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

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.