Abstract Title Synergistic effect of silver nanoparticles and antibiotics in enhancing antimicrobial activity against ocular pathogens

Background Ocular infections, such as endophthalmitis and keratitis, remain one of the leading causes of irreversible blindness, worldwide. Despite advances in antibiotic therapy, increasing antimicrobial resistance among pathogens limit treatment options. We sought to develop dual antibacterial nanoformulations using silver and antibiotics and evaluated their antimicrobial efficacy against ocular pathogens.

Methods Silver nanoparticles were synthesized via a chemical reduction reaction, then loaded with vancomycin and coated with chitosan. Minimum inhibitory concentration (MIC) of silver nanoparticles against staphylococcus aureus (SA) and methicillin-resistant staphylococcus aureus (MRSA) were determined via double dilution antibacterial assay with optical density measured at 600 nanometers. Ex vivo porcine eyes, ex vivo human vitreous, cultured human retinal Müller glial (MIO-M1) cells, and in vivo mice eyes were then intravitreally infected with SA. Chitosan-silver-nanoparticles (CAgNPs), chitosan-silver-vancomycin-nanoparticles (CAgVNPs), and vancomycin alone were separately administered to the above testing groups post-6 hours of infection. The samples were harvested and plated for colony-forming units (CFUs). Statistical significance was performed using one-way ANOVA. Lastly, the inflammatory response and cytotoxicity of the nanoformulations were assessed: inflammatory markers of SA-infected mice eyes were evaluated via ELISA, cytotoxicity of nanoformulations in SA-infected MIO-M1 cells were evaluated quantitatively via LDH assay and qualitatively via Calcein assay, and cytotoxicity of nanoformulations in SA-infected mice eyes were evaluated via TUNEL assay and H&E staining.

Results CAgVNPs had a two-fold reduction in MIC compared to vancomycin. Comparing CAgVNP to vancomycin, there was an 8-fold reduction in CFUs in ex vivo porcine eyes, a 3.5-fold reduction in ex vivo human vitreous, and a 103-fold reduction in in vivo mice eyes. Levels of inflammatory markers IL-1β, IL-6, and KC were decreased in SA-infected mice eyes treated with CAgVNPs compared to vancomycin. LDH values were the same as the control group without SA infection. Calcein assay showed that CAgVNPs were able to save MIO-M1 cells from SA infection. TUNEL assay of mice eye sections that were treated with CAgVNPs were histologically the same as the control group without SA infection.

Conclusion This study demonstrates that silver nanoparticles have potent antimicrobial activity against ocular pathogens ex vivo, in vitro, and in vivo. More importantly, silver nanoparticles have synergistic effects with antibiotics to reduce MIC as well as enhance antimicrobial activity. Lastly, silver nanoparticles do not demonstrate any cytotoxicity, indicating their potential use for treatment of ocular infections.