In vitro bioactivity evaluation of synthesized 18F-FDG grayanotoxin-gadolinium-nanoparticles

In this study, Grayanotoxin III (GTX-III) compounds which can be found naturally in Rhododendron ponticum L. (purple coloured forest rose plant, which belongs to Ericaceae family) and ''mad honey'', were extracted from the leaves and flowers of the forest rose and mad honey. The extracted compound then conjugated with Gadolinium nanoparticles (GdO@Si-NP). Synthesized Gadolinium nanoparticles were conjugated and radiolabelled with 2deoksi-2-(F-18) floro-D-glukoz (18F-FDG). Afterwards, bioactivity of the conjugation product was investigated by applying to Breast Adenokarsinom (MCF7) and Prohormone Konvertaz 3 (PC3) cell lines and evaluated together with the MR image contrasts. Grayanotoxin-III compounds were romeved from dried leaves and flowers of the forest rose extracted with methanol and crystalized with etil acetate and then purified by High Performance Liquid Chromatography (HPLC). However, mad honey was mixed with methanol and, the extract was passed through solide phase extraction (SPE) cartridge for seperation of the compounds and then the extract was purified by HPLC. Grayanotoxin-III (GTX-III) compound was identified with Liquid Chromatography – Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) spectroscopy method. Gadolinium nanoparticles (Gd-NP) were synthesized by co-precipitation method and silicated. Size of the nanoparticles was measured by using Dynamic Light Scattering (DLS) and characterization of the nanoparticles was defined by using Scanning Electron Microscopy (SEM). By using 1,1'carbonyldiimidazole/N-Hydroxysuccinimide (CDI/NHS) conjugation method, synthesized gadolinium nanoparticles (GdO@Si-NP) were conjugated with GTX-III-Yaprak (GTX-III-Y) GTX-III-Çiçek (GTX-III-Ç) ve GTX-III-Bal (GTX-III-B) compounds. The yield of the conjugation process was determined with HPLC analysis and found to be100 %. Bioactivity of the conjugated nanoparticles (GTX-III-Y-GdO@Si-NP, GTX-III-Ç-GdO@Si-NP ve GTX-III-B-GdO@Si-NP) was investigated by carrying out cytotoxicity and incorporation tests on PC3 and MCF7 cell lines. Cytotoxicity tests were done with 0.25-0.5-1-5-10-25 μg/mL substance concentrations, after 24 for hours of observation in the incubation process, IC50 values were determined. In the incorporation studies, 0.2 mL of the substance concentrated with 10 μg/mL were added into the 96 well plate which was having PC3 and MCF7 cells. The cells were observed for the 1st, 4th, 8th and 24th hours and incorporation values were evaluated by using contrast values from MR image of the PC3 and MCF-7 cell cultures. Conjugation of 18F-FDG with GTX-III-Y-GdO@Si-NP, GTX-III-Ç-GdO@Si-NP and GTX-III-B-GdO@Si-NP, were caryyied out with CDI/NHS method and yield of the conjugation process was determined by Thin Layer Radiochromatography (TLRC) and HPLC analysis. In conclusion, GTX-III compounds extracted from forest rose and mad honey, were found to be cytotoxic to MCF7 and PC3 cell lines. However, GdO@Si-NP decreased the cytotoxicity to MCF7 while increasing PC3 cells. It was shown that incorporation studies can be carried out by using Gadolinium NP contrasts of MCF7 and PC3 cell lines, that was determined MR image technique. CDI/NHS method may be used for conjugation of GTX-III-GdO@Si-NP with 18F-FDG . As a result of the study, the synthesized 18F-FDG-GTX-III-GdO@Si-NP compounds have the potential of being used as theranostic agents.