Updated on 2022/08/17

写真a

 
TAKEOKA, Shinji
 
Affiliation
Faculty of Science and Engineering, School of Advanced Science and Engineering
Job title
Professor

Concurrent Post

  • Faculty of Science and Engineering   Graduate School of Advanced Science and Engineering

  • Affiliated organization   Global Education Center

Research Institute

  • 2020
    -
    2022

    理工学術院総合研究所   兼任研究員

Education

  •  
    -
    1991

    Waseda University   Graduate School of Science and Engineering   Major in Applied Chemistry  

  •  
    -
    1986

    Waseda University   School of Science and Engineering   Department of Applied Chemistry  

Degree

  • Waseda University   Dr. Engr.

Research Experience

  • 1990
    -
    1991

    Fellow,JapanSocietyforPromotionofScience

Professional Memberships

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    The Society of Blood Substitutes, Japan

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    Japanese Society for Biomaterials:

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    The Japan Society of Drug Delivery System

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    Japanese Society for Artificial Organs:

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    American Chemical Society

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    The Chemical Society of Japan

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    The Chemical Society of Japan

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    The Chemical Society of Japan

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    The Chemical Society of Japan

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    The Society of Polymer Science, Japan

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    The Society of Polymer Science, Japan

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    The Society of Polymer Science, Japan

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    The Society of Polymer Science, Japan

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    The Society of Polymer Science, Japan

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    The Society of Blood Substitutes, Japan

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    The Society of Blood Substitutes, Japan

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    The Society of Blood Substitutes, Japan

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    The Society of Blood Substitutes, Japan

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    The Society of Blood Substitutes, Japan

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    The Society of Polymer Science, Japan

▼display all

 

Research Areas

  • Biomaterials

  • Biomedical engineering

Research Interests

  • Function, Properties and Materials in Chemistry,Functions and Properties of Macromolecules or Molecular Assemblies,Medical Engineering and Bio-related Materials,Polymer Syntheses

Papers

  • In situ transplantation of adipose tissue-derived stem cells organized on porous polymer nanosheets for murine skin defects.

    Nishiwaki K, Aoki S, Kinoshita M, Kiyosawa T, Suematsu Y, Takeoka S, Fujie T

    Journal of biomedical materials research. Part B, Applied biomaterials    2018.09  [Refereed]

    DOI PubMed

  • Lysine-containing cationic liposomes activate the NLRP3 inflammasome: Effect of a spacer between the head group and the hydrophobic moieties of the lipids

    Tianshu Li, Jieyan He, Gabor Horvath, Tomasz Próchnicki, Eicke Latz, Shinji Takeoka

    Nanomedicine: Nanotechnology, Biology, and Medicine   14 ( 2 ) 279 - 288  2018.02  [Refereed]

     View Summary

    Cationic lipids containing lysine head groups and ditetradecyl, dihexadecyl or dioctadecyl glutamate hydrophobic moieties with/without propyl, pentyl or heptyl spacers were applied for the preparation of cationic liposomes using a simple bath type-sonicator. The size distribution, zeta potential, cellular internalization, and cytotoxicity of the liposomes were characterized, and the innate immune stimulation, e.g., the NLRP3 inflammasome activation of human macrophages and THP-1 cells, was evaluated by the detection of IL-1β release. Comparatively, L3C14 and L5C14 liposomes, made from the lipids bearing lysine head groups, ditetradecyl hydrophobic chains and propyl or pentyl spacers, respectively, were the most potent to activate the NLRP3 inflammasome. The possible mechanism includes endocytosis of the cationic liposomes and subsequent lysosome rupture without significant inducement of reactive oxygen species production. In summary, we first disclosed the structural effect of cationic liposomes on the NLRP3 inflammasome activation, which gives an insight into the application of nanoparticles for improved immune response.

    DOI PubMed

  • The efficacy of basic fibroblast growth factor-loaded poly(lactic-co-glycolic acid) nanosheet for mouse wound healing

    Shimpo Aoki, Mao Fujii, Toshinori Fujie, Keisuke Nishiwaki, Hiromi Miyazaki, Daizoh Saitoh, Shinji Takeoka, Tomoharu Kiyosawa, Manabu Kinoshita

    Wound Repair and Regeneration   25 ( 6 ) 1008 - 1016  2017.11  [Refereed]

     View Summary

    Although human recombinant basic fibroblast growth factor (bFGF) is widely used for wound healing, daily treatment with bFGF is required because of its short half-life. An effective controlled-release system of bFGF is, therefore, desired in clinical settings. To investigate the efficacy of a bFGF-loaded nanosheet for wound healing, focusing on the controlled-release of bFGF, bFGF-loaded poly(lactic-co-glycolic acid) (PGLA) nanosheets were developed, and their in vitro release profile of bFGF and their in vivo efficacy for wound healing were examined. A polyion complex of positively charged human recombinant bFGF and negatively charged alginate was sandwiched between PLGA nanosheets (70 nm thick for each layer). The resulting bFGF-loaded nanosheet robustly adhered to silicon skin by observation using a microscratch test. bFGF was gradually and continuously released over three days in an in vitro incubation study. Treatment with the bFGF-loaded nanosheets (every 3 day for 15 days) as well as with a conventional bFGF spray effectively promoted wound healing of mouse dorsal skin defects with accelerated tissue granulation and angiogenesis, although the dose of bFGF used in the treatment with the bFGF nanosheets was approximately 1/20 of the sprayed bFGF. In conclusion, we developed a bFGF-loaded nanosheet that sustained a continuous release of bFGF over three days and effectively promoted wound healing in mice.

    DOI PubMed

  • Adhesive and robust multilayered poly(lactic acid) nanosheets for hemostatic dressing in liver injury model

    Takuya Komachi, Hideaki Sumiyoshi, Yutaka Inagaki, Shinji Takeoka, Yu Nagase, Yosuke Okamura

    JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS   105 ( 7 ) 1747 - 1757  2017.10  [Refereed]

     View Summary

    Freestanding biodegradable nanosheets composed of poly(l-lactic acid) (PLLA) have been developed for various biomedical applications. These nanosheets exhibit unique properties such as high adhesiveness and exquisite flexibility; however, they burst easily due to their nanometer thickness. We herein describe a freestanding, multilayered nanosheet composed of PLLA fabricated using a simple combination procedure: (i) multilayering of PLLA and alginate, (ii) gelation of the alginate layers, (iii) fusion-cut sealing, and (iv) elution of the alginate layers. The multilayered nanosheets not only reinforced the bursting strength but also provided a high level of adhesive strength. In fact, they were found to show potential as a hemostatic dressing, and they tended to show reduced tissue adhesion that accompanies liver injury. Therefore, we propose this biomaterial as a candidate for an alternative to conventional therapy in hemorrhage. (c) 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1747-1757, 2017.

    DOI PubMed

  • On the injectability of free-standing magnetic nanofilms

    Silvia Taccola, Virginia Pensabene, Toshinori Fujie, Shinji Takeoka, Nicola M. Pugno, Virgilio Mattoli

    BIOMEDICAL MICRODEVICES   19 ( 3 ) 51  2017.09  [Refereed]

     View Summary

    Free-standing films with sub-micrometric thickness, composed of soft polymers and functional nanostructures are promising candidates for many potential applications in the biomedical field, such as reduced port abdominal surgery. In this work, freely suspended poly(L-lactic acid) nanofilms with controlled morphology embedding superparamagnetic iron oxide nanoparticles were fabricated by spin-coating deposition. The mechanical properties of magnetic nanofilms were investigated by Strain-Induced Elastic Buckling Instability for Mechanical Measurements (SIEBIMM) test. Our results show that these freely suspended nanocomposite nanofilms are highly flexible and deformable, withYoung's moduli of few GPa. Since they can be handled in liquid with syringes, a quantitative description of the nanofilms behavior during the manipulation with clinically applicable needles has been also provided. These magnetic nanofilms, remotely controllable by external electromagnetic fields, have potential applications in minimally invasive surgery as injectable nanopatches on inner organs wall.

    DOI PubMed

  • Preparation, Characterization, and Preliminary In Vitro Testing of Nanoceria-Loaded Liposomes

    Agostina Grillone, Tianshu Li, Matteo Battaglini, Alice Scarpellini, Mirko Prato, Shinji Takeoka, Gianni Ciofani

    NANOMATERIALS   7 ( 9 )  2017.09  [Refereed]

     View Summary

    Cerium oxide nanoparticles (nanoceria), well known for their pro-and antioxidant features, have been recently proposed for the treatment of several pathologies, including cancer and neurodegenerative diseases. However, interaction between nanoceria and biological molecules such as proteins and lipids, short blood circulation time, and the need of a targeted delivery to desired sites are some aspects that require strong attention for further progresses in the clinical application of these nanoparticles. The aim of this work is the encapsulation of nanoceria into a liposomal formulation in order to improve their therapeutic potentialities. After the preparation through a reverse- phase evaporation method, size, Z-potential, morphology, and loading efficiency of nanoceria-loaded liposomes were investigated. Finally, preliminary in vitro studies were performed to test cell uptake efficiency and preserved antioxidant activity. Nanoceria-loaded liposomes showed a good colloidal stability, an excellent biocompatibility, and strong antioxidant properties due to the unaltered activity of the entrapped nanoceria. With these results, the possibility of exploiting liposomes as carriers for cerium oxide nanoparticles is demonstrated here for the first time, thus opening exciting new opportunities for in vivo applications.

    DOI PubMed

  • Effect of the nanoformulation of siRrNAa-lipid assemblies on their cellular uptake and immune stimulation

    Kohei Kubota, Kohei Onishi, Kazuaki Sawaki, Tianshu Li, Kaoru Mitsuoka, Takaaki Sato, Shinji Takeoka

    International Journal of Nanomedicine   12   5121 - 5133  2017.07  [Refereed]

     View Summary

    Two lipid-based nanoformulations have been used to date in clinical studies: lipoplexes and lipid nanoparticles (LNPs). In this study, we prepared small interfering RNA (siRNA)-loaded carriers using lipid components of the same composition to form molecular assemblies of differing structures, and evaluated the impact of structure on cellular uptake and immune stimulation. Lipoplexes are electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic siRNA in an aqueous environment, whereas LNPs are nanoparticles embedding siRNA prepared by mixing an alcoholic lipid solution with an aqueous siRNA solution in one step. Although the physicochemical properties of lipoplexes and LNPs were similar except for small increases in apparent size of lipoplexes and zeta potential of LNPs, siRNA uptake efficiency of LNPs was significantly higher than that of lipoplexes. Furthermore, in the case of LNPs, both siRNA and lipid were effectively incorporated into cells in a co-assembled state
    however, in the case of lipoplexes, the amount of siRNA internalized into cells was small in comparison with lipid. siRNAs in lipoplexes were thought to be more likely to localize on the particle surface and thereby undergo dissociation into the medium. Inflammatory cytokine responses also appeared to differ between lipoplexes and LNPs. For tumor necrosis factor-a, release was mainly caused by siRNA. On the other hand, the release of interleukin-1β was mainly due to the cationic nature of particles. LNPs released lower amounts of tumor necrosis factor-a and interleukin-1β than lipoplexes and were thus considered to be better tolerated with respect to cytokine release. In conclusion, siRNA-loaded nanoformulations effect their cellular uptake and immune stimulation in a manner that depends on the structure of the molecular assembly
    therefore, nanoformulations should be optimized before extending studies into the in vivo environment.

    DOI PubMed

  • Construction and evaluation of pH-sensitive immunoliposomes for enhanced delivery of anticancer drug to ErbB2 over-expressing breast cancer cells

    Tianshu Li, Takuya Amari, Kentaro Semba, Tadashi Yamamoto, Shinji Takeoka

    NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE   13 ( 3 ) 1219 - 1227  2017.04  [Refereed]

     View Summary

    1,5-Dihexadecyl N, N-diglutamyl-lysyl-L-glutamate (GGLG) liposomes were previously developed to enhance drug delivery efficiency in tumor cells owing to its pH-responsive properties. Herein, we report the modification of GGLG liposomes by conjugating a Fab' fragment of an ErbB2 antibody to the terminus of PEG (polyethylene glycol)-lipid (Fab'-GGLGliposomes). The conjugation of Fab' fragments did not affect the antibody activity, drug (doxorubicin, DOX) encapsulation efficiency, stability during storage or pH-sensitivity. However, the binding affinity of Fab'-GGLG liposomes was enhanced to ErbB2-overexpressing HCC1954 cells specifically, and the cell association increased 10-fold in comparison to GGLG liposomes. Consequently, intracellular DOX delivery was enhanced, with an increased cytotoxicity in HCC1954 cells (i.e., IC50 of 1.17 and 3.08 mu g/mL for Fab'-GGLG-DOX and GGLG-DOX liposomes, respectively). Further, a significantly enhanced tumor growth inhibition was obtained in an ErbB2-overexpressing breast cancer-bearing mouse model. Therefore, a potent anticancer drug delivery system was constructed by the immunological modification of pH-sensitive liposomes. (C) 2016 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Correction to "Glue-Free Stacked Luminescent Nanosheets Enable High-Resolution Ratiometric Temperature Mapping in Living Small Animals".

    Miyagawa T, Fujie T, Ferdinandus, Vo Doan TT, Sato H, Takeoka S

    ACS applied materials & interfaces   9 ( 7 ) 6652  2017.02  [Refereed]

    DOI PubMed

  • Glue-Free Stacked Luminescent Nanosheets Enable High-Resolution Ratiometric Temperature Mapping in Living Small Animals

    Takuya Miyagawa, Toshinori Fujie, Ferdinandus, Tat Thang Vo Doan, Hirotaka Sato, Shinji Takeoka

    ACS APPLIED MATERIALS & INTERFACES   8 ( 49 ) 33377 - 33385  2016.12  [Refereed]

     View Summary

    In this paper, a microthermograph, temperature mapping with high spatial resolution, was established using luminescent molecules embedded ultrathin polymeric films (nanosheets), and demonstrated in a living small animal to map out and visualize temperature shift due to animal's muscular activity. Herein, we report super flexible and self-adhesive (no need of glue) nanothermosensor consisting of stacked two different polymeric nanosheets with thermosensitive (Eu-tris (dinaphthoylmethane)-bis-trioctylphosphine oxide: EuDT) and insensitive (Rhodamine 800) dyes being embedded. Such stacked nanosheets allow for the ratiometric thermometry, with which the undesired luminescence intensity shift due to focal drift or animal's z-axis displacement is eliminated and the desired intensity shift solely due to the temperature shift of the sample (living muscle) can be acquired. With the stacked luminescent nanosheets, we achieved the first-ever demonstration of video filming of chronologically changing temperature-shift distribution from the rest state to the active state of the muscles in the living animal. The polymer nanosheet engineering and in vivo microthermography presented in the paper are promising technologies to microscopically explore the heat production and heat transfer in living cells, tissues, and organisms with high spatial resolution beyond what existing thermometric technologies such as infrared thermography have ever achieved.

    DOI PubMed

  • Fibrinogen gamma-Chain Peptide-Coated Adenosine 5 ' Diphosphate-Encapsulated Liposomes Rescue Mice From Lethal Blast Lung Injury via Adenosine Signaling

    Kohsuke Hagisawa, Manabu Kinoshita, Hiroki Miyawaki, Shunichi Sato, Hiromi Miyazaki, Shinji Takeoka, Hidenori Suzuki, Keiichi Iwaya, Shuhji Seki, Satoshi Shono, Daizoh Saitoh, Yasuhiro Nishida, Makoto Handa

    CRITICAL CARE MEDICINE   44 ( 9 ) E827 - E837  2016.09  [Refereed]

     View Summary

    Objectives: Fibrinogen gamma-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes can accumulate via dodecapeptide HHLGGAKQAGDV interactions at bleeding sites where they release adenosine 5'-diphosphate that is rapidly metabolized to adenosine, which has tissue-protective effects. We investigated the efficacy of fibrinogen.-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes to treat blast lung injury, with a focus on adenosine signaling.
    Design: Controlled animal study.
    Setting: University research laboratory.
    Subjects: Adult male C57BL/6 mice.
    Interventions: Mice were pretreated with fibrinogen.-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes, dodecapeptide HHLGGAKQAGDV-(phosphate-buffered saline)-liposomes, adenosine 5' diphosphateliposomes, or phosphate-buffered saline-liposomes. Five minutes after treatment the mice received a single laser-induced shock wave (1.8 J/cm(2)) that caused lethal blast lung injury, and their survival times and lung injuries were then assessed. We also evaluated the therapeutic effect of posttreatment with fibrinogen gamma-chain (dodecapeptide HHLGGAKQAGDV)coated adenosine 5'-diphosphate-encapsulated liposomes or H12-(phosphate-buffered saline)-liposomes 1 minute after laser-induced shock wave exposure. To examine the effect of adenosine signaling, adenosine A(2A) receptor (ZM241385) or adenosine A(2B) receptor (PSB 1115) antagonists were administered to the mice 1 hour before the pretreatment with fibrinogen gamma-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes that was followed by laser-induced shock wave exposure.
    Measurements and Main Results: Pre-and posttreatment with fibrinogen.-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes significantly increased mouse survival [fibrinogen gamma-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes: 58% survival vs H12-(phosphate-buffered saline)-liposomes: 8%; p < 0.05 (posttreatment)] and mitigated pulmonary tissue damage/hemorrhage and neutrophil accumulation after laser-induced shock wave exposure. fibrinogen gamma-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes accumulated at pulmonary vessel injury sites after laser-induced shock wave exposure with both pre-and posttreatment. Furthermore, pretreatment with fibrinogen.-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes reduced albumin and macrophage inflammatory protein-2 levels in bronchoalveolar lavage fluid. Although fibrinogen gamma-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes pretreatment did not affect blood coagulation activity in the injured mice, its beneficial effect on blast lung injury was significantly abrogated by A(2A) or A(2B) adenosine receptor antagonists (A(2A) antagonist: 17% survival; A(2B) antagonist: 33% vs dimethyl sulfoxide control: 80%; p < 0.05, respectively).
    Conclusions: Fibrinogen gamma-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes may be effective against blast lung injury by promoting tissue-protective adenosine signaling and could represent a novel controlled-release drug delivery system.

    DOI PubMed

  • Phospholipase C beta 1 induces membrane tubulation and is involved in caveolae formation

    Takehiko Inaba, Takuma Kishimoto, Motohide Murate, Takuya Tajima, Shota Sakai, Mitsuhiro Abe, Asami Makino, Nario Tomishige, Reiko Ishitsuka, Yasuo Ikeda, Shinji Takeoka, Toshihide Kobayashi

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   113 ( 28 ) 7834 - 7839  2016.07  [Refereed]

     View Summary

    Lipid membrane curvature plays important roles in various physiological phenomena. Curvature-regulated dynamic membrane remodeling is achieved by the interaction between lipids and proteins. So far, several membrane sensing/sculpting proteins, such as Bin/amphiphysin/Rvs (BAR) proteins, are reported, but there remains the possibility of the existence of unidentified membrane-deforming proteins that have not been uncovered by sequence homology. To identify new lipid membrane deformation proteins, we applied liposome-based microscopic screening, using unbiased-darkfield microscopy. Using this method, we identified phospholipase C beta 1 (PLC beta 1) as a new candidate. PLC beta 1 is well characterized as an enzyme catalyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). In addition to lipase activity, our results indicate that PLC beta 1 possessed the ability of membrane tubulation. Lipase domains and inositol phospholipids binding the pleckstrin homology (PH) domain of PLC beta 1 were not involved, but the C-terminal sequence was responsible for this tubulation activity. Computational modeling revealed that the C terminus displays the structural homology to the BAR domains, which is well known as a membrane sensing/sculpting domain. Overexpression of PLC beta 1 caused plasma membrane tubulation, whereas knockdown of the protein reduced the number of caveolae and induced the evagination of caveolin-rich membrane domains. Taken together, our results suggest a new function of PLC beta 1: plasma membrane remodeling, and in particular, caveolae formation.

    DOI PubMed

  • Massive Fabrication of Polymer Microdiscs by Phase Separation and Freestanding Process

    Hong Zhang, Mao Fujii, Yosuke Okamura, Li Zhang, Shinji Takeoka

    ACS APPLIED MATERIALS & INTERFACES   8 ( 25 ) 16296 - 16302  2016.06  [Refereed]

     View Summary

    We present a facile method to fabricate polymer thin films with tens of nanometers thickness and several micrometers size (also called "microdiscs" herein) by applying phase separation of polymer blend. A water-soluble supporting layer is employed to obtain a freestanding microdisc suspension. Owing to their miniaturized size, microdiscs can be injected through a syringe needle. Herein, poly(D,L-lactic acid) microdiscs were fabricated with various thicknesses and sizes, in the range from ca. 10 to 60 nm and from ca. 1.0 to 10.0 mu m, respectively. Magnetic nanoparticles were deposited on polymer microdiscs with a surface coating method. The magnetic manipulation of microdiscs in a liquid environment under an external magnetic field was achieved with controllable velocity by adjusting the microdisc dimensions and the loading amount of magnetic components. Such biocompatible polymer microdiscs are expected to serve as injectable vehicles for targeted drug delivery.

    DOI PubMed

  • Development of a ubiquitously transferrable silver-nanoparticle-loaded polymer nanosheet as an antimicrobial coating

    Keisuke Ito, Akihiro Saito, Toshinori Fujie, Hiromi Miyazaki, Manabu Kinoshita, Daizoh Saitoh, Shinya Ohtsubo, Shinji Takeoka

    JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS   104 ( 3 ) 585 - 593  2016.04  [Refereed]

     View Summary

    Ultra-thin polymer films (nanosheets) fabricated by a layer-by-layer (LbL) method possess unique properties such as high flexibility, adhesive strength, and transparency, and can be peeled off from a substrate and attached to various surfaces via a water-soluble supporting film. Therefore, flexible and transferrable LbL nanosheets are convenient tools as coating materials. Here, we fabricated a novel antimicrobial coating material by embedding silver nanoparticles (AgNPs) in an LbL nanosheet composed of layers of chitosan and sodium alginate (Ag-LbL nanosheet) by means of a photo-reduction method. Optimizing the amount of irradiated energy applied led to robust antimicrobial efficacy against methicillin-resistant Staphylococcus aureus (MRSA), sufficient to meet ISO standards (ISO 22196), while maintaining the flexibility and adhesive potency of the LbL nanosheet. Thus, the Ag-LbL nanosheet is a promising coating material that can provide antimicrobial efficacy to various surfaces. (c) 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 585-593, 2016.

    DOI PubMed

  • Stretchable, adhesive and ultra-conformable elastomer thin films

    Nobutaka Sato, Atsushi Murata, Toshinori Fujie, Shinji Takeoka

    SOFT MATTER   12 ( 45 ) 9202 - 9209  2016  [Refereed]

     View Summary

    Thermoplastic elastomers are attractive materials because of the drastic changes in their physical properties above and below the glass transition temperature (T-g). In this paper, we report that free-standing polystyrene (PS, T-g: 100 degrees C) and polystyrene-polybutadiene-polystyrene triblock copolymer (SBS, T-g: -70 degrees C) thin films with a thickness of hundreds of nanometers were prepared by a gravure coating method. Among the mechanical properties of these thin films determined by bulge testing and tensile testing, the SBS thin films exhibited a much lower elastic modulus (ca. 0.045 GPa, 212 nm thickness) in comparison with the PS thin films (ca. 1.19 GPa, 217 nm thickness). The lower elastic modulus and lower thickness of the SBS thin films resulted in higher conformability and thus higher strength of adhesion to an uneven surface such as an artificial skin model with roughness (R-a = 10.6 mu m), even though they both have similar surface energies. By analyzing the mechanical properties of the SBS thin films, the elastic modulus and thickness of the thin films were strongly correlated with their conformability to a rough surface, which thus led to a high adhesive strength. Therefore, the SBS thin films will be useful as coating layers for a variety of materials.

    DOI PubMed

  • Focal calcium monitoring with targeted nanosensors at the cytosolic side of endoplasmic reticulum

    Yanyan Hou, Satoshi Arai, Yoshiaki Takei, Atsushi Murata, Shinji Takeoka, Madoka Suzuki

    SCIENCE AND TECHNOLOGY OF ADVANCED MATERIALS   17 ( 1 ) 293 - 299  2016  [Refereed]

     View Summary

    Ca2+ distribution is spatially and temporally non-uniform inside cells due to cellular compartmentalization. However, Ca2+ sensing with small organic dyes, such as fura-2 and fluo-4, has been practically applied at a single cell level where the averaged signal from freely diffusing dye molecules is acquired. In this study, we aimed to target azide-functionalized fura-2 (N-3-fura-2) to a specific site of subcellular compartments to realize focal Ca2+ sensing. Using scAVD (single-chain avidin)-biotin interaction and a copper-free click reaction system, we linked N-3-fura-2 to specifically-targeted scAVD protein fused with a red fluorescent protein mCherry, so that Ca2+ sensors conjugated with four N-3-fura-2 dyes with dibenzocyclooctyne (DBCO)-PEG4-biotin as a linker were generated at subcellular compartments in living cells. In cytoplasm, N-3-fura-2 showed a prolonged retention period after binding to scAVD. Furthermore, the reacted N-3-fura-2 was retained inside cells even after free dyes were washed out by methanol fixation. When scAVD was overexpressed on endoplasmic reticulum (ER) membranes, N-3-fura-2 was accumulated on ER membranes. Upon histamine stimulation, which increases cytosolic Ca2+ concentration, ER-localized N-3-fura-2 successfully sensed the Ca2+ level changes at the cytosolic side of ER membrane. Our study demonstrated specific targeting of N-3-fura-2 to subcellular compartments and the ability of sensing focal Ca2+ level changes with the specifically targeted Ca2+ sensors.
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    DOI PubMed

  • Pharmacokinetic Properties of Single and Repeated Injection of Liposomal Platelet Substitute in a Rat Model of Red Blood Cell Transfusion-Induced Dilutional Thrombocytopenia

    Mai Hashimoto, Kazuaki Taguchi, Shigeru Ogaki, Hiroshi Watanabe, Manabu Kinoshita, Kahoko Nishikawa, Shinji Takeoka, Yasuo Ikeda, Makoto Handa, Masaki Otagiri, Toru Maruyama

    JOURNAL OF PHARMACEUTICAL SCIENCES   104 ( 11 ) 3968 - 3976  2015.11  [Refereed]

     View Summary

    A preclinical study of dodecapeptide ((400)HHLGGAKQAGDV(411)) (H12)-(adenosine diphosphate, ADP)-liposomes for use as a synthetic platelet (PLT) substitute under conditions of red blood cell (RBC) transfusion-induced dilutional thrombocytopenia is limited to pharmacological effect. In this study, the pharmacokinetics of H12-(ADP)-liposomes in RBC transfusion-induced dilutional thrombocytopenic rats were evaluated. As evidenced by the use of C-14, H-3 double-radiolabeled H12-(ADP)-liposomes in which the encapsulated ADP and liposomal membrane were labeled with C-14 and H-3, respectively, the H12-(ADP)-liposomes remained intact in the blood circulation for up to 3 h after injection, and were mainly distributed to the liver and spleen. The encapsulated ADP was mainly eliminated in the urine, whereas the outer membrane was mainly eliminated in the feces. These successive pharmacokinetic properties of the H12-(ADP)-liposomes in RBC transfusion-induced dilutional thrombocytopenic rats were similar to those in healthy rats, except for the shorter retention time in the circulation. When H12-(ADP)-liposomes were repeatedly injected into RBC transfusion-induced dilutional thrombocytopenic rats at intervals of 5 days at a dose of 10 mg lipids/kg, the second dose of injected H12-(ADP)-liposomes were rapidly cleared from the circulation, namely, via the accelerated blood clearance phenomenon. These novel pharmacokinetic findings provide useful information for the further development of H12-(ADP)-liposomes as a PLT substitute. (c) 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3968-3976, 2015

    DOI PubMed

  • Patchwork Coating of Fragmented Ultra-Thin Films and Their Biomedical Applications in Burn Therapy and Antithrombotic Coating

    Yosuke Okamura, Yu Nagase, Shinji Takeoka

    MATERIALS   8 ( 11 ) 7604 - 7614  2015.11  [Refereed]

     View Summary

    We have proposed free-standing centimeter-sized ultra-thin films (nanosheets) for biomedical applications. Such nanosheets exhibit unique properties such as transparency, flexibility, and good adhesiveness. However, they are only easily adhered to broad and flat surfaces due to their dimensions. To this end, we recently proposed an innovative nanomaterial: the nanosheets fragmented into submillimeter-size pieces. Intriguingly, such fragmented nanosheets could be adhered to uneven and irregular surfaces in addition to flat surfaces in a spread-out patchwork manner. We herein review the fabrication procedure and characterization of fragmented nanosheets composed of biodegradable polyesters and thermostable bio-friendly polymers, and their biomedical applications in burn therapy and antithrombotic coating using a patchwork coating.

    DOI PubMed

  • Effect of Repeated Injections of Adenosine Diphosphate-Encapsulated Liposomes Coated with a Fibrinogen -Chain Dodecapeptide Developed as a Synthetic Platelet Substitute on Accelerated Blood Clearance in a Healthy and an Anticancer Drug-Induced Thrombocytopenia Rat Model

    Kazuaki Taguchi, Mai Hashimoto, Shigeru Ogaki, Hiroshi Watanabe, Shinji Takeoka, Yasuo Ikeda, Makoto Handa, Masaki Otagiri, Toru Maruyama

    JOURNAL OF PHARMACEUTICAL SCIENCES   104 ( 9 ) 3084 - 3091  2015.09  [Refereed]

     View Summary

    Adenosine diphosphate (ADP)-encapsulated liposomes coated with a fibrinogen -chain dodecapeptide [H12 (dodecapeptide ((400)HHLGGAKQAGDV(411)))-(ADP)-liposome] is a synthetic platelet substitute, in which the surface is covered with polyethylene glycol (PEG). It has been reported that repeated injections of PEGylated liposomes induce an accelerated blood clearance (ABC) phenomenon, which involves a loss in the long-circulation half-life of the material when administered repeatedly to the same animals. The objective of this study was to determine whether the ABC phenomenon was induced by repeated injections of H12-(ADP)-liposome in healthy and anticancer drug-induced thrombocytopenia model rats. The findings show that the ABC phenomenon was induced by healthy rats that were repeatedly injected with H12-(ADP)-liposomes at the interval of 5 days at a dose of 10 mg lipids/kg. The ABC phenomenon involves the production of anti-H12-(ADP)-liposome immunoglobulin M (IgM) and complement activation. On the other hand, when thrombocytopenia model rats were repeatedly injected with H12-(ADP)-liposomes under the same conditions, no ABC phenomenon, nor was any suppression of anti-H12-(ADP)-liposome IgM-mediated complement activation observed. We thus conclude that the repeated injection of H12-(ADP)-liposome treatment in rat model with anticancer drug-induced thrombocytopenia did not induce the ABC phenomenon. (c) 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3084-3091, 2015

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  • Establishment of a total liquid ventilation system using saline-based oxygen micro/nano-bubble dispersions in rats

    Kenta Kakiuchi, Kenichi Matsuda, Norikazu Harii, Keitaro Sou, Junko Aoki, Shinji Takeoka

    JOURNAL OF ARTIFICIAL ORGANS   18 ( 3 ) 220 - 227  2015.09  [Refereed]

     View Summary

    Micro/nano-bubbles are practical nanomaterials designed to increase the gas content in liquids. We attempted to use oxygen micro/nano-bubble dispersions as an oxygen-rich liquid as a means for total liquid ventilation. To determine the oxygen content in the bubble dispersion, a new method based on a spectrophotometric change between oxy- and deoxy-hemoglobin was established. The oxygen micro/nano-bubble dispersion was supplied to an experimental total ventilation liquid in anesthetic rats. Though the amount of dissolving oxygen was as low as 6 mg/L in physiological saline, the oxygen content in the oxygen micro/nano-bubble dispersion was increased to 45 mg/L. The positive correlation between the oxygen content and the life-saving time under liquid ventilation clearly indicates that the life-saving time is prolonged by increasing the oxygen content in the oxygen micro/nano-bubble dispersion. This is the first report indicating that the oxygen micro/nano-bubbles containing a sufficient amount of oxygen are useful in producing oxygen-rich liquid for the process of liquid ventilation.

    DOI PubMed

  • Sustainable antimicrobial effect of silver sulfadiazine-loaded nanosheets on infection in a mouse model of partial-thickness burn injury

    Keisuke Ito, Akihiro Saito, Toshinori Fujie, Keisuke Nishiwaki, Hiromi Miyazaki, Manabu Kinoshita, Daizoh Saitoh, Shinya Ohtsubo, Shinji Takeoka

    ACTA BIOMATERIALIA   24   87 - 95  2015.09  [Refereed]

     View Summary

    Partial-thickness burn injury has the potential for reepithelialization and heals within 3 weeks. If the wound is infected by bacteria before reepithelization, however, the depth of disruption increases and the lesion easily progresses to the full-thickness dermal layers. In the treatment of partial-thickness burn injury, it is important to prevent the wound area from bacterial infection with an antimicrobial dressing. Here, we have tested the antimicrobial properties of polymeric ultra-thin films composed of poly(lactic acid) (termed "PLA nanosheets"), which have high flexibility, adhesive strength and transparency, and silver sulfadiazine (AgSD), which exhibits antimicrobial efficacy. The AgSD-loaded nanosheet released Ag+ for more than 3 days, and exerted antimicrobial efficacy against methicillin-resistant Staphylococcus aureus (MRSA) in an in vitro Kirby-Bauer test. By contrast, a cell viability assay indicated that the dose of AgSD used in the PLA nanosheets did not show significant cytotoxicity toward fibroblasts. In vivo evaluation using a mouse model of infection in a partial-thickness burn wound demonstrated that the nanosheet significantly reduced the number of MRSA bacteria on the lesion (more than 10(5)-fold) and suppressed the inflammatory reaction, thereby preventing a protracted wound healing process. (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

    DOI PubMed

  • Treatment with fibrinogen gamma-chain peptide-coated, adenosine 5 '-diphosphate-encapsulated liposomes as an infusible hemostatic agent against active liver bleeding in rabbits with acute thrombocytopenia

    Kohsuke Hagisawa, Kahoko Nishikawa, Rempei Yanagawa, Manabu Kinoshita, Mami Doi, Hidenori Suzuki, Keiichi Iwaya, Daizoh Saitoh, Shuhji Seki, Shinji Takeoka, Makoto Handa, Yasuhiro Nishida

    TRANSFUSION   55 ( 2 ) 314 - 325  2015.02  [Refereed]

     View Summary

    BackgroundWe evaluated the hemostatic efficacy of H12-(adenosine 5-diphosphate [ADP])-liposomes in the setting of active liver bleeding in rabbits with dilutional thrombocytopenia after massive transfusion.
    Study Design and MethodsAcute thrombocytopenia (platelet [PLT] count<50x10(9)/L) was induced in rabbits by repeated blood withdrawal and isovolemic transfusion of autologous washed red blood cells. Liver hemorrhage was initiated by a penetrating liver injury. Subsequently, the animals received tamponade treatment for the liver hemorrhage for 5 minutes and were intravenously administered H12-(ADP)-liposomes with PLT-poor plasma (PPP), PLT-rich plasma (PRP), PPP alone, H12-(phosphate-buffered saline [PBS])-liposome/PPP, or H12-(ADP)-liposomes/PPPplusfibrinogen concentrate during the tamponade.
    ResultsAdministration of H12-(ADP)-liposomes/PPP rescued 60% of the rabbits from the liver hemorrhage; PRP administration rescued 50%. In contrast, rabbits receiving PPP or H12-(PBS)-liposome/PPP achieved only 10 or 17% survival, respectively, for the first 24 hours. H12-(ADP)-liposomes/PPP as well as PRP consistently reduced bleeding volumes and shortened clotting times (CTs) in comparison to PPP administration. Specifically, bleeding volumes in the initial 5 minutes averaged 11mL (H12-(ADP)-liposomes/PPP) and 17mL (PRP) versus 30mL (PPP; p<0.05); CTs averaged 270 and 306 seconds versus 401 seconds (p<0.05). H12-(ADP)-liposomes were observed at the bleeding site with thrombus formation, suggesting an induction of thrombi. Neither macro- nor microthrombi were detected in the lung, kidney, spleen, or liver in rabbits treated with H12-(ADP)-liposomes. Supplementation of fibrinogen to H12-(ADP)-liposomes/PPP did not significantly improve rabbit survival.
    ConclusionsH12-(ADP)-liposomes might be a safe and effective therapeutic tool during damage control surgery for trauma patients with acute thrombocytopenia and massive bleeding.

    DOI PubMed

  • A ratiometric fluorescent molecular probe for visualization of mitochondrial temperature in living cells

    Mitsumasa Homma, Yoshiaki Takei, Atsushi Murata, Takafumi Inoue, Shinji Takeoka

    CHEMICAL COMMUNICATIONS   51 ( 28 ) 6194 - 6197  2015  [Refereed]

     View Summary

    Mitochondrial thermodynamics is the key to understand cellular activities related to homeostasis and energy balance. Here, we report the first ratiometric fluorescent molecular probe (Mito-RTP) that is selectively localized in the mitochondria and visualize the temperature. We confirmed that Mito-RTP could work as a ratiometric thermometer in a cuvette and living cells.

    DOI PubMed

  • A Cu-free clickable fluorescent probe for intracellular targeting of small biomolecules

    Kento Yamagishi, Kazuaki Sawaki, Atsushi Murata, Shinji Takeoka

    CHEMICAL COMMUNICATIONS   51 ( 37 ) 7879 - 7882  2015  [Refereed]

     View Summary

    We synthesized a novel cyclooctyne-based clickable fluorescent probe with versatile properties such as high cell-membrane permeability and free diffusibility in the cell. Our probe "FC-DBCO'' was conjugated to an azide-modified mannose via a Cu-free click reaction in living HeLa cells and displayed intracellular specific fluorescence imaging with low background signals.

    DOI PubMed

  • Micro-thermography in millimeter-scale animals by using orally-dosed fluorescent nanoparticle thermosensors

    Satoshi Arai, Ferdinandus, Shinji Takeoka, Shin'ichi Ishiwata, Hirotaka Sato, Madoka Suzuki

    ANALYST   140 ( 22 ) 7534 - 7539  2015  [Refereed]

     View Summary

    We propose an instant micro-thermography method using a fluorescent-nanoparticle thermosensor capable of reporting temperature as the fluorescence intensity ratio of the temperature-sensitive dye to the reference. We demonstrate "temperature mapping" inside a fruit fly larva that was orally dosed with nanoparticle thermosensors.

    DOI PubMed

  • Homeotropic alignment of dendritic columnar liquid crystal induced by hydrogen-bonded triphenylene core bearing fluoroalkyl chains.

    Shinsuke Ishihara, Yusuke Furuki, Jonathan P Hill, Katsuhiko Ariga, Shinji Takeoka

    Journal of nanoscience and nanotechnology   14 ( 7 ) 5130 - 7  2014.07  [Refereed]  [International journal]

     View Summary

    A 1:3 molar complex of the fluoroalkyl side chain-substituted 2,6,10-tris-carboxymethoxy-3,7,11-tris(4,4,5,5,6,6,7,7,7-nonafluoroheptyloxy)triphenylene (TPF4) with the second generation dendron 3,5-bis(3,4-bis-dodecyloxybenzyloxy)-N-pyridin-4-yl-benzamide (DN) assembled through complementary hydrogen bonding to form a supramolecular columnar liquid crystal, which exhibited homeotropic alignment when sandwiched between octadecyltrichlorosilane (OTS)-coated or indium tin oxide (ITO)-coated glass plates due to specific interactions between the fluoroalkyl side chains and the substrates.

    DOI PubMed

  • Enhanced cellular uptake of maleimide-modified liposomes via thiol-mediated transport

    Tianshu Li, Shinji Takeoka

    International Journal of Nanomedicine   9 ( 1 ) 2849 - 2861  2014.06  [Refereed]

     View Summary

    With a small amount of maleimide modification on the liposome surface, enhanced cellular uptake of liposomes and drug-delivery efficiency can be obtained both in vitro and in vivo. Herein, we describe the mechanisms underlying this enhanced cellular uptake. Suppression of the cellular uptake of maleimide-modified liposomes (M-GGLG, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate [GGLG]/cholesterol/poly(ethylene glycol) - 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [PEG5000-DSPE]/maleimide [M]-PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03) caused by temperature block and addition of serum was alleviated compared with that of liposomes without maleimide modification (GGLG liposomes, composed of GGLG/cholesterol/PEG5000-DSPE/PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03). When 0.01 nM N-ethylmaleimide was used to pre-block cellular thiols, the cellular uptake of M-GGLG liposomes was decreased to approximately 70% in HeLa, HCC1954, MDA-MB-468, and COS-7 cell lines. Moreover, inhibition of a thiol-related reductase such as protein disulfide isomerase resulted in a 15%-45% inhibition of the cellular uptake of M-GGLG liposomes, whereas GGLG liposomes were not influenced. Further, single and mixed inhibitors of clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis did not efficiently inhibit the cellular uptake of M-GGLG liposomes. Using confocal microscopy, we verified that M-GGLG liposomes were localized partially in lysosomes after inhibition of the mentioned conventional endocytic pathways. Therefore, it was hypothesized that the mechanisms underlying the enhanced cellular uptake of liposomes by maleimide modification was thiol-mediated membrane trafficking, including endocytosis and energy-independent transport. © 2014 Li and Takeoka.

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  • Periosteum-Mimetic Structures Made from Freestanding Microgrooved Nanosheets

    Xuetao Shi, Toshinori Fujie, Akihiro Saito, Shinji Takeoka, Ying Hou, Yiwei Shu, Mingwei Chen, Hongkai Wu, Ali Khademhosseini

    ADVANCED MATERIALS   26 ( 20 ) 3290 - +  2014.05  [Refereed]

     View Summary

    A "sticker-like" PLGA nanosheet with microgrooved patterns is developed through a facile combination of spin coating and micropatterning techniques. The resulting microgrooved PLGA nanosheets can be physically adhered on flat or porous surfaces with excellent stability in aqueous environments and can harness the spatial arrangements of cells, which make it a promising candidate for generating biomimic periosteum for bone regenerative applications.

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  • Intracellular delivery of universal proteins using a lysine headgroup containing cationic liposomes: Deciphering the uptake mechanism

    Satya Ranjan Sarker, Ryosuke Hokama, Shinji Takeoka

    Molecular Pharmaceutics   11 ( 1 ) 164 - 174  2014.01  [Refereed]

     View Summary

    An amino acid-based cationic lipid having a TFA counterion (trifluoroacetic acid counterion) in the lysine headgroup was used to deliver functional proteins into human cervical cancer cells, HeLa, in the presence of serum. Proteins used in the study were fluorescein isothiocyanate (FITC) labeled bovine serum albumin, mouse anti-F actin antibody [NH3], and goat anti mouse IgG conjugated with FITC. The formation of liposome/protein complexes was confirmed using native polyacrylamide gel electrophoresis. Furthermore, the complexes were characterized in terms of their size and zeta potential at different pH values and found to be responsive to changes in pH. The highest delivery efficiency of the liposome/albumin complexes was 99% at 37 °C. The liposomes effectively delivered albumin and antibodies as confirmed by confocal laser scanning microscopy (CLSM). Inhibition studies showed that the cellular uptake mechanism of the complexes was via caveolae-mediated endocytosis, and the proteins were subsequently released from either the early endosomes or the caveosomes as suggested by CLSM. Thus, lysine-based cationic liposomes can be a useful tool for intracellular protein delivery. © 2013 American Chemical Society.

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  • A Nanoparticle-Based Ratiometric and Self-Calibrated Fluorescent Thermometer for Single Living Cells

    Yoshiaki Takei, Satoshi Arai, Atsushi Murata, Masao Takabayashi, Kotaro Oyama, Shin'ichi Ishiwata, Shinji Takeoka, Madoka Suzuki

    ACS NANO   8 ( 1 ) 198 - 206  2014.01  [Refereed]

     View Summary

    The homeostasis of body temperature and energy balance is one of the major principles in biology. Nanoscale thermometry of aqueous solutions is a challenging but crucial technique to understand the molecular basis of this essential process. Here, we developed a ratiometric nanothermometer (RNT) for intracellular temperature measurement in real time. Both the thermosensitive fluorophore, beta-diketonate chelate europium(III) thenoyltrifluoroacetonate, and the thermoinsensitive fluorophore, rhodamine 101, which was used as a self-reference, are embedded in a polymeric particle that protects the fluorophores from intracellular conditions. The ratiometric measurement of single RNT spots is independent of the displacement of the RNT along the z-axis. The temperature is therefore determined at the location of each RNT under an optical microscope regardless of the dynamic movement of living cells. As a demonstration of the spot-by-spot intracellular thermometry, we successfully followed the temperature change in individual RNT spots in a single cell together with the Ca2+ burst induced by the Ca2+ ionophore ionomycin. The temperature increases differently among different spots, implying heterogeneous heat production in the cell. We then show that, in some spots, the temperature gradually decreases, while in others it remains high. The average temperature elevation within a cell is positively correlated to the increase in Ca2+, suggesting that the activity and/or number of heat sources are dependent on the Ca2+ concentration.

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  • Cationic amino acid based lipids as effective nonviral gene delivery vectors for primary cultured neurons

    Yumiko Aoshima, Ryosuke Hokama, Keitaro Sou, Satya Ranjan Sarker, Kabuto Iida, Hideki Nakamura, Takafumi Inoue, Shinji Takeoka

    ACS Chemical Neuroscience   4 ( 12 ) 1514 - 1519  2013.12  [Refereed]

     View Summary

    The delivery of specific genes into neurons offers a potent approach for treatment of diseases as well as for the study of neuronal cell biology. Here we investigated the capabilities of cationic amino acid based lipid assemblies to act as nonviral gene delivery vectors in primary cultured neurons. An arginine-based lipid, Arg-C3-Glu2C14, and a lysine-based lipid, Lys-C3-Glu2C14, with two different types of counterion, chloride ion (Cl-) and trifluoroacetic acid (TFA -), were shown to successfully mediate transfection of primary cultured neurons with plasmid DNA encoding green fluorescent protein. Among four types of lipids, we optimized their conditions such as the lipid-to-DNA ratio and the amount of pDNA and conducted a cytotoxicity assay at the same time. Overall, Arg-C3-Glu2C14 with TFA- induced a rate of transfection in primary cultured neurons higher than that of Lys-C 3-Glu2C14 using an optimal weight ratio of lipid-to-plasmid DNA of 1. Moreover, it was suggested that Arg-C 3-Glu2C14 with TFA- showed the optimized value higher than that of Lipofectamine2000 in experimental conditions. Thus, Arg-C3-Glu2C14 with TFA- is a promising candidate as a reliable transfection reagent for primary cultured neurons with a relatively low cytotoxicity. © 2013 American Chemical Society.

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  • A novel application of maleimide for advanced drug delivery: In vitro and in vivo evaluation of maleimide-modified pH-sensitive liposomes

    Tianshu Li, Shinji Takeoka

    International Journal of Nanomedicine   8   3855 - 3866  2013.10  [Refereed]

     View Summary

    Maleimide is a stable and easy-to-handle moiety that rapidly and covalently conjugates thiol groups of cysteine residues in proteins or peptides. Herein, we use maleimide to modify the surface of liposomes in order to obtain an advanced drug delivery system. Employing a small amount (0.3 mol%) of maleimide-polyethylene glycol (PEG) to modify the surface of the liposomes M-GGLG-liposomes, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate (GGLG)/cholesterol/poly(ethylene glycol) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG5000-DSPE)/maleimide-PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03, drug delivery efficiency was remarkably improved both in vitro and in vivo compared to unmodified liposomes (GGLG-liposomes, composed of GGLG/cholesterol/PEG5000-DSPE/PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03). Moreover, this modification did not elicit any detectable increase in cytotoxicity. The maleimide-modification did not alter the physical characteristics of the liposomes such as size, zeta potential, pH sensitivity, dispersibility and drug encapsulation efficiency. However, M-GGLG-liposomes were more rapidly (≥2-fold) internalized into HeLa, HCC1954, and MDA-MB-468 cells compared to GGLG-liposomes. In vivo, M-GGLG-liposomes encapsulating doxorubicin (M-GGLG-DOX-liposomes) also showed a more potent antitumor effect than GGLG-DOX-liposomes and the widely used 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-DOX-liposomes after two subcutaneous injections around breast cancer tissue in mice. The biodistribution of liposomes in this model was observed using an in vivo imaging system, which showed that M-GGLG-liposomes were present for significantly longer at the injection site compared to GGLG-liposomes. The outstanding biological functions of the maleimide-modified liposomes as a novel drug delivery system make them ideally suited to a wide range of applications. © 2013 Li and Takeoka.

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  • Application of nanosheets as an anti-adhesion barrier in partial hepatectomy

    Daisuke Niwa, Masatsugu Koide, Toshinori Fujie, Nobuhito Goda, Shinji Takeoka

    JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS   101 ( 7 ) 1251 - 1258  2013.10  [Refereed]

     View Summary

    Postoperative adhesion often causes serious adverse effects such as bowl obstruction, chronic abdominal pain, pelvic pain, and infertility. We previously reported that a poly-L-lactic acid (PLLA) nanosheet can efficiently seal a surgical incision without scarring. In this report, we examined whether the PLLA nanosheet can form an effective anti-adhesion barrier in partial hepatectomy accompanied by severe hemorrhaging in rats. To evaluate the anti-adhesive property of the nanosheet, the liver wound surface was covered with TachoComb((R)), a well-known hemostat material used in clinical procedures, and then with the PLLA nanosheet. Dressing the wound surface with TachoComb((R)) alone caused severe adhesion with omentum and/or residual parts of the liver. By contrast, combinational usage of TachoComb((R)) and the PLLA nanosheet significantly reduced such adhesion, presumably by inhibiting the permeation of oozing blood cells and the infiltration of fibroblastic cells. Moreover, the nanosheet displayed low permeability against serum proteins as well as cells in vitro, supporting the notion that the PLLA nanosheet has anti-adhesive properties in vivo. These results strongly suggested that the PLLA nanosheet is a promising material for reducing unwanted postoperative adhesion. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 101B: 1251-1258, 2013.

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  • Pharmacokinetic Study of the Structural Components of Adenosine Diphosphate-Encapsulated Liposomes Coated with Fibrinogen gamma-Chain Dodecapeptide as a Synthetic Platelet Substitute

    Kazuaki Taguchi, Hayato Ujihira, Shigeru Ogaki, Hiroshi Watanabe, Atsushi Fujiyama, Mami Doi, Yosuke Okamura, Shinji Takeoka, Yasuo Ikeda, Makoto Handa, Masaki Otagiri, Toru Maruyama

    DRUG METABOLISM AND DISPOSITION   41 ( 8 ) 1584 - 1591  2013.08  [Refereed]

     View Summary

    Fibrinogen gamma-chain (dodecapeptide HHLGGAKQAGDV, H12)-coated, ADP-encapsulated liposomes [H12-(ADP)-liposomes] were developed as a synthetic platelet alternative that specifically accumulates at bleeding sites as the result of interactions with activated platelets via glycoprotein IIb/IIIa and augments platelet aggregation by releasing ADP. The aim of this study is to characterize the pharmacokinetic properties of H12-(ADP)-liposomes and structural components in rats, and to predict the blood retention of H12(ADP)-liposomes in humans. With use of H12-(ADP)-liposomes in which the encapsulated ADP and liposomal membrane cholesterol were radiolabeled with C-14 and H-3, respectively, it was found that the time courses for the plasma concentration curves of C-14 and H-3 radioactivity showed that the H12-(ADP)-liposomes remained intact in the blood circulation for up to 24 hours after injection, and were mainly distributed to the liver and spleen. However, the C-14 and H-3 radioactivity of H12-(ADP)-liposomes disappeared from organs within 7 days after injection. The encapsulated ADP was metabolized to allantoin, which is the final metabolite of ADP in rodents, and was mainly eliminated in the urine, whereas the cholesterol was mainly eliminated in feces. In addition, the half-life of the H12-(ADP)-liposomes in humans was predicted to be approximately 96 hours from pharmacokinetic data obtained for mice, rats, and rabbits using an allometric equation. These results suggest that the H12-(ADP)-liposome has potential with proper pharmacokinetic and acceptable biodegradable properties as a synthetic platelet substitute.

    DOI PubMed

  • Development of Latanoprost-Loaded Biodegradable Nanosheet as a New Drug Delivery System for Glaucoma

    Kenji Kashiwagi, Keisuke Ito, Hiroki Haniuda, Shinya Ohtsubo, Shinji Takeoka

    INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE   54 ( 8 ) 5629 - 5637  2013.08  [Refereed]

     View Summary

    PURPOSE. We investigated the IOP reduction and safety of latanoprost-loaded biodegradable nanosheet (LBNS) as a new antiglaucoma drug delivery system (DDS).
    METHODS. We fabricated a 40 nm thick multilayered biodegradable nanosheet that is composed of chitosan and sodium alginate by means of the layer-by-layer method. Latanoprost isopropyl ester was loaded on the nanosheet to prepare 25, 2.5, and 0.25 mu g/cm(2) LBNSs. A nanosheet without latanoprost isopropyl ester (NS) and 0.005% latanoprost ophthalmic solution were prepared as controls. LBNSs or NS was applied to rat cornea, and IOP was monitored for 9 days. Local adverse effects and eye scratching movement also were investigated. The amount of latanoprost acid in aqueous humor and was measured in rabbits.
    RESULTS. The 0.25 mu g/cm(2) LBNS and 0.005% latanoprost ophthalmic solution showed significant IOP reduction only for 1 day after application, whereas the IOP reduction rates of 2.5 mu g/cm(2) LBNS at 1, 2, 4, 7, and 9 days after application were -27.0% +/- 14.8%, -22.0% +/- 16.7%, -25.8% +/- 18.0%, -22.7% +/- 20.9%, and -6.6% +/- 17.0%, respectively. The 25 mu g/cm(2) LBNS reduced IOP in a similar manner. The 25 mu g/cm(2) LBNS induced transient hyperemia, whereas the 0.25 and 2.5 mu g/cm(2) LBNSs did not exert any local adverse effects. The eye scratching movement test showed that application of 25 mu g/cm(2) LBNS did not cause any irritation of the eye. Latanoprost acid was detected in aqueous humor up to 6 days after application of 2.5 mu g/cm(2) LBNS.
    CONCLUSIONS. LBNS may be used as a novel antiglaucoma DDS.

    DOI PubMed

  • Effective control of massive venous bleeding by "multioverlapping therapy" using polysaccharide nanosheets in a rabbit inferior vena cava injury model.

    Kohsuke Hagisawa, Akihiro Saito, Manabu Kinoshita, Toshinori Fujie, Naoki Otani, Satoshi Shono, Young-Kwang Park, Shinji Takeoka

    Journal of vascular surgery. Venous and lymphatic disorders   1 ( 3 ) 289 - 97  2013.07  [Refereed]  [International journal]

     View Summary

    OBJECTIVE: To investigate the efficacy of multioverlapping therapy using a polysaccharide nanosheet having 75-nm thickness for sealing and stopping massive venous hemorrhage. METHODS: The hydrostatic durability of the polysaccharide nanosheet was evaluated in vitro when secured to an incised silicon tube. For in vivo studies, the inferior vena cava (IVC) of rabbits was cut longitudinally, and multiple polysaccharide nanosheets were overlapped onto the injured IVC. RESULTS: The mechanical hydrostatic durability of the nanosheets was gradually augmented by an increasing number of multilayered nanosheets in vitro. This durability was saturated at 80 ± 6 mm Hg by four layers of nanosheets, which was robust enough to seal injured vessel walls of the large IVC. Multioverlapping therapy using nanosheets effectively sealed and stopped bleeding from the injured IVC in vivo. One month later, no inflammatory tissue response was observed around the nanosheet attachment sites of the IVC, while conventional suturing repair in control rabbits showed a severe inflammatory response around the sutured area. CONCLUSIONS: The multioverlapping therapy using the polysaccharide nanosheets will effectively stop massive venous bleeding without adverse effects in the immediate or chronic postoperative setting.

    DOI PubMed

  • [Development of nanoparticle for coagulant].

    Okamura Y, Takeoka S

    Nihon Jibiinkoka Gakkai kaiho   116 ( 6 ) 673 - 678  2013.06  [Refereed]

    DOI PubMed

  • Arginine-based cationic liposomes for efficient in vitro plasmid DNA delivery with low cytotoxicity

    Satya Ranjan Sarker, Yumiko Aoshima, Ryosuke Hokama, Takafumi Inoue, Keitaro Sou, Shinji Takeoka

    International Journal of Nanomedicine   8   1361 - 1375  2013.04  [Refereed]

     View Summary

    Background: Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt) in the arginine head group. Methods: Cationic lipids were hydrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000. Results: We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt) that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular. Arginine-based cationic liposomes with HCl-salt showed the highest transfection efficiency in PC-12 cells. However, arginine-based cationic liposomes with TFA salt showed the highest transfection efficiency in HeLa cells, regardless of the presence of serum, with very low associated cytotoxicity. Conclusion: The gene delivery efficiency of amino acid-based cationic assemblies is influenced by the amino acids (ie, arginine or lysine) present as the hydrophilic head group and their associated counterions. © 2013 Sarker et al, publisher and licensee Dove Medical Press Ltd.

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  • Tapping-mode AFM study of tip-induced polymer deformation under geometrical confinement

    Hong Zhang, Yukio Honda, Shinji Takeoka

    Langmuir   29 ( 5 ) 1333 - 1339  2013.02  [Refereed]

     View Summary

    The morphological stability of polymer films is critically important to their application as functional materials. The deformation of polymer surfaces on the nanoscale may be significantly influenced by geometrical confinement. Herein, we constructed a mechanically heterogeneous polymer surface by phase separation in a thin polymer film and investigated the deformation behavior of its nanostructure (∼30 nm thickness and ∼100 nm average diameter) with tapping-mode atomic force microscopy. By changing different scan parameters, we could induce deformation localized to the nanostructure in a controllable manner. A quantity called the deformation index is defined and shown to be correlated to energy dissipation by tip-sample interaction. We clarified that the plastic deformation of a polymer on the nanoscale is energy-dependent and is related to the glass-to-rubber transition. The mobility of polymer chains beneath the tapping tip is enhanced, and in the corresponding region a rubberlike deformation with the lateral motion of the tip is performed. The method we developed can provide insight into the geometrical confinement effects on polymer behavior. © 2013 American Chemical Society.

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  • Application of poly-l-lactic acid nanosheet as a material for wound dressing

    Shimpo Aoki, Manabu Kinoshita, Hiromi Miyazaki, Akihiro Saito, Toshinori Fujie, Keiichi Iwaya, Shinji Takeoka, Daizoh Saitoh

    Plastic and Reconstructive Surgery   131 ( 2 ) 236 - 240  2013.02  [Refereed]

     View Summary

    The authors evaluated the efficacy of an ultrathin nanosheet consisting of poly-L-lactic acid (75 nm thick) as a wound dressing material. A full-thickness skin defect was made on the backs of mice and overlapped with or without the poly-L-lactic acid nanosheet. Wound healing was more rapidly improved by overlapping with the nanosheet, especially in the early healing period (at 4 to 6 days). The remaining wound area in the treatment group was significantly smaller at 4 days than in the control group. Histologically, a clear layer was observed over the granulation layer by the nanosheet therapy at 4 days. Thus, overlapping therapy with the poly-L-lactic acid nanosheet accelerated wound healing and formed a clear layer just above the granulation tissue. The poly-L-lactic acid nanosheet may have potential as a novel wound dressing to promote wound healing. Copyright © 2013 by the American Society of Plastic Surgeons.

    DOI PubMed

  • Novel therapeutic use of polysaccharide nanosheets for arachnoid plasty and enhancement of venous tensile strength in rat microneurosurgery.

    Otani N, Kinoshita M, Fujie T, Saito A, Takeoka S, Saitoh D, Hagisawa K, Nawashiro H, Shima K

    Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia   20 ( 2 ) 301 - 305  2013.02  [Refereed]

    DOI PubMed

  • Fragmentation of Poly(lactic acid) Nanosheets and Patchwork Treatment for Burn Wounds

    Yosuke Okamura, Koki Kabata, Manabu Kinoshita, Hiromi Miyazaki, Akihiro Saito, Toshinori Fujie, Shinya Ohtsubo, Daizoh Saitoh, Shinji Takeoka

    ADVANCED MATERIALS   25 ( 4 ) 545 - 551  2013.01  [Refereed]

     View Summary

    Freestanding poly(L-lactic acid) (PLLA) nanosheets are mass-produced by a simple combination of a spin-coating-assisted multi-layering process and a peeling technique. The resulting PLLA nanosheets are fragmented by homogenization and then reconstructed into a "patchwork" sheet on various surfaces without any adhesive reagents. The patchwork is shown to offer excellent protection against burn wound infection with Pseudomonas aeruginosa, and may therefore be an alternative to conventional burn therapy for prevention of infection.

    DOI PubMed

  • Selective Molecular Permeability Induced by Glass Transition Dynamics of Semicrystalline Polymer Ultrathin Films

    Toshinori Fujie, Yuko Kawamoto, Hiroki Haniuda, Akihiro Saito, Koki Kabata, Yukio Honda, Eriko Ohmori, Toru Asahi, Shinji Takeoka

    MACROMOLECULES   46 ( 2 ) 395 - 402  2013.01

     View Summary

    Most polymers solidify below a glass transition temperature (;), which is important for the fabrication of polymeric materials. The glass transition dynamics (GTD) of polymers alters their physical properties and therefore the range of applications suitable for the particular materials. In this regard, most GTD studies were oriented to the thermodynamics of amorphous polymer systems, while little studies were known for semicrystalline polymers. Here, we focus on the glassy and crystalline properties of semicrystalline polymers such as poly(L-lactic acid) (PLLA) and envisage to control the nanostructure of free-standing PLLA ultrathin films (referred as "PLLA nanosheets"), via thermodynamic rearrangement of polymer chains entangled in a quasi-two-dimensional interface during the GTD process. The annealing process on the PLLA nanosheets (<100 nm thick) resulted in the formation of semicrystalline domains and microscopic apertures with polymer chains (similar to 100 nm in size). Such nanostructure surprisingly induced selective molecular permeability, which was controlled as a function of film thickness and inherent crystallinity. The present methodology demonstrates the direct conversion of thermodynamic properties of semicrystalline polymers into the functional nanostructured polymeric materials.

    DOI

  • Intracellular click reaction with a fluorescent chemical Ca2+ indicator to prolong its cytosolic retention

    Yoshiaki Takei, Atsushi Murata, Kento Yamagishi, Satoshi Arai, Hideki Nakamura, Takafumi Inoue, Shinji Takeoka

    CHEMICAL COMMUNICATIONS   49 ( 66 ) 7313 - 7315  2013  [Refereed]

     View Summary

    The powerful strategy of "intracellular click reaction" was used to retain a chemical Ca2+ indicator in the cytosol. Specifically, a novel clickable Ca2+ indicator "N-3-fura-2 AM" was coupled with dibenzylcyclooctyl-modified biomacromolecules via copper-free click reaction in living cells and Ca2+ oscillation was observed for an extended period of time.

    DOI PubMed

  • Pharmacokinetic Study of Adenosine Diphosphate-Encapsulated Liposomes Coated with Fibrinogen γ-Chain Dodecapeptide as a Synthetic Platelet Substitute in an Anticancer Drug-Induced Thrombocytopenia Rat Model

    Kazuaki Taguchi, Hayato Ujihira, Hiroshi Watanabe, Atsushi Fujiyama, Mami Doi, Shinji Takeoka, Yasuo Ikeda, Makoto Handa, Masaki Otagiri, Toru Maruyama

    Journal of Pharmaceutical Sciences   102 ( 10 ) 3852 - 3859  2013  [Refereed]

     View Summary

    A fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV, H12)-coated, adenosine diphosphate (ADP)-encapsulated liposome [H12-(ADP)-liposome] was designed to achieve optimal performance as a homeostatic agent and expected as a synthetic platelet alternative. For the purpose of efficient function as platelet substitute, H12-(ADP)-liposomes should potentially have both acceptable pharmacokinetic and biodegradable properties under conditions of an adaptation disease including thrombocytopenia induced by anticancer drugs. The aim of this study was to characterize the pharmacokinetics of H12-(ADP)-liposomes in busulphan-induced thrombocytopenic rats using 14C, 3H double radiolabeled H12-(ADP)-liposomes, in which the encapsulated ADP and liposomal membrane (cholesterol) were labeled with 14C and 3H, respectively. After the administration of H12-(ADP)-liposomes, they were determined to be mainly distributed to the liver and spleen and disappeared from organs within 7 days after injection. The encapsulated ADP was mainly eliminated in the urine, whereas the outer membrane (cholesterol) was mainly eliminated in feces. The successive dispositions of the H12-(ADP)-liposomes were similar in both normal and thrombocytopenic rats. However, the kinetics of H12-(ADP)-liposomes in thrombocytopenic rats was more rapid, compared with the corresponding values for normal rats. These findings, which well reflect the clinical features of patients with anticancer drug-induced thrombocytopenia, provide useful information for the development of the H12-(ADP)-liposomes for future clinical use. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3852-3859, 2013.

    DOI PubMed

  • Novel therapeutic use of polysaccharide nanosheets for arachnoid plasty and enhancement of venous tensile strength in rat microneurosurgery

    Naoki Otani, Manabu Kinoshita, Toshinori Fujie, Akihiro Saito, Shinji Takeoka, Daizoh Saitoh, Kohsuke Hagisawa, Hiroshi Nawashiro, Katsuji Shima

    Journal of Clinical Neuroscience Corresponding   20 ( 2 ) 301 - 305  2012.12

    DOI

  • Ability of fibrinogen gamma-derived dodecapeptides with different sequences to bind to rat platelets

    Koji Tokutomi, Toshiaki Tagawa, Maki Korenaga, Masatoshi Chiba, Tomohiro Asai, Naohide Watanabe, Shinji Takeoka, Makoto Handa, Yasuo Ikeda, Naoto Oku

    INTERNATIONAL JOURNAL OF PHARMACEUTICS   438 ( 1-2 ) 296 - 301  2012.11  [Refereed]

     View Summary

    A dodecapeptide (gamma 400-411) derived from a fibrinogen gamma-chain carboxyl-terminal sequence recognizes specifically the active form of GPIIb/IIIa on the surface of activated platelets. For the purpose of efficient hemostasis, we previously developed ADP-encapsulated liposomes modified with human-dodecapeptide (HHLGGAKQAGDV, human-H12). On the other hand, the amino-acid sequence of H12 from rats is HHMG-GSKQVGDM, having only 67% homology to that from humans. Here, we investigated the ability of rat-H12 in comparison with human-H12 to bind to platelets. Firstly, rat platelets were activated with phorbol-12-myristate-13-acetate (PMA), and the activation was confirmed by flow cytometry. Next, we evaluated the dissociation constant (K-d) of human-H12 and rat-H12 for dissociation from rat platelets by using FACS. As a result, the K-d of human-H12 and rat-H12 with respect to rat platelets was 2.78 +/- 0.21 and 2.91 +/- 0.22 mu M, respectively. Furthermore, H12 from both species inhibited quite similarly the aggregation of rat platelets in platelet-rich plasma (PRP). These results suggest that H12 from different species with different amino acid sequences interacts similarly with GPIIb/IIIa on platelets. (C) 2012 Published by Elsevier B.V.

    DOI PubMed

  • Fibrinogen gamma-chain peptide-coated, ADP-encapsulated liposomes rescue thrombocytopenic rabbits from non-compressible liver hemorrhage

    K. Nishikawa, K. Hagisawa, M. Kinoshita, S. Shono, S. Katsuno, M. Doi, R. Yanagawa, H. Suzuki, K. Iwaya, D. Saitoh, T. Sakamoto, S. Seki, S. Takeoka, M. Handa

    JOURNAL OF THROMBOSIS AND HAEMOSTASIS   10 ( 10 ) 2137 - 2148  2012.10

     View Summary

    . Background: We developed a fibrinogen ?-chain (dodecapeptide HHLGGAKQAGDV [H12])-coated, ADP-encapsulated liposome (H12-[ADP]-liposome) that accumulates at bleeding sites via interaction with activated platelets via glycoprotein IIbIIIa and augments platelet aggregation by releasing ADP. Objective: To evaluate the efficacy of H12-(ADP)-liposomes for treating liver hemorrhage in rabbits with acute thrombocytopenia. Methods: Thrombocytopenia (platelets < 50 000 mu L-1) was induced in rabbits by repeated blood withdrawal (100 mL kg-1 in total) and isovolemic transfusion of autologous washed red blood cells. H12-(ADP)-liposomes with platelet-poor plasma (PPP), platelet-rich plasma (PRP), PPP, ADP liposomes with PPP or H12-(PBS)-liposomes/PPP, were administered to the thrombocytopenic rabbits, and liver hemorrhage was induced by penetrating liver injury. Results: Administration of H12-(ADP)-liposomes and of PRP rescued all thrombocytopenic rabbits from liver hemorrhage as a result of potent hemostasis at the liver bleeding site, although rabbits receiving PPP or ADP liposomes showed 20% survival in the first 24 h. Administration of H12-(ADP)-liposomes and of PRP suppressed both bleeding volume and time from the site of liver injury. H12-(phosphate-buffered saline)-liposomes lacking ADP also improved rabbit survival after liver hemorrhage, although their hemostatic effect was weaker. In rabbits with severe thrombocytopenia (25 000 platelets mu L-1), the hemostatic effects of H12-(ADP)-liposomes tended to be attenuated as compared with those of PRP treatment. Histologic examination revealed that H12-(ADP)-liposomes accumulated at the bleeding site in the liver. Notably, neither macrothombi nor microthrombi were detected in the lung, kidney or liver in rabbits treated with H12-(ADP)-liposomes. Conclusions: H12-(ADP)-liposomes appear to be a safe and effective therapeutic tool for acute thrombocytopenic trauma patients with massive bleeding.

    DOI

  • Coordinative Nanoporous Polymers Synthesized with Hydrogen-Bonded Columnar Liquid Crystals

    Shinsuke Ishihara, Yusuke Furuki, Jonathan P. Hill, Katsuhiko Ariga, Shinji Takeoka

    JOURNAL OF NANOSCIENCE AND NANOTECHNOLOGY   12 ( 10 ) 7885 - 7895  2012.10  [Refereed]

     View Summary

    In this paper, we report the development of nanoporous polymer which demonstrates the coordination property toward zinc porphyrin. A hydrogen-bonded columnar liquid crystalline precursor composed of a triphenylene template and three equivalent of the surrounding dendric amphiphile bearing a pyridyl head group and a polymerizable aliphatic chain, was covalently fixed by photo-polymerization, and then the subsequent selective removal of the template successively resulted in a nanoporous polymer in which the pore wall is modified with pyridyl groups. The nanoporous polymer reflected the conformation of template, and displayed considerable coordination ability of the pyridyl groups towards zinc porphyrin. The coordinative nanoporous polymer is promising as a nano-scaled scaffold for the organization of dyes into functional supramolecular architectures.

    DOI PubMed

  • Heterofunctional nanosheet controlling cell adhesion properties by collagen coating

    Daisuke Niwa, Toshinori Fujie, Thorsten Lang, Nobuhito Goda, Shinji Takeoka

    JOURNAL OF BIOMATERIALS APPLICATIONS   27 ( 2 ) 131 - 141  2012.08

     View Summary

    Recently, biomaterials have been widely used in a variety of medical applications. We previously reported that a poly-l-lactic acid (PLLA) nanosheet shows anti-adhesive properties and constitutes a useful biomaterial for preventing unwanted wound adhesion in surgical operations. In this article, we examine whether the PLLA nanosheet can be specifically modified with biomacromolecules on one surface only. Such an approach would endow each side of the nanosheet with discrete functions, that is anti-adhesive and pro-healing properties. We fabricated two distinct PLLA nanosheets: (i) collagen cast on the surface of a PLLA nanosheet (Col-Cast-PLLA) and (ii) collagen spin-coated on the nanosheet (Col-Spin-PLLA). In the Col-Spin-PLLA nanosheet, the collagen layer had a thickness of 5-10 nm on the PLLA surface and displayed increased hydrophilicity compared to both PLLA and Col-Cast-PLLA nanosheets. In addition, atomic force microscopy showed disorganized collagen fibril formation on the PLLA layer when covered using the spin-coating method, while apparent bundle formations of collagen were formed in the Col-Cast-PLLA nanosheet. The Col-Spin-PLLA nanosheet provided a microenvironment for cells to adhere and spread. By contrast, the Col-Cast-PLLA nanosheet displayed reduced cell adhesion compared to the Col-Spin-PLLA nanosheet. Consistent with these findings, immunocytochemical analysis clearly showed fine networks of actin filaments in cells cultured on the Col-Spin-PLLA, but not the Col-Cast-PLLA nanosheet. Therefore, the Col-Spin-PLLA nanosheet was shown to be more suitable for acting as a scaffold. In conclusion, we have succeeded in developing a heterofunctional nanosheet comprising a collagen modified side, which has the ability to rapidly adhere cells, and an unmodified side, which acts as an adhesion barrier, by using a spin-coating technique.

    DOI

  • Therapeutic efficacy of an antibiotic-loaded nanosheet in a murine burn-wound infection model

    Akihiro Saito, Hiromi Miyazaki, Toshinori Fujie, Shinya Ohtsubo, Manabu Kinoshita, Daizoh Saitoh, Shinji Takeoka

    ACTA BIOMATERIALIA   8 ( 8 ) 2932 - 2940  2012.08  [Refereed]

     View Summary

    Polymeric ultra-thin films (nanosheets) possess unique properties that make them suitable materials for various biomedical applications. In our previous study, we assessed the use of an antibiotic (tetracycline, TC)-loaded nanosheet (or "TC-nanosheet") for the treatment of gastrointestinal tissue defects. The nanosheet consisted of three functional layers: layer-by-layer nanosheet as a stable platform, TC as an antimicrobial agent with autofluorescence for tracing, and a poly(vinyl acetate) nanosheet to act as a protecting layer. The TC-nanosheet has high flexibility, adhesive strength and transparency. Here, we evaluated the effectiveness of the TC-nanosheet in preventing full thickness burn-wound infections. In an in vivo study, murine dorsal skin was injured by full-thickness burns and then infected with Pseudomonas aeruginosa (P. aeruginosa), a common bacterium causing burn-associated infections. The wound site was treated either with a TC-nanosheet, TC-unloaded nanosheet or left untreated. Wound management was facilitated by the high transparency of the TC-nanosheet. The TC-nanosheet significantly improved burn-wound infection by P. aeruginosa in mice. Indeed, all mice treated with the TC-nanosheet survived, whereas the other treatment groups displayed increased rates of mortality due to bacterial infection. According to histological analyses and viable bacterial counting in the liver (bacterial translocation), the TC-nanosheets were able to prevent not only the local inflammation but also systemic inflammation. We conclude that the TC-nanosheet can act as an effective treatment for full-thickness burn-wound infection. Hence, the TC-nanosheet is a promising therapeutic tool for burn-wound management in severely burn-injured patients. (c) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

    DOI PubMed

  • Mass Spectrometric Screening of Ligands with Lower Off-Rate from a Clicked-Based Pooled Library

    Satoshi Arai, Shota Hirosawa, Yusuke Oguchi, Madoka Suzuki, Atsushi Murata, Shin'ichi Ishiwata, Shinji Takeoka

    ACS COMBINATORIAL SCIENCE   14 ( 8 ) 451 - 455  2012.08  [Refereed]

     View Summary

    This paper describes a convenient screening method using ion trap electrospray ionization mass spectrometry to classify ligands to a target molecule in terms of kinetic parameters. We demonstrate this method in the screening of ligands to a hexahistidine tag from a pooled library synthesized by click chemistry. The ion trap mass spectrometry analysis revealed that higher stabilities of ligand-target complexes in; the gas phase were related to lower dissociation rate constants, i.e., off-rates in solution. Finally, we prepared a fluorescent probe utilizing the ligand with lowest off-rate and succeeded in performing single molecule observations of hexahistidine-tagged myosin V walking on actin filaments.

    DOI PubMed

  • An ultrathin poly(L-lactic acid) nanosheet as a burn wound dressing for protection against bacterial infection

    Hiromi Miyazaki, Manabu Kinoshita, Akihiro Saito, Toshinori Fujie, Koki Kabata, Etsuko Hara, Satoshi Ono, Shinji Takeoka, Daizoh Saitoh

    WOUND REPAIR AND REGENERATION   20 ( 4 ) 573 - 579  2012.07  [Refereed]

     View Summary

    Burn wounds are highly susceptible to bacterial infection due to impairment of the skin's integrity. Therefore, prevention of bacterial colonization/infection in the wound is crucial for the management of burns, including partial-thickness burn injuries. Although partial-thickness burn injuries still retain the potential for reepithelialization, the complication of wound infection severely impairs the reepithelialization even in such superficial burn injuries. We recently developed a biocompatible nanosheet consisting of poly(l-lactic acid) (PLLA). The PLLA nanosheets have many useful and advantageous biological properties for their application as a wound dressing, such as sufficient flexibility, transparency, and adhesiveness. We herein investigated the suitability of the PLLA nanosheets as a wound dressing for partial-thickness burn wounds in mice. The PLLA nanosheets tightly adhered to the wound without any adhesive agents. Although wound infection with Pseudomonas aeruginosa in the controls significantly impaired reepithelialization of burn wounds, dressing with the PLLA nanosheet markedly protected against bacterial wound infection, thereby improving wound healing in the mice receiving partial-thickness burn injuries. The PLLA nanosheet also showed a potent barrier ability for protecting against bacterial penetration in vitro. The ultrathin PLLA nanosheet may be applied as a protective dressing to reduce environmental contamination of bacteria in a partial-thickness burn wound.

    DOI PubMed

  • Morphological Evolution within Spin-Cast Ultrathin Polymer Blend Films Clarified by a Freestanding Method

    Hong Zhang, Shinji Takeoka

    MACROMOLECULES   45 ( 10 ) 4315 - 4321  2012.05

     View Summary

    Polymer phase separation has established a series of bottom-up nanofabrication methods. Owing to the intrinsic immiscibility of most polymer blends, phase separation is typically produced by rapid quenching during spin-casting. However, the full sequence of events that occur during this process is still unclear, especially in the case of ultrathin polymer blend films. Herein, a freestanding method is first introduced to obtain morphological information from the bottom side of the film. We demonstrate that when the thickness of ultrathin film is comparable to the dimensional scale of the phase separation domains, it is feasible to prepare perforated films with uniform nanopores via selective solvent etching. Our results also provide direct evidence that the spinodal decomposition mechanism plays an important role in determining the final morphology within the ultrathin polymer blend films. These findings are of practical value in the fabrication of desired nanostructures by polymer phase separation.

    DOI

  • Evaluation of the influence of ionization states and spacers in the thermotropic phase behaviour of amino acid-based cationic lipids and the transfection efficiency of their assemblies

    Satya Ranjan Sarker, Satoshi Arai, Motohide Murate, Hiroshi Takahashi, Masaki Takata, Toshihide Kobayashi, Shinji Takeoka

    International Journal of Pharmaceutics   422 ( 1-2 ) 364 - 373  2012.01

     View Summary

    The influence of both the ionization states and the hydrocarbon chain spacer of a series of amino acid-based cationic lipids was evaluated in terms of gene delivery efficiency and cytotoxicity to the COS-7 cell line and compared with that of Lipofectamine™ 2000. We synthesized a series of amino acid-based cationic lipids with different ionization states (i.e., -NH 2, -NH 3+Cl - or -NH 3+TFA -) in the lysine head group and different hydrocarbon chain spacers (i.e., 0, 3, 5 or 7 carbon atoms) between the hydrophilic head group and hydrophobic moieties. In the 3-carbon series, the cationic assemblies formed a micellar structure in the presence of -NH 3+Cl - and a vesicular structure both in the presence of -NH 2 and -NH 3+TFA -. Differential scanning calorimetry (DSC) data revealed a significantly lower (8.1°C) gel-to-liquid crystalline phase transition temperature for cationic assemblies bearing -NH 3+TFA - when compared to their -NH 2 counterparts. Furthermore, the zeta potential of cationic assemblies having -NH 3+TFA - in the hydrophilic head group was maximum followed by -NH 3+Cl - and -NH 2 irrespective of their hydrocarbon chain spacer length. The gene delivery efficiency in relation to the ionization states of the hydrophilic head group was as follows: -NH 3+TFA - > -NH 3+Cl - > -NH 2. © 2011 Elsevier B.V.

    DOI PubMed

  • Walking nanothermometers: spatiotemporal temperature measurement of transported acidic organelles in single living cells

    Kotaro Oyama, Masao Takabayashi, Yoshiaki Takei, Satoshi Arai, Shinji Takeoka, Shin'ichi Ishiwata, Madoka Suzuki

    LAB ON A CHIP   12 ( 9 ) 1591 - 1593  2012

     View Summary

    We fabricated fluorescent nanoparticles which monitor temperature changes without sensitivity to pH (4-10) and ionic strength (0-500 mM). The nanothermometers spontaneously enter living HeLa cells via endocytosis, enclosed in acidic organelles, i.e., endosome/lysosome, and then transported along microtubules in a temperature-dependent manner, working as "walking nanothermometers".

    DOI

  • A TEMPO-conjugated fluorescent probe for monitoring mitochondrial redox reactions

    Shota Hirosawa, Satoshi Arai, Shinji Takeoka

    CHEMICAL COMMUNICATIONS   48 ( 40 ) 4845 - 4847  2012  [Refereed]

     View Summary

    We report a mitochondrial targeted redox probe (MitoRP) that comprises a nitroxide radical (TEMPO) moiety and coumarin 343. Using isolated mitochondria in the presence/absence of substrates and inhibitors of oxidative phosphorylation, we demonstrated that MitoRP is a useful probe to monitor the electron flow associated with complex I.

    DOI PubMed

  • 高分子系ナノシートの外科手術用創傷被覆材、癒着防止剤としての可能性

    武岡 真司, 藤枝 俊宣

    MATERIAL STAGE   12   16 - 19  2012

  • 同種皮膚マイクロスキングラフティングによる新規熱傷治療法の基礎研究

    宮﨑裕美, 永野穂高, 野原良介, 木下 学, 齋藤晃広, 小野 聡, 武岡真司, 齋藤大蔵

    熱傷   38 ( 1 ) 31 - 38  2012

  • 生体投与可能なナノ構造体の構築と血小板代替物への応用

    岡村陽介, 武岡真司

    人工血液   20 ( 2 ) 38 - 47  2012

  • Ultrastructural analysis of thrombin-induced interaction between human platelets and liposomes carrying fibrinogen gamma-chain dodecapeptide as a synthetic platelet substitute

    Hidenori Suzuki, Yosuke Okamura, Yasuo Ikeda, Shinji Takeoka, Makoto Handa

    THROMBOSIS RESEARCH   128 ( 6 ) 552 - 559  2011.12

     View Summary

    Background: The dodecapeptide HHLGGAKQAGDV (H12) in the carboxy-terminus of the fibrinogen.-chain is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated liposomes carrying fibrinogen.-chain dodecapeptide as a synthetic platelet substitute. Objectives: We examined the interaction between human platelets and H12-liposomes during thrombin-induced activation using flow cytometry and electron microscopy (EM).
    Methods and results: After thrombin-activation, a remarkable time-dependent increase in binding of the H12-liposomes to platelets was found by flow cytometry. A large-sized swollen open canalicular system (OCS) was observed in the spheroidal platelets from 60 sec to 5 min after thrombin-activation, but intact H12-liposomes were not evident by conventional EM. Cryoultramicrotomy and immunogold staining with anti-H12 antibody were successful in identifying the liposomes; they appeared as small particles with a unit membrane around 0.2 to 0.4 mu m in diameter, and gold labels representing H12 were distributed homogeneously on the surface. Abundant H12-liposomes were localized not only on the surface membrane but also in the lumen of the large sized swollen OCS in the platelets at 60 sec after thrombin-activation. The formation of the large-sized swollen OCS was inhibited by pre-incubation with unbound H12, EDTA or anti-GPIIb/IIIa antibody. In thrombin-induced platelet aggregates we observed electron-transparent areas between adherent platelets, in which abundant H12-liposomes were distributed.
    Conclusions: We demonstrate morphologically that H12-liposomes bind to thrombin-activated platelets and accumulate between adherent platelets like fibrinogen, leading to large-scale aggregation. (C) 2011 Elsevier Ltd. All rights reserved.

    DOI

  • Novel technique of overlaying a poly-l-lactic acid nanosheet for adhesion prophylaxis and fixation of intraperitoneal onlay polypropylene mesh in a rabbit model

    Keiichi Fujino, Manabu Kinoshita, Akihiro Saitoh, Hidekazu Yano, Kahoko Nishikawa, Toshinori Fujie, Keiichi Iwaya, Minoru Kakihara, Shinji Takeoka, Daizoh Saitoh, Yuji Tanaka

    SURGICAL ENDOSCOPY AND OTHER INTERVENTIONAL TECHNIQUES   25 ( 10 ) 3428 - 3436  2011.10

     View Summary

    Background One problem with polypropylene mesh (PPM) used to repair abdominal wall hernias is dense adhesions to the visceral surface. The authors developed the biocompatible poly-l-lactic acid (PLLA) nanosheet (thickness < 100 nm), which has the unique ability to adhere tightly to tissues but not to opposing tissues. This study investigated the antiadhesive and fixative characteristics of the PLLA nanosheet after placement of intraperitoneal onlay PPM (IPOM) overlaid with a PLLA nanosheet on intact peritoneum.
    Methods The PLLA nanosheet was fabricated by the spin-coating method and peeling technique with polyvinyl alcohol (PVA) as a supporting film. Two 1.5-cm-square pieces of mesh were implanted on each peritoneal side of the midline incision. The mesh was fixed to the peritoneum with a suture and then overlaid with a 4-cm-square piece of Seprafilm or nanosheet. To examine the fixative property, mesh was overlaid with Seprafilm or nanosheet without a fixed suture. After 4 weeks, mesh adhesion, inflammatory reaction, fixation, and dislocation of mesh were evaluated.
    Results Nanosheet-overlaid meshes were flexible and fit over the peritoneum. Adhesion was observed in 10% of the nanosheet-overlaid meshes and in 50% of the Seprafilm-overlaid meshes. The adhesion tenacity grade was significantly lower with the nanosheet-overlaid meshes (0.1 +/- A 0.1) than with the Seprafilm-overlaid meshes (1.0 +/- A 0.4) (p = 0.029), and the percentage of the adhesion area also was lower with the nanosheet-overlaid meshes (1.0 +/- A 1.0% vs 8.5 +/- A 3.2%; p = 0.037). The mean inflammatory cell counts were lower with the nanosheet-overlaid meshes (p = 0.0023). Regarding the fixative property, 37.5% of the nanosheet-overlaid meshes were fixated on the peritoneum, but no Seprafilm-overlaid mesh was fixated.
    Conclusions Overlaying of a PLLA nanosheet was effective for adhesion prophylaxis of intraperitoneal mesh. It also may have a possible beneficial effect on fixation of mesh.

    DOI

  • Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ)

    Takuji Nabetani, Asami Makino, Francoise Hullin-Matsuda, Taka-aki Hirakawa, Shinji Takeoka, Nozomu Okino, Makoto Ito, Toshihide Kobayashi, Yoshio Hirabayashi

    JOURNAL OF LIPID RESEARCH   52 ( 6 ) 1294 - 1302  2011.06

     View Summary

    Ceramides play a crucial role in divergent signaling events, including differentiation, senescence, proliferation, and apoptosis. Ceramides are a minor lipid component in terms of content; thus, highly sensitive detection is required for accurate quantification. The recently developed isobaric tags for relative and absolute quantitation (iTRAQ) method enables a precise comparison of both protein and aminophospholipids. However, iTRAQ tagging had not been applied to the determination of sphingolipids. Here we report a method for the simultaneous measurement of multiple ceramide and monohexosylceramide samples using iTRAQ tags. Samples were hydrolyzed with sphingolipid ceramide N-deacylase (SCDase) to expose the free amino group of the sphingolipids, to which the N-hydroxysuccinimide group of iTRAQ reagent was conjugated. The reaction was performed in the presence of a cleavable detergent, 3-[3-(1,1-bisalkyloxyethyl)pyridine-1-yl]propane-1-sulfonate (PPS) to both improve the hydrolysis and ensure the accuracy of the mass spectrometry analysis performed after iTRAQ labeling. This method was successfully applied to the profiling of ceramides and monohexosylceramides in sphingomyelinase-treated Madin Darby canine kidney (MDCK) cells and apoptotic Jurkat cells.-Nabetani, T., A. Makino, F. Hullin-Matsuda, T. Hirakawa, S. Takeoka, N. Okino, M. Ito, T. Kobayashi, and Y. Hirabayashi. Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ). J. Lipid Res. 2011. 52: 1294-1302.

    DOI

  • Aqueous Colloidal Mesoporous Nanoparticles with Ethenylene-Bridged Silsesquioxane Frameworks

    Chihiro Urata, Hironori Yamada, Ryutaro Wakabayashi, Yuko Aoyama, Shota Hirosawa, Satoshi Arai, Shinji Takeoka, Yusuke Yamauchi, Kazuyuki Kuroda

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   133 ( 21 ) 8102 - 8105  2011.06

     View Summary

    Aqueous colloidal mesoporous nanoparticles with ethenylene-bridged silsesquioxane frameworks with a uniform diameter of similar to 20 nm were prepared from bis(triethoxysilyl)ethenylene in a basic aqueous solution containing cationic surfactants. The nanopartides, which had higher hydrolysis resistance under aqueous conditions, showed lower hemolytic activity toward bovine red blood cells than colloidal mesoporous silica nanoparticles.

    DOI

  • Free-Standing Poly(L-lactic acid) Nanofilms Loaded with Superparamagnetic Nanoparticles

    Silvia Taccola, Andrea Desii, Virginia Pensabene, Toshinori Fujie, Akihiro Saito, Shinji Takeoka, Paolo Dario, Arianna Menciassi, Virgilio Mattoli

    LANGMUIR   27 ( 9 ) 5589 - 5595  2011.05

     View Summary

    Freely suspended nanocomposite thin films based on soft polymers and functional nanostructures have been widely investigated for their potential application as active elements in microdevices. However, most studies are focused on the preparation of nanofilms composed of polyelectrolytes and charged colloidal particles. Here, a new technique for the preparation of poly(L-lactic acid) free-standing nanofilms embeddidng superparamagnetic iron oxide nanoparticles is presented. The fabrication process, based on a spin-coating deposition approach, is described, and the influence of each production parameter on the morphology and magnetic properties of the final structure is investigated. Superparamagnetic free-standing nanofilms were obtained, as evidenced by a magnetization hysteresis measurement performed with a superconducting quantum interference device (SQUID). Nanofilm surface morphology and thickness were evaluated by atomic force microscopy (AFM), and the nanoparticle dispersion inside the composites was investigated by transmission electron microscopy (TEM). These nanofilms, composed of a biodegradable polyester and remotely controllable by external magnetic fields, are promising candidates for many potential applications in the biomedical field.

    DOI

  • A Few Immobilized Thrombins Are Sufficient for Platelet Spreading

    Yosuke Okamura, Roman Schmidt, Ines Raschke, Maik Hintze, Shinji Takeoka, Alexander Egner, Thorsten Lang

    BIOPHYSICAL JOURNAL   100 ( 8 ) 1855 - 1863  2011.04

     View Summary

    Eukaryotic cells respond to signaling molecules with picomolar to nanomolar sensitivities. However, molar concentrations give no suggestion of the sufficient number of molecules per cell and are confusing when referring to physiological situations in which signaling molecules act in an immobilized state. Here, we studied platelet adhesion by thrombin, a key step in normal hemostasis and pathological arterial thrombosis. We generated a biofunctional nanosheet surface to mimic the in vivo solid-state interaction between platelets and thrombin at sites of injured tissues. We observed that <10 molecules readily activate platelets with high specificity, resulting in platelet adhesion and spreading. This number is much lower than expected from previous experiments in solution, in which the sole activation of platelets required a >1000-fold stoichiometric excess of thrombin. We conclude that immobilizing thrombin apposed to the membrane receptor allows platelets to respond with very high sensitivity. Moreover, we propose that irreversible cell activation may require several ligands to avoid activation by single, mislocalized signaling molecules.

    DOI

  • Decoration of fibrinogen gamma-chain peptide on adenosine diphosphate-encapsulated liposomes enhances binding of the liposomes to activated platelets

    Koji Tokutomi, Toshiaki Tagawa, Maki Korenaga, Masatoshi Chiba, Tomohiro Asai, Naohide Watanabe, Shinji Takeoka, Makoto Handa, Yasuo Ikeda, Naoto Oku

    INTERNATIONAL JOURNAL OF PHARMACEUTICS   407 ( 1-2 ) 151 - 157  2011.04

     View Summary

    For the purpose of efficient hemostasis, we previously developed ADP-encapsulated liposomes modified with a dodecapeptide (HHLGGAKQAGDV, H12), H12-(ADP)Lipo. This liposome actually enhanced platelet aggregation in vitro, and showed significant hemostatic effect in vivo. Since fibrinogen (Fbg) is abundant in the bloodstream, it is unclear why this liposome binds platelets so efficiently, overcoming the competition with Fbg. Therefore, we investigated the relationship between H12 density on the liposome and the binding ability to platelets, and evaluated the inhibitory effect of Fbg on the binding of H12-(ADP)Lipo to platelets. As a result, the binding ability to platelets steeply increased depending on H12 density until it reached about 3 x 10(15) H12 molecules/m(2). The 50% inhibition concentration of Fbg on the binding of H12-(ADP)Lipo to platelets was about 25-fold over the concentration of H12 molecules on the liposome. Moreover, almost no inhibition by Fbg was observed at the physiological concentration of it. This result suggests that the ability of H12 to bind to GPIIb/IIIa increased overwhelmingly by the anchoring to the liposome that enabled the cooperative binding of H12 peptides to the platelets. (C) 2011 Elsevier B.V. All rights reserved.

    DOI

  • Chirality Sensing by Nonchiral Porphines

    Jan Labuta, Shinsuke Ishihara, Atsuomi Shundo, Satoshi Arai, Shinji Takeoka, Katsuhiko Ariga, Jonathan P. Hill

    CHEMISTRY-A EUROPEAN JOURNAL   17 ( 13 ) 3558 - 3561  2011.03

    DOI

  • Increased viscosity of hemoglobin-based oxygen carriers retards NO-binding when perfused through narrow gas-permeable tubes

    Hiromi Sakai, Naoto Okuda, Shinji Takeoka, Eishun Tsuchida

    MICROVASCULAR RESEARCH   81 ( 2 ) 169 - 176  2011.03

     View Summary

    Increased fluid viscosity of a solution of hemoglobin-based oxygen carriers (HBOCs) reduces vasoconstrictive effects because increased shear stress on the vascular wall enhances the production of vasorelaxation factors such as NO. Nevertheless, on a microcirculatory level, it remains unclear how viscosity affects the reaction of HBOCs and NO. In this study, different HBOCs were perfused through narrow gas-permeable tubes (25 mu m inner diameter at 1 mm/s centerline velocity; hemoglobin concentration [Hb] = 5 g/dL). The reaction was examined microscopically based on the Hb visible-light absorption spectrum. When immersed in a NO atmosphere, the NO-binding of deoxygenated Hb solution (viscosity, 1.1 cP at 1000 s(-1)) in the tube occurred about twice as rapidly as that of red blood cells (RBCs): 1.6 cP. Binding was reduced by PEGylation (PEG-Hb, 7.7 cP), by addition of a high molecular weight hydroxyethyl starch (HES) (2.8 cP), and by encapsulation to form Hb-vesicles (HbVs, 1.5 cP; particle size 279 nm). However, the reduction was not as great as that shown for RBCs. A mixture of HbVs and HES (6.2 cP) showed almost identical NO-binding to that of RBCs. Higher viscosity and particle size might reduce lateral diffusion when particles are flowing. The HbVs with HES showed the slowest NO-binding. Furthermore, Hb encapsulation and PEGylation, but not HES-addition, tended to retard CO-binding. Increased viscosity reportedly enhances production of endothelium NO. In addition, our results show that the increased viscosity also inhibits the reaction with NO. Each effect might mitigate vasoconstriction. (C) 2010 Elsevier Inc. All rights reserved.

    DOI

  • 高分子ナノシートの物性と医療展開

    齊藤晃広, 武岡真司

    未来材料   11 ( 3 )  2011.03

  • 分子集合科学による高分子超薄膜(ナノシート)の調製とナノ絆創膏としての医療展開

    武岡真司

    応用物理   80 ( 2 ) 133 - 136  2011.02

  • Fluorescent "Turn-on" system utilizing a quencher-conjugated peptide for specific protein labeling of living cells

    Satoshi Arai, Su-In Yoon, Atsushi Murata, Masao Takabayashi, Xiaoyu Wu, Yixin Lu, Shinji Takeoka, Miwako Ozaki

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   404 ( 1 ) 211 - 216  2011.01

     View Summary

    A specific protein fluorescent labeling method has been used as a tool for bio-imaging in living cells. We developed a novel system of switching "fluorescent turn on" by the recognition of a fluorescent probe to a hexahistidine-tagged (His-tag) protein. The tetramethyl rhodamine bearing three nitrilotriacetic acids, which was used as a fluorescent probe to target a His-tagged protein, formed a reversible complex with the quencher, (Dabcyl)-conjugated oligohistidines, in the homogeneous solution, causing fluorescence of the fluorophore to be quenched. The complex when applied to living cells (COS-7) expressing His-tagged proteins on the cell surface caused the quencher-conjugated oligohistidines to be dissociated from the complex by specific binding of the fluorescent probe to the tagged protein, resulting in the fluorescent emission. The complex that did not participate in the binding event remained in the quenched state to maintain a low level of background fluorescence. (C) 2010 Elsevier Inc. All rights reserved.

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  • Convenient method for surface modification by patching a freestanding anti-biofouling nanosheet

    Toshinori Fujie, Hiroki Haniuda, Shinji Takeoka

    JOURNAL OF MATERIALS CHEMISTRY   21 ( 25 ) 9112 - 9120  2011

     View Summary

    A convenient method for surface modification is crucially important for the preparation of a functional interface. The precise control of surface properties by chemical and physical approaches is expected to enhance the potential application of innovative nanomaterials. In this study, we propose a methodology for convenient surface modification using a freestanding anti-biofouling ultra-thin film (nanosheet) bearing poly(2-methacryloyloxyethyl phosphorylcholine: MPC) (i.e., pMPC-nanosheets). The physicochemical properties of the pMPC-nanosheet such as physiological stability, surface wettability and anti-biofouling were precisely controlled by means of thermal crosslinking of the nanosheet, the grafting amount of MPC and tuning the thickness of pMPC brushes. These approaches revealed physicochemical insight into the pMPC-nanosheet and its remarkable biological response. The pMPC-nanosheet was prepared via a spin-coating assisted layer-by-layer method in combination with atom transfer radical polymerization of MPC. The freestanding pMPC-nanosheet, comprising an 11 nm-thick pMPC brushed layer with a hydrophilic surface, was easily transferred by tweezers, cut by scissors, patterned by a needle and patched onto various interfaces with the aid of a water-soluble supporting film. The pMPC-nanosheets patched on cell culture substrates displayed anti-biofouling properties such as anti-coagulant activity against human whole blood as well as the potential to microscopically pattern murine fibroblast cells. The present study not only reveals physicochemical properties of the surface-functionalized nanosheet for biological application, but also constitutes an alternative approach to conventional lithographic techniques using photoresists and micro-patterned molds; thereby providing a new tool in the field of nanobiotechnology.

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  • Ultra-thin conductive free-standing PEDOT/PSS nanofilms

    Francesco Greco, Alessandra Zucca, Silvia Taccola, Arianna Menciassi, Toshinori Fujie, Hiroki Haniuda, Shinji Takeoka, Paolo Dario, Virgilio Mattoli

    SOFT MATTER   7 ( 22 ) 10642 - 10650  2011

     View Summary

    Free-standing conductive ultra-thin films based on poly(3,4-ethylenedioxythiophene)/poly (styrenesulfonate) (PEDOT/PSS) are realized. A fabrication process based on a modified Supporting Layer technique is proposed that provides for the easy production of conductive nanofilms having a very large surface area with typical thickness of tens of nanometres. The proposed free-standing nanofilms can be manipulated, folded and unfolded in water many times without suffering from cracks, disaggregation or from loss of conductive properties. After collecting them onto rigid or soft substrates, they retain their functionality. Structural and functional properties of the nanofilms are described by means of their thickness, topography, conductivity and Young's modulus. Strong dependences of these properties on residual water, post-deposition treatments and environmental moisture are clearly evidenced. Possible applications are foreseen in the field of sensing and actuation, as well as in the biomedical field, e. g. as smart substrates for cell culturing and stimulation.

    DOI

  • Release abilities of adenosine diphosphate from phospholipid vesicles with different membrane properties and their hemostatic effects as a platelet substitute

    Yosuke Okamura, Shunsuke Katsuno, Hidenori Suzuki, Hitomi Maruyama, Makoto Handa, Yasuo Ikeda, Shinji Takeoka

    JOURNAL OF CONTROLLED RELEASE   148 ( 3 ) 373 - 379  2010.12

     View Summary

    We have constructed phospholipid vesicles with hemostatic activity as a platelet substitute. The vesicles were conjugated with a dodecapeptide (HHLGGAKQAGDV, H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411). We have recently exploited these vesicles as a potential drug delivery system by encapsulation of adenosine 5'-diphosphate (ADP) (H12-(ADP)-vesicles).
    Here we explore the relationship between the ADP release from H12-(ADP)-vesicles with different membrane properties and their hemostatic effects. In total, we prepared five kinds of H12-(ADP)-vesicles with different lamellarities and membrane flexibilities. By radioisotope-labeling, we directly show that H12-(ADP)-vesicles were capable of augmenting platelet aggregation by releasing ADP in an aggregation-dependent manner. The amount of ADP released from the vesicles was dependent on their membrane properties. Specifically, the amount of ADP released increased with decreasing lamellarity and tended to increase with increasing membrane flexibility. Our in vivo results clearly demonstrated that H12-(ADP)-vesicles with the ability to release ADP exert considerable hemostatic action in terms of correcting prolonged bleeding time in a busulphan-induced thrombocytopenic rat model.
    We propose a recipe to control the hemostatic abilities of H12-(ADP)-vesicles by modulating ADP release based on membrane properties. We believe that this concept will be invaluable to the development of platelet substitutes and other drug carriers. (C) 2010 Elsevier B.V. All rights reserved.

    DOI

  • Construction of a 'turn-on' fluorescent probe system for His-tagged proteins

    Atsushi Murata, Satoshi Arai, Su-In Yoon, Masao Takabayashi, Miwako Ozaki, Shinji Takeoka

    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS   20 ( 23 ) 6905 - 6908  2010.12

     View Summary

    Hexahistidine ((His)(6)) tags are used to purify genetically engineered proteins. Herein, we describe the construction of a 'turn-on' fluorescent probe system that consists of the fluorescence quencher, Dabcyl, conjugated to (His)(6), and fluorescent tetramethylrhodamine conjugated to nitrilotriacetic acid, which, in the presence of Ni2+, can bind (His)(6). The system is turned off when Dabcyl-(His)(6) is bound to the fluorescent nitrilotriacetic acid derivative. The binding strength of this system was assessed using electrospray ionization mass spectrometry, fluorescence correlation spectroscopy, and fluorescence intensity distribution analysis-polarization. Although there was no significant enhancement in fluorescence after addition of an equimolar amount of ubiquitin, the fluorescence increased from 14% to 40% of its initial intensity when an equimolar amount of (His)(6)-ubiquitin was added. Therefore, this system should be able to specifically recognize (His)(6)-proteins with good resolution and has the additional advantage that a washing step is not required to remove fluorescent probe, that is, not bound to the (His)(6)-protein. (C) 2010 Elsevier Ltd. All rights reserved.

    DOI

  • 高分子超薄膜(ナノシート)の医用材料としての展開

    藤枝 俊宣, 武岡 真司

    高分子   59 ( 12 ) 930 - 931  2010.12

  • FLUORESCENT "TURN-ON" SYSTEM UTILIZING THE QUENCHER-CONJUGATED PEPTIDE TOWARD SPECIFIC PROTEIN LABELLING IN LIVE CELL

    S. Yoon, S. Arai, A. Murata, M. Takabayashi, X. Wu, Y. Lu, S. Takeoka, M. Ozaki

    JOURNAL OF NEUROCHEMISTRY   115   27 - 28  2010.10

  • Adhesion and proliferation of skeletal muscle cells on single layer poly(lactic acid) ultra-thin films

    Leonardo Ricotti, Silvia Taccola, Virginia Pensabene, Virgilio Mattoli, Toshinori Fujie, Shinji Takeoka, Arianna Menciassi, Paolo Dario

    BIOMEDICAL MICRODEVICES   12 ( 5 ) 809 - 819  2010.10

     View Summary

    An increasing interest in bio-hybrid systems and cell-material interactions is evident in the last years. This leads towards the development of new nano-structured devices and the assessment of their biocompatibility. In the present study, the development of free-standing single layer poly(lactic acid) (PLA) ultra-thin films is described, together with the analysis of topography and roughness properties. The biocompatibility of the PLA films has been tested in vitro, by seeding C2C12 skeletal muscle cells, and thus assessing cells shape, density and viability after 24, 48 and 72 h. The results show that free-standing flexible PLA nanofilms represent a good matrix for C2C12 cells adhesion, spreading and proliferation. Early differentiation into myotubes is also allowed. The biocompatibility of the novel ultra-thin films as substrates for cell growth promotes their application in the fields of regenerative medicine, muscle tissue engineering, drug delivery, and-in general-in the field of bio-hybrid devices.

    DOI

  • Synthesis and self-assembling behavior of a porphyrin bearing multiple meso-conjugated barbiturates

    Satoshi Arai, Toshiya Okamura, Shinji Takeoka

    TETRAHEDRON LETTERS   51 ( 39 ) 5177 - 5180  2010.09

     View Summary

    An efficient procedure through deprotection of 2,4,6-trimethoxypyrimidine derivative afforded porphyrinato zinc bearing multi-barbiturates acting as multiple hydrogen bonding sites at the meso positions. Addition of excess amphiphilic triaminopyrimidine derivative, as a complementary motif to the barbiturate, in an aqueous solution of porphyrin conjugated with multiple barbiturates at the meso positions resulted in precipitation. The cast film from the chloroform solution of the precipitate indicated the formation of a well-defined porphyrin assembly. (c) 2010 Elsevier Ltd. All rights reserved.

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  • Dual therapeutic action of antibiotic-loaded nanosheets for the treatment of gastrointestinal tissue defects

    Toshinori Fujie, Akihiro Saito, Manabu Kinoshita, Hiromi Miyazaki, Shinya Ohtsubo, Daizoh Saitoh, Shinji Takeoka

    BIOMATERIALS   31 ( 24 ) 6269 - 6278  2010.08

     View Summary

    An ultra-thin polymer film (nanosheet) composed of polysaccharides (i.e., polysaccharide nanosheet) provides sufficient adhesiveness, flexibility and robustness to act as an effective wound dressing. We have recently demonstrated the sealing effect of a nanosheet on a murine cecal puncture. Nevertheless, a small percentage of bacteria penetrated the nanosheet because of its ultra-thin structure. Here, we have developed an antibiotic-loaded nanosheet to inhibit bacterial penetration and investigated its therapeutic efficacy using a model of a murine cecal puncture. Tetracycline (TC) was sandwiched between a poly(vinylacetate) (PVAc) layer and the polysaccharide nanosheet (named PVAc-TC-nanosheet). Under physiological conditions, TC was released from the nanosheet for 6 h. Microscopic observation between the interface of the PVAc-TC-nanosheet and bacteria demonstrated how its potent anti-microbial effect was achieved. In vivo studies show that overlapping therapy with the PVAc-TC-nanosheet (thickness: 177 nm) significantly increases mouse survival rate after cecal puncture as well as suppressing an increase in the intraperitoneal bacterial count and leukocyte count. (C) 2010 Elsevier Ltd. All rights reserved.

    DOI

  • Sealing effect of a polysaccharide nanosheet for murine cecal puncture

    Toshinori Fujie, Manabu Kinoshita, Satoshi Shono, Akihiro Saito, Yosuke Okamura, Daizoh Saitoh, Shinji Takeoka

    SURGERY   148 ( 1 ) 48 - 58  2010.07

     View Summary

    Background. Recent developments in nanobiotechnology have led us to develop a method of producing a free-standing polymer nanosheet composed of polysaccharides (ie, polysaccharide nanosheet) with a thickness of tens of nanometers. Owing to its enormous aspect ratio, the polysaccharide nanosheet is semi-absorbent and has a physical adhesive strength 7 5-fold greater than that of conventional films of > 1 mu m thickness. Herein, we have investigated the therapeutic sealing effect of this polysaccharide nanosheet on murine cecal puncture as a wound dressing material.
    Methods. Murine cecum was punctured and then overlapped with the polysaccharide nanosheet Thereafter, we evaluated its sealing effect on bacterial peritonitis as well as the protection offered by the polysaccharide nanosheet against bacterial permeability using an in vitro transmembrane assay
    Results. The 39-nm-thick polysaccharide nanosheet overlapped tightly the perforated recant. No adhering agents were required because of the ability of the polysaccharide nanosheet to adhere to the tissue surface by physical adsorption (eg, van der Waals interaction) Sealing the perforated cecum with the polysaccharide nanosheet increased survival rate without postoperative adhesion by comparison with untreated mice (90 vs 30%, P < .01) These data were supported by the improvement in peritonitis related to bacterial counts, white blood cell counts, and the serum tumor necrosis factor level. Moreover, using an. in vitro transmembrane assay, we showed that the polysaccharide nanosheet inhibited effectively bacterial penetration.
    Conclusion. We have demonstrated the potential clinical benefits of the nanosheet-type biomaterial that can be used for repairing a cecal colotomy without chemical bonding agents (Surgery 2010;148 48-58)

    DOI

  • 穿孔性腹膜炎に対するナノシートの応用

    藤枝俊宣, 木下学, 齋藤大蔵, 武岡真司

    Surgery Frontiea   17   37 - 43  2010.06

  • Visualization of liposomes carrying fibrinogen gamma-chain dodecapeptide accumulated to sites of vascular injury using computed tomography

    Yosuke Okamura, Kaoruko Eto, Hitomi Maruyama, Makoto Handa, Yasuo Ikeda, Shinji Takeoka

    NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE   6 ( 2 ) 391 - 396  2010.04

     View Summary

    We have constructed liposomes with hemostatic activity as a platelet substitute using moderately thrombocytopenic rats. The liposomes were conjugated with the dodecapeptide (HHLGGAKQAGDV: H12), which is a fibrinogen gamma-chain C-terminal sequence (gamma 400-411). To visualize liposome accumulation at the site of vascular injury by in vivo computed tomography, a water-soluble contrast dye, N,N'-bis[2-hydroxy-1-(hydroxylmethyl)ethyl]-5-[(2S)-2-hydroxylpropanoylamino]-2,4,6-triiodoisophthalamide (iopamidol), was encapsulated into the H12-conjugated liposomes. We achieved direct visualization of specific accumulation of the H12(iopamidol)liposomes at the jugular vein injured by ferric chloride and succeeded in semiquantitative analyses of the accumulated amount of H12-liposomes in the injured site. We therefore propose that H12-liposomes that are specifically recruited to, and exert their hemostatic activity at the site of vascular injury, have a significant potential as a carrier and/or as an ideal platelet substitute. Furthermore, the H12-(iopamidol)liposomes would also be clinically useful as diagnostic agents for pathological thrombus detection and as contrast dyes for hepatosplenography.
    From the Clinical Editor: The authors have constructed liposomes with hemostatic activity as a platelet substitute using moderately thrombocytopenic rats. They propose that H12-liposomes that are specifically recruited to, and exert their hemostatic activity at the site of vascular injury, have a significant potential as a carrier and/or as an ideal platelet substitute. Furthermore, the H12-(iopamidol)liposomes would also be clinically useful as diagnostic agents for thrombus detection and as contrast dyes for hepatosplenography. (C) 2010 Elsevier Inc. All rights reserved.

    DOI

  • Hemoglobin encapsulation in vesicles retards NO and CO binding and O-2 release when perfused through narrow gas-permeable tubes

    Hiromi Sakai, Naoto Okuda, Atsushi Sato, Tatsuya Yamaue, Shinji Takeoka, Eishun Tsuchida

    AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY   298 ( 3 ) H956 - H965  2010.03

     View Summary

    Sakai H, Okuda N, Sato A, Yamaue T, Takeoka S, Tsuchida E. Hemoglobin encapsulation in vesicles retards NO and CO binding and O-2 release when perfused through narrow gas-permeable tubes. Am J Physiol Heart Circ Physiol 298: H956-H965, 2010. First published December 31, 2009; doi:10.1152/ajpheart.00741.2009.-Intravenous administration of cell-free Hb induces vasoconstriction and circulatory disorders, presumably because of the intrinsic affinities to endogenous nitric oxide (NO) and carbon monoxide (CO) as vasorelaxation factors and because of the facilitated O-2 release that might induce autoregulatory vasoconstriction. We examined these gas reactions when Hb-containing solutions of four kinds were perfused through artificial narrow tubes at a practical Hb concentration (10 g/dl). Purified Hb solution, polymerized bovine Hb (Poly(B)Hb), encapsulated Hb [Hb-vesicles (HbV), 279 nm], and red blood cells (RBCs) were perfused through a gas-permeable narrow tube (25 mu m inner diameter) at 1 mm/s centerline velocity. The level of reactions was determined microscopically based on the visible-light absorption spectrum of Hb. When the tube was immersed in NO and CO atmospheres, both NO binding and CO binding of deoxygenated Hb (deoxy-Hb) and Poly(B)Hb in the tube was faster than those of HbV and RBCs, and HbV and RBCs showed almost identical binding rates. When the tube was immersed in a N-2 atmosphere, oxygenated Hb and Poly(B)Hb showed much faster O-2 release than did HbV and RBCs. Poly(B)Hb showed a faster reaction than Hb because of the lower O-2 affinity of Poly(B)Hb than Hb. The diffusion process of the particles was simulated using Navier-Stokes and Maxwell-Stefan equations. Results clarified that small Hb (6 nm) diffuses laterally and mixes rapidly. However, the large-dimension HbV shows no such rapid diffusion. The purely physicochemical differences in diffusivity of the particles and the resulting reactivity with gas molecules are one factor inducing biological vasoconstriction of Hb-based oxygen carriers.

    DOI

  • Evaluation of pH-responsive liposomes containing amino acid-based zwitterionic lipids for improving intracellular drug delivery in vitro and in vivo

    Yosuke Obata, Shoji Tajima, Shinji Takeoka

    JOURNAL OF CONTROLLED RELEASE   142 ( 2 ) 267 - 276  2010.03

     View Summary

    We developed pH-responsive liposomes containing synthetic glutamic acid-based zwitterionic lipids and evaluated their properties both in vitro and in vivo with the aim of constructing an efficient liposome-based systemic drug delivery system. The glutamic acid-based lipids; 1,5-dihexadecyl N-glutamyl-L-glutamate (L1) and 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate (L2) were synthesized as a pH-responsive component of liposomes that respond to endosomal pH. The zeta potential of liposomes containing L1 or L2 was positive when the solution pH was below 4.6 or 5.6, respectively, but negative at higher pH values. The pH-responsive liposomes showed improved fusogenic potential to an endosome-mimicking anionic membrane at acidic pH, where the zeta potential of the liposomes was positive. We then prepared doxorubicin (DOX)-encapsulating liposomes containing L1 or 12, and clarified by confocal microscopic studies that the contents were rapidly transferred into both the cytoplasm and nucleus. Release of DOX from the endosomes mediated by the pH-responsive liposomes dramatically inhibited cancer cell growth. The L2-liposomes were slightly more effective than L1-liposomes as a drug delivery system. Intravenously injected L2-liposomes displayed blood persistence comparable to that of conventional phospholipid (PC)-based liposomes. Indeed, the antitumor efficacy of L2-liposomes was higher than that of PC-based liposomes against a xenograft breast cancer tumor in vivo. Thus, the high performance of L2-liposomes results from both efficient intracellular drug delivery and comparable blood persistence in comparison with the conventional PC-based liposomes in vitro and in vivo. (C) 2009 Elsevier B.V. All rights reserved.

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  • Evaluation of cationic liposomes composed of an amino acid-based lipid for neuronal transfection

    Yosuke Obata, Gianni Ciofani, Vittoria Raffa, Alfred Cuschieri, Arianna Menciassi, Paolo Dario, Shinji Takeoka

    NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE   6 ( 1 ) 70 - 77  2010.02

     View Summary

    We investigated the ability of cationic liposomes composed of 1,5-dihexadecyl N-arginyl-L-glutamate (Arg-Glu2C(16)) to carry nucleic acids into neuronal cells. Such liposomes have been shown to have a remarkable capacity for transfecting immortalized cell lines. Lipoplexes between the Arg-Glu2C(16) liposomes and plasmid DNA encoding green fluorescent protein (GFP) were analyzed in terms of lipoplex formation, intracellular DNA trafficking, transfection efficiency, and cytotoxicity in neuronal SH-SY5Y cells. A maximum number of cells expressing GFP was obtained with lipoplexes at a lipid-to-DNA ratio of 15. With these lipoplexes, 16% of the cells were GFP- positive, which was approximately fourfold higher than the level obtained with a commercially available transfection reagent, Lipofectamine 2000. Furthermore, as a result of the low cytotoxicity of the Arg-Glu2C(16) lipoplexes, the proportion of GFP- positive cells could be increased to 25% by increasing the concentration of lipoplexes that was applied to the cells. We have demonstrated that Arg-Glu2C(16), as a model cationic amino acid-based lipid, has a high capability as a gene carrier, even for neuronal transfection.
    From the Clinical Editor: In this study, specific cationic liposomes were characterized as nucleic acid transfection agents for neuronal cells. A fourfold higher transfection rate with low cytotoxicity was reported compared to Lipofectamine 2000, a commercial reagent. The authors conclude that the studied cationic liposomes have a high capability as a gene carrier for neuronal transfection. This may become clinically significant in future gene therapy efforts of neuronal diseases. (C) 2010 Elsevier Inc. All rights reserved.

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  • Basic consideration on tissue adhesion and sealing of ultra-thin nanobiomaterial in visceral pleural defect repair

    Matsutani, N, Fujie, T, Ozeki, Y, Takeoka, S

    JACSURG(日本呼吸器外科学会雑誌),   1   2 - 7  2010

  • A nano-fibrous assembly of collagen-hyaluronic acid for controlling cell-adhesive properties

    Toshinori Fujie, Sho Furutate, Daisuke Niwa, Shinji Takeoka

    SOFT MATTER   6 ( 19 ) 4672 - 4676  2010

     View Summary

    We report an artificially fabricated extracellular matrix (ECM)-like nanostructure, which utilizes self-assembly of collagen and hyaluronic acid in a quasi two-dimensional space of tens-of-nm thickness, displaying different cellular adhesive characteristics depending on the structural properties between the fibril and non-fibril elements. This nano-fibrous assembly will facilitate a mechanobiological approach for the development of tissue-engineering scaffolds.

    DOI

  • Free-Standing Biodegradable Poly(lactic acid) Nanosheet for Sealing Operations in Surgery

    Yosuke Okamura, Koki Kabata, Manabu Kinoshita, Daizoh Saitoh, Shinji Takeoka

    ADVANCED MATERIALS   21 ( 43 ) 4388 - +  2009.11

     View Summary

    A free-standing biodegradable nanosheet composed of poly(L-lactic acid) (PLLA) was shown to have excellent sealing efficacy for a gastric incision as a novel wound dressing material that did not require adhesive agents, and the PLLA nanosheet-induced wound repair showed neither scars nor tissue adhesion. This material may, therefore, be an ideal alternative to conventional tissue repairing procedures using suture/ligation in surgery.

    DOI

  • Free-Standing Biodegradable Poly(lactic acid) Nanosheet for Sealing Operations in Surgery

    Yosuke Okamura, Koki Kabata, Manabu Kinoshita, Daizoh Saitoh, Shinji Takeoka

    ADVANCED MATERIALS   21 ( 43 ) 4388 - +  2009.11

     View Summary

    A free-standing biodegradable nanosheet composed of poly(L-lactic acid) (PLLA) was shown to have excellent sealing efficacy for a gastric incision as a novel wound dressing material that did not require adhesive agents, and the PLLA nanosheet-induced wound repair showed neither scars nor tissue adhesion. This material may, therefore, be an ideal alternative to conventional tissue repairing procedures using suture/ligation in surgery.

    DOI

  • Novel Platelet Substitutes: Disk-Shaped Biodegradable Nanosheets and their Enhanced Effects on Platelet Aggregation

    Yosuke Okamura, Yoshihito Fukui, Koki Kabata, Hidenori Suzuki, Makoto Handa, Yasuo Ikeda, Shinji Takeoka

    BIOCONJUGATE CHEMISTRY   20 ( 10 ) 1958 - 1965  2009.10

     View Summary

    We have studied biocompatible spherical carriers carrying a dodecapeptide, HHLGGAKQAGDV (H12), on their surface as platelet substitutes. This peptide is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411) and specifically recognizes the active form of glycoprotein IIb/IIIa on activated platelets. Our purpose is to assess the possibility of making a novel platelet substitute consisting of disk-shaped nanosheets having a large contact area for the targeting site, rather than conventional small contact area spherical carriers. ne H12 peptide was conjugated to the surface of the free-standing nanosheets made of biodegradable poly(D,L-lactide-co-glycolide) (PLGA). These H12-PLGA nanosheets were fabricated onto 3 mu m disk-shaped patterned hydrophobic octadecyl regions on a SiO(2) substrate. By way of comparison, spherical H12-PLGA microparticles with the same surface area and conjugation number of H12 were also prepared. The resulting H12-PLGA nanosheets specifically interacted with the activated platelets adhered on the collagen surface at twice the rate of the H12-PLGA microparticles under flow conditions, and showed platelet thrombus formation in a two-dimensional spreading manner. Thus, H12-PLGA nanosheets might be a suitable candidate novel platelet alternative substitute for infused human platelet concentrates for the treatment of bleeding in patients with severe thrombocytopenia.

    DOI

  • Novel Platelet Substitutes: Disk-Shaped Biodegradable Nanosheets and their Enhanced Effects on Platelet Aggregation

    Yosuke Okamura, Yoshihito Fukui, Koki Kabata, Hidenori Suzuki, Makoto Handa, Yasuo Ikeda, Shinji Takeoka

    BIOCONJUGATE CHEMISTRY   20 ( 10 ) 1958 - 1965  2009.10

     View Summary

    We have studied biocompatible spherical carriers carrying a dodecapeptide, HHLGGAKQAGDV (H12), on their surface as platelet substitutes. This peptide is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411) and specifically recognizes the active form of glycoprotein IIb/IIIa on activated platelets. Our purpose is to assess the possibility of making a novel platelet substitute consisting of disk-shaped nanosheets having a large contact area for the targeting site, rather than conventional small contact area spherical carriers. ne H12 peptide was conjugated to the surface of the free-standing nanosheets made of biodegradable poly(D,L-lactide-co-glycolide) (PLGA). These H12-PLGA nanosheets were fabricated onto 3 mu m disk-shaped patterned hydrophobic octadecyl regions on a SiO(2) substrate. By way of comparison, spherical H12-PLGA microparticles with the same surface area and conjugation number of H12 were also prepared. The resulting H12-PLGA nanosheets specifically interacted with the activated platelets adhered on the collagen surface at twice the rate of the H12-PLGA microparticles under flow conditions, and showed platelet thrombus formation in a two-dimensional spreading manner. Thus, H12-PLGA nanosheets might be a suitable candidate novel platelet alternative substitute for infused human platelet concentrates for the treatment of bleeding in patients with severe thrombocytopenia.

    DOI

  • Adhesive, Flexible, and Robust Polysaccharide Nanosheets Integrated for Tissue-Defect Repair

    Toshinori Fujie, Noriyuki Matsutani, Manabu Kinoshita, Yosuke Okamura, Akihiro Saito, Shinji Takeoka

    ADVANCED FUNCTIONAL MATERIALS   19 ( 16 ) 2560 - 2568  2009.08

     View Summary

    Recent developments in nanotechnology have led to a method for producing free-standing polymer nanosheets as a macromolecular organization. Compared with bulk films, the large aspect ratio of such nanosheets leads to unique physical properties, such as transparency, noncovalent adhesion, and high flexibility. Here, a biomedical application of polymer nanosheets consisting of biocompatible and biodegradable polysaccharides is reported. Micro-scratch and bulge tests indicate that the nanosheets with a thickness of tens of nanometers have sufficient physical adhesiveness and mechanical strength for clinical use. A nanosheet of 75 nm thickness, a critical load of 9.1 x 10(4) N m(-1), and an elastic modulus of 9.6 GPa is used for the minimally invasive repair of a visceral pleural defect in beagle dogs without any pleural adhesion caused by wound repair. For the first time, clinical benefits of sheet-type nano-biomaterials based on molecular organization are demonstrated, suggesting that novel therapeutic tools for overlapping tissue wounds will be possible without the need for conventional surgical interventions.

    DOI

  • Mechanism of Flocculate Formation of Highly Concentrated Phospholipid Vesicles Suspended in a Series of Water-Soluble Biopolymers

    Hiromi Sakai, Atsushi Sato, Shinji Takeoka, Eishun Tsuchida

    BIOMACROMOLECULES   10 ( 8 ) 2344 - 2350  2009.08

     View Summary

    Polyethylene glycol-modified vesicles (liposomes) encapsulating hemoglobin (HbV) are artificial oxygen carriers that have been developed as a transfusion alternative. The HbV suspension in an albumin solution is nearly Newtonian; other biopolymers, hydroxyethyl starch (HES), dextran (DEX), and modified fluid gelatin, induce flocculation of HbVs through depletion interaction and render the suspensions as non-Newtonian. The flocculation level increased with hydrodynamic radius (R(h)) or radius of gyration (R(g)) of series of HES or DEX with different molecular weights at a constant polymer concentration (4 wt %). However, the flocculation level differed markedly among the polymers. A crowding index (C(i)) representing the crowding level of a polymer solution is defined as (excluded volume of one polymer) x (molar concentration) x Avogadro's number, using R(h) or R(g). All polymers' flocculation level increases when C(i) approaches 1: when the theoretical total excluded Volumes approach the entire solution Volume, the excluded HbV particles are forced to flocculate.

    DOI

  • Peculiar flow patterns of RBCs suspended in viscous fluids and perfused through a narrow tube (25 mu m)

    Hiromi Sakai, Atsushi Sato, Naoto Okuda, Shinji Takeoka, Nobuji Maeda, Eishun Tsuchida

    AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY   297 ( 2 ) H583 - H589  2009.08

     View Summary

    Sakai H, Sato A, Okuda N, Takeoka S, Maeda N, Tsuchida E. Peculiar flow patterns of RBCs suspended in viscous fluids and perfused through a narrow tube (25 mu m). Am J Physiol Heart Circ Physiol 297: H583-H589, 2009. First published June 5, 2009; doi:10.1152/ajpheart.00352.2009.-Red blood cells (RBCs) generally deform to adopt a parachute-like, torpedo-like, or other configuration to align and flow through a capillary that is narrower than their major axis. As described herein, even in a narrow tube (25 mu m) with diameter much larger than that of a capillary, flowing RBCs at 1 mm/s align axially and deform to a paraboloid shape in a viscous Newtonian fluid (505 kDa dextran medium) with viscosity of 23.4-57.1 mPa.s. A high-speed digital camera image showed that the silhouette of the tip of RBCs fits a parabola, unlike the shape of RBCs in capillaries, because of the longer distance of the RBC-free layer between the tube wall and the RBC surface (similar to 8.8 mu m). However, when RBCs are suspended in a "non-Newtonian" viscous fluid (liposome-40 kDa dextran medium) with a shear-thinning profile, they migrate toward the tube wall to avoid the axial lining, as "near-wall-excess," which is usually observed for platelets. This migration results from the presence of flocculated liposomes at the tube center. In contrast, such near-wall excess was not observed when RBCs were suspended in a nearly Newtonian liposome-albumin medium. Such unusual flow patterns of RBCs would be explainable by the principle; a larger particle tends to flow near the centerline, and a small one tends to go to the wall to flow with least resistance. However, we visualized for the first time the complete axial aligning and near-wall excess of RBCs in the noncapillary size tube in some extreme conditions.

    DOI

  • Hydrodynamic Transformation of a Freestanding Polymer Nanosheet Induced by a Thermoresponsive Surface

    Toshinori Fujie, Jin Young Park, Atsushi Murata, Nicel C. Estillore, Maria Celeste R. Tria, Shinji Takeoka, Rigoberto C. Advincula

    ACS APPLIED MATERIALS & INTERFACES   1 ( 7 ) 1404 - 1413  2009.07

     View Summary

    Freestanding quasi-two-dimensional ultrathin films (e,g., 41 nm thick polymer nanosheets) were produced. on which stimuli-responsive 47 nm chick polymer brushes were constructed by atom transfer radical polymerization (ATRP) of poly(N-isopropylacrylamide). The resulting surfaces of the multilayered polysaccharide ultrathin films were evaluated by ellipsometry, IR imaging, In situ variable-temperature atomic force microscopy (AFM), and contact angle measurements. The morphological transformation of the freestanding polymer nanosheet bearing thermoresponsive polymer brushes was observed macroscopically through reversible structural color changes at the air-water interface. The dynamic shape change of the nanosheetwas also monitored with the addition of a surfactant such as sodium n-dodecylsulfate to reduce the hydrophobicity of the surface, It was then demonstrated chat the highly flexible freestanding polymer nanosheet is capable of acting as a unique platform for inducing stimuli-responsive I behavior in nanomaterials.

    DOI

  • Magnetic nanosheet adhesion to mucosal tissue

    V. Pensabene, V. Mattoli, A. Menciassi, S. Takeoka, T. Fujie, P. Dario

    Proceeding of 9th International Conference on Nanotechnology, IEEE Nano 2009    2009.07

  • Plasmid DNA-encapsulating liposomes: Effect of a spacer between the cationic head group and hydrophobic moieties of the lipids on gene expression efficiency

    Yosuke Obata, Shunsuke Saito, Naoya Takeda, Shinji Takeoka

    BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES   1788 ( 5 ) 1148 - 1158  2009.05

     View Summary

    We have synthesized a series of cationic amino acid-based lipids having a spacer between the cationic head group and hydrophobic moieties and examined the influence of the spacer on a liposome gene delivery system. As a comparable spacer, a hydrophobic spacer with a hydrocarbon chain composed of 0, 3, 5, 7, or 11 carbons, and a hydrophilic spacer with an oxyethylene chain (10 carbon and 3 oxygen molecules) were investigated. Plasmid DNA (pDNA)-encapsulating liposomes were prepared by mixing an ethanol solution of the lipids with an aqueous solution of pDNA. The zeta potentials and cellular uptake efficiency of the cationic liposomes containing each synthetic lipid were almost equivalent. However, the cationic lipids with the hydrophobic spacer were subject to fuse with biomembrane-mimicking liposomes. 1,5-Dihexadecyl-N-lysyl-N-heptyl-L-glutamate, having a seven carbon atom spacer, exhibited the highest fusogenic potential among the synthetic lipids. Increased fusion potential correlated with enhanced gene expression efficiency. By contrast, an oxyethylene chain spacer showed low gene expression efficiency. We conclude that a hydrophobic spacer between the cationic head group and hydrophobic moieties is a key component for improving pDNA delivery. (C) 2009 Elsevier B.V. All rights reserved.

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  • Biocompatibility Study of Hemoglobin Vesicles, Cellular-Type Artificial Oxygen Carriers, with Human Umbilical Cord Hematopoietic Stem/Progenitor Cells Using an In Vitro Expansion System

    Miki Yamaguchi, Mitsuhiro Fujihara, Shinobu Wakamoto, Hiromi Sakai, Shinji Takeoka, Eishun Tsuchida, Hirofumi Hamada, Hiroshi Azuma, Hisami Ikeda

    ASAIO JOURNAL   55 ( 3 ) 200 - 205  2009.05

     View Summary

    Hemoglobin vesicles (HbVs), liposomal oxygen carriers containing human hemoglobin, are candidates for development of a clinically useful transfusion alternative. Our previous in vivo animal studies of massive HbV dosages demonstrated the lack of any suppressive effect on hematopoiesis. Before starting clinical trials, we aimed to examine the details of the biocompatibility of HbVs with human hematopoietic stem/progenitor cells. We investigated the effect of HbVs at a concentration of up to 3 vol/vol (%) on expansion of human umbilical cord (CB) hernatopoietic stem/progenitor cells, using a coculture system of human TERT-transfected bone marrow stromal cells and CD34(+) cells in vitro. The exposure of CB CD34(+) cells to HbVs up to 14 days suppressed the expansion of total cells and the CD34(+) cells themselves, whereas the empty liposomes, that did not contain Hb, had modestly inhibitory effects on the expansion of these cells. As a result, the number of colonies obtained from the expanded CD34(+) cells was inhibited by the exposure to HbVs. In contrast, exposure to HbVs for 3 days had no effect on the expansion of CD34(+) cells and only slightly decreased the number of total cells. Our in vitro experimental condition does not fully recreate the physiological condition, and the effect of the direct contact of HbV would be magnified because of the absence of shielding by the vasculature and the lack of the reticuloendothelial system and blood stream. However, the present data raise some concern regarding hematopoiesis, and one has to pay attention to this in future human clinical trials. ASAIO Journal 2009; 55:200-205.

    DOI

  • Fabrication of free-standing albumin-nanosheets having heterosurfaces

    Yosuke Okamura, Takahiro Goto, Daisuke Niwa, Yoshihito Fukui, Masanobu Otsuka, Norikazu Motohashi, Tetsuya Osaka, Shinji Takeoka

    JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A   89A ( 1 ) 233 - 241  2009.04

     View Summary

    Sheet-shaped carriers, having both obverse and reverse surfaces and thus a large contact area for targeting a site, have several advantages over spherical-shaped carriers, which have an extremely small contact area for targeting sites. Here, we proposed a novel method to prepare a free-standing ultrathin and biocompatible nanosheet having heterosurfaces, by a combination of four processes: (1) specific adsorption of recombinant human serum albumin (rHSA) molecules onto a patterned octadecyltrimethoxysilane self-assembled monolayer region (ODS-SAM), (2) preparation of nanosheets of rHSA molecules bearing thiol groups (SH-rHSA) via two-dimensionally disulfide crosslinking, (3) surface modification of the resulting nanosheet, and. (4) preparation of the free-standing nanosheet by detachment from the ODS-SAM. The SH-rHSA molecules at pH 5.0 and a concentration of 1 mu g/ml, were specifically adsorbed on the patterned ODS-SAM regions by hydrophobic interaction, and were two-dimensionally crosslinked in the presence of copper ion as an oxidant. The rHSA-nanosheets were then simply detached from the ODS-SAM by treatment with surfactant. We succeeded in the preparation of rectangular (10 mu m X 30 mu m) and ultrathin (4.5 +/- 1.0 nm) rHSA-nanosheets on a patterned ODS-SAM, and could also obtain free-standing rHSA-nanosheets having heterosurfaces by Surface modification with fluorescent latex beads. Thus, the rHSA-nanosheets having heterosurfaces could be regarded as a new biomaterial for drug carriers, hemostatic reagents, wound dressing for burn injury, and so forth. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 89A: 233-241, 2009

    DOI

  • Development of fibrinogen gamma-chain peptide-coated, adenosine diphosphate-encapsulated liposomes as a synthetic platelet substitute

    Y. Okamura, S. Takeoka, K. Eto, I. Maekawa, T. Fujie, H. Maruyama, Y. Ikeda, M. Handa

    JOURNAL OF THROMBOSIS AND HAEMOSTASIS   7 ( 3 ) 470 - 477  2009.03

     View Summary

    Background: The dodecapeptide HHLGGAKQAGDV (H12), corresponding to the fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411), is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated H12-coated nanoparticles (polymerized albumin or liposome) as platelet function-supporting synthetic products. Objectives: To strengthen the hemostatic ability of H12-coated particles as a platelet substitute, we exploited installation of a drug delivery function by encapsulating adenosine diphosphate (ADP) into liposomes [H12-(ADP)-liposomes]. Methods and results: Via selective interaction with activated platelets through GPIIb/IIIa, H12-(ADP)-liposomes were capable of augmenting agonist-induced platelet aggregation by releasing ADP in an aggregation-dependent manner. When intravenously injected into rats, liposomes were readily targeted to sites of vascular injury as analyzed on computed tomography. In fact, comparable to fresh platelets, liposomes exhibited considerable hemostatic ability for correcting prolonged bleeding time in a busulphan-induced thrombocytopenic rabbit model. In addition, the liposomes showed no activating or aggregating effects on circulating platelets in normal rabbits. Conclusion: H12-(ADP)-liposome may thus offer a promising platelet substitute, being made with only synthetic materials and exerting hemostatic functions in vivo via reinforcement of primary thrombus formation by residual platelets in thrombocytopenia at sites of vascular injury, but not in circulation.

    DOI

  • Boron Nitride Nanotubes: A Novel Vector for Targeted Magnetic Drug Delivery

    Gianni Ciofani, Vittoria Raffa, Jun Yu, Ying Chen, Yosuke Obata, Shinji Takeoka, Arianna Menciassi, Alfred Cuschieri

    CURRENT NANOSCIENCE   5 ( 1 ) 33 - 38  2009.02

     View Summary

    Whereas several biomedical applications of carbon nanotubes have been proposed, the use of boron nitride nanotubes (BNNTs) in this field has been largely unexplored despite their unique and potentially useful properties. Our group has recently initiated an experimental program aimed at the exploration of the interactions between BNNTs and living cells. In the present paper, we report on the magnetic properties of BNNTs containing Fe catalysts which confirm the feasibility for their use as nanovectors for targeted drug delivery. The magnetisation curves of BNNTs characterised by the present study are typical of superparamagnetic materials with important parameters, including magnetic permeability and magnetic momentum, derived by employing Langevin theory. In-vitro tests have demonstrated the feasibility for influencing the uptake of BNNTs by living cells by exposure to an external magnetic source. A finite element method analysis devised to predict this effect produced predictive data with close agreement with the experimental observations.

    DOI

  • Realization, characterization and functionalization of lipidic wrapped carbon nanotubes

    Gianni Ciofani, Yosuke Obata, Izumi Sato, Yosuke Okamura, Vittoria Raffa, Arianna Menciassi, Paolo Dario, Naoya Takeda, Shinji Takeoka

    JOURNAL OF NANOPARTICLE RESEARCH   11 ( 2 ) 477 - 484  2009.02

     View Summary

    Mass-produced carbon nanotubes (CNTs) are strongly aggregated and highly hydrophobic, and processes to make them water soluble are required for biological applications. Both covalent and non-covalent strategies are pursued for obtaining stable, highly concentrated CNT aqueous dispersions. Covalent functionalization has the great disadvantage of producing an irreversible chemical modification of nanotubes, thus alterating their mechanical, chemical and electric properties. On the other hand, non-covalent functionalization is often obtained by employing surfactants that sensibly affect cell viability. Moreover, derivatization with biological moieties is often impossible through non-covalent CNT dispersion. This paper proposes a non-covalent dispersion of multi-wall CNT based on a lipidic mixture that can guarantee high concentration and high stability as well as high cytocompatibility. Moreover, CNTs wrapped with a lipid membrane are realized to demonstrate that the proposed CNTs can be functionalised with a dodecapeptide that specifically recognizes activated platelets without chemical modification of the nanotube itself.

    DOI

  • Selective surface modification of free-standing polysaccharide nanosheet with micro/nano-particles identified by structural color changes

    Toshinori Fujie, Yosuke Okamura, Shinji Takeoka

    COLLOIDS AND SURFACES A-PHYSICOCHEMICAL AND ENGINEERING ASPECTS   334 ( 1-3 ) 28 - 33  2009.02

     View Summary

    The chemical and physical modification of a material surface is important for practical applications. In this study, we utilized the bilateral nature of a free-standing polysaccharide nanosheet as a platform for surface modification, selectively modifying the upper and lower surfaces with different sizes of latex beads. By means of a water-soluble sacrificial layer, we adsorbed latex beads with the diameter of 200 rim onto the obverse surface of the polysaccharide nanosheet as well as latex beads with that of 2 mu m onto the reverse side. The optical characteristics of the nanosheet were analyzed on the basis of 'thin film interference theory' by making stepwise increments in thickness through sequentially overlaying free-standing 30nm polysaccharide nanosheets onto a SiO(2) substrate. The surface of the polysaccharide nanosheet was quite flat and smooth. Having confirmed by optical and scanning electron microscopy that we had achieved selective surface modification of the polysaccharide nanosheet with micro/nano-latex beads, we noted a unique optical property when the nanosheet was adsorbed on the SiO(2) substrate. The localization of the beads on the nanosheet surface produced structural colors in the films due to an optical interference. This selective surface modification technique will be utilized to create 'smart' nanocomposites possessing different surface functions. (C) 2008 Elsevier B.V. All rights reserved.

    DOI

  • FLUID RESUSCITATION WITH ARTIFICIAL OXYGEN CARRIERS IN HEMORRHAGED RATS: PROFILES OF HEMOGLOBIN-VESICLE DEGRADATION AND HEMATOPOIESIS FOR 14 DAYS

    Hiromi Sakai, Yasushi Seishi, Yosuke Obata, Shinji Takeoka, Hirohisa Horinouichi, Eishun Tsuchida, Koichi Kobayashi

    SHOCK   31 ( 2 ) 192 - 200  2009.02

     View Summary

    Polyethylene glycol (PEG)-modified hemoglobin (Hb) vesicles (HbVs) are artificial oxygen carriers encapsulating a concentrated Hb solution in phospholipid vesicles. In our previous studies, HbV showed a sufficient resuscitative effect comparable to that of red blood cells in hemorrhagic shock animal models during several hours' observation. However, the profiles of the recovery, including hematopoiesis and elimination of HbV, remain unknown. This study conducted 14-day observations of Wistar rats after hemorrhagic shock and fluid resuscitation with HbV suspended in recombinant human serum albumin. Shock was induced by 50% blood withdrawal from a femoral artery. The rats showed hypotension, metabolic acidosis, and hyperventilation. After 15 min, they received HbV or shed autologous blood through a femoral vein. Both groups showed rapid recovery of hemodynamic and blood gas parameters. No meaningful difference was found between groups. After decannulation and awakening, the rats were housed in cages. The reduced hematocrit of the HbV group returned to the original level in 7 days. Plasma enzyme levels were slightly higher in both groups at 1 day because of systemic reperfusion injury. Splenomegaly was considerable in the HbV group because of the HbV accumulation and extramedullar hematopoiesis, but it subsided within 14 days. Along with the HbV elimination in the spleen and liver, immunohistochemistry with anti-PEG antibody revealed that PEG-conjugated lipid had disappeared within 14 days. In conclusion, HbV showed a sufficient resuscitative effect comparable to that of red blood cell transfusion. Phagocytized HbV disappeared within 14 days. Elevated hematopoiesis contributed to complete hematocrit recovery within 7 days.

    DOI

  • Motion of polymerized albumin particles in a model arteriole in the presence of red blood cells

    Tetsuya Tsuji, Shinji Takeoka, Yosuke Okamura, Ryo Sudo, Yasuo Ikeda, Kazuo Tanishita

    Journal of Biorheology   23 ( 1 ) 29 - 34  2009

     View Summary

    Polymerized albumin particles (poly Alb) with recombinant glycoprotein Ibα (rGPIbα-poly Alb) are a promising candidate for a platelet substitute. Thus, we focused on the lateral motion of poly Alb in the presence of red blood cells, because the lateral motion plays an important role in aggregate formation. We visualized the microscopic motion of poly Alb toward the immobilized ligand (von Willebrand factor, VWF) surface in a model arteriole with red blood cells with a high-speed camera. At a higher shear rate of 1,500 s-1, the concentration profile of poly Alb appeared to peak near the wall. This profile enhances the interaction between the particles and wall. Particularly the migration angle, being the angle of the poly Alb velocity vector, was enlarged near the wall and contributed to transfer of poly Alb toward the immobilized VWF surface. This tendency is desirable to achieve the adhesion of particles on the wall. © Japanese Society of Biorheology 2009.

    DOI

  • Fabrication and characterization of ultra-thin magnetic films for biomedical applications

    V. Mattoli, V. Pensabene, T. Fujie, S. Taccola, A. Menciassi, S. Takeoka, P. Dario

    PROCEEDINGS OF THE EUROSENSORS XXIII CONFERENCE   1 ( 1 ) 28 - +  2009

     View Summary

    Polymeric ultra-thin films, so called nanosheets, show peculiar properties making them potentially useful for several applications in biomedicine. Moreover, the possibility to functionalize these films by using different agents opens new and partially unexplored scenarios, including micro/nano sensing and actuation. This paper compares two different methods for the preparation of free-standing nanosheets, loaded with magnetic particles for no-contact manipulation in liquid environment. Morphology and functionalities of the two types of nanosheets have been characterized and compared. These magnetic nanosheets could find applications as free-standing carriers to be released and controlled in endoluminal surgery or as plasters with nanometric thickness to be delivered in situ on surgical incisions.

    DOI

  • Deformability and adhesive force of artificial platelets measured by atomic force microscopy

    Toru Wada, Yosuke Okamura, Shinji Takeoka, Ryo Sudo, Yasuo Ikeda, Kazuo Tanishita

    Journal of Biorheology   23 ( 1 ) 35 - 40  2009

     View Summary

    Platelet glycoprotein GPIaIIa is an adhesive protein that recognizes collagen. We have investigated polymerized albumin particles conjugated with recombinant GPIaIIa (rGPIaIIa-poly Alb) for their platelet-like function. To evaluate the feasibility of these particles to achieve the hemostatic process, we measured the deformability (Young's modulus and spring constant) and the adhesive force of the particles using atomic force microscopy, which can measure the mechanical properties of individual cells. Our results showed that the Young's modulus of these particles was 2.3-fold larger than that of natural platelets and 12-fold larger than that of human red blood cells. The Young's modulus of the particles may have been determined by the properties of the polymerized albumin particle, although the glycoprotein of the platelet surface also contributed to the higher modulus. Our results also showed that the adhesive force of the rGPIaIIa-poly Alb with the collagen ligand was 52% of that of natural platelets. These two mechanical properties (deformability and adhesive force) of cells or particles, such as rGPIaIIa-poly Alb, are important specifications for the optimum design of platelet substitutes. © Japanese Society of Biorheology 2009.

    DOI

  • ナノメディシンに向けた医用材料の開発の現状と展望

    武岡 真司

    東京女子医科大学雑誌     96 - 102  2009

  • Development of biodegradable nanosheets as nanoadhesive plaster

    Shinji Takeoka, Yosuke Okamura, Toshinori Fujie, Yoshihito Fukui

    PURE AND APPLIED CHEMISTRY   80 ( 11 ) 2259 - 2271  2008.11

     View Summary

    Sheet-shaped carriers having both obverse and reverse surfaces (thus, a large contact area for targeting a site and adhesive properties without any chemical cross-linker onto tissue surface) have several advantages as surgical dressings. These advantages include active targeting over spherically shaped carriers, which thus have an extremely small contact area for targeting sites. Here, we propose a novel methodology for preparation of a freestanding, ultra-thin, and biocompatible polymer nanosheet having heterosurfaces, fabricated through macromolecular assembly. In the context of biomedical applications, the targeted properties include injectable sheet-shaped drug carriers having precisely controlled size by exploiting micropatterned substrate, and giant polymer nanosheets composed of biocompatible polysaccharides. A huge aspect ratio, in excess of 10(6), is particularly applicable for novel surgical dressings. These biocompatible polymer nanosheets having heterosurfaces can thus be regarded as new biomaterials for minimally invasive treatment.

    DOI

  • NO and CO binding profiles of hemoglobin vesicles as artificial oxygen carriers

    Hiromi Sakai, Atsushi Sato, Peter Sobolewski, Shinji Takeoka, John A. Frangos, Koichi Kobayashi, Marcos Intaglietta, Eishun Tsuchida

    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS   1784 ( 10 ) 1441 - 1447  2008.10

     View Summary

    Hemoglobin vesicles (HbVs) are artificial oxygen carriers encapsulating purified and concentrated Hb solution in phospholipid vesicles (liposomes). We examined in-vitro reaction profiles of a formulation of HbV with NO and CO in anaerobic and aerobic conditions using stopped-flow spectrophotometry and a NO electrode. Reaction rate constants of NO to deoxygenated and oxygenated HbV were considerably smaller than those of cell-free Hb because of the intracellular NO-diffusion barrier. The reaction of CO with deoxygenated HbV was slightly slower than that of cell-free Hb solely because of the co-encapsulated allosteric effector, pyridoxal 5'-phosphate. The NO depletion in an aerobic condition in the presence of empty vesicles was monitored using a NO electrode, showing that the hydrophobic bilayer membrane of HbV, which might have higher gas solubility, does not markedly facilitate the O-2 and NO reaction, and that the intracellular Hb is the major component of NO depletion. In conclusion, HbV shows retarded gas reactions, providing some useful information to explain the absence of vasoconstriction and hypertension when they are intravenously injected. (c) 2008 Elsevier B.V. All rights reserved.

    DOI

  • Helical arrangement of a hydrogen-bonded columnar liquid crystal induced by a centered triphenylene derivative bearing chiral side-chains

    Shinsuke Ishihara, Yusuke Furuki, Shinji Takeoka

    POLYMERS FOR ADVANCED TECHNOLOGIES   19 ( 8 ) 1097 - 1104  2008.08

     View Summary

    A hydrogen-bonded helical columnar liquid crystal was synthesized, in which the helical structure is induced by a centered triphenylene derivative bearing chiral side-chains. The triphenylene derivative, 2,6,10-tris(carboxymethoxy)-3,7,11-tris((S)-(-)-2-methyl-1-butanoxy)triphenylene (TPC4(S)), and a dendric amphiphile, 3,5-bis-(3,4-bis-dodecyloxy-benzyloxy)-N-pyridine-4-yl-benzamide (DenC12), were mixed in a 1:3 ratio to obtain a complex, TPC4(S)-DenC12. Analyses by H-1-NMR spectroscopy, diffusion ordered spectroscopy (DOSY), CD spectroscopy, infrared (IR) spectroscopy, polarized optical microscopy (POM), differential scanning calorimetry (DSC), and X-ray diffractometry revealed that TPC4(S)-DenC12 self-assembles to form helical columnar stacks in solution and a helical columnar liquid crystal in bulk. The hydrogen bonding between TPC4(S) and DenC12 is essential for the helical columnar organization, and the preference for a one-handed helical conformation is likely derived from the steric interaction between the chiral side-chains and the dendric amphiphiles in the packing of the hydrogen-bonded columnar assemblies. Copyright C(-) 2008 John Wiley & Sons, Ltd.

    DOI

  • Haemostatic effects of polymerized albumin particles carrying fibrinogen gamma-chain dodecapeptide as platelet substitutes in severely thrombocytopenic rabbits

    Y. Okamura, T. Fujie, M. Nogawa, H. Maruyama, M. Handa, Y. Ikeda, S. Takeoka

    TRANSFUSION MEDICINE   18 ( 3 ) 158 - 166  2008.06

     View Summary

    Our purpose was to produce a platelet substitute that could enhance haemostatic ability using rabbits with severe thrombocytopenia. We have developed polymerized albumin particles (polyAlb) for treatment of bleeding and focused on a dodecapeptide, HHLGGAKQAGDV (H12), as a useful ligand for activated platelet. This sequence occurs only at the carboxy-terminus of the fibrinogen gamma-chain (gamma 400-411). H12 was conjugated to the surface of polyAlb modified with poly(ethylene glycol) (PEG) chains to produce blood-compatible particles (H12-PEG-polyAlb) that had prolonged blood residence time and enhanced stability in vitro and in vivo. The H12-PEG-polyAlb was administered intravenously to rabbits with severe thrombocytopenia, and the ear bleeding time was measured in order to evaluate the haemostatic effect. The H12-PEG-polyAlb significantly shortened the ear bleeding time of severely thrombocytopenic rabbits and showed no effect on the inhibition or promotion of endogenous and exogenous coagulation activities. Furthermore, we could assess the haemostatic capacity of the H12-PEG-polyAlb, based on the relationship between transfused platelet count and the bleeding time. The H12-PEG-polyAlb may be a suitable candidate for an alternative to human platelet concentrates infused to treat bleeding in patients with severe thrombocytopenia.

    DOI

  • Evaluation of cationic assemblies constructed with amino acid based lipids for plasmid DNA delivery

    Yosuke Obata, Daisuke Suzuki, Shinji Takeoka

    BIOCONJUGATE CHEMISTRY   19 ( 5 ) 1055 - 1063  2008.05

     View Summary

    We synthesized cationic lipids bearing lysine, histidine, or arginine as a cationic headgroup for use in gene transfer studies. The cationic assemblies formed from lysine- or arginine-type lipids gave unilamellar vesicles (similar to 100 nm diameter), whereas the morphology of the histidine-type lipids was tube-like. The competences of the cationic assemblies were sufficient to form lipoplexes, and the resulting lipoplexes were evaluated in terms of gene expression efficiencies with COS-7 cells. The lysine- or arginine-type lipids exhibited higher gene expression efficiencies than that of Lipofectamine2000, a conventional transgenic reagent, indicating that stable lipoplexes could be prepared between spherical cationic assemblies and plasmid DNA. The gene expression efficiency in relation to the cationic headgroup of the lipids was as follows: lysine >= arginine > histidine. In addition, gene expression efficiency was enhanced by decreasing the length of the alkyl chain of the hydrophobic moiety. Unlike Lipofectamine2000, no reduction in transfection efficiency in the presence of fetal bovine serum was observed for the lipoplexes formed using synthetic cationic lipids. Moreover, the synthetic cationic lipids revealed remarkably low cytotoxicity compared with Lipofectamine2000. In conclusion, cationic assemblies formed from 1,5ditetradecyl-N-lysyl-L-glutamate or 1,5-ditetradecyl-N-arginyl-L-glutamate can be used as an effective plasmid DNA delivery system.

    DOI

  • Magnetic driven alginate nanoparticles for targeted drug delivery

    Gianni Ciofani, Vittoria Raffa, Yosuke Obata, Arianna Menciassi, Paolo Dario, Shinji Takeoka

    CURRENT NANOSCIENCE   4 ( 2 ) 212 - 218  2008.05

     View Summary

    The aim of this paper is to develop highly magnetized, biodegradable and biocompatible, polymeric nanoparticles for drug delivery in cell therapy. Alginate magnetic nanoparticles are realized by an emulsion/reticulation technique, after the dispersion of magnetite in an alginate solution. Such nanoparticles are characterized in terms of external morphology (FIB imaging), microstructure (TEM imaging), size distribution, zeta potential, magnetic properties (SQUID analysis) and drug release behaviour. Magnetization curves show the typical trend of superparamagnetic materials. Important parameters, such as magnetic permeability and magnetic momentum, are derived by employing Langevin theory. Experimental results reveal that a bi-exponential model fully describes the drug release. Finally, in vitro experiments on NIH/3T3 cells are carried out and demonstrate that our magnetic alginate nanoparticles can effectively drive the drug delivery towards an external magnetic field source.

    DOI

  • Fabrication of free-standing nanoparticle-fused nanosheets and their hetero-modification using sacrificial film

    Yosuke Okamura, Saori Utsunomiya, Hidenori Suzuki, Daisuke Niwa, Tetsuya Osaka, Shinji Takeoka

    COLLOIDS AND SURFACES A-PHYSICOCHEMICAL AND ENGINEERING ASPECTS   318 ( 1-3 ) 184 - 190  2008.04

     View Summary

    Sheet-shaped carriers, having obverse and reverse surfaces and thus a large contact area as a targeting site, have several advantages over spherical-shaped carriers, which have an extremely small contact area. Herein is proposed a novel method for the preparation of free-standing nanoparticle-fused nanosheets having uniform micrometer shape, nanometer thickness and heterogenous surfaces, using a water-soluble sacrificial film. This was achieved by combination of four processes: (1) specific adsorption of latex beads at pH 5.0 and a concentration of 1.0 x 10(11) mL(-1) onto a patterned dodecyltrimethoxysilane self-assembled monolayer (DTS-SAM) region by a conventional dry patterning process, (2) fabrication of the latex bead-sheet via thermal-fusion at 110 degrees C for 60s, (3) preparation of the free-standing nanosheet by detachment from the DTS-SAM, and (4) hetero-modification of the resulting nanosheet using a water-soluble poly(acrylic acid) as a sacrificial supporting film. Thus, this sheet-shaped carrier having hetero-surfaces can be regarded as a new material for delivery of drugs, hemostatic reagents and as wound dressings for bum injury, etc. (C) 2007 Elsevier B.V. All rights reserved.

    DOI

  • Loading of curcumin into macrophages using lipid-based nanoparticles

    Keitaro Sou, Shunsuke Inenaga, Shinji Takeoka, Eishun Tsuchida

    INTERNATIONAL JOURNAL OF PHARMACEUTICS   352 ( 1-2 ) 287 - 293  2008.03

     View Summary

    Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, Cm) is a natural compound which possesses antioxidant, anti-inflammatory and anti-tumor ability. Here, phospholipid vesicles or lipid-nanospheres embedding Cm (CmVe or CmLn) were formulated to deliver Cm into tissue macrophages through intravenous injection. Cm could be solubilized in hydrophobic regions of these particles to form nanoparticle dispersions, and these formulations showed ability to scavenge reactive oxygen species as antioxidants in dispersions. At 6 h after intravenous injection in rats via the tail vein (2 mg Cm/kg bw), confocal microscopic observations of tissue sections showed that Cm was massively distributed in cells assumed as macrophages into the bone marrow and spleen. Taken together, these results indicate that the lipid-based nanoparticulates provide improved intravenous delivery of Cm to tissues macrophages, specifically bone marrow and splenic macrophages in present formulation, which has therapeutic potential as an antioxidant and anti-inflammatory. (C) 2007 Elsevier B.V. All rights reserved.

    DOI

  • Encapsulation of concentrated hemoglobin solution in phospholipid vesicles retards the reaction with NO, but not CO, by intracellular diffusion barrier

    Hiromi Sakai, Atsushi Sato, Kaoru Masuda, Shinji Takeoka, Eishun Tsuchida

    JOURNAL OF BIOLOGICAL CHEMISTRY   283 ( 3 ) 1508 - 1517  2008.01

     View Summary

    One physiological significance of the red blood cell (RBC) structure is that NO binding of Hb is retarded by encapsulation with the cell membrane. To clarify the mechanism, we analyzed Hb-vesicles (HbVs) with different intracellular Hb concentrations, [Hb] in, and different particle sizes using stopped-flow spectrophotometry. The apparent NO binding rate constant, k(on)'((NO)), of HbV at [Hb](in) = 1 g/dl was 2.6 x 10(7) M-1 s(-1), which was almost equal to k(on)((NO)) of molecular Hb, indicating that the lipid membrane presents no obstacle for NO binding. With increasing [Hb] in to 35 g/dl, k(on)'((NO)) decreased to 0.9 x 10(7) M-1 s(-1), which was further decreased to 0.5 x 10(7) M-1 s(-1) with enlarging particle diameter from 265 to 452 nm. For CO binding, which is intrinsically much slower than NO binding, k(on)'((CO)) did not change greatly with [Hb] in and the particle diameter. Results obtained using diffusion simulations coupled with elementary binding reactions concur with these tendencies and clarify that NO is trapped rapidly by Hb from the interior surface region to the core of HbV at a high [Hb] in, retarding NO diffusion toward the core of HbV. In contrast, slow CO binding allows time for further CO- diffusion to the core. Simulations extrapolated to larger particles (8 mu m) showing retardation even for CO binding. The obtained k(on)'((NO)) and k(on)'((CO)) yield values similar to those reported for RBCs. In summary, the intracellular, not extracellular, diffusion barrier is predominant due to the rapid NO binding that induces a rapid sink of NO from the interior surface to the core, retarding further NO diffusion and binding.

    DOI

  • Fibrinogen γ-chain peptide-coated, adenosine diphosphate-incorporated liposomes as a synthetic platelet substitute

    Handa, M, Okamura, Y, Takeoka, S, Ikeda, Y

    Transfusion   48   6A  2008

  • 医用材料としての高分子超薄膜(ナノシート)の構築

    藤枝 俊宣, 岡村 陽介, 武岡 真司

    コンバーティック   10   137  2008

  • 高分子ナノシートの構築と新しい医用材料としての展開

    岡村 陽介, 藤枝 俊宣, 武岡 真司

    化学工業   59   1 - 7  2008

  • Novel Polymeric Nanosheets for Gerontic Applications

    Takeoka, S, Fukui, Y, Fujie, T, Okamura, Y

    Gerontechnology   7   220 - 224  2008

  • 血液の仕組みと人工血液(血液代替物)へのアプローチ

    武岡 真司, 岡村 陽介

    化学と教育   56   2 - 5  2008

  • 人工血小板の開発

    岡村 陽介, 武岡 真司, 半田 誠, 池田 康夫

    Medical Science Digest,   3   138 - 140  2008

  • Ubiquitous transference of a free-standing polysaccharide nanosheet with the development of a nano-adhesive plaster

    Toshinori Fujie, Yosuke Okamura, Shinji Takeoka

    ADVANCED MATERIALS   19 ( 21 ) 3549 - +  2007.11

     View Summary

    A free-standing polysaccharide nanosheet prepared by using a spin-coating-assisted layer-by-layer method is transferred from silicone rubber onto human skin (see figure) by fabricating a three-layered 'nano-adhesive plaster' involving a water-soluble sacrificial layer. By using this novel technique, conventional ultrathin films can be made without the need for a solid substrate.

    DOI

  • Rheological properties of hemoglobin vesicles (artificial oxygen carriers) suspended in a series of plasma-substitute solutions

    Hiromi Sakai, Atsushi Sato, Shinji Takeoka, Eishun Tsuchida

    LANGMUIR   23 ( 15 ) 8121 - 8128  2007.07

     View Summary

    Hemoglobin vesicles (HbV) or liposome-encapsulated Hbs are artificial oxygen carriers that have been developed for use as transfusion alternatives. The extremely high concentration of the HbV suspension (solutes, ca. 16 g/dL; volume fraction, ca. 40 vol %) gives it an oxygen-carrying capacity that is comparable to that of blood. The HbV suspension does not possess a colloid osmotic pressure. Therefore, HbV must be suspended in or co-injected with an aqueous solution of a plasma substitute (water-soluble polymer), which might interact with HbV. This article describes our study of the rheological properties of HbV suspended in a series of plasma substitute solutions of various molecular weights: recombinant human serum albumin (rHSA), dextran (DEX), modified fluid gelatin (MFG), and hydroxylethyl starch (HES). The HbV suspended in rHSA was nearly Newtonian. Other polymersHES, DEX, and MFGinduced HbV flocculation, possibly by depletion interaction, and rendered the suspensions as non-Newtonian with a shear-thinning profile (10(-4)-10(3) s(-1)). These HbV suspensions showed a high storage modulus (G') because of the presence of flocculated HbV. However, HbV suspended in rHSA exhibited a very low G'. The viscosities of HbV suspended in DEX, MFG, and high-molecular-weight HES solutions responded quickly to rapid step changes in shear rates of 0.1-100 s(-1) and a return to 0.1 s(-1), indicating that flocculation is both rapid and reversible. Microscopically, the flow pattern of the flocculated HbV that perfused through microchannels (4.5 mu m deep, 7 mu m wide, 20 cmH(2)O applied pressure) showed no plugging. Furthermore, the time required for passage was simply proportional to the viscosity. Collectively, the HbV suspension viscosity was influenced by the presence of plasma substitutes. The HbV suspension provides a unique opportunity to manipulate rheological properties for various clinical applications in addition to its use as a transfusion alternative.

    DOI

  • Prolonged hemostatic ability of polyethylene glycol-modified polymerized albumin particles carrying fibrinogen gamma-chain dodecapeptide

    Yosuke Okamura, Toshinori Fujie, Hitomi Maruyama, Makoto Handa, Yasuo Ikeda, Shinji Takeoka

    TRANSFUSION   47 ( 7 ) 1254 - 1262  2007.07

     View Summary

    BACKGROUND: Second-gene ration platelet (PLT) substitutes for treatment of bleeding were studied and the focus was on a dodecapeptide, HHLGGAKQAGDV (H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411) and exists only in a fibrinogen domain.
    STUDY DESIGN AND METHODS: H12 was conjugated to the surface of polymerized albumin particles (polyAlb) modified with polyethylene glycol (PEG) chains to produce biocompatible particles (H12-PEG-polyAlb) that had prolonged blood circulation t(1/2) and were more stable in vitro and in vivo compared with H12-polyAlb (not modified with PEG). H12-PEG-polyAlb was administered intravenously into thrombocytopenic rats and the t(1/2) of the particles and the tail bleeding time were measured to evaluate the prolongation in the hemostatic effect.
    RESULTS: H12-PEG-polyAlb particles modified with PEG prolonged the t1/2 and maintained specific binding ability to activated PLTs. The particles dose dependently shortened the tail bleeding time of thrombocytopenic rats 6 hours after injection.
    CONCLUSION: H12-PEG-polyAlb may be a suitable candidate for treatment of bleeding into thrombocytopenic patients as an alternative to PLT concentrate transfusion.

    DOI

  • Synthesis of porphyrins bearing uracyl groups and their assembly induced by melamine derivatives

    Satoshi Arai, Hiroki Ohshiro, Hiroyuki Nishide, Shinji Takeoka

    POLYMERS FOR ADVANCED TECHNOLOGIES   18 ( 6 ) 497 - 501  2007.06

     View Summary

    Self-assembled porphyrins via noncovalent bonding have attracted wide-ranging researchers in material science. We reported herein the synthesis of the tetraphenyl porphyrin derivatives bearing uracyl groups as acceptor-donor-acceptor (ADA) type hydrogen bonding units, through the condensation of 5,10- or 5,15-bis (3-amino-4-ethylhexylphenyl) porphyrin derivatives with 6-carboxyuracyl derivatives. When two porphyrins having uracyl groups at the different substituted positions were respectively mixed with a melamine derivative in benzene, H-1 NMR spectra showed that the 5,15 substituted uracyl porphyrin formed a hydrogen-bonded suprastructure with the melamine derivative as a complementary molecule to the uracyl moiety, although the other 5,10-substituted uracylporphyrin could not form such a structure. The SEM observation indicated that the mixture with the 5,15-substituted uracyl porphyrin and the melamine with long alkyl chains formed a sheet-like structure. Copyright (C) 2007 John Wiley & Sons, Ltd.

    DOI

  • Selective uptake of surface-modified phospholipid vesicles by bone marrow macrophages in vivo

    Keitaro Sou, Beth Goins, Shinji Takeoka, Eishun Tsuchida, William T. Phillips

    BIOMATERIALS   28 ( 16 ) 2655 - 2666  2007.06

     View Summary

    An advantage of using vesicles (liposomes) as drug delivery carriers is that their pharmacokinetics can be controlled by surface characteristics, which can permit specific delivery of the encapsulated agents to organs or cells it? vivo. Here we report a vesicle formulation which targets the bone marrow after intravenous injection in rabbits. Surface modification of the vesicle with an anionic amphiphile; L-glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5- dihexadecyl ester (SA) results in significant targeting of vesicles to bone marrow. Further incorporation of as little as 0.6 mol% of poly(ethylene glycol)-lipid (PEG-DSPE) passively enhanced the distribution of SA-vesicles into bone marrow and inhibited hepatic uptake. In this model, more than 60% of the intravenously injected vesicles were distributed to bone marrow within 6 h after administration of a small dose of lipid (15 mg/kg b.w.). Histological evidence indicates that the targeting was achieved due to uptake by bone marrow macrophages (BMM phi). The efficient delivery of encapsulated scintigraphic and fluorescent imaging agents to BMM phi suggests that vesicles are promising carriers for the specific targeting of BMM phi and may be useful for delivering a wide range of therapeutic agents to bone marrow. (c) 2007 Elsevier Ltd. All rights reserved.

    DOI

  • A hydrogen-bonded supramolecular hexagonal columnar liquid crystal composed of a tricarboxylic triphenylene and monopyridyl dendrons

    Shinsuke Ishihara, Yuusuke Furuki, Shinji Takeoka

    CHEMISTRY LETTERS   36 ( 2 ) 282 - 283  2007.02

     View Summary

    Tricarboxylic triphenylene (TPC5) and monopyridyl dendron (DenC12) were mixed in 1:2, 1:3, and 1:4 molar ratios, and investigation by IR, DSC, and XRD studies proved that TPC5 and DenC12 self-assembled to form a hexagonal columnar liquid crystal with 1:3 molar stoichiometry via complementary hydrogen-bonds.

    DOI

  • Enzymatic elimination of hydrogen peroxide by a mthemoglobin/L-tyrosine system

    Tomoyasu Atoji, Hirotaka Yatami, Motonari Aihara, Shinji Takeoka

    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND BIOTECHNOLOGY   35 ( 6 ) 555 - 567  2007

     View Summary

    We studied the peroxidase activity of ferrylhemoglobin radical (Hb(Fe4+ = 0*)) generated by the reaction of metHb (Hb(Fe3+)) with hydrogen peroxide (H2O2). To clarify the behaviors of ferrylHb radical, it was isolated from the reaction mixture of metHb and H2O2 by GPC at 4 degrees C. The radical species underwent rapid autoreduction to metHb at 37 degrees C accompanied with denaturation; however, it was stable for several minutes at VC. In ESR measurements, the signal of the ferrylHb radical immediately disappeared in the presence of L-Tyrosine (L-Tyr), and simultaneously, the signal of the ferric heme increased. This suggested that the ferrylHb radical immediately converted to metHb by L-Tyr even at 4 degrees C. Furthermore, dimerized L-Tyr was detected in the reaction mixture. This showed that the ferrylHb radical was reduced to metHb by electron donation from L-Tyr. The enzymatic reaction using L-Tyr as the substrate resulted in the elimination of H2O2 in this system.

    DOI

  • Effects of hemoglobin vesicles, a liposomal artificial oxygen carrier, on hematological responses, complement and anaphylactic reactions in rats

    Hideki Abe, Hiroshi Azuma, Miki Yamaguchi, Mitsuhiro Fujihara, Hisami Ikeda, Hiromi Sakai, Shinji Takeoka, Eishun Tsuchida

    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND BIOTECHNOLOGY   35 ( 2 ) 157 - 172  2007

     View Summary

    Hemoglobin vesicle (HbV), a liposomal oxygen carrier containing human hemoglobin, was intravenously infused into rats. After the infusion of saline, the HbV or empty vesicle (EV), numbers of red cells, leukocytes and platelets in peripheral blood were unchanged during the observation period of one week in addition to each time point among three groups. However, the lymphocyte ratio transiently decreased and the granulocyte ratio increased in the HbV and EV groups at 6 h after the infusion. Those changes returned to the initial value one day after the infusion and those were maintained for the subsequent observation period. No dramatic change was seen in the ratio of CD4(+)/CD8(+) T cells. A transient decrease of the complement titer was observed three days after the infusion of HbV and EV, although the consumption of complement titer was not detected in rat serum by mixing HbV or EV in vitro, indicating that the transient decrease of complement titer in vivo was not due to the consumption of complement due to the interaction with HbV or EV. Multiple infusions of HbV caused the decrease of complement titer only after the first infusion and no allergic reaction was observed. No anaphylactic shock was observed in rats administered with EV several times, while ovalbumin (OVA) sensitized rats died with symptoms of respiratory distress after the second OVA administration. These results indicate that HbV could be administered without serious clinical symptoms or adverse reactions.

    DOI

  • Atropisomers of meso-conjugated uracyl porphyrin derivatives and their assembling structures

    Satoshi Arai, Daisuke Niwa, Hiroyuki Nishide, Shinji Takeoka

    ORGANIC LETTERS   9 ( 1 ) 17 - 20  2007.01

     View Summary

    We have synthesized a 5,15 meso-substituted methyluracyl porphyrin derivative bearing 6-methyluracyl units directly at the meso positions. The atropisomerization was regulated by steric replusion between the methyl substituents. When the atropisomers were mixed with alkylated melamine as a complementary hydrogen-bonding unit, the hydrogen-bonded assemblies were analyzed by diffusion-ordered spectroscopy (DOSY) in solution, which clarified that the alpha alpha isomer formed a face-to-face dimer, whereas the alpha beta isomer took a zigzag structure.

    DOI

  • ヘモグロビン小胞体(HbV)-リコンビナントアルブミン分散溶液による40%交換輸血:ラット脾臓内HbV代謝と造血に関する2週間の観察

    酒井宏水, 堀之内宏久, 山本学, 池田 栄二, 武岡真司, 高折益彦, 土田英俊, 小林紘一

    日本輸血細胞治療学会誌(二次掲載)    2007

  • Development of Nano-Biotechnology; from Artificial Platelets to Nano-Adhesive Plasters

    S. Takeoka

    The 4th 21COE International Symposium on “Practical Nano-Chemistry”, Tokyo   Program and Abstracts   5 - 5  2006.12

  • ナノ絆創膏の開発-ヘテロな表裏を持つ生分解ナノシートの開発-

    武岡真司

    第28回生体膜と薬物の相互作用シンポジウム ランチョンセミナー, 静岡    2006.11

  • Hemoglobin vesicles containing methemoglobin and L-tyrosine to suppress methemoglobin formation in vitro and in vivo

    Tomoyasu Atoji, Motonari Aihara, Hiromi Sakai, Eishun Tsuchida, Shinji Takeoka

    BIOCONJUGATE CHEMISTRY   17 ( 5 ) 1241 - 1245  2006.09

     View Summary

    Hemoglobin (Hb) vesicles have been developed as cellular-type Hb-based O-2 carriers in which a purified and concentrated Hb solution is encapsulated with a phospholipid bilayer membrane. Ferrous Hb molecules within an Hb vesicle were converted to ferric metHb by reacting with reactive oxygen species such as hydrogen peroxide (H2O2) generated in the living body or during the autoxidation of oxyHb in the Hb vesicle, and this leads to the loss of O-2 binding ability. The prevention of metHb formation by H2O2 in the Hb vesicle is required to prolong the in vivo O-2 carrying ability. We found that a mixed solution of metHb and L-tyrosine (L-Tyr) showed an effective H2O2 elimination ability by utilizing the reverse peroxidase activity of metHb with L-Tyr as an electron donor. The time taken for the conversion of half of oxyHb to metHb (T-50) was 420 min for the Hb vesicles containing 4 g/dL (620 mu M) metHb and 8.5 mM L-Tyr ((metHb/L-Tyr) Hb vesicles), whereas the time of conversion for the conventional Hb vesicles was 25 min by stepwise injection of H2O2 (310 mu M) in 10 min intervals. Furthermore, in the (metHb/L-Tyr) Hb vesicles, the metHb percentage did not reach 50% even after 48 h under a pO(2) of 40 Torr at 37 degrees C, whereas T-50 of the conventional Hb vesicles was 13 h under the same conditions. Moreover, the T-50 values of the conventional Hb vesicles and the (metHb/L-Tyr) Hb vesicles were 14 and 44 h, respectively, after injection into rats (20mL/kg), confirming the remarkable inhibitory effect of metHb formation in vivo in the (metHb/L-Tyr) Hb vesicles.

    DOI

  • Preparation of freestanding nano-adhesive plasters having hetero-surfaces

    S. Takeoka

    232nd ACS Fall National Meeting in San Francisco   poly   234  2006.09

  • Hemostatic effects of polymerized albumin nano-particles carrying fibrinogen g-chain dodecapeptide as platelet substitutes

    Yosuke Okamura, Toshinori Fujie, Makoto Handa, Yasuo Ikeda, Shinji Takeoka

    232nd ACS Fall National Meeting in San Francisco   poly   481  2006.09

  • Novel Approaches to Develop Artificial Platelets / Platelet Substitutes (S12-2)

    S. Takeoka

    The 4th Asian-Pacific Congress on Thrombosis and Hemostasis, Suzhou   Abstract Book   48  2006.09

  • 分子集合科学を利用した人工血球の創製とその応用

    武岡真司

    第43回茅コンファレンス、山梨    2006.08

  • 血小板代替物の開発

    武岡真司, 岡村陽介

    バイオインダストリー   23 ( 7 ) 65 - 71  2006.07

  • 分子集合化学を用いた人工血小板の研究展開—ナノバイオテクノロジーへの挑戦—

    武岡真司

    第3回ニューマテリアル研究会、滋賀    2006.06

  • “Design and Preparation of Blood Substitutes Using Molecular Assembling Technologies”,

    S. Takeoka

    3rd IUPAC-sponsored International Symposium on Macro- and Supramolecular Architectures and Materials (MAM-06), Tokyo   Program and Abstracts   71  2006.05

  • Development of Nano Band-Aid Adhereing To A Wound From Inside of A Blood Vessel

    S. Takeoka

    BASF Workshop Nanostructured Surfaces, Shanghai   Abstract   84  2006.05

  • Design and Preparation of Blood Substitutes Using Molecular Assembling Technologies

    S. Takeoka

    3rd IUPAC-sponsored International Symposium on Macro- and Supramolecular IUPAC-sponsored International Symposium on Macro- and Supramolecular Architectures and Materials (MAM-06), Waseda Univ., Tokyo, JAPAN   Program and Abstracts   71  2006.05

  • 血管の内側から傷口に貼るナノ絆創膏の開発

    武岡真司

    国際BIO EXPO バイオ アカデミック フォーラム、東京ビックサイト    2006.05

  • 分子集合科学を利用した人工血液の創製

    武岡真司

    人工血液   13 ( 4 ) 136 - 147  2006.04

  • 血小板代替物の開発の現状

    岡村陽介, 藤枝俊宣, 半田誠, 池田康夫, 武岡真司

    人工血液   13 ( 4 ) 155 - 160  2006.04

  • 1.人工血液はどこまで出来ているのか、2.早稲田大学理工学部の新たな出発

    武岡真司

    「夢のたまご塾」飛騨アカデミー、ホテル季古里    2006.03

  • 薄膜状高分子構造体とその調製 方法-ナノ絆創膏への挑戦-

    武岡真司

    Technology Link in W.T.L.O2    2006.03

  • Acute 40 percent exchange-transfusion with hemoglobin-vesicles (HbV) suspended in recombinant human serum albumin solution: degradation of HbV and erythropoiesis in a rat spleen for 2 weeks

    H Sakai, H Horinouchi, M Yamamoto, E Ikeda, S Takeoka, M Takaori, E Tsuchida, K Kobayashi

    TRANSFUSION   46 ( 3 ) 339 - 347  2006.03

     View Summary

    BACKGROUND: Hemoglobin-vesicles (HbVs; diameter, 251 +/- 81 nm) are artificial O-2 carriers. Their efficacy for acute exchange transfusion has been characterized in animal models. However subsequent profiles of recovery involving the degradation of HbV in the reticuloendothelial system (RES) and hematopoiesis remain unknown.
    STUDY DESIGN AND METHODS: Isovolemic 40 percent exchange transfusion was performed in 60 male Wistar rats with HbV suspended in 5 g per dL recombinant human serum albumin (rHSA; HbV/rHSA, [Hb] = 8.6 g/dL), stored rat RBCs suspended in rHSA (sRBC/rHSA), or rHSA alone. Hematological and plasma biochemical analyses and histopathological examination focusing on the spleen were conducted for the subsequent 14 days.
    RESULTS: The reduced hematocrit (Hct) level (26%) for the HbV/rHSA and rHSA groups returned to its original level (43%) in 7 days. Plasma erythropoietin was elevated in all groups: the rHSA group showed the highest value on Day 1 (321 +/- 123 mIU/mL) relating to the anemic conditions (HbV/rHSA, 153 +/- 22; sRBC/rHSA, 63 +/- 7; baseline, 21 +/- 3). Simultaneously, splenomegaly occurred in all the groups as HbV/rHSA > rHSA > sRBC/rHSA. Histopathologically, the accumulated HbV in the spleen was undetectable by Day 14, but hemosiderin was deposited in slight quantities for both the HbV/rHSA and sRBC/rHSA groups. Considerable amounts of erythroblasts were apparent in the spleens of both the rHSA and the HbV/rHSA groups.
    CONCLUSION: HbVs were phagocytized and degraded in RES, a physiological compartment for the degradation of RBCs, and the elevated erythropoietic activity resulted in the complete recovery of Hct within 7 days in the rat model.

    DOI

  • Modulation of Nanoparticles for Blood Substitutes

    S. Takeoka

    21COE International Symposium, UNIVERSITE MONTPELLIER II   Abstract   7  2006.02

  • Interaction of hemoglobin vesicles, a cellular-type artificial oxygen carrier, with human plasma: Effects on coagulation, kallikrein-kinin, and complement systems

    H Abe, M Fujihara, H Azuma, H Ikeda, K Ikebuchi, S Takeoka, E Tsuchida, H Harashima

    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND BIOTECHNOLOGY   34 ( 1 ) 1 - 10  2006

     View Summary

    Hemoglobin vesicles (HbVs), cellular-type artificial oxygen carriers containing human hemoglobin, were assessed for their biocompatibility by mixing with human plasma in vitro . Among three kinds of HbVs (PEG-DPEA-HbV, PEG-DPPG-HbV and DPPG-HbV), PEG-DPEA-HbV did not affect the extrinsic or intrinsic coagulation activities of the plasma, while PEG-DPPG-HbV and DPPG-HbV tended to shorten the intrinsic coagulation time. The kallikrein-kinin cascade of the plasma was slightly activated by PEG-DPPG-HbV and DPPG-HbV, but not by PEG-DPEA-HbV. The complement consumption of the plasma was observed by incubation with DPPG-HbV, but not with PEG-DPEA-HbV or PEG-DPPG-HbV. These results indicate that PEG-DPEA-HbV has a higher biocompatibility with human plasma.

    DOI

  • Modulation of Nanoparticles for Blood Substitutes (Red Blood Cell Substitutes and Platelet Substitutes)

    S. Takeoka

    Waseda-Korea Universities' Joint Symposium on "Nanoscience and Nanotechnology"   Abstract  2006.01

  • Cooperative Effects of Platelets Aggregation of Platelet Substitutes at High Shear Rates;Mixed System of Fibrinogen gamma-Chain Dodecapeptide- or Glycoprotein Ibalpha-Conjugated Latex Beads under Flow Conditions

    Okamura, Y, Suzuki, H, Tsuchida, E, Handa, M, Ikeda, Y, Takeoka, S

    J. Artif. Organs   9   251 - 258  2006

    DOI

  • Host-guest assembly of pyridinium-conjugated calix[4]arene via cation-pi interaction

    S Ishihara, S Takeoka

    TETRAHEDRON LETTERS   47 ( 2 ) 181 - 184  2006.01

     View Summary

    25-(4-Pyridiniumbutoxy)-26,27,28-trihydroxycalix[4]arene bromide I was designed to have both host and guest units in one molecule, and was assembled to become an oligomer via a cation-pi interaction. X-ray diffraction (XRD) crystallographic study of I revealed that it oriented in a one-dimensional structure. Titrimetric H-1 NMR analysis and electrospray ionization mass spectrometry (ESI-MS) analysis indicated that I formed an oligomer in solution, and the restraint of the segmental motion would lead to the stabilization of the cation-pi interaction compared with a bi-molecular complex composed of 25,26,27,28-tetrahydroxycalix[4]arene and N-butylpyridinium bromide. (c) 2005 Elsevier Ltd. All rights reserved.

    DOI

  • Porphyrin capped with calix[4]arene derivative via hydrogen bonds

    S Arai, H Ohkawa, S Ishihara, T Shibue, S Takeoka, H Nishide

    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN   78 ( 11 ) 2007 - 2013  2005.11

     View Summary

    A calix[4]arene-porphyrin duplex was prepared by mixing equimolar amounts of hydroxy or carboxy calix[4]arene derivatives and pyridyl or o-aminophenylporphyrin derivatives. Electrospray ionization mass spectrometry (ESI-MS) was applied to screen a suitable pair of a calix[4]arene and a porphyrin. meso-Tetra(2-pyridyl)porphyrin formed a duplex with tetrahydroxy or tetrahydroxymethylcalix[4]arene via triple or quadruple hydrogen bonds. H-1 NMR spectra showed that the tetrahydroxycalix[4]arene was symmetrically located upon the porphyrin ring, whereas trihydroxycalix[4]arene was slanted on the porphyrin ring. The duplex of meso-tetra(2-pyridyl)porphyrinatocobalt(II) and tetrahydroxymethylcalix[4]arene formed a complex with benzylimidazole, which was capable of reversible dioxygen-binding. The capping structure upon porphyrin provided by calix[4]arene raised the life-time of dioxygen-adduct compared with the porphyrin without calix[4]arene.

    DOI

  • Hemostatic effects of phospholipid vesicles carrying fibrinogen gamma chain dodecapeptide in vitro and in vivo

    Y Okamura, Maekawa, I, Y Teramura, H Maruyama, M Handa, Y Ikeda, S Takeoka

    BIOCONJUGATE CHEMISTRY   16 ( 6 ) 1589 - 1596  2005.11

     View Summary

    We studied prototypes of platelet substitutes that bear on their surface a dodecapeptide, HHLG-GAKQAGDV (1112). The peptide is a fibrinogen gamma chain carboxy-terminal sequence (gamma 400-411) and recognizes specifically the active form of glycoprotein (GP) IIb/IIIa on the surface of activated platelets. We conjugated H12 to the end of poly(ethylene glycol) chains on the surface of a phospholipid vesicle with an average diameter of 220 nm to prepare H12-PEG-vesicles. The half-life of the H12-PEG-vesicles was significantly prolonged by PEG modification, and the ability of H12 on the surface of the vesicle to recognize GPIIb/IIIa was maintained even though the surface was modified with PEG chains. The H12-PEG-veiscles enhanced the in vitro thrombus formation of platelets that were adhering to a collagen-immobilized plate, when thrombocytopenia-imitation blood was passed over the plate. Based on the flow cytometric analyses of PAC-1 binding and P-selectin expression, the H12-PEG-vesicles were shown not to cause platelet activation. Furthermore, the H12-PEG-vesicles dose-dependently shortened the tail bleeding time of thrombocytopenic rats. It was confirmed that the H12-PEG-vesicles had a hemostatic effect and may be a suitable candidate for an alternative to human platelet concentrates transfused into thrombocytopenic patients.

    DOI

  • 第12回 日本血液代替物学会年次大会を開催して

    武岡真司

    人工血液   13 ( 3 ) 98 - 99  2005.11

  • 赤血球代替物、血小板代替物の開発の現状と将来展望

    武岡真司

    第49回 日本輸血学会北海道支部会例会、北海道赤十字血液センター    2005.09

  • 分子集合科学を利用した人工赤血球の創製とその応用

    武岡真司

    治験促進啓発シンポジウム、軽井沢万平ホテル   日本医師会治験促進センター発刊   29 - 31  2005.09

  • 分子集合科学を利用した人工血液の創製とその応用

    武岡真司

    第43回 茅コンファレンス、大泉高原八ヶ岳ロイヤルホテル   予稿集   29  2005.08

  • Hemostatic effects of fibrinogen gamma-chain dodecapeptide-conjugated polymerized albumin particles in vitro and in vivo

    Y Okamura, S Takeoka, Y Teramura, H Maruyama, E Tsuchida, M Handa, Y Ikeda

    TRANSFUSION   45 ( 7 ) 1221 - 1228  2005.07

     View Summary

    BACKGROUND: Prototypes of platelet (PLT) substitutes have been studied and the focus was on a dodecapeptide, HHLGGAKQAGDV (H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411) and exists only in the fibrinogen domain.
    STUDY DESIGN AND METHODS: H12 was conjugated to the surface of polymerized albumin particles (polyAlb) as biocompatible and biodegradable particles with a mean diameter of 260 +/- 60 nm, and the hemostatic ability of H12-conjugated polyAlb (H12-polyAlb) under flow conditions and thrombocytopenic rats have been studied
    RESULTS: H12-polyAlb enhanced the in vitro thrombus formation of activated PLTs on a collagen-immobilized plate when exposed to the flowing thrombocytopenic imitation blood. Furthermore, the analysis of the tail bleeding time of rats that were made thrombocytopenic by busulfan injection showed that H12-polyAlb had a hemostatic effect. Based on the bleeding time and the amount injected, the hemostatic capacity of 20 H12-polyAlb was estimated to correspond to that of one PLT.
    CONCLUSION: These results were important first steps toward the development of PLT substitutes and indicated that H12-polyAlb may be a suitable candidate for an alternative to human PIT concentrates transfused into thrombocytopenic patients in the future.

    DOI

  • Hemostatic Effects of Polymerized Albumin Particles Carrying Recombinant Glycoprotein Iba As Platelet Substitutes in vitro and in vivo

    Hemostatic Effects of Polymerized Albumin Particles Carrying Recombinant Glycoprotein Iba As Platelet Substitutes in vitro, in vivo

    The Xth International Symposium on Blood Substitutes, Brown Univ., Providence, RI, USA   Program and Abstracts   92  2005.06

  • 第53回日本輸血学会、止血機能を有する微粒子系(血小板代替物)の設計と機能評価

    武岡真司

    日本輸血学会雑誌   51 ( 2 ) 140  2005.05

  • 人工赤血球・人工血小板の開発の現状

    武岡真司

    臨床麻酔   29   721 - 726  2005.04

  • New generation of hemoglobin-based oxygen carriers evaluated for oxygenation of critically ischemic hamster flap tissue

    C Contaldo, J Plock, H Sakai, S Takeoka, E Tsuchida, M Leunig, A Banic, D Erni

    CRITICAL CARE MEDICINE   33 ( 4 ) 806 - 812  2005.04

     View Summary

    Objectives: The aim of this study was to investigate and compare the effects of a traditionally formulated, low-viscosity, right-shifted polymerized bovine hemoglobin solution and a highly viscous, left-shifted hemoglobin vesicle solution (HbV-HES) on the oxygenation of critically ischemic peripheral tissue.
    Design. Randomized, prospective study.
    Setting. University laboratory.
    Subject. A total of 40 male golden Syrian hamsters.
    Interventions: Island flaps were dissected from the back skin of anesthetized hamsters. The flap included a critically ischemic, hypoxic area that was perfused via a collateralized vasculature. One hour after completion of the preparation, the animals received a 33% blood exchange with 6% hydroxyethyl starch 200/0.5 (HES, n = 9), HbV suspended in HES (HbV-HES, n = 8), or polymerized bovine hemoglobin solution (n = 9).
    Measurements and Main Results., Three hours after the blood exchange, microcirculatory blood flow (laser-Doppler flowmetry) was increased to 262% of baseline for HbV-HES (p < .01) and 197% for polymerized bovine hemoglobin solution (p < .05 vs. baseline and HbV-HES). Partial tissue oxygen tension (bare fiber probes) was only improved after HbV-HES (9.4 torr to 14.2 torr, p < .01 vs. baseline and other groups). The tissue lactate/pyruvate ratio (microdialysis) was elevated to 51 in the untreated control animals, and to 34 +/- 8 after HbV-HES (p < .05 vs. control) and 38 +/- 11 after polymerized bovine hemoglobin solution (not significant).
    Conclusions. Our study suggests that in critically ischemic and hypoxic collateralized peripheral tissue, oxygenation may be improved by normovolemic hemodilution with HbV-HES. We attributed this improvement to a better restoration of the microcirculation and oxygen delivery due to the formulation of the solution.

    DOI

  • 機能性分子素子としての人工赤血球・人工血小板の構築

    武岡真司

    日本顕微鏡学会関東支部 第29回 講演会   予稿集   15  2005.03

  • Oxygen transport by low and normal P50 Hb-vesicles in extreme hemodilution

    Cabrales, P, Sakai, H, Tsai, AG, Takeoka, S, Tsuchida, E, Intaglietta, M

    Am. J. Physiol. Heart Circ. Physiol.   288   H1885 - H1892  2005

    DOI

  • Effects of hemoglobin vesicles on resting and agonist-stimulated human platelets in vitro

    S Wakamoto, M Fujihara, H Abe, M Yamaguchi, H Azuma, H Ikeda, S Takeoka, E Tsuchida

    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND BIOTECHNOLOGY   33 ( 2 ) 101 - 111  2005

     View Summary

    Hemoglobin vesicles (HbV) are artificial oxygen carriers that encapsulate a concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane. The oxygen transporting ability of HbV in vivo has been demonstrated by the transfusion of HbV into hemorrhagic shock rodent models. However, the compatibility of HbV with human blood cells must be evaluated. Preincubation of platelets with concentrations of 20% or 40% HbV had no effect on the binding of PAC-1, a monoclonal antibody that detects activation-dependent conformational changes in alpha(IIb)beta(3) on platelets, or the surface expression of CD62P in whole blood. ADP-induced increases in PAC-1 binding were significantly enhanced by exposing the platelets to concentrations of either 20% or 40% HbV, whereas the ADP-induced increases in CD62P expression were not affected by HbV treatment at either concentration. Preincubation of platelet-rich plasma (PRP) with HbV minimally reduced the spontaneous release of TXB2 and RANTES, but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with ADP. Similarly, preincubation of PRP with HbV minimally reduced the spontaneous release of RANTES but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with collagen, although collagen-induced serotonin release tended to decrease with HbV pretreatment. These data suggest that the exposure of human platelets to high concentrations of HbV (up to 40%) in vitro did not cause platelet activation and did not adversely affect the formation and secretion of prothrombotic substances or proinflammatory substances triggered by platelet agonists, although one of the earliest events in ADP-induced platelet activation was slightly potentiated by HbV pretreatment at the doses tested. Taken together, these results imply that HbV, at concentrations of up to 40%, do not have any aberrant interactions with either unstimulated or agonist-induced platelets.

    DOI

  • Developmental Trend of Artificial Blood (Artificial Red Blood Cells)

    Takeoka, S

    JMAJ   48 ( 3 ) 1 - 5  2005

  • Hydrogen Peroxide Elimination Activity of Methemoglobin by L-Tyrosine and Application to Hemoglobin Vesicles

    Atoji, T, Aihara, H, Sakai, H, Tsuchida, H, Takeoka, S

    Bioconjugate Chemistry   17   1241 - 1245  2005

    DOI

  • Physiological capacity of the reticuloendothelial system for the degradation of hemoglobin vesicles (artificial oxygen carriers) after massive intravenous doses by daily repeated infusions for 14 days

    H Sakai, Y Masada, H Horinouchi, E Ikeda, K Sou, S Takeoka, M Suematsu, M Takaori, K Kobayashi, E Tsuchida

    JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS   311 ( 3 ) 874 - 884  2004.12

     View Summary

    A hemoglobin vesicle (HbV; diameter 252 +/- 53 nm) or liposome-encapsulated Hb is an artificial oxygen carrier developed for use as a transfusion alternative, and its oxygen-transporting capacity has been well characterized, although critical physiological compartments for the Hb degradation after a massive infusion of HbV and the safety outcome remain unknown. In this study, we aimed to examine the compartments for its degradation by daily repeated infusions (DRI) of HbV, focusing on its influence on the reticuloendothelial system ( RES). Male Wistar rats intravenously received the HbV suspension at 10 ml/kg/day for 14 consecutive days. The cumulative infusion volume ( 140 ml/kg) was equal to 2.5 times the whole blood volume ( 56 ml/kg). The animals tolerated the DRI well and survived, and body weights continuously increased. One day after DRI, hepatosplenomegaly occurred significantly through the accumulation of large amounts of HbV. Plasma clinical chemistry was overall normal, except for a transient elevation of lipid components derived from HbV. These symptoms subsided 14 days after DRI. Hemosiderin deposition and up-regulation of heme oxygenase-1 coincided in the liver and spleen but were not evident in the parenchyma of these organs. Furthermore, the plasma iron and bilirubin levels remained unchanged, suggesting that the heme-degrading capacity of the RES did not surpass the ability to eliminate bilirubin. In conclusion, phospholipid vesicles for the encapsulation of Hb would be beneficial for heme detoxification through their preferential delivery to the RES, a physiological compartment for degradation of senescent RBCs, even at doses greater than putative clinical doses.

    DOI

  • 血小板代替物の展開

    武岡真司, 岡村陽介

    Molecular Medicine   41   1494 - 1500  2004.12

  • Physicochemical characterization of cross-linked human serum albumin dimer and its synthetic heme hybrid as an oxygen carrier

    T Komatsu, Y Oguro, Y Teramura, S Takeoka, J Okai, M Anraku, M Otagiri, E Tsuchida

    BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS   1675 ( 1-3 ) 21 - 31  2004.11

     View Summary

    The recombinant human serum albumin (rHSA) dimer, which was cross-linked by a thiol group of Cys-34 with 1,6-bis(maleimido)hexane, has been physicochemically characterized. Reduction of the inert mixed-disulfide of Cys-34 beforehand improved the efficiency of the cross-linking reaction. The purified dimer showed a double mass and absorption coefficient, but unaltered molar ellipticity, isoelectric point (pl: 4.8) and denaturing temperature (65degreesC). The concentration dependence of the colloid osmotic pressure (COP) demonstrated that the 8.5 g dL(-1) dimer solution has the same COP with the physiological 5 g dL(-1) rHSA. The antigenic epitopes of the albumin units are preserved after bridging the Cys-34, and the circulation lifetime of the I-125-labeled variant in rat was 18 h. A total of 16 molecules of the tetrakis{(1-methylcyclohexanamido)phenyl}porphinatoiron(II) derivative (FecycP) is incorporated into the hydrophobic cavities of the HSA dimer, giving an albumin-heme hybrid in dimeric form. It can reversibly bind and release O-2 under physiological conditions (37degreesC, pH 7.3) like hemoglobin or myoglobin. Magnetic circular dichroism (CD) revealed the formation of an O-2-adduct complex and laser flash photolysis experiments showed the three-component kinetics of the O-2-recombination reaction. The O-2-binding affinity and the O-2-association and -dissociation rate constants of this synthetic hemoprotein have also been evaluated. (C) 2004 Elsevier B.V. All rights reserved.

    DOI

  • 3-Dithiolthione-substituted polythiophene and its redox activities

    Takemura, I, T Iwasaki, S Takeoka, H Nishide

    CHEMISTRY LETTERS   33 ( 11 ) 1482 - 1483  2004.11

     View Summary

    We synthesized 3-dithiolthione-substituted polythiophene, poly [3-(1',2-dithiol-3'-thione-4'-yl)thiophene], by cyclization reaction of poly [3-bis(methoxycaronyl)methyl thiophene]. Its redox behavior and electronic states showed that the oxidation of the polythiophene followed the oxidation of the dithiolthione ring to the radical cation of the dithiolthione ring.

    DOI

  • Recognition Properties of Polymerized Albumin Particles and Phospholipid Vesicles Bearing Recognition Proteins

    S. Takeoka, Y. Okamura, Y. Teramura, E. Tsuchida, Y. Okamura, Y. Teramura, E. Tsuchida, M. Handa, Y. Y. Teramura, E. Tsuchida, M. Handa, Y. Ikeda

    Japan-UK Platelet Conference, Oxford UK   Abstract   26  2004.09

  • Reduction of methemoglobin via electron transfer from photoreduced flavin: Restoration of O-2-binding of concentrated hemoglobin solution coencapsulated in phospholipid vesicles

    H Sakai, Y Masada, H Onuma, S Takeoka, E Tsuchida

    BIOCONJUGATE CHEMISTRY   15 ( 5 ) 1037 - 1045  2004.09

     View Summary

    Ferric methemoglobin is reduced to its ferrous form by photoirradiation either by direct photoexcitation of the heme portion to induce electron transfer from the surrounding media (Sakai at al. (2000) Biochemistry 39, 14595-14602) or by an indirect electron transfer from a photochemically reduced electron mediator such as flavin. In this research, we studied the mechanism and optimal condition that facilitates photoreduction of flavin mononucleotide (FMN) to FMNH2 by irradiation of visible light, and the succeeding reduction of concentrated metHb in phospholipid vesicles to restore its 02 binding ability. Visible light irradiation (435 nm) of a metHb solution containing FMN and an electron donor such as EDTA showed a significantly fast reduction to ferrous Hb with a quantum yield (Phi) of 0.17, that is higher than the method of direct photoexcitation of heme ( Phi = 0.006). Electron transfer from a donor molecule to metHb via FMN was completed within 30 ns. Native-PAGE and IEF electrophoresis indicated no chemical modification of the surface of the reduced Hb. Coencapsulation of concentrated Hb solution (35 g/dL) and the FMN/EDTA system in vesicles covered with a phospholipid bilayer membrane (Hb-vesicles, HbV, diameter: 250 nm) facilitated the metHb photoreduction even under aerobic conditions, and the reduced HbV restored the reversible O-2 binding property. A concentrated HbV suspension ([Hb] = 8 g/dL) was sandwiched with two glass plates to form a liquid layer with the thickness of about 10 mum (close to capillary diameter in tissue, 5 mum), and visible light irradiation (221 mW/cm(2)) completed 100% metHb photoreduction within 20 s. The photoreduced FMNH2 reacted with O-2 to produce H2O2, which was detected by the fluorescence measurement of the reaction of H2O2 and p-nitrophenylacetic acid. However, the amount of H2O2 generated during the photoreduction of HbV was significantly reduced in comparison with the homogeneous Hb solution, indicating that the photoreduced FMNH2 was effectively consumed during the metHb reduction in a highly concentrated condition inside the HbV nanoparticles.

    DOI

  • Hemorrhagic shock resuscitation with an artificial oxygen carrier, hemoglobin vesicle, maintains intestinal perfusion and suppresses the increase in plasma tumor necrosis factor-alpha

    A Yoshizu, Y Izumi, S Park, H Sakai, S Takeoka, H Horinouchi, E Ikeda, E Tsuchida, K Kobayashi

    ASAIO JOURNAL   50 ( 5 ) 458 - 463  2004.09

     View Summary

    It is known that damage to the intestinal mucosa followed by systemic inflammatory response is one of the leading causes of shock related morbidity and mortality. In this study, we examined the ability of an artificial oxygen carrier hemoglobin vesicle (HbV) to sustain systemic and intestinal perfusion during hemorrhagic shock. In rabbits, hemorrhagic shock (40% of the estimated blood volume) was resuscitated with 5% albumin (alb group), HbV suspended in 5% albumin (HbValb group), or washed red blood cells suspended in 5% albumin (RBCalb group). Plasma tumor necrosis factor (TNF)-alpha level was measured in rats under the same experimental protocol. No significant intergroup differences were seen in systemic hemodynamics. In contrast, parameters of intestinal perfusion significantly deteriorated in the alb group but were equally well sustained in the HbValb and RBCalb groups. Also, a significant increase in plasma TNF-alpha level was seen in the alb group but not in the RBCalb or HbValb groups. These results indicate the proficient oxygen transporting capability of HbV and its potential efficacy in shock resuscitation.

    DOI

  • Metabolism of hemoglobin-vesicles (artificial oxygen carriers) and their influence on organ functions in a rat model

    H Sakai, H Horinouchi, Y Masada, S Takeoka, E Ikeda, M Takaori, K Kobayashi, E Tsuchida

    BIOMATERIALS   25 ( 18 ) 4317 - 4325  2004.08

     View Summary

    Phospholipid vesicles encapsulating Hb (Hb-vesicies: HbV) have been developed for use as artificial O-2 carriers (250 nm phi). As one of the safety evaluations, we analyzed the influence of HbV on the organ functions by laboratory tests of plasma on a total of 29 analytes. The HbV Suspension ([Hb] = 10 g/dl) was intravenously infused into male Wistar rats (20 ml/kg; whole blood = 56 ml/kg). The blood was withdrawn at 8 h, and 1, 2, 3, and 7 days after infusion, and the plasma was ultracentrifuged to remove HbV in order to avoid its interference effect oil the analytes. Enzyme concentrations, AST, ALT, ALP, and LAP showed significant, but minor changes. and did not show a sign of a deteriorative damage to the liver that was one of the main organs for the HbV entrapment and the succeeding metabolism. The amylase and lipase activities showed reversible changes, however, there was no morphological changes in pancreas. Plasma bilirubin and iron did not increase in spite of the fact that a large amount of Hb was metabolized in the macrophages. Cholesterols, phospholipids, and beta-lipoprotein transiently increased showing the maximum at 1 or 2 days, and returned to the control level at 7 days. They should be derived from the membrane components of HbV that are liberated from macrophages entrapping HbV. Together with the previous report of the prompt metabolism of HbV in the reticuloendothelial system by histopathological examination, it can be concluded that HbV infusion transiently modified the values of the analytes without any; irreversible damage to the corresponding organs at the bolus infusion rate of 20 ml/kg. (C) 2003 Elsevier Ltd. All rights reserved.

    DOI

  • Detection of lipopolysaccharide in hemoglobin-vesicles by Limulus amebocyte lysate test with kinetic-turbidimetric gel clotting analysis and pretreatment of surfactant

    H Sakai, S Hisamoto, Fukutomi, I, K Sou, S Takeoka, E Tsuchida

    JOURNAL OF PHARMACEUTICAL SCIENCES   93 ( 2 ) 310 - 321  2004.02

     View Summary

    A method to quantitatively measure the bacterial endotoxin content (lipopolysaccharide, LPS) in phospholipid vesicles or liposomes is necessary because the conventional Limulus amebocyte lysate (LA-L) test does not provide an accurate measurement due to the hydrophobic interaction of LPS and vesicles that shields the activity of LPS to clot the LA-L coagulant. This interference was evident from isothermal titration calorimetry results in our study that clearly demonstrated the insertion of the LPS molecule into the phospholipid bilayer membrane. Hemoglobin-vesicles (HbVs; particle diameter = 251+/-80 nm; [Hb] = 10 g/dL) are artificial oxygen carriers encapsulating a conc. Hb solution in phospholipid vesicles, and their oxygen transporting ability has been extensively studied. To accurately measure the LPS content in the HbV suspension, we tested the solubilization of HbV with deca(oxyethylene) dodecyl ether (C12E10), used to release the LPS entrapped in the vesicles, as a pretreatment for the succeeding LAL assay of the kinetic-turbidimetric gel clotting (detecting wavelength, 660 nm). The C12E10 surfactant interferes with the gel clotting in a concentration-dependent manner, and the optimal condition was determined in terms of minimizing the dilution factor and C12E10 concentration. We clarified the condition that allowed the measurement of LPS at > 0.1 endotoxin units (EU)/mL in the HbV suspension. Moreover, the utilization of histidine-immobilized agarose gel effectively concentrated the trace amount of LPS from the C12E10-solubilized HbV solution and washed out C12E10 as an inhibitory element. The LA-L assay with the LPS-adsorbed gel resulted in the detection limit of 0.0025 EU/mL. Pretreatment with C12E10 would be applicable not only to HbVs but also to other drug delivery systems using phospholipid vesicles encapsulating or incorporating functional molecules. (C) 2004 Wiley-Liss, Inc.

    DOI

  • Hemoglobin-vesicles suspended in recombinant human serum albumin for resuscitation from hemorrhagic shock in anesthetized rats

    H Sakai, Y Masada, H Horinouchi, M Yamamoto, E Ikeda, S Takeoka, K Kobayashi, E Tsuchida

    CRITICAL CARE MEDICINE   32 ( 2 ) 539 - 545  2004.02

     View Summary

    Objective: Hemoglobin-vesicle (HbV) has been developed to provide oxygen-carrying ability to plasma expanders. Its ability to restore the systemic condition after hemorrhagic shock was evaluated in anesthetized Wistar rats for 6 hrs after resuscitation. The HbV was suspended in 5 g/dL recombinant human serum albumin (HbV/rHSA) at an Hb concentration of 8.6 g/dL.
    Design: Prospective, randomized, controlled trial.
    Setting: Department of Surgery, School of Medicine, Keio University.
    Subjects: Forty male Wistar rats.
    Interventions: The rats were anesthetized with 1.5% sevoflurane inhalation throughout the experiment. Polyethylene catheters were introduced through the right jugular vein into the right atrium for infusion and into the right common carotid artery for blood withdrawal and mean arterial pressure monitoring.
    Measurements and Main Results: Shock was induced by 50% blood withdrawal. The rats showed hypotension (mean arterial pressure = 32 +/- 10 mm Hg) and significant metabolic acidosis and hyperventilation. After 15 mins, they received HbV/rHSA, shed autologous blood (SAB), washed homologous red blood cells (wRBC) suspended in rHSA (wRBC/rHSA, [Hb] = 8.6 g/dL), or rHSA alone. The HbV/rHSA group restored mean arterial pressure to 93 +/- 8 mm Hg at 1 hr, similar to the SAB group (92 +/- 9 mm Hg), which was significantly higher compared with the rHSA (74 9 mm Hg) and wRBC/rHSA (79 8 mm Hg) groups. There was no remarkable difference in the blood gas variables between the resuscitated groups; however, two of eight rats in the rHSA group died before 6 hrs. After 6 hrs, the rHSA group showed significant ischemic changes in the right cerebral hemisphere relating to the ligation of the right carotid artery followed by cannulation, whereas the HbV/rHSA, SAB, and wRBC/rHSA groups showed less changes.
    Conclusions: HbV suspended in recombinant human serum albumin provides restoration from hemorrhagic shock that is comparable with that using shed autologous blood.

    DOI

  • Recognition properties of platelet membrane protein (rGPIb alpha)-conjugated particles made of polymerized albumin and phospholipids under flow conditions.

    Takeoka S, Tsuchida E, Ikeda Y

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   227, 10-POLY Part 2   U348 - U348  2004

  • Stability of porphyrin-calix[4]arene complexes analyzed by electrospray ionization mass spectrometry

    S Arai, S Ishihara, S Takeoka, H Ohkawa, T Shibue, H Nishide

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY   18 ( 18 ) 2065 - 2068  2004

     View Summary

    The stability of some porphyrin-calix[4]arene sodium-ion complexes were determined by a collision-activated decomposition (CAD) method utilizing electrospray ionization mass spectrometry (ESI-MS). Comparing the values of E-1/2, the collision energy at which the relative intensity of the complex ion is 0.5, we found that the porphyrin-calix[4]arene complex with the higher value of E-1/2 corresponded to that with the larger association constant (K-ass), as measured by H-1-NMR in CDCl3. Both our ESI-MS and NMR studies proved that the number of hydrogen bonds and the rigidity of the calix[4]arene stabilized the complex. The ESI-MS technique could be successful in screening the binding affinity in host-guest systems with a small amount of sample. Copyright (C) 2004 John Wiley Sons, Ltd.

    DOI

  • 血小板代替物の担体設計からDrug Local Deliveryの可能性

    武岡真司, 岡村陽介, 土田英俊, 池田康夫

    日本血栓止血学会誌   15   21 - 26  2004

    DOI

  • 話題の焦点、実用化に近づく人工赤血球(人工酸素運搬体),

    武岡真司

    アニムス   34   34 - 38  2004

  • 輸血の有効性と安全性-血液新法施行下での輸血医療の方向,輸血医療のトピックス,人工血液(人工赤血球)の開発動向,

    武岡真司

    日本医師会雑誌   131   907 - 909  2004

  • 止血機能を有する微粒子系の設計と機能評価

    武岡真司, 岡村陽介

    Cardiovascular Anesthesia   8   35 - 42  2004

  • Function of fibrinogen gamma-chain dodecapeptide-conjugated latex beads under flow

    S Takeoka, Y Okamura, Y Teramura, N Watanabe, H Suzuki, E Tsuchida, M Handa, Y Ikeda

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   312 ( 3 ) 773 - 779  2003.12

     View Summary

    In order to perform a fundamental study of platelet substitutes, novel particles that bound to activated platelets were prepared using two oligopeptides conjugated to latex beads. The oligopeptides were CHHLGGAKQAGDV (H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411), and CGGRGDF (RGD), which contains a fibrinogen alpha-chain sequence (alpha 95-98 RGDF). Both peptides contained an additional amino-terminal cysteine to enable conjugation. Human serum albumin was adsorbed onto the surface of latex beads (average diameter 1 mum) and pyridyldisulfide groups were chemically introduced into the adsorbed protein. H 12 or RGD peptides were then chemically linked to the modified surface protein via disulfide linkages. H12- or RGD-conjugated latex beads prepared in this way enhanced the in vitro thrombus formation of activated platelets on collagen-immobilized plates under flowing thrombocytopenic-imitation blood. Based on the result of flow cytometric analyses of agglutination, PAG-1 binding, antiP-selectin antibody binding, and annexin V binding, the H12-conjugated latex beads showed minimal interaction with non-activated platelets. These results indicate the excellent potential of H12-conjugated particles as a candidate for a platelet substitute. (C) 2003 Elsevier Inc. All rights reserved.

    DOI

  • Prolonged oxygen-carrying ability of hemoglobin vesicles by coencapsulation of catalase in vivo

    Y Teramura, H Kanazawa, H Sakai, S Takeoka, E Tsuchida

    BIOCONJUGATE CHEMISTRY   14 ( 6 ) 1171 - 1176  2003.11

     View Summary

    Hemoglobin (Hb) vesicles (particle diameter, ca. 250 nm) have been developed as Hb-based oxygen carriers in which a purified Hb solution is encapsulated with a phospholipid bilayer membrane. The oxidation of Hb to nonfunctional ferric Hb (metHb) was caused by reactive oxygen species, especially hydrogen peroxide (H2O2), in vivo in addition to autoxidation. We focused on the enzymatic elimination of H2O2 to suppress the metHb formation in the Hb vesicles. In this study, we coencapsulated catalase with Hb within vesicles and studied the rate of metHb formation in vivo. The Hb vesicles containing 5.6 x 10(4) unit mL(-1) catalase decreased the rate of metHb formation by half in comparison with Hb vesicles without catalase. We succeeded in prolonging the oxygen-carrying ability of the Hb vesicle in vivo by the coencapsulation of catalase.

    DOI

  • Effective encapsulation of proteins into size-controlled phospholipid vesicles using freeze-thawing and extrusion

    K Sou, Y Naito, T Endo, S Takeoka, E Tsuchida

    BIOTECHNOLOGY PROGRESS   19 ( 5 ) 1547 - 1552  2003.09

     View Summary

    We are aiming to improve the encapsulation efficiency of proteins in a size-regulated phospholipid vesicle using an extrusion method. Mixed lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-dipalmitoyl-L-glutamate-N-succinic acid (DPEA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5,000)] (PEG-DSPE) at a molar ratio of 5, 5, 1, and 0.033 were hydrated with a NaOH solution (7.6 mM) to obtain a polydispersed multilamellar vesicle dispersion (50 nm to 30 mum diameter). The polydispersed vesicles were converted to smaller vesicles having an average diameter of ca. 500 nm with a relatively narrow size distribution by freeze-thawing at a lipid concentration of 2 g dL(-1) and cooling rate of -140 degreesC min(-1). The lyophilized powder of the freeze-thawed vesicles was rehydrated into a concentrated protein solution (carbonyl hemoglobin solution, 40 g dL(-1)) and retained the size and size distribution of the original vesicles. The resulting vesicle dispersion smoothly permeated through the membrane filters during extrusion. The average permeation rate of the freeze-thawed vesicles was ca. 30 times faster than that of simple hydrated vesicles. During the extrusion process, proteins were encapsulated into the reconstructed vesicles with a diameter of 250 20 nm.

    DOI

  • Effective encapsulation of proteins into size-controlled phospholipid vesicles using freeze-thawing and extrusion

    K Sou, Y Naito, T Endo, S Takeoka, E Tsuchida

    BIOTECHNOLOGY PROGRESS   19 ( 5 ) 1547 - 1552  2003.09

     View Summary

    We are aiming to improve the encapsulation efficiency of proteins in a size-regulated phospholipid vesicle using an extrusion method. Mixed lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-dipalmitoyl-L-glutamate-N-succinic acid (DPEA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5,000)] (PEG-DSPE) at a molar ratio of 5, 5, 1, and 0.033 were hydrated with a NaOH solution (7.6 mM) to obtain a polydispersed multilamellar vesicle dispersion (50 nm to 30 mum diameter). The polydispersed vesicles were converted to smaller vesicles having an average diameter of ca. 500 nm with a relatively narrow size distribution by freeze-thawing at a lipid concentration of 2 g dL(-1) and cooling rate of -140 degreesC min(-1). The lyophilized powder of the freeze-thawed vesicles was rehydrated into a concentrated protein solution (carbonyl hemoglobin solution, 40 g dL(-1)) and retained the size and size distribution of the original vesicles. The resulting vesicle dispersion smoothly permeated through the membrane filters during extrusion. The average permeation rate of the freeze-thawed vesicles was ca. 30 times faster than that of simple hydrated vesicles. During the extrusion process, proteins were encapsulated into the reconstructed vesicles with a diameter of 250 20 nm.

    DOI

  • Hemostatic effects of polymerized albumin particles bearing rGPIa/IIa in thrombocytopenic mice

    Y Teramura, Y Okamura, S Takeoka, H Tsuchiyama, H Narumi, M Kainoh, M Handa, Y Ikeda, E Tsuchida

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   306 ( 1 ) 256 - 260  2003.06

     View Summary

    The recombinant fragment of the platelet membrane glycoprotein Ia/IIa (rGPIa/IIa) was conjugated to the polymerized albumin particles (polyAlb) with the average diameter of 180 nm. The intravenous administration of rGPIa/IIa polyAlb to thrombocytopenic mice ([platelet] = 2.1 +/- 0.3 x 10(5) particles/muL) with three doses of ca. 2.4 x 10(10), 7.2 x 10(10), and 2.4 x 10(11) particles/kg, respectively, significantly reduced their bleeding time to 426 +/- 71, 378 +/- 101, and 337 +/- 46 s, respectively, whereas that of the control groups (PBS) was 730 +/- 198 s. The injection of rGPIa/IIa-polyAlb (2.4 x 10(11) particles/kg) was approximately equal to the effect of the injection of the mouse platelets at a dose of 2.0 x 10(10) particles/kg. It was confirmed that rGPIa/IIa-polyAlb had a recognition ability against collagen and could contribute to the hemostasis in the thrombocytopenic mice as a platelet, substitute. (C) 2003 Elsevier Science (USA). All rights reserved.

    DOI

  • Normovolemic hemodilution with Hb vesicle solution attenuates hypoxia in ischemic hamster flap tissue

    Erni, D, Wettstein, R, Schramm, S, Contaldo, C, Sakai, H, Takeoka, S, Tsuchida, E, Leunig, M, Banic, A

    Am.J.Physiol.-Heart Circ. Physiol.   284   H1702 - H1709  2003.05

  • Treatment of serum containing hemoglobin vesicles (oxygen carriers) to prevent their interference in laboratory tests

    Sakai, H, Tomiyama, K, Masada, Y, Takeoka, S, Horinouchi, H, Kobayashi, K, Tsuchida, E

    Clin. Chem. Lab. Med.   41   222 - 231  2003.02

    DOI

  • A duplex of tetra(2-pyridyl)porphyrin and tetrahydroxylealix[4]arene

    AOhkawa, H, Arai, S, Takeoka, S, Shibue, T, Nishide, H

    Chem. Lett.   32 ( 11 ) 1052 - 1053  2003

    DOI

  • Improved oxygenation in ischemic hamster flap tissue is correlated with increasing hemodilution with Hb vesicles and their O2 affinity

    Contaldo C, Schramm S, Wettstein R, Sakai H, Takeoka S, Tsuchida E, Leunig M, Banic A, Erni D

    Am. J. Physiol. Heart Circ. Physiol.   285 ( 3 ) H1140 - H1147  2003

  • O2 release from Hb vesicles evaluated using an artificial, narrow O2-permeable tube: comparison with RBCs and acellular Hbs

    Sakai, H, Suzuki, Y, Kinoshita, M, Takeoka, S, Maeda, N, Tsuchida, E

    Am. J. Physiol.-Heart Circ. Physiol.   285 ( 6 ) H2543 - H2551  2003

  • DDSの基礎技術:キャリア ③リポソーム

    武岡真司, 寺村裕治, 池田康夫, 土田英俊

    血液・免疫・腫瘍   8   30 - 35  2003

  • 酸素輸液(人工赤血球)の安全度と体組織への酸素供給

    土田英俊, 宗慶太郎, 酒井宏水, 小松晃之, 武岡真司, 堀之内宏久, 末松誠, 小林紘一

    麻酔   53   s55 - 56  2003

  • フィブリノーゲンγ鎖ドデカペプチド結合粒子の血小板代替物の展開

    岡村陽介, 寺村裕治, 武岡真司, 土田英俊, 鈴木英紀, 渡辺直英, 半田 誠, 池田康夫

    人工血液   11   205 - 210  2003

  • 酸素輸液ヘモグロビン小胞体に混在するリポポリサッカライドの定量法

    久本秀治, 酒井宏水, 福富一平, 宗慶太郎, 武岡真司, 土田英俊

    人工血液   11 ( 3 ) 173 - 178  2003

  • 酸素輸液(人工赤血球)

    土田英俊, 酒井宏水, 武岡真司, 宗慶太郎, 小林紘一

    医学のあゆみ   205   558 - 566  2003

  • Effect of Hb-encapsulation with vesicles on H2O2 reaction and lipid peroxidation

    S Takeoka, Y Teramura, T Atoji, E Tsuchida

    BIOCONJUGATE CHEMISTRY   13 ( 6 ) 1302 - 1308  2002.11

     View Summary

    We encapsulated a purified and concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane to form Hb vesicles (particle diameter, ca. 250 nm) for the development of artificial oxygen carriers. Reaction of Hb inside the vesicle with hydrogen peroxide (H2O2) is one of the important safety issues to be clarified and compared with a free Hb solution. During the reaction of the Hb solution with H2O2, metHb (Fe-III) and ferrylHb (Fe-IV=O) are produced, and H2O2 is decomposed by the catalase-like reaction of Hb. The aggregation of discolored Hb products due to heme degradation is accompanied by the release of iron (ferric ion). On the other hand, the concentrated Hb within the Hb vesicle reacts with H2O2 that permeated through the bilayer membrane, and the same products as the Hb solution are formed inside the vesicle. However, there is no turbidity change, no particle diameter change of the Hb vesicles, and no peroxidation of lipids comprising the vesicles after the reaction with H2O2. Furthermore, no free iron is detected outside the vesicle, though ferric ion is released from the denatured Hb inside the vesicle, indicating the barrier effect of the bilayer membrane against the permeation of ferric ion. When vesicles composed of egg york lecithin (EYL) as unsaturated lipids are added to the mixture of Hb and H2O2, the lipid peroxidation is caused by ferrylHb and hydroxyl radical generated from reaction of the ferric iron with H2O2, whereas no lipid peroxidation is observed in the case of the Hb vesicle dispersion because the saturated lipid membrane of the Hb vesicle should prevent the interaction of the ferrylHb or ferric iron with the EYL.

    DOI

  • Rolling properties of rGPIb alpha-conjugated phospholipid vesicles with different membrane flexibilities on vWf surface under flow conditions

    S Takeoka, Y Teramura, Y Okamura, E Tsuchida, M Handa, Y Ikeda

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   296 ( 3 ) 765 - 770  2002.08

     View Summary

    The recombinant fragment of the platelet membrane glycoprotein, rGPIbalpha, was conjugated to phospholipid vesicles with the average diameter of ca. 1 mum using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). We used five kinds of rGPIbalpha-vesicles with different fluorescent anisotropies of 1,6-diphenyl-1,3,5-hexatriene (DPH) to study the rolling properties of the vesicles on the von Willebrand factor (vWf)-immobilized surface. Under flow conditions, the rolling velocity of the rGPIbalpha-vesicles decreased with the increasing membrane flexibility. It is considered that the vesicles with a high membrane flexibility have a high deformability and can be flattened to a high degree during rolling on the vWf surface, thus resulting in the large contact area. We obtained a recipe to control the rolling velocity of artificial platelets by membrane flexibility. (C) 2002 Elsevier Science (USA). All rights reserved.

    DOI

  • Characteristics of bovine hemoglobin as a potential source of hemoglobin-vesicles for an artificial oxygen carrier

    H Sakai, Y Masada, S Takeoka, E Tsuchida

    JOURNAL OF BIOCHEMISTRY   131 ( 4 ) 611 - 617  2002.04

     View Summary

    Hemoglobin-vesicles (HbV) have been developed for use as artificial O-2 carriers in which a purified Hb solution is encapsulated within a phospholipid bilayer membrane. In this study, bovine Hb (BHb) was tested as a source of HbV instead of human Hb (HHb). We compared the preparation process and characteristics of BHbV with those of HHbV. The purification of BHb was effectively performed simply with an ultrafiltration system including a process for removing virus and scrapie agent. The removal ratio of the phospholipid components of bovine red blood cells was over 99.99%, and the protein purity was over 99.9%. The deoxygenated and carbonylated BHb showed denaturation transition temperatures at 83 and 87degreesC, respectively, which are higher than those of HHB (80 and 78degreesC, respectively), and resistant to pasteurization (60degreesC, 10 h). The purified BHb was concentrated to over 40 g/dl, and encapsulated in a phospholipid bilayer membrane to form BHbV with a diameter of about 280 nm. The O-2 affinity (P-50) of the BHbV was regulated by coencapsulation of an appropriate amount of Cl- (as NaCl), which binds to BHb as an allosteric effector, in the range 16-28 Torr, comparable to human blood (P-50 = 28 Torr). This is quite simple in comparison with HHb which requires phosphate derivatives such as pyridoxal 5-phosphate as a replacement for 2,3-diphoshoglyceric acid. The viscosity and colloid osmotic pressure of the BHbV when suspended in 5% human serum albumin are 3.5 cP and 20 Torr, respectively, comparable to those of human blood. In conclusion, BHb can be used as a source for the production of HbV, not only because of its abundance in the cattle industry, but also because of the physicochemical advantages of the purification process, thermal stability, and regulation of O-2 affinity in comparison with HHb.

  • Complete deoxygenation from a hemoglobin solution by an electrochemical method and heat treatment for virus inactivation

    YB Huang, S Takeoka, H Sakai, H Abe, J Hirayama, K Ikebuchi, H Ikeda, E Tsuchida

    BIOTECHNOLOGY PROGRESS   18 ( 1 ) 101 - 107  2002.01

     View Summary

    Hemoglobin (Hb) has been widely studied as a raw material for various types of oxygen carriers. In the purification of Hb from red blood cells including virus inactivation and denaturation of other proteins and the long-term storage of Hb vesicles (HbV), a deoxygenation process is one of the important processes because of the high stability of deoxygenated Hb to heating and metHb formation. Though an oxygenated Hb solution can be deoxygenated with an artificial lung, it is difficult to reduce the oxygen partial pressure of the Hb solution to less than 10 Torr. We developed an electrochemical system for complete deoxygenation of the Hb solution at the cathode compartment using hydrogen containing nitrogen gas at the anode compartment. Oxygen in the Hb solution was reduced to OH- at the cathode compartment within several minutes at a potential value of - 1.67 V and was finally converted to water by neutralization with H+ from the anode in the whole system. The resulting completely deoxygenated Hb could tolerate heat treatment at 62 degreesC for 10 h with no denaturation of deoxygenated Hb. The metHb formation rate of reoxygenated Hb at 37 degreesC was not changed after heat treatment. Furthermore, vesicular stomatitis virus (VSV) could be inactivated at an inactivation degree of more than 5.96 log by heat treatment.

    DOI

  • Systemic and microvascular responses to hemorrhagic shock and resuscitation with Hb vesicles

    Sakai, H, Takeoka, S, Wettstein, R, Tsai, A.G, Intaglietta, M, Tsuchida, E

    Am. J. Physiol. Heart Circ. Physiol.   283 ( 3 ) H1191  2002

  • Carbonylation of oxyhemoglobin solution (HbO2 → HbCO) using a membrane oxygenator

    I. Fukutomi, H. Sakai, S. Takeoka, E. Tsuchida, K. Sakai

    Journal of Artificial Organs   5 ( 2 ) 102 - 107  2002

     View Summary

    In the purification process of hemoglobin (Hb) from red blood cells, we stabilized Hb as carbonylhemoglobin (HbCO) against pasteurization at 60°C. In this study, the process of carbonylation (HBO2 → HbCO) was tested with a membrane oxygenator (CX-II08
    membrane area, 0.8m2
    maximum circulation rate, 1.21/min) under the conditions of a solution flow rate of 100-1000 ml/min and a CO gas flow rate of 30-100 ml/min. Comparing the overall O2 transfer coefficient of carbonylation with that of deoxygenation (N2 flow) revealed that the resistance to O2 transfer of carbonylation was about 35 times smaller, indicating that carbonylation hindered O2 rebinding (deoxyHb → HbO2). On the other hand, the O2 released in the course of carbonylation hindered carbonylation at the beginning, because rebinding of O2 is competitive with carbonylation. The time required for carbonylation was significantly shortened from 1000 to 150s when the solution flow rate was increased from 50 to 400 ml/min
    however, the CO gas flow rate did not affect it very much. Increasing the Hb concentration from 7.5 to 15 g/dl accelerated carbonylation by 1.3 times. Even though further study is necessary to select a suitable polymer membrane to avoid protein adsorption, a membrane oxygenator will be effective for the large-scale carbonylation of Hb as a starting material of HbV in the production process.

    DOI

  • Study of virus removal from hemoglobin solution using PLANOVATM-15N

    Naito, Y, Fukutomi, I, Masada, Y, Sakai, H, Takeoka, S, Tsuchida, E, Abe, H, Hirayama, J, Ikebuchi, K, Ikeda, H

    J. Artif. Organs   5   141 - 145  2002

    DOI

  • 酸素輸液(ヘモグロビン小胞体)を含有する血清の生化学的検査

    酒井宏水, 富山賢一, 政田陽平, 武岡真司, 堀之内宏久, 小林紘一, 土田英俊

    人工血液   10   47 - 53  2002

  • 酸素輸液の設計と開発動向

    武岡真司

    医薬の門   41;528-532   528 - 532  2001.10

  • Synthesis of disulfide-containing aniline and copolymerization with aniline

    JS Cho, S Sato, S Takeoka, E Tsuchida

    MACROMOLECULES   34 ( 9 ) 2751 - 2756  2001.04

     View Summary

    A novel disulfide-containing aniline, 1,4-dihydrobenzo[d] [1,2] dithiin-5-ylamin (1), was synthesized by the alkylbromination of 1,2-dimethyl-3-nitrobenzene, followed by the formation of a disulfide bond via thioesterification and then the reduction of the nitro group. The monomer showed a reductive and oxidative potential of the disulfide bond at -0.63 and 1.25 V (vs Ag/Ag+), respectively. Although the monomer showed the oxidative potential of aniline at 0.85 V (vs SCE), the electro- or chemical-oxidative polymerization of the monomer resulted in the formation of only the oligomer, suggesting that the disulfide-containing condensed ring would prevent further propagation of the polymerization due to steric hindrance. However, the nearly ideal copolymerization of 1 with aniline (An) was carried out by chemical oxidative polymerization. The resulting copolymers (1/An = 1/1 and 2/1, by mole) had weight-averaged molecular weights ((M) over bar (w)) of 10 000 and 6600, respectively and good redox activities coupled with those of the polyaniline and disulfide group. The copolymer showed conductivities of 5.1 x 10(-2) S/cm for 1/1 and 1.3 x 10-2 S/cm for 2/1 after being redoped with camphorsulfonic acid. It is suggested that these copolymers might be good candidates for the cathode materials of secondary polymer batteries.

    DOI

  • Synthesis of disulfide-containing aniline and copolymerization with aniline

    JS Cho, S Sato, S Takeoka, E Tsuchida

    MACROMOLECULES   34 ( 9 ) 2751 - 2756  2001.04

     View Summary

    A novel disulfide-containing aniline, 1,4-dihydrobenzo[d] [1,2] dithiin-5-ylamin (1), was synthesized by the alkylbromination of 1,2-dimethyl-3-nitrobenzene, followed by the formation of a disulfide bond via thioesterification and then the reduction of the nitro group. The monomer showed a reductive and oxidative potential of the disulfide bond at -0.63 and 1.25 V (vs Ag/Ag+), respectively. Although the monomer showed the oxidative potential of aniline at 0.85 V (vs SCE), the electro- or chemical-oxidative polymerization of the monomer resulted in the formation of only the oligomer, suggesting that the disulfide-containing condensed ring would prevent further propagation of the polymerization due to steric hindrance. However, the nearly ideal copolymerization of 1 with aniline (An) was carried out by chemical oxidative polymerization. The resulting copolymers (1/An = 1/1 and 2/1, by mole) had weight-averaged molecular weights ((M) over bar (w)) of 10 000 and 6600, respectively and good redox activities coupled with those of the polyaniline and disulfide group. The copolymer showed conductivities of 5.1 x 10(-2) S/cm for 1/1 and 1.3 x 10-2 S/cm for 2/1 after being redoped with camphorsulfonic acid. It is suggested that these copolymers might be good candidates for the cathode materials of secondary polymer batteries.

    DOI

  • Carbon monoxide from heme catabolism protects against hepatobiliary dysfunction in endotoxin-treated rat liver

    Takanori Kyokane, Shinji Norimizu, Hisashi Taniai, Tokio Yamaguchi, Shinji Takeoka, Eishun Tsuchida, Makoto Naito, Yuji Nimura, Yuzuru Ishimura, Makoto Suematsu

    Gastroenterology   120 ( 5 ) 1227 - 1240  2001

     View Summary

    Background &amp
    Aims: Liver is a major organ for heme detoxification under disease conditions, but its self-protective mechanisms against the toxicity are unknown. This study aimed to examine roles of carbon monoxide (CO), the gaseous product of heme oxygenase (HO), in ameliorating hepatobiliary dysfunction during catabolism of heme molecules in endotoxemic livers. Methods: Vascular resistance and biliary flux of bilirubin-IXα, an index of HO-derived CO generation, were monitored in perfused livers of endotoxemic rats. Livers were perfused with HbO2, which captures nitric oxide (NO) and CO, or metHb, a reagent trapping NO but not CO. Results: In endotoxin-pretreated livers where inducible NO synthase and HO-1 overproduced NO and CO, HbO2 caused marked vasoconstriction and cholestasis. These changes were not reproduced by the NO synthase inhibitor aminoguanidine alone, but by coadministration of zinc protoporphyrin-IX, an HO inhibitor. CO supplementation attenuated the events caused by aminoguanidine plus zinc protoporphyrin-IX, suggesting that simultaneous elimination of these vasorelaxing gases accounts for a mechanism for HbO2-induced changes. This concept was supported by observation that metHb did not cause any cholestasis
    the reagent captures NO but triggers CO overproduction through rapid degradation of the heme by HO-1. Conclusions: These results suggest protective roles of CO against hepatobiliary dysfunction caused by heme overloading under stress conditions.

    DOI PubMed

  • Development of the Albumin Polymers with the Hemostotic Ability

    Blood・Immunity・Cancer   6 ( 1 ) 46  2001

  • Effects of poly(ethyleneglycol)-modified hemoglobin vesicles on N-formyl-methionyl-leucylphenylalanine-induced responses of polymorphonuclear neutrophils in vitro

    T Ito, M Fujihara, H Abe, M Yamaguchi, S Wakamoto, S Takeoka, H Sakai, E Tsuchida, H Ikeda, K Ikebuchi

    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND IMMOBILIZATION BIOTECHNOLOGY   29 ( 6 ) 427 - 437  2001

     View Summary

    [Poly(ethylene-lycol)]-modified hemoglobin vesicles (PEG-HbV), a type of encapsulated hemoglobin, have been developed as artificial oxygen carriers and it is important to evaluate their blood compatibility. We studied the effects of PEG-HbV on human polymorphonuclear neutrophils (PMNs) in vitro, focusing on the functional responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP) as an agonist. The pretreatment of the PMNs with PEG-HbV up to a concentration of 60 mg/dl Hb did not affect the fMLP-triggered chemotactic activity. In parallel to these results, the fMLP-induced upregulation of CD11b (Mac-1) levels on the PEG-MV-pretreated PMNs was comparable to that of untreated cells. Furthermore, the pretreatment of the PMNs with the PEG-HbV even at 600 mg/dl Hb did not affect the gelatinase B (Matrix methalloproteinase-9 (MMP-9)) release, suggesting that the fMLP-induced release of secondary and tertiary granules was normal. In addition, the fMLP-triggered superoxide production of the PMNs was unchanged by the pretreatment with the PEG-HbV at 600 mg/dl Hb. Thus. these results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on the fMLP-triggered functions of human PMNs.

    DOI

  • Hemoglobin-vesicles as oxygen carriers - Influence on phagocytic activity and histopathological changes in reticuloendothelial system

    Sakai, H, Horinouchi, H, Tomiyama, K, Ikeda, E, Takeoka, S, Kobayashi, K, Tsuchida, E

    Am. J. Pathol.   159 ( 3 ) 1079 - 1088  2001

  • Virus inactivation in hemoglobin solution by heat treatment

    H Abe, K Ikebuchi, J Hirayama, M Fujihara, S Takeoka, H Sakai, E Tsuchida, H Ikeda

    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND IMMOBILIZATION BIOTECHNOLOGY   29 ( 5 ) 381 - 388  2001

     View Summary

    To increase the safety of stroma-free hemoglobin solution (SFH) as an artificial oxygen carrier source, we investigated the effect of heat treatment on virus inactivation in hemoglobin solution. The hemoglobin solution spiked with vesicular stomatitis virus (VSV) was treated at 60 degreesC for 1 hr under either an air or CO atmosphere. VSV was inactivated at >5.8 log(10) and >6.0 log(10) under the air and CO atmosphere, respectively. Although the methemoglobin rate increased after the heat treatment under the air atmosphere, no methemoglobin formation was observed by the treatment under the CO atmosphere. Isoelectric focusing analysis revealed the denaturation of hemoglobin after the heat treatment under the air, while hemoglobin banding was not altered in the carbonylated condition. Some protein bands other than hemoglobin were weakened or disappeared on SDS-PAGE after the heat treatment under both conditions. In addition, the hemoglobin concentration in the SFH was higher after the heat treatment than before the treatment. These findings indicate that the heat treatment under the CO atmosphere inactivates viruses without hemoglobin denaturation, and hence, this method is a promising approach to prepare a safer SFH as artificial oxygen carriers.

    DOI

  • Fibrinogen-conjugated albumin polymers and their interaction with platelets under flow conditions

    S. Takeoka, Y. Teramura, Y. Okamura, M. Handa, Y. Ikeda, E. Tsuchida

    Biomacromolecules   2 ( 4 ) 1192 - 1197  2001

     View Summary

    Albumin polymers, having an average diameter of 1020 ± 250 nm, were prepared by the disulfide polymerization of recombinant human serum albumin (rHSA) by controlling of the pH and temperature. Fibrinogen could be conjugated on the surface of an albumin polymer using N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). Under flow conditions, the fibrinogen-conjugated albumin polymers (fibrinogen-albumin polymers) were irreversibly attached to the platelet-immobilized surface in the reconstituted blood at a low platelet concentration ([platelet] = 5.0 × 104/μL, a 5-fold diluted platelet concentration), and the attachment was suppressed by the addition of anti-GPIIb/IIIa monoclonal antibodies. It was confirmed that fibrinogen-albumin polymers specifically interacted with GPIIb/IIIa expressed on the surface of the activated platelets. Although platelets with a low platelet concentration were hardly attached to the platelet-immobilized surface under the flow conditions, the addition of fibrinogen-albumin polymers enhanced the attachment of the remaining platelets to the surface, indicating that the fibrinogen-albumin polymers would help the hemostatic ability of platelets at the site of vascular injury of patients in thrombocytopenia.

    DOI PubMed

  • Effects of poly(ethyleneglycol)-modified hemoglobin vesicles on agonist-induced platelet aggregation and RANTES release in vitro

    S Wakamoto, M Fujihara, H Abe, H Sakai, S Takeoka, E Tsuchida, H Ikeda, K Ikebuchi

    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND IMMOBILIZATION BIOTECHNOLOGY   29 ( 3 ) 191 - 201  2001

     View Summary

    We studied the effects of hemoglobin-vesicles modified with PEG (PEC-HbV), a type of liposome-encapsulated hemoglobin (LEH), on human platelet functions in vitro. The effect of a low concentration of PEG-HbV (Hb; 5.8 mg/dl) was assessed by examining an agonist-induced aggregation response, and that of relatively high concentrations of PEG-HbV (Hb; 0.29, 1 and 2 g/dl) by measuring the release of RANTES (Regulated upon activation, normal T-cell expressed and presumably secreted) from platelets, which is regarded as a marker of platelet activation. The preincubation of platelets with PEG-HbV at 5.8 mg/dl of Hb did not affect platelet aggregation induced by collagen, thrombin and ristocetin. The pretreatment of platelet-rich plasma (PRP) with PEG-HbV at concentrations up to 2 g/dl of Hb had no aberrant effects on the collagen-induced RANTES release. Furthermore, the collagen-induced release of RANTES from PRP was not affected by longer incubation with PEG-HbV at 2 g/dl of Hb. The basal levels of RANTES from PRP were unchanged in the presence of PEG-HbV. These results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on platelet functions in the presence of plasma.

    DOI

  • 止血能を有するアルブミン重合体の開発.

    武岡真司, 寺村裕治, 土田英俊, 池田康夫

    血液・免疫・腫瘍/メディカルレビュー社   6   46 - 50  2001.01

  • 粒子径を制御したアルブミン重合体の合成とGPIbα結合体の評価.

    寺村裕治, 武岡真司, 土田英俊, 池田康夫

    人工血液   8   90 - 95  2000.12

  • Photoreduction of methemoglobin by irradiation in the near-ultraviolet region

    H Sakai, H Onuma, M Umeyama, S Takeoka, E Tsuchida

    BIOCHEMISTRY   39 ( 47 ) 14595 - 14602  2000.11

     View Summary

    Ferric metHb can be photoreduced to the ferrous state by direct photoexcitation in the near-ultraviolet region. In this research, we studied the mechanism and facilitating conditions for the photoreduction and the resulting restoration of O-2 binding. MetHb in phosphate-buffered saline or pure water in a CO atmosphere was photoreduced to form HbCO by illuminating the N band (365 nm), one of the porphyrin pi --> pi* transitions, whereas the photoreduction did not occur in Ar, N-2, or O-2. The transient absorption spectrum exhibited the generation of deoxyHb within 30 ns in both the CO and Ar atmospheres; however, only in CO did the subsequent CO binding inhibit the back reaction. The photoreduction rate was dependent on the pH and ligand anions, showing that aquametHb in the high-spin state was predominant for the photoreduction. Axial ligand-to-metal charge-transfer (LMCT) bands overlap with the Soret and Q bands in metHb; however, the excitation of these bands showed little photoreduction, indicating that the contribution of these LMCT bands is minimal. Excitation of the N band significantly contributes to the photoreduction, and this is facilitated by the external addition of mannitol, hyaluronic acid, Trp, Tyr, etc. Especially, Trp allowed the photoreduction even in an Ar atmosphere, and the reduced Hb can be converted to HbO(2) by O-2 bubbling. One mechanism of the metHb photoreduction that is proposed on the basis of these results consists of a charge transfer from the porphyrin ring to the central, ferric iron to form the porphyrin pi cation radical and ferrous iron by the N band excitation, and the contribution of the amino acid residues in the globin chain as an electron donor or an electron pathway.

    DOI

  • Synthesis of multiacyl poly(ethylene glycol) for the conjugation of cytochrome c to phospholipid vesicle

    H Ohkawa, Y Teramura, S Takeoka, E Tsuchida

    BIOCONJUGATE CHEMISTRY   11 ( 6 ) 815 - 821  2000.11

     View Summary

    To conjugate water-soluble macromolecules on the surface of phospholipid vesicles, we synthesized a poly(ethylene glycol) (PEG)-lipid having four acyl chains using a lysine (Lys)-type monodendron structure. One end of the diamino-PEG was amidified with Lys, and then two amino groups of the Lys moiety were amidified with two Lys derivatives which had been acylated with two stearoyl groups. The other end of the PEG was activated with a triazine group or a pyridyldithio group. The hydrate of the lipid mixture of dipalmitoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol, and the PEG-lipid at a molar ratio of 5/5/1/0.3 was extruded in order to prepare the phospholipid vesicles with the average diameter of 270 +/- 20 nm. The coupling ratio of cytochrome c with the PEG-lipid was monitored by HPLC, detecting the pyridyl 2-thione liberated from the pyridyldithio group and determining it to be 26% on the basis of the incorporated PEG-lipid.

    DOI

  • Synthesisi and physicochemical characterization of Hb-based O2 carriers: Comparison between cellular and acelluar types

    8th International Symposium on Blood Substitutes    2000.11

  • Synthesis and assembly of poly(ethylene glycol)-lipids with mono-, di-, and tetraacyl chains and a poly(ethylene glycol) chain of various molecular weights

    S Takeoka, K Mori, H Ohkawa, K Sou, E Tsuchida

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   122 ( 33 ) 7927 - 7935  2000.08

     View Summary

    We synthesized a series of amphiphiles with poly(ethylene glycol) [MW 2000 (PEG20), 5000 (PEG50), 12 500 (PEG125)] as a headgroup and one, two, or four palmitoyl chains (1C16, 2C16, or 4C16), using a lysine monodendron as a connector. The relationship between the hydrophilic-hydrophobic balance of the multiacyl PEG-lipids and the physicochemical characteristics in self- or co-assembly with phospholipids were studied. The PEG-lipids were easily synthesized by combination of a general liquid-phase peptide synthesis and the acylation of an amino acid. The PEG part of the PEG-lipid films was crystallized to show a typical spherulite pattern. The thermal properties and microscopic observation revealed the phase separation of PEG and acyl chain parts. The critical micelle concentrations (cmcs) mainly depend on the number of acyl chains rather than the molecular weight of the PEG chain, although the area per molecule is dependent on the molecular weight of the PEG chain rather than the number of the acyl chains. The gel-to-liquid crystalline phase transition temperature was increased with the increasing number of acyl chains and the decreasing molecular weight of the PEG chain. The PEG-lipids in the aqueous dispersions assemble to take fibrous structures with bimolecular thickness because of the intermolecular hydrogen bonding. The PEG-lipids were immobilized onto the surface of the phospholipid vesicles by simply adding their aqueous dispersions to the vesicle dispersion; however, they dissociated from the vesicles on dilution of the mixed dispersion because they were incorporated into the vesicles in an equilibrium state. To prevent the dissociation of the PEG-lipids, at least two and four acyl chains were required for PEG with M-W 5000 and 12 500, respectively. The aggregation of the vesicles by the addition of water-soluble polymers was significantly inhibited with the increasing molecular weight of the PEG chain. For the tight immobilization of the PEG-lipids with the long PEG chain onto the vesicular surface, an increased number of acyl chains is necessary, and the surface modification with the long PEG chains significantly increases the dispersion stability of the vesicles.

    DOI

  • Papers presented at PAT '99 - Tokyo - Editorial

    Polym. Adv. Technol.   11   369  2000.08

  • Proton-conduction in poly(alkylenecarbonate)/poly(thiophenylenesulfonic acid) composites

    JS Cho, Y Hayashino, K Miyatake, S Takeoka, E Tsuchida

    POLYMERS FOR ADVANCED TECHNOLOGIES   11 ( 8-12 ) 548 - 552  2000.08

     View Summary

    Highly sulfonated poly(thiopenylenesulfonic acid) (PTPSA) was prepared from a soluble polysulfonium salt precursor and mixed with poly(alkylenecarbonate) (PAC, alkylene ethylene, PEG; propylene, PPC; butylene, PBC). The PTPSA and PAC were Employed as a proton source for conduction and a flexible base matrix :for proton dissociation and conduction pathway, respectively. For the composites of PTPSA/PAC (3/1, 2/1, 2/1 and 1/2, by weight), the effect of alkyl chain lengths of PAC, temperature dependence of proton-conductivity, and water content on the composites was studied. The proton-conductivity of the PTPSA/PAC composites Increased with the increasing PTPSA portion. The PTPSA/PBC (3/1) composite shows the highest proton-conductivity of 1.5 x 10(-6) Scm(-1) at 30 degreesC. However, the increased incorporation of PTPSA does not contribute to producing the higher proton conduction the composites. Moreover, the PTPSA/PBC composite including 15 wt% of water retained the proton-conductivity of 7.2 x 10(-2) Scm(-1) at 240 degreesC, which could be the conductivity comparative to the Nafion membrane and thermal stability. Copyright (C) 2000 John Wiley & Sons, Ltd.

    DOI

  • Poly(ethylene glycol)-modification of the phospholipid vesicles by using the spontaneous incorporation of poly(ethylene glycol)-lipid into the vesicles

    K Sou, T Endo, S Takeoka, E Tsuchida

    BIOCONJUGATE CHEMISTRY   11 ( 3 ) 372 - 379  2000.05

     View Summary

    The critical micelle concentrations of 1,2-dipalmitoyl-sn-glycero-3-phosphoethaanolamine-N-[monomethoxy poly(ethyleneglycol) (5000)] (PEG-DPPE) and its distearoyl analogue (PEG-DSPE) were 70 and 9 mu M, respectively, in buffer solutions ([Tris] = 20 mM, [NaCl] = 140 mM, pH 7.4) at 37 degrees C. When these PEG-Lipid micelle dispersions were mixed with the dispersions of phospholipid vesicles comprised of a C16 membrane, of which the carbon number is 16, or a C18 membrane, the PEG-lipid micelles were dissociated into monomers and then spontaneously incorporated into the surface of the preformed vesicles. The incorporation rates and the enthalpy changes during incorporation were measured with an isothermal titration microcalorimeter. The incorporation rate of PEG-DPPE was faster than that of PEG-DSPE, because the dissociation rate of the PEG-DPPE micelles was faster than that of PEG-DSPE micelles. The incorporation equilibrium constant of PEG-DSPE was larger than that of PEG;DPPE due to its slow dissociation rate from the membrane, caused by the stronger hydrophobic interaction. The combination of PEG-DSPE and the C18 membrane was the most thermodynamically stabilized pair. Furthermore, the dispersion stability of the surface-modified vesicles prepared by this spontaneous incorporation was analyzed by using the critical molecular weight of the polymer for the aggregation of vesicles. The aggregation of the vesicles was successfully supressed with an increase in the molecular weight of the PEG in the PEG-lipid and its incorporation ratio.

    DOI

  • Poly(ethylene glycol)-conjugation and deoxygenation enable long-term preservation of hemoglobin-vesicles as oxygen carriers in a liquid state

    H Sakai, K Tomiyama, K Sou, S Takeoka, E Tsuchida

    BIOCONJUGATE CHEMISTRY   11 ( 3 ) 425 - 432  2000.05

     View Summary

    The stability of hemoglobin vesicles (HbV) as an oxygen infusion was tested during the storage for 1 year at 4, 23, and 40 degrees C. The surface of the HbV was modified with poly(ethylene glycol) (PEG), and the suspension was deoxygenated with nitrogen bubbling. The samples stored at 4 and 23 degrees C showed a stable dispersion state for 1 year, though the sample stored at 40 degrees C showed the precipitation and decomposition of vesicular components, a decrease in pH, and 4% leakage of total Hb after 1 year. The PEG chains on the vesicular surface stabilize the dispersion state and prevent the aggregation and fusion due to their steric hindrance. The original metHb content (ca. 3%) before the preservation gradually decreased to less than 1% in all the samples after 1 month due to the presence of homocysteine inside the vesicles which consumed the residual oxygen and gradually reduced the trace amount of metHb. The rate of metHb formation was strongly dependent on the partial pressure of oxygen, and no increase in metHb formation was observed due to the intrinsic stability of the deoxygenated Hb. Preservation at 4 and 23 degrees C slightly reduced P-50 (increased the oxygen affinity) from 38 Torr to 32 and 31 Torr, respectively. These results indicate the possibility that HbV suspension can be stared at room temperature for at least I year.

    DOI

  • 酸素輸液の開発

    武岡真司

    日本医工学治療学会第14回学術大会/日本医工学治療学会    2000.02

  • Proton-conduction of perfluorooctanesulfonic acid/poly(propylene carbonate) composites

    Cho, JS, Hayashino, Y, Miyatake, K, Takeoka, S, Tsuchida, E

    Chem. Lett.   2000/1,44 ( 1 ) 44 - 45  2000

    DOI

  • Synthesis and physicochemical characterization of a series of hemoglobin-based oxygen carriers: Objective comparison between cellular and acellular types

    H Sakai, M Yuasa, H Onuma, S Takeoka, E Tsuchida

    BIOCONJUGATE CHEMISTRY   11 ( 1 ) 56 - 64  2000.01

     View Summary

    A series of hemoglobin (Hb)-based O(2) carriers, acellular and cellular types, were synthesized and their physicochemical characteristics were compared. The acellular type includes intramolecularly cross-linked Hb (XLHb), polyoxyethylene (POE)-conjugated pyridoxalated Hb (POE-PLP-Hb), hydroxyethylstarch-conjugated Hb (HES-XLHb), and glutaraldehyde-polymerized XLHb (Poly-XLHb). The cellular type is Hb-vesicles (HbV) of which the surface is modified with POE (POE-HbV). Their particle diameters are 7 +/- 2, 22 +/- 2, 47 +/- 17, 68 +/- 24, and 224 +/- 76 nm, respectively, thus all the materials penetrate across membrane filters with 0.4 mu m pore size, though only the POE-HbV cannot penetrate across the filter with 0.2 mu m pore size. These characteristics of permeability are important to consider-an optimal particle size in microcirculation in vivo. POE-PLP-Hb ([Hb] = 5 g/dL) showed viscosity of 6.1 cP at 332 s(-1) and colloid osmotic pressure (COP) of 70.2 Torr, which are beyond the physiological conditions (human blood, viscosity = 3-4 cP, COP = ca. 25 Torr). XLHb and Poly-XLHb showed viscosities of 1.0 and 1.5 cp, respectively, which are significantly lower than that of blood. COP of POE-HbV is regulated to 20 Torr in 5% human serum albumin (HSA). HES-XLHb and POE-HbV/HSA showed comparable viscosity with human blood. Microscopic observation of human red blood cells (RBC) after mixing blood with POE-PLP-Hb or HES-XLHb disclosed aggregates of RBC, a kind of sludge, indicating a strong interaction with RBC, which is anticipated to modify peripheral blood flow in vivo. On the other hand, XLHb and POE-HbV showed no rouleaux or aggregates of RBC. The acellular Hbs (P(50) = 14-32 Torr) have their specific O(2) affinities determined by their structures, while that; of the cellular POE-HbV is regulated by coencapsulating an appropriate amount of an allosteric effector (e.g., P(50) = 18, 32 Torr). These differences in physicochemical characteristics between the acellular and cellular types indicate the advantages of the cellular type from the physiological points of view.

    DOI

  • Conjugation of von Willebrand factor-binding domain of platelet glycoprotein Ibα to size-controlled albumin microspheres

    Shinji Takeoka, Yuji Teramura, Haruki Ohkawa, Yasuo Ikeda, Eishun Tsuchida

    Biomacromolecules   1 ( 2 ) 290 - 295  2000

     View Summary

    Albumin microspheres (AMS), of which the average diameter is 240 ± 10 nm, were prepared by pH control and heat treatment. Cytochrome c and rGPIbα
    a water-soluble fragment of the α chain of a recombinant platelet glycoprotein (GP)Ib containing a von Willebrand factor (vWf)-binding site were selected as a receptor protein. Cytochrome c was used as a probe protein for monitoring. Onto the surface of the AMS and those proteins, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was reacted through the amide linkage to obtain PD-AMS, PD-cytochrome c, and PD-rGPIbα, respectively. The latter two were further reduced to SH-cytochrome c and SH-rGPIbα by dithiothreitol and conjugated with PD-AMS by a thiol - disulfide exchange reaction. The resulting AMS contain cytochrome c or rGPIbα of about 25000 or 2500 molecules, respectively. The addition of ristocetin to the rGPIbα-AMS in the presence of vWf caused specific aggregation. Furthermore, the rGPIbα-AMS enhanced the ristocetin induced platelet aggregation in a low platelet concentration (4.0 × 107/mL).

    DOI PubMed

  • Molecular dimensions of Hb-based O-2 carriers determine constriction of resistance arteries and hypertension

    Sakai, H, Hara, H, Yuasa, M, Tsai, AG, Takeoka, S, Tsuchida, E, Intaglietta, M

    Am. J. Physiol.-Heart Circul. Physiol.   279   H908 - H915  2000

  • Preparation and properties of polyaniline doped with poly(thiophenylenesulfonic acid)

    K Miyatake, JS Cho, S Takeoka, E Tsuchida

    MACROMOLECULAR CHEMISTRY AND PHYSICS   200 ( 12 ) 2597 - 2601  1999.12

     View Summary

    A novel polyaromatic acid, poly(thiophenylenesulfonic acid) (PTPSA), prepared from a soluble precursor of poly(arylenesulfonium salt) was employed as a dopant for polyaniline (PAn). The electro-oxidative polymerization of aniline in PTPSA aqueous solution gives an electroactive PAn/PTPSA composite with three redox couples at 0.16, 0.53, and 0.71V (vs. SCE). Composites of different molar ratio ([SO3H]/[An] = 0.10, 0.21, 0.42, 0.50, 0.83, 1.67) was prepared in order to confirm the transformation from the emeraldine base to its PTPSA salt, where a quantitative doping reaction was observed by means of UV-VIS and IR measurements. The thennogravimetric analysis revealed that the composites do not decompose up to 200 degrees C in nitrogen atmosphere. The composite ([SO3H]/[An] = 0.50) shows an electrical conductivity of 1.5 S . cm(-1) at 30 degrees C and 3.0 x 10(-1) S . cm(-1) at 170 degrees C. The composite retains its conductivity after heating at 150 degrees C for 24 h.

    DOI

  • ポリオキシエチレン修飾と脱酸素化による酸素輸液(ヘモグロビン小胞体)の長期保存

    人工血液/日本血液代替物学会   7;105-110  1999.12

  • Carbon monoxide overproduced by heme oxygenase-1 causes a reduction of vascular resistance in perfused rat liver

    Wakabayaski, Y, Takamiya, R, Mizuki, A, Kyokane, T, Goda, N, Yamaguchi, T, Takeoka, S, Tsuchida, E, Suematsu, M, Ishimura, Y

    Am. J. Physiol.-Gastroint. Liver Physiol.   277   G1088 - G1096  1999.11

  • Non-invasive observation of resistance arteries in conscious hamsters fitted with dorsal skinfold windows

    Microcirculation Annual 1999/日本医学館   pp.91-92  1999.11

  • 人工血液の製剤化技術とその物性評価

    第11回界面科学部会/日本油化学会    1999.11

  • 分子集合を利用した細胞型人工赤血球の創製とその評価

    第10回バイオマテリアル研究会/高分子学会    1999.11

  • ポリオキシエチレン結合デンドロン型脂質の合成と物性

    第48回高分子討論会/高分子学会    1999.10

  • Hemoglobin-based oxygen carriers (1): Synthesis and physicochemical characterization of crosslinked, polymerized, polymer-conjugated and encapsulated hemoglobin

    The 5th International Symposium on Polymers for Advanced Technologies (Tokyo)    1999.09

  • 活性酸素から考慮したヘモグロビン利用酸素輸液のセル構造の重要性

    第6回日本血液代替物学会年次大会/日本血液代替物学会    1999.09

  • ヘモグロビンを用いる酸素輸液の合成と物性比較

    日本医工学治療学会第13回学術大会/日本医工学治療学会    1999.09

  • ヘモグロビン小胞体存在下におけるヒト末梢血単核細胞の各種サイトカイン産生の挙動

    人工血液/日本血液代替物学会   7; 1, 27-32  1999.04

  • Photoexcitation and electron transfer reactions of zinc lipidporphyrins in DMSO

    T Ohgushi, ZC Li, FM Li, T Komatsu, S Takeoka, E Tsuchida

    JOURNAL OF PORPHYRINS AND PHTHALOCYANINES   3 ( 1 ) 53 - 59  1999.01

     View Summary

    The photophysical and photochemical properties of 5,10, 15,20-tetrakis{alpha,alpha,alpha,alpha-o-[2',2'-dimethyl-20'-((2"-(trimethylammonio)ethyl) phosphonatoxy)alkanamido]phenyl}porphinatozinc(II) (zinc lipidporphyrins, ZnLPs (C-10, C-18)) have been studied in homogeneous DMSO solution and compared with those of 5, 10,15,20-tetrakis{alpha,alpha,alpha,alpha-o-pivalamidophenyl}porphinatozinc(II) (ZnTpivPP) and tetrakis-phenylporphinatozinc(II) (ZnTPP). The fluorescence quantum yields of the ZnLPs were lower than that of ZnTPP, but their fluorescence lifetimes were relatively long. The electron transfer reactions from the photoexcited states of these Zn porphyrin complexes to several quinone derivatives were evaluated by fluorescence spectroscopy and laser hash photolysis. The efficiencies of oxidative quenching of the excited porphyrins via a dynamic process were significantly decreased by the presence of the bulky substituents on one side of the porphyrin macrocycle. This steric effect of the porphyrin side-chains was quantitatively examined by the Marcus classical treatment. (C) 1999 John Wiley & Sons, Ltd.

    DOI

  • Nonaqueous proton conduction in poly(thiophenylenesulfonic acid)/poly(oxyethylene) composite

    Miyatake, K, Fukushima, K, Takeoka, S, Tsuchida, E

    Chem. Mat.   11 ( 5 ) 1171 - 1174  1999

    DOI

  • Microvascular Responses to Hemodilution with Hb-vesicles as Red Cell Substitutes : Influences of Oz Affinity

    Sakai, H, Tsai, AG, Rohlfs, RJ, Hara, H, Takeoka, S, Tsuchida, E

    Am. J. Physiol.-Heart Circul. Physiol.   276   H553 - H562  1999

  • The Heme Oxygenase System in Liver Microcirculation: A Key Mechanism for Hemoglobin Degradation

    Blood Substitutes/Elsevier Science   Chapt. 19, pp241-250  1998.12

  • 酸素輸液の現状

    武岡真司

    組織培養工学/ニューサイエンス社   24;13, pp.488-492  1998.09

  • Physical properties and packing states of molecular assemblies of synthetic glycolipids in aqueous dispersions

    S Takeoka, K Sou, C Boettcher, JH Fuhrhop, E Tsuchida

    JOURNAL OF THE CHEMICAL SOCIETY-FARADAY TRANSACTIONS   94 ( 15 ) 2151 - 2158  1998.08

     View Summary

    Amidic glycolipids, 1,5-bis-O-aIkyl-N-maltooligonoyl-L-glutamate (1), having various lengths of two hydrocarbon chains (carbon number, m: 14, 16, 18) and maltooligotose with (glucose unit, n: 3, 5, 7) and a N-glycosidic lipid, 1,5-bis-O-octadecyl-N-maltopentaonosyl-L-glutamate (2) have been synthesized. The assembling structures were analyzed by microscopic observation, such as negatively stained TEM, cryo-TEM, and AFM. The glycolipid 1a (rn,n. 14,5) showed a fiber-like structure in all the observed temperatures, while 1b (16,5) showed a fiber-like structure when the hydrating temperature was above the gel-to-liquid crystalline phase transition temperature (T-c; 45 degrees C) and a large disk-like structure when incubated below the T-c. The glycolipid 1c (18,5) took a large disk-like structure after hydration of the powder above the T-c. The glycolipids 1d (18,3) and 1e (18,7) showed a mixture of large disks and large vesicles and a mixture of small disks and micelles, respectively. The N-glycosidic lipid, 2, with no amide linkage made a vesicular structure only. The preparation procedure using high shear stress, such as extrusion and sonication, converted the large disk of 1c to smaller assemblies, such as small disk-, cone-, and granule-like assemblies, depending on the preparation conditions. The glycolipid molecules in the planer part of the disk were packed so tightly that molecular mobility was very low even above the T-c(58 degrees C), and the reactivity of the saccharide chain against Concanavalin A was also very low, indicating that the high reactivity probably comes from the loose packing of saccharide chains around the edge part of the assemblies.

    DOI

  • Subcutaneous microvascular responses to severe hemodilution with hemoglobin-vesicles as red cell substitutes

    Sakai, H, Hara, H, Kerger, H, Tsai, AG, Park, SI, Takeoka, S, Nishide, H, Tsuchida, E, Intaglietta, M

    J. Vasc. Res.   35   261  1998.08

  • 酸素輸液展開の現状

    日本人工臓器学会セミナー    1998.07

  • 人工赤血球開発の現状—長期保存と棚置きの可能性

    第25回日本低温医学会総会    1998.07

  • 酸素輸液の現状

    土田英俊, 武岡真司

    組織培養工学   24 ( 13 ) 488 - 492  1998.06

  • Subcutaneous microvascular responses to hemodilution with a red cell substitute consisting of polyethyleneglycol-modified vesicles encapsulating hemoglobin

    H Sakai, AG Tsai, H Kerger, SI Park, S Takeoka, H Nishide, E Tsuchida, M Intaglietta

    JOURNAL OF BIOMEDICAL MATERIALS RESEARCH   40 ( 1 ) 66 - 78  1998.04

     View Summary

    Phospholipid vesicles encapsulating purified hemoglobin [Hb vesicles (HbV); diameter 259 +/- 82 mm; oxygen affinity 31 mm Hg; [Hb] 5 and 10 g/dL] were developed to provide oxygen-carrying capacity to plasma expanders. Their function as a blood replacement was tested in the subcutaneous microvasculature of awake hamsters during severe hemodilution in which 80% of the red blood cell mass was substituted with suspensions of the vesicles in 5% human serum albumin (HSA) solution. Vesicles were tested with membranes that were unmodified (HbV/HSA) or conjugated with polyethyleneglycol (PEG) on the vesicular surface (PEG-HbV/HSA). The viscosity of 10 g/dL HbV/HSA was 8 cP at 358 s(-1) owing to the intervesicular aggregation, while that of 10 g/dL PEG-HbV/HSA was 3.5 cP, since PEG chains inhibit aggregation. Both materials yielded normal mean arterial pressure, heart rate, and blood gas parameters at all levels of exchange, which could not be achieved with HSA alone. Subcutaneous microvascular studies showed that PEG-HbV/HSA significantly improved microhemodynamic conditions (flow rate, functional capillary density, vessel diameter, and oxygen tension) relative to unmodified HbV/HSA. Even though the enhancement of PEG modification did not achieve the functional characteristics of the blood-perfused microcirculation, PEG reduced vesicular aggregation and viscosity, improving microvascular perfusion relative to the unmodified type. These results highlight the significance of microvascular analysis in the design of red cell substitutes and the necessity of surface modification of HbV to prevent aggregation. (C) 1998 John Wiley & Sons, Inc.

    DOI

  • Disklike assemblies of a synthetic glycolipid and their association with Concanavalin A

    S Takeoka, K Sou, T Ohgushi, E Tsuchida

    SUPRAMOLECULAR SCIENCE   5 ( 1-2 ) 159 - 162  1998.03

     View Summary

    A synthetic glycolipid, 1,5-bis-O-octadecyl-N-maltopentaonoyl-L-glutamate (1), was dispersed in aqueous media above its phase transition temperature to form sheet-like assemblies. They were changed to disk- and ellipsoid-like assemblies by extrusion and sonication, respectively. The structures, stabilities, and thermal properties were studied with microscopic, optical, and calorimetric methods. The disk-like assemblies were homogeneous and the most stable. They formed a unique complex with Concanavalin A (ConA) to become a rouleaux-like structure. (C) 1998 Elsevier Science Limited.

    DOI

  • Distribution of heme oxygenase isoforms in rat liver -Topographic basis for carbon monoxide-mediated microvascular relaxation

    Goda, N, Suzuki, K, Naito, M, Takeoka, S, Tsuchida, E, Tametani, T, Suematsu, M

    J. Clin. Invest.   101 ( 3 ) 604 - 612  1998.02

  • Antioxidative dopant for thermal-resisting polypyrrole and its mechanism

    Takeoka, S, Hara, T, Fukushima, K, Yamamoto, K, Tsuchida, E

    Bull. Chem. Soc. Jpn.   71 ( 6 ) 1471 - 1476  1998

    DOI

  • Effects of the pH-controlled hemoglobin vesicles by CO2 gas

    S Park, T Kose, M Hamasaki, S Takeoka, H Nishide, E Tsuchida

    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND IMMOBILIZATION BIOTECHNOLOGY   26 ( 5-6 ) 497 - 506  1998

     View Summary

    The hemoglobin vesicle (HbV) is a red cell substitute encapsulating purified concentrated Hb in a phospholipid vesicle. In order to improve the oxygen carrying capability of HbV, the pH value of the Hb solution should be adjusted to 7.0 in the HbV preparation, and then the pH value should be adjusted to 7.4 where HbV functions as an oxygen carrier, because the maximum value of [Hb]/[Lipid] was obtained in which the pH of the Hb solution was 7.0, and the metHb formation rate was suppressed in the pH 7.4. Generally, the pH control of the inner aqueous phase of HbV is difficult by changing the pH in the outer phase. We could control the pH of the Hb solution from 7.4 to 7.0 by dissolving CO2 into the Hb solution, and after the preparation of HbV, the pH of HbV is changed to 7.4 by reducing the pressure. The resulting pH-controlled HbV by CO2 gas showed a high [Hb]/[Lipid] value of 1.7 with a low rate of metHb formation.

  • Oxygen releasing from cellular hemoglobin

    N Kawai, H Ohkawa, H Maejima, S Takeoka, H Nishide, E Tsuchida

    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND IMMOBILIZATION BIOTECHNOLOGY   26 ( 5-6 ) 507 - 517  1998

     View Summary

    The oxygen-releasing behavior of hemoglobin vesicles (HbV) was measured in order to study the difference in oxygen dynamics inside and outside the cellular Hb using a conventional stopped now method and a newly developed stopped flow flash photolysis method. The partial pressure of oxygen in the solution outside the HbV was monitored with the lifetime of the triplet state of meso-tetraphenylporphinatozinc(II) bound to human serum albumin excited by the laser flash. The change in the partial pressure of oxygen outside the HbV showed a biphasic profile and was slower than that inside the HbV. The first phase shows the oxygen-releasing process from Hb near the phospholipid bilayer membrane, and the second phase is considered the process in which oxygen diffuses to the bulk aqueous region and reaches the equilibrium value.

  • Human serum albumin-bound synthetic hemes as an oxygen carrier: Determination of equilibrium constants for heme binding to host albumin

    T Komatsu, K Hamamatsu, S Takeoka, H Nishide, E Tsuchida

    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND IMMOBILIZATION BIOTECHNOLOGY   26 ( 5-6 ) 519 - 527  1998

     View Summary

    Human serum albumin (HSA) incorporating synthetic tetraphenylporphinatoiron(II) derivatives (FeP1 or FeP2) can bind and release oxygen reversibly under physiological conditions (in aqueous media, pH 7.4, 37 degrees C). The maximal binding ratio of FeP1/HSA was estimated to be eight, and the stepwise equilibrium constants for FeP1 binding to HSA (K-1-K-8) ranged from 1.2x10(6) to 1.3x10(4) M-1. The major binding sites of FeP1 are presumably identical to those of hemin, bilirubin and long-chain fatty acids. The O-2-binding ability of the HSA-FeP can be regulated by changing the molecular structure of the incorporated hemes. The half-lifetime of the O-2-coordinated FeP2 in HSA was significantly longer than that of HSA-FeP1.

  • Cancellation of nitric oxide- and carbon monoxide-dependent vasorelaxation elicited by acellular hemoglobin derivatives in endotoxemic rat liver

    Kyokane, T, Suematsu, M, Naito, M, Nimura, Y, Ishimura, Y, Takeoka, S, Tsuchida, E

    Hepatology   28   1138  1998

  • Differences in Particle Size and Oxygen-Binding Affinity Between Cross-linked Hemoglobin and Hemoglobin Vesicle

    Oxygen Homeostasis and Its Dynamics/Springer   1  1997.10

  • Differences in Oxygen Transport and Physical Properties Between Cellular and Acellular Hemoglobins

    Proceedings of VII International Symposium on Blood Substitutes   3B-05  1997.09

  • Hemoglobin-Vesicles Prepared by Controlling the Interaction of Hemoglobin and Phospholipid Membrane

    Proceedings of VII International Symposium on Blood Substitutes   p39-b  1997.09

  • Properties of and oxygen binding by albumin-tetraphenylporphyrinatoiron(II) derivative complexes

    Eishun Tsuchida, Katsutoshi Ando, Hiromitsu Maejima, Noriyuki Kawai, Teruyuki Komatsu, Shinji Takeoka, Hiroyuki Nishide

    Bioconjugate Chemistry   8 ( 4 ) 534 - 538  1997.07

     View Summary

    A hydrophobic tetraphenylporphyrinatoiron(II) derivative bearing a covalently bound axial imidazole [Fe(II)P] was efficiently and noncovalently bound into human serum albumin (HSA) up to an average of eight Fe(II)P molecules per HSA molecule. The aqueous solutions of the HSA-Fe(II)P complex provided a reversible and relatively stable oxygen adduct under physiological conditions (pH 7.4 and 37 °C). The half-life of the oxygen adduct (τ( 1/4 )) was 1 h at 37 °C in an air atmosphere. With Fe(II)-TpivPP (the so-called 'picket-fence heme') having no axial base, an oxygenated HSA-Fe(II)TpivPP complex was obtained using a 20-fold molar excess of 1,2-dimethylimidazole, but the τ( 1/4 ) was very short (ca. 10 min at 37 °C). The oxygen affinity [P( 1/4 )(O2)] and oxygen transporting efficiency (OTE) of HSA-Fe(II)P at 37 °C were 30 Torr and 22%, respectively. Furthermore, the oxygen-binding and dissociation rate constants (k(on) and ko(off)) are extremely high in comparison with those of hemoglobin. The HSA molecule binding eight Fe(II)P molecules can transport about 3.4 mL/dL of oxygen under physiological conditions, corresponding to about 60 % of the oxygen transporting amount of human blood.

    DOI PubMed

  • Methemoglobin formation in hemoglobin vesicles and reduction by encapsukated thiols

    Shinji Takeoka, Hiromi Sakai, Takehiro Kose, Yuichi Mano, Yuriko Seino, Hiroyuki Nishide, Eishun Tsuchida

    Bioconjugate Chemistry   8 ( 4 ) 539 - 544  1997.07

     View Summary

    The hemoglobin vesicle (HbV) is a red cell substitute encapsulating purified concentrated Hb in a phospholipid vesicle. In order to suppress metHb formation or autoxidation, for the long-term maintenance of the oxygen transporting capability, a series of thiols (cysteine, Cys
    glutathione, GSH
    homocysteine, Hcy
    and acetylcysteine, Acy) were studied as reductants of metHb. Hcy and GSH showed a good suppressive effect on metHb formation, while Cys adversely accelerates the metHb formation at a rate twice that of the Hb solution without any reductants and Acy showed no change. The significant suppression by the coaddition of superoxide dismutase (SOD) and catalase to Cys indicated that Cys was easily oxidized by oxygen and simultaneously generates a large amount of active oxygens. The effective suppression of metHb formation by SOD and catalase was not observed for HbV containing no reductants, indicating that the generation of active oxygens from Hb itself is not significant. The coencapsulation of Hcy with Hb resulted in a low rate of metHb formation in HbV (initial rate, 1%/h) in vitro at oxygen partial pressure (P(O2)) of 142 Torr. The rate increased with decreasing P(O2), showed a maximum (2.2%/h) around P(O2) = 23 Torr, and then decreased to 0%/h at 0 Torr. From these results, it is suggested that the fast metHb formation rate in the blood circulation of Wistar rats injected with 20 vol % of the HbV solution would be mainly caused by the exposure of HbV to the low P(O2).

    DOI PubMed

  • Evaluation of the capabilities of a hemoglobin vesicle as an artificial oxygen carrier in a rat exchange transfusion model

    Y Izumi, H Sakai, T Kose, K Hamada, S Takeoka, A Yoshizu, H Horinouchi, R Kato, H Nishide, E Tsuchida, K Kobayashi

    ASAIO JOURNAL   43 ( 4 ) 289 - 297  1997.07

     View Summary

    Encapsulation of hemoglobin within a liposome is one of the strategies in the development of artificial oxygen carriers. It maintains the oxygen transporting properties of hemoglobin and, at the same time, eliminates the side effects of cell free hemoglobin. Hemoglobin vesicles (HbV) are a type of liposome encapsulated hemoglobin. They have a particle size of approximately 250 nm, a hemoglobin concentration of 10 g/dl, and the oxygen affinity, P50, is regulated to 32 Torr. In this study the authors examined the oxygen transporting capability of HBV in vivo, by performing exchange transfusions in rats. Exchange transfusion (90% of the estimated circulatory volume) with HbV suspended in 5% albumin (containing 160 mEq/L, sodium and 107 mEq/L, chloride) was carried out in mate Wistar rats. Mean arterial pressure and heart rate were monitored through the arterial catheter. Arterial blood samples for gas analyses were also obtained from the arterial catheter. Abdominal aortic blood flow was measured by an ultrasonic pulsed Doppler flowmeter as an indicator of cardiac output. The oxygen tension of blood withdrawn from the right atrium was measured as an indicator of mixed venous oxygen tension. These values were employed to calculate oxygen delivery and consumption. Renal cortical and skeletal muscle tissue oxygen tensions were monitored as indicators of tissue perfusion. Five percent albumin and washed rat red blood cells suspended in 5% albumin containing 10 g/dl of hemoglobin, were employed as controls. At the completion of a 90% exchange transfusion, renal cortical and skeletal muscle tissue oxygen tensions, along with oxygen delivery and consumption, were sustained almost equally well with the HbV suspension compared to the washed rat red blood cell suspension, but declined significantly with the albumin suspension. The results indicate that the oxygen transporting capability of HbV was almost equivalent to that of rat red blood cells.

    DOI

  • 臨床利用を目指す人工赤血球

    第46回高分子学会年次大会講演予行集   IIA16IL  1997.05

  • Construction of Artificial Methemoglobin Reduction Systems in Hb Vesicles

    Artificial Cells,Blood Substitutes,and Immobilization Biotechnology/Marcel Dekker   25;1  1997.02

    DOI

  • Surface modification of hemoglobin vesicles with poly(ethylene glycol) and effects on aggregation, viscosity, and blood flow during 90% exchange transfusion in anesthetized rats

    H Sakai, S Takeoka, SI Park, T Kose, H Nishide, Y Izumi, A Yoshizu, K Kobayashi, E Tsuchida

    BIOCONJUGATE CHEMISTRY   8 ( 1 ) 23 - 30  1997.01

     View Summary

    Poly(ethylene glycol) (PEG(5000))-conjugated phosphatidylethanolamine was introduced onto the surface of hemoglobin vesicles (HbV); phospholipid vesicles encapsulating concentrated Hb (d = 0.257 +/- 0.087 mu m; P-50 = 32 Torr). The obtained PEG-modified HbV (HbV-PEG) was studied for use as a red cell substitute from the viewpoint of rheology, surface properties, and hemodynamics. The viscosity of the unmodified HbV suspended in saline ([Hb] = 10 g/dL) was 2.6 cP (shear rate = 358 s(-1), 37 degrees C), less than that of human blood (4 cP). However, when suspended in a 5 g/dL albumin solution (HbV/albumin), it increased to 8 cP due to the molecular interaction between albumin and vesicles, and the viscosity increased with decreasing shear rate, e.g., 37 cP at 0.58 s(-1). As for the HbV-PEG/albumin, on the other hand, the viscosity was 3.5 cP at 358 s(-1) and was comparable with that of human blood. Optical microscopy showed formless flocculated aggregates of the unmodified HbV, while no aggregates were confirmed for the HbV-PEG. The steric hindrance of PEG chains seemed to be effective in preventing intervesicular access and the resulting aggregation To estimate the flow profiles in the capillaries, the suspensions were allowed to penetrate through isopore membrane filters (pore size = 0.4-8 mu m, cf. capillary diameter = 4-10 mu m). The penetration rate of the HbV-PEG/albumin was higher than that of the unmodified HbV/albumin due to the suppression of aggregation, whereas both of them were significantly higher than that of human blood due to the smaller size of vesicles than RBC. Ninety percent exchange transfusion was performed with the HbV-PEG/albumin or HbV/albumin in anesthetized Wistar rats (n = 6). The blood flow in the abdominal aorta increased 1.5 times, and the total peripheral resistance decreased in the HbV-PEG/albumin-administered group in comparison with the HbV/albumin group. As for the blood gas parameters, the base excess and pH remained at higher levels in the HbV-PEG/albumin group, and the O-2 tension in mixed venous blood for the HbV-PEG/albumin group tended to be maintained at a higher level than that for the HbV/albumin group. Thus, the PEG modification of HbV reduced the viscosity by the suppression of aggregation and resulted in prompt blood circulation in vivo.

    DOI

  • Reduction of methemoglobin via electron transfer across the bilayer membrane of Hb vesicles

    Takeoka, S, Ohgushi, T, Nishide, H, Tsuchida, E

    Bull. Chem. Soc. Jpn.,   70 ( 5 ) 1171 - 1178  1997

    DOI

  • Construction of artificial methemoglobin reduction systems in Hb vesicles

    S Takeoka, T Ohgushi, H Sakai, T Kose, H Nishide, E Tsuchida

    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND IMMOBILIZATION BIOTECHNOLOGY   25 ( 1-2 ) 31 - 41  1997

     View Summary

    The hemoglobin vesicle (HbV) is a red cell substitute encapsulating purified concentrated Hb in a phospholipid vesicle. In order to suppress metHb formation for the long term maintenance of oxygen transporting capability in vivo, thiols (cysteine, Cys; homocysteine, Hey) were studied as reductants of metHb. Hey showed a suppressive effect on metHb formation, while Cys adversely accelerates metHb formation at the rate of twice the Hb solution without any reductants. The suppression of Cys-induced metHb formation by the addition of superoxide dismutase (SOD) and catalase indicated that Cys was easily oxidized by oxygen and simultaneously generated a large amount of active oxygens. The rate of metHb formation was influenced by PO2 and pH. Furthermore, the reducing systems (methylene blue (MB), NADH or ascorbic acid) were added to the outer aqueous phase of HbV, and the artificial reduction systems constructed through the bilayer membrane were evaluated.

    DOI

  • The oxygen carrying capability of hemoglobin vesicles evaluated in rat exchange transfusion models

    K Kobayashi, Y Izumi, A Yoshizu, H Horinouchi, SI Park, H Sakai, S Takeoka, H Nishide, E Tsuchida

    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND IMMOBILIZATION BIOTECHNOLOGY   25 ( 4 ) 357 - 366  1997

     View Summary

    To evaluate the oxygen transporting capability of Hemoglobin vesicles (HbV) the physiological responses to 40% and 90% exchange transfusions with HbV in anesthetized rat were observed. Hb concentration of HbV dispersions is 10g/dL. HbV dispersed in phosphate buffered saline and HbV dispersed in 5% albumin solution were used as samples for 40% and 90% exchange transfusions, respectively. HbV surface-modified with polyoxyethylene (HbV-Poe) was also used in the 90% exchange transfusion. As controls, phosphate buffered saline, 5% albumin solution, and HbV containing methemoglobin and therefore deprived of oxygen transporting capabilities (metHbV) were administered as non-oxygen carrying fluids and washed rat red blood cells (ratRBC) as an oxygen carrying fluid. Measurements included mean arterial pressure, arterial blood gas analyses, aortic blood flow and renal cortical tissue oxygen tension. At the completion of the exchange transfusion renal cortical tissue oxygen tensions along with oxygen delivery and consumption were sustained almost equally well with the HbV dispersion compared to the washed rat red blood cell dispersion, but declined significantly in the phosphate buffered saline and albumin solutions. These results indicated that the oxygen transporting capability of HbV was almost equivalent to that of rat red blood cells. in the HbV-Poe group, aortic blood flow was sustained higher in comparison to the HbV group. As for the blood gas parameters, pH and venous oxygen tensions in the HbV-Poe group tended to be higher than those in the HbV group.

    DOI

  • Critical molecular weight effects in the aggregation of phospholipid vesicles triggered by water-soluble polymers and an integrated glycolipid

    S Takeoka, K Sou, S Arase, T Ohgushi, E Tsuchida

    MACROMOLECULES   29 ( 25 ) 8132 - 8136  1996.12

     View Summary

    The intervesicular aggregation of phospholipid vesicles is induced by the addition of water-soluble polymers such as poly(ethylene glycol), dextran, etc. due to the interaction between the vesicular surface and the water-soluble polymers. The interaction can be expressed by the critical molecular weight (M(c)) of the water-soluble polymers for the aggregation of vesicles. The surface modification of vesicles with glycolipids (O-1,O-5-bis(octadecyl) N-maltooligonoyl-L-glutamate) accelerates the aggregation of vesicles induced by dextran; therefore, M(c) significantly decreased due to the surface modification. No dependence of phospholipid concentration and dextran concentration in an aqueous phase on the M(c) indicates that dextran does not act as a cross-linking agent among the vesicles. A clear dependence of the density of the saccharide chains on the vesicular surface on the M(c) suggests that dextran should adsorb on the surface of the vesicles by the interaction with the oligosaccharide chains on the surface and cause vesicular aggregation. A lower critical solution temperature was observed for this kind of interaction, and the critical temperature was controlled by changing the molecular weight of dextran.

    DOI

  • Differences in Oxygen Transport and Physical Property Between Encapsulated Hemoglobin and Cell-free Hemoglobin

    Keio University International Symposia for Life Sciences and Medicine    1996.12

  • Physiologic responses to exchange transfusion with hemoglobin vesicles as an artificial oxygen carrier in anesthetized rats: changes in mean arterial pressure and renal cortical tissue oxygen tension

    Izumi, Y, Sakai, H, Hamada, K, Takeoka, S, Yamahata, T, Kato, R, Nishide, H, Tsuchida, E, Kobayashi, K

    Critical Care Medicine   24 ( 11 )  1996.11

    DOI

  • Current Issues in Blood Substitute Research and Development

    人工血液/日本サイマルインターナショナル   4;3  1996.09

  • Methemoglobin Reduction of Hemoglobin-Vesicles by Thiols

    International Symposium on High-Tech Polymers and Polymer Complexes/蘭州    1996.08

  • Construction of Artificial Reduction Systems in Hb-vesicles

    6th International Symposium on Blood Substitutes/Montreal    1996.08

  • Functional Evaluation of hemoglobin- and lipidheme-vesicles as red cell substitutes

    H Sakai, K Hamada, S Takeoka, H Nishide, E Tsuchida

    POLYMERS FOR ADVANCED TECHNOLOGIES   7 ( 8 ) 639 - 644  1996.08

     View Summary

    The two kinds of veri cell substitutes, hemoglobin-vesicles (HbV) and lipidheme-vesicles (LihV, totally synthetic oxygen carrier), were evaluated in terms of physicochemical properties such as binding and dissociating reactions of ligands (CO, O-2 and NO), rheological and structural properties. Carbonylation of Hb during the purification of Hb and the preparation of HbV is effective to prevent Hb denaturation. The rates of oxygenation of both HbV and LihV are faster than that of I ed blood cells (RBC). Their oxygen affinities (P-50, HbV, 32 mmHg; LihV, 43 mmHg, cf. RBC, 28 mmHg) can be controlled to transport a sufficient amount of oxygen comparable with that of RBC. The smaller sizes of vesicles are advantageous for prompt ligand reaction and low viscosity. Both HbV and RBC show about 100 times less vasoconstrictive effects than stripped Hb. HbV shows only one sixth of the slow binding rate of NO (=endothelial derived relaxation factor) in comparison with stripped Hb. Inhibition of vasoconstriction by those vesicles is discussed from the kinetic data.

    DOI

  • Layer-controlled hemoglobin vesicles by interaction of hemoglobin with a phospholipid assembly

    S Takeoka, T Ohgushi, K Terase, T Ohmori, E Tsuchida

    LANGMUIR   12 ( 7 ) 1755 - 1759  1996.04

     View Summary

    Hemoglobin vesicles, which encapsulated concentrated hemoglobin (Hb) with a bilayer of dipalmitoylphosphatidylcholine/cholesterol/palmitic acid, were prepared under various preparation conditions in order to decrease the number of bilayers (n) constructing the vesicle and increase the Ho concentration in the interior of the vesicle ([Hb](in)). n decreased when the surface potential of the bilayer became more negative because of the electrostatic repulsion between the bilayers, while with a changing xi-potential of Hb from positive to negative, [Hb](im) showed a precipitous fall because of the electrostatic repulsion between Hb and the surface of the bilayer. A temperature decrease leads to a quality increase in the Hb vesicles ([Hb]/[lipid]) in spite of the [Hb](in) decrease by the viscosity increase of the Hb solution. This is explained by the effective reduction of n due to the reduction in membrane fluidity and the protonation of Hb.

    DOI

  • Oxygen Transporting Ability and Solution Properties of Stabilized Hemoglobin-Vesicles (HbV)

    24th Congress of the International Society of Blood Transfusion/東京    1996.04

  • 赤血球代替物の近況

    医学のあゆみ/医歯薬出版   117;2  1996.02

  • Physical properties of hemoglobin vesicles as red cell substitutes

    H Sakai, K Hamada, S Takeoka, H Nishide, E Tsuchida

    BIOTECHNOLOGY PROGRESS   12 ( 1 ) 119 - 125  1996.01

     View Summary

    Hemoglobin vesicles (HbV) as red cell substitutes were prepared from a purified carbonylhemoglobin (HbCO) solution and a lipid mixture composed of phospholipids, cholesterol, and cr-tocopherol. The diameter was controlled to 251 +/- 87 nm using an extrusion method; the vesicles penetrated through the membrane filters with regulated pore sizes. After the ligand exchanging reaction (HbCO --> HbO(2)), the oxygen affinity (P-50) Of HbV was 32 Torr, which was controlled with the coencapsulation of pyridoxal 5'-phosphate. The rate of metHb formation in HbV was nonenzymatically reduced with the coencapsulation of DL-homocysteine. The Hb concentration of the HbV suspension, which was dispersed in a phosphate buffered saline solution (pH 7.4), was controlled at 10 g/dL. At this concentration, the total lipid concentration was 6.2 g/dL and the viscosity, 2.6 cP (230 s(-1)), was lower than that of the blood (4.4 cP). The HbV suspension showed a typical non-Newtonian flow for a particle dispersion and agreed well with the Casson model. The viscosity at shear rates lower than 23 s(-1) showed a maximum with increasing the mixing ratio of human blood, plasma, or albumin, while no maximum was observed for the mixture with washed red blood cells. The aggregates of HbV are formed by interaction with plasma proteins, including albumin, while the aggregates reversibly dissociate at higher shear rate.

    DOI

  • Physical properties of hemoglobin vesicles as red cell substitutes

    H Sakai, K Hamada, S Takeoka, H Nishide, E Tsuchida

    BIOTECHNOLOGY PROGRESS   12 ( 1 ) 119 - 125  1996.01

     View Summary

    Hemoglobin vesicles (HbV) as red cell substitutes were prepared from a purified carbonylhemoglobin (HbCO) solution and a lipid mixture composed of phospholipids, cholesterol, and cr-tocopherol. The diameter was controlled to 251 +/- 87 nm using an extrusion method; the vesicles penetrated through the membrane filters with regulated pore sizes. After the ligand exchanging reaction (HbCO --> HbO(2)), the oxygen affinity (P-50) Of HbV was 32 Torr, which was controlled with the coencapsulation of pyridoxal 5'-phosphate. The rate of metHb formation in HbV was nonenzymatically reduced with the coencapsulation of DL-homocysteine. The Hb concentration of the HbV suspension, which was dispersed in a phosphate buffered saline solution (pH 7.4), was controlled at 10 g/dL. At this concentration, the total lipid concentration was 6.2 g/dL and the viscosity, 2.6 cP (230 s(-1)), was lower than that of the blood (4.4 cP). The HbV suspension showed a typical non-Newtonian flow for a particle dispersion and agreed well with the Casson model. The viscosity at shear rates lower than 23 s(-1) showed a maximum with increasing the mixing ratio of human blood, plasma, or albumin, while no maximum was observed for the mixture with washed red blood cells. The aggregates of HbV are formed by interaction with plasma proteins, including albumin, while the aggregates reversibly dissociate at higher shear rate.

    DOI

  • Thermal stabilization of polypyrrole by incorporation of aromatic sulfonate derivatives as dopants

    Takeoka, S, Hara, T, Yamamoto, K, Tsuchida, E

    Chem. Lett.   1996 ( 2 ) 253 - 254  1996

    DOI

  • ポリエチレングリコール型脂質および糖脂質によるヘモグロビン小胞体(HbV)の表面修飾効果

    朴 晟翼, 宗 慶太郎, 酒井宏水, 武岡真司, 西出宏之, 土田英俊

    人工血液   4   9 - 14  1996

  • Rheological Characteristics of the Phospholipid Vesicles Surface-Modified with Oligosaccharide

    Pacific Polymer Conference (Hawaii)    1995.12

  • DESTRUCTION AND RECONSTITUTION OF POLYPHOSPHOLIPID VESICLES WITH SIZE MEMORY

    S TAKEOKA, T OHGUSHI, E TSUCHIDA

    MACROMOLECULES   28 ( 23 ) 7660 - 7666  1995.11

     View Summary

    1,2-Bis(2,4-octadecadienoyl)-sn-glycero-3-phosphocholine (DODPC), constructing the bilayer membrane of a vesicle, was polymerized by UV irradiation. The freeze-dried vesicles were completely dissolved in chloroform to obtain cross-linked phospholipid polymer (polyDODPC). The polyDODPC consisted of high molecular weight components and oligomers. The polyDODPC was dispersed by sonication, and two size distributions of the reconstituted vesicles were confirmed, larger vesicles from high molecular weight polyDODPC and smaller vesicles from oligomers. When the polyDODPC vesicles of various sizes and unimodal size distribution were reconstituted by an extrusion method, the vesicles of the same size as the vesicles at polymerization showed excellent stability compared with the other sizes. Interestingly, the original size of the polyDODPC vesicles could be completely restored by a cholate dialysis method. It was also clarified that vesicles larger than the original size were reconstituted from the higher molecular weight component, and the smaller vesicles were from the oligomers. Furthermore, a linear phospholipid polymer obtained from 1-palmitoyl-2-(2,4-octadecadienoyl)-sn-glycero-3-phosphocholine could not restore the original size. Those results indicate that the original size of the vesicles can be restored completely by all polyDODPCs having the original composition of large and small molecular weights and cross-linked structure.

    DOI

  • INTERMOLECULAR INTERACTIONS OF LITHOCHOLIC ACID-DERIVATIVES WITH PHOSPHOLIPID AS BILAYER-MEMBRANE

    ZC LI, FM LI, S ARASE, S TAKEOKA, E TSUCHIDA

    LANGMUIR   11 ( 8 ) 3161 - 3166  1995.08

     View Summary

    Three single chain lithocholic acid derivatives with a steroidal moiety in the center of the hydrocarbon chain and two head groups at both ends of the chain were synthesized and characterized. Due to their high compatibility with phospholipids, they can be incorporated homogeneously into the phospholipid bilayer membrane. The resulting mixed vesicles showed high stability against aggregation by monitoring the turbidity change in the vesicle suspensions. The entire membrane-spanning packing thus locates the steroidal moiety at the center of the bilayer. The interaction of the steroidal moiety with Lipid molecules at the center of the bilayer resulted in higher mobility of lipid molecules below the T-c of the host phospholipid membrane as clarified by temperature-dependent H-1 NMR, fluorescence depolarization anisotropy, and differential scanning calorimetry (DSC). The results were compared with those of cholesterol, whose steroidal moiety is located near the surface of the membrane.

    DOI

  • Modification of hemoglobin-vesicles with Oligosccharide Chains

    Artif. Organs Today/VSP   4;4  1995.04

  • LIGAND-EXCHANGE REACTION OF CARBONYLHEMOGLOBIN TO OXYHEMOGLOBIN IN A HEMOGLOBIN LIQUID MEMBRANE

    JE CHUNG, K HAMADA, H SAKAI, S TAKEOKA, E TSUCHIDA

    NIPPON KAGAKU KAISHI   1995 ( 2 ) 123 - 127  1995.02

     View Summary

    In order to change carbonylhemoglobin (HbCO) to oxyhemoglobin(HbO(2)), the photodissociation of HbCO and O-2-binding to hemoglobin were studied using Hb liquid membrane system in O-2 atmosphere. The reduction rate of HbCO increased with illuminance and saturated nearly at 2.0 . 10(5) 1x. under a constant O-2 pressure of 760 Torr. The reduction rate of HbCO was a 5-fold low when the partial O-2 pressure was decreased from 760 to 0 Torr under the illuminance. In the static liquid membrane the exchanging rate of CO ligand in HbCO was decreased with the increment of the thickness of the liquid membrane, while the stirred liquid membrane showed a reverse result. The ligand exchange apparatus was made based on the obtained results, and the high flow rate of Hb solution enhanced the reduced amount of HbCO. Moreover, when the ligand exchange of Hb-vesicle dispersion was compared with an Hb solution, it proceeded more efficiently due to the encapsulation of concentrated Hb into a small compartment dispersed in a buffer solution.

    DOI

  • COORDINATION BEHAVIOR OF O-2 AND CO IN A SOLID FILM CONSISTING OF HEMOGLOBIN AND MALTOSE

    JF CHUNG, H SAKAI, S TAKEOKA, H NISHIDE, E TSUCHIDA

    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN   68 ( 3 ) 1006 - 1011  1995

     View Summary

    Solid hemoglobin (Hb) films were prepared by casting from a mixed solution of human hemoglobin and maltose (wt ratio of Hb/maltose is 60/40). The behavior of O-2 and CO in coordinating to Hb in the films was then observed. The coordination equilibrium constants of both ligands were smaller by about an order of magnitude, and the rate constants (K-on, and k(off)) were smaller by about 13 orders of magnitude, than those of aqueous solutions of Hb. Interestingly, O-2 and CO saturation was limited to 52% and 86%, respectively, under a partial pressure of 760 Torr (1 Torr=133.322 Pa) of each gas, in spite of the one-to-one ligand binding ratio in aqueous solutions. This is due to the restraint of the structural change from a tense to a relaxed state in the solid phase. The limited saturation of the coordination in the solid phase was found to increase to 100% when a small amount (5.9%) of water was added; a significant structural change from the tense to the relaxed states followed.

    DOI

  • Electronic Processes in Macromolecular Metal Complexes

    Macromolecular Metal Complexes/Springer-Verlag   Chapt.4  1995

  • Stabilized Hemoglobin Vesicles

    Artificial Red Cells, Materials, Performances and Clinical Studyas Blood Substitutes/John Wiley & Sons   Chapt.3  1995

    DOI

  • ヘモグロビン液膜のカルボニルヘモグロビンからオキシヘモグロビンへの配位子交換

    鄭 主恩, 濱田健一, 酒井宏水, 武岡真司, 土田英俊

    日化誌   2   123 - 127  1995

    DOI

  • 赤血球代替物(NRC)の成分定量

    濱田健一, 巨勢丈裕, 大串 建, 酒井宏水, 武岡真司, 西出宏之, 土田英俊

    人工血液   3   96 - 101  1995

  • ヘモグロビン液膜のカルボニルヘモグロビンからオキシヘモグロビンへの配位子交換

    鄭 主恩, 濱田健一, 酒井宏水, 武岡真司, 土田英俊

    日化誌   2   123 - 127  1995

    DOI

  • 赤血球代替物(NRC)の成分定量

    濱田健一, 巨勢丈裕, 大串 建, 酒井宏水, 武岡真司, 西出宏之, 土田英俊

    人工血液   3   96 - 101  1995

  • ヘモグロビン小胞体の酸素輸送に関するラット交換輸血試験

    酒井宏水, 泉 陽太郎, 山畑 健, 濱田健一, 武岡真司, 西出宏之, 小林紘一, 土田英俊

    人工血液   3   81 - 86  1995

  • EFFECT OF A LITHOCHOLIC ACID-DERIVATIVE ON THE MOLECULAR PACKING AND STABILITY OF PHOSPHOLIPID-VESICLES

    ZC LI, FM LI, S ARASE, S TAKEOKA, E TSUCHIDA

    CHEMISTRY LETTERS   1994 ( 12 ) 2199 - 2202  1994.12

     View Summary

    A lithocholic acid derivative having high compatibility with phospholipid was synthesized and incorporated to the phospholipid bilayer membrane. A significant influence of it on the molecular packing and the stability enhancement of the vesicle suspension were clarified by a fluorescence depolarization method and a turbidity measurement, respectively.

    DOI

  • SUPPRESSION OF METHEMOGLOBIN FORMATION BY GLUTATHIONE IN A CONCENTRATED HEMOGLOBIN SOLUTION AND IN A HEMOGLOBIN-VESICLE

    H SAKAI, S TAKEOKA, Y SEINO, E TSUCHIDA

    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN   67 ( 4 ) 1120 - 1125  1994.04

     View Summary

    The suppression of methemoglobin (metHb) formation by glutathione (GSH) is difficult because GSH is oxidized not only by the reduction of metHb, but also by oxygen to generate active oxygen species, such as superoxide (O2-.) and hydrogen peroxide (H2O2), which contribute to metHb formation. An effective nonenzymatic reduction of metHb was achieved at a high concentration of Hb (40 wt%, 2.48 x 10(-2) M subunits (1 M = 1 mol dm-3)) because the reduction of metHb was accelerated, and at a low partial pressure of oxygen (pO2 = 40 Torr (1 Torr = 133.322 Pa)) because the oxidation of GSH was effectively suppressed. Hb-vesicles, which encapsulate concentrated Hb (40 wt%, 2.48 x 10(-2) M subunits), transport oxygen in the blood stream at relatively low pO2 (110 Torr in artery and 40 Torr in venous, 58 Torr in average). The rate of metHb formation in Hb-vesicles was effectively decreased from 3.8 x 10(-7) to 1.6 x 10(-7) M s-1 by coencapsulating GSH at 58 Torr.

    DOI

  • INTERACTION BETWEEN PHOSPHOLIPID ASSEMBLIES AND HEMOGLOBIN (HB)

    S TAKEOKA, K TERASE, H YOKOHAMA, H SAKAI, H NISHIDE, E TSUCHIDA

    JOURNAL OF MACROMOLECULAR SCIENCE-PURE AND APPLIED CHEMISTRY   A31 ( 1 ) 97 - 108  1994

     View Summary

    In order to prepare Hb-vesicles having a high oxygen-transporting ability, it is important to encapsulate concentrated Hb with a mixed lipid membrane having low lamellarity (n). The concentration of Hb in the Hb-vesicles ([Hb](in)) increased with the Hb concentration used for the preparation of Hb-vesicles. Hb-vesicles with smaller lamellarity could be obtained y lowering the preparation temperature. The encapsulation of Hb was significantly influenced by the ionic strength and the solution pH because lipids and Hb are electrolytes. The lamellarity decreases with increasing solution pH because of electrostatic repulsion between the surfaces of lipid membranes. On the other hand, [Hb](in) increases when the solution pH is lowered below the pI of Hb (7.0 at 25 degrees C) due to electrostatic interaction between Hb and the surface of the membrane. This interaction decreases with the ionic strength of the Hb solution, leading to a lower [Hb](in).

  • Interaction between phospholipids assembly and globin proteins

    Takeoka, S, Yokohama, H, Terase, K, Tsuchida, E

    Abstr. Pap. Am. Chem. Soc.   207   145  1994

  • Extraction of purified Hb from red-cell by coexistence of organic media including polymers

    Tsuchida, E, Chung, JE, Seino, Y, Sakai, H, Takeoka, S

    Abstr. Pap. Am. Chem. Soc.   141   207  1994

  • CHARACTERISTICS OF HB-VESICLES AND ENCAPSULATION PROCEDURE

    S TAKEOKA, H SAKAI, K TERASE, H NISHIDE, E TSUCHIDA

    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND IMMOBILIZATION BIOTECHNOLOGY   22 ( 3 ) 861 - 866  1994

     View Summary

    The performance of Hb-vesicles depends on the weight ratio of Hb to lipid ([Hb]/[Lipid]). This value is improved by lowering the number of bilayer membrane of the vesicle and raising the concentration of Hb in the interior of the vesicle. Maximum [Hb]/[Lipid] ratio was obtained at ca. pH 7, that would relate to the isoelectric point (pl) of Hb at 25 degrees C. On the other hand, the [Hb]/[Lipid] ratio decreased with ionic strength and increased with lowering temperature. The Hb-vesicles encapsulating 40 g/dl Hb with only one bilayer membrane were isolated by using the difference in the density of the vesicles.

    DOI

  • Convenient method of purify hemoglobin

    Sakai, H, Takeoka, S, Nishide, H, Tsuchida, E

    Artif. Cells Blood Substit. Immobil. Biotechnol.   22   651 - 656  1994

    DOI

  • グルタチオンによるHb小胞体のmetHb生成抑制

    横浜裕明, 清野由里子, 鄭 主恩, 酒井宏水, 武岡真司, 西出宏之, 土田英俊

    人工臓器   23 ( 3 ) 868 - 871  1994

     View Summary

    To increase the amount of oxygen transported by Hb-vesicles, it is important to suppress metHb formation. α-Tocopherol decreased lipid peroxidation in Hb-vesicles composed of EYL, whereas the Hb-vesicles composed of saturated phospholipids, such as DPPC, showed less metHb formation. To suppress metHb formation in a highly purified Hb solution, glutathione (GSH) was studied as a reductant. GSH is oxidized not only by metHb reduction but also by O2 to generate active oxygen species, which react with Hb to form metHb. Effective nonenzymatic metHb reduction was observed at a high [Hb] because of the acceleration of metHb reduction, and at lower partial pressure of O2 (pO2) because of suppression of GSH oxidation. Hb-vesicles encapsulating 40g/dl Hb with pyridoxal 5'-phosphate showed high oxygen transporting efficiency. The rate of metHb formation at a low pO2 in the blood stream was decreased by GSH.

    DOI CiNii

  • Hb小胞体のオリゴ糖修飾効果

    酒井宏水, 滝貞幹正, 鄭 主恩, 武岡真司, 土田英俊

    人工臓器   23 ( 3 ) 864 - 867  1994

     View Summary

    Synthetic glycolipids were introduced into lipid membranes of Hb-vesicles in order to modify the surface of the vesicles with oligosaccharide chains. The resulting suspension of the Hb-vesicles showed good rheological properties and high structural stability. Intervesicular aggregation induced by the addition of Ca2+(5mM) or dextran (Mw. 40, 000) was suppressed due to the excluded volume effect of the oligosaccharide chains which inhibited the access of the surrounding vesicles. After freeze-thawing of the Hb-vesicles with oligosaccharide chains, no diameter change was observed, and leakage of Hb was also suppressed. No change in oxygen binding properties indicates that small molecules or ions as allosteric effectors were not leaked from the vesicles. Therefore, the Hb-vesicles can be stored in a frozen state by the surface modification of the vesicles with the oligosaccharide chains.

    DOI CiNii

  • PURIFICATION OF CONCENTRATED HEMOGLOBIN USING ORGANIC-SOLVENT AND HEAT-TREATMENT

    H SAKAI, S TAKEOKA, H YOKOHAMA, Y SEINO, H NISHIDE, E TSUCHIDA

    PROTEIN EXPRESSION AND PURIFICATION   4 ( 6 ) 563 - 569  1993.12

    DOI

  • SINTERED-TANTALUM ELECTRODE MODIFIED WITH POLYPYRROLE BY ELECTROPOLYMERIZATION

    YS PARK, T OHTA, S TAKEOKA, K YAMAMOTO, E TSUCHIDA

    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN   66 ( 8 ) 2449 - 2451  1993.08

     View Summary

    Polypyrrole (PPy) formation in the interior of a porous electrode by electropolymerization was considered to be impossible. When a concentrated pyrrole solution was soaked into a sintered-Ta electrode and electrolyzed in a pyrrole-free solution, PPy was formed in the interior. PPy occupied 70% of the inner space, and no defect was observed by SEM.

    DOI

  • 人工血液研究の最近の進歩

    土田英俊, 武岡真司

    最新医学   48 ( 7 ) 1069 - 1077  1993.07

  • Cryoprotection of synthetic glycolipids for phospholipid-vesicles

    Sakai, H, Takisada, M, Takeoka, S, Tsuchida, E

    Chem. Lett.   1993   1981 - 1984  1993

    DOI

  • Preparation conditions of human hemoglobin resicles covered with lipid membranes

    Takeoka, S, Sakai, H, Nishide, H, Tsuchida, E

    Artificial Organs Today   3 ( 3 ) 129 - 136  1993

  • IN-SITU FORMATION OF PPY/TA2O5/TA STRUCTURE BY ELECTROPOLYMERIZATION AND ITS ELECTRICAL-PROPERTIES

    YS PARK, S TAKEOKA, K YAMAMOTO, E TSUCHIDA

    MOLECULAR CRYSTALS AND LIQUID CRYSTALS   227   219 - 229  1993

     View Summary

    Both PPy film and Ta2O5 layer are simultaneously formed on Ta-electrode by one process of electrical oxidation in an aqueous solution. Prepara tion of the structure (PPy/Ta2O5/Ta) can also takes place by the electrolysis using Ta-electrode on which PPy is formed previously by electropolymerization. Since PPy film obtained by the former method has higher electric conductivity (110 S/cm) and better adhesion than the later, (PPy/Ta2O5/Ta)structure formed simul taneously is confirmed better electrical properties as a capacitor.

    DOI

  • Oxidation of a PPy‐modified tantalum electrode

    Young‐Seo Park, Kimihisa Yamamoto, Shinji Takeoka, Tomoyuki Ohta, Eishun Tsuchida

    Polymers for Advanced Technologies   4 ( 5 ) 329 - 334  1993

     View Summary

    A tantalum electrode on which polypyrrole (PPy) had been previously formed by electropolymerization was galvanostatically electrolyzed in an aqueous solution of 0.01 wt% phosphoric acid. This process contains the irreversible oxidation of a PPy film, the decomposition of solvent, and the formation of Ta2O5 by the reaction of OH− coming through the PPy film, with Ta electrodes. A three layer‐structure (PPy/Ta2O5/Ta) was confirmed by electron spectroscopy for chemical analysis (ESCA). A PPy film containing CIO4− as dopant [PPy(CIO4−)] was significantly deteriorated in comparison with PPy(TsO−) at the electrolysis. Therefore, the (PPy(TsO−)/Ta2O5/Ta) system showed better electrical characteristics as a capacitor than the (PPy(CIO4−)/Ta2O5/Ta) system showed better electrical characteristics as a capacitor than the (PPy(ClO4−)/Ta2O5/Ta) system. Copyright © 1993 John Wiley &amp
    Sons, Ltd.

    DOI

  • 脂質分子膜で被覆したHb小胞体の調製条件

    武岡真司, 酒井宏水, 西出宏之, 土田英俊

    人工臓器   22 ( 2 ) 566 - 569  1993

     View Summary

    The quality of artificial red cells (ARC); Hb-vesicles (200nmφ), can be evaluated from the lamellarity (n) of Hb-vesicles and the concentration of Hb in lipid membrane vesicles ([Hb]in) as well as oxygen binding properties of Hb vesicles. It is important to prepare Hb-vesicles with higher concentration of Hb ([Hb]in) and lower lamellarity (n). [Hb]in increased with the concentration of Hb used for the preparation of Hb-vesicles. The Hb-vesicles with smaller lamellarity could be obtained with lowering the preparation temperature. Covering of lipid membrane to the concentrated Hb solution was also influenced by the ionic strength and the pH of the Hb solution because lipids and Hb are electrolytes. The highest [Hb]/[Lipid] ratio was obtained when the pH was at around isoelectric point of Hb (pI=7.0). The lamellarity decreased with decreasing the concentration of NaCl.

    DOI CiNii

  • オリゴ糖修飾ヘモグロビン小胞体の分散安定度

    大串 健, 鄭 主恩, 酒井宏水, 武岡真司, 土田英俊

    人工血液   2   97 - 101  1993

  • INHIBITION EFFECT OF AGGREGATION OF PHOSPHOLIPID-VESICLES BY INCORPORATION OF GLYCOLIPIDS

    S TAKEOKA, H SAKAI, H OHNO, K YOSHIMURA, E TSUCHIDA

    JOURNAL OF COLLOID AND INTERFACE SCIENCE   152 ( 2 ) 351 - 358  1992.09

    DOI

  • INHIBITION OF INTERVESICULAR AGGREGATION OF PHOSPHOLIPID-VESICLES BY INCORPORATION OF DIALKYL OLIGOSACCHARIDE GLYCEROL

    S TAKEOKA, H SAKAI, M TAKISADA, E TSUCHIDA

    CHEMISTRY LETTERS   1992 ( 9 ) 1877 - 1880  1992.09

     View Summary

    1,2-Di-O-octadecyl-3-O-alpha(beta)-maltopentaosyl-rac-glycerol (DOMPG) was incorporated into lipid vesicles to modify the surface of the vesicles. The aggregation of small unilamellar vesicles below the gel-to-liquid crystalline phase transition temperature and the aggregation of negatively-charged vesicles by the addition of calcium ion were effectively inhibited.

    DOI

  • SIMULTANEOUS FORMATION OF A POLYPYRROLE FILM AND A TANTALUM OXIDE LAYER BY ELECTROCHEMICAL OXIDATION

    YS PARK, K YAMAMOTO, S TAKEOKA, E TSUCHIDA

    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN   65 ( 7 ) 1860 - 1865  1992.07

     View Summary

    Electropolymerization of pyrrole on tantalum (Ta) electrodes was carried out in Britton-Robinson buffer solutions containing 0.1 M (1 M=1 mol dm-3) potassium chloride (KCl) under galvanostatic conditions. A polypyrrole (PPy) film and a tantalum oxide (Ta2O5) layer are formed on a Ta electrode by one electrochemical oxidation process. The conditions of this simultaneous formation were investigated in respect with the current density, pyrrole concentration, pH, and total charge. Under certain conditions ([py]=0.5 M, pH=1.8, current density=3.5 mA cm-2, charge 1 C), a 5.6-mu-m thick PPy film was efficiently formed on a homogeneous 45 nm thick Ta2O5 layer.

    DOI

  • INTERACTION BETWEEN POLYNUCLEAR AROMATIC-HYDROCARBONS AND SODIUM-ION IN POLY(OXYETHYLENE)

    S TAKEOKA, K HORIUCHI, E TSUCHIDA

    SOLID STATE IONICS   50 ( 1-2 ) 175 - 179  1992.01

     View Summary

    The ion-ion interaction of the ion pairs between radical anions of pi-conjugative molecules (polynuclear aromatic hydrocarbons, AR) such as anthracene, phenanthrene or naphthalene, and sodium ion in CH3(OCH2CH2)xOCH3 was evaluated from ionic conductivity measurement. The rate of ion-dissociation increased as either the number of (oxyethylene) unit(x) or the electron affinity of AR increased. This indicates that the continuous (oxyethylene) units are more favorable for ion-dissociation and a molecule having higher ability to become a radical anion shows higher rate of dissociation.

  • OXYGEN-TRANSPORT AND SOLUTION PROPERTIES OF POLYLIPID HB VESICLES (ARC)

    S TAKEOKA, E HASEGAWA, H NISHIDE, E TSUCHIDA, S SEKIGUCHI

    BIOMATERIALS ARTIFICIAL CELLS AND IMMOBILIZATION BIOTECHNOLOGY   20 ( 2-4 ) 399 - 404  1992

     View Summary

    Polymerized phospholipid vesicle encapsulating Hb (polylipid/Hb vesicle) was prepared from a mixture of unsaturated phospholipid, cholesterol and unsaturated fatty acid and polymerization by gamma-ray irradiation. The average radius of resulting vesicles was 203 +/- 39 nm and concentrated Hb (30 wt%) was efficiently encapsulated. gamma-Ray polymerization proceeds theoretically at low temperature (4-degrees-C). P50 and oxygen transporting efficiency were adjusted to 40 mmHg and 40%, respectively. Oncotic pressure and solution viscosity can be controlled to the same values as blood.

  • Encapsulation of Hb into unsaturated lipid vesicles and γ‐ray polymerization

    Hiromi Sakai, Shinji Takeoka, Hiroaki Yokohama, Hiroyuki Nishide, Eishun Tsuchida

    Polymers for Advanced Technologies   3 ( 7 ) 389 - 394  1992

     View Summary

    A carbonyl hemoglobin (HbCO) solution was stirred with a mixed powder of polymerizable 1,2‐bis(2,4‐octadecadienoyl)‐sn‐glycero‐3‐phosphocholine (DODPC), cholesterol and stearic acid (7/7/2 by mol). The mixture was extruded through polycarbonate membrane filters (final pore size = 0.2 μm Ø). The average diameter of the resulting vesicles was 203 ± 39 nm. The [Hb]/[Lipid] ratio (the weight ratio of Hb in vesicle to lipid) increased with the Hb concentration, and decreased with the NaCl concentration. A maximum [Hb]/[lipid] ratio was observed at pH 6.9, which was the same as the isoelectric point of Hb. The vesicles were stabilized by γ‐irradiation (60Co) because the bilayer lipids bound each other to yield polyphospholipids. Denaturation of Hb by γ‐irradiation was not detected. These polyphospholipid vesicles encapsulating Hb were stable even against the freeze–thaw cycles and the freeze‐drying procedure. Copyright © 1992 John Wiley &amp
    Sons, Ltd.

    DOI

  • Preparation of dehydrated powder of hemoglobin vesicles

    Lin Wang, Shinji Takeoka, Eishun Tsuchida, Satoru Tokuyama, Takahiro Mashiko, Tadashi Satoh

    Polymers for Advanced Technologies   3 ( 1 ) 17 - 21  1992

     View Summary

    The carbonyl hemoglobin (CO‐Hb), which was used to prevent denaturation (metHb) during the preparation of samples, was encapsulated into lipid vesicles constituted from unsaturated phospholipid, cholesterol and unsaturated fatty acid. Unsaturated components were polymerized by γ‐irradiation to enhance the stability of bilayer membrane. An aqueous dispersion of resulting Hb vesicles was freeze‐dried in the presence of saccharides (50–200 mM) to obtain a dehydrated powder of Hb vesicles. Change in the vesicle size, the leakage of encapsulated Hb and the oxidation of Hb to metHb were not observed. Therefore, the long‐term storage of Hb vesicles can be realized as a dry powder. Copyright © 1992 John Wiley &amp
    Sons, Ltd.

    DOI

  • A lithium secondary battery using a thin film of polymer electrolyte as a separator

    Tomoyuki Ohta, Shinji Takeoka, Eishun Tsuchida, Han Yu Feng, Zheng Sheng Fu, Yun Pu Wang

    Polymers for Advanced Technologies   3 ( 8 ) 433 - 436  1992

     View Summary

    Ion‐conductive polymer which shows an ionic conductivity (σi) of 1.4 × 10−4S/cm at 25°C when mixed with LiClO4 (molar ratio in Li/OE = 0.05) was used as a separator of electrodes in a lithium secondary battery. The effect of high ionic conductivity on the performance of the battery was studied. The polymer structure was $$ \\left[ {\\rm H}\\rlap{-} ({\\rm CH}_2 {\\rm CHR}\\rlap{-} )_{\\rm n} {\\rm H},{\\rm R} = - {\\rm CH}_2 ({\\rm OCH}_{\\rm 2} {\\rm CH}_{\\rm 2} )_6 {\\rm OCH}_3 \\right],$$ and the cathode was comprised of poly(1,3,4‐thiadiazole disulfide), graphite powder and the polymer electrolyte. The cell [(−)Li/polymer electrolyte/graphite–poly(disulfide) (+)] had an open circuit voltage (Voc) of 3.25 V, a plateau voltage of 2.75 V, a discharge density (id) of 0.05 mA/cm2 with the cathode utilization of 63%, and achieved over several tens of cycles at 25°C. Copyright © 1992 John Wiley &amp
    Sons, Ltd.

    DOI

  • ELECTROPOLYMERIZATION OF PYRROLE ON A TANTALUM ELECTRODE

    K YAMAMOTO, YS PARK, S TAKEOKA, E TSUCHIDA

    JOURNAL OF ELECTROANALYTICAL CHEMISTRY   318 ( 1-2 ) 171 - 181  1991.11

     View Summary

    Electropolymerization of pyrrole was carried out on tantalum as a base metal electrode. The Ta electrode retards polymerization at lower pyrrole concentrations because of the formation of insulating Ta2O5. However, at high concentrations of pyrrole, the polymerization proceeds efficiently to yield a polypyrrole (PPy) film, similar to that on a Pt electrode. The PPy film on a Ta electrode shows the same electroactivity and electric conductivity as that on a Pt electrode. Electropolymerization on Ta is accompanied by a decrease in electrode area due to the formation of the Ta2O5, the behavior of which is the only difference from that on Pt.

    DOI

  • 高分子イオン伝導体の設計とその応用

    武岡真司, 土田英俊

    化学   46 ( 10 ) 68 - 69  1991.10

  • SODIUM-ION CONDUCTION OF PERFLUOROSULFONATE IONOMER POLY(OXYETHYLENE) COMPOSITE FILMS

    S TAKEOKA, K HORIUCHI, S YAMAGATA, E TSUCHIDA

    MACROMOLECULES   24 ( 8 ) 2003 - 2006  1991.04

     View Summary

    A composite film of Na+-form Nafion and diendoacetylated poly(oxyethylene) (MBAR(w) = 400) (POE400E) was cast from a dimethylformamide solution at 120-degrees-C. The ionic conductivity of this thin film was measured. The resulting film showed good flexibility, transparency, and no leakage of POE400E up to the POE incorporation of 55 wt%. At this composition, sodium ionic conductivity was 1.0 x 10(-6) S cm-1, which is relatively high as a single-ion conductor because of the high dissociation of the sodium perfluorosulfonate groups. The ionic conductivity increases with POE incorporation. This can be explained from a decrease in the glass transition temperature (T(g)) of POE regions and the connection of ion clusters swollen by POE.

    DOI

  • POE Incorporation into Ionic Clusters of Ionomer and Ion-conduction Behaviors

    S. Takeoka, H. Sakai, H. Shin, T. Ohta, E. Tsuchida

    POLYMERS FOR ADVANCED TECHNOLOGIES   2 ( 2 ) 69 - 74  1991.04

     View Summary

    Poly(oxyethylene) (POE) was incorporated into the ionic clusters of ionomer, ethylene and methacrylic acid (7.2% neutralized with KOH) copolymer membrane. The changes of properties were studied from SAXS, DSC, IR and ionic conductivity. The IR study suggested that the coordinated structures in ionic clusters of the membrane were destroyed by POE incorporation, and also SAXS suggested that ionic clusters were swollen by POE incorporation. The ionic conductivity, a carrier being K+ in this system, increases from 10(-16) S/cm to 10(-9) S/cm at 30 degrees C by the incorporation of POE (20.5 wt%). On the other hand, a large amount of POE (63 wt%) could be incorporated into ionomer membrane by the esterification of methacrylic acid groups (93%) with POE. When LiClO4 was added, ionic conduction occurred in the phase-separated POE domain, which had a low glass transition temperature (-55.2 degrees C), showing an ionic conductivity 2.6 x 10(-6) S/cm at 25 degrees C.

    DOI

  • PHASE-SEPARATION OF POLYMERIZED MIXED LIPOSOMES - ANALYSIS OF RELEASE BEHAVIOR OF ENTRAPPED MOLECULES WITH SKELETONIZATION

    S TAKEOKA, H SAKAI, H OHNO, E TSUCHIDA

    MACROMOLECULES   24 ( 6 ) 1279 - 1283  1991.03

     View Summary

    The mixed liposomes, composed of a polymerizable lipid, 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phosphorylcholine (DODPC), and nonpolymerizable membrane constituents, DPPC, cholesterol, and sodium didodecyl phosphate, were prepared by an extrusion method. After polymerization, nonpolymerizable constituents were removed to obtain the polymerized framework of the liposome (the skeletonized liposome). The release of small molecules from the skeletonized liposomes through resulting holes was analyzed. A 5(6)-carboxyfluorescein (CF) and saccharides with various molecular weights were applied as release molecules. The molecular weight of dextran, whose retention ratio in the skeletonized liposomes is 50%, relates to the apparent size of the holes, i.e., the apparent domain size of the phase separation of a polymerized mixed liposome. The size of the holes increases with an increase in the mole fraction of nonpolymerizable lipids. This also depends on the polymerization temperature and the structure of nonpolymerizable lipids.

    DOI

  • Ion dissociation and conduction of Nafion/modified oligo(oxyethylene) composite films

    Yun Pu Wang, Han Yu Feng, Zheng Sheng Fu, Eishun Tsuchida, Shinji Takeoka, Tomoyuki Ohta

    Polymers for Advanced Technologies   2 ( 6 ) 295 - 299  1991

     View Summary

    In order to improve the ionic conductivity of solid polymer electrolyte by controlling ion(alkali metal ion)–dipole(ether oxygen) interaction, two kinds of modification were adopted on oligo(oxyethylene) (OOE). One is the capping of terminal hydroxyl groups of OOE with methyl or acetyl groups. The other is the replacement of the center ethylene group of OOE with methylene or propylene group. Ion–dipole interaction was analyzed by measuring the ion dissociation, ion conduction and Tg of Nafion/modified OOE composite films. The modification of the end groups was more effective than that of the center group in increasing ionic conductivity. The methyl group is superior to the acetyl group as the end group of OOE for lithium ion conduction. Copyright © 1991 John Wiley &amp
    Sons, Ltd.

    DOI

  • DISSOCIATION AND CONDUCTION OF ALKALI-METAL ION IN NAFION CROWN-ETHER COMPOSITE FILMS

    S TAKEOKA, H SAKAI, E TSUCHIDA

    CHEMISTRY LETTERS   1990 ( 9 ) 1539 - 1542  1990.09

    DOI

  • LARGER CATIONS CAN MOVE FASTER IN SOLID POLYMER ELECTROLYTES

    H OHNO, N KOBAYASHI, S TAKEOKA, H ISHIZAKA, E TSUCHIDA

    SOLID STATE IONICS   40-1   655 - 658  1990.08

    DOI

  • LIPOSOME FORMATION OF SELECTIVELY-POLYMERIZED DIENE-CONTAINING PHOSPHOLIPIDS AND THEIR POSTPOLYMERIZATION

    S TAKEOKA, H OHNO, H IWAI, E TSUCHIDA

    JOURNAL OF POLYMER SCIENCE PART A-POLYMER CHEMISTRY   28 ( 4 ) 717 - 730  1990.03

    DOI

  • POLYMERIZATION OF LIPOSOMES EVALUATED FROM MOLECULAR-WEIGHT DISTRIBUTION OF DIENE-TYPE PHOSPHOLIPID POLYMERS

    S TAKEOKA, N KIMURA, H OHNO, E TSUCHIDA

    POLYMER JOURNAL   22 ( 10 ) 867 - 874  1990

    DOI

  • 「エネルギー利用関連新素材」(IV) 高分子固体電解質と電池

    武岡真司, 土田英俊

    燃料協会誌   69 ( 1 ) 56 - 64  1990.01

    DOI

  • CONTROL OF RELEASE OF ENCAPSULATED MOLECULES FROM POLYMERIZED MIXED LIPOSOMES INDUCED BY PHYSICAL OR CHEMICAL STIMULI

    S TAKEOKA, H OHNO, N HAYASHI, E TSUCHIDA

    JOURNAL OF CONTROLLED RELEASE   9 ( 2 ) 177 - 186  1989.07

    DOI

  • EFFECT OF PHASE-TRANSITION ON PHOTOSENSITIZED RADICAL POLYMERIZATION OF DIENE-CONTAINING LIPIDS AS LIPOSOMES .7.

    H OHNO, S TAKEOKA, H IWAI, E TSUCHIDA

    MACROMOLECULES   22 ( 1 ) 61 - 66  1989.01

    DOI

  • DIENE-CONTAINING LIPIDS AS PROBES FOR PHASE-TRANSITION BEHAVIOR OF LIPIDS IN LIPOSOMES

    S TAKEOKA, H IWAI, H OHNO, E TSUCHIDA

    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN   62 ( 1 ) 102 - 108  1989.01

    DOI

  • STUDY ON THE PHASE-TRANSITION BEHAVIOR OF POLYMERIZED LIPOSOMES THROUGH THE INTERACTION OF DIENE-GROUPS IN THEIR ACYL CHAINS

    S TAKEOKA, H SAKAI, L WANG, H OHNO, E TSUCHIDA

    POLYMER JOURNAL   21 ( 8 ) 641 - 648  1989

    DOI

  • POLYMERIZATION OF DIENE-CONTAINING LIPIDS AS LIPOSOMES BY RADICAL INITIATORS .6. POLYMERIZATION OF 1,3-BIS(2,4-OCTADECADIENOYL)-RAC-GLYCERO-2-PHOSPHORYLCHOLINE

    S TAKEOKA, E HASEGAWA, H OHNO, E TSUCHIDA

    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN   61 ( 6 ) 2007 - 2012  1988.06

    DOI

  • POLYMERIZATION OF DIENE-CONTAINING LIPIDS AS LIPOSOMES BY RADICAL INITIATORS .4. EFFECT OF LIPID PACKING ON THE POLYMERIZATION PROFILE

    H OHNO, S TAKEOKA, H IWAI, E TSUCHIDA

    MACROMOLECULES   21 ( 2 ) 319 - 322  1988.02

    DOI

  • POLYMERIZATION OF LIPOSOMES COMPOSED BY DIENE-CONTAINING LIPIDS BY RADICAL INITIATORS .2. POLYMERIZATION OF MONODIENE-TYPE LIPIDS AS LIPOSOMES

    H OHNO, S TAKEOKA, H IWAI, E TSUCHIDA

    JOURNAL OF POLYMER SCIENCE PART A-POLYMER CHEMISTRY   25 ( 10 ) 2737 - 2746  1987.10

    DOI

  • POLYMERIZATION OF DIENE CONTAINING LIPIDS AS LIPOSOMES BY RADICAL INITIATORS .3. UNEQUIVALENT CHEMICAL ENVIRONMENT OF DIENE GROUPS IN 1-ACYL AND 2-ACYL CHAINS OF POLYMERIZABLE LIPIDS ANALYZED BY RADICAL POLYMERIZATION

    H OHNO, S TAKEOKA, E TSUCHIDA

    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN   60 ( 8 ) 2945 - 2951  1987.08

    DOI

  • INCORPORATION OF FLUORESCENT-PROBES TO THE INNER AQUEOUS PHASE OF PREVIOUSLY POLYMERIZED LIPOSOMES

    H OHNO, S TAKEOKA, N HAYASHI, E TSUCHIDA

    MAKROMOLEKULARE CHEMIE-RAPID COMMUNICATIONS   8 ( 5 ) 215 - 218  1987.05

    DOI

  • SKELETONIZED HYBRID LIPOSOMES

    H OHNO, S TAKEOKA, E TSUCHIDA

    POLYMER BULLETIN   14 ( 6 ) 487 - 490  1985.12

  • Rheological properties of PEG-modified HB-vesicles (HBVS) and their oxygen transporting capacity in vivo

    Sakai H, Sou K, Takeoka S, Kobayashi K, Tsuchida E

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   227, 339-PMSE Part 2   U541 - U542

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Books and Other Publications

  • Functional Polymer Films, Chapt 29 Fabrication, Properties, and Biomedical Applications of Nanosheets

    Toshinori Fujie, Yosuke Okamura, Shinji Takeoka

    WILEY-VCH  2011 ISBN: 9783527321902

  • 遺伝子治療、DDS

    武岡真司

    生命・医療・福祉ハンドブック、早稲田大学生命・生体・福祉研究所編、コロナ社  2007.02

  • 動く臓器としての血液に学ぶ -人工赤血球・人工血小板への挑戦-

    武岡 真司, 岡村 陽介, 阿閉 友保

    “ファイバー” スーパーバイオバイオメティクス 〜近未来の新技術創成〜 本宮 達也 監修, NTS  2006.10

  • Hemoglobin-Vesicles as a Molecular Assembly: Characteristics of Preparation Process and Performances as Artificial Oxygen Carriers

    H. Sakai, K. Sou, S. Takeoka, K. Kobayashi, E. Tsuchida

    Blood Substitutes, Elsevier, Amsterdam  2006

  • 血液疾患ハンドブック、人工血液の開発の現状と展望

    武岡真司

    再生つばさの会  2005.06

  • 図解 高分子新素材のすべて、国武豊喜監修、人工赤血球の仕組み

    武岡真司

    工業調査会  2005.05

  • Organ Microcirculation, Design and Modification of Nanoparticles for Blood Substitutes

    Takeoka,S

    Springer  2005

  • Artificial Oxygen Carrier, Hemoglobin-Vesicles (HbV) as Artificial Oxygen Carriers

    Sakai, H, Sou, K, Takeoka, S, Kobayashi, K, Tsuchida, E

    Springer  2005

  • 「医療用マテリアルと機能膜」,第5章人工赤血球

    酒井宏水, 宗慶太郎, 武岡真司, 小林紘一, 土田英俊

    (株)シーエムシー出版  2005

  • 機能性脂質のフロンティア、2 リン脂質二分子膜小胞体を利用した赤血球代替物

    武岡真司

    シーエムシー出版  2004.12

  • 最近の化学工学56 先端医療における化学工学 人工血液(赤血球、血小板)の最近の進歩

    武岡真司

    化学工業社  2004.12

  • バイオロジクス-生物由来物質を用いた製品開発-

    武岡真司, 後藤洋子, 川上浩良

    高分子学会編  2004.09

  • 周術期輸液の最前線、IV 酸素輸液の展望1)小胞体型人工赤血球の開発動向

    宮尾秀樹編, 武岡真司

    真興交易㈱医書出版部  2004

  • 新訂版・表面科学の基礎と応用、第12節, 人工赤血球

    土田英俊, 武岡真司, 小松晃之, 酒井宏水

    エヌ・ティー・エス社  2004

  • ぼくもノーベル賞をとるぞ!!

    高分子学会編著, 武岡真司ほ

    朝日出版社  2001

  • バイオミメティックスハンドブック、酸素輸液としての人工赤血球(第7章(分泌と体内輸送系)第6節)

    長田義仁, 土田英俊, 酒井宏水, 武岡真司

    エヌ・ティー・エス社  2000

  • Evaluation of the Oxygen Trasnporting Capability of Hemoglobin Vesicles "Blood Substitutes-Present and Future Perspectives" chapt.14

    Elsevier Science  1998

  • Stabilized Hemoglobin Vesicles

    "Artificial Red Cells" John Wiley & Sons  1995

  • Interpolymer Complexes and their Ion-Conduction

    "Macromolecular Complexes in Chemistry and Biology" Springer-Verlag  1994

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Works

  • Secretary, Program Committee, VII International Symposium on Blood Substitutes

  • Secretary, Executive Committee, 5th International Symposium on Polymers for Advanced Technologies(PAT)

  • Program Committee, Executive Committee, IX International Symposium on Blood Substitutes

  • Executive Committee, Internal Symposium on 21-COE Practical Nano-Chemistry

  • Program Committee, 13th Keio University International Symposia for Life Sciences and Medicine, Research and Development of Artificial Oxygen Carrier-Its Frontline-A Gateway to Diagnostic and Therapeutic Interventions

  • Organizing Committee, 14th Keio University International Symposia for Life Sciences and Medicine, Organ Microcirculation

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Awards

  • 大隈記念学術褒章(奨励賞)

    2011.10  

Research Projects

  • Construction of Monomolecular Membrane with Different Surface Properties and Electron Transfer

  • Research for Molecular Assembly of Heme Derivatives and Their Electronic Process

  • New preparation of Oxygen-Permselective Membranes and their Application for the Air Battery of Next Generation

  • Preclinical Evaluation Tests of Red Cell Substitutes by Microcirculatory Dynamical Analyzes

  • One-Step Multi-electron Transfer for Molecular Conversion

  • 赤血球代替物の非酵素的還元システムに関する研究

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(基盤研究(C))

  • 止血機能を有し、血管障害部位に特異的に集積する蛋白質高分子量体の設計

    科学研究費助成事業(慶應義塾大学)  科学研究費助成事業(萌芽的研究)

  • Establishment of production process of cellular- type oxygen carriers

  • Development of artificial oxygen carriers under consideration of microvascular homeostasis

  • Intelligent approach to construct intravenously injectable nanoparticles by interface transference properties

  • Construction of Hemoglobin-vesicle with long-term in vivo oxygen transporting ability

  • Development of functional polymer nanosheet for the treatment of burn injury and gastrointestinal tissue defects.

  • Construction of Removel System of Cesium Ion by Cosedimentation with Organic Macrocyclic Molecules

  • Compromised hostへの重度侵襲対策

    科学研究費助成事業(防衛医科大学校(医学教育部医学科進学課程及び専門課程、動物実験施設、共同利用研究)  科学研究費助成事業(基盤研究(B))

  • ナノシートとマイクロスキンを用いた重症熱傷救命のための治療開発研究

    科学研究費助成事業(防衛医科大学校(医学教育部医学科進学課程及び専門課程、動物実験施設、共同利用研究)  科学研究費助成事業(基盤研究(C))

  • 相分離貫通型ナノシートとナノディスクを利用する新規なナノフィルム製剤の創製

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(基盤研究(B))

  • Research on Protein-Conjugated Polymers

  • Research on Organic Electron Conductive Materials

  • Research on Glycolipids and Application

  • 微生物機能を利用した資源循環型水環境プロセスの構築

    文部科学省 

  • 人工臓器素材開発

    文部科学省 

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Presentations

  • Antibiotic-loaded nanosheets for the treatment of gastrointestinal tissue defects

    Presentation date: 2010.09

  • Antibiotic-loaded nanosheets for the treatment of gastrointestinal tissue defects

    Presentation date: 2010.08

  • Approach of Nanomedicine Using Functional Nanoparticles and Nanofilms

    Presentation date: 2010.06

  • Properties of Polymer Ultra-Thin Films (Nanosheets) and Medical Development of Nanoadhesive Plasters

    Presentation date: 2010.06

  • Properties of Nanosheets and Medical Applications

    Presentation date: 2010.06

  • Medical Development of Polymer Ultra-thin Films (Nanosheets)

    Presentation date: 2010.05

  • Medical Development of Polymer Ultra-thin Films (Nanosheets)

    Presentation date: 2010.04

  • Introduction of Bio-molecular Assembly Science Lab.

    Presentation date: 2010.03

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Specific Research

  • 生体分子イメージング用ナノシートの構築

    2019  

     View Summary

    申請者は、機能性蛍光分子を固定したナノシートを用いて生体表面の温度やpHのレシオ型イメージングに成功した。本特定課題研究では、生体分子を蛍光イメージングとして捉えるために、フッ素系高分子インクでナノシート表面にパターンを印刷して自己展開する技術を確立した。また、アンモニアやドーパミンを電気化学的にセンシングする方針に変更して検討を行った。測定対象を呼気に含まれるアンモニアガスとしてPEDOT/PSS膜が吸着アンモニアガスを酸化する際の電流値の変化を測定すると共に、神経伝達物質であるドーパミンを対象としてドーパミンを鋳型に重合したPPy膜がドーパミンを選択的に酸化する際のシグナルの変化を測定している。

  • 微小環境イメージング用蛍光ナノシートの開発

    2018   藤枝 俊宜

     View Summary

    報告者は、ナノ粒子やナノシートに温度応答性の蛍光色素と標準用蛍光色素を利用したレシオ型ナノ温度計を開発し、細胞内エンドソームや筋組織の温度を蛍光顕微鏡で計測することに成功している。この場合、蛍光色素はナノシート内部に封入され温度以外の因子の影響は受けず、ナノシートは表面密着性が高いため高い時空間分解能にて表面温度を計測できた。そのデモンストレーションとして、カブトムシの飛翔筋組織に温度応答ナノシートと温度非感受性ナノシートを重ね合わせることによって、レシオ型な組織の温度イメージング像をリアルタイムに捉えることに成功した。また酸素応答性ポルフィリン誘導体を導入した薄膜型センサーをナノシート化した場合、酸素濃度の変化を感度良く捉えることもできている。この様に温度やガスのバイオイメージングでは、なるべく厚さが薄いナノシート程、時間分解能が高くなる。但し、感度を上げるためにはより広い領域で強度を稼ぐ必要があるために、空間分解能を下げざるを得なくなる。他方、pHの測定は、溶液中の濃度測定であるために、水溶性のポリアニオンに検出用蛍光物質を共有結合で固定し、ポリカチオンとの静電的なコンプレックスの形成(layer-by-layer 法)させた後熱架橋によって水中での安定化を確保することでpHセンサー部の構築に成功した。そのデモストレーションとしては昆虫の様な動物の組織のpH変化は時間軸が複雑であるために難易度が高い。そこで、植物に着目し植物の葉の組織にpH非感受性ナノシートと共に密着させて貼付し、高い塩化物イオンの水溶液に葉を曝すとシンポ―トによって塩化物イオンと共にプロトンを細胞内に取り込むことによって起こる、アポプラストpH変化をイメージング像として感度高く捉えることを示した。更に、膜厚はプロトン拡散に影響を及ぼすため薄膜化することで応答速度が速くなることを本研究で実証できた。

  • 脂質ライブラリー/マイクロ流路デバイスを用いたゲノム編集用ナノ粒子の開発

    2018   李 天舒

     View Summary

     リポソームは貪食細胞にエンドサイトーシスにて積極的に取り込まれる。報告者は、カチオン性アミノ酸型脂質のライブラリーを保有し、遺伝子やタンパク質の運搬体とした研究を推進してきた。その結果、脂質の構造(極性頭部、スペーサー長、アルキル鎖長、カウンターイオン)や、遺伝子やタンパク質との混合比や濃度によって、細胞への導入効率・遺伝子発現効率が異なることを示してきた。更には、細胞の種類によってもそれらの条件が異なることも示してきた。 ボン大学LIMESとの共同研究により、異なる構造のカチオン性アミノ酸型脂質からなるリポソームと免疫細胞との相互作用の分子機構を明らかにしてきた。その結果、ある特定の構造の脂質が特に自然免疫系の免疫細胞の賦活化ならびに獲得免疫系におけるアジュバント効果において、高い活性を示すことを初めて見出した。更に、あるカチオン性アミノ酸型脂質のアルキル鎖長が炭素数で2個異なるだけで、通常のエンドサイトーシス経路から膜融合の経路でリポソームが取り込まれることを見出した。膜融合は、標的細胞に対して、短時間に高効率でリポソームに担持させた薬物を細胞側に移行させることができる。 また、アニオン性アミノ酸型脂質リポソームにおいては、活性化された血小板との相互作用において、脂質の構造の相違がリポソームのζ電位の相違に繋がり、更に血小板凝集塊に取り込まれる効率が異なることも明らかにした。 この様な脂質構造と細胞における生理活性との相違をより高い効率でスクリーニングするために、混合脂質のエタノール溶液と担持させたい生体高分子を溶解させた水溶液とをマイクロ流路中で混合して、複合体ナノ粒子を構築し、限外ろ過によってエタノールやナノ粒子未担持の生体高分子を除去した後に、細胞との相互作用を評価する方法を検討している。

  • カチオン性リポソームの膜融合による生理活性高分子の細胞内送達システムの構築

    2017   Tianshu Li

     View Summary

    当研究室にて保有しているカチオン性アミノ酸型脂質ライブラリーを用いて、カチオン性アミノ酸型脂質リポソームを調製し、これをTHP-1細胞やマクロファージにアプライした。リポソームの粒子径やゼータ電位は殆ど相違しないにもかかわらず、脂質の分子構造が僅かに異なるだけで細胞との相互作用が大きく異なることが明らかとなった。主としてエンドサイトーシス機構で取り込まれるもの、膜融合機構で取り込まれるもの、殆ど細胞に取り込まれないものに分類された。膜融合機構で取り込まれるカチオン性リポソームはアニオン性のsiRNAやタンパク質など生理活性高分子を高い効率で運搬することが示唆され、従来にない生理活性高分子の細胞内運搬が見込まれる。

  • ナノシートのアジ化による可視化と分解挙動の解析

    2017  

     View Summary

    医用高分子の薄膜(ナノシート)は、創傷被覆用”ナノ絆創膏“として注目されている。本計画では、生分解性ナノシートの分解特性を、外見観察による形態や大きさの変化、顕微鏡観察による表面のラフネスの増大や膜厚の変化、構成分子量の低下などから解析した。37℃の生理食塩水中に自己支持状態で震盪させたところ、PDLLAナノシートでは3週間ごろからこより状になったが破断は見られず、7週間でも膜厚は変化しなかった。PLGA5050ナノシートでは膜表面が粗くなり日数と共に小さな孔が大きくなって4週間で破断につながった。酵素Proteinase K溶液による分解試験では、多孔質化と共にナノシートの膜厚の減少が認められ、その程度はPLGA>PDLLA>ポリカプロラクトンの順であった。

  • 生細胞内小器官の温度を計測するレシオ型分子温度計の創製

    2015   井上 貴文

     View Summary

    生細胞のミトコンドリアに集積して温度を計測するレシオ型蛍光プローブ(Mito-RTP)を開発した。本プローブは、温度感受性の無いNIR-rhodamine 101と温度感受性を示すRhodamine Bを共有結合させており、ミトコンドリア集積能を持つと共に蛍光強度比によって絶対温度が計測できる。HeLa細胞にMito-RTPを添加すると30分以内にミトコンドリアに集積し、培地温度を上昇させた際の温度依存性は-2.72%/℃であった。ミトコンドリアの脱共役剤を添加すると、約2.5 ℃の熱産生が引き起こされていた。

  • 生細胞内クリックケミストリーによる生体関連分子イメージング法の開発

    2014  

     View Summary

    細胞膜透過性を有する無触媒クリック型蛍光色素FC-DBCO(Fluorescein-Conjugated DiBenzoCycloOctyne)を分子設計し、有機合成した。本プローブは蛍光分子であるフルオレセインの誘導体と無触媒クリック分子であるジベンゾシクロオクチンをカップリングした構造であり、細胞膜を自由に透過できる性質を持っている。従って、もし生細胞内にアジド基を有する標的分子があればそれと結合して蛍光標識するが、標的分子がない場合にはそのまま細胞を透過してしまい、培地の洗浄によりほぼ完全に細胞内から除去される。また、本実験条件では細胞毒性は認められなかった。N-アセチルマンノサミン誘導体(アジド化マンノース)とFC-DBCOを用いて生細胞内クリック反応を評価したところ、アジド化マンノースの細胞内動態(細胞質→酸性小胞体→ゴルジ体→細胞膜)を蛍光イメージングすることができ、生細胞内アジド基修飾分子の動態解析に対する本プローブの有用性を実証した。

  • 細胞内小器官の温度を計測する新規な蛍光プローブの創製

    2014   井上貴文

     View Summary

    生細胞のミトコンドリア温度を測定するために、ミトコンドリアに局在するレシオ型温度応答性蛍光プローブを分子設計を行い、有機合成した。本プローブは温度応答性を示すローダミンBと温度に不感応なローダミン101のスペーサーにて結合させた構造であり、ローダミンBの蛍光強度/ローダミン101の蛍光強度の比は、温度上昇に比例して減少するので、濃度変化や焦点ブレに関係なく温度変化のみを検出できる。試験管レベルで検量線を作成したところ、2.72%/℃の温度感受性を有することが明らかとなった。また、本プローブは細胞に振り掛け約30分静置すると細胞膜を透過してミトコンドリアに集積する性質を持っているので、測定したいタイミングでミトコンドリアのみの温度を計測できる。また、測定条件では細胞毒性は認められなかった。脱共役剤(FCCP)をHeLa細胞の培地に添加したところ添加約5分後に有意な温度上昇が観測された。

  • バイオ医薬品運搬システム構築のための薬物担持リピッドナノシートの創製

    2011  

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    研究代表者は、リン脂質二分子膜小胞体(リポソーム)を用いたドラッグデリバリーシステムの研究と開発を行っている。特に、リポソームに抗体を結合させたり、遺伝子を担持させる研究を進めてきた。一般にバイオ医薬品(抗体、酵素、遺伝子など)は高価であり、これをリポソームに内包させるためには内包効率を如何に向上させるかが課題となる。他方、研究代表者は、医用高分子からなるナノシートの開発に成功し、ナノ絆創膏などとして医療応用を目指している。本研究では、犠牲膜法でナノシートを構築する手法をヒントに、ポリビニルアルコール(PVA)の様な水溶性犠牲膜上に当研究室で保有しているカチオン性アミノ酸型脂質、ポリエチレングリコール結合脂質をコーティングしてナノシート(約5~10nm厚)を形成させ、そこに反対電荷のバイオ医薬品(アニオン性の成長因子、酵素、遺伝子)水溶液をキャストし、さらにカチオン性脂質のナノシート(約5~10nm厚)を形成させて3層構造を形成する方法を検討した。これを基板ごと水槽に浸漬させることで、犠牲膜であるPVA層が水に溶解し、3層構造の薬物担持リピッドナノシートが遊離する。リピッドナノシートは分子集合体であるため、水和と共に高速撹拌などでずり応力を加えると分子集合が再構築され、薬物を内包した二分子膜小胞体構造が得られ、ずり応力を撹拌速度やホモジナイザー、超音波照射によって強くするとそれに応じてリポソームの粒子径が小さく制御される。本法の利点は、アニオン性のバイオ医薬品の担持効率を究極に高めることであり、若干の漏出は溶解したPVAを限外濾過にて除去する際に除去できる。検討の結果、この方法の最大の課題はバイオ医薬品(例えば成長因子)のキャスト後の状態であった。スピンコート法で形成されたリピッドナノシートに成長因子をキャストした場合、凝集や相分離など不均一な状態で乾燥するとその上に平滑なリピッドナノシートを構築することができない。成長因子をスピンコートすることで平滑なナノシートを構築することができるものの、スピンコート時に相当量の成長因子が損失してしまうことも明らかとなった。ダイコーターなどで成長因子をコートし、更にその上にリピッドナノシートを構築する方法が有効と思われた。しかし、そのためにはマイクログラビアなどの薄膜連続作成装置ならびに多量のリピッドとバイオ医薬品(例えば成長因子)を必要とするので、経済的な理由で進めることができなかった。しかし、この検討により、アニオン性バイオ医薬品をカチオン性多糖類とポリイオンコンプレックスを作らせてナノシートに担持させる手法の契機になり、次に繋がる展開となった。

  • ナノスケールレベルで形態を制御したアルブミン超薄膜の構築と血小板代替物への応用

    2005  

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     リソグラフィー加工によって得られたナノスケールのパターンを利用した多様な形状の高分子ナノシート(ポリスチレンナノシート、アルブミンナノシート、多糖類電解質ナノシート)を大量に構築する技術を確立する。そこに表裏に異なるリガンドやレセプターを担持させ、血流中における形態と血小板代替物としての機能について、既に確立されているin vitro, in vivo評価系と血管損傷モデルを用いて明らかにすることを目的としている。 平成17年度は、親水部(SiO2)と疎水部(オクタデシルトリメトキシシラン; ODMS)からなる親-疎水マイクロパターン基板(ODMS-SiO2基板)上に、チオール基を複数導入させたrHSA(SH-rHSA)を吸着、架橋により2次元高分子体とし、rHSAナノシートの剥離体を構築した。更に、シートの片面に蛍光標識ラテックスビーズ(LB)を結合させ、ヘテロな表裏を持つナノシートを共焦点顕微鏡にて観察した。ODMS-SiO2基板(10μm x 30μmが7千個)にローダミン標識SH-rHSA(pH 5.0, 1 g/mL)を蜜に吸着させ、未吸着SH-rHSAを除去後、銅(II)イオンを含む酢酸緩衝液中に静置した。界面活性剤(C12E10)処理により剥離させた分散系をフィルターに捕捉し、共焦点顕微鏡にて観察したところ、ほぼパターン通りの形状を維持したシート(膜厚約7nm)が鮮明に3次元観察できた。NBD蛍光標識させたLBにて片面を修飾したrHSAナノシートを543 nmで励起させ、検出波長(>560 nm)として共焦点顕微鏡観察したところ、湾曲したシート全面が赤色に発光した。458 nmで励起させNBDのみの検出波長(505~530 nm)に設定したところ、シート状に黄色発光したが湾曲部が消光(蛍光エネルギー移動)した様子が観察できた。これは、湾曲部の裏面にLBが存在しているためと考えられる。 以上より、シートの片面のみを修飾できたことを共焦点顕微鏡観察によって初めて証明できた。

  • 生理不活性な合成脂質を利用した新しいヘモグロビン小胞体の創製

    2002  

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    高濃度Hbをリン脂質二分子膜にて内包したヘモグロビン小胞体を人工赤血球として研究開発している。リン脂質としては、両イオン性のホスファチジルコリン型脂質、アニオン性ホスファチジルグリセロール型脂質、ポリエチレングリコール(PEG)結合ホスファチジルエタノールアミン型脂質の混合体を用いているが、特にアニオン性脂質による血小板や白血球の活性化が課題となっていた。本特定課題研究では、ジアルキル L-グルタメートを基本骨格とし、これに無水コハク酸をアミド結合させたアニオン性グルタミン酸型脂質や、片末端をカルボン酸としたPEGを結合させたPEG結合グルタミン酸型脂質、更にはグルタミン酸をα位やγ位を選択的に結合させた両イオン性脂質を合成した。これらを混合してヘモグロビン小胞体を調製し、血液適合性を検討した結果、以下の知見を得た。(1) グルタミン酸型脂質は簡便合成可能で、対応するリン脂質の置き換えが可能であった。(2) アニオン性の新規グルタミン酸型脂質脂質は血小板の活性化を全く認めず、従来のホスファチジルグリセロール型脂質を使用による副作用を解決できた。(3) 従来のポリエチレングリコール(PEG)結合ホスファチジルエタノールアミン型脂質は、白血球、特に好中球の活性化を認めたが、新規のPEG結合グルタミン酸型脂質では白血球の活性化が認められなかった。(4) 両イオン性のグルタミン酸型脂質では、α位とγ位の結合で分子集合挙動には大きな相違が認められ、前者では単独で小胞体構造、後者では単独でリボン状構造が形成された。コレステロールとの混合により両者とも小胞体構造を形成するものの、前者の方が安定であった。しかし、前者の場合、白血球数の増減に影響が認めれられた。結論として、アニオン性脂質、PEG結合性脂質を新規のグルタミン酸型脂質に置換することに成功したが、両イオン性の脂質には更なる分子設計が必要と思われた。恐らく、コレステロールとの配置を考慮してグルタミン酸とグルタミン酸との間に何らかのスペーサーが必要であると推測される。今回の研究成果により非常に安価なグルタミン酸型脂質により高価なリン脂質の代替と、それによる血液適合性の向上が実現でき、リポソーム製剤の安定度向上に対して大きな進展となった。

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