CN111748120A - 一种聚多巴胺掺杂葡聚糖水凝胶多孔支架及制备方法及应用 - Google Patents
一种聚多巴胺掺杂葡聚糖水凝胶多孔支架及制备方法及应用 Download PDFInfo
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Abstract
本发明涉及一种聚多巴胺掺杂葡聚糖水凝胶多孔支架及制备方法及应用,用于细胞体外3D培养及伤口修复,水凝胶支架由以下方法制备得到:使用氢氧化钠溶液溶解葡聚糖,制备葡聚糖溶液,将盐酸多巴胺加入到氢氧化钠溶液氧化聚合生成聚多巴胺溶液,两者按一定比例混合均匀后加入交联剂乙二醇缩水甘油醚,置于37℃恒温箱中12小时后形成凝胶,本发明中所用的葡聚糖是一种细菌分泌的高水溶性天然多糖,具有良好的生物相容性和可降解性,形成凝胶后生成大量微孔结构,具有的大比表面积可为聚多巴胺提供大量的负载位点,有利于细胞进入至凝胶内部,与聚多巴胺产生黏附作用,是一种优异的多孔水凝胶支架材料,在伤口修复及生物组织工程上具有广阔的应用前景。
Description
技术领域
本发明涉及生物支架领域,尤其是涉及一种聚多巴胺掺杂葡聚糖水凝胶多孔支架及制备方法及应用。
背景技术
葡聚糖是某些微生物在生长过程中分泌的一种以葡萄糖为单糖组成的微生物多糖,其中葡萄糖单元之间以糖苷键连接。葡聚糖具有很好的生物相容性和可降解性,但是葡聚糖凝胶的黏附能力较弱,在实际应用中易与机体脱落,从而阻碍其进一步应用。
研究发现,海洋生物贻贝通过其足腺分泌的具有超强黏附性能的蛋白,可在海水等潮湿环境中牢固黏附在各种材料的表面,聚多巴胺是一种贻贝仿生类材料,具有与贻贝黏附蛋白相似的结构和超强的黏附性能。
水凝胶支架应用于生物医学领域,不仅需要优异的生物相容性,还需要具有一定的力学性能和生物学功能,以适用于不同的医用领域和临床需求。开发一种简单快捷、成本低廉的多功能水凝胶多孔支架的需求仍然十分迫切,也是当前水凝胶多孔支架应用于临床实践亟待解决的技术问题。
发明内容
针对现有技术存在的不足,本发明的目的在于提供一种聚多巴胺掺杂葡聚糖水凝胶多孔支架及制备方法及应用。
为实现上述目的,本发明提供一种聚多巴胺掺杂葡聚糖水凝胶多孔支架的制备方法,将盐酸多巴胺固体溶于1.2M的氢氧化钠水溶液中,在常温条件下搅拌进行氧化反应制备含30mg/mL的盐酸多巴胺氢氧化钠水溶液,将葡聚糖溶于1.2M的氢氧化钠水溶液中,在常温条件下搅拌进行氧化反应制备含15%(w/v)葡聚糖溶液,将盐酸多巴胺氢氧化钠水溶液和葡聚糖溶液混合搅拌,加入与混合液体积比为1∶10的交联剂,所述盐酸多巴胺氢氧化钠水溶液和葡聚糖溶液体积比为1-5∶5。
进一步的,所述交联剂为乙二醇缩水甘油醚。
本发明还提供上述制备方法制备的聚多巴胺掺杂葡聚糖水凝胶多孔支架。
本发明还提供上述聚多巴胺掺杂葡聚糖水凝胶多孔支架的应用,聚多巴胺掺杂葡聚糖水凝胶多孔支架用于制备细胞支架的应用。
作为本发明的一种应用方式,所述细胞支架的孔隙为5~30μm。
本发明还提供上述聚多巴胺掺杂葡聚糖水凝胶多孔支架的应用,聚多巴胺掺杂葡聚糖水凝胶多孔支架用于制备皮肤修复凝胶的应用。
作为本发明的一种应用方式,所述皮肤修复凝胶负载有细胞生长因子、抗菌剂、保湿剂、防腐剂、抗氧剂、乳化剂、增稠剂、甜味剂、胶浆剂、芳香剂以及矫味剂中的一种或多种。
本发明具有如下优点:葡聚糖具有很好的生物相容性和可降解性,成胶后生成的多孔葡聚糖凝胶能为细胞提供生长位点,且多糖材料能为细胞的生长提供一定的营养物质,是一种理想的细胞支架材料,在细胞3D培养和伤口修复中具有很好的应用前景,聚多巴胺(PDA)由于具有与贻贝的足肌蛋白相似的官能团(邻苯二酚基团),可以显著增强材料的粘合性能,聚多巴胺的邻苯二酚基团可以通过氢键,静电相互作用和π-π堆积与各种表面相互作用,使得凝胶具有显着的组织粘附特性,将其掺杂至葡聚糖凝胶中后,有望能够促进细胞在凝胶中的黏附及防止凝胶载体在机体上的脱离,此外,聚多巴胺的掺杂能够增强葡聚糖凝胶的力学性能,调节凝胶孔径大小,结合葡聚糖与聚多巴胺的优点,有望构筑性能优异且实用性强的水凝胶多孔支架,本发明所构筑的水凝胶多孔支架在细胞3D培养和伤口修复等生物医学领域具有广阔的应用前景。
附图说明
图1为葡聚糖/聚多巴胺水凝胶支架的储能模量;
图2为葡聚糖/聚多巴胺水凝胶支架的扫描电镜图;
图3为成纤维细胞与葡聚糖/多巴胺水凝胶共培养后LIVE/DEAD染色分析结果;
图4为空白组(灭菌PBS)和水凝胶组(D4)对大鼠的伤口修复照片。
具体实施方式
下面将结合实施例和效果例对本发明做进一步的详述,而非限制本发明的范围。
实施例1:葡聚糖/聚多巴胺多孔水凝胶细胞支架的制备
其制备方法步骤如下:
1)盐酸多巴胺氢氧化钠水溶液是通过将30g盐酸多巴胺固体溶于1L的1.2M氢氧化钠水溶液中,在空气条件下(25℃,磁力搅拌24h)进行氧化反应来制备;
2)使用氢氧化钠溶液(1.2M)溶解葡聚糖,制备浓度为15%(w/v)的葡聚糖溶液;
3)取2mL步骤1中的盐酸多巴胺氢氧化钠水溶液和5mL步骤2中葡聚糖溶液以及3mL的1.2M的氢氧化钠溶液混合在一起,室温搅拌均匀。
4)向步骤(3)中所制备的混合溶液中加入1.0mL交联剂EGDE,搅拌均匀后置于37℃恒温箱中,交联12h形成凝胶,记为D2。
实施例2:葡聚糖/聚多巴胺多孔水凝胶细胞支架的制备
其制备方法步骤如下:
1)盐酸多巴胺氢氧化钠水溶液是通过将30g盐酸多巴胺固体溶于1L的1.2M氢氧化钠水溶液中,在空气条件下(25℃,磁力搅拌24h)进行氧化反应来制备;
2)使用氢氧化钠溶液(1.2M)溶解葡聚糖,制备浓度为15%(w/v)的葡聚糖溶液;
3)取3mL步骤1中的盐酸多巴胺氢氧化钠水溶液和5mL步骤2中葡聚糖溶液以及2mL的1.2M的氢氧化钠溶液混合在一起,室温搅拌均匀。
4)向步骤(3)中所制备的混合溶液中加入1.0mL交联剂EGDE,搅拌均匀后置于37℃恒温箱中,交联12h形成凝胶,记为D3。
实施例3:葡聚糖/聚多巴胺多孔水凝胶细胞支架的制备
其制备方法步骤如下:
1)盐酸多巴胺氢氧化钠水溶液是通过将30g盐酸多巴胺固体溶于1L的1.2M氢氧化钠水溶液中,在空气条件下(25℃,磁力搅拌24h)进行氧化反应来制备;
2)使用氢氧化钠溶液(1.2M)溶解葡聚糖,制备浓度为15%(w/v)的葡聚糖溶液;
3)取5mL步骤1中的盐酸多巴胺氢氧化钠水溶液和5mL步骤2中葡聚糖溶液混合在一起,室温搅拌均匀。
4)向步骤(3)中所制备的混合溶液中加入1.0mL交联剂EGDE,搅拌均匀后置于37℃恒温箱中,交联12h形成凝胶,记为D4。
实施例4:葡聚糖/聚多巴胺水凝胶支架的力学性能测试
其评估步骤如下:
设置对照组,使用氢氧化钠溶液(1.2M)溶解葡聚糖,制备浓度为15%(w/v)的葡聚糖溶液,取5mL葡聚糖溶液和5mL的1.2M的氢氧化钠溶液混合在一起,加入1.0mL交联剂EGDE,搅拌均匀后置于37℃恒温箱中,交联12h形成凝胶,记为D1。
通过使用流变仪对四组D1、D2、D3、D4不同多巴胺掺杂量的凝胶进行力学性能测试发现,随着聚多巴胺掺杂量的增加,复合凝胶的储能模量显著增加,由780Pa增加到1600Pa,其结果如图1所示。扫描电镜测试复合凝胶的表面形貌发现每种凝胶孔径均一,且孔径大小能够通过聚多巴胺掺杂量调节,孔径在5~30微米之间,如图2所示,随着聚多巴胺掺杂量的增加,孔径逐渐变小,对照组的凝胶孔隙最大,并且整体结构疏松,D2的孔径直径在15~25微米之间;D3的孔隙直径10-20微米之间;D4的孔隙直径在3-10微米之间,使其能够应用于不同细胞种类的支架,模拟不同细胞环境,具有更多样的应用。
实施例5:葡聚糖/聚多巴胺多孔水凝胶细胞支架与小鼠成纤维细胞共培养
其评估步骤如下:
设置对照组,使用氢氧化钠溶液(1.2M)溶解葡聚糖,制备浓度为15%(w/v)的葡聚糖溶液,取5mL葡聚糖溶液和5mL的1.2M的氢氧化钠溶液混合在一起,加入1.0mL交联剂EGDE,搅拌均匀后置于37℃恒温箱中,交联12h形成凝胶,记为D1。
取D1、D4的水凝胶,分成32份,每份干重约为5~7mg,放入孔板中75%酒精消毒12h,后用磷酸缓冲盐溶液(PBS)浸泡冲洗酒精1~2天,每天换液2~3次。
在共聚焦碟子中接种一定量的小鼠成纤维细胞(NIH3T3),使用DMEM培养基,在5%CO2气体条件下37℃中孵育24h,之后加入D1、D4水凝胶样品,在成纤维细胞与水凝胶样品共培养1天、3天、5天后分别对共聚焦碟子中的成纤维细胞状态通过细胞活/死染色进行评价。实验中的细胞每两天换一次培养基。
图3结果显示,细胞伪足在纤维表明粘附延伸,细胞呈三角型、多角形以及梭型等多种形态。这意味着本发明者构筑的水凝胶多孔支架适合细胞粘附增殖和生长。相比于单纯的葡聚糖水凝胶(D1),多巴胺掺杂的水凝胶(D4)在培养5天后细胞更加密集,说明多巴胺的掺杂可以有效促进细胞的粘附和增殖,该水凝胶支架为细胞的3D培养提供了一种新的可能。
实施例6:聚多巴胺掺杂的葡聚糖水凝胶支架用于伤口修复
其评估步骤如下:
设置对照组,使用氢氧化钠溶液(1.2M)溶解葡聚糖,制备浓度为15%(w/v)的葡聚糖溶液,取5mL葡聚糖溶液和5mL的1.2M的氢氧化钠溶液混合在一起,加入1.0mL交联剂EGDE,搅拌均匀后置于37℃恒温箱中,交联12h形成凝胶,记为D1。
将4月龄的SD大鼠随机分为3组,用戊巴比妥麻醉后,在鼠背部中间线皮肤处构建一个直径为8mm的圆形伤口。实验组在鼠背部中间贴上8mm大小的D4水凝胶薄片,空白对照组大鼠在伤口处贴上8mm大小的D1水凝胶薄片。术后观察时间点为0、7、14、21天,在对伤口愈合情况进行拍照记录后,采用戊巴比妥麻醉,对大鼠实施安乐死。
图4结果显示,相比于对照组(D1),多巴胺掺杂的水凝胶组(D4)在培养21天后能有效地促进和加速大鼠的伤口愈合。本发明中所构筑的多巴胺掺水凝胶为临床医用伤口敷料提供了一种新的可能。且该水凝胶支架可以进一步的装载一些生长因子和抗菌药物等,使得这种材料具有修复皮肤伤口的潜在应用价值。
最后有必要在此说明的是:以上实施例只用于对本发明的技术方案作进一步详细地说明,不能理解为对本发明保护范围的限制,本领域的技术人员根据本发明的上述内容作出的一些非本质的改进和调整均属于本发明的保护范围。
Claims (7)
1.一种聚多巴胺掺杂葡聚糖水凝胶多孔支架的制备方法,其特征在于:将盐酸多巴胺固体溶于1.2M的氢氧化钠水溶液中,在常温条件下搅拌进行氧化反应制备含30mg/mL的盐酸多巴胺氢氧化钠水溶液,将葡聚糖溶于1.2M的氢氧化钠水溶液中,在常温条件下搅拌进行氧化反应制备含15%(w/v)葡聚糖溶液,将盐酸多巴胺氢氧化钠水溶液和葡聚糖溶液混合搅拌,加入与混合液体积比为1∶10的交联剂,所述盐酸多巴胺氢氧化钠水溶液和葡聚糖溶液体积比为1-5∶5。
2.一种聚多巴胺掺杂葡聚糖水凝胶多孔支架的制备方法,其特征在于:所述交联剂为乙二醇缩水甘油醚。
3.一种聚多巴胺掺杂葡聚糖水凝胶多孔支架,其特征在于,所述水凝胶多孔支架为根据权利要求1或2所述的一种聚多巴胺掺杂葡聚糖水凝胶多孔支架的制备方法制备的凝胶。
4.一种聚多巴胺掺杂葡聚糖水凝胶多孔支架的应用,其特征在于,根据权利要求3所述的一种聚多巴胺掺杂葡聚糖水凝胶多孔支架用于制备细胞支架的应用。
5.根据权利要求4所述的一种聚多巴胺掺杂葡聚糖水凝胶多孔支架的应用,其特征在于,所述细胞支架的孔隙为5~30μm。
6.一种聚多巴胺掺杂葡聚糖水凝胶多孔支架的应用,其特征在于,根据权利要求3所述的一种聚多巴胺掺杂葡聚糖水凝胶多孔支架用于制备皮肤修复凝胶的应用。
7.根据权利要求6所述一种聚多巴胺掺杂葡聚糖水凝胶多孔支架的应用,其特征在于,所述皮肤修复凝胶负载有细胞生长因子、抗菌剂、保湿剂、防腐剂、抗氧剂、乳化剂、增稠剂、甜味剂、胶浆剂、芳香剂以及矫味剂中的一种或多种。
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