A previous report about polypeptide-Dox nanoparticles was referred (40). Aldoxorubicin, Dox-SA, or Dox-CBD-SA was injected into FVB mice with established tumors at 4.16 mg/kg via tail vein. Tumor was collected, weighed, and put on ice for 2 or 24 hours after injection. Tumor tissues were suspended in 1 ml of acidified isopropanol and homogenized using Lysing Matrix D and FastPrep-24 5G (MP Biomedical) for 40 s at 5000 beats/min. After homogenization, samples were protected from light and incubated overnight at 4°C. Samples were centrifuged (5000g, 5 min), and the supernatants were loaded onto a 96-well black plate (100 μl/well, triplicate). Fluorescence was measured to quantify the amount of Dox in tissue extracts as described above. Tumors from untreated mice were also processed, and serial dilutions of tissue extracts were measured to obtain the standard curves of tissue-derived autofluorescence.

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