A supramolecular pyroptosis nanogenerator based on non- covalent interactions for cancer immunotherapy

Abstract Background Pyroptosis has emerged as a potent immunogenic cell death mechanism with great potential in cancer immunotherapy. However, strategies for its precise induction and the elucidation of their structure-activity relationships remain elusive. Methods A supramolecular pyroptosis nanogenerator (SNPs@CaCO 3 ) was engineered through synergistic host–guest recognition, multiple hydrogen bonds, and electrostatic templating. The molecular glue Ad-G incorporates an adamantane domain for β-cyclodextrin (β-CD) encapsulation and a guanine group that stabilizes DAC through programmable multivalent hydrogen bonds. CUR is concurrently loaded into the hydrophobic β-CD cavity, while carboxylate groups on both β-CD and poly(ethylene glycol)-block-poly(methacrylic acid) (PEG-b-PMAA) electrostatically template Ca 2+ ions for controlled CaCO 3 biomineralization. Biodistribution, antitumor efficacy, pyroptosis induction, and immune activation were evaluated using fluorescence imaging, histological analysis, Western blotting, ELISA, and multicolor flow cytometry in orthotopic 4T1 breast tumor models and lung metastasis models. Results SNPs@CaCO 3 exhibited favorable physicochemical properties with acid-triggered release behavior, maintaining stability at physiological pH while enabling payload release under acidic conditions. In the orthotopic 4T1 model, SNPs@CaCO 3 treatment significantly suppressed tumor growth, enhanced CD8⁺ T cell infiltration, and reduced regulatory T cells. Mechanistically, Western blotting confirmed the synergistic activation of caspase-3 and GSDME cleavage, while ELISA revealed substantial secretion of pro-inflammatory cytokines, consistent with robust pyroptosis. DAC epigenetically upregulated GSDME expression through DFNA5 promoter demethylation, driving caspase-3/GSDME-mediated pyroptosis, as evidenced by DAMPs release and efficient dendritic cell maturation. Crucially, combining SNPs@CaCO 3 with αPD-L1 checkpoint blockade nearly eradicated lung metastases and significantly prolonged survival without detectable systemic toxicity. Conclusions This supramolecular engineering strategy provides a molecular blueprint with a defined structure-activity relationship to precisely trigger pyroptosis, advancing the frontier of cancer immunotherapy. The nanogenerator effectively addresses the coordinated delivery of chemically distinct payloads, overcomes epigenetic silencing, and remodels the immunosuppressive tumor microenvironment, offering a promising platform for combination immunotherapy.