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Lipoplexes with different surface charge were prepared from a short oligonucleotide (20 mer, dsAT) inserted into liposomes of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE). The starting liposomes were prepared by two different procedures, i.e. progressive dsAT addition starting from plain liposomes (titration) and direct mixing of dsAT with pure liposomes (point to point preparation). Lipoplexes were characterized from a molecular point of view by Electron Spin Resonance (ESR) of a cationic spin probe and by Nuclear Magnetic Resonance. Structural and surface features were analysed by Zeta potential (zeta) measurements and Cryo-TEM micrographs. The complete set of results allowed to demonstrate that: i) the interactions between dsAT and cationic lipids were strong and occurred at the liposome surface; ii) the overall shape and physicochemical properties of liposomes did not change when short nucleic acid fragments were added before surface charge neutralization; iii) the bilayer structure of the lipids in lipoplexes was substantially preserved at all charge ratios; iv) the physical status of lipoplexes with electrical charge far from neutrality did not depend on the preparation method.

Cationic liposomes-DNA complexes (lipoplexes) are largely used in gene delivery. Deciphering specific chemical and physical properties of lipoplexes is a necessary step to unravel the mechanisms underlying transfection and to improve transfection efficacy in each experimental model. In the present paper we investigated the physico-chemical features of lipoplexes containing a plasmid encoding for the GFP protein, in order to correlate these results with transfection efficacy. Cationic unilamellar vesicles (mean diameter 100 nm) were prepared, from the cationic DC-Chol lipid and the zwitterionic phospholipid DOPE. The two components of the liposome bilayer were used at molar ratio close to unity. ESR spectra were recorded and zeta potential zeta was measured on liposomes complexed with the plasmid. One of the main points of interest in this paper resided in the fact that both kinds of measurements were carried out in the same conditions (i.e. lipid concentration, medium composition, and pH) employed for cell transfection experiments. Transfection was performed on CHO cells; the percentage of fluorescent cells was evaluated and compared with the above physico-chemical features. It emerged that the composition and pH of the medium, the lipoplex/cell ratio, as well as the amount of lipoplex added to the cell culture were critical parameters for transfection efficacy. Finally, lipoplex surface charge played a fundamental role to achieve a high transfection level.