Abstract
This study was designed to assess the in vitro gene expression efficiency and therapeutic effectiveness of polymer mediated transfection of primary myoblasts. Autologous primary myoblast transplantation may improve the function of infarcted myocardium via myogenesis. In addition, primary myoblasts can carry exogenous angiogenic genes that encode angiogenic factors to promote therapeutic angiogenesis. Viral vectors have limited clinical application due to the induction of inflammatory reactions, tumorigenic mutations and genome integration. To overcome these problems, two new biodegradable poly(disulfide amine)s, poly(cystaminebisacryamide-diaminohexane) [poly(CBA-DAH)] and poly(cystaminebisacryamide-diaminohexane-arginine) [poly(CBA-DAH-R)], were synthesized as polymer carriers for gene delivery. In this study, primary myoblasts were isolated and purified from rat skeletal muscles. Based on an optimized polymer mediated transfection procedure using a luciferase assay and confocal microscopy, these two poly(disulfide amine)s induced up to 16-fold higher luciferase expression and much higher green fluorescence protein expression than branched poy(ethylenimine) (bPEI, 25kDa) in primary myoblasts. By flow cytometry, poly(CBA-DAH) and poly(CBA-DAH-R) promote rates of cellular uptake of florescence-labeled polymer/pDNA complexes of 97% and 99%, respectively, which are rates higher than that of bPEI 25kDa (87%). Both poly(disulfide amine)s were much less cytotoxic than bPEI 25kDa. The in vitro time-course and co-culture experiments verified that polymer engineered primary myoblasts have the ability to stimulate endothelial proliferation. These data confirmed that poly(disulfide amine)s are the safe and feasible polymeric gene carriers to transfect VEGF(165) into primary myoblasts. Polymer engineered primary myoblasts have potential for therapeutic application in the treatment of ischemic heart diseases.