Fig. 2. Proposed mechanism of endosomal escape of delivered mRNA.

Endosomal escape of delivered mRNA is largely dependable on interactions between ionizable lipids and naturally occurring anionic phospholipids in the endosomal membrane.⁴³ Prior to membrane fusion, ionizable lipids in lipid nanoparticles (LNPs) and anionic lipids in the endosomal membrane adopt a cylindrical conformation which is compatible with molecular packing in a bilayer phase. The acidic environment in endosomes facilitates protonation of ionizable lipids into cationic lipids. Cationic and anionic lipids generate ion pairs whose combined cross-sectional headgroup area is smaller than the total of individual headgroup areas before membrane fusion. Consequently, the ion pair adopts a conical shape which promotes the formation of inverted, non-bilayer phases, such as the hexagonal shape illustrated above. Thus, the formation of ion pairs between lipids promotes membrane fusion and disruption, allowing mRNA to escape from endosomes. This figure is created with BioRender.com