Study Of Hyaluronic Acid-modified Extracellular Vesicles Delivering Pirfenidone For Hepatic Fibrosis Inhibition

Hepatic fibrosis is a wound-healing response process resulting from chronic injury to the liver.The damaged hepatocytes secrete various cytokines and chemokines that induce hepatic stellate cells（HSC）to differentiate into myofibroblasts.Activated HSC produce extracellular matrix in the surrounding.The accumulation of extracellular matrix leads to the formation of fibrous scarring,which will disrupt the liver’s normal structure and lead to the dysfunction of the whole organ in the end.Currently,the majority of drugs inhibiting hepatic fibrosis are mainly targeted at the treatment of HSC.However,HSC exist between hepatocytes and hepatic sinusoidal endothelial cells in the Disse space.Other hepatocytes easily uptake drugs before reaching the target site,resulting in poor therapy.Therefore,developing active targeted drug delivery systems is particularly important for treating hepatic fibrosis.Pirfenidone（PFD）is a small molecule medicine of pyridine with broad-spectrum anti-fibrotic and anti-inflammatory effects.Currently,PFD has been shown to take activated HSC as a therapeutic target and ameliorate hepatic fibrosis by inhibiting TGF-β1-mediated cell signaling pathways.However,the special physiological location of HSC lead to low bioavailability when administered alone.It will cause more serious gastrointestinal side effects and skin-related diseases such as rash and photoallergic reactions in a few patients,which limit its further application.Extracellular vesicles（EVs）are natural nanocarriers with a phospholipid bilayer structure,secreted from the intracellular to the extracellular environment.EVs can deliver endogenous substances between cells with highly biocompatible.Moreover,exogenous substances such as small molecules of chemicals,nucleic acids,and proteins can also be effectively delivered to the diseased tissue to ameliorate or suppress the related disorders.CD44receptor is highly expressed on the surface of activated HSC,which is an effective target for hepatic fibrosis.Using the ligand-receptor binding effect can effectively improve the active targeting of drugs.DSPE-PEG₂₀₀₀-HA is formed by coupling hyaluronic acid（HA）and the amphiphilic molecule DSPE-PEG₂₀₀₀,which can be embedded in the lipid bilayer of EVs through hydrophobic interaction.It can target activated HSC and improve the therapeutic effect of hepatic fibrosis by the property of HA specifical binding CD44 receptor.Based on this,a hyaluronic acid-modified extracellular vesicle with drug-loading（HA@EVs-PFD）was designed for the treatment of hepatic fibrosis.Its physicochemical properties were investigated and anti-liver fibrosis ability was evaluated in vitro and in vivo.In this paper,the following aspects were investigated.1.Establishment of in vitro analytical method for PFDIn this study,PFD was quantitatively analyzed by high-performance liquid chromatography and the methodological validation was performed.Based on the results of the full wavelength scan of the UV-Vis spectrometer,315 nm was selected as the detection wavelength of PFD.The methodological results showed that the linearity of the PFD standard curve had good linearity,and the spiked recovery was within the standard range.The analytical method had good specificity and precision,and the drug properties were stable.The experimental results met the relevant requirements and the method can be used to detect the content of PFD.2.Preparation and characterization of HA@EVs-PFDIn this study,EVs were purified by differential centrifugation and ultrafiltration combined with polyethylene glycol precipitation.PFD was encapsulated by membrane permeation through ultrasound.The HA@EVs-PFD was subsequently prepared by attaching DSPE-PEG₂₀₀₀-HA to the surface of EVs by low-temperature incubation.The morphology,particle size,zeta potential,and surface characteristic proteins were identified by transmission electron microscopy,particle size analysis,and Western Blot.The experimental results showed that the physicochemical properties of HA@EVs-PFD were well and could be studied in vitro and in vivo anti-fibrotic studies.3.In vitro evaluation of HA@EVs-PFDIn vitro studies of HA@EVs-PFD were evaluated by two cells,rat hepatic stellate cells HSC-T6 and rat normal hepatocytes BRL.The cytotoxicity results showed that HA@EVs-PFD had almost no effect on the survival rate of two cells,indicating its high biosafety.The activated model in vitro was constructed by TGF-β1 stimulation of HSC-T6.Then,the cellular uptake and anti-liver fibrosis ability in vitro of HA@EVs-PFD were evaluated.The results showed that HA@EVs-PFD was taken up by activated HSC-T6 in a time-dependent within 4 h.Its fluorescence intensity was approximately4.52-fold higher than that of blank EVs under the same conditions of 4 h incubation and increased 13-fold compared to the uptake in BRL cells,demonstrating the significant active targeting of HA@EVs-PFD to activated HSC.The results of Western Blot showed that HA@EVs-PFD could downregulate the expression of collagen synthesis-related proteins and had a stronger ability to inhibit HSC-T6 activation compared to free PFD.4.In vivo evaluation of HA@EVs-PFDThe anti-liver fibrosis effect of HA@EVs-PFD was evaluated in vivo by establishing a rat liver fibrosis model through intraperitoneal injection of CCl₄.After 4weeks of treatment,the collagen area in the rat liver was reduced,the hepatocyte status was significantly improved,and hepatic fibrosis was ameliorated.After 4 h of tail vein injection,HA@EVs-PFD could effectively accumulate in the fibrotic liver.The results of the hemolysis assay showed that HA@EVs-PFD did not occur hemolytic effect and had good biocompatibility.In addition,the results of histopathological sections and determination of liver and kidney function factors in rat serum showed that HA@EVs-PFD did not significantly damage the major organs of rats and improved liver and kidney function after treatment.In summary,HA@EVs-PFD with active targeting ability was prepared in this study,and its physicochemical properties were suitable.The results of in vivo and in vitro experiments showed that it targeted the liver obviously and had a significant anti-fibrotic effect.Therefore,HA@EVs-PFD is a drug delivery system that inhibits hepatic fibrosis by targeting and inhibiting activated HSC,and has good clinical application prospects.