Ar-23 Derivatives With High Endosomal Disrupting Ability Enhance Poly(L-lysine)-mediated Gene Transfection

Background：pH-sensitive peptides are a relatively new strategy for conquering poor endosomal release of cationic polymer-mediated transfection. Modification of antimicrobial peptides (AMPs) by exchanging positively-charged residues with negatively-charged glutamic acid residues (Glu) greatly improved its lytic activity at the endosomal pH, which could improve cationic polymer-mediated transfection.Methods：In the present study, we investigate the effect of the number of Glu substituted for positively-charged residues on the endosomal escape activity of AR-23 and the ability of mutated AR-23 for enhancing cationic polymer-mediated transfection. Three analogs were synthesized by replacing the positively-charged residues in AR-23's sequence with Glu one-by-one. The pH-sensitive lysis ability of the peptides, the effect of peptides on the physicochemical characteristics, the intracellular trafficking，the transfection efficiency, and the cytotoxicity of the polyplexes were determined.Results：Increased lytic activity of peptides was observed with the increased number of Glu replacement in the AR-23 sequence at acidic pH. The number of Glu substituted for positively-charged residues of AR-23 dramatically affects its lysis ability at neutral pH. Triple-Glu substitution in AR-23's sequence greatly improved PLL-mediated gene transfection efficiency while maintaining low cytotoxicity.Conclusion：The results indicate that replacement of positively-charged residues with enough Glu residues may be considered a method for designing pH-sensitive peptides, which could be applied as potential enhancers for improving cationic polymer-mediated transfection.