石墨烯基纳米复合材料抗菌及清除环境耐药基因的研究

Antibiotic resistance of bacteria seriously threatens human health in many aspects, including reducing effectiveness, aggravating resistance and environmental pollution of conventional antibiotics or disinfectants, so it is very necessary to develop novel materials and technologies with highly efficient antibacterial activities. In recent years, nano-antibacterial materials have been extensively studied and highlighted promising approaches for combating bacterial resistance. However, current nano-materials have not yet combined the activities of antibacterial and environmental antibiotic resistance genes elimination，as well as exerted the risk of inducing bacterial resistance to nanoparticles and promoting horizontal transfer of the resistance genes. Here, we will base on the excellent carrying and adsorption properties of graphene materials, through functional modification, structure-activity relationship study and optimization of the components and preparation method, to improve the synergistic effect among multiple components, synthesizing the graphene-based nanocomposites with activities of anti-drug resistant bacteria and environmental antibiotic resistance genes elimination/ destruction. Moreover, the nanocomposites would also possess the characteristics of not easily inducing resistance, high stability and low biological toxicity. The evaluation of anti-resistant bacteria ability and resistant genes elimination/ destruction efficacy of nanocomposite in various media will be performed against a variety of drug-resistant pathogens. The investigation of nanocomposite inducing bacterial nanomaterial-resistance would be conducted, as well as explore solutions. The toxicity of nanocomposite will also be evaluated. This study aims to provide scientific reference and technical support for combating drug-resistant bacteria and controlling environmental contamination by antibiotic resistance genes.

细菌耐药严重威胁人类健康，传统抗生素及消毒剂存在效力下降、加剧耐药产生及导致环境污染等问题，亟需发展高效抗菌新材料和新技术。近年来纳米抗菌材料成为研究热点并展现了良好的应用前景，但当前报道材料不能兼备高效抗菌和清除环境耐药基因的能力，且存在诱导抗性、促进细菌耐药基因水平转移的风险。本项目基于石墨烯材料优异的运载及吸附性能，进行功能化化学活性修饰，研究材料构效关系并优化组分配比和制备方法，提升多元组分的协同效应，合成兼备抗耐药菌及环境耐药质粒清除/破坏能力的纳米复合材料，同时具有不易诱导抗性、高稳定性及低生物毒性等特点。选取多种代表性耐药菌，在多介质下系统评价其抗耐药菌能力、耐药基因清除/破坏效力并探索作用机理；研究其是否诱导细菌产生纳米材料抗性并探究解决方案；系统评估其生物毒性。本研究拟为应对细菌耐药问题、控制环境耐药基因污染提供科学参考和技术支持。

细菌耐药严重威胁人类健康，传统抗生素存在效力下降、加剧耐药产生及导致环境污染等问题，亟需发展高效抗菌新手段。近年来纳米抗菌材料成为研究热点，但当前报道材料不能兼备高效抗菌和清除环境耐药基因的能力。本项目合成了2种石墨烯基纳米复合材料氧化石墨烯-聚乙烯亚胺-纳米银（GO-PEI-Ag）及氧化石墨烯-聚乙二醇-纳米银（GO-PEG-Ag），具备高效抗耐药菌及不诱导病原产生纳米抗性的优点，尤其是GO-PEI-Ag兼备了良好的耐药质粒清除能力。主要结果如下：1. 改进方法制备了高洁净度单分散的GO，为功能化纳米复合材料的合成提供了良好的基质与骨架。2. 建立了简便、环保的负载纳米银颗粒（AgNPs）的石墨烯基纳米复合材料的合成方法。创新采用微波加热法无需有毒还原剂或表面活性剂的添加，利用GO自身的还原及骨架作用使AgNPs高密度、小粒径均匀地负载在GO表面；通过功能化修饰使纳米抗菌复合材料在细胞培养液、生理盐水等多种溶液中可稳定分散性，解决了石墨烯材料在电解质溶液中聚集沉淀的问题。3.纳米复合材料具备高效的抗菌能力且未诱导纳米抗性产生。包括耐药菌在内的多种病原菌测试，10μg/mL的GO-PEI-Ag对革兰氏阴、阳性菌的杀灭率分别为100%及≥97.4%；10μg/mL的GO-PEG-Ag对革兰氏阴、阳性菌的杀灭率为≥99.5%和≥95%。纳米材料对耐药菌及标准菌杀灭能力相同，不受耐药基因影响，破坏细菌结构导致细胞内容物泄露是主要杀菌机理。对革兰氏阳性菌的杀灭率略低于对革兰氏阴性菌的杀灭率，推测原因主要是革兰氏阳性菌具有较厚细胞壁，对纳米材料的物理破坏起到了一定的缓冲。1μg /mL低浓度纳米复合材料刺激，病原菌压力传代培养20代，最低抑菌浓度（MIC）及杀菌率无明显变化。4. GO-PEI-Ag具备良好的耐药质粒清除能力，20μg /mL的GO-PEI-Ag对mph(A)、mcr-1、arr-3、blaNDM-1 四种耐药质粒的清除率均≥99.9%。5.具备较低的细胞毒性，经50 μg/mL的2种纳米复合材料作用8h后HeLa细胞活性≥80%。6.良的好抗菌持久性及抗有机物干扰能力，10μg/ml的GO-PEI-Ag喷洒物表24h及48h后，对自然下落菌杀灭率分别为100%及80.9%；体系内血清浓度为50%时纳米材料对大肠杆菌的杀菌率未受到无明显影响。

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