Name: using magnetometry to monitor cellular incorporation and subsequent biodegradation of chemically synthetized iron oxide nanoparticles
Uploaded: 2021-02-27T13:30:00
Description: Watch this Scientific Journal Video about Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles at JoVE.com

这项工作得到了欧洲联盟（ERC-2014-CoG项目马蒂斯#648779）的支持。作者想承认巴黎大学13号的CNanoMat物理化学特征平台。

1. 磁纳米粒子的合成
<ol><li>核心合成–微波辅助<ol>
<li>溶解 400 毫克铁 （III） 乙酰乙酰酮 （+99.9%）在 10 mL 苯酒精 （BA， 99.8%）在30毫升单波玻璃瓶内。</li>
		<li>在20分钟内将悬架的温度从25°C提高到250°C（以11.25°C/min的速度），并使用微波反应器将其保持在250°C30分钟。</li>
		<li>将苯醇中悬浮的纳米粒子转移到玻璃瓶中，使用新磁铁分离纳米粒子30分钟。</li>
		<li>用10毫升二氯甲烷、1 M氢氧化钠?...

<ol>
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使用快速高效的微波合成，磁性纳米粒子可以轻松合成，控制大小，并进一步涂上给定分子。关键的一步是在真空下储存铁盐和苯基醇，以保持小的分散大小。苯基醇同时作为溶剂和配体，允许直接获得校准裸氧化铁，而无需额外的配体。纳米粒子在水中转移后，裸露的磁性纳米粒子可以很容易地涂上多种配体。这种涂层具有纳米粒子和纳米粒子细胞相互作用的特性，可以影响其细胞内化，生物?...

人们越来越关注氧化铁纳米粒子的磁性特征，用于广泛的生物医学应用。他们对磁共振的反应使得它们成为磁共振成像（MRI）的可靠对比剂，这是再生医学的一个优势，在植入1后，可以跟踪带有磁纳米粒子的细胞。利用磁场，也可以在远处引导细胞：这样，细胞球体2，3，环4，或表5可以进行磁力工程，也可以远程刺激6，在无脚手架组织发展的资产。这些纳米粒子的可能性范围还包括药物输送系统7，8和磁性和光诱导的高温治疗杀死癌细胞9，10，11。对于所有这些应用，纳米粒子通过静脉注射或直接内化在细胞中集成在生物环境中，然后留在细胞内，这使人对其细胞内的命运产生疑问。
在体内分析传达了对纳米粒子在生物体中命运的一般理解：在血液中注射后，它们首先被肝脏（库普弗细胞）、脾脏和骨髓的巨噬细胞捕获，逐渐退化，并加入生物体12、13、14、15、16、17、18、19的铁池。只有由于纳米粒子在整个生物体中的循环，才能进行定性观测。通常，传输电子显微镜 （TEM） 可用于直接观察纳米粒子，并且可以通过剂量确定器官中铁的存在。最近，他们的命运被直接评估在细胞池，这意味着在近距离电路没有铁逃逸，允许定量测量他们的生物转化在细胞水平20，21，22。通过分析与其结构完整性密切相关的纳米粒子的磁性，可以进行此类测量。振动样品磁力计 （VSM） 是一种定期振动样品的技术，因此所诱导的通量线圈测量提供了应用磁场样品的磁时刻。这种同步检测允许快速测量，这是确定大量样品20，21，22，23的磁时刻的资产。然后，VSM检索到的宏观磁信号对与纳米粒子的大小和结构直接相关的整个生物样本进行了定量概述。特别是，它提供了样品饱和时的磁时刻（以 emu 表示），这是直接量化样品中存在的磁性纳米粒子的数量，分别到其特定的磁性。
结果表明，磁性纳米粒子的细胞内处理与其结构特征紧密相连。这些功能可以通过最佳的合成协议进行控制。每个协议都具有优点和局限性。氧化铁纳米粒子通常通过共生铁离子24在液态溶液中合成。为克服纳米粒子尺寸多分散性的局限性，已开发出聚醇介质溶胶法等其他合成方法25种。通过热分解的无声方法导致生产非常校准的氧化铁纳米粒子26。然而，使用大量的表面活性剂，如烯酰胺或油酸，使其功能化和生物医学应用的水转移复杂化。因此，我们通过一条无糖溶胶路线合成这种磁性纳米粒子，从而达到高晶度、纯度和可重复性。该协议产生控制良好的大小纳米粒子，可以通过温度变化28调整。然而，微波辅助的非水溶胶路由的纳米粒子的上限约为12纳米。此程序将不适用于在室温下使用铁氧颗粒的应用。除了核心合成，另一个主要功能要考虑的是涂层。涂层位于纳米粒子表面，充当锚定分子，帮助纳米粒子有针对性地内化，或者保护纳米粒子免受降解。由于苯乙醇同时作为氧气源和配体，光裸纳米粒子的产生无需额外的表面活性剂或配体。然后，纳米粒子在没有表面活性剂交换过程的情况下，在合成后很容易表面功能化。
其中，评估了两种纳米粒子，它们的核心相同，涂层也不同。该核心采用快速高效的微波技术合成。比较的两种涂层包括柠檬酸，这是生物医学应用中用作涂层剂最多的涂层之一，29、30和聚丙烯酸 （PAA）， 聚合物涂层具有大量的溷合功能。然后，VSM磁力计测量首先用于量化细胞吸收的纳米粒子，然后作为干细胞内化后纳米粒子结构完整性的直接评估。结果表明，孵化浓度影响纳米粒子的吸收，涂层影响其降解，PAA的大量锚定分子保护核心免受降解。

<table>
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA (1x)</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl alcohol for synthesis</td>
			<td>Sigma Aldrich</td>
			<td>8.22259</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>Prepare a 1 mM stock solution diluted in Ethanol 100% and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Dichloromethane &#8805;99% stabilised, GPR RECTAPUR</td>
			<td>VWR Chemicals</td>
			<td>23367</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with Glutamax I</td>
			<td>Life Technologies</td>
			<td>31966-021</td>
			<td>No sodium pyruvate, no HEPES</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>VWR</td>
			<td>20821.310</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution 10% neutral buffered</td>
			<td>Sigma</td>
			<td>HT5012</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 1.0N Standardized Solution</td>
			<td>Alfa Aesar</td>
			<td>35640</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron(III) acetylacetonate (&#62; 99.9%)</td>
			<td>Sigma Aldrich</td>
			<td>517003</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS Premix Universal Culture Supplement (20x)</td>
			<td>Corning</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid 2-phosphate</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td>Prepare a fresh concentrated solution (25 mM) diluted in distilled water</td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Sigma</td>
			<td>P5607</td>
			<td>Prepare a 175 mM stock solution diluted in distilled water and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Mesenchymal Stem Cell (MSC)</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>Monowave glass vial</td>
			<td>Anton Paar</td>
			<td>82723_us</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave reactor</td>
			<td>Anton Paar</td>
			<td>Monowave 300</td>
			<td></td>
		</tr>
		<tr>
			<td>MSCGM BulletKit medium</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td>For the complete medium, add the provided BulletKit (containing serum, glutamine and antibiotics) to the MSCGM medium</td>
		</tr>
		<tr>
			<td>PBS w/o CaCl2 w/o MgCl2</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin (10.000U/mL)/Streptomicin (10.000&#181;g/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(acrylic acid, sodium salt)</td>
			<td>Sigma Aldrich</td>
			<td>416010</td>
			<td>MW = 1200 g/mol</td>
		</tr>
		<tr>
			<td>RPMI medium 1640, no Glutamine</td>
			<td>Life Technologies</td>
			<td>31870-025</td>
			<td>No sodium pyruvate, no HEPES</td>
		</tr>
		<tr>
			<td>Sodium hydroxide, 1.0N Standardized Solution</td>
			<td>Alfa Aesar</td>
			<td>35629</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate solution 100mM</td>
			<td>Sigma</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile conical centrifuge tube</td>
			<td>Falcon</td>
			<td>352097</td>
			<td>15 mL tubes</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-sodium citrate</td>
			<td>VWR</td>
			<td>33615.268</td>
			<td>Prepare a 1 M stock solution diluted in distilled water and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Tri-Sodium Citrate Dihydrate, Certified AR for Analysis</td>
			<td>Sigma Aldrich</td>
			<td>10396430</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra centrifugal filter</td>
			<td>Amicon</td>
			<td>AC S510024</td>
			<td></td>
		</tr>
	</tbody>
</table>

using magnetometry to monitor cellular incorporation and subsequent biodegradation of chemically synthetized iron oxide nanoparticles

由氧化铁制成的磁性纳米粒子对广泛的生物医学应用有着特殊的兴趣，它们经常被内化在细胞中，然后留在细胞内。一个挑战是用可靠和精确的方法评估他们在细胞内环境中的命运。在此，我们介绍了振动样品磁力计 （VSM） 的使用，通过测量其磁力矩精确量化细胞内磁性纳米粒子的完整性。干细胞首先被标记为两种类型的磁性纳米粒子：纳米粒子具有通过快速高效的微波无味溶胶合成产生的相同核心，其涂层也不同：常用柠檬酸分子与聚丙烯酸相比。然后通过离心实现3D细胞球体的形成，用VSM在不同时间测量这些球体的磁力矩。获得的时刻是纳米粒子完整性的直接指纹，其值下降表明纳米粒子退化。对于两个纳米粒子来说，磁性时刻会随着培养时间的减少而减少，揭示它们的生物降解。与柠檬酸相比，还显示了聚丙烯酸涂层的保护作用。

使用微波辅助合成，产生单分散 8.8 ± 2.5 纳米核心尺寸的磁性纳米粒子，并涂上柠檬酸盐或 PAA（图 1A）。然后，干细胞与这些纳米粒子一起在培养基中以给定的浓度分散30分钟，导致其内分泌和细胞内分泌（图1B）内。然后，磁性干细胞悬浮在介质、离心体中，形成的细胞颗粒培养长达21天（图1C）。获得的球体是固定的，例如停?...

Watch this Scientific Journal Video about Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles at JoVE.com

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

氧化铁纳米粒子通过无味溶胶程序合成，并涂有离子短分子或聚合物。利用磁力计（VSM）来监测人类干细胞内磁纳米粒子的整合和生物转化。

使用磁力学监测化学合成氧化铁纳米粒子的细胞合并和后续生物降解

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...

本视频介绍了磁纳米粒子合成和利用磁力学拍摄生物环境中转化的快速高效方法。磁性纳米粒子已经引起了生物医学应用越来越多的关注，重要的是要明白，命运一旦内化在细胞中。这是通过测量细胞磁性在这里实现的。对于磁性纳米粒子的合成和测量，结果可能受到污染的影响。使用清洁良好的化学小瓶，确保磁力计设备不受污染。磁力学样品架通常用于粉末样品。我们展示了如何使持有人的使用适应液体或生物样品。在30毫升的马纳波玻璃瓶中，将400毫克铁三乙酰乙酰酮溶解在10毫升苯甲酸酯中。使用微波反应器，将悬浮液以每分钟11.25摄氏度的速度加热至250摄氏度，并将悬浮液保持在250摄氏度。要分离纳米粒子，请应用新钛磁铁30分钟。要对纳米粒子进行颗粒，使用 10 毫升指定的洗涤液进行一系列洗涤，并应用新钛磁铁5分钟。取出过滤液，在减去两摩尔盐酸中加入12毫升10。离心悬架和 100 千吨过滤器在 2，200 倍 G 10 分钟。然后将纳米粒子在10毫升10到负2摩尔盐酸中重新悬索。准备手稿中描述的涂层分子溶液。在涂层分子和纳米粒子之间以5比1的质量将10毫升纳米粒子液分散到涂层分子溶液中。然后使用磁搅拌器在室温下搅拌混合物两小时。反应完成后，使用一个摩尔氢氧化钠将混合物的pH数调整为7。当人类间质干细胞的培养处于第五和90%的交汇点时，去除介质，用无血清RPMI冲洗细胞，而无谷氨酰胺。每150平方厘米培养瓶，加入10毫升纳米粒子悬浮液。在37摄氏度和5%的二氧化碳下孵化烧瓶30分钟。然后丢弃含有纳米粒子悬浮液的介质，用无血清RPMI 1640清洗细胞一次。添加 DMEM 补充 10% FBS 和 1% 青霉素链霉素。在37摄氏度和5%的二氧化碳下孵化过夜，使纳米粒子完全内化。在每个磁性标记的 MSC 瓶中加入 10 毫升 0.05% 的 EDTA 预制温度为 37 摄氏度。两到三分钟后，当细胞分离时，通过添加三分之一的预先警告补充DMEM的体积，立即灭活试金刚一。将分离的细胞以260倍G为基数，为期5分钟。吸气介质，在小体积的细胞球体培养介质中重新悬生细胞，浓度约为每50微升20万细胞。将一毫升新鲜制备的细胞球体培养介质添加到 15 毫升无菌圆锥形离心管中。然后添加相当于20万个细胞的细胞悬浮体积。将这些磁性标记的细胞在180倍G下离心三分钟，以便在管子底部形成细胞颗粒。然后将管子放置在37摄氏度和5%的二氧化碳孵化器中，保持瓶盖稍微打开以进行气体交换。将一定体积的磁性纳米粒子或固定的单细胞球体悬浮物放入振动样品磁力计或 VSM 样本支架中。如有必要，PTFE 胶带可用于防止任何泄漏。将样本持有人插入 VSM 并扫描样本偏移。将样品放置在与磁化最大值对应的位置。以每秒 20 欧尔斯泰特的速度在负 1，500 Oersteds 和正 1，500 Oersteds 之间的低磁场进行首次测量。以每秒200欧尔斯泰特的速度，在零奥尔斯泰特和正30，000欧尔斯泰特之间的高磁场进行第二次测量。在振动样品磁力计中测量了分散在液态溶液中的10微光片。曲线第二部分的坡度是诊断常数，表示来自水和样本持有人的诊断信号。这种诊断常数是从溶液的磁性时刻中减去的，只获取纳米粒子的磁力矩。然后确定饱和时的磁性时刻。涂层纳米粒子内化在干细胞中。电子显微镜图像显示，柠檬酸盐涂层和PAA涂层的纳米粒子被限制在内体中。细胞通过离心弹丸后，形成了一种细胞球体，可以长时间保存在培养中。还使用 VSM 测量了单个细胞球体，并利用饱和磁矩来计算细胞样本中的纳米粒子数量。纳米粒子的吸收取决于它们的孵化浓度。随着时间的推移，细胞球体的磁性下降表明纳米粒子的生物降解，电子显微镜证实了这一点。与PAA涂层的纳米粒子相比，引文涂层纳米粒子的降解更为严重。利用磁力学，可以精确量化细胞内磁纳米粒子的降解，并评估纳米粒子的未来对对原始行为感兴趣的人的影响。在此协议中，磁力学测量是在细胞球体上进行的，但它们可以扩展到细胞中，进入悬浮物或器官。通过上述方法，您可以探索新设计的磁纳米粒子在不同细胞系统中的相互作用，还可以跟踪其体内生物分布和命运。

Research

JoVE Journal

Bioengineering

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

科研

生物工程

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

细胞标记和超顺磁性氧化铁纳米粒子靶向

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Bioprinted蜂窝构造的可行性使用三分配器笛卡尔打印机

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光​​谱遥感制图

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

动脉粥样硬化的快速检测的氧化铁纳米颗粒微波驱动综合

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

非规范氨基酸选择性压力合成抗菌肽的研究

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...