双靶向肿瘤多模态纳米分子探针用于手术导航技术指导脑胶质瘤精确切除的研究

The early and accurate diagnosis of glioma is significant for clinical therapy and prognosis. Traditional imaging methods such as MR and CT showed limitations for medium and low grade glioma. Result of high normal brain uptake of 18F-FDG PET and too short half-life time of 11C-methionine to transportation, a new kind of high targeting, fit half-life time and broad-spectrum tracer is needed. Based on our past work and literatures, many tumor cells have positive endocytosis of activable cell penetrating peptide (ACPP). Although the stability of nano-micells are poor, the in-vivo test demonstrated that elevated targeting tumor imaging was attributed with nanoparticles labeled with ACPP as molecular imaging probes. We designed that 18F (as positron imaging tracer module), Cy5.5 (as infrared imaging tracer module) and ACPP ( as active targeting tumor module) have attached with polylysine nanoparticles (as passive targeting tumor module) by NHS ester which prefers to react with ammonia displayed on the surface of polylysine. A positron and optical molecular imaging probe targeting glioma is synthesized, so as to navigate the precise site of glioma for accuracy of surgery therapy.

术中能否彻底切除肿瘤是提高脑胶质瘤患者生存率的关键问题。常用影像学方法对中低级胶质瘤显示不佳且较难用于术中成像；术中神经导航肿瘤荧光显像剂具有需要术前避光和荧光淬灭的缺点。因此，利用分子影像技术开发出具有高敏感性、特异性和靶向的正电子纳米探针可更好地指导手术切除。本课题的前期工作及文献报道发现，肿瘤细胞靶向多肽（ACPP）连接到纳米胶束分子探针可提高肿瘤靶向成像效果，但纳米胶束存在稳定性差的缺点。本课题拟采用结构更稳定的多聚赖氨酸树状纳米为载体，利用NHS酯在温和条件下易与氨基反应的特性，将ACPP主动靶向、18F正电子显像、Cy5.5光学成像（末端均连接NHS酯）等单元分别连接至纳米载体（含氨基），合成一种对胶质瘤高敏感性、特异性的双靶双模纳米探针用于术前诊断、术中实时探测瘤体实施精确切除。为建立一种分子影像探针引导下的术中神经导航胶质瘤精确切除方法奠定了实验基础。

术中能否彻底切除肿瘤是提高脑胶质瘤患者生存率的关键问题，因此利用分子影像技术开发出具有高敏感性、特异性和靶向的正电子纳米探针可更好地指导手术切除。本研究成功制备了两种前体NOTA-DGL及NOTA-DGL-PEG-RGDyC，首次对树状大分子多聚赖氨酸DGL的衍生物进行了68Ga正电子核素放射性标记，合成68Ga-NOTA-DGL和68Ga-NOTA-DGL-PEG-RGDyC两种显像剂，为正电子核素68Ga标记树状纳米DGL提供了一条通用的、方便高效地标记方法，并成功通过小动物PET/CT活体成像观察到纳米探针DGL的体内分布特征。显像剂可在15min内可制得，放化产率在50%~70%之间，分离纯化后其放化纯度均＞98%。pH值介于4.0~4.2之间为无色、透明、澄清的溶液，无其他重金属污染。脂水分配系数测定，68Ga-NOTA-DGL的LogP=-1.62，68Ga-NOTA-DGL-PEG-RGDyC的LogP= -3.037，表明两种纳米分子探针均呈水溶性。显像剂分别在0.1 M PBS及小牛血清中在37℃下孵育至120min后放化纯均＞95%，说明两种纳米分子探针的体外稳定性良好，比活度达30 GBq/μmol。A549及U87MG细胞能快速、高效地摄取显像剂，随时间的延长流出速度缓慢下降，且在2h末仍有显像剂滞留。显像剂在正常小鼠血液中清除速度较快，放射性摄取主要分布在肝脏和脾脏，其他主要脏器放射性摄取较少。荷瘤鼠分别注射68Ga-NOTA-DGL及68Ga-NOTA-DGL-PEG-RGDyC 1小时后，U87MG肿瘤组织的摄取值分别为（0.19±0.02）%ID/g和（0.27±0.09）%ID/g，高于肌肉组织。荷瘤鼠经尾静脉分别注射68Ga-NOTA-DGL及68Ga-NOTA-DGL-PEG-RGDyC后，Micro-PET显像显示放射性摄取均主要分布在肝脏，瘤结节见少量摄取。瘤内注射实验结果提示了纳米分子探针具有良好的瘤内滞留效应，这为肿瘤的治疗带来了新的思路。