| Summary: | 碩士 === 長庚大學 === 醫學影像暨放射科學系 === 99 === Apoptosis is one of the programmed cell death and plays an important role in the regulation of normal tisssue growth, development, and immunity mechanism. Apoptosis is marked by the expression of phosphatidylserine (PS) on the outer layer of apoptotic cell membrane which can be detected by annexin V (ANV), an endogenous protein possessing high binding affinity to PS, labeled with fluorescent dyes or radionuclides.
Tc-99m-HYNIC-ANV was the most widely studied radiolabeled ANV for imaging apoptosis in vivo. However, high renal uptake of the tracer and suboptimal binding affinity of ANV to PS under physiological calcium concentration may limit the clinical use of the Tc-99m-HYNIC-ANV. Another Tc-99m labeled ANV conjugated with L,L-ethylenedicysteine (EC) showed promising characteristics of relatively low renal uptake. An ANV- Kunitz protease inhibitor (KPI) fusion protein, ANV-6L15, was proved to possess better PS binding affinity than ANV. The combination of EC technology and ANV-6L15 may be possible to develop a Tc-99m based radiopharmaceutical superior to Tc-99m-HYNIC-ANV for imaging apoptosis. The aims of this study were: (1) to optimize the conjugation of EC to ANV and ANV-6L15; (2) to optimize the Tc-99m radiochemistry of EC conjugates of ANV and ANV-6L15.
EC was conjugated to ANV and ANV-6L15, respecyively, with conditions of a conjugation time of 2 hours or 24 hours at room temperature, in combination with an EC: protein molar ratio of 20 or 2,000. After purification with dialysis, the EC conjugates of ANV and ANV-6L15, prepared with various conditions, were labeled with fluorescent FITC and Tc-99m, respectively. The identities of labeled EC-potein conjugates were verified by size-exclusion HPLC as well as SDS-PAGE. The radiochemical purity (> 95%) of either radiolabeld protein was determined by instant thin-layer chromatography (ITLC). The binding affinity of each FITC- or Tc-99m-labeled conjugates was characterized on erythrocyte ghosts, served as an apoptotic cell model, at calcium levels of 2.5 mM and 5 mM, respectively.
The results of binding assay revealed that either FITC-EC-ANV or 99mTc-EC-ANV showed similar binding affinity to erythrocyte ghosts whether the EC-protein conjugate was prepared with EC: protein molar ratio of 20 or 2,000, and/or with the conjugation time of 2 hours or 24 hours. The binding ratio of each FITC-EC-protein conjugate was significantly higher than that of corresponding Tc-99m-EC-protein. The binding to erythrocyte ghosts was generally calcium-concentration-dependent among EC-proptein conjugates and their Tc-99m labeled analogs. The difference between binding ratios of Tc-99m-EC-ANV and Tc-99m-EC-ANV-6L15 to erythrocyte ghosts was insignificant.
According to the in vitro binding results, we suggest to use less chelator (EC: ANV = 20) and 2 hours for conjugation of ANV derivatives with EC. Tc-99m-EC-ANV-6L15 did not show its superiority to Tc-99m-EC-ANV on binding to erythrocyte ghosts. Further works were required to improve the binding for development of apoptosis targeting tracers.
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