聚乙二醇修饰的铂钴磁性纳米颗粒作为新型造影剂的磁共振成像及长期毒性研究
发布时间：2019-03-16 14:36
【摘要】：目的开发一种新型超顺磁性纳米材料Pt3Co，探讨其作为T2造影剂用于活体磁共振成像（magnetic response imaging，MRI）的可行性及价值。 方法①合成磁性纳米颗粒Pt3Co，采用十八碳烯聚乙二醇（polyethylene glycol，PEG）修饰其水溶性得到Pt3Co-PEG，并对其稳定性、表征及磁性特征进行研究。②将小鼠乳腺癌细胞4T1、人乳腺癌细胞sk-br-3、正常人肝细胞HL-7702及人肾上皮细胞293T分别与不同浓度Pt3Co-PEG结合，通过MTT比色法、乳酸脱氢酶（LDH）泄露试验及细胞内活性氧（ROS）检测实验进行Pt3Co-PEG体外毒性研究。③将荷4T1肿瘤的两组雌性Balb/c小鼠分别经尾静脉注射相同剂量的Pt3Co-PEG和Resovist（商用MR T2造影剂），于不同时间点（2h、6h、24h）进行小鼠肿瘤组织及肝脏的T2成像，并测量其T2信号值。④经尾静脉注射一定量Pt3Co-PEG的5组（包括1组对照组）同周龄健康雌性Balb/c小鼠，于注射后不同时间（1d、7d、30d、60d）用摘眼球法取血，，进行血常规及血生化分析；同时取其包括肝脏、脾脏、肾脏、心脏、肺脏、小肠等在内的主要器官组织进行苏木精和伊红（HE）染色；以此来实现活体长期毒性监测。⑤在④中每组小鼠取过血及主要脏器后，再取其肝脏、脾脏、肾脏、心脏、肺脏、小肠、骨骼、肌肉及皮肤等器官组织，通过烧ICP法检测Pt、Co元素在小鼠体内的生物分布状况。 结果①透射电子显微镜下，Pt3Co呈均匀分布的颗粒状，粒径约6nm；经PEG修饰后，Pt3Co-PEG在不同生理溶液中均有较好的稳定性；磁滞曲线显示Pt3Co-PEG具有超顺磁性；近红外图显示吸收波长3000cm-1处有修饰的十八碳烯聚乙二醇分布；3.0T磁共振成像仪测得Pt3Co-PEG的T2弛豫率可达451.2mM/s，远远高于商用T2造影剂Resovist（其T2弛豫率约为193.4mM/s）；②系列体外毒性实验显示Pt3Co-PEG在较高浓度时亦未见明显细胞毒性；③两组荷4T1小鼠模型尾静脉注射相同剂量Pt3Co-PEG和Resovist后，随着时间的推移，Pt3Co-PEG组小鼠肿瘤区域的T2信号值降低幅度明显大于Resovist组，而两组的肝脏部位T2信号变化趋势相似；④活体长期毒性实验结果显示，血生化、血常规指标均在正常范围内；主要脏器HE染色与对照组相比未见明显差异；⑤长期生物分布结果显示，在小鼠尾静脉注射一定量的Pt3Co-PEG60d以后，Pt、Co元素在肝、脾及小肠中仍有较高量的残留，该问题有待通过改善Pt3Co-PEG磁性纳米颗粒的表面修饰来解决。 结论超顺磁性纳米颗粒Pt3Co-PEG在生理环境下具有较好的稳定性和极强的顺磁性。小鼠体内MR成像结果显示Pt3Co-PEG能明显降低肝脏及肿瘤组织的T2信号，尤其在肿瘤部位的成像效果优于目前商用造影剂Resovist。经过长期系统的体内外毒性实验及生物分布结果显示，虽然Pt3Co-PEG在生物体内有一定量的残留，但没有明显的生物毒性。因此，本实验组合成的Pt3Co-PEG有望成为新一代的高效MR T2造影剂，用于肿瘤诊断。
[Abstract]:Objective to develop a new type of superparamagnetic nanomaterials (Pt3Co,) to explore the feasibility and value of using as T2 contrast agent for in vivo magnetic resonance imaging (magnetic response imaging,MRI). Methods 1Magnetic nanoparticles Pt3Co, were synthesized by modifying their water solubility with octadecene polyethylene glycol (polyethylene glycol,PEG) to obtain Pt3Co-PEG, and their stability, characterization and magnetic properties were studied. 2Murine breast cancer cells 4T11, Human breast cancer cell sk-br-3, normal liver cell HL-7702, and human renal epithelial cell 293T, combined with different concentrations of Pt3Co-PEG, were determined by MTT colorimetry. Lactate dehydrogenase (LDH) leakage test and intracellular reactive oxygen species (ROS) assay were used to study the toxicity of Pt3Co-PEG in vitro. 3 the same dose of Pt3Co-PEG was injected into the tail vein of two groups of female Balb/c mice bearing 4T1 tumor. And Resovist (commercial MR T2 contrast agent), At different time points (2 h, 6 h, 24 h), T2 imaging of tumor tissue and liver of mice was performed and their T2 signal values were measured. 4 healthy female Balb/c mice of the same week age were injected with a certain amount of Pt3Co-PEG via caudal vein (including a control group). At different time after injection (1 d, 7 d, 30 d, 60 d), the blood was extracted by eyeball extraction method, and the blood routine and blood biochemical analysis were carried out. At the same time, the main organs including liver, spleen, kidney, heart, lung and small intestine were stained with hematoxylin and eosin (HE). 5 after blood and main organs were taken from each group of mice, the liver, spleen, kidney, heart, lung, small intestine, bone, muscle and skin and other organs and tissues were taken to detect Pt, by burning ICP method. Biodistribution of Co in mice. Results 1 under the transmission electron microscope, Pt3Co showed a uniform particle size of about 6 nm. After PEG modification, Pt3Co-PEG had good stability in different physiological solutions, and the hysteresis curve showed that Pt3Co-PEG had superparamagnetism. Near infrared images showed that there was a modified octadecene polyethylene glycol distribution at the absorption wavelength of 3000cm-1. The T2 relaxation rate of Pt3Co-PEG measured by 3.0T MRI was 451.2 mm / ml, which was much higher than that of commercial T2 contrast agent Resovist (the T2 relaxation rate was about 193.4mM/s). A series of in vitro toxicity tests showed that there was no obvious cytotoxicity of Pt3Co-PEG at higher concentration. 3After the same dose of Pt3Co-PEG and Resovist were injected into the tail vein of the 4T1-bearing mice in the two groups, the decrease of T2 signal value in the tumor area of the Pt3Co-PEG group was significantly higher than that of the Resovist group with the passage of time, while the trend of T2 signal change in the liver of the two groups was similar. (4) the results of long-term toxicity test in vivo showed that the blood biochemical and routine indexes were within the normal range, and there was no significant difference in HE staining between the main organs and the control group. 5 the results of long-term biodistribution showed that after a certain amount of Pt3Co-PEG60d was injected into the tail vein of the mice, there were still high levels of Pt,Co residues in the liver, spleen and small intestine. This problem needs to be solved by improving the surface modification of Pt3Co-PEG magnetic nanoparticles. Conclusion superparamagnetic nanoparticles Pt3Co-PEG have good stability and strong paramagnetism in physiological environment. The results of MR in mice showed that Pt3Co-PEG could significantly decrease the T2 signal of liver and tumor tissue, especially in the tumor site, which was better than that of commercial contrast agent Resovist. at present. After long-term systematic in vitro and in vivo toxicity test and biological distribution results, although there is a certain amount of Pt3Co-PEG residues in organisms, there is no obvious biotoxicity. Therefore, the combined Pt3Co-PEG is expected to be a new generation of high efficient MR T2 contrast agent for tumor diagnosis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R445.2
本文编号：2441563
[Abstract]:Objective to develop a new type of superparamagnetic nanomaterials (Pt3Co,) to explore the feasibility and value of using as T2 contrast agent for in vivo magnetic resonance imaging (magnetic response imaging,MRI). Methods 1Magnetic nanoparticles Pt3Co, were synthesized by modifying their water solubility with octadecene polyethylene glycol (polyethylene glycol,PEG) to obtain Pt3Co-PEG, and their stability, characterization and magnetic properties were studied. 2Murine breast cancer cells 4T11, Human breast cancer cell sk-br-3, normal liver cell HL-7702, and human renal epithelial cell 293T, combined with different concentrations of Pt3Co-PEG, were determined by MTT colorimetry. Lactate dehydrogenase (LDH) leakage test and intracellular reactive oxygen species (ROS) assay were used to study the toxicity of Pt3Co-PEG in vitro. 3 the same dose of Pt3Co-PEG was injected into the tail vein of two groups of female Balb/c mice bearing 4T1 tumor. And Resovist (commercial MR T2 contrast agent), At different time points (2 h, 6 h, 24 h), T2 imaging of tumor tissue and liver of mice was performed and their T2 signal values were measured. 4 healthy female Balb/c mice of the same week age were injected with a certain amount of Pt3Co-PEG via caudal vein (including a control group). At different time after injection (1 d, 7 d, 30 d, 60 d), the blood was extracted by eyeball extraction method, and the blood routine and blood biochemical analysis were carried out. At the same time, the main organs including liver, spleen, kidney, heart, lung and small intestine were stained with hematoxylin and eosin (HE). 5 after blood and main organs were taken from each group of mice, the liver, spleen, kidney, heart, lung, small intestine, bone, muscle and skin and other organs and tissues were taken to detect Pt, by burning ICP method. Biodistribution of Co in mice. Results 1 under the transmission electron microscope, Pt3Co showed a uniform particle size of about 6 nm. After PEG modification, Pt3Co-PEG had good stability in different physiological solutions, and the hysteresis curve showed that Pt3Co-PEG had superparamagnetism. Near infrared images showed that there was a modified octadecene polyethylene glycol distribution at the absorption wavelength of 3000cm-1. The T2 relaxation rate of Pt3Co-PEG measured by 3.0T MRI was 451.2 mm / ml, which was much higher than that of commercial T2 contrast agent Resovist (the T2 relaxation rate was about 193.4mM/s). A series of in vitro toxicity tests showed that there was no obvious cytotoxicity of Pt3Co-PEG at higher concentration. 3After the same dose of Pt3Co-PEG and Resovist were injected into the tail vein of the 4T1-bearing mice in the two groups, the decrease of T2 signal value in the tumor area of the Pt3Co-PEG group was significantly higher than that of the Resovist group with the passage of time, while the trend of T2 signal change in the liver of the two groups was similar. (4) the results of long-term toxicity test in vivo showed that the blood biochemical and routine indexes were within the normal range, and there was no significant difference in HE staining between the main organs and the control group. 5 the results of long-term biodistribution showed that after a certain amount of Pt3Co-PEG60d was injected into the tail vein of the mice, there were still high levels of Pt,Co residues in the liver, spleen and small intestine. This problem needs to be solved by improving the surface modification of Pt3Co-PEG magnetic nanoparticles. Conclusion superparamagnetic nanoparticles Pt3Co-PEG have good stability and strong paramagnetism in physiological environment. The results of MR in mice showed that Pt3Co-PEG could significantly decrease the T2 signal of liver and tumor tissue, especially in the tumor site, which was better than that of commercial contrast agent Resovist. at present. After long-term systematic in vitro and in vivo toxicity test and biological distribution results, although there is a certain amount of Pt3Co-PEG residues in organisms, there is no obvious biotoxicity. Therefore, the combined Pt3Co-PEG is expected to be a new generation of high efficient MR T2 contrast agent for tumor diagnosis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R445.2
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本文编号：2441563
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