钌配合物功能化金纳米粒子的制备及其生物活性研究

发布时间：2018-03-01 07:29

本文关键词： 抗肿瘤药物金纳米粒子钌配合物生物活性　出处：《深圳大学》2017年硕士论文　论文类型：学位论文

【摘要】：研发抗肿瘤药物是科研研究的难点和重点,该项研究不仅结合了化学、生物、药学、医学等领域,而且将对人类生命健康产生了重大影响。在金属抗肿瘤药物中,铂配合物是临床医学上广泛使用的抗肿瘤药物,然而,铂配合物作为抗肿瘤药物具有明显的毒副作用,如肾毒性、肝毒性、神经毒性以及产生耐药性等,这使得人们把目光转向开发其它的金属抗肿瘤药物。钌配合物具有稳定的化学结构和良好的物理、化学性质,具有潜力成高效低毒的新型抗肿瘤药物。钌配合物是六配位八面体的结构,配体的可改变性使其结构具有多样性,可满足不同的设计要求。但其缺乏肿瘤靶向性且穿膜能力差,降低其抗肿瘤活性。而金纳米粒子(AuNPs)由于具有良好的生物相容性,被广泛用于药物载体、生物探针、荧光成像等应用。因此,本论文通过将钌配合物负载到金纳米粒子表面,结合二者优异的性质,提高其生物活性。本论文主要通过柠檬酸钠还原法合成AuNPs,利用同时含巯基和氨基活性基团的分子修饰AuNPs,形成Au-S键,构建稳定性良好的金纳米材料体系,该体系的NH2基团用于与含羧基的钌配合物进行酰胺化反应,最终得到钌-金纳米复合体系。研究表征了钌-金纳米复合材料的形貌特点,光谱学性质和抗肿瘤活性,具体的研究内容如下:1、采用柠檬酸钠还原法改变还原剂的比例,合成了纳米尺寸为15 nm、30 nm、80nm、120 nm的金纳米粒子。为提高金纳米粒子的稳定性,采用巯基配体通过Au-S的共价吸附作用修饰AuNPs,以提高其稳定性。通过紫外分光光度计、Zetas电位仪、纳米激光粒度仪对HS-PEG2000和LA-mPEG5000两种巯基聚乙二醇,以及巯基乙胺、巯基丙酸、半胱氨酸三种巯基小配体进行筛选,选出了LA-mPEG5000和巯基乙胺是最适合用来对金纳米粒子进行稳定修饰的双配体,其中,LA-mPEG5000可以提高金纳米粒子的稳定性,巯基乙胺可以使其金纳米粒子表面具有活性基团,方便其后序应用。修饰后的金纳米粒子可在不同浓度的盐溶液和不同pH值的缓冲液中稳定存在。2、我们选取了具有抗肿瘤活性且带有羧基的钌配合物[Ru(OPDA)(4-CTPY)NO]3+(Ru-NO)与经过巯基乙胺修饰后表面带有氨基的金纳米粒子通过酰胺化反应结合,使Ru-NO负载到金纳米粒子表面,再通过紫外分光光度计、红外光谱仪、能谱仪来表征构建的钌-金纳米复合体系(Au-Ru-NO),以确保Ru-NO已成功负载到金纳米粒子表面;经过ICP-MS测量,可知钌-金纳米复合体系中每个金原子表面负载的钌原子个数约为3.28×102。将钌配合物负载到金纳米粒子表面,结合二者优异的物化性能,提高其生物活性,可以更好的应用到生物医学领域中。3、通过酰胺化反应成功构建钌-金纳米复合体系后,采用多种细胞方法对Ru-NO和Au-Ru-NO的生物活性进行研究。通过MTT法测定可知,Ru-NO作用于Hep-2细胞24h后的细胞毒性约为8±2μM(即Ru-NO作用于Hep-2细胞的IC50浓度),而Au-Ru-NO作用于Hep-2细胞的IC50浓度约为17±2μM,说明钌配合物功能化金纳米粒子后可降低细胞毒性。此外,通过Annexin V/PI双染法,活性氧含量的测定,线粒体膜电位的变化、蛋白质免疫印迹法等来探索细胞凋亡机制。通过以上方法可知Ru-NO主要通过诱导细胞内活性氧含量增加、线粒体膜电位降低、Bcl-2蛋白下调、p53和Bax蛋白上调来促使Hep-2细胞凋亡;而Au-Ru-NO在引起细胞内活性氧含量增加、线粒体膜电位降低的同时,促使p53蛋白下调、Bax蛋白上调来诱导细胞凋亡。激光共聚焦显微镜可观察到Ru-NO和Au-Ru-NO主要分布在细胞质,少量可进入细胞核。通过Ru-NO和Au-Ru-NO的实验结论对比,可知Au-Ru-NO在引起细胞凋亡的同时可降低系统的整体毒性,有作为活性高毒性低抗肿瘤药物的潜能。
[Abstract]:The development of anticancer drugs is the key point of the research, the study is not only a combination of chemical, biological, pharmaceutical, medical and other fields, but also had a significant impact on human health. The metal anticancer drugs, platinum complexes are widely used in clinical antitumor drugs. However, platinum complex has obvious side effects as antitumor drugs, such as kidney toxicity, liver toxicity, neurotoxicity and drug resistance, which makes people turn to the development of other metal anticancer drugs. Ruthenium complexes have stable chemical structure and good physical and chemical properties, has the potential to new anticancer drugs with high efficiency and low toxicity the ruthenium complexes are six coordinated structure with eight sides, the ligand can change the structure of diversity, can meet different design requirements. But the lack of tumor targeting and penetrating ability, To reduce its antitumor activity. The gold nanoparticles (AuNPs) with good biocompatibility, is widely used in drug carrier, biological probe, fluorescent imaging applications. Therefore, the ruthenium complexes supported gold nanoparticles surface, with two excellent properties, improve the biological activity of the thesis. The sodium citrate reduction method for the synthesis of AuNPs, using molecular modification of AuNPs thiol containing amino group and at the same time, the formation of Au-S bond, construct the good stability of the gold nano material system, and a carboxyl group containing ruthenium complexes by amidation reaction for NH2 groups of the system, finally get the ruthenium nano gold characterization of composite system. The morphology characteristics of ruthenium Au nanocomposites, spectroscopic properties and antitumor activity, the specific contents are as follows: 1, using sodium citrate reduction method changes the reducing agent ratio, synthesis of nano scale 15 inch nm, 30 nm, 120 80nm, nm gold nanoparticles. In order to improve the stability of gold nanoparticles, using thiol ligands modified by covalent AuNPs adsorption of Au-S, in order to improve its stability. By UV spectrophotometer, Zetas potential instrument, laser nanometer particle size analyzer for HS-PEG2000 and LA-mPEG5000 two kinds of polyethylene glycol well, Mercaptoethylamine, 3-mercaptopropionic acid, cysteine three thiol small ligands were screened, selected LA-mPEG5000 and Mercaptoethylamine is the most suitable for the dual ligand, stable modification of gold nanoparticles, LA-mPEG5000 can improve the stability of gold nanoparticles, Mercaptoethylamine can make the surface of gold nanoparticles with active groups, then the order of convenient application..2 stable buffer modified gold nanoparticles in salt solutions of different concentrations and different pH values, we selected the anti-tumor activity and With carboxyl ruthenium complexes [Ru (OPDA) (4-CTPY) NO]3+ (Ru-NO) combined with the Mercaptoethylamine modified with amino on the surface of gold nanoparticles through amidation reaction, the Ru-NO load to the gold nanoparticle surface, by UV spectrophotometer, infrared spectrometer, capable of characterizing constructing spectrometer of ruthenium gold nano composite system (Au-Ru-NO), to ensure that the Ru-NO has successfully loaded onto the gold nanoparticles surface; after ICP-MS measurement, the number of gold atoms each ruthenium atom surface load is about 3.28 x 102. ruthenium complexes supported gold nanoparticles surface in the ruthenium - gold nano composite system, combined with the physical and chemical properties of two excellent and to improve its biological activity and can be better applied to the field of biomedical.3, successfully constructed through amidation reaction to ruthenium gold nano composite system, using various methods to study the Ru-NO cells and the biological activity of Au-Ru-NO The determination by MTT method, the cytotoxicity of Ru-NO in Hep-2 cells after 24h is about 8 + 2 M (the concentration of IC50 in Hep-2 cells is Ru-NO, and IC50) concentration in Hep-2 cell Au-Ru-NO is about 17 + 2 M, that ruthenium complexes functionalized gold nanoparticles can be reduced cell toxicity. In addition, the Annexin V/PI double staining method for determination of active oxygen content, mitochondrial membrane potential changes, Western blotting to investigate the apoptosis mechanism. Through the above method shows that Ru-NO mainly through the active oxygen content in cells induced decrease in mitochondrial membrane potential, the downregulation of Bcl-2 protein, p53 protein and Bax protein up to promote the apoptosis of Hep-2 cells; whereas Au-Ru-NO increase in ROS content caused by cells, mitochondrial membrane potential decreased at the same time, the downregulation of p53 protein, Bax protein expression to induce cell apoptosis. Laser confocal microscope can be observed by Ru -NO and Au-Ru-NO are mainly distributed in cytoplasm, and a small amount can enter the nucleus. Compared with the experimental results of Ru-NO and Au-Ru-NO, Au-Ru-NO can induce cell apoptosis and reduce the overall toxicity of the system. It has potential as an active, highly toxic and low anti-tumor drug.

【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O641.4;TB383.1

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