Female BALB/c mice (age: 8 weeks, weight: 20−22 g) were purchased from the Army Medical University Animal Center (Chongqing, China) under protocols approved by the Animal Ethics Committee of the Army Medical University. The study design and all animal experimental procedures were performed in accordance with the Guide and Use of Laboratory Animals (Institute of Laboratory Animal Resources), and the IACUC for Medical Research at Army Medical University. Tumor-bearing mice were prepared by subcutaneously injecting a suspension of 12×104 4T1 cells in PBS (100 μL) into the right lateral hind limbs of female mice.

When the tumor size reached 2.5×2.5 mm, 30 μL of PHSA-ICG-TAT (equivalent to 15 μM of ICG) was injected into the tail vein of the mice for 24 h. Subsequently, in vivo fluorescence imaging was performed. The heart, liver, spleen, lung, and kidney tissues and tumor tissues of mice were dissected for ex vivo fluorescence imaging. The fluorescence was recorded using the Maestro all-optical imaging system (Caliper Life Sciences, Hopkinton, MA, USA).

When tumor sizes reached 2×2 mm, 60 μL of PHSA-ICG and PHSA-ICG-TAT (corresponding to 30 μM ICG) or 30 μM ICG was intratumorally injected into the tail veins of mice. After 24 h, the tumor of each mouse was exposed to a light source at a wavelength of 808 nm for 5 min (power density: 1 W/cm2; irradiation power: 2.5 W; irradiation area: 2.5 cm2). Images were captured to record changes in tumor size. Tumor sizes were measured using a caliper every day after the treatment. Tumor volumes (V) were calculated using the following equation: V = X × Y2/2, where X and Y are the longer and shorter diameters (mm) of the tumor, respectively. Seven days after laser irradiation, tumor tissues of mice were collected for the pathological observation of H&E staining.

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