2.3. Preparation of mRNA-LNPs

The mRNA-LNPs used here were based on the lipid composition of a clinically approved BNT162b2 formulation [21], and prepared using a generalized protocol collated from multiple sources [22,23]. Briefly, ionizable cationic lipid ALC-0315 (#HY-138170, MedChemExpress LLC, Monmouth Junction, NJ, USA), neutral lipid 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC; #850365P, Avanti Polar Lipids, Inc., Birmingham, AL, USA), cholesterol (#C8667, Sigma Aldrich, St. Louis, MO, USA), and PEGylated lipid ALC-0159 (#HY-138300, MedChemExpress) were mixed at a molar ratio of 46.3:9.4:42.7:1.6 mol% in ethanol. OVA mRNA was prepared in 10 mM citrate buffer at a nitrogen/phosphate ratio of 6. Both the solutions were mixed together at a total flow rate of 12 mL/min and a flow rate ratio of 3:1 v/v (aqueous/organic phase) using the NanoAssemblr Ignite (Precision NanoSystems Inc., Cytiva, Vancouver, BC, Canada). Newly encapsulated mRNA-LNPs were dialyzed and concentrated using 100 K Amicon Ultra centrifugal filters (#UFC/910096, Merck Millipore, Burlington, MA, USA). The LNP particle size and zeta potential were checked using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The encapsulated mRNA content in the mRNA-LNPs was determined using the Quant-iT RiboGreen RNA Reagent and Kit (#11490, Invitrogen, Thermo Fisher Scientific Inc., Darmstadt, Germany). The final mRNA-LNP vaccine was prepared by diluting mRNA-LNP to a final volume of 40 µL, containing 1 µg of mRNA in 10% sucrose solution.