세포투과펩타이드를 이용한 HEK293T 세포에서의 Plasmid DNA Transfection 연구

Abstract

Cell penetrating peptide (CPP) is a non-viral carrier for the delivery of several important therapeutic and diagnostic cargos. One of its wider applications is the intracellular delivery of plasmid DNA. It has been found that CPP (bearing positive charge) makes non-covalent complexes with plasmid DNA (bearing negative charge) when combined at an appropriate charge/charge ratio in a suitable buffer. The non-covalent complex adopts a unique structure with positive surface charge that has the potential to penetrate the cellular membrane. Followed by the cellular entry, CPP/pDNA complexes have to escape endosome and achieve a successful nuclear import for gene expression. In this study, we have used our novel CPPs to make non-covalent complexes with plasmid DNA (pEGFP-N1) in autoclaved distilled water at different N/P ratios. The best N/P ratio during gel retardation assay and zeta analysis, was chosen to be used for transfection of HEK293T cells. For particle stabilization and unique structural features, we have added calcium chloride to CPP/pDNA complexes. In order to avoid endosomal breakdown, we have used Chloroquine with our complexes during invitro experiments. Our results indicate that CPP/pDNA complexes can penetrate the cell membrane but are trapped in endosome, hence no transfection was observed. Adding calcium chloride to CPP/pDNA complexes show some improvement in transfection but not comparable to Lipofactamine 2000. CPP/pDNA +CQ show enhanced CPP signal but negligible transfection. However, CPP+CaCl2+CQ brings about a transfection that is higher than Lipofactamine 2000. Our results demonstrate that the combined effect of calcium chloride and chloroquine with CPP/pDNA is better than CPP/pDNA alone or CPP/pDNA +CaCl2 or CPP/pDNA +CQ. Our data suggest that Endosomal entrapment is the rate-limiting step in CPP mediated transfection. A unique CPP based carrier system with inbuilt endosomal escape mechanism similar to Chloroquine and the net structural configuration of the complexes similar to calcium chloride condensed particles, could be a universal delivery system for nucleic acid delivery.