Addition of ascorbic acid to the extracellular environment activates lipoplexes of a ferrocenyl lipid and promotes cell transfection

Abstract

The level of cell transfection mediated by lipoplexes formed using the ferrocenyl lipid bis(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA) depends strongly on the oxidation state of the two ferrocenyl groups of the lipid (reduced BFDMA generally mediates high levels of transfection, but oxidized BFDMA mediates very low levels of transfection). Here, we report that it is possible to chemically transform inactive lipoplexes (formed using oxidized BFMDA) to “active” lipoplexes that mediate high levels of transfection by treatment with the small-molecule 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). Characterization of lipoplexes by small-angle neutron scattering (SANS) and by cryogenic transmission electron microscopy (cryo-TEM) 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. The results of this study provide guidance for the design of redox-active lipids that could lead to methods that enable spatial and/or temporal control of cell transfection.

Redox-Based Control of the Transformation and Activation of siRNA Complexes in Extracellular Environments Using Ferrocenyl Lipids

“…We used serum-free media for all of the cell-based studies reported here to permit comparison to the results of past studies on BFDMA/DNA lipoplexes and to facilitate biophysical characterization. 29–31,33,35,36 …”

“…We demonstrated in past studies that the addition of small-molecule chemical reducing agents (such as glutathione 31 or ascorbic acid, AA 33 ) to lipoplexes of oxidized BFDMA and plasmid DNA can be used to activate these otherwise “inactive” lipoplexes and promote higher levels of cell transfection. Additional results shown in Figure 2 (white bars) demonstrate that this approach can also be used as a practical means to activate lipoplexes of oxidized BFDMA and siRNA.…”

“…Additional results shown in Figure 2 (white bars) demonstrate that this approach can also be used as a practical means to activate lipoplexes of oxidized BFDMA and siRNA. We used AA as a reducing agent in these experiments because it reduces oxidized BFDMA rapidly, 33 and because we have demonstrated that it can be used to activate DNA-based lipoplexes directly (and in the presence of cells) by simple addition of aliquots of AA to lipoplex-containing cell culture media. 33 For all experiments described below, aliquots of AA were added to BFDMA/siRNA lipoplexes after diluting them in culture media and adding them to cells (see Materials and Methods for additional details).…”

“…30,31,33–36 For example, whereas lipoplexes of DNA and reduced BFDMA have multilamellar nanostructures, inactive lipoplexes of DNA and oxidized BFDMA were found to be amorphous and have zeta potentials that were substantially more negative. The results of this current study demonstrate that this ferrocene-containing cationic lipid can be used to prepare lipoplexes of siRNA that promote effective gene silencing in cells and, significantly, that features of redox-specific “on/off” control of the biological activity observed using lipoplexes formed from plasmid DNA are largely preserved and maintained in lipoplexes formed using siRNA – without the need to further optimize lipid/siRNA charge ratios or change other aspects of nanoparticle formulation.…”

Control of Dual Stimuli-Responsive Vesicle Formation in Aqueous Solutions of Single-Tailed Ferrocenyl Surfactant by Varying pH and Redox Conditions

“…Dodecyltrimethylammonium bromide DTAB was added to the dilute solutions to eliminate cloudy solutions of 11-FAB 29,30 . DTAB does not absorb visible light wavelength 400-700 nm .…”