Deep Eutectic Solvents As Rapamycin Delivery System To Inhibit Corneal Allograft Rejection

Objective:Rapamycin(RAPA)is an effective and specific inhibitor of the m TOR pathway that can inhibit corneal immune rejection via a variety of mechanisms.However,the disadvantage of low water solubility limits its application in ophthalmology.Deep eutectic solvents(DESs)are undisputed green and sustainable organic solvents that can significantly improve the solubility of active pharmaceutical ingredient(API)and have been developed as drug delivery systems to treat a wide range of clinical diseases.This study aims to improve the solubility and bioavailability of RAPA by preparing RAPA-NADES drug delivery system,and to study its effect on the immune rejection of corneal transplantation.Methods:(1)Preparation and selection of DESs.Four kinds of choline chloride(Ch Cl)based DESs were prepared by heating method.Cytotoxicity test(CCK-8 test)was used to evaluate in vitro biocompatibility of the prepared DESs using human corneal epithelial cells(HCECs),while mouse eye and rabbit eye irritation tests were adopted to assess its in vivo biocompatibility.(2)Characterization of Ch Cl/Carbohydrates-based DES.Water content,viscosity,p H value,thermal stability and structural properties of three kinds of Ch Cl/Carbohydratesbased DESs were measured.(3)Preparation of RAPA-NADES drug delivery system.HPLC was used for quantitative analysis of rapamycin in methanol.0.1% RAPA-NADES drug delivery system was prepared and the dissolution curve of rapamycin in Ch Cl/Fructose-based DES was drawn.(4)Corneal transplantation model.Heterogeneic penetrating keratoplasty model was constructed in mice.Mice(n=40)were randomly divided into blank group,control group,NADES group and RAPA-NADES group.Eye drop therapy was performed on the second day after operation,with a dose of 5 μl per time.Slit lamp photos were taken every two days to observe the condition of allograft rejection,record the time of corneal allograft rejection and draw the graft survival curve.(5)Anti-corneal allograft rejection experiments of RAPA-NADES drug delivery system.At 16 days post operation,corneal allograft of the four groups were taken,and the severity of allograft inflammation in each group was observed and compared by HE staining.The expression levels of TNF-α,IL-6,IL-1β,IL-10,IL-12 and IL-17α in cornea were detected by real-time PCR.Results:(1)Preparation and selection of DESs.The stable,uniform,and bright solutions were obtained after cooling.Then Ch Cl/carbohydrates-based DESs are selected to be used as drug carrier solvent after biocompatible evaluation.(2)Characterization of Ch Cl/carbohydrates-based DES.The viscosity and p H value of the solvent decreased with the increase of water content.DSC shows that exothermic peaks in the mixture is between 65 and 85℃,indicating strong interactions between Ch Cl and carbohydrate.FTIR shows that strong hydrogen bonds are formed between chloride ions and hydroxyl groups in the prepared solvent.(3)Preparation of RAPA-NADES drug delivery system.The cumulative drug release rate of as-prepared 0.1% RAPA-NADES drug delivery system is about 20% in 5 hours.(4)Clinical observation of corneal allograft rejection in mice treated by Allo+RAPANADES drug delivery system.The mean survival time of Allo+RAPA-NADES group(23.6±6.2 days)was significantly longer than those of Allo+NADES group(15.6±1.1 days)and control group(16.4±1.1 days),with statistical significance(p<0.05).Kaplan-Meier analysis showed that the survival rate in Allo+RAPA-NADES group was better than those in the control group and NADES group with the statistically significant difference(p<0.001).(5)Evaluation of the anti-immune rejection effect of RAPA-NADES drug delivery system.HE staining results show no inflammatory response of the blank group,strong inflammatory response of the control group and Allo+NADES group,and mild inflammation response of the Allo+RAPA-NADES group.The expression levels of inflammatory factors including TNF-α,IL-6,IL-1β,IL-10,IL-12 and IL-17α in each group were detected by real-time PCR,and the results show that the expression levels of inflammatory factors such as IL-6,IL-1β and IL-10 were gradually weakened in the following order,Allo+PBS group,Allo+NADES group,Allo+RAPA-NADES group and blank group(p< 0.05).Conclusion:(1)Ch Cl/Carbohydrates-based DES deserves to be developed as a delivery system due to its easy preparation,good biocompatibility,and excellent solubility.(2)The study also confirmed that the as-prepared RAPA-NADES drug delivery system can effectively delay the occurrence of immune rejection after corneal transplantation in mice and greatly extend the survival period of the allgraft.