In Vitro Photothermal Radiotherapy

Firstly, the in vitro photothermal performance of the NSs was investigated. MoSe₂@BSA NSs and FA-MoSe₂@BSA NSs with the same Mo concentrations cultured with cells for 3 h and then irradiated by NIR irradiation for 5 min (808 nm, 1 W/cm²). The temperature of the treated cells in each well were detected with an infrared thermal camera (Fluke TI25, USA), respectively.

Next, for in vitro photothermal therapy, adherent 4T1 cells were cultured with different concentration of MoSe₂@BSA NSs and FA-MoSe₂@BSA NSs for 3 h. The NSs outside the cells were removed. The cells were then treated with or without NIR (808 nm, 1 W/cm², 5 min) and different dosage of X-ray irradiation (RT, 0–5 Gy, 0.084 Gy/s). After another 24-h incubation, cell viability was detected by a standard CCK-8 assay. The treated cells above were further co-stained by calcein-AM/PI to detect the live and dead cells and then imaged by a confocal laser scanning microscope (calcein-AM: Ex = 488 nm, Em = 515 nm; PI: Ex = 535 nm, Em = 617 nm). Moreover, the treated cells were also analyzed by γ-H2AX immunofluorescence. After the treatment above, the cells were fixed by 4% paraformaldehyde for 10 min and permeabilized with methanol for 15 min at − 20 °C and washed with PBS. Afterwards, the cells were mixed with a blocking buffer (1% BSA in PBS solution) for 1 h at 25 °C and further incubated with anti-phospho-histone γ-H2AX mouse monoclonal antibody (dilution 1:500) overnight at 4 °C. After PBS washing, the fluorescence of the cells was observed by confocal laser scanning microscope.