CN115300666B - 一种生物启发协同抗菌水凝胶敷料及其制备方法 - Google Patents
一种生物启发协同抗菌水凝胶敷料及其制备方法 Download PDFInfo
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Abstract
本发明公开一种生物启发协同抗菌水凝胶敷料及其制备方法，包括DCS‑OSA席夫碱交联生物相容性水凝胶基底，其中，OSA连接Cu‑MOF并且掺入rPDA NPs；其制备方法包括步骤：(1)分别制备DCS、OSA、Cu‑MOF、rPDA NPs；(2)将OSA和Cu‑MOF合成OSA‑MOF；(3)将OSA‑MOF和DCS溶液混合后，加入rPDA NPs后搅拌，最终得到DCS‑OSAM‑rPDA水凝胶。
Description
技术领域
本发明涉及一种水凝胶伤口敷料，尤其涉及一种受生物行为启发设计的、具有协同抗菌作用的伤口敷料及其制备方法。
背景技术
近年来，皮肤伤口在人类生活中的频繁出现导致伤口治疗成为了一个重要的健康问题，往往消耗着高昂的经济和社会成本。伤口愈合是一个复杂、动态的生物学过程，涉及多组织细胞的协同作用，包括止血、炎症、增殖和重塑这四个独特而又重叠的阶段。在伤口愈合的初期，止血是面临的第一个重要问题；之后，细菌感染会导致严重的组织损伤、恶化伤口愈合；中性粒细胞和巨噬细胞在伤口部位聚集并产生大量活性氧(ROS)，ROS在伤口愈合的增殖和重塑阶段影响成纤维细胞向伤口的迁移和肉芽组织的形成。针对上述挑战，多种生物材料已被开发用于伤口愈合，包括静电纺丝纤维、聚合物薄膜、多孔泡沫和水凝胶。水凝胶是由壳聚糖、聚乙二醇等亲水聚合物的物理或化学键交联组成的三维(3D)网络，不溶性亲水结构显示出吸收伤口渗出物的显着潜力，可以为伤口提供湿润环境并允许氧气扩散以加速愈合。虽然水凝胶伤口敷料的使用在一定程度上促进了伤口愈合，但其应用只针对上述伤口愈合中的单个问题，未能在伤口愈合的全过程中产生协同作用，这极大地影响了每个阶段都高度协调的伤口愈合的最终效果。此外，这些水凝胶中的大多数只能修复单一类型的伤口，这限制了它们在临床中的广泛应用。因此，亟待开发出兼具止血、抗感染、抗氧化等多种功能于一体的新型水凝胶伤口敷料，能够在不同伤口的整个愈合过程中发挥协同作用，用于全过程促进伤口愈合。
细菌感染所引起的组织恶化是在伤口治疗过程中所面临的最棘手的问题，因此抗菌无疑是伤口愈合过程中最关键且被关注最多的部分。抗生素是对抗细菌感染的常用方法，但是抗生素的使用存在许多弊端。因此，迫切需要开发安全、高效、环境友好的无抗生素治疗方法。条件触发抗菌策略和主动抗菌策略能够很好的规避上述安全性问题，引起了人们极大的兴趣。常用的条件触发抗菌策略主要包括光热疗法(PTT)和光动力疗法(PDT)，但二者都高度依赖光激发纳米材料的合成和特定波长光源等外加条件，在实际应用中操作复杂且难以控制。主动抗菌策略可以自发的直接接触或间接释放小分子物质对细菌外膜进行破坏进而杀灭细菌，在实际应用中抗菌效果虽然不强但不需复杂的外加条件。为了解决某种单一抗菌剂的性能缺陷，开发具有效率高、疗效久、环境污染小、生物相容性好、不易产生耐药性的新型有机/无机复合抗菌材料，在伤口愈合等实际抗感染应用中具有巨大的潜力。
金属有机骨架(MOF)是一类由金属离子或与多齿有机配体组成的配位多孔材料，具有超高比表面积、可调且均匀的孔隙结构和良好的热稳定性等特点，在药物递送、生物传感、生物成像等生物医学领域具有广泛的应用。有趣的是，MOF是由金属离子-无机材料和有机配体-有机材料复合而成的新型无机/有机多孔复合材料，它兼具了有机抗菌剂的高效性和无机抗菌剂的稳定性，通过协同作用能够大幅提升抗菌性能和适用范围，具有耐热性高、安全性好、抗菌效率高、抗菌实效快、抗菌持续性好、稳定性好、不易产生耐药性等优点，非常适合用于医疗抗菌。HKUST-1通过释放破坏细菌细胞膜的Cu2+对金黄色葡萄球菌和大肠杆菌具有显着的抗菌活性。最近报道了一种基于IRMOF-3有机配体合成后修饰的新方法以引入抗菌功能，对大肠杆菌、金黄色葡萄球菌等多种细菌的抗菌性能得到了提高，并且优于商业抗生素的抗菌效果。MOF的抗菌机制主要依托在溶液环境中抗菌成分释放后的自由运动，进而与细菌进行接触杀灭。然而，伤口环境具有复杂、多变的特性，伤口敷料需要在固体皮肤层、液体分泌物和外部空气等不同环境内对细菌进行破坏，如活性氧(ROS)等抗菌物质的寿命较短、扩散距离有限，再加上细菌的运动的干扰，这就导致了细菌与抗菌物质之间的接触效率明显降低，大大减弱了MOF的抗菌效果，使得伤口感染治疗效果不理想。因此，仅仅靠MOF的细菌捕获能力和杀菌效率仍然不足以用于伤口环境抗感染及促进伤口愈合的临床应用。
自然选择的过程导致了小到分子结构、大到生物行为的演变，这些材料、结构、模型、系统和过程已针对多种多样的特定功能进行了优化。基于大自然设计原则的仿生学及生物启发，被用来设计和构建各种高度复杂精密的工程模型，并利用丰富的科学知识来解决人类面临的问题及挑战。蜘蛛作为一类优秀的猎手，长立于昆虫类动物食物链的顶端。其狩猎过程大致分为三步：首先通过蜘蛛网将猎物进行固定，接下来通过獠牙咬破猎物的外层皮肤，最后在猎物体内释放毒液以对猎物进行全过程协同杀灭。受蜘蛛捕获猎物的生物行为启发，包封在水凝胶中的MOF等功能性纳米颗粒类似于不灵活但却具有杀伤性的“蜘蛛”，而水凝胶网络则可以设计成具有锚定、捕获细菌功能的“蜘蛛网”。因此，将Cu-MOF和rPDA NPs引入水凝胶中作为功能成分，通过整合“蛛网捕获猎物”、“獠牙破坏外膜”、“毒液内部杀伤”等多部分功能来设计高效协同抗菌的功能性纳米颗粒-水凝胶复合伤口敷料，兼具止血、抗感染、抗氧化等多种功能于一体，用于全过程促进伤口愈合。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题包括：
一、单一种类抗菌剂性能较弱。
二、仅靠MOF的细菌捕获能力和杀菌效率仍然不足以用于伤口环境抗感染及促进伤口愈合的临床应用。
三、单一功能的水凝胶未能在伤口愈合的全过程中产生协同促进作用。
为实现上述目的，本发明首先提供了一种生物启发协同抗菌水凝胶敷料，包括DCS-OSA席夫碱交联生物相容性水凝胶基底，其中，OSA连接Cu-MOF并且掺入rPDA NPs。
本发明还提供了一种生物启发协同抗菌水凝胶敷料的制备方法，包括步骤：
(1)分别制备DCS、OSA、Cu-MOF、rPDA NPs；
(2)将OSA和Cu-MOF合成OSA-MOF；
(3)将OSA-MOF和DCS溶液混合后，加入rPDA NPs后搅拌，最终得到DCS-OSAM-rPDA水凝胶。
进一步地，步骤(1)中，DCS通过如下方式制备：将壳聚糖溶于乙酸水溶液中，室温搅拌；为使反应更均匀，在乙醇中加入十二醛，然后将此溶液加入壳聚糖溶液中，搅拌直至溶解；然后将过量的氰化硼氢化钠缓慢加入壳聚糖溶液中，在室温下连续搅拌反应后，用氢氧化钠溶液将pH调节至中性后，用乙醇沉淀DCS；沉淀用70％-100％乙醇洗涤，直至pH值为中性；将产物真空干燥至恒重，最后将所得固体研磨，得到白色细粉。
进一步地，步骤(1)中，OSA通过如下方式制备：将海藻酸钠溶于超纯水中，配成1％海藻酸钠水溶液，然后在海藻酸钠溶液中加入NaIO4，室温避光搅拌反应后，通过在搅拌下向反应溶液中加入乙二醇来淬灭氧化；反应结束后，将溶液在超纯水中用透析膜进行透析，多次换水直至透析液中不含NaIO4；将透析液冷冻干燥，得到OSA。
进一步地，步骤(1)中，Cu-MOF通过如下方式制备：将Cu(NO3)2·2.5H2O和3-氨基-5-巯基-1,2,4-三氮唑(ATMA)溶解在DMF/C2H5OH混合溶剂中，将混合溶液加热反应，反应结束后，取出反应液洗涤后，将沉淀物重新溶于DMF/C2H5OH混合溶剂中，在真空下继续反应后再次洗涤，得到最终产物Cu-MOF。
进一步地，步骤(1)中，rPDA NPs通过如下方式制备：首先合成了聚多巴胺纳米粒子：将盐酸多巴胺溶解在超纯水和乙醇的混合溶液中，然后滴加氨水到溶液体系中；将反应液连续搅拌反应结束后，溶液用超纯水洗涤，得到PDA纳米颗粒；然后，将制备的PDA与抗坏血酸混合反应后洗涤确保体系中不再有未反应的抗坏血酸后得到还原型聚多巴胺纳米颗粒。
进一步地，步骤(2)中，OSA-MOF通过如下方式制备：首先，在两个容器中加入PH值4.5～6的MES缓冲液，然后分别加入EDC和NHS，将两种溶液加入OSA 中，活化羧基的反应结束后，向溶液中加入Cu-MOF继续反应；最后，洗涤反应产物得到OSAM。
进一步地，步骤(3)中，将25％w/v OSAM、1.5％w/v DCS溶液混合，加入一定浓度的rPDA NPs，然后在室温下搅拌，使溶液由液态变为凝胶状，最终得到DCS-OSAM-rPDA水凝胶。
本发明的技术效果有：
一、生物启发协同抗菌水凝胶整合了“蛛网捕获猎物”、“獠牙破坏外膜”、“毒液内部杀伤”等多部分功能，对其生物启发协同抗菌机制进行了深入的研究。水凝胶具有优异的抗菌效果，对于金黄色葡萄球菌和大肠杆菌均有99.9％以上的抗菌率。
二、通过功能性材料在水凝胶体系中的引入，使得复合水凝胶在伤口愈合的全部过程中做到初期止血、中期抗感染和消炎、后期抗氧化和促进愈合，开发出兼具止血、抗感染、抗氧化等多种功能于一体的新型水凝胶伤口敷料，能够在不同伤口的整个愈合过程中发挥协同作用，解决了了单一功能水凝胶在促进伤口愈合过程中的限制，从而用于全过程促进伤口愈合，对于伤口愈合临床应用具有潜在价值。建立感染的大鼠全层皮肤损伤模型之后进行实体评价，复合水凝胶处理10天后伤口闭合率高达90％。
三、该水凝胶材料来源广泛，成本较低，制备工艺简单，是一种具有临床转化前景的新型抗菌水凝胶敷料。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个较佳实施例中的生物启发协同抗菌水凝胶宏观合成过程及自愈合性能验证示意图；
图2是本发明的一个较佳实施例中的功能性纳米颗粒及水凝胶合成的表征实验示意图；
图3是本发明的一个较佳实施例中的生物启发协同抗菌水凝胶体外抗菌性能验证示意图。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例，使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现，本发明的保护范围并非仅限于文中提到的实施例。
如图1-2所示，本实施例中以DCS(十二烷基壳聚糖)-OSA(氧化海藻酸钠)的席夫碱交联作为生物相容性水凝胶基底，连接上Cu-MOF(铜基金属有机框架)并且掺入rPDA NPs(还原聚多巴胺纳米颗粒)，通过简单反应制备了一种具有仿生协同抗菌功能的水凝胶敷料，并可以用于全过程促进伤口愈合。其具体制备和验证步骤如下：
(1)功能性纳米颗粒的合成：
Cu-MOF的合成：将Cu(NO3)2·2.5H2O(3mmol)和3-氨基-5-巯基-1,2,4-三氮唑(ATMA)(4.5mmol)溶解在100mL DMF/C2H5OH中混合溶剂(DMF：C2H5OH＝1:1)，将混合溶液加热至120℃，继续反应12h。反应结束后，取出反应液，以8000rpm洗涤。洗涤后，将沉淀物重新溶于DMF/C2H5OH混合溶剂(DMF：C2H5OH＝1:1)中，在120℃真空下继续反应24h，反应后再次洗涤，得到最终产物Cu-MOF。
rPDA NPs的合成：首先合成了聚多巴胺(PDA)纳米粒子：将400mg盐酸多巴胺溶解在90mL超纯水和30mL乙醇的混合溶液中，然后快速滴加1.5mL氨水到溶液体系中。将反应液连续搅拌24小时，可以观察到溶液由透明变为黄色，最后变为深棕色。反应结束后，溶液用超纯水洗涤两次，每次15000rpm离心10分钟，得到PDA纳米颗粒。其次，PDA被抗坏血酸还原生成还原型聚多巴胺纳米颗粒(rPDA NPs)：将制备的PDA与适量的抗坏血酸(300mM)混合30分钟。反应后，溶液至少洗涤3次，以确保体系中不再有未反应的抗坏血酸。
(2)生物启发协同抗菌水凝胶敷料的制备：
十二烷基改性壳聚糖(DCS)的合成：将1g壳聚糖(CS)溶于50mL 2％乙酸水溶液中，室温搅拌。为使反应更均匀，在40mL乙醇中加入一定量的十二醛，然后将此溶液加入上述壳聚糖溶液中，搅拌4小时直至溶解。然后将过量的氰化硼氢化钠(NaCNBH4:CS＝3:1)缓慢加入溶液体系中，在室温下连续搅拌14h。反应后，用氢氧化钠溶液将pH调节至7.0，用乙醇沉淀DCS。沉淀用70％-100％乙醇洗涤4次以上，直至pH值为中性。将产物在40℃真空干燥至恒重，最后将所得固体研磨，得到白色细粉。
氧化海藻酸钠(OSA)的合成：1g海藻酸钠溶于100mL超纯水中，配成1％海藻酸钠水溶液，然后在海藻酸钠溶液中加入8g NaIO4，室温避光搅拌过夜。反应24小时后，通过在搅拌下向反应溶液中加入2mL乙二醇0.5小时来淬灭氧化。反应结束后，将溶液在超纯水(5L)中用透析膜(MWCO～3500Da)进行透析，多次换水直至透析液中不含高碘酸钠。通过将0.5mL透析液等分试样添加到0.5mL 1％AgNO3溶液中并确保没有沉淀物来检查NaIO4的存在。透析72小时后，将透析液冷冻干燥，得到OSA。
氧化海藻酸钠-MOF(OSAM)的合成：羧基通过EDC和NHS活化，使OSA和Cu-MOF通过形成酰胺键连接。首先，在两个试管中加入250μL MES缓冲液(PH：4.5～6)，然后分别加入4mgEDC和6mg NHS，将两种溶液各200μL加入到100mg OSA中，活化羧基的反应持续30分钟。反应结束后，向溶液中加入10μL Cu-MOF(2.5mg/mL)，继续反应2小时。最后，洗涤反应产物得到OSAM。
水凝胶的合成：为了合成DCS-OSAM-rPDA水凝胶，将25％w/v OSAM、1.5％w/v DCS溶液混合，加入3.5-140μg/mL的rPDA NPs，然后在室温下搅拌15分钟。观察到溶液由液态变为凝胶状，最终得到DCS-OSAM-rPDA水凝胶。将水凝胶在-80℃冷冻干燥24小时。将冻干的水凝胶样品粘贴在样品板上的导电胶带上，喷金(喷涂时间为100s)，使干燥的水凝胶导电。然后用扫描电子显微镜拍照进行测试。通过FT-IR验证合成反应的成功性来对水凝胶及其组分进行表征。
(3)生物启发协同抗菌水凝胶协同抗菌性能的验证：
如图3所示，以革兰氏阴性菌的代表菌株大肠杆菌和革兰氏阳性菌的代表菌株金黄色葡萄球菌为模型菌，研究材料的抗菌性能。根据中华人民共和国国家标准《GB/T20944.3-2008》，抗菌性能试验采用振荡法。将2mL初接种菌悬液加入9mL营养肉汤中，混匀；将1mL上述菌液混入9mL营养肉汤中；1mL混合在9mL PBS缓冲溶液中；将5mL混入45mL PBS缓冲溶液中，成功制备104CFU/mL菌悬液，向锥形瓶中加入70mL PBS缓冲液，分别加入样品和对照，再加入5mL菌悬液，将培养物在37℃和130r/min下摇床18h。摇匀后，从锥形瓶中取出100μL菌液，铺在营养琼脂培养基上。37℃培养24小时后，计算每个平板上的菌落数。阴性对照中获得的菌落数为NC，样品中获得的菌落数为NS，抗菌率(R，％)按下式计算：R＝(NC–NS)/NC×100％。根据标准，采用抑制圈法(ZOI)验证水凝胶及其成分的抗菌性能：将100μL上述菌液滴于琼脂板上，均匀涂抹。在其上加入水凝胶及其组分并在37℃下孵育18小时，最后观察ZOI的大小。通过将Cu-MOF的浓度设置为2.5、5、10、25、50μg/mL，将初始菌液和抗菌材料溶液按照1:1的体积比混合过夜，滴加100μL菌液于琼脂平板上均匀涂抹，按标准方法计算最低抑菌浓度(MIC)。两种细菌在抗菌材料处理前后取样，戊二醛固定，酒精梯度脱水。用扫描电镜观察细菌形态。
(4)生物启发协同抗菌水凝胶全过程促进伤口愈合性能的验证：
小鼠断尾止血实验：用4％水合氯醛腹腔注射麻醉小鼠。麻醉后将小鼠置于实验操作台上，对尾部进行消毒。切断一半的尾巴，自由流血10s。10s后擦去渗出的血液，将尾巴插入纱布和水凝胶中进行止血治疗，并建立相同伤口而不做外界干预的小鼠阴性对照组。每10s取出止血材料，观察出血是否仍在继续。当伤口止血时，记录从伤口处理到伤口止血的时间。在检测失血量的实验中，处理前称量止血材料质量m0，实验完成后称量材料质量m1。失血量的计算公式为：失血量＝m1-m0。
对氧化应激损伤细胞的保护效果验证：为了验证rPDA NPs对细胞的氧化应激保护作用，测量了水凝胶处理后用H2O2培养的293T的细胞活力。首先将293T细胞接种在6孔板中并培养过夜。去除细胞培养基，然后将贴壁细胞与水凝胶在37℃温育24小时。去除多余的水凝胶，将细胞与H2O2在37℃下孵育24小时，并通过CCK-8试剂盒评估细胞活力。
细胞毒性试验：293T细胞在96孔细胞培养皿中培养24小时，每孔4000个细胞(每孔100μL)，以便在实验前附着。然后，将不同的材料添加到孔中并再孵育24小时。CCK-8试剂盒用于确定实验组的细胞活力，并表示为相对于对照细胞的细胞活性百分比。
伤口愈合体内实验：SD雄性小鼠(平均体重180克)通过腹腔注射4％水合氯醛(0.1毫升/10克)进行麻醉，并用电动理发器剃毛。然后在每只小鼠的背部剪成全层皮肤缺损伤口(圆形，直径1厘米)。之后，用100μL 108CFU/mL的金黄色葡萄球菌感染伤口24小时，形成感染性伤口。用生理盐水洗涤后，实验组用水凝胶样品处理，而对照组只用生理盐水处理。术后0d、1d、2d、4d、6d、8d、9d、10d用相机在固定高度拍摄不同组别照片，记录创面大小(A)及图像，并通过公式计算伤口闭合率(％)：伤口愈合率(％)＝(A0–AT)/A0×100％。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
Claims (7)
1.一种生物启发协同抗菌水凝胶敷料的制备方法，其特征在于，包括步骤：
(1)分别制备十二烷基壳聚糖(DCS)、氧化海藻酸钠(OSA)、Cu-MOF、rPDA NPs；
(2)将氧化海藻酸钠(OSA)和Cu-MOF合成Cu-OSA-MOF；
(3)将Cu-OSA-MOF和十二烷基壳聚糖(DCS)溶液混合后，加入rPDANPs后搅拌，最终得到DCS-OSAM-rPDA水凝胶。
2.如权利要求1所述的生物启发协同抗菌水凝胶敷料的制备方法，其中，步骤(1)中，十二烷基壳聚糖(DCS)通过如下方式制备：将壳聚糖溶于乙酸水溶液中，室温搅拌；为使反应更均匀，在乙醇中加入十二醛，然后将此溶液加入壳聚糖溶液中，搅拌直至溶解；然后将过量的氰化硼氢化钠缓慢加入壳聚糖溶液中，在室温下连续搅拌反应后，用氢氧化钠溶液将pH调节至中性后，用乙醇沉淀十二烷基壳聚糖(DCS)；沉淀用70％-100％乙醇洗涤，直至pH值为中性；将产物真空干燥至恒重，最后将所得固体研磨，得到白色细粉。
3.如权利要求1所述的生物启发协同抗菌水凝胶敷料的制备方法，其中，步骤(1)中，氧化海藻酸钠(OSA)通过如下方式制备：将海藻酸钠溶于超纯水中，配成1％海藻酸钠水溶液，然后在海藻酸钠溶液中加入NaIO4，室温避光搅拌反应后，通过在搅拌下向反应溶液中加入乙二醇来淬灭氧化；反应结束后，将溶液在超纯水中用透析膜进行透析，多次换水直至透析液中不含NaIO4；将透析液冷冻干燥，得到氧化海藻酸钠(OSA)。
4.如权利要求1所述的生物启发协同抗菌水凝胶敷料的制备方法，其中，步骤(1)中，Cu-MOF通过如下方式制备：将Cu(NO3)2·2.5H2O和3-氨基-5-巯基-1,2,4-三氮唑(ATMA)溶解在DMF/C2H5OH混合溶剂中，将混合溶液加热反应，反应结束后，取出反应液洗涤后，将沉淀物重新溶于DMF/C2H5OH混合溶剂中，在真空下继续反应后再次洗涤，得到最终产物Cu-MOF。
5.如权利要求1所述的生物启发协同抗菌水凝胶敷料的制备方法，其中，步骤(1)中，rPDANPs通过如下方式制备：首先合成了聚多巴胺纳米粒子：将盐酸多巴胺溶解在超纯水和乙醇的混合溶液中，然后滴加氨水到溶液体系中；将反应液连续搅拌反应结束后，溶液用超纯水洗涤，得到PDA纳米颗粒；然后，将制备的PDA与抗坏血酸混合反应后洗涤确保体系中不再有未反应的抗坏血酸后得到还原型聚多巴胺纳米颗粒。
6.如权利要求1所述的生物启发协同抗菌水凝胶敷料的制备方法，其中，步骤(2)中，Cu-OSA-MOF通过如下方式制备：首先，在两个容器中加入PH值4.5～6的MES缓冲液，然后分别加入EDC和NHS，将两种溶液加入氧化海藻酸钠(OSA)中，活化羧基的反应结束后，向溶液中加入Cu-MOF继续反应；最后，洗涤反应产物得到Cu-OSA-MOF。
7.如权利要求1所述的生物启发协同抗菌水凝胶敷料的制备方法，其中，步骤(3)中，将25％w/v Cu-OSA-MOF、1.5％w/v十二烷基壳聚糖(DCS)溶液混合，加入3.5-140μg/mL的rPDANPs，然后在室温下搅拌，使溶液由液态变为凝胶状，最终得到DCS-OSAM-rPDA水凝胶。
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