Nanoassemblr ignite

The mRNA was encapsulated with lipid nanoparticles as previously described [10 (link)]. Briefly, the mRNA was diluted with an aqueous solution of mRNA at pH = 4.0 to a final concentration of 100 μg /ml. Then the solution of mRNA was rapidly mixed with lab-prepared lipids dissolved using Nano Assemblr Ignite (PrecisionNanoSystems). Lab-prepared lipids dissolved contained an ionizable cationic lipid, phosphatidylcholine, cholesterol, and PEG-lipid, and the ratio of which was 50:10:38.5:1.5 mol/mol. The mRNA-LNP formulations were diluted with Phosphate Buffered Saline (PBS) and concentrated by AmiconUltra Centrifugal Filter Unit (Millipore) to desired concentrations. The concentration and encapsulation rate of mRNA were measured by Quant-it RiboGreen RNA Assay Kit (Invitrogen, #R11490). The formulations were passed through a 0.22 μm filter and stored at 4°C. Lmix vaccine, an equal mass of multiple mpox virus antigen mRNA, was mixed after being encapsulated by lipid nanoparticles. Rmix vaccine, an equal mass of multiple mpox virus antigen line linearized plasmids were mixed, and then the mixture was transcribed into mRNA that formed a mixture of antigens mRNA.

The mRNA was encapsulated with lipid nanoparticles as previously described [10 (link)]. Briefly, the mRNA was diluted with an aqueous solution of mRNA at pH = 4.0 to a final concentration of 100 μg /ml. Then the solution of mRNA was rapidly mixed with lab-prepared lipids dissolved using Nano Assemblr Ignite (PrecisionNanoSystems). Lab-prepared lipids dissolved contained an ionizable cationic lipid, phosphatidylcholine, cholesterol, and PEG-lipid, and the ratio of which was 50:10:38.5:1.5 mol/mol. The mRNA-LNP formulations were diluted with Phosphate Buffered Saline (PBS) and concentrated by AmiconUltra Centrifugal Filter Unit (Millipore) to desired concentrations. The concentration and encapsulation rate of mRNA were measured by Quant-it RiboGreen RNA Assay Kit (Invitrogen, #R11490). The formulations were passed through a 0.22 μm filter and stored at 4°C. Lmix vaccine, an equal mass of multiple mpox virus antigen mRNA, was mixed after being encapsulated by lipid nanoparticles. Rmix vaccine, an equal mass of multiple mpox virus antigen line linearized plasmids were mixed, and then the mixture was transcribed into mRNA that formed a mixture of antigens mRNA.

mRNAs were encapsulated in lipid nanoparticles by NanoAssemblr Ignite microfluidic cartridge technology (Precision Nanosystems). In brief, lipids were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 (ionizable lipid:DSPC:cholesterol:PEG2000 PE) and mRNA was prepared in 25 mM sodium acetate buffer (pH5.0) at 0.1 mg/ml. Then the lipid mixture and mRNA were mixed at an N/P ratio of 4 through the “Y” shape microfluidic cartridge at a total flow rate of 6 ml/min and a flow rate ratio of 3:1 (aqueous phase:organic phase). After formulation, the mRNA-LNP was dialyzed three times against sterile and Ca2 + /Mg²⁺ free PBS (Cat# 17-516F, Lonza) through a 3.5 K MWCO Slide-A-Lyzer dialysis cassette (Cat# 66330, Thermo Scientific), followed by concentration through a centrifugal filter YM30 (Cat# MRCF0R030, EMD Millipore). The mRNA-LNP was adjusted to 50 μg/ml with or without 10% sucrose (w/v) and aliquoted for different storage conditions. At least two independent preparations for each mRNA-LNP type were used in all experiments.

Lipid nanoparticles (LNPs) were prepared according to a reported protocol^{48 (link)}. Briefly, all lipid components were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 (SM-102; distearoylphosphatidylcholine cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), and mRNAs were dissolved in sodium citrate buffer (50 mM; pH 4) solution at a charge ratio of N/P = 6. LNPs were formulated using NanoAssemblr® IgniteTM (Precision Nanosystems, Vancouver, Canada) by mixing aqueous and organic solutions at a ratio of 3:1 and a total flow rate of 10 mL/min. The LNP solution was concentrated by ultrafiltration using an Amicon Ultracentrifugal filter (UFC9030, Merck Millipore, Billerica, MA, USA), following the manufacturer’s instructions.

LNPs were prepared as per a reported protocol^{36 (link)}. Briefly, all lipid components were dissolved in ethanol at a molar ratio of 25:25:10:38.5:1.5 (SM-102; 6,6′-trehalose dioleate; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamin (DOPE); butyl lithocholate; and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000)), and mRNAs were dissolved at a charge ratio of N/P = 3 in sodium citrate buffer (50 mM; pH 4) solution³⁷ . LNPs were formulated using NanoAssemblr® Ignite^TM (Precision Nanosystems, BC, Canada) by mixing the aqueous and organic solutions at a ratio of 3:1 and a total flow rate of 10 mL/min. The solution of LNPs was concentrated by ultrafiltration using Amicon Ultra centrifugal Filter (UFC9030, Merck Millipore, MA, USA) following the manufacturer’s instructions.