目的制备一种新型 68Ga标记靶向脊髓灰质炎病毒受体相关蛋白4(PVRL4，又称Nectin－4)双环肽，并对其用于乳腺癌显像的可行性进行评价。
方法合成生物素(Biotin)修饰的靶向Nectin－4双环肽(简写为BMIC)Biotin－BMIC，通过体外细胞染色实验对其靶向性进行初步评价。双环肽BMIC经1，4，7－三氮杂环壬烷－1，4－二乙酸(NODA)修饰制得标记前体NODA－BMIC，经一步法标记制备靶向Nectin－4探针 68Ga－NODA－BMIC。应用荷乳腺癌小鼠活体microPET显像结合体外实验对该探针显像性能进行考察。采用两独立样本 t检验、重复测量方差分析处理数据。
结果细胞荧光染色初步表明，荧光标记的双环肽Biotin－BMIC在Nectin－4阳性BT474乳腺癌细胞上较Nectin－4阴性MDA－MB－231乳腺癌细胞高度聚集。 68Ga－NODA－BMIC未校正产率可达(71.5±2.2)%，放化纯>95%。比活度>3 GBq/μmol。温育10、30、60和120 min后，BT474乳腺癌细胞较MDA－MB－231乳腺癌细胞均有高放射性摄取( F＝1 302.00， P<0.001)。荷瘤鼠 68Ga－NODA－BMIC microPET显像表明，BT474移植瘤较MDA－MB－231移植瘤显影清晰，且对比度良好。注射探针后10、30、60和120 min，BT474与MDA－MB－231移植瘤摄取差异有统计学意义( F＝1 826.00， P<0.001)，其中注射后60 min BT474移植瘤的摄取值为(5.03±0.14)每克组织百分注射剂量率(%ID/g)，显著高于MDA－MB－231移植瘤对应值[(0.19±0.04) %ID/g； t＝79.40， P<0.001]。BT474肿瘤/肌肉(T/M)比值高于MDA－MB－231( F＝222.00， P<0.001)，其中注射后60 min前者T/M比值为24.75±3.10，显著优于后者对应值(1.30±0.15； t＝14.31， P＝0.002)。体内显像结果与离体免疫组织化学分析一致。
结论新型靶向Nectin－4双环肽PET探针 68Ga－NODA－BMIC，合成简便，标记产率和放化纯满意。探针体内显像性能佳，靶组织显影清晰，可能在乳腺癌诊疗中发挥独特作用。
ObjectiveTo prepare a novel 68Ga-labeled bicyclic peptide targeting poliovirus receptor related protein 4 (PVRL4, Nectin-4), and evaluate its feasibility for breast cancer imaging via in vitro and in vivo experiments.
MethodsA Biotin-modified bicyclic peptide targeting Nectin-4, Biotin-BMIC, was synthesized, and its targeting properties were preliminarily evaluated by in vitro cell staining experiments. BMIC was modified by 1, 4, 7-triazonane-1, 4-diacetic acid (NODA) and the labeling precursor NODA-BMIC was prepared. A potential PET probe targeting Nectin-4, 68Ga-NODA-BMIC was prepared by one-step labeling strategy. The imaging properties of the probe were investigated by in vivo microPET imaging and in vitro experiments in mice bearing breast tumors. Data were analyzed by independent-sample t test and repeated measures analysis of variance.
ResultsFluorescence staining of the cells showed that the fluorescently labeled bicyclic peptide, Biotin-BMIC, was highly aggregated in Nectin-4 positive BT474 breast cancer cells compared to those in Nectin-4 negative MDA-MB-231 cells. The uncorrected yield of 68Ga-NODA-BMIC was (71.5±2.2)% and the radiochemical purity was greater than 95%. The specific activity was greater than 3 GBq/μmol. After incubation 10, 30, 60 and 120 min, higher radioactivity uptakes were found in BT474 breast cancer cells compared to those in MDA-MB-231 breast cancer cells respectively ( F=1 302.00, P<0.001). MicroPET imaging showed that the BT474 xenograft tumors were clearly visible with favorable contrast. A significant statistical difference in uptakes between BT474 and MDA-MB-231 xenograft tumor uptake at 10, 30, 60, and 120 min after probe injection respectively was existed ( F=1 826.00, P<0.001). At 60 min postinjection, the uptake value of BT474 tumors was (5.03±0.14) percentage activity of injection dose per gram of tissue (%ID/g), which was significantly higher than that of MDA-MB-231 tumors ((0.19±0.04) %ID/g; t=79.40, P<0.001). Meanwhile, the tumor-to-muscle ratios in the former were also greater than those in the latter ( F=222.00, P<0.001). At 60 min postinjection, the tumor-to-muscle ratio in the former was significantly higher than that in the latter (24.75±3.10 vs 1.30±0.15; t=14.31, P=0.002). The results were consistent with the immunohistochemistry staining.
ConclusionsA novel bicyclic peptide PET probe targeting Nectin-4, 68Ga-NODA-BMIC, is easy to be synthesized and owns satisfactory labeling yield and radiological purity. The imaging performance is good and the target tissues could be visualized. It may play a unique role in the diagnosis and treatment of breast cancer.
李励琦,徐悦,潘栋辉,等. 68Ga标记靶向Nectin－4双环肽的制备及乳腺癌显像研究 [J]. 中华核医学与分子影像杂志,2024,44(12)：741-747.
DOI:10.3760/cma.j.cn321828-20240703-00244版权归中华医学会所有。
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李励琦：研究设计、研究实施、论文撰写；徐悦、王辛宇、王立振：研究实施、数据采集；潘栋辉、严骏杰、陈重阳：数据采集与分析；杨敏：统计学分析、研究指导；徐宇平：论文修改、统计学分析、经费支持、研究指导
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