro an% in vivo stu%ies* ' Control Release 5@@:M 4@:A4H5B64@;H44>* 4=* 0emper EM) +ooger% ,) T&uis I) +ei.nen 'H) van Tellingen ?* Mo%ulation o! t&e #loo%H #rain #arrier in oncology6 t&erapeutic opportunities !or t&e treatment o! #rain tumoursT Cancer Treat Rev 5@@9M 8@A:B694:H958* 4>* C&e$&onin 2P) V&ir$ov 3A) Gurina ?I) et al* PEGylate% immunoliposomes %irecte% against #rain astrocytes* Drug Deliv 5@@:M 45A4B64H;* 5@* ?livier 'C* Drug transport to #rain (it& targete% nanoparticles* NeuroR" 5@@:M 564@=* 54* Mog&imi SM) Hunter AC) Murray 'C* ongEcirculating an% targetEspeci!ic nanoparticles6 t&eory to practice* P&armacol Rev 5@@4M :865=8* 55* Mog&imi SM) S/e#eni '* Stealt& liposomes an% long circulating nanoparticles6 critical issues in p&armaco$inetics) opsoni/ation an% proteinE#in%ing properties* Prog ipi% Res 5@@8M 9569;8* 58* Ga#i/on A) S&mee%a H) +aren&ol/ 3* P&armaco$inetics o! pegylate% liposomal %o"oruE #icin6 revie( o! animal an% &uman stu%ies* Clin P&armaco$inet 5@@8M 95694>* 59* +ic$el U) 3os&i$a(a T) Par%ri%ge ,M* Delivery o! pepti%es an% proteins t&roug& t&e #loo%H#rain #arrier* A%v Drug Deliv Rev 5@@4M 9;659<* 5:* Tsu.i A) Tamai II* CarrierEme%iate% or speciali/e% transport o! %rugs across t&e #loo%H #rain #arrier* A%v Drug Deliv Rev 4>>>M 8;A5H8B65<<H5>@* 5;* T&ole M) No#manna S) Hu(yler ') +artmann A) -ric$er G* Upta$e o! cationi/ie% al#umin couple% liposomes #y culture% porcine #rain microvessel en%ot&elial cells an% intact #rain capillaries* ' Drug Target 5@@5M 4@A9B688<H899* 5<* u ,) Tan 3V) Hu 0 ) 'iang WG* Cationic al#umin con.ugate% pegylate% nanoparticle (it& its transcytosis a#ility an% little to"icity against #loo%H#rain #arrier* Int ' P&arm 5@@:M 5>:A4H5B659<H5;@* 5=* ?mori N et al* Targeting o! postEisc&emic cere#ral en%ot&elium in rat #y liposomes #earing polyet&ylene glycolEcouple% trans!erring* Neurol Res 5@@8M 5:65<:* 5>* Sauer I et al* An apolipoprotein EE%erive% pepti%e me%iates upta$e o! sterically sta#ili/e% liposomes into #rain capillary en%ot&elial cells* +ioc&emistry 5@@:M 9965@54* 8@* Hu(yler ') ,u D) Par%ri%ge ,M* +rain %rug %elivery o! small molecules using immunoliposomes* Proc Natl Aca% Sci USA 4>>;M >86494;9H494;>* 84* Sc&lac&et/$i - et al* Gene t&erapy o! t&e #rain6 t&e transEvascular approac&* Neurology 5@@9M ;5645<:*

5>@

?livier an% Pereira %e ?liveira

85* V&ang 3) Sc&lac&et/$i -) Par%ri%ge ,M* Glo#al nonEviral gene trans!er to t&e primate #rain !ollo(ing intravenous a%ministration* Mol T&er 5@@8M <644* 88* V&ang 3) Sc&lac&et/$i -) V&ang 3-) +oa%o R') Par%ri%ge ,M* Normali/ation o! striatal tyrosine &y%ro"ylase an% reversal o! motor impairment in e"perimental par$insonism (it& intravenous nonviral gene t&erapy an% a #rainEspeci!ic promoter* Hum Gene T&er 5@@9M 4:A9B688>H8:@* 89* ?livier 'C) Huertas R) ee H') Calon -) Par%ri%ge ,M* Synt&esis o! pegylate% immunonE anoparticles* P&arm Res 5@@5M 4>6448<H4498* 8:* Me&nert ,) Ma%er 0* Soli%Hlipi% nanoparticles6 pro%uction) c&aracteri/ation an% applications* A%v Drug Deliv Rev 5@@4M 9<64;:* 8;* -un%aro A) Cavalli R) +argoni A) 2ig&etto D) Vara GP) Gasco MR* NonEstealt& an% stealt& lipi% nanoparticles AS NB carrying %o"oru#icin6 p&armaco$inetics an% tissue %istri#uE tion a!ter i*v* a%ministration to rats* P&armacol Res 5@@@M 95A9B688<H898* 8<* ,ang 'W) Sun W) V&ang VR* En&ance% #rain targeting #y synt&esis o! 8u):uE%ioctanoylE:E !luoroE5uE%eo"yuri%ine an% incorporation into soli%Hlipi% nanoparticles* Eur ' P&arm +iop&arm 5@@5M :965=:* 8=* Vara GP) Cavalli R) +argoni A) -un%aro A) 2ig&etto D) Gasco MR* Intravenous a%minisE tration to ra##its o! nonEstealt& an% stealt& %o"oru#icinEloa%e% soli%Hlipi% nanoparticles at increasing concentrations o! stealt& agent6 p&armaco$inetics an% %istri#ution o! %o"oru#icin in #rain an% ot&er tissues* ' Drug Target 5@@5M 4@A9B685<H88:* 8>* 0o/iara 'M) oc$man PR) Allen DD) Mumper R'* Paclita"el nanoparticles !or t&e potential treatment o! #rain tumors* ' Control Release 5@@9M >>A5B65:>H5;>* 9@* 0o/iara 'M) oc$man PR) Allen DD) Mumper R'* In situ #loo%H#rain #arrier transport o! nanoparticles* P&arm Res 5@@8M 5@A44B64<<5H4<<=* 94* oc$man PR) 0o/iara 'M) Mumper R') Allen DD* Nanoparticle sur!ace c&arges alter #loo%H#rain #arrier integrity an% permea#ility* ' Drug Target 5@@9M 45A>H4@B6;8:H;94* 95* oc$man PR) 0o/iara ') Ro%er 0E) et al* In vivo an% in vitro assessment o! #aseline #loo%H#rain #arrier parameters in t&e presence o! novel nanoparticles* P&arm Res 5@@8M 5@A:B6<@:H<48* 98* 0reuter '* Nanoparticulate systems !or #rain %elivery o! %rugs* A%v Drug Deliv Rev 5@@4M 9<6;:H=4* 99* MJller RH) 0ec$ CM* Drug %elivery to t&e #rainRreali/ation #y novel %rug carriers* ' Nanosci Nanotec&nol 5@@9M 969<4* 9:* 0reuter ' et al* ApolipoproteinEme%iate% transport o! nanoparticleE#oun% %rugs across t&e #loo%H#rain #arrier* ' Drug Target 5@@5M 4@684<* 9;* Sun ,) Wie C) ,ang H) Hu 3* Speci!ic role o! polysor#ate =@ coating on t&e targeting o! nanoparticles to t&e #rain* +iomaterials 5@@9M 5:A4:B68@;:H8@<4* 9<* Alyau%tin RN) Reic&el A) o#en#erg R) Ramge P) 0reuter ') +egley D'* Interaction o! polyA#utylcyanoacrylateB nanoparticles (it& t&e #loo%H#rain #arrier in vivo an% in vitro* ' Drug Target 5@@4M >65@>H554* 9=* ?livier 'C) -enart ) C&auvet R) Pariat C) Cecc&elli R) Couet ,* In%irect evi%ence t&at %rug #rain targeting using polysor#ate =@Ecoate% poly#utylcyanoacrylate nanoparticles is relate% to to"icity* P&arm Res 4>>>M 4;A45B64=8;H4=95*

4=

Nanoparticles !or Gene Delivery6 -ormulation C&aracteristics

'aspreet 0* 2asir an% 2ino% a#&aset(ar
Department o! P&armaceutical Sciences) University o! Ne#ras$a Me%ical Center) ?ma&a) Ne#ras$a) U*S*A*

INTR?DUCTI?N T&e recent a%vances in t&e !iel% o! molecular #iology &ave &ig&lig&te% gene t&erapy as a promising approac& !or t&e treatment o! numerous %iseases inclu%ing genetic %isor%ers) suc& as cystic !i#rosis) &emop&iliaM many somatic %iseases suc& as tumors) neuro%egenerative %iseasesM an% severe viral in!ections suc& as AIDS* Despite t&e consi%era#le interest generate% in gene t&erapy an% t&e p&enomenal pace at (&ic& researc& &as a%vance%) Q%eliveryS o! genes to t&e target cells still remains t&e most !ormi%a#le c&allenge* Polynucleoti%e molecules Ae*g*) DNA or RNAB are large) &y%rop&ilic) macromolecules (it& a net negative c&arge* Unli$e ot&er %rugs) t&ese are very la#ile in t&e #iological environment an% %o not cross #iological mem#ranes e!!ectively* T&us) t&e nee% !or an e!!ective an% sa!e geneE%elivery system is Cuite o#vious* In general) gene %elivery1e"pression vectors can #e #roa%ly categori/e% into t&e viral an% nonviral vectors* 2iral vectors inclu%e t&e use o! genetically engineere% retroviruses) a%enoviruses) a%enoEassociate% viruses) an% ot&er viruses t&at &ave #een use% !or geneEtrans!er proce%ures* Alt&oug& t&e viruses are &ig&ly e!!icient !or gene trans!er to cells) t&eir pote ntial to in%uce %rastic immune responses suc& as (it& a%enE ovirus or t&e ris$ o! insertional mutagenesis in t&e &ost genome (it& retroviral vectors A4B &as spar$e% a ma.or %e#ate over t&eir sa!ety !or &uman gene t&erapy* T&us) t&e current consensus is to %evelop suita#le vector systems (&ic& are minimally invasive Asa!eB an% &ig&ly e!!icient !or gene t&erapy in &umans* T&is &as steere% researc& to(ar%s t&e %evelopment o! nonviral vectors !or gene %elivery* Nonviral vectors inclu%e nanoparticles ANPsB) liposomes) an% comple"es prepare% eit&er using cationic lipi%s Alipople"esB or polymers Apolyple"esB) an% also mec&aniE cal met&o%s suc& as electroporation or micronee%le in.ections o! plasmi%) especially !or trans!ection t&roug& t&e s$in sur!ace* Among polymeric gene e"pression systems) #io%egra%a#le NPs o!!er certain a%vantages !or gene %elivery* T&ese can #e !orme% #y encapsulating DNA into polymers or #y comple"ing DNA to t&e sur!ace o! pre!orme% NPs A-ig* 4B* Plasmi% DNA can #e encapsulate% in polymeric NPs AiB alone as na$e% plasmi% DNA A5)8B) AiiB con%ense% (it& some cationic polymers A9B) or AiiiB in noncon%ense% !orm (it& some protective e"cipients A:B* Polynucleoti%e molecules can also #e comple"e% (it& positively c&arge% entities Acationic sur!actants or polysacc&ari%esB gra!te% on t&e sur!ace o! pre!orme% polymeric NPs A;B* T&us) t&ere is a great %egree o! !le"iE #ility in %eveloping #io%egra%a#le NPs as gene e"pression vectors*

P? 3A ACTIC ACIDB1P? 3AD) E ACTIDEEC?EG 3C? IDEBE+ASED NPS PolyAlactic aci%B AP AB an% polyA%)lElacti%eEcoEglycoli%eB AP GAB are t&e #io%eE gra%a#le an% #iocompati#le polymers use% !or !ormulating NPs an% are approve%
5>4

5>5

2asir an% a#&aset(ar

P?

3 M E R IC N A N ? P A R T IC -?R G E N E D E I2 E R 3

ES

C?NDENSE DNA ,ITH P? 3MERS AP? 3P EWESB

ENCAPSU ATE DNA IN P? 3MERIC MAT RIW ?R RESER2?IR

C?MP EW DNA T? THE SUR-ACE ?P? 3MERIC NAN?PARTIC ES

P? 3 ETH3 ENIMINE P? 3E E 3SINE P? 3 AMID?AMINE P? 3 f AMIN? ESTERS CATI?NIC DENDRIMERS CHIT?SAN

P? 3 ACTIC ACID P? 3 ACTICEC?EG 3C? IC ACID P? 3 f AMIN? ESTERS CHIT?SAN

P? 3 A 03 C3AN?ACR3 ATE P? 3 ACTIC ACID P? 3 ACTICEC?EG 3C? IC ACID CHIT?SAN AGra!te% (it& cationic polysacc&ari%es or sur!actantsB

E EE EEo E
DNA

o o o o

o o o o

EE E Eo EE
Polyple" Matri" Reservoir

EE E E oE oE
o

o o o o

oE

Polymer

o Cationic sur!actant

EE E E E

-IGURE 4 Sc&ematic representation s&o(ing %i!!erent types o! polymeric nanoparticles !or gene %elivery*

!or &uman use #y t&e U*S* -oo% an% Drug A%ministration* P A1P GA NPs (it& entrappe% plasmi% DNA are o! special interest !or gene %elivery %ue to t&eir nonE viral an% nonimmunogenic nature* Plasmi% DNA is entrappe% into t&e polymeric matri") (&ic& not only protects DNA !rom nucleases) #ut also allo(s a control over t&e DNAErelease $inetics !rom NPs* T&e main a%vantage o! t&ese NPs is t&e slo( release o! DNA !rom t&e NPs (&ic& !acilitates sustaine% levels o! gene e"pression* Moreover) t&e %uration an% levels o! gene e"pression can #e easily mo%ulate% #y altering !ormulation parameters suc& as DNA6polymer ratio) or polymer molecular (eig&t) an% composition* P GA NPs are generally !ormulate% using Q(aterEinEoilEinE(aterS %ou#leE emulsion solvent evaporation tec&niCue) using polyvinyl alco&ol AP2AB as an emulE si!ier A5B* In #rie!) an aCueous solution o! plasmi% DNA is emulsi!ie% into t&e polymer solution in organic solvents Ausually c&loro!orm or met&ylene c&lori%eB eit&er #y &ig&Espee% &omogeni/ation or sonication* T&is (aterEinEoil emulsion is t&en mi"e% into a secon% aCueous p&ase Ausually an aCueous solution o! sur!actant1 emulsi!ier) typically P2A is use%B* T&e %ou#le (aterEinEoilEinE(ater emulsion is t&en !orme% using sonication or &omogeni/ation* T&e organic solvent is t&en allo(e% to evaporate #y stirring t&e emulsion at room temperature !or appro"imately 4= to 5@ &ours* T&is results in t&e !ormation o! polymeric NPs (it& entrappe% plasmi% DNA (&ic& are recovere% #y ultracentri!ugation) (as&e% (it& %istille% (ater to remove P2A an% unentrappe% DNA* T&e NPs are suspen%e% in (ater an% lyop&ili/e% to !orm a %ry po(%er* NPs prepare% using t&is proce%ure are usually aroun% 4@@ nm in %iameter an% &ave a negative /eta potential A<B* DNA loa%ing in t&ese NPs &as

Nanoparticles !or Gene Delivery

5>8

#een reporte% aroun% @*:[ to 5*:[ A(1(B A8B* T&e e!!iciency o! DNA encapsulation using t&is process %epen%s on t&e polymer composition) molecular (eig&t o! t&e polymer) an% t&e nature an% concentration o! emulsi!ier use% in t&e process A5B* T&ere are some concerns regar%ing t&e sta#ility o! DNA %uring t&e encapsulation process) %ue to t&e use o! &ig&Es&ear !orces Agenerate% using &ig&Espee% &omogeniE /ation or sonicationB an% e"posure o! DNA to t&e organic solvents A=)>B* T&ese conE %itions may result in t&e trans!ormation o! DNA !rom its supercoile% !orm to t&e openEcircular or linear !orms* Preservation o! t&e structural integrity o! DNA is important) (&ereas t&e supercoile% !orm &as t&e &ig&est #ioactivity an% t&e linear !orm is least active A4@B* Stu%ies &ave %emonstrate% t&at t&ere is only a minimal loss o! activity o! DNA !ollo(ing nanoencapsulation as t&e DNA is mostly present in t&e supercoile% or openEcircular !orms in t&e NPs A44B* A cryopreparation met&o% !or t&e microencapsulation o! plasmi% DNA &as #een %escri#e%) to re%uce t&e loss o! t&e supercoile% !orm o! DNA %uring !ormuE lation an% to improve t&e encapsulation o! DNA A45B* T&is met&o% involves !ree/ing t&e aCueous p&ase o! t&e primary emulsion containing t&e plasmi% DNA #e!ore su#.ecting it to &omogeni/ation* T&is &as #een s&o(n to preserve t&e content o! t&e supercoile% !orm o! DNA) as DNA !ro/en in t&e aCueous p&ase is e"pose% to miniE mum s&ear stress* A%%ition o! sacc&ari%es as cryoprotectants in t&e primary emulE sion can also prevent t&e structural loss o! DNA* Sacc&ari%es prevent crystal !ormation !rom t&e #u!!er salts Apresent in t&e primary emulsionB %uring lyop&iliE /ation an% t&us conseCuently prevent nic$ing o! DNA #y suc& salt crystals* ?ster et al* A48B &ave %escri#e% a gentle solvent %isplacement met&o%) (it&out t&e use o! &ig&Espee% &omogeni/ation !or t&e encapsulation o! DNA* A ne( class o! #io%egra%a#le polymers consisting o! amineEmo%i!ie% P2A #ac$#one gra!te% (it& P GA si%e c&ains &as #een use% in t&is proce%ure* T&e tertiary amino groups in t&e polymer #ac$#one interact (it& DNA #y electrostatic interactions an% !acilitate NP !ormation %ue to t&eir amp&ip&ilic c&aracter* T&ese NPs e"&i#ite% positive /eta potential an% &ig& trans!ection e!!iciencies in cell culture compara#le to polyet&ylenimineHDNA comple"es* T&e &ig& trans!ection e!!iciency may #e %ue to t&e rapi% polymer %egra%ation rates an% t&e preservation o! DNA integrity an% #ioactivity %uring t&e NP !ormulation proce%ure* ?(ing to t&eir small si/e) P GA NPs are ta$en up #y cells Amainly #y en%ocyE tosisB an% !acilitate intracellular %elivery o! plasmi% DNA* As NPs come in %irect contact (it& t&e cell mem#ranes) t&e sur!ace properties o! NPs are critical in %etermining t&eir intracellular !ate an% can potentially in!luence t&e gene trans!ecE tion e!!iciencies A-ig* 5B A5B* T&ese inclu%e t&e sur!aceEassociate% P2A in NPs) &y%roE p&ilicity) an% t&e sur!ace c&arge A/eta potentialB o! NPs* It &as #een s&o(n t&at !or NPs !ormulate% using t&e %ou#leEemulsion solvent evaporation) a !raction o! P2A use% in t&e !ormulation remains associate% (it& t&e NP sur!ace) an% cannot #e remove% even #y multiple (as&ings A49B* T&is resi%ual P2A on NP sur!ace can alter its p&ysical properties an% a!!ect t&e cellular upta$e o! NPs* NPs (it& lo(er amounts o! sur!aceEassociate% P2A s&o( a#out t&ree!ol% &ig&er cellular upta$e in vascular smoot& muscle cells t&an t&e NPs (it& &ig&er resi%ual P2A A4:B* T&is coul% #e %ue to s&iel%ing o! t&e sur!ace c&arge reversal o! NPs #y t&e presence o! &ig&er amount o! sur!aceEassociate% P2A) (&ic& coul% a!!ect t&e en%osomal escape o! NPs* -urt&er) t&e amount o! P2A associate% (it& t&e NP sur!ace %epen%s on its concentration use% as an emulsi!ier %uring !ormulation) its molecular (eig&t) an% %egree o! &y%ro"ylation A5B* Cellular internali/ation o! NPs also %epen%s on t&eir particle si/e an% t&us &as #een s&o(n to a!!ect t&e gene trans!ection* T&e smaller si/e

5>9

2asir an% a#&aset(ar

NPs

Protein
Cellular upta$e D Particle si/e D Sur!ace properties

PE

RE
Gene e"pression mRNA D Nuclear transport o! DNA DStructural integrity o! DNA release% !rom NPs

Nucleus

DNA

IntraEcellular release o! DNA D En%osomal escape D Polymer composition D DNA6polymer ratio D DNA loa%ing in NPs D Inclusion o! e"cipients

-IGURE 5 ASee color insert*B -ormulation !actors in!luencing nanoparticleEme%iate% gene e"presE sion* A##reviations6 NPs) nanoparticlesM PE) primary en%osomesM RE) recycling en%osomes*

Aless t&an 4@@ nmB o! NPs s&o(e% 5<E!ol% &ig&er gene trans!ection t&an t&e larger si/e Amore t&an 4@@ nmB NPs A<B* T&us) t&e smaller si/e (it& a uni!orm particle si/e %istri#ution is e"pecte% to increase t&e gene trans!ection e!!iciency o! NPs* ?t&er important !ormulation parameters (&ic& in!luence t&e gene trans!ecE tion a#ility o! NPs inclu%e t&e molecular (eig&t o! polymer an% its composition Alacti%e to glycoli%e ratioB A5B* NPs !ormulate% (it& &ig&erEmolecularE(eig&t polyE mer s&o(e% en&ance% gene trans!ection* T&is may #e attri#ute% to t&e relatively &ig&er DNA loa%ing an% its release !rom NPs prepare% (it& &ig&EmolecularE(eig&t polymer A-ig* 8B* Hig&er viscosity an% #etter emulsi!ying properties o! t&e polymer solution !acilitate &ig&er loa%ing o! DNA in NPs an% also lea% to lo(er particle si/e o! NPs* Polymer composition can a!!ect t&e &y%rop&o#icity o! t&e polymer an% t&us can a!!ect t&e DNA loa%ing an% release o! DNA !rom t&e NPs* NPs prepare% using more &y%rop&o#ic polymers Apolylacti%esB %emonstrate% lo(er trans!ection t&an t&ose !ormulate% using copolymers o! polylacti%e an% glycoli%e A-ig* 9B* T&e slo( rate o! release o! DNA !rom t&e &y%rop&o#ic polymeric matri" may #e responsi#le !or t&e lo(er levels o! gene trans!ection* Alt&oug& t&e levels o! gene e"pression (it& NPs are lo(er t&an t&at ac&ieve% (it& lipi%E#ase% gene %elivery) t&ey are sustaine% !or a prolonge% perio% o! time* -urt&er NPEme%iate% gene trans!ection is not a!!ecte% #y t&e presence o! serum in t&e cell culture me%ia an% t&us P GA NPs constitute a potential gene %elivery vector !or in vivo gene %elivery* Pra#&a an% a#&aset(ar A4;B &ave s&o(n slo( intracellular release o! plasmi% DNA !rom t&e P GA NPs) (&ic& results in susE taine% retention o! DNA insi%e t&e cells A-ig* :B* NPs loa%e% (it& (tEp:8 gene %emE onstrate% &ig&er mRNA levels !or p:8 as compare% to t&at (it& a liposomal !ormulation at !ive %ays a!ter trans!ection o! MDAEM+E98:S #reast cancer cells* T&e sustaine% p:8 e"pression levels resulte% in greater an% sustaine% in&i#ition o! cell proli!eration in vitro as compare% to t&at (it& liposomal !ormulation or plasmi%

Nanoparticles !or Gene Delivery

5>:

-IGURE 8 E!!ect o! molecular (eig&t o! P GA on AAB in vitro release o! DNA !rom NPs an% trans!ection o! NPs in A+B MC-E< cells* Cells A8:)@@@ per (ell in 59E(ell plateB (ere incu#ate% (it& NPs A999 _g1m 1(ellB !or one %ay a!ter (&ic& t&e me%ium in t&e (ells (as replace% (it& !res& me%ium A(it&E out NPsB* Me%ium (as c&ange% on alternate %ays t&erea!ter* NPs s&o(e% sustaine% gene trans!ection in MC-E< cell line* Data as mean e S*E*M*) n ^ ;* A##reviation6 NPs) nanopartiE cles* Source6 -rom Re!* 5*

DNA alone* Co&en et al* A8B &ave s&o(n t&at %espite t&e lo(er trans!ection levels o#serve% in vitro (it& NPs as compare% to liposomal !ormulations) t&e in vivo gene trans!ection (it& NPs (as one to t(o or%ers o! magnitu%e greater t&an t&at (it& liposomes at seven %ays a!ter an intramuscular in.ection in mice* T&eir stu%ies %emE onstrate% gene e"pression sustaining over 5= %ays in vivo (it& a single %ose o! intramuscular in.ection o! NPs* Suc& sustaine% gene e"pression is a%vantageous especially i! t&e &al!Eli!e o! t&e e"presse% protein is very s&ort an%1or a c&ronic gene %elivery is reCuire% !or #etter t&erapeutic e!!icacy* Alt&oug& t&ese NPs &ave #een s&o(n to release plasmi% DNA at slo(er rates insi%e t&e cell) it is important t&at t&e DNA release% as a result o! polymer %egra%aE tion retains its #ioactivity* T&ere are reports s&o(ing t&at t&e %egra%ation o! P GA1 P A polymers into lactic an% glycolic aci%s can pro%uce &ig&ly aci%ic microenviE ronments in t&e NPs) (&ic& can compromise t&e sta#ility an% activity o! DNA release% !rom NPs A4<B* A%%itional e"cipients suc& as polyet&ylene o"i%e APE?B can #e coencapsulate% (it& plasmi% DNA in polymeric NPs in or%er to prevent t&e generation o! e"tremely aci%ic microenvironments insi%e NPs on polymer %egra%ation A4=B* Polymer #len%s o! P GA an% polyo"yet&ylene %erivatives &ave

5>;

2asir an% a#&aset(ar

-IGURE 9 E!!ect o! polymer comE position on AAB in vitro DNA release !rom NPs an% A+B trans!ection o! NPs in MC-E< cells* Cells A8:)@@@1(ell in 59E(ell plateB (ere incu#ate% (it& NPs A999 _g1m 1(ellB !or one %ay) an% t&en t&e me%ium (as replace% (it& !res& me%ium A(it&out NPsB* Me%ium (as c&ange% on every alterE nate %ay t&erea!ter) an% trans!ection levels (ere %etermine% at one) t&ree) !ive) an% seven %ays postEtrans!ecE tion in MC-E< cell line* T&is !igure represents lacti%e6glycoli%e ratio* Data s&o(n as mean e S*E*M*) n ^ ;* A##reviation6 P A) PolyAlactic aci%B* Source6 -rom Re!* 5*

4*5 { 4 -luorescence intensity @*= @*; @*9 @*5 @ @*@= 4 8 Time ADayB : < Nanoparticles DNA

-IGURE : 7uantitative %etermiE nation o! intracellular DNA levels cells trans!ecte% (it& 3?3?E la#ele% DNAEloa%e% nanopartiE cles %emonstrate% sustaine% an% increase% intracellular DNA levels as oppose% to transient DNA levels in t&e cells trans!ecte% (it& na$e% DNA* Data are represente% as t&e mean e t&e stan%ar% error o! t&e mean An ^ ;M P c @*@@4 !or points mar$e% (it& asteris$sB* Source6 -rom Re!* 4;*

Nanoparticles !or Gene Delivery

5><

#een use% to prepare NPs entrapping plasmi% DNA) using a mo%i!ie% emulsi!icationE solvent %i!!usion tec&niCue A:B* P GA an% polo"amer or polo"amine (ere mi"e% in %i!!erent ratios an% %issolve% in met&ylene c&lori%e* An aCueous solution o! plasmi% DNA can t&en #e emulsi!ie% into t&is organic p&ase containing polymers using vorE te"ing* T&e emulsion is t&en poure% into a polar p&ase un%er mo%erate magnetic stirring) to allo( imme%iate precipitation o! t&e polymer* T&us) t&is mo%i!ie% tec&E niCue avoi%s t&e use o! &ig& s&ear !orces %uring nanoencapsulation o! DNA) (&ic& can cause structural %amages to t&e DNA* Moreover) t&e use o! polo"amers an% polo"amine !acilitate% DNA encapsulation an% resulte% in an increase in t&e encapE sulation o! DNA (it&in t&e NPs* T&e nature an% H + value A&y%rop&ilic1lipop&ilic #alanceB o! t&e sur!actant Apolo"amer1polo"amineB can possi#ly a!!ect t&e interacE tion o! DNA (it& t&e &y%rop&o#ic polymers* Hig&er encapsulation o! DNA (it& polymer #len%s ma%e (it& sur!actants o! interme%iate H + values can #e attri#E ute% to t&e improve% compati#ility o! &y%rop&ilic plasmi% DNA (it& t&e &y%roE p&o#ic P GA polymer* T&e rate o! release o! plasmi% DNA !rom t&e NPs also %epen%s on t&e H + value o! t&e polyo"yet&ylene %erivative use% in t&e polymer #len%* NPs prepare% using &y%rop&ilic polo"amers ATetronic >@=) H + ^ 8@*@B s&o(e% a continuous an% !ast release o! DNA (it&in one (ee$) (&ereas t&e ones prepare% (it& a more &y%rop&o#ic sur!actant ATetronic >@9) H + ^ 49*:B s&o(e% slo( release o! DNA over a perio% o! t(o (ee$s A-ig* ;B* Attempts &ave #een ma%e to improve t&e encapsulation o! DNA in #ioE %egra%a#le NPs* Con%ensing plasmi% DNA prior to encapsulation can increase t&e encapsulation as compare% to encapsulation o! uncon%ense% DNA* Plasmi% DNA con%ense% (it& polycations suc& as polyElElysine AP B or (it& cationic %en%rons &as also #een encapsulate% in t&e P GA NPs* T&is allo(s !or t&e protection o! DNA insi%e t&e polymeric matri" an% controlle% %elivery o! plasmi% DNA* Ri#eiro et al* A9B &ave reporte% t&e encapsulation o! plasmi% DNA con%ense% (it& cationic P E#ase% lipi%ic %en%rons A%en%riple"esB into P GA NPs #y %ou#leEemulsion met&o%* T&e si/e o! P GAE%en%riple" particles (as a !unction o! t&e molar c&arge ratios at (&ic& DNA (as con%ense% (it& t&e %en%riple"es* Plasmi% DNA coul% #e

-IGURE ; Release pro!iles o! plasmi% DNA !rom P GA6Tetronic >@= AsCuaresB an% P GA6 Tetronic >@9 AtrianglesB #len% NPs an% !rom t&e control P GA AstarsB NPs Amean e S*D*) n ^ 8B* A##reviations6 P GA) polyAD) Elacti%eEcoEglycoli%eBM NPs) nanoparticles* Source6 -rom Re!* :*

5>=

2asir an% a#&aset(ar

!ully con%ense% (it& cationic %en%rons at &ig& A4@64B molar c&arge ratios) resulting in %en%riple"es (it& a positive /eta potential an% a DNA loa%ing o! 4:*@[ A(1(B* P GA NPs can #e mo%i!ie% to generate cationic sur!aces) to (&ic& negatively c&arge% DNA molecules can #e con%ense%* An emulsionH%i!!usionHevaporation tec&niCue using P2AHc&itosan #len% as a sta#ili/er &as #een %escri#e% to pro%uce cationically mo%i!ie% P GA NPs A4>B* P2A &elps in t&e sta#ili/ation o! t&e particles) (&ereas c&itosan o(ing to its positive c&arge provi%es a cationic sur!ace to t&e NPs* Plasmi% DNA can #e comple"e% to t&e sur!ace o! t&ese pre!orme% cationic NPs #y means o! electrostatic interactions) t&us avoi%ing t&e involvement o! DNA %uring t&e NP !ormulation an% &ence preserving DNA integrity* +len%s o! t&e polymers P A1P GA (it& polyet&ylenimine APEIB &ave also #een use% to prepare positively c&arge% NPs #y a %ia!iltration met&o% A5@B* T&e use o! %ia!iltration tec&niCue avoi%s t&e use o! any sur!actant in t&e NP !ormulation as t&e &y%rop&ilic nature o! PEI e!!ectively re%uces t&e inter!acial energy #et(een t&e &y%rop&o#ic NP sur!aces an% t&e aCueous me%ia* Particle si/e an% t&e /eta potential o! t&ese NPs can #e controlle% #y t&e amount o! PEI use% in t&e polymer #len%s* Imine groups o! PEI can #e use% to comple" DNA to t&e sur!ace o! cationic NPs* T&e NPs pro%uce% using t&is met&o% s&o(e% &ig& a%sorption capacity !or plasmi% DNA an% a &ig& %ispersive sta#ility* -urt&er) t&e sur!ace o! polymeric NPs #ase% on P GA1P A can #e !unctionaE li/e% to improve t&eir #io%istri#ution an% also to con.ugate targeting ligan%s (&ic& can %irect NPs to speci!ic cells1tissues (&ere gene %elivery is %esire%* Sur!ace mo%i!iE cation o! NPs is ac&ieve% eit&er #y a%sor#ing amp&ip&ilic e"cipients onto pre!orme% NPs or #y covalently lin$ing e"cipients to t&e coreE!orming polymer prior to NP !ormulation* +io%egra%a#le NPs &ave #een prepare% using P Egra!tEpolysacc&ari%e copolymers an% polyA%)lElactic aci%B #y using a solvent evaporation met&o% or t&e %ia!iltration met&o% A;B* NPs prepare% using t&ese copolymers &a% #i!unctional sur!aces (it& positively c&arge% amino groups o! P an% t&e polysacc&ari%e moiE eties* T&is increase% t&e a%sorption capacity o! NPs !or polynucleoti%es an% allo(e% intro%uction o! ligan% Acar#o&y%rateB moieties on t&e NP sur!ace !or ligan%E me%iate% recognition o! speci!ic receptors* T&e !ormulation resulte% in NPs as small as ;@ nm in %iameter) an% s&o(e% e"cellent %ispersive sta#ility in p&osp&ateE #u!!ere% saline* A pre!erential %istri#ution o! %e"tran moieties over P (as o#serve% in t&e outer sur!ace o! NPs using a P Egra!tE%e"tran copolymer* Presence o! %e"tran c&ains on NP sur!ace can #e potentially use!ul to prevent nonspeci!ic interE actions (it& serum proteins an% also !or receptorEme%iate% targeting to speci!ic cells* Su#seCuent to intravenous in.ection o! t&e NPs) t&e ma.or pro#lem is to avoi% t&eir opsoni/ation #y plasma proteins an% su#seCuent upta$e #y t&e p&agocytic cells* PE?1PEG &as #een use% to coat t&e polymeric NPs to provi%e a protective &y%rop&ilic s&eat&) (&ic& prevents t&e rapi% opsoni/ation o! t&e ot&er(ise &y%roE p&o#ic NPs #y reticuloen%ot&elial system ARESB an% t&us prolong t&e circulation time o! NPs in #loo% stream A54B* T&e &y%rop&o#ic part o! PE?1PEG polymers can a%sor# to NP sur!ace) (&ereas t&e &y%rop&ilic c&ains protru%e to(ar%s t&e aCueous me%ium* PEG coats on NP sur!ace also provi%e an attractive opportunity to c&emiE cally con.ugate activeEtargeting ligan%s to t&e NP sur!ace A55)58B* T&ese coatings can mo%i!y t&e #io%istri#ution o! NPs (&en in.ecte% into t&e systemic circulation* Ho(ever) it &as #een argue% t&at some o! t&ese polymers can #e easily %isplace% #y serum proteins) (&ic& can lea% to aggregation o! NPs A59B* T&us) alternative approac&es o! synt&esi/ing copolymers o! P A1P GA (it& PEG A5:)5;B an% coenE capsulation o! PEG (it& plasmi% DNA insi%e P GA NPs &ave #een trie% A5<B*

Nanoparticles !or Gene Delivery

5>>

CHIT?SAN NAN?PARTIC ES C&itosan is a linear cationic polysacc&ari%e) o#taine% #y partial al$aline %eacetyE lation o! c&itin) a polymer !oun% in t&e e"os$eleton o! crustaceans* C&itosans are #io%egra%a#le an% #iocompati#le polymers an% t&us can #e potentially sa!e an% nonto"ic carriers !or gene %elivery* Moreover) %erivatives o! c&itosan can #e synt&eE si/e%) (it& relative ease) to &y%rop&o#ically mo%i!y c&itosan or to con.ugate %i!!erent c&emical entities !or targeting c&itosan NPs to speci!ic tissues or organs* NPs !or gene %elivery can #e prepare% #y AiB comple"ing plasmi% DNA (it& c&itosan A5=B or AiiB #y encapsulating DNA insi%e c&itosan NPs A5>B* T&e &ig& %enE sity o! amino groups present in t&e glucosamine #ac$#one o! c&itosan can #e proE tonate% an% t&us o!!ers t&e opportunity to comple" c&itosans (it& negatively c&arge% DNA molecules) o(ing to t&e electrostatic interactions* Sel!Eassem#ling c&itosan1DNA comple"es) in t&e si/e range o! 4:@ to :@@ nm) (ere !irst prepare% #y mi"ing a solution o! c&itosan (it& plasmi% DNA A8@B* T&e particle si/es o! t&e comple"es %epen% on t&e molecular (eig&t o! c&itosan use%) #ut not on t&e #u!!er compositions* C&itosan) &y%rop&o#ically mo%i!ie% using %eo"yc&olic aci%) can !orm sp&erical sel!Eaggregates (it& a mean %iameter o! 4;@ nm in aCueous me%ia A5=B* C&arge comple"es !orme% #et(een t&ese sel!Eaggregates o! mo%i!ie% c&itosan an% plasmi% DNA &ave #een s&o(n to trans!ect C?SE4 cells in culture* T&e trans!ection e!!iciency o! c&itosan sel!Eaggregate1DNA comple"es (as reporte% to #e more t&an t&at ac&ieve% (it& plasmi% DNA alone) #ut lo(er t&an liposomal !ormulations* DNA can #e encapsulate% in c&itosan NPs) prepare% #y a comple" coacerE vation met&o%) yiel%ing particle si/es in t&e range o! 5@@ to :@@ nm* Mao et al* A5>B &ave stu%ie% t&e in!luence o! various parameters o! NP preparation on t&e si/e o! c&itosanHDNA NPs* T&e si/e o! t&e particles (as optimi/e% to 4@@ to 5:@ nm using an N1P ratio o! t&ree to eig&t* T&e /eta potential o! t&ese particles (as o45 to o4= m2 at a pH lo(er t&an ;*@ an% nearly /ero at pH <*5* C&itosan NPs coul% partially proE tect DNA !rom nucleases* T&e trans!ection e!!iciency (as !oun% to #e cellE%epen%ent an% lo(er t&an t&at ac&ieve% (it& ipo!ectamineHDNA comple"es in HE0 5>8 cells* Ho(ever) t&e presence o! serum in t&e cell culture me%ium %i% not inter!ere (it& t&e trans!ection using c&itosan NPs* C&itosanHDNA NPs are also consi%ere% a very appealing carrier c&oice !or oral geneE%elivery strategies) o(ing to t&e mucoE a%&esive properties o! c&itosan* ?rally a%ministere% c&itosanHDNA NPs can a%&ere to t&e gastrointestinal epit&elium an% trans!ect epit&elial or immune cells present in t&e gutEassociate% lymp&oi% tissue A84B*

P? 3AfEAMIN? ESTERBE+ASED NAN?PARTIC ES PolyAfEamino estersB constitute a relatively ne( class o! synt&etic #io%egra%a#le cationic polymers (it& tertiary amines in t&eir #ac$#one* Unli$e ot&er cationic polymers) t&ese are &y%rolytically %egra%a#le polymers an% are generally less cytoE to"ic t&an t&e polycations suc& as PEI A85B* Using MTT assay) t&ese polymers an% t&eir %egra%ation pro%ucts &ave #een s&o(n to #e nonto"ic relative to PEI in NIH 8T8 cell line* T&ese polymers &ave #een s&o(n to possess pHE%epen%ent solu#ility) an% t&us are suita#le !or t&e preparation o! DNA NPs (&ic& can trigger t&e polymer %egra%ation an% release o! encapsulate% DNA in t&e aci%ic pH in en%osomal vesicles insi%e cells A88B* ?(ing to t&eir polycationic nature) t&ese polymers can con%ense

8@@

2asir an% a#&aset(ar

DNA into comple"es o! t&e or%er o! :@ to 5@@ nm an% t&us can #e use% !or gene trans!ection* P? 3AA 03 C3AN?ACR3 ATEB NAN?PARTIC ES PolyAal$ylcyanoacrylateB APACAB NPs (ere !irst prepare% #y Couvreur et al* in 4><> A89B* PACA NPs &ave #een use% !or intracellular %elivery o! various oligonucleoti%e seCuences* PACA NPs can #e coate% (it& cationic sur!actants Acetyltrimet&ylamE monium #romi%eB or polymers ADEAEE%e"tranB (&ic& can #e use% !or comple"ing oligonucleoti%es via electrostatic interactions A8:B* Alternatively) oligonucleoti%es can #e associate% (it& t&e PACA NPs #y covalently lin$ing to a &y%rop&o#ic molecule Ac&olesterolB (&ic& can t&en #e anc&ore% at t&e NP sur!ace A8;B* In earlier stu%ies) it &as #een s&o(n t&at t&e PACA NPs are ta$en up #y t&e RES system o! t&e #o%y an% t&us can #e use% !or targeting DNA %elivery to t&e RES organs suc& as liver) spleen) lungs) an% #one marro( A8<B*

MAGNETIC NAN?PARTIC ES Magnetic NPs usually incorporate magneticEresponsive materials suc& as magnetite) iron) nic$el) co#alt) an% so on* In contrast to t&e conventional polymeric NPs) magnetic NPs can #e %irecte% an% pre!erentially locali/e% in t&e %isease% tissue upon application o! an e"ternal locali/e% magnetic !iel%* T&e magnetic NPs can #e mo%i!ie% (it& %i!!erent c&emical compoun%s to !acilitate a c&ange in t&e sur!ace c&arge or particle si/e o! t&e NPs* Magnetic NPs !or gene %elivery involve associating na$e% plasmi% DNA or DNA vectors Asuc& as DNA comple"e% (it& polycations or pac$age% in viral vectorsB to t&e sur!ace o! t&e NPs* T&is tec&niCue o! gene trans!ection is commonly re!erre% to as QmagnetoE !ection*S T&e magnetic !iel% can improve t&e vector concentration near t&e cells to #e trans!ecte% an% t&us &as #een s&o(n to re%uce t&e vector %ose an% incu#ation time (it& vector reCuire% to ac&ieve &ig& trans!ection e!!iciency in vitro A8=B* T&e sur!ace o! superparamagnetic ironEo"i%e NPs coate% (it& PEI &as #een use% to associate PEIHDNA comple"es A8=B* T&e #asic principle use% !or t&e !ormulation o! t&ese magnetic NPs is t&at o! saltEin%uce% aggregation o! colloi%al particles* PEIEcoate% magnetic NPs) PEI) an% plasmi% DNA are mi"e% in saltEcontaining soluE tions to !orm t&e magnetic vectors !or gene trans!ection* PEIHDNA comple"es Apolyple"esB associate% (it& superparamagnetic NPs can #e gui%e% to accumulate selectively in t&e target tissue) #y t&e use o! an e"ternal magnetic !iel% !ocuse% at t&e tissue* MagneticE!iel%Egui%e% local trans!ection in t&e gastrointestinal tract an% in #loo% vessels &as also #een reporte% A8=B* Anot&er report o! using magnetic NPs !or gene %elivery involves association o! magnetic Amag&emiteB NPs (it& t&e DNA vector &emagglutinating virus o! 'apanEenvelope AH2'EEB A8>B* T&is can potentially en&ance t&e gene trans!ection e!!iciency o! t&e vector #y promoting its cell association) using magnetic !orce* T&e magnetic NPs (ere coate% eit&er (it& protamine sul!ate or &eparin to pro%uce a cationically c&arge% sur!ace o! t&e NPs* T&e DNAEcarrying vectorRH2'EER(as associate% (it& t&e mo%i!ie% NPs an% use% !or gene trans!ection* T&ese magnetic NPs promote gene trans!ection) me%iate% #y cell !usion o! t&e H2'EE vectors* Cationically c&arge% magnetic NPs can #e speculate% to promote greater associaE tion o! t&e vector (it& t&e cells) (&ic& t&en carry DNA into t&e cells un%er t&e in!luE ence o! magnetic !orce) t&us en&ancing t&e trans!ection e!!iciency o! t&e vectors*

Nanoparticles !or Gene Delivery

8@4

T&us) t&e use o! magnetic NPs in con.unction (it& DNA alone or DNA polyple"es is an emerging gene trans!ection tec&niCue) (&ic& can e!!ectively increase t&e gene trans!ection e!!iciency o! t&ese vectors* Ho(ever) it is important t&at suc& magnetic NPs !or gene trans!ection #e (aterE#ase%) #iocompati#le) nonto"ic) an% nonimmunogenic*

SUMMAR3 +io%egra%a#le NPs constitute sa!er an% versatile geneE%elivery systems t&at allo( ligan% con.ugation !or targete% gene t&erapy as (ell as mo%ulation o! t&e DNA release to control t&e level an% %uration o! gene e"pression* Alt&oug& (e &ave ac&ieve% some success in overcoming t&e cellular #arrier o! DNA %elivery) t&e nuclear mem#rane still stan%s as a rateElimiting !actor !or e!!icient gene e"pression using nonviral vectors A9@B* Incorporation o! cationic pepti%es suc& as nuclear locaE li/ing signaling pepti%es) protein trans%uction %omain !rom t&e transactivator o! transcription) an% protamine &ave #een s&o(n to ena#le nuclear transport o! DNA) an% &ence con.ugating t&ese to a carrier system may prove to #e an e!!ective strategy to en&ance gene e"pression o! nonviral systems A94B* -or in vivo applications) it is important to restrict t&e gene trans!ection to t&e treate% AtargetB tissue) an% t&us) targeting o! NPs can potentially increase t&e e!!icacy an% re%uce t&e to"icity o! gene t&erapy* Approac&es suc& as %ecorating t&e sur!ace o! NPs (it& cellEspeci!ic ligan%s an% use o! magnetic !orce to target t&e NPs to speci!ic tissues or organs !or targete% gene %elivery) t&oug& in early stages o! %evelopment) constitute promising e!!orts at improving gene %elivery* To success!ully %evelop suc& !ormulation approac&es !or targete% gene %elivery) it is essential to a%% to our #asic un%erstan%ing o! t&e cellular #arriers an% mec&anisms o! intracellular sorting o! NPs) an% ultimately %e!ine rational !ormulation strategies to overcome t&ese #arriers A95B*

AC0N?, EDGMENTS Grant support !rom t&e National Institutes o! Healt& AR@4 E+@@8><:B an% a pre%octoral !ello(s&ip to '*0*2* !rom t&e American Heart Association) Heartlan% A!!iliate AA(ar% k@:4:9=>VB* Aut&or A2* *B ac$no(le%ges &is !ormer la#oratory mem#ers) Dr* S(ayam Pra#&a an% Dr* San.ee# 0* Sa&oo) (&ose %ata are cite% in t&is c&apter*

RE-ERENCES
4* i V) Dullmann ') Sc&ie%lmeier +) et al* Murine leu$emia in%uce% #y retroviral gene mar$ing* Science 5@@5M 5>;A::;<B69><* 5* Pra#&a S) a#&aset(ar 2* Critical %eterminants in P GA1P A nanoparticleEme%iate% gene e"pression* P&arm Res 5@@9M 54A5B68:9H8;9* 8* Co&en H) evy R') Gao ') et al* Sustaine% %elivery an% e"pression o! DNA encapsulate% in polymeric nanoparticles* Gene T&er 5@@@M <A55B64=>;H4>@:* 9* Ri#eiro S) Hussain N) -lorence AT* Release o! DNA !rom %en%riple"es encapsulate% in P GA nanoparticles* Int ' P&arm 5@@:M 5>=A5B68:9H8;@* :* Csa#a N) Caamano P) Sanc&e/ A) Domingue/ -) Alonso M'* P GA6polo"amer an% P GA6 polo"amine #len% nanoparticles6 ne( carriers !or gene %elivery* +iomacromolecules 5@@:M ;A4B65<4H5<=* ;* Maruyama A) Is&i&ara T) 0im 'S) 0im S,) A$ai$e T* Nanoparticle DNA carrier (it& polyAlElysineB gra!te% polysacc&ari%e copolymer an% polyA%)lElactic aci%B* +iocon.ug C&em 4>><M =A:B6<8:H<95*

8@5

2asir an% a#&aset(ar

<* Pra#&a S) V&ou ,V) Panyam ') a#&aset(ar 2* Si/eE%epen%ency o! nanoparticleE me%iate% gene trans!ection6 stu%ies (it& !ractionate% nanoparticles* Int ' P&arm A5@@5BM 599A4H5B64@:H44:* =* 0uo 'H) 'an MS) Sung 0C* Evaluation o! t&e sta#ility o! polymerE#ase% plasmi% DNA %elivery systems a!ter ultrasoun% e"posure* Int ' P&arm 5@@8M 5:<A4H5B6<:H=9* >* engs!el% CS) Anc&or%oCuy T'* S&earEin%uce% %egra%ation o! plasmi% DNA* ' P&arm Sci 5@@5M >4A<B64:=4H4:=>* 4@* Mi%%aug& CR) Evans R0) Montgomery D ) Casimiro DR* Analysis o! plasmi% DNA !rom a p&armaceutical perspective* ' P&arm Sci 4>>=M =<A5B648@H49;* 44* a#&aset(ar 2) +ona%io ') Gol%stein S) evy R'* Gene trans!ection using #io%egra%a#le nanosp&eres6 results in tissue culture an% a rat osteotomy mo%el* Colloi% Sur! + 4>>>M 4;65=4H5>@* 45* An%o S) Putnam D) Pac$ D,) anger R* P GA microsp&eres containing plasmi% DNA6 preservation o! supercoile% DNA via cryopreparation an% car#o&y%rate sta#ili/ation* ' P&arm Sci 4>>>M ==A4B645;H48@* 48* ?ster CG) ,ittmar M) Unger -) et al* Design o! amineEmo%i!ie% gra!t polyesters !or e!!ecE tive gene %elivery using DNAEloa%e% nanoparticles* P&arm Res 5@@9M 54A;B6>5<H>84* 49* Mura$ami H) 0o#ayas&i M) Ta$euc&i H) 0a(as&ima 3* Preparation o! polyA%)lElacti%eE coEglycoli%eB nanoparticles #y mo%i!ie% spontaneous emulsi!ication solvent %i!!usion met&o%* Int ' P&arm 4>>>M 4=<A5B6498H4:5* 4:* Sa&oo S0) Panyam ') Pra#&a S) a#&aset(ar 2* Resi%ual polyvinyl alco&ol associate% (it& poly A%)lElacti%eEcoEglycoli%eB nanoparticles a!!ects t&eir p&ysical properties an% cellular upta$e* ' Control Release 5@@5M =5A4B64@:H449* 4;* Pra#&a S) a#&aset(ar 2* NanoparticleEme%iate% (il%Etype p:8 gene %elivery results in sustaine% antiproli!erative activity in #reast cancer cells* Mol P&arm 5@@9M 4A8B6544H54>* 4<* -u 0) Pac$ D,) 0li#anov AM) anger R* 2isual evi%ence o! aci%ic environment (it&in %egra%ing polyAlacticEcoEglycolic aci%B AP GAB microsp&eres* P&arm Res 5@@@M 4<A4B64@@H4@;* 4=* To#io M) Nolley ') Guo 3) Mciver ') Alonso M'* A novel system #ase% on a polo"amer1 P GA #len% as a tetanus to"oi% %elivery ve&icle* P&arm Res 4>>>M 4;A:B6;=5H;==* 4>* Ravi 0umar MN) +a$o(s$y U) e&r CM* Preparation an% c&aracteri/ation o! cationic P GA nanosp&eres as DNA carriers* +iomaterials 5@@9M 5:A4@B64<<4H4<<<* 5@* 0im IS) ee S0) Par$ 3M) et al* P&ysicoc&emical c&aracteri/ation o! polyAlElactic aci%B an% polyA%)lElacti%eEcoEglycoli%eB nanoparticles (it& polyet&ylenimine as gene %elivery carrier* Int ' P&arm 5@@:M 5>=A4B65::H5;5* 54* Re%&ea% HM) Davis SS) Illum * Drug %elivery in polyAlacti%eEcoEglycoli%eB nanoE particles sur!ace mo%i!ie% (it& polo"amer 9@< an% polo"amine >@=6 in vitro c&aracteriE sation an% in vivo evaluation* ' Control Release 5@@4M <@A8B68:8H8;8* 55* ?tsu$a H) Nagasa$i 3) 0atao$a 0* PEGylate% nanoparticles !or #iological an% p&armaE ceutical applications* A%v Drug Deliv Rev 5@@8M ::A8B69@8H94>* 58* +enns 'M) 0im S,* Tailoring ne( gene %elivery %esigns !or speci!ic targets* ' Drug Target 5@@@M =A4B64H45* 59* Neal 'C) Stolni$ S) Sc&ac&t E) et al* In vitro %isplacement #y rat serum o! a%sor#e% ra%ioE la#ele% polo"amer an% polo"amine copolymers !rom mo%el an% #io%egra%a#le nanoE sp&eres* ' P&arm Sci 4>>=M =<A4@B64595H459=* 5:* Stolni$ S) Dunn SE) Garnett MC) et al* Sur!ace mo%i!ication o! polyAlacti%eEcoEglycoli%eB nanosp&eres #y #io%egra%a#le polyAlacti%eBHpolyAet&ylene glycolB copolymers* P&arm Res 4>>9M 44A45B64=@@H4=@=* 5;* Ha(ley AE) Illum ) Davis SS* Preparation o! #io%egra%a#le) sur!ace engineere% P GA nanosp&eres (it& en&ance% lymp&atic %rainage an% lymp& no%e upta$e* P&arm Res 4>><M 49A:B6;:<H;;4* 5<* Pere/ C) Sanc&e/ A) Putnam D) et al* PolyAlactic aci%BHpolyAet&ylene glycolB nanoparticles as ne( carriers !or t&e %elivery o! plasmi% DNA* ' Control Release 5@@4M <:A4H5B6 544H559* 5=* ee 03) 0(on IC) 0im 3H) 'o ,H) 'eong S3* Preparation o! c&itosan sel!Eaggregates as a gene %elivery system* ' Control Release 4>>=M :4A5H8B6548H55@*

Nanoparticles !or Gene Delivery

8@8

5>* Mao H7) Roy 0) TroungE e 2 ) et al* C&itosanHDNA nanoparticles as gene carriers6 synt&esis) c&aracteri/ation an% trans!ection e!!iciency* ' Control Release 5@@4M <@A8B6 8>>H954* 8@* Mumper R') ,ang ') Claspell 'M) Rollan% AP* Novel polymeric con%ensing carriers !or gene %elivery* Proc Int Symp Control Rel +ioact Mater 4>>:M 5564<=H4<>* 84* +ern$opESc&nurc& A) 0ra.ice$ ME* Mucoa%&esive polymers as plat!orms !or peroral pepti%e %elivery an% a#sorption6 synt&esis an% evaluation o! %i!!erent c&itosanHEDTA con.ugates* ' Control Release 4>>=M :@A4H8B654:H558* 85* ynn DM) anger R* Degra%a#le polyA#etaEamino estersB6 synt&esis) c&aracteri/ation) an% sel!Eassem#ly (it& plasmi% DNA* ' Am C&em Soc 5@@@M 45564@<;4H4@<;=* 88* ynn DM) Ami.i MM) anger R* pHEresponsive polymer microsp&eres6 rapi% release o! encapsulate% material (it&in t&e range o! intracellular pH* Ange( C&em Int E% Engl 5@@4M 9@A>B64<@<H4<4@* 89* Couvreur P) 0ante +) Rolan% M) et al* Polycyanoacrylate nanocapsules as potential lysoE somotropic carriers6 preparation) morp&ological an% sorptive properties* ' P&arm P&armacol 4><>M 84A:B6884H885* 8:* C&avany C) e Doan T) Couvreur P) Puisieu" -) Helene C* Polyal$ylcyanoacrylate nanoparticles as polymeric carriers !or antisense oligonucleoti%es* P&arm Res 4>>5M >A9B6 994H99>* 8;* Go%ar% G) +outorine AS) SaisonE+e&moaras E) Helene C* Antisense e!!ects o! c&olesE terolEoligo%eo"ynucleoti%e con.ugates associate% (it& polyAal$ylcyanoacrylateB nanoparticles* Eur ' +ioc&em 4>>:M 585A5B69@9H94@* 8<* enaerts 2) Nagel$er$e '-) 2an +er$el T') et al* In vivo upta$e o! polyiso#utyl cyanoacrylate nanoparticles #y rat liver 0up!!er) en%ot&elial) an% parenc&ymal cells* ' P&arm Sci 4>=9M <8A<B6>=@H>=5* 8=* Sc&erer -) Anton M) Sc&illinger U) et al* Magneto!ection6 en&ancing an% targeting gene %elivery #y magnetic !orce in vitro an% in vivo* Gene T&er 5@@5M >A5B64@5H4@>* 8>* Moris&ita N) Na$agami H) Moris&ita R) et al* Magnetic nanoparticles (it& sur!ace mo%iE !ication en&ance% gene %elivery o! H2'EE vector* +ioc&em +iop&ys Res Commun 5@@:M 889A9B64454H445;* 9@* Mun$onge -M) Dean DA) Hillery E) Griesen#ac& U) Alton E,* Emerging signi!icance o! plasmi% DNA nuclear import in gene t&erapy* A%v Drug Deliv Rev 5@@8M ::A;B6<9>H<;@* 94* Par$ 3') iang '-) 0o 0S) 0im S,) 3ang 2C* o( molecular (eig&t protamine as an e!!icient an% nonto"ic gene carrier6 in vitro stu%y* ' Gene Me% 5@@8M :A=B6<@@H<44* 95* a#&aset(ar 2* Nanotec&nology !or %rug an% gene t&erapy6 t&e importance o! un%erE stan%ing molecular mec&anisms o! %elivery* Curr ?pin +iotec&nol 5@@:M 4;A;B6;<9H;=@*

4>

Gastrointestinal Applications o! Nanoparticulate DrugEDelivery Systems

Maria Rosa Gasco
Nanovector srl) Torino) Italy

EWCITING REAS?NS -?R STUD3ING THE APP ICATI?N ?NAN?PARTICU ATE S3STEMS T? THE GASTR?INTESTINA TRACT T&e upta$e o! nanoparticulate systems ANPSsB t&roug& t&e gastrointestinal tract AGITB is to%ay a (ellE$no(n an% accepte% p&enomenon) an% e"cellent revie(s o! t&e intestinal upta$e o! particles &ave #een pu#lis&e% A4H:B* NPS upta$e !rom t&e gut can provi%e an a%%itional %rug a%ministration routeM eac& system &as its o(n p&armaco$inetic parameters an% speci!ic %rugEcarrying a#ility* T&e #ioactive moleE cule is transporte% into t&e GIT #y carriers (&ose c&emicop&ysical c&aracteristics must #e ta$en into account) alt&oug& t&e c&emicop&ysical an% p&armacological c&aracteristics o! t&e %rug remain intact* T&is c&apter (ill concentrate particularly on t&e translocation o! NPSs via t&e lymp&atic system) an% #rie!ly consi%er t&e &it&erto less (i%ely stu%ie% colonic targeting o! NPSs*

ymp&atic Targeting PeyerPs patc&es are t&e most important structural units o! t&e gut associate% (it& lymp&oi% tissue AGA TBM t&ey are c&aracteri/e% #y MEcells t&at overlie t&e lymE p&oi% tissue an% are speciali/e% !or en%ocytosis an% transport into intraepit&elial spaces an% a%.acent lymp&oi% tissue* Nanoparticulates #in% to t&e apical mem#rane o! MEcells) a!ter (&ic& t&ey are rapi%ly internali/e% an% Qs&uttle%S to t&e lymp&oE cytes A8):B* T&e lymp&atic a#sorption o! a %rug via t&e GA T &as an a%vantage over t&e portal #loo% route) #ecause it avoi%s presystemic meta#olism #y t&e liver A&epatic !irstEpass e!!ectB* -lorence A;B &as suggeste% a simpli!ie% %iagram o! preE an% posta#sorption processes A-ig* 4B* T&e reCuisites ena#ling NPS to #e a#sor#e% via t&e GA T not only concern t&e loa%e% %rug) #ut also an% more speci!ically t&e carrierPs p&ysical c&aracteristics) suc& as si/e) s&ape) speci!ic sur!ace) sur!ace c&arge) c&emical sta#ility o! #ot& NPS an% loa%e% %rug) potential interactions (it& gut contents) transit time t&roug& t&e GIT) transport t&roug& t&e mucosa) a%&esion to epit&elial sur!aces) an% aggregation o! t&e particulates in contact (it& t&e !lui% content o! t&e gut* T&e transit an% transE location o! NPSs %epen% to a consi%era#le e"tent on t&eir mean %iameter) sur!ace c&arge) an% release c&aracteristics A5)8B* Some p&enomena) suc& as aggregation) a%sorE ption) an% a%&esion) can alter t&e /eta potential) &y%rop&ilicity) an% si/e o! t&e NPS* -rom t&e p&armaceutical stan%point) t&e measure o! any !easi#le application o! a %rugE%elivery system is its e!!icacy) t&at is) its a#ility to e"ert a p&armacological e!!ect* In%ee%) #esi%es t&e %i!!erent properties o! NPS liste% a#ove) a critical aspect is t&e loa%ing capacity o! t&e nanoparticulates* T&e &ig&er t&e loa%ing) t&e &ig&er is t&e #ioavaila#ility per particle a#sor#e% A5B* Anot&er important issue is #iocompatiE #ility an% #io%egra%a#ility o! t&e NPS components*

8@:

8@;

Gasco

-IGURE 4 A simpli!ie% sc&ematic o! preEa#sorption an% postEa#sorption processes in nanoparticleE %epen%ent %rug %elivery to gastrointestinal site% !ollo(ing oral a%ministration) &ig&lig&ting t&e variety o! processes involve% in t&e .ourney o! a nanoparticle !rom %elivery to target* Source6 -rom Re!* ;*

ymp& targeting o! NPSs can permit A5)8):B6 AiB oral %elivery o! la#ile molecules protecte% #y t&e carrierM AiiB oral %elivery o! poorly solu#le molecules !ollo(ing t&eir nanosolu#ili/ation in NPSsM AiiiB improve% #ioavaila#ility o! %rugs (it& poor a#sorption c&aracteristics) %ue #ot& to t&e large speci!ic sur!ace o! NPSs an% to t&eir increase% resi%ence timeM AivB oral %elivery o! vaccine antigens to gutEassociate% lymp&oi% tissueM AvB translocation o! antineoplastic %rugs !or treatment o! lymp&oE masM AviB %elivery o! %iagnostics !or t&e lymp&atic systemM AviiB sustaine%1controlle% %rug release) particularly important !or to"ic %rugs Ae*g*) antineoplastic %rugsBM AviiiB re%uction o! %rugErelate% GI mucosa irritationM an% Ai"B avoi%ance o! t&e &epatic !irstEpass e!!ect*

Colonic Targeting ymp&atic tissue in t&e colon) rectum) an% appen%i" vermi!orm is usually not !oun% in %iscrete patc&es) #ut is %i!!usely present in aggregate masses at irregular intervals* Despite t&e !act t&at t&e colon an% rectum contain t&e most a#un%ant lymp&oi% intestinal tissue) t&ere is a lac$ o! researc& e"amining lymp&atic a#sorption !rom t&ese sites A5B* T&e presence o! MEcells A<B suggests t&at NPSs may also #e a#sor#e% in t&is region o! t&e GIT* ColonEspeci!ic %rug %elivery systems can #e use% to improve %rug #ioavaila#ility at t&e colon) particularly in t&e case o! proteins an% pepti%es) targeting t&em to t&e less proteolytically active colon A=B* Nanoparticulates targete% to t&e colon &ave #een stu%ie% (it& t&e aim o! treating some types o! colon %isease) #ut also to investigate transport an% release o! some la#ile molecules suc& as pepti%es an% proteins as some early stu%ies s&o(e%* Colonic targeting o! nanoparticulates can permit t&erapeutic intervention in pat&ological processes o! t&e gut) suc& as ulcerative colitis or Cro&nPs %isease A5B) an% possi#le targeting o! la#ile molecules Asuc& as pepti%es an% small proteinsB to t&e colon A=B*

DE2E ?PMENT ?- NAN?PARTICU ATE S3STEMS -?R 3MPHATIC TARGETING T&e !ollo(ing NPSs are consi%ere%*

Gastrointestinal Applications o! Nanoparticulate DrugEDelivery Systems

8@<

Den%rimers Alt&oug& t&e !irst reports on %en%rimers (ere pu#lis&e% more t&an t(o %eca%es ago) t&eir potential #iological applications &ave only #een e"plore% in t&e past !e( years A5)>)4@B* Den%rimer c&emistry &as #een use% to prepare mono%isperse nanoparticles A%iameter #elo( :@ nmB in or%er to investigate t&e relation #et(een nanoparticle %iameter an% upta$e !rom t&e GIT A8B* T&e stu%y e"amine% #ot& %en%rimers (it& lipi%ic e"ternal c&aracter an% a series o! cationic %en%rimers) an% investigation o! %en%rimer a#sorption in rats t&roug& PeyerPs patc&es an% enterocytes s&o(e% t&eir pre!erential upta$e t&roug& PeyerPs patc&es* :E-luorouracil A44B can #e entrappe% in polyami%oamine %en%rimers t&at &ave #een coate% (it& p&osp&olipi%sM in vivo stu%ies in al#ino rats &ave s&o(n p&osp&olipi%Ecoate% %en%rimers to #e more e!!ective orally t&an !ree %rug* ymp&atic upta$e also increase%) in%icating a#sorption o! t&e !ormulation via t&e lymp&atic route*

Polymeric Nanoparticulates ANanoparticles an%1or NanocapsulesB Polymeric nanoparticulates are particles o! %iameter #elo( 4 _m) prepare% !rom natural or synt&etic polymers* Natural polymers Ai*e*) proteins or polysacc&ari%esB &ave not #een (i%ely use% !or t&is purpose) as t&ey may vary in purity an% o!ten reCuire crossElin$ing) (&ic& coul% %enature t&e em#e%%e% %rug A:B* Synt&etic #io%egra%a#le polymers &ave receive% muc& more attentionM t&e most (i%ely use% polymers &ave #een polyAlactic aci%B) polyAglycolic aci%B) t&eir copolymers polyAlacti%eEcoEglycoli%e aci%B AP GAB A45B) an% polyal$ylcyanoacrylates APACAB A48B* T&e #ioactives are (ell protecte% in t&ese polymers) (&ic& o!!er t&e a%vantage o! %elivering %rugs in a sustaine% !as&ion) avoi%ing repeate% a%ministration* Nanoparticles (ere !irst %evelope% !or t&e parenteral routeM more recently) t&ey &ave also #een stu%ie% as oral %elivery ve&icles* T&e ma.or interest is in t&e lymE p&atic upta$e o! nanoparticles #y PeyerPs patc&es in t&e GA T A45B* Microparticles remain in PeyerPs patc&es) (&ile nanoparticles are %isseminate% systemically A:B* Clearly) a (i%e variety o! %rugs can #e %elivere% via t&e oral route using polymeric nanoparticulate carriers* Muc& o! t&e researc& &as !ocuse% on t&e a#sorption en&ancement o! pepti%es A49B) proteins) an% vaccine antigens* Mucoa%&esive a#ility can #e con!erre% to NPSs #y coating t&eir sur!ace (it& microa%&esive polymers suc& as c&itosan or Car#opol bM action is more e!!ective an% prolonge%) con!irming t&e use!ulness o! mucoa%&esive properties A4:B* Insulin entrappe% in PACA nanosp&eres %isperse% in an oily p&ase containing a sur!actant pro%uces prolonge% &ypoglycemia A4;B* T&e !ate o! polyAiso#utylcyanoacrylateB nanocapsules carrying insulin a%ministere% to rats (as monitore% #y !luorescence an% transmission electron microscopy ATEMB an% evi%ence% intestinal a#sorption t&roug& t&e epit&elial mucosa A4<B* A luminescent polymer (as use% as a visi#le tracer to monitor t&e oral !ate o! P GA microsp&eres containing insulin A4=B* T&e t&erapeutic e!!ects o! anot&er pepti%e) octreoti%e) can #e improve% an% prolonge% on incorporation into PACA nanocapsules A4>B* +io%egra%a#le P GA nanoparticles containing salmon calcitonin AsCTB) comE ple"e% (it& amp&ip&ilic molecules) a!!or% &ig& loa%ing e!!iciencyM a#sorption (as goo% on oral a%ministration to rats A5@B* Positively c&arge% nanoparticles incorpoE rating cyclosporin A A54B &ave #een prepare% #y t&e emulsi!ication solvent %i!!usion met&o%M t&e #ioavaila#ility in #eagle %ogs increase% 4*5E!ol% over t&at o! Neoral b*

8@=

Gasco

T&e same pepti%e (as encapsulate% #y nanoprecipitation (it&in non#io%egra%a#le polymers) an% %i!!erent !ormulations (ere prepare%M oral a#sorption o! t&e pepti%e nanoparticulates in ra##its (as compare% to t&at o! Neoral b capsule* T&e relative #ioavaila#ility o! cyclosporin A !rom various !ormulations range% !rom 5@[ to 8:[ t&at o! Neoralb A55B* Heparin &as no oral #ioavaila#ility) an% is t&us generally a%ministere% #y t&e parenteral route* HeparinEloa%e% polymeric nanoparticles) prepare% (it& #io%eE gra%a#le polyElEcaprolactone an% P GA an% non#io%egra%a#le positively c&arge% polymers) use% alone or in com#ination) (ere a%ministere% orally to ra##its A58BM (it& eac& !ormulation antiEWa activity an% activate% partial t&rom#oplastin time AaPTTB (ere %etecte%* In particular) antiEWa activity (as %etecte% !or a longer perio% t&an (&en a &eparin solution (as a%ministere% intravenously* A %elivery system allo(ing oral a%ministration o! vaccines (oul% #e i%eal* An antigen is ta$en up into t&e PeyerPs patc&es mostly t&roug& t&e MEcells) (&ic& appear to #e t&e main site !or upta$e o! antigens a!ter oral a%ministration) an% it is generally accepte% t&at limite% %oses o! antigen are su!!icient !or mucous immuniE /ation* ?ral %elivery o! antigens may #e consi%ere% t&e most convenient means o! pro%ucing an IgA anti#o%y response A59B* T&e use o! microE an% nanoparticles !or t&e oral %elivery o! antigens can #e e!!icient i! t&ese systems are a#le to protect t&e antigen molecule* ?ral a%ministration o! antigens incorporate% in particulates in%uces a stronger antigenEspeci!ic immune response t&an %o antigens in t&e (aterE solu#le !orm) #ecause incorporation in NPSs protects t&em !rom t&e lo( pH o! t&e stomac& an% !rom proteolytic en/ymes A5:)5;B* T&e immune response &as #een stu%ie% a!ter su#cutaneous) oral) an% intranasal a%ministration to mice o! P GA nanosp&eres loa%e% (it& #ovine serum al#umin A+SAB) evaluating some !actors a!!ecting t&e immune response) suc& as si/e) sur!ace &y%rop&o#icity) /eta potential) a%.uvants) or e"cipients use% in t&e !ormulations A5<B*

iposomes ?ral %elivery o! liposomes (oul% #e t&e simplest an% most convenient route !or %rug a%ministration A5=)5>B) #ut it is important to e"amine t&eir sta#ility in t&e aci% pH o! t&e stomac& environment an% in t&e gastrointestinal me%ium* Sta#ility in t&e p&ysiological con%itions a!!ecting oral %rug %elivery &as #een stu%ie% to %etermine (&ic& components &ave t&e #est c&ance o! surviving t&roug& t&e GIT A8@B* iposomes coate% (it& polyet&ylene glycol containing recom#inant &uman epi%ermal gro(t& !actor (ere a%ministere% orally to rats an% t&e area un%er t&e concentrationHtime curve AAUCB (as evaluate% an% compare% to t&at o! t&e solution* It increase% 4*<E an% 5*:E!ol% !or p&osp&ati%ylc&oline an% %ipalmitoylp&osE p&ati%ylc&oline liposomes) respectively A84B* iposomes containing an e"tract o! natural marine lipi%s containing large amounts o! NE8Epolyunsaturate% !atty aci%s APU-AB (ere prepare% (it& t&e aim o! increasing PU-A #ioavaila#ility* T&oracic lymp& %uctEcannulate% rats (ere !e% intragastrically (it& t&ese liposomes) an% a#sorption o! !atty aci%s (as &ig&er t&an (it& !is& oil A85B* T&e e!!ect o! liposomes coate% (it& c&itosan or Car#opol b on ratsP intestinal a#sorption o! calcitonin &as #een stu%ie%) testing negatively an% positively c&arge% liposomes* T&e overall p&armacological e!!icacy o! coate% liposomes (as more t&an %ou#le t&at o! noncoate% liposomes A88B* A#sorption o! sCT !rom t&e intestine is poor #ecause o! its &ig& molecular (eig&t an% rapi% %egra%ation #y en/ymes in t&e

Gastrointestinal Applications o! Nanoparticulate DrugEDelivery Systems

8@>

GIT* Dou#le liposomes Ai*e*) liposomes containing smaller liposomesB (it& %i!!erent c&arges (ere investigate% as carriers o! sCT) a%ministere% to rats) an% compare% to t&e solution* T&e &ig&est &ypocalcemic e!!ect (as o#taine% (it& cationic c&arge% liposomes A89B*

Sel!EAMicroBEmulsi!ying DrugEDelivery Systems Sel!EAmicroBemulsi!ying DrugEDelivery Systems ASAMBEDDSsB are isotropic mi"tures o! oils) sur!actants) solvents) an% cosolvents1sur!actantsM t&ey are use% solely !or t&e purpose o! improving oral a#sorption o! &ig&ly lipop&ilic %rugs* T&e principal c&aracteristic o! t&ese systems is t&eir a#ility to !orm !ine oilEinE(ater emulsion or microemulsion upon mil% stirring) !ollo(ing %ilution #y t&e aCueous p&ase A8:)8;B* A supersatura#le ASESEDDSB !ormulation o! paclita"el) %evelope% using &y%ro"ypropylcellulose as precipitation in&i#itor A8<B) in a p&armaco$inetic stu%y on rats ac&ieve% !ive!ol% oral #ioavaila#ility versus an orally %ose% Ta"ol b !ormulation* An SAMBEDDS o! paclita"el (as a%ministere% to rats (it& an% (it&out t&e use o! PEglycoprotein in&i#itors A8=B* Compare% to commercial Ta"ol b) t&e oral #ioavaila#ility o! paclita"el SAMBEDDS increase% at t&e various %oses* T&e #ioavailE a#ility (as signi!icantly improve% !or paclita"el SAMBEDDS (&en cyclosporin A) as in&i#itor o! PEglycoprotein) (as coa%ministere%* An SAMBEDDS system loa%e% (it& simvastatin) orally a%ministere% to #eagle %ogs) increase% #ioavaila#ility 4*:E!ol% versus t&e conventional oral ta#let A8>B*

Soli%H ipi% Nanoparticles Soli%Hlipi% nanoparticles AS NsB are in t&e su#micron si/e range an% usually consist o! #iocompati#le an% #io%egra%a#le materials) suc& as triglyceri%es an% !atty aci%sM t&e pro%uction an% properties o! S Ns &ave #een revie(e% A9@H95B* 2arious preparaE tion met&o%s are possi#le6 one %epen%s on t&e application o! mec&anical &ig&E pressure &omogeni/ation to a melte% lipi% %isperse% in an aCueous solution o! sur!actants A9@B* ,&en camptot&ecinEloa%e% S Ns) pro%uce% #y &ig&Epressure &omogeni/ation) (ere a%ministere% perorally to rats) t&e concentrationHtime curves in plasma an% in organ tissues s&o(e% en&ance% availa#ility o! t&e %rug compare% to solution6 AUC an% mean resi%ence time AMRTB increase% signi!icantly A98B* Piri#e%il S Ns) a%ministere% orally to ra##its) increase% %rug #ioavaila#ility more t&an t(o!ol% compare% to pure piri#e%il A99B* S Ns loa%e% (it& allEtrans retinoic aci%) as a poorly solu#le mo%el %rug) can #e prepare% #y &ig&Epressure &omogeni/aE tionM a%ministere% orally to rats) t&ey signi!icantly en&ance% trans retinoic aci% a#sorption A9:B* T&e #ioavaila#ility o! clo/apine !ormulate% in S Ns prepare% #y t&e &ot &omogeni/ation met&o% (as %etermine% a!ter intravenous AI2B an% intra%uo%eE nal a%ministration an% compare% to t&ose o! a clo/apine suspension* T&e area un%er t&e curve AAUCB increase% #y a#out t&ree times in t&e case o! t&e I2 route) an% up to 9*: times !or t&e intra%uo%enal route A9;B* Clear solutions o! cyclosporin A) a soli% triglyceri%e) a (aterEmisci#le organic solvent) an% a mi"ture o! sur!actants an% emulsi!iers (ere %isperse% #y mi"ing in (ater to pro%uce a suspension o! nanoparE ticles o! si/es #et(een 5: an% 9@@ nm %epen%ing on t&e !ormulations* T&e oral #ioavaila#ility (as %etermine% on &umansM t&e #est results (ere similar to t&e Neoralb re!erence !ormulation an% (ere o#taine% !or t&e !ormulations !orming nanoparticles #elo( ;@ nm A9<B* ?t&er preparation met&o%s !or S Ns are t&e solvent emulsi!ication tec&niCue A9=B an% t&e solvent %i!!usion met&o% A9>B*

84@

Gasco

S Ns can also #e pro%uce% #y %ispersing (arm microemulsions in col% (ater A:@):4B* S Ns are t&e soli%i!ie% %roplets o! microemulsions (it& a mean %iameter o! =@ to 5@@ nmM t&ey can incorporate #ot& &y%rop&ilic an% lipop&ilic %rugs* Many %rugs &ave #een incorporate% in S Ns) an% %i!!erent a%ministration routes &ave #een stu%ie% in la#oratory animals* Stealt& S Ns &ave #een prepare% !or t&e purpose o! avoi%ing reticuloen%ot&elial system recognitionM a!ter I2 a%ministration) S Ns an% stealt& S Ns are a#le to cross t&e #loo%H#rain #arrier) increasing t&e MRT Ato a greater e"tent (it& stealt& t&an (it& nonstealt& S NsB o! t&e loa%e% %rug compare% to solution A:5B* S Ns are ta$en up (it&in a !e( minutes #y neoplastic A:8):9B an% nonneoplastic cell lines A::B* T&e gastrointestinal upta$e an% transport in t&e lymp&atic circulation o! %rugE unloa%e% S Ns (as initially stu%ie%M la#ele% an% unla#ele% S Ns (ere a%minisE tere% %uo%enally to rats) at t(o %i!!erent amounts in eCual volumes* A!ter a !e( minutes) TEM evi%ence% S Ns in t&e lymp&M t&e si/e o! t&e particles in t&e lymp& A48@H49@ nmB (as practically unc&ange% a!ter a%ministration* a#ele% S Ns (ere use% to evaluate lymp&atic upta$e an% t&e ra%ioactivity %ata con!irme% transport o! S Ns in lymp& an% #loo%* S N concentration versus time increase% out o! proportion to t&e increase% amount a%ministere%) #eing very muc& &ig&er (it& t&e &ig&er concentration o! S Ns A:;B* Successively) to#ramycin (as incorporate% in S Ns as a mo%el %rugM it (as c&osen #ecause it is not a#sor#e% at t&e gastrointestinal level an% is t&ere!ore still a%ministere% #y t&e parenteral route* To#ramycinEloa%e% S Ns ATo#raES NB (ere a%ministere% to rats into t&e %uo%enum an% compare% to To#raES N an% to#ramycin solution) #ot& a%ministere% I2 A:<B* T&e AUC o! I2 To#raES N (as 4<4@ min _g m Z4) (&ereas t&at o! To#raEsolution a%ministere% I2 (as 8:5 min _g m Z4* T&e AUC o! To#raES Ns a%ministere% %uo%enally (as over 4@@ times t&at o! t&e solution an% over 5@ times t&at o! To#raES N) #ot& a%ministere% I2* P&armaco$inetic parameters s&o(e% a mar$e% %i!!erence #et(een To#raES Ns a%ministere% %uo%enally an% I2) in t&e !ormer case provi%ing a su!!iciently &ig& level o! t&e %rug even a!ter 59 &ours* Most o! t&e %i!!erence #et(een t&e t(o a%minE istration routes is %ue to t&e transmucosal transport o! S Ns to t&e lymp& rat&er t&an to t&e #loo%* To#raES N a%ministere% %uo%enally act as a sustaine%Erelease system* S Ns containing t&ree %i!!erent percentages A4*5:[) 5*:[) an% :[B o! to#raE mycin (ere prepare%) an% t&e same %ose o! To#ra o! eac& o! t&e t&ree types (as a%ministere% intra%uo%enally to rats A:=B* -igure 5 s&o(s t&e to#ramycin plasma concentrations versus time !or t&e t&ree types o! S Ns* T&e p&armaco$inetic

-IGURE 5 To#ramycin plasma concentrations versus time e stan%ar% %eviation a!ter %uo%enal a%ministration o! t&e t&ree types o! To#raES N* A##reviation6 S N) soli%Hlipi% nanoparticle* Source6 -rom Re!* :=*

Gastrointestinal Applications o! Nanoparticulate DrugEDelivery Systems

844

p&armaco$inetic parameters are so name% #ecause t&ey re!er to t&e %rug incorporate% into S N an% not to t&e !ree %rugM %ata e"presse% as mean e SDM MRT) meanresi%ence timeM T415f) @*@@5%)! e @*@@@4@*@@9 elimination &al!Eli!eM AUC) area un%er t&e curve o! plasma e @*@@@5@*@4@ ie concentration versus time) Cl) total #o%y clearanceM 2 %) volume o! Cl A @*@@@8 1&B %istri#utionM statistical signi!icanEces areMS N 4*5: versus S N 5*:@#p c @*@:*cp c @*@4*%p c @*@@4MS N 4*5: versus S N :*@@ep c @*@4*!p c @*@@4MS N 5*: versus S N :*@@gp c @*@:*&p c @*@4*ip c @*@@4*A##reviations6 AUC) area un%er t&e curveM S N) soli%Hlipi% nanoparticle*Source6 -rom Re!* :<*

@*@>9 e @*@4@@*@>; e @*@@>@*@<8 e @*@5@ 2% A B

<9==@%)! e 5@9<8<9;> AUC e 4:4; Amin*mg148=4< B & e <@<

4>@4c)! e 84@ >>= e 545>4g e 4; T415fAminB

5>;9c)! e =:=4:99 e 8@998>g e 48= MRTAminB Apparent P&armaco$inet ic Parametersa

84*:#)e e 4*>5=*: e Cma" @*98;*8& e 4*5 Amg1 B

To#raES N 4*5:To#raES N -ormulation 5*:@To#raES N :*@@ aQApparentS TA+ E 4

845

Gasco

parameters varie% consi%era#ly (it& t&e percentage o! To#ra ATa#le 4B* T&is !in%ing is pro#a#ly %ue to t&e %i!!erences among t&e t&ree types o! S Ns) in particular) t&e num#er o! S Ns a%ministere%) average %iameter) total sur!ace area) an% %rug conE tent in eac& nanoparticle* T&e &ig&est percentage o! To#ra in S Ns correspon%e% to t&e &ig&est release rate) (&ereas t&e lo(est percentage pro%uce% t&e most prolonge% release* Sustaine% %rug release #y t&e oral route coul% t&us #e o#taine% #y approE priately mi"ing S Ns loa%e% (it& %i!!erent percentages o! t&e %rug* To#ramycin (as still %etermine% in lymp& mesenteric no%es 54 &ours a!ter %uo%enal a%minisE tration o! 5*:[ To#ra S Ns) as con!irme% also #y TEM* T&e amounts o! To#ra in t&e $i%neys a!ter To#raES N a%ministration) %uo%enally or I2) (ere lo(er t&an a!ter I2 a%ministration o! t&e solution A:>B* I%aru#icin is an ant&racycline a%ministere% #y t&e I2 or t&e oral route* Its e!!icacy &as #een %emonstrate% in t&e treatment o! %i!!erent tumors* S Ns incorE porating i%aru#icin AI%aES NsB A;@B (ere a%ministere% I2 an% %uo%enally to rats in a comparative stu%y versus solution) t&e aim #eing to %etermine (&et&er %rug #ioE availa#ility can #e improve%* A!ter %uo%enal a%ministration o! I%aES N an% o! I%a solution) i%aru#icin an% its main meta#olite i%aru#icinol (ere %etermine% over time) as s&o(n in -igure 8A an% +M t&e AUC an% elimination &al!Eli!e o! i%aru#icin (ere) respectively) a#out 54 an% 8@ times &ig&er t&an t&ose (it& solution* Again t&e AUC o! I%aES Ns a%ministere% intravenously (as lo(er t&an (&en a%ministere% %uo%enally* P&armaco$inetics an% #io%istri#ution o! I%aES Ns %i!!er !rom t&ose o! t&e solution* T&ese c&anges coul% #e e"ploite% to re%uce to"icity an% increase clinical e!!icacy o! t&e %rug*

-IGURE 8 AAB I%aru#icin plasma concentrations versus time a!ter %uo%enal a%ministration o! t&e t(o !ormulations* A+B I%aru#icinol plasma concentrations versus time a!ter %uo%enal a%ministration o! t&e t(o !ormulations*

Gastrointestinal Applications o! Nanoparticulate DrugEDelivery Systems

848

C? ?NIC DRUG TARGETING USING NAN?PARTICU ATES ?ne (ay to target t&e colon is to incorporate %rugs in appositely stu%ie% nanoparE ticulates* In one suc& stu%y) coate% calciumEalginateEgel #ea%s (ere use% to entrap liposomes A;4B* +ee venom pepti%e (as ta$en as a mo%el %rug) an% investigate% in vitro an% t&en in vivo6 gammaEscintigrap&y (as use% in a &uman stu%y to %etermine colonic arrival time) (&ic& (as !oun% to #e !our to !ive &ours* +ioa%&esion o! NPSs to in!lame% colonic mucosa (as s&o(n to #e si/eE %epen%ent) using !luorescent particles) in a rat stu%y ta$ing nonulcerate% tissue as comparison A;5B* P GA nanoparticles A;8B carrying an antiin!lammatory %rug) Rolipram) (ere t&us stu%ie% !or t&e treatment o! in!lammatory #o(el %isease* E"perimental colitis (as in%uce% in rats (it& trinitro#en/enesul!onic aci%) (&ic& pro%uce% signi!icant %amage o! t&e intestinal tissue* Nanoparticles containing Rolipram (it& a %iameter #et(een 5@@ an% :@@ nm (ere prepare%M !or #etter targeE ting) t&ey com#ine% e!!icient %rug loa%ing (it& an appropriate si/e range A;9B* T&e particles (ere a%ministere% !or !ive %ays an% compare% (it& t&e %rug solution* A!ter !ive %ays) (&en %rug treatment (as %iscontinue%) t&e %rug solution group un%er(ent a severe relapse) (&ereas t&e nanoparticle group continue% to s&o( re%uce% in!lammation levelsM t&e solution group &a% a &ig&er a%verse e!!ect in%e" t&an t&e nanoparticle group*

SPECI-IC AD2ANTAGES Many a%verse con%itions in t&e GIT must #e overcome to o#tain t&e %esire% results a!ter a%ministration o! nanoparticulates* +ot& aca%emia an% in%ustry &ave ma%e great e!!orts in t&is %irection over t&e last 4@ to 4: years) #ut more stu%ies are reCuire% to ac&ieve t&e results t&at so many groups are (or$ing !or* -rom t&e p&arE maceutical stan%point) most nanoparticulates targete% to t&e lymp& &ave increase% #ioavaila#ility versus t&e re!eree %rugM t&is is particularly apprecia#le !or la#ile %rugs an% molecules (it& poor solu#ility* ?ne !actor reCuiring improvement !or some NPSs is incorporation e!!iciency) (&ic& must #e increase% in or%er to reac& t&e reCuire% %ose* Stu%ies are also necessary (it& regar% to storage sta#ility* ,it& regar% to t&e %i!!erent NPSs) t&e &istory o! some o! t&em) suc& as %en%rimers) is s&ort an% t&us t&ere are relatively !e( in vivo on t&e oral route* Some polymeric #io%egra%a#le nanoparticles are alrea%y on t&e mar$et) alt&oug& to %ate only !or t&e parenteral route* Stu%ies on t&e oral route &ave c&ie!ly a%%resse% t&e a%ministration o! vaccine antigens) pepti%es) an% small proteins* Provi%e% t&at t&e polymer is selecte% appropriately Atype) molecular (eig&t) $in% o! preparationB) t&e results o#taine% in vivo &ave #een promising* ipi%E#ase% systems &ave ac&ieve% some o! t&e %esire% resultsM some antiprotease %rugs) ritonavir) an% saCuinavir) carrie% #y SAMBEDDS) are no( on t&e mar$et an% provi%e #etter #ioE availa#ility t&an t&e re!eree %rug A89B* Some pro%uction met&o%s o! S Ns %o not use any solvent) t&eir !easi#ility is relatively goo%) an% t&eir components are p&ysioE logically compati#le* S Ns loa%e% (it& a %rug ATo#ramycin or I%aru#icinB an% a%ministere% %uoE %enally s&o( #etter p&armaco$inetic parameters t&an t&e same %rug a%ministere% I2 as a solution* To#raES N a%ministere% %uo%enally permitte% a#sorption o! to#ramycin #y t&e GIT) interestingly) as t&is is a %rug still only a%ministere% #y t&e parenteral route* Interest in t&e oral a%ministration o! c&emot&erapeutics &as #een stimulate% #y t&e %iscovery t&at oral !luoropyrimi%ines &ave at least eCuivalent e!!icacy) as

849

Gasco

(ell as t&e potential to re%uce to"icity) compare% to a%ministration #y t&e I2 routeM using rational nanoparticulate %esign) several antineoplastic %rugs coul% #e %evelE ope% !or oral use A;:B* Alt&oug& stu%ies on nanoparticulates targeting t&e colon are relatively !e() some interesting results &ave #een ac&ieve% using liposomes A;4B an% P GA nanoparticles A;8B*

E2A UATI?N AND -UTURE PERSPECTI2ES T&e main goal o! a%ministration o! NPSs #y t&e oral route is to lo(er t&e %ose o! t&e %rug Aan% conseCuently to %iminis& its to"icityB as (ell as to improve patient comE pliance an% supply an easy a%ministration route* ?t&er aims may #e to %ecrease t&e !e%1!aste% varia#ility an% patientEtoEpatient varia#ility* E!!icient incorporation o! #ioactive molecules in NPSs reCuires inE%ept& stu%y* -actors t&at must #e e"amine% in or%er to o#tain t&e #est !ormulation !or t&e type o! NPSs selecte% are6 AiB su!!icient %rug loa%ing to ac&ieve t&erapeutic levelsM AiiB goo% translocation o! NPSs to lymp& Ai*e*) small si/e) #iocompati#le) #io%egra%a#le components) c&emicop&ysical sta#ility o! carrier an% %rug) /eta potential) etc*BM AiiiB sustaine%1controlle% %rug release !rom NPSsM an% AivB increase% oral #ioavaila#ility to en&ance e!!icacy* Many steps &ave alrea%y #een ta$en in t&ese %irections) #ut researc& must continue) varying t&e sur!ace properties o! NPSs) improving t&eir %rugEloa%ing capacity) an% testing ne( materials (it& little or no to"icity !or use as carriersM t&is last reCuisite is particularly important !or %rugs to #e ta$en c&ronically* An innovaE tive oralE%elivery re!ormulation o! a %rug coul% e"ten% its patent li!eM %evelopment o! ne( %elivery systems !or ol%) (ellE$no(n molecules) (&et&er natural or out o! patent) coul% lea% to re%uce% si%e e!!ects an% to more e!!icient t&erapy) (it& evi%ent social #ene!its*

RE-ERENCES
4* 0reuter '* Peroral a%ministration o! nanoparticles* A%v Drug Deliv Rev 4>>4M <6<4* 5* Hussain N) 'aitely +) -lorence AT* Recent a%vances in un%erstan%ing t&e upta$e o! micE roparticulates across t&e gastrointestinal lymp&atics* A%v Drug Deliv Rev 5@@4M :@64@<* 8* -lorence AT) Hussain N* Trancytosis o! nanoparticle an% %en%rimer systems6 evolving vistas* A%v Drug Deliv Rev 5@@4M :@6S;>* 9* Nis&io$a 3) 3os&ino H* ymp&atic targeting (it& nanoparticulate systems* A%v Drug Deliv Rev 5@@4M 9<6::* :* Hans M ) o(man AM* +io%egra%a#le nanoparticles !or %rug %elivery an% targeting* Curr ?pin Sol St Mater Sci 5@@5M ;684>* ;* -lorence AT* Issues in oral nanoparticles6 %rug carrier upta$e an% targeting* ' Drug Target 5@@9M 456;:* <* Uc&i%a '* Electron microscopic stu%y o! micro!ol% cells AM cellsB in normal an% in!lame% &uman appen%i"* Gastroenterol 'pn 4>==M 5865:4* =* ,atts P') Illum * Colonic %rug targeting* Drug Dev In% P&arm 4>><M 586=>8* >* Patri A0) Ma.oros I') +a$er 'R* Den%ritic polymer carriers !or %rug %elivery* Curr ?pin C&em +iol 5@@5M ;69;;* 4@* Gillies ER) -rac&et 'M* Den%rimers an% %en%ritic polymers in %rug %elivery* Drug Discov To%ay 5@@:M 4@68:* 44* Tripat&i P0) 0&opa%e AS) Nagaic& S) et al* Den%rimer gra!ts !or %elivery o! :E!luorouracil* P&arma/ie 5@@5M :<65;4* 45* +ala I) Hari&aran S) 0umar R* P GA nanoparticles in %rug %elivery6 t&e state o! t&e art* Crit Rev T&er Drug Carrier Syst 5@@9M 5468=<* 48* 2aut&ier C) Du#ernct C) -attal E) et al* PolyAal$ylcyanoacrylatesB as #io%egra%a#le materials !or #iome%ical applications* A%v Drug Deliv Rev 5@@8M ::6:4>*

Gastrointestinal Applications o! Nanoparticulate DrugEDelivery Systems

84:

49* Delie -) +lancoEPrieto M'* Polymeric particulates to improve oral #ioavaila#ility o! pepti%e %rugs* Molecules 5@@:M 4@6;:* 4:* Ta$eu$i H) 3amamoto H) 0a(as&ima 3* Mucoa%&esive nanoparticulate systems !or pepti%e %rug %elivery* A%v Drug Deliv Rev 5@@4M 9<68>* 4;* Damge C) 2ran$s H) +alsc&mit P) et al* PolyAal$ylcyanoacrylateB nanosp&eres !or oral a%ministration o! insulin* ' P&arm Sci 4>><M =;649@8* 4<* PintoEAlp&an%ary H) A#ou#a$ar M) 'allar D) et al* 2isuali/ation o! insulin loa%e% nanoE capsules6 in vitro an% in vivo stu%ies a!ter oral a%ministration to rats* P&arm Res 5@@8M 5@64@<4* 4=* i 3) 'ang H ) 'in 'E) et al* +ioa%&esive !luorescent microsp&eres as visi#le carriers !or local %elivery o! %rugsRupta$e o! insulin loa%e% PCE-+1P GA microsp&eres #y t&e gastrointestinal tract* Drug Deliv 5@@9M 44688:* 4>* Damge C) Ap&ra&amian) Marc&ais H) et al* PolyAal$ylcyanoacrylateB nanocapsules as a %elivery system !or octreocti%e) a long acting somatostatin analogue* ' P&arm P&armacol 4>><M 9>6>9>* 5@* Sang 3oo H) G(an Par$ T* +io%egra%a#le nanoparticles containing proteinH!atty aci% comple"es !or oral %elivery o! salmon calcitonin* ' P&arm Sci 5@@9M >869==* 54* ElES&a#ouri MH* Positively c&arge% nanoparticles !or improving t&e oral #ioavaila#ility o! cyclosporinEA* Int ' P&arm 5@@5M 59>64@4* 55* U#ric& N) Sc&mi%t C) +o%meier R) et al* ?ral evaluation in ra##its o! cyclosporinEloa%e% Eu%ragit RS or R nanoparticles* Int ' P&arm 5@@:M 5==64;>* 58* 'iao 3) U#ric& N) Marc&an%EArvier M) et al* In vitro an% in vivo evaluation o! oral &epaE rinEloa%e% polymeric nanoparticles in ra##its* Circulation 5@@5M 4@:658@* 59* -oster N) Hirst +H* E"ploiting receptor #iology !or oral vaccination (it& #io%egra%a#le particulates* A%v Drug Deliv Rev 5@@:M :<6984* 5:* -oo$s AR* Development o! oral vaccine !or &uman use* Curr ?pin Mol T&er 5@@@M 56=@* 5;* Ta#ata 3) Inoue 3) I$a%a 3* Si/e e!!ect on systemic an% mucosal immune responses in%uce% #y oral a%ministration o! #io%egra%a#le microsp&eres* 2accine 4>>;M 4964;;<* 5<* Gutierro I) Hernan%e/ RH) Igartua H) et al* Si/e %epen%ent immune response a!ter su#cutaneous) oral an% intranasal a%ministration o! +SA loa%e% nanosp&eres* 2accine 5@@5M 546;<* 5=* Allen TM* iposomes opportunity in %rugE%elivery* Drugs 4>><M :9Asuppl 9B6=* 5>* +arratt GM* T&erapeutic applications o! colloi%al %rug carriers6 p&ysical structure to t&erapeutic applications* P&arm Sci Tec&nol To%ay 5@@@M 864;8* 8@* Taira MC) C&iaramonti NS) -ecuc& 0M) et al* Sta#ility o! liposomal !ormulations in p&ysiological con%itions !or oral %rug %elivery* Drug Deliv 5@@9M 446458* 84* Cansell M) Nac$a -) Com#e N* Marine lipi%E#ase% liposomes increase in vivo -A #ioavaila#ility* ipi%s 5@@8M 8=6::4* 85* Ta$euc&i H) Matsui 3) 3amamoto H) et al* Mucoa%&esive properties o! car#opol or c&itosanE coate% liposomes an% t&eir e!!ectiveness in oral a%ministration o! calcitonin to rats* ' Control Release 5@@8M =;658:* 88* i H) Son 'H) Par$ 'S) et al* Polyet&ylene glycolEcoate% liposomes !or oral %elivery o! recom#inant &uman epi%ermal gro(t& !actor* Int ' P&arm 5@@8M 5:=644* 89* 3ama#e 0) 0ato 3) ?nis&i H) et al* Potentiality o! %ou#le liposomes containing salmon calcitonin as an oral %osage !orm* ' Control Release 5@@8M =>695>* 8:* Gers&ani$ T) +enita S* Sel!E%ispersing lipi% !ormulations !or improving oral a#sorption o! lipop&ilic %rugs* Eur ' P&arm +iop&arm 5@@@M :@64<>* 8;* a(rence M') Rees GD* MicroemulsionE#ase% me%ia as novel %elivery systems* A%v Drug Deliv Rev 5@@@M 9:6=>* 8<* Gao P) Rus& PD) P!un% ,) et al* Development o! a supersatura#le SEDDS !ormulation o! paclita"el (it& improve% #ioavaila#ility* ' P&arm Sci 5@@8M >5658=;* 8=* Gursoy RN) +enita S* Sel!Eemulsi!ying %rug %elivery systems ASEDDSB !or improve% oral %elivery o! lipop&ilic %rugs* +iome% P&armacot&er 5@@9M :=64<8* 8>* 0ang +0) ee 'S) C&ron S0) et al* Development o! sel!Emicroemulsi!ying %rug %elivery systems ASMEDDSB !or oral #ioavaila#ility en&ancement o! simvastatin in #eagle %ogs* Int ' P&arm 5@@9M 5<96;:* 9@* Muller RH) 0arsten M) Sven G* Soli%Hlipi% nanoparticles AS NB !or controlle% %rug %eliveryRa revie( o! t&e state o! t&e art* Eur ' P&arm +iop&arm 5@@@M :@64;4*

84;
94* +ummer PM* C&emical consi%eration o! lipi%E#ase% oral %rug %eliveryRsoli%Hlipi% nanoparticles* Crit Rev T&er Drug Carrier Syst 5@@9M 5464* 95* Man.unat& 0) Re%%y 'S) 2en$ates(arlu 2* Soli%Hlipi% nanoparticles as %rug %elivery systems* Met&o%s -in% E"p Clin P&armacol 5@@:M 5<645<* 98* 3ang S) V&u ') u 3) et al* +o%y %istri#ution o! camptot&ecin soli%Hlipi% nanoparticles a!ter oral a%ministration* P&arm Res 4>>>M 4;6<:4* 99* Demirel M) 3a/an 3) Muller RH) et al* -ormulation an% in vitroHin vivo evaluation o! piri#e%il soli%Hlipi% micro an% nanoparticles* ' Microencapsul 5@@4M 4=68:>* 9:* Hu ) Tang W) Cui -* Soli%Hlipi% nanoparticles to improve #ioavaila#ility o! poorly solu#le %rugs* ' P&arm P&armacol 5@@9M :;64:5<* 9;* Man.unat& 0) 2en$ates(arlu 2* P&armaco$inetics) tissue %istri#ution an% #ioavaila#ility o! clo/apine soli%Hlipi% nanoparticles a!ter intravenous an% intra%uo%enal a%minisE tration* ' Control Release 5@@;M496;85 9<* +e$erman T) Golenser ') Dom# A* Cyclosporin nanoparticulate liposp&eres !or oral a%ministration* ' P&arm Sci 5@@9M >8645;9* 9=* Sie$man +) ,estesen 0* Investigation on soli%Hlipi% nanoparticles prepare% #y precipiE tation in o1( emulsion* Eur ' P&arm +iop&arm 4>>;M 9864@9* 9>* Hu -7) 3uan H) V&ang RH) et al* Preparation o! soli%Hlipi% nanoparticles (it& clo#etasol propionate #y a novel solvent %i!!usion met&o% in aCueous system an% p&ysicoc&emical c&aracteri/ation* Int ' P&arm 5@@5M 58>6454* :@* Gasco MR* Met&o% !or pro%ucing soli%Hlipi% microsp&eres &aving narro( si/e %istri#uE tion* US Patents 4>>8M:)5:@)58;* :4* Gasco MR* Soli%Hlipi% nanosp&eres !rom (arm microemulsions* P&arm Tec& Eur 4>><M >6:5* :5* Gasco MR* Soli%Hlipi% nanoparticles !or %rug %elivery* P&arm Tec& Eur 5@@@M 48685* :8* Serpe ) aurora S) Pi//imenti S) et al* C&olesteryl #utyrate soli%Hlipi% nanoparticles as a #utyric aci% proE%rug6 e!!ects on cell proli!eration) cellEcycle %istri#ution an% cEmyc e"pression in &uman leu$emic cells* Anticancer Drugs 5@@9M 4:6:5:* :9* Serpe ) Catalano MG) Cavalli R) et al* Cytoto"icity o! anticancer %rugs incorporate% in soli%Hlipi% nanoparticles on HTE5> colorectal cancer cell line* Eur ' P&arm +iop&arm 5@@9M :=6;<8* ::* Dian/ani C) Cavalli R) Vara GP) et al* C&olesteryl #utyrate nanoparticles en&ances #utyrate in&i#ition o! neutrop&ils a%&esion to en%ot&elium* +r ' P&armacol* 5@@;M49=6;9= :;* +argoni A) Cavalli R) Caputo ?) et al* Soli%Hlipi% nanoparticles in lymp& an% plasma a!ter %uo%enal a%ministration to rats* P&arm Res 4>>=M 4:6<9:* :<* Cavalli R) Vara GP) Caputo ?) et al* Transmucosal transport o! to#ramycin incorporate% in S N a!ter %uo%enal a%ministration to rats* Part I* A p&armaco$inetic stu%y* P&armacol Res 5@@@M 956:94* :=* Cavalli R) +argoni A) Po%io 2) et al* Duo%enal a%ministration o! soli%Hlipi% nanoparticles loa%e% (it& %i!!erent percentages o! to#ramycin* ' P&arm Sci 5@@8M >564@=:* :>* +argoni A) Cavalli R) Vara GP) et al* Transmucosal transport o! to#ramycin incorporate% in soli%Hlipi% nanoparticles AS NB a!ter %uo%enal a%ministration to rats* Part II* Tissue %istri#ution* P&armacol Res 5@@4M 9869><* ;@* Vara GP) +argoni A) Cavalli R) et al* P&armaco$inetics an% tissue %istri#ution o! i%aruE #icin loa%e% soli%Hlipi% nanoparticles a!ter %uo%enal a%ministration to rats* ' P&arm Sci 5@@5M >464@59* ;4* Wing ) Da(ei C) iping W) et al* ?ral colonEspeci!ic %rug %elivery !or #ee venom pepti%e6 %evelopment o! a coate% calcium alginate gel #ea%sEentrappe% liposome* ' Control Release 5@@8M >865>8* ;5* amprec&t A) U#ric& N) 3amamoto H) et al* Si/e %epen%ent targeting o! micro an% nanoparticulate carriers to t&e in!lame% colonic mucosa* P&arm Res 5@@4M 4=6<==* ;8* amprec&t A) U#ric& N) 3amamoto H) et al* Design o! rolipram loa%e% nanoparticles6 comparison o! t(o preparation met&o%s* ' Control Release 5@@4M <465><* ;9* amprec&t A) U#ric& N) 3amamoto H) et al* +io%egra%a#le nanoparticles !or targete% %rug %elivery in treatment o! in!lammatory #o(el %isease* ' P&armacol E"p T&er 5@@4M 5>>6<<:* ;:* Royce ME) Ho!! PM) Pa/%ur R* Novel oral c&emot&erapy agents* Curr ?ncol Rep 5@@@M 5684*

Gasco

5@

Nanoparticles as A%.uvantE2ectors !or 2accination

Socorro Espuelas
Department o! P&armacy an% P&armaceutical Tec&nology) University o! Navarra) Pamplona) Spain

Carlos Gama/o
Department o! Micro#iology) University o! Navarra) Pamplona) Spain

MarXa 'osG +lancoEPrieto an% 'uan M* Irac&e

Department o! P&armacy an% P&armaceutical Tec&nology) University o! Navarra) Pamplona) Spain

INTR?DUCTI?N oo$ing !or success!ul vaccines &as #ecome one o! t&e %riving !orces in glo#al &ealt&* T&e &istory o! vaccination is ric& in many trials to treat numerous in!ectious %iseases* 2accinations are responsi#le !or appro"imately 5:[ o! glo#al mortality) especially in c&il%ren younger t&an !ive years A4B* ive attenuate% vaccines are still #y !ar t&e most utili/e% !or t&eir e!!iciency (it& respect to inactivate% Ai*e*) #acterinsB an% acellular or su#unit ones) #ut is it necessary to run t&e ris$ o! using live vaccinal strainsT Are t&ere not ot&er sa!er alternativesT ConseCuently) t&e nee% !or #etter vaccines clearly e"ists) #ut in spite o! t&e progress ma%e %uring t&e last !e( years) Qt&e i%eal vaccineS against many severe in!ectious %iseases still is not availa#le* T&e i%eal live vaccine &as to comply) at least) (it& t&e !ollo(ing reCuisites6 AiB innocuous !or t&e vaccinate% &ost) AiiB provi%e longEterm protection (it& a single vaccination) AiiiB minimi/e t&e longEterm pro%uction o! anti#o%ies) (&ic& may interE !ere (it& t&e current sero%iagnosis tests o! natural !iel% in!ections) AivB avoi% t&e contamination o! !oo% or any e!!ect in t&e environment) AvB nonpat&ogenic !or &umans or any ot&er plant or animal &ost) an% AviB #e #iologically sta#le* In conseCuence) t&e actual ten%encies are oriente% to(ar%s t&e %evelopment o! ne( vaccines containing per!ectly c&aracteri/e% antigens) rigorously controlle% %uring all t&e steps concerning t&eir preparation) an% sa!e* Ho(ever) t&e ne( vaccines o! t&e ne( #iotec&nology era su!!er) in general) !rom immunogenicity) reCuiring t&e use o! a%.uvants A5)8B* T&e a%.uvants &ave various !unctions) suc& as !acilitate t&e passage t&roug& cellular #arriers an% t&e stimulation o! t&e cells o! t&e immune system* T&e nanoparE ticles may #e consi%ere% as a%.uvants t&at can accomplis& t&ese reCuisites) #eing a#le even to in%uce a response at t&e mucosa level given t&eir capacity to interact (it& it* ,e (ill revie( t&ese carrier a%.uvants in t&is c&apter*

IMMUN?AD'U2ANTS A%.uvants are compoun%s t&at en&ance or mo%ulate t&e immunogenicity o! coa%E ministere% antigens* Speci!ic antigen1a%.uvant com#inations pre!erentially in%uce type 4 or type 5 cyto$ine responses* T&e T&4 su#set secretes cyto$ines) inclu%ing interleu$inE5 AI E5B an% inter!eronEq AI-NEqB) to assist in cellEme%iate% immune
84<

84=

Espuelas et al*

response* T&e T&5 su#set assists pre!erentially in anti#o%y immune responses a!ter secreting cyto$ines inclu%ing interleu$inE9 AI E9B* T&e mec&anism o! t&e a%.uvant action is in some cases) still un$no(n A9B* In spite o! large lists o! compoun%s an% strategies %escri#e% as a%.uvants) t&e only -DAEapprove% a%.uvant is alum) a genE eral name !or t&e aluminumE#ase% mineral salt A:B* It yiel%s a reasona#le anti#o%y response AT&5B) #ut it %oes not in%uce a T&4 pro!ile* T&4 immunity is essential !or protection against many in!ective organisms Ae*g*) intracellular parasites) inclu%ing virus an% some pro$aryotic an% eu$aryotic microorganismsB an% even to limit allerE genic processes A;B* Moreover) aluminum a%.uvants &ave s&o(n limitations in t&eir applica#ility in vaccines #ase% on smallEsi/e% pepti%es or antigenEe"pressing DNA A<)=B* Anot&er limitation lies in t&e !act t&at aluminumEa%sor#e% vaccines are !rostE sensitive an% t&us not lyop&ili/a#le* T&ere!ore) a large num#er o! ne( a%.uvants are un%er investigation) suc& as t&e particulateE%elivery systems* It is (ell esta#lis&e% t&at a%.uvants can en&ance t&e speci!ic immune response o! t&e coa%ministere% antigens #y t(o ma.or mec&anisms A8)>B6

4* T&ey increase t&e antigen upta$e #y antigenEpresenting cells AAPCsB AQ%eliverySB* T&e a%.uvants are %irectly engul!e% #y APCs or t&ey !orm a %epot o! t&e antigens t&at prolong t&eir e"posure an% so t&e c&ance to #e ta$en up #y APCs* 5* Anot&er group o! a%.uvants AQimmunopotentiatorsSB %irectly activates innate immune cells* To t&is category #elong in!lammatory stimuli A!reCuently associate% (it& vaccine a%ministrationB) cyto$ines) CD9@ ) an% patternEassociate% molecular pat&ogens APAMPs6 molecular patterns s&are% #y multiple microorganisms) not presente% in mammalian cells) t&at activate immune cells t&roug& interaction (it& patternErecognition receptorsB* ipopolysacc&ari%e) murine) !lagellin) an% ot&er main structural components o! #acterial cells are e"amples o! PAMPs use% empirically !or a long time A9B*

ParticulateE%elivery systems #elong to t&e !irst category o! a%.uvants A4@)44B) alt&oug& t&eir activity as immunopotentiators cannot #e completely %iscar%e%* NAN?PARTIC ES AND ANTIGENEPRESENTING CE S

Numerous reports in%icate t&at particulate antigens are naturally capture% #y APCs) #ut also t&at si/e is a %ecisive !actor !or an a%eCuate upta$e* Den%ritic cells ADCsB) a para%igm o! APCs) are a#le to capture particles in vitro an% in vivo #ut to a lesser e"tent t&an macrop&ages an% t&e optimal si/e is smaller t&an t&at %escri#e% !or macrop&ages Ain t&e range o! :@@ nm or #elo(B A45)48B* In a%%ition) t&e sur!ace c&arge an% &y%rop&o#icity o! nanoparticles also mo%i!y t&eir upta$e A49B* ?nce inoculate%) t&e particle sur!ace c&anges #y t&e a%sorption o! en%ogenous proteins presente% in serum an% in t&e interstitial !lui% at t&e a%ministration site* T&e more &y%rop&o#ic t&e sur!ace is) t&e easier t&e coating process #y t&e %ominant proteins Aopsonins as IgG or C8 complement !actorsB can #e A4:B* Macrop&ages an% DCs &ave receptors !or -c !raction o! IgG) C8 complement component) or t&e CElectins !amily) suc& as mannose receptors A4;B* Coating nanocarriers (it& t&ese ligan%s &as #een proven a goo% strategy !or improving antigen %elivery an% targeting* -urt&ermore) t&ese ligan%s &ave in!luence in t&e pro!ile o! t&e elicite% immune response A4<B*

THE -ATE ?- NAN?PARTIC ES A-TER ADMINISTRATI?N Parenteral Immuni/ation Nanoparticles a%ministere% intramuscularly AIMB or su#cutaneously ASCB are e"pecte% to remain essentially at t&e site o! a%ministration an% control t&e release o!

Nanoparticles as A%.uvantE2ectors !or 2accination

84>

t&e loa%e% antigen* Slo(ly %egra%a#le polymers Ni*e*) polyAlacti%e aci%B AP AB) polyAlacti%eEcoEglycoli%eB AP GAB) polyAmet&ylmet&acrylateB APMMAB) polyAal$ylc yanoacrylatesB) or caprolactone polymers APECBO are suita#le !or t&is application as t&ey provi%e a%eCuately prolonge% antigen release* Nanoparticles o! PMMA (ere claime% to #e #io%egra%a#le a!ter SC or IM in.ection an% s&o(n to e"&i#it very po(er!ul a%.uvant properties !or a num#er o! antigens* T&us) t&ey (ere a#le to improve t&e anti#o%y response an% t&e protection against in!luen/a c&allenge in comparison to t&e classical a%.uvant aluminum &y%ro"i%e A4=B* Desai an% co(or$ers A4>B &ave %emonstrate% a%.uvant properties o! P GA nanoparticles containing encapsulate% stap&ylococcal enteroto"oi% +* Ho(ever) t&e systemic anti#o%y immune response elicite% in t&e animals in.ecte% SC (it& t&ese nanoparticles (as compara#le to t&at o#taine% !ollo(ing in.ection o! alum* T&e same group A5@B s&o(e% in a !urt&er stu%y (it& tetanus to"oi% ATTB) t&at coin.ecting su#cutaneously to rats TTEalum along (it& TTEloa%e% P GA nanoparticles in%uces a synergistic immune response* T&e com#ination in%uce% a !our!ol% greater mean serum antiETT IgG response t&an a single in.ection o! TT nanoparticles alone* In anot&er stu%y) a single immuni/ation o! P A nanoparticles entrapping immunoreactive TT a%ministere% IM to rats elicite% antiETT anti#o%ies t&at perE siste% !or more t&an !ive mont&s A54B* Recently) Sc&Fll et al* A55)58B %emonstrate% t&e e!!ectiveness o! P GA nanoparE ticles in immunot&erapy o! allergy to #irc& pollen A+etEv4B in +A +1c mice* P GA nanoparticles loa%e% (it& +etEv4) t&e ma.or allergen) en&ance% t&e rig&t immunoE genicity o! t&e allergen an% %i% not lea% to novel sensiti/ation o! nave animals* T&e aut&ors assume% t&at vaccination (it& P GA nanoparticles can counter#alance an ongoing T&5 response to +etEv4) rea%%ressing it to a T&4Eprotective response*

Mucosal Immuni/ation Alt&oug& most vaccines tra%itionally &ave #een a%ministere% #y IM or SC in.ection) mucosal a%ministration o! vaccines o!!ers a num#er o! important a%vantages) inclu%ing easier a%ministration) re%uce% a%verse e!!ects) an% t&e &ig&er mucosal immune response ac&ieve%* Anot&er important a%vantage is t&at t&e vast ma.ority o! in!ections are !rom a mucosal sur!ace an% protection at t&is port o! entry level is essential Agastrointestinal) respiratory) an% urogenital tractsB*

?ral Route T&e most attractive) easiest) an% most accepta#le route !or mucosal immuni/ation is t&e oral one* Ho(ever) as a result o! t&e aci%ity in t&e stomac&) an e"tensive range o! %igestive en/ymes in t&e intestine) an% a protective coating o! mucus t&at limits access to t&e mucosal epit&elium) t&e oral %elivery o! vaccines remains a c&allenge (&ere it is %i!!icult to ac&ieve &ig& an% repro%uci#le e!!ects* In or%er to solve t&ese %i!!iculties) nanoparticles may #e use!ul to protect antigens an% !acilitate t&e interE action (it& components o! t&e gut mucosa #y a mec&anism o! #ioa%&esion* In mice) oral immuni/ation (it& P GA nanoparticles &as #een s&o(n to in%uce potent mucosal an% systemic immunity to entrappe% antigens A59)5:B* -or instance) t&e encapsulation o! Helyco#acter pylori antigens in P GA nanopartiE cles in%uce% signi!icantly speci!ic mucosal IgA response as (ell as serum IgG4 an% IgG5# responses A59B* Anot&er interesting stu%y (as #ase% on t&e use o! TT associate% (it& sul!o#utylate%polyAvinyl alco&olBEgra!tEpolyAlacti%eEcoEglycoli%eB nanoparticles) t&at a given #y peroral AP?B or intranasal AINB route in%uce% speci!ic IgG an% IgA in mice in a repro%uci#le manner A5;B*

85@

Espuelas et al*

?ral vaccination using c&itosan nanoparticles as vector !or To"oplasma gon%ii GRA 4 pDNA prime% an anti#o%yEme%iate% immune response) alt&oug& not t&e cellular protective one A5:B* Similarly) it (as s&o(n t&at mucosal immunity to(ar%s Tat protein coul% #e triggere% in mice #y oral or rectal route (&en t&is protein is loa%e% in c&itosan nanoparticles* Tat protein plays an essential role in HI2E4 replicaE tion an% also participates in TEcell immunosuppression A5<B* In !act) sera !rom mice immuni/e% (it& c&itosan nanoparticles in&i#ite% t&e activity o! Tat protein) (&ic& is a prereCuisite !or t&e %evelopment o! an antiEAIDSEprotective vaccine A5=B* In a%%ition) t&is vaccine also in%uce% cellEme%iate% immunity A5=B* More recently) c&itosan nanoparticles &ave also #een prove% e!!ective as a%.uE vant immunot&erapy A5>)8@B* T&us) DNA enco%ing allergens !rom &ouse mites (ere comple"e% (it& c&itosan an% %elivere% orally) in%ucing a success!ully speci!ic T&4E immune response A8@B* -inally) o! special interest in immunot&erapy o! allergy an% treatment o! %iseases in%uce% #y a %is#alance in t&e T&41T&5 response suc& as autoimmune %isor%ers) is t&e capacity o! t&e a%.uvant to in%uce I E4@ A84)85B* T&us) pegylate% nanoparticles containing oval#umin (ere a#le to in%uce signi!icant an% sustaine% levels o! I E4@ !or at least si" (ee$s in mice A88B* In any case) it appears t&at QconventionalS nanoparticles o!!er a limite% a#ility as a%.uvants !or oral vaccination* An interesting strategy to solve t&is pro#lem may #e t&e use o! nanoparticles coate% (it& #acterial a%&esins in or%er to o#tain a simiE lar %istri#ution (it&in t&e gut to t&at o#serve% !or t&e pat&ogen* In t&is conte"t) (e &ave evaluate% t&e properties o! Gantre/ nanoparticles coate% (it& !lagellin !rom Salmonella enteriti%is A89B* In vivo) t&ese carriers %isplaye% a similar %istri#ution (it&in t&e gut as t&e (&ole #acteria interacting strongly (it& t&e enterocytes an% PeyerPs patc&es A-ig* 4B*

Nasal Route In contrast to oral a%ministration) nasally a%ministere% vaccines &ave to #e transE porte% over a very small %istance an% are not e"pose% to lo( pH values an% %egra%E ing en/ymes* In a%%ition) t&e nasal mucosa s&o(s a relatively goo% permea#ility !or proteins) compare% to t&e gastrointestinal tract* -urt&ermore) nasal %elivery o! antigens allo(s a %irect access to t&e nasalEassociate% lymp&oi% tissue ANA TB) t&e ma.or site o! antigen upta$e an% presentation) (&ereas oral %elivery reCuires %i!!uE sion o! t&e antigen carriers to e"tensively (i%esprea% lymp&oi% aggregates* 2arious aut&ors &ave stu%ie% t&e nasal mucosa as a potential route o! vaccinaE tion against tetanus) as nanoparticles &ave #een %emonstrate% to #e a use!ul vector

-IGURE 4 ASee color insert*B 2isuali/ation o! !lagellinEcoate% nanoparticles Are% %otsB in t&e !ollicleEassociate% epit&elium o! PeyerPs patc&es #y !luorescence* -lagellin o! S* enteriti%is (as use% to coat Gantre/ nanoparticles Ala#ele% (it& r&o%amine + isot&iocyanateB an%) t&us) o#taine% a%.uvant vectors a#le to sprea% (it&in t&e gut in a (ay similar to t&e (&ole #acteria* Source6 -rom Re!* 89*

Nanoparticles as A%.uvantE2ectors !or 2accination

854

system !or TT* T&e intranasal application o! t&at !ormulation resulte% in signi!iE cantly increase% IgG an% IgA levels compare% to nontreate% animals A5;)8:)8;B* -or ot&er clinically relevant antigens) it (as !oun% t&at) in general) intranasal vaccinaE tion reCuires !e(er a%ministrations at lo(er %osing levels to stimulate immune responses t&an orally* T&is !act can #e e"plaine% #y a lo(er e!!icient particle upta$e !rom t&e gut an% a more %estructive gastrointestinal environment !or proteins A5;B* Ta#le 4 summari/es t&ese e"perimental (or$s*

TA+ E 4 Some o! Stu%ies Using Nanoparticles as 2accine A%.uvants !or Nasal A%ministration -ormulation S+EP2A EgE P GA NP Antigen Species Results Re!erences A5=B

TetanusSmallest to"oi% particles in%uce% Mice t&e most signi!icant anti#o%y responses Tetanus to"oi%RatIntranasal a%ministration o!C&itosan NP) c&itosan NP !ormulationCSEP GA NP) provi%e% &ig& an% longEP AHPEG NP lasting immune response P A NP) P AH Tetanus to"oi%MicePegylation increases t&e PEG NPsta#ility o! resulting nanoparticles an% elicite% &ig&er anti#o%y levels C&itosan NPTetanus to"oi%Mice; mont&s posta%ministration) t&e IgA response (as signi!icantly &ig&er t&an !or a control vaccine P AHPEG NPTetanus to"oi%RatA %ecrease in t&e si/e improves t&e transport across t&e nasal mucosa P GA NPIn!luen/a virusRatSmaller nanoparticles antigensprovi%e% #etter immuni/aE tion t&an larger ones MiceEncapsulation o! antigen inP GA NP)+ovine paraE P GA NP provi%e% &ig&erPMMA NPin!luen/a type 8 levels o! speci!ic anti#o%iesproteins t&an a%sorption in PMMA NP Polystyrene NP Concanavalin AEMice)2accines provi%e% &ig& inactivate% HI2E4macaCueimmune responses an% provi%e% signi!icant protection against intravagiE nal c&allenge MiceIn%uction o! protectiveCalciumHerpes simple" immunity against intravagiEp&osp&ate NPvirus typeE5 nal c&allengeglycoprotein MiceImmuni/ation in%uce%C&itosan NPpDNA e"pressing protection against c&allengeeit&er &emagglutiE (it& in!luen/a A virusnin HA or nuclear protein o! t&e in!luen/a A virus

A8<B

A8;B

A8=B

A8:B

A8>B

A9@B

A94)95B

A98B

A99B

A##reviations6 CSEP GA NP) c&itosanEcoate% polyAlactic aci%Hglycolic aci%B nanoparticlesM P A NP) polyAlactic aci%B nanoparticlesM P AHPEG) polyAet&ylene glycolBEcoate% polyAlactic aci%B nanoparticlesM P GA) polyAlacti%eE coEglycoli%eB nanoparticlesM PMMA NP) polymet&ylmet&acrylate nanoparticlesM S+EP2A EgEP GA NP) sul!o#utylate% polyAvinyl alco&olBEgra!tEpolyAlacti%eEcoEglycoli%eB nanoparticles*

855

Espuelas et al*

In contrast) pegylation appears to #e a goo% strategy to en&ance t&e a#ility o! nanoparticles as a%.uvants !or nasal vaccination* In !act) IgG levels elicite% #y TT loa%e% in P AHPEG nanoparticles (ere signi!icantly &ig&er an% longerElasting t&an t&ose correspon%ing to #ot& t&e !lui% vaccine an% conventional nanoparticles A9:B* Moreover) t&is potential o! pegylate% nanoparticles (as also %emonstrate% !or a mo%el DNA nasal vaccine) (&ere a single nasal %ose o! DNA PEGEnanoparticles le% to a signi!icant anti#o%y response to t&e enco%e% protein A8<)8=B* All o! t&ese results concerning pegylate% nanoparticles can #e %irectly relate% to t&e a#ility o! PEG coating to !acilitate t&e internali/ation o! nanoparticles t&roug& a given epit&elium A88)8<)9;B* It appears t&at t&e PEG coating &as a role in sta#ili/ing nanoparticles in mucosal !lui%s) !acilitating t&e transport o! t&e nanoencapsulate% antigen an%) &ence) eliciting a &ig& an% longElasting immune response* Comparing PEGEnanoparticles (it& c&itosanEcoate% nanoparticles) it (as o#serve% t&at PEGEnanoparticles (ere more e!!icient t&an c&itosanEcoate% nanoparE ticles in !acilitating t&e transport o! t&e associate% antigen ATTB t&roug& t&e mucosa* T&e e"planation (as !oun% in t&e %i!!erent interaction mec&anisms o! #ot& types o! mo%i!ie% nanoparticles (it& t&e nasal epit&elium A8:B* In particular) PEGE nanoparticles %i% not interact (it& t&e mucin) (&ereas t&ose coate% (it& c&itosan (ere %esigne% to stic$ to t&e mucus layer A9<)9=B) impe%ing t&e transport across t&e nasal epit&elium* Again) mo%i!ication o! t&e p&ysicoc&emical c&aracteristics o! nanoparticles may !acilitate t&e %i!!usion o! nanoparticles t&roug& t&e protective mucus layer an% t&eir interaction (it& t&e NA T* In t&is conte"t) it &as #een %escri#e% t&at a %ecrease on t&e si/e o! P GA nanoparticles resulte% in an increase in t&eir a#ility to reac& t&e NA T an% to potentiate t&e immune response A8>B* In anot&er interesting (or$) t(o %i!!erent types o! nanoparticles (ere evaluate% as a%.uvants !or nasal immuni/ation against #ovine parain!luen/a type 8 virus* In t&is (or$) viral proteins (ere eit&er encapsulate% in P GA nanoparticles or a%sor#e% in PMMA nanoparticles A99B* Mice immuni/e% (it& a single %ose o! P GA nanoparticles %evelope% &ig&er levels o! virusEspeci!ic anti#o%y t&an mice immuE ni/e% (it& t&e PMMA vaccine or (it& t&e viral proteins alone* In any case) t&ese results can #e %ue to t&e met&o% o! association #et(een antigen an% nanoparticle Aencapsulation vs* a%sorptionB or to t&e intrinsic a#ility o! t&e polymer to act as a%.uvant AP GA vs* PMMAB A99B* Miya$e an% co(or$ers %escri#e% t&e a#ility o! polystyrene nanoparticles coate% (it& Concanavalin A to e!!iciently capture HI2E4 particles A9@B* A!ter intranaE sal immuni/ation) t&is !ormulation in%uce% a &ig& vaginal IgA anti#o%y response in mice* In macaCues) a!ter a series o! si" intranasal immuni/ations (it& t&is !ormuE lation an% intravaginal c&allenge) only 88[ o! animals #ecame in!ecte%* Similar results (ere o#taine% (it& a %erive% vaccine o#taine% #y mi"ing ConAENP (it& inactivate% HI2E4 particles A94B* In mice) nasal immuni/ation (it& &erpes simple" virus typeE5 glycoprotein in calcium p&osp&ate nanoparticles in%uce% protective immunity against an intravagE inal c&allenge A95B* Also in mice) mucosal immuni/ation (it& c&itosanHDNA nanoparticles in%uce% protection against c&allenge (it& in!luen/a A virus A98B* Recently) a c&itosanEin!luen/a vaccine (as !oun% e!!ective in &umans (&en a%minE istere% #y t&e nasal route A9>B* ?verall) it is reasona#le to consi%er t&at t&e nasal route seems to #e more promising !or vaccination in mice t&an t&e oral one !or nanoparticle vaccine a%minE istration A5;B*

Nanoparticles as A%.uvantE2ectors !or 2accination

858

?t&er Mucosal Routes -e( recent stu%ies &ave !ocuse% on pulmonary immuni/ation an% t&e involvement o! t&e pulmonary immune system in eliciting protective immune responses against in&ale% pat&ogens* In t&is conte"t) t&e pulmonary a%ministration o! pDNA Aenco%E ing protective epitopes !rom Myco#acterium tu#erculosisB loa%e% in c&itosan nanoE particles in%uce% increasing levels o! inter!eronEgamma compare% to eit&er a control !ormulation or t&e more !reCuently use% intramuscular immuni/ation route A:@B* In contrast) calcium p&osp&ate ACPB nanoparticles) containing HS2E5 viral proteins) &ave #een prove% e!!ective in en&ancing t&e mucosal immune response against t&e virus* In mice) immuni/e% intravaginal immuni/ation (it& HS2E5 loa%e% in CP nanoparticles in%uce% &ig& levels o! HS2Especi!ic mucosal IgA an% IgG in vaginal lavage !lui%s* -urt&ermore) mice vaccinate% (it& t&is !ormulation (ere protecte% against c&allenge) (it& &ig&er survival rates an% less clinical in!ecE tion t&an unvaccinate% controls A95B* In summary) nanoparticles containing entrappe% or a%sor#e% antigens are #eing investigate% as vaccine a%.uvant alternatives to t&e currently use% alum (it& t&e o#.ective o! %eveloping #etter vaccine a%.uvants an% minimi/ing t&e !reCuency o! immuni/ation* Ho(ever) little success &as #een prove% #y t&e oral a%ministraE tion o! antigens or allergens in nanoparticles* Accumulate% e"perimental evi%ence suggests t&at simple encapsulation o! vaccines into nanoparticles is unli$ely to result in t&e success!ul %evelopment o! oral vaccines) an% improvements in t&e current tec&nology are clearly nee%e%* In contrast) t&e nasal route seeme% to #e more promising !or vaccination t&an t&e oral one !or nanoparticle vaccineE#ase% a%ministration*

RE-ERENCES
4* 0ieny MP) E"cler ' ) Girar% M* Researc& an% %evelopment o! ne( vaccines against in!ecE tious %iseases* Am ' Pu#lic Healt& 5@@9M >964>84* 5* ima 0M) %os Santos SA) Ro%rigues 'M) et al* 2accine a%.uvant6 it ma$es t&e %i!!erence* 2accine 5@@9M 55658<9* 8* ?PHagan DT) 2aliante NM* Recent a%vances in t&e %iscovery an% %elivery o! vaccine a%.uvants* Nat Rev Drug Discov 5@@8M 56<5<* 9* S&ei$& NA) alES&amisi M) Morro( ,'* Delivery systems !or molecular vaccination* Curr ?pin Mol T&er 5@@@M 568<* :* Clements C') Gri!!it&s E* T&e glo#al impact o! vaccines containing aluminium a%.uvants* 2accine 5@@5M 5@659* ;* in%#la% E+) El&ay M') Silva R) et al* A%.uvant mo%ulation o! immune responses to tu#erculosis su#unit vaccines* In!ect Immun 4>><M ;:6;58* <* -rancis M') -ry CM) Ro(lan%s D') et al* Immune response to uncouple% pepti%es o! !ootE an%Emout& %isease virus* Immunology 4>=<M ;464* =* 0(issa M) in%#la% E+) Sc&irm#ec$ R) et al* CoE%elivery o! a DNA vaccine an% a protein vaccine (it& aluminium p&osp&ate stimulates a potent an% multivalent immune response* ' Mol Me% 5@@8M =46:@5* >* Sc&i.ns 2E* Immunological concepts o! vaccine a%.uvant activity* Curr ?pin Immunol 5@@@M 4569:;* 4@* 0ersten G) Hirsc&#erg H* Antigen %elivery systems* E"pert Rev 2accines 5@@9M 869:8* 44* Ulmer '+* En&ancement o! vaccine potency t&roug& improve% %elivery* E"pert ?pin +iol T&er 5@@9M 964@9:* 45* T&iele ) Mer$le HP) ,alter E* P&agocytosis o! synt&etic particulate vaccine %elivery systems to program %en%ritic cells* E"pert Rev 2accines 5@@5M 4654:* 48* T&iele ) Mer$le HP) ,alter E* P&agocytosis an% p&agosomal !ate o! sur!aceEmo%i!ie% microparticles in %en%ritic cells an% macrop&ages* P&arm Res 5@@8M 5@6554H55=*

859
49* Reece 'C) 2ar%a"is N') Mars&all 'A) et al* Upta$e o! HI2 an% late" particles #y !res& an% culture% %en%ritic cells an% monocytes* Immunol Cell +iol 5@@4M <>65::* 4:* Allemann E) Gravel P) erou" 'C) et al* 0inetics o! #loo% component a%sorption on polyA%)lElactic aci%B nanoparticles6 evi%ence o! complement C8 component involvement* ' +iome% Mater Res 4>><M 8<655>* 4;* Gei.ten#ee$ T+) van 2liet S') Engering A) et al* Sel!E an% non sel!Erecognition #y CEtype lectins on %en%ritic cells* Annu Rev Immunol 5@@9M 55688* 4<* -oge% C) Sun%#la% A) Hovgaar% * Targeting vaccines to %en%ritic cells* P&arm Res 5@@5M 4>655>* 4=* Desai M) Hil!inger ') Ami%on G) et al* Immune response (it& #io%egra%a#le nanosp&eres an% alum6 stu%ies in ra##its using stap&ylococcal enteroto"in +Eto"oi%* ' Microencapsul 4>>>M 4<654:* 4>* 0atare 30) Pan%a 0) al(ani 0) et al* Potentiation o! immune response !rom polymerE entrappe% antigen6 to(ar% %evelopment o! single %ose tetanus to"oi% vaccine* Drug Deliv 5@@8M 4@6584* 5@* 0reuter '* NanoparticleE#ase% %rug %elivery systems* ' Control Release 4>>4M 4;64;>* 54* Rag&uvans&i R') Mistra A) Tal(ar GP) et al* En&ance% immune response (it& a com#ination o! alum an% #io%egra%a#le nanoparticles containing tetanus to"oi%* ' Microencapsul 5@@4M 4=6<58* 55* Sc&FllI),eissen#Fc$A)-FrsterE,al%lE)etal*AllergenEloa%e%#io%egra%a#lepolyA%)lElacticEcoE glycolicB aci% %o(nEregulate an ongoing T&5 response in t&e +A +1c mouse mo%el* Clin E"p Allergy 5@@9M 89684:* 58* Sc&Fll I) +olt/ENitulescu G) 'ensenE'arolim E* Revie( o! novel particulate antigen %elivery systems (it& special !ocus on treatment o! type I allergy* ' Control Release 5@@:M 4@964* 59* Ensoli +) +arillari G) Sala&u%%in SV) et al* Tat protein o! HI2E4 stimulates gro(t& o! cells %erive% !rom 0aposiPs sarcoma lesions o! AIDS patients* Nature 4>>>M 89:6=9* 5:* e +uanec H) 2etu C) ac&gar A) et al* In%uction in mice o! antiETat mucosal immunity #y t&e intranasal an% oral routes* +iome% P&armacot&er 5@@4M ::684;* 5;* 0im S3) Do& H') 'ang MH) et al* ?ral immuni/ation (it& Helico#acter pyloriEloa%e% polyA%)lElacti%eEcoEglycoli%eB nanoparticles* Helico#acter 4>>>M 9688* 5<* +ivasE+enita M) aloup M) 2erstey&e S) et al* Generation o! To"oplasma gon%ii GRA4 protein an% DNA vaccine loa%e% c&itosan particles6 preparation) c&aracteri/ation) an% preliminary in vivo stu%ies* Int ' P&arm 5@@8M 5;;64<* 5=* 'ung T) 0amm ,) +reiten#ac& A) et al* Tetanus to"oi% loa%e% nanoparticles !rom sul!oE #utylate% polyAvinyl alco&olBEgra!tEpolyAlacti%eEcoEglycoli%eB6 evaluation o! anti#o%y response a!ter oral an% nasal application in mice* P&arm Res 5@@4M 4=68:5* 5>* C&e( ' ) ,ol!o(ic/ C+) Mao H7) et al* C&itosan nanoparticles containing plasmi% DNA enco%ing &ouse %ust mite allergen) Der p 4 !or oral vaccination in mice* 2accine 5@@8M 5465<5@* 8@* Roy 0) Mao H7) Huang S0) et al* ?ral gene %elivery (it& c&itosanHDNA nanopartiE cles generates immunologic protection in a murine mo%el o! peanut allergy* Nat Me% 4>>>M :68=<* 84* A%orini * Cyto$ineE#ase% immunointervention in t&e treatment o! autoimmune %isE eases* Clin E"p Immunol 5@@8M 48564=:* 85* +laser 0) A$%is A* Interleu$inE4@) T regulatory cells an% speci!ic allergy treatment* Clin E"p Allergy 5@@9M 89685=* 88* 3onc&eva 0) Gme/ S) Campanero MA) et al* +ioa%&esive properties o! pegylate% nanoparticles* E"pert ?pin Drug Deliv 5@@:M 565@:* 89* Salman H) Gama/o C) Campanero MA) et al* SalmonellaEli$e #ioa%&esive nanoparticles* ' Control Release 5@@:M 4@;64* 8:* 2ila A) Sanc&e/ A) Evora C) et al* P AHPEG particles as nasal protein carriers6 t&e in!luE ence o! t&e particle si/e* Int ' P&arm 5@@:M 5>5698* 8;* 2ila A) Sanc&e/ A) Evora C) et al* PEGHP A nanoparticles as carriers !or nasal vaccine %elivery* ' Aerosol Me% 5@@9M 4<64<9* 8<* 2ila A) Sanc&e/ A) To#io M) et al* Design o! #io%egra%a#le particles !or protein %elivery* ' Control Release 5@@5M <=64:*

Espuelas et al*

Nanoparticles as A%.uvantE2ectors !or 2accination

85:

8=* 2ila A) Sanc&e/ A) 'anes 0) et al* o( molecular (eig&t c&itosan nanoparticles as ne( carriers !or nasal vaccine %elivery in mice* Eur ' P&arm +iop&arm 5@@9M :<6458* 8>* emoine D) Desc&uyteneer M) Hogge -) et al* Intranasal immuni/ation against in!luen/a virus using polymeric particles* ' +iomater Sci Polym E% 4>>>M 4@6=@:* 9@* S&ep&ar% M') To%% D) A%air +M) et al* Immunogenicity o! #ovine parain!luen/a type 8 virus proteins encapsulate% in nanoparticle vaccines) !ollo(ing intranasal a%minisE tration to mice* Res 2et Sci 5@@8M <964=<* 94* A$agi T) 0a(amura M) Ueno M) et al* Mucosal immuni/ation (it& inactivate% HI2E4E capturing nanosp&eres in%uces a signi!icant HI2E4Especi!ic vaginal anti#o%y response in mice* ' Me% 2irol 5@@8M ;>64;8* 95* Miya$e A) A$agi T) Enose 3) et al* In%uction o! HI2Especi!ic anti#o%y response an% protection against vaginal SHI2 transmission #y intranasal immuni/ation (it& inactiE vate% SHI2Ecapturing nanosp&eres in macaCues* ' Me% 2irol 5@@9M <868;=* 98* He 7) Mitc&ell A) Morcol T) et al* -ree in PMC calcium p&osp&ate nanoparticles in%uce mucosal immunity an% protection against &erpes simple" virus type 5* Clin Diagn a# Immunol 5@@5M >64@54* 99* Illum ) 'a##alEGill I) Hinc&cli!!e M) et al* C&itosan as a novel nasal %elivery system !or vaccines* A%v Drug Deliv Rev 5@@4M :46=4* 9:* 2ila A) Gill H) McCallion ?) et al* Transport o! P AHPEG particles across t&e nasal mucosa6 e!!ect o! particle si/e an% PEG coating %ensity* ' Control Release 5@@9M >=6584* 9;* 2ila A) Snc&e/ A) PGre/ C) et al* P AHPEG nanosp&eres6 ne( carriers !or transmucosal %elivery o! proteins an% plasmi% DNA* Polym A%v Tec&nol 5@@5M 486:4* 9<* To#io M) Gre! R) Sanc&e/ A) et al* Stealt& P AHPEG nanoparticles as protein carriers !or nasal a%ministration* P&arm Res 4>>=M 4:65<@* 9=* +e&rens I) Pea AI) Alonso 'M) et al* Comparative upta$e stu%ies o! #ioa%&esive an% nonE#ioa%&esive nanoparticles in &uman intestinal cell lines an% rats6 t&e e!!ect o! mucus on particle a%sorption an% transport* P&arm Res 5@@5M 4>644=:* 9>* +ivasE+enita M) van Mei.gaar%en 0E) -ran$en 0 ) et al* Pulmonary %elivery o! c&itosanH DNA nanoparticles en&ances t&e immunogenicity o! a DNA vaccine enco%ing H AE A{@5@4Erestricte% TEcell epitopes o! Myco#acterium tu#erculosis* 2accine 5@@9M 5564;>* :@* Rea% RC) Naylor SC) Potter C,) et al* E!!ective nasal in!luen/a vaccine %elivery using c&itosan* 2accine 5@@:M 58698;<*

54

Trans%ermal Applications o! Nanoparticulates

'ong(on S&im
Nanotec&nology Researc& Team) S$in Researc& Institute) RYD Center) Amorpaci!ic Corporation) 0younggi) Sout& 0orea

INTR?DUCTI?N As t&e principal purpose o! using nanoparticulates lies on increasing #ioavaila#ility o! %rugs) t&e more accurate %e!inition o! trans%ermal applications s&oul% #e t&e applications o! trans%ermal %rug %elivery (it& nanoparticulates* T&e p&armaceutiE cal in%ustries &ave applie% t&ese nanoparticulates to %eliver %rugs reCuiring acute treatment or to !ormulate t&e nonsolu#le %rugs into t&e practical !ormula ena#ling t&e in vivo applications* ?! course) t&ere are some topical applications o! nanoparE ticulates targete% to t&e s$in organ) #ut most o! t&e p&armaceutical applications (it& nanoparticulates &ave #een %evelope% to give systemic e!!ects to t&e #o%y #y penetrating t&e !ull t&ic$ness o! s$in layers A4)5B* -or t&ese purposes) t&e %rugs encapsulate% #y nanoparticulates &ave to penetrate various regions o! t&e #o%y* Many mec&anical an% electrical %elivery systems !or nanoparticulates are availa#le using in.ection) patc&) an% electrop&oresis met&o%s ot&er t&an t&e simple topical a%ministration o! sprea%ing t&e %rug onto s$in sur!ace A8B* Ho(ever) t&e use o! nanoparticulates in t&e cosmetic in%ustry &as le% to e"pectation o! t&e e!!icacy o! %rugs on t&e s$in itsel! in most cases* It is use% to ma$e t&e s$in tones elegant) to prevent t&e !ormation o! melanin #y U2 lig&t) to %egra%e callous layers) to remove (rin$les) an% to moisturi/e s$in* T&e typical %i!!erence o! a nanoparticulate system applie% in t&e cosmetic in%ustry !rom t&at o! t&e p&armaceutical in%ustry is t&at t&e ma.or %elivery met&o% is very limite% to t&e topical application o! sprea%ing onto s$in sur!aces) an% it also serves t&e emotional !unction o! provi%e% #eauty an% psyc&ological satis!action* +ut it is certain t&at t&e current ma.or tren% o! inter%isciplinary researc& is con%ucte% (it&out any limitations) an% t&e same tren% is applie% in all in%ustries* -rom t&e a#ove) t&e application o! nanoparticulates &as #een %iscusse% #y using t(o in%ustry categories !or convenience) #ut it is true t&at suc& classi!ication seems to get more o#scure as time goes #y* T&e ne(ly emerging cosmeceutical pro%ucts in t&e cosmetic in%ustry propose more en&ance% e!!icacy t&an previous pro%ucts* T&ese pro%ucts are availa#le not only !or s$in (&itening) &air removal) &air gro(t&) anti(rin$le) anticellulite) an% acne treatments) #ut also !or atopic %ermatitis an% cosmetic supplementary !oo%s !or s$in &ealt&* T&e same tren% is going on in p&arE maceutical in%ustry in t&e !orm o! noninvasive treatment in t&e %ermatological area !or cosmetic purposes* Namely) t&e treatment met&o%s suc& as some laser t&erapy !or t&e poreEtig&tening) c&emical peeling) an% ionEtreatment met&o%s &ave #een per!orme% to en&ance t&e s$in con%ition or te"ture an% to provi%e a semipermanent ma$eup #y penetrating colorants in epi%ermal layers* In t&e past) only t&e e!!icacy o! a pro%uct (as appreciate% in p&armaceutical pro%uct %evelopment #ut) no() t&e %evelopment o! p&armaceutical pro%ucts t&at (ere only %i!!erentiate% #y color)

85<

85=

S&im

-IGURE 4 ASee color insert*B CryoEsectione% images o! t&e !luorescent la#ele% nanoparticulate (it& porcine epi%ermal s$in layer a!ter in vitro s$in permeation test* AAB Stratum corneum outer layer a%sorption o! 5@@ nmEsi/e% polysturene nanoparticulate* A+B Percutaneous a#sorption o! 9@ nmE si/e% polystyrene nanoparticulate* ACB Epi%ermal layer penetration o! t&e mo%i!ie% nanoparticulate* A##reviations6 EP) epi%ermisM SC) stratum corneum*

!ragrance) taste) an% te"ture are currently e"pecte% to meet t&e consumerPs comple" %eman%s* Accor%ingly) it seems not %esira#le to classi!y t&e nanoparticulate system !or trans%ermal %elivery in cosmetics #y terms use% to %escri#e its trans%ermal %elivery o! %rugs* So) t&e current c&apter is inten%e% to %escri#e t&e trans%ermal applications o! nanoparticulate #y t&e %ept& o! s$in to (&ic& nanoparticulates are %elivere%6 stratum corneum a%sorption) percutaneous a#sorption) an% (&ole s$in penetration A-ig* 4B*

C ARI-ICATI?NS IN THIS CHAPTER Permeation T&e terminology in s$in %elivery in%icates t&e p&enomenon o! molecular moveE ment o! active ingre%ients into s$in* As in t&e case o! %elivering some ingre%ients #y sprea%ing onto a s$in sur!ace) t&ese molecules are generally %issolve% in some type o! liCui%Ep&ase ve&icles or t&ey s&oul% &ave !lui%ity #y t&emselves) an% t&is mass trans!er !ollo(s -ic$Ps %i!!usion la(* T&e reCuire% %riving !orce !or t&e %i!!usion is t&e t&ermo%ynamic energy o! t&e concentration gra%ient A9B*

Penetration T&e term QpenetrationS in%icates t&e s$in %elivery o! particulates #y colloi%al moveE ment) an% t&is particulate is &omogeneously %isperse% in some me%ium* T&e

Trans%ermal Applications o! Nanoparticulates

85>

status o! particulates %isperse% in a me%ium coul% #e %epen%ent on t&e particulate properties inclu%ing its si/e an% mass* As i! particulates #elong to t&e region t&at is not a!!ecte% #y gravity li$e molecules) t&ey seem to %i!!use #y t&ermo%ynamic energy an% t&e %i!!usion is in a %irection t&at can re%uce t&e overall entropy value o! t&e system*

Nanoparticulates Nanoparticulates in%icate various types o! su#si%iary concepts o! particulates t&at can #e represente% as capsules) aggregates) po(%ers) crystals) micelles) emulsions) comple") an% vesicles* It is usually use% to %esignate a particulate (&ose range si/e reac&es a su#micron level* Ho(ever) t&e current c&apter uses t&e terminology not only to in%icate particulates (it& su#micron si/e) #ut also to in%icate t&e system s&o(ing a colloi%al %ispersion (it&in appropriate ve&icles*

STRATUM C?RNEUM ADS?RPTI?N T&e stratum corneum is t&e location (&ere t&e trans%ermal %elivery is initiate%) an% as in t&e most molecules) it is also po(e!ul #arrier to nanoparticulates A:B* P&ysically consi%ere%) s$in &as irregular sur!aces s&o(ing elevation %i!!erences #et(een sur!aces) (&ic& ma$es it %i!!icult !or large materials greater t&an t&e elevaE tion %i!!erences to &ave enoug& contact area on t&e s$in sur!aces) an% an e"ternal !orce can easily %etac& t&em A;B* Primarily) t&ere are application areas t&at utili/e t&e small si/es o! nanoparticulates to ac&ieve even %ispersion an% strong a%&esion onto t&e s$in sur!ace A<B* T&ese nanoparticulate applications are (i%ely %evelope% in t&e cosmetic in%ustry to #e use% as organic or inorganic pigments to manu!acture cosE metics !or color ma$eup) an% t&ere are inorganic nanoparticulates propose% !or U2 lig&t protection* Ho(ever) it is never %esira#le !or suc& inorganic nanoEsi/e% materials to #e penetrate% un%er t&e stratum corneum) #ut rat&er it is recommen%e% t&ey #e remove% naturally #y t&e turnEover perio% o! stratum corneum or #y (as&ing A=H4@B* In t&e aspect o! %rug %elivery) it is most i%eal !or %rugs %issolve% in a liCui%E p&ase ve&icle at t&e molecular level to #e evenly sprea% onto s$in sur!aces to ac&ieve t&e &ig& trans%ermal %elivery e!!iciency* +ut i! suc& i%eal solvents or ve&icles %o not e"ist or t&ese molecules are repulsive to t&e s$in sur!ace) it is a%vantageous to use a nanoparticulate system t&at &as &ig& a!!inity !or s$in* So) t&e use o! soli%Hlipi% nanoparticles or a polymeric nanoparticulate t&at (as mo%i!ie% to &ave a sur!ace to #e easily a%sor#e% #y s$in sur!ace is appropriate A44)45B* +y using suc& materials) t&e nanoparticulate encapsulating t&e %rugs can #e evenly sprea% onto s$in surE !aces) an% a!ter t&e me%ium is eit&er evaporate% or a#sor#e%) t&e strong a%&esion !orce #et(een nanoparticulates pro%uces mem#raneEli$e su#strates) (&ic& !unction as a type o! patc& (it& &ig& %rug concentration A44B* ?n t&e #asis o! t&is) t&e %rug concentration in t&e s$in sur!ace is rapi%ly elevate% an% t&e strong %riving !orce o! t&e concentration gra%ient stimulates t&e %rug %elivery* -or suc& a mec&anism) it is essential to assume t&at t&e %rug encapsulate% #y t&e nanoparticulates s&oul% #e release% into s$in easily* T&e appropriate $in% o! %rugs !or suc& a nanoparticulate application is %etermine% #y t&e c&aracteristics o! molecular (eig&t or &y%rop&ilic property o! t&e %rugs) (&ic& &ave a mo%erate range o! s$in a#sorption availa#ility) #ut no a!!inity (it& s$in sur!ace* T&e %rugs !or commonly use% p&armaceutical patc& pro%ucts coul% #e t&e appropriate level o! %rugs t&at can apply in t&is type o! nanoparticulate applications*

88@

S&im

PERCUTANE?US A+S?RPTI?N In cases (&en t&e %rug molecules can penetrate stratum corneum) an% molecules s&o(ing rapi% molecular %ispersion in (ater p&ase coul% #e %isperse% into (&ole s$in (it&out many pro#lems %ue to t&e &ig& moisture level (it& lo( cell %ensity un%er t&e epi%ermal layer A49B* So) t&e penetration o! %rugs or particulates into straE tum corneum #ecomes t&e most important tas$ in t&e trans%ermal %elivery) an% currently t(o approac&es are availa#le6 4* T&e use o! materials t&at can %istur# or remove t&e #arrier !unction o! stratum corneum or t&e mi"e% use o! particulates (it& mec&anical %evices or (it& electrical %evices* 5* T&e noninvasive applications o! special particulates t&at can en&ance t&e penetration (it&out %amaging t&e #arrier !unction o! s$in* T&e !irst met&o% coul% #e reclassi!ie% into passive an% active approac&es* T&e passive met&o% %elivers t&e %rug molecules t&roug& simple %i!!usion #y %istur#ing t&e #arrier !unction o! t&e stratum corneum (it& permeation en&ancers t&at can pertur#) e"tract) an% e"c&ange t&e molecules (&ic& comprise t&e lipi% #ilayer o! stratum corneum) (it& solvents t&at can solu#ili/e lipi%s) or (it& sur!aceEactive agents appropriate to s$in use A4:)4;B* As t&e most representative !ormulations in cosmetic pro%ucts) emulsions an% micelles can #e t&e particulate systems t&at apply to t&e !irst met&o%* As t&ey are comprise% #y oils an% sur!aceEactive agents &aving a!!inity to s$in) t&e e!!ective pertur#ation o! stratum corneum is availa#le (&en #eing sprea% onto s$in) (&ic& (ill !acilitate easier %rug %elivery %o(n into t&e epi%ermal layer* T&e %rug property t&at !its t&is %elivery system can #e (aterEsoluE #le or oilEsolu#le ingre%ients) or t&ey can #e micelli/e% #y using sur!actants an% appropriate solvents* -urt&ermore) i! t&e %roplet si/e o! t&e emulsion particulate can #e uni!ormly ma%e to nanoscale) t&is emulsion o! nanoparticulates can en&ance %rug permeation an% systemic e!!iciency o! %rugs A4<)4=B* Ho(ever) #ecause o! t&e %i!!iculty o! isolating %rugs !rom e"ternal environments !or t&ese emulsion or micelle particulates) t&e a#ility to prevent t&e %rug %egra%ation can #e &ar%ly e"pecte%* So) i! t&e %rug to #e capture% is unsta#le in oil or (ater p&ase) it is not t&e appropriate system to #e use%* -or suc& %rugs) various sta#ili/ers an% antio"i%ants s&oul% #e #len%e% to acCuire enoug& sta#ility to ac&ieve e!!ective %rug %elivery %uring t&e perio% o! use* In a%%ition) it is &ar% to ac&ieve t&e en&ancement o! %rug %elivery un%er t&e epi%ermal layers #y t&e active transport o! particulates #ecause t&ese t(o types o! particulates release %rug as t&ey contact s$in sur!ace #y t&e a#sorption o! sur!actants or oils onto s$in sur!ace) or #y losing t&eir particulate s&ape %ue to t&e sur!ace energy c&ange A4>B* Mean(&ile) t&e active approac& enaE #les t&e po(%er type o! %rug particulates to #e %irectly transporte% #y applying instantaneously strong pressure o! !lui% onto s$in sur!ace* Also) t&e use o! sur!aceE c&arge% particulates an% t&e use o! iontop&oresis or electrop&oresis to en&ance t&e penetration o! %rugs are also availa#le* Alt&oug& t&ese types o! met&o%s coul% en&ance t&e %rugPs s$in %elivery very e!!ectively #y minimi/ing t&e %amage on s$in stratum corneum) it is a#solutely necessary to use specially manu!acture% instruE ments !or t&e operations) an% t&e a%%itional cost an% t&e usa#ility %epreciation &ave to #e consi%ere% A5@)54B* Contrary to t&e emulsion or micelle system) t&e secon% met&o% is a %rugE%elivery met&o% (it&out causing s$in %amage #y using nanoparE ticulate (&ic& %oes not lose its original property o! particulate (&ile penetrating t&e stratum corneum o! t&e s$in sur!ace* T&e special types o! liposomes an%

Trans%ermal Applications o! Nanoparticulates

884

polymeric nanoparticulates coul% #e suggeste% as its most representative e"amples* As a special type o! liposome) t&ere is lipi% vesicle system t&at coul% #e represente% as Trans!ersomed* It &as #een $no(n as t&e vesicle mem#rane t&at &as ultra%eE !orma#ility an% can overcome t&e insta#ility o! typical liposomes in s$in sur!ace) (&ic& can penetrate s$in appen%ages an% even t&e intracellular space o! s$in smaller t&an t&e si/e o! vesicle itsel! A55)58B* As in t&e case o! polymeric nanoparticE ulate %isperse% in a (ater p&ase) recent stu%ies &ave reveale% t&at most o! t&e particulates penetrate t&roug& t&e s$in appen%ages an% s&unts t&at &ave #een $no(n to #e t&e pat&(ays !or large &y%rop&ilic molecules A59)5:B* Especially t&e &air !ollicles (ere regar%e% as t&e ma.or pat&(ay) an% transport e!!iciency seems to #e %etermine% #y t&e average particulate si/e* So) t&e smaller si/e o! nanoparticuE late coul% transport t&e %rug more easily un%er t&e epi%ermal layer A5;H5=B A-ig* 5B* As t&e polymeric nanoparticulates &ave %i!!erent a#sorption c&aracteristics %ue to t&e material property o! polymers) t&e %rug encapsulation an% release #e&avior (it&in t&e nanoparticulate s&oul% #e e"pecte% to #e %i!!erent !rom t&at o! microparE ticulate systems* Previously) t&e s$in a#sorption ten%ency o! t&e %rug #y nanoparE ticulate (as en&ance% or %elaye% %epen%ing upon t&e %rug types) Cuestioning t&e true e!!icacy o! t&e nanoparticulate A5>H84B) #ut it is more appropriate to interpret t&e pro#lem #y t&e (ay t&at t&e c&emical property or sur!ace c&aracteristics li$e t&e partition coe!!icient o! capture% molecules cannot #e completely covere% #y t&e nanoparticulate encapsulation* +ut yet) t&ere are no proven results regar%ing t&is matter) an% !urt&er stu%ies to resolve t&e pro#lem s&oul% #e ma%e* I! any o! t&ese met&o%s &as #een selecte% to %eliver t&e %rug to t&e epi%ermal layer) t&e suita#le %rugs s&oul% #e active materials (it& similar !unctions as $eratinocytes an% melanoE cytes in cosmetic pro%ucts* Namely) t&ese active materials can %egra%e melanin or in&i#it t&e !ormation o! it !rom melanocytes* As a p&armaceutical pro%uct) t&e steroi%al %rug o! &y%rocortisone coul% #e suggeste% as its e"ample) (&ic& alleviates t&e atopic symptom #y t&e action on anger&ans cells e"isting in t&e epi%ermal layer* It also s&o(s its e!!ectiveness on eryt&ema) psoriasis) an% %ermatorr&ea* Ho(ever) t&e longEterm use o! t&is %rug can causes severe si%e e!!ects* So) t&e %eliE cate %esign &as to #e ma%e to ma"imi/e its e!!icacy #y using an e!!ective %elivery system an% to minimi/e t&e si%e e!!ects*

-IGURE 5 ASee color insert*B C SM images o! t&e nanoparticulate penetration (it& a guinea pig s$in AcryoEsectione%B* -luorescentEla#elle% nanoparticulate Agreen regionB) nucleiEla#ele% DAPI A#lue regionB* AAB 9@ nm si/e o! nanoparticles) A+B 48@ nm si/e o! nanoparticles A4@ pieces o! /E%irection sectioning image o! cross sectione% tissue are merge%B* Nanoparticulate6 PolycaprolacE toneEpolyet&yleneglycol #loc$ copolymer aggregates* -luorescent6 Ru#rene* Source6 -rom Re!* 5<*

885

S&im

-IGURE 8 ASee color insert*B Images o! t&e liposome nanoparticulate (it& !luorescent #y in vitro permeation test (it& a guinea pig s$in* AAB RITC saturate% solution A+B RITC (it& t&e mo%i!ie% liposome* ARe% regionB) &y%rop&ilic !luorescent %ye ARITCBM A#lue regionB) DAPI !or nucleiM #asal layer A(&ite arro(sB*

As mentione% previously) i! all material %elivery an% %i!!usion can #e easily ac&ieve% a!ter penetrating t&e stratum corneum) it seems to #e not reCuire% !or nanoparticulates to penetrate t&e epi%ermal layer* Ho(ever) some $in% o! %rugs coul% not penetrate t&e epi%ermal permea#ility #arrier AEP+B (&ic& &as t&e #asal layer A%ermalHepi%ermal .unction layerB* EP+ is t&oug&t to act as t&e #arrier to (ater loss) intestinal !lui%) an% in!ection o! microorganisms A85B* It is suppose% t&at t&e &ig& cell %ensity an% a#un%ant amounts o! materials comprising t&e e"traE cellular matri" an% tig&t .unction #et(een cells in t&is layer mig&t a!!ect %rugE permeation #e&avior* So) t&e particulate t&at can penetrate t&e (&ole epi%ermal layer s&oul% #e use% (&en %elivering %rug to t&e %ermal layer A-ig* 8B* As !or t&e cosmetics to #e use% on t&e %ermal layer level) t&e anti(rin$le application to en&ance t&e collagen synt&esis an% t&e anticellulite application to #e use% to %estroy an% to remove t&e irregularly presenting su#!ats un%er t&e %ermis coul% #e suggeste% as appropriate pro%ucts !or use) an% applications !or %eep (oun% &ealing coul% #e suggeste% !or e!!ective use in p&armaceutical pro%ucts* Alt&oug& it is %i!!erentiate% #y t&e %ept& o! (oun% an% t&e %egree o! its seriousness) i! a part o! s$in (as in.ure%) t&e in.ure% part is initially #loc$e% #y #loo% coagulation to prevent #loo% an% #o%y !lui%s loss) an% rapi% epi%ermal cell %ivisions are carrie% out to recover t&e in.ure% s$in sur!ace* +ut t&e recovery !or t&e in.ure% %ermis ta$es more time to progress* At t&at time) t&e insu!!iciency or overEmultiplication o! %ermal cells can cause t&e scar in t&e s$in* So) i! t&e nanoparticulates are %irectly %elivere% %o(n to t&e %ermal layer to release t&e %rug in concentrative manner) t&e resulting rapi% multiplication o! !i#ro#last cells (ill increase cell %ensity) an% can stimulate t&e normal synt&esis o! collagen to &ave t&e (rin$leE!ree s$in promise% #y cosmetic pro%uctsM t&e application o! it seems appropriate !or t&e restoration o! scarE!ree s$in as promote% in p&armaceutical pro%ucts*

,H? E S0IN PENETRATI?N T&e particulate system %esigne% !or (&ole s$in penetration &as t&e purpose o! en&ancing t&e overall #ioavaila#ility o! %rug molecules (&ic& are reCuire% to #e %elivere% to t&e target organs in spite o! lo( permea#ility %ue to large molecular (eig&t an% lo( partition coe!!icient on s$in* -or t&e nanoparticulate systems)

Trans%ermal Applications o! Nanoparticulates

888

nee%e% to penetrate t&e s$in) nearly i%entical particulate c&aracteristics are reCuire% to %eliver t&e %rugs to %ermal or epi%ermal layer as .ust %iscusse% a#ove* Consi%ere% !rom t&e aspect o! %rugs to #e a#sor#e%) material partitioning into s$in can #e in&i#ite% #y corneocytes an% #y various compositions o! lipi%s an% !atty aci%s t&at !ill up t&e gap* T&at is (&y t&e particulate system is primarily !ormulate% to penetrate t&e stratum corneum) #ut once penetration #elo( stratum corneum is accomplis&e%) !ree %i!!usion is allo(e% #y t&e a#un%ant amount o! &umoral !lui% in s$in tissue t&at can #e %elivere% into t&e (&ole #o%y t&roug& t&e circulation system* Alt&oug& t&e nanoparticulate system may succee% in penetratE ing t&e stratum corneum) t&e e!!iciency o! %elivering t&e %rug into t&e circulation system can #e %o(ngra%e% i! t&e %rug cannot permeate t&e epi%ermal an% %ermal layer or cannot avoi% various types o! e"isting %e!ense systems in s$in* ?t&er(ise) i! t&e %rugs &ave lipop&ilic property an% lo( molecular (eig&t) t&e initial partiE tioning onto lipi% compositions in t&e outermost layer o! s$in can #e easily ac&ieve%) #ut %elivery un%er t&e stratum corneum layer (it& t&e relatively &ig& moisture content in epi%ermal an% %ermal layers (ill get more %i!!icult !or t&e mass trans!er o! t&e molecules* So) t&ese molecules (ill #e clusteri/e% %ue to t&e %ecrease% solu#ility in t&e environment) an% eventually t&e mo#ility o! t&ese molecules coul% #e nearly %isa#le% #y %i!!usion* Even i! nanoparticulates can penetrate (&ole s$in layer an% can avoi% various immune systems presenting in t&e s$in organ a!ter %ispersion) t&ey &ave to !ace t&e &ig&ly %evelope% an% more comple" %e!ense system in t&e #o%y suc& as t&e #loo%H #rain #arrier) anger&ans cells an% TEcells in liver) PeyerPs patc&es in t&e gutEassociE ate% lymp&oi% tissue) an% so on A88)89B* So) a &ig& %elivery e!!iciency coul% not #e e"pecte% unless a proper counterplan !or t&is is prepare%* As in t&e case o! percutaE neous a#sorption) t&e particulate !or s$in penetration s&oul% not #e %estroye% #y e"ternal environments #et(een t&e early steps o! s$in penetration an% t&e %elivery into t&e circulation system) an% must &ave material properties t&at can suppress %rug release an% avoi% %egra%ation o! t&e encapsulate% %rugs* Alt&oug& t&ere is muc& unclari!ie% in!ormation) i! t&e particulate types o! micelles) emulsions) an% generally manu!acture% liposome vesicles (ere applie% #y sprea%ing onto s$in surE !aces) it is %i!!icult to maintain t&eir original particulate s&ape %ue to t&e a#sorption #e&avior #et(een s$in sur!ace an% molecules comprising t&e particulates an% #y t&e %i!!erence o! sur!ace energy A8:B* In some cases o! t&eir practical uses) it (as reporte% t&at t&e particulates (ere %estroye% #y t&e salts e"isting on e"u%ates or s(eat %ucts in t&e s$in sur!ace* T&ere are many cases t&at cannot maintain t&e sta#ility o! t&e system %ue to t&e rapi% evaporation o! (ater or ot&er solvents in t&e s$in sur!ace) an% i! t&e pro%uct reCuires sprea%ing action #y users) t&e e"ternal p&ysical stress o! sprea%ing may cause t&e structural %estruction o! t&e particuE late to occur* So) t&ese systems are not an appropriate system #ecause t&e particulate cannot #e maintaine% until it reac&es t&e %estination %ue to #urstEreleasing t&e %rug premature A-ig* 9B* To avoi% suc& pro#lems) t&e most easily applie% met&o% is in.ection a%minisE tration* T&e material is %elivere% %o(n to t&e %ermal layer in t&e s$in #arrier #y using a mec&anical %evice* T&is met&o% is not strictly restricte% #y particulate si/e) an% it also allo(s %irect in.ection into #loo% vessels to &ave imme%iate e!!iciency o! t&e %rug* A%%itionally) it can avoi% t&e particulate %isruption cause% #y environE mental c&ange an% #y t&e primary protection mec&anisms e"isting in s$in* Ho(ever) t&e in.ection a%ministration met&o% is stress!ul to t&e user an% its use is %i!!icult) (&ic& seems %isa%vantageous in longEterm an% repeate% use* T&e met&o% &as

889

S&im

-IGURE 9 ASee color insert*B -luorescent images o! in vivo permeation stu%y (it& Al#ino Hartley guinea pig* AAB ?1, emulsion !ormulation* A+B Mo%i!ie% polymeric nanoparticulate*

#een improve% to create t&e patc& !ormulation met&o%) #ut it also &as some %isa%E vantages %ue to t&e action o! #arrier !unction in s$in #y s(elling an% #y t&e aspect o! &aving relatively lo( %elivery e!!iciency* As !or t&e system to #e use% easily) to minimi/e s$in %amage an% to %eliver a &ig&er concentration o! %rug to t&e circulaE tion system) t&e use o! particulate t&at can penetrate t&e (&ole s$in layer suc& as t&e polymeric particulate) ot&er soli% type nanoparticulates) or special lipi% vesicle particulate seems appropriate* Et&osomed &as #een $no(n as a liposome system t&at can maintain &ig& et&anol content) (&ic& can e!!ectively %istur# t&e s$in lipi%s (it&out losing its particulate property to en&ance t&e %rugE%elivery e!!iciency A8;)8<B* In a%%ition) t&e previously mentione% Trans!ersomed &as s&o(n &ig& e!!iciency o! %rug %elivery !or (&ole #o%y application in a%%ition to t&e !unction o! %elivering %rugs to s$in layers* Ho(ever) it is still %i!!icult to !in% a commercialE i/e% polymeric nanoparticulate !or e"ternal s$in use as (ell as !or (&ole #o%y !unctions o! a %rug* Its limite% %elivery pat&(ay ot&er t&an &air !ollicles ma$e it %i!!icult to increase t&e %elivery e!!iciency) its ina#ility to capture &y%rop&ilic %rugs) an% t&e very limite% amount o! applica#le materials are t&oug&t to #e t&e appropriate reasons*

THE IMITATI?NS ?- NAN?PARTICU ATE S3STEM -?R TRANSDERMA APP ICATI?NS -irst) t&ere are limitation o! acCuiring sta#le materials* Most o! t&e nanoparticuE lates an% t&eir %e#ris t&at penetrate %eep into s$in layers mig&t cause an immune response* So) materials t&at can #e %isintegrate% #y appropriate mec&anisms to #e a#sor#e% into #o%y &ave to #e use%* Ho(ever) very limite% num#ers o! materials e"ist !or a nanoparticulate system) (&ic& can #e %egra%e% (it&in s$in to #e a#sor#e% naturally into t&e #o%y* T&is coul% #e suggeste% as t&e ma.or topic t&at researc&ers speculate) t&e conventionally use% U*S* -oo% an% Drug A%ministration A-DABEapprove% materials o! polyAlactic aci%B AP AB) polyAglycolic aci%B APGAB) an% poly%io"anone APDSB t&at are using lecit&in or #iocompati#le polymeric #iomaterialsM t&ese &ave nearly reac&e% t&eir ma"imum capacity to enlarge t&eir !iel% o! application* Similar %egra%a#le polyesterEtype polymers o! polyAan&y%ri%eB) polyp&osp&onate) polyami%e an% polyiminocar#onate) polyE caprolactone) polyp&osp&a/ene) an% polyAp&osp&ate esterB are availa#le) #ut

Trans%ermal Applications o! Nanoparticulates

88:

cannot #e use% #ecause t&eir o!!icial approvals &ave not #een grante%* -or t&ese reasons) many investigators currently ma$e every e!!ort to !in% an% to apply materials t&at can #e as sa!e as lecit&in in t&e &uman #o%y an% materials &aving clear %egra%ation mec&anisms t&at can #e synt&esi/e% an% puri!ie% suc& as P A A8=)8>B* Alt&oug& t&ese nanoparticulates (ill #e !ormulate% !rom sa!e materials !or &uman #o%y) consi%eration !or a %i!!erent aspect o! t&e material not s&o(n in t&e #ul$ p&ase mig&t #e necessary (&en t&ese materials are !ormulate% in t&e !orm o! nanoparticulates an% (&en t&ese nanoparticulates are actually use% #y encapsulating %rugs* As an e"ample) some $in% o! nanoparticulate system is proE pose% !or s$in penetration) an% t&e en&ance% s$in a#sorption rate &as t&e ris$ o! causing strong irritation or immune response %ue to t&e overstimulation o! t&e s$in immune system #y t&e %rug* So) t&e nanoparticulate system !or trans%ermal %elivery s&oul% #e investigate% in t&e appropriate concentration ranges to repreE sent its sa!ety an% e!!icacy !rom t&e !ollo(ing !our aspects6 %rugs) ra( materials comprising nanoparticulate) %rugEencapsulate% nanoparticulate) an% t&e empty nanoparticulate* Secon%ly) a limite% %elivery pat&(ay coul% #e suggeste%* In spite o! t&e e"cellent s$in a#sorption rate o! general s$in pro%ucts o! e"ternal use) t&ere are still some pro#lems remaining to #e solve%* Especially !or t&e nanoparticulates to penetrate lo(er t&an t&e stratum corneum) use in.ure% s$in parts) &air !ollicles) an% au"iliary s$in systems as t&e pat&(ay to move* +ut t&e area o! t&e pat&(ays provi%e% #y t&ese au"iliary s$in systems ma$e up a very small portion compare% to t&e total s$in area* T&e s$in a#sorption o! t&e %rug using t&e limite% pat&(ays essentially reCuires a larger range o! a%ministration an% repeate% use to reac& t&e %esire% %rug concentration in a #o%y compare% to t&e oral an% in.ection a%minisE tration met&o%s* T&ir%ly) t&ere is an a#sence o! proper sta#ili/ation !unctions !or unsta#le active ingre%ients* +ecause o! t&e relatively t&in (all t&ic$ness o! nanoparticulate comE pare% to microparticulate) it is #eyon% t&e capa#ility o! nanoparticulate to prevent t&e material %egra%ation #y reactive o"ygen species) U2 lig&t) an% &eat* Practically) t&e active ingre%ients o! cosmetic pro%ucts are easily %egra%a#le #y o"ygen an% &eat A9@B* I! a nanoparticulate system is applie% to en&ance #ioavaila#ility o! actives to t&e s$in) it is o!ten necessary to a%% various a%%itives) li$e antio"i%ants an% antiE !a%ing agents) or &ig&ly airEtig&t containers s&oul% #e use%*

THE -UTURE ?- NAN?PARTICU ATE APP ICATI?NS -?R TRANSDERMA DE I2ER3 Simply consi%ere%) t&e researc& can overcome t&e limitation o! nanoparticulate systems as %escri#e% in t&is c&apter* As mentione% a#ove) t&e a%vantages o! nanoparticulate applications s&oul% #e ma"imi/e% an% t&e %isa%vantages s&oul% #e overcome* T&e ultimate nanoparticulate applications !or trans%ermal %elivery t&at (ill #e %evelope% in t&e !uture can %eliver nearly all types o! %rugs inclu%ing macromolecules into target sites #y simply sprea%ing onto t&e s$in sur!ace to proE vi%e concentrative an% longEterm release o! t&e %rug* To enlarge t&e applica#le scopes o! nanoparticulate !or trans%ermal %elivery) it is a#solutely necessary to %evelop ne( nanoparticulate systems to en&ance t&e s$in a#sorption an% not #e a!!ecte% #y t&e active ingre%ients to #e encapsulate%* A#ove all) t&e material %iverE si!ication o! nanoparticulate #y t&e %evelopment o! material science s&oul% #e solve% initially) an% t&e analysis met&o%s to analy/e t&e c&emical an% p&ysical

88;

S&im

properties o! nanoparticulate s&oul% #e %evelope% along (it& t&e %evelopment o! analytical instruments (&ic& (ill ena#le t&e a#ove goals* RE-ERENCES
4* Muller RH) 'aco#s C) 0ayser ?* Nanosuspensions as particulate %rug !ormulations in t&erapy rationale !or %evelopment an% (&at (e can e"pect !or t&e !uture* A%v Drug Deliv Rev 5@@4M 9<68H4<* 5* Panyam ') a#&aset(ar 2* +io%egra%a#le nanoparticles !or %rug an% gene %elivery to cells an% tissue* A%v Drug Deliv Rev 5@@8M ::685>H89<* 8* +arry +,* Is trans%ermal %rug %elivery researc& still important to%ayT DDT 5@@4M ;A4>B6>;<H><4* 9* Ha%gra!t '* S$in %eep* Eur ' P&arm +iop&arm 5@@9M :=A5B65>4H5>>* :* S&a& 2P) -lynn G ) 3aco#i A) et al* +ioeCuivalence o! topical %ermatological %osage !ormsRmet&o%s o! evaluation o! #ioeCuivalence* S$in P&armacol Appl S$in P&ysiol 4>>=M 44644<H459* ;* -ie%ler M) Meier ,ED) Hoppe U* Te"ture analysis o! t&e sur!ace o! t&e &uman s$in* S$in P&armacol 4>>:M =65:5H5;:* <* Muller RH) Ra%t$e M) ,issing SA* Soli%Hlipi% nanoparticles AS NB an% nanostructure% lipi% carriers AN CB in cosmetic an% %ermatological preparations* A%v Drug Deliv Rev 5@@5M :9Asuppl 4B6S484HS4::* =* Auton TR) ,est&ea% DR) ,oolen +H) Scott RC) ,il$s M-* A p&ysiologically #ase% mat&ematical mo%el o! %ermal a#sorption in man* Hum E"p To"icol 4>>9M 486:4H;@* >* Re%%y M+) Guy R) +unge A * Does epi%ermal turnover re%uce percutaneous penetraE tionT P&arm Res 5@@@M 4<A44B64949H494>* 4@* 0(on SS) Nam 3S) ee 'S) et al* Preparation an% c&aracteri/ation o! coen/yme 74@E loa%e% PMMA nanoparticles #y a ne( emulsi!ication process #ase% on micro!lui%i/aE tion* Colloi% Sur! A 5@@5M 54@6>:H4@9* 44* Sc&reier H) +ou(stra '* iposomes an% niosomes as topical %rug carriers6 %ermal an% trans%ermal %rug %elivery* ' Control Release 4>>9M 8@64H4:* 45* Morganti P) Ruocco E) ,ol! R) Ruocco 2* Percutaneous a#sorption an% %elivery systems* Clin Dermatol 5@@4M 4>6 9=>H:@4* 48* an Honey(ellENguyen P) De Graa!! AM) ,outer Groenin$ H,) +ou(stra 'A* T&e in vivo an% in vitro interactions o! elastic an% rigi% vesicles (it& &uman s$in* +ioc&im +iop&ys Acta 5@@5M 4:<8648@H49@* 49* Ritsc&el ,A) Hussain AS* T&e principles o! permeation o! su#stances across t&e s$in* Met&o%s -in% E"p Clin P&armacol 4>==M 4@A4B68>H:;* 4:* Smit& E,) Mai#ac& HI) e%s* Percutaneous Penetration En&ancers* Ne( 3or$6 CRS Press) 4>>:* 4;* Ha%gra!t '* Mo%ulation o! t&e #arrier !unction o! t&e s$in* S$in P&armacol Appl S$in P&ysiol 5@@4M 496<5H=4* 4<* Stiess M) e%* Mec&anisc&e 2er!a&renstec&ni$ 4* +erlin6 Springer) 4>>:6:>H;5* 4=* %e 2ringer T) %e Ron%e HAG* Preparation an% structure o! a (aterEinEoil cream containE ing lipi% nanoparticles* ' P&arm Sci 4>>:M =969;;H9<5* 4>* MJller RH) MI%er 0) Go&la S* Soli%Hlipi% nanoparticles AS NB !or controlle% %rug %eliveryRa revie( o! t&e state o! t&e art* Eur ' P&arm +iop&arm 5@@@M :@64;4H4<<* 5@* +arry +,* +reac&ing t&e s$inPs #arrier to %rugs* Nat +iotec&nol 5@@9M 55A5B64;:H4;<* 54* Mar$ R) Prausnit/) Mitragotri S) anger R* Current status an% !uture potential o! trans%ermal %rug %elivery* Nat Rev Drug Discov 5@@9M 8644:H459* 55* Cevc G) +lume G) Sc&at/lein A* Trans!ersomesEme%iate% transepi%ermal %elivery improves t&e regionEspeci!icity an% #iological activity o! corticosteroi%s in vivo* ' Control Release 4>><M 9:6544H55;* 58* Cevc G) Sc&at/lein A) +lume G* Trans%ermal %rug carriers6 #asic properties) optimi/aE tion an% trans!er e!!iciency in t&e case o! epicutaneously applie% pepti%es* ' Control Release 4>>:M 8;68H4;* 59* Cevc G* ipi% vesicles an% ot&er colloi%s as %rug carriers on t&e s$in* A%v Drug Deliv Rev 5@@9M :;6;<:H<44*

Trans%ermal Applications o! Nanoparticulates

88<

5:* +arry +,* Drug %elivery routes in s$in6 a novel approac&* A%v Drug Deliv Rev 5@@5M :9A4B6S84HS9@* 5;* Alvare/ERoman R) Nai$ A) 0alia 3N) Guy RH) -essi H* S$in penetration an% %istri#ution o! polymeric nanoparticles* ' Control Release 5@@9M >>6:8H;5* 5<* S&im ') 0ang HS) Par$ ,ES) Han SEH) 0im ') C&ang IES* Trans%ermal %elivery o! mino"iE %il (it& #loc$ copolymer nanoparticles* ' Control Release 5@@9M ><69<<H9=9* 5=* Pra#&a S) V&ou ,EV) Panyam ') a#gaset(ar 2* Si/eE%epen%ency o! nanoparticleE me%iate% gene trans!ection6 stu%ies (it& !ractionate% nanoparticles* Int ' P&arm 5@@5M 59964@:H44:* 5>* Muller +) 0reuter '* En&ance% transport o! nanoparticle associate% %rugs t&roug& natural an% arti!icial mem#ranesRa general p&enomenonT Int ' P&arm 4>>>M 4<=658H85* 8@* Mar$us '* Cappel) 0reuter '* E!!ect o! nanoparticles on trans%ermal %rug %elivery* ' Microencapsul 4>>4M =A8B68;>H8<9* 84* Hassan #outaunne 'eanE-ranois C&aulet) C&ristine Ploron) -ranois -alson an% -a#rice Pivot* Sustainel e" vivo s$in antiseptic activity o! c&lor&e"i%ine in polyAlEcaprolactoneB nanocapsule encapsulate% !rom as a %igluconate* ' Control Release 5@@5M =5684>H889* 85* Troya TEC) Ra&#ara R) Ara#/a%e& A) C&eunga RME0) Tur$sena 0* Delaye% epi%ermal permea#ility #arrier !ormation an% &air !ollicle #errations in InvECl%n; mice* Mec& Dev 5@@:M 4556=@:H=4>* 88* -lorence AT* T&e oral a#sorption o! microE an% nanoparticulates6 neit&er e"ceptional nor unusual* P&arm Res 4>><M 49A8B65:>H5;;* 89* -lorence AT) Hussain N* Transcytosys o! nanoparticle an% %en%rimer %elivery systems6 evolving vistas* A%v Drug Deliv Rev 5@@4M :@6;>H=>* 8:* Vellmer S) P!eil ,) asc& '* Interaction o! p&osp&ati%ylc&oline liposomes (it& t&e &uman stratum corneum* +ioc&im +iop&ys Acta 4>>:M 458<64<;H4=5* 8;* Touitou E) Dayan N) +ergelson ) Go%in +) Elia/ M* Et&osomesEnovel vesicular carriers !or en&ance% %elivery6 c&aracteri/ation an% s$in penetration properties* ' Control Release 5@@@M ;:69@8H94=* 8<* Touitou E) Go%in +) ,eiss C* En&ance% %elivery o! %rugs into an% across t&e s$in #y et&osomal carriers* Drug Dev Res 5@@@M :@69@;H94:* 8=* Drotle!!a S) ung(it/a U) +reuniga M et al* +iomimetic polymers in p&armaceutical an% #iome%ical sciences* Eur ' P&arm +iop&arm 5@@9M :=68=:H9@<* 8>* Soppimat&a 0S) Amina#&avia TM) 0ul$arnia AR) Ru%/ins$i ,E* +io%egra%a#le polymeric nanoparticles as %rug %elivery %evices* ' Control Release 5@@4M <@64H5@* 9@* 'enning 2) Gysler A) Sc&a!erE0orting M) Go&la SH* 2itamin A loa%e% soli%Hlipi% nanoE particles !or topical use6 occlusive properties an% %rug targeting to t&e upper s$in* Eur ' P&arm +iop&arm 5@@@M 9>A8B6544H54=*

In%e"

A#ra"aneb parenteral !ormulations c&aracteri/ation an% manu!acture) 99 clinical trials) 99H9: p&armaco$inetics) 99 vs* Ta"olb) 99 A#sorptiveEme%iate% transcytosis) 5;; Acetylsalicylic aci% SEM) 45: Acrylates) 5<9H5<; %erivatives) 5<: Active tumor targeting metallic nanoparticle %rugE%elivery systems) 49>H4:@ Actively targete% t&erapies %rugE%elivery system nanoengineering) 4@; A%.uvantEvector vaccination) 84<H858 A%riamycinb) 5<= Aerosol !lo( reactor met&o%) o! multicomponent %rug nanoE an% microparticle synt&esis) 4@4H45; e"perimental) 449H44< instrumentation an% c&aracteri/ation) 44;H44< materials) 449 particle pro%uction) 449 precursor solutions) 449 results) 44< nanostructure% %rug microparticles) 454H45: pepti%e coating particles) 454H459 reactor %rug crystalli/ation) 454H459 polymeric %rug nanoparticles) 44= Agitator #ea% mill) << Al#umin) 5<5 nanosp&eres) 4; Alen%ronate liposomal) 59@ Alginates) 49) :5 Al$ylcyanoacrylates monomer inter!acial polymeri/ation) 4:< Alumina nanosp&eres) 4=H4> Am+isomeb) <5

5EAminoc&romone UE=;=>8 AU=;B local %elivery !or restenosis) 59; Amorp&ous materials electrospinning) ;8H;9 Amorp&ous mo%el) 54> Amp&ip&ilic #loc$ copolymers) 48 Amp&otericin liposomal) >4 Anest&etics) 9@ parenteral !ormulations) 9@H9: Angiogenic tumor #loo% vessels tumorEtargeting metallic nanoparticle %rugE%elivery systems) 49>H4:@ Anti#o%yEcoate% nanosp&eres) 55 Anticancer parenteral !ormulations) 9@H9: AntiE-l$ anti#o%yEcoate% >@3 nanoparticles) 4;> Anti!ungal parenteral !ormulations) 8=H8> clinical stu%y) 8>H9@ preclinical stu%ies (it& %rugEresistant !ungal stain) 8> (it& %rugEsuscepti#le !ungal stain) 8> AntigenEpresenting cells AAPCB) 84= AntiEHER5 anti#o%yEtargete% gol%1silicon nanoparticles) 4;> Antimicro#ial nanoemulsions) 4=4 Antitranspirants) 55< APC* See AntigenEpresenting cells AAPCB Apolipoprotein E) <: Arecai%ine propargyl ester AAPEB) 5<: Arterial locali/ation %o"oru#icin) 59=

+alloon in.ury caroti% artery) 599 +++* See +loo%E#rain #arrier A+++B +eclomet&asone %ipropionate po(%ers pro%uction) 454) 455 SEM) 45: +ee venom pepti%e) 848

88>

89@
+erner type lo(Epressure impactor A+ PIB) 44: +iocon.ugate nanoparticleEaptamer) 4;> +io%egra%a#le polylacti%e nanosp&eres) 4= +isp&osp&onate) 5:@H5:5 +loo%E#rain #arrier A+++B) 4@<) 4><) 5=4H5=8 pat&ologic alterations) 5=8 + PI* See +erner type lo(Epressure impactor A+ PIB +rain compartment) 5=9H5=; +rain en%ot&elial cells) 58=H5=< +rain tumors +++) 5=8 iron o"i%e nanoparticles) 4=@ C&olesteryl myristate ACMB) 48@H484 supercoole% smectic nanoparticles properties an% p&ase #e&avior) 48@H485 Clinical nano%iagnostics) 4<= C I? paramagnetic nanoparticles) 4;> Clo%ronate liposomal) 59@ Clo!a/imine nanosuspension vs* liposomal !orm) <: liposomal nanosuspension) <: Clotrima/ole release pro!iles) 558 CM* See C&olesteryl myristate ACMB CMC* See Critical micelle concentration ACMCB CNS* See Central nervous system ACNSB Collagen) 49 Colonic targeting NPDDS) 8@:) 8@;H845 ComputerEcontrolle% molecular tools) 4=4 Controlle%Erelease systems %rugE%elivery system nanoengineering) >>H4@5 Cosmetics lipi% nanoparticles) 548H55> N C) 549) 55> Creams N C) 55> Critical micelle concentration ACMCB) > Cryopreparation met&o% DNA microencapsulation) 5>8 C2D* See C&emical vapor %eposition AC2DB Cyclo%e"trins ACDB) <:) 4@8 Cyclop&osp&ami%eEloa%e% poly#utylcyanoacrylate) 4;5 Cyclosporine A) 5<8 HPC) 45 Cyp&er) 58; C3TE;@>4) 4:4H4:9 Cytara#ine liposomal) >4 Cytoplasm) 4:>H4;@ Cytos$eleton) 4;@ Cytosol) 4;@

In%e"

Caely"b) 5=9 Calcium car#onate nanoparticles) 49 Calcium o"i%e po(%er antimicro#ial properties) 4=@ Camptot&ecin) 55 Cancer* See ?ncology Car#o&y%rates cells) 4;4 Car#on nanotu#es) 5@) 4;> Car%iovascular %isease nano#iotec&nology) 4=@ Caroti% artery #alloon in.ury) 599 Carrageenans) 5<9 gelatin) 5<9 Cationic liposomes) 59@H595 !ormulation strategies) >4H>9 CD* See Cyclo%e"trins ACDB Cells c&emical composition) 4;@H4;5 macromolecules) 4;4H4;5 small organic molecules) 4;4 structure) 4:> Central nervous system ACNSB) 4=:H4=;) 5=4H5=9 %o"oru#icin) 5=9 nano#iotec&nology) 4=@ Ceramic nanoparticles) 4;> C-C* See C&loro!luorocar#on AC-CB C&emical vapor %eposition AC2DB) 4;9 C&itosan) 49) :5) 5<5 C&itosanE#ase% nanoparticulate %rugE%elivery systems) 49 C&itosan nanoparticles gene %elivery) 5>> C&loro!luorocar#on AC-CB Montreal Protocol) <;

Dalargin) 4>: Dantrolene malignant &ypert&ermia) 94) 95 Dative #in%ing) 495 Daunoru#icin liposomal) >4 DC* See Den%ritic cells ADCB Den%rimerE#ase% %rugE%elivery systems) 49 lymp&atic targeting) 5:9

In%e"

894
NDrugE%elivery system nanoengineeringO polymeric strategies !or %rug action prolongation) 4@9H4@; sustaine% an% triggere% releases lipi%E#ase%) 4@4 p&ysicoc&emical properties) 4@5H4@8 polymeric) >>H4@9 targete% t&erapies) 4@;H4@< transport across tig&t en%ot&elial .unctions) 4@< DrugEeluting stents) 58: Drug nanocrystals) <4H=: clinical applications) com#ination tec&nologies) =4H=5 %e!ine%) <5 %rug %iscovery) 4<: !inal !ormulations) =9 &ig&Epressure &omogeni/ation) <>H=9 me%ia milling processes) <;H<= micro!lui%i/er tec&nology) <> milling) =8 nanoe%ge tec&nology) =4H=5 nanopure tec&nology) =4 WP) =5H=8 ocular %elivery) <; oral %elivery) <9H<: parenteral a%ministration) <: particle si/e re%uction tec&niCues) <;H=9 p&ysicoc&emical properties) <8H<9 pistonEgap &omogeni/ation in (ater) <>H=@ precipitation) <=H<> pulmonary %rug %elivery) <; SEM) =: Drug po(%ers nanoscale antimicro#ial properties) 4=@ pro%uction micronsi/e%) 448) 44; nano) 44;) 44< DSC* See Di!!erential scanning calorimetry ADSCB DSPEEPEG* See 4)5EDistearoylE8E p&osp&ati%ylet&anolamine ADSPEEPEGB DTA* See Dip&t&eria to"in A ADTAB

Den%rimers) 4;<H4;>) 4>>H5@4 %rug %iscovery) 4<:H4<< Den%ritic cells ADCB) 84= Deo%orants) 55< Dermal lipi% nanoparticles) 548H55> Dermal nanosuspensions) <; De"amet&asone) 4=>) 4>>) 5<= local %elivery !or restenosis) 598H59: De"tran nanosp&eres magnetic plain) 4> De"trans) 49 Di!!erential scanning calorimetry ADSCB) :; Dioleoyl p&osp&ati%ylet&anolamine AD?PEB) >5) >8 Dip&t&eria to"in A ADTAB) >8 DipEpen nanolit&ograp&y) 4;9H4;: Direct compress tec&nology) =: Dispatc& cat&eter) 5@< Dissocu#es tec&nology) <> Dissolution velocity) <4 4)5EDistearoylE8Ep&osp&ati%ylet&anolamine ADSPEEPEGB) 5<< D +en%ing Tester) ;<H;= DNA microencapsulation cryopreparation met&o%) 5>8 plasmi% P GA) 5>4H5>= polymer composition) 5>; D?CSPER) 594 D?PE* See Dioleoyl p&osp&ati%ylet&anolamine AD?PEB Dou#le emulsionEsolvent evaporation met&o%) 599 Do"ilb) 5=9 Do"oru#icin) 4>= CNS) 5=5 liposomal) >4 local %elivery !or arterial locali/ation) 59< TS ) 4@4H4@5 Drug a%ministration common routes) 4;9 DrugE%elivery system nanoengineering) >>H4@< actively targete% t&erapies) 4@;H4@< activity %uration en&ancement) 4@8H4@< controlle%E an% triggere%Erelease systems) >>H4@8 %rug structural sta#ility) 4@8H4@; !ormulation processing strategies) 4@9 !uture %irections) 4@< passive tissue targeting) 4@;

E!!lu" pumps protection) :> Electrospinning) ;4 amorp&ous materials) ;9 Electrospun !i#ers sustaine%Erelease nano!i#erE#ase% %rug %elivery) ;: Electrospun nano!i#ers) ;9H;: %rugE%elivery systems) 58

895
Emulsi!icationE%i!!usion tec&niCue) 54< Emulsi!icationEevaporation met&o%) 54< Emulsion %roplets vs* lipi% nanoparticles) 55: En%oplasmic reticulum) 4;@ En%osomolytic liposomes !ormulation strategies) >4H>5 En%ot&elial .unctions) tig&t transport across) 4@< En&ance% permea#ility an% retention AEPRB e!!ect) 4;< EnvelopeEtype nano%evice) = En/ymes) 4>;H4>< Epi%ermal permea#ility #arrier AEP+B) 885 EPR* See En&ance% permea#ility an% retention AEPRB e!!ect Et&ium #romi%e %isplacement assay) 5@4 Etomi%ate supercoole% smectic nanoparticles) 489 Eu%ragitb 4@@ particles) 449) 44; DSC t&ermograms) 45@ p&ysical properties) 454 Evaporative precipitation) 444 Gene e"pression nanoparticleEme%iate% !ormulation !actors) 5>9 Gene t&erapy NPDDS) 4==H4>@ Gene trans!ection NP) 5>9 Geometric num#er mean %iameter AGNMDB) 44;H458 Geometric stan%ar% %eviation AGSDB) 44;H454 G-P* See Green !luorescent protein AG-PB Glia%in) :8 Glial cells) 4<> GNMD* See Geometric num#er mean %iameter AGNMDB Gol% nanoparticles antiEHER5 anti#o%yEtargete%) 4;> %rug %iscovery) 4<: molecular %iagnostics) 4<9 tumorEtargeting metallic nanoparticle %rugE%elivery systems) 49>H4:5 Gol% nanos&ells) 4;> Gol% nanosp&eres) 4> Golgi #o%ies) 4;@ Green !luorescent protein AG-PB) 49;) 49< GSD* See Geometric stan%ar% %eviation AGSDB Gut associate% (it& lymp&atic tissue AGA TB) 8@:

In%e"

-ace po(%ers) 55< -acial ma$eup pro%ucts) 55< -i#ro#last gro(t& !actor A-G-B) 58: :E-luorouracil) 5<< -ullerene molecules %rug %iscovery) 4<8H4<; -usigenic liposome &emagglutinating virus o! 'apanEliposomes A2irosomeB) 594H595 -usogenic liposomes !ormulation strategies) >4H>5

GA A) >5 GA T* See Gut associate% (it& lymp&atic tissue AGA TB Ganciclovir) 5<5 Gastrointestinal applications NPDDS) 8@:H84= Gelatin) 49) :5 carrageenan) 5<5 nanosp&eres) 4; Gels N C) 549 Gene %elivery c&itosan nanoparticles) 5>> nanoparticles !ormulation c&aracteristics) 5>4H5>> PACA) 8@@ polymeric nanoparticles sc&ematic representation) 5>5

Hair %ressings) 55< Heparin) 8@= Hig&Epressure &omogeni/ation) =4) 444) 54;) 559 %rug nanocrystals) <;H<= Hig&Es&ear !orces) 444 HP#etaCD* See Hy%ro"ypropylE#etaE cyclo%e"trin AHP#etaCDB HPC* See Hy%ro"ypropyl cellulose AHPCB Human immuno%e!iciency virus AHI2B) 4= Human serum al#umin AHSAB) > Hy%rocar#on ammonium compoun% antimicro#ial properties) 4=@ Hy%rogel matrices) 49 Hy%rogenE#ase% nanoparticulate %rugE%elivery systems) 45 Hy%rop&ilic HC salt %rugs !lu" en&ancement) 4@8 Hy%rop&o#ic #in%ing) 4:4 Hy%rosol tec&nology) <= Hy%rot&ermal process) 4;9 Hy%ro"ypropylE#etaEcyclo%e"trin AHP#etaCDB t&ermogravimetric curves) 4@:

In%e"

898
0etocona/ole) 559 0etopro!en) 454 p&ysical properties) 455 actose SEM) 459 aser pyrolysis) 4;9 eucine SEM) 459 HERHEtargete% silicaEcoate% lipi% micelles) 4;> ipe" e"tru%er) >@ ipi%AsB cells) 4;4 %rug e"pulsion) 54: !ree/e %rie% nanocapsules) < liposome !ormulation strategies) >4H>5 ocular applications) 5<4H5<> ipi%E#ase% colloi%al nano%rugE%elivery systems) ;H< liposomes %rug carriers) >4 !ormulation strategies) >4H>: ipi%E#ase% %rugE%elivery system nanoengineering !or sustaine% an% triggere% releases) >>H4@8 nanoparticulate) =>H>: liposome preparation an% c&aracteri/ation) =>H>4 ipi% carriers* See Nanostructure% lipi% carriers AN CB ipi%Eencapsulate% per!luorocar#on nanoemulsions) 4;> ipi% micelles HERHEtargete% silicaEcoate%) 4;> ipi% nanoparticles cosmetic) %ermal an% trans%ermal applications) 545H55> vs* emulsion %roplets) 55: liposome !ormulation strategies) >: ipo!ectamine) 594 ipop&ilic %rugs liposome preparation) =>H>@ iposomal alen%ronate) 5:5 iposomal amp&otericin) >4 iposomal clo%ronate) 5:5 iposomal cytara#ine) >4 iposomal %aunoru#icin) >4 iposomal %o"oru#icin) >@ iposomal nanosuspension clo!a/imine nanosuspension) <: iposomal vertepor!in) >4

Hy%ro"ypropyl cellulose AHPCB cyclosporine A) 45 Hypert&ermia malignant %antrolene) 94

I#upro!en supercoole% smectic nanoparticles) 48:H48; ICAME4* See Intracellular a%&esion moleculeE4 AICAME4B I%aru#icin) 845H848 IDDEPTM) <> I-P* See Intestinal !lui% pressure AI-PB I E;* See Interleu$inE; AI E;B I P* See Isolate% lim# protocol AI PB Immuni/ation mucosal) 84> nasal) 85@H855 oral) 84>H85@ parenteral) 84=H84> Immunoa%.uvants) 84<H84= Immunoliposomes) 4>5 Imper!ect crystal mo%el) 54> In!ections nano#iotec&nology) 4<>H4=4 In.ecta#le nanosuspension monocyte p&agocytic system) 9@ in vivo %istri#ution) as !unction o! particle si/e) 8> Insulin) 4;< Interleu$inE; AI E;B) 594 Interpenetrating polymer net(or$s AIPNB PAA) 5@@ Intestinal !lui% pressure AI-PB) 49<H49= Intestinal permea#ility) <4 Intracellular a%&esion moleculeE4 AICAME4B) 594 Intramuscular %rug a%ministration) 4;9 Intravenous %rug a%ministration) 4;9 Intravenous parenteral !ormulations) 94 Ionic #in%ing) 4;4 IPN* See Interpenetrating polymer net(or$s AIPNB Iron nanoparticles tumorEtargeting metallic nanoparticle %rugE%elivery systems) 49>H4:@) 4:4 Iron o"i%e nanoparticles #rain tumors) 4=@ Isolate% lim# protocol AI PB) 4:8 Isopropylacrylami%e polyacrylic aci%) 5@@ Itracona/ole) 98 vs* Sporano"b) 98

899
iposomeAsB) <5) 4;=) 4;> cationic) 59@H595 cryoETEM microscopy) 5:5 %iscovery) 4;8 !ormulation strategies) >4H>: cationic liposomes) >8H>9 !usogenic an% en%osomolytic liposomes) >5H>8 lipi% composition !or increase% sta#ility in vivo) >4H>5 lipi% nanoparticles) >9H>: pHEsensitive liposomes) >5 sterically sta#ili/e% liposomes) >5 targete% liposomes) >9 temperatureEsensitive liposomes) >8 lipi%E#ase% colloi%al nano%rugE%elivery systems) ;H< lipi%E#ase% nanoparticulate %rugE%elivery systems) => preparation an% c&aracteri/ation) =>H>@ lipop&ilic %rugs) >@ lo( temperature t&ermosensitive %o"oru#icin) 4@4) 4@5 nanocarriers) !or restenosis) 58 puri!ication) >@ restenosis local %rug %elivery) 58< iposomeE#ase% %rugE%elivery systems lymp&atic targeting) 8@:H8@; ipstic$s) 55< Eleucine) 445H449 pro%uction) 449 SEM) 44< o( temperature t&ermosensitive liposomes A TS B %o"oru#icin) 4@5 uci!erase protein HE0E5>8 cell line) 59: ymp&atic targeting %rugE%elivery systems %en%rimerE#ase%) 8@< liposomeE#ase%) 8@=H8@> NPDDS) 8@: ysosomes) 4;@ Malignant &ypert&ermia %antrolene) 94H95 Mannitol malignant &ypert&ermia) 94 Mass me%ian aero%ynamic %iameter AMMADB) 4< Mec&anical integrity testing nano!i#erE#ase% %rug %elivery) ;4H;> Me%ia milling processes %rug nanocrystals) <:H<= Megaceb) =9 Melamine nanosp&eres) 4= Meta#olomics) 4;< Metallic nanoparticle %rugE%elivery systems tumorEtargeting) 4:@H4:9 Met&ylmet&acrylate AMMAB) 5<: Micelles) 4;> %rug carriers nanoparticulate polymeric) >H4@ HERHEtargete% silicaEcoate% lipi%) 4;> polymeric %rug solu#ili/ation) 4@ researc& tren%s) 45 Micona/ole supercoole% smectic nanoparticles) 48: Microencapsulation DNA cryopreparation met&o%) 5>8 Micro!lui%ics) :H; %rug nanocrystals) <=H<> Microni/ation) <5 Micronsi/e% %rug po(%ers pro%uction) 448 Microparticles preparation) 44: Mi!epristone) = Milling %rug nanocrystals) =8H=9 Mitoc&on%ria) 4;@ M 2* See Multilamellar vesicles AM 2B MMA* See Met&ylmet&acrylate AMMAB MMAD* See Mass me%ian aero%ynamic %iameter AMMADB Molecular con%ensation) 4;: Molecular %iagnostics nano#iotec&nology) 4<8H4<: Molecular nanotec&nology) 4;5 Monoclonal anti#o%ies) 49< Monocytes) 59>H5:4 p&agocytic system in.ecta#le nanosuspension) 8=

In%e"

Macrop&ages) 59>H5:8) 84= Magnesium o"i%e po(%er antimicro#ial properties) 4=@ Magnetic nanoparticles) 4;> gene %elivery) 8@@H8@4 molecular %iagnostics) 4<8 Magnetic plain %e"tran nanosp&eres) 4>

In%e"

89:
NNano!i#erE#ase% %rug %eliveryO %issolution en&ancement !or imme%iateErelease %osage) ;8H;9 largeEscale manu!acturing) ;:H;> mec&anical integrity testing) ;<H;> sustaine%Erelease electrospun !i#ers) ;9H;: Nano!i#ers electrospun) ;4H;8 Nano&y#ri%s) 55H58 Nanolit&ograp&y %ipEpen) 4;9H4;: Nanomaterials c&aracteristics) 4H8 manu!acture) 8H; measurement) 4H8 Nanome%icine) 4<8H4=5) 4<<H4=5 t(entyE!irst century) 4<= Nanometer lengt& scale) 5 Nanometrology) 4H8 NanoparticleAsB* See also Soli%Elipi% nanoparticles AS NBM Supercoole% smectic nanoparticles a!!ecting #iological per!ormance) :;H:= analytical c&aracteri/ation) 9 #iocompati#ility) := an% #io%egra%a#ility) :: #iological researc&) 4;> #ottomEup manu!acturing) 9H: calcium car#onate) 49 ceramic) 4;>) 4;> c&aracteri/ation) :9H:= C I? paramagnetic) 4;> commercially availa#le) 4=H5@ copolymer ratio) :: crystallinity) :: %issolution pro!ile) :< %rug %iscovery) 4<:H4<; %rug loa%ing an% loa%ing e!!iciency) :< %rugEpolymer interactions) :; !uture %irections) 59 gene %elivery !ormulation c&aracteristics) 5>4H8@4 gol% %rug %iscovery) 4<:H4<; molecular %iagnostics) 4<8 &y%rop&o#icity) :: internali/ation into cells) := iron o"i%e #rain tumors) 4=@ lipi% vs* emulsion %roplets) 55:

Montreal Protocol C-C) <; Mucoa%&esive) 4><H4>= Mucoa%&esive nanoparticulate %rugE%elivery systems) 4><H4>= Mucosal immuni/ation) 84> Multi!unctional nanoparticle sc&ematic representation) 495 Multilamellar vesicles AM 2B) => Multiple mo%el) 54>

Nano#iotec&nology car%iovascular %isease) 4=@ central nervous system %isor%ers) 4<>H4=@ %rug %elivery) 4<;H4<< %rug %iscovery) 4<:H4<; !uture prospects) 4=4H4=5 in!ections) 4=@H4=4 molecular %iagnostics) 4<8H4<9 oncology) 4<> personali/e% me%icine) 4=4 t&erapeutic applications) 4;=H4<@ Nano#o%ies %rug %iscovery) 4<; Nanocapsules) 4;>) 58; lipi% !ree/e %rie%) < polymeric %rug carriers) 4< Nanocarriers restenosis) 58:H5:; Nanocontainer tec&nology) 58 Nanocrystal* See also Drug nanocrystals tec&nology) <;H<= Nanocrystallites) 55 Nano%evices envelopeEtype) > !a#rication) 4;8H4;; synt&etic) 4<< Nano%iagnostics) 4<8) 4<<H4<= clinical) 4<< Nano %rug po(%ers pro%uction) 448H44; Nanoe%ge tec&nology %rug nanocrystals) =4H=5 Nanoemulsions antimicro#ial) 4=4 lipi%Eencapsulate% per!luorocar#on) 4;> Nanoen%oscopy) 4<<H4<= Nanoengineere% prost&etics) 54 Nano!i#erE#ase% %rug %elivery) ;4H;; automate% !i#er si/ing) ;;H;<

89;
NNanoparticleAsBO magnetic) 4;> gene %elivery) 5>>H8@4 molecular %iagnostics) 4<8 molecular (eig&t) :9H:: natural #io%egra%a#le polymers) :5H:8 neurosurgery) 4>@ non#io%egra%a#le polymers) :9 particle si/e) :;H:< p&ysical properties) :9H:;) 44> polyA#etaEamino esterBE#ase% gene %elivery) 5>>H8@@ poly#utylcyanoacrylate) 5== polyester polysacc&ari%e) 4; polylacti%eEcoEglycoli%e scanning electron microscopic images) :< polymeric carriers) :4H:8 polyvinyl caprolactone) :9 preparation) 44: protective against pat&ogens) 54 r&o%amineEcontaining) 58= route o! a%ministration) :; silicon antiEHER5 anti#o%yEtargete%) 4;> solu#ility) :: structure) 4=; sur!ace mo%i!ication) := synt&etic #io%egra%a#le polymers) :8H:9 tagging) 5@ targete% %elivery) :> t&erapeutic applications) 4;<H4;= t&ermosensitive) :9 t&iamineEtargete%) 4;> t&iomer) 54 topE%o(n manu!acturing) :H; /eta potential) :< NanoparticleEaptamer #iocon.ugate) 4;> Nanoparticle %rug %elivery systems ANPDDSB) 44H45) 4=:H4>8 a#sorptiveEme%iate% transcytosis) 5=; a%vantages) 4=;H4=< al$ylcyanoacrylate monomer inter!acial polymeri/ation) 4=< applications) 4==H5@5 #rain compartment) 5=:H5=; en%ot&elial cells) 5=;H5=< #rainEtargete% %elivery) 5=9H5=: cancer) 4>:H4>; central nervous system) 5=4H5== colonic targeting) 8@;) 848 NNanoparticle %rug %elivery systems ANPDDSBO %rugs) 4=>H4>@ en/ymes) 4>;H4>< !ormulation applications) 4>@ gastrointestinal applications) 8@:H849 a%vantages) 848H849 evaluation) 849 !uture perspectives) 849 gene t&erapy) 4>>H5@5 &y%rogenE#ase%) 45H48 lymp&atic targeting) 8@:H8@; %evelopment) 8@;H8@= manu!acturing tec&niCues) 4=<H4== mucoa%&esive) 4><H4>= ocular applications) 4>8) 5<4H5<> p&armaceutical applications) 4=:H5@8 polymer inter!acial %eposition) 4=<H4== proteins an% pepti%es) 4==H4>8 pulmonary treatment) 4>8H4>: receptorEme%iate% transcytosis) 5=;H5=< ris$s) 5@5H5@8 vascular t&rom#osis) 4>=H4>> NanoparticleEme%iate% gene e"pression !ormulation !actors) 5>9 Nanoparticulate %e!ine%) 85<H85= %rug carriers polymeric micelle) >H45 protamineE#ase%) 49H4:) 4>< %rugE%elivery systems polymerE#ase%) 45H4= Nanoprecipitation) 444 Nanopro#es plaCue) 4=@ Nanopure tec&nology) =4H=5 %rug nanocrystals) =4 WP) =5H=8 Nanoscale %rug po(%ers antimicro#ial properties) 4>@ Nanoscience) 4 Nanos&ells gol%) 4;> oncology) 4<> Nanosp&eres) 4;> al#umin) 4;H4< alumina) 4>H5@ anti#o%yEcoate%) 55 #io%egra%a#le polylacti%e) 4=H4> gelatin) 4;H4< gol%) 4> magnetic plain %e"tran) 4> melamine) 4=

In%e"

In%e"

89<
NNanosuspensions parenteral %eliveryO pre%ecessor tec&nology) 88 pre%iction o! sta#ility) 8;H8= in vitro %issolution) 8< Ta"olb) 89) 99 Nanotec&nology) 4 %evelopment) 4;5H4;8 %iverse an% emerging tren%s) 5@H58 Nanot&erapeutic applications #iological reCuirements) 4:>H4<@ Nanot&erapeutic %evices) 4;;H4;= Nanotu#es car#on) 5@) 4;> cellular manipulation) 54 Nano(ires siliconE#ase%) 4;> Napro"en p&ysical properties) 44= Nasal immuni/ation) 84>H855 Natural an% synt&etic polymers) :4H:> in vitro cell culture) :=H:> Ne( c&emical entities ANCEB) << Nitric o"i%e eluting stents nano!i#ers) 4=@ N0>44) 4=@ N C* See Nanostructure% lipi% carriers AN CB Nonviral vectors) 5@4H5@5 Normali/ation (in%o() 49= NP gene trans!ection) 5>8 polyAD) Elactic aci%BE#ase% !ormulation c&aracteristics) 5>4H5>= restenosis local %rug %elivery) 58< nanocarriers) 58< polymeric gene %elivery) 595H598 NPDDS* See Nanoparticle %rug %elivery systems ANPDDSB Nucleic aci%s) 4;4H4;5

NNanosp&eresO nanocarriers !or restenosis) 58< plain polymet&yl met&acrylate) 4=H4> polystyrene) 4= silica) 4> silver) 4> Nanostructure% %rug microparticles aerosol !lo( reactor met&o% multicomponent %rug synt&esis) 448H449 pepti%e coating particles) 455H45: reactor %rug crystalli/ation) 454H455 Nanostructure% lipi% carriers AN CB) 549H54; c&emical protection) 559 controlle%Erelease properties) 555H558 cosmetic pro%ucts) 548) 54:) 55;H55> creams) 554H555 entrapment e!!iciency) 555H558 gels) 55@H554 &y%ration) 559H55; loa%ing capacity) 555H558 morp&ology an% structure) 54=H54> occlusive e!!ects) 559H55; penetration en&ancement) 55; p&ysical sta#ility) 554H555 pro%uction) 55@H554 s$in e!!ects) 554H55; trans%ermal pro%ucts) 54>H554) 55<H55> types) 54=H54> Nanostructure% materials) 54 Nanostructure% monolit&s) 55 Nanosuspensions clo!a/imine) <: vs* liposomal !orm) <: %ermal) <; in.ecta#le monocyte p&agocytic system) 9@ liposomal) <: parenteral %elivery) 88H9; com#ination o! component tec&nologies) 89 com#ine% precipitation1&omogeni/ation) 8; !ormulation approac&es) 89H8; !ormulation selection) 8; &istorical intro%uction) 88H89 &omogeni/ation) 8: in.ecta#le nanosuspension sa!ety) 8=H9@ intrat&ecal %elivery) 9; manu!acturing met&o%s) 89H8; molecular %eterminants o! particle si/e) 8;H8< parenteral !ormulation applications) 9@H9: p&armaco$inetic pro!iles) 8<H8= p&armaco$inetics) 99 precipitation) 8:

?ctylglucosi%e) >@ ?cular applications %rug nanocrystals) <; lipi%s) 5<= NPDDS) 4>8H4>9) 5<4H5<> ?ilEinE(ater emulsion) 444 ?ligo%eo"yri#onucleoti%es A?DNB) >8 ?ncology) 4><H4>= nano#iotec&nology) 4<; p&oto%ynamic t&erapy) 49 ?p&t&almic %rugs) 4>8

89=
?psini/ation) 495 ?ptical t(ee/ers) 8 ?ral %elivery %rug nanocrystals) <9 ?ral %rug a%ministration) 4;9 ?ral immuni/ation) 84> ?s(al% ripening) <= Pectin) 5<5) 5<8 PEC2D* See PlasmaEen&ance% vapor %eposition APEC2DB PEG* See Polyet&ylene glycol APEGB PEI* See Polyet&ylenimine APEIB Penetration %e!ine%) 85<H85= PE?* See Polyet&ylene o"i%e APE?B Pepti%e coating particles nanostructure% %rug microparticles) 454H455 Pepti%es NPDDS) 4==H4>8 Percutaneous a%sorption) 85= Percutaneous coronary intervention APCIB) 58: restenosis) 4=@ Per!ume) 55= Permeation %e!ine%) 85= Pero"isomes) 4;@ Personali/e% me%icine nano#iotec&nology) 4=4 PeyerPs patc&es) 8@:) 8@;) 85@ P&agocytic system monocytes in.ecta#le nanosuspension) 9@ P&aseEinversionE#ase% tec&niCue) 54< P&ase separation) 444 P&osp&ati%ylc&olines APCB) >5 P&osp&orotioate% oligo%eo"ynucleoti%es APTE?DNB) 58>H59@) 599 P&oto%ynamic t&erapy cancer) 49 P&otosensiti/ers) 49 P&otot&ermal tumor a#lation) 4<> PillCamd) 4<= PistonEgap &omogeni/ation in (ater %rug nanocrystals) <> Plain polymet&yl met&acrylate nanosp&eres) 4=H4> Plasma cell mem#rane) 4:> PlasmaEen&ance% vapor %eposition APEC2DB) 4;; Plasmi% DNA P GA) 5>< PlateletE%erive% gro(t& !actor APDG-B) 58:) 598H599) 5;8 Platelets) 499H49: P G* See Polyglycoli%e AP GB P GA* See Polylacti%eEcoEglycoli%e AP GAB P GAEPEG* See Polylacti%eEcoEglycoli%e polyet&ylene glycol AP GAEPEGB

In%e"

PAA* See Polyacrylic aci% APAAB PACA* See Polyal$ylEcyanoacrylates APACAB Paclita"el) 59> local %elivery !or arterial locali/ation) 59=H59> parenteral !ormulations) 9@H9: c&aracteri/ation an% manu!acture) 99 supersatura#le !ormulation) 8@> Palm oil) 4@4 Parenteral A#ra"aneb c&aracteri/ation an% manu!acture) 99 clinical trials) 99H9: p&armaco$inetics) 99 Parenteral !ormulations anticancer) 99H9: anti!ungal) 95H98 clinical stu%y) 98H99 %rug nanocrystals) <9 intravenous) 94H95 preclinical stu%ies %rugEresistant !ungal stain) 95H98 %rugEsuscepti#le !ungal stain) 95 regional anest&etics) 8>H94 Parenteral immuni/ation) 84=H84> Parenteral paclita"el) 84=H84> c&aracteri/ation an% manu!acture) 99 Parenteral Ta"olb clinical trials) 99H9: Particle !ormation e"perimental parameters) 44= Passive tissue targeting %rugE%elivery system nanoengineering) 4@; Passive tumor targeting metallic NDDS) 49> P+CA* See Poly#utylcyanoacrylate AP+CAB PC* See P&osp&ati%ylc&olines APCB PEc&annel M?S-ET !a#rication) 4;: PCI* See Percutaneous coronary intervention APCIB PCNA* See Proli!erating cell nuclear antigen APCNAB PDG-* See PlateletE%erive% gro(t& !actor APDG-B

In%e"

89>
Polymeric nanoparticulateE#ase% %rugE%elivery systems aerosol !lo( reactor met&o%) 448H449 gene %elivery sc&ematic representation) 5;> lymp&atic targeting) 8@: preparation) :; Polymet&acrylate APMAB) :8 Polymet&yl met&acrylate APMMAB) :8 Polystyrene nanosp&eres) 4= Polyvinyl caprolactone nanoparticles) :8 Poorly solu#le %rugs solu#ili/ation) <4H=: Precipitation %rug nanocrystals) <=) =8H=9 evaporative) 444 Progesterone supercoole% smectic nanoparticles) 48:H48; Proli!erating cell nuclear antigen APCNAB) 58> Propo!ol) 4< Prost&etics nanoengineere%) 54 ProtamineE#ase% nanoparticulate %rug carriers) 49H4: Protective nanoparticles against pat&ogens) 54 Proteins cells) 4;5 NPDDS) 4==H4>8 Proticles) 49H4:) 4>< PTE?DN* See P&osp&orotioate% oligo%eo"ynucleoti%es APTE?DNB Pullulan) :8) 5@5 Pulmonary %rug %elivery) 4>8H4>: %rug nanocrystals) >;

PMA* See Polymet&acrylate APMAB PMMA* See Polymet&yl met&acrylate APMMAB PolyAD) Elactic aci%B !ormulation c&aracteristics) 5<<H5<= PolyAD) Elacti%eEcoEglycoli%eB) 5<<H5<= PolyAepsilonEcaprolactonB) 5<; Polyacrylic aci% APAAB) 5<: IPN) 5@@ isopropylacrylami%e) 5@@ Polyal$ylEcyanoacrylates APACAB) :9) 5<9H5<< gene %elivery) 5>> Polyan&y%ri%es) :8 PolyA#etaEamino esterBE#ase% nanoparticles gene %elivery) 5>> PolyAD) Elactic aci%BE#ase% NP !ormulation c&aracteristics) 5>4H5>< Poly#utylcyanoacrylate AP+CAB cyclop&osp&ami%eEloa%e%) 4>8 nanoparticles) 5== Polycar#onate mem#rane) >@ PolyElEcaprolactones) :8 Polyester polysacc&ari%e nanoparticles) 4; Polyet&ylene glycol APEGB) 498 Polyet&ylene o"i%e APE?B) 5>: Polyet&ylenimine APEIB) 5>= Polyglycoli%e AP GB) 58< Polylacti%e) :8 Polylacti%eEcoEglycoli%e AP GAB) :8) 4=;H4=<) 5><) 8@< molecular (eig&t) 5>: nanoparticles) SEM) :< plasmi% DNA) 5>< Polylacti%eEcoEglycoli%e polyet&ylene glycol AP GAEPEGB !ormulation) 4>; PolymerAsB natural origin) 5<5H5<9 PolymerE#ase% nanoparticulate %rugE%elivery systems) 45H4= Polymeric %rug nanoparticles aerosol !lo( reactor met&o%) 449H44; Polymeric gro(t& in&i#itors) <= Polymeric micelles %rug solu#ili/ation) 4@H44 researc& tren%s) 45 Polymeric nanocapsularE#ase% %rugE%elivery systems lymp&atic targeting) 8@;H845 Polymeric nanocapsules %rug carriers) 4<

7uantum %ots) 4;> %rug %iscovery) 4<:H4<; molecular %iagnostics) 4<8H4<9 Rapamuneb) <= Rapamycin) 58; Reactor %rug crystalli/ation nanostructure% %rug microparticles) 454H455 ReceptorEme%iate% transcytosis) 5=;H5=< Regional anest&etics parenteral !ormulations) 9@H94 RES* See Reticuloen%ot&elial system ARESB Restenosis) 58: %e"amet&asone) 59:H59; gene t&erapy) 58>H59@ liposomal gene %elivery) 59@H595

8:@
NRestenosisO local %elivery !ormulation) 59; local %rug %elivery) 58<H58=) 598H59; nanocarriers) 58:H5:; polymeric NP gene %elivery) 595H598 systemic %rug %elivery) 59=H5:: arterial locali/ation) 59=H59> !or systemic target) 59>H5:4 Reticuloen%ot&elial system ARESB) > Retinoic aci%) 5<= R&o%amine) 5:8 nanoparticles containing) 58= RHEtargete% silicaEcoate% lipi% micelles) 4;> Ri#osomes) 4:> Rolipram) 848 Roug& en%oplasmic reticulum) 4:>H4;@ So%ium leucine SEM) 45: SolEgel process) 4;9H4;: Soli%Elipi% nanoparticles AS NB) :) :9) 4==) 549 #rain) 5=<H5== c&emical protection) 559 controlle%Erelease properties) 555H559 cosmetic pro%ucts) 54>H554 creams) 554 entrapment e!!iciency) 555H559 gels) 55@H554 &y%ration) 559H55; loa%ing capacity) 555H559 lymp&atic targeting) 8@= morp&ology an% structure) 54=H54> occlusive e!!ects) 559H55; ocular applications) 5<= penetration en&ancement) 55: p&ysical sta#ility) 554H555 pro%uction) 54;H54= researc& tren%s) <H> s$in e!!ects) 554H55> trans%ermal pro%ucts) 54>H55@ types) 54> Solvent %isplacement met&o%) 54< SPI?* See Superparamagnetic iron o"i%e ASPI?B SPM* See Scanning pro#e microscopy ASPMBM Sul!opropylmet&acrylate ASPMB Sporano"b) 95H99) <5 vs* itracona/ole) 98 parenteral a%ministration) <: SprayE%rying) =9) 445H448 Starc&) 49 Starc& acetate) 5<5 StemEcellE#ase% t&erapies nano#iotec&nology) 4<= Stents %rugEeluting) 58: Sterically sta#ili/e% liposomes !ormulation strategies) >5 Stratum corneum a%sorption) 85= Su#cutaneous %rug a%ministration) 4;9 Sul!opropylmet&acrylate ASPMB) 5<: Supercoole% smectic nanoparticles) 45>H48= CM nanoparticles properties an% p&ase #e&avior) 488H48< ultrastructure) 48;) 48= etomi%ate) 48: i#upro!en) 48: matri" composition) 488H48: mo%el %rugs) 48:H48; preparation met&o%s) 485

In%e"

SaltingEout) 444 Scanning electron microscopy ASEMB) 8 Scanning pro#e microscopy ASPMB) 8 Sel!E%iagnostics) 4=4 Sel!EAmicroB emulsi!ying %rugE%elivery systems lymp&atic targeting) 8@>H845 SEM* See Scanning electron microscopy ASEMB S&aving ai%s) 55< SilicaEcoate% lipi% micelles HERHEtargete%) 4;> Silica nanosp&eres) 4> SiliconE#ase% nano(ires) 4;> Silicone nanoporeEmem#raneE#ase% %rugE%elivery system) 4: Silicon nanoparticles antiEHER5 anti#o%yEtargete%) 4;> Silver nanosp&eres) 4> Silver po(%er antimicro#ial properties) 4=4 SingleE#eam gra%ient trap) 8 SingleEmolecule %etection) 54 SiRNA) >8 S$in aging) 55< permeation test) 85<) 885) 889 S N* See Soli%Elipi% nanoparticles AS NB SmallEscale cyclone) 44; SMC* See Smoot& muscle cells ASMCB Smectic nanoparticles preparation met&o%s) 485 Smoot& en%oplasmic reticulum) 4;@ Smoot& muscle cells ASMCB) 58: So%ium c&lori%e SEM) 45:

In%e"

8:4
TriCorb) <= Triggere%Erelease% systems %rugE%elivery system nanoengineering) >>H4@8 mo%ulation o! %rug environment an% p&ysicoc&emical properties %rugE%elivery system nanoengineering) 4@5H4@8 polymeric %rugE%elivery system %rugE%elivery system nanoengineering) >>H4@@ TumorAsB #loo% vessels) 49: p&otot&ermal a#lation) 4<> TumorEassociate% macrop&ages ATAMB) 49: Tumor necrosis !actor ATN-B) 49=) 594 TumorEtargeting metallic nanoparticle %rugE%elivery systems) 494 active vs* passive tumor targeting) 49> angiogenic tumor #loo% vessels) 49;H49< c&emistry) 4:4 cyto$ineEme%iate% re%uction o! interstitial !lui% pressure) 4:@ gol% nanoparticles) 4:@ stu%ies) 4:4H4:5 intratumor #arriers) 49> iron nanoparticles) 4:@ stu%ies) 4:5 multi!unctional nanoparticles) 4:8H4:9 RES clearance) 495H499 sa!ety) 4:@H4:4 t&ermal a#lation) 4:5 tumor angiogenesis an% interstitial !lui% pressure) 49= an% vasculari/ation) 499H49: tumor mo%els) 49>H4:@ vascular en%ot&elial gro(t& !actor) 49:H49; T(ee/ers optical) 8 Tyrp&ostins local %elivery !or restenosis) 598H59:

NSupercoole% smectic nanoparticlesO sta#ili/er system) 485H488 t&ermotropic mesop&ases) 48@ Superparamagnetic iron o"i%e ASPI?B) 4:@ Sustaine%Erelease% systems %rugE%elivery system nanoengineering) >>H4@4) 4@5H4@8 mo%ulation o! %rug environment) 4@5H4@8 p&ysicoc&emical properties) 4@5H4@8 polymeric %rugE%elivery system) >>H4@4 Sustaine%Erelease electrospun !i#ers nano!i#erE#ase% %rug %elivery) ;9H;: Synt&etic nano%evices) 4<<

TAM* See TumorEassociate% macrop&ages ATAMB Targete% %rug %elivery) 4<; Targete% liposomes !ormulation strategies) >9 Ta"olb vs* A#ra"aneb) 99 nanosuspensions) 88H89 parenteral !ormulations clinical trials) 99H9: TAWUSb) 58; TEM* See Transmission electron microscopy ATEMB TemperatureEsensitive liposomes !ormulation strategies) >8 TGA* See T&ermogravimetric analysis ATGAB T&ermal a#lation tumorEtargeting metallic nanoparticle %rugE%elivery systems) 4:5H4:8 T&ermogravimetric analysis ATGAB) :; T&ermosensitive nanoparticles) :9 T&ermotropic calamitic mesop&ases structure) 48@ T&iamineEtargete% nanoparticles) 4;> T&iomer nanoparticles) 54 Tissue !actor) 58< TN-* See Tumor necrosis !actor ATN-B TNT system) 4;> To#ramycin) 84@) 845H848 S N) 5<= Transcytosis receptorEme%iate%) 5=;H5=< Trans%ermal applications) 85<H88: !uture) 88: limitations) 889H88: Trans!ersomes) 4;> Transmission electron microscopy ATEMB) 8 Triacylglycerols t&reeE%imensional structure) 54:

U=;* See 5EAminoc&romone UE=;=>8 AU=;B UltraEsmall superparamagnetic iron o"i%e AUSPI?B) 4:@ Ultraviolet #loc$ers) 55<) 55=

2accination a%.uvantEvector) 84<H858 2ascular en%ot&elial gro(t& !actor A2EG-B tumorEtargeting metallic nanoparticle %rugE%elivery systems) 49:H49;) 4:@

8:5
2ascular smoot& muscle cells A2SMCB) 58> 2ascular t&rom#osis) 4>=H4>> 2asculogenesis) 49; 2asoactive intestinal pepti%e A2IPB) 4>9 2ectors nonviral) 5@4 2EG-* See 2ascular en%ot&elial gro(t& !actor A2EG-B 2ertepro!in liposomal) >@ 2esicular vacuolar organelles A22?B) 49: 2IP* See 2asoactive intestinal pepti%e A2IPB 2irosome) 594H595 von ,ille#ran% !actor) 499 22?* See 2esicular vacuolar organelles A22?B ,aterEinEoilEinE(ater %ou#le emulsion) 5>5 ,aterEsolu#le amp&ip&ilic nanocarriers !ree/e %rying) 44 ,et milling) 444 ,&ole s$in penetration) 885H889

In%e"

Vel%o"b) <5 Veta potential nanoparticles) :<

-IGURE 4*4 engt& scale s&o(ing t&e nanometer in conte"t* T&e lengt& scale o! interest !or nanoscience an% nanotec&nologies is !rom4@@ nm %o(n to t&e atomic scale appro"imately @*5 nm* Source6 -rom Re!* 4*

-IGURE 8*5

Sc&ematic %iagram o! speci!ic receptor targeting o! nanosp&eres using ligan%s*

-IGURE 9*;

Results o! automate% !i#er si/e analysis*

-IGURE :*: T(o nanosuspenE sions compose% similarly) pro%uce% #y &ig&Epressure &omogeni/ation) conventional met&o% Ale!t B versus translucent nanosuspension ApartiE cle si/e (ell #elo( 4@@ nmB resulting !rom H>; tec&nology* T&e re% laser #eam is re!lecte% #y t&e tiny nanoE crystals* Source 6 -rom Re!* 9@*

-IGURE 44*4 Structure o! a typical cell*

-IGURE 48*4 Di!!erent types o! nanoparticulate structures*

-IGURE 49*4 +asic types o! soli% nanoparticles an% nanostrucE ture% lipi% center* A##revations6 N C) nanostructure% lipi% carrierM S N) soli%Hlipi% nanoparticle* Source6 -rom Re!* 99*

-IGURE 4:*4 Con!ocal images o! #alloonEin.ure% rat caroti% arteries a!ter intaluminal %elivery o! r&o%amine solution an% r&o%amineEcontaining nanoparticles* T&e arteries (ere &arveste% >@ minutes AA an% DB) eig&t &ours A+ an% EB) an% one %ay AC an% -B a!ter %elivery* Source6 -rom Re!* 4@=*

-IGURE 4:*5 AAB In&i#ition o! neointimal !ormation 49 %ays a!ter #alloon in.ury to rat caroti% artery an% intraluminal instillations (it& 5@ _M A4nmoleB antisense or scram#le% partially p&osp&orot&ioate% oligo%eo"ynucleoti%es APTE?DNsB encapsulate% in P GA nanoparticles Atotal o! :@ _ suspensionB* P&otomicrograp&s o! representative &istological sections* 2erc&oe!!Ps elastin stain) magni!ication `45*:* A+B -luorescence micrograp&s o! #lan$ an% PTE?DNEloa%e% P GA nanoparticles* T&e PTE ?DNs (ere covalently la#ele% (it& -ITC prior encapsulation* Note !luorescence signal in over >@[ o! t&e particles* A##reviations6 A) a%ventitiaM ) l(nenM M) me%iaM N) neointimaM NP) nanoparticlesM SCENP) scram#le% NPM ASENP) antisense NP* Source6 -rom Re!s* 4>= an% 5@4*

-IGURE 4:*8 ACB o(E an% &ig&Epo(er p&otomicrograp&s o! &yperc&olesterolemic ra##it iliac artery stents at 5= %ays A2er&oe!! stainingB o! control AI an% IIB an% o! animals treate% (it& A 8 mg1$g AIII an% I2B an% ; mg 1$g A2 an% 2IB* A##reviation6 A) liposomal alen%ronate* Source 6 -rom Re!s* 5;> an% 5>5*

-IGURE 4=*5 -ormulation !actors in!luencing nanoparticleEme%iate% gene e"pression* A##reviations6 NPs) nanoparticlesM PE) primary en%osomesM RE) recycling en%osomes*

-IGURE 5@*4 2isuali/ation o! !lagellinEcoate% nanoparticles Are% %otsB in t&e !ollicleEassociate% epiE t&elium o! PeyerPs patc&es #y !luoE rescence* -lagellin o! S* enteriti%is (as use% to coat Gantre/ nanoE particles Ala#ele% (it& r&o%amine + isot&iocyanateB an%) t&us) o#tainE ing a%.uvant vectors a#le to sprea% (it&in t&e gut in a similar (ay t&an t&e (&ole #acteria* Source6 -rom Re!* 89*

-IGURE 54*4 CryoEsectione% images o! t&e !luorescent la#ele% nanoparticulate (it& porcine epi%ermal s$in layer a!ter in vitro s$in permeation test* AAB Stratum comeum outer layer a%sorption o! 5@@ nmEsi/e% polysturene nanoparticulate* A+B Percutaneous a#sorption o! 9@ nmEsi/e% polyE styrene nanoparticulate* ACB Epi%ermal layer penetration o! t&e mo%i!ie% nanoparticulate* A##reviations6 EP) epi%ermisM SC) stratum comeum*

-IGURE 54*5 C SM images o! t&e nanoparticulate penetration (it& a guinea pig s$in AcryoE sectione%B* -luorescentEla#elle% nanoparticulate Agreen regionB) nucleiEla#ele% DAPI A#lue regionB* AAB 9@ nm si/e o! nanoparticles) A+B 48@ nm si/e o! nanoparticles A4@ pieces o! /E%irection sectioning image o! cross sectione% tissue are merge%B* Nanoparticulate6 PolycaprolactoneEpolyet&yleneglycol #loc$ copolymer aggregates* -luorescent6 Ru#rene* Source6 -rom Re!* 5<*

-IGURE 54*8 Images o! t&e liposome nanoparticulate (it& !luorescent #y in vitro permeation test (it& a guinea pig s$in* AAB RITC saturate% solution A+B RITC (it& t&e mo%i!ie% liposome* ARe% regionB) &y%rop&ilic !luorescent %ye ARITCBM A#lue regionB) DAPI !or nucleiM #asal layer A(&ite arro(sB*

-IGURE 54*9 -luorescent images o! in vivo permeation stu%y (it& Al#ino Hartley guinea pig* AAB ?1, emulsion !ormulation* A+B Mo%i!ie% polymeric nanoparticulate*