title: Metal contaminants promote degradation of lipid/DNA complexes during lyophilization.
abstract: Oxidation reactions represent an important degradation pathway of nucleic acid-based pharmaceuticals. To evaluate the role of metal contamination and chelating agents in the formation of reactive oxygen species (ROS) during lyophilization, ROS generation and the stability of lipid/DNA complexes were investigated. Trehalose-containing formulations were lyophilized with different levels of transition metals. ROS generation was examined by adding proxyl fluorescamine to the formulations prior to freeze-drying. Results show that ROS were generated during lyophilization, and both supercoil content and transfection rates decreased as the levels of metal-induced ROS increased. The experiments incorporating chelators demonstrated that some of these agents (e.g., DTPA, desferal) clearly suppress ROS generation, while others (e.g., EDTA) enhance ROS. Surprisingly, there was not a strong correlation of ROS generated in the presence of chelators with the maintenance of supercoil content. In this study, we demonstrated the adverse effects of the presence of metals (especially Fe(2+)) in nonviral vector formulations. While some chelators attenuate ROS generation and preserve DNA integrity, the effects of these additives on vector stability during lyophilization are difficult to predict. Further study is needed to develop potent formulation strategies that inhibit ROS generation and DNA degradation during lyophilization and storage.
authors.affiliation: Center for Pharmaceutical Biotechnology, University of Colorado Health Sciences Center School of Pharmacy, C238, Denver, CO 80262, USA. Marion.Molina@uchsc.edu
authors.forename: Marion d C
authors.initials: Md
authors.lastname: Molina
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Anchordoquy
mesh_terms: D000818:Animals; D019556:COS Cells; D002614:Chelating Agents / Q000737:chemistry; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism*; D003676:Deferoxamine / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D005290:Ferric Compounds / Q000737:chemistry*; D005450:Fluorescamine; D005612:Freeze Drying / Q000379:methods; D007202:Indicators and Reagents; D007502:Iron Chelating Agents / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D004369:Pentetic Acid / Q000737:chemistry; D010618:Phenanthrolines / Q000737:chemistry; D017382:Reactive Oxygen Species / Q000378:metabolism; D012996:Solutions / Q000737:chemistry; D014162:Transfection; D014199:Trehalose / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D002614:Chelating Agents; D005290:Ferric Compounds; D007202:Indicators and Reagents; D007502:Iron Chelating Agents; D008081:Liposomes; D010618:Phenanthrolines; D017382:Reactive Oxygen Species; D012996:Solutions; D014867:Water; C017049:bathophenanthroline disulfonic acid; D005450:Fluorescamine; D004369:Pentetic Acid; D004247:DNA; D004492:Edetic Acid; D014199:Trehalose; D003676:Deferoxamine