Additional file 1 of Ultrasound initiated tumor catalytic PANoptosis by mesoporous piezoelectric nanocatalysts

Additional file 1. Methods. Fig. S1 Transmission electron microscopy image of cBTO NPs. Fig. S2 X-ray diffraction spectra of cBTO NPs and partially enlarged spectrum with 2θ range between 44° and 48°. Fig. S3 X-ray diffraction spectra of tBTO NPs and partially enlarged spectrum with 2θ rangebetween 44° and 48°. Fig. S4 Scanning electron microscopy images of tBTO NPs treated with H2O2 at 110 °C for 2 h (a) and 125 °C for 1 h (b). Fig. S5 Hydrodynamic diameter profiles of mtBTO-1, mtBTO-2, and mtBTO-3 NPs. Fig. S6 The N2 adsorption/desorption isotherms and pore size distribution of mtBTO-1, mtBTO-2, and mtBTO-3 NPs. Fig. S7 X-ray diffraction spectra of mtBTO-1, mtBTO-2, and mtBTO-3 NPs with magnified 2θ range between 44° and 48°. Fig. S8 Atomic force microscopy images of tBTO, mtBTO-1, mtBTO-2, and mtBTO-3 NPs. Fig. S9 HR-TEM characterization of mtBTO-1 NPs. Fig. S10 HAADF-STEM characterization of mtBTO-1 NPs. Fig. S11 FTIR spectra of tBTO (blue line) and mtBTO-1 NPs (red line). Fig. S12 XPS spectra of tBTO and mtBTO-1 NPs and corresponding spectrum for Ti2p and Ba3d. XPS X-ray photoelectron spectroscopy. Fig. S13 Band structure determination of mtBTO-1 NPs. Fig. S14 US-initiated degradation kinetics of MB after varied treatments under pH 6.0. Fig. S15 Temperature curve of 1 ml PBS during ultrasound treatment for 3 min at varied power densities (0.5, 1.0, 1.5, and 2.0 W/cm2) and duty ratio (50 and 100%). Fig. S16 Cell viability of B16 cells after being treated with US at 1.0 W/cm2 and 50% duty ratio for a total US time of 1 min at varied US duration parameters. Fig. S17 CLSM images of B16 cells stained with H2DCF-DA probe after incubation with mtBTO, ZnO2@mtBTO, and SNO-ZnO2@mtBTO NPs with or without ultrasound treatments. Fig. S18 Statistical analysis of DCF fluorescence intensity of B16 cells after incubation with mtBTO, ZnO2@mtBTO, and SNO-ZnO2@mtBTO NPs with or without ultrasound treatments. Fig. S19 CLSM images of B16 cells stained with SOSG probe after incubation with mtBTO, ZnO2@mtBTO, and SNOZnO2@ mtBTO NPs with or without ultrasound treatments. Fig. S20 CLSM images of B16 cells stained with DHE probe after incubation with mtBTO, ZnO2@mtBTO, and SNO-ZnO2@mtBTO NPs with or without ultrasound treatments. Fig. S21 CLSM images of B16 cells stained with DAX-J2 PON probe after incubation with mtBTO, ZnO2@mtBTO, and SNO-ZnO2@mtBTO NPs with or without ultrasound treatments. Fig. S22 Statistical analysis of fluorescence intensity of JC-1 in B16 cells after incubation with PBS, mtBTO NPs, ZnO2@mtBTO NPs, and SNO-ZnO2@mtBTO NPs with or without ultrasound treatments. Fig. S23 Cell viability of RAW264.7 after varied treatments. Fig. S24 Flow cytometry analysis on RAW264.7 cells after incubating with B16 tumor cells which were pretreated with PBS, ZnO2@mtBTO NPs, SNO-ZnO2@mtBTO NPs, and NZCB NPs (50 μg/ml) with or without US irradiation (1.0 W/cm2, 1 min). Fig. S25 Body weight of mice in each group after treatments of saline, US, mtBTO NPs + US, ZnO2@mtBTO NPs + US, SNOZnO2@ mtBTO NPs + US, and NZCB NPs + US. Fig. S26 Tumor volume curves of saline, US, mtBTO NPs + US, ZnO2@mtBTO NPs + US, SNOZnO2@ mtBTO NPs + US, and NZCB NPs + US groups during the evaluation timeframe. Fig. S27 Digital photos of mice from each group on day 12 after treatment of saline, US, mtBTO NPs + US, ZnO2@mtBTO NPs + US, SNO-ZnO2@mtBTO NPs + US, and NZCB NPs + US. Fig. S28 TUNEL immunofluorescence microscopic images of tumor sections from different groups. Fig. S29 H&E stained images of major organs of mice including heart, liver, spleen, lung and kidney after treatments of saline, US, mtBTO NPs + US, ZnO2@mtBTO NPs + US, SNO-ZnO2@mtBTO NPs + US, and NZCB NPs + US (Scale bar = 200 μm). Fig. S30 Biosafety analysis of mice via the complete blood data analysis including white blood cells (WBC), lymphocyte (LYM), granulocytes (GRA), hemoglobin concentration (HGB), mean corpuscular hemoglobin (MCH), red blood cells (RBC), platelet (PLT), albumin (ALB), creatine kinase (CK), creatine kinase-MB (CK-MB), blood creatinine (CREA) and glucose (GLU) markers. Fig. S31 Biodistribution assay of major organs of mice after intratumoral injection of NZCB NPs (in Ba concentration) at 10 mg/kg for 2, 24, and 48 h. Fig. S32 Representative gating strategy of spleen lymphocytes for M1-macrophage assay. Fig. S33 Representative gating strategy of spleen lymphocytes for CD8 T cell assay. Fig. S34 Statistical analysis of CD86 cells in CD11b F4/80 cells and CD8 T cells in CD3 T cells isolated from spleen tissues of mice after treatments of saline, US, mtBTO NPs + US, ZnO2 mtBTO NPs + US, SNO-ZnO2@mtBTO NPs + US, and NZCB NPs + US. Fig. S35 Expression of apoptosis-associated genes including BAX, CYCS, CASP3, TNF-α, PRF1 and GZMB between control group and NZCB + US group. Fig. S36 Relative protein expression of PANoptosis-related proteins after different treatments. Fig. S37 Western blotting and quantitative analysis of proteins of Fas, FasL, cleaved CASP8, ZBP1, IRF1, and p-STING in different groups.