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Abstract
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 the 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 co-stimulatory 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 in vivo.
Supplementary materials
Title
Supplementary Information for "In Vitro Evaluation of Flame-Made Calcium Phosphate Nanoparticles for Antigen Delivery and Immunostimulation"
Description
Table S1 summarizes synthesis parameters for three CaP nanoparticle sizes (small, medium, large), including flame conditions, precursor concentrations, specific surface area, primary particle size (BET), hydrodynamic size, and zeta potential.
Figure S1 shows TEM-based size distributions for medium and large CaP primary particles with log-normal fitting.
Figure S2 presents OVA loading efficiency across CaP nanoparticle sizes and protein concentrations.
Figures S3 and S4 detail the flow cytometry gating for CD11c⁺ bone marrow-derived dendritic cells (BMDCs) and assess antigen uptake and processing via fluorescently labeled OVA conjugated with CaP nanoparticles.
Figure S5 illustrates flow plots showing co-stimulatory marker expression (CD86, CD80, CD40) on BMDCs post-stimulation, using fluorescence-minus-one (FMO) controls for accurate gating.
Figure S6 quantifies fold changes in marker upregulation after stimulation with CaP nanoparticles ± OVA, with statistical significance noted.
Figure S7 presents cytokine quantification results cytokine levels (IL-12p40, TNF-α) in BMDC supernatants post-treatment, measured by ELISA..
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