有机金属框架纳米载药系统的构建

发布时间：2018-07-29 14:04

【摘要】：金属-有机骨架(Metal-Organic Frameworks, MOFs)是近年来新型的多孔式有机-无机杂化的功能性的材料。与其他功能性材料相比,M O F s具有结构可变性和易功能化的特征,可同时实现较高的载药量和适宜的药物控制释放的能力。本课题构建了纳米有机金属框架Cu-BTC载药系统并成功将抗肿瘤代表性药物5-氟尿嘧啶(5-FU)包封于载体中,初步探索了有机金属框架纳米载药系统构建的可行性及规律。本文首先考察载体Cu-BTC的制备,以粒径、载体形态、分散性为指标,单因素考察法分别考察溶剂热法和配位调控法的影响因素。结果表明,本文所用的配位调控法制备出的载体粒径均为纳米级,且分散度较好。下一步对配位调控法中反应物浓度、搅拌速度、滴定时间用正交试验进一步优化,优化后确定最佳因素条件为:反应物浓度为二倍浓度(即:醋酸铜0.36g,苯甲酸4g,均苯三甲酸0.8g)、滴定时间为1.5h、搅拌速度为1000转/分钟。综合试验结果得出最优的制备工艺为:将0.36g醋酸铜和4g苯甲酸(调控剂)溶于30mL 正丁醇中,将0.8g均苯三甲酸溶于30mLDMF溶液当中,搅拌两种溶液至完全溶解。室温搅拌,同时将配体溶液滴入到金属盐溶液中(边滴加边搅拌),滴定时间约为1.5h,然后搅拌30min。搅拌速度为1000r/min,将产物离心,用乙醇洗涤2次,超声分散。所得产物常温干燥,称量。对所合成的纳米载体进行了 XRD定性鉴别,结果表明样品X射线衍射图谱和由单晶结构模拟的Cu-BTC标准物质十分吻合。SEM和粒径分布仪查看载体形态,测定粒径分布。结果SEM测得载体粒径大小在50～1 OOnm之间, 而粒径分布仪测定粒径在270nm左右。这是由于载体分散于水中时,水分子包裹在载体周围,使粒径增大。此外,从SEM图谱可见载体形态呈规整的圆球形,不粘连,分散性好。TEM和氮气吸附试验描述了载体中孔径的大小及分布,经证实,载体中孔径大小约为2到5nm。适宜载入粒径小于此范围的药物。热重分析曲线证明160℃以内为最佳活化温度,当温度升至310℃左右时,框架结构发生改变,故本载体有较高的热稳定性,与文献报道的热稳定性基本吻合[84]。最后,我们对载体在血液中的溶血性进行考察,相关图片证明,载体具有较好的血液相容性。本课题用响应面法考察最佳载药工艺,首先以载药量为指标,通过单因素法筛选载药的影响因素及各因素水平值的最优区间。相关数据及图谱表明,药载比、载药时间、乙醇浓度对载药量影响较大。以这三个因素为自变量,通过响应面法考察最优载药工艺。三维曲面图和等高线图得到优化后条件为乙醇浓度70%;药载比7:1;载药时间96h时,载药量最高。工艺验证结果,载药量均达到30%以上,证明优化后的工艺稳定,操作简便,结果可信。对载药后有机金属框架进行表征,SEM表征结果显示载药前后载体大小及形态均无明显改变,说明载药过程对载体形态无影响;XRD广角衍射表征结果载药前后图谱无明显差异;小角衍射可见载药后载体孔道发生变化,证明药物载入载体的孔道中;差示扫描量热法中DTA曲线证明载药过程对纳米有机金属骨架的热稳定性不产生影响。体外释放试验是通过对比正向动态透析法和反向动态透析法得到的,并分别以零级动力学方程、一级动力学方程和Higuchi方程对上述两种方法测得的体外释药数据进行拟合。结果表明,两种方法测得的数据均较符合一级动力学方程,两种方法释放曲线均显示出在刚开始的1小时表现为突释,这是由于吸附在载体表面的药物经扩散进入释放介质所致,并随后出现平稳的释放效果。MTT法测定FITC-5-FU-Cu-BTC体外细胞毒性,结果表明,空载体的毒性和5-FU药物的毒性相当,FITC-Cu-BTC毒性较高;荧光倒置显微镜测定细胞对FITC-5-FU-Cu-BTC的摄取可知,载药载体可被细胞吸收,并随着样品浓度的增加吸收的量明显加大;由流式细胞仪测定的结果可见,FITC-5-FU-Cu-BTC细胞摄取过程呈饱和性和温度依赖性,初步推测是通过内吞途径被细胞摄取的。
[Abstract]:Metal-Organic Frameworks (MOFs) is a new porous organic-inorganic hybrid functional material in recent years. Compared with other functional materials, M O F s has the characteristics of structural variability and functionalization, which can simultaneously achieve higher drug loading and appropriate drug control release ability. This topic has been constructed. The nano organic metal frame Cu-BTC drug loading system was successfully encapsulated in the carrier of the antitumor drug 5- fluorouracil (5-FU). The feasibility and regularity of the construction of the organic metal frame nano drug loading system were preliminarily explored. First, the preparation of the carrier Cu-BTC was investigated with the particle size, the carrier morphology and dispersion as the index, and the single factor investigation method was used. The influence factors of the solvent heat method and coordination regulation method are not investigated. The results show that the size of the carrier prepared by the coordination method in this paper is all nanoscale, and the degree of dispersion is good. The next step is to optimize the concentration of reactants, stirring speed and titration time in the coordination control method with orthogonal test. The concentration is two times concentration (i.e., copper acetate 0.36g, 4G benzoic acid, 0.8g of benzoic acid three formic acid), the titration time is 1.5h, and the stirring speed is 1000 rpm. The optimum preparation process is that 0.36g copper acetate and 4G benzoic acid (regulators) are dissolved in 30mL Ding Chunzhong, and 0.8g mean three formic acid is dissolved in 30mLDMF solution. Two solutions are completely dissolved. At room temperature, at the same time, the ligand solution is dripped into the metal salt solution (side drop and agitation), the titration time is about 1.5h, then the stirring speed of 30min. is 1000r/min, the product is centrifuged, 2 times washed with ethanol and ultrasonic dispersion. The product is dry at normal temperature and weighed. The synthesized nano carrier is XRD The results show that the X ray diffraction pattern of the sample and the Cu-BTC standard material simulated by the single crystal structure are in good agreement with the.SEM and the particle size distribution instrument to examine the carrier morphology and determine the particle size distribution. Results the size of the carrier particle size of the carrier is between 50~1 OOnm and the particle size distribution meter is about 270nm. This is due to the dispersion of the carrier in the water. In addition, the water molecules are wrapped around the carrier to make the particle size increase. In addition, it can be seen from the SEM map that the shape of the carrier is a regular round ball and does not adhere. The size and distribution of the pore size in the carrier is described by the good dispersibility.TEM and nitrogen adsorption test. It is proved that the size of the pore size of the carrier is about 2 to 5nm. and is suitable for carrying the drug with smaller size smaller than this range. The analysis curve proves that the optimum activation temperature is within 160 C. When the temperature rises to about 310 C, the frame structure changes. Therefore, the carrier has high thermal stability, which basically coincides with the thermal stability reported in the literature [84].. Finally, we examine the hemolysis of the carrier in the blood. The related pictures show that the carrier has better blood. The best drug loading process was investigated by response surface method. First, the influence factors of drug loading and the optimal range of the level of each factor were selected by the single factor method. The related data and Atlas showed that the drug load ratio, the time of drug loading and the concentration of ethanol had great influence on the drug loading. These three factors were the independent variables and passed the noise. The optimum drug loading process was investigated by surface method. The optimized conditions were ethanol concentration 70%, drug loading ratio 7:1, and drug loading time 96h, the drug loading was the highest. The results of the process validation were more than 30%, which proved that the optimized process was stable, the operation was simple and the results were credible. The organic metal frame after the drug loading was characterized, The results of SEM showed that the size and morphology of the carrier before and after the drug loading had no obvious changes, indicating that the drug loading process had no influence on the carrier morphology, and there was no obvious difference in the spectrum of the carrier before and after the XRD wide angle diffraction; the small angle diffraction showed that the carrier channel changes after the drug loading, which proved that the drug loaded into the channel of the carrier, and the DTA curve in the differential scanning calorimetry. It is proved that the drug loading process has no effect on the thermal stability of the nanoscale organometallic skeleton. In vitro release test is obtained by comparing the forward dynamic dialysis method and the reverse dynamic dialysis method, and the above two methods are fitted with zero order kinetic equation, first order dynamic equation and Higuchi equation. The results showed that the data obtained by the two methods were all in accordance with the first order kinetic equation, and the release curves of the two methods showed a sudden release at the beginning of the first 1 hours, which was due to the diffusion of drugs adsorbed on the surface of the carrier and into the release medium, and then the.MTT method was used for the determination of FITC-5-FU-Cu-BTC cells in vitro. Toxicity, the results showed that the toxicity of the empty carrier was equal to the toxicity of the 5-FU drug, and the toxicity of the FITC-Cu-BTC was high. The fluorescence inversion microscope determined the uptake of FITC-5-FU-Cu-BTC by cells, and the amount of the carrier could be absorbed by the cell and increased with the increase of the concentration of the sample; the results measured by the flow cytometer can be seen, FITC-5-FU-Cu-B The uptake process of TC cells is saturated and temperature dependent. It is presumably presumed to be absorbed by cells through endocytosis pathway.
【学位授予单位】：黑龙江中医药大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R943

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