【作者】 王进华；

【导师】 龚洪翰； Shawn Chen；

【作者基本信息】 南昌大学 ， 外科学， 2009， 博士

【摘要】 背景与目的分子成像是一个迅速崛起的生物医学研究手段和临床学科,其目标是无创,定量可视化发生在细胞和亚细胞水平的体内分子过程。一般来说,分子成像的应用要求快速、敏感、高分辨率力的影像技术,结合使用对比剂从而使细胞和亚细胞水平生物过程能够可视化。至目前为止,单光子发射计算机断层扫描(SPECT),正电子发射计算机断层扫描(PET)和光学成像(Optical imaging)技术仍然是体内临床前分子影像最常用的影像方法,其主要原因是这些方法高的敏感性。然而核成像技术(PET/SPECT)使用电离辐射,没有提供解剖信息且空间分辨率力低,而光学成像空间分辨力低且深度穿透力受限。因此,近年来磁共振(MR)被引入作为替代方法,由于这个技术提供极好的内在高空间分辨力的软组织对比和无电离辐射。此外,该技术能够在单次试验通过结合不同的成像系列提供解剖信息和弥散,灌注和血流等生理参数。MR已经成为分子影像的一个重要工具,然而MRI的缺点是对于MR造影剂的低敏感性,因此采用不同策略提高造影剂的效力,如研发新颖的纳米颗粒将使MR成像更有利于适合分子影像。应用MRI结合铁氧颗粒(IONP)为基础的细胞标记技术提供一个良好解决在宿主器官内非侵袭性追踪移植细胞的方法,但这个技术的主要挑战是诱导充足数量的颗粒进入细胞内以弥补细胞分裂所引起的稀释效应,而不影响正常细胞功能。对于细胞标记,尽管SPIO颗粒很容易被巨噬细胞吞噬进入细胞内,然非吞噬细胞及分裂慢的细胞铁氧磁性纳米颗粒吸入却很少,因此有效的细胞标记需要高浓度的纳米颗粒,但这经常引起细胞毒性。当前,提高对比剂内化的一些方法已经提出并进行了研究,最常用方法是利用商业化Feridex结合转染试剂,然而,其缺少通用的公式,试验冗长和单细胞系要获得最优化转染效果易发生错误。鉴于上述因素,本研究应用MR及新颖高磁矩的磁共振分子成像对比剂-人血清白蛋白-多巴胺-四氧化三铁氧纳米颗粒(Human serum albumin-Dopamine-Fe3O4 Nanoparticles, HSA-IONPs)进行分子影像学研究,通过与商业应用的Feridex纳米颗粒对比研究,研究HSA-IONPs磁特性,各种不同细胞系HSA-IONPs(终浓度为20μg·ml-1铁)细胞标记的有效性,并在不同动物模型中应用HSA-IONPs标记神经干细胞、巨噬细胞行MR分子成像。目的合成HSA-Dopamine-Fe3O4纳米颗粒(HSA-IONPs)-新颖的磁共振分子成像对比剂并研究其磁特性。方法油酸涂层铁氧颗粒(15nm)来自Ocean NanoTech公司。对于表面修饰,大约50mg油酸涂层铁氧颗粒分散在5ml氯仿内,20mg多巴胺溶解在2ml DMSO内并加入上述油酸涂层铁氧颗粒的氯仿内,形成一均质溶液,混和溶液加热至70℃后一小时,凉至室温。已烷作为不良溶剂用于沉淀纳米颗粒,15000g离心15min收集纳米颗粒后,用氮气吹干,在超声波的作用下重新分散纳米颗粒进入DMSO。另外,把20mg HSA(人血清白蛋白)溶解在硼酸盐缓冲剂(50nM,PH8.5),在超声作用下,把溶解在DMSO的纳米颗粒滴加至HSA溶液内。合成产物在PBS(PH 7.4)溶液中透析(MWCO 12000-14000)24h以便交换缓冲液并除去游离的多巴胺,此后,得到的纳米颗粒再次离心,30000g离心20min以去除游离的HSA,产物重分散于PBS缓冲液内,小部分聚集物通过注射过滤器(0.22μm)移除。采用电感耦合等离子体发射光谱仪(ICP-AMS)、透射电子显微镜(TEM)、动态光散射(DLS)、Zeta电位及MRI对纳米颗粒进行定量和特征,尾静脉注射Feridex和HSA-Dopamine-Fe3O4纳米颗粒(lOmg Fe/kg)进入正常裸鼠体内后,在不同时间点行肝脏MR T2W成像。结果DLS测量分散状态的HSA-IONPs流体动力学结果是28.4nm,TEM显示合成的纳米颗粒呈单分散单晶体和圆形结构,无团块聚集现象。Zeta电位结果显示HSA-Dopamine-Fe3O4纳米颗粒呈负电荷(-9.46±1.86MV)。MR Phantom结果显示HSA-IONPs的R2值(R2值=314.5mM-1S-1)明显高于常规应用的Feridex纳米颗粒(Feridex R2值=123.6mM-1S-1),HSA-IONPs显示更好的T2对比,HSA-IONPs可应用于肝脏成像。结论HSA-Dopamine-Fe3O4纳米颗粒具有更高的磁矩,优于商业应用的铁氧颗粒(Feridex)。目的研究新型的磁共振分子成像对比剂HSA-IONPs细胞标记的有效性。方法在不同时间点应用HSA-IONPs和Feridex (20μg Fe-ml-1)纳米颗粒孵育人前列腺癌PC3细胞、人头颈磷状细胞癌UMSCC-22B细胞、神经干细胞、间充质细胞、人胚胎干细胞、巨噬细胞。行普鲁氏蓝染色,核固红反染。MTT分析用于评估纳米颗粒的毒性作用。神经干细胞和巨噬细胞吞噬的纳米颗粒应用ICP-AMS、TEM及MR Phantom成像进行定量和特征。双侧大脑基底节立体定向注射纳米颗粒标记的神经干细胞(1×104)后6h,行MR T2W扫描；建立MCAO脑中风模型,静脉注射巨噬细胞后在不同时间点(扫描前、4h、1d、2d、5d、7d)行MR扫描。结果低浓度20μg/ml HSA-IONPs即可有效孵育细胞,且不需要转染试剂。TEM见纳米颗粒位于细胞内的内涵体／溶酶体内,纳米颗粒有聚集改变。HSA-IONPs对研究细胞系的生长和分裂没有显著性毒性影响(P<0.05)。与Feridex组相比,ICP-AMS结果显示神经干细胞和巨噬细胞HSA-IONPs组呈现更高的纳米颗粒吸收。HSA-IONPs或Feridex加转染试剂(50μg Fe·ml-1+Lipofactamine 2000)孵育神经干细胞24h,与Feridex相比纳米颗粒吸收有显著性差异。神经干细胞纳米颗粒的孵育浓度与细胞吸收的铁量呈相关依赖性。MR细胞Phantom成像显示T2WI信号强度有显著性差异,未饱和前,R2值与细胞数量呈良好的线性相关关系。体内MR及病理证实HSA-IONPs孵育的神经干细胞比Feridex孵育吸收更多的铁量。MR扫描可用来追踪局灶性脑缺血中风后标记的巨噬细胞迁移。结论HSA-Dopamine-Fe3O4细胞标记有效且优于Feridex。目的探讨肿瘤相关巨噬细胞(TAMs) MR成像可行性。方法应用HSA-IONPs(20μg/ml Fe)孵育巨噬细胞(RAW264.7)24h,经尾静脉注射5×106标记巨噬细胞至皮下4T1荷瘤BALB/c小鼠和22B荷瘤裸鼠,在不同时间点(扫描前、注射后6h、1、2、3或7d),采用7.0 T MR扫描仪行T2W横、冠状位扫描,动物处死后肿瘤及脏器行病理检查。结果普鲁士蓝染色、电镜及细胞Phantom成像证实细胞标记有效。注射后6h4T1肿瘤坏死囊变中心周围见明显低信号影,24h见坏死区与肿瘤实质区交界部位有不规则环形低信号影；22B肿瘤注射后6h MRI见多发散在灶性低信号影,24h点片状低信号影缩小；两种肿瘤注射后2、3或7d MRI形态、位置和24h相似,但信号强度递减,病理证实标记的巨噬细胞存在,与影像结果一致。结论TAMs MR成像可行。注射后24h T2WI是显示肿瘤巨噬细胞迁移、浸润肿瘤内的最佳时机,注射后6h可能反映病变局部血流灌注及新生血管的状态,不同的肿瘤之间巨噬细胞分布方式有差异。更多还原

【Abstract】 Background and purposeMolecular imaging is a rapidly growing biomedical research tool and clinical discipline aimed at noninvasive,quantitative visualization of in vivo molecular processes occurring at cellular and subcellular levels.In general, molecular imaging applications require fast, sensitive and high-resolution imaging techniques, combined with the use of contrast agents that enable the visualization of biological processes at the cellular and molecular level.Until recently, positron emission tomography (PET), single photon emission computed tomography (SPECT) and optical imaging techniques were the most frequently used imaging modalities for pre-clinical molecular imaging in vivo. This is predominantly attributed to the high sensitivity of these modalities,However, nuclear imaging techniques (PET/SPECT) use ionizing radiation, provide no anatomical information and have a low spatial resolution. And Optical imaging suffers from limited depth penetration and relatively low spatial resolution.Therefore,magnetic resonance imaging (MRI) has been recently introduced as an alternative, as this technique provides excellent intrinsic soft-tissue contrast at a high spatial resolution without using ionizing radiation. Moreover, this technology has the advantage that both anatomical information and physiological parameters, like diffusion, perfusion and flow, can be obtained within one single experiment by combining different imaging sequences.MR has become an important tool for molecular imaging.The drawback of MRI is however its low sensitivity for MR contrast agents. Nevertheless, different strategies can be applied to improve the efficiency of such agents, and thus make MRI suitable for molecular imaging purposes. The application of magnetic resonance imaging in conjunction with iron oxide nanoparticle (IONP) based cell labeling techniques, has provided an excellent solutionto the non-invasive tracking of the implanted cells in the host organism. One major challenge for this technique, however, is to induce sufficient amount of particles into the cells to compensate the dilution effect caused by cell division, while not affecting the normal cellular functions. For cell labeling, although SPIO particles are easily internalized by macrophages, uptake by nqnphagocytic and slow-dividing cells is poor, so that high concentrations which often cause cell toxicity are needed for efficient cell labeling.Currently, Several approaches have been raised and investigated to improve the internalization of the contrast agent, with the mostly utilized one being the employment of commercial Feridex in combination with transfection agents. However, lacking of a universal formula, tedious trials and errors have to be made with individual cell line to achieve optimal transfection.In view of these factors, In the study, we conducted the study of molecular imaging with MR imaging and a novel magnetic resonance contrast agent with high magnetic moment-HSA-Dopamine-Fe3O4 nanoparticles (HSA-IONPs) for. through comparative study of Feridex and HSA-Dopamine-Fe3O4 nanoparticles, we investigaed magnetic properties of HSA-IONPs and cell labeling efficiency for different types of cell lines (final concentration of 20μg·ml-1 Fe), and performed molecular magnetic resonance Imaging using neural stem cells,macrophages labled with HSA-IONPs in different animal models.Purpose To synthesize HSA-Dopamine-Fe3O4 nanoparticles (HSA-IONPs)-a novel magnetic resonance contrast agent for molecular imaging and study its magnetic properties.Methods Olecate coated iron oxide nanoparticles (15 nm) were obtained from Ocean NanoTech (Springdale, AR). For surface modification, about 50 mg of oleate coated nanoparticles were dispersed in 5 ml of chloroform. Into the solution,20 mg of dopamine in 2 ml DMSO was added, forming a homogeneous solution. The mixture was heated at 70℃for 1 hour, and was then cooled down to r.t. Hexanes was added as poor solvent to precipitate the nanoparticles, and the nanoparticles were collected by centrifuging at 15,000 g for 15 minutes. Afterwards, they were blow dried with nitrogen, and were redispersed in DMSO with the aid of sonication. On the other hand,20 mg of HSA was dissolved in borate buffer (50 nM, pH 8.5). And with sonication, the nanoparticles in DMSO were dropwise added to the HSA solution. get rid of free dopamine and free HSA.then,were redipersed in PBS buffer. Nanoparticle were quantified and characterized by ICP-AMS、TEM、DSL(dynamic light scattering) and MRI. After normal nude mice were injected with Feridex and HSA-IONPs (10mg Fe/kg) via tail vein.T2W MR scan were performed for studying liver imaging in vivo at different time points.Results Dynamic light scattering (DLS) measured the hydrodynamic diameter of the HSA-IONPs in their dispersion state, The measurement results in water were 28.4 nm. TEM images showed Nanoparticles were monodisperse and single crystalline with circular structure, no aggregated clumps. The results of zeta potential measurements revealed that the HSA-Dopamine-Fe3O4 nanoparticles exhibited negative zeta potential (-9.46±1.86MV).MR phantom results indicated that R2 value of HSA-IONPs(R2=314.5mM-1S-1) was significent higher than that of the conventiona-lly utilized Feridex nanoparticles (R2=123.6mM-1S-1), those HSA-Dopamine-Fe3O4 show better T2 contrast and can be used for liver imaging.Conclusion HSA-IONPs have higher magnetic moment and are better than commercial SPIO nanoparticles(Feridex).Purpose To study cell labeling efficiency of HSA-IONPs as a novel magnetic resonance contrast agent for molecular imaging. Methods HSAIO-NPs or Feridex(final concentration of 20μg·ml-1 Fe) was used to label human prostate cancer PC-3 cells、human head and neck squamous cell carcinoma UMSCC-22B cells、neural stem cells、mesenchymal stem cells、human embryonic stem cells and macrophages respectively at different time points (2、9、12、24h or 48h), All Cells were stained by prussian blue and then counterstained with nuclear fast red. MTT assay was used to evaluate the in vitro cytotoxic effects of nanoparticles. Both nanoparticles phagocytosed by neural stem cells and macrophages were quantified and characterized by ICP-AMS、TEM、and MR Phantom imaging.T2W MR scans were performed 6h after labeled neural stem cells were implanted into bilateral basal ganglia of nude mouse brains by stereotactic injection.24h after the onset of MCAO, macrophages labeled by HSA-IONPs were injected into rats via tail vein and MR scan were performed at different time points (prescan,4h, 1d,2d,5d,7d).Results All cells were incubated with merely 20μgml particles without transfection agent for efficient cell labeling.TEM analysis results found populations of nanoparticles within endosomes/lysosoms and some aggregation could be seen. no toxicity and no harm on cell growth and division was found for the studied cell lines (P<0.05).The results of ICP-AMS assay showed that neural stem cells and macrophages labeled with HSA-IONPs presented higher uptake of nanoparticles than those labled with Feridex. Neural stem cells were incubated with HSA-IONPs or Feridex plus transfection reagents(50μg Fe·ml-1 plus Lipofactamine 2000) for 24h. Compared with Feridex nanoparticles, neural stem cells labeled with HSA-IONPs and transfection agents presented higher uptake of nanoparticles. There was strong dependence on the concentration of nano-particles and iron uptake of neural stem cells. MR Phantom images showed a significant difference between T2WI signal intensity of cell labled with HSA-IONPs and feridex. There was strong linear correlation between R2 values and number of cells before the saturation limit.In vivo MR imaging and pathological examinations confirmed neural stem cells incubated with HSA-IONPs absored more iron than those incubated with Feridex. MR scans could be used to track macrophage migration after focal cerebral ischemic stroke.Conclusion Cell labeling efficiency of HSA-IONPs has an advantage over those of Feridex nanoparticle.Purpose To explore the feasibility of MR imaging for tumor-associated macrophages.Methods Macrophages(RAW264.7)were incubated with HSA-IONPs (20μg/ml Fe)for 24 hours,Then,5×106 labeled macrophages were injected into subcutaneous 4T1 tumor-bearing BALB/c mice and 22B tumor-bearing nude mice via tail vein. At different time points(0h、6h、1d、2d、3d or 7d), all animals performed transverse and coronal T2-weighted MR scans using a 7.0-T MR imaging unit..After sacrifice at different time points, Tumor、liver、spleen and other organs were removed and processed for histological examination.Results Prussian blue staining、TEM and cell phantom imaging confirm efficient cell labeling. in 4T1 tumor model,MR images showed significant low signal intensity on T2-weighted imaging surrounding the center of necrosis and cystic degeneration 6 hours after macrophage injection.,irregular ring low signal enhancement could be seen at the junction of necrotic and parenchyma area at 24 hours. In 22B tumor model,MR images presented distinct multiple small scattered foci of low signal intensity on T2-weighted imaging around the center of necrosis and cystic degeneration 6 hours after macrophage injection. at 24 hours, reduced multiple small scattered foci of low signal intensity could be observed.2、3 or 7days after macrophage injection, MR morphology and location of low signal intensity was similar to those at 24 hours after injection on T2-weighted imaging.But, signal intensity gradually dereased over time.pathological examination verified the presence of labeled macrophages, pathological findings were consistent with imaging results.Conclusion MR imaging for tumor-associated macrophages is feasible.24 hours after macrophage injection is the optimal time point for studing macrophage migrating and infiltrating into tumor on T2-weighted imaging. MR images may indicate the perfusion of local blood flow and the status of neovascularization 6 hours after macrophage injection, The pattern of TAM distribution differed between different tumors.更多还原