叶酸受体介导的磁性纳米给药系统的构建及用于肿瘤诊治的实验研究

发布时间：2018-01-22 09:50

本文关键词： 纳米载体诊断治疗叶酸受体 MRI造影剂化疗靶向传输　出处：《中国科学技术大学》2017年博士论文　论文类型：学位论文

【摘要】：从全世界范围看,肿瘤尤其是恶性肿瘤极大地威胁着人类的健康,如何有效的治疗肿瘤、提高患者的生存质量是肿瘤研究领域亟待解决的问题。肿瘤的早期诊断和治疗对提高肿瘤治愈率非常关键。传统的肿瘤诊断和治疗是两个分开的独立过程,早期肿瘤很难被发现,往往需要借助影像学技术辅助诊断。核磁共振成像MRI是一项先进的医学影像技术,被广泛用于肿瘤的早期诊断,临床50%以上的磁共振成像需要造影剂,但是临床常用的钆基造影剂存在靶向性差、肝肾毒副作用大、体内存留时间短等缺陷,严重制约了其在临床的应用。肿瘤的治疗方法包括手术治疗、放射治疗、化学药物治疗等,其中化学药物治疗被认为是目前临床上最有效的治疗手段。然而传统的化疗药物存在组织选择性差、药物毒副作用大、肿瘤多耐药性等缺点,严重影响了化疗的效果。近年来,肿瘤"诊疗一体化"概念被提出,旨在将诊断和治疗相结合,在提高药物效果,减小药物副作用的同时,监控病变发展与治疗效果,及时调整治疗方案,避免对疾病的治疗不足或过度治疗。随着纳米技术的飞跃发展,与生物医学相结合的纳米医药成为研究的热点,纳米材料由于具有特殊的物理和化学性质,容易进行表面修饰和功能化设计,被用作药物载体。本论文以肿瘤的诊治为出发点,旨在构建以纳米材料为载体的肿瘤诊疗一体化平台。利用纳米材料尺寸和结构的特殊性,对纳米载体进行修饰改性,将抗肿瘤药物和MR成像造影剂接载到一个纳米载体中,同时完成药物输送和MR成像,实现多功能的整合。人正常组织是pH为7.4左右的中性环境,肿瘤组织为弱酸性微环境,利用这种生理差异可以设计pH敏感的纳米药物传输系统,pH可控释放药物。肿瘤细胞表面往往特异性表达一些肿瘤标志物如叶酸受体(Folate Receptor,FR),叶酸受体是靶向治疗最常用的靶点,纳米给药体系通过偶联叶酸(Folic Acid,FA),可以特异性识别肿瘤细胞,与叶酸受体特异性结合并由受体介导内化,完成靶向肿瘤诊治。在本论文中,我们选择了磁性氧化铁纳米材料作为药物载体,构建叶酸受体介导的载药磁性纳米pH敏感靶向给药系统。磁性氧化铁纳米材料由于自身的超顺磁性和良好的生物相容性,既可用作药物载体又是良好的MR成像造影剂,被广泛用于肿瘤的诊断和治疗研究中。本论文内容具体包括以下几个部分:1.回顾肿瘤研究的文献资料,核磁共振成像技术在肿瘤的早期诊断治疗中有重要的作用,钆元素和铁元素分别是T1、T2造影剂的理想材料选择,已有研究证实超顺磁性氧化铁纳米颗粒作为药物载体和MR成像造影剂有很好的应用前景。在本论文的第二章中,我们对氧化铁纳米颗粒Fe304进行修饰改性,通过溶剂热降解反应制备出钆掺杂氧化铁纳米材料GFON,由于钆离子的引入,能够有效提高磁共振成像的T1和T2信号,是良好的T1-T2双模式造影剂。阿霉素(Doxorubicin,DOX)是常用的广谱抗肿瘤药物,化疗效果显著,选择钆铁氧化物GFON做药物载体,通过化学键在该纳米载体表面接载叶酸配体FA和化疗药物阿霉DOX,构建出兼具磁共振成像和抗肿瘤治疗的多功能纳米医药FA-GFON-DOX。材料性能检测显示FA-GFON-DOX纳米颗粒具有良好的MR成像性能和pH敏感的药物释放性能;通过MTT法、流式细胞术、共聚焦实验等细胞实验验证了该纳米探针可以导致HeLa细胞、SCC3细胞的的凋亡或坏死,对正常的牙周膜干细胞PDLSC细胞毒性小;可以特异性识别并靶向进入HeLa细胞、SCC3细胞内累积。2.体内环境的复杂性,是阻碍许多纳米药物用于肿瘤治疗的限制因素。为了验证FA-GFON-DOX多功能纳米给药系统对在体肿瘤的成像增强和抑制效果,我们利用BALB/c免疫缺陷裸鼠分别建立了人宫颈癌HeLa细胞系、口腔鳞癌SCC3细胞系的肿瘤异种移植模型,进行了体内实验研究。通过荷瘤裸鼠的MR成像实验证实FA-GFON-DOX纳米探针可以显著增强在体肿瘤的MR成像效果;荷瘤裸鼠体内药代动力学分析的的结果显示,FA-GFON-DOX纳米药物可以很好的抑制宫颈癌、口腔鳞癌异种移植肿瘤的浸润生长,减少化疗药物DOX对机体的毒副作用,提高了化疗效果。3.口腔鳞癌的发病率在常见恶性肿瘤中排名第六位,由于肿瘤所在位置的特殊性很难做到早期诊断治疗。化疗是口腔鳞癌的主要治疗手段之一,顺铂(Cisplatin,CDDP)是口腔鳞癌化疗中使用最多的药物,但是顺铂具有明显的肝肾毒性、骨髓抑制等副作用。已有研究表明叶酸受体在鳞状细胞癌中高度表达,因此可以选择叶酸受体作为口腔鳞癌靶向治疗的靶点。在本论文的第四章,我们致力于寻找一种新型的多功能纳米探针用于口腔鳞癌的靶向诊断和治疗。在第二章中,已经通过实验证实钆铁氧化物纳米材料是良好的药物载体和MR成像造影剂,在本论文的第四章,通过对纳米材料的进一步修饰改性制备出多孔核壳结构的钆铁氧化物纳米颗粒CSGFN用作药物载体,多孔核壳结构由于比表面积高、孔隙率高可装载更多的药物。通过化学反应接载叶酸配体FA和抗肿瘤药物顺铂CDDP到该纳米载体上,得到叶酸受体介导的的载顺铂磁性纳米pH敏感靶向给药系统FA-CSGFN-CDDP用于口腔鳞癌的诊断和治疗。体外实验结果显示,顺铂药物CDDP的释放是嗜酸性、pH敏感依赖型;细胞毒理实验的结果验证了它对口腔鳞癌SCC3细胞毒性大,流式细胞术、共聚焦实验验证了该纳米探针可以特异性识别并进入SCC3细胞累积;我们使用BALB/c免疫缺陷裸鼠建立了口腔鳞癌SCC3细胞系的肿瘤模型,荷瘤裸鼠的MR成像实验表明该纳米探针可以显著增强在体肿瘤的成像效果;药物代谢实验的结果说明FA-CSGFN-CDDP可以有效抑制SCC3细胞的异常增殖,控制口腔鳞癌的体内浸润发展,同时降低顺铂药物的副作用。纳米载体的使用,诊疗一体化概念的推出,使纳米医药朝着多功能诊断治疗的方向发展。可以预测叶酸受体介导的磁性纳米靶向给药系统在肿瘤的MRI诊断和靶向治疗方面具有巨大的应用前景,使肿瘤的可视化诊治、个性化治疗成为可能。
[Abstract]:From a worldwide perspective, tumor, especially malignant tumors which threaten human health, how to effectively treat tumor, improve the quality of life of patients is an urgent problem in the field of cancer research. Early diagnosis and treatment of tumors to improve the cure rate of cancer is very important. The diagnosis and treatment of tumor is the traditional two independent process separate, early cancer was found to be difficult, often need the help of imaging diagnosis. Magnetic resonance imaging MRI is an advanced medical imaging technique is widely used in the early diagnosis of cancer, clinical magnetic resonance imaging more than 50% to contrast agent, but the clinical commonly used gadolinium based contrast agent has poor targeting side effects, liver and kidney, defects such as short retention time in vivo, seriously restricts its clinical application. The treatment of cancer includes surgery, radiotherapy, chemotherapy and other treatment, The chemical medicine is considered to be the most effective treatment in clinic at present. However, the traditional chemotherapy drugs are selective tissue, drug toxicity, tumor multidrug resistance and other shortcomings, has seriously affected the effect of chemotherapy. In recent years, the tumor "theranostics" concept was proposed, aimed at diagnosis and treatment combination and in improving the drug effect, reduce the side effects of drugs at the same time, to monitor the lesion development and treatment effect, adjust treatment, avoid the treatment of disease is insufficient or excessive treatment. With the rapid development of nanotechnology, combined with biomedical nano medicine has become a hot research topic, nano materials with special physical and chemical properties easy, surface modification and functional design, was used as a drug carrier. In this paper, the diagnosis and treatment of cancer as a starting point, to construct nano materials as the carrier of the swollen The diagnosis and treatment of tumors. Using special integrated platform of nano size and structure, the modification of nano carrier, the antitumor drugs and MR imaging contrast agent to pick up a nano carrier, while the completion of drug delivery and MR imaging, to achieve the integration of multi functions. The normal group is neutral environment pH about 7.4 of the tumor tissue is weak acidic microenvironment, the physiological differences can design a pH sensitive nano drug delivery system, pH controlled release drug. Tumor cells often specific expression of some tumor markers such as folate receptor (Folate Receptor, FR), folate receptor targeted therapy is the most commonly used. Nano drug delivery system by coupling (Folic Acid, FA) of folic acid, can specifically recognize tumor cells, combined with folate receptor specificity and by receptor-mediated internalization, complete targeting tumor diagnosis and treatment. In this paper, we choose Magnetic iron oxide nano materials as drug carrier, construction of drug loaded magnetic nanoparticles of pH sensitive target folate receptor mediated drug delivery system. The magnetic iron oxide nano materials because of their superparamagnetic magnetic compatibility and good biocompatibility, which can be used as a drug carrier is good MR imaging contrast agent, is widely used in research and diagnosis the treatment of tumor. The content of this thesis includes the following parts: 1. review of tumor research literature, magnetic resonance imaging at the early stage of the tumor plays an important role in diagnosis and treatment of GD element and iron element are T1, ideal material selection T2 contrast agent, studies have confirmed superparamagnetic iron oxide nanoparticles as a drug carrier and MR imaging contrast agent has very good application prospects. In the second chapter of this thesis, we modified the iron oxide nanoparticles Fe304 by solvent thermal degradation Prepared by the reaction of GD doped iron oxide nano material GFON, due to the introduction of gadolinium ions, can effectively improve the magnetic resonance imaging of the T1 and T2 signals, is a good T1-T2 dual mode contrast agent. Adriamycin (Doxorubicin, DOX) is a commonly used broad-spectrum anti-tumor drugs, chemotherapy effect, selection of gadolinium iron oxide GFON as drug carrier and by chemical bonds in the nano surface of the carrier carrying folate ligand FA and chemotherapy drugs adriamycin DOX, constructed with magnetic resonance imaging and multifunctional nano medicine FA-GFON-DOX. materials performance testing of anti-tumor therapy showed that FA-GFON-DOX nanoparticles have good imaging performance of MR and pH sensitive drug release properties; by MTT method, flow cytometry, confocal experiment cell experimental results show that the nano probe can lead to HeLa cell, SCC3 cell apoptosis or necrosis, stem cell PDLSC cell toxicity of normal periodontal ligament; Specific recognition and targeted into HeLa cells, SCC3 cells,.2. cumulative complexity of internal environment, hinder many nano drug for tumor therapy and limitation. In order to verify the FA-GFON-DOX multifunctional nano drug delivery system for in vivo imaging enhancement and inhibition effect, we human cervical cancer HeLa cells were established by using BALB/c immunodeficient nude mice, tumor xenograft model of oral squamous cell carcinoma SCC3 cell line, was studied by MR. In vivo imaging experiment in nude mice demonstrated that FA-GFON-DOX nanoparticles can significantly enhance the MR imaging effect in tumor bearing nude mice; pharmacokinetics analysis results showed that FA-GFON-DOX nanoparticles can be very good the inhibition of cervical carcinoma, oral squamous cell carcinoma xenograft tumor invasion, reduce toxicity and side effect of DOX chemotherapy, improve the chemotherapy effect of.3. The incidence rate of oral squamous cell carcinoma in common malignant tumors ranked sixth, due to the special location of the tumor is difficult to achieve the early diagnosis and therapy. Chemotherapy is the major treatment of oral squamous cell carcinoma, cisplatin (Cisplatin, CDDP) is the most used drug in the chemotherapy of oral squamous cell carcinoma, but cisplatin has obvious liver and kidney toxicity, bone marrow inhibition of other side effects. Studies have shown that high expression of folate receptor in squamous cell carcinoma, so we can choose the folate receptor as oral squamous cell carcinoma targeted therapy targets. In the fourth chapter, we are committed to finding a new multifunctional nano probe for oral squamous cell carcinoma targeted in the diagnosis and treatment. In the second chapter, has been confirmed by gadolinium iron oxide nano materials is a good drug carrier and MR imaging contrast agent, in the fourth chapter, based on the further modification of nano materials Preparation of gadolinium iron oxide nano particles of CSGFN porous core-shell structure used as a drug carrier was modified, the porous shell structure with high specific surface area, porosity and high loading more drugs. Through chemical reaction to pick up folate ligand FA and anticancer drugs cisplatin CDDP to the nanometer carrier, get folate receptor mediated cisplatin loaded magnetic nano pH sensitive targeting drug delivery system of FA-CSGFN-CDDP for the diagnosis and treatment of oral squamous cell carcinoma in vitro. The experimental results show that cisplatin CDDP release is eosinophilic, pH sensitive type; cell toxicity test results show its high toxicity on oral squamous cell carcinoma SCC3 cells, flow cytometry, confocal experiment the nano probe can specifically recognize and enter the SCC3 cell accumulation; we use the BALB/c tumor model in immunodeficient athymic mice established oral squamous cell carcinoma SCC3 cell line in nude mice, MR like. The results show that the nanoparticles can significantly enhance the imaging effect of in vivo drug metabolism; experimental results show that FA-CSGFN-CDDP can effectively inhibit the proliferation of SCC3 cells, control of oral squamous cell carcinoma infiltration in vivo development, while reducing the side effects of cisplatin. The use of nano carrier, and launched the concept of integration, the nano medicine towards multi the function of diagnosis and treatment direction. Magnetic nanoparticles can predict target folate receptor mediated drug delivery system in MRI diagnosis and targeted therapy of tumor has great application prospect, the visualization of the tumor diagnosis, individualized treatment has become possible.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R73-3;TB383.1

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