Mechanistic and functional aspects of the interaction of AR-23 with mammalian cell membrane and improvement of branched polyethylenimine-mediated gene transfection

Background Previous studies have suggested that reducing the positive charge of melittin could increase endosomal release activity and improve branched polyethylenimine (BPEI)‐mediated transfection. AR‐23 is a melittin‐related peptide from Rana tagoi, which shows 81% sequence identity with melittin but has less positively‐charged residues than melittin. The present study aimed to investigate the mechanistic and functional aspects of the interaction of AR‐23 with mammalian cells and thus improve BPEI‐mediated gene transfection. Methods AR23 and two AR‐23 analogs (AR‐20 without positively‐charged residues and AR‐26 with the same positively‐charged residues as melittin) were analyzed. Circular dichroism (CD) spectrometry was used to analyze the secondary structures of the peptides. Peptide‐induced depolarization of cell membrane, the membrane‐lytic activity of the peptides, and their potency with respect to enhancing the cellular uptake of calcein were evaluated. The physicochemical characters of complexes were measured and the effect of the peptides on BPEI‐mediated transfection was determined. Results The CD spectra results indicated that a positive charge in AR‐23 played a crucial role in maintaining the α‐helical conformation, whereas an extra positive charge could not increase α‐helical formation. AR‐23 displayed a similar depolarization ability to melittin. However, AR‐23 showed a lower membrane lytic activity under physiological conditions and a higher lytic activity at endosomal pH than melittin and AR‐26, which possess more positive charges. Compared to melittin and AR‐26, AR‐23, with a higher endosomal escaping activity, resulted in a higher enhancement of BPEI‐mediated gene transfection, as well as the maintainance of a lower cytotoxicity. Conclusions We suggest that AR‐23 may be considered as a potential enhancer for improving the transfection efficiency of cationic polymers. Copyright © 2013 John Wiley & Sons, Ltd.