4T1 cells were first seeded onto six-well plates at an initial cell density of 105 U per well, and the incubation conditions were kept the same with the in vitro experiments above. When the cell confluence reached around 70%, the previously added culture media were replaced by new ones containing TCPS (blank control), ACC@DOX.Fe2+-CaSi-PEG, ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG, or MMP-2–pretreated ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (MMP-2 concentration, 400 ng/ml). The concentration of nanosamples was 34 μg/ml, and the equivalent DOX concentration was maintained at 1 μg/ml. The incubation would continue for 12 or 24 hours. Subsequently, the culture medium in all wells was removed, and the cells were washed three times with PBS. The cells were then detached by trypsin without EDTA-Na and purified twice by repetitive centrifugation/PBS washing. Cell lysate (containing 1% SDS, 1% Triton X-100, and 40 mM tris acetate) was eventually added to lyse the cells, and the resultant solution was sonicated to ensure compete cell disintegration. The iron level was detected by ICP emission spectroscopy on an iCAP 6300 Duo (Thermo Fisher Scientific).

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