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Swelling Behavior and Morphological Evolution of Mixed Gelatin/Silk Fibroin Hydrogels Eun S. Gil,† David J. Frankowski,‡ Richard J. Spontak,‡,§ and Samuel M. Hudson*,† Fiber and Polymer Science Program and Departments of Chemical & Biomolecular Engineering and Materials Science & Engineering, North Carolina State University, Raleigh, North Carolina 27695 Received June 10, 2005; Revised Manuscript Received July 21, 2005

Mixed protein-based hydrogels have been prepared by blending gelatin (G) with amorphous Bombyx mori silk fibroin (SF) and promoting β-crystallization of SF via subsequent exposure to methanol or methanol/ water solutions. The introduction of β crystals in SF serves to stabilize the hydrogel network and extend the solidlike behavior of these thermally responsive materials to elevated temperatures beyond the helixfcoil (hfc) transition of G. In this work, we investigate the swelling and protein release kinetics of G/SF hydrogels varying in composition at temperatures below and above the G hfc transition. At 20 °C, G and G-rich mixed hydrogels display evidence of moderate swelling with negligible mass loss in aqueous solution, resulting in porous polymer matrixes upon solvent removal according to electron microscopy. When the solution temperature is increased beyond the G hfc transition to body temperature (37 °C), these gels exhibit much higher swelling with considerable mass loss due to dissolution and release of G. The extent to which these properties respond to temperature decreases systematically with increasing SF content. The unique temperature- and composition-dependent properties of G/SF hydrogels dictate the efficacy of these novel materials as stimuli-responsive delivery vehicles. Introduction Hydrogels consist of three-dimensional cross-linked macromolecular networks wherein the constituent polymer species are hydrophilic.1 Upon sufficient chemical or physical stabilization, hydrogels swell, but do not dissolve, in an aqueous environment. Due to their biocompatible and often stimuli-responsive nature, hydrogels are used extensively in biomedical research involving, but not limited to, soft contact lenses,2 tissue engineering scaffolds,3 controlled drug-release vehicles,4 mechanical actuators,5 and liquid chromatographic columns.6 Various synthetic polymers exhibit sufficient hydrophilicity to be used in the preparation of hydrogels,7,8 but emerging biomedical and pharmaceutical technologies require responsive and functional hydrogels exhibiting both biocompatibility and biodegradability.9 Some biomacromolecules already implemented for this purpose include polysaccharides,10 cellulose derivatives,11-13 and proteins.14,15 Hydrogels prepared from proteins are of particular interest, due to their ability to form complex hierarchical structures, as well as their inherent thermal and pH sensitivity.16,17 For these and related reasons, protein-based hydrogels have also been successfully applied in drug-delivery systems,18 as sealants for vascular prostheses19 and artificial tissue matrices.20,21 Two proteins that have attracted much attention as biomaterial candidates are gelatin (G), generated by breaking the triple-helix conformation of collagen into single-stranded macromolecules, and silk fibroin (SF), recovered directly from silk worms and spiders. * To whom correspondence should be addressed. E-mail: Sam_Hudson@ ncsu.edu. † Fiber and Polymer Science Program. ‡ Department of Chemical & Biomolecular Engineering. § Department of Materials Science & Engineering.

At temperatures below ∼30 °C in an aqueous environment, G spontaneously transforms back from random-coil to triplehelix conformation, thereby introducing physical cross-link sites that stabilize the formation of a polymer network at sufficiently high G concentrations. Because the helixfcoil (hfc) conformational transition encountered upon reheating dissolves the reformed helix-stabilized network, G hydrogels are classified as thermally reversible.21 At elevated temperatures (near body temperature, 37 °C), the mechanical properties exhibited by these hydrogels are poor, but can be improved by chemical means.22,23 While chemical modification of G hydrogels is often accompanied by increased toxicity, UV-induced cross-linking provides an alternative route by which to improve the mechanical properties of G hydrogels without the need for chemical cross-linking agents.24 Silk fibroin is another protein that has attracted significant attention as a biomedical material on the basis of its intrinsic properties.25-28 Although it continues to be considered for a wide range of biotechnologies, such as surgical sutures,29 cosmetics,30 enzyme immobilization,31 wound covering materials,32 cell-growth substrates,26 drugdelivery vehicles,33 and tissue engineering scaffolds,25 SF does not behave as a stimuli-responsive material due to the presence of β crystals that ensure dimensional stability over a wide temperature range. We have previously reported,34 however, that mixed G/SF hydrogels stabilized by β crystals retain the G hfc transition and therefore display thermal responsiveness. In this work, G and SF are prepared under different conditions and at different ratios to produce macroscopically homogeneous bioconjugate blends35 and, upon hydration, mixed hydrogels. Amorphous SF is generated upon dissolu-

10.1021/bm050396c CCC: $30.25 © 2005 American Chemical Society Published on Web 09/07/2005

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tion of native (semicrystalline) SF in a solvent composed of calcium nitrate and methanol (MeOH).36 Subsequent immersion of amorphous SF in MeOH or an aqueous MeOH solution induces a conformational transformation from random coil to β-sheet (silk II), which introduces physical cross-link sites into the G/SF hydrogels under investigation. Our prior analysis34 of these mixed hydrogels has explored their unique thermal and rheological properties before and after β-crystal formation. The purpose of the present study is to establish the composition-dependent swelling behavior and morphological evolution of G/SF hydrogels at temperatures below and above the G hfc transition as the initial step required to establish the controlled release efficacy of these novel protein-based hydrogels. Experimental Section Materials. Grade 5A raw Bombyx mori silk with an average denier of 20.86 was obtained from Fiac¸ a`o de Seda Bratac S.S. (Brazil), and gelatin (type A from porcine skin, 175 bloom) was acquired from Sigma Chemicals. All other chemicals used in this study (see the following section) were purchased from Fisher Scientific or Aldrich Chemicals and used directly without further purification. Methods. 1. Preparation. A master batch of amorphous SF solution was prepared according to the dissolution method previously reported by Ha et al.36 The dried silk was degummed with 0.25% w/v sodium lauryl sulfate and 0.25% w/v sodium carbonate in boiling water (bath ratio of 1:10 w/v). After 1 h, the SF was removed and thoroughly washed in deionized water. The sericin fraction was calculated as 0.229. Dried SF (10 wt %) was subsequently dissolved in a 75/25 w/w Ca(NO3)2·4H2O/MeOH solution. The resultant SF solution was dialyzed in a cellulose membrane tube (MWCO 6000-8000) for 4 days with the deionized water changed daily. The concentration of the dialyzed SF solutions, estimated as 4.2 wt %, was diluted to 4 wt % by adding deionized water to the dialyzed SF solution. Dried G was suspended at a concentration of 4 wt % in deionized water, and the suspension was subsequently dissolved under agitation at 40 °C. The composition of each G/SF blend, denoted by G/SFW (where W denotes the weight percent of G in the blend), was controlled by isothermally mixing predetermined quantities of the parent G and SF solutions. Corresponding films were prepared by solvent casting the G/SF solutions on polystyrene Petri dishes at 10 °C, followed by storage under vacuum for 1 week. The resultant films, measuring ca. 200 µm thick, were treated with MeOH or an aqueous MeOH solution for 5-1200 min at 20 °C to induce SF β-crystallization in the presence of the G triple-helix conformation and then dried under vacuum for 24 h. 2. Analysis. A 1 cm × 1 cm specimen was cut from each film and weighed prior to immersion in a phosphate-buffered saline (PBS) solution (pH 7.4) at either 20 or 37 °C. Swollen films were removed at predetermined exposure times and immediately weighed. Once the films were air-dried and vacuum-dried for an additional 24 h, the films were reweighed. This procedure was repeated in triplicate to ensure reliable results. Two different ratios were obtained from these

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gravimetric measurements, viz., Qo ) (mS - mDo)/mDo

(1a)

Qf ) (mS - mDf)/mDf

(1b)

and

where mS is the weight of a swollen film, and mDo and mDf correspond to the weight of the dried film before and after swelling, respectively. Calculated values of Qo yielded solvent-induced changes in hydrogel mass inclusive of protein release, whereas Qf provided a more conventional measure of hydrogel swelling under isothermal conditions. In the limit that a hydrogel remained completely waterinsoluble, mDo ) mDf, so Qo ) Qf. Otherwise, the percent of mass retained (R) by a hydrogel upon exposure to the PBS solution for a specific time was determined from R ) 100% × (Qo - 1)/(Qf - 1)

(2)

Complementary scanning electron microscopy (SEM) images of the protein-based hydrogels under investigation were acquired from select specimens after exposure to PBS solution at 20 and 37 °C. Vitrified specimens were freezedried to avoid substantial morphological damage and subsequently Au-coated to increase conductivity and reduce charging. Secondary-electron images of surfaces and fractures produced in liquid nitrogen were collected on a Hitachi S-3200N microscope operated at an accelerating voltage of 5 kV. To discern the extent of protein extraction, mixed G/SF films weighing 20 mg were immersed in 6 mL of the PBS solution at 20 and 37 °C, and 200-µL aliquots of the solution were collected at regular exposure intervals. To maintain constant volume, 200 µL of PBS was added for each aliquot removed. The concentration of extracted protein (primarily G, especially at high SF concentrations) was determined by colorimetric assay with a bicinchoninic acid protein assay kit (Sigma Chemicals). Each 200-µL aliquot was mixed with 2 mL of assay solution, and the resultant solutions were incubated at ambient temperature for 24 h. The absorbance at 562 nm was measured by spectrophotometry and used to calculate the mass of protein released at each exposure interval on the basis of an intensity-concentration calibration curve generated from five solutions differing in G concentration due to progressive dilution of the parent G solution. The viscoelastic nature of several hydrogels was investigated by dynamic shear rheology conducted on a REOLOGICA rheometer with 25-mm parallel plates. The dynamic elastic modulus (G′) of hydrogels differing in MeOH treatment was measured at a constant oscillatory frequency of 1 Hz, shear strain of 0.1%, and normal force of 2.0 N, as detailed elsewhere.34 Results and Discussion Crystallization Conditions. Previous spectroscopic analysis35 of nonhydrated G/SF blends reveals that the MeOHinduced crystallization of amorphous SF is rapid, reaching saturation in just a few minutes. The swelling data evaluated at 20 °C and presented in Figure 1a confirm this behavior

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Figure 1. The degree of G/SF hydrogel swelling (Qf) at 20 °C as functions of (a) treatment time in a 75/25 w/w MeOH/H2O solution and (b) hydrogel composition. The hydrogels in part a differ in composition (W, in wt % G): 90 (O), 75 (b), 60 (4), 40 (2) 25 (]), and 0 ([). The solid and dashed lines serve to connect the data, and the error bars, which are smaller than the symbols employed here, denote the standard error. The inset provided in part a is an enlargement showing the data collected from SF-rich systems over the course of 60 min (shaded area).

for hydrogels varying in composition from 0 to 90 wt % G. In this case, the composition of the crystallization solution is 75/25 w/w MeOH/H2O and the treatment time extends up to 1200 min. Since the values of Qf are nearly independent of treatment time, it follows that most of the β-crystallization in SF occurs relatively quickly (within ∼5 min). In G-rich hydrogels, this observation reflects the permeability of MeOH within the hydrogel matrices. Because of the relatively low degree of SF crystallization in these hydrogels, the diffusivity and solubility of MeOH are expected to be high, as is the accessibility of individual SF molecules for dehydration and subsequent crystallization. Close examination of the swelling data collected from the pure SF and SF-rich hydrogels and displayed in the inset in Figure 1a, on the other hand, reveals that Qf decreases (by as much as 24%) over the first 60 min of MeOH treatment before finally reaching a steady value. Such transient behavior is attributed to crystal-induced reductions in MeOH permeability and SF accessibility. Included in Figure 1b is the composition dependence of Qf determined as an average quantity from the data provided in Figure 1a. This figure clearly demonstrates that Qf increases systematically with increasing G content (and decreasing SF cross-link density) in mixed G/SF hydrogels. We recognize, however, that at sufficiently low SF crosslink densities, non-networked SF may likewise leach from the hydrogels. The influence of MeOH solution composition on G/SF hydrogel property development after a crystallization treatment of 2 h at 20 °C is displayed in Figure 2. In Figure 2a, values of Qf measured from the G/SF75 hydrogel in PBS

Figure 2. Dependence of (a) G/SF75 hydrogel swelling (Qf) and (b) the dynamic elastic modulus (G′) on the composition of aqueous MeOH solutions used to induce SF crystallization over a period of 2 h. The data correspond to two temperatures (in °C)s20 (b) and 37 (O)sand the solid and dashed lines serve to connect the data.

Figure 3. Swelling (Qo) kinetics of G/SF hydrogels varying in composition (W, in wt % G)s100 (O), 90 (b), 75 (4), and 60 (2)sin PBS solution at 20 °C. The solid and dashed lines correspond to regressions of eq 3 to the data. Values of parameters acquired from these regressions are listed in Table 1. The inset is an enlargement showing the data collected over the course of 1 day. Table 1. Kinetic Parameters Discerned by Regression Analysis of the Hydrogel Swelling Data temp (°C)

W (wt % G)

A

20

100 90 75 60 75 60 40 25 0

0.00 0.00 0.00 0.00 3.03 1.49 0.94 0.54 0.27

37

a

k1 (days-1) 126 158 192 371 -10.3 -3.64 -2.06 -0.46

k2 (days-1) 9.04 26.5 76.2 277 4.50 3.76 4.30 2.73

Qo,∞a 13.9 5.96 2.52 1.34 0.74 0.52 0.46 0.38 0.27

Calculated from A + k1/k2.

solution after an equilibration period of 48 h decrease with increasing MeOH concentration up to 75 wt % MeOH and then increase thereafter at both 20 and 37 °C. Note that this variation in Qf is more pronounced at the elevated temper-

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Figure 4. Swelling (Qo) kinetics of G/SF hydrogels varying in composition (W, in wt % G)s75 (O), 60 (b), 40 (4), 25 (2), and 0 (])sin PBS solution at 37 °C. The solid and dashed lines correspond to regressions of eq 3 to the data. Values of parameters acquired from these regressions are included in Table 1. The inset is an enlargement showing the data collected over the course of 1 day.

Figure 5. (a) Mass retention (R) kinetics of G/SF hydrogels varying in composition (W, in wt % G)s75 (O), 60 (b), 40 (4), 25 (2), and 0 (])supon exposure to PBS solution at 37 °C. The solid and dashed lines correspond to regressions of eq 3 to the data. (b) Long-time mass retention (R∞) of G/SF hydrogels in PBS at 37 °C as a function of W determined by fitting eq 3 to the data in part a (0) and the data measured after 6 days (9). The solid line is a linear regression to the data.

ature due to conformational differences in G. At 20 °C, G possesses a triple-helix conformation that permits limited swelling with very little protein release. Increasing the temperature to 37 °C, however, permits the G chains to behave as expandable random coils. These swelling data are consistent with spectroscopic results35 that indicate aqueous MeOH solutions with ∼25-50 wt % water are most effective at inducing the β-sheet conformation of SF in G/SF blends, although the chemical origin of this response is not yet known. In addition, corresponding values of the dynamic elastic modulus are included as a function of MeOH concentration in Figure 2b and show that the solidlike character of the G/SF75 hydrogel is maximized when the blend is treated with a 75/25 w/w MeOH/H2O solution.

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Figure 6. Long-time swelling behavior of G/SF hydrogels after 6 days of exposure to PBS solution at 20 °C (filled symbols) and 37 °C (open symbols) presented in terms of Qf (circles, dashed lines) and Qo (squares, solid lines). Also included are regressed values of Qo,∞ (triangles) from Table 1 for comparison. The solid and dashed lines serve to connect the data.

Figure 7. Released protein from G/SF hydrogels differing in composition (W, in wt % G)s100 (O), 90 (b), 75 (4), 60 (2) 40 (]), 25 ([), and 0 (0)sinto PBS solution at two different temperatures (in °C): (a) 20 and (b) 37. The solid and dashed lines serve to connect the data.

Comparison of parts a and b of Figure 2 confirms that Qf and G′ are inversely related, since both constitute metrics of material rigidity.11 As Qf decreases (increases) due to variation in MeOH concentration, G′ is observed to increase (decrease). Similar behavior is apparent as the exposure temperature is elevated from 20 to 37 °C. As this occurs, G undergoes its hfc transition and transforms from a rigid, triple-helix conformation (high G′) to a soft, random-coil conformation (low G′). On the basis of these results, SF is crystallized in a 75/25 w/w MeOH/H2O solution for 2 h at 20 °C (to avoid premature dissolution of G) throughout the remainder of this study. Swelling and Release Kinetics. The swelling response of G/SF hydrogels differing in composition to PBS solutions at 20 °C is featured in Figure 3. Hydrogels composed of pure G swell substantially over the course of 1 day (see the inset in Figure 3a) and then continue to evolve slowly until they ultimately reach saturation. Similarly, the G/SF90



Figure 9. Fracture (a) and surface (b) SEM images of pure SF upon exposure to PBS solution for 48 h at 20 °C. Note the relatively featureless surface in part b. Similar morphologies are observed under comparable conditions at 37 °C and are not included here for that reason.

general kinetic expression of the form Qo ) A + Figure 8. SEM images acquired from pure G upon exposure to PBS solution for (a) 30 min and (b) 2 h at 20 °C. The enlargements provided in part a illustrate the platelike morphology observed.

hydrogel exhibits rapid, but less pronounced, swelling after 8 h of exposure and eventually attains saturation in the same fashion as the pure G hydrogel. The slow progressive increase of Qo in these two systems suggests that the swollen physical networks, stabilized for the most part by the triplehelix conformation of G, continue to loosen so that they accommodate additional water molecules as the exposure time increases. In contrast, the remaining hydrogels displayed in Figure 3 exhibit accelerated swelling and attain full saturation typically within 1 h. This feature is attributed to the β-sheet conformation of SF. The semicrystalline SF networks within mixed G/SF hydrogels are impermeable to water and likewise promote dimensional stability, resulting in rigidified hydrogels and markedly lower values of Qo. Since the values of mDo and mDf measured from these swelling experiments are comparable, the corresponding Qf(t) data are quantitatively similar to the Qo(t) results in Figure 3 and are not included here for that reason. The swelling data in this and subsequent figures can be represented by a

k1t 1 + k2t

(3)

where A, k1, and k2 are fitting parameters. Values of the fitting parameters extracted from the data in Figure 3 via nonlinear regression analysis are listed in Table 1. If the temperature of the PBS solution is increased to body temperature (37 °C), the G molecules comprising the G/SF hydrogels adopt a random-coil conformation and are subject to dissolution and extraction from the hydrogel matrix. For this reason, the swelling data provided in Figure 4 are likewise reported in terms of Qo, rather than Qf, and exclude the pure G and G/SF90 hydrogels. In all cases, the hydrogels begin at Qo ) 0 and then rapidly increase to a compositiondependent value after about 1 h. For clarity, the initial increase in Qo from Qo ) 0 is only featured in the inset included in Figure 4. After about 2 h, Qo(t) decreases abruptly. The value of Qo measured from the G/SF75 hydrogel gradually and continually changes over the entire course of the time range investigated (6 days), whereas Qo values determined from the remaining hydrogels tend to reach a plateau in less than 3 days. These results indicate that water permeation, followed by hydrogel swelling, occurs rapidly within the first hour of exposure and initially dominates the hydrogel response to the surrounding aqueous

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Figure 10. Fracture (a,c) and surface (b,d) SEM images of G/SF25 hydrogels after exposure to PBS solution for 48 h at 20 °C (a,b) and 37 °C (c,d). Except for a few indentations measuring several micrometers across in part d, these images closely resemble those presented for pure SF in Figure 9.

environment. Over the next hour or so, permeation and swelling compete with protein extraction due to G dissolution. After this time, protein extraction from the hydrogels becomes the dominant process as the swollen hydrogels irreversibly lose mass upon continued exposure. It is important to recognize that this reduction in hydrogel mass at 37 °C is not observed at 20 °C (cf. Figure 3), since the helix-stabilized G molecules remain virtually water-insoluble below their hfc transition. The curves included in Figure 4 correspond to regressions of eq 3 to the data (with the initial time assigned to 1 h), and the fitting parameters obtained from these regressions are listed in Table 1. The Qo data in Figure 4 can alternatively be presented in terms of mass retention (R) according to eq 2 by assuming that the initial weight occurs after 1 h of exposure. The time dependence of R for G/SF hydrogels exposed to PBS solution at 37 °C is evident in Figure 5a and confirms that the G-rich hydrogels undergo extensive mass loss (∼70% for the G/SF75 hydrogel after 6 days) at this temperature. As the SF concentration in the hydrogels is increased, the extent to which mass is retained in the hydrogel increases systematically, with the pure SF hydrogel exhibiting almost negligible mass change over the full course of 6 days. The curves featured in Figure 5a are generated from a regression analysis of a kinetic expression identical in form to the one provided in eq 3. Values of hydrogel mass retention at long exposure time (R∞), obtained from (i) plateau values derived from regression analysis (where R∞ ) A + k1/k2, as in Table 1)

and (ii) experimental data measured after an exposure time of 6 days, are displayed as a function of hydrogel composition in Figure 5b. The results in this figure exhibit a surprisingly linear reduction in long-time mass retention with increasing G content. It is interesting, and perhaps fortuitous, that the regressed linear relationship in Figure 5b predicts complete protein extraction from pure G hydrogels at 37 °C. To explore further the competition between hydrogel swelling and protein extraction at 20 and 37 °C, values of long-time swelling discerned from regression of eq 3 to the data (Qo,∞, calculated from A + k1/k2), as well as experimental data acquired after 6 days, are shown as functions of hydrogel composition in Figure 6. As mentioned earlier, Qf(t) and Qo(t) are comparable at 20 °C, verifying that little, if any, protein is extracted from the G/SF hydrogels at this temperature. At 37 °C, however, Qf values consistently become larger, while Qo values become smaller than their respective values at 20 °C, confirming that the hydrogels gain more water and lose more mass at 37 °C than at 20 °C. The most probable reason for such behavior is selective extraction of G at 37 °C, since the pure SF hydrogels show little mass loss during exposure to PBS solution at this temperature (cf. Figure 5a). The amount of protein released from the G/SF hydrogels into PBS solution at 20 and 37 °C has also been measured with the colorimetric assay described in the Experimental Section. Resultant protein release profiles are presented in Figure 7a,b. At 20 °C, only the pure G and G/SF90 hydrogels


Figure 11. SEM images of fractured G/SF75 hydrogels after exposure to PBS solution for (a) 30 min and (b) 48 h at 20 °C. Largescale porosity reminiscent of that observed for G in Figure 8 is evident.

display evidence of continued protein release after the hydrogels reach their fully swollen state. According to the data reported in Figure 7a, the remaining hydrogels remain nearly insoluble after their initial swelling at this temperature. At 37 °C, however, almost all the G/SF hydrogels continue to release protein after they are fully swollen (cf. Figure 7b). Since the G molecules adopt a random-coil conformation and readily dissolve in aqueous solutions at this temperature (which is the reason why the G/SF100 specimen is not included in Figure 7b), they are most likely responsible for the released protein, although we do not discount the possibility that a small (but expectedly negligible) quantity of SF may also be released into solution at both temperatures. Silk fibroin is reported to consist of a mixture of long and short chains with molecular weights of ca. 350 and 25 kDa, respectively. Although long SF chains can form complex supramolecular structures,37 they are expected to crystallize more readily than short SF chains, which may instead adopt a globular structure and remain amorphous even after MeOH treatment. Although the short chains constitute about 50 mol % of SF, they appear to remain within the mixed hydrogel networks, since little SF is released into the PBS solution at either 20 or 37 °C. This observation strongly suggests that the Cys residues in the N- and C-termini of the SF long chains promote the formation of disulfide bonds with the SF short chains, thereby resulting in a covalent linkage between the long and short chains and effective immobilization of the short chains upon exposure to solution.


Morphological Evolution. The morphological characteristics of select G/SF hydrogels after exposure to PBS solution and subsequent freeze-drying have been examined by SEM. Figure 8 is a series of SEM images acquired from pure G hydrogels at 20 °C. After just 30 min of exposure to solution (Figure 8a), the hydrogel is observed to swell substantially, resulting in a highly porous polymer upon solvent removal, in agreement with the swelling kinetics featured in Figure 3. The 10× enlargements included in Figure 8a facilitate scrutinization of the fine sheetlike microstructure presumably afforded by the triple-helix conformation of G. The thickness of the sheets is estimated between 250 and 400 nm. After 2 h of exposure to solution (Figure 8b), the hydrogel has continued to swell, since the pores generated upon solvent removal are larger and more fully formed than those evident in Figure 8a. Complementary images of the G hydrogel could not be collected at 37 °C due to dissolution considerations. In marked contrast, pure SF hydrogels show no evidence of swelling after 48 h at 20 °C, due most likely to (i) the intrinsic hydrophobicity of SF and (ii) the presence of rigidifying β crystals. The corresponding SEM images of a specimen fracture (Figure 9a) and surface (Figure 9b) verify that the aqueous solution has little effect on SF hydrogel morphology, although some small surface pits measuring ca. 500 nm in diameter are visible in Figure 9b. Qualitatively similar results have been obtained at 37 °C and are not reproduced here for that reason. Thus, pure G and SF hydrogels exhibit markedly different morphologies upon exposure to PBS solution at 20 and 37 °C. At intermediate hydrogel compositions, the extent to which the G/SF hydrogel morphology evolves due to swelling is anticipated to be highly composition-dependent. In the SF-rich G/SF25 hydrogel, for instance, fracture and surface morphologies appear virtually identical to each other at 20 and 37 °C (Figure 10, parts a,b and c,d, respectively), as well as to the pure SF hydrogel at 20 °C in Figure 9, after 48 h. It is important to recognize that there is no evidence of pores remaining upon solvent removal from a highly swollen hydrogel matrix in Figure 10. This observation is consistent with the data featured in Figure 4, wherein the swelling behavior of the G/SF25 hydrogel is seen to be comparable to that of the pure SF hydrogel at 37 °C. A G-rich hydrogel, on the other hand, is expected to be much more sensitive to solvent-induced swelling. An image of a fractured G/SF75 hydrogel acquired after just 30 min of hydration at 20 °C (cf. Figure 11a) clearly reveals the onset of swelling, whereas the same hydrogel exhibits profound swelling (and pore formation upon solvent removal) after 48 h at the same temperature (cf. Figure 11b). Note that the pores in Figure 11b (G/SF75, 48 h) are smaller than those in Figure 8b (G/ SF100, 2 h), despite the substantially longer exposure time under isothermal conditions. Increasing the solution temperature to 37 °C yields the highly porous fracture and surface images of the G/SF75 hydrogel displayed in Figure 12. After just 30 min, the hydrogel exhibits pronounced swelling and internal pore formation, as well as selective extraction through the specimen surface (cf. Figure 12b). Upon further exposure (2 h), the internal morphology does not change appreciably, but the pores extending through the specimen

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Figure 12. Fracture (a,c) and surface (b,d) SEM images of G/SF75 hydrogels after exposure to PBS solution for (a,b) 30 min and (c,d) 2 h at 37 °C. Note the formation of surface pores that grow in size with increasing exposure time due to protein release.

Figure 13. Schematic diagram illustrating the competitive swelling and protein release mechanisms encountered by pure G and G/SF hydrogels at both ambient and body temperatures.

surface enlarge due to continual protein release, as evidenced by the protein release data reported in Figure 7b. These images therefore provide an accurate physical depiction of the morphological changes that accompany the competitive mechanisms of swelling and release in mixed G/SF hydrogels at various compositions and solution temperatures. Conclusions Novel protein-based mixed hydrogels have been produced by blending G with amorphous SF and subsequently inducing

SF crystallization, which serves to stabilize the hydrogels at elevated temperatures. In the present study, we report on the swelling and protein release kinetics of G/SF hydrogels varying in composition at temperatures below and above the G hfc transition. The swelling behavior of these hydrogels reveals that β-crystallization is (i) virtually complete after only ∼5 min of exposure to MeOH in a 75/25 w/w MeOH/ H2O solution and (ii) sensitive to MeOH concentration in the aqueous MeOH solutions used to induce crystallization. The extent to which G/SF hydrogels swell is consistently higher at 37 °C than at 20 °C, indicating that the dissolved triple-helix conformation of G in the mixed hydrogel networks permits greater water sorption. For similar reasons, swelling kinetics confirm that the G/SF hydrogels undergo little mass loss at 20 °C but continuously lose protein as G molecules are slowly released into the surrounding aqueous solution at 37 °C. The illustration presented in Figure 13 depicts these two scenarios and shows the importance of the triple-helix and β-sheet conformations as physical cross-link sites that together stabilize the hydrogel networks at 20 °C. At 37 °C, however, the triple-helix conformation is replaced by random coils that expand and dissolve into the surrounding aqueous environment, in which case the β crystals of SF become solely responsible for stabilizing the G/SF hydrogels at elevated temperatures. Due to their tailorable composition- and temperature-dependent swelling and release properties, these mixed G/SF hydrogels are anticipated to constitute attractive biomaterials that are ideally suited for thermally responsive biomedical and pharmaceutical ap-


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