CN114533947B - 一种具有抗菌活性和加速伤口愈合能力的碳基双金属纳米簇的制备方法及应用 - Google Patents

一种具有抗菌活性和加速伤口愈合能力的碳基双金属纳米簇的制备方法及应用 Download PDF

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CN114533947B
CN114533947B CN202210160838.1A CN202210160838A CN114533947B CN 114533947 B CN114533947 B CN 114533947B CN 202210160838 A CN202210160838 A CN 202210160838A CN 114533947 B CN114533947 B CN 114533947B
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黄昊文
汪子祺
朱佳怡
邓克勤
阳秀梅
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Hunan University of Science and Technology
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Abstract

本发明公开一种具有抗菌活性和加速伤口愈合能力的碳基双金属纳米簇的制备方法及应用。本发明利用微波辅助法成功地将金、银以纳米簇的形式嵌入碳骨架中，含金银碳点将金纳米簇、银纳米簇和碳点三者性质结合。进一步通过紫外照射处理，转换银元素在碳基双金属纳米簇中的价态赋予其不同的抑菌性质，可用于区别筛选耐药菌和普通细菌，消除伤口中的不同程度的炎症反应。同时，碳基双金属纳米簇对生物系统具有良好的生物相容性，通过MTT的验证，含金银碳点对正常细胞有增殖作用，改善了一般纳米材料低毒性的特点。

Description

一种具有抗菌活性和加速伤口愈合能力的碳基双金属纳米簇 的制备方法及应用
技术领域
本发明属于伤口愈合以及生物医用敷料领域，具体涉及具有抗菌活性和加速伤口愈合能力的碳基双金属纳米簇的制备方法及应用。
背景技术
近年来，耐甲氧西林金黄色葡萄球菌(MRSA)、耐药结核病(TB)、耐碳青霉烯的肠杆菌科(CRE)等多重耐药菌在世界快速传播，直接证明了在抗生素的治疗下，细菌仍能够成功地进化和生存，继而导致伤口难以快速愈合。理想的愈合系统应该要满足以下几个方面：快速关闭伤口，减少感染，模仿细胞外基质特征，润湿伤口，减少疤痕形成等。但目前的产品、设备和技术均不能在保留皮肤功能的同时满足以上几个方面。快速应用发展的纳米材料，呈现了抑菌材料的独特性质，为伤口愈合治疗和根除抗性细菌这两方面提供了契机，提供了加速伤口愈合的解决方案。
基于结合不同治疗性纳米材料的协同作用，已经制定了两个领域的策略：将具有抗菌特性的纳米材料嵌入各种基于天然聚合物/无机材料的混合支架中，或使用纳米载体来封装活性剂。碳点(CDs)是一类尺寸小于10纳米的碳基荧光纳米材料。由于其优越的光学特性、易于合成、良好的生物相容性、抗光漂白作用和出色的水溶性，使得它们在生物医学应用中备受关注。银纳米粒子(AgNPs)具有卓越的抗菌特性，金纳米粒子(AuNPs)具有良好化学稳定性和生物相容性。文献中描述，AgNPs可以释放调节抗炎细胞因子，促进伤口快速闭合，还可以通过角质细胞的增殖来刺激表皮的再表皮化。AuNPs能够促进伤口愈合并抑制微生物活性，低剂量时还能增强角质细胞的生长和分化，这使得它们在抑菌，伤口治疗方面备受欢迎。因此，为了提供具有抗菌活性以及伤口愈合作用的设计，本发明将纳米金银与碳量子点结合在一个碳基双金属纳米簇结构中，获得了一系列新的特性。
发明内容
本发明的目的是提供一种具有抗菌活性和加速伤口愈合能力的碳基双金属纳米簇的制备方法及应用。合成一种兼具良好抑菌性和促进伤口细胞增殖作用的碳基双金属纳米簇材料，利用纳米银、纳米金具有抗菌活性和刺激伤口愈合等突出特性，碳量子点具有良好的水分散性、易于合成和表面修饰化等特点将二者结合以实现协同效应；开发了纳米材料在加速愈合伤口方面的应用潜力；开启了纳米材料在筛选并杀死耐药菌应用的新领域，制备简单、操作方便，是一款极具潜力适应普及的新型双金属纳米材料。
本发明的目的是通过下述方式实现的：
一种具有抗菌活性和加速伤口愈合能力的碳基双金属纳米簇的制备方法，包括如下步骤：
(1)将体积比为3-5：1的等浓度HAuCl4与AgNO3混合后，加入谷胱甘肽至谷胱甘肽的浓度为7-8mmol/L；
(2)向步骤(1)所得双金属盐溶液中加入葡萄糖粉末，搅拌至溶解；
(3)将步骤(2)所得溶液进行微波加热，并加入去离子水溶解产物；
(4)对步骤(3)所得产物溶液进行透析，去除未反应物质，然后进行冻干处理，得到黄棕色粉末状的碳基双金属纳米簇簇复合材料，即含金银碳点。
进一步地，还包括含金银碳点的紫外处理，具体为：将步骤(4)所得含金银碳点置于紫外灯下照射2～6h，得到紫外处理的含金银碳点(UV)。
进一步地，步骤(1)中，HAuCl4与AgNO3的浓度为10-30mmol/L。
进一步地，步骤(3)中，微波加热的时间为20-30min。
进一步地，步骤(4)中，所述透析采用截留量为8000-14000Da的透析袋。
上述碳基双金属纳米簇材料具有过氧化物模拟酶性质，测试方法为TMB显色：向体系中加入TMB溶液，TMB浓度为5mmol/L，两种溶液的体积比为1：1，确定仅有TMB和含金银碳点存在时，TMB也能变色，说明含金银碳点具有较强的过氧化物模拟酶性质。
上述碳基双金属纳米簇材料在溶液中能与溶解氧形成活性氧，具有抑菌作用，检验含金银碳点的抑菌性的细菌浓度为106CFU/mL；细菌以大肠杆菌，大肠杆菌O157:H7；金黄色葡萄球菌，耐甲氧西林金黄色葡萄球菌为例。
上述碳基双金属纳米簇通过紫外照射处理后，对不同分型的细菌表现出不同的抑菌活性，可以区别同种类型中不同亚型的细菌，因此能够应用于筛选致病菌和耐药菌中。
上述碳基双金属纳米簇材料既有抑菌作用又有促进伤口细胞增殖作用，能够促进人和其它动物伤口细胞增殖作用达到加速伤口愈合的目的。检验含金银碳点细胞增殖作用的细胞为人皮肤成纤维细胞(BJ)、人胚胎肾细胞(HEK-293)。
依据碳基双金属纳米簇的抑菌和促进细胞增殖的双重性质，制备性质稳定的、能快速使伤口愈合的碳基双金属纳米簇水凝胶，所述碳基双金属纳米簇水凝胶的制备方法为：
(a)分别配置M/G摩尔比为1：3-4(优选1:3.21)的海藻酸钠水溶液(海藻酸钠主要由两种糖醛酸单体组成，一种是赶路糖醛酸(简称M)，另一种是古洛糖醛酸(简称G))和质量分数为4-6％(优选5％)的无水氯化钙溶液，搅拌均匀完全溶解，备用；
(b)空白模具中加入1-2mL海藻酸钠溶液；
(c)将模具在无水氯化钙溶液中浸泡2-3小时；
(d)用无菌水冲洗水凝胶，在无菌水中浸泡1-1.5小时；
(e)取24孔板，孔中加入含金银碳点溶液，转移步骤(d)所得凝胶至孔板内完全浸泡16-32小时；
(f)塑封4℃以下保存。
有益效果
与现有技术相比，本发明的有益效果是：
(1)本发明是应用于抑耐药菌，伤口愈合双重功能的新型碳基双金属纳米簇材料。利用微波辅助法成功地将金、银以纳米簇的形式嵌入碳骨架中，含金银碳点将金纳米簇、银纳米簇和碳点三者性质结合。进一步通过紫外照射处理，转换银元素在碳基双金属纳米簇中的价态赋予其不同的抑菌性质，可用于区别筛选耐药菌和普通细菌，消除伤口中的不同程度的炎症反应。同时，碳基双金属纳米簇对生物系统具有良好的生物相容性，通过MTT的验证，含金银碳点对正常细胞有增殖作用，改善了一般纳米材料低毒性的特点。
(2)本发明基于碳基双金属纳米簇优异的水溶性，海藻酸钠的高吸水性进而成功制备了加速伤口愈合的水凝胶敷料。
(3)本发明突破了纳米材料在伤口愈合应用中常见的性质即抑菌性，打破了纳米材料因对人体细胞具有毒性使伤口愈合速度慢的僵局。操作方法简便的同时还可以有效筛选抑制耐药菌活性，具有广阔的应用前景。
附图说明
图1：a为含金银碳点的透射电子显微镜、粒径分布图，b为含金银碳点的电位图。
图2：为含金银碳点的紫外可见吸收光谱、荧光发射光谱。
图3：a为含金银碳点的X射线光电子能谱，b为含金银碳点(UV)的X射线光电子能谱。
图4：为验证含金银碳点具有过氧化物模拟酶性质的显色图片。从左至右分别是TMB+H2O2，TMB+含金银碳点和TMB+含金银碳点+H2O2作用12h后的显色图。
图5：为验证含金银碳点对革兰氏阴阳氏菌抑菌性的平板涂布图。
图6：为含金银碳点、含金银碳点(UV)对不同分型的细菌响应不同的平板涂布抗菌实验。
图7：为检测与含金银碳点作用后致病菌和普通细菌体内ROS变化实验。
图8：a、b为验证含金银碳点、含金银碳点(UV)具有细胞增殖性的细胞增值率图片。
图9：为验证含金银碳点对伤口细胞的增殖性质，检测与含金银碳点作用后细胞内ATP的变化柱状图。
图10：为碳基双金属纳米簇水凝胶的样品图。
图11：为本发明实施例碳基双金属纳米簇处理SD大鼠背部伤口后伤口愈合的照片。
图12：为SD大鼠实验组的伤口愈合后实验组对照组伤口周围组织切片图。
具体实施方式
以下实施例旨在说明本发明而不是对本发明的进一步限定。
实施例1
含金银碳点、含金银碳点(UV)的制备：
将1mL新鲜配置的谷胱甘肽溶液，4mL的AgNO3和1ml的HAuCl4(两者浓度均为20mmol/L)于干净烧杯中混合均匀。待混合物溶液颜色从黄褐色变成乳白色后，加入二次蒸馏水稀释混合物至50mL，混合5分钟后再加入2g葡萄糖至完全溶解。用高火微波混合物20分钟左右取出。迅速加入50mL二次蒸馏水摇匀使产物重新溶于水中。待溶液体系稳定后，用截留量为8000-14000的透析袋透析48小时后，即可得到浅黄色的含金银碳点溶液。含金银碳点是粒径为3纳米左右分散均匀呈电负性的碳基双金属纳米簇复合材料，其形貌粒径电位如图1所示；图2含金银碳点的荧光光谱中双发射峰表征了其优越的光学特性。该复合物具有过氧化物模拟酶性质，如图4所示，在仅含有含金银碳点和TMB的情况下，含金银碳点也能使其变色。含金银碳点在紫外灯照射下6h左右得到含金银碳点(UV)。通过XPS探索含金银碳点、含金银碳点(UV)之间的差异，结果如图3所示。
含金银碳点水凝胶的制备：
称取海藻酸钠粉末0.4g(M:G＝1:3.21)。混合均匀后溶于10mL无菌二次蒸馏水中，搅拌成均匀的粘稠溶液备用。取质量分数为1％的无水氯化钙无菌溶液置于玻璃皿中备用。用吸样器向空白模具中加入海藻酸钠溶液，使其完全浸泡在CaCl2中2小时左右。凝胶定型后取出，用无菌水清洗3次后，用无菌水浸泡1小时。取24孔板，孔中加入含金银碳点溶液，转移凝胶至孔板内完全浸泡。浸泡24小时左右取出，真空包装4℃低温无菌保存。含金银碳点水凝胶实物图如图10所示。
实施例2
含金银碳点区别同类不同型细菌的检测：
使用LB液体培养基将菌液稀释至106CFU/mL浓度，将稀释菌液1mL与样品(对照组用无菌纯水)以1:1的体积比分组添加。放于恒温培养箱37℃培养18h。培养完成后取100uL稀释液均匀涂布于LB固体培养基上。放于37℃恒温培养箱中培养18h取出拍照并记录菌落数。实验选取大肠杆菌、大肠杆菌O157：H7；金黄色葡萄球菌、MRSA为例。由图5可知，含金银碳点对革兰氏阴性菌、革兰氏阳性菌均有一定的抑菌性能。由图6可知，含金银碳点(UV)对于普通细菌的抑菌性能较好；含金银碳点对致病菌和耐药性菌抗菌性能较好。检测菌内ROS的变化，验证上述实验现象，从图7中可以看出ROS变化表达的结果与平板涂布呈现的一致。
实施例3
含金银碳点细胞增殖作用检测：
细胞增殖作用通过细胞毒性实验进行测试，将细胞接种于96孔板中，细胞量为0.5万/孔。培养24h至细胞恢复正常生长状态后，弃去原培养液，重新加入样品与新配培养液的混合液，对照组用无菌水与培养液的混合液。培养1-3天，将混合液移除，加入新配培养液200uL，MTT 20uL，继续培养4h后，吸去空中全部液体，加入150uL DMSO，150rpm震荡10分钟。用酶标仪测590nm处吸光度。如图8所示，含金银碳点促进人皮肤成纤维细胞(BJ)、人胚胎肾细胞(HEK-293)生成。进一步检测细胞内的ATP变化(图9)，与含金银碳点作用后的细胞ATP含量提高，表示体内在加速新陈代谢，含金银碳点对细胞是有增殖作用的。
实施例4
含金银碳点水凝胶应用—活体实验：
选取4只健康的5-6周大的SD雄性大鼠。在大鼠后腿两侧对称地建立两个直径为8毫米、厚度为3毫米的切除性伤口。两个伤口中的一个放置水凝胶用作治疗，另一个被用作对照，用3M胶带封闭伤口。每两天拍摄一次伤口照片，并更换水凝胶和胶带。伤口愈合后，用4％多聚甲醛固定伤口和周围组织，对小鼠的创伤性皮肤进行切片，进行病理检查。实验组和对照组伤口的恢复情况如图11所示，在实验第8天，实验组伤口完全愈合而对照组伤口未完全恢复。病理分析如图12所示，对照组的角质层是脱落的，真皮层的胶原纤维稀少松散；实验组角质层贴合紧密，真皮层胶原纤维数量多排列紧密。碳基双金属纳米簇通过小鼠伤口愈合试验充分的验证了含金银碳点的两个性质。验证了该水凝胶的可行性。

Claims (7)

1.一种具有抗菌活性和加速伤口愈合能力的碳基双金属纳米簇的制备方法，其特征在于，包括如下步骤：
（1）将体积比为3-5：1的等浓度HAuCl4与AgNO3混合后，加入谷胱甘肽至谷胱甘肽的浓度为7-8mmol/L；
（2）向步骤（1）所得双金属盐溶液中加入葡萄糖粉末，搅拌至溶解；
（3）将步骤（2）所得溶液进行微波加热，并加入去离子水溶解产物；
（4）对步骤（3）所得产物溶液进行透析，去除未反应物质，然后进行冻干处理，得到黄棕色粉末状的碳基双金属纳米簇簇复合材料，即含金银碳点；
还包括含金银碳点的紫外处理，具体为：将步骤（4）所得含金银碳点置于紫外灯下照射2~6 h，得到紫外处理的含金银碳点。
2.根据权利要求1所述的具有抗菌活性和加速伤口愈合能力的碳基双金属纳米簇的制备方法，其特征在于，步骤（1）中，HAuCl4与AgNO3的浓度为10-30 mmol/L。
3.根据权利要求1所述的具有抗菌活性和加速伤口愈合能力的碳基双金属纳米簇的制备方法，其特征在于，步骤（3）中，微波加热的时间为20-30min。
4.根据权利要求1所述的具有抗菌活性和加速伤口愈合能力的碳基双金属纳米簇的制备方法，其特征在于，步骤（4）中，所述透析采用截留量为8000-14000Da的透析袋。
5.权利要求1至4任一项所述的具有抗菌活性和加速伤口愈合能力的碳基双金属纳米簇的制备方法得到的碳基双金属纳米簇在筛选致病菌和耐药菌中的应用。
6.权利要求1至4任一项所述的具有抗菌活性和加速伤口愈合能力的碳基双金属纳米簇的制备方法得到的碳基双金属纳米簇在制备加速伤口愈合的水凝胶敷料中的应用。
7.一种碳基双金属纳米簇水凝胶的制备方法，其特征在于，包括如下步骤：
（a）分别配置M/G摩尔比为1：3-4的海藻酸钠水溶液和质量分数为4-6%的无水氯化钙溶液，搅拌均匀完全溶解，备用；
（b）空白模具中加入1-2mL海藻酸钠溶液；
（c）将模具在无水氯化钙溶液中浸泡2-3小时；
（d）用无菌水冲洗水凝胶，在无菌水中浸泡1-1.5小时；
（e）取24孔板，孔中加入权利要求1至5任一项所述制备方法得到的含金银碳点溶液，转移步骤（d）所得凝胶至孔板内完全浸泡16-32小时；
（f）塑封4℃以下保存。
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