Additional file 1 of Intracellular marriage of bicarbonate and Mn ions as “immune ion reactors” to regulate redox homeostasis and enhanced antitumor immune responses
Creators: name: Feng, Yushuo
name: Liu, Yaqing
name: Ma, Xiaoqian
name: Xu, Lihua
name: Ding, Dandan
name: Chen, Lei
name: Wang, Zongzhang
name: Qin, Ruixue
name: Sun, Wenjing
name: Chen, Hongmin
Publisher: name: figshare
Year: 2022
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Figure S1. Typical TEM images of MnCO3-ICG complexes prepared at different feeding ratios of ICG and MnCl2·4H2O. Figure S2. (a) the absorption spectra of MnCO3-ICG complexes at different time. (b) Typical TEM images of MnCO3-ICG complexes prepared at different time. Figure S3. (a) TEM imaging of MnCO3-ICG-PAH complexes prepared with different feeding ratios of MnCO3-ICG and PAH. (b) Photographs of MnCO3-ICG-PAH complexes after being incubated in H2O, PBS (7.4), cell medium (DMEM), and serum (FBS) for 12 h. (c) The hydrodynamic diameter of MnCO3-ICG-PAH complexes with different feeding ratios. Figure S4. (a) X-ray powder diffraction pattern of the MnCO3-ICG and the re-assembled nanoformulation after incubation in the acidic buffer solutions. (b) XPS spectra for MnCO3-ICG and the re-assembled nanoformulation after incubation in the acidic buffer solutions. XPS spectra of Mn2p for (c) for MnCO3-ICG and (d) the re-assembled nanoformulation after incubation in the acidic buffer solutions. Figure S5. (a) Ultrasound images of the generation of CO2 after CaCO3 incubation with different time (pH 5.8 + H2O2). (b) Ultrasound signal intensity of the generation of CO2 after CaCO3 incubation with different time (pH 5.8 + H2O2). (c) The typical chromatograms of CO2 generated after MnCO3-ICG incubation with acidic environment. Figure S6. (a) Colorimetric analysis of the Fenton-like reaction for MB decolorization with different concentration of HCO3− in acidic environment. (b) Colorimetric analysis of the Fenton-like reaction for MB decolorization with different treatment. (c) Colorimetric analysis of the Fenton-like reaction for MB decolorization after different reaction time with acidic environment (pH 5.8). Figure S7. (a) Colorimetric analysis of the Fenton-like reaction for MB decolorization after pulling a vacuum. (b) Bar plot showing the of MB after different treatments. Figure S8. The absorption spectra of ICG, Mn-ICG and MnCO3-ICG. Figure S9. (a) Hep G2 cells and (b) U87MG cells incubated with different concentrations of MnCO3-ICG with or without 808 nm laser (0.5 W/cm2, 5 min) irradiation. Figure S10. Relative cellular viabilities of normal L02 and 3T3 cells incubated with different concentrations of MnCO3-ICG. Figure S11. Intracellular ·OH generation after incubation with Mn-ICG detected by DCFH-DA probe (scale bar, 50 μm). Figure S12. Lipid damage assessment measured by lipid peroxidation assays (*p &lt; 0.05). Figure S13. (a) CLSM observation on the intracellular distribution of lipoperoxides in 4T1 cells after incubation with PBS and Mn-ICG for 24 h. The red fluorescence is the lipid ROS ROS in cells and membranes after the staining with BODIPY-C11 (scale bar, 50 μm). (b) lipoperoxides, based on BODIPY staining resuts in panel (a). Figure S14. (a) CLSM observation on the changes in the mitochondrial membrane potential of 4T1 cells after incubation with different concentration of Mn-ICG (scale bar, 50 μm). (b) The membrane potential (ΔΨm) changes, assessed by JC-1staining. Figure S15. LDH release assay after incubation with different concentration of Mn-ICG. Figure S16. T1-relaxation rate (r1) and T1-weighted MR images of MnCO3-ICG. Figure S17. Ex vivo fluorescence images of the organs harvested in BALB/C tumor-bearing mice at 24 h post-injection. Figure S18. Body photos in different formulations after the 14-day treatment period. Groups 1, 2, 3 and 4 were used to represent PBS, MnCO3-ICG ([Mn]: 2 mg/kg, four dose), 2 × MnCO3-ICG ([Mn]: 4 mg/kg, four dose), 4 × MnCO3-ICG ([Mn]: 8 mg/kg, four dose), respectively. Figure S19. Images of representative tumors taken from mice in different formulations after the 14-day treatment period. Groups 1, 2, 3, 4, 5 and 6 were used to represent PBS, MnCO3-ICG ([Mn]: 2 mg/kg, four dose), 2 × MnCO3-ICG ([Mn]: 4 mg/kg, four dose), 4 × MnCO3-ICG ([Mn]: 8 mg/kg, four dose), MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), one dose) and MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), three dose), respective. Figure S20. The tumor growth inhibition curves of BALB/C tumor-bearing mice exposed to different formulations after the treatment period. Groups 1, 2, 3, 4, 5 and 6 were used to represent PBS, MnCO3-ICG ([Mn]: 2 mg/kg, four dose), 2 × MnCO3-ICG ([Mn]: 4 mg/kg, four dose), 4 × MnCO3-ICG ([Mn]: 8 mg/kg, four dose), MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), one dose) and MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), three dose), respective. Figure S21. The T2-MR imaging and digital photos of mice BALB/C tumor-bearing after the 21-day treatment period (group 6). Groups 6 was used to represent MnCO3-ICG([Mn]: 2 mg/kg + L (0.5 W/cm2), three dose), respectively. Figure S22. Representative immunofluorescence staining of nucleus (blue) and vessel (green) on the tumor slices collected 14-day after laser irradiation (scale bar, 50 μm). Figure S23. Representative immunofluorescence staining of TUNEL (nucleus (blue) and apoptotic cells (red)) and ROS (nucleus (blue) and ROS (green)) on the tumor slices. (scale bar, 200 μm). Figure S24. The flow cytometric histograms showing the intratumor infiltration of the effector T cells (CD8 + T cells). Groups 1, 2, 3, 4, 5 and 6 were used to represent PBS, MnCO3-ICG ([Mn]: 2 mg/kg, four dose), 2 × MnCO3-ICG ([Mn]: 4 mg/kg, four dose), 4 × MnCO3-ICG ([Mn]: 8 mg/kg, four dose), MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), one dose) and MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), three dose), respectively. Figure S25. Body weight curves of BALB/C tumor-bearing mice after various treatments (n = 5). Groups 1, 2, 3, 4, 5 and 6 were used to represent PBS, MnCO3-ICG ([Mn]: 2 mg/kg, four dose), 2 × MnCO3-ICG ([Mn]: 4 mg/kg, four dose), 4 × MnCO3-ICG ([Mn]: 8 mg/kg, four dose), MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), one dose) and MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), three dose), respectively. Figure S26. Hematoxylin &amp; eosin (H&amp;E)-stained of the organ harvested from mice in different formulations after the 14-day treatment period. Groups 1, 2, 3, 4, 5 and 6 were used to represent PBS, MnCO3-ICG ([Mn]: 2 mg/kg, four dose), 2 × MnCO3-ICG ([Mn]: 4 mg/kg, four dose), 4 × MnCO3-ICG ([Mn]: 8 mg/kg, four dose), MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), one dose) and MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), three dose), respectively, (scale bar, 100 μm). Figure S27. (a) Intracellular ·OH generation after incubation with MnCO3-ICG in different buffer solutions (pH 7.4, pH 6.5, and pH 6.5 with laser) detected by DCFH-DA probe. The blue and green fluorescence indicate cell nucleus and DCFH-DA, respectively (scale bar, 50 μm). (b) The fluorescence intensity of ·OH,based on DCFH-DA staining resuts in panel (a) (**P &lt; 0.05). Figure S28. (a) CLSM observation on the intracellular distribution of lipoperoxides in Hep 1–6 cells after incubation with PBS and MnCO3-ICG for 24 h. The red fluorescence is the lipid ROS in cells and membranes after the staining with BODIPY-C11 (scale bar, 50 μm). (b) lipoperoxides, based on BODIPY staining results in panel (a) (***P &lt; 0.001). Figure S29. (A) CLSM observation on the changes in the mitochondrial membrane potential of Hep 1–6 cells after incubation with different concentration of MnCO3-ICG (scale bar, 50 μm). (B) The membrane potential (ΔΨm) changes, assessed by JC-1staining (***P &lt; 0.001). Figure S30. LDH release assay of Hep 1–6 cells after incubation with different concentration of MnCO3-ICG (***p &lt; 0.001).