贵金属纳米粒子的体外生物学效应评估及其相关研究

发布时间：2018-05-27 01:32

本文选题：贵金属纳米粒子 + 生物学效应　；参考：《兰州大学》2016年硕士论文

【摘要】：随着纳米技术的快速发展及其产品的广泛应用,纳米材料将通过各种可能途径释放到环境中,并通过不同途径进入人体内。然而,对纳米材料与生物系统相互作用的认识还不深入,其生物安全性还未得到全面分析和评价。目前,纳米材料的生物安全效应评估几乎都是以传统生物学实验的方法获取数据加以说明,然而鉴于纳米材料特殊的理化性质可能对这些生物实验方法产生干扰并未加以排除,实际上,最终的安全性评估结果并不真实可靠。为了准确全面的评估纳米粒子的生物安全性,十分有必要系统地确认纳米粒子对于测试方法本身的干扰。另外,纳米材料的生物效应还来源于纳米材料与生物分子的相互作用。纳米粒子一旦与生物液体(如血液、细胞液)接触,其表面会迅速吸附一层或多层蛋白质分子,形成蛋白质冕。纳米粒子在生物体中的性质主要由表面蛋白壳决定,即纳米粒子表面吸附的生物分子的种类和多少赋予了纳米材料在生物环境中新的生物学特性。蛋白质冕的研究能为预测纳米粒子在生物体系中的行为以及设计特定功能的纳米粒子提供最基础的物理化学信息。本论文围绕纳米材料的体外生物效应以及纳米材料与生物分子相互作用,开展了纳米银(AgNPs)在体外生物效应检测中纳米材料对检测方法的干扰,以及纳米材料与蛋白质相互作用的荧光相关光谱法研究,具体研究结果总结如下:1、从光吸收、纳米粒子表面吸附及与化学试剂发生反应等三个方面系统的、定量的研究不同粒径和表面官能团的纳米银(AgNP-PVP-20,AgNP-CIT-20及AgNP-CIT-110)对几种常见的细胞毒性评估实验的干扰,如LDH释放、MTS测定、NO产生及ROS形成的检测。研究结果表明AgNPs确实对上述检测实验产生了干扰,尤其是LDH实验,42.8μg mL-1 AgNP-PVP-20导致0.5 U m L-1 LDH标液体系中LDH活力值下降50%,而42.8μg m L-1 AgNP-CIT-20下降了约70%。结果暗示我们在对AgNPs或其他纳米材料的生物安全效应评估前,须确认纳米粒子是否会对检测实验方法产生干扰。2、探究不同表面包被层的纳米银(AgNP-PVP-20,AgNP-CIT-20)对Hep G2产生的细胞毒性、ROS产生水平及在细胞内AgNPs的分布。MTS、LDH、NO及ROS实验均显示CIT包被的AgNPs产生的细胞毒性明显高于PVP包被的AgNPs,且浓度越大其细胞毒性越强。实验结果证实AgNPs可引起细胞膜破裂、LDH泄露、细胞增殖受抑制及氧化应激反应,从而导致不同程度细胞坏死或凋亡现象。3、基于荧光寿命相关光谱模型,利用金属增强荧光效应且伴随荧光分子荧光寿命变化原理,分离不同荧光寿命物种之间的关联信息,研究纳米金(AuNPs)与蛋白质分子的动力学过程,并获得AuNPs与蛋白相互作用的吸附和脱附速率常数等动力学信息。实验过程中合成了约65 nm的AuNPs,选用柔性HS-PEG-NH2对其进行了功能化,控制金属与荧光团的距离,最终用Alexa Fluo 594酯荧光标记。研究结果表明,在预实验条件下,不同分子量的HS-PEG-NH2对Alexa Fluo594酯荧光寿命改变影响不大。
[Abstract]:With the rapid development of nanotechnology and the wide application of its products, nanomaterials will be released into the environment through various possible pathways and enter the human body in different ways. However, the understanding of the interaction between nanomaterials and biological systems is not thorough, and the biological safety of nanomaterials has not been fully analyzed and evaluated. The assessment of biological safety effects is almost all of the data obtained by traditional biological experiments. However, in view of the special physical and chemical properties of nanomaterials, the possible interference with these biological methods is not eliminated. In fact, the final results of safety assessment are not true and reliable. The biological safety of particles is very necessary to systematically confirm the interference of nanoparticles to the test method itself. In addition, the biological effects of nanomaterials are also derived from the interaction between nanomaterials and biomolecules. Once the nanoparticles are exposed to a biological liquid (such as blood, cell fluid), the surface will quickly adsorb a layer of or multilayer protein. The nature of the nanoparticles in the organism is determined mainly by the surface protein shell, that is, the species and the number of biomolecules adsorbed on the surface of the nanoparticles endow the new biological properties of nanomaterials in the biological environment. The study of the protein corona can predict the behavior and design of nanometers in the biological system. Specific functional nanoparticles provide the most basic physical and chemical information. In this paper, the biological effects of nanomaterials in vitro and the interaction between nanomaterials and biomolecules, the interference of nano materials on detection methods in the detection of biological effects in vitro, and the interaction of nanomaterials and proteins in the detection of biological effects in vitro, are carried out in this paper. The research results of light correlation spectroscopy are summarized as follows: 1, from three aspects such as light absorption, surface adsorption of nanoparticles and reaction with chemical reagents, quantitative study on the interference of different particle sizes and surface functional groups (AgNP-PVP-20, AgNP-CIT-20 and AgNP-CIT-110) to several common cytotoxicity assessment experiments Such as LDH release, MTS determination, NO production and the detection of ROS formation. The results show that AgNPs does produce interference with the above detection experiments, especially the LDH experiment. The 42.8 mu g mL-1 AgNP-PVP-20 results in a decrease of 50% of the activity value in the 0.5 U m L-1 standard liquid system. Before evaluating the biosafety effect of nanomaterials, it is necessary to confirm whether nanoparticles can interfere with the testing methods of.2, explore the cytotoxicity of AgNP-PVP-20 (AgNP-CIT-20) to Hep G2, the level of ROS production and the distribution of AgNPs in the cell,.MTS, LDH, NO, and ROS experiments. The cytotoxicity of S was significantly higher than that of the PVP coated AgNPs, and the greater the concentration was, the stronger the cytotoxicity. The experimental results confirmed that AgNPs could cause cell membrane rupture, LDH leakage, cell proliferation inhibition and oxidative stress reaction, resulting in cell necrosis or apoptosis.3, based on fluorescence life correlation spectral model, using metal enhancement The fluorescence effect and the principle of fluorescence lifetime change, separate the correlation information between different fluorescent life species, study the kinetic process of AuNPs and protein molecules, and obtain the kinetic information of adsorption and desorption rate constant of AuNPs and protein interaction. In the experiment, about 65 nm AuNPs was synthesized. It was functionalized with flexible HS-PEG-NH2 to control the distance between metal and fluorescein, and the fluorescence of Alexa Fluo 594 ester was finally marked. The results showed that, under the pre experimental conditions, the HS-PEG-NH2 of different molecular weights had little effect on the change of the fluorescence lifetime of Alexa Fluo594 ester.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TB383.1

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