Integrin靶向拉曼共振活性金纳米探针的研制及其在神经胶质瘤手术中的应用研究

Malignant glioma is the most common and most lethal type of brain tumor, which is characterized by high recurrence and infiltrative nature. The extent of tumor resection (percentage of tumor removed) directly correlates with the length of survival. The current difficulty in visualizing the true extent of infiltrating tumor during surgical resection represents one of the major reasons for the poor prognosis of glioma patients. In this application, we propose to develop a molecular imaging probe to delineate the microscopic invasive tumor margins of gliomas. Taking advantage of the ultra-sensitivity of Raman spectroscopy, we have developed a Raman-active nanoparticle to detect the orthotopically implanted human glioma xenograft. Based on the 1st generation of nanoparticle, an ultra-bright RGD peptide-conjugated molecular Raman-imaging probe (termed here Surface-Enhanced Resonance Raman Scattering (SERRS)-Nanostars) will be generated to specifically bind the integrins overexpressed by the RCAS-PDGF/pNestin-TVA glioma, which is the state-of-the-art genetically engineered mouse model closely mimicking human tumors. We expect that the integrin-targeting SERRS-Nanostars can selectively accumulate in the tumor tissues, improve tumor-to-normal-tissue ratio of Raman signal, enable high-resolution tumor border delineation and benefit the prognosis of glioma patients through more complete tumor resection. If successful, a principle for clinical translation will be established to accurately identify the microscopic tumor and to optimize therapeutic regimen.

恶性神经胶质瘤是最常见也是死亡率最高的脑部肿瘤。研究表明肿瘤切除程度与患者生存期直接相关。但由于该肿瘤的高浸润性，使常规手术中肿瘤边界的界定非常困难。本研究拟研制能在微观水平识别浸润性肿瘤边界的分子影像探针。利用拉曼光谱成像的超高灵敏性，我们前期已开发出带拉曼活性的金纳米颗粒，并用于原位种植神经胶质瘤小鼠模型。在此基础上，我们计划开发偶联了RGD多肽的具有拉曼共振活性的金纳米材料，使其能特异性靶向结合在神经胶质瘤细胞上过量表达的integrins。为更好地模拟神经胶质瘤的高浸润特性，将采用RCAS-PDGF/pNestin-TVA病毒诱导的神经胶质瘤转基因小鼠模型。我们预期integrin靶向拉曼探针较非靶向探针能更多地在肿瘤区的滞留，从而提高肿瘤/正常组织的拉曼信号比值，更易于确定肿瘤的边界，增加肿瘤组织的完全切除率并改善预后。本研究将为肿瘤的精确诊断定位和手术治疗方案优化提供科学依据。

手术切除仍是治疗恶性神经胶质瘤的不可替代的方法。术中由于不能准确界定神经胶质瘤的浸润性边界，遗留的肿瘤组织是其高复发、高致死的主要原因。为此本课题开展了研制能在微观水平识别浸润性肿瘤边界的分子影像探针的研究：拟利用神经胶质瘤细胞系、神经胶质瘤转基因小鼠模型和原位注射模型在多层面证明integrin靶向的拉曼共振纳米金探针能达到很高的肿瘤/正常组织摄取比值。利用拉曼成像技术的高灵敏性，在临床前体内测试模型中更准确地检测浸润性神经胶质瘤肿瘤边界，以提高传统手术的准确率。..本课题已完成：1. 具有integrin靶向的拉曼共振活性纳米金探针（RGD-SERRS-NP）与非靶向的拉曼共振活性纳米金探针(RAD-SERRS-NP)的制备与物化性质鉴定：cRGDyK多肽被用作特异结合integrin的靶向分子共价连接在具有拉曼共振活性的纳米金颗粒（SERRS-NP）上；而 cRADyK被用作非特异性结合的对照多肽。2. 在integrin高表达的U87-MG神经胶质瘤细胞中，RGD-SERRS-NP在50pM的浓度以下并未检测到明显细胞毒性，拉曼信号的检测下限为1飞摩尔，而且展示出比RAD-SERRS-NP更高的细胞摄取量。3. 在RCAS-PDGFB神经胶质瘤小鼠模型中，基于RGD-SERRS-NP拉曼光谱成像结果不但清楚地展示了肿瘤原发灶，而且能界定浸润性肿瘤边界，指示肿瘤转移的路径，还可检测出微卫星肿瘤转移灶（检测下限为15微米）。.总之，本课题在体外和体内系统都证实了RGD-SERRS-NP探针的对integrin高表达肿瘤识别的超高灵敏性和特异性。如能成功转化入临床应用，将显著提高神经胶质瘤的完全切除率，进而提高病患的生存率。我们已按照研究计划顺利完成各项研究目标，主要的研究成果作为封面文章发表在2016年8月Theranostics杂志（IF=8.8）上，相关的其它成果另发表论文2篇。