【作者】 周惠云；

【导师】 陈西广；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2007， 博士

【摘要】 壳聚糖具有很好的生物相容性和生物可降解性,且其降解产物对人体健康无害,近年来在药物制剂中的应用越来越广泛,本文建立了壳聚糖复合微球和壳聚糖温敏水凝胶两种缓释体系,并对两种体系进行了特性分析。壳聚糖、醋酸纤维素可以通过W/O/W复乳化法制得复合微球,试验中不需要添加化学交联剂,所制备微球无毒副作用,由亲水的壳聚糖内核和疏水的醋酸纤维素外被组成。复合微球是圆形的、可自由流动的无粘连的球状结构。微球大小、表面结构及雷尼替丁载药效率和体外缓释效果受壳聚糖浓度、壳聚糖分子量、醋酸纤维素浓度及CA/CS比例的影响;微球的粒径随着壳聚糖溶液的浓度及壳聚糖分子量的增大而增大,同时表面结构越来越致密;随着醋酸纤维素浓度的增大(由1%升高到5%)微球粒径也逐步增大,微球的表面变得更加皱褶,微球的结构也更加致密;而CA/CS比例对微球粒径的影响比较复杂,在最佳CA/CS时,微球具有最小的粒径。随着壳聚糖浓度的升高微球载药效率逐步降低,而载药量随壳聚糖浓度升高而升高;小分子量壳聚糖(47,145KD)和大分子量壳聚糖(1130KD)的载药效率相对较高,而中等分子量壳聚糖(308,499KD)的载药效率较低;雷尼替丁的载药效率随着醋酸纤维素浓度和CA/CS比例的升高逐步提高。雷尼替丁载药微球的体外释放受微球不同制备条件的影响。微球的释放效率不受释放介质pH值的影响;微球粒径大小影响模型药物的体外释放,粒径大的微球具有较慢的释放效率;壳聚糖溶液的浓度变化明显影响雷尼替丁的体外释放,由较高浓度壳聚糖溶液所制备微球具有较低的释放效率,即随着壳聚糖浓度的增大微球的缓释作用逐步增强;随着壳聚糖分子量的增大,盐酸雷尼替丁的释放速度逐步减慢,由高分子量壳聚糖(1130KD)制备微球的释放速度最慢;由2%醋酸纤维素制备的复合微球具有最慢的盐酸雷尼替丁释放效率,而由较低浓度(1%)或较高浓度(3%、5%)醋酸纤维素所制备微球均具有较快的释放速率;CA/CS比例对盐酸雷尼替丁的体外释放的影响与醋酸纤维素浓度的影响相似,CA/CS为3/1时制备微球具有最佳的缓释效果。由壳聚糖的醋酸缓冲液与αβ-甘油磷酸制备壳聚糖水凝胶具有温敏性,具有在37℃条件性由溶胶状转变为凝胶状的温敏特性。壳聚糖水凝胶体系在4℃条件下可贮存至少3个月。醋酸缓冲液的pH值、离子强度、壳聚糖/αβ-甘油磷酸比例、壳聚糖的浓度、分子量及脱乙酰度对壳聚糖水凝胶的特性包括水凝胶的pH值、浊度及旋转粘度均有影响。缓冲液pH4.6、离子强度0.15mo/L、壳聚糖/αβ-甘油磷酸8.8/1.2、壳聚糖(1360KD、75.4%DD、浓度2.0%)为最佳水凝胶制备条件。壳聚糖水凝胶体系在37℃凝胶化后其表面结构更加皱褶,表面的孔状结构基本消失,而不同溶剂条件(pH值、离子强度)、壳聚糖/αβ-甘油磷酸比例及不同特性的壳聚糖(浓度、分子量、脱乙酰度)所制备的水凝胶具有不同的表面结构和特征。壳聚糖水凝胶体系是一理想的缓释体系,并且其释放速率可以由制备条件的改变来调节。由不同方法制备的载药水凝胶具有不同的释放速率;不同醋酸缓冲液及不同分子量壳聚糖所制备水凝胶对模型药物的体外释放具有不同的影响;壳聚糖水凝胶体系对于亲水性药物比疏水性药物释放速率慢。模型药物的特性显著影响壳聚糖微球的载药效率,微球的载药效率随着模型药物的疏水性提高而提高。释放介质pH值对不同载药微球的体外释放均没有影响,说明释放介质的影响取决于微球的组成,而与模型药物的性质无关。微球对不同的模型药物均具有较好的缓释效果,并随着模型药物疏水性的提高释放速率逐步减慢,即对于疏水性药物具有更好的缓释效果。壳聚糖微球具有较强的粘附性,与胃粘膜结合比较紧密;与肠粘膜具有良好的吸附性,但随着时间的延长,微球的吸附率逐步降低;模型药物的加入对复合微球的吸附性几乎没有影响。体内粘附性试验结果表明,壳聚糖/醋酸纤维素微球具有较强的胃粘膜吸附性,可以延长微球胃滞留时间,50%微球滞留于胃部的时间为2.63h左右,50%微球到达结肠的时间是3.93h左右,说明壳聚糖复合微球具有作为延长胃滞留时间的复合粘附系统的潜力。用直接接触法检测壳聚糖复合微球及壳聚糖水凝胶的溶血性。溶血试验结果表明不同微球、水凝胶及其材料的溶血率均小于5%,即壳聚糖的两种缓释体系均符合溶血试验要求,试验材料无溶血性。用MEF细胞的相对增殖率检测壳聚糖水凝胶的细胞相容性。壳聚糖水凝胶浸提液在不同浓度及不同浸提时间对MEF细胞的相对增殖率均高于80%,甚至达到100%,说明壳聚糖水凝胶具有良好的细胞相容性,且生物相容性不受浸提时间及浸提液浓度的影响。壳聚糖/醋酸纤维素复合微球埋植于大鼠背部肌肉,壳聚糖水凝胶注射植入大鼠背部原位形成凝胶状结构,分别于不同时间取样检测两种缓释体系的生物相容性。植入后周围组织无明显炎症,未发现组织变性和坏死,可在组织内缓慢降解,具有良好的组织相容性,6周后反应接近外科缝合丝线组。各取样点血液各项指标及血清各项生化指标检测结果均在正常值范围内,且与正常对照和手术缝合线组相比没有明显差异,说明两种壳聚糖缓释体系具有较好的生物相容性。壳聚糖微球在PBS溶液中随着时间的延长逐渐降解,空白微球和三种不同载药微球随时间延长的降解趋势相同,均以比较缓慢的速度降解;在溶菌酶溶液中壳聚糖微球在开始的10天内降解的速度较快;10天以后壳聚糖微球均以较平缓的趋势降解,且空白微球的降解速度稍快于巯嘌呤载药微球。壳聚糖温敏凝胶在PBS溶液中随时间逐步降解,在最初10天内迅速降解,剩余重量达到40%;而10天后呈缓慢的趋势降解;PBS的不同离子强度对凝胶的降解没有明显影响,而0.15M PBS中水凝胶降解相对较慢;壳聚糖凝胶的浓度对降解有显著影响,随着壳聚糖凝胶浓度的提高,降解速度逐渐减慢。壳聚糖水凝胶的酶解呈现相同的降解趋势,在最初的7天很快降解,然后降解呈一平缓的曲线,以缓慢的速度继续降解;溶菌酶浓度影响壳聚糖水凝胶的降解,随着溶菌酶浓度的提高会加快水凝胶的降解;壳聚糖凝胶浓度同样影响水凝胶的酶解,当底物浓度较低时(0.5%-2.0%),壳聚糖水凝胶呈现相同的降解程度;当壳聚糖水凝胶的浓度超过2.0%,则随着浓度的提高,壳聚糖的降解速度逐渐减慢。总之,壳聚糖缓释微球及温敏水凝胶具有良好的缓释效果和生物相容性,可以作为理想的活性物质的载体系统。更多还原

【Abstract】 Chitosan (CS) has been extensively examined in the pharmaceutical industry for its potential in the development of controlled release of drug delivery due to its excellent biocompatibility, biodegradability, bioactivity and nontoxicity. New chitosan microspheres and thermosensitive hydrogel were prepared and the characteristics were evaluated.New microspheres containing hydrophilic core and hydrophobic coating as controlled release system with no toxic reagents is proposed. W/O/W emulsion and solvent evaporation methods are used to make chitosan/cellulose acetate microspheres (CCAM). The appearance of microspheres was spherical, free-flowing and non-aggregated. Chitosan concentration, chitosan molecular weight, cellulose acetate (CA) concentration and the ratio of CA/chitosan (CS) had influence on the CCAM size, appearance, ranitidine hydrochloride loading and releasing efficiency in vitro. The microsphere size became bigger and the appearance of microspheres became more compacted with the increasing of chitosan concentration and chitosan molecular weight. While the CA concentration was increased from 1% to 5%, the microsphere size became bigger and the appearance of microspheres became more corrugated and the structure became more compact. The optimal condition for preparation of the microspheres was the ratio of CA/CS at 3/1.The loading efficiency of ranitidine decreased with the increasing of chitosan concentration. The CCAM prepared with high molecular weight (1130KD) and lower molecular weight (47, 145KD) chitosan had relatively high loading efficiency, however the loading efficiency was relatively low at middle molecular weight(308,499KD). The loading efficiency increased with the increasing of cellulose acetate concentration and the ratio of CA/CS. The release efficiency of ranitidine from the CCAM wasn’t affected by the pH values of release medium. The size of microspheres affected the release of ranitidine and the bigger microspheres released slower than that of the smaller parts. Molecular weight and concentration of chitosan affected the release in vitro. The release rate became slower with the increasing of chitosan concentration and molecular weight and the release from CCAM prepared with highest molecular weight chitosan (1130KD) was the slowest. The optimal condition for preparation of the microspheres was CA concentration at 2% and the ratio of CA/CS at 3/1 which had the slowest release rate in vitro.A thermosensitive hydrogel was prepared with chitosan (dissolving in the acetic acid/sodium acetate buffer solution) andαβ-mixtured-glycerophosphate (αβ-GP) which could be transformed into gel at 37℃. The thermosensitive characteristics, appearance and structure of hydrogel were affected by the pH values, ion intensity and ratio of CS/αβ-GP and concentration, molecular weight (MW) and DD of chitosan. The pH 4.6, ion intensity 0.15mol/L, ratio of CS/αβ-GP 8.8/1.2, chitosan solution of 2%, MW of 1360KD and DD of 75.4% were the optimal condition to prepare CS-αβ-GP thermosensitive hydrogel. The appearance of hydrogel incubated at 37℃became more corrugated and the granule was almost disappeared. The appearance and characteristics of hydrogel changed with the different solvent condition and different characteristics of chitosan. The CS-αβ-GP hydrogel system was an ideal sustained release system and the release rate could be adjusted by the change of preparation conditions. Model drugs released from CS-αβ-GP hydrogel prepared by methodⅠwas slower than that from hydrogel prepared by methodⅡ. The CS-αβ-GP hydrogel made from different pH values of buffer solution and different molecular weight of chitosan had different release efficiency of adriamycin (ADR) in vitro. The hydrophilic model drug (ADR) released 60% to 70% during 24 hours which was slower than that of hydrophobic drug (6-MP).CCAM loaded different model drugs were prepared by the method of W/O/W emulsion. The loading efficiency was affected by the characteristics of model drugs and it increased with the increasing of hydrophilicity of model drugs. The release of different model drugs wasn’t affected by the pH values of medium, so it illustrated that the affection of medium on the release was depended on the materials of microspheres but not the model drugs. The CCAM system had good effect on the controlled release in vitro of all model drugs of different hydrophobicity. However, the release rate became slower with the increasing of the hydrophobicity of model drugs.CCAM had good mucoadhensiveness and could adhere to the gastric mucosa tightly. The delivery system of CCAM had good adhesive ability to small intestine but the remaining percent decreased with the time lasting. The adding of model drugs made little difference in mucoadhensiveness from the blank CCAM. The mucoadhesive tests in vivo showed that CCAM could retain in gastrointestinal tract for an extended period of time. The time that 50% of CCAM remained in stomach after administered was 2.63h and that of 50% of CCAM get to colon after administered was 3.93h. These results suggest that CCAM is a useful dosage form targeting the gastric mucosa or prolonging gastric residence time as a multiple-unit mucoadhesive system.The hemolysis of CCAM and chitosan hydrogel samples were tested by direct contact methods, according to ISO 10 993-4 (1992). Under the conditions of this study, blood in contact with tested samples would not be considered hemolytic, showing hemolysis value less than 5%.The cell compatibility of chitosan hydrogel was evaluated by measuring the relative growth rate of MEF cell. The RGR of MEF cell at different extract concentration and different extract time were all more than 80% and even to 100%. The results showed that the hydrogel had good cell compatibility and the compatibility isn’t affectted by the extract concentration and time.CCAM were implanted into muscles of rats and chitosan hydrogel were injected at the back of rats which transited into gel in situ. After a definite time interval, the CCAM or gel implanted were observed to evaluate the biocompatibility. It was observed that the two sustained release systems had no toxic effects on surrounding tissue and was slowly degraded after implanted into rat back muscular porch. The present study suggests that the two systems have good biocompatibility and biodegradation. A number of blood biochemical parameters were all in the normal range and had no obvious difference with constant group which illustrated that the two chitosan systems had good blood compatibility.CCAM was degraded gradually with the time lasting in PBS solution. Blank CCAM and CCAM loaded different model drugs had the same degraded trend and degraded in a slow speed. Lysozyme degradation rates of all kinds of CCAM was fast in the first 10 days. The Lysozyme degradation rates became slower after 10 days and the CCAM loaded 6-MP was degraded slower than that of blank CCAM. Chitosan thermosensitive hydrogel degraded with the time lasting in PBS solution. It degraded quickly in the first 10d and the remaining weight reached 40%. The ion intensity affected the degradation a little. The concentration of hydrogel had obvious affection on the degradation rate which decreased with the increasing of hydrogel concentration. The Lysozyme degradation of hydrogel was fast in the first week and then degraded in a slower speed in the following. The lysozyme degradation rate became quick with the increasing of lysozyme concentration. In the mean time, the lysozyme degradation of hydrogel was affected by the hydrogel concentration. The degradation rate maintained the same at low concentration(0.5%-2.0%)and decreased with the increasing of hydrogel concentration when it was higher than 2.0%.CCAM and chitosan thermosensitive hydrogel had good sustained release and good biocompatibility which could be the ideal delivery systems of active biomaterials.更多还原