Optimization of PEGylation for cationic triacyl lipid-based siRNA lipoplexes prepared using the modified ethanol injection method for tumor therapy.

We previously developed a modified ethanol injection (MEI) method to construct small interfering RNA (siRNA) lipoplexes by mixing a lipid-ethanol solution with an siRNA-containing phosphate-buffered saline solution. Here, we constructed siRNA lipoplexes with 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-,,-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12), 1,2-dioleoyl--glycero-3-phosphoethanolamine, and poly(ethylene glycol) (PEG)-lipid using our MEI method. The siRNA lipoplexes were PEGylated with 1, 3, 5, and 10 mol% PEG cholesteryl ether (PEG-Chol), 1,2-dimyristoyl--glycero-3-methoxypolyethylene glycol (mPEG-DMG), or 1,2-distearoyl--glycero-3-phosphoethanolamine--(methoxy[polyethylene glycol]) (mPEG-DSPE). PEGylation of siRNA lipoplexes with PEG-Chol did not attenuate the inhibitory effects of Luc and polo-like kinase 1 (PLK1) siRNA lipoplexes on the luciferase (Luc) activity and proliferation of human cervical carcinoma HeLa-Luc, human ovarian cancer SK-OV-3-Luc, and human breast cancer MCF-7-Luc cells stably expressing Luc. In contrast, PEGylated lipoplexes with 10 mol% mPEG-DMG inhibited Luc activity by Luc siRNA but considerably attenuated the PLK1 siRNA-mediated cytotoxic effects. For PEGylated siRNA lipoplexes with mPEG-DSPE, inhibitory effect of Luc siRNA on Luc activity decreased with increasing amounts of PEG modification, and PLK1 siRNA-mediated cytotoxic effects disappeared at more than 3 mol% PEGylation. Erythrocyte aggregation and hemolysis induction by the siRNA lipoplexes were effectively inhibited by 10 mol% PEGylation, irrespective of the PEG-lipid. Compared to those with 1 mol% PEG-Chol, PEGylated siRNA lipoplexes with 10 mol% PEG-Chol potently reduced siRNA accumulation in mouse lungs post-intravenous administration. Overall, TC-1-12-based siRNA lipoplexes with 10 mol% PEG-Chol exerted PLK1 siRNA-mediated cytotoxic effects, without inducing hemolysis and erythrocyte aggregation.