(M1130-03-15) Novel Lipid Nanoparticles for Potential mRNA Delivery Vaccine Applications

Purpose: The outbreak of the COVID-19 pandemic enlarged the investigation and development to deliver mRNAs for disease prevention, especially after the FDA approval of Pfizer and Moderna vaccines. However, the delivery system of such a drug modality is still in high demand due to mRNA degradation. Therefore, lipid nanoparticles were designed as mRNA delivery platforms by penetrating amphiphilic cell membranes, releasing drug payloads through endosomal escape, and ultimately triggering immune responses through cascaded signaling pathways. As the linkage between innate and adaptive immunity, dendritic cells have attracted much attention and are present as popular targeting candidates. These trending cell lines, if applied to mRNA vaccines, are of great promise to prevent cancers and serve to treat autoimmune diseases. Herein, we aim to screen the ionizable lipid library to optimize the potent candidates for mRNA delivery into JAWS II cell lines. Then we will characterize the lipid nanoparticles and analyze the safety parameters, together with the further mRNA vaccine application encoding specific sequences.

Methods: Prepare LNPs by rapidly pipetting the mRNA-containing ethanol phase into the lipid ethanol phase. Briefly, an aqueous phase containing luciferase-encoding mRNA was prepared in a 10 mM citrate buffer (pH 3). The ethanol phase is prepared by mixing ionizable lipids, phospholipids, cholesterol, and PEG in predetermined molar ratios. The JAWS II cell line was cultured in Alpha-MEM medium containing 2 mM L-glutamine, 5 ng/mL GM-CSF, and 20% FBS. Cells were seeded in luciferase 96-well plates at 20,000 cells per well. After a 24-hour pre-incubation at 37 degrees and 5% CO2, 5 uL of formulated nanoparticles were added to each well, followed by an additional 18-hour incubation. After processing, add 95 μl of pre-thawed Bright-Glo Luciferase Substrate to each well using a multichannel pipette. Shake the plate for 3-5 min in the dark, then measure the relative luminescence intensity on a plate reader.

Results: All nanoparticle size ranges from 150 nm to 180 nm in diameter (data not shown here). For the luciferase assay, lipid V6 showed the highest relative luminescence intensity. The intensity of lipid V6 was 7 and 6 folds higher than lipid V9 and lipid V7, respectively. Furthermore, lipid V6 surpassed the FDA-approved ALC-0315 lipid formulation by more than 100 folds.

Conclusion: The result shows that vaccine lipid V6 showed the highest Luciferase intensity among all candidates for transfecting luciferase-encoding mRNA into JAWS II cell lines. Although the optimization of lipid ratios and lipid-mRNA ratios are still under investigation, this lipid V6 is promising to build a systemic platform for delivering mRNAs for preventative vaccine applications.

Acknowledgments: Authors disclose no conflicts.

Figure 1. The relative luminescence intensity of lipid vaccine candidates/ Luci-mRNA transfection in JAWS II cell line.