CN108721647B - 一种用于指导胶质瘤精准切除的多模态纳米探针 - Google Patents

一种用于指导胶质瘤精准切除的多模态纳米探针 Download PDF

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CN108721647B
CN108721647B CN201710257572.1A CN201710257572A CN108721647B CN 108721647 B CN108721647 B CN 108721647B CN 201710257572 A CN201710257572 A CN 201710257572A CN 108721647 B CN108721647 B CN 108721647B
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陈亮
毛颖
岳琪
高西辉
李聪
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Abstract

本发明属于分子影像探针领域，涉及一种用于指导胶质瘤精准切除的多模态纳米探针，尤其涉及具有靶向EGFRvIII阳性胶质瘤作用的磁共振/拉曼纳米金颗粒探针及其中间体的合成，及其在胶质瘤术前精确规划和术中实时导航切除中的应用。所述纳米探针以球形金纳米颗粒(AuNS)为基底，在其表面连接顺磁性螯合剂Gd3+‑DTPA和拉曼信号分子IR783B，并以PEG为载体连接靶向EGFRvIII的多肽FALGEA。本纳米探针有助于保证术前规划和术中切除的一致性，能克服现有技术的影像探针循环时间短、无靶向性、难以透过完整的血脑屏障等缺点，实现胶质瘤，特别是EGFRvIII阳性胶质瘤的高信噪比示踪及精准切除。

Description

一种用于指导胶质瘤精准切除的多模态纳米探针
技术领域
本发明属于分子影像探针领域，涉及多模态纳米探针，具体涉及一种用于指导胶质瘤精准切除的多模态纳米探针，尤其涉及具有靶向EGFRvIII阳性胶质瘤作用的磁共振/拉曼纳米金颗粒探针及其中间体的合成，及其在胶质瘤术前精确规划和术中实时导航切除中的应用。
背景技术
现有技术公开了在原发性颅内肿瘤中，脑胶质瘤发病率最高，接近40.5％，具有生存期短、复发率及死亡率高等特点；尤其是胶质母细胞瘤，尽管近年来的综合治疗(包括手术、放疗、化疗)取得了很大进步，但其中位生存期仅为14.6个月。目前，手术仍然是大多数胶质瘤患者的首选治疗手段，具有减瘤、颅内减压、明确病理等作用，已有研究表明，胶质瘤手术切除程度和患者预后呈正相关，术后肿瘤残余是导致早期复发的关键因素。然而，胶质瘤呈浸润性生长，术中较难判断肿瘤边界，过度切除可能损及周围正常脑组织，尤其是对于毗邻功能区及大血管的肿瘤，可能导致难以逆转的功能障碍。因此，如何实现胶质瘤的精准切除，在保留正常脑组织的同时最大程度切除肿瘤组织，是平衡胶质瘤患者生存期和生活质量的前提，而精准示踪胶质瘤边界则是其中的关键技术。
磁共振成像(MRI)具有组织分辨率高、无电离辐射等优点，是目前最常用的脑肿瘤术前诊断手段。临床实践显示，神经导航技术基于术前MRI资料，旨在术中精确定位病灶，但开颅后难以避免的脑组织移位会导致定位偏差，从而降低了神经导航的准确程度。因此，近年来，术中MRI(iMRI)和荧光染料等新技术开始被应用于脑肿瘤手术，然而，iMRI具有操作复杂、费用高昂、麻醉时间延长等缺点，从而使其应用仅限于较大的临床治疗中心，难以大范围推广，且目前使用的Gd3+-DTPA等造影剂半衰期短，需要反复注射，可能会加重造影剂的肾毒性；5-ALA荧光染料可在肿瘤细胞内产生发出蓝色荧光的代谢物，新近已被FDA批准用于胶质瘤手术，但背景荧光强、潜在的光毒性、淬灭时间短等限制了其进一步应用。因此，当前胶质瘤手术仍然缺乏效果佳、缺点少的示踪技术。
研究报道了光学成像和磁共振成像相结合优势互补的多模式影像技术能够提供较单一影像技术更加灵敏和高分辨率的示踪效果；拉曼光谱成像是一种基于光子散射反映分子特征的成像技术，与荧光成像不同，拉曼光谱利用单次激发即可完成多种分子检测，且光淬灭少、内源性分子引起的背景信号弱；有研究报道，原有的拉曼光谱信号较弱，限制了在生物领域的进一步应用，表面增强拉曼光谱(Surface-enhanced Raman scattering,SERS)是近年来新兴的拉曼技术，通过在贵金属粗糙表面结合拉曼信号分子使得信号放大106-1010倍，从而极大地提高了检测灵敏度；此外，当拉曼信号分子的激发波长与入射光波长一致产生共振时，可进一步放大信号2-3倍，即表面增强共振拉曼光谱(Surface-enhanced resonance Raman scattering,SERRS)；纳米金颗粒生物相容性好、易于修饰、共振区域处于可见光区，因此是目前最常用的SERS和SERRS底物；有研究报道了一种融合MRI、SERS和光声成像的多模态纳米金颗粒探针，该探针穿过破坏的血脑屏障并利用EPR(enhanced permeability and retention)效应进入肿瘤区域，可高灵敏地指导胶质瘤手术切除，但胶质瘤的血脑屏障存在异质性，不同分型及级别的血脑屏障破坏程度不同，因此，仅仅依赖EPR效应的探针并不能准确描绘胶质瘤边界，理想的探针尚需具备针对胶质瘤的特异性靶向作用。
特异度强、表达程度高的肿瘤标志物是实现靶向作用的根本，已有较多研究探索胶质瘤的肿瘤标志物，其中，EGFRvIII是跨膜蛋白EGFR(epidermal growth factorreceptor)的变体，在胶质瘤中呈不同程度的表达而在正常脑组织中不表达，因此是目前最为公认的胶质瘤特异性标记。更重要的是，有研究发现，EGFRvIII也可表达于肿瘤周边血管内皮细胞，有望介导纳米探针跨越完整的血脑屏障并进而靶向脑肿瘤。已有研究报道EGFRvIII抗体修饰的氧化铁纳米颗粒可准确勾勒胶质瘤边界，并通过阻遏EGFR磷酸化及下游信号通路从而抑制肿瘤进展。EGFRvIII多肽也已作为肿瘤特异性抗原，被用于针对胶质母细胞瘤患者的临床试验。因此，EGFRvIII是目前理想的胶质瘤示踪靶点。
目前尚无靶向EGFRvIII的多模态金颗粒探针用于胶质瘤成像及手术的报道，基于现有技术的现状，本申请的发明人针对现有技术存在的缺陷，拟提供一种用于指导胶质瘤精准切除的多模态纳米探针。
发明内容
本发明的目的是针对现有技术存在的缺陷，提供一种用于指导胶质瘤精准切除的多模态纳米探针，具体涉及用于指导胶质瘤切除的跨血脑屏障靶向多模态纳米探针，尤其是具有EGFRvIII靶向作用的MR/SERRS金纳米颗粒探针。本纳米探针能克服现阶段影像探针循环时间短、无靶向性、难以透过完整的血脑屏障等缺点，实现胶质瘤，特别是EGFRvIII阳性胶质瘤的高信噪比示踪及精准切除。
具体而言，本发明的用于指导胶质瘤切除的跨血脑屏障靶向多模态纳米探针是一种对EGFRvIII阳性的胶质瘤具有靶向功能的多模态示踪剂AuP-FAL，其特征在于，以球形金纳米颗粒(AuNS)为基底，在其表面连接顺磁性螯合剂Gd3+-DTPA和拉曼信号分子IR783B，并以PEG为载体连接靶向EGFRvIII的多肽FALGEA。
本发明中，FALGEA多肽的氨基酸序列为Phe-Ala-Leu-Gly-Glu-Ala，PEG为聚乙二醇，用于桥连多肽的双功能化PEG的两端分别是N-羟基琥珀酰亚胺酯和巯基，两者首先合成为PEG衍生物。
本发明中，球形金纳米颗粒(AuNS)由氯金酸和柠檬酸钠合成。
本发明中，Gd3+-DTPA作为磁共振造影剂，提供磁共振成像，DTPA双酸酐和氨基乙烷硫醇首先反应，再修饰到AuNS表面，继而螯合钆离子，从而实现Gd3+-DTPA和AuNS的连接。
本发明中，IR783B作为拉曼信号分子，提供SERRS成像，巯基修饰的IR783B修饰到已连接Gd3+-DTPA的AuNS表面。
本发明中，结合FALGEA多肽的PEG衍生物和修饰Gd3+-DTPA、IR783B的AuNS反应，完成AuP-FAL的合成。
本发明中，为验证目标探针对胶质瘤的靶向示踪灵敏度及信噪比，还制备了无肿瘤靶向基团的参比影像探针AuP-PEG，由巯基PEG和连接Gd3+-DTPA、IR783B的AuNS合成。
本发明中，拉曼光谱显示，AuP-FAL在509和541cm-1处存在尖锐双峰，可作为该探针的特异性标志。
本发明中，多模态指磁共振成像和拉曼光谱成像，AuP-FAL可被磁共振成像和拉曼成像同时动态监测。
本发明中，AuP-FAL可精准指导对EGFRvIII阳性胶质瘤的手术切除；根据拉曼光谱上的特征双峰，采用手持拉曼仪实时、多角度检测可能的肿瘤残余，最终组织学证实肿瘤完全切除。
本发明利用EGFRvIII在胶质瘤细胞表面及肿瘤周边血管内皮细胞表面均有高度表达特性，以及FALGEA多肽对EGFRvIII有很高的结合常数的特性，提出跨血脑屏障靶向胶质瘤的示踪方法，其中FALGEA、Gd3+-DTPA、IR783B在金纳米颗粒表面，静脉注射后，探针首先通过FALGEA多肽主动靶向并富集到肿瘤新生血管，探针在脑血管内皮细胞表面浓度的增加将提高跨血脑屏障作用，由于EGFRvIII在胶质瘤细胞表面高表达，穿越血脑屏障后的探针将再次靶向到肿瘤细胞上；所述的跨血脑屏障二级靶向机制可得到较单一靶向或EPR效应更佳的肿瘤示踪效果。
本发明的用于指导胶质瘤精准切除的跨血脑屏障靶向多模态纳米探针具有的临床转化前景，其优点有：第一，手持式拉曼仪具有操作简单、多角度检测、实时分析等优点，经临床实践应用于人胶质瘤手术结果显示，AuP-FAL在手持式拉曼仪的帮助下，可以实现术中实时、迅速判断肿瘤边界；第二，相比现有技术的5-ALA荧光引导的手术，AuP-FAL所依赖的SERRS效应具有更高的灵敏度和更弱的自体荧光；第三，动物实验证实AuP-FAL尚未表现出器官毒性，为进一步应用于人体打下了基础。
附图说明
图1.靶向纳米探针和对照探针的合成路线图。
图2.纳米探针的透射电镜图。
图3.纳米探针的粒径和电位图。
图4.纳米探针的拉曼光谱图。
图5.纳米探针靶向EGFRvIII阳性细胞的磁共振和拉曼光谱图。
图6.纳米探针体内磁共振成像图和HE验证图。
图7.纳米探针在拉曼光谱下引导肿瘤切除及HE验证图。
图8.纳米探针示踪肿瘤边界的透射电镜图和拉曼光谱图。
图9.纳米探针的器官毒性评估HE图。
具体实施方式
本发明结合实施例和相应附图做进一步阐释说明，以下实施例仅用于说明目的，不用于限制本发明范围。
实施例1、靶向纳米探针和对照探针的合成
合成路线如图1所示，聚乙二醇SH-PEG-NHS(化合物1，40mg,0.02mmol)和多肽FAL(12.12mg,0.02mmol)溶于0.3mL DMF中，室温搅拌反应2h得到PEG衍生物2。500mL的0.294mM四氯金酸溶液，煮沸，快速加入5mL的38.8mM的柠檬酸钠水溶液，回流反应5min，反应产物过经截流分子量10000的透析袋纯化后得到柠檬酸修饰的金纳米粒3，粒径约为60nm。二亚乙基三胺五乙酸二酸酐(10mg,28μmol)和2-巯基乙胺(1mg,28μmol)溶于0.5mL DMF中，室温搅拌反应4h得到化合物4。化合物4逐滴加入10mL金纳米粒3(0.13μM)中，室温搅拌反应过夜得到金纳米粒5。GdCl3(7.0mg,26μmol)溶于200μL去离子水中(pH 7.4)，加入10mL金纳米粒5(0.13μM)的水溶液中，室温搅拌反应过夜，产物透析纯化后得到金纳米粒6。巯基修饰的IR783B(3mg,3μmol)溶于0.2mL DMF中，逐滴加入10mL金纳米粒6(0.1μM)中，室温搅拌反应过夜得到金纳米粒7。化合物2和PEG-SH(79mg,39.5μmol)溶于0.5mL去离子水中，加入10mL金纳米粒7(0.13μM)的水溶液中，室温搅拌反应过夜，产物透析纯化后得到纳米探针AuP-FAL。PEG-SH(80mg,40μmol)溶于0.5mL去离子水中，加入10mL金纳米粒7(0.13μM)的水溶液中，室温搅拌反应过夜，产物透析纯化后得到纳米探针AuP-PEG。
实施例2、纳米探针的透射电镜图
取适量纳米探针，滴加在透射电镜专用铜网上，置于40度烘箱中烘干后，通过透射电镜采集TEM图，如图2显示，纳米探针粒径约为60nm，分散均匀，金核心外包裹有半透明的聚乙二醇层。
实施例3、对纳米探针的流体动力学粒径分布和Zeta电位的测定
在室温下用动态光散射的方法测定目标纳米探针AuP-FAL和参比探针AuP-PEG的流体动力学半径；用溶于蒸馏水的2.0mg/ml的牛血清白蛋白标准溶液进行设备校准，样品用0.45μm的滤头过滤并用pH 7.4的1X PBS稀释到100g/mL，流体动力学半径和大小分布通过规则化的公式进行运算；在测定纳米探针的表面电荷时，用一种Zeta电位为-50mV的标准溶液校准设备，纳米探针药物溶液用0.45μm的滤头过滤并用10mM的NaCl溶液稀释到200g/mL；
图3显示了AuP-FAL的平均直径是61.2nm和60.7nm，平均Zeta电位是-23.3mV。
实施例4、检测纳米探针的拉曼光谱
使用QE65Pro手持式拉曼检测仪及785nm入射激光进行拉曼光谱检测，如图4拉曼光谱显示，AuP-FAL在509和541cm-1处存在尖锐双峰，可作为该探针的特异性标志。
实施例5、体外靶向作用实验
采用ELISA方法检测AuP-FAL对EGFRvIII的靶向作用，在AuP-FAL上连接生物素，96孔板、37℃培养U87-EGFRvIII(图5A验证)、野生型U87和人血管内皮细胞系bEnd.3过夜，10mg/ml牛血清蛋白封闭，每孔加入16μM AuP-FAL，4℃孵育1小时并用PBST洗涤，每孔加入100μL辣根过氧化物酶标记的链霉亲和素，室温孵育1小时并用PBST洗涤，加入100μL缓冲液，避光孵育20分钟，每孔加入100μL硫磺酸(1.0M)，在492nm处检测吸光度，结果显示(如图5B所示)，AuP-FAL对EGFRvIII阳性胶质瘤细胞的结合能力是EGFRvIII阴性胶质瘤细胞的1.6倍、血管内皮细胞的2.2倍；
在此基础上检测细胞摄取。37℃用0.5nM AuP-FAL或AuP-PEG孵育U87-EGFRvIII24小时，细胞离心后采用Bruker Biospec 7.0T磁共振仪检测T1map，AuP-FAL处理后的EGFRvIII阳性胶质瘤细胞的T1值明显下降，而AuP-PEG处理后的EGFRvIII阳性胶质瘤细胞的T1值无明显改变(如图5C所示)；此外，PBS洗涤并用4％福尔马林固定5分钟，采用Renishaw inVia拉曼显微镜、785nm入射激光采集拉曼信号，AuP-FAL处理后的EGFRvIII阳性胶质瘤细胞可检测到特征性的双峰拉曼信号，而AuP-PEG处理后的EGFRvIII阳性胶质瘤细胞则检测不到(如图5D、E所示)。
实施例6、磁共振成像研究实验
本发明的所有的动物实验依照复旦大学伦理委员会评估和认可的指南进行；EGFRvIII阳性的U87MG胶质母细胞瘤细胞(1.0×106细胞重悬于5μL PBS)在有小鼠衔接器的立体定位仪的协助下被接种到裸鼠的右侧纹状体(前囟点旁开1.8mm，往前0.6mm，深3mm)；接种后14－18天，颅内肿瘤长到直径0.2－0.5mm大小，用以进行显像实验；
在Bruker Biospec 7.0T磁共振仪上获取活体磁共振成像，实验前，将自制的三通导管埋入小鼠尾静脉，该导管系统通过T形接头(Cole-Parmer,Vernon Hills,IL)进行调节，小鼠通过异氟烷(0.5－2％)及氧气混合气体予以麻醉，麻醉后的小鼠头部被固定在自制的表面线圈中，小鼠在磁体线圈中的体温通过水浴加热进行维持，呼吸则通过BrukePhysioGard系统进行持续监控，每只小鼠从尾静脉注射0.05mmol/kg[Gd3+]对应的AuP-FAL或AuP-PEG，共0.25mL体积的纳米影像药物PBS溶液，采集注射前、后脑部动态T1加权像(自旋回波脉冲序列，视野(FOV)2cm×2cm，矩阵128×128，TR＝300ms，TE＝11ms，NA＝8)，三维T1加权像用一个快速小角度激发成像序列(FLASH)来获得，flip angle＝45°,FOV＝1.5cm×1.5cm×1.5cm,矩阵128×128×32,TR＝35ms,TE＝6.2ms,NA＝8，目标区域(ROI)在不同时间点的增强强度(IE)通过以下公式表示IE＝(RI(t)–RI(0))/RI(0)×100％，其中，RI(t)对应于在各个时间点测定的标准化的信号强度，而RI(0)是纳米探针注射前标准化的信号强度，时间依赖的肿瘤和周围正常脑组织之间的荧光强度比(T/N比值)用纳米影像药物注射前的值进行标准化；
如图6所示，注射AuP-FAL的EGFRvIII阳性胶质瘤模型在MRI T1序列上的信噪比明显高于注射AuP-PEG的模型；AuP-FAL在体内循环时间较长，注射24小时后仍可看到清醒的图像。
实施例7、拉曼指导的手术研究实验
如图7所示，上述肿瘤鼠模型在术前24小时经尾静脉注射AuP-FAL，术中依据切除程度适时采用手持式拉曼仪检测拉曼信号，直至检测不出AuP-FAL特征性拉曼峰，术后用小鼠大脑被离体并浸没在formalin和PFA的混合液中(体积1：9混合)固定适当时间，固定好的组织用石蜡包埋并切成3－4μm厚，切片进行H&E染色，并用Leica MZ75高性能立体显微镜2.5X和5.0X的物镜观察，最终组织学证实肿瘤完全切除。
实施例8、肿瘤边界示踪研究实验
如图8所示，采用上述电镜及拉曼显微镜方法检测获得的小鼠脑切片，电镜结果显示，注射AuP-FAL后的EGFRvIII阳性胶质瘤模型肿瘤内存在纳米颗粒、正常脑组织内无纳米颗粒；拉曼光谱显示，注射AuP-FAL后的EGFRvIII阳性胶质瘤模型瘤内和近肿瘤边界处均检测到特征信号，正常脑组织未检测到特征信号。
实施例9、器官毒性评估实验
健康小鼠经尾静脉注射PBS或AuP-FAL，1天或7天后处死小鼠，解剖取出心、肝、脾、肺、肾、脑等主要脏器，按上述实施例所述方法行HE染色，评估器官受损程度，结果如图9所示，未发现明显细胞坏死、水肿等毒性表现。

Claims (6)

1.一种用于指导胶质瘤精准切除的多模态纳米探针，其特征在于，以球形金纳米颗粒AuNS为基底，在其表面连接顺磁性螯合剂Gd3+-DTPA和拉曼信号分子IR783B，以PEG为载体连接靶向EGFRvIII的多肽FALGEA，制成多模态纳米探针AuP-FAL。
2.按权利要求1所述的用于指导胶质瘤精准切除的多模态纳米探针，其特征在于，所述的多模态指T1加权磁共振成像和表面增强共振拉曼散射成像。
3.按权利要求1所述的用于指导胶质瘤精准切除的多模态纳米探针，其特征在于，所述的FALGEA多肽的氨基酸序列为Phe-Ala-Leu-Gly-Glu-Ala，该多肽特异性识别胶质瘤细胞上的表皮生长因子受体变体EGFRvIII。
4.按权利要求1所述的用于指导胶质瘤精准切除的多模态纳米探针，其特征在于，用于桥连多肽的双功能化聚乙二醇的两端分别是N-羟基琥珀酰亚胺酯和巯基。
5.按权利要求1所述的用于指导胶质瘤精准切除的多模态纳米探针，其特征在于，拉曼光谱显示在509和541 cm-1处存在尖锐的特征双峰。
6.权利要求1所述的用于指导胶质瘤精准切除的多模态纳米探针在用于制备胶质瘤术前精确规划和术中实时导航切除方案中所用药物的用途。
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