Study On Curcumin Loaded Liposomes Based On The Recognition Of Fn14 To Treat Hepatic Fibrosis

Background Hepatic fibrosis is caused by long-term liver injury which leading to extracellular matrix over-deposition in body wound repair response.Hepatic stellate cells(HSCs)play an important role in the process of liver fibrosis.Inhibit the proliferation and activation of HSCs could be an effective strategy for the treatment of hepatic fibrosis.The incidence and mortality of liver fibrosis and cirrhosis has been high in those years globally.But there is no effective treatment of drugs or means for patients in clinic.Therefore,we need to develop anti-fibrosis agents for which are safe and effective to solve the clinical difficulties and control the condition of patients effectively.Objective The specific binding peptide of HSCs(sequence: ~DC~DH~DP~DR~DE~DV~DD~DV~DE~DL~DY~DS~DT~DV~DF~DG~DH)was screened by mRNA display based on the early research achievement of our research group.The purpose of our study is to research an effective anti-fibrosis preparation to target the site of hepatic fibrosis.Hence we use the active targeted drug delivery strategy for the treatment of liver fibrosis.The main contents of this study including the three aspects: the preparation and characterization of D-FNB peptide modified liposomes,in vitro evaluation of D-FNB peptide modified liposomes and in vivo evaluation of D-FNB peptide modified liposomes.The methods and results of our study are as follows:Methods and Results Preparation and Characterization of D-FNB Peptide Modified Targeted Liposomes.Firstly the binding ability of D-FNB peptide to the extracellular domain protein of Fn14 was investigated by SPR technique and molecular docking approach.The results showed that the D-FNB peptide(sequence: ~DC~DH~DP~DR~DE~DV~DD~DV~DE~DL~DY~DS~DT~DV~DF~DG~DH,Fn14-binding peptide,D-FNB)had a strong affinity for the extracellular domain protein of Fn14.Thereafter,D-FNB-PEG3400-DSPE was synthesized by the reaction of maleimide in MAL-PEG3400-DSPE with the thiol group in D-FNB peptide.The reaction was monitored by TLC and then purified by CL-4B.The results showed that the D-FNB-PEG3400-DSPE has been synthesized successfully by the method.CUR-loaded liposomes were prepared by a thin-film hydration method.The obtained liposomes were homogeneous(size: 144.21 ± 1.14 nm)and dispersed well.The shape of the liposomes were complete under the transmission electron microscope(TEM),the EE% of liposomes was 94.86 ± 2.14%,and the in vitro release of CUR showed that there had no differences between D-FNB-LIP/CUR and mPEG-LIP/CUR.In Vitro Evaluation of D-FNB Peptide Modified Liposomes.Various model cell lines were examined by immunofluorescent staining to detect the expression of biomarkers.Then,the cellular internalization of D-FNB-LIP was investigated qualitatively and quantitatively via the detection of coumarin-6-positive cells by a fluorescence microscope and flow cytometry.D-FNB-LIP/coumarin-6 were more efficiently integrated by HSC-T6 cells than mPEG-LIP/coumarin-6,the fluorescence intensity was approximately 2.54-fold higher in the HSC-T6 cells treated with the D-FNB-modified vesicles than in those treated with mPEG-LIP.However,there were no significant differences in the vesicle uptake among BRL-3A cells.Preincubation of HSC-T6 cells with the free D-FNB peptide and TWEAK greatly inhibited the uptake of the targeting vesicles by the cells.These data indicated that the D-FNB peptide specifically recognized Fn14-overexpress-ing cells,which supported the potential use of D-FNB-LIP for drug delivery into activated HSCs in vivo.In vitro cytotoxicity of the peptide-modified CUR formulation for target cells was detected and quantified by the MTT assay.As expected,FNB-LIP/CUR(IC50 = 7.3 ?g/mL)demonstrated significantly higher cytotoxicity against HSCs than mPEG-LIP/CUR(IC50 = 18.6 ?g/mL),while normal hepatocyte cells,BRL-3A,showed no clear selectivity for the different treatments.Finally,the in vitro anti-fibrosis effect of CUR formulations was further evaluated by the TUNEL and western blot assay.Compared to the mPEG-LIP/CUR treated group,the D-FNB-LIP /CUR treatment could induce more target cell apoptosis and also significantly alleviate the expression of ?-SMA,suggesting the inhibition of HSCs activation.In Vivo Evaluation of D-FNB Peptide Modified Liposomes.Firstly,the levels of hepatic fibrosis were evaluated in the model mice using several assessment methods,including H&E and Sirius red staining and biochemical assays.The serum levels of ALT and AST,which are the markers of hepatic damage,were significantly higher in the model mice,especially in the groups with long-term CCl4 induction than in the normal mice.In addition,the livers of the normal mice had a smooth surface with homogeneous and flexible textures,while those of the model mice(8 weeks of CCl4 treatment)were covered with fibrous capsules,and their textures became sclerosal.As shown in H&E staining,after 8 weeks of continuous treatment with CCl4,central necrosis of the hepatic lobule and inflammation with central vein bridging necrosis were obvious,and Sirius red+ collagen appeared in fibrous septa and portal areas in the model mice.These results demonstrated that the liver fibrosis model was successfully established.Accordingly,in vivo imaging assays were performed in the normal and CCl4-treated model mice with different formulations.It was observed that the D-FNB-modified liposomes could significantly improve the distribution of encapsulated fluorescence in the fibrotic liver compared to the non-modified vesicles at the same time points.A similar trend was also detected by the quantitative analysis of in vivo drug distribution in the normal and model mice after a single dose of the CUR formulations.Based on the results,FNB-LIP/CUR was more prominently accumulated in liver tissues than mPEG-LIP/CUR.Furthermore,the strong co-localization of fluorescence signals in coumarin-6-loaded D-FNB-LIP as well as the presence of ?-SMA-and Fn14-positive areas in fibrotic liver tissue indicated that the modification of liposomes with D-FNB peptide remarkably enhanced the liver-targeting efficiency of the drug delivery systems,especially their ability to precisely hit the diseased sites.The therapeutic effects were assessed by the evaluation of pathology,immunofluorescence staining,and biochemical factors.H&E and Sirius red staining showed that the treatment with CUR-loaded FNB-LIP could alleviate the hepatic fibrosis,unlike the treatments with CUR-loaded mPEG-LIP and the Saline.The expression of Fn14 and the fibroblast marker ?-SMA in liver tissues were detected after the therapy with the different formulations of CUR.The FNB-LIP/CUR remarkably decreased the infiltration of Fn14+ and ?-SMA+ myofibroblasts in the fibrotic livers of the treated mice.Furthermore,the serum levels of ALT and AST were significantly lower in the mice treated with the targeting formulation compared with the mPEG-LIP/CUR and the Saline-treated groups,showing a notable curative effect of hepatic fibrosis by D-FNB modified liposome.Conclusion In summary,our study has designed and prepared a D-FNB peptide mediated targeting drug delivery system and we solve the problems that drug could not reach the site effectively with the delivery strategy of active targeting.Through the in vitro and in vivo anti-fibrosis research in D-FNB peptide modified liposomes,confirmed the targeted and therapeutic effect of the treatment.Inhibit the proliferation of hepatic stellate cell effectively.Anti-inflammatory and reduce the deposition of extracellular matrix is the research objectives of anti-fibrosis drugs.Our subject prepared the functional peptide modified and CUR-loaded TDDS successfully and suggesting a promising potential for targeting treatment in liver fibrosis.