The yin of exofacial protein sulfhydryls and the yang of intracellular glutathione in in vitro transfection with SS14 bioreducible lipoplexes

Although redox-sensitive transfectants have been considered hitherto as the Holy Grail of gene delivery because of their ability to restrict the release of nucleic acids to intracellular compartments, the reasons for their sometimes lackluster performance do not seem likewise clear. To ascertain the possible influence of extracellular soluble thiols, exofacial protein sulfhydryls (EPTs) and glutathione (GSH) on the overall efficacy of bioreducible lipoplexes, we utilized a cationic gemini surfactant in which the two single-chain amphiphiles are held together by a suitable redox-sensitive linkage. We herein draw a big picture whereby the interaction of bioreducible lipoplexes with cells and their internalization are tightly coupled events that ultimately do affect transgene expression. Specifically, we provide evidence that in plain DMEM EPTs entail the reduction-triggered disruption of bioreducible lipoplexes, thereby resulting in a considerable ~30% waste of nucleic acids and low transgene expression. The release of DNA from lipoplexes can be partially overcome (~16%) by transfecting cells in complete medium and fully reverted by preincubating bioreducible lipoplexes in the same culture supernatant for 1 h prior to transfection (i.e. preconditioning), thus increasing transfection efficiency by ~3-fold and ~10-fold, respectively. These results lead to the proposal of the protein corona as the central mediator in shielding bioreducible lipoplexes from the action of exofacial sulfhydryls in the early phase of delivery and provide a smart solution as to how to increase their efficacy. Finally, we pinpoint associations between intracellular GSH levels, known to be particularly high in cancer cells, and the extent of transfection.