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Articles Volume Phase Transition in Interpenetrating Networks of Poly(N-isopropylacrylamide) with Gelatin† Dibakar Dhara, G. V. N. Rathna, and Prabha R. Chatterji* Speciality Polymers Group, Organic Coatings and Polymers Division, Indian Institute of Chemical Technology, Hyderabad 500 007, India Received January 21, 1999. In Final Form: November 17, 1999 Fully interpenetrating polymer networks (IPNs) of gelatin and poly(N-isopropylacrylamide) (PNIPA) are synthesized. The IPNs exhibit volume phase transition in aqueous medium and re-entrant type behavior in mixed solvent systems similar to pure PNIPA hydrogels. Neither the hydrophilic ambience of the gelatin network nor the additional topological constraints imposed by the IPN morphology restrain the characteristic features of PNIPA suggesting short-range interactions are responsible for the volume phase transition (VPT) phenomenon. Thus it could very well be that the conditions of the planarity of the amide group together with the directionality of the H bonds with the solvent decide the phenomenon of lower critical solution temperature (LCST)/VPT in PNIPA polymers.
1.0. Introduction Among stimuli responsive hydrogels, poly(N-isopropylacrylamide) (PNIPA) containing networks constitute one of the most extensively studied systems because of its well-defined volume phase transition (VPT) in water around 32-34 °C.1-5 It has been shown that when external conditions such as temperature, solvent composition, or additive concentration change, a gel reversibly swells or shrinks discontinuosly and this is termed as volume phase transition. Besides temperature, VPT can be induced by several other stimuli such as changes in solvent composition,6-11 presence of inorganic or organic additives,12-15 etc. Attempts are being made to exploit the VPT for the development of novel temperature-sensitive devices16 for * To whom correspondence should be addressed. E-mail:
[email protected]. Fax: 91-40-7173757. † IICT Communication No. 3968. (1) Hirokawa, Y.; Tanaka, T. J. Chem. Phys. 1984, 81, 6379. (2) Taylor, L. D.; Cerankowski, L. D. J. Polym. Sci., Polym. Chem. Ed. 1975, 13, 255. (3) Katayama, S.; Ohata, A. Macromolecules 1985, 18, 2781. (4) Hirotsu, S.; Hirokawa, Y.; Tanaka, T. J. Chem. Phys. 1987, 87, 1392. (5) Hoffman, A. S.; Afrassiabi, A.; Dong, L. C. J. Controlled Release 1986, 4, 213. (6) Hirotsu, S. J. Chem. Phys. 1988, 88, 427. (7) Katayama, S.; Hirokawa, V; Tanaka, Y. Macromolecules 1984, 17, 2641. (8) Mukae, K.; Sawamura, S.; Makino, K.; Kim, S. W.; Ueda, I.; Shirahama, K. J. Phys. Chem. 1993, 97, 737. (9) Schild, H. G.; Muthukumar, M.; Tirrell, D. A. Macromolecules 1991, 24, 948. (10) Winnik, F. M.; Ringsdrof, H.; Venzmer, J. Macromolecules 1990, 23, 2415. (11) Hirotsu, S.; Okajima, T.; Yamamoto, T. Macromolecules 1995, 28, 775. (12) Park, T. G.; Hoffman, A. S. Macromolecules 1993, 26, 5045. (13) (a) Wada, N.; Kajima, Y.; Yagi, Y.; Inomata, H.; Saito, S. Langmuir 1993, 9, 46. (b) Bae, Y.; Okano, T.; Hsu, R.; Kim, S. Macromol. Chem., Rapid. Commum. 1986, 19, 493. (14) Saito, S.; Konno, M.; Inomata, H. In Advances in Polymer Sciences; Springer-Verlag: Berlin Handerberg, 1993; Vol. 109, p 207. (15) Sakai, M.; Satoh, N.; Tsujii, K.; Zhang, Y.-Q.; Tanaka, T. Langmuir 1995, 11, 2493. (16) Freitas, R. E. S.; Cussler, E. L. Sep. Sci. Technol. 1987, 22, 911.
protein phase separation,17 controlled drug delivery,18,19 solute separation,17,20 size selective extraction solvent,20 enzyme activity,21 temperature-controlled cell culture,22 etc. In an attempt to combine stimuli responsiveness with biodegradability, we have synthesized an interpenetrating polymer network of gelatin and PNIPA. Gelatin, a degradation fragment of the structural protein collagen, is completely biodegradable and is extensively used in food and pharmaceutical formulations. This well-characterized hydrophilic protein fragment forms a gel when hot, concentrated (>5%) aqueous solutions are cooled.23 The synthetic routes and gelling properties of interpenetrating networks of gelatin with poly(acrylamide) and sodium carboxymethyl cellulose have been studied extensively in our laboratory.24-27 We have also made thorough analysis of the controlled delivery systems based on these interpenetrating polymer networks (IPNs).28 It was then a logical step forward to combine stimuli responsiveness with bioerodibility. Recently Bomberg29 has reported grafting of PNIPA chains to zein protein. In comparison, the [PNIPA-gelatin] system is simpler in preparation and more versatile with respect to compositional variation and above all does not in anyway alter the chemistry of the PNIPA chain. (17) Freitas, R. E. S.; Cussler, E. L. Chem. Eng. Sci. 1987, 42, 97. (18) Okano, T.; Bae, Y.; Kim, S. J. Controlled Release 1989, 9, 271. (19) Dong, L. C.; Hoffman, A. S. J. Controlled Release 1990, 13, 21. (20) Feil, H.; Bae, Y.; Feijen, J.; Kim, S. J. Membr. Sci. 1991, 64, 283. (21) Dong, L. C.; Hoffman, A. S. J. Controlled Release 1986, 4, 223. (22) Yamada, N.; Okano, T.; Sakai, H.; Karikusa, F.; Sawasaki, Y.; Sakurai, Y. Macromol. Chem., Rapid. Commum 1990, 11, 571. (23) Veis, A. In The Macromolecular Chemistry of Gelatin; Horecker, B., Kaplan, N. O., Scheraga, H. A., Eds.; Academic Press: New York, 1964. Vol. 5. (24) Kaur, H.; Chatterji, P. R. Macromolecules 1990, 23, 4868. (25) Chatterji, P. R. Macromolecules 1991, 24, 4214. (26) Rathna, G. V. N.; Mohan Rao, D. V.; Chatterji, P. R. Macromolecules 1994, 27, 7920. (27) Chatterji, P. R. J. Appl. Polym. Sci. 1990, 40, 401. (28) Rathna, G. V. N.; Mohan Rao, D. V.; Chatterji, P. R. Manuscript in preparation. (29) Bomberg, L. J. Phys. Chem. B 1997, 101, 504.
10.1021/la990065j CCC: $19.00 © 2000 American Chemical Society Published on Web 01/15/2000
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Table 1. Compositions of the [PNIPA-Gelatin] IPNs Synthesized (total polymer concentration in all the IPN hydrogels was 10% (w/v)) weight fraction in hydrogel sample code
gelatin
PNIPA
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12
1.00 0.95 0.90 0.85 0.80 0.60 0.40 0.20 0.15 0.10 0.05 0.00
0.00 0.05 0.10 0.15 0.20 0.40 0.60 0.80 0.85 0.90 0.95 1.00
Besides all these, we felt an IPN is an interesting system to probe the phenomenon of volume phase transition in PNIPA hydrogels. It has been easy to explain the VPT in PNIPA gels induced by temperature and solvent composition.1-11 All interpretations focus on the structural features of PNIPA: the nonpolar hydrophobic main chain with a hydrophobic pendant connected through a polar hydrophilic amide functionality. It has been suggested that the extensive hydrogen-bonding network of amide groups with water is responsible for keeping the linear polymer in solution or the gel swollen. As the temperature rises or the water content in the solvent mixture decreases, this network is disrupted and the linear polymer precipitates or the gel deswells. It has also been alluded that this is similar to coil globule transitions commonly encountered in proteins. What will be the status of PNIPA chains when entangled in an IPN with gelatin? It is well established that the main properties of the gelatin network are governed by water-mediated hydrogen bonds. How effectively will the hydrophobic forces operate when the PNIPA segments are embedded in an overwhelmingly hydrophilic network? This chemistry together with the topological constraints in an IPN appeared uniquely suited to provide significant clues toward understanding the role of intermolecular interactions and molecular structure in modulating volume phase transition. 2.0. Experimental Section 2.1. Materials. NIPA from Aldrich was recrystallized from hexane; N,N′- methylenebis(acrylamide), ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), and gluteraldehyde (as 25% aqueous solution), special grade from SRL, Bombay, India, were used without further purification. Gelatin (from Loba Chemie, Bombay) has a molecular weight of 1.24 × 105. All other chemicals and solvents were of analytical grade. Double distilled water was used for all the experiments. 2.2. Preparation of Hydrogels. 2.2.1. Simple Networks. PNIPA gels were prepared by free radical polymerization of NIPA and bisacrylamide (2.7 wt % of NIPA) triggered by APS-TEMED mixture in water at 4 °C.12 Gelatin gels were prepared by the method reported by Chatterji.27 In both cases the pregel solution was poured between two siliconised glass plates separated by Teflon spacers (6 mm) and left overnight for setting. Uniform disks were punched out of the slab using a cork borer. Gelatin gels were subsequently cross-linked by glutaraldehyde treatment. 2.2.2. Interpenetrating Networks. The method earlier reported24,25 for [poly(acrylamide)-gelatin] IPN hydrogels was adopted for [PNIPA-gelatin] IPNs too, except employing a low temperature (8 °C) to prevent phase separation of PNIPA. Samples S1 to S12 (please see Table 1) were prepared by dissolving varying amounts of gelatin and NIPA in water keeping to a final total concentration of 10% (w/v). To this solution (100 mL) cross-linker bisacrylamide (2.7 wt % of NIPA), the initiator
APS (50 mg), and accelerator TEMED (50 µL) were added. The cross-link density of the PNIPA network turns out to be 17 mmol of cross-links per gram of the sample. The solution was thoroughly stirred and poured into the glass mould (6 mm spacing) immediately. The gelled slabs were removed carefully, and cut by a cork borer and the samples were placed in 1% glutaraldehyde solution for 6 h for cross-linking of gelatin. After cross-linking the gels were dialyzed against water with frequent changing of water. After 7 days the gels were removed and air-dried to constant weight. Network formation in gelatin was ascertained by amino acid analysis of the IPN samples, which registered almost complete modification of lysine residues.24-25,30 2.3. Swelling Studies. Swelling was monitored as a function of composition (PNIPA to gelatin ratio), temperature (8 °C to 42 °C), NaCl concentration (0 M to 4 M), and solvent composition. If not mentioned otherwise, the swelling studies were carried out at ambient temperature (23-25 °C). In a typical case, the dried gel disk was weighed (wi) and transferred to the required medium. At regular intervals the disk was removed from the solvent, its surface was pressed gently with tissue paper to remove the excess solvent, weighed (wt), and then returned to the medium. This process of swelling and weighing was continued until the disk attained a constant final weight (wf). For temperature dependence studies, the samples were incubated in a thermostated water bath for 24 h after confirming that this was well over equilibration time. Water content and degree of swelling were calculated in the usual way.12,31
degree of swelling ) water content (%) )
swollen weight - dry weight dry weight
swollen weight - dry weight × 100 swollen weight
3.0. Results It has been suggested that temperature-induced phase transition of PNIPA in aqueous medium is mainly driven by increased interaction between hydrophobic segments on the polymer, caused by a reduced structuring of water around hydrophobic groups with increasing temperature.32-35 It has also been implied that this results in a conformational transition of PNIPA chains from a hydrophilic to hydrophobic form. Studies on the copolymerization of NIPA with other monomers indicate that ionic, hydrophilic comonomers increase the lower critical solution temperature (LCST) presumably by improving the hydrophilic character of the polymer thus forcing the polymers to stay in solution or the gel to remain swollen.36-39 In the [PNIPA-gelatin] IPN, the individual chemistry of the PNIPA chains is not affected, but the ambience is. It is possible that increased topological constraints such as entanglements within the gelatin network would restrict the flexibility of the PNIPA segments. We set out to investigate how these changes would affect the transition phenomenon. Table 1 lists the composition of hydrogels studied. From S1 to S12, gelatin weight fraction changes from 1.0 to 0 and PNIPA weight fraction changes from 0 to 1.0. All (30) Chatterji, P. R. J. Appl. Polym. Sci. 1989, 37, 2203. (31) Padmavathi, N. Ch.; Chatterji, P. R. Macromolecules 1996, 29, 1976. (32) Otake, K.; Inomata, H.; Konno, M.; Saito, S. Macromolecules 1990, 23, 283. (33) Privalov, P. L.; Gill, S. J. Adv. Protein Chem. 1988, 39, 191. (34) Privalov, P. L. Annu. Rev. Biophys. Chem. 1989, 18, 47. (35) Murphy, K. P.; Privalov, P. L.; Gill, S. J. Science 1990, 247, 559. (36) Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W. Macromolecules 1993, 26, 2496. (37) Ilavski, M.; Hrouz, J.; Havlicek, I. Polymer 1985, 26, 1514. (38) Beltran, S.; Hooper, H. H.; Blanch, H. W.; Prausnitz, J. M. J. Chem. Phys. 1990, 92, 2061. (39) Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W. Macromolecules 1992, 25, 5528.
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Figure 2. Dynamic swelling behavior of the [PNIPA-gelatin] IPN hydrogels at 42.0 °C.
Figure 1. Dynamic swelling behavior of the [PNIPA-gelatin] IPN hydrogels at 27.0 °C: A, initial stages; B, until equilibrium.
samples were clear and transparent, indicating the absence of macroscopic phase separation. The color of samples ranged from pale brown to amber with increasing gelatin content. This is because the cross-linking reaction between gelatin and glutaraldehyde results in a chromophoric Schiff base linkage24 and the intensity of color depends on the degree of cross-linking which can be quantitatively estimated from amino acid analysis. Because glutaraldehyde cross-linking of gelatin takes place through the amino groups of lysine residues, estimation of free lysine content in the native and cross-linked samples quantifies the degree of cross-linking. Gelatin used in this study had a molecular weight of 35 000 with a lysine content of ∼7 nmol/µg. This makes it roughly 24 lysine residues per chain. Amino acid analysis of the native gelatin and the IPN samples indicated that all the lysines have been modified. 3.1. Dynamic Swelling Behavior of the IPNs. Figure 1 and Figure 2 show the dynamic swelling of the IPNs for temperatures below and above the LCST, respectively. The rate of swelling of the IPNs depends on the temperature as well as the composition. At 27 °C below the VPT temperature of PNIPA, the IPNs with higher PNIPA content swell faster and more. As expected at 42 °C, above the LCST, the trend is reversed. The obvious fact is that between 27 and 42 °C, while gelatin gels register moderate swelling, the swelling and shrinkage characteristics of
Figure 3. Comparative swelling of pure PNIPA and gelatin gels at 27 and 42 °C.
PNIPA hydrogels vary by a factor of 8 (Figure 3). This factor shoots to 15 when the lower limit is 8 °C. This enables us to monitor the fluctuations in the PNIPA network while the gelatin chains provide a uniform hydrated network in the temperature range studied. 3.2. Volume phase Transition. Figure 4 shows the swelling of these IPNs at different temperatures as a function of composition. This figure brings into focus several interesting results. The transition point is 32 °C. At this temperature the composition of the IPN has only a marginal effect on swelling. Below this temperature the swelling increases as we increase the PNIPA concentration, and above this temperature shrinking sets in. Another important observation is that even at as low as 0.1 weight fraction PNIPA makes its presence felt by registering a slight increase in swelling at 8 and 27 °C, shrinking at 37 and 40 °C. The low-temperature curves (8 and 27 °C) have a special feature. At PNIPA weight fraction of 0.75, there is a break in the curve. The IPNs do exhibit VPT at the same temperature as pure PNIPA gels (Figure 5). This is indeed significant especially in view of the fact that copolymerization of NIPA with other hydrophilic ionic monomers raises the LCST.36-39 Copolymerization would invariably modify the periodicity of
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Figure 4. Equilibrium degree of swelling of the [PNIPAgelatin] IPN hydrogels as a function of PNIPA content.
Figure 5. Temperature-induced volume phase transition in [PNIPA-gelatin] IPN hydrogels.
the interaction of NIPA monomer units with the molecules in the swelling medium. That the VPT is not affected per se except the magnitude implies that the forces triggering VPT are probably operative at dimensions smaller than the entanglement lengths. 3.3. Effect of Sodium chloride on VPT. Park and Hoffman12 were first to demonstrate that aqueous sodium chloride could induce volume phase transition in nonionic PNIPA hydrogels. They monitored the effect of a series of sodium salts and concluded that the chloride ion is responsible for bringing about this transition by interfering with the water structure around the polymer chain. Several reports13-15 have appeared on the effect of additives on the volume phase transition in pure nonionic PNIPA hydrogels. Saito et al.14 tried to extend the theory further by alluding to the primary and secondary water clusters. They suggested that hydrophobic interactions outweigh the hydrophobic hydration as the salt concentration in the aqueous medium increases. Salt has practically no effect on the swelling of gelatin gel.40 We felt that since the PNIPA network is entwined with the more hydrophilic gelatin network, the dehydration of
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Figure 6. NaCl-induced volume phase transition in [PNIPAgelatin] IPN hydrogels at 25 °C.
Figure 7. Re-entrant behavior of PNIPA hydrogel in mixed solvents at 25 °C.
PNIPA chains would be hindered. Indeed our attempts to probe the VPT phenomenon in PNIPA hydrogels using hydrotropes revealed the importance of structural requirements.41 However the IPNs exhibit VPT at the same salt concentration as pure PNIPA hydrogels. The water content of the IPNs in the presence of NaCl is plotted in Figure 6. As the salt concentration rises above 0.8 M, the gel shrinks, and below this concentration it remains swollen. 3.4. Effect of Solvent Composition. Pure PNIPA hydrogels exhibit a re-entrant type of behavior in acetone/ water, DMSO/water, and methanol/water mixtures7-10 (Figure 7). This has been interpreted by assuming that the mixing free energy between polymer segments is not a linear function of the solvent composition. Investigating this phenomenon Schild et al.9 point out that a mechanism that involves local polymer-solvent interactions is more plausible. Increasing content of organic solvent turns the medium less compatible for gelatin hydrogels as shown (40) Rathna, G. V. N.; Mohan Rao, D. V.; Chatterji, P. R. J. Macromol. Sci. Pure. Appl. Chem. 1996, A33, 1199. (41) Dhara, D.; Chatterji, P. R. Langmuir 1999, 15, 930.
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Figure 8. Behavior of gelatin hydrogels in mixed solvents at 25 °C.
Figure 10. Swelling behavior of [PNIPA-gelatin] IPN hydrogels in [water-DMSO] mixture at 25 °C.
Figure 9. Swelling behavior of the [PNIPA-gelatin] IPN hydrogels in [water-acetone] mixture at 25 °C.
Figure 11. Swelling behavior of the [PNIPA-gelatin] IPN hydrogels in [water-methanol] mixtures at 25 °C.
in Figure 8. We monitored the swelling behavior of the IPNs in all these solvent mixtures. Figures 9-11 show the results. The re-entrant type behavior of PNIPA is evident in the IPNs too.
in the ∆H for VPT for PNIPA gel in water by deuterium substitution. The quickness of the transition indicates that PNIPA chains can change its shape very effectively to suit its environment. The amide groups can reorganize themselves to form either intermolecular hydrogen bonds with solvents or intramolecular hydrogen bonds between successive amide groups. This special feature can explain the solubility characteristics of the polymer. At temperatures below LCST, the intermolecular hydrogen bonds with solvent, like water, predominate and the polymer is solubilized. However above LCST the increased molecular motion of the solvent molecules perturbs this state and the polymer deswells. Addition of organic solvents such as DMSO and methanol gradually (Figures 7 and 9-11) decreases the solubility of the polymer from 20% v/v onward rather sharply; however, with the continuous addition of solvent, its solubility increases gradually. This could be attributed with more polar solvents such as DMSO and methanol; the water molecules are rearranged such that hydrogen bonding interactions with the organic polar solvents are better optimized leaving the polymer stabilized by intramolecular hydrogen bonds. At this juncture, its solu-
4.0. Discussion The results presented above indicate that interpenetrating morphology does not constrain the conformational transition in the PNIPA network. All the characteristic features of pure PNIPA hydrogels, i.e., VPT induced by temperature, by NaCl, and by the re-entrant type behavior in mixed solvent systems, are exhibited by the IPNs too. This implies that intrachain or interchain interactions responsible for the VPT are effective over shorter stretches. The reversibility of the transition and the small ∆H associated with it42,43 also imply the same. That hydration and hydrogen bonding would be predominant factors for the swelling of PNIPA below 32 °C have been established categorically by Shirota et al.44 by measuring the changes (42) Urry, D. W. Prog. Biophys. Mol. Biol. 1992, 57, 23. (43) Vadnere, M.; Amidon, G.; Lindenbaum, S. L.; Haslam, J. L. Int. J. Pharm. 1984, 22, 5528. (44) Shirota, H.; Endo, T.; Horie, K. Chem. Phys. 1998, 238, 487.
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bility decreases. However, with the continuous addition of organic solvent, intermolecular hydrogen bonds are optimized more effectively with solvents again making the polymer again more soluble. Monitoring the photophysical behavior of dansyl-labeled PNIPA hydrogels in water-methanol mixed solvent systems, Asano et al.45 suggest preferential adsorption of one of the solvents to the polymer or the gel being responsible for triggering VPT. Ilmain et al.,46 while emphasising that cooperativity is the essential driving force for VPT, suggest that the VPT in interpenetrating networks of poly(acrylamide) and poly(acrylic acid) is controlled by cooperative zipping interactions between molecules which result from hydrogen bonding. The LCST behavior of a two-component system is attributed to highly directional short-range interactions, such as the H bond which requires specific orientations of the molecules involved.47-50 In the case of PNIPA, the planarity of the amide group together with the directionality of the H bonds it forms with the solvent would impose severe limitations on the number of available energy states in the solvated state. Any slight perturbation could disrupt this delicate spatial arrangement leading to deswelling of the polymer. Since all the polymer molecules respond in concert to the changes in the energy of interaction with (45) Asano, M.; Winnik, F. M.; Yamashita, T.; Horie, K. Macromolecules 1995, 28, 5861. (46) Ilmain, F.; Tanaka, T.; Kokufuta, E. Nature 1991, 349, 400. (47) Wheeler, J. C. J. Chem. Phys. 1975, 62, 433. (48) Hirschfelder, J.; Stevenson, D.; Eyring, H. J. Chem. Phys. 1937, 5, 896. (49) Barker, J. A.; Fock, W. Discuss. Faraday Soc. 1953, 15, 188. (50) Lang, J. C.; Morgan, R. D. J. Chem. Phys. 1980, 73, 5849.
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the solvent, this becomes a collective phenomenon. The perturbation could be van der Waals, electrostatic, and/ or hydrophobic interaction. The observation that the transition is equally affected by a variety of additives of widely different chemical and structural characteristics, such as simple salts, ionic and nonionic surfactants, low molecular weight alcohols, hydrotropes, etc., is consistent with this possibility. Conclusions The swelling behavior of PNIPA-gelatin interpenetrating networks emphasizes the role of short-range interactions between PNIPA and the solvent in the LCST/ VPT phenomenon. The planarity of the amide group together with the highly directional nature of the H bonds with the solvent could impose severe limitations on the number of available energy states in the solvated state. This would make the system susceptible to a variety of perturbations, such as van der Waals, electrostatic, and hydrophobic interactions, which could disrupt this delicate spatial arrangement in the solvated state, leading to deswelling. Acknowledgment. We are grateful to Professor John M. Prausnitz, Department of Chemical Engineering, University of California, Berkeley, CA, and the reviewers for critical comments and useful suggestions. D.D. acknowledges financial assistance from UGC, New Delhi, India, in the form of a Senior Research Fellowship. LA990065J
