低分子肝素pH敏感巯基壳聚糖纳米粒的研究

发布时间：2018-03-03 11:04

本文选题：低分子肝素　切入点：巯基壳聚糖　出处：《山西医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：随着生物技术的飞速进展，越来越多的大分子如蛋白质、多肽、多糖和核酸类药物应用于临床。这类药物毒副作用小、疗效确切，但大都具有多重胃肠道吸收屏障而限制了口服应用。低分子肝素(LMWH)是治疗深静脉血栓的大分子药物，在胃酸中不稳定，小肠中相对稳定，水溶性强、分子量大及荷负电荷的特点使其难以透过胃肠粘膜吸收，临床只能静脉注射给药。巯基壳聚糖(TCS)是壳聚糖(CS)的衍生物，具有生物可降解、低毒、抑酶、促粘膜吸收及生物粘附等特性，但生物粘附作用比CS更强。本文以大分子药物LMWH为模型药物，，以TCS为载体材料，羟丙甲基纤维素邻苯二甲酸酯(HP-55)为交联剂，采用离子交联技术制备LMWH pH敏感TCS纳米粒。以期能通过pH敏感纳米粒酸性条件下不溶解及空间障碍保护低分子肝素免受胃酸和消化酶降解；利用TCS促进渗透、增强粘附、抑酶作用，以增加LMWH的粘膜透过，达到提高其口服生物利用度的目的。本文主要研究内容如下： 1、TCS的合成与表征以CS为载体材料，碳二亚胺为催化剂，与巯基乙酸反应合成TCS。建立Ellman’s试剂紫外分光光度法，测定TCS中巯基含量为(396.97±54.54) μmol/g。以巯基含量为指标考察影响合成的因素，用红外光谱分析法表征。 2、LMWH pH敏感TCS纳米粒的制备及表征以TCS为载体材料、HP-55为交联剂，采用离子交联法制备LMWH pH敏感TCS纳米粒。采用透射电子显微镜观察纳米粒形态为类圆球形；粒度仪测定纳米粒的平均粒径(329.6±7.3) nm、PDI (0.185±0.01)、Zeta电势(34.5±2.25)mV。 3、LMWH体外分析方法的建立建立天青A紫外分光光度法测定LMWH含量。并进行了重复性、精密度和回收率等方法学考察。分析方法准确可靠，可用于LMWH体外样品的分析。测定LMWH pH敏感TCS纳米粒载药量为(12.463±0.003) IU/mg、包封率为(97.75±0.02)%。 4、LMWH pH敏感TCS纳米粒的体外评价透析法考察纳米粒体外药物释放，实验结果表明pH敏感TCS纳米粒在酸性条件下能显著减少药物的释放；Franz扩散池法考察纳米粒药物肠粘膜透过作用，药物肠粘膜累积透过量：pH敏感巯基壳聚糖纳米粒、巯基壳聚糖纳米粒＞pH敏感壳聚糖纳米粒＞药物溶液；Mett毛细玻管法考察纳米粒的抑酶作用，pH敏感巯基壳聚糖纳米粒抑酶作用强于pH敏感壳聚糖纳米粒和药物溶液；体外孵化实验考察纳米粒对药物的保护作用，pH敏感巯基壳聚糖纳米粒对药物的保护作用强于巯基壳聚糖纳米粒和药物溶液。 5、LMWH pH敏感TCS纳米粒的体内评价激光扫描共聚焦显微分析技术考察大鼠口服荧光标记纳米粒后肠粘膜粘附作用，pH敏感TCS纳米粒肠粘膜荧光强度明显高于药物溶液、pH敏感CS纳米粒和TCS纳米粒；大鼠口服pH敏感TCS纳米粒后，活化部分凝血活酶时间（APTT）显著延长，生物利用度显著提高，表明抗凝作用增强、LMWH口服吸收量提高。
[Abstract]:With the rapid development of biotechnology, more and more macromolecules such as proteins, peptides, polysaccharides and nucleic acids are used in clinic. Low molecular weight heparin (LMWH) is a macromolecular drug for the treatment of deep venous thrombosis, which is unstable in gastric acid, relatively stable in small intestine and strong in water solubility. The characteristics of high molecular weight and negative charge make it difficult to be absorbed through gastrointestinal mucosa, so it can only be given intravenously in clinic. Thioglycol-chitosan (TCSs) is a derivative of chitosan (CSS), which is biodegradable, low toxic and enzyme suppressive. Promote mucosal absorption and biological adhesion, but the biological adhesion is stronger than CS. In this paper, macromolecular drug LMWH was used as model drug, TCS as carrier material and hydroxypropylcellulose phthalate (HP-55) as crosslinking agent. LMWH pH sensitive TCS nanoparticles were prepared by ion crosslinking technique, which could protect low molecular weight heparin from gastric acid and digestive enzyme degradation through pH sensitive nanoparticles in acidic condition. In order to increase the mucosal permeability of LMWH and increase its oral bioavailability. The main contents of this paper are as follows:. Synthesis and characterization of TCS. Using CS as carrier material and carbodiimide as catalyst, TCS was synthesized by reaction with thioglycolic acid. The UV spectrophotometric method of Ellman's reagent was established. The content of thiol in TCS was 396.97 卤54.54 渭 mol / g. It was characterized by infrared spectroscopy. Preparation and characterization of LMWH pH sensitive TCS nanoparticles. Using TCS as carrier material, LMWH pH sensitive TCS nanoparticles were prepared by ion crosslinking method. The morphology of LMWH nanoparticles was observed by transmission electron microscopy (TEM), and the average particle size of LMWH nanoparticles was determined by particle size analyzer. The average particle size of LMWH was 329.6 卤7.3) nmPDI 0.185 卤0.01nmPe (34.5 卤2.25mV). Establishment of in vitro analytical method for LMWH. A UV spectrophotometric method for the determination of LMWH was established. The methods of repeatability, precision and recovery were investigated. The method was accurate and reliable. It can be used for the analysis of LMWH samples in vitro. The drug loading capacity of LMWH pH sensitive TCS nanoparticles is 12.463 卤0.003). The encapsulation efficiency is 97.75 卤0.02%. In vitro evaluation of TCS nanoparticles with LMWH pH sensitivity. In vitro drug release of nanoparticles was investigated by dialysis. The results showed that pH sensitive TCS nanoparticles could significantly reduce drug release under acidic conditions. Franz diffusion cell method was used to investigate the intestinal mucosal permeability of nanoparticles. Drug intestinal mucosal cumulative transmittance: ph sensitive mercapto chitosan nanoparticles, The inhibition effect of mercapto chitosan nanoparticles > pH sensitive chitosan nanoparticles > drug solution / mett capillary tube method was studied. The inhibition effect of pH sensitive chitosan nanoparticles was stronger than that of pH sensitive chitosan nanoparticles and drug solutions. In vitro incubation experiments were conducted to investigate the protective effect of nanoparticles on drugs. The protective effect of pH sensitive mercapto chitosan nanoparticles was stronger than that of mercapto chitosan nanoparticles and drug solutions. In vivo evaluation of TCS nanoparticles sensitive to LMWH pH. Laser scanning confocal microscopy study on intestinal mucosal adhesion of TCS nanoparticles after oral fluorolabeling in rats the fluorescence intensity of pH sensitive TCS nanoparticles was significantly higher than that of pH sensitive CS nanoparticles and TCS nanoparticles in drug solution. After oral administration of pH sensitive TCS nanoparticles, the activated partial thromboplastin time (APTT) was significantly prolonged and the bioavailability was significantly increased, which indicated that the anticoagulant effect was enhanced and the oral absorption of LMWH was increased.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R944.9

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