2.5. Establishment of orthotopic osteosarcoma model

4-week-old female BALB/c nude mice were purchased from the Charles River Laboratories (Beijing, China). All animals were kept following the Guide for the Care and Use of Laboratory Animals, and all experiments about the animals were permitted by the Animal Care and Use Committee of Jilin University. To establish an orthotopic osteosarcoma model, 20.0 μL of cell suspension containing 2.0 × 10⁶ 143B cells was injected into the right tibial bone marrow cavity of mouse. The first treatment was implemented when the volume of the tumor region was approaching 200 mm³. Free DOX, NP/DOX, or ^CaNP/DOX were injected by tail vein at consistently DOX dose (5.0 mg (kg BW)⁻¹) on day 0, 4, 8, and 12. The control group was treated with PBS. The growth of osteosarcoma was recorded until four days after the last treatment. The volume of the orthotopic osteosarcoma region was calculated concerning Equation (1).

AP represented the value measured at the apex of the knee joint, and L represented the value measured along the longitudinal axis of the tibia.