Characterization of ph- and thermosensitive hydrogel as a vehicle for controlled protein delivery

Characterization of pH- and Thermosensitive Hydrogelas a Vehicle for Controlled Protein Delivery

WENPING SHI,1 YANWEN JI,1 XINGE ZHANG,2 SHUJUN SHU,2 ZHONGMING WU3

1Tianjin Institute of Sexually Transmitted Diseases, General Hospital, Tianjin Medical University, Tianjin 300052, China

2Key Laboratory of Functional Polymer Materials Ministry of Education, Institute of Polymer Chemistry, Nankai University,Tianjin 300071, China

3Key Laboratory of Hormones and Development Ministry of Health, Metabolic Diseases, Hospital, Tianjin Medical University,Tianjin 300070, China

Received 20 December 2009; revised 21 June 2010; accepted 30 June 2010

Published online 22 September 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22328

ABSTRACT: N-[(2-Hydroxy-3-trimethylammonium)propyl] chitosan chloride (HTCC) waschemically modified using glycidyltrimethylammonium chloride (GTMAC). A new compositehydrogel was prepared using the mixture of HTCC and "-$-glycerophosphate ("-$-GP). Thegelation of HTCC/GP mainly depended on the concentration and proportion of HTCC and GP.Thermogravimetric analysis exhibited high stability of HTCC/GP hydrogels. Surface morphol-ogy assay demonstrated that HTCC/GP hydrogels were well constructed with three-dimensional(3D) porous structures in the range of 5 of 40 :m. The insulin was entrapped during the for-mation of hydrogel. In vitro, the insulin release was controlled by modifying the composition,drug loading, and pH condition. The hydrogel dissolved and released drug quickly under acidiccondition, whereas it absorbed water and released drug slowly under neutral or basic condi-tions. The hydrogels were biocompatible, and the cells could adhere to and then migrated tothe hydrogels. Furthermore, these cells were viable and retained 3D morphology inside thehydrogels. Interestingly, HTCC/GP hydrogel showed both thermo- and pH-sensitive proper-ties. There are potential applications in tissue engineering, cell encapsulation, and intelligentdrug delivery systems. © 2010 Wiley-Liss, Inc. and the American Pharmacists AssociationJ Pharm Sci 100:886–895, 2011Keywords: hydrogels; polyelectrolytes; controlled release; biocompatibility; proteins

INTRODUCTION

Hydrogels based on both natural and synthetic poly-mers are widely used in tissue engineering anddrug-controlled release system.1–3 The most com-monly studied hydrogels having environmental sen-sitivity are responsive to either pH or temperature.4–6

Among biopolymers of interest, chitosan (CS), a natu-ral polysaccharide of $-(1,4)-linked 2-amino-2-deoxy-D-glucopyranose, is a nontoxic and biodegradablebiopolymer that has been widely used as a support-ing material for tissue engineering applications, con-trolled drug delivery, and cell culture.7–12 However,the low solubility is the main drawback of CS. It can

Correspondence to: Yanwen Ji (Telephone: +86-22-60363193;Fax: +86-22-60363193; E-mail: [email protected]); ZhongmingWu (E-mail: [email protected])Journal of Pharmaceutical Sciences, Vol. 100, 886–895 (2011)© 2010 Wiley-Liss, Inc. and the American Pharmacists Association

be dissolved only in dilute inorganic and organic acidsolutions when pH is lower than 6.5,13 which limit itsdirect applications in many fields.

The poor water solubility of CS can be improvedby attaching a quaternary ammonium moiety to CS.Usually, quaternized CS was prepared by reacting CSwith quaternized reagents in strong basic environ-ment at high temperature. The quaternized reagentsused were mainly methyl iodide and glycidyltrimethy-lammonium chloride (GTMAC),14,15 accordingly, N-[(2-hydroxy-3-trimethylammonium)propyl] chitosanchloride (HTCC) is obtained by reacting CS with GT-MAC in the present work. HTCC shows better watersolubility, moisture retentiveness, antimicrobial ac-tivity, absorptive property, and cell proliferative ca-pacity than CS.16,17

HTCC solution was neutralized with a polyol coun-terion dibasic salt such as "-$-GP, and the neutral-ized solution remained liquid for long periods of time

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PH- AND THERMOSENSITIVE HYDROGELS FOR CONTROLLED PROTEIN DELIVERY 887

at room temperature and turned into gels as temper-ature increased.18 Because of the stronger cationicproperty of quaternary amino groups, as comparedwith amino groups, the hydrogel prepared by quater-nized CS showed more distinct pH sensitivity. There-fore, the current work evaluated the thermo- and pH-sensitive hydrogels formed by HTCC and "-$-GP foruse in protein drug delivery system. GP, an organiccompound that is naturally found in the body, is al-ready approved by the Food and Drug Administration.It is used as a source of phosphate in the treatmentof imbalance in calcium and phosphate metabolism.The main purpose of this study was to characterizein vitro release kinetics of model protein insulin en-capsulated in the hydrogels and evaluate their poten-tial use as a scaffold for cell culture.

EXPERIMENTAL

Materials

Chitosan (MW = 300 kDa), with a degree of deacety-lation of 95%, was purchased from Zhejiang YuhuanOcean Biochemical (Zhejiang, China). The mixtureof "-GP and $-GP ("-$-GP) was provided by WuhanXinhuayuan Science and Technology Co. Ltd. (Hubei,China). GTMAC was obtained from the laboratory.Pure crystalline porcine insulin (nominal activity =28 IU/mg), used without further purification, was ob-tained from Xuzhou Wanbang Biochemical Co. Ltd.(Jiangsu, China). All other reagents were of analyticgrade.

Analytical Instrumentation

FT-IR spectra were obtained using a Bio-Rad FTS-6000 FT-IR single-beam spectrometer (Cambridge,MA, USA) set at a 4 cm−1 resolution. UV–vis spectraldata were recorded using a UV spectrometer (Shi-madzu UV-2550 (Kyoto, Japan). Proton NMR spec-tral data were obtained at 400 MHz, using a Var-ian INOVA 400 NMR (Salt Lake City, Utach, USA).To examine thermal stability of hydrogels, the hy-drogel samples were analyzed by thermogravimet-ric analysis (TGA, NETZSCH TG 209, NETZSCH-Geratebau GmbH, Selb, Germany). Decompositionprofiles of TGA were recorded at a heating rateof 10◦C /min in nitrogen between 20◦C and 650◦C.The freeze-dried hydrogels were fractured carefully,and their interior morphologies were visualized us-ing a scanning electron microscope (SEM, SS-550;Shimadzu, Kyoto, Japan). Before the SEM obser-vation, the hydrogel samples were fixed on alu-minum stubs and coated with gold. Cell proliferationwas determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 492 nm,which was recorded on an OPTI Max MicroplateReader (Molecular Devices, Sunnyvale, California).

Synthesis of Quaternized Chitosan (HTCC)

HTCC was prepared following the previous report.18

Briefly, CS (1.0 g, 6.2 mmol) was dispersed indeionized water (20.0 mL) at 80◦C. GTMAC (3.8 g,24.8 mmol) was dissolved in aqueous solution andadded to the CS suspension. The molar ratio ofGTMAC to amino group of CS was 4:1. After the mix-ture was continuously stirred for 24 h at 80◦C, theturbid and yellowish reaction solution was collectedby filtration. To obtain purer HTCC, the product waspurified by dialysis for 2 to 3 days.

Preparation of HTCC/GP Hydrogel

HTCC (36.0 mg) was dissolved in 0.1 M of lactic acidsolution (0.5 mL) and "-$-GP (100.0 mg) was added todeionized water (0.5 mL) at room temperature. Afterboth of the solutions were cooled at 4◦C for 15 min,the GP solution was added dropwise with a syringepump to HTCC solution in an ice bath under stirring,and the obtained solution was stirred for 10 min. Thehydrogel was formed by heating HTCC/GP solutionin a water bath at 37◦C for several minutes. Simi-larly, CS/GP hydrogel was prepared according to themethod mentioned earlier.

Physical Characterization of Hydrogel

Swelling

The swelling behavior was determined by immers-ing the dried hydrogel in 5 mL of buffer solutionswith desired pH value at room temperature. At pre-determined time intervals, the samples were takenout and blotted carefully with filter paper to re-move the surface-adhered liquid droplets and thenweighed on an electronic microbalance (AE 240,Mettler, Greifensee, Switzerland) to an accuracy of±0.01 mg. The percentage of equilibrium water up-take was calculated as follows:

Water, uptake, (%) = Ws−W0

W0× 100%

where Ws is the weight of hydrogel at various swellingtimes and W0 is the initial weight of hydrogel.

Rheological Evaluation

The rheological properties were examined by StressTech rheometer (Reologica Instruments AB, Lund,Sweden) with the standard steel parallel-plate geom-etry of 25-mm diameter. The test methods employedwere oscillatory stress sweep and frequency sweep.The stress sweep was performed on the hydrogel todetermine the linear viscoelastic region profiles, thestorage modulus (G′), and the loss modulus (G′′) un-der the same physical condition. The test tempera-ture was increased from 25◦C to 60◦C, with a fre-quency sweep at a fixed shear stress (10 Pa) and the

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011

888 SHI ET AL.

oscillatory frequency (1 Hz). The stress sweep was setup by holding the temperature (25◦C) and frequency(1 Hz) constant while increasing the stress level from0.1 to 100 Pa. The hydrogels were also subjected toa frequency sweep at a fixed shear stress (10 Pa)and temperature (25◦C), the oscillatory frequency wasincreased from 0.01 to 20 Hz, and G′ and G′′ wererecorded. The plots of G′ and G′′ versus shear stressor frequency or temperature from the two sweep testswere obtained directly from the software controllingthe rheometer.

Incorporation of Insulin

The insulin was dissolved in 0.1 M of lactic acid so-lution at three final concentrations (100, 200, and300 :g/mL). HTCC and GP were successively addedto the above-mentioned solution under stirring andthen incubated at 37◦C for 2 h to form hydrogels.The samples were freeze-dried and stored at 4◦Cbefore use.

In Vitro Release Studies

The insulin release was determined by incubationinsulin-loaded hydrogels at 37 ± 0.1◦C in 2 mLof phosphate-buffered saline (PBS) with horizontalshaking. At predetermined time points, 100 :L of thissolution was taken out and replenished by the freshbuffer solutions. The amount of free insulin was ana-lyzed by the Bradford method and a calibration curvewas obtained using nonloaded hydrogel to correct forthe intrinsic absorption of the polymer.

Analysis of Release Profile

To know the drug release mechanism and comparethe release profiles among hydrogels, the drug re-leased amount versus time was used. The release datawere analyzed with the following Korsmeyer–Peppasmodel:

Mt/M∞ = ktn

where Mt /M∞ is the fractional amount of the drugreleased at time t, n is a diffusion exponent indicat-ing the release mechanism, and k is a characteris-tic constant of the system. Kinetic parameters n andk are obtained by fitting the release data to theabove-mentioned equation.19 For a hydrogel, whenn = 0.5, the drug release mechanism is Fickian dif-fusion. When n = 1, case II transport occurs, leadingto zero-order release. When the value of n is between0.5 and 1, anomalous transport is observed.

Silver Stain of Encapsulated Protein

A NuPAGE Novex 20% Bis-Tris gel was employed.Each protein sample (7.5 :L) was treated with Nu-PAGE 4 × LDS loading buffer (2.5 :L) and heated at

99◦C for 5 min to denature the protein. Each sample(5 :L) was loaded onto the gel, which was then run at100 V for 1 h. The solution was changed three timesto remove the remaining detergent ions as well asfixation acid from the gel. The cold silver staining so-lution was then added to the gel, which was immersedfor 20 min to allow the silver ions to bind to proteins.The silver stain solution was poured out, and the gelwas washed quickly with deionized water. The gel wasimmersed in an ice-cold developer solution (200 mL)until optimal image intensity was obtained. The de-veloping process was stopped by immersing the gel in7.5% ice-cold glacial acetic acid.

Cytotoxicity and Cell Viability Analysis

The cytotoxicity of HTCC/GP and CS/GP hydro-gels was measured in 96-well plate by the MTTmethod. The viability of NIH3T3 cells cultured in amedium where the hydrogel was previously immersedwas evaluated. In particular, the hydrogel was in-cubated in RPMI-1640 medium at 37◦C for 5 daysunder orbital stirring at 120 rpm. After incubation,the medium ‘‘conditioned’’ by the hydrogel was cen-trifuged at 16,000 g 4◦C for 30 min, and then filteredto remove the hydrogel. The cells were seed in 96-well plates with a seeding density of 1 × 104 cells/well in a medium volume of 100 :L, and the plateswere returned to the incubator (5% CO2) at 37◦C for24 h. The culture medium was then replaced with the“conditioned” medium. Cell viability was determinedon second and fourth days, respectively. After 48 and96 h of incubation, 20 :L of MTT (5 mg/mL in PBS)was added to the wells and the plates were then re-turned to the incubator for further 4-h incubation.After this incubation period, the medium and MTTwere discarded and then 150 :L of dimethylsulfox-ide was added to each well to dissolve the formazancrystals. The reaction plate was stirred at 800 rpm for1 min and the optical density was read on a microplatereader at 492 nm. Cell viability was determined as apercentage of the positive control (untreated cells).

Morphological Observation of Cells

For morphological observation, NIH3T3 cells werecultivated with polymer films at 37◦C for 3 days. Af-ter being rinsed with PBS twice, they were fixed withglutaraldehyde solution (2.5%, w/v) in 0.2 M of phos-phate buffer (pH 7.4) at 4◦C for 2 h, stained with1% osmium tetroxide for 12 h, and then washed withPBS and gradually dehydrated by treatment withethanol solution (from 25% to 90%, v/v). These spec-imens were freeze-dried for 6 h after replacement ofethanol with t-butyl alcohol and then gold-coated us-ing a sputter coater. Their surface was examined bySEM (SS-550; Shimadzu).

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011 DOI 10.1002/jps


Table 1. The Preparation of HTCC/GP Hydrogel

SampleHTCC(wt %)a

"-$-GP Concentration(wt %)a

Gelation Time(min)

HTCC/GP1 6.7 16.7 Fast b

HTCC/GP2 6.7 10 5HTCC/GP3 4 16.7 22CS/GP 4 10 28

aThe concentration is the initial concentration.bFast means about 1 min.

RESULTS AND DISCUSSION

Preparation and Characterization of HTCC

HTCC was prepared by reacting CS with GTMACin water. GTMAC readily reacts with the compound-containing amino groups to give quaternary aminogroups. The structure of HTCC was confirmed by 1HNMR spectrum in deionized water. The chemical shiftat 3.2 ppm is for the methyl group on the quaternarynitrogen, which does not appear in the spectrum ofCS. As can be seen from FT-IR spectrum of HTCC(Fig. 1), the peak appears at 1600 cm−1, representingweakened NH2 deformation and the appearance of anew peak at 1482 cm−1, which can be attributed tothe methyl groups of the ammonium group. The C–Ostretching vibration between 1068 and 1027 cm−1

does not change in HTCC compared with CS, indi-cating the introduction of quaternary amino groupsinto the CS chains.20

Preparation and Characterization of HTCC/GP

The mixture of HTCC and CS with "-$-GP was ini-tially an aqueous solution and gradually turned intoa hydrogel as the temperature increased to 37◦C. Butthe hydrogel did not revert to initial liquid state whenthe temperature decreased. The composition of theHTCC/GP hydrogel is shown in Table 1.

Figure 1 shows the FT-IR spectra of GP, HTCC,and HTCC/GP hydrogel. The spectrum of HTCC/GP

Figure 1. FT-IR spectra of GP, HTCC and HTCC/GP.

hydrogel is different from that of HTCC matrix. Theintensity of 1482 cm−1 peak of methyl groups of qua-ternary hydrogen is weaker than that of HTCC, indi-cating that the phosphate groups of GP are associatedwith trimethylammonium groups of HTCC throughelectrostatic interactions to form the polyelectrolytecomplexes.

To examine the microstructure of HTCC/GP hy-drogel, it was incubated in PBS at 37◦C andthen instantaneously plunged into liquid nitrogen,freeze-fractured, and examined by SEM. In general,hydrogel exhibits porous microstructures with differ-ent chamber diameters and distributions. The av-erage chamber diameter ranges from 5 to 40 :m.The larger chamber diameters qualitatively correlatewith swelling ratio of the hydrogels. The pore sizedecreases with an increase in cross-linker concentra-tion, which is caused by the decreased distance be-tween polymer chains. Compared with CS/GP hydro-gel, HTCC/GP hydrogel shows more porous structure(see Fig. 2). This is probably due to the decreased crys-tallinity and increased uniformity of HTCC as com-pared with CS. With the decline of the HTCC, thesurface structure of the HTCC/"-$-GP becomes moreloose, which allows small molecules and cells to movein the network easily. The porous structures of thehydrogels suggest their potential as scaffolds for cellinfiltration and growth.

Thermogravimetric Analysis

Thermogravimetric (TG-DTG) curves for GP, HTCC,and HTCC/GP hydrogels are shown in Figure 3. ForHTCC, weight loss takes place in two stages. The firstone with a weight loss of 5% is assigned to free wa-ter and water linked through hydrogen bonds andreaches a maximum at 87◦C; The second stage ofweight loss of 40%, up to 273◦C, corresponds to thedecomposition (thermal and oxidative) of CS and va-porization and elimination of volatile products. It isknown that pyrolysis of polysaccharides starts by arandom split of the glycosidic bonds, followed by fur-ther decomposition to produce acetic and butyric acidsand a series of lower fatty acids, where C2, C3, andC6 predominate.21 Variations in the peak area andposition related to water loss are expected to reflectphysical and molecular changes caused by the modifi-cation of the polysacchardies.22 HTCC/GP investiga-tion of Figure 3 reveals that there ware differences inpeak area and position, indicating that there are dif-ferences in the water-holding capacity and strength ofwater–polymer interaction for HTCC/GP hydrogels.The second stage of degradation displayed in Figure 3shows peaks with maxima at 280◦C for HTCC/GP,indicating an increase in hydorgel thermal stabilitycompared with HTCC and the interaction betweenthe two polymers.


890 SHI ET AL.

Figure 2. SEM micrographs of HTCC/GP and CS/GP hy-drogels.

Rheology of the HTCC/GP Hydrogel

The modulus of an HTCC/GP network displayed thesame overall relaxation profile at all tested deforma-tions. The modulus of HTCC/GP1, HTCC/GP2, andHTCC/GP3 remained independent of strain ampli-tude for strains up to 15, 6, and 2 Pa, respectively,which defined the extent of the linear regime (seeFig. 4a). However, the modulus decayed slowly with

Figure 3. TG (A) and DTG (B) curves for GP, HTCC andHTCC/GP hydrogel.

increasing strain, indicating that the bending rigid-ity of HTCC/GP was responsible for this strain-hardening phenomenon. The characteristic “yield”strain was defined here as the strain at which themodulus started decreasing (onset of shear soften-ing). Therefore, HTCC/GP resisted further deforma-tion, which gave rise to strain hardening. For largeshear deformations, the HTCC/GP network eventu-ally broke and/or flowed, indicated by a drop in G′.According to the previous reports, the measured G′ in-creased with an increase in the intermolecular cross-links in the hydrogel network.23–25 Therefore, the evo-lution of G′ as a function of cross-linking density wasmonitored to assess the extent of effective intermolec-ular cross-links formed in the hydrogel networks. Ob-viously, HTCC/GP1 hydrogel had a higher G′ thanHTCC/GP2 hydrogel, indicating that increasing thecontent of GP in HTCC/GP hydrogel with the sameconcentration of HTCC enhanced the cross-linkingdensity.

Frequency sweep tests are widely used to ob-tain information about the stability of three-dimensional cross-linked networks.23,26,27 Figure 4bshows the corresponding frequency spectra of G′ for



Figure 4. Variations with stress (A), frequency (B) andtemperature (C) of the storage modulus G0’ of HTCC/GPhydrogels: HTCC/GP1 (filled square), HTCC/GP2 (filled tri-angle) and HTCC/GP3 (filled circle).

HTCC/GP gels. Both systems showed typical gel spec-tra, with G′ showing only very little frequencydependence, which could be asserted that the storagemodulus would have a finite value even at low fre-quencies (i.e., it showed solid-like properties). How-ever, at higher frequencies, all the hydrogels showeda slow decrease in G′ in response to decreased rigidityof the network strands. Moreover, the results showed

that the variation in chemical composition betweenthe samples significantly affected G′ values.

Admixing GP to HTCC aqueous solution, as an ex-ample of the new system, increased the pH of thesolution as a result of the neutralizing effect of thephosphate groups. In the presence of this salt, how-ever, HTCC solutions remained liquid below roomtemperature, even with pH values within a physiolog-ically acceptable neutral range from 6.8 to 7.2. Thesenearly neutral HTCC/GP aqueous solutions would gelquickly when heated. Figure 4c showed the rheolog-ical behavior of HTCC/GP neutral solution duringheating between 25◦C and 65◦C. The sharp rise inthe elastic modulus upon heating, curves of HTCC/GP1 and HTCC/GP3, clearly indicated that the liquidsolution turned into a solid-like gel in the vicinity of37◦C. For HTCC/GP2 hydrogel, however, the elasticmodulus showed an increase in the vicinity of 35◦C.Such a tendency toward complete thermoreversibilitybecame pronounced for HTCC/GP solutions.

The results showed that the temperature of incip-ient gelation increased as the content of HTCC de-creased, while the proportion of GP showed the effecton the temperature of gelation.

Swelling Analysis

The swelling behaviors of HTCC/GP hydrogels areshown in Figure 5. The swelling ratio of differ-ent proportions of HTCC/GP hydrogels after im-mersed in the same pH buffer for 24 h is plotted inFigure 5a. It is clearly indicated that the swellingratio increased with a decrease in HTCC. As can beseen from Figure 5b, the water uptake of the gelsreached the maximal value within approximately 3 hunder these conditions. However, HTCC/GP hydrogeldissolved rapidly in acidic solution (data not shown),which was attributed to the quaternized CS chainsbearing positive charges. The swelled network al-lowed more acid solution to enter the interior. As a re-sult, the protonated quaternized CS chains dissolvedquickly. It is shown that HTCC/GP hydrogel has bet-ter pH sensitivity. Moreover, the hydrogels still main-tain the integrity after longer exposure time (after 2weeks) but their weight decreased, which was due tothe fact that HTCC was partially degraded and someof GP dissolved.

In Vitro Release of Protein

Insulin release from HTCC/GP hydrogels is displayedin Figure 6. The initial rate of release of proteinis rapid and then slows down after several hours.This indicate that proteins on the surface of hydro-gels diffuse rapidly from the initial swelling of thegel. Later, protein is released slowly from HTCC/GPhydrogels. The remaining protein in hydrogels can-not be released until HTCC/GP hydrogels are com-pletely degraded or dissolved in release medium. This


892 SHI ET AL.

Figure 5. Swelling behavior of HTCC/GP hydrogel: (A)different HTCC/GP hydrogels; (B) HTCC/GP1 in differentpH conditions at 37 ◦C (mean ± SD, n = 3).

is mostly attributed to the presence of protein–gelinteractions, which might be based on electrostaticinteractions and/or hydrogen bonding.

A series of HTCC/GP hydrogels studied is shown inFigure 6a. The amount of released insulin decreaseswith increasing content of HTCC or GP in the hy-drogels. This is most likely because the cross-linkingdensity of HTCC/GP hydrogel that increases as HTCCor GP is increased. As a result, the release rate of in-sulin becomes slow.

Figure 6b illustrates the in vitro release behavior ofHTCC/GP1 hydrogel with different insulin amounts.The results show that the higher the drug loading, theless release of HTCC/GP1 hydrogel. Approximately42.9% of the drug was released with 100 :g/mL con-centration of insulin during 72 h. When the trappedinsulin concentration increased to 200 and 300 :g/mL, only 26.0% and 22.7%, respectively, were detectedin the supernatant during 72 h. Because of the pres-ence of amino groups and carboxyl groups on insulinchains, it can form hydrogen bonds or establish elec-trostatic interactions with GP or HTCC, which maystrengthen cross-linked network and slow down the

Figure 6. In vitro percentage release of insulin fromHTCC/GP hydrogels: (A) different HTCC/GP hydrogels con-taining the same insulin content (100 :g/ml); (B) HTCC/GP1 hydrogel loading various amount insulin; (C) differentpH conditions. Triplicates for each hydrogel were analyzedand each datum point represents the mean value ± SD(n = 3).

drug release. Therefore, the drug release slowed downwith the increased drug loading, which is very similarto the results reported by Wu et al. for an HTCC/PEG/GP hydrogel system.28



Table 2. Drug Release Kinetic Data for HTCC/GP Hydrogels Obtained from Fitting Drug Release Experimental Data to theKorsmeyer–Peppas Equation

Korsmeyer–Peppas Model

SampleInsulin Content

(:g/mL) pHDiffusion

Exponent, nKinetic

Constant, kCorrelation

Coefficient, R2 Transport Mechanism

100 7.4 0.1947 12.2011 0.8769 Fickian diffusion5.7 0.254 24.2172 0.83296.5 0.2272 18.9744 0.8076

HTCC/GP1 200 7.0 0.2212 11.5981 0.9144 Fickian diffusion7.4 0.1951 12.0066 0.88578.0 0.1550 6.4196 0.8943

300 7.4 0.3135 12.2504 0.9075 Fickian diffusionHTCC/GP2 200 7.4 1.5988 0.5617 0.9947 Fickian diffusion and polymer chain relaxationHTCC/GP3 200 7.4 1.3557 0.7733 0.9841 Fickian diffusion and polymer chain relaxation

Owing to pH sensitivity of HTCC/GP hydrogel,insulin release properties were evaluated underdifferent pH conditions. As shown in Figure 6c, at pH5.7, drug was released rapidly from the hydrogel andalmost 65% of insulin was released in 3 days. In basicor neutral condition, however, the drug was releasedslowly. At pH 7.4, about 26% of insulin was released.When pH value was raised to 8, much slower releasewas observed and only 12% insulin was released. Theinsulin release behaviors corresponded well with thepH sensitivity of the hydrogel.

In this study, Korsmeyer–Peppas model Mt/M∞ =ktn was used to analyze the release data, including theinitial burst region as shown in Figures 6a–c. The ex-perimental data had a fairly good fit to the Korsmey-er–Peppas equation for insulin release from initialburst stage up to 60% release. The diffusional expo-nent (n), correlation coefficient (R2), and release ratecoefficient (k) obtained ae summarized in Table 2. Agood correlation coefficient (R2) approaching 0.90 wasobtained in all cases. It was shown that the exponentn values for the release of insulin from HTCC/GP1were slightly less than 0.5, suggesting a Fickian re-lease behavior. However, the exponent n values forthe release of insulin from HTCC/GP2 and HTCC/GP3 were more than 0.5, indicating Fickian releasebehavior and polymer chain relaxation.

Integrity of the Encapsulated Proteins

In terms of protein delivery system for the clinicalapplications, silver stain of two-dimensional gel elec-trophoresis is adopted to study the stability of pro-teins, which is a combination of high-resolution elec-trophoresis/isoelectric focusing electrophoresis andpolyacrylamide gel electrophoresis. The analysis ofreleased insulin revealed that neither protein frag-mentation nor covalent dimerization occurred duringour encapsulation process (Fig. 7). The results sug-gested that HTCC/GP hydrogel could maintain theintegrity of protein.

Figure 7. SDS-PAGE of insulin (Mw 5700) released fromhydrogel HTCC/GP2 in pH 7.4 PBS. Lane1: insulin stan-dard. Lanes 2, 3, 4 and 5: insulin released from hydrogeldeposited after 10, 20, 40 and 72 h, respectively.

Cytotoxicity Assay

A series of experiments was performed to evaluateHTCC/GP hydrogels potential toxicity. The MTT re-sults obtained with NIH3T3 cells are presented inFigure 8. As shown in Figure 8a, HTCC/GP hydro-gel present less cytotoxicity than hydrogel CS/GP. Adose–response analysis was carried out by exposingNIH3T3 cells to various concentrations of hydrogel for4 days (Fig. 8b). No cytotoxicity (>80%) was obtainedwith an increase in the concentration of HTCC/GP(from 1 to 10 mg/mL). Also, time-course experimentsconfirmed a slight increase in cell viability from 48to 96 h. The results indicated that cells proliferatedon hydrogels after 4 days of culture and the cell num-ber still increased on the fourth day. This shows thatHTCC/GP hydrogel had no apparent cytotoxicity. Theunique properties suggest that hydrogel HTCC/GP,especially HTCC/GP2, is a potential candidate for awide range of biomedical applications in wound heal-ing and tissue repair due to its biocompatibility.

Adhesion and Morphology of NIH3T3 Cells

The adhesion behavior of NIH3T3 cells, mainlymacrophages on hydrogels, was investigated by SEM


894 SHI ET AL.

Figure 8. Cytotoxicity of HTCC/GP and CS/GP hydrogelsquantified via MTT-assay: (A) 2 days; (B) 4 days. The con-centration is of HTCC/GP and CS/GP hydrogels incubatedin RPMI-1640 medium. Each datum point represents themean value ± SD (n = 6).

as shown in Figure 9. The distribution of NIH3T3 cellsencapsulated by HTCC/GP media was assessed after3 days. Each micrograph reveals that a few cells onthe surface of cleavages of HTCC/GP hydrogel main-tain their polygonal shape despite the fact that dehy-dration and drying steps of sample preparation maylead to morphological changes in hydrogels and celldropout. Polygonal cells bearing a multitude of mi-crospikes cover the surface of HTCC/GP. Some of themprotrude forming pseudopodia whose tips seem to con-tact the surface of the material as shown in Figure 9.The remaining microspikes are flattened on the sur-face, suggesting the a strong force of attachment ontothe hydrogel and maintaining the expansion of themechanically stronger scaffolds.

CONCLUSION

The new composite hydrogels were prepared by theionic interaction between HTCC and "-$-GP. Their

Figure 9. SEM photomicrographs of adherent cells on hy-drogel surfaces. Cell line was cultured on the hydrogel net-works for 3 days and the hydrogel was not swollen in PBSprior to cell loading.

mechanical and swelling properties were readily con-trolled by pH and the content of HTCC and GP.The gel could easily incorporate drug in the solutionstate, which was stable below or at room temperatureand became transparent at 37◦C. In vitro, the releaseproperty depended on loading efficiency, pH value,and the composition of HTCC/GP hydrogel. Moreover,drug bioactivity was not affected during the formationof hydrogel. HTCC/GP hydrogels possessed good bio-compatibility, and the cells could adhere and migrateinside the hydrogel networks. The thermo- and pH-sensitive HTCC/GP hydrogel can be well applied tocell encapsulation and protein delivery.

ACKNOWLEDGMENTS

Financial support from the PhD Programs Foun-dation for New Teachers of Ministry of Educa-tion of China (grant no. 20091202120005), TianjinHealth Bureau Scientific Research Found (grantno. 07kz047), and the Science and Technology Devel-opment Foundation of Tianjin Municipal EducationCommission of China (grant no. 20070219) are grate-fully acknowledged.

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Characterization of ph- and thermosensitive hydrogel as a vehicle for controlled protein delivery