Arginine‐Rich Cell‐Penetrating Peptides Induce Lipid Rearrangements for Their Active Translocation across Laterally Heterogeneous Membranes

Abstract 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 (R 9 )‐mediated membrane reorganization that facilitates the translocation of peptides across laterally heterogeneous membranes is directly visualized. The electrostatic binding of cationic R 9 to anionic phosphatidylserine (PS)‐enriched domains on a freestanding lipid bilayer induces lateral lipid rearrangements; in particular, in real‐time it is observed that R 9 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 R 9 through L d domains without pore formation, even when L o domains are more negatively charged. Indeed, whenever R 9 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.