Time evolution of PEG-shedding and serum protein coronation determines the cell uptake kinetics and delivery of lipid nanoparticle formulated mRNA

Abstract

Development of efficient lipid nanoparticle (LNP) vectors remains a major challenge towards broad clinical translation of RNA therapeutics. New lipids will be required, but also better understanding LNP interactions with the biological environment. Herein, we model protein corona formation on PEG-ylated DLin-MC3-DMA LNPs and identify time-dependent maturation steps that critically unlock their cellular uptake and mRNA delivery. Uptake requires active serum proteins and precedes after a significant (∼2 hours) lag-time, which we show can be eliminated by pre-incubating LNPs for 3-4 hours in serum-containing media. This indicates an important role of protein corona maturation for the pharmacokinetic effects of these LNPs. We show, using single-nanoparticle imaging, NMR diffusometry, SANS, and proteomics, that the LNPs, upon serum exposure, undergo rapid PEG-shedding (∼30 minutes), followed by a slower rearrangement of the adsorbed protein layer. The PEG-shedding coincides in time with high surface abundance of Apolipoprotein A-II, whereas the LNPs preferentially bind Apolipoprotein E when their maximum uptake-competent state is reached. Finally, we show that pre-incubation of the LNPs enables rapid uptake and allows pulse-chase video-microscopy colocalization experiments with sufficiently short pulse durations to gain improved mechanistic understanding of how intracellular trafficking events determine delivery efficacy, emphasizing early endosomes as important delivery-mediating compartments.

Interaction Kinetics of Individual mRNA-Containing Lipid Nanoparticles with an Endosomal Membrane Mimic: Dependence on pH, Protein Corona Formation, and Lipoprotein Depletion

“…Instead, there was a significant shift in the onset of LNP binding to lower pH, and a more than 50% reduction in the rate of LNP binding at all pH conditions assayed. These results suggest that the FBS-induced protein corona formation on the surface of the LNPs, addressed in detail elsewhere, 14 reduces the MC3mediated pH-induced electrostatic attraction to the negatively charged SLB. However, there was no reduction in the rate of LNP binding even at the lowest pH (as observed for pristine LNPs, Figure 2), which suggests that protein corona formation at least in part prevents appreciable LNP collapse and lipid accumulation in the contact zone and/or exchange with the endosomal membrane mimic.…”

“…These observations are curious in the context of the reported significance of nanoparticle clustering for modulating cellular uptake and endosomal escape. , Further, even though lipoprotein depletion was not observed to influence the tendency of LNPs to undergo collapse and/or aggregation, lipoproteins seem to hamper the electrostatic attraction of LNPs to the endosomal membrane, which is, in turn, expected to drive the critical endosomal escape event. Since protein corona formation precedes and has even been shown to be required for efficient cellular uptake, − these findings call for complementary live cell studies to gain deeper insight into the influence of protein corona formation in general, and lipoproteins in particular, not only on uptake efficiency, but also with respect to maintained coronation in the endosomal lumen and the impact of coronal proteins on endosomal escape.…”

“…However, investigations of this type are complicated by the fact that prior to cellular uptake, LNPs become spontaneously coated with proteins from biological fluids in a complex and far from understood manner. , This so-called protein corona on the surface of LNPs is in fact often a requirement for efficient cellular uptake, and lipoproteins, in particular apolipoprotein E (ApoE), have been identified as particularly crucial, likely by mediating binding to LDL receptors residing in plasma membranes. , Significantly less is known, however, regarding the possible impact of the protein corona on endosomal processing of LNPs, and how endosomal acidification and enzymatic degradation of the protein corona may influence the critical escape events. We have recently shown that LNP-mediated mRNA uptake and subsequent protein translation depend on the duration of LNP-serum preincubation, and hence protein corona maturation, prior to cellular exposure, which suggests that the protein corona formation deserves special attention.…”

“… 12 , 13 Significantly less is known, however, regarding the possible impact of the protein corona on endosomal processing of LNPs, and how endosomal acidification and enzymatic degradation of the protein corona may influence the critical escape events. We have recently shown that LNP-mediated mRNA uptake and subsequent protein translation depend on the duration of LNP-serum preincubation, and hence protein corona maturation, prior to cellular exposure, 14 which suggests that the protein corona formation deserves special attention.…”

Physiology, pathology and the biomolecular corona: the confounding factors in nanomedicine design

“…This results in progressive but surprisingly fast absorption of biomolecules to the nanomedicine surfaces once they are in contact with complex biological fluids. 3 Corona formation is also a very dynamic process that evolves over time 17,23,24 and, it is also related to the aggregation state 25 and disintegration 26 of the nanomedicines. A corona that forms quickly may consist of more abundant (Fig.…”