Preparation and in vitro evaluation of novel lipopeptide transfection agents for efficient gene delivery

Gene therapy by delivery of nonviral expression vectors is highly desirable, due to their safely, stability, and suitability for production as bulk pharmaceuticals. However, low transfection efficiency remains a limiting factor in application on nonviral gene delivery. Despite recent advances in the field, there are still major obstacles to overcome. In an attempt to construct more efficient nonviral gene delivery vectors, we have designed a series of novel lipopeptide transfection agents, consisting of an alkyl chain, one cysteine, 1 to 4 histidine and 1 to 3 lysine residues. The lipopeptides were designed to facilitate dimerization (by way of the cysteine residues), DNA binding at neutral pH (making use of charged lysine residues), and endosomal escape (by way of weakly basic histidine residues). DNA/lipopeptide complexes were evaluated for their biophysical properties and transfection efficiencies. The number and identity of amino acids incorporated in the lipopeptide construct affected their DNA/lipopeptide complex forming capacity. As the number of lysine residues in the lipopeptide increased, the DNA complexes formed became more stable, had higher zeta potential (particle surface charge), and produced smaller mean particle sizes (typically 110 nm at a charge ratio of 5.0 and 240 nm at a charge ratio of 1.0). The effect of inclusion of histidines in the lipopeptide moiety had the opposite effect on complex formation to lysine, but was necessary for high transfection efficiency. In vitro transfection studies in COS-7 cells revealed that the efficiency of gene delivery of the luciferase encoding plasmid, pCMV-Lue, mediated by all the lipopeptides, was much higher than poly(L-lysine) (PLL), which has no endosomal escape system, and in two cases was slightly higher than that of branched polyethylenimine (PEI). Lipopeptides with at least two lysine residues and at least one histidine residue produced spontaneous transfection complexes with plasmid DNA, indicating that endosomal escape was achieved by incorporation of histidine residues. These low molecular weight peptides can be readily synthesized and purified and offer new insights into the mechanism of action of transfection complexes. © 2008 American Chemical Society.