Poster X19 - Impact of Protein Corona Formation on Hydrophobic-Core Polymer Nanoparticles: A Study of Altered Immunogenicity.

Introduction: Nanoparticles have become indispensable in the realm of drug delivery and immune modulation due to their versatile applications. However, it's crucial to acknowledge that nanoparticles possess intrinsic properties capable of either enhancing or suppressing immune responses, contingent upon factors like their size, shape, charge, and hydrophobicity. Importantly, when nanoparticles traverse the bloodstream, they interact with serum proteins, leading to the formation of a protein corona. This corona, in turn, undergoes dynamic changes, thereby altering the biological and chemical identity of the nanoparticles.

While the influence of the protein corona on nanoparticle interactions with immune cells is acknowledged, a comprehensive understanding of its specific effects on inflammasome activation remains elusive. This study endeavors to address this knowledge gap by synthesizing and meticulously characterizing two distinct polymer nanoparticles with varying degrees of core hydrophobicity. Our initial focus lies in delineating the impact of differing core hydrophobicity levels on NLRP3 inflammasome activation, a pivotal component of immune response regulation.

Subsequently, we deliberately induce protein corona formation on both nanoparticle variants, aiming to pinpoint the pivotal proteins constituting the hard corona layer. Moreover, we embark on a quest to unravel the precise role played by these identified proteins in shaping the immunogenicity of the nanoparticles. To augment our understanding, we delve deeper into the mechanistic intricacies by scrutinizing the changes in the stress signals elicited by the nanoparticles, such as lysosomal rupture due to the protein corona formation.

Materials and

Methods: The supramolecular polymers were synthesized using the "living" RAFT polymerization technique where the hydrophilic and hydrophobic counterparts were carefully modified, rendering the appropriate amphiphilicity. These polymers were self-assembled into micellar nanoparticles employing dialysis methodology to produce uniform monomodal size SNPs. After the SNP formation, they were further incubated with rat serum at a concentration of 4:1 (v/v) at room temperature under constant agitation to form the protein corona. The size and the zeta potential of the nanoparticles and the corona complexes was determined by dynamic light scattering using a Malvern Nanozetasizer ZS90. The protein corona composition was characterized using nano-flow EASY-nLC 1000 UHPLC coupled to an Orbitrap Fusion mass spectrometer with a PepMap RSLCnano column.

For IL-1B ELISA and ASC Specks confocal microscopy, engineered immortalized bone-marrow-derived macrophages (iBMDMs) were primed with 100 ng/mL of ultrapure LPS for 4 h followed by SNPs/corona complexes addition at a final 500 μg/mL polymer concentration. Cell viability was measured by MTT assay at the same working concentrations of SNPs.

For lysosomal rupture confocal microscopy, DiR-dye encapsulated SNPs/corona were added to the cells for 4 h, which were then stained with NucBlue (2 drops/mL) and Lysotracker Red DnD-99 (0.1 μM) for 30 min before the imaging was performed using a Nikon Crest V2 Spinning Disk Confocal Microscope. For studying nanoparticle release, iBMDMs were treated with SNPs/corona for 4 hours after which the SNP-containing supernatant was replaced with fresh media and internalization was measured by flow cytometry at 0, 4, 8, and 24 hrs.

Results, Conclusions, and Discussions: 1. The two polymer nanoparticles were synthesized by varying the length of the repeating units from y=100 to 400, and the alkyl side chain lengths from x=1 to 5 (C4 to C12). They were named as BM100 and DM400 respectively The size of both the nanoparticles were in the range of 100-150 nm with a narrow PDI ( < 0.3). The protein corona for both the nanoparticles resulted in a slight increase in the size which is mostly because of the protein adsorption phenomenon.

2. As a next step, the hard corona composition was determined by LC-MS. It was noticed that the top 20 proteins formed the major part of the corona contributing to around 70% of the entire corona composition. Also, the abundance of the proteins in the hard corona was not found to be proportional to the abundance of proteins in the serum, indicating differential rate of protein adsorption to the SNPs. Moreover, the corona composition for the two SNPs were found to vary only slightly, suggesting the surface charge as the primary factor determining the protein adsorption.

3. Upon performing ELISA, it was evident that DM400, having a higher core hydrophobicity induced more IL-1B release on iBMDMs suggesting a higher NLRP3 inflammasome activation as compared to BM100. Interestingly, the IL-1B secretion was found to reduce significantly after corona formation. To gain further mechanistic insights, we used imaging techniques to visualize and quantify ASC speck formation in iBMDMs expressing CFP-tagged ASC protein upon exposure to signal-1 and -2, necessary for NLRP3 inflammasome activation The minimal specks formation upon corona formation further corroborates our previous findings by exhibiting a significant decrease in NLRP3 inflammasome activation due to the protein–nanoparticle complex.

4. Subsequently, when examining lysosomal rupture, it became evident that the formation of a protein corona substantially diminishes the nascent SNPs' capacity to efficiently breach the lysosomal membrane. Furthermore, a thorough investigation into cellular uptake elucidated the intracellular segregation of the protein corona and the SNPs, leading to the premature exocytosis of the isolated SNPs. This process prompts the SNPs to evade the lysosomal pathway, resulting in minimal lysosomal rupture.

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