Varying the hydrophobic core composition of polymeric nanoparticles affects NLRP3 inflammasome activation†

Understanding the interactions of nanoparticle carriers with innate immune cells is crucial for informing the design and efficacy of future nano-immunotherapies. An intriguing aspect of their interaction with the immune system has recently emerged, i.e., their ability to activate the NLRP3 inflammasome, a key component of the innate immune response. While the effect of the 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. The impact of modulating SNP core hydrophobic properties was investigated in macrophages by evaluating their cellular internalization, cytokine release, lysosomal rupture-calcium signaling, calcium flux-mitochondrial ROS production and their ability to activate the NLRP3 inflammasome, providing mechanistic insights into inflammasome activation. We established a direct correlation between increasing the side chain length of the polymer construct, thereby increasing the core hydrophobicity of SNPs and enhanced NLRP3 complex formation, as indicated by 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. Specifically, the nanoparticle with the longest alkyl side chain effectuated NLRP3 activation preferentially through the mitochondrial damage pathway. In vivo evaluation of SNPs in C57BL/6 mice confirmed elevated proinflammatory cytokines, notably with the SNP having the longest C₁₂-alkyl side chain. This confirms that the higher core hydrophobicity composition of the SNP results in inflammasome activation in vivo. 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.