HSA (50 mg) was accurately weighed, dissolved in 9 mL of pure water, and stirred magnetically in a 20 mL reaction flask. Approximately 20–40 mg of EDC and NHS were added into the reaction flask, and the duration of the activation reaction was 15 min. Next, NH2-PEG4000-NH2 (150 mg) and PBS (1 mL) were added to the reaction flask for 24 h. Following reaction, the material was placed in ultrapure water for dialysis and purification to obtain PHSA and stored in a refrigerator at 4°C.

After adding 2 mL of PHSA liquid and 10 μL of DDT to the reaction flask, the reaction was stirred for 15 min at 25°C. Subsequently, 30 μL of ICG fluorescent dye was added, and the reaction was stirred for 12 h in the dark at 25°C. After the reaction, the pH of the purified water was adjusted to 7.0–7.5 to be used as dialysis solution with PBS, and the solution in the reaction bottle was dialyzed against the light for 24 h in the adjusted pH aqueous solution. The dialysis-purified PHSA-ICG was placed in a refrigerator at 4°C for subsequent use.

After adding 1 mL of 2.5 mg/mL TAT into the reaction bottle, 5 mg of EDC and NHS were added, and the solution was stirred at 25°C for 10 min. At the same time, 1 mL of PHSA-ICG solution and 200 μL of Tris-HCl (pH 8.8) solution were added to a new reaction bottle, and the mixture was stirred for 15 min in the dark. Subsequently, 1 mL of the activated TAT solution was added to the PHSA-ICG solution and the reaction was continued for 12 h in the dark. After the reaction, the product was dialyzed in ultrapure water for 8 h to obtain purified PHSA-ICG-TAT and stored in a refrigerator at 4°C.

The morphology of the nanoparticles was characterized using a transmission electron microscope (TEM; Tecnai G2 F20 S-TWIN TEM) with negative electron staining of the phosphotungstic acid protein. Furthermore, the size of the nanoparticles was tested by dynamic light scattering using a Brookhaven Zeta PALS analyzer. The ICG concentration of the nanomaterials was determined by the ultraviolet absorption method. Prior to the tests, different ICG standard concentration working solutions were prepared (0, 2.5, 5, 7.5, and 10 μM) to construct a standard curve and measure the content of ICG in PHSA-ICG-TAT and PHSA-ICG. In addition, 5 μM of ICG, PHSA, PHSA-ICG, and PHSA-ICG-TAT were dissolved in 50% ethanol for 200–900 nm ultraviolet spectrum analysis.

For the detection of dispersion stability, 10 μM PHSA-ICG-TAT and PHSA-ICG were added to 1 mL of PBS, RPMI 1640 medium, and FBS. After mixing, the solution was placed at 25°C in the dark for 24 h, centrifuged at 7,000 rpm for 10 min, and inspected for the presence of any precipitation.

The photodynamic property of PHSA-ICG-TAT was evaluated by determining the generation of 1O2 according to the fluorescence signal of Singlet Oxygen Sensor Green (SOSG). SOSG was added to different solutions of ICG (5 μM), PHSA-ICG-TAT, and PHSA-ICG (PHSA-ICG and PHSA-ICG-TAT containing ICG equivalent to 5 μM) in PBS and irradiated with an 808 nm laser (power density: 2.0 W/cm2; irradiation power: 5 W; irradiation area: 2.5 cm2; 5 min). SOSG fluorescence was excited with a light source at a wavelength of 494 nm to a maximum of 525 nm following irradiation. The level of SOSG in the samples was evaluated by comparing the enhanced SOSG fluorescence measurement to that obtained for the control sample or background.

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