Name: fabrication and characterization of griffithsin-modified fiber scaffolds for prevention of sexually transmitted infections
Uploaded: 2017-10-31T14:00:00.000+00:00
Duration: 12 min 21 s
Description: Watch this Scientific Journal Video about Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections at JoVE.com

我们感谢犹太遗产基金为这项研究提供资金。我们感谢 Dr. 斯图尔特 II 慷慨地提供使用静电纺丝系统。我们还感谢 Dr. 为我们提供 Griffithsin。此外, 我们还感谢 Dr. 之场和他的实验室在 GRFT ELISA 工作中训练我们。

1. 纺纤维脚手架的制备和制造 警告: 所有与溶剂或聚合物溶液的工作应在化学油烟机上执行 。在开始协议之前, 请参阅每个试剂的材料安全数据表. 
<ol> <li> electrospin 3 毫升 15% w/w PLGA 聚合物溶液, 重720毫克50:50 聚 (乳酸-羟基酸) (plga; 0.55 到 0.75 dL/克, 31-57 kDa) 成10毫升闪烁瓶。解决方案的体积是基于当前研究中使用的典型批次大小. 
	注: 要添加到给定体积的溶剂中的聚合物质量, 必须首先确定用于溶解聚合物的溶剂的密度。溶剂 Hexafluoro-2-propanol (HFIP) 的密度是1.59 克/毫升。因此, 根据需要的3.0 毫升 HFIP 的体积, 溶剂的重量是4.8 克 (3.0 毫升 x 1.59 克/毫升)。对于 plga 的15% 瓦特/w 分数的 HFIP, 720 毫克 plga 必须添加到3.0 毫升 HFIP (0.15 x 4800 毫克 = 720 毫克)。使用% 瓦特/w 聚合物/溶液, 而不是% 瓦特/v 的好处是, 这提供了一个定义的重量最终解决方案。在步骤1.3 中溶剂蒸发的情况下, 这种定义的重量能够更准确地替代溶剂. </li>
	<li> 使用血清玻璃吸管将3.0 毫升 HFIP 添加到含有 PLGA (从步骤 1.1) 的玻璃闪烁瓶中。用塑料薄膜盖住瓶子, 然后测量并记录瓶子的质量. </li>
	<li> 在37和 #176 的夜间孵育聚合物悬浮液, 以确保聚合物完全溶解。如果任何溶剂蒸发, 减少瓶子质量, 添加 HFIP, 直到瓶达到其原始质量在步骤 1.2. </li>
	<li> 孵化后, 准备静电纺丝设备 ( 图 2 A )。尽管可以使用任何尺寸的芯轴, 但这里使用的是旋转的 25 mm 外直径不锈钢芯棒作为收集器. 
	注: 考虑到相同体积的静电纺丝溶液, 大芯轴直径会降低纤维厚度. </li>
	<li> 将聚合物溶液吸入3毫升注射器. </li>
	<li> 连接一个钝的18口径, 和 #189; 英寸针尖到注射器和免除过剩的解决方案 (通常是0.25 毫升), 以消除空顶部在针尖. </li>
	<li> 将注射器放在注射器泵上, 并将仪器流量设置为2.0 毫升/小时. 
	注: 此流量以前是根据聚合物粘度对此配方进行优化的. </li>
	<li> 将电源连接到注射器针, 并使用 +27 伏的电压 electrospin 聚合物溶液。针和收集器之间的距离应设置为大约25厘米 ( 图 2 B )。
	注意: 静电纺丝过程产生溶剂蒸气。使用通风罩或封闭式设备 ( 图 2 ) 删除有害气体 . </li>
	<li> 一旦整个解决方案是纺, 关闭电源, 并允许卷筒旋转额外的30分钟, 以充分蒸发溶剂. </li>
	<li> 关闭旋转芯轴收集器, 并使用刀片式服务器从芯轴上切断光纤。使用刀片轻轻从芯轴剥离纤维. </li>
	<li> 将纺 PLGA 纤维收集到标记的培养皿中, 并在 desiccatorovernight 中放置以除去残余溶剂. </li>
</ol> 2。GRFT 纤维的表面改性 <ol> <li> 准备磷酸盐缓冲盐水 (PBS) 和 2-(n-吗) 磺酸 (MES 缓冲液) 的溶液。准备 PBS 通过溶解 8 g 氯化钠, 0.2 g 氯化钾, 1.44 g Na 2 HPO 4 , 和 0.24 g 在 2 L 的 4 PO 1 在超纯水中。同样地, 在超纯水的 1 L 中溶解 19.52 g mes (游离酸、兆瓦 195.2) 和29.22 克氯化钠, 以制备 mes 缓冲器。确保每个溶液的最终 ph 值分别介于 7.2-7.5 和 5.0-6.0 之间, 使用 ph 计. </li>
	<li> 准备单独的工作解决方案 (2 mm) 和 NHS (5 mm). 
	<ol> <li> 从冷冻库中取出平衡和 NHS, 并在称量前允许它们在室温下进行加热. </li>
		<li> 在1.5 毫升离心管中重4毫克. </li>
		<li> 重6毫克 NHS 到另一离心管. </li>
		<li> 向每个管道添加1毫升的 MES 缓冲区。涡流两个管大力确保试剂完全溶解. </li>
	</ol> </li> <li> 通过将70毫克重达50毫升圆锥离心管来制备羟胺的溶液. </li>
	<li> 将20毫升 PBS 添加到羟胺和漩涡中以溶解. </li>
	<li> 将适量的 PLGA 纤维放入15毫升锥形离心管中。通常, 每个反应批次使用75毫克的纤维. </li>
	<li> 将8毫升的 MES 缓冲区添加到 15 ml 管中. </li>
	<li> 添加1毫升的每一个产品和 NHS 的解决方案早准备的管。溶液的最终体积应为10毫升。最终浓度的亚胺和 NHS 应分别为0.4 毫克/毫升和0.6 毫克/毫升. </li>
	<li> 关闭并密封15毫升管与塑料薄膜和位置在一个转子上, 使解决方案轻轻倒置15分钟室温 ( 图 3 B )。这一步激活聚合物上的羧基, 允许与 GRFT 蛋白 ( 图 3 A ) 共价修饰. </li>
	<li> 活化后, 通过加入14和 #181, 仔细淬火反应; L #946;-基到管子翻转管几次, 以确保完全混合. 
	警告: 和 #946-基是剧毒的, 只应用于化学油烟机. </li>
	<li> 丢弃上清液, 用10毫升 PBS 冲洗两次 PLGA 纤维, 去除残留的 #946;-基. </li>
	<li> 冲洗后, 在管中添加适量的 GRFT 库存溶液。例如, 5 nmol GRFT/毫克光纤将需要6.35 和 #160; 和 #181; GRFT 库存解决方案 (从10毫克/毫升的股票) 每毫克的纤维。因此, 75 毫克纤维样品将需要476.25 和 #160; 和 #181; 10 毫克/毫升 GRFT 库存解决方案. 
	注: GRFT 纤维的理论载荷为 0.05, 0.5 和 5 nmol GRFT 每毫克纤维制造. </li>
	<li> 添加足够的 PBS, 使最终的音量为8毫升, 关闭和倒置管, 以确保彻底混合. </li>
	<li> 用塑料薄膜密封管, 再将其放置在转子上, 此时为2小时. </li>
	<li> 在 2 h 孵化后, 通过在15毫升离心管中加入2毫升羟胺溶液来淬火反应。根据制造商的说明, 在淬火反应中羟胺的最终浓度应为0.7 毫克/毫升. </li>
	<li> 将溶液拌匀并丢弃上清液。用10毫升超纯水冲洗表面改性 PLGA 纤维两次, 去除任何游离 GRFT. </li>
	<li> 将光纤转移到培养皿中, 并将其放入干燥中, 直到纤维完全干燥为止。将培养皿转移到4和 #176; C 用于存储. </li>
</ol> 3。GRFT 表面改性纤维的 sem 表征 <ol> <li> 在 sem 标本上放置一条双面碳带。使用永久标记标识信息的示例安装的底部标记. </li>
	<li> 从一个表面改性纤维中切割三样品, 并将它们放在单独的标本上。每个样品的厚度大约是0.5 毫米. </li>
	<li> 溅射涂层样品使用电子诱导粒子沉积从一个金板。溅射大衣九十年代, 在2.4 伏. 
	注: 溅射涂层时间可能因设备参数而异, 包括电压和安培量. </li>
	<li> 图像在 8 kV 的样本, 放大范围从1000到 5,000X. </li>
</ol> 4。从表面改性纤维中提取 GRFT 的研究 <ol> <li> 将2毫克的纤维一式三份放入1.5 毫升的离心管中. </li>
	<li> 将1毫升二甲基亚砜 (亚砜) 添加到管中, 然后在室温下进行涡流和孵育1分钟, 以完全溶解纤维. </li>
	<li> 孵育后, 稀释10和 #181; 我分从步骤4.2 的亚砜纤维溶液, 至少100倍于三 EDTA (TE) 缓冲 (pH = 8.0). </li>
	<li> 将样本存储在-20 和 #176; C 直到加载特性与 ELISA. </li>
</ol> 5。从纤维中测量 GRFT 解吸 <ol> <li> 评估从纤维中释放或解的 GRFT 量, 重 5-10 毫克的表面修饰纤维, 并在离心管中放置。记录每管中的纤维质量. </li>
	<li> 添加1毫升的适当的解决方案, 模拟生理环境 ( 如 , PBS, TE 缓冲区, 模拟阴道液 (特别), 等 ), 以每个样本. </li>
	<li> 在 200 rpm、37和 #176 ° C 的旋转振动筛上孵育 1 h 的样品. </li>
	<li> 孵化后, 从瓶子中取出大约1毫升的 TE 缓冲液, 解 GRFT, 分到集束管。存储在-20 和 #176; C 直到蛋白质定量. </li>
	<li> 将样品转移到新的离心管中, 在离心管内的纤维上加入1毫升的新鲜缓冲液, 并孵育至下一时间点. </li>
	<li> 用于测量发布的典型时间点包括: 1、2、4、6、8、24、48、72 h 和1周. 在这些研究中观察到了 4 h 后可忽略的解吸现象. </li>
</ol> 6。酶联免疫吸附 GRFT 提取和解吸的定量化 <ol> <li> 用0.1 毫升的 HA (10 和 #181; g/毫升) 涂上96孔的 elisa 板, 每个井都有前面描述的 51 。用塑料薄膜封住盘子, 在晚上4和 #176 上孵育; C ( 图 4 ). </li>
	<li> 卸下涂层缓冲, 并在 PBS 中添加0.3 毫升阻断缓冲器 (2-3% 牛血清白蛋白 (BSA), 0.05% 聚 20)。在室温下孵育至少2小时的板材. </li>
	<li> 孵育后, 用1x 和 #160;P bs 0.1% 聚 20 (PBS P) 冲洗钢板3次。漂洗后, 将0.1 毫升的样品、标准或 PBS 作为负控制注入各自的井中。室温下再孵育1小时的钢板. </li>
	<li> 用 PBS-P 再次冲洗盘子3次。冲洗后, 将0.1 毫升的主抗体 (山羊抗 GRFT 血清) 放入每一个井中, 并在室温下孵育至少1小时。通常, 初级抗体溶液在 PBS 中稀释 1:10,000. </li>
	<li> 在用主抗体孵化样品后再冲洗盘子3次与 PBS P。加入0.1 毫升的二次抗体 (辣根过氧化物酶 (HRP)-共轭兔山羊 IgG), 并在室温下孵育1小时。二级抗体溶液在 PBS 中稀释 1:10,000. </li>
	<li> 洗盘子3次。每井添加0.1 毫升 TMB 2-过氧化物酶基底。监视颜色的开发 (大约2分钟), 然后添加 0.1 mL H 2 , 以便 4 (1 N) 以淬火反应。阅读平板阅读器在 450 nm. </li>
	<li> 平均背景 OD 值 (仅接收 PBS 的水井), 并将其从实验组中减去. </li>
</ol>

作者没有什么可透露的。

由于其多孔结构和大面积, EFs 已发现在医疗保健的各种应用, 其中包括服务作为治疗性分娩的车辆。在 EFs 中可以将药物和其他活性剂纳入可调谐的分娩, 而生物制剂和化学配体可以被共轭到纤维表面, 用于特定于单元的目标52或传感53。这里描述了 GRFT 表面修饰的 PLGA EFs 的制作, 作为一个预防 HIV 感染的递送支架。采用静电纺丝法合成了 GRFT-EFs, 相对于其他纤维生产方法, 具有成本低、生产率高的优点49,53。
协议中的关键步骤 EFs 的形成严重依赖于聚合物溶液的性质, 特别是溶液或溶剂粘度54。影响聚合物溶液粘度的因素包括聚合物分子量、聚合物浓度和所用溶剂的类型。溶液或溶剂粘度通常通过改变聚合物与溶剂的比值来调节, 以获得所需的聚合物浓度。每一个制造, 必须保持体积在夜间孵化, 以保持适当的 polymer-to-solvent 比 (粘度)。在足够高的聚合物浓度下, 聚合物分子在静电纺丝过程中缠绕在溶液中以产生纤维。在静电纺丝过程中, 珠将形成在喷尖端, 如果有足够的聚合物纠缠, 液体射流将爆发从这一点在一个关键的电压, 并加快以鞭状的方式向收集芯棒55。溶剂蒸发后, 将导致射流细化, 产生纤维的线程, 因为他们在收集芯线沉积。一旦合成, 应通过扫描电镜分析, 以验证正确的形态和一致的纤维直径。串珠状 EFs 的存在可能是由于低溶液粘度56, 极高的应用电压,57, 聚合物进给速率58, 或所有三因素的组合。如果观察到这一点, 应提高聚合物浓度, 并调整应用电压和进给率, 以达到纤维状的形态。
为了将蛋白质共轭到 EF 表面, PLGA 羧基在这里的反应是使用琥珀酰亚胺二交联化学59。在改性过程中, 纤维在 NHS 的存在下被短暂地孵育, 导致羧酸转化为稳定胺反应酯。在 two-step 共轭过程中, 关键的是在每个步骤中使用的缓冲器具有最佳的 pH 值, 在制造商的说明中注明, 以确保最大的共轭效率。琥珀酰亚胺酯的半衰期从四到五小时在中性 pH 值和戏剧性地减少在更加基本的情况60。因此, 第一反应应在 MES 缓冲区中的 ph 值 5-6, 然后将活化纤维转移到 PBS 缓冲区 (ph 7.2-7.5), 随后和立即与 GRFT 的反应。同样重要的是, 通过添加 2-基, 并充分冲洗后的纤维羧酸盐活化, 以灭活。这将有助于防止蛋白活化和联在第二反应期间, 这可能会降低共轭效率。
修改和故障排除 虽然我们以前的工作证明了各种 GRFT 改性纤维对 HIV-1 感染的功效, 但某些工艺改变可以考虑定制 EF 的形态学或改善 GRFT (或其他蛋白质) 共轭效率或纤维生成29。特别是, EF 表面积可以通过降低纤维直径来增加, 以使更大的表面积进行共轭。以往的研究表明, 降低聚合物的浓度和黏度产生较小的纤维直径61,62。然而, 当浓度低于阈值时, 这种方法受到串珠纤维形成的限制。为了减小纤维直径而不改变溶液粘度, 可以利用双溶剂系统来降低表面张力, 或添加盐, 增加溶液电导率56,57。这两种方法将使更大的静电纺丝喷气机的拉伸, 可能产生较小的纤维直径。此外, 低分子量聚合物可用于制造较小直径的纤维。降低光纤直径还提供了产生较小孔隙的额外好处, 这可能使 EFs 更有效地成为病毒渗透的屏障, 对于 microbicide-based 应用程序63。最后指出, 湿度会影响 EF 的产量。在较高的湿度下, 由于纤维的形成具有不同寻常的宏观形态, 导致产量趋于下降。因此, 在静电纺丝室内安装湿度控制系统, 可以方便生产出具有一致产量的 EFs, 如果这给制造带来了挑战的话。
为了提高 GRFT 共轭效率, 还可以考虑 functionalizable 组的选择和定位。例如, 如果感兴趣的蛋白质的主要胺类位于三维蛋白质结构的内部, 空间位阻可能阻止 EFs 上的活化羧基与原胺相互作用, 从而降低反应的可能性。这一挑战可以通过氨基酸取代蛋白质来产生一个更接近蛋白质表面的初级胺基团来克服。然而, 由于 GRFT 和其他蛋白质依赖于其结合位点的功能的具体活动, 蛋白质构象的改变应在功能分析中进行彻底的测试, 然后再进行共轭。
限制 GRFT 表面改性的一个主要限制是利用二交联化学进行低共轭效率的可能性。如果感兴趣的抗病毒蛋白有一个高集成电路50, 低共轭效率可能不会提供足够的保护 (在这些应用) 病毒感染。但是, 对于 GRFT (或其他修改过的) EFs、蛋白质和活性剂, 它们的共价键不能EFs 可能仍会吸附到表面。这些吸附剂通过瞬时增加可用于病毒结合的局部浓度, 提供了补充共轭 GRFT 活性的潜能。在本例中, 解 GRFT 可能绑定到不直接与 EFs 联系的 HIV-1 病毒, 并可提供与前 4 h (pericoital) 管理有关的另一种保护机制。因此, 共轭和表面吸附的 GRFT 可能有助于提供统一的预防性传播感染。
尽管从蛋白质共轭和解吸中得到了保护, 但其他具有潜在更高效率的表面修饰策略可能会被追求。例如, PLGA 终止与胺而不是羧基可用于共轭活化 GRFT。另外, 可以采用不同的表面修饰策略来提高蛋白质的共轭效率。由 EFs 组成的纤维膜已通过二交联化学的方式功能化了素 (64。法或添加链球菌 (激进分子-他的亲酰-谷氨酸-赖氨酸) 通过重组可能使修改后的蛋白质形成强大的和极端稳定的价与素的互动。而价, 素-生物素的联系是最强的价键, 具有 femtomolar 亲和力, 可能导致稳定的共轭纤维表面。对于任何表面修饰策略, 都应考虑空间位阻, 以最大限度地提高共轭效率。
最后, 我们设想, 表面修改的 EFs 将提供一种替代策略, 以提供主动代理封装的保护模式。在单 EF 平台上结合封装和表面修饰技术的一个考虑因素是在表面改性过程中减少活性剂的过早释放。对于在水溶液中需要较长的孵育时间的表面修饰反应, 由于聚合物水解, 有很大比例的活性剂被加载可能会丢失。
关于现有方法和替代方法的意义 在以前的工作中, 我们观察到, 未修改的空白 EFs 能够在 transwell 插入上述 infectible 单元格29的情况下, 通过 ~ 38% 抑制 HIV 感染。这一观察结合了生物相容性, 类似网络的显微组织, 和曲折的 efs, 与观察到的强力抗病毒和粘附性的 GRFT, 促进了表面改性的 efs 的发展, 在这里描述。
相对于目前在杀菌剂应用中使用的其他交付技术, 由于其体系结构和定制能力, EFs 具有广泛的潜在应用。在其他工作中, EFs 的纤维形态使它们能够提供活性剂, 并模拟 ECM, 使其成为组织工程的合适支架。EFs 也已进行了表面修改, 以增强生物相容性并增强持续释放65,66。
为了增强生物相容性或通过特定的结合活动提供功能, 存在着多种方法, 允许将化合物附着到 EFs 表面, 如等离子体处理、湿化学方法和表面接枝聚合50。在 GRFT 的情况下, 具体绑定到 HIV-1 的病毒糖蛋白, 湿化学方法的琥珀酰亚胺是最理想的, 因为有能力穿透纤维深的网格, 同时也保留 GRFT 功能50。GRFT 固定在 EFs 中的可持续性地交付给首次登记税, 并立即提供预防艾滋病毒感染的保护。
与现有的策略, 在 EFs 内封装疗法, 以抑制性传播疾病, 共价共轭提供了明显的优势, 增加潜在的亲和力之间的 GRFT 和 HIV 病毒。通过表面修饰将 GRFT 固定到 EFs, 可以实现高度局部化的 GRFT 浓度, 这增加了多与 HIV 结合的机会。此外, EF 固定的 GRFT, 相对于自由 GRFT 在解决方案中, 可能通过阻碍细胞内化来防止 GRFT 池的枯竭。此外, 由于 GRFT 作为稳定和粘合剂进入抑制剂的独特作用机制, 共价键表面结合使表面改性 EFs 提供了物理屏障, 病毒渗透, 除了强大的抗病毒性能。
此方法的未来应用 表面修饰的使用为交付提供了机会在一个 EF 平台中集成多个活动代理。在未来, 我们寻求开发一种多用途技术 (邮电部), 其中各种活性剂可以封装在和共轭到 EFs。这些 MPTs 的设计可以通过将疗法与不同的作用机制结合起来, 来保护更广泛的病原体。通过利用 efs 的表面和内部来提供具有不同操作机制的活动代理, 将最大限度地发挥 efs 防范病毒感染的潜能。

使用 EFs 作为一个专题传送平台有可能大大减少性传播感染。目前, 有超过3600万人感染艾滋病毒, 仅在2015年就有超过200万新病例报告为1,2。此外, 单纯疱疹病毒2型 (HSV-2) 感染影响到全世界数亿人, 并已证明, 以提高艾滋病毒的获得 2-5 倍3。由于 HSV-2 感染和艾滋病毒的获取之间的这种关系, 有很大的兴趣开发新的活性药物, 同时提供预防多种性传播疾病的保护。此外, 开发新的车辆, 以改善这些抗病毒药物的运送提供了进一步加强保护和治疗功效的潜力。为了达到这个目标, EFs 被作为一个新的交付平台来进行研究, 以减少 HIV-1 和 HSV-2 感染的流行。
在过去的二十年中, EFs 在药物传递和组织工程领域中得到了广泛的应用4。通常, 生物相容性聚合物被选择容易转化为治疗性应用。为了制造聚合物 EFs, 所选的聚合物在有机溶剂或水溶液中溶解, 这取决于聚合物疏水性的程度5。在静电纺丝过程之前, 有兴趣的活性剂被添加到溶剂或水溶液中。然后, 聚合物溶液被吸入到注射器中, 并在受到电流的同时慢慢喷射出来。这个过程通常会导致聚合物纤维与板料或圆柱形宏观 (图 1) 和光纤直径不等, 从微米到纳米级的6。对于大多数的治疗性应用, 活性剂在纤维纺丝过程中被整合在一起, 通过扩散和随后的纤维降解从纤维中释放出来。通过使用不同类型的聚合物或聚合物共混物来改变降解或释放的速率, 以建立一个理想的释放剖面, 传授独特的化学和物理性质7, 并促进几乎所有的封装复合.因此, EFs 已证明有利于小分子药物和生物制剂的交付, 包括蛋白质、肽、寡核苷酸和生长因子6,8,9。
在 STI 预防领域, 最近使用 EFs 来合并和提供抗病毒药物的持续或诱导释放10,11,12,13,14 ,15,16,17,18,19。在最早的一项研究中, pH 反应纤维的开发, 以释放活性剂, 以响应环境变化的女性生殖道 (首次登记税), 作为一种按需保护的方法, 反对 HIV-111。因为, 其他研究已经调查了由聚乙烯氧化物和聚 l-乳酸 (PLLA) 组成的聚合物共混物, 以评估抗病毒药物和避孕剂在 HIV-1 预防和避孕方面的可调谐释放,体外12. 其他研究显示了 EFs 提供以下内容的可行性: 小分子抗病毒药物的长时间释放14, 强大和灵活的机械性能20, 3 维交付体系结构21, 抑制精子穿透性12, 以及与其他交付技术合并的能力13。最后, 以前的工作已经评估了聚合物纤维的持续交付抗病毒剂抗常见 co-infective 病毒, HSV-2 和 HIV-114。在这项研究中, 高分子纤维通过保持其结构长达1月, 并为病毒进入提供了物理屏障, 从而为抗病毒药物的传递提供了补充活性。从这些结果中可以看出, EFs 可能被用于物理和化学上阻碍病毒感染。
虽然可调谐释放特性使聚合 efs 成为一个诱人的杀菌剂交付平台, 在其他应用中开发了 efs 作为表面修饰的支架7。EFs 已被用来模仿细胞外基质 (ECM) 的形态学, 通常充当支架以改善细胞再生22, 并增强其在组织工程中的效用23,24。由聚ε-内 (PCL) 和 PLLA 等聚合物组成的纤维经过表面修饰后, 在静电纺丝作用下, 通过生长因子和蛋白质来传授类似于 ECM 的特性, 包括增加细胞黏附和增殖25,26. 此外, 还对抗菌表面改性的 EFs 进行了评估, 以防止特定致病细菌的生长,27,28。由于这种多功能性和诱导生物效应的能力, EF 技术继续在各种领域扩展, 以提供 multi-mechanistic 的功能。然而, 尽管它们在多种应用中都有效用, 但表面改性纤维最近才在杀菌剂领域29中进行了探索。
在开发新的预防和治疗性传播疾病的新技术的同时, 还开发了新型的生物疗法。最有前途的杀菌剂候选者之一是粘合剂抗病毒凝集素, GRFT30。最初来源于一种红藻, GRFT 已证明活动作为一个强有力的抑制剂的艾滋病毒, HSV-2, SARS, 以及丙肝病毒31,32,33,34,35,36. 事实上, 在 biologically-based 抑制剂中, GRFT 具有最有力的抗 HIV 活性, 几乎在接触30时就立即灭活 HIV-1, 同时在培养基中维持稳定和活动, 从阴道微生物最多10天37。最近, 0.1% GRFT 凝胶被证明保护小鼠免受阴道 HSV-2 的挑战, 使它成为对 HSV-2 和 HIV-1 的第一行保护的有希望的候选者32,38. 对于 HIV 病毒, GRFT 抑制感染的物理结合 gp120 或终端甘露糖 n-链接的糖残留病毒的信封表面, 以防止进入38,39,40,41 ,42。这种抑制是非常强大的, 与 IC50的接近 3 ng/mL43。除了抑制艾滋病毒感染, 研究还表明, GRFT 通过抑制病毒的细胞传播来防止 HSV-2 感染 (32。在所有情况下, GRFT 已经证明是对病毒颗粒的粘合剂, 同时显示出高抗变性。最后, GRFT 已经证明了协同活动与诺福韦 (TFV) 和其他抗病毒药物的组合44, 使它可行, 并可能有利于 co-administer 与 EFs。GRFT 的强大特性使它成为一种极好的 biologically-based 抗病毒候选物, 通过 EF 技术可以提高分娩的效率。
利用这一知识的粘合剂和固有的抗病毒性能的 GRFT, 一个高分子纤维脚手架设计, 整合这些属性, 提供第一层病毒进入抑制29。找到灵感的方式, 宫颈粘液阻碍病毒传输主要通过粘粘蛋白相互作用, 我们假设, 通过使用 EFs 作为脚手架和共价修改表面与 GRFT, 高密度表面共轭 GRFT 会在其入口45,4647中削弱和禁用病毒。在这里, EFs 被开发为一个固定的脚手架, 提供一个蛋白质, 病毒性的粘合剂钝化屏障平台。我们试图结合的强大的抗病毒性能的 GRFT 与一个生物相容, 可修改, 耐用的高分子平台, 创造一个新的病毒 "陷阱"。
为了实现这些目标, 由 PLGA 组成的纤维是纺, 并使用琥珀酰亚胺化学, 以随后修改 EF 表面与 GRFT。PLGA 作为一个模型聚合物, 由于其广泛使用的静电纺丝48, 结合其生物相容性和 cost-effectiveness。此外, 表面修改利用了 EFs 的大面积, 并提供了一个有用的替代方案, 可与封装结合, 以最大程度地提高光纤实用程序49。与传统的封装方法不同的是, 只有部分 GRFT 是可用的 (而且只有在首次登记时才会出现), 表面修饰可以使 GRFT 在整个治疗期间保持最大的生物活性。此外, 采用传统的静电纺丝方法, 如蛋白质等亲水性化合物的加入, 可能会导致较低的封装效率和蛋白质活性的丧失50。因此, GRFT 表面改性纤维可以提供一种有前途的替代交付方法, 可以单独使用或与静电纺丝结合, 以加强预防性病感染。

<table><tbody><tr><td>Poly(Lactide-co-Glycolide) (PLGA) 50:50</td><td>Lactel</td><td>B6013-2P</td><td></td></tr><tr><td>1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP)</td><td>Thermo Scientific&amp;nbsp;</td><td>147541000</td><td></td></tr><tr><td>Blunt Dispensing Needle 18g X 1/2</td><td>Brico Medical Supplies</td><td>BN1815</td><td></td></tr><tr><td>BD 3mL Syringe Luer-lok tip</td><td>VWR</td><td>309657</td><td></td></tr><tr><td>Parafilm (plastic film)</td><td>Sigma Aldrich</td><td>P7793</td><td></td></tr><tr><td>2-(N-Morpholino)ethanesulfonic acid (MES Buffer)</td><td>Sigma Aldrich</td><td>M3671&amp;nbsp;</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>S7653</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma Aldrich</td><td>P9333</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma Aldrich</td><td>S7907</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P0662</td><td></td></tr><tr><td>Hydroxysuccinimide (NHS)</td><td>Thermo Scientific&amp;nbsp;</td><td>24500</td><td></td></tr><tr><td>1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)</td><td>Thermo Scientific&amp;nbsp;</td><td>22980</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Fisher</td><td>BP176</td><td></td></tr><tr><td>Griffithsin (GRFT)</td><td>Kentucky Bioprocessing</td><td>NA</td><td>custom made, no product number</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Milipore</td><td>317275</td><td></td></tr><tr><td>Polyethylene glycol sorbitan monolaurate (Polysorbate, Tween 20)</td><td>Sigma Aldrich</td><td>P9416&amp;nbsp;</td><td></td></tr><tr><td>Tris EDTA Buffer</td><td>Sigma Aldrich</td><td>93283</td><td></td></tr><tr><td>Flat-Bottom Immuno Nonsterile 96-Well Plates</td><td>Thermo Scientific&amp;nbsp;</td><td>3355</td><td></td></tr><tr><td>Influenza Hemagglutinin (HA)</td><td>Kentucky Bioprocessing</td><td>NA</td><td>custom made, no product number</td></tr><tr><td>Goat Anti-GRFT (Primary Antibody)</td><td>Kentucky Bioprocessing</td><td>NA</td><td>custom made, no product number</td></tr><tr><td>Rabbit anti-goat IgG-HRP (Secondary Antibody)</td><td>Santa Cruz</td><td>2056</td><td></td></tr><tr><td>Sure Blue TMB Microwell Peroxidase Substrate</td><td>KPL</td><td>52-00-00</td><td></td></tr></tbody></table>

fabrication and characterization of griffithsin-modified fiber scaffolds for prevention of sexually transmitted infections

纺纤维 (EFs) 已广泛应用于各种治疗应用中;然而, 它们只是最近才被用作预防和治疗性传播感染 (sti) 的技术。此外, 许多 EF 技术的重点是封装活性剂, 相对于利用表面传授 biofunctionality。在这里, 我们描述了一种方法制造和表面修饰聚 (乳酸-羟基) 酸 (PLGA) 纺纤维, 具有强大的抗病毒凝集素 Griffithsin (GRFT)。PLGA 是一种经 FDA 批准的聚合物, 由于其优异的化学和生物相容性, 已被广泛应用于药物的传递。GRFT 是一种天然的、有效的、安全的凝集素, 对包括人体免疫缺陷病毒1型 (HIV-1) 在内的多种病毒具有广泛的活性。当结合, GRFT 改性纤维已经证明了强力灭活的 HIV-1在体外。这份手稿描述的方法来制造和表征 GRFT 修改的 EFs。首先, PLGA 是纺制造纤维支架。纤维随后表面修饰与 GRFT 使用 1-乙基 3-(3-dimethylaminopropyl) 二 (和 n-羟基琥珀酰亚胺 (NHS) 化学。扫描电子显微镜 (SEM) 用于评估表面修饰的配方的大小和形貌。此外, 基于 gp120 或血 (HA) 的 ELISA 可以用来量化 GRFT 共轭的数量, 以及 GRFT 解吸从纤维表面。该协议可以更广泛地应用于制造表面修饰的各种不同蛋白质的纤维。

纤维形态对表面改性 EFs 的抗病毒能力有显著影响。虽然静电纺丝是一种方便而直接的方法, 但优化聚合物的配方可能会导致不规则的纤维形态 (图 5B-C)。静电纺丝条件的改变, 形成串珠状或无定形的垫样形态, 通常是由溶剂-聚合物不相容, 低聚合物粘度, 流速, 或其他静电纺丝条件造成的。由此产生的纤维结构变化可能导致不同的药物合并纤维的释放剖面或共轭功效的不一致, 改变纤维的能力, 物理或化学阻碍病毒渗透。使用所描述的静电纺丝条件制备的 PLGA EFs 应导致不同的纤维形貌, 直径范围介于1.5 和2.8 µm (图 6)。要确定纤维的形态和直径, 应在其他特性或改性步骤之前用 SEM 检查 EFs。
空白、0.05、0.5 和 5 nmol GRFT 纤维的 SEM 图像在纤维形态 (图 6A-D) 中没有显著差异, 表明 GRFT 修饰对纤维形态没有影响。为了确定每一个 EF 配方的平均直径, 至少50随机测量从 SEM 图像的视野。在 ImageJ 中测量和计算 EF 配方的平均直径, 如图 6E所示。所有 EF 配方都有类似的平均直径约1.9 µm, 证明了不被修改的纤维制造过程的一致性, 在分批。
为了确定与 efs 共轭的 GRFT 的数量, GRFT-efs 在亚砜中被溶解, 然后在 TE 缓冲器中进行100倍的稀释, 以从纤维中提取 GRFT。用 ELISA 法定量测定了每毫克纤维的 GRFT 共轭量。每个修改密度 (0.05, 0.5 和 5 nmol GRFT 每毫克纤维), 十复制被评估。对于 5, 0.5 和 0.05 nmol GRFT/毫克 ef 修改, 每 ef 有 373, 165 和 42 ng GRFT 每毫克 ef, 导致共轭效率分别为 0.6, 4.2 和6.9%。这些结果表明, GRFT-EFs 共轭与较高的理论表面修饰密度, 导致更多的 GRFT 共轭的纤维 (图 7A)。但是, 与由此产生的共轭效率29存在反向关联。
为了评估与吸附的纤维表面共价共轭的 GRFT 的数量, GRFT-EFs 在特别中孵化, 以确定 GRFT 释放的数量。在前 4 h, 113, 25 和 10 ng GRFT 每毫克 EF 被检测到在特别 5, 0.5, 和 0.05 nmol 理论修改浓度, 分别。这些值对应于 GRFT 共轭到5、0.5 和 0.05 nmol GRFT-EFs 的30%、41% 和24% 的数量。4小时后, 在释放洗中检测到所有三配方中可忽略的 GRFT。在图 7B中显示了1、2和 4 h 后的 GRFT 释放。在一起, 这些数据表明, 大多数 GRFT 是共价键绑定到 EFs, 和表面吸附 GRFT 释放在前4小时。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56492/56492fig1.jpg"> 
图 1:纺光纤的宏观形态学。所示的纺纤维是用4毫米 (圆筒) 和25毫米 (片) 直径的心轴分别制造的。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56492/56492fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56492/56492fig2.jpg"> 
图 2:静电纺丝设备(A) 聚合物沉积液流的收集芯棒, 以及 (B) 全静电纺丝装置。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56492/56492fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56492/56492fig3.jpg"> 
图 3:英孚修改的 GRFT 使用了琥珀酰亚胺化学的示意图.(A) PLGA 上的羧基与 NHS 发生反应, 形成胺反应酯, 随后形成稳定的胺键, 与 GRFT 的主要胺。(B) 在8毫升的 MES 缓冲液中, 用2毫升/琥珀酰亚胺试剂将两个毫克纤维圆盘或片进行切割和孵化, 并旋转15分钟.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56492/56492fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56492/56492fig4.jpg"> 
图 4:使用 ELISA 法演示 GRFT 量化的示意图.96井 immunoplate 涂有 gp120 或 HA, 以捕捉和固定 GRFT。对 GRFT 的主要抗体, 与辣根过氧化物酶相关的二级抗体, 和 ELISA 基质依次添加到量化 GRFT。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56492/56492fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56492/56492fig5.jpg"> 
图 5:溶剂选择对 PLGA EF 形态学的影响(A) HFIP 中的 15% w/w PLGA EFs 显示了理想的线状形态。(B) 在氯仿和胺中的 15% w/w PLGA EFs, 由于非溶剂选择或聚合物浓度 (粘度) 而无法形成。(C) 15% w/w 和 (D) 20% w/w TFE 的 PLGA EFs 显示了高分子粘度的重要性。在配方中形成具有较低聚合物浓度 (C) 的珠子, 同时提高聚合物浓度 (溶剂粘度) 导致了定义良好的纤维形态 (D)。注意,图 5B是在较低的放大倍数下拍摄的, 以显示类似席子的形态。缩放条形图 = 10 µm。<a href="//ecsource.j href=""></a href="//ecsource.j>ove.com/files/ftp_upload/56492/56492fig5large.jpg "目标 =" _blank ">> 请点击这里查看更大版本的这个数字。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56492/56492fig6.jpg"> 
图 6:裸和 GRFT 修改后的 EFs 的 SEM 图像和光纤直径.(A) 裸 plga efs 和 plga efs 表面-修改了 (B) 0.05 nmol, (C) 0.5 nmol, 和 (D) 5 nmol GRFT 每毫克纤维。缩放条形图 = 10 µm (E) 未修改的和 GRFT 的直径 (EFs)。误差线代表平均± SEM。未被修改的和 GRFT 的纤维直径无统计学差异。图 6E已从新郎et al.改编29 <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56492/56492fig6large.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/56492/56492fig7v2.jpg"> 
图 7: 从 GRFT 修改的 EF 中共轭的 GRFT 和解的数量.(A) 随着 GRFT 反应物浓度的增加, 与 EF 纤维的每毫克 GRFT 共轭的数量增加。(B) 从每根纤维中释放出的 GRFT 的数量在特别的0.05、0.5 和 5 nmol 配方中显示为1、2和 4 h 孵化。误差线代表平均± SEM。这个数字已经适应了新郎et al.29 <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56492/56492fig7v2large.jpg">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections at JoVE.com

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Griffithsin 修饰纤维支架预防性传播感染的制备与表征

这份手稿描述了制造和表征 Griffithsin 修饰聚 (乳酸-羟基酸) 纺纤维的程序, 证明有效的粘合剂和抗病毒活性对抗人体免疫缺陷病毒1型感染体外。本文介绍了用于合成、表面修饰和表征表面改性纤维 Griffithsin 的形态、共轭和解吸的方法。

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

fabrication-characterization-griffithsin-modified-fiber-scaffolds-for

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.4K 次观看.  University of Louisville. 这份手稿描述了制造和表征 Griffithsin 修饰聚 (乳酸-羟基酸) 纺纤维的程序, 证明有效的粘合剂和抗病毒活性对抗人体免疫缺陷病毒1型感染体外。本文介绍了用于合成、表面修饰和表征表面改性纤维 Griffithsin 的形态、共轭和解吸的方法。

视频: Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

科研

生物工程

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

实验室规模的人工合成蜘蛛丝生产

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

微流控干纺及再生丝素纤维的表征

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

化学

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

静电生长因子释放到微纤维支架

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

静电纳米纤维支架与渐变的纤维组织

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

界面聚电解质纤维的生物分子在时间控释复合支架

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

的简便和环保路线制造聚乳酸支架与分级孔径

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells

Genetic engineering of T cells for CAR-T cell therapy has come to the forefront of cancer treatment over the last few years. CAR-T cells are produced by viral gene transfer into T cells. The current gold standard of viral gene transfer involves spinoculation of retronectin-coated plates, which is expensive and time-consuming. There is a significant need for efficient and cost-effective methods to generate CAR-T cells. Described here is a method for fabricating inexpensive, dry macroporous alginate scaffolds, known as Drydux scaffolds, that efficiently promote viral transduction of activated T cells. The scaffolds are designed to be used in place of gold standard spinoculation of retronectin-coated plates seeded with virus and simplify the process for transducing cells. Alginate is cross-linked with calcium-D-gluconate and frozen overnight to create the scaffolds. The frozen scaffolds are freeze-dried in a lyophilizer for 72 h to complete the formation of the dry macroporous scaffolds. The scaffolds mediate viral gene transfer when virus and activated T cells are seeded together on top of the scaffold to produce genetically modified cells. The scaffolds produce &gt;85% primary T cell transduction, which is comparable to the transduction efficiency of spinoculation on retronectin-coated plates. These results demonstrate that dry macroporous alginate scaffolds serve as a cheaper and more convenient alternative to the conventional transduction method.

Herein is a protocol for creating dry macroporous alginate scaffolds that mediate efficient viral gene transfer for use in genetic engineering of T cells, including T cells for CAR-T cell therapy. The scaffolds were shown to transduce activated primary T cells with &gt;85% transduction.

干式大孔海藻酸盐支架的制备和使用用于T细胞的病毒转导

Genetic engineering of T cells for CAR-T cell therapy has come to the forefront of cancer treatment over the last few years. CAR-T cells are produced by ...

<meta charset="utf8"></head><body>FN-Silk 膜的形成和附着到细胞培养物的插入物上

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.

<meta charset="utf8"></head><body>作者聚焦：使用重组功能化蜘蛛丝膜增强体外细胞培养模型

Tissue culture inserts with plastic membranes are the golden standard in cell culture labs as permeable supports to establish cell layers and models of barrier tissues. Herein, we present a simple method to replace the plastic membrane with a more biologically relevant membrane made from a recombinant functionalized spider silk protein.

纳米原纤维基底膜模拟物，由重组功能化蜘蛛丝制成，用于定制组织培养小室

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using ...