Study On A New Apoptosis Imaging Agent^99mTc-Tat_49-57-DEVD In Vitro And Vivo

ObjectiveThe aim of this study was to explore the preparation method of 99mTc-labeled Caspase-3's substrate peptide analogs,conjugated with hydrazinonictinamide (HYNIC) and to evaluate its biodistribution in normal mice; to study of the uptake about the new probe in human breast carcinoma apoptotic cell in vitro and the apoptotic imaging in tumor bearing nude mice.In this study we investigated the feasibility of 99mTc-labeled Caspase-3's substrate peptide analogs (RKKRRQRRRDEVD, Tat49-57-DEVD) for molecular apoptosis imaging diagnosis on tumor in vivo.MethodsStudy on the labeling condition of 99mTc-HYNIC-Tat49-57-DEVD and its biodistribution test in normal mice:â‘ Caspase-3's substrate peptides was modified by the cell-penetrating peptides HIV-TAT49-57 (named Tat49-57-DEVD) conjugating the bifunctional chelator-HYNIC was synthesized. The peptide was labeled with 99mTc through an indirect method using tiricine/EDDA as co-ligands, then the labeled mixture, Radiochemical was purified through Sep-Pak C18 column. Then the preparation of 99mTc-HYNIC-Tat49₅7-DEVD was completed. The radiolabeling efficiency, radiochemical purity and stability of the 99mTc-HYNIC-Tat49-57-DEVD were assessed.â‘¡Its biodistribution in normal mice were studied. There were five groups of KM mice, and 1μg of radio labeled 99mTc-HYNIC-Tat49-57-DEVD in 100μl eluent was injected into each mouse via a tail vein. At different time point post-injection, Whole blood samples were collected and the mice were sacrificed by cervical dislocation. The myocardium, liver, spleen, lung, kidney, brain, stomach, intestines, muscle, and bone were harvested, rinsed, weighed and counted on a y counter. The percent uptake per gram tissue were calculated.Study of the uptake in carcinoma cell in vitro:MCF-7 cell lines were induced apoptosis by Cyclophosphamide (CTX) in different concent rations,then analyzed by flow cytometry assay with fluorescein isothiocyanate-conjugated Annexin V (FITC-Annexin V) and propidium iodide (PI) staining. Meanwhile,the peptides HYNIC-Tat49-57-DEVD is radiolabele the 99mTc.The cell uptake test were carried out. The correlation of apoptotic rate detected by flow cytomet ry and cell uptake was analyzed. The expression of Caspase-3 proteins was detected with immunohistochemical analysis of apoptotic tumor cell.Study of apoptosis imaging in tumor bearing nude mice:â‘ Balb/c nu/nu nude mice bearing human breast cancer cancer MCF-7 xenografts were studied. The mice were randomized to receive a single dose of CTX (100mg/kg, intraperitoneally).After having received CTX 72 hours,the mice were injected the radiotracers through tail vein.Then static acquisitions were performed at different time point with a gammar camera fitted with a low-energy high-resolution collimator using a 256×256 matrix with a 20% energy window set at 140 keV. The radioactivity ratios of tumor to chest and contralateral limbs were calculated using ROI technique.â‘¡Five tumor model mice were sacrificed 90min after radiotracer injection, tumors and contralateral limbs were harvested, rinsed, weighed and counted on aγcounter. The study of immunohistochemical analysis of tumor histological paraffin section:Caspase-3 expression was examined immunohistochemically by using SP method. The expression of Caspase-3 proteins was detected with immunohistochemical analysis of apoptotic tumor cell by using SP method.The Caspase-3 polyclonal antibody of negative control group was instead by PBS.ResultsStudy of the labeling condition of 99mTc-HYNIC-Tat49-57-DEVD and its biodistribution test on normal mice:The radiochemical purity of the 99mTc-HYNIC-Tat49-57-DEVD was 91%. After incubation with fresh human serum for 30min and 120min, the radiochemical purities were 91.5% and 90.8%, and analyzed by Sep-Pak C18 column, the radioactivity peak showed no shift to early or late fractions. The reaction mixture was incubated at room temperature for 5h, and the radiochemical purity was 90%. The biodistribution in normal mice showed it quickly removed from blood and excreted mainly through kidney and liver.Study of the uptake in carcinoma cell in vitro:CTX at different concent rations can induce cell apoptosis in a dose-dependent manner, internalization ration of 99mTc-HYNIC-Tat49-57-DEVD to apoptotic cells isa high.The uptake rate of radiolabeled activity linearly correlated to total fluorescence as observed by FITC-Annexinâ…¤flow cytometry assay (r=0.856,P< 0.01). The expression of Caspase-3 protein in apoptotic tumor cells were seen in the endochylema of apoptotic tumor cells.Study of apoptotic imaging in tumor bearing nude mice:Imaging of tumor bearing nude mice:Tumor lesions were clearly visible 90 min after injection and the ratios of tumor/contralateral limbs were 2.23±0.13 respectively. Biodistribution of tumor bearing nude mice:the ratios of tumor/contralateral limbs were 3.29±0.82. The expression of Caspase-3 were mainly seen in the endochylema of tumor cells, and there were brown granulocorpuscle or diffuse distribution. But in negative-control group that using Sodium Chloride instead, the endochylema of tumor cells did not observe staining.ConclusionsThe labeling method of Tat49-57-DEVD conjugated by HYNIC has been proved successfully. It was an optimal method with simple steps and high radiolabeling efficiency and well stability. Radiochemical was mainly excreted through liver and kidney. The study of imaging and biodistribution in vivo showed that 99mTc-HYNIC-Tat49-57-DEVD remains fine biological activity, and it is well worth researching as a promising Caspase-3's substrate peptides radiopharmaceutical with radionuclide labeled, which provided a basis for the further radionuclide apoptosis imaging study.