Polyfluoroalkyl‐Tagged Cell‐Penetrating Peptide‐Additives Enhance Intracellular Protein Delivery via Sustained Monomeric Lipid Interaction

Abstract

ABSTRACT Recent advances in cell‐penetrating peptide (CPP)‐mediated intracellular protein delivery emphasized the critical role of sustained membrane association in enhancing delivery efficiency. Here, we report cell‐surface‐reactive, polyfluoroalkyl‐tagged polyarginine peptides with varying fluorine content as CPP‐additives that significantly enhance protein delivery in living cells. At low micromolar concentrations (2.5 µM), CPP‐additives containing 11–13 fluorine atoms enhanced intracellular protein delivery over 2‐fold relative to a tagless control without observable cytotoxicity. Live‐cell time‐lapse fluorescence imaging revealed that a CPP‐additive with 13 fluorine atoms showed prolonged membrane association (>5 min) relative to a tagless control and facilitated rapid protein internalization within 10 min. Remarkably, surface‐enhanced infrared absorption spectroscopy (SEIRAS) with POPC membranes showed that fluorous CPP‐additives initially interacted with the lipid bilayer predominantly as aggregates but subsequently inserted into the membrane interior as monomers without fluorous tag‐tag association. Complementary molecular dynamics simulations of the initial membrane‐association step provided atomistic insight, showing partial lipid insertion of a monomeric CPP‐additive with 13 fluorine atoms while no insertion was observed for a tagless control within the same time scale. Collectively, our findings establish polyfluoroalkyl‐tagged CPP‐additives as potent, non‐cytotoxic vectors for intracellular protein delivery and provide mechanistic detail regarding the molecular basis of their lipid bilayer interactions.