Leaflet dependent effect of anionic phospholipids on the permeation of cell penetrating peptides into model membranes

Cell-penetrating peptides (CPPs) are often short, highly positively charged molecules that cross the cell membrane without requiring any protein transport. In eukaryotic and bacterial membranes, negatively charged lipids such as phosphatidylglycerol (PG) and phosphatidylserine (PS) are often distributed asymmetrically between the two bilayer leaflets. Notably, changes in anionic lipid asymmetry are implicated in antimicrobial resistance and disease states. Thus, a physical understanding of how CPPs interact with and permeate asymmetric membranes could reveal design principles for future CPPs as drugs or drug conjugates. Here, we systematically investigate the effect of anionic lipid asymmetric leaflet distribution on interactions of two CPPs, NAF-144-67 and RW9, using large unilamellar vesicles (LUVs) as a model system. LUVs with varied inner and outer leaflet distributions of PS, phosphatidylglycerol (PG), and phosphatidic acid (PA) were generated using methyl-β-cyclodextrin exchange and incubated with RW9 or Dansyl-NAF-144-67 at a 1/100 peptide/lipid ratio. Fluorescence spectroscopy was used to probe the chemical environment of the peptides at equilibrium using the solvatochromic properties of tryptophan and Dansyl. As anionic lipids were doped into the outer leaflet, more CPP permeated into the membrane interior for both peptides while inner leaflet anionic leaflets had minimal effect on peptide-membrane permeation. The effect of anionic lipid gradient between leaflets was also explored and the partition coefficient of each peptide into the hydrophobic interior of the membrane was quantified, with PA in the outer leaflet resulting in the most membrane-partitioning by both peptides. These results suggest that positively charged CPPs may permeate to a greater extent into cells with anionic outer leaflet lipids, such as cancer cells, while cells with inner leaflet anionic lipids may not produce the same effect, thus informing the design of CPPs for selective drug delivery.