e3MPH16: A D-Glutamic Acid-Substituted Peptide for Efficient and Low-Cytotoxicity Cytosolic Delivery of Macromolecules.

Antibody-based therapeutics have shown remarkable success in targeting extracellular molecules, yet their application to intracellular targets remains largely unexplored due to the absence of efficient delivery systems. The large molecular weight and hydrophilicity of immunoglobulin G (IgG) make cytosolic delivery particularly challenging. Previously, we developed a cytosolic delivery peptide, E3MPH16, based on a modified Mastoparan X sequence, which enabled efficient delivery of macromolecules such as dextran with minimal cytotoxicity. However, effective intracellular delivery of antibodies required high concentrations, limiting its practical utility. In this study, we aimed to enhance delivery efficiency while preserving low toxicity by introducing d-amino acid substitutions into E3MPH16. The resulting peptide, e3MPH16, incorporates d-glutamic acid residues at the N-terminus to improve serum stability and protease resistance. Functional analyses demonstrated that e3MPH16 significantly improves cytosolic delivery of Cre recombinase and antibodies compared with the original E3MPH16, without increasing membrane-lytic activity or cytotoxicity. These results underscore the potential of d-amino acid-substituted peptides such as e3MPH16 as a promising platform for the intracellular delivery of unmodified functional antibodies.