Abstract
Objectives: AnnexinV l led either with radionuclide with emission or with fluorescein has been known to be capable as the agent for detecting apoptosis..99mTc by Tc(V)O3+ l led Annexin V via hydrazinonicotin- amide as a bifunctional chelator (99mTc(V)HYNIC-Annexin V )has been reported previously as the apoptosis imaging agent. In this study, the 99mTc(I) tricarbonyl ion, 99mTc(I) (CO)3+ ion, instead of 99mTc(V)O3+, l led HYNIC-Annexin V is developed to image apoptosis. Since [99mTc(CO)3(OH2) 3] +is easily and kinetically fast complexed with HYNIC with 2N atom donors, the novel[99mTc(CO)3(OH2) 3+] l ling to HYNIC-Annexin V may lead to a better apoptosis imaging agent. Methods: The conjugate of AnnexinV coupled with HYNIC and [99mTc(CO)3(OH2) 3] +were prepared respectively according to the previous reports[Blankenberg et al. PNAS 11(1995)6349 and Alberto et al. J Am Chem Soc 120(1998) 7987]. [99mTc(CO)3(OH2) 3] +labeled HYNIC- Annexin V was prepared by mixing the[99mTc(CO)3(OH2) 3] +solution at pH7.4 with HYNIC-Annexin V at 25˚C. The obtained 99mTc(I)HYNIC- Annexin V was further purified by a centrifugal concentrator. Radioanalytical methods were executed including size-exclusion HPLC, TLC and electrophoresis to characterize the 99mTc(I)HYNIC-AnnexinV prepared. Apoptotic cell binding ability was assessed by a competitive binding assay with fluorescein-Annexin V in HepG2 human hepatoma cells undergoing apoptosis in starvation. Balb/c mice were hepatocyte apoptosis induced by administrating antiFas antibody and thereafter were injected with 99mTc(I)HYNIC-Annexin V. At 10, 30, 60, 120 and 180 min postinjection, the mice were sacrificed and the major tissues and organs were excised, rinsed in saline, weighed, and transferred to an auto counter for activity measurement. Results: The prepared 99mTc(I)HYNIC-Annexin V was assayed by a size- exclusion HPLC and found to have a radiochemical purity as high as 90% . 99mTc(I)HYNIC-Annexin V was observed to be stable either in PBS or in serum within 1h but to decrease to a purity at 70% at 4h after preparation. In the biodistribution study, the major uptake and retention of 99mTc(I)HYNIC-Annexin V was found in liver and kidneys, with other organs showing very much lower level of radioactivity. The uptake of 99mTc(I)HYNIC-Annexin V in the liver of the mice bearing with hepatocyte apoptosis was apparently at least two fold higher than the case in the normal mice. Conclusions: [99mTc(CO)3(OH2) 3] +can be efficiently and stably labeled with HYNIC-Annexin V. The effectiveness of the novel prepared 99mTc(I)-HYNIC-Annexin V for imaging apoptosis has been evidenced by in vitro and in vivo experiments in this study .