Published papers

We prepared a cellulose nanofiber (CNF)-based porous membrane with three dimensional cellular structures. CNF was concentrated via a surfactant-induced assembly by mixing CNF with a cationic surfactant, domiphen bromide (DB). Furthermore, they were accumulated by centrifugation to obtain a CNF-DB sol. Next, when the CNF-DB sol was naturally dried, a membrane composed of densely packed CNF was obtained. On the other hand, when the CNF-DB sol was freeze-dried, a porous membrane with the anisotropic cellular structure could be obtained. The interspace between layered CNF sheets was tunable by the DB concentration in the assembly process and the centrifugal force in the accumulation process. FT-IR analysis of the porous membrane showed the formation of hydrogen bonds between the CNF, resulting in facilitation of crosslinking of the CNF and formation of the cellular structures. The obtained CNF-DB membrane exhibited high water resistance. They showed a high ability to absorb hydrophobic dyes such as Nile red and rhodamine B (RhB) due to the presence of the hydrophobic core of DB micelles. Then, the release of RhB could be controlled by the ionic strength in the medium. In addition, they possessed a high ability to adsorb cationic metals such as Ag ions due to the presence of carboxyl moieties of CNF. Next, in situ synthesis of silver nanoparticles (AgNPs) was carried out by employing the CNF-DB membrane as a template for Ag ion adsorption and reduction. Deposition of AgNPs could be observed on the CNF-DB membrane, which suppressed aggregation of AgNPs. Almost all AgNPs were arrayed apart from each other to generate the hotspots, which could enhance surface-enhanced Raman scattering (SERS) of AgNPs. Such an AgNPs-CNF composite membrane could be applied for a label-free analysis of adsorbed RhB.

We prepared biobased nanocapsules by the polymer deposition over the ceramide-containing liposomes to render abilities to load versatile substances and to release them in response to enzymatic degradation of the capsule wall. First, we carried out phosphorylation of chitosan (CHI) to make it amphoteric. Then, we prepared a liposome containing glucosylceramide (GlcCer) and carried out deposition of phosphorylated chitosan (PCHI). The obtained capsule showed high structural stability against solubilization by surfactants and organic solvents, where it maintained its size even in 90 vol% DMSO. Next, we attempted to load various substances into the capsule. A lipophilic Nile red dissolved in 50 vol% DMSO could be loaded into the capsule without rupturing. The loading capacity of PCHI-deposited liposomes was almost three times higher than that of bare liposomes. Its release was suppressed to less than 10 % in an aqueous solution, whereas it could be released to more than 40 % when a small amount of surfactant was added to mimic a lipophilic environment. Also, an amphoteric nature of the capsule derived from PCHI allowed for loading of both cationic and anionic substances. Furthermore, lysozyme (Lyso) was deposited onto the capsule to provide the capsule wall with the enzymatic degradability. Although the release of substances from the capsule was suppressed to less than 10 % without Lyso, it was increased to approximately 50 % by enzymatic degradation of the PCHI layer.

We investigated one-pot synthesis of gold nanoparticles (AuNPs) and AuNPs-polymer hybrid nanoparticles in a water-in-oil (W/O) miniemulsion. Water nanodroplets were dispersed as a reactor in cyclohexane using dimethicone as a surfactant. First, we intended to generate AuNPs in water nanodroplets, which contained HAuCl4 with a mixture of 1-vinylpyrrolidone (VP) and N,N’-methylenebisacrylamide (MBAAm). Several AuNPs with the size of approximately 10–20 nm were generated in each nanodroplet via reduction of AuCl4- by dimethicone. On the other hand, AuNPs was scarcely generated in water nanodroplets containing HAuCl4 with 2-hydroxyethyl methacrylate (HEMA). Next, we carried out polymerization in water nanodroplets to encapsulate AuNPs into polymer nanoparticles. Several AuNPs were encapsulated within polymer particles by copolymerization of VP and MBAAm with an oil-soluble initiator (V-601), and some AuNPs were present in the oil phase. The encapsulation efficiency of AuNPs was 34%. The encapsulation efficiency of AuNPs was dramatically increased by increasing the concentration of the initiator. The same result was obtained by increasing the concentration of the crosslinker due to rapid gelation of nanodroplets. On the other hand, monodisperse particles could be obtained by polymerization of HEMA using a water-soluble initiator (V-65). After polymerization for 5 d at 65ºC, the color of the suspension changed from opaque blue to blue purple and the encapsulation efficiency of AuNPs (5–20 nm) increased to 99 wt%. The average number of AuNPs encapsulated in a PHEMA nanoparticle increased from 1.5 to 4.6 particle. Furthermore, the size of AuNPs could be tuned by increasing the HAuCl4 concentration, and the corresponding color of the suspensions such as blue, purple, and red could be observed. The number and location of AuNPs in polymer nanoparticles could also be tuned by additives such as L-histidine and polyvinylpyrrolidone. By drop-casting the obtained hybrid nanoparticles, we could obtain gold-polymer hybrid films where AuNPs were evenly distributed within a film. The films exhibited different colors corresponding to their LSPR of AuNPs.

We prepared keratin nanoparticles, which could respond to temperature and pH changes and to redox reactions, by electrostatic association of keratin and a cationic surfactant, dioctadecyl dimethyl ammonium chloride (DODAC). Solubilized keratin chains were assembled into nanoparticles (Ker-DODAC) and then crosslinked by disulfide formation. The resultant nanoparticles (Ker-DODACss) possessed a hydrophobic core composed of DODAC, enabling hydrophobic compounds to be loaed and retained in the core. By increasing the temperature, the fluidity of the core was increased, resulting in Ker-DODACss swelling and burst release of loaded compounds. Next, cholesterol was loaded into Ker-DODACss to modify the inner environment, such as the hydrophobicity and fluidity of the core. The nanoparticles (Ker-DODACss-Chol) exhibited reduced fluidity of the core and suppressed release of the payload. Furthermore, the release could be triggered by decreasing the electrostatic interaction between keratin and DODAC at pH values lower than 7.4. It was also found that Ker-DODACss-Chol possessed the ability to release payloads by breaking disulfide bonds. As a further internal modification, we incorporated a lipophilic antioxidant, α-tocopherol, into Ker-DODACss-Chol. It was confirmed that Ker-DODACss-Chol could act as a physical barrier to prevent α-tocopherol from undergoing oxidative degradation.

We developed a W/O miniemulsion reactor system enabling the preparation of biopolymer nanoparticles and control of their morphologies and inner microstructures. First, the W/O miniemulsion was prepared by suspending water nanodroplets containing agarose in oil. Then, we produced agarose hydrogel nanoparticles (AgarH) by the gelation of agarose in nanodroplets. Next, we prepared agarose xerogel nanoparticles (AgarX) by precisely tuning the water evaporation from AgarH. We controlled the morphologies (solid and hollow) and crystal structure of AgarX by changing the pressure and temperature during water evaporation. In fact, AgarX with a hollow structure was generated through rapid evaporation at a low pressure. AgarH was dissolved in Milli-Q water when the temperature was raised to 90 °C. On the other hand, AgarX remained a solid particle under the same conditions, likely because of its high crystallinity. We expect that this technique can be applied to prepare xerogels of diverse biomass-based polymers for the replacement of synthetic polymers.

We developed a miniemulsion templating method to prepare porous polymeric membranes. First, water nanodroplets were suspended in an oil phase by using a nonionic and polymeric surfactant to form a water-in-oil (W/O) miniemulsion. After the nanodroplets accumulated by centrifugation, a small amount of monomer was added as an oil phase to resuspend the water nanodroplets in a monomer phase. Then, photopolymerization of the monomer phase was conducted to generate pores in the polymeric matrix. The size of the nanodroplets was tuned by the surfactant concentration to control the pore size of the membranes. We could produce pore morphologies such as closed-cellular, open-cellular, and bicontinuous structures by tuning the volume fraction of the nanodroplets. Alternatively, nanodroplets were accumulated by centrifugation, and then further surfactants were added to the monomer to suppress the coalescence of nanodroplets. This enabled us to generate a highly porous open-cellular structure while maintaining the size and spherical shape. Next, HAuCl4 was reduced by using the surfactant displayed at the inner surface of the pore wall as the reducing agent. Gold nanoparticles were produced in the inner pores of the polymeric membrane, showing coloration derived from local surface plasmon resonance.

We studied the influence of the chemical properties of the polymer particle and the polymer substrate on particle indentation. The polymer particles were arrayed over the polymer substrate and heat treatment (annealing) was conducted above the glass transition temperature (Tg) of bulk polymer to indent polymer particles into the polymer substrate. Surface topologies of particle-indented surfaces were observed with a field-emission scanning electron microscope (FE-SEM) and the degree of particle indentation (DI) was evaluated with a scanning probe microscope (SPM). The particle-arrayed substrate was placed upside down and then heat treatment was carried out. It was found that there was no effect of gravity on particle indentation. To prepare polymer particles with different chemical properties, surface grafting of hydrophobic, hydrophilic, cationic and anionic polymer chains was carried out by iniferter-initiated living radical polymerization. We found that the indentation of these particles was influenced by the outermost surface of the particle. Next, to investigate the effect of the substrate surface on particle indentation, sulfonation of the polystyrene (PS) substrate was carried out with concentrated sulfuric acid. We found that the surface property of the substrate also influenced particle indentation. From these results, we concluded that particle indentation would be determined by the interaction between the particle surface and the substrate surface. Finally, we intended to analyze the viscoelastic property of the substrate surface by particle indentation. We could estimate Tg values of PS and polyvinyl chloride (PVC) substrates by observing the particle indentation at the different annealing temperatures.

We prepared biobased nanocapsules with enzymatic degradability, which were generated by the layer-by-layer deposition of enzymes and polypeptide over the liposomal surface. Here, we demonstrate two different systems based on the enzymatic degradation of polymer layers. First, the deposition of trypsin and polyarginine (PArg), which is cleavable by trypsin, was carried out over a negatively charged liposome. The enzymatic cleavage of PArg resulted in exposure of the lipid membrane, which facilitated release of the cargo. Next, we attempted to degrade the outer polymer layer of the multilayered capsule wall to display the inner polymer layer by enzymatic degradation. This approach enabled the accumulation and targeting of the nanocapsules through the affinity between the displayed polymer layer and the target hydroxyapatite (HAp). The polymer wall was constructed with an inner layer consisting of poly-L-glutamic acid (PGlu) and an outer layer consisting of trypsin and PArg onto the liposome. The degradation of the outer PArg by trypsin allowed the surface to display the inner PGlu, which has bone-targeting ability. In addition, the polymer wall was constructed from an inner layer of PArg and an outer layer of pepsin and PGlu. The degradation of the outer PGlu by pepsin led to inner PArg on the surface to achieve cell-penetrating activity.

Herein, we focused on mucin, which is a large viscous glycoprotein in terms of materials science, and reported preparation of mucin gel particles and incorporation of enzymes to provide the particle with self-degradable and releasable properties. To expose the hydrophobic peptide cores, trimming of sugar moieties was carried out by β-elimination reaction under alkaline conditions (tMucin). Nano-sized tMucin particles were prepared by the assembly of tMucin with the aid of a cationic surfactant. Then, cross-linking of tMucin particles was carried out via heat treatment (annealing) to induce thermal aggregation of the polypeptide chains. The hydrodynamic diameter of tMucin particles reversibly changed in response to calcium ions. Next, in an attempt to render the particle degradable, lysozyme was incorporated into the tMucin particles for the hydrolysis of oligosaccharide chains. These particles were gradually degraded upon enzymatic cleavage of the mucin molecules, facilitating the release of their incorporated substances. Also, the degradation of the mucin particles and the release of lysozyme were tunable by environmental conditions, such as temperature and calcium ions, in addition to the degree of cross-linking of the particles.

We intended to create a free-standing and organic–inorganic hybrid colloidal membrane. The colloidal membrane was assembled from polymeric capsules, which were prepared by coating of polymer layers over a liposome (liponanocapsules). In this study, two approaches were employed for mineralization over the membrane with calcium phosphate (CaP) to control its mechanical robustness and biodegradability (hybrid bioscaffold). One approach was based on CaP deposition over the liponanocapsules and their assembly into the hybrid membrane. CaP deposition was conducted via the counter-diffusion of phosphate ions and calcium ions across the capsule wall to obtain hybrid nanocapsules. Then, free-standing hybrid membrane was obtained by utilizing hybrid nanocapsules as building blocks for drying-mediated assembly. The obtained hybrid membrane was degraded into individual nanocapsules and degradation could be tuned by the crystal structure of CaP. In another approach, the free-standing membrane was assembled from DNA-coated liponanocapsules and then the counter-diffusion of ions was carried out across each assembled nanocapsule for CaP mineralization. The mechanical robustness of the membrane was significantly improved and its degradation was suppressed by CaP mineralization. This is probably because mineral cross-linkages were formed in the interspace between each nanocapsule. Fluorescent substances could be incorporated in each nanocapsule of the membrane and their release could be tuned by the control in crystal properties of CaP.

We developed a miniemulsion-based nanoreactor system, which can allow for morphological tuning of CaCO3 crystals in nanodroplets and their high encapsulation efficiency in polymer particles. We first obtained nano-sized CaCO3 by using the aqueous nanodroplet as a nanoreactor. In addition, it was found that crystal growth and morphological transformation of CaCO3 from spherical to rod-like crystals were induced by adding 2-hydroxyethyl methacrylate (HEMA) in nanodroplets. Such transformation was also promoted by raising the incubation temperature or increasing the number of nanodroplets in the presence of HEMA. We speculated that crystal growth of CaCO3 crystals was induced by coalescence of nanodroplets and morphological transformation to rod-like shape was undertaken with an assistance of HEMA. In this way, we could tune the shapes of CaCO3 with the well-controlled size and aspect ratio by tuning environments in nanodroplets. Then, subsequent polymerization of HEMA, which was used as a monomer, was carried out for encapsulation of CaCO3 with rod-like morphologies inside polymer nanoparticles. As a result, spherical and spheroidal hybrid nanoparticles, which possessed homogeneity in size and shape, were obtained. Then, by spin-coating of hybrid nanoparticles onto the glass substrate, we could obtain a closely-packed particle monolayer.

We carried out surface-grafting from proteins and their assembling into objects with unique nanostructured materials (nano-objects). To immobilize polymer-initiating sites, amino groups of bovine serum albumin (BSA) were allowed to react with iniferter groups (BSA-i). Then, graft polymerization of N-isopropyl acrylamide (NIPAM) was performed by light-initiated living radical polymerization from immobilized iniferter moieties of BSA-i. The polymer-grafted BSA (BSA-g-PNIPAM) was assembled into nano-objects through the precipitation of PNIPAM graft chains and their sizes and morphologies were tuned by the chain length, the density and the chemical structure of graft polymers in addition to the environmental conditions such as temperature and pH. It was possible to retain the structures of nano-objects by thermal denaturation via heat treatment. Fluorescent substances were encapsulated in particulate nano-objects (nanoparticles) assembled from PNIPAM-g-BSA and their release could be regulated by tuning pH and temperature. Next, further graft polymerization from PNIPAM-grafted BSA was carried out by living radical polymerization of a cationic monomer, N,N-dimethylamino propyl acrylamide methyl chloride quaternary (DMAPAAQ). The grafted polymer was composed of a block copolymer of PNIPAM and a cationic polymer (PDMAPAAQ) and the gel-like nano-object was generated by increasing temperature. In contrast to PNIPAM-g-BSA, it became insoluble even when the temperature decreased, probably due to the electrostatic association between anionic regions of BSA and cationic regions of graft polymers. Coating of BSA-g-P(NIPAM-b-DMAPAAQ) enabled to form a uniform thin layer over a human hair. A free-standing membrane could be obtained by peeling from a water repellent substrate to create a porous membrane.

We developed a mineral cross-linking strategy to prepare a biopolymer-based nanoparticle using calcium phosphate (CaP) as a cross-linker. Nanoparticles were first formed by mixing deoxyribonucleic acid (DNA) with cationic surfactants, and were cross-linked by CaP precipitation. After removal of the surfactants, we carried out the alternative dialysis of nanoparticles against CaCl2 aqueous solution and phosphate buffered solution for further mineral cross-linking. XRD and FT-IR studies revealed that the resultant nanoparticles were produced by mineral cross-linkages of hydroxyapatite (HAp) and the crystal amount and properties such as morphology and crystallinity could be well-controlled by the reaction conditions. Chemical dyes could be incorporated into nanoparticles via their affinities with crystal faces of HAp and DNA. Their release was tunable by crystal amount and properties of mineral cross-linkages. Also, the release could be triggered by mineral dissolution in response to pH. Such a mineral cross-linking will open up a potential way to provide a nanoparticle with versatile functions such as cleavable cross-linking, binding affinity for cargos, and pH-responsive release.

We developed a particle nanoimprinting technique assisted by the array of core-shell particles. Core-shell particles composed of a solid core of polystyrene and a soft shell were prepared by soap-free emulsion polymerization and subsequently seeded polymerization. By the Langmuir-Blodgett method, particles were arranged into a closely packed 2D array over the water surface and transferred onto a polystyrene (PS) substrate at a regular interval. The PS substrate was heated up above its glass transition temperature (Tg) by either UV irradiation using a high-pressure Hg lamp or heat treatment in a temperature-controlled incubator. It could be observed that a nanopatterned indented surface was formed through the denting of particles into the PS substrate (particle nanoindenting). By the detachment of particles from the substrate by ultrasonication in ethanol, nanoholes were produced over the surface (particle nanoimprinting). The depth and the wall of nanoholes and their interval were tunable by the shell thickness and the 2D packing ratio of core-shell particle monolayers. The contact angle decreased from 70 degrees of the pristine particle monolayer to 13 degrees by the particle nanoindenting, and again increased to 50 degrees by detaching the particles from the substrate to create the nanoholes. The use of nanoholes as zepto-litter volume vessels enabled us to produce and arrange nanocrystals, such as NaCl and CaCO 3 (zepto-reactor). © 2014 American Chemical Society.

We developed a new method to prepare aggregates of specific cells and immobilize cells on a substrate with specific shapes by using a synthetic multifunctional tool, which consisted of a cell adhesive Arg-Gly-Asp (RGD) sequence, a photoreactive phenyl azido group and a biotin group. This chemical nanotool, RGD-2-(6-[biotinamido]-2-(p-azidobenzamido)-hexaneamido)ethyl-1, 3′-dithio-proprionate (RGD-BED) was added to human umbilical vein endothelial cells to bind to receptors via the ligand-receptor interaction. Next a photoimmobilization of the binding RGD-BED was carried out by UV irradiation to covalently couple a phenyl azido moiety of RGD-BED with the neighboring site of the integrin receptor. We found that not only the migration distance of RGD-BED immobilized cells was diminished, but also the cell morphology was fixed on the substrate due to the blocking of integrin receptors by RGD-BED. In contrast, the addition of biocytin-containing polymers, poly(N-methacryloyloxy biocytin-co-dimethylacrylamide), and avidin to the RGD-BED-immobilized cells led to restore cellular migration behavior, probably arising from the increase in the rigidity of the environment surrounding the cells. Furthermore, by the addition of avidin to the RGD-BED-immobilized cells, three-dimensional cell aggregates were formed due to the cross-linking of the biotin moieties of RGD-BED. These results show that RGD-BED is a potential nanotool not only to label and collect targeted cells by the formation of cell aggregates but also to suppress mobility and morphologies of specific cells to be applicable for medical treatments.

We developed a route for one-pot synthesis of fluorescent hybrid nanoparticles and their assembly into a nanofilm by utilizing a fusion and fission-triggered nanoreactor system based on water-in-oil "mini- emulsion". A fluorescent nanocrystal, ZnS:Mn2+, was produced in a confined system via fusion and fission of nanodroplets induced by ultrasonication and the amount of nanocrystals formed in each nanoreactor was easily controlled. Then, in situ polymerisation allowed for encapsulation of nanocrystals inside polymer nanoparticles, leading to an efficient surface capping of nanocrystals with polymers and hence a strong light emission under UV irradiation. Also, hybrid nanoparticles with different colours could be created by tuning of Mn2+ doping into ZnS nanocrystals, which can give rise to an energy transfer from carboxylic groups of surface capping polymers to the Mn2+. These hybrid nanoparticles were then spin-coated to form hybrid nanofilms where nanocrystals were uniformly distributed. The multicolour tuning of nanofilms was achieved simply by mixing nanoparticles with ZnS-derived blue emission and those with ZnS:Mn2+-derived orange emission. We believe that our nanoreactor system would offer a viable way in creating fluorescent nanomaterials. © 2013 The Royal Society of Chemistry.

Thermosensitive nanoparticles were prepared by mimicking protein folding where polymer aggregates were formed by precipitation of thermosensitive polymer chains followed by disulfide formation of their thiol groups. N-Isopropylacrylamide (NIPAM) and methacryloxy succini-mide (SuMA) were co-polymerized and then cysteamine was allowed to react with succinimide moieties of the polymer to render thiol moieties. A polymer aqueous solution precipitated to form nano-sized aggregates by increasing temperature above its lower critical solution temperature (LCST), and their sizes were monodispersed and tunable by the polymer concentration. The aggregates were cross-linked to produce nanoparticles by oxidation of thiol groups in a manner similar to formation of a disulfide bond of protein. As a result, the cross-linked nanoparticles exhibited swelling by decreasing temperature below the LCST of the copolymer. Fluorescein and bovine serum albumin (BSA) were chosen as a small and a large substance, respectively, and were encapsulated into the swollen nanoparticles at 25 °C. Fluorescein was rapidly released from both swollen and shrunken nanoparticles. Although BSA exhibited little release at any temperatures, it was released from nanopar-ticles by adding the reducing agent to dissociate the disul-fide cross-linking and incubating below the LCST.

We developed a novel route to control both mineralization and polymerization inside a nanoreactor based on a miniemulsion system to create organic-inorganic hybrid nanomaterials with diverse structures and morphologies. Nanodroplets of calcium nitrate (Ca(NO 3) 2)/monomer and nanodroplets of sodium carbonate (Na 2CO 3) were separately prepared and suspended in the oil phase. They were mixed through the fusion and fission process triggered by ultrasonication to precipitate a biomineral only inside the nanodroplets. In the presence of monomer, 2-hydroxyethyl methacrylate (HEMA), we could obtain unique structures of nano-sized CaCO 3 particles, which were crystalline with spherical or rod-like shapes or amorphous. By subsequent polymerization, we could encapsulate nano-CaCO 3 inside the polymeric nanoparticles at a desired stage of crystal growth. The preincubation period for CaCO 3 nucleation and growth and the polymerization rate were important factors to manipulate the structures and polymorphs of nano-CaCO 3 inside the hybrid nanoparticles, owing to inhibition of crystal growth and transformation by polymerization of HEMA. Also, the growth of rod-like CaCO 3 inside the nanoparticles led to formation of spheroidal hybrid nanoparticles. By using hybrid nanoparticles as building blocks, we could produce nanofilms containing nano-CaCO 3 with a variety of sizes and structures. Crystal transformation of amorphous CaCO 3 was induced inside the nanofilm by increasing the temperature to above the T g of PHEMA. © 2012 The Royal Society of Chemistry.