The Construction Of Mesenchymal Stem Cells Derived Exosomes Loaded With Dexamethasone And Its Function On Autoimmune Hepatitis

Objective: Autoimmune hepatitis is an immune-mediated inflammatory disease caused by unknown etiology.At present,symptomatic support treatment is still the basic approach to treat AIH.Glucocorticoid could sometimes be used in appropriate timing due to its side effects,and liver transplantation could only be considered as a last option as a result of the shortness of liver supply.Exosomes derived from mesenchymal stem cells are rich in bioactive substances,which have great potential to repair damaged cells and tissues,and thus it can be used in the treatment of AIH.The purpose of this study is to design a drug delivery system based on the exosomes secreted by mesenchymal stem cells.Dexamethasone was loaded into the membrane of exosomes and thus was named Exo@DEX.In the meantime,in cellular and animal experiments,we observed the phenotypic characteristics of Exo@DEX,the liver targeting of Exo@DEX,the therapeutic effect in AIH and safety evaluation of Exo@DEX.Methods:(1)The whole bone marrow method was used to extract mouse bone marrow mesenchymal stem cells.After being cultured to the third generation,flow cytometry was used to identify the surface protein of the cells.Cell supernatant was collected to separate and purify exosomes by ultracentrifugation and DEX was loaded into exosomes by pulsed ultrasound.The exosome protein markers CD9 and CD63 on both Exo and Exo@DEX were identified by Western blot.The morphology of Exo and Exo@DEX was observed by transmission electron microscope.The size distribution and zeta potential of Exo and Exo@DEX were analyzed by nanoparticle tracking analysis and dynamic light scattering.HPLC was used to quantify the amount of DEX in Exo@DEX.(2)At the cellular level,CCK-8 method was used to detect the effect of Exo@DEX on the viability of RAW264.7 and L02 cells.Apoptosis kit was used to detect the effect of Exo@DEX on cell apoptosis.Laser confocal microscope and flow cytometry were used to observe the uptake of Exo@DEX by different cells at different time points.(3)At the animal level,the liver targeting ability,organ distribution and liver retention time of Exo and Exo@DEX were investigated by an in vivo animal imaging system.The enrichment of Exo and Exo@DEX in liver was verified by flow cytometry and frozen sections.The murine model of autoimmune hepatitis was constructed by injection of concanavalin A.8 h later,serum was obtained to detect transaminase level.In addition,liver tissues were collected for H&E section as well as the detection of inflammatory cytokines.Healthy mice were then administered with Exo@DEX for biosafety detection.H&E sections of several organs were collected,and the blood routine and biochemical analysis were carried out to investigate the biological safety of Exo@DEX.Results:(1)The purity of mesenchymal stem cells was more than 95%;Exo and Exo@DEX both expressed CD9 and CD63 and showed a round like vesicle structure under TME.The size distribution of both nanoparticles was around 100 nm.Zeta potential of Exo was about-10 mV,and the zeta potential of Exo@DEX was about-6 mV.In addition,the typical peak of DEX was detected by HPLC.(2)Compared with DEX group,Exo@DEX treatment downregulated the viability of RAW264.7 cells,promoted the occurrence of cell apoptosis,while barely had any effect on L02 cells.After co-incubation of Exo@DEX with both cells,Exo@DEX was observed to be taken up by both cells in a time-dependent way,with RAW264.7 taking up more nanoparticles.(3)Both Exo and Exo@DEX were able to target the liver,peaking at 2 h and staying for 24 h.In AIH murine model,Exo@DEX was able to effectively reduce the liver damage,downregulated the level of liver transaminase and inflammatory cytokines.Furthermore,Exo@DEX was found no significant toxicity to main organs,and there is barely any side effect in blood routine test and biochemical analysis.Conclusions(1)Exosomes derived from MSC were successfully incorporated with DEX and the formed nanoparticle was called Exo@DEX(2)The therapeutic effect of Exo@DE on autoimmune hepatitis was preliminarily obtained.(3)A liver targeted drug delivery system with great biosafety was explored.