作者要感谢美国陆军医学研究和装备司令部（批准＃W81XWH-10-1-0806）和国立卫生研究院（批准＃U19青年AI091031-01和赠款＃1R01GM090280）的财政支持。国民承认化学与生物工程Balloun教授和NLBP的承认威尔金森教授跨学科的工程。我们感谢她协助执行的纳米官能实验朱莉娅贝拉。

1。高通量碳水化合物的合成<ol><li>之前自动dimannoside合成，适当保护的糖捐助，通常三氯，受体，主要是氟烯酒精，合成在板凳上顶。 </li><li>一个编写程序自动化合成为的dimannoside的。在图2给出一个基本的自动化程序示意图。在节目中，它是保证，前发起人此外，供体和受体的混合物搅拌至少30分钟。 </li><li>在二氯甲烷合成捐助，承兑人trimethylsilyltrifluoromethanesulfonate的的的解决方案。甲苯和二氯甲烷使用最频繁的糖基化反应。 </li><li>此外，准备的临时保护团体在80％和100％的甲醇甲醇脱试剂的解决方案。 </li><li>方案开始之前，确保在1.902香港专业教育学院室内的湿度是自动化室在30％或更低。高湿度是不利的糖基化反应。 </li><li>一旦程序启动，机械臂的顺序转移到反应瓶体和受体的解决方案。然后将混合物搅拌30分钟。 </li><li>下一步机械臂的混合物转移到0.2 0.3等值trimethylsilyltrifluoromethanesulfonate，，通常在室温下虽然较低的温度，如-20°C可以达到。反应混合物中，搅拌30分钟。 </li><li> 30分钟后，反应停止并拆除一个小等分，以监测反应进程。如果不完整的，可以继续反应，并最终可以修改所需要的时间。 </li><li>一旦反应完成后，被转移到反应混合物的氟固相萃取（FSPE）墨盒含C 8 F 17改性硅胶净化。 </li><li>购物车脊先洗净，用80％甲醇 - 水（8毫升），以获得摆脱非氟分数。 </li><li>然后墨盒100％的甲醇洗涤获得的氟标签所需的产品。如果需要额外的净化，可以停止机器的反应产物（S）为净化除去额外的手段。 </li><li>净化周期结束后，机械臂分配进入反应瓶甲醇钠。搅拌反应2小时。如果反应不完全，可以再次将持续一段较长的时间，最终可以修改程序所需的时间。 </li><li>反应完成后，该产品是由FSPE纯化，然后溶解在无水甲苯随后通过蒸发除去残留的水。 </li><li>然后重复周期（从6至13步）所需的链长，直到获得目标分子。 </li><li>自动化获得保护的产品是日EN进一步纯化和充分，如核磁共振（NMR）光谱技术的特点。最终的目标分子的完全脱（去除所有余下保护组），然后完成外自动化平台作为一项规则，因为它通常涉及爆炸的氢气和钯。最后脱步骤进行台式以外的自动化平台。第一步是臭氧分解氧化产生的醛到羧酸氟标记的双债券。该产品是由柱层析纯化。最后一步是由钯催化氢化脱苄醚组。该产品通过硅藻土垫摆脱钯得到纯净的最终产品。 </li></ol> 2。高通量的纳米粒子表面官能<ol><li>进行高通量的高分子合成和纳米制造出来后，相同PRotocol和机器人设置共聚物粒子制造系统的基础上癸二酸（SA）和1,6 -双（ 对羧基苯氧）正己烷（CPH）和1,8 -二（Petersen 等 19描述。第羧基苯氧）-3,6 dioxaoctane（CPTEG）和本院。一个机器人沉积设备使用的示意图，在图1b。 </li><li>随着纳米制造，持有含纳米粒子库管复位的直线驱动器阶段。 </li><li>酸酐颗粒表面附着的碳水化合物，胺羧酸偶联反应20连续两个反应组成的执行。 </li><li>第一反应，在第一个可编程的注射泵的注射器充满10当量（公式）（颗粒表面的平均摩尔羧酸浓度等值）1 - 乙基-3 - （3 - 二甲氨基）-CArbodiimide盐酸盐（EDC）和10式。在水溶液中的乙二胺，而在第二个可编程的注射泵的注射器装12式。 N-羟基琥珀酰亚胺（NHS）在水溶液中。 </li><li>使用LabVIEW程序，试剂悬浮存入纳米库*。 </li><li>接下来，每个样品超声在40赫兹（30秒）和管人脱离机器人平台。 </li><li>纳米悬浮液在4°C孵育为9小时**不断旋转</li><li>反应时间完成后，管离心（12000 XG为5分钟），返回到机器人站，执行两个步骤洗涤。 </li><li>用于洗涤，仍然是空的，并在加载第一个可编程的注射泵注射器，而在第二次注射泵的注射器用冷水填补。在每个管上清撤回到空的注射器和第二泵存款冷水。 </li><li>纳米的均匀化执行步骤2.6中所述的颗粒悬浮。管离心（12000 XG为5分钟）和第二次的洗涤步骤进行步骤2.9中所述。 </li><li>对于第二个反应，两个沉积步骤。在第一沉积步骤，12式。东区区议会都装有一台泵和12式。第二泵的NHS加载。 </li><li>第二沉积步骤，包括10式。第一和第二泵（即乳糖，半乳糖，或DI-甘露糖）***，并与10式第三泵的具体糖类。乙醇酸（作为对照****）。 </li><li>纳米悬浮液匀浆所述步骤2.6在4°C孵育不断旋转的Ĥ9 </li><li>反应时间完成后，一洗步骤执行步骤2.8，2.9和2.10。 </li><li>功能化纳米粒子库，然后放置在真空室中干燥至少2小时。 </li><li>功能化纳米粒子，然后字符通过X射线光电子能谱和高吞吐量的苯酚 - 硫酸试验，以确定表面组成和糖的浓度分别ized。利用扫描电子显微镜和动态光散射，以确定颗粒大小，粒度分布，表面电荷。 </li></ol>注：*沉积量每管中的纳米粒子的质量有所不同。 **第一个和第二个反应的反应时间是可以改变的调整最终糖浓度。 ***每个糖类沉积取决于所需的组成测试管。 ****乙醇酸对于碳水化合物的附件在本研究采用的具体反应，是作为一个连接器的控制因为地deprotected糖已经有这种分子共价连接，可以进一步附着到纳米粒子表面。 3。代表结果在FULLY保护dimannoside 如图2所示，合成使用的自动化平台。合成的化合物进行了表征，通过1 H NMR，1 VXR 400兆赫作为溶剂使用CDCL 3谱仪。 NMR谱如图3所示。 利用高通量的纳米制造和酸酐官能纳米此处所述，附件dimannose，乳糖和半乳糖已经进行了成功的10，11。使用此设置，确定最佳反应条件（即反应温度和时间），以达到预期的纳米功能化和形态。当反应进行，在4°C间，而不是室温，减少纳米粒子聚集SEM观察（数据未显示）。表1显示了代表性成果特性官能50:50 CPTEG：要么DI-甘露糖或本院纳米乳糖，在4°C的合成数据表明，由于功能化的纳米直径平均小幅增长。虽然非功能化纳米粒子有一个约负Zeta电位。 -20 mV时，官能颗粒表现出了积极的Zeta电位值，证明成功的纳米粒子表面官能。乳糖和DI-甘露糖都是中性糖，但是，从乙烯胺组二胺利用附加糖的链接可能是积极的zeta电位。 反应时间是另一个变量，可能会影响纳米粒子的最终形态和糖取得的依恋程度。通过调整反应时间，最终连接到纳米粒子表面的糖浓度可控制在如图4A所示。正如所料，增加上50:50 CPTEG的表面浓度dimannose：本院纳米总的反应时间和18小时后达到最高。 24小时的总反应时间与功能的纳米粒子被用来评价自己的能力为目标，对小鼠骨髓衍生的树突状细胞（DCS）CLRS。流式细胞仪是用来评估两个发光受体的表达（即，中再国际（CD209，DC-SIGN的）和甘露糖受体（CD206））与非官能刺激后，乳糖和DI-甘露糖功能化纳米粒子（ 图4B）。获得较高的两种受体的表达，这是一个有效的定位指示，乳糖和DI-甘露糖功能化纳米粒子时，细胞与刺激。然而，DI-甘露糖功能化粒子表现出较高的水平，表明这种配体特异性受体研究的一种表达。 <table border="1"><tbody><tr><td> 纳米粒子类型 </td><td> 平均粒径（nm）的 </td><td> 大道愤怒颗粒ζ电位（毫伏） </td></tr><tr><td>非官能</td><td> 162±43 </td><td> -20±0.6 </td></tr><tr><td>乳糖</td><td> 235±34 </td><td> 26±2.4 </td></tr><tr><td> DI-甘露糖</td><td> 243±32 </td><td> 30±4.2 </td></tr></tbody></table> 表1纳米表征。非功能化和功能化的特点是准弹性光散射和zeta电位测量。粒度数据表示平均值±标准偏差（SD）的动态光散射在三个独立的实验中收集到的数据。 Zeta电位数据代表三个独立的读数平均值±标准差。改变zeta电位的迹象表明，糖是有效结合的的50:50 CPTEG：青山医院的纳米粒子的表面。 <img alt="图1" src<html> =“/ files/ftp_upload/3967/3967fig1.jpg”/&gt; 图1。（一）图形表示碳水化合物酸酐纳米功能化和功能化纳米粒子库，可与所描述的高通量的方法设计的一个例子与追求的方法。 （二）利用粒子的功能化，其中包括（一）三个东北1000泵（二）机器人阶段集成两个驱动器（Zaber）：在x方向的运动之一，和其他自动沉积器具的示意图表示在y方向的运动;（三）两个相邻的机架（适当的管和试管）三个驱动器，每个方向（X，Y，Z）组成的机器人第二阶段。串联泵和一共有5个驱动器。执行器和泵的操作使用LabVIEW软件的计算机。此图是不是规模。arge.jpg“目标=”_blank“&gt;点击这里查看大图。 <img alt="图2" src="/files/ftp_upload/3967/3967fig2.jpg" /> 图2。自动图形表示迭代使用甘露糖作为一个例子的碳水化合物的合成。 <img alt="图3" src="/files/ftp_upload/3967/3967fig3.jpg" /> 图3。1 H核磁共振的保护dimannoside的。 <img alt="图4" src="/files/ftp_upload/3967/3967fig4.jpg" /> （一） 图4。 反应时间对纳米粒子表面糖的浓度。中显示的数据，50:50 CPTEG：本院纳米功能与dimannose在不同的反应时间和反应进行了在4°C所示的两个独立的功能化实验的平均值和标准误差。 （二）乳糖和DI-甘露糖功能化纳米粒子有效目标的DC-SIGN（CIRE，CD209）和甘露糖受体（CD206）在骨髓来源的树突状细胞刺激后官能50:50 CPTEG这两个标记的表达增强表明：青山医院纳米时所获得的表达比较非官能粒子。

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Soc. 132, 11428-11430 (2010).</li><li>Hakamori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+glycosylation+in+tumor+and+tumor+associated+carbohydrate+antigens.">Aberrant glycosylation in tumor and tumor associated carbohydrate antigens.</a> Adv. Cancer Res. 59, 257-331 (1989).</li><li>Atherton, T., Sheppard, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Solid-phase peptide synthesis: a practical approach. , (1999).</li><li>Caruthers, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+synthesis+machines:+DNA+chemistry+and+the+uses.">Gene synthesis machines: DNA chemistry and the uses.</a> Science. 230, 281-285 (1985).</li><li>Plante, O. J., Palmacci, E. R., Seeberger, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+solid-+phase+synthesis+of+oligosaccharides.">Automated solid- phase synthesis of oligosaccharides.</a> Science. 291, 1523-1527 (2001).</li><li>Ko, K. -. S., Park, G., Yu, Y., Pohl, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+group-based+colorimetric+monitoring+of+fluorous-phase+and+solid-phase+synthesis+of+oligoglucosamines.">Protecting group-based colorimetric monitoring of fluorous-phase and solid-phase synthesis of oligoglucosamines.</a> Org. Lett. 10, 5381-5384 (2008).</li><li>Pohl, N. L., Chen, X. H. R., Wang, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+solution-phase+oligosaccharide+synthesis+and+carbohydrate+microarrays:+development+of+fluorous-based+tools+for+glycomics.">Automated solution-phase oligosaccharide synthesis and carbohydrate microarrays: development of fluorous-based tools for glycomics.</a> Chemical Glycobiology. , 272-287 (2008).</li></ol>

NLBP创始人和在碳水化合物公司LuCella的生物科学公司持有权益

作为靶向制剂直接纳米粒子的相互作用，免疫细胞碳水化合物的疗效已证实10，11。在我们的实验室以前的研究已经表明，酸酐纳米粒子的特定糖能够针对不同的抗原提呈细胞（APC）CLRS，从而提高免疫细胞的活化，这可能是为进一步的T细胞活化10日，11日的重要。然而，针对几个参数，如酸酐化学，大小，密度糖型糖或表面需要进行优化，从而增加吞吐量挖掘这样一个多参数的系?...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>电动XYZ阶段：3倍的T​​-LSM050A，每轴50毫米的旅行</td><td> zaber技术</td><td>的T-XYZ-LSM050A-KT04 </td></tr><tr><td> NE-1000单注射泵</td><td>新时代热泵系统</td><td> NE-1000 </td></tr><tr><td>耐热* Vista *的无框可重复使用的玻璃培养管</td><td>康宁</td><td> 07-250-125 </td></tr><tr><td>屈臣氏集团1000 </td><td> chemspeed技术</td><td></td></tr><tr><td> LabVIEW的</td><td>国家仪器</td><td> 776671-35 </td></tr><tr><td>上海黄金交易所的气密注射器，鲁尔禄</td><td> Sigma Aldrich公司</td><td> 509507 </td></tr><tr><td> XL-2000 Sonicator </td><td> qsonica </td><td> Q55 </td></tr><tr><td>微型管肩</td><td> Fisher Scientific则</td><td> 05-450-127 </td></tr></tbody></table>

high-throughput synthesis of carbohydrates and functionalization of polyanhydride nanoparticles

涉及的领域，如材料设计，纳米技术，化学和免疫学的跨学科的方法必须利用合理设计有效的疫苗载体。纳米粒子为基础的平台，可以延长疫苗抗原的持久性，它可以提高疫苗的免疫原性。作为疫苗运载工具1几种可生物降解的聚合物已被研究，特别酸酐颗粒表现出的能力，提供持续稳定的蛋白抗原的释放和激活抗原呈递细胞和调节免疫反应2-12。 这些疫苗载体的分子设计需要集成的聚合物性能的合理选择以及适当的靶向制剂纳入。高通量靶向配体和官能颗粒制造自动化是一个功能强大的工具，这将提高学习能力，广泛ŕ昂热物业，并会导致重现疫苗输送装置的设计。 能够被免疫细胞上的特异性受体的靶向配体除了已被证明是调节和裁缝10,11,13 C型凝集素受体 ​​（CLRS）承认碳水化合物对目前的模式识别受体（PRRS）的免疫反应表面的病原体。刺激免疫细胞通过CLRS可以增强国际化，为进一步14,15 T细胞活化抗原和随后介绍。因此，糖类分子在免疫反应的研究中发挥重要作用，然而，使用这些生物分子经常遭受从可用性的结构定义和纯碳水化合物缺乏。基于迭代的解决方案相反应的一个自动化平台可以使快速和控制这些综合有挑战性的分子的合成，显着较低的Building比传统固相法16,17块的数量。 我们在此报告一个自动化的解决方案相如甘露糖为基础的靶向配体中间净化与氟固相提取低聚糖的合成协议。自动化的方法，使碳水化合物为主的靶向剂的发展后，我们描述酸酐纳米粒子的表面采用机器人自动化集由LabVIEW经营如前所述10附件方法。碳水化合物与表面官能针对的CLRS 10,11和增加吞吐量挖掘与多参数系统的复杂性，将具有极大的价值（ 图1a）的制造方法的疗效。

Watch this Scientific Journal Video about High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles at JoVE.com

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

在这篇文章中，高通量方法，为进一步利用针对特定抗原提呈细胞受体合成寡糖及其附件酸酐纳米粒子的表面。

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

high-throughput-synthesis-carbohydrates-functionalization

Research

JoVE Journal

Bioengineering

11.3K 次观看.  Iowa State University. 在这篇文章中，高通量方法，为进一步利用针对特定抗原提呈细胞受体合成寡糖及其附件酸酐纳米粒子的表面。

视频: High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

科研

生物工程

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

组合合成和高通量的蛋白质释放的聚合物薄膜和纳米粒子库

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

高吞吐量的单细胞和多细胞微胶囊

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

动脉粥样硬化的快速检测的氧化铁纳米颗粒微波驱动综合

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

医疗器械细菌高通量鉴定驱蚊聚合物

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues 用于可注射再生疗法和高通量药物筛选

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...