(324a) Chemical Activation of Lipoplexes Prepared From DNA and a Ferrocene-Containing Lipid: Toward Active Control Over the Delivery of DNA to Cells

Cationic lipids have been investigated broadly as non-viral agents for the delivery of DNA to cells in vitro and in vivo. One broad challenge for the design of cationic lipid-based DNA delivery systems is the development of lipids that can be used to exert levels of spatial and/or temporal control over the delivery of DNA to cells (e.g., at specific times or to specific populations of cells within a larger population). The design of the lipids that offer active and external control over the timing and the locations at which DNA is available to cells and tissues could potentially find use in a broad range of fundamental and applied contexts, ranging from basic biomedical research, tissue engineering, and, potentially, for the development of new gene-based therapies.

In this presentation, we report a step toward the design of new principles for spatial and temporal control over the lipoplex-mediated delivery of DNA to cells.  Our approach is based on the use of a redox-active ferrocene-containing cationic lipid, bis(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA). Unlike conventional cationic lipids used for cell transfection, BFDMA can be transformed reversibly between a reduced state (net charge of +1) and an oxidized state (net charge of +3) by the electrochemical or chemical oxidation/reduction of the ferrocene groups located in the hydrophobic tails of the lipid.  Our past studies have demonstrated that the oxidation state of BFDMA significantly affects the interactions of this lipid with DNA. For example, in those past studies we identified lipid concentrations over which lipoplexes formed from reduced BFDMA are able to transfect cells with high efficiency, but for which lipoplexes formed from oxidized BFDMA and DNA yield negligible levels of transgene expression. Physical characterization experiments also demonstrated that the oxidation state of BFDMA significantly influences the zeta potentials and nanostructures of lipoplexes formed from BFDMA and DNA. The results of these past studies suggest a redox-based approach that could be used to provide control over the ‘activation’ or ‘inactivation’ of these lipoplexes.

Here, we demonstrate that it is possible to transform ‘inactive’ lipoplexes (formed using oxidized BFDMA) to ‘active’ lipoplexes using the small-molecule chemical reducing agent ascorbic acid (vitamin C). Our results demonstrate that this transformation can be conducted in cell culture media and in the presence of cells by addition of ascorbic acid to lipoplex-containing media in which cells are growing. Treatment of lipoplexes of oxidized BFDMA with ascorbic acid resulted in lipoplexes composed of reduced BFDMA, as characterized by UV/vis spectrophotometry, and lead to activated lipoplexes that mediated high levels of transgene expression in the COS-7, HEK 293T/17, HeLa, and NIH 3T3 cell lines.

Characterization of internalization of DNA by confocal microscopy and measurements of the zeta potentials of lipoplexes suggested that these large differences in cell transfection result from (i) differences in the extents to which these lipoplexes are internalized by cells and (ii) changes in the oxidation state of BFDMA that occur in the extracellular environment (i.e., prior to internalization of lipoplexes by cells). Additional physicochemical investigation of lipoplexes before and after activation using cryogenic transmission electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS) revealed changes in the nanostructures of lipoplexes upon the addition of ascorbic acid, from aggregates that were generally amorphous, to aggregates with a more extensive multilamellar nanostructure.

When combined, the results of this study provide guidance for the design of redox-active lipids for cell transfection and provide the basis of an approach for the extracellular activation of lipoplexes that could lead to new methods for exerting spatial and/or temporal control over the transfection of cells in vitro.