Construction Of Novel Empagliflozin Nano-micellar Eye Drops And Study On Its Effectiveness In The Treatment Of Corneal Chemical Injury

Objective:Corneal chemical injury,especially the alkali burn,can lead to extremely serious acute eye injury.When corneal alkali burn occurs,timely on-site irrigation and first aid is important to relief corneal damages.However,clinical practise including drug treatment and corneal transplantation,were highly limited in late stage of treatment.The researchand development of new drugs for the treatment of corneal alkali burn is highly desired in ophthalmology.In ths study,two marketed drugs,dipotassium glycyrrhizinate and empagliflozin,were used for developping nanomicelle eye drops for the treatment of corneal alkali burn.This novel eye drops were prepared,in vitro and in vivo evaluated,and therapeutic effect evaluated with corneal alkali burned mice.Methods:1.Empagliflozin and dipotassium glycyrrhizinate nanomicelle eye drops were prepared by thin film hydration method.The encapsulation rate,potential,particle size,PDI and apparent solubility of the eye drops were determined.2.The storage stability of nano micelle eye drops at 25℃ was investigated.The antioxidant capacity was determined by FRAP method.The in vitro release characteristics were evaluated by dialysis method.The eye safety of the micellar eye drops was evaluated with rabbit eyes.The absorption characteristics of nanomicelle eye drops to the eye were qualitatively evaluated.3.Healthy male C57BL/6J mice were randomly divided into six groups,namely PBS treatment group,0.1% sodium hyaluronate treatment group,empagliflozin suspension group,dipotassium glycyrrhizinate group,dipotassium glycyrrhizinate &empagliflozinmixture group,Empagliflozin and dipotassium glycyrrhizinate nanomicelle eye drops group.A mice model of corneal alkali burn was established.The alkali burn mice in each group were given the corresponding eye drops listed above.At the corresponding time points of the experiment,the fluorescein sodium staining of mice cornea was recorded to judge the condition of corneal epithelial repair.e Neovascularization and corneal turbidity were observed.Corneal nerve sensitivity of mice was measured by corneal perception meter.Pathological sections of corneal tissue were prepared for analysis.Inflammatory factors such as HMGB1,IL-6,IL-1β,NF-κB and TNF-α were detected by ELISA kit.The m RNA expression of HMGB1,RAGE,TLR-2,NF-κB and other cytokines was determined by q RT-PCR.Results:1.The encapsulation rate,average particle size,polydispersity index and Zeta potential of the optimized Empagliflozin-dipotassium glycocyrrhizate nanomicellar eye drops were 97.91±0.50%,6.659±0.196 nm,0.226±0.059 and-(4.103±0.157)m V.Its apparent solubility in PBS was 22.403±0.713 mg/m L.XRD and FT-IR results show that there was no chemical reaction between Empagliflozin and dipotassium glycocyrrhizate,and their physical and chemical properties were stable.2.Empagliflozin and dipotassium glycyrrhizinate nanomicellar eye drops showed high stability under short-term storage conditions of 25℃ for 12 weeks.It had strong antioxidant activity.The Empagliflozin encapsulated in dipotassium glycyrrhizinate nanomicelles significantly accelerated its release in vitro.The eye drops were well tolerated in rabbit eyes.Micellar eye drops not only promoted the absorption of empagliflozin into corneal surface,but also had a high ability to penetrate the posterior segment.3.In the mouse model of corneal alkali burn established by sodium hydroxide injuries,the effect of accelerating corneal wound healing was much stronger to the empagliflozin and dipotassium glycyrrhizinate nanomicellar eye drops group than to all other groups.It could significantly inhibit the degree of corneal opacity and angiogenesis.It could improve the degree of corneal nerve insensitivity caused by corneal alkali burn.Nanomicellar eye drops also effectively repaired damaged corneal tissue structure;ELISA results showed that the levels of inflammatory factors such as HMGB1,IL-6,IL-1β,NF-κB and TNF-α were significantly reduced in the nanomicelle eye drops treated group.q RT-PCR results showed that nanomicelle eye drops decreased the m RNA expression levels of HMGB1,RAGE and other cytokines.Conclusion:Empagliflozin and dipotassium glycyrrhizinate nanomicelle eye drops had characters of high safety,and significantly improved the absorption and penetration of insoluble empagliflozin to eyes;It could prompt repairing of damaged corneal nerve and tissue,and inhibit the formation of corneal neovascularization in corneal alkali burn mice model.The therapeutic effect of this micelle eye drops on corneal alkali burn was related with the inhibition of HMGB1 signaling pathway.