鞣酸与Al（Ⅲ）配位微囊用于pH响应性疫苗递送的研究

Tannic acid-Aluminum （Ⅲ） coordination-enabled microcapsules for pH-responsive protein vaccine delivery

目的 建立简便温和的抗原负载方法,制备具有pH响应性和可生物降解性微囊,实现抗原的有效递送.方法 先采用共沉淀法将鸡卵白蛋白（OVA）包埋于CaCO3颗粒内,然后以此CaCO3颗粒为模板,使鞣酸（TA）与Al（Ⅲ）通过金属有机配位键在模板表面形成涂层,再采用乙二胺四乙酸二钠除去CaCO3模板,得到载有OVA的TA-Al（Ⅲ）微囊[OVA@TA-Al（Ⅲ）微囊].采用场发射扫描电子显微镜、透射电子显微镜、X射线能谱仪和原子力显微镜对微囊进行表征,激光扫描共聚焦显微镜观察OVA的分布情况,考察微囊在不同pH值磷酸盐缓冲液中OVA的累积释放率,噻唑蓝实验检测微囊对永生化的小鼠树突状细胞DC2.4的细胞毒性,激光扫描共聚焦显微镜观察DC2.4细胞对微囊的吞噬情况.结果 场发射扫描电子显微镜和透射电子显微镜结果显示,OVA@TA-Al（Ⅲ）微囊的结构完整,直径约4μm,呈中空、塌陷状态.X射线能谱图显示,微囊共存在C、O、Al、Si和Na 5种元素,其中C、Al及部分O元素属于微囊的组成.原子力显微镜图显示,微囊囊壁厚度均一（约16 nm）,具有超薄的囊壁.激光扫描共聚焦显微镜结果显示,OVA均匀分布于CaCO3颗粒中.配位键的pH敏感性使得OVA@TA-Al（Ⅲ）微囊具有pH响应性,此外微囊还具有良好的生物相容性,DC2.4细胞对微囊亦具有较好的吞噬能力.结论 建立了一种简便温和的抗原负载方法,制备的微囊可实现抗原的有效负载和pH响应性递送,有望作为一种新型抗原递送载体用于临床研究.

Objective To establish a simple and gentle antigen loading method to prepare pH-responsive and biodegradable microcapsules for efficient antigen delivery.Methods Co-precipitation method was used to embed chicken egg albumin （OVA） in CaCO3 particles.Then,TA and Al （Ⅲ） were coated on the surface of CaCO3 particles template by metal-organic coordination bonds.The CaCO3 template was removed from disodium edetate to obtain TA-Al（Ⅲ） microcapsules carrying OVA,i.e.the OVA@TA-Al（Ⅲ） microcapsules.The microcapsules were characterized by field emission scanning electron microscopy,transmission electron microscopy,X-ray energy spectrometry and atomic force microscopy.The distribution of OVA in the microcapsules was observed by laser scanning confocal microscopy.The cumulative release rate of OVA in the microcapsules at different pH phosphate buffers was also investigated.The cytotoxicity of the microcapsules on immortalized mouse dendritic cells DC2.4 was observed by thiazolyl blue assay.The phagocytosis of the microcapsules by DC2.4 cells was observed by laser scanning confocal microscopy.Results The results of field emission scanning electron microscope and transmission electron microscopy showed that the OVA@TA-Al（Ⅲ） microcapsules have a intact structure and a hollow and collapsed appearance with a diameter of about 4 μm.X-ray energy spectrum showed that there are five kinds of elements,i.e.C,O,Al,Si and Na,in the microcapsules,among which C,Al and some O elements belong to the composition of the microcapsules.Atomic force microscopy showed that the microcapsules have an ultra-thin wall,and the walls of the microcapsules are uniform in thickness （about 16 nm）.Laser scanning confocal microscopy showed that OVAs were evenly distributed in the CaCO3 particles.Moreover,the pH sensitivity of the coordination bond makes the OVA@TA-Al（Ⅲ） microcapsules have pH responsiveness.In addition,the microcapsules also have good biocompatibility,and the DC2.4 cells also have good phagocytic ability to the microcapsules.Conclusion A simple and gentle antigen-encapsuling method was developed to achieve effective antigen payload and pH responsive delivery.The prepared microcapsules are expected to be used as a novel antigen delivery vector for clinical research.