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Methods: The ionizable cationic lipids were synthesized by enzyme-assist reactions and purified by silica gel column. High-throughput screening was performed in vitro to determine the best ionizable cationic lipid candidate following an orthogonal array experimental design which aimed to screen the optimal molar ratio of the lipids for CLC-003. After identifying the optimized formulation in vitro, the LNP3 were synthesized by a microfluidic chip device with firefly luciferase encoded mRNA for in vivo studies. Meanwhile, the LNP3 were formulated with different concentration of cytoprotectant to evaluate the long-term storage capabilities. After a comprehensive evaluation of LNP3 using a reporter gene in vitro and in vivo, we encapsulated the spike protein encoded mRNA into our newly developed LNP3 via microfluidic device to obtain COVID-19 vaccines which maintained function for at least 3 months storage at-20 C after testing. The mice (n= 5) of each group were injected with the LNP3 on a prime-boost manner and the blood and tissue samples were tested accordingly. Materials: All the materials used for synthesis were purchased from Sigma-Aldrich. The mRNAs were obtained from TriLink Biotechnologies and System Biosciences. All other general reagents and kits were all commercially available.

Results: Through the orthogonal design, the best formulation of LNP3 was determined, which exhibited much higher transfection efficacy than FDA approved MC3 LNPs. Notably, the bioluminescence generated by both fresh and 3-month stored luciferase-mRNA encapsulating LNP3 reached~ 2.4* 108 p/s in vivo after intramuscular injection …