Takahisa Anada, Michiharu Kawahara, Taisei Shimada, Ryotaro Kuroda, Hidenori Okamura, Daiki Setoyama, Fumi Nagatsugi, Yuya Kunisaki, Eriko Kage-Nakadai, Shingo Kobayashi and Masaru Tanaka : A Nucleic Acid Prodrug That Activates Mitochondrial Respiration, Promotes Stress Resilience, and Prolongs Lifespan, Journal of the American Chemical Society, 147, 25, 22161-22175, 2025.
(Summary)
Mitochondrial dysfunction caused by aging leads to decreased energy metabolism, resulting in functional decline and increased frailty in multiple tissues. Strategies for protecting and activating mitochondria under stressful conditions are required to suppress aging and age-related diseases. However, it is challenging to develop drugs capable of boosting mitochondrial respiration and compensating for the reduced intracellular adenosine triphosphate (ATP) levels. In this study, we developed a prodrug that stimulates the metabolism of intracellular adenine nucleotides (AXP: adenosine monophosphate (AMP), adenosine diphosphate (ADP), and ATP). It enhances AMP-activated protein kinase activity, fatty acid oxidation, oxidative stress resistance, and mitochondrial respiration, thereby increasing the intracellular ATP levels. Furthermore, this prodrug markedly extended the lifespan of Caenorhabditis elegans. AXP-driven stimulation of cellular energy metabolism proposed herein represents a novel geroprotective strategy and paves the way for the development of bioenergetic-molecule therapeutics.
Panyue Wen, Anjaneyulu Dirisala, Haochen Guo, Xueying Liu, Shingo Kobayashi, Hiroaki Kinoh, Takahisa Anada, Masaru Tanaka, Kazunori Kataoka and Junjie Li : Engineering durable antioxidative nanoreactors as synthetic organelles for autoregulatory cellular protection against oxidative stress, Journal of Controlled Release, 382, 113683, 2025.
(Summary)
Polymersomes, which are polymer vesicles containing an aqueous cavity enclosed in a polymer membrane, hold enormous potential for biomedical applications. In recent years, enzyme-loaded polymersomes, serving as therapeutic nanoreactors, have drawn substantial interest. A crucial requirement for effective catalytic function is to impart semipermeability to the vesicular membrane while maintaining its role as a protective barrier for encapsulated enzymes. However, achieving both long-term stability and optimal membrane permeability for sustained functionality remains a challenge in many reported examples. In this study, we introduce ROS-responsive polyion complex vesicles (PICsomes) loaded with antioxidant enzymes (catalase) as antioxidative nanoreactors. The intrinsic semipermeability and crosslinked network structure of the membrane enable long-lasting catalytic function of catalase. The nanoreactor exhibits inherent cell-protective properties against oxidative stress in fibroblasts due to the ROS-scavenging ability of polymers. Notably, triggered by ROS, the nanoreactor demonstrates autoregulatory control of redox homeostasis. This is because the cysteamine released by PICsomes not only acts as a free radical scavenger but also facilitates the transport of L-cysteine into cells, thereby enhancing glutathione (GSH) biosynthesis. The results further demonstrate significant long blood circulation of PICsomes loaded with catalase and strong protection effects against bloodstream oxidative stress, paving the way for the further development of truly effective in vivo therapeutics. These findings underscore the potential of the engineered antioxidative nanoreactor with durable functionality as synthetic organelles for cellular protection against oxidative stress.
Kaito Yokota, Sari Usuda, Tomoya Nishimura, Rintaro Takahashi, Yusuke Taoka, Shingo Kobayashi, Masaru Tanaka, Kazuaki Matsumura and Ichi Shin Yusa : Self-Assembly and Drug Encapsulation Properties of Biocompatible Amphiphilic Diblock Copolymers, Langmuir, 41, 1, 765-773, 2025.
(Summary)
To prepare amphiphilic diblock copolymers (M100Pm), a controlled radical polymerization approach was employed, incorporating hydrophilic poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) with hydrophobic poly(3-methoxypropyl acrylate) (PMPA). The synthesized diblock copolymers feature a PMPC block with a degree of polymerization (DP) of 100 and a PMPA block with DP (=m) values of 171 and 552. The hydrophilic PMPC block exhibits biocompatibility, such as inhibition of platelet and protein adsorption, because of its hydrophilic pendant zwitterionic phosphorylcholine groups that have the same chemical structure as cell membrane surfaces. The PMPA block exhibits hydrophilicity because of its hydrophilic ether groups; however, it is predominantly hydrophobic. In addition, PMPA exhibits biocompatibility. Because both blocks of M100Pm are biocompatible, M100Pm has potential applications in the biomedical field as an innovative material. Because of the hydrophobicity of the PMPA blocks, which were surrounded by hydrophilic PMPC shells, M100Pm aggregated when dispersed in water. M100P171 and M100P552 formed spherical micelles and vesicles, respectively. As the DP of the PMPA block increased, the aggregate size and number also increased. Doxorubicin was successfully encapsulated within the M100Pm aggregates. Given their biocompatible properties, M100Pm aggregates have potential applications in drug delivery systems.
Natsuki Hayakawa, Masahito Nishiura, Takahisa Anada, Shingo Kobayashi, Toshiki Sawada, Takeshi Serizawa and Masaru Tanaka : Suspension Culture System for Isolating Cancer Spheroids using Enzymatically Synthesized Cellulose Oligomers, ACS Applied Bio Materials, 7, 1, 306-314, 2023.
(Summary)
Isolating cancer cells from tissues and providing an appropriate culture environment are important for a better understanding of cancer behavior. Although various three-dimensional (3D) cell culture systems have been developed, techniques for collecting high-purity spheroids without strong stimulation are required. Herein, we report a 3D cell culture system for the isolation of cancer spheroids using enzymatically synthesized cellulose oligomers (COs) and demonstrate that this system isolates only cancer spheroids under coculture conditions with normal cells. CO suspensions in a serum-containing cell culture medium were prepared to suspend cells without settling. High-purity cancer spheroids could be separated by filtration without strong stimulation because the COs exhibited antibiofouling properties and a viscosity comparable to that of the culture medium. When human hepatocellular carcinoma (HepG2) cells, a model for cancer cells, were cultured in the CO suspensions, they proliferated clonally and efficiently with time. In addition, only developed cancer spheroids from HepG2 cells were collected in the presence of normal cells by using a mesh filter with an appropriate pore size. These results indicate that this approach has potential applications in basic cancer research and cancer drug screening.
(Keyword)
3D cell culture / cancer spheroids / cell isolation / cellulose / coculture
Riku Saeki, Shingo Kobayashi, Rena Shimazui, Teruki Nii, Akihiro Kishimura, Takeshi Mori, Masaru Tanaka and Yoshiki Katayama : Characterization of polypropyleneimine as an alternative transfection reagent, Analytical Sciences/Supplements, 39, 6, 1015-1020, 2023.
(Summary)
Polypropyleneimine (PPI) was examined as a transfection reagent comparing with most widely used polymer, polyethyleneimine (PEI). PPI had better responsiveness to the endosomal pH and showed more condensation ability of plasmid DNA than PEI. Although the cytotoxicity of PPI was somewhat higher than PEI, the transfection efficacy of PPI was comparable with PEI or higher than PEI in some cell line. Thus, PPI would be an alternative transfection reagent.
Shingo Kobayashi, Atsushi Sugasaki, Yosuke Yamamoto, Yuta Shigenoi, Airi Udaka, Aki Yamamoto and Masaru Tanaka : Enrichment of Cancer Cells Based on Antibody-Free Selective Cell Adhesion, ACS Biomaterials Science & Engineering, 8, 10, 4547-4556, 2022.
(Summary)
Blood-compatible and cell-adhering polymer materials are extremely useful for regenerative medicine and disease diagnosis. (Meth)acryl polymers with high hydrophilicity have been widely used in industries, and attempts to apply these polymers in the medical field are frequently reported. We focused on crosslinked polymer films prepared using bifunctional monomers, which are widely used as coating materials, and attempted to alter the cell adhesion behavior while maintaining blood compatibility by changing the chemical structure of the crosslinker. Four bifunctional monomers were studied, three of which were found to be blood-compatible polymers and to suppress platelet adhesion. The adhesion behavior of cancer cells to polymer films varied; moreover, the cancer model cells MCF-7 [EpCAM(+)] and MDA-MB-231 [EpCAM (-)], with different expression levels of epithelial cell adhesion molecule (EpCAM), showed distinct adhesion behavior for each material. We suggest that a combination of these materials has the potential to selectively capture and enrich highly metastatic cancer cells.
(Keyword)
blood-compatible polymer / crosslinked polymeric material / early detection and diagnosis of cancer / liquid biopsy / multifunctional monomer / nonthrombogenic polymer
Wonryung Lee, Hwan Seung Jeong, Woo Young Lim, Hyunhwan Lee, Joohyuk Kang, Hyunjae Lee, Injun Lee, Seop Hyung Han, Shingo Kobayashi, Masaru Tanaka and Soo Byeong Bae : Conformable microneedle pH sensors via the integration of two different siloxane polymers for mapping peripheral artery disease, Science Advances, 7, 48, eabi6290, 2021.
(Summary)
Flexible microneedles are important tools that allow access to the inside of biological tissue from the outside without surgery. However, it had been hard to realize microneedle sensor arrays on flexible substrates because of the difficulty of attaining a needle with a high Young's modulus for a selected area on a thin or soft substrate. In this work, we developed a microneedle sensor on a hybrid substrate based on high Young's modulus epoxy siloxane for the microneedles and low Young's modulus polydimethylsiloxane for the conformable substrate. Polyaniline was deposited on the microneedle for pH sensing. The mechanical durability of the device was assessed by insertion into pig skin 1000 times. Last, the flexible microneedle pH sensors showed their utility for monitoring pH distribution in rats in a peripheral artery diseases model.
Shichen Liu, Shingo Kobayashi, nosuke Shin Nishimura, Tomoya Ueda and Masaru Tanaka : Effect of pendant groups on the blood compatibility and hydration states of poly(2-oxazoline)s, Journal of Polymer Science, 59, 21, 2559-2570, 2021.
(Keyword)
blood compatibility / hydration state / intermediate water / poly(2-oxazoline)s
Kei Nishida, Takahisa Anada, Shingo Kobayashi, Tomoya Ueda and Masaru Tanaka : Effect of bound water content on cell adhesion strength to water-insoluble polymers, Acta Biomaterialia, 134, 313-324, 2021.
(Summary)
Adhesion of cells on biomaterials plays an essential role in modulating cellular functions. Although hydration of biomaterials occurs under biological conditions, it is challenging to systematically evaluate the correlation of hydrated water content in biomaterials with the cell adhesion strength. In this report, we investigated the effect of bound water content on the adhesion strength of cells on poly(2-methoxyethyl acrylate) (PMEA) analogue substrates. Water-insoluble PMEA analogues were synthesized to fabricate substrates with a systemically controlled bound water content. To assess the surface properties of their substrates, contact angle measurement, atomic force microscopy (AFM), and fluorescence measurement were conducted. To reflect the effect of bound water of PMEA analogues, the relationship between the bound water content and cell adhesion behavior was evaluated under serum-free condition. From the single cell force spectrometry (SCFS) and microscopic analysis, it revealed that the increment of bound water content on the substrates decreased cell adhesion strength and cell spreading on the substrates. The bound water content exhibited a good correlation with adhesion strength, spreading area, circularity, and aspect ratio of cells. Our findings indicate that the bound water content could contribute to the development of a novel biomaterial and evaluation of cell behaviors on biomaterials. STATEMENT OF SIGNIFICANCE: For coordinating cell functions, such as growth, mobility, and differentiation, modulating the adhesion strength between cells and their environments is important. Although the hydration to biomaterials has been reported to be closely related to a antifouling property, the effect of hydration water on the cell adhesion behavior is not well understood. We present the first demonstration of essential relationship between cell adhesion strength and hydrated water on a biomaterials surface using the water-insoluble polymers with different hydrated water content. The results reveal that the hydrated water content of polymer substrates strong correlation with adhesion strength of cells. Collectively, the hydrated water content of the biomaterials will be a predominant factor affecting the cell adhesion strength and behavior.
Shichen Liu, Shingo Kobayashi, Toshiki Sonoda and Masaru Tanaka : Poly(tertiary amide acrylate) Copolymers Inspired by Poly(2-oxazoline)s: Their Blood Compatibility and Hydration States, Biomacromolecules, 22, 6, 2718-2728, 2021.
(Summary)
Modifying the side chain of poly(meth)acrylate with moieties originating from biocompatible polymers can be an effective method for developing novel blood-compatible polymers. Inspired by biocompatible poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx), four water-soluble poly(tertiary amide acylate) analogues bearing a pendant tertiary amide were synthesized. The results of hemolysis and cell viability tests showed that all the poly(tertiary amide acylate) analogues were compatible with red blood cells, HeLa cells, and normal human dermal fibroblasts as PMeOx or PEtOx. Among the four poly(tertiary amide acylate) analogues, poly[2-(N-methylpropionamido)ethyl acrylate] (PPEA) and poly[2-(N-ethylacetamido)ethyl acrylate] (PEAE) showed thermosensitivity in aqueous solution; especially, PPEA (10 mg mL-1) exhibited a lower critical solution temperature of 37 °C. Water-insoluble copolymers were prepared to investigate the possibility of applying these synthesized polymers as blood-compatible coatings. The poly[n-butyl methacrylate70-co-2-(N-methylacetamido)ethyl methacrylate30] (coPAEM) coatings significantly suppressed plasma protein adsorption, denaturation degree of adsorbed fibrinogen, and platelet adhesion. Intermediate water (IW), whose content can generally indicate the blood compatibility of polymers, was found in all hydrated homopolymers and copolymers by differential scanning calorimetry. The present work demonstrated that the tertiary amide moiety in the side chain of poly(meth)acrylate was an effective contributor to blood compatibility and IW.
(Keyword)
Acrylates / Amides / Biocompatible Materials / HeLa Cells / Humans / Oxazoles / Polymers / Water
Tsung An Kuo, Shingo Urata, Ryohei Koguchi, Toshiki Sonoda, Shingo Kobayashi and Masaru Tanaka : Effects of Side-Chain Spacing and Length on Hydration States of Poly(2-methoxyethyl acrylate) Analogues: A Molecular Dynamics Study, ACS Biomaterials Science & Engineering, 7, 6, 2383-2391, 2021.
(Summary)
Hydration states of polymers are known to directly influence the adsorption of biomolecules. Particularly, intermediate water (IW) has been found able to prevent protein adsorption. Experimental studies have examined the IW content and nonthrombogenicity of poly(2-methoxyethyl acrylate) analogues with different side-chain spacings and lengths, which are HPx (x is the number of backbone carbons in a monomer) and PMCyA (y is the number of carbons in-between ester and ether oxygens of the side-chain) series, respectively. HPx was reported to possess more IW content but lower nonthrombogenicity compared to PMCyA with analogous composition. To understand the reason for the conflict, molecular dynamics simulations were conducted to elucidate the difference in the properties between the HPx and PMCyA. Simulation results showed that the presence of more methylene groups in the side chain more effectively prohibits water penetration in the polymer than those in the polymer backbone, causing a lower IW content in the PMCyA. At a high water content, the methoxy oxygen in the shorter side chain of the HPx cannot effectively bind water compared to that in the PMCyA side chain. HPx side chains may have more room to contact with molecules other than water (e.g., proteins), causing experimentally less nonthrombogenicity of HPx than that of PMCyA. In summary, theoretical simulations successfully explained the difference in the effects of side-chain spacing and length in atomistic scale.
Tsung An Kuo, Shingo Urata, Ryohei Koguchi, Toshiki Sonoda, Shingo Kobayashi and Masaru Tanaka : Molecular Dynamics Study on the Water Mobility and Side-Chain Flexibility of Hydrated Poly(ω-methoxyalkyl acrylate)s, ACS Biomaterials Science & Engineering, 6, 12, 6690-6700, 2020.
(Summary)
Intermediate water (IW) is known to play an important role in the antifouling property of biocompatible polymers. However, how IW prevents protein adsorption is still unclear. To understand the role of IW in the antifouling mechanism, molecular dynamics simulation was used to investigate the dynamic properties of water and side-chains for hydrated poly(ω-methoxyalkyl acrylate)s (PMCxA, where x indicates the number of methylene carbons) with x = 1-6 and poly(n-butyl acrylate) (PBA) in this study. Since the polymers uptake more water than their equilibrium water content (EWC) at the polymer/water interface, we analyzed the hydrated polymers at a water content higher than that of EWC. It was found that the water molecules interacting with one polymer oxygen atom (BW1), of which most are IW molecules, in PMC2A exhibit the lowest mobility, while those in PBA and PMC1A show a higher mobility. The result was consistent with the expectation that the biocompatible polymer with a long-resident hydration layer possesses good antifouling property. Through the detailed analysis of side-chain binding with three different types of BW1 molecules, we found that the amount of side-chains simultaneously interacting with two BW1 molecules, which exhibit the highest flexibility among the three kinds of side-chains, is the lowest for PMC1A. The high mobility of BW1 is thus suggested as the main factor for the poor protein adsorption resistance of PMC1A even though it possesses enough IW content and relatively flexible side-chains. Contrarily, a maximum amount of side-chains simultaneously interacting with two BW1 molecules was found in the hydrated PMC3A. The moderate side-chain length of PMC3A allows side-chains to simultaneously interact with two BW1 molecules and minimizes the hydrophobic part attractively interacting with a protein at the polymer/water interface. The unique structure of PMC3A may be the reason causing the best protein adsorption resistance among the PMCxAs.
Toshiki Sonoda, Shingo Kobayashi, Keisuke Herai and Masaru Tanaka : Side-Chain Spacing Control of Derivatives of Poly(2-methoxyethyl acrylate): Impact on Hydration States and Antithrombogenicity, Macromolecules, 53, 19, 8570-8580, 2020.
Yoshihide Toyokawa, Shingo Kobayashi, Haruka Tsuchiya, Tomokazu Shibuya, Makiko Aoki, Jun Sumiya, Shun Ooyama, Tetsuya Ishizawa, Naohiko Makino, Yoshiyuki Ueno and Masaru Tanaka : A fully covered self-expandable metallic stent coated with poly (2-methoxyethyl acrylate) and its derivative: In vitro evaluation of early-stage biliary sludge formation inhibition, Materials Science and Engineering. C, Materials for Biological Applications, 120, 111386, 2020.
(Summary)
The adhesion and deformation behavior of proteins at the inner surface of fully covered, self-expandable metallic stents coated with biocompatible polymers, poly(2-methoxyethyl acrylate) (PMEA) and poly(3-methoxypropyl acrylate) (PMC3A), were analyzed. Model bile solution, proteins, and bacteria were used to unravel the inhibitory ability of the polymer coatings. Adsorbance of proteins and adherence of bacteria were both strongly inhibited by the polymer coatings. Circulation tests were performed under clinical conditions using human bile from patients. Adsorption/deformation of proteins and early-stage sludge formation were inhibited on stent surfaces coated with PMEA derivatives. The present study revealed that early-stage biliary sludge formation on PMEA- and PMC3A-coated stents was suppressed due to the strong resistance of the polymers to protein adsorption/deformation, brought about by intermediate water in hydrated polymer coatings, which is not present in conventional coating materials, such as silicone and polyurethane.
Tsung An Kuo, Toshiki Sonoda, Shingo Urata, Ryohei Koguchi, Shingo Kobayashi and Masaru Tanaka : Elucidating the Feature of Intermediate Water in Hydrated Poly(ω-methoxyalkyl acrylate)s by Molecular Dynamics Simulation and Differential Scanning Calorimetry Measurement, ACS Biomaterials Science & Engineering, 6, 7, 3915-3924, 2020.
(Summary)
Intermediate water (IW) has been reported to play an important role in nonthrombogenicity of biomaterials. However, clear insights into the IW in the hydrated polymer are still debated. In this study, a series of molecular dynamics simulations was performed to identify the IW structure in hydrated poly(ω-methoxyalkyl acrylate)s (PMCxAs, where x indicates the number of methylene carbons) with x = 1-6. Through the quantitative comparison with experimental measurements, IW molecules were suggested to mainly come from the water interacting with an oxygen atom of the polymers, while most of the nonfreezing water (NFW) molecules corresponded to the water interacting with two polymer oxygen atoms. In addition, the IW molecules were found to effectively enhance the flexibility of the PMCxA side chains in comparison with the NFW molecules. The variations of the saturated IW content and the side-chain flexibility with the methylene carbon chain length of PMCxA were also found to be correlated with the experimental nonthrombogenicity of PMCxA, suggesting that the polymer with the more saturated IW content and higher chain flexibility possesses better nonthrombogenicity. Furthermore, through the analyses of the interplays between the IW and polymer and between IW and its adjacent water, we found that the presence of the unique interaction between IW and its adjacent water in the hydrated poly(2-methoxyethyl acrylate) (PMEA) is the main factor causing different cold crystallization behaviors of PMEA from the other PMCxAs rather than the interaction between water and the PMCxA matrix. The findings will be useful in the development of new nonthrombogenic materials.
Ryohei Koguchi, Katja Jankova, Yuki Hayasaka, Daisuke Kobayashi, Yosuke Amino, Tatsuya Miyajima, Shingo Kobayashi, Daiki Murakami, Kyoko Yamamoto and Masaru Tanaka : Understanding the Effect of Hydration on the Bio-inert Properties of 2-Hydroxyethyl Methacrylate Copolymers with Small Amounts of Amino- or/and Fluorine-Containing Monomers, ACS Biomaterials Science & Engineering, 6, 5, 2855-2866, 2020.
(Summary)
Materials exhibiting "bio-inert properties" are essential for developing medical devices because they are less recognized as foreign substances by proteins and cells in the living body. We have reported that the presence of intermediate water (IW) with the water molecules loosely bound to a polymer is a useful index of the bio-inertness of materials. Here, we analyzed the hydration state and the responses to biomolecules of poly(2-hydroxyethyl methacrylate) (PHEMA) copolymers including small amounts of 2-(dimethylamino)ethyl methacrylate (DMAEMA) (N-series) or/and 2,2,2-trifluoroethyl methacrylate (TFEMA) (F-series). The hydration structure was analyzed by differential scanning calorimetry (DSC), the molecular mobility of the produced copolymers by temperature derivative of DSC (DDSC), and the water mobility by solid 1H pulse nuclear magnetic resonance (NMR). Although the homopolymers did not show bio-inert properties, the binary and ternary PHEMA copolymers with low comonomer contents showed higher bio-inert properties than those of PHEMA homopolymers. The hydration state of PHEMA was changed by introducing a small amount of comonomers. The mobility of both water molecules and hydrated polymers was changed in the N-series nonfreezing water (NFW) with the water molecules tightly bound to a polymer and was shifted to high-mobility IW and free water (FW) with the water molecules scarcely bound to a polymer. On the other hand, in the F-series, FW turned to IW and NFW. Additionally, a synergetic effect was postulated when both comonomers coexist in the copolymers of HEMA, which was expressed by widening the temperature range of cold crystallization, contributing to further improvement of the bio-inert properties.
(Keyword)
amino and fluorine / bio-inert / fluorinated polymers / hydration state / intermediate water / poly(2-hydroxyethyl methacrylate)
Yu Meng Tsai, Fumihiro Aratsu, Shogo Sekida, Shingo Kobayashi and Masaru Tanaka : Blood-Compatible Poly(2-methoxyethyl acrylate) Induces Blebbing-like Phenomenon and Promotes Viability of Tumor Cells in Serum-Free Medium, ACS Applied Bio Materials, 3, 4, 1858-1864, 2020.
(Summary)
Circulating tumor cells (CTCs) are highly related to tumor metastasis and an effective technique of detecting/isolating CTCs has been expected to be established for tumor treatments. Previously, we reported that platelets cannot adhere but tumor cells can adhere on poly(2-methoxyethyl acrylate) (PMEA) substrate. In this study, we report that cell viability of human fibrosarcoma (HT-1080) cells was promoted on PMEA substrate in serum-free medium. The significant blebbing-like phenomenon and spontaneous formation of cell aggregation were observed on PMEA substrate. Moreover, cell viability was promoted by activation of Akt signaling pathway via N-cadherin-mediated cell-cell contact. These results suggest that not only capture efficiency and purity but also high cell viability of CTCs can be accomplished by using PMEA substrate. PMEA substrate can be a promising candidate for medical applications such as in vitro screening of anticancer drugs and is an excellent platform for studies and diagnoses of tumor migration and metastasis.
Katja Jankova, Irakli Javakhishvili, Shingo Kobayashi, Ryohei Koguchi, Daiki Murakami, Toshiki Sonoda and Masaru Tanaka : Hydration States and Blood Compatibility of Hydrogen-Bonded Supramolecular Poly(2-methoxyethyl acrylate), ACS Applied Bio Materials, 2, 10, 4154-4161, 2019.
(Summary)
The practical use of the viscous liquid polymer, poly(2-methoxyethyl acrylate) (PMEA), was expanded from thin films with excellent blood compatibility to thick coatings and free-standing films without essentially sacrificing its blood compatibility. This was undertaken by creating multiple hydrogen-bonding polymer networks by introducing a functional methacrylic monomer bearing a 6-methyl-2-ureido-4[1H]-pyrimidone group in the PMEA backbone via free radical copolymerization. The hydrogen-bonded PMEA (H-PMEA) contained about 6 mol % of the functional monomer in the copolymer. These functional monomers as physical cross-links are distributed in the PMEA matrix with a Tg of -35 °C, making H-PMEA a solid rubber-like material with recoverable tensile strain. Additionally, mechanical tests revealed its tensile strength, and thermogravimetric analyses confirmed its higher thermostability. The dry and hydration states of H-PMEA were assessed by differential scanning calorimetry, contact angle, and atomic force microscopy measurements. Comparison with viscous PMEA was made. For the first time, we included PVC alongside PET, the surface we usually use as a negative control, in the platelet adhesion test with human blood, and found out that 1.5 times more platelets adhered onto the PVC surface than onto the PET surface, while H-PMEA proved to have a clear edge. Thus, H-PMEA may serve as a suitable replacement for polymers in products contacting blood as it shows potential for making free-standing films, thick coatings, implants, and articles with various geometries for the medicinal industry.
(Keyword)
2-{3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)ureido}ethyl methacrylate / blood compatible polymer / free-standing film / hydrogen bonding / intermediate water / poly(2-methoxyethyl acrylate)
J. Brian Ree, Shingo Kobayashi, Kyuyoung Heo, Joon Taek Lee, Toshifumi Satoh, Takashi Ishizone and Moonhor Ree : Nanoscale film morphology and property characteristics of dielectric polymers bearing monomeric and dimeric adamantane units, Polymer, 169, 225-233, 2019.
(Keyword)
Adamantane-containing polymers / Amorphous morphology / Critical angle / Electron density / Isotropic dielectric constant / Mass density / Nanoscale thin films / Optical isotropy / Poly(adamantane)s / Random chain orientation / Refractive index / Zero birefringence
Neural interfaces enabling light transmittance rely on optogenetics to control and monitor specific neural activity, thereby facilitating deeper understanding of intractable diseases. This study reports the material strategy underlying an optogenetic neural interface comprising stretchable and transparent conductive tracks and capable of demonstrating high biocompatibility after long-term (5-month) implantation. Ag/Au core-shell nanowires contribute toward improving track performance in terms of stretchability (<60% strain), transparency (<83%), and electrical resistance (15 Ω sq-1 ). The neural interface integrated with gel-coated exterior microelectrodes preserves low impedance (1.1-3.2 Ω cm2 ) in a saline solution over the evaluated 5-month period. Besides the use of efficient conductive materials, surface treatment using antithrombogenic polymer tends to prevent the growth of granulation tissue, thereby facilitating clear monitoring of electrocorticograms (ECoG) in a rodent during chronic implantation. The flexible and transparent neural interface pathologically exhibits noncytotoxicity and low inflammatory response while efficiently recording evoked ECoG in a nonhuman primate via optogenetic stimulation. The proposed highly reliable interface can be employed in multifaceted approaches for translational research based on chronic implants.
Wonryung Lee, Shingo Kobayashi, Masase Nagase, Yasutoshi Jimbo, Itsuro Saito, Yusuke Inoue, Tomoyuki Yambe, Masaki Sekino, G. George Malliaras, Tomoyuki Yokota, Masaru Tanaka and Takao Someya : Nonthrombogenic, stretchable, active multielectrode array for electroanatomical mapping, Science Advances, 4, 10, eaau2426, 2018.
(Summary)
High-precision monitoring of electrophysiological signals with high spatial and temporal resolutions is one of the most important subjects for elucidating physiology functions. Recently, ultraflexible multielectrode arrays (MEAs) have been fabricated to establish conformal contacts with the surface of organs and to measure propagation of electrophysiological signals with high spatial-temporal resolution; however, plastic substrates have high Young's modulus, causing difficulties in creating appropriate stretchability and blood compatibility for applying them on the dynamically moving and surgical bleeding surface of the heart. Here, we have successfully fabricated an active MEA that simultaneously achieves nonthrombogenicity, stretchability, and stability, which allows long-term electrocardiographic (ECG) monitoring of the dynamically moving hearts of rats even with capillary bleeding. Because of the active data readout, the measured ECG signals exhibit a high signal-to-noise ratio of 52 dB. The novel stretchable MEA is carefully designed using state-of-the-art engineering techniques by combining extraordinarily high gain organic electrochemical transistors processed on microgrid substrates and a coating of poly(3-methoxypropyl acrylate), which exhibits significant antithrombotic properties while maintaining excellent ionic conductivity.
Ferdous Khan, Fumihiro Aratsu, Shingo Kobayashi and Masaru Tanaka : A simple strategy for robust preparation and characterisation of hydrogels derived from chitosan and amino functional monomers for biomedical applications, Journal of Materials Chemistry. B, Materials for Biology and Medicine, 6, 31, 5115-5129, 2018.
(Summary)
Molecular interactions of amino functional (AF) monomers with chitosan (CS) lead to the formation of external stimuli responsive hydrogels (HGs). These have the potential to produce biomaterials with novel properties by a simple blending approach. Six independent AF monomers such as diethylenetriamine (DETA), bis(3-aminopropyl)amine (BAPA), 3,3'-diamino-N-methyldipropyleamine (DAMPA), hexamethylenediamine (HMDA), N,N-dimethylethylamine (DMEA) and diethylamine (DEA) with distinct functional groups and chain lengths were designed to form stable HGs at physiological pH. Such AF monomers are able to form HGs within a short time (in the range from 10 to 19 seconds) by physically interacting with CS. This is an alternative to the covalently crosslinking reaction process, providing cost effective HG biomaterials. HG complexes were characterized by rheometry, differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) spectroscopy. The interaction between AF monomers and the CS polymer has been discussed and the results have been confirmed by FTIR analysis. The storage modulus (G'), loss modulus (G'') and complex viscosity (η*) were evaluated for all HGs using a rheometer, and the ratios of CS and the particular AF monomer were optimized for stable HG formation. The swelling ratio was evaluated using a simple method and was found to be directly related to the structure of the AF monomer, pH and temperature. These HGs were utilised for encapsulation, and the release of active molecules (e.g., reactive red 120 (RR120) as a model compound) was measured at low pH 5.5, physiological pH 7.4 and high pH 9.5. The cell viability and cellular compatibility of the HGs were evaluated in vitro cell culture, demonstrating that all the five different types of HGs support cellular compatibility, attachment and growth. The physical mixing of AF monomers with CS is expedited for the development of new bespoke economically viable biomaterials.
Daiki Murakami, Yoko Kitahara, Shingo Kobayashi and Masaru Tanaka : Thermosensitive Polymer Biocompatibility Based on Interfacial Structure at Biointerface, ACS Biomaterials Science & Engineering, 4, 5, 1591-1597, 2018.
(Summary)
The interfacial structure of a thermosensitive biocompatible polymer, poly[2-(2-methoxyethoxy)ethyl methacrylate] (PMe2MA), at the polymer/phosphate-buffered saline (PBS) interface was investigated by atomic force microscopy. A number of nanometer scale protrusions appeared at 37 °C and disappeared at 22 °C, reversibly. This structural change occurred above the lower critical solution temperature of PMe2MA in PBS (19 °C), indicating that the formation of protrusions was explained by the microphase separation of polymer and water at the interfacial region. The protein adsorption and platelet adhesion onto PMe2MA interface were drastically restrained at 22 °C compared to that at 37 °C. Detachment of NIH3T3 cells accompanied by the dissipation of protrusions on the PMe2MA interface was also demonstrated. These results indicate that the protrusions act as the scaffold for the protein adsorption and cell adhesion.
(Keyword)
atomic force microscopy / biocompatible polymer / interface / intermediate water
Shingo Kobayashi, Miyuki Wakui, Yukihisa Iwata and Masaru Tanaka : Poly(ω-methoxyalkyl acrylate)s: Nonthrombogenic Polymer Family with Tunable Protein Adsorption, Biomacromolecules, 18, 12, 4214-4223, 2017.
(Summary)
A series of polyacrylates with different n-alkyl side chain lengths (1 to 6, and 12 carbons) and a ω-methoxy terminal group (poly(ω-methoxyalkyl acrylate): PMCxA) were prepared to study their nonthrombogenicity using human platelet adhesion, micro bicinchoninic acid (micro BCA) protein assay, and enzyme-linked immunosorbent assay (ELISA) tests. In all cases, human platelet adhesion was suppressed on the PMCxA-coated substrates, and the number of human platelets adhered to the PMC3A (poly(3-methoxypropyl acrylate))-coated surface was comparable to that of commercialized nonthrombogenic coating agent poly(2-methoxyethyl acrylate) (PMEA, equal to PMC2A). The amount of protein adsorbed onto the PMCxA was investigated by micro BCA using bovine serum albumin (BSA) and human fibrinogen (hFbn), revealing that PMC3A exhibited significantly high resistance to nonspecific BSA adsorption. Additionally, the amount of hFbn adsorbed onto the PMC3A was suppressed to the same extent as PMEA. The exposure degree of platelet adhesion sites in adsorbed hFbn was evaluated using an ELISA test, and the degree on the PMCxA with three to six methylene carbons was comparable to the PMEA. The hydration water structure in the hydrated PMCxA was also characterized using differential scanning calorimetry (DSC). The amount of intermediate water, which is the hydration water molecules that moderately interact with the polymer matrix, was maximum in the PMEA with two methylene run lengths, whereas the amount decreased by increasing the number of methyelnes in the side chain. The amount of adsorbed protein increased with a decrease in the amount of intermediate water, suggesting that the protein adsorption amount is tunable by simply changing the number of methylene carbons in the side chain. The present study revealed that poly(ω-methoxyalkyl acrylate)s are useful for blood-contacting medical devices, and PMC3A is the best mode of PMCxA to apply as an antiprotein adsorption coating agent.
Kazuhiro Sato, Shingo Kobayashi, Asuka Sekishita, Miyuki Wakui and Masaru Tanaka : Synthesis and Thrombogenicity Evaluation of Poly(3-methoxypropionic acid vinyl ester): A Candidate for Blood-Compatible Polymers, Biomacromolecules, 18, 5, 1609-1616, 2017.
(Summary)
A poly(vinyl acetate) derivative, poly(3-methoxypropionic acid vinyl ester) (PMePVE), was synthesized to develop a new candidate for blood compatible polymers. The monomer MePVE was synthesized by a simple two-step reaction, and then the MePVE was polymerized via free radical polymerization to obtain PMePVE. Human platelet adhesion tests were performed to evaluate the thrombogenicity, and the platelet adhesion was suppressed on the PMePVE-coated substrate. To determine the expression of the nonthrombogenicity of the PMePVE, the plasma protein adsorption and a conformationally altered state of fibrinogen were analyzed by a microBCA assay and enzyme-linked immunosorbent assay. The adsorption and denaturation of the plasma proteins were inhibited on the PMePVE; thus, PMePVE exhibited blood compatibility. A distinctive hydration water structure in the nonthrombogenic polymer, intermediate water (IW), was observed in the hydrated PMePVE by differential scanning calorimetry analysis. The nonthrombogenicity of PMePVE is considered to be brought about by the presence of IW.
Yusuke Inoue, Tomoyuki Yokota, Tsuyoshi Sekitani, Akiko Kaneko, Taeseong Woo, Shingo Kobayashi, Tomokazu Shibuya, Masaru Tanaka, Hiroyuki Kosukegawa, Itsuro Saito, Takashi Isoyama, Yusuke Abe, Tomoyuki Yambe, Takao Someya and Masaki Sekino : Antithrombotic Protein Filter Composed of Hybrid Tissue-Fabric Material has a Long Lifetime, Annals of Biomedical Engineering, 45, 5, 1352-1364, 2017.
(Summary)
There are recent reports of hybrid tissue-fabric materials with good performance-high biocompatibility and high mechanical strength. In this study, we demonstrate the capability of a hybrid material as a long-term filter for blood proteins. Polyester fabrics were implanted into rats to fabricate hybrid tissue-fabric material sheets. The hybrid materials comprised biological tissue grown on the fabric. The materials were extracted from the rat's body, approximately 100 days post-implantation. The tissues were decellularized to prevent immunological rejection. An antithrombogenicity test was performed by dropping blood onto the hybrid material surface. The hybrid material showed lesser blood coagulation than polysulfone and cellulose. Blood plasma was filtered using the hybrid material to evaluate the protein removal percentage and the lifetime of the hybrid material in vitro. The hybrid material showed a comparable performance to conventional filters for protein removal. Moreover, the hybrid material could work as a protein filter for 1 month, which is six times the lifetime of polysulfone.
Kohei Osawa, Shingo Kobayashi and Masaru Tanaka : Synthesis of Sequence-Specific Polymers with Amide Side Chains via Regio-/Stereoselective Ring-Opening Metathesis Polymerization of 3-Substituted cis-Cyclooctene, Macromolecules, 49, 21, 8154-8161, 2016.
Ken Kono, Hitomi Hiruma, Shingo Kobayashi, Yoji Sato, Masaru Tanaka, Rumi Sawada and Shingo Niimi : In vitro endothelialization test of biomaterials using immortalized endothelial cells, PLoS ONE, 11, 6, e0158289, 2016.
(Summary)
Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs) can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC) and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.
Kazuhiro Sato, Shingo Kobayashi, Miho Kusakari, Shogo Watahiki, Masahiko Oikawa, Takashi Hoshiba and Masaru Tanaka : The Relationship between Water Structure and Blood Compatibility in Poly(2-methoxyethyl Acrylate) (PMEA) Analogues, Macromolecular Bioscience, 15, 9, 1296-1303, 2015.
(Summary)
Six types of poly(2-methoxyethyl acrylate) (PMEA) analogues were synthesized and the water structure in the hydrated polymers was characterized using differential scanning calorimetry (DSC). The hydrated PMEA analogues exhibited the different amounts of intermediate water. Non-thrombogenicity evaluation was performed on PMEA analogues for platelet adhesion and protein adsorption. Platelet adhesion was suppressed on PMEA analogues. In addition, the protein adsorption and deformation were suppressed by increasing the amount of intermediate water. This study demonstrates that the amount of intermediate water might play a key role in expressing the blood compatibility of polymeric materials.
(Keyword)
biomaterial / blood compatibility / intermediate water / protein adsorption / water structure
Shingo Kobayashi, Hyunwoo Kim, W. Christopher MacOsko and A. Marc Hillmyer : Functionalized linear low-density polyethylene by ring-opening metathesis polymerization, Polymer Chemistry, 4, 4, 1193-1198, 2013.
Jie Song, Anne Bringuier, Shingo Kobayashi, M. Adam Baker and W. Christopher Macosko : Adhesion between polyethylenes and different types of polypropylenes, Polymer Journal, 44, 9, 939-945, 2012.
Jie Song, M. Christopher Thurber, Shingo Kobayashi, M. Adam Baker, W. Christopher MacOsko and Craig H. Silvis : Blends of polyolefin/PMMA for improved scratch resistance, adhesion and compatibility, Polymer, 53, 16, 3636-3641, 2012.
Shingo Kobayashi, M. Louis Pitet and A. Marc Hillmyer : Regio- and stereoselective ring-opening metathesis polymerization of 3-substituted cyclooctenes, Journal of the American Chemical Society, 133, 15, 5794-5797, 2011.
Hyunwoo Kim, Shingo Kobayashi, A. Mohd Abdurrahim, J. Minglun Zhang, Albina Khusainova, A. Marc Hillmyer, A. Ahmed Abdala and W. Christopher MacOsko : Graphene/polyethylene nanocomposites: Effect of polyethylene functionalization and blending methods, Polymer, 52, 8, 1837-1846, 2011.
Shingo Kobayashi, Jie Song, Craig H. Silvis, W. Christopher MacOsko and A. Marc Hillmyer : Amino-functionalized polyethylene for enhancing the adhesion between polyolefins and polyurethanes, Industrial & Engineering Chemistry Research, 50, 6, 3274-3279, 2011.
Shingo Kobayashi, Hiroshi Kataoka and Takashi Ishizone : Synthesis of well-defined poly(ethylene-alt-1-vinyladamantane) via living anionic polymerization of 2-(1-Adamantyl)-1,3-butadiene,followed by hydrogenation, Macromolecules, 42, 14, 5017-5026, 2009.
Shingo Kobayashi, Cheng Lu, R. Thomas Hoye and A. Marc Hillmyer : Controlled polymerization of a cyclic diene prepared from the ring-closing metathesis of a naturally occurring monoterpene, Journal of the American Chemical Society, 131, 23, 7960-7961, 2009.
Shingo Kobayashi and Masaru Tanaka : Design of biomaterials through direct ring-opening metathesis polymerisation of functionalised cyclic alkenes, Molecular Systems Design & Engineering, 8, 8, 960-991, Jun. 2023.
Masaru Tanaka, Shingo Kobayashi, Daiki Murakami, Fumihiro Aratsu, Aki Kashiwazaki, Takashi Hoshiba and Kazuki Fukushima : Design of Polymeric Biomaterials: The "Intermediate Water Concept", Bulletin of the Chemical Society of Japan, 92, 12, 2043-2057, Jan. 2019.
Hsuan Ken Liao, Shingo Kobayashi, Hyunwoo Kim, A. Ahmed Abdala and W. Christopher Macosko : Influence of functionalized graphene sheets on modulus and glass transition of PMMA, Macromolecules, 47, 21, 7674-7676, Nov. 2014.
Shingo Kobayashi, W. Christopher MacOsko and A. Marc Hillmyer : Model linear low density polyethylenes from the ROMP of 5-hexylcyclooct-1-ene, Australian Journal of Chemistry, 63, 8, 1201-1209, Aug. 2010.
K. Sugiyama, R. Kakuchi, Shingo Kobayashi, Akira Hirao, J. Zhang, T. Hoye and C. Macosko : Synthesis of star-branched polymers by means of reactive melt blending of end-functionalized polymers, Polymer Preprints Japan, 55, 2, 2579-2580, Dec. 2006.
(Keyword)
Amino group / Anhydride moiety / End-functionalized polymer / Living anionic polymerization / Reactive melt blending / Star-branched polymer
Shingo Kobayashi and Takashi Ishizone : Synthesis of polystyrene derivatives containing pendant adamantyl groups via anionic polymerization, Polymer Preprints Japan, 54, 2, 2504, Dec. 2005.
Design and Synthesis of Polymeric Materials Exhibiting Immunosuppressive Activity in Response to Reactive Oxygen Species (ROS) (Project/Area Number: 25K15911 )
Development of Reversible Double-Layer Polymer Modification Technology to Break the Safety/Efficacy Trade-Off of Delivering Enzyme (Project/Area Number: 23K28429 )
Development of label free separation and recovery technology for circulating tumor cells (Project/Area Number: 22H00591 )
Elucidation of polymer structure-biocompatibility relationship using model polymers with controlled side-chain sequences (Project/Area Number: 21K12687 )
Development of synthesis strategies for controlling the biocompatibility of polymeric materials (Project/Area Number: 15K05512 )
Design and synthesis of tumor cell adherent blood compatible materials (Project/Area Number: 26282134 )
Synthesis of bio-compatible polymers via regio- and stereoselective ring-opening metathesis polymerization (Project/Area Number: 24750097 )