Research reported in this publication was supported by The Johns Hopkins Applied Physics Laboratory&#8217;s Research and Exploratory Development Department, Office of Technology Transfer, and Stuart S. Janney Fellowship Program, in addition to the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number R21HL127355.

1.处理前哨淋巴结<ol><li>前哨淋巴结的合成 注意：使用PIT方法1,17,20制备亚20纳米前哨淋巴结1。 <ol><li>使用无菌技术，生物试剂或细胞培养级试剂和消毒材料（即，吸液管，刮铲，小瓶等 ）。 </li><li>使用生物安全柜前哨淋巴结的合成（ 图2）。 </li><li>结合荧光染料的0.6毫克（尼罗红（NIR），NIR浓度约为0.3毫克/毫升）和0.10克Heneicosane（脂质，5重量/重量％的脂质浓度）到小瓶（15ml）中，共熔体在90℃，搅拌。 </li><li>添加0.11克聚氧乙烯（10）油基醚（表面活性剂，5.5重量/重量％的表面活性剂浓度）。在90℃和搅拌共熔化所得混合物。 </li><li>添加1.79克无菌水到混合物中，加热至90℃，并搅拌直至得到透明纳米乳剂形成。 注意：在继续搅拌和控制冷却，SLNS创建。这些条件使水包油乳液的反转成油在水中的乳液。亲脂性分子，例如NIR分区到脂质小滴。 </li><li>准备在平行的纳米乳剂不添加荧光染料，以充当对照样品。结合0.10克Heneicosane和0.11g聚氧乙烯（10）油基醚到小瓶（15毫升）共熔在90℃，并搅拌。 <ol><li>添加1.79克无菌水到混合物中，加热至90℃，并搅拌直至得到透明纳米乳剂形成。 </li></ol></li><li>进一步用消毒无菌0.2微米的过滤器每一个纳米乳液。 </li></ol><img alt="图2" src="/files/ftp_upload/53102/53102fig2.jpg" /> 图2.合成前哨淋巴结的。 <a href="https://www.jove.com/files/ftp_upload/53102/53102fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </li><li>粒度和Polydispersity <ol><li>使用动态光散射（DLS）来测量粒径用玻璃试管的前哨淋巴结1和多分散性，并设计用于测量粒度的仪器。 </li><li>在此之前的样品的测量结果，测量两个已知标准粒径根据制造商的协议为标准制备和测量。 </li><li>填充玻璃试管用1.5ml标准溶液，并使用相同的测量条件样品测量的标准溶液。 注：上一步重复每个标准。 </li><li>测量样品的制备。对于每一个，使用下列实验参数：100秒，水（1.33）的折射率，水粘度的运行时间，在20℃（1.002毫帕·秒）。 注意：这种技术使用频移的光来测量纳米粒子的尺寸。 </li><li>报告的平均粒径杂色的和多分散第一百粒径分布。 </li></ol></li><li>熔点，熔化潜热<ol><li>使用差示扫描热量计（DSC）配备有自动取样器和液氮冷却源来调查前哨淋巴结1的热行为。 </li><li>吸取所制备的前哨淋巴结成40μl的铝盘的质量为约25毫克，并使用通用的卷曲机压榨，以减少在DSC扫描时水分流失气密地密封盘。 </li><li>测量从5至80℃的每个纳米乳剂。 </li><li>报告的熔点，并从DSC图，其中，谷表示熔点和区，在DSC图由材料的量除以曲线下的积分熔化的潜热表示熔化的潜热。 </li></ol></li></ol>与成纤维细胞和树突状细胞2.前哨淋巴结相互作用<ol><li>人类纤维原细胞培养<ol><li>培养初级人网络成纤维细胞根据制造商的在液体介质中对人真皮成纤维细胞（介质106），补充有低血清生长补充（LSGS）试剂盒的培养说明书。当达到80％汇合维持细胞在湿润气氛中，在37℃，5％的CO 2和传代培养。 </li><li>对于这两种MTT法和图像分析，种子细胞在96孔板中，每孔2×10 4个细胞的密度，并允许以平衡在37℃CO / N之前SLN曝光。 </li><li>在零时间，稀释在完全培养基前哨淋巴结所指示（图4），相对于脂质浓度，并添加每孔10微升的每个复制井在96孔板中。 </li><li>维持细胞在37℃，5％的CO 2，在潜伏期。 </li></ol></li><li> MTT毒力测定<ol><li>后孵育前哨淋巴结的24小时，确定细胞活力使用MTT测定法，根据制造商的协议。&lt;/ LI&gt; <li>一旦洗涤细胞并添加新鲜培养基到每个孔中（100μl/孔）。通过在10分钟为70％的甲醇溶液孵育用新鲜培养基更换之前杀死对照孔。 </li><li>添加的MTT试剂（每孔10微升）到微孔板的每个孔中，并培育O / N在37℃。 </li><li> O / N培养后，溶解与所提供的洗涤剂溶液内福尔马肼晶体（每孔100μl）根据制造商的协议。 </li><li> 3小时温育的在RT后，获得的吸光度值以使用酶标仪570nm处测定波长。 注意：用于应小于1％（重量/体积）3-（4,5- dimethylthiazolyl-2）-2，5-二苯基四唑鎓溴化物的MTT试剂。 </li></ol></li><li>荧光显微镜<ol><li>在以下的成纤维细胞与SLN给药特定的时间点，用无菌磷酸盐缓冲盐水（PBS）洗两次的成纤维细胞和固定10分钟用冷的70％甲醇中。 </li><li> 10分钟后，使用倒置显微镜替换用PBS，采集图像甲醇，使用带通（BP）五十〇分之六百九十零纳米滤波器集合。 </li></ol></li><li>小鼠骨髓树突状细胞的培养<ol><li>诱导小鼠骨髓来源的树突细胞（BMDC）先前所描述的23，24。简言之，将C57BL / 10骨髓100毫米培养皿以2×10 6个/板用重组鼠粒细胞-巨噬细胞集落刺激因子（GM-CSF，300U / ml）和重组鼠IL-4板单细胞悬浮液（B细胞刺激因子，200U / ml）中。更换介质每3天。 </li><li>第7天，以0.5和5.0微克/毫升脂质的终浓度在37℃添加NIR加载前哨淋巴结的菜肴和维持细胞，5％的CO 2培养箱中暴露的希望的长度。 </li><li>收集从餐具细胞通过吸所有媒体中的板，它收集在一50ml管中，随后在板的彻底清洗（撞出松散广告海伦特细胞）用5ml磷酸盐缓冲盐水（PBS）中含有4mM乙二胺四乙酸（EDTA）。收集所得到的细胞悬液以相同的50ml管中。 </li></ol></li><li>通过流式细胞仪前哨淋巴结公司注册评估<ol><li>确定在点2.4.3中获得的细胞悬浮液的浓度和分布3×10 5细胞成12毫米×75毫米的圆底FACS管。 </li><li>通过加入1ml的PBS中，然后离心管5分钟，在400×g离心，在4℃的温度下洗细胞。 </li><li>弃去上清，重悬用含有抗CD11c-FITC单克隆抗体（0.25微克/毫升终浓度）加入100μl的PBS / 2mM EDTA的/ 0.1％牛血清白蛋白（BSA）（染色缓冲液）中沉淀。孵育在冰上30分钟，在黑暗中。 </li><li>用1毫升的PBS洗涤细胞，然后离心管5分钟，在400×g离心，在4℃的温度下进行。 </li><li>弃上清，重悬每个沉淀用400μl的染色缓冲液中。这些细胞现在已经准备好收购用流式细胞仪（可能配有蓝色和红色激光）样品。 </li><li>使用流式细胞仪分析软件，通过门控上的CD11c +细胞（FITC阳性）的人口和比较前哨淋巴结相关的荧光强度（在PerCP-经Cy5.5或APC通道上测得）评估前哨淋巴结注册成立由DC的水平。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这项研究中，前哨淋巴结的合成和它们的适用性的细胞内靶向应用进行了探索。这些生物相容性纳米粒子希望作为运载工具多种应用，包括药物输送，基因沉默，和疫苗技术25-30。合成超小型前哨淋巴结用一种简便方法，以及它们与初级皮肤细胞和初级免疫细胞相互作用进行了探讨。前哨淋巴结被设计为包括荧光染料（NIR），其中担任一个模型治疗货物的封装。 PIT...

纳米颗粒为基础的运载工具显示细胞内定位的应用具有很大的承诺，提供了一个机制，专门改变细胞信号传导和基因表达。这些车辆可装载药物，蛋白质，并设计成影响的细胞反应，并实现在靶组织中的期望的效果的核酸。许多类型的纳米载体的已探索用于治疗和诊断益处包括脂质，聚合物，硅，和磁性材料。这些系统是有吸引力的，因为它们的潜力用于局部药物递送，增加的治疗浓度在靶组织，并降低全身毒性。 固体脂质纳米颗粒（前哨淋巴结）是已成为一种有希望的药物递送载体，近年来的纳米颗粒递送系统的充分研究的实例。前哨淋巴结可以容易地配制成用于多种应用中，包括生物传感1，化妆品2，和叔herapeutic交付3-7。其效用源于这样一个事实，他们是完全由可再吸收的，无毒的脂质，从而增强了生物相容性。在合成过程中，亲脂性药物可以掺入SLN车辆，从而提高药物的溶解度和适宜用于肠胃外施用。 SLN汽车还有助于稳定封装的治疗，减少其降解和清除，并最大限度地发挥治疗作用。这些车辆特别适合于长效，控制释放制剂由于在体温3,4,8,9其稳定性。重要的是，药物在脂质纳米粒封装改变药物分子的内在药动学曲线。这提供了一个潜在的优点，允许药物具有窄治疗指数的控制释放。的前哨淋巴结-掺入治疗剂的释放速率可以基于所述脂质的降解率或药物扩散速度在调谐脂类基质。 前哨淋巴结通常改造以积聚在特定靶组织。例如，它们的大小（通常大于10nm）potentiates保留在循环中，其中肿瘤组织的泄漏脉管促进沉积。此外，颗粒施用途径已经显示出改变生物分布与潜在的目标特定生理结构如淋巴结10,11。一旦沉积在靶组织，以获得适当的细胞相互作用和纳米颗粒的最终的内化是具有挑战性，因为细胞膜为选择性地控制离子和分子的流入和流出所述单元 12的能力。为了便于细胞吸收，这是可以修改的纳米载体与特定的配体，包括肽，小分子，和单克隆抗体13,14。一些机制，包括被动渗透和纳米粒子的主动运输穿过细胞膜先前已经描述3,12,15。在一般情况下，它已经证明，细胞的纳米粒子相互作用由纳米颗粒的物理化学性质，包括大小，形状，表面电荷和表面化学的影响，除了特定小区参数诸如细胞类型或细胞周期时相12。 先前的调查显示亚10纳米前哨淋巴结的合成使用相转化温度（PIT）17的方法，局部16和生物标志物检测的应用1。这是一个温和的合成方法，其中2的组合物保持不变，同时使温度逐渐变化。加热的溶液连续搅拌下，在冷却至RT的结果中的纳米乳剂。这个过程的结果在前哨淋巴结的合成与较小粒度的1比以前报道使用各种方法对脂质楠的合成oparticles 17-22。所得大小比例，小于20纳米，提供的优点为胞内靶向应用，由于增加的表面积和用于增强细胞相互作用的潜力。 示意图前哨淋巴结，旨在提供一种荧光染料或治疗， 显示在图1的前哨淋巴结组成的脂质内部（例如，线性烷）使亲脂性化合物（例如，染料或治疗剂）的掺入和表面活性剂外的（ 如线性离子表面活性剂），四面环水。在这项研究中，前哨淋巴结装载用荧光染料和用作模型来研究粒子 - 细胞相互作用。原代人真皮成纤维细胞和小鼠的树突细胞暴露于染料加载SLN随时间以表征相对于毒性和颗粒摄取的相互作用。甲3-（4,5-二甲基吡啶-2-基）-2,5- diphenylphenyltetrazolium溴化物（MTT）法是utili捷思，以确定合适的剂量水平。荧光显微镜，流式细胞术用来检查颗粒吸收体外两种方法。 <img alt="图1" src="/files/ftp_upload/53102/53102fig1.jpg" /> 图1.原理SLN的展示的主要成分。 <a href="https://www.jove.com/files/ftp_upload/53102/53102fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Nile Red (NiR)</td>
			<td>Sigma</td>
			<td>19123</td>
			<td>BioReagent, suitable for fluorescence, &#8805;98.0%</td>
		</tr>
		<tr>
			<td>Heneicosane</td>
			<td>Aldrich</td>
			<td>286052</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>Brij O10</td>
			<td>Sigma</td>
			<td>P6136</td>
			<td>Brij 97, C18-1E10, Polyoxyethylene (10) oleyl ether</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td>Sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Syringe Filter 0.2 &#181;m Supor Membrane Low Protein Binding</td>
			<td>Life Sciences</td>
			<td>PN4612</td>
			<td>Non-Pyrogenic</td>
		</tr>
		<tr>
			<td>Nanotrac Ultra&#160;</td>
			<td>Microtrac</td>
			<td>serial number U1985IS</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Differential Scanning Calorimeter</td>
			<td>Mettlet-Toledo</td>
			<td>----</td>
			<td>Instument</td>
		</tr>
		<tr>
			<td>Primary human fibroblasts&#160;</td>
			<td>Life Technologies</td>
			<td>C-004-5C</td>
			<td>Neonatal (HDFn)</td>
		</tr>
		<tr>
			<td>Medium 106</td>
			<td>Life Technologies</td>
			<td>M-106-500</td>
			<td>A sterile, liquid medium for the culture of human dermal fibroblasts.</td>
		</tr>
		<tr>
			<td>Low Serum Growth Supplement Kit (LSGS Kit)</td>
			<td>Life Technologies</td>
			<td>S-003-K</td>
			<td>All the components of complete LSGS</td>
		</tr>
		<tr>
			<td>MTT Cell Proliferation Assay Kit</td>
			<td>Trevigen</td>
			<td>4890-025-K</td>
			<td>Sensitive kit for the measurement of cell proliferation based upon the reduction of the tetrazolium salt, 3,[4,5-dimethylthiazol-2- yl]-2,5-diphenyl-tetrazolium bromide (MTT)</td>
		</tr>
		<tr>
			<td>Safire2 microplate reader</td>
			<td>Tecan</td>
			<td>----</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline&#160;</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>For molecular biology</td>
		</tr>
		<tr>
			<td>Recombinant murine GM-CSF&#160;</td>
			<td>R&#38;D Systems</td>
			<td>415</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
		<tr>
			<td>Recombinant murine IL-4&#160;</td>
			<td>R&#38;D Systems</td>
			<td>404</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
	</tbody>
</table>

solid lipid nanoparticles (slns) for intracellular targeting applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

PIT中方法用于合成前哨淋巴结和相转化温度测定利用水浴中。将样品缓慢加热，轻轻搅拌直至溶液呈清晰。相转化温度使用heneicosane脂质所作的前哨淋巴结是45℃。 表1总结了粒径，多分散性，熔点和熔化的前哨淋巴结的潜热。使用上述的加工条件中合成的前哨淋巴结导致控制前哨淋巴结和NIR加载前哨淋巴结用18.59和16.87毫微米的平均粒径并用5.83和4.47，多分散性分别（ 见图3）。...

Watch this Scientific Journal Video about Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications at JoVE.com

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

solid-lipid-nanoparticles-slns-for-intracellular-targeting

Research

JoVE Journal

Bioengineering

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

16.8K 次观看.  The Johns Hopkins Applied Physics Laboratory. In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions. 

视频: Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

科研

生物工程

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

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 ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

生产和目标单价量子点

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofunctionalized普鲁士蓝纳米粒子多式联运分子成像应用

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

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 ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

纳米丝绸制造和药物输送应用

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

人骨髓间充质干细胞吸收含有法卡林多醇的新型脂质包覆纳米颗粒

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

使用微流体混合平台为基因传递制订和定性脂质纳米粒子

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...