Sondeo in situ de los efectos de la densidad de empaquetamiento de lípidos y la concentración de CPP en el proceso...

Cell-penetrating peptides (CPPs) exhibit concentration-dependent efficiency limitations in transmembrane delivery; however, elucidating the molecular mechanism of this concentration dependence from the perspective of interface interaction with phospholipid membranes remains unclear. Here, we employed in situ high-resolution broadband sum-frequency generation vibrational spectroscopy (HR-BB-SFG-VS/SFG-VS) to probe molecular-level interactions between penetrating peptide (PEN) and egg sphingomyelin (ESM) monolayers at the air-water interface. Our study reveals three interconnected mechanisms to govern PEN-ESM interfacial evolution related to this concentration dependence. PEN insertion exhibits a nonmonotonic concentration threshold effect that balances structural ordering promotion and disruption while dictating efficiency transitions. Simultaneously, asymmetric chain reorganization occurs with sphingosine terminal methyl orientation shifts modulated by lipid packing density and PEN concentration, showing angular variations from 32° to 55°, whereas N-alkyl chain terminal methyl angles remain stable between 32° and 38°. Furthermore, the lipid packing density and PEN concentration synergistically regulate interfacial hydrogen-bond networks and adsorption states. At high lipid density such as 30 mN/m, elevated hydrogen-bond network proportions correlate with non-hydrogen-bonded PEN carbonyl states. Conversely, low density conditions such as 10 mN/m reduce network proportions and promote hydrogen-bonded adsorption. Crucially, efficient CPP translocation requires balancing amphipathic domain interactions with dynamic bilayer restructuring, with nonlinear ordering transitions identifying critical thresholds for transmembrane insertion. Lipid packing density and PEN concentration jointly orchestrate interfacial perturbation modes, demonstrating their pivotal role in governing molecular transport efficiency. Our work also demonstrates the unique capability of SFG-VS for resolving such interfacial dynamics, which can offer fundamental insights for designing functional membrane systems.