Drug loading efficiency and in vitro drug release

To determine the drug loading content (DLC) and encapsulation efficiency (EE), the optical spectra of supernatants collected after each washing step were measured using a Shimadzu UV-1800 spectrophotometer. The amount of ICG and DOX in the supernatants were determined according to the corresponding calibration curves (absorbance intensity vs. concentration) at 488 nm and 780 nm, respectively. Then, the DLC and EE for both drugs were calculated according to formulas (4) and (5), respectively.

where W_{Fed drug} is the initial amount of fed drug, W_{Drug in supernatant} is the amount of drug in the supernatants after centrifugation, and W_{SMID NPs} is the amount of as-synthesized SMID nanoparticles.

Drug release from SMID nanoparticles was evaluated under different pH or H₂O₂ conditions based on a traditional dialysis method in vitro ⁶⁹. Specifically, 2 mL of SMID nanoparticle dispersion (1.5 mg·mL^-1) was loaded into each dialysis bag (MWCO = 3500) and then submerged into 78 mL of 1×PBS as releasing medium under different pH conditions (pH = 7.4, 6.8 or 5.0). H₂O₂ concentration was set as 100 μM to reflect the condition of tumor microenvironment in the H₂O₂ positive groups. The setup was placed in a shaker at a constant temperature of 37 °C or 43 °C under the dark condition. To monitor the release kinetics, 3 mL of releasing medium was sampled at predesigned time points, and fresh medium of equivalent volume was supplemented to the releasing system. To analyze the photothermal response of drug release, the dialysis bag was exposed to an NIR laser (808 nm, 2 W·cm^-2) for several on/off cycles (5 min of irradiation per cycle). The amount of released drug was quantified according to a calibration curve measured by florescence spectroscopy (λ_ex: 488 nm, λ_em: 560 nm).