Investigation of safety and efficacy of ZnO nanoparticle-loaded alginate-hyaluronic acid hydrogel for wound dressings: In vitro and in vivo studies

• Small Zinc oxide nanoparticles (ZnO-NPs) demonstrated significant antibacterial activity in Minimum Biofilm Eradication Concentration (MBEC™) assay®. • ZnO-NPs at high doses increased toxicity but enhanced bacterial death. • Pure alginate-hyaluronic acid (Alg/HA) hydrogels showed strong antibacterial action, enhanced cell growth and accelerated wound healing. • ZnO-loaded hydrogels delayed tissue healing and decreased fibroblast migration in animal model. • Pure hydrogels performed superior to ZnO-loaded ones in terms of biocompatibility and healing. The integration of nanotechnology and biomaterials is valuable for the future of wound care. However, concerns regarding the biosafety of the nanoparticles remain unresolved. This study aims to evaluate the activity and safety of the alginate-hyaluronic acid (Alg/HA) hydrogel-loaded zinc oxide nanoparticles (ZnO-NPs) of three different size ranges (10-30, 35-45, 80-200 nm) and concentrations (5, 10, 20 mg/mL). Fourier Transform Infrared (FTIR) Spectroscopy, and X-ray fluorescence (XRF) Spectroscopy were used to confirm the successful loading and uniform distribution of ZnO-NPs within the hydrogel matrix. Minimum Biofilm Eradication Concentration (MBEC™) assay® against Staphylococcus aureus and Pseudomonas aeruginosa was used to evaluate antibacterial activity. The ZnO-NPs (10–30 nm, 20 mg/mL) loaded hydrogel and pure hydrogel achieved the highest antibacterial efficacy in planktonic states. However, MTT and scratch assays of ZnO-NPs loaded hydrogels indicated a significant reduction in cell viability and migration. The ZnO-NPs loaded hydrogel reduced cell viability up to 85% compared to 100% in the pure Alg/HA hydrogel. In scratch assay, the pure hydrogel showed 68% wound closure within 24 h compared to 55.77% for control group. The pure Alg/HA demonstrated superior in vitro performance. In vivo study showed promising efficacy of the pure Alg/HA hydrogel, promoting complete epithelial regeneration by day 15 (resulting in 95.68% wound closure), while ZnO-NPs hydrogels delayed healing (63.88% of wound closure). Overall, while ZnO-NPs show notable antibacterial properties, their potential cytotoxicity at higher concentrations limits their suitability for wound healing, which urges the reconsideration of using ZnO-NPs for wound dressing applications.