CN114796598A - 一种促难愈性创面愈合的抗菌水凝胶及应用 - Google Patents
一种促难愈性创面愈合的抗菌水凝胶及应用 Download PDFInfo
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Abstract
本发明属于生物医用高分子材料和医疗器械领域，具体涉及一种促难愈性创面愈合的抗菌水凝胶及应用。所述方法包括：制备单宁酸复合纳米粒子，将单宁酸复合纳米粒子与天然高分子聚合物溶液混合，最后通过特异性酶促交联反应一步制备纳米复合的水凝胶。由该方法制备的水凝胶具有多重功能，包括适配的机械性能、优异的抗菌性能、促胞内一氧化氮生成性能、优异的生物相容性以及促血管生成性能等，可广泛用于调节各类难愈性创面的病理微环境，加速创面愈合。
Description
技术领域
本发明属于生物医用高分子材料和医疗器械领域，具体涉及一种促难愈性创面愈合的抗菌水凝胶及应用。
背景技术
本发明公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
难愈性创面，特别是糖尿病创面，会经历异常的炎症浸润，还容易受到细菌的侵蚀。细菌感染诱发的慢性皮肤损伤更加难以修复，加重的炎症反应最终会导致细胞死亡和组织坏死，并抑制伤口的修复过程。此外细菌感染会加剧创面由于缺血、缺血管诱发的氧气匮乏症状，不利于伤口修复。因此，抑制创面的细菌感染和过度炎症是治疗难愈性创面的重要一环。
近期，基于“湿性愈合理论”发展起来的水凝胶敷料，越来越广泛的应用于辅助伤口治疗。抗菌水凝胶常通过引入抗生素来获取抗菌性能，虽然抗生素抑菌的功能被广泛应用，然而其滥用会增强细菌的耐药性，不建议长期使用。同时，单一的抗菌功能来源会限制水凝胶的抗菌效果。因此，亟需新一代抗菌水凝胶敷料来促进难愈性创面的伤口修复。
发明内容
为了解决上述技术的问题，本发明提供了一种促难愈性创面愈合的抗菌水凝胶及应用。使用具有内源抗菌性能的材料，同时向其中均匀分散具有光热响应性的功能性纳米粒子，通过温和可控的反应条件合成水凝胶，将功能性纳米粒子的光热抗菌疗法与抗菌材料结合使用，使水凝胶显示出增强的抗菌活性。该抗菌水凝胶还具有适配的机械性能和良好的生物相容性，可以通过抵御细菌感染，降低炎症水平，促进血管新生来改善伤口难愈的状况，可以广泛应用于难愈性皮肤修复、组织再生等领域。
为实现上述目的，本发明采用以下的技术方案：
(1)制备单宁酸复合纳米粒子；
(2)将具有内源抗菌性能的天然高分子材料A溶解在特异性酶溶液中制得溶液一；
(3)将天然高分子材料B溶解在水溶液中制得溶液二；
(4)将复合纳米粒子均匀分散到步骤(3)制得的溶液二中得到溶液三；
(5)均匀混合溶液一及溶液三后静置一段时间使酶促反应完全，天然高分子材料A与B之间会形成三维互穿网络，制得抗菌水凝胶。
优选的，步骤(1)包括以下步骤：通过超声分散金属盐溶液及单宁酸溶液，混合后调节溶液pH为6～8，超声反应6～24小时，待反应完全后离心并洗涤产物，真空干燥8～36小时，得到单宁复合纳米粒子。其中金属盐包括但不限于三氯化铁、硫酸铜和氯化锰，金属盐溶液浓度为0.1～5mol/L，单宁酸溶液浓度为0.5～10g/L。
优选的，步骤(2)中所述天然高分子材料A包括但不限于聚赖氨酸、壳聚糖及其衍生物。
优选的，步骤(2)中所述特异性酶溶液包括但不限于谷氨酰胺转氨酶溶液；特异性酶溶液浓度为50～200U/mL，溶液一的浓度为2～20g/L。
优选的，步骤(3)中所述天然高分子材料B包括但不限于丝素蛋白和明胶；溶液二的浓度为10～20g/L。
优选的，步骤(4)中所述的溶液三的纳米粒子浓度为0.01～0.1g/mL。
优选的，步骤(5)中所述的溶液一与溶液三的体积比为4∶1～1∶5。
本发明还提供由上述方法制备的抗菌水凝胶。
本发明还提供由上述方法制备的抗菌水凝胶调节各类难愈性创面的病理微环境，加速创面愈合，以及在组织工程、临床治疗领域的广泛应用。
本发明的有益效果在于：
本发明引入生物相容性良好、吸湿能力优异的天然生物材料聚氨基酸和聚多糖为主体材料，并向其中均匀分散单宁酸复合纳米粒子，通过酶促反应简易高效地一步构建水凝胶支架，反应条件温和可控。通过天然材料固有的抗菌特性与纳米粒子的光热响应性结合，赋予水凝胶支架优异的联合抗菌性能，有较好的抑菌杀菌效果，可以有效避免创面遭受细菌感染以及一系列并发症。同时，聚氨基酸具有与天然蛋白质相似的二级结构，可模拟细胞外基质的功能结构，构建组织工程多孔支架，能够有效促进损伤后细胞的迁移分化与组织的再生重建，另外，水凝胶可以促进细胞胞内一氧化氮的生成从而能够促进伤口的血管新生。该抗菌水凝胶在医用伤口敷料、细胞支架等生物医疗领域具有广阔的应用价值。
附图说明
图1是本发明所述抗菌水凝胶的成胶过程图。
图2是本发明所述抗菌水凝胶的孔径图片(右图为局部放大图)。
图3是本发明所述抗菌水凝胶对大肠杆菌作用后的细菌活/死染色结果。
图4是本发明所述抗菌水凝胶的细胞相容性实验结果。
图5是本发明所述抗菌水凝胶胞内一氧化氮生成的实验结果。
图6是本发明所述抗菌水凝胶促进细菌感染的糖尿病小鼠伤口愈合的实验结果。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例中所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详述的本发明。
实施例1
(1)将单宁酸粉末超声溶解在去离子水中，配置成浓度为1g/L的溶液。将三氯化铁粉末通过超声溶解在去离子水中，配置成浓度为0.5mol/L的溶液。将单宁酸溶液与三氯化铁溶液混合，调节溶液pH至7.0，继续超声反应12小时后，离心处理，并分别使用乙醇和去离子水洗涤离心后所得的固体三次后，将所得固体60℃真空干燥24小时，得到铁/单宁酸复合纳米粒子。
(2)将谷氨酰胺转氨酶粉末溶解在PBS缓冲液中，配置成浓度为50U/mL的特异性酶溶液。使用新制的酶溶液溶解聚赖氨酸粉末，制成浓度为10g/L聚赖氨酸酶溶液。
(3)将明胶颗粒加入去离子水中，60℃水浴溶解，制成浓度为10g/L明胶水溶液。
(4)将步骤(1)制得的铁/单宁酸复合纳米粒子添加至明胶水溶液中，通过超声溶解4小时，制得浓度为0.05g/mL的铁/单宁酸复合纳米粒子的明胶水溶液。
(5)将步骤(2)制得的聚赖氨酸酶溶液与步骤(3)制得的铁/单宁酸复合纳米粒子的明胶水溶液按体积比1∶1混合，震荡后静置待酶促反应完全，聚赖氨酸和明胶通过酶促反应形成三维互穿网络，制得水凝胶样品1(图1为水凝胶样品1从溶液向凝胶转变的过程图)。
实施例2
(1)将单宁酸粉末超声溶解在去离子水中，配置成浓度为3g/L的溶液。将三氯化铁粉末通过超声溶解在去离子水中，配置成浓度为2mol/L的溶液。将单宁酸溶液与三氯化铁溶液混合，调节溶液pH至7.0，继续超声反应18小时后，离心处理，并分别使用乙醇和去离子水洗涤离心后所得的固体三次后，将所得固体60℃真空干燥12小时，得到铁/单宁酸复合纳米粒子。
(2)将谷氨酰胺转氨酶粉末溶解在PBS缓冲液中，配置成浓度为100U/mL的特异性酶溶液。使用新制的酶溶液溶解聚赖氨酸粉末，制成浓度为20g/L聚赖氨酸酶溶液。
(3)将丝素蛋白粉末加入去离子水中溶解，制成浓度为20g/L丝素蛋白水溶液。
(4)将步骤(1)制得的铁/单宁酸复合纳米粒子添加至丝素蛋白水溶液中，通过超声溶解4小时，制得浓度为0.01g/mL的铁/单宁酸复合纳米粒子的丝素蛋白水溶液。
(5)将步骤(2)制得的聚赖氨酸酶溶液与步骤(4)制得的铁/单宁酸复合纳米粒子的丝素蛋白水溶液按体积比1∶1混合，震荡后静置待酶促反应完全，聚赖氨酸和丝素蛋白通过酶促反应形成三维互穿网络，制得水凝胶样品2。
实施例3
(1)将单宁酸粉末超声溶解在去离子水中，配置成浓度为1g/L的溶液。将硫酸铜颗粒溶解在去离子水中，配置成浓度为2mol/L的溶液。将单宁酸溶液与硫酸铜溶液混合，调节溶液pH至6.0，继续超声反应24小时后，离心处理，并分别使用乙醇和去离子水洗涤离心后所得的固体三次后，将所得固体60℃真空干燥12小时，得到铜/单宁酸复合纳米粒子。
(2)将谷氨酰胺转氨酶粉末溶解在PBS缓冲液中，配置成浓度为100U/mL的特异性酶溶液。使用新制的酶溶液溶解壳聚糖粉末，制成浓度为2g/L壳聚糖酶溶液。
(3)将丝素蛋白粉末加入去离子水中溶解，制成浓度为10g/L丝素蛋白水溶液。
(4)将步骤(1)制得的铜/单宁酸复合纳米粒子添加至丝素蛋白水溶液中，通过超声溶解4小时，制得浓度为0.1g/mL的铜/单宁酸复合纳米粒子的丝素蛋白水溶液。
(5)将步骤(2)制得的壳聚糖酶溶液与步骤(4)制得的铜/单宁酸复合纳米粒子的丝素蛋白水溶液按体积比1∶2混合，震荡后静置待酶促反应完全，壳聚糖和丝素蛋白通过酶促反应形成三维互穿网络，制得水凝胶样品3。
实施例4
(1)将单宁酸粉末超声溶解在去离子水中，配置成浓度为5g/L的溶液。将氯化锰颗粒快速搅拌溶解在去离子水中，配置成浓度为2mol/L的溶液。将单宁酸溶液与氯化锰溶液混合，调节溶液pH至7.0，继续超声反应12小时后，离心处理，并分别使用乙醇和去离子水洗涤离心后所得的固体三次后，将所得固体60℃真空干燥12小时，得到锰/单宁酸复合纳米粒子。
(2)将谷氨酰胺转氨酶粉末溶解在PBS缓冲液中，配置成浓度为100U/mL的特异性酶溶液。使用新制的酶溶液溶解聚赖氨酸粉末，制成浓度为20g/L聚赖氨酸酶溶液。
(3)将明胶颗粒加入去离子水中，60℃水浴溶解，制成浓度为10g/L明胶水溶液。
(4)将步骤(1)制得的锰/单宁酸复合纳米粒子添加至明胶水溶液中，通过超声溶解4小时，制得浓度为0.1g/mL的锰/单宁酸复合纳米粒子的明胶水溶液。
(5)将步骤(2)制得的聚赖氨酸酶溶液与步骤(4)制得的锰/单宁酸复合纳米粒子的明胶水溶液按体积比1∶2混合，震荡后静置待酶促反应完全，聚赖氨酸和明胶通过酶促反应形成三维互穿网络，制得水凝胶样品4。
实施例5
(1)将单宁酸粉末超声溶解在去离子水中，配置成浓度为1g/L的溶液。将氯化锰颗粒快速搅拌溶解在去离子水中，配置成浓度为2mol/L的溶液。将单宁酸溶液与氯化锰溶液混合，调节溶液pH至7.0，继续超声反应12小时后，离心处理，并分别使用乙醇和去离子水洗涤离心后所得的固体三次后，将所得固体60℃真空干燥12小时，得到锰/单宁酸复合纳米粒子。
(2)将谷氨酰胺转氨酶粉末溶解在PBS缓冲液中，配置成浓度为200U/mL的特异性酶溶液。使用新制的酶溶液溶解聚赖氨酸粉末，制成浓度为20g/L聚赖氨酸酶溶液。
(3)将丝素蛋白粉末加入去离子水中溶解，制成浓度为20g/L丝素蛋白水溶液。
(4)将步骤(1)制得的锰/单宁酸复合纳米粒子添加至丝素蛋白水溶液中，通过超声溶解4小时，制得浓度为0.3g/mL的锰/单宁酸复合纳米粒子的丝素蛋白水溶液。
(5)将步骤(2)制得的聚赖氨酸酶溶液与步骤(4)制得的锰/单宁酸复合纳米粒子的丝素蛋白水溶液按体积比1∶1混合，震荡后静置待酶促反应完全，聚赖氨酸和丝素蛋白通过酶促反应形成三维互穿网络，制得水凝胶样品5。
实施例6
(1)将单宁酸粉末超声溶解在去离子水中，配置成浓度为2g/L的溶液。将三氯化铁粉末通过超声溶解在去离子水中，配置成浓度为1mol/L的溶液。将单宁酸溶液与三氯化铁溶液混合，调节溶液pH至7.0，继续超声反应18小时后，离心处理，并分别使用乙醇和去离子水洗涤离心后所得的固体三次后，将所得固体60℃真空干燥12小时，得到铁/单宁酸复合纳米粒子。
(2)将谷氨酰胺转氨酶粉末溶解在PBS缓冲液中，配置成浓度为200U/mL的特异性酶溶液。使用新制的酶溶液溶解壳聚糖粉末，制成浓度为2g/L壳聚糖酶溶液。
(3)将丝素蛋白粉末加入去离子水中溶解，制成浓度为20g/L丝素蛋白水溶液。
(4)将步骤(1)制得的铁/单宁酸复合纳米粒子添加至丝素蛋白水溶液中，通过超声溶解4小时，制得浓度为0.5g/mL的铁/单宁酸复合纳米粒子的丝素蛋白水溶液。
(5)将步骤(2)制得的壳聚糖酶溶液与步骤(4)制得的铁/单宁酸复合纳米粒子的丝素蛋白水溶液按体积比4∶1混合，震荡后静置待酶促反应完全，壳聚糖和丝素蛋白通过酶促反应形成三维互穿网络，制得水凝胶样品6。
实施例7
以实施例1制备的纳米复合水凝胶进行一系列测试：
(1)抗菌水凝胶的电镜扫描
将实施例1构建的抗菌水凝胶冷冻干燥后，通过扫描电子显微镜观察水凝胶冻干样的微观孔径结构，如图2所示，水凝胶呈现出均匀的孔径，这有利于应用过程中氧气、营养物质的输送，以及相关细胞的迁移。
(2)抗菌水凝胶的的细菌活/死染色测试
将大肠杆菌与抗菌水凝胶共孵育，使用活/死染色试剂盒对大肠杆菌进行染色。以PBS溶液加激光(XL)照射作为对照组，Gel/PL是不添加纳米粒子的水凝胶，Gel/PL@FeIIITA是按照实施例1构建的抗菌水凝胶，Gel/PL@FeIIITA+XL是按照实施例1构建的抗菌水凝胶加激光照射。如图3所示，抗菌水凝胶显示出良好的抗菌效果，激光照射引发的光热响应性进一步提升了水凝胶的抗菌性能。
(3)抗菌水凝胶的细胞毒性测试
在三天的时间中，通过MTT法测试抗菌水凝胶对小鼠成纤维细胞(L929)的细胞毒性大小，以评估其生物安全性。分别测试DMEM+XL(仅培养基加激光照射)，Gel+PL(明胶和聚赖氨酸溶液混合不加谷氨酰胺转氨酶)，Gel/PL，Gel/PL@FeIIITA以及Gel/PL@FeIIITA+XL组的细胞毒性。如图4所示，与抗菌水凝胶共孵育后的细胞长势良好且基本都高于纯DMEM的对照组，即细胞存活率都达到95％以上，展现出抗菌水凝胶良好的生物相容性，
(4)抗菌水凝胶的胞内一氧化氮生成测试
将细胞与抗菌水凝胶共孵育后，使用一氧化氮探针(DAF-FM DA)检测胞内一氧化氮的生成水平，胞内荧光强度与一氧化氮的含量成正比。如图5所示，实验结果表明，随着谷氨酰胺转氨酶的引入，胞内荧光强度有了明显的提升，这表明胞内一氧化氮浓度的提升，这对于促进难愈性创面的血管新生和伤口愈合是有益的。
(5)抗菌水凝胶治疗细菌感染的糖尿病创面效果测试
首先，构建糖尿病小鼠模型并构建全层皮肤缺损伤口。接着，构建细菌感染创面。向创面施用抗菌水凝胶来测试其的创面治疗效果。如图6所示，结果表明，通过联合抗菌作用，施加激光的抗菌水凝胶展现出突出的促进伤口愈合效果。
综上所述，本发明制备的抗菌水凝胶，成胶过程便捷且环境友好，条件温和可控，水凝胶具有良好的杀菌和抑菌性能，适宜的孔径结构，优异的生物相容性，且具有潜在的促血管新生能力，可以显著促进糖尿病伤口愈合，在未来的临床应用中有广阔的前景。
需要说明的是：以上所述仅为本发明优选的实验方案，并非用于限定本发明的权利范围；同以上描述，对于相关技术领域的专门人士应可明了及实施，因此其他未脱离本发明所揭示的精神下所完成的等效改变或修饰，均应包含在申请专利范围中。
Claims (9)
1.一种促难愈性创面愈合的抗菌水凝胶及应用，其特征在于，该方法包括以下步骤：
(1)制备单宁酸复合纳米粒子；
(2)将具有内源抗菌性能的天然高分子材料A溶解在特异性酶溶液中制得溶液一；
(3)将天然高分子材料B溶解在水溶液中制得溶液二；
(4)将复合纳米粒子均匀分散到步骤(3)制得的溶液二中得到溶液三；
(5)均匀混合溶液一及溶液三后静置一段时间使酶促反应完全，天然高分子材料A与B之间会形成三维互穿网络，制得抗菌水凝胶。
2.根据权利要求1所述的制备方法，其特征在于，步骤(1)包括以下步骤：
通过超声分散金属盐溶液及单宁酸溶液，混合后调节溶液pH为6～8，超声反应6～24小时，待反应完全后离心并洗涤产物，真空干燥8～36小时，得到单宁酸复合纳米粒子。其中金属盐包括但不限于三氯化铁、硫酸铜和氯化锰，金属盐溶液浓度为0.1～5mol/L，单宁酸溶液浓度为0.5～10g/L。
3.根据权利要求1所述的制备方法，其特征在于，步骤(2)中所述天然高分子材料A包括但不限于聚赖氨酸、壳聚糖及其衍生物。
4.根据权利要求1所述的制备方法，其特征在于，步骤(2)中所述特异性酶溶液包括但不限于谷氨酰胺转氨酶溶液，酶溶液浓度为50～200U/mL，溶液一的浓度为2～20g/L。
5.根据权利要求1所述的制备方法，其特征在于，步骤(3)中所述天然高分子材料B包括但不限于丝素蛋白和明胶，溶液二的浓度为10～20g/L。
6.根据权利要求1所述的制备方法，其特征在于，步骤(4)中所述的溶液三的纳米粒子浓度为0.01～0.1g/mL。
7.根据权利要求1所述的制备方法，其特征在于，步骤(5)中所述的溶液一与溶液三的体积比为4∶1～1∶5。
8.根据权利要求1～7所述的制备方法即可制备抗菌水凝胶。
9.根据权利要求8制备的抗菌水凝胶具有适配的机械性能、优异的抗菌性能、促胞内一氧化氮生成性能、优异的生物相容性以及促血管生成性能等，可广泛用于调节各类难愈性创面的病理微环境，加速创面愈合，可广泛用于组织工程以及临床治疗领域。
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