This study is supported by a grant from Comunidad de Madrid (S2010/BMD-2326, Inmunothercan-CM), by Fundacio La Marato de TV3 (70/C/2012) and by and by Spanish Economy Ministry (MAT2013-47303 P).

1.试剂的制备<ol><li>制备1mM的HEPES缓冲液中溶解23.8毫克HEPES在100毫升蒸馏水中。将pH调节至7。 </li><li>制备10％的硫酸氢钠3将10g加入NaHSO 3在100毫升蒸馏水中。搅拌15分钟该混合物。 </li><li>制备NaOH溶液中溶解1g氢氧化钠在100毫升水中。搅拌10分钟。 </li><li>制备的10mM磷酸盐缓冲液在1升水中溶解600毫克的NaH 2 PO 4的。加入仔细0.34毫升磷酸，搅拌30分钟。调整pH值至2.9（可接受的范围2.7-3.0）。 </li><li>制备的10mM磷酸盐缓冲液中溶解269毫克的NaH 2 PO 4的和1.09克Na 2 HPO 4的蒸馏水，使1升的体积调整pH值至7.2。 </li></ol> 2.纳米涂层油酸合成（OA-IONP） <ol><li>在微波适应瓶中加0.5克铁（ACAC）3，1.4 olei毫升C酸，0.6毫升油胺和1.19将1,2- hexadodecanediol的。用刻度吸量管加入10 mL苯基醚通过精心的瓶壁。 </li><li>介绍在微波反应器中的烧瓶中，并启动微波协议。 注:微波软件使选择像温度，压力，搅拌速度，功率和反应时间不同的参数的大小。此外，它在相同的协议，允许调谐合成加载三个不同阶段的可能性。一旦装合成协议启动，微波炉加热快样本尽可能（斜坡过程中），在所选择的反应时间（运行进程）维护它。功率选举决定斜坡的时间。 </li><li>装载在微波炉动态研究。该协议包含三个阶段: <ol><li>第1阶段:将温度设定为60℃，时间2分钟，压力250psi的150瓦的功率。搅拌速度必须处于很高的位置，并在最大功率。 </li><li>麈e 2的:温度设定为200℃，时间20分钟，压力250磅和300瓦的功率。搅拌速度必须处于很高的位置，并在最大功率。 </li><li>第3阶段:将温度设定为250℃，时间10分钟，压力250磅和300瓦的功率。搅拌速度必须处于很高的位置，并在最大功率。 </li></ol></li><li>完成该协议后，让烧瓶在室温下冷却。 注:冷却下来的过程可以具有或不具有气流来完成。这两种情况都提供了相同的结果。骨料出现在搅拌棒，并在烧瓶壁上，用乙醇洗，并把它放到锥形。 </li><li>使用玻璃吸管将反应混合物转移至锥形并加入10毫升EtOH中98％。把一个钕 - 铌 - B系磁铁烧瓶下面，等待5分钟，并用玻璃吸管除去上清液。 </li><li>加入10 mL EtOH中，超声处理在RT样品2分钟，40千赫，将样品上的磁铁和消除上清液。在重复执法机关这一步吨三次。 </li><li>分散油酸纳米颗粒在30毫升CHCl 3中，声处理的，在40千赫兹，在室温5分钟。检查流体力学尺寸的激光粒度仪按照制造商的说明。把0.5毫升的OA-IONP在玻璃比色杯中，并添加0.5 氯仿的溶液中。验收范围7-10纳米表现为强的Z平均大小。 注:OA-IONP可以很好地分散于己烷中。 </li></ol> 3.壬二酸纳米颗粒的合成（壬酸IONP） <ol><li>溶解44.3毫克的KMnO 4的和在H 2 O的混合物150.4毫克BTACl的:三氯甲烷（3:2）溶液中。加所得到的溶液，以OA-IONP的5ml的等分试样在微波适于烧瓶中。 </li><li>启动壬酸IONP微波协议。设定温度为105℃，时​​间9分钟，压力250磅和功率在300W。放10毫升磷酸盐缓冲液的pH = 2.9到烧瓶中并重复微波协议。冷却步骤后，恢复纳米粒子使用磁铁和消除上清液。 </li><li>在40千赫溶于5ml 10％硫酸氢钠3添加到Erlenmeyer烧瓶中，超声处理2分钟，在25℃，收集用磁铁粒子和消除上清液。 （该步骤重复2次。）三次，用1％的NaOH洗涤纳米颗粒和在5ml磷酸盐缓冲液pH的= 7.2最后再分散它。 </li><li>检查流体动力学大小和ζ电位。把0.7毫升壬-IONP成一次性折叠毛细血管细胞并将其插入到激光粒度仪。 注:验收范围大小25-35纳米表现为强的Z平均大小。验收范围Z-潜在-45±5 mV的。较大的纳米颗粒（〜70纳米），可以用磷酸盐缓冲液pH&gt; 7代替pH值= 2.9（参照化学欧元J 2008）获得。 </li></ol> 4.奈立纳米颗粒的合成（奈立-IONP） <ol><li>在离心机添加12毫克EDC和15毫克硫代NHS的2毫升等分壬-IONP的。放在在RT涡旋35分钟该混合物。 </li><li>把离心机下方的磁铁动摇纳米颗粒，吸出上清液，并用1.5的HEPES 1mM的pH值为7的缓冲毫升洗涤颗粒。 （重复此步骤两次。）之后，加入5毫克奈立和动摇旋涡混合物2小时。 </li><li>单独的纳米颗粒与用1mM HEPES pH值为7的缓冲磁铁和洗涤（3×2毫升）中。最后，分散奈立-IONP在2ml 1mM的HEPES pH值为7的缓冲。 </li><li>检查流体动力学大小和ζ电位。把0.7毫升奈立-IONP成一次性折叠毛细血管细胞并将其插入到激光粒度仪（见设备安装）。 注:验收范围大小40-45纳米表现为强的Z平均大小。验收范围Z-潜在-20±5 mV的。 </li></ol> 5. 在 ApoE基因粥样硬化斑块的体内检测- / -小鼠通过MRI <ol><li>对于MRI收购准备 注:前几个所需的动物实验TRA系统。因此，这将需要: <ol><li>使用合适的设备，麻醉动物。 </li><li>获得与外部暖空气一闭路循环的温水系统，以保持动物稳定的温度。 注意:在这种情况下，MRI兼容的监测和浇注系统注册该MRI磁体内部的动物的温度。 </li><li>监测该动物的附近的外部温度，所述动物，在MRI控制台使用集成接口位于胸部附近的动物的身下的呼吸传感器的主体（直肠温度计）的温度。 </li></ol></li><li> MRI实验<ol><li> （在两，三分钟，为MRI实验过程中维持1-1.5％的感应2％）与100％氧气麻醉线与蒸发异氟醚的动物。 </li><li>放置动物在与配置文件获取的帮助下磁体的中心。 </li><li>步骤5.2.2之后，调谐RF线圈300兆赫（7 T）的共振频率与匹配线圈的特性阻抗50欧姆为最佳的信号的接收。 注:需要注意的是外部接线和连接将通过适配器/分路器的测量系统，发射线圈的每个部分单独（在我们的情况下，它是一种正交线圈）。 </li><li>调整和匹配线圈后，将线圈放入扫描仪。 </li><li>射频脉冲校准（形状和长度）和中心频率调整执行这两个脉冲校准和手动频率的中心。手动执行90°脉冲校准，粗匀场（见下文），中心频率和接收器的增益的调整。 </li><li>执行使用梯度回波（FLASH或GRE）定位扫描（3飞机侦察获取准确位置:轴向，冠状位，也被称为tripilot。 </li><li>执行磁铁垫补优化磁场均匀的岑磁体之三。手动执行此步骤中，使用一种或单脉冲FID序列（见5.2.3），调整包括在系统中的第一和第二订单垫片或任何自动匀场序列 注:还有匀磁场容易认可和T2 *测量（如越大越好）或光谱窄FWHM。 </li></ol></li><li>斑块5 MRI数据采集<ol><li>注入100微升（1毫克的[Fe]毫升-1）奈立纳米粒子进入静脉注射尾静脉图像采集后1小时注射。加载MRI收购粥样硬化斑块的看着腹主动脉（肾分叉）的参数。 </li><li>放多片，10〜20片在交错模式，以尽量减少工件。 </li><li>获得高分辨率的快速自旋回波T1加权的MRI在冠状或轴向视图，其参数如下:FOV 60×30毫米（冠状），30×30毫米（轴向），切片厚度一般在0.8毫米（与小梯度线圈共nfiguration这可以减小到0.6mm），400毫秒TR，8毫秒的TE，256×256采集和重建矩阵数据，6（大梯度）至8（小梯度）信号的平均值为5-8分钟平均采集时间。 注:TE是尤其关键的，血液信号和流量和化学位移的工件，可以限制应用程序的存在。在这种情况下不区分大小写流快速自旋回波T2-IR可以帮助减少文物和补充数据在相同的位置收购可以帮助表征牌匾。另外，预饱和脉冲可以用于减少周围动脉壁的壁和化学位移伪像减少的外边界的更好的圈定的脂肪组织。 </li><li>传输的标准格式，如DICOM和查看图像在适当的软件（ 如 Osirix成像软件或酰胺:医学影像数据考官）5。量化对比效果手动划定VESSEL区域，壁厚，管腔面积和斑块负荷5。 </li></ol></li></ol>

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氧化铁纳米颗粒（IONP）是最重要的纳米材料之一，它已被用于从很久以前不同的应用。使用这些材料作为造影剂用于磁共振成像（MRI）是一种行之有效的字段。然而，合成路线往往需要几个时间，设置复杂。由于极大地降低反应时间，并提高再现性利用微波驱动的合成似乎是为生产高品质的纳米颗粒的一个很好的选择。另外，在上述协议中，微波技术已被用于两个不同的纳米粒子的合成。微波允...

动脉粥样硬化是动脉壁从失调的脂质代谢和有缺陷的炎症反应所得的一种多因素慢性炎性疾病。由于患病和这个和有关的心血管疾病的经济和社会成本有在处理新的工具，其中纳米是最有前途的之一的病理学越来越感兴趣。1-3然而有快速例子非常少生产和探针表征这是基本的翻译到诊所4在这个协议中，我们使用的氧化铁纳米颗粒的微波合成用于进一步官能化与双膦酸酯，并在对ApoE动脉粥样硬化的体内检测- / -小鼠在1小时5氧化铁纳米颗粒（IONP）是一个公知的纳米材料及其作为造影剂用于磁共振成像（MRI）使用已建立用于检测不同疾病的■在过去几年6-8 微波合成（MWS），允许具有高重复性和增强的产量合成纳米粒子在极短的时间9,10在我们的协议，我们得到IONP斑块三步定位功能。最后一个是氨基二膦酸，奈立，这是由于它的钙结合性能是我们战略的关键附件。由于它们的天然类似物焦磷酸（PPI），奈立已在成骨不全（OI）和骨的佩吉特氏病（PDB），用于其向骨矿物高亲和力的用于治疗11-13 三个步骤的协议概括在方案1的步骤一和二中进行使用微波技术。第一步公布的方法的修改提供油酸涂酸氧化铁纳米颗粒（OA-IONP）14的协议是一个适应的tradit的微波合成有理热分解合成15,16含有的Fe（ACAC）混合物3，油酸，油胺和1,2-十二烷二醇溶解于苄醇，并在两个加热过程进行。纯化进行用EtOH洗涤，并用的Nd-Fe-B磁体中收集颗粒，以消除过量上清液中的表面活性剂的。然后，OA-IONP都在CHCl 3中稳定化。正如预期的那样，由于非常快速的加热，预期的结果表明，通过微波合成的纳米颗粒的核心（3.7±0.8 nm），而与传统的热分解比较流体力学尺寸（7.5纳米）的角度更小;然而，纳米颗粒仍呈现优良的结晶度。 第二步包括在双键，存在于油酸的直接化学修饰，使用类似的KMnO 4，开发中的基团被修改为MW条件原始方法一种强氧化剂。17第一阶段形成的MnO 4之间的复合物-和双键。然后，在酸性条件下的第二阶段，产生使壬酸IONP油酸分子的切割。的每9分钟这两个阶段后，将样品进行纯化，第一次洗涤用加入NaHSO 3 1％至过量的MnO 4的减少-到的MnO 2，然后用NaOH的1％，以中和酸。 在纯化步骤之后，壬-IONP都在10mM磷酸盐缓冲液pH = 7.2稳定。这个缓冲区是粒子类似原，热反应发生了什么事胶体稳定性的最佳环境。18载于OA-IONP双键的直接氧化利用微波是的优势很好的例子在纳米颗粒的合成采用这种技术。与反应需要24小时的经典方法，微波的利用率降低reacti时间到18分钟。此外，微波驱动的协议表现出优异的再现性后4重复给予纳米颗粒流体动力学大小为30±5纳米。除了在流体动力学大小的变化的，ζ电位是一个很好的参数来快速检查成功反应。由于壬-IONP新羧酸基团的存在，对于ζ电位的值大约是-44毫伏，非常类似于由热的方法得到的值。 对奈立的对壬-IONP该附件中，使用传统的EDC /硫代NHS缀合。19这种合成方法是行之有效的，因为使用的活化羧酸与磺基-NHS反应期间确保胶体稳定性。磷酸盐缓冲液的消除与奈立膦反应在1毫米的HEPES缓冲液中进行后（pH约7）。该反应使奈立-IONP具有40±4毫微米在窄大小颇的流体动力学大小ibution和zeta电位的-24.1 mV的。 该过程为用于动脉粥样硬化斑块的体内可视IONP的快速合成T2加权对比剂的MRI中描述尽管该方法的可行性允许与游离胺的任何肽/抗体的附接，使用相同的条件下，用于不同的目的领域。

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			<td height="21" style="height:21px;width:395px;">Microwave Explorer/Discover Hybrid-12</td>
			<td style="width:176px;">CEM Corporation, USA</td>
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			<td style="width:311px;">Any microwave for chemical synthesis can be used</td>
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			<td height="21" style="height:21px;">Disposable PD-10 desalting columns&#160;</td>
			<td>GE Healthcare life sciences</td>
			<td>17-0851-01</td>
			<td>Any size exclusion column will work</td>
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			<td height="21" style="height:21px;">Amicon&#174;Ultra-0.5 ml&#160;</td>
			<td>Merck Millipore Ltd</td>
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			<td></td>
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			<td height="21" style="height:21px;">Calibrated pH meter&#160;</td>
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			<td height="21" style="height:21px;">Neodymium magnet&#160;</td>
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			<td>ND010B</td>
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			<td height="21" style="height:21px;">Vortex Genius 3&#160;</td>
			<td>IKA</td>
			<td>3340000</td>
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			<td height="21" style="height:21px;">ZetaSizer Nano ZS&#160;</td>
			<td>Malvern Instruments</td>
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			<td></td>
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			<td height="21" style="height:21px;">Standard (macro) cell Optical glass&#160;</td>
			<td>Labbox</td>
			<td>11718</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer nanoseries disponsable folded capillary cells DTS1070</td>
			<td>Malvern</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Bruker Minispec mq60</td>
			<td style="width:176px;">Bruker</td>
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microwave-driven synthesis of iron oxide nanoparticles for fast detection of atherosclerosis

一种快速和可重复的微波驱动的协议已被开发用于奈立膦官能纳米粒子的合成。从疏水性纳米粒子的合成开始，我们的方法是基于从热分解法对微波驱动合成的适应。的新方法产生的反应时间与传统的程序相比的降低。此外，使用微波技术的提高了反应的再现性，一些从视图的临床应用的点重要。这种铁氧化物纳米颗粒的新颖性是奈立膦酸盐的附着。使用这种分子的引线朝提供钙在动脉粥样硬化斑块结合在体外和选择性积累特性体内纳米粒子的外侧的二膦酸盐部分。该协议允许在大约3小时的合​​成和斑块的检测，因为从organi最初的合成Ç前体。其在动脉粥样硬化区积累在不到1小时提供了对比剂特别适合于临床应用。

在这个协议中，三个不同的IONP的合成进行说明。从疏水OA-IONP开始，用微波驱动综合的帮助下获得稳定的水性纳米。在一个非常窄的尺寸分布（ 图1c）中的所有纳米颗粒呈现超小流体力学尺寸（DH &lt;50纳米）。利用微波技术的呈现在核心规格方面超小的纳米粒子。由于微波产生一个快速加热，在与其他的方法在纳米颗粒的核心给予较小的尺寸比较成核增加率。...

Watch this Scientific Journal Video about Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis at JoVE.com

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

微波技术使动脉粥样硬化斑块特征的氧化铁纳米颗粒极快的合成。在所述纳米颗粒的外部侧使用氨基二膦酸的提供了在动脉粥样硬化区快速积累。

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

microwave-driven-synthesis-iron-oxide-nanoparticles-for-fast

Research

JoVE Journal

Bioengineering

10.4K 次观看.  Centro Nacional de Investigaciones Cardiovasculares Carlos III and CIBER de Enfermedades Respiratorias. 微波技术使动脉粥样硬化斑块特征的氧化铁纳米颗粒极快的合成。在所述纳米颗粒的外部侧使用氨基二膦酸的提供了在动脉粥样硬化区快速积累。 

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

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

科研

生物工程

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

纳米谐波再生研究

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

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

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

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 ...

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

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

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 ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

蛋白质的MS检测快速酶法处理与流通型微反应器

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

单核靶向肽 Amphiphile 胶束合成动脉粥样硬化的研究

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

银纳米粒子改性多壁碳纳米管的合成及其抗菌活性和细胞毒性的评价

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

阴离子氧化铈纳米粒子催化清除体内植物活性氧的研究

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...