A New Peptide-Lipid Formulation Capable of Achieving Transfection Efficiency with Low Toxicity

Abstract Delivering genetic material to mammalian cells is a major challenge in fundamental research and therapeutic applications, especially when balancing transfection efficiency and cytotoxicity. We describe here a nonviral gene delivery system based on the cationic peptide (KH)9-BP100 and the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), with or without superparamagnetic Fe3O4–polyethylenimine nanoparticles (Fe3O4–PEI), under various solvent conditions. We studied the physicochemical properties and biological performance of these systems. Preparing the (KH)9-BP100 formulation in ethanol enabled efficient transfection of roGFP-encoding plasmid DNA into HEK293 cells while maintaining low cytotoxicity at relevant concentrations. Ethanol was the most favorable solvent among those evaluated, resulting in improved transfection performance compared with DMSO and THF formulations. Flow cytometry confirmed high transfection efficiencies for both peptide-based formulations, with mean values of 95.67 ± 1.31% for (KH)9-BP100/DNA and 98.35 ± 0.62% for (KH)9-BP100/Fe3O4–PEI/DNA, compared with 90.13 ± 4.11% for Lipofectamine 3000/DNA. Although the Fe3O4–PEI-containing formulation exhibited the highest mean transfection efficiency, the overall differences among formulations did not reach statistical significance (p = 0.0526). Cell viability remained high across all groups, with no statistically significant differences detected among the formulations (p = 0.5335). The incorporation of Fe3O4–PEI adds a magnetic component to the peptide–lipid system while maintaining high transfection efficiency and cell viability, providing a basis for future studies of magnetically guided gene delivery. The (KH)9-BP100/Fe3O4–PEI formulation provides a multifunctional nonviral gene delivery system that warrants further investigation, including potential magnetically guided delivery.