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: mzhang@u.washington.edu (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.

[12] D.A. Rees, E.J. Welsh, Secondary and tertiary structure of

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

versus timolol ophthalmic solution in adults with open-angle

glaucoma or ocular hypertension: a six-month, double-

masked, multicenter study, Clin. Ther. 23 (2001) 440450.

[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