Suppression of NLRP3/Caspase-1/GSDMD Mediated Corneal Epithelium Pyroptosis Using Melatonin-Loaded Liposomes to Inhibit Benzalkonium Chloride-Induced Dry Eye Disease

Introduction: Benzalkonium chloride (BAC) is widely employed as a preservative in eye drops, which will cause the death of corneal epithelial cells due to ROS production, DNA strand breakage, and mitochondrial dysfunction, resulting in dry eye disease (DED)-like changes in ocular surface tissues.In this study, Melatonin (MT) liposomes (TAT-MT-LIPs) designed by loading MT into TAT-modified liposomes have been developed, characterized, and used for inhibiting BAC-induced DED (BAC-DED).Methods: The TAT was chemically grafted onto the Mal-PEG 2000 -DSPE by Michael's addition between the sulfhydryl group in TAT and the maleimide group in Mal-PEG 2000 -DSPE.TAT-MT-LIPs were prepared using film dispersion followed by the extrusion method and topically treated in rats once a day.BAC-DED was induced in rats by topical administration with 0.2% BAC twice daily.Defects, edema, and inflammation of the corneas, as well as IOP, were examined.Histologic analyses of corneas were performed to assess the change of mitochondrial DNA oxidation and NLRP3/Caspase-1/GSDMD signaling transduction.Results: After topical administration, TAT-MT-LIPs significantly alleviated DED-clinical symptoms of experimental animals by inhibiting tissue inflammation and preventing the loss of the corneal epithelium and conjunctival goblet cells.Our data suggested continuous ocular surface exposure of BAC-induced NLRP3/Caspase-1/GSDMD mediated corneal epithelium pyroptosis, which was not reported before.BAC caused substantial mt-DNA oxidation, which promoted the transduction of NLRP3/Caspase-1/GSDMD and consequent corneal epithelium pyroptosis.TAT-MT-LIPs could efficiently suppress the BAC-induced corneal epithelium pyroptosis and inflammation by inhibiting mt-DNA oxidation and the subsequent signal transmission.Conclusion: NLRP3/Caspase-1/GSDMD mediated corneal epithelium pyroptosis is involved in the development of BAC-DED.The present study provided new insights into the adverse effects of BAC, which can serve as a new target for protecting corneal epithelium when applying BAC as a preservative in eye drops.The developed TAT-MT-LIPs can efficiently inhibit BAC-DED and give great potential to be developed as a new DED treatment.