Two tumor cell lines were used in this study, which are the 4T1 breast cancer cells and A375 melanoma cells. They were both cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Gibco), penicillin (100 U/ml), and streptomycin (100 μg/ml). The temperature was set at 37°C, and the incubation atmosphere contained 5% CO2. The culture media were refreshed every 2 days. The 4T1 cells were seeded onto a 96-well plate at density of 104 U per well and subsequently incubated overnight at 37°C under an atmospheric CO2 level of 5%. The incubation media were then replaced with fresh ones containing ACC-CaSi-PAMAM-FA/mPEG, DOX, ACC@DOX-CaSi-PAMAM-FA/mPEG, ACC@ DOX.Fe2+-CaSi-PAMAM-FA/mPEG, and MMP-2–pretreated ACC@DOX.Fe2+-CaSi -PAMAM-FA/mPEG (the concentration of MMP-2 was 400 ng/ml), while the tissue culture polystyrene (TCPS) group was used as the blank control. The concentration of nanosamples was 34 μg/ml, and the equivalent DOX concentration was maintained at 1 μg/ml. Each sample group contained six wells, and the incubation periods were set to 12, 24, and 48 hours. Fresh media containing MTT agents (0.5 mg/ml) were added into each well when the incubation was complete and incubated for 4 more hours. Dimethyl sulfoxide (100 μl) was then added to dissolve the emerging formazan crystals. The optical density of the samples at the excitation wavelength of 490 nm was measured on a SpectraMax i3x microplate reader.

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