Efficient encapsulation of DNA plasmids in small neutral liposomes inducedby ethanol and calcium

Efficient encapsulation of DNA plasmids inside small, neutral liposomes com posed of 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), DOPC/DOPE (1,2-dioleoy l-sn-phosphatidylethanolamine) (1:1) and DOPC/DOPE/cholesterol (1:1:1) was achieved by the addition of ethanol and calcium chloride to an aqueous mixt ure of small unilamellar vesicles (SUVs) and plasmid. Following dialysis ag ainst low-salt buffer, the neutral lipid complexes (NLCs) had average effec tive diameters less than 200 nm and encapsulated up to 80% of the DNA. Opti mum Ca2+ and ethanol concentrations for each lipid mixture were determined by statistically designed experiments and mathematical modeling of trapping efficiency. NLCs are unilamellar, have neutral surface potentials, and ret ain entrapped DNA at pH 4.0 and in serum at 37 degrees C. The circulation a nd clearance properties of the complexes following intravenous administrati on in mice are similar to empty neutral liposomes, and the toxicity of NLCs are expected to be significantly reduced compared to other non-viral gene- delivery systems. The NLC encapsulation method, if it can be combined with effective targeting and endosome-release technologies to achieve efficient and tissue-specific transfection, may represent an important alternative to current systemic gene therapy approaches. (C) 2000 Elsevier Science B.V. A ll rights reserved.