INTRODUCTION

()
The application of click chemistry to polymer science has led to
improvements in bioconjugation techniques. Click reaction are modular
coupling reactions tolerant to a wide range of functional groups, simple to
perform and can be conducted in very high yield. The broadly applied Cu(I)catalyzed Huisgen dipolar cycloaddition between azides and alkynes
provides 1,2,3-triazole rings with 1,4 regioselectivity and quantitative
transformation under mild conditions, due to the orthogonal nature of the
azide-alkyne reactive pair. Triazole moieties are also interesting conjugation
entities as they are proven to be relatively stable to the metabolic
degradation and the triazole ring also can participate in the hydrogen
bonding, which enhances the biological importance of this specific click
coupling reaction. Therefore, this highly effective and selective click
reaction was used to overcome problems of low yield and poor selectivity
frequently encountered in grafting to reactions involving polysaccharides
and synthetic hydrophobic polymers. Maynard described an aminooxy end
functionalized pNIPAAm immobilized by click chemistry on a gold surface
after reduction of the trithiocarbonate end-group. The pNIPAAm surface was
then incubated with an aldehyde modified heparin to yield the
polysaccharide-functionalized surface.
Heparin is a linear polysaccharide consisting of alternating1,4-linked
uronic acid and a-D-glucosamine residues. Heparin has numerous important
biological activities, associated with its interaction with a diverse range of
proteins. It is mainly used as an anticoagulant agent and as a regulator of
the complementary activity in inflammatory diseases. Furthermore, heparins
regulate the activity of heparin-dependent growth factors, such as FGF and
VEGF, resulting in the modulation of angiogenesis and even tumor
development. Yamaguchi, described the assembly of functionalized heparin
to hydrogels via the non-covalent interaction. The modulated interaction of
heparin with VEGF was demonstrated. It indicated the potential for targeted
delivery of growth factors and erosion of hydrogels on the basis of ligandreceptor interaction that occur between cells and the extra-cellular matrix.
Huh described a heparin based bioconjugate with retinoic acid by chemical
conjugation, giving rise to an active metabolite which was used for targeting
therapies.
However the use of unfractionated heparin in medical treatments
leads to undesirable side effects, such as bleeding complications or heparininduced thrombocytopenia. These complications led to use of low-molecular
weight heparins, which show more predictable pharmacological actions,
sustained activity and better biological proprieties. UFH has been replaced
in the last few years by LMWH which retains the biological and chemical
properties of UFH and decreases the side effects. Kiick, reported a long-term
delivery method for LMWH via subcutaneous injection of long-lasting
poly() hydrogels. LMWH was modified with reactive maleimide groups so
that it can be crosslinked into continuous networks with four-arm thiolated
poly. Bemiparin, a second generation LMWH, was used in this work because
it provides the longest half-life and highest anti-FXa/anti-FIIa activity ratio of

an second-generation LMWH. It was obtained by the chemical b-elimination

depolymerization of UFH to give a LMWH with molecular weight of 3.6kDa.
Thermoresponsive, water-soluble polymers exhibiting a lower critical
solution temperature have been increasingly investigated for
nanotechnology and biotechnology application. Recently, the predominance
of PNIPAM, as the general example of a thermoresponsive polymer, was
challenged by the discovery that random copolymers of 2-()ethyl
methacrylate and oligo-methacrylate can be designed to exhibit a LCST in
water, which can be finely tuned between 26 and 90C depending on
OEOMA content. In contrast to PNIPAM the value for the LCST of the
copolymers is almost independent of molar mass, concentration, and ionic
strength. In addition, since the addition of oligo() sequence to responsive
polymers was shown to decrease cytotoxicity, these poly()- based
copolymers are expected to be non-toxic and non-immunogenic.
Another polymer that exhibits LCST behavior is poly() ethyl
methacrylate. PDMAEMA is a water-soluble, temperature-sensitive and pHsensitive polymer, due to the inherent amine protonation in physiological
media which makes the polymer an interesting component in a polybasic
drug delivery system. The synthesis of thermally responsive homopolymer
and random copolymer brushes using DMAEMA or OEOMA was reported.
Well-defined asymmetric hydrophilic graft chitosan grafted with both
PDMAEMA and PNIPAM were synthesized by ATRP and click chemistry.
Amide bonds are formed by the union of the carbonyl group on
carboxylic acid and nitrogen of amines. It is usually necessary to first
activate the carboxylic acid to efficiently perform this ligation, a process that
usually takes place by converting the OH of the acid into a good leaving
group prior to treatment with the amine. The choice of the activator agent is
critical and carbodiimides are highly popular class of activators. However,
they form secondary products that are usually very difficult to remove and
the functionalization reactions were not always conducted in high yield. 4()-4- methylmorpholinium chloride is a triazine derivate, which has the
particular advantage of promoting amide synthesis in alcohols or aqueous
media, without ester formation and with selectivity comparable to 1-()-3ethylcarbodiimide hydrochloride or any other carbodiimides. Furthermore,
the yields obtained with DMTMM are higher than those obtained with
carbodiimide based reagents.
The synthetic strategy applied in this work is depicted in scheme.
Azido-functionalized methacrylate polymers were synthesized. In parallel,
the carboxylic groups of bemiparin were modified with propargylamine to
generate alkynyl side groups along the polysaccharide backbone using
DMTMM. The product formed with DMTMM was compared with the product
obtained using a carboiimide reagent. The targeted bioconjugates,
bemiparin-g-poly, were prepared by click coupling reaction using Cu(I)
catalyzed azide-alkne dipolar cycloaddition.
Creating bioconjugates by combining stimuli responsive polymers
with proteins and polysaccharides can result in a synergistic combination of
properties of the individual components and overcome their separate
limitations. The protein or polysaccharide component can impact
(bio)functional properties to the bioconjugate, whereas the polymer
component can improve protein or polysaccharide stability, solubility and
biocompatibility. The synthetic polymer can also introduce new properties to

the bioconjugate such as stimuli responsiveness, self-assembly, phase

separation behavior, and can even modulate protein or polysaccharide
activity. Recent developments in polymer chemistry have advanced the
synthesis of materials in which synthetic polymers obtained by controlled
radical polymerization, such as reversible addition-fragmentation chain
transfer or ATRP, are immobilized to biological (macro)molecules to enhance
the solubility, stability, activity, or therapeutic utility of the biological entity.
ATRP and RAFT have proven especially proficient for the synthesis of
conjugates by direct polymerization of vinyl monomers from biological
components functionalized to contain a group capable of intiating chain
growth. Nicolas described in their review several tailor-made
macromolecular moieties using relatively simple processes such as ATRP
and click chemistry. They made special focus on end-functional polymers
with a whole range of biological recognition abilities, obtaining them by
combination of living radical polymerization with proteins and peptides in
the rapidly expanding field of bioconjugation.
The motivation of this work is double: the coupling of bemiparin with
a biocompatible and thermoresponsive polymer part provides a stabilization
of the low molecular weight heparin, decreasing its solubility in physiological
condictions and modulating the direct interaction of bemiparin functional
groups with the well known growth factors FGF and VEGF. The presence of
the thermoresponsive polymeric component provides an additional
stabilization of the system with a clear hydrophobic/hydrophilic equilibrium
which is dependent on the temperature, and therefore the modulation of
interactions with the bioactive growth factors.
Materials and methods
The low Molecular weight heparina, bemipairn was kindly donted by Rovi
Pharmaceuticals Laboratories . 2-(Dimethylamino)ethyl methacrylate ,
di(thkene glycol)methyl ether meyhacrylate and poly(ethylene glycol)methyl
ether methacrylate were purified by passing the monomer throught a
column of basic aluminum oxide. Copper (II) bromide and tris[(2pyridyl)methyl]amine were stored under vacuum and used withoutfurther
purification. Propargylamine , 4-(.)-4- methylmorpholinium chloride, Nhydroxysuccinimide , ascorbic acid , copper sulphate pentahydrate, tin 2ethylhexanoate , alumininum oxide basic, toluene, tetrahydrofuran and
ethyle ether were used as received.

Functionalization of bemiparin with alkyne groups

Prepartion of alkynyl-functionalized bemiparin was carried out by
modification of carboxylic groups present on bemiparin with propargylamine
using 4-()-4- methylmorpholinium choride of 1- ethyl-3- () carbodiimide.
This was accomplished by mixing bemiparin with 10 molar equivalent of
propargylamine and DMTMM uin 5 ml of distilled water. The reaction mixture
was kept under magnetic stirring at room temperature for 5 hours. The
same reaction was carried out usin EDC and N-Hydroxysuccinimide instead
of DMTMM. Both products were purified by dialysis against water for 3 days,

chaging the water twice a day and finally freeze-dryong to obtain the
purified bemparinalkyne.

Synthesis of temperature and pH sensitive polymers by ATRP

The synthesis of the azido ATRP initiator was carried out as previously
reported.
Synthesis of N3-PDMAEMA using AGET ATRP.N3-PEG3-BPA, CuBr2 TPMA and
DMAEMA were dissolved in 9.4mL of toluene in 25mL Schlenk flask. The
flask was sealed and the solution was bubbled with nitogen for 20minutes.
The reaction mixture was placed in an oil bath preheated to 40C and 0.40
ml of a degassed Sn(II) EtHex solution was injected into the flask to reduce
the catalyst complex and start the polymerization. The reaction was stopped
after one hour by opening the flask to air and addition of 10ml of THF,
before the solution was passed over a short column of basic alumina and
polymer isolated after precipitation into ethyl ethet three toimes. H NMR 500
MHz: 0.8-1.2 methylene protons of the methacrylic group, 1.25()
methylene protons linked to azide function in the initiatorm 1.5()
methylene protons of the main polymeric chain, 2.2 () both methyl groups
of DMAEMA, 2.5() methylene protons linked to the tertiary amino group of
DMAEMA, 3.1() methylene groups of the initiator linked to the ether
function, 3.9 () methylene protons of DMAEMA linked to the ether function.

Synthesis of N3-P() using AGET ATRP. N3-PEG3-BPA, CuBr2, TPMA,

OEOMA300 and MEO2MA were dissolved in 9.4mL of toluene in a 25mL
Schlenk flask. The flask was sealed and bubbled with nitrogen for 20min.
The reaction mixture was placed in an oil bathe preheated to 40C and
0.4ml of degassed SN(II) EtHex solution was injected into the reaction
mixture to reduce the copper catalyst complex and start the polymerization.
After one hour the reaction was stopped and 10ml of THF was added to the
flask then the solution was passed through a short column of basic alumina
before the polymer was precipitated by addition to ethyl ether three times.
H NMR 500MHz : 0.8() methyl protons of the methacrylic functions,
1.25() methylene protons linked to azide function in the initiator, 1.6
merhylene protons of the main polymeric chin . 3.2 methyl protons of the
methscrylic functions of the graft chain of both MEO2MA and OEO300MA,
3.2 methylene groups of the initiator linked to the ether function plus
methylene groups of the graft chain of both MEO2MA and OEO300MA, 3.9
methylene protons of graft chains of MEO2MA and OEO300MA linked of the
ether function

Prepration of graft copolymers by click chemistry

The axido-functional polymer and the alkynyl-functionalized bemiparin were
dissolved in 15ml distilled water. The catalyst system, CuSO4.5H2O and
sodium ascorbate were added to the solution and the mixture was degassed
by purging with nitrogen of 2 hours. The reaction mixture was then stirred
for 24 hours at room temperature. The catalytic complex was removed by

passing the final reaction mixture through a basic alumina column and
purified by dialysis against water for 4 days and isolated by freeze-drying.
Characterization
Structural characterization of the polymer was carried out by NMR and
Fourier transform infrared spectroscopy. H, C and Heteronuclear Multiplebond correlation spectroscopy NMR analysis were performed in deuterium
oxide solutions at room temperatue using a Varian XL-500 spectrometer and
the spectra were recorded. Sample concentration was 15mg ml for H and
100 mg ml for C and HMBC spectra. Spectra were analyzed with the
MestreNova v 6.1 software.
FTIR spectra were obtained in the attenuated total reflection mode on a
Perkin Elmer Spectrum One FT-IR spectrometer at room temperature by 32
scans, and with a resolution of 4cm.
Thermogravimetric analysis was performed on a TGA Q500, working under
50mL min nitrogen flow, and at a heating rate of 10C min, from 30 to
600C.
The evolution of number and weight average molecular weight with
monomer conversion was determined by size exclusion chromatography in a
perkin-elmer apparatus equipped with an isocratic pump serial 200
connected to a differential refractometric detector. Two resipore columns
were conditioned at 70C and used to elute the samples at 0.3ml min HPLC
grade N,N- dimethylformamide supplemented with 0.1 % v/v LiBr.
Calibration SEC was carried out with monodisperse standard poly()
samples in the range of .obtained from polymer laboratories with sample
injection volumes of 20 uL
Lower Critical solution temperature of all the polymers: . Were measured
by UV spectroscopy, using a scanning temperature program to see the
aggregation of the system when the LCST is reached. All the systems were
analyzes at a concentration of 1 mg ml aqueous solution. The temperature
sweep was carried out from 20C to 90C at 1C min and transmittance
measured at 450nm. LCST was defined at the temperature of the onset of
aggregation measured by the transmittance/temperature diagram, during
the heating ramp of the aqueous polymer solutions.

RESULTS

A series of novel heparin-based bioconjugates were prepared by a

combination of functionalization of bemiparin and preparation of
homogeneous functional polyacrylics chains by ARGET ATRP, and grafting to
by the click chemistry strategy, according to Scheme 1. The methodology
provides a procedure for preparation o clean homogeneous polymers with
specific properties, such as controlled composition, molecular weight and
molecular weight distribution, and allows the preparation of bioconjugates
under mild conditions.

PREPARATION OF HEPARIN-ALKYNE DERIVATIVES

Aqueous amidation of polysaccharides in water with carbodiimides is one of
the most widely used methods for polysaccharides modification. Due to its
water solubility EDC is the preferentially used carbodiimide. There are two
steps to the amidation reaction with EDC: the firt one consists of activation
of the carboxylic acid group forming an O-acyl isourea intermediate. The
second step involves nucleophilic attack of the activated carboxylic group by
the amine which leads to the formation of an amide bond. The reaction is
strongly pH-dependnt but the optimal pH for each step is different. Indeed,
carboxylic acid activation by EDC is best performed in an acidic
environment, whereas amide formation is best conducted at higher pH,
when the amine is deprotonated. However, EDC is rapidly hydrolyzed into an
N-acyl urea by- product at such a high pH and no amidation can occur.
Therefore, although carbodiimides are widely used as activated coupling
reagents, their synthetic byproducts can be difficult to remove. After
functionalization of bemiparin with propargylamine the EDC residue was
difficult to remove due to the fornation of ammonium sulfate salts between
the O-acly urea byproducts and the sulfate groups of bemiparin. Indeed
removal of this byproduct resulted in degradation of the polysaccharide due
to washing that was required at a basic pH Furthermore, the major product
obtained using EDC and NHS as activator reagents was an ionic salt rather
than formation of the desired product with a covalent amide bond.
Recently,- 4methylmorpholinium chloride was developed to facilitate an
efficient one-step amide functionalization of both small molecules and
polymers. Favorable attributes of DMTMM are easy removal of excess
reagent and byproducts from the reaction by dialysis against distilled water,
compatibility with many solvents including water, alcohols, diethyl ether,
ethyl acetate, and tetrahydrofuran, high reaction yields, and relatively low
cost. DMTMM can be adapted for use in a wide range of pH and this
compatible with several bioconjugation strategies, and DMTMM has been
employed for modification of a number of polymers including
polysaccharides. Scheme 2 provides a comparison of use of both activation
agents for preparation of alkynyl functionalized bemiparin.
The products were characterized by H NMR, C NMR, HMBC and FTIR.
These techniques confirmed the effective functionalization of bemiparin.
Under the experimental conditions reported in this work selective amidation
of the carboxylic group of bemiparin occurred whenDMTMM was used as the
activator. However, the use of EDC carbodiimide and the succinimide NHS
protocol predominately formed ionic carboxylate and sulfate salts instead of
the desired covalent product. This was clearly demonstrated by FTIR and
NMR spectroscopy as reported The FTIR spectrum does not show the most
characteristic C-H stretching band of the alkyne function because this band
is overlapped by bemiparin hydroxyl groups; however the presence of a
band at 2165 cm that corresponds to the secondary characteristic c=c
stretching band of the alkyne function in the conjugate bemiprin-alkyne is
observed. The band is not present in the dialzed product prepared with EDC,
and the spectrum corresponds to the formation of an ionic sulfate
ammonium salt as indicted in Scheme 2A. It is clear from this figure that the
modification of bemiparin with proprgylamine activated by DMTMM gives a
product with a spectrum showing modification of the band at 2940cm, which

can be assigned to the C-H aliphatic stretching vibration. Logically this band
is the contribution of the components and the covalent product, but the
change in the profile and the intensity makes clear that the modification was
produced bu the addition of the propargyl moiety to the heparin chains. A
detailed analysis of the pattern of the signal at 1600cm shows a slight shift
at lower frequency, which could be indicative of the formation of the amide
function corresponding to the conjugation of bemiparin and propargylamine.
However, the resolution is not enough to consider the corresponding
contribution quantitatively. It is interesting to high-light the bands at
1430cm and 1384cm, carboxylate carbonyl symmetric stretching, that can
be assigned to the ionized carboxylate functionality, according to other work
on carboxylate salt derivatives of heparin. The ration of the band at 1430cm
with respect to the band at 1640cm, carbonyl stretching in carboxylic acids
and amides, decreases and the band ar 1384 cm clearly disappears after
the amidation of the carboxylic groups by propargylamine, whereas the
product of the reaction with EDC corresponds to the formation of an ionic
salt, as shown in Scheme 2A.
The H NMR spectra, show two peaks that could be assigned to the
alkynyl proton, those at 2.54 and 2.80 ppm. When the bemiparin was
functionalized using EDC and NHS, only one peak appeared, at 2.80ppm.
However, when DMTMM was used at the activator product of the reaction
presents two different peaks, one at 2.54 at the other at 2.80 ppm. These
peaks can be assigned to the covalent conjugate (amide), 2.54ppm, and the
ionic salt, 2.80ppm. An HMBC NMR experiment was carried out to confirm
the formation of the amide bond. The HMBC spectrum of the bemiparinalkylne functionalized using DMTMM confirmed the presence of the
propargylamine unit covalently attached to bemiparin. Namely, the
appearance of a peak at 2.54ppm, assigned to the alkyne proton, correlated
with signals at 79.2 and 28.3 ppm belonging to methinyl and methylene
carbons of propargylamine, respectively. Furthermore, the methylene
protons of propargylamine at 3.93ppm were correlated with signals at 71.4,
79.2 and 164ppm due to the methylene and methane carbons of
propargylamine and the carboxylic carbon of bemiparin, respectively, which
indicates that selective amidation occurred. However, when EDC and NHS
were used, no correlation between the methylene protons of propargylamine
and the carboxylic carbon of bemiparin was detected. Besides, after both
products were dialyzed against water at pH 10 for 48 h, the signal at
2.80ppm disappears confirming that signal belongs to the ionic association
of propargylamine with bemiparin and after dialysis at ph >9.8, the amine
group of the proparglyamine is not protonated and thus the ionic bonds are
dissociated.
Applying equation I-V to the chemical structure of bemiparin, there are no
average 7 sulfate residues, 2 acetamide groups and 1 free amine function
for each sequence of 10 disaccharide repeating units, as indicated in fig.1.
This allows the determination of the degree of propargyl functionalization by
comparing the integrated intensities of the resonance signal of the
acetamide group, NHCOCH3, 1.90ppm, and the signal of the alkyne group at
2.54ppm of propargylamine. The resulting degree of alkyne functionalization
%.
SYNTHESIS OF N3- FUNCTIONALIZED POLYMERS BY ATRP

Atom transfer radical polymerization (ATRP) was used for the synthesis of
well-defined polymer chains with well-controlled molecular weight, narrow
molecular weight distruibution and functional chain-ends. Functional
oligomers initiators can be used in ATRP for the preparation of well-defined
clickable polymers. The use of an initiator containing an azide and an
bromide provided a well defined azido functionalized polymer Brterminated responsive polymer which is clickable to alkyne-functionalized
bemiparin to from pH and thermo sensitive bioconjugates.
SYNTHESIS OF N3-PDMAEMA. ATRP of DMAEMA was performed under
relatively mild conditions in a variety of organic solvents or in aqueous
solution. In this work the azido PDMAEMA was prepared in toluene at 40C in
the presence od CuBr2/TPMA as the precursor of the catalytic system and
using N3-PEG3-BPA as the intiator. Sn(II) EtHex was added to the
reactionmixture to reduce Cu(II) to Cu(I). This catalytic system allowed the
application of a very low concentration of copper as is simultaneously
provides an excellent medium to avoid the oxidation of the in situ generated
Cu(I). SEC results show the formation of polymers with monomodal
molecular weight distributions and polydispersity indexes lower than 1.4.
The polymers displayed an increase of molecular weight with conversion
while maintaining narrow molecular weight distributions, which indicates
that the polymerization was carried out under controlled conditions.
Structural characterization of the N3-PDMAEMA polymer confirmed the
incorporation of fragments of the functionalized initiator at both polymer
chain ends. H NMR spectra show the signal from the initiator protons at
3.2ppm. The number- average molecular weight was calculated on the basic
of the ratio of the methyl protons of the polymer at 0.8-1.2 ppm to the
initiator protons at 3.2ppm. Further more , FTIR analysis shows an
absorption band at 2194cm corresponding to the asymmetric stretching
vibration of the azide group. The number- average molecular weight of the
N3-PDMAEMA determined by NMR was in good agreement with the value
obtained by SEC for the same polymer.
N3-P () SYNTHESIS . Copolymerization of di()methyl ether
methacrylate with poly()methyl ether methacrylate by ATRP was reported.
The LCST of the copolymers depends on the composition and increases with
increasing mole fraction of OEOMA in the polymer chain. A statistical
copolymer of MEO2MA with OEOMA300 was prepared by ATRP with the
same N3-PEG3-BPA initiator targeting a copolymer that displayed a LCST
close to the biological temperature. The copolymerization proceeded under
controlled conditions leading to formation of a monodisperse copolymer with
narrow molecular weight distribution. The number of monomer units in the
final copolymer was estimated to be 36 from H NMR spectroscopy inn d6DMSO from the relative intensities of phenyl-initiator protons at 7.2ppm and
methyl group protons from both MEO2MA and OEOMA300 at 0.9-1.2 ppm .
The fina composition of the copolymer was calculated, considering the
relative intensity of methylene signals at 3.77ppm. Talking into account that
MEO2MA contains 3 methylene groups,the other 9 belonging to the
OEMA300, this provides a final molar fraction of 0.75 MEO2MA and 0.25
OEOMA300 in the copolymer chains. The evidence of polymerization from
azido-initiator also was demonstrated by FTIR analysis where an absorption
band at 2204cm was observed corresponding to the asymmetric stretching
vibration of the azide group.

PREPARATION OF BEMIPARIN-G-POLY(METH)ACRYLATE COPOLYMERS

BY CLICK REACTIONS
The alkyne-functionalized bemiparin was used for grafting to click Cu(I)catalyzed dipolar cycloaddition reaction with N3-end-functionalized of 2ethyl methacrylate and the copolymer of dimethyle ether methacrylate and
polymethyl ether methacrylate. The reaction was carried out in
homogeneous aqueous phase media using an equimolar amout of the azide
polymer with respect to the alkynyl groups on bemiparin. The reaction was
carried out at 25C for 24h, and the product was dialyzed against distilled
water for 3 days and isolated by freeze-drying. H NMR and FTIR spectra of
the isolated products were recorded to characterize the bemiparin- polymer
bioconjugate.
According to the FTIR spectrum, both the characteristic C=O stretching
band of polycrylates at 1728 cm and C=O stretching band of bemiparin t
1640 are present. The complete disappearance of the N3 stretching band at
21494 cm and at 2204 cm and the alkynyl stretching band at 2165 cm in
the FTIR spectrum of the click product, plus the appearance of an H NMR
resonance signal at 7.4 ppm, that can be assigned to the triazole ring
proton, proved the formation of the bemiparin-g- PDMAEMA and bemiparing-P copolymer conjugates by azide-alkyne click cycloaddition.
SEC CHARACTETIZATION
The polysaccharide recovered from the dialysis had a SEC Mn=3300 Da and
a PDI= 1.19, essentially the same as the unprocessed bemiparin-polymer,
confirming that no polysaccharide degradation occurs under the adopted
click reaction conditions. In both cases, SEC characterization of the diblock
copolymers showed monomodal traces and polydispersity index less than
1.4. values of molar masses by SEC were relative to poly(..) standards used
to calibrate the columns. An increase in molecular weight was observed
when bemiparin was incorporated into the polymer system, keeping low
polydispersity index.
THERMOGRAVIMETRIC ANALYSIS
The weight loss curve recorded from the TGA analysis of the bemiparin graft
copolymer systems showed two well-resolved degradation steps (EST),
which allowed quantitative evaluation of the composition, taking into
account the total weight loss percentage and the residue left after thermal
degradation. The bemiparin-alkyne and azido-polymer weight percentages
were determined from TGA curves by considering the original graft
copolymers and the residue of the bioconjugates after thermal degradation.
The final molar composition was calculated after taking into account the
fractional weight percentages and the molecular weight of the products. The
amount of bemiparin in the bioconjugates was determined to be 23 wt% in
the PDMAEMA homopolymer and 26 wt% in the P() copolymer, giving a
molar composition of 1:1 for both the bemiparin-PDMAEMA and the
bemiparin-P systems.
THERMAL PROPERTIES
The LCST of a copolymer is defined as the critical temperature at which the
polymer solution undergoes phase separation from a one phase to a two

phase system, one rich and one poor in polymer. Below the LCST, enthalpic
interaction, related to the hydrogen bonding between the polymer and the
water molecules, are responsible for the polymer dissolution. When the
temperature is raised above the LCST, entropy dominates leading to
polymer aggregation/precipation. The LCST of polymers in aqueous solutions
can be modulated by incorporating hydrophilic or hydrophobic moieties into
the copolymer backbones and incorporation of the hydrophilic bemiparin
segment in the bioconjugates structure has a significant effect on the
temperature-sensitive phase transition behavior.
In acidic media, a PDMAEMA-N3 polymer is fully ionized and increasing
the temperature has no significant effect on the swelling ration up to pH 7.
However at pH 7.4, increasing the temperature causes a gradual decrease in
the swelling ratio of the polymer and above 43C the polymer precipitated.
Increasing the pH decreases the degree of ionization of the amino group
along the polymer chain, leading to an increase of the hydrophobicity of the
system, which in turn decreases the phase transition temperature from 43C
to 35C. At lower pH, the system remains soluble in all temperature ranges.
At a constant pH, the grafted bemiparin increases the hydrophilicity of
the system resulting in a corresponding increase of LCST from 35C to 44C
at pH 10, and from 43 C to 65C at pH 7.4.
The properties of PEG polymers in aqueous medium vary depending on
the molecular structure of the monomer units, the nature of the
polymerizable moiety, the length of the PEG side chain and end-group of the
PEG chain. In fact, the balance between hydrophilic and hydrophobic
moieties in the molecular structure of the polymers determines their
solubility properties. In the case of oligo()methyl ether methacrylates the
ether oxygen of PEG from active H-bonds with water, whereas the non-polar
carbon-carbon backbone leads to a competitive hydrophobic effect. Thus,
polymers with very short PEG side-chains are either not water soluble or
only weakly hydrophilic. On the other hand, polymers with longer PEG side
chains, such as OEOMA300, are solube in water, even at higher
temperatures. Random copolymers of MEO2MA and OEOMA300 exhibit LCST
that can be precisely adjusted by varying the ration of comonomers. In this
work, the comonomer composition was adjusted to obtain a LCST of 3233C, which is close to the physiological temperature of 37C. Changes in
the pH of the medium did not appear to affect the LCST of the P() system
due to the absence of acidic or basic groups. However the grafting to the
copolymer to bemiparin increased the hydrophilicity of the system, which in
the turn increased the phase transition temperature from 32C to 41C at
pH 7.4 and from 33 C to 40C at pH10
CONCLUSION
AGET ATRP was employed to prepare azido-containing pH responsive and
temperature sensitive polymer systems with predictable molecular weight
and low polydispersity. Alkyne functionalized bemiparin was prepared by
amidation of carboxylic acid group on the polysaccharide mediated by 4()-4- methylmorpholinium chloride. Well-defined hybrid polysaccharide
structures were prepared by grafting to experiments using Cu(I) catalyzed
azide- alkyne dipolar cycloaddition (click) reactions, resulting in the
formation of bemiparin bioconjugates with advanced functional properties.
The coupling reactions proved to be quantitative, on the basic of size

exclusion chromatography and NMR and FTIR spectroscopy, yieldind pure

diblock copolymers composed of bemiparin and methacrylic polymers. The
biocojugates were both temperature and pH-sensitive water-soluble and
polymeric building blocks. This simple reaction leads to formation of hybrid
double hydrophilic block copolymers that combine biocompatibility, water
solubility and both pH and temperature sensitivity. The thermal properties
of the bioconjugates were studied in aqueous solution by UV-vis
spectroscopy. The obtained LCST values scaled linearly with the composition
of the bioconjugate heparin systems.