Review Papers (Misc) - KOUMOTO Kazuya
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Cholesterol-appended beta-(1 -> 3)-D-glucan schizophylllan for antisense oligonucleotides delivery to enhance the cellular uptake
K Koumoto, M Mizu, T Anada, T Nagasaki, S Shinkai, K Sakurai
BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 78 ( 10 ) 1821 - 1830 2005.10
Publisher：CHEMICAL SOC JAPAN
Schizophyllan is a natural beta-(1 -> 3)-D-glucan existing as a triple helix in water and as a single chain in dimethyl sulfoxide (DMSO), respectively. As we already reported, when a homo-polynucleotide is added to a schizophyllan solution. the single chain of schizophyllan forms a complex with the polynucleotide. One of the potential applications of this novel complex is an antisense-oligonucleotide (AS ODN) carrier. The present paper describes a modification technique that enables us to introduce a cholesterol group only to the side chain of schizophyllan. We prepared four cholesterol-appended schizophyllans with different modification levels. Using these compounds, we made complexes and carried out an in vitro antisense assay, administrating a phosphorothioate AS ODN to the several cell lines to depress their c-myb mRNA. When we used 2.2-2.3 mol % modified schizophyllan as the carrier, the antisense effect was most enhanced among others. The addition of beta-cyclodextrin improved the complexation ability as well as the up-take for highly modified samples. Furthermore, the cytotoxicity for these modified schizophyllan samples was negligibly as small as the natural (unmodified) schizophyllan. The present work has thus clarified that schizophyllan can act as a new potential candidate for AS ODN carriers.
DOI： 10.1246/bcsj.78.1821
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PEG-appended beta-(1 -> 3)-D-glucan schizophyllan to deliver antisense-oligonucleotides with avoiding lysosomal degradation
R Karinaga, K Koumoto, M Mizu, T Anada, S Shinkai, K Sakurai
BIOMATERIALS 26 ( 23 ) 4866 - 4873 2005.8
Publisher：ELSEVIER SCI LTD
Schizophyllan is a natural beta-(1 -> 3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO). As we already reported, when a homo-polynucleotide [e.g., poly(dA) or poly(C)] is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. One of the potential applications for this novel complex is an antisense-oligonucleotide (AS ODN) carrier. The present paper describes a modification technique that enabled us to introduce PEG only to the side chain of schizophyllan. This technique consisted of periodate oxidation of the glucose side chain and subsequent reaction between methoxypolyethylene glycol amine and the formyl terminate, followed by reduction with NaBH4. Subsequently, we made a complex from PEG-appended schizophyllan and an AS ODN sequence, and carried out an in vitro antisense assay, administrating the AS ODN complex to depress A375 c-myb mRNA of A375 melanoma cell lines. The PEG-SPG/AS ODN complex showed more enhanced antisnese effect than naked AS ODN dose, i.e., the same level as that of RGD-appended SPG. Here, the RGD system has been shown one on the most effective AS ODN carrier (Science 261 (1993) 1004-1012). When we added nigericin to the assay system, the antisense effect was not affected in the PEG-SPG system, on the other hand, it was almost eliminated in the RGD system. Nigericin is well known to interrupt transport from endosome to lysosome. Therefore, the difference between the PEG and RGD complexes indicates that, in the PEG system, AS ODN was able to escape from lysosomal degradation. The present work has thus proposed a new strategy to delivery AS ODN using schizophyllan as a new carrier. (C) 2004 Elsevier Ltd. All rights reserved.
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Parallel vs. anti-parallel orientation in a curdlan/oligo(dA) complex as estimated by a FRET technique
M Numata, K Koumoto, M Mizu, K Sakurai, S Shinkai
ORGANIC & BIOMOLECULAR CHEMISTRY 3 ( 12 ) 2255 - 2261 2005.6
Publisher：ROYAL SOC CHEMISTRY
We already found that beta-1,3-glucan polysaccharides form polymeric complexes with certain polynucleotides, but the parallel vs. anti-parallel orientation in those complexes had remained unsolved. In this paper, this controversial problem has been discussed for curdlan/oligo(dA) complexes utilizing two different energy transfer techniques. The first system consists of a combination of fluorescein-labeled curdlan and 3'-(or 5'-)tetramethyl-rhodamine (TAMRA)-labeled oligo(dA). The second system utilizes gold nanoparticles: that is, two curdlan chains were linked by a disulfide bond and after complexation with oligo(dA), the complex was immobilized on gold nanoparticles. In this system, TAMRA was attached to the 3' (or 5') end of oligo(dA) and the gold particle acted as a fluorescence quencher (energy acceptor). These experiments have led us to conclude that in the curdlan/oligo(dA) complex, parallel orientation is more favourable than anti-parallel orientation. These findings have enabled us to envision a clearer image for the complexation mode between beta-1,3-glucan polysaccharides and polynucleotides.
DOI： 10.1039/b500156k
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Gene delivery system using polysaccharide schizophyllan and poly(DA) tailed DNA.
T Anada, R Karinaga, K Koumoto, M Mizu, S Shinkai, K Sakurai
ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 229 U1160 - U1160 2005.3
Publishing type：Research paper, summary (international conference) Publisher：AMER CHEMICAL SOC
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In situ monitoring of polysaccharide-polynucleotide interaction using a schizophyllan-immobilized QCM device
DH Yang, AH Bae, K Koumoto, SW Lee, K Sakurai, S Shinkai
SENSORS AND ACTUATORS B-CHEMICAL 105 ( 2 ) 490 - 494 2005.3
Publisher：ELSEVIER SCIENCE SA
Schizophyllan belongs to the beta-(1 -> 3)-D-glucan family and is well known as a triple helix polysaccharide. It is already known that the single chain of schizophyllan (s-SPG) can specifically form a complex with particular polynucleotides. As an application of this recognition capability, a novel schizophyllan derivative connected with a disulfide bond through along methylene spacer was synthesized, and its interaction with polynucleoticles was directly monitored with a self-assembled monolayer on QCM. The gold-coated QCM electrode was covered in ca. 60% as a monolayer of s-SPG. The QCM resonator showed sensitive mass increases for poly(C) and poly(U) in the presence of K+ ion, which are consistent with our previous results. The present results indicate that the schizophyllan-immobilized QCM resonator can be a new sensing device for biomolecules. (c) 2004 Elsevier B.V. All rights reserved.
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Competition between polysaccharide/polynucleotide complexation vs., polynucleotide hybridization; salt concentration dependence of the reaction direction
Ryouji Karinaga, Kazuya Koumoto, Jusaku Minari, Masami Mizu, Takahisa Anada, Kazuo Sakurai, Seiji Shinkai
e-Journal of Surface Science and Nanotechnology 3 38 - 45 2005.2
Schizophyllan is a natural β-(1→3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when a homo-polynucleotide [ex. poly (A) or poly(dA)] was added to the schizophyllan/DMSO solution and subsequently DMSO was exchanged for water, the single chain of schizophyllan formed a complex with the polynucleotide. In this paper, using an adenine-thymine (or uracil) double strand as a model system, we explored whether s-SPG can unzip the polynucleotide duplexes, examining the competition between the schizophyllan/polynucleotide complexation and the polynucleotide hybridization with circular dichroism and fluorescence spectroscopy at various NaCl concentrations. The unzipping reaction happened for the hetero-duplexes (i.e., RNA-DNA duplexes) at low salt concentrations, on the other hand, it was not observed for the homo-duplexes at all the salt concentrations. When we compared dissociation temperatures, the unzipping reaction of the homo-duplexes was expected at low salt concentrations. This discrepancy between the experimental results and the expectation, as well as the different behavior between the hetero- and homo- duplexes, can be ascribed to the difference in the activation energy of intermediate states. © 2005 The Surface Science Society of Japan.
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Poly(N-substituted-acrylamide)-branched schizophyllans are useful for selective recovery of homopolynucleotides through convenient and quick precipitation procedures
T Matsumoto, M Umeda, M Numata, T Hasegawa, K Sakurai, K Koumoto, S Shinkai
POLYMER JOURNAL 37 ( 3 ) 177 - 185 2005
Publisher：SOC POLYMER SCIENCE JAPAN
Schizophyllans carrying poly(N-substituted-acrylamide)-branches were synthesized from native schizophyllan through Ce(IV)-initiated radical homo- or co-polymerization of N-iso-propyl- and N-sec-butyl-acrylamide. A series of structural analyses including elemental analysis, gel permeation chromatography, H-1 NMR, and enzymatic degradation using β-1,3-glucanase revealed that schizophyllans (150kDa) bearing long poly (N-substituted-acrylamide)-branches (ca. 290 kDa) are obtained. These SPG-conjugates form macromolecular complexes with certain homopolynucleotides such as poly(C), poly(dA), and poly(A) and then efficiently co-precipitate them on heating. For example, schizophyllan carrying poly(N-iso-propylacrylamide)-branches can selectively recover poly(C) from a mixture of poly(C) and poly(U) by simple heating (36° C) followed by centrifugation. HPLC analysis of the recovered polynucleotide revealed the high poly(C)-selectivity (poly(C):poly(U) = 95:5). Similarly, schizophyllan carrying more hydrophobic poly(N-iso-propylacrylamide-co-N-sec-butylacrylamide) -branches precipitates at lower temperature (ca. 20° C) and is applicable to isolation of poly(A) that forms less stable macromolecular complexes with SPG and SPG-derivatives (the dissociation temperatures are around 30° C).
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Transferrin-appended β-(1→3)-D-glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake
Takahisa Anada, Ryouji Karinaga, Masami Mizu, Kazuya Koumoto, Takahiro Matsumoto, Munenori Numata, Seiji Shinkai, Kazuo Sakurai
e-J. Surface Sci. Nanotech. 3 195 - 202 2005
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Removal of the side-chain glucose groups from schizophyllan improves the thermal stability of the polycytidylic acid complexes under the physiological conditions
K Koumoto, R Karinaga, M Mizu, T Anada, K Sakurai, T Kunitake, S Shinkai
BIOPOLYMERS 75 ( 5 ) 403 - 411 2004.12
Publisher：JOHN WILEY & SONS INC
Thermal stabilization of the complex between polycytidylic acid [poly(Q] and the modified schizophyllan (SPG) whose hydrophilic side-chain glucose groups are selectively removed utilizing mild Smith-degradation has been investigated. With the decrease in the side-chain glucose groups of schizophyllan, the complex with poly(C) can be considerably stabilized compared with unmodified SPG; for example, the T. value after the removal of the side-chain glucose groups from 33.3 (unmodified) to 1.0 is enhanced by 14degreesC In addition, the thermal stabilization effect is even operative under the physiological conditions ([NaCl] = 0.15 mol dm(-3)). This effect is exerted owing to the construction of the hydrophobic atmosphere around the complex. Although schizophyllan lost the side-chain glucose groups, it still kept the protection effect of the bound poly(C) chain against RNaseA-mediated hydrolysis as observed for unmodified schizophyllan. The assessment of the cytotoxicity for A375:human malignant melanoma, and HL60:human promyelocytic leukemia revealed that the modified schizophyllan scarcely increases the cytotoxicity. These results indicate that the present modification for schizophyllan is of great significance in a viewpoint to develop the practical gene carriers operative even under the physiological conditions. (C) 2004 Wiley Periodicals, Inc.
DOI： 10.1002/bip.20148
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Proposal of new modification technique for linear double-stranded DNAs using the polysaccharide schizopyllan
T Anada, H Matsunaga, R Karinaga, K Koumoto, M Mizu, K Nakano, S Shinkai, K Sakurai
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 14 ( 22 ) 5655 - 5659 2004.11
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
A natural polysaccharide schizophyllan (SPG) has been known to form a stable complex with poly(dA). We attached a poly(dA)(80) tail to the both ends of a linear double-stranded DNA, which had been prepared from a plasmid DNA vector. The poly(dA) tailed DNA verified to form complex with SPG by gel electrophoresis and atomic force microscopy (AFM). AFM images indicated that the complexes exhibit a dumbbell-like architecture, that is, quite similar to that of adenovirus genome. The complex demonstrated excellent exonuclease resistance, probably because of the protection effect by SPG complexation. (C) 2004 Elsevier Ltd. All rights reserved.
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A polysaccharide carrier for immunostimulatory CpG DNAs to enhance cytokine secretion
M Mizu, K Koumoto, T Anada, T Matsumoto, M Numata, S Shinkai, T Nagasaki, K Sakurai
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 126 ( 27 ) 8372 - 8373 2004.7
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Antisense oligonucleotides bound in the polysaccharide complex and the enhanced antisense effect due to the low hydrolysis
M Mizu, K Koumoto, T Anada, K Sakurai, S Shinkai
BIOMATERIALS 25 ( 15 ) 3117 - 3123 2004.7
Publisher：ELSEVIER SCI LTD
Schizophyllan is a beta-(1--> 3)-D-glucan and can form a novel complex with some single-chains of DNAs. As the preceding paper revealed, the polynucleotide bound in the complex is more stable to nuclease-mediated hydrolysis than the polynucleotide itself (i.e., naked polynucleotide). This paper examined possibility to apply this complex to an antisense DNA carrier, using an in vitro (cellfree) transcription/translation assay. In this assay, we used a plasmid DNA coding a green fluorescence protein (GFP) and an antisense DNA designed to hybridize the ribosome-binding site in the GFP-coded mRNA. When the antisense DNA was administered as the complex, a lower GFP expression efficiency (or higher antisense effect) is observed over naked DNA. This is because the antisense DNA in the complex is protected from the attack of deoxyribonuclease. When exonuclease I, which specifically hydrolyzes single DNA chains, was present in the GEP assay system, the antisense effect was not changed for the complex while being weakened in the naked antisense DNA system. These results imply that the exonuclease I cannot hydrolyze the antisense DNA in the complex, while it can hydrolyze naked DNA to reduce its antisense effect. (C) 2003 Elsevier Ltd. All rights reserved.
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Protection of polynucleotides against nuclease-mediated hydrolysis by complexation with schizophyllan
M Mizu, K Koumoto, T Kimura, K Sakurai, S Shinkai
BIOMATERIALS 25 ( 15 ) 3109 - 3116 2004.7
Publisher：ELSEVIER SCI LTD
Schizophyllan is a beta-(1-->3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when some homo-polynucleotide (for example, poly(dA) or poly(C)) is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan (s-SPG) forms a complex with the polynucleotide. The present work demonstrates that the polynucleotide bound in the complex is more stable to nuclease-mediated hydrolysis than the polynucleotide itself (i.e., naked polynucleotide), using high-performance liquid chromatography and ultraviolet absorbance technique. A kinetic study for the hydrolysis clarified that the simple Michaelis-Menten relation is held and the maximum velocity for the complex is one-sixth as small as that of the naked polynucleotide. This low hydrolysis rate for the complex suggests that s-SPG is applicable to a carrier for antisense oligonucleotides. (C) 2003 Elsevier Ltd. All rights reserved.
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Long-term expression with a cationic polymer derived from a natural polysaccharide: Schizophyllan
T Nagasaki, M Hojo, A Uno, T Satoh, K Koumoto, M Mizu, K Sakurai, S Shinkai
BIOCONJUGATE CHEMISTRY 15 ( 2 ) 249 - 259 2004.3
Publisher：AMER CHEMICAL SOC
Among the various synthetic gene carriers based on biomaterials, cationic polymers with polysaccharide backbones have long been studied as nonviral vectors due to their low immunogenicity and high water solubility. Schizophyllan, a beta-(1,3)-glucan, is one of the various polysaccharides that are clinically administered. Furthermore, its safety in the human body has already been confirmed. Various functional groups can be selectively introduced into the side chain, not into the main chain of schizophyllan. Therefore, we have synthesized various oligoamine conjugates from schizophyllan. It was confirmed that their in vitro transfection efficiencies are superior to that of polyethylenimine by adjusting the molecular weight and the degree of amination of cationic schizophyllan. While it was possible to reduce cytotoxicity by adjusting the amount of DNA complex per cell, as seen with poly-L-lysine, polyethylenimine, and chitosan, PEGylation was the most effective means of reducing toxicity. Furthermore, using cationic schizophyllan carriers, it was also possible to express a reporter protein for a long period of time due to a long residence time of plasmid DNA in cells.
DOI： 10.1021/bc034178x
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Lactose-appended schizophyllan is a potential candidate as a hepatocyte-targeted antisense carrier
T Hasegawa, M Umeda, T Matsumoto, M Numata, M Mizu, K Koumoto, K Sakurai, S Shinkai
CHEMICAL COMMUNICATIONS ( 4 ) 382 - 383 2004.2
Publisher：ROYAL SOC CHEMISTRY
A schizophyllan (beta-1,3-glucan) derivative carrying lactose-appendages prepared by reductive amination can form stable macromolecular complexes with polynucleotides, shows excellent affinity with a lactose-binding lectin, and effectively mediates gene transfection into hepatocytes.
DOI： 10.1039/b313426a
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Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake efficiency
T Matsumoto, M Numata, T Anada, M Mizu, K Koumoto, K Sakurai, T Nagasaki, S Shinkai
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1670 ( 2 ) 91 - 104 2004.1
Publisher：ELSEVIER SCIENCE BV
Schizophyllan is a natural beta-(1 --> 3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when some homo-phosphodiester polynucleotide (for example, poly(dA) or poly(C)) is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. Furthermore, we have already demonstrated that one of the potential applications of this novel complex is an antisense-oligonucleotide (AS ODN) carrier. This work describes a versatile and universal modification technique which enables us to introduce various functional groups only to the side chain of schizophyllan. This technique consists of periodate oxidation of the glucose side chain (it does not react with the main chain because of the absence of the 1,2-diol group in beta-(1 --> 3)-glucan) and subsequent introduction of the functional groups into the formyl terminate. In the present work, the introduced functional groups were spermine, octa-arginine (R8), arginine-glycine-aspartic acid tripeptide (RGD) and some amino or alpha-amino acid compounds. Using these compounds, we made the complexes and carried out an in vitro antisense assay for them, administrating a phosphorothioate AS ODN to the melanoma A375 or leukemia HL-60 cell lines to depress their c-myb mRNA. When we used the R8 or RGD modified schizophyllan as the antisense carrier, the antisense effect was most enhanced among others. Their superiority can be ascribed to enhancement of endocytosis due to these functional peptides. Furthermore, the cytotoxicity for these two modified schizophyllans was negligibly as small as the natural (unmodified) schizophyllan. One of the peculiar features of our system is that the complex (i.e., carrier + AS ODN) is charged negatively in total, which is different from the conventional systems. The present work has thus clarified that schizophyllan can act as a new potential candidate for AS ODN carriers. (C) 2004 Elsevier B.V. All rights reserved.
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Low M-W sulfated curdlan with improved water solubility forms macromolecular complexes with polycytidylic acid
K Koumoto, M Umeda, M Numata, T Matsumoto, K Sakurai, T Kunitake, S Shinkai
CARBOHYDRATE RESEARCH 339 ( 1 ) 161 - + 2004.1
Publisher：ELSEVIER SCI LTD
The water solubility of curdlan was enhanced by partial sulfation at O-6. Circular dichroism measurements suggest that the sulfated curdlan with the degree of substitution (DS) from 0 to 8.7 mol % forms macromolecular complexes with polycytidylic acid (poly(C)). Although the thermal stability of the complexes decreased with increase in DS, this could be overlapped by addition of NaCl in the concentration above that of serum. The results clearly indicate that the drawback arising from the electrostatic repulsion between the anionic charges can be partially compensated by the presence of salt. Furthermore, the polynucleotide chain complexed with the sulfated curdlan was protected from the enzymatic hydrolysis, corroborating the assumption that the sulfated curdlan has an ability to bind poly(C). (C) 2003 Elsevier Ltd. All rights reserved.
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Enhancement of antisense effect by reducing interactions between proteins and ODNs
Masami Mizu, Kazuya Koumoto, Takahisa Anada, Ryouji Karinaga, Taro Kimura, Kazuo Sakurai, Takeshi Nagasaki, Seiji Shinkai
Bull. Chem. Soc. Jpn. 77 1101 - 1110 2004
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Schizophyllans carrying oligosaccharide appendages as potential candidates for cell-targeted antisense carrier
T Hasegawa, T Fujisawa, M Numata, T Matsumoto, M Umeda, R Karinaga, M Mizu, K Koumoto, T Kimura, S Okumura, K Sakurai, S Shinkai
ORGANIC & BIOMOLECULAR CHEMISTRY 2 ( 21 ) 3091 - 3098 2004
Publisher：ROYAL SOC CHEMISTRY
Schizophyllans carrying beta-lactoside and alpha-mannoside appendages were prepared from native schizophyllan through NaIO4 oxidation followed by reductive amination using aminoethyl-beta-lactoside and alpha-mannoside, respectively. The resulting schizophyllans form stable macromolecular complexes with polynucleotides, such as poly(C) and poly(dA). Specific affinity between these macromolecular complexes and saccharide-binding proteins was demonstrated by surface plasmon resonance and agarose gel staining assays. beta-Lactoside-appended schizophyllan enhanced an antisense activity in hepatocytes which express lactoside-binding proteins on their cell surfaces.
DOI： 10.1039/b412124b
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Polysaccharide/polynucleotide complexes. Part 6. Complementary-strand-induced release of single-stranded DNA bound in the schizophyllan complex
K Koumoto, M Mizu, K Sakurai, T Kunitake, S Shinkai
CHEMISTRY & BIODIVERSITY 1 ( 3 ) 520 - 529 2004
Publisher：VERLAG HELVETICA CHIMICA ACTA AG
Spectroscopic properties of single-stranded DNA/schizophyllan ternary complexes (ss-DNA - 2s-SPG), induced by addition of either complementary or noncomplementary strands, have been investigated. The addition of the complementary strands to ss-DNA - 2s-SPG induced the quick release of the bound ss-DNA to the complementary strands (both DNA and RNA), whereas the ternary complex was unaffected upon addition of noncomplementary strands. Our experiments imply that SPG has complexation properties indispensable to the gene carriers. As far as we know, there is no report on exploitation of such nonviral gene carriers that can accomplish an intelligent release of the bound ss-DNA toward the complementary strands. We believe, therefore, that SPG, a natural and neutral polysaccharide, has a great potential to become a new ss-DNA carrier.