Staphylococcus aureus is a leading cause of ocular keratitis worldwide, and the upsurge of Methicillin-resistant Staphylococcus Aureus (MRSA) strains necessitated the development of new antimicrobial agents. D-limonene is the major constituent of oil extracted from citrus peel, which has been utilized for its gastroprotective, antifungal, antitumor, and antibacterial effects. The present study aimed to develop an effective in-situ ocular limonene-based nanoemulgel to enhance the efficacy of fluoroquinolones against MRSA associated ocular biofilm infection. The nanoemulsion composed of limonene, Tween®80, propylene glycol at a ratio of 5:4:1 loaded with levofloxacin. The formulated levofloxacin-loaded limonene-based nanoemulsion physiochemical properties namely; droplet size, polydispersity index, zeta potential, and in-vitro drug release were studied and stability over three months was assessed. Furthermore, in-vitro antimicrobial susceptibility was investigated on biofilm-forming MRSA strain through kinetics of killing and biofilm assay. The in-situ nanoemulgel ocular irritation was studied by HET-CAM test. The results demonstrated that levofloxacin-loaded limonene-based nanoemulsion had a particle size of 119 ± 0.321 nm with improved eradicating efficacy of MRSA biofilm, where the MIC of the loaded nanoemulgel was 3.12 mg/ml significantly less than that of drug alone (6.25 mg/ml). HET-CAM test showed no signs of hemorrhage, coagulation, or lysis for the loaded nanoemulgel same as sodium chloride solution (negative control) where its irritation score was zero compared to 9.87 for the positive irritant group (1%w/v sodium lauryl sulfate). In conclusion, the current investigation provided a strong foundation for further studies of limonene nanoemulgel as a potential complementary therapeutic agent against resistant bacterial strains.
Keywords: Biofilm; D-limonene; Levofloxacin; MRSA; Nanoemulgel; Staphylococcus aureus.