CN108157387B - 一种银纳米线抗菌气凝胶及其制备方法和应用 - Google Patents

一种银纳米线抗菌气凝胶及其制备方法和应用 Download PDF

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CN108157387B
CN108157387B CN201711210615.7A CN201711210615A CN108157387B CN 108157387 B CN108157387 B CN 108157387B CN 201711210615 A CN201711210615 A CN 201711210615A CN 108157387 B CN108157387 B CN 108157387B
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姚琛
周宾
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Abstract

本发明公开了一种银纳米线抗菌气凝胶，该气凝胶由银纳米线以及组装在银纳米线表面的抗菌短肽组成，抗菌短肽的氮端由半胱氨酸修饰后固定在银纳米线表面；其中，抗菌短肽的序列为RIVVIRVA、KIWVIRWR、RIWVIRWR、RIWVIWRR、RRWVIWRR、IVVIRVA、IWVIRWR、RWVIWRR、VVIRVA、WVIRWR或WVIWRR中的一种。本发明还公开了上述银纳米线抗菌气凝胶的制备方法和应用。本发明银纳米线抗菌气凝胶以银纳米线为基础模块，抗菌短肽的氮端经半胱氨酸修饰后，组装在银纳米线表面，以银纳米线/抗菌短肽组装体构建三维结构抗菌气凝胶，得到的银纳米线抗菌气凝胶具有高比表面积、高孔隙率以及多活性位点的优点，本发明银纳米线抗菌气凝胶能够在短时间内实现高的杀菌率。

Description

一种银纳米线抗菌气凝胶及其制备方法和应用
技术领域
本发明涉及一种银纳米线抗菌气凝胶，还涉及上述银纳米线抗菌气凝胶的制备方法和应用，属于生物医用材料技术领域。
背景技术
早在7000年前，银就开始作为抗菌材料被用于防治细菌传播的疾病，帕拉塞尔苏斯的《炼金术》中提到：银因其具有独特的杀菌功效，常被作为治疗传染病的药物。二十世纪初，Albert C.Barnes发明了一种名为“Argyrol”(希腊语“银”的意思)的蛋白银溶液，用于预防眼部传染病。长期以来，银抗菌材料因其高活性、长效稳定性和安全低毒等优点引起了人们的普遍关注和应用。特别是随着纳米材料研究的兴起，银纳米材料成为抗菌剂领域研究的重要热点之一。银纳米材料不仅具备银单质本身稳定的理化特性，还具备纳米粒子量子效应、表面效应、量子隧道效应和小尺寸效应等四个特性，导致其在光学、热学、电学、化学和生物学性能等方面较普通材料具有明显优势。
银纳米材料在临床各科都有广泛研究和潜在应用，如骨科使用的纳米银骨水泥的材料，通过体外实验证实对耐药菌株具有明显抑制作用；皮肤科以纳米银结合一般化妆品被认为是替代糖皮质激素治疗过敏性皮炎的潜在治疗手段；心血管科使用银纳米材料能够降低静脉针留置病人受感染的风险；烧伤科对病人进行植皮手术后，以纳米银作处理能够有效提高网状上皮移植的上皮化率，缩短烧伤创面的愈合时间，显示出一定的促修复再生作用。除此之外，一些纳米银医用材料和医疗器械也已经得到广泛应用，如载纳米银的抗菌口罩、防护服、纳米银抗菌纤维制成的烧烫伤敷料、医用绷带、纳米银外用乳膏、凝胶制剂、纳米银系不锈钢器械、纳米银聚氨酯心室导管、纳米银无菌导管、纳米银镀层的药物传送导管等。
银纳米材料的性质不仅受到尺寸大小的影响，还与纳米材料的形状密切相关，如纳米粒、纳米线、纳米管、纳米棒、纳米带、同轴纳米电缆等。银纳米线常被用作纳米激光器、单电子量子计算机的存储元件、扫描隧道显微镜的针尖、纳米器件和超大规模集成电路中的连线、光导纤维、微电子学方面的微型钻头以及复合材料的增强剂等。近年来，人们开始关注到银纳米线作为基础构建模块制备有序的三维材料。研究表明，银纳米线与聚苯乙烯混合制备所得的三维纳米复合材料表现出良好的连续性与刚性，其导电渗滤性能优于碳纳米管(Advanced Functional Materials，2010，20：2709-2716.)。Chen等在银纳米颗粒中掺杂银纳米线，由于纤维状银纳米线的存在，可以连接导电填料中分散的银纳米颗粒以提高导电性能并起稳定作用(Journal ofMaterialsScience，2007，42：3172-3176.)。Cui等以棉花为载体，结合银纳米线与碳纳米管制备成滤水装置，可高速有效地对高浓度含菌水进行灭菌处理(Nano Letters，2010，10：3628-3632.)。
制备新型抗菌材料的思路之一是将无机抗菌剂与有机物、无机多孔材料等通过化学键合或物理吸附等方法复合，通常以有机物/无机多孔材料为骨架，无机抗菌剂分散其中，因此，其主要成分往往是非抗菌成分/低抗菌成分。因此一种既具有三维网状结构，并且骨架和通过化学键合分散在骨架中的物质均具有高效抗菌成分的新型抗菌材料的开发很有必要。
发明内容
发明目的：本发明所要解决的技术问题是提供一种银纳米线抗菌气凝胶，该银纳米线抗菌气凝胶以无机抗菌剂(银纳米线)为基础模块，将两个维度限制在微纳米尺度内，第三维延伸到较大尺寸，构建出具有高比表面积、高孔隙率的三维网状结构，并在其二维微纳米结构上多位点复合(键合)抗菌短肽，最终得到的抗菌材料具有高活性、低密度和长效安全性。
本发明还要解决的技术问题是提供上述银纳米线抗菌气凝胶的制备方法。
本发明最后要解决的技术问题是提供上述银纳米线抗菌气凝胶在制备抗菌材料或抗菌药物中的应用。
为解决上述技术问题，本发明所采用的技术方案为：
一种银纳米线抗菌气凝胶，所述气凝胶由银纳米线以及组装在银纳米线表面的抗菌短肽组成，所述抗菌短肽的氮端由半胱氨酸修饰后与银纳米线键合；其中，所述抗菌短肽的序列为RIVVIRVA、KIWVIRWR、RIWVIRWR、RIWVIWRR、RRWVIWRR、IVVIRVA、IWVIRWR、RWVIWRR、VVIRVA、WVIRWR或WVIWRR中的一种。
其中，所述气凝胶的密度为5～50mg/crm3。
其中，所述银纳米线的直径为50～150nm，长度为30～100μm。
其中，所述抗菌短肽为阳离子三肽～九肽，所带净电荷为+2～+6。
上述银纳米线抗菌气凝胶的制备方法，由如下方法制得：将所需量的银纳米线与氮端由半胱氨酸修饰后的抗菌短肽于硼砂缓冲液中组装一段时间，将组装后的银纳米线-抗菌短肽水溶液冷冻干燥后即可得到银纳米线抗菌气凝胶。
其中，具体包括如下步骤：
步骤1，醇热法制备银纳米线：将所需量的硝酸银、氯化铜、氯化钠和聚乙烯吡咯烷酮溶于一定量的乙二醇中，于油浴下加热，然后往混合溶液中加入硝酸银/乙二醇溶液，充分反应后将所得的产物经离心分离、水洗后，加入一定量丙酮，收集沉淀，即为银纳米线；
步骤2，将所需量的氮端由半胱氨酸修饰后的抗菌短肽与步骤1制得的银纳米线于硼砂缓冲液中组装24～48小时；
步骤3，气凝胶的制备：将步骤2制得的一定浓度的银纳米线-抗菌短肽水溶液置于液氮预冷的反应装置中，冷冻干燥后得到银纳米线抗菌气凝胶。
其中，所述银纳米线的浓度为5～50mg/mL，抗菌短肽的浓度为1～200μg/mL。
其中，所述抗菌短肽的序列为RIVVIRVA、KIWVIRWR、RIWVIRWR、RIWVIWRR、RRWVIWRR、IVVIRVA、IWVIRWR、RWVIWRR、VVIRVA、WVIRWR或WVIWRR中的一种，
上述银纳米线抗菌气凝胶在制备抗菌材料或抗菌药物中的应用。
相比于现有技术，本发明的技术方案所具有的有益效果为：
本发明的银纳米线抗菌气凝胶以银纳米线为基础模块，抗菌短肽的氮端经半胱氨酸修饰后，组装在银纳米线表面，以银纳米线/抗菌短肽组装体构建三维结构抗菌气凝胶，得到的银纳米线抗菌气凝胶具有高比表面积、高孔隙率以及多活性位点的优点，本发明银纳米线抗菌气凝胶不仅具有高的抗菌活性，而且还能降低抗菌肽的最低抑菌浓度；进一步说，本发明银纳米线抗菌气凝胶基于阳离子抗菌短肽的抗菌特性和对细菌外排机制的干扰，复合传统银纳米抗菌材料，使银纳米线抗菌气凝胶材料具有抗菌谱广、活性高、不易产生耐药性的特点；最后，本发明以液相多元醇法、丙酮沉淀法制备、分离银纳米线，重复性、可操作性好，得到的银纳米线结构规整、纯度高。
附图说明
图1为本发明银纳米线抗菌气凝胶的微观结构图；
图2为图1的局部放大图。
具体实施方式
以下结合附图对本发明的技术方案做进一步说明，但是本发明要求保护的范围并不局限于此。
本发明银纳米线抗菌气凝胶，气凝胶由银纳米线以及组装在银纳米线表面的抗菌短肽组成，抗菌短肽的氮端由半胱氨酸修饰后与银纳米线键合；其中，抗菌短肽的序列为SEQNO.1：RIVVIRVA、SEQNO.2：KIWVIRWR、SEQNO.3：RIWVIRWR、SEQNO.4：RIWVIWRR、SEQNO.5：RRWVIWRR、SEQNO.6：IVVIRVA、SEQNO.7：IWVIRWR、SEQNO.8：RWVIWRR、SEQNO.9：VVIRVA、SEQNO.10：WVIRWR或SEQNO.11：WVIWRR中的一种。
实施例1
本发明的银纳米线抗菌气凝胶，是由银纳米线堆砌而成的具有高度取向结构的气凝胶(呈三维网状结构)，其密度为5～50mg/cm3，由如下方法制备而成：
步骤1，配制100mL含0.0014mM硝酸银、0.017mM氯化铜、0.05mM氯化钠、200mM聚乙烯吡咯烷酮(分子量55000)的乙二醇溶液，将乙二醇溶液于油浴185℃下加热1小时，随后逐滴加入30mL新鲜配制的0.12M硝酸银/乙二醇溶液，充分搅拌，冷却至室温，所得产物经离心分离、三次水洗后，加入30mL丙酮，收集白色沉淀，得到银纳米线；经扫描电子显微镜表征，制得的银纳米线平均直径约84nm，平均长度约63μm；
步骤2，将初始浓度为32μg/mL的抗菌短肽CKIWVIRWR与初始浓度为20mg/mL步骤1制得的银纳米线在硼砂缓冲液中组装48小时；
步骤3，将组装后的银纳米线/抗菌短肽水溶液置于液氮预冷的金属容器中，冷冻干燥后得到银纳米线抗菌气凝胶。
采用平板涂布法，以不同作用时间下细菌悬浮液中活细菌的浓度的对数值与作用时间关系评价银纳米线气凝胶对***金黄色葡萄球菌(S.aureus)和革兰氏阴性细菌大肠杆菌(E.coli)的抗菌性能，结果见表1。
表1为不同作用时间下实施例1制得的银纳米线气凝胶对***金黄色葡萄球菌(S.aureus)和革兰氏阴性细菌大肠杆菌的杀菌率：
Figure BDA0001482970560000041
表1说明实施例1制得的银纳米线抗菌气凝胶对***金黄色葡萄球菌(S.aureus)和革兰氏阴性细菌大肠杆菌1小时的杀菌率达到100％。
实施例2
本发明的银纳米线抗菌气凝胶，是由银纳米线堆砌而成的具有高度取向结构的气凝胶，其密度为5～50mg/cm3，由如下方法制备而成：
步骤1，配制50mL含0.0014mM硝酸银、0.017mM氯化铜、0.05mM氯化钠、40mM聚乙烯吡咯烷酮(分子量200000)的乙二醇溶液，将乙二醇溶液于油浴185℃下加热1小时，随后逐滴加入20mL新鲜配制的0.08M硝酸银/乙二醇溶液，充分搅拌，冷却至室温，所得产物经离心分离、三次水洗后，加入50mL丙酮，收集白色沉淀，得到银纳米线；经扫描电子显微镜表征，制得的银纳米线平均直径约45nm，平均长度约58μm；
步骤2，将初始浓度为128μg/mL的抗菌短肽CWVIWRR与初始浓度为50mg/mL步骤1制得的银纳米线在硼砂缓冲液中组装24小时；
步骤3，将组装后的银纳米线/抗菌短肽水溶液置于液氮预冷的金属容器中，冷冻干燥后得到银纳米线抗菌气凝胶。
以含苯唑西林和氯化钠的Mueller-Hinton(MH)肉汤分别增殖培养耐甲氧西林金黄色葡萄球菌(MRSA)，将实施例2制得的银纳米线气凝胶以平板涂布法评价其抗菌性能。结果表明，实施例2制得的银纳米线抗菌气凝胶与MRSA作用10min后，细菌浓度由8.72*107±0.05*107CFU/mL降至5.11*102±0.03*102CFU/mL。对照组不含抗菌短肽CWVIWRR的银纳米线气凝胶(该银纳米线气凝胶由与实施例2对应的步骤2～3采用相同初始浓度为50mg/mL的银纳米线制得)与MRSA作用30min后，细菌浓度由8.72*107±0.05*107CFU/mL降至7.33*106±0.07*106CFU/mL。另一对照组抗菌短肽CWVIWRR(初始浓度为128μg/mL)与MRSA作用30min后，细菌浓度由8.72*107±0.05*107CFU/mL降至9.05*104±0.01*104CFU/mL。
实施例3
本发明的银纳米线抗菌气凝胶，是由银纳米线堆砌而成的具有高度取向结构的气凝胶，其密度为5～50mg/cm3，由如下方法制备而成：
步骤1，配制200mL含0.0014mM硝酸银、0.017mM氯化铜、0.05mM氯化钠、500mM聚乙烯吡咯烷酮(分子量55000)的乙二醇溶液，将乙二醇溶液于油浴185℃下加热1小时，随后逐滴加入30mL新鲜配制的0.02M硝酸银/乙二醇溶液，充分搅拌，冷却至室温，所得产物经离心分离、三次水洗后，加入100mL丙酮，收集白色沉淀，得到银纳米线；经扫描电子显微镜表征，制得的银纳米线平均直径约32nm，平均长度约84μm；
步骤2，将初始浓度为64μg/mL的抗菌短肽CWVIWRR与初始浓度为10mg/mL步骤1制得的银纳米线在硼砂缓冲液中组装48小时；
步骤3，将组装后的银纳米线/抗菌短肽水溶液置于液氮预冷的金属容器中，冷冻干燥后得到银纳米线抗菌气凝胶。
以含苯唑西林和氯化钠的MH肉汤培养增殖耐甲氧西林表皮葡萄球菌(MRSE)，将实施例3制得的银纳米线气凝胶以平板涂布法评价其抗菌性能。结果表明，实施例3制得的银纳米线抗菌气凝胶对MRSE 30min的杀菌率达99.896％。
本发明的银纳米线抗菌气凝胶以银纳米线为基础模块，将两个维度限制在微纳米尺度内，第三维延伸到较大尺寸，构建出具有高比表面积、高孔隙率的三维网状结构，并在其二维微纳米结构上多位点复合抗菌短肽(抗菌短肽的序列为RIVVIRVA、KIWVIRWR、RIWVIRWR、RIWVIWRR、RRWVIWRR、IVVIRVA、IWVIRWR、RWVIWRR、VVIRVA、WVIRWR或WVIWRR中的一种)，最终得到具有高活性(所有成分均为高效抗菌成分)、低密度和长效安全低毒的抗菌材料，该抗菌材料在水体过滤净化、生物材料及器械表面处理、嵌入式医用材料等领域均有潜在的应用前景。
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Claims (8)

1.一种银纳米线抗菌气凝胶，其特征在于：所述气凝胶由银纳米线以及组装在银纳米线表面的抗菌短肽组成，所述抗菌短肽的氮端由半胱氨酸修饰后固定在银纳米线表面；其中，所述抗菌短肽的序列为RIVVIRVA、KIWVIRWR、RIWVIRWR、RIWVIWRR、RRWVIWRR、IVVIRVA、IWVIRWR、RWVIWRR、VVIRVA、WVIRWR或WVIWRR中的一种；所述气凝胶具有三维网状结构。
2.根据权利要求1所述的银纳米线抗菌气凝胶，其特征在于：所述气凝胶的密度为5~50mg/cm3。
3.根据权利要求1所述的银纳米线抗菌气凝胶，其特征在于：所述银纳米线的直径为50~150 nm，长度为30~100 μm。
4.根据权利要求1所述的银纳米线抗菌气凝胶，其特征在于：所述抗菌短肽为阳离子三肽~九肽，所带净电荷为＋2~＋6。
5.一种权利要求1所述的银纳米线抗菌气凝胶的制备方法，其特征在于，由如下方法制得：将所需量的银纳米线与氮端由半胱氨酸修饰后的抗菌短肽于硼砂缓冲液中组装一段时间，将组装后的银纳米线-抗菌短肽水溶液冷冻干燥后即可得到银纳米线抗菌气凝胶。
6.根据权利要求5所述的银纳米线抗菌气凝胶的制备方法，其特征在于，具体包括如下步骤：
步骤1，醇热法制备银纳米线：将所需量的硝酸银、氯化铜、氯化钠和聚乙烯吡咯烷酮溶于一定量的乙二醇中，于油浴下加热，然后往混合溶液中加入硝酸银/乙二醇溶液，充分反应后将所得的产物经离心分离、水洗后，加入一定量丙酮，收集沉淀，即为银纳米线；
步骤2，将所需量的氮端由半胱氨酸修饰后的抗菌短肽与步骤1制得的银纳米线于硼砂缓冲液中组装24~48小时；
步骤3，气凝胶的制备：将步骤2制得的一定浓度的银纳米线-抗菌短肽水溶液置于液氮预冷的反应装置中，冷冻干燥后得到银纳米线抗菌气凝胶。
7.根据权利要求5所述的银纳米线抗菌气凝胶的制备方法，其特征在于：所述银纳米线的浓度为5~50 mg/mL，抗菌短肽的浓度为1~200μg/mL。
8.权利要求1~4任一所述的银纳米线抗菌气凝胶在制备抗菌材料或抗菌药物中的应用。
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