Degradation of lyophilized lipid/DNA complexes during storage: the role of lipid and reactive oxygen species.

摘要

The presence of trace amounts of metal ions in nonviral vector formulations can significantly affect the stability of lipid/DNA complexes (lipoplexes) during acute freeze-drying. The goal of the present study was to evaluate the generation of reactive oxygen species (ROS) in dried formulations of lipoplexes and in their individual components (lipid or naked DNA). The experiments were conducted in the presence or absence of a transition metal (Fe2+). Lipoplexes and their individual components were formulated in trehalose and subjected to lyophilization and stored for a period of up to 2 months at +60 degrees C. Physico-chemical characteristics and biological activity were evaluated at different time intervals. Generation of ROS during storage was determined by adding a fluorescence probe to the formulations prior to freeze-drying. We also monitored the formation of thiobarbituric reactive substances (TBARS). Our results show that ROS and TBARS form during storage in the dried state. Our findings also suggest that degradation is more rapid in the presence of lipid, even in the absence of metal. We also showed that dried naked DNA formulations are more stable without the lipid component. Effective strategies are then needed to minimize the formation and accumulation of oxidative damage of lipoplexes during storage.