示踪胰腺肿瘤间质FAP-α超微纳米探针MR/双荧光成像与化疗增敏研究

Abundant interstitial fibroblasts constitute the tumor microenvironment for pancreatic cancer cells. Based on the previous research, one protein named fibroblast-activating protein (FAP-α) is over-expressed in interstitial fibroblasts of malignant pancreatic tumor microenvironment. The protein of FAP-α is closely related to the occurrence, development, invasion and metastasis of pancreatic tumors. FAP-α is not expressed in normal tissues and organs. It is of great significance to trace FAP-α for distinguishing benign and malignant of pancreatic tumors, and estimate prognosis. In this study, FAP-α was used as the activation target to contrast nanoprobes. Firstly, ultra-small super-paramagnetic Fe3O4 nanoparticles were synthesized. MR/ double fluorescence imaging or drug loading probes were built with polypeptide chain coupled fluorescent reagent of ANNA or fraxinellone (Frax). Polymer compound of PEI-β-PEG were modified for long circulation in vivo and responding to tumor acidic microenvironment. Polypeptide chain was hydrolyzed by FAP-α to activate fluorescent reagent of ANNA or to release drug of Frax. The purpose of MR/ dual-fluorescence imaging to quantify FAP-α and to target chemotherapy-sensitization was achieved. Secondly, transmission electron microscopy (TEM), dynamic light scattering (DLS) and other detection methods were used to verify the physicochemical property of the nanoprobes. The targeting drug release effect of nanoprobes was verified by in vivo and in vitro imaging experiments. Finally, after in vivo treatment, molecular, histopathological, and immunohistochemical testing were performed to compare and evaluate the chemotherapy-sensitization effect of the combination of probe and gemcitabine as well as the efficacy of the gemcitabine monotherapy group. In this project, we designed and constructed FAP-α tracing imaging and FAP-α targeted drug release probes to provide a clinical basis for the early treatment and diagnosis of pancreatic cancer.

胰腺肿瘤细胞生长微环境中丰富的间质纤维母细胞高表达成纤维活化蛋白(FAP-α)，其与胰腺肿瘤发生发展、侵袭转移过程密切相关，正常组织无FAP-α表达，示踪FAP-α对判别胰腺肿瘤良恶性和预后具有重要意义。本研究拟以FAP-α为激活靶点，构建修饰可体内长循环且肿瘤酸性微环境响应的PEI-β-PEG高分子链的超小超顺磁性纳米Fe3O4负载多肽链偶联荧光剂或梣酮药物的MR/双荧光成像或载药探针。FAP-α水解多肽链调控荧光剂活化或药物释放，达到MR/双荧光定量FAP-α成像和靶向化疗增敏之目的。透射电镜、动态光散射等检测方法验证探针性能；体内外验证探针靶向性成像和释药效果；体内疗效通过分子病理免疫组化等检测验证，对比评估探针和吉西他滨联合用药组的化疗增敏效果以及吉西他滨单药组化疗效果。本项目设计构建的示踪FAP-α成像和靶向释药增敏探针，期望为临床提供诊疗依据，早日攻克胰腺癌这一难题。

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，从而具有重要意义。本项目以胰腺肿瘤细胞生长微环境中丰富的间质纤维母细胞高表达成纤维活化蛋白(FAP-α)为靶点，以超小超顺磁性纳米Fe3O4为载体，以青蒿琥酯为破坏微环境间质的药物，首先“破坏”纤维间质，然后进一步化疗增敏的双模态可视化纳米探针（PEG-Art-VSPIO），对该纳米探针的物化性质进行性能表征，表明该探针粒径较小，探针溶液稳定性佳，成像参数在预期范围，体内外分子成像研究表明此纳米探针具有靶向成像效果。最后，对该纳米探针的体内外毒性、生物学相容性、化疗增敏疗效进行了验证。体外细胞学显示，此探针有一定的抑制胰腺纤维细胞增殖的作用，体内毒性实验结果显示，和对照组相比，各组小鼠体重，血液常规指标，肝、肾功能及胆固醇血糖生物标记物指标，均未发现显著变化。体内体内成像结果显示纳米探针累积至肿瘤部位的信号强度在注射2h后即逐渐增强，4h大约达到峰值。在治疗期间，荷瘤鼠体重有些许的变化，其中单化疗药组小鼠体重略有下降，而纳米探针加化疗药组小鼠体重下降后再次恢复；治疗后，纳米探针加化疗药组肿瘤体积远低于其他组，说明该纳米探针具有良好的化疗增敏效果。以上结果显示，本项目研究制备的超小超顺磁性纳米探针是一个较为理想的胰腺癌微环境靶向分子探针，有望为临床提供诊疗依据，早日攻克胰腺癌这一难题。