CN111643731A - 全氟十四酸修饰的多孔支架材料及其制备方法 - Google Patents

全氟十四酸修饰的多孔支架材料及其制备方法 Download PDF

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CN111643731A
CN111643731A CN201910166946.8A CN201910166946A CN111643731A CN 111643731 A CN111643731 A CN 111643731A CN 201910166946 A CN201910166946 A CN 201910166946A CN 111643731 A CN111643731 A CN 111643731A
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王淑芳
史杰
沙成花
王恺
吴静
孙玥
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Nankai University
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Abstract

本发明公开了一种全氟十四酸修饰的多孔纤维支架材料及其制备方法，其特点在于利用双喷头静电对纺技术制备聚己内酯和明胶复合纤维多孔材料，通过EDC/NHS反应将全氟十四酸负载到材料表面，形成负载全氟十四酸的多孔纤维支架材料。所构建的多孔支架材料具有良好的力学性能、生物相容性和生物可降解性，全氟十四酸在低氧条件下可以释放氧气，提高组织工程支架材料内部氧含量，有利于细胞在材料上的黏附、迁移和增殖，进而有利于组织再生。

Description

全氟十四酸修饰的多孔支架材料及其制备方法
技术领域
本发明属于组织工程与再生医学领域，具体说是可以提高氧含量以利于组织再生的负载全氟十四酸的聚己内酯-明胶复合纤维多孔支架材料及其制备方法。
背景技术
组织再生中缺氧现象的存在是影响再生性能的重要原因。氧气在组织再生中具有重要作用，支架材料中心部位缺氧影响细胞增殖和分化。同正常氧(21％O2)环境下的细胞相比，在缺氧环境中生长的细胞产生更多的自由基，这些自由基需要15倍以上的葡萄糖才能产生相同数量的三磷酸腺苷(ATP)。因而缺氧细胞会由于脱氧核糖核酸(DNA)损伤，细胞周期停滞和葡萄糖快速消耗而死亡。全氟十四酸(PFTA)是一种很好的氧气载体，具有低极化率，可与非极性气体混溶(O2，CO，CO2和NO)。PFTA乳液中氧分压与氧溶解度呈线性关系，因而在低氧条件下PFTA可以迅速提高环境中氧气，进而可以解决材料内部缺氧问题。
为了解决材料内部缺氧问题，本发明采用双喷头静电对纺技术制备聚己内酯(PCL)和明胶复合纤维多孔支架，通过EDC/NHS反应将全氟十四酸(PFTA)负载到多孔纤维材料表面，该发明为构建可募集氧气的血管支架提供技术支持。
发明内容
本发明是利用双喷头静电对纺技术制备聚己内酯和明胶复合纤维多孔材料，通过EDC/NHS反应将全氟十四酸负载到材料表面，形成全氟十四酸修饰的多孔纤维支架材料；具体制备方法包括以下步骤：
1)配制静电纺丝溶液：将聚己内酯溶于氯仿/甲醇混合溶剂，其中氯仿与甲醇的体积比为5∶1，溶液在磁力搅拌机上搅拌过夜，得到质量/体积浓度为25％聚己内酯纺丝溶液；明胶溶于六氟异丙醇，溶液在磁力搅拌器上搅拌4h，得到质量/体积浓度为6％明胶纺丝溶液；
2)双喷头静电对纺的条件为：PCL流速1-8mL/h，电压为10-16kV，接收距离为15cm，收集时间为20min；明胶流速4mL/h，电压18kV，接受距离10cm，收集时间20min；得到膜状纤维材料，将得到的纤维膜置于真空干燥箱中干燥；
3)PFTA溶液配制：配制30mM N-(3-二甲基氨丙基)-N’-乙基碳二亚胺盐酸盐(EDC)，7.5mM N-羟基琥珀酰亚胺(NHS)反应液；以75％乙醇和100％乙醇两种溶剂溶解EDC和NHS，之后称取的PFTA溶解于EDC/NHS混合溶液中，使其充分溶解；
4)PFTA负载：将制备好纤维膜放入含100μg/ml的PFTA溶液中，然后将多孔纤维膜置于4℃冰箱反应12h，反应结束后，用蒸馏水洗涤膜，每10分钟换一次水，重复10次，最后将其置于摇床上蒸馏水洗6h，充分除去未反应的物质，中间每隔2小时换一次水；洗涤结束后，将多孔膜材料放在滤纸上自然晾干；
5)载氧能力检测：将PFTA负载前后的纤维膜分别放置在两个15ml离心管中，添加5mL生理盐水，并向离心管中通5min氧气；随后用血气分析仪检测溶液中氧气的含量；因为PFTA与氧气可通过范德华力结合，因此负载PFTA纤维膜的溶液中氧分压远远高于未负载PFTA的纤维膜的溶液。
本发明与现有技术相比突出优点在于
1)材料选取上，脂肪族聚酯材料拥有较好的生物相容性和力学性能，明胶亲水，容易降解。
2)制备工艺上，利用双喷头静电对纺技术制备纤维材料。静电纺丝工艺可制备多种不同材料，纺丝条件相对温和，参数易控制。由静电纺丝制备的材料，更加接近细胞外基质环境，有利于细胞的黏附、迁移与增殖。
3)功能上，静电纺丝制备的纤维材料纤维形貌和力学性能良好，粗丝大孔有利于细胞浸润，明胶改善了材料的亲水性，提高了材料的生物相容性，全氟十四酸在低氧条件下可以提高材料内部氧含量，进而促进材料上细胞增殖和黏附，有利于组织再生。
具体实施例
实施例1：
1)配制静电纺丝溶液：量取15mL三氯甲烷和3mL甲醇(混合液体积比5∶1)，接着称取4.5g聚己内酯放入玻璃瓶中，溶液在磁力搅拌机上搅拌过夜，得到质量/体积浓度为25％聚己内酯纺丝溶液；量取20ml六氟异丙醇，称取1.2g明胶，溶液在磁力搅拌器上搅拌4h，得到质量/体积浓度为6％明胶纺丝溶液；
2)双喷头静电对纺的条件为：聚己内酯流速8mL/h，电压16kV，收集距离15cm，纺丝时间为20min；明胶流速4mL/h，电压15kV，收集距离10cm；得到膜状纤维多孔材料，将得到的多孔纤维膜材料置于真空干燥箱中干燥；
3)配制PFTA溶液：以100mL75％乙醇为溶剂配制30mM EDC，7.5mM NHS溶液，之后称取10mg PFTA溶解于EDC/NHS混合溶液中；
4)负载PFTA：将制备好的多孔纤维膜材料放入含100μg/ml的PFTA溶液中，然后再将膜材料置于4℃冰箱反应12h；反应结束后，用蒸馏水洗涤膜，每10min换一次水，重复10次，最后将其置于摇床上蒸馏水洗6h，充分除去未反应的物质，中间每隔2h换一次水；洗涤结束后，将膜放在滤纸上自然晾干；
5)载氧能力检测：将PFTA负载前后的纤维膜分别放置在两个15ml离心管中，添加5mL生理盐水，并向离心管中通5min氧气；随后用血气分析仪检测溶液中氧气的含量；因为PFTA与氧气可通过范德华力结合，因此负载PFTA纤维膜的溶液中氧分压远远高于未负载PFTA的纤维膜的溶液。
实施例2：
1)配制静电纺丝溶液：量取15mL三氯甲烷和3mL甲醇(混合液体积比5∶1)，接着称取4.5g聚己内酯放入玻璃瓶中，溶液在磁力搅拌机上搅拌过夜，得到质量/体积浓度为25％聚己内酯纺丝溶液；量取20ml六氟异丙醇，称取1.2g明胶，溶液在磁力搅拌器上搅拌4h，得到质量/体积浓度为6％明胶纺丝溶液；
2)双喷头静电对纺的条件为为：聚己内酯流速4mL/h，电压12kV，收集距离15cm，纺丝时间为20min；明胶流速4mL/h，电压15kV，收集距离10cm得到膜状纤维材料，将得到的纤维膜置于真空干燥箱中干燥；
3)配制PFTA溶液：以100mL75％乙醇为溶剂配制30mM EDC，7.5mM NHS溶液；之后称取10mg PFTA溶解于EDC/NHS混合溶液中；
4)负载PFTA：将制备好纤维膜放入含100μg/ml的PFTA溶液中；然后将膜置于4℃冰箱反应12h；反应结束后，用蒸馏水洗涤膜，每10min换一次水，重复10次，最后将其置于摇床上蒸馏水洗6h，充分除去未反应的物质，中间每隔2h换一次水，洗涤结束后，将膜放在滤纸上自然晾干；
5)载氧能力检测：将PFTA负载前后的纤维膜分别放置在两个15ml离心管中，添加5mL生理盐水，并向离心管中通5min氧气；随后用血气分析仪检测溶液中氧气的含量；因为PFTA与氧气可通过范德华力结合，因此负载PFTA纤维膜的溶液中氧分压远远高于未负载PFTA的纤维膜的溶液。
实施例3：
1)配制静电纺丝溶液：量取15mL三氯甲烷和3mL甲醇(混合液体积比5∶1)，接着称取4.5g聚己内酯放入玻璃瓶中，溶液在磁力搅拌机上搅拌过夜，得到质量/体积浓度为25％聚己内酯纺丝溶液；量取20ml六氟异丙醇，称取1.2g明胶，溶液在磁力搅拌器上搅拌4h，得到质量/体积浓度为6％明胶纺丝溶液；
2)双喷头静电对纺的条件为：聚己内酯流速8mL/h，电压16kV，收集距离15cm，纺丝时间为20min；明胶流速4mL/h，电压15kV，收集距离10cm得到膜状纤维材料，将得到的纤维膜置于真空干燥箱中干燥；
3)配制PFTA溶液：以100mL100％乙醇为溶剂配制30mM EDC，7.5mM NHS溶液；之后称取10mg PFTA溶解于EDC/NHS混合溶液中；
4)PFTA负载：将制备好纤维膜放入含100μg/ml的PFTA溶液中，然后将膜置于4℃冰箱反应12h；反应结束后，用蒸馏水洗涤膜，每10min换一次水，重复10次，最后将其置于摇床上蒸馏水洗6h，充分除去未反应的物质，中间每隔2h换一次水，洗涤结束后，将膜放在滤纸上自然晾干；
5)载氧能力检测：将PFTA负载前后的纤维膜分别放置在两个15ml离心管中，添加5mL生理盐水，并向离心管中通5min氧气；随后用血气分析仪检测溶液中氧气的含量；因为PFTA与氧气可通过范德华力结合，因此负载PFTA纤维膜的溶液中氧分压远远高于未负载PFTA的纤维膜的溶液。
实施例4：
1)配制静电纺丝溶液：量取15mL三氯甲烷和3mL甲醇(混合液体积比5∶1)，接着称取4.5g聚己内酯放入玻璃瓶中，溶液在磁力搅拌机上搅拌过夜，得到质量/体积浓度为25％聚己内酯纺丝溶液；量取20ml六氟异丙醇，称取1.2g明胶，溶液在磁力搅拌器上搅拌4h，得到质量/体积浓度为6％明胶纺丝溶液；
2)双喷头静电对纺的条件为：聚己内酯流速4mL/h，电压12kV，收集距离15cm，纺丝时间为20min；明胶流速4mL/h，电压15kV，收集距离10cm得到膜状纤维材料，将得到的纤维膜置于真空干燥箱中干燥；
3)配制PFTA溶液：以100mL，100％乙醇为溶剂配制30mM EDC，7.5mM NHS溶液。之后称取10mg PFTA溶解于EDC/NHS混合溶液中；
4)PFTA负载：将制备好纤维膜放入含100μg/ml的PFTA溶液中；然后将膜置于4℃冰箱反应12h；反应结束后，用蒸馏水洗涤膜，每10min换一次水，重复10次，最后将其置于摇床上蒸馏水洗6h，充分除去未反应的物质，中间每隔2h换一次水，洗涤结束后，将膜放在滤纸上自然晾干。
5)载氧能力检测：将PFTA负载前后的纤维膜分别放置在两个15ml离心管中，添加5mL生理盐水，并向离心管中通5min氧气；随后用血气分析仪检测溶液中氧气的含量；因为PFTA与氧气可通过范德华力结合，因此负载PFTA纤维膜的溶液中氧分压远远高于未负载PFTA的纤维膜的溶液。
实施例5：
使用模具将纤维膜制成直径为1cm的圆片，放入48孔板中，紫外照射过夜灭菌。将人脐静脉内皮细胞(HUVEC)和人平滑肌细胞(SMC)分别接种于预先灭好菌的膜上，HUVEC和SMC接种数量均为1*104个/孔，然后将48孔放置于低氧(2％O2)培养箱中培养24小时。弃掉培养基，用PBS洗涤2次，加入2.5％戊二醛4℃过夜固定，第二天利用梯度酒精脱水，真空干燥后，用SEM观察细胞状态。结果发现内皮细胞和平滑肌细胞在PFTA修饰后的纤维膜可以完全铺展生长，而在未负载PFTA的膜上，两种细胞呈皱缩状态。这表明在低氧条件下PFTA可以促进血管内皮细胞和平滑肌细胞在材料上铺展，利于细胞功能的实现。
实施例6：
利用Calcein-AM/PI活细胞/死细胞双染试剂盒对细胞在材料上存活情况进行检测。Calcein-AM(引入乙酰甲氧基甲酯的钙黄绿素)仅用于活细胞染色，PI(碘化丙啶)用于死细胞染色。使用模具将纤维膜制成直径为1cm的圆片，放入48孔板中，紫外照射过夜灭菌。将人脐静脉内皮细胞(HUVEC)和人平滑肌细胞(SMC)分别接种于纤维膜上，HUVEC和SMC接种数量均为1*104个/孔，然后将细胞放置于低氧(2％O2)培养箱中培养，在第3天按照说明书步骤对细胞进行染色，用激光共聚焦显微镜观察。结果发现：内皮细胞在PFTA修饰后的纤维膜上生长状态良好；平滑肌细胞在PFTA的纤维膜上生长状态明显优于未负载组。因此PFTA修饰后的纤维膜对内皮细胞具有良好的生物相容性，且其可以促进平滑肌增殖，利于组织重塑。

Claims (2)

1.全氟十四酸修饰的多孔纤维支架材料，是利用双喷头静电对纺技术制备聚己内酯和明胶复合纤维多孔材料，通过EDC/NHS反应将全氟十四酸负载到材料表面所形成的多孔纤维支架材料。
2.全氟十四酸修饰的多孔纤维支架材料的制备方法，具体包括以下步骤：
1)配制静电纺丝溶液：将聚己内酯溶于氯仿/甲醇混合溶剂，其中氯仿与甲醇的体积比为5∶1，溶液在磁力搅拌机上搅拌过夜，得到质量/体积浓度为25％聚己内酯纺丝溶液；明胶溶于六氟异丙醇，溶液在磁力搅拌器上搅拌4h，得到质量/体积浓度为6％明胶纺丝溶液；
2)双喷头静电对纺的条件为：PCL流速1-8mL/h，电压为10-16kV，接收距离为15cm，收集时间为20min；明胶流速4mL/h，电压18kV，接受距离10cm，收集时间20min；得到膜状纤维材料，将得到的纤维膜置于真空干燥箱中干燥；
3)PFTA溶液配制：配制30mM N-(3-二甲基氨丙基)-N’-乙基碳二亚胺盐酸盐(EDC)，7.5mM N-羟基琥珀酰亚胺(NHS)反应液；以75％乙醇和100％乙醇两种溶剂溶解EDC和NHS，之后称取的PFTA溶解于EDC/NHS混合溶液中，使其充分溶解；
4)PFTA负载：将制备好纤维膜放入含100μg/ml的PFTA溶液中，然后将多孔纤维膜置于4℃冰箱反应12h，反应结束后，用蒸馏水洗涤膜，每10分钟换一次水，重复10次，最后将其置于摇床上蒸馏水洗6h，充分除去未反应的物质，中间每隔2小时换一次水；洗涤结束后，将多孔膜材料放在滤纸上自然晾干；
5)载氧能力检测：将PFTA负载前后的纤维膜分别放置在两个15ml离心管中，添加5mL生理盐水，并向离心管中通5min氧气；随后用血气分析仪检测溶液中氧气的含量；因为PFTA与氧气可通过范德华力结合，因此负载PFTA纤维膜的溶液中氧分压远远高于未负载PFTA的纤维膜的溶液。
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