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Biomacromolecules 2005, 6, 1635-1641 1635 Structure-Property Relationships in Poly(ethylene glycol)-Protein Hydrogel Systems Made from Various Proteins Kirill I. Shingel* and Marie-Pierre Faure Bioartificial Gel Technologies Inc., 400 Maisonneuve West, Suite 1156, Montreal, H3A 1L4 Quebec, Canada Received November 29, 2004; Revised Manuscript Received February 15, 2005 A series of poly(ethylene glycol)-protein hydrogels were synthesized with different proteins, and the resultant structures were characterized in terms of swelling behavior and mechanical, optical, and drug release properties. Irrespectively of the protein involved in polymerization with poly(ethylene glycol), all studied systems were found to be loosely cross-linked networks, where both polymer and protein are completely solvated, enabling as high as 96% water content. Changes in the apparent transparency of the hydrogels synthesized with different proteins were attributed to the ability of the protein component to self-associate via hydrophobic interactions. The polyelectrolyte nature of the protein component governs the pH responsiveness of the network, which manifested itself in a pH-dependent mechanism of swelling and drug release. It was demonstrated that there is great opportunity to modulate the final characteristics of the hydrogel system to fit the need of specific biomedical application. 1. Introduction On the basis of original research on poly(ethylene glycol) (PEG)-bovine serum albumin conjugates by G. Fortier’s group,1,2 we created a concept of protein-based biocompatible hydrogels for use in various cosmetic and pharmaceutical applications.3 This hydrogel represents a network of the protein cross-linked with PEG macromolecules. Due to the hydrophilic nature of both components, this material may contain as much as 96% water in their structure, thus forming an excellent liquid compartment for sustained drug release. Although PEG is widely used in hydrogel chemistry and readily available in numerous activated forms,4 PEG-protein drug delivery systems are less known. T. J. Sanborn et al. has recently synthesized peptide-PEG hydrogels5 which, however, are not homologous to our material since only a short peptide chain derived from fibrin was taken in that study, as opposed to relatively high molecular weight proteins used in this work for gel preparation. High water content combined with the presence of a natural polymer makes protein-containing hydrogels inherently biocompatible, which is an obvious advantage of such hydrogels over widely used HEMA or polyacrylamide materials. It should be noted that the influence of the molecular weight of PEG and its content on the swelling behavior and mechanical and drug-releasing properties of the gel was investigated in detail,6,7 whereas effects of the protein component attracted less attention. Therefore, to better understand how the molecular properties of the protein influence the physical appearance and structural peculiarities of the materials, we synthesized a series of hydrogels from soy protein, hydrolyzed soy protein, * To whom correspondence should be addressed. Fax: (514) 280-7805. E-mail: [email protected]. pea albumin, and casein. All these proteins became popular and accepted in recent years as additives for cosmetic formulation. Pea albumin, for example, is used to improve the elasticity of the skin by decreasing its surface tension.8 A direct administration of bioactive proteins, however, may be associated with allergic reactions and skin irritation. A pegylation approach used in preparing protein-based hydrogels serves to eliminate these undesirable effects. It should be mentioned that the protein hydrogel is expected to possess complex patterns of physicochemical characteristics because of the great diversity in the physicochemical properties of proteins which are taken or potentially available for synthesis. This gives an opportunity to modulate the final characteristics of the hydrogel, which, in turn, may accelerate development of the optimal design of this drug delivery system. Important factors that may influence hydrogel structure and properties are the polyelectrolyte nature of the biopolymers and availability of hydrophobic regions for intermolecular interactions.9 The idea that all these properties of the proteins would inevitably govern the properties of the resultant hydrogel prompted us to perform this study. 2. Materials and Methods 2.1. Materials. Soy protein isolate (SPI) with protein content 96% was used “as received” from Archer Daniels Midland Co. (Decatur, IL). Pea albumin was purchased from Coletica (France). Casein of cosmetic grade was from American Casein Co. (Burlington, NJ). Poly(ethylene glycol) bis(p-nitrophenyl carbonate) or “activated PEG” was synthesized by the reaction of PEG-8000 with p-nitrophenyl chloroformate in dichloromethane (CH2Cl2) as described elsewhere,10 and was used as a macromolecular cross-linking agent of proteins. 10.1021/bm0492475 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/19/2005 1636 Biomacromolecules, Vol. 6, No. 3, 2005 All other chemicals purchased from Sigma or J.T. Baker were of analytical, ACS reagent, or equivalent grade. 2.2. Protein Solubilization. It is known that the formation of the solid hydrogel material upon cross-linking with activated PEG is possible only at a certain optimal concentration of the protein6 dependent on the molar ratio of activated carbonate groups of PEG and accessible amino groups of the protein. Preliminary experiments revealed that the optimal concentrations for pea albumin, SPI, casein, and hydrolyzed soy protein are 80, 100, 125, and 120 mg/mL, respectively, when the concentration of activated PEG is kept constant at 220 mg/mL. For the hydrogel synthesis, protein solutions in which the protein concentration was 100 mg/mL for SPI, 125 mg/mL for casein, and 120 mg/mL for hydrolyzed soy protein were prepared in 0.16 M sodium hydroxide. The suspension obtained upon dissolution of crude pea albumin in 0.16 M sodium hydroxide was centrifuged, and only the supernatant fraction diluted to the protein concentration of 80 mg/mL was used for gel synthesis. For analytical assays, soluble protein fractions from both hydrolyzed soy protein (HSP) and SPI was extracted by 0.1 M Na2HPO4 at pH 9.3. The suspensions were incubated at 45 °C to facilitate protein extraction. After 3 h of incubation, the suspensions were separated by centrifugation. Supernatants containing protein were filtered through a 0.22 µm membrane filter and used for analysis. 2.3. Gel Synthesis. An aqueous solution of activated PEG was mixed with an equal volume of the prepared protein solutions, and the resultant mixture was placed between two glasses to obtain a gel with a thickness of about 1.8 or 1.0 mm. The hydrogel matrix reticulation is known to appear due to formation of the urethane links between free amino groups of protein and PEG-carbonate moieties.6 After the polymerization was completed, the gel was placed in PBS buffer to wash out p-nitrophenol that was formed as a byproduct of the cross-linking reaction. Here, the hydrogels are coded as SOYGEL, PEAGEL, CASGEL, and HSOYGEL for the samples prepared with soy protein, pea albumin, casein, and hydrolyzed soy protein, respectively. 2.4. Gel Characterization. Preweighed disks of the gels (about 250 mg) were hydrolyzed in 3 mL of 1.0 M sodium hydroxide at 45 °C. The solution thus obtained was subjected to protein and PEG content determination by means of bicinchoninic acid and iodine colorimetric assays, respectively,6 using a “Benchmark” microplate spectrophotometer from BioRad. All the measurements were performed in triplicate, and the data were expressed as an average. The equilibrium water content (EWC) was estimated from gravimetric measurements of the dry and completely swollen hydrogels as follows: EWC (%) ) 100 (Ws - Wd)/Wd, where Ws and Wd stand for the weights of the swollen and dry gels, respectively. Transparency of the gel was characterized by indexes of absorbance of 2.25 mm thick pastilles at 450 nm. Additional measurements were performed for 1.18 mm thick hydrogel samples. The swelling behavior of the SOYGEL and PEAGEL samples at different pHs was studied to determine the Shingel and Faure correlation between the solubility of the protein and the ability of the corresponding gels to absorb water. In these experiments, the solubility of the proteins in the range of pH 3.5-9.0 were determined as the percentage of the soluble fraction of the protein after reconstitution in a solvent, incubation, and centrifugation of the corresponding initial protein solutions. In osmotic deswelling experiments preweighed gel samples were covered with a cellulose membrane for dialysis (molecular weight cutoff limit 10000). The samples were incubated in the solutions of PEG 20000 with polymer concentration ranging from 0.25 to 25.0 g/dL. After the equilibrium between gel pastilles and the surrounding solutions was reached, the samples were weighed again. The values of the polymer volume fraction were calculated from gravimetric measurements, and the equilibrium swelling pressure was then determined, as described by Stubbe et al.11 Similar experiments were performed with hydrogel samples that were loaded with a 1% (w/v) solution of caffeine. Mechanical properties of the hydrogel samples prepared with different proteins were tested by using the Mach-1 compression system (Biosensing Inc., Laval, Quebec, Canada) equipped with the 150 g load cell, which measured the load (g) as a function of displacement (µm) during compression and had a working surface of 0.503 cm2. The hydrogel pastilles of known thickness (measured by a micrometric magnifier) were subjected to 10-17 subsequent compression ramps with an amplitude of the load cell displacement of 100 µm (about 5% of the initial pastille thickness) and a compression velocity of 50.0 µm/s. The relaxation time after compression was found to be 30 s, and this value was used for all studied samples. The values of the load for each compression-relaxation step was recorded, presented as a function of the load cell displacement, and used for calculation of the elasticity modulus E: E ) (∆N/∆L)h0 (1) where N is the load at a displacement L and h0 is the initial thickness of the hydrogel sample. 2.5. Drug Release. Samples were incubated in a 1% (w/v) solution of caffeine in PBS buffer for 24 h to integrate the drug into the hydrogel. The volume ratio of the gel samples to caffeine solution was ∼1:100. After 6 h of incubation the medium was changed to a new aliquot of a fresh solution of the drug. The concentration of caffeine in the gel samples after incubation was determined to be 0.98% ( 0.04%, indicating that the partition coefficient of caffeine between the gel and bulk solution at equilibrium was close to 1.0. The gel pastilles (about 250 mL) with integrated 1% (w/v) caffeine were then immersed in PBS solution, pH 7.4, for caffeine release. Aliquots taken at certain intervals of time were subjected to chromatographic analysis for caffeine quantification. The amount of caffeine released from the hydrogels (Mt) was expressed as a percentage of the total amount of the drug incorporated into the samples (Mt/M0). These data were analyzed on the basis of Fick’s law of the solute diffusion from the slab of thickness l: Mt/M0 ) 4(Dt/πl2)1/2. Diffusion coefficients Dcaff (cm2/s) for caffeine were derived from the slope of the linear part of the plot PEG-Protein Hydrogels Mt/M0 versus t0.5. Similar experiments were performed in determining the rate constants of p-nitrophenol release, kpNP (min-1). The permeability of the hydrogels was studied using streptomycin, caffeine, and methylparaben as model solutes. Hydrogel samples were soaked separately in 1% (w/v) solutions of the drugs, and release of the molecule was monitored in the experiments with static cells,12 as reported previously.13 Quantification of the drugs in the release media was carried out by means of HPLC, and the data were analyzed in terms of the quantity of the drug that was loaded (C0) and released from the gels at time t (Ct). Permeability coefficients (P, cm2/s) were calculated from the plots of (Vδ/2A) ln(1 - (2Ct/C0) versus t, where V designates the volume ratio of the gel and receptor medium, A is the surface area, and δ is the thickness of the gel slab.14 2.6. Chromatography. The molecular weight of the proteins (native and hydrolyzed soy proteins, pea albumin, and casein) was estimated by means of size-exclusion chromatography (SEC). SEC was carried out by using a Hewlett-Packard 1050 chromatography system (Agilent Technologies, Inc., Delaware). The molecular weights of native soy proteins are known to vary considerably, from 10000 to 100000; therefore, to achieve satisfactory separation within this range, we used a TSK G4000PWXL (300 × 7.8 mm i.d., 5 µm particle size) column packed with a methacrylate-based hydrophilic stationary phase (TosoBiosep, Stuttgart, Germany). To eliminate any undesirable effects of protein sorption on the polymeric phase, a solution of 0.05 M potassium phosphate (pH 9.0) containing 20% (v/v) methanol was used as the mobile phase. The molecular weights of the proteins under investigation were derived from the calibration curve constructed using the following proteins: aprotinin, Mr ) 6500; R-lactalbumin, Mr ) 14200; carbonic anhydrase, Mr ) 36000; ovalbumin, Mr ) 45000; bovine serum albumin, Mr ) 66000; fructose-6-phosphate kinase, Mr ) 84000; β-galactosidase, Mr ) 116000; myosin, Mr ) 205000. Denaturing SEC was performed with 0.05 M potassium phosphate (pH 9.0), where urea was added up to a final concentration of 0.5 M. In both SEC experiments the flow rate was 1.0 mL/min. The component composition of the proteins was also studied by means of reversed-phase chromatography (RP-HPLC). RP-HPLC was performed in the gradient mode on a system consisting of a type 600E pump, a 776 autosampler, and a 996 photodiode array detector, all obtained from Waters (Milford, MA). The column used was an Ace C4 (150 × 3.9 mm i.d., particle size 5 µm) silica-based column thermostabilized at 40 °C. The mobile phase composed of 0.1% TFA in water (eluent system A) and 0.075% TFA in 60% (v/v) acetonitrile (eluent system B) was pumped at a flow rate of 1.0 mL/min, and the column effluent was measured within the wavelength range 210-300 nm at a 4.8 nm resolution. The elution was carried out with a linear gradient of B from 0 to 90% for 30 min followed by isocratic elution in 90% B for 5 min. Before chromatography, the mobile phases were filtered through membrane filters (pore diameter 0.22 µm) (Millipore) and degassed for 20 min. Biomacromolecules, Vol. 6, No. 3, 2005 1637 Figure 1. Size-exclusion chromatograms of soy protein (a), hydrolyzed soy proteins (b, c), and pea albumin (d) used for gel synthesis in 0.05 M potassium phosphate (pH 9.0) with 20% (v/v) methanol (TSK G4000PWXL column, flow rate 1.0 mL/min). Analysis of caffeine, streptomycin, and methylparaben in drug release experiments was carried out on a HewlettPackard 1050 chromatography system (Agilent Technologies) in isocratic mode on an Ace C18 reversed-phase column, using a 20% (v/v) methanol or 25% (v/v) acetonitrile solution in 0.025 M phosphoric acid as the mobile phase. Detection was performed at 272 nm (caffeine), 210 nm (streptomycin), 254 nm (methylparaben), or 317 nm (p-nitrophenol), followed by quantification of the drug, using a five-point standard calibration curve. 3. Results and Discussion 3.1. Molecular Weight of Proteins. Size-exclusion analysis revealed that all proteins are not homogeneous with respect to molecular weight (see Figure 1). Native soy protein (SPI) was found to contain two major components eluted in the volumes that corresponded to values of 92800 and 34600. Similar results were obtained recently for β-conglycinin and glycinin components of this protein from SDS-PAGE experiments,15 although the values of 18000016 and 302000-37500017 are more commonly known for soybean proteins in the aggregated state. Acid hydrolysis of proteins leads to a decrease of the molecular weight of the proteins, which was indeed found for hydrolyzed soy protein (see Figure 1). The chromatographic profiles of hydrolyzed soy protein samples corresponded to a molecular weight of 38000-43000, although some minor high molecular weight species (about 210000) were also observed, indicating that the hydrolysis was not complete. These estimates are in good agreement with the results of SDS-PAGE electrophoresis of the hydrolyzed soy protein (M. Robert, M. P. Faure, unpublished data) and outcomes of SEC in denaturing conditions (data not shown here). The molecular weight of pea albumin components was equal to 38000 and 23600, whereas casein was eluted as a single peak with Mr ) 26000. In both cases, a satisfactory correlation was found between the molecular weight values estimated by means of SEC and those reported in the literature for casein18 and pea albumin fractions.18,19 1638 Biomacromolecules, Vol. 6, No. 3, 2005 Shingel and Faure Table 1. Component Compositions, Initial and Equilibrium Water Contents, and Diffusion Characteristics of the Hydrogels Synthesized with Different Proteins (Mean Value ( SD, n ) 3) code protein Mr [PEG], mg/g of gel protein concn, mg/g of gel initial water concn, % EWC, % Dcaff, cm2/s × 10-7 SOYGEL-1 SOYGEL-2 HSOYGEL-1 HSOYGEL-2 PEAGEL CASGEL-1 CASGEL-2 92800, 34600 92800, 34600 38000 43000 38000, 23600 26000 26000 13.78 ( 2.77 16.27 ( 5.17 19.05 ( 4.83 17.60 ( 3.95 13.37 ( 3.49 14.19 ( 2.12 15.13 ( 3.17 24.85 ( 1.12 29.14 ( 4.20 50.08 ( 1.20 50.14 ( 1.23 22.55 ( 1.95 13.78 ( 1.80 11.91 ( 1.84 68.0 68.0 64.0 64.0 70.0 63.5 63.5 95.78 ( 0.32 95.06 ( 0.24 92.69 ( 0.29 92.68 ( 0.16 95.60 ( 0.24 96.27 ( 0.25 96.16 ( 0.19 6.73 ( 1.25 6.54 ( 1.12 4.57 ( 1.47 4.69 ( 0.83 7.48 ( 2.21 6.78 ( 1.03 7.85 ( 1.64 Figure 2. Optical density of the hydrogels measured at 450 nm as a characteristic of the transparency of the 2.25 mm thick pastilles of poly(ethylene glycol)-protein material. 3.2. General Properties of the Hydrogels and Physical Appearance. To achieve a maximal water-absorbing capacity of the hydrogel as a necessary prerequisite for successful drug delivery application, proteins with different molecular weights were cross-linked with activated PEG. The component composition and physical characteristics of these gels are summarized in Table 1 and Figure 2. The values of EWC found from hydrogel swelling experiments (EWC ) 92-97%) (see Table 1) are in good agreement with the values obtained from summation of the PEG and protein contents, but exceed the values calculated from the initial amounts of PEG and proteins used for gel synthesis (initial water content 64-70%). This indicates that the gels immediately after synthesis are not at the equilibrium swelling level and are able to absorb solvent upon subsequent incubation in aqueous solution. It should be noted that the content of protein and PEG is expressed in Table 1 as a mass ratio to the weight of fully swollen gel, i.e., when the network imbibes the maximal volume of water and additional swelling is precluded by the covalent linkages and/or physical interactions between the protein and PEG. A statistically meaningful variation (p < 0.05) in EWC data is observed for hydrogels synthesized from different proteins, for example, SOYGEL (EWC ) 95-96%) and HSOYGEL (EWC ) 92.7%) samples (Table 1). Although the concentrations of the proteins taken for synthesis were close, we found that HSOYGEL samples have 2 times higher protein content than SOYGEL. As is known, the ability of a hydrogel to swell is dependent on the density of cross-links and/or occurrence of noncovalent interactions between components included in a network. Therefore, taking into account that all studied samples comprise about the same amount of PEG, the difference in the EWC values (Table 1) may be attributed to the structural differences of hydrogels formed by different proteins and their affinity toward water and also ability to bind solvent. 3.3. Influence of pH on the Swelling of HSOYGELs and PEAGELs. The solubility of proteins in varied pHs was found to be another important factor governing the swelling behavior of the studied materials. The solubility of proteins is known to be a function of numerous factors, among which are the net charge of the macromolecules and its distribution along the backbone and the character of protein-solvent and protein-protein interactions. For example, at pH 4.5 soy protein has a lower solubility, while high pH denatures the protein, facilitating its dissolution.17 This behavior, partially explained by the presence of a large proportion of acidic amino acids in the structure of this protein,20,21 was also seen in this study (see Figure 3). It is interesting to note that the difference in the solubility of proteins impacts the swelling capacity of the corresponding hydrogels at varied pHs (see Figure 3). Generally, the swelling capacity of a HSOYGEL increases with increasing pH, and the dependence of the swelling capacity on pH shows a trend similar to that obtained for the solubility of non-cross-linked protein. Close to the isoelectric point (pI ) 4.5) soy protein has the lowest solubility, and respectively, the swelling capacity of SOYGEL is minimal at this pH. At pH higher than 7.0, the network is completely ionized, since the pH is greater than the pK of the carboxylic groups of the biopolymer. This ionization increases the water-binding capacity of the hydrogel structure, leading to a higher swelling power. The changes in the ionization of the HSOYGEL are accompanied by the structural alterations, leading to the pH-controlled diffusion of the solute through the hydrogel matrix (see Figure 3a). A contrasting trend of pH-dependent solubility was observed for pea albumin (see Figure 3b). The solubility of this protein increases with decreasing pH; however, the corresponding gel showed no pH responsiveness for swelling capacity. To obtain this gel, the suspension obtained by dispersing pea albumin at pH 11.5 was centrifuged so that only the alkaline-soluble fraction of pea protein was crosslinked with PEG. It is interesting to note that this alkalinesoluble fraction precipitates from the solution at neutral pH, but the corresponding hydrogel shows no pH-dependent structural changes (Figure 3b). 3.4. Hydrophobicity of Proteins and Transparency of the Gels. Transparency of the hydrogel is important both esthetically and practically, as this property permits visual inspection of the site of application in various biomedical PEG-Protein Hydrogels Biomacromolecules, Vol. 6, No. 3, 2005 1639 Figure 4. Reversed-phase chromatograms of soy protein (a) and hydrolyzed soy protein (b) on a C4 reversed-phase column in a linear gradient of acetonitrile concentration. Figure 3. Solubility of hydrolyzed soy protein (a) and pea albumin (b), and the swelling capacity of the hydrogels synthesized thereof as a function of pH. The rate constants of p-nitrophenol release, kpNP, are shown to demonstrate the pH-dependent mechanism of the solute diffusion in hydrogel. applications. The colorimetric determination of the intensity of the light scattered by the protein aggregates is shown in Figure 2. PEAGEL is completely transparent, as it has the lowest index of absorbance at 450 nm, whereas other samples are characterized by an opalescence, which increases in the order PEAGEL > CASGEL > HSOYGEL > SOYGEL (see Figure 2). The poor transparency of the soy protein gels can be explained on the basis of the inherent ability of this protein for self-association and self-assembly in solution due to hydrophobic interaction and formation of disulfide bridges (-S-S-) between cysteine residues in response to different conditions including pH, temperature, ionic strength, etc.17 Hence, the degree to which these macromolecules are prone to form aggregates could be evaluated by measuring the surface hydrophobicity of the protein. In this study the hydrophobicity of soy proteins was evaluated from RP-HPLC experiments. Typical elution profiles of native and hydrolyzed soy proteins are shown in Figure 4. The major components of SPI are eluted at 13 and 22-26 min, whereas, in contrast, the peaks found for hydrolyzed soy protein are shifted toward shorter retention times and appear between 18 and 23 min, indicating that these macromolecules after hydrolysis are less retentive on the reversed-phase column. The retention time of soy protein fragments was found to increase with increasing hydrophobic moiety content (not shown), and this strongly evidences the occurrence of a hydrophobicity-based mechanism of protein separation. Thus, the chromatographic behavior found for hydrolyzed soy proteins suggests a lowered hydrophobicity of the biopolymer as a result of the acid treatment. Hydrolysis of highly aggregative native soy protein19 is believed to reduce the ability for self-association, which results in the formation of a more transparent hydrogel upon polymerization with PEG (Figure 2). These findings illustrate the existence of the relationships between the processes of the self-association and the physical appearance of the hydrogels, in particular their visual transparency. 3.5. Mechanical and Drug Release Properties. Typical stress-relaxation curves obtained for all studied hydrogels are depicted in Figure 5. In these experiments, a load cell is brought into contact with the surface of the gel, thus generating the load data as a function of the cell displacement. A set of 10-17 compression ramps were performed thus that once a certain displacement is achieved, the load recedes and the material starts to relax to an equilibrium level (see Figure 5). For all 10 cycles, the relaxation time did not exceed 30 s; however, the amplitude of the load generated by the constant 100 µm displacement was found to increase with increasing number of compression steps (see Figure 5). This can be explained by structural alterations of the original network, which become denser as the load cell advances 1640 Biomacromolecules, Vol. 6, No. 3, 2005 Shingel and Faure Figure 5. Stress-relaxation curves obtained for HSOYGEL-1 (a) and PEAGEL (b) samples upon the application of multiple compression ramps with an amplitude of the load cell displacement of 100 µm and a compression velocity of 50.0 µm/s. The values of the elasticity modulus of HSOYGEL-1 (1) and PEAGEL (4), as calculated for each compression step with the use of eq 1, are plotted in the inset. progressively into the gel. The elasticity modulus also increased with increasing number of compression steps, demonstrating nonlinear elasticity of the material (see the inset in Figure 5) upon compression. The relatively short relaxation times are symptomatic for the loosely cross-linked network, where the motion of macromolecules is only lightly restricted by cross-links. Indeed, for the hydrogels with such high water content the theory predicts a relatively low crosslinking density.22 Interestingly, a linear elastic response of the material was observed recently in the experiments with the hydrogel samples undergoing tensile deformation (unpublished results). The disparity observed between compressive and tensile properties of the protein-containing hydrogel requires further investigations, being supposedly associated with the specific microstructure of the network. Water or aqueous solution represents a primary transport vehicle in the studied systems. Therefore, an analysis of solvent-network interactions is important for understanding the mechanism of drug release. The affinity of the studied hydrogels toward aqueous solvent was studied in osmotic deswelling experiments, which allowed calculation of the osmotic pressure of the hydrogel, Πsw, as a function of the polymer volume fraction φ (see Figure 6). Dependences of Πsw on φ were then analyzed by using the equation23 ∏sw ) Aφn - Aφn-1/3φ1/3 (2) where n is a constant showing a thermodynamic quality of the solvent. The calculated values of n are summarized in Table 2 for several gels in pure water and in a 1% (w/v) solution of caffeine. The values of n in the range of 2.24-2.42 obtained in pure solvent are indicative of good solvent conditions,23 and this serves as strong evidence of the high affinity of the network toward an aqueous solvent. It appears that the protein component of the hydrogel has a strong influence on its diffusion barrier properties. This effect is illustrated in Table 1 by the apparent diffusion coefficient of caffeine, which was taken as an example of a Figure 6. Osmotic swelling pressure of the hydrogels as a function of the polymer volume fraction. The dashed lines represent the best fit approximating experimental points, as calculated by eq 2. Table 2. Values of Exponent n and Correlation Coefficient R Obtained from the Least-Squares Fits to Eq 2 for Hydrogels in Pure Solvent (naq) and 1% (w/v) Caffeine Solution (ncaff) sample naq R ncaff R Soygel-1 Hsoygel-1 Casgel-1 Peagel 2.241 2.424 2.295 2.252 0.998 0.982 0.999 0.999 3.097 2.550 2.408 2.352 0.996 0.997 0.997 0.999 nonionic drug. HSOYGEL samples are characterized by the smallest diffusion coefficients, as can be expected from the structural peculiarities discussed above and the EWC values of this hydrogel (Table 1). Another factor responsible for the observed retardation of the diffusion in this gel is believed to be the interaction between caffeine and hydrophobic moieties of the protein. This was confirmed by measuring the permeability of the HSOYGEL for streptomycin, caffeine, and methylparaben. Interestingly, the value of the permeability coefficient for streptomycin (Pstr ) 6.49 × 10-5 cm2/s) exceeds those of methylparaben (Pmp ) 0.96 × 10-5 cm2/s) or caffeine (Pcaff ) 2.51 × 10-5 cm2/s) despite the larger hydrodynamic size of the streptomycin molecule. These results thus demonstrate that there are probably strong interactions between the solutes and the protein network which are favored by the pronounced hydrophobic character of both caffeine and methylparaben. For all studied systems the diffusion coefficients are much smaller than the self-diffusion coefficient of bulk water, D ) 2.8 × 10-5 cm2/s. This indicates that, in the studied gels, the diffusion/transport of both water and drug is governed by a diffusive flow mechanism,24 which is possible owing to the high porosity of the matrixes. 4. Conclusion In this study we synthesized a series of PEG-protein hydrogels using soy protein, hydrolyzed soy protein, pea albumin, and casein. The component composition of the hydrogel was determined, and the composites obtained were PEG-Protein Hydrogels characterized in terms of swelling behavior and mechanical and drug release properties. From the experimental results it appeared that the individual molecular properties of proteins influence the physicochemical characteristics of the resultant hydrogel. Studied systems were found to represent a lightly cross-linked network, where both PEG and protein are completely solvated, enabling as high as 96% equilibrium water content. As was found for HSOYGEL and SOYGEL samples, the polyelectrolyte nature of the biopolymer governs the pH responsiveness of the corresponding network, which manifested itself in a pH-dependent mechanism of swelling and drug release. This property of the soy protein gels makes them excellent candidates as pH-controlled drug release material. The disadvantages of this system, such as fragility and poor transparency, can be overcome by utilization of other proteins such as pea albumin or casein in combination with soy protein in polymerization with PEG, since both pea albumin and casein were shown to form a transparent hydrogel. Thus, the result of this work demonstrated that there is a great opportunity to modulate the final characteristics of the system to fit the need of specific biomedical applications. References and Notes (1) (2) (3) (4) D’Urso, E. M.; Fortier, G. Biotechnol. Tech. 1994, 8, 71-76. Gayet, J.-C.; Fortier, G. J. Controlled Release 1996, 38, 177-184. Fortier, G. U.S. Patent No. 5,733,563, 1998. (a) Zalipsky, S. Bioconjugate Chem. 1995, 6, 150-165. (b) Hoffman, A. AdV. Drug DeliVery ReV. 2002, 43, 3-12. (c) Roberts, M. J.; Bentley, M. D.; Harris J. M. AdV. Drug DeliVery ReV. 2002, 54, 459476. (5) Sanborn, T. J.; Messersmith, P. B.; Barron, A. E. Biomaterials 2002, 23, 2703-2710. (6) D’Urso, E. 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