A Melittin-Derived Peptide with Improved Cytosolic Delivery Efficiency through Caveolae- and Actin-Mediated Endocytosis.

Efficient cytosolic delivery of functional proteins such as therapeutic antibodies remains a major challenge in drug development. In this study, we sought to optimize the cytosolic delivery peptide Mel-V8G12, a melittin derivative, through structure-guided design and functional screening of its amino acid substitutions. Among seven derivatives, VG-6, featuring A10L, T11E, and S18K substitutions demonstrated superior cytosolic delivery efficiency compared with the parental Mel-V8G12, while maintaining low cytotoxicity. Notably, VG-6 exhibited enhanced membrane-lytic activity toward neutral lipid membranes, yet did not increase cellular toxicity, suggesting a delivery mechanism distinct from conventional pH-responsive endosomolytic peptides. Mechanistic studies revealed that, in contrast to Mel-V8G12 which predominantly utilizes actin-mediated endocytosis, VG-6 additionally engages caveolae-mediated endocytosis, contributing to its enhanced cytosolic delivery. Furthermore, VG-6 enabled successful cytosolic delivery of functional Cre recombinase and immunoglobulin G (IgG), facilitating biological activity and subcellular targeting. These findings suggest that VG-6 is a promising tool for intracellular delivery of protein therapeutics via a unique membrane-interacting and endocytic pathway.