Abstract: This study was mainly aimed to evaluate the potential use of a novel ocular drug delivery system, organic-inorganic hybrid nanocomposites based on chitosan derivatives and layered double hydroxides (LDH).Organic polymers of chitosan-glutathione (CG) and pre-activated chitosan-glutathione (CG-2MNA) were successfully synthesized and characterized.LDH with intercalated phacolysin (PCL), including larger hexagonal LDH-PCL (Lh-LDH-PCL), larger spherical LDH-PCL (Ls-LDH-PCL), smaller hexagonal LDH-PCL (Sh-LDH-PCL), CG hybrid LDH-PCL (LDH-PCL-CG), and CG-2MNA hybrid LDH-PCL (LDH-PCL-CG-2MNA), were preparedThe nanocomposites with particle size of 107.2-274.9 nm were characterized by powder X-ray diffraction, Fourier transform IR, transmission electron micrographs, etc.In vivo precorneal retention studies showed that the detectable time of all nanocomposites was prolonged from 2 to 6 h in comparison to PCL saline.Accordingly, the AUC0-6h values of Lh-LDH-PCL, Ls-LDH-PCL, Sh-LDH-PCL, LDH-PCL-CG, and LDH-PCL-CG-2MNA nanocomposites were increased by 2.27-, 2.08-, 3.08-, 4.67-, and 3.36-fold, resp.The Draize test and hematoxylin and eosin staining demonstrated that modified LDH had no eye irritation after single and repeated administration.These results indicated that chitosan derivatives-LDH hybrid nanocomposite dispersion could be a promising ocular drug delivery system to improve precorneal retention time of drugs.Graphical Abstract: Thiolated chitosan-LDH hybrid nanocomposite dispersion could be a promising ocular drug delivery system to improve precorneal retention time of drugs and may facilitate penetration of drugs into tissues of the eyes.