Layered Double Hydroxides-Loaded Sorafenib For The Treatment Of Liver Fibrosis

Objective:In this study,layered double hydroxide were used as sorafenib（SRF）drug carrier to deliver SRF in the treatment of liver fibrosis.The mechanism of action of nanoagent LDHs-SRF on cell proliferation,apoptosis and migration were verified in vitro,and the therapeutic effect of LDHs-SRF on CCl₄-induced liver fibrosis rat model was explored in vivo,providing new perspectives and ideas for the treatment of liver fibrosis.Methods:1.LDHs and LDHs-SRF were prepared by hydrothermal and co-precipitation method;2.The morphology of LDHs-SRF was observed by TEM;3.FTIR and XRD were used to detect the nano preparation for phase characterization;4.LDHs-SRF particle size distribution and Zeta potential were measured by Malvern laser particle size analyzer;5.The drug loading of LDHs-SRF was determined by UV spectrophotometer;6.The uptake of FITC-labeled LDHs-SRF by HSC-T6 in rat hepatic stellate cells was observed under confocal laser scanning microscope and flow cytometry;7.CCK-8 assay was used to detect the effect of LDHs-SRF on proliferation inhibition of HSC-T6 cells;8.The effect of LDHs-SRF on HSC-T6 cell migration was detected by cell scratch and Transwell method;9.Flow cytometry was used to detect the effects of nano-agents on apoptosis and cycle change of HSC-T6 cells;10.H＆E,Masson,Sirius red staining and immunohistochemical staining were used to detect the anti-fibrosis effect of LDHs-SRF on CCl₄-induced rat liver fibrosis model;11.Western blot was used to detect the effects of LDHs-SRF on proteins and expressions of hepatic fibrosis related signaling pathways in HSC-T6 cells.Results:1.Layered double hydroxide loaded sorafenib（LDHs-SRF）was successfully constructed by hydrothermal and co-precipitation method;2.TEM showed that LDHs and LDHs-SRF showed polygonal sheet structure and irregular rod shape,respectively;3.FTIR results showed that no new chemical bonds were formed between LDHs and LDHs-SRF,XRD data show the crystal structure integrity of LDHs-SRF;4.The average particle sizes of LDHs and LDHs-SRF are 105.3 nm and 120.1 nm,respectively,and the average Zeta potentials of LDHs and LDHs-SRF are+34.7 m V and+40.1 m V,respectively;5.UV spectrophotometer detected and calculated LDHs-SRF drug loading about 25%;6.Cell uptake experiment showed that LDHS-SRF had been absorbed by HSC-T6 at 1h,and the uptake rate reached 91.75%;7.Compared with SRF group,LDHs-SRF could effectively inhibit the proliferation of HSC-T6 cells;8.Compared with SRF group,LDHs-SRF group could effectively inhibit the migration ability of HSC-T6cells;9.LDHs-SRF group could induce apoptosis of HSC-T6 cells and arrest the cell cycle in G2/M phase;10.Rat liver fibrosis model was successfully established by using carbon tetrachloride CCl₄,and it was observed that liver inflammation infiltration Collagen fiber deposition was significantly reduced in LDHs-SRF group,and the expressions of liver fibrosis markersα-SMA protein and ECM Collagen I protein were significantly reduced;11.Western blot results showed the protein expression levels of p-AKT,TGF-β1,p-Smad2/3,vimentin,snail1,N-cadherin,α-SMA and collagenⅠwere highly decreased,while the protein expression levels of E-cadherin,smad7 were significantly increased in HSC-T6 after LDHs-SRF treatment.Conclusion:In this study,LDHs-SRF loaded with sorafenib was successfully synthesized.The results showed that LDHs-SRF inhibited the proliferation and activation of HSC-T6 cells through TGF-β1/Smad/EMT signaling pathway and AKT pathway.In addition,LDHs-SRF treatment of hepatic fibrosis rats significantly reduced liver inflammatory infiltration and collagen fiber deposition,and improved thetherapeutic effect of anti-fibrosis in vivo.Therefore,LDHs-SRF can be used as a potential strategy for the treatment of hepatic fibrosis.