Differential efficacy of DOTAP enantiomers for siRNA delivery in vitro

DOTAP, as a racemic mixture, is a cationic lipid and a widely used transfection reagent. In this study, the effect of DOTAP's stereochemical structure on transfection efficiency was evaluated in vitro. Racemic and enantiomerically pure DOTAP were used in lipoplex formulations to deliver siRNA to MCF-7 cells, targeting the aromatase enzyme. At the 50 nM siRNA concentration and lipid-to-RNA charge ratios of 4 and 5, the R enantiomer of DOTAP was found to perform better than either the S- or the racemic agent. In addition, at 10 nM siRNA concentration and a charge ratio of 3, the R- lipoplex formulation silenced aromatase by ∼50% whereas the S and racemic formulations caused no significant target downregulation. Differences in lipid packing were modeled using membrane simulations. The results showed that, when combined with cholesterol, pure R-DOTAP and S-DOTAP enantiomers had 105% and 115% of lipid density relative to racemic DOTAP, respectively. These findings suggest an important role of lipid chirality in future development of lipid based siRNA delivery systems.