Liposome for mRNA

LNP formulations were prepared as described previously (Richner et al., 2017). Briefly, lipids were dissolved in ethanol at molar ratios of 50:10:38.5:1.5 (ionizable lipid: DSPC: cholesterol: PEG-lipid). The lipid mixture was combined with mRNA at a ratio of 3:1 (aqueous:ethanol) using a microfluidic mixer (Precision Nanosystems). Formulations were dialyzed against PBS (pH 7.4), concentrated using Amicon Ultra Centrifugal Filters (EMD Millipore), passed through a 0.22-µm filter and stored at 4°C until use. All formulations were tested for particle size, RNA encapsulation, and endotoxin and were found to be between 80 to 100 nm in size, with greater than 90% encapsulation and <1 EU/ml of endotoxin.

Briefly, lipids were dissolved in ethanol at molar ratios of 50:10:38.5:1.5 (ionizable lipid: DSPC: cholesterol: PEG-lipid). The lipid mixture was combined with a 50 mm citrate buffer (pH 4.0) containing mRNA at a ratio of 3:1 (aqueous:ethanol) using a microfluidic mixer (Precision Nanosystems, Vancouver, BC). Formulations were dialyzed against PBS (pH 7.4) in dialysis cassettes for at least 18 hr. Formulations were concentrated using Amicon Ultra Centrifugal Filters (EMD Millipore, Billerica, MA), passed through a 0.22-μm filter and stored at 4°C until use. All formulations were tested for particle size, RNA encapsulation, and endotoxin and were found to be between 80 to 100 nm in size, with greater than 90% encapsulation and < 1 EU/ml of endotoxin.