CN112868668B - 一种Fe3O4-DA-AMP纳米复合抗菌材料及其制备方法和应用 - Google Patents
一种Fe3O4-DA-AMP纳米复合抗菌材料及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于金属纳米材料技术领域，具体公开了一种Fe3O4‑DA‑AMP纳米复合抗菌材料及其制备方法和应用。本发明通过Fe与DA的酚羟基之间的配位键将DA标记的抗菌肽(DA‑己二酸‑GKRWWKWWRR)连接到Fe3O4上，己二酸作为连接分子。制备得到的Fe3O4‑DA‑AMP纳米复合抗菌材料，具有优异的抗菌性能，并且Fe3O4具有良好的生物相容性，对机体细胞的正常生理活动干扰小，毒性低，安全性高。
Description
技术领域
本发明属于金属纳米材料技术领域，具体涉及一种Fe3O4-DA-AMP纳米复合抗菌材料及其制备方法和应用。
背景技术
随着科技的发展，生活水平的提高，人们对自身居住、工作、生活的环境卫生要求也日益提高，由此促进了抗菌技术和抗菌材料的快速发展，纳米抗菌材料便是其中重要的一种。纳米抗菌材料是一类具备抑菌性能的新型材料，具有比表面积大、反应活性高等优点，可以使微生物包括细菌、真菌、酵母菌、藻类以及病毒等的生长和繁殖保持较低的水平，从而大幅提高材料的抗菌性。近些年大量耐药细菌及多重耐药细菌不断的出现。细菌自发的耐药突变与抗生素的筛选及病原菌的环境适应能力，是临床耐药细菌和多重耐药细菌产生的基础。抗菌肽(AMPs)有着传统抗生素无可比拟的优势，其抗菌机制独特，杀菌作用迅速且不易引发细菌的耐药性，是一类极具潜力的抗菌药物。
现有纳米复合抗菌材料存在一定的细胞毒性并且制备成本高(例如金纳米粒和银纳米粒等)。
磁纳米材料是一种具有尖晶石结构的粉末材料，与一般的纳米材料相同，也具有四个基本的纳米尺寸效应。磁纳米材料的生物相容性较好，可进行生物降解，对人体不存在潜在的细胞毒性，同时也表现出一些有趣的特性：超顺磁性、高饱和磁场、低居里温度以及独特的理化性质，并且表面容易修饰生物功能基团，因此在各种分离技术、药物输送***、磁共振成像显影剂、磁热疗、食品微生物检测等方面得到广泛的应用，已经成为纳米材料中的研究热点之一。
发明内容
本发明的目的在于提供一种Fe3O4-DA-AMP复合纳米抗菌材料及其制备方法和应用，本发明制备的抗菌复合材料有较好的抗菌效果，在抑菌、杀菌领域均有广泛的应用前景。
本发明具体技术方案如下所述：
本发明提供的纳米复合抗菌材料由Fe3O4磁纳米颗粒和DA修饰的抗菌肽组成，通过Fe与DA的酚羟基之间的配位键将DA标记的抗菌肽(DA-己二酸-GKRWWKWWRR)连接到Fe3O4上，己二酸作为连接分子。
其中，Fe3O4磁性纳米颗粒，由FeCl2和FeCl3共沉淀制得，粒径为143nm，电位为-7.5mV。
DA修饰的抗菌肽DA-己二酸-GKRWWKWWRR由固相合成法制得，即固相树脂上被Fmoc保护的单体氨基酸去保护后露出氨基，通过缩合反应与溶液中氨基酸的羧基形成肽键，从而将氨基酸连接到树脂上，使肽链从C端向N端延伸，直至合成所需肽链。具体操作见实施例。
DA修饰的抗菌肽(DA-AMP)经过HPLC进行纯化。
通过Fe与DA的酚羟基之间的配位键将DA标记的抗菌肽(DA-己二酸-GKRWWKWWRRC)连接到Fe上。
Fe3O4-DA-AMP纳米复合抗菌材料的粒径为170nm，电位为14mV。
Fe3O4-DA-AMP纳米复合抗菌材料制备方法步骤如下：
1)合成Fe3O4；
2)合成DA-己二酸-GKRWWKWWRR；
3)合成Fe3O4-DA-AMP
采用DA-AMP的配体交换反应将DA-AMP和Fe3O4结合。
Fe3O4-DA-AMP纳米复合抗菌材料用于制备磁性纳米抗菌探针，用于抑菌、杀菌，可用于体外血液消毒。
与现有技术相比，本发明具有以下有益效果：
本发明方法制备得到的Fe3O4-DA-AMP纳米复合抗菌材料，具有较高的抗菌活性，可广泛应用于抑菌、杀菌领域中；本发明公开的制备工艺简单，原料来源广泛，反应条件温和，易于合成，适于推广使用。
附图说明：
图1为Fe3O4-DA的粒径图；
图2为Fe3O4-DA-AMP纳米复合抗菌材料的粒径图；
图3为Fe3O4-DA和Fe3O4-DA-AMP纳米复合抗菌材料的电位图；
图4为DA-己二酸-GKRWWKWWRRC的HPLC图；
图5为DA-己二酸-GKRWWKWWRRC的质谱图；
图6为Fe3O4-DA-AMP、Fe3O4和AMP的抗菌图；
图7为Fe3O4-DA-AMP的MIC测定细菌涂板图；
图8为Fe3O4-DA-AMP的MIC定量图。
具体实施方式
以下结合实施例对本发明进行详细阐述，但本发明不局限于这些实施例。
实施例1
1、Fe3O4-DA-AMP纳米复合抗菌材料的合成
1)Fe3O4的合成
将FeCl2和FeCl3(摩尔比为1：2)溶于超纯水中，通过加入NH4OH共沉淀制备OA-Fe3O4纳米颗粒。
在500mL的圆底烧瓶中，将0.86g FeCl2和2.35g FeCl3溶解在40mL去离子水中。然后在剧烈搅拌下，快速加入3.75mL浓度为25％的NH4OH。再将溶液温度升高至80℃的过程中，注入0.3mL的油酸(OA)。混合1小时后，在剧烈搅拌下将30mL甲苯和过量的NaCl加入溶液中，获得分散在甲苯相中的OA-Fe3O4纳米颗粒。分离甲苯相，并加入30mL丙酮后通过磁分离法收集疏水的Fe3O4纳米颗粒。将收集的Fe3O4纳米颗粒在40℃下真空干燥过夜，然后以10mg/mL的浓度分散在甲苯中。
注：所有的玻璃器皿都要洗干净，水都是超纯水。
2)DA-己二酸-GKRWWKWWRR的合成
多肽是基于带有Fmoc保护基团的2-Chlorotrityl Chloride树脂，通过固相多肽合成技术得到。
分别称取相当于树脂5倍摩尔量的氨基酸、HBTU、HOBT，溶于DMF中，加入DIEA耦合30min。然后加入20％哌啶反应30min脱去Fmoc保护基，重复此步骤直达合成所需的肽链(GKRWWKWWRR)。分别称取相当于树脂(合成有多肽的树脂)5倍摩尔量的己二酸、HBTU、HOBT溶于DMF中，加入DIEA耦合30min，反应两次。DA的修饰方式与己二酸一致。最后用切割液将标记的多肽从树脂上裂解下来，HPLC纯化，分子量通过LC-MS确定，纯化图和质谱图如附图4和图5所示。
3)Fe3O4-DA-AMP的合成
采用DA-AMP的配体交换反应将DA-AMP和Fe3O4结合。
将5mg的DA-AMP溶解在pH＝4.0的水溶液中，然后加入500μL的OA-Fe3O4甲苯溶液，并震荡10分钟以促进配体交换。添加过量的丙酮，通过外加磁铁可观察到分散颗粒的沉淀。所获得的沉淀物可以容易地分散在水中，形成稳定的胶体。随后沉淀-分散过程重复3次进行，洗去游离的DA-AMP。
对照组DA-Fe3O4合成方法同Fe3O4-DA-AMP的合成，具体如下所示：
称取DA溶解在pH＝4.0的水中，然后加入OA-Fe3O4甲苯溶液振荡促进两者结合。
2、Fe3O4-DA-AMP纳米复合抗菌材料的表征
1)Fe3O4-DA-AMP纳米复合抗菌材料的水合粒径和Zeta电位的测定实验
分别取20μL合成好的DA-Fe3O4、Fe3O4-DA-AMP，用去离子水稀释至2mL。其中，1mL用于测水合粒径，1mL用于测Zeta电位。每组样品平行测定三次，并监测整个过程的变化。效果见附图1和附图2。
根据测定DA-Fe3O4、Fe3O4-DA-AMP的水合粒径和Zeta电位的变化情况，来判断每一步是否成功。由图可以发现，从最初的DA-Fe3O4纳米颗粒到最终的Fe3O4-DA-AMP纳米探针，其水合粒径增大，证明了DA-AMP的成功偶联。图3是Zeta电位的变化情况，Zeta电位的变化也证明了Fe3O4纳米颗粒连接上了DA-AMP。
3、Fe3O4-DA-AMP纳米复合抗菌材料的抗菌性能测试
1)Fe3O4-DA-AMP对金黄色葡萄球菌的抗菌效果
为了探究Fe3O4-DA-AMP纳米复合抗菌材料对细菌生长的影响，吸取过夜培养的S.aureus细菌100μL，加入到5mL TSB培养基中，37℃培养6h，此时菌浓度为108CFU。将菌液稀释50倍，然后分别取250μL Fe3O4-DA-AMP、DA-Fe3O4、DA-AMP和1mL稀释后的菌液混合，180rpm、37℃共培养1h。取出共培养液稀释20000倍，取70μL加到TSA固体培养基上，用涂布器涂布均匀，倒扣放入培养箱，37℃培养16h。取出培养皿计数菌落并计数，用origin软件作图。
根据Fe3O4-DA-AMP纳米复合抗菌材料与Fe3O4和DA-AMP抗菌效果的对比，可以看出Fe3O4-DA-AMP表现出明显的抗菌效果(图6)。
2)Fe3O4-DA-AMP对金黄色葡萄球菌的MIC测定
为了探究Fe3O4-DA-AMP纳米探针对金黄色葡萄球菌的MIC，吸取过夜培养的S.aureus细菌100μL，加入到5mL TSB培养基中，37℃培养6h，此时菌浓度为108CFU。将菌液稀释50倍，然后分别取不同浓度的Fe3O4-DA-AMP纳米探针250μL和1mL稀释后的菌液混合，180rpm、37℃共培养1h。取出共培养液稀释20000倍，取70μL加到TSA固体培养基上，用涂布器涂布均匀，倒扣放入培养箱，37℃培养16h。取出培养皿计数菌落并计数，用origin软件作图。
根据不同浓度Fe3O4-DA-AMP纳米探针的抗菌效果可以看出，Fe3O4-DA-AMP纳米探针的MIC50约为0.35mM(图7、8)。
Claims (6)
1.一种Fe3O4-DA-AMP纳米复合抗菌材料，其特征在于，所述的纳米复合抗菌材料由Fe3O4磁纳米颗粒、DA修饰的抗菌肽DA-己二酸-GKRWWKWWRR组成，通过Fe与DA的酚羟基之间的配位键将DA修饰的抗菌肽连接到Fe3O4上，己二酸作为连接分子；
DA-己二酸-GKRWWKWWRR的合成方法为：多肽是基于带有Fmoc保护基团的2-Chlorotrityl Chloride树脂，通过固相多肽合成得到；
分别称取相当于树脂5倍摩尔量的氨基酸、HBTU、HOBT，溶于DMF中，加入DIEA耦合30min，然后加入20％哌啶反应30min脱去Fmoc保护基，重复此步骤直达合成所需的肽链GKRWWKWWRR；分别称取相当于合成有多肽的树脂5倍摩尔量的己二酸、HBTU、HOBT溶于DMF中，加入DIEA耦合30min，反应两次，DA的修饰方式与己二酸一致，最后用切割液将标记的多肽从树脂上裂解下来。
2.根据权利要求1所述的Fe3O4-DA-AMP纳米复合抗菌材料，其特征在于，所述Fe3O4磁性纳米颗粒，由FeCl2和FeCl3共沉淀制得，粒径为143nm，电位为-7.5mV。
3.根据权利要求1所述的Fe3O4-DA-AMP纳米复合抗菌材料，其特征在于，所述DA修饰的抗菌肽DA-己二酸-GKRWWKWWRR由固相合成法制得。
4.根据权利要求1所述的Fe3O4-DA-AMP纳米复合抗菌材料，其特征在于，所述Fe3O4-DA-AMP纳米复合抗菌材料的粒径为170nm，电位为14mV。
5.一种根据权利要求1所述的Fe3O4-DA-AMP纳米复合抗菌材料的制备方法，其特征在于，所述Fe3O4-DA-AMP纳米复合抗菌材料的制备方法步骤如下：
1)合成Fe3O4；
2)合成DA-己二酸-GKRWWKWWRR；
3)合成Fe3O4-DA-AMP
采用DA-AMP的配体交换反应将DA-AMP和Fe3O4结合。
6.一种根据权利要求1所述的Fe3O4-DA-AMP纳米复合抗菌材料的应用，其特征在于，所述Fe3O4-DA-AMP纳米复合抗菌材料用于抑菌、杀菌领域。
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