RESUMEN
Hyperglycemia-induced protein glycation and formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of diabetic complications and pathological biomineralization. Receptors for AGEs (RAGEs) mediate the generation of reactive oxygen species (ROS) via activation of NADPH-oxidase. It is conceivable that binding of glycated proteins with biomineral particles composed mainly of calcium carbonate and/or phosphate enhances their neutrophil-activating capacity and hence their proinflammatory properties. Our research managed to confirm this hypothesis. Human serum albumin (HSA) was glycated with methylglyoxal (MG), and HSA-MG was adsorbed onto mineral microparticles composed of calcium carbonate nanocrystals (vaterite polymorph, CC) or hydroxyapatite nanowires (CP). As scopoletin fluorescence has shown, H2O2 generation by neutrophils stimulated with HSA-MG was inhibited with diphenyleneiodonium chloride, wortmannin, genistein and EDTA, indicating a key role for NADPH-oxidase, protein tyrosine kinase, phosphatidylinositol 3-kinase and divalent ions (presumably Ca2+) in HSA-MG-induced neutrophil respiratory burst. Superoxide anion generation assessed by lucigenin-enhanced chemiluminescence (Luc-CL) was significantly enhanced by free HSA-MG and by both CC-HSA-MG and CP-HSA-MG microparticles. Comparing the concentrations of CC-bound and free HSA-MG, one could see that adsorption enhanced the neutrophil-activating capacity of HSA-MG.
Asunto(s)
Activación Neutrófila , Piruvaldehído , Carbonato de Calcio , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Peróxido de Hidrógeno , Minerales , NADP , NADPH Oxidasas/metabolismo , Piruvaldehído/farmacología , Albúmina Sérica , Albúmina Sérica Humana/química , Albúmina Sérica GlicadaRESUMEN
While the enteral delivery of proteolytic enzymes is widely established for combating many diseases as an alternative to antibiotic treatment, their local delivery only emerges as administration route enabling sustained release in a controlled manner on site. The latest requires the development of drug delivery systems suitable for encapsulation and preservation of enzymatic proteolytic activity. This study proposes hybrid microspheres made of mucin and biodegradable porous crystals of calcium carbonate (CC) as the carriers for chymotrypsin (CTR) delivery. CTR is impregnated into CC and hybrid CC/mucin (CCM) microspheres by means of sorption without any chemical modification. The loading of the CC with mucin enhances CTR retention on hybrid microspheres (adsorption capacity of ≈8.7 mg g-1 vs 4.7 mg g-1 ), recharging crystal surface due to the presence of mucin and diminishing the average pore diameter of the crystals from 25 to 8 nm. Mucin also retards recrystallization of vaterite into nonporous calcite improving stability of CCM microspheres upon storage. Proteolytic activity of CTR is preserved in both CC and CCM microspheres, being pH dependent. Temperature-induced inactivation of CTR significantly diminishes by CTR encapsulation into CC and CCM microspheres. Altogether, these findings indicate promises of hybrid mucin-vaterite microspheres for mucosal application of proteases.