Journal
JOURNAL OF PHARMACEUTICAL SCIENCES
Volume 93, Issue 9, Pages 2259-2273Publisher
JOHN WILEY & SONS INC
DOI: 10.1002/jps.20138
Keywords
nonviral vectors; lyophilization; storage stability; reactive oxygen species; gene delivery; dehydration; cationic lipids; DNA
Funding
- NIGMS NIH HHS [GM60587-01] Funding Source: Medline
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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, 60degreesC). 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., -20degreesC). 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. (C) 2004 Wiley-Liss, Inc. and the American Pharmacists Association.
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