Benzaldehyde‐Tagged Cell‐Penetrating Peptides Enable Efficient Cytosolic Delivery of Bioactive Cargos via Dynamic Covalent Anchoring

Cytosolic delivery of biomacromolecules constitutes an indispensable component in cell therapies, genome editing, and a diversity of fundamental research applications but remains challenging. Delivery by cell-penetrating peptides (CPPs) has been extensively investigated, especially owing to their advantages in minimal cytotoxicity and low transcriptional interference. However, for the vast majority of CPPs, cellular entry occurs predominantly via endocytic uptake, leading to endosomal entrapment and cargo degradation. Herein, we demonstrate that benzaldehyde-tagged cationic CPPs are highly effective in accessing the cytosol of different human cell lines in an energy-independent manner even at low micromolar concentrations. Live-cell reductive amination coupled with membrane fractionation and fluorescence imaging confirms imine-mediated covalent interaction between benzaldehyde-tagged CPP and native membrane proteins. Comparative interactors profiling using CPP-based photo-crosslinking probes further identifies cell membrane partners for CPPs and reveals a spatial interaction network consistent with the observed subcellular distributions. Furthermore, benzaldehyde-tagged CPPs form more stable delivery complexes with protein cargos via transient imine bonds and other non-covalent interactions. Finally, we showcase the efficient delivery of diverse bioactive proteins and peptides into the cytosol of various cell lines, enabling apoptosis induction or actin filament staining. This imine anchoring technique would open up new avenues for CPPs-based intracellular delivery of biomacromolecules.