Construction And Biological Evaluation For Renal Delivery System Of Immunoliposomes Modified By Anti-integrin-αlpha8

Objective To explore the synergistic effects of emodin(EMO)and glycyrrhizin(GL)against renal fibrosis and screen for optimal molar ratio.To construst PEGylated long-circulating liposomes for evading the reticulo-endothelial system uptake and improving the circulating time of Emodin and Diammonium Glycyrrhizinate in vivo.Anti-integrin αlpha8 Conjugated Immunoliposomes were prepared with the purpose of Targeting Delivery of Emodin and Diammonium Glycyrrhizinate(DAG)against Renal Fibrosis.Methods NIH3T3 was treated with EMO,GL and EMO/GL at different molar ratios(1:1,1:2,1:5,1:10)for 48 h,respectively.Then,cell viability was assessed using MTT assay.Moreover,the myofibroblast transdifferentiation cell line,induced by transforming growth factor-β1(TGF-β1),was treated with EMO,GL and EMO/GL(molar ratio at 1:5)for 48 h,respectively,followed by Western blot analysis ofα-smooth muscle actin(α-SMA)、transforming growth factor-β1(TGF-β1)、collagen type 1(Col1)and fibronectin(FN).Long-circulating liposomes were prepared according to calcium acetate gradient method.Immunoliposomes tailoted to target Glomerular mesangial cells were developed by post-insertion method.Mean particle size and Zeta potential were estimated by laser particle size analyzer.Morphology of long-circulating liposomes and Immunoliposomes was observed using a transmission electronmicroscope and entrapment efficiency was investigated by ultrafiltration method.In vitro drug release from long-circulating liposomes was measured by dialysis method.An MTT assay was applied to examine the cytotoxicity of drug-free carriers on NIH3T3 and HBZY-1 cells.The cellular uptake of Di I-labeled long-circulating liposomes and Immunoliposomes in HK-2 and HBZY-1 cells were investigated by laser confocal mocroscopy and flow cytometry.MTT assay was used to explore the cytotoxicity of Immunoliposomes and the anti-fibrosis effect in vitro.The molecular mechanism of Immunoliposomes against renal fibrosis was investigated by Western blot analyses through observing the protein(α-SMA、COL-1,FN)express of fibrortic marks and phosphorylated protein Smad3 express.Results The cell proliferation could be significantly inhibited when the mole ratio of EMO/GL range from 1:2 to 1:10.Furthemore,in comparison with control group,the protein expression levels of α-SMA,TGF-β1,Col1 and FN were significantly decreased in emodin group and combination group(P<0.05).Especially,the protein expressions of the above mentioned factors except TGF-β1were significantly downregulated in combination group compared to emodin group(P<0.05).The morphology of Immunoliposomes was spherical with good overall dispersibility.The Immunoliposomes exhibited an mean partical size of(92.37±0.36)nm with the Zeta potential of(4.62±0.362)m V.Entrapment efficiency of emodin was 45.48±1.96% and diammonium glycyrrhizinate was 44.29±1.13%.The release behavior of emodin in vitro showed an apparently sustained release feature in the tested mediaand diammonium glycyrrhizinate could be completely released from the long-circulating liposomes over a 48 h period.In vitro,the drug-free carriers showed good biocompatibility and low toxicity on NIH3T3 and HBZY-1 cells.The immunoliposomes exhibited similar cytotoxicity against NIH3T3 and HBZY-1 cells to EMO/DAG.The down-regulated expression of α-SMA、COL-1,FN protein in immunoliposomes was slightly higher than that of EMO / DAG,which may be the result of the differences between the uptake rates of free and immunoliposomes by HBZY-1 cells.The former are usually taken up by endocytosis,a process relatively slower compared to that of transmembrane diffusion of free drugs.But those protein expression of immunoliposomes down-regulated lower compared with long-circulating liposomes may be caused by the specific binding of anti-Integrin-αlpha8 to HBZY-1 cells.While the modefication of Anti-integrin-αlpha8could increase the Glomerular mesangial cells uptake to a certain extent.Conclusion EMO and GL synergistically inhibited proliferation and myofibroblast differentiation,as well reduced the synthesis of extracellular matrix at ratio of range from 1:2 to 1:10.Anti-integrin-αlpha8 Conjugated Immunoliposomes for Targeted Delivery of Emodin and Diammonium Glycyrrhizinate were successfully developed,increasing the HBZY-1 cell uptake and finally boosting the anti-renal fibrosis in vitro.