Development and evaluation of nanofibers and the nanostructured lipid carrier associated with hesperitin as a therapeutic potential in the repair of periodontal tissues

Abstract

The development of membranes for bone or tissue regeneration, with good mechanical properties, predictable rate of degradation, low cost, and structure similar to the extracellular matrix is of great interest to the area of nanobiotechnology and tissue engineering. Within this context, scaffolds of nanofibers present morphological characteristics capable of promoting greater cell/matrix-cell interaction, as well as the incorporation of bioactive molecules, reinforcing their biological properties. Hesperitin (a bioactive molecule) is a hesperidin metabolite belonging to the subgroup of flavonones with effective therapeutic properties such as antioxidant, anti-inflammatory, anticancer, neuroprotective, analgesic and beneficial effects on bone metabolism, thus being considered as a new alternative form of prevent and/or treat infections. The objective of STUDY 1 will be to develop a nanofiber scaffold associated with hesperitin in order to obtain a biocompatible and bioactive material for application in periodontal regeneration. Initially, in objective 1, the action of hesperitin in the expression of inflammatory mediators, inflammatory genes and signaling pathways in cell culture like RAW 264.7 macrophages stimulated by LPS will be evaluated by Bioplex 200, RT-PCR and Luminex Multi-pathway Signaling Kit. In sequence, in objective 2, expression of 23 genes regulating bone metabolism in osteogenesis and osteoclastogenesis will be evaluated in MC3T3-E1 pre-osteoblastic cells and analyzed in RT2 Profiler PCR microarray, formation of mineralized nodules, organization and maturation of collagen, differentiation of osteoclasts by immunohistochemistry of TRAP and activity of osteoclasts and reabsorption points. In objective 3, scaffolds of poly (- caprolactone) nanofibers will be characterized using the electrospinning technique. In order to evaluate the viability, adhesion and cell spread on nanofiber scaffolds, pre-osteoblastic MC3T3-E1 cells will be used. Finally, in objective 4, hesperitin will be added to the scaffolds and the expression of inflammatory mediators, as well as the differentiation and metabolic activity of osteoblasts and osteoclasts will be evaluated as described in objectives 1 and 2. The objective of STUDY 2 will be to develop a nanostructured lipid carrier containing hesperitin capable of promoting its use for regeneration and treatment of periodontal disease. Initially, in objective 1, nanostructured lipid systems loaded with hesperitin (SLN-HT) will be synthesized and characterized. Next, in objective 2, human osteoblast cell lines will be treated with non-cytotoxic doses of SLN-HT and their effects on osteogenesis will be determined through the expression of genes regulating bone metabolism, formation of mineralized nodules, organization and maturation of collagen. Finally, in objective 3, the action of SLN-HT on the expression of inflammatory mediators and matrix metalloproteinases, in addition to its impact on the NF-kB signaling pathways, will be evaluated in human monocytes / macrophages stimulated by Fusobacterium nucleatum. The numerical data , obtained by application of the laboratory protocols, will be submitted to the specific statistical analysis using the software GraphPad Prism 6, and all the tests of this study will be applied with significance level of 5% (p <0.05). (AU)