The 4T1 cells (5×105 cells/well) were seeded in six-well plates and incubated overnight at 37°C in a humidified 5% CO2 atmosphere. ICG, PHSA-ICG, and PHSA-ICG-TAT (10 μM), as well as PBS (Control), were added to the cells, and the solutions were incubated for 24 h. The cells were irradiated with light at a wavelength of 808 nm (power density: 2.0 W/cm2) for 5 min and cultured further for 24 h.

The cells in each group were washed with PBS, and the apoptosis was measured using an Annexin FITC/PI apoptosis detection kit and flow cytometer BD FACSCalibur (BD Pharmingen).

The comet assay was performed as previously described.30 Briefly, molten 0.6% N-methyl-1,3-propanediamine was used to soak the frosted glass slide, which was left to dry naturally. Then, 70 μL of 0.7% LMPA at (37°C) was added to 10 μL of 4T1 cell suspension from each group to reach a cell number of 6,000. The solution was mixed and rapidly dripped on the frosted glass slide preheated at 37°C, immediately covered with a coverslip, and fixed at 4°C for 10 min. Subsequently, the coverslip was removed, and the cells were lysed in pre-cooled cell lysate at 4°C for 1 h in the dark. Electrophoresis was performed after unwinding for 30 min in alkaline electrophoresis buffer (electrophoresis parameters: 25 V, 300 mA, 25 min). After electrophoresis, the slides were rinsed and 20 ug/mL of EB 10 μL was added dropwise. The slides were covered with a coverslip and observed under a fluorescence microscope (DP80; Olympus). The extent of DNA damage was analyzed using the CASP software and evaluated as the percentage of DNA in the tail of the comet (Tail DNA%).

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