Formulation strategies to minimize oxidative damage in lyophilized lipid/DNA complexes during storage.
It has been shown that degradation of lipid/DNA complexes (lipoplexes) continues in the dried state during storage. The goal of this study was to evaluate the ability of various strategies to minimize the formation of reactive oxygen species (ROS) in lyophilized lipoplexes during storage, including metal removal from reagents, air displacement, and fortification with chelator/antioxidant agents. Formulations containing individual chelator (DTPA) and antioxidants (L-methionine or alpha-tocopherol), or in combination, were subjected to lyophilization. Accelerated storage conditions were investigated and physico-chemical characteristics and biological activity of samples were monitored at different time intervals. Generation of ROS during storage was determined by adding proxyl fluorescamine to the formulations prior to freeze-drying. Lipid peroxidation was assessed by monitoring the formation of thiobarbituric reactive substances (TBARS) and lipid hydroperoxides. We also assessed the effect of increased moisture content on the chemical and biological stability of lipoplexes containing additives. Our results show that both ROS and TBARS are generated in lyophilized cakes during storage, and that agents such as DTPA or alpha-tocopherol are efficient in protecting lipid/DNA complexes against oxidative damage in the dried state. Our experiments also indicate that higher residual moisture has a deleterious effect on the stability of lipid/DNA complexes during storage.
Molina
Marion D C
MD
Nucleonics Inc., Horsham, PA 19044, USA. mmolina@nucleonicsinc.com
Anchordoquy
Thomas J
TJ
D000818:Animals; D019556:COS Cells; D002152:Calorimetry, Differential Scanning; D002614:Chelating Agents / Q000737:chemistry; D002626:Chemistry, Pharmaceutical; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry*; D005612:Freeze Drying; D008055:Lipids / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry; D010084:Oxidation-Reduction; D004369:Pentetic Acid / Q000737:chemistry; D017382:Reactive Oxygen Species / Q000737:chemistry; D017392:Thiobarbituric Acid Reactive Substances; D024502:alpha-Tocopherol / Q000737:chemistry
D002614:Chelating Agents; D008055:Lipids; D017382:Reactive Oxygen Species; D017392:Thiobarbituric Acid Reactive Substances; D004369:Pentetic Acid; D004247:DNA; D008715:Methionine; D024502:alpha-Tocopherol