Mechanistic study of plasmid DNA delivery by Magainin 2-derived stapled peptides

In this study, the mechanism of the plasmid DNA (pDNA) delivery system using Magainin 2-derived stapled peptides (st7-5 and st7-5_R) was analysed. The effect of different cationic residues (Lys and Arg) on the stability and intracellular transport efficiency of the peptide/pDNA complex was assessed, with both peptides forming stable α-helical structures under neutral conditions and st7-5_R showing higher helical strength under acidic conditions. Agarose gel shift assays and PicoGreen staining showed that both peptides formed complete complexes at N/P ratios of 1.5-2 and protected pDNA from nucleases. However, in the presence of heparin sulfate, the st7-5_R/pDNA complex maintained higher stability than the st7-5/pDNA complex. Interaction analysis with lipid membranes indicated that st7-5 with Lys residues interacted strongly at pH 7.4 and st7-5_R with Arg residues at pH 5.5, suggesting that st7-5_R was highly capable of facilitating its escape from endosomes. The Förster resonance energy transfer (FRET) signal observed by confocal laser scanning microscopy (CLSM) indicated that the st7-5_R/pDNA complex disintegrated over time after intracellular introduction, releasing the encapsulated pDNA into the cell. These results indicate that Magainin 2-derived stapled peptides are promising carriers for efficient intracellular nucleic acid delivery, especially st7-5_R with its excellent stability and delivery efficiency. The findings of this study will contribute to the design of drug delivery system carrier peptides and the improvement of nucleic acid therapeutics.