(512e) Protein Corona Formation on Lipid Nanoparticles Affects the NLRP3 Inflammasome

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 corona composition which eventually controls 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). It has been widely studied that the new identity in the form of protein corona can affect the nanoparticle-cell interaction by exhibiting novel interactions with cell receptors (Oh, J. Y. Nat. Commun. 2018). It is also elucidated that the corona proteins can interact with innate immune system to control cellular stress and damage signals via regulation of inflammasomes (Pavlin, M. et al. Int. J. Mol. Sci. 2022). 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.

Results and Discussion: Two different systems of lipid nanoparticles (LNPs) were synthesized via ethanol dilution method by varying the molar concentration of five key lipid constituents. Upon performing experiments to quantify the activation of NLRP3 inflammasome in LPS primed iBMDMs, LNP2, having 50 molar percent ionizable lipid was found to show a significantly higher activation than LNP1 which had no ionizable lipid (Fig-1A). Both the LNPs were then incubated with rat serum to form the protein corona. The proteins adsorbed on both the nanoparticles were identified and quantified (Fig-1E) by using liquid chromatography tandem mass spectrometry (LC-MS/MS). It is interesting to note that the top 20 most abundant proteins were the same for LNP1 and LNP2, but the order of abundance varied significantly for certain proteins. It implies to the idea that certain proteins are found in abundance in all nanomaterials, but the abundant proteins are not always the same. To evaluate the effect of corona proteins on NLRP3 inflammasome complex, we quantified the IL-1β concentration by ELISA and observed a significant reduction of NLRP3 activation upon the formation of corona layer (Fig-1A). The results were further validated by measuring the ASC specks formation (Fig-1B) which can be used as a simple upstream readout for inflammasome activation (Nagar, A et al. Front. Immunol. 2021).

As a next step, we evaluated the surface interactions of LNPs by quantifying the change in cellular uptake in macrophages because of the corona formation. In the absence of any corona, LNP2 showed a higher internalization than LNP1. This is possibly because of the high content of the ionizable lipid which can foster electrostatic attraction and promote nonspecific binding with the negatively charged cell membrane. But after corona was formed, a significant reduction in the internalization was observed for both the LNPs. However, as less internalization has not always been found directly corresponding to inflammasome activation, we wanted to explore specific mechanisms besides the dwindling cellular uptake of protein corona complexes which result in the NLRP3 inflammasome suppression. Three signaling pathways (mitochondrial reactive oxygen species (ROS), Calcium and Potassium ion influx, and lysosomal damage) were studied for the possible route of protein corona mediated NLRP3 inflammasome suppression. Corona formation did not significantly result in a change of the ROS production or the ion influx for the LNPs. Next, the lysosomal rupture was investigated which is a key driving force for NLRP3 inflammasome activation via cathepsin-B maturation (Nandi, D. et al. ACS Appl. Mater. Interfaces 2021). A sequential trend of internalization as well as the lysosomal rupture was noticed on comparing the LNPs in the presence and absence of corona proteins. The LNP ingenuity to efficiently rupture the lysosome was found to get reduced significantly upon corona formation in iBMDMs (Fig-1C,D). These studies collectively indicate that the corona adsorption on nanoparticles is negatively correlated with NLRP3 inflammasome. And the shielding effect of adsorbed proteins resulting in inefficient cellular uptake and minimized lysosomal rupture are the primary mechanism for the observed inhibiting effect.

Previous studies also suggested that the protein corona formation can hinder particle recognition by macrophages (Caracciolo, G et al. Langmuir, 2015) . Thus, a mechanistic approach to study the factors that determine the endocytic pathway including LNP formulation as well as the multilayered corona composition is a key step to answer questions relating to circulation time, biodistribution, toxicity, and most importantly the particle interaction with the immune system. To study the functional changes due to the corona formation and to characterize the uptake mechanisms of LNPs in the presence and absence of serum proteins, different inhibitors of endocytosis were used each corresponding to a specific pathway. Overall, the results suggest the involvement of multiple pathways of internalization for LNPs in macrophages. Also, the lipid constituents play a vital role in determining the potential endocytic mechanism. Moreover, the corona formation altered the LNP recognition by cell receptors, thus effecting the internalizing pathway.

Conclusion: In conclusion, we were able to demonstrate the inverse correlation of protein corona with the NLRP3 inflammasome complex. A systematic approach to the study helped us to uncover the underlying mechanism for the inhibiting effect of the corona layer. 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. Not only the protein coating decides the fate of LNPs once inside the cell. But our observations suggest the involvement of protein corona in the very first interaction between nanoparticles and macrophages. 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.