Biophysical and transfection studies of the diC14-amidine/DNA complex

Liposomes of the synthetic cationic lipid, N-t-butyl-N'-tetradecylamino-propionamidine (diC(14)-amidine), efficiently ports DNA into mammalian cells in the absence of other (neutral) lipids. The compositional simplicity of this transfection mix makes it attractive from a formulation perspective. We have used low- and wide-angle x-ray diffraction and polarized light microscopy to characterize the thermotropic phase behavior and microstructure of diC(14)-amidine and of the lipid/DNA (circular plasmid, 5.4 kb) complex with a view to understanding the structure of the complex and its role in transfection. Upon heating, the lipid in buffer undergoes a lamellar crystalline (L-c, d(001) = 41.7 Angstrom)-to-lamellar liquid crystal (L-alpha(C), d(001) depends on hydration and T) transition at similar to40degreesC. Sonicated lipid vesicles with a reported transition temperature of similar to23degreesC complex with DNA. Complex formation is complete at a DNA/lipid mole ratio (rho) of 0.8. Adding DNA to the lipid causes d(001) of the multilayered complex to drop from 52 to 49 Angstrom as p rises from 0.03 to 1.64. The minimal DNA-DNA duplex separation observed is 26 Angstrom, consistent with the close packing of B-DNA. Lipid bilayers in the complex undergo a lamellar gel (L-beta(C))-to-L-alpha(C) (superscript c refers to complex) transition at similar to23degreesC. Transfection efficiency was maximized at rho = 0.4. The structure and transfection data combined suggest that densely packaged DNA in a net positively charged complex is essential for transfection.