Figure 1.

(A) A formulation of lipid nanoparticles is synthesized by mixing the aqueous phase containing the mRNA and the ethanol phase containing the lipophilic compounds, using a microfluidic device. The ionizable lipid complexes with the negatively charged mRNA at low pH and can both facilitate endocytosis and endosomal escape. Phospholipid provides structural integrity to the bilayers and can assist with endosomal escape of the mRNA to the cytosol. Cholesterol helps stabilize the LNPs and promotes membrane fusion. The lipid-anchored polyethylene glycol prevents LNP aggregation and reduces nonspecific interactions. (B) Size analysis of formulation B-11. Diameter distribution of the LNPs comprising the vaccine solution formulated with OVA mRNA, as determined using dynamic light scattering (DLS). (C) Cryogenic transmission electron microscopy image of the LNP solution suggests that the LNPs have a spherical shape and consist of a multilamellar structure. (D) In the first phase of the optimization (Library A; empty columns), different components were investigated, each at a constant molar composition. Percentages of OVA specific CD 8 T cells 7 days after the injection of 10 μg of total mRNA per mouse are plotted for each formulation, including the original formulation (hatched column). The data are presented as mean + SD, n = 5. The three components, C14-PEG2000 (A-12), cKK-E12 (A-2), and SLS (A-18) in Library A were identified for further investigation in Library B (black columns). Combinations of C14-PEG2000, cKK-E12, and SLS in different concentrations were tested to afford the optimized formulation B-11 (patterned column).