Optimization of Lipid Nanoformulations for Effective mRNA Delivery

Abstract

Introduction: Since the coronavirus disease 2019 (COVID-19) pandemic, the value of mRNA vaccine has been widely recognized worldwide. Messenger RNA (mRNA) therapy platform provides a promising alternative to DNA delivery in non-viral gene therapy. Lipid nanoparticles (LNPs), as effective mRNA delivery carriers, have been highly valued by the pharmaceutical industry, and many LNPs have entered clinical trials.

Methods: We developed an ideal lipid nanoformulation, named LNP3, composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol, and observed its release efficiency, sustained release, organ specific targeting and thermal stability.

Results: In vitro studies showed that the transfection efficiency of LNP3 was higher than that of LNPs composed of DOTAP-DOPE and DOTAP-cholesterol. The positive to negative charge ratio of LNPs is a determinant of mRNA transfer efficiency in different cell lines. We noted that the buffer affected the packaging of mRNA LNPs and identified sodium potassium magnesium calcium and glucose solution (SPMCG) as a favorable buffer formulation. LNP3 suspension can be lyophilized into a thermally stable formulation to maintain activity after rehydration both in vitro and in vivo. Finally, LNP3 showed sustained release and organ specific targeting.

Conclusion: We have developed an ideal lipid nanoformulation composed of DOTAP, DOPE and cholesterol for effective mRNA delivery.

Keywords: 1,2-dioleoyl-3-trimethylammonium-propane; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; cholesterol; lipid nanoparticles; mRNA delivery.

Figure 1

Investigation of cellular association and transfection efficiency of mRNA-LNPs in vitro. (A and B) Green fluorescent imaging of HEK293T and DC2.4 cells after transfection of RNA-LNP. The RNA-LNP constituted with DOTAP/DOPE/cholesterol lipoplex and EGFP-RNA at various charge ratios. Fluorescent images were collected after transfection from three time points, 24, 48 and 72 h. (C) The fluorescent images of HEK293T and DC2.4 cells after simultaneous transfection of three RNA-LNP, ie DOTAP-DOPE, DOTAP-Chol and LNP3, respectively. (D) Green fluorescent imaging of HEK293T cells after transfection of RNA-LNP3 modified with different molar quantities of DSPE-PEG₂₀₀₀, and the images were collected at three time points, 24, 48 and 72 h. The scale bar was 100 μm.

Figure 2

Investigation of buffer-formulated mRNA-LNP3 complex transfection effect. (A) Comparison of different solutions as formulated buffers for mediating mRNA-LNP3 transfection to HEK293T cells. (B) Optimization and development of different formulated buffers based on SPMCG to explore the transfection effect of mRNA-LNP3 complex. The fluorescent images were collected from three time points after transfection, 24, 48 and 72 h. The scale bar was 100 μm.

Figure 3

Transfection activity investigation of mRNA-LNP3 after lyophilization and rehydration using Opti-mem as formulated buffer. (A) Effect of sucrose concentration on EGFP mRNA delivery and expression to HEK293T cells mediated by LNP3 during lyophilization and rehydration. (B) Effect on EGFP mRNA-LNP3 transfection to HEK293T cells after lyophilization and storage for a week at different temperatures, in this work, sucrose concentration was 10% (w/v). w/v represents mass to volume ratio, RT represent room temperature. The fluorescent images were collected from three time points after transfection, 24, 48 and 72 h. The scale bar was 100 μm.

Figure 4

Investigation of biodistribution, sustainability and organ targeting characteristics of lipoplexes in mice following subcutaneous, intramuscular and intravenous administration. (A) Bioluminescence imaging of Balb/c nude mice after SC and IM injection of Fluc mRNA-LNP3. The images collected after transfection at time points of 12, 24, 48, 72 and 96 h. (B) Bioluminescence imaging of Balb/c nude mice after IV injection of Fluc mRNA-LNP3 and Fluc mRNA-LNP4 from two time points, 24 and 48 h, after transfection. (C) Bioluminescence imaging of liver, lung, kidney, spleen and skin in Balb/c mice after IV injection of Fluc mRNA-LNP3 and Fluc mRNA-LNP4, the results were collected 48 h later after transfection.