1.1 Materials and Animals
RPMI-1640 medium (SERVICEBIO), FBS(EVERY GREEN),TEFAPI-06 precursor༈Boomray Pharmaceuticals Co., Ltd.༉,sodium acetate༈Sigma-Aldrich༉,177LuCl3(Chengdu Syncor Pharmaceutical Co.,Ltd), HCl༈Sigma-Aldrich༉,Sep-Pak Light C18 cartridge(Waters),silica gel(Merck), Radio-Thin-Layer Chromatography (RTLC) ( Scan-RAM, LabLogic), γ-counter( Beijing Nuclear Instrument Factory), live small animal imaging device (In Vivo-Xtreme, Bruker), 5-part differential hematology analyzer(BC-5000vet, Mindray), veterinary V-52D dilution(5.5 L*2, Mindray), Veterinary V-52 DIFF hemolytic(300 mL, Mindray), Veterinary V-52LH hemolytic(90mL, Mindray), veterinary V-52D dilution(20L, Mindray), Veterinary probe washing solution(50mL, Mindray), Automatic biochemical analyzer(Chemray 240, Rayto), Microplate Reader(Epoch, BioTeK)
Female BALB/c mice (6–8 weeks of age,20 ± 2g) and Specific-pathogen-free (SPF) grade Sprague-Dawley (SD) rats(200 ± 20g) were purchased from the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences
1.2.1 Cell culture and animal models.
The CT26 murine colon carcinoma was cultured in RPMI-1640 medium with 10% FBS at 37°C in a humidified atmosphere containing 5% CO2. To establish the CT26 tumor–bearing mouse models, we inoculated 1×106 tumor cells subcutaneously into the right hind back of BALB/c mice. Female BALB/c mice (6–8 weeks of age,20 ± 2g) and Specific-pathogen-free (SPF) grade Sprague-Dawley (SD) rats(200 ± 20g) were purchased from the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. Randomly assign animals to different groups after numbering them. All animals were housed under a 12-h light/dark cycle in a temperature-controlled animal room and allowed free access to food and tap water. Animal experiments were performed according to the institutional guidelines. The study protocol was approved by the Lanzhou University Second Hospital Committee for Animal Care and Use.
1.2.2 Radiolabeling of TEFAPI-06
100µg of the TEFAPI-06 precursor(MW:1689.38.) was dissolved in 1 ml of 0.25M sodium acetate buffer. Subsequently, 1110MBq 177LuCl3 solution was added. Adjust the pH to 4.0-5.5 using 0.1M HCl, and the reaction was conducted at 100°C for 30 minutes. Post-cooling, the reaction mixture was diluted into 5 mL saline in a syringe and purified by using a pre-conditioned Sep-Pak Light C18 cartridge activated by ethanol (5 mL) and water (5 mL). The final product was eluted into a vial through the C18 column, followed by sterile filtration using a 1 ml ethanol solution. The radiochemical purity and Quality control of 177Lu-TEFAPI-06 were checked with polymer-backed silica gel Thin-Layer Chromatography (TLC) ( Scan-RAM, LabLogic) using a 1 : 1 mixture of 10M ammonium acetate and methanol as mobile phase. The final solution was then passed through a 0.22 µm filter and pH was adjusted to 5–7 by addition of 0.25M sodium acetate buffer.
1.2.3 Biological Distribution Study and Tumor Enrichment
Sprague-Dawley (SD) rats(n = 4)were administered an intravenous injection of 177Lu-TEFAPI-06 at a dose of 22.2MBq per rat for the in vivo biological distribution study. The rats were euthanized at specific time points post-injection (0.5h, 2h, 24h, 48h, 72h, and 144h). In addition, each tumor bearing mouse was euthanized at a specific time point(24h,72h,96h,144h) after intravenous injection of 177Lu TEFAPI-06 at a dose of 3.7MBq. Subsequently, the uptake of radioactivity in various organs was quantified using γ-counter. This measurement facilitated the calculation of the percentage of the injected dose per gram of tissue (% ID/g). In parallel, tumor-bearing mice(n = 3) were injected intravenously of 177Lu-TEFAPI-06 at a dose of 3.7MBq per mouse. Post-injection, tumor imaging was performed with live small animal imaging device ( exposure time:2.0s ). The software that comes with the instrument was used for data processing.
1.2.4 Biotoxicity experiment
Sprague-Dawley (SD) rats were allocated into four groups, each comprising six individuals, with an equal distribution of males and females. The study was designed with three dosage levels: low (0.2 mg/kg), medium (0.6 mg/kg), and high (2 mg/kg). Observational assessments were conducted 5 minutes before and after dosing, and subsequently at 0.5h, 1h, 2h, 4h, and 6h post-dosing. Daily monitoring included morning and afternoon observations, recording dietary intake, activity levels, excretion patterns, fur condition, behavior, and any mortality events. Blood samples were collected from the orbit and heart respectively on the 3rd and 15th day after intravenous injection for routine blood analysis (red blood cell count, hemoglobin concentration, white blood cell count, platelet count) and blood biochemistry (aspartate aminotransferase, alkaline phosphatase, total bilirubin, creatinine, urea, albumin, glucose, total cholesterol, triglycerides), following the guidelines for new drug research. The above blood samples were sent to Wuhan Servicebio Biotechnology Co., Ltd. for testing, and the kit used was provided by the company. Fifteen days post-drug administration, the rats were dissected and organ tissues (heart, liver, spleen, kidney, brain, gonads - testes/ovaries, thymus) were weighed for organ index calculations and some of organs were preserved in 10% neutral buffered formalin. Following fixation, tissue processing was carried out in accordance with standard operating procedures (SOPs), encompassing sample collection, dehydration, embedding, sectioning, and hematoxylin and eosin (HE) staining.