超超磁氧化铁纳米探针在肺外结核检测的合成、表征及应用
1Department of Pulmonary and Critical Care Medicine,Taipei Medical University-Shuang Ho Hospital, 2Department of Research and Department of Dentistry,Taipei Medical University / Shuang-Ho Hospital, 3Department of Pathology,Taipei Medical University, 4Department of Urology, School of Medicine, College of Medicine,Taipei Medical University, 5McLean Imaging Center,McLean Hospital/Harvard Medical School, 6Department of Physical Medicine and Rehabilitation, School of Medicine, College of Medicine,Taipei Medical University, 7Department of Medicine,Mackay Medical College, 8Department of Biological Science and Technology,National Chiao Tung University, 9Graduate Institute of Clinical Medicine,Taipei Medical University
Summary
为了改进结核分枝杆菌抗原的血清学诊断测试，我们开发了超副磁性氧化铁纳米探针来检测肺外结核病。
Abstract
合成了一种分子成像探针，包括超副磁性氧化铁（SPIO）纳米颗粒和结核分枝杆菌表面抗体（MtbsAb），以提高肺外结核（ETB）的成像灵敏度。合成了SPIO纳米探针，并与MtbsAb结合。纯化SPIO-MtbsAb纳米探针采用TEM和NMR进行特征。为了确定探针的靶向能力，SPIO-MtbsAb纳米探针与Mtb一起孵育，用于体外成像测定，并注射到Mtb接种小鼠体内进行磁共振（MR）的体内调查。Mtb和THP1细胞磁共振成像（MRI）的对比度增强降低与SPIO-MtbsAb纳米探针浓度成正比。在30分钟静脉注射SPIO-MtbsAb纳米探针注射Mtb感染小鼠后，与接受PBS注射的小鼠相比，在T2加权MR图像中，肉芽肿位位的信号强度提高了14倍。MtbsAb 纳米探针可用作 ETB 检测的新方式。
Introduction
在全球范围内，肺外结核病（ETB）占结核病病例的很大比例。然而，ETB诊断往往错过或延迟，因为它的阴险的临床表现和诊断测试的不良表现;错误的结果包括痰涂片对酸快杆菌的阴性，缺乏组织病理学上的肉芽肿组织，或未能培养结核分枝杆菌（Mtb）。与典型病例相比，ETB发生频率较低，并且很少涉及 Mtb 杆菌的释放。此外，它通常被本地化在难以接近的部位，如淋巴结，胸膜，和骨关节区域1。因此，侵入性程序，以获得足够的临床标本，这使得细菌确认的风险和困难，是必不可少的2，3，4。
市售的ETB抗体检测测试在临床检测中不可靠，因为它们的灵敏度范围广泛（0.00-1.00）和所有肺外位点结合的特异性（0.59-1.00）。5 。干扰素β、培养性滤蛋白（CFP）和早期分泌抗原靶点（ESAT）的酶相关免疫点（ELISPOT）检测已用于诊断潜伏和活性结核病。然而，结果因诊断ETB6、7、8的不同疾病部位而异。此外，皮肤PPD（纯化蛋白衍生物）和昆蒂弗龙-结核病经常提供假阴性结果9。QuantiFERON-TB-2G是一种全血免疫反应测定，不需要从受影响的器官标本，这可能是一个替代诊断工具6，10，11。其他通常用于结核病脑膜炎的诊断方法，如聚合酶链反应，仍然过于麻木不仁，无法自信地排除临床诊断12，13。这些常规测试表明，没有足够的诊断信息来发现肺外感染部位。因此，临床上需要新的诊断模式。
分子成像旨在设计新的工具，可以直接筛选体内疾病过程的特定分子靶点14，15。超副磁性氧化铁（SPIO），一种T2加权NMR造影剂，可以显著提高磁共振（MR）成像（MRI）16、17的特异性和灵敏度。这种新的功能成像模式可以通过配体受体相互作用在分子水平上精确绘制组织变化草图。在这项研究中，合成了一种新的分子成像探针，包括SPIO纳米粒子，与Mtb表面抗体（MtbsAb）结合，用于ETB诊断。SPIO纳米探针是微创对组织和身体在检查18，19。此外，这些纳米探针由于其顺磁特性，可以在低浓度下显示精确的MR图像。此外，SPIO纳米探针似乎引起最少的过敏反应，因为铁离子的存在是正常生理的一部分。在这里，在细胞和动物模型中评估了针对ETB的SPIO-MtbsAb纳米探针的灵敏度和特异性。结果表明，纳米探针可作为超敏感成像剂用于ETB诊断。
Protocol
所有有关动物实验的协议均遵循美国国家卫生研究院《实验室动物护理和使用指南》（2011 年第 8 版）的标准实验室动物育种操作程序，并经机构动物护理和使用委员会。 1. SPIO纳米粒子合成 在室温下大力搅拌 Dextran T-40 （5 mL; 50% w/w） 和水性 FeCl3±6H2O （0.45 g; 2.77 mmol） 和 FeCl2×4H2O （0.32 g; 2.52 mmol） 溶液的混合物，制备涂有氧化铁的磁?…
Representative Results
SPIO-MtbsAb 纳米探针合成和表征SPIO纳米粒子被设计成与MtbsAb结合。稳定在SPIO纳米粒子表面的dextran被上氯二苯并联。SPIO纳米粒子随后与EDBE结合，以激活dextran端的初级胺功能组。SA 然后被结合形成 SPIO-EDBE-SA。SPIO-MtbsAb 纳米探针在耦合剂存在的情况下，通过 MtbsAb 与 SPIO-EDBE-SA 结合，在最后一步中形成。SPIO-MtbsAb纳米探针的TEM图像（图1）表明SPIO-MtbsAb纳米探针的外…
Discussion
与相关研究类似，我们对SPIO-MtbsAb纳米探针的发现表明Mtb27、28具有显著特异性。在小鼠模型中注射TB后1个月，发现了皮下Mtb肉芽肿。典型的结核肉芽肿学发现包括淋巴细胞渗透、上皮性巨噬细胞的存在和新血管化。酸速杆菌分散在结核病变中，证实了MtbsAb免疫性化学发现。这表明Mtb表面抗原和MtbsAb之间的免疫反应。柏林蓝突出显示了与MtbsAb相同的区域?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢台湾经济部（NSC-101-2120-M-038-001，MOST 104-2622-B-038-007，MOST 105-2622-B-038-004）的财政支持，以开展此项研究工作。这份手稿由华莱士学术编辑编辑编辑。
Materials
| (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate | Sigma-Aldrich | ||
| 1-hydroxybenzotriazole | Sigma-Aldrich | ||
| dextran(T-40) | GE Healthcare Bio-sciences AB | ||
| epichlorohydrin, 2,2'-(ethylenedioxy)bis(ethylamine) | Sigma-Aldrich | ||
| ferric chloride hexahydrate | Fluka | ||
| ferrous chloride tetrahydrate | Fluka | ||
| Human monocytic THP-1 | |||
| M. bovis BCG | Pasteur Mérieux | Connaught strain; ImmuCyst Aventis | |
| MRI | GE medical Systems | 3.0-T, Signa | |
| NH4OH | Fluka | ||
| NMR relaxometer | Bruker | NMS-120 Minispec | |
| Sephacryl S-300 | GE Healthcare Bio-sciences AB | ||
| Sephadex G-25 | GE Healthcare Bio-sciences AB | ||
| SPECTRUM molecular porous membrane tubing, 12,000 -14,000 MW cut off | Spectrum Laboratories Inc | ||
| TB surface antibody- Polyclonal Antibody to Mtb | Acris Antibodies GmbH | BP2027 | |
| transmission electron microscope | JEOL | JEM-2000 EX II |
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Cite This Article
Lee, C., Chiu, L., Fang, C., Yeh, S., Zuo, C. S., Chen, S., Kuo, L., Wang, Y., Lai, W. T. Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection. J. Vis. Exp. (156), e58227, doi:10.3791/58227 (2020).