负载两性霉素B壳聚糖-聚乳酸纳米粒的制备及其释药性能*☆

背景：两性霉素B为治疗深部真菌感染的首选药物，但该药无法通过血脑屏障而对隐球菌性脑膜炎的治疗效果甚微。利用纳米粒子作为药物载体的优势，通过相分离透析技术制备负载两性霉素B的壳聚糖-聚乳酸纳米粒子，有望克服两性霉素B的不足。 目的：对负载两性霉素B的壳聚糖-聚乳酸纳米粒进行表征，分析其体外药物释放能力。 设计、时间及地点：重复测量设计，于 2008-11/2009-04 在国家纳米科学中心纳米医学与生物实验室完成。 材料：壳聚糖，平均相对分子质量为3.4×105，脱乙酰度为93%，为上海卡伯工贸有限公司产品。两性霉素B为Sigma公司产品。 方法：在二甲基亚砜溶液中，在三乙胺存在下，通过壳聚糖和D,L-丙交酯的开环聚合反应能够生成壳聚糖-聚乳酸共聚物。该共聚物由亲水壳聚糖段和疏水聚乳酸段组成，在水中能够组装形成纳米粒子。两性霉素B通过相分离透析技术包载于纳米粒子中。 主要观察指标：激光粒度分析仪测定纳米颗粒的粒径大小、粒径分布，环境扫描电镜观察纳米颗粒的外观形态，紫外光谱分析负载两性霉素B的壳聚糖-聚乳酸纳米粒的包封率、载药量和释药性能。 结果：壳聚糖-聚乳酸纳米粒和负载两性霉素B的壳聚糖-聚乳酸纳米粒，其粒径分别为114 nm和153 nm(当丙交酯与壳聚糖摩尔比为11∶1时)。纳米粒子粒径分布较窄，呈球形。共聚物中丙交酯与壳聚糖摩尔比影响药物的包封率和载药量，随着丙交酯与壳聚糖摩尔比从11∶1到20∶1，包封率从(62.3±3.5)%增加到(90.7±2.8)%，载药量从(7.8±1.2)%增加到(12.3±1.4)%。随着聚乳酸段质量比增加，纳米粒子尺寸、包封率和载药量增加，而药物释放降低。 结论：开环聚合制备壳聚糖-聚乳酸共聚物及用相分离透析方法制备负载两性霉素B纳米粒简便可靠，负载两性霉素B后纳米粒径明显变大，且纳米粒对两性霉素B有很高的包封率，体外释药具有明显的缓释作用。 关键词：两性霉素B；壳聚糖；聚乳酸；纳米粒子；包封率；体外释放

Preparation and drug-release property of amphotericin B-loaded chitosan-polylactic acid nanoparticles

BACKGROUND: Amphotericin B is firstly used to treat deeply fungous infection; however, the therapeutic effect is poor due to inability to cross the blood-brain barrier (BBB). Chitosan-poly (lactide) (chitosan-co-PLA) copolymer were synthesized by phase separation dialysis technique to overcome the shortage of amphotericin B. OBJECTIVE: To study the representation and drug-release ability of amphotericin B-loaded chitosan-co-PLA nanoparticles. DESIGN, TlME AND SETTING: A repeated measurement design was performed at the Nano-medicine and Nano-biotech Laboratory of National Center for Nanoscience and Technology from November 2008 to April 2009. MATERIALS: Chitosan with the average molecular weight of 3.4×105 and 93% deacetylation degree was produced by KaBo BiochemicaI Co., Shanghai. Amphotericin B was purchased from Sigma, USA. METHODS: Chitosan-polylactide copolymers were synthesized by ring-opening polymerization of DL-lactide (DLLA) and water soluble chitosan in dimethyl sulfoxide solution in the presence of triethylamine. The chitosan-polylactide copolymers, consisting of hydrophilic chitosan and hydrophobic polylactide segments, could form nanoparticles in water. Amphotericin B was incorporated into chitosan-polylactide polymeric nanoparticles by the phase separation-dialysis method. MAIN OUTCOME MEASURES: Particles size and polydispersity index were detected by laser particle size analyzer. The morphology of amphotericin B-loaded chitosan-polylactide nanoparticles was observed by environmental scanning electron microscopy. In addition, encapsulation capability, loading content and in vitro drug release of the amphotericin B nanoparticles was analyzed by UV-spectrophotometer. RESULTS: The mean diameters of chitosan-polylactide nanoparticles and amphotericin B-loaded nanoparticles were 114 nm and 153 nm (D, L-lactide/chitosan molar ratio 11:1), respectively. The nanoparticles possessed a uniform particles size distribution and spherical in shape. The entrapment efficiency and drug loading depended mainly on the copolymer composition ratio of D, L-lactide to chitosan oligomer. The encapsulation efficiency increased from (62.3±3.5)% to (90.7±2.8)%, drug loading increased from (7.8±1.2) % to (12.3±1.4) % with increasing the molar ratio of D, L-lactide to chitosan oligomer from 11 to 20. As polylactide weight ratio increased, the nanoaprticles size, encapsulation efficiency and drug-loading content increased, and the drug release rate decreased. CONCLUSION: Both ring-opening polymerization and phase separation-dialysis method are reliable and simple. The particle diameter is large, the encapsulation efficiency is great, and the in vitro drug release is sustained.