新型膜乳化法制备粒径均一聚乳酸微球及其作为疫苗佐剂的应用
文献类型：学位论文
| 作者 | 卫强 |
| 学位类别 | 博士 |
| 答辩日期 | 2008-06-08 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 过程工程研究所 |
| 导师 | 马光辉 |
| 关键词 | 快速膜乳化 乙肝疫苗 控释微球 单甲氧基聚乙二醇-聚乳酸共聚物 治疗性疫苗 |
| 其他题名 | Study on preparation of PLA microspheres by novel membrane emulsification technique and their application as vaccine adjuvant |
| 学位专业 | 生物化工 |
| 中文摘要 | 乙型肝炎是严重威胁人类健康的传染病之一，但迄今为止，对于乙型肝炎尚无有效的治疗药物，接种疫苗是目前预防和控制乙肝病毒的主要手段。将抗原包埋于聚乳酸微球内部免疫接种后，抗原在体内缓慢释放，由此产生持续的高抗体水平，可以减少接种次数，提高免疫覆盖率，而且以聚乳酸微球作为佐剂能够增强抗原被抗原提呈细胞吞噬的能力，增强抗原诱导的细胞免疫应答。但是现有的制备方法所得的微球粒径不均一、批次间重复性差，很难研究粒径与免疫效果的关系，也很难确定微球佐剂最佳的粒径范围；另外当前的研究中仍存在着抗原易失活和包埋率偏低等问题。本论文发展新型膜乳化技术与溶剂挥发/萃取法相结合的工艺，旨在制备在体内可以靶向抗原提呈细胞的聚乳酸微球佐剂，并进行高包埋率和高生物活性保持的载药微球和新型治疗性乙肝疫苗制剂的研究。 本论文工作分为三个部分。第一部分探讨新型膜乳化技术结合单乳-溶剂去除法制备粒径均一的空白微球的可行性，采用不同膜乳化技术来制备得到了不同粒径的聚乳酸微球和纳米微球，并调节制备工艺实现了对聚乳酸微球的结构控制。不同粒径和结构的聚乳酸微球的制备，为后续单剂疫苗微球制剂的开发和治疗性乙肝疫苗微球制剂的开发打下基础。第二部分采用快速膜乳化技术结合复乳-溶剂去除法制备粒径均一且高包埋率和高活性保持的载药微球。以溶菌酶为模型蛋白，采用乙酸乙酯作为有机溶剂制备的微球对溶菌酶的包埋率和活性保持高达97.5%和93.2%。在此基础上，开展了疫苗微球的研究工作。三种不同组成的材料：PLA40k、PELA40k(2kMPEG) 和 PELA40k(2kPEG) 制备微球的对乙肝表面抗原的包埋率分别是61.3%、75.4%和90.4%。两嵌段结构的PELA40k(2kMPEG)能更好的稳定初乳，因而获得最高的抗原包埋效率。乳液的固化速率影响内外水相的融合时间，因而也影响微球的包埋率。微球的粒径分布系数CV值在19%左右，平均粒径为1~2 µm，能够靶向抗原提呈细胞。复乳溶剂法制备载药微球时药物的损失可以内外水相的融合机制来分析，调节膜材的组成和乳液的固化速率是提高微球包埋效率的两种有效策略。第三部分采用第一部分工作中制备得到的聚乳酸微球作为抗原佐剂制备新型治疗性乙肝疫苗制剂及冻干粉剂。采用ELISPOT方法和CTL 杀伤试验作为评价细胞免疫效果的方法。以PLA微球为佐剂能同时显著增强HBsAg诱导的IFN-γ分泌水平和特异性CTL杀伤作用，呈现出较强的佐剂作用，而以铝盐为佐剂的商品化疫苗尽管对HBsAg诱导的IFN-γ分泌有一定的增强作用，但同时抑制HBsAg诱导的特异性CTL杀伤作用；在体液免疫方面，以PLA为佐剂的抗原制剂的也能达到铝佐剂疫苗的效果。 研究表明，用聚乳酸微球作为乙肝疫苗的佐剂，可以制备得到高包埋率和长效释放的乙肝疫苗微球制剂和治疗性乙肝疫苗制剂，为抗原的免疫接种和免疫治疗提供了一个新的思路。 |
| 英文摘要 | Hepatitis B is one of the most serious infectious diseases in the world. At present, no effective drug has been developed yet for chronic hepatitis B virus infection therapy, and immunization represents the only known way to prevent the spread of the virus. A single-shot vaccine can be made using microspheres as adjuvant, from which the encapsulated antigens are released under control over prolonged period, which would reduce the number of injections as well as enhance the immune response. Moreover, the microspheres with a diameter under 5 μm would be ideal for passive targeting of antigen presenting cells, and activated specific T cells to induce high antigen specific cellular immunization response. However, the size distribution of microspheres obtained by conventional methods is very broad, which is unfavorable for efficient adsorption in vivo and the precise control of antigen release. The objectives of this dissertation are to develop a novel membrane emulsification technique for preparation of uniform-sized polylactide (PLA) microspheres with high drug encapsulation efficiency, high bioactivity preservation, as well as to investigate the application of PLA microspheres in novel hepatitis B therapeutic vaccine. The dissertation is divided into three parts. The first part focused on using membrane emulsification techniques to prepare uniform-sized microspheres as vaccine adjuvant. PLA microspheres and nanoparticles could be effectively prepared by premix membrane emulsification technique. Porous microspheres and hollow microspheres were also prepared successfully. The second part focused on preparation of drug containing microspheres with high encapsulation efficiency and bioactivity preservation. Lysozyme was selected as a model protein, and the encapsulation efficiency and bioactivity preservation of entrapped lysozyme were 97.5% and 93.2%, respectively. Hepatitis B antigen was then encapsulated into microspheres to develop single-shot vaccine. The size of microspheres was about 1~2µm, and the coefficient of variation value (CV) was less than 20%. Compared with the triblock copolymer PELA40k(2kPEG), the diblock copolymer PELA40k(2kMPEG) yielded a more stable interfacial layer at the interface of oil and water phase, and thus was more suitable to stabilize primary emulsion and to protect the coalescence of inner water phase and external water phase, resulting in high encapsulation efficiency of antigen (90.4%). On the other hand, solidification rate affected on coalescence during microspheres, and the encapsulation efficiency increased with the increase of solidification rate of microspheres. The loss of encapsulated protein was ascribed to that the protein in the inner droplets tended to merge with external water phase. Improving the polymer properties and solidification rates were considered as two effective strategies to yield high encapsulation. The third part focused on preparation of novel hepatitis B therapeutic vaccine using PLA microspheres as adjvant. The enzyme linked immunosorbent spot (ELISPOT) assay and cytotoxic assay were used to evaluate the mice’s cellular immune response specific induced by HBsAg. The microspheres vaccine could effectively stimulate the mice splenocytes and CD8+ T lymphocytes to secret HBsAg-specific IFN-γ, and the levels of IFN-γ secretion were higher compared with commercial alum vaccine. The cytolytic assays also demonstrated that microspheres vaccine could enhance the cytotoxic activity induced by HBsAg. In addition, the level of anti-HBsAg titer obtained from microspheres vaccine reached to those of alum-vaccine. In conclusion, the uniform-sized PLA microspheres prepared by membrane emulsification technique have great potentials as adjuvant for single-shot vaccine and therapeutic vaccine. |
| 语种 | 中文 |
| 公开日期 | 2013-09-13 |
| 页码 | 142 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1119]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 卫强. 新型膜乳化法制备粒径均一聚乳酸微球及其作为疫苗佐剂的应用[D]. 过程工程研究所. 中国科学院过程工程研究所. 2008. |
入库方式： OAI收割
来源：过程工程研究所
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