Published papers

We exploited three approaches to fabricate urea nanofibers by using an array of core-shell particles as a template. Core-shell particles were prepared by seeded polymerization and used as an atto-liter scale incubator (atto-incubator) to form nanofibers of urea crystal. Firstly, the mixture of the particle suspension and a urea aqueous solution was spin-coated to promote arrangement of core-shell particles and urea crystallization simultaneously (mix-spin method). Secondly, core-shell particles were arranged by a Langmuir-Blodgett apparatus and then the spin-coating of a urea aqueous solution was performed (LB-spin method). Then, the nano-network of urea crystals was produced by heat treatment at 80 °C. The interparticle distance and the amount of the remaining solution were important to determine the shapes, orientation and amounts of urea crystals. In the LB-natural drying method, one-directionally oriented nanocrystals were obtained over the wide range of the interparticle distance. As preliminary attempts, the obtained nanofibers were used as a template for the production of nanoscale metallic materials. Au nanofibers, nanotubes and nanorods could be obtained by gold sputtering deposition on the urea nanofiber and dissolving urea crystals. Ag-urea hybrid nanofibers were also produced using a mixture of urea and Ag nanoparticles by the modified LB-spin method. © 2012 Elsevier B.V.

We created a free-standing membrane as a novel bioscaffold through the assembly of polymer-coated liposomes. Polyarginine (PArg) possessing a cell-penetrating activity was used to form the polymer layer onto a negatively charged liposome (lipo-PArg). The capsule wall of PArg over liposomes made it possible to improve the mechanical property of capsules and to display deoxyribonucleic acid (DNA) over the vesicle surface through the electrostatic attraction (lipo-PArg-DNA). The release rates of a fluorescent probe encapsulated in lipo-PArg and lipo-P Arg-DNA were tunable by the number of polymeric layers of the capsule walls. To investigate the cell-membrane permeability of lipo-P Arg-DNA, polymer-coated liposomes were incubated with human umbilical vein endothelial cells (HUVECs) at 4 °C. It was found that lipo-P Arg underwent a significant cellular uptake, whereas bare liposomes and liposomes modified with chitosan were incapable of overcoming the plasma membrane barrier. To prepare a free-standing membrane composed of polymer-coated liposomes, a suspension of lipo-PArg-DNA was cast over a mesh hole and dried up. SEM observation revealed that a free-standing membrane was obtained through drying-mediated assembly process without rupturing polymer-coated liposomes inside the membrane. On the other hand, it was not possible to obtain a complete membrane from a mixture of lipo-PArg and DNA. In summary, lipo-PArg-DNA capsules possess versatile functions as a drug carrier, and their assembly enables us to create a free-standing membrane applicable as a bioscaffold. © 2011 American Chemical Society.

Biocompatible polymer-magnetite hybrid nanoparticles were prepared by means of in situ synthesis of magnetite within polysaccharide hydrogel nanoparticles. Hydrogel nanoparticles were first fabricated by blending high-molecular-weight carboxymethyl cellulose as an anionic polymer, and low-molecular-weight chitosan as a cationic polymer to form polyion complexes (CC particles). These polyion complexes were then chemically crosslinked using genipin, a bio-based cross-linker, to form stable nanoparticles having a semi-IPN structure (CCG particles). Magnetite was lastly synthesized within CCG particles by the coprecipitation method to obtain polymer-magnetite hybrid nanoparticles (CCGM particles). The formations of CC, CCG and CCGM particles were mainly observed by transmittance, absorbance of genipin and TEM, respectively, and their hydrodynamic diameters and zeta-potentials were analyzed. It was confirmed that the hydrodynamic diameters and the zeta-potentials of these particles were significantly influenced by pH of the suspension, which was attributed to the charges of polymers. The diameters of CCGM particles were smaller than 200. nm at any pH conditions, suggesting the possibility to apply them as drug delivery carriers. CCGM particles exhibited the responsiveness to a magnetic field in addition to their high dispersion stability, indicating their potential to be utilized as a biomaterial for hyperthermia. © 2011 Elsevier B.V.

We created an organic-inorganic hybrid nanocapsule by utilizing polysaccharide-coated liposomes as a reaction site for the deposition of calcium phosphate (CaP). Phosphate ions were encapsulated in a liposome, followed by the layer-by-layer deposition of chitosan (CHI), dextran sulfate (DXS), or DNA onto the liposome surface. Calcium ions were added to an aqueous suspension of the phosphate ion-incorporated nanocapsules to prepare the nanocapsules that provide a variety of walls for the counter-diffusion of ions and the surface for CaP deposition. As a result, control in biomineralization, such as thickness and crystal properties, over the nanocapsules was achieved by tuning the counter-diffusion of the calcium ions and the phosphate ions through the capsule wall and the surface chemical composition of nanocapsules. Furthermore, we carried out DNA adsorption onto CaP-coated liposomes. DNA was releasable from the nanocapsules because of the dissolution of CaP under acidic conditions. © 2011 American Chemical Society.

We intended to combine the liposomal preparation and the layer-by-layer deposition to prepare a nanosized capsule. Chitosan (CHI) was deposited to form the cationic polymeric layer onto a negatively charged liposomal surface and further deposition was carried out using anionic polymers dextran sulfate (DXS) or deoxyribonucleic acid (DNA). ζ-Potentials of nanocapsules changed between positive and negative charges at each deposition. FE-TEM revealed that the liposome remained a spherical shape even after the layer-by-layer (LbL) deposition. The capsule wall showed a dramatic increase in stability against the surfactant Triton X-100 compared to a bare liposome, and the stability was controllable by the adsorption amount of the polymer. These suggest that the polymer multilayer was generated on the liposome surface by the layer-by-layer depositions of polysaccharides. The three kinds of chemical substances with different charges, 1-hydroxy pyrene-3,6,8-trisulfonic acid (HPTS), alendronate, and glucose, were encapsulated into nanocapsules and the release was suppressed by the polymeric capsule wall irrespective of charges. The release from DNA-deposited nanocapsules (liponano-CHI-DNA) was clearly increased by raising temperature from 25 to 60 °C. This indicates that the temperature-dependent release was achieved by applying DNA denaturation as a temperaturedependent "switch", which influenced the permeability of the capsule wall. © 2009 American Chemical Society.

We studied the topographical effect of roughness displayed by a closely packed particle monolayer on formation of a cell monolayer (cell sheet). Particle monolayers were prepared by Langmuir-Blodgett deposition using particles, which were 527 nm (SA053) and 1270 nm (SA127) in diameter. Human umbilical vein endothelial cells (HUVECs) were seeded at a high density (2.0 × 105 cells/cm2) onto particle monolayers. It was found that cells gradually became into contact with adjacent cells on the SA053 monolayer and the formed cell sheet could be readily detached from the particle monolayer by gentle pipetting. On the other hand, cells adhering onto the tissue culture polystyrene (TCPS) and the SA127 particle monolayer were difficult to peel off. At a low cell seeding density (5.0 × 104 cells/cm2), pre-coating with bovine plasma fibronectin (FN) allowed cell growth on an SA053 particle monolayer, and a confluent monolayer was able to be peeled as a cell sheet from the particle monolayer just by pipetting. By immunostaining of human fibronectin, we found that fibronectin was secreted and concentrated onto the substrate side of a cell sheet. The obtained cell sheet adhered and grew on the TCPS again within 20 min. © 2008 Elsevier B.V. All rights reserved.

We prepared two-dimensional (2D) arrays composed of nanoscale hydrogel particles as a model of suprastructured assemblages. Poly(N-isopropylacrylamide) (PNIPAM) hydrogel particles and the core-shell particles, which possess a rigid polystyrene core encased in a PNIPAM hydrogel shell, were selected as components for arrays. Below and above 32°C, these two kinds of particles swelled and shrank, respectively, due to temperature-dependent solubility of a PNIPAM chain. The particle dispersion was spread and then compressed to produce particle monolayers closely packed at the air-water interface. To study thermal behavior of 2D arrays, surface pressure and surface area for obtained 2D arrays were measured at 40 and 25°C with leaving the monolayer on the water surface. Both of them varied in response to temperature, indicating that each particle of 2D arrays swelled and shrank at the water surface. Although the pressure and the area for the 2D array of PNIPAM hydrogel particles exhibited great responses at the beginning, they gradually decreased and eventually disappeared with repeating the temperature cycle, due to particle coalescence. On the other hand, the 2D array of core-shell particles maintained the thermosensitive response without relaxation though its variation between 40 and 25°C was still small. By assembling the mixture of PNIPAM and core-shell particles into the monolayer, the 2D array with large and steady responses could be obtained.

Hollow nanoparticles (bionanocapsules) were prepared using liposomes as a template for the layerby-layer deposition of biopolymers. We carried out the alternative deposition of poly-L-lysine (PLys) and poly-L-aspartic acid (PAsP) onto a negatively charged liposome, which was prepared from dilauroyl phosphatidic acid (DLPA) and dimyristoylphosphatidylcholine (DMPC). Efficient separation of nanocapsules from unbound polymers was achieved by ultrafiltration. ζ-Potentials of nanocapsules changed between positive and negative charges at each deposition. FE-TEM revealed that the liposome was covered with a thin polymeric layer. A fluorescent probe, l-hydroxypyrene-3,6,8- trisulfonic acid (HPTS), was encapsulated into nanocapsules. The release of HPTS was suppressed by the polymer deposition, and the release rate was tunable by coverage of the first PLys layer and the ratio of DLPA. © 2007 American Chemical Society.

We developed a small incubator of an atto-liter scale (atto-incubator) for producing and arraying nanosized materials. The core-shell particles with a hydrogel shell were used as an incubator to isolate individual droplets of the solution and to specify the nucleation sites for crystallizing NaCl and urea solutes. Their array was deposited on a polystyrene dish by the spin-coating of a particulate dispersion, and then dried to remove any residual solvent. When the array was exposed to a solution, the hydrogel shell absorbed the solution and swelled. Nucleation of the nanocrystals at the particles occurred as the arrays were dried again. We obtained single crystals as a rectangular solid with a uniform shape at 8 wt.% NaCl. By the use of atto-incubators for urea crystallization, we obtained nanosized fibers of a single crystal, which were aligned in the direction of the flow. The atto-incubator could allow us to control the nucleation region and the size and shape of the crystals. © 2006 Elsevier B.V. All rights reserved.

We studied topological effects of subcellular roughness displayed by a closely packed particle monolayer on adhesion and growth of endothelial cells. Poly(styrene-co-acrylamide) (SA) particles were prepared by soap-free emulsion copolymerization. Particle monolayers were prepared by Langmuir-Blodgett deposition using particles, which were 527 (SA053) and 1270 nm (SA127) in diameter. After 24-h incubation, cells tightly adhered on a tissue culture polystyrene dish and randomly spread. On the other hand, cells attached on particle monolayers were stretched into a narrow stalk-like shape. Lamellipodia spread from the leading edge of cells attached on SA053 monolayer to the top of the particles and gradually gathered to form clusters. This shows that cell-cell adhesion became stronger than cell-substrate interaction. Cells attached to SA127 monolayer extended to the reverse side of a particle monolayer and engulfed particles. They remained immobile without migration 24 h after incubation. This shows that the inhibition of extensions on SA127 monolayer could inhibit cell migration and cell proliferation. Cell growth on the particle monolayers was suppressed compared with a flat TCPS dish. The number of cells on SA053 gradually increased, whereas that on SA127 decreased with time. When the cell seeding density was increased to 200,000 cells cm-2, some adherent cells gradually became into contact with adjacent cells. F-actin condensations were formed at the frame of adherent cells and the thin filaments grew from the edges to connect each other with time. For the cell culture on SA053 monolayer, elongated cells showed a little alignment. Cells showed not arrangement of actin stress fibers but F-actin condensation at the contact regions with neighboring cells. Interestingly, the formed cell monolayer could be readily peeled from the particle monolayer. These results indicate that endothelial cells could recognize the surface roughness displayed by particle monolayers and the response was dependent on the pitch of particle monolayers. © 2006 Elsevier B.V. All rights reserved.

To control the cellular uptake of drugs and genes, we synthesized a liposomal carrier possessing membrane-permeable ligands and a detachable poly(ethylene glycol) (PEG) coating. For the detachable coating, a lipid having a thiolytic cleavable spacer (PEG-S-S-DOPE) was synthesized by the reaction of dioleoylphosphatidylethanolamine (DOPE) with a PEG chain via a disulfide linkage. The liposomes were prepared from a mixture of dipalmitoylphosphatidylcholine (DPPC), DOPE, PEG-S-S-DOPE, and cholesteryl hemisuccinate (CHEMS). The octamer (R8 peptide) of arginine was chosen as the membrane-permeable ligand and covalently immobilized onto the CHEMS portion of the liposome surface (PEG-S-S-R8-liposome). The disulfide bond of the PEG chain was cleaved to display the R8 peptides on the liposome surface by adding a reducing agent such as l-cysteine, and thereby internalization of the liposomes was significantly facilitated. When l-cysteine was added to the mixture of cells and the liposome that incorporated plasmids encoding the enhanced green fluorescence protein (pEGFP), the expression of EGFP was low but could be observed in almost 100% of the cells. © 2006 Elsevier B.V. All rights reserved.

A fundamental study of the usage of peptide nucleic acid (PNA) and DNA at the surface of a surface plasmon resonance (SPR) biosensor for detecting a specific point mutation in the K-ras gene for early detection of cancers is reported. The extents of hybridization using DNA analytes of different length which are targets were studied at 25 and 40°C. The 15mer PNA ligand immobilized to the surface of the sensor had a high ability to detect a 11mer DNA analyte point mutation at 25 and 40°C. On the contrary, when DNA was used as a ligand, detection was only confirmed at 40°C which indicates that the difference of melting temperature (Tm) between the complementary and mismatched analyte was a key factor for point mutation detection. This also implied the decrease of Tm by immobilizing a probe to a surface since it was below the one in solution. Therefore, effects from the surface are seen in this system and the surrounding environment makes a difference to the discrimination process. To understand the process in detail, kinetic analysis was performed and association constants were found to be larger for DNA ligands implying the effect coming from the sensor surface. In this case negative charges of the carboxyl groups of the matrix had a tendency to repel negatively charged DNA ligands. Affinity constants (KA) were calculated and as expected, the ratio of KA between the complementary and mismatched analyte was of the order of 102 for PNA interacting with 11mer analytes, making it a promising probe for genetic diagnosis. These results indicate the importance of the effect of analyte length, temperature and the environment surrounding the ligand for accomplishing an all or none single base mismatched detection system. © 2002 Elsevier Science B.V. All rights reserved.

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.

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.

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.

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.

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.

Poly(dimethylsiloxane)-containing macro-azo-initiator was used in dispersion polymerization of methyl methacrylate. Stable microspheres were obtained when the solvents had solubility parameters in the range of 7.4-8.2. The microspheres were stabilized by a stabilizer formed in-situ, that is, polydimethylsiloxane-block-poly(methyl methacrylate). Copyright (C) 1999 Elsevier Science B.V. All rights reserved.

Many synthetic microspheres are spherical and symmetrical because of the thermodynamical limitations of the reaction systems. We developed modification techniques to prepare spherical microspheres with intrinsic unsymmetry, for example, a microsphere with both an anionic and a cationic part. The modification was performed at the liquid-solid interface. Reactive microspheres with p-nitrophenyl moieties were settled onto the IgG-preadsorbed substrate, and the reaction between the activated ester moieties and IgG molecules proceeded only at the interface upon attachment. After the reaction, the modified microspheres were detached from the substrate with ultrasonication. In another modification at the air-water interface, the dispersion of reactive microspheres in ethanol was first spread on a water surface to produce the monolayer of microspheres, and then hydrolysis at the water side of the monolayer was performed by adding a NaOH aqueous solution to the subphase. After this unsymmetrical hydrolysis, we carried out IgG immobilization with the remaining reactive moieties. In both interface methods, the immobilized IgG molecules were labeled with colloidal gold particles conjugated with anti-IgG antibodies. Transmission electron microscopy indicated that gold particles could be observed on only the immobilized side. Small microspheres were coupled to the reactive microsphere, and its unsymmetry was confirmed by scanning electron microscopy. Such unsymmetrical microspheres will be applicable to the electrical rheology, diagnosis, display technology, and creation of functional devices through the assembling of microspheres.