研究者詳細 - 金澤　秀子

論文 - 金澤　秀子

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Effective Separation for New Therapeutic Modalities Utilizing Temperature-responsive Chromatography

Maekawa Y., Ayano E., Nagase K., Kanazawa H.

Analytical Sciences 37 （ 5 ） 651 - 660 2021年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 09106340

In recent years, drug discovery and therapeutics trends have shifted from a focus on small-molecule compounds to biopharmaceuticals, genes, cell therapy, and regenerative medicine. Therefore, new approaches and technologies must be developed to respond to these changes in medical care. To achieve this, we applied a temperature-responsive separation system to purify a variety of proteins and cells. We developed a temperature-responsive chromatography technique based on a poly(N-isopropylacrylamide) (PNIPAAm)-grafted stationary phase. This method may be applied to various types of protein and cell separation applications by optimizing the properties of the modified polymers used in this system. Therefore, the developed temperature-responsive HPLC columns and temperature-responsive solid-phase extraction (TRSPE) columns can be an effective separation tool for new therapeutic modalities such as monoclonal antibodies, nucleic acid drugs, and cells.

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Thermoresponsive Cationic Block Copolymer Brushes for Temperature-Modulated Stem Cell Separation

Kenichi Nagase, Ayumu Ota, Tadashi Hirotani, Sota Yamada, Aya Mizutani Akimoto, Hideko Kanazawa

Macromolecular Rapid Communications 2000308 2020年08月

研究論文（学術雑誌）, 共著, 査読有り

Design of two complementary copolymers that work as a glue for cell-laden collagen gels

Taihei Nishimoto, Ko Matsukawa, Kenichi Nagase, Hideko Kanazawa, Aya Mizutani Akimoto, Ryo Yoshida

Chemical Communications 46 （ 10565 ） 10548 2020年08月

研究論文（学術雑誌）, 査読有り

Antibody drug separation using thermoresponsive anionic polymer brush modified beads with optimised electrostatic and hydrophobic interactions

Kenichi Nagase, Saki Ishii, Koji Ikeda, Sota Yamada, Daiju Ichikawa, Aya Akimoto, Yutaka Hattori, Hideko Kanazawa

Scientific Reports 10, 11896, (2020) 10 11896 2020年07月

研究論文（学術雑誌）, 共著, 査読有り

Simultaneous analysis of multiple oligonucleotides by temperature-responsive chromatography using a poly(N-isopropylacrylamide)-based stationary phase

Yutaro Maekawa, Kaichi Yamazaki, Miwa Ihara, Kenichi Nagase, Hideko Kanazawa

Analytical and Bioanalytical Chemistry 412 5341 - 5351 2020年06月

研究論文（学術雑誌）, 共著

Green analytical method for the simultaneous analysis of cytochrome P450 probe substrates by poly(N-isopropylacrylamide)-based temperature-responsive chromatography

Yutaro Maekawa, Naoya Okamoto, Yuji Okada, Kenichi Nagase, Hideko Kanazawa

Scientific Reports10(1), 8828, (2020) 10 （ 1 ） 8828 2020年06月

研究論文（学術雑誌）, 共著, 査読有り

Mixed polymer brush as a functional ligand of silica beads for temperature-modulated hydrophobic and electrostatic interactions

Kenichi Nagase, Sakiko Kitazawa, Sota Yamada, Aya Mizutani Akimoto, Hideko Kanazawa

Analytica Chimica Acta 1095 1 - 13 2020年01月

研究論文（学術雑誌）, 共著, 査読有り

Thermoresponsive anionic copolymer brush-grafted surfaces for cell separation

Nagase K., Uchikawa N., Hirotani T., Akimoto A., Kanazawa H.

Colloids and Surfaces B: Biointerfaces （Colloids and Surfaces B: Biointerfaces） 185 110565 2020年01月

ISSN 09277765

© 2019 Elsevier B.V. Cell separation methods that do not require surface modification of cells are needed in tissue engineering and regenerative medicine. We developed thermoresponsive anionic polymer brushes for cell separation without modification of the cell surfaces. Copolymer brush poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-tert-butyl acrylate, P(NIPAAm-co-tBAAm-co-tBA), was prepared on a cover glass plate through activator regenerated by electron transfer atom transfer radical polymerization (ARGET-ATRP). The tert-butyl group of the copolymer brush was then deprotected and a P(NIPAAm-co-tBAAm-co-acrylic acid (AAc)) brush-modified glass surface was obtained. ARGET-ATRP synthesis achieved polymers with low polydispersity. The negative surface charge of the polymer brush-modified substrates was evaluated using zeta potential measurements and the phase transition temperature of the polymer was modulated between 37–20 °C to perform cell adhesion and detachment, respectively. The adhesion and detachment behavior of cells used in cardiovascular tissue engineering on the thermoresponsive anionic polymer brushes was investigated. Normal human umbilical vein endothelial cells (HUVEC) exhibited prompt detachment from the thermoresponsive anionic polymer brush surfaces. In addition, normal human aortic smooth muscle cells (SMC) exhibited relatively high adhesion on thermoresponsive anionic polymer brush-modified surfaces compared with those modified with thermoresponsive polymer brushes without anionic groups. By utilizing the difference in the cell adhesion and detachment properties of the cell types, a mixture of HUVEC and SMC was separated simply by altering the applied temperature. This result indicated that the prepared thermoresponsive anionic polymer brush-modified glass surface could be used as a tool for the separation of cells in cardiovascular tissue engineering.

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刺激応答性高分子を用いたバイオセパレーション

長瀬健一, 金澤秀子

高分子 69 （ 9 ） 472 - 473 2020年

研究論文（学術雑誌）, 共著, 査読有り

Characteristic differences of cell sheets composed of mesenchymal stem cells with different tissue origins

Nakao M., Inanaga D., Nagase K., Kanazawa H.

Regenerative Therapy （Regenerative Therapy） 11 34 - 40 2019年12月

© 2019 The Japanese Society for Regenerative Medicine Introduction: Stem cell therapy with mesenchymal stem cells (MSCs)has been widely used in many clinical trials, and therapy with MSC sheets shows promise for patients. However, there are few reports characterizing MSC sheets. In the present study, the properties of MSC sheets derived from bone marrow, adipose tissue, and umbilical cord were evaluated. Methods: Cell sheets were fabricated with MSCs from different tissue origins in temperature-responsive cell culture dishes with and without pre-coating of fetal bovine serum (FBS). MSC adhesion behavior in the culture dish was observed. Secretion of cytokines related to cell proliferation and immune regulation from MSC sheets was investigated by ELISA. The adhesion properties of the MSC sheets were investigated by time-lapse microscopy. Results: Different cell adhesion and proliferation rates in temperature-responsive cell culture dishes were observed among the three types of MSCs. FBS pre-coating of the dishes enhanced cell attachment and proliferation in all cell types. Harvested cell sheets showed high attachment capacity to tissue culture polystyrene dish surfaces. Conclusions: MSC sheets can be fabricated from MSCs from different tissue origins using temperature-responsive cell culture dishes. The fabricated MSC sheets could be useful in cell transplantation therapies by choosing appropriate types of MSCs that secrete therapeutic cytokines for the targeted diseases.

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Phenotypic traits of mesenchymal stem cell sheets fabricated by temperature-responsive cell culture plate: structural characteristics of MSC sheets.

Nakao M, Kim K, Nagase K, Grainger DW, Kanazawa H, Okano T

Stem cell research & therapy （Stem Cell Research and Therapy） 10 （ 1 ） 353 2019年11月

© 2019 The Author(s). Background: In most stem cell therapy strategies reported to date, stem cells are introduced to damaged tissue sites to repair and regenerate the original tissue structure and function. MSC therapeutic efficacies are inconsistent, largely attributed to transplanted MSC difficulties both in engrafting at tissue sites and in retaining their therapeutic functions from suspension formulations. MSC functional components, including cell adhesion and cell-cell junction proteins, and ECM that contribute to essential cellular therapeutic effects, are damaged or removed by proteolytic enzymes used in stem cell harvesting strategies from culture. To overcome these limitations, methods to harvest and transplant cells without disrupting critical stem cell functions are required. Cell sheet technology, exploiting temperature-responsive cell culture surfaces, permits cell harvest without cell protein damage. This study is focused on phenotypic traits of MSC sheets structurally and functionally to understand therapeutic benefits of cell sheets. Methods/results: This study verified cleaved cellular proteins (vinculin, fibronectin, laminin, integrin β-1, and connexin 43) and increased apoptotic cell death produced under standard trypsin harvesting treatment in a time-dependent manner. However, MSC sheets produced without trypsin using only temperature-controlled sheet harvest from culture plastic exhibited intact cellular structures. Also, MSCs harvested using enzymatic treatment (i.e., chemical disruption) showed higher pYAP expression compared to MSC sheets. Conclusion: Retention of cellular structures such as ECM, cell-cell junctions, and cell-ECM junctions is correlated with human umbilical cord mesenchymal stem cell (hUC-MSC) survival after detachment from cell culture surfaces. Retaining these proteins intact in MSC cultures using cell sheet technology is proposed to enhance stem cell survival and their function in stem cell-based therapy.

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Temperature-modulated cell-separation column using temperature-responsive cationic copolymer hydrogel-modified silica beads

Nagase K., Inanaga D., Ichikawa D., Mizutani Akimoto A., Hattori Y., Kanazawa H.

Colloids and Surfaces B: Biointerfaces （Colloids and Surfaces B: Biointerfaces） 178 253 - 262 2019年06月

ISSN 09277765

© 2019 Elsevier B.V. There is strong demand for cell separation methods that do not decrease cell activity or modify cell surfaces. Here, new temperature-modulated cell-separation columns not requiring cell-surface premodification are described. The columns were packed with temperature-responsive cationic polymer hydrogel-modified silica beads. Poly(N-isopropylacrylamide-co-n-butyl methacrylate-co-N,N-dimethylaminopropyl acrylamide) hydrogels with various cationic moieties were attached to silica-bead surfaces by radical polymerization using N,Nʹ-methylenebisacrylamide as a crosslinking agent. The beads were packed into solid-phase extraction columns, and temperature-dependent cell elution from the columns was found using HL-60 and Jurkat cells. The retention HL-60 and Jurkat cells in columns containing cationic beads at 37 °C was 95.3% to 99.6% and 95.0% to 98.8%, respectively. By contrast, beads without cationic properties exhibited low cell retention (20.6% for HL-60 and 32.5% for Jurkat cells). The cells were mainly retained through both electrostatic and hydrophobic interactions. The retained HL-60 (4.9%) and Jurkat cells (40%) were eluted at 4 °C from the column with a low composition of cationic monomer (DMAPAAm, 1 mol% in copolymer), because the temperature-responsive hydrogels on the beads became hydrophilic, decreasing the hydrophobic interactions between the cells and the beads. A higher number of Jurkat cells than HL-60 cells were eluted because of differences in their electrostatic properties (Jurkat cells: −2.53 mV; HL-60 cells: −20.7 mV). The results indicated that cell retention by the hydrogel-coated beads packed in a solid phase extraction column could be modulated simply by changing the temperature.

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Adsorption-Desorption Control of Fibronectin in Real Time at the Liquid/Polymer Interface on a Quartz Crystal Microbalance by Thermoresponsivity

Li J., Kaku T., Tokura Y., Matsukawa K., Homma K., Nishimoto T., Hiruta Y., Akimoto A., Nagase K., Kanazawa H., Shiratori S.

Biomacromolecules （Biomacromolecules） 20 （ 4 ） 1748 - 1755 2019年04月

ISSN 15257797

Copyright © 2019 American Chemical Society. The cell manipulation technique using thermoresponsive polymers is currently attracting much attention for applications in the medical field. To achieve arbitrary and accurate cell control, it is necessary to intensely research fibronectin behavior. A smart surface, which has thermoresponsive wettability and which can adsorb or desorb fibronectin repeatedly without the presence of cells, was fabricated by an electrospinning method. The fabricated coating changed its structure as the temperature was changed, and this transformation could substitute for the pulling force generated by the cytoskeletal contraction of cells. Moreover, a coated quartz crystal microbalance was able to detect the fibronectin behavior as frequency shifts, which could be used in the estimation of the mass shift with the aid of suitable equations. This coating and measurement system can contribute greatly not only to the development in the medical field centered on biomaterial manipulation technologies, but also to the improvement of medical instruments.

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LAT1-Targeting Thermoresponsive Liposomes for Effective Cellular Uptake by Cancer Cells

Maekawa-Matsuura M., Fujieda K., Maekawa Y., Nishimura T., Nagase K., Kanazawa H.

ACS Omega （ACS Omega） 4 （ 4 ） 6443 - 6451 2019年04月

© 2019 American Chemical Society. L-type amino acid transporter 1 (LAT1) is a transporter that is more highly expressed in cancer cells compared with normal cells. In the present study, liposomes, composed of egg phosphatidylcholine (EPC) and dioleoyl phosphatidylethanolamine, were modified with LAT1-targeting thermoresponsive polymer, l-tyrosine-conjugated poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)). The cellular uptake of the prepared LAT1-targeting liposomes was evaluated using HeLa cells as a cancer cell model. At temperatures above the polymer's lower critical solution temperature, uptake of the liposomes into cells was observed because the polymer at the liposome surface became hydrophobic and interacted with the cell membrane. Flow cytometry analysis suggested that l-tyrosine-P(NIPAAm-co-DMAAm)-liposomes exhibited markedly increased cellular uptake by HeLa cells compared with that of liposomes not modified with l-tyrosine. This result indicated that cellular uptake of liposomes can be enhanced by the affinity between l-tyrosine and the LAT1 of HeLa cells. The developed functional liposomes, which exhibit both thermoresponsive and LAT1-targeting properties, would be appropriate for temperature-modulated drug delivery and imaging with good targeting ability.

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Liposomes with temperature-responsive reversible surface properties

Nemoto R., Fujieda K., Hiruta Y., Hishida M., Ayano E., Maitani Y., Nagase K., Kanazawa H.

Colloids and Surfaces B: Biointerfaces （Colloids and Surfaces B: Biointerfaces） 176 309 - 316 2019年04月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 09277765

© 2019 Elsevier B.V. Liposomes composed of egg phosphatidylcholine and cholesterol were modified with the temperature-responsive polymer poly(N-isopropylacrylamide-co-N, N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)), and exhibited reversible surface properties with temperature. Completely reversible liposome aggregation due to P(NIPAAm-co-DMAAm) hydration/dehydration was demonstrated over four successive cycles of heating and cooling. The P(NIPAAm-co-DMAAm) polymer was hydrated during cooling, which dispersed the liposomes. The rigidity of the liposomal membrane was one of the factors in the reversible aggregation, as was the modification density of the polymer on the liposomes. A low density on relatively rigid liposomes could maintain the polymer property of reversible hydrated layers below critical solution temperature (LCST) boundary. Above the LCST, temperature-responsive polymers could also transport negatively charged liposomes into cells. The reversible behavior of the temperature-responsive polymer-modified liposomes has not been reported previously and could enable new applications for switching deposit forms as alternative drug carriers.

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Effect of polymer phase transition behavior on temperature-responsive polymer-modified liposomes for siRNA transfection

Nagase K., Hasegawa M., Ayano E., Maitani Y., Kanazawa H.

International Journal of Molecular Sciences （International Journal of Molecular Sciences） 20 （ 2 ） 430 2019年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 16616596

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Small interfering RNAs (siRNAs) have been attracting significant attention owing to their gene silencing properties, which can be utilized to treat intractable diseases. In this study, two temperature-responsive liposomal siRNA carriers were prepared by modifying liposomes with different polymers—poly(N-isopropylacrylamide-co-N,N-dimethylaminopropyl acrylamide) (P(NIPAAm-co-DMAPAAm)) and poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) P(NIPAAm-co-DMAAm). The phase transition of P(NIPAAm-co-DMAPAAm) was sharper than that of P(NIPAAm-co-DMAAm), which is attributed to the lower co-monomer content. The temperature dependent fixed aqueous layer thickness (FALT) of the prepared liposomes indicated that modifying liposomes with P(NIPAAm-co-DMAPAAm) led to a significant change in the thickness of the fixed aqueous monolayer between 37 ◦ C and 42 ◦ C; while P(NIPAAm-co-DMAAm) modification led to FALT changes over a broader temperature range. The temperature-responsive liposomes exhibited cellular uptake at 42 ◦ C, but were not taken up by cells at 37 ◦ C. This is likely because the thermoresponsive hydrophilic/hydrophobic changes at the liposome surface induced temperature-responsive cellular uptake. Additionally, siRNA transfection of cells for the prevention of luciferase and vascular endothelial growth factor (VEGF) expression was modulated by external temperature changes. P(NIPAAm-co-DMAPAAm) modified liposomes in particular exhibited effective siRNA transfection properties with low cytotoxicity compared with P(NIPAAm-co-DMAAm) modified analogues. These results indicated that the prepared temperature-responsive liposomes could be used as effective siRNA carriers whose transfection properties can be modulated by temperature.

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Mixed polymer brush as a functional ligand of silica beads for temperature-modulated hydrophobic and electrostatic interactions

Nagase K., Kitazawa S., Yamada S., Akimoto A., Kanazawa H.

Analytica Chimica Acta （Analytica Chimica Acta） 1095 1 - 13 2019年

ISSN 00032670

© 2019 Elsevier B.V. We developed a mixed polymer brush, which consists of temperature-responsive polymer and cationic polymer modified beads as functional chromatographic matrices, for temperature-modulated multiple hydrophobic and electrostatic interactions. The mixed polymer brush was modified on silica beads through the combination of reversible addition−fragmentation chain transfer (RAFT) polymerization of N,N-dimethylaminopropyl acrylamide (DMAPAAm) and surface initiated atom transfer radical polymerization (ATRP) of N-isopropylacrylamide (NIPAAm). Zeta potential measurement of the mixed polymer brush revealed that the zeta potential increased with increasing temperature, which was attributed to the exposed PDMAPAAm on the beads arising from the shrinking of PNIPAAm upon increasing the temperature. The prepared beads were used as the packing material of high performance liquid chromatography (HPLC) columns, and the elution behavior of steroids, adenosine nucleotides, and proteins through the column was observed. The retention time of steroids increased with increasing the column temperature because of the dehydration of PNIPAAm in the mixed polymer brush. Adenosine nucleotides were also retained by the columns, which was attributed to the electrostatic interaction with PDMAPAAm in the mixed polymer brush. Several proteins were adsorbed on the column at elevated temperatures because of the enhanced electrostatic interaction of exposed PDMAPAAm and the enhanced hydrophobic interaction resulting from the dehydration of PNIPAAm. By exploiting this unique property, mixtures of proteins could be separated by simply changing the column temperature. These results indicate that the developed mixed polymer brush modified beads would be useful as functional chromatographic packing matrices for thermally-modulated multiple hydrophobic and electrostatic interactions.

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Temperature-responsive mixed-mode column containing temperature-responsive polymer-modified beads and anionic polymer-modified beads

Nagase K., Watanabe M., Zen F., Kanazawa H.

Analytica Chimica Acta （Analytica Chimica Acta） 1079 220 - 229 2019年

ISSN 00032670

© 2019 Elsevier B.V. We developed temperature-responsive mixed-mode columns packed with poly(N-isopropylacrylamide) (PNIPAAm)-modified beads and poly(2-acrylamido-2-methylpropane sulfonic acid) (PAMPS)-modified beads in various ratios. The PNIPAAm-modified silica beads and PAMPS-modified silica beads were prepared by surface-initiated atom transfer radical polymerization of N-isopropylacrylamide and 2-acrylamido-2-methylpropane sulfonic acid, respectively. We confirmed polymer modification of the silica beads by CHN elemental analysis, FTIR, zeta-potential measurements, and SEM. To determine the column separation efficiency, we examined the elution behaviors of cold medicine active ingredients and monoamines from each column. Analyte separation occurred on columns with PNIPAAm to PAMPS ratios of 1:20 and 1:10, whereas a column containing only PNIPAAm-modified beads did not retain the analytes. The analytes were retained on the columns through hydrophobic and electrostatic interactions with PNIPAAm and PAMPS, respectively. The separation performance improved with increasing column temperature because of dehydration of PNIPAAm and enhancement of hydrophobic interactions at elevated temperatures. The mixed-mode columns will be useful for separating basic bioactive compounds because the retention of analytes can be modulated by changing the column temperature and the composition.

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Crosslinked Poly(N-Isopropylacrylamide)-Based Microfibers as Cell Manipulation Materials with Prompt Cell Detachment

Konishi T., Mizutani Akimoto A., Nishimoto T., Tokura Y., Tenjimbayashi M., Homma K., Matsukawa K., Kaku T., Hiruta Y., Nagase K., Kanazawa H., Shiratori S.

Macromolecular Rapid Communications （Macromolecular Rapid Communications） 40 （ 23 ） e1900464 2019年

ISSN 10221336

© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Stimuli-responsive smart materials are a key to the realization of next-generation medical technologies. Among them, the temperature-responsive polymer poly(N-isopropylacrylamide) (PNIPAAm) is attracting particular attention because it is easy to use in physiological conditions. PNIPAAm-grafted surfaces can undergo temperature-modulated cell adhesion and detachment without proteolytic enzymes, and can be used as cell-separating materials through selective cell adhesion/detachment. However, cell detachment at reduced temperatures is problematic because it takes several hours. A novel thermoresponsive crosslinked microfiber system that can greatly reduce the cell detachment time is introduced in this study. The crosslinked fibers provide temperature-dependent volume change, and enable cell detachment within 10 min of reducing the temperature, which is one-sixth of the time required in previous studies. The prompt cell detachment is thought to arise from a completely new mechanism derived from fiber swelling. This system will make a significant contribution as a novel cell manipulating system for next-generation medical technology.

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CD44-targeting nanocarriers for cancer treatment

Yamanouchi T., Kanazawa H.

Drug Delivery System （Drug Delivery System） 34 （ 1 ） 38 - 45 2019年

ISSN 09135006

© 2019, Japan Society of Drug Delivery System. All rights reserved. Recently, the theory of cancer stem cell (CSC) is gaining increasing attention from researchers and has become an important focus of cancer research. Since the discovery that the marker molecule is over-expressed in a variety of solid tumors, many studies have focused on methods for targeting the hyaluronan receptor CD44. Hyaluronic acid, the primary CD44 binding molecule, has proved a significant factor in developing nanocarriers that demonstrate preferential tumor accumulation and increased cell uptake. Research is frequently conducted to add further functions to nanocarriers targeting CD44. It is expected that more effective drug delivery will be possible by combining with existing technology. In this review, we introduce research approaches from the current literature that take advantage of CD44-targeting ability and describe the possible advantages for each approach. Finally, we describe our recent progress in hyaluronic acid-modified thermoresponsive liposome aiming for CD44-targeting and combined with oncothermia.

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論文 - 金澤　秀子