靶向Endoglin的CL-PEG-MnFe_2O_4探针构建及体外实验

Synthesis and in vitro experiment of Endoglin targeted CL-PEG-MnFe_2O_4 nanomicelle

目的:合成靶向细胞内皮糖蛋白(Endoglin)的CL-PEG-Mn Fe_2O_4分子探针,并探讨其在肿瘤新生血管及新生淋巴管双重靶向显像中的可行性。方法:将与Endoglin特异性结合的多肽CL-1555与超敏感的聚乙二醇两亲片段修饰的Mn Fe_2O_4(PEG-PCL-Mn Fe_2O_4)纳米胶束耦联制备靶向Endoglin的CL-PEG-Mn Fe_2O_4纳米胶束。用CL-PEG-Mn Fe_2O_4标记肿瘤源性血管内皮细胞(VECs)及淋巴管内皮细胞(LECs)。通过普鲁士蓝染色、透射电子显微镜及核磁共振成像了解纳米粒内皮细胞的结合情况及MRI信号改变。结果:在相同铁浓度下,CL-PEG-Mn Fe_2O_4标记的肿瘤源性内皮细胞的标记率较PEG-PCL-Mn Fe_2O_4高。在铁浓度为0、0.5、1.0、2.0、5.0及10.0μg/m L时,靶向CL-PEG-Mn Fe_2O_4和非靶向PEG-PCLMn Fe_2O_4标记诱导后VECs的标记率分别为0%、(27.75±3.84)%、(61.63±3.12)%、(83.43±3.76)%、100.00%、100.00%和0%、(14.56±3.24)%、(37.53±2.62)%、(51.62±3.32)%、(88.36±4.26)%、100.00%。在T1WI,信号强度呈缓慢升高;在T2WI,信号强度逐渐下降,T2*WI下降更明显。相对于非靶向PEG-PCL-Mn Fe_2O_4纳米胶束标记肿瘤源性内皮细胞和靶向CLPEG-Mn Fe_2O_4纳米胶束标记未诱导内皮细胞,靶向CL-PEG-Mn Fe_2O_4纳米胶束标记的肿瘤源性内皮细胞信号改变更明显,但随着标记铁浓度的增加,该差异逐渐减小。结论:CL-PEG-Mn Fe_2O_4纳米粒在体外能与肿瘤源性VECs和LECs特异性结合,并可通过核磁共振成像进行检测,这为肿瘤新生血管及新生淋巴管的双重靶向显像提供了实验基础。

Objective To synthesize Endoglin targeted CL-PEG-Mn Fe_2O_4 probe and discuss on its feasibility in dual target imaging for tumor angiogenesis and lymphangiogenesis. Methods Endoglin targeted polypeptide CL-1555 was synthesized and bound to the surface of PEG-PCL-Mn Fe_2O_4 nanomicelle to construct Endoglin targeted CL-PEG-Mn Fe_2O_4 nanomicelle. The tumorderived vascular endothelial cells（VECs） and lymphatic endothelial cells（LECs） were co-cultured with CL-PEG-Mn Fe_2O_4 nanomicelle. The intracytoplasmic nanoparticles and MR signals were confirmed by Prussian blue iron staining, transmission electron microscopy and MRI. Results With the same iron concentration, the tumor-derived endothelial cell labeling ratio with CL-PEG-Mn Fe_2O_4 nanomicelle was significantly higher than that with PEG-PCL-Mn Fe_2O_4 nanomicelle. At iron concentrations of 0, 0.5, 1.0, 2.0, 5.0, 10.0 ug/m L, the labeling ratios of targeted CL-PEG-Mn Fe_2O_4 and off-targeted PEG-PCL-Mn Fe_2O_4 for induced VECs were respectively 0%,（27.75±3.84）%,（61.63±3.12）%,（83.43±3.76）%, 100.00% and 100.00%; 0%,（14.56±3.24）%,（37.53±2.62）%,（51.62±3.32）%,（88.36±4.26）% and 100.00%. Signal intensity gradually increased in T1 WI, and decreased in T2 WI and T2＊WI, especially in T2＊WI. Compared with the tumor-derived endothelial cells with off-targeted PEG-PCL-Mn Fe_2O_4 nanomicelle and non-induced endothelial cells with targeted CL-PEG-Mn Fe_2O_4 nanomicelle, the tumor-derived endothelial cells with targeted CL-PEG-Mn Fe_2O_4 nanomicelle showed more significant signal changes, and the significant differences were gradually decreased with the increasing of iron concentration. Conclusion The CL-PEG-Mn Fe_2O_4 nanomicelle can specifically bind to tumor-derived VECs and LECs, and be detected by MRI, providing potential experimental basis for dual target MRI for tumor angiogenesis and lymphangiogenesis.