Evaluation of the Effect of the Introduction of Disubstituted Amino Acids Aib on the Lipid Membrane into Amphipathic Helical Peptides K9L9

Cell-penetrating peptides (CPPs) have attracted considerable attention as carriers that facilitate the intracellular delivery of biomacromolecules. In this study, the amphipathic antimicrobial peptide K9L9 was used as a model, and a series of peptides incorporating the non-proteinogenic amino acid α-aminoisobutyric acid (Aib) were designed and synthesized to investigate the relationship between peptide structure, membrane interaction, and cellular uptake behavior. Circular dichroism analysis revealed that K9L9 adopted a β-sheet–like conformation in an aqueous solution, whereas the Aib-containing peptide formed a stable α-helical structure. In liposome leakage assays, peptides containing two Aib residues exhibited enhanced membrane-disruptive activity, whereas the peptide containing four Aib residues exhibited reduced activity. Furthermore, intracellular delivery studies using fluorescein isothiocyanate-dextran (FITC-dextran) demonstrated that delivery efficiency was significantly enhanced when the peptides were preincubated with FITC-dextran, suggesting that peptide–cargo complex formation plays a critical role in the delivery process. In addition, K9L9 exhibited rapid cellular uptake at early time points, whereas the Aib-containing peptides showed a gradual increase in uptake over time, indicating distinct uptake kinetics. These results demonstrate that Aib-induced helix stabilization modulates the membrane interaction and cellular uptake behavior of amphipathic peptides.