Box-Behnken-Optimized Bacterial Nanocellulose-Silver and Zinc Oxide Nanocomposites: Bactericidal Activity,...

Bacterial nanocellulose (BNC) nanocomposites functionalized with silver (BNC-AgNC) and zinc oxide (BNC-ZnONC) nanoparticles were developed and optimized using Box-Behnken design (BBD) for potential wound dressing applications. Quadratic models exhibited excellent predictability (R² = 0.9765 and 0.9811, respectively), identifying optimal loading conditions of 3% nanoparticle concentration, 4 h loading time, and 32.5 °C, yielding maximum inhibition zone diameters of 2.81 cm (BNC-AgNC) and 2.65 cm (BNC-ZnONC). Nanocomposite formation was confirmed by UV-Vis spectroscopy, XRD, FTIR, SEM-EDX, DLS, and TGA, collectively demonstrating successful nanoparticle incorporation without disruption of the cellulose crystalline backbone. Both composites exhibited pronounced antibacterial activity against Gram-positive (S. aureus, S. pyogenes) and Gram-negative (S. marcescens, P. aeruginosa) pathogens, with BNC-AgNC showing bactericidal potency (MIC: 8-32 µg/mL) compared to BNC-ZnONC (MIC: 32-128 µg/mL). Cytotoxicity assessment on human fibroblasts revealed that BNC-ZnONC maintained > 97% cell viability across all tested concentrations (IC₅₀ >125 µg/mL), whereas BNC-AgNC exhibited concentration-dependent toxicity (IC₅₀ =63 µg/mL). Scratch wound healing assays demonstrated that BNC-ZnONC promoted fibroblast migration comparable to pristine BNC (81.84% closure), while BNC-AgNC significantly impaired wound closure even at sub-lethal concentrations. DPPH radical scavenging and hydration studies further supported the biocompatibility profile of BNC-ZnONC. These findings establish a functional dichotomy: BNC-AgNC offers potent bactericidal activity with a narrow therapeutic window, while BNC-ZnONC represents a more favorable candidate combining antimicrobial efficacy, cytocompatibility, and pro-healing properties for biomedical wound management.