纤维连接蛋白亚型胞外结构域特异性探针^(18)F-AlF-NOTA-PEG_(4)-ZD2的制备及microPET显像

Preparation and microPET imaging of extradomain-B fibronectin specific probe 18F-AlF-NOTA-PEG4-ZD2

目的制备针对纤维连接蛋白亚型胞外结构域B(EDB-FN)的特异性分子探针18F-AlF-1,4,7-三氮杂环壬烷-1,4,7-三乙酸-(聚乙二醇)4-ZD2(18F-AlF-NOTA-PEG4-ZD2),并进行体内外理化性质评价。方法通过Al18F一步螯合标记的方法制备18F-AlF-NOTA-PEG4-ZD2,通过高效液相色谱(HPLC)测定其放化纯和体外稳定性,测定脂水分配系数(logP),并行细胞摄取实验[将三阴性乳腺癌MDA-MB-231细胞(1×106/管)分为3组(各3管),分别为阳性组、抑制组和控制对照组]。取MDA-MB-231荷瘤裸鼠(n=3;实验组)行18F-AlF-NOTA-PEG4-ZD2 microPET显像(30、60、90和120 min),并与阻断组(n=3;注射NOTA-PEG4-ZD2后0.5 h再注射18F-AlF-NOTA-PEG4-ZD2)进行对照。采用两独立样本t检验分析数据。结果成功制备18F-AlF-NOTA-PEG4-ZD2,优化后的放化产率为(33.8±2.1)%(未行衰变校正,n=8),放化纯>96%;37 ℃放置120 min,18F-AlF-NOTA-PEG4-ZD2在人血清和PBS中的放化纯均>93%,体外稳定性好;产物比活度为(11.1±3.2) GBq/μmol;logP为-1.43±0.05。注射18F-AlF-NOTA-PEG4-ZD2后120 min,阳性组肿瘤细胞摄取为(1.77±0.28)百分加样活度(%AR)/106个细胞,抑制组细胞摄取为(0.76±0.07)%AR/106个细胞(t=4.30,P=0.032)。荷瘤裸鼠microPET显像示,18F-AlF-NOTA-PEG4-ZD2主要经肝肾代谢,注射后60 min实验组肿瘤摄取为(1.94±0.21)每克组织百分注射剂量率(%ID/g),注射后90 min肿瘤/肌肉比值为3.80±0.25;阻断组注射后60 min肿瘤摄取为(0.43±0.09) %ID/g(t=3.18,P=0.006)。结论 18F-AlF-NOTA-PEG4-ZD2制备简单、标记率高、体外稳定性好,microPET显像肿瘤摄取和靶本比高,特异性良好,有较长的肿瘤滞留时间,在EDB-FN高表达的三阴性乳腺癌中具有良好的应用前景。

Objective To prepare specific molecular probe 18F-AlF-1,4,7-triazacylononane-1,4,7-triacetic acid-(polyethylene glycol)4-ZD2(18F-AlF-NOTA-PEG4-ZD2)for targeting extradomain-B fibronectin(EDB-FN),and evaluate its properties in vitro and in vivo.Methods 18F-AlF-NOTA-PEG4-ZD2 was prepared by one-step chelation labeling with Al18F.The radiochemical purity and in vitro stability were determined by high performance liquid chromatography(HPLC).The partition coefficient(logP)of 18F-AlF-NOTA-PEG4-ZD2 was evaluated,and the cell uptake experiment was carried out(triple-negative breast cancer(MDA-MB-231)cells(1×106/tube)were divided into 3 groups(n=3 per group);positive group,inhibition group,control group).MicroPET imaging was performed on MDA-MB-231 bearing nude mice(n=3)after 18F-AlF-NOTA-PEG4-ZD2 injection(30,60,90,120 min)and compared with blocking group(n=3,NOTA-PEG4-ZQ2 was preinjected at 0.5 h before 18F-AIF-NOTA-PEGa-ZD2 injection).Independent-sample t test was used to analyze the data.Results 18F-AlF-NOTA-PEG4-ZD2 was successfully prepared.The optimized radiochemical yield was(33.8±2.1)%(undecay corrected,n=8)and the radiochemical purity was>96%.After incubating 120 min at 37℃,the radiochemical purity of 18F-AlF-NOTA-PEG4-ZD2 in human serum and PBS was>93%,indicating its good stability in vitro.The specific activity was(11.1±3.2)GBq/μmol,and logP was-1.43±0.05.The uptake value of tumor cells was(1.77±0.28)percentage applied activity(%AR)/106 cells at 120 min post-injection in positive group,and the total uptake value of the inhibition group was(0.76±0.07)%AR/106 cells(t=4.30,P=0.032).MicroPET imaging in tumor bearing nude mice showed that 18F-AlF-NOTA-PEG4-ZD2 was mainly metabolized by the liver and kidneys.The tumor uptake value was(1.94±0.21)percentage activity of injection dose per gram of tissue(%ID/g)at 60 min post-injection and the tumor/muscle ratio was 3.80±0.25 at 90 min post-injection in the experimental group,while the tumor uptake value of tumor bearing nude mice in the blocking group was(0.43±0.09)%ID/g at 60 min post-injection(t=3.18,P=0.006).Conclusions 18F-AlF-NOTA-PEG4-ZD2 can be prepared simply with high labeling rate and good stability in vitro,with high tumor uptake and tumor/muscle ratio in microPET imaging,and good specificity and long tumor residence time.The probe has good application prospect in breast cancer with high expression of fibronectin subtype EDB-FN.