Synthetic gene transfer vectors based on zwitterionic nanoliposome-DNA assemblies (nanolipoplexes), formed by the mediation of magnesium ions, were prepared by a scalable method without employing volatile solvents, high-shear force treatments or extrusion. The zwitterionic nanolipoplexes (NLP) were formulated with PC (phosphatidylcholine) and DPPC (a natural lung surfactant) incorporating different amounts of cholesterol (CHOL). The resulting structures were characterised in terms of their morphology, size and DNA content. In addition, the toxicity and transfection efficiency of the nanolipoplexes were evaluated in cultured Chinese hamster ovary-K1 (CHO-K1) cells. The effects of the multivalent cation Mg(2+) on nanoliposome-DNA transfection potency were evaluated. Formulations containing 10% CHOL showed maximum transfection efficiency and the optimum amount of Mg(2+) ions for transfection with minimum cytotoxicity was ca. 20 mM. The zwitterionic formulations showed significantly less cytotoxicity compared to a commercially available cationic liposome reagent or polyethylenimine (PEI) while they were superior in terms of gene transfer potency. The zwitterionic vectors formulated in this study avoid the use of toxic cationic lipids as well as toxic solvents and may have potential application in gene therapy. The new method will enable scale-up and manufacture of safe and efficacious transfection vehicles required for preclinical and clinical studies. Based on the advantages and superiority of the formulated nanolipoplexes, this method allows for the acceleration of nanolipoplex formulation, enabling the rapid development and evaluation of novel carrier systems for genes and other drugs.