4T1 or A375 cells were seeded into six-well plates as described above and treated with various samples when the cell confluence reached 70%. The samples included TCPS (blank control), 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 (MMP-2 concentration, 400 ng/ml). The incubation lasted for 24 hours, and the concentration of nanosamples was 34 μg/ml, and the DOX concentration was at 1 μg/ml, which is to maintain both the nanoparticle and drug dosage at an equivalent level. When the incubation was complete, the cells were washed twice with PBS and incubated with DOPIBY C11 (Lipoperoxide indicator; concentration, 5 μM) for 30 min. The intracellular level of lipoperoxides was monitored using a CytoFLEX flow cytometry system (Beckman Coulter). A same experimental setup was also used for the CLSM observations.

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