Nobutaka Hanagata

Hokkaido University, Sapporo, Hokkaidō, Japan

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Publications (163)558.89 Total impact

  • K. Shiba · M. Tagaya · T. Sugiyama · N. Hanagata
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    ABSTRACT: To design non-cytotoxic controlled release system is one of the major challenges in such a field as biomedicine. For this purpose, well-defined, luminescent and cytocompatible titania/dye hybrid nanoparticles (NPs) are synthesized. Hybrid NPs with a uniform size/shape are achieved through the precisely controlled microfluidic hydrolysis and co-condensation reactions of titanium tetraisopropoxide and 3-aminopropyltriethoxysilane in the presence of octadecylamine (ODA). By adding the cell-staining luminescent dye of fluorescein isothiocyanate (FITC) molecules to the starting solution, the spherical titania/aminopropyl-silica/ODA/FITC hybrid NPs (average diameter: 400 nm, coefficient of variation: 9.5%) are successfully prepared. Interestingly, the NPs demonstrate enhanced luminescence after treating them with phosphate buffered saline (PBS) and a possible mechanism is discussed. These NPs are not dispersed in PBS during the treatment due to their hydrophobic surface, which is an important feature to avoid cytotoxicity caused by an endocytic pathway. The NIH3T3 fibroblast viability test revealed that the hybrid NPs are not cytotoxic, realizing the clear imaging of the cellular shapes by the cellular gradual reactions mainly with the FITC released from the hybrid NPs floating at the interface. Therefore, the well-defined hybrid NPs with tailored surface/release properties are promising candidates for slowly-controlling cellular environmental systems (e.g., drug delivery systems).
    No preview · Article · Dec 2015 · RSC Advances
  • Song Chen · Xinxin Du · Lan Jia · Haixin Chang · Toshiyuki Ikoma · Nobutaka Hanagata
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    ABSTRACT: Combination of silica component with other materials is one of the current strategies to design bone regenerative materials. In this study, novel reduced graphene oxide (RGO)-aminosilica hybrid nanosheets with enhanced osteo-compatibility were synthesized from a mixture of 3-aminopropyltriethoxysilane (APTES), graphene oxides (GO) and water. The presence of APTES in the mixture not only caused the conversion of GO to RGO, but also led to the hydrolysis and condensation of itself. It was for the first time reported the reducing role of APTES in the conversion of GO to RGO. It was found that the silicon (IV) ions were released from the hybrid nanosheets in a sustained way. The in vitro osteo-compatibility was evaluated by incubating the hybrid nanosheets with osteoblast MC3T3-E1 cells. A water soluble tetrazolium salt assay quantitatively indicated that the hybrid nanosheets had no significant toxicity and exhibited good biocompatibility. An alkaline phosphatase assay quantitatively indicated that the hybrid nanosheets enhanced the osteoblast differentiation compared to the GO nanosheets. An immunochemical assay further qualitatively indicated that the hybrid nanosheets stimulated the production of osteopontin as typical marker for osteoblast differentiation. Thus, the resultant hybrids nanosheets had a potential application in the bone regeneration.
    No preview · Article · Dec 2015
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    ABSTRACT: Gene delivery is often accomplished by the forward or reverse transfection protocol. In either protocol, a transfection reagent (usually cationic) is added to increase the delivery efficiency. In this study, we employed a series of nanosheet networks to facilitate the delivery of naked plasmid DNA into human mesenchymal stem cells (hMSCs). By adding different chemicals into the reaction mixture for etching the silica glass, we were able to fabricate inorganic/organic hybrid nanosheet networks with different physico-chemical characteristics. We then analyzed the transfection efficiency on different nanosheets and the possible dependence of the transfection efficiency on the physico-chemical parameters of nanosheets. The results showed that all nanosheet networks were noncytotoxic and demonstrated a high cell survival rate (∼90%) after transfection. The transfection efficiency was critically determined by the aspect ratio (height/thickness of the wall) of the nanosheets. The effects of chemistry or other surface properties were not significant. Moreover, the transfection efficiency may be successfully predicted by the initial cell migration rate and the activation of integrin β3 on the nanosheets. Compared to the conventional method, transfection using concurrent cell/plasmid seeding on the nanosheets is not only more effective but also much safer. Future efforts may focus on combining the inorganic/organic hybrid nanosheets with soft substrates for in situ transfection.
    No preview · Article · Sep 2015 · Physical Chemistry Chemical Physics
  • Nobutaka Hanagata · Hiromi Morita
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    ABSTRACT: Contradictory results have been reported for in vitro evaluations of whether zinc oxide nanoparticles (ZnO NPs) are cytotoxic. Though there have been reports of ZnO NPs cytotoxicity due to Zn ions released from the nanoparticles, there have also been reports concluding that Zn ions are not cytotoxic. This inconsistency is mostly attributed to the types of cells used. In this research, we investigated the difference in the level of ZnO NPs cytotoxicity due to culturing conditions. The sensitivity of human lung epithelial cells to ZnO NPs cytotoxicity differed depending on the dispersing medium, physiological state of the cells resulting from their growth stage, and composition of the medium. Further, with regard to the toxicity of ZnO NPs, NPs internalized into cells had a greater cytotoxic effect than Zn ions released from ZnO NPs. Instead of inducing cell death, ZnO NPs internalized into cells slowed the rate of cell proliferation. Furthermore, the cytotoxicity of ZnO NPs depended greatly on the concentration of calcium ions (Ca(2+)) in the medium. When the concentration of Ca(2+) was low, the cytotoxicity of ZnO NPs increased markedly. However, the toxicity of ZnO NPs was mitigated by the addition of CaCl2 to the medium. Global gene expression analysis revealed that Ca(2+)-induced upregulation of cell cycle functions could be attributable to the mitigation of ZnO NP toxicity by Ca(2+).
    No preview · Article · Sep 2015 · The Journal of Toxicological Sciences
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    ABSTRACT: Toll-like receptor 9 recognizes CpG oligodeoxynucleotides (ODNs) and induces immune-mediator cytokines. Natural phosphodiester class B CpG ODNs induce interleukin-6 (IL-6), but not interferon-α (IFN-α). We prepared silicon nanoparticles (Si NPs) with different positive surface charge density and bound negatively charged class B CpG ODN molecules electrostatically. No significant differences in the amount of class B CpG ODN molecules or negative surface charge after binding of the molecules onto naked NPs was observed. However, the profile of cytokine induction from peripheral blood mononuclear cells was correlated with a positive surface charge density of naked NPs prior to binding of CpG ODN molecules. The level of IL-6 induction slightly decreased as the positive surface charge density was increased, while the IFN-α induction significantly increased as the positive surface charge density was increased. This observation demonstrates that the bifurcated cytokine induction can be regulated by the surface charge of naked NPs.
    Full-text · Article · Sep 2015 · Journal of Drug Delivery Science and Technology
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    ABSTRACT: Background Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood–brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. Method To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. Results A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm2. After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes. Conclusion These results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes.
    Preview · Article · Sep 2015 · International Journal of Nanomedicine
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    Huijie Zhang · Shini Feng · Ting Yan · Chunyi Zhi · Xiao-Dong Gao · Nobutaka Hanagata
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    ABSTRACT: CpG oligodeoxynucleotides (ODNs) stimulate innate and adaptive immune responses. Thus, these molecules are promising therapeutic agents and vaccine adjuvants against various diseases. In this study, we developed a novel CpG ODNs delivery system based on polyeth-yleneimine (PEI)-functionalized boron nitride nanospheres (BNNS). PEI was coated on the surface of BNNS via electrostatic interactions. The prepared BNNS–PEI complexes had positive zeta potential and exhibited enhanced dispersity and stability in aqueous solution. In vitro cytotoxicity assays revealed that the BNNS–PEI complexes with concentrations up to 100 μg/mL exhibited no obvious cytotoxicity. Furthermore, the positively charged surface of the BNNS– PEI complexes greatly improved the loading capacity and cellular uptake efficiency of CpG ODNs. Class B CpG ODNs loaded on the BNNS–PEI complexes enhanced the production of interleukin-6 and tumor necrosis factor-α from peripheral blood mononuclear cells compared with CpG ODNs directly loaded on BNNS. Contrary to the free CpG ODNs or CpG ODNs directly loaded on BNNS, class B CpG ODNs loaded on the BNNS–PEI complexes induced interferon-α simultaneously. PEI coating may have changed the physical form of class B CpG ODNs on BNNS, which further affected their interaction with Toll-like receptor 9 and induced interferon-α. Therefore, BNNS–PEI complexes can be used to enhance the immunostimulatory effect and therapeutic activity of CpG ODNs and the treatment of diseases requiring interleukin-6, tumor necrosis factor-α, and interferon-α.
    Full-text · Article · Aug 2015 · International Journal of Nanomedicine
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    Yi Xu · Peter Claiden · Yufang Zhu · Hiromi Morita · Nobutaka Hanagata
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    ABSTRACT: Abstract In this study, we proposed to modify mesoporous silica nanoparticles (MSNs) with 3-aminopropyltriethoxysilane (NH2-TES), aminoethylaminopropyltriethoxysilane (2NH2-TES) and 3-[2-(2-aminoethylamino)ethylamino] propyl-trimethoxysilane (3NH2-TES) for binding of cytosine-phosphate-guanosine oligodexynucleotides (CpG ODN), and investigated the effect of different amino groups of MSNs on the CpG ODN delivery. Serum stability, in vitro cytotoxicity, and cytokine interleukin-6 (IL-6) induction by MSN-NH2/CpG, MSN-2NH2/CpG and MSN-3NH2/CpG complexes were investigated in detail. The results showed that three kinds of aminated-MSN-based CpG ODN delivery systems had no cytotoxicity to RAW264.7 cells, and binding of CpG ODN to MSN-NH2, MSN-2NH2 and MSN-3NH2 nanoparticles enhanced the serum stability of CpG ODN due to protection by the nanoparticles. However, three aminated MSN-based CpG ODN delivery systems exhibited different CpG ODN delivery efficiency, and MSN-NH2/CpG complexes had the highest ability to induce IL-6 secretion.
    Full-text · Article · Aug 2015 · Science and Technology of Advanced Materials
  • Song Chen · Qiqing Zhang · Lan Jia · Xinxin Du · Nobutaka Hanagata
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    ABSTRACT: A controllable length of inorganic nanotubes as a drug delivery system is crucial to understanding internalization mechanisms and designing new biomedical applications. In this study, silica nanotubes (SiNTs) with controlled length ranging from several hundred nanometers to several micrometers were firstly fabricated via a facile and effective acid-degradation collagen template route and then functionalized with chitosan (ChSiNTs) to deliver immunostimulatory cytosine-phosphodiester-guanine oligodeoxynucleotides (CpG ODNs). It was found that the length of SiNTs could be well controlled through the adjustment of the acid-treatment temperature. Cytotoxic assessment indicated that SiNTs exhibited good biocompatibility when separately incubated with four types of cell lines: 293XL-hTLR9, A549, NIH3T3, and C2C12. The cellular uptake of SiNTs was strongly affected by their length and cell type. A decrease in the length led to an increase in the cellular uptake of SiNTs, while a significantly higher cellular uptake by C2C12 cells was observed in comparison with A549 and NIH3T3 cells. An immunochemical assay revealed that SiNTs were located in the endolysosomes after cellular internalization. ChSiNTs were positive and well complexed with negative CpG ODNs to produce a ChSiNT/CpG ODN complex (CpG-ChSiNT) via electric force. ChSiNTs were located in the endolysosomes after internalization and enhanced the cellular uptake of CpG-ODNs. CpG ODNs could be released from CpG-ChSiNTs in a sustained way and specifically recognized by the TLR9 receptor in 293XL-hTLR9 cells. The amount of interleukin-6 cytokine stimulated by CpG-ChSiNT against peripheral blood mononuclear cells was higher than that by free CpG ODNs and ChSiNTs significantly enhanced the immunostimulatory response of CpG ODNs.
    No preview · Article · Aug 2015 · Journal of Materials Chemistry B
  • Nobutaka Hanagata
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    ABSTRACT: Using global gene expression analysis, the effects of biomaterials and nanomaterials can be analyzed at the genetic level. Even though information obtained from global gene expression analysis can be useful for the evaluation and design of biomaterials and nanomaterials, its use for these purposes is not widespread. This is due to the difficulties involved in data analysis. Because the expression data of about 20,000 genes can be obtained at once with global gene expression analysis, the data must be analyzed using bioinformatics. A method of bioinformatic analysis called gene ontology can estimate the kinds of changes on cell functions caused by genes whose expression level is changed by biomaterials and nanomaterials. Also, by applying a statistical analysis technique called hierarchical clustering to global gene expression data between a variety of biomaterials, the effects of the properties of materials on cell functions can be estimated. In this chapter, these theories of analysis and examples of applications to nanomaterials and biomaterials are described. Furthermore, global microRNA analysis, a method that has gained attention in recent years, and its application to nanomaterials are introduced. © 2015 S. Karger AG, Basel.
    No preview · Article · Jul 2015 · Frontiers of oral biology
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    ABSTRACT: In the present study, the interaction of 5-Fluorouracil with herring sperm DNA is reported using spectroscopic and molecular modeling techniques. This binding study of 5-FU with hs-DNA is of paramount importance in understanding chemico-biological interactions for drug design, pharmacy and biochemistry without altering the original structure. The challenge of the study was to find the exact binding mode of the drug 5-Fluorouracil with hs-DNA. From the absorption studies, a hyperchromic effect was observed for the herring sperm DNA in the presence of 5-Fluorouracil and a binding constant of 6.153 × 103M-1 for 5-Fluorouracil reveals the existence of weak interaction between the 5-Fluorouracil and herring sperm DNA. Ethidium bromide loaded herring sperm DNA showed a quenching in the fluorescence intensity after the addition of 5-Fluorouracil. The binding constants for 5-Fluorouracil stranded DNA and competitive bindings of 5-FU interacting with DNA-EB systems were examined by fluorescence spectra. The Stern-Volmer plots and fluorescence lifetime results confirm the static quenching nature of the drug-DNA complex. The binding constant Kb was 2.5 × 104 L mol-1 and the number of binding sites are 1.17. The 5-FU on DNA system was calculated using double logarithmic plot. From the Forster nonradiative energy transfer study it has been found that the distance of 5-FU from DNA was 4.24 nm. In addition to the spectroscopic results, the molecular modeling studies also revealed the major groove binding as well as the partial intercalation mode of binding between the 5-Fluorouracil and herring sperm DNA. The binding energy and major groove binding as -6.04 kcal mol-1 and -6.31 kcal mol-1 were calculated from the modeling studies. All the testimonies manifested that binding modes between 5-Fluouracil and DNA were evidenced to be groove binding and in partial intercalative mode.
    Full-text · Article · Jul 2015 · Biophysical Reviews and Letters
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    ABSTRACT: A versatile method for the rapid fabrication of aligned fullerene C60 nanowhiskers (C60NWs) at the air-water interface is presented. This method is based on the vortex motion of a sub-phase (water), which directs floating C60 nanowhiskers (C60NWs) to align on the water surface according to the direction of rotational flow. Aligned C60NWs could be transferred onto many different flat substrates and, in this case, aligned C60NWs on glass substrates were employed as a scaffold for cell culture. Bone forming human osteoblast MG63 cells adhered well to the C60NWs and their growth was found to be oriented with the axis of the aligned C60NWs. Cells grown on aligned C60NWs were more highly oriented with the axis of alignment than when grown on randomly oriented nanowhiskers. A study of cell proliferation on the C60NWs implied their non-toxicity indicating their potential for use in biomedical applications.
    No preview · Article · Jun 2015 · ACS Applied Materials & Interfaces
  • Nobutaka Hanagata
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    ABSTRACT: Interferon-induced transmembrane protein 5 (IFITM5) is an osteoblast-specific membrane protein that has been shown to be a positive regulatory factor for mineralization in vitro. However, Ifitm5 knockout mice do not exhibit serious bone abnormalities, and thus the function of IFITM5 in vivo remains unclear. Recently, a single point mutation (c.-14C>T) in the 5′ untranslated region of IFITM5 was identified in patients with osteogenesis imperfecta type V (OI-V). Furthermore, a single point mutation (c.119C>T) in the coding region of IFITM5 was identified in OI patients with more severe symptoms than patients with OI-V. Although IFITM5 is not directly involved in the formation of bone in vivo, the reason why IFITM5 mutations cause OI remains a major mystery. In this review, the current state of knowledge of OI pathological mechanisms due to IFITM5 mutations will be reviewed.
    No preview · Article · Jun 2015 · Journal of Bone and Mineral Metabolism
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    ABSTRACT: Background Extrahepatic cholangiocarcinoma is very difficult to diagnose at an early stage, and has a poor prognosis. Novel markers for diagnosis and optimal treatment selection are needed. However, there has been very limited data on the proteome profile of extrahepatic cholangiocarcinoma. This study was designed to unravel the proteome profile of this disease and to identify overexpressed proteins using mass spectrometry-based proteomic approaches.Methods We analyzed a discovery set of formalin-fixed paraffin-embedded tissues of 14 extrahepatic cholangiocarcinomas employing shotgun mass spectrometry, and compared proteome profiles with those of 7 controls. Then, selected candidates were verified by quantitative analysis using scheduled selected reaction monitoring-based mass spectrometry. Furthermore, immunohistochemical staining employed a validation set of 165 cases.ResultsIn total, 1,992 proteins were identified and 136 proteins were overexpressed. Verification of 58 selected proteins by quantitative analysis revealed 11 overexpressed proteins. Immunohistochemical validation for 10 proteins showed positive rates of S100P (84%), CEAM5 (75%), MUC5A (62%), OLFM4 (60%), OAT (42%), CAD17 (41%), FABPL (38%), AOFA (30%), K1C20 (25%) and CPSM (22%) in extrahepatic cholangiocarcinomas, which were rarely positive in controls.Conclusions We identified 10 proteins associated with extrahepatic cholangiocarcinoma using proteomic approaches. These proteins are potential targets for future diagnostic biomarkers and therapy.
    Full-text · Article · Apr 2015 · Journal of Hepato-Biliary-Pancreatic Sciences
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    Shanmugavel Chinnathambi · Nobutaka Hanagata

    Full-text · Conference Paper · Mar 2015
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    ABSTRACT: The interaction of antimetabolite 5-fluorouracil (5FU) with bovine serum albumin (BSA) under UVC (253.7 nm) irradiation was investigated in the present study using UV-Vis spectroscopy, steady state/time resolved fluorescence spectroscopic techniques. The stability of protein was found to be very strong when BSA gets bind to 5FU and moreover it is compared with the free BSA under UVC irradiation. From the fluorescence spectroscopic study, the stability of the complex was found to acquire 2-fold stronger than free protein. From the molecular modelling studies, we came to know the hydrogen bonds between BSA and antimetabolite 5FU are strong, up to 70.4 J/m 2 under UVC irradiation.
    Full-text · Article · Jan 2015 · International Journal of Spectroscopy
  • Cuilian Tao · Yufang Zhu · Xianglan Li · Nobutaka Hanagata
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    ABSTRACT: We developed a potential cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) delivery system by binding of CpG ODN onto aminated mesoporous silica nanoparticles (MSNs) to form CpG/MSN-NH2 complexes for Toll-like receptor 9 (TLR9)-mediated induction of cytokines. Serum stability, in vitro cytotoxicity, cellular uptake, and interleukin-6 (IL-6) induction of CpG/MSN-NH2 complexes were investigated. The results showed that MSN-NH2 nanoparticles had no cytotoxicity to Raw 264.7 cells, and binding of CpG ODN to MSN-NH2 nanoparticles enhanced serum stability of CpG ODN due to the protection by nanoparticles. Furthermore, CpG/MSN-NH2 complexes could be efficiently taken up by cells due to small particle size and good dispersity. Most importantly, CpG/MSN-NH2 complexes significantly enhanced the level of IL-6 induction, stimulated by interaction between CpG ODN and TLR9 in endolysosomes. Therefore, MSNs would be a promising carrier for enhancing the delivery efficiency of CpG ODN.
    No preview · Article · Nov 2014 · Microporous and Mesoporous Materials
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    ABSTRACT: Theranostic nanoparticles currently have been regarded as an emerging concept of 'personalized medicine' with diagnostic and therapeutic dual-functions. Eu3+ doped hydroxyapatite (HAp) has been regarded as a promising fluorescent probe for in vivo imaging applications. Additionally, substitution of Ca2+ with Fe3+ in HAp crystal may endow the capability of producing heat upon exposure to a magnetic field. Here we report a preliminary study of doping mechanism and photoluminescence of Eu3+ and Fe3+ doped HAp nanoparticles (Eu/Fe:HAp). HAp with varied concentration of Eu3+ and Fe3+ doping are presented as Eu(10 mol%):HAp, Eu(7 mol%)-Fe(3 mol%):HAp, Eu(5 mol%)-Fe(5 mol%):HAp, Eu(3 mol%)-Fe(7 mol%):HAp, and Fe(10 mol%):HAp in the study. The results showed that the HAp particles, in nano-size with rod-like morphology, were successfully doped with Eu3+ and Fe3+, and the particles can be well suspended in cell culture medium. Photoluminescence analysis revealed that particles have prominent emissions at 536 nm, 590 nm, 615 nm, 650 nm and 695 nm upon excitation at a wavelength of 397 nm. Moreover, these Eu/Fe:HAp nanoparticles belonged to B-type carbonated HAp, which has been considered an effective biodegradable and biocompatible drug/gene carrier in biological applications.
    Full-text · Article · Sep 2014 · Science and Technology of Advanced Materials
  • Cuilian Tao · Yufang Zhu · Xianglan Li · Nobutaka Hanagata
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    ABSTRACT: We developed a potential cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) delivery system based on magnetic mesoporous silica (MMS) nanoparticles by binding of CpG ODN onto aminated MMS (MMS-NH2) nanoparticles to form CpG/MMS-NH2 complexes for Toll-like receptor 9 (TLR9)-mediated induction of cytokines. Magnetization, serum stability, in vitro cytotoxicity, cellular uptake, and interleukin-6 (IL-6) induction of CpG/MMS-NH2 complexes were evaluated. The results showed that MMS nanoparticles exhibited superparamagnetic behavior with a saturation magnetization of 6.5 emu/g. Also, MMS-NH2 nanoparticles had no cytotoxicity to Raw 264.7 cells, and CpG/MMS-NH2 complexes enhanced serum stability of CpG ODN and could be localized in the endolysosomes after endocytosis by cells. Importantly, CpG/MMS-NH2 complexes significantly enhanced the TLR9-mediated IL-6 induction compared to free CpG ODN. Therefore, CpG/MMS-NH2 complexes could be magnetic targeted delivery and significantly enhance the TLR9-mediated cytokine induction for stimulating immune responses.
    No preview · Article · Sep 2014 · RSC Advances
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    ABSTRACT: T cell receptor (TCR) phosphorylation requires the kinase Lck and phosphatase CD45. CD45 activates Lck by dephosphorylating an inhibitory tyrosine of Lck to relieve autoinhibition. However, CD45 also dephosphorylates the TCR, and the spatial exclusion of CD45 from TCR clustering in the plasma membrane appears to attenuate this negative effect of CD45. To further investigate the role of CD45 in signal initiation, we reconstituted membrane TCR clusters in vitro on supported lipid bilayers. Fluorescence microscopy of single clusters showed that incorporation of CD45 enhanced phosphorylation of TCR clusters, but only when Lck co-clustered with TCR. We found that clustered Lck autophosphorylated the inhibitory tyrosine and thus could be activated by CD45, whereas diffusive Lck molecules did not. In the TCR-Lck clusters and at low CD45 density, we speculate that the effect of Lck activation may overcome dephosphorylation of TCR, resulting in a net positive regulation. The CD45 density in physiological TCR clusters is also low because of the exclusion of CD45. Thus, we propose that the spatial organization of TCR/Lck/CD45 in T cell membranes is important not only for modulating the negative role of CD45 but also for creating conditions in which CD45 has a positive role in signal initiation.
    Preview · Article · Aug 2014 · Journal of Biological Chemistry

Publication Stats

3k Citations
558.89 Total Impact Points


  • 2010-2015
    • Hokkaido University
      • Graduate School of Life Science
      Sapporo, Hokkaidō, Japan
  • 2006-2015
    • National Institute for Materials Science
      • • Nanotechnology Innovation Station
      • • International Center for Young Scientist (ICYS)
      Tsukuba, Ibaraki, Japan
  • 2013
    • Anna University, Chennai
      • Department of Medical Physics
      Chennai, Tamil Nādu, India
  • 2003-2006
    • Tokyo University of Technology
      • School of Bionics
      Edo, Tōkyō, Japan
  • 1993-2006
    • The University of Tokyo
      • Research Center for Advanced Science and Technology
      Tokyo, Tokyo-to, Japan
  • 1998
    • Research Institute of Innovative Technology for the Earth
      Kioto, Kyoto, Japan