CN114159613A - 一种抗菌止血复合敷料及其制备方法 - Google Patents

一种抗菌止血复合敷料及其制备方法 Download PDF

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CN114159613A
CN114159613A CN202111323317.5A CN202111323317A CN114159613A CN 114159613 A CN114159613 A CN 114159613A CN 202111323317 A CN202111323317 A CN 202111323317A CN 114159613 A CN114159613 A CN 114159613A
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chitosan
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CN114159613B (zh
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孙嘉华
王风华
刘磊
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Jiangsu University
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Abstract

本发明属于医用材料领域，公开了一种抗菌止血敷料及其制备方法。复合敷料包括基底层和纳米纤维修饰层，基底层为壳聚糖水刺无纺布，纳米纤维修饰层为烷基化壳聚糖/酸化碳纳米纤维/聚乙烯醇纳米纤维薄膜，通过静电纺丝的方式将纳米纤维修饰层材料修饰在基底层的壳聚糖水刺无纺布上，制备得到具有抗菌止血功能的复合敷料。本发明制备得到的复合敷料的基底层能够快速凝血，纳米纤维修饰层能够杀死细菌，减少血液的过度流失以及促进凝血。基底层和纳米纤维修饰层协同凝血，加快凝血级联反应，从而实现快速止血。该复合敷料具有良好的生物相容性、快速止血功能和杀菌性能，有望用于临床急救、战场救护等领域。

Description

一种抗菌止血复合敷料及其制备方法
技术领域
本发明属于医用材料领域，涉及一种医用的抗菌止血复合敷料及其制备方法。
背景技术
机械创伤、烧伤、外科手术、战斗创伤等皆可造成机体产生创面及出血。在伤口较轻的情况下，出血可以由人体自身止血机制停止。但是当出血不可控时，往往会造成伤者的死亡或者是截肢，并且这类死亡占全世界所有创伤死亡的30％以上，其中大多数发生在接受紧急治疗之前，因此早期介入止血对重大创伤的生存和最佳恢复起着决定性作用。目前市面上有以下几种止血材料：(1)蛋白质(纤维蛋白原/凝血酶)，(2)铝硅酸盐(高岭土和沸石)，(3)多糖(纤维素，葡聚糖和壳聚糖)。虽然蛋白类材料具有高效的凝血能力，但是使用不便并且价格高昂，而铝硅酸盐材料往往具有一定毒性。目前使用最多的多糖包括纤维素和壳聚糖，而纤维素的凝血能力有限，因此壳聚糖成为高效，安全且成本相对低廉的凝血产品。壳聚糖水刺无纺布已被广泛用在创伤止血等领域，但是其依然会存在止血过程中吸收大量的血液，止血效果和抗菌效果有限等问题。近年来的诸多研究表明，通过对壳聚糖进行烷基化改性大大提高了壳聚糖的凝血能力，其止血机理与单纯的壳聚糖不同，主要通过烷烃链与血细胞的细胞膜相互作用形成网状凝胶，利用烷基化壳聚糖制备的纳米纤维膜具有显著的止血性能，但其抗菌性能不佳。
由于抗生素的广泛使用造成了耐药菌的出现，多药耐药细菌(MDR)来源的伤口感染加剧了愈合的难度，因为用于杀死这些细菌的有效抗菌剂较少。光热疗法(PTT)对机体损伤小，不易产生耐药性，是预防伤口感染的最佳选择。在目前的研究中，各种光热剂用于伤口敷料，如氧化石墨烯(GO)，聚多巴胺纳米颗粒(PDA-NPs)，碳纳米纤维(CNF)。其中碳纳米纤维因为其优异的光热转换效率被广泛用于光热杀菌。
发明内容
针对现有技术的不足，本发明提供了一种医用抗菌止血复合敷料及其制备方法，其基底层为快速止血的壳聚糖水刺无纺布层，纳米纤维修饰层为烷基化壳聚糖/酸化碳纳米纤维/聚乙烯醇组成的止血抗菌，防止血液损失的纳米纤维层。该复合敷料具有快速止血、防止血液损失、高效抗菌的功效。
为了实现上述目的，本发明采用如下技术方案。
一种抗菌止血复合敷料，包括基底层和纳米纤维修饰层组成，所述的基底层为壳聚糖水刺无纺布，所述的纳米纤维修饰层是由以下原料经静电纺丝制备而成的纳米纤维膜：烷基化壳聚糖、酸化碳纳米纤维、聚乙烯醇。其中聚乙烯醇辅助烷基化壳聚糖进行电纺，从而提高纺丝液的可纺性，对碳纳米纤维进行酸化从而减少碳纳米纤维在纺丝液中的聚集，提高纺丝液的均一性。
所述抗菌止血复合敷料的制备方法，包括如下步骤：
步骤1：将壳聚糖水刺无纺布固定在静电纺丝的滚筒收集器上；
步骤2：将烷基化壳聚糖的乙酸水溶液、酸化碳纳米纤维的乙酸悬浮液、聚乙烯醇的水溶液混合均匀制备得到纺丝液，置于真空环境脱气泡后将纺丝液置于推进泵中，调节静电纺丝参数得到复合敷料；将获得的复合敷料置于真空环境中干燥，除掉未挥发的有机溶剂。
步骤1中，壳聚糖水刺无纺布中，壳聚糖的含量为30％-70％，优选为50％。
步骤2中，烷基化壳聚糖的乙酸水溶液、酸化碳纳米纤维的乙酸悬浮液、聚乙烯醇的水溶液的体积比为8:12:1。
步骤2中，烷基化壳聚糖的乙酸水溶液中，烷基化壳聚糖的质量百分浓度为3wt％。
进一步地，所述烷基化壳聚糖，是采用十二醛对壳聚糖修饰得到的，所述的烷基化壳聚糖的取代度为8％-10％，优选为9.42％；其中，壳聚糖的脱乙酰度为90％-99％，优选95％；分子量为5000-20000，优选20000。
步骤2中，所述酸化碳纳米纤维的乙酸悬浮液中，酸化碳纳米纤维的质量分数为0-0.3wt％。
步骤2中，所述聚乙烯醇的水溶液中，聚乙烯醇的聚合度为1700±50，聚乙烯醇的质量分数为10wt％。
步骤2中，所述静电纺丝参数为：25℃，小于50％的湿度对静电纺丝液进行静电纺丝，电压20-25kv，针头20G，推进速度为6-10μl/min，针头到收集器的距离为15-20cm。
步骤2中，所述真空环境中干燥温度为30-50℃。
与现有技术相比，本发明的优点是：
(1)纳米纤维修饰层的烷基化壳聚糖/酸化碳纳米纤维/聚乙烯醇纳米纤维膜能够减少伤口处的血液损失，加速凝血级联反应并实现光热杀菌。
(2)纳米纤维修饰层和基底层起到协同止血的作用，能够形成血凝块从而快速封堵伤口，减少血液的流失，挽救伤者的性命。
(3)制备方法简单，成本低廉，止血抗菌防失血效果明显。
附图说明
图1是本发明的抗菌止血敷料的结构示意图；其中，1.壳聚糖水刺无纺布基底层，2.烷基化壳聚糖/酸化碳纳米纤维/聚乙烯醇纳米纤维修饰层。
图2是本发明的全血凝固时间的实验。
图3是本发明的敷料防止血液损失的实验。
图4是本发明的体外光照辅助抗菌实验；其中，a为不同组别加入大肠杆菌后经过近红外光照处理，培养的大肠杆菌的相对存活率，b为不同组别加入金黄色葡萄球菌后经过近红外光照处理，培养的金黄色葡萄球菌的相对存活率，c为不同组别分别加入大肠杆菌、金黄色葡萄球菌后，经过近红外光照处理，培养的大肠杆菌、金黄色葡萄球菌的平板菌落照片。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
如图1所示，本发明的抗菌止血敷料的结构包括壳聚糖水刺无纺布基底层1，烷基化壳聚糖/酸化碳纳米纤维/聚乙烯醇纳米纤维修饰层2。
实施例1
将20cm*15cm的壳聚糖水刺无纺布缠绕在滚筒收集器上，其中，壳聚糖水刺无纺布中，壳聚糖的含量为50％。
将取代度为9.42％的烷基化壳聚糖0.12g溶于4ml的90％的乙酸溶液中，制备得到3.0wt％的溶液，随后加入10wt％的聚乙烯醇水溶液6ml，搅拌均匀后将纺丝液装入推进泵中，调节参数进行纺丝，将烷基化壳聚糖/聚乙烯醇纳米纤维修饰在壳聚糖水刺无纺布上。静电纺丝完毕进行真空处理，除去残余的有机溶剂。
实施例2
将20cm*15cm的壳聚糖水刺无纺布缠绕在滚筒收集器上，其中，壳聚糖水刺无纺布中，壳聚糖的含量为50％。
将取代度为9.42％的烷基化壳聚糖0.12g溶于4ml的90％的乙酸溶液中，制备得到3.0wt％的溶液，随后加入10wt％的聚乙烯醇水溶液6ml，1wt％的酸化碳纳米纤维乙酸悬浮液0.5ml，搅拌6h后将纺丝液装入推进泵中，调节参数进行纺丝，将烷基化壳聚糖/酸化碳纳米纤维/聚乙烯醇纳米纤维修饰在壳聚糖水刺无纺布上。静电纺丝完毕进行真空处理，除去残余的有机溶剂。
实施例3
将20cm*15cm的壳聚糖水刺无纺布缠绕在滚筒收集器上，其中，壳聚糖水刺无纺布中，壳聚糖的含量为50％。
将取代度为9.42％的烷基化壳聚糖0.12g溶于4ml的90％的乙酸溶液中，制备得到3.0wt％的溶液，随后加入10wt％的聚乙烯醇水溶液6ml，1.5wt％的酸化碳纳米纤维乙酸悬浮液0.5ml，搅拌6h后将纺丝液装入推进泵中，调节参数进行纺丝，将烷基化壳聚糖/酸化碳纳米纤维/聚乙烯醇纳米纤维修饰在壳聚糖水刺无纺布上。静电纺丝完毕进行真空处理，除去残余的有机溶剂。
实施例4
将20cm*15cm的壳聚糖水刺无纺布缠绕在滚筒收集器上，其中，壳聚糖水刺无纺布中，壳聚糖的含量为50％。
将取代度为9.42％的烷基化壳聚糖0.12g溶于4ml的90％的乙酸溶液中，制备得到3.0wt％的溶液，随后加入10wt％的聚乙烯醇水溶液6ml，2wt％的酸化碳纳米纤维乙酸悬浮液0.5ml，搅拌6h后将纺丝液装入推进泵中，调节参数进行纺丝，将烷基化壳聚糖/酸化碳纳米纤维/聚乙烯醇纳米纤维修饰在壳聚糖水刺无纺布上。静电纺丝完毕进行真空处理，除去残余的有机溶剂。
性能测试
(1)体外凝血实验。
测试溶液使用加抗凝剂枸橼酸钠的抗凝羊血，模拟人体伤口流出的血液。由新鲜羊血添加10％(V/V)的枸橼酸钠水溶液(2.5wt％)搅拌均匀配制而成。
测试方法为取6支离心管分别加入测试溶液1ml，在37℃恒温水浴中加热10min，分别加入实施例1～4得到的复合敷料、壳聚糖水刺无纺布和什么都不加(10mm*10mm)，接着加入0.2M的CaCl2溶液100μl继续恒温加热，每隔15s钟倾斜一次试管，直至试管中的血液不流动为止，分别记录各组试管中测试溶液的凝固时间。
由图2可知，相较于别的实验组，实施例4的全血凝固时间最短，表明实施例4能够实现快速止血，有作为止血敷料应用的潜力。
(2)防止血液损失实验。
通过对实施例4得到的样品进行防止血液损失实验。
测试方法为取2只10ml离心管，在1/3处制造一个直径3mm的孔，采用复合敷料对其进行覆盖，并用医用胶带进行固定，然后将6ml血液倒入离心管观察血液损失情况。
由图3可知，相较于普通的壳聚糖水刺无纺布，复合敷料在防止血液损失的过程中，能够起到明显的减少血液损失的作用，表明复合敷料具有作为止血敷料的潜力。
(3)体外光照辅助抗菌实验
对实施例1-4、壳聚糖水刺无纺布、不加任何材料的空白对照组进行光热辅助杀菌实验。
测试方法为取1cm*1cm的实施例1-4和壳聚糖水刺无纺布于2ml的离心管中，在其表面滴加50μl的大肠杆菌悬浮液(106CFU/ml)随后采用近红外光照射(808nm，2W/cm2)15min。接着将1ml PBS缓冲液加入每一个离心管中充分震荡得到细菌悬浮液，随后将细菌悬浮液用PBS缓冲液稀释20倍,滴加100μl稀释过的细菌悬浮液在LB培养基上，37℃孵育24h，计算每一个培养基上的菌落数，从而得到细菌的相对存活率。金黄色葡萄球菌采用同样的测试方法。
由图4可知，相较于普通的壳聚糖水刺无纺布和空白对照组，实施例1-4的光照辅助杀菌效果随着碳纳米纤维含量的增加而增加，并且实施例4的抗菌效果可以达到90％，表明实施例4具有用于良好的抗菌功能，可作为抗菌止血敷料使用。

Claims (10)

1.一种抗菌止血复合敷料，其特征在于，包括基底层和纳米纤维修饰层；所述基底层为壳聚糖水刺无纺布，所述纳米纤维修饰层为烷基化壳聚糖/酸化碳纳米纤维/聚乙烯醇纳米纤维薄膜。
2.根据权利要求1所述的抗菌止血复合敷料的制备方法，其特征在于，包括如下步骤：
步骤1：将壳聚糖水刺无纺布固定在静电纺丝的滚筒收集器上；
步骤2：将烷基化壳聚糖的乙酸水溶液、酸化碳纳米纤维的乙酸悬浮液、聚乙烯醇的水溶液混合均匀制备得到纺丝液，置于真空环境脱气泡后将纺丝液置于推进泵中，调节静电纺丝参数得到复合敷料；将获得的复合敷料置于真空环境中干燥。
3.根据权利要求2所述的制备方法，其特征在于，步骤1中，壳聚糖水刺无纺布中，壳聚糖的含量为30％-70％。
4.根据权利要求2所述的制备方法，其特征在于，步骤2中，烷基化壳聚糖的乙酸水溶液、酸化碳纳米纤维的乙酸悬浮液、聚乙烯醇的水溶液的体积比为8:12:1。
5.根据权利要求2所述的制备方法，其特征在于，步骤2中，烷基化壳聚糖的乙酸水溶液中，烷基化壳聚糖的质量百分浓度为3wt％。
6.根据权利要求5所述的制备方法，其特征在于，所述烷基化壳聚糖，是采用十二醛对壳聚糖修饰得到的，所述的烷基化壳聚糖的取代度为8％-10％；其中，壳聚糖的脱乙酰度为90％-99％，分子量为5000-20000。
7.根据权利要求2所述的制备方法，其特征在于，步骤2中，所述酸化碳纳米纤维的乙酸悬浮液中，酸化碳纳米纤维的质量分数为0-0.3wt％。
8.根据权利要求2所述的制备方法，其特征在于，步骤2中，所述聚乙烯醇的水溶液中，聚乙烯醇的聚合度为1700±50，聚乙烯醇的质量分数为10wt％。
9.根据权利要求2所述的制备方法，其特征在于，步骤2中，所述静电纺丝参数为：25℃，小于50％的湿度对静电纺丝液进行静电纺丝，电压20-25kv，针头20G，推进速度为6-10μl/min，针头到收集器的距离为15-20cm。
10.根据权利要求2所述的制备方法，其特征在于，步骤2中，所述真空环境中干燥温度为30℃-50℃。
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