Plasmid DNA is Protected against Ultrasonic Cavitation-Induced Damage when Complexed to Cationic Liposomes

Abstract

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.

Synergistic effect of ultrasound and PEI on DNA transfection in vitro

“…These data also suggest that sonication did not damage the DNA, which can occur at conditions more extreme than those used in this study [40]. Although not observed here, previous work has shown that complexing DNA with cationic polymers and lipids can increase the stability of plasmid DNA during sonication [18,40,45,46].…”

Analysis of Cationic Liposome-mediated Interactions of Plasmid DNA with Murine and Human Melanoma Cells in Vitro

“…This may, in turn, be an important factor in designing effective lipid-based DNA delivery systems (3). However, the degree to which aggregation occurs following addition of DNA to cationic liposomes varies with different liposomal lipid composition (6), and the lipid structures generated as a consequence of the aggregation events are also dependent on the cationic liposomes used. Differences in lipid composition did not, however, appear to influence the level of DNA delivery in vitro.…”

“…It is evident, however, that the liposome-DNA aggregates are heterogeneous with respect to size and shape (5,6) and generate highly complex fused structures as shown by electron microscopy (7,8). Transfection can be achieved using this heterogeneous population of aggregates, but the complex that is directly responsible for mediating the transfection process is still unknown.…”

Uptake Pathways and Subsequent Intracellular Trafficking in Nonviral Gene Delivery

“…At high positive or negative charge ratios, relatively small complexes are formed, whereas large aggregates are usually formed when the net charge is close to neutrality (Eastman et al, 1997;Almofti et al, 2003). The cationic liposomes used are typically small (ϳ100 nm) before adding to DNA; however, complexes formed with DNA exhibit diameters that range from as small as 200 nm to structures as large as 2 m (Wasan et al, 1996). The formation of lipoplexes is generally difficult to control, and different structures are produced in the same lipoplex preparation.…”