Structure–Function Relationships of Amphipathic (Arg–Arg–Aib)_n Peptides: Impact of Chirality and Chain Length on Membrane Permeability and Nucleic Acid Delivery

Abstract

Cell-penetrating peptides (CPPs) have been widely applied as carriers in drug delivery systems (DDS) capable of transporting diverse biomolecules, including nucleic acids and highly hydrophilic low-molecular-weight compounds, into cells. Amphipathic CPPs composed of arginine and the α,α-disubstituted amino acid Aib (2-aminoisobutyric acid) have been investigated for their potential application as carrier peptides. In addition, the incorporation of d-amino acids into CPPs has been utilized as a strategy to confer resistance against proteolytic degradation, which is one of the major challenges associated with CPPs. In this study, we evaluated the structure, membrane permeability, and plasmid DNA (pDNA) delivery capability of (Arg–Arg–Aib)_n peptides with different combinations of l/d-Arg residues. Secondary structures were analyzed by circular dichroism (CD) spectroscopy, and their correlation with membrane permeability was examined. Consequently, α-helical peptides exhibited enhanced membrane permeability with increasing peptide chain length. In comparison between the same chain length of α-helical peptides and random-coil peptides, the difference in membrane permeabilities decreased as the peptide chain length increased. Notably, the peptide (l-Arg–d-Arg–Aib)₄ exhibited the highest protease resistance despite containing l-Arg residues and demonstrated pDNA transfection efficiency comparable to that of an α-helical peptide composed entirely of d-Arg residues. Optimization of l/d-Arg combinations for membrane permeability and gene delivery efficiency in (Arg–Arg–Aib)_n is useful for rationally designing amphipathic CPPs with l/d-amino acids.