Introductionpost-operative endophthalmitis (POE) following cataract surgery remains a significant concern due to its impact on vision and the associated socio-economic burden. S-Nitrosoglutathione (GSNO) shows promise due to its stability and antimicrobial properties. Hydroxypropyl methylcellulose (HPMC) is commonly used in eye drops for drug retention, while methacrylic anhydride (MA) modification enhances adhesion. In our study, we aimed to develop a novel GSNO-loaded HPMC-MA hydrogel, and used an ex-vivo corneal keratitis model to test the anti-inflammatory and anti-microbial effects of HPMC-MA hydrogels containing GSNO. Additionally, the study evaluated the irritative potential of the eye drop formulation containing GSNO-loaded HPMC-MA hydrogels on rabbit eyes. MethodWe prepared and tested the GSNO-loaded HPMC-MA hydrogels characters. Fourier Transform Infrared (FTIR) spectroscopy measurement to verify modification, cell viability assays, osmolality tests, in-vivo ocular irritation assessments, inflammatory cells genes expression using RT-PCR, bacterial concentration analysis with an ex-vivo S. aureus keratitis model were conducted. ResultsFTIR spectroscopy confirmed successful MA modification and GSNO incorporation. Cell viability experiments suggested optimal GSNO concentration at 80 μM. Osmolality tests indicated no significant changes in osmolality post-modification. Ocular irritation tests on rabbits showed no adverse reactions. In the ex-vivo keratitis model, GSNO-HPMC-MA reduced inflammation and inhibited bacterial growth, as reflected in gene expression and bacterial concentration analyses. ConclusionThe study demonstrates the successful development of GSNO-loaded HPMC-MA hydrogels with potential as a topical formulation for endophthalmitis treatment. This novel approach shows promise in addressing challenges related to drug delivery efficiency and antimicrobial efficacy in ocular infections.