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Nobuo Yoshimoto,
Akiko Kida,
Xu Jie,
Masaya Kurokawa,
Masumi Iijima,
Tomoaki Niimi,
Andrés D Maturana,
Itoshi Nikaido,
Hiroki R Ueda,
Kenji Tatematsu, Katsuyuki Tanizawa,
Akihiko Kondo,
Ikuo Fujii,
Shun'ichi Kuroda
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ABSTRACT: When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms.
Scientific Reports 01/2013; 3:1191.
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ABSTRACT: Quinohemoprotein amine dehydrogenase (QHNDH), an αβγ heterotrimer present in the periplasm of several Gram-negative bacteria, catalyzes the oxidative deamination of various aliphatic amines such as n-butylamine for assimilation as carbon and energy sources. The γ subunit of mature QHNDH contains a protein-derived quinone cofactor, cysteine tryptophylquinone, and three intrapeptidyl thioether cross-links between Cys and Asp or Glu residues. In its cytoplasmic nascent form, the γ subunit has a 28-residue N-terminal leader peptide that is necessary for the production of active QHNDH but must be removed in the following maturation process. Here, we describe the role of a subtilisin-like serine protease encoded in the fifth ORF of the n-butylamine-utilizing operon of Paracoccus denitrificans (termed ORF5) in QHNDH biogenesis. ORF5 disruption caused bacterial cell growth inhibition in n-butylamine-containing medium and production of inactive QHNDH, in which the γ subunit retained the leader peptide. Supply of plasmid-encoded ORF5 restored the cell growth and production of active QHNDH, containing the correctly processed γ subunit. ORF5 expressed in Escherichia coli but not its catalytic triad mutant cleaved synthetic peptides surrogating for the γ subunit leader peptide, although extremely slowly. The cleaved leader peptide remained unstably bound to ORF5, most likely as an acyl enzyme intermediate attached to the active-site Ser residue. These results demonstrate that ORF5 is essential for QHNDH biogenesis, serving as a processing protease to cleave the γ subunit leader peptide nearly in a disposable manner.
Journal of Biological Chemistry 02/2012; 287(9):6530-8. · 4.77 Impact Factor
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ABSTRACT: Copper amine oxidases (CAOs) catalyse the oxidation of various aliphatic amines to the corresponding aldehydes, ammonia and hydrogen peroxide. Although CAOs from various organisms share a highly conserved active-site structure including a protein-derived cofactor, topa quinone (TPQ), their substrate specificities differ considerably. To obtain structural insights into the substrate specificity of a CAO from Arthrobacter globiformis (AGAO), we have determined the X-ray crystal structures of AGAO complexed with irreversible inhibitors that form covalent adducts with TPQ. Three hydrazine derivatives, benzylhydrazine (BHZ), 4-hydroxybenzylhydrazine (4-OH-BHZ) and phenylhydrazine (PHZ) formed predominantly a hydrazone adduct, which is structurally analogous to the substrate Schiff base of TPQ formed during the catalytic reaction. With BHZ and 4-OH-BHZ, but not with PHZ, the inhibitor aromatic ring is bound to a hydrophobic cavity near the active site in a well-defined conformation. Furthermore, the hydrogen atom on the hydrazone nitrogen is located closer to the catalytic base in the BHZ and 4-OH-BHZ adducts than in the PHZ adduct. These results correlate well with the reactivity of 2-phenylethylamine and tyramine as preferred substrates for AGAO and also explain why benzylamine is a poor substrate with markedly decreased rate constants for the steps of proton abstraction and the following hydrolysis.
Journal of biochemistry 02/2012; 151(2):167-78. · 1.95 Impact Factor
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ABSTRACT: Hepatitis B virus (HBV) envelope L proteins, when synthesized in yeast cells, form a hollow bionanocapsule (BNC) in which genes (including large plasmids up to 40 kbp), small interfering RNA (siRNA), drugs, and proteins can be enclosed by electroporation. BNCs made from L proteins have several advantages as a delivery system: Because they display a human liver-specific receptor (the pre-S region of the L protein) on their surface, BNCs can efficiently and specifically deliver their contents to human liver-derived cells and tissues ex vivo (in cell culture) and in vivo (in a mouse xenograft model). Retargeting can be achieved simply by substituting other biorecognition molecules such as antibodies, ligands, receptors, and homing peptides for the pre-S region. In addition, BNCs have already been proven to be safe for use in humans during their development as an immunogen of hepatitis B vaccine. This protocol describes the loading of BNCs and their use in cell culture and in vivo.
Cold Spring Harbor Protocols 01/2012; 2012(6):702-5. · 4.63 Impact Factor
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ABSTRACT: A bio-nanocapsule (BNC) is a hollow nanoparticle consisting of an approximately 100-nm-diameter liposome with about 110 molecules of hepatitis B virus (HBV) surface antigen L protein embedded as a transmembrane protein. BNC can encapsulate various drugs and genes and deliver them specifically to human hepatic cells based on the ability of HBV to recognize human hepatocyte, which is integrated in the N-terminal region of L protein. However, it is elusive whether the cellular attachment and entry into hepatic cells of BNC utilize the early infection mechanism of HBV. In this study, we have found that while all human hepatic cells show distinct affinities for BNC compared to non-hepatic cells, primary hepatocytes shows the highest efficiency for cellular binding and incorporation of BNC. Amounts of BNCs bound weakly and strongly to cell membranes and those entered into the cells varied significantly depending on the types of human hepatic cells. The weak and strong binding modes of BNC are likely mediated through binding to two distinct HBV receptors (heparin-mediated low-affinity and unidentified high-affinity receptors), which play major roles in the early infection mechanism of HBV. The rates of cellular uptake of BNC are similar to those reported for HBV. The BNCs incorporated into the cells are swiftly sorted to either early endosomes or macropinosomes and then to late endosomes and/or lysosomes. These findings strongly suggest that BNC is bound to and incorporated into human hepatic cells according to the early infection mechanism of HBV.
Journal of Controlled Release 11/2011; 160(2):322-9. · 5.73 Impact Factor
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ABSTRACT: Simultaneous detection of multiple antigens by conventional immunological methods has been limited by the source of primary antibodies. Each antibody should be derived from a different host species (or subclass of immunoglobulin (Ig)) for suppressing the cross-reactions of secondary antibodies. Here we describe an innovative method for simultaneous, rapid, and sensitive detection of multiple antigens using ∼30-nm bio-nanocapsules (BNCs) displaying IgG Fc-binding Z domains derived from Staphylococcus aureus protein A (ZZ-BNC). When Cy2-labeled ZZ-BNC (Cy2-ZZ-BNC) was used instead of Cy2-labeled secondary antibody in western blot analysis, both sensitivity and signal intensity were significantly increased. The complex of Cy5-ZZ-BNC and mouse IgG2a (which shows moderate affinity to the Z domain) was not dissociated, even in the presence of 8-fold excess of free mouse IgG2a. In addition, crosslinking with BS(3) (bis-sulfosuccinimidyl suberate) efficiently stabilized the interaction. The ZZ-BNCs labeled with various Cy dyes facilitated the simultaneous detection of multiple antigens using primary antibodies derived from the same host species, by western blot analysis, immunocytochemistry and flow cytometry, which could expand the possibility of bio-imaging probes in various immunofluorescence techniques.
Biomaterials 08/2011; 32(34):9011-20. · 7.40 Impact Factor
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ABSTRACT: Bio-nanocapsules (BNCs) are hollow particles (approx. 50 nm diameter) consisting of hepatitis B virus surface antigen (HBsAg) large (L, pre-S1+pre-S2+S) proteins embedded in a unilamellar liposome, sharing the same transmembrane S region with an immunogen of hepatitis B vaccine (i.e., HBsAg small (S) protein particle). BNCs can incorporate drugs and genes into the hollow space and systemic administration of the BNCs can deliver the products to human liver via the human hepatocyte-specific receptor within the pre-S (pre-S1+pre-S2) region displayed on BNC's surface. Thus, BNCs are expected to offer efficient and safe non-viral nanocarriers to deliver human liver-specific genes and drugs. To date, BNCs have been purified from the crude extract of BNC-overexpressing yeast cells by fractionation with polyethylene glycol followed by one CsCl equilibrium and two sucrose density gradient ultracentrifugation steps. However, the process was inefficient in terms of yield and time, and was not suitable for mass production because of the ultracentrifugation step. Furthermore, trace contamination with yeast-derived proteinases degraded the pre-S region, which is indispensable for liver-targeting, during long-term storage. In this study, we developed a new purification method involving heat treatment and sulfated cellulofine column chromatography to facilitate rapid purification, completely remove proteinases, and enable mass production. In addition, the BNCs were functional for at least 14 months after lyophilization with 5% (w/v) sucrose as an excipient. This new process will significantly contribute to the development of forthcoming BNC-based nanomedicines as well as hepatitis B vaccines.
Protein Expression and Purification 08/2011; 78(2):149-55. · 1.59 Impact Factor
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Seikagaku. The Journal of Japanese Biochemical Society 08/2011; 83(8):691-703. · 0.04 Impact Factor
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Masumi Iijima,
Hiroyasu Kadoya,
Satoko Hatahira,
Shingo Hiramatsu,
Giman Jung,
Aaron Martin,
John Quinn,
Joohee Jung,
Seong-Yun Jeong,
Eun Kyung Choi,
Takeshi Arakawa,
Fumiyo Hinako,
Masanobu Kusunoki,
Nobuo Yoshimoto,
Tomoaki Niimi, Katsuyuki Tanizawa,
Shun'ichi Kuroda
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ABSTRACT: To enhance the sensitivities and antigen-binding capacities of immunosensors, oriented immobilization of antibodies on the surface of the sensor chip is critical, but to date, this has not been adequately achieved. We describe a way of adsorbing immunoglobulin (Ig) proteins onto 32-nm bio-nanocapsules (BNCs) through IgG Fc-binding domains derived from Staphylococcus aureus protein A (ZZ-BNC). This arrangement permits approximately 60 molecules of mouse total IgG bind to ZZ-BNC and all the IgG Fv regions to be displayed outwardly for the effective binding of antigens. ZZ-BNCs adsorbed onto the gold surface of the sensor chip of the quartz crystal microbalance (QCM) could markedly enhance the sensitivity and antigen-binding capacity of the chip. On the sensor chip of surface plasmon resonance (SPR), antibodies on the ZZ-BNCs showed higher affinities to each antigen than those on protein A. The BNC-coated sensor chip is very stable, and should prove useful for various immunosensor applications due to oriented immobilization of antibodies.
Biomaterials 02/2011; 32(6):1455-64. · 7.40 Impact Factor
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Katsuyuki Tanizawa
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ABSTRACT: Hydrogen sulfide (H(2)S) has been established as the third gaseous signaling molecule following nitric oxide and carbon monoxide and participates in a variety of cellular functions such as modulation of neuronal transmission, endothelium-dependent vasorelaxation, stimulation of angiogenesis and regulation of insulin release. Although cystathionine β-synthase and cystathionine γ-lyase have been regarded as the main producers of H(2)S in many tissues including brain, liver and kidney, Kimura and his colleagues have recently communicated that 3-mercaptopyruvate sulphurtransferase coupled with cysteine (aspartate) aminotransferase is responsible for the production of H(2)S in the vascular endothelium of the thoracic aorta [Shibuya et al. (2009) J. Biochem. 146, 623-626]. This finding provides a new insight into the production of the physiologically important signaling molecule.
Journal of biochemistry 02/2011; 149(4):357-9. · 1.95 Impact Factor
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ABSTRACT: To reveal the chemical changes and geometry changes of active-site residues that cooperate with a reaction is important for understanding the functional mechanism of proteins. Consecutive temporal analyses of enzyme structures have been performed during reactions to clarify structure-based reaction mechanisms. Phenylethylamine oxidase from Arthrobacter globiformis (AGAO) contains a copper ion and topaquinone (TPQ(ox)). The catalytic reaction of AGAO catalyzes oxidative deaminations of phenylethylamine and consists of reductive and oxidative half-reactions. In the reduction step, TPQ(ox) reacts with a phenylethylamine (PEA) substrate giving rise to a topasemiquinone (TPQ(sq)) formed Schiff-base and produces phenylacetaldehyde. To elucidate the mechanism of the reductive half-reaction, an attempt was made to trap the reaction intermediates in order to analyze their structures. The reaction proceeded within the crystals when AGAO crystals were soaked in a PEA solution and freeze-trapped in liquid nitrogen. The reaction stage of each crystal was confirmed by single-crystal microspectrometry, before X-ray diffraction measurements were made of four reaction intermediates. The structure at 15 min after the onset of the reaction was analyzed at atomic resolution, and it was shown that TPQ(ox) and some residues in the substrate channel were alternated via catalytic reductive half-reactions.
Journal of Synchrotron Radiation 01/2011; 18(1):58-61. · 2.73 Impact Factor
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ABSTRACT: Enigma homolog protein 1 (ENH1) acts as a scaffold that selectively associates protein kinases and transcription factors with cytoskeletal elements. ENH1 comprises an N-terminal PDZ domain and three C-terminal LIM domains. Through the LIM domains ENH1 interacts with the N-terminal region of protein kinase C βI (PKCβI). Here, we show that when ENH1 is co-expressed, PKCβI is translocated from the cytoplasm to the plasma membrane in the absence of any other stimulation. Moreover expression of ENH1 markedly increases PKCβI activity in the absence of PKC activators. A similar activation of PKCβI was observed with co-expression of Cypher1 or Enigma, but not other LIM proteins. The region including the three LIM domains of ENH1 (residues 415-591) appears to be sufficient for this PKCβI activation. Finally, interaction with ENH1 also increases the activity of PKCα and PKCγ, whereas it reduces PKCζ activity. These findings provide strong evidence that ENH1 activates conventional PKCs by directly binding through its LIM domains. Thus, LIM domains have a novel molecular function: the regulation of PKC activities in a PKC isoform-specific manner.
Cellular signalling 01/2011; 23(5):928-34. · 4.09 Impact Factor
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ABSTRACT: Recently, our group has proposed a novel gene-regulation system responding to cAMP-dependent protein kinase (PKA) that has been applied to living cells. In this study, human liver-specific bionanocapsules (BNCs) are used as a gene-delivery system to increase transfection efficiency and to target specific cell types. BNCs can efficiently deliver a target gene to human hepatocytes and hepatoma cells in vitro or in vivo. The combination of a signal-responsive gene-delivery system with BNCs led to an increase in the transfection efficiency and selectivity for hepatoma cells. Expression from the delivered gene was identified from PKA-activated hepatoma cells (HepG2), but not from colon tumor cells (WiDr). These results show that the combination of a gene-regulation system responding to an intracellular signal with BNC can be used for the selective treatment of human hepatoma cells.
International journal of pharmaceutics 08/2010; 396(1-2):174-8. · 2.96 Impact Factor
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Jeong-Hun Kang,
Jun Oishi,
Jong-Hwan Kim,
Moeko Ijuin,
Riki Toita,
Byungdug Jun,
Daisuke Asai,
Takeshi Mori,
Takuro Niidome, Katsuyuki Tanizawa,
Shun'ichi Kuroda,
Yoshiki Katayama
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ABSTRACT: Hepatoma (hepatocellular carcinoma) is the most common type of malignant tumor originating in the liver and has a relatively low 5-year survival rate. The development of hepatoma-targeted therapy is needed to increase treatment efficiency and to reduce the incidence of undesirable side effects. In this study we developed a novel hepatoma-targeted gene delivery system. The gene delivery system was prepared by combining a human liver cell-specific bionanocapsule (BNC) and a tumor cell-specific gene regulation polymer, which responds to hyperactivated protein kinase C alpha in hepatoma cells. The complex of the polymer-DNA with BNCs was delivered into cells and tissues. The developed system showed increased transfection efficiency and resulted in cell-specific gene expression in hepatoma cells and tissues (HuH-7), but no gene expression in normal human hepatocytes or human epidermoid tumor cells (A431). The combination of a tumor cell-specific gene regulation system responding to protein kinase C alpha and BNCs showed excellent potential for the selective treatment of hepatomas. The system could be a useful method with applications in hepatoma-specific gene therapy and molecular imaging. From the clinical editor: Hepatocellular carcinoma is the most common type of malignant tumor in the liver with a low 5-year survival rate. In this study, a novel hepatoma-targeted gene delivery system was prepared by combining a human liver cell-specific bionanocapsule and a tumor cell-specific gene regulation polymer, which responds to hyperactivated protein kinase C (PKC)a in hepatoma cells. The system could be a useful in hepatoma-specific gene therapy and molecular imaging.
Nanomedicine: nanotechnology, biology, and medicine 02/2010; 6(4):583-9. · 5.44 Impact Factor
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ABSTRACT: A bacterial two-component signal transduction system, WalK/WalR, is essential to the cell viability of Gram-positive bacteria and is therefore a potential target for the development of a new class of antibiotics. We have solved the X-ray crystal structure of the DNA-binding domain of the response regulator WalR (WalRc) from a Gram-positive pathogen Staphylococcus aureus, currently causing serious problems in public health through the acquisition of multi-drug resistance. The structure contains a winged helix-turn-helix motif and closely resembles those of WalRs of Bacillus subtilis and Enterococcus faecalis, and also that of PhoB of Escherichia coli. Gel mobility shift assays with mutant WalRs revealed specific interactions of WalR with the target DNA, as elaborated by in silico modeling of the WalRc-DNA complex.
Bioscience Biotechnology and Biochemistry 01/2010; 74(9):1901-7. · 1.28 Impact Factor
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ABSTRACT: Macromolecules that can assemble a large number of enzyme and antibody molecules have been used frequently for improvement of sensitivities in enzyme-linked immunosorbent assays (ELISAs). We generated bionanocapsules (BNCs) of approximately 30nm displaying immunoglobulin G (IgG) Fc-binding ZZ domains derived from Staphylococcus aureus protein A (designated as ZZ-BNC). In the conventional ELISA using primary antibody and horseradish peroxidase-labeled secondary antibody for detecting antigen on the solid phase, ZZ-BNCs in the aqueous phase gave an approximately 10-fold higher signal. In Western blot analysis, the mixture of ZZ-BNCs with secondary antibody gave an approximately 50-fold higher signal than that without ZZ-BNCs. These results suggest that a large number of secondary antibody molecules are immobilized on the surface of ZZ-BNCs and attached to antigen, leading to the significant enhancement of sensitivity. In combination with the avidin-biotin complex system, biotinylated ZZ-BNCs showed more significant signal enhancement in ELISA and Western blot analysis. Thus, ZZ-BNC is expected to increase the performance of various conventional immunoassays.
Analytical Biochemistry 10/2009; 396(2):257-61. · 3.00 Impact Factor
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ABSTRACT: The nuclear-cytoplasmic shuttling protein RBCC protein interacting with protein kinase C1 (RBCK1) possesses transcriptional and ubiquitin ligase activities. We have recently reported that RBCC protein interacting with protein kinase C2 (RBCK2), a RING-in-between-RING fingers domain-lacking splice variant of RBCK1, lacks transcriptional activity, but rather represses the RBCK1-mediated transcriptional activity as a cytoplasmic tethering protein for RBCK1. In this study, we have found that RBCK2 overexpressed in human embryonic kidney 293 cells interacts with the polyubiquitin chain and the polyubiquitin-interacting subunit S5a, and significantly increases the intracellular amount of polyubiquitinated proteins. These results strongly suggested that RBCK2 functions as an adaptor protein for the polyubiquitinated protein and the S5a subunit in 26S proteasome through its novel zinc finger motif and ubiquitin-like domain, respectively, presumably delivering the polyubiquitinated proteins to the entrance of the 26S proteasome catalytic domain for degradation.
FEBS Journal 10/2009; 276(21):6375-85. · 3.79 Impact Factor
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ABSTRACT: We found that eosinophil cationic protein (ECP) stimulated the growth of mouse Balb/c 3T3 fibroblasts. ECP-treated 3T3 cells were more flattened and exhibited enhanced stress fiber formation. The enhancement of cytoskeleton after addition of recombinant ECP appeared stable and was able to inhibit disassembly of actin filaments that was induced by fibroblast growth factor-2. The ROCK inhibitor, Y-27632, abrogated this enhancement on stress fiber formation that was induced by ECP indicating the involvement of Rho/ROCK signaling pathway. The effect of ECP was assessed on the differentiation of primary cardiomyocytes derived from rat neonatal heart since the development of actin filaments is significantly related with organization of stress fibers. As the result, both beating rate and the expression of cardiac muscle specific markers such as atrial natriuretic factor were enhanced in the presence of ECP. Thus ECP may also function as a cardiomyocyte differentiation factor.
Growth factors (Chur, Switzerland) 07/2009; 27(4):228-36. · 2.47 Impact Factor
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ABSTRACT: A bio-nanocapsule (BNC) is an ~50-nm hepatitis B virus (HBV) subviral particle comprising HBV envelope L proteins and a lipid bilayer, and is synthesized in recombinant Saccharomyces cerevisiae. When BNCs are administered intravenously in a mouse xenograft model, they can accumulate specifically in human liver-derived tissues and enter cells efficiently by the HBV-derived human liver-specific infection machinery, localized at the outer-membrane pre-S region of the L protein. BNC specificity for the human liver can be altered to other tissues by substituting the pre-S region using targeting molecules (e.g., antibodies, lectins, cytokines). BNCs can spontaneously form complexes with liposomes (LPs) by the membrane fusogenic activity of the pre-S region. LPs containing various therapeutic materials (e.g., chemicals, proteins, DNA, RNA) can therefore be covered with BNCs to form an ~150-nm BNC-LP conjugate. BNC-LP conjugates injected intravenously can deliver incorporated materials to target tissues specifically and efficiently by utilizing the HBV-derived infection machinery. The stability of BNC-LP conjugates in the blood circulation is similar to that of PEGylated LPs. In this chapter, we describe the preparation and in vivo application of BNC-LP conjugates, and the potential of BNC-LP conjugates as in vivo pinpoint drug delivery systems.
Methods in enzymology 01/2009; 464:147-66. · 1.90 Impact Factor
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ABSTRACT: A bio-nanocapsule (BNC) is an ~50-nm hepatitis B virus (HBV) subviral particle comprising HBV envelope L proteins and a lipid bilayer, and is synthesized in recombinant Saccharomyces cerevisiae. When BNCs are administered intravenously in a mouse xenograft model, they can accumulate specifically in human liver-derived tissues and enter cells efficiently by the HBV-derived human liver-specific infection machinery, localized at the outer-membrane pre-S region of the L protein. BNC specificity for the human liver can be altered to other tissues by substituting the pre-S region using targeting molecules (e.g., antibodies, lectins, cytokines). BNCs can spontaneously form complexes with liposomes (LPs) by the membrane fusogenic activity of the pre-S region. LPs containing various therapeutic materials (e.g., chemicals, proteins, DNA, RNA) can therefore be covered with BNCs to form an ~150-nm BNC–LP conjugate. BNC–LP conjugates injected intravenously can deliver incorporated materials to target tissues specifically and efficiently by utilizing the HBV-derived infection machinery. The stability of BNC–LP conjugates in the blood circulation is similar to that of PEGylated LPs. In this chapter, we describe the preparation and in vivo application of BNC–LP conjugates, and the potential of BNC–LP conjugates as in vivo pinpoint drug delivery systems.
Methods in Enzymology - METH ENZYMOLOGY. 01/2009; 464:147-166.
