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Supplementary information for NIR-triggered on-site NO/ROS/RNS nanoreactor: Cascade-amplified photodynamic/photothermal therapy with local and systemic immune responses activation |
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Illustration showing the preparation and use of the RNS nanoreactor. The PBNO-Ce6 nanoparticles heat up (PTT) and release NO when exposed to NIR laser. At the same time, ROS are generated (PDT) and further transform the NO into RNS. The combined effect of PTT and RNS-enhanced PDT causes cell apoptosis and promotes strong activation of local and systemic immune responses.
Bioinformatics analysis of breast cancer patients. (a) Immunohistochemistry images showing HIF1A, VEGFA, and GLUT1 staining in breast cancer tumor tissue and normal breast tissue. Scale bar: 100 μm. (b) HIF1A, VEGFA, and GLUT1 gene expression profiles across TCGA/GTEx breast cancer datasets. (c) Differential expression of HIF1A, VEGFA, and GLUT1 in TNBC compared to non-TNBC. (d) Kaplan-Meier survival curves for OS and DMFS in patients with high vs low immune scores. (e) Single cell analysis of T cells in TNBC and non-TNBC tumors. (Tn: naive T cells; Tef: effector T cells; Tex: exhausted T cells; Tm: memory T cells; Tef: effector memory T Cell.) (f) Kaplan-Meier survival curves for OS and PFS in TNBC and non-TNBC patients.
Characterization of PBNO-Ce6 nanoparticles. (a) Particle size distribution and TEM image of PBNO. (b) FT-IR spectra of PB, PBNO and SNP. (c) N2 adsorption-desorption isotherms of PBNO. (d) Loading capacity and efficiency of Ce6 on PBNO. (e) Size and zeta potential of PBNO and PBNO-Ce6. (f) UV-vis absorption spectra of PBNO, Ce6 and PBNO-Ce6. (g) Thermographs of H2O, PB and PBNO irradiated by 660-nm laser under 0.4 W cm−2. (h) Corresponding photothermal heating curves of H2O, PB and PBNO. (i) Linear time vs -lnθ plots obtained from the cooling period of PB and PBNO.
NIR triggered generation of NO/ROS/RNS by PBNO-Ce6. (a) Scheme showing NO/ROS/RNS generation. (b) NO release detected after laser irradiation of PBNO. (c) ROS generation detected by SOSG fluorescence of PB, PBNO, PB-Ce6, and PBNO-Ce6 after laser irradiation. (d) RNS generation detected by BBoxiProbe® O56 fluorescence of PB, PBNO, PB-Ce6, and PBNO-Ce6 after laser irradiation. (e) BBoxiProbe® O56 fluorescence intensity over time. (f) Fluorescence images of 4T1 cells stained with probes for NO (DAM-FM DA), ROS (DCFH-DA) and RNS (BBoxiProbe® O52). Scale bar: 50 μm. (g) Corresponding flow cytometry analysis of 4T1 cells stained with DAM-FM DA, DCFH-DA and BBoxiProbe® O52.
In vitro therapeutic efficacy of PBNO-Ce6. (a) Viability of 4T1 cells after 24 h incubation with PB, PBNO and PBNO-Ce6 without laser irradiation. (b) Viability of 4T1 cells after 24 h incubation with PB, PBNO, PB-Ce6 or PBNO-Ce6 and irradiated at 660 nm (0.4 W·cm−2, 5 min). (c) Viability of 4T1 cells incubated with PBNO and PBNO-Ce6 (with or without ice bath cooling) for 24 h and irradiated at 660 nm. (d) Fluorescence imaging of Calcein-AM and PI stained 4T1 cells after different treatments (concentration: 50 μg·mL−1). Green: Calcein-AM; Red: PI. Scale bar: 50 μm. (e) Flow cytometry analysis of apoptosis in 4T1 cells after different treatments (concentration: 25 μg·mL−1).
In vivo therapeutic efficacy of PBNO-Ce6. (a) Fluorescence images of mice at different time points after intravenous injection of free Ce6 or PBNO-Ce6. (b) Thermal images of mice during laser irradiation. (c) Temperature changes at the tumor area after irradiation. (d) Fluorescence imaging of NO, ROS and RNS generation in tumors. (e) TUNEL and H&E staining of tumors after treatment. Scale bar: 50 μm. (f) Tumor growth curves of mice after different treatments. (g) Kaplan-Meier survival curves of mice in different treatment groups. (*p< 0.05, **p< 0.01,***p< 0.001, ****p< 0.0001)
Local and systemic antitumor immune responses induced by PBNO-Ce6 treatment. (a) Tumor growth curves of mice after tumors rechallenge in different treatment groups. (b, c) Flow cytometry analysis of immunosuppressive Tregs (CD4+Foxp3+CD25+) and antitumor CTLs (CD3+CD8+) in (b) primary tumors and (c) spleens. (d, e) Analysis of IFN-γ+ and Ki67+ expression in CTLs in (d) primary tumors and (e) spleens. (*p< 0.05, **p< 0.01,***p< 0.001, ****p< 0.0001)
Biocompatibility of PBNO-Ce6 in vivo. (a) H&E images of heart, liver, spleen, lung, and kidney tissues from mice at 14 days post-injection. Scale bar: 50 μm. (b) Routine blood cell count of mice at 14 days post-injection. WBC: white blood cells; RBC: red blood cells; MPV: mean platelet volume; MCHC: mean corpuscular hemoglobin concentration; MCH: mean corpuscular hemoglobin; HCT: hematocrit; HGB: hemoglobin; PLT: platelet count. The gray areas indicate the normal range of the indicators. (c) Blood biochemistry analysis of mice at 14 days post-injection. ALT: alanine aminotransferase; ALP: alkaline phosphatase; BUN: blood urea nitrogen; CRE: creatinine; AST: aspartate aminotransferase; GLU: glucose.