Preparation of liposomal gene therapy vectors by a scalable method without using volatile solvents or detergents

A scalable and safe method was developed to prepare liposomal carriers for entrapment and delivery of genetic material. The carrier systems were composed of endogenously occurring dipalmitoylphosphatidylcholine (DPPC), negatively charged dicetylphosphate (DCP), cholesterol (CHOL) and glycerol (3%, v/v). Liposomes were prepared by a modified and improved version of the heating method in which no harmful chemical or procedure is involved. Anionic lipoplexes were formed by incorporating plasmid DNA (pCMV-GFP) to the liposomes by the mediation of calcium ions. Transfection efficiency and toxicity of the lipoplexes were evaluated in CHO-K1 cells using flow cytometry and MTT assay, respectively. Controls included DNA-Ca2+ complexes (without lipids), anionic liposome–DNA complexes (with no Ca2+), and a commercially available cationic liposomal formulation. Results indicated fast and reproducible formation of non-toxic lipoplexes that possess long-term stability, high DNA entrapment capacity (81%) and high transfection efficiency. The lipoplex preparation method has the potential of large-scale manufacture of safe and efficient carriers of nucleic acid drugs.