LNP Preparation and Characterization

LNPs were synthesized by mixing one volume of lipid mixture of ionizable lipid, DSPC, cholesterol, and DMG-PEG (40:10.5:47.5:2 mol ratio) in ethanol and three volumes of mRNA (1:23 (w/w) mRNA to lipid) in acetate buffer. Lipids and mRNA were injected in to a microfluidic mixing device Nanoassemblr (Precision Nanosystems, Vancouver, BC, Canada) at a combined flow rate of 12 mL/min. The resultant mixture was dialyzed against phosphate buffered saline (PBS; pH 7.4) for 16 h to remove ethanol.

Particles in PBS were analyzed for size and uniformity by dynamic light scattering (DLS, Malvern Instruments Ltd., Worcestershire, U.K.). Intensity-size data can be found in Figure S3. RNA encapsulation in LNPs was calculated according to Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher, Waltham, MA, USA).