Naoji Kubota

A simple preparation of half N-acetylated chitosan highly soluble in water and aqueous organic solvents

A simple and improved method of preparing highly soluble chitosan (half N-acetylated chitosan) was developed using a series of chitosan samples of low molecular weights, and the solubility of the half N-acetylated chitosan in water and organic solvents was investigated in detail. To reduce the molecular weight, chitosan was treated with NaBO3 under the condition that chitosan was homogeneously dissolved in aqueous acetic acid. Weight-average molecular weights of the obtained chitosan samples were determined using a size-exclusion chromatography system equipped with a low-angle laser light-scattering photometer. Each chitosan sample was then N-acetylated with acetic anhydride under the condition that chitosan was homogeneously dissolved in aqueous acetic acid again. The water solubility of the half N-acetylated chitosan thus prepared increased with decreasing molecular weight. From 1H NMR spectroscopy, it was suggested that the sequence of N-acetylglucosamine and glucosamine residues was random. The solubility of the half N-acetylated chitosan of low molecular weight was rather high even in aqueous dimethylacetamide and dimethylsulfoxide.

Recovery of serum proteins using cellulosic affinity membrane modified by immobilization of CU2+ ion

An affinity membrane was prepared from a porous cellulose membrane, and adsorption and recovery of serum proteins were investigated from the viewpoint that affinity membranes are efficacious against separation and purification of biomaterials. Into the cellulose membrane, iminodiacetate (IDA) group that acts as a ligand to metal ions was introduced (Cell–IDA membrane), and then Cu2+ ion was immobilized (Cell–IDA–Cu membrane). Bovine serum albumin (BSA) and γ-globulin (BγG), which are the major proteins in blood, were adopted as model proteins to be separated. The Cell–IDA–Cu membrane had large adsorption capacity for these proteins despite the low degree of modification. The amounts of proteins adsorbed on the Cell–IDA–Cu membrane increased with increasing pH, and BγG was adsorbed more than BSA. High protein recoveries from the Cell–IDA–Cu membrane were obtained. The separation of these proteins was also conducted under the optimum conditions of adsorption and recovery, and BγG was concentrated more than BSA although the initial concentration of BγG was lower than that of BSA.

Synthesis of glycosides of resveratrol, pterostilbene, and piceatannol, and their anti-oxidant, anti-allergic, and neuroprotective activities.

Resveratrol was glucosylated to its 3- and 4′-β-glucosides by cultured cells of Phytolacca americana. On the other hand, cultured P. americana cells glucosylated pterostilbene to its 4′-β-glucoside. P. americana cells converted piceatannol into its 4′-β-glucoside. The 3- and 4′-β-glucosides of resveratrol were further glucosylated to 3- and 4′-β-maltosides of resveratrol, 4′-β-maltoside of which is a new compound, by cyclodextrin glucanotransferase. Resveratrol 3-β-glucoside and 3-β-maltoside showed low 2,2-diphenyl-1-picrylhydrazyl free-radical-scavenging activity, whereas other glucosides had no radical-scavenging activity. Piceatannol 4′-β-glucoside showed the strongest inhibitory activity among the stilbene glycosides towards histamine release from rat peritoneal mast cells. Pterostilbene 4′-β-glucoside showed high phosphodiesterase inhibitory activity. Graphical Abstract Stilbenes were glucosylated to the corresponding β-glucosides by cultured cells of Phytolacca americana. Stilbene glucosides were converted into β-maltosides by cyclodextrin glucanotransferase.

Mechanism of introduction of exogenous genes into cultured cells using DEAE-dextran-MMA graft copolymer as non-viral gene carrier.

Comparative investigations were carried out regarding the efficiency of introduction of exogenous genes into cultured cells using a cationic polysaccharide DEAE-dextran-MMA (methyl methacrylate ester) graft copolymer (2-diethylaminoethyl-dextran-methyl methacrylate graft copolymer; DDMC) as a nonviral carrier for gene introduction. The results confirmed that the gene introduction efficiency was improved with DDMC relative to DEAE-dextran. Comparative investigations were carried out using various concentrations of DDMC and DNA in the introduction of DNA encoding luciferase (pGL3 control vector; Promega) into COS-7 cells derived from African green monkey kidney cells. The complex formation reaction is thought to be directly proportional to the transformation rate, but the complex formation reaction between DDMC and DNA is significantly influenced by hydrophobic bonding strength along with hydrogen bonding strength and Coulomb forces due to the hydrophobicity of the grafted MMA sections. It is thought that the reaction is a Michaelis-Menten type complex formation reaction described by the following equation: Complex amount = K1 (DNA concentration)(DDMC concentration). In support of this equation, it was confirmed that the amount of formed complex was proportional to the RLU value.

Mechanism of the Introduction of Exogenous Genes into Cultured Cells Using DEAE-Dextran-MMA Graft Copolymer as a Non-Viral Gene Carrier. II. Its Thixotropy Property

From comparative investigations regarding the efficiency of introducing exogenous genes into cultured cells using DEAE-dextran and DEAE-dextran-MMA (methyl methacrylate ester) graft copolymer (2-diethylaminoethyl-dextranmethyl methacrylate graft copolymer; DDMC) as a nonviral carrier, we have confirmed that the gene transfection efficiency of DDMC is higher than that of DEAE-dextran. Comparative investigations in which DNA encoding luciferase (pGL3 control vector; Promega) was introduced into COS-7 cells derived from African green monkey kidney cells with and without the use of an incubator shaker were also carried out using various concentrations of DDMC. Without an incubator shaker, the transfection efficiency results were reversed, namely that the gene introduction efficiency of DDMC was inferior to that of DEAE-dextran. The aqueous solution of the cationic graft-copolymer displays thixotropic properties, which is why a strong shear stress is needed for it to flow and wet the cells. The reaction between DNA and DDMC is thought to be a Michaelis-Menten type complex formation reaction that can be described by the following equation: Complex amount = K1 (DNA concentration) (DDMC concentration).The complex formation reaction is thought to involve Coulomb forces between DDMC and DNA and is also significantly influenced by hydrogen bonding strength along with hydrophobic bonding strength due to the hydrophobicity of the grafted MMA sections.

Permeability properties of isometrically temperature-responsive poly(acrylic acid)-graft- oligo(N-isopropylacrylamide) gel membranes

A series of temperature-responsive hydrogels were prepared by grafting oligo(N-isopropylacrylamide) (ONIPAAm) chains onto a crosslinked poly(acrylic acid) (PAAc) network, intending an application to drug delivery systems (DDS). Cloud points and swelling ratios of the obtained PAAc-graft-ONIPAAm gels were measured as a function of temperature under various pH conditions. At pH > 4.5, the clear cloud points were observed at 31–33°C, which were almost the same values as that of poly(N-isopropylacrylamide) (PNIPAAm), whereas swelling/shrinkage phenomenon was not observed in the temperature range 25–45°C. It seemed that grafted ONIPAAm chains underwent the coil-to-globule transition, while the crosslinked PAAc network remained unchanged, due to the anionic charges on the main chains. In the presence of NaCl in the buffer solution, the phase transition temperature was slightly lowered. To assess the applicability of these temperature-responsive PAAc-graft-ONIPAAm gels to DDS, permeability of theophylline through the gel membranes was measured as a function of temperature. At a temperature below the cloud point, the permeability of theophylline was low, whereas it was high at an elevated temperature above the cloud point.

Temperature-responsive properties of poly(acrylic acid-co-acrylamide)-graft-oligo(ethylene glycol) hydrogels

A series of temperature- and pH-responsive hydrogels were prepared from acrylic acid (AAc), acrylamide (AAm), oligo(ethylene glycol)monoacrylate (OEGMA), and oligo(ethylene glycol)diacrylate by varying the AAc:AAm molar ratio and the OEGMA content. Phase-transition temperatures and swelling ratios of the obtained poly(AAc-co-AAm)-graft-OEG gels were measured as a function of temperature and pH. At pH < 5, the obvious transition temperatures ranging from 5 to 35°C were obtained as the AAc: AAm molar ratio was varied. The highest transition temperature was obtained at the AAc: AAm ratios of 5: 5 and 6: 4, and the sharp transition curves were observed at the AAc: AAm ratios from 5: 5 to 8: 2. The transition temperature further increased with increasing OEGMA content. It was suggested that OEG graft chains with a large mobility played an important role for the formation of hydrogen bonding in the hydrogels. The gels prepared here showed obvious reproducibility of the phase transition in response to temperature changes, which suggests the feasibility of their practical applications.

Ionically conductive polymer gel electrolytes prepared from vinyl acetate and methyl methacrylate for electric double layer capacitor

In order to obtain highly conductive polymer electrolytes for an electric double layer capacitor, three kinds of polymer gel electrolytes were prepared. Vinyl acetate (VAc) and methyl methacrylate (MMA) were copolymerized with divinyl adipate (DA) and ethylene glycol dimethacrylate (EGDMA), respectively, in propylene carbonate (PC) containing tetraethylammonium tetrafluoroborate (TEATFB) to form network polymer gel electrolytes. MMA was also copolymerized with butylene glycol DMA for comparison. The polymer gel electrolytes obtained were characterized by means of thermogravimetry, complex impedance analysis, and cyclic voltammetry for use in the electric double layer capacitor. The ionic conductivities of the polymer gel electrolytes were dependent on the TEATFB concentration, temperature, and crosslinking degree. The polymer gel electrolytes in the VAc-DA system exhibited higher room temperature conductivities (10−2 S/cm) than those in the MMA-EGDMA system. Further, the polymer gel electrolytes in the VAc-DA system showed good electrochemical stability windows ranging from −4.0 to 4.0 V versus Ag. Thermal analysis revealed that the polymer gel electrolytes in both systems were stable up to 150°C.

Anticancer efficacy of a supramolecular complex of a 2-diethylaminoethyl–dextran–MMA graft copolymer and paclitaxel used as an artificial enzyme

Summary The anticancer efficacy of a supramolecular complex that was used as an artificial enzyme against multi-drug-resistant cancer cells was confirmed. A complex of diethylaminoethyl–dextran–methacrylic acid methylester copolymer (DDMC)/paclitaxel (PTX), obtained with PTX as the guest and DDMC as the host, formed a nanoparticle 50–300 nm in size. This complex is considered to be useful as a drug delivery system (DDS) for anticancer compounds since it formed a stable polymeric micelle in water. The resistance of B16F10 melanoma cells to PTX was shown clearly through a maximum survival curve. Conversely, the DDMC/PTX complex showed a superior anticancer efficacy and cell killing rate, as determined through a Michaelis–Menten-type equation, which may promote an allosteric supramolecular reaction to tubulin, in the same manner as an enzymatic reaction. The DDMC/PTX complex showed significantly higher anticancer activity compared to PTX alone in mouse skin in vivo. The median survival times of the saline, PTX, DDMC/PTX4 (particle size 50 nm), and DDMC/PTX5 (particle size 290 nm) groups were 120 h (treatment (T)/control (C), 1.0), 176 h (T/C, 1.46), 328 h (T/C, 2.73), and 280 h (T/C, 2.33), respectively. The supramolecular DDMC/PTX complex showed twice the effectiveness of PTX alone (p < 0.036). Above all, the DDMC/PTX complex is not degraded in cells and acts as an intact supramolecular assembly, which adds a new species to the range of DDS.

Synthesis, oxygen radical absorbance capacity, and tyrosinase inhibitory activity of glycosides of resveratrol, pterostilbene, and pinostilbene

The stilbene compound resveratrol was glycosylated to give its 4′-O-β-D-glucoside as the major product in addition to its 3-O-β-D-glucoside by a plant glucosyltransferase from Phytolacca americana expressed in recombinant Escherichia coli. This enzyme transformed pterostilbene to its 4′-O-β-D-glucoside, and converted pinostilbene to its 4′-O-β-D-glucoside as a major product and its 3-O-β-D-glucoside as a minor product. An analysis of antioxidant capacity showed that the above stilbene glycosides had lower oxygen radical absorbance capacity (ORAC) values than those of the corresponding stilbene aglycones. The 3-O-β-D-glucoside of resveratrol showed the highest ORAC value among the stilbene glycosides tested, and pinostilbene had the highest value among the stilbene compounds. The tyrosinase inhibitory activities of the stilbene aglycones were improved by glycosylation; the stilbene glycosides had higher activities than the stilbene aglycones. Resveratrol 3-O-β-D-glucoside had the highest tyrosinase inhibitory activity among the stilbene compounds tested. Graphical abstract Synthesis and evaluation of stilbene glucosides.