To increase cationic liposome-mediated intravenous DNA delivery extruded DOTAP:cholesterol liposomes were used to form complexes with DNA, resulting in enhanced expression of the chloramphenicol acetyltransferase gene in most tissues examined. The DNA:liposome ratio, and mild sonication, heating, and extrusion steps used for liposome preparation were crucial for improved systemic delivery. Size fractionation studies showed that maximal gene expression was produced by a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size. Cryo-electron microscopy examination demonstrates that the DNA:liposome complexes have a novel morphology, and that the DNA is condensed on the interior of invaginated liposomes between two lipid bilayers. This structure could account for the high efficiency of gene delivery in vivo and for the broad tissue distribution of the DNA:liposome complexes. Ligands can be placed on the outside of this structure to provide for targeted gene delivery.

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The elastic area compressibility modulus, Ka, of lamellar liquid crystalline bilayers was determined by a new experimental approach using 2H-NMR order parameters of lipid hydrocarbon chains together with lamellar repeat spacings measured by x-ray diffraction. The combination of NMR and x-ray techniques yields accurate determination of lateral area per lipid molecule. Samples of saturated, monounsaturated, and polyunsaturated phospholipids were equilibrated with polyethylene glycol (PEG) 20,000 solutions in water at concentrations from 0 to 55 wt % PEG at 30 degrees C. This procedure is equivalent to applying 0 to 8 dyn/cm lateral pressure to the bilayers. The resulting reductions in area per lipid were measured with a resolution of +/-0.2 A2 and the fractional area decrease was proportional to applied lateral pressure. For 1,2-dimyristoyl(d54)-sn-glycero-3-phosphocholine, 1-stearoyl(d35)-2-oleoyl-sn-glycero-3-phosphocholine (SOPC-d35), and 1-stearoyl(d35)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC-d35) cross-sectional areas per molecule in excess water of 59.5, 61.4, and 69.2 A2 and bilayer elastic area compressibility moduli of 141, 221, and 121 dyn/cm were determined, respectively. Combining NMR and x-ray results enables the determination of compressibility differences between saturated and unsaturated hydrocarbon chains. In mixed-chain SOPC-d35 both chains have similar compressibility moduli; however, in mixed-chain polyunsaturated SDPC-d35, the saturated stearic acid chain appears to be far less compressible than the polyunsaturated docosahexaenoic acid chain.