Preparation Of Targeted Liposomes Encapsulating Promoting Hepatocyte Growth Factor And Evaluation Of Their Anti-fibrosis Effect On Liver Fibrosis

Liver fibrosis and advanced fibrosis(liver cirrhosis) represent the final common pathway of virtually all chronic liver diseases.The hepatic stellate cell(HSC) is the primary cell-type in the liver responsible for excess collagen synthesis during hepatic fibrosis,so this cell is the major target for anti-fibrosis treatment.The anti-fibrosis effect will be enhanced by enhancing drugs target to HSC.Cyclic RGD(Arg-Gly-Asp) peptides recognize specially the typeâ…¥collagen receptor which is on the surface of activated HSC,so they can be used to modify the drugs which have effects on activated HSC.After modified with polyethylene glycol (PEG),sterically stabilized liposomes(SSL) have the ability to escape the recognization and clearance of reticuloendothelial system so that they can prolong the half-life phase of drug which is encapsulated.Studies in vitro and in vivo have shown that promoting hepatocyte growth factor(pHGF) has the anti-fibrosis effect on liver fibrosis,but pHGF has not shown a satisfactory anti-fibrosis effect in clinical application because of its short half-life phase and its adverse events caused by affecting other cells,for example promoting the development of liver tumor.Therefore,the purpose of our study is to synthesis a kind of SSL which is encapsulating pHGF and modified with cyclic RGD peptides(RGD-SSL-pHGF). Thus the half-life period of pHGF will be prolonged and its target to activated HSC will be enhanced.The anti-fibrosis effect of RGD-SSL-pHGF and the mechanism of action will be evaluated.This study includes four parts:to compare the effect of pHGF and recombinant hepatocyte growth factor on HSC;to synthesis RGD-SSL-pHGF and evaluate their physico-chemical properties;to evaluate the effect of RGD-SSL-pHGF on culture-activated HSC;to evaluate the treatment effect of RGD-SSL-pHGF on liver fibrosis in rats.Part One Compare the effect of pHGF and recombinant hepatocyte growth factor on HSC.Aims:To compare the effect of pHGF and recombinant human hepatocyte growth factor(rh-HGF) on the growth and apoptosis of culture-activated HSC.Methods:HSC were isolated from normal male SD rats(400～450g,14 weeks old) by continuous infusion with pronase E and typeâ…£collagenase and purified with density gradient centrifugation.After cultured and passaged,the expression ofα-smooth muscle actin(α-SMA) in HSC was evaluated.Then the growth of HSC inhibited by pHGF and rh-HGF were compared with 3-(4,5-dimethylthiazo(-z-yl)-3,5 diphenytetrazoliumromide(MTT) method and the apoptosis of activated HSC promoted by pHGF and rh-HGF were compared with TdT-mediated dUTP Nick-End Labeling(TUNEL) method.Results:The harvest rate of HSC was about 2×10⁷ per rat,and the viability was about 96%.The cells cultured for 24 hours after passaged were proved to be activated HSC based on the appearance and the expression ofα-SMA.Both pHGF and rh-HGF inhibited the growth of HSC,and this effect was increasing following with the increased concentration.The inhibition effect of rh-HGF was superior to that of pHGF at the same concentration.The IC₅₀ of pHGF was 165.67±7.09ng/ml,higher than that of rh-HGF(132.00±4.20ng/ml)(P=0.02).pHGF induced 33.67%activated HSC to apoptosis,while rh-HGF induced 43.96%activated HSC to apoptosis at the concentration of 150ng/ml(P=0.05).Conclusion:Both pHGF and rh-HGF inhibited the growth of HSC and promoted the apoptosis of activated HSC,but rh-HGF was superior to pHGF.Part Two Synthesis RGD-SSL-pHGF and evaluate their physico-chemical properties.Aims:To synthesis RGD-SSL-pHGF and evaluate their physico-chemical properties.Methods:SSL encapsulating pHGF were synthesized,and modified with cyclic RGD peptides which were conjugated with DOPE-PEG-MAL on the surface of SSL. Then the properties of RGD-SSL-pHGF were evaluated,including size,encapsulated percentage of pHGF and stability.In addition,SSL encapsulating calcein(CF) modified with or without RGD(RGD-SSL-CF or SSL-CF) were synthesized.After cultured with these liposomes and CF solution for 24 hours,the fluorescence intensity in HSC cytoplasm was evaluated with flow cytometry in order to evaluate the target of these agents to HSC.Results:The mean size of RGD-SSL-pHGF was 92.7nm and their surface potential was—18.49mv.The encapsulated percentage of pHGF was 32.38%,that was to say,there were about 5μg pHGF and 10nmol RGD per milliliter liposomes solution.These liposomes were stable at below 4℃.6.58%phospholipids were loss in the synthesize process.After cultured with RGD-SSL-CF,SSL-CF and CF solution for 24 hours,the fluorescence intensity in RGD-SSL-CF group was highest(P＜0.05).Conclusion:RGD-SSL-pHGF were synthesized.The encapsulation in SSL modified with RGD prolonged the action time of pHGF and enhanced its target to activated HSC.Part Three The effect of RGD-SSL-pHGF on culture-activated HSCAims:To compare the effects of pHGF encapsulated in SSL modified with or without RGD(RGD-SSL-pHGF or SSL-pHGF),pHGF and SSL modified with RGD (RGD-SSL) on cultured-activated HSC.Methods:RGD-SSL-pHGF,SSL-pHGF and RGD-SSL were synthesized.After cultured with these liposomes and pHGF,the growth of HSC,the expression of mRNA and the production ofα-SMA and typeâ… collagen in HSC were compared.The apoptosis of HSC was compared,too.Results:After culturing with HSC for 48 hours,pHGF promoted the apoptosis of HSC and inhibited the expression ofα-SMA,transforming growth factor-β1, procollagenα₁(â… ),tissue inhibitor of metalloproteinase-1 and -2,and matrix metalloproteinase-2 mRNA.It also inhibited the production ofα-SMA and typeâ… collagen.Compared with SSL-pHGF,pHGF and RGD-SSL,RGD-SSL-pHGF has the most significant effects on HSC.Conclusion:After encapsulated into SSL modified with RGD,pHGF showed more significant anti-fibrosis effect on activated HSC.Part Four The treatment effect of RGD-SSL-pHGF on liver fibrosis in ratsAims:To evaluated the treatment effect and the action mechanism of RGD-SSL-pHGF on liver fibrosis in rats.Methods:Liver fibrosis was induced in rats by injecting thioacetamide intraperitoneally for 10 weeks.After resting for another 3 weeks,these rats were divided into 6 groups(n=10) according to injected agents:RGD-SSL-pHGF, SSL-pHGF,pHGF,RGD-SSL,pHGF plus RGD-SSL and saline.After injected these agents every other days for 4 weeks,the histologic stage,the fibrotic area percentage, the hydroxyproline amount per gram hepatic tissue,the apoptotic rate ofα-SMA-positive cells and the expression of procollagenα₁(â… ) andα₁(â…¢) mRNA in every group were compared.Results:The resolution of liver fibrosis was the most significant in RGD-SSL-pHGF group.There were the lowest fibrotic area percentage and the least hydroxyproline amount in RGD-SSL-pHGF group,too.The injection of RGD-SSL-pHGF also promoted the mostα-SMA-positive cells to apoptosis and inhibited most significantly the expression of procollagenα₁(â… ) mRNA.Conclusion:RGD-SSL-pHGF promoted the resolution of liver fibrosis by promoting the apoptosis ofα-SMA-positive cells and the digestion of existing fiber collagen and inhibiting the production of typeâ… collagen.