新型荧光纳米材料碳点的毒性及作用机制研究

发布时间：2018-03-26 01:01

本文选题：碳点　切入点：急性毒性　出处：《中国人民解放军军事医学科学院》2014年博士论文

【摘要】：将纳米材料用于生物标记与诊断成像，是当前纳米科技应用于肿瘤医学新兴的研究领域，如何应用纳米材料独特的小尺寸效应、表面效应、量子效应、光、电、磁效应等性质来设计生物相容性良好的肿瘤诊断分子探针，提高肿瘤标志物检测的特异性与灵敏度，是目前肿瘤医学对纳米科技提出的极具挑战性的问题。纳米材料要转化为医药产品应用于临床，其生物安全性是一个必须解决的关键问题。迄今为止，国际上尚未制定出一套用于纳米材料评价特定的指导原则。多数纳米医药制品的安全评价依然采用常规毒理学的评价指导原则。已有的研究表明，纳米材料的尺寸形状、表面电荷及其表面修饰等性质均会对其生物学行为与生物效应产生影响。近年来一些纳米医药产品，经常规毒理学评价定为无毒，但在使用过程中却因为检测到新的毒作用而被美国FDA撤出临床。常规毒理学越来越不能适应对纳米生物医药制品安全评价的要求。制定一套客观并能实时监测纳米颗粒在暴露过程中对机体可能得毒性作用的评价方法迫在眉睫，纳米毒理学所肩负的任务任重而道远。碳点是一种新型的荧光碳纳米颗粒，，其粒径一般在10nm以下，具有良好的分散性和荧光稳定性。这种碳纳米材料具有传统荧光染料所不具备的诸多优良性质，如：荧光强度高、抗光漂白能力强、激发光谱连续等。此外，碳点具有很好的水溶性，其表面的羧基易于进行生物偶联与功能化修饰。碳点本身不含有任何有毒元素，与半导体量子点相比，展现出卓越的生物兼容性和环境友好性鉴于上述诸多优良的理化性质，碳点极具制备肿瘤诊断探针的潜质，但是目前针对于碳点安全性评价的研究却非常稀少，评价内容也不全面、不系统，对碳点可能的毒性作用机制更是缺乏深入的研究。本课题承接了国家973项目——“应用纳米技术解决胃癌预警与早期诊断中的关键科学问题”中碳点（一种用于标记胃癌早期诊断探针的纳米材料）对生物体的毒理效应及其作用机制的评价研究任务，我们以国家食品药品监督管理局药物评价指南为基础和指导，参照国际化学物毒理学检测指南对碳点的生物学效应和致毒机制进行了系统性评价。研究内容包括： (1)碳点的理化性质表征。采用原子力显微镜、紫外可见光谱仪、荧光光度计等仪器分别对硝酸氧化法制备碳点的形貌、粒径、吸收光谱、荧光光谱及荧光量子产率等参数进行了表征。 (2)碳点的急性、亚急性毒性研究。分别选用BALB/C小鼠和Wistar大鼠，一次性尾静脉注射染毒，记录了受试动物在给药后不同时间点一般状况、食物利用率、体重、血液学、血清生化指标的变化及主要脏器的病理改变。 (3)碳点的遗传毒性评价。选用沙门氏菌回复突变试验（受试菌株为鼠伤寒沙门氏菌TA97、TA98、TA100、TA102）、染色体畸变试验、碱性单细胞凝胶电泳试验及骨髓微核试验四个组合试验对碳点的致基因突变、染色体畸变和DNA损伤效应进行了测定。 (4)碳点对免疫功能的影响。选用BALB/C小鼠作为模式动物，以尾静脉单次及多次注射的方式染毒，在给药后多个时间点，采用脾细胞增殖试验、淋巴细胞种类及亚群分析、巨噬细胞中性红吞噬试验、免疫相关细胞因子分泌及表达水平检测、免疫器官病理形态分析等方法分别研究了碳点对小鼠巨噬细胞功能、T淋巴细胞与B淋巴细胞增殖活性及其介导的免疫功能和相关免疫器官病理变化的影响。试验结果如下： (1)碳点的理化性质表征结果表明，碳点的颗粒直径在5～10nm，紫外、红外光谱证实了碳点表面富含羧基和羟基基团，从结构上阐明了碳点具有很好的水溶性。荧光光谱分析表明碳点随着激发光的不同可以发不同颜色的荧光，裸碳点在340nm激发，可以发射强的蓝色荧光，其荧光量子产率在0.85%左右。 (2)碳点以单次静脉注射方式染毒对模式动物没有明显的急性、亚急性毒性。动物的一般状况良好；食物利用率、体重、血液学、血清生化指标均没有出现异常变化；主要脏器无明显的病理改变。 (3)遗传毒性组合试验评价结果显示，碳点在受试剂量内不能诱导鼠伤寒沙门氏菌发生突变，对CHL细胞的染色体无致畸变作用，无明显的DNA损伤效应，不能诱发受试动物的骨髓红细胞发生微核突变，说明碳点无明显的遗传毒性。 (4)免疫毒性研究结果表明，碳点尾静脉注射单次染毒，在暴露早期，巨噬细胞被激活功能增强， IFN-γ等细胞因子的分泌水平增加，但未见淋巴细胞种群与数量的显著变化，未诱发明显的免疫效应；但在染毒后第9天，碳点能显著促进T淋巴细胞的活化增殖，提高IFN-γ与TNF-的分泌水平，增强Th1细胞介导的免疫效应。碳点多次染毒，T淋巴细胞亦出现明显增殖，IFN-γ与TNF-相关细胞因子分泌水平增加，且在基因水平的表达上调。碳点的持续暴露作用诱发了机体的氧化应激损伤，破坏了机体的免疫平衡状态，导致相关靶器官出现了可代偿的病理病变。本论文立题于当前极具挑战的纳米材料安全性研究方向，通过一系列分子、细胞和动物水平的试验，首次对碳点的生物安全性做了较系统、全面地评价。一般毒性与遗传毒性评价结果表明碳点在受试剂量内没有明显的毒性作用；免疫毒性研究结果显示，碳点在单次染毒时可以促进Th1细胞介导的免疫效应，但在多次给药的情况下，碳点可诱发机体相关免疫器官发生可代偿的病理病变。上述试验结果为碳点在生物医药领域及临床上的应用奠定了安全性实验基础。
[Abstract]:The nano materials for biomarkers and diagnostic imaging, the application of nanometer technology in cancer medicine new research field, how to use the small size effect, unique nano material surface effect, quantum effect, optical, electrical, magnetic effect, biocompatibility properties to design good molecular probe to improve tumor diagnosis, tumor marker specific and the sensitivity of detection, is the medical oncology of nano technology put forward a challenge.
Nano materials for conversion of pharmaceutical products in clinical application, the biological safety is a key problem that must be solved. So far, the world has yet to develop a set of guidelines for the specific evaluation of nanometer materials. Most of the safety evaluation of pharmaceutical products is still using nano evaluation routine toxicology studies have shown that the guiding principle. The shape, size of nano materials, surface charge and surface modification properties will have an impact on the biological behavior and biological effects. In recent years, some nano pharmaceutical products, routine toxicological evaluation is non-toxic, but in the use of the process but because the detected toxic action by the United States and the new FDA clinical routine toxicology and withdrawal. Can not adapt to the evaluation of nano bio pharmaceutical products safety requirements. Develop a set of objective and real-time monitoring of nanoparticles may on the body during their exposure The evaluation of toxic effects is imminent, and the task of nanotoxicology is a long way to go.
Carbon is a new type of fluorescent carbon nanoparticles, the particle size is generally less than 10nm, with good dispersibility and stability of fluorescence. The carbon nano material has many excellent properties, the traditional fluorescent dyes do not have such as high fluorescence intensity, anti photobleaching ability, luminescence spectrum. In addition carbon, good water solubility, the surface carboxyl easy bioconjugation with functionalization. Carbon itself does not contain any toxic elements, compared with the semiconductor quantum dots exhibit excellent biological compatibility and environmental friendliness in view of many physical and chemical properties of the excellent, very little carbon preparation tumor diagnosis probe potential, but the current research for carbon safety evaluation of needle is very scarce, the evaluation content is not comprehensive, systematic, toxic effects on the mechanism of carbon potential is the lack of in-depth research.
This topic to undertake a national 973 project, "the application of nanotechnology to solve key scientific problems in early warning and early diagnosis of gastric cancer (a point in carbon nano materials used for early diagnosis of gastric cancer probe evaluation of toxicological effects on organisms) and the mechanism of the task, we use the State Food and drug Administration for drug evaluation guide for reference and reference to the biological effects of international guidelines on chemical toxicology testing point carbon and the toxic mechanism of the system of evaluation.
The research contents include:
(1) physical and chemical characterization of carbon dots. The morphology, particle size, absorption spectrum, fluorescence spectrum and fluorescence quantum yield of carbon dots were characterized by atomic force microscopy, UV VIS spectrometer and fluorometer.
(2) acute point carbon, subacute toxicity study. We used BALB/C mice and Wistar rats, intravenous injection were recorded in animal subjects administered at different time points after the general situation, food utilization rate, body weight, hematology, pathological changes of serum biochemical indexes and main organs.
(3) the genetic toxicity evaluation of carbon dots. Selected Salmonella reverse mutation test (test strains of Salmonella typhimurium TA97, TA98, TA100, TA102), chromosome aberration test, single cell gel electrophoresis test and micronucleus test of four combination test of carbon point gene mutation, chromosome aberration and DNA damage was measured.
(4) effects of carbon on the immune function. BALB/C mice were used as model animal, with tail vein single and multiple injection way exposure, after the administration of multiple time points, the spleen cell proliferation assay, cell type and subgroup analysis, macrophage phagocytosis of neutral red test, immune related cytokines and the expression level of detection, immune organ pathological analysis and other methods were studied carbon on macrophage function, affect the immune function of T lymphocytes and B lymphocytes proliferation and its mediated immune organs and related pathological changes.
The results of the test are as follows:
(1) characterization results show that carbon carbon, the particle diameter of 5 ~ 10nm, ultraviolet, infrared spectroscopy confirmed that the carbon rich surface carboxyl and hydroxyl groups from the structure to clarify the point carbon has good water solubility. The fluorescence spectrum analysis showed that carbon dots with fluorescence excitation can be different color emitting different, bare carbon at 340nm excitation, can emit strong blue fluorescence, the fluorescence quantum yield of around 0.85%.
(2) a single intravenous injection of carbon dots did not show acute and subacute toxicity to the model animals. The general condition of the animals was good. There was no abnormal change in food utilization, body weight, hematology and serum biochemical indexes. There was no obvious pathological change in the main organs.
(3) evaluate the genetic toxicity of combined test results show that the carbon in dose not induced by Salmonella typhimurium mutation on CHL cell chromosome aberration free, no obvious effect of DNA damage, unable to induce animal subjects bone marrow erythrocyte micronucleus mutation occurred, indicating no obvious genotoxicity of carbon.
(4) the immune toxicity results showed that carbon intravenous injection of single exposure, in the early exposure, enhance the function of macrophages are activated, the secretion level of IFN- gamma and other cytokines increased, but no significant changes in population and the number of lymphocytes, did not induce obvious immune effect; but in ninth days after exposure, carbon can significantly promote the proliferation and activation of T lymphocytes, improve the secretion level of IFN- gamma and TNF-, enhance the immune effect of Th1 cells mediated by carbon. Multiple exposure, T also appeared obvious lymphocyte proliferation, IFN- gamma and TNF- related cytokines secretion increased, and the level of expression of up-regulated genes. The effect of continuous exposure of carbon induced the oxidative stress injury, damage the immune balance of the body, resulting in target organs appeared pathological lesions can be compensated.
The direction of research on safety of nano materials to choose this subject to the current challenges, through a series of molecular, cellular and animal level test, for the first time the biological safety of carbon point to do a more comprehensive evaluation system, evaluation of general toxicity and genetic toxicity. The results indicate that the carbon in the subjects had no obvious toxicity dose; immune toxicity results showed that carbon can promote the immune response mediated by Th1 cells in a single exposure, but in the case of multiple dosing, carbon induced immune organ pathological lesions can be compensated. The results lay a foundation for the application of safety experiment in carbon the biological medicine field and clinic.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114

【相似文献】