CN114177369A - 封堵性防粘连膜材料及其制备方法 - Google Patents
封堵性防粘连膜材料及其制备方法 Download PDFInfo
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- CN114177369A CN114177369A CN202111551984.9A CN202111551984A CN114177369A CN 114177369 A CN114177369 A CN 114177369A CN 202111551984 A CN202111551984 A CN 202111551984A CN 114177369 A CN114177369 A CN 114177369A
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- Prior art keywords
- endothelialization
- membrane
- adhesion
- electrostatic spinning
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- Materials For Medical Uses (AREA)
Abstract
本发明属于生物医学材料技术领域，具体涉及封堵性防粘连膜材料及其制备方法。本发明提供的封堵性防粘连膜材料的制备方法，通过直接涂抹进行结合，直接成型，成型方法简单，选取的材料均为安全无毒的可将降解材料，从而为制备的封堵性防粘连膜材料的安全性提供了保证。内皮细胞在抗凝血、调控细胞增殖和抑制血栓形成方面有重要作用，防粘连材料表面内皮细胞化，可以减少血栓的形成和血小板激活，对材料血液相容性的改善具有显著的影响。本发明提供的封堵性防粘连膜材料，外层的止血凝胶与内层的内皮化静电纺丝防粘连膜降解时间不同，满足前期具有止血效果，后期具有防粘连的效果，满足临床需求。
Description
技术领域
本发明属于生物医学材料技术领域，具体涉及封堵性防粘连膜材料及其制备方法。
背景技术
近些年来，国内外心血管外科迅速发展，心脏手术的例数与日俱增，需要再次心脏手术的比例也明显上升。外科手术中出血是常见的现象，针对出血常用的材料包括止血微球、止血纱布、止血海绵和止血密封剂，它们可以极大减少术中的出血量和术后并发症，从而提升手术效果。其中止血密封剂，其强度高，具有一定的组织粘附性，能通过物理封堵完成止血，不受抗凝药物的影响。
目前的医用止血封闭剂有纤维蛋白胶，α-氰基丙烯酸酯、明胶、壳聚糖类。理想的止血封闭剂必须满足以下条件：与组织相容性好，能被机体代谢或生物降解，无异物残留；有一定的黏性和流动性，能快速黏接或阻止吻合口两端缘的分离；能迅速润湿和填充吻合口的裂隙；常温下，能迅速成膜、固化，形成不溶于水的膜状物，从而有效阻隔。单独的密封剂使用后的异物反应可能会存在粘连，因此需要止血密封剂与防粘连材料复合。但是，密封剂材料在完成止血作用后粘附在防粘连膜上容易丧失防粘连的功能。
发明内容
针对上述现有技术的不足，本发明提供了封堵性防粘连膜材料及其制备方法，目的是为了解决现有技术中出血后，使用单独的密封剂后的异物反应可能会存在粘连，而兼具止血功能的密封剂材料在完成止血作用后粘附在防粘连膜上容易丧失防粘连的功能的技术问题。
本发明提供的封堵性防粘连膜材料的制备方法，具体技术方案如下：
封堵性防粘连膜材料的制备方法，制备静电纺丝膜与内皮化材料结合获得内皮化静电纺丝膜内皮化静电纺丝膜，以及制备止血凝胶溶液，将止血凝胶溶液附着在所述内皮化的静电纺丝膜两侧，获得封堵性防粘连膜材料。
在某些实施方式中，所述内皮化静电纺丝膜具体的制备步骤如下：
步骤1，将高分子材料溶解在有机溶剂混合中，获得纺丝液；所述高分子材料为聚乳酸-羟基乙酸共聚物、聚乳酸、聚乳酸-聚乙二醇嵌段共聚物、聚己内酯-羟基乙酸共聚物和聚己内酯中一种或多种，所述有机溶剂为N,N-二甲基甲酰胺、六氟异丙醇、丙酮、四氢呋喃中的一种或两种；
步骤2，向步骤1中纺丝液中添加内皮化材料，获得内皮化纺丝液；所述内皮化材料为抗体、多肽、生长因子或者他汀类药物，所述抗体为CD34，所述多肽为RGD、YIGSR、REDV、CAG或SVVYGLR，所述生长因子为VEGF、EGF、FGF或PDGF，所述他汀类药物为替米沙坦；
步骤3，将步骤2中内皮化纺丝液进行静电纺丝处理，获得内皮化静电纺丝膜。
优选地，步骤1中，所述高分子材料的分子量为5-50w，所述纺丝液的浓度为20-50％。
优选地，步骤2中，所述静电纺丝的温度为20-30℃，湿度为40-55％，电压为15-40kv，推进速率10-50μL/min。
步骤3中，所述内皮化静电纺丝膜的纤维直径为1-2μm，所述内皮化静电纺丝膜的厚度为70-170μm。
在某些实施方式中，所述内皮化静电纺丝膜具体的制备步骤如下：
步骤A，将高分子材料溶解在有机溶剂混合中，获得纺丝液；所述高分子材料为聚乳酸-羟基乙酸共聚物、聚乳酸、聚乳酸-聚乙二醇嵌段共聚物、聚己内酯-羟基乙酸共聚物和聚己内酯中一种或多种，所述有机溶剂为N,N-二甲基甲酰胺、六氟异丙醇、丙酮、四氢呋喃中的一种或两种；
步骤B，将步骤A中纺丝液进行静电纺丝处理，获得静电纺丝膜；
步骤C，向步骤B中电纺丝膜接枝内皮化材料，获得内皮化静电纺丝膜；所述内皮化材料为多肽，所述多肽为RGD、YIGSR、REDV、CAG或SVVYGLR。
优选地，步骤A中，所述高分子材料的分子量为5-50w，所述纺丝液的浓度为20-50％。
优选地，步骤B中，所述静电纺丝的温度为20-30℃，湿度为40-55％，电压为15-40kv，推进速率10-50μL/min。
优选地，步骤C中，所述接枝内皮化材料的处理包括如下步骤：
(1)将静电纺丝膜表面通过偶联剂或引入活性基团进行表面修饰，获得改性纤维膜，所述活性基团为氨基或羧基；
(2)将步骤(1)中改性纤维膜浸入内皮化材料溶液并静置24h，获得内皮化静电纺丝防粘连膜。
步骤C中，所述内皮化静电纺丝膜的纤维直径为1-2μm，所述内皮化静电纺丝膜的厚度为70-170μm。
在某些实施方式中，所述止血凝胶为聚氨酯或聚乙二醇。
本发明提供了第二个技术方案-封堵性防粘连膜材料，利用上述的方法制备的封堵性防粘连膜材料。
本发明具有以下有益效果：本发明提供的封堵性防粘连膜材料的制备方法，通过直接涂抹进行结合，直接成型，成型方法简单，选取的材料均为安全无毒的可将降解材料，从而为制备的封堵性防粘连膜材料的安全性提供了保证。内皮细胞在抗凝血、调控细胞增殖和抑制血栓形成方面有重要作用，防粘连材料表面内皮细胞化，可以减少血栓的形成和血小板激活，对材料血液相容性的改善具有显著的影响。本发明提供的封堵性防粘连膜材料，外层的止血凝胶与内层的内皮化静电纺丝防粘连膜降解时间不同，满足前期具有止血效果，后期具有防粘连的效果，满足临床需求。
附图说明
图1是本发明提供的封堵性防粘连膜材料的结构示意图；
图2是本发明实施例2的封堵性防粘连膜材料的实物图；
图3是本发明实施例3的封堵性防粘连膜材料的实物图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图1-3，对本发明进一步详细说明。
实施例1
本实施例提供的封堵性防粘连膜材料的制备方法，具体技术方案如下：
(1)静电纺丝法防粘连膜的制备
配制PLGA溶液，PLGA(PLA/PGA＝75/25，分子量60，000)聚合物含量为50％(w/v)，添加5*10-5g/mLVEGF，溶剂为DMF与丙酮的混合溶液(体积比为1/1)。将溶液置于磁力搅拌器上室温搅拌3-5小时，转移进注射器后进行纺丝。纺丝温度25℃左右，湿度控制在50％左右，电压在15-20kv，推进速率20-50μL/min，得到70-170μm厚度的纤维膜，纤维直径1-2μm。室温下，置于真空干燥箱中保存。
(2)止血凝胶的制备
将聚四氢呋喃120℃减压真空除水3h，冷却至40℃，加入3倍摩尔比的左旋赖氨酸二异氰酸酯，升温至75℃反应3h得到A组分。止血凝胶B组分选用1，4-丁二胺。
(3)三层复合结构
将止血凝胶A组分与B组分按照1：1混合涂到防粘连膜两侧得到复合结构。
本实施例还提供了利用上述方法制备的封堵性防粘连膜材料。
实施例2
本实施例提供的封堵性防粘连膜材料的制备方法，具体技术方案如下：
(1)静电纺丝法防粘连膜的制备
配制PLGA和PCL(两者质量比为90/10)溶液，总聚合物含量为30％(w/v)，溶剂为DMF与丙酮的混合溶液(体积比为1/1)。将溶液置于磁力搅拌器上室温搅拌3-5小时，转移进注射器后进行纺丝。纺丝温度20℃左右，湿度控制在40％左右，电压在15-20kv，推进速率20-50μL/min，得到70-170μm厚度的纤维膜，纤维直径1-2μm。室温下，置于真空干燥箱中保存。
将静电纺丝膜浸入KMnO4的H2SO4(1.2N)溶液中进行氧化处理，并依次用HCl和纯水冲洗后干燥，获得氧化纤维膜，预处理纤维膜浸入0.2M EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和0.1M NHS(N-羟基琥珀酰亚胺)混合溶液中活化半小时，反应体系为0.1M MES缓冲液(2-(N-吗啉)-乙磺酸)，然后在纯化水中冲洗，获得预处理纤维膜，纤维膜浸入RGD多肽溶液中37℃反应24h，干燥得内皮化防粘连膜。
(2)止血凝胶的制备
将聚乙二醇二丙烯酸酯PEGDA加入水中，配成浓度为10-50％(w/v)的水溶液，在40℃中搅拌溶解，得到均匀溶液；将光引发剂Irgacure 2959溶解到混合溶液中，光引发剂在水中的浓度为0.5-3％(w/v)，得到均匀溶液。
(3)三层复合结构
将溶液涂到防粘连膜两侧用UV光照射1-10min得到三层复合结构。
本实施例还提供了利用上述方法制备的封堵性防粘连膜材料。如图2所示，本实施例提供的封堵性防粘连膜材料表面止血凝胶材料分布均匀。
实施例3
本实施例提供的封堵性防粘连膜材料的制备方法，具体技术方案如下：
(1)静电纺丝法防粘连膜的制备
配制PLA和PLGA(两者质量比为90/10)溶液，总聚合物含量为20％(w/v)，溶剂为DMF与丙酮的混合溶液(体积比为1/1)。将溶液置于磁力搅拌器上室温搅拌3-5小时，转移进注射器后进行纺丝。纺丝温度30℃左右，湿度控制在55％左右，电压在15-20kv，推进速率20-50μL/min，得到70-170μm厚度的纤维膜，纤维直径1-2μm。室温下，置于真空干燥箱中保存。
将防粘连纤维膜浸入溶解在无水己烷中的10％(v/v)硅烷(APTES)溶液中，在N2气氛下将氨基硅烷链共价结合到表面。反应结束后，无水己烷冲洗3次，真空干燥。
醛肽的合成：REDV四肽(精氨酸-谷氨酸-天冬氨酸-缬氨酸)7-氨基庚酸，苯丙氨酸使用自动合成器和固相法合成。精氨酸的侧链保护使用五甲基-2,3-二氢苯并呋喃-5-磺酰基(Pbf)，天冬氨酸和谷氨酸的侧链保护使用叔丁基(OBut)。
在搅拌下用三氟乙酸(TFA)去除侧链保护1小时，在搅拌下用乙酸、水、甲醇和二氯甲烷(10:5:21:63)的溶液裂解树脂1小时得到醛肽，冻干保存。
将冻干肽以10-5M(原液)的起始浓度溶解在PBS中加入还原剂氰基硼氢化钠(2.19mg NaBH3CN对应1mg肽)。将膜放入肽溶液功能化24小时。随后样品在PBS中洗涤，干燥后得到内皮化防粘连膜。
(2)止血凝胶的制备
四臂聚乙二醇琥珀酰胺活性酯的合成：将0.5g4-arm-PEG-COOH、110mg NHS和184mg EDC溶解在25ml二氯甲烷中，40℃搅拌24h，然后用2M盐酸水溶液、饱和氯化钠水溶液和去离子水洗涤去除单体，用午睡硫酸钠进行干燥，真空干燥得到PEG-NHS。
4-arm-PEG-NHS和4-arm-PEG-NH2分别溶解在PBS中，制备成浓度为15wt％的溶液。
(3)三层复合结构
将两种溶液按照1：1的比例混合涂到防粘连膜两侧得到三层复合结构。
本实施例还提供了利用上述方法制备的封堵性防粘连膜材料。如图3所示，本实施例提供的封堵性防粘连膜材料表面止血凝胶材料分布均匀。
上述仅本发明较佳可行实施例，并非是对本发明的限制，本发明也并不限于上述举例，本技术领域的技术人员，在本发明的实质范围内，所作出的变化、改型、添加或替换，也应属于本发明的保护范围。
Claims (8)
1.封堵性防粘连膜材料的制备方法，其特征在于，制备静电纺丝膜与内皮化材料结合获得内皮化静电纺丝膜，以及制备止血凝胶溶液，将止血凝胶溶液附着在所述内皮化的静电纺丝膜两侧，获得封堵性防粘连膜材料。
2.根据权利要求1所述的封堵性防粘连膜材料的制备方法，其特征在于，所述内皮化静电纺丝膜具体的制备步骤如下：
步骤1，将高分子材料溶解在有机溶剂混合中，获得纺丝液；所述高分子材料为聚乳酸-羟基乙酸共聚物、聚乳酸、聚乳酸-聚乙二醇嵌段共聚物、聚己内酯-羟基乙酸共聚物和聚己内酯中一种或多种，所述有机溶剂为N,N-二甲基甲酰胺、六氟异丙醇、丙酮、四氢呋喃中的一种或两种；
步骤2，向步骤1中纺丝液中添加内皮化材料，获得内皮化纺丝液；所述内皮化材料为抗体、多肽、生长因子或者他汀类药物，所述抗体为CD34，所述多肽为RGD、YIGSR、REDV、CAG或SVVYGLR，所述生长因子为VEGF、EGF、FGF或PDGF，所述他汀类药物为替米沙坦；
步骤3，将步骤2中内皮化纺丝液进行静电纺丝处理，获得内皮化静电纺丝膜。
3.根据权利要求1所述的封堵性防粘连膜材料的制备方法，其特征在于，所述内皮化静电纺丝膜具体的制备步骤如下：
步骤A，将高分子材料溶解在有机溶剂混合中，获得纺丝液；所述高分子材料为聚乳酸-羟基乙酸共聚物、聚乳酸、聚乳酸-聚乙二醇嵌段共聚物、聚己内酯-羟基乙酸共聚物和聚己内酯中一种或多种，所述有机溶剂为N,N-二甲基甲酰胺、六氟异丙醇、丙酮、四氢呋喃中的一种或两种；
步骤B，将步骤A中纺丝液进行静电纺丝处理，获得静电纺丝膜；
步骤C，向步骤B中电纺丝膜接枝内皮化材料，获得内皮化静电纺丝膜；所述内皮化材料为多肽，所述多肽为RGD、YIGSR、REDV、CAG或SVVYGLR。
4.根据权利要求2或3所述的封堵性防粘连膜材料的制备方法，其特征在于，所述高分子材料的分子量为5-50w，所述纺丝液的浓度为20-50％。
5.根据权利要求2或3所述的封堵性防粘连膜材料的制备方法，其特征在于，所述静电纺丝的温度为20-30℃，湿度为40-55％，电压为15-40kv，推进速率10-50μL/min，所述内皮化静电纺丝膜的纤维直径为1-2μm，所述内皮化静电纺丝膜的厚度为70-170μm。
6.根据权利要求3所述的封堵性防粘连膜材料的制备方法，其特征在于，所述接枝内皮化材料的处理包括如下步骤：
(1)将静电纺丝膜表面通过偶联剂或引入活性基团进行表面修饰，获得改性纤维膜，所述活性基团为氨基或羧基；
(2)将步骤(1)中改性纤维膜浸入内皮化材料溶液并静置24h，获得内皮化静电纺丝防粘连膜。
7.根据权利要求1所述的封堵性防粘连膜材料的制备方法，其特征在于，所述止血凝胶为聚氨酯或聚乙二醇。
8.封堵性防粘连膜材料，其特征在于，利用权利要求1-7任一项所述的方法制备的封堵性防粘连膜材料。
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