CN116726241B - 一种胶原蛋白止血抗菌敷料及其制备方法 - Google Patents
一种胶原蛋白止血抗菌敷料及其制备方法 Download PDFInfo
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- CN116726241B CN116726241B CN202311008967.XA CN202311008967A CN116726241B CN 116726241 B CN116726241 B CN 116726241B CN 202311008967 A CN202311008967 A CN 202311008967A CN 116726241 B CN116726241 B CN 116726241B
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- antibacterial dressing
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Abstract
本发明提供一种胶原蛋白止血抗菌敷料及其制备方法，包括以下组分：间苯二酚改性聚氨基酸、人源胶原蛋白、凝胶赋形剂和中药提取物；该止血敷料具有快速止血、溶血率低、无毒、弹性好的优势，得益于敷料能能优异的介孔结构；另外，敷料吸液后，间苯二酚改性聚氨基酸一方面能够螯合红细胞中血红蛋白中的铁离子，在伤口表面形成胶状物起到封堵止血作用，另一方面与胶原蛋白复合后提高了胶原蛋白的活性，提高了血小板活化效应，从而加快伤口凝血；进一步对止血海绵浸绩中药溶液，进一步提高了止血敷料的抗菌性和止血性能。
Description
技术领域
本发明属于合成生物医学材料技术领域，具体涉及一种胶原蛋白止血抗菌敷料及其制备方法。
背景技术
介孔材料具有出色的止血能力，广泛应用于医学敷料领域，具体包括无机止血材料和有机止血材料。无机止血材料有沸石、蒙脱土和硅酸盐等颗粒，这些材料主要是通过在与血液接触后快速吸水能够浓缩凝血因子和血小板形成凝块；有机止血介孔材料主要包括高分子聚合物或天然生物基材料通过冷冻干燥得到，天然生物基材料由于具有环保、无毒、自然来源广等优点得到广泛的研究，主要包括由明胶、纤维素、海藻酸盐、壳聚糖、透明质酸等。天然生物基介孔止血敷料要兼顾生物相容性、介孔性、力学性能、止血能力和皮肤粘结性。
胶原蛋白是动物体内的一种纤维蛋白，富含于皮肤、筋腱、骨骼中，具有良好的生物相容性，常被用作重要的生物医用材料原料，可作为吸收性创面止血材料，常利用胶原蛋白被交联后的多孔结构吸收血液膨胀，形成凝血的网架，封闭血管裂口或者创面，并激活血小板，达到止血的目的。但目前基于胶原蛋白制备介孔止血材料的研究较少。
发明内容
基于上述技术现状，本发明提供一种胶原蛋白止血抗菌敷料。
本发明的技术方案如下：
一种胶原蛋白止血抗菌敷料，包括以下组分：邻苯二酚改性聚氨基酸、人源胶原蛋白、凝胶赋形剂和中药提取物。
进一步的，所述聚氨基酸包括半胱氨酸及赖氨酸、精氨酸、组氨酸、色氨酸中的至少一种；
作为优选，所述聚氨基酸为（赖氨酸-半胱氨酸-酪氨酸-组氨酸）共聚物。
进一步的，所述凝胶赋形剂为氧化刺槐豆胶与卡拉胶的复合物；所述复合物中，氧化刺槐豆胶与卡拉胶的质量比例为(2:8)~(4:6)。
进一步的，所述中药成分可选自：三七素、积雪草苷、白芨多糖中的一种或多种；作为优选，所述中药提取物为积雪草苷。
进一步的，按质量百分比计算，所述一种胶原蛋白止血抗菌敷料，包括以下组分：30~40%邻苯二酚改性聚氨基酸、20~30%人源胶原蛋白、30~60%凝胶赋形剂。
所述人源胶原蛋白为重组人源化胶原蛋白及其衍生物，包括但不限于：I型重组人源胶原蛋白、II型重组人源胶原蛋白、Ⅲ型重组人源胶原蛋白或/和其衍生物中的一种或多种。
本发明还提供上述一种胶原蛋白止血抗菌敷料的制备方法，包括以下步骤：
制备聚氨基酸：以三乙胺为引发剂，DMF为溶剂、氨基酸环内酸酐单体进行开环聚合反应，反应物用无水乙醚沉降析出、洗涤干燥后得到带有保护基的聚氨基酸；将带有保护基的聚氨基酸溶解于三氟乙酸中，加入溴化氢/乙酸混合液，反应除去保护基，粗产物经无水乙醚沉降析出、洗涤干燥、透析纯化后得到聚氨基酸；
所述氨基酸环内酸酐可选自L-酪氨酸-NCA、苄氧羰基-L-赖氨酸-NCA、L-半胱氨酸-NCA、苄氧羰基-L-精氨酸-NCA、苄氧羰基-L-组氨酸-NCA、苄氧羰基-L-色氨酸-NCA、甘氨酸-NCA；
制备邻苯二酚改性聚氨基酸：在DMPA的催化下，聚氨基酸与4-烯丙基邻苯二酚在THF/甲醇混合溶剂中进行紫外光照射反应，产物透析、纯化、干燥得到邻苯二酚改性聚氨基酸。
制备复合凝胶：分别配制邻苯二酚改性聚氨基酸水溶液、凝胶赋形剂水溶液并灭菌备用，另外配置人源胶原蛋白的水溶液；首先混合苯二酚改性聚氨基酸水溶液和人源胶原蛋白的水溶液，室温下搅拌1h后静置12h；然后继续加入凝胶赋形剂水溶液，搅拌均匀后升温至45±2℃下搅拌反应6h，得到的凝胶产物。
制备止血海绵：将凝胶产物放入模具中，在冷干机中冷冻干燥得止血海绵；
所述冷冻温度为-50~-80℃，时间为12~24h。
浸绩含中药成分的甲醇溶液得到止血抗菌敷料：配置含中药的甲醇溶液，将得到的止血海绵浸绩于溶液中，在常温下吹干得到止血抗菌敷料。
本发明的有益效果：
本发明制备得到的胶原蛋白止血抗菌敷料具有快速止血、溶血率低、无毒、弹性好的优势，得益于敷料能能优异的介孔结构，孔径大小为80~300μm，孔隙率为70%以上，拉伸强度为0.18~0.22MPa；另外，敷料吸液后，邻苯二酚改性聚氨基酸一方面能够螯合红细胞中血红蛋白中的铁离子，在伤口表面形成胶状物起到封堵止血作用，另一方面与胶原蛋白复合后提高了胶原蛋白的活性，提高了血小板活化效应，从而加快伤口凝血；进一步对止血海绵浸绩中药溶液，进一步提高了止血敷料的抗菌性和止血性能。
附图说明
图1为实施例3、对比例1和2制备的止血敷料的SEM图。
图2为止血敷料的抑菌活性试验结果。
图3为止血敷料的小鼠断尾出血量试验结果。
图4为止血敷料的小鼠肝脏出血量试验结果。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
实施例1
制备邻苯二酚改性聚氨基酸。
制备聚氨基酸
部分原料来源许下：
以L-半胱氨酸-NCA为例，以L-半胱氨酸制备L-半胱氨酸-NCA，制备方法如下：将L-半胱氨酸用THF溶解后，在氮气保护下加热至55℃，然后滴加三光气的THF溶液，滴完后升温至65℃反应，待溶液变澄清透明后，保温继续反应1h，减压蒸馏除去溶剂后，以无水THF和正己烷重结晶得到白色固体，即L-半胱氨酸-NCA。L-半胱氨酸与三光气的投料摩尔比为1:2。
按化学计量比往反应瓶中加入三乙胺、无水DMF和正己烷的混合溶液（V/V=10:1）和氨基酸反应单体，打开氮气保护装置，在35℃下持续反应72h，得到黄色透明的粘稠产物，用无水乙醚沉降、洗涤、过滤，重复三次后进行真空干燥后得到含有保护基的聚氨基酸。
将含有保护基的聚氨基酸溶解于三氟乙酸中，缓慢加入溴化氢/乙酸混合溶液，于30℃搅拌反应1 h后，在无水乙醚中沉降、洗涤、过滤，重复三次后进行真空干燥，将得到的固体溶解于少量的水，置于截留分子量为3500Da的透析袋中，在蒸馏水中透析纯化，冷冻干燥，得到白色固体为聚氨基酸。
各反应物的投料比例列于表2。
制备邻苯二酚改性聚氨基酸
称取上述制备得到的聚氨基酸溶于THF/甲醇（v/v=1/1）的混合溶液中，加入计量比的安息香二甲醚（DMPA），搅拌均匀后，加入4-烯丙基邻苯二酚，置于紫外反应箱中，光照并搅拌反应3h，得到的反应液浓缩后置于截留分子量为3500Da的透析袋中，在蒸馏水中透析纯化，冷冻干燥，得到邻苯二酚改性聚氨基酸。聚氨基酸、4-烯丙基邻苯二酚、DMPA、溶剂的投料比例为1g:0.4g:2g:15mL。
不同聚氨基酸制备得到的邻苯二酚改性聚氨基酸分别命名为PO-1、PO-2、PO-3、PO-4。
实施例2
制备氧化刺槐豆胶。
将20g天然刺槐豆胶和15g的高碘酸钠溶于200mL蒸馏水中，用锡箔纸包裹遮光，在室温下磁力搅拌8h；将5g乙二醇加入混合液中搅拌1h以消除未反应的高碘酸钠，得到的反应液浓缩后置于3500Da透析袋中透析3d，冷冻干燥得到氧化刺槐豆胶粉末。
实施例3
制备胶原蛋白止血抗菌敷料。
分别配制0.52g/mLPO-1邻苯二酚改性聚氨基酸水溶液、氧化刺槐豆胶与卡拉胶的混合溶液（氧化刺槐豆胶和卡拉胶的质量比为3:7，总的质量浓度为15.2wt%），并对上述溶液进行灭菌备用，另外配置20.05wt%的人源胶原蛋白的水溶液。
按体积比混合苯二酚改性聚氨基酸水溶液和人源胶原蛋白的水溶液，室温下搅拌1h后静置12h；然后继续加入氧化刺槐豆胶与卡拉胶的混合溶液，搅拌均匀后升温至45±2℃下搅拌反应6h，得到的凝胶产物；将凝胶产物放入模具中，在冷干机中-60℃冷冻干燥20h得止血海绵。所述各溶液的混合体积比如下，PO-1水溶液：氧化刺槐豆胶与卡拉胶的混合溶液：人源胶原蛋白水溶液=1:5:2。
配置10.21mg/mL的积雪草苷甲醇溶液，将得到的止血海绵浸绩于溶液中，超声1h后取出，在常温下吹干得到止血抗菌敷料。
部分原材料来源如下：
卡拉胶为k型，购于上海麦克林生化科技股份有限公司；
刺槐豆胶购于广州天佳生物科技有限公司；
积雪草苷购于兰州沃特莱斯生物科技有限公司；
人源胶原蛋白为人源III型胶原蛋白，购于华北生物制药有限公司。
对比例1
一种止血敷料的制备方法，包括如下步骤：
分别配制0.51g/mL P1聚氨基酸水溶液、氧化刺槐豆胶与卡拉胶的混合溶液（氧化刺槐豆胶和卡拉胶的质量比为3:7，总的质量浓度为15.2wt%），并对上述溶液进行灭菌备用，另外配置20.05wt%的人源胶原蛋白的水溶液。
按体积比混合P1水溶液和人源胶原蛋白的水溶液，室温下搅拌1h后静置12h；然后继续加入氧化刺槐豆胶与卡拉胶的混合溶液，搅拌均匀后升温至45±2℃下搅拌反应6h，得到的凝胶产物；将凝胶产物放入模具中，在冷干机中-60℃冷冻干燥20h得止血海绵。所述各溶液的混合体积比如下，P1水溶液：氧化刺槐豆胶与卡拉胶的混合溶液：人源胶原蛋白水溶液=1:5:2。
配制10.21mg/mL的积雪草苷甲醇溶液，将得到的止血海绵浸绩于溶液中，超声1h后取出，在常温下吹干得到止血抗菌敷料。
对比例2
一种止血敷料的制备方法，包括如下步骤：
分别配制0.52g/mL PO-1聚氨基酸水溶液、氧化刺槐豆胶与卡拉胶的混合溶液（氧化刺槐豆胶和卡拉胶的质量比为3:7，总的质量浓度为15.2wt%），并对上述溶液进行灭菌备用。
按体积比混合PO-1水溶液和氧化刺槐豆胶与卡拉胶的混合溶液，搅拌均匀后升温至45±2℃下搅拌反应6h，得到的凝胶产物；将凝胶产物放入模具中，在冷干机中-60℃冷冻干燥20h得止血海绵。所述各溶液的混合体积比如下，PO-1水溶液：氧化刺槐豆胶与卡拉胶的混合溶液=1:5。
配制10.21mg/mL的积雪草苷甲醇溶液，将得到的止血海绵浸绩于溶液中，超声1h后取出，在常温下吹干得到止血抗菌敷料。
对比例3
一种止血敷料的制备方法，包括如下步骤：
分别配制0.52g/mLPO-1邻苯二酚改性聚氨基酸水溶液、氧化刺槐豆胶与卡拉胶的混合溶液（氧化刺槐豆胶和卡拉胶的质量比为3:7，总的质量浓度为15.2wt%），并对上述溶液进行灭菌备用，另外配置20.05wt%的人源胶原蛋白的水溶液。
按体积比混合苯二酚改性聚氨基酸水溶液和人源胶原蛋白的水溶液，室温下搅拌1h后静置12h；然后继续加入氧化刺槐豆胶与卡拉胶的混合溶液，搅拌均匀后升温至45±2℃下搅拌反应6h，得到的凝胶产物；将凝胶产物放入模具中，在冷干机中-60℃冷冻干燥20h得止血海绵。所述各溶液的混合体积比如下，PO-1水溶液：氧化刺槐豆胶与卡拉胶的混合溶液：人源胶原蛋白水溶液=1:5:2。
对上述实施例3和对比例1~3制备的止血敷料进行如下测试：
测试例1 孔结构表征
（1）孔结构特征分析：通过美国康塔Poremaster GT60全自动孔隙度压汞分析仪测定，采用连续扫描模式，室温测试。
SEM：常规方法喷金后，采用JEOLJSM-6380LV 扫描电子显微镜分析止血海绵的微观结构。
实施例3和对比例1~2制备的止血海绵的横截面SEM参阅附图1。
测试例3 抗菌性能表征
对敷料进行抑菌活性测试：将止血海绵消毒后裁剪成直径15mm的圆片，放置在装有固体培养基的培养皿中央。用移液枪吸取20μL菌液（分别为：大肠杆菌、金黄葡萄球菌，浓度均为1×109CFU/mL）涂布于圆片上。将培养基置于37℃培养箱中培养24h后取出，在无菌条件下移除圆片后利用打孔器取出圆片正下方直径为10mm的固体培养基，置于离心管中，并加入2000μL PBS缓冲液，超声4min后得到原菌液。将原菌液分别稀释104倍得到稀释液，分别取150μL各浓度稀释液涂布在装有固体培养基的培养皿中。将其置于37℃培养箱中培养24h后取出，观察菌落数。
实施例3和对比例1~3制备得到的止血海绵的抑菌测试结果参阅附图2。
测试例4 动物止血实验
用10wt%水合氯醛(1ml/300g)腹腔注射麻醉大鼠，固定在手术板上。
对大鼠断尾模型，用灭菌外科手术剪，剪断等长的尾巴(1/3),放置在预先称重滤纸上。切割后，将尾巴暴露在空气中10秒，确保正常失血。然后，用止血材料轻微覆盖出血部位。记录止血时间和出血量，直至无出血。
对于大鼠肝损伤模型，用灭菌手术刀经腹部切开，暴露大鼠肝脏，在肝脏下方放置一张预先称重的滤纸。接着，用针头穿刺肝脏，确保正常失血10秒。然后，用止血材料轻微覆盖出血部位。记录止血时间和出血量，直至无出血。
两种模型下小鼠的出血量如图3和图4所示。
【制备不同配方的止血抗菌敷料】
一种胶原蛋白止血抗菌敷料，按质量百分比计算，包括如下组分：
上述溶液的浓度说明：
邻苯二酚改性聚氨基酸溶液，浓度为0.5±0.1g/mL；
凝胶赋形剂溶液，即氧化刺槐豆胶与卡拉胶的混合溶液，氧化刺槐豆胶和卡拉胶的质量比为3:7，总的质量浓度为15.2wt%；
人源胶原蛋白溶液为20.05wt%的人源胶原蛋白的水溶液。
对上述止血敷料进行孔结构分析、力学性能测试、抗菌性测试、细胞毒性测试和溶血测试，结果列于表5。
力学性能分析具体为：将干燥的试样按GB /T 634-1996切成标准样条，采用INSTRON 5560型万能材料实验机测试止血海绵的力学性能。
溶血测试具体为：取抗凝后的老鼠血以1000 rmp/min离心10min，获得红细胞。随后，用PBS洗涤红细胞3次，最后稀释至5%(v/v)。然后，将红细胞溶液滴在材料表面，在37℃的摇床中孵育1h。以去离子水做为阳性对照，PBS为阴性对照。随后，将样品转移到1.5ml EP管中，以1000 rmp/min离心10min。用酶标仪在450nm波长处测定上清液的吸光度，并计算溶血率。
细胞毒性测试具体为：取一管冻存的3T3细胞逐步解冻，置于细胞培养基中孵育3天。将3T3细胞接种于96孔板中，密度为3000个/孔。第二天细胞贴壁后，分别取材料的浸提液来替换细胞培养液，并在37℃培养箱内共培养24h。在预设时间，用cck8染色液与细胞孵育2h，在酶标仪450nm处测定每个孔的吸光度，计算细胞存活率。
抑菌活性测试具体为：按照上述测试例3的方法得到菌落数后，根据以下公式I还原稀释前原菌液中细菌/真菌的个数N，再根据公式II得到抑菌率。
式中，n为培养皿中的菌落数，D为稀释倍数。
式中，N0为空白组的菌个数，Ni为试验组的菌个数。
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 (9)
1.一种胶原蛋白止血抗菌敷料的制备方法，其特征在于，所述敷料包括以下组分：邻苯二酚改性聚氨基酸、人源胶原蛋白、凝胶赋形剂和中药提取物；
所述聚氨基酸包括半胱氨酸及赖氨酸、精氨酸、组氨酸、色氨酸中的至少一种；所述凝胶赋形剂为氧化刺槐豆胶与卡拉胶的复合物；
所述胶原蛋白止血抗菌敷料的制备方法如下：
制备聚氨基酸：以三乙胺为引发剂，DMF为溶剂、氨基酸环内酸酐单体进行开环聚合反应，反应物用无水乙醚沉降析出、洗涤干燥后得到带有保护基的聚氨基酸；将带有保护基的聚氨基酸溶解于三氟乙酸中，加入溴化氢/乙酸混合液，反应除去保护基，粗产物经无水乙醚沉降析出、洗涤干燥、透析纯化后得到聚氨基酸；
制备邻苯二酚改性聚氨基酸：在DMPA的催化下，聚氨基酸与4-烯丙基邻苯二酚在THF/甲醇混合溶剂中进行紫外光照射反应，产物透析、纯化、干燥得到邻苯二酚改性聚氨基酸；
制备复合凝胶：分别配制邻苯二酚改性聚氨基酸水溶液、凝胶赋形剂水溶液并灭菌备用，另外配置人源胶原蛋白的水溶液；首先混合邻苯二酚改性聚氨基酸水溶液和人源胶原蛋白的水溶液，室温下搅拌1h后静置12h；然后继续加入凝胶赋形剂水溶液，搅拌均匀后升温至45±2℃下搅拌反应6h，得到的凝胶产物；
制备止血海绵：将凝胶产物放入模具中，在冷干机中冷冻干燥得止血海绵；
浸绩含中药成分的甲醇溶液得到止血抗菌敷料：配置含中药的甲醇溶液，将得到的止血海绵浸绩于溶液中，在常温下吹干得到止血抗菌敷料。
2.根据权利要求1所述的一种胶原蛋白止血抗菌敷料的制备方法，其特征在于，所述聚氨基酸为赖氨酸-半胱氨酸-酪氨酸-组氨酸的共聚物。
3.根据权利要求2所述的一种胶原蛋白止血抗菌敷料的制备方法，其特征在于，所述聚氨基酸，各氨基酸的质量百分比含量为：半胱氨酸、赖氨酸、酪氨酸、组氨酸=14% : 7% :14% : 65%。
4.根据权利要求1所述的一种胶原蛋白止血抗菌敷料的制备方法，其特征在于，所述凝胶赋形剂中，氧化刺槐豆胶与卡拉胶的质量比例为(2:8)~(4:6)。
5.根据权利要求1所述的一种胶原蛋白止血抗菌敷料的制备方法，其特征在于，所述中药成分提取物为三七素、积雪草苷、白芨多糖中的一种或多种。
6.根据权利要求1或5所述的一种胶原蛋白止血抗菌敷料的制备方法，其特征在于，所述中药提取物为积雪草苷。
7.根据权利要求1所述的一种胶原蛋白止血抗菌敷料的制备方法，其特征在于，按质量百分比计算，包括以下组分：30~40%邻苯二酚改性聚氨基酸、20~30%人源胶原蛋白、30~60%凝胶赋形剂。
8.根据权利要求1所述的一种胶原蛋白止血抗菌敷料的制备方法，其特征在于，所氨基酸环内酸酐单体选自L-酪氨酸-NCA、苄氧羰基-L-赖氨酸-NCA、L-半胱氨酸-NCA、苄氧羰基-L-精氨酸-NCA、苄氧羰基-L-组氨酸-NCA、苄氧羰基-L-色氨酸-NCA、甘氨酸-NCA中的多种。
9.根据权利要求1所述的一种胶原蛋白止血抗菌敷料的制备方法，其特征在于，所述冷冻温度为-50~-80℃，时间为12~24h。
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| CN109731127A (zh) * | 2019-02-27 | 2019-05-10 | 西南交通大学 | 一种多孔止血海绵及其制备方法 |
| CN113214476A (zh) * | 2021-05-11 | 2021-08-06 | 上海交通大学 | 一种仿生糖聚肽水凝胶及其制备方法和应用 |
| CN114984300A (zh) * | 2022-05-30 | 2022-09-02 | 浙江大学 | 一种强韧抗菌水凝胶敷料及其制备方法 |
| CN114835892A (zh) * | 2022-06-02 | 2022-08-02 | 南方科技大学 | 一种阳离子型共聚氨基酸及其制备方法 |
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