兰州大学机构库 >药学院
新型半导体硒化铜铁纳米材料的制备 及抗菌性能与机制研究
Alternative TitleSynthesis of Novel Semiconductor Copper Iron Selenide Nanomaterial and Study on Antimicrobial Properties and Mechanisms
Wang Meng
Subtype硕士
Thesis Advisor刘映前
2023-05-29
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学硕士
Degree Discipline药学
Keyword硒化铜铁 Copper iron selenide 纳米抗菌材料 Anti-microbial nanomaterial 内容物的泄露 Leakage of intracellular components 活性氧的产生 Reactive oxygen species generation 纳米刀片锋利的边缘效应 Sharp edges effect of nano- knives 生物相容性 Biocompatibility
Abstract

抗菌相关药物的严重短缺和抗生素滥用导致的多药耐药性使得新型抗菌药物的研发迫在眉睫。纳米材料因其本身尺寸优点具有较强的跨膜能力、抑制外排泵的功能及不易诱发耐药性等特点,作为抗肿瘤药物已经有着诸多研究,但其用于抗真菌方面是近几年新的研究课题,迄今为止硒化铜铁纳米材料还未被应用于抗真菌研究中。

本课题采用温和、绿色、无污染的水相沉淀法制备出了硒化铜铁纳米材料。通过高分辨率等离子体发射光谱仪、X-射线光电子能谱仪、X射线衍射仪、傅里叶变换红外光谱仪、透射电子显微镜、扫描电子显微镜、紫外光电子能谱等手段测定了纳米材料的元素含量及价态、化学组成、形貌、晶型等物理化学性质,确定了其结构式为CuFeSe2-PVP。

对这种独特的半导体CuFeSe2-PVP纳米材料进行了体内外抗真菌活性及清除生物被膜能力的检测,通过96孔板实验测定了CuFeSe2-PVP对三种白色念珠菌珠(常用菌株ATCC10231,敏感菌株ATCC14053,耐药菌株BNCC359501)的MIC90值低至1.56 μg/mL,其抗真菌效果优于阳性对照药物(特比萘芬、氟康唑、5-氟胞嘧啶)和传统抗微生物型纳米材料(AgNPs、AuNPs、TiO2NPs),又通过涂板实验验证了CuFeSe2-PVP的体外抗真菌效果。结晶紫染色实验证明,即使低浓度的CuFeSe2-PVP也能有效清除已长成的生物被膜,且超过同浓度下阳性对照药物的清除率,又通过SYTOTM 9/PI双染色验证了其高效的生物被膜清除作用。在体动物实验有力地证明了CuFeSe2-PVP纳米材料优于5-氟胞嘧啶的治疗效果,H&E和Masson染色显示CuFeSe2-PVP纳米材料治疗的真菌感染小鼠病灶处皮肤已恢复至健康水平。接下来进行了CuFeSe2-PVP杀真菌机制的研究,通过绘制杀菌曲线、检测真菌胞内核酸和蛋白质的泄露、测定暗示着细胞内容物泄漏的钾离子的流出表明了真菌是在短时间内大量死亡的;通过扫描电子显微镜和透射电子显微镜观察到CuFeSe2-PVP纳米刀片自身锋利的边缘可以插入真菌体内,从而引起真菌死亡;通过体外电子自旋共振谱分析证实在过氧化氢存在下,纳米材料可以产生活性氧(ROS),在DCFH-DA染色的给药真菌体内检测到了大量ROS的存在,ROS也是被广泛报导的杀微生物机制之一。以上几种机制相互协同,赋予了CuFeSe2-PVP纳米材料在短时间内高效杀真菌的能力。生物相容性是材料应用于体内的前提条件,从材料的选择上,选取的元素为人体必需的微量元素,广泛应用的无毒无害辅料聚乙烯吡咯烷酮(PVP)作为形貌导向剂,制备的纳米材料本身无毒无害;体外实验检测了大鼠心肌细胞H9C2的存活率和兔红细胞的溶血率,体内实验通过小鼠心、肝、脾、肺、肾器官的H&E染色检测了材料的动物毒性,以上实验均初步证明了CuFeSe2-PVP纳米材料的生物相容性。

体外抑菌实验测定了CuFeSe2-PVP对金黄色葡萄球菌(ATCC29213)和表皮葡萄球菌(ATCC12228)的MIC90值分别低至6.25 μg/mL和1.56 μg/mL,又通过涂板实验验证了其抗细菌效果,结果表明其抗细菌效果超过传统抗菌型纳米颗粒(AgNPs、AuNPs、TiO2NPs)且等同于抗生素(万古霉素、环丙沙星、噻孢霉素);结晶紫染色实验证明了即使在低浓度下(MIC90),CuFeSe2-PVP也能有效清除已长成的金黄色葡萄球菌和表皮葡萄球菌生物被膜;通过SYTOTM 9/PI双染色验证了其优异的生物被膜清除作用。在体动物实验中,进一步证明了CuFeSe2-PVP的治疗效果,H&E和Masson染色显示了CuFeSe2-PVP纳米材料治疗的小鼠病灶处皮肤已经恢复至健康水平。后续进行了CuFeSe2-PVP杀细菌机制的研究,通过检测细菌体内核酸和蛋白质的泄露及测定钾离子的流出表明了细菌是在短时间内大量死亡的;扫描电子显微镜和透射电子显微镜观察可见菌体模糊甚至看不到细菌的存在,在细菌“尸体”周围观察到了纳米材料;在过氧化氢存在下纳米材料能够产生大量的ROS,这主要与Cu+的释放有关;以上几种机制相互协同,使CuFeSe2-PVP纳米材料表现出优异的杀细菌性能。

最终本课题得到了制备简便、内在抗菌能力强、稳定性高、生物相容性好、易于进一步转化的新型纳米材料CuFeSe2-PVP。这项研究不仅提供了一种新型的杀菌候选药物,而且对未来抗菌型纳米药物的设计有所启发。

Other Abstract

With the severe shortage of antimicrobial-related drugs and multidrug resistance due to antibiotic abuse, it become increasingly urgent for us to develop novel antimicrobial drugs. Nanomaterials have already been studied as anti-tumor drugs because of their unique size, strong transmembrane ability, inhibition of efflux pump function and less prone to resistance, but their antifungal applications are a new topic research in recent years. Copper iron selenide nanomaterials have hitherto not been used in antifungal research.

In this thesis, the copper iron selenide nanomaterial is facilely synthesized by a mild, green and non-polluting aqueous precipitation method. Its physical and chemical properties such as elemental content and valence, chemical composition, morphology and crystal forms were tested by ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer), XPS (X-Ray Photoelectron Spectroscopy), XRD (X-ray Diffraction), FTIR (Fourier Transform Infrared Spectrometer), TEM (Transmission Electron Microscope), SEM (Scanning Electron Microscope), UPS (Ultraviolet Photoelectron Spectroscopy) and so forth. Eventually, the structural formula was determined to be CuFeSe2-PVP.

This unique semiconductor CuFeSe2-PVP demonstrates superior antifungal activity than the positive control drugs (terbinafine, fluconazole, 5-fluorocytosine) and traditional antimicrobial nanoparticles (AgNPs, AuNPs, TiO2NPs) in both planktonic fungi and biofilm. The antifungal effect of CuFeSe2-PVP on Candida albicans (ATCC10231, ATCC14053, BNCC359501) was determined by inhibition rate test in microplate with MIC90 as low as 1.56 μg/mL and was verified by the spread-plate experiment. Then, crystal violet assay and live/dead staining were usually used to quantify the anti-biofilm efficacy, and CuFeSe2-PVP with the concentration of 1.56 μg/mL attained a nearly complete biofilm eradication even facing the pre-formed Candida albicans biofilm. Animal experiments in vivo have strongly demonstrated that the CuFeSe2-PVP nanomaterial had higher therapeutic efficacy than 5-fluorocytosine, and mice treated with the CuFeSe2-PVP nanomaterial had completely recovered to healthy levels by analyzing H&E and Masson staining of skin wound. In the study of the fungicidal mechanism, the fact that massive fungi were killed in a short period was demonstrated by making time-dependent fungal inhibition assay, detecting the leakage of nucleic acid and protein, and measuring the leakage of K+ which implied the leakage of intracellular components. It was observed by SEM and TEM that the fungal death was directly bound up with the fact that the nano-knives derived from the action of sharp edges itself could be inserted into the fungal body. ROS generation is also extensively reported as bactericidal mechanisms. Encouragingly, CuFeSe2-PVP could generate ROS (Reactive Oxygen Species) detected by ESR (Electron Spin Resonance) experiments and DCFH-DA staining assay. In conclusion, the ability of CuFeSe2-PVP to effectively killing fungi in a short duration is achieved through combining aforementioned mechanisms. Furthermore, biocompatibility is a prerequisite for the application of all materials in vivo. At first, from the perspective of selected materials, elements in this nanomaterial are the essential metals for humans and PVP is widely used for stabilizing and shape-directing agent, thus the obtained nanomaterial is inherently safe. In vitro experiments, the survival rate of H9C2 and the rate of hemolysis in rabbit erythrocyte were examined. And the animal toxicity of the nanomaterial was tested by H&E staining of the heart, liver, spleen, lung and kidney organs in vivo experiments. All of the above experiments have preliminary demonstrated the biocompatibility of the CuFeSe2-PVP nanomaterial.

At the same time, the bactericidal effects of CuFeSe2-PVP in vitro on Staphylococcus aureus (ATCC29213) and Staphylococcus epidermidis (ATCC12228) were determined by inhibition rate test in microplate with MIC90 as low as 6.25 μg/mL and 1.56 μg/mL, respectively. The antibacterial effect was also verified by spread-plate experiment. The above results revealed that the antibacterial effect exceeded that of conventional antibacterial nanoparticles (AgNPs, AuNPs, TiO2NPs) and was equivalent to that of antibiotics (vancomycin, ciprofloxacin, thiosporine). Then, crystal violet assay and live/dead staining were often used to quantify the anti-biofilm efficacy. Intriguingly, CuFeSe2-PVP with low concentrations could attain a nearly complete biofilm eradication even facing the pre-formed bacterial biofilms. In vivo studies, the therapeutic efficacy of CuFeSe2-PVP has robustly been demonstrated, where H&E and Masson staining of skin samples from wounds showed that mice cured with CuFeSe2-PVP nanomaterial had recovered to healthy levels. In addition, On the research of the bactericidal mechanism, the fact that large number of bacteria were killed in a short period was illustrated by detecting the leakage of nucleic acid and protein and measuring the leakage of K+. Besides, the body of bacteria was found by SEM and TEM to be obscured or even invisible, while nanomaterials were observed surrounding the bacterial “corpse”. Last but not least, CuFeSe2-PVP can generate massive ROS, which is primarily associated with the release of Cu+. Generally speaking, the superior bactericidal properties of CuFeSe2-PVP nanomaterial are achieved by combining above mechanisms.

At last, a novel nanomaterial CuFeSe2-PVP is obtained which has simple synthesis method, high intrinsic anti-fungal and anti-bacterial ability, excellent stabilization, and outstanding biocompatibility, making it easier to be further transformed. This study not only provides a novel fungicidal and bactericidal drug candidate, but also inspires the future design of anti-fungal and antibacterial nanomedicines.

MOST Discipline Catalogue医学 - 药学(可授医学、理学学位)
URL查看原文
Language中文
Other Code262010_220200942230
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/535710
Collection药学院
Affiliation
兰州大学药学院
Recommended Citation
GB/T 7714
Wang Meng. 新型半导体硒化铜铁纳米材料的制备 及抗菌性能与机制研究[D]. 兰州. 兰州大学,2023.
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