Methylene blue-loaded flexible liposomes improve the efficacy of photodynamic therapy for the treatment of bacterial keratitis

Photodynamic therapy is emerging as a promising alternative to combat antibiotic-resistant microorganisms causing keratitis. Since photosensitizer penetration through bacterial cell walls limits efficacy, nanovesicles offer a viable solution to enhance effectiveness. This study evaluated the antimicrobial efficacy of flexible liposomes loaded with methylene blue against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa. Liposomes were prepared using the film hydration method, optimizing lecithin type, solvent, and edge activator. The antimicrobial activity of the liposomal systems was assessed using two methylene blue concentrations and two light energy fluences. Incorporating methylene blue into the liposomes enhanced the growth inhibition of MRSA compared to free methylene blue. For P. aeruginosa, significant increases in growth inhibition were observed with higher irradiance, application time, and methylene blue concentration in liposomes compared to the photosensitizer solution (free form). Reactive oxygen species production after photoactivation was quantified by fluorescein photobleaching, showing a significant increase with the liposomal system compared to free methylene blue. Although reactive oxygen species production reached saturation at higher methylene blue concentrations, bacterial growth inhibition continued to increase with prolonged exposure, suggesting accumulated oxidative damage. Cell viability studies with HaCaT cells indicated that at 20 mW/cm2, diluted samples (1:100 and 1:50) were non-cytotoxic, while a 1:10 dilution (20 mu g/mL) significantly reduced viability. All samples were cytotoxic at 60 mW/cm2, likely due to their strong membrane affinity. These findings highlight the need for optimized photodynamic therapy conditions to balance antimicrobial efficacy with host cell safety.