Hepatitis: Tanizawa K

Jung J, Kasuya T, Tanizawa K, Kuroda S. · The Institute of Scientific and Industrial Research, Osaka University, Ibaraki City, Japan. · Yakugaku Zasshi. · Pubmed #17473521 links to  free full text

Abstract: To maximize the beneficial effects and minimize the side effect of drugs, DDS (drug delivery system) has been attracted many researchers in the recent drug development. Especially, the in vivo pinpoint delivery system for drugs is very important and key technology for developing the next generations of anti-cancer drugs and gene therapies. Bio-nanocapsule (BNC) is recombinant yeast-derived hepatitis B virus surface antigen particle, which has been used as a recombinant hepatitis B vaccine for the last 20 years in the world. BNC can incorporate various materials (chemical compounds, proteins, genes, siRNA, etc) by the fusion with liposome, and deliver them to the organs and tissues in vivo specifically by the action of bio-recognition molecules on the BNC's surface. The transfection efficiency is significantly higher than that of liposome, because BNC harbors the complete set of hepatitis B virus infection machinery. Recently, we succeeded in the in vivo retargeting of BNC by displaying either antibody or homing peptide, less than 10 amino acid residues for in vivo targeting. BNC is a hybrid of liposome and virus, and very flexible system for in vivo retargeting. BNC might be very promising carriers in the next generation of DDS.

Yu D, Fukuda T, Tuoya, Kuroda S, Tanizawa K, Kondo A, Ueda M, Yamada T, Tada H, Seno M. · Graduate School of Natural Science and Technology, Okayama University, 3.1.1 Tsushima-Naka, Okayama 700-8530, Japan. · IUBMB Life. · Pubmed #16596748 No free full text.

Abstract: The bio-nanocapsule (BNC) is our concept of artificial hollow nanoparticles that have been designed and produced through biotechnological procedures. We proposed an empty virus-like particle, which consists of a recombinant L envelope protein of hepatitis B virus (HBV) and a lipid derived from the host cell, as an engineered BNC. Although this BNC was first developed as an immunogen of hepatitis B vaccine, the pre-S1 region in N-terminus of L envelope protein confers hepatocyte specific infectivity of HBV on the BNC. This recombinant BNC is now being developed as a novel platform of drug delivery system (DDS) vector for selective delivery.

Yamada T, Ueda M, Seno M, Kondo A, Tanizawa K, Kuroda S. · Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047, Japan. · Curr Drug Targets Infect Disord. · Pubmed #15180463 No free full text.

Abstract: The hepatitis B virus (HBV) surface antigen (HBsAg) L particle is a hollow nano-scale particle. HBsAg L particles have many properties that make them useful for in vivo gene transfer vectors and drug delivery systems. Gene therapy so far has required the in vivo pinpoint delivery of genetic materials into the target organs and cells. Gene transfer by HBsAg L particles might be an attractive method, since their tropism is the same as that of HBV. The HBsAg L particles are able to deliver therapeutic payloads with high specificity to human hepatocytes. In addition, the specificity of L particle can be altered by displaying various cell-binding molecules on the surface. Our results indicate that the L particle is suitable for a cell- and tissue-specific gene/drug transfer vector. In this review, we discuss HBsAg L particles as a gene/drug transfer vector and its potential for the treatment of infectious diseases.

Kasuya T, Nomura S, Matsuzaki T, Jung J, Yamada T, Tatematsu K, Okajima T, Tanizawa K, Kuroda S. · Department of Structural Molecular Biology, The Institute of Scientific and Industrial Research, Osaka University, Japan. · FEBS J. · Pubmed #18959756 No free full text.

Abstract: We previously developed the bio-nanocapsule, which consists of hepatitis B virus envelope L proteins. The bio-nanocapsule can be used to deliver genes and drugs specifically to the human liver-derived tissues in xenograft models, presumably by utilizing the human liver-specific mechanism of hepatitis B virus infection. The hepatitis B virus tropism is highly restricted to humans and higher primates. Thus, to evaluate the in vivo therapeutic effects of forthcoming bio-nanocapsule-based medicines, it will be crucial to develop an animal model whose liver is susceptible to both bio-nanocapsule and hepatitis B virus. In the present study, we aimed to establish a bio-nanocapsule-susceptible animal model using transgenic rats expressing squamous cell carcinoma antigen-1 (SCCA1), which has been proposed to be a receptor for hepatitis B virus, interacting with the hepatitis B virus envelope protein and enhancing the cellular uptake of hepatitis B virus. We show that the recombinant SCCA1 protein interacts directly with bio-nanocapsule and inhibits its attachment to the cultured human liver-derived cells. Furthermore, we have established a transgenic rat that specifically expresses SCCA1 in the liver and also demonstrate that the amount of bio-nanocapsule accumulated in the liver is significantly increased by the SCCA1 expression. Histological analysis suggests that bio-nanocapsule is preferentially incorporated into the SCCA1-expressing hepatocytes but not into macrophages, such as Küppfer cells, nor into endothelial cells. Therefore, this animal model is expected to be useful for the development of bio-nanocapsule-based medicines.

Kasuya T, Jung J, Kadoya H, Matsuzaki T, Tatematsu K, Okajima T, Miyoshi E, Tanizawa K, Kuroda S. · Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan. · Hum Gene Ther. · Pubmed #18717644 No free full text.

Abstract: Metastasis is a key aspect of tumor malignancy, and several malignant tumors show expression of various mature N-type glycans. In particular, beta1-6 branching N-acetylglucosamine (GlcNAc) is abundantly expressed as a part of high-mannose glycans in various highly metastatic cancers. Phaseolus vulgaris agglutinin-L(4) isolectin (L(4)-PHA), which adheres to beta1-6 GlcNAc specifically, has been used for in situ cancer diagnosis. Bionanocapsules (BNCs), hollow particles with a diameter of approximately 80 nm and composed of hepatitis B surface antigen (HBsAg) and a lipid bilayer, have been developed as human liver-specific nanocapsules for in vivo drug delivery system. In this study, we have generated L(4)-PHA-displaying BNCs (PHA-BNCs) and examined whether L(4)-PHA could retarget the BNCs to malignant tumors as a "biosensor" distinguishing tumor metastaticity. Fluorescence-labeled PHA-BNCs injected systemically into a mouse xenograft model were found to accumulate in beta1-6 GlcNAc-expressing malignant tumors. The PHA-BNCs were able to deliver DNA to the malignant cancer cells. These results open up the possibility of using L(4)-PHA lectin as a targeting molecule in a drug delivery system, and of using PHA-BNCs as a novel nanodevice for malignant tumor-specific bioimaging and drug delivery.

Kasuya T, Yamada T, Uyeda A, Matsuzaki T, Okajima T, Tatematsu K, Tanizawa K, Kuroda S. · Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan. · J Biosci Bioeng. · Pubmed #18691539 links to  free full text

Abstract: Human hepatocyte-specific delivery of green fluorescent protein was succeeded in the mouse xenograft model by fusion with hepatitis B virus surface antigen pre-S regions (pre-S(1+2)), not with each pre-S region. The entire pre-S region would be useful for human liver-specific delivery of therapeutic proteins and bio-imaging fluoroproteins in biomedical field.

Jung J, Matsuzaki T, Tatematsu K, Okajima T, Tanizawa K, Kuroda S. · Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan. · J Control Release. · Pubmed #18207275 No free full text.

Abstract: Bio-nanocapsules (BNCs) consisting of hepatitis B virus (HBV) surface antigen (HBsAg) are approximately 50-nm hollow particles displaying a human hepatocyte-recognizing molecule (pre-S1 peptide). They have been used as an HB vaccine for the last two decades. Original BNC can incorporate various payloads (e.g., drugs, genes) by electroporation and deliver them to human hepatocytes specifically by utilizing the HBV infection mechanism. Here, we developed a new BNC conjugated with liposomes and succeeded in incorporating large materials (100-nm fluorescence-labeled polystyrene beads and >30 kbp plasmids) into the BNC-liposome complex. The complex delivered these large materials to human hepatocytes specifically ex vivo and in vivo. The transfection efficiency of the BNC-liposome complex was significantly higher than that of the original BNC. These results indicated that BNC confers the tissue- and cell-specificity on the conventional liposomes and raises new possibilities for drug delivery systems, gene delivery systems, and bio-imaging systems in vivo.

Nagaoka T, Fukuda T, Yoshida S, Nishimura H, Yu D, Kuroda S, Tanizawa K, Kondo A, Ueda M, Yamada H, Tada H, Seno M. · Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka Okayama, Japan. · J Control Release. · Pubmed #17306405 No free full text.

Abstract: The bio-nanocapsules (BNCs) composed of the recombinant envelope L-protein of hepatitis B virus constitute efficient delivery vectors specifically targeting human hepatocytes. Here, we have tried to enhance the stability of the BNCs because the L-proteins in the BNCs were aggregated due to random disulfide bridging when stored for a long period at 4 degrees C. The envelope protein contains fourteen cysteine residues in the S domain. Aggregation of the envelope proteins might be avoided if unessential cysteine residues are replaced or removed because the irreversible alkylation of the free sulfhydryl group protects against the aggregation and enhances the efficiency of encapsulation. In this study, the possibility of reducing the number of cysteine residues in the S domain to enhance the stability of the BNCs was assessed. The replacement of each cysteine residue by site-directed mutation showed that nine of fourteen cysteine residues were not essential to obtaining BNCs secreted into the culture media. Furthermore, upon evaluating the combination of these mutations, it was found that eight residues of replacement were acceptable. The mutant BNCs with replaced eight cysteine residues were not only more resistant against trypsin, but also more effective in transducing genes into human hepatoma-derived HepG2 cells than the original type BNC. Thus, we demonstrated that the minimized number of cysteine residues in the S domain could enhance the stability of the BNCs.

Iwasaki Y, Ueda M, Yamada T, Kondo A, Seno M, Tanizawa K, Kuroda S, Sakamoto M, Kitajima M. · Department of Surgery, School of Medicine, Keio University, Tokyo, Japan. · Cancer Gene Ther. · Pubmed #16990844 No free full text.

Abstract: The development of safe and efficient liver-specific gene delivery approaches offers new perspectives for the treatment of liver disease, in particular, liver cancer. We evaluated the therapeutic potential of hepatotropic nanoparticles for gene therapy of liver tumor. These nanoparticles do not contain a viral genome and display the hepatitis B virus L antigen, which is essential to confer hepatic specificity. It has not been shown whether a therapeutic effect could be obtained using L nanoparticles in a human liver tumor xenograft model. Rats bearing human hepatic (NuE) and non-hepatic tumors were injected with L nanoparticles containing a green fluorescent protein (GFP) expression plasmid. GFP expression was observed only in NuE-derived tumors but not in the non-hepatic tumor. The potential for treatment of liver tumors was analyzed using L nanoparticles containing the herpes simplex virus thymidine kinase gene, in conjunction with ganciclovir pro-drug administration. The growth of NuE-derived tumors in L particle-injected rats was significantly suppressed, but not of the non-hepatic tumor control. In summary, this is the first demonstration that nanoparticles could be used for delivery of therapeutic genes with anti-tumor activity into human liver tumors. This intravenous delivery system may be one of the major advantages as compared to many other viral vector systems.

Shishido T, Muraoka M, Ueda M, Seno M, Tanizawa K, Kuroda S, Fukuda H, Kondo A. · Faculty of Engineering, Kobe University, Kobe 657-8501, Japan. · Appl Microbiol Biotechnol. · Pubmed #16847600 No free full text.

Abstract: Bionanocapsules (BNCs) are hollow nanoscale particles composed of L protein of the hepatitis B virus surface antigen that represent specific affinity for human hepatocytes. BNCs can transfer genes and drugs into human hepatocytes efficiently and specifically. BNC can be expressed in yeast cells. In this study, we developed a new L particle production system using a stably transfected insect cell line. For this purpose, we established a host-vector system using the Trichoplusia ni insect cell line. L particles were efficiently secreted by the overexpression of the L protein, which was fused to the secretion signal peptide. The concentration of L particles was reached approximately 1.7 microg/ml in 5 days during cultivation in a serum-free medium without antibiotic selective pressure. The production of L particles was maintained for at least 75 days. The secretory production of L particles facilitated their easy purification by chromatography. Furthermore, it was demonstrated that purified L particles can transfect only human hepatocytes. Therefore, an insect cell expression system is an attractive tool for the production of BNC.

Maniwa K, Tanaka E, Inoue T, Sakuramoto M, Minakuchi M, Maeda Y, Tanizawa K, Takeda T, Okamoto M, Komatsu M, Taguchi Y. · Department of Respiratory Medicine, Tenri Hospital. · Kansenshogaku Zasshi. · Pubmed #16444978 No free full text.

Abstract: A 70-year-old man with liver cirrhosis and previous gastrectomy admitted for fever, coughing, and bloody sputum soon after convalescing from pulmonary tuberculosis had a peripheral white blood cell count of 9,900/microL, C-reactive protein of 14.1mg/dL, serum albumin of 2.0g/dL, and serum positive for antiaspergillus and beta-D glucan antibodies. Chest radiography showed thickening of the walls of the large residual cavities with previous tuberculosis lesions and infiltrates around them. On day 2 of hospitalization, Aspergillus fumigatus without other bacillus was detected in sputum culture taken on admission. Despite immediate treatment with intravenous micafungin and oral itraconazole and improved brief initial improvement, his general condition abruptly deteriorated into frequent massive hemoptysis and he developed of shock, respiratory failure, and severe malnutrition, dying 30 days later. Autopsy findings showed pulmonary aspergillosis in and around the large cavities and on the other side of the lungs. Pulmonary aspergillosis without hematological malignanciy and immunosuppression can thus be abruptly severe and fatal due to malnourishment stemming from pre-existing conditions such as chronic hepatitis despite prompt, ordinarily adequate medical treatment.

Yu D, Amano C, Fukuda T, Yamada T, Kuroda S, Tanizawa K, Kondo A, Ueda M, Yamada H, Tada H, Seno M. · Graduate School of Natural Science and Technology, Okayama University, Japan. · FEBS J. · Pubmed #16008564 links to  free full text

Abstract: A bio-nanocapsule (BNC), composed of the surface antigen (sAg) of the hepatitis B virus, is an efficient nanomachine with which to accomplish the liver-specific delivery of genes and drugs. Approximately 110 molecules of sAg are associated to form a BNC particle with an average diameter of 130 nm. The L protein is an sAg peptide composed mainly of preS and S regions. The preS region, with specific affinity for human hepatocytes, is localized in the N-terminus. The S region following the preS has two transmembrane regions responsible for the formation of particles. In this study, the fusion of emerald green fluorescent protein (EGFP) at the C-terminus of the S region was designed to deliver proteins to human hepatocytes. Truncation of the C-terminus of the S region was required to obtain sufficient expression levels in Cos7 cells. The nanoparticles that were produced delivered EGFP to human hepatoma cells, displaying the EGFP moiety outside, or enclosing it inside. However, only a single orientation characterizes the particle, so that either type of L fusion particle could be effectively and independently separated by an antibody affinity column. The dual C-terminal topologies of the L fusion particles designed in this study could be applied to various proteins for the C-terminal moiety of the L fusion proteins, depending on the character of the proteins, such as cytoplasmic proteins, as well as cytokines or ligands to cell surface receptors. We suggest that this fusion design is the most efficient way to prepare a BNC that delivers proteins to specific cells or tissues.

Yamada T, Iwasaki Y, Tada H, Iwabuki H, Chuah MK, VandenDriessche T, Fukuda H, Kondo A, Ueda M, Seno M, Tanizawa K, Kuroda S. · Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan. · Nat Biotechnol. · Pubmed #12833071 No free full text.

Abstract: Hepatitis B virus envelope L particles form hollow nanoparticles displaying a peptide that is indispensable for liver-specific infection by hepatitis B virus in humans. Here we demonstrate the use of L particles for the efficient and specific transfer of a gene or drug into human hepatocytes both in culture and in a mouse xenograft model. In this model, intravenous injection of L particles carrying the gene for green fluorescent protein (GFP) or a fluorescent dye resulted in observable fluorescence only in human hepatocellular carcinomas but not in other human carcinomas or in mouse tissues. When the gene encoding human clotting factor IX was transferred into the xenograft model using L particles, factor IX was produced at levels relevant to the treatment of hemophilia B. The yeast-derived L particle is free of viral genomes, highly specific to human liver cells and able to accommodate drugs as well as genes. These advantages should facilitate targeted delivery of genes and drugs to the human liver.

Kanno T, Yamada T, Iwabuki H, Tanaka H, Kuroda S, Tanizawa K, Kawai T. · Department of Atomic Scale Science, Institute of Scientific and Industrial Research (SANKEN), Osaka University, Mihogaoka, Ibaraki, Osaka 567-0047, Japan. · Anal Biochem. · Pubmed #12413451 No free full text.

Abstract: Vesicles have been utilized as nanoscale vehicles for reagents including potential drug delivery systems. When used to deliver drugs, vesicle size and the size distribution are important factors in the determination of the dosage, cell specificity, and rate of clearance from the body. Current size measurement techniques for vesicles are electron microscopy and dynamic light scattering, but their results are not equal. Therefore atomic force microscopy was attempted as another size measurement technique. After adsorption of the vesicles from a low-concentration solution of vesicles on mica substrate, each vesicle is generally found as a flattened structure. The diameters of vesicles in these solutions and their distribution have been successfully estimated from the surface area of the flattened structure of each vesicle. At higher concentrations, we have found a monolayer crammed with dome-shaped vesicles on the substrate. The diameters of vesicles in these solutions have also been successfully estimated from the surface area of the dome-shaped structure of each vesicle. Diameters of vesicles in solution estimated from two different vesicle concentrations are not close to those reported by electron microscope studies but are close to those reported by dynamic light scattering studies.

Yamada T, Iwabuki H, Kanno T, Tanaka H, Kawai T, Fukuda H, Kondo A, Seno M, Tanizawa K, Kuroda S. · Department of Structural Molecular Biology, Institute of Scientific and Industrial Research (SANKEN), Osaka University, 8-1 Mihogaoka, 567-0047, Ibaraki, Japan. · Vaccine. · Pubmed #11312011 No free full text.

Abstract: The hepatitis B virus (HBV) envelope (env) protein is composed of three regions; the 108- or 119-residue pre-S1 region involved in the direct interaction with hepatocytes, the 55-residue pre-S2 region associated with the polymerized albumin-mediated interaction, and the major 226-residue S protein region. Thus, to improve the immunogenic potency of conventional HB vaccines, development of a new vaccine containing the entire pre-S1 region in addition to pre-S2 and S is desired. We previously reported the efficient production of the HBV env L (pre-S1 + pre-S2 + S) protein in the recombinant yeast cells [J Biol Chem 267 (1992) 1953]. In this study, the HBV env L protein produced as nano-particles in yeast has been purified and characterized. By equilibrium sedimentation, an average molecular weight of L particle was estimated to be approximately 6.4 x 10(6), indicating that about 110 molecules of L proteins are assembled into an L particle. By atomic force microscopy in a moist atmosphere, the L particles were observed as large spherical particles with a diameter of 50-500 nm. The L particles were stable on short-time heating at a high temperature and long-time storage at a low temperature but rather unstable on repeated freezing and thawing and treatment with dithiothreitol. When immunized in mice, L particles elicited efficiently and simultaneously the anti-S, anti-pre-S2, and anti-pre-S1 antibodies. The ED(50) values in mice for the anti-S and anti-pre-S2 antibodies were similar to those elicited by the M (pre-S2 + S) particles. Furthermore, the anti-pre-S1 rabbit antibodies were found to recognize various segments of the pre-S1 region, including the pre-S1 (21-47) segment. These results show the high ability of L particles to induce all antibodies against HBV env proteins, hence promising the future application of L particles for the next generation HB vaccine.