Control of the Chemical Cross-Linking of Gelatin by a Thermosensitive Polymer: Example of Switchable Reactivity Chantal Boudet, ² Ilias Iliopoulos,* ,² Olivier Poncelet, ‡ and Michel Cloitre ² Matie ` re Molle et Chimie (UMR-7167, ESPCI-CNRS), 10 rue Vauquelin, 75231 Paris Cedex 05, France and KODAK Industrie, Centre de Recherches, Route de Demigny, 71102 Chalon-sur-Sao ˆ ne Cedex, France Received June 10, 2005; Revised Manuscript Received August 29, 2005 Chemical cross-linking of gelatin is achieved using a thermosensitive reactive copolymer based on N-isopropylacrylamide (NIPAM). The copolymer bears 5 mol % acrylic acid units which form amide bonds with the amino groups of gelatin in the presence of a water-soluble carbodiimide. The cross-linking reaction occurs only below the LCST =34 °C (lower critical solution temperature), i.e., when the copolymer is in the coil conformation. Above the LCST the copolymer adopts a globule conformation and its ability to react with gelatin is drastically reduced. By setting the temperature above or below the LCST it is possible to switch off or on the reactivity of the system and control the gelation process. The switch temperature can be set at the desired value by adjusting the composition of the thermosensitive copolymer. Introduction Gelatin is a polypeptide derived by hydrolytic degradation of collagen. It has unique gelling properties and is used in photographic, food, pharmaceutical, and cosmetics industry. It is soluble in water at temperatures above T gel and forms reversible physical gels below T gel . Gel formation is due to the partial recovering of the triple-helix conformation of the native collagen. 1-4 T gel depends on the origin of gelatin and is ≈30 °C for the most common mammalian gelatins. Irreversible, chemical cross-linking of gelatin can easily be achieved by reaction with several types of reagents, which are able to react with the functional side groups of the polypeptide chain (OH, NH 2 , COOH). 1,5 This chemical cross- linking is widely used in applications like, for instance, photographic films 5-7 or biomedical materials where me- chanical stability is required at temperatures above 35 °C. 8-10 Chemical cross-linking of gelatin can also occur upon reaction with a second reactive polymer. 11,12 In that case mixed gels are formed, and their properties depend on the gel composition and the nature of the second polymer. The temperature at which the chemical cross-linking occurs is another important parameter that controls the relative con- tribution of physical and chemical bonds to the properties of the final gel. 11-13 Schacht et al. 11 showed that the chemical cross-linking between gelatin and dextran dialdehyde is by far more efficient when done at 20 °C than at 4 °C. This was attributed to the differences in the gelatin conformation, which in turn controls the relative contribution of physical and chemical cross-linking. Similar conclusions were re- ported for gelatin/hyaluronan mixed gels cross-linked by enzymatic reaction. 12 Among the mixed systems based on gelatin, of special interest are those containing poly(N-isopropylacrylamide) (PNIPAM). 14,15 PNIPAM exhibits inverse solubility behavior in aqueous solution (lower critical solution temperature, LCST); it is soluble below the LCST and phase separates at temperatures above the LCST. 16,17 At the LCST (≈32 °C) the PNIPAM chain undergoes a coil-to-globule transition, and this thermosensitive behavior is of interest for the development of stimuli-responsive aqueous formulations and gels. 17 The properties of gelatin and PNIPAM have been combined in graft copolymers, 18,19 interpenetrating net- works, 15 or composite membranes. 14 However, to our knowl- edge PNIPAM has never been used as a polymeric ther- mosensitive cross-linker for gelatin. In this work we explore the possibility to control the chemical cross-linking and properties of gelatin gels using a thermosensitive copolymer based on NIPAM. The copoly- mer bears a low fraction, 5 mol %, of acrylic acid (AA) units (see Table 1 and Figure 1) and is used as the minor component of the system with respect to gelatin. Gelatin/ PNIPAM cross-linking occurs by amide bond formation between the AA groups of the copolymer and the amino groups of gelatin in the presence of a water soluble carbodiimide (EDC), 20,21 Figure 2. We show that the copolymer reactivity, and consequently the chemical cross- linking, can be controlled by the copolymer conformation. The reactivity is off above the LCST and turned on by lowering the temperature below the LCST. Such switchable reactivity can be of interest when gelatin-based liquid formulations need to be chemically fixed after molding or coating. For the sake of comparison, we also report data obtained with a nonthermosensitive copolymer based on N,N- dimethylacrylamide (DMAM). Experimental Section Materials. Photographic-grade gelatin was a gift from Kodak Industrie (France). It was a lime-treated bovine ossein gelatin with a weight-average molecular weight of 1.8 × * To whom correspondence should be addressed. E-mail: Ilias.Iliopoulos@espci.fr. ² Matie `re Molle et Chimie. ‡ KODAK Industrie. 3073 Biomacromolecules 2005, 6, 3073-3078 10.1021/bm0503928 CCC: $30.25 © 2005 American Chemical Society Published on Web 10/11/2005