Formulation and Investigation of the In Vitro Immunostimulatory Potential of Microparticulate Guanine-α-D-Fructose in Vaccine Candidates.

BACKGROUND/OBJECTIVES: Particulate vaccine formulations require delivery systems and immunostimulatory components that support antigen-presenting cell interaction while maintaining formulation stability and cytocompatibility. This study examines Guanine-α-D-Fructose (GDF), a guanine-fructose small-molecule immunostimulatory candidate, incorporated into poly(lactic acid-co-glycolic acid) (PLGA) microparticles and benchmarked in vitro against established adjuvant formulations containing Alum or AddaVax. METHODS: GDF-loaded microparticles were prepared and characterized for particle size, morphology, surface charge, entrapment efficiency, and release behavior. Their in vitro immunostimulatory activity was evaluated using murine dendritic cells. To explore formulation compatibility, GDF MPs were tested in combination with multiple particulate antigen formulations, including measles, gonorrhea, SARS-CoV-2, influenza A (H3N2), and Zika. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was utilized to measure cell viability, while nitric oxide generation was measured with the Griess assay. Dendritic cell activation-associated surface marker expression was analyzed by flow cytometry using MHC I, MHC II, CD80, and CD40. RESULTS: The particles showed consistent spherical morphology, sustained GDF release, and favorable cytocompatibility at concentrations up to 250 µg/mL, with reduced viability at higher concentrations. GDF MPs increased nitric oxide production and activation-associated marker expression, with responses benchmarked against Alum and AddaVax and comparable in several assay conditions. Increased autophagosome-associated fluorescence was also observed, suggesting modulation of autophagy-related cellular activity; however, direct antigen-processing or antigen-presentation assays are still required. CONCLUSIONS: Overall, these findings support GDF-loaded PLGA microparticles as an in vitro immunostimulatory particulate formulation suitable for further mechanistic and in vivo evaluation in vaccine-related systems.