^99Tc^m标记人表皮生长因子受体2小分子靶向结合蛋白的制备及体外结合特性

Preparation and characterization of ^99Tc^m-labeled human epidemal growth factor type 2 affibody molecule in vitro

目的探讨^99Tc^m标记人表皮生长因子受体2（HER2）小分子靶向结合蛋白ZHER2：342的制备方法，分析其在体外与HER2的结合特洼。方法利用配体交换法进行ZHER2：342的^99Tc^m标记，分析不同质量的SnCl2和NaOH、不同反应时间对标记率的影响，探讨最佳的标记方法。用HPLC测定标记物的标记率与放化纯，并分析其体外稳定性。分析标记物在体外与HER2高表达的人卵巢癌SKOV-3细胞的结合率及滞留率，并与HER2低表达的人乳腺癌MDA—MB-231细胞进行对比。在体外用过量未标记的ZHER2：342对SKOV-3细胞HER2进行封闭后，与未封闭组进行对比，研究该分子探针与HER2结合的特异性。采用单因素方差分析及两样本t检验分析数据。结果^99Tc^m标记ZHER2：342的最佳条件为：反应体系中依次加人ZHER2：342 5μg（1g／L）、NaOH 5μg（1g／L）、SnCl2 8．8μg（1g／L）、^99Tc^mO4^-1 150μl（37MBq），轻微振荡后室温静置1h.^99Tc^m-ZHER2：342标记率为（98.10±1.73）％，放化纯〉98％；体外稳定性好，与生理盐水及新鲜人血清混合24h后放化纯均〉85％。在体外与HER2高表达的SKOV-3细胞具有较高的结合率，混合后6h最高，结合率为（995±1．02）％，且各时间点细胞结合率均明显高于HER2低表达的MDA—MB-231细胞（5．68～9．88与0．56～2．11；t=-34．50—-13．14，均P〈0．01）。此外，加入过量的未标记的ZHER2：342进行受体封闭时，^99Tc^m-ZHER2：342与SKOV-3细胞的结合率从（9．95±1．02）％降到（2．11±0．27）％（t=-13．14，P〈0．01）。结论^99Tc^m标记ZHER2：342方法简单，标记率高；标记产物体外稳定性好，在体外可与HER2高表达的人卵巢癌SKOV-3细胞特异性结合，是有潜力的HER2靶向分子影像探针。

Objective To prepare the ^99Tc^m-labeled human epidermal growth factor receptor type 2 （HER2） affibody molecule ZHER2：342 and evaluate its receptor binding specificity in vitro. Methods The molecular ZHER2：342 was labeled with ^99Tc^m using the ligand exchange method. The labeling efficiency and radiochemical purity were measured by HPLC. The major factors, such as the mass of SnCl2 and NaOH and reaction time were analyzed, and the optimal method was summarized. Cell binding kinetics and cellular retention of the probe were investigated in HER2-expressing SKOV-3 cells and MDA-MB-231 cells with low HER2 expression respectively. HER2 binding specificity of ^99Tc^m-ZER2：342 was analyzed by a pre-injection of excess unlabeled ZHER2：342 to saturate HER2 receptors. One-way analysis of variance and two-sample t test were used. Results The optimal labeling procedure was as follows： 5μg （ 1 g/L） of ZHER2：342 was mixed with 5μg of NaOH （1 g/L）, then 8.8 μg SnCl2（ 1 g/L, solution） was added, followed by 150 μl （37 MBq） ^99TcmO4^- solution, and finally the mixture was slightly vortexed and incubated for 1 h at room temperature. ^99Tc^m- ZER2,342 was stable in vitro with a high labeling efficiency of （98.10± 1.73）%. The radiochemlcal purity was 〉98%, and was more than 85% after the incubation for 24 h in saline and fresh human serum. The cell bind- ing of ^99Tc^m-ZHER2：342 with HER2-expressing SKOV-3 cells gradually increased over time with a peak of （9.95±1.02） % at 6 h. The binding of ^99Tc^m-ZHER2：342 in SKOV-3 cells was significantly higher than that in MDA-MB-231 ceils at every time point （5.68-9.88 vs 0.56-2.11 ; t： from -34.50 to -13.14, all P〈0.01）. The labeled molecular probe retained the capacity to bind specifically to HER2-expressing SKOV-3 cells since the ceU binding decreased from （9.95±1.02）% to （2.11±0.27）% after receptor saturation （t=-13.14, P〈0.01）. Conclusions ^99Tc^m-ZHER2：342 has a high labeling efficiency, good stability and optimal binding specificity. These characteristics enable it to be a promising molecular probe for HER2-targeting imaging.