This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIP (Nos. 2014R1A2A1A01006527 and 2011-0030075).

1.母模通过光刻水动力聚焦微流控芯片（手足口病）的制备<ol><li>设计用于根据制造商的方案使用计算机辅助设计（CAD）软件的手足口病（ 图1a）的光掩模。 </li><li>冲洗用丙酮，异丙醇（IPA），和去离子（DI）水4&#39;的硅晶片，从晶片去除有机和无机灰尘。 </li><li>清洁带O 2等离子体的硅晶片在100瓦的功率为5分钟，以增加晶片和SU-8之间的接合强度。 </li><li>旋涂将4ml负光致抗蚀剂的SU-8 2150到晶片以3000rpm进行30秒以实现厚度150μm（在图1b b1）中 。 </li><li>放置在电炉上的SU-8涂布的晶片在65℃5分钟，设定温度至95℃，然后离开热板在晶片30分钟，软烘烤。 </li><li>放置设计光掩模在晶片上，并在一个掩模对准暴露于UV光（260毫焦耳厘米-2，26秒，10毫瓦厘米-2）（在图 B2 1b）中 。 </li><li>执行曝光后烘烤在电炉上（65℃5分钟，然后95℃，12分钟）。 </li><li>开发通过在10分钟的SU-8显影剂浴浸渍晶片，然后将其转移到新鲜的显影剂5秒，以获得清洁的表面。 </li><li>冲洗晶片20秒用DI水并用N 2气干燥它为10秒（在图 B3 1b）中 。使用制造晶片在第2节的主模聚二甲基硅氧烷（PDMS）铸造。 </li></ol> 2.通过PDMS铸造加工的手足口病<ol><li>使用1节作为主模铸造PDMS获得的图形晶片。 </li><li>混合的PDMS预聚物和在10的重量比的固化剂均匀地:​​1;例如，使用1克固化剂为10克PDMS预POLYM的呃。 </li><li>倒入PDMS预聚合物到主模和在真空室中脱气它为1小时（B4在图1b）。 </li><li>母模与PDMS预聚物放入烘箱中在65℃下3小时。 </li><li>切割该固化的PDMS成用锋利的解剖刀单个芯片的规模。小心剥离用手从母模的固化的PDMS复制品。 </li><li>重复步骤2.2至2.5，以获得相同的PDMS复制品。 </li><li>入口冲床和出口孔成使用空穴冲床具有比连接管的外径稍小直径的副本中的一个。 </li><li>应用空气等离子体处理，使用一个电晕每个副本的接合面积。34 注意:使用电晕在通风良好的地方，以避免臭氧积聚。 </li><li>下降5微升甲醇到空气等离子处理的区域。精细对准两个相同的PDMS副本用手manipu制造的手足口病<html>特征研，并通过显微镜（B5 图1b）检查调整。 注意:空气等离子体处理过的PDMS复制品是相当粘且难以操纵。因此，5微升甲醇加入到空气等离子处理的表面以用作润滑剂。 </li><li>放置手足口病在设定至65℃过夜烘箱加强（B6在图1b）二PDMS复制品之间的结合。键的两个相同的PDMS复制品来增加手足口病的微通道的高度和操作过程中避免在微流体通道的微滴的堵塞。 </li></ol><img alt="图1" src="/files/ftp_upload/52813/52813fig1.jpg" /> 图1:手足口病的制备过程概述 （a）该光掩模为手足口病的设计参数。为手足口病的制作过程（ 二 ）插图。ftp_upload / 52813 / 52813fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 3.的NIPAAm丰富的制备（N-丰富）的NIPAAm和贫（N-差）由超饱和的NIPAAm相分离阶段<ol><li>使用涡旋混合器1;:以1 AW / w比例溶解于DI水中的NIPAAm单体例如，溶解在10ml去离子水将10克的NIPAAm的（ 图2a的第一图像）。 注意:一旦的NIPAAm单体在室温下充分溶解，溶液出现混浊（ 图2a的第二图像）。这种现象是第一个线索已经成功发生过饱和的NIPAAm单体的那溶解度诱导相分离。 </li><li>允许单体溶液以在垂直位置在室温下静置至少15分钟。顶部相是富N相，和更密集底部相是N差相（ 图2a的第三图像）。个的密度简丰富和N-差相分别为0.93±0.01和0.99±0.01克厘米-3。15 </li><li>当分离两相界面变得清晰，轻轻取出加入2ml从富N和N-差相单体溶液通过不使用吸管扰乱这个接口。 </li><li>加入4毫克N，N&#39;甲基双丙烯酰胺（MBAAm）作为交联剂和4mg 4-（2-羟基乙氧基）苯基- （2-羟基-2-丙基）酮作为光引发剂到N-丰富和N所提取的国定贫困单体溶液以制备用于低交联剂浓度芯的液体1和2（2毫克毫升-1）样品（b1和图2b b2）中。 </li><li>重复先前的步骤3.3，并添加80毫克MBAAm和4mg 4-（2-羟基乙氧基）苯基 - （2-羟基-2-丙基）酮向每个提取的N-丰富和N-差单体溶液以制备芯流体图1和2对于高交联剂浓度（40毫克毫升-1）样品（b3和图 B4URE 2b）中。 </li><li>溶解油表面活性剂的10％（重量）到矿物油来制备鞘液（b5在图2b）。 </li></ol><img alt="图2" src="/files/ftp_upload/52813/52813fig2.jpg" /> 图2:对杰纳斯Microhydrogel合成材料准备 （ 一 ）通过的NIPAAm过饱和相分离富N和N-差单体溶液制备方法。 （ 二 ）材料，并在协议中使用实验装置的详细信息。 <a href="https://www.jove.com/files/ftp_upload/52813/52813fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 使用手足口病剑锋Microhydrogels 4.合成<ol><li>负载2 ml的核心的流体1和2（B1，B2或B3，B4在连接古尔2b）和所述鞘液（b5在图2b）成三个独立3毫升注射器。 </LI&gt; <li>装入注射器到注射器泵和用管（ 图（b）中 。使用管将手足口病的流体出口连接至收集储存各注射器连接到手足口病的适当的流体入口。 </li><li>设定注射器泵和在2,2，和10微升分钟-1的流速注入芯流体1和流体2和鞘液，分别。 </li><li> （可选的）调谐芯流体1和流体2的流速以调节的Janus微滴的每一侧的相对体积比。 </li><li> UV光源垂直约1cm从收集容器离开的位置。切换UV光源和视觉监控连续生产剑锋microhydrogels的。 注意:使用UV监测microhydrogel生产时保护护目镜。 </li><li>收集制造剑锋microhydrogels成一个锥形管，并使用IPA清洗。然后，离心锥形管（780克5分钟），以解决microhydrogels。 </li><li>重复步骤4.6几次以除去完全包围的Janus microhydrogels矿物油。 </li><li>重复步骤4.6但使用去离子水为0.005％的水的表面活性剂（体积/体积）代替IPA以除去剩余的IPA周围的Janus microhydrogels。 </li><li>在10ml的小瓶含去离子水储存充分洗涤剑锋microhydrogels。 </li></ol> 5.剑锋Microhydrogels的各向异性热反应性分析<ol><li>使用移液管，从第4节合成的Janus microhydrogels放入一个24孔板中。允许直到在孔的底部表面上形成的单层的microhydrogels沉降15秒。 </li><li>获得使用具有5X物镜直立光学显微镜在24℃下实现Ja​​nus microhydrogel的图像。 </li><li>设置孔板下的热电模块和控制该模块的电压，以增加包含的Janus微将溶液的温度水凝胶至32℃。 </li><li>在32℃下再次通过使用直立光学显微镜用5X物镜获取的Janus microhydrogel的图像。 </li><li>重复步骤5.2-5.4 24次，同时注意选择不同的剑锋microhydrogel进行统计分析。 </li><li>从在24和32℃下不同的Janus microhydrogels的25幅图像，测量根据制造商的说明使用图像分析软件的Janus microhydrogels的富PN和PN-差部分的半径。 </li></ol>

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Commun. 47 (9), 2634-2636 (2011).</li><li>Hauber, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab chip. 6 (12), 1548-1549 (2006).</li><li>Nisisako, T., Torii, T., Takahashi, T., Takizawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+monodisperse+bicolored+Janus+particles+with+electrical+anisotropy+using+a+microfluidic+co-flow+system.">Synthesis of monodisperse bicolored Janus particles with electrical anisotropy using a microfluidic co-flow system.</a> Adv. Mater. 18 (9), 1152-1156 (2006).</li><li>Seiffert, S., Romanowsky, M. B., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Janus+microgels+produced+from+functional+precursor+polymers.">Janus microgels produced from functional precursor polymers.</a> Langmuir. 26 (18), 14842-14847 (2010).</li><li>Peppas, N. A., Hilt, J. Z., Khademhosseini, A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+in+biology+and+medicine:+from+molecular+principles+to+bionanotechnology.">Hydrogels in biology and medicine: from molecular principles to bionanotechnology.</a> Adv. Mater. 18 (11), 1345-1360 (2006).</li></ol>

The authors declare that they have no competing financial interests.

两种不混溶基材料通常用于合成的Janus microhydrogels。直到最近，由相同的基本材料制成的Janus microhydrogels很少报道和所报告的Janus microhydrogels没有一个明确的内部的形态，由于所引起的元件材料的可混溶性的紊乱。35，36在这个协议中，我们展示了一个方法合成完全在单一基体材料，PNIPAAm的组成，具有明确条块结构的Janus microhydrogels。 如以合成的Janus microhydrogels一个关键...

Hydrogels are a network of hydrophilic polymer chains.1 An increasing amount of research in the field of hydrogels has promoted significant advances and revealed their similarity to biological tissues; the properties of hydrogels allow the uptake of large amounts of water while maintaining their structure. Environmentally responsive hydrogels have also been studied extensively because of their ability to swell or shrink reversibly in response to external stimuli.2 Several triggers, including temperature,3-5 pH,6,7 light,8,9 electric fields,10,11 and specific molecules, such as glucose,12,13 have been suggested to control the geometric shape of hydrogels. Among the many environmentally responsive hydrogels currently available, poly(N-isopropylacrylamide) (PNIPAAm), a well-known thermo-responsive hydrogel, exhibits volume shrinkage above a low critical solution temperature (LCST) of 32 &#176;C.14 A recent study by Sasaki et al.15 reported the intriguing liquid-liquid phase separation of supersaturated NIPAAm, which is the monomer of PNIPAAm. According to this report, supersaturated NIPAAm was dissolved with a 10-fold molar excess of H2O, and soon after, the solution separated into two liquid phases when allows to stand at a temperature above 25 &#176;C; by contrast, dilute NIPAAm was dissolved homogeneously under the same conditions.
Microparticles made of environmentally responsive hydrogels are fascinating candidates for application in drug delivery,16,17 catalysis,18 sensing,19,20 and photonics.21 Traditional synthetic methods including emulsion polymerization, are used to produce hydrogel microparticles with polydispersity.22,23 However, certain applications require microparticles with a narrow size distribution, for example, to stabilize the pharmacokinetics of drug delivery.24 Irregularly shaped or polydisperse embolic microparticles aggregate proximally into clusters, leading to chronic inflammatory responses in embolic particles for cancer therapeutic treatment.25,26
The microfluidic approach is at the forefront of research as a means of fabricating micro-sized particles with narrow size distributions and complex shapes.27-31 The advantages of fabricating microparticles in the microfluidic device are predicated by the small characteristic length of the microfluidic device, which results in a low Reynolds number. In contrast to traditional bulk emulsification where drops are formed in parallel, microdroplets produced in microfluidic devices are generated in series and subsequently polymerized into microparticles upon exposure to UV irradiation. The fundamental principle of droplet formation using a microfluidic device is balance between the interfacial tension and the shear force of the sheath fluid acting on the core fluid. 
Despite the obvious advantages of microfluidic fabrication of droplets/particles, Janus droplets/particles consisting of the same base material are rarely reported because the internal morphology of these droplets/particles is generally disturbed by the diffusion and perturbation of the core fluids. To circumvent this intrinsic limitation, two groups recently reported the preparation of the Janus microparticles by employing heat-induced phase separation of colloidal nanoparticles and UV-directed phase separation.32,33
To this end, we report a microfluidic approach to synthesize Janus microhydrogels entirely composed of a single base material and obtain a product with clearly compartmentalized morphology. Our approach is based on the primary concept of liquid-liquid phase separation of supersaturated NIPAAm monomer. The resulting Janus microhydrogels were found to possess unique properties including anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability.

<table><tbody><tr><td>Silicon wafer</td><td>LG Siltron</td><td>4&quot;, Test grade</td><td>Wafer for master mold fabrication</td></tr><tr><td>Acetone</td><td>Samchun Pure Chemical</td><td>A0097</td><td>Cleaning silicon wafer</td></tr><tr><td>Isopropyl alcohol (IPA)</td><td>Daejung Chemicals &amp;amp; Metals</td><td>5035-4404</td><td>Cleaning silicon wafer</td></tr><tr><td>Water purification system</td><td>Merck Millipore</td><td>EMD Millipore RIOs Essential 5</td><td>Prepering&amp;nbsp; deionized water</td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt; plasma machine</td><td>Femto Science</td><td>VITA-A</td><td>Cleaning silicon wafer</td></tr><tr><td>SU-8 2150 negative photoresist</td><td>MicroChem</td><td>Y111077 0500L1GL</td><td>Photoresist for master mold fabrication</td></tr><tr><td>Hot plate</td><td>Misung Scientific</td><td>HP330D, HP150D</td><td>Baking SU-8</td></tr><tr><td>SU-8 developer</td><td>Microchem</td><td>Y020100 4000L1PE</td><td>Developing SU-8</td></tr><tr><td>Mask aligner system for photolithograpy</td><td>Shinu Mst Co.</td><td>CA-6M</td><td>Photolithography</td></tr><tr><td>Sylgard 184 silicone elastomer kit</td><td>Dow Corning</td><td>1064891</td><td>PDMS casting</td></tr><tr><td>Laboratory Corona Treater</td><td>Electro-technic Products Inc.</td><td>Model BD-20AC</td><td>PDMS air plasma treatment&amp;nbsp;</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-isopropylacrylamide (NIPAAm)</td><td>Sigma-Aldrich</td><td>415324-50G</td><td>Monomer</td></tr><tr><td>&lt;em&gt;N,N&#039;&lt;/em&gt;-methylenebisacrylamide (MBAAm)</td><td>Sigma-Aldrich</td><td>146072-100G</td><td>Crosslinker of NIPAAm</td></tr><tr><td>4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone, Irgacure 2959</td><td>BASF</td><td>55047962</td><td>Photoinitiator of NIPAAm</td></tr><tr><td>ABIL EM 90</td><td>Evonik Industries</td><td>201109</td><td>Sufactant for oil</td></tr><tr><td>Vortex mixer</td><td>Scientific Industries Inc.</td><td>Vortex-Genie 2</td><td>Mixing</td></tr><tr><td>Tygon tubing</td><td>Saint-Gobain</td><td>I.D. 1/32&quot;, O.D. 3/32&quot;, Wall 1/32&quot;</td><td>Connecting tube between syringes and HFMD</td></tr><tr><td>UV light source</td><td>Hamamatsu</td><td>Spot light source LC8</td><td>Polymerization from NIPAAm to PNIPAAm</td></tr><tr><td>Syringes, NORM-JECT (3 ml)</td><td>Henke-Sass Wolf GmbH</td><td>22767</td><td>Loading of materials</td></tr><tr><td>Syringe pump</td><td>KD Scientific</td><td>KDS model 200</td><td>Perfusion of materials</td></tr><tr><td>Tegitol Type NP-10</td><td>Sigma-Aldrich</td><td>NP10-500ML</td><td>Surfactant for water</td></tr><tr><td>Oil red O</td><td>Sigma-Aldrich</td><td>O0625-25G</td><td>Dye for N-rich phase</td></tr><tr><td>Oil Blue N</td><td>Sigma-Aldrich</td><td>391557-5G</td><td>Dye for N-rich phase</td></tr><tr><td>Yellow food dye</td><td>Edentown F&amp;amp;B</td><td>NA</td><td>Dye for N-poor phase</td></tr><tr><td>Green food dye</td><td>Edentown F&amp;amp;B</td><td>NA</td><td>Dye for N-poor phase</td></tr><tr><td>Power supply</td><td>Agilent</td><td>E3649A</td><td>Power source for thermoelectric module</td></tr><tr><td>Thermoelectric module</td><td>Peltier</td><td>FALC1-12710T125</td><td>Temparature control</td></tr><tr><td>Centrifuge machine</td><td>Labogene</td><td>1248R</td><td>Settling down microhydrogels</td></tr><tr><td>24-well plate</td><td>SPL Life Sciences</td><td>32024</td><td>Reservoir for observation</td></tr><tr><td>Optical microscope</td><td>Nikon</td><td>ECLIPSE 80i</td><td>Optical observation</td></tr><tr><td>Image analysis software</td><td>IMT i-Solution Inc.</td><td>iSolutions DT</td><td>Measurement of radius</td></tr></tbody></table>

synthesis of poly(n-isopropylacrylamide) janus microhydrogels for anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this unique property, Janus microparticles have been recognized as a new class of materials, thereby attracting a great deal of attention from various research fields. The versatility of these microparticles has been exemplified through their uses as building blocks for self-assembly, electrically responsive actuators, emulsifiers for painting and cosmetics, and carriers for drug delivery. This study introduces a detailed protocol that explicitly describes a synthetic method for designing novel Janus microhydrogels composed of a single base material, poly(N-isopropylacrylamide) (PNIPAAm). Janus microdroplets are firstly generated via a hydrodynamic focusing microfluidic device (HFMD) based on the separation of a supersaturated aqueous NIPAAm monomer solution and subsequently polymerized through exposure to UV irradiation. The resulting Janus microhydrogels were found to be entirely composed of the same base material, featured an easily identifiable compartmentalized morphology, and exhibited anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability. We believe that the proposed method introduces a novel hydrogel platform with the potential for advanced synthesis of multi-functional Janus microhydrogels.

图3a给出用于通过手足口病，合成的Janus microhydrogels实验装置的示意图。富 - N和N-差相恰恰注入手足口病为核心的流体1和2，然后合并并在由矿物油的，因为瑞利毛细不稳定性的鞘流体孔口分成的Janus微滴。因此， 如图3b中已成功生成富N和N-差相构成的Janus微滴。微滴的直径为190微米具有小于2％的变化（CV）的系数。观察到的Janus微滴的清晰?...

Watch this Scientific Journal Video about Synthesis of PolyN-isopropylacrylamide Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability at JoVE.com

Synthesis of PolyN-isopropylacrylamide Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

We present a protocol to synthesize Janus microhydrogels composed entirely of the same base material, poly(N-isopropylacrylamide) (PNIPAAm), with a clearly compartmentalized structure base on the phase separation of a supersaturated NIPAAm monomer solution. The synthesized Janus microhydrogels show unique properties such as anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability.

合成聚（<em&gt;ñ</em&gt; -isopropylacrylamide）剑锋Microhydrogels各向异性热响应能力和亲有机/亲水承载能力

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this ...

synthesis-poly-n-isopropylacrylamide-janus-microhydrogels-for

Research

JoVE Journal

Chemistry

Synthesis of Poly(N-isopropylacrylamide) Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

9.9K 次观看.  Pohang University of Science and Technology (POSTECH). We present a protocol to synthesize Janus microhydrogels composed entirely of the same base material, poly(N-isopropylacrylamide) (PNIPAAm), with a clearly compartmentalized structure base on the phase separation of a supersaturated NIPAAm monomer solution. The synthesized Janus microhydrogels show unique properties such as anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability.

视频: Synthesis of PolyN-isopropylacrylamide Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

保利的微波辅助功能化（乙二醇）和On-树脂链聚合反应，并形成水凝胶的肽使用

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

科研

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 ...

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

去耦力学传递线索的影响制备羟基聚丙烯酰胺水凝胶的

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

生物工程

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

可注射药物加载超分子凝胶局部导管注入猪心

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

简单的聚丙烯酰胺为基础的多井刚度检测方法的刚度依赖的细胞反应的研究

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

温敏纳米结构表面的组织工程准备

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

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.), ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

磁性和热敏性聚（<em&gt;ñ</em基于异丙基丙烯酰胺）的微凝胶用于磁触发控制释放

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...

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 ...

合成聚（

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