Highly Efficient Cationic Ethylphosphatidylcholine siRNA Carrier for GFP Suppression in Modified Breast Cancer Cells

The therapeutic potential of RNA interference (RNAi) is considerable because it could allow for selective gene silencing in vivo with high specificity and potency. A major challenge in successfully realizing the full potential of RNAi therapeutics, however, is the efficient delivery of siRNA, the molecules that mediate RNAi. The physicochemical features of siRNA, namely high molecular weight, negative charge and hydrophilicity, impede passive diffusion across the plasma membrane. Additionally, a series of barriers, such as association with serum proteins, uptake by the reticuloendothelial system, kidney filtration, and degradation by endogenous nucleases, obstruct efficient siRNA delivery in vivo. Therefore, transfection agents are used to carry the siRNA molecule through the cell membrane and later detach from it once inside the cell, where it is then loaded into the RNA-induced silencing complex. Synthetic cationic lipids are being developed as non-viral carriers of nucleic acids into cells (1–4), and they currently represent one of the most widely used strategies for in vivo gene delivery. They readily form complexes (lipoplexes) with polyanionic nucleic acids, protecting the latter until entry into cells. However, a critical obstacle for clinical application of lipid-mediated nucleic acid delivery is its unsatisfactory efficiency. At present, a growing number of synthetic lipid transfection agents are becoming commercially available (5–9). Recent reports for successful delivery of siRNA to the liver using cationic lipids (10, 11) are promising for the therapeutic potential of these vectors. Our work is focused on a particularly attractive cationic lipid class, cationic ethylphosphatidylcholines (ePCs) (12, 13). These lipids are slowly metabolized and exhibit low toxicities (12, 13). They are derived from the natural phosphatidylcholines (PCs), in which the zwitterionic phosphocholine headgroup is converted into a cation by esterification of the phosphate group. An additional advantage of this class of cationic lipids is that they do not require a helper lipid for successful delivery. Lipids of this type have demonstrated reasonably good transfection efficiencies for plasmid DNA, both in vitro and in vivo, as well as in antitumor and anti-cystic fibrosis gene therapies (14–20). In a recent molecular structure – activity study, we demonstrated that the hydrophobic lipid moiety is a powerful modulator of transfection efficiency (21, 22). By assessing a set of ~30 cationic ePCs, we showed that hydrocarbon chain variations of these lipids modulate their gene transfection efficiency for human umbilical artery endothelial cells (HUAECs) by over two orders of magnitude (21–24). The observed structure – activity relationship manifests in a well-expressed transfection increase with decreasing chain length and increasing chain unsaturation. Maximum transfection has been found for cationic PCs with monounsaturated 14:1 chains (C14-ePC in Figure 1). Figure 1 Structures of the cationic ethylphosphatidylcholines (ePCs) used in the study. A, Dimyristoleoyl-ePC (C14); B, dioleoyl-ePC (C18); C, dilauroyl-ePC (C12). Except for optimizing the hydrophobic moiety of a single lipid species, another strategy for enhancing transfection efficiency of ePCs by fine-tuning their hydrophobic parts has been found in using combinations of cationic ePCs with different hydrocarbon chains. Thus, the binary mixture of dilauroyl- (C12) and dioleoyl-(C18) ePCs at a 6:4 ratio (C12/18) has been identified as a particularly efficient agent for gene transfection into HUAECs, exceeding more than 30-fold the efficiency of both separate compounds (C12-ePC:C18-ePC in Figure 1) (25, 26). These two nucleic acid vectors, C14-ePC and C12-ePC:C18-ePC, which exhibited particularly high efficiency in transfecting plasmid DNA, together with our best studied benchmark ePC – the dioleoyl ePC (C18-ePC in Figure 1), were tested in the present work for their ability to deliver siRNA for the purposes of gene silencing. They have been compared to the gold standard in gene silencing, Lipofectamine RNAiMAX, in their efficiency for green fluorescent protein (GFP) suppression in modified breast cancer cells.