Mechanisms of protection of cationic lipid-DNA complexes during lyophilization

Gene therapy using nonviral vectors offers advantages over viral methods. However, the instability of aqueous suspensions of cationic lipid-DNA complexes is a major problem that must be overcome to develop this therapeutic modality on a pharmaceutical scale. Disaccharides have been reported to protect lipid-DNA complexes during lyophilization, and recovery of transfection correlates with the retention of particle size. However, the mechanism by which disaccharides achieve this protection is not known. The purpose of this study was to investigate the protective mechanism by lyophilizing cationic lipid-DNA complexes with a variety of solutes that have different physical behaviors during the lyophilization process. In agreement with previous studies, disaccharides conferred protection to lipid-DNA complexes. By contrast, a large polymeric sugar, hydroxyethyl starch, did not protect as well. The level of protection by additives, such as mannitol, that crystallized during lyophilization was also less than that of the disaccharides, but some protection was nonetheless observed. These data suggest that water replacement plays a significant role in protecting complexes during lyophilization. A second mechanism that prevents aggregation by diluting complexes within freeze-concentrated solutions or dried cakes may also contribute to protection. Sample vitrification did not correlate with maintenance of transfection efficiency. Elucidation of the mechanism(s) by which cationic lipid-DNA complexes are protected during lyophilization will permit a rational approach to the development of stable, lyophilized formulations.