Name: synthesis, characterization, and application of superparamagnetic iron oxide nanoprobes for extrapulmonary tuberculosis detection
Uploaded: 2020-02-16T12:00:00
Description: Watch this Scientific Journal Video about Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection at JoVE.com

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。

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
<ol><li>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?...

<ol>
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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-hydroxybenzotriazole</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dextran(T-40)</td>
			<td>GE Healthcare Bio-sciences AB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>epichlorohydrin, 2,2&#39;-(ethylenedioxy)bis(ethylamine)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ferric chloride hexahydrate</td>
			<td>Fluka</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ferrous chloride tetrahydrate</td>
			<td>Fluka</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human monocytic THP-1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M. bovis BCG</td>
			<td>Pasteur M&#233;rieux</td>
			<td>Connaught strain; ImmuCyst Aventis</td>
			<td></td>
		</tr>
		<tr>
			<td>MRI</td>
			<td>GE medical Systems</td>
			<td>3.0-T, Signa</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4OH</td>
			<td>Fluka</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NMR relaxometer</td>
			<td>Bruker</td>
			<td>NMS-120 Minispec</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephacryl S-300</td>
			<td>GE Healthcare Bio-sciences AB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex G-25</td>
			<td>GE Healthcare Bio-sciences AB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SPECTRUM molecular porous membrane tubing, 12,000 -14,000 MW cut off</td>
			<td>Spectrum Laboratories Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TB surface antibody- Polyclonal Antibody to Mtb</td>
			<td>Acris Antibodies GmbH</td>
			<td>BP2027</td>
			<td></td>
		</tr>
		<tr>
			<td>transmission electron microscope</td>
			<td>JEOL</td>
			<td>JEM-2000 EX II</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis, characterization, and application of superparamagnetic iron oxide nanoprobes for extrapulmonary tuberculosis detection

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。

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...

Watch this Scientific Journal Video about Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection at JoVE.com

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

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。

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

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

在全球范围内，肺外结核病诊断往往被推迟，因为它需要手术干预以确认，因为非特定的临床表现和诊断测试的不良表现。为了改进结核杆菌抗原的血清学诊断测试，我们开发了用于检测肺外结核的超顺磁氧化铁纳米蛋白。TB-SPIO 纳米蛋白由于其顺磁特性，可以低浓度提供精确的磁共振图像，而不会引起任何自身免疫反应。TB-SPIO纳米蛋白能够定位和检测结核分枝杆菌感染。它们也可以用作 MRI 对比剂，用于检测其他疾病。在尝试协议之前，请务必仔细研究协议，因为实验的每个部分都使用特定的核心技术。该方法的视觉演示可以帮助学生了解如何实现这个过程，特别是对于一些更复杂的实验。为准备脱硝层涂层氧化铁磁性纳米粒子，在室温下大力将5毫升脱氧核糖核糖T40与45毫克水性氯化铁六水合物和32毫克氯化铁四水合物溶液混合。快速添加 10 毫升氢氧化铵，并使用搅拌棒在室温下搅拌产生的黑色悬浮液一小时。在搅拌孵育结束时，将溶液离心并取出集料。为了将最终的SPIO产品与未绑定的脱硝剂T40分离，将整个五毫升反应混合物装入2.5 x 33厘米的凝胶过滤色谱柱中，用0.1摩尔醋酸钠和0.15摩尔氯化钠缓冲液将德克斯特兰吸气。然后，在空隙体积中收集纯化的脱衣涂层氧化铁磁性纳米粒子，并按照标准规程，在330纳米和490纳米时检测铁和脱氧核糖核酸柱的脱硫。为了产生SPIO结合结核分枝杆菌表面抗体，首先在室温下将SPIO-EBDE碱性溶液的10毫升与1克沙奇氢化物混合24小时，合成SPIO-EDBE-succinic氢化物。第二天，使用12，000至4，000个分子量截止分子多孔膜管，以每变化26小时改变两升蒸馏水，对溶液进行透析。上次更换后，将产生的溶液的100微升加入400微升，每毫升TB表面抗体4.5毫克，然后加入1-羟基苯二甲醇和苯并三甲醇-1-二氧三叶草磷六氟磷酸作为催化剂，在室温下搅拌24小时。使用 PBS 作为缓冲液，通过凝胶过滤色谱将溶液与未绑定抗体分离。要确定平均粒径、形态和大小分布，请将复合分散物滴入 200 网状铜网格，并在室温下风干样品，然后加载到传输电子显微镜上，以进行 100 千伏成像。要测量纳米刺激的松弛时间值，请使用20兆赫和37摄氏度的核磁共振松弛计加减1度。对于纳米蛋白脉冲细胞成像，首先根据标准细胞培养协议培育人类THP-1单核细胞。当细胞达到适当的激活阶段时，将1倍10对7个单核细胞进行预孵化，用1倍10到分枝杆菌BCG的第六个菌落形成单元，进行一小时。在孵育结束时，将细胞转移到一毫升微离心管中，并在每管中加入两毫摩尔的SPIO结核分枝杆菌表面抗体结合纳米蛋白，在37摄氏度和5%的二氧化碳下进行一小时的孵育。在孵育结束时，通过离心收集细胞，并在200微升新鲜介质中重新供应颗粒。然后，使用快速梯度回声脉冲序列通过 3.0-T 磁共振成像扫描样品，以确定纳米刺激的特异性和灵敏度。要用M.bovis BCG为实验动物接种疫苗，首先在索顿的培养基中重组冻干疫苗或细菌库存，然后用盐水稀释重组的库存溶液。接下来，将溶液的100微升装入每只动物一毫升注射器中，将全部细菌内，以热处理方式注射到每只小鼠的左背或右背肩皮中。对于活纳米探针动物体内磁共振成像，在将两个悬浮在200微升盐水中的SPIO-TB抗体探针注入每只小鼠的尾静脉之前，获取每个麻醉实验动物的T2加权快速自旋图像。然后，在注射后立即对动物进行图像，并每隔五分钟对随后 30 分钟进行一次成像。在成像会话结束时，使用信号强度定量分析所有磁共振图像，以测量 M.tuberculosis 颗粒瘤中心与粒体区域相邻的背肌的可比位置的指定感兴趣区域。然后，使用该公式在注入对比剂之前和注射后 3 小时使用信号强度测量来计算相对信号增强。接种一个月后，从每只动物的皮内接种地点采集组织，用赤氧素和 eosin、Ziehl-Neelsen 染色用于酸性快细菌的 Ziehl-Neelsen 污渍以及用于铁铁的柏林蓝色，将正式固定石蜡嵌入 5 至 10 微米厚的组织样本。SPIO-TB表面抗体纳米蛋白的透射电子显微镜成像显示，纳米蛋白具有分散性，平均尺寸为每核3.8正负0.4纳米。M.bovis BCG 和酸快细菌菌株可以通过 Ziehl-Neelsen 染色检测。在用含有铁铁的探针进行分离和培养后，可以通过柏林蓝色染色来识别细菌。SPIO-TB表面抗体纳米蛋白的结核靶向程度可以通过T2加权磁共振成像确定，如在以浓度依赖的方式发生的纳米刺激存在时信号强度降低所示。在纳米注射动物中，M.tuberculosis颗粒瘤区域信号减少的T2加权增强比对照点高约14倍。感染一个月后，在纳米蛋白注射的C57黑色6小鼠中可以观察到有组织的皮下粒体瘤，这些病变中注意到新的血液血管化和淋巴细胞和上皮巨噬细胞聚集体。阳性M.tuberculosis表达也观察到在粒状区域酸快杆菌染色阳性在病变部位。柏林蓝，铁铁阳性污渍，也可以用来确定探头对结核的敏感性。对于生命体内磁共振图像，纳米刺激注射动物在注射SPIO-TB抗体探针之前，获取每个麻醉实验动物的基线T2加权快速自旋图像。现在，我们已经建立了S单位检测肺外结核病的技术，这些抗体可以用来专门诊断肺外结核病。

Research

JoVE Journal

Medicine

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Production and Detection of Reactive Oxygen Species ROS in Cancers

在癌症的生产和检测活性氧（ROS）的

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

科研

医学

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Ferumoxytol，一个FDA批准的氧化铁纳米粒子标记干细胞

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

Analytical Techniques for Assaying Nitric Oxide Bioactivity

为含量测定一氧化氮生物活性分析技术

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

癌症治疗监测的切伦科夫发光成像（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

建立和UTI和CAUTI表征在小鼠模型

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

板基检测血液中一氧化氮的 vasp 磷酸化

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

聚乙胺包覆氧化铁纳米粒子作为载体, 在体外和体内向巨噬细胞输送小干扰 rna

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

Assessment and Characterization of Hyaloid Vessels in Mice

小鼠透明血管的评估与表征

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

跟踪超副磁性氧化铁标记的美感血干细胞使用MRI后，创伤性脑损伤Murine模型内伤分娩

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Synthesis and Characterization of Multi-Modal Phase-Change Porphyrin Droplets

多模相变化波菲林液滴的合成和特征

Phase-change droplets are a class of ultrasound contrast agents that can convert into echogenic microbubbles in situ with the application of sufficient ...