【作者】 朱丽英；

【导师】 姚善泾；

【作者基本信息】 浙江大学 ， 生物化工， 2011， 博士

【摘要】 口服结肠定位给药系统(oral colon-specific drug delivery system),是指通过适当的方法,使药物经口服后避免在胃和小肠中释放,运送至回盲部后才开始释放,从而发挥局部或全身治疗作用的一种新型给药系统。通过结肠靶向给药,使药物能够直接作用于病变部位,减少由于药物在胃肠道上端吸收而引起的副作用,并使生物大分子类药物能够实现口服给药。在口服结肠定位给药系统中,载体材料的选择十分关键,在活性功能成分的释放、导向等方面具有决定性作用。其中,天然多糖具有良好的生物相容性和生物降解性,越来越多地用于口服结肠定位给药材料的研究。壳聚糖和纤维素硫酸钠都是来源广泛的天然多糖衍生物,两者结合能够形成不溶于水的聚电解质复合物。有研究发现,壳聚糖/纤维素硫酸钠(NaCS)聚电解质复合物在口服结肠定位给药系统中具有很大的应用潜力。本论文以壳聚糖和NaCS为材料制备的复合膜为对象,围绕复合膜的生物降解、机械、溶胀和药物渗透性能等内容进行展开,并且以壳聚糖和NaCS为主要材料制备微球,对其的制备和在载蛋白药物的应用进行了初探。针对在标定NaCS性能方法准确性方面存在的问题,本论文确定了分别用凝胶渗透色谱法、BaSO4比浊法和酶降解法来测定NaCS的分子量、取代度和生物降解性能,并由此考察了NaCS制备条件对NaCS性质影响。结果表明,反应时间越长,NaCS的分子量越小；而分子量分布系数随反应时间而变化,先逐渐增加,在6h达到最大值(5.16),继续增加反应时间,分子量分布系数却迅速下降。NaCS的取代度随着反应时间先逐渐增大,到6h后,取代度保持在0.5左右。通过纤维素酶降解实验可知,NaCS的降解性随着其制备时间的增加而呈现逐渐下降的趋势。这些结果说明制备条件对产品NaCS的性质有较大影响并将直接影响其应用。考察了壳聚糖与NaCS的质量比对壳聚糖/NaCS复合膜表面形态、机械性能(强度、韧性等)和溶胀性能的影响。结果发现,当壳聚糖/NaCS质量比为1：2时,该复合膜的表面较光滑,但膜表面有分散的不规则小孔,说明壳聚糖/NaCS复合膜是多孔膜。机械性能和溶胀性能的测定结果证明,壳聚糖和NaCS的最佳质量比在1：2左右。通过改变壳聚糖分子量、NaCS分子量和取代度,可以调节该复合膜的机械性质和溶胀性能。壳聚糖分子量越高,该复合膜的断裂伸长率越大；而NaCS分子量和取代度的增大则会导致该膜的溶胀率降低。此外,溶液pH对壳聚糖/NaCS复合膜溶胀率也有显著的影响。其次,采用胃肠道中的主要酶,包括胃蛋白酶、α-淀粉酶、胰蛋白酶、脂肪酶和纤维素酶,对壳聚糖、NaCS和壳聚糖/NaCS复合膜的降解性能进行了系统的研究。结果显示,胃蛋白酶、淀粉酶、胰蛋白酶和脂肪酶对壳聚糖都具有显著的水解活性,但不能降解NaCS.然而,纤维素酶表现出了较高的NaCS水解活性,壳聚糖水解活性却比较低。在壳聚糖/NaCS复合膜的降解研究中,发现其降解速率与壳聚糖和NaCS的分子量密切相关。此外,本论文还通过在pH分别为1.5、7.4和6.4的溶液中添加酶来模拟胃液、小肠液和结肠液,以此探讨壳聚糖/NaCS聚电解质复合物在胃肠道的降解特性。体外降解实验的结果表明,壳聚糖/NaCS复合膜的配方将直接影响其在胃肠道系统中的崩解时间和位置。如当壳聚糖分子量为563.3 kDa, NaCS分子量为169.7 kDa时,两者所形成的复合膜在模拟胃液中处理3h将崩解,而当壳聚糖和NaCS分子量分别为563.3 kDa和710.8 kDa时,复合膜在经历3h的胃液处理和6h的小肠液处理后,仍保持其表面的完整。由此可见,壳聚糖/NaCS聚电解质在胃肠道定位给药系统中具有良好的应用潜能。为研究壳聚糖/NaCS复合膜的药物控制释放特性,本论文还研究了该复合膜的药物渗透性能。采用包括扑热息痛、5-氨基水杨酸(5-ASA)和大肠杆菌蛋白质混合物等三种物质作为模型药物,对该复合膜的药物渗透性能进行了研究。结果表明,壳聚糖/NaCS复合膜的渗透性能与其溶胀性能密切相关。通常情况下,高溶胀率将导致高渗透系数。并且,该复合膜的渗透性能深受壳聚糖和NaCS的分子量以及pH的影响。此外,本论文还以扑热息痛作为模型药物研究了壳聚糖/NaCS复合膜在模拟胃肠液中对药物的渗透性能。结果表明,通过调整该膜的配方,可以使该膜分别实现胃、小肠和结肠定位释药的目的。鉴于壳聚糖/NaCS复合物在蛋白质定位释药方面表现出了较好的潜能,本论文以壳聚糖、NaCS和三聚磷酸钠(TPP)为原料,采用滴定法制备了壳聚糖/NaCS/TPP双层微球。通过FTIR分析了双层微球的内外层成分,发现其外层由壳聚糖、NaCS和TPP三种物质组成,而内层主要成分为壳聚糖和TPP。微球的制备时间和TPP浓度对微球的形态有显著的影响。TPP浓度较低时,该微球表面都较为光滑。但TPP浓度越高,反应时间越长,双层微球表面越容易皱缩,当反应时间为10 min时,所得到的双层微球表面最为光滑。此外,本论文以牛血清蛋白(BSA)为模型药物研究了该微球的包封率、载药量、溶胀性能和释药特性。TPP浓度对BSA包封率和载药量有重要的影响。未加TPP时,壳聚糖/NaCS微球具有较高的药物包封率(99.8%±0.1%)和载药量(37.5%±1.9%)。壳聚糖/TPP微球的载药量非常低,仅有4.5%±2.7%。与这两种微球相比,壳聚糖/NaCS/TPP双层微球也具有较高的药物包封率(79%以上)和载药量(18%以上)。为了研究壳聚糖/NaCS/TPP双层微球的溶胀性能和BSA体外释放性能,本论文选取pH分别为2.0、7.4和6.4的Na2HPO4-.柠檬酸缓冲液模拟胃液、小肠液和结肠液。结果表明,当TPP浓度在1.0%(w/v)或以上时,壳聚糖/NaCS/TPP双层微球出现了两步溶胀行为。而该微球的BSA释放特性与其溶胀实验结果并不一致。当TPP浓度为1.5%(w/v)时,双层微球在pH为2.0和6.4的缓冲溶液中几乎不释放BSA,但在pH 7.4下,经4h后该微球能够释放不足25%的BSA。在pH 6.4的缓冲溶液中加入适量纤维素酶能够使该微球的BSA释放量显著增大,在120 min时由原来的1.9%提高到50%以上。为了制备粒径均一的双层微球,本论文还采用微流控法研究了微球的制备。研究发现,采用2%(w/v)的壳聚糖和含5% Span 80的液体石蜡溶液,能够制备出粒径在100-200μm,粒径分布系数小于6%的双层微球。这为壳聚糖/NaCS复合物在制剂学上的发展提供了参考。更多还原

【Abstract】 Oral colon-specific drug delivery system is a novel drug delivery system, which targets drug to the colon for treatment of local or systemic disorders with proper methods. With colon-specific drug delivery system, drug could act directly on the lesion site, reduce side effect induced by drug release in the upper gastrointestinal tract, and enable biomacromolecules to be used for oral administration. Carriers play the key role in the oral colon-specific drug delivery systems. They have decisive effect on the release, as well as the target sites of active components. Among various carriers, natural polysaccharides are investigated more frequently for oral colon-specific delivery systems, owing to their good biocompatibility and biodegradability. Chitosan and sodium cellulose sulfate (NaCS) are both derivatives of natural polysaccharides with abundant resources. The combination of the two polymers could form polyelectrolyte complex (PEC) insoluble in water. It was reported that chitosan/NaCS PEC had great potential for colon-specific drug delivery systems. This work will focus on the mechanical and swelling properties, biodegradation and drug permeation of chitosan/NaCS complex film. In addition, we explored the preparation of microspheres based on chitosan and NaCS and its application in the delivery of proteinic drugs.Considering the problems in the measurement of NaCS properties, gel permeation chromatograph, BaSO4 turbidimetry and enzymatic degradation were employed to determine the molecular weight, structure and degradation properties of NaCS, as well as the influence of the preparation conditions. The results showed that increasing the reaction time would lead to lower molecular weight. Meanwhile, the polydispersity changed with the reaction time. With the increase of the reaction time, the polydispersity increased and obtained the maximum (5.16) at 6 h. However, further increase would cause a sharp decrease of polydispersity. When it came to the substitution degree of NaCS, it increased with increase of reaction time gradually, and kept about 0.5 from 6 h. In addition, the degradation of NaCS by cellulose decreased with the increase of the preparation time of NaCS. The results showed that the preparation conditions have great influence on the properties and application of NaCS.Effects of the weight of chitosan to NaCS on the morphology, the mechanical (strength and brittleness) and swelling properties of the complex film were studied. Obviously, chitosan/NaCS complex films were porous membrane. When chitosan/ NaCS ratio was 1:2, the complex film was smooth, with diverse, irregular pores. It was confirmed by mechanical and swelling studies that the optimum chitosan/NaCS ratio was about 1:2. The mechanical and swelling properties of chitosan/NaCS films could be controlled by adjusting chitosan Mw, and the Mw and substitution degree of NaCS. Higher elongation at break of the complex films would be obtained with chitosan of higher Mw. Meanwhile, the increase of the Mw and substitution degree of NaCS would cause the decrease of the swelling ratios. In addition, pH of the solution has great influence on the swelling ratios of chitosan/NaCS films.The biodegradation characteristics of chitosan, NaCS and chitosan/NaCS PEC films were investigated with main gastrointestinal enzymes, including pepsin, trypsin, lipase,α-amylase, and cellulase. The results showed that pepsin, amylase, trypsin and lipase have the appreciable hydrolytic activity to chitosan, but can not degrade NaCS. However, cellulase showed high cellulosic activity and low chitosanolytic activity. For the hydrolysis of chitosan/NaCS films, the degradation rates were greatly influenced by the molecular weights of chitosan and NaCS. Furthermore, in order to study biodegradation behaviors of chitosan/NaCS complex in gastrointestinal tract, the simulated gastric fluids (SGF), simulated intestinal fluids (SIF) and simulated colonic fluids (SCF) were prepared by adding enzymes to the solutions, respectively, with pH 1.5,7.4 and 6.4. In vitro tests showed that different formulations caused diverse disintegration time of the films through the gastrointestinal tract. For example, when the Mw of chitosan and NaCS were 563.3 and 169.7 kDa, respectively, the complex film would disintegrate after treatment in SGF for 3 h. However, when the Mw of chitosan and NaCS were 563.3 and 710.8 kDa, respectively, the complex film would keep intact after treatment in SGF for 3 h and SIF for 6 h. The results indicated the PEC based on chitosan and NaCS showed good potential for the gastrointestinal delivery systems.In order to assess the controlled drug release behavior of films composed of chitosan and sodium cellulose sulfate (NaCS), the permeability of drugs across chitosan/NaCS films were measured. The permeability experiments were conducted with three substances, including paracetamol,5-aminosalicylic acid (5-ASA) and protein mixture from Escherichia coli. The results showed that the permeability of chitosan/NaCS films was closely related to the swelling properties. In addition, both the permeability and swelling properties of chitosan/NaCS films were markedly influenced by the weight ratio of chitosan to NaCS, the molecular weight (Mw) of chitosan and NaCS as well as pH values. Furthermore, the permeability of paracetamol across the film was conducted in simulated gastrointestinal solutions for in vitro tests. The results showed that the films have the gastro-, intestine-and colon-targeted drug release properties, respectively, when they were exposed to the simulated gastrointestinal fluids in sequence.Chitosan/NaCS was found to have great potential in the delivery of protein in ourwork. So double-walled microspheres based on chitosan, NaCS and sodium tripolyphosphate (TPP) were prepared by dipping method. It was found by fourier transform infrared spectroscopy (FTIR) that the shell of the microspheres was composed of chitosan, NaCS and TPP, while the inner layer was based on chitosan and TPP. The concentration of TPP and the reaction time had marked influence on the morphology of the double-walled microspheres. When the concentration of TPP was low, the surface of the microspheres seemed smooth. Nevertherless, higher concentration of TPP and increase of the reaction time would cause shrinkage of the surface of the microspheres. When the reaction time was 10 min, smooth microspheres would be obtained.Furthermore, encapsulation efficiency (EE) and loading efficiency (LE) and the release behavior of the microspheres were investigated with bovine serum albumin (BSA) as the model drug. The concentration of TPP had great influence on EE and LE. Without addition of TPP, rather high EE (99.8%±0.1%) and LE (37.5%±1.9%) could be achieved. However, quite low LE (4.5%±2.7%) would be got with chitosan/TPP microspheres. As for chitosan/NaCS/TPP double-walled microspheres, high EE (above 79%) and LE (above 18%) could also be obtained. In order to assess in vitro release of BSA from chitosan/NaCS/TPP double-walled microspheres, the environment of SGF, SIF and SCF were simulated with solutions of pH 2.0,7.4 and 6.4, respectively. Two-step swelling performance was observed with concentration of TPP≥1.0%(w/v). However, the release of BSA from the microspheres was not in conformance with the swelling studies. It should be noted that almost no BSA released when the microspheres with 1.5% TPP was at pH 2.0 and 6.4. Less than 25% BSA was released even after treatment in the solution of pH7.4 for 4 h. Addition of cellulose to the solution of pH 6.4 would cause marked increase of BSA from the microsphers, from 1.9% to over 50% after 120 min.To prepare uniform double-walled microspheres, microfluid control was employed. Chitosan microspheres with diameter 100-200μm and distribution efficiency≤6% could be obtained with 2% (w/v) chitosan and liquid paraffin (5% Span 80). This work will be useful for the development of chitosan/NaCS complex in the technology of pharmaceutics.更多还原