Fig 1: CuS/EPDA tumor targeting mechanism. (A) Cell viability assay of lung metastatic H-1993 cells after treatment with CuS (2mg/mL) or CuS/EPDA (2–4mg/mL) for 4h (Scale bar, 20 μm). (B and C) cytotoxicity evaluation of CuS/EPDA NCs in H-1993 and A549 cells under neutral or hypoxic pH. Cells were treated with CuS/EPDA NCs (0.5–10μg/mL−1) for 4h. Data represent the mean±SD of three independent experiments (*P < 0.05; **P < 0.01). (D–F) Flow cytometry analysis of intracellular cytokines TNF-A, IL-6 and IL-12 levels in NC-treated mice. Serum from mice was isolated 48–72h following treatment. Data represent the mean ±SD of three independent experiments. (G and H) DC maturation analysis using flow cytometry after staining with CD11c, CD80 and CD86. Data represent the mean ±SD of three independent experiments (**P < 0.01). (I and J) Immunofluorescence staining of immunosuppression biomarkers iNOS and Arg1 in tumor tissues after CuS and CuS/EPDA incubation. Confocal microscopy images of Arg1 (green). iNOS labeled cells (red) and DAPI (blue). Images were captured using Carl Zeiss fluorescence confocal microscope. Data represent the mean±SD of three independent experiments (*P < 0.01). (K) Intracellular ROS assay (Deepred, ab186029, Abcam) of lung metastatic H-1993 cells after treatment with CuS (2mg/mL) or CuS/EPDA (2–4mg/mL) for 4 h (scale bar, 20 μm). Differences were considered statistically significant at p < 0.05. Statistically significant data are indicated by asterisks.