biodegradable thermoresponsive hydrogels for aqueous encapsulation and controlled release of...

Biodegradable Thermoresponsive Hydrogels for AqueousEncapsulation and Controlled Release of

Hydrophilic Model Drugs

Xiao Huang† and Tao Lu Lowe*,†,‡,§

Departments of Surgery, Bioengineering, and Materials Science and Engineering, Pennsylvania StateUniversity, 500 University Drive, Hershey, Pennsylvania 17033

Received February 14, 2005; Revised Manuscript Received April 15, 2005

A series of hydrogels with both thermoresponsive and completely biodegradable properties was developedfor aqueous encapsulation and controlled release of hydrophilic drugs in response to temperature change.The hydrogels were prepared in phosphate-buffered saline (pH 7.4) through free radical polymerization ofN-isopropylacrylamide (NIPAAm) monomer and a dextran macromer containing multiple hydrolyticallydegradable oligolactate-2-hydroxyethyl methacrylate units (Dex-lactateHEMA). Swelling measurementresults demonstrated that four gels with feeding weight ratios of NIPAAm:Dex-lactateHEMA) 7:2, 6:3,5:4, and 4:5 (w/w) were thermoresponsive by showing a lower critical solution temperature at approximately32 °C. The swelling and degradation of the hydrogels strongly depended on temperature and hydrogelcomposition. An empirical mathematical model was established to describe the fast water absorption at theearly stage and deswelling at the late stage of the hydrogels at 37°C. Two hydrophilic model drugs, methyleneblue and bovine serum albumin, were loaded into the hydrogels during the synthesis process. The molecularsize of the drugs, the hydrophilicity and degradation of the hydrogels, and temperature played importantroles in controlling the drug release.

Introduction

Hydrogels have attracted wide research interest as con-trolled release devices due to their tunable chemical andthree-dimensional physical structure, high water content,good mechanical properties, and biocompatibility.1 Biore-sponsive, “intelligent” or “smart” hydrogels can regulate drugrelease through responding to environmental stimuli byswelling and deswelling. Various bioresponsive hydrogelshave been developed for drug delivery based on thermore-sponsive polymer poly(N-isopropylacrylamide) (PNIPAAm)due to its unique volume phase transition at a lower criticalsolution temperature (LCST) in water around 32°C, whichis close to body temperature.2-13 They swell and collapsesignificantly in an aqueous environment at temperaturesbelow and above the LCST, respectively. Several investiga-tions, including our own, have shown that the phase behaviorand mechanical properties of PNIPAAm hydrogels can bemodified by the addition of more hydrophobic or hydrophilicmonomers for desired drug delivery.3,6,7,9,13-19 PNIPAAm-based polymers may allow aqueous loading of protein drugs,protecting the drug from a hostile environment,20 andmodulate drug release in response to temperature change.21

However, many current thermoresponsive PNIPAAm hy-drogels have problems in nonbiodegradability and nonsus-tained drug release under physiological conditions.22,23

Degradation of the hydrogel matrix not only circumventsremoval of the empty device but also can be used to modulatethe release of encapsulated drugs for a long period of time.24

Hydrogels composed of poly(lactic acid) (PLA)-based anddextran-based polymers have been extensively studied forsustained release of protein drugs in recent years, becausePLA is hydrolytically degradable and hydrophobic,24-27

dextran, a natural polysaccharide, is enzymatically degradableand hydrophilic,24-29 and both polymers are biocompatible.When hydrogels consist of both PLA and dextran, they maycontrol the sustained release of drugs by adjusting the ratiosbetween PLA and dextran and through degradation by bothhydrolysis and dextranase.24-27,30 However, many currentlyavailable biodegradable hydrogels have problems in thetradeoff of aqueous loading of protein drugs (to avoid usingorganic solvents for the loading, which may cause instabilityand denaturation of the protein drugs) and mechanicalstrength of the hydrogels,26,30 and lack of response tophysiological changes as well.

In view of these aspects, a promising strategy for designingnovel hydrogel drug delivery systems is to combine themerits of both bioresponsive and biodegradable hydrogels.In the literature, PNIPAAm-based polymers have beenchemically incorporated with enzymatically biodegradablepoly(amino acid)12 or dextran.3,8,10,31However, the enzymesneeded to decompose these polymers are localized in limitedbiological systems, and hence, the application of such devicesmay sometimes be limited. In our previous work, wedesigned and synthesized a series of hydrogel systemscomposed of PNIPAAm, poly(L-lactic acid), and dextran in

* To whom correspondence should be addressed. Phone: (717) 531-8602. Fax: (717) 531-4464. E-mail: [email protected].

† Department of Surgery.‡ Department of Bioengineering.§ Department of Materials Science and Engineering.

2131Biomacromolecules 2005,6, 2131-2139

10.1021/bm050116t CCC: $30.25 © 2005 American Chemical SocietyPublished on Web 05/21/2005

dimethylformamide (DMF), and the hydrogels were ther-moresponsive and partially hydrolytically degradable.32 Tosynthesize thermoresponsive and completely hydrolyticallydegradable hydrogels in aqueous solutions to incorporate avery high amount of protein drugs into the hydrogels,enhance the stability of the loaded drugs, and avoid futuresurgical removal of the hydrogels, in this study we havecopolymerized NIPAAm monomer with a dextran macromercontaining multiple hydrolytically degradable oligolactate-2-hydroxyethyl methacrylate units (Dex-lactateHEMA) atdifferent feeding weight ratios in phosphate-buffered saline(PBS) (pH 7.4) solutions. The dextran macromer wassynthesized on the basis of the method developed by Henninkand co-workers.33,34 We have characterized the chemicalstructures of the synthesized hydrogels by the FTIR tech-nique. We have also studied the following properties of thehydrogels: thermoresponsive and swelling properties at 25and 37°C, below and above the LCST, respectively, byweight measurements, and degradation properties at 37°Cby weight loss measurements and the FTIR technique. Wehave loaded two hydrophilic model drugs of very differentmolar masses, methylene blue (Mw ) 320 g‚mol-1) andbovine serum albumin (BSA;Mw ) 67000 g‚mol-1), intothe hydrogels during the hydrogel synthesis process in PBS(pH 7.4) solutions. We have investigated the in vitro releasekinetics of these two drugs at both 25 and 37°C. Althoughnot addressed in this paper, the enzymatic degradation ofthe designed hydrogels by dextranase could be expected tobring more versatility for the materials as drug deliverydevices. This work provides insight for designing multifunc-tional biomaterials for organic-solvent-free encapsulation ofhydrophilic drugs with high loading efficiency and sustainedrelease of protein drugs at physiological temperature.

Experimental Section

Materials. Dextran (Mw ) 15000-20000 g‚mol-1) andmethylene blue (Mw ) 320 g‚mol-1) were purchased fromPolysciences, Inc., Warrington, PA, and Acros, Inc., MorrisPlains, NJ, respectively. The following materials wereobtained from Sigma-Aldrich, Inc., St. Louis, MO: NIPAAm,2-hydroxethyl methacrylate (HEMA), 4-(N,N-diethylamino)-pyridine (DMAP),N,N′-carbonyldiimidazole (CDI),L-lactide,stannous octoate (SnOct2), tetrahydrofuran (THF), dimethylsulfoxide (DMSO), N,N,N′,N′-tetramethylethylenediamine(TEMED), potassium peroxydisulfate (KPS), BSA (Mw )67000 g‚mol-1), and Bradford reagent. All the chemicalswere used as received. Deionized distilled water was usedin all the experiments.

Synthesis of Dex-lactateHEMA Macromers. Dex-lactateHEMA was synthesized according to the methoddeveloped by Hennink and co-workers33,34 with slightmodification. Briefly, at first,L-lactide (4.32 g, 30 mmol)and HEMA (3.90 g, 30 mmol) were reacted in the presenceof nitrogen and catalyst SnOct2 (121.5 mg, 1 mol % withrespect to HEMA) at 110°C for 1 h. HEMA-lactate productwas collected by dissolving the cooled reaction mixture inTHF, precipitating in ice-cold water, dissolving in ethylacetate, drying over MgSO4, and concentrating under reduced

pressure. The yield of the above reaction was 23%. Next,CDI (1.76 g, 10.8 mmol) was dissolved in THF (100 mL)in a nitrogen atmosphere and reacted with the aboveHEMA-lactate (3.74 g, 10.8 mmol) at room temperaturefor 16 h. The resulting HEMA-lactate-imidazolyl carbam-ate (HEMA-lactateCI) was obtained by solvent evaporationunder reduced pressure, and the reaction yield was 100%.Finally, dextran (10.0 g, 61.8 mmol of glucopyranoseresidues) was dissolved in DMSO (90 mL) in a nitrogenatmosphere. After dissolution of DMAP (2.0 g), HEMA-lactateCI (3.78 g, 8.60 mmol) was added, and the mixturewas stirred at room temperature for 4 days. The Dex-lactateHEMA product was obtained by precipitating thereaction mixture in a large excess volume of cold dry2-propanol, washed several times with 2-propanol, and driedin a vacuum for at least 24 h. The yield of the final reactionwas 87%.

Synthesis of Thermoresponsive and BiodegradableHydrogels.NIPAAm and Dex-lactateHEMA (total amount450 mg) at different weight ratios, NIPAAm:Dex-lactate-HEMA ) 7:2, 6:3, 5:4, and 4:5 (w/w), were dissolved in2.28 mL of PBS (pH 7.4) solvent under nitrogen. Afterdissolution, KPS in PBS (50 mg/mL, 270µL) and TEMEDin PBS (20% (v/v), 150µL) were added as initiator andaccelerator, respectively. The mixture was mixed thoroughlyand quickly injected into the space between two glass plateswrapped by Teflon films (thickness 1.6 mm), and the gelationwas allowed to proceed at room temperature for 1 h. Disk-shaped gel samples (∼8 mm in diameter) were then cut off,washed in a large volume of a 50:50 (v/v) ethanol/watermixture for 30 min, and dried in the air till no further weightloss occurred. Hydrogels were denoted as gels 7/2, 6/3, 5/4,and 4/5 corresponding to their initial NIPAAm:Dex-lactateHEMA feeding ratios of 7:2, 6:3, 5:4, and 4:5 (w/w),respectively. The dried 7/2 gel appeared to be transparent,while the other dried gels were semitransparent, possiblybecause the hydrogels increased in heterogeneity withincreasing Dex-lactateHEMA component (decreasing theNIPAAM component).19,35 The synthesis yields of the gels7/2, 6/3, 5/4, and 4/5 were 95%, 99%, 97%, and 92%,respectively.

Chemical Structure Characterization. To confirm thesuccess of the syntheses of the Dex-lactateHEMA mac-romer, the chemical shifts of the dextran, lactateHEMA, andDex-lactateHEMA which matched their nuclear spins in amagnetic field were measured using a proton nuclearmagnetic resonance (1H NMR, Bruker DPX-300, Ettlingen,Germany) spectrophotometer. For lactateHEMA, deuteratedchloroform (CDCl3, 99.9%, Aldrich) was used as solvent,and the chloroform-d1 resonance was set at 7.27 ppm. Fordextran and Dex-lactateHEMA macromer, DMSO-d6 (99.9%,Aldrich) was used as solvent, and the central DMSO linewas set at 2.50 ppm. Samples for the1H NMR measurementswere prepared by dissolving approximately 10 mg of eachmaterial in 1 mL of corresponding solvent.

To confirm the successful syntheses of the hydrogels, wecharacterized the chemical structures of the synthesizedhydrogels through studying their infrared absorption bandswhich match their natural vibrational modes using an

2132 Biomacromolecules, Vol. 6, No. 4, 2005 Huang and Lowe

attenuated total reflection (Pike Technologies, Madison, WI)Fourier transform infrared (ATR-FTIR) spectroscope (Ther-mo Nicolet Avetar 370, Madison, WI). The dry gels 7/2,6/3, 5/4, and 4/5 grounded into pieces were compressed ontothe ZnSe crystal, and FTIR spectra were recorded in thewavenumber range of 4000-650 cm-1.

Physical Properties of Hydrogels. 1. ThermoresponsiveProperties. Weighed dry hydrogel samples were immersedin PBS (pH 7.4) at 10°C. The temperature was raised every1 h, ranging from 10 to 60°C. Before each temperatureadjustment, the swollen gel samples were removed from thesolvent, the surface water was carefully absorbed by filterpaper, and the gel samples were weighed. The swelling ratioq was defined as

in which Wt andW0 are the weights of the swollen and drygels, respectively.

2. Swelling Properties. Dry hydrogel samples wereimmersed in PBS (pH 7.4) at 37°C. Weights of the wethydrogel samples were measured at selected time points.Equation 1 was used to calculate the swelling ratioq. PBS(pH 7.4) solvent was replaced every day.

3. Degradation Properties. The degradation of thesynthesized hydrogels should be dependent on both envi-ronmental pH and the presence of enzyme dextranase dueto the existence of poly(L-lactide) and dextran segments,respectively. In this paper we only focused on the degradationstudy under normal physiological conditions by using PBS(pH 7.4) as the degradation medium. Dry hydrogel sampleswere immersed in PBS (pH 7.4) at 25 and 37°C, respec-tively. At predetermined time points, samples were removedfrom the solvents, dried in the air overnight, and weighed todetermine weight loss. After weighing, the samples wereplaced back in PBS (pH 7.4) for continuous degradation.PBS solvent was replaced every day. The FTIR spectra ofthe degrading hydrogels were also recorded by ATR-FTIRspectroscopy at selected time points. The wet hydrogels werecompressed onto the ZnSe crystal, and three positions of eachhydrogel sample were chosen randomly for the FTIRmeasurements. The FTIR spectra were processed with abackground subtraction of the PBS (pH 7.4) solvent.

Drug Loading and in Vitro Release.Hydrophilic modeldrugs methylene blue (Mw ) 320 g‚mol-1) and protein BSA(Mw ) 67000 g‚mol-1) were loaded into the hydrogels duringthe hydrogel synthesis. In detail, methylene blue or BSA(22.5 mg, 5 wt % with respect to the polymerizationprecursors) was dissolved in NIPAAm/Dex-lactateHEMAin PBS (pH 7.4) solutions before the addition of initiatorKPS and accelerator TEMED (see the above text for thedetailed hydrogel synthesis). After synthesis, the gels weredried directly in the air without washing. In vitro releasestudies were conducted by immersing dried methylene blue-or BSA-loaded gels in 3 mL of PBS (pH 7.4) solvent at 25and 37°C, respectively. At selected time intervals duringone month, 1 mL release solutions were collected andreplaced with fresh PBS (pH 7.4) solvent. Methylene blueand BSA amounts were quantified by a UV/vis spectropho-tometer (PerkinElmer Lamda 25, Shelton, CT) at a wave-length of 668 nm and Bradford protein assay, respectively.

Results and Discussion

Synthesis of Dex-lactateHEMA Macromers. The chemi-cal structures of Dex-lactateHEMA macromer andPNIPAAm-co-Dex-lactateHEMA hydrogels are sketched inFigure 1.24 1H NMR spectra presented in Figure 2 confirmedthe successful synthesis of lactateHEMA and Dex-lactate-HEMA macromer. As shown in Figure 2B,C, the charac-teristic chemical shift peaks of HEMA residues atδ ≈ 6.0(a, CH2dC<), δ ≈ 5.6 (a, CH2dC<), δ ≈ 1.9 [b, CH2dC(CH3)-], and δ ) 4.4-4.2 [c, d, -OCH2CH2O-; g,-O(OdC)CHCH3OH] ppm were observed in both thelactateHEMA and Dex-lactateHEMA. Parts B and C ofFigure 2 also demonstrated the characteristic peaks of thelactyl residues of both the lactateHEMA and Dex-lactate-HEMA at δ ≈ 5.2 [e,-O(OdC)CHCH3O-] andδ ) 1.6-1.4 [f, h, -O(OdC)CHCH3O-] ppm. Importantly, theappearance of the chemical shift peaks of the-O(OdC)-CHCH3OH of the lactateHEMA in Figure 2B,C atδ ≈ 4.4nm instead ofδ ≈ 3.8 nm clearly indicated that HEMA wasindeed esterified with the lactate graft.34 After lactateHEMAwas reacted with dextran, the successful synthesis of Dex-lactateHEMA macromer was confirmed by the characteristicpeaks of the glycopyranose backbone of dextran (Figure 2A)

Figure 1. Schematic chemical structure of Dex-lactateHEMA (a) and NIPAAm-co-Dex-lactateHEMA (b) hydrogels.

q ) (Wt - W0)/W0 (1)

Hydrogels for Encapsulation and Controlled Release Biomacromolecules, Vol. 6, No. 4, 2005 2133

at δ ≈ 4.6 ppm for anomeric protons,δ ) 5.0-4.8 ppm forprotons of -OH, and δ ) 4.5-4.4 ppm for protons of-CH2- and-CH<, as well as the disappearance of the peakat δ ≈ 2.7 ppm for the protons of the hydroxyl group [i,-O(OdC)CHCH3OH] of the lactateHEMA. The averagelength (DPav) of the lactate spacer graft in lactateHEMAcould be calculated from the ratio of the integral of He tothat of Ha (Figure 1B). The degree of substitution (DS;amount of methacrylate groups per 100 dextran glucopyra-nose residues) of Dex-lactateHEMA could be estimated asthe ratio between the average integral of the protons of vinyl-ending groups and the integral of the anomeric protons (Ha:H1). In this study, DPav and DS were equal to 3 and 4.6,respectively.

Synthesis of Thermoresponsive and BiodegradableHydrogels. The chemical compositions of the synthesized

gels 7/2, 6/3, 5/4, and 4/5 were confirmed by FTIRmeasurements. As shown in Figure 3, the presence of thecharacteristic bands of Dex-lactateHEMA at 1760 (CdOstretching from the lactateHEMA group) and 1010 (C-OHstretching from the dextran group) cm-1 were observed inthe IR spectra of all four hydrogels. The typical amide I andII bands at 1650 and1540 cm-1, respectively, and dividedbands of symmetric C-H bending from the-CH(CH3)2

group at 1385 and 1370 cm-1 from PNIPAAm were alsoobserved in the IR spectra of all four hydrogels. Anotherstrong piece of evidence to confirm that Dex-lactateHEMAand dextran were indeed successfully incorporated into allfour hydrogels was that the relative intensities of thecharacteristic bands of Dex-lactateHEMA with respect tothose of PNIPAAm increased in the order gel 7/2< gel 6/3< gel 5/4< gel 4/5, with increasing feeding ratios of Dex-

Figure 2. 1H NMR spectra of dextran (A), lactateHEMA (B), and Dex-lactateHEMA (C).

Figure 3. FTIR spectra of gels 7/2 (a), 6/3 (b), 5/4 (c), and 4/5 (d): peak 1, CdO stretching; peak 2, amide I; peak 3, amide II; peaks 4 and5, C-H bending from -CH(CH3)2; peak 6, C-OH stretching.


lactateHEMA macromer to NIPAAm monomer. It is alsoworth mentioning that the intensities of the two peaks at 1385and 1370 cm-1 of PNIPAAm are usually identical; however,in our four gels, the band at 1370 cm-1 became strongerand stronger than the one at 1381 cm-1 with increasing Dex-lactateHEMA amount in the gels. The reason was thatdextran also had an IR vibration at these two bands and itsband was stronger at 1370 cm-1 than at 1381 cm-1.

Thermoresponsive Properties of Hydrogels.The ther-moresponsive properties of hydrogels 7/2, 6/3, 5/4, and 4/5were studied by measuring their swelling ratios in PBS (pH7.4) as a function of temperature. As shown in Figure 4, allfour hydrogels demonstrated a volume phase transition atapproximately 32°C, which is the typical LCST of PNIPAAmpolymers. At temperatures below the LCST, the swellingratios of the four hydrogels decreased with increasing Dex-lactateHEMA component due to the increasing amount ofthe cross-linkers. It was surprising that at temperatures belowthe LCST the swelling ratios of all four hydrogels slightlyincreased with temperature, which was opposite the typicalbehavior of PNIPAAm-based thermoresponsive hydrogels.The reason might be that the increased temperature acceler-ated the degradation rate of the cross-linker oligolactate ofthe hydrogels and thus favored more water uptake.36,37 Astemperature reached the LCST, strong hydrophobic interac-tions between the PNIPAAm chains due to the presence oftheir isopropyl groups occurred, so that a significant amountof water was dispelled from the hydrogels, and phaseseparation and a substantial decrease of the swelling ratiosof the hydrogels were observed. The phase transition becameless sharp with the fold of the swelling ratios decreasing inthe order gel 7/2 (20-fold)< gel 6/3 (16-fold)< gel 5/4≈gel 4/5 (8-fold) with a decrease of the thermoresponsivePNIPAAm moiety. While the temperature continuouslyincreased above the LCST, the swelling ratios of the fourhydrogels increased with an increase of the Dex-lactate-HEMA component, and decreased with temperature, whichwas opposite those at temperatures below the LCST. Apossible explanation might be that the decreased effects ofthe enhanced hydrophobicity of the hydrogels on the swellingratios outperformed the opposite effects of the decreased

cross-link amount with an increase of the PNIPAAm moietyand temperature.

Degradation Properties of Hydrogels.At 25 °C, belowthe LCST, gels 7/2, 6/3, 5/4, and 4/5 were observed to startto disintegrate after 1, 2, 4, and 5 days of incubation in PBS(pH 7.4), respectively. They then gradually dissolved withintwo weeks. As discussed in the previous section, all thehydrogels were highly hydrophilic and swollen at tempera-tures below the LCST. Therefore, abundant water couldeasily access and hydrolyze the ester bonds of oligolactatecross-links so that the hydrogels were broken down after acertain time. It was also understandable that hydrogels withhigher Dex-lactateHEMA component possessed more cross-links and ester bonds thus needed longer time to degrade.

In contrast, at 37°C, above the LCST, the degradation ofall four gels 7/2, 6/3, 5/4, and 4/5 was much slower than at25 °C. There was no observable disintegration of the fourhydrogels during 8 days of incubation in PBS (pH 7.4)solvent at 37°C. The weight of the four hydrogels decreasedmore than 10% within the first day, and reached a stablestage after 1 and 2 days for gels 7/2 and 6/3, respectively(Figure 5). The weights of gels 5/4 and 4/5 continuouslydecreased with time for 8 days. The driving force for theweight loss was the hydrolysis of the oligolactate cross-linksof the hydrogels. As the cross-links broke down, the dextranand PNIPAAm chains linked with the degraded cross-linkswere freed up and diffused into the buffer solutions. Sincedextran chains were hydrophilic and PNIPAAm chains werehydrophobic at 37°C, the freed dextran chains might diffusefaster than the PNIPAAm chains. With more and more freeddextran diffusing out, the remaining hydrogel networksbecame more and more hydrophobic despite the decreasednumber of cross-links with time. As a result, water diffusionand access to the polymer chains became more and moredifficult with time, so that the weight loss of gels 7/2 and6/3 became negligible after 1 and 2 days, respectively, andgels 5/4 and 4/5 degraded slower with time. Figure 5 alsodemonstrated that the weight losses and degradation ratesof the four hydrogels increased in the order gel 7/2< gel6/3 < gel 5/4< gel 4/5 with increasing Dex-lactateHEMAcomponent, due to the hydrophilicity of dextran chains.

To support the above hypothesis of the hydrogel degrada-tion mechanism, the ATR-FTIR spectra of degrading hy-

Figure 4. Thermoresponsive properties of gels 7/2 (b), 6/3 (2), 5/4(9), and 4/5 ([) determined by measuring swelling ratios as a functionof temperature from 10 to 60 °C after equilibrium for 1 h at eachtemperature interval in PBS (pH 7.4) solvent.

Figure 5. Weight loss of gels 7/2 (b), 6/3 (2), 5/4 (9), and 4/5 ([)in PBS (pH 7.4) solvent at 37 °C.


drogels (in 37 °C PBS) at selected time points weremeasured, and the relative peak intensities at 1010 cm-1

characterizing C-OH stretching from the dextran backbone(IC-OH) to those at 1650 cm-1 featuring amide I of thePNIPAAm segment (Iamide I) were calculated. To avoidexperimental errors caused by the nonuniformity of hydrogelsamples, the ATR-FTIR measurements ofIC-OH and Iamide I

at three random positions of the hydrogels were averaged ateach time point. As clearly shown in Figure 6, the ratios ofIC-OH to Iamide I decreased with time, confirming the fasterdiffusion of the freed dextran segments than PNIPAAmduring degradation.

Swelling Property of Hydrogels.The swelling ratios ofthe four gels 7/2, 6/3, 5/4, and 4/5 in PBS (pH 7.4) solutionsagainst time at 25°C are plotted in Figure 7. The swellingratios of all four hydrogels increased dramatically andreached values more than 10-fold of the dry weight of thehydrogels within 2 h. With time, the differences betweenthe swelling ratios of the four hydrogels became more andmore obvious, and at 2 h, the swelling ratios of the hydrogelsdecreased in the order gel 7/2> gel 6/3 > gel 5/4 > gel4/5. The reason was that both PNIPAAm and dextran

moieties in the hydrogels were very hydrophilic at 25°Cand the cross-linking density (hydrophobic oligolactate) ofthe hydrogels increased in the order gel 7/2< gel 6/3< gel5/4 < gel 4/5. From the time beyond 2 h to thetime whenthe hydrogels started disintegration, all four hydrogelscontinuously absorbed water and swelled to higher and higherlevels. The mechanical strength of the hydrated samplesbecame weak, and thus, the handling of the samples becamedifficult after 2 h. Therefore, the swelling ratio measurementswere stopped at 2 h.

The swelling ratios of the four gels 7/2, 6/3, 5/4, and 4/5in PBS (pH 7.4) solutions against time at 37°C are depictedin Figure 8, with the time axis on the logarithmic scale. Allfour hydrogels had much lower swelling ratios at 37°C thanat 25 °C on the same time scale because the PNIPAAmmoiety was hydrophobic at 37°C. The swelling ratios of allfour hydrogels at 37°C increased quickly, reached maximumvalues within the first 4 h, and increased in the order gel7/2 < gel 6/3 < gel 5/4 < gel 4/5 with increasing Dex-lactateHEMA component due to the hydrophilicity of dextranchains. From 4 h to 1day, the swelling ratios of all fourgels 7/2, 6/3, 5/4, and 4/5 started to decrease dramaticallyand reached relatively stable levels at around 3, 7, 12, and>20 days, respectively. The swelling ratios decreased in theorder gel 7/2≈ gel 6/3, gel 5/4< gel 4/5 between 1 and4 days, and oppositely started to increase in the order gel7/2 > gel 6/3g gel 5/4g gel 4/5 between 6 and 18 days.In the previous section, we have discussed that, as thedegradation of the hydrogels proceeded, the freed hydrophilicdextran segments quickly entered into the surrounding buffersolutions, not only causing a decrease in hydrogel masses,but also leaving behind hydrogel networks that became moreand more hydrophobic. As a result, water was expelled frominside the gels, leading to gel deswelling. The reduction ofboth water and hydrogel masses may contribute to thedecrease of swelling ratios after the maximum values arereached. Since the decreases of polymer masses due todegradation within 8 days were only∼15-50% of theoriginal dry hydrogel weights (Figure 5), hydrogel deswellingwas the dominant factor that caused swelling ratios todecrease at the later stage. It is easy to understand thathydrogels with higher amounts of the Dex-lactateHEMA

Figure 6. FTIR peak intensity at 1010 cm-1 (C-OH stretching fromthe dextran backbone) normalized by that at 1650 cm-1 (amide I fromthe PNIPAAm segment): IC-OH/Iamide I as a function of time for gels7/2 (b), 6/3 (2), 5/4 (9), and 4/5 ([) that degraded in PBS (pH 7.4)solvent at 37 °C.

Figure 7. Swelling ratios of gels 7/2 (b), 6/3 (2), 5/4 (9), and 4/5([) in PBS (pH 7.4) solvent at 25 °C as a function of time.

Figure 8. Swelling ratios of gels 7/2 (b), 6/3 (2), 5/4 (9), and 4/5([) in PBS (pH 7.4) solvent at 37 °C as a function of time: symbols,experimental data; dashed lines, fitting curves based on eq 5.


moiety had faster initial swelling and reached highermaximum swelling ratios due to their higher hydrophilicity.Previously, we also discussed that the mass loss rates of thehydrogels decreased with time. When the mass losses enteredinto slower stages, changes in hydrogel hydrophilicity alsobecame less dramatic, resulting in lower deswelling rates andeventually minimal changes of swelling ratios with time.

On the basis of the above discussion, to further understandand interpret the mechanisms behind the biphase swellingratio data depicted in Figure 8, we developed an empiricalmathematical model. The increasing phase of the swellingratio curves at the early stage before 4 h inFigure 8 mightbe fit by the power law, which has been suggested as a simplemodel to describe solvent uptake and swelling of nonbio-degradable hydrogels:38,39

whereq1 is the swelling ratio,t is time, andk1 and n areconstants. When the exponentialn ) 0.5, solvent diffusionfollows Fickian diffusion, whenn ) 1.0, the diffusion iscase II diffusion, and when 0.5< n < 1.0, the diffusion isa combination of Fickian and case II diffusion and is usuallycalled anomalous diffusion.38 On the other hand, the decreas-ing phase of the swelling ratio kinetics might be fit by anexponential decay function:40

whereq2 is the swelling ratio andk2 and m are constants.Here we combined eqs 2 and 3 to establish a new equation

for the description of the whole range of swelling ratiochange of the degradable hydrogel systems designed in thisstudy:

or

whereq is the swelling ratio andK1, K2, n, andm are allconstants. When the value oft is small,K1t-n is the dominantterm in the denominator, so that eq 5 is similar to eq 2; onthe other hand, whent takes a large value,K2 exp(mt) makesa major contribution to the denominator, and eq 5 may berestored to the form of eq 3.

Fitting curves are plotted together with the experimentaldata in Figure 8, and the values of the parameters aretabulated in Table 1. The results suggested that the developed

equation fit the swelling ratio data of gels 4/5, 5/4, and 6/3well. With a decrease of the hydrophilicity of the hydrogels,the exponential indexn decreased from 1.236 for gel 4/5 to0.9686 for gel 5/4 and to 0.6837 for gel 6/3, correspondingto so-called super case II, perfect case II, and anomaloustransport, respectively.41 Oppositely, the parameterm in-creased in the order gel 6/3> gel 5/4> gel 4/5, which mightindicate that water inside the hydrogel networks was expelledat a higher rate with increasing hydrogel hydrophobicity. Thefitting of the swelling data of gel 7/2 (Figure 6) wasunsatisfactory by using the developed model, which mightbe owed to the relatively too high hydrophobicity and rigidityof gel 7/2 at 37°C. Equation 5 was established based on eq2 for swellable hydrogels and eq 3 for shrinking hydrogels,so that when hydrogels were hydrophobic and slightlyswellable, more or less like homo-PNIPAAm gels, such asgel 7/2, eq 5 was not applicable to model their swellingbehavior anymore.

In Vitro Release of Model Drugs from 5/4 Gels.Todevelop the designed hydrogels as controlled drug releasedevices, two hydrophilic model drugs, methylene blue andBSA, were loaded into the hydrogels during the synthesisprocess in PBS (pH 7.4) solutions. Methylene blue waschosen because its molar mass is small (Mw ) 320 g‚mol-1)and its concentration can be easily detected by UV/visspectroscopy without interference from degraded polymermaterials. BSA was employed because it is widely used asa model protein drug, its molar mass (Mw ) 67000 g‚mol-1)is much higher than that of methylene blue, and itsconcentration can be easily quantified by Bradford proteinassay. In vitro release profiles of methylene blue in PBS (pH7.4) solvent were studied at temperatures below (25°C) andabove (37°C) the LCST. Figure 9 demonstrates the fractionalreleaseMt/M∞ of methylene blue against time, whereMt andM∞ are the cumulative amount of drug released at timetand equilibrium, respectively. At 25°C, data were collectedbefore the hydrogels started disintegrating. The fractionalrelease of methylene blue showed more than 80% initial burstrelease within the first 1 h at 37°C, and reached equilibriumafter 1 and 4 days at 37 and 25°C, respectively. Surprisingly,the release rate was higher at 37°C than at 25°C, suggestingthat the release was not dominantly controlled by the swelling

Table 1. Fitting Parameters in Equation 5 for the Swelling RatioKinetics of Hydrogels at 37 °C

hydrogelsample n m K1 K2 R2

gel 4/5 1.236 0.4326 0.001114 0.1784 0.994gel 5/4 0.9686 0.5082 0.00549 0.1778 0.988gel 6/3 0.6837 0.7576 0.03219 0.2642 0.911gel 7/2 1.742 0.04721 0.0007231 0.7019 0.640

q1 ) k1tn (2)

q2 ) k2 exp(-mt) (3)

1q

) 1q1

+ 1q2

(4)

q ) 1

K1t-n + K2 exp(mt)

(5)

Figure 9. Fractional release of methylene blue from gel 5/4 in PBS(pH 7.4) solvent at 25 (O) and 37 (b) °C.


and degradation of the hydrogel. The possible reasons mightbe as follows: (1) Methylene blue is a small hydrophilicmolecule with molar mass 320 g‚mol-1, so that its diffusioninto the release medium was hardly hindered by the hydrogelnetwork. (2) The hydrogel network was more hydrophobicat 37 °C than at 25°C, which would be favorable for thesmall hydrophilic molecules to enter into the aqueous releasemedium. (3) The hydrogel size was bigger at 25°C than at37 °C, so that the concentration gradients of methylene blueinside the hydrogels were lower at 25°C than at 37°C.42,43

It is worth mentioning that 90% of loaded methylene bluewas released from the hydrogels at equilibrium time at both25 and 37°C.

Figure 10 demonstrates the fractional releaseMt/M∞

profiles of model protein BSA. At a temperature below theLCST, 25°C, the BSA release was completed within 4 daysbefore the hydrogels started to disintegrate. At a temperatureabove the LCST, 37°C, the release curve was featured by amoderate initial burst followed by a sustained release withdiminishing rate for up to 15-20 days. The BSA releasereached a plateau with around 40% of the initially loadedBSA retained inside gel 5/4 after 15 days according to theaccumulative release data (data not shown). The BSAfractional release at 37°C for up to 15 days could be fitby the well-known power equation, in the same format aseq 2:39

The above equation has been used to analyze drug releasefrom both swellable and nonswellable polymeric deliverysystems.43 Fickian diffusion (n ) 0.5) and case II transport(n ) 1) are often obtained when drugs are released fromdiffusion-controlled and swelling-controlled systems, respec-tively. A system controlled by both diffusion and swellingusually generates 0.5< n < 1. In our system, after datafitting, we obtainedk ) 0.531,n ) 0.2595, andR2 ) 0.991.The reason thatn was smaller than 0.5 might be due to theincreasing hydrophobicity of the hydrogel networks withtime. Due to the fast swelling and degradation of gel 5/4 at25 °C, eq 6 could not be used to fit the release data of BSA

from gel 5/4 at 25°C. The release rate of BSA from gel 5/4was higher at 25°C than at 37°C, which was opposite thetemperature-dependent trend of the release rate of methyleneblue. This might be because the diffusion of a high molarmass molecule such as BSA (Mw ) 67000 g‚mol-1) couldbe hindered and thus controlled by the degradation of gel5/4 at 37°C when gel 5/4 is relatively hydrophobic and denseat 37°C.

Conclusions

A series of novel thermoresponsive and biodegradablehydrogels were designed and prepared in aqueous PBS (pH7.4) solvent by copolymerizing NIPAAm and Dex-lactate-HEMA, a dextran methacrylate macromer containing ahydrolyzable oligolactate spacer, at different feeding weightratios: NIPAAm:Dex-lactateHEMA) 7:2, 6:3, 5:4, and4:5 (w/w). All four synthesized hydrogels were temperaturesensitive by showing an LCST at approximately 32°C. Theirswelling ratios increased and oppositely decreased at tem-peratures below and above the LCST, respectively, withdecreasing Dex-lactateHEMA moiety (or increasingPNIPAAm moiety). The four hydrogels swelled less anddegraded slower in PBS (pH 7.4) solutions at 37°C (abovethe LCST) than at 25°C (below the LCST), and the networkhydrophobicity increased while the hydrogels were degradedat 37°C. The dynamic weight loss amounts and rates of thehydrogels at 37°C increased with increasing Dex-lactate-HEMA moiety (or decreasing PNIPAAm moiety). Theswelling ratios of the hydrogels against time at 37°C showedbiphase curves, and were fit by an empirical mathematicalequation (5) combining power eq 2 and exponential eq 3when the hydrogels were not highly hydrophobic such asgels 6/3, 5/4, and 4/5. Equation 2 was based on the maincontribution of the solvent diffusion and hydrogel swellingat the early increasing swelling stage, and exponential eq 3was based on the main contribution of the increased hydrogelhydrophobicity with time at the late decreasing swellingstage.

Two hydrophilic model drugs, low molar mass methyleneblue (Mw ) 320 g‚mol-1) and high molar mass BSA (Mw )67000 g‚mol-1), were loaded into gel 5/4 during the synthesisprocesses in aqueous PBS (pH 7.4) solutions. The releaseof methylene blue from gel 5/4 was slower at 25°C (belowthe LCST) than at 37°C (above the LCST), while the releaseof BSA at these two temperatures showed the opposite trend.These results suggest that the drug release kinetics stronglydepend on environmental temperature, the swelling anddegradation of the hydrogel, and the interactions of the loadeddrugs with the hydrogel networks. BSA release data at 37°C were fit by a traditional power equation (6), generatinga diffusion exponentn < 0.5, which might be due to theincreasing hydrogel hydrophobicity with time.

Currently, we are carrying out further aqueous synthesisof the thermoresponsive and biodegradable hydrogels bychanging the length of the oligolactate spaces, the numberof dextran’s -OH pending groups substituted by cross-linkable groups, and the feeding weight ratios betweenNIPAAm and dextran macromer for controlled release of a

Figure 10. Fractional release of BSA from gel 5/4 in PBS (pH 7.4)solvent at 25 (O) and 37 (b) °C. The dashed line is the fitting curvegenerated from eq 6.

Mt

M∞) ktn (6)


variety of drugs in response to temperature changes. We arealso investigating the biosafety of the developed hydrogelsand the stability of loaded protein drugs.

Acknowledgment. We are grateful to Dr. ShengshengLiu for his generous help with the NMR analysis and theWhitaker Foundation and Penn State Surgery FeasibilityGrant for financial support.

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