Name: facile preparation of internally self-assembled lipid particles stabilized by carbon nanotubes
Uploaded: 2016-02-19T13:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes at JoVE.com

我们要感谢马修·贝克J.博士，现在斯特拉斯克莱德大学，格拉斯哥与拉曼实验和尼克·冈特先生的支持，他之前这个项目的工作。

注意:在此工作中使用的CNT是在相比其散装同行可具有额外的危害纳米颗粒形式。石墨吸入，天然和合成，可引起尘肺62相似，煤工尘肺。此外，已经有关于碳纳米结构的毒性和一些以前的研究的关注建议与碳纳米管63-68的吸入有关的急性和慢性毒性。因此，避免了细CNT粉吸入，并且十分小心处理。如吸入，移至空气新鲜处。如呼吸困难，用纯氧代替，并就医。碳纳米管的溶液/分散液的配方是相当安全的。 注意:脂质和本研究中使用的表面活性剂是食品级材料和一般因而无危险，但是它们刺激眼睛和皮肤，并且也高度易燃。因此，请使用所有适当的安全实践，例如使用的ENgineering控制（通风橱）和个人防护装备（护目镜，手套，实验室外套，全长裤，封闭趾鞋）办理或准备时，纳米粒子样本。在与皮肤或眼睛，应立即冲洗眼睛或皮肤用大量的水至少15分钟的接触情况。如果需要就医。 1.脂质/散装水相的制备注意:在4℃存放在冰箱中的脂质。纯级脂质应贮存在冰箱中（-20℃）。它们分装成小玻璃瓶中，以避免整个股市和处理方便的污染。其他化学物质，包括碳纳米管和表面活性剂可以储存在室温下，但让他们远离阳光直射。 <ol><li>保持脂质， 即的Dimodan U（DU）和植烷三醇（PT）在RT之前，以避免湿气凝结开瓶/小瓶的盖子15-20分钟。 （注:杜是一个蒸馏甘油酯含有96％的单酸甘油酯和其余的二甘油酯和游离脂肪酸。在杜两个主要单甘油酯组分是亚油酸（62％）和油酸（25％）。因此，DU的疏水部分主要含有C18链（91％），其确切的组合物的是如下; C18:2（61.9％），C 18:1（24.9％），和C18:0（4.2％），其中C 18表示18C-链和冒号后的数字表示的C = C键数目。 PT是3,7,11,15-四甲基-1,2,3- hexadecanetriol光学异构体的混合物。它不含有酯官能团，而是由高度支化的phytanyl尾巴与三羟基首基的。既杜和PT的形成的过量的水存在下这也是稳定的脂质颗粒13，45）的芯的情况下立方相。 </li><li>通过将小瓶在热水浴中或60℃以上保持一含有烧杯水融化脂质（加热磁力搅拌器:230伏，50赫兹，630 W或类似被用于加热水在烧杯中）。 </li><li>使用块加热器或者热小瓶。不加热直接包含在热板上小瓶脂质，以避免温度梯度和随后的脂质分解。 </li><li>称量500毫克的熔化的脂类的，在预先称重的离心管（带锥形卡扣帽，1.5毫升），用巴斯德玻璃吸管用胶乳灯泡。 </li><li> 500毫升超纯水（水电阻率= 18.2MΩ·厘米）添加到上述离心管中。 </li><li>手动混合组分使用微小（定制）锅铲15分钟。通过平坦化用钳子注射器针头（0.9毫米×40毫米套管长度）的尖端做出这样的刮刀。 </li><li>离心机以2,000×g的速度的脂质/水混合物10分钟。再次手动搅拌该混合物10分钟，然后平衡它24小时。表征样品之前，鼓动他们为5分钟，然后让它们在室温。 </li><li>以确保在整个管的平衡脂质相的形成，进行大约10次冻融循环和INTE<html> rmittently进行离心步骤定义如上。两者的杜和PT形式高度粘稠散装脂质相使得难以手动处理它们（图1）。 注:上述协议（第1部分），仅需要，如果一个人想比较纳米结构的行为（晶格型和自组装的尺寸）分散颗粒与散装脂质相和/或使用它作为一个控制确认原来的纳米结构的保留。 </li></ol><img alt="图1" src="/files/ftp_upload/53489/53489fig1.jpg" /> 图1.使用高能量输入（超声波），并使用不同的CNT-稳定剂，即SWCNT，与从高粘度脂类相流体一致性O / W颗粒乳液的制备碳纳米管-OH，MWCNT-COOH（图来自参考转载[50]从化学的英国皇家学会的许可）。_upload / 53489 / 53489fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 2.表面活性剂的制备稳定的脂质颗粒<ol><li>制备出0.2％（W ​​/ W）表面活性剂（普朗尼克F127）的水溶液中。 <ol><li>通过搅拌，约20-30分钟（使用磁力搅拌棒的磁性板）溶解200毫克在100毫升的超纯水的表面活性剂（白色蓬松粉末）。普朗尼克F127是一种非离子表面活性剂和通常用作乳化稳定剂。它是PEO 99 -PPO 67 -PEO 99的三嵌段共聚物，因此需要很长的时间在水中溶解。 </li></ol></li><li> 500毫克熔融杜或PT（使用巴斯德玻璃吸管）添加到玻璃小瓶（闪烁钠钙装有箔衬里尿素帽，20ml）中。 </li><li>加上9.5克0.2％F127的解决方案。 </li><li>探头超声机，紧紧夹住瓶的铁架台颚（铁架台与设置站，钳，碱，棒，橡胶3颚和bosshead），使得它能够承受通过超声处理产生的振动。 </li><li>插入连接于细胞超声仪的实心钛合金探针英寸（13毫米直径×139毫米长度）。调整小瓶的高度和位置，以确保它的侧面和底部没有接触到探针。的探针和玻璃小瓶的底部在0.5厘米的距离给出了良好的结果。 </li><li>超声处理在1秒脉冲的脉冲模式，在35％的功率（最大的）1秒的延迟时间调节为10分钟该混合物。小瓶会变得非常热，由于超声处理过程中产生的热量。因此，允许它取下来钳前冷却至室温。 </li><li>存储在RT乳白色形成的分散至少24小时之前，进一步使用。这是为了确保其对相分离的稳定性。 注意:在与使用探头后，用纸巾用丙酮清洗，干燥，然后用超纯水的冲洗ð再一次擦干。 </li></ol> 3.水纯碳纳米管的分散体的制备<ol><li>在两个独立的烧杯，权衡4毫克粉末状的MWCNT-OH和MWCNT-COOH，这两者都是黑色的。 </li><li> 500毫升超纯水添加到每个烧杯中。使用探针超声波发生器在40％超声处理2分钟的混合物以连续脉冲模式功率（最大的）。所得的碳纳米管分散液的浓度为8微克/毫升（原液）。 </li><li>稀释碳纳米管原液用超纯水的适当量达到6.25，5,4，2微克/毫升MWCNT分散体。 </li><li>超声振荡如前所述，这些分散体（见3.2）。 </li><li>同样地，在500毫升的超纯水分散3毫克粉末状的单壁碳纳米管的（也黑色的），以使一个6微克/毫升SWCNT分散液（原液）。 </li><li>稀释SWCNT原液和超声处理他们以上（见图3.2）所描述获得0.5，0.4，0.3125，0.2微克/毫升SWCNT dispersions。 注意:所有的分散体都清楚约30分钟，在此之后，碳纳米管在底部开始沉降。 </li></ol> 4.制备CNT-中稳定的纳米脂质颗粒的（图1） <ol><li>称重在500毫克熔融杜到玻璃小瓶中。 </li><li>添加9.5的6微克/毫升的单壁碳纳米管分散液的小瓶中的溶液。 </li><li>使用相同的参数超声处理在CNT-DU混合物作为用于制备纯的CNT分散体（见3.2）。在冷却至RT，用保守内部自组装纳米结构的碳纳米管稳定脂质颗粒将准备。 </li><li>以类似的方式，使用0.4微克/毫升和0.2微克/毫升的单壁碳纳米管分散液制备所述脂质颗粒。 </li><li>按照协议4.1到4.4，以使使用MWCNT-OH和MWCNT-COOH，但使用不同浓度的，即8,4和2微克/毫升的CNT的脂质颗粒。 </li><li>同样，使用准备4微克/毫升MWCNT-OH和多壁碳纳米管三种不同的CNT-PT分散-COOH以及0.4微克/毫升的单壁碳纳米管。注意，CNT-PT分散体需要较少功率（最大的35％），但在连续脉冲模式较长的时间（15分钟）。冷却分散体RT和表征之前离开他们24小时。 注意:超声参数可能对于不同的脂类不同（如在DU和PT这里）和不同的组合物;它们需要被优化，以实现良好的稳定化的分散体。 </li></ol> 5.监控CNT稳定脂质分散体的稳定性<ol><li>监控分散体通过视觉观察稳定性:检查分散体不稳定或肿块于分散体已经形成。 </li><li>定期取（用数码相机）的照片。例如，采取分散的照片每天都在第一个星期，然后每隔一天一个星期后每周一次，接下来的两个星期，最后每月一次按要求。 </li></ol>

我们什么都没有透露。

脂质颗粒的稳定 三种不同的碳纳米管用于稳定脂质分散体;其中两个是多壁和用-OH和-COOH基团官能化的，一种是单壁和非官能（原始）。碳纳米管的尺寸变化如下（直径×长度）:MWCNT-COOH:9.5纳米×1.5微米;碳纳米管-OH:8-15纳米×50微米; SWCNT:1-2纳米点¯x1-3微米。粉状碳纳米管通过探针的超声波处理分散在水中。碳纳米管的上述大小可能进一步下降，由于超超声，尽管并不均衡。在纯净?...

在过去的几十年中，纳米技术已成为一个强大的工具，特别是在医学打击臭名昭著疾病如癌症1的临床前开发的领域。在这种情况下，用尺寸&lt;1000nm的广泛探索作为各种活性生物分子如药物，蛋白质，核酸，基因和诊断显像剂1-4的递送载体的纳米级结构。这些生物分子的纳米颗粒内的任一胶囊或缀合到纳米粒子的表面和由触发器如pH或温度5,6-在作用部位被释放。尽管在尺寸非常小，这些纳米颗粒的表面积大证明是活性生物分子的靶向递送大大有利的。在粒度和生物相容性的控制是最重要的，以优化治疗效果，因此纳米颗粒7,8的适用性。9-13脂质，聚合物14,15，16,17的金属和碳纳米管18,19已被广泛用作纳米载体各种生物医学和制药应用。 此外，基于脂质的自组装纳米结构纳米载体的应用在许多其它领域，包括食品和化妆品行业20,21宽的意义。例如，它们在蛋白质结晶22，生物分子23的分离的使用，如食品稳定剂例如在甜点24，和在活性分子如营养物质，风味剂和香料25-31的交付。自组装的脂质纳米结构不仅必须释放生物活性分子在受控和靶向方式32-38的能力，但他们也能保护功能分子从化学和酶降解39,40。虽然平面流体双层是最通讯由两亲脂质分子在水存在下形成的纳米结构，其他的结构，例如六方和立方通常也观察到20,41,42。纳米结构体的类型取决于脂质"分子形状的结构，在水中的脂质组合物，以及对温度和压力43采用这样的物理化学条件。非平面脂质纳米结构的适用性特别是立方相的，是因为它们的高粘度和非均相域一致性的限制。这些问题是由分散在大量的水中的脂质纳米结构，以形成油包水（O / W）含有微米或亚微米大小的脂质颗粒乳剂克服。在这种方式下，低粘度的合适的产品能够在保持分散颗粒内的原始脂质自组装结构来制备。这些内部自组装的颗粒的形成（简称为ISAsomes 44 </sup&gt; 例如，从立方相和六角相hexosomes）cubosomes通常需要高能量输入步骤以及加入稳定剂，例如表面活性剂或聚合物的组合。在这个方向最近的研究表明，包括二氧化硅纳米颗粒46，粘土47-49和碳纳米管50前述乳液，适当称为皮克林51或拉姆斯登-Pickering乳液52的稳定各种固体颗粒45的应用程序。 近年来，碳基纳米结构如单壁碳纳米管（单壁碳纳米管），多壁碳纳米管（多壁碳纳米管）和富勒烯已收到的极大关注作为新型生物材料53,54。主要关注的是其毒性55-58，不溶于水，59和因此他们的生物相容性56。解决这些问题的有效方法是在表面功能alization采用无毒和生物相容分子如脂类。在水存在，脂类的方式与碳纳米管相互作用，碳纳米管的疏水表面由极性水性介质屏蔽而脂质的亲水性头部基团有助于在水中60,61它们的溶解度或分散体。脂质是细胞器以及一些食品材料的组成成分，因此其装饰应理想降低CNT的体内毒性 。在碳纳米管18,19和脂质纳米结构9-13基于独立医学领域的应用正在广泛开展，但是，结合两者的性能应用尚未充分探讨。 在这项工作中，我们使用两种不同类型的脂质和三种类型的碳纳米管，其中单壁碳纳米管是在纯净的形式，而多壁碳纳米管与羟基和羧酸基团官能化。我们已经使用非常低浓度的碳纳米管制备的分散体稳定性取决于几个因素如脂质的类型，碳纳米管的种类，脂质至CNT使用的，以及对电力和持续时间采用这种超声处理参数比。此视频协议提供的动力学稳定使用各种CNT-稳定脂质纳米粒的方法的技术细节。

<table><tbody><tr><td>Dimodan U</td><td>Danisco</td><td>15312</td><td>Store at 4&amp;nbsp;&amp;deg;C. Non-hazardous. Irritant to eyes and skin.</td></tr><tr><td>Phytantriol (&gt; 95%, GC)</td><td>TCI Europe N.V.</td><td>P1674</td><td>Store at 4&amp;nbsp;&amp;deg;C. Non-hazardous. Irritant to eyes and skin.</td></tr><tr><td>Single walled Carbon Nanotubes (90%)</td><td>Nanostructured &amp;amp; Amorphous Materials, Inc.&amp;nbsp;</td><td>1246YJS</td><td>Store at room temperature. Away from direct light. Irritating to eyes, skin and respiratory system.</td></tr><tr><td>Multi-walled carboxylic acid functionalized Carbon Nanotubes (&gt;&amp;nbsp;80% Caron basis, &gt;&amp;nbsp;8% carboxylic acid functionalized)</td><td>Sigma-Aldrich Co. LLC&amp;nbsp;</td><td>755125</td><td>Store at room temperature. Away from direct light. Causes serious eye irritation. May cause respiratory irritation.</td></tr><tr><td>Graphitized Multi-walled hydroxy functionalized Carbon Nanotubes (99.9%)</td><td>Nanostructured &amp;amp; Amorphous Materials, Inc. (NanoAmor)&amp;nbsp;</td><td>1224YJF</td><td>Store at room temperature. Away from direct light. Irritating to eyes, skin and respiratory system.</td></tr><tr><td>Pluronic F127</td><td>Sigma-Aldrich Co. LLC&amp;nbsp;</td><td>P2443</td><td>BioReagent, suitable for cell culture. Not a hazardous substance or mixture. Store at room temperature.</td></tr><tr><td>Acetone (99.5%)</td><td>Fisher Scientific&amp;nbsp;</td><td>10134100</td><td>Highly flammable liquid. Causes serious eye irritation. May cause drowsiness or dizziness.</td></tr><tr><td>Jars with loose, enfolding lids (375&amp;nbsp;ml)</td><td>VWR International Ltd</td><td>216-3308</td><td></td></tr><tr><td>Beaker, 1,000 ml</td><td>Fisher Scientific&amp;nbsp;</td><td>12942161</td><td>heavy duty, low form, with spout and graduations</td></tr><tr><td>Pasteur glass pipette (150 mm length) with latex bulb</td><td>Fisher Scientific&amp;nbsp;</td><td>10006021</td><td></td></tr><tr><td>Microcentrifuge tube conical snap cap 1.5&amp;nbsp;ml</td><td>Fisher Scientific&amp;nbsp;</td><td>11558232</td><td></td></tr><tr><td>Spatula</td><td>Fisher Scientific&amp;nbsp;</td><td>11352204</td><td></td></tr><tr><td>Heating magnetic stirrer</td><td>Fisher Scientific&amp;nbsp;</td><td>11715704</td><td></td></tr><tr><td>Magnetic stirrer bars (cylindrical, opaque PTFE, 30&amp;nbsp;mm x 7&amp;nbsp;mm (l x diameter))</td><td>Fisher Scientific&amp;nbsp;</td><td>10011792</td><td></td></tr><tr><td>Needle (0.9 mm x 40&amp;nbsp;mm cannula length)</td><td>Terumo UK Ltd</td><td>MN-2038MQ</td><td></td></tr><tr><td>Retort Stand Set - With stand, clamp, base, rod, rubber 3 jaw and bosshead</td><td>Camlab Ltd, UK</td><td>1177157</td><td></td></tr><tr><td>Millipore water equipment</td><td>Barnstead Nanopure, Thermoscientific, USA</td><td></td><td></td></tr><tr><td>Progen Genfuge 24D Digital Microcentrifuge</td><td>Progen Scientific</td><td>C-2400</td><td></td></tr><tr><td>Probe ultra-sonicator, with 13 mm&amp;nbsp;</td><td>SONICS, Vibracell,&amp;nbsp; USA</td><td></td><td></td></tr><tr><td>5 MP camera with auto-focus and LED flash</td><td>Samsung Galaxy Fame Mobile camera</td><td></td><td></td></tr><tr><td>Raman Spectrometer</td><td>Horiba Jobin-Yvon LabRAM HR800 spectrometer</td><td></td><td></td></tr><tr><td>Mastersizer 3000</td><td>&amp;nbsp;Malvern Instruments Ltd, Malvern, United Kingdom</td><td></td><td></td></tr><tr><td>Small angle X-ray scattering (SAXS)</td><td>SAXSpace camera (Anton Paar, Graz, Austria), X-ray generating equipment (ISO-DEBYEFLEX3003, GE Inspection Technologies GmbH), closed water circuit (Chilly 35, HYFRA, Germany).&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

facile preparation of internally self-assembled lipid particles stabilized by carbon nanotubes

我们提出了一种简便的方法，制备由碳纳米管（CNT）稳定的纳米结构的脂质颗粒。单壁（原始）和多壁（官能化）碳纳米管被用作稳定剂，以产生皮克林类型油包水（O / W）乳剂。脂质即的Dimodan U和植烷三醇用作乳化剂，其在过量的水自组装成的双连续立方Pn3m相。高粘度的阶段被分成使用传统的表面活性剂稳定剂或为这里所做的碳纳米管的存在探头超声波发生器更小的粒子。最初，碳纳米管（粉末形式）分散在水中，然后进一步超声与熔化的脂类以形成最终乳液。在此过程中的CNT得到涂覆有脂质分子，这反过来又被假定为包围脂滴，以形成微粒乳状液是稳定数月。 CNT-中稳定的纳米结构的脂质颗粒的平均尺寸是在亚微米ř法兰，这与颗粒比较好使用常规的表面活性剂稳定化。相比于纯脂质相（散装状态）小角X射线散射数据证实原始Pn3m立方相在CNT-中稳定的脂质分散液的滞留。蓝移和在特性G和拉曼光谱观察到碳纳米管的G&#39;带的强度的降低表征碳纳米管表面和脂质分子之间的相互作用。这些结果表明，在碳纳米管和脂质之间的相互作用是负责在水溶液中相互稳定。作为稳定化所用的碳纳米管的浓度是非常低的和脂质分子能够官能化碳纳米管，碳纳米管的毒性预计是微不足道，而他们的生物相容性大大提高。因此，本方法发现在各种生物医学应用的巨大潜力，例如用于开发混合纳米载体系统m的输送ultiple功能分子如在联合治疗中或polytherapy。

下面的结果表示）的分散体的稳定性，二）脂质颗粒的尺寸分布，三）自组装以及d的类型）对于CNT的脂质包衣的证据。分散体的稳定性（ 图2）用一个500万像素的摄像头，支持自动对焦和LED闪光灯监控。 <img alt="图2" src="/files/ftp_upload/53489/53489fig2.jpg" /> 图2.得到仅在一定区域内的稳定乳液的CNT...

Watch this Scientific Journal Video about Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes at JoVE.com

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

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 ...

facile-preparation-internally-self-assembled-lipid-particles

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

9.6K 次观看.  University of Central Lancashire. 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.

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

A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and scalable technique efficiently functionalizes metallic nanoparticles with thiol-ligands in a miscible water/organic solvent mixture allowing for rapid grafting of thiol groups onto the gold nanoparticle surface. The hydrophobic ligands on the nanoparticles then quickly phase separate the nanoparticles from the aqueous based suspension and confine them to the air-fluid interface. This drives the ligand-capped nanoparticles to form monolayer domains at the air-fluid interface.&#160; The use of water-miscible organic solvents is important as it enables the transport of the nanoparticles from the interface onto template-free substrates.&#160; The flow is mediated by a surface tension gradient3,4 and creates macroscopic, high-density, monolayer nanoparticle-ligand films.&#160; This self-assembly technique may be generalized to include the use of particles of different compositions, size, and shape and may lead to an efficient assembly method to produce low-cost, macroscopic, high-density, monolayer nanoparticle films for wide-spread applications.

A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.

一种技术，功能化和自组装纳米宏观 - 配体单层膜模板上无基材

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and ...

科研

化学

Preparation of Carbon Nanosheets at Room Temperature

Amphiphilic molecules equipped with a reactive, carbon-rich "oligoyne" segment consisting of conjugated carbon-carbon triple bonds self-assemble into defined aggregates in aqueous media and at the air-water interface. In the aggregated state, the oligoynes can then be carbonized under mild conditions while preserving the morphology and the embedded chemical functionalization. This novel approach provides direct access to functionalized carbon nanomaterials. In this article, we present a synthetic approach that allows us to prepare hexayne carboxylate amphiphiles as carbon-rich siblings of typical fatty acid esters through a series of repeated bromination and Negishi-type cross-coupling reactions. The obtained compounds are designed to self-assemble into monolayers at the air-water interface, and we show how this can be achieved in a Langmuir trough. Thus, compression of the molecules at the air-water interface triggers the film formation and leads to a densely packed layer of the molecules. The complete carbonization of the films at the air-water interface is then accomplished by cross-linking of the hexayne layer at room temperature, using UV irradiation as a mild external stimulus. The changes in the layer during this process can be monitored with the help of infrared reflection-absorption spectroscopy and Brewster angle microscopy. Moreover, a transfer of the carbonized films onto solid substrates by the Langmuir-Blodgett technique has enabled us to prove that they were carbon nanosheets with lateral dimensions on the order of centimeters.

We present the synthesis of an amphiphilic hexayne and its use in the preparation of carbon nanosheets at the air-water interface from a self-assembled monolayer of these reactive, carbon-rich molecular precursors.

碳纳米片在室温下准备

Amphiphilic molecules equipped with a reactive, carbon-rich "oligoyne" segment consisting of conjugated carbon-carbon triple bonds self-assemble into ...

Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous solution. In a carbon nanotube/PEO105-PPO70-PEO105 (poloxamer 407) aqueous solution, the amphiphilic poloxamer 407 adsorbs onto the carbon nanotube surfaces and self-assembles into continuous layers, driven by intermolecular interactions between constituent molecules. The addition of 5-methylsalicylic acid changes the self-assembled structure from spherical-micellar to a cylindrical morphology. The fabricated poloxamer 407/carbon nanotube hybrid particles exhibit thermo-responsive structural features so that the density and thickness of poloxamer 407 layers are also reversibly controllable by varying temperature. The detailed structural properties of the poloxamer 407/carbon nanotube particles in suspension can be characterized by small-angle neutron scattering experiments and model fit analyses. The distinct curve shapes of the scattering intensities depending on temperature control or addition of aromatic additives are well described by a modified core-shell cylinder model consisting of a carbon nanotube core cylinder, a hydrophobic shell, and a hydrated polymer layer. This method can provide a simple but efficient way for the fabrication and in-situ characterization of carbon nanotube-based nano particles with a structure-tunable encapsulation.

A method for the functionalization of carbon nanotubes with structure-tunable polymeric encapsulation layers and structural characterization using small-angle neutron scattering is presented.

单壁碳纳米管与热可逆嵌段共聚物及表征功能化小角度中子散射

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

体外支持性脂质双层自组织蛋白模式的重组

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

生物化学

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

生物膜的微阵列用刮墨刀型组合方式形成

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

生物工程

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

在水/空气界面上掺杂与疏水有机分子混合脂膜的自组装

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

人工脂质纳米管网作为内质神经的底部模型的自发形成与重排

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

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 ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

使用滑动运动蛋白运动度测定法制造多组分脂质纳米管网络

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

使用 CIJ 和 μMIVM 混合器技术合成脂质纳米颗粒

Synthesizing Lipid Nanoparticles by Turbulent Flow in Confined Impinging Jet Mixers

Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of two COVID-19 messenger RNA (mRNA) vaccines. On a small scale, LNPs are often made using microfluidics; however, the limitations of these devices preclude their use on a large scale. The COVID-19 vaccines are manufactured in large quantities using confined impinging jet (CIJ) turbulent mixers. CIJ technology enables production at a laboratory scale with the confidence that it can be scaled to production volumes. The key concepts in CIJ mixing are that the mixing length and time scale are determined by the turbulence intensity in the mixing cavity and that the nanoparticle formation occurs away from walls, eliminating the problem of deposition on surfaces and fouling. This work demonstrates the process of making LNPs using confined impinging jet mixer technology with two geometries: the two-jet CIJ and the four-jet multi-inlet vortex mixer (MIVM). The advantages and disadvantages of each mixing geometry are discussed. In these geometries, LNPs are formed by rapid mixing of an organic solvent stream (usually ethanol containing the ionizable lipids, co-lipids, and stabilizing PEG-lipids) with an aqueous anti-solvent stream (aqueous buffer containing RNA or DNA). The operating parameters for the CIJ and MIVM mixers are presented to prepare reproducible LNPs with controlled size, zeta potential, stability, and transfection effectiveness. The differences between LNPs made with poor mixing (pipetting solutions) compared to CIJ mixing are also presented.

作者聚焦：通过在受限几何形状中通过湍流混合增强脂质纳米颗粒的形成

A detailed protocol for synthesizing lipid nanoparticles (LNPs) using confined impinging jet (CIJ) mixer technologies, including a two-jet CIJ and a four-jet multi-inlet vortex mixer (&#181;MIVM), is demonstrated. The CIJ mixers generate reproducible, turbulent micro-mixing environments, resulting in the production of monodisperse LNPs.

在受限冲击射流混合器中通过湍流合成脂质纳米颗粒

Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of ...