Cationic Liposome–Plasmid DNA Complexes Used for Gene Transfer Retain a Significant Trapped Volume

□ The goal of this study is to determine whether cationic liposomes retain any trapped volume after their complexation to plasmid DNA. This serves two purposes: to further the understanding of the physical nature of liposome/plasmid DNA complexes used in gene therapy and to investigate the potential for codelivery of other encapsulated molecules with the liposome–DNA complexes. Cationic liposomes composed of N,N-dioleoyl-N,N-dimethylammonium chloride and dioleoylphosphatidylethanolamine (DODAC/DOPE, 50/50mol %) encapsulating an aqueous trap marker were used to prepare liposome–DNA complexes at various charge ratios. The trapped volume before and after DNA binding was measured by two methods: dialysis and filtration. The effect of tissue culture medium on trapped volume was also investigated. A lipid-mixing assay was employed to further characterize the aggregation events that influence trap volume. The trapped volume (Vt) of neutral control liposomes was 1.1±0.04μL/μmol, which was not affected by the addition of DNA. For cationic liposomes in the absence of DNA the Vt was 1.45±0.46 and 1.54±0.08μL/μmol, as measured by the filtration and dialysis methods, respectively. After addition of DNA, the residual trapped volume (RVt) decreased to 0.43±0.1μL/μmol and 0.47±0.05μL/μmol, as determined by each method, respectively. RVt increased as the ratio of cationic lipid to DNA (nmol of lipid/mg of DNA) was increased above 10, a ratio that corresponds to a charge ratio (positively charged lipids to negatively charged phosphate groups) of 1.62. Aggregation and lipid-mixing were greatest at charge ratios coinciding with the lowest trapped volume. in the presence of tissue culture medium, the Vt of cationic liposomes but not neutral liposomes was reduced, suggesting that the salts have a direct effect on cationic liposomes in the absence of DNA. The RVt of both neutral and cationic liposomes in the presence of DNA, however, was not different from that of the liposomes in the absence of DNA. These results suggest that a significant trapped volume is retained by cationic liposomes after binding to plasmid DNA. This is an important finding with regard to the potential use of DNA/liposome complexes in the codelivery of other bioactive molecules at the time of cell transfection.