Fine-Tuning and Enhancement of pH-Dependent Membrane Permeation of Cyclic Peptides by Utilizing Noncanonical Amino Acids with Extended Side Chains

The development of cyclic peptides that exhibit pH-sensitive membrane permeation is a promising strategy for tissue-selective drug delivery. Here, we investigated the pH-dependent interactions of designed cyclic peptides bearing noncanonical amino acids of long acidic side chains with lipid membranes, including surface binding, insertion into the bilayers, and translocation across the membrane. It was found that as the length of the side chain of acidic amino acid increased, the binding affinity of the peptides to phosphatidylcholine bilayer surfaces decreased, while the pH for the 50% insertion of the peptides into the bilayers increased. pH-dependent membrane permeation kinetics were examined using a fluorescence-based liposomal assay. The pH for membrane permeation of the peptides was found to increase with the side chain length, resulting in specific membrane permeation at pH ~6.5. In addition, the longer side chain of acidic amino acids improved the maximum rate of membrane permeation at low pH. The activation thermodynamic parameters of the permeation process suggested that both entropic and enthalpic contributions affected the permeation. It was also demonstrated that the cyclic peptide bearing the longest 2-aminosuberic acid residue showed intracellular delivery of cargo molecules into living cells in a pH-dependent manner. This study provides useful clues in the rational design of membrane-permeable peptides.