Experimental Investigatian On Liver-targeting Hepatitis B Immuneglobulin Nanoparticles

Background Hepatitis B virus (HBV) infection is one of the most prevalent diseases in the world. According to WHO report, one third of the world's population (2 billion people) has been infected with HBV, and more than 400 million people are chronically infected by the HBV. Despite the availability of an effective vaccine, hepatitis B still continues to be a significant health problem. Updated antiviral therapy of chronic hepatitis B could not eliminate HBV infection. The aims of treatment of chronic hepatitis B are only to achieve sustained suppression of HBV replication and remission of liver disease. For this reason, the discovery of new and effective antiviral agents against HBV infection is of considerable importance. Hepatitis B immunoglobulin (HBIG) is a passive immunoprophylaxis for HBV. Earlier studies have demonstrated the therapeutic effect of HBIG is believed to be due to high-affinity binding with HBs-containing particles and neutralization of HBV in the circulation. Recently, Ralf Schilling has demonstrated that HBIG was internalized in the hepatocytes, can reduce the excretion of HBV or interfere with the envelopment of viral nucleocapsids containing HBV DNA intermediates. HBIG can protect the transplanted liver against HBV reinfection. Therefor, HBIG may be a new and poteintial antiviral agent against HBV infection.Nanoparticles are small polymeric particles in the nanometer size range. Usually, they are easily taken up by phagocytic cells and accumulate in the organs of the reticuloendothelial system (RES) such as the liver and the spleen. As a drug carrier system, it has the ability of Organ, tissue or cell targeting, changing the biodistribution of drugs, controlling drug release, decreasing the drug adverse effect and enhancing the efficacy of drugs. Therefore, Study on the hepatic targeted drug delivery system (TDDS) will promote treatment for liver diseases.Objective Based on the pharmacologic action of HBIG and the characteristic of nanoparticle drug delivery system, We study:1. The liver-targeted poly(butylcynaoacrylate) nanoparticles of HBIG (HBIG-PBCA-NP) were prepared by emulsion polymerization method with butylcyanoacylate (BCA) as carrier material and the technical conditions for preparing the nanoparticles at pH＞5.0 aqueous medium were optimized.2. To evaluate the safety and the liver-targeted and sustained release characteristics of HBIG-PBCA-NP in vitro and in vivo.3. Using a panel of hepatocyte-derived cell lines, we investigated in vitro what are differences between HBIG or HBIG-PBCA-NP on the drug internalization of hepatocytes and inhibition of HBsAg release and HBV replication in the cells. Depended on the results we evaluated the possibility of HBIG-PBCA-NP as a new antiviral agent for treatment of chronic hepatitis B.Methods1. Inhibition the secretion of HBsAg and HBV DNA secretion by HBIG in vitro: QSG7701 cells were cultured with DMEM and different concentrations of HBIG. At different time points after culture, the supernatant was collected for HBsAb quantitative detection by time-resolved immunofluorometric assay (IFMA) and calculated the amount of intracellular HBIG. HepG 2.2.15 cells were cultured with DMEM in the absence or present of different concentrations of HBIG. The supernatant at some days interval was collected for HBsAg quantitative detection by IFMA and HBV DNA quantitative detection by real-time fluorogenic quantitative PCR(RTFQ-PCR). MTT assay was used to evaluate the cytotoxicity of HBIG.2. Optimization of the technical conditions for preparing HBIG-PBCA-NP: HBIG-PBCA-NP is prepared by emulsion polymerization method with butylcyanoacrylate as the polymeric carrier. The preparing technology is optimized by single factor test, uniform design and orthogonal design test with nanoparticle morphology, size and size distribution, polydisperse index and the entrapment efficiency as the test standard.3. Studies of the liver-targeted and sustained release characteristics of HBIG-PBCA-NP in vitro and in vivo: In vitro experiments the procedure were as follows: (1)QSG7701 cells and HepG2.2.15 cells were cultured with DMEM and HBIG-PBCA-NE At 72h after incubation the cells was collected for detection of the nanoparticle by transmission electron microscope (TEM); (2)QSG7701 cells were cultured with DMEM and different concentrations of HBIG-PBCA-NP, At different time points after culture the supernatant was collected for HBsAb quantitative detection by IFMA and calculated the amount of intracellular HBIG. In vivo experiments the procedure were as follows: (1) HBIG-PBCA-NP and/or HBIG were administrated to the mice by the tail intravenous injection. After 30 min the mice were killed and the liver tissiue were collected for detection the nanoparticle by TEM and HBIG by immunohistochemistry; (2) ¹²⁵I-HBIG was prepared by Iodonen labelling method and ¹²⁵I-HBIG-PBCA-NP prepared by the technical conditions for preparing HBIG- PBCA-NP. After ¹²⁵I-HBIG-PBCA-NP and ¹²⁵I-HBIG were administrated to the mice by the tail intravenous injection, ¹²⁵I radioactivity in mouse's organs were determined at some hours interval. Target index (TI) was calculated to value the organ's distribution of ¹²⁵I-HBIG-PBCA-NP.4. Study of HBIG-PBCA-NP safety in vitro and in vivo: In vitro experiments were done as follows: (1) QSG7701 cells and HepG2.2.15 cells were cultured with DMEM and HBIG-PBCA-NP. At 72h after incubation the cells was collected for detection the morphology change by light microscope(LM) and the pathological change by TEM; (2) QSG7701 cells, HepG2.2.15cells and PBMCs were cultured with DMEM and the different concentrations of HBIG-PBCA-NP,HBIG and blank nanoparticles, At different times, The cell proliferation rate was measured by MTT assay and levels of Lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were detected in the supernatant of QSG7701 cells by biochemical analysis. In vivo experiments were done as follows: (1) HBIG-PBCA-NP and/or HBIG were administrated to the mice by the tail intravenous injection. After 30 min the mice were killed and the liver tissiue were collected for detection the morphology change (HE dyeing) by LM and the pathological change by TEM and serum ALT levels by biochemical analysis; (2) In vitro hemolysis of HBIG-PBCA-NP and blank nanoparticles was tested by test tube method. 5. Inhibition of the excretion of HBsAg and HBV DNA in vitro by HBIG-PBCA-NP : HepG 2.2.15 cells were cultured with DMEM in the absence or present of different concentrations of HBIG-PBCA-NR The supernatant at some days interval was collected for HBsAg quantitative detection by IFMA and HBV DNA quantitative detection by RTFQ-PCR, and as compared with the same concentration of HBIG. The cytotoxicity of HBIG-PBCA-NP was evaluated by MTT.Results1. (1)38～46% of HBIG was endocytosed into QSG7701 after 48 hours of culture. (2) HBIG in the concentration from 0.01 IU/mL to 10.0IU/mL had no cytotoxicity to the cells of QSG7701 and HepG2.2.15.(3) HBsAg and HBV DNA in supernatants of HepG2.2.15 cultured with 0.1, 1.0 or 10.0 IU of HBIG/ml was reduced significantly than that of HepG2.2.15 cultured without HBIG after 3, 6, 9 days incubation,respectively(P＜0.01). (4) HBsAg and HBV DNA in supernatants of HepG2.2.15 with 0.1, 1.0 or 5.0 IU of HBIG/ml was reduced significantly,after three days incubation(P＜0.01). The amount of HBsAg secreted in supernatants was reduced continuingly at 5th and 7th days after HBIG removed (P＜0.01), but rebound at 9th (P＜0.01) and 11th days after HBIG removed, respectively, as compared to the control. The amount of HBV DNA secreted in supernatants was reduced continuingly At 5th days after HBIG removed (P＜0.01), but rebound at 7th, 9th and 11th days after HBIG removed, respectively, as compared to the control.2. The HBIG-PBCA-NP emulsified solution showed that HBIG-PBCA-NP was round, smooth and not adhesion, according to the optimized condition preparation. The average diameter of nanoparticle is (119.87±8.01) nm, range from 50 to 220 nm, and the entrapment efficiency of the drug was (90.10±3.66) %.3. After inoculation of HBIG-PBCA-NP with QSG7701 and HepG2.2.15 cells, the nanoparticles were in the cytoplasm and nucleus by TEM photograph. 65～73% of HBIG was endocytosed into QSG7701 after 48 hours of culture with different concentration of HBIG-PBCA-NP and is 1.5 times higher than that after cultured of QSG7701 with the same concentration of HBIG. The HBIG in mouse liver cells detected by immunohistochemistry showed much more intensity in the HBIG-PBCA-NP groups than that of HBIG groups(P＜0.01). TEM photographs of mouse liver tissue showed many nanoparticles in liver cells. The specific radioactivity of ¹²⁵I-HBIG was 152.25μci/mg HBIG , the Labelling efficiency was 61.3%. The average diameter, entrapment efficiency of the ¹²⁵I-HBIG-PBCA-NP was (123.85±7.61) nm and (86.3±5.1) %, respectively. ¹²⁵I radioactivity (CPM% and CPM%/g) in ¹²⁵I-HBIG-PBCA-NP treatment groups was higher in the mice liver and lower in kidney and lung, as compared with that in ¹²⁵I-HBIG treatment groups. The DTI derived from radioactivity of ¹²⁵I-HBIG-PBCA-NP/ ¹²⁵I-HBIG in liver and spleen was over one, and the DTI of liver over spleen.4. There were no morphology and pathological changes in QSG7701 and HepG2.2.15 cells after cultured with HBIG-PBCA-NP, HBIG and blank nanoparticles by LM and TEM photographs. No cytotoxicity(0～1 grade) was showed when QSG7701, HepG2.2.15 and PBMC cultured with HBIG-PBCA-NP, HBIG and blank nanoparticles in the study range of concentration. The LDH and ALT levels of HBIG-PBCA-NP, HBIG and blank nanoparticles treatment groups from QSG7701 cells have no difference. TEM and LM photographs of mouse liver tissue in HBIG-PBCA-NP, HBIG and blank nanoparticles treatment groups showed no morphology and pathological changes and serum ALT levels have no difference. HBIG-PBCA-NP did not induce hemolysis.5. (1) HBsAg and HBV DNA in supernatants of HepG2.2.15 with 0.1, 1.0 or 10.0 IU of HBIG-PBCA-NP/ml was reduced significantly as compared to HepG2.2.15 without HBIG-PBCA-NP after 3, 6, 9 days incubation, respectively(P＜0.01), but no difference as compared with the same concentration of HBIG(p＞0.05). (2) HBsAg and HBV DNA in supernatants of HepG2.2.15 with 0.1, 1.0 or 5.0 IU of HBIG-PBCA-NP /ml was reduced significantly, as compared to the control after three days incubation, respectively (P＜0.01). The inhibition of HBsAg excretion was sustained at 5th and 7th days after HBIG-PBCA-NP removed (P＜0.01), but rebound at 9th and 11th days after HBIG-PBCA-NP removed (P＜0.01), respectively, as compared to the control. And there are difference between HBIG-PBCA-NP groups and HBIG groups at 9th～ 11th days(P＜0.01). The amount of HBV DNA secreted in supernatants was reduced continuingly At 5th days after HBIG-PBCA-NP removed (P＜0.01), but rebound at 7th (P＜0.01), 9th and 11th days after HBIG-PBCA-NP removed, respectively, as compared to the control. And there are difference between HBIG-PBCA-NP groups and HBIG groups at 7th～11th days (P＜0.05).Conclusion1. HBIG can be endocytosed into hepatocytes and can inhibit the secretion of HBsAg and HBV DNA in vitro.2. HBIG-PBCA-NP had been successfully prepared at pH5.2 aqueous medium according to the optimum conditions. The preparation of HBIG-PBCA-NP was feasible and reproducible, which could obtain desired nanoparticle morphology, size and size distribution, polydisperse index and the entrapment efficiency, etc.3. HBIG-PBCA-NP has the good liver target tropism and sustained release characteristics.4. The safety of HBIG-PBCA-NP is pretty good and the PBCA-NP has good biocompatibility.5. HBIG-PBCA-NP can be endocytosed into hepatocytes and can inhibit the secretion of HBsAg and HBV DNA in vitro. HBIG-PBCA-NP can enchance the pharmacologic action of HBIG and has sustained release characteristics in vitro.