Hesperetin-Loaded Nanoemulsion: Development, Characterization, Pro-Osteogenic and Anti-Inflammatory Activities

Objectives: The objective of this study was to develop and characterize a new hesperetin-loaded nanoemulsion (HT-NE) and evaluate in vitro its effect on I) the differentiation, metabolic activity and gene expression of osteoblasts and, II) the secretion of proinflammatory cytokines and matrix metalloproteinases (MMPs) in macrophages.
Methods: The HT-NE was obtained by the sonication method and characterized by dynamic light scattering, its encapsulation efficiency was analyzed by high-performance liquid chromatography and the particle size was registered by transmission electron microscopy. To evaluate its pro-osteogenic activity, Saos-2 osteoblasts were treated with non-cytotoxic concentrations of HT-NE and cell proliferation was measured by Alamar Blue assay. Mineralized calcium nodule formation was assessed by Alizarin Red, alkaline phosphatase (ALP) activity using a colorimetric assay and collagen organization by Picrosirius Red. Additionally, gene expression of Runx2, ALPL, and TGF-β were analyzed by RT-qPCR. In order to evaluate its anti-inflammatory effect, monocytes/macrophages U937 cells were stimulated with Fusobacterium nucleatum and treated with non-cytotoxic concentrations of HT-NE. The expression of IL-6, IL-8, IL-1β, TNF-α, MMP-1, MMP-3 and MMP-9 were measured by ELISA.
Results: The HT-NE formulation showed a colloidal stability, satisfactory efficiency of encapsulation with values of 74.07 ± 5,33%, values of droplet size less than 98 ± 1.33 nm, polydispersity index less than 0.58 ± 0.03 and zeta potential greater than -38.03 ± 0.80 mV. Non-cytotoxic concentrations showed a significant increase in cell proliferation, mineralization nodules formation, ALP activity, collagen formation and gene expression (p<0.05). Moreover, HT-NE significantly decreased cytokines and MMPs levels produced by macrophages (p<0,05).
Conclusions: Our study showed a great colloidal stability of a new HT-NE developed and suggests its promise pro-osteogenic and anti-inflammatory effects to treating inflammatory diseases involving bone. More studies exploring in vivo behavior of this new nanoemulsion are necessary to complement our findings.