Lipid Nanoparticle (LNP) Formulations

Ionizable lipid [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino) butanoate (Dlin-MC3-DMA; #34365) and 1,2-Dimyristoyl-rac-glycero-3-PE-methoxy-Polyethyleneglycol-2000 (DMPE-2kPEG; #34649) were purchased from Cayman Chemical. Cholesterol was purchased from Sigma Aldrich (St. Louis, MO). 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). LNP was produced under RNase-free conditions. First, the glass bottle and stir bar were washed with chloroform and dried with nitrogen gas. 100 µg of mRNA was added to 600 µL of 50 mM citrate buffer (Sigma-Aldrich, St. Louis, MO) in the bottle. The mRNA solution was then mixed with the 200 µL of ethanolic lipid solution and stirred simultaneously. The lipid formulation was composed of DLin-MC3-DMA, DSPC, cholesterol, and DMPE-2kPEG at a ratio of 50: 10: 38.5: 1.5.^31,32 After evaporation for 4 hr in a vacuum chamber under gentle stirring, the solution was diluted in 10% sucrose buffer (Fisher Scientific, Waltham, MA) and condensed with the Amicon^® centrifugal filter with a 100 kDa molecular-weight cut-off (Millipore Sigma, Burlington, MA). (Merck, Darmstadt, Germany) at 600 × g at 4°C, the mRNA-LNPs were stored at −80°C with a final concentration from 0.25 to 1.00 µg/µL and kept from repeated thawing and freezing.