Biodegradable microspheres containing leukotriene B(4) and cell-free antigens from Histoplasma capsulatum activate murine bone marrow-derived macrophages.

Because of the potential protective role of leukotrienes (LTs) in histoplasmosis and the therapeutic and prophylactic effects of cell-free antigens from Histoplasmacapsulatum (CFAgs), the aim of this study was to develop and characterise biodegradable LTB(4)/CFAgs-loaded microspheres (MS) that could promote cellular activation for future immunisation purposes. LTB(4)/CFAgs-loaded MS that were developed through a double emulsion/extraction process were characterised according to their size, zeta potential, morphology, entrapment efficiency and in vitro release kinetics. We evaluated the uptake of LTB(4)/CFAgs-loaded MS by bone marrow derived-macrophages (BMDM). The TNF-α and chemokines, and nitrite production, in the supernatant of BMDM cultures were analysed by enzyme-linked immunosorbent assay (ELISA) and Griess reaction, respectively. We found an instantaneous release of CFAgs and a prolonged release of LTB(4) from the poly-(d,l-lactide-co-glycolide) (PLGA) MS. The microencapsulation process did not alter the zeta potential nor the spherical morphology of the MS. The appropriate size of the LTB(4)/CFAgs-loaded MS (smaller than 10µm) enabled the efficient uptake by BMDM and also induced TNF-α, CXCL1/KC, CCL2/MCP-1, CCL5/RANTES and nitrite oxide release by these cells. In conclusion, the biodegradable LTB(4)/CFAgs-loaded MS were able to efficiently activate murine BMDM and thereby have the potential to be used in an effective vaccine against H. capsulatum infection.

The uptake of PLGA micro or nanoparticles by macrophages provokes distinct in vitro inflammatory response.

Biodegradable micro/nanoparticles generated from PLGA have recently attracted attention due to their clinically proven biocompatibility, especially for immunization purposes. These polymeric particulate delivery systems are able to present antigens and activate both humoral and cellular responses. Many studies have discussed the ideal size of these particles in contributing to the generation of the different types of immune response. However, these studies do not demonstrate the effect of micro or nanoparticles, without any encapsulated bioactive, on phagocytic cells after the uptake process. In this context, the aim of this study was to analyze the in vitro inflammatory behavior of J774 murine macrophages after particles' uptake, since nano/microparticles per se can differently activate phagocytic cells, using or not appropriate receptors, inducing distinct inflammatory responses. An o/w emulsion solvent extraction-evaporation method was chosen to prepare the particles. We determined their diameters, zeta potential and morphology. Fluorescent particles' uptake by J774 murine "macrophage-like" cells was also analyzed. To evaluate the in vitro inflammatory profile of these cells after micro or nanoparticles' uptake, we conducted NF-κB translocation assay by confocal microscopy and also determined the pro-inflammatory cytokines production provoked by the particles.

Characterization and in vitro activities of cell-free antigens from Histoplasma capsulatum-loaded biodegradable microspheres.

In the last decades, the incidence of histoplasmosis, a pulmonary fungal disease caused by Histoplasma capsulatum, has increased worldwide. In this context, vaccines for the prevention of this infection or therapies are necessary. Cell-free antigens (CFAgs) from H. capsulatum when administered for murine immunization purposes are able to confer protection and control of the infection, since they activate cellular immunity. However, the most of vaccination procedures need several antigens administrations and immunoadjuvants, which are not approved for use in humans. The aim of this study was to develop and characterize a vaccination approach using biodegradable PLGA microspheres (MS) that could allow the controlled and/or sustained release of the encapsulated antigens from H. capsulatum. CFAgs-loaded MS presented a size less than 10 microm, were marked engulfed by bone marrow-derived macrophages (BMDM phi) and induced the nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by these cells. Our data show that CFAgs-loaded MS induce cell activation, suggesting an immunostimulant effect to be further investigated during immunization procedures. CFAgs-loaded MS present potential to be used as vaccine in order to confer protection against H. capsulatum infection.