Time-Resolved Inspection of Ionizable Lipid-Facilitated Lipid Nanoparticle Disintegration and Cargo Release at an Early Endosomal Membrane Mimic.

Advances in lipid nanoparticle (LNP) design have contributed notably to the emergence of the current clinically approved mRNA-based vaccines and are of high relevance for delivering mRNA to combat diseases where therapeutic alternatives are sparse. LNP-assisted mRNA delivery utilizes ionizable lipid-mediated cargo translocation across the endosomal membrane driven by the acidification of the endosomal environment. However, this process occurs at a low efficiency, a few percent at the best. Utilizing surface-sensitive fluorescence microscopy with a single LNP and mRNA resolution, we have investigated pH-controlled interactions between individual LNPs and a planar anionic supported lipid bilayer (SLB) formed on nanoporous silica, mimicking the electrostatic conditions of the early endosomal membrane. For LNPs with an average diameter of 140 nm, fusion with the anionic SLB preferentially occurred when the pH was reduced from 6.6 to 6.0. Furthermore, there was a delay in the onset of LNP fusion after the pH drop, and upon fusion, a significant fraction (>70%) of mRNA was released into the acidic solution representing the endosomal lumen, while a fraction of mRNA remained bound to the SLB even after reversing the pH to neutral cytosolic conditions. Finally, a comparison of the fusion efficiency of two LNP formulations with different surface concentrations of gel-forming lipids correlated with differences in the protein translation efficiency previously observed in human primary cell transfection studies. Together, these findings emphasize the relevance of biophysical investigations of ionizable lipid-containing LNP-assisted mRNA delivery mechanisms while potentially also offering means to optimize the design of LNPs with enhanced endosomal escape capabilities.