Fig. 4: Studies on the formation and intracellular delivery mechanisms of liposomal LNP mRNA systems.
From: Liposomal lipid nanoparticles for extrahepatic delivery of mRNA
Liposomal LNP with RB/I = 4 (nor-MC3/ ESM: cholesterol/ PEG-DMG; 20/40:40/1.5 mol/mol) or RB/I = 2.3 (nor-MC3/ESM/cholesterol/PEG-lipid; 30/35/35/1.5 mol/mol) were formulated containing Firefly Luciferase mRNA (N/P = 6), n = 3. A Cryo-TEM micrographs generated following the mixing stage at pH 4 (after dialysis against 25 mM NaOAc pH 4 to remove ethanol) and after dialysis against PBS to raise the pH to 7.4, micrograph has been reproduced twice. The white arrows in the pH 4 preparations indicate the presence of electron dense structures interpreted as complexes of mRNA with ionizable lipid. The white arrows in the pH 7.4 micrograph indicate the electron dense oil droplet structures formed by the ionizable lipid in the neutral form. B Cryo-TEM micrographs of LNP mRNA systems (originally in PBS pH 7.4 buffer) that were subsequently dialyzed against lower pH buffers to mimic endosomal pH environments (pH 6.4: early endosomes, pH 5.6: late endosomes, pH 5.0: lysosomes), micrograph has been reproduced twice. The white arrows in the pH 5.6 and pH 5 micrographs show that the electron dense regions decrease in size quickly as the pH is lowered, consistent with a partitioning of the charged form into the lipid bilayer.
