【作者】 张雪琼；

【导师】 郑化；

【作者基本信息】 武汉理工大学 ， 材料学， 2010， 博士

【摘要】 肝炎、肝硬化和肝癌等慢性肝脏疾病,极大地危害人体的健康。尽管目前某些药物能够达到预期的治疗效果,但由于给药系统难以将药物准确传递到肝脏病灶细胞而降低了其生物利用度,并产生较大毒副作用,限制了其临床应用。解决这个问题的有效办法就是靶向给药系统。靶向给药系统能将药物传递到病灶部位,减少用药剂量和给药次数,提高药物治疗指数,避免或减轻药物不良反应。二硫键对谷胱甘肽的浓度刺激具有强的响应性,由于血液中谷胱甘肽的浓度较低(微摩尔级),二硫键很稳定,能维系纳米粒形态结构的稳定性,控制纳米粒内部包封的药物不被释放；而在细胞中,由于谷胱甘肽的浓度相对较高(毫摩尔级),二硫键能被谷胱甘肽分解,纳米粒结构受到破坏,内部药物被释放出来。课题通过半乳糖配基对羧甲基壳聚糖氨基的修饰赋予其主动肝靶向性,通过羧甲基壳聚糖羧基的巯基化,在组装纳米粒的过程中发生二硫键交联,赋予纳米粒高稳定性及智能性(即对谷胱甘肽浓度的刺激响应性)。主要研究内容和结果如下：(1)以包封率和粒径为指标,采用正交设计优化羧甲基壳聚糖纳米粒制备工艺,考察了羧甲基壳聚糖分子量对纳米粒粒径及包封率的影响,随着分子量的增大,纳米粒的粒径及包封率都增大。体外释放试验表明,羧甲基壳聚糖纳米粒可对甘草酸起到缓释作用,分子量较大的羧甲基壳聚糖制备的纳米粒的体外释放较慢。药动学研究表明,羧甲基壳聚糖纳米粒较甘草酸溶液分布快药理作用强,能延长体内作用时间。小鼠体内组织分布表明,将甘草酸制成纳米粒后,在肝脏中的分布增大,在肾脏中的分布减少,有利于提高疗效,降低毒副作用。(2)以O-羧甲基壳聚糖为原料,采用还原胺化法合成得到半乳糖羧甲基壳聚糖(LAC-CMC)。在LAC-CMC纳米粒的制备过程中,采用单因素法考察了LAC-CMC分子量、甘草酸浓度、LAC-CMC浓度对包封率及粒径的影响。体外释放试验表明,分子量较大的LAC-CMC制备的纳米粒的体外释放较慢。药动学研究表明,半乳糖羧甲基壳聚糖纳米粒较羧甲基壳聚糖纳米粒药理作用强。小鼠体内组织分布表明,引入肝靶向基团后,纳米粒的肝靶向性增强,毒副作用减弱。(3)半乳糖羧甲基壳聚糖在EDC和NHS催化下,与半胱胺反应,得到N-半乳糖-O-巯基化羧甲基壳聚糖。采用Ellman’s试剂测定了巯基的含量,并考察了半胱胺的用量、反应的pH值、反应时间对巯基含量的影响。体外释放实验表明,交联纳米粒在模拟血液中72小时的释放较少,而在模拟细胞液中72小时能将药物大量的释放出来,充分证明了交联纳米粒的智能释药性。药代动力学实验表明：将甘草酸制成交联纳米粒,可延长甘草酸在体内的作用时间,在一定程度上克服甘草酸注射液代谢太快的缺点,提高药物疗效；组织分布试验表明：交联纳米粒能延长甘草酸在肝中的药物浓度、具有明显的肝靶向性,有利于提高甘草酸对肝组织的疗效,交联纳米粒能明显降低甘草酸在肾组织中的蓄积,因而能有效降低其肾毒性。因此,N-半乳糖-O-巯基化羧甲基壳聚糖所制备的交联纳米粒具有智能释药和肝靶向的特点,有望作为纳米药物载体。更多还原

【Abstract】 Chronic hepatic disease such as hepatitis, hepatocirrhosis and hepatoma was a major class of serious disease that threating the health of the people. At present, some drugs can reach the anticipative curative effect, but drugs were difficult to be transferred to the targeting site specifically and accurately by the drug delivery system, which caused a low bioavailbility, toxicity, side-effect and limited drugs clinic application. An effective approach to overcome this critical issue is the development of targeted drug delivery systems.Targeted drug delivery systems can release the drugs at the desired site of action, reduce drug dosage and administration times, increase therapeutic efficacy, and reduce side-effect. Covalently bonded disulfides can be formed spontaneously by autoxidation of sulfhydryls, primarily via oxidation upon exposure to air, which can reversibly be cleaved in the presence of reducing agents. The advantage of the nanoparticles with a disulfide crosslinkage in drug delivery is that the disulfide bond is stable in the blood, but cleaved inside the cell based on the fact that the concentrations of glutathione, the most abundant reducing agent in most cells including mammals, are in a millimolar range inside the cells, whereas those in blood plasma are in a micromolar range.Galactose group was coupled with carboxymethyl chitosan to synthesis lactosaminated carboxymethyl chitosan (LAC-CMC) for liver specificity. Through the sulfhydrylation of LAC-CMC, the nanoparticles were crosslinked through disulfide bond for intelligence. The main contents and conclusions of study are as follows:The preparation of CMC nanoparticle was optimized by the method of orthogonal design, according to encapsulation efficency and size. The impact of CMC molecular weight to nanoparticle size and encapsulation efficency was also studied, and as a result, encapsulation efficency and size of nanoparticles increased when the molecular weight increased. The in vitro release experiment showed that the nanoparticles with larger molecular weight had more apparent effect to slow down the release. The pharmacokinetics study demonstrated that CMC nanoparticles deliveried faster and had stronger pharmacological effect to lengthen the active time. The study of tissue delivery in rats indicated that the concentration of glycyrrhizic acid in liver increased while in kidney it decreased, after glycyrrhizic acid was loaded in nanoparticles, which can boost the curative effect and lower the toxic and side-effect.LAC-CMC was synthesized from O-CMC, by reductive amination reaction. While preparing the LAC-CMC nanoparticles, the influence of the molecular weight of LAC-CMC, the concentration of glycyrrhizic acid and the concentration of LAC-CMC to size and encapsulation coefficency was studied by the method of single factor. The in vitro release experiment showed that LAC-CMC nanoparticles with larger molecular weight can delay the release of glycyrrhizic acid. The pharmacokinetics study indicated that LAC-CMC nanoparticles acted stronger pharmacological effect than CMC nanoparticles. The study of tissue delivery in rats showed the liver targeting efficiency was increased and side effect was decreased after the targeting group was introduced into the molecule.Thiolated LAC-CMC was synthesized by LAC-CMC and cysteamine under the catalyst of N-(3-dimethyl amino-propyl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccimide (NHS) through the acylation. Ellman’s reagent was employed to determine the content of-SH. To study the factor to impact the content of-SH, the amount of cysteamine, the pH and the reaction time were investigated. The in vitro release experiment showed that the crosslinked nanoparticles released more drug in mimic cytoplasm than in mimic blood after 72h, which proved the intelligent release of crosslinked nanoparticles. The pharmacokinetics study showed that the reactive time of glycyrrhizic acid was lengthened after it was loaded in crosslinked nanoparticles, which, to some degree, overcame the fast metabolization of glycyrrhizic acid solution and boosted the curative effort. The study of tissue showed that crosslinked nanoparticles can increase the concentration of drug in liver and manifest a obvious liver targeting property, which can reinforce the drug’s curative effect, meanwhile, the crosslinked nanoparticles can reduce the content of glycyrrhizic acid in kidney so as to lower its renal toxicity.Therefore, the crosslinked nanoparticles may be used as a potential drug delivery system with hepatic targeting and intelligent release.更多还原