This exploratory/developmental proposal seeks to develop methods for achieving active control over the transfection of cells using a ferrocene-containing cationic lipid that can be transformed electrochemically. The approach is founded on preliminary observations that lipid/DNA complexes prepared from a ferrocene- containing cationic lipid lead to high levels of cell transfection when the lipid is in a reduced redox state, but very low levels of transfection when the lipid is in the oxidized state. This critical observation suggests the basis of a general and facile approach that could be used to achieve active control over the transfection of cells in vitro or in vivo though the in situ electrochemical activation of lipoplexes. The proposed research seeks to test the hypothesis that a redox-active, ferrocene-containing cationic lipid can be used to formulate lipoplexes that are either active or inactive toward cell transfection, and that the activation of `inactive' formulations can be can be controlled externally and actively through the application of low electrical potentials. The following Specific Aims are designed to evaluate this hypothesis and determine the feasibility of this approach. They are: 1) To characterize levels of transfection and cytotoxicity in a panel of cell lines using lipoplexes formed from plasmid DNA and either the completely oxidized or completely reduced forms of a model ferrocene-containing cationic lipid (BFDMA). A second goal of this Aim seeks to evaluate differences in the ability of reduced and oxidized BFDMA to mediate transgene expression in vivo when administered to mice by tail vein injection; 2) To measure the rates at which changes in the oxidation state of BFDMA can be effected within lipoplexes and to characterize the rates of reorganization of lipoplexes upon transformation. A second goal of this Aim seeks to characterize the integrity of DNA within lipoplexes upon exposure to the electrochemical potentials used to transform the lipid; and 3) To demonstrate that electrochemical transformation of DNA/BFDMA lipoplexes in situ leads to changes in the extent of transfection of cells in vitro and in vivo. We envisage the potential outcomes of this exploratory/developmental research as having substantial fundamental and applied impacts in at least two ways: first, through the introduction of new tools for basic biological and biomedical research, and, secondly, through the introduction of methods that could ultimately be used to achieve spatial and temporal control of transfection in therapeutic or clinical contexts. ? ? ?
