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载重组人生长激素聚乳酸-聚乙二醇微囊的制备与应用研究
Alternative TitleStudy on preparation and application of sustained release PELA microcapsules loading rhGH
韦祎
Subtype博士
Thesis Advisor马光辉
2012-05-23
Degree Grantor中国科学院研究生院
Degree Discipline生物化工
Keyword快速膜乳化技术 聚乳酸-聚乙二醇共聚物 重组人生长激素 缓控释微囊 粒径均一性
Abstract重组人生长激素(rhGH)在临床上广泛应用于治疗矮小症、严重烧伤、艾滋病患者的脂肪代谢障碍等多种疾病。但rhGH的半衰期短,必须频繁注射才能达到有效的血药浓度,造成患者顺应性差。针对此问题,科研工作者们对rhGH缓释微囊进行了大量的研究,但目前仍普遍存在以下问题:微囊粒径不均一、药物突释率高(大于20%)、释放不完全、药物稳定性差等,至今仍无应用于临床的rhGH长效缓释微囊上市。为了攻克以上难题,本论文采用两亲性的聚乳酸-聚乙二醇共聚物(PELA)包埋生长激素,并使用快速膜乳化法与复乳法相结合的策略,旨在制备粒径均一性好、药物包埋率高、突释率低、可持续释放和药物活性保持良好的缓释微囊,并对制备过程的关键因素、PELA微囊保持药物稳定性的微观机理以及PELA微囊在体内的有效性及安全性进行研究。 本论文分为四个部分。第一部分对快速膜乳化法结合复乳法过程中影响微囊粒径均一性和表面形貌的因素进行了系统研究,发现过膜压力和油相溶剂种类分别对微囊的粒径均一性和表面形貌影响最为关键,选用乙酸乙酯作为油相溶剂,同时对油水比、外水相稳定剂PVA浓度以及固化时的搅拌速率进行优化,得到了粒径分布系数(CV值)在15.0%以下的光滑微囊,并可以通过选择不同孔径的膜来制备不同粒径的PELA微囊,实现了粒径可控。 第二部分是采用上述优化条件对rhGH进行了包埋,深入研究了PEG嵌段的比例对药物释放及稳定性的影响,并引入接触角结合原子力显微镜和石英晶体微天平技术从微观界面上观察材料对蛋白的吸附,用激光共聚焦显微镜和荧光探针技术监控微囊内部微环境的变化。结果表明,当PELA的PEG嵌段比例增加时,利于药物的持续释放,因为PELA亲水性的增长减少了材料与蛋白间的非特异性吸附,加快后期药物的释放速度,同时也利于维持蛋白药物的稳定性。进一步的动物实验结果表明PELA亲水性的增加大大改善了rhGH的药代动力学和药效学性质,同时增强了安全性。第三部分采用上述优化后的最佳的PELA膜材,进一步研究了初乳化制备方法和微囊粒径对释药行为的影响。结果表明:随着初乳化速度升高,微囊的包埋率增加,突释效应及蛋白释放速度明显降低。另外,微囊粒径也会影响药物包埋率和释放行为:随着微囊粒径的减小,包埋率降低,突释率升高,后期累积释放率也较高。在优化条件下,药物包埋率可以达到90%以上,同时获得最佳释放行为。论文第四部分从包埋率、体外释放、体外活性保留等方面,采用优化后的制备条件和PELA分子量,制备了最佳粒径的PELA微囊,并与传统的PLA 、PLGA微囊进行了对比。结果表明:相对于PLA和PLGA而言,PELA微囊具有高包埋率、低突释、持续释放且释放出的生长激素结构与构象保持较好的优点。进一步的动物实验结果表明注射PELA微囊的大鼠,其曲线下面积明显高于注射rhGH溶液组、PLA微囊组和PLGA微囊组;反应rhGH活性的因子IGF-1和IGFBP-3也呈现出同样的趋势,摘除垂体的大鼠模型实验进一步验证了PELA微囊的有效性。载药PELA微囊不表现出免疫原性,在机体内不产生炎症反应,对心、肝、肾等主要脏器也没有毒副作用,是一种安全的缓释载体。研究表明,采用快速膜乳化法与复乳法相结合的工艺制备粒径均一的两亲性PELA微囊不仅适用于包埋rhGH,还适用于其他蛋白多肽类药物,是一种很有应用前景的缓控释载体。
Other AbstractrhGH is widely used in clinical for treatment of pediatric short stature, serious burn and fat metabolism of AIDS patients. Due to its short half-life, rhGH therapy still suffers the burden of daily injection. However, presently, none of rhGH sustained release microcapsules is on the market due to several drawbacks including broad particle size distribution, a high burst release (above 20%), incomplete release, and protein denaturation after administration. Therefore, the overall goal of this research is to prepare amphiphilic PELA microcapsules with narrow size distribution by combining double emulsion method with premix membrane emulsification technique for sustained release of rhGH and realize high encapsulation efficiency, low initial burst and stability of rhGH. The dissertation is divided into four parts. The first part focused on the preparation of PELA microcapsules with uniform size and smooth surface. It was found that the transmembrane pressure and type of oil phase played important role on the size distribution and surface properties of microcapsules, respectively. By optimizing volume ratio of W1/O and O/W2, PVA concentration in external water phase, stirring rate and oil phase, smooth microcapsules of various sizes with CV value below 15% could be obtained using the membrane with different pore size. The second part investigated the influence of polymer hydrophilicity on rhGH incomplete release and stability profile. Contact-angle combined with QCM-D and AFM techniques were used to study the interaction between PELA and rhGH to elucidate the mechanism for incomplete protein release. Furthermore, the mechanisms responsible for rhGH stability were examined by monitoring the microenvironment pH change using CLSM and fluorescent probe. We further investigated the efficacy and safety of rhGH-PELA microcapsules in vivo. The more hydrophilic PELA microcapsules not only showed the highest amount of rhGH, IGF-1 and IGFBP-3 released, but also more efficacious and safer profiles. The third part investigated the effects of primary emulsion preparation methods and particle size on drug encapsulation efficiency and release profiles by using optimized PELA. It was found that encapsulation efficiency increased, initial release and cumulative release decreased when primary emulsification rate enhanced. In addition, with the decrease of particle size, the encapsulation efficiency declined but initial release and cumulative release increased. By optimizing the process parameters, the drug encapsulation efficiency of microcapsules prepared by SPG membrane emulsification technique could exceed 90%. The fourth part showed that PELA microcapsules exhibited advantages of high encapsulation efficiency, low initial burst, sustained release, and stability of compared with PLA and PLGA microcapsules. In addition, PELA microcapsules appear to be more efficacious in vivo in terms of weight gain, and elevation in IGF-1 and IGFBP-3 levels. Furthermore, rhGH PELA did not appear to be immunogenic with no or minimal inflammatory response or toxic effects in the body. All these results strongly suggested that PELA microcapsules have great potential as a clinically effective and safe system for sustained delivery of rhGH. In conclusion, the novel membrane emulsification technique, combined with W/O/W double emulsion method, is a potential technique to prepare amphiphilic microcapsules containing therapeutic protein and peptide drugs including rhGH.
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/1835
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
韦祎. 载重组人生长激素聚乳酸-聚乙二醇微囊的制备与应用研究[D]. 中国科学院研究生院,2012.
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