2.4. In vitro drug release assay

The drug release properties of DOX from our synthesized delivery system were investigated (DOX amount: 200 µg) using a dialysis bag (MWCO 3500) containing 4 mL of a drug-loaded nanoparticle suspension immersed in 10 mL of a Tris–HCl solution (pH = 5.3, 6.5, and 7.4). The samples were continuously oscillated in a shaking water bath at a speed of 150 rpm at a constant temperature of 37 °C. One milliliter of the sample was taken from the solution at different time points for fluorescence spectroscopy, and then replaced with the same volume of fresh medium. The concentration of released DOX was measured using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Santa Clara, CA). The fluorescence intensity of the blank Tris–HCl solution (pH = 5.3, 6.5, and 7.4) was also measured and used for correction. The experimental procedure performed was as previously described by Xu et al. (2019).