Name: forming giant-sized polymersomes using gel-assisted rehydration
Uploaded: 2016-05-26T15:00:00.000+00:00
Duration: 8 min 45 s
Description: Watch this Scientific Journal Video about Forming Giant-sized Polymersomes Using Gel-assisted Rehydration at JoVE.com

We would like to gratefully acknowledge Dr. Ian M. Henderson, Andrew Gomez and Dr. Walter F. Paxton for their technical expertise, advice and help in this work. This work was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering (BES-MSE). ACG, DYS and GDB were supported by BES-MSE. This work was performed, in part, at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science (user project number RA2015A0004, PI: ACG).

1.聚合物和琼脂糖准备注:手套和白大褂在任何时候都应在本协议佩戴。护目镜与任何有机溶剂或可能飞溅任何一步工作中同样需要。 <ol><li>制备在氯仿中的5mg / ml的聚合物溶液。 1毫升氯仿添加至5mg固体聚（环氧乙烷）的- B-聚（丁二烯）（PEO-PBD，EO 22 -BD 37）在玻璃小瓶中并涡旋充分溶解二嵌段共聚物。执行化学通风柜用氯仿所有步骤。 </li><li>在99.5％（摩尔）未标记的聚合物的0.5％（摩尔）的最终浓度混合在荧光标记的脂质（购已经溶解在氯仿）。 <ol><li>例如，吸管〜12微升的氯仿1毫克/毫升的荧光标记的脂质成997.58微升的PEO-PBD，EO 22 -BD 37聚合物的氯仿（与2950道尔顿的分子量）为0的最终浓度。5摩尔％标记的脂质混合，99.5摩尔％的聚合物。 </li><li>与在-20℃的耐氯仿 - 盖密封的管形​​瓶中商店溶液。裹在瓶边缘水管工的磁带，其中盖子拧在小瓶中并固定盖子小瓶。包裹在盖的外侧另一块管道工胶带的最后缠绕在带的外封口膜，以确保最小的蒸发。 </li></ol></li><li>通过在250毫升锥形瓶中的50毫升水（或者50μL的100mM蔗糖）中混合0.5毫克标准的琼脂糖制成1％w / v琼脂糖溶液。沸腾在标准微波〜1分钟的琼脂糖溶液（或直至完全溶解作为由溶液的结算说明）。 注意:琼脂糖溶液可以冷却几分钟后使用，或者使其固化，储存和使用相同的过程再次熔化。 </li></ol> 2.琼脂糖膜的制备<ol><li>切到底掀起了1000微升枪头，以防止堵塞。使用切尖，吸管300微升融化的琼脂糖溶液到25平方毫米的玻璃直接从制造商盖玻片。 </li><li>允许琼脂糖冷却至65-75〜℃下之前沉积。如果琼脂糖太酷了，就开始在传播过程中表面上聚集。相反，如果琼脂糖太热，这将需要更多的扩频粘附琼脂糖到表面。 </li><li>持用戴手套的手指盖玻片只是边缘，并使用另一个1000微升枪头的长边均匀地横跨盖玻片的表面上扩散的琼脂糖。移动枪头来回盖玻片直到琼脂糖完全附着并覆盖盖玻片。 </li><li>放置琼脂糖涂覆盖玻片到一张封口膜的与琼脂糖朝上。一旦盖玻片所需数量的涂覆，放置封口膜与盖玻片成37℃培养箱脱水琼脂糖到表面上至少1小时。 注:脱水通过琼脂糖的可见层的消失来确定;盖玻片应看平，清晰。一旦电影完全脱水，储存与琼脂糖面朝上的一次性塑料培养皿盖玻片。 </li></ol> 3.高分子成膜<ol><li>吸移管将30μl制备的聚合物溶液到琼脂糖膜。 </li><li>持用戴手套的手指盖玻片只是边缘并使用18G的针的长边跨使用圆形扩频运动琼脂糖膜散布溶液。继续传播，直到溶液蒸发。 注意:请小心针的尖底的。 </li><li>放置在一个塑料培养皿聚合物面朝上的聚合物膜，并把培养皿成覆盖铝箔标准室内真空干燥器（防止标记的脂质的漂白）至少1小时，以完全除去任何残留的溶剂。 </li></ol> 4.波尔的形成ymersomes <ol><li>坚持一个coverwell，无论是自制聚二甲基硅氧烷（PDMS）以及（与中间的约1cm直径固化的PDMS的约0.5厘米厚的块冲压出）或市售很好地涂覆有聚合物膜的盖玻片。轻轻按下coverwell到朝上直到紧密密封在coverwell和盖玻片之间形成的聚合物的聚合物涂覆的盖玻片。要小心，不要太用力按下，打破盖玻片。 </li><li>添加200-500微升补液溶液（水是好的，但缓冲剂或媒体的阵列也作品）至腔室（补液体积取决于附着在coverwell的大小）。 </li><li>创建一个湿度室以降低补液溶液的蒸发。放置沿着玻璃培养皿的边缘的轧制，湿的Kimwipe。与附着coverwell聚合物薄膜放入湿度室中，并用盖子覆盖。盖培养皿用铝箔，以尽量减少漂白。地点培养皿上热板设定为40℃至少30分钟。 </li><li>从24-70℃，调整加热板的再水化过程中的温度来调整所形成的小泡（ 图5）的大小。 70℃继续超出这个温度时，靠近琼脂糖的Tm 值所以应谨慎使用。 </li></ol> 5.漂白后表征荧光恢复聚合物囊泡（FRAP） <ol><li>移动盖玻片与附着coverwell直接到达摄像倒置显微镜。 </li><li>选择基于包含在聚合物溶液中的荧光脂质适当滤波器集合。罗丹明标记的脂质掺杂到聚合物溶液中，使用一个二十五分之五百六十零激发滤光器和三十六分之六百零七nm发射滤波器，或类似过滤器组。 </li><li>用100X油物镜聚焦，其中聚合物囊泡将附着在盖玻片的顶表面上。如果聚合物囊泡特别大（&gt; 20微米），或者如果聚合物膜太厚，较低功率的目标（ 例如，40X）应该被用于更好地可视化的聚合物囊泡。 </li><li>识别使用荧光显微镜的聚合物囊泡。由特性中空，球形囊泡直径&gt; 5微米的识别聚合物囊泡。 </li><li>表征利用荧光恢复细胞膜的流动性含有一个可调节冷凝器的荧光显微镜漂白（FRAP）之后。由于附着的性质和基材聚合物囊泡严密包装，聚合物囊泡的自然运动是有限的，增加FRAP成像质量。 <ol><li>专注于感兴趣的聚合物囊泡显微镜。关闭冷凝器的小区域，并确保一个聚合物囊泡的边缘是感兴趣的成像区域内。 </li><li>增加相机曝光，并确保所有的中性密度过滤器除去以有效光漂白的感兴趣的区域。允许膜FL<html>荧光强度光漂白30-60秒或直至荧光强度显著降低。 </li><li>关闭灯泡并完全打开冷凝器。减少曝光回到起始设置立即开始捕获图像，每30秒3分钟。 </li><li>计算使用标准方法12膜扩散系数。如果漂白区域重新获得3-5分钟内的荧光强度，这表明该膜是流体。 </li></ol></li></ol> 6.表征聚合物囊泡尺寸<img alt="图6" src="/files/ftp_upload/54051/54051fig6.jpg" /> 图6.使用映像软件的聚合物囊泡的大小分析过程。一步一步如何测量形成的小泡的直径指令。用户必须知道使用的显微镜​​的像素/微米的校准单元。ove.com/files/ftp_upload/54051/54051fig6large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <ol><li>打开聚合物囊泡的所获取的图像中的图像分析软件13。 </li><li>通过点击分析下拉框，并在点击"设置规模"设置为图像的规模。 <ol><li>校准刻度使用标准方法显微镜（即微米）。 </li><li>填写校验台，并单击"确定"。 </li></ol></li><li>点击在线工具盒，画一条线跨越泡的直径。 </li><li>通过点击分析下拉框，并点击"测量"收集长度测量。 </li><li>继续测量遵循上面的程序和每一个新的测量将被添加到测量数据窗口个别囊泡。 </li><li>按"Ctrl + D"绘制每一行，并测量长度，印画上线后，图像使其更容易追踪这些囊泡进行了分析。 </li></ol>

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Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

聚合物囊泡正在成为广泛地探讨作为生物膜模仿。聚合物的稳健和灵活的性能使得它们对需要膜功能化，长寿和调整的响应研究广泛的吸引力。形成巨型的聚合物囊泡9,10（&gt; 4微米），传统的方法是劳动密集和需要昂贵和专用设备。这里，我们提出的第一次中，用于形成使用标准便宜实验室试剂和设备巨尺寸聚合物囊泡一种快速，灵活和可靠的方法。 使用凝胶辅助补液，单层聚合物囊泡可以迅速地形成（&lt;30分钟），在各种补液溶液（包括细胞培养基）中，用各种不同的聚合物（未示出数据）。多层或不对称囊泡的形成使用这种技术，没有观察到。贯穿本工作中，我们使用的聚（氧化乙烯） - B-聚（丁二烯）（PEO-PBD，EO 22 -BD 37）中性二嵌段共聚物。许多不同的聚合物组合物（包括带电嵌段共聚物）工作良好使用此方法（未示出）。然而，一些可商购的三嵌段共聚物和较高分子量的二嵌段共聚物（〜&gt; 5,000道尔顿）不形成不同的聚合物囊泡。对于所有在此手稿的实验中，标记的脂质的低浓度掺杂到仅用于可视化目的的聚合物溶液。还可以用于可视化的其他方法，包括直接官能化用荧光染料的聚合物。聚合物囊泡同样可以用亮视野显微镜成像，虽然荧光显微镜提供更高的分辨率。 最少量修改的协议通常不改变的结果。例如，在分散到盖玻片的聚合物溶液的浓度小的差异不改变聚合物f的形成ILM形成。虽然没有测定完整浓度范围内，将成功地用一个大范围的聚合物膜的浓度（ 例如 ，1-10毫克/毫升）的发生聚合物囊泡形成。然而，也有一些协议改变那些聚合物囊泡的形成产生不利影响。最值得注意的是在形成非常差聚合物囊泡这一轮盖玻片（而不是正方形）的结果。我们认为这是极为甚至这实际上阻碍了聚合物囊泡形成的玻璃涂层琼脂糖。 一个该技术的最显着的挑战是要以高收率回收从表面聚合物囊泡的能力。有某些情况下，其中从原始表面去除聚合物囊泡可以是有利的。由于从脱水的聚合物膜的高背景荧光，移除个别的聚合物囊泡，并坚持他们打扫盖玻片将增加的成像质量和表征（汉邦FRAP分析过程中cularly）。要做到这一点，温和移液用移液管尖端，其中端已被切断将从表面解吸聚合物囊泡（虽然回收囊泡的数量比那些最初形成显著低级）。然后聚合物囊泡可以放置在修改盖玻片的表面，使聚合物囊泡与新盖玻片交互。通常情况下，为中性的PEO-PBD聚合物囊泡，盖玻片用臭氧处理15分钟，使聚合物囊泡向下落到表面用于成像。不同表面改性需要不同聚合物囊泡的组合物（ 例如，带负电或带正电荷的聚合物）。 在这个协议中使用的大多数材料是成功地存储并用于天至数周。固化琼脂糖可再沸腾和重复使用，直到琼脂糖开始有聚集体即使煮沸后，或凝固琼脂糖开始干燥。用干燥的琼脂糖膜盖玻片可以存储和使用无限期D（ 例如 ，月）。溶于氯仿的聚合物可以储存在-20℃下几个月。一旦聚合物薄膜上琼脂糖膜干燥，然而，膜需要房子在真空下被储存和使用的两个周（未直接测定长期贮存中，但也有在聚合物囊泡从聚合物膜年长超过两个形成明显的差异周）。 使用这里介绍的凝胶辅助补液协议，数百均匀状聚合物囊泡与使用标准设备和廉价的实验室试剂劳动短短几个小时内迅速形成。此外，可以以各种的生理缓冲溶液和从不同的聚合物组合物（ 未示出）来形成聚合物囊泡。次要的改变的方法不负面改变聚合物囊泡的形成，呈现凝胶辅助补液一个通用和方便的技术来与科学家不同的技术电子xpertise。 容易地对细胞的大小尺度创建巨型聚合物囊泡的能力是用于构建人工细胞样系统是必不可少的。凝胶辅助补液的使用和多功能性的易用性使这些聚合物囊泡提供了仿生领域的巨大进步为建立强大的细胞膜模仿的。例如，使用这种技术，对于不同的细胞内成分，具有细胞膜蛋白和膜转运蛋白的掺入的聚合物的官能化，封装策略仅举几个，可以设计成基于聚合物囊泡人工细胞。

合成细胞大小，巨单层囊泡（GUVs）的创建是在不同的细胞过程的体外重建越来越大的兴趣，以建立用于人造细胞样系统1,2的产生的框架。而脂膜组成GUVs是天然的，生物膜的优良模拟物，它们对环境波动的不稳定，并具有相当短的货架寿命。由于这些限制，两亲性嵌段共聚物已经被用作脂质模拟物以形成聚合物囊泡，或聚合物囊泡。在此范围内，聚合物囊泡是在仿生细胞系统的发展有利，因为它们增加的稳定性3，化学多功能4,5和修改的物理性状6 - 8。 以形成目前的方法巨型的聚合物囊泡包括electroformation 9和模板补液10，两个W的HICH是费时，劳动强度大，需要专门的设备和产生完好巨型聚合物囊泡的产量低。一个简单的凝胶辅助补液方法最近被用于生产脂质GUVs 11的研制。这里，我们描述了凝胶辅助补液技术的适应来创建具有不同的聚合物组合物巨聚合物囊泡，受控的大小，和在各种缓冲组合物。 简言之，将1％w / v的标准DNA凝胶电泳琼脂糖膜脱水到玻璃盖玻片。在氯仿制备的聚合物溶液然后通过脱水琼脂糖膜扩散并且使其蒸发。以下完全去除溶剂，聚合物膜被再水化中选择的，在温和加热（40-70℃）和巨（&gt; 4微米）大小的聚合物囊泡都在30分钟内形成的缓冲。这种方法产生快速使用标准实验室设备和reagen数百完全完好，结构良好的聚合物囊泡TS以最低的成本。 <img alt="图1" src="/files/ftp_upload/54051/54051fig1.jpg" /> 图1原理图描绘了凝胶辅助补液方法。二嵌段共聚物组成的巨聚合物囊泡是补液的约30分钟后形成的。亲水块表示在品红和疏水块在绿色表示。 <a href="https://www.jove.com/files/ftp_upload/54051/54051fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>125 ml Erlenmeyer flask</td><td>VWR</td><td>89000-360</td><td></td></tr><tr><td>18 guage needle</td><td>VWR</td><td>BD-305196</td><td></td></tr><tr><td>25 mm square coverslips</td><td>VWR</td><td>48366-089</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9539</td><td>A standard agarose for DNA gel electrophoresis</td></tr><tr><td>Chloroform</td><td>Sigma</td><td>288306-2L</td><td></td></tr><tr><td>Commerical coverwell</td><td>Electron Microscopy Sciences</td><td>70336</td><td>Press-to-Seal Silicone Isolators</td></tr><tr><td>Fiji Image Analysis Software</td><td>ImageJ</td><td></td><td>Free Download: http://fiji.sc/Fiji&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Glass vial with Teflon Lid</td><td>VWR</td><td>66009-556</td><td>1.9 ml capacity, case of 144</td></tr><tr><td>Liss-rhodamine B PE Lipid</td><td>Avanti Polar Lipids</td><td>810150C</td><td>1 mg of lipid at 1 mg/ml concentration in chloroform</td></tr><tr><td>Parafilm</td><td>VWR</td><td>52858-000</td><td>10.2 cm x 38.1 m</td></tr><tr><td>poly(ethylene oxide)-b-poly(butadiene) or PEO-PBD (with a molecular weight of 2,940)</td><td>Polymer Source</td><td>P2904-BdEO</td><td>&lt;a href=&quot;http://polymersource.com/dataSheet/P2904-BdEO.pdf&quot;&gt;http://polymersource.com/dataSheet/P2904-BdEO.pdf&lt;/a&gt;</td></tr><tr><td>Pastic Petri Dish</td><td>VWR</td><td>25384-092</td><td></td></tr><tr><td>Teflon tape</td><td>VWR</td><td>300001-057</td><td>1/2&quot; width</td></tr></tbody></table>

forming giant-sized polymersomes using gel-assisted rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

聚合物囊泡用上述（ 图1）和图2中所示的普通实验室设备所概述的程序形成。聚合物囊泡用去离子水（ 图3）再水合和上成像用100X油物镜在落射荧光倒置显微镜。请注意，如果聚合物囊泡是不可见的，他们可能在规模过大与100X油目标捕捉形成;较低的功率目标可能需要被替代使用。之一的使用凝胶辅助补液的优点是在各种补液溶液形成聚合物囊泡的多功能性。被成功地形成在去离子水中，一个完整的哺乳动物细胞培养基，如在图4中所示的蔗糖溶液和两个生理学相关的缓冲液（磷酸盐缓冲的盐水（PBS）或Tris缓冲盐水（TBS）），聚合物囊泡。在标准条件下（补液与在40℃下1小时）水，大于〜70聚合物囊泡通常每视图40×40微米2区域中。虽然从表面上看生产聚合物囊泡是不是均匀的，有几十个与该销售代表产量的视场。此外，聚合物囊泡是在溶液中稳定至少24小时。 聚合物囊泡大小很容易通过在不同温度下再水化聚合物膜调谐。在RT形成在去离子水聚合物囊泡具有2.9±0.7微米的平均直径。作为复水过程中的温度升高，聚合物囊泡的平均尺寸也增加（ 见表1）。在高温（&gt; 60℃），甚至大于100微米（ 图5）大小形成的聚合物囊泡。 所有的图像处理是使用开源的成像软件（ 图6）完成。采集到的图像在软件程序被打开。校准的像素大小订立设置比例框。使用行工具，系横跨直径绘制。每个单独的线绘制之后，所述标定长度进行测定，并加入到结果框。数据可以然后选择（ 如 Excel中，棱镜，产地等 ）的数学软件绘制<img alt="图2" src="/files/ftp_upload/54051/54051fig2.jpg" /> 图2.所需聚合物囊泡的形成所需要的项目的共同和廉价的实验材料的图片有:石蜡膜中，将18.5g针，聚合物在氯仿或其它合适的溶剂中，1000微升枪头，锥形瓶中，镊子，琼脂糖25平方毫米的盖玻片，PDMS成像井和一个玻璃培养皿。 <a href="https://www.jove.com/files/ftp_upload/54051/54051fig2large.jpg" target="_blank">请点击这里</a>查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54051/54051fig3.jpg" /> 图3.图片凝胶辅助补液的方法。琼脂糖溶解散布在25平方毫米的盖玻片，直到连薄膜覆盖整个盖玻片。然后盖玻片放入37℃培养箱和电影脱水。聚合物溶液沉积在脱水琼脂糖膜和使用以圆周运动的针传播。然后将盖玻片放置在干燥器中O / N充分蒸发的任何溶剂的残留物。最后，成像室粘附于基片和聚合物再水合以选择的溶液，并置于培养皿中，在40℃下进行至少25分钟，以允许聚合物囊泡的形成。 <a href="https://www.jove.com/files/ftp_upload/54051/54051fig3large.jpg" target="_blank">请点击查看的这个更大的版本数字。</a> </p<html> &gt; <img alt="图4" src="/files/ftp_upload/54051/54051fig4.jpg" /> 图4.聚合物囊泡可以形成在各种不同的缓冲器。PEO-PBD聚合物膜在40℃与所指示的缓冲器再水合1小时。比例尺= 10微米。 <a href="https://www.jove.com/files/ftp_upload/54051/54051fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54051/54051fig5.jpg" /> 图5.复水过程中增加温度增加了聚合物囊泡的大小。在所示补液温度在水中形成聚合物囊泡的代表性落射荧光图像。在较大的聚合物囊泡温度升高的结果。比例尺= 10微米。_blank"&gt;请点击此处查看该图的放大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>温度（℃） </td><td>平均尺寸（微米） </td></tr><tr><td> 24 </td><td> 2.93±0.7 </td></tr><tr><td> 40 </td><td> 5.76±2.5 </td></tr><tr><td> 50 </td><td> 6.65±2.4 </td></tr><tr><td> 60 </td><td> 11.46±5.8 </td></tr><tr><td> 70 </td><td> 14.04±7.0 </td></tr></tbody></table> 表1增加期间增加聚合物囊泡大小补液结果的温度。平均直径为大于100聚合物囊泡在每进行了计算不同温度条件的水和这里被描绘。错误是标准偏差。

Watch this Scientific Journal Video about Forming Giant-sized Polymersomes Using Gel-assisted Rehydration at JoVE.com

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

形成用凝胶辅助补液巨型的聚合物囊泡

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

forming-giant-sized-polymersomes-using-gel-assisted-rehydration

Research

JoVE Journal

Bioengineering

9.3K 次观看.  Sandia National Laboratories. We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

视频: Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

巨囊泡封装微球通过一个水包油乳液的离心制备

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

科研

生物化学

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

使用Photoredox催化剂蠕虫状胶束的简便合成可见光介导的分散聚合

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

化学

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

通过反应挤出、微流体、自组装和电纺丝制备多长尺度可降解 Thermoresponsive 水凝胶

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

生物工程

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

快速, 可伸缩的组装和负载的生物活性蛋白和免疫增强剂到不同的合成 Nanocarriers 通过闪光 Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

用于复杂蛋白质网络的脂质囊泡包封方案

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are&#160;trapped in microfluidic devices for analysis by optical microscopy.

作者聚焦:应对合成细胞工程中的挑战

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

在纳米和微米大小的囊泡中构建失衡代谢网络

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, ...

Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure

Polymersomes are membrane-bound, bilayer vesicles created from amphiphilic block copolymers that can encapsulate both hydrophobic and hydrophilic payloads for drug delivery applications. Despite their promise, polymersomes are limited in application due to their spherical shape, which is not readily taken up by cells, as demonstrated by solid nanoparticle scientists. This article describes a salt-based method for increasing the aspect ratios of spherical poly(ethylene glycol) (PEG)- based polymersomes. This method can elongate polymersomes and ultimately control their final shape by adding sodium chloride in post-formation dialysis. Salt concentration can be varied, as described in this method, based on the hydrophobicity of the block copolymer being used as the base for the polymersome and the target shape. Elongated nanoparticles have the potential to better target the endothelium in larger diameter blood vessels, like veins, where margination is observed. This protocol can expand therapeutic nanoparticle applications by utilizing elongation techniques in tandem with the dual-loading, long-circulating benefits of polymersomes.

Polymersomes are self-assembled polymeric vesicles that are formed in spherical shapes to minimize Gibb's Free Energy. In the case of drug delivery, more elongated structures are beneficial. This protocol establishes methods to create more rod-like polymersomes, with elongated aspect ratios, using salt to induce osmotic pressure and reduce internal vesicle volumes.

使用渗透压力调节聚酯聚合物的形状

Polymersomes are membrane-bound, bilayer vesicles created from amphiphilic block copolymers that can encapsulate both hydrophobic and hydrophilic payloads ...

形成用凝胶辅助补液巨型的聚合物囊泡

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巨囊泡封装微球通过一个水包油乳液的离心制备

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

使用Photoredox催化剂蠕虫状胶束的简便合成可见光介导的分散聚合

将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中

通过反应挤出、微流体、自组装和电纺丝制备多长尺度可降解 Thermoresponsive 水凝胶

快速, 可伸缩的组装和负载的生物活性蛋白和免疫增强剂到不同的合成 Nanocarriers 通过闪光 Nanoprecipitation

在纳米和微米大小的囊泡中构建失衡代谢网络

在纳米和微米大小的囊泡中构建失衡代谢网络

在纳米和微米大小的囊泡中构建失衡代谢网络

使用渗透压力调节聚酯聚合物的形状