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 ...

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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. 