靶向TSPO显像剂99Tcm-DTPA-CB86的制备及其对关节炎症的SPECT/CT显像研究

目的制备99Tcm标记的转运蛋白（TSPO）配体CB86[99Tcm-DTPA（二亚乙基三胺五乙酸）-CB86]，探讨其作为TSPO靶向关节炎症显像新型分子探针的可行性。方法通过偶联双功能螯合剂制备DTPA-CB86，进行99Tcm标记，经高效液相色谱纯化，测定其放化纯度和体外稳定性。选用巨噬细胞RAW264.7进行体外细胞结合实验，测定99Tcm-DTPA-CB86的结合率和外排率。采用弗氏佐剂建立左踝关节炎症小鼠模型，对其行99Tcm-DTPA-CB86 Micro SPECT/CT显像，并观察探针的体内分布情况。采用SPSS 18.0统计软件对符合正态分布及方差齐性的数据进行t检验。结果99Tcm-DTPA-CB86的标记率为（95.86 ±2.45）%，放化纯度为（97.45 ±0.69）%，其在室温下的磷酸盐缓冲液中的稳定性良好，放置4 h后，其标记率仍>90%。99Tcm-DTPA-CB86能与巨噬细胞RAW264.7特异性结合，3 h的摄取率达到最高峰[（36.45 ±2.18）%]，在加入过量未标记的DTPA-CB86后，RAW264.7细胞对99Tcm-DTPA-CB86的摄取明显受到抑制[（10.43 ±2.01）%]，与未阻断时相比差异具有统计学意义（t=6.217，P < 0.05）；RAW264.7细胞对99Tcm-DTPA-CB86外排较少，摄取率从4.5 h时的（33.31 ±2.34）%到8 h时的（19.32 ±2.01）%，减少了13.99%。Micro SPECT/CT显像结果显示，99Tcm-DTPA-CB86在小鼠左踝关节炎症部位清晰可见，且能被过量的DTPA-CB86明显抑制；体内生物学分布结果表明，其具有较好的炎症靶向性；注射后3 h踝关节炎症部位的摄取仍可达（2.35 ±0.10）% ID/g。结论99Tcm标记CB86易于制备，具有较好的理化性质及体内代谢学性质，同时具有较好的关节炎症摄取，有望发展成新的TSPO靶向SPECT关节炎症显像分子探针。

Preparation and imaging of arthritis of 99Tcm-DTPA-CB86 for TSPO targeted imaging

ObjectiveTo develope a novel radiolabeled translocator protein(TSPO) ligand CB86 targeting agent 99Tcm-diethylene-triaminepentaacetic acid (DTPA)-CB86 and evaluate its biological properties.MethodsDPTA-CB86 was prepared by coupling with a bifunctional chelating agent, and then labeled with 99Tcm to obtain 99Tcm-DTPA-CB86. The labeling efficiency, radiochemical purity, and stability were determined in vitro. In vitro cellular uptake and efflux were performed using RAW264.7 macrophage cells. Biodistribution and micro-SPECT/CT images were investigated on Freund's adjuvant-induced left arthritis in mice. SPSS 18.0 analysis software(t-test) was used to fit the normal distribution and homogeneity of variance.ResultsThe labeling yields and radiochemical purity of 99Tcm-DTPA-CB86 were (95.86±2.45)% and (97.45±0.69)%, respectively. 99Tcm-DTPA-CB86 displayed good stability, with a radiochemical purity of more than 90%, in phosphate-buffered solution(PBS) at 4 h. It also exhibited high specific TSPO binging in RAW264.7 macrophage cells in vitro. The highest uptake ratio was (36.45±2.18)% at 3 h after incubation, which then decreased significantly[(10.43 ±2.01)%; t=6.217, P < 0.05)] after adding excessive unlabeled DTPA-CB86. The difference was significant. Cell efflux analysis showed that 99Tcm-DTPA-CB86 had good cell retention by RAW264.7 cells, with only about 13.99%[decreased from (33.31±2.34)% to (19.32±2.01)% of total input radioactivity] of 99Tcm-DTPA-CB86 efflux observed during 4.5 h to 8 h of incubation. Biodistribution and SPECT/CT imaging demonstrated that the uptake of 99Tcm-DTPA-CB86 in the left arthritic ankles was significantly increased compared with that in contralateral normal ankles. Uptake in the arthritic ankles could be largely blocked by an excess of DTPA-CB86.Conclusion99Tcm-DTPA-CB86 can be readily synthesized and clearly visualize arthritis with low background, thus demonstrating its potential as a promising molecular probe targeting TSPO for arthritic SPECT imaging.