Benzotriazole–1,2,4-triazole–fluoroquinolone-conjugated chitosan sponges: Antimicrobial activity and in vitro/in vivo wound-healing evaluation

Novel benzotriazole–1,2,4-triazole-fluoroquinolone-conjugated chitosan sponges were developed as multifunctional wound-healing biomaterials. Ciprofloxacin-derived heterocyclic compounds (UP7 and UP8) were synthesized and covalently grafted onto chitosan to obtain Cs-UP7 and Cs-UP8. Structural characterization by FTIR, NMR, MALDI-TOF/MS, XRD, SEM, and TGA confirmed successful conjugation and enhanced thermal stability. The modified sponges exhibited high porosity (94.34–96.66%) and mean 5-h swelling ratios of approximately 1580–1650%, exceeding the corresponding values of native chitosan. Both conjugated formulations produced significantly larger growth-inhibition zones than neat chitosan under the agar-diffusion conditions, with total zone diameters reaching 38 mm. In vitro studies demonstrated high L929 fibroblast viability and a concentration- and time-dependent modulation of net scratch-wound closure under serum-containing conditions. Furthermore, in vivo wound-healing experiments showed favorable tissue regeneration, collagen organization, and dermal remodeling at the day-14 histopathological endpoint. However, the serum-containing in vitro net scratch-wound-closure assay did not show a consistent advantage of the conjugated sponges over pristine chitosan; in several dose–time conditions, pristine chitosan produced higher wound closure, whereas the highest Cs-UP8 concentration showed reduced closure at later time points. Among the formulations, Cs-UP8 showed the most favorable in vivo histopathological outcome, particularly with respect to granulation-tissue formation and qualitative dermal remodeling, although re-epithelialization and angiogenesis scores did not differ significantly among groups. These findings support Cs-UP8 as a promising antimicrobial wound-dressing candidate for further evaluation, while indicating that its regenerative advantage is endpoint- and assay-dependent rather than universal.