Effective mRNA transfection of tumor cells using cationic triacyl lipid‑based mRNA lipoplexes

Previously, it was reported that mRNA/cationic liposome complexes (mRNA lipoplexes) composed of the cationic triacyl lipid, 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-<i>N</i>,<i>N</i>,<i>N</i>- trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12), with 1,2-dioleoyl-<i>sn-</i>glycero-3-phosphoethanolamine and poly(ethylene glycol) cholesteryl ether, induce high protein expression in human cervical carcinoma HeLa cells. In the present study, the authors aimed to optimize mRNA transfection using TC-1-12-based mRNA lipoplexes. mRNA lipoplexes were prepared at various charge ratios (+:-) using modified ethanol injection (MEI) and thin-film hydration (TFH) methods and compared the protein expression efficiency after transfection of HeLa cells with the developed mRNA lipoplexes. Firefly luciferase (FLuc) and enhanced green fluorescent protein (EGFP) mRNA lipoplexes prepared using the MEI method exhibited higher Luc and EGFP expression levels in cells than those prepared using the TFH method. Moreover, FLuc mRNA lipoplexes prepared using the MEI and TFH methods at charge ratios of 3:1 and 4:1, respectively, exhibited the highest Luc expression in cells. However, transfection with mRNA lipoplexes using the MEI and TFH methods induced moderate cytotoxicity in HeLa cells (46 and 57% cell viability, respectively). Furthermore, Cy5-labeled mRNA lipoplexes, which were prepared using the MEI method, showed higher cellular uptake of mRNA than those prepared using the TFH method. In the transfection of FLuc mRNA lipoplexes prepared using the MEI method, the storage of the lipid-ethanol solution at 37˚C for 4 months did not decrease Luc expression in HeLa cells. Additionally, FLuc mRNA lipoplexes prepared using the MEI method, induced relatively high Luc expression in human prostate carcinoma PC-3 and human liver cancer HepG2 cells with low cytotoxicity (103 and 81% cell viability, respectively). Overall, the results highlighted the potential of TC-1-12-based mRNA lipoplexes prepared using the MEI method for efficient mRNA delivery to cells.