本文選題：低分子肝素　切入點：巰基殼聚糖　出處：《山西醫(yī)科大學》2014年碩士論文　論文類型：學位論文

【摘要】：隨著生物技術的飛速進展，越來越多的大分子如蛋白質、多肽、多糖和核酸類藥物應用于臨床。這類藥物毒副作用小、療效確切，但大都具有多重胃腸道吸收屏障而限制了口服應用。低分子肝素(LMWH)是治療深靜脈血栓的大分子藥物，在胃酸中不穩(wěn)定，小腸中相對穩(wěn)定，水溶性強、分子量大及荷負電荷的特點使其難以透過胃腸粘膜吸收，臨床只能靜脈注射給藥。巰基殼聚糖(TCS)是殼聚糖(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更強。 本文以大分子藥物LMWH為模型藥物，，以TCS為載體材料，羥丙甲基纖維素鄰苯二甲酸酯(HP-55)為交聯(lián)劑，采用離子交聯(lián)技術制備LMWH pH敏感TCS納米粒。以期能通過pH敏感納米粒酸性條件下不溶解及空間障礙保護低分子肝素免受胃酸和消化酶降解；利用TCS促進滲透、增強粘附、抑酶作用，以增加LMWH的粘膜透過，達到提高其口服生物利用度的目的。 本文主要研究內容如下： 1、TCS的合成與表征 以CS為載體材料，碳二亞胺為催化劑，與巰基乙酸反應合成TCS。建立Ellman’s試劑紫外分光光度法，測定TCS中巰基含量為(396.97±54.54) μmol/g。以巰基含量為指標考察影響合成的因素，用紅外光譜分析法表征。 2、LMWH pH敏感TCS納米粒的制備及表征 以TCS為載體材料、HP-55為交聯(lián)劑，采用離子交聯(lián)法制備LMWH pH敏感TCS納米粒。采用透射電子顯微鏡觀察納米粒形態(tài)為類圓球形；粒度儀測定納米粒的平均粒徑(329.6±7.3) nm、PDI (0.185±0.01)、Zeta電勢(34.5±2.25)mV。 3、LMWH體外分析方法的建立 建立天青A紫外分光光度法測定LMWH含量。并進行了重復性、精密度和回收率等方法學考察。分析方法準確可靠，可用于LMWH體外樣品的分析。測定LMWH pH敏感TCS納米粒載藥量為(12.463±0.003) IU/mg、包封率為(97.75±0.02)%。 4、LMWH pH敏感TCS納米粒的體外評價 透析法考察納米粒體外藥物釋放，實驗結果表明pH敏感TCS納米粒在酸性條件下能顯著減少藥物的釋放；Franz擴散池法考察納米粒藥物腸粘膜透過作用，藥物腸粘膜累積透過量：pH敏感巰基殼聚糖納米粒、巰基殼聚糖納米粒＞pH敏感殼聚糖納米粒＞藥物溶液；Mett毛細玻管法考察納米粒的抑酶作用，pH敏感巰基殼聚糖納米粒抑酶作用強于pH敏感殼聚糖納米粒和藥物溶液；體外孵化實驗考察納米粒對藥物的保護作用，pH敏感巰基殼聚糖納米粒對藥物的保護作用強于巰基殼聚糖納米粒和藥物溶液。 5、LMWH pH敏感TCS納米粒的體內評價 激光掃描共聚焦顯微分析技術考察大鼠口服熒光標記納米粒后腸粘膜粘附作用，pH敏感TCS納米粒腸粘膜熒光強度明顯高于藥物溶液、pH敏感CS納米粒和TCS納米粒；大鼠口服pH敏感TCS納米粒后，活化部分凝血活酶時間（APTT）顯著延長，生物利用度顯著提高，表明抗凝作用增強、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 鹵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 鹵7.3) nmPDI 0.185 鹵0.01nmPe (34.5 鹵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 鹵0.003). The encapsulation efficiency is 97.75 鹵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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R944.9

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