LyP-1修饰磁纳米颗粒介导的肿瘤磁感应靶向热疗

【作者】 张洁英；

【导师】 唐劲天；

【作者基本信息】 北京中医药大学 ， 微生物与生化药学， 2012， 硕士

【摘要】 研究目的本课题旨在研究主动靶向磁纳米粒子(LyP-1-MNPs)的制备方法,并在细胞水平上完成磁感应靶向热疗的生物相容性研究和靶向性验证,在动物水平上研究LyP-1-MNPs磁感应靶向热疗的安全性与有效性。研究方法1.LyP-1-MNPs的制备：采用一步法制备PEG修饰的Fe3O4磁纳米粒子,经活化后在其表面偶联细胞穿膜肽LyP-1。利用傅立叶红外光谱仪(FTIR)、热重分析(TGA)、透射电镜(TEM)、zeta电位与粒度分析仪等分析方法对所制备的PEG修饰的Fe304磁纳米粒子的粒径、形态、蛋白偶联质量比及zeta电位分析等进行表征检测,并将制备样品放置于交变磁场中检测其升温性能,并在室温条件下观察其稳定性和分散性。2.细胞实验：采用RT-PCR和Western Blot的研究方法对高低表达P32蛋白的肿瘤细胞P32蛋白的表达量进行验证；采用CCK-8法研究靶向性磁纳米粒子的细胞毒性；甲苯胺蓝染色和透射电镜(TEM)观察磁纳米粒子在不同细胞内的聚集状态；激光共聚焦(LCSM)检测磁纳米粒子在细胞内的具体定位情况；电感耦合等离子发射光谱仪(ICP-MS)检测单个细胞对磁纳米粒子的摄取量。3.动物实验：制作荷瘤鼠模型；施行实验动物分组,随机分为3组：阴性对照组、热疗组、靶向热疗组。实验组肿瘤原位注射磁流体,热疗后称量体重,绘制体重变化曲线；记录肿瘤体积,绘制肿瘤体积变化曲线。热疗30天后对荷瘤鼠进行安乐死,取心、肝、脾、肺、肾、肿瘤,进行组织切片,HE染色观察靶向磁纳米粒子的体内生物相容性；普鲁士蓝染色观察靶向磁纳米粒子在体内的生物分布情况。研究结果1.制备的磁纳米粒子稳定性和磁响应性良好,经细胞穿膜肽LyP-1修饰后呈规整的球形,粒径有所增加,约15nm,分散性更好；交变磁场下具有良好的升温能力；一步法制备的PEG修饰的磁纳米粒子表面带负电荷,经LyP-1修饰后表面带正电荷。2. RT-PCR和Western-Blot验证了人乳腺癌细胞株MCF-7为一种高表达P32蛋白的细胞株,小鼠结直肠癌细胞株CT-26为一种相对低表达P32蛋白的细胞株。3.所制备的靶向磁纳米粒子无显著细胞毒性,毒性分级为0级或1级,生物相容性好。靶向磁纳米粒子进入细胞的数目高于PEG修饰的非靶向磁纳米粒子进入相同细胞的数目,且高表达P32蛋白的癌细胞MCF-7对靶向磁纳米粒子的摄取量明显高于相对低表达P32蛋白的细胞株CT-26对靶向磁纳米粒子的摄取量。靶向磁纳米粒子在细胞内定位有两种：一部分存在于细胞质中,聚集程度高,推测是吞饮作用或吞噬作用介导的胞吞途径而进入胞内；一部分分散的存在于胞质中或细胞核中,分散性强,猜测是P32蛋白受体介导的内吞途径进入胞内。4.动物实验结果显示,实验组荷瘤鼠的肿瘤体积均得到有效抑制,热疗组荷瘤鼠的治愈率为30%,靶向热疗组荷瘤鼠治愈率为60%,治愈率提高了一倍。病理切片观察显示,磁纳米粒子在生物体内的生物组织相容性良好,未见明显的免疫反应或炎症反应,普鲁士蓝染色观察磁纳米粒子在体内的分布与代谢情况表明磁纳米粒子在治愈后期主要出现在肿瘤部位或肿瘤消退后的原肿瘤部位,在脏器中也有聚集,主要存在于脾、肺、肝中,而又以脾的铁含量最高。研究结论LyP-1修饰的靶向磁纳米粒子具有良好的分散性、磁响应性和升温性能,且生物相容性好,并能有效提高高表达P32蛋白的细胞对靶向纳米粒子的摄取能力。细胞实验和动物实验结果均表明靶向磁性纳米粒用于磁感应靶向热疗是安全的、可行的,磁感应靶向热疗效果是显著的。更多还原

【Abstract】 ObjectiveThe aims of the present study are to investigate the preparation of LyP-1modified magnetic nanoparticles (LyP-1-MNPs) which could home to the cancer overexpressing protein P32, to study the biocompatibility and targeting properties of LyP-1-MNPs in vitro, and to test the targeting therapy of LyP-1-MNPs in vivo.Methods1. Preparation and characterization ofLyP-1modified MNPsFirstly, Polyethylene glycol(PEG)-coated magnetic nanoparticals (PEG-MNPs) were synthesized by "single-step method", which is chemical co-precipitation method substantially, but it is more simple and cost less time. PEG-MNPs, modified by special agent, were conjugated with cell-penetrating peptide LyP-1. The size, zeta potential, and coating characterizations of LyP-1-MNPs were analyzed by transmission electron microscope (TEM), zetaplus zeta potential measurement, and fourier transform infrared spectroscopy (FTIR).2. In vitro studyThe expressions of P32of cell lines were detected by RT-PCR and western-blot. Cytotoxicity of functional magnetic nanoparticles was evaluated by CCK-8assay, and the aggregation state of LyP-1-MNPs in cells was studied by Prussian blue staining and transmission electron microscopy (TEM). Subcellular localization of LyP-1-MNPs in different cell lines was observed by Laser Scanning Confocal Microscope(LCSM).The iron content of cellular uptake was measured by Inductively coupled plasma mass spectrometry (ICP-MS).3. In vivo studyThe tumor-bearing mice model was built by transplanting human breast cancer cell line MCF-7cells into the subcutaneous tissue of the right hind limb of BALB/c nude mouse. And then all animals were divided into three groups randomly:negative control group, hyperthermia group, and targeting therapy group. The experimental groups were injected with magnetic fluid, after magenetic hyperthermia treatment, the body weight of animals and the tumor volume were measured, then the body weight curves and the tumor volume curves were plotted. When30days had passed after therapy, the mice were euthanized with an intraperitoneal injection of pentobarbital sodium(120mg/kg), the important organs, such as heart, liver, spleen, lungs, kidneys and tumor,were obtained and prepared for histological analysis, then the samples were cut into about5μ m thick section and the sections were stained with either hematoxylin and eosin (H&E) or Prussian blue. Biocompatibility of MNPs was analysed by H&E staining, and biodistrbution of MNPs was observred by Prussian blue staining.Results1. Magnetic nanoparticles had good stability, well magnetism response, and great dispersibility. When exposed to alternating magnetic field, MNPs had good warming performance. The morphology of particals modified with LyP-1was approximately spherical and the mean size of the magnetic nanoparticles was about15nm.After being coated by Polyethylene glycol (PEG), the surface of MNPs displayed the negative charge properties, and when LyP-1was conjugated with the PEG-MNPs, the zeta potential was changed into positive charge from negative charge.2. We found that human breast cancer MCF-7is a P32-high-expressing cell line and relatively,colorectal cancer cell line CT-26is a P32-low-expressing cell line using RT-PCR and western-blot assay.3. The result of CCK-8assay showed that PEG-MNPs modified with LyP-1had no significant cytotoxicity at a concentration of less than60μg/mL, cytotoxicity grade had a narrow range between0grade and1grade. The cellular uptake of targeting nanoparticals was higher than that of Polyethylene glycol (PEG)-coated magnetic nanoparticals (PEG-MNPs) which has no targeting properties, and the cellular uptake of LyP-1-MNPs in overexpressing P32cell MCF-7was much higher than that in low-expressing P32cell CT-26. The targeting nanoparticals mainly localized in the cytoplasm, and some were found in the nucleus, we speculated that the distribution of LyP-1-MNPs maybe effected by different mechanisms of uptake routines, because of the size of LyP-1-MNPs, the most MNPs aggregated and were uptaked through cells swallowed way into the cytoplasm, while some that had good dispersibility and small size could be transported across the cell wall and nucleus wall through the receptor-mediated swallowed way. 4. The result of in vivo experiment demonstrated that the tumor volume of the experimental groups was suppressed effectively,and the curative ratio of hyperthermia group was30%(n=10), and the curative ratio of targeting therapy group was60%(n=10),which was the twice of that of hyperthermia group. Histological analysis indicated that the biocompatibility of magnetic nanoparticals was very well, because there was no any inflammation and immunoreaction. Prussian blue staining of sections of samples illustrated that the biodistribution of MNPs in body mainly accumulated in the tumors and also showed in the spleen and lungs.ConclusionsMagnetic nanoparticles had good stability, well magnetism response, great dispersibility, good warming performance, nice biocompatibility. After modified with LyP-1, the LyP-1modified magnetic nanoparticles could home to the overexpressing P32cell lines MCF-7, and the cellular uptake of magnetic nanoparticals was much higher than the non-targeting nanoparticals significantly. We also found that targeting magnetic nanoparticals had a greater inhibitive effect on tumor growth than hyperthermia group or control group.更多还原