The Stability of lyophilized lipid/DNA complexes during prolonged storage

The Stability of lyophilized lipid/DNA complexes during prolonged storage
June 2004
Marion d.C. Molina, Taylor K. Armstrong, Ye Zhang, Mayank M. Patel, Yvonne K. Lentz, Thomas J. Anchordoquy
Center for Pharmaceutical Biotechnology, University of Colorado School of Pharmacy, Denver, Colorado 80262
Journal of Pharmaceutical Sciences, Volume 93, Issue 9 , Pages 2259 - 2273
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Abstract
It is well known that excipients are required to protect nonviral vectors during the lyophilization process. The goal of this study is to describe the stability of lyophilized nonviral vector preparations on pharmaceutically relevant timescales and provide insight into the factors that govern long-term stability of vectors in the dried state. Lipid/DNA complexes were lyophilized in glucose, sucrose, or trehalose and stored for a period of up to 2 years at five different temperatures (-20, 4, 22, 40, 60?C). We evaluated simultaneously the physico-chemical characteristics (size, zeta potential, ethidium bromide (EtBr) accessibility, supercoiled DNA content) and the ability of vector formulations to transfect COS-7 cells at different time intervals. In addition, a fluorescence assay was utilized to assess levels of ROS in the dried cake after storage. The physical state of each formulation was evaluated by determination of the glass transition temperature and residual moisture content, before and after storage. Results from our stability study show that a progressive degradation of lipid/DNA complexes occurs in terms of transfection rates, particle size, dye accessibility, and supercoil content, even when samples are stored at low temperatures (e.g., -20?C). Furthermore, our preliminary results on the quantification of free radicals in rehydrated formulations emphasize the importance of developing strategies to prevent the formation of reactive oxygen species (ROS) during prolonged storage in the dried state. ? 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:2259-2273, 2004