Fusion-dependent formation of lipid nanoparticles containing macromolecular payloads

The success of Onpattro™ (patisiran) clearly demonstrates the utility of lipid nanoparticle (LNP) systems

for enabling gene therapies. These systems are composed of ionizable cationic lipids, phospholipid,

cholesterol, and polyethylene glycol (PEG)-lipids, and are produced through rapid-mixing of an ethanolic-

lipid solution with an acidic aqueous solution followed by dialysis into neutralizing buffer. A detailed

understanding of the mechanism of LNP formation is crucial to improving LNP design. Here we use cryogenic

transmission electron microscopy and fluorescence techniques to further demonstrate that LNP are

formed through the fusion of precursor, pH-sensitive liposomes into large electron-dense core structures

as the pH is neutralized. Next, we show that the fusion process is limited by the accumulation of PEGlipid

on the emerging particle. Finally, we show that the fusion-dependent mechanism of formation

also applies to LNP containing macromolecular payloads including mRNA, DNA vectors, and gold

nanoparticles.