Oligonucleotide-cationic liposome interactions. A physicochemical study.

Cationic liposomes are effective in delivering antisense oligonucleotides into cells in culture, but their interactions with the oligonucleotides are poorly understood. We studied the aggregation and fusion reactions during the formation of cationic lipid/oligonucleotide complexes in solution and their interactions with lipid bilayers. Phosphorothioate oligonucleotides (15-mer) were complexed with cationic liposomes composed of dimethyldioctadecylammonium bromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE) at 8:15 molar ratio or of a commercial formulation DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammoniummethylsul fate), at different ratios with apparent -/+ charge ratios of 0.03-5.6. Mean size of the complexes increased with -/+ ratio so that at charge ratios 0.4-2.0 the size increased by at least an order magnitude due to the oligonucleotide induced aggregation. Resonance energy transfer experiments showed that in addition to aggregation oligonucleotides induced fusion of cationic liposomes, but the fusion was rate-controlled by the initial aggregation step. Rate constants for oligonucleotide induced aggregation were dependent on lipid concentration and were in the range of (0.2-1).10(7) M-1 s-1 and (1-10).10(7) M-1 s-1 for DDAB/DOPE and DOTAP, respectively. Increase in oligonucleotide concentration induced the aggregation and fusion until at high -/+ ratios electrostatic repulsion of negative surfaces inhibited further aggregation and fusion. DOTAP/oligonucleotide complexes did not induce leakage of calcein from neutral EPC liposomes, but did cause leakage at -/+ charge ratios of < 0.7 and > 2.0 from EPC/DOPE liposomes. Also at -/+ charge ratios below 0.8 DOTAP/oligonucleotide complexes induced leaking from negatively charged DPPC/DPPG liposomes. These results indicate that either phosphatidylethanolamine or negative charge are required in the cell membrane for fusion of cationic liposome-oligonucleotide complexes. The ratio of oligonucleotide to cationic lipid is critical in determining the physicochemical properties of the mixture.