| Chronic hepatitis B(CHB)is a kind of viral infectious disease in the world,especially in China.There are no radical drugs in clinic.The existing drugs have many problems,such as a long course of treatment,high dosage,low efficacy,drug resistance and relapse after drug withdrawal.New target gene drugs for the life cycle of hepatitis B virus have become a research hotspot.Among them,the gene drug 10-23 DNAzyme has a strong ability to specifically inhibit the expression of the hepatitis B virus antigen,but it has some problems such as high molecular weight and difficult drug delivery.In this study,hepatitis B virus C gene(expressing HBeAg)and S gene(expressing HBsAg)specific cleavage enzyme DrzBC and DrzBS were used as therapeutic gene.Three functional glycolipid graft micelles,namely chitosan oligosaccharide-stearic acid graft CSSA,chitosan oligosaccharide-stearic acid disulfide bond graft CSSO and galactosylated chitosan oligosaccharide-stearic acid disulfide bond graft Gal-CSSO,were synthesized and used to construct gene delivery systems by electrostatic combination with DrzBC and DrzBS to suppress hepatitis B virus C gene and S gene respectively for anti-hepatitis B treatmentTo achieve effective cytoplasmic delivery of therapeutic genes DrzBC and DrzBS,the CSSA gene delivery system was constructed.Low molecular weight chitosan(Chitosan oligosaccharide)obtained by enzymatic hydrolysis was chemically grafted with carboxyl group of stearic acid and active amino group of chitosan oligosaccharide by the EDC method to synthesize CSSA.The diameter of CSSA was 67.3±3.4 nm,the amino substitution degree(SD%)of CSSA was 37.9±0.62%,and the critical micelle concentration(CMC)was 75.02 ± 1.27 μg/mL.By gel retardation electrophoresis analysis,CSSA/DrzBC and CSSA/DrzBS systems were constructed at the optimized N/P of 10.HBV-transfected cell lines HepG2.2.15 were incubated with gene delivery system.The inhibition rates of CSSA/DrzBC and CSSA/DrzBS on HBeAg and HBsAg expression increased within 72 h.The maximum inhibition rates were 82.51 ± 1.28% and 84.11±2.62%at 72 h,respectively.After 72 h,the inhibition rates decreased slightly.The inhibition rate of Lipo2000/DrzBC and Lipo2000/DrzBS on HBeAg and HBsAg expression increased within 48 h and reached the maximum inhibition rate at 48 h,which were 47.29 ± 1.86%and 33.58 ± 0.72%,respectively.After 48 h,the inhibition rate decreased.Compared with Lipo2000 gene delivery system,the inhibition rates of CSSA/DrzBC and CSSA/DrzBS on the expression of HBeAg and HBsAg were significantly increased at the same incubation time,and the peak inhibition rates increased by 0.74 times and 1.50 times,respectively.Through cell uptake investigation,it was found that CSSA could be rapidly absorbed by cells and CSSA with high amino substituted(37.9±0.62%)could concentrate in the cytoplasm,which was beneficial for 10-23DNAzyme to exert effect in cytoplasm.Measured by the MTT method,the IC50 values of CSSA and Lipo2000 incubated with HepG2.2.15 for 72 h were 412.5 μg/mL and 6.8 μg/mL respectively,indicating that the cytotoxicity of CSSA was significantly lower than that of Lipo2000,and the safety of CSSA was good.In order to accelerate the drug release of the gene delivery system,CSSO gene delivery system was designed as a new system which was sensitive to the high concentration of GSH in hepatocytes.CSSO was synthesized by two-step amide reaction with chitosan oligosaccharides and stearate amine crosslinked by disulfide bond.The diameter of CSSO was 123.00 ± 10.98 nm,the SD%of CSSO was 6.79±0.15%,and the CMC was 65.84 ± 1.11 μg/mL.By gel retardation electrophoresis analysis,CSSO/DrzBC and CSSO/DrzBS systems were constructed at the optimized mass ratio of 60.The inhibition rates on the HepG2.2.15 expression of HBeAg and HBsAg by CSSO/DrzBC and CSSO/DrzBS reached 73.86 ± 1.77%and 67.80 ± 2.51%at 48 h,and further rose to 83.83 ± 2.34%and 76.79±2.18%at 72 h.The inhibition rates were significantly higher than those of the positive control of Lipo2000 gene delivery system,and the peak inhibition rates increased by 0.77 times and 1.29 times,respectively.Compared with the CSSA gene delivery system,the efficacy of CSSO gene delivery system was significantly improved at 24 h,indicating the rapid-onset characteristic of CSSO gene delivery system.Fluorescence labeling and confocal microscopy showed that CSSO gene delivery system could effectively escape lysosomes and then release gene rapidly responding to high GSH concentration in hepatocytes.CSSO micelles and its gene delivery system were incubated with cells for 48 h and the cell survival rate was>80%,exhibiting a better safety than CSSAConsidering that the main host of HBV infection is hepatocytes,improving the targeting of gene delivery system to hepatocytes is conducive to the effective gene therapy in vivo.Using the specificity of hepatocytes specific asialoglycoprotein receptor(ASGPR)to recognize galactosyl groups,galactosyl groups were selected as active targeting ligands for hepatocytes.Galactosylated CSSO(Gal-CSSO)was synthesized by linking the amino group of CSSO with the galactosyl group of lactose acid by the EDC method.The diameter of Gal-CSSO was 138.33±3.68 nm,the SD%of Gal-CSSO was 11.91 ± 0.21%,and the CMC was 81.06 ± 1.32 pg/mL.Gal-CSSO/DrzBC and Gal-CSSO/DrzBS systems were constructed at the optimized mass ratio of 60.The inhibition rates on the HBeAg and HBsAg expression of HepG2.2.15 by Gal-CSSO/DrzBC and Gal-CSSO/DrzBS at 48 h,reached 75.39± 1.81%and 69.34 ±1.95%,respectively,and further rised to 85.89±2.57%and 79.86±1.87%at 72 h.The inhibition rates were significantly higher than those of the positive control group of Lipo2000 drug delivery system,and the peak inhibition rates increased by 0.82 times and 1.38 times,respectively.Fluorescence labeling and confocal microscopy showed that the performance of Gal-CSSO gene delivery system,such as hepatocyte uptake rate,lysosome escape and intracellular gene drug release,was significantly better than that of CSSO system.The cytotoxicity study on HepG2.2.15 showed that after incubation with Gal-CSSO micelles and its gene delivery system,the cell survival percentages were still above 80%,indicating a low toxicity and high safety of Gal-CSSO micelles and its gene delivery system.Further,Gal-CSSO gene delivery system was constructed with Cy5 fluorescent labeled 10-23DNAzyme.Distribution in vivo and cell targeting study of liver tissue showed that Gal-CSSO gene delivery system had a high concentration in liver tissue,and was mainly targeted to hepatocyte of liver tissue.The uptake of hepatocyte was 7.78 times higher than that of Kupffer cells.It showed that the Gal-CSSO gene delivery system had better liver targeting and hepatocytes selectivity in vivo than the CSSO system.Compared with the blank control group,no significant effect on body weight of mice,and no significant changes in major tissues were found after administration of Gal-CSSO gene delivery system,suggesting the potential for efficient and safe therapeutic application in vivo. |
