Nanoparticle formulation and characterization

LNPs were formulated via microfluidic mixing of one-part ethanol phase (containing the lipids) and three parts aqueous phase (containing the mRNA). The ethanol phase contained the MC3, DSPC, DMG-PEG2k, and cholesterol at a molar ratio of 50:10:1.5:38.5, respectively. For synthesizing novel particles, the percentage of DMG-PEG2k was changed from 1.5% to 0.5%, 3% or 5% while cholesterol was changed from 38.5% to 39.5%, 37.0% or 35%, respectively. For the second set of particles, DMG-PEG2k varied from 0.5% to 5% while DSPC was changed from 10% to 11%, 8.5% or 6.5%, respectively (Table 1). The aqueous phase consisted of the mRNA in 50 mM Citrate buffer pH 4. Following microfluidic mixing, the nanoparticle solution was subjected to buffer exchange with PBS (pH 7.2) and concentrated using Amicon Ultra-4100k MWCO (EMD Millipore) centrifugal filters. The nanoparticles were stored at 4°C until diluted for injections. The LNPs were characterized for hydrodynamic radius (nm) and polydispersity index (PDI) using dynamic light scattering (Zetasizer Nano ZSP, Malvern Instruments), while mRNA encapsulation efficiency was measured using a modified Quant-iT RiboGreen RNA reagent (Life Technologies).

LNP-Lipid nanoparticle, DMG-PEG-1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, DSPC-1,2-distearoyl-sn-glycero-3-phosphocholine, MC3-Dlin-MC3-DMA.