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The present study focuses on the synthesis and evaluation of neomycin-loaded hydrogels as potential substrate for wound healing application. Herein, genipin crosslinked gelatin interpenetrated diosgenin-modified nanocellulose (DGN-NC) hydrogels were synthesized. The hydrogels' chemical structures as well as surface morphology, mechanical property, and thermal behavior were characterized. Swelling analysis and gelation kinetics of the hydrogels were studied and the results obtained showed good swelling capacity as well as high gel yield. In addition, the prepared loaded hydrogels were evaluated for antibacterial activity against human pathogenic E. coli and S. aureus bacteria with inhibition capacity determined in the range of 50-88%. In vitro cytocompatibility and drug release studies were also explored under simulated physiological conditions achieving high cell viability and release percentage >80% and >90% after 24 h, respectively. In effect, the design hydrogels in the present study possess adequate incorporated antibacterial properties with significant potentials towards wound dressing and healing applications., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)