Distinct Adsorption Behavior and Structures of Cell-Penetrating Peptides at a Model Lipid Membrane Interface: A Heterodyne-Detected Vibrational Sum Frequency Generation Spectroscopy Study

Abstract

Arginine-rich cell-penetrating peptides (CPPs) are widely used as molecular delivery vectors, yet the molecular mechanism of their membrane activity and how they penetrate a cell remains unclear. Here, we investigate the interfacial structures of positively charged octa-arginine (R8) and its hydrophobically modified analogue, stearyl-octa-arginine (SR8), at negatively charged lipid monolayers using phase-resolved heterodyne-detected vibrational sum-frequency generation (HD-VSFG) spectroscopy. HD-VSFG measurements show that R8 significantly decreases the intensity of the positive OH stretch band of interfacial water, while SR8 changes it to a negative band. This suggests that the hydrophobic stearyl moiety in SR8 promotes higher adsorption efficiency, inducing charge inversion. In the amide I region, R8 exhibits two spectral components at ~1640 and ~1680 cm-1, whereas SR8 only shows a dominant component near ~1640 cm-1, indicating that stearylation substantially affects the interfacial peptide conformation and/or its orientational distribution. Furthermore, from the analysis of the polarization dependence of the lipid CO stretch band, it is suggested that the lipid carbonyls adopt a broader orientational distribution upon SR8 adsorption than upon R8 adsorption. These results demonstrate that hydrophobic modification affects not only the adsorption efficiency of arginine-rich CPPs but also their interfacial structure and peptide-lipid interactions, at a charged lipid interface.