Evaluation of Smad4 mRNA Delivery in Colorectal Cancer

All experiments of animal were in accordance with the guidelines on the use and care of laboratory animals for biomedical research published by the National Institutes of Health, and approved by the Ethics Committee of Shanghai University (No. 2022-238). Female BALB/c nude mice (purchased from Shanghai SLAC Laboratory Animal Company, Shanghai, China), 4–5 weeks, housed in a barrier facility on a 12 h light/dark cycle at 22–24 °C and 45–55% humidity. The maximal tumor size is limited to 2 cm in length and the maximal tumor burden was not exceeded in all the animal experiments. For the subcutaneous tumor model, each nude mouse was subcutaneously inoculated with 2 × 10⁶ SW480 cells under aseptic conditions. After 3 weeks of inoculation, the nude mice were divided into three groups randomly and injected with saline, naked Smad4 mRNA, and nano-lantern, respectively. For the orthotopic xenograft tumor model, BALB/c nude female mice were anesthetized through isoflurane. 2 × 10⁶ SW480-Luc cells were mixed with matrigel (1:1, v/v) and then were inoculated into the cecal wall with an insulin gauge syringe through a midline incision. IVIS system was adopted to monitor the tumor growth via tail vein injection of D-luciferin (10 mg/mL). After the luminescence intensity reach about 1.0 × 10⁹, interventions started followed by intraperitoneal injection of saline, naked Smad4 mRNA, and nano-lantern, respectively. Mouse body weight and tumor volumes (smaller diameter² × larger diameter × 0.5) were measured twice a week. After the tumor was harvested, tumor tissues and organs, including the heart, liver, spleen, lung, and kidney, were fixed in formaldehyde solution for H&E staining. Proteins were extracted from the tumor tissues for Western blot analysis.