Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes.

Significance: Neurodegenerative diseases are increasing among the world's population, but there are no cures. These disorders all involve proteins that assemble into amyloid fibers which results in brain cell death. Evidence suggests that association of these proteins with lipid membranes is crucial for both functional and pathological roles. In Parkinson's disease, the involved protein, α-synuclein, is thought to function in trafficking of lipid vesicles in the brain. In search of mechanistic origins, increasing focus is put on identifying neurotoxic reactions induced by membrane interactions. To contribute new clues to this question, we here employed a new surface-sensitive scattering microscopy technique. With this approach, we discovered that α-synuclein perturbs vesicles in a stepwise and lipid-dependent fashion already at very low protein coverage.