CN107383409B - 一种制备促进血液相容性pdms基底的方法 - Google Patents

一种制备促进血液相容性pdms基底的方法 Download PDF

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CN107383409B
CN107383409B CN201710615366.3A CN201710615366A CN107383409B CN 107383409 B CN107383409 B CN 107383409B CN 201710615366 A CN201710615366 A CN 201710615366A CN 107383409 B CN107383409 B CN 107383409B
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薛鹏
李倩
许志刚
孙利红
康跃军
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Abstract

本发明涉及生物材料的表面修饰，更具体地设计基于多巴胺与PDMS具有良好的粘附性，同时，透明质酸具有很好的血液相容性，而且多巴胺与透明质酸可以通过氢键和静电相互作用进行整合。所以基于以上特性，首先在PDMS上连接上多巴胺，然后再在相应条件下修饰上透明质酸。通过一系列的表征实验证明该方法制备的PDMS基底不仅具有良好的抗凝作用，而且更具有很好的血液相容性。并且这种经过多巴胺和透明质酸修饰的PDMS材料有望用于医疗器械材料研究的基底材料。

Description

一种制备促进血液相容性PDMS基底的方法
技术领域
本发明涉及生物材料领域，具体涉及探究经过PDA-HA修饰后的PDMS的血液相容性。
背景技术
透明质酸（ hyaluronic acid， HA， 分子式：（C14H21NO11)n）是一种酸性粘多糖，1934年美国哥伦比亚大学眼科教授Meyer等首先从牛眼玻璃体中分离出该物质。透明质酸以其独特的分子结构和理化性质在机体内显示出多种重要的生理功能，如润滑关节，调节血管壁的通透性，调节蛋白质，水电解质扩散及运转，促进创伤愈合等。尤为重要的是，透明质酸具有特殊的保水作用，是目前发现的自然界中保湿性最好的物质，被称为理想的天然保湿因子。
多巴胺（dopamine, DA, 化学结构式：C6H3(OH)2-CH2-CH2-NH2， CAS号：62-31-7）是一种神经传导物质，用来帮助细胞传送脉冲的化学物质。这种脑内分泌物和人的情欲、感觉有关，它传递兴奋及开心的信息。近年来，在生物医学及生物材料学方面广为应用。它在水溶液中很容易被溶解氧氧化，继而引发自聚交联反应，可以在几乎任何一种固体材料表面形成紧密附着的复合层。将待改进的固体材料浸润在新鲜配制的多巴胺水溶液中，即可得到表面附着聚多巴胺交联复合层的改性基底材料。经研究表明，当用PH=8.5的Tris盐酸配置多巴胺溶液时，其自聚效果更好。
聚二甲基硅氧烷（Polydimethylsiloxane, PDMS，化学结构式：（C2H6OSi)n，CAS号：9016-00-6）无色或浅黄色液体，无味，透明度高，具有耐热性、耐寒性、黏度随温度变化小、防水性、表面张力小、具有导热性，透光性好，具有优良的物理特性，比如它具有良好的透光性，便于观察；弹性好，有益于延展；同时具有穿透性，可以让CO2、O2等气体分子通过；背景荧光小，便于荧光观察；低毒，价廉等特性。因此在PDMS上可以实现体外培养细胞，从而观察细胞的生长情况，及其体外模拟一些复杂体内的代谢环境，这对研究生物体内细胞的作用机制具有重要的意义。
发明内容
基于透明质酸具有良好的生物相容性，但其很难粘附于固体材料表面。然而，多巴胺与固体表面具有牢固的粘合力，并且透明质酸和多巴胺之间可以通过氢键和静电相互作用进行结合。所以本文旨在用PDMS为基底，首先在PDMS表面coating一层多巴胺，然后再通过氢键和静电作用固定上更多的透明质酸，从而探究该基底的血液相容性。
本发明的技术方案具体如下：
一种制备促进血液相容性PDMS基底的方法包括以下步骤：
(1)制备PDMS基底：首先，将弹性剂与固化剂以质量比为10:1混合配置成PDMS，搅拌均匀，然后将其倒入组织培养板中，倒入体积为0.5-1 mL，在真空干燥箱内真空除气泡1h，然后70℃加热2 h，即得到了未经任何修饰的PDMS基底；
(2)对PDMS基底进行PDA的修饰：用PH=8.5的Tris盐酸溶液配置浓度为2 mg/mL的多巴胺(DA)溶液，然后将其倒入步骤(1)中得到的未经任何修饰的PDMS上，倒入体积为1-2ml,具体倒入溶液体积以盖住PDMS基底为准，然后将其置于25℃环境中，作用24 h，然后用去离子水清洗三遍，洗掉PDMS上未粘附以及粘附不牢固的多巴胺，即得到聚多巴胺（PDA）修饰过的PDMS基底；
(3)对PDA-PDMS基底进行HA的修饰：配置浓度为1-5 mg/mL的透明质酸(HA)溶液，然后将透明质酸溶液倒入步骤(2)中得到的聚多巴胺修饰过的PDMS基底中，置于37℃环境中，作用12 h，然后用去离子水清洗三遍，洗掉PDMS上未粘附以及粘附不牢固的透明质酸，即得到促进血液相容性的HA-PDA-PDMS基底。
优选的，所述步骤(3)中的透明质酸(HA)溶液的浓度为2mg/mL。
进一步的，制备促进血液相容性PDMS基底的方法在体外细胞培养、抗凝和血液相容性和医疗器械材料的应用。
本发明主要优点有：
1. 针对目前透明质酸具有良好的血液相容性，而且PDMS是一种很好的生物材料，但是透明质酸在PDMS上的粘附力不强，所以很难将其稳定的固定于PDMS基底表面。本项目创造性地用多巴胺对PDMS具有较强的粘附力，且作用条件及其简单等优势，首先将多巴胺固定于PDMS上，然后再利用多巴胺和透明质酸之间的氢键和静电作用，从而将透明质酸牢固的固定于PDMS基底之上。
2. 本项目中，不仅利用多巴胺在PDMS上较强的粘附性将透明质酸固定于PDMS基底上，而且还用血小板，人脐静脉内皮细胞以及巨噬细胞等分别探究了该修饰后的PDMS基底的抗凝作用，以及抗炎症性也是本项目的最大特色之一。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图：
图1为本发明实施例1中制备促进血液相容性PDMS基底的示意图。
图2为本发明实施例1中在PDMS上固定的多巴胺和透明质酸的量的测量结果。
图3为本发明实施例2中不同PDMS基底上凝聚的血小板情况。
图4为本发明实施例2中不同PDMS基底上人脐静脉内皮细胞的增值情况。
图5为本发明实施例2中不同PDMS基底上的炎症反应结果。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1 制备具有血液相容性的PDMS基底
（1）制备PDMS基底：首先，将弹性剂与固化剂以质量比为10:1混合配置成PDMS，搅拌均匀，然后将其倒入24孔板中，倒入体积为1 mL每孔，然后放入真空干燥箱内真空除气泡1 h， 随后在70℃条件下加热2 h 进行固化，即得到了未经任何修饰的PDMS基底。
（2）对PDMS基底进行PDA的修饰：用PH=8.5的Tris盐酸溶液配置浓度为2 mg/mL的多巴胺溶液。然后将多巴胺溶液倒入步骤（1）中得到的未经任何修饰的PDMS上，倒入体积为1 mL每孔。然后将培养板置于25℃环境中， 24 h 后，取出培养板，倒掉多巴胺溶液，然后用去离子水冲洗3遍，每遍约10 S，洗掉没有粘附上或者粘附不牢固的多巴胺。然后放置过夜待其自然晾干。
（3）对PDA-PDMS基底进行HA的修饰：用去离子水分别配置浓度为1 mg/L, 2 mg/mL, 5 mg/mL 的透明质酸溶液，然后将不同浓度的透明质酸溶液分别倒入（2）中得到的多巴胺修饰过的 PDMS基底中，置于37℃环境中， 12 h 后，取出培养板，倒掉透明质酸溶液，用去离子水冲洗3遍，每遍约10 S，洗掉没有粘附上或者粘附不牢固的透明质酸。然后放置过夜待其自然晾干。其制作过程见图1。然后又检测了不同的PDMS基底上最终固定的多巴胺以及透明质酸的量，其结果见图2，结果显示利用本实验方法不仅成功的将PDA固定在了PDMS上而且又进一步地提高了透明质酸的粘附量以及稳定性和持久性。
实施例2 对制备的具有血液相容性的PDMS基底进行血液相容性的检测
（1）从新鲜的血液中分离出血小板，将血小板分别倒入不同的PDMS基底中，在37℃条件下孵育1 h ,然后用1×PBS清洗五遍，再分别利用GMP-140和LDH试剂盒对不同基底上凝聚的血小板的活性及数量进行半定量。结果如图3，从图中可以看出经过修饰的PDMS上凝聚的血小板明显减少，从而说明本试验方法得到的PDMS基底具有良好的抗凝作用。
（2）将人脐静脉内皮细胞分别接种于不同的PDMS基底上，然后分别在培养4 h, 24h,72 h ,然后测定细胞的活性及其增值情况，结果如图4，从图中可以看出，经过修饰的PDMS上细胞的活性以及增殖情况都降低了，这从另一个方面也反映出本实验方法得到的PDMS基底具有良好的血液相容性。
（3）将巨噬细胞分别接种于不同的PDMS基底上，培养24 h 后，分别利用ELISA试剂盒检测TNF-α, IL-6 和 IL-12这些炎症因子的释放情况，其结果如图5，从图中可以看出经过修饰的PDMS不会引起明显的炎症反应。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其做出各种各样的改变，而不偏离本发明权利要求书所限定的范围。

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1.一种制备促进血液相容性PDMS基底的方法，其特征在于：包含以下步骤：
(1)制备PDMS基底：首先，将弹性剂与固化剂以质量比为10:1混合配置成PDMS，搅拌均匀，然后将其倒入组织培养板中，倒入体积为0.5-1 mL，在真空干燥箱内真空除气泡1 h，然后70℃加热2 h，即得到了未经任何修饰的PDMS基底；
(2)对PDMS基底进行PDA的修饰：用PH=8.5的Tris盐酸溶液配置浓度为2 mg/mL的多巴胺(DA)溶液，然后将其倒入步骤(1)中得到的未经任何修饰的PDMS上，倒入体积为1-2 ml,具体倒入溶液体积以盖住PDMS基底为准，然后将其置于25℃环境中，作用24 h，然后用去离子水清洗三遍，洗掉PDMS上未粘附以及粘附不牢固的多巴胺，即得到聚多巴胺（PDA）修饰过的PDMS基底；
(3)对PDA-PDMS基底进行HA的修饰：配置浓度为1-5 mg/mL的透明质酸(HA)溶液，然后将透明质酸溶液倒入步骤(2)中得到的聚多巴胺修饰过的PDMS基底中，置于37℃环境中，作用12 h，然后用去离子水清洗三遍，洗掉PDMS上未粘附以及粘附不牢固的透明质酸，即得到促进血液相容性的HA-PDA-PDMS基底。
2.根据权利要求1所述的一种制备促进血液相容性PDMS基底的方法，其特征在于：所述步骤(3)中的透明质酸(HA)溶液的浓度为2mg/mL。
3.由权利要求1所述的制备促进血液相容性PDMS基底的方法在体外细胞培养、抗凝和血液相容性和医疗器械材料的应用。
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