Role of Polymeric Endosomolytic Agents in Gene Transfection:A Comparative Study of Poly(l-lysine) Grafted withMonomeric l-Histidine Analogue and Poly(l-histidine)

摘要

Endosomal entrapment is one of the main barriers that must be overcome for efficient gene expression along with cell internalization, DNA release, and nuclear import. Introducing pH-sensitive ionizable groups into the polycationic polymers to increase gene transfer efficiency has proven to be a useful method; however, a comparative study of introducing equal numbers of ionizable groups in both polymer and monomer forms, has not been reported. In this study, we prepared two types of histidine-grafted poly(l-lysine) (PLL), a stacking form of poly(l-histidine) (PLL-g-PHis) and a mono- l-histidine (PLL-g-mHis) with the same number of imidazole groups. These two types of histidine-grafted PLL, PLL-g-PHis and PLL-g-mHis, showed profound differences in hemolytic activity, cellular uptake, internalization, and transfection efficiency. Cy3-labeled PLL-g-PHis showed strong fluorescence in the nucleus after internalization, and high hemolytic activity upon pH changes was also observed from PLL-g-PHis. The arrangement of imidazole groups fromPHis also provided higher gene expression than mHis due to its abilityto escape the endosome. mHis or PHis grafting reduced the cytotoxicityof PLL and changed the rate of cellular uptake by changing the quantityof free ε-amines available for gene condensation. The subcellularlocalization of PLL-g-PHis/pDNA measured by YOYO1-pDNAintensity was highest inside the nucleus, while the lysotracker, whichstains the acidic compartments was lowest among these polymers. Thus,the polymeric histidine arrangement demonstrate the ability to escapethe endosome and trigger rapid release of polyplexes into the cytosol,resulting in a greater amount of pDNA available for translocationto the nucleus and enhanced gene expression.