NLRP3 inflammasome is affected by Protein Corona formation on Lipid Nanoparticles

Introduction:: Nanoparticles, when in the blood stream absorb proteins in the surroundings to form a complex termed as protein corona. This protein adsorption process is a complex phenomenon where the size, shape, hydrophobicity, and surface charge of the nanoparticles all combinedly determine the toxicity, biodistribution, cellular uptake, and the interfacial properties of nanoparticles, thus giving them a distinct biological identity (Rampado, R et al. Front. Bioeng. Biotechnol. 2020). While it has been elucidated that the new identity in the form of protein corona can affect the nanoparticle-cell interaction in several ways (Oh, J. Y. Nat. Commun. 2018), but the effect of protein corona on innate immune system is yet to be explored. In this study, we described the potential impacts of the corona formation on surface interactions as well as intracellular signaling pathways by explaining the shift in immunogenicity involved in lipid nanoparticles (LNPs).


Materials and Methods:: Two systems of LNPs were synthesized via ethanol dilution method, by varying the molar ratios of each constituent lipids. The protein corona was formed by incubating the LNPs with normal rat serum and the composition was quantified by LC-MS. The LNPs and their corona complexes were then screened for NLRP3 activation in LPS-primed iBMDMs by measuring IL-1β cytokine release via ELISA and NLRP3 oligomerization via ASC speck live cell imaging. To discern the mechanisms for inflammasome activation, we measured the mitochondrial ROS, calcium influx and lysosomal rupture. Finally, we evaluated the effect of lipid composition and the protein corona in endocytic pathways by quantifying the internalization in iBMDMs using different pharmacological inhibitors.


Results, Conclusions, and Discussions:: Results and Discussion: We reported LNP2, having 50 molar percent ionizable lipid to significantly activate higher NLRP3 inflammasome on LPS primed iBMDMs as compared to LNP1, which has no ionizable lipid (Fig-1A). Interestingly, the corona formation caused a notable decrease in the inflammasome activation. This was verified by IL-1β release from ELISA (Fig-1A) and ASC specks formation from confocal microscopy (Fig-1B). The reduced lysosomal rupture was described as the major mechanism behind the suppressed NLRP3 activation (Fig-1C,D). The corona composition was identified by LCMS where the top 20 proteins formed around 70 percent of the entire protein corona for both the LNPs. As a next step, the cellular uptake studies helped us to realize the involvement of multiple endocytic pathways of LNPs in macrophages. Also, the lipid constituents and the corona formation were found to be major factors in deciding the potential internalization pathways.

Conclusion: In conclusion, we were able to demonstrate the inverse correlation of protein corona with the NLRP3 inflammasome complex. The reduced lysosomal disruption by the corona as compared to pristine LNPs was reported as the primary cause of the corona based NLRP3 inflammasome suppression. The findings described the mechanism of internalization followed by the nanoparticles in the absence and presence of corona. Overall, the results suggest the lipid formulation and corona composition to play a vital role in regulating internalization mechanisms as well as intracellular signaling pathways.


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