O19 - NLRP3 Inflammasome Activation potentiated by varying Ionizable groups in Star Polymer Platform

Introduction: : Cancer nanovaccines (N.V.s) represent a groundbreaking class of immunotherapeutic agents that capitalize on the distinctive attributes of nanoparticles to evoke a potent immune response against malignant cells. Understanding the nanomaterial-immune system interactions is pivotal for establishing safe delivery vehicles and vaccine adjuvants. Physicochemical properties of nanomaterials have been investigated as triggers for inflammasome activation. Among these parameters, hydrophobicity and surface charge have been studied extensively but their combined effect is not studied. Here, we engineered star-shaped random copolymers incorporating both hydrophobic and ionizable groups on a single polymer backbone, where the hydrophobic moiety was fixed while varying the ionizable monomers. To do so, we employed a 4-arm polyethylene glycol as the chain transfer agent for the RAFT polymerization of the hydrophobic (D.M.) and ionizable monomers (DMA, DiPA, TBM, MEM). Even though the hydrophobic group was constant across all the four-star N.P.s, there was elevated IL-1β expression in two of the star polymer N.P.s (DM-DMA and DM-TBM). This was reiterated by the higher ASC speck formation that directly correlates to the NLRP3 complex formation. A thorough mechanistic evaluation was also carried out to determine the downstream signaling cascades acting as signal 2 towards NLRP3 activation (detailed schematic in Figure 1a). This study established that the presence of hydrophobicity and ionizable groups in a single nanocarrier platform could result in a synergistic or antagonistic effect on NLRP3 activation. Thus, by simply tweaking these parameters, we can design nanocarriers with the requisite immunostimulation capability to enhance their efficacy as N.P.s without causing immunotoxicity.

Materials and

Methods: : The polymers were synthesized using living RAFT polymerization and characterized in depth using NMR. Further, dialysis methodology was employed for nanoparticle synthesis followed by physicochemical characterization using DLS, 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 NPs. 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.

Results, Conclusions, and Discussions:: Results and

Discussion: We have designed a multiparametric star polymer platform to investigate the effect of two crucial nanoparticle-associated molecular patterns (NAMPs), namely hydrophobicity and ionizable groups in a single polymer backbone (schematic in Fig 1b). The IL-1β concentration in iBMDMs were significantly high in the case of polymers DM-DMA and DM-TBM, that can be attributed to their higher surface zeta potential (Fig 1c). In coherence with previous reports, there was a direct correlation observed between surface charge and inflammasome activation. This was reiterated by the ASC specks imaging analysis (Fig 1d, e). We further investigated the downstream signaling pathways acting as Signal 2 in the NLRP3 inflammasome activation, including the lysosomal rupture-cathepsin B, calcium influx, and mitochondrial ROS production (Fig 1f). Cationic nanoparticles are thought to be more effective at causing lysosomal rupture due to the stronger interaction between the anionic membrane of the lysosome and the positively charged surface of the nanoparticle upon internalization, leading to its destabilization. As expected, DM-DMA and DM-TBM showed augmented lysosomal rupture as depicted quantitatively from the ratio of the lysotracker signal normalized by the DiR dye signal from the N.P.s (TRITC/Cy5). Among the auxiliary Signal 2 pathways, DM-DMA demonstrated ameliorated calcium influx, whereas DM-TBM resulted in elevated mitochondrial damage.

Conclusion: We engineered a unique star polymer platform as a tool to evaluate the combined effect of hydrophobicity and cationic surface charge of N.P.s on the NLRP3 inflammasome activation. The nanoparticles DM-DMA and DM-TBM with higher zeta potential resulted in elevated IL-1β expressions in iBMDMs. Further corroborated by augmented ASC speck formation. The mechanistic investigation into the downstream signaling pathways revealed that both DM-DMA and DM-TBM caused considerably high lysosomal rupture but variability in auxiliary signaling cascade, wherein DM-DMA elevated calcium influx and DM-TBM led to higher mitochondrial ROS production, respectively. This comprehensive study established that the consolidated effect of hydrophobic and ionizable groups can be synergistic or antagonistic on inflammasome activation. The current investigation provides crucial information that tweaking the physicochemical properties of nanoparticles can help in designing the ideal vaccine adjuvants or delivery vehicles with no immunotoxicity.

Acknowledgements and/or References (Optional)::