CN115475642A - V2N MXene的合成方法及其应用 - Google Patents
V2N MXene的合成方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种V2NMXene的合成方法及其应用,通过光热增强的纳米酶催化活性对抗细菌治疗的应用,属于抗菌纳米材料研究领域,该纳米材料以V2AlN为前体,通过氢氟酸蚀刻去除材料的铝层,最后超声剥离得到单层的氮化钒MXene,本发明的氮化钒MXene在光热增强的纳米酶作用下能够产生大量的活性氧,从而有效清除细菌及其生物膜和促进小鼠皮下脓肿的修复,同时具有良好的生物相容性,因此,该氮化钒MXene纳米材料在抗感染治疗领域有良好的应用前景。
Description
技术领域
本发明涉及抗菌生物活性领域,尤其是涉及MXene纳米材料对金黄色葡萄球菌抑菌效果研究,具体涉及一种氮化钒MXene的合成方法及应用。
背景技术
金黄色葡萄球菌(Staphylococcusaureus,S.aureus),是一种常见的临床致病菌,对公众健康造成危险。随着抗菌药物在临床上的广泛使用,使得菌株对药物出现不同程度的耐药性,导致许多种类的抗菌药物已经不能满足临床治疗需求,给治疗带来很大困扰。与此同时,相关监管机构批准的新型抗生素数量一直在下降,因此开发一种不易产生耐药性的新型抗菌药物迫在眉睫。近年来,纳米酶(Nanozymes)凭借其广谱抗菌性被称为“新一代抗生素”。然而,较低的催化活性导致单一的纳米酶治疗难以取得令人满意的抗菌效果,从而限制了其在抗菌领域的应用。
其中MXene是一类金属碳氮化物新型纳米二维片层,其已被广泛应用于晶体管、储能设备、海水淡化、电催化剂、电磁干扰屏蔽、电磁屏蔽织物、电化学超级电容器、锂离子电池、钾离子电池、锌离子电池、导电薄膜,部分MXene及其复合材料对大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌具有抗菌活性,因此MXene目前也在生物医药中有应用,作为抗菌材料。现有研究中,研究人员对Ti2C和Ti3C2对大肠杆菌的抗菌性能进行研究,发现Ti2C相对细菌的生存能力没有影响,而Ti3C2相具有抗菌活性。目前研究中对其他单纯MXene材料的抗菌活性并没有深入研究。
但是现有技术中单纯的MXene抗菌性能不高,更多的是将MXene作为现有抗菌药物的载体改进抗菌性能,或者对其进行改性,提高抗菌活性。如专利公开号CN111184908 A所述的,将Mxene加入纳米铌酸盐中,在Mxene表面原位负载纳米铌酸盐,得到Mxene复合压电材料,具备抗菌性能。
因此本发明的目的在于,寻找一种新的具有优异抗菌活性的MXene,拓展作为抗菌药物的MXene的种类。
发明内容
本发明的目的在于提供一种V2N MXene的新用途以及其合成方法,还提供一种基于V2N的抗菌药物。
为实现上述发明目的,
本发明具有提供一种MXene在制备仿酶制剂的应用,所述MXene分子式为V2N,所述仿酶制剂为仿氧化酶制剂或仿过氧化物酶制剂。
本发明具有提供V2N MXene在制备抗菌药物的应用。
进一步地,所述MXene联合NIR光热组合体系在制备抗菌药物中的应用。
进一步地,所述MXene与H2O2作为组合物在制备抗菌药物中的应用。
进一步地,所述MXene联合H2O2以及NIR光热组合体系在制备抗菌药物中的应用。
进一步地,所述抗菌药物为抗金黄色葡萄球菌的药物。
本发明具体提供一种抗菌药物组合物,所述组合物包括V2N MXene和H2O2。
本发明具体提供一种MXene联合近红外光热的协同纳米抗菌组合体系,包括V2NMXene、H2O2及近红外激光器。
进一步地,所述V2N MXene的溶液浓度为25-100μg·mL-1,所述H2O2的浓度为0.1-0.2mM,所述近红外激光器为近红外功率密度为1W·cm-2,进行5-10分钟光照。
本发明具体提供一种氮化钒MXene的制备方法,以V2AlN为前体,通过酸蚀,超声剥离的方法,合成了一种具有生物活性的纳米材料。
在室温下将V2AlN粉末缓慢浸入50wt%HF水溶液中并且搅拌12小时,随后,将得到的溶液进行离心并冲洗数次,直到上清液的pH达到6,将离心得到的材料与TPAOH水溶液混合12小时,离心,将材料洗涤清除残留的TPAOH,超声处理,离心得到所需的V2N MXene。
在本发明一个具体的实施方式中,在室温下将V2AlN粉末缓慢浸入20mlHF水溶液(50wt%)中并且磁力搅拌12小时,随后,将得到的溶液进行离心(4000rpm)并冲洗数次,直到上清液的pH达到6,将离心得到的材料与40mLTPAOH水溶液混合12小时,最后,通过离心(12000rpm),将材料洗涤三次清除残留的TPAOH,并超声处理1小时,并将溶液静置过夜,离心得到所需的氮化钒MXene。
本发明具有如下优点:
本发明以V2AlN为前体,通过酸蚀,超声剥离的方法,合成了二维氮化钒MXene纳米材料。证实了V2N MXene具有良好的光热效应和纳米酶催化活性。本发明发现了单纯V2NMXene具备抗菌活性。利用V2N MXene的纳米酶催化活性,与双氧水联用产生活性氧进行杀菌。利用V2N MXene的具有良好的光热效应,能联用光热治疗手段,也就是近红外光线照射条件下,通过将光能转化成热能,从而杀死细菌。并且本发明提供的V2N MXene光热效应,使得局部升温的温度不会过高,避免了现有技术部分具有光热效应的纳米材料升温过高(>50℃)损伤感染周围的正常组织。本发明提供的V2N MXene物理特性的治疗机制使其在治疗细菌感染时可以避免耐药性的产生。进一步的,V2N MXene、H2O2和近红外光共同作用下,通过光热增强的纳米酶催化活性产生大量活性氧进行杀菌,具有显著的抑菌效果。
在测试材料抗菌活性中,发现其对金黄色葡萄球菌及其生物膜有显著的清除效果。本发明还通过活体实验来进一步研究了氮化钒MXene对金黄色葡萄球菌的灭菌活性,发现其能有效促进小鼠皮下脓肿的愈合,对细菌感染的小鼠皮下脓肿模型有很好的促进修复功能,同时具有良好的生物安全性。
关于本发明的使用术语的定义:除非另有说明,本文中术语提供的初始定义适用于整篇说明书的该术语;对于本文没有具体定义的术语,应该根据公开内容和上下文,给出本领域技术人员能够给予它们的含义。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
附图说明
图1A为实施例1中V2AlN粉末的微观结构图,1B为实施例1中V2N MXene的合成中间体微观结构图,1C为实施例1中V2N MXene的微观结构图;
图2A为实施例2中V2N MXene的吸收光谱图,2B为近红外光照射不同浓度的V2NMXene溶液(10分钟)溶液温度的变化结果统计图,2C为V2N MXene光热转换效率结果图;
图3A为实施例3中V2N MXene的氧化酶催化活性,3B为实施例3中V2N MXene的过氧化物酶催化活性,图3C为温度对V2N MXene对TMB催化活性的影响,图3D为温度对V2N MXene对H2O2催化活性的影响;
图4A为实施例4中V2N MXene平板涂布对金黄色葡萄球菌的影响,4B为实施例4中V2N MXene以及其他实验组平板涂布对金黄色葡萄球菌的影响统计结果,4C为实施例4中V2NMXene以及其他实验组平板涂布对金黄色葡萄球菌的形态学实验;
图5A为实施例5中V2N MXene清除金黄色葡萄球菌生物膜实验,5B为实施例5中V2NMXene以及其他实验组处理后金黄色葡萄球菌生物膜的量结果统计图;
图6A为小鼠皮下脓肿模型建立流程图,6B为实施例6中V2N MXene以及其他实验组处理后小鼠皮下脓肿照片,6C为收集小鼠皮下脓肿处皮肤组织进行菌落计数,6D小鼠皮下脓肿相关感染面积统计结果图;6E收集小鼠皮下脓肿处皮肤组织进行菌落计数统计结果图;
图7为实施例7中的V2N MXene生物相容性实验结果。
具体实施方式
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1V2N MXene的合成路线
室温下将1gV2AlN粉末,其微观结构图如图1A所示,缓慢浸入20mlHF水溶液(50wt%)中并且磁力搅拌12小时,随后,将得到的溶液进行离心(4000rpm)并冲洗数次,直到上清液的pH达到6,将离心得到的材料,其微观结构如图1B所示,与40mLTPAOH水溶液混合12小时,最后,通过离心(12000rpm),将材料洗涤三次清除残留的TPAOH,并超声处理1小时。并将溶液静置过夜,离心得到所需的氮化钒MXene,产物的微观结构图如图1C所示。
实施例2V2N MXene的光热性能
为了研究V2N的光热性能,使用紫外-可见光-近红外光谱仪测定不同浓度下(6.25、12.5、25、50、100μg·mL-1)V2N MXene的吸收光谱(图2A)。使用1064nm激光器(1.0W·cm-2)在室温下照射V2N MXene(0,12.5,25,50μg·mL-1)溶液10分钟,并且使用红外热成像仪,记录下溶液温度的变化(图2B)。表明使用1064nm激光器(1.0W·cm-2)在室温下照射50μg·mL-1 V2N MXene照射5-10分钟其升温到合适的杀菌温度。最后,根据公式计算得出V2NMXene光热转换效率为31.67%(图2C)。
实施例3V2N MXene的纳米酶催化活性
为了研究V2N MXene的纳米酶催化活性。使用抗坏血酸(AA,1mM)测试材料氧化酶功能,加入100μg·mL-1 V2N MXene随着反应的进行AA在292nm处的吸收峰持续降低,说明V2N MXene具有氧化酶功能,产生活性氧氧化AA(图3A)。使用3,3',5,5'-四甲基联苯胺TMB(1mM)测试材料过氧化物酶功能,加入100μg·mL-1 V2N MXene,V2N+H2O2+TMB组在652nm处的吸收峰最为明显,说明V2N MXene具有过氧化物酶功能,分解H2O2,产生活性氧(图3B)。同时在室温下以及50℃(模拟近红外光照射)环境下测定了酶的动力学催化过程,计算得到TMB(图3C),H2O2(图3D)Km和Vmax值,验证光热能增强纳米酶催化活性。
实施例4 V2N MXene清除金黄色葡萄球菌实验
复苏金黄色葡萄球菌,培养至对数期后,稀释到OD=0.05进行体外抗菌实验。共设置PBS组、PBS+NIR-II组、H2O2组、H2O2+NIR-II组、V2N组、V2N+NIR-II组、V2N+H2O2组,和V2N+H2O2+NIR-II组(n=3)。其中V2N和H2O2浓度分别为50μg·mL-1和0.1mM,加入量为100μL。NIR-II组在加入V2N和H2O2后给予1064nm的NIR-II激光照射(1W·cm-2,10min)。
在37℃培养箱中孵育2h后,稀释,涂布在胰酪大豆胨液体培养基(TSB)平板上,培养18h后,对菌落进行计数。如图4A,B所示,V2N+NIR-II组、V2N+H2O2组细菌数量与对照组相比有统计学差异,V2N+H2O2+NIR-II组细菌数量与对照组相比有显著的统计学差异。
形态学实验是将各组的细菌低温离心收集,用PBS轻轻清洗3次,用2.5%戊二醛溶液重悬和浸泡,放置4℃冰箱固定过夜。然后,去除固定液,用不同浓度的乙醇(30%,50%,70%,90%,95%,100%)进行梯度脱水处理,每次10min。将细菌滴在硅片上,进行烘干。烘干后的样品,进行喷金处理,最后上机,观察细菌形态。如图4C所示,对照组和H2O2处理组,细菌形态结构完好。V2N MXene处理的各组的细菌形态结构均出现不同程度的破坏。尤其是V2N+H2O2+NIR-II组细菌,细菌结构完全崩塌,甚至可以看到细菌内容物流出。
实施例5V2N MXene清除金黄色葡萄球菌生物膜实验
复苏金黄色葡萄球菌,培养至对数期后,稀释到OD=0.05,以96孔板为载体,每孔中加入200μL菌液,静置于37℃培养箱48h。每隔12h更换TSB培养液。48h后,吸尽孔板中的培养基,然后用PBS缓冲液清洗2次,洗去未黏附的菌体。
共设置PBS组、PBS+NIR-II组、H2O2组、H2O2+NIR-II组、V2N组、V2N+NIR-II组、V2N+H2O2组,和V2N+H2O2+NIR-II组(n=3)。其中V2N MXene溶液的浓度均为50μg·mL-1,加入量为200μL,H2O2浓度0.1mM,加入量为200μL。NIR-II组在加入V2N和H2O2后给予1064nm的NIR-II激光照射(1W·cm-2,10min)。
每个组别3个重复孔,在37℃培养箱静置2h。然后移除每孔中的溶液,自然干燥后,加入200μL0.1mg·mL-1的结晶紫染液进行染色20min,再用PBS冲洗,室温静置干燥,除去多余水分,随后加入200μL33%乙酸脱色15min,结果如图5A所示,对照组染色最深,生物膜完整,V2N+H2O2+NIR组颜色最浅。最后用酶标仪测定OD590的值衡量生物膜的量,结果如图5B所示。表明光热增强的纳米酶催化活性,对细菌生物膜有明显的清除效果。
实施例6V2N MXene促进小鼠皮下脓肿的愈合实验
采用BALB/c小鼠(雌性,6周龄)建立小鼠皮下脓肿模型,模型建立过程如图6A所示。具体过程为:去除小鼠背部的皮毛,并且在脊柱两侧皮下注射50μL金黄色葡萄球菌(1×109CFUmL-1)。24h后脓肿形成,将小鼠随机分为5组:PBS组、V2N组、V2N+NIR-II组、V2N+H2O2组、V2N+H2O2+NIR-II组(n=5),V2N MXene和H2O2浓度分别为50μg·mL-1和0.1mM,对小鼠分别注射200μLV2N MXene和H2O2。NIR-II组小鼠给予NIR-II激光照射(1W·cm-2,10min)。分别于0、1、3、5、7、10天拍摄小鼠脓肿照片,记录脓肿的愈合情况(图6B,D),结果V2N+NIR-II组、V2N+H2O2组、V2N+H2O2+NIR-II组相对对照组具有显著差异。并且在10天后处死小鼠,收集皮肤组织进行菌落计数(图6C,E),结果V2N+NIR-II组、V2N+H2O2组、V2N+H2O2+NIR-II组相对对照组具有显著差异。
实施例7 V2N MXene生物相容性测试
通过检测小鼠腹腔注射不同浓度(5μg·mL-1至100μg·mL-1)氮化钒MXene后,通过检测小鼠的血生化指标溶血率检测氮化钒MXene的生物相容性;结果如图7所示,(5μg·mL-1至100μg·mL-1)氮化钒MXene溶血率均小于5%,符合生物材料医用标准。
综上,本发明提供了一种基于V2N MXene纳米材料的仿酶制剂。该材料具有良好的仿氧化酶活性和仿过氧化物酶活性,能够用来制备高催化活性的仿酶制剂。该材料不仅在体外对金黄色葡萄球菌具有良好的抗菌活性,还能在体内有效地杀死金黄色葡萄球菌并促进金黄色葡萄球菌感染的动物皮肤伤口愈合;在低浓度的H2O2存在下,本发明基于V2NMXene纳米材料的仿酶制剂对金黄色葡萄球菌具有优异的体内抗菌活性,并能加速伤口愈合。本发明提供的基于V2N MXene纳米材料是一种非抗生素材料,而且具有优良的生物相容性,其能够解决抗生素滥用带来的细菌耐药问题,在制备仿生材料和抗菌药物中具有广阔的应用前景。
Claims (10)
1.一种MXene在制备仿酶制剂的应用,其特征在于:所述MXene分子式为V2N,所述仿酶制剂为仿氧化酶制剂或仿过氧化物酶制剂。
2.一种MXene在制备抗菌药物的应用,其特征在于:所述MXene分子式为V2N。
3.根据权利要求2所述的MXene的应用,其特征在于:所述MXene联合NIR光热组合体系在制备抗菌药物中的应用。
4.根据权利要求2所述的MXene的应用,其特征在于:所述MXene与H2O2作为组合物在制备抗菌药物中的应用。
5.根据权利要求2所述的MXene的应用,其特征在于:所述MXene联合H2O2以及NIR光热组合体系在制备抗菌药物中的应用。
6.根据权利要求2-5任一所述的MXene的应用,其特征在于:所述抗菌药物为抗金黄色葡萄球菌的药物。
7.一种抗菌药物组合物,其特征在于:所述组合物包括V2N MXene和H2O2。
8.一种MXene联合近红外光热的协同纳米抗菌组合体系,其特征在于,包括V2N MXene、H2O2及近红外激光器。
9.根据权利要求8所述的一种MXene联合近红外光热的协同纳米抗菌组合体系,其特征在于,所述V2N MXene的溶液浓度为25-100μg·mL-1,所述H2O2的浓度为0.1-0.2mM,所述近红外激光器为近红外功率密度为1W·cm-2,进行5-10分钟光照。
10.一种V2N MXene的制备方法,具体包括如下步骤:
在室温下将V2AlN粉末缓慢浸入50wt%HF水溶液中并且搅拌12小时,随后,将得到的溶液进行离心并冲洗数次,直到上清液的pH达到6,将离心得到的材料与TPAOH水溶液混合12小时,离心,将材料洗涤清除残留的TPAOH,超声处理,离心得到所需的V2N MXene。
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