肿瘤血管靶向性纳米氧化锰对比剂的制备、成像及生物学效应的研究

发布时间：2018-06-24 23:05

本文选题：氧化锰纳米粒子 + 分子影像　；参考：《第二军医大学》2014年博士论文

【摘要】：【研究背景】肿瘤是目前全球最常见疾病之一，其发病率和死亡率都非常高。据世界卫生组织报道，每年全球有超过1000万新发病例，约700万人死亡。美国2013年的新发癌症病例为166万，其中58万癌症死亡病例。在中国，到2012年底，新的癌症病例每年为350万，因癌症死亡的人数是250万。“早期发现”，“及时诊断”和“早期正确治疗”是提高患者生存率的关键。如何在（小）肿瘤的启动或早期阶段，及时发现和确诊，是实现“三早”的重要途径。肿瘤生长进展需要依靠新生血管供应细胞代谢所必需的营养和氧气，因此肿瘤的发生、发展及转移与肿瘤血管生成密切相关。因此如能够定量化、可视化地检测并研究肿瘤新生血管，将有助于癌症的早期诊断。而且，对于临床肿瘤抗新生血管治疗，能够进行全程监测，观察新生血管的状态，评估治疗效果。肿瘤分子生物学研究表明，在肿瘤的血管生成和转移等生物学事件中，肿瘤新生血管表面或某些肿瘤细胞表面存在的一种粘附分子超家族成员—整合素有着重要作用。目前，在肿瘤早期诊断的肿瘤新生血管靶点研究中，研究较多的是αvβ3整合素受体。整合素αvβ3受体在增殖的肿瘤新生血管内皮细胞高度表达，而在静息血管内皮细胞上无表达，是肿瘤新生血管的标志分子之一。αvβ3受体能够结合特异性识别并结合到细胞外基质中的甘-精-天冬氨酸序列（RGD序列），，为在探针的设计和修饰方面，使其向肿瘤新生血管内皮细胞靶向性聚合，提供了重要依据。目前在评价肿瘤血管的成像模式中，核磁共振MR成像是一种理想的、不断发展的成像方式。它不仅可以多个参数、多种序列、轴冠矢状多方位成像，而且无电离辐射，还可以较高的空间分辨率采集组织结构解剖、生理病理及相关理化信息。MRI对比剂，尤其是靶向对比剂的研发和应用，在提高MRI的疾病诊断准确率的同时，赋予其反映病灶代谢特征、评估治疗效果的能力，具有广阔的临床应用前景。磁共振分子成像是分子影像学研究热点。第一部分磁共振靶向对比剂（RGD-mPEG-DA-MnO）的制备构建和表征【目的】制备整合素αvβ3介导的磁共振靶向对比剂偶联RGD包被聚乙二醇并连接多巴胺的氧化锰纳米粒子(RGD-mPEG-DA-MnO)并进行体外表征。【材料和方法】以四水合氯化锰和油酸为原料，采用热裂解法合成超小MnO纳米粒子，对其进行mPEG和DA的表面修饰和RGD连接。使用透射电子显微镜镜（TEM）、动态光散射（DLS）、氢质子磁共振波谱（1HNMR）、傅里叶变换红外光谱仪（FTIR）、电感耦合等离子体原子发射光谱仪（ICP-AES）及核磁共振分别对制备的靶向对比剂进行形态、粒径、结构、稳定性、密度、样品锰元素含量及弛豫效能进行体外表征。【结果】新合成的mPEG-DA-MnO磁性纳米粒子水合粒径在150nm左右，粒径分布比较均一，稳定性高，其锰元素含量为2.48×10-3mg/MG（每毫克对比剂里面有2.48×10-3mg的锰）。五种不同样品的表面修饰物mPEG-DA的密度范围为6.51-17.2mmol/m2，其相应r1弛豫效能的范围为16.14mM-1S-1到5.98mM-1S-1，r1最大者约为医用钆对比剂r1弛豫效能的3倍。成功偶联RGD的RGD-mPEG-DA-MnO靶向磁性纳米粒子对比剂具备高纯度。【结论】以四水合氯化锰和油酸为原料的热裂解法可成功合成超小MnO纳米粒子，经mPEG和DA的表面修饰能够制备纳米化、单分散相、高纯度、稳定性、弛豫效能的纳米粒子mPEG-DA-MnO，并偶联RGD肽成功制备靶向磁性纳米粒子RGD-mPEG-DA-MnO。第二部分对比剂的毒性和造影效果的体外研究【目的】探讨新合成对比剂（RGD-mPEG-DA-MnO）的细胞毒性和造影效果。【材料和方法】MTT法检测新合成对比剂（RGD-mPEG-DA-MnO）在体外对A549细胞生存率的影响。采用PhillipAchieva3.0T磁共振对新合成对比剂进行T1WI及T1Mapping成像，T1加权成像信号强度、T1map值分别评价所制备不同粒径和修饰物密度的对比剂的成像对比度和弛豫效能。【结果】对比剂浓度在500ug/ml时，其细胞存活率仍较高，其IC50（半抑制浓度）为818.9ug/ml。随着锰离子浓度的增大，粒径5nmMnO核心的RGD-mPEG-DA-MnO造影图像逐渐变亮。粒径5nmMnO核心的RGD-mPEG-DA-MnO的r1弛豫效能为11.6s-1mM-1。【结论】RGD-mPEG-DA-MnO磁性纳米对比剂在一定浓度范围内对细胞无明显毒性。其成像对比度较高，r1弛豫效能约为医用Gd-DTPA弛豫效能的2倍。第三部分磁共振对比剂的动物在体成像研究【目的】研究MR靶向对比剂RGD-mPEG-DA-MnO靶向荷人肺癌裸鼠、实现肿瘤血管及细胞靶向显影的可行性，探讨该磁性纳米对比剂的成像特点。【材料和方法】 1、非靶向磁共振对比剂(mPEG-DA-MnO)对荷人肺腺癌裸鼠的MRI研究BALB/C昆明白鼠经尾静脉注入靶向磁共振对比剂（mPEG-DA-MnO），观察注射对比剂前后不同时间点肝脏、肾脏和肌肉的T1WI信号的变化。 2、磁共振靶向对比剂(RGD-mPEG-DA-MnO)对荷人肺腺癌裸鼠的MRI研究人肺腺癌A549细胞株传代并对裸鼠皮下接种，制成荷人肺腺癌的裸鼠模型。通过尾静脉注入（RGD-mPEG-DA-MnO）靶向磁共振对比剂，观察注射前后不同时间点荷瘤鼠的瘤体、肝脏及颈背部肌肉T1WI信号值变化。 3、磁共振靶向对比剂(RGD-mPEG-DA-MnO)与非靶向对比剂(mPEG-DA-MnO)荷人肺癌裸鼠MRI成像对照研究按照随机对照原则，将8只荷瘤裸鼠平均分为两组，实验组4只和对照组4只，实验组通过尾静脉注入靶向对比剂RGD-mPEG-DA-MnO，对照组注射mPEG-DA-MnO，观察并比较在注射对比剂前后不同时间点两组动物的肿瘤和肌肉T1WI信号强度值变化。【结果】注射mPEG-DA-MnO对比剂15min后，其肝、肾脏区域有明显增强效果，1h后增强效果减弱。2h后对比剂代谢进入膀胱。在靶向荷A549肺腺癌细胞肿瘤裸鼠MRI研究中，对比剂RGD-mPEG-DA-MnO有向瘤体局部聚集的趋势，强化峰值出现在注射对比剂后约2小时左右，最大强化率达109.9％；肝脏也表现出明显强化，强化峰值出现在注射对比剂后约1小时左右，之后呈现出比较缓慢的回落趋势；和非靶向的mPEG-DA-MnO进行对照，(1)所制备的靶向对比剂RGD-mPEG-DA-MnO具有明显的靶向瘤体效果，实验组肿瘤各扫描时间点的信号强度值差异的统计学意义显著(F=166.83，P0．0001)，强化峰值出现时间约注入对比剂后2小时；无靶向对比剂mPEG-DA-MnO组的瘤体各扫描时间点信号值差异亦有显著统计学意义，强化高峰出现在注射后约1小时；靶向组（RGD-mPEG-DA-MnO）及无靶向组（mPEG-DA-MnO）的肌肉各扫描时间点信号强度值总体差异不大。【结论】靶向对比剂RGD-mPEG-DA-MnO能与整合素αvβ3受体特异性结合，对荷瘤裸鼠的肿瘤新生血管MR成像的强化效果具持续性和特异性，使MR靶向显像及靶向诊断具有可行性。第四部分聚乙二醇包被的磁性氧化锰纳米粒子的体外生物学效应探讨【目的】探讨聚乙二醇包被的磁性氧化锰纳米粒子对比剂对人脐静脉内皮细胞（HUVECs）和人肺腺癌A549细胞的生物学效应及相关机制。【方法】采用多种细胞生物学研究方法探讨聚乙二醇包被的磁性氧化锰纳米粒子标记脐静脉血管内皮细胞(HUVECs)和A549人肺腺癌细胞后，对其增殖、迁移、侵袭、分化、细胞骨架及细胞亚器官等生物学行为的影响。【结果】浓度超过500ug/L的包被聚乙二醇的氧化锰磁性纳米粒子可抑制内皮细胞HUVECs增殖、迁移和侵袭，此抑制能力表现出浓度依赖性；氧化锰纳米粒子可抑制内皮细胞的管腔分化形成能力；可引起细胞骨架发生重构，粘着斑数目降低，细胞粘附能力呈浓度依赖性降低；去粘附可进一步引起细胞增殖、迁移、侵袭、分化能力的损害及抑制。高浓度的氧化锰纳米粒子降低细胞线粒体膜电位，并诱导活性氧产生氧化应激反应。【结论】高浓度的聚乙二醇包被的磁性氧化锰纳米粒子（mPEG-DA-MnO）可通过诱导氧化应激造成细胞毒作用。
[Abstract]:[research background]
Cancer is one of the most common diseases in the world and its incidence and mortality are very high. According to the WHO, there are more than 1000 million new cases in the world every year, about 7 million people die. In the United States, 1 million 660 thousand of new cases of cancer in 2013, of which 580 thousand are cancer deaths. In China, by the end of 2012, new cancer cases are 3 million 500 thousand a year of 3 million 500 thousand. The number of deaths due to cancer is 2 million 500 thousand. "Early detection", "timely diagnosis" and "early correct treatment" are the key to improve the survival rate of patients. How to find and diagnose in the start or early stage of (small) tumor is an important way to realize the "three early". The growth of tumor needs to rely on the supply of cell metabolism of the neovascularization. Essential nutrients and oxygen are necessary, so the occurrence, development and metastasis of tumor are closely related to the angiogenesis of tumor. Therefore, it will be helpful to the early diagnosis of cancer if it can be quantified and visualized and studied the neovascularization of tumor. Moreover, the whole course monitoring and observation of the neovascularization for the anti neovascularization of the clinical tumor The molecular biological study of tumor molecular biology shows that in the biological events such as angiogenesis and metastasis of tumor, a kind of adhesion molecule superfamily member of the tumor cell surface or some tumor cells has an important role. In the study, most of the research is the alpha v beta 3 integrin receptor. The integrin alpha v beta 3 receptor is highly expressed in the proliferating neovascular endothelial cells, but is not expressed on the resting vascular endothelial cells. It is one of the biomarkers of the neovascularization of the tumor. The alpha v beta 3 receptor can be combined with the specific recognition and combined with the glycinine aspartate in the extracellular matrix. The acid sequence (RGD sequence) provides an important basis for the design and modification of the probe into the targeting polymerization of neovascular endothelial cells in tumor cells. In the evaluation of tumor angiogenesis, magnetic resonance MR imaging is an ideal and constantly developing imaging mode. It can not only have multiple parameters, multiple sequences, and axial crown sagittal. Multidimensional imaging, without ionizing radiation, can also acquire tissue anatomy, physiological and pathological and related physical and chemical.MRI contrast agents, especially target contrast agents, to improve the diagnostic accuracy of MRI, and give them the ability to reflect the metabolic characteristics of the lesions and evaluate the therapeutic effect. Magnetic resonance molecular imaging (MRI) is a hot research area in molecular imaging.
Part 1 Preparation and characterization of magnetic resonance targeted contrast agent (RGD-mPEG-DA-MnO).
[Objective] to prepare an integrin - alpha v beta 3 - mediated magnetic resonance targeting contrast agent coupled with RGD - coated peg and dopamine - based manganese oxide nanoparticles (RGD-mPEG-DA-MnO) and to characterize in vitro.
[materials and methods] super small MnO nanoparticles were synthesized with four hydrated manganese chloride and oleic acid by thermal splitting. The surface modification of mPEG and DA and RGD connection were used. The transmission electron microscope (TEM), dynamic light scattering (DLS), hydrogen proton magnetic resonance spectroscopy (1HNMR), Fourier transform infrared spectrometer (FTIR), inductance coupling, etc. were used. The morphology, particle size, structure, stability, density, manganese content and relaxation efficiency of the target contrast agent prepared by atomic emission spectrometer (ICP-AES) and nuclear magnetic resonance (NMR) were characterized respectively.
[results] the diameter of the newly synthesized mPEG-DA-MnO magnetic nanoparticles is about 150nm, the particle size distribution is uniform, the stability is high, the manganese content is 2.48 x 10-3mg/MG (2.48 10-3mg Mn in the contrast agent per milligram). The density range of the surface modifier of five different samples is 6.51-17.2mmol/m2, and its corresponding R1 relaxation The range of efficiency is from 16.14mM-1S-1 to 5.98mM-1S-1, and the largest of R1 is about 3 times the relaxation efficiency of the medical gadolinium contrast agent R1. The successful coupling of RGD's RGD-mPEG-DA-MnO target to the magnetic nanoparticle contrast agent has high purity.
[Conclusion] ultra small MnO nanoparticles can be successfully synthesized with four hydrated manganese chloride and oleic acid as raw materials. By surface modification of mPEG and DA, nano, monodisperse, high purity, stable and relaxor nanoparticles mPEG-DA-MnO are prepared, and the target magnetic nanoparticles RGD-mPEG-DA-MnO. can be prepared by coupling RGD peptide.
The second part: the toxicity and contrast effect of contrast agents in vitro.
[Objective] to explore the cytotoxicity and contrast effect of the new synthetic contrast agent (RGD-mPEG-DA-MnO).
[materials and methods] MTT method was used to detect the effect of new synthetic contrast agent (RGD-mPEG-DA-MnO) on the survival rate of A549 cells in vitro. T1WI and T1Mapping imaging of new synthetic contrast agents were performed by PhillipAchieva3.0T magnetic resonance (MRI), T1 weighted imaging signal intensity, and T1map value to evaluate the imaging of contrast agents with different particle sizes and modifier densities respectively. Contrast and relaxation efficiency.
[results] the cell survival rate of the contrast agent at 500ug/ml was still high, and its IC50 (semi inhibitory concentration) was 818.9ug/ml. with the increase of the concentration of manganese ions, the RGD-mPEG-DA-MnO contrast image of the kernel of the particle size 5nmMnO gradually brightened. The R1 relaxation efficiency of the RGD-mPEG-DA-MnO of the particle size 5nmMnO core was 11.6s-1mM-1.
[Conclusion] RGD-mPEG-DA-MnO magnetic nano contrast agent has no obvious toxicity to cells in a certain concentration range. Its imaging contrast is high, and the relaxation efficiency of R1 is about 2 times of the efficacy of medical Gd-DTPA relaxation.
The third part is animal imaging in vivo.
[Objective] to study the feasibility of MR targeted contrast agent RGD-mPEG-DA-MnO targeting human lung cancer in nude mice, to explore the feasibility of tumor angiogenesis and cell targeting, and to explore the imaging characteristics of the magnetic nano contrast agent.
[materials and methods]
1, MRI study of non targeted magnetic resonance contrast agent (mPEG-DA-MnO) in nude mice bearing human lung adenocarcinoma BALB/C mice were injected with target magnetic resonance contrast agent (mPEG-DA-MnO) into the tail vein of Kunming white mice, and the changes of T1WI signals in the liver, kidney and muscle were observed at different time points before and after the injection of contrast agent.
2, magnetic resonance targeting contrast agent (RGD-mPEG-DA-MnO) for human lung adenocarcinoma MRI research human lung adenocarcinoma A549 cell line and subcutaneous inoculation of nude mice into nude mice bearing lung adenocarcinoma. Through the tail vein injection (RGD-mPEG-DA-MnO) target magnetic resonance contrast agent, the tumor body, liver and neck were observed at different time points before and after injection. The T1WI signal value of the back muscles changes.
3, magnetic resonance targeting contrast agent (RGD-mPEG-DA-MnO) and non target contrast agent (mPEG-DA-MnO) nude mice with lung cancer MRI imaging control study, according to the random control principle, 8 tumor bearing nude mice were divided into two groups, 4 experimental group and 4 control group, the experimental group was injected into the target contrast agent RGD-mPEG-DA-MnO through the tail vein, and the control group was injected mPEG-DA-MnO, The T1WI and signal intensity of two groups of animals before and after injection of contrast agent were observed and compared.
[results] after the injection of mPEG-DA-MnO contrast agent 15min, the liver and kidney region had an obvious enhancement effect. After 1h, the enhancement effect weakened the.2h contrast agent metabolism into the bladder. In the MRI study on A549 lung adenocarcinoma cell tumor, the contrast agent RGD-mPEG-DA-MnO had the trend of local aggregation of the tumor to the tumor body, and the enhancement peak appeared after the injection contrast agent. About 2 hours or so, the maximum intensification rate was 109.9%, the liver also showed obvious enhancement, and the intensification peak appeared about 1 hours after the injection contrast agent, and then showed a slow downward trend, compared with the non target mPEG-DA-MnO, and (1) the target contrast agent RGD-mPEG-DA-MnO has obvious target tumor effect. The statistical significance of the difference of signal intensity of each time point of the tumor was statistically significant (F=166.83, P0.0001), and the peak time of the enhancement was about 2 hours after the injection of contrast agent; the difference of the signal value of each scanning time point of the tumor body without target contrast agent mPEG-DA-MnO group also had significant statistical significance, and the peak of the intensities appeared about 1 hours after the injection. The signal intensity values of target group (RGD-mPEG-DA-MnO) and non target group (mPEG-DA-MnO) at different scanning time points were not significantly different.
[Conclusion] target contrast agent RGD-mPEG-DA-MnO can be specifically combined with integrin alpha v beta 3 receptor. The enhancement effect of MR imaging in tumor neovascularization of tumor bearing nude mice is continuous and specific, which makes MR target imaging and target diagnosis feasible.
The fourth part of polyethylene glycol coated magnetic manganese oxide nanoparticles in vitro biological effects
[Objective] to investigate the biological effect and mechanism of the magnetic manganese oxide nanoparticle contrast agent coated with polyethylene glycol (PEG) on human umbilical vein endothelial cells (HUVECs) and human lung adenocarcinoma A549 cells.
[Methods] the effects of polyethylene glycol coated magnetic manganese oxide nanoparticles on the proliferation, migration, invasion, differentiation, cytoskeleton and cell suborgans of the umbilical vein endothelial cells (HUVECs) and A549 human lung adenocarcinoma cells were investigated by multiple cell biological methods. [results] the concentration was more than 500ug/L The manganese oxide magnetic nanoparticles coated with polyethylene glycol can inhibit the proliferation, migration and invasion of endothelial cell HUVECs, and the inhibition ability shows a concentration dependence. Manganese oxide nanoparticles can inhibit the formation of endothelium cells, cause the remodeling of the cytoskeleton, the number of adhesion spots and the concentration of cell adhesion. Dependence can be reduced; adhesion can further induce cell proliferation, migration, invasion, differentiation and inhibition. High concentrations of manganese oxide nanoparticles reduce the cell mitochondrial membrane potential and induce reactive oxygen species to produce oxidative stress reactions. [Conclusion] high concentration of polyethylene glycol coated magnetic manganese oxide nanoparticles (mPEG-DA-MnO) can be used. Cytotoxic effects are induced by inducing oxidative stress.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R943;R96

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