Bi-layered nanofibers loaded with pomegranate flowers extract as a novel wound dressing: Fabrication, characterization, and in vivo healing promotion

Prolonged and impaired wound healing is a serious complication, and there is an emerging demand for strategies to overcome this problem. In this study, we developed electrospun bilayer nanofibers consisting of a chitosan/PVA active layer loaded with pomegranate flower extract (PFE) and a PCL inactive back layer. Structural, morphological, and physicochemical properties of nanofibers were characterized and wound healing effects of PFE-loaded nanofibers were assessed by in vitro and in vivo experiments, including antibacterial tests, wound closure measurements, and histological and immunohistochemical (IHC) analyses. Nanofibers with optimum formulation showed good hydrophilicity, a mean diameter of 404 nm, and an average roughness of 217 nm. TGA and FTIR studies exhibited typical absorption characteristics of PFE, PCL, and PVA and the compatibility of PFE with the components of bilayer nanofibers. The cumulative amount of PFE released from the monolayer and bilayer fibers after 6 h was measured to be 100% and 64%, respectively, indicating the more sustained release of PFE from bilayer scaffolds. Based on mechanical studies, a more ductile behavior was observed for bilayer fibers than monolayer fibers. The antibacterial assay for PFE-loaded nanofibers exhibited an inhibition zone with a diameter of 19.0 ± 0.5 mm and 15.8 ± 0.3 mm for S. aureus and P. aeruginosa, respectively. PFE-loaded nanofiber exposure contributed to faster wound closure (88% in 14 days) and a significant rise in the expression of α-SMA, CD31, collagen I, and vimentin, indicating a higher tissue regeneration rate for PFE-loaded nanofibers over other treatment groups. In conclusion, bilayer nanofibers containing PFE have a promising potential for wound healing acceleration.