Subcutaneous transplantation cancer model in BALB/c nude mice and experiment procedures

A total of 16 BALB/c nude mice (female, weighed 13–16 g) were used in the experiment, and were separated into four groups randomly. 1 × 10⁷ A2780 tumor cells suspended in PBS (100 μL)were subcutaneously inoculated into the right flank of every nude mouse. When the tumor volume was about 100 mm³ (about 2 weeks), grouping treatments was performed as follows: (1) Control; (2) FeNx@GOx@M; (3) UTMD; (4) FeNx@GOx@M + UTMD. 100 μL nanoparticles (1 mg/mL) or SonoVue solution were injected intratumorally according to the grouping. For the control group, 0.9% saline was injected intratumorally instead. The detailed procedures of different groups were shown in Table ​Table1.1. According to the previous study [48], the in vivo experimental ultrasonic irradiation conditions were set as: frequency 1 MHz, sound intensity 1 W/cm², duty cycle 20%, irradiation time 3 min. The day of the first treatment was recorded as day 0, and the treatment was repeated every other day for 7 consecutive times. The mice were housed in SPF-level animal laboratories. All of the animal procedures were performed in compliance with the guidelines of the Animal Research Committee of Sichuan University.

Experimental procedures in vivo

Weight of mice was recorded and tumor size was measured by portable ultrasound before each treatment. After finishing the last treatment, the mice were sacrificed, the tumors were removed and then tumors’ weight were measured. HE staining, Ki-67 immunohistochemical (IHC) staining and apoptosis testing were performed on the tumor tissues. Meanwhile, the main organs of mice, including heart, liver, spleen, lung and kidney, were taken out for HE staining.