形貌调控与镀层修饰结合制备口服疫苗载体*

口服疫苗因其具有接种方便、能产生黏膜免疫等优点而备受关注,但胃肠道屏障、酸性环境和蛋白酶等不利条件制约了口服疫苗免疫效果的发挥。为提升其免疫效果,将形貌调控与镀层修饰策略结合制备新型口服疫苗载体,具体为将溶剂蒸发法与快速膜乳化法结合制备聚乳酸-羟基乙酸共聚物(PLGA)杆状颗粒,并采用能够增强免疫反应的β-葡聚糖及具有更高降解pH的硫醇化修饰的羟丙基甲基纤维素苯二甲酸酯(T-HPMCP)对PLGA杆状颗粒镀层修饰。在制备PLGA杆状颗粒时,通过对外水相条件的摸索制备出了适合小肠上皮细胞摄取的长度在2~4 μm、宽度在1~2 μm的PLGA杆状颗粒。体外实验结果表明通过T-HPMCP修饰的疫苗载体在酸性环境下保持稳定有利于抗原活性保护,同时能够在pH≥7.4时分解而使抗原释放。细胞和动物实验结果表明其特殊的杆状形貌可实现较高的肠道上皮摄取速率及转运效率,并且β-葡聚糖的修饰能活化树突状细胞(DC),提升OVA特异性IgA和IgG抗体水平。综上,制备的镀层PLGA杆状颗粒作为口服疫苗载体可提升机体免疫应答,为口服疫苗的研究提供了新的材料和思路。

Preparation of Oral Vaccine Carriers by Combining Morphology Control and Plating Modification

Oral vaccines have attracted much attention due to their advantages of high patient compliance, reduced generation of harmful waste, convenient vaccination, and their ability to cause mucosal immunity. However, unfavorable conditions such as the acidic environment of the stomach, proteases, intestinal mucus and tight junctions between intestinal epithelial cells make the gap between oral vaccines and injectable vaccines too large, which restricts the immune effect of oral vaccines. In order to improve the immune effect of oral vaccines, we combined morphology control and coating modification strategies to prepare a new type of oral vaccine carrier. Specifically, polylactic acid-glycolic acid copolymer (PLGA) rod-shaped particles were prepared by combining emulsion solvent evaporation method with fast membrane emulsification method, then β-glucan that enhances immune response and thiolated hydroxypropyl methyl cellulose phthalate (T-HPMCP) with higher degradation pH and stronger adhesion with intestinal epithelium were used for coating modification of PLGA rod-shaped particles. In the preparation of PLGA rod-shaped particles, the effects of PBS concentration and polyvinyl alcohol (PVA) concentration in the outer aqueous phase on the preparation of PLGA rod-shaped particles were explored. It is found that the deformation degree of PLGA particles first increased and then decreased with the increase of PBS concentration, but the deformation degree of PLGA particles always increased with the increase of PVA concentration. This is because PBS forms an electric double layer with -COO^- of PLGA, which makes the emulsion more stable and makes deformation more difficult to occur. Finally, the optimal formula was determined to prepare PLGA rod-shaped particles with a length of 2-4 μm and a width of 1-2 μm suitable for the uptake of small intestinal epithelial cells. It is found that the protein entrapment rate of PLGA rod-shaped particles is higher than that of PLGA spherical particles because the electric double layer makes the emulsion more stable. The results of in vitro experiments show that the vaccine carrier modified by T-HPMCP is stable in an acidic environment and the amount of released protein is negligible, which can prevent the antigen from being corroded by the acidic environment and is conducive to the protection of antigen activity. It can be decomposed at pH ≥ 7.4 and then release antigen. The results of cell experiments show that its special rod-shaped morphology can be taken up by Caco-2 cells faster and can be rapidly transported by the Caco-2 cell monolayer model constructed in transwell chambers, and the modification of β-glucan can also promote dendritic cells (DCs) secretion of surface molecules MHC-I, MHC-II and CD80 to activate DCs. The results of animal experiments showed that PLGA rod-shaped particles modified by β-glucan and T-HPCMP could increase the levels of OVA-specific IgA and IgG antibodies, which reached their maximum on the 28th day, and they promoted immune central memory T cells and CD8⁺ effects generation of memory T cells. In conclusion, the prepared coated PLGA rod-shaped particles can improve the immune response of the body as an oral vaccine carrier, thereby producing a better immune effect and providing new materials and ideas for the research of oral vaccines.