Yoshihiro Sasaki

Tokyo Medical and Dental University, Edo, Tōkyō, Japan

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Publications (48)116.38 Total impact

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    ABSTRACT: The effects of the supramolecular polyrotaxane (PRX) structure on cellular internalization are investigated by flow cytometry and confocal laser scanning microscopy. AF-545-labeled aminated PRXs (APRXs) containing different numbers of threaded α-cyclodextrins (CDs) and amino groups are synthesized; their cellular uptakes are analyzed using HeLa cells in serum. The APRX threaded CD number is discovered to be a more critical factor for enhancing cellular internalization than the APRX amine content. Additionally, APRXs are demonstrated to be more easily internalized than conventional linear cationic macromolecules. Because increased numbers of threaded CDs are related to increased PRX rigidity, the PRX rigid frame resulting from CD molecules threaded on a poly(ethylene glycol) (PEG) chain is suitable for intracellular tools in therapy and diagnosis.
    Macromolecular Bioscience 03/2014; 14(3). · 3.74 Impact Factor
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    ABSTRACT: Apert syndrome is an autosomal dominantly inherited disorder caused by missense mutations in fibroblast growth factor receptor 2 (FGFR2). Surgical procedures are frequently required to reduce morphological and functional defects in patients with Apert syndrome; therefore, the development of noninvasive procedures to treat Apert syndrome is critical. Here we aimed to clarify the etiological mechanisms of craniosynostosis in mouse models of Apert syndrome and verify the effects of purified soluble FGFR2 harboring the S252W mutation (sFGFR2IIIcS252W) on calvarial sutures in Apert syndrome mice in vitro. We observed increased expression of Fgf10, Esrp1, and Fgfr2IIIb, which are indispensable for epidermal development, in coronal sutures in Apert syndrome mice. Purified sFGFR2IIIcS252W exhibited binding affinity for fibroblast growth factor (Fgf) 2 but also formed heterodimers with FGFR2IIIc, FGFR2IIIcS252W, and FGFR2IIIbS252W. Administration of sFGFR2IIIcS252W also inhibited Fgf2-dependent proliferation, phosphorylation of intracellular signaling molecules, and mineralization of FGFR2S252W-overexpressing MC3T3-E1 osteoblasts. sFGFR2IIIcS252W complexed with nanogels maintained the patency of coronal sutures, whereas synostosis was observed where the nanogel without sFGFR2S252W was applied. Thus, based on our current data, we suggest that increased Fgf10 and Fgfr2IIIb expression may induce the onset of craniosynostosis in patients with Apert syndrome and that the appropriate delivery of purified sFGFR2IIIcS252W could be effective for treating this disorder.
    PLoS ONE 01/2014; 9(7):e101693. · 3.53 Impact Factor
  • RSC Advances 10/2013; 3(48):25716–25718. · 3.71 Impact Factor
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    ABSTRACT: The intracellular delivery of enzymes is an essential methodology to extend their therapeutic application. Herein, we have developed dissociable supermolecule-enzyme polyelectrolyte complexes based on reduction-cleavable cationic polyrotaxanes (PRXs) for the reactivation of delivered enzymes. These PRXs are characterized by their supramolecular frameworks of a polymeric chain threading into cyclic molecules, which can form polyelectrolyte complexes with anionic enzymes while retaining their three dimensional structure, although their enzymatic activity is reduced. Upon the addition of a reductant, the PRXs dissociate into their constituent molecules and release the enzymes, resulting in a complete recovery of enzymatic activity. Under the intracellular environment, the PRX-based enzyme complexes showed the highest intracellular enzymatic activity and efficient activation of anticancer prodrugs to induce cytotoxic effects in comparison with the non-dissociable complexes and the commercial cell-penetrating peptide-based reagents. Thus, the intracellularly dissociable supermolecules are an attractive system for delivering therapeutic enzymes into living cells.
    Scientific Reports 07/2013; 3:2252. · 5.08 Impact Factor
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    ABSTRACT: Polysaccharide nanogels are one of the most attractive carriers for drug delivery systems. Nanogels encapsulate proteins in their hydrated polymer networks, and minimize the denaturation of proteins. In this study, we demonstrated the cross-linking of acryloyl group-modified polysaccharide nanogels via photopolymerization, which allowed the formation of novel hydrogel particles and macrogels. The mechanical properties of the resultant hydrogels depended on the concentrations of the nanogels and the cross-linkers. The most significant property of the nanogel-cross-linked hydrogel was the ability to encapsulate insulin via hydrophobic interactions. After incubation of the hydrogel containing insulin in water, the hydrogel was degraded by hydrolysis, and insulin was gradually released from the hydrogels over a period of 1 week. According to these results, this nanogel-cross-linked hydrogel prepared via photopolymerization has potential for innovative biomaterials.
    Reactive and Functional Polymers 07/2013; 73(7):958–964. · 2.51 Impact Factor
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    ABSTRACT: New hybrid poly(ethylene glycol) (PEG) hydrogels crosslinked with both nanogels and nanogel-coated liposome complexes are obtained by Michael addition of the acryloyl group of a cholesterol-bearing pullulan (CHP) nanogel to the thiol group of pentaerythritol tetra(mercaptoethyl) polyoxyethylene. The nanogel-coated liposome complex is stably retained after gelation and the complexes are well dispersed in the hybrid gel. Microrheological measurements show that the strength and gelation time of the hybrid hydrogel can be controlled by changing the liposome:nanogel ratio. The hydrogel is gradually degraded by hydrolysis under physiological conditions. In this process, the nanogel is released first, followed by the nanogel-coated liposomes. Hybrid hydrogels that can incorporate various molecules into the nanogel and liposomes, and release them in a two-step controllable manner, represent a new functional scaffold capable of delivering multiple drugs, proteins or DNA.
    Advanced healthcare materials. 11/2012; 1(6):722-8.
  • Yoshihiro Sasaki, Kazunari Akiyoshi
    ChemInform 07/2012; 43(31).
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    ABSTRACT: We propose a simple method to prepare lipid nanotubes, which can be used to transport biological molecules. By applying shear stress to surface-immobilized liposomes on a solid substrate, we obtained long lipid nanotubes arranged in a well-controlled direction.
    RSC Advances 03/2012; 2(7):2682-2684. · 3.71 Impact Factor
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    ABSTRACT: A supramolecular system that can activate an enzyme through photo-isomerization was constructed by using a liposomal membrane scaffold. The design of the system was inspired by natural signal transduction systems, in which enzymes amplify external signals to control signal transduction pathways. The liposomal membrane, which provided a scaffold for the system, was prepared by self-assembly of a photoresponsive receptor and a cationic synthetic lipid. NADH-dependent L-lactate dehydrogenase, the signal amplifier, was immobilized on the liposomal surface by electrostatic interactions. Recognition of photonic signals by the membrane-bound receptor induced photo-isomerization, which significantly altered the receptor's metal-binding affinity. The response to the photonic signal was transmitted to the enzyme by Cu(2+) ions. The enzyme amplified the chemical information through a catalytic reaction to generate the intended output signal.
    Chemistry 03/2012; 18(11):3258-63. · 5.93 Impact Factor
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    Masaru Mukai, Yoshihiro Sasaki, Jun-ichi Kikuchi
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    ABSTRACT: A nanosensory membrane device was constructed for detecting liposome fusion through changes in an enzymatic activity. Inspired by a biological signal transduction system, the device design involved functionalized liposomal membranes prepared by self-assembly of the following molecular components: a synthetic peptide lipid and a phospholipid as matrix membrane components, a Schiff's base of pyridoxal 5'-phosphate with phosphatidylethanolamine as a thermo-responsive artificial receptor, NADH-dependent L-lactate dehydrogenase as a signal amplifier, and Cu(2+) ion as a signal mediator between the receptor and enzyme. The enzymatic activity of the membrane device was adjustable by changing the matrix lipid composition, reflecting the thermotropic phase transition behavior of the lipid membranes, which in turn controlled receptor binding affinity toward the enzyme-inhibiting mediator species. When an effective fusogen anionic polymer was added to these cationic liposomes, membrane fusion occurred, and the functionalized liposomal membranes responded with changes in enzymatic activity, thus serving as an effective nanosensory device for liposome fusion detection.
    Sensors 01/2012; 12(5):5966-77. · 2.05 Impact Factor
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    ABSTRACT: Molecular chaperones selectively trap heat-denatured proteins or their intermediates, primarily by hydrophobic interactions, to prevent irreversible aggregation resulting from macromolecular host (molecular chaperone)-guest (protein) interactions. The molecular chaperone function is an important concept that is expected to lead to breakthroughs in drug delivery systems, especially for protein or peptide delivery in regenerative medicine, such as bone regeneration. We have reported that polysaccharide nanogels act as artificial molecular chaperones. To further clarify the molecular chaperone function of nanogels as protein carriers, the elucidation of nanogel-protein interactions are especially important. Here, we investigated the interaction of a protein with a polysaccharide nanogel using fluorescence correlation spectroscopy at variable temperatures, using fluorescence-labeled bovine serum albumin (BSA) as a model protein. In particular, thermodynamic parameters of the heat-induced complexation of protein with CHP nanogels were evaluated using the van't Hoff plot. The plot shows that the CHP nanogels strongly complexed with heat-denatured BSA. The increased hydrophobicity of the denatured, unfolded protein may prefer complexation with amphiphilic hydrogel nanoparticles over complexation with the completely folded native protein. Thermodynamic parameters suggest that the complexation is entropically driven, rather than enthalpically, under the conditions studied.
    Current Drug Discovery Technologies 06/2011; 8(4):308-13.
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    ABSTRACT: Protein-crosslinked nanogels were prepared by introducing vitamin B6 (pyridoxal) to hydrophilic polysaccharide. The pH dependence of Schiff base formation was utilized to generate a pH-sensitive nanogel system. This approach represents a new method to prepare hybrid nanogels crosslinked with biomolecules.
    Polym. Chem. 05/2011; 2(6):1267-1270.
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    ABSTRACT: Cell-free protein synthesis is a promising technique for the rapid production of proteins. However, the application of the cell-free systems requires the development of an artificial chaperone that prevents aggregation of the protein and supports its correct folding. Here, nanogel-based artificial chaperones are introduced that improve the folding efficiency of rhodanese produced in cell-free systems. Although rhodanese suffers from rapid aggregation, rhodanese was successfully expressed in the presence of the nanogel and folded to the enzymatically active form after addition of cyclodextrin. To validate the general applicability, the cell-free synthesis of ten water-soluble proteins was examined. It is concluded that the nanogel enables efficient expression of proteins with strong aggregation tendency.
    Macromolecular Bioscience 03/2011; 11(6):814-20. · 3.74 Impact Factor
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    ABSTRACT: Molecular communication is a novel communication paradigm inspired by information processing in biological systems using molecules as information medium. In this study, we achieved the selective propagation of transport vesicles which is an essential technique to establish a molecular communication system using artificial cells. The hybridisation of DNA was employed to conduct a signal-induced delivery of transport vesicles from a sender to a receiver vesicle formed with phospholipids. Oligonucleotide lipids each with a different single-stranded DNA, which acts as recognition tags, were embedded in the sender, receiver and transport vesicles. The addition of a DNA signal with a complementary sequence to connect two oligonucleotide lipids induced the assembly of two types of large unilamellar vesicles as well as recombination of the vesicles. In addition, the selective delivery of transport vesicles between sender and receiver giant vesicles is discussed, based on the results of fluorescence microscopic observations.
    Supramolecular Chemistry 03/2011; 23(3–4):218-225. · 1.55 Impact Factor
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    ABSTRACT: We constructed a supramolecular system on a liposomal membrane that is capable of activating an enzyme via DNA hybridization. The design of the system was inspired by natural signal transduction systems, in which enzymes amplify external signals to control signal transduction pathways. The liposomal membrane, providing a platform for the system, was prepared by the self-assembly of an oligonucleotide lipid, a phospholipid and a cationic synthetic lipid. The enzyme was immobilized on the liposomal surface through electrostatic interactions. Selective recognition of DNA signals was achieved by hybridizing the DNA signals with the oligonucleotide lipid embedded in the liposome. The hybridized DNA signal was sent to the enzyme by a copper ion acting as a mediator species. The enzyme then amplified the event by the catalytic reaction to generate the output signal. In addition, our system demonstrated potential for the discrimination of single nucleotide polymorphisms.
    Organic & Biomolecular Chemistry 02/2011; 9(7):2397-402. · 3.57 Impact Factor
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    ABSTRACT: The thermal stabilization and refolding of horseradish peroxidase (HRP) upon heating were investigated using an artificial molecular chaperone consisting of cholesterol-bearing pullulan (CHP) nanogels. The CHP nanogels inhibited the aggregation of HRP under heating by complexation with the denatured HRP. The enzyme activity of HRP complexed with CHP nanogels was not detected. However, the enzyme activity recovered up to 80% of native HRP after the addition of cyclodextrin (CD) to the complex. The dissociation of CHP nanogels was induced by the formation of an inclusion complex of cholesterol groups of CHP with CD. The enzyme activity of HRP was only significantly recovered by the addition of β-CD or its derivatives. Natural molecular chaperones, such as GroEL/ES, trap, fold, and release the nonnative proteins by changing the hydrophobicity of the specific sites of the molecular chaperone that interact with the nonnative protein. The functional mechanism of the nanogel chaperon system is similar to that of natural molecular chaperones. The nanogel chaperone system is a useful tool to aid the refolding and thermal stabilization of unstable proteins for post-genome research, and in medical and biological applications. KeywordsArtificial chaperone–Protein refolding–Nanogel–Hydrophobic interaction–Cyclodextrin
    Colloid and Polymer Science 01/2011; 289(5):685-691. · 2.16 Impact Factor
  • Yoshihiro Sasaki, Kazunari Akiyoshi
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    ABSTRACT: Nanosize hydrogels (nanogels) are polymer nanoparticles with three-dimensional networks, formed by chemical and/or physical cross-linking of polymer chains. Recently, various nanogels have been designed, with a particular focus on biomedical applications. In this review, we describe recent progress in the synthesis of nanogels and nanogel-integrated hydrogels (nanogel cross-linked gels) for drug-delivery systems (DDS), regenerative medicine, and bioimaging. We also discuss chaperone-like functions of physical cross-linking nanogel (chaperoning engineering) and organic-inorganic hybrid nanogels.
    The Chemical Record 12/2010; 10(6):366-76. · 4.38 Impact Factor
  • Yoshihiro Sasaki, Kazunari Akiyoshi
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    ABSTRACT: Molecular chaperones in living systems inspired us to explore new concepts for assisting protein refolding. The chaperone selectively interacts with a non-native protein by hydrophobic interaction to prevent irreversible aggregation and releases the protein in its refolded form with the aid of ATP and another co-chaperone. Cyclodextrins have been used to simulate the function of the chaperones by controlling the hydrophobic interaction with proteins. In this chapter, we review the cyclodextrin (CD)-related protein refolding systems.
    Current pharmaceutical biotechnology 02/2010; 11(3):300-5. · 3.40 Impact Factor
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    ABSTRACT: Polysaccharide nanogels have been demonstrated to aid the refolding processes of chemically or thermally denatured proteins, a function that is similar to that of natural molecular chaperones. In this study, we examined the possibilities of using the nanogel chaperone system to mediate protein folding in a cell-free (in vitro) protein synthesis system containing transcription/translation factors. High-performance liquid chromatography showed that a polysaccharide nanogel comprising cholesteryl group-bearing pullulan (CHP) trapped unfolded or partially folded green fluorescent protein (GFP) expressed in the cell-free system. The protein release and refolding processes, which are induced by ATP in natural molecular chaperone systems, were also simulated by methyl-β-cyclodextrin (M-β-CD). The CHP nanogels dissociate on complexation with M-β-CD to yield dissociated CHP. Thus, the dissociation of the CHP nanogel–protein complex subsequently allows for the release and folding of GFP. The folding kinetics in the presence of the CHP nanogel and M-β-CD was comparable to that of spontaneous folding in the absence of CHP/M-β-CD, indicating that the CHP nanogels did not affect protein synthesis in the cell-free system, providing correctly folded active proteins.
    Polymer Journal 01/2010; · 1.50 Impact Factor
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    ABSTRACT: This paper describes construction of a nanosensory device for amplified detection of biologically important amines as chemical signals. The device was inspired by a biological signal transduction system, and was fabricated on an artificial cell membrane through self-organization of the molecular components, such as a synthetic receptor and a natural enzyme. Selective recognition of biologically important amines was achieved by a synthetic receptor with a pyridoxal moiety, as evaluated by means of electronic absorption spectroscopy. The selectivity in detecting amines as chemical signals mainly depends on hydrophobicity of the amines. The event upon detecting the chemical signals was transmitted to an enzyme by a metal ion acting as a mediator species, and then the enzyme amplified the event by the catalytic reaction to obtain signal output. This paper is realization of a biomimetic signal transduction system using amines as chemical signals and may provide a useful guidepost for designing integrated nanosystems.
    Biotechnology and Bioengineering 10/2009; 105(1):37-43. · 4.16 Impact Factor

Publication Stats

246 Citations
116.38 Total Impact Points


  • 2008–2014
    • Tokyo Medical and Dental University
      • Institute of Biomaterials and Bioengineering
      Edo, Tōkyō, Japan
  • 2006–2013
    • Kyoto University
      • • Department of Polymer Chemistry
      • • Department of Synthetic Chemistry and Biological Chemistry
      Kioto, Kyōto, Japan
  • 2001–2012
    • Nara Institute of Science and Technology
      • Graduate School of Materials Science
      Ikoma, Nara, Japan
  • 2007
    • Kyushu University
      • Department of Applied Chemistry
      Fukuoka-shi, Fukuoka-ken, Japan