Facile fabrication of varisized calcium carbonate microspheres as vaccine adjuvants

Functional calcium carbonate (CaCO₃) particles of micron and submicron sizes used in catalysis and biomedicine have attracted considerable attention for decades. In this paper, the process parameters for CaCO₃ crystallization were systematically investigated. Our experimental results demonstrated the significance of temperature during fabrication. Under the optimized conditions, various uniform-sized and spherical CaCO₃ microparticles (MPs) with average diameters from 0.8 μm to 5 μm were facilely and rapidly fabricated via different mixing strategies including mechanical stirring, homogenization, and ultrasonication. The physicochemical characteristics of the CaCO₃ microspheres were evaluated. And, the hepatitis B surface antigen (HBsAg) used as a model antigen was encapsulated into the particles (1 μm and 4 μm) for investigating the immune responses elicited after vaccination. In vitro, dendritic cells (DCs) were significantly activated by the MP-based vaccine formulations with up-regulated co-stimulatory molecules expression of CD40 and CD83. After immunization, CaCO₃ MPs loaded with HBsAg induced greater lymphocyte activation, more cytokine secretion, higher antigen-specific IgG titers and more memory T cell generation to protect against reinfection. Therefore, the CaCO₃ MPs, especially the 1 μm particles, could induce strong cellular and humoral immune responses, probably because of easier uptake and more efficient antigen-presentation by DCs. With the advantages of good biocompatibility, high loading capacity and easy preparation, they could be potentially useful as vaccine adjuvants. These results might provide further design principles for potent inorganic particulate adjuvant and delivery systems.