葡糖基聚合物修饰的金纳米粒子的设计、制备及蛋白识别

发布时间：2018-04-30 06:21

本文选题：金纳米粒子 + 含葡糖基聚合物　；参考：《东华大学》2017年硕士论文

【摘要】：近年来,纳米级的金颗粒具有荧光性、超分子与分子识别等特殊的物理化学性能使其在搭载核酸及糖类大分子中被用来进行免疫、标定、示踪领域备受关注。多功能共聚物胶束因其具有优异的生物相容性以及形貌结构易于控制、响应条件易于调节等特点被作为一种新型的高分子材料,将此类多功能共聚物胶束搭载到金纳米粒子上,已逐渐为生物医药领域的研究热点。本实验课题在大量先前研究的基础上,通过“一锅法”将制备的金纳米粒子(AuNPs)与通过可逆加成-断裂链转移(Reversible addition-fragmentation chain transfer,RAFT)聚合方法合成的一系列基于温敏单体DEGMA的聚合物进行接枝,经过多种表征方法确认其分子的化学结构,利用分子间相互作用力产生的自组装行为获得了温度敏感性含葡萄糖基聚合物@AuNPs胶束,系统研究了其在温度响应性、生物相容性、对蛋白质的特异性识别功能及其在促进肝癌细胞死亡的研究。论文第一章介绍了AuNPs的研究现状以及基于PDEGMA温敏性含糖聚合物@AuNPs胶束的合成方法及其生物应用研究,并概述了本课题的研究背景。第二章,主要内容是利用RAFT聚合技术和“一锅法”设计合成一系列基于不同结构的温敏性含糖物及其@AuNPs,并使用多种表征手段进行分子化学结构的确定。为了得到同时具有生物活性和温敏性的聚合物@AuNPs,我们先利用“酶促法”制备的含糖单体6-O-乙烯基壬二酸-D-葡萄糖酯(OVNGlu)和温度敏感性单体二聚乙二醇单甲醚甲基丙烯酸酯(DEGMA)分别通过RAFT聚合生成不同结构的温敏含糖聚合物P(DEGMA-co-OVNGlu)、PDEGMA-b-POVNGlu。再通过“一锅法”将温敏含糖基聚合物与氯金酸在还原剂、保护剂的作用下合成P(DEGMA-co-OVNGlu)@AuNPs、PDEGMA-b-POVNGlu@AuNPs。利用核磁共振氢谱(~1H NMR)、傅里叶红外光谱(FT-IR)、紫外可见光光谱(UV-Vis)和凝胶渗透色谱(GPC)对聚合物的分子化学结构进行了表征。结果表明,温敏性含葡萄糖聚合物的化学组成与实验设计之初的用料比例相一致;制备温敏性含糖聚合物中PDEGMA的分子量与单体转化率呈线性关系,这使得温敏性含糖聚合物的分子量可以通过RAFT聚合技术有效调控;利用RAFT聚合技术与“一锅法”技术可以制备出结构规整的温敏含糖聚合物@AuNPs。第三章,我们将温敏性含糖聚合物@AuNPs溶解于水中,对不同结构温敏性含糖聚合物及其@AuNPs溶液的温敏性和胶束自组装机理进行研究。使用紫外可见光分光光度计测定聚合物、聚合物@AuNPs的低临界溶解温(LCST),聚合物P(DEGMA-co-OVNGlu)的LCST值为34℃,PDEGMA-b-OVNGlu的LCST值在35℃,P(DEGMA-co-OVNGlu)@AuNPs的LCST值是36℃;PDEGMA-b-OVNGlu@AuNPs的LCST值为37℃。结果表明,在相同单体比例的条件下,接枝金纳米粒子聚合物的LCST值比未接枝金纳米粒子聚合物的LCST值高。通过动态、静态激光散射实验分别测定了不同温度下聚合物胶束及聚合物@AuNPs胶束在水溶液状态下的流体力学半径(Dh)值和均方根旋转半径(Rg)值。发现其数值,开始随温度变化保持不变,中间阶段开始变大,最后达到一个稳定数值。最终通过透射电子显微镜(TEM)观察制备的聚合物胶束以及聚合物@AuNPs胶束的形貌,观察所得聚合物具有规整的球形结构且分散均匀。实验表明:制备的温敏性含葡糖基聚合物能在水溶液中自组装形成近球形的纳米粒子,将AuNPs与温敏性含葡糖基聚合物接枝后同样可以在水溶液中自组装成球形纳米粒子,其LCST值可以通过聚合物的分子结构加以调整。本研究将温敏性含葡糖基聚合物与AuNPs接枝,丰富了含糖聚合物在生物应用领域的研究,同时拓展了金纳米粒子的表面修饰分子的种类。第四章,当通过化学技术合成新的物质在面向生物学应用时,其生物相容性是一个不可避免的重要问题。我们分别研究不同结构的聚合物@AuNPs后与凝集素的识别能力、外界温度变化对其识别凝集素的影响;通过生物相容性实验验证所聚合物对正常细胞的毒性影响;不同结构的温敏性含葡糖基聚合物与凝集素识别后对肝癌细胞生长的影响。结果表明,结构规整的温敏性含葡萄糖嵌段聚合物@AuNPs与凝集素的识别效果最佳,同时比未接枝AuNPs的聚合物识别效果好;随着环境温度的变化,分子表面暴露的糖基位点越多,胶束与凝集素的识别效率也将有所提高;所合成的聚合物材料对正常细胞均具有良好的生物相容性;聚合物@AuNPs胶束在与凝集素识别后对肝癌细胞细胞的正常生长具有抑制作用。第五章,对课题中的主要内容进行总结。通过本课题的研究,对未来深入研究进行展望。
[Abstract]:In recent years, nanoscale gold particles have the characteristics of fluorescence, supramolecular and molecular recognition and other special physical and chemical properties, which have been used for immunization, calibration and tracing in nucleic acid and saccharide macromolecules. Multifunction copolymer micelles have excellent biocompatibility and easy control of morphology and structure. As a new type of polymer material, it is a new type of polymer material. This kind of multifunctional copolymer micelle is attached to gold nanoparticles. It has gradually become a hot research field in the field of biological medicine. On the basis of a large number of previous studies, the gold nanoparticles (AuNPs) prepared by one pot method can be reversibly added by the "one pot method". A series of polymers based on thermosensitive monomer DEGMA synthesized by Reversible addition-fragmentation chain transfer (RAFT) polymerization were grafted. The chemical structure of the molecules was confirmed by a variety of characterization methods. The temperature sensitive glucose based polymer was obtained by the self-assembly behavior of intermolecular interaction force. @AuNPs micelles are studied systematically in temperature response, biocompatibility, specific recognition of protein and the research on promoting the death of hepatoma cells. Chapter 1 introduces the research status of AuNPs and the synthesis methods based on PDEGMA thermosensitive polymer @AuNPs micelles and their biological applications. The second chapter is the second chapter. The main content is to use the RAFT polymerization technology and one pot method to synthesize a series of temperature sensitive sugars and their @AuNPs based on different structures, and use a variety of characterization methods to determine the molecular chemical structure. In order to obtain the simultaneous bioactivity and temperature sensitivity of the polymer @AuNPs, I The glycosylated 6-O- vinyl nonandiacid -D- glucose ester (OVNGlu) and the temperature sensitive monomer two polyethylene glycol monomethyl ether methacrylate (DEGMA) were prepared by the enzyme method, and the temperature sensitive sugar containing polymer P (DEGMA-co-OVNGlu) of different structures was formed by RAFT polymerization, respectively, and PDEGMA-b-POVNGlu. was then carried out by "one pot method" by the "one pot" method of Wen Min. P (DEGMA-co-OVNGlu) @AuNPs was synthesized with glycosyl polymer and chlorochloric acid under the action of reductant and protectant. PDEGMA-b-POVNGlu@AuNPs. using nuclear magnetic resonance spectroscopy (~1H NMR), Fourier infrared spectroscopy (FT-IR), UV visible light spectroscopy (UV-Vis) and gel permeation chromatography (GPC) were used to characterize the molecular structure of the polymer. The results showed that the molecular structure of the polymer was characterized by the UV spectroscopy (FT-IR), UV visible light spectroscopy (UV-Vis) and gel permeation chromatography (GPC). The chemical composition of the thermosensitive glucose containing polymer is in accordance with the proportion of the material at the beginning of the experimental design; the molecular weight of PDEGMA in the preparation of thermosensitive sugar containing polymers has a linear relationship with the monomer conversion rate, which makes the molecular weight of the thermosensitive polymer containing sugar can be effectively regulated by the RAFT polymerization technology; and the RAFT polymerization technology and "one pot method" are used. The thermo sensitive sugar polymer @AuNPs. third chapter is prepared by the technique. We dissolve the thermosensitive polymer @AuNPs in water and study the temperature sensitivity and the micellar self-assembly mechanism of different structure temperature sensitive polymers and their @AuNPs solutions. The UV spectrophotometer is used to determine the polymer and polymer. The low critical dissolution temperature (LCST) of @AuNPs, the LCST value of the polymer P (DEGMA-co-OVNGlu) is 34, the LCST value of PDEGMA-b-OVNGlu is 35, P (DEGMA-co-OVNGlu) @AuNPs LCST is 36, and the PDEGMA-b-OVNGlu@AuNPs is 37 C. The result shows that the graft gold nanoparticles polymer is more than the graft gold under the same monomer ratio. The LCST value of the nanoparticle polymer is high. By dynamic and static laser scattering experiments, the hydrodynamic radius (Dh) value and the root mean square rotation radius (Rg) value of polymer micelles and polymer @AuNPs micelles at different temperatures are measured respectively. Finally, a stable value was reached. The morphology of the polymer micelles and polymer @AuNPs micelles prepared by the transmission electron microscope (TEM) was observed. The polymers with a regular spherical structure and uniform dispersion were observed. The results showed that the prepared thermosensitive glucosylated polymers could self assemble in aqueous solution to form nearly spherical. Nanoparticles, after grafting AuNPs with thermosensitive glucoside polymers, can also be self assembled into spherical nanoparticles in aqueous solution, and their LCST values can be adjusted by the molecular structure of the polymer. This study has grafted the thermosensitive glucoside polymer with AuNPs, enriching the study of the sugar containing polymers in the field of biological applications. The type of surface modifier of gold nanoparticles is extended. In the fourth chapter, when a new substance is synthesized by chemical technology in biological application, its biocompatibility is an unavoidable problem. We study the recognition ability of the polymer @AuNPs and the agglutinin of different structures, and the external temperature change to identify the coagulant. The effects of the biocompatibility test on the toxic effects of the polymers on normal cells; the effects of the temperature sensitive glucosyl polymers and lectin on the growth of hepatoma cells in different structures. The results show that the structural regularity of the temperature sensitivity of @AuNPs and agglutinin is best. The recognition efficiency of the polymer was better than that of the ungrafted AuNPs; the more glycosyl sites exposed on the surface of the molecule, the recognition efficiency of the micelles and agglutinin will also be improved; the synthesized polymer materials have good biocompatibility to normal cells, and the polymer @AuNPs micelles are fine for liver cancer after identification with the agglutinin. The normal growth of cell cells has inhibitory effect. The fifth chapter summarizes the main contents of the subject. Through the research of this topic, the future research is prospected.

【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TB383.1;R735.7

【参考文献】