【作者】 李娜；

【导师】 余家会；

【作者基本信息】 华东师范大学 ， 高分子化学与物理， 2010， 硕士

【摘要】 核磁共振造影剂自从20世纪80年代被发现和应用以来,在核磁共振成像中有着独特的位置,对现代医学检测的发展起着重要的作用。为了实现早检测、早发现,本文在树形大分子基础上合成了一种新型的靶向性大分子造影剂FA-PEG (4000Da) PAMAM-Gds,初步了研究了其体外细胞毒性(MTT、FCM实验),组织急慢性毒性(组织切片),细胞靶向性(cell-TEM);体内MR成像,在MR试验中研究了大分子造影剂的驰预率,最佳成像浓度,最佳成像时间以及其在裸鼠体内的代谢等。具体内容如下：第一章绪论核磁共振造影剂是用来缩短成像时间、提高成像对比度和清晰度的一种成像增强对比剂。它能改变体内局部组织中水质子的驰预速率,提高正常与患病部位的成像对比度,从而显示体内器官的功能状态。核磁共振造影剂的繁荣发展,使得目前存在的MR造影剂多种多样,其分类方法亦然。四氧化三铁等超顺磁性造影剂、水溶性顺磁钆螯合物磁共振造影剂等都得到深入的研究和发展。新型的靶向大分子造影剂一般指分子量大于20000Da的造影剂复合物,是一类非常具有研究前景的MR造影剂。我们合成并研究了,树形大分子(PAMAM)为载体主体的,聚乙二醇(PEG)调控分子粒径,叶酸组织靶向型,水溶性顺磁性钆螯合物磁共振造影剂：FA-PEG-PAMAM-Gds.第二章靶向大分子造影剂(FA-PEG-PAMAM-Gds)的合成采用传统的发散法,以乙二胺为核多步迈克尔加成制备了4.0G的树形大分子聚合物聚酰-胺胺(PAMAM)。将此大分子与丁二酸酐反应得到端基为羧基的PAMAM-COOH。叶酸与聚乙二醇反应制得一端为氨基的分子量不同的叶酸靶向缩合物：H2N-PEG(1000 Da)-NH-FA, H2N-PEG (2000 Da)-NH-FA和H2N-PEG (4000 Da)-NH-FA.靶向缩合物与端基为羧基的PAMAM-COOH部分缩合反应,制备得到靶向大分子载体PAMAM-PEG-FA,同时制备了两种不具靶向性的大分子载体mPEG (5000 Da)-PAMAM和mPEG (2000 Da)-PAMAM。大分子载体与GdCl3·6H2O螯合,得靶向型大分子造影剂复合物FA-PEG-PAMAM-Gds和无靶向性的造影剂复合物Gds-PAMAM-PEG。1H-NMR、FT-IR等表征了酰胺键缩合等反应,证明了合成实验的成功实现。SEM、TEM、DLS等手段对大分子造影剂复合物进行了分子形貌和粒径表征。第三章靶向大分子造影剂的体外实验研究本章主要研究靶向型造影剂复合物FP (4000 Da) PGds的体外细胞毒性、生物相容性和细胞表面的靶向性富集。KB、L929、7702等6种细胞的MTT实验,以及辅助性的A549细胞的FCM实验,结果表明了大分子造影剂复合物FP (4000 Da) PGds的基本无细胞毒性、生物相容性高。’心脏、肺部,等组织器官的切片观察,表明大分子造影剂复合物FP (4000 Da) PGds的无急性和慢性组织毒性。细胞株KB和L929的cell-TEM观察结果,表明大分子造影剂复合物FP (4000 Da) PGds在叶酸受体表达成阳性的细胞表面靶向性富集的良好效果。第四章靶向大分子造影剂的MRI效果评价FA-PEG(4000 Da)-PAMAM-Gds造影剂复合物的MR实验均在西门子3.0T Trio Tim磁共振成像仪上完成了。完成动物体内/皮下肿瘤接种的基础上,设计并完成了驰预率的测定、体内最佳成像时间确定、代谢规律初步研究和肿瘤处靶向富集成像等实验,对造影剂复合物FA-PEG (4000 Da)-PAMAM-Gds的一些基本性质做了初步的研究。更多还原

【Abstract】 Magnetic resonance imaging contrast agents have played an important role in medical development, and also have been further developed and studied since its born. In order to develop potential tumor-targeted circulation-prolonged macromolecular magnetic resonance imaging (MRI) contrasts agent without the use of low molecular gadolinium (Gd) ligands, a new macromolecular contrast targeting agent (FP (4oooDa) P-Gds),based on the dendritic polymer, was synthesized. We did lots of vitro and vivo experiments to test the quality of MR contrast agent:FP (4000Da) P-Gds.In vitro cytotoxicity (MTT, FCM test), tissue toxicity (biopsy)and so on,we carried out to prove its none cell toxicity. In vivo experiments, the MR was used to study the relaxation rate of the macromolecular contrast agent. Additionly, a series of tests were taken out to find its best imaging concentration, optimal imaging time and so on. so,we got a few initial knowledge about the metabolizability of the macromolecular contrast agent.Chapter 1:IntroductionMagnetic resonance imaging contrast agents are used to improve MR image which can reduce the proton’relaxation time in an organization. If also can increase MR image distinctness between the normal and diseased organs, which make the disease found earlier and easier. Till now, tens of and hundreds of MRI contrast agents have been synthesized and tested, There’re many classification methods about the MRI contrast agents,such as the superparamagnetic Fe3O4 contrast agents, water-soluble paramagnetic gadolinium chelate agent and so on. Macromolecular MR contrast agents refers to a new kind of MRI contrast agents,whose molecular weight are generally bigger than 20000Da. In our study, we designed and synthesized a dendrimer (PAMAM) based, as a polymer carrier, water-soluble paramagnetic gadolinium chelate magnetic resonance contrast agent:FA-PEG-PAMAM-Gds,whose molecular size wased controlled by polyethylene glycol(PEG).With conjugated folate, FA-PEG-PAMAM-Gds could be used as a tumor targeted MR contrast agents.Chapter 2:Synthesis of Dendrimer-based tumor targeted Macromolecular MR contrast agent (FA-PEG-PAMAM-Gds)Based on the traditional tracing method, through multi-step Michael addition, the dendritic polymer with ethylenediamine core was synthesized. We got PAMAM-COOH as a result of carboxyl reaction which refers to succinic anhydride and polymer polyamidoamine(PAMAM, dendrimer) 4.0G. H2N-PEG(1000Da)-NH-FA、H2N-PEG (2oooDa)-NH-FA and H2N-PEG (4oooDa)-NH-FA were synthesized from FA and H2N-PEG-NH2 through the acylation reaction. And according to the condensation reaction, H2N-PEG (n)-NH-FA reacted with PAMAM-COOH, formed a tumor targeted carrier PAMAM-PEG(1000Da)-FA. In the same way, another two tumor targeted carrier:PAMAM-PEG(2oooDa)-FA and PAMAM-PEG(4oooDa)-FA were also prepared. Two different types of non-targeted molecular carrier:mPEG (2oooDa)-PAMAM, and mPEG (5000Da)-PAMAM were also prepared. Then all the five macromolecular carriers were chelated with GdCl3·6H2O. So,we got dendrimer-based tumor targeted Macromolecular MR contrast agent FA-PEG(1000Da)-PAMAM-Gds、FA-PEG(2000Da)-PAMAM-Gds and FA-PEG(4oooDa)-PAMAM-Gds and non-targeted MR contrast agents mPEG(2oooDa)-PAMAM-Gds、mPEG(5oooDa)-PAMAM-Gds. The chemical structure of the Synthetic compounds all were confirmed by nuclear magnetic resonance (’H-NMR) or Fourier transform infrared (FT-IR) spectra etc,and the mass percentage content of Gd (Ⅲ) in FA-PEG-PAMAM-Gd was measured by inductively coupled plasma-atomic emission spectrometer (ICP-AES). SEM, TEM and so on were used to describe the surface characters of the macromolecular MR contrast agents.Chapter 3:The vitro study of the macromolecular MR contrast agentsOur vitro studies focuses on the drug cytotoxicity and biocompatibility of the MR contrast agents, as while we studied its enrichment on the surface of the target cellulars. KB, L929,7702 and other three kinds of cells were taken out in MTT test,together with A549 cells’FCM experiments, macromolecular MR contrast agent FP (4oooDa) PGds were proved has no cell toxicity no matter to tumor or normal calls. Preliminary study on the organization’toxicity,reflects the macromolecular MR contrast agent FP (4oooDa)PGds of non-acute and chronic tissue toxicity,"The vitro tumor targeted enrichment of MR contrast agent FP (4000Da)PGds " refers to KB and L929 two kinds of cells’cell-TEM experimentation, which reflects the high tumor targeted ability of the macromolecular MR contrast agent. That little cytotoxicity-. high mass percentage of Gd and enough enrichment in tumor surface, suggestted its great potential as a tumor-targeted macromolecular MRI contrast agent.Chapter 4:The vivo MR evaluation of the macromolecular MRI contrast agentsMR experiments were carried out on the Siemens 3.0T Trio Tim magnetic resonance image system which is the most advanced examine instruments in ShangHai.In this chapter,we found, of all the five MR contrast agents contrast agent,FP (4000Da)PGds had the highest relaxation rate. The the best injection concentration of the FA-PEG(4oooDa)-PAMAM-Gds contrast agent was 6.2266g/L.The best body imaging time was 50min later after the injection of FA-PEG(4oooDa)-PAMAM-Gds. About the tumor targeted and metabolism of the FA-PEG(4oooDa)-PAMAM-Gds agent, we do a preliminary study and characterization through nude mice and normal white mice.MR results displayed that it can targeted enrich around the tumor,and make its MR imaging more legible.更多还原