来自加拿大自然科学和工程研究理事会 (NSERC) 的资助, NSERC 创建-同上 (细胞外基质的综合设计) 计划, 20/20: NSERC 眼科生物材料研究网, 和安大略研究部和创新早期研究员奖项目被认可。

1. 酰肼功能高分子的合成
注: 为 PNIPAM 仿 thermoresponsive POEGMA 前体聚合物 (PO10) 提供了以下特定配方, 30 (摩尔) 酰肼功能化。不同相变温度的 PNIPAM 和 POEGMA 前体聚合物可以使用相同的一般方法制备, 但修改所用的核心单体的类型和比例 (见1.2 部分, 用于各种 POEGMA 聚合物的修改)21,25,27。
<ol><li>称37毫克 22 '-偶 (2-丁酸) (AIBMe, 引发剂), 3.1 克甲基丙烯酸二甘醇 (oligoethyleneglycol ()2MA), 0.9 克甲基丙烯酸甲酯 (OEGMA475, 475 克/摩尔 n=7-8 环氧乙烷重复单位), 523 µL丙烯酸 (AA, 单体), 和7.5 µL 的 thiolglycolic 酸 (TGA, 链转移剂) 成20毫升玻璃闪烁瓶。</li>
	<li>对于 PO0 (室温转换温度 POEGMA), 使用 4.0 g M () 2 MA (无 OEGMA 475).对于 PO100 (无过渡温度 POEGMA), 请使用 4.0 g OEGMA475 (无 M ()2MA)。 注意: 根据鲁兹 et和 OEGMA475中的中间混合物的使用, 可以实现中间相转变温度, 这是基于 M () 2 MA 和。23
</li>
	<li>在一个或多个颈部的圆底烧瓶中溶解所有的二恶烷 (5 毫升/克总单体) 试剂。</li>
	<li>用氮气 (超高压级) 流量净化反应30分钟。</li>
	<li>一旦清除, 将烧瓶放在一个预热的油浴保持在75°c 为4小时在氮气和 400 rpm 磁性搅拌。</li>
	<li>4小时后, 使用旋转蒸发器设置为50°c 和 200 rpm, 去掉溶剂。</li>
	<li>在150毫升的去离子水中溶解产生的聚合物产物。</li>
	<li>将己二酸 dihydrizide (抗利尿激素) 添加到聚合物中的 AA 残留量的五倍摩尔上 (在本例中, aa 包括 29 (摩尔) 的单体单位在所生产的聚合物, 如每电导滴定)。</li>
	<li>用0.1 米 HCl 将溶液 ph 值调整为 ph 值4.75。</li>
	<li>一旦 pH 值稳定, 添加n-(3-dimethylaminopropyl)-n'-ethylcarbodiimide (EDC), 在5倍摩尔过剩的 AA 残留数量)。</li>
	<li>保持反应 pH 值4.75 与滴状加法 0.1 M HCl 4 h。</li>
	<li>让反应在一夜之间引起轰动。</li>
	<li>将产品溶液倒入三 ~ 30 厘米长的透析管 (3500 大分子量切断, 1 英寸厚度), 使用漏斗尽量减少溢出。使用夹钳关闭前管底部的填充折叠一个小 (~ 2 厘米) 段的管, 以提高钳的完整性;在填充完成后, 在顶部重复 (按下以去除气泡)。将管子放在100倍多的去离子水中, 然后离开至少6小时, 完全替换六循环透析的水以达到理想的纯度。</li>
	<li>Lyophilize 透析样品获得最终的干高分子产品。</li>
</ol>2. 醛功能性聚合物的合成
<ol><li>22-Dimethoxyethyl 甲基丙烯酸甲酯 (DMEMA) 醛-前驱单体的合成<ol>
<li>将200毫升的20% 瓦特/v 氢氧化钠溶液放入500毫升3颈的圆底烧瓶中。</li>
		<li>冷却溶液在冰浴和保持温度0°c 与冰在反应期间。</li>
		<li>将50毫升的 aminoacetyl 醛二甲缩醛加入冷却的氢氧化钠溶液中。</li>
		<li>添加0.1 克的速度 (22, 66-Tetramethylpiperidin 1 基) oxyl) 和搅拌在 400 rpm 使用磁搅拌杆, 直到节拍完全溶解。</li>
		<li>添加48毫升的甲基丙烯酰氯化滴, 使用滴定管超过2小时。</li>
		<li>2小时后, 用铝箔覆盖反应容器, 并在一夜之间进行搅拌。</li>
		<li>在 1 L 分离漏斗、震动、脱气和丢弃顶层时, 将反应产物添加到75毫升的石油醚中提取产物。</li>
		<li>重复步骤2.1.7 三次, 方法是将底层产品从每个提取步骤添加到下一个提取周期。</li>
		<li>拆卸最后的底层产品, 并转移到100毫升烧杯。</li>
		<li>添加〜5克硫酸镁 (Mg2所以4) 到烧杯与单体, 直到 "雪地球" 的效果被观察到。</li>
		<li>通过100毫升傅书礼漏斗过滤, 以删除 Mg2, 因此4。</li>
		<li>每两次用〜75毫升的叔丁基甲基醚冲洗烧杯, 每次都要通过漏斗浇出漂洗液。</li>
		<li>将产品转移到500毫升的圆底烧瓶, 并在室温 200 RPM 的旋转蒸发器中蒸发溶剂以收集最终产品。</li>
	</ol>
</li>
	<li>醛功能高分子聚合物的合成 注: 为 PNIPAM 仿 POEGMA 前体聚合物 (PO10) 提供了以下特定配方, 其中 30 (摩尔) 醛功能化。不同相变温度的 PNIPAM 和 POEGMA 前体聚合物可以使用相同的一般方法制备, 但要修改所用的核心单体的类型和比例 (见1.2 部分, 用于各种 POEGMA 聚合物的修改)21,25,27。<ol>
<li>称37毫克 22 '-偶 (2-丁酸) (AIBMe), 3.10 g 二乙二醇甲基丙烯酸甲酯 (ethyleneglycol)2MA, 0.1 克寡聚甲基丙烯酸酯 (OEGMA475, 475 克/摩尔, n=7-8 环氧乙烷重复单位), 1.30 克 n (22-dimethoxyethyl) 丙烯酰胺 (DMEMA) 和7.5 µL thiolglycolic 酸 (TGA) 成20毫升玻璃闪烁瓶。</li>
		<li>对于 PO0 (室温转换温度 POEGMA), 使用 4.0 g M () 2 MA (无 OEGMA 475).对于 PO100 (无过渡温度 POEGMA), 请使用 4.0 g OEGMA475 (无 M ()2MA)。 注: 根据鲁兹 et和 OEGMA475中的中间混合物的使用, 可以实现中间相转变温度。23
</li>
		<li>在一个或多个颈部的圆底烧瓶中溶解所有的二恶烷 (5 毫升/克总单体) 试剂。</li>
		<li>用氮气 (超高压级) 流量净化反应30分钟。</li>
		<li>清洗后, 将烧瓶放在预加热的油浴中, 在氮气和 400 rpm 磁性搅拌下维持在75°c 4 小时。</li>
		<li>4小时后, 使用旋转蒸发器设置为50°c 和 200 rpm, 去掉溶剂。</li>
		<li>将所得的聚合物产品溶解在100毫升的去离子 H2O。</li>
		<li>在溶解溶液中加入50毫升1米 HCl, 在磁性搅拌 (400 RPM) 下搅拌24小时, 充分水解 DMEMA 中的缩醛功能。</li>
		<li>反应完成后, 按照步骤1.13 将聚合物溶液转化为透析油管。</li>
		<li>Lyophilize 透析样品获得最终的干高分子产品。</li>
	</ol>
</li>
</ol>3. 腙交联散装水凝胶的制备
<ol><li>将酰肼和醛功能性聚合物分别溶于10毫米磷酸盐缓冲盐水 (PBS) 或任何所需的水缓冲液中, 以创建所需浓度的溶液。 注: 质量浓度在 5-40 wt% 之间通常使用, 与凝胶在较低的浓度可能如果更高的功能组分数是存在的聚合物。</li>
	<li>使用单筒注射器转移溶液, 将每个前体溶液 (每1毫升) 装入一个双筒注射器 (2.5 毫升容积, 1:1 比注射器) 的单独桶中, 连接到静态混合器 (1.5 "长度) 和 (可选) 注射器 (通常为18克,1.5 "长度为体外研究) 和 (可选) 注射器 (典型地 18 G, 1.5" 长度为体外研究)。</li>
	<li>通过冲孔进入硅橡胶板, 制备所需的厚度、形状和直径的模具。 注: 在一个典型的实验中, 一个标准的冲床设置是用来打7毫米直径圆柱孔内1/16 英寸厚的硅橡胶板 (总容积的水库 ~ 300 µL)。</li>
	<li>在标准的玻璃显微镜幻灯片上安装硅胶模具, 使模具中穿孔的孔完全由玻璃支撑。 在安装硅胶模具之前, 建议使用0.1 米 HCl 清洗玻璃。</li>
	<li>通过静态搅拌机共挤出双桶注射器的内容完全填充 (或略溢出, 与半月板在顶部) 硅胶模具。 注: 可在一个挤出样品中制备多个样品, 条件是胶凝时间与填充多个模具所需的总时间相同, 或较长。</li>
	<li>将另一个标准的玻璃显微镜幻灯片放在模具的顶部, 等待凝胶完成。 注: 在 < 1 分钟内合成切片中所述的标准配方;相对于 M (OEGMA) 2MA (POEGMA 水凝胶), 在较低的官能团密度、较低的聚合物浓度和/或更高分数的475中观察到较慢的凝胶时间 (因而需要更长的等待时间)。</li>
	<li>取下顶部的显微镜滑梯, 用刮刀将水凝胶从硅橡胶模具中推开。</li>
	<li>从下显微镜滑块中提起模具, 回收水凝胶进行进一步测试。</li>
</ol>4. 腙交联凝胶微粒的制备
<ol><li>微流控芯片的研制<ol>
<li>将硅晶片 (D = 76.2 毫米, 380 µm 厚度, 掺磷, < 100 > 方向) 通过加热在热板上, 在200摄氏度为5分钟脱水。</li>
		<li>将晶片放在旋转涂布机上, 涂上100µm 厚层的 SU-8 100 光刻胶, 应用〜7毫升的 SU-8 抵抗, 以 500 rpm/秒的速度将旋转速度提高到 3000 rpm, 然后以 3000 rpm 的速度保持30秒。</li>
		<li>预烘烤的涂层在65°c 10 分钟, 然后软烘烤涂层在95°c 30 分钟。</li>
		<li>用图 2a定义的微流控形状的透明度打印光掩模, 这样透明截面是聚合光刻胶层所需的图案。</li>
		<li>将光刻胶涂层的硅晶片和光掩模在面罩光刻中, 并在九十五年代将晶片曝光至 365 nm (6.5 W 曝光功率)。</li>
		<li>烘烤的图案晶圆10分钟在95摄氏度, 首先通过放置在一个热板在65°c, 随后加热电热板到95°c 在10°c/分钟。</li>
		<li>将晶片从热板上取下, 在500毫升烧杯中放置100毫升 SU-8 显影液, 至少10分钟, 在溶液中缓慢旋转晶片, 以除去未曝光的光刻胶。10分钟后, 用异丙醇冲洗图案的晶片, 用空气干燥。在阴凉、干燥的环境中, 在不使用光的情况下, 将图案化的晶片存放在柔软的光刻副本成型上。</li>
		<li>将花纹微流控模放入培养皿中。在芯片的入口和插座上放置10毫米的 L/秒13的硅胶管。</li>
		<li>倒入10毫升的聚 (二甲基硅氧烷) (硅橡胶), 通过混合有机硅弹性体基和硅胶弹性体固化剂在10:1 的比例) 在芯片顶部, 小心避免纳入任何在放置的硅胶管内的。</li>
		<li>将培养皿放在真空室中10分钟, 以除去在固化过程中保持图案结构的气泡。</li>
		<li>通过将含有花纹模和成灾的培养皿放在85摄氏度的热板上, 以2-3 小时的温度将其固化。</li>
		<li>小心剥离已固化的硅晶片, 以暴露的软平版图案的微流控模复制。</li>
		<li>将阵列的中和玻璃滑倒在高功率等离子清洗器与空气饲料。九十年代将等离子应用于 200 mTorr 和 45 W, 将其与玻片结合, 并创建最终的微流控芯片。</li>
	</ol>
</li>
	<li>凝胶微粒的合成<ol>
<li>在0.056 毫升无水乙醇中溶解 NIPAM (4.5g)、丙烯酸 (0.5 克-15 (摩尔) 总单体)、巯基乙酸酸 (TGA、80µL) 和 22-azobisisobutyric 酸二甲酯 (AIBME、20克), 制备酰肼功能性 PNIPAM (PNIPAM-Hzd), 并随后采取步骤 1.4-1.14 完成综合, 虽然改变反应温度到56°c 在步骤1.5。</li>
		<li>通过溶解 NIPAM (4 克) 制备醛功能化 PNIPAM (PNIPAM), n-(22-dimethoxyethyl) 甲基丙烯酸酯 (DMEMA, 0.95 克 13.4 (摩尔) 总单体), 巯基乙酸酸 (TGA, 80 µL), 22-azobisisobutyric 酸二甲酯 (AIBME, 0.056 克) 在20毫升乙醇和随后的步骤 2.2. 4-2. 2.10 完成合成, 虽然改变反应温度到56°c 在步2.2.5。</li>
		<li>溶解 PNIPAM-Hzd 和 PNIPAM 在 6 wt% 在去离子水和负载成单独的标准5毫升注射器。</li>
		<li>在重石蜡油中溶解 1 wt% 非离子表面活性剂 (例如, 跨度 80), 并将溶液装入标准的60毫升注射器中。</li>
		<li>将两个前体聚合物溶液注射器分别连接到微流控芯片上的两个单独的聚合物进气道和石蜡油溶液, 通过 1/32 "ID 硅胶管 (每入口30厘米长,每出口45厘米长)。</li>
		<li>使用两个单独的输液注射器泵 (一个用于上游的油, 一为油在喷嘴以后增加), 在1.1 毫升/小时和5.5 毫升/小时之间的流动率交付油到芯片, 不用开始聚合物流程质数芯片并且保证芯片是无缺陷的和 操作 (通常保持在30分钟期间)。</li>
		<li>使用单独的输液注射器泵, 以0.03 毫升/小时的流速将每种水性聚合物溶液输送到芯片上。</li>
		<li>在初始稳定期后, 以确保流平衡和均匀颗粒形成 (30 分钟-1 小时), 收集颗粒在磁性搅拌圆底烧瓶。</li>
		<li>收集粒子直到所有的油被消耗 (12-55 小时, 取决于流量)。停止注射器泵, 如果需要, 立即泵水到位的前体聚合物溶液通过芯片清洗。 然而, 鉴于这些材料的快速原位凝胶, 当流停止时, 建议使用一个新的芯片为每个单独的实验。</li>
		<li>关闭磁搅拌, 允许凝胶微粒沉淀。用吸管醒酒所有可用的石蜡油。</li>
		<li>要除去剩余的石蜡油, 用戊烷清洗凝胶微粒 (每0.5 毫升的微粒量为10毫升), 大力混合乳液1分钟, 允许凝胶微粒重新沉淀1-2 小时, 醒酒关闭使用吸管的剩余有机相。重复至少5次, 以确保完整的石蜡油清除。</li>
		<li>并用重悬在20毫升玻璃闪烁瓶内的10毫升去离子水中凝胶微粒, 并在一夜之间用氮气清除瓶子以除去任何残留的戊烷。</li>
	</ol>
</li>
</ol>5. 腙交联磁性的制备
<ol><li>在去离子水中溶解 PNIPAM-Hzd (1 w/v%) 和 PNIPAM (1 w/v%) 的库存溶液。分别按照4.2.1 和4.2.2 的说明, 准备 PNIPAM Hzd 和 PNIPAM 的限期任用。</li>
	<li>加热一个5毫升整除的 PNIPAM-Hzd 库存解决方案70˚C 使用一个油浴下的磁性搅拌 (350 RPM) 内20毫升玻璃闪烁瓶。 注: 溶液应变得不透明 (即温度超过 PNIPAM-Hzd 的低临界溶液温度), 但不应形成可见沉淀。</li>
	<li>添加一个0.25 毫升整除的 PNIPAM-限期 (5-20 wt% 的质量 PNIPAM-Hzd 目前在种子解决方案) 下降明智的加热 PNIPAM Hzd 解决方案在一段时间内 5-10 s。</li>
	<li>在闪烁瓶中继续混合溶液15分钟, 然后从油浴中取出样品, 让产品在一夜之间冷却到室温。</li>
	<li>透析产生的磁性超过6x6 小时周期 (使用 3500 kDa 截留分子量透析膜) 对去离子水去除任何非交联聚合物。如果需要, lyophilize 存储。</li>
</ol>6. 腙交联纳米纤维的制备
<ol><li>通过溶解37毫克二甲基 22 '-偶 (2-丁酸) (AIBMe), 4.0 克 oligoethyleneglycol 甲基丙烯酸酯 (OEGMA 475, 475 克/摩尔, n=7-8 环氧乙烷重复单元) 和0.25 克丙烯酸, 制备酰肼功能化 POEGMA (POEGMA-Hzd)(AA) 在20毫升二恶烷和以下步骤 1.3-1.14 完成合成。</li>
	<li>通过溶解50毫克二甲基 22 '-偶 (2-丁酸) (AIBMe), 4.0 克 oligoethyleneglycol 甲基丙烯酸酯 (OEGMA 475, 475 克/摩尔, n=7-8 环氧乙烷重复单元) 和0.60 克 n (22-POEGMA) 来制备醛功能化的 POEGMA-dimethoxyethyl) 甲基丙烯酸甲酯 (DMEMA) 在20毫升二恶烷和以下步骤 2.2. 3-2. 2.10 完成合成。</li>
	<li>在分离的去离子水溶液中溶解 POEGMA-Hzd (15 wt%) 和 POEGMA-限期 (15 wt%)。</li>
	<li>在去离子水中溶解聚 (环氧乙烷) (乙烯基, Mw= 600x103克/摩尔, 5 wt%)。 将1毫升的聚氧乙烯溶液与步骤6.3 中制备的每个反应性 POEGMA 溶液混合, 以创建 7.5 wt% POEGMA 前体聚合物和 2.5 wt% 氧乙烯的最终前驱体溶液。</li>
	<li>将两个解决方案加载到3节 (也包括 1.5 "静态混合器") 中描述的同一双筒注射器的单独桶中, 并在输液注射器泵上安装双筒注射器。</li>
	<li>将静态混合器和钝头18G 针连接到双筒注射器上。</li>
	<li>将高压电源连接至钝尖针, 接地在收集器上。 注: 收集器包括10毫米 x 10 毫米的铝箔或10毫米直径铝盘纺纱速度为 200 rpm, 两个安装垂直的针在距离从针的一端10厘米。</li>
	<li>启动注射器泵的速率为0.48 毫升/小时, 同时, 开关高电压8.5 伏, 以执行静电纺丝和创建纳米纤维。</li>
	<li>继续静电纺丝, 以使不同厚度的脚手架, 或直到进口解决方案用尽。</li>
	<li>要去除聚氧乙烯纺丝助剂, 请在去离子水中浸泡24小时的收集脚手架。</li>
</ol>

<ol>
	<li>Heskins, M., Guillet, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+Properties+of+Poly(N-isopropylacrylamide).">Solution Properties of Poly(N-isopropylacrylamide).</a> J. Macromol. Sci. A. 2 (8), 1441-1455 (1968).</li><li>Lutz, J. -. F., Akdemir, &. #. 2. 1. 4. ;., Hoth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point+by+Point+Comparison+of+Two+Thermosensitive+Polymers+Exhibiting+a+Similar+LCST:+Is+the+Age+of+Poly(NIPAM)+Over.">Point by Point Comparison of Two Thermosensitive Polymers Exhibiting a Similar LCST: Is the Age of Poly(NIPAM) Over.</a> J. Am. Chem. Soc. 128 (40), 13046-13047 (2006).</li><li>Pelton, R. 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作者没有什么可透露的。

我们已经成功地将所有这些制造技术应用到多个聚合物系统中, 只使用了上面详细描述的 PNIPAM 和 POEGMA 方法的细微变化;但是, 这些协议的使用者必须认识到当其他聚合物被替换到这些过程中可能出现的问题。特别是, 增加前体聚合物的粘度会对加工 (特别是微流控法) 和两种前驱体聚合物的混合效率产生负面影响。此外, 聚合物的凝胶时间必须以依赖于形态学的速率来控制, 以避免过早的凝胶作用来抑制流动或防止反应性预聚合物的互扩散, 必要的是形成理想的均匀凝胶结构。下面介绍了每种策略的具体局限性, 以及我们用来适应这些方法在每个制造长度范围内处理这些限制的方法。
双桶注射器共挤出散装水凝胶 凝胶时间是控制的关键变量, 以保证双筒注射器技术在大体积水凝胶成型中的有效性。聚合物, 凝胶太快后, 接触 (< 1-2 s) 可以堵塞静态搅拌机, 要么停止流动完全或导致非化学计量的数量的两个前体聚合物挤出从注射器。我们发现, 凝胶时间 > 5 s 是可取的 (虽然不是必需的) 使用这种技术;这是特别重要的, 如果复制水凝胶正在进行物理或机械分析, 以确保每一个水凝胶铸造具有相同的成分。通过改变一个或两个前体聚合物 (较低的官能团密度导致较慢的凝胶化) 或改变用于形成凝胶的前体聚合物的浓度, 可以很容易地改变凝胶时间 (低浓度导致凝胶化)21。交替地, 取代 (更反应性) 醛组与 (无反应) 酮组作为亲电性在胶凝对显著降低凝胶时间, 而不显著改变的组成, 产生的水凝胶35;用醛和酮单体前体混合物制备的聚合物, 可用于调整所需的凝胶时间, 而不改变所使用的前体聚合物的浓度 (因而形成的凝胶中固体的质量百分比)。
我们还注意到, 第一个水凝胶铸件并不总是具有与随后的水凝胶铸造相同的性质, 这一观察归因于两个桶的内容实际达到静态混合器的速率稍有差异。因此, 我们通常通过挤出一个小 (< 0.3 毫升) 凝胶的一部分, 在启动铸造过程, 以尽量减少这种变异的双桶注射器。最后, 虽然在使用寡聚合成预聚合物时通常不会有问题, 但一个或多个前体聚合物溶液的粘度在这种技术的背景下可能构成挑战, 无论是用简单的拇指凹陷来促进流动。以及在静态混合器中促进有效的混合。然而, 有些令人惊讶的是, 即使是具有明显不同粘度的前体聚合物溶液, 使用部件列表中描述的静态混合器附件 (例如, PNIPAM 具有高分子重量碳水化合物26), 表明由于不匹配的粘度而导致低效混合的担忧可能至少在大体积范围内并不显著。如果需要, 使用注射器泵 (而不是拇指) 来驱动流量和/或使用一个更大的测量针在出口可以帮助克服与 extrudability 在这些系统中的问题。
微尺度水凝胶通过反应微流体 与微流体方法有关的关键步骤是凝胶微粒的制备是微流体芯片与两种反应聚合物的启动。如果聚合物以不同的压力或不同的速率传递到芯片中, 则差压可以推动一个前体聚合物溶液回流到储层 (或至少朝向储层) 的其他前驱体聚合物。这就导致了从粒子形成到上游的凝胶化, 有效地堵塞了流量, 从而需要芯片处理。在每个储层与混合点之间留下的曲折路径, 会对回流产生明显的阻力;然而, 即使是训练有素的操作员也会偶尔在稳定的流量机制实现之前对芯片进行凝胶。根据我们的经验, 在1-2 分钟之间通常需要稳定的流动后的水滴形成 (时间相对多分散凝胶微粒生产);如果在操作的前5-10 分钟内没有发现任何问题, 则可能会连续数小时完成单分散粒子的生产。使用具有相对较好的粘度的前体聚合物以及非瞬时凝胶时间 (至少 > 十五年代优选), 大大有助于避免此类问题, 并促进稳定流动的形成。
请注意, 不同的流速范围从 0.01-0. 1 毫升/小时的水相中和 1.1-5.5 毫升/小时的油相中已经测试使用这个芯片设计, 导致制造的大小范围的 ~ 25-100 µm 根据剪切应用在流聚焦连接;更快的流速等同于更高的剪切, 因而更小的微粒形成了31,32。改变油流量, 同时保持总水流量低 (0.03 毫升/小时, 如协议所述) 被发现是最有效的控制凝胶微粒的大小, 而不损害任何单分散性或寿命的设备, 两者都是观察到在所引用的总水流量的较高端明显减少。更大的油流量 (> 5.5 毫升/小时), 以创建较小的粒子是可能的, 但增加了芯片剥离的风险 (一个常见的限制, 与等离子结合的微流控芯片)。使用另一种方法粘合芯片可以使流速更快, 从而减少凝胶微粒的产生, 这是我们目前正在探索的一种策略。减小喷嘴的大小也可能有助于减少可能产生的微粒的大小, 尽管在粒子形成之前, 过早凝胶的风险增加了。流速慢往往导致流动不稳定, 因而更高的 polydispersities 和增加芯片凝胶的风险;采用多通道微流控系统, 其稳定性和分辨率高于本协议中使用的标准注射器泵, 可以克服这种局限性。
石油的选择对于这项协议的成功至关重要, 因为较重的油 (在收集后防止凝胶微粒聚集的有利条件) 导致喷嘴中的颗粒形成的一致性远远低于所报告的轻质硅油。《议定书》。我们假设这种减少的重现性是由于更低的浓度的注射器泵浦较重的油, 导致更多的可变剪切在搅拌点。避免凝胶微粒聚集在收集瓶也是一个挑战, 特别是立即在出口从微流控装置在点就地凝胶不完整和大量可用的反应功能小组是可利用的形成桥梁在碰撞的微粒之间在汇集浴。这一挑战的解决方法是: 增加微流控芯片本身的出口通道长度, 保持凝胶微粒在层流流中较长的时间, 以促进更完整的凝胶;在喷嘴后添加侧通道, 将更多的油注入芯片中, 从而更好地将凝胶微粒在该后混合通道中分离, 而不会影响喷嘴本身或颗粒生产速率的剪切场;并在收集瓶中加入磁性搅拌机, 以避免凝胶微粒沉淀, 保持相邻颗粒间的平均分离。虽然非常缓慢的胶凝聚合物可能会改善设备的稳定性和最小化的问题与启动, 这类系统也被观察到大大增加了凝胶微粒聚集的风险, 作为更多的反应性功能组保持反应 (从而能够形成粒子之间的桥梁) 在较长的时间内。因此, 15-60 s 顺序的凝胶时间似乎是最佳的这一技术: 足够慢, 使启动, 但足够快, 以确保大多数反应功能组被消耗之前的凝胶微粒退出层流通道进入收集瓶。
最后, 删除模板油是必不可少的, 以确保所产生的粒子保持预期的智能性能的基础上添加预聚合物的组成, 并使这些粒子在生物医学的环境中使用。所描述的戊烷洗涤过程在这方面是非常有效的一般凝胶微粒生产。然而, 这种技术在直接生物医学环境中的应用 (例如, 片上细胞封装) 需要重新评估此协议。我们还探讨了橄榄油的使用, 建议在接触细胞36的情况下, 作为分散剂的一种更惰性的油。虽然粒子的形成是可能的, 凝胶微粒的数量比可以达到与矿物油, 至少与目前的芯片设计多分散。因此, 虽然该芯片似乎适用于合成聚合物和天然高分子凝胶微粒形成31, 可能需要一个修改的设计, 以更广泛地利用这种技术在所有可能的材料组合。
反应自组装纳米水凝胶 磁性已形成使用非常广泛的加工条件, 包括不同浓度的种子聚合物 (0.5-2 wt%), 不同的交联比例: 种子聚合物 (0.05-0. 2), 不同的温度 (40-80 °c), 不同的混合速度 (200-800 rpm), 和不同的加热时间后加入交联剂聚合物 (2-60 分钟)28。就浓度而言, 观察到的趋势一般都是预测的, 因为种子聚合物浓度越高, 交联剂的磁性和比例越高: 种子聚合物导致磁性的交联密度较高, 从而降低thermoresponsivities。应该强调的是, 增加种子聚合物浓度太高最终导致大块聚集而不是 nanoaggregation, 符合在常规自由基沉淀过程中观察到的形成thermoresponsive 磁性3。更短的加热时间也被发现有利于形成更小和更分散的颗粒。我们假设, 在温度高于 LCST 一个或两个前体聚合物时, nanoaggregate 在较长时间内会增加聚集在 nanogel 碰撞上的概率, 而腙键的疏水性则相对于无论是前体醛或肼功能基团, 使这种聚集更有可能随着交联程度的提高。最后, 从工艺角度来看, 较短的加热时间是有利的, 因为在交联聚合物添加后, 单分散 nanogel 种群可以在2分钟内形成;10分钟被发现是最长的时间, 可持续生产单分散磁性, 同时也允许生产更高交联磁性。有趣的是, 这种方法对混合非常不敏感, 几乎完全相同的颗粒大小和粒度分布是由于混合在不同的速度, 甚至扩展到较大的体积。虽然最初对这一结果感到惊讶, 但它可能会提到热力学在调节 nanogel 生产中的主要作用。
为了达到低 polydispersities, nanoaggregate 的胶体稳定性和水化程度似乎是关键变量。例如, nanoaggregates 准备使用更亲水性的酰肼功能高分子作为种子, 而不是亲水性较低的醛功能性聚合物, 导致磁性显著降低 polydispersities。实验装配温度与种子聚合物 LCST 的差异也至关重要。在种子聚合物 LCST (T-LCST) < 5 °c 以上的温度下操作, 可提供单分散 nanogel 形成的最高概率;在 LCST 以上的操作产生更多的疏水和折叠 nanoaggregates, 更有可能聚集和不太可能的交联, 而在 LCST 以下操作导致一个相对不紧凑的种子聚合物, 不能有效或重现联。为了对粒子单分散性进行最佳预测, 我们建议首先进行紫外/可见光扫描, 以测量种子聚合物的起始 LCST, 然后在该 LCST 之上的温度1-2 摄氏度下执行自组装过程。
请注意, 使用这种方法生产的磁性可以是冻干和 redispersed, 不改变胶体的稳定性, 往往是不可能的自组装结构和我们认为, 由于我们的交联稳定方法。我们还预计, 只有种子聚合物需要 thermoresponsive 这种方法来工作;使用不响应或响应其他刺激的交联聚合物, 可能进一步扩大这种技术的最终适用性。最后, 由于两种反应前体聚合物的混合在这种情况下是被动的, 而不是活性的, 胶凝时间在过程控制方面相对于所描述的其他制造策略来说要重要得多。然而, 即使在这种技术, 保持总交联时间 < 30 分钟是可取的, 以尽量减少粒子聚集的风险。
纤维结构水凝胶的活性静电纺丝 控制反应性预聚合物的凝胶时间是凝胶碳纤维生产成功的关键。特别是, 近似地匹配静态混合器中前体聚合物的停留时间 (通过改变双筒注射器溶液的流速和静态混合器的长度和扭曲控制) 与大块胶凝前体聚合物的时间是必不可少的, 既保持可纺性, 以及确保有效地交联的纺纤维之间的针和收藏家。快速的胶凝导致无效的泰勒锥体发展和因而可怜的可纺性, 而较慢的胶凝导致水溶液, 而不是凝胶击中收集器, 导致传播和最终形成薄膜凝胶而不是纳米。在停留时间稍低于散装凝胶时间也被发现是有效的 (确实比减少针头堵塞的风险), 因为水蒸发, 因为溶液是纺有效地集中的前体聚合物在流, 从而加快了纺丝过程中的凝胶动力学。在同样的静脉, 在较高的针到收集器的距离 (> 10 厘米) 在这个过程中通常是有利的, 因为更短的距离减少了水蒸发的时间, 因此需要更严格的控制之间的关系停留时间和凝胶时间, 以保持纤维结构产品。
请注意, 使用聚氧乙烯 (或其他高分子量和容易静电纺丝聚合物) 在本议定书中是必不可少的, 以促进碳纤维的形成, 因为短和高度分枝 POEGMA 低聚物不能单独达到足够程度的纠缠, 以诱导纺丝;相反, 电喷雾结果在所有的工艺条件下测试的 POEGMA 配方 (虽然这可能也有应用, 使可降解凝胶颗粒使用这种化学)。要保持一个完全纤维结构的形态, 必须要有 1 wt% (1 MDa 分子量) 的最小聚氧乙烯浓度。注意, 在不破坏纤维结构网络完整性的情况下, 在简单浸泡过程 (去离子水 24 h) 下, 聚氧乙烯可以从纤维中除去;这样, 聚氧乙烯作为一种瞬态静电纺丝助剂, 比最终纤维结构产品的重要组成部分更具作用。还要注意的是, 各种类型的收藏家, 包括简单的铝箔 (创建薄层水凝胶, 可以分层从收集器浸泡) 以及旋转铝盘 (创建更厚的脚手架) 可以与此相同的结合使用技术, 提供了控制凝胶速率、静电纺丝速率、静电纺丝过程中水分蒸发速率不变的其它工艺变量。
有趣的是, 根据用于制备不同形貌的方法, 在同一水凝胶前体制备的水凝胶的降解时间上有显著的差异。例如, POEGMA 纤维结构水凝胶的降解速度比散装 POEGMA 水凝胶具有相同的成分, 尽管其表面积明显较高, 从而获得水水解腙键。我们将这些差异与所描述的协议在混合前体聚合物的几何形状方面的内在反差联系在一起, 这可能导致内部凝胶 homogeneities 和/或明显不同的形态和/或在原位聚合物前体的浓度与凝胶的同一时间尺度, 特别是在静电纺丝中, 由于在这一过程中观察到的同时的水蒸发和交联。虽然这可能有点复杂的选择前体聚合物如果一个聚合物的目标是在每个议定书中使用, 它也可能提供一个技术机会, 使水凝胶与一个化学成分, 但非常不同的物理性质。
总的来说, 所描述的方法提供了一个战略, 以制造可降解 (或至少 renally clearable) 类似的 thermoresponsive 聚合物在多长尺度 (散装, 微, 和纳米) 和多种类型的内部结构 (颗粒或纤维)。这些议定书解决了将常规制备的合成 thermoresponsive 材料成功地翻译成生物医学领域的关键障碍: 可注射性和降解性。我们正在继续探讨这些材料在药物输送和组织工程应用中的应用, 从肿瘤的物理靶向、药物在血脑屏障的转运、蛋白质的治疗交付眼球的背面、组织的定向生长以及细胞的 thermoreversible 黏附和分化等应用。

智能材料由于其对外部和/或环境信号的可逆 "按需" 响应的潜力而引起了极大的关注。温度响应材料由于其较低的临界溶液温度 (LCST) 行为而引起了特别的兴趣, 从而导致温度驱动降水在温度 T > LCST1,2上。在 thermoresponsive 水凝胶的背景下, 这种较低的临界溶液温度行为表现为可逆膨胀/消胀事件, 导致温度可调谐的体积大小 (较大的 T < LCST)3, 孔隙大小 (大于 t< LCST)4和界面属性 (在 T < LCST 上更亲水)5。这种转变已广泛应用于药物传递 (外部或环境 triggerable 药物释放4,6,7), 组织工程和细胞培养 (为 thermoreversible 细胞黏附力/分层8,9,10), 分色 (用于可切换的膜孔隙度和渗透或热回收诊断支持11,12,13)、微流控过程 (用于调节流14、15) 和流变修饰符 (用于温度可调谐的粘度16)。最常见的 thermoresponsive 水凝胶是以聚 (n-丙烯酰胺) (PNIPAM)17 为基础的, 尽管在聚 (oligoethylene 乙二醇甲基丙烯酸酯) (POEGMA) 2 中也进行了显著 (和增加) 的工作. ,18和保利 (vinylcaprolactam) (PVCL)19,20。POEGMA 已经吸引了最近的兴趣, 因为它预期改进的生物相容性21,22和它的轻便到调谐 LCST 行为, 其中线性可预测的混合单体与不同数量的环氧乙烷重复单位在他们的侧链可以改变 LCST 从20°c 到 > 90 °c2,23。然而, 这些聚合物都是由自由基聚合制备的, 因此含有碳碳骨架, 在生物医学应用中大大限制了这种聚合物的潜在效用和可译性, 其中降解 (或至少通过肾脏过滤清除的能力) 通常是一个要求。
针对这一限制, 我们最近广泛地报道了腙化学 (i. e) 的应用情况, 即酰肼与醛功能性预聚合物的反应, 以制备 thermoresponsive 的可降解类似物。水凝胶24,25,26,27,28,29。酰肼与醛类在功能性前体聚合物混合过程中的快速可逆反应30同时启用原位凝胶 (允许在不需要手术的情况下对这些材料进行简便的注射。植入或任何类型的外部聚合刺激, 如紫外线照射或化学启动), 以及水解降解网络的速率控制的化学和密度的交联地点。此外, 通过保持前聚合物的分子量, 用于制备低于肾脏过滤极限的水凝胶, 使用这种方法的水凝胶会降解回寡聚前体聚合物, 可以从体内清除 25 ,27,28。再加上这些材料所诱发的低细胞毒性和低炎症组织反应25,26,27, 此方法为使用 thermoresponsive 提供了一种潜在的可翻译方法。智能水凝胶在医学, 特别是如果有良好控制的可降解类似物的水凝胶在所有长度尺度 (散装, 微, 和纳米) 可以制造。
在本协议中, 我们描述了合成 thermoresponsive 预聚合体功能的方法, 可控数量的酰肼和醛类, 以及应用这些聚合物创建水凝胶具有良好定义的维度各种长度刻度。特别是, 这篇手稿描述了四种不同的方法, 我们已经开发, 以控制反应肼和醛功能性预聚合物的混合, 从而创建 thermoresponsive 水凝胶网络, 具有良好定义的几何和形貌:
要创建具有定义大小的可降解散装水凝胶, 描述了一种模板化策略, 该方法将反应性预聚合体装入与静态混合器一起安装在其插座上的双筒注射器的单独桶中, 随后将其共挤成带有所需水凝胶形状和尺寸的硅胶模具21,27 (图 1)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54502/54502fig1.jpg"> 图 1: 散装水凝胶形成示意图.酰肼和醛功能聚合物溶液 (在水或水中的缓冲) 被装入单独桶的双桶注射器, 然后通过静态搅拌机共挤出成圆柱形硅胶模具。快速的就地凝胶在混合形成腙交联水凝胶, 是自由站立 (一旦模子被去除) 在秒到分钟之内, 取决于前体聚合物的浓度和功能群密度。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54502/54502fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了在微米尺度上制造可降解的凝胶微粒, 本文描述了一种反应微流体方法, 即前体聚合物溶液采用软光刻-模板化微流控芯片设计同时混合和乳化, 使混合反应聚合物液滴的形成随后凝胶原位形成凝胶微粒, 其尺寸由乳液 (图 2)31,32组成。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54502/54502fig2.jpg"> 图 2: 通过反应微流体形成凝胶微粒的示意图。(A、B)酰肼和醛功能聚合物溶液 (水或水缓冲液) 由注射器泵送入单独的水库, 连接在下游的一个曲折系列的通道, 旨在创造一个压力梯度防止回流。然后, 聚合物被混合在被石蜡油从双方流动 (也由注射器泵驱动) 的剪切和强制通过喷嘴, 导致流动聚焦生产水 (聚合物溶液) 液滴在连续的石蜡油阶段(参见 (B) 为喷嘴区域和雾滴形成过程的例证)。另外两个石蜡油入口位于喷嘴后, 以进一步分离收集通道中的水滴, 以允许在颗粒去除层流之前完成凝胶, 随后产生的微粒凝胶收集在一个磁性搅拌烧杯;(C) 喷嘴的水滴生成过程的图片 (注意, 酰肼聚合物被标记为蓝色, 以说明混合)
为了在纳米尺度上制造可降解的凝胶微粒, 本文描述了一种热驱动的反应自组装方法, 其中一种反应前体聚合物 ("种子" 聚合物) 的溶液在其 LCST 之上加热, 形成稳定的 nanoaggregate,随后交联的补充反应前体聚合物 ("交联" 聚合物);由此产生的腙交联 nanogel 的大小由 nanoaggregate (图 3)28直接进行模板。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/54502/54502fig3v2.jpg"> 图 3: 通过热驱动的反应自组装 nanogel 形成示意图.含 (thermoresponsive) 酰肼官能化聚合物的水溶液在其低临界溶液温度以上加热, 以形成稳定的 uncrosslinked nanoaggregate。接着, 通过腙键形成, 加入醛功能聚合物, 从而使 nanogel 颗粒在 LCST 下冷却后稳定 nanoaggregate。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54502/54502fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了制造可降解的纳米纤维, 本文描述了一种反应静电纺丝技术, 在其出口 (用于制造散装水凝胶) 的双筒注射器连接到标准的静电纺丝平台 (图4)33。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/54502/54502fig4.jpg"> 图 4: 用活性静电纺丝法制备凝胶碳纤维形成示意图.一个双桶注射器与静态混合器 (加载如描述散装水凝胶, 但也包括一小部分的高分子量聚乙烯 (环氧乙烷) 作为静电纺丝辅助) 安装在注射器泵, 针在年底的注射器连接到高压电源。腙交联发生在纤维纺纱过程中, 以便当流击中收集器 (无论是铝箔或旋转铝盘) 的纤维结构形态保持。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54502/54502fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
该协议使用 PNIPAM 或 POEGMA 作为感兴趣的聚合物, 证明了这种方法在创建可降解智能水凝胶网络中的应用;然而, 所描述的基本方法可以转化为任何水溶性聚合物, 尽管对粘度有适当的调整, (在自组装 nanogel 制造方法的情况下) 预聚合物在形成种子时的稳定性nanoaggregate。

<table border="0" cellpadding="0" cellspacing="0" style="width:1525px;" width="1525">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Chemicals</td>
			<td style="width:268px;"></td>
			<td style="width:287px;"></td>
			<td style="width:477px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">2,2 - azobisisobutryic acid dimethyl ester</td>
			<td style="width:268px;">Wako Chemicals</td>
			<td style="width:287px;">101138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Di(ethylene glycol) methyl ether methacrylate (M(EO)2MA)</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">447927</td>
			<td>188.2 g/mol, n=2 ethylene oxide repeat units</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Oligo (ethylene glycol) methyl ether methacrylate (OEGMA475)</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">447943</td>
			<td>475 g/mol, n=8-9 ethylene oxide repeat units</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Acrylic acid (AA), 99%</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">147230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Thioglycolic acid (TGA), 98%</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">T3758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Dioxane, 99%</td>
			<td style="width:268px;">Caledon Labs</td>
			<td style="width:287px;">360481</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Nitrogen, UHP grade</td>
			<td style="width:268px;">Air Liquide</td>
			<td style="width:287px;">Alphagaz1 765A-44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Adipic acid dihydrazide (ADH), 98%</td>
			<td style="width:268px;">Alfa Aesar</td>
			<td style="width:287px;">A15119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">N&#39;-ethyl-N-(3- dimethylaminopropyl)-carbodiimide (EDC, x%)</td>
			<td style="width:268px;">Carbosynth</td>
			<td style="width:287px;">FD05800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Hydrochloric acid (HCl), 37%</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">320331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Sodium hydroxide (NaOH), 97%</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">221465</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aminoacetyl aldehyde dimethyl acetal, 99%</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">121967</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">4-Hydroxy-TEMPO, 97%</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">176141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Methacryloyl chloride,97x%</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">523216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Petroleum ether, 95%</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">32047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Magnesium sulfate, 99.5%</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">tert-Butyl methyl ether, &#62;99.0%</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">443808</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Phosphate buffered saline</td>
			<td style="width:268px;">BioShop</td>
			<td style="width:287px;">PBS405.1</td>
			<td>1x, pH 7.3-7.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">N-isopropylacrylamide, 99%</td>
			<td style="width:268px;">J&#38;K Scientific</td>
			<td style="width:287px;">258717</td>
			<td>Recrystallized from 60% hexanes/40% toluene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Ethanol, anhydrous</td>
			<td style="width:268px;">Commerical Alchols</td>
			<td style="width:287px;">P016EAAN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Span 80</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">S6760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Heavy paraffin oil</td>
			<td style="width:268px;">Caledon Labs</td>
			<td style="width:287px;">1326197</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Pentane, reagent grade</td>
			<td style="width:268px;">Caledon Labs</td>
			<td style="width:287px;">1/10/7800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Poly (ethylene oxide) average Mv 600,000</td>
			<td style="width:268px;">Sigma Aldrich</td>
			<td style="width:287px;">182028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Supplies essential for synthesis and hydrogel fabrication</td>
			<td style="width:268px;"></td>
			<td style="width:287px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Rotary evaporator</td>
			<td>Heidolph</td>
			<td style="width:287px;">G3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Dialysis tubing (3500 Da molecular weight cut-off)</td>
			<td>Spectrum Labs</td>
			<td style="width:287px;">28170-166</td>
			<td>Vol/length= 6.4mL/cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Double barrel syringe</td>
			<td style="width:268px;">Medmix</td>
			<td style="width:287px;">L series</td>
			<td>L series, 2.5 mL, 1:1 volume ratio</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Static mixer</td>
			<td style="width:268px;">Medmix</td>
			<td style="width:287px;">L series</td>
			<td>L series, 2.5 mL, 1:1 volume ratio, 1.5&#34; length</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Silicone rubber sheet, 1/16&#34; thickness</td>
			<td style="width:268px;">McMaster-Carr</td>
			<td style="width:287px;">9010K12, 30A Durometer (Super Soft)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Syringe pump</td>
			<td style="width:268px;">KD Scientific</td>
			<td>KDS Legato 200</td>
			<td>Infuse Only Dual Syringe Pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">High voltage power supply</td>
			<td style="width:268px;">Spellman</td>
			<td style="width:287px;">230-20R</td>
			<td>0 to 20 kV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Microfluidic Chip Fabrication</td>
			<td style="width:268px;"></td>
			<td style="width:287px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Silicon wafer</td>
			<td style="width:268px;">University Wafer</td>
			<td style="width:287px;">2080</td>
			<td>D = 76.2 mm; 380 &#181;m thickness; P-doped; &#60;100&#62; orientation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">SU-8 100</td>
			<td style="width:268px;">MicroChem</td>
			<td style="width:287px;">Y131273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">SU-8 Developer</td>
			<td style="width:268px;">MicroChem</td>
			<td style="width:287px;">Y020100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Custom 2.5&#34; spincoater</td>
			<td style="width:268px;">Built in-house</td>
			<td style="width:287px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Mask Aligner</td>
			<td style="width:268px;">KARL SUSS</td>
			<td style="width:287px;">MJB3 UV400 (with a 276 W lamp)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Masterflex L/S 13 Silicone Tubing</td>
			<td style="width:268px;">Cole Parmer</td>
			<td>OF-96400-13</td>
			<td>Peroxide-cured</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dow Corning Sygard 184 Silicone Elastomer Base&#160;</td>
			<td>Ellsworth Adhesives</td>
			<td style="width:287px;">4019862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dow Corning Sygard 184 Silicone Elastomer Curing Agent&#160;</td>
			<td>Ellsworth Adhesives</td>
			<td style="width:287px;">4019862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">High Power Plasma Cleaner&#160;</td>
			<td style="width:268px;">Harrick</td>
			<td style="width:287px;">PDC-002-HP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Characterization Instruments</td>
			<td style="width:268px;"></td>
			<td style="width:287px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Mach 1 micromechanical tester</td>
			<td style="width:268px;">Biomomentum</td>
			<td style="width:287px;">LB007-EN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Cellstar tissue culture 12 well plate</td>
			<td style="width:268px;">Greiner Bio-one</td>
			<td style="width:287px;">665 180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Cell culture insert for 12 well plate</td>
			<td style="width:268px;">Corning</td>
			<td style="width:287px;">08-771-12</td>
			<td>8 &#181;m pore size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Optical microscope</td>
			<td>Olympus BX51 optical microscope</td>
			<td style="width:287px;">BX51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Temperature-controlled microscope stage</td>
			<td>Linkam Scientific</td>
			<td style="width:287px;">THMS600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Gel permeation chromatograph (GPC)</td>
			<td style="width:268px;">Waters</td>
			<td style="width:287px;">590 HPLC Pump</td>
			<td>Waters Styragel columns (HR2, HR3, HR4; 30 cm x 7.8 mm (ID); 5 mm particles), Waters 410 refractive index detector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Dynamic light scattering (DLS)</td>
			<td>Brookhaven</td>
			<td style="width:287px;">90Plus Particle Size Analyzer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Transmission electron microscopy (TEM)</td>
			<td>TEMSCAN</td>
			<td style="width:287px;">JEOL 1200EX</td>
			<td>Accelerating voltage 100 kV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Scanning electron microscopy (SEM)</td>
			<td style="width:268px;">Tescan</td>
			<td style="width:287px;">Vega II LSU</td>
			<td>Accelerating voltage 10 kV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Microsquisher</td>
			<td style="width:268px;">CellScale Biomaterials Testing</td>
			<td style="width:287px;">MS-50M-01</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabricating degradable thermoresponsive hydrogels on multiple length scales via reactive extrusion, microfluidics, self-assembly, and electrospinning

虽然为各种生物医学应用探索了各种智能材料 (例如、药物输送、组织工程、bioimaging、等), 但由于缺乏与生物相关的技术, 它们的最终临床应用受到阻碍。大多数智能材料的降解情况。对于温度敏感的水凝胶, 这一点尤其适用, 因为它几乎是基于功能上不可降解的聚合物 (例如、聚 (n-丙烯酰胺) (PNIPAM) 或聚 (oligoethylene 乙二醇) (POEGMA) 而一致的。).因此, 为了有效地将 thermoresponsive 水凝胶的潜能转化为远程控制或代谢调节药物的挑战, 细胞骨架可调谐的细胞材料相互作用, theranostic 材料的潜力对于影像学和药物传递, 以及其他类似的应用, 需要一种方法, 使水凝胶 (如果不能完全降解) 至少能够在所需的生命周期内的肾脏清除。为此, 本议定书描述了基于酰肼和醛功能化 PNIPAM 或 POEGMA 寡聚物与分子的反应, 在多长尺度上制备水解作用可降解腙交联水凝胶的研究重量低于肾脏过滤限值。具体来说, 方法制造可降解的 thermoresponsive 散装水凝胶 (使用双桶注射器技术), 水凝胶微粒 (在两个微型通过使用微流体平台促进同时混合和采用热驱动自组装和交联方法对前体聚合物和纳米尺度进行乳化, 并介绍了水凝胶纳米纤维 (采用反应静电纺丝策略)。在每种情况下, 水凝胶的温度响应特性类似于通过传统的自由基交联过程实现, 但腙交联网络可以退化一段时间, 以重新形成寡聚前体聚合物和允许清除。因此, 我们预计这些方法 (这可能是一般适用于任何合成水溶性聚合物, 而不仅仅是智能材料) 将使合成智能材料更容易地转化为临床应用。

从双筒注射器挤出到硅胶模具的散装水凝胶符合模具的尺寸, 并在脱模后变得自由站立;凝胶通常发生秒至几分钟后, 共挤出取决于使用聚合物前体。典型通过肿胀 (测量浓度使用细胞培养插入容易地去除水凝胶从肿胀解答), thermoresponsivity (用相同的技术测量, 但循环孵化温度上面和在相变温度下), 降解 (使用相同的技术测量, 但较长的时间段), 剪切或压缩模数 (用2毫米厚和7毫米直径模压样品测量) 证明了水凝胶的调谐反应取决于前体聚合物的化学 (特别是 POEGMA, 用于制备水凝胶的短到长链 OEGMA 单体的比例), 前体聚合物上官能团的摩尔分数, 以及它们的浓度前体聚合物 (图 5)27。
微流体导致形成明确定义的凝胶微粒的大小规模25-100 µm, 以大小可控的基础上的流量的油和/或联合水聚合物相 (图6A) 31.热阶段光学显微镜证实, 凝胶微粒保持 thermoresponsive 的性质的散装水凝胶, 显示可逆的温度依赖性肿胀-deswelling 只有轻微滞后在周期 1 (归因于折叠状态34中相邻酰胺组之间的不可逆氢键形成与散装 PNIPAM 水凝胶 (图 6B)32中观察到的结果一致。此外, 凝胶微粒随着时间的推移退化回其寡聚前体, 使肾脏清除 (图6C) 32.
由酰肼功能化的 PNIPAM 聚合物的 nanoaggregation 驱动的自组装在加热溶液中, 其次是与醛功能化的 PNIPAM 聚合物交联, 结果在高度单分散磁性 (多分散性 < 0.1) 上大小范围为 180-300 nm, 具体取决于所使用的过程条件 (图 7A)28。磁性保留传统的自由基交联 PNIPAM 磁性的典型 thermoresponsive 行为, 随着更多交联聚合物的加入, 热 deswelling 度降低 (图 7B)。磁性可以冻干和 redispersed, 而不改变粒子大小 (图 7C), 并通过水解降解一段时间, 重新形成用于制定磁性的寡聚前体聚合物 (图7 D)。
活性静电纺丝创建了一个纤维结构水凝胶结构 (图8 a), 碳纤维直径为 300 nm, 而没有可见的 electrosprayed 粒子呈现 33.浸泡在水中的 POEGMA 的纳米纤维导致快速水化 (大约两个数量级的速度比达到相同成分的大块凝胶,图 8B), 但保持纤维结构形态学超过8-10 周之前,在生理条件下水解降解;由于酸性催化腙键降解的可能性 (图 8C), 在酸性催化环境中观察到更快的降解。纤维结构结构在多周期的干燥和肿胀状态下也具有机械健壮性, 能够轻松处理和重复紧张 (图8 D).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/54502/54502fig5v2.jpg"> 图 5: 属性原位-凝胶散装可降解 thermoresponsive 水凝胶.(A) 有代表性的 POEGMA 凝胶网络显微组织和体积水凝胶图像, 以相应的凝胶时间作为摩尔在前体聚合物中加入 OEGMA475的功能;(B)PO100水凝胶的贮存模量 (B) 前体聚合物浓度和 (C) 摩尔% 官能团, 每个前驱体聚合物的组成;(D F)POEGMA 水凝胶 OEGMA 的物理化学性质475摩尔% 合并: (D) 存储模数 (E) 在1米 HCl 中的降解剖面, (F) 体积相变温度, 响应范围20-60 °c 的温度变化。所有误差线表示 n=4 复制测量的标准偏差。从引用27中修改, 并具有 Elsevier 的权限。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54502/54502fig5v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/54502/54502fig6.jpg"> 图 6: 活性微流体可降解凝胶微粒的性能。(A) 石蜡油流速对水中 (纯化) 凝胶微粒尺寸的影响;(B) 在体积相变温度高于和低于单热循环的情况下, 在水中 Thermoresponsivity 纯化的凝胶微粒;(C) 视觉评估 (照片) 和凝胶渗透色谱跟踪 (图) 确认凝胶微粒退化回其前体聚合物组分 (这里, 在1米 HCl 中, 以促进成像时间尺度的加速退化);缩放条 = 100 µm. 根据参考32改编。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54502/54502fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/54502/54502fig7v2.jpg"> 图 7: 活性自组装的可降解磁性的性质.(A) 用不同醛制备的磁性的粒度分布: 由动态光散射产生的肼聚合物质量比 (嵌入: 透射电子显微图像确认磁性的球形性质);(B) 温敏自组装粒子作为醛的质量比值的函数: 用于制备磁性的酰肼聚合物 (从动态光散射), 用误差条表示 n=4 复制的标准偏差;(C) 视觉上确认在冻干前后缺乏 nanogel 聚集;(D) 磁性酸催化降解的视觉确认 (在1米的 HCl 中, 与上文的其他研究一致);(E) 凝胶渗透色谱仪 nanogel 降解产物的痕迹, 表明它们与酰肼和醛功能性前体聚合物相似。与引用28的权限相适应。版权所有 2015, 美国化学学会。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54502/54502fig7v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/54502/54502fig8v2.jpg"> 图 8: 活性静电纺丝可降解纳米纤维的性能.(A) 扫描电子显微图像中的纳米纤维在干燥状态 (左), 半浸在水中 (中间, 薄膜), 并充分浸泡在水中过夜 (右, 厚脚手架);(B) 纤维结构水凝胶 (红色) 相对于同一成分的大块水凝胶 (蓝色) 肿胀, 误差条代表 n=4 复制的标准偏差;(C) 扫描电子显微镜和 (嵌入) 视觉图像跟踪酸性催化降解纳米纤维在 1M HCl;(D) 干燥的拉伸循环 (80 个周期, 20% 伸长/周期) 和肿胀 (325 个周期, 10% 伸长/周期在10毫米 PBS) 静电纺丝纤维结构水凝胶。图从参考33修改并在皇家化学学会许可下转载。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54502/54502fig8v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning at JoVE.

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

介绍了一种基于腙聚合物寡聚物在块状、微尺度和纳米尺度上交联的可降解 thermoresponsive 水凝胶的制备规程, 后者用于制备凝胶纳米粒子和纳米纤维。

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.), ...

fabricating-degradable-thermoresponsive-hydrogels-on-multiple-length

Nanyang Technological University

Research

JoVE Journal

Bioengineering

13.3K 次观看.  McMaster University. 介绍了一种基于腙聚合物寡聚物在块状、微尺度和纳米尺度上交联的可降解 thermoresponsive 水凝胶的制备规程, 后者用于制备凝胶纳米粒子和纳米纤维。

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

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

静电基础:优化解决方案和设备参数

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

科研

生物工程

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

固相Submonomer Peptoid聚合物及其自组装成高度有序的纳米片的合成

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

制造超疏水高分子材料生物医学应用

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

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 ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

制造棉分析仪器

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 &#176;C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

The side stream from plant oil production of Crambe abyssinica has limited value. The purpose of this study was to find methods for extruding materials based on this side stream, demonstrating that products with a higher value can be produced. The extrudates were found to have promising properties.

薄膜挤出<em&gt;海甘蓝abyssinica</em&gt; /谷朊粉共混物

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...