Utilizing Proteolytic‐Resistant Nano‐Short Peptide Based on Naphthyl Tail‐Anchored to Combat Bacterial Infections

Abstract The extensive application of antimicrobial peptides (AMPs) as viable alternatives to antibiotics is constrained by their high susceptibility to enzymatic degradation by proteases. In this study, a series of potent nano‐short peptides are engineered based on the short peptide anti‐enzymolysis motif “R D RRP” by introducing different hydrophobic groups using different strategies. The validity of the self‐assembly system triggered by naphthyl tail anchoring is confirmed through a comprehensive analysis of the bioactivity and nanoproperties of these nano‐short peptides. The naphthyl tail‐anchored N 4 peptide (Nal‐Nal‐Nal‐Nal‐R D ‐R‐R‐P) could self‐assemble into nanofibers in aqueous solutions, exhibiting potent and broad‐spectrum antimicrobial activity with excellent biocompatibility (Geometric Mean of the Minimum Inhibitory Concentration (GM MIC ) = 5.04, Geometric Mean of the Selectivity Index (GM SI ) = 50.8) and remarkable biostability against physiological challenges (salt concentrations, serum components, and various proteases). More importantly, the low resistance propensity for N 4 is attributed to multiple antimicrobial mechanisms combining physical membrane‐breaking and energy metabolism disruption. Its efficacy is substantiated in both Escherichia coli ( E. coli ) induced murine peritonitis‐sepsis and Methicillin‐Resistant Staphylococcus aureus ( MRSA) mediated skin infection models in mice. In summary, these findings advance the design of AMPs with enhanced protease resistance and the development of peptide‐based nanomaterials for biomedical applications.