論文 - 藤本　啓二

Amphoteric initiators suitable for emulsifier-free emulsion polymerization and the properties of the resulting latices

Fang S., Fujimoto K., Kondo S., Shiraki K., Kawaguchi H.

Colloid and Polymer Science （Colloid and Polymer Science）  279 （ 6 ） 589 - 596 2001年

研究論文（学術雑誌）, 査読有り,  ISSN  0303402X

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The soap-free emulsion polymerizations of styrene (St) and acrylamide (AAm) or methyl methacrylate (MMA) were carried out in the presence of three kinds of amphoteric initiators, and the polymerization kinetics and the colloidal properties of the latices produced were emphatically investigated. It was found that the number of carbon atoms between the amidino and carboxyl groups in each initiator exhibited an appreciable effect on the dissociation as well as on the solubility of the initiator in water, and therefore, that the properties of the colloidal particles depended on the structure of the initiators used. All the copolymerization runs, except for the polymerization using (2,2′-azobis(N-(2-caboxyethyl)-2-methylpropionamidine) under a strongly alkali condition, gave amphoteric latices, which indicated higher critical flocculation concentrations at lower or higher pHs than at a medium pH. The surface charge density measured by titration for poly(St/MMA) particles was about 3-10 times as high as that for the poly(St/AAm) ones, though these were prepared under the same conditions other than the monomer composition. The influence of the polymerization pH on both the polymerization rate and the surface charge density of the resulting latices was negligible even if the constants of the decomposition rate and the dissociation of the amphoteric initiators strongly depended on the pH of the medium.

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Preparative techniques of nanoparticles for drug delivery

Fujimoto K.

Drug Delivery System 16 （ 3 ） 155 - 164 2001年

ISSN  09135006

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This review presents the outstanding contributions and preparative techniques in mainly polymeric nanoparticles used as drug delivery systems. Nanoparticles discussed here vary in the diameter from 10 to 1000 nm. The formation of nanoparticles composed of one polymer chain and many polymer chains is reviewed in terms of the intrapolymer and interpolymer self-association and methods of preparation and drug loading are briefly described. Several methods to prepare nanoparticles by dispersing the preformed polymers are also demonstrated, e. g., solvent evaporation method and solvent diffusion method. Particles can be produced via a polymerization process from monomers. The preparative techniques for nanoparticles include emulsion polymerization, miniemulsion polymerization, and microemulsion polymerization. For drug incorporation, preparation of core-shell nanoparticles is also described. As a novel approach to fabricate nanoparticles, supra-molecular structured materials such as dendrimers are introduced. Finally, our attempts to utilize nanoparticles for cell-death induction are stated and the concept of ‘cell-death programmed cell’ is proposed in this literature. © 2001, THE JAPAN SOCIETY OF DRUG DELIVERY SYSTEM. All rights reserved.

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Novel preparation of nonsymmetrical microspheres using the Langmuir-Blodgett technique

Nakahama K., Kawaguchi H., Fujimoto K.

Langmuir （Langmuir）  16 （ 21 ） 7882 - 7886 2000年10月

研究論文（学術雑誌）, 査読有り,  ISSN  07437463

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We have utilized the Langmuir-Blodgett (L-B) technique to develop the generic technology that can prepare nonsymmetrical microspheres. The dispersion of the microspheres in butanol was spread on a water surface and compressed by a L-B apparatus to obtain a microsphere monolayer. Then, an amphiphilic and reactive polymer was prepared by terpolymerization of N-octadecyl acrylamide, p-nitrophenyl acrylate, and 2,2,2,-trifluoroethyl methacrylate. A stable polymer monolayer was transferred onto the microsphere monolayer by the L-B technique. It was found from contact angle measurement that the surface became hydrophobic with deposit of the terpolymer. X-ray photoelectron spectroscopy data demonstrated that only the top side of modified microspheres was covered with the terpolymer.

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Emulsifier-free emulsion copolymerization of styrene and acrylamide using an amphoteric initiator

Fang S., Fujimoto K., Kondo S., Shiraki K., Kawaguchi H.

Colloid and Polymer Science （Colloid and Polymer Science）  278 （ 9 ） 864 - 871 2000年09月

研究論文（学術雑誌）, 査読有り,  ISSN  0303402X

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Emulsifier-free emulsion copolymerization of styrene (St) and acrylamide (AAm) has been investigated in the presence of an amphoteric water-soluble initiator, 2,2'-azobis[N-(2-carboxyethyl)-2-2-methylpropionamidine]hydrate (VA057). The kinetics of polymerization and the colloidal properties of the resulting latices were studied and compared with the cases using ionic initiators. When adopting the amphoteric initiator at pHs lower than 10, stable amphoteric poly (St/AAm) latices, evidenced by the electrophoretic mobility, were prepared directly. Meanwhile, almost the same conversion versus time curves appeared and there were no apparent differences in the final particle sizes for those polymerizations, whereas in the polymerization at pH 10, a much lower rate of copolymerization and a larger size of particles were observed. The surface charge density and the growth rate of latex particles produced with VA057 at pH<10 were comparable to those of the particles with a cationic initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride, but were apparently lower than those with an anionic initiator, potassium persulfate, when the polymerizations were carried out under corresponding conditions. The number of initiator fragments incorporated onto the particle surfaces was independent of polymerization pH, except for pH 10. The abnormal performance of VA057 at pH 10 was attributed to its degradation due to hydrolysis.

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Inspiration provided by the living world and design of synthetic materials

Fujimoto K.

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 45 （ 11 ） 1859 - 1864 2000年08月

ISSN  00399450

Modification and functionalization of cell surfaces by polymer chains

6th World Biomaterial Congress 2000年05月

単著, 査読有り

Emulsifier-free emulsion copolymerization of styrene and acrylamide using an amphoteric initiatorsl

Fang,S.-J., Fujimoto,K., Kondo,S., Shiraki,K., and Kawaguchi,H.,

Colloid Polym. Sci., 278,(2000)    864-871 2000年

共著, 査読有り

Renaturation of Reduced Ribonuclease A with Microsphere-Induced Refolding System

Shimizu,H., Fujimoto,K., and Kawaguchi,H

Biotechnology Progress 16, (2000).    248-253 2000年

共著, 査読有り

Improved refolding of denatured/reduced lysozyme using disulfide-carrying polymeric microspheres

Shimizu,H., Fujimoto,K., and Kawaguchi,H.,

Colloids and Surfaces B: Biointerfaces （Colloids and Surfaces B: Biointerfaces）  18 （ 2 ） 137-144 - 144 2000年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  09277765

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Many proteins tend to aggregate during refolding, especially at high protein concentrations. To reduce aggregation and improve the refolding yields, denatured/reduced lysozyme at 0.5 mg ml-1 was refolded using disulfide-carrying microspheres. The extent of renaturation was evaluated by measuring the content of recovered soluble protein and its enzymatic activity. Addition of the dispersion of the modified microspheres to the aqueous solution of reduced lysozyme resulted in the binding of the proteins on the microsphere surface, followed by the spontaneous release of the bound proteins. As a consequence, a 10% active enzyme was obtained. However, no renaturation occurred without the microspheres due to aggregation. Moreover, when glutathione was added 20 h after incubation with the microspheres, the refolding yields could increase to ~40%. These results indicate that the disulfide moieties of the microspheres play an important role in preventing the aggregation process and catalyzing the refolding reaction through the thiol-disulfide interchange reactions. Copyright (C) 2000 Elsevier Science B.V.

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A novel preparation of non-symmetrical microspheres using the Langmuir-Blodgett technique

Nakahama,K., Kawaguchi,H., and Fujimoto,K.,

Langmuir 16, (2000)    7882-7886 2000年

共著, 査読有り

Control of cell death by the smart polymeric vehicle

Fujimoto,K., Iwasaki1,C., Arai1,C., Kuwako,M., and Yasugi,E.,

Biomacromolecules （Biomacromolecules）  1 （ 4 ） 515-518 - 518 2000年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  15257797

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We report a novel drug delivery system for apoptosis induction by a "smart" polymer vehicle possessing thermosensitivity and bioaffinity. The polymer chain was prepared by copolymerization of N-isopropyl-acrylamide and N-methacryloyloxysuccinimide. Cell-adhesive RGDS peptide was conjugated with the copolymer as a ligand model for bioaffinity. When the temperature was increased, nanoscale aggregates precipitated from a copolymer aqueous solution. Either dolichyl phosphate (dol-p), which is an apoptotic inducer, or dolichol was added to aggregates at around the precipitation temperature (31°C), and the temperature was raised to 37°C for incorporation. Aggregates incorporating dol-p or dolicol were added to a human promonocytic leukemia U937 cell suspension at 37°C. When the temperature was lowered to 25 °C, cells underwent apoptosis in the presence of Ca2+. Probably, copolymer vehicles were concentrated on a cell surface through the binding of RGDS and integrin and the release of lipid inducers was caused by the disruption of vehicles in response to temperature.

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Preparation of partly hydrophobized, crosslinked polyacrylamide particles by terpolymerization of acrylamide/ N,N-methylenebisacrylamide/styrene in inverse microemulsion

Bartoň J., Kawamoto S., Fujimoto K., Kawaguchi H., Capek I.

Polymer International （Polymer International）  49 （ 4 ） 358 - 366 2000年

研究論文（学術雑誌）, 査読有り,  ISSN  09598103

　概要を見る

Free-radical polymerization of a termonomer system comprising acrylamide (AAm) N,N′-methylenebisacrylamide (MBAAm) and styrene (S) initiated by water-soluble ammonium peroxodisulphate (APS) or by toluene-soluble dibenzoyl peroxide (DBP) in inverse microemulsion (toluene/ S/AOT//water//AAm/MBAAm), leads to the formation of partly hydrophobized crosslinked polymer particles of tailored chemical composition, degree of crosslinking and polymer particle size. Styrene strongly decreases the rate of terpolymerization, while the presence of MBAAm has almost no effect on the polymerization rates observed. This conclusion is valid for both APS and DBP initiators. Increase of the S/T mass ratio (T is toluene) in inverse microemulsion leads to an increase of polymer particle diameter from about 20 nm to about 50 nm attributed to toluene swelling of the styrene-rich structural moieties of AAm-co-S copolymer located on the surface of polymer particles. Polymerization kinetics measurements pointed to the important role of exiting water soluble AAm and MBAAm monomer radicals generated by thermal decomposition of APS in water pools of inverse micelles for initiation of polymerization reactions of sparingly water-soluble S monomer in the oil-phase of the inverse microemulsion. It was shown that the polymerization and copolymerization reactions of S in the presence of AAm and/or MBAAm are effectively initiated by water-soluble APS and also by oil-soluble DBP initiators. During dialysis the polymerized single-phase water/oil Winsor IV inverse microemulsion gradually converts itself into a two-phase oil/water Winsor I dispersion system with volume fraction of aqueous phase Φaw≈0.950. The water phase contains water swelled, crosslinked polymer particles of diameters 80-300 nm. During dialysis, toluene and the sodium salt of bis(2-ethyl hexyl)sulphosuccinic acid (AOT) partition between the oil phase of the dialysed dispersion system and the water dialysate. After evaporation of water from the dialysed inverse microemulsion, solid, dried, crosslinked polymer particles in the form of a transparent film, almost uncontaminated by AOT surfactant, were obtained. © 2000 Society of Chemical Industry.

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Versatility of thermosensitive particles

Kawaguchi H., Kisara K., Takahashi T., Achiha K., Yasui M., Fujimoto K.

Macromolecular Symposia （Macromolecular Symposia）  151   591 - 598 2000年

研究論文（学術雑誌）, 査読有り,  ISSN  10221360

　概要を見る

Poly(N-isopropylacrylamide) (PNIPAM) particles are highly swollen below the transition temperature in water so that they become very soft and hydrophilic, but collapsed above the transition temperature to become hard and hydrophobic. Such changes bring about the discontinuous temperature dependence of adsorbability of protein, activity of immobilized enzyme, cell activating ability of the particles. The transition temperature of PNIPAM was changed by binding something to the chain end and this property was used to control the enzyme activity with temperature. Sudden change in the topology of the particle surface by quick temperature change gave excess stimulus to cells when the change was transmitted straightly to the cells.

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Renaturation of reduced ribonuclease A with a microsphere-induced refolding system

Shimizu H., Fujimoto K., Kawaguchi H.

Biotechnology Progress （Biotechnology Progress）  16 （ 2 ） 248 - 253 2000年

研究論文（学術雑誌）, 査読有り,  ISSN  87567938

　概要を見る

We intended to refold reduced ribonuclease A (RNase A) using polymeric microspheres. Polymeric microspheres were allowed to react with dithiothreitol (DTT) to immobilize the disulfide and thiol moieties on their surface. The fully reduced RNase A was added to the dispersion of the modified microspheres. Protein refolding and renaturation were estimated by the change in the number of disulfide bonds of RNase A and the recovery of the enzymatic activity, respectively. Without microspheres, the activity gradually recovered with the increase in the number of disulfide bonds. However, the formation of disulfide bonds of reduced RNase A was accelerated by adding the modified microspheres, and the rate of renaturation was increased depending on the amount of charged DTT and the reaction time of the immobilization. These results indicate that modified microspheres significantly catalyze the recovery of active RNase A from the reduced form. The protein adsorption data demonstrated that the disulfide moieties of the modified microspheres react with the thiol moieties of the reduced RNase A to form a mixed disulfide. The thiol/disulfide exchange reaction can possibly proceed at the microsphere/protein interface, resulting in the formation of a correct three-dimensional structure.

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Using Polymeric Microspheres for Renaturation of Reduced Proteins

The Fourth Asian Symposium on Biomedical Materials in Singapore (Kobunshi Kankokai)    106-107 1999年12月

共著, 査読有り

Cell Surface Modification by Reactive Polymer Chains and Control of Cell Recognition and Response

The Fourth Asian Symposium on Biomedical Materials in Singapore (Kobunshi Kankokai)    106-107 1999年12月

共著, 査読有り

DNA-carrying latex particles for DNA diagnosis 3. Detection of point mutant DNA using an indirect labeled probe

Hatakeyama M., Iwato S., Hanashita H., Nakamura K., Fujimoto K., Kawaguchi H.

Colloids and Surfaces A: Physicochemical and Engineering Aspects （Colloids and Surfaces A: Physicochemical and Engineering Aspects）  153 （ 1-3 ） 445 - 451 1999年08月

研究論文（学術雑誌）, 査読有り,  ISSN  09277757

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We have developed DNA-carrying affinity latex particles for distinction of point-mutational genes, but point-mutational DNA was not full length in the sample obtained from patients and had to be labeled. To overcome this disadvantage, we used an indirect labeled probe in this study. The indirect labeled probe was complementary with a 5' end region of 70mer DNA involved in the K-ras gene. The indirect labeled probe did not play a role of direct detection of point-mutational DNA, but the complex of 70mer DNA and an indirect labeled probe hybridized with single-stranded DNA immobilized on latex particles. This study suggests practical DNA diagnosis by using DNA-carrying affinity latex. Copyright (C) 1999 Elsevier Science B.V. All rights reserved.

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Cell membrane dynamics and the induction of apoptosis by lipid compounds

Fujimoto,K., Iwasaki,C., Kawaguchi,H., Yasugi,E., and Oshima,M.

FEBS Letters （FEBS Letters）  vol.446 （ 1 ） 113-116 - 116 1999年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00145793

　概要を見る

特定の脂質は細胞にアポトーシスを誘導した。これらの脂質は培養液中でサブミクロンサイズのエマルションを形成して細胞膜に分配することがわかった。さらにこの分配に際して細胞膜の動的な変化が極めて短時間で起こり、アポトーシス発現のマーカーとなることがわかった。

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Refolding of protein using thiol-carrying latex particles

Shimizu H., Fujimoto K., Kawaguchi H.

Colloids and Surfaces A: Physicochemical and Engineering Aspects （Colloids and Surfaces A: Physicochemical and Engineering Aspects）  vol.153 （ 1-3 ） 421-427 - 427 1999年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  09277757

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Mis-folded ribonucleaseA (RNaseA) could be refolded by the use of microspheres onto which sulfhydryl and disulfide groups were introduced. Modified microspheres were first added to a mis-folded RNaseA aqueous solution. After incubation, proteins were separated from the solution by centrifugation and the enzymatic activity of recovered RNaseA was measured. The refolding was clearly induced by the modified microspheres. On the contrary, virtually no refolding was observed on the unmodified microspheres. Possibly, sulfhydryl groups on the microsphere can preferentially attack disulfide bonds in mis-folded RNaseA, and this attack can trigger protein refolding through thiol-disulfide exchange reactions at the microsphere-protein interface. Copyright (C) 1999 Elsevier Science B.V. All rights reserved.

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Cell response to micropatterned surfaces produced with polymeric microspheres

Miyaki M., Fujimoto K., Kawaguchi H.

Colloids and Surfaces A: Physicochemical and Engineering Aspects （Colloids and Surfaces A: Physicochemical and Engineering Aspects）  Vol.153 （ 1-3 ） 603-608 - 608 1999年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  09277757

　概要を見る

Surface topography plays an important role in cell function. We fabricated microstructured patterns from monolayers of polymeric microspheres, which were arranged on a flat polystyrene dish. Such patterns could be quickly formed by spin-coating microspheres and were easily controlled by changing the size of the settled microspheres. Polystyrene (PS) microspheres having different sizes were prepared by soap-free emulsion polymerization in the presence of sodium chloride with different concentrations. The zeta potential of these microspheres was approx. -25 mV regardless of their diameter. Electron microscopic observations revealed that the microspheres were deposited on a PS dish in hexagonally-packed monolayers with a pitch equal to the diameter of each microsphere. The water contact angles gradually decreased with an increase of the pitch. To investigate the topographical effect of these surfaces on the neutrophil-like cell function, microscopic observations and measurements of active oxygen released from cells were carried out. Microscopic observation revealed that spreading cells adhered on PS microsphere-settling surfaces as well as on a PS film. As for active oxygen release, cell response to a PS microsphere-settling surface depended on the diameter of settling microspheres and showed a maximum for diameters of approx. 1 μm. This tendency was observed in phosphate-buffered solutions both with and without serum. Continuous activation which followed by the initial attachment seems to relate to the change of cytoskeletal systems. These results indicate that cells recognize the roughness of the micropatterned surface covered with PS microspheres, and a pitch between the tops of settled microspheres is an important factor for cell recognition. Copyright (C) 1999 Elsevier Science B.V. All rights reserved.

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