Enantiomeric lysine-based cationic lipids: Design, synthesis, and characterization for in vitro gene delivery

• Lysine-based cationic lipid synthesis was a one-step reaction and easy to purify. • Transfection efficiency rose with longer aliphatic chains in lysine-based lipids. • Lysine configuration had no effect on transfection efficiency. • Lysine-based cationic lipid formulations showed no cytotoxicity. • Lysine-based cationic lipids are safe and effective for in vitro gene delivery. Nucleic acid delivery is crucial for gene therapy, vaccination, and cancer treatment. Despite advances in cationic lipid-based vectors, their transfection efficiency is often limited by structural complexity. In this study, we synthesized lysine-based cationic lipids by esterifying d - or l -lysine with alcohols of varying chain lengths. The compounds were characterized by infrared spectroscopy and mass spectrometry, incorporated into liposomes, and evaluated for transfection efficiency, particle size, morphology, and cytotoxicity in vitro. Transfection performance increased with chain length, peaking at 20 carbons, with no significant differences between d - and l -enantiomers. Compared to Lipofectamine 2000, the optimized liposomes showed superior gene delivery while preserving cell viability. They displayed predominantly spherical to oval morphologies, particle sizes of 30–130 nm, and negligible cytotoxicity. These results suggest lysine-based cationic lipids are promising, safe, and effective nonviral vectors for nucleic acid delivery.