<i>In Vitro</i> Evaluation of Flame-Made Calcium Phosphate Nanoparticles for Antigen Delivery and Immunostimulation

The use of nanoparticles in vaccine formulations has become increasingly prevalent, with the rise of subunit vaccines. However, the production of nanoparticles is often not scalable, presenting a significant challenge for large-scale vaccine manufacturing. Additionally, these nanoparticle delivery systems often require additional immunopotentiators to elicit a robust immune response, further complicating vaccine formulation. In this study, we explore the potential of flame-synthesized calcium phosphate (CaP) nanoparticles, produced via flame spray pyrolysis (FSP) (a highly reproducible and scalable method), as vaccine adjuvants capable of both antigen delivery and immunostimulation. We produced three different CaP nanoparticles with controlled crystallinity and size to screen for their immunostimulatory properties and evaluated their capacity to load and protect the model antigen ovalbumin (OVA) from enzymatic degradation. Our results show that all three CaP nanoparticles significantly enhance antigen internalization and processing by bone marrow-derived dendritic cells (BMDCs), critical for effective T cell activation. OVA conjugated with amorphous CaP nanoparticles outperformed crystalline CaP in increasing the expression of costimulatory markers CD86 and CD80 on BMDCs, as well as enhancing IL-6 production, indicating their potential as effective immunopotentiators. This dual functionality, in addition to the facile synthesis process, could simplify vaccine formulations by obviating the need for separate immunostimulatory agents. This work lays the foundation for further research to establish the flame-made CaP nanoparticle effectiveness and safety as adjuvants <i>in vivo</i>.