| 题名 | 壳聚糖温敏水凝胶作为鼻粘膜免疫递送系统的研究 |
| 作者 | 吴有斌 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-05-23 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 马光辉 |
| 关键词 | 鼻黏膜免疫 壳聚糖 温敏水凝胶 凝胶颗粒 抗原递送系统 |
| 其他题名 | Study of thermal-sensitive hydrogel based on chitosan as nasal immune delivery system |
| 学位专业 | 生物化工 |
| 中文摘要 | 鼻黏膜免疫具有一系列的优势，受到日益广泛的关注。但是，鼻黏膜纤毛的高频摆动能引起抗原的快速清除，同时，紧密排列的鼻黏膜上皮细胞层也构成了阻止抗原进入机体的天然屏障。为了克服上述鼻黏膜免疫的缺陷，本文基于壳聚糖这一天然材料，发展了一种新型的温敏水凝胶抗原递送系统。这种水凝胶在室温下为溶液状态，但在鼻腔温度下能发生胶凝。将该水凝胶作为鼻腔抗原递送系统，对不同类型的抗原进行了动物免疫实验评价。同时，利用其温敏性能，结合快速膜乳化技术和自固化方法，制备得到一种pH敏感的壳聚糖凝胶颗粒，研究了其作为鼻黏膜免疫载体的可行性。首先，将壳聚糖与环氧丙基三甲基氯化铵（GTMAC）反应，通过优化实验条件，制备得到了一系列具有精确季铵取代度的壳聚糖季铵盐（HTCC），将HTCC与甘油磷酸钠（GP）复配制备得到温度敏感性水凝胶，并探索了HTCC和GP的浓度，以及季铵取代度（QD）对水凝胶体系粘度和胶凝时间的影响。结合鼻腔清除异物的半衰期，优选出了具有最佳粘度和胶凝时间的凝胶体系组成：HTCC，0.35 g；GP，1.0 g；QD：79.5%。在此基础上，进一步对水凝胶的安全性进行了系统的评价，结果表明，该水凝胶毒性低，具有良好的生物相容性。将温敏水凝胶体系作为H5N1流感裂解抗原的鼻黏膜免疫递送系统，在动物水平进行了评价。结果显示，水凝胶-抗原混合物能诱导显著的系统IgG（包括IgG1和IgG2a亚型）和呼吸道黏膜（鼻腔和肺）IgA的抗体反应，诱导产生了高水平的Th1/Th2细胞因子，且不改变细胞和体液免疫反应的平衡，并能明显激活NALT淋巴细胞，刺激产生NALT细胞的免疫记忆。温敏水凝胶抗原体系免疫小鼠后产生的抗体以及细胞因子的分泌水平优于或者能达到商品化的MF59佐剂组水平。进一步的机理研究发现，水凝胶通过溶液-凝胶的转变显著延长抗原在鼻腔的停留时间，并借助其可逆性破坏紧密连接蛋白ZO-1的能力打开上皮细胞间的通道，从而增强抗原的渗透，实现鼻腔抗原的高效利用。为了验证温敏水凝胶对不同类型鼻腔免疫的可行性，采用人腺病毒为载体的埃博拉膜蛋白抗原（Ad-GPZ）进行了小鼠滴鼻免疫，结果表明HTCC取代度为79.5%和60%的水凝胶诱导产生最高的系统和黏膜抗体水平。与H5N1的平衡的Th1/Th2免疫结果不同，水凝胶病毒抗原体系使得机体免疫显著偏向细胞免疫方向（Th1），然而免疫后产生的细胞因子水平仍然很低。进一步通过黏膜和注射的组合免疫方式，成功地诱导高水平的系统和黏膜免疫水平，并同时增强了细胞因子介导的T细胞活化。基于温敏水凝胶良好的pH敏感性和温度敏感性，进一步将该体系与快速膜乳化法相结合，制备得到粒径均一的凝胶颗粒（CHPs），粒径为1.6 μm左右。装载H5N1流感裂解抗原后，在pH 5环境下，抗原能快速释放，中性或碱性环境释放缓慢。以髓源DC为平台进行的细胞水平评价结果表明，CHPs借助其pH敏感性在溶酶体实现靶向抗原释放，激活且上调细胞共刺激分子的表达，并通过溶酶体逃逸进而增强抗原特异性的细胞免疫应答。本论文针对鼻黏膜免疫的问题及传统鼻腔疫苗剂型的不足，基于壳聚糖材料发展出了一种新型的鼻腔抗原递送系统，显示出了对不同类型抗原的鼻黏膜递送能力。此外，利用快速膜乳化技术结合自固化方法制备出了壳聚糖水凝胶颗粒，并展示出了良好的细胞免疫水平的增强能力，对病毒或胞内感染性疾病的免疫以及治疗性疫苗的开发具有显著的优势。 |
| 英文摘要 | Nasal immunization attracted increasing attention in recent years because of its excellent advantages. However, few of nasal vaccines achieved success in clinical use due to several reasons. Firstly, nasal cilia can beat in a high frequency, leading to the short antigen residence time in nasal cavity. Secondly, the tightly arranged epithelial cell forms a hard barrier to prevent the foreign materials from taking root in nasal mucosa. To solve these problems, we developed a novel antigen delivery system based on chitosan thermal-sensitive hydrogel, which can undergo sol-gel transition at body temperature. As nasal antigen delivery system, it showed potential application for different types of vaccines. Furthermore, a kind of pH sensitive nanogel particle was prepared based on this hydrogel by SPG membrane emulsification technique and self -solidification method. As antigen adjuvant, it showed the ability of enhancing cellular response by in vivo DC evaluation. At the first, a series of quarternized HTCC was synthesized from chitosan with GTMAC. After that, the thermal-sensitive hydrogel was prepared by simply mixing the HTCC and GP. The influence of GP concentration, HTCC concentration, and QD on the viscosity and gelation time was discussed, and then an ideal composition which was the most suitable for nasal administration was selected. Besides, safety evaluation of this hydrogel displayed little toxic to the nasal epithelial cell, nasal mucosa, and other organs. Subsequently, H5N1 influenza split antigen was used to evaluate the efficiency of the selected hydrogel as nasal delivery system. It suggested that hydrogel significantly enhanced the system IgG responses and mucosal IgA responses, as well as Th1 and Th2 cytokines level without change the quality of immunity, and it also boosted the nasal immune memory, which could reach or exceed the level induced by MF59 adjuvant. Further exploration displayed that hydrogel could significantly prolong the antigen residence time in nasal cavity and enhance the antigen penetration through the nasal mucosa by opening the cell space between nasal epithelial cells. This phenomenon was caused by the ability of hydrogel’s reversible damage to the ZO-1 protein, a tight conjugation localized in nasal mucosa. Next, we discussed the potential of hydrogel as the delivery system of Ad-GPZ antigen. It showed that QD-79.5 and QD-60 hydrogels which quaternization degrees were 79.5% and 60%, respectively, boosted the highest system and mucosal antibody responses and biased the immunity to Th1 type response. To achieve the same high cytokines level and antibody responses as intramuscular (IM) immunization, a combined procedure was adopted by mixing IM and intranasal (IN) immune routes. Finally, we successfully achieved the double enhancement of cellular and humoral immune responses. Lastly, chitosan was further formulated into a new hydrogel particulate adjuvant with a particle size of 1.6 μm by SPG membrane emulsification technique. It displayed that loaded antigen can release quckly under pH 5.5, while kept slow release at pH 7.4. This adjuvant up regulated the expression of costimulatory molecules of DCs, suggestting the ability of enhancing cellular immune responses. The obvious lysosome-escaping of antigens by CLSM observation confirmed this result. In conclusion, a novel nasal antigen delivery system and a potent nasal adjuvant were developed in this thesis. The thermal-sensitive hydrogel showed a universal ability of delivering different kinds of nasal antigens. The particulate adjuvant exhibited a promising picture for the vaccines that need strong cellular immune responses. |
| 语种 | 中文 |
| 公开日期 | 2013-09-25 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1836]  |
| 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 | 吴有斌. 壳聚糖温敏水凝胶作为鼻粘膜免疫递送系统的研究[D]. 中国科学院研究生院. 2012. |
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