論文 - 金澤　秀子

Protein purification using solid-phase extraction on temperature-responsive hydrogel-modified silica beads

Okubo K., Ikeda K., Oaku A., Hiruta Y., Nagase K., Kanazawa H.

Journal of Chromatography A （Journal of Chromatography A）  1568   38 - 48 2018年09月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00219673

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© 2018 Elsevier B.V. Recently, the importance of biopharmaceuticals in medical treatments has been increasing, and effective protein purification methods are strongly required for their production. In the present study, a temperature-responsive solid-phase extraction (SPE) column was developed for the purification of proteins without affecting their bioactivity. A temperature-responsive polymer hydrogel-modified stationary phase was prepared by coating aminopropyl silica beads (average diameter, 40–64 μm) with poly(N-isopropylacrylamide) (PNIPAAm)-based thermoresponsive hydrogels. n-Butyl methacrylate and acrylic acid were copolymerized with PNIPAAm as hydrophobic and anionic monomers, respectively. Using these temperature-responsive SPE columns, targeted proteins were retained on the thermoresponsive hydrogel at 40 °C through hydrophobic and electrostatic interactions. After the temperature was reduced from 40 °C to 4 °C, the retained proteins were successfully eluted from the column. Using the temperature-responsive SPE system, lysozyme was successfully separated from ovalbumin without any loss in bioactivity (99.7 ± 0.1%). Rituximab, a monoclonal antibody, was also purified from BSA or hybridoma cell culture medium using the prepared SPE column. Denaturation of rituximab was not observed in the rituximab fraction eluted from the SPE column. These results demonstrate that temperature-responsive polymer-based SPE can be applied in biomedical purifications, while maintaining the biological activity of the proteins.

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Comparison of plasma propofol concentration for apnea, response to mechanical ventilation, and airway device between endotracheal tube and supraglottic airway device in Beagles

Tomoya IIZUKA, Kenichi MASUI, Hideko KANAZAWA, Ryohei NISHIMURA

Journal of Veterinary Medical Science 80 （ 9 ） 1420 - 1423 2018年07月

研究論文（学術雑誌）, 共著, 査読有り

LAT1-targeting thermoresponsive fluorescent polymer probes for cancer cell imaging

Matsuura M., Ohshima M., Hiruta Y., Nishimura T., Nagase K., Kanazawa H.

International Journal of Molecular Sciences （International Journal of Molecular Sciences）  19 （ 6 ） 1646 2018年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  16616596

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© 2018 by the authors. Licensee MDPI, Basel, Switzerland. L-type amino acid transporter 1 (LAT1) is more highly expressed in cancer cells compared with normal cells. LAT1 targeting probes would therefore be a promising tool for cancer cell imaging. In this study, LAT1-targeting thermoresponsive fluorescent polymer probes based on poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)) were synthesized and their affinity for LAT1 was evaluated. The synthesized polymer probes interacted with LAT1 on HeLa cells, and inhibition of L-[3 H]-leucine, one of the substrates for LAT1 uptake, was investigated. L-Tyrosine-conjugated P(NIPAAm-co-DMAAm) inhibited the uptake of L-[3 H]-leucine, while P(NIPAAm-co-DMAAm) and L-phenylalanine-conjugated P(NIPAAm-co-DMAAm) did not. This result indicated that L-tyrosine-conjugated polymer has a high affinity for LAT1. The fluorescent polymer probes were prepared by modification of a terminal polymer group with fluorescein-5-maleimide (FL). Above the polymer transition temperature, cellular uptake of the polymer probes was observed because the polymers became hydrophobic, which enhanced the interaction with the cell membrane. Furthermore, quantitative analysis of the fluorescent probe using flow cytometry indicated that L-tyrosine-conjugated P(NIPAAm-co-DMAAm)-FL shows higher fluorescence intensity earlier than P(NIPAAm-co-DMAAm)-FL. The result suggested that cellular uptake was promoted by the LAT1 affinity site. The developed LAT1-targeting thermoresponsive fluorescent polymer probes are expected to be useful for cancer cell imaging.

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Mesenchylmal stem cell culture on poly(N-isopropylacrylamide) hydrogel with repeated thermo-stimulation

Akimoto A., Niitsu E., Nagase K., Okano T., Kanazawa H., Yoshida R.

International Journal of Molecular Sciences （International Journal of Molecular Sciences）  19 （ 4 ） 1253 2018年04月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  16616596

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© 2018 by the authors. We prepared thermoresponsive hydrogels by mixing poly(N-isopropylacrylamide) (PNIPAAm) derivatives as the main chain components, octa arm polyethylene glycol (PEG) as a crosslinker, and the Arg-Gly-Asp-Ser (RGDS) peptides as cell adhesion units. Human bone marrow derived mesenchymal stem cells (hbmMSCs) were cultured on the hydrogels. The PNIPAAm gel prepared by the post crosslinking gelation method was revealed to be cytocompatible and showed temperature dependent changes in mechanical properties. Repeated changes in the swelling ratio of the PNIPAAm gel affected the shape of the hbmMSCs. With respect to both cytocompatibility and reversibility of changes in mechanical properties, the PNIPAAm gel system could be potentially useful for the analysis of cell mechanobiology.

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Poly(N-isopropylacrylamide) based thermoresponsive polymer brushes for bioseparation, cellular tissue fabrication, and nano actuators

Nagase K., Okano T., Kanazawa H.

Nano-Structures and Nano-Objects （Nano-Structures and Nano-Objects）  16   9 - 23 2018年04月

研究論文（学術雑誌）, 共著

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© 2018 Elsevier B.V. Many thermoresponsive surfaces for biomedical applications have been developed using various techniques for modifying substrates with thermoresponsive polymers. In addition, surface-initiated living radical polymerization methods such as atom-transfer radical polymerization (ATRP) and reversible addition–fragmentation chain-transfer (RAFT) polymerization have been developed as nano structure controlled surface modification methods for modifying thermoresponsive polymers to substrates. These polymerization techniques allow the accurate control of polymerization and the formation of densely packed polymer brush structures. The present review article summarizes a variety of thermoresponsive polymer brushes, mainly comprising poly(N-isopropylacrylamide) (PIPAAm), prepared by surface-initiated ATRP and RAFT polymerization. Self-oscillating polymer brushes as new nano-actuators are also discussed.

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Design of functional thermoresponsive polymer brushes and their application to bioseparation

Nagase K., Okano T., Kanazawa H.

Kobunshi Ronbunshu （Kobunshi Ronbunshu）  75 （ 2 ） 143 - 154 2018年03月

ISSN  03862186

　概要を見る

©2018, The Society of Polymer Science, Japan. Protein drugs and therapeutic cells are among promising current and future therapies for patients. The importance of these proteins and cells has increased in the clinical field, and protein and cell purification methods, without losing their potency or biological activity, are urgently needed. We prepared thermoresponsive polymer brushes as a new protein and cell separation tool because interactions between the polymer brushes and targeted proteins and cells can be modulated by changing the external solution temperature without using other agents that might contaminate these biologics or disrupt their function. Using these new thermoresponsive polymer brushes, a variety of different bio-separations have been performed by simple temperature changes in aqueous solutions. The present review describes the recent achievement in bioseparation using thermoresponsive polymer brushes.

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Development of nanocarriers functionalized with stimuli-responsive polymer for controlled cellular uptake

Yamanouchi T., Katsuyama N., Hiruta Y., Ayano E., Kanazawa H.

Kobunshi Ronbunshu （Kobunshi Ronbunshu）  75 （ 2 ） 116 - 127 2018年03月

ISSN  03862186

　概要を見る

©2018, The Society of Polymer Science, Japan. Drug delivery system (DDS) is a technology that controls sustained release of medicines in the body and localized delivery to diseased tissues. Therefore, it is possible to keep the concentration of medicine at therapeutically effective concentrations for a long time and to reduce side effects. We have achieved controlled intracellular uptake, and release of medicines by nanocarriers modified with stimuli-responsive polymers based on poly(N-isopropylacrylamide), which undergoes a reversible phase transition in response to external temperature stimulation. In this article, we introduce our recent research on the design of temperature- and pH-responsive polymers, which enables selective intracellular uptake with external stimulation, and applications to DDS of an anti-tumor agent and siRNA by micelle and liposomes modified with those polymers.

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Poly(N-isopropylacrylamide)-based thermoresponsive surfaces provide new types of biomedical applications

Nagase K., Yamato M., Kanazawa H., Okano T.

Biomaterials （Biomaterials）  153   27 - 48 2018年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  01429612

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© 2017 The Authors Thermoresponsive surfaces, prepared by grafting of poly(N-isopropylacrylamide) (PIPAAm) or its copolymers, have been investigated for biomedical applications. Thermoresponsive cell culture dishes that show controlled cell adhesion and detachment following external temperature changes, represent a promising application of thermoresponsive surfaces. These dishes can be used to fabricate cell sheets, which are currently used as effective therapies for patients. Thermoresponsive microcarriers for large-scale cell cultivation have also been developed by taking advantage of the thermally modulated cell adhesion and detachment properties of thermoresponsive surfaces. Furthermore, thermoresponsive bioseparation systems using thermoresponsive surfaces for separating and purifying pharmaceutical proteins and therapeutic cells have been developed, with the separation systems able to maintain their activity and biological potency throughout the procedure. These applications of thermoresponsive surfaces have been improved with progress in preparation techniques of thermoresponsive surfaces, such as polymerization methods, and surface modification techniques. In the present review, the various types of PIPAAm-based thermoresponsive surfaces are summarized by describing their preparation methods, properties, and successful biomedical applications.

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Reversible conformational changes in parallel type G-quadruplex structure inside a thermoresponsive hydrogel

Erika Hasuike, Aya Mizutani Akimotoa, Reiko Kurodab, Ko Matsukawa, Yuki Hiruta, Hideko Kanazawa, Ryo Yoshida

Chemical Communications 53   3142 - 3144 2017年02月

研究論文（学術雑誌）, 共著, 査読有り

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Dual temperature- and pH-responsive polymeric micelle for selective and efficient two-step doxorubicin delivery

Yuki Hiruta, Yuki Kanda, Naoya Katsuyama, Hideko Kanazawa.

RSC Adv 7   29540 - 29549 2017年

研究論文（学術雑誌）, 共著, 査読有り

Transcutaneous drug delivery by liposomes using fractional laser technology

Takahiro Fujimoto, Jian Wang, Kazuki Baba, Yuka Oki, Yuki Hiruta, Masayuki Ito, Shinobu Ito, Hideko Kanazawa

Lasers in Surgery & Medicine 49   525 - 532 2017年

研究論文（学術雑誌）, 共著

The use of a temperature-responsive column for the direct analysis of drugs in serum by two-dimensional heart-cutting liquid chromatography

Toshiyasu Mikuma, Ryo Uchida, Mizuki Kajiya, Yuki Hiruta, Hideko Kanazawa

Analytical and Bioanalytical Chemistry 409   1059 - 1065 2017年

研究論文（学術雑誌）, 共著, 査読有り

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Tunable Surface Properties of Temperature-Responsive Polymer Modified Liposomes Induce Faster Cellular Uptake

Jian Wang, Eri Ayano, Yoshie Maitani, Hideko Kanazawa

ACS OMEGA 2 （ 1 ） 316 - 325 2017年

研究論文（学術雑誌）, 共著, 査読有り

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Propofol-fentanyl interaction in Beagles - Apnea, response to mechanical ventilation, endotracheal tube, and tetanic stimulation

T. Iizuka, K. Masui, T. Miyabe-Nishiwaki, H. Kanazawa, R. Nishimura

Research in Veterinary Science 115   34 - 42 2017年

研究論文（学術雑誌）, 共著

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Fractional laser-assisted percutaneous drug delivery via temperature-responsive liposomes

Takahiro Fujimoto, Masayuki Ito, Shinobu Ito, Hideko Kanazaw

Journal of Biomaterials Science, Polymer Edition 28 （ 7 ） 679 - 689 2017年

研究論文（学術雑誌）, 共著, 査読有り

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Enhanced Cellular Uptake and Gene Silencing Activity of siRNA Using Temperature-Responsive Polymer-Modified Liposome.

Wang J, Ayano E, Maitani Y, Kanazawa H

Int J Pharm 523   217 - 228 2017年

研究論文（学術雑誌）, 共著, 査読有り

Design and synthesis of temperature-responsive polymer/silica hybrid nanoparticles and application to thermally controlled cellular uptake

Hiruta Y, Nemoto R, Kanazawa H

Colloids Surf B Biointerfaces 153   2 - 9 2017年

研究論文（学術雑誌）, 共著, 査読有り

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Intracellular localization and delivery of plasmid DNA by biodegradable microsphere-mediated femtosecond laser optoporation

Atsuhiro Ishii, Yuki Hiruta, Dag Heinemann, Alexander Heisterkamp, Hideko Kanazawa, Mitsuhiro Terakawa

Journal of Biophotonics 10   1723 - 1731 2017年

研究論文（学術雑誌）, 共著, 査読有り

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Local Release of VEGF Using Fiber Mats Enables Effective Transplantation of Layered Cardiomyocyte Sheets

Kenichi Nagase, Yuhei Nagumo, Miri Kim, Hee-Jung Kim, Hei-Won Kyung, Hye-Jin Chung, Hidekazu Sekine, Tatsuya Shimizu, Hideko Kanazawa, Teruo Okano, Seung-Jin Lee, Masayuki Yamato

Macromolecular Bioscience 17   1700073 2017年

研究論文（学術雑誌）, 査読有り

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超高速液体クロマトグラフィーを用いた向精神薬の複数同時迅速分析.

芳川満輝、内田亮、黒木巽、三熊敏靖、蛭田勇樹、永田佳子、金澤秀子

分析化学 65 （ 5 ） 173 - 179 2016年

研究論文（学術雑誌）, 共著, 査読有り