Dry eye syndrome (DES) is a common eye disease. Artificial tears (AT) are used to treat DES, but they are not effective. In this study, we assessed the anti-inflammatory effect of AT containing epigallocatechin gallate (EGCG) and hyaluronic acid (HA) on DES. Human corneal epithelial cells (HCECs) were used in the WST-8 assay to determine the safe dose of EGCG. Lipopolysaccharide-stimulated HCECs showing inflammation were treated with EGCG/HA. The expression of IL-1ß, IL-6, IL-8, and TNF-α was assessed by real-time PCR and AT physical properties such as the viscosity, osmolarity, and pH were examined. AT containing EGCG and HA were topically administered in a rabbit DES model established by treatment with 0.1% benzalkonium chloride (BAC). Tear secretion was assessed and fluorescein, H&E, and TUNEL staining were performed. Inflammatory cytokine levels in the corneas were also examined. The non-toxic optimal concentration of EGCG used for the treatment of HCECs in vitro was 10 μg/mL. The expression of several inflammatory genes, including IL-1ß, IL-6, IL-8, and TNF-α, was significantly inhibited in inflamed HCECs treated with 10 μg/mL EGCG and 0.1% (w/v) HA (E10/HA) compared to that in inflamed HCECs treated with either EGCG or HA alone. AT containing E10/HA mimic human tears, with similar osmolarity and viscosity and a neutral pH. Fluorescence examination of the ocular surface of mouse eyes showed that HA increased drug retention on the ocular surface. Topical treatment of DES rabbits with AT plus E10/HA increased tear secretion, reduced corneal epithelial damage, and maintained the epithelial layers and stromal structure. Moreover, the corneas of the E10/HA-treated rabbits showed fewer apoptotic cells, lower inflammation, and decreased IL-6, IL-8, and TNF-α levels. In conclusion, we showed that AT plus E10/HA had anti-inflammatory and mucoadhesive properties when used as topical eye drops and were effective for treating DES in rabbits.

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Application of autologous serum eye drops (SEDs) is a recognized means to treat severe dry-eye syndrome (DES). Due to the inconvenience and difficulty of preparing SEDs from some patients, producing SEDs from allogeneic blood donations is gaining popularity. A major safety concern associated with allogeneic blood is virus transmission. We therefore herein evaluated the possibility of applying a solvent/detergent (S/D) treatment to inactivate viruses and studied the impacts of such treatment of SEDs to resolve DES in a rabbit model. Sera prepared from the blood of five rabbits were pooled and divided into two sub-pools. One was untreated (SEDs), while the other was virally-inactivated with 1% Tri-n-butyl phosphate/1% Triton X-45 at 31°C for 1 h (S/D-SEDs). DES was induced in rabbits using 0.1% benzalkonium chloride (BAC). Rabbits were divided into five groups of two rabbits each. One group was untreated (control), three were treated twice daily for 3 weeks using PBS, SEDs, or S/D-SEDs, and the last received an additional 0.1% BAC (as the negative control). The DES condition was determined by measuring aqueous tear secretion (Schirmer’s test), corneal fluorescein staining, a corneal histologic examination, TUNEL stain apoptosis, and corneal inflammatory marker (tumor necrosis factor-α, interleukin (IL)-1β, IL-8, and IL-6) expressions. We first confirmed that SEDs and S/D-SEDs had similar protein profiles and transforming growth factor (TGF)-β contents. Animal experiments showed that tear secretion did not significantly differ between the SED and S/D-SED groups but was significantly higher than in the PBS group. Eye fluorescein staining revealed dramatic improvements in epithelial defects in groups treated with SEDs or S/D-SEDs, and hematoxylin/eosin staining revealed microscopic epithelial layers similar to those of the untreated controls. Inflammatory markers and TUNEL studies showed that healthy epithelium had been restored in groups treated with SEDs or S/D-SEDs. In conclusion, this preclinical study supports the possibility of using S/D virally inactivated SEDs to treat DES and restore a normal epithelium.

Dry eye syndrome (DES) is one of the most common types of ocular diseases. There is a major need to treat DES in a simple yet efficient way. Artificial tears (AT) are the most commonly used agents for treating DES, but are not very effective. Herbal extractions of ferulic acid (FA), an anti-oxidant agent, and kaempferol (KM), an anti-inflammatory reagent, were added to buffer solution (BS) to replace ATs for DES treatment. The cytotoxicity and anti-inflammatory effects were examined in vitro by co-culture with human corneal epithelial cells (HCECs) to obtain the optimal concentration of KM and FA for treating HCECs. Physical properties of BS, such as pH value, osmolality, and refractive index were also examined. Then, rabbits with DES were used for therapeutic evaluation. Tear production, corneal damage, and ocular irritation in rabbits’ eyes were examined. The non-toxic concentrations of KM and FA for HCEC cultivation over 3 days were 1 µM and 100 µM, respectively. Live/dead stain results also show non-toxicity of KM and FA for treating HCECs. Lipopolysaccharide-stimulated HCECs in inflammatory conditions treated with 100 µM FA and 1 µM KM (FA100/KM1) showed lower IL-1B, IL-6, IL-8, and TNFα expression when examined by real-time PCR. The BS with FA100/KM1 had neutral pH, and a similar osmolality and refractive index to human tears. Topical delivery of BS + FA100/KM1 showed no irritation to rabbit eyes. The corneal thickness in the BS + FA100/KM1 treated group was comparable to normal eyes. Results of DES rabbits treated with BS + FA100/KM1 showed less corneal epithelial damage and higher tear volume than the normal group. In conclusion, we showed that the combination of FA (100 µM) and KM (1 µM) towards treating inflamed HCECs had an anti-inflammatory effect, and it is effective in treating DES rabbits when BS is added in combination with these two herbal supplements and used as a topical eye drop.

Neovascularization (NV) of the cornea disrupts vision which leads to blindness. Investigation of antiangiogenic, slow-release and biocompatible approaches for treating corneal NV is of great importance. We designed an eye drop formulation containing gelatin/epigallocatechin-3-gallate (EGCG) nanoparticles (NPs) for targeted therapy in corneal NV. Gelatin-EGCG self-assembled NPs with hyaluronic acid (HA) coating on its surface (named GEH) and hyaluronic acid conjugated with arginine-glycine-aspartic acid (RGD) (GEH-RGD) were synthesized. Human umbilical vein endothelial cells (HUVECs) were used to evaluate the antiangiogenic effect of GEH-RGD NPs in vitro. Moreover, a mouse model of chemical corneal cauterization was employed to evaluate the antiangiogenic effects of GEH-RGD NPs in vivo. GEH-RGD NP treatment significantly reduced endothelial cell tube formation and inhibited metalloproteinase (MMP)-2 and MMP-9 activity in HUVECs in vitro. Topical application of GEH-RGD NPs (once daily for a week) significantly attenuated the formation of pathological vessels in the mouse cornea after chemical cauterization. Reduction in both vascular endothelial growth factor (VEGF) and MMP-9 protein in the GEH-RGD NP-treated cauterized corneas was observed. These results confirm the molecular mechanism of the antiangiogenic effect of GEH-RGD NPs in suppressing pathological corneal NV.