构建透明质酸功能化三氧化二铋纳米颗粒用于肿瘤靶向成像及其放疗增敏研究

发布时间：2017-12-28 23:05

本文关键词：构建透明质酸功能化三氧化二铋纳米颗粒用于肿瘤靶向成像及其放疗增敏研究　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:恶性肿瘤是一种难治性疾病,严重威胁到人类的健康最。放射治疗是目前临床上治疗恶性肿瘤最常用的手段之一。然而,由于影像学在肿瘤类疾病的诊断及定位上的局限性,临床治疗时常出现放射治疗的剂量未达肿瘤组织的致死剂量、定位不够精确而对周围正常组织造成放疗损伤,从而引起放疗副反应;同时由于各类肿瘤组织自身存在着不同程度的放疗抗性,降低了肿瘤细胞对放射治疗的敏感性,从而进一步削弱了放疗的临床治疗效果。为了克服在肿瘤类疾病的诊疗过程中所出现的影像学诊断误差及病灶组织的放疗抵抗,本课题旨在利用水热聚醇法构建一种多功能的新型纳米颗粒—透明质酸功能化三氧化二铋纳米颗粒(HA-Bi_2O_3 NPs),用于肿瘤类疾病的靶向性CT造影成像引导下的放疗增敏。方法:利用透明质酸钠、六水氯化铋(Bi Cl3·6H2O)为前驱物通过水热聚醇法制备HA-Bi_2O_3 NPs;通过透射电子显微镜/高分辨透射电子显微镜(TEM/HR-TEM)、傅氏转换红外线光谱分析仪(FT-IR)、Nano DLS高敏度粒径分析仪、X射线光电子能谱学分析仪(XPS)和X射线衍射仪(XRD)揭示HA-Bi_2O_3的理化特性;通过流式细胞术、半定量PCR和细胞成像来研究HA介导的HA-Bi_2O_3 NPs细胞吞噬行为;利用细胞增值率检测、溶血实验、病理切片和电感耦合等离子体质谱(ICP-MS)来分析HA-Bi_2O_3 NPs的生物相容性;利用ICR小鼠皮下肿瘤模型CT平扫检测HA-Bi_2O_3 NPs的肿瘤靶向性CT成像;利用细胞增值率检测、克隆形成实验、活-死细胞染色、流式细胞术(凋亡与周期)、ICR小鼠皮下肿瘤模型体内实验检测HA-Bi_2O_3 NPs的放疗增敏作用。结果:1.HA-Bi_2O_3 NPs的制备及表征利用水热聚醇法成功制备HA-Bi_2O_3 NPs,Nano DLS高敏度粒径分析仪显示HA-Bi_2O_3 NPs粒径分布均匀,大小约为45nm,在PBS缓冲液中保存8天后HA-Bi_2O_3 NPs粒径大小无明显变化;TEM/HR-TEM提示HA-Bi_2O_3 NPs的水溶性、分散性好,粒径大小均一,晶格条纹明显,晶面间距为3.3±0.2?;XRD显示HA-Bi_2O_3 NPs的物相结构符合Bi_2O_3的特征,为多相异质结构。FT-IR提示HA-Bi_2O_3 NPs,表面具有羧基、羟基、羰基等亲水基团;XPS提示HA-Bi_2O_3 NPs主要是由铋、氧、碳等元素组成。2.透明质酸(HA)介导的HA-Bi_2O_3 NPs的细胞吞噬行为通过定量PCR提示CD44抗原的表达在人肝癌细胞株SMMC-7721中比在人乳腺癌细胞株MCF7细胞高,与相关文献的报道相一致;利用流式细胞术揭示HA-Bi_2O_3 NPs可被SMMC-7721细胞和MCF7细胞吞噬,但在CD44抗原高表达的SMMC-7721细胞中,HA-Bi_2O_3 NPs的吞噬效率较CD44抗原低表达的MCF7细胞高。3.HA-Bi_2O_3 NPs的生物相容性的表征检测细胞的增殖抑制率显示,不同浓度的HA-Bi_2O_3 NPs(0-400μg/m L)对SMMC-7721、VSMCs和MCF7的细胞活性均无明显影响(p0.05);溶血实验提示,不同浓度的HA-Bi_2O_3 NPs(0-400μg/ml)均不引起明显的溶血反应;ICP-MS显示,ICR小鼠经尾静脉注射HA-Bi_2O_3 NPs后,心、肝、脾、肺、肾对HA-Bi_2O_3 NPs均有摄取,其中以肝脏(摄取量:27.80mg/g)和肾脏(摄取量:7.40mg/g)对HA-Bi_2O_3 NPs的摄取为主;组织切片提示HA-Bi_2O_3 NPs对小鼠重要脏器无明显毒性,未引起明显的病理变化。4.HA-Bi_2O_3 NPs在肿瘤CT靶向性成像的研究体外CT成像结果提示相较于已用于临床的CT造影剂碘海醇,HA-Bi_2O_3 NPs具有更加优秀的CT造影成像效果。皮下荷瘤小鼠经尾静脉注射HA-Bi_2O_3 NPs,10min后小鼠各脏器及皮下肿瘤的CT造影信号均有所增强,其中肾脏、膀胱及皮下肿瘤处的信号增强尤为明显;30min后,肾脏的信号强度开始下降而膀胱及皮下肿瘤的信号增强仍十分明显。5.HA-Bi_2O_3 NPs的放疗增敏效果的实验研究单纯HA-Bi_2O_3 NPs对SMMC-7721细胞的增殖率的无显著影响,而当HA-Bi_2O_3 NPs联合放射治疗时,则明显抑制了SMMC-7721细胞的增殖。随着提高HA-Bi_2O_3 NPs的浓度或加大放射治疗的强度,SMMC-7721细胞增殖率所受到的抑制越发明显,与剂量呈正相关性,尤其是在400μg/m L浓度的HA-Bi_2O_3 NPs联合9Gy的放疗剂量时,SMMC-7721细胞的增值率显著降低至23.5%(p0.05);克隆形成实验提示,单纯HA-Bi_2O_3 NPs组与对照组的细胞增值率无明显差异,单纯放疗组在6Gy的放疗剂量下,细胞增值率为46.7%,而实验组在6Gy的放疗剂量及200μg/m L浓度的HA-Bi_2O_3 NPs下,细胞增值率显著降低至29.8%;活-死细胞染色实验中,相较于对照组、单纯HA-Bi_2O_3 NPs组和单纯放疗组,HA-Bi_2O_3 NPs联合放疗组中可被FDA染色(绿色)的存活细胞明显减少,并与HA-Bi_2O_3 NPs浓度成负相关,可被PI染色(红色)的凋亡细胞明显增多,并与HA-Bi_2O_3 NPs浓度成正相关;如流式细胞术(凋亡)所示,对照度及单纯HA-Bi_2O_3 NPs组中的早期凋亡率及晚期凋亡率无明显差异,单纯放疗组中,早期凋亡率升高至30.4%,晚期凋亡率升至5.89%,而实验组在6Gy的放疗剂量联合200μg/m L浓度的HA-Bi_2O_3 NPs下,早期和晚期凋亡率均明显升高,分别达到41.2%和14.1%;流式细胞术(周期)提示,相比于对照组与单纯HA-Bi_2O_3 NPs组,单纯放疗组(6Gy)引起G2/M期阻滞(19.87%),Sub-G1期峰值升高至2.34%,实验组在6Gy的放疗剂量下,随着HA-Bi_2O_3 NPs浓度的升高,G2/M期阻滞越发明显、Sub-G1期峰值逐渐升高,特别是HA-Bi_2O_3 NPs的浓度为200μg/m L时,G2/M期升高为33.00%,Sub-G1期峰值为21.59%;ICR小鼠体内实验提示,对照组和单纯HA-Bi_2O_3 NPs组荷瘤小鼠的皮下肿瘤体积在10d时增长至270%,生存曲线提示对照组和单纯HA-Bi_2O_3 NPs组小鼠的存活时间中位数分别为为17d和20d;单纯放疗组的肿瘤体积增长受到抑制,在10d时缩小了约14.6%,存活时间中位数为24d;HA-Bi_2O_3 NPs联合放射治疗则明显抑制了肿瘤生长,第10d时肿瘤体积缩小了33.9%,存活时间中位数为35d。结论:1.本研究以六水氯化铋作为铋源材料,利用透明质酸进行修饰,通过水热聚醇法成功构建了HA-Bi_2O_3 NPs。2.HA-Bi_2O_3 NPs的水溶性、分散性和稳定性良好,粒径大小均一;物相结构为多层异质性,晶格条纹明显,晶格间距为3.3±0.2?,符合Bi_2O_3的特征;HA-Bi_2O_3 NPs主要是由铋、氧、碳等元素组成,颗粒表面官能团主要为羧基、甲基、羰基等;生物相容性良好。3.HA-Bi_2O_3 NPs相较于已用于临床的CT造影剂碘海醇,具有更加优秀的CT造影成像效果,同时,纳米材料具有EPR效应和体内滞留时间长等特性,使得HA-Bi_2O_3 NPs可以用于肿瘤靶向性影像诊断。4.HA-Bi_2O_3 NPs可以增强放射射线对肿瘤的抑制和杀伤作用,具有成为临床放疗增敏剂的潜力。
[Abstract]:Objective: malignant tumor is a refractory disease, which is a serious threat to human health. Radiation therapy is one of the most commonly used methods to treat malignant tumors at present. However, due to limitations in the diagnosis and localization of tumor diseases on imaging, clinical treatment often dose not tumor lethal dose, positioning is not accurate and the surrounding normal tissue caused by radiation damage, causing the side effect of radiotherapy; at the same time, because of many kinds of tumors has different resistance to radiotherapy the degree of reduced tumor cell sensitivity to radiation therapy, so as to further weaken the curative effect of radiotherapy. In order to diagnose errors and radiotherapy lesions appeared overcome in the process of diagnosis and treatment of diseases such as tumor imaging resistance, the purpose of this study is to construct a new nanoparticles by hydrothermal multifunctional polyol method of hyaluronic acid functionalized three oxidation two bismuth nanoparticles (HA-Bi_2O_3 NPs), radiotherapy for cancer diseases the targeted CT imaging guided sensitization. Methods: using chloride sodium hyaluronate, six water (Bi Cl3 6H2O) bismuth precursor by hydrothermal synthesis of HA-Bi_2O_3 NPs poly alcohols; by transmission electron microscopy and high-resolution transmission electron microscopy (TEM/HR-TEM), Fourier transform infrared spectroscopy (FT-IR), Nano DLS high sensitivity particle size analyzer, X ray photoelectron spectroscopy analyzer (XPS) and X ray diffraction (XRD) reveals the physicochemical characteristics of HA-Bi_2O_3; to study the HA mediated HA-Bi_2O_3 NPs cells by semi quantitative PCR and cell imaging and flow cytometry, cell proliferation rate by phagocytic behavior; test, hemolysis test, pathological sections and inductively coupled plasma mass spectrometry (ICP-MS) to analyze the biological compatibility of HA-Bi_2O_3 NPs; tumor using ICR mice subcutaneous tumor model CT scan detection of HA-Bi_2O_3 NPs to CT imaging; rate of detection, clone formation assay, using live cell proliferation Dead cell staining, flow cytometry (apoptosis and cycle), and ICR mice model of subcutaneous tumor were tested in vivo to detect the sensitization effect of HA-Bi_2O_3 NPs. Results: the prepared HA-Bi_2O_3 NPs 1.HA-Bi_2O_3 preparation and characterization of NPs by hydrothermal polyol method, Nano DLS high sensitivity HA-Bi_2O_3 NPs particle size analyzer showed uniform particle size distribution, the size is about 45nm, save 8 days HA-Bi_2O_3 the particle size of NPs had no obvious change in PBS buffer; TEM/HR-TEM HA-Bi_2O_3 NPs water solubility, good dispersibility, uniform particle size, lattice fringe is obvious, the interplanar spacing is 3.3 + 0.2?; XRD display HA-Bi_2O_3 NPs phase structure with the characteristics of Bi_2O_3 multiphase heterogeneous structure. FT-IR indicates HA-Bi_2O_3 NPs, which has hydrophilic groups such as carboxyl, hydroxyl and carbonyl groups; XPS indicates that HA-Bi_2O_3 NPs is mainly composed of bismuth, oxygen, carbon and other elements. 2. hyaluronic acid (HA) expression of HA-Bi_2O_3 mediated by NPs cell phagocytosis behavior showed CD44 antigen by quantitative PCR in human hepatocellular carcinoma cell line SMMC-7721 than in human breast cancer cell line MCF7 cells, consistent with relevant reports; HA-Bi_2O_3 revealed by NPs flow cytometry by SMMC-7721 cells and MCF7 phagocytosis, but high expression of CD44 antigen in SMMC-7721 cells, HA-Bi_2O_3 NPs phagocytosis low expression efficiency compared with CD44 antigen of MCF7 cells. Characterization of 3.HA-Bi_2O_3 cell compatibility of NPs biological inhibition rate showed that different concentrations of HA-Bi_2O_3 NPs (0-400 g/m L) had no significant effect on cell activity of SMMC-7721, VSMCs and MCF7 (P0.05); hemolysis test indicated that different concentrations of HA-Bi_2O_3 (NPs 0-400 g/ml) were not obvious hemolytic reaction; ICP-MS display, ICR mice by tail vein injection of HA-Bi_2O_3 after NPs, heart, liver and spleen, lung and kidney of HA-Bi_2O_3 NPs were among the liver uptake (intake: 27.80mg/g) and kidney (intake: 7.40mg/g) of HA-Bi_2O_3 NPs uptake; tissue sections showed that HA-Bi_2O_3 NPs had no obvious toxicity to the important in mice, did not cause obvious pathological changes. The study of 4.HA-Bi_2O_3 NPs in targeted imaging of tumor CT showed that compared with the CT contrast agent used in clinic, CT imaging showed better CT imaging effect than HA-Bi_2O_3 NPs. Subcutaneous tumor bearing mice by intravenous injection of HA-Bi_2O_3 NPs, CT contrast signals are organs and subcutaneous tumors in mice increased after 10min, including kidney, bladder and subcutaneous tumor signal enhancement at 30min after the start, very obvious; signal intensity decreased and the signal of the bladder and kidney skin tumor enhancement is still very obvious. The effect of 5.HA-Bi_2O_3 NPs on the radiosensitizing effect was studied. HA-Bi_2O_3 NPs alone had no significant effect on the proliferation rate of SMMC-7721 cells, but when HA-Bi_2O_3 NPs combined with radiotherapy, it significantly inhibited the proliferation of SMMC-7721 cells. With the increase of the concentration of HA-Bi_2O_3 NPs or increasing the radiation intensity of treatment, the inhibition rate of SMMC-7721 cell proliferation is more and more obvious, and the dose was positively correlated, especially in the radiation dose 400 g/m L concentration of HA-Bi_2O_3 NPs combined with 9Gy, the added value of SMMC-7721 cells significantly reduced to 23.5% (P0.05); cloning experiments suggest simple, no difference in the rate of HA-Bi_2O_3 in NPs group and control group cell proliferation, radiotherapy group in the radiation dose of 6Gy, cell proliferation rate was 46.7%, while the experimental group in the radiotherapy dose of 6Gy and 200 g/m L concentration of HA-Bi_2O_3 NPs, cell proliferation was significantly reduced to 29.8%; the live - dead cell staining in the experiment, compared with control group, HA-Bi_2O_3 NPs group and simple radiotherapy group, radiotherapy group HA-Bi_2O_3 NPs by FDA staining (green) cell survival was significantly reduced, and the concentration of NPs and HA-Bi_2O_3 negative Related by PI staining (red) the number of apoptotic cells was significantly increased, and positively correlated with HA-Bi_2O_3 concentration of NPs; flow cytometry (apoptosis) in control and HA-Bi_2O_3 alone in group NPs
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730

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