Mouse SA or CBD-SA was solubilized in PBS containing 2 mM EDTA. Four molar equivalents of Traut’s reagents solved in PBS containing 2 mM EDTA were added and incubated for 1 hour at room temperature in the dark. Excess Traut’s reagents were removed by a Zeba spin desalting column (Thermo Fisher Scientific). Fifteen molar equivalents of aldoxorubicin (MedChemExpress) dissolved in 10 mM sodium phosphate buffer (pH 5.9) was added and incubated for 1 hour at room temperature and overnight at 4°C in the dark. To quench the reaction, 20 molar equivalents of l-cysteine [dissolved in PBS containing 2 mM EDTA; Sigma-Aldrich (pharma-grade)] against aldoxorubicin was added. Unreacted Dox precipitates were removed by centrifugation (10,000g, 5 min). Supernatant was further purified by a Zeba spin desalting column, followed by ultrafiltration using Amicon Ultra (Merck, 10 kDa molecular mass cutoff). Concentration of Dox in the final product was quantitated by absorbance at 495 nm, using a molar extinction coefficient of 10,650 (L mol−1 cm−1). The concentration of protein content was measured by Pierce BCA (bicinchoninic acid assay) Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

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