CN113209363B - 一种粘附止血可注射壳聚糖凝胶及其制备方法与应用 - Google Patents
一种粘附止血可注射壳聚糖凝胶及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种粘附止血可注射壳聚糖凝胶及其制备方法与应用。本发明首先以壳聚糖为原料,在其表面引入邻苯二酚官能团制得壳聚糖‑氢化咖啡酸,然后进一步引入二苯并环辛炔基团制得壳聚糖‑氢化咖啡酸‑二苯并环辛炔,最后利用邻苯二酚官能团的氧化交联作用以及二苯并环辛炔基团与叠氮化的聚乙二醇的无铜催化叠氮炔环加成反应合成凝胶材料,即所述粘附止血可注射壳聚糖凝胶。本发明把邻苯二酚结构化合物接枝到壳聚糖上,可提高壳聚糖材料湿组织粘附性、促凝血性和快速止血性;把多臂聚乙二醇混入敷料结构内部,可提高壳聚糖的机械性能。本发明制得的凝胶有潜力作为一种新型生物材料在细胞、组织工程、药物递送等生物医学领域得到广泛应用。
Description
技术领域
本发明属于医用敷料领域,具体涉及一种粘附止血可注射壳聚糖凝胶及其制备方法与应用。
背景技术
由于创伤和病理生理状况的增加,皮肤创伤修复成为了主要的医疗保健问题。皮肤是人体重要的屏障组织,一旦形成伤口,发生感染的几率会大大增加。严重的皮肤伤口,例如大面积的、深达真皮层的伤口通常会导致疼痛、感染甚至截肢,极大地影响了患者的生活质量。皮肤慢性伤口的护理昂贵,导致医疗资源消耗,为了降低成本,找到更经济、简单、有效的皮肤创口敷料显得尤为重要。现今各种创口敷料被相继开发出来,但现有的皮肤伤口敷料缺乏柔韧性、粘附性和止血性,而前者限制了其在复杂伤口或关节(手腕,肘部,脚踝等)等部位的贴合性,导致潜在的细菌入侵,从而使伤口愈合受阻甚至使伤口状况恶化。因此急需研制出一种合适的创伤敷料,使其同时具有可注射性、良好的机械性能、快速止血性以及强组织粘附性促进创口愈合。
壳聚糖(CS)是甲壳素的脱乙酰基产物,是自然界中少见的带正电的高分子化合物,本身具有非凡的生物学特性,例如可生物降解,抗菌活性,生物相容性以及良好的细胞结和能力。近期壳聚糖水凝胶已经被应用于各种生物医学应用中,包括药物递送、伤口敷料和组织工程支架等。但单一组分的壳聚糖材料,机械性能和湿组织粘附性较差限制了CS在医学领域中的应用。为了改善CS的物理化学性质,通常会对CS进行改性来提高其性能。
贻贝通过足丝可以在湿润的条件下附着到各种基材表面,足丝中的主要成分是富含邻苯二酚(儿茶酚)基团的黏性蛋白,在贻贝蛋白的交联和粘附过程中发挥着重要作用。通过邻苯二酚改性的壳聚糖材料的组织粘附性要明显优于纯壳聚糖以及其它壳聚糖衍生物。
聚乙二醇(PEG)是具有可调机械性能的亲水性聚醚化合物。具有直链或支链(多臂或星形)结构的PEG大分子单体的末端可以有各种官能团,例如甲氧基,羧基,氨基,硫醇基,叠氮化物,这使其更易于用于改性其它材料。通过在天然高分子材料中加入PEG可以增强材料的生物学性能,特别是机械性能。
基于以上背景,可制得一种同时接枝邻苯二酚和PEG的壳聚糖,这有望制备一种同时具有良好的机械性能、湿组织粘附性和快速止血性的可注射壳聚糖凝胶敷料。
发明内容
本发明的首要目的是优化现有的医用敷料,提供一种具有良好的机械性能、强组织粘附性、促凝血性、快速止血性的可注射壳聚糖水凝胶的制备方法,通过在壳聚糖材料上接枝邻苯二酚提高其组织粘附性和快速止血性,通过进一步接枝多臂聚乙二醇进一步提高其机械性能。
本发明的另一目的在于提供一种由上述方法制得的粘附止血可注射壳聚糖凝胶。
本发明的再一目的在于提供上述粘附止血可注射壳聚糖凝胶的应用。
本发明目的通过以下技术方案实现:
一种粘附止血可注射壳聚糖凝胶的制备方法,包括以下步骤:首先以壳聚糖为原料,在其表面引入邻苯二酚官能团制得壳聚糖-氢化咖啡酸(CS-HA),然后进一步引入二苯并环辛炔基团制得壳聚糖-氢化咖啡酸-二苯并环辛炔,最后利用邻苯二酚官能团的氧化交联作用以及二苯并环辛炔基团与叠氮化的聚乙二醇的无铜催化叠氮炔环加成(SPAAC)反应合成凝胶材料,即所述粘附止血可注射壳聚糖凝胶。
进一步的,所述的壳聚糖-氢化咖啡酸制备步骤如下:在壳聚糖盐酸溶液中加入氢化咖啡酸,壳聚糖与氢化咖啡酸的质量比为1:0.5-1:3,室温反应1-3小时,经去离子水透析、真空冻干,即可得到壳聚糖-氢化咖啡酸(CS-HA)。
进一步的,所述的壳聚糖-氢化咖啡酸-二苯并环辛炔制备步骤如下:将壳聚糖-氢化咖啡酸溶于PBS缓冲溶液,加入二苯并环辛炔-聚乙二醇-活性酯,室温下反应4-6小时,二苯并环辛炔-聚乙二醇-活性酯与壳聚糖-氢化咖啡酸材料质量比为1:50-150;经去离子水透析、真空冻干得到壳聚糖-氢化咖啡酸-二苯并环辛炔(CS-HA-DBCO)。
进一步的,壳聚糖-氢化咖啡酸溶于PBS缓冲溶液获得的溶液浓度为2wt%。
进一步的,制得壳聚糖-氢化咖啡酸-二苯并环辛炔后,进一步的操作步骤如下:取CS-HA-DBCO溶于PBS溶液后,按照CS-HA-DBCO与聚乙二醇叠氮摩尔比1:10-1:50,与聚乙二醇叠氮溶液充分混匀,加入高碘酸盐,混合均匀,高碘酸盐与CS-HA-DBCO的质量比为1:10-20,在室温下2-5分钟后形成新的大分子生物医学材料即所述粘附止血可注射壳聚糖凝胶。
进一步的,CS-HA-DBCO溶于PBS溶液后获得的溶液浓度为2-3wt%。
所述的二苯环辛炔-聚乙二醇-活性酯优选为二苯并环辛炔-聚乙二醇-活性酯可以是二苯并环辛炔-四聚乙二醇-活性酯,二苯并环辛炔-五聚乙二醇-活性酯或是二苯并环辛炔-十二聚乙二醇-活性酯的至少一种。
所述的聚乙二醇叠氮优选为四臂、六臂、八臂聚乙二醇叠氮中的至少一种。
所述高碘酸盐为高碘酸钠或高碘酸钾。
所述壳聚糖分子量为3万-20万,脱乙酰度为50%-95%。
本发明主要通过以下步骤实现:
(1)配制壳聚糖溶液,加入氢化咖啡酸,搅拌一段时间;然后加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)溶液(溶剂为体积比1:1的去离子水和酒精),反应1小时,透析后冻干制得CS-HA;所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液浓度为25-50mg/mL;
(2)将步骤(1)中制得的CS-HA溶解于PBS溶液,加入二苯并环辛炔-聚乙二醇-活性酯反应4-6小时,透析后冻干制得CS-HA-DBCO;
(3)将步骤(2)中制得的CS-HA-DBCO溶解于PBS溶液,加入高碘酸盐和聚乙二醇叠氮溶液,室温下2-5分钟后交联形成所述粘附止血可注射壳聚糖凝胶。
进一步的,步骤(1)中壳聚糖溶液浓度为1wt%,通过1mol/L NaOH溶液调节壳聚糖溶液的pH为5.4。
进一步的,步骤(1)中加入氢化咖啡酸后搅拌1小时。
上述方案制备得到的一种粘附止血可注射壳聚糖凝胶,兼具良好的机械性能,湿组织粘附性,促凝血性以及快速止血性。
所述粘附止血可注射壳聚糖凝胶可以制成冻干海绵、水凝胶等临床使用的敷料或器械。
所述粘附止血可注射壳聚糖凝胶可以作为止血、水性皮肤等用来治疗和预防创伤和疾病。
本发明相对于现有技术具有以下优点:
1)本发明把邻苯二酚结构化合物接枝到壳聚糖上,可提高壳聚糖材料湿组织粘附性、促凝血性和快速止血性。
2)本发明把多臂聚乙二醇混入敷料结构内部,可提高壳聚糖的机械性能。
3)这种具有组织粘附性、快速止血性、可注射壳聚糖凝胶有潜力作为一种新型生物材料在细胞、组织工程、药物递送等生物医学领域得到广泛应用。
附图说明
图1是实施例1中的CS-HA-DBCO的合成图;
图2是凝胶的压缩应力-应变图(A),以及组织粘附性能图(B);
图3是凝胶的促凝血性能图;
图4是凝胶的止血性能图;
图2-4中的改性壳聚糖凝胶敷料是指实施例2中制备的CS-PEG-HA凝胶,普通壳聚糖凝胶敷料是指参照文献(Li H,Wijekoon A,Leipzig ND,3Ddifferentiation of neuralstem cells in macroporous photopolymerizable hydrogel scaffolds[J].2012,7(11):e48824.)制备的凝胶。
具体实施方式
下面结合实施例和附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。本发明涉及的原料均可从市场上直接购买。对于未特别注明的工艺参数,可参照常规技术进行。
实施例中使用的壳聚糖分子量为5万,脱乙酰度为75%,4臂聚乙二醇叠氮的分子量为5000。
实施例1:壳聚糖-氢化咖啡酸-二苯并环辛炔(CS-PEG-HA)的合成
(1)称取0.5g壳聚糖,加入到45.5mL(pH=1.6)的去离子水中,制成1wt%壳聚糖溶液,并将最终pH调为5.4。
(2)称取0.4g氢化咖啡酸(HA)加入壳聚糖溶液中,搅拌并使最终pH保持在3.7-4.0。
(3)称取1.2448g EDC,配制50mL体积比为1:1的去离子水和乙醇作为溶剂,将EDC溶液加入到壳聚糖溶液中搅拌反应1小时。加入EDC能活化氢化咖啡酸中的羧基,使其可以与壳聚糖中的氨基进行反应。
(4)将步骤(3)的最终反应物在pH=3.5的酸性溶液中透析48小时,离心过滤除菌后得到CS-HA。
(5)将步骤(4)合成的CS-HA溶于PBS溶液(2wt%,pH 7.4)。
(6)加入二苯并环辛炔-四聚乙二醇-羟基琥珀酰亚胺酯(与CS-HA的质量比为1:50(北京普益华科技有限公司)并在室温下高速搅拌反应4小时,生成CS-HA-DBCO,最后将反应产物透析后冻干储存。
实施例2:粘附止血可注射壳聚糖凝胶敷料的制备方法。
(1)CS-HA-DBCO溶液的制备:将实施例1制备的邻苯二酚和二苯并环辛炔接枝的壳聚糖(CS-HA-DBCO)称取15mg溶于5mL PBS溶液中,制备成3%的CS-HA-DBCO溶液。
(2)4臂聚乙二醇叠氮溶液的制备(4arm-PEG-Azide):称取4arm-PEG-Azide200mg置于2mL玻璃瓶中,加入1mL去离子水,制备成20wt%的4arm-PEG-Azide溶液。
(3)高碘酸钾溶液的制备:称取高碘酸钾5mg、溶于1mL去离子水中,制备成0.5wt%的高碘酸钾溶液。
(4)CS-PEG-HA水凝胶的制备:首先取0.5mL步骤(1)制备的CS-HA-DBCO溶液置于玻璃瓶中;然后取0.1mL步骤(2)中制备的4arm-PEG-Azide溶液和0.02mL步骤(3)中制备的高碘酸钾溶液置于反应瓶中,电磁搅拌充分混匀,获得CS-PEG-HA凝胶前溶液。最后通过医用注射器吸取并注射到模具中,室温下2-5分钟后交联形成即得到本发明所述的具有组织粘附性、快速止血性、可注射壳聚糖水凝胶(CS-PEG-HA凝胶)。
实施例3:粘附止血可注射壳聚糖凝胶的力学性能测试
将实施例2制备的CS-PEG-HA凝胶样品(n=3)置于万能试验机上(型号为ELF3220),以2mm/min的压缩速度进行压缩试验,水凝胶压缩量为60%。保存数据做应力-应变图,结果见图2中的A图。
实施例4:粘附止血可注射壳聚糖凝胶的组织粘附性能测试。
(1)凝胶的组织粘合强度使用搭接剪切测试,以猪皮为基材进行测试。
(2)将新鲜的猪皮切成长方形(50mm×10mm),并将实施例2中的CS-PEG-HA凝胶前溶液转移到皮下脂肪,然后将另一片猪皮放在上面,粘合面积为1cm2。
(3)用500g的砝码压约24小时后,然后用万能试验机在5mm/min的拉伸速率下测量粘合强度。结果见图2中的B图。
实施例5:粘附止血可注射壳聚糖凝胶的促凝血性测试
(1)将100μL实施例2制备的CS-PEG-HA凝胶置于离心管中并在37℃水浴中孵育5分钟。
(2)加入0.5mL抗凝兔血(上海迈瑞尔化学技术有限公司)和0.1mLCaCl2溶液(0.2mol/L)。每20秒倒置一次,直至血液完全停止流动,记录时间。结果见图3。
实施例6:粘附止血可注射壳聚糖凝胶的止血性能测试
(1)使用大鼠(南方医科大学实验动物中心)肝脏出血模型测试凝胶止血性能。
(2)将大鼠麻醉后,用称量过重量的滤纸放在肝脏下方,并用2mm的活检打孔器打孔,产生出血。
(3)立即在出血处,注射200μL实施例2中的CS-PEG-HA凝胶,2分钟后称取滤纸的重量,计算出血量,结果见图4。
实施例3-6的测试中,同时参照文献(Li H,Wijekoon A,Leipzig ND,3Ddifferentiation of neural stem cells in macroporous photopolymerizablehydrogel scaffolds[J].2012,7(11):e48824.)制备普通壳聚糖凝胶敷料并进行测试比对。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种粘附止血可注射壳聚糖凝胶的制备方法,其特征在于包括以下步骤:首先以壳聚糖为原料,在其表面引入邻苯二酚官能团制得壳聚糖-氢化咖啡酸,然后进一步引入二苯并环辛炔基团制得壳聚糖-氢化咖啡酸-二苯并环辛炔,最后利用邻苯二酚官能团的氧化交联作用以及二苯并环辛炔基团与叠氮化的聚乙二醇的无铜催化叠氮炔环加成反应合成凝胶材料,即所述粘附止血可注射壳聚糖凝胶。
2.根据权利要求1所述的一种粘附止血可注射壳聚糖凝胶的制备方法,其特征在于,所述的壳聚糖-氢化咖啡酸制备步骤如下:在壳聚糖盐酸溶液中加入氢化咖啡酸,壳聚糖与氢化咖啡酸的质量比为1:0.5-1:3,室温反应1-3小时,经去离子水透析、真空冻干,即可得到壳聚糖-氢化咖啡酸。
3.根据权利要求1所述的一种粘附止血可注射壳聚糖凝胶的制备方法,其特征在于,所述的壳聚糖-氢化咖啡酸-二苯并环辛炔制备步骤如下:将壳聚糖-氢化咖啡酸溶于PBS缓冲溶液,加入二苯并环辛炔-聚乙二醇-活性酯,室温下反应4-6小时,二苯并环辛炔-聚乙二醇-活性酯与壳聚糖-氢化咖啡酸材料质量比为1:50-150;经去离子水透析、真空冻干得到壳聚糖-氢化咖啡酸-二苯并环辛炔。
4.根据权利要求1所述的一种粘附止血可注射壳聚糖凝胶的制备方法,其特征在于,制得壳聚糖-氢化咖啡酸-二苯并环辛炔后,进一步的操作步骤如下:取壳聚糖-氢化咖啡酸-二苯并环辛炔溶于PBS溶液后,按照壳聚糖-氢化咖啡酸-二苯并环辛炔与聚乙二醇叠氮摩尔比1:10-1:50,与聚乙二醇叠氮溶液充分混匀,加入高碘酸盐,混合均匀,高碘酸盐与壳聚糖-氢化咖啡酸-二苯并环辛炔的质量比为1:10-20,在室温下2-5分钟后形成所述粘附止血可注射壳聚糖凝胶。
5.根据权利要求1所述的一种粘附止血可注射壳聚糖凝胶的制备方法,其特征在于,所述的二苯并环辛炔基团为二苯并环辛炔-聚乙二醇-活性酯。
6.根据权利要求4所述的一种粘附止血可注射壳聚糖凝胶的制备方法,其特征在于,所述的聚乙二醇叠氮为四臂、六臂、八臂聚乙二醇叠氮中的至少一种;
所述高碘酸盐为高碘酸钠或高碘酸钾。
7.根据权利要求1所述的一种粘附止血可注射壳聚糖凝胶的制备方法,其特征在于,具体制备步骤如下:
(1)配制壳聚糖溶液,加入氢化咖啡酸,搅拌一段时间;然后加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液,反应1小时,透析后冻干制得壳聚糖-氢化咖啡酸;壳聚糖与氢化咖啡酸的质量比为1:0.8,所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液浓度为25-50 mg/mL;
(2)将步骤(1)中制得的壳聚糖-氢化咖啡酸溶解于PBS溶液,加入二苯并环辛炔-聚乙二醇-活性酯反应4-6小时,透析后冻干制得壳聚糖-氢化咖啡酸-二苯并环辛炔;二苯并环辛炔-聚乙二醇-活性酯与壳聚糖-氢化咖啡酸的质量比为1:50;
(3)将步骤(2)中制得的壳聚糖-氢化咖啡酸-二苯并环辛炔溶解于PBS溶液,加入高碘酸盐和聚乙二醇叠氮溶液,室温下2-5分钟后交联形成所述粘附止血可注射壳聚糖凝胶;所述壳聚糖-氢化咖啡酸-二苯并环辛炔与聚乙二醇叠氮摩尔比1:10-1:50,所述高碘酸盐与壳聚糖-氢化咖啡酸-二苯并环辛炔的质量比为1:10-20。
8.根据权利要求7所述的一种粘附止血可注射壳聚糖凝胶的制备方法,其特征在于,步骤(1)中壳聚糖溶液浓度为1wt%,通过1mol/L NaOH溶液调节壳聚糖溶液的pH为5.4;步骤(1)中加入氢化咖啡酸后搅拌1小时;
步骤(2)中的二苯并环辛炔-聚乙二醇-活性酯为二苯并环辛炔-四聚乙二醇-活性酯、二苯并环辛炔-五聚乙二醇-活性酯或二苯并环辛炔-十二聚乙二醇-活性酯中的至少一种。
9.一种由权利要求1-8任一项所述方法制备得到的粘附止血可注射壳聚糖凝胶。
10.权利要求9所述粘附止血可注射壳聚糖凝胶在制备冻干海绵、水凝胶中的应用。
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