Genistein and SnCl2·2H2O were purchased from Sigma-Aldrich, and the solvents used were purchased from Merck.
Instant thin layer chromatography‑silica gel (ITLC‑SG) (Agilent, Technologies), dose calibrator (Victoreen), Single Channel Analyzer (ORTEC). The Technetium-99m radioisotope was obtained from the Enviro Technetium-99m Generator, manufactured by Enviro Korea Co., Ltd.
Method for preparation of 99mTc genistein kit. The preparation of 99mTc genistein compound was carried out by optimizing the formulation of all parameters: genistein solution as ligand, SnCl2.H2O solution as reducing agent, pH value, and incubation time. In addition, sodium pertechnetate solution [99mTc] with an activity 7.4 - 17 MBq was added in the process of forming 99mTc genistein. The radiochemical purity (RCP) of the complex formation is monitored by TLC and must fulfill the USP requirement of > 90% 14-15.
Thin‑layer chromatography (TLC) procedure. Determination of the radiochemical purity of 99mTc genistein from impurities 99mTcO2 and 99mTcO4- using the TLC method. This quality control method is simple and practical to use. The stationary phase used is TLC SGF 254 with a mobile phase of NaCl solution. This TLC system aims to separate TcO4- impurities, where TcO4- will migrate and 99mTc genistein compound will remain at the starting point.
The separation of 99mTcO2 was carried out with the stationary phase ITLC‑SG and the mobile phase a mixture of C2H5OH : H2O : NH4OH (2:5:1). As a result, the impurity of 99mTcO2 will migrate carried away by the mobile phase onto the TLC plate, and the 99mTc genistein compound will remain at the starting point. The strips were measured for their radioactive activity using SCA (Single Channel Analyzer), and the calculation of RCP of 99mTc genistein is based on the equation:
% 99mTc-Genistein = 100 % - (% 99mTcO2 + % 99mTcO4-) 16
Stability of 99mTc-genistein. The in vitro stability of the 99mTc-genistein kit was determined at room temperature. Measurements will be observed the percentage of radiochemical purity by TLC every 1 hour for 5 hours of observation time and its physical appearance.
Electric charge measurement. Determination of the charge of the 99mTc genistein kit was carried out by electrophoresis method using Whatman no.1 paper (GE Healthcare). Whatman paper (37cm x 1cm) is marked every 1 cm apart, with 0 in the middle. On the right, the scale of 18 is written for positive values, and the anode is placed, while the scale is 18 on the left for negative values and the cathode is placed. At the zero point, the 99mTc genistein kit is spotted and the paper was immersed in 0.02 N phosphate buffer solution, pH 7.5. The electrophoresis device was supplied with a voltage of 350 Volts, 11-16 mA. After 1 hour, Whatman paper pieces were measured by SCA.
Determination of Lipophilicity. The test was conducted by adding 2 mL of 1-octanol solution and 2 mL of 0.9% NaCl (saline solution) pH 7.4 into centrifuge tubes and adding 10-50 μL of 99mTc-genistein solution. The solution was homogenized by vortexing for 1 minute and centrifuged at 3000 rpm for 10 minutes. Each 100μL of the 1-octanol fraction and saline solution was counted for radioactivity by SCA. The partition coefficient was determined by calculating the radioactivity ratio of 1-octanol and saline solution.
Measurement of plasma protein binding. Determination of plasma protein binding was carried out by precipitation method using TCA solution. A total of 500 μL of blood samples were added to 50 μL of radiopharmaceutical 99mTc genistein and homogenized by vortex for 1 minute. The mixture was incubated for 10 minutes at 37°C, then 1 mL of 0.9% NaCl solution and 1 mL of 5% TCA solution were added. This solution was centrifuged at 3000 rpm for 15 minutes. The precipitate formed is then separated from the supernatant. The supernatant solution was added again with 1 mL of TCA solution, the process of precipitation and separation was repeated. The precipitate fraction was washed again with the addition of 1 mL of 0.9% NaCl solution, centrifuged and separated again. Radioactivity of the precipitate fraction (a) and the supernatant (b) will be measured by SCA and the plasma protein binding value is calculated by the following equation.