Delivery Of Camptothecin With The Retinol-conjugated Micelles For Suppression Of The Glycolysis Of Hepatic Stellate Cells And Liver Fibrogenesis In Mice

Myofibroblastic HSCs are considered as key target cells for liver fibrosis therapy,because they are the dominant contributors to ECM production in experimental fibrosis.Deregulated accumulation of myofibroblasts drived from quiescent hepatic stellate cells(HSCs)contributes to the pathogenesis of liver fibrosis and cirrhosis.We have known that the glycolytic activity in myofibroblasts connects to the development of liver fibrosis.Camptothecin(CPT)was chosen and examined to decrease the hypoxia induced factor 1?(HIF-1-?)protein and expression of its downstream targets in HSCs including the critical glycolytic rate-limiting enzyme gene,further more inhibit the MF phaenotype of activated HSCs.In this study,we demonstate a retinol-conjugated nanoparticles physically enveloped Camptothecin(CPT)that selectively target to HSCs,suppresses the glycolysis of the active HSCs and thus reverses the myo fibroblastic HSCs to quiescent status in vivo,infiltration of inflammatory cells and collagen protein accumulation in the CCl4 injured mouse liver.CPT serving as a promising drug for the mild safe and effective therapy for liver fibrosis.Firstly,we synthesis PEG-PCL copolymer with retinolligand then prepare micellar and the PEG-PCL copolymer without ligand was set as control.The VA-PEG-PCL and PEG-PCL micelles were fabricated by the solvent evaporation method.The physiochemical properties of materials and micellar were characterization through 1HNMR,FT-IR,DLS and TEM.Cytotoxicity analysis and hemolysis rate were then measured and illustrated favourable bioconpatibility of VA-PEG-PCL and PEG-PCL micelles.Then we prepared the micellar which enveloped CPT and evaluated their physicochemical properties.The result evidenced that the syntheses of the PEG-PCL copolymers with or without the retinol modification were acconplished;average diameter of the generated nanoparticles was 82.7 nm for the VA-PEG-PCL and 90.3 nm for the PEG-PCL,respectively and both of them possessed round shape;both micellar preparations were obtained with pretty low CMC values,low toxicity and excellent hemo-compatibility;In vitro drug release profiles indicated that the drug release profile of the micelles was stable and partially controlled in the pH-dependent manner.We studied the cellular uptake of the Nile red-loaded VA-PEG-PCL or PEG-PCL micellar against the activative HSCs,primary HSCs and primary HCs in vitro.Compared to the PEG-PCL nanoparticles,the VA-PEG-PCL micellar significantly enhance cellular uptake both in the immortalized active HSC-T6 cells and the culture-activated primary HSC cells.Further more,biodistribution studies with the CCl4 acute injured mice revealed that the VA-PEG-PCL micellar were illustrious accumulated in the injured liver,further indicating that retinol-conjugated polymer nanoparticles provided not only the excellent actively targeting to HSCs in vitro but also much better enrichment of the loads in the fibrotic liver.CPT provided excellent inhibition to HSCs in vitro,as shown either by cytotoxicity assays or by more downregulation of several fibrotic genes such as ?-Sma and Collagen lal.The same tendency was found in the study of CPT-loaded micelles against culture-activated primary HSCs too.And because of the target profile of VA,the VA-PEG-PCL micelle shown the stronger inhibition than PEG-PCL micelles.To investigatived the therapeutic efficacy of naked CPT or encapsulated CPT,fibrotic mice induced by the chronic carbon tetrachloride(CCl4)injection were then given systemically the naked or encapsulated camptothecin simultaneously,and resolution of fibrosis by CPT was comprehensively assessed with the biochemical,histological and molecular biolological assays.Only the CPT which was physically enveloped in VA-PEG-PCL micellar greatly improved the CCl4 injury induced fibrosis as shown in effectively inhibited the glycolysis and suppress expand:ing of the MF-HSCs in fibrotic mouse liver,consequently inhibited fibrogenesis induced by the CCl4 so that ameliorated hepatic fibrosis.Quite the contrary,naked CPT and PEG-PCL loaded CPT seems to be no obviously therapeutic effect,that might because of the CPT toxicity lead to apoptosis of liver cells so that promote the occurrence of hepatic fibrosis.To further evaluate the therapeutic efficacy of FA-PEG-PCL loaded CPT in liver fibrosis,we used the same fibrosis model described above.And the result showed that only FA-PEG-PCL loaded CPT can effectively protect CPT during circulation,direct its active attacks to the HSCs,suppress the glycolysis of MF-HSCs and lessen fibrosis extent in mice.So that ameliorated hepatic fibrosis and restore liver function at the same time.Such findings suggest that this CPT drug delivery system,designed to selectively target to HSCs,can serve as a promising drug for the treatment of suppression the occurrence and development of hepatic fibrosis,which could be a mild safe and effective effect.