Understanding cell-penetrating peptide mechanisms using computational electrophysiology simulations.

Cell-penetrating peptides (CPPs) can enter cells without inducing cytotoxicity and can be coupled with cargo molecules to be used to deliver drugs, DNA, or nanoparticles into cells. The peptide-membrane interactions driving the internalization mechanism are not completely understood. In this study, we introduce Computational Electrophysiology (CompEL) as a tool for the computational investigation of CPP and membrane interaction leading to internalization, focusing on cationic CPPs such as Arg9, MAP, TP10, and TP2. CompEL induces membrane stress through ion imbalance, prompting the membrane to alleviate this stress via pore formation. Using double bilayer molecular dynamics (MD) simulations with one or eight peptides, we show that CPPs can use these pores to translocate, whereas non-CPP nonaleucine peptide fails to cross the membrane and instead contributes to pore stabilization. In the eight-peptide systems we observe that some peptides can cooperate to reach translocation or to foster pore stabilization. This study introduces CompEL as a powerful tool for CPP research, shedding light into the molecular peptide-membrane interactions governing CPP translocation, and offering valuable insights for the design of next-generation delivery systems.