Name: solid lipid nanoparticles (slns) for intracellular targeting applications
Uploaded: 2015-11-17T14:00:00.000+00:00
Duration: 8 min 19 s
Description: Watch this Scientific Journal Video about Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications at JoVE.com

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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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Nanoparticulate+Drug+Delivery+Systems:+A+Revolution+in+Dosage+Form+Design+and+Development.">Lipid Nanoparticulate Drug Delivery Systems: A Revolution in Dosage Form Design and Development.</a> Recent Advances in Novel Drug Carrier Systems. , 107-140 (2012).</li></ol>

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><tbody><tr><td>Nile Red (NiR)</td><td>Sigma</td><td>19123</td><td>BioReagent, suitable for fluorescence, &amp;ge;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 &amp;micro;m Supor Membrane Low Protein Binding</td><td>Life Sciences</td><td>PN4612</td><td>Non-Pyrogenic</td></tr><tr><td>Nanotrac Ultra&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Sigma</td><td>P5493</td><td>For molecular biology</td></tr><tr><td>Recombinant murine GM-CSF&amp;nbsp;</td><td>Peprotech</td><td>315-03</td><td>&gt;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td></tr><tr><td>Recombinant murine IL-4&amp;nbsp;</td><td>Peprotech</td><td>214-14</td><td>&gt;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

17.0K 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

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

内部自组装脂质颗粒的简易制备碳纳米管稳定

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

科研

化学

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

生物工程

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

含纳米片生物活性剂的配制与表征

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

生物化学

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

阳离子纳米脂质体的生成, 用于高效输送体外转录信使 rna

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

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 ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

用于细胞质 siRNA 传递的带电反应性聚合物纳米粒子的制备

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

生物活性亲水球状蛋白的纳米脂质体制备与表征

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

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 ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

长期沉默的1S Intersectin的在小鼠肺的特异性siRNA，通过阳离子脂质体重复交货。通过电子显微镜的击倒效果评价

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...