Language：English   Publishing type：Research paper (scientific journal)  

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腸内細菌叢を破壊することが知られる抗生物質（バンコマイシン）を腸間で特異的に吸着する粒子を開発し、実際に、腸内細菌を保護することを示した。また細菌叢の破壊に由来する感染症からマウスを保護することができた。

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

beta-ガラクトシダーゼを酵素として、一細胞の抗原を高感度に増感検出する方法（蛍光がターンオンし、細胞に共有結合して脱離しない）の開発に初めて成功した。

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

抗体医薬を低分子化合物で置き換えることを企図し、がん細胞と内在性の抗体を架橋する低分子を開発した。この分子は、in vitro, in vivoで抗がん活性を示した。

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PNA-peptideコンジュゲートにより、腫瘍において選択的にRNAiを生じるシステムを開発した。腫瘍で高活性化しているcathepsin Bを標的にしたシステムであるが、一般性が高く、望みのプロテアーゼに対して設計可能である。

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Language：English   Publisher：Analytical Sciences  

This study investigates the impact of hydrophobic modification on the immunogenicity, cytotoxicity, and inflammatory response of Alaska pollock gelatin (ApGltn) microparticles (MPs). Gelatin, known for its inherent biocompatibility, was modified with decyl group (C10) to explore potential alterations in its interaction with the immune system. Immunogenicity was evaluated through the measurement of material-specific IgM and IgG responses, indicating no significant increase post-modification. Cytotoxicity against Caco-2 cell lines and NF-κB-mediated LPS-induced inflammation were also assessed, revealing no exacerbation by the modified MPs. Furthermore, C10 modification with different types of linkage such as secondary amine and amide structure did not influence immune reactivity. These findings suggest that C10 modification maintains the non-immunogenicity and biocompatibility of gelatin MPs, supporting their potential use in biomedical applications. Graphical abstract: (Figure presented.).

DOI： 10.1007/s44211-024-00643-2

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Language：English   Publisher：Analytical Sciences  

Saccharomyces cerevisiae, a widely studied yeast known for its industrial applications, is increasingly recognized for its potential in immunomodulation. This study aimed to systematically analyze and compare the immune-modulating properties of various S. cerevisiae strains under controlled experimental conditions. Three essential signals crucial for immune response activation were evaluated to elucidate the immunological responses elicited by these strains, i.e., dendritic cells (DC) cytokine secretion profiles, maturation status, and T cell polarization. Analysis of DC cytokine secretion profiles and maturation status revealed that all tested yeast strains induced DC activation, characterized by significant IL-6 secretion and modest IL-10 induction, as well as upregulation of MHC II molecules. Additionally, strain-specific effects were observed, particularly, strain AJM109 and Y1383 uniquely enhanced CD86 and PD-L1 expression, respectively, suggesting differential impacts on DC co-stimulatory signaling. Furthermore, strain Y1383 showed a unique capacity to support Treg-mediated immune suppression, demonstrating its potential in immune tolerance induction. These findings underscore the complexity of S. cerevisiae-based immune modulation and emphasize the importance of standardized evaluation methods to distinguish their specific immunological effects. Graphical abstract: (Figure presented.)

DOI： 10.1007/s44211-024-00641-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

This study introduces the α-rhamnose (Rham)-conjugated prodrug of SN-38 (Rham-SN-38) as a promising alternative to irinotecan. α-rhamnosidase, responsible for SN-38 release from Rham-SN-38, does not express in human cells, minimizing individual variability and side effects. The injection of the α-rhamnosidase into the tumor tissues makes it possible, for the first time, to activate the Rham-SN-38. Furthermore, α-rhamnosidase demonstrates significantly higher activity than carboxylesterase, the specific enzyme activating irinotecan. SN-38 release mediated by α-rhamnosidase completes within 2 h, with a kcat/Km value approximately 5.0 × 104-fold higher than that of irinotecan. The 50% inhibition concentration (IC50) of Rham-SN-38 against three types of cancer cells and one normal cell exceeds 4.5 × 103 nM. The addition of α-rhamnosidase significantly increases cytotoxicity, with IC50 comparable to free SN-38. The QIC50, an index reflecting the difference in cytotoxicity with and without α-rhamnosidase, exceeds approximately 1.0 × 102-fold. Rham-SN-38, synthesized in this study, demonstrates significant potential as a prodrug for cancer therapy. Graphical abstract: (Figure presented.)

DOI： 10.1007/s44211-024-00593-9

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Language：English   Publisher：Analytical Sciences  

Effect of drugs on the intracellular activity of lactate dehydrogenase (LDH) has been measured by using water-soluble tetrazolium (WST). Because the assay is usually conducted in the presence of dead cells, net activity of live cells is not evaluated. Here, we reported the assay of the net intracellular LDH activity of live cells by counting the live cells using fluorescent staining of nucleus. By using a deep red fluorescent dye, dual measurements of fluorescence signal of nucleus and absorbance of WST could be conducted with transparent 96-well-plates. We found that conventional assay in the presence of dead cells overestimate the effect of drugs on the LDH activity. Graphical Abstract: (Figure presented.)

DOI： 10.1007/s44211-024-00631-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

In nanomedicine, PEGylation of nanomaterials poses a dilemma owing to its inhibition in interacting with target cells and retention in target tissues despite its biocompatibility and nonspecific internalisation suppression. PEGylated...

DOI： 10.1039/d3tb02049e

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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Abstract

The proto‐oncogene c‐Src is a protein‐tyrosine kinase that is associated with increased risk of acute and chronic kidney diseases, cardiovascular diseases, neurological diseases, and cancers. The purpose of this study was to develop a gene delivery carrier responding to activated c‐Src and to evaluate its efficacy in vitro and in vivo. The polymeric gene carrier consists of a neutral main chain bearing a peptide substrate of c‐Src or negative control peptide. Complexes of the polymer containing c‐Src‐targeting peptides and DNA were phosphorylated in the presence of c‐Src, resulting in release of the DNA. Green fluorescent protein (GFP) expression was observed after microinjection of complexes of polymer containing c‐Src‐targeting peptides and GFP‐encoding DNA into A431 cells at the nitrogen/phosphate (N/P) ratio of 1.0. GFP was not expressed in cells microinjected with negative control polymer/DNA complex. Direct injection of complexes of polymer containing c‐Src‐targeting peptides and luciferase‐encoding DNA at N/P ratios of 1.0 and 2.0 into tumor‐bearing mice resulted in luciferase expression in A431 tumors but not in normal skin tissues. No luciferase expression was observed in A431 tumors or skin tissues after injection of control polymer/DNA complex. These results indicate that our gene delivery carrier enables activated c‐Src‐specific gene expression.

DOI： 10.1002/slct.202303331

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Language：English   Publisher：Analytical Methods  

Identification as well as quantification of ammonia are required in some analytical fields including forensic science. For this purpose, gas chromatography/mass spectrometry (GC/MS) analysis is one of the most suitable techniques. Although ammonia needs to be derivatized for GC/MS analysis, conventional derivatization reagents require anhydrous conditions because they are highly reactive with water. Here, we investigated ethenesulfonyl fluoride (ESF) as a selective reagent for ammonia derivatization in aqueous media to develop a rapid identification method for ammonia in aqueous media. The Michael addition reaction of ammonia with ESF rapidly produced a tri-ESF derivative suitable for GC/MS analysis. We optimized the derivatization reaction conditions and extraction solvent. With the optimized protocol, the detection limit for aqueous ammonia was 0.05 μg mL−1. The calibration curve showed good linearity (R2 = 0.9998) in the range of 0.10-100.0 μg mL−1, and the accuracy (% bias) and the precision (% relative standard deviation) for concentrations of 0.10, 0.25, 10.0, and 75.0 μg mL−1 were within ± 10% (intra- and inter-day). The proposed ESF-based method could quantify ammonia in samples containing interfering nucleophilic substances. This method was successfully applied to ammonia-containing commercial products.

DOI： 10.1039/d3ay01071f

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Publisher：Journal of Controlled Release  

Herein, we report engineered macrophages, termed “MacTrigger,” acting as a trigger to induce an inflammatory environment only in tumor tissues. This led to intensive anti-tumor effects based on the removal potential of foreign substances. The strength of this study is the utilization of two unique functions of macrophages: (1) their ability to migrate to tumor tissues and (2) polarization into the anti-inflammatory M2 phenotype in the presence of tumor tissues. The MacTrigger accelerated the release of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), when it was polarized to the M2 phenotype. When the MacTrigger was administered to tumor-bearing mice, tumor growth was significantly inhibited compared with the non-treatment group, the un-transfected macrophages group, and the group with engineered macrophages capable of randomly releasing TNF-α. Additionally, the ratio of the M1 phenotype to the M2 phenotype in tumor tissues was >1 only in the MacTrigger group. Moreover, the ratios of natural killer cells and CD8+T cells in tumor tissues were increased compared with other groups. These results indicate that MacTrigger can induce inflammation in tumor tissues, leading to effective anti-tumor effects. In normal tissues, especially the liver, notable side effects were not observed. This is because, in the liver, the MacTrigger was not polarized to the M2 phenotype and could not induce inflammation. These results suggest that the MacTrigger is a “trigger” that can induce inflammation only in tumor tissues, then allowing the body to attack tumor tissues through the innate immunity system.

DOI： 10.1016/j.jconrel.2023.04.010

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DOI： 10.1016/j.jconrel.2023.04.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

This study presents a simple strategy for the sequestration of globular proteins as clients into synthetic polypeptide-based complex coacervates as a scaffold, thereby recapitulating the scaffold-client interaction found in biological...

DOI： 10.1039/d3sc00993a

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Repository Public URL： https://hdl.handle.net/2324/7178520

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

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. Graphical abstract: [Figure not available: see fulltext.].

DOI： 10.1007/s44211-023-00284-x

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Other Link： https://link.springer.com/article/10.1007/s44211-023-00284-x/fulltext.html

Language：English   Publisher：Royal Society of Chemistry (RSC)  

This study presents a simple strategy for the sequestration of globular proteins as clients into synthetic polypeptide-based complex coacervates as a scaffold, thereby recapitulating the scaffold-client interaction found in biological condensates. Considering the low net charges of scaffold proteins participating in biological condensates, the linear charge density (σ) on the polyanion, polyethylene glycol-b-poly(aspartic acids), was reduced by introducing hydroxypropyl or butyl moieties as a charge-neutral pendant group. Complex coacervate prepared from the series of reduced-σ polyanions and the polycation, homo-poly-L-lysine, could act as a scaffold that sequestered various globular proteins with high encapsulation efficiency (>80%), which sometimes involved further agglomerations in the coacervates. The sequestration of proteins was basically driven by electrostatic interaction, and therefore depended on the ionic strength and charges of the proteins. However, based on the results of polymer partitioning in the coacervate in the presence or absence of proteins, charge ratios between cationic and anionic polymers were maintained at the charge ratio of unity. Therefore, the origin of the electrostatic interaction with proteins is considered to be dynamic frustrated charges in the complex coacervates created by non-neutralized charges on polymer chains. Furthermore, fluorescence recovery after photobleaching (FRAP) measurements showed that the interaction of side-chains and proteins changed the dynamic property of coacervates. It also suggested that the physical properties of the condensate are tunable before and after the sequestration of globular proteins. The present rational design approach of the scaffold-client interaction is helpful for basic life-science research and the applied frontier of artificial organelles.

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

Enzymes are used to amplify signals for detection of antigen proteins in biological samples. However, the enzymes conventionally used for this purpose have limitations, such as the presence of the...

DOI： 10.1039/d3an00314k

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DOI： 10.1016/j.jconrel.2023.04.010

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Language：English  

DOI： 10.1016/j.jconrel.2023.04.010

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Language：English   Publishing type：Research paper (scientific journal)  

Repository Public URL： https://hdl.handle.net/2324/7173543

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Language：English   Publishing type：Research paper (scientific journal)  

Repository Public URL： https://hdl.handle.net/2324/7178526

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jconrel.2023.04.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

The fabrication of regular hexagonal PIC nanosheets was achieved via induction of the α-helix secondary structure in a PEGylated catiomer promoted by complexation with polyphosphates.

DOI： 10.1039/d2cc05137k

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Repository Public URL： https://hdl.handle.net/2324/7178534

Publisher：Chemistry Letters  

Polynucleotide-loaded nanoparticles are potentially useful for the regulation of intracellular nucleotide levels. Polyadenylic acid and polyguanylic acid were used as model compounds for encapsulation into poly(lactic-co-glycolic acid) nanoparticles. The properties of the produced nanoparticles, as well as the effect of the polynucleotide molecular size on encapsulation into the nanoparticles were investigated.

DOI： 10.1246/cl.220318

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Language：English   Publisher：Cell Reports  

Clostridioides difficile causes nosocomial antibiotic-associated diarrhea on a global scale. Susceptibility to C. difficile infection (CDI) is influenced by the composition and metabolism of gut microbiota, which in turn are affected by diet. However, the mechanism underlying the interplay between diet and gut microbiota that modulates susceptibility to CDI remains unclear. Here, we show that a soy protein diet increases the mortality of antibiotic-treated, C. difficile-infected mice while also enhancing the intestinal levels of amino acids (aas) and relative abundance of Lactobacillus genus. Indeed, Ligilactobacillus murinus-mediated fermentation of soy protein results in the generation of aas, thereby promoting C. difficile growth, and the process involves the anchored cell wall proteinase PrtP. Thus, mutual interaction between dietary protein and the gut microbiota is a critical factor affecting host susceptibility to CDI, suggesting that dietary protein sources can be an important determinant in controlling the disease.

DOI： 10.1016/j.celrep.2022.111332

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Developing nanovehicles for delivering antibiotics is a promising approach to overcome the issue of antibiotic resistance. This study aims to utilize a polyion complex (PICs) system for developing novel nanovehicles for polymyxin-type antibiotics, which are known as last resort drugs. The formation of antibiotic-based PIC nanostructures is investigated using colistimethate sodium (CMS), an anionic cyclic short peptide, and a series of block catiomers bearing different amounts of guanidinium moieties on their side chains. In addition, only the modified catiomer, and not the unmodified catiomer, self-assembles with CMS, implying the importance of the guanidine moieties for enhancing the interaction between the catiomer and CMS via the formation of multivalent hydrogen bonding. Moreover, micellar and vesicular PIC nanostructures are selectively formed depending on the ratio of the guanidine residues. Size-exclusion chromatography reveals that the encapsulation efficiency of CMS is dependent on the guanidinium modification ratio. The antimicrobial activity of the PIC nanostructures is also confirmed, indicating that the complexation of CMS in the PICs and further release from the PICs successfully occurs.

DOI： 10.1002/marc.202200316

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フローサイトメトリーを高感度化するために、酵素反応を利用して細胞に対する蛍光標識の強度を増強させる方法を開発した

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Enzyme-catalyzed amplification of fluorescent immunolabeling of a single cell for high-sensitive flow cytometry

Language：English   Publishing type：Research paper (scientific journal)  

Applications of enzymes are intensively studied, particularly for biomedical applications. However, encapsulation or immobilization of enzymes without deactivation and long-term use of enzymes are still at issue. This study focuses on the polymeric vesicles "PICsomes" for encapsulation of enzymes to develop a hecto-nanometer-scaled enzyme-loaded reactor. The catalytic activity of a PICsome-based enzyme nanoreactor is carefully examined to clarify the effect of compartmentalization by PICsome. Encapsulation by PICsome provides a stability enhancement of enzymes after 24 h incubation at 37 °C, which is particularly helpful for maintaining the high effective concentration of β-galactosidase. Moreover, to control the microenvironment inside the nanoreactor, a large amount of dextran, a neutral macromolecule, is encapsulated together with β-galactosidase in the PICsome. The resulting dextran-coloaded nanoreactor contributes to the enhancement of enzyme stability, even after exposure to 24 h incubation at -20 °C, mainly due to the antifreezing effect.

DOI： 10.1002/mabi.201600542

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Inflammatory activation of macrophages is a key factor in chronic inflammatory diseases such as ulcerative colitis. The excessive production of reactive oxygen species (ROS)/reactive nitrogen species (RNS) by macrophages causes oxidative stress during the inflammatory response and exaggerates inflammatory lesions in ulcerative colitis. Inhibition of the inflammatory activation of macrophages is a promising treatment for chronic inflammatory diseases. Here, we prepared self-filling polymer-lipid hybrid nanoparticles (PST-PLNPs) consisting of poly dl-lactic acid as a hydrophobic biodegradable polymer core encapsulating α-tocopherol (T) and phosphatidylserine (PS) both on the surface and interior of the particle. We confirmed the anti-inflammatory response of these hybrid nanoparticles in activated murine macrophages. PS has anti-inflammatory effects on macrophages by modulating the macrophage phenotype, while α-tocopherol is an antioxidant that neutralizes ROS. We found that PS-containing (PS-PLNPs) and PS plus α-tocopherol-containing (PST-PLNPs) polymer-lipid hybrid nanoparticles significantly increased the viability of lipopolysaccharide (LPS)-treated macrophages compared with phosphatidylcholine-containing PLNPs. PST-PLNPs had a better effect than PS-PLNPs, which was attributed to the synergy between PS and α-tocopherol. This synergic action of PST-PLNPs reduced NO and pro-inflammatory cytokine (IL-6) production and increased anti-inflammatory cytokine (TGF-β1) production when incubated with activated macrophages. Thus, these self-filling biodegradable polymer-lipid hybrid nanoparticles (PST-PLNPs) containing anti-oxidant and anti-inflammatory molecules might be potential alternative drug carriers to liposomes and polymeric nanoparticles for the treatment of chronic inflammatory diseases such as ulcerative colitis.

DOI： 10.1039/c7md00174f

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細胞膜に膜貫通した状態で分子を修飾する方法を世界ではじめて開発した。

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Language：English   Publishing type：Research paper (scientific journal)  

Reversal of efflux of an anticancer drug in human drug-resistant breast cancer cells by inhibition of protein kinase Cα (PKCα) activity.
P-glycoprotein (Pgp) is a 170-kDa transmembrane protein that mediates the efflux of anticancer drugs from cells. Pgp overexpression has a distinct role in cells exhibiting multidrug resistance (MDR). We examined reversal of drug resistance in human MDR breast cancer cells by inhibition of protein kinase Cα (PKCα) activity, which is associated with Pgp-mediated efflux of anticancer drugs. PKCα activity was confirmed by measurement of phosphorylation levels of a PKCα-specific peptide substrate (FKKQGSFAKKK-NH2), showing relatively higher basal activity in drug-resistant MCF-7/ADR cells (84 %) than that in drug-sensitive MCF-7 cells (63 %). PKCα activity was effectively suppressed by the PKC inhibitor, Ro-31-7549, and reversal of intracellular accumulation of doxorubicin was observed by inhibition of PKCα activity in MCF-7/ADR cells compared with their intrinsic drug resistance. Importantly, increased accumulation of doxorubicin could enhance the therapeutic efficacy of doxorubicin in MDR cells significantly. These results suggest a potential for overcoming MDR via inhibition of PKCα activity with conventional anticancer drugs.

DOI： 10.1007/s13277-015-3963-4

Language：Japanese   Publishing type：Research paper (scientific journal)  

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We synthesized polymeric gene carriers consisting of poly-L-lysine (PLL) main chain modified both with substrate peptide for protein kinase C (PKC) and alkanethiol (pentadecanethiol). Due to the grafted substrate peptide, the polyplex prepared from these carriers is expected to show gene expression triggered by the phosphorylation of the peptide by intracellular PKC. The modified alkanethiol on the main chain stabilized the polyplex both via disulfide crosslinking and hydrophobic interaction. The polyplex found to show gene expression in vitro when the alkanethiol content in the main chain was enough low (4-mol%-modification of PLLs ε-amine group) to minimize cytotoxic effect. Even though the content of alkanethiol is low, the polyplex had significant stability in a model serum solution and showed longer blood circulation in vivo. The polyplex clearly accumulated in tumor after intravenous injection.

DOI： 10.1080/09205063.2015.1054922

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Amphiphilic polymers bearing hydrophobic alkyl groups are expected to be applicable for both ligand presentation on the cell surface and intercellular crosslinking. To explore the optimum design for each application, we synthesized eight different acyl-modified dextrans with varying molecular weight, alkyl length, and alkyl modification degree. We found that the behenate-modified polymers retained on the cell surface longer than the palmitate-modified ones. Since the polymers were also modified with biotin, streptavidin can be presented on the cell surface through biotin-streptavidin recognition. The duration of streptavidin on the cell surface is longer in the behenate-modified polymer than the palmitate-modified one. As for the intercellular crosslinking, the palmitate-modified polymers were more efficient than the behenate-modified polymers. The findings in this research will be helpful to design the acyl-modified polymers for the cell surface engineering.

DOI： 10.1080/09205063.2015.1007414

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Small interfering RNA (siRNA) is regarded as a promising tool for cancer therapy because of the wide applicability to various cancer-related genes. However, non-specific delivery of siRNA is one of the major causes of adverse effects. To access the issue, here we designed a new siRNA system which turns on RNAi responding to a cancer cell-specific protease, cathepsin B. The system uses a peptide nucleic acid (PNA)-peptide conjugate to provide this protease-responsive activation. The PNA-peptides were found to form hybrids with double-stranded RNAs with complementary protruding regions, which then affected the susceptibility of dsRNA to Dicer. The dsRNA/PNA-peptide hybrids were activated in cancer cells with a high cathepsin B activity to show RNAi.

DOI： 10.1039/c5ra17737e

Language：English   Publishing type：Research paper (scientific journal)  

We reported here a preparation of a cancer-specific gene carrier by native chemical ligation of a substrate peptide of protein kinase Cα (PKCα), which highly expresses in many types of cancer. This carrier is a tetramer of the substrate peptide connected with adequate spacer to maintain reactivity toward PKCα. The carrier successfully regulated the reporter gene expression responding to the phosphorylation of its serine residues.

DOI： 10.1246/cl.141121

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In order to enhance the interactions between macrophages and cancer cells, thiol-terminated nucleic acid aptamers were immobilized on methacryloyl-functionalised carbohydrates of macrophages. The adhesion of cancer cells on the surface modified macrophages was significantly accelerated.

DOI： 10.1039/c5cc06211j

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We reported here an alternative strategy of antibody internalization by crosslinker-mediated endocytosis. This cross-linker consists of IgG binding peptide and folic acid as cancer targeting ligands toward the folate receptor, which highly expresses in many cancer cells surface. This crosslinker successfully facilitated antibody internalization into cancer cell by the folate receptor-mediated endocytosis.

DOI： 10.1246/cl.141157

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A rational design strategy has been developed for the construction of stable peptide-based anchors for the efficient modification of cell surfaces. Six types of peptide composed of five residues with divalent hydrophobic groups have been designed using this new strategy. Among them, a peptide with a sequence of NBD-Lys-Lys(X)-Lys-Lys-Lys(X)-NH₂ (NBD: fluorophore, Lys(X): N-ε-palmitoyl-L-lysine) was found to show the highest modification efficacy and longevity in culture medium. The good performance of this peptide was attributed to (1) its high aqueous solubility, which allowed it to partition from the medium to the cell surface, and (2) the high binding affinity of the saturated palmitoyl groups to the cell membrane. We found that the distribution of the peptide was affected by recycling endosome, which enabled the representation of the peptide following its endocytotic disappearance from the cell membrane. Biotin was also presented on the cell surface using this peptide-based anchor to examine its recognition by streptavidin. The efficacy of the recognition process increased as the length of the oligoethylene glycol spacer increased, indicating that it was necessary for the biotin tag to move away from the membrane glycoproteins on the cell surface to facilitate its efficient recognition by streptavidin. (Figure Presented).

DOI： 10.1021/bc500465j

Language：English   Publishing type：Research paper (scientific journal)  

We examined a series of linear polyethylenimine (LPEI)-based nanocarriers that activate transgene expression in response to cancer-specific protein kinase Cα (PKCα). Eight types of LPEI-peptide conjugate differing in peptide content and number were synthesized using click chemistry. The conjugates could form polyplexes with pDNA through electrostatic interaction, but the degree of pDNA condensation, sizes, and surface charges of the resulting polyplexes depended on the pendant-peptide content and number. None of the polyplexes showed significant cytotoxicity toward human hepatoma cells (HepG2). Furthermore, pendant peptide content and number markedly affected transgene activation in response to PKCα. To achieve an all-or-none response to PKCα, we determined the optimum peptide content and number in LPEI-peptide conjugates as ≈6. mol% and ≈40. peptides/conjugate.

DOI： 10.1016/j.colsurfb.2014.09.004

Language：English   Publishing type：Research paper (scientific journal)  

To deliver anti-cancer drugs to tumors, a hydrophobic cavity was prepared in the dendritic molecule, dendritic poly(L-lysine) of sixth generation (KG6), which was used as a drug carrier. The dendritic molecule was modified with polyethylene glycol (PEG)-linked hydrophobic penta-phenylalanine or penta-Alanine. The hydrophobic cavity was formed between the KG6 and PEG chains. The penta-phenylalanine peptide was better in encapsulating doxorubicin (DOX) in the cavity compared with penta-Alanine. The loaded DOX was slowly released from the cavity, and it depended on pH. After intravenous injection, the DOX-loaded dendrimers accumulated in the tumor by the enhanced permeability and retention effect, and showed significant suppression of tumor growth without loss of body weight. These results indicate that hydrophobic oligopeptides can be used for forming a hydrophobic cavity in a dendritic molecule for delivery of anti-cancer drugs to tumor sites.

DOI： 10.1080/09205063.2014.938979

Language：English   Publishing type：Research paper (scientific journal)  

Overhauser-enhanced magnetic resonance imaging (OMRI), which is a double resonance technique, creates images of free radical distribution in animals by enhancing the water proton signal intensity by the overhauser effect. In this study, we constructed a contrast agent by combining PROXYL groups that have nitroxyl radicals with PEG-modified dendritic poly(l-lysine) that accumulates in the tumor by enhanced permeability and retention (EPR) effect. Addition of the PROXYL groups at the PEG chains termini on KG6 was advantageous in OMRI, because the ESR signal of the nitroxyl radical was maintained without decay caused by mobility restriction, even if the PROXYL groups were attached at 25 mol% on one molecule. After intramuscular injection of the molecule modified at 25 mol%, that is, PR₂₅-PEG-KG6, a significant OMRI signal was observed at the injected site. However, no signal was detected in the tumor after intravenous injection of PR₂₅-PEG-KG6 to a tumor-bearing mouse, although PR ₂₅-PEG-KG6 itself accumulated in the tumor. The reason was that the nitroxyl radicals were immediately reduced in the blood after the injection, suggesting that use of stable nitroxyl radicals will enable detection of tumors by OMRI after intravenous injection.

DOI： 10.1080/09205063.2014.943538

Language：English  

Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

We demonstrate a polyion complex (PIC) nanoparticle that contains both a responsive fluorophore and an "internal standard" fluorophore for quantitative measurement of protein kinase (PK) activity. The PK-responsive fluorophore becomes more fluorescent with PK-catalyzed phosphorylation of substrate peptides incorporated in the PIC, while fluorescence from the internal standard remains unchanged during phosphorylation. This new concept will be useful for quantitative PK assays and the discovery of PK inhibitors.

DOI： 10.1021/bc500142j

Language：English   Publishing type：Research paper (scientific journal)  

Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

Purpose Nitrate-layered double hydroxide material (nitrate-LDH) matrix can be considered as a potential formulation of delivering nitrogen into soil in a sustained manner.
Materials and methods The nitrate-LDH matrix was formulated by a co-precipitation technique and subsequently characterized by scanning electron microscopy, X-ray analysis, and infrared spectroscopy. The release of nitrate was monitored in different buffer mediums: buffer A as a simulated acidic soil solution and buffer B as a simulated neutral soil solution.
Results and discussion The stability of nitrate-LDH against thermal decomposition was evaluated by thermal gravimetric analysis. The nitrate-LDH supported a sustained controlled release process of nitrate during 16 days into acidic soil at 15 degrees C, while the release was continued to 20 days into neutral soil at the same temperature. The increase of soil temperature slightly enhanced the release of nitrate.
Conclusions We offered a potential management strategy of soil nitrogen leaching process. The nitrate form of layered double hydroxide material was used as a nitrogen fertilizer in order to monitor the release of nitrate anion into soil at different conditions.

DOI： 10.1007/s11368-013-0766-3

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Protein kinase (PK)-responsive gene carriers modified with polyethylene glycol (PEG) chains using an acid-labile linker were developed. These carriers were obtained by modifying the PEG chains and substrate peptides for the PKs (PKA or PKCα) on the branched polyethyleneimine main chain. Polyplexes formed from these carriers and plasmid DNA (pDNA) were stably dispersed under neutral pH medium. The polyplexes were also taken up by cells on the release of the PEG chains under the slightly acidic extracellular pH associated with cancer cells. The polyplexes taken up by cells resulted in gene expression when the substrate peptides were phosphorylated by the intracellular PKs to release pDNA from the polyplexes. These novel gene carriers are expected to be promising for cancer-specific gene therapy via intravenous administration. © 2013 National Taiwan University.

DOI： 10.4015/S101623721340005X

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Here, we synthesized dextrans modified with trivalent cationic or anionic groups. Aqueous solutions of the cationic and anionic dextrans were then mixed resulting in the formation of polyion complex nanogels (PIC-NGs), which have physically crosslinked salt bridges formed between the cationic and anionic groups. To prepare PIC-NGs in high yield, the content of ionic groups in the cationic and anionic dextrans should be low to avoid interparticle salt bridge formation. The structure of the PIC-NGs is easily affected by the ionic strength of the solution because of shielding of the charges in the ionic groups. However, the conversion of a small amount of the physical crosslinks to covalent crosslinks significantly improved the stability of the PIC-NGs. The covalent crosslinking also stabilized the PIC-NGs against pH change, which will destabilize the salt bridges. These results indicated that the conversion of the small amount of physical crosslinks to covalent crosslinks stabilized the other salt bridges.

DOI： 10.1016/j.jcis.2012.09.015

Language：English   Publishing type：Research paper (scientific journal)  

The management of irrigation water presents a great challenge for the agriculture field. In view of increasing soil water-holding capacity and increasing water-use efficiency, an efficient strategy of managing irrigation water based on formulating highly absorbent polymer-inorganic clay composite (polyacrylic acid-layered double hydroxide: PAA-LDH) was offered. The PAA-LDH composite was synthesized by an incorporation/in situ polymerization technique. Scanning electron microscopy, X-ray analysis and infrared spectroscopy were used to confirm the composite structure. The thermal gravimetric analysis was applied to investigate the polymer thermal stability after the composite formation. The irrigation experiments were conducted in a wooden soil box with a transparent plexiglas side by using a subsurface drip irrigation system. The X-ray patterns and infrared spectra confirmed the incorporation of acrylic acid monomer (AA) into the gallery of LDH. The SEM images emphasized the composite structure of PAA-LDH and indicated its ability to absorb and keep water. The stability of PAA was promoted against the thermal decomposition after the composite formation. The composite structure of PAA-LDH worked as water barrier and secondary water source during the irrigation process. The soil moisture distribution patterns were enhanced after the application of PAA-LDH composites as a soil conditioner. (C) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jhydrol.2012.08.051

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Gold nanorods that have an absorption band in the near-infrared region and a photothermal effect have been used as nanodevices for near-infrared imaging and thermal therapy. Choice of the optimal shape of gold nanorods which relates optical properties and in vivo biodistribution is important for their applications. In the present study, to investigate the relationship between the shape of gold nanorods and their biodistribution after intravenous injection, we first prepared two types of gold nanorods that had distinct aspect ratios but had the same volume, zeta potential, and PEG density on the gold surface. Biodistributions of the two types of gold nanorods after intravenous injection into tumor-bearing mice were then compared. Although a slight difference in accumulation in the spleen was observed, no significant difference was observed in the liver, lung, kidney, and tumors. These results suggest that biodistribution of the gold nanorods in the aspect ratio range of 1.7 to 5.0, diameter of 10 to 50 nm, and volume of approximately 4 × 103 nm3 was dependent mainly on surface characteristics, PEG density, and zeta potential.

DOI： 10.1186/1556-276X-7-565

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

In this work we designed a novel nano carrier, a linear polyethylenimine (LPEI)-peptide conjugate, for cancerspecific expression of transgenes. The conjugate was easily synthesized by using a click chemistry scheme orthogonal to the reactive side groups of the peptide, which is the substrate of protein kinase Cα (PKCα). Polyplexes of the conjugates with plasmid DNA (pDNA) were intact and stably dispersed even in the presence of cell lysate. Despite this stability, the polyplexes readily dissociated upon phosphorylation of the grafted peptides by PKCα. Because of its endosomal escape ability and adequate susceptibility to PKCα, the polyplexes
showed an all-or-none type response to PKCα activity in transgene expression in vitro. The polyplexes achieved cancer tissuespecific
transgene expression even for a tumor with a relatively low PKCα activity. Thus the LPEI−peptide conjugate has high potential as a nanocarrier for cancer-targeted gene therapy.

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Target delivery and controlled release of the chemopreventive drug sulindac that possesses low water solubility present a great challenge for its pharmaceutical industry. Here, we offered an advanced nanomatrix formulation system of sulindac based on layered double hydroxide materials. The X-ray analysis and infrared spectroscopy confirmed the incorporation of sulindac into the gallery of the layered double hydroxides. The incorporation ratios of sulindac were recorded to be 45, 31 and 20 for coprecipitation, anion-exchange and reconstruction techniques, respectively. The scanning electron microscopy showed a nanomatrix-structure of similar to 50 nm. The release studies of sulindac-nanomatrix showed a 96% controlled release at the small intestine solution during 3 h(s), indicating an enhancement in the dissolution profile of sulindac after the matrix formation. The layered structure of the matrix supplied sulindac with a well-ordered structure and a relatively hydrophobic microenvironment that controlled the guest hydrolysis and reactivity during the release process. The laminar structure of layered double hydroxides offered a safe preservation for sulindac against photodecarboxylation, and enhanced the drug thermal stability from 190 to 230A degrees C. The ionic electrostatic interaction of sulindac through its acidic group with layered double hydroxides demolished the gastrointestinal ulceration.

DOI： 10.1007/s10856-012-4566-x

Language：English  

Language：English  

Language：English  

Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Research paper (scientific journal)  

Protein kinases (PKs) play crucial roles in intracellular signal transduction and they are one of the representative targets of drug development because their hyper-activation is related to many diseases including cancer. Kinomics is a technique to evaluate the activities of whole cellular PKs and will be important for drug development and diagnosis for personalized medicine. Peptide microarray is one of the representative methods to evaluate kinomics. We have proposed several techniques to improve the detection of peptide microarrays and have achieved quantitative evaluation of cellular PKs based on both fluorescence and SPR detection. We have also succeeded in screening of highly reactive substrate peptides of target PKs by using our microarrays.

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

To investigate the correlation between the counts per minute (CPM) by radioactivity assay and the phosphorylation ratio by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, we prepared 136 peptide substrates. The correlation coefficient of phosphorylation ratios to CPM was 0.77 for all samples. However, the more the numbers of positively charged amino acids increased, the more the correlation coefficient increased. Although positively charged amino acids can have an effect on the correlation results, MALDI-TOF MS analysis is a useful means for monitoring phosphorylated peptide and protein kinase activity instead of radioactivity assays.

DOI： 10.1016/j.ab.2011.08.035

Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

Recently, we have proposed a new system of gene regulation called 'drug or gene delivery system responding to cellular signals' (D-RECS). In this system, transgene expression is activated in response to intracellular target protein kinases or proteases for safe, cell-specific gene delivery by using peptide-polymer conjugates. Here we applied this system to an intracellular Rho-associated coiled-coil kinase (ROCK) signal, which is activated abnormally in cardiovascular diseases. A ROCK responsive polymer consisting of neutral polymers in main chain and cationic ROCK substrate peptides in side chains was prepared and could form the complex with plasmid DNA. The complex was transferred into NIH3T3 cells with or without L-α-lysophosphatidic acid (LPA) that increases ROCK activity. At an N/P ratio of 2.0, a significant increase of the gene expression was identified in LPA-treated NIH3T3 cells, but was disappeared in NIH3T3 cells treated with ROCK specific inhibitor, Y-27632. These results suggest that the ROCK responsive polymer can regulate gene expression in response to ROCK activity.

DOI： 10.1016/j.jconrel.2011.05.002

Language：English   Publishing type：Research paper (scientific journal)  

We recently developed a novel tumor-targeted gene delivery system responding to hyperactivated intracellular signals. Polymeric carrier for gene delivery consists of hydrophilic neutral polymer as main chains and cationic peptide substrate for target enzyme as side chains, and was named polymer-peptide conjugate (PPC). Introduction of chondroitin sulfate (CS), which induces receptor-medicated endocytosis, into polymers mainly with a high cationic charge density such as polyethylenimine can increase tumor-targeted gene delivery. In the present study, we examined whether introduction of CS into PPC containing five cationic amino acids can increase gene expression in tumor cells. Size and zeta potential of plasmid DNA (pDNA)/PPC/CS complex were <200 nm and between -10 and -15 mV, respectively. In tumor cell experiments, pDNA/PPC/CS complex showed lower stability and gene regulation, compared with that of pDNA/PPC. Moreover, no difference in gene expression was identified between positive and negative polymer. These results were caused by fast disintegration of pDNA/PPC/CS complexes in the presence of serum. Thus, we suggest that introduction of negatively charged CS into polymers with a low charge density may lead to low stability and gene regulation of complexes.

DOI： 10.1186/1556-276X-6-532

Language：English   Publishing type：Research paper (scientific journal)  

Protein kinase (PK)-responsive nanoparticles (NPs) comprising a hydrophobically modified peptide substrate for PKs and a fluorescein-labeled polyanion (pA-F) were reported for monitoring PK activity via fluorescence intensity measurements. In this system, the formation of NPs by mixing lipopeptides and pA-Fs results in fluorescence quenching, while the quenched fluorescence recovered following dissociation of the NPs owing to the phosphorylation reaction of PKs. Eleven lipopeptides with different hydrophobic moieties (hydrocarbon and lithocholic acid) and four pA-Fs having main chains with differing flexibilities and fluorescein contents were synthesized and used to fabricate a series of twenty-four PK-responsive NP probes. The responses of the PK-responsive NP probes to PKs were evaluated to screen the most suitable NP probes. The assay system was then used to determine the IC(50) values for five inhibitors, the results of which were very similar to those previously reported. Thus, PK-responsive NPs are useful tools for high-throughput screening (HTS) of PK inhibitors.

DOI： 10.1021/bc200066w

Language：English   Publishing type：Research paper (scientific journal)  

In chromatin, gene transcription is regulated through posttranslational modifications on the histone N-terminal tail sequences, typically an acetyl group modification on lysine residues. To realize a simple model of the gene regulation of chromatin, we designed a hydrophilic polymer grafted with histone H3 tail peptides. The polyplex formed from the polymer and DNA suppressed the gene expression effectively although the polyplex was weaker than the polyplex of poly-L-lysine and DNA. This weaker polyplex afforded the acetylation of the lysine residue of the grafted peptides by histone acetyltransferase. Subsequently, the gene expression was activated due to the relaxation of the polyplex which was brought by a cationic charge decrease in the grafted peptides. This molecular system is the first functional model of the gene regulation of the chromatin.

DOI： 10.1016/j.bmc.2011.05.011

Language：English   Publishing type：Research paper (scientific journal)  

Language：English  

Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Gold nanorods have strong absorption bands in the near-infrared region, in which light penetrates deeply into tissues. The absorbed light energy is converted into heat by gold nanorods, the so-called 'photothermal effect'. Hence, gold nanorods are expected to act not only as on-demand thermal converters for photothermal therapy but also as controllers of a drug-release system responding to irradiation by near-infrared light. To achieve a controlled-release system that can be triggered by light irradiation, double-stranded DNA (dsDNA) was modified on gold nanorods. When the dsDNA-modified gold nanorods were irradiated by near-infrared light, the single-stranded DNA (ssDNA) was released from gold nanorods due to the photothermal effect. The amount of released ssDNA was dependent upon the power and exposure time of light irradiation. Release of ssDNA was also observed in tumors grown on mice after light irradiation. Such a controlled-release system of oligonucleotide triggered by the photothermal effect could expand the applications of gold nanorods that have unique optical characteristics in medicinal fields. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2011.02.042

Language：Others  

Oligonuleotide delivery with dendritic poly(L-lysine) for treatments of the liver disorders
A cationic peptide dendrier, dendritic poly(L-lysine), forms complexes with oligonucleotides and can deliver them to liver after intravenous injection. Here, we tried to deliver apolipoprotein B-specific siRNA for the treatment of hypercholesterolemia and NFκB decoy for the hepatitis treatment. Significant therapeutic effects in those disease model mice were observed after intravenous injection of the oligonucleotides complexes with dendritic poly(L-lysine). © 2010 Materials Research Society.

Language：English   Publishing type：Research paper (scientific journal)  

For safe and efficient gene therapy, the development of gene delivery systems to specifically target tumor cells is one of the most important issues regarding present gene delivery methodologies. Recently, we have developed a novel drug or gene delivery system responding to cellular signals (D-RECS) that can activate transgenes in response to hyperactivated cellular signals. Especially, a protein kinase C (PKC)α-responsive polymeric carrier (polymer-peptide conjugate, PPC(S)) showed highly specific gene expression to tumor cells and tissues. In the present study, we have applied the PKCα-responsive polymeric carrier to tumor gene therapy. PPC(S) consists of a polyacrylamide backbone and cationic peptide side chains, which together make PPC(S) as a positive polymer, a PKCα-specific substrate. A negative control polymer, PPC(A), was also prepared by replacing a serine residue at the phosphorylation site of the peptide side chains of PPC(S) with alanine. A complex of PPC(S) with caspase-8 or the herpes simplex virus-thymidine kinase (HSV-TK) gene as therapeutic genes was transfected into certain tumor cells or tissues. The prodrug ganciclovir (GCV) was then intraperitoneally injected into PPC(S)/HSV-TK complex-transfected mice. The PPC(S)/gene complex showed significant cytotoxicity toward the tumor cells and suppression of tumor growth, compared with those of the PPC(A)/gene complex or PBS. These results indicate that the PKCα-responsive polymeric carrier is applicable for tumor-targeted gene therapy.

DOI： 10.1016/j.jconrel.2010.08.017

Language：English  

DOI： 10.1016/j.jconrel.2010.07.027

Language：English  

We describe a powerful peptide microarray for profiling protein kinase substrates that combines the merits of chemoselective immobilization of peptides to achieve high density spots with the advantages of fluorescence-based analysis of phosphorylation for nonhazardous detection. For detection of on-chip phosphorylation, we used a fluorescence-labeled antiphosphotyrosine antibody to detect phosphotyrosine and a biotinylated Phostag, which was subsequently bound with a fluorescence-labeled streptavidin for phosphoserine/threonine. More than 290 kinds of Tyr peptides and over 1,100 kinds of Ser/Thr peptides were chemoselectively immobilized onto a glass surface in a high-density format to profile a panel of protein kinases, including c-Src, c-Abl, EGFR, JNK1, ERK2, p38α, and PKA. Many novel, highly reactive and specific peptides were identified as substrates for each protein kinase. Most substrates had the consensus motifs that have been reported previously but some new motifs were also found. The identification of two designed peptides that have higher reactivity than the famous PKA substrate (Kemptide) indicates that analysis of the amino acid biases of substrates is very helpful to the design of new substrates with high reactivity. Thus, the high-density peptide microarray is expected to be a powerful approach for high-throughput discovery of potential substrates for protein kinases.

Language：English   Publishing type：Research paper (scientific journal)  

To design a nanocomposite formulation system of lipid-regulating drugs with versatile approaches using layered double hydroxide (LDH) material.
The co-precipitation technique has been used to prepare the selected drugs [bezafibrate (BZF) and clofibric acid (CF)]-LDH nanocomposites. The nanocomposite materials (BZF-LDH and CF-LDH) were characterized by X-ray powder diffraction, infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy. The in vitro study was investigated in simulated gastrointestinal solutions at 36.8A degrees C.
X-ray measurement and spectroscopic analysis indicated the formation of fully monophase drug-nanocomposites. The nanocomposites' gallery heights were calculated to be 23.5 and 16.3 for BZF and CF, respectively. The new gallery heights indicated that BZF and CF drugs have been stacked into LDH as a monolayer with a staggered inter-digitated arrangement. The size of the nanocomposites described by SEM microscopy was similar to aEuro parts per thousand 0.1 mu m. The nanocomposite formulation has improved the drugs properties (thermal stability, dissolution, and controlled release) beside the achievement of drug target delivery.
Nanocomposites composed of lipid-regulating drugs (BZF and CF) with LDH were successfully synthesized as a new formulation system of this drug category. The LDH nanocomposite formulation system has improved the drugs release properties.

DOI： 10.1007/s11095-010-0175-x

Language：English  

Gold nanorods, rod-shaped gold nanoparticles, have strong absorbance in the near-infrared region, and the absorbed light energy can be converted to heat, the so-called photothermal effect. The gold nanorods were coated with thermoresponsive polymers, which have different phase transition temperatures that were controlled by adding comonomers, N,N-dimethylacrylamide (DMAA) or acrylamide (AAm) to N-isopropylacrylamide (NIPAM). The phase transition temperatures of poly(NIPAM-DMAA) and poly(NIPAM-AAm)-coated gold nanorods were 38 and 41 °C, respectively, while polyNIPAM-coated gold nanorods showed phase transition at 34 °C. Irradiation of the coated gold nanorods using the near-infrared laser induced a decrease in their sizes due to a phase transition of the polymer layers. Poly(NIPAM-AAm)-coated gold nanorods stably circulated in the blood flow without a phase transition after intravenous injection. Irradiation of near-infrared light at a tumor after the injection resulted in the gold specifically accumulating in the tumor. This novel accumulation technique which combines a thermoresponsive polymer and the photothermal effect of the gold nanorods should be a powerful tool for targeted delivery in response to light irradiation.

DOI： 10.1021/bc100284s

Language：Others  

High-throughput detection of protein kinase activities in cell lysate based on the aggregation of gold nanoparticles with peptides
Cationic peptides were used as coagulant of gold nano-particle (GNP, 20 nm) for the monitoring cellular protein kinase activities. If cationic peptides, which are designed as specific substrates to target protein kinases, were added into the GNP dispersion, the color of the dispersion changed from red to blue due to the aggregation. On the other hand, if the peptide was phosphorylated with target protein kinase, addition of the phosphorylated peptide didn&#039;t change the color of the GNP dispersion. Thus, this system can be a rapid assay of protein kinase activity. The method was applicable to the assay of intracellular protein kinase by using cell lysate. In addition, we applied the system for high-throughput screening of protein kinase inhibitor using 1300 kinds of chemical library. The result indicated that the method was also useful for the drug discovery. © 2010 Materials Research Society.

Language：English   Publishing type：Research paper (scientific journal)  

To offer a multifunctional and applicable system of the high-value biotechnological phytohormone gibberellic acid (GA), a nanohybrid system of GA using the inorganic Mg-Al layered double-hydroxide material (LDH) was formulated. The ion-exchange technique of LDH was applied to synthesize the GA-LDH hybrid. The hybrid structure of GA LDH was confirmed by different spectroscopic techniques. The nanohybrid size was described by SEM to be similar to 0.1 mu m. The GA-LDH nanohybrid structure was the key parameter that controlled GA properties. The layered molecular structure of LDH limited the interaction of GA molecules in two-dimensional directions. Accordingly, GA molecules did not crystallize and were released in an amorphous form suitable for dissolution. At various simulated soil solutions, the nanohybrids showed a sustained release process following Higuchi kinetics. The biodegradation process of the intercalated GA showed an extended period of soil preservation as well as a slow rate of degradation.

DOI： 10.1021/jf102501n

Language：English   Publishing type：Research paper (scientific journal)  

Hepatoma (hepatocellular carcinoma) is the most common type of malignant tumor originating in the liver and has a relatively low 5-year survival rate. The development of hepatoma-targeted therapy is needed to increase treatment efficiency and to reduce the incidence of undesirable side effects. In this study we developed a novel hepatoma-targeted gene delivery system. The gene delivery system was prepared by combining a human liver cell-specific bionanocapsule (BNC) and a tumor cell-specific gene regulation polymer, which responds to hyperactivated protein kinase C alpha in hepatoma cells. The complex of the polymer-DNA with BNCs was delivered into cells and tissues. The developed system showed increased transfection efficiency and resulted in cell-specific gene expression in hepatoma cells and tissues (HuH-7), but no gene expression in normal human hepatocytes or human epidermoid tumor cells (A431). The combination of a tumor cell-specific gene regulation system responding to protein kinase C alpha and BNCs showed excellent potential for the selective treatment of hepatomas. The system could be a useful method with applications in hepatoma-specific gene therapy and molecular imaging. From the clinical editor: Hepatocellular carcinoma is the most common type of malignant tumor in the liver with a low 5-year survival rate. In this study, a novel hepatoma-targeted gene delivery system was prepared by combining a human liver cell-specific bionanocapsule and a tumor cell-specific gene regulation polymer, which responds to hyperactivated protein kinase C (PKC)a in hepatoma cells. The system could be a useful in hepatoma-specific gene therapy and molecular imaging.

DOI： 10.1016/j.nano.2010.01.007

Language：English   Publishing type：Research paper (scientific journal)  

Recently, our group has proposed a novel gene-regulation system responding to cAMP-dependent protein kinase (PKA) that has been applied to living cells. In this study, human liver-specific bionanocapsules (BNCs) are used as a gene-delivery system to increase transfection efficiency and to target specific cell types. BNCs can efficiently deliver a target gene to human hepatocytes and hepatoma cells in vitro or in vivo. The combination of a signal-responsive gene-delivery system with BNCs led to an increase in the transfection efficiency and selectivity for hepatoma cells. Expression from the delivered gene was identified from PKA-activated hepatoma cells (HepG2), but not from colon tumor cells (WiDr). These results show that the combination of a gene-regulation system responding to an intracellular signal with BNC can be used for the selective treatment of human hepatoma cells.

DOI： 10.1016/j.ijpharm.2010.06.012

Language：English   Publishing type：Research paper (scientific journal)  

We reported here the full characterization of the hysteresis of the phase transition behavior of an aqueous solution of poly(N-ethyl-2-propionamidoacrylamide) (PNEPA), which has a unique alpha,alpha-disubstituted structure, by using microcalorimetry and FT-IR. Phase transition temperatures near the thermodynamic equilibrium were determined by extrapolating the scanning rate of the microcalorimetry to zero. The calculated hysteresis from the phase transition temperature was unusually very large (similar to 8 degrees C). FT-IR analysis indicated that the large hysteresis of PNEPA resulted from a coupling of intra-/intermonomeric unit hydrogen bonds, which is known to occur in a beta-sheet of proteins but has never been reported in temperature-responsive polymers. The effects of the molecular weight and polymer concentration on the hysteresis were studied by using fractionated PNEPAs and it was found that a low molecular weight and a low concentration enhanced the hysteresis.

DOI： 10.1021/jp102681q

Language：English  

Three temperature-responsive polymers which are alpha,alpha-disubstituted vinyl polymers having two amphiphilic groups (ethylamide or ethylester) per monomeric unit were designed. Two of these polymers showed unusually large hysteresis in their phase transition temperatures between a heating and a cooling process. This hysteresis resulted from the extremely slow kinetics of the dissolution process of the aggregated polymer chains in the cooling process due to intra- and interchain interactions including hydrogen bonding and hydrophobic interaction. The high density of the amphiphilic substituents on the polymer chain due to the alpha,alpha-disubstituted structure enhanced these intra- and interchain interactions. The large hysteresis was also observed in the volume change of a corresponding hydrogel. These new classes of temperature-responsive polymers are interesting materials because their large hystereses can be regarded as erasable memory function.

DOI： 10.1021/la100020t

Language：English  

Gold nanorods exhibit strong absorbance of light in the near infrared region, which penetrates deeply into tissues. Since the absorbed light energy is converted into heat, gold nanorods are expected to act as a contrast agent for in vivo bioimaging and as a thermal converter for photothermal therapy. To construct a gold nanorod targeted delivery system for tumor a peptide substrate for urokinase-type plasminogen activator (uPA), expressed specifically on malignant tumors, was inserted between the PEG chain and the surface of the gold nanorods. In other words, we constructed PEG-peptide-modified gold nanorods. After mixing the gold nanorods with uPA, the PEG chain was released from the surface of the gold and subsequently nanorod aggregation took place. The formation of the aggregation was monitored as a decrease in light absorption at 900 nm. Tumor homogenate induced a significant decrease in this absorption. Larger amount of the PEG-peptide-modified gold nanorods bound to cells expressing uPA in vitro compared with control gold nanorods, which had scrambled sequence of the peptide. The PEG-peptide-modified gold nanorods showed higher accumulation in tumor than the control after they were injected intravenously into tumor-bearing mice, however, the density of the peptide on the surface of the gold nanorods was a key factor of their biodistributions. This targeted delivery system, which responds to uPA activity, is expected to be a powerful tool for tumor bioimaging and photothermal tumor therapy.

DOI： 10.1016/j.bmc.2010.04.070

DOI： 114

Language：English  

A novel gold nanoparticle (GNP)-based colorimetric assay was developed for cancer diagnosis. This system is based on the noncrosslinking aggregation mechanism with a cationic protein kinase C (PKC) alpha-specific peptide substrate, which is used as a coagulant of citrate-coated GNP with anionic surface charges. The phosphorylation of peptide substrates by PKCalpha suppressed GNP aggregation, resulting in a red color, but in the case of non-phosphorylation, the color of the GNP solution changed from red to blue, indicating particle aggregation. Moreover, a correlation between the color change of the GNP dispersions and the level of activated PKCalpha was identified from experiments using cancer cell lines, or xenografted mouse cancer and normal mouse tissues. When our system was applied to human breast cancers and normal human breast tissues, cancer tissue lysates became red in color, indicating GNP dispersion, while all lysates from normal tissue turned the GNP solution blue. MALDI-TOF MS analysis and Western blotting experiment confirmed that these different results between cancer and normal tissues reflected the difference in PKCalpha activity. This study is the first report on the application of the GNP-based colorimetric assay to the diagnosis of cancer.

DOI： 10.1016/j.bios.2009.12.022

Language：English  

Gene therapy is a promising strategy for the treatment of HIV infection, but cell specificity remains an issue. Recently we have developed a new concept for a drug or gene delivery system responding to cellular signals (D-RECS) to achieve cell-specific transgene expression using a non-viral polymer-based vehicle. According to this concept, intracellular signaling enzymes, which are activated specifically in target cells, are used to trigger transgene expression. We previously applied this concept to HIV-1 protease and showed that the recombinant protease could act as a suitable signal. Here we further developed this system to achieve highly specific transgene expression in HIV-infected cells. We prepared a polymeric gene regulator grafted with a cationic peptide containing the HIV-Tat peptide via a specific substrate for HIV-1 protease. The regulator formed a stable polyplex with the transgene, suppressing its transcription. HIV-1 protease cleaved the peptide and released the transgene, which was consequently expressed specifically in activated HIV-infected cells, but remained unreleased and inactive in uninfected cells. The validity of this approach was further confirmed by applying it to the CVB1 2A protease of coxsackievirus (Picornaviridae family). This strategy should be widely applicable for specific expression of a variety of therapeutic genes in virus-infected cells.

DOI： 10.1016/j.jconrel.2009.08.025

Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

Recently, there is a growing interest in the intracellular signal-targeting gene therapy or diagnosis, mainly by using the reaction of targeting enzymes with peptide substrates. In the present study, we proved the importance of target intracellular signal-specificity peptide substrate for intracellular signals-targeting gene therapy or diagnosis. Protein kinase C (PKC) was used as a trigger to activate the transgene expression. Two peptides, a positive peptide showing phosphorylation levels on several PKC isozymes (PKCalpha, betaII, gamma, epsilon, eta, zeta, and iota/lambda) and a negative peptide in which the phosphorylation site was destroyed by changing from serine to alanine, were designed. Moreover, two polymers possessing each peptide as a pendant chain, a PKC-responsive conjugate [PPC(S)] and a negative control conjugate [PPC(A)], were synthesized. After the introduction of complexes into cells or tissues, gene expression for PPC(S)/DNA complexes was higher that for PPC(A)/DNA complexes. However, no difference in gene expression between B16 melanoma tumors and normal skin tissues was identified. These results suggest that a peptide substrate specific to a target intracellular signal is very important for intracellular signals-targeting gene therapy or diagnosis.

DOI： 10.1016/j.bmcl.2009.09.034

Publishing type：Research paper (scientific journal)  

Language：English  

Gold nanorods have strong absorbance in the near infrared region, which penetrates deeply into tissues, where the absorbed light energy is converted into heat. Therefore, gold nanorods are expected to act as an effective contrast agent for in vivo bioimaging and as a thermal converter for photothermal therapy. We grafted various amounts of polyethylene glycol (PEG) onto the surface of gold nanorods and investigated the effects of grafting level and injection dose on the biodistribution in the tumor-bearing mice after intravenous injection and enhanced permeability and retention (EPR). Higher PEG grafting levels were advantageous for reticuloendothelial system (RES) avoidance and for suppression of aggregation of the gold nanorods in the circulation. Modification with a PEG:gold molar ratio of 1.5 was sufficient to show both prolonged circulation and the EPR effect. When the injection dose was increased above 19.5 microg of gold, the RES uptake in the liver was saturated and surplus gold nanorods were distributed to other tissues, especially the spleen and the tumor. The results of this study will provide an important basis for the development of cancer therapies mediated by the photothermal effect of gold nanorods.

DOI： 10.1016/j.jconrel.2009.06.006

Language：English   Publishing type：Research paper (scientific journal)  

We recently proposed a novel gene regulation system responding to specifically and abnormally activated intracellular enzymes in diseased cells. In the present study, we focused on protein kinase C (PKC)alpha, which is hyper-activated in most tumor cells, as a trigger for transgene regulation. We prepared cationic copolymers comprising hydrophilic and neutral polymers in main chains and cationic peptide substrates with different contents in side chains. Our copolymer with high peptide content (>3 mol%) condensed with pDNA more weakly than with poly(L-lysine) (pLL) having a similar molecular weight, but gene suppression was nearly identical to that of pLL, probably due to the steric hindrance of the main chains in our copolymer. Steric hindrance of the main chains barely affected the phosphorylation reaction of the pendant peptide. In cell and mouse experiments, higher gene expression was observed in complexes of pDNA with copolymers pended PKC alpha-specific substrate peptide than that in complexes with negative copolymers pended peptide substituted phosphorylation site of serine residues with alanine. These results indicate that our system can recognize intracellular PKC alpha as a trigger to regulate transgene expression, and may be useful for tumor gene therapy.

DOI： 10.1016/j.jconrel.2009.06.011

Language：English   Publishing type：Research paper (scientific journal)  

We describe a label-free method for the kinase inhibition assay toward discovery of kinase inhibitors. The surface plasmon resonance (SPR) imaging analysis using zinc(II) compound was adopted on the on-chip phosphorylation analysis. In this study, following three subjects were focused: (1) to monitor the inhibition of three inhibitors supporting by their specific inhibition mechanisms, (2) to quantify the inhibitory activities, and (3) to prove the reliability of the obtained 50% inhibition concentration (IC(50)) value. First, the inhibitory activities of Amide 5-24, H-89 and Gö6983 on PKA and PKCdelta were determined, and specific inhibitions for two kinases could be observed quantitatively. Second, the inhibition curves of Amide 5-24, Amide 14-22 and H-89 were obtained, and the results supported the two previous reports: (1) the inhibition efficiency of Amide 5-24 was much higher than that of Amide 14-22, and (2) the inhibitory activity of H-89 followed ATP-binding site blocking mechanism. Last, the obtained IC(50) values by the SPR imaging were almost corresponded to those by the solution assay, although on-chip phosphorylation efficiency was low (approximately 12%). In conclusion, validation of the on-chip phosphorylation analysis for kinase inhibitors was achieved. This label-free method might be applied for discovery of kinase inhibitors.

DOI： 10.1016/j.biosystems.2009.04.007

Language：English  

BACKGROUND: Control of inflammation is essential for the clinical management of many common human diseases. However, there are few generally applicable strategies to convert an abnormal intracellular signal into a gene expression that leads to normalization of the intracellular environment. Recently, we proposed a novel strategy termed D-RECS (i.e. drug or gene delivery system responding to cellular signals) to convert an intracellular signal to transgene expression. In the present study, we applied this concept to inflammatory cells using Ikappa-B kinase as a signal molecule that triggers the gene expression. METHODS: Candidate cationic substrates of Ikappa-B kinase (IKK)beta were synthesized and their reactivity was investigated. Then, polymers grafted with these peptides were prepared by radical polymerization. Polymer/DNA complexes (polyplexes) were prepared by mixing plasmid DNAs with the polymers. The behaviour of these polyplexes by adding IKKbeta was examined. Furthermore, changes of gene expression were evaluated after the microinjection of polyplex into living cells under conditions of nuclear factor (NF)-kappaB activation. RESULTS: Synthetic peptides with additional lysine residues were well phosphorylated by IKKbeta. Both the polymer and the polyplex were also phosphorylated by IKKbeta. The results of gel shift assay showed that the polyplex was disintegrated and free DNA was released in the presence of IKKbeta. The polyplex comprising-green fluorescent protein plasmid DNA and the polymer expressed the transgene in living cells exposed to a pro-inflammatory stimulus. CONCLUSIONS: Our concept of cell-specific gene expression was demonstrated to work in inflammatory cells. This method may provide a unique strategy for gene therapy exclusively in inflammatory cells.

DOI： 10.1002/jgm.1342

Language：English  

Language：English  

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

DOI： 10.1016/j.bbrc.2009.03.129

Language：English  

Language：English  

Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

The fabrication of peptide microarrays using hyperbranched polymers (HBPs) to improve the signal-to-noise ratio was demonstrated. Due to a high density of reactive groups at the chain ends of the HBPs, as well as to their spherical shape, HBPs can be used as linkers to increase the amount of immobilized peptides through raising the specific surface area of the glass substrate. A zwitterionic HBP was used as a blocking agent to reduce the noise level of the peptide microarrays. The zwitterionic HBP shows comparably excellent blocking ability to a commercially available BSA-based blocking agent. Thus, it was concluded that HBPs have high potential for the fabrication of highly sensitive peptide microarrays.

DOI： 10.1063/1.3116124

Language：English   Publishing type：Research paper (scientific journal)  

Radical polymerization of N-isopropylacrylamide (NIPAAm) in toluene at low temperatures, in the presence of fluorinated-alcohols, produced heterotactic polymer comprising an alternating sequence of meso and racemo dyads. The heterotacticity reached 70% in triads when polymerization was carried out at -40 degrees C using nonafluoro-tert-butanol as the added alcohol. NMR analysis revealed that formation of a 1:1 complex of NIPAAm and fluorinated-alcohol through C=O center dot center dot center dot H-O hydrogen bonding induces the heterotactic specificity. A mechanism for the heterotactic-specific polymerization is proposed. Examination of the phase transition behavior of aqueous solutions of heterotactic poly(NIPAAm) revealed that the hysteresis of the phase transition between the heating and cooling cycles depended on the average length of meso dyads in poly(NIPAAm). (C) 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 2539-2550, 2009

DOI： 10.1002/pola.23338

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Language：English  

Monitoring and targeting protein kinases is widely accepted as a promising approach for disease diagnosis and drug discovery. For this purpose, the authors have developed an original type of peptide array as a high-throughput screening assay for quantitatively evaluating kinase activity. A volume of 2 nL of peptide solution was spotted onto a formyl group-modified glass slide by using an arrayer, which was designed for use with protein chip technology. The phosphorylation was recognized by fluorescence-label antibody and detected with an automatic microarray scanner widely used in DNA chip technology. The system needs low sample volume, provides a high-density peptide array, and supplies high reproducibility. It provided enough sensitivity for inhibitor screening, even though a relatively low concentration of purified kinase was employed. The assay also proved useful for the detection of intracellular kinase activity as well as for the measurement of the fluctuations of intracellular protein kinase activity with drug stimulation. Thus, this peptide array would be applicable for kinase-targeted diagnosis, cell-based drug screening, and signal pathway investigation.

DOI： 10.1177/1087057108329348

Language：English  

Gold nanorods can be used as photothermal converters, permitting near-infrared (NIR) light to be transmitted deep into tissues without causing damage. We prepared hybrid nanorods with a core-shell structure, i.e., a single gold nanorod encapsulated in a poly (N-isopropylacrylamide) nanogel. Hybrid nanorods demonstrated remote, reversible, pulsatile phase transition and in vivo action after irradiation using a NIR laser.

DOI： 10.1021/bc800480k

Publishing type：Research paper (scientific journal)  

Language：English  

Syndiotactic poly(N-n-propylacrylamide)s (PNNPAM) with various racemo (r) diad contents were synthesized, and their phase transition behaviors were studied by high-sensitivity differential scanning calorimetry and FT-IR spectroscopy. The cooperativity of the phase transition increased with the increase in the r diad content. Compared with the atactic PNNPAM, the number of cooperative units, which shows the monomeric units length undergoing cooperative phase transition, increased 2.5- to 4-fold in the syndiotactic polymers. From the results of FT-IR spectroscopy and quantum chemical calculations, it was determined that the high cooperativity of the syndiotactic polymers resulted from local formation of an ordered structure in a dehydrated state.

DOI： 10.1021/la8034837

Language：English  

Hsp16.5, a small heat-shock protein (sHSP) from hyperthermophilic archaeon, forms a homogeneous complex comprised of 24 subunits with a molecular mass of 400 kDa. This complex self-organizes under physiological conditions, and the structure of the complex is a nanoscale spherical capsule with small pores. Furthermore, this natural nanocapsule exhibits very high thermal stability. In this paper, we functionalized the nanocapsule to control the structure in response to external stimuli such as a protease signal and temperature. For this purpose, several mutations (Mut1-10) to create a cleavage site for a specific protease, Factor Xa, were introduced on the outer surface of the nanocapsule using a genetic engineering strategy. The resulting mutants were expressed to high levels in Escherichia coli. One of these mutants, Mut6, which has the most accessible cleavage site located at the triangular pore on the surface of the capsule, formed a spherical assembly similar to that observed for the wild-type protein. Mut6 showed the highest sensitivity to Factor Xa, and the structure of the protease digested Mut6 disassembled irreversibly after heating. In contrast, the nanocapsule comprising the wild-type Hsp16.5 was not influenced by the dual stimuli. These results suggest that Mut6 acts as a stimulus-responsive nanocapsule. Such a characteristic of the protein-based nanocapsule has attractive potential as a versatile intelligent system.

DOI： 10.1016/j.bmc.2008.11.013

Language：English   Publishing type：Research paper (scientific journal)  

We succeeded in cancer cell specific gene expression by using a polyplex responsive to protein kinase Calpha, which is activated in various types of cancer cells.

DOI： 10.1021/ja805364s

Language：English  

Naproxen (NP) and flurpibrofen (FB) as non-steroidal anti-inflammatory drugs (NSAIDs) of 2-arylpropionic acid derivatives have been used as host organic drugs to be intercalated into layered double hydroxide (LDH) applying reconstruction and co-precipitation techniques. The obtained NP-LDH and FB-LDH nanocomposites were characterized by X-ray powder diffraction, infrared and thermogravimetric analyses. From drug loading, thermal analysis and X-ray measurements we can decide that coprecipitaion technique is better than reconstruction technique to obtain intercalated monophase nanocomposites. In acidic medium LDH dissolved and the intercalated drug starts to release in a molecular form which is suitable for adsorption. The drug solubility has been investigated before and after intercalation. It has been found that LDH improves the drug solubility and its dissolution rate.

DOI： 10.1016/j.ejps.2008.08.006

Language：English   Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

The radical polymerizations of N-alkylacrylamides, such as N-methyl-(NMAAm), N-n-propyl-(NNPAAm), N-benzyl-(NBnAAm), and N-(1-phenylethyl)acrylamides (NPhEAAm), at low temperatures were investigated in the absence or presence of hexamethylphosphoramide (HMPA) and 3-methyl-3-pentanol (3Me3PenOH), which induced the syndiotactic specificities in the radical polymerization of N-isopropylacrylamide (NIPAAm). In the absence of the syndiotactic-specificity inducers, the syndiotacticities of the obtained polymers gradually increased as the bulkiness of the N-substituents increased. Both HMPA and 3Me3PenOH induced the syndiotactic specificities in the NNPAAm polymerizations as well as in the NIPAAm polymerizations. The addition of 3Me3PenOH into the polymerizations of NMAAm significantly induced the syndiotactic specificities, whereas the tacticities of the obtained polymers were hardly affected by adding HMPA. In the polymerizations of bulkier monomers, such as NBnAAm and NPhEAAm, HMPA worked as the syndiotactic specificity inducer at higher temperatures, whereas 3Me3PenOH hardly influenced the stereospecificity, regardless of the temperatures. The phase-transition behaviors of the aqueous solutions of poly(NNPAAm)s were also investigated. It appeared that the poly (NNPAAm) with racemo dyad content of 70% exhibited unusual large hysteresis between the heating and cooling processes. (C) 2008 Wiley Periodicals, Inc.

DOI： 10.1002/pola.22797

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

The purpose of this study was to find protein kinase C (PKC) isozyme-specific peptides. A peptide library containing 1772 sequences was designed using Scansite and screened by MALDI-TOF MS and kinase activity assays for PKC isozyme-specificity. A peptide (Alphatomega; H-FKKQGSFAKKK-NH(2)) with high specificity for PKC alpha relative to other isozymes was identified. The peptide was phosphorylated to a greater extent by tissue lysates from B16 melanoma, HepG2, and human breast cancer, which had higher levels of activated PKC alpha, when compared to normal skin, liver, and human breast tissue lysates, respectively. Moreover, addition of Ro-31-7549, an inhibitor with great specificity for PKC alpha, to the phosphorylation reaction caused a dose-dependent reduction in phosphorylation, but no inhibition was identified with the addition of rottlerin and H-89. These results show that this peptide has great potential as a PKC alpha-specific substrate.

DOI： 10.1002/pmic.200701045

Language：English   Publishing type：Research paper (scientific journal)  

Language：English  

We developed a peptide microarray based on surface plasmon resonance (SPR) imaging for monitoring protein kinase activities in cell lysates. The substrate peptides of kinases were tethered to the microarray surface modified with a self-assembled monolayer of an alkanethiol with triethylene glycol terminus to create a low nonspecific binding surface. The phosphorylation of the substrate peptides immobilized on the surface was detected with the following phosphate specific binders by amplifying SPR signals: anti-phosphotyrosine antibody for tyrosine kinases and Phos-tag biotin (a phosphate-specific ligand with biotin tag) for serine/threonine kinases. Using the microarray, 9 kinds of protein kinases were evaluated as a pattern of phosphorylation of 26 kinds of substrate peptides. The pattern was unique for each protein kinase. The microarray could be used to evaluate the inhibitory activities of kinase inhibitors. The microarray was applied successfully for kinase activity monitoring of cell lysates. The chemical stimuli responsive activity changes of protein kinases in cell lysates could also be monitored by the peptide microarray. Thus, the peptide microarray based on SPR imaging would be applicable to cell-based drug discovery, diagnosis using tissue lysates, and biochemical studies to reveal signal transduction pathways.

DOI： 10.1016/j.ab.2007.12.011

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese  

Protein Kinase Assay Based on Aggregation of Gold Nanoparticles

DOI： 10.1295/kobunshi.56.841

Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English  

Little is known on mechanisms involved in transport of complex of DNA and gene carrier molecules from the cytosol to the nucleus. We aimed to identify cytosolic proteins interacting with the polyethylenimine (PEI)/DNA complex, using 2-D gel electrophoresis and peptide mass fingerprinting. Fifteen proteins including actin, beta-tubulin, and other metabolic proteins were identified. They demonstrated various molecular weights and isoelectric points, and were categorized into 3 groups: early binding, late binding, and transient binding proteins. Protein binding caused DNA release from the PEI/DNA complex with a cation/anion (C/A) ratio of 2, where complex formation was weak. Knowledge on interactions between cytosolic proteins and DNA/carrier complexes will help understand intracellular gene delivery mechanisms.

DOI： 10.1016/j.jconrel.2006.12.027

Language：English   Publishing type：Research paper (scientific journal)  

In this study, we prepared gold nanorod (NR)-embedded N-isopropylacrylamide (NIPAM) hydrogels and studied their volume phase transition behavior induced by near-infrared (near-IR) laser irradiation utilizing the photothermal conversion characteristics of the NRs. When poly(ethylene glycol)-modified NRs were used for the preparation of composite gels, the NRs showed marked dispersion stability in the gel. Near-IR laser irradiation of the gel (cylindrical shape, diameter = 140 microm) under the following conditions, NR concentrations in the gel > or =100 microM and laser irradiation power > or =490 mW, resulted in shrinkage of the gel in the following manner: (1) waist formation around the irradiation spot and (2) growth of the waist along the axial directions of the gel. The gel shrinking induced by near-IR irradiation occurred much more rapidly than that afforded by a temperature jump, because the former was not accompanied by the skin layer formation, which disturbs the rapid shrinking of the gels. When a composite gel containing the model drug (rhodamine-labeled dextran) was irradiated with a near-IR laser, the rapid release of the drug was observed. Taking advantage of the high spatial resolution of the irradiation point, we further achieved the irradiation-point-specific release of the drug from one such gel.

DOI： 10.1021/la0627967

Language：English  

DNA microarray enables the analysis of DNA or mRNA expression levels, but it has not been possible to completely understand life using obtained information. Consequently, protein or peptide arrays have attracted much interest. Since the development of a practical protein microarray is still far away in light of handling difficulties, the peptide microarray is a promising tool for analyzing protein functions. We have developed a peptide microarray to detect protein kinase activity in cell lysate. All substrate peptides for kinases were immobilized chemoselectively on amino-coated glass slides. After phosphorylation of the immobilized peptides, phosphorylation was detected by fluorescence imaging. We detected the protein kinase activities, including that in cell lysate, in response to drug stimulation. Therefore, this peptide microarray would be useful for a high-throughput kinase assay of intracellular signals and would be applicable to drug screening.

DOI： 10.2116/analsci.23.271

Language：English   Publishing type：Research paper (scientific journal)  

The temperature-responsive poly(dehydroalanine derivative)s having two substituents at the alpha-position were successfully synthesized. By varying the combination of the structures of the two substituents, the relatively large diversity of their phase transition temperatures could be achieved.

DOI： 10.1246/cl.2007.334

Language：English  

A nonamer peptide containing a diene group in the center of the sequence was synthesized. When the peptide forms an antiparallel beta-sheet, the diene groups align ca. 5 A apart on the beta-sheet. The diene groups successfully photopolymerized without distorting the beta-sheet structure. The obtained beta-sheet showed high stability against acid denaturation and addition of 1,1,1,3,3,3-hexafluoroisopropanol.

DOI： 10.1021/bm060835n

Language：English   Publishing type：Research paper (scientific journal)  

This study aimed to investigate the relationships between structures of gene carrier molecules and their activities for gene delivery into cells. We compared 2 types of poly(L-lysine) as carriers, that is, dendritic poly(L-lysine) (KG6) and linear poly(L-lysine) (PLL). KG6 formed a neutral DNA complex, and its DNA compaction level was weaker than that of PLL. The amount of DNA binding and uptake into cells mediated by PLL was 4-fold higher than that with KG6. However, KG6-mediated gene expression was 100-fold higher than that by PLL. Since pK(a) values of terminal amines of KG6 were lowered even though small amounts of DNA were internalized into cells, sufficient DNA amounts for effective gene expression escaped to the cytosol due to the proton sponge effect in the endosome. In addition, weakly compacted DNA with KG6 was advantageous in accessing RNA polymerase in the cell nucleus. On the other hand, PLL did not show the proton sponge effect in the endosome and resulted in strong compaction of DNA. Even though large DNA amounts were internalized into cells, most of the DNA would not take part in gene expression systems in the nucleus. Amount of induced cytokine production after intravenous injection of DNA complexes with KG6 and PLL was low, and was similar to the case when DNA was injected alone. Therefore, no significant difference in effects on cytokine production was observed between KG6 and PLL. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2006.09.033

Language：English   Publishing type：Research paper (scientific journal)  

A novel mass spectrometry-based assay system for determining protein kinase activity employing mass-tagged substrate peptide probes was used for the diagnosis of tumors. Two peptide probes (H-type and D-type) were synthesized containing the same substrate peptide sequence for protein kinase C (PKC). The molecular weights of the two probes differ because of the incorporation of deuterium into the acetyl groups of the D-type probe. The lysates of the normal and tumor tissue were prepared and reacted with the H- and D-type peptide probes, respectively. The PKC activities of the normal and tumor tissues can be compared simply and directly by calculating the phosphorylated ratio to each peptide probe, obtained from the peak intensity of the mass spectrum after mixing of the two reaction solutions. The phosphorylation ratio for the reaction of the H-type peptide probe with the tumor tissue lysate (B16 melanoma) was more than three times higher than that of the D type peptide probe with the normal skin tissue lysate. These results show that the novel assay system for detecting protein kinase activity using mass-tag technology can be a simple and useful means to profile protein kinase activity for cell or tissue lysate samples, and can be applied to the diagnosis of tumors.

DOI： 10.1016/j.jasms.2006.09.004

Language：English  

We have previously reported artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) using a cationic polymer. However, this polymer alone cannot deliver any gene into living cells. In the present work, we modified the signal-responsive polymer to the RGD peptide for the introduction of a polymer/DNA complex into living cells and succeeded in regulating the gene expression responding to intracellular PKA activation.

DOI： 10.1016/j.bmcl.2006.08.096

Language：English   Publishing type：Research paper (scientific journal)  

In the parallel and anti-parallel beta-sheet structures, hydrogen bonding arises between the amide bonds of the peptide chains to arrange them with a distance of ca. 5 angstrom. That distance matched with the repeating unit distance of polydiacetylene. In this study, the effectiveness of the beta-sheet as a template for the polymerization of diacetylene was examined by using diacetylene-introduced oligopeptides. The diacetylene-introduced amino acid (Thr(DA)) was synthesized from L-threonine. Though peptides Ac-Thr(DA)-NHMe and Ac-[Thr(DA)](2)-NHMe formed anti-parallel beta-sheet, they showed slight or no polymerization in both of the solid and the solution states. On the other hand, Ac-[Thr(DA)](5)-NHMe and 11mer peptide with a Thr(DA) in the center of the sequence contained anti-parallel beta-sheet structure and formed polydiacetylene of high degree of polymerization with high conversion during the cleavage process of the peptide from resin in the solution. This result indicated that the preorganization of the peptide through the beta-sheet formation was necessary for the polymerization of diacetylene group. Thus, the beta-sheet motif was effective template for the polymerization of diacetylene.

DOI： 10.1142/S0217979206040519

Language：English   Publishing type：Research paper (scientific journal)  

The electrorheological (ER) properties were studied with ternary suspensions containing solid and liquid dispersoids in silicone oil (DMS). The solid dispersoid used was polyether/montmorillonite nanocomposite particles in which polyether molecules were intercalated between the layers of montmorillonite. The liquid dispersoid was urethane-modified polyether prepared from 4,4'-diphenylmethane diisocyanate (MDI) and poly(propylene glycol) (PPG). The steady shear viscosity and dynamic viscoelasticity were measured with a rotating parallel disc rheometer equipped with ER measurement system. The binary and ternary mixtures of these solid and liquid dispersoids with DMS, i.e. composite particle/DMS suspension, polyether/DMS liquid blend, and ternary blend containing both dispersoids, showed an increase of viscosity in response to an electric field (positive ER effect). The ER effect of a ternary suspension containing the particles and polyether liquids was larger than those of binary suspension or blend with one of these dispersoids. The ER effect was found to be improved by combination of the solid and liquid dispersoids, although the recovery time of viscosity after removing the electric field became rather longer, which would be due to the partly irreversible macroscopic aggregation of the dispersoids.

DOI： 10.1142/S0217979206040738

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Multi-step precipitation separation system was developed by using aqueous mixtures of some thermosensitive polymers. The following three polymers were used here; poly(N-n-propylacrylamide), poly(N-isopropylacrylamide), and poly(N-isopropylmethacrylamide). A mixture of the three polymers showed three endothermic peaks, and the peak top temperatures were almost consistent with that of the each polymer solution. The polymers were purified by thermal precipitation to obtain fractions which can respond in narrow temperature ranges prior to use. In the case of the precipitation separation of two polymers mixtures, purities of the obtained precipitate and supernatant fractions became high comparing with the case in which the unpurified polymers were used. Parts of the polymers which were not the precipitation targets were also precipitated by the separation procedures. This was caused not only by insolubilization of the non-targeted polymers due to their phase transitions but also by their non-specific entanglement with the targeted polymers. The purities of the fractions also improved when the difference of the phase transition temperature between two polymers was large enough to avoid the coprecipitation. In the case of the precipitation separation of mixtures of the three polymers, purities of each fraction also improved when the purified polymers were used.

DOI： 10.1007/s00289-005-0486-y

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/la0356194

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1295/polymj.34.624

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/1097-0290(20010205)72:3<261::AID-BIT2>3.0.CO;2-7

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English  

Event date： 2007.7

Country：Japan  

Presentation type：Oral presentation (general)  

Venue：東京  

Presentation type：Oral presentation (general)  

Venue：横浜市   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：横浜市  

Presentation type：Oral presentation (general)  

Venue：横浜市  

Presentation type：Oral presentation (general)  

Venue：山形市  

Presentation type：Oral presentation (general)  

Venue：山形市  

Presentation type：Oral presentation (general)  

Venue：Auckland   Country：New Zealand  

Presentation type：Oral presentation (general)  

Venue：Auckland   Country：New Zealand  

Presentation type：Oral presentation (general)  

Venue：Auckland   Country：New Zealand  

Presentation type：Oral presentation (general)  

Venue：Auckland   Country：New Zealand  

Presentation type：Oral presentation (general)  

Venue：福岡市  

Presentation type：Oral presentation (general)  

Venue：Honolulu   Country：United States  

Presentation type：Oral presentation (general)  

Venue：Viena   Country：Austria  

Presentation type：Oral presentation (general)  

Venue：名古屋  

Presentation type：Oral presentation (general)  

Venue：名古屋  

Presentation type：Oral presentation (general)  

Venue：名古屋  

Presentation type：Oral presentation (general)  

Venue：東京  

Language：Japanese  

Language：Japanese  

細胞内シグナル応答型遺伝子医薬送達システムの複合体安定化の試み

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese  

細胞内シグナルに応答したsiRNAデリバリーシステムの開発

Language：Japanese  

高分子‐ペプチド複合体による遺伝子の転写制御

Language：Japanese  

ポリマー/ペプチドコンジュゲートを用いるがんイメージング剤―前立腺がん特異的プロテアーゼ蛍光プローブ

Language：Others  

The combination of drug or gene delivery system responding to cellular signals (D-RECS) and sonoporation system for effective and safe gene delivery
We have developed new gene expression-regulating polymer that can activate transgene expression in response to target intracellular signals. Here, we tried applying sonoporation system to this gene regulation system to enhance the gene expression efficacy. Sonoporation is the method for effective gene transfection in vitro and in vivo. Therefore, the method might enhance the transfection efficiency in our polymer and realize an efficient and safe gene delivery system. Results suggested that the combination of our polymer and sonoporation could improve the gene expression compared to the system using only our polymer that transfers genes into cells via endocytosis. It also kept the ability of the gene regulation responding to cellular signals. © 2010 Materials Research Society.

Language：Japanese  

ペプチドグラフト型LPEIを用いたD‐RECS法における癌特異性の向上

Language：Others  

Language：Japanese  

がん診断を目指した蛍光物質含有超音波造影剤の開発

Language：Others  

The effect of PEG grafted on gold nanorods and their injection dose on biodistribution in tumor-bearing mice
Gold nanorods have strong surface plasmon band in the near-infrared region, at which light penetrates deeply into tissues, and convert the absorbed light energy into heat. Therefore, gold nanorods are expected to act as an effective contrast agent for in vivo bioimaging and as a photosensitizer for photothermal therapy. In order to efficiently achieve these applications without side effects, gold nanorods should be selectively accumulated in the target organ. In this study, we optimized the length of the poly (ethylene glycol) (PEG) chain to stabilize the PEG-modified gold nanorods in blood circulation, and investigated the effects of PEG grafting level and injection dose on the biodistribution and enhanced permeability and retention (EPR) effect after intravenous injection into mice. PEG 5,000 - , PEG 10,000 - and PEG 20,000 - modified gold nanorods showed higher blood circulation stability compared with PEG 2,000 - modifed gold nanorods. Higher PEG grafting levels were advantageous for the reticuloendothelial system (RES) avoidance and suppression of aggregation of the gold nanorods in the blood circulation. Modification with a PEG 5,000 : gold molar ratio of 1.5 was sufficient to show both prolonged circulation and the EPR effect. When the injection dose was increased above 39.0 μg of gold, the RES uptake in the liver was saturated and surplus gold nanorods were distributed to other tissues, especially the spleen and tumor. This information is important key to provide the successful application of gold nanorods in the field of nanomedicine. © 2010 Materials Research Society.

Language：Japanese  

がん細胞特異的に遺伝子を活性化するシグナル応答型遺伝子キャリヤー

Language：Japanese  

ペプチド複合体を用いるプロテインキナーゼ計測用蛍光分子プローブ

Language：Japanese  

がん細胞内の異常シグナルを利用した遺伝子デリバリーシステム

Language：Japanese  

ペプチドアレイを用いた表面プラズモン共鳴(SPR)イメージングによる網羅的なOn‐chipリン酸化解析への展開

Language：Japanese  

プロテインキナーゼ活性検出のための蛍光プローブの作製と機能評価

Language：Japanese  

細胞内シグナル酵素応答型ポリマーによる遺伝子発現制御

Language：Japanese  

クリックケミストリーによるポリマーへの修飾

Language：Japanese  

Gene delivery system use of sonoporation with gene carriers responding to cellular signals

Language：English  

Development of Gold Nanorods That Accumulatate into Tumor

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：2

Number of peer-reviewed articles in Japanese journals：3

Type：Competition, symposium, etc. 

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：7

Type：Competition, symposium, etc. 

Number of participants：50

Type：Competition, symposium, etc. 

Number of participants：50

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：9

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：12

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：9

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：12

Type：Competition, symposium, etc. 

Number of participants：150