The 4T1 or A375 cells were established by injecting 100 μl of PBS containing 107 U of 4T1 cells into the subcutaneous tissue of the mice. Various samples were then administrated when the tumor size reached 60 mm3, and the initial weight of all mice was maintained at 18.2 ± 0.2 g. Briefly, 30 4T1 tumor-bearing mice were randomly divided into five groups (each with six mice). The sample groups are PBS, ACC-CaSi-PAMAM-FA/mPEG, DOX, ACC@DOX-CaSi-PAMAM-FA/mPEG, and ACC@ DOX.Fe2+-CaSi-PAMAM-FA/mPEG. All samples were injected through the tail vein at an equivalent DOX concentration of 5 mg/kg. The injection was repeated every other day, and the body weight and tumor volume of nude mice were both recorded. The tumor volume was calculated as Vtumor = LW2/2 (L, maximum diameter of the tumor; W, minimum diameter of the tumor, both were measured using a digital vernier caliper). After 21 days of treatment, all mice were euthanized, and the tumors and major organs were harvested for the subsequent analysis. Typically, the organs and tissues were sectioned and embedded into paraffin after being fixed with 10% formalin at 4°C for 24 hours, and then, paraffin-embedded sections were stained with H&E to monitor the cytotoxicity induced by various samples. In addition, the tumor sections were also stained by the colorimetric TUNEL Apoptosis Assay Kit to determine the therapeutic effect. Both the H&E- and TUNEL-stained tissue sections were then observed with a microscope.

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