CN112741930A - 一种酶改性的抗凝血瓣膜及其制备方法 - Google Patents
一种酶改性的抗凝血瓣膜及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种酶改性的抗凝血瓣膜及其制备方法。其制备方法为：(1)将生物瓣膜材料置于多酚化合物与可溶性金属盐的混合溶液中，于20～40℃反应5～60min，取出清洗；(2)以步骤(1)所得产物为基底，重复步骤(1)1～20次后，取出清洗并在室温下干燥；(3)将步骤(2)所得产物置于浓度为0.1～2.5％的戊二醛溶液中活化2～10h后，置于浓度为0.01～10mg/mL，能利用葡萄糖产生过氧化氢的酶溶液中，于1～15℃活化4～12h后，取出洗净即可。本发明利用瓣膜自身特点，修饰多酚‑金属螯合物与葡萄糖氧化酶，简便高效地实现瓣膜体内长效抗凝血的要求，降低瓣膜失效率。
Description
技术领域
本发明属于生物医学工程功能材料技术领域，具体涉及一种酶改性的抗凝血瓣膜及其制备方法。
背景技术
针对瓣膜功能不全或失效的临床现状，开发可用于治疗瓣膜反流性疾病的人工瓣膜极具市场价值。生理状态下的瓣膜较薄，且容易受到瓣叶周围血流压力的影响，目前导致瓣膜失效的主要原因是血栓问题。瓣膜植入体内后会立刻引发一系列凝血反应，如凝血因子与黏着蛋白沉积，纤维蛋白原吸附变性，血小板聚集与激活等，因此对瓣膜进行抗凝修饰对维持瓣膜长期服役能力至关重要。
内皮衍生舒张因子一氧化氮(NO)是内皮细胞分泌的重要的抗凝因子，可维持血管血压、抑制血小板激活与聚集。NO抗凝材料常有两类，即NO供体类与NO催化类，虽然NO的有效释放能抑制材料植入体内后的凝血反应，但此类材料也面临诸多限制，尤其是NO供体本身不稳定，外源性供体前体物质致癌，内源性NO供体在体内浓度低且变化大，反应过程中也会产生毒性物质。生理条件下，NO是由一氧化氮合酶在NADPH和O2(或过氧化氢)存在下催化L-精氨酸产生的，此外当L-精氨酸或过氧化氢浓度受限的情况下，一氧化氮合酶会催化生成NO的单电子还原形式硝酰(HNO)，与NO一样具有抗凝血作用。HNO的来源是体内稳定且含量丰富的L-精氨酸，其反应毒性、稳定性均优于NO。然而一氧化氮合酶作为天然酶，虽然高度专一且高效，但价格高，难以长期储存的问题限制了其产业应用。
发明内容
针对现有技术中的上述不足，本发明提供一种酶改性的抗凝血瓣膜及其制备方法，本申请中多酚-金属螯合物提供类一氧化氮合酶的功能，与稳定交联在瓣膜中的葡萄糖氧化酶组成酶串联系统高效催化生成抗凝血物质硝酰，具有制备工艺简单，成本较低且抗凝效果理想的特点。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
一种酶改性的抗凝血瓣膜的制备方法，包括以下步骤：
(1)将生物瓣膜材料置于多酚化合物与可溶性金属盐的混合溶液中，于20～40℃反应5～60min，取出清洗；
(2)以步骤(1)所得产物为基底，重复步骤(1)1～20次后，取出清洗并在室温下干燥；
(3)将步骤(2)所得产物置于浓度为0.1～2.5％的戊二醛溶液中活化2～10h后，置于浓度为0.01～10mg/mL，能利用葡萄糖产生过氧化氢的酶溶液中，于1～15℃活化4～12h后，取出洗净即可。
进一步地，多酚化合物与可溶性金属盐的质量比为1:0.1～1。
进一步地，多酚化合物与可溶性金属盐的质量比为1:0.1。
进一步地，步骤(1)中反应温度为30℃，反应时间为10min。
进一步地，进一步地，多酚化合物为具有邻酚结构的多酚化合物。
进一步地，具有邻酚结构的多酚化合物为多巴胺、单宁酸、表没食子儿茶素没食子酸酯EGCG、表儿茶素没食子酸酯ECG、表儿茶素EC、表没食子儿茶素EGC、邻苯二酚及邻苯三酚中的至少一种。
进一步地，可溶性金属盐为氯化铁、氯化铜、氯化银、氯化锰或氯化银。
进一步地，酶为葡萄糖氧化酶、柠檬酸修饰的金纳米颗粒、金/三氧化二铝纳米颗粒、金/氧化钛颗粒、含金的双金属/三金属纳米颗粒中的一种。
进一步地，步骤(3)中活化温度为4℃，活化时间为4h。
进一步地，生物瓣膜材料为主动脉瓣、肺动脉瓣、静脉瓣、二尖瓣或三尖瓣。
本发明的有益效果为：
1、天然酶可催化反应在温和条件下高效进行，且对底物有高度专一性，但价格高，易失活不易长期储存的问题限制了它在工业领域的应用。本申请中制备的多酚-金属螯合物既具有类似一氧化氮合酶的催化能力，又相比一氧化氮合酶更稳定，易制备且成本更低。此外，多酚金属螯合物更加稳定，易于制备，适合工业化推广且不会产生单分子易聚集失去活性的问题。
2、将葡萄糖氧化酶以交联的方式与瓣膜结合相比于表面修饰更加稳定及持久地发挥作用。
3、多酚-金属螯合物联合葡萄糖氧化酶可形成酶串联通路，瓣膜中通过戊二醛交联而稳定且大量存在的葡萄糖氧化酶可利用血液中的葡萄糖产生局部高浓度的过氧化氢，随后被多酚金属螯合物利用并催化L-精氨酸产生抗凝血物质HNO，可避免传统NO涂层具有潜在毒性及供体来源不稳定的缺陷，从而高效持久地维持瓣膜的抗凝血性能。本发明利用瓣膜自身特点，修饰多酚-金属螯合物与葡萄糖氧化酶，简便高效地实现瓣膜体内长效抗凝血的要求，降低瓣膜失效率。
附图说明
图1为传统戊二醛瓣膜的半体血液相容性测试结果；
图2为本申请实施例2制备得到的瓣膜材料的半体血液相容性测试结果。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
实施例1
一种酶改性的抗凝血瓣膜的制备方法，包括以下步骤：
(1)将瓣膜放入浓度为1mg/ml的多巴胺与浓度为0.5mg/ml的氯化铁的混合水溶液中，25℃反应20min，取出瓣膜清洗3次，完成一个反应周期；
(2)重复反应周期10次，干燥；
(3)将经步骤(2)处理的材料浸泡在浓度为0.5％的戊二醛溶液中活化4h后洗净；
(4)将步骤(3)所得材料放入浓度为0.15mg/ml的葡萄糖氧化酶的PBS溶液中，4℃条件下活化4h，洗净即得目标材料。
实施例2
一种酶改性的抗凝血瓣膜的制备方法，包括以下步骤：
(1)将瓣膜放入浓度为1mg/ml的单宁酸与浓度为0.1mg/ml的氯化铁的混合水溶液中，30℃反应10min，取出瓣膜清洗3次，完成一个反应周期；
(2)重复反应周期5次，干燥；
(3)将经步骤(2)处理的材料浸泡在浓度为0.15％的戊二醛溶液中活化2h后洗净；
(4)将步骤(3)所得材料放入浓度为0.2mg/ml的葡萄糖氧化酶的PBS溶液中，4℃条件下活化4h，洗净即得目标材料。
实施例3
一种酶改性的抗凝血瓣膜的制备方法，包括以下步骤：
(1)将瓣膜放入浓度为1mg/ml的单宁酸与浓度为0.1mg/ml的氯化铜的混合水溶液中，30℃反应20min，取出瓣膜清洗3次，完成一个反应周期；
(2)重复反应周期5次，干燥；
(3)将经步骤(2)处理的材料浸泡在浓度为0.2％的戊二醛溶液中活化2h后洗净；
(4)将步骤(3)所得材料放入浓度为0.2mg/ml的葡萄糖氧化酶的PBS溶液中，4℃条件下活化4h，洗净即得目标材料。
实施例4
一种酶改性的抗凝血瓣膜的制备方法，包括以下步骤：
(1)将瓣膜放入浓度为1mg/ml的EGCG与浓度为0.1mg/ml的氯化铁的混合水溶液中，30℃反应10min，取出瓣膜清洗3次，完成一个反应周期；
(2)重复反应周期5次，干燥；
(3)将经步骤(2)处理的材料浸泡在浓度为0.1％的戊二醛溶液中活化2h后洗净；
(4)将步骤(3)所得材料放入浓度为0.5mg/ml的葡萄糖氧化酶的PBS溶液中，4℃条件下活化4h，洗净即得目标材料。
实施例5
一种酶改性的抗凝血瓣膜的制备方法，包括以下步骤：
(1)将瓣膜放入浓度为0.8mg/ml的单宁酸与浓度为0.1mg/ml的氯化银的混合水溶液中，25℃反应60min，取出瓣膜清洗3次，完成一个反应周期；
(2)重复反应周期5次，干燥；
(3)将经步骤(2)处理的材料浸泡在浓度为0.15％的戊二醛溶液中活化2h后洗净；
(4)将步骤(3)所得材料放入浓度为0.15mg/ml的葡萄糖氧化酶的PBS溶液中，4℃条件下活化4h，洗净即得目标材料。
实施例6
一种酶改性的抗凝血瓣膜的制备方法，包括以下步骤：
(1)将瓣膜放入浓度为1mg/ml的多巴胺与浓度为0.1mg/ml的氯化银的混合水溶液中，30℃反应20min，取出瓣膜清洗3次，完成一个反应周期；
(2)重复反应周期5次，干燥；
(3)将经步骤(2)处理的材料浸泡在浓度为1％的戊二醛溶液中活化2h后洗净；
(4)将步骤(3)所得材料放入浓度为0.2mg/ml的葡萄糖氧化酶的PBS溶液中，4℃条件下活化4h，洗净即得目标材料。
实验例
以戊二醛瓣膜为对照组，实施例2制备得到的抗凝血瓣膜为实验组，分别检测其半体血液相容性，其结果分别见图1和图2。
在图1中，可明显看到有与瓣膜不相容的血细胞，而图2中则未见与瓣膜不相容的血细胞，可以看出本申请制备得到的瓣膜材料具有优异的生物相容性和抗凝血性能。
Claims (7)
1.一种酶改性的抗凝血瓣膜的制备方法，其特征在于，包括以下步骤：
(1)将生物瓣膜材料置于多酚化合物与可溶性金属盐的混合溶液中，于20～40℃反应5～60min，取出清洗；
(2)以步骤(1)所得产物为基底，重复步骤(1)1～20次后，取出清洗并在室温下干燥；
(3)将步骤(2)所得产物置于浓度为0.1～2.5％的戊二醛溶液中活化2～10h后，置于浓度为0.01～10mg/mL，能利用葡萄糖产生过氧化氢的酶溶液中，于1～15℃活化4～12h后，取出洗净即可。
2.根据权利要求1所述的酶改性的抗凝血瓣膜的制备方法，其特征在于，所述多酚化合物与可溶性金属盐的质量比为1:0.1～1。
3.根据权利要求1或2所述的酶改性的抗凝血瓣膜的制备方法，其特征在于，所述多酚化合物为具有邻酚结构的多酚化合物。
4.根据权利要求3所述的酶改性的抗凝血瓣膜的制备方法，其特征在于，所述具有邻酚结构的多酚化合物为多巴胺、单宁酸、表没食子儿茶素没食子酸酯EGCG、表儿茶素没食子酸酯ECG、表儿茶素EC、表没食子儿茶素EGC、邻苯二酚及邻苯三酚中的至少一种。
5.根据权利要求1或2所述的酶改性的抗凝血瓣膜的制备方法，其特征在于，所述可溶性金属盐为氯化铁、氯化铜、氯化银、氯化锰或氯化银。
6.根据权利要求1所述的酶改性的抗凝血瓣膜的制备方法，其特征在于，所述酶为葡萄糖氧化酶、柠檬酸修饰的金纳米颗粒、金/三氧化二铝纳米颗粒、金/氧化钛颗粒、含金的双金属/三金属纳米颗粒中的一种。
7.根据权利要求1所述的酶改性的抗凝血瓣膜的制备方法，其特征在于，所述生物瓣膜材料为主动脉瓣、肺动脉瓣、静脉瓣、二尖瓣或三尖瓣。
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