Mechanically enhanced microcapsules for cellular gene therapy
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Microcapsules bearing a covalently cross-linked coating have been developed for cellular gene therapy as an improvement on alginate-poly(L-lysine)-alginate (APA) microcapsules that only have ionic cross-linking. In this study, two mutually reactive polyelectrolytes, a polycation (designated C70), poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride-co-2-aminoethyl methacrylate hydrochloride) and a polyanion (designated A70), poly(sodium methacrylate-co-2-(methacryloyloxy)ethyl acetoacetate), were used during the microcapsule fabrication. Ca-alginate beads were sequentially laminated with C70, A70, poly(L-lysine) (PLL), and alginate. The A70 reacts with both C70 and PLL to form a approximately 30 microm thick covalently cross-linked interpenetrating polymer network on the surface of the capsules. Confocal images confirmed the location of the C70/A70/PLL network and the stability of the network after 4 weeks implantation in mice. The mechanical and chemical resistance of the capsules was tested with a "stress test" where microcapsules were gently shaken in 0.003% EDTA for 15 min. APA capsules disappeared during this treatment, whereas the modified capsules, even those that had been retrieved from mice after 4-weeks implantation, remained intact. Analysis of solutions passing through model flat membranes showed that the molecular weight cut-off of alginate-C70-A70-PLL-alginate is similar to that of alginate-PLL-alginate. Recombinant cells encapsulated in APA and modified capsules were able to secrete luciferase into culture media. The modified capsules were found to capture some components of regular culture media used during preparation, causing an immune reaction in implanted mice, but use of UltraCulture serum-free medium was found to prevent this immune reaction. In vivo biocompatibility of the new capsules was similar to the APA capsules, with no sign of clinical toxicity on complete blood counts and liver function tests. The increased stability of the covalently modified microcapsules coupled with the acceptable biocompatibility and permeability demonstrated their potential for use as immunoisolation devices in gene therapy.