j. Cosmet.

Sci., 55,253-264 (May/June2004)

Ethosomesand liposomesas topical vehiclesfor

azelaic acid: A preformulationstudy
ELISABETTA ESPOSITO, ENEA MENEGATTI,

and

RITA CORTESI, Department

of Pharmaceutical
Sciences,
University
of
Ferrara,1-441O0 Ferrara,Italy.
Accepted
for publication
April 6, 2004.
Synopsis

The basicpropertiesand the in vitroreleaserate kineticsof azelaicacid (AA), alternativelyvehiculatedin

differentphospholipid-based
vesiclessuchas ethosomes
or liposomes,were investigated.Ethosomeswere
producedby a simple methodbasedon addition of an aqueousphaseto an ethanolsolution(comprised
between20% and 45%, v/v) of soyphosphatidylcholine(5%, w/w) and AA (0.2%, w/w) under mechanical
stirring.Liposomes
wereobtainedby the samecompositionin the absence
of ethanolwith the reverse-phase
evaporationmethod. Vesicle size was measuredby photon correlationspectroscopy
(PCS), evidencing
smaller mean diametersand narrowerdimensionaldistributionsin the caseof ethosomeswith respectto
liposomes.
In orderto obtainhomogeneously
sizedvesicles,both ethosomaland liposomaldispersions
were
extrudedthroughpolycarbonate
membranes
with poresof calibrateddiameter(400 nm and 200 nm).
Vesiclemorphologywascharacterized
by freeze-fracturescanningelectronmicroscopy(SEM) showingthe
presence
of unilamellarvesiclesboth in liposome-and in ethosome-based
dispersions.
Free energymeasurementsof the vesiclebilayerswere conductedby diffbrentialscanningcalorimetry(DSC). AA diffusion
fromethosomal
or liposomaldispersions
andfromethosomes
andliposomes
incorporated
in a viscous
gel was
investigatedby a Franzcell assembled
with syntheticmembranes.
The releaserate wasmorerapid from
ethosomalsystemsthan from liposomalsystems.In particular,ethosomes
producedby the highestethanol
concentration
released
AA morerapidly,and the sametrend wasfoundusingviscousforms.

INTRODUCTION

Azelaic acid (AA) is a saturateddicarboxylicacid found naturally in wheat, rye, and

barley.It is a competitiveinhibitor of mitochondrialoxidoreductases
and of 5 alphareductase,
inhibiting the conversion
of testosterone
to 5-dehydrotestosterone
(1). AA is
an anti-keratinizingagent,displayingantiproliferativecytostaticeffectson keratinocytes
and modulatingthe early and terminal phasesof epidermaldifferentiation.It also
possesses
bacteriostaticactivity toward both aerobicand anaerobicbacteria,including
Propionibacterium
aches
(2).
Acheis a skin disorderoccuringwhenexcess
oil (sebum)productioncombinedwith dead
skin cellsclogspores.Bacteriaform in the pores,resultingin red, inflamedpimples,
Addressall correspondence
to ElisabettaEsposito.
253

254

JOURNAL OF COSMETIC SCIENCE

pus-filledwhiteheads,or blackheads.
There are many differentformsof acne,but acne
vulgarisis the most commonform. The physiopathologic
mechanismof acneseemsto
be dependenton four main factors:(a) sebumproductionand excretion,(b) type of
keratinizationof the follicular channel,(c) microbialcolonizationof the pilosebaceous
unit, and (d) inflammatoryreactionof the perifolliculararea. AA is effectivein the
treatmentof acnebecauseit possesses
an activity againstall of thesefactors(3,4). AA is
alsoefficacious
in the treatmentof alopeciaaerata,a highly unpredictableautoimmune
skin diseaseresultingin the lossof hair on the scalpand elsewhere
on the body(1).
In the presentstudythe in vitroreleaserate kineticsof AA alternativelyvehiculatedin
differentphospholipid-based
vesicles,suchasethosomes
or liposomes,
wereinvestigated.
Ethosomes
couldbe describedaslipid vesicularsystems
embodyingethanolin relatively
high concentrations.
These"softvesicles"
representnovelvesicularcarriersfor enhanced
deliveryto/throughthe skin (5). Ethosomeshavea particlesizethat can be modulated
from tensof nanometersto microns.One main featureof this new type of vesicleis its
soft structure,which carriesthe incorporated
activeagentinto the skin lipid bilayers,
enablingfacilitateddelivery.The useof ethosomalcarriersresultsin deliveryof high
concentrations
of activeto/throughthe skin, regulatedby the systemcompositionand
their physicalcharacteristics.
Vesicle characterization
can be assessed
by DSC, DLS,
NMR, ultracentrifugation,and electronmicroscopy.Dayan and Touitou (6) havedemonstratedthe major potentialof ethosomes
to promotedrug penetrationthroughskin
with respectto liposomes.
In vivoexperimentsand clinicaltrialshavedemonstrated
that a rangeof moleculessuch
as testosterone,acyclovir(Zovirax; Glaxo Wellcome plc), and insulin can be delivered
effectivelythroughthe cell .membranes
of animaland humanskin. An alterationof the
ethosome
formulationcanmodulatethe levelof penetration(restrictingdrugdeliveryto
the skin only, as requiredfor herpeslabialistreatmentwith Zovirax, or allowing full
dermalpenetration,as requiredfor insulintherapy)(7). Anothermolecule,trihexyphenidyl hydrochloride,incorporatedin ethosomes,
is proposedfor transdermaladministration in Parkinsonpatients,from which the geriatricpopulationmay greatlybenefit
(6). Transdermalabsorptionof polypeptidesis currentlyunder investigation(8). The
high interestin ethosomes
in the designof new therapieshas beeninvestigatedwith
otherdrugssuchaspropranolol;in this respectethosomes
haveshowntheir potentialas
transdermaldosageforms for prophylaxisof migraine (9). Moreover,the ability of
ethosomesto deliver compoundsto cells in culture was investigated(10).
The enhanceddeliveryof activesusingethosomes
overliposomescanbe ascribedto an
interactionbetweenethosomes
and skin lipids. A possiblemechanismfor this interaction hasbeenproposed.It is thought that the first part of the mechanismis due to the
"ethanoleffect,"wherebyintercalationof ethanolinto intercellularlipids enhances
lipid
fluidity and decreases
the density of the lipid multilayer. This is followed by the
"ethosome
effect,"which includesinterlipid penetrationandpermeationby the opening
of new pathwaysdue to the malleabilityand fusionof ethosomes
with skin lipids,
resultingin the releaseof the drug in deeplayersof the skin (6).

The purposes
of thispaperare(a) the productionof ethosomes
andliposomes
asvehicles
for azelaicacid;(b) the characterization
of the vesiclesby freeze-fracture
scanningelectron microscopy
(SEM), photoncorrelationspectroscopy
(PCS),and differentialscanning

ETHOSOMAL

AND

LIPOSOMAL

DISPERSIONS

255

calorimetry(DSC); (c) the determinationof azelaicacid diffusion from ethosomaland

liposomaldispersions;
and (d) the determinationof azelaicacid diffusionfrom ethosomes
and liposomesincorporatedin a hydrophilicgel.

EXPERIMENTAL
MATERIALS

Azelaicacid (AA) waspurchasedfrom Sigma-Aldrich(Milan, Italy). Soyphosphatidyl

choline,Phospholipon
90 (PC)waspurchased
fromNattermannPhospholipid
GmbH
(Cologne,
Germany).Carbopol
934 P (Carbomer
934) wasfromBiochim(Milan,Italy).
All other materialsand solventsof the high purity grade were from Sigma-Aldrich.

PRODUCTION

OF ETHOSOMES

For the preparationof ethosomes,

PC (50 mg/ml) and AA (2 mg/ml) werefirst dissolved
in ethanol(comprisedbetween20% and 45%, v/v). An aqueousphaseconstitutedof

isotonicPalitzschbuffer(IPB) (5 mM Na2B407,180 mM H3BO3, 18 mM NaCI) was

then slowlyaddedto the ethanolicsolutionunder continuousstirring at 700 rpm by a
Eurostardigital IKA Labotechnik(Sardo;Turin, Italy). Mechanicalstirring was performedfor 5 min at room temperature.
PRODUCTION

OF LIPOSOMES

Liposomeswere preparedby the reverse-phase

evaporationmethod (REV) (11). The
aqueousphaseconsistedof 5 ml of IPB, and the organicphasewasa solutionof PC (50
mg/ml) and AA (2 mg/ml) in 20 ml of diethyl ether.The biphasicsystemwasvortexed
and sonicatedat 0C for 5 min in a bath-type Branson2200 sonicator(BransonUltrasonicsCo., Danbury, CT). The ether present in the obtained stable emulsionwas
removedby rotaryevaporationunderreducedpressureat roomtemperature,resultingin
a turbid, white liposomedispersion.

PHYSICAL

CHARACTERIZATION

OF ETHOSOMES

AND

LIPOSOMES

In orderto obtain homogeneously

sizedvesicles,both ethosomaland liposomaldispersionswereextrudedwith an ExtruderTM(Nipex Biomembranes
Inc., Vancouver,Canada)
through polycarbonatemembranes(NucleoporeCorp., Pleasanton,CA) with poresof
calibrateddiameter(12). In particular,ethosomes
and liposomeswere subjectedto one
extrusioncyclethroughtwo stacked400-nm-pore-sizefilters (LIPO ex 400, ETHO ex
400) followedby three extrusioncyclesthrough two stacked200-nm-pore-sizemembranes(LIPO ex 200, ETHO ex 200).

Electronmicroscopy
analyses
wereperformedby the freezefracturingtechnique.Briefly,
ethosomalor liposomaldispersions
werefrozenby the "propanejet" technique,cryofixed

andmicrofractured
at 108K(Balzen
BAF300)witha 10-5 Papression,
andreplicas
werereproducedby a platinum/carbone
matrix. For the electronmicroscopy
analysis,a

256

JOURNAL OF COSMETIC SCIENCE

Philips EM 301 was employedat 1000 KV. Photographswere taken on Agfa Scientia
23D56 film and developedby a GeratoneG5C instrumentfor 3.5 min at 293K.

Photo,corre/atio,
spectroscopy
(PCS). Submicronparticlesizeanalysiswasperformedusing
a Zetasizer 3000 PCS (Malvern Instruments, Malvern, UK) equipped with a 5 mW
helium neonlaserwith a wavelengthoutput of 633 nm. Glassware
wascleanedof dust
by washingwith detergentand rinsingtwice with water for injections.Measurements
were made at 25C at an angle of 90 . Data were interpreted using the "method of
cumulants."

DifJrentialscanning
calorimetry
(DSC). The transitiontemperaturesof both ethosomes
and liposomeswere measuredby DSC techniqueusinga ShimadzuTA-60WS in an
aluminum pan, undera constantnitrogenstream.Measurements
were performedat a
temperaturerangebetween-40C and 120Cat a heatingrate of 30 per min. The
sampleweight was about 3 mg, and PC concentrationwas the samefor all samples
tested.

PRODUCTION

OF HYDROPHILIC

GELS

For the preparationof the hydrophilicgel, the carboxyvinylpolymercarbomer(Carbopol 934 P) (1.5%, w/w) was addedto bidistilledwater and left to swell at room
temperatureto obtain a homogeneous
and liquified mixture. After an overnightincubation, triethanolaminewas added to neutralizethe solution.The obtainedgel was
alternatively diluted (1.1%, w/w) with ethanol solution or ethosomalor liposomal
suspensions,
resultingin a final AA concentration
of 1 mg/ml and a carbomerconcentration of 0.75%, w/w.

IN VITRO

DIFFUSION

STUDY

The experimentswere carriedon usinga standardglassFranz releaserate cell (13) with

a 1-cm diameterorifice(0.78 cm2 area)assembled

with a celluloseester-based
membrane,0.6-pm poresize(Schleicher
& Schuell,Germany).As receptorphasea solution
of IPB/EtOH 70:30 (v/v) wasused.The receptorphasewasalwaysdegassed
beforeuse
and pouredin the cell bodyto overflowing,in orderto avoidair bubbleformation.To
studythe AA releaserate, 1 ml of drugsolutionor 1 g of the formulationto beanalyzed
was placed into the donor cell compartmentand tamped down on the membrane,
previouslymoistenedwith the receptorphase.The upperpart of the chamberwassealed
to avoidevaporation.The receptorphasewasstirredby meansof a constantlyspinning
bar magnetandthermostated
at 37C.At predetermined
time intervalsbetweenoneand
six hours,samples(0.15 ml) of the receptorphasesolutionwerewithdrawnand the AA
concentrationin the receptorphasewas measuredusing HPLC. Each removedsample
wasreplacedwith an equalvolumeof simplereceptorphase.

Theamount
ofAA released
perunitarea(pg/cm
2)wasplottedagainst
thesquare
root
of time. The slopeof the line (regression)
represents
the releaserate of the drug. The
releaserate coefficientswere expressed
both as experimentallyobservedfluxes(Fo) and
asnormalizedfluxes(Fn) or diffusioncoefficient(D) (D=Fn--Fo/C, where C is the azelaic
acid concentrationexpressed
in mg/ml). All the obtainedD valueswere determinedsix

ETHOSOMAL

AND

LIPOSOMAL

DISPERSIONS

257

times in independentexperiments,and the mean values +_standarddeviationswere

calculated.

HPLC analysis.
The quali-quantitativeanalysisof azelaicacid wasperformedby HPLC
using a Jascoapparatusconsistingof a two-plungersalternativepump, a variablewavelengthUV detector,a RheodineInc. 7125 injectionvalve, and a Shimadzuintegrator. For the analysisof azelaicacid, a Hypersil BDS-ultrasphereC18 column (15 x
0.46 cm) packedwith 5-pm particleswasemployed.Elutionwasconductedat 215 nm
with an isocraticmobile phaseconsistingof acetonitrile:Naphosphate0.05 M, pH 3
(25:75, v/v) at a flow rate of 1 ml/min.

RESULTS
PRODUCTION

AND

DISCUSSION

OF ETHOSOMAL

AND

LIPOSOMAL

DISPERSIONS

As describedin the Experimentalsection,ethosomes

were spontaneously
producedby
dissolutionof PC and AA in ethanolfollowedby slow additionof an aqueousbuffer
under continuousstirring at room temperature.During preparation,dispersionsdisplayed initial optical transparencydue to the high ethanol concentration'sability to
maintainPC in solution.By addingincreasingconcentrations
of IPB buffer, dispersions
becameturbid. Turbidity of the systemis ascribedto a reorganization
of PC molecules
within the system,resulting in ethosomalvesiclesformation. Ethosomalsuspensions
wereproducedwith increasing
amountsof ethanol,namely20%, 30%, 40%, and45%,
v/v; PC concentrationwas always 5%, p/v. In all casesthe use of different ethanol
concentrationsresultedin white milky suspensions
with no significantdifferencesin
macroscopicaspect.Ethosomalsuspensions
with ethanol 20% (ETHO 20) and 40%
(ETHO 40), v/v, were selectedas standard formulations.

Liposomeswere producedby a reverse-phase

evaporationmethod. In particular, the
methodwasbasedon sonicationof a biphasicsystemconstitutedof IPB and a solution
of PC/azelaicacid in diethyl ether,resultingin a stableemulsion.Afterwards,a turbid,
white liposomedispersionwas obtainedthrough removalof the ether presentin the
emulsionby rotaryevaporation
underreducedpressure.
Table I reportsmeandiameters
of emptyliposomalandethosomal
vesicles,
asdeterminedby PCSandexpressed
both as
Table

VesicleMean Diameterand Polydispersity

of Empty Liposomes
and Ethosomes
as Determinedby PCS
Vesicles

Z Average(nm)

Polydispersity

Intensity (nm)

LIPO

790.5

1.00

785.4

ETHO 20
ETHO 30

338.4
286.7

0.28
0.17

328.8
280.0

ETHO 40
ETHO 45

259.9
397.8

0.22
0.14

254.9
394.9

LIPO: liposomalsuspension.
ETHO 20: ethosomalsuspension
producedusing ethanol20% by volume.
ETHO 30: ethosomalsuspension
producedusingethanol30% by volume.ETHO 40: ethosomalsuspension
produced
usingethanol40% by volume.ETHO 50: ethosomal
suspension
produced
usingethanol45% by
volume.

Data are the meanof four determinations

on differentdispersions.
SD: -+5%.

258

JOURNAL OF COSMETIC SCIENCE

Z average
and asintensity.
One canobservethe largermeandiameterof liposomes(790.5
nm) with respectto ethosomes
(ranging from 260 to 398 rim). The mean size of
ethosomalvesicleswas independentfrom the percentageof ethanol employedfor their
production.Moreover,the high polydispersityindex of liposomes(PI = 1) reflecteda
broad dimensionaldistribution. Conversely,ethosomepolydispersityindicesdid not
exceed0.28, indicating narrow size distributions.

After production,AA-containingethosomaland liposomalsuspensions

were subjected
to extrusionthrough calibratedmembranesin order to size the vesicles.In particular,
extrusionwasperformed(a) by one cyclethroughtwo stackedmembranes
of 400-nm
pore sizeand (b) by three cyclesthrough two stackedmembranesof 200-nm pore size.
Mean diametersof vesiclesbeforeand after extrusionwere measuredby PCS and are
reportedin Table II asboth Z average
and intensity.
It is to be notedthat both in the case
of liposomaland of ethosomalvesiclesthe presence
of AA resultedin an increasein mean
diameter.Moreover,as found for empty vesicles,beforethe extrusionliposomalvesicles
were characterizedby a larger mean diametercomparedto ethosomalvesicles.In particular,vesiclemeandiameterswere817 nm in the caseof liposomes,
440 nm in the case
of ETHO 20, and 527 nm in the caseof ETHO 40.
After the extrusionboth liposomaland ethosomalvesicleswere characterized
by mean
diametersreflectingthe sizeof the membranepore employedfor the extrusion.It is to
be noticed that, after each extrusioncycle,polydispersityindiceshave been lowered,
demonstratingthat the extrusionstepcan improvethe sizedistribution.
PHYSICAL

CHARACTERIZATION

OF ETHOSOMES

AND

LIPOSOMES

Figure 1 showsmicrographsof liposomes,ETHO 20, and ETHO 40 obtainedby the

freeze-fracture
electronmicroscopic
techniquefollowedby the extrusionsteps.One can
observethe presenceof unilamellarvesiclesboth in liposome-and in ethosome-based
dispersions.
Vesiclesarecharacterized
by regularspheroidalmorphologyand by the same
dimensional range.
Table

II

AzelaicAcid-containingVesicleMean Diameter and Polydispersity

asa Functionof ExtrusionCycle
Vesicles

Z Average(nm)

LIPO
LIPO ex 400
LIPO ex 200

817.8
233.0
165.1

ETHO

20

ETHO 20 ex 400
ETHO 20 ex 200

Polydispersity

Intensity (nm)

1.00
0.31
0.14

814.9
224.7
162.3

440.8

0.28

428.8

305.9
179.5

0.14
0.09

260.0
177.2

40

527.5

0.22

531.0

ETHO 40 ex 400
ETHO 40 ex 200

274.9
173.9

0.16
0.02

244.9
174.9

ETHO

LIPO: liposomalsuspension.
ETHO 20: ethosomal
suspension
producedby the useof ethanol20%. ETHO
40: ethosomalsuspension
producedby the useof ethanol40%. ex 400: vesiclesextrudedthrough400nm-pore-size
polycarbonate
membranes.
ex200: vesicles
extrudedthrough400-nm-pore-size
polycarbonate
membranes
and through200-nm-pore-size
polycarbonate
membranes.
Data are the mean of four determinationson differentdispersions.
SD: +5%.

ETHOSOMAL

AND

LIPOSOMAL

DISPERSIONS

2 59

Figure 1. Freeze-fracture
electronmicrographs
of liposomal
dispersion
(panelA), ethosomal
20% dispersion(panelB), andethosomal
40% dispersion
(panelC). Liposomal
andethosomal
dispersions
weresubjectedto extrusion
through200-rim-pore-size
membranes.
The barequals510 nm.

Afterproduction
of dispersions,
freeazelaicacidwasnotseparated
fromtheencapsulated
one sinceazelaicacid, due to its amphiphilicnature,aims towardpartitioningrapidly
betweenthe internal and externalspacesof ethosomes
or liposomes.During the sepa-

rationprocess,
performed
by gel permeation
chromatography,
the concentration
gradient,dueto the removalof thenonencapsulated
drug,leadsto leakageof azelaicacidfrom
the ethosomes
and/orliposomes,
thuspreventingthe correctevaluationof encapsulation
efficiency
and finally resultingin a progressive
decrease
in the total azelaicacid concentration.For thesereasons,
the wholepreparationwasemployedfor DSC and in vitro
release rate studies.

DSC studies.
In orderto gain someinformationaboutethosome
properties,freeenergy
measurements
of the vesiclebilayerswereconducted
by DSC studies.It is well known
that DSCmeasures
the temperature
dependence
of the excess
heatcapacityof a system
due to thermal capacity.The heat capacitycurvesof liposomesthat undergosuch
transitionscontaininformationon the enthalpy and entropy of thesetransitions.In
particular,the main transitiontemperature
(Tin) is the gel-to-lipid-crystal
transition,
whichis a ratherrapidtransitionthat canbe described
in equilibriumthermodynamic
termsduringDSC experiments.
Ts is a verysubgeltransition(Ts - Tm-30C) that is
not verywell characterized
in molecularterms(14). Table III reportstransitiontemperatures
(Tin andTs) of emptyandAA-containing
vesicles,
ascalculated
from DSC
thermograms.
In particular,the Tm of emptyliposomes
wasfoundhigherthan 2.7C
and 5.8Cwith respectto ETHO 20 and ETHO 40, respectively.
Theseresultsare in
agreement
with the findingsof Touitouandcolleagues
(5,6), suggesting
that the ethoTable

III

TransitionTemperatures
of VesiclesasDeterminedby DSC
Tm

Empty vesicles
LIPO

Ts/Tm

Azelaic acid-containingvesicles

+ 11.5C

ETHO

20

+8.8C

ETHO

40

+ 5.7C

Tm: main thermal transitiontemperature.Ts: subtransitiontemperature.

- 10.8C/+

11.5C

- 10.2C/+9.0C
-6.7C/+7.5C

260

JOURNAL OF COSMETIC SCIENCE

somes are in a more fluid

state than vesicles in the absence of ethanol.

The lower Tm

displayed
by ETHO 40 (5.7C)with respectto ETHO 20 (8.8C)canbe ascribed
to the
higher ethanolconcentrationthat providesincreased
vesiclemalleability.Moreover,one
can noticethat empty vesiclesdisplaypositiveTm valuesboth for liposomesand for
ethosomes,while in the caseof AA-containing vesicles,thermogramsshow both a
negativeTs and a positiveTm, indicatingthe presenceof an endothermicaswell asan
esothermitpeak. This result is consistentwith the hypothesisthat the presenceof AA
within the dispersions
canaffectvesiclestructure,leadingto somechanges
in theirsoftness.
3500

'A
3000

2500

2000

1500
lOOO

[]

0 , , , , I , ,[,,I , , I .... I , , , , I
0

10

15

20

25

square root of time (minutes)

3500

B
3000

2500
2000

1500
lOOO
500

o
o

10

15

20

25

square root of time (minutes)

Figure2. In vitrorelease
ratekineticsof azelaicacidvehiculated
in anethanolsolution(/) or in liposomal
(V), ethosomal
20% (O), andethosomal
40 % ([-) dispersions.
Experiments
wereperformed
by a cellulose
estermembranewith 0.6-pm poresizeandIPB/ethanol70:30 (v/v) asreceptor
phase.The reportedresults
representthe meanvalues+ SD of six independentexperiments.

ETHOSOMAL

PRODUCTION

OF VISCOUS

ETHOSOMAL

AND

AND

LIPOSOMAL

LIPOSOMAL

DISPERSIONS

261

FORMULATIONS

For the designof a formulationconstitutedof phospholipidvesicles,suchasethosomes

or liposomes,the choiceof lipid compositionand additivesmust be donecarefullyto
guaranteevesicleintegrity in the final product.The ethosomalor liposomalsuspension
shouldhavea viscosity-enabling
permanence
on the skin surface;nevertheless
the presence of surfactantsin emulsionformulationscan be a considerable
problem for the
stabilityof etho-liposomalvesicles(15). It is well known that surfactantscan interact
with the double layer of liposome/ethosome
vesicles,leading to formation of mixed
vesiclesand finally to disruptionof liposome/ethosome
organization.Theseinstability
problemshavesuggested
the useof liposomal/ethosomal
formulationsbasedon colloidal
agents,ableto conferadequateviscosityto the mediastabilizingthe physicalproperties
of liposomalsystems(16,17). In particular,in the presentstudy,surfactant-free
formulations basedon carbomerhydrophilicgel have been produced,able to adjust the
viscosityof the ethosomaland liposomalsuspensions
without altering the vesiclestability. After dilution with ethosomalor liposomalsuspensions,
the use of carbomer
resultedin the formationof transparentformulationswith an adequateviscosityfor
administration

DIFFUSION

on the skin.

EXPERIMENTS

In order to investigateazelaicacid diffusionfrom the different phospholipid-based

vehicles,an in vitrotest basedon a percutaneous
absorptionglasscell (Franztype) was
employed(13). Using the diffusioncell with a syntheticmembrane,the methodcanbe
utilized to determinethe drug releasecharacteristics
from the ethosome/liposome-based
formulationsandthuscanbe usedasa qualitycontrolprocedureto assurebatch-to-batch
uniformity(18). A celluloseestermembranewith 0.6-[m poresizeand a receptorphase
constitutedof IPB/ethanol70:30 (v/v) wereused.This kind of receptorphasewaschosen
in orderto guaranteeadequatesolubilityand unrestrictedaccess
to AA. Actually, the use
Table

IV

In Vitro ReleaseRate Coefficientsof AzelaicAcid Incorporatedin Different TopicalForms

Formulation
'
EtOH solution

Fopg/cm
rain'5

logD

15.55

1.19

EtOH/carbomergel

49.31

4.1

0.61

LIPO

59.63

4.97

0.70

LIPO gel

13.88

1.9

0.28

ETHO

87.79

7.31

0.86

0.51

20

ETHO 20 gel
ETHO 40

ETHO 40 gel

186.6

D cm/min
'5 103

38.62

3.22

119.96

9.99

0.10

54.77

4.56

0.66

EtOH/carbomer
gel:ethanolsolutionincorporated
in carbomer-based
gel.LIPO: liposomesuspension.
LIPO
gel: LIPO incorporatedin carbomer-based
gel. ETHO 20: ethosomalsuspension
producedby the useof
ethanol20%. ETHO 20 gel: ETHO 20 incorporatedin carbomer-based
gel. ETHO 40: ethosomalsuspensionproducedby the useof ethanol40%. ETHO 40 gel: ETHO 40 incorporated
in carbomer-based
gel.
Azelaicacid concentrationwas always12 rag/mi.
Experiments
wereperformed
by a Franzrelease
ratecellassembled
with a cellulose
estermembrane
(0.6-pm
pore size)and IPB/ethanol 70:30 (v/v) as receptorphase.
Data are the mean of six determinations. SD: -+8%.

262

JOURNAL OF COSMETICSCIENCE

3500

2000

3O00

1600

2500
-o

1200

1500

800

1000

....,,, ,,, ...., n-400

5O0

10

15

20

25

square root of time (minutes)

2O0O

10

15

20

25

square root of time (minutes)

2000
D

600

1600

1200

1200

800

800

400

n- 400

10

15

20

squarerootoftime(minutes)

25

10

15

20

25

squarerootoftime(minutes)

Figure3. In vitrorelease
ratekinetics
of azelaic
acidincorporated
in theformulation
reported
below.
Experiments
wereperformed
bya cellulose
estermembrane
with 0.6-pmporesizeandIPB/ethanol
70:30

(v/v)asreceptor
phase.
(A)Ethanol
solution
(/); ethanol
solution
incorporated
in carbomer-based
gel(A).
(B)Liposomal
suspension
(V); liposomal
suspension
incorporated
in carbomer-based
gel('). (C)Ethosomal
20%suspension
(O); ethosomal
20%suspension
incorporated
in carbomer-based
gel(O). (D)Ethosomal

40%suspension
([); ethosomal
40%suspension
incorporated
in carbomer-based
gel(1). Thereported
resultsrepresent
the meanvalues+ SD of sixindependent
experiments.

of pureIPB or IPB/ethanol
80:20did notleadto a goodAA solubilization
(datanot
shown).

Theamount
ofAA released
perunitarea(pg/cm
2)wasplotted
against
thesquare
root
oftime.Thecumulative
amount
ofAAreleased
waslinearandproportional
tothesquare
rootof time.Theslope,whichrepresents
therelease
rate,steady-state
flux,wascalculatedby linearregression.
Correlation
coefficients
of the regression
linewerebetween
0.971

and 0.998

in all studies.

It isknown
thatthemembrane
should
notimpede
orcontrol
therelease
rateofthedrug
fromtheformulation
intothereceptor
phase,
actingonlyasa support
(18,19).Thiswas
demonstrated
by measuringthe diffusionof AA ethanolicsolution(D coefficient15.55

cm/min
'5103)compared
to AAdiffusion
fromliposomal
andethosomal
systems
(Figure2). AA concentrations
from the solutionwerealwayslargerthan from the

vesicular
systems,
suggesting
thatthemembrane
employed
wasnotrate-limiting.
The
calculated
D coefficients
for AA incorporated
intothedifferent
topicalformsarereported in Table IV.

ETHOSOMAL

AND

LIPOSOMAL

DISPERSIONS

263

The highestD coefficientwasfoundin the caseof ETHO 40, followedby ETHO 20.

Themorerapidrelease
displayed
byAA fromETHO 40 (9.99cm/min
'5' 103)with
respect
to ETHO 20 (7.31cm/min
103)canbeattributed
to themorefluidstateof
ETHO 40 vesicles,possiblypromoting diffusion of AA through the vehicle. The D
coefficientof AA from liposomeswas approximatelyhalf the D coefficientfrom ethosomes.

Figure3 reportsa comparison

betweenthe releasekineticsof AA solubilizedin ethanol
or ethosomalor liposomaldispersionsand AA vehiculatedin the sameformulations
viscosized
in carbomergels(namelyEtOH carbomergel, LIPO gel, ETHO 20 gel, and
ETHO 40 gel). One shouldobservethat the trend of AA diffusionfrom low-viscosity
vehicleswas confirmedwhen viscosizedformswere studied,even if the incorporationof
AA ethanolicsolutionor AA ethosomalor liposomaldispersionsin carbomergel resuitedin a slowerreleaseof the drug. This behaviorcanbe attributedto the viscosityof
the gel, which actsasan impedimentto the releaseof AA. The 1owe_st
D-coefficientwas

foundin the caseof LIPO gel(1.9 cm/min

103).TheD dofficient
of AA from
ETHO 40 gel waslargerwith respectto the D coefficientfrom EtOH carbomergel.
With regardto ethosomal
dispersions,
the presence
of ethanolmakesthe lipid membrane
lesstightly packedthan the conventionalvesiclesand confersa softer,more malleable
structureto the ethosomes,
possiblypromotingAA diffusionthrough the vehicle.

CONCLUSIONS

The resultshere presentedhavedemonstratedthat AA-containingethosomalvesicles,

characterized
by a morphologysimilar to that of liposomalones,can be producedby a
simple method. Extrusionof the dispersionsallowsone to obtain unilamellar vesiclesof
calibratedsize. AA-containingethosomesare characterizedby a soft and malleable
structure(as demonstratedby DSC studies)as a function of the ethanolconcentration
employedfor their production.The more fluid stateof ETHO 40 could be responsible
for the higher AA diffusionthrough this vehiclewith respectto ETHO 20 and LIPO.

ACKNOWLEDGMENTS

This work wassupportedby the Ministry of Education,UniversityandResearch

of Italy
(MIUR) FIRB project.

REFERENCES

(1) A. S. Breatnach,Azelaic acid--Biological activitiesand therapeuticapplications,DrugsToday,25,

463-472 (1989).

(2) Q. H. NguyenandT. P. Bui, Azelaicacid:Pharmacokinetic

and pharmacodynamic
propertiesand its
therapeuticrole in hyperpigmentary
disordersand acne,Int. J. Dermato/.,34, 75-84 (1995).
(3) A. Fitton and K. L. Goa, Azelaicacid:A reviewof its pharmacological
propertiesand therapeutic
efficacyin acneand hyperpigmentary
skin disorders,
Drugs,41, 780-798 (1991).
(4) S. Passi,M. Picardo, C. De Luca, and M. Nazzaro-Porro,Mechanismof azelaicacid action in acne,
Dermatol.Venereol.,
124, 455-463 (1989).

264

JOURNAL OF COSMETIC SCIENCE

(5) E. Touitou, N. Dayan,L. Bergelson,

B. Godin,and M. Eliaz, Ethosomes--Novelvesicularcarriersfor

enhanced
delivery:Characterization
andskinpenetrationproperties,J.Controlled
Release,
65,403-418
(2OOO).

(6) N. DayanandE. Touitou,Carriersfor skindeliveryof trihexyphenidylHCI: Ethosomes

vs.liposomes,
Biomaterials,21, 1879-1885 (2000).
(7) E. Horwitz, S. Pisanty, R. Czerninski,M. Helser, E. Eliav, and E. Touitou, A clinical evaluationof a

novelliposomalcarrierfor acyclovirin the topicaltreatmentof recurrentherpeslabialis,OralSurg.Oral

Med. Oral Pathol. Oral Radiol. Endod.,87, 700-705 (1999).
(8) E. Touitou,B. Godin,and C. Weiss,Enhanced
deliveryof drugsinto andacross
the skinby ethosomal

carriers,Drug Devel.Res.,50, 406-415 (2000).

(9) P. Akkaramongkolporn,
K. Terada,and E. Yonemochi,Molecularpropertiesof propranololhydro-

chloridepreparedasdrug-resincomplexes,
DrugDev.Ind. Pharm.,27, 359-364 (2001).
(lO) E. Touitou, B. Godin, N. Dayan,C. Weiss,A. Piliponsky,and F. Levi-Schaffer,
Intracellulardelivery

mediatedby an ethosomalcarrier,Biomateria&22, 3053-3059 (2001).

(11) F. Szokaand D. Papahadjopoulos,
Procedurefor preparationof liposomes
with largeinternalaqueous

spaceand high captureby reversephaseevaporation,Proc.Natl. Acad.Sci. USA, 75, 4194-4198

(1978).
(12)
(13)

(14)
(15)

(16)
(17)

(18)
(19)

L. D. Mayer, M.J. Hope, and P. R. Cullis, Vesiclesof variablesizeproducedby a rapid extrusion

procedure,
Biochim.
Biophys.
Acta,858, 161-168 (1986).
C. Nastruzzi, E. Esposito,C. Pastesini,R. Gainbari, and E. Menegatti, Comparativestudyon the
releasekineticsof methyl nicotinatefrom topic formulations,
Int. J. Pharm.,90, 43-50 (1993).
R. L. BiltonenandD. Lichtenberg,The useof differentialscanningcalorimetryasa tool to characterize
liposomepreparations,
Chem.Phys.Lipids,64, 129-142 (1993).
C. Nastruzzi,E. Esposito,E. Menegatti,and P. Walde, Useand stabilityof liposomein dermatological
preparations,J.Appl. Cosmetol.,
11, 77-91 (1993).
G. W. Y. Tin, Vesiclestabilization.Eur. PatentAppl.EP 162724 (1985).
K Thomaand U.E. Jocham,"Liposome
Dermatics:Assessment
of Long-TermStability,"in Liposome
Dermatics,
0. Braun-Falco,H. C. Korting, and H. I. Maibach,Eds. (Springer-Verlag,Berlin, Heidelberg, 1992), pp. 150-166.
P. V. Shah,J. S. Elkins,J. Hanus,C. Noorizadeh,andJ.P. Skelly,In vitro releaseof hydrocortisone
from topicalpreparations
and automatedprocedure,Pharm.Res.,8, 55-59 (1991).
P.V. Shah,J. S. Elkins, and R. L. Williams, In vitro drug releasemeasurements
for topical glucocorticoidcreams,Pharmacopeial
Forum,19, 5048-5060 (1993).