Incorporation of 2,3-diaminopropionic acid into linear cationic amphipathic peptides produces pH-sensitive vectors.

Nonviral vectors that harness the change in pH in endosomes, are increasingly being used to deliver cargoes, including nucleic acids, into mammalian cells. Here we present evidence that the pK(a) of the beta-NH(2) in 2,3-diaminopropionic acid (Dap) is sufficiently lowered, when Dap is incorporated into peptides, that its protonation state is sensitive to the pH changes that occur during endosomal acidification. The lowered pK(a) of around 6.3 is stabilized by the increased electron-withdrawing effect of the peptide bonds, by intermolecular hydrogen bonding and from contributions arising from the peptide conformation. These include mixed polar/apolar environments, Coulombic interactions and intermolecular hydrogen bonding. Changes in the charged state are therefore expected between pH 5 and 7, and large-scale conformational changes are observed in Dap-rich peptides, in contrast to analogues containing lysine or ornithine, when the pH is altered through this range. These physical properties confer a robust gene-delivery capability on designed cationic amphipathic peptides that incorporate Dap.

Design and evaluation of histidine-rich amphipathic peptides for siRNA delivery.

PURPOSE: Short linear peptides have a high potential for delivering various drugs with therapeutic potential, including nucleic acids. Recently, we have shown that the cationic amphipathic histidine-rich peptide LAH4 (KKALLALALHHLAHLALHLALALKKA) possesses high plasmid DNA delivery capacities. Since such peptides are thought to efficiently disrupt endosomal membranes, we have tested their ability to deliver small interfering RNA (siRNA) into mammalian cells. METHODS: Using a human cell line stably transfected with a luciferase-encoding expression vector, we have evaluated the ability of LAH4 and five derivatives thereof to deliver siRNAs and silence gene expression. RESULTS: The six peptides are all efficient siRNA delivery vehicles whose efficiency in mediating gene silencing in 911-Luc cells was greater than that of commercially available compounds including Lipofectamine, DOTAP and polyethylenimine. In addition, by using the proton pump inhibitor bafilomycin A1, we show that efficient siRNA delivery to the cytosol requires acidification of the endosomes. CONCLUSIONS: The LAH4 histidine-rich cationic amphipathic peptides represent an interesting and promising family of compounds for siRNA delivery.