A Supramolecular Thermal Switch for Precision Pyroptosis via Host−Guest Recognition and Electrostatic Interactions

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  SCHEME 1. Schematic illustration of a supramolecular thermal switch designed for precision pyroptosis induction and cascade‐amplified cancer therapy. (A) Fabrication process of the supramolecular thermal switch for precision pyroptosis. The system is constructed via host−guest complexation of I3 − within the cavity of β‐CD, followed by Ca2+ stabilization on the carboxylate‐functionalized surface and subsequent CaCO3 biomineralization. (B) Therapeutic mechanism of supramolecular thermal switch for cascade‐amplified cancer therapy. Following cellular uptake, the lysosomal acidity dissolves the CaCO3 matrix, co‐releasing Ca2+ and I3 −. Upon NIR irradiation, the BODIPY‐based thermal switch is activated, converting I3 − into I2 and initiating a destructive cascade of oxidative stress, mitochondrial damage and Ca2+ dyshomeostasis. The endogenous Ca2 + dyshomeostasis and exogenous Ca2 + release synergistically induce Ca2+ overload, activating the caspase‐3/GSDME‐mediated pyroptosis. The release of DAMPs promotes dendritic cell maturation and T lymphocyte recruitment, thereby stimulating a potent systemic antitumor immunity to suppress tumor growth and metastasis.

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  FIGURE 1. Characterization of the SNPs and their assembly process. (A) Synthetic route of B‐CD‐COOH. (B) UV–vis absorption and fluorescence emission spectra of B‐CD‐COOH. (C) 1H NMR spectra (400 MHz, 298 K, DMSO‐d6 ) of β‐CD (1.00 mM) and the β‐CD⊃I3 − host–guest complex (1.00 mM). (D) FTIR spectra of a physical mixture (β‐CD + I3 −) and the host–guest complex (β‐CD⊃I3 −). (E) Zeta potential of B‐CD‐COOH, B‐CD‐COOH with Ca2+ and the final SNPs. (F) ITC titration profile for the host−guest complexation between β‐CD and I3 −. (G) TEM images and (H) hydrodynamic size distributions of nanoparticles prepared under varying biomineralization conditions. I: 6.18 mg mL−1 CaCl2, 2.95 mg mL−1 Na2CO3, stirred 24 h; II: 6.18 mg mL−1 CaCl2, 5.90 mg mL−1 Na2CO3, stirred 12 h; III: 6.18 mg mL−1 CaCl2, 5.90 mg mL−1 Na2CO3, stirred 24 h; IV: 6.18 mg mL−1 CaCl2, 5.90 mg mL−1 Na2CO3, stirred 48 h; V: 6.18 mg mL−1 CaCl2, 11.8 mg mL−1 Na2CO3, stirred 24 h.

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  FIGURE 2. Comprehensive physicochemical characterization and proposed assembly mechanism of the SNPs. (A) Element mapping analysis of the SNPs. (B) XPS spectrum of the SNPs. (C) XRD patterns of the SNPs, with reference patterns for calcite CaCO3, KI and NaCl. (D) Time‐dependent structural disintegration of SNPs in an acidic environment (pH 5.6), as visualized by TEM images and quantified by the evolution of DLS over 12 h. (E) Comparative analysis of DLC and EE for I3 − across different carries. Cumulative release profiles of (F) Ca2+ and (G) I3 − from SNPs under different simulated physiological conditions. (H) Molecular structures of the key building blocks: B‐CD‐COOH, Ca2+ and CO3 2−. (I) Proposed time‐resolved self‐assembly mechanism: (I) formation of the core B‐CD‐COOH complex, (II, III) electrostatic coordination of Ca2+ ions with carboxyl groups, (IV, V) oriented growth of a CaCO3 mineral shell via reaction with CO3 2−, leading to the final SNPs. Data in (E‐G) are presented as mean ± SD (n ≥ 3). Statistical significance was calculated via ordinary one‐way ANOVA with a Tukey's test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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  FIGURE 3. The SNPs initiate a destructive cascade of oxidative stress, mitochondrial damage and calcium buffering system collapse, leading to specific Ca2+ overload and ultimately culminating in caspase‐3/GSDME‐mediated pyroptosis and potent ICD. (A) CLSM images of 4T1 cells incubated with SNPs for 2 h, with lysosomal location (Lyso‐Tracker) and nuclear staining (DAPI). (B) Time‐dependent cellular uptake of SNPs quantified by flow cytometry. (C) Cellular ROS levels after different treatments. (D) Cell viability of 4T1 cells post‐treatments. (E) WB analysis of GSDME‐N and C‐CAS‐3. (F) Quantification of LDH release into cell culture supernatants. (G) Secretion levels of IL‐18 and IL‐1𝛽 in supernatants. Quantification of (H) CRT exposure and (I) HMGB1 efflux. (J) CLSM images of JC‐1‐stained 4T1 cells following different treatments. (K) Quantification of ATP release into cell culture supernatants. (L) Bio‐TEM images of mitochondrial ultrastructure. (M) Intracellular Ca2+ levels detected by Fluo‐4 AM staining. (N) Pyroptosis frequency across treatment groups. (o) Bright‐field and CLSM micrographs of 4T1 cells post‐treatments. Data are presented as mean ± SD (n ≥ 3). Statistical significance was…

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  FIGURE 4. In vivo pharmacokinetics, antitumor efficacy, and mechanism validation of the SNPs (L). (A) Pharmacokinetic profiles of B‐CD‐COOH and SNPs, as determined by monitoring BODIPY‐derived UV–vis absorption spectroscopy over time. (B) In vivo fluorescence whole‐body imaging and corresponding ex vivo organ biodistribution at 1, 2, 4, 12, and 24 h post‐injection. (C) The tissue distribution of I2 characterized by UV–vis absorption spectroscopy. (D) Therapeutic regimen for primary tumor therapy. (E) Representative images of excised tumors at the study endpoint. (F) Tumor growth curves across different treatment groups. (G) WB analysis of GSDME‐N and C‐CAS‐3 expression in tumor tissues following different treatments. (H) Quantitative analysis of the protein levels shown in (G). (I) H&E, Ki67, CRT and HMGB1 staining of tumor tissues following different treatments. Data are presented as mean ± SD (n ≥ 3). Statistical significance was calculated via ordinary one‐way ANOVA with a Tukey's test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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  FIGURE 5. The SNPs‐induced immunogenic cell death orchestrates a comprehensive antitumor immune response, linking innate activation to adaptive immunity. (A,B) Flow cytometry analysis of DCs maturation in TDLNs. Flow cytometry analysis of (C,D) CD4+ T cells (CD3+CD4+), (E,F) CD8+ T cells (CD3+CD8+) and (G,H) Tregs (CD25+Foxp3+) in treated tumor tissues. (I) Serum levels of immunocompetent cytokines including IL‐6, IFN‐𝛾 and TNF‐𝛼 following different treatments. (J) Immunofluorescence staining of tumor‐infiltrating Foxp3+ Tregs and CD56+ NK cells across treatment groups. Data are presented as mean ± SD (n ≥ 3). Statistical significance was calculated via ordinary one‐way ANOVA with a Tukey's test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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  FIGURE 6. Anti‐metastasis efficacy and mechanism validation of the SNPs. (A) Therapeutic regimen for bilateral tumor therapy. Tumor growth curves of (B) primary and (C) abscopal tumors across different treatment groups. (D,E) Flow cytometry analysis of DCs maturation in TDLNs. Flow cytometry analysis of (F,G) CD8+ T cells (CD3+CD8+) and (H,I) Tregs (CD25+Foxp3+) in abscopal tumor tissues. Data are presented as mean ± SD (n ≥ 3). Statistical significance was calculated via ordinary one‐way ANOVA with a Tukey's test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.