The Synthesis And Biological Evaluation Of Dendrimer-based Tumor Targeted MRI Contrast Agent

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.