Authors: L. Brito, S. Little, R. Langer, M. Amiji
Affilation: Northeastern University, United States
Pages: 365 - 368
Keywords: Gene delivery, stent, nanoparticle, restenosis
Non-viral gene delivery and transfection in primary human vascular endothelial cells (EC) and smooth muscle cells (SMC) has tremendous potential for cardiovascular diseases, such as in treatment of coronary restenosis. Using a combination of synthetic cationic poly(beta amino ester) (PBAE) (Figure 1a) and phospholipids (DOTAP) (Figure 1b), we have designed a nanovector system that can transfect EC and SCM cells with very high efficiency. Methods: The lipopolyplexes were made by pre-condensing plasmid DNA expressing enhanced green fluorescent protein (EGFP-N1) with PBAE. After DNA condensation with the polymer, DOTAP liposomes were added to the suspension creating positively-charged lipopolyplexes. Following characterization for size and surface charge, the lipopolyplexes were tested for cytotoxicity, cell uptake and trafficking behavior, and transfection efficiency in EC and SMC. Quantitative transfection of green fluorescent protein (GFP) was confirmed by flow cytometry analysis and measuring intracellular GFP with enzyme-linked immunosorbent assay (ELISA). Qualitative transfection was determined by fluorescence microscopy. Lipopolyplexes were also embedded in a gelatin matrix and loaded onto a stainless steel mesh. The mesh was placed on top of cells to see the transfection efficiency of the lipoplexes embedded on a stainless steel matrix. Results: Addition of DOTAP to PBAE-condensed DNA resulted in formation of nano-sized lipopolyplexes with a net positive charge. The lipopolyplexes did not induce significant cytotoxicity, especially at the doses that were necessary for transfection in both EC and SMC. When fluorescently-labeled lipopolyplexes were administered to the cells, the microscopy results showed increased particle uptake as a function of time up to a maximum by 6 hours of incubation. High efficiency of GFP transfection was confirmed by quantitative analysis. For instance, the flow cytometry results showed that GFP transfection efficiency was 33.4% and 23.6% for EC and SMC, respectively, at 24 hours post-administration using a 1 g DNA dose. The GFP expression was also confirmed by ELISA assay (Figure 2). Conclusions: The results of this study show that PBAE-DOTAP-plasmid DNA lipopolyplexes are a promising non-viral vector system for gene delivery and transfection in EC and SMC. Based on these results, there is tremendous potential for use of these lipopolyplexes in cardiovascular diseases, such as coronary restenosis by embedding them onto a stent.
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