Arginine-Rich Cell-Penetrating Peptides Induce Lipid Rearrangements for Their Active Translocation across Laterally...

Arginine-rich cell-penetrating peptides (CPPs) have emerged as valuable tools for the intracellular delivery of bioactive molecules, but their membrane perturbation during cell penetration is not fully understood. Here, nona-arginine (R₉)-mediated membrane reorganization that facilitates the translocation of peptides across laterally heterogeneous membranes is directly visualized. The electrostatic binding of cationic R₉ to anionic phosphatidylserine (PS)-enriched domains on a freestanding lipid bilayer induces lateral lipid rearrangements; in particular, in real-time it is observed that R₉ fluidizes PS-rich liquid-ordered (L_o) domains into liquid-disordered (L_d) domains, resulting in the membrane permeabilization. The experiments with giant unilamellar vesicles (GUVs) confirm the preferential translocation of R₉ through L_d domains without pore formation, even when L_o domains are more negatively charged. Indeed, whenever R₉ comes into contact with negatively charged L_o domains, it dissolves the L_o domains first, promoting translocation across phase-separated membranes. Collectively, the findings imply that arginine-rich CPPs modulate lateral membrane heterogeneity, including membrane fluidization, as one of the fundamental processes for their effective cell penetration across densely packed lipid bilayers.