Tumor Growth Inhibition Assay

When the tumor volume reached approximately 50 mm³, the mice were randomly divided into 5 groups, i.e., the PBS, ST, ST-AS, ST-Si, and ST-AS&Si groups. Each mouse was injected with 100 μL of nanodrug solution via the tail vein (AS dose, 60 μg/kg body weight; siRNA dose, 250 μg/kg body weight). The width and length of the tumor size were measured using a vernier caliper, and each mouse was weighed. Over a total of seven treatments for each mouse, the injection was performed every 3 days while the tumor size and weight of mice were recorded accordingly. The day of first administration was denoted as Day 1. When the tumor volumes reached 2000 mm³, the mice underwent euthanasia as the humanitarian end point of the study. During the therapeutic process, 100 μL of d-luciferin solution (10 mg mL^–1) was intraperitoneally injected into mice for bioluminescence imaging to monitor the tumor growth and metastasis at 1, 10, and 19 days. At 19 days, the mice receiving d-luciferin solution were sacrificed, and tumors and organs were excised. The metastatic tumors in the liver and lung were observed through bioluminescence imaging in vivo and ex vivo. The excised tumors, livers, and lungs were fixed in 4% paraformaldehyde for at least 24 h, and then, the paraffin sections of 2 μm or frozen section of 5 μm was prepared. The tumor nodules were observed from H&E images of the liver and lung to assess the tumor metastasis in different animal groups.

The antitumor efficacy was further tested in one additional syngeneic orthotopic tumor model. The mice were randomly divided into 4 groups for the treatments of PBS, STscr-AS&Si, NT-AS&Si, and ST-AS&Si. The tumor size, mouse body weight, and cumulative survival were recorded.