Hesperetin-Loaded Nanoemulsion: In Vitro Modulation of Osteoclastogenesis and Periodontal Inflammatory Responses

Several adjunctive therapies have emerged for the treatment of periodontal diseases. Among them, nanotechnology combined with natural products has attracted considerable interest. Hesperetin is a flavonoid known for its biological activities, and its nanoencapsulation improves its therapeutic benefits by enhancing its physicochemical characteristics and stability. Therefore, this in vitro study aimed to evaluate the effects of a hesperetin-loaded nanoemulsion (HT-NE) against osteoclastogenesis and on inflammatory response induced by Fusobacterium nucleatum. First, the reproducibility and long-term stability of the formulation were assessed by determining particle size, polydispersity index, and zeta potential. Additionally, morphology was assessed using transmission electron microscopy (TEM). Then, the effect of HT-NE at the selected concentrations on osteoclasts differentiation and cellular metabolic activity were evaluated by fluorescence microscopy, analysis of osteoclastogenesis-related gene expression (Nfatc1, Ctsk, Oscar, MMP-9, Trap), and Western Blot analysis to Nfatc1 protein expression. To further investigate the anti-inflammatory potential of HT-NE, monocytes/macrophages were exposed to F. nucleatum,followed by ELISA-based quantification of IL-1β, IL-8, IL-6, TNF-α, and the matrix metalloproteinases MMP-1, MMP-3, and MMP-9. The formulation exhibited appropriate physicochemical properties. Additionally, HT-NE was able to inhibit the multinucleated osteoclast formation and reduce the expression of all genes tested and Nfatc1 protein. At the selected concentrations without significant effects on metabolic activity, HT-NE reduced osteoclastogenesis. Finally, treatment with HT-NE resulted in decreased levels of the evaluated cytokines and MMPs. HT-NE has effects against osteoclastogenesis and may inhibit pro-inflammatory cytokines and MMPs expressions, demonstrating its potential to modulate osteoinflammatory responses in vitro.