基于无机纳米粒子构建新型基因载体及其生物学效应研究

发布时间：2018-10-24 11:59

【摘要】：基因治疗作为一种全新的、革命性的治疗手段，在过去几十年取得了巨大发展，成为治疗疾病的新技术，越来越受到关注。而基因治疗的关键在于如何构建生物安全性好、基因递送效率高的输运载体。金纳米笼（Au NCs）具有特殊的局域表面等离子体共振(LSPR)现象，可吸收近红外波段(700～1200nm)的光波，将光能转换为热能，可实现对疾病的热疗效应。聚乙烯亚胺(PEI)分子上含有大量氨基基团而显正电性，可负载负电性基因实现目标基因的递送，从而达到基因治疗的效果。基于此，本研究通过制备银纳米立方体，经氯金酸刻蚀得到金纳米笼（AuNCs）；将PEI进行巯基化修饰，通过Au-S键将PEI锚定到金纳米笼表面，得到一种新型的基因载体(AuNCs-PEI)，并初步研究了该载体对质粒DNA的运载效果。结果如下：1、银纳米立方体及金纳米笼的制备及表征通过控制硫化钠的浓度、滴加量，及通入体系的氩气速率，得到边长为45nm的单分散银纳米立方体颗粒，其形貌单一且粒径均匀；同时控制氯金酸的滴加体积和浓度，可制得LSPR峰在810nm处的金纳米笼。2、AuNCs-PEI的构建及其运载基因效果的研究将巯基化修饰的1.8KDa PEI和10KDa PEI，与AuNCs表面的PVP进行配体交换，得到相应的基因载体，分别记为AuNCs-PEI(1.8)和AuNCs-PEI(10)。采用波长为800nm的飞秒激光器照射AuNCs-PEI分散液，可检测到分散液的温度明显上升，说明该载体具有明显的热效应。凝胶阻滞实验结果显示，AuNCs-PEI与EGFP(绿色荧光蛋白质粒)质量比(Au/DNA)高于1：1时，可将DNA全部结合到载体上；MTT毒性检测结果显示，AuNCs-PEI (10)在Au/DNA质量比为15时，293T细胞仍保持较高的活力(85%)，同时AuNCs-PEI (1.8)处理的细胞活力与对照组相当，说明二者均具有较高的生物相容性；倒置荧光显微镜及流式细胞仪检测结果发现，AuNCs-PEI (10)在与DNA质量比为15时，，细胞转染效率可达15%以上，说明AuNCs-PEI (10)载体具有一定基因递送能力。因此，AuNCs-PEI(10)在基因治疗相关疾病方面，具有一定的应用前景。此外，为阐明特殊形貌(如方形、棒状、线型等)的纳米银颗粒在印刷电子领域可能存在的应用前景，我们比较了银纳米立方体与同粒径球形银纳米颗粒的导电性，结果显示采用球形银纳米颗粒印制的导电图样，其电导率高于银纳米立方体的电导率。另外，由于球形银纳米颗粒合成方法更为简单，且可大批量合成；而银纳米立方体合成条件苛刻，批次产量小。说明基于银纳米立方体制备的导电银浆实现实际应用较为困难。
[Abstract]:Gene therapy, as a new and revolutionary therapy, has made great progress in the past few decades, and has become a new technology in the treatment of diseases, which has attracted more and more attention. The key of gene therapy is how to construct transport vector with good biological safety and high gene delivery efficiency. Gold nano-cage (Au NCs) has a special local surface plasmon resonance (LSPR) phenomenon. It can absorb the near infrared (700~1200nm) light wave and convert the light energy into heat energy, which can realize the effect of hyperthermia on disease. Polyethyleneimide (PEI) molecule contains a large number of amino groups and is positively charged, which can load negative electricity gene to realize the delivery of the target gene, thus achieving the effect of gene therapy. Based on this, the silver nanocrystals were prepared, and the gold nanocages (AuNCs); was prepared by chlorauric acid etching. The PEI was modified by sulfhydrylation, and PEI was anchored to the surface of the gold nanowires by Au-S bond. A novel gene vector (AuNCs-PEI) was obtained, and the effect of the vector on carrying plasmid DNA was preliminarily studied. The results are as follows: 1. The preparation and characterization of silver nanocrystals and gold nanometers were studied. By controlling the concentration of sodium sulphide, the amount of drops and the rate of argon gas flowing into the system, the monodisperse silver nanocrystalline particles with side length of 45nm were obtained. Its morphology is single and its particle size is uniform. At the same time, the drop volume and concentration of chlorgold acid are controlled. The construction of AuNCs-PEI with LSPR peak at 810nm and its effect on gene delivery were studied. The ligand exchange of mercapto-modified 1.8KDa PEI and 10KDa PEI, with PVP on the surface of AuNCs was carried out, and the corresponding gene vectors were recorded as AuNCs-PEI (1.8) and AuNCs-PEI (10), respectively. Using femtosecond laser with wavelength of 800nm to irradiate AuNCs-PEI dispersions, the temperature of the dispersions can be detected to rise obviously, which indicates that the carrier has obvious thermal effect. The results of gel block test showed that when the mass ratio of AuNCs-PEI and EGFP (green fluorescent protein particle) (Au/DNA) was higher than 1:1, all DNA could be bound to the carrier. The results of MTT toxicity test showed that 293T cells maintained high activity (85%) when the Au/DNA mass ratio was 15:00, and the cell viability of AuNCs-PEI (1.8) was similar to that of the control group, indicating that both of them had high biocompatibility. The results of reverse fluorescence microscopy and flow cytometry showed that the transfection efficiency of AuNCs-PEI _ (10) was more than 15% when the mass ratio of AuNCs-PEI _ (10) to DNA was 15:00, indicating that the AuNCs-PEI _ (10) vector had the ability of gene delivery. Therefore, AuNCs-PEI (10) has a certain application prospect in gene therapy related diseases. In addition, in order to elucidate the potential applications of silver nanoparticles with special morphologies (such as square, rod, line, etc.) in the field of printed electronics, we compared the electrical conductivity of silver nanometers with spherical silver nanoparticles of the same size. The results show that the electrical conductivity of spherical silver nanoparticles is higher than that of silver nanocrystals. In addition, the synthesis method of spherical silver nanoparticles is simpler and can be synthesized in large quantities, while the synthesis conditions of silver nanocrystals are harsh and the batch yields are small. It is difficult to realize the practical application of conductive silver paste based on silver nano cube.
【学位授予单位】：北京印刷学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TB383.1

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