Mechanisms of protection of cationic lipid-DNA complexes during lyophilization

Gene therapy using nonviral vectors offers advantages over viral methods. H owever, the instability of aqueous suspensions of cationic lipid-DNA comple xes is a major problem that must be overcome to develop this therapeutic mo dality on a pharmaceutical scale. Disaccharides have been reported to prote ct 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 th is study was to investigate the protective mechanism by lyophilizing cation ic lipid-DNA complexes with a variety of solutes that have different physic al behaviors during the lyophilization process. In agreement with previous studies, disaccharides conferred protection to lipid-DNA complexes. By cont rast, 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 so me protection was nonetheless observed. These data suggest that; water repl acement plays a significant role in protecting complexes during lyophilizat ion. A second mechanism that; prevents aggregation by diluting complexes wi thin freeze-concentrated solutions or dried cakes may also contribute to pr otection. Sample vitrification did not correlate with maintenance of transf ection efficiency. Elucidation of the mechanism(s) by which cationic lipid- DNA complexes are protected during lyophilization will permit a rational ap proach to the development of stable, lyophilized formulations. Copyright (C ) 2000 Wiley-Liss, Inc.