Extracellular Vesicles-mediated Lefty1 MRNA Delivery For Hepatic Fibrosis Inhibition

Hepatic fibrosis(HF)is a pathological process of overhealing after liver damage,which will progressively develop into more serious diseases,such as cirrhosis and cancer.Activated hepatic stellate cells(HSC),a type of cells that mainly causes HF,constantly secrete extracellular matrix(ECM)to trigger a large amount of collagen fibers,changing the structure of liver and leading to HF.Transforming growth factorbeta(TGF-β)1 is one of the important cytokines for inducing HSC activation.Although small molecule inhibitors TGF-β1 have been approved for the treatment of idiopathic pulmonary fibrosis,their hepatotoxicity is likely to increase the burden on the liver and limit their application in HF therapy.mRNA therapy is a safe and effective strategy by transfecting mRNA into cells and translating proteins.Thus,inhibition of HSC activation by blocking TGF-β1 signal transduction can be an effective strategy to treat HF using mRNA therapy.However,there are some problems of mRNA therapy in the treatment of HF.(1)mRNA is poor in stability and easily degraded by nucleases.(2)The pathological structure of HF hinders the delivery of mRNA.For example,overdeposited ECM is easily to isolate mRNA.HSC that located in a narrow perisinus space makes mRNA difficult to reach it.Moreover,the capture and clearance of liver macrophages to drugs is a barrier to mRNA delivery.In view of this,we constructed extracellular vesicles(EVs)to deliver therapeutic Left/Right determination factor(Lefty)1 mRNA for HF inhibition.Firstly,rat HSC-T6 cells were transfected with Lefty1 plasmid by electroporation to secrete EVs loaded with Lefty1 mRNA.We investigated the potential mechanism of increasing EVs secretion by electroporation.Secondly,we prepared engineered EVs with surface modification by "Don’t eat me" ligand differentiation cluster 47(CD47)and cyclic peptide cRGDfK,so that EVs shielded the phagocytosis of liver macrophages and actively targeted to activate HSC.Finally,we evaluated the antifibrotic effect of engineered EVs delivering Lefty1 mRNA both in vitro and in vivo.The research contents are as follows:1.Study on inhibition effect of Lefty1 to activated HSC-T6In order to study the inhibition effect of Lefty1 on activated HSC-T6,we transfected Lefty1 plasmid into activated HSC-T6 by electroporation,detected and analyzed proteins changing in HSC-T6 by western blot.The results showed that the expression of activated HSC-T6 proteins,alpha-smooth actin(α-SMA)and vimentin(Vim),was significantly reduced,possibly through the classical TGF-β/SMAD signaling pathway inhibiting the activation of HSC-T6.2.Study on the effect of electroporation on cell secretion of EVsIn order to study the effect of electroporation on the secretion of EVs,we detected the yield and properties of EVs,and analyzed the secretion mechanism of EVs by electroporation.The results showed that the number of EVs particles secreted by HSCT6 after electroporation was 7.68 times that of EVs collected after normal culture,and that secreted by hepatocytes BRL after electroporation was 3.15 times.The morphology,particle size,potential and marker protein of EVs collected after electroporation were not significantly different from those of no-treated EVs.Proteomic analysis found that lysosomal-associated transmembrane protein 4B(Laptm4B)significantly increased by2.39 times after electroporation,which can bind ceramides and accelerate the transport of multivesicles bodies to the plasma membrane,promoting the secretion of EVs.After electroporation of the HSC-T6 with Laptm4 B knockdown,EVs secretion did not increase significantly.Thus,electroporation may increase the secretion of EVs in cells by affecting the pathway of Laptm4 B binding to ceramides.3.Antifibrosis evaluation of Lefty1 mRNA delivered by EVs in vitroTo investigate the antifibrotic effects of EVs delivering Lefty1 mRNA(LEVs)in vitro,we characterized their physicochemical properties and investigated their effect on HSC-T6 activation.The results showed that the selection of HSC-T6 as a cellular source of LEVs promoted the uptake of LEVs by activated HSCs.The physicochemical characterization results showed that LEVs was 113.0 ± 19.6 nm,with negative potential and vesicle morphology,expressing EVs marker protein,and the Lefty1 mRNA loading was 3.06 times than that of blank EVs,and had good stability.Cell evaluation showed that LEVs had no obvious cytotoxicity and were significantly uptaken within 4 h by activated HSC-T6,and mainly internalized through macropinocytosis and lipid raft pathways.Finally,cellular immunofluorescence and western blot results showed that LEVs significantly reduced the expression of α-SMA,downregulated the expression of type I collagen,tissue inhibitor of metalloproteinase(TIMP)-1 significantly,and upregulated the expression of matrix metalloproteinase(MMP)-1,demonstrating that LEVs inhibited HSC activation,reduced collagen secretion and promoted collagen degradation.Therefore,LEVs have demonstrated good antifibrotic effect in vitro.4.Evaluation of anti-fibrosis in LEVs in vivoIn order to investigate the anti-fibrosis effect of LEVs further in vivo,we established a rat HF model to evaluate the effect of LEVs in the treatment of fibrosis.The results showed that LEVs reduced the content of collagen volume fraction and hydroxyproline in liver tissues after 4 weeks of treatment,and downregulated the expression of α-SMA,which proved that they reduced the production of collagen fibers and showed the effect of inhibiting HSC activation in vivo.However,LEVs did not exhibit significant antifibrotic effects compared to EVs.We observed the distribution of LEVs was by immunofluorescence in liver tissues.The results showed that LEVs colocalized with HSC,indicating that it had the ability to penetrate the ECM barrier to reach HSC cells.It was also found that LEVs colocalized with hepatic macrophages,indicating that LEVs were uptaken by hepatic macrophages.The co-culture of hepatic macrophages with HSC-T6 verified that the inhibition of LEVs to activated HSC-T6 was inhibitied by hepatic macrophages uptaken.5.Antifibrotic evaluation of Lefty1 mRNA delivered by engineered EVsIn order to enhance the role of EVs delivering Lefty1 mRNA in the treatment of HF,we developed engineered EVs to evade macrophage phagocytosis and target HSCs actively,and evaluated the antifibrotic effect of Lefty1 mRNA delivered by engineered EVs in vitro and in vivo.Firstly,we constructed HSC-T6 cells that overexpressed CD47,and then transfected the Lefty1 plasmid by electroporation,and collected EVs(CLEVs).Untransfected Lefty1 plasmid EVs named CEVs.The targeted peptide,cRGDfKDSPE-PEG,was linked to CLEVs to obtain modified EVs(RCEVs and RCLEVs).The results of physicochemical characterization showed that RCLEVs were 114.0 ± 50.8nm with negative potential and vesicle structure,expressed marker proteins,and significantly expressed CD47 compared with LEVs.The results showed that the uptake of RCEVs and CEVs were significantly reduced by hepatic macrophages compared with NEVs,indicating that CD47 and cRGDfK modifications reduced the phagocytosis of EVs by hepatic macrophages.Compared with CEVs,the uptake of RCEVs by activated HSC-T6 was significantly increased,indicating the enhanced ability to target activated HSC.Further,we investigated the anti-fibrotic effect of RCLEVs in vivo.Compared with unmodified and single-modified EVs,RCLEVs reduce collagen fiber volume in liver tissue and reduce collagen secretion significantly.The results of distribution showed that RCLEVs were able to evade phagocytosis of hepatic macrophages and increase colocalization with HSC more significantly.The safety evaluation of RCLEVs showed that they had no obvious organ toxicity and they had the effect of protecting liver.In summary,this study found that Lefty1 had an inhibitory effect on TGF-β1-induced HSC activation through TGF-β/SMADsignaling;Electroporation increases EVs secretion by influencing the pathway of Laptm4 B binding to ceramides;EVs delivering Lefty1 mRNA prepared based on electroporation showed obvious antifibrotic effects in vitro and in vivo.RCLEVs modified by CD47 and cRGDfK were further developed,which can avoid being phagocytosed by hepatic macrophages and target to HSC proactively,with enhanced anti-fibrotic effect and good biosafety.Therefore,the delivery of Lefty1 mRNA by engineered EVs provides a new therapeutic idea in the treatment of HF and has great clinical application value.