Engineering lipid nanoparticles (LNP) with hydroxycholesterol substitution: motivation, design, and synthesis. (A) Schematic of LNP components, formulation, and expected structure. An ethanol phase containing lipid-anchored PEG (polyethylene glycol), cholesterol, X-hydroxycholesterol, DOPE (1,2-Dioleoyl-snglyeero-3-phosphoethanolamine), and C14–4 ionizable lipid and an aqueous phase containing mRNA are mixed in a microfluidic device to produce LNPs. (B) Diagram of LNP delivery into a T cell and the endosomal trafficking mechanisms involving the Rab family of proteins. Rab5, Rab7, and Rab11 associate with the early, late, and recycling endosomes, respectively. (C) Design of an LNP library incorporating the substitution of various hydroxycholesterols for unmodified cholesterol.