4.5. HT-29 Xenograft Mouse Model

Animal studies were performed in accordance with the guidelines approved by the Chonnam National University Animal Research Committee (CNU IACUC-H-2020-19). Adult (6-week-old, ≈25 g) male athymic nude mice were purchased from OrientBio (Gwangju, South Korea). HT-29 cancer cells were cultured and suspended in 100 μL of PBS before being subcutaneously inoculated in the right flank of each mouse (1 × 10⁶ cells per mouse). When tumor sizes reached about 1 cm in diameter between 8 to 10 days post-inoculation, the MTX-ZW solution (10 μg/100 μL in PBS; 100 μM) was administered intravenously. Animals were euthanized for in vivo NIR fluorescence imaging within a designated period of time.