自荧光介孔二氧化硅纳米粒子的制备及其用于多功能药物载体的研究

发布时间：2018-05-31 22:16

  本文选题：自荧光 + 介孔二氧化硅　； 参考：《兰州大学》2016年博士论文

【摘要】：近年来,药物载体作为降低药物毒副作用、改善药物体内分布与代谢、提高药物治疗效率的一种手段,已经成为生物医学领域的研究热点之一。其中,介孔二氧化硅纳米粒子由于其具有有序的介孔结构、比表面积大、生物相容性好、药物负载率高等优点,引起了人们的广泛关注,且被认为是最有前景的药物载体之一。然而,该纳米粒子在实际应用中尚存在一些亟待解决的问题,如在水中的分散性差、易聚集;功能相对单一,现有的修饰方法存在潜在毒性;且各种功能分子之间的分配存在竞争性矛盾;实现不同药物之间的协同作用的方法可控性低等。本论文针对上述问题展开了自荧光介孔二氧化硅纳米粒子的制备、表征及其作为药物载体的应用等研究。本论文制备了一系列自荧光介孔二氧化硅纳米粒子,采用傅立叶变换红外光谱(FTIR)和热重分析(TGA)对介孔二氧化硅纳米粒子的修饰过程进行了监测,采用透射电子显微镜(TEM)对介孔二氧化硅纳米粒子修饰前后的形貌变化及粒径变化进行了观察,通过N2吸附-脱附对介孔二氧化硅纳米粒子修饰前后的孔径、孔容及比表面积进行了测定,采用荧光分光光度计、荧光显微镜及共聚焦荧光显微镜对介孔二氧化硅纳米粒子的荧光性质及荧光成像能力进行了评价,采用四甲基偶氮唑盐微量酶反应比色法(MTT法)初步考察了介孔二氧化硅纳米粒子的体外细胞毒性,通过体外释放实验考察了介孔二氧化硅纳米粒子作为药物载体在刺激响应性药物控制释放行为方面的可行性,具体研究内容及结果如下:(1)采用PAMAM树枝体硅烷偶联剂修饰介孔二氧化硅纳米粒子,制备了具有pH敏感性和自荧光的药物载体,研究结果发现经PAMAM树枝体硅烷偶联剂修饰后,介孔二氧化硅纳米粒子表面含有大量的伯胺基团,有利于进一步进行修饰,引入其他官能团分子,如靶向分子;同时,经PAMAM树枝体硅烷偶联剂修饰后,介孔二氧硅纳米粒子的载药量得到了进一步提高。此外,研究还发现,通过改变溶液的pH值,可以改变PAMAM树枝状分子的构象,进而改变其在粒子外层的密度,实现药物的控制释放,并可以防止药物在中性条件下的突释;荧光发射强度和荧光颜色可随着药物负载率和药物分子的释放而发生变化,从而具有在跟踪或检测方面应用的潜力。研究结果表明,PAMAM树枝体可同时作为ph敏感的封堵剂和荧光标记物。(2)基于硫酸软骨素(cs)和pamam树枝体采用层层自组装的方法制备了多功能介孔二氧化硅纳米粒子药物载体。研究发现,在中性条件下,pamam/cs层层自组装膜可以有效地将药物进行封堵,抑制了介孔中药物的提前释放;该药物载体具有刺激响应性药物控制释放性质,即模型药物阿霉素和姜黄素在中性ph值条件下释放缓慢,在酸性(ph5.5)条件下释放较快;考察了pamam/cs层层自组装膜对粒子分散性质的影响,结果表明,层层自组装膜能使介孔二氧化硅纳米载体具有更好的分散性;通过溶血实验发现层层自组装膜可以有效屏蔽硅羟基与血红细胞膜的相互作用,从而提高该纳米粒子的血液相容性;荧光光谱和荧光显微镜结果初步表明该纳米粒子具有荧光成像的能力;cs的靶向功能可以促进该纳米粒子药物载体进入肿瘤细胞,增强其细胞毒性。(3)采用负载了荧光抗癌药物姜黄素的f127胶束(cf127)作为介孔二氧化硅纳米粒子的封堵剂和荧光标记物,制备了多功能介孔二氧化硅纳米粒子药物载体。研究结果表明,cf127可以作为介孔二氧化硅纳米粒子的封堵剂;两种药物载体(介孔二氧化硅纳米粒子和胶束)的组合提供了一种普适性的设计制备多功能药物载体的方法,可同时实现刺激性控制药物释放和荧光成像功能;体外药物释放实验结果表明,cf127胶束通过席夫碱键键接在介孔二氧化硅纳米粒子表面后,可以实现药物的ph响应性控制释放,这可以归因于席夫碱对ph的敏感性,即在中性条件下,席夫碱键可以较稳定存在,因此可以阻止药物释放,而在ph值为5.5时,席夫碱键断裂,释放药物;荧光实验结果发现,cf127胶束聚集在介孔二氧化硅纳米颗粒表面后,cf127胶束的荧光强度会明显增强,该现象有助于荧光成像;此外,通过考察该载体在连续紫外激发光照射下的荧光强度变化,发现cf127胶束具有良好的抗光漂白性,这些结果显示cf127胶束可作为介孔二氧化硅纳米粒子药物载体的荧光标记物。(4)采用抗癌药物姜黄素分子作为介孔二氧化硅纳米粒子的封堵剂和荧光成像剂,制备了具有自荧光功能和刺激响应性的药物载体(lpcc-c-f127)。研究结果表明,姜黄素封堵剂可以有效地把药物分子保留在介孔里,避免药物分子进入细胞前的提前泄露;体外药物释放实验结果表明,在ph5.5和谷胱甘肽(gsh)存在下,姜黄素上的β-硫酯可以被有效水解,从而使姜黄素分子从介孔上脱落,实现药物的刺激响应性控制释放;采用荧光分光光度计分析后发现,通过多步功能化修饰后,纳米药物载体的荧光强度随着姜黄素封堵剂上姜黄素分子的增加而增强;分散性实验结果表明,f127膜层可以通过聚乙二醇(peg)壳层提高该药物载体的分散性,同时还可创建一个疏水的微环境提高该药物载体的荧光强度;mtt法实验结果表明,所制备的药物载体具有良好的细胞生物相容性。(5)基于姜黄素分子采用一锅法制备了姜黄素聚合物壳封堵的多功能介孔二氧化硅纳米粒子药物载体。体外药物释放实验结果表明,姜黄素聚合物壳可以有效地将药物分子封堵在介孔里,并能实现介孔的开关,实现介孔内药物分子的控制释放;降解实验结果表明,姜黄素聚合物壳可以被谷胱甘肽(GSH)有效降解,并且其降解速度与GSH的浓度成正比;通过荧光分光光度计和荧光显微镜测定发现姜黄素聚合物壳可以使该纳米粒子药物载体具有荧光功能;分散性实验结果发现,所得姜黄素聚合物壳可以改善介孔二氧化硅药物载体在磷酸缓冲液中的分散性;通过体外细胞实验结果证实该药物载体能够有效杀死A549癌细胞。
[Abstract]:In recent years, as a means to reduce the side effects of drug toxicity, improve the distribution and metabolism of drugs and improve the efficiency of drug treatment, it has become one of the hot spots in the field of biomedicine. It has attracted wide attention and is considered to be one of the most promising drug carriers. However, there are still some problems to be solved in its practical application, such as poor dispersivity and easy aggregation in water, relatively single function, potential toxicity of existing modification methods, and various functional molecules. In this thesis, a series of self fluorescent mesoporous silica nanoparticles have been prepared. The modification process of mesoporous silica nanoparticles was monitored by Fu Liye transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). The changes of morphology and particle size of mesoporous silica nanoparticles were observed by transmission electron microscopy (TEM), and the mesoporous silica nanoparticles were adsorbed by N2 adsorption and desorption. The pore size, Kong Rong and specific surface area were measured before and after the subsurface modification. Fluorescence spectrophotometer, fluorescence microscope and confocal fluorescence microscope were used to evaluate the fluorescence properties and fluorescence imaging ability of mesoporous silica nanoparticles. The mesopore was preliminarily investigated by four methyl azazolase Microenzyme reaction Colorimetry (MTT method). The cytotoxicity of silica nanoparticles in vitro was investigated in vitro. The feasibility of mesoporous silica nanoparticles as drug carriers in stimulating response to drug control release behavior was investigated. The specific content and results were as follows: (1) modified mesoporous silica nanoparticles with PAMAM dendrane coupling agent, The drug carriers with pH sensitivity and self fluorescence were prepared. The results showed that the surface of mesoporous silica nanoparticles had a large number of primary amine groups modified by PAMAM dendritic silane coupling agent, which was beneficial to the further modification and the introduction of other functional groups, such as target molecules, and modified by the PAMAM dendranes silane coupling agent. After that, the drug loading of mesoporous two oxygen silicon nanoparticles has been further improved. In addition, it is found that by changing the pH value of the solution, the conformation of PAMAM dendrites can be changed and the density of the particles in the outer layer of the particles can be changed to control the release of drugs, and the release of the drugs under neutral conditions can be prevented; the intensity of fluorescence emission and the intensity of fluorescence emission can be prevented. The fluorescence color changes with the drug loading rate and the release of drug molecules, and thus has the potential to be used for tracking or detection. The results show that PAMAM dendrites can be used as pH sensitive plugging agent and fluorescent marker. (2) a layer self-assembly method based on chondroitin sulfate (CS) and PAMAM branches is prepared. A multifunction mesoporous silica nanoparticle drug carrier. It is found that, under neutral conditions, pamam/cs layer self assembled monolayers can effectively block drugs and inhibit the early release of drugs in the mesoporous. The drug carrier has the properties of stimulating response drug control release, that is, the model drug adriamycin and curcumin in neutral pH value. The release was slow under the condition of acid (pH5.5) condition. The effect of pamam/cs layer self assembled film on the dispersion properties of particles was investigated. The results showed that the layered self assembled monolayer could make the mesoporous silica nanoscale have better dispersion, and the hydroxyl group and blood red fine could be effectively shielded by the hemolysis experiment. The interaction of the cell membrane to improve the blood compatibility of the nanoparticles; the fluorescence and fluorescence microscopy results showed that the nanoparticle had the ability of fluorescence imaging; the targeting function of CS could promote the nanoparticle drug carrier to enter the tumor cells and enhance its cytotoxicity. (3) a fluorescent anti-cancer drug, Jiang Huang, was used. F127 micelle (cf127) is used as the plugging agent and fluorescent marker of mesoporous silica nanoparticles, and the drug carrier of multifunction mesoporous silica nanoparticles is prepared. The results show that cf127 can be used as a plugging agent for mesoporous silica nanoparticles, and the combination of two kinds of drug carriers (mesoporous silica nanoparticles and micelles) A universal method for the preparation of multifunctional drug carriers is provided, which can simultaneously achieve stimulant control of drug release and fluorescence imaging. In vitro drug release experiment results show that cf127 micelles can be controlled by the pH response of the mesoporous silica nanoparticles after the Schiff base bond is connected to the mesoporous silica nanoparticles. It can be attributed to the sensitivity of Schiff base to pH, that is, under the neutral condition, the Schiff base bond can be more stable and thus can prevent the release of the drug. When the pH value is 5.5, the Schiff base bond breaks and releases the drug. The fluorescence test results show that the fluorescence intensity of the cf127 micelles will be evident after the cf127 micelles are aggregated on the mesoporous two oxygen silicon nanoparticles. In addition, the cf127 micelles have good anti photobleaching properties by examining the changes in the fluorescence intensity of the carrier in continuous UV irradiation. These results show that the cf127 micelle can be used as a fluorescent marker for the carrier of the mesoporous silica nanoparticles. (4) the anti-cancer drug Jiang Huang is used. As the plugging agent and fluorescent imaging agent of mesoporous silica nanoparticles, a drug carrier (lpcc-c-f127) with self fluorescence function and stimulus responsiveness is prepared. The results show that the curcumin plugging agent can effectively keep the drug molecules in the mesoporous and avoid the advance leakage of drug molecules before the cells; in vitro drugs. The results of the release experiment showed that the beta thioester on Curcumin could be hydrolyzed effectively in the presence of pH5.5 and glutathione (GSH), so that the curcumin molecules fell off the mesoporous and controlled the response of the drug to be controlled and released. The fluorescence spectrophotometer was used to detect the fluorescence of the nano drug carrier after multi-step functionalization. The strength was enhanced with the increase of curcumin molecules on the curcumin plugging agent. The results of dispersion experiment showed that the F127 film could improve the dispersibility of the drug carrier by the polyethylene glycol (PEG) shell, and also create a hydrophobic microenvironment to improve the fluorescence intensity of the drug carrier. The results of the MTT method showed that the prepared drug was loaded. The body has good biocompatibility. (5) the drug carrier of multifunction mesoporous silica nanoparticles is prepared by one pot method based on the curcumin molecule. The results of drug release in vitro show that the curcumin polymer shell can effectively block the molecule in the mesoporous and realize the mesoporous. The results showed that the curcumin polymer shell could be effectively degraded by glutathione (GSH), and the degradation rate was proportional to the concentration of GSH. The fluorescence spectrophotometer and fluorescence microscope showed that the gingiber polymer shell could make the nanoparticle drug carrier. The results showed that the curcumin polymer shell could improve the dispersibility of the mesoporous silica drug carrier in the phosphoric acid buffer solution, and the results of cell experiment in vitro proved that the drug carrier could effectively kill the A549 cancer cells.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TQ127.2;TB383.1
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本文编号：1961494