CN113712044A - 一种改性金纳米棒光热抑菌制剂及其制备方法与应用 - Google Patents
一种改性金纳米棒光热抑菌制剂及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种改性金纳米棒光热抑菌制剂及其制备方法与应用;本制备方法采用金纳米棒溶胶、金属离子水溶液、多酚化合物水溶液以及Tris‑HCl缓冲液在常温下振荡反应获得改性金纳米棒光热抑菌制剂,试验过程仅需约10分钟,所得改性金纳米棒光热抑菌制剂具有均一的核壳结构。本发明制备方法简单快速、绿色安全,不需要使用有机试剂和昂贵复杂的仪器设备,所用试剂易获得,且均无毒无污染,符合绿色化学理念,不会对环境造成污染。本发明通过高生物安全性的金属多酚网络置换单纯金纳米棒表面存在的高生物毒性的十六烷基三甲基溴化铵,获得具有良好生物相容性的改性金纳米棒光热抑菌制剂,使其在医药、食品、生物抗菌等领域显示出巨大优势。
Description
技术领域
本发明涉及抑菌制剂及其工艺制备,尤其涉及一种改性金纳米棒光热抑菌制剂及其制备方法与应用。
背景技术
传统对抗细菌的方法主要以抗生素、金属离子和季铵离子为主,但是价格昂贵、有毒且对环境不友好的缺点,限制了它们的进一步使用和转化。
而且,过度使用抗生素会导致细菌耐药性的产生,治疗效果不可避免地降低,甚至导致“超级细菌”的产生。因此,探索一种新的治疗策略以更有效、更安全地对抗细菌感染而没有明显的副作用以及由此产生的耐药性具有重要意义。
目前,随着纳米技术的发展,许多纳米材料被开发用于抗菌,包括金属离子型纳米抗菌材料、光催化型纳米抗菌材料、季铵盐或季磷盐修饰改性的表面接触型纳米抗菌材料以及纳米载体负载抗生素型纳米抗菌材料。
另外,其他新型抗菌材料、抗菌光动力疗法和光热裂解方法也被开发以解决细菌耐药性问题。其中,光热疗法是一种新型的杀菌疗法,它利用光热剂将近红外光转化为光热来破坏不同类型的病原体和微生物,被认为是一种新型的杀菌疗法。
由于其微创性和高选择性,成为最有前途的治疗方法。迄今为止,具有良好光热响应的材料主要包括贵金属纳米材料(金、铂、银等)、碳纳米材料(氧化石墨烯、碳纳米管)、过渡金属硫族化合物纳米材料(硫化铜、硫化锌等)、以及有机染料纳米材料。
金纳米棒(GNRs)由于可以吸收近红外(NIR)区域的激光波长,是生物组织的透明窗口,可以有效地将吸收的NIR光能转化为热量,已成为光热疗法的优秀候选材料。
但是,有毒表面活性剂十六烷基三甲基溴化铵难以去除,使得金纳米棒具有较高的毒性,生物安全性差。
此外,单纯的金纳米棒光热转化效率低、抗菌效果差,使其在医疗、食品、生物抗菌等各领域的应用受到限制。因此需要对金纳米棒进行功能化修饰以提高光热转化效率和生物相容性并用于微生物的高效光热抗菌。酚类化合物是存在于多种生物体中的一大类天然分子,被认为是非常安全的化合物,可作为食品添加剂添加到食品中。从真菌到细菌,从植物到动物,这些无处不在的化合物因其独特的理化特性而获得了极大的研究关注。
金属多酚网络(MPNs)是由金属离子和多酚,通过配位作用形成的超分子网络结构,金属多酚网络本身具有一定的光热效应,在置换金纳米棒表面的高毒性十六烷基三甲基溴化铵时,不会因为将其功能化修饰在金纳米棒的表面而影响金纳米棒的光热效应,特别是,金属多酚网络的高粘合性使其易于涂覆在金纳米棒表面,使金纳米棒能够与金属多酚网络发挥协同光热效应,光热转换效率大大提高,显著高于单独金纳米棒、单独金属多酚网络。
因此,利用金属多酚网络功能化修饰金纳米棒,制备一种改性金纳米棒抑菌制剂,不仅能够改善金纳米棒生物安全性差、生物毒性高的缺点,扩展其应用范围,可广泛用作绿色安全抗菌剂。
其次,利用金属多酚化合物的功能化修饰还能够同时发挥金纳米棒与金属多酚网络的协同光热作用,使该抑菌制剂的光热效率得到显著提高,能够更高效的杀死细菌。
发明内容
本发明的目的在于克服上述现有技术的缺点和不足,提供一种简单、绿色、安全的改性金纳米棒光热抑菌制剂及其制备方法与应用。
本发明改性金纳米棒光热抑菌制剂,利用高生物安全性的金属多酚网络置换金纳米棒表面高生物毒性的十六烷基三甲基溴化铵,得到具有良好生物相容性的改性金纳米棒光热抑菌制剂。
利用本发明基于改性金纳米棒光热抑菌制剂,通过发挥金纳米棒与金属多酚网络的协同光热作用,使得改性金纳米棒光热转化效率增强3~4倍,达到高效抑菌、广谱杀菌的目的。
本发明通过下述技术方案实现:
一种改性金纳米棒光热抑菌制剂的制备方法,其包括如下步骤:
首先,振荡条件下于10mL离心管依次加入水、金纳米棒溶胶、金属离子、多酚化合物水溶液、Tris-HCl缓冲液,常温条件下振荡反应,反应结束进行离心,除去上清液,得到金属多酚网络修饰的金纳米棒(GNRs@MPNs)用作改性金纳米棒光热抑菌制剂,所得改性金纳米棒光热抑菌制剂用Tris-HCl缓冲液重新分散,保存。
优选的,所述金纳米棒溶胶、金属离子、多酚化合物水溶液、Tris-HCl缓冲液的浓度分别为:(150~200)ppm、(1~2)mg/mL、(2~4)mg/mL、(10~20)mM。
优选的,所述Tris-HCl缓冲液的PH为8.0。
优选的,所述金纳米棒溶胶、金属离子水溶液、多酚化合物水溶液加入的时间间隔是1min。
优选的,所述金属离子为:Fe3+、Cu2+、Mn2+中的至少一种。
优选的,所述多酚水溶液中的多酚化合物为:表没食子儿茶素没食子酸酯(EGCG)、单宁酸(TA)、原花青素(OPC)中的至少一种。
优选的,所述Tris-HCl缓冲液加入后,振荡反应5min。
更优选的,向10mL离心管中依次加入1~2mL的超纯水、1~2mL摩尔浓度为200ppm的金纳米棒溶胶、300~500uL浓度为1mg/mL的金属离子水溶液、300~500uL浓度为4mg/mL的多酚化合物水溶液、1~2mL浓度为20mM的Tris-HCl缓冲液。
优选的,所述振荡的频率为600~800rpm;所述室温为20~25℃;所述离心的转速为8000~10000rpm,离心时间为5~10min,温度为20~25℃。
优选的,所述改性金纳米棒光热抑菌制剂于4℃以下保存。
本发明相对于现有技术,具有如下的优点及效果:
(1)本发明制备方法简单、合成速度快、成本低廉,无需使用有机试剂和昂贵复杂的仪器设备;
所用试剂易获得,且均无毒无污染,符合绿色化学理念,对环境无污染。
(2)本发明制备的改性金纳米棒光热抑菌制剂,利用高生物安全性的金属多酚网络置换金纳米棒表面高生物毒性的十六烷基三甲基溴化铵,得到具有良好生物相容性的改性金纳米棒光热抑菌制剂,改善了金纳米棒存在毒性、生物相容性差的劣势,提高了抑菌制剂的生物安全性,拓宽了抑菌使用范围,使其在医药、食品、生物抗菌等领域具有显著优势。
(3)本发明改性金纳米棒光热抑菌制剂的制备方法,利用金属多酚网络的高粘合性涂覆在金纳米棒表面实现功能化修饰,能够最大化发挥金纳米棒与金属多酚网络的协同光热效应,而且选用金属多酚网络改性金纳米棒,极大地提高了光热转换效率,相比于单纯的金纳米棒,光热转换效率提高了3~4倍,能够高效杀死细菌,且具有广谱杀菌效果,避免细菌耐药性的产生。
(4)本发明所制备的改性金纳米棒光热抑菌制剂,对革兰氏阳性细菌和革兰氏阴性细菌,例如金黄色葡萄球菌和大肠杆菌,在微量使用的情况下,其抑菌率高达近100%,抑菌效果显著。
附图说明
图1为本发明实施例中基于单宁酸改性的金纳米棒抑菌制剂的透射电子显微镜(TEM)图。
图2为本发明实施例中金纳米棒和一种改性金纳米棒光热抑菌制剂光热转换效率对比效果图。
图3为本发明实施例中改性金纳米棒光热抑菌制剂的大肠杆菌杀菌效果柱状图。
图4为本发明实施例中金纳米棒和改性金纳米棒光热抑菌制剂的生物毒性对比效果图。
具体实施方式
下面结合具体实施例对本发明作进一步具体详细描述。
实施例1
一种改性金纳米棒光热抑菌制剂及其制备方法,步骤如下:
在室温条件下,于10mL离心管中加入1mL超纯水并置于振荡器上振荡,振荡器的振荡频率为600rpm/min,依次向此离心管中加入1mL摩尔浓度为200ppm的金纳米棒溶胶、500uL浓度为1mg/mL的MnSO4水溶液、500uL摩尔浓度为4mg/mL的原花青素水溶液,时间间隔均为1min,最后,向其中继续加入2mL浓度为20mM的Tris-HCl缓冲液(PH8.0),室温振荡反应5min。反应结束后,将离心管取下,在8000rpm转速,25℃下离心5min后,除去上清液,加入5mL超纯水重新分散,得到改性金纳米棒光热抑菌制剂;将制备得到的抑菌制剂置于4℃冰箱中保存。
光热杀菌:取0.99mL大肠杆菌悬浮液与0.01mLGNRs@Fe-OPC溶液中,用漩涡震荡仪混合3min后,静置。用1.5W/cm2的808nm近红外激光照射器照射10min,用PBS缓冲液(1×)稀释102-105倍,取稀释后的悬浮液0.1mL进行平板涂布,在37℃培养箱中培养12-18h,计算菌落数,以此得到杀菌率。
按照实施例1,合成的改性金纳米棒光热抑菌制剂的整个试验过程,仅需10分钟,且所用多酚化合物(表没食子儿茶素没食子酸酯、单宁酸、原花青素)来自于植物,属于天然化合物,无毒无害,对环境无污染,符合绿色、环保、安全的理念。
图1是基于单宁酸修饰的改性金纳米棒光热抑菌制剂的TEM图;由图1可以看出,在金纳米棒的表面形成具有一定厚度的壳层,意味着金属多酚网络成功涂覆在金纳米棒表面,置换十六烷基三甲基溴化铵的毒性,表明一种改性金纳米棒光热抑菌制剂的成功合成。
图2是金纳米棒和改性金纳米棒光热抑菌制剂光热转换效率对比图;由图2可以看出,改性金纳米棒光热抑菌制剂的光热转换效率得到了极大程度地提高,与单纯的金纳米棒的光热效率相比提高3~4倍。
图3是金纳米棒和改性金纳米棒光热抑菌制剂分别对大肠杆菌的杀菌效果柱状图;由图3可知,金纳米棒对大肠杆菌的杀死率为32%,而改性金纳米棒光热抑菌制剂对大肠杆菌的杀死率达到89%~99%左右,抗菌效果显著高于单纯金纳米棒。
图4为本发明实施例中金纳米棒和改性金纳米棒光热抑菌制剂的生物毒性对比效果图;由图4可知,经过金纳米棒处理细胞后,细胞存活率仅为20%左右,表明金纳米棒具有很高的生物毒性,而改性金纳米棒光热抑菌制剂处理细胞后,细胞存活率为99.1%~100%,表明改性金纳米棒光热抑菌制剂具有很好的生物相容性。
实施例2
一种改性金纳米棒光热抑菌制剂及其制备方法,步骤如下:
在室温条件下,于10mL离心管中加入2mL超纯水并置于振荡器上振荡,振荡器的振荡频率为700rpm/min,依次向此离心管中加入1mL摩尔浓度为200ppm的金纳米棒溶胶、500uL浓度为1mg/mL的FeCl3水溶液、500uL摩尔浓度为4mg/mL的单宁酸水溶液,时间间隔均为1min,最后,向其中继续加入1mL浓度为20mM的Tris-HCl缓冲液(PH8.0),室温振荡反应5min。反应结束后,将离心管取下,在9000rpm转速,25℃下离心7min后,除去上清液,加入5mL超纯水重新分散,得到改性金纳米棒光热抑菌制剂;将制备得到的抑菌制剂置于4℃冰箱中保存。
光热杀菌:取0.99mL大肠杆菌悬浮液与0.01mLGNRs@Fe-TA溶液中,用漩涡震荡仪混合2min后,静置。用2W/cm2的808nm近红外激光照射器照射6min,用PBS缓冲液(1×)稀释102-105倍,取稀释后的悬浮液0.1mL进行平板涂布,在37℃培养箱中培养12-18h,计算菌落数,以此得到杀菌率。
实施例3
一种改性金纳米棒光热抑菌制剂及其制备方法,步骤如下:
在室温条件下,于10mL离心管中加入1.4mL超纯水并置于振荡器上振荡,振荡器的振荡频率为800rpm/min,依次向此离心管中加入1mL摩尔浓度为200ppm的金纳米棒溶胶、300uL浓度为1mg/mL的CuCl2水溶液、300uL摩尔浓度为4mg/mL的表没食子儿茶素没食子酸酯水溶液,时间间隔均为1min,最后,向其中继续加入1mL浓度为20mM的Tris-HCl缓冲液(PH8.0),室温振荡反应5min。反应结束后,将离心管取下,在10000rpm转速,20℃下离心5min后,除去上清液,加入5mL超纯水重新分散,得到改性金纳米棒光热抑菌制剂;将制备得到的抑菌制剂置于4℃冰箱中保存。
光热杀菌:取0.99mL大肠杆菌悬浮液与0.01mLGNRs@Fe-EGCG溶液中,用漩涡震荡仪混合1min后,静置。用3W/cm2的808nm近红外激光照射器照射5min,用PBS缓冲液(1×)稀释102-105倍,取稀释后的悬浮液0.1mL进行平板涂布,在37℃培养箱中培养12-18h,计算菌落数,以此得到杀菌率。
本发明实施例中未注明具体条件者,按照常规条件或者制造商建议的条件进行。所用未注明生产厂商者的原料、试剂等,均为可以通过市售购买获得的常规产品。
本发明制备工艺简单快速、绿色安全,不需要使用有机试剂和昂贵复杂的仪器设备,所用试剂易获得,且均无毒无污染,符合绿色化学理念,不会对环境造成污染。
本发明通过高生物安全性的金属多酚网络置换单纯金纳米棒表面存在的高生物毒性的十六烷基三甲基溴化铵,获得具有良好生物相容性的改性金纳米棒光热抑菌制剂,使其在医药、食品、生物抗菌等领域显示出巨大优势。
其次,本发明利用金属多酚网络的高粘合性,涂覆在金纳米棒表面实现功能化修饰,能够最大化发挥金纳米棒与金属多酚网络的协同光热效应,而且选用金属多酚网络改性金纳米棒,极大地提高了光热转换效率,相比于单纯的金纳米棒,光热转换效率提高了3~4倍,能够高效杀死细菌,且具有广谱杀菌效果,避免细菌耐药性的产生。
此外,本发明改性金纳米棒光热抑菌制剂具有广谱杀菌的功能,不会产生细菌耐药性,在对抗革兰氏阳性菌、抗革兰氏阴性菌以及耐药菌方面均具有很好抑菌效果。
如上所述,便可较好地实现本发明。
本发明的实施方式并不受上述实施例的限制,其他任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种改性金纳米棒光热抑菌制剂的制备方法,其特征在于包括如下步骤:
振荡条件下于10mL离心管,依次加入水、金纳米棒溶胶、金属离子水溶液、多酚化合物水溶液和Tris-HCl缓冲液,常温条件下振荡反应;
反应结束进行离心,除去上清液,得到金属多酚网络修饰的金纳米棒,用作改性金纳米棒光热抑菌制剂,所得改性金纳米棒光热抑菌制剂用Tris-HCl缓冲液重新分散,保存。
2.根据权利要求1所述改性金纳米棒光热抑菌制剂的制备方法,其特征在于,所述金纳米棒溶胶、金属离子水溶液、多酚化合物水溶液、Tris-HCl缓冲液的浓度依次为:(150~200)ppm,(1~2)mg/mL,(2~4)mg/mL,(10~20)mM。
3.根据权利要求2所述改性金纳米棒光热抑菌制剂的制备方法,其特征在于,所述Tris-HCl缓冲液的PH为8.0。
4.根据权利要求3所述改性金纳米棒光热抑菌制剂的制备方法,其特征在于,所述金纳米棒溶胶、金属离子水溶液和多酚化合物水溶液加入的时间间隔依次是1min。
5.根据权利要求4所述改性金纳米棒光热抑菌制剂的制备方法,其特征在于,所述金属离子水溶中的金属离子为Fe3+、Cu2+、Mn2+中的至少一种;
所述多酚水溶液中的多酚化合物为:表没食子儿茶素没食子酸酯、单宁酸、原花青素中的至少一种。
6.根据权利要求5所述改性金纳米棒光热抑菌制剂的制备方法,其特征在于,所述Tris-HCl缓冲液加入后,振荡反应5min。
7.根据权利要求6所述改性金纳米棒光热抑菌制剂的制备方法,其特征在于,所述振荡的频率为600~800rpm;所述室温为20~25℃;所述离心的转速为8000~10000rpm,离心时间为5~10min,温度为20~25℃。
8.一种改性金纳米棒光热抑菌制剂,其特征在于由权利要求1-7中任一项所述制备方法获得。
9.权利要求8所述改性金纳米棒光热抑菌制剂的应用,其特征在于,将改性金纳米棒光热抑菌制剂,用于光热抗菌,步骤如下:
将体积比为99:1的细菌溶液和改性金纳米棒抑菌制剂溶液混合,通过漩涡震荡仪混合均匀1-3min,静置;然后在经过近红外光照射,用PBS缓冲液(1×)稀释102-105倍,取稀释后的悬浮液进行平板涂布,在37℃培养箱中培养,计算菌落数,以此计算杀菌率。
10.权利要求9所述一种改性金纳米棒光热抑菌制剂的应用,其特征在于:所述近红外光的激光照射器的功率为1.5W/cm2-3W/cm2,近红外光的波长为808nm,照射时间为5-10min。
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