基于GPC1的载冬凌草甲素靶向多模态分子探针对胰腺癌诊疗一体化的研究

The mortality of pancreatic cancer accounts for the fourth in malignant tumors，early diagnosis at molecular level is the key to improving prognosis. GPC1 is highly expressed in pancreatic cancer, and low expression or no expression in normal pancreatic tissues and benign lesions. Oridonin can inhibit the proliferation of tumor cells, leading to the apoptosis of tumor cells. Choosing GPC1 as a specific target of pancreatic cancer, and the gold cage used as the carrier, to establish the targeted probe loading Oridonin with fluorescence, CT, MRI imaging, controlled by the PH/ enzyme sensitive hyaluronic acid. The qualities and functions of the probe were tested by using transmission electron microscopy, dynamic light scattering and so on. Pancreatic cancer cells and pancreatic ductal epithelial cells with probes were taking series experiments of in vitro toxicity and pharmacokinetic, taking technical tests，such as MTT, transwell, flow cytometry and so on. The probes were injected into the mouse model of pancreatic cancer, subjected to fluorescence, CT and MRI imaging in vivo, and then the tumor tissues and the surrounding tissues were tested by molecular pathology. Setting up the loaded gemcitabine targeted probe group, unloaded drug targeted probe group and gemcitabine group as control groups. Finally, the imaging and pathological results were compared. The in vitro and in vivo study of ORI loading multiprobes with targeted molecular imaging is to realize site-specific delivery and therapy, which is expecting to diagnose and treat pancreatic cancer at early stage.

胰腺癌死亡率占恶性肿瘤第4位，在分子水平进行早期诊疗是改善预后的关键。GPC1在胰腺癌中高表达，在正常胰腺组织及良性病变中低表达或不表达。冬凌草甲素（ORI）能抑制肿瘤细胞的增殖、诱导凋亡。选择以GPC1为胰腺癌特异性靶点，以金纳米笼为载体，构建载ORI，以PH/酶敏感的透明质酸控制给药闸门的荧光、CT和MR靶向探针，利用投射电镜、动态光散射等对其性能进行检测；对探针与胰腺癌细胞和胰腺导管上皮细胞等进行系列体外毒性和药代动力学实验，采用MTT、Transwell等技术检测。将探针注入胰腺癌小鼠模型，进行活体荧光、CT、MR成像，取肿瘤和瘤周组织进行分子病理学检测；并和载吉西他滨探针组、未载药探针组、单纯吉西他滨组对照；最后，将影像和病理结果进行对比分析。本研究通过载ORI靶向多模态探针的体内外实验，在靶向分子成像的同时，进行靶向药物递送、定点释放、靶向治疗，希望实现胰腺癌早期诊疗一体化。

胰腺癌恶性程度高，临床进展快，死亡率高。对胰腺癌的早期诊断可以提高其术后生存质量及生存期，因此具有重要意义。本项目以金纳米笼AuNC为载体，以胰腺癌中高表达的GPC1为特异性靶点，以冬凌草甲素为治疗药物，以透明质酸酶及pH敏感的透明质酸为控制给药阀门，合成了多模态纳米诊疗探针（ORI-GPC1-NPs），对该纳米探针的物化性质进行性能表征，表明该探针粒径合适，稳定性佳，成像参数理想，具有pH值/酶敏感的控制性释放能力。随后，通过体内外多模态分子成像研究了此多模态纳米探针（ORI-GPC1-NPs）的靶向成像效果，包括离体细胞、活体动物原位移植瘤模型的荧光、MRI成像。最后，利用人胰腺癌裸鼠原位移植瘤模型对此多模态纳米探针（ORI-GPC1-NPs）的体内毒性、靶向药物递送的疗效进行了评价。体外细胞学实验结果显示ORI-GPC1-NPs可抑制胰腺癌细胞增殖，促进细胞凋亡，阻滞细胞周期，抑制细胞侵袭。体内毒性实验结果显示，和阴性对照组相比，高、中、低剂量ORI-GPC1-NPs组裸鼠体重，血液常规指标，肝、肾功能生物标记物指标，均未发现显著变化。体内成像结果显示多模态纳米探针ORI-GPC1-NPs组肿瘤部位在注射12h后可检测到NIRF/MR信号。随着时间的延长，肿瘤部位的信号强度逐渐增强，在注射24h后达到峰值。离体NIRF和ICP-MS结果显示，此多模态纳米探针ORI-GPC1-NPs主要积累在肝脏、肿瘤、肾脏内。治疗期间，荷瘤小鼠体重均未出现显示变化；治疗后，ORI-GPC1-NPs组肿瘤体积和重量远低于其他对照组，具有良好的治疗效果。上述结果表明本项目研究制备的多模态纳米诊疗探针是一个较为理想的胰腺癌双模态靶向分子探针，有望成为在分子水平早期诊断胰腺癌的重要手段，达到早期诊断胰腺癌的目的。