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Alternative TitleA rheumatoid arthritis magnetic resonance imaging contrast based on folic acid conjugated PEG-b-PAA@SPION
Thesis Advisor张欣
Degree Grantor中国科学院研究生院
Degree Discipline生物工程
Keyword原位合成   超顺磁性氧化铁纳米粒子   类风湿性关节炎   核磁共振成像   巨噬细胞靶向
Abstract核磁共振成像(Magnetic Resonance Imaging,MRI)是目前类风湿性关节炎(rheumatoid arthritis,RA)的临床诊断中最有效的方法之一,作为磁共振成像的阴性造影剂,超顺磁性氧化铁纳米粒子(Superparamagnetic iron oxide nanoparticle,SPION)在RA的MRI诊断中具有独特的优势。在本文研究中,我们设计了一种基于多元醇方法(polyol method)的叶酸(folic acid,FA)靶向,两嵌段共聚物PEG-b-PAA原位修饰的SPION(FA-PEG-b-PAA@SPION)。在该体系中PEG分子赋予SPION良好的生理稳定性和生物相容性,高温的多元醇反应使得纳米粒子具有很好的磁性能,而叶酸则可以靶向于RA部位炎症细胞大量表达的叶酸受体,起到主动分子靶向的作用。本文的主要内容如下: 1. 利用原子转移自由基聚合(ATRP)反应可控合成了PAA聚合度分别为18,36和57的三种PEG-b-PAA两嵌段共聚物,并以此作为表面配体分别原位合成了尺寸为52 nm,17 nm和9 nm的PEG-b-PAA@SPION。1H NMR 表征了三组聚合物的结构,透射电子显微镜(TEM)和动态光散射(DLS)则表征了PEG-b-PAA@SPION尺寸与形态并通过DLS测定了三组SPION的血清稳定性。通过上述测试优选出PEG-b-PAA36@SPION 和PEG-b-PAA57@SPION 用于后续实验。随后利用DCC/DMAP反应将叶酸偶联到PEG-b-PAA@SPION,利用XPS和UV-vis 光谱验证了FA连接的成功,磁滞回线和r2弛豫率则表征了不同粒径大小SPION的磁响应能力,并证明了FA连接不影响磁性能。 2. 通过体外的巨噬细胞吞噬实验验证了FA-PEG-b-PAA@SPION对于炎症激活状态的巨噬细胞的特异性靶向性。结合磁性能结果我们优选出FA-PEG-b-PAA36@SPION用于后续实验与表征。 3. 利用AIA(antigen induced arthritis)模型的MRI成像实验测试了FA-PEG-b-PAA36@SPION和PEG-b-PAA36@SPION对关节部位的造影能力。结果表明,FA-PEG-b-PAA36@SPION造影部位信号明显减弱且减弱程度大于PEG-b-PAA36@SPION。该结果证明了FA-PEG-b-PAA36@SPION的关节靶向性和RA部位良好的成像能力。 综上所述,本文初步探讨了FA-PEG-b-PAA36@SPION作为RA的MRI 造影剂的可行性,为其进一步应用于生物医学领域奠定基础。
Other AbstractMagnetic Resonance Imaging (MRI) is one of the most effective ways for the diagnosis of rheumatoid arthritis (RA). As the negative imaging contrast agent of MRI, superparamagnetic iron oxide nanoparticle (SPION) offers unique properties for RA imaging. However, targeting imaging of rheumatoid arthritis in vivo requires a specific, magnetic sensitive and ultra-stable MRI contrast agent. In this study, SPION with preferable colloid stability and optimized size was obtained by using in situ polyol method with diblock copolymer PEG-b-PAA acting as surface ligand. PEG coating was incorporated to offer good colloidal stability for magnetic iron oxide particles and proper length PAA block was used to control the core size of the nanoparticles. Folic acid was conjugated onto PEG-b-PAA@SPION for its target binding to recruit and activate macrophages in synovium. The main results were summarized as follows: Firstly, we employed atom transfer radical polymerization (ATRP) to obtain three kinds of PEG-b-PAA co-polymer with PAA DP (degree of polymerization) to be 18, 36 and 57, respectively. Increasing the DP of PAA from 18, 36 to 57 led to a decreasing size of iron oxide nanoparticles from 52 nm, 17 nm to 9 nm respectively. 1H NMR was used to verify the synthesis of PEG-b-PAA co-polymer. Transmission electron microscope (TEM) and Dynamic light scattering (DLS) results indentified the size and morphology of the nanoparticles. Colloid stability was assessed by measuring their hydrodynamic sizes of the nanoparticles in DMEM cell culture medium containing 10% FBS in different time. From the above test we selected PEG-b-PAA36@SPION and PEG-b-PAA57@SPION for further evaluation. Afterwards, Folic acid was conjugated onto PEG-PAAX@SPION as specific targeting molecule for activated macrophages in rheumatoid arthritis joint. We employed x-ray photoelectron spectroscopy (XPS) and UV-vis spectrum to verify and quantify the conjugation of folic acid. Hysteresis loops and r2 relaxivities indentified the magnetic property of the SPION. Secondly, we used in vitro endocytosis experiments to confirm the better performance of folic acid conjugated SPION than non-folic acid modified SPION. Based on the result of endocytosis experiments and magnetic property test of the SPION, we selected FA-PEG-b-PAA36@SPION for In vivo MRI experiment. Thirdly, in vivo MRI clearly demonstrated the significant signal diminishment of arthritis joint in antigen induced arthritis (AIA) rats by intravenous injection of the optimized nanoparticle FA-PEG-b-PAA36@SPION. These results indicated that the FA-PEG-b-PAA36@SPION could serve as a promising candidate for MRI of rheumatoid arthritis. In sum, these results indicated that the FA-PEG-b-PAA36@SPION could serve as a promising candidate for MRI of rheumatoid arthritis, which may lay the foundation for further biomedical application.
Document Type学位论文
Recommended Citation
GB/T 7714
钟砚琦. 叶酸连接的PEG-b-PAA@SPION用于类风湿性关节炎MRI的研究[D]. 中国科学院研究生院,2014.
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