مقالات پذیرفته شده در نهمین کنگره بین المللی زیست پزشکی
Evaluation of Biological Effects of RGD Peptide Labeled with Indium-113 m Radionuclide on Cancer Cells Expressing αvβ3 Integrin
Evaluation of Biological Effects of RGD Peptide Labeled with Indium-113 m Radionuclide on Cancer Cells Expressing αvβ3 Integrin
Fatemeh Badipa,1Safar Farajnia,2Behrouz Alirezapuor,3,*Elham Kamalkazemi,4
1. Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran 2. Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 3. Radiation Application Research School, Nuclear Science and Technology Research Institute, Tehran, Iran 4. Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
Introduction: The phenomenon of angiogenesis, as a key factor in the growth and spread of cancer cells, involves molecular interactions between the components of the extracellular matrix and vascular cells. Among the various integrins expressed on the surface of cells, the αvβ3 Integrin exhibits a distinct expression pattern during angiogenesis. Integrin αvβ3 is a critical receptor that regulates tumor growth, invasion, metastasis, and angiogenesis. In this study, our goal is to label the RGD dimer with Indium-113 m. This reaction has been investigated from the perspective of various parameters, including pH and temperature, as well as different concentrations of peptide and radionuclide. The optimal conditions for these parameters have been determined during the research. To verify the correctness of the labeled compound's performance, various tests were conducted, including quality control using a radiochemical purity test, stability studies in phosphate buffer, saline buffer, and human serum, as well as a biodistribution test.
Methods: For the In113m labeling, 20 nmol of DOTA-RGDFK peptide was dissolved in 20 μL of ultrapure water and incubated with four mCi (148 MBq) of In113m for 15 min at 95°C, pH 3.5 (pH was adjusted with 2.5 M sodium acetate). After 15 minutes, the reaction was stopped by adding 8 mL of deionized water. For further purification, the product was passed through a C18 cartridge (previously activated with 5 mL of ethanol/ 5ml WFI water).
In 113 m, analytical RTLC was then used to purify the DOTA-RGDFK product, and the radioactive peak containing the desired product was collected. After removal of the solvent by rotary evaporation, the activity was then reconstituted in PBS and passed through a 0.22-μm Millipore filter into a sterile multidose vial for in vitro and in vivo experiments. The labeling was done with 100% decay-corrected yield.
Results: In the quality control department, using RTLC paper chromatography, the percentage of radiochemical purity was determined to be 100% in the stability studies section, which was performed in PBS buffer and human blood serum at 4-time intervals (0, 60, 90, and 120 minutes). In general, the stability of the labeled peptide in PBS buffer over time is higher than in human serum. However, overall, the stability of both compounds has decreased over time, with 96% stability in PBS buffer and 94% in human serum after 120 minutes. The Immunoreactivity of the radiolabeled In-113 m –DOTA-RGDFK towards the U87-MG cell line (positive cell line for expression of αvβ3 receptors) and the CHO cell line (negative cell line for expression of αvβ3 receptors) was determined by using the Lindmo assay protocol. Under these conditions, the Immunoreactivity of the radioimmunoconjugate was found to be 82% for U87-MG and 9% for CHO. In the biodistribution test, four normal Balb/c mice were studied. 50 mCi of the labeled peptide was injected into all the mice through a vein, and at time intervals (15, 30, 60, and 120) minutes after the injection, each of the mice was killed separately, and first, they were imaged and dissected in all of them. The absorption rate of each organ is studied in terms of ID per gram percent (%).
Conclusion: In 113 m –DOTA-RGDFK, a potential compound for molecular imaging of SPECT for the diagnosis of cancer cells that express αvβ3 receptors.