Tumor xenograft mouse studies

Five- to six-week-old Balb/c female nude mice were subcutaneously administered approximately 1 × 10⁷ SJSA-1 cells (overexpressing luciferase) into the right hind leg to establish xenografted tumors. To test the anti-osteosarcoma stem cell activity of NP_J4+Apa, 1 × 10⁷ SJSA-1 spheroid-derived cells were subcutaneously injected into the right hind leg to establish osteosarcoma stem cell-derived xenografted tumors. Different formulations were intravenously administrated once every other day. The animal weights and tumor volumes were measured every day. Tumor size was calculated as the tumor volume = (tumor length) × (tumor width)²/2. For in vivo fluorescence imaging, the mice were injected with d-luciferin and anesthetized. The fluorescence signals were measured using the Xenogen IVIS Spectrum (Caliper Life Sciences, USA). After the mice were sacrificed, the tumor tissues were fixed and stained with hematoxylin and eosin (H&E) for histopathological evaluation.

For in vivo fluorescence imaging of DiR, 100 μL saline, 10 μg near infrared dye (DiR) in 100 μL saline, or 10 μg DiR-loaded NPs (NP_DiR) in 100 μL saline was intravenously injected into tumor-bearing nude mice. The mice were anesthetized, and fluorescence signals were measured using the Xenogen IVIS Spectrum at 12 and 24 h post-injection. Three mice were used in each group. Then, fluorescence images of the whole bodies and tumors dissected from nude mice were individually taken as above.