Abstract
Aim: Cationic ethylphosphatidylcholines (ePCs) were evaluated for the delivery of siRNA in modified breast cancer cells. Materials and Methods: Dimyristoleoyl-ePC (C14), dioleoyl-ePC (C18), and dilauroyl-ePC (C12) nanoparticles were complexed with siRNA for green fluorescent protein (GFP) suppression in modified MCF-7 breast cancer cells. The kinetics of GFP suppression were followed over the course of 72 hours. Results: C14, which has been previously found to be particularly effective in gene transfection into primary human umbilical artery endothelial cells, was also remarkably effective as siRNA carrier, with an efficacy exceeding that of Lipofectamine RNAiMAX. The C14 toxicity remained comparable to that of RNAiMAX. The efficacy of the other tested cationic ePC formulations was less than that of C14 and RNAiMAX. Conclusion: The cationic lipid C14 is a highly efficient siRNA carrier that could be used for the development of new formulations for siRNA delivery into cancer cells. A valuable advantage of the C14 formulations is the fact that they are simple, and do not require adjuvants or complex preparation procedures.
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).
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.
Materials and Methods
Dimyristoleoyl-ePC, dioleoyl-ePC, and dilauroyl-ePC were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Lipofectamine RNAiMAX was from Invitrogen (Grand Island, NY, USA). The lipids were used as siRNA carriers for GFP suppression in modified breast cancer cells. MCF-7 cell line was purchased from the American Type Culture Collection (Rockville, MD, USA). Cells were grown in phenol red free Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 5% fetal bovine serum (FBS) and 100 μg/ml streptomycin at 37°C, in a humidified atmosphere of 5% CO2/95% air. To establish phrGFP-transfected cells, breast cancer cells were transfected with phrGFP II-1 plasmid (Stratagene, CA, USA), in LipofectAMINE 2000 (Invitrogen, Carlsbad, CA, USA), as described in the manufacturer's standard procedure. Stably transfected cells were selected with G418 and characterized by fluorescence microscopy. The cell line was established from a single colony and named MCF-7phrGFP-s22. The siRNA targeting the overexpressing phrGFP (5’-CAGGAGACAUGAGCUUCAAGGUGAA-3’ and 5’-UUCACCUUGAAGCUCAUGUCUCCUG-3’), and scrambled control siRNA (sense: 3’-UUCUCCGAACGUGUCACGUTT-5’, antisense: 3’-TTAAGAGGCUUGCACAGUGCA-5’) were custom-synthesized by Invitrogen. Lipid nanoparticles were prepared in sterile deionized water by overnight hydration of the dry lipid followed by vortexing and short (30 s) ultrasonication. The siRNA/lipid complexes were prepared by a standard procedure including pipetting the siRNA solution into the lipid dispersion and vortex mixing, 20-30 min prior to their application to cells. The transfection time was 16 h in all experiments and the kinetics of GFP suppression by siRNA were followed over the course of 72 h. The effects were quantified by measuring the GFP fluorescence in 96-well plates with ~20 000 cells and typically 20 or 40 pmol siRNA applied per well. The results are represented as means±standard deviation (SD).
Results
A total of 10 experiments with 160 samples (6 wells per sample) were carried out to compare the effect of cationic ePCs and RNAiMAX at different lipid and siRNA concentrations. In all samples, the cationic lipid was in excess relative to siRNA base pairs and their molar ratio varied in the range 2-4 lipid molecules per base pair. Optimum results for C14 were obtained at lipid concentrations of 2-3 μg per well, which displayed negligible toxicity. Higher C14 concentrations resulted in marked non-specific suppression of the GFP fluorescence, up to ~80% at 5-10 μg of C14 per well. The C12:C18 mixture (6:4 molar ratio) and C18 preparations were non-toxic, however, their complexes with siRNA were less efficient than the C14/siRNA complex; optimum results were attained at 4 μg per well. Best results for the RNAiMAX/siRNA complexes were obtained at 0.2 μl RNAiMAX/well, in accordance with the manufacturer's recommendation. Controls with higher RNAiMAX concentrations (0.4-0.6 μl/well) also resulted in marked non-specific GFP suppression.
The basic results of this work are shown in Figures 2, 3 and 4 and may be summarized as follows: i) The cationic ePC C14, which was previously found to be particularly effective in gene transfection into HUAECs (23-25), was also remarkably effective as an siRNA carrier. Most notably, its efficacy exceeded that of RNAiMAX (Figures 2 and 3), while the efficacy of the other tested cationic ePC formulations (C12/18 mixture and C18) was less than that of both C14 and RNAiMAX (Figure 4). ii) The toxicity of C14 remained comparable to that of RNAiMAX in the concentration range in which C14 displayed higher efficiency, while the toxicity of the C18 was lower relative to C14 and RNAiMAX. The higher fusogenicity of C14 (23) is the suggested reason for its higher toxicity in relation to the longer-chain C18 lipid.
Green fluorescent protein (GFP) suppression by siRNA complexes with the cationic lipid carriers C14 and RNAiMAX in breast cancer cell line MCF-7phrGFP-s22. The GFP fluorescence intensity was measured in 96-well plates and normalized by the ‘cells only’ control.
Conclusion
In conclusion, it appears on the basis of the present results that the cationic lipid dimyristoleoyl-ePC (C14) is a highly efficient siRNA carrier that could be used for the development of new formulations for siRNA delivery into cancer cells. A valuable advantage of the C14 formulations is the fact that they are simple and do not require adjuvants or complex preparation procedures. The results presented here are in accordance with our previous studies (21-24) on the structure – activity relationships of cationic PCs, which imply that the high efficacy of C14 in transfection stems from its high fusogenicity and ability to induce non-lamellar phases in lipid bilayers.
Kinetics of green fluorescent protein (GFP) suppression by siRNA complexes with C14 and RNAiMAX over the course of 72 hours.
Green fluorescent protein (GFP) suppression by siRNA complexes with the cationic lipid carriers C18 and C12/18 and RNAiMAX in breast cancer cell line MCF-7phrGFP-s22. The GFP fluorescence intensity was measured in 96-well plates and normalized by the ‘cells only’ control.
Acknowledgements
BT and RK thank Professor R.C. MacDonald (Northwestern University) for useful discussions. This work was supported in part by DOD Grants W81XWH-08-0610 and W81XWH-08-1-0521. It was also supported in part by Award UL1RR025755 form the National Center for Advancing Translational Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Advancing Translational Science of the National Institutes of Health.
- Received May 13, 2012.
- Revision received May 31, 2012.
- Accepted May 31, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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