Surface grafting of poly(ethylene glycol) in the presence of cells, a strategy to construct double-layered mammalian cells containing microcapsules

grantor: University of TorontoSurface grafting of poly(ethylene glycol), (PEG) was performed in close proximity to live PC12 cells while maintaining a sufficient number of viable cells in vitro. The purpose of the study was to improve the biocompatibility of microencapsulated cells by performing a surface graft and constructing a double layered microcapsule by manipulating the surface chemistry. Several hydrogels that contain reactive primary amines intended for the inner-layer were synthesized. Poly(acrylic acid-co-acrylamide-co-methyl methacrylate-co-vinylamine) was synthesized via Hofmann degradation with a maximum of 8 mole% vinylamines but it was mechanically insufficient. On the other hand, crosslinked poly(acrylamide-co-vinylamine) films, synthesized in a similar manner, had 50 mole% primary amine on the surface and were reacted with tresylated PEG to form a modified outer layer: XPS showed $\sim$2% of amines coupled with PEG. Covalent attachment of PEG occurred in addition to the passive physical adsorption which was also observed by mixing the film with non-activated PEG. To evaluate if the reaction could be applied to microcapsules, the effect of the reaction on the cells was studied by model experiments using crosslinked films because of the difficulty in fabricating the amine containing polymers into microcapsules. The cells retained $\sim$80% viability (trypan blue) while seeded on top of films which being reacted with 10 $\mu$mole/g tresylated PEG in 0.1 M (N-tris hydroxymethyl-2-aminoethane sulfonic acid) buffer for 2 hours at 37$\sp\circ$C. Surprisingly, the reaction itself was not considered to be the major cause of cell death. Rather it appeared that the combined effect of the cytotoxicity of the leaving groups and the changes in osmotic pressure were more significant. Consistent with these observations, the activity of cells in unreactive poly(hydroxyethyl methacrylate-co-methyl methacrylate) microcapsules did not decrease after 2 hours of incubation with activated PEG according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide) assay. Surface modifying the microcapsules was considered possible by optimizing the reaction conditions so that the diffusion of the tresyl group into the capsule would be limited to keep the capsule core concentration $<$20 $\mu$mole/g. Novel microcapsules containing live cells with different surface and bulk composition may be created by further refinement of this study.Ph.D