本文關(guān)鍵詞： 抗腫瘤藥物 金納米粒子 釕配合物 生物活性　出處：《深圳大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研發(fā)抗腫瘤藥物是科研研究的難點(diǎn)和重點(diǎn),該項(xiàng)研究不僅結(jié)合了化學(xué)、生物、藥學(xué)、醫(yī)學(xué)等領(lǐng)域,而且將對(duì)人類(lèi)生命健康產(chǎn)生了重大影響。在金屬抗腫瘤藥物中,鉑配合物是臨床醫(yī)學(xué)上廣泛使用的抗腫瘤藥物,然而,鉑配合物作為抗腫瘤藥物具有明顯的毒副作用,如腎毒性、肝毒性、神經(jīng)毒性以及產(chǎn)生耐藥性等,這使得人們把目光轉(zhuǎn)向開(kāi)發(fā)其它的金屬抗腫瘤藥物。釕配合物具有穩(wěn)定的化學(xué)結(jié)構(gòu)和良好的物理、化學(xué)性質(zhì),具有潛力成高效低毒的新型抗腫瘤藥物。釕配合物是六配位八面體的結(jié)構(gòu),配體的可改變性使其結(jié)構(gòu)具有多樣性,可滿足不同的設(shè)計(jì)要求。但其缺乏腫瘤靶向性且穿膜能力差,降低其抗腫瘤活性。而金納米粒子(AuNPs)由于具有良好的生物相容性,被廣泛用于藥物載體、生物探針、熒光成像等應(yīng)用。因此,本論文通過(guò)將釕配合物負(fù)載到金納米粒子表面,結(jié)合二者優(yōu)異的性質(zhì),提高其生物活性。本論文主要通過(guò)檸檬酸鈉還原法合成AuNPs,利用同時(shí)含巰基和氨基活性基團(tuán)的分子修飾AuNPs,形成Au-S鍵,構(gòu)建穩(wěn)定性良好的金納米材料體系,該體系的NH2基團(tuán)用于與含羧基的釕配合物進(jìn)行酰胺化反應(yīng),最終得到釕-金納米復(fù)合體系。研究表征了釕-金納米復(fù)合材料的形貌特點(diǎn),光譜學(xué)性質(zhì)和抗腫瘤活性,具體的研究?jī)?nèi)容如下:1、采用檸檬酸鈉還原法改變還原劑的比例,合成了納米尺寸為15 nm、30 nm、80nm、120 nm的金納米粒子。為提高金納米粒子的穩(wěn)定性,采用巰基配體通過(guò)Au-S的共價(jià)吸附作用修飾AuNPs,以提高其穩(wěn)定性。通過(guò)紫外分光光度計(jì)、Zetas電位儀、納米激光粒度儀對(duì)HS-PEG2000和LA-mPEG5000兩種巰基聚乙二醇,以及巰基乙胺、巰基丙酸、半胱氨酸三種巰基小配體進(jìn)行篩選,選出了LA-mPEG5000和巰基乙胺是最適合用來(lái)對(duì)金納米粒子進(jìn)行穩(wěn)定修飾的雙配體,其中,LA-mPEG5000可以提高金納米粒子的穩(wěn)定性,巰基乙胺可以使其金納米粒子表面具有活性基團(tuán),方便其后序應(yīng)用。修飾后的金納米粒子可在不同濃度的鹽溶液和不同pH值的緩沖液中穩(wěn)定存在。2、我們選取了具有抗腫瘤活性且?guī)в恤然尼懪浜衔颷Ru(OPDA)(4-CTPY)NO]3+(Ru-NO)與經(jīng)過(guò)巰基乙胺修飾后表面帶有氨基的金納米粒子通過(guò)酰胺化反應(yīng)結(jié)合,使Ru-NO負(fù)載到金納米粒子表面,再通過(guò)紫外分光光度計(jì)、紅外光譜儀、能譜儀來(lái)表征構(gòu)建的釕-金納米復(fù)合體系(Au-Ru-NO),以確保Ru-NO已成功負(fù)載到金納米粒子表面;經(jīng)過(guò)ICP-MS測(cè)量,可知釕-金納米復(fù)合體系中每個(gè)金原子表面負(fù)載的釕原子個(gè)數(shù)約為3.28×102。將釕配合物負(fù)載到金納米粒子表面,結(jié)合二者優(yōu)異的物化性能,提高其生物活性,可以更好的應(yīng)用到生物醫(yī)學(xué)領(lǐng)域中。3、通過(guò)酰胺化反應(yīng)成功構(gòu)建釕-金納米復(fù)合體系后,采用多種細(xì)胞方法對(duì)Ru-NO和Au-Ru-NO的生物活性進(jìn)行研究。通過(guò)MTT法測(cè)定可知,Ru-NO作用于Hep-2細(xì)胞24h后的細(xì)胞毒性約為8±2μM(即Ru-NO作用于Hep-2細(xì)胞的IC50濃度),而Au-Ru-NO作用于Hep-2細(xì)胞的IC50濃度約為17±2μM,說(shuō)明釕配合物功能化金納米粒子后可降低細(xì)胞毒性。此外,通過(guò)Annexin V/PI雙染法,活性氧含量的測(cè)定,線粒體膜電位的變化、蛋白質(zhì)免疫印跡法等來(lái)探索細(xì)胞凋亡機(jī)制。通過(guò)以上方法可知Ru-NO主要通過(guò)誘導(dǎo)細(xì)胞內(nèi)活性氧含量增加、線粒體膜電位降低、Bcl-2蛋白下調(diào)、p53和Bax蛋白上調(diào)來(lái)促使Hep-2細(xì)胞凋亡;而Au-Ru-NO在引起細(xì)胞內(nèi)活性氧含量增加、線粒體膜電位降低的同時(shí),促使p53蛋白下調(diào)、Bax蛋白上調(diào)來(lái)誘導(dǎo)細(xì)胞凋亡。激光共聚焦顯微鏡可觀察到Ru-NO和Au-Ru-NO主要分布在細(xì)胞質(zhì),少量可進(jìn)入細(xì)胞核。通過(guò)Ru-NO和Au-Ru-NO的實(shí)驗(yàn)結(jié)論對(duì)比,可知Au-Ru-NO在引起細(xì)胞凋亡的同時(shí)可降低系統(tǒng)的整體毒性,有作為活性高毒性低抗腫瘤藥物的潛能。
[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.

【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：O641.4;TB383.1

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本文編號(hào)：1550961