CN111388753A - 负载Cu-MOFs的多孔纤维支架材料及其制备方法和用途 - Google Patents

负载Cu-MOFs的多孔纤维支架材料及其制备方法和用途 Download PDF

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CN111388753A
CN111388753A CN202010101404.5A CN202010101404A CN111388753A CN 111388753 A CN111388753 A CN 111388753A CN 202010101404 A CN202010101404 A CN 202010101404A CN 111388753 A CN111388753 A CN 111388753A
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王淑芳
王志红
张祥云
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Nankai University
Institute of Biomedical Engineering of CAMS and PUMC
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Abstract

本发明公开了一种负载Cu‑MOFs的多孔纤维支架材料及其制备方法和用途，是采用静电纺丝技术制备的含Cu‑MOFs纳米粒子的生物可降解高分子多孔纤维支架材料，可以用于人工血管构建。采用静电纺丝技术制备的生物可降解高分子纤维对Cu‑MOFs起到很好的支持和包裹作用，减缓Cu‑MOFs的降解，有效提高Cu‑MOFs的催化寿命，同时静电纺丝制备的支架材料纤维形貌和力学性能良好，更加符合细胞外基质环境，有利于细胞的粘附，迁移和增殖。实现Cu‑MOFs的长期催化性能，可以稳定可控催化生成NO。作为小口径人工血管移植材料，可以提高其血液相容性以及加速材料的内皮化，促进血管组织再生。

Description

负载Cu-MOFs的多孔纤维支架材料及其制备方法和用途
技术领域：
本发明所属技术领域为生物材料与组织工程，具体说是负载生物活性物质的生物材料及其制备方法和用途。
技术背景
金属有机框架结构(MOF)的多孔性和高比表面积的特点使其具有优良的催化活性和催化使用寿命。人体血液中存在S-亚硝基硫醇等一氧化氮供体，它们可以被特定的催化剂加速分解并释放一氧化氮(NO)。本发明以铜离子为基础的金属有机框架结构(Cu-MOFs)作为催化剂催化内源性一氧化氮供体分解产生一氧化氮(NO)，其中一氧化氮在防止急性血栓、抑制内膜增生、促进内皮细胞生长，从而维持血管长期畅通等方面具有重要作用，因此，Cu-MOFs可以作为NO催化剂应用于心血管材料以及其他抗凝血材料的制备。然而，Cu-MOFs在蛋白质溶液中不能稳定存在导致其催化寿命缩短，同时如果存在突释现象的话，高浓度铜离子会对机体产生毒性。因此，针对Cu-MOFs在蛋白质溶液中容易降解的问题，本发明采用静电纺丝的方法将Cu-MOFs负载并包裹到聚己内酯(PCL)纤维中，提高Cu-MOFs的负载稳定性，实现长期稳定催化生成NO的目的。
本发明的技术内容
本发明所述支架是采用静电纺丝技术制备负载Cu-MOFs纳米粒子的生物可降解高分子多孔纤维支架材料；所述生物可降解高分子包括聚己内酯(PCL)，聚乳酸(PLA)，聚丙交酯-乙交酯共聚物(PLGA)，聚羟基脂肪酸酯(PHA)，聚己内酯-丙交酯共聚物(PLCL)中的一种或多种生物可降解高分子组成的混合物。其具体制备方法包括以下步骤：
1)配置静电纺丝溶液：将生物可降解高分子聚合物溶于氯仿/甲醇混合溶剂，其中氯仿与甲醇的比例为5∶1，或者溶于六氟异丙醇溶剂，质量体积浓度为10％，室温搅拌约6小时，充分混匀；取溶于乙醇中的浓度为10mg/mL的Cu-MOFs溶液进行离心，去除上清乙醇，用无水甲醇将Cu-MOFs重悬，将重悬的Cu-MOFs溶液加入到充分溶解的生物可降解高分子溶液中，Cu-MOFs的用量是生物可降解高分子用量的0.125％；继续搅拌1h，获得充分溶解的生物可降解高分子和Cu-MOFs的混合溶液；
2)静电纺丝，条件为：混合纺丝溶液的流速2mL/h，电压为18kV或21kV，接收距离为15cm，收集时间为2h或0.5h；真空干燥箱中干燥48小时，即得到负载Cu-MOFs的多孔纤维支架膜材料或管材料。
负载Cu-MOFs的多孔纤维支架材料，可以用于与血液接触的抗凝血材料，特别优选用于人工血管支架材料。
本发明与现有技术相比突出优点在于：
1)材料选取上，生物可降解高分子拥有较好的生物相容性和力学性能。Cu-MOFs催化剂应用领域广阔，与无机铜离子催化剂相比，因其介孔表面积大使其具有更优良的催化性能。
2)制备工艺上，与传统的通过直接生长晶体，电化学，层层自组装等将Cu-MOFs以表面固定方式固定到材料表面相比，利用静电纺丝技术可以有效解决Cu-MOFs和基质材料相互作用弱，容易分离而产生毒性、催化寿命缩短等问题。本研究采用生物可降解高分子和Cu-MOFs混合静电纺丝的方式制备负载Cu-MOFs的生物可降解高分子纤维支架，其中生物可降解高分子纤维对Cu-MOFs起到很好的支持和包裹作用，减缓Cu-MOFs的降解，有效提高Cu-MOFs的催化寿命，同时静电纺丝制备的支架材料纤维形貌和力学性能良好，更加符合细胞外基质环境，有利于细胞的粘附，迁移和增殖。
3)功能上，静电纺丝制备的负载Cu-MOFs的生物可降解高分子纤维支架，有效减缓了Cu-MOFs的降解，实现稳定可控的NO释放和Cu-MOFs的长期催化性能。作为小口径人工血管移植材料，提高血液相容性以及加速材料的内皮化，促进血管组织再生。
具体实施方式
实施例1：
1)配置静电纺丝溶液：量取三氯甲烷和甲醇混合液2mL(混合液体积比5∶1)，称取0.2g相对分子质量为80000的PCL放入玻璃瓶中，在磁力搅拌器上搅拌过夜，得到浓度为10％的充分溶解均一的PCL纺丝溶液；吸取10mg/mL的Cu-MOFs溶液25μL，1500rpm离心5min，弃上清，用200μL甲醇重悬后，加入到充分溶解的PCL溶液中，继续搅拌1h，充分溶解。将上述溶液加入到直径为14.9mm的微量注射器中，将高压直流电源与微量注射器的针头相连，用铝箔包裹在圆柱形接收器上，将接收器与地线相连，微量注射器正对接收器的中央；2)静电纺丝，条件为：微量注射器中的溶液流速2mL/h，电压18kV，收集距离15cm，纺丝时间为2h；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48小时，即为负载Cu-MOFs的多孔纤维支架膜材料。
注：本发明所采用的Cu-MOF配体为1，3，5苯三甲酸(H3BTC)。
实施例2：
1)配置静电纺丝溶液：量取三氯甲烷和甲醇混合液2mL(混合液体积比5∶1)，称取0.2g相对分子质量为80000的PCL放入玻璃瓶中，在磁力搅拌器上搅拌过夜，得到浓度为10％的充分溶解均一的PCL纺丝溶液；吸取10mg/mL的Cu-MOFs溶液25μL，1500rpm离心5min，弃上清，用200μL甲醇重悬后，加入到充分溶解的PCL溶液中，继续搅拌1h，充分溶解。将上述溶液加入到直径为14.9mm的微量注射器中，将高压直流电源与微量注射器的针头相连，将直径为2mm的不锈钢棒安装到接收器上，将接收器与地线相连，微量注射器正对接收器的中央；
2)静电纺丝的条件为：微量注射器中的溶液流速2mL/h，电压21kV，收集距离15cm，纺丝时间为0.5h；得到管状纤维材料，将得到的纤维管置于真空干燥箱中干燥48小时，即为负载Cu-MOFs的多孔纤维人工血管支架材料。
实施例3：
1)配置静电纺丝溶液：量取六氟异丙醇溶液2mL，称取0.2g PLCL(分子量80000)放入玻璃瓶中，在磁力搅拌器上搅拌过夜，得到浓度为10％的充分溶解均一的PLCL纺丝溶液；吸取10mg/mL的Cu-MOFs溶液25μL，1500rpm离心5min，弃上清，用200μL甲醇重悬后，加入到充分溶解的PLCL溶液中，继续搅拌1h，充分溶解。将上述溶液加入到直径为14.9mm的微量注射器中，将高压直流电源与微量注射器的针头相连，用铝箔包裹在圆柱形接收器上，将接收器与地线相连，微量注射器正对接收器的中央；
2)静电纺丝，条件为：微量注射器中的溶液流速2mL/h，电压18kV，收集距离15cm，纺丝时间为2h；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48小时，即得负载Cu-MOFs的多孔纤维支架膜材料。
实施例4：
称取10mg的血管支架材料(PCL和PCL-0.125％MOF)放入到10mL的工作液中(100μMGSNO和100μM GSH)，37℃，振荡条件下避光反应1，2，3，4，5和6h。反应结束后，用NO检测试剂盒检测NO生成量，具体操作为：将待测溶液取出50μL加入96孔板中，再依次加入50μLGriess试剂I和Griess试剂II，静置15min后在酶标仪上读取540nm处的吸光值，根据标准曲线计算NO生成量。更换工作液，重复三次，测试其催化稳定性。结果如表1显示，负载Cu-MOFs的人工血管有效地加速了GSNO的分解，更换工作液后，PCL-0.125％MOF人工血管仍然具有加速GSNO分解产生NO的作用。
实施例5：
取SD大鼠的新鲜血清，将制备好的10mg人工血管支架材料(PCL和PCL-0.125％MOF)置于500μL新鲜血清中，1，2和3h，37℃，避光条件下轻轻震荡。在预定的时间点，采用NO检测试剂盒(氧化还原酶法)进行测定，在550nm读取吸收值。蒸馏水为空白对照，100μmol/LNaNO2为标准品，根据公式计算得到血清中NO的浓度。结果显示PCL-0.125％MOF人工血管明显加速了血清中NO供体的分解。
实施例6：
将长度为1cm的PCL和PCL-0.125％MOF人工血管支架材料及连接管在肝素钠溶液中浸泡，再用经抗凝处理的塑料管将血管支架连成并联管路使用。剪开SD大鼠腹部皮肤，剥离腹主动脉和下腔静脉，使用留置针分别插入两根血管中，将管路的两头分别连接到两根留置针上，打开留置针的开关使血液持续畅通。循环2小时后，考察材料的抗凝血性能。结果如表1显示，PCL-0.125％MOF人工血管管腔内具有更低质量的血栓形成以及更少的血小板黏附，证明PCL-0.125％MOF人工血管具有很好的抗凝血的功能。
实施例7：
将负载0.125％Cu-MOFs的10％PCL的人工血管原位移植到大鼠的腹主动脉，进行动物实验评价。将未负载Cu-MOFs的10％PCL的人工血管作为空白对照，利用大鼠腹主动脉人工血管替代模型，将负载Cu-MOFs的人工血管移植到大鼠腹主动脉，2周和4周后考察材料的通畅性，并取材做冰冻切片，用免疫荧光染色的方法和en-face染色，检测内皮化的情况。
结果：人工血管腹主动脉移植模型评价结果发现，将负载Cu-MOFs的人工血管，利用取材后组织切片进行CD31染色，观察内皮细胞在材料表面的覆盖率。如表1所示，植入4周后，通过免疫荧光染色发现未负载Cu-MOFs人工血管表面内皮覆盖率为56.72％，而负载Cu-MOFs的人工血管表面内皮细胞的覆盖率达到82.67％，通过en-face染色发现实验组内皮沿血流方向排布，证明我们负载Cu-MOFs的人工血管通过催化血液中NO供体的分解产生NO起到了促进内皮化的作用。
表1.PCL和PCL-0.125％MOF材料性质比较
Figure BSA0000202030480000051

Claims (4)

1.一种负载Cu-MOFs的多孔纤维支架材料，其特征在于是由Cu-MOFs纳米粒子和生物可降解高分子经静电纺丝工艺制成的复合纤维多孔支架材料。
2.根据权利要求1所述的负载Cu-MOFs的多孔纤维支架材料的制备方法，其特征在于采用静电纺丝技术经以下步骤制备而成：
1)配置静电纺丝溶液：将生物可降解高分子聚合物溶于氯仿/甲醇混合溶剂，其中氯仿与甲醇的比例为5∶1，或者溶于六氟异丙醇溶剂，质量体积浓度为10％，室温搅拌约6小时，充分混匀；取溶于乙醇中的浓度为10mg/mL的Cu-MOFs溶液进行离心，去除上清乙醇，用无水甲醇将Cu-MOFs重悬，将重悬的Cu-MOFs溶液加入到充分溶解的生物可降解高分子溶液中，Cu-MOFs的用量是生物可降解高分子用量的0.125％；继续搅拌1h，获得充分溶解的生物可降解高分子和Cu-MOFs的混合溶液；
2)静电纺丝，条件为：混合纺丝溶液的流速2mL/h，电压为18kV或21kV，接收距离为15cm，收集时间为2h或0.5h；真空干燥箱中干燥48小时，即得到负载Cu-MOFs的多孔纤维支架膜材料或管材料。
3.负载Cu-MOFs的多孔纤维支架材料的用途，用于与血液接触的抗凝血材料，特别优选用于人工血管支架材料构建。
4.权利要求1所述的生物可降解高分子包括聚己内酯(PCL)，聚乳酸(PLA)，聚丙交酯-乙交酯共聚物(PLGA)，聚羟基脂肪酸酯(PHA)，聚己内酯-丙交酯共聚物(PLCL)，及两种及两种以上生物可降解高分子组成的混合物。
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