N-琥珀酰壳聚糖的合成及其包载槐定碱微球制备工艺的研究

【作者】 张玲；

【导师】 吴清；

【作者基本信息】 北京中医药大学 ， 中药制药， 2007， 硕士

【摘要】 目的:壳聚糖及其衍生物是各种药物可控释放载体中很有前途的候选辅料,但因壳聚糖仅溶于一些酸性介质,而不能溶于水,在很大程度上限制了它的推广应用。N-琥珀酰壳聚糖是壳聚糖琥珀酰化的衍生物,其钠盐形式可溶于水,具有更大的适用性。生物学研究结果表明水溶性N-琥珀酰壳聚糖较壳聚糖具有更低的毒性、更长的体内半衰期,在体内的降解速度较慢,并能选择性地蓄积于肿瘤组织,是一种优良的抗肿瘤药物的靶向载体。由于市场上尚未有N-琥珀酰壳聚糖出售,因此本课题的目的是探讨N-琥珀酰壳聚糖的合成工艺,并考察其对几种肿瘤细胞的亲和性,最后进行N-琥珀酰壳聚糖作为水溶性药物槐定碱微球包载材料的可行性研究。方法:(1)N-琥珀酰壳聚糖的合成工艺研究:用化学法降解得到不同分子量范围的壳聚糖,与丁二酸酐反应生成不同分子量范围的N-琥珀酰壳聚糖,通过其取代度、溶解性和红外光谱的测定,验证并确定N-琥珀酰壳聚糖的合成工艺。(2)N-琥珀酰壳聚糖与肿瘤细胞亲和性实验的研究:不同分子量的N-琥珀酰壳聚糖用异硫氰酸荧光素进行荧光标记后,分别与人胚肺成纤维细胞、肺癌、乳腺癌、肝癌、胰腺癌、结肠癌细胞共孵育,通过荧光显微镜和流式细胞仪检测细胞的荧光强度来确定其与肿瘤细胞亲和性的强弱。(3)槐定碱N-琥珀酰壳聚糖微球制备工艺的研究:通过单因素考察和星点设计-效应面法优化了用乳化-交联法制备槐定碱微球的制备工艺,并对所得微球进行了体外释药机制研究。结果:(1)确定的N-琥珀酰壳聚糖的合成工艺:壳聚糖用醋酸溶解后用丙酮稀释,滴加丁二酸酐的丙酮溶液常温下反应5小时,壳聚糖与丁二酸酐的配比为1:2,过滤后滤饼用无水乙醇分散,NaOH调pH值到8～10,过滤即得,样品的平均取代度为0.369,水溶性符合要求,红外图谱表明合成了N-琥珀酰壳聚糖。(2)除了分子量最大的N-琥珀酰壳聚糖样品外,其余四种分子量的N-琥珀酰壳聚糖与肺癌A549细胞的亲和性随着分子量的降低而逐渐增强。N-琥珀酰壳聚糖与肺癌A549细胞的亲和性强于正常人肺细胞及其它肿瘤细胞,可选择性地与肺癌细胞结合。在实验中确定的安全剂量下未发现对各种细胞有任何毒性反应。(3)优化的用乳化-交联法制备槐定碱微球的最佳工艺为:药物与N-琥珀酰壳聚糖投药比为1:10,2%N-琥珀酰壳聚糖为水相,液体石蜡为油相,水/油相比例为1:8,2%Span-80为乳化剂,乳化45min ,2.5%的甲醛为交联剂,40℃加热交联固化5h,冰浴硬化2h。最佳工艺制备的微球平均粒径为12.49μm,包封率为73.2%,开始释放较快,2小时释放累积达36%,后释放缓慢,24小时累积释放达70%,但24小时后,释放趋于平缓,体外释放符合Higuchi方程。结论:证明了合成的N-琥珀酰壳聚糖与肺癌A549细胞有较强亲和性,优选了槐定碱N-琥珀酰壳聚糖微球的制备工艺,为N-琥珀酰壳聚糖作为一种新型的水溶性药物的靶向载体,尤其是肺靶向载体提供了实验依据。更多还原

【Abstract】 Objective:Chitoson and it’s derivatives are ideal candidates for sustained and controlled drugrelease formulations. They have excellent prorerties. such as, biocompatibility, biodegraded,non-toxicity, adsorption properties, etc. N-Succinyl-chitosan is one of the derivative ofchitoson, it is water-soluble, so it is more useful. This paper’s objective was to compoundN-Succinyl-chitosan,andthentotesttheaffinitiesbetweenN-Succinyl-chitosanandsomekindof cancer cells. At last , we wanted to test that if it was feasible to prepare sophoridinemicrospherewithN-Succinyl-chitosan.Method:(1) A series of chitosans with different molecular weight were first prepared bychemical degradation. They were then used to synthesize N-Succinyl-chitosan with thesuccinate according to different proportion.The production separately be maded to the KBrcompressed tablet to do the infrared spectrum to adopt the conductometric titration todetermine the degree of substitution. According to the degree of substitution to make sure thecompounding method. (2) N-Succinyl-chitosan labeled with a fluorescein FITC. They wereincubated with A549 lung cancer cells and other kind of cancer cells for affinity test .Fluorescence intensity of cells was determined by flow cytometry to assess the affinity ofN-Succinyl-chitosan for A549 lung cancer cells.(3) Optimized the preparation technology byusing the emulsifying-chemical cross-linking method through the separate factors testing andthecentralcompositedesign-responsesurfacemethodology.Result:(1)We confirmed the optimum synthesis condition and the process flow ofN-Succinyl-chitosan. the average degree of substitution is 0.35.(2)The results showed that theaffinity of N-Succinyl-chitosan for A549 lung cancer cells was strong and declined withincreasing their molecular weight except the sample of the largest molecular weight.(3) Theoptimum preparation technology was: the N-succinyl-chitosan concentration was 2%,the ratioofW/O volume was 1:8, the emulsifyingagent concentration was 2%Span-80,the emulsifyingtime was 45 min,the cross-linking agent concentration was 2.5% formaldehyde,thecross-linkingtemperatureandtimewas40℃and 5h,2hice-hardness.Theaverageparticlesizewas 12.49μm andtheloadingefficiency was 73.2%.Thereleaseof drugfrom themicrospheresindicatedthatthe microspheresplayedaroleinsustainedrelease.Conclusion: N-Succinyl-chitosan is promising in the carriers of lung-targeting drug deliverysystem.更多还原