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Several FDA-approved adjuvants signal through the NLRP3 inflammasome and IL-1β release. Identifying small molecules that induce IL-1β release could allow targeted delivery and structure-function optimization, thereby improving safety and efficacy of next-generation adjuvants. In this work, we leverage our existing high throughput data set to identify small molecules that induce IL-1β release. We find that ribociclib induces IL-1β release when coadministered with a TLR4 agonist in an NLRP3- and caspase-dependent fashion. Ribociclib was formulated with a TLR4 agonist into liposomes, which were used as an adjuvant in an ovalbumin prophylactic vaccine model. The liposomes induced antigen-specific immunity in an IL-1 receptor-dependent fashion. Furthermore, the liposomes were coadministered with a tumor antigen and used in a therapeutic cancer vaccine, where they facilitated rejection of E.G7-OVA tumors. While further chemical optimization of the ribociclib scaffold is needed, this study provides proof-of-concept for its use as an IL-1 producing adjuvant in various immunotherapeutic contexts.

Cationic polymers are widely used materials in diverse biotechnologies. Subtle variations in these polymers' properties can change them from exceptional delivery agents to toxic inflammatory hazards. Conventional screening strategies optimize for function in a specific application rather than observing how underlying polymer-cell interactions emerge from polymers' properties. An alternative approach is to map basic underlying responses, such as immunogenicity or toxicity, as a function of basic physicochemical parameters to inform the design of materials for a breadth of applications. To demonstrate the potential of this approach, we synthesized 107 polymers varied in charge, hydrophobicity, and molecular weight. We then screened this library for cytotoxic behavior and immunogenic responses to map how these physicochemical properties inform polymer-cell interactions. We identify three compositional regions of interest and use confocal microscopy to uncover the mechanisms behind the observed responses. Finally, immunogenic activity is confirmed in vivo . Highly cationic polymers disrupted the cellular plasma membrane to induce a toxic phenotype, while high molecular weight, hydrophobic polymers were uptaken by active transport to induce NLRP3 inflammasome activation, an immunogenic phenotype. Tertiary amine- and triethylene glycol-containing polymers did not invoke immunogenic or toxic responses. The framework described herein allows for the systematic characterization of new cationic materials with different physicochemical properties for applications ranging from drug and gene delivery to antimicrobial coatings and tissue scaffolds., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)