1107. Cellular Internalization and Distribution of DNA Complexes from Cell-Adhesive Substrates

Enhancing localized gene expression is requisite to successful application of gene delivery in gene therapy, tissue engineering and functional genomics. Our approach to enhancing localized expression involves improving the delivery system. Substrate-mediated delivery strives to overcome barriers in localized gene transfer by immobilizing DNA in the cellular microenvironment, thereby increasing the DNA concentration proximal to the cells. DNA is first mixed with cationic lipids or polymers to form complexes and then immobilized on a cell-adhesive substrate. The challenge in substrate-mediated delivery is to immobilize complexes for retention on the substrate while also allowing complex release for subsequent cellular internalization.We hypothesized that influencing the interaction between complex and substrate would enhance transgene expression by increasing cellular internalization and improving the cellular distribution of DNA complexes. Coating polystyrene substrates with serum yielded up to 1500-fold enhancement in expression with polyethylenimine (PEI) polyplexes when compared to delivery from a non-coated substrate. The delivery of Lipofectamine 2000 lipoplexes from serum-coated substrates enhanced the number of cells expressing reporter gene. Confocal microscopy of substrates with complexes and cells indicated that polyplexes delivered from serum-coated substrates were able to localize to cell surfaces, but remained distributed across the surface when the substrate was not coated.The distribution of internalized complexes in the serum-coated and uncoated systems was examined to elucidate the differences in expression. Flow cytometry with FITC-labeled plasmid was utilized to quantify the distribution of internalization. After 24 hours, 100% of cells had internalized polyplexes from serum-coated substrates, whereas only 30% of cells had internalized polyplexes from uncoated tissue culture polystyrene substrates. Lipoplexes delivered from the substrates had similar internalization distribution. These studies demonstrate the importance of designing immobilization that allows for internalization in attaining efficient localized gene expression. Understanding the mechanisms in substrate-mediated delivery will enhance the application of gene delivery in vitro and in vivo.