LEUKOTRIENE B4-LOADED MICROSPHERES: A NEW THERAPEUTIC STRATEGY TO MODULATE CELL ACTIVATION

TF14

Nicolete, R., Rius, C. , Piqueras,L., Jose, P.J., Sanz , M.J., Faccioli, L.H. 

Departamento de Análises Clínicas, Toxicológicas e Bromatológicas

Faculdade de Ciências Farmacêuticas de Ribeirão Preto

Universidade de São Paulo 

Ribeirão Preto-SP

Introduction: Leukotriene B₄ (LTB₄) is a potent inflammatory mediator that also stimulates the immune response. In addition, it promotes polymorphonuclear leukocyte phagocytosis, chemotaxis, chemokinesis and modulates cytokines/chemokines release. Regarding chemical instability of the leukotriene molecule, in the present study we assessed the immunomodulatory activities conferred by LTB₄ released from biodegradable poly-lactic coglycolic acid (PLGA) microspheres (MS). A previous oil-in-water emulsion solvent extraction-evaporation method was chosen to prepare LTB₄-loaded MS. Objective: To assess the activity of microencapsulated LTB₄ during in vitro and in vivo assays. Methodology: Previously characterized LTB₄-MS were employed to assess the inflammatory effects of microencapsulated LTB₄ within the mice cremasteric microcirculation by the use of intravital microscopy (IM). To extend these events to human, human umbilical vein and artery endothelial cells (HUVECs and HUAECs, respectively) were stimulated with LTB₄-MS or LTB₄ in solution. Nitrites and MCP-1 production were measured by Griess reaction and ELISA, respectively. Peroxisome proliferator-activated receptor-a (PPAR-a) expression was detected by Western Blot. Results: IM within the mice cremasteric microcirculation showed that after 4 h intraescrotal injection of 0.1 ml of LTB₄-MS, significant increases in leukocyte rolling flux (72.0 ± 5.2 vs. 46.0 ± 1.7 cells/min), adhesion (4.0 ± 1.0 vs. 1.2 ± 0.5 cells per 100 mm vessel) and emigration (7.3 ± 0.6 vs. 1.3 ± 1.0 cells per field) followed by significant decreases in the leukocyte rolling velocity (14.1 ± 0.9 vs. 25.9 ± 3.3 mm/s) were detected vs. values obtained in the control group (saline). HUVECs stimulation with LTB₄-MS for 4h provoked a significant increase in nitrites and MCP-1 levels when compared with those obtained with medium or unloaded MS. Similar results were obtained after 4 and 24h HUAECs stimulation. LTB₄-MS also activated PPAR-a receptor in a different manner when compared to unloaded MS or LTB₄ in solution, especially when a specific antagonist of the membrane receptor was used. Conclusion: LTB₄-loaded MS preserve the biological activity of the encapsulated mediator indicating their use as a new strategy to modulate cell activation, especially in the innate immune response.