Efficient mRNA Delivery with mRNA Lipoplexes Prepared Using a Modified Ethanol Injection Method

Abstract

Messenger RNA (mRNA)-based therapies are a novel class of therapeutics used in vaccination and protein replacement therapies for monogenic diseases. Previously, we developed a modified ethanol injection (MEI) method for small interfering RNA (siRNA) transfection, in which cationic liposome/siRNA complexes (siRNA lipoplexes) were prepared by mixing a lipid-ethanol solution with a siRNA solution. In this study, we applied the MEI method to prepare mRNA lipoplexes and evaluated the in vitro and in vivo protein expression efficiencies. We selected six cationic lipids and three neutral helper lipids to generate 18 mRNA lipoplexes. These were composed of cationic lipids, neutral helper lipids, and polyethylene glycol-cholesteryl ether (PEG-Chol). Among them, mRNA lipoplexes containing N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16) or 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl) propan-2-yl) amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12) with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and PEG-Chol exhibited high protein expression in cells. Furthermore, mRNA lipoplexes composed of DC-1-16, DOPE, and PEG-Chol exhibited high protein expression in the lungs and spleen of mice after systemic injection and induced high antigen-specific IgG1 levels upon immunization. These results suggest that the MEI method can potentially increase the efficiency of mRNA transfection, both in vitro and in vivo.

Keywords: delivery; lipoplex; mRNA; mRNA vaccine.

Figure 1

Structure of cationic lipids, neutral helper lipids, and the PEG-lipid used in this study. DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt; DC-1-14: N,N-dimethyl-N-tetradecyltetradecan-1-aminium bromide; DC-1-16: N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide; DDAB: N,N-dimethyl-N-octadecyloctadecan-1-aminium bromide; DC-6-14: 2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium chloride; TC-1-12: 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine; Chol: cholesterol; PEG-Chol: polyethylene glycol-cholesteryl ether.

Figure 2

Effect of cationic and neutral helper lipids in mRNA lipoplexes on luciferase expression in HeLa cells after transfection with FLuc mRNA lipoplexes. mRNA lipoplexes with FLuc mRNA were added to HeLa cells at 0.5 μg/mL mRNA, and luciferase assays were carried out 24 h after incubation. Lipofectamine^® MessengerMAX^TM was used as a control for mRNA transfection. Each column represents the mean + S.D. (n = 3).

Figure 3

Effect of cationic lipids in mRNA lipoplexes on EGFP expression in HeLa cells after transfection with EGFP mRNA lipoplexes. mRNA lipoplexes with EGFP mRNA were added to HeLa cells at 0.5 μg/mL mRNA, and EGFP expression (green) was observed 24 h after incubation. Scale bar = 100 μm. EGFP: enhanced green fluorescent protein.

Figure 4

Effect of cationic lipids in mRNA lipoplexes on cellular uptake in HeLa cells after transfection with Cy5-mRNA lipoplexes. mRNA lipoplexes with Cy5-mRNA were added to HeLa cells at 0.5 μg/mL mRNA, and localization of Cy5-mRNA (green) was observed 3 h after incubation. Scale bar = 100 μm.

Figure 5

Effect of incubation time and mRNA concentration on luciferase expression in HeLa cells after transfection with FLuc mRNA lipoplexes. LP-DC-1-14/DOPE, LP-DC-1-16/DOPE, and LP-TC-1-12/DOPE lipoplexes with FLuc mRNA were added to HeLa cells. (A) Luciferase activities were measured 24 h after transfection of mRNA lipoplexes at 0.5, 1, or 2 μg/mL mRNA. (B) Luciferase activities were measured 4, 24, or 48 h after transfection of mRNA lipoplexes with FLuc mRNA at 0.5 μg/mL mRNA. Each column represents the mean + S.D. (n = 3).

Figure 6

Effect of cationic lipids in mRNA lipoplexes on cell viability 24 h after transfection of FLuc mRNA lipoplexes into HeLa cells. FLuc mRNA lipoplexes were added to HeLa cells at 0.5 μg/mL mRNA. Each column represents the mean + S.D. (n = 6). *** p < 0.001, compared to untreated cells.

Figure 7

Effect of cell types on luciferase expression and viability in A549 and PC-3 cells after transfection with FLuc mRNA lipoplexes. LP-DC-1-14/DOPE, LP-DC-1-16/DOPE, and LP-TC-1-12/DOPE lipoplexes with FLuc mRNA were added to A549 (A,C) and PC-3 cells (B,D) at 0.5 μg/mL mRNA, and luciferase activity (A,B) and cell viability (C,D) were measured 24 h after incubation. Each column represents the mean + S.D. (n = 3 for A and B, n = 6 for C and D). * p < 0.05, ** p < 0.01, compared to untreated cells.

Figure 8

mRNA biodistribution and protein expression in mice after IM injection of mRNA lipoplexes. (A) mRNA lipoplexes with 5 μg of Cy5-mRNA were intramuscularly injected into mice. The mice were sacrificed 1 or 24 h after injection, and Cy5 fluorescence images of the tissues were acquired with an exposure time of 5 s. Scale bar = 1 cm (B) mRNA lipoplexes with 5 μg of FLuc mRNA (5moU) were administered intramuscularly into mice. The mice were sacrificed 4 or 24 h after injection, and the luciferase activity of the tissues was measured. Each column represents the mean + S.D. (n = 4–6).

Figure 9

mRNA biodistribution and protein expression in mice after IV injection of mRNA lipoplexes. (A) mRNA lipoplexes with 10 μg of Cy5-mRNA were intravenously injected into mice. The mice were sacrificed 1 h after injection, and Cy5 fluorescence images of the tissues were acquired with an exposure time of 5 s. Scale bar = 1 cm. (B) mRNA lipoplexes with 20 μg of FLuc mRNA (5moU) were administered systemically into mice. The mice were sacrificed 4 or 24 h after injection, and the luciferase activity of the tissues was measured. Each column represents the mean + S.D. (n = 4–5).

Figure 10

Induction of anti-OVA IgG1 after IV injection of OVA mRNA lipoplexes. LP-DC-1-16/DOPE lipoplexes with 20 μg FLuc mRNA (5moU) or OVA mRNA (5moU) were systemically injected into the mice on days 0 and 14. Blood was collected from immunized mice on day 28, and OVA-specific IgG1 in serum was quantified. Each column represents the mean + S.D. (n = 4). OVA: ovalbumin.