Slide-A-Lyzer MINI Dialysis Device (Thermo Fisher Scientific, 10 kDa molecular mass cutoff) was used to determine the release profile of Dox from conjugates. Dox conjugates were diluted in either PBS (pH 6.5 or 7.4) or 0.1 M acetate buffer (pH 5.0) to a final concentration of 100 μM (Dox basis). Each sample (150 μl) was loaded into the device and dialyzed with buffer (50 ml). Dialysis was performed using a magnetic stirrer. The temperature of the stage was set to 37°C, and samples were protected from light during dialysis. Dialysate was collected at various time points and stored at 4°C until the sample collection was finished. Dialysate was loaded onto a 96-well black plate in duplicate (90 μl/well). Fluorescence was determined using excitation at 495 nm and emission at 590 nm. Serial dilution of Dox hydrochloride was prepared in the same buffer to create a standard curve.

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