多功能“薄皮大腔”PLGA纳微囊佐剂的构建及其作用机制

In recent year, microparticles and nanoparticles are widely studied as vaccine adjuvant and delivery system, which demonstrated significant advantages in terms of inducing cellular immunity. However, there still exist two major challenges in inducing ideal immune responses, one is the lower antigen uptake and transport efficiency by antigen presenting cells (APC), the other is the poorer controllable release of antigen in APC. In this project, multifunctional PLGA microcapsule with novel structure is fabricated to deal with these challenges. The high antigen-loading PLGA microcapsule with "thin wall and great cavity" and targetity will be prepared, which not only can contain more antigen in "great cavity" to increase antigen loading efficiency, but also can be rapidly degraded due to "thin wall". Furthermore, the addition of antigen release couple of ammonium bicarbonate (NH4CO3) in PLGA microcapsule can regulate antigen release in endosome and lysosome of APC. The hydrogen ions (H+) in endosome and lysosome of APC can penetrate into microcapsule and react with ammonium bicarbonate (NH4CO3) to generate gas of ammonia and carbon dioxide, which can break the thin wall of microcapsule leading to rapid antigen release. In addition, biomimetic material of dopamine will be employed for modification of PLGA microcapsule to introduce reactive group for coupling targeting ligand (anti-DEC-205) of dendritic cell (DC), which will greatly improve antigen-loading microcapsule uptake and transport efficiency by APC. At the same time, the action mechanism of PLGA microcapsule as vaccine adjuvant and delivery system will also be clarified.

近年来，纳微颗粒作为疫苗佐剂与递送系统被广泛研究，其在诱导细胞免疫方面具有显著优势，但存在被抗原提呈细胞（APC，如DC等）摄取与转运效率低、抗原在胞内可控释放性差的问题，导致免疫应答效果不理想。为此，本项目拟构建抗原载量高、可控释放、靶向呈递的"薄皮大腔"PLGA纳微囊。通过结构调控制备载抗原"薄皮大腔"PLGA纳微囊，可装载更多抗原，在纳微囊内结合使用抗原释放伴侣碳酸氢铵（NH4HCO3），其在APC胞内内体/溶酶体的弱酸环境下，H+进入纳微囊内与碳酸氢铵（NH4HCO3）反应生成气体，撑破薄壁，实现抗原的快速释放；在此基础上，通过仿生材料多巴胺对PLGA纳微囊表面进行镀层，在温和条件下引入活性基团并接枝靶向配体（anti-DEC-205），使纳微囊靶向DC等抗原提呈细胞，提高APC对载抗原纳微囊的摄取与转运。同时，研究这种多功能新结构PLGA纳微囊作为疫苗佐剂与递送系统的作用机制。

经过四年的研究，成功构建了“薄皮大腔”、具有pH敏感释放行为的PLGA纳微囊，被DC摄取后，能够通过pH敏感行为在胞内快速释放抗原，促进抗原向胞质分布，诱导强有力的细胞免疫；体内免疫结果证实所构建的纳微囊能够诱导B细胞、CD4+T细胞、CD8+T细胞的活化，并诱导强有力的CTL，显著增强细胞免疫，同时也能诱导优于铝盐佐剂的抗体分泌；在此基础上，在纳微囊表面通过镀层多巴胺偶联DC靶向配体，进一步提升了DC对抗原的摄取，进一步提升体液与细胞免疫水平；机制研究表面，纳微囊胞内pH敏感的胞内快速释放抗原行为，大大增强了抗原的MHCⅠ提呈，显著提升细胞免疫水平。