Additional file 1 of Synergistic combination of targeted nano-nuclear-reactors and anti-PD-L1 nanobodies evokes persistent T cell immune activation for cancer immunotherapy

Additional file 1: Methods. Fig. S1. SEM image of Fe-PHCN. Scale bar: 1 μm. Fig. S2. TEM image of Fe-PHCN@DOX. The scale bar represents 100 nm. Fig. S3. Size of Fe-PHCN by dynamic light scattering (DLS) measurement. Fig. S4. Energy-dispersive X-ray spectroscopy (EDX) of Fe-PHCN. Fig. S5. Zeta potential of Fe-HCN and Fe-PHCN by DLS measurement. Fig. S6. FT-IR spectra of Fe-HCN and Fe-PHCN. Fig. S7. UV-vis absorbance spectra of the Fe-PHCN in NIR I and NIR II region. Fig. S8. Digital photograph of DOX, Fe-PHCN, and Fe-PHCN@DOX. Fig. S9. The size changes of Fe-PHCN@DOX in water, PBS, and DMEM medium containing 10% FBS for 6 days. Fig. S10. UV−vis spectra (A) and photographs (B) of MB aqueous solution under different concentrations of Fe-PHCN. Fig. S11. Flow cytometry analysis of uptake level of the Fe-PHCN@DOX in the CT26 cells with/without HA treatment. Fig. S12. Fluorescence images of DCF-stained CT26 cells under PHCN and Fe-PHCN treatment. Fig. S13. Flow cytometry gating strategy for the analysis of Annexin V-FITC/PI co-staining cell apoptosis. Fig. S14. Coomassie blue staining was performed after SDS-PAGE running: M represents marker; 1 represents anti-PD-L1 antibody; 2 represents anti-PD-L1 nanobody. Fig. S15. Confocal microscopy images (A) and fluorescence intensity (B) of anti-PD-L1 nanobodies and antibody distribution in tumor sections after injection. The scale bar is 50 μm. Fig. S16. The binding activity of PD-L1 receptor on CT26 cells with BSA, anti-PD-L1 nanobody (Nab), and anti-PD-L1 antibody (ab) was measured by flow cytometry. Fig. S17. The levels of cytokines released from T cells after different treatment. Fig. S18. The quantification (A) and percentage (B) of CD69+CD8+ T cells after different treatment. Fig. S19. The quantification (A) and percentage (B) of M1-type macrophages (CD11b+F4/80+CD86+CD206-) and M2-type macrophages (CD11b+F4/80+CD86-CD206+) in vitro after different treatment by flow cytometry. Fig. S20. Flow cytometry gating strategy for the analysis of DCs maturation in vitro. Fig. S21. ELISA analysis of the levels of cytokines TNF-⍺ secreted by DC2.4 cells in the medium. Fig. S22. The GSH depletion in tumor tissues after under different concentrations of Fe-PHCN@DOX. Fig. S23. Tumor growth curve of CT26 tumor-bearing mice after different treatments in 12 days. Fig. S24. Flow cytometry gating strategy for the analysis of DCs maturation in vivo. Fig. S25. Flow cytometry gating strategy for the analysis of M1-TAMs and M2-TAMs in vivo. Fig. S26. Flow cytometry gating strategy for the analysis of CD8 cells and CD4 cells in vivo. Fig. S27. The quantification of CD4+ helper T (CD3+CD4+) cells by flow cytometric analyses after various treatments. Fig. S28. The quantification of IFN-γ+ CD8+ T cells (A), Granzyme B+ CD8+ T cells (B) and Perforin+ CD8+ T cells (C) by flow cytometric analyses after different treatments. Fig. S29. The quantification of IFN-γ+ CD4+ T cells (A) and TNF-α+ CD4+ T cells (B) by flow cytometric analyses after different treatments. Fig. S30. The quantification of Treg cells (CD3+CD4+Foxp3+) by flow cytometric analyses after different treatments. Fig. S31. The quantification (A) and percentage (B) of infiltrating T cells in tumors by flow cytometric analyses after different treatment. Fig. S32. Flow cytometry gating strategy for the analysis of effector memory T cells in vivo. Fig. S33. ELISA analysis of the levels of cytokines IFN-γ in serum of mice after PBS and Fe-PHCN@DOX plus laser plus nanobody treatments. Fig. S34. Hemolytic percent of red blood cells incubated with Fe-PHCN@DOX at various concentrations. Fig. S35. H&E-stained images of heart, liver, spleen, lung, and kidney of the mice treated with PBS and Fe-PHCN@DOX plus laser plus nanobody. Scale bar stands for 100 μm. Fig. S36. Serum levels of ALT (A), AST (B), and IL-1β (C) were measured after the Fe-PHCN@DOX treatments. (D) H&E staining of the major organs including heart, liver, spleen, lung, and kidney after treatments. Scale bar stands for 200 μm.