Plasmid DNA is protected against ultrasonic cavitation-induced damage when complexed to cationic liposomes.
Cationic liposomes bound to plasmid DNA are currently used for in vitro and in vivo gene therapy applications, but such complexes readily form large, heterogeneous aggregates that are not appropriate for pharmaceutical development. More importantly, size heterogeneity makes studies focused on optimizing gene transfer to cells difficult to conduct or understand. For this reason we have evaluated the effect of microprobe sonication on these complexes in an effort to achieve process-controlled size homogeneity. Complexes were prepared using a 7.2 kb reporter plasmid and the following liposomal lipid combinations: DDAB/DOPE (50:50 mol %), DDAB/DOPE/PEG-PE (50:45:5 mol %), DDAB/EPC (50:50 mol %), DDAB/EPC/PEG-PE (50:45:5, 50:40:10, 50:35:15 mol %), DODAC/DOPE (50:50 mol %), and DODAC/EPC (50:50 mol %) (DDAB, dimethyldioctadecylammonium bromide; DOPE, dioleoylphosphatidylethanolamine; PEG-PE, monomethoxypolyethylene glycol2000 succinate- distearoylphosphatidylethanolamine; EPC, egg phosphatidylcholine; DODAC, dioleoyldimethylammonium chloride). The influence of complex composition and lipid:DNA ratio was evaluated. Particle size was determined before and after complexation and again after sonication using the quasi-elastic light scattering technique. DNA integrity was assessed via agarose gel electrophoresis. Finally, gene transfection was evaluated using CHO cells that were transfected in vitro with sonicated and unsonicated complexes. It is established in this study that size reduction can occur, but this is dependent on cationic and neutral lipid composition and, in some cases, lipid:DNA ratio. Surprisingly, the process of sonication leaves a significant percentage of the plasmid DNA intact and capable of in vitro transfection. This study shows that plasmid DNA can be protected from damage due to sonication by liposome complex formation. This may indicate that more common pharmaceutical methods for size reduction which subject particles to mechanical stress may be applicable in preparation of liposome/DNA formulations for in vivo application.
Wasan
E K
EK
Medical Oncology Advanced Therapeutics, British Columbia Cancer Agency, Vancouver, Canada.
Reimer
D L
DL
Bally
M B
MB
D000818:Animals; D016466:CHO Cells; D002412:Cations / Q000737:chemistry; D006224:Cricetinae; D004249:DNA Damage; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry*; D014162:Transfection; D014465:Ultrasonics; D001616:beta-Galactosidase / Q000235:genetics
D002412:Cations; D008055:Lipids; D008081:Liposomes; D001616:beta-Galactosidase