Synergistic beeswax-based nano-formulation for enhanced chronic wound healing and antibacterial potency

Chronic wounds, affecting over 10.5 million U.S. Medicare beneficiaries and exacerbated by diabetes, represent a critical healthcare challenge due to multifactorial pathophysiology, antimicrobial resistance, and inadequate therapeutic delivery. To address these limitations, we developed a novel beeswax-encapsulated emulsified formulation (F4) combining delafloxacin (DLX), lidocaine (LIDO), and sodium hyaluronate (SHA). F4 demonstrated high encapsulation efficiency (87.6-91.3%), optimal nanoparticle characteristics (115.68 nm, PDI 0.26, zeta potential -31.70 mV), and pH-responsive sustained release (94-100% cumulative release at 24 h, pH 7.4). Kinetic modeling confirmed a diffusion-controlled release mechanism (Korsmeyer-Peppas R ² > 0.989), and comprehensive forced degradation studies coupled with in silico toxicological profiling (following ICH M7 guidelines) confirmed the formulation's stability and low impurity risk. Critically, F4 exhibited significantly enhanced antibacterial activity versus free DLX: larger inhibition zones against all tested pathogens (Gram-positive: S. aureus, B. subtilis; Gram-negative: E. coli, P. aeruginosa), 4-fold lower MICs against S. aureus (0.031 vs. 0.125 µg mL^-1) and E. coli (0.031 vs. 0.125 µg mL^-1), and potent bactericidal effects (MBC/MIC ≤ 2). F4 also disrupted biofilms at sub-MIC levels (1/16-1/64 MIC), comparable to ciprofloxacin, and achieved complete bactericidal killing of planktonic S. aureus within 12 h at 4× MIC. Furthermore, time-kill kinetics demonstrated a concentration-dependent bactericidal effect. Cytotoxicity assays confirmed enhanced biocompatibility (CC₅₀: 872.40 µg mL^-1), preserving >80% fibroblast viability at therapeutic doses. This multimodal platform concurrently addresses infection control (including biofilm resistance), analgesia, tissue regeneration, and controlled delivery, offering a transformative strategy for chronic wound management.