Poster CC12 - Induction of Supramolecular Polymer Nanoparticles as NLRP3 agonist

Introduction: Understanding how the nanoparticles interact with innate immune cells is pivotal towards the design and efficacy of future nano-immunotherapies. In this domain, the interaction of these carriers with the immune system followed by their ability to activate a key component of innate immunity, i.e. the NLRP3 inflammasome is an emerging field. While the effect of surface properties of nanoparticles has been extensively investigated in the context of nanoparticle-immune cell interactions, the influence of core composition remains largely unexplored, particularly regarding its impact on inflammasome activation. To shed light on these interactions, we developed a library of supramolecular polymer nanoparticles (SNPs) with different core compositions, varying their hydrophobic quotient by virtue of the side chain length and the repeating units in the polymer construct. A direct correlation was established between increasing the side chain length of the polymer construct, thereby increasing the core hydrophobicity of SNPs and enhanced NLRP3 complex formation, as insinuated by the ASC speck imaging analysis and the elevated 1L-1β expression. Furthermore, the results demonstrated that the inflammasome signaling cascades and kinetics varied based on the SNP's hydrophobic side chain length and repeating units. In vivo evaluation of SNPs in C57BL/6 mice confirmed elevated expressions of proinflammatory cytokines, specifically with the SNP having the longest C12-alkyl side chain. In summary, this study established SNP core composition as a novel nanoparticle-associated molecular pattern (NAMP) responsible for NLRP3 inflammasome activation, shedding light on intricate cellular pathways for informed nanoparticle design in immunotherapy and vaccine applications.

Materials and

Methods: The supramolecular polymers were synthesized using living RAFT polymerization and characterized in depth using NMR and Gel permeation chromatography (GPC) to deduce the number-average (Mn) and weight-average molecular weights (Mw) and their polydispersity index (PDI = Mw/Mn). Further, dialysis methodology was employed for nanoparticle synthesis followed by physicochemical characterization using DLS, FE-SEM, HR-TEM and Zeta. The immortalized bone marrow-derived macrophages (iBMDMs) were employed for all in vitro assays investigating the inflammasome activation mechanism. The expression of pro-inflammatory cytokine IL-1β was determined using quantitative ELISA upon incubation of lipopolysaccharide (LPS) primed-cells with SNPs (LPS acts as signal 1 followed by signal 2 triggered by SNP to activate NLRP3). The immunofluorescence staining protocol was employed to visualize ASC specks to gain insight into the NLRP3 complex formation. The effect of SNPs on lysosomal rupture, Ca2+ influx and mitochondrial ROS production (Signal 2) were assessed using confocal microscopy and flow cytometry. To investigate the capability of polymer nanoparticles to activate inflammasome in vivo, the C57BL/6 mice were employed as the mouse model. These mice were intravenously injected with the respective SNP series at a 40 mg/kg dose via tail-vein.

Results, Conclusions, and Discussions: Results and

Discussion: We have designed a unique polymer platform to elucidate the effect of core composition of NPs on NLRP3 activation by selectively tweaking multiple core parameters in a single polymer without varying the surface properties. The core composition of NPs was modulated by varying the alkyl (Cn) side chain length and repeating units of the hydrophobic monomer (Fig 1a). As can be seen in Fig 1b, the IL-1β concentration in iBMDMs significantly increased with an increase in core hydrophobicity quotient, and this was reiterated by the changes in ASC-speck formation (important indicator of NLRP3 inflammasome assembly, Fig 1c). We continued to investigate the involvement of downstream signaling pathways associated with NLRP3 activation, including the lysosomal rupture-cathepsin B, calcium influx, and mitochondrial damage. Notably, we discerned variations in inflammasome signaling cascades attributed to the length and repeating units of the alkyl chains. The SNPs containing the octyl side chain demonstrated NLRP3 inflammasome activation by the lysosomal destabilization-calcium influx and mitochondrial disruption-calcium release pathways (Fig 1d, e). The nanoparticle with the C12 alkyl chain "DM400" induced NLRP3 activation through the mitochondrial damage pathway (Fig 1e). But these SPNs did not result in pyroptosis. A preliminary in vivo study carried out in male C57BL/6 mice revealed escalated levels of 2 important pro-inflammatory cytokines, IL-1β (Fig 1f) and TNF-α in the case of the SNP DM400, the polymer nanoparticle with the highest core hydrophobicity quotient.

Conclusion: We designed a multiparametric polymer platform and successfully identified core composition of the nanoparticles as a novel NAMP modulating NLRP3 inflammasome activation. The elevated IL-1β expression and visualization of ASC specks established a direct link between the alkyl chain length core parameter and NLRP3 complex formation. The comprehensive in vitro mechanistic study revealed that SNPs induced inflammasome activation variably through different intracellular pathways, namely, lysosomal rupture, calcium influx and mitochondrial damage; without inducing pyroptosis. The SNP with the longest “C12” alkyl chain and the highest repeating units (DM400) resulted in elevated IL-1β and TNF-α expressions in vivo. This is a pivotal study in informing design strategies towards nanoparticle development as vaccine adjuvants or for immunotherapeutic applications.

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