多功能纳米金颗粒研制及其用于子宫内膜癌靶向腹膜后淋巴结转移灶显像与放疗增敏的实验研究

Endometrial cancer with retroperitoneal lymph node metastasis currently lack effective assessment and management methods. Most patients have to receive systemic retroperitoneal lymphadenectomy unnecessarily. Nano-targeting technology offers a fascinating diagnosis and therapeutic option in cancer due to the unique biological properties of nanoparticles. In this project we are going to prepare one kinds of multi-functional Gold Nanoparticles(mf-GNPs). Three different functional molecules would be coupled on the surface of GNPs particles, including glucose(Glu), polyethyleneglycol (PEG) and indocyanine green(ICG). These molecules could offer mf-GNPs a variety of distinctive properties such as lymphatic targeting, tumor targeting, fluorescence imaging as well as X-ray based imaging sensitization and radiotherapy enhancement. Human endometrial cancerous cell line with high specific glucose receptors expression level on the surface of cytomembrane would be identified for further studies. Cellular uptake and X-ray irradiation experiments in vitro would be performed to confirm the targeting and radiosensitization properties of mf-GNPs to tumor cells. A mice endometrial cancer retroperitoneal lymph-node metastasis animal model would be established for in vivo studies. Drug distribution and pharmacokinetic studies in animal tissues following mf-GNPs administration would be performed to confirm the lymph and tumor targeting properties of mf-GNPs in vivo. Eventually, micro-CT imaging in vivo would be used to explore the diagnostic accuracy of mf-GNPs based CT imaging for retroperitoneal lymph node metastatic lesions. In vivo bioluminescence imaging would be used to identify the metastatic lymph nodes to enable accurate surgical removal of the target lesion during operation. Metastatic lymph nodes X-ray irradiation study in vivo would be performed to confirm the radiotherapy enhancement property of mf-GNPs. In this project we integrate multiple functions into a single GNPs particle to realize the simultaneous cancer diagnosis and therapy. The established gold nanoplatform promises a powerful tool for imaging sensitization, in vivo bioluminescence imaging and radiotherapy enhancement to retroperitoneal lymph node metastatic lesions in endometrial cancer. The translation of mf-GNPs into the clinic will also have significant impact on the care of cancer patients using precise image-guided precise interventions in the near future.

子宫内膜癌缺乏有效的腹膜后淋巴结转移评估与治疗手段，多数患者接受了不必要的淋巴结切除术。课题组针对此临床困境，基于前期工作基础，利用纳米技术制备葡萄糖、聚乙二醇和吲哚菁绿三种功能分子修饰的多功能纳米金颗粒，使其具备淋巴靶向、肿瘤靶向、荧光显像及X线显像/放疗增敏特性。课题组筛选葡萄糖受体高表达内膜癌细胞系，体外实验验证多功能纳米金颗粒的肿瘤细胞靶向、放疗增敏和荧光显像特性。建立内膜癌小鼠腹膜后淋巴结转移瘤模型，体内药物分布和药代学实验验证其淋巴靶向和肿瘤靶向性。荷瘤小鼠术前Micro-CT扫描探讨其淋巴结转移瘤显像增敏功能；术中活体荧光成像明确其病灶实时荧光显像指导手术的功能；术后X线照射淋巴结转移瘤验证其放疗增敏特性。本课题利用一个平台同时实现腹膜后淋巴结病灶的CT显像增敏、实时荧光显像和放疗增敏，实现基于精确显像基础上的精确治疗，避免系统性淋巴结切除术的弊端，有望实现肿瘤诊治一体化。

近年来子宫内膜癌（Endometrial Cancer，EA）发病率呈上升趋势，但缺乏有效的精准病情评估和诊治方案。本课题基于此临床难题，利用纳米材料独特的生物学性质，制备一种兼具术前影像学增敏、术中实时显像和术后放疗增敏多种功能的纳米金颗粒。该纳米载体由四部分组成1）葡萄糖分子，其与内膜癌细胞高表达葡萄糖转运蛋白受体（GLUT1)特异性结合,选择性识别内膜癌细胞；2）荧光分子吲哚菁绿，进入肿瘤实现术中组织实时显像；3）聚乙二醇（PEG），稳定纳米金同时降低网状内皮系统的吸收；4）颗粒中心纳米金粒子，利用金属元素特性实现放疗增敏和显像增敏。课题组成功制备上述多功能纳米金颗粒，该材料大小均匀、分散度好，理化性质稳定。分子生物学技术筛选出葡萄糖受体高表达的子宫内膜癌细胞系（ISK）并构建其干扰和过表达细胞系作为对照，体外纳米颗粒细胞摄取实验结果表明干扰组、正常组、过表达组摄取该纳米金荧光强度分别为11.318au，5.51au和27.216（P<0.05)，体外验证其肿瘤靶向性；细胞毒性实验验证多功能纳米金颗粒的低毒性特点；X照射下细胞放疗增敏比为1.35，体外验证该材料放疗增敏效应；细胞功能实验验证该材料可降低肿瘤细胞迁移侵袭能力，并通过放疗诱导肿瘤细胞凋亡；成功构建内膜癌腹腔和皮下荷瘤小鼠模型，体内注射纳米金颗粒后行组织分布实验验证了体内肿瘤靶向特性；CT显像和活体荧光显像探讨其术中实时荧光显像指导手术精准切除的可行性；最后X线照射腹腔和皮下荷瘤小鼠，PBS组肿瘤大小是多功能纳米金组（X线）的2.3倍，在受照荷瘤小鼠的中位生存期，多功能纳米金组放射增敏比是无照射纳米组的的1.2倍，体内验证纳米金颗粒的放疗增敏特性。综上所述，本课题制备的多功能纳米金颗粒理化性质符合生物学应用要求，体内体外证实其肿瘤靶向特性、肿瘤放疗增敏特性，同时具有显像增敏和实时显像潜力；该多功能纳米金颗粒有望实现肿瘤诊疗一体化，实现基于精准诊断基础上的精确治疗，符合转化医学研究宗旨，具有良好的转化应用前景。