Response Surface Methodology-Guided Optimization of Flucytosine Nanoemulsion for Enhanced Antifungal Performance

Abstract Although flucytosine is a powerful antifungal drug, formulation-related issues frequently restrict its clinical usage, and the growing prevalence of invasive fungal infections caused by Candida and Cryptococcus species poses a serious public health problem. This work designed, optimized, and assessed a flucytosine-loaded nanoemulsion using a Box–Behnken experimental design to improve physicochemical stability and antifungal activity. Pre-formulation studies confirmed the purity, crystallinity, and compatibility of flucytosine with selected excipients. A total of 17 nanoemulsion formulations were prepared by varying the oil concentration, surfactant–co-surfactant (Smix) ratio, and homogenization time, with particle size, polydispersity index (PDI), and entrapment efficiency (%EE) as key responses. The optimized formulation (F7) exhibited a small droplet size (~104 nm), low PDI (<0.3), high entrapment efficiency (~95%), suitable pH, and a stable negative zeta potential, indicating excellent physical stability. Transmission electron microscopy confirmed spherical and uniformly dispersed droplets. In vitro drug release studies demonstrated a sustained release profile over 24 hours, with the optimized formulation showing significantly higher cumulative drug release compared to other batches. Antifungal evaluation against Candida albicans revealed a markedly larger zone of inhibition for the nanoemulsion compared to plain drug solution, indicating enhanced antifungal efficacy. Stability studies conducted under International Council for Harmonisation (ICH) conditions confirmed the formulation's stability over 3 months. Overall, the proposed flucytosine nanoemulsion demonstrated higher antifungal activity, prolonged drug release, better physicochemical features, and good stability, suggesting its potential as a successful antifungal therapeutic delivery method.