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Featured researches published by Tooru Ooya.


Journal of the American Chemical Society | 2006

Biocleavable polyrotaxane-plasmid DNA polyplex for enhanced gene delivery.

Tooru Ooya; Hak Soo Choi; Atsushi Yamashita; Nobuhiko Yui; Yuko Sugaya; Arihiro Kano; Atsushi Maruyama; Hidetaka Akita; Rie Ito; Kentaro Kogure; Hideyoshi Harashima

A biocleavable polyrotaxane, having a necklace-like structure consisting of many cationic alpha-cyclodextrins (alpha-CDs) and a disulfide-introduced poly(ethylene glycol) (PEG), was synthesized and examined as a nonviral gene carrier. The polyrotaxane formed a stable polyplex having positively charged surface even at low charge ratio. This is likely to be due to structural factors of the polyrotaxane, such as the mobile motion of alpha-CDs in the necklace-like structure. Rapid endosomal escape was observed 90 min after transfection. The positively charged surface and the good buffering capacity are advantageous to show the proton sponge effect. The pDNA decondensation occurred through disulfide cleavage of the polyrotaxane and subsequent supramolecular dissociation of the noncovalent linkages between alpha-CDs and PEG. Transfection of the DMAE-SS-PRX polyplex is independent of the amount of free polycation. Those properties played a key role for delivery of pDNA clusters to the nucleus. Therefore, the polyplex nature and the supramolecular dissociation of the polyrotaxane contributed to the enhanced gene delivery.


Journal of Controlled Release | 1999

Synthesis of theophylline-polyrotaxane conjugates and their drug release via supramolecular dissociation

Tooru Ooya; Nobuhiko Yui

Theophylline-polyrotaxane conjugates were synthesized by coupling theophylline with alpha-cyclodextrins (alpha-CDs) in the polyrotaxane. The polyrotaxane is a molecular assembly in which many alpha-CDs are threaded onto a poly(ethylene glycol) (PEG) chain capped with L-phenylalanine (L-Phe). Theophylline-7-acetic acid was activated by coupling with 4-nitrophenol, and then ethylenediamine was allowed to react with the active ester in order to obtain N-aminoethyl-theophylline-7-acetoamide. This derivative was coupled with a 4-nitrophenyl chloroformate-activated polyrotaxane to obtain the theophylline-polyrotaxane conjugates. The conjugates formed a specific association under physiological conditions, depending upon interactions between the theophylline molecules and/or the terminal l-Phe moiety in the conjugates. In vitro degradation of the conjugates revealed that theophylline-immobilized alpha-CDs were completely released by hydrolysis of the terminal peptide linkage in the polyrotaxane. This result indicates that the association of the conjugates does not induce the steric hindrance but rather enhances the accessibility of enzymes to the terminal peptide linkages. It is suggested that our designed drug-polyrotaxane conjugates can release the drugs via the dissociation of the supramolecular structure without steric hindrance of enzymatic accessibility to the terminal peptide linkages.


Journal of Controlled Release | 1999

Hyaluronic acid grafted with poly(ethylene glycol) as a novel peptide formulation.

Kazuteru Moriyama; Tooru Ooya; Nobuhiko Yui

Hyaluronic acids (HA) grafted with poly(ethylene glycol) (PEG) (PEG-g-HA) were synthesized. The materials characterization, enzymatic degradability and peptide (insulin) release from solutions of the copolymers were examined. Distribution of bioactive peptides within the polymer chain is well-known for combinations of PEG and polysaccharides as aqueous polymer two-phase systems. Insulin was preferentially partitioned into the PEG phase in a PEG/HA solution system. Enzymatic degradation of the copolymers was strongly dependent on the PEG content. Thermal analysis revealed that PEG-g-HA exhibited a variation in phase-separated structures depending on the PEG content. The solution of PEG-g-HA enabled insulin to remain in the PEG moieties dispersed in the HA matrix. Leakage of insulin from the copolymers was dependent upon the PEG content. Leakage rate of insulin from copolymer containing between 7 and 39% by weight of PEG were similar. A dramatic increase in leakage rate occurred when the PEG content was increased to greater than 39% by weight. It is considered that the loaded insulin was partitioned into the PEG moieties and became entangled with the PEG chains. The conformational change of insulin was effectively prevented in PEG-g-HA solutions, although insulin was denatured in storage of both phosphate buffered solution and HA solution. Such a heterogeneous-structured polymeric solution may be advantageous as an injectable therapeutic formulation for ophthalmic or arthritis treatment.


Macromolecular Chemistry and Physics | 1999

Synthesis and characterization of a polyrotaxane consisting of β-cyclodextrins and a poly(ethylene glycol)-poly(propylene glycol) triblock copolymer

Hiroaki Fujita; Tooru Ooya; Nobuhiko Yui

A polyrotaxane in which many β-cyclodextrins (β-CyDs) are threaded onto a triblock copolymer of poly(ethylene glycol) (PEG) and poly(propylene glycol) (PPG) capped with fluorescein-4-isothiocyanate (FITC) was synthesized as a model of stimuli-responsive molecular assemblies for nanoscale devices. β-CyDs threaded onto the triblock copolymer enhance the solubility of the polyrotaxane and presumably contribute to the prevention of aggregation between PPG segments. The interaction of β-CyDs with a terminal FITC moiety was observed to be significant at 10°C, however, with increasing temperature, the interaction of β-CyDs with a PPG segment becomes prominent. From these results, it is concluded that the majority of β-CyDs move toward the PPG segment with increasing temperature although some β-CyDs may reside on PEG segments.


Journal of Biomaterials Science-polymer Edition | 1997

Synthesis and characterization of biodegradable polyrotaxane as a novel supramolecular-structured drug carrier.

Tooru Ooya; Nobuhiko Yui

Polyrotaxanes were synthesized as novel biodegradable polymers with supramolecular assembly and their properties evaluated in vitro. The synthesis of biodegradable polyrotaxanes consists of three steps: preparation of an inclusion complex consisting of alpha-cyclodextrins (alpha-CDs) and amino-terminated poly(ethylene glycol) (PEG); introduction of L-phenylalanine (L-Phc) at each complex terminal via peptide linkages: and hydroxypropylation of alpha-CDs in the polyrotaxanes. Succinimide ester of benzyloxycarbonyl-L-Phe was condensed with the terminal amino groups of the inclusion complex. 1H-NMR and GPC results showed that alpha-CDs were threaded onto a PEG chain and L-Phe moieties were introduced at each terminal of the PEG chain. Further, the amount of threaded alpha-CDs was found to be governed by the molecular weight of PEG. The hydroxypropylation of alpha-CDs improved the solubility of the polyrotaxanes in PBS (pH 7.4). The hydroxypropylated (HP-) polyrotaxanes were characterized by terminal peptide cleavage using papain. In vitro degradation of HP-polyrotaxanes revealed that HP-alpha-CDs threaded onto a PEG chain were released only when terminal peptide linkages were cleaved. Moreover, threaded HP-alpha-CDs onto a PEG chain was found to be completely released. Kinetics of terminal peptide cleavage were also evaluated by catalytic efficiency (kcat/K(m)). The kcat/K(m) values were found to be independent of the molecular weight of HP-polyrotaxanes but to be affected by terminal hydrophobic moieties. It is proposed that our designed polyrotaxanes are feasible as novel drug carriers.


Journal of Controlled Release | 2008

Supramolecular control of polyplex dissociation and cell transfection: Efficacy of amino groups and threading cyclodextrins in biocleavable polyrotaxanes

Atsushi Yamashita; Daizo Kanda; Ryo Katoono; Nobuhiko Yui; Tooru Ooya; Atsushi Maruyama; Hidetaka Akita; Kentaro Kogure; Hideyoshi Harashima

A novel strategy for gene delivery using biocleavable polyrotaxanes, in which dimethylaminoethyl-modified alpha-cyclodextrins (DMAE-alpha-CDs) are threaded onto a poly(ethylene glycol) (PEG) chain capped with benzyloxycarbonyl-L-tyrosine via disulfide linkages (DMAE-SS-PRX), involves the formation of a stable polyion complex (polyplex) against a counter polyanion and the intracellular plasmid DNA (pDNA) release from the polyplex accompanied by the supramolecular dissociation of DMAE-SS-PRXs. In this study, we prepared biocleavable polyrotaxanes with different numbers of threading alpha-CD and amino (DMAE) groups to enhance the transfection activity of DMAE-SS-PRXs. 29DMAE-alpha18-SS-PRX, in which the numbers of alpha-CD molecules and amino groups were 18 and 29 respectively, exhibited a high transfection activity compared with other PRXs. The transfection activity of DMAE-SS-PRXs seems to be related to the efficacy of pDNA release from those polyplexes, which was controlled by the number of alpha-CD and/or amino groups in the polyrotaxane carrier. Most of the DMAE-SS-PRX polyplexes released the pDNA only in the presence of both 10 mM DTT and of the counter-polyanion, as expected, except for 14DMAE-alpha18-SS-PRX, which released pDNA in the absence of dextran sulfate once the DTT had been added to the polyplex solution. The transfection activity of 14DMAE-alpha18-SS-PRX was significantly lower than that of 29DMAE-alpha18-SS-PRX regardless of the above features. Confocal laser scanning microscopic (CLSM) observation suggested that the specific result for 14DMAE-alpha18-SS-PRX might be due to a premature release of pDNA from the most dissociative 14DMAE-alpha18-SS-PRX polyplex in the cytosol. Therefore, transfection activity seems to be related to an appropriate timing of pDNA release.


Journal of Controlled Release | 2002

Multivalent interactions between biotin-polyrotaxane conjugates and streptavidin as a model of new targeting for transporters.

Tooru Ooya; Nobuhiko Yui

Kinetic analysis of interactions between biotin-polyrotaxane or biotin-alpha-cyclodextrin (biotin-alpha-CD) conjugates and streptavidin was carried out as a model of new targeting to transporters using the surface plasmon resonance (SPR) technique. The biotin-polyrotaxane conjugates, in which biotin-introduced alpha-CDs are threaded onto a poly(ethylene oxide) chain capped with bulky end-groups, are expected to increase the valency of biotin from monovalent to multivalent binding. The number of biotins conjugated with one polyrotaxane molecule varied from 11 to 78, and apparently increased the association equilibrium constant (K(a)), assuming pseudo-first-order kinetics. A detailed dissociation kinetics was analyzed and the re-binding of the biotin-polyrotaxane conjugates was observed on the streptavidin-deposited SPR surface. The magnitude of the re-binding is likely to become larger with increasing the number of biotins, suggesting multivalent interaction on the SPR surface. To quantify the effect of valency, competitive inhibition assay was performed in terms of the supramolecular structure of the polyrotaxane. The inhibitory potency of the biotin-polyrotaxane conjugate was found to be 4-5 times greater than that of the biotin-alpha-CD conjugate. Therefore, the biotin-polyrotaxane conjugates by supramolecular formation of the biotin-alpha-CD conjugate significantly switches from monovalent to multivalent bindings to the model binding protein, streptavidin.


Nature Protocols | 2007

Synthesis of a biocleavable polyrotaxane-plasmid DNA (pDNA) polyplex and its use for the rapid nonviral delivery of pDNA to cell nuclei

Atsushi Yamashita; Nobuhiko Yui; Tooru Ooya; Arihiro Kano; Atsushi Maruyama; Hidetaka Akita; Kentaro Kogure; Hideyoshi Harashima

This protocol provides a method for synthesizing a biocleavable polyrotaxane/plasmid DNA (pDNA) polyplex and for using it to deliver pDNA into cell nuclei. The biocleavable polyrotaxane is synthesized in four steps: (i) introduction of disulfide linkages at both terminals of PEG, (ii) preparation of an inclusion complex between disulfide-containing PEG and α-cyclodextrins (α-CDs), (iii) synthesis of polyrotaxane and (iv) modification of α-CDs in the polyrotaxane with dimethylethylenediamine. A polyplex of pDNA with the polyrotaxane is formed when the two compounds are dissolved together in a phosphate buffer. Subcellular localization of rhodamine-labeled pDNA in fluorescently labeled organelles is quantified by Z-series of confocal images captured by confocal laser scanning microscopy. Significant amounts of pDNA delivered to the nucleus can be expected as well as high transfection activity of the polyplex. This protocol can be completed in 23–32 d.


Biosensors and Bioelectronics | 2010

Fluorescent protein recognition polymer thin films capable of selective signal transduction of target binding events prepared by molecular imprinting with a post-imprinting treatment.

Hirobumi Sunayama; Tooru Ooya; Toshifumi Takeuchi

The functional monomer bearing three functional groups for protein imprinting was designed, which has a structure consisting of a polymerizable methacryloyl group, a secondary amine group for fluorescent dye conjugation by a post-imprinting treatment, and a benzoic acid moiety capable of interacting with a target protein. Lysozyme-imprinted polymer thin films were prepared on the initiator-immobilized glass substrates by radical polymerization in the presence of lysozyme, the designed functional monomer, a co-monomer(s) and a crosslinker. After the removal of lysozyme, fluorescein isothiocyanate was introduced into the secondary amine group of the functional monomer residues in the imprinted thin film as a fluorescent reporter dye (post-imprinting treatment). Lysozyme was selectively bound to the thin film with a binding constant of ca. 10(6) M(-1). Since the reporter dye can be only introduced into the binding cavity, the fluorescent response can be detected only when the guest is bound to the cavity, namely only specific binding events can be transduced as fluorescence spectral change. Compared with the SPR measurement, selective binding to the imprinted cavity can be more precisely detected by the proposed method, enabling us to prepare a new class of protein recognizable materials with the ability of the specific signal transduction of protein binding events.


International Journal of Pharmaceutics | 2002

Carboxyethylester-polyrotaxanes as a new calcium chelating polymer: synthesis, calcium binding and mechanism of trypsin inhibition

Tooru Ooya; Masaru Eguchi; Atsushi Ozaki; Nobuhiko Yui

A carboxyethylester-polyrotaxane was synthesized as a novel calcium chelating polymer in the field of oral drug delivery and characterized in terms of mechanism of trypsin inhibition. Here, carboxyethylester (CEE) groups are introduced to all the primary hydroxyl groups in alpha-cyclodextrins (alpha-CDs), which are threaded onto a poly(ethylene glycol) chain capped with bulky end-groups (polyrotaxane). The solubility of the CEE-polyrotaxane in physiological conditions increased with pH, indicating ionization-related solubility similar to conventional polyacrylates. The ability of calcium (Ca2+) chelation was found to increase in the order of poly(acrylic acid) (PAA)>CEE-polyrotaxanez.Gt;CEE-alpha-CD, suggesting that the increased density of carboxyl groups enhances the Ca2+ chelating ability. The activity of trypsin was inhibited by these compounds in the same order of the calcium chelation. However, the inhibitory effect of CEE-polyrotaxane was reduced by adding excess Ca2+ without precipitation that was observed in the presence of PAA. Such the reduced inhibition and precipitation by CEE-alpha-CD was not observed. Therefore, the inhibitory effect of CEE-polyrotaxane is due to Ca2+ chelation from trypsin without non-specific interaction.

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Nobuhiko Yui

Tokyo Medical and Dental University

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Kang Moo Huh

Chungnam National University

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Won Kyu Lee

Japan Advanced Institute of Science and Technology

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Shintaro Sasaki

Japan Advanced Institute of Science and Technology

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Atsushi Yamashita

Japan Advanced Institute of Science and Technology

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Atsushi Maruyama

Tokyo Institute of Technology

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