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transformed to a semisolid hydrogel at body temperature. After an initial burst release in the first 5 h, a steady linear release
of protein from the hydrogel was achieved for a period of ~70 h. Prolonged quasi-linear release of protein up to 40 days was
1. Introduction
Thermoreversible hydrogels are of great interest
Journal of Controlled Release 103T Corresponding author. Department of Materials Science andachieved by crosslinking the hydrogel with genipin in situ, in a fashion suitable for protein encapsulation while maintaining
the injectability of the hydrogel. The crosslinkage transformed the copolymer from a physical gel to an insoluble chemical
gel and substantially reduced the initial burst release of protein. Both high performance liquid chromatography (HPLC) and
gel electrophoresis indicated that the primary structure of BSA released from the hydrogels with or without genipin-
crosslinking was generally conserved. The hydrogel can be prepared in solutions with a physiological pH, allowing the safe
incorporation of bioactive molecules for a broad range of medical applications, particularly for sustained in vivo drug release
and tissue engineering.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Thermoreversible gels; Chitosan; PEG; Biocompatible; Protein releasePEG-grafted chitosan as an injectable thermosensitive
hydrogel for sustained protein release
Narayan Bhattaraia, Hassna R. Ramaya, Jonathan Gunna,
Frederick A. Matsenb, Miqin Zhanga,b,TaDepartment of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USAbDepartment of Orthopaedics and Sports Medicine, University of Washington, Seattle, WA 98195, USA
Received 11 September 2004; accepted 22 December 2004
Abstract
Thermosensitive polymer hydrogels that undergo a sol-to-gel transition in response to temperature changes are of great
interest in therapeutic delivery and tissue engineering as injectable depot systems. A chitosan-based, injectable thermogel was
prepared by grafting an appropriate amount of PEG onto the chitosan backbone and studied for drug release in vitro using
bovine serum albumin (BSA) as a model protein. When more than ~40 wt.% of PEG was grafted to chitosan chains via
covalent bonding, the aqueous solution of the resultant copolymer was an injectable liquid at low temperature and0168-3659/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2004.12.019
Engineering, Un
Tel.: +1 206 616 9356; fax: +1 206 543 3100.
E-mail address: [email protected] (M. Zhang).(2005) 609624
www.elsevier.com/locate/jconrelulation, and tissuein drug delivery, cell encapsiversity of Washington, Seattle, WA 98195, USA.engineering [1,2]. Early research in the field focused
on synthesis of thermosensitive gel materials includ-
repair and regeneration through controlled and
sustained release of loaded drugs. PEG is a neutral,
ontroling poly(ethylene glycol)/poly(propylene glycol)
block copolymers (poloxamers), poly(ethylene gly-
col)/poly(butylenes glycol) block copolymers, polo-
xamer-g-poly(acrylic acid) and copolymers of N-
isopropylacrylamide that exhibited a sol-to-gel
transition in aqueous solutions [36]. Such materials
are generally not biodegradable, limiting their
practicality for use in the clinic. Diblock copoly-
mers of poly(ethylene oxide) (PEG) and poly(lactic
acid) (PLA), and triblock copolymers of PEG-
PLGA-PEG were introduced later as alternative
hydrogels that would provide biodegradable and
injectable drug-delivery systems under physiological
conditions [79]. The aqueous phases of these PEG-
based copolymers exhibited thermoreversible tran-
sitions from a sol at high temperature to a gel at
body temperature.
Some natural polymers including gelatin, agar-
ose, amylase, amylopectin, cellulose derivatives,
carrageenans, and gellan, exhibit thermoreversible
gelation behavior [1014]. They assume a random
coil conformation at high temperature but change
conformation to form double helices and aggregates
that act as knots at decreased temperaturesthe
process of forming physical gels [15]. Some
cellulose derivatives of natural polymers, such as
methyl cellulose and hydroxypropyl cellulose,
exhibit reverse thermogelation behavior (gelation
at elevated temperatures). Cellulose is not soluble in
water, but by introducing hydrophilic moieties the
cellulose derivatives become water soluble, and at
an optimum balance of hydrophilic and hydro-
phobic moieties, they can undergo a sol-to-gel
transition in water [16].
Chitosan, a polysaccharide derived from naturally
abundant chitin, is currently receiving a great deal of
interest for medical and pharmaceutical applications
that utilize chitosan in various chemical and physical
gel forms [17]. Chitosan, with about an 85% degree of
deacetylation, is insoluble in solutions at neutral or
alkaline pH and exhibits a gel-like precipitate when
the solution pH is brought close to neutral. Recently, a
hydrogel made of chitosan solution neutralized with a
polyol counterionic monohead salt was developed,
which allowed the chitosan solution to remain in a
liquid state at low temperature and transform into a
N. Bhattarai et al. / Journal of C610gel at body temperature [18]. A typical hydrogel
solution was obtained by mixing a chitosan (91%water soluble, non-toxic polymer. It is one of only a
small number of synthetic polymers approved by the
FDA for internal consumption and injection in a
variety of foods, cosmetics, personal care products
and pharmaceuticals, and has been used in a wide
range of biomedical applications [19]. In this study,
bovine serum albumin (BSA) was used as a model
protein to study the drug release behavior of PEG-g-
chitosan hydrogel. Prolonged protein release
(weeks), which is essential for a number of tissue
engineering applications, was achieved by cross-
linking the PEG-g-chitosan hydrogel with genipin in
situ under physiological conditions. Genipin has
recently drawn great interest in tissue engineering
due to its excellent tissue compatibility. It was
estimated that genipin is approximately 5000
10,000 times less cytotoxic than commonly used
glutaraldehyde [20].
2. Materials and methods
2.1. Materials
Chitosan from crab shells with 85% deacetyla-
tion (weight average molar mass Mw 190 kDa
and Brookfield viscosity 200800 cps in 1%
solution with 1% acetic acid), methoxy poly
(ethylene glycol) (PEG) (Mn=2000) and bovine
serum albumin (BSA) were obtained from Aldrich
Chemical Co. (St. Louis, NO), and used as
received. Genipin was obtained from Challengedeacetylation) solution (200 mg in 9 ml HCl solution
[0.1 M]) with a glycerophosphate disodium salt
solution (560 mg in 1 ml distilled water). Although
the gel was capable of maintaining the bioactivity of
loaded bone protein and viability of chondrocytes
entrapped in the gel, the excess use of glycerophos-
phate salt may need to be avoided in a number of
biomedical applications.
This study aims to develop a chitosan-based
injectable, thermosensitive system based upon PEG-
grafted chitosan (PEG-g-chitosan), that can serve as
a therapeutic drug-delivery system promoting tissue
led Release 103 (2005) 609624Bioproducts Co., Taiwan. All other reagents were
chemical grade and used as received.
ontrol2.2. Synthesis of PEG-g-chitosan
The PEG-g-chitosan was prepared by the method
described byHarris et al. [21]. First, PEG-aldehydewas
prepared by oxidation of PEG with DMSO/acetic
anhydride. After PEG completely dissolved in anhy-
drous DMSO/chloroform (90/10 v/v), acetic anhydride
was added into the mixture under a nitrogen atmos-
phere. The molar ratio of acetic anhydride to PEG was
12. The mixture was stirred for 12 h at room temper-
ature under a nitrogen atmosphere and precipitated
with excess diethyl ether. The precipitate was separated
from the solution and reprecipitated twice from chloro-
form solution with diethyl ether. After drying under
vacuum, white PEG-aldehyde powder was obtained.
PEG-grafted chitosan (PEG-g-chitosan) was pre-
pared by alkylation of chitosan followed by Schiff
base formation. PEG-aldehyde and chitosan with a
molar ratio of 0.4/1 were added into a mixture of
acetic acid and methanol (2/1 v/v). Aqueous cyano-
borohydride (NaCNBH3) solution was then added
dropwise into the mixture of chitosan and PEG-
aldehyde at pH 6 with a molar ratio of 0.02/0.3 for
NaCNBH3/PEG-aldehyde. The resultant mixture was
dialyzed with a dialysis membrane (MW 12000
14000 cut) against distilled water and 0.05 M aqueous
NaOH solution, and the solution was subsequently
freeze-dried. PEG-grafted chitosan was obtained by
removal of unreacted PEG from the freeze-dried
samples with excess acetone. By changing the molar
ratio of PEG-aldehyde to sodium cyanoborohydride,
samples with different wt.% of grafted PEG were
obtained.
2.3. Characterization of PEG-g-chitosan copolymer
NMR and infrared spectroscopy techniques were
used to characterize PEG-g-chitosan. 1H NMR spectra
acquired with a Bruker AV-301 spectrometer at 50 8Crevealed the chemical structure of PEG-g-chitosan and
the degree of substitution of PEG on chitosan.
Samples of 1020 mg each were prepared, and
dissolved in 0.7 ml of D2O containing 0.5 M DCl/
D2O.1H NMR data of chitosan and PEG-g-chitosan
was obtained as follows. The assignments and
chemical shifts of chitosan and PEG-g-chitosan are:
N. Bhattarai et al. / Journal of Cd 4.95.2 (1H br, H-1), 3.74.2 (br, H-3, H-4, H-5, H-6 and H-6V), 3.4 (0.85H, br s, H-2), 2.2 ppm (0.4H, brs, NHAc). PEG-g-chitosan: d 55.3 (1H, br, H-1 ofGlcN), 5.38 (br, 0.15H, H-1 of N-alkylated GlcNAc),
3.74.3 (m, H-3, H-4, H-5, H-6, H-6V, NCH2b ofN-alkylated PEG and singlet of OCH2 of PEG), 3.6
(OCH3), 3.43.52 (0.85H, br s, H-2), 2.25 ppm
(0.4H, br s, NHAc). The peak intensities of H-2, H-3,
H-4, H-5, and H-6 of PEG-g-chitosan did not match
the number of hydrogen atoms, because the peak of
PEG methylene was overlapped with those of H-2, H-
3, H-4, H-5, and H-6 of monosaccharide residues.
For infrared spectroscopic analysis, a dried sample
of 5 mg was mixed with 300 mg dry KBr and pressed
into a pellet using a macro KBr die kit. The solid
pellet was placed in a magnetic holder, and the system
was purged with nitrogen before testing. Polarized
Fourier Transformed Infrared (FTIR) spectra of 200
scans at 4 cm1 resolution were obtained using aNicolet 5DX spectrometer equipped with a DTGS
detector and a solid transmission sample compart-
ment. Spectrum analyses and display were performed
using standard Nicolet and Microcal Origin software.
2.4. Hydrogel preparation and gelation study
PEG-g-chitosan copolymers were mixed with
doubly distilled water in different polymer weight
percentages and left overnight in a refrigerator at 4 8C.The mixtures were vortexed several times and
centrifuged to remove air bubbles, and 2 ml of each
solution (13 wt.%) was placed in a 10 ml tube and
tightly capped with a rubber septum. The solutions
were maintained between 5 and 10 8C prior to solgeltransition studies. A simple test tube inverting method
was employed to determine the occurrence of solgel
transition [8]. The sol phase was defined as flowing
liquid and the gel phase as non-flowing gel when the
hydrogel solution in the test tube was inverted.
Thermoreversible behavior of the hydrogels was
examined for the temperature range of 1045 8C.The gelation experiment was also performed using
phosphate buffered saline (PBS) at pH 7.4. No
significant effect from the salts of the PBS was
observed on gelation behavior.
2.5. Viscosity measurements
led Release 103 (2005) 609624 611Thermoreversible gelation behavior of PEG-g-
chitosan was further studied by measuring the
ontrolsolution viscosity of the samples at neutral pH.
Viscosities of aqueous PEG-g-chitosan solutions
were measured as a function of time and temper-
ature using a Hakke Viscometer (VT550) equip-
ped with SP2P sensors. The solution in a rotor
was thermostated with a water bath circulator.
Shear viscosity measurements were made at a
temperature range of 1045 8C at a fixed shearrate of 30 s1.
2.6. In vitro protein release study
Different amounts of BSA were dissolved in 1.5
ml deionized distilled water to obtain BSA sol-
utions with final concentrations ranging from 200
to 1000 Ag/ml. Solutions were prepared in 15 mlpolypropylene tubes wherein 35 mg of the PEG-g-
chitosan was mixed into each BSA solution and the
mixtures were left overnight in a refrigerator at 4
8C. After light vortexing of the polymer/proteinmixtures, air bubbles were removed by centrifuga-
tion. The solutions containing PEG-g-chitosan and
BSA were incubated at 37 8C for 10 min to formgels, and 7 ml of PBS (pH 7.4) was added to each
tube. The gels stuck on the walls of the tubes were
removed gently with a spatula and transferred into
the release media. At specified sample collection
times, 1 ml solution out of 7 ml total solution was
removed and transferred to a siliconized 1.5 ml
microcentrifuge tube, and the medium in the tube
was replenished with 1 ml of fresh PBS. Protein
content of each sample was analyzed with modified
Coomassie blue protein assay (BioradR) in a 96-well plate using UV spectroscopy at 590 nm. A
calibration curve was generated at each time
interval using a non-loaded gel in order to correct
for the intrinsic absorbance of the polymer.
Samples in triplicate were analyzed for each
experiment.
To achieve prolonged protein release, PEG-g-
chitosan hydrogels containing BSA were cross-
linked with genipin. 1.5 ml of PEG-g-chitosan/
BSA solution was mixed with 0.5 mM genipin
solution at 4 8C and the mixture was maintained at37 8C for either 10 min or 24 h, before PBS wasadded to the mixture. Protein release studies were
N. Bhattarai et al. / Journal of C612carried out at 37 8C for the hydrogels of bothcrosslinking times.2.7. Microscopy analysis
Samples for the protein release study were frozen
in liquid nitrogen and freeze-dried for 24 h. The
samples were coated with gold/palladium and the
morphology was examined using a scanning electron
microscope (SEM) (JEOL 5200).
2.8. Analysis of released proteins by high performance
liquid chromatography (HPLC) and gel electropho-
resis (SDS-PAGE)
To examine the stability of the protein in the
hydrogel environment, and the possible influence of
the crosslinking agent on protein integrity (or aggre-
gation), the protein released from the hydrogels was
analyzed using a high-performance liquid chromatog-
raphy (HPLC) system equipped with a Rheodyne
7725i injection valve (Beckman Coulter, Rheodyne,
Rohnert Park, CA), a System Gold Solvent Module
(126), and UV Detector (168; Beckman Coulter,
Fullerton, CA). A strong anion-exchange chromato-
graphic column, Biosuitek Q 10 Am AXC, 757.5mm (Waters, Milford, MA), was used. The stationary
phase had a pore size of 1000 2 and the proteincapacity was specified at 331 mg/column. Detection
was performed with UV absorbance at 280 nm. The
mobile phases were 20 mM TrisHCl pH 8.0 (Eluent
A) and Eluent Awith 1 M sodium chloride (Eluent B).
The flow rate was 0.8 ml/min and the gradient was 0
80% of Eluent B over 15 min. The sample volume
was 20 Al. The concentration of the samples wasmaintained at ~2mg/ml. Duplicate measurements were
made for each sample. The experiments were per-
formed at ambient room temperature. All calculations
were performed using 32 Karat Software (Beckman
Coulter, Fullerton, CA.).
The structural integrity of BSA released from PEG-
g-chitosan hydrogels with and without genipin cross-
link was also examined using a Bio-Rad Mini-Protean
III electrophoresis system. All the BSA solutions
prepared for the HPLC experiments were used for the
415% SDS-PAGE study. The BSA sample solutions
were directly loaded into the wells with a micro-
pipette, and the electrophoresis was performed at 200
V, 100 mA. The gel was stained with 0.1% Coomassie
led Release 103 (2005) 609624Brilliant Blue to visualize protein bands. The study
was conducted according to the manufacturers pro-
tocol. The gel pictures were taken with a scanner after
wiping off all the water from the gel membrane.
3. Results
3.1. Synthesis of PEG-g-chitosan
Fig. 1 shows the chemical scheme for modifying
chitosan with a PEG-aldehyde to yield an imine
(Schiff base) and subsequently converting it to PEG-
grafted chitosan (PEG-g-chitosan) through reduction
with sodium cyanoborohydride (NaCNBH3) [22]. The
final purified product of the PEG-g-chitosan was
analyzed by 1H NMR. Compared to 1H NMR
spectrum of chitosan, peaks in PEG-g-chitosan
spectrum in the range of 3.64.5 ppm were not well
separated due to the overlapping of a more intense
peak of PEG methylene group and peaks of saccha-
rine backbone of chitosan. Methyl group of PEG was
clearly observed at 3.6 ppm. Furthermore, the H-1
proton signal from chitosan shifted from d=4.9 to 5.2ppm after the chitosan was grafted with PEG, and the
OOH
N. Bhattarai et al. / Journal of ControlNHCH2CH2(OCH2CH2)mOCH3
OHO nOO
NH2
OH
HOn
1)CH3O(CH2CH2CHO)mCH2CHO
2) NaCNBH3
OO
OH
HO n
AcOH, MeOH, pH 3-5
CHCH2(OCH2CH2)mOCH3N
AcOH, MeOH, pH 5-5.5Fig. 1. Chemical scheme for grafting PEG onto chitosan.H-2 proton signal shifted from d=3.4 to 3.5 ppm.These shifts correspond to N-alkylation of chitosan
[23]. The degree of PEG substitution (DS) was
evaluated from the relative peak intensities of the
methylene group of PEG and the H-1 of mono-
saccharide residue in 1H NMR spectra [24]. By
changing the molar ratio of PEG-aldehyde to sodium
cyanoborohydride, samples with different wt.% of
PEG grafted were obtained (Table 1). The data shown
in Table 1 indicates that by keeping the amount of
NaCNBH3 roughly constant (G45 through G68), the
amount of grafted PEG increased with the increase of
the PEG-aldehyde to chitosan ratio. On the other
hand, excess NaCNBH3 reduced the PEG grafting
(G36), presumably because the excess amount of
NaCNBH3 made the solution more basic in which
chitosan was less soluble, hindering its chemical
reaction with PEG.
Results in Table 1 also show that by grafting an
appropriate amount of PEG onto the chitosan back-
bone, PEG-g-chitosan soluble in water was obtained.
All of the samples except G36, which had the lowest
amount of grafted PEG, were soluble in water at
physiological pH. Viscosities of all these soluble
polymers were also measured at two different temper-
atures. Viscosity differences at the two temperatures
shown in the last column of Table 1 indicate that the
viscosities of two as-synthesized polymer samples
(G45 and G55) differ significantly at temperatures 37
and 10 8C, and the viscosities increased withincreasing temperature. This suggests an inversed
thermal relation between solution viscosity and
temperature, which is the basis of formation of a
thermoreversible gel. Thus, only these two samples
(i.e., G45 and G55) were extensively studied as
potential candidates of injectable hydrogels.
A comparative IR spectrum study of PEG-g-
chitosan, chitosan, and PEG, as shown in Fig. 2,
confirmed the success of grafting PEG to chitosan.
The chitosan IR spectrum exhibited characteristic
bands of 1664 (amide I), 1580 (amide II) and 1380
cm1 (amide III). The absorption bands at 1160 cm1
(asymmetric stretching of COC bridge), 1075 and
1033 cm1 (CO stretching) were characteristics of itssaccharine structure [22,25,26]. NH and OH
stretching vibrations were characterized by the broad1
led Release 103 (2005) 609624 613band in the region of 32003500 cm . Pure PEG has
characteristic peaks at 1280, 947, and 843 cm1
gels reverted back to solutions when temperature
dropped to 10 8C or below. This behavior wasobserved by tilting or inverting the test tube contain-
ing the hydrogel at different temperatures. The typical
so-to-gel transition time was 10F4 min.Solgel transition behavior of PEG-g-chitosan was
further illustrated by rheological analysis. Fig. 3 shows
the variation in viscosity over time at fixed temper-
atures of 10 and 37 8C, respectively, for solutions ofpure chitosan and PEG-g-chitosan samples G45 and
G55. The chitosan solution was prepared in dilute
acetic acid, and the pH was maintained at 5.7F0.2 byslowly adding a dilute solution of NaOH, whereas
solutions of G45 and G55 were prepared in doubly
distilled water at pH 7.4F0.5. By studying gelation
ontrolled Release 103 (2005) 609624[25,26]. For the PEG-g-chitosan sample, the peaks
corresponding to the hydroxyl group, amino group
and amide group of chitosan shifted slightly, and their
intensities were significantly reduced as a result of
PEG grafting. Compared to the amide I peak at 1664
cm1, the peak intensity of amide II significantlydecreased. This resultant spectrum shows that the
NH2 groups of chitosan were partially grafted with
Table 1
Samples prepared with different molar ratios of reagents
Sample
no.
[PEG-aldehyde]/
[chitosan]
[NaCNBH3]/
[PEG-aldehyde]aDSb Graft
wt.%cDgd
(Pa. S)
G36 1 2 0.08 36
G45 0.4 0.3 0.16 45 2.2
G55 0.6 0.3 0.25 55 5.2
G64 1 0.3 0.26 64 0.02
G68 1 0.1 0.30 68 0.03
a A 5 M stock solution of NaCNBH3 in 1 M NaOH was used after
diluted with water to 3 times of the original volume.b Degree of PEG substitution (DS) on chitosan backbone as
determined from 1H NMR spectra.c Graft weight.% was calculated from the relation: (WtWc)/
Wt100, where Wt is the weight of freeze-dried graft copolymer,and Wc is the weight of chitosan in feed.d Viscosity difference of the aqueous solution (pH=7.5) of PEG-g-
chitosan at two temperatures, 10 and 378C. The concentration of thesolution was ranged from 1.35 to 3 wt.%. Viscosity was measured
by a Haake Viscomer at a fixed shear rate.
N. Bhattarai et al. / Journal of C614PEG. If the chitosan were fully grafted, the peaks
corresponding to NH2 groups at 1580 cm1 would
disappear and form a single peak after completion of
the reaction [22,25,26]. The characteristic peaks
associated with PEG in PEG-g-chitosan at 1280,
947, and 843 cm1 were significantly decreased.The peaks at 1120 and 2880 cm1 in PEG-g-chitosanwere attributable to the superposition of CO and C
H stretching vibrations of chitosan and PEG.
3.2. Thermoreversible gelation behavior
Both G45 and G55 samples (Table 1) of PEG-g-
chitosan, with 45 and 55 wt.% of PEG grafted to
chitosan, respectively, underwent an apparent sol-to-
gel transition in the solutions with polymer concen-
trations ranging from 1.3 to 3 wt.%. Below the
transition temperature, the solutions were viscous
liquids that flowed easily and were injectable through
a 20-gauge needle. As the solutions were heated to
body temperature, they transformed into gels. Thebehavior of PEG-g-chitosan solutions of various
polymer concentrations, it was found that solutions
with high polymer concentrations gelled faster than
those with low polymer concentrations. Samples G45
and G55 shown in Fig. 4 are representatives of those
polymers whose PEG-to-chitosan ratios led to ther-
moreversible gelation when the solutions were pre-
pared with proper polymer concentrations. Although
the viscosity data for 3 wt.% pure chitosan solution is
shown, no apparent phase transition was observed.
3.3. BSA release from hydrogels
Hydrogels made from G55 and G45 PEG-g-
chitosan were used for the BSA release study. Fig. 4
shows the percent cumulative release profiles of the
3500 3000 2500 2000 1500 1000
0.0
0.2
0.4
0.6
0.8
1.0
1.2
----------------------------------------13
80
--------------------------------------11
20
-------------------------------------------28
80
---------------------------------15
80
-------------------------------------------34
40
-----------------------------------------16
64
b
a
Abso
rban
ce
Wavenumber(cm-1)
cFig. 2. FTIR spectra of (a) PEG, (b) chitosan, and (c) PEG-g-
chitosan (sample G45 in Table 1).
hydrogel matrices loaded with BSA at differen
concentrations ranging from 200 to 1000 Ag/mlTwo distinctive release characteristics were seen for
hydrogels made from G55 and G45. The G55 ge
showed a release of 5267% of BSA in the first 5 h
whereas the G45 gel showed a release of 1058% of
BSA in the same time period. Both copolymers
showed slow BSA release in the period of 570 h
and no apparent release thereafter. Clearly, after 70 h
the remaining BSA was trapped in the gel matrix and
could not be completely released until the gel matrix
was dissolved in media.
Typically, G55 gels dissolved in PBS (pH 7.4) in
around 2 weeks and G45 gels in around 3 weeks. Fig
0 20 40 60 80 100 120 1400
Time (h)
0 20 40 60 80 100 120 1400
10
20
30
40
50
60
70
80
90
100B
200 400 600 800 1000C
umm
ulat
ive re
leas
e (%
)
Time (h)
Fig. 4. In vitro cumulative percent release of BSA from
thermoreversible hydrogels of (A) G55 and (B) G45 loaded with
different concentrations of BSA. Each point represents the mean
valueFSEM (n=3).
0 500 1000 1500 2000 2500 3000 35000
1
2
3
4
5
At 37C
At 10C
Visc
osity
(Pa.S
)
Time (second)
A
0 500 1000 1500 2000 2500 3000 35000
1
2
3
4
5
Visc
osity
(Pa.S
)
Time (second)
B
At 37C
At 10C
0 500 1000 1500 2000 2500 3000 35000
1
2
3
4
5
6
7CAt 37C
At 10C
Visc
osity
(Pa.S
)
Time (second)
Fig. 3. Aqueous solution viscosities of (A) pure chitosan, (B) G45
and (C) G55 of PEG-g-chitosan as a function of time at two
temperatures 10 and 37 8C. The filled symbols refer to the solutionsat 37 8C and the open symbols refer to the solutions at 10 8C.Polymer concentration of solutions for (A), (B) and (C) were 3, 1.3
and 3 wt.%, respectively.
N. Bhattarai et al. / Journal of Controlt
.
l
,
,10
20
30
40
50
60
70
80
90
100
110A
Cum
mul
ative
rele
ase
(%)
200 400 600 800 1000
led Release 103 (2005) 609624 615.
5 is one of the representative curves showing the
dissolution of G55 hydrogel immersed in PBS within
2 weeks time. PEG-g-chitosan hydrogels were placed
into glass vials and thermostated under the same
conditions used in the release studies. At predeter-
mined time intervals, the gels were separated out from
the release medium, washed with DI water and gently
blotted. Then, they were freeze-dried for 48 h and
weighed again. Approximately 18% of the dry weight
was lost in first 48 h, and only about 10% during the
following week (Fig. 5). The most significant weight
loss occurred around 2 weeks. The release of BSA
was thought to be largely due to diffusion and
accelerated by the weight loss of the gel. However,
this argument cannot be fully justified when compar-
N. Bhattarai et al. / Journal of Control616ing the release kinetics (Fig. 4) with the weight loss
profile (Fig. 5), at least at the initial stage of the drug
release. In the first 5 h, more than 65% of the BSA
was released from the gel while only a negligible
amount of the gel matrix was lost. This suggests that
the BSA release was dominated by diffusion and not
by gel erosion in the early stage of the release. The
high BSA diffusion might be due to the large amount
of water present in the gel, and the low erosion rate
might be due to the intermolecular interaction of the
three-dimensional physical gel.
In general, hydrogels loaded with BSA of different
concentrations exhibited a similar trend in accumu-
lative BSA release, except for the initial bburstQrelease which exhibited an accumulative release
proportional to BSA loading. Clearly, hydrogels of
0 2 4 6 8 10 12 140
10
20
30
40
50
60
70
80
90
100
Wei
ght l
oss
(%)
Time (days)Fig. 5. Weight loss of hydrogel (G55) as a function of immersion
time in PBS (pH=7.4) at 37 8C.this type are suitable for short-term drug release,
including treatment plans measured in hours or days.
3.4. Genipin-treated PEG-g-chitosan hydrogel
Genipin solution with a concentration of 0.5 mM
was added to the PEG-g-chitosan solution and mixed
at 10 8C under constant stirring. The mixture was thenthermostated at 37 8C. The crosslinking of thehydrogel completed within several hours, character-
ized by a change in color from transparent to light
yellow to deep blue. The color change was due to the
formation of a crosslinked network by the reaction of
chitosan fragments in PEG-g-chitosan with genipin
[27], and Fig. 6 shows the possible chemical scheme.
As the reaction proceeded the viscosity of the solution
increased. FTIR and viscosity measurements were
made to estimate the networking reaction time after
the genipin treatment. IR spectra of hydrogels treated
with genipin for different time periods are shown in
Fig. 7. The spectra of the genipin treated samples as
compared to the spectra of non-treated samples
showed a significant decrease in adsorption at 1570
cm1, which may be attributable to the absorption ofNH2 group as a result of the reaction between
chitosan segment of PEG-chitosan and genipin. This
decrease in adsorption is particularly significant after
a 3 h reaction. Besides all the characteristic peaks
corresponding to PEG and chitosan segments, the new
peak at ~1380 cm1 is attributable to the ring-stretching of heterocyclic amine in the hydrogel
network.
The crosslinking reaction and the reaction time
frame were further studied by measuring the viscosity
of the hydrogel solution during gelation. The result is
shown in Fig. 8, along with the viscosity profile for a
chitosan solution with genipin under the same
conditions for comparison. The viscosity of the
genipin-crosslinked PEG-g-chitosan solution in-
creased noticeably at two distinct stages as opposite
to the single stage exhibited by the chitosan solution.
The first rapid increase in viscosity for the PEG-g-
chitosan solution was due to its thermoreversible
nature, whereas the second increase was due to the
networking reaction set by genipin. The viscosity of
chitosan solution started to increase abruptly after a 2
led Release 103 (2005) 609624h reaction, whereas the second increase in viscosity
for PEG-g-chitosan solution started about 4 h after
2HN
NH2
NH2
2HN
2HN
2HN O
C
OHHOH2C
O OCH3
+ +O
ONH2
O
O
chitosan
genipin
PEG
N. Bhattarai et al. / Journal of ControlNH2
NH2
NH
2HN N
OHHOH2C
O
N
Oreaction. This result shows that the gelation rate due
to the networking reaction was faster with chitosan
solution than with PEG-g-chitosan. The slower
reaction rate in PEG-g-chitosan was probably due
to the presence of fewer reactive amine groups and
the steric hindrance created by the PEG segments.
were mixed with genipin solution at 4 8C, and proteinrelease of the mixtures was monitored upon gelation
at 37 8C. BSA release profiles of three types ofsamples for a 50 h period are shown in Fig. 9: (1) G55
hydrogel loaded with 100 Ag/ml BSA after a 10 mingelation, (2) G55 hydrogel loaded with 100 Ag/mBSA and 0.5 mM genipin after a 10 min gelation, and
(3) G55 hydrogel loaded with 100 Ag/ml BSA and 0.5mM genipin after a 24 h gelation. In each case, the
original gel volume and BSA concentration was the
same. The addition of genipin did not seem to affec
the injectability of the hydrogel solution after the
mixture was cooled to 4 8C for 24 h. However, thesolutions with incorporated genipin lost thermorever-
sibility at 37 8C, and the color of the gels changedfrom transparency to yellow within 2 h and later
changed to blue. This is not a problem for the
intended applications where only injectability of the
hydrogel solution at low temperature and the gelation
process at body temperature are concerned.
As expected, crosslinking hydrogels with genipin
prolonged the BSA release profile of the hydrogels
1800 1600 1400 1200
t=0 ht=0.5 ht=1 h
t=3 h
t=12 h
t=6 h
t=24 h
Abso
rban
ce (A
.U.)
Wavenumber (cm-1)
Fig. 7. FTIR spectra of PEG-g-chitosan (G55) hydrogel treated with
genipin at different reaction times.
Fig. 6. Chemical scheme of crosslinking chitosan with genipin.l
t3.5. BSA release from PEG-g-chitosan hydrogels
crosslinked with genipin
To achieve prolonged protein release, PEG-g-
chitosan hydrogels were crosslinked with genipin in
situ. PEG-g-chitosan solutions pre-loaded with BSA
0 50 100 150 200 250 300 350 4000
1
2
3
4
5
6
7
8
9
10
Vis
cosi
ty (
Pa.S
)
Time (minutes)
G55
chitosan
Fig. 8. Viscosity changes of PEG-g-chitosan (G55) and pure
chitosan during gelation in the presence of genipin as a function
of time. Polymer concentrations of PEG-g-chitosan and chitosan
solutions were both 3 wt.%, and the final concentration of genipin
was 0.5 mM. Solutions were prepared at 10 8C and the viscositieswere measured at 37 8C.
led Release 103 (2005) 609624 617.
proteins in PBS at 37 8C for different time periods.The samples collected were frozen in liquid nitrogen
and dried by freeze-drying. After 24 h of BSA release,
the hydrogel exhibited a pore size of 1530 Am. Forboth hydrogels (G45 and G55), no apparent changes
in surface morphology were observed in the first 24 h
of protein release in PBS (images are not shown).
Both morphologies exhibited larger pore sizes and
rougher surfaces after the hydrogels were immersed in
PBS for 2 weeks.
Drastic changes in porosity were observed after the
hydrogels were treated with genipin. These hydrogels
showed relatively low porosity after immersion in
PBS for both 1 day and 2 weeks (Figs. 11C and D).
Between the genipin-treated samples with different
immersion times in PBS, no substantial change in
porosity was found in a period of 1 month.
3.7. BSA structural integrity
ontrolled Release 103 (2005) 6096240 10 20 30 40 500
20
40
60
80
100
Cum
mul
ative
rele
ase
(%)
Time (h)
A B C
Fig. 9. In vitro percent cumulative release of BSA from PEG-g-
chitosan hydrogels: (A) G55, (B) G55 treated with genipin for 10
min, and (C) G55 treated with genipin for 24 h. All the hydrogels
contain the same amount of BSA (1000 Ag/ml), and BSA wasreleased in PBS with pH=7.4. The concentration of genipin was 0.5
mM. Triplicates for each hydrogel were analyzed and each data
point represents the mean valueFSEM.
N. Bhattarai et al. / Journal of C618The hydrogel without genipin released more than 70%
of BSA in the first 5 h, while the gel crosslinked with
genipin for 24 h released only ~12% of BSA in the
first day and another 30% in 1 week (release profile
for extended period is shown in Fig. 10). For the
hydrogel treated with genipin for only 10 min, ~15%
of BSA was released within the first day and another
~25% of BSA in 2 days.
Fig. 10 shows the cumulative BSA release
profiles of G55 hydrogels (after 24 h genipin
crosslinking) with different BSA loading concentra-
tions over a period of 40 days. The BSA release rate
rose with an increase in the amount of BSA loaded
in the gel. The release profiles exhibited a fast
release rate in the first 5 h, followed by a virtually
linear release over a 40-day period. In contrast with
unlinked PEG-g-chitosan hydrogels, the crosslinked
hydrogels are potentially suitable for long-term drug
release applications.
3.6. Morphology of freeze-dried hydrogels by SEM
Material structures of PEG-g-chitosan copolymers
were examined by SEM. Fig. 11 shows the SEM
micrographs of G45 and G55 hydrogels after releasingExposure of BSA to the ionic solution and cross-
linking agents could affect protein structure and
stability [28]. Possible detrimental effects of this
process include protein denaturation, aggregation,
hydrolysis, and reaction with the crosslinking agents,
all of which could decrease the activity of proteins
encapsulated in hydrogels. Therefore, the effect of the
0 10 20 30 400
20
40
60
80
100
Cum
ulat
ive R
elea
se (%
)
Time (days)
1000 800 600 400 200
Fig. 10. In vitro percent cumulative release of BSA from G55
hydrogels with BSA concentrations ranging from 200 to 1000 Ag/ml. All the hydrogels were pre-treated with genipin for 24 h. The
release study was performed in PBS with pH=7.4. Concentration ofgenipin was 0.5 mM. Triplicates for each hydrogel were analyzed
and each data point represents the mean valueFSEM.
BD
fter im
5 trea
ontrolled Release 103 (2005) 609624 619A
C
Fig. 11. Scanning electron micrographs of G55 and G45 hydrogels a
immersion time 24 h, (B) G45 with immersion time 2 weeks, (C) G5
genipin and immersion time 2 weeks. The scale bar is 10 Am.
N. Bhattarai et al. / Journal of Chydrogel environment on the integrity of encapsulated
BSA was investigated using HPLC and SDS-PAGE.
Both experiments were carried out on the BSA
released from hydrogels of G45 for 3 days (with
and without genipin treatment) and compared with the
original BSA in solution (i.e., a BSA standard). Fig.
12 shows the HPLC chromatograms of the standard
BSA (A), the BSA released from a non-crosslinked
gel (B) and from a genipin-crosslinked gel (C).
It was noted that for all the samples there is a major
and a minor component peak, labeled as a and hregions, respectively. Comparing the elution pattern
with those obtained for standard albumin as reported
by the column manufacturer [29] as well as with those
from the literature [30], the major component peak
was identified to be BSA monomer and the minor
component peak to be the BSA oligomers (or mixture
of BSA dimer, trimer, tetramer etc.). The results
showed that the major portion BSA released from the
hydrogels was monomer, although the ratio of BSA
monomer to oligomer in standard BSA is higher than
in the release solutions. This suggests that the
majority of BSA released from hydrogels retain their
integrity.mersion in PBS (pH=7.4, 37 8C) for different times: (A) G45 withted with genipin and immersion time 24 h, and (D) G55 treated withFig. 13 shows the SDS-PAGE results of the
released BSA and commercial BSA. Molecular
weight markers are shown in Lane I and IV, and the
commercial BSA (Lane II and V) exhibited clear
4 6 8 10 12 14 16 18
Elution Time (min)
BSA Standard BSA Non-crosslinked BSA Crosslinked
Fig. 12. Anion-exchange chromatograms of (A) BSA standard, (B
BSA released from non-crosslinked hydrogel, and (C) BSA released
from genipin-crosslinked hydrogel.)
bands at 66 kDa. The SDS-PAGE gel banding patterns
of the BSA released from the hydrogels without and
with genipin treatment are shown in Lane III and VI,
respectively. It is seen that the both BSA solutions
that a small portion of the protein was multimers,
which is consistent with the results obtained by
HPLC. No bands corresponding to lower molecular
weights were observed, suggesting that the released
BSA did not undergo hydrolysis.
4. Discussion
The first step in developing chitosan-based inject-
able hydrogels is to improve its solubility at neutral/
physiological pH. This was achieved in this study by
grafting an appropriate amount of PEG onto the
chitosan backbone. The required minimum amount of
PEG necessary to make the graft copolymer soluble at
neutral pH was found to be 40F4% w/w. Themechanism of the thermoreversible solgel transition
for the PEG-g-chitosan copolymer is illustrated in Fig.
14(A and B). At low temperature, the intermolecular
forces of the PEG-g-chitosan are dominated by the
hydrogen bonding between hydrophilic groups of
250 150 100 75
50 37 25
Markers, Mw (kDa)
I II III IV V VI
Fig. 13. Coomassie-stained SDS-PAGE gel of BSA. Lanes (I), (II),
(III), (IV), (V), (VI) are, respectively, the molecular weight markers,
BSA standard, BSA released from noncrosslinked hydrogel,
molecular weight markers, BSA standard and BSA released from
genipin-crosslinked hydrogel.
N. Bhattarai et al. / Journal of Controlled Release 103 (2005) 609624620have distinct dark bands present at 66 kDa, indicating
that the integrity of the released protein is largely
retained. However, the presence of the faint bands
corresponding to higher molecular weights suggestedFig. 14. A schematic representation of thermoreversible hydrogel formulat
transformation into thermoirreversible form with addition of genipin (C). (A
of hydrophobic interactions between hydrophobic moieties on chitosan b
some of the amine residue of chitosan with genipin (chemical reaction sc
corresponding to the chemical structures shown in (A), (B), and (C), respion of aqueous PEG-g-chitosan solution (A and B) and subsequently
) Stretched chitosan backbone in solution, (B) gel formed as a result
ackbone, (C) thermoirreversible hydrogel formation by reaction ofPEG and water molecules, leading to dissolution of
the polymer chains (Fig. 14A). The hydrophobic
interactions between the polymer chains prevail as
temperature increases [3133], leading to the associ-heme shown in Fig. 6), (D), (E), and (F): photographs of hydrogels
ectively.
ontrolation of chitosan segments and the reduction of the
mobility of PEG molecules. As a result, a long-range
networka gel is formed (Fig. 14B). As reported for
thermogels based on chitosan/glycerol phosphate
[18,34], at neutral pH chitosanchitosan hydrophobic
interactions play the major role in hydrogel formation
when the solution temperature is increased. Depend-
ing on the degree of deacetylation of chitosan and the
pH of solution, the mixture of chitosan with excess
glycerol phosphate salt will vary in gelation rate and
transition temperature. This type of thermosensitive
gelation has also been observed in other cellulose
derivatives grafted with hydrophilic moieties. In this
study, the thermoreversible solgel transition of PEG-
g-chitosan was achieved only at an optimum balance
of hydrophilic and hydrophobic moieties in PEG-g-
chitosan. Grafting PEG to chitosan renders the
polymer soluble in watera prerequisite for gelation.
There was a minimum amount of grafted PEG under
which the polymer was insoluble in water even at low
temperatures (e.g., ~10 8C). However, excess PEGgrafting (e.g., PEG wt.%N55) suppressed hydrophobicinteractions between the chitosan chains, resulting in a
non-gellable solution at 37 8C (results not shown). Inthe present study, the injectable, thermoreversible
hydrogel formed when the chitosan grafted with a
PEG concentration ranging from 45 to 55 wt.%. Other
factors including PEG molecule weight can influence
the effective range of this concentration.
One of the key applications of this novel thermor-
eversible hydrogel is in controlled drug delivery. A
mixture of PEG-g-chitosan and drugs can be prepared
as an aqueous solution in normal physiological pH
below the gelation temperature to form a drug
delivery liquid. This liquid can be administrated into
a warm-blooded animal where the liquid transforms
into a gelled drug depot in situ for sustained release.
With BSA as a model protein, cumulative release of
BSA from the thermosensitive hydrogel was found to
be rapid in the first 5 h, slow down afterwards, and
continue for up to 70 h. BSA carried a net negative
charge since all the release experiments were carried
out at pH=7.4 which is greater than the isoelectric
point (pI=4.7) of BSA. The charged BSA occupied
the water channels of the hydrogel from which it
would be released by diffusion along the concen-
N. Bhattarai et al. / Journal of Ctration gradient between the gel matrix and release
medium. The thermoreversible hydrogel developed inthis study contains a relatively large amount of water
and less polymeric mass, which is favorable for high-
concentration drug loading. However, this also leads
to rapid drug release as a result of high mobility of
drug molecules in solution within the hydrogel.
All the thermoreversible hydrogels presented in
this study exhibited a dose-dependent release profile.
Hydrogels with a higher amount of loaded BSA
produced a greater concentration gradient and
exhibited faster release than hydrogels loaded with
lower doses (Fig. 4). However, independent of the
amount of BSA loaded, all the hydrogels of this type
released ~90% of BSA in 70 h. The remaining BSA
might have interacted with very few free amine
groups on the chitosan segments at the neutral pH
[35] and consequently were released only when the
hydrogel matrix was completely eroded or dissolved.
The faster BSA release rate of G55 than that of G45
may be attributable to the material density. With a
denser matrix, G45 hydrogel may entrap BSA in the
matrix for a longer period of time. These BSA release
rates are comparable with previous thermoreversible
hydrogels made of chitosan/glycerol phosphate [34].
In an attempt to extend the duration of protein
release from chitosan/PEG hydrogels for applications
that require prolonged drug release, an approach was
sought to fine-tune the drug release rate while
preserving the injectability of the hydrogel. A number
of methods have been studied to decrease the
diffusivity of loaded-drugs in matrix materials by,
for example, decreasing the hydrophilicity [36] or
diffusivity [37] in the hydrogel structure, or covalently
linking a protein to the hydrogel matrix [38]. Each of
these methods has been applied to the modification of
chitosan for drug delivery applications [17,39]. In a
previous report on a chitosan-based thermoreversible
hydrogel, a 24 h drug release time was extended to a
few weeks by incorporating liposomes in the hydrogel
[40]. The approach presented here to reducing gel
material dissolution and drug release at body temper-
ature is by crosslinking the hydrogel with genipin in
situ to render the hydrogel insoluble in water. The use
of genipin is an alternative to the several common
crosslinking agents developed for crosslinking various
biopolymers having primary amine groups [27,41].
When the PEG-g-chitosan thermoreversible hydro-
led Release 103 (2005) 609624 621gel was treated with genipin it maintained injectability
for at least 24 h at 4 8C, but the PEG-g-chitosan/
pH, including physiological pH (7.4) at which
bioactive species can be safely and uniformly
ontrolgenipin hydrogel effectively decreased the BSA
release rate as compared to the unlinked PEG-g-
chitosan hydrogel (Fig. 9). The mechanism and
kinetics of the crosslinking reaction between biopol-
ymer containing primary amine groups and genipin
has been recently studied [27,41,42]. In the present
PEG-g-chitosan thermoreversible hydrogel, not all the
amine groups of chitosan reacted with PEG molecules
during the grafting process as illustrated above, and
the remaining amine groups reacted with genipin to
form an insoluble network (Fig. 6). Upon reaction
with genipin, the transparent thermo reversible hydro-
gel turned into a thermo irreversible blue-colored
hydrogel (Fig. 14 D, E, F). BSA release profiles for
hydrogels treated with genipin for 10 min and 24 h,
respectively, were compared in Fig. 9. It was observed
that increasing genipin treatment time had little effect
on the release profile. This may be due to the highly
porous structure of the graft polymer where the matrix
material can effectively react with the crosslinking
agent in a relatively short time period due to a large
overall surface area, and consequently, prolonged
exposure will not make a substantial difference due
to increased crosslinking. The structures shown in Fig.
11C and D were formed after the hydrogels treated
with genipin were immersed in the release media for
24 h and 2 weeks, respectively. As expected, the BSA/
PEG-g-chitosan/genipin hydrogel extended the dura-
tion of protein release in a dose-dependent fashion
(Fig. 10).
The results in Figs. 12 and 13 suggest that
majority of BSA released from both noncrosslinked
and crosslinked hydrogels was in its monomer form
suggesting that the integrity of the protein released
from the hydrogels was largely retained. However,
the hydrogel may have selectively released the
lower molecular weight monomer while retaining
larger aggregates in its matrices. A similar notion
has been suggested for PLGA microspheres and
other hydrogels [43]. The SDS-PAGE of the
untreated BSA had major smearing as compared
with the genipin-treated BSA. This might be due to
the presence of more free chitosan fragments
present in the release medium for the untreated
hydrogel than for the genipin-treated hydrogel
where all the chitosan chains were tightly bound
N. Bhattarai et al. / Journal of C622by the crosslinking. When the BSA was released
from the hydrogel under gel electrophoresis, freeincorporated; (2) the produced hydrogel has a gelation
temperature well below, and thus gels readily at, body
temperature, making it ideally suited to serve as an
injectable depot for sustained drug delivery; (3) the
hydrogel has favorable drug release profiles: after an
initial short burst release, virtually linear release
profiles can be obtained for all the protein loadings
studied so far. A short-term (several days) protein
release investigation for a non-crosslinked PEG-g-
chitosan system and a quasi-linear long-term (several
weeks) drug release study for a crosslinked PEG-g-
chitosan showed positive results. Although present
research is targeted at controlled drug release, the
hydrogels developed also find application in tissue
engineering, such as tissue repair and regeneration.
Since all the component materials involved have been
proven to be tissue-compatible, the copolymer hydro-chitosan fragments may have migrated and been
subsequently stained with Coomassie Brilliant blue.
Large chitosan fragments would be retarded by gel
electrophoresis while small fragments would
migrate to the bottom of the acryl amide gel [44].
The data presented here does not preclude the
possibility that the BSA could be bound to the
hydrogel matrix via reaction with the crosslinking
reagents [41] and the reaction between amino acids
in the protein and amine groups in the chitosan.
The BSA could have also non-specifically formed
the complexes with PEG segments [45]. Further
investigation of the BSA structure within the
hydrogel may help to clarify this issue, which is
beyond the scope of this study in view of the major
concern being the functionality of the released
proteins in therapeutics.
5. Conclusions
An injectable, thermoreversible hydrogel was
fabricated by chemically grafting monohydroxy PEG
on chitosan chains. The approach presented in this
study has demonstrated the following favorable
attributes: (1) it provides the flexibility to easily
prepare the hydrogel in solutions of a wider range of
led Release 103 (2005) 609624gel is potentially suited for a wide range of in vivo
biomedical applications.
[11] S. Arnott, A. Fulmer, W.E. Scott, I.C.M. Dea, R. Moorhouse,
N. Bhattarai et al. / Journal of Controlled Release 103 (2005) 609624 623D.A. Rees, Agarose double helix and its function in agarose-
gel structure, J. Mol. Biol. 90 (1974) 269.
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polysaccharides in solutions and gels, Angew Chem., Int. Ed.
16 (1977) 214223.
[13] A. Shedden, J. Laurence, R. Tipping, T.X.S. Grp, Efficacy and
tolerability of timolol maleate ophthalmic gel-forming solution
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[14] G. Franz, Polysaccharides in pharmacy, Adv. Polym. Sci. 76Acknowledgements
The authors would like to acknowledge the
funding support from the University of Washington
Engineered Biomaterials (UWEB) Center funded by
National Science Foundation (NSF-EEC 9529161).
The authors would also like to acknowledge the lab
assistance from Shirley Chou and Haiyan Zhang.
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PEG-grafted chitosan as an injectable thermosensitive hydrogel for sustained protein releaseIntroductionMaterials and methodsMaterialsSynthesis of PEG-g-chitosanCharacterization of PEG-g-chitosan copolymerHydrogel preparation and gelation studyViscosity measurementsIn vitro protein release studyMicroscopy analysisAnalysis of released proteins by high performance liquid chromatography (HPLC) and gel electrophoresis (SDS-PAGE)
ResultsSynthesis of PEG-g-chitosanThermoreversible gelation behaviorBSA release from hydrogelsGenipin-treated PEG-g-chitosan hydrogelBSA release from PEG-g-chitosan hydrogels crosslinked with genipinMorphology of freeze-dried hydrogels by SEMBSA structural integrity
DiscussionConclusionsAcknowledgementsReferences