Effects of arginine derivatives and oligopeptides on the physical properties of model membranes

Abstract

Cell-penetrating peptides (CPPs) are often rich in arginine. They are able to cross cell membranes and carry other molecules into cells without being cytotoxic. The mechanisms by which they accomplish this are still controversial, although endocytotic pathways or spontaneous entry mechanisms have both been proposed. This work takes a bottom-up approach to the role of arginine in CPPs, by comparing an arginine side chain mimic with arginine oligopeptides of different length, with respect to their influence on membrane model systems of various lipid compositions. Simulations as well as breakthrough force spectroscopy experiments showed that the arginine sidechain mimic increased likelihood of pore formation in negatively charged membranes. This destabilisation of membranes was not found for hexa-arginine, a known CPP, for any lipid composition or peptide concentration, neither in breakthrough force spectroscopy nor by liposome leakage assays. However, hexa-arginine was shown to induce membrane stack formation on negatively charged solid-supported lipid bilayers at low peptide concentration. It was also found that arginine peptides of any length, as well as hexa-lysine, were able to cause stack formation in these membrane compositions, provided that peptide concentration was high enough. Our findings combined with literature mention of lamellarisation of model membranes and cell membranes caused by arginine-rich peptides suggest that membrane stack formation and lamellarisation is likely an important aspect of oligoarginine CPPs’ mode of action. However, our data do not support membrane disruption or pore formation as a means of cell entry. Future work should determine whether our findings are reproducible for longer oligoarginines, and provide final proof to reject the hypothesis of pore formation. Overall, this work points to a non-pore forming mode of action of CPPs, at least for shorter oligoarginines.