聚赖氨酸树枝状分子的制备及其负载纳豆激酶用于靶向溶血栓的研究

发布时间：2018-01-29 09:40

本文关键词： 聚赖氨酸树枝状分子纳豆激酶溶血栓血栓靶向磁性纳米颗粒体外溶栓　出处：《兰州大学》2017年硕士论文　论文类型：学位论文

【摘要】：纳豆激酶(NK)作为一种新型的溶栓剂,因具备直接溶栓、激活体内细胞产生尿激酶和内源组织型纤溶酶原激活剂等优势引起了研究者们的高度重视。然而,NK作为蛋白酶类药物仍存在一些缺点,如容易受到外部环境的影响、对温度和pH的变化较为敏感。与传统的溶栓治疗相比,利用药物载体包埋溶栓药物已经吸引了研究者们的注意。经药物载体包埋NK之后,能够更好地保护NK不失活,并提高其耐温度和pH的稳定性。聚赖氨酸树枝状分子具有可控的尺寸、可功能化的表面官能团、良好的水溶性、生物降解性及生物相容性,被认为是一种理想的药物载体。因此,本学位论文使用赖氨酸为主要原料,制备了一系列不同代数的聚赖氨酸树枝状分子,研究了其作为药物载体包埋NK在溶血栓方面的性质。为了实现靶向性溶血栓的目的,引入了磁靶向和血栓部位靶向,前者是借助外部磁场达到靶向血栓部位的目的;后者是利用精氨酸-甘氨酸-天冬氨酸(RGD肽)与血小板GP IIb/IIIa受体特异性结合的能力,从而具备血栓靶向性。本学位论文主要包括以下两个方面:1.通过发散-收敛法制备了二代、三代和四代聚赖氨酸树枝状分子(PLLD Gn),使其与溶栓药物NK复配形成纳米复合物。当NK与PLLD G4的摩尔比为1:30时,形成的NK/PLLD纳米复合物具有最高的NK酶活(高达117%)。荧光光谱研究结果表明,NK与PLLD之间的作用力为氢键作用和范德华力,并且NK/PLLD纳米复合物具有较好的耐温度和p H的稳定性。体外溶栓研究结果表明,在12 h内NK/PLLD纳米复合物溶栓率达到50%,与游离NK相比,NK/PLLD纳米复合物能起到缓释NK的作用。溶血实验和MTT实验研究结果表明,PLLD具有更低的细胞毒性和良好的生物相容性。2.使用共沉淀法制备了Fe_3O_4磁性纳米颗粒,用3-氨丙基三乙氧基硅烷(APTES)修饰后,再用三代聚赖氨酸树枝状分子(PLLD)修饰,最后用RGD修饰得到新型磁性纳米颗粒Fe_3O_4-PLLD-RGD,再通过离子吸附作用将NK固定到Fe_3O_4-PLLD-RGD表面,得到Fe_3O_4-PLLD-RGD/NK。体外靶向研究结果表明,经RGD修饰的磁性纳米颗粒具有好的血栓靶向能力和磁靶向能力。体外溶栓研究结果表明,在2.5 h内,Fe_3O_4-PLLD-RGD/NK磁性纳米颗粒溶栓率达到50%;在5.5 h内,游离NK和Fe_3O_4-PLLD/NK磁性纳米颗粒的溶栓率均达到50%。以上结果表明,Fe_3O_4-PLLD-RGD/NK磁性纳米颗粒具有良好的血栓靶向能力和快速溶栓能力。溶血实验和MTT实验研究结果表明,Fe_3O_4-PLLD-RGD具有更低的细胞毒性和良好的生物相容性。
[Abstract]:As a new type of thrombolytic agent, nattokinase (NKK) has attracted great attention due to its advantages of direct thrombolysis, activation of urokinase and endogenous tissue plasminogen activator. NK as a protease drug still has some disadvantages, such as easy to be affected by external environment, sensitive to temperature and pH changes, compared with traditional thrombolytic therapy. The use of drug carrier embedded thrombolytic drugs has attracted the attention of researchers. After the drug carrier embedding NK, it can better protect NK from inactivation. The poly (lysine) dendrimer has controllable size, functional surface functional group, good water solubility, biodegradability and biocompatibility. It is considered to be an ideal drug carrier. Therefore, a series of dendritic molecules with different algebras have been prepared using lysine as the main raw material in this dissertation. In order to achieve the purpose of targeted thrombolytic thrombus, magnetic targeting and thrombus site targeting were introduced. The former uses external magnetic field to reach the target thrombus. The latter is the ability to specifically bind to platelet GP IIb/IIIa receptor using arginine-glycine-aspartic acid peptide. This dissertation mainly includes the following two aspects: 1.The second generation, the third generation and the fourth generation of Poly (lysine) dendrimer (PLLD Gnn) were prepared by divergen-convergence method. When the molar ratio of NK to PLLD G4 is 1:30. The formed NK/PLLD nanocomposites have the highest NK enzyme activity (up to 1177.The fluorescence spectra show that the interaction force between NK and PLLD is hydrogen bond and van der Waals force). The results of in vitro thrombolytic study showed that the thrombolysis rate of NK/PLLD nanocomposites reached 50% within 12 hours. Compared with free NK, NK-P / PLLD nanocomposites can play the role of sustained-release NK. The results of hemolysis experiment and MTT experiment show that NK-P / PLLD nanocomposite can release NK slowly. PLLD has lower cytotoxicity and better biocompatibility. 2. Fe_3O_4 magnetic nanoparticles were prepared by coprecipitation method. After modified with 3-aminopropyl triethoxy silane (APTES), then modified with three generation poly-lysine dendrimer (PLLDD). Finally, the novel magnetic nanoparticles Fe3O4-TiPLLD-RGDwere prepared by RGD modification, and NK was immobilized to the Fe_3O_4-PLLD-RGD surface by ion adsorption. Fe3O4-PLLD-RGD / NK. the results of in vitro targeting study showed that Fe3O4-PLLD-RGD- NK. The magnetic nanoparticles modified by RGD had good thrombus targeting ability and magnetic targeting ability. The results of thrombolysis in vitro showed that the thrombolytic activity was within 2.5 h. The thrombolysis rate of Fe_3O_4-PLLD-RGD/NK magnetic nanoparticles was 50%. Within 5.5 h, the thrombolysis rates of both free NK and Fe_3O_4-PLLD/NK magnetic nanoparticles were 50%. Fe_3O_4-PLLD-RGD/NK magnetic nanoparticles have good thrombolytic targeting ability and rapid thrombolytic ability. The results of hemolysis experiment and MTT experiment show that. Fe_3O_4-PLLD-RGD has lower cytotoxicity and good biocompatibility.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96

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