Name: facile and efficient preparation of tri-component fluorescent glycopolymers via raft-controlled polymerization
Uploaded: 2015-06-19T14:00:00.000+00:00
Duration: 10 min 54 s
Description: Watch this Scientific Journal Video about Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization at JoVE.com

This work was supported by the Experiment Station Chemical Laboratories of the University of Missouri, and by the Cystic Fibrosis Association of Missouri.

1.合成Glycomonomer 2- Lactobionamidoethyl甲基丙烯酰胺<ol><li>溶解2克乳糖酸3.0毫升无水甲醇和缓慢下降明智的时尚加无水乙醇直到溶液刚变浑浊，然后通过旋转蒸发除去溶剂。 </li><li>溶解残渣，从步骤1.1，溶在3.0ml无水甲醇中，并再次，慢慢加入无水乙醇直至刚好混浊，然后经由旋转蒸发蒸去溶剂。重复此步骤3次，得到lactobiono -1,5-内酯（1.94克，98％产率）。此产品具有足够的纯度在下面反应中使用。 </li><li> 3.0毫升甲醇中添加1.0克lactobionolactone与N - （2-氨基乙基）甲基丙烯酰胺（AEMA，0.58克）和氢醌单甲醚（MEHQ，1.0毫克），自聚合的抑制剂，2.0毫升甲醇中，接着以1.0毫升三乙胺。在RT搅拌48小时。 </li><li>加20 500ml的去离子H 2澳（DH 2 </子&gt; O）的反应烧瓶中，然后通过蒸发除去甲醇和自由三乙胺通过旋转蒸发浓缩至干。 </li><li>以除去任何剩余的乳糖酸，加20的dh 2 O中的溶液，然后通过阴离子交换柱的水溶液（OH -型 ，10毫米×20毫米）到含有1.0毫克的MEHQ接收烧杯中。 </li><li>除去三乙胺，在步骤1.5中产生的，通过蒸发，通过旋转蒸发浓缩至干。 </li><li>添加20毫升的dh 2 O，并通过缓慢加入阳离子交换树脂（H +型 ）的1毫克等分试样直至不再茚三酮反应性材料是可检测的除去未反应AEMA。监视除去通过取该溶液的1微升等份各树脂加入后，将其应用到一个薄层层析板，然后喷涂板在乙醇溶液中的2％茚三酮。当没有深蓝色的颜色被观察到的开发时，板被加热到90℃保持1分钟，终点已经到达。 </li><lI&gt;通过多孔玻璃漏斗过滤溶液，将滤液转移到一个试管中，冷冻的样品在-80℃下，然后冷冻干燥。 </li><li>从样品中取出的MEHQ通过在甲醇（约0.5毫升）的最低量溶解的冷冻干燥物，然后添加冷的无水丙酮（-20℃，15毫升）以沉淀产物。用烧结玻璃漏斗收集沉淀，通过过滤，然后干燥该沉淀物在干燥器中在真空下，得到2- lactobionamidoethyl甲基丙烯酰胺（LAEMA），为灰白色粉末（0.94克，68％产率）。此产品具有足够的纯度在下面反应中使用。 </li></ol> 2.合成单体2- Gluconamidoethyl甲基丙烯酰胺注:2-gluconamidoethyl甲基的制备（GAEMA），它不具有侧链的糖，改编自发布方法15。 <ol><li>添加2.0克AEMA的溶解在10毫升甲醇中的溶液的D-葡萄糖酸内酯（1.6克）在30毫升甲醇中，并在搅拌下，慢慢地添加1.6毫升三乙胺。 </li><li>在RT搅拌24小时，进行反应。 </li><li>用烧结玻璃漏斗过滤沉淀出的产品，并冲洗沉淀三次，每次10毫升异丙醇中，然后用10毫升无水丙酮。干燥在真空下干燥器沉淀的产物。 </li></ol> 3. R​​AFT Glycopolymer合成<ol><li>以除去抑制剂的MEHQ存在于商业的N - （2-羟基乙基）丙烯酰胺（HEAA），加入1 ml HEAA至2ml微量离心管中，随后加入0.5克三氧化二铝纳米颗粒。离心管中，在300×g离心30秒，并使用顶层HEAA在以下的反应。 </li><li>小心加入32.8毫克LAEMA的（70.0微摩尔），1.7毫克AEMA的（10.5微摩尔）和27.5微升HEAA（270微摩尔），全部溶解在0.4的dh 2 O毫升，以良好的清洁1毫升Schlenk管，从而有一个monom3:77 20 ER摩尔比。 </li><li>在平行的反应中，为了产生对照聚合物，其不具有任何挂件糖，以代替在步骤3.2采用LAEMA的，在反应代替21.4毫克GAEMA的（70.0微摩尔）。 </li><li>到相应的Schlenk管（ 即 3.2或3.3），依次添加含0.53毫克（4-氰基戊酸）-4-二硫代苯甲酸酯（1.9微摩尔，RAFT试剂）和50微升DMF中含有250微克的DMF中的50微升4,4&#39;-偶氮双（4-氰基戊酸）（0.9微摩尔，引发剂）。通过手指轻轻拍打混合。 </li><li>冻结包含在Schlenk管内容采用干冰:乙醇浴（75克干冰在100ml乙醇中），内10-50毫乇施加真空，然后关闭的Schlenk阀并允许溶液缓慢解冻至室温。重复此冷冻撤离冻融循环两次。确保所有的试剂，最后解冻后解散。 </li><li>将舒伦克管插入密封塑料BA克，疏散袋的空气，然后密封。含有Schlenk管袋转移到水浴预热在70℃孵育24小时。 </li><li>在Schlenk管中的溶液小心地转移到准备好的透析袋（MWCO = 3500），和透析针对卫生署2 O（10×2升）24小时，改变的dh 2 O每隔一小时的前8个小时。以下透析，从透析管转移样品到一试管中，冷冻的样品在-80℃下，然后冻干它。 </li></ol>注意:得到的统计的聚甲基丙烯酰胺/丙烯酰胺（PMA）含有侧链4-ö-β-D-吡喃半乳糖基-D-葡糖酰胺（lactobionamide）（来自步骤3.2）或D-葡糖酰胺（来自步骤3.3）共聚物，分别是获得。为便于讨论，这两个glycopolymers简写为PMA-LAEMA和PMA-GAEMA分别。 4.后期修饰Glycopolymers与荧光团</P&gt; <ol><li>溶解5.0毫克含有〜0.9微摩尔的伯胺官能团的glycopolymer PMA-LAEMA或PMA-GAEMA在0.9磷酸盐缓冲盐水（PBS，0.1M磷酸钠，0.15M氯化钠，pH值7.5）中的溶液，分别</li><li>慢慢在100μlDMF中添加0.6毫克的羧基琥珀酰亚胺基酯向快速搅拌下的解决方案。轻轻搅拌该反应16小时，在黑暗中在室温。 </li><li>虽然避光，装载样品放入事先准备好的透析管（MWCO = 3500），并针对透析卫生署2 O（2升）16小时，更换透析液每隔一小时前8小时。以下透析，从透析管转移样品到一试管中，冷冻的样品在-80℃下，然后冻干它。 </li></ol>注意:按照冻干，荧光glycopolymers PMA-LAEMA荧光素和PMA-GAEMA荧光素，分别被获得。 在甘氨酸5表征共聚物<ol><li>确定数均分子量（M n）时，重均分子量（M w）的与glycopolymers的上装有凝胶渗透色谱法（GPC）的软件，其GPC商用HPLC系统的分散度（M W / M n）的列适合分子量的兴趣，和折射率检测器，用0.1M的Tris / 0.1M的氯化钠缓冲液（pH 7）作为洗脱剂以0.6ml / min的14的流速。使用聚乙二醇标准作为分子量标准（分子量:200-1,200,000克/摩尔）。 </li><li>量化glycopolymers 16内伯胺官能团的实际浓度。根据公布的方法分析17总碳水化合物的含量合成glycopolymers的。 </li><li>通过核磁共振光谱执行14结构组成和glycomonomers LAEMA纯度测试，GAEMA和glycopolymers PMA-LAEMA，PMA-GAEMA在D 2 O中。 </li></ol>合成Glycopolymers与6.结合凝集素试验涂琼脂糖珠<ol><li>添加1.5毫升PBS中至50μl悬浮液的鸡冠刺桐凝集素（ECL）包被的琼脂糖珠，离心机在300×g离心1分钟，并小心地取出和处理上清液。重复此步骤两次，然后重悬珠子在0.5ml PBS中。 </li><li>加入3 PMA-LAEMA荧光素或PMA-GAEMA荧光素（阴性对照）在6微升PBS至珠粒悬浮微克，孵育混合物，在黑暗中，在室温进行1小时。 </li><li>用1.5毫升PBS清洗3次，并重新悬浮珠0.2毫升的PBS洗涤混合物。加载一个等分试样（4微升）到一个井上的免疫荧光显微镜载玻片（聚四氟乙烯涂层），盖上盖玻片，并使用FITC滤波用荧光显微镜观察（激发波长:467-498纳米，发射波长:513- 566纳米）和10X客观的检查T的结合他荧光glycopolymers与珠14。 </li></ol>

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The authors have nothing to disclose.

一种简便，高效的协议进行的RAFT基三组分荧光glycopolymers，具有和不具有侧链的碳水化合物，以及它们在一个凝集素结合试验使用，证明本报告中。该协议开始的glycomonomers LAEMA和GAEMA的准备。通过单步的RAFT控制共聚，glycopolymers具有可重复的产率，预测的单体组合物和低分散​​性，能得到。以下修正后glycopolymers用羧基琥珀酰亚胺酯，将所得各荧光标记glycopolymer的结合是很容易可测试其凝集素结合特异性。 在初始制备的步骤是在随后glycopolymer合成所采用的glycomonomers的，容易获得的乳糖酸和葡糖酸内酯被利用。从理论上讲，任何感兴趣的碳水化合物，从单糖到复杂的寡糖，可以转换器具d，来glycomonomers由缀合的靶糖到伯羟基上的葡萄糖的C6。以下的氧化还原葡萄糖残基，和其后续的脱水内酯，产物然后可以容易地与上AEMA的伯胺反应形成相应的glycomonomer反应。这条路线的进一步的实例可以看出，在最近的一份报告14。应当指出的是，在启动任何聚合步骤之前，MEHQ，一种有效的聚合抑制剂，必须从所有单体和glycomonomer制剂在使用前除去。这可容易的通过使用甲醇的最小量以溶解该具有MEHQ然后立即用丙酮把它在-20℃以沉淀在高收率抑制剂无产品的glycomonomer完成。 至关重要的自由基聚合计划，注重细节和单体纯度的强调。由于是典型在RAFT聚合体系，它由自由基源，在RAFT试剂，单体和溶剂。在这种可视呈现，单步RAFT聚合系统进行说明，着重于生产从反应混合物具有在水溶液中三种不同的单体生成的统计共聚物。两个独立的RAFT介导的反应被呈现，其中一个利用glycomonomer一个拥有一个挂件，非还原性糖类末端（ 即，β-D-半乳糖），以及其他的，具有无界糖残基的多元醇。常见的两种RAFT介导的反应是单体具有在作为间隔分子奇异羟基，而另一个具有一个游离胺为修正后与氨基反应的荧光团。 因为氧在反应混合物中，环境中的存在是有害的RAFT介导的聚合，其去除跟踪水平通过几个冷冻EVA是容易实现cuate - 解冻循环，同时保持在高真空下的Schlenk管反应容器中。 应当指出的是，在反应的不同单体的摩尔比可根据需要进行调整。另外，通过改变RAFT试剂的使用量，所得到的聚合物的长度，可以控制18。然而，在RAFT试剂与引发剂的摩尔比应始终大于二，以确保产品的低分散度。在这些条件下，共聚的演变是稳定，并且该反应的再现性是非常高的。如此说来，这是不可能的，可以得到所有参与单体的统计共聚物内的完全均匀分布，由于它们的不同的聚合速度。表征不同单体的聚合物中的分布仍是非常具有挑战性的。 修正后的方法，在这里提出，是既简单，更amenabl以e为使用更宽选择的荧光标记，比施加到标签glycopolymers 2,11其他协议。这些将包括许多水溶性胺 - 反应性荧光团，量子点，生物素，和其他。合成，标记glycopolymers的结合特异性可容易地核查使用外源凝集素与已知结合亲和力。 PMA-GAEMA属没有挂件糖是一个合适的阴性对照。 Glycopolymers经由这个路线来制备不同的荧光标记物已被成功地用于在的凝集素介导的细菌结合14调查。所提出，此简单而且有效的统计荧光glycopolymers准备应于各种各样glycobiological研究提供的巨大潜力。

在过去的二十年里，研究合成glycopolymers经历了缓慢但持续的发展，在研究，包括研究侧重于凝集素识别过程1-3传染机制证明显著的潜力。因为合成glycopolymers具有多价的糖部分表现出高得多的凝集素结合功效相比，单价碳水化合物，它们在糖生物学领域3很大的需求。在临床研究中特别感兴趣的是使用荧光glycopolymers的表征的凝集素介导的细菌可用对人体呼吸道细胞表面和粘膜糖蛋白的碳水化合物结合。早在体外研究中细菌的结合试验中使用可商购的聚丙烯酰胺系glycopolymers。其中一些探头表现出令人鼓舞的结果，但对于，obtainability，和很多到很多差异在这两个POL引发市场担忧ymer分子量和glycoepitope内容。一种经济的在实验室协议被开发这将提供对结构的含量，大小和靶向细菌凝集素合成glycopolymers的纯度令人满意的控制。 在寻 ​​找一个合适的合成方法来glycopolymers，一个相对较新的聚合技术，使用采用可逆加成-断裂链转移（RAFT）试剂4一型受控的自由基聚合的测试。这样的RAFT试剂最近已应用在几glycopolymer制剂5-7。与其他glycopolymer制备协议相比，RAFT介导的聚合中表现出一些优点，包括耐多种单体的结构和反应条件，用水溶液潜在的兼容性，以及所需的聚合产物8,9-低尺寸分散性。值得注意的令人感兴趣的是制备RAFT-BA的协议SED三组分glycopolymers，允许不同的单体，其中每一个可具有不同的功能10-13的组合物的控制。然而，大多数的以前的研究工作要么缺乏异头挂件碳水化合物10，或采用阶梯导致三嵌段共聚物的聚合反应，即包括共价连接的均聚物，这往往用于不同的目的不是统计聚合物，其是共聚物，其中单体的序列残留遵循统计规律9-13。 最近，采用硫羰基硫基RAFT化合物（4-氰基戊酸）-4-二硫代苯甲酸酯在含水环境中，一组的制备RAFT基含有特定挂件糖及其在应用线性三组分统计glycopolymers凝集素介导的细菌结合测试报告14。该方法中，以视觉方式呈现的总体目标，是制备三组分通过RAFT控制共聚统计荧光glycopolymers。因为易于一步法聚合协议的，精细地控制聚合物长度和组合物，以及该反应的高再现性，该协议可以很容易地应用于glycopolymers具有所需结构的其它的RAFT基合成。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Lactobionic acid</td><td>Sigma-Aldrich</td><td>153516</td><td></td></tr><tr><td>D-Gluconolactone&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G2164</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(2-hydroxyethyl) acrylamide (HEAA)</td><td>Sigma-Aldrich</td><td>697931</td><td></td></tr><tr><td>Orange II sodium salt</td><td>Sigma-Aldrich</td><td>O8126</td><td></td></tr><tr><td>Hydroquinone monomethyl ether (MEHQ)</td><td>Sigma-Aldrich</td><td>54050</td><td>Polymerization inhibitor</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(2-aminoethyl) methacrylamide hydrochloride (AEMA)</td><td>Polysciences, Inc</td><td>24833-5</td><td></td></tr><tr><td>Triethylamine</td><td>Fisher Scientific</td><td>BP-616</td><td></td></tr><tr><td>Anion-exchange resin IRN-78 hydroxide-form, 80 mesh</td><td>Sigma-Aldrich</td><td>10343-U</td><td></td></tr><tr><td>Cation-exchange resin 50Wx8, 200 mesh</td><td>Sigma-Aldrich</td><td>217514</td><td></td></tr><tr><td>Aluminum oxide, ~150 mesh&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A1522</td><td>Type WN-6, Neutral, Activity Grade Super I</td></tr><tr><td>Ninhydrin</td><td>Sigma-Aldrich</td><td>N4876</td><td>An ethanol solution of 0.2% ninhydrin was used in the test</td></tr><tr><td>4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid</td><td>Sigma-Aldrich</td><td>722995</td><td>RAFT agent</td></tr><tr><td>4,4&amp;prime;-Azobis(4-cyanovaleric acid)</td><td>Sigma-Aldrich</td><td>11588</td><td>Polymerization initiator</td></tr><tr><td>Carboxyfluorescein succinimidyl ester&amp;nbsp;</td><td>Life Technologies</td><td>C1157</td><td></td></tr><tr><td>Erythrina Cristagalli lectin coated agarose bead</td><td>Vector Laboratories</td><td>AL-1143&amp;nbsp;</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Solvent&lt;/strong&gt;</td></tr><tr><td>dH2O</td><td></td><td></td><td>Produced by Barnstead water purification system, 18 megOhm-cm</td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>A461-4</td><td>ACS grade or better</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A454-4</td><td>ACS grade or better</td></tr><tr><td>Absolute ethanol</td><td>Fisher Scientific</td><td>BP2818-100</td><td>ACS grade or better</td></tr><tr><td>Dimethylformamide</td><td>Sigma-Aldrich</td><td>22705</td><td>ACS grade or better</td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A929-4</td><td>ACS grade or better</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Dialysis membrane (MWCO: 3,500)</td><td>Spectrum Labs</td><td>132720</td><td></td></tr><tr><td>Polyethylene glycol analytical standard standard</td><td>Sigma-Aldrich</td><td>O2393</td><td></td></tr><tr><td>Schlenk tube, 1 ml</td><td>Quark Glass</td><td></td><td>Customized</td></tr><tr><td>TSK-GEL G4000 PWxl&amp;nbsp;</td><td>Tosoh Bioscience&amp;nbsp;</td><td>8022</td><td>Used for GPC analysis of the glycopolymers</td></tr><tr><td>Empower 3 with GPC/SEC package</td><td>Waters Corporation</td><td></td><td></td></tr><tr><td>Waters Alliance HPLC system&amp;nbsp;</td><td>Waters Corporation</td><td></td><td>Equipped with refractive index detector (Waters 2414) and fluorescence detector (Waters 2475)</td></tr><tr><td>Avance III 800 MHz NMR Spectrometer</td><td>Bruker Corporation</td><td></td><td></td></tr><tr><td>BX43 fluorescence microscope</td><td>Olympus Corporation</td><td></td><td>Used with FITC filter in the glycopolymer binding test</td></tr><tr><td>Rotavap / Rotoevaporator</td><td>Heidolph</td><td></td><td></td></tr><tr><td>Fritted disc funnel</td><td>Fisher Scientific</td><td>10-310-109</td><td></td></tr><tr><td>Lyophilizer</td><td>Labconco</td><td></td><td></td></tr><tr><td>Immunofluorescence microscope slide</td><td>Polysciences</td><td>18357-1</td><td></td></tr><tr><td>Revco Ultima Plus -80&amp;nbsp;&amp;#176;C Freezer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Plastic Vacuum Bag and Hand Pump</td><td>Ziploc</td><td></td><td></td></tr><tr><td>Vacuum Pump, Direct Drive, Maxima C Plus</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Vacuum Gauge</td><td>Sargent-Welch</td><td></td><td></td></tr></tbody></table>

facile and efficient preparation of tri-component fluorescent glycopolymers via raft-controlled polymerization

合成glycopolymers是各种生化和生物医学研究领域使用的工具和灵活的工具。使用可逆加成 - 断裂链转移控制良好的荧光统计glycopolymers的简便而有效的合成的一例（RAFT）基聚合是证明。该合成开始于由lactobionolactone 和 N的反应得到β -半乳糖-含glycomonomer -2- lactobionamidoethyl甲基丙烯酰胺的制备- （2-氨基乙基）甲基丙烯酰胺（AEMA）。 2- Gluconamidoethyl甲基丙烯酰胺（GAEMA）被用作结构类似物缺乏一个终端β-D-半乳糖苷。下面RAFT介导的共聚合反应涉及三个不同的单体:N - （2-羟乙基）丙烯酰胺作为间隔物，AEMA作为靶进行进一步荧光标记，和glycomonomers。宽容含水系统，在反应中使用的RAFT试剂是（4-氰基戊酸）-4-二硫代苯甲酸酯。低分散度（≤1.32），可预测的共聚物组合物，以及高的聚合再现性的产品中观察到。荧光聚合物是通过修改glycopolymers用羧基琥珀酰亚胺酯靶向伯胺官能团上AEMA获得。所得glycopolymers的凝集素结合特异性通过用涂有特定glycoepitope识别外源凝集素相应琼脂糖珠测试验证。因为易于合成的，严密控制的产品组合物和反应的重现性好，该协议可以被翻译朝向其它制备RAFT基glycopolymers具有特定的结构和组合物，根据需要。

glycomonomer的合成 乳糖酸被本文用作glycomonomers的制备的一个例子。使用在LAEMA 11合成的初步报告的方法，不同的收益率在准备与纯度不理想，观察。使用阳离子和阴离子交换树脂的改性的纯化方法，除去提供稳定的产品产率和高纯度的未反应的起始原料，它是由1 H和13 C-NMR光谱（ 图1）证实。 RAFT glycopolymer合成和修改后的glycopolymers与荧光 与此相反，通过台阶的RAFT聚合制备的嵌段glycopolymers，这一步共聚协议提供在整个聚合物主链均匀glycomonomer分布。这里示出的glycopolymers含有20摩尔％GL的ycomonomer，77摩尔％HEAA作为间隔物，和3摩尔％AEMA的作为后修饰（参见图2）。1 H和13 C-NMR光谱法的靶确认PMA-LAEMA和PMA-GAEMA的结构（ 图3和4）。如图5所示 ，当对没有RAFT合成的glycopolymer的GPC洗脱曲线作图，二者PMA-LAEMA和PMA-GAEMA具有低分散度，证明了RAFT方法的功效。正如预期的那样，PMA-GAEMA有Mn为比PMA-LAEMA由于PMA-GAEMA缺乏挂件糖较小。碳水化合物和伯胺官能团的RAFT glycopolymers的内容的分析表明，单体在产物glycopolymers的比率为与开始在RAFT介导的聚合反应（ 表1）使用的单体的化学计量比是一致的。这标志着单体compositio的严格控制ns的合成glycopolymers，作为设计的。 伯胺官能基团与活化的荧光团反应是在蛋白质标记一种广泛使用的技术。这种技术就是在这里采用与羧基标签纯化glycopolymers。以下修正后，荧光聚合物获得（ 图6）。通过GPC分析法检测反应中的荧光素标记的聚合物的不降解（数据未显示）。 结合合成glycopolymers测试与外源凝集素涂层琼脂糖珠 以评估合成glycopolymers的凝集素结合特异性，凝集素包被的琼脂糖珠与已知的糖结合特异性使用。 鸡冠刺桐凝集素（ECL），在实验中使用的，具有朝向β-D-半乳糖苷的结合特异性。 图7A清楚地表明，PMA-LAEMA-FLuorescein，其中包含β-D-半乳糖苷作为侧碳水化合物，表现出很强的与ECL凝集素结合。与此相反，负结合于glycopolymer PMA-GAEMA荧光素，它不具有一个侧挂糖的ECL， 如图7B所示 。该结果例证了合成荧光glycopolymer的结合效力和亲和力。 <img alt="图1" src="/files/ftp_upload/52922/52922fig1.jpg" /> 图1.分配1 H-（a）和13 C-NMR（B）光谱（D 2 O）为LAEMA。（这个数字已经被修改Wang 等 14） <a href="https://www.jove.com/files/ftp_upload/52922/52922fig1large.jpg" target="_blank">请点击这里查看本一个更大的版本图。</a> &lt;P类="j​​ove_content"FO:保持together.within页="总是"&gt; <img alt="图2" src="/files/ftp_upload/52922/52922fig2.jpg" /> 图2.含β半乳糖苷作为吊坠糖荧光glycopolymer PMA-LAEMA的合成示意图。 <a href="https://www.jove.com/files/ftp_upload/52922/52922fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52922/52922fig3.jpg" /> 图3.分配1 H-（A）和13 C-NMR（B）光谱（D 2 O）的PMA-LAEMA glycopolymer。（这个数字已经被修改Wang 等 14） <a href="https://www.jove.com/files/ftp_upload/52922/52922fig3large.jpg" target="_blank">辩诉交易</a>SE点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/52922/52922fig4.jpg" /> 图4.分配1 H-（A）和13 C-NMR（B）光谱（D 2 O）的PMA-GAEMA。（这个数字已经被修改Wang 等 14） <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alt="图5" src="/files/ftp_upload/52922/52922fig5.jpg" /> RAFT基PMA-GAEMA和PMA-LAEMA图5.凝胶渗透色谱痕迹使用和不使用RAFT试剂制备的。与此相反的PMA-LAEMA而不RAFT试剂（蓝色）中制备，RAFT-<html>基于PMA-LAEMA（绿色）具有低得多的分散度（M W / M n）的。筏基PMA-GAEMA（红色）和PMA-LAEMA有类似的GPC曲线，但前者具有较小的Mn为由于没有任何挂件糖。 <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52922/52922fig6.jpg" /> 白色非标记glycopolymer（左管）相比图6 PMA-LAEMA前和修正后用荧光团之后。（A）中 ，荧光素标记的PMA-LAEMA显示强的黄色（右管）。 （B）在紫外线，非标记的PMA-LAEMA（左管，1毫克/毫升在PBS中）是黑暗，并与无荧光呈现，而荧光素标记的PMA-LAEMA（右管，1毫克/毫升在PBS中）示出了强烈的绿色荧光。://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图7" src="/files/ftp_upload/52922/52922fig7.jpg" /> 图7. 鸡冠刺桐凝集素（ECL）涂层琼脂糖珠结合含glycopolymersβ-D-半乳糖，而不是那些不具备一个挂件糖。（A）PMA-LAEMA荧光素（3微克）表现出很强的结合与ECL ，而在（B）的 PMA-GAEMA荧光素，其具有无挂件β-D-半乳糖苷残基，显示出与凝集素包被的珠无结合。 比例尺= 100微米。 表1.定向值的合成参数和glycopolymers的实际组成。一）定位值，值期望的产品; B）DP，聚合度; C）NA，无法使用。 <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody style="vertical-align:middle;text-align:center"><tr><td style="width:120px"></td><td style="width:40px"> DP b </td><td style="width:70px"> 分散度 </td><td style="width:120px"> glycomonomers的实际内容 摩尔％ </td><td> 伯胺的实际内容 摩尔％ </td></tr><tr><td> 指定值 </td><td> 100 </td><td> &lt;1.3 </td><td> 20 </td><td> 3 </td></tr><tr><td> PMA-LAEMA </td><td> 99 </td><td> 1.26 </td><td> 19 </td><td> 3.2 </td></tr><tr><td> PMA-GAEMA </td><td> 89 </td><td> 1.32 </td><td> NAÇ </td><td> 2.7 </td></tr></tbody></table>

Watch this Scientific Journal Video about Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization at JoVE.com

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

通过RAFT控制聚合轻便和高效制备三组分荧光Glycopolymers

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and ...

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JoVE Journal

Chemistry

9.6K 次观看.  University of Missouri. An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

视频: Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

密度梯度多层聚合（dGMP的）：创建多室的，可定制的一种新技术，组织工程支架

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

科研

生物工程

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

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

可控合成和高度统一聚荧光追踪（<em&gt;ñ</em&gt; -isopropylacrylamide）微凝胶

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this ...

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.

A protocol for the synthesis and characterization of diffusive motion of cyclic polymers at the single molecule level is presented.

环状聚合物的合成及其在熔融状态扩散运动的表征在单分子水平

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This work presents the preparation of methionine functionalized biocompatible block copolymers via RAFT polymerization. Firstly, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-aminopropyl)methacrylamide (BNHEMA-b-APMA, BA) was synthesized via RAFT polymerization using 4,4&#8217;-azobis(4-cyanovaleric acid) (ACVA) as an initiating agent and 4-cyanopentanoic acid dithiobenzoate (CTP) as the chain transfer agent. Subsequently, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-guanidinopropyl)methacrylamide (methionine grafted BNHEMA-b-GPMA, mBG) was prepared by modifying amine groups in APMA with methionine and guanidine groups. Three kinds of block polymers, mBG1, mBG2, and mBG3, were synthesized for comparison. A ninhydrin reaction was used to quantify the APMA content; mBG1, mBG2, and mBG3 had 21%, 37%, and 52% of APMA, respectively. Gel permeation chromatography (GPC) results showed that BA copolymers possess molecular weights of 16,200 (BA1), 20,900(BA2), and 27,200(BA3) g/mol. The plasmid DNA (pDNA) complexing ability of the obtained block copolymer gene carriers was also investigated. The charge ratios (N/P) were 8, 16, and 4 when pDNA was complexed completely with mBG1, mBG2, mBG3, respectively. When the N/P ratio of mBG/pDNA polyplexes was higher than 1, the Zeta potential of mBG was positive. At an N/P ratio between 16 and 32, the average particle size of mBG/pDNA polyplexes was between 100-200 nm. Overall, this work illustrates a simple and convenient protocol for the block copolymer carrier synthesis.

This work presents the preparation of methionine functionalized biocompatible block copolymers (mBG) via the reversible addition-fragmentation chain transfer (RAFT) method. The plasmid DNA complexing ability of the obtained mBG and their transfection efficiency were also investigated. The RAFT method is very beneficial for polymerizing monomers containing special functional groups.

用于靶向质粒DNA输送的美聚氨酸功能化生物相容性块共聚物

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

RGD功能化水凝胶作为治疗应用的工具合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

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.), ...

3D Printing and In Situ Surface Modification via Type I Photoinitiated Reversible Addition-Fragmentation Chain Transfer Polymerization

3D printing provides facile access to geometrically complex materials. However, these materials have intrinsically linked bulk and interfacial properties dependent on the chemical composition of the resin. In the current work, 3D printed materials are post-functionalized using the 3D printer hardware via a secondary surface-initiated polymerization process, thus providing independent control over the bulk and interfacial material properties. This process begins with preparing liquid resins, which contain a monofunctional monomer, a crosslinking multifunctional monomer, a photochemically labile species that enables initiation of polymerization, and critically, a thiocarbonylthio compound which facilitates reversible addition-fragmentation chain transfer (RAFT) polymerization. The thiocarbonylthio compound, known commonly as a RAFT agent, mediates the chain growth polymerization process and provides polymeric materials with more homogeneous network structures. The liquid resin is cured in a layer-by-layer fashion using a commercially available digital light processing 3D printer to give three-dimensional materials having spatially controlled geometries. The initial resin is removed and replaced with a new mixture containing functional monomers and photoinitiating species. The 3D printed material is then exposed to light from the 3D printer in the presence of the new functional monomer mixture. This allows photoinduced surface-initiated polymerization to occur from the latent RAFT agent groups on the surface of the 3D printed material. Given the chemical flexibility of both resins, this process allows a wide range of 3D printed materials to be produced with tailorable bulk and interfacial properties.

3D Printing and In Situ Surface Modification via Type I Photoinitiated Reversible Addition-Fragmentation Chain Transfer Polymerization

The present protocol describes the digital light processing-based 3D printing of polymeric materials using type I photoinitiated reversible addition-fragmentation chain transfer polymerization and the subsequent in situ material post-functionalization via surface-mediated polymerization. Photoinduced 3D printing provides materials with independently tailored and spatially controlled bulk and interfacial properties.

通过I型光引发可逆加成-碎片链转移聚合进行3D打印和原位表面改性

3D printing provides facile access to geometrically complex materials. However, these materials have intrinsically linked bulk and interfacial properties ...

Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N&#39;,N&#39;-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.

Nanogels are an excellent and versatile nanoparticle platform for the delivery of biologics. Stimuli-responsive poly(ethylene) glycol-based polymeric nano-gels, capable of encapsulating protein-based payloads, were synthesized using a one-step cross-linking co-nanopolymerization strategy in aqueous conditions. The optimal fabrication and characterization of these novel nanoparticles are presented here.

使用水性一步交联和共纳米聚合合成刺激响应纳米凝胶

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

来自微凝胶棒的互连大孔3D支架

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...