7thSNM2013 7th International Symposium

on Nanomedicine

November 7-9, 2013

At Nakamura Centenary Hall

of Kyushu Institute of Technology

(Kitakyushu)

Organized by Kyushu Institute of technology

Society of Nanoscience and Technology Innovative Area “NanoMedicine Molecular Science”

JST-CREST Japan Nanomedicine Society

Cooperated by

Surface Science Society of Japan

Financial Support by Program for Promoting Inter-University Collaborative Education, Ministry of Education, Culture, Sports, Science and Technology

Financial support available for

Academic conference and meetings, West Japan Industry and Trade Convention Association, Kitakyushu

Konica Minolta Science and Technology Foundation

1

Scientific Organizing Committee Chair

Shigeori TAKENAKA (Kyushu Inst. Tech.) Tsuneo URISU (Nagoya Univ.)

Steering Committee

Chair Kosuke GONDA (Tohoku Univ.) Miwa SUGIURA (Ehime Univ.)

Yasuhisa FUJITA (Shimane Univ.) Yumiko KINOSHITA (JNMS)

Members Atsushi NATSUME (Nagoya Univ.) Hideo HIGUCHI (The Univ. Tokyo)

Hiroo IWATA (Kyoto Univ.) Kanta TSUMOTO (Mie Univ.)

Kazuhiko ISHIHARA (The Univ. Tokyo) Keiichi TORIMITSU (Tohoku Univ.) Manabu TOKESHI (Hokkaido Univ.) Masahiro HIRAOKA (Kyoto Univ.)

Masahito YAMAZAKI (Shizuoka Univ.) Masako YUDASAKA (AIST)

Noriaki OHUCHI (Tohoku Univ.) Shinae KONDOH (Tokyo Tech.)

Shinobu SATO (Kyushu Inst. Tech.) Takahisa TAGUCHI (NICT)

Takeaki OZAWA (The Univ. Tokyo) Takahisa DEWA (Nagoya Inst. Tech.)

Tyuji HOSHINO (Chiba Univ.) Tomomi NEMOTO (Hokkaido Univ.) Tomohiro KONNO (The Univ. Tokyo)

Toshio OGINO (Yokohama Nation. Univ.) Yoko MATSUMOTO (Sojo Univ)

Yoko Urabe (JNMS) Yumiko KINOSHITA (JNMS)

Symposium SecretariatShinobu Sato Kyushu Institute of Technology

2

-----Welcome ----- Welcome to the 7th International Symposium on Nanomedicine (ISNM2013) arranged by

Prof. Shigeori Takenaka of Kyushu Institute of Technology - the host university and also the

special session “Brain nanomedicine,” is arranged by Prof. Tsuneo Urisu of Japan

Nanomedicine Society. This Symposium was started seven years ago to discuss the latest

results of research on Nanomedicine and related technologies. We are delighted to have the

seventh one today and I would like to express our sincere thanks to all of the participants.

The combination of top-down and bottom-up nano-technologies evoked a real revolution

in various fields of science and technology. Medical treatment is one of the promising fields

where we will find important applications of this new technology. Once recognized, we can

expect these new technologies to be used for developing novel devices or methods for

treatment of cancer, neurodegenerative diseases and many other intractable diseases.

The aims of this Symposium are (1) to provide a platform on which to discuss the future

prospects for these science-technologies and construct an international human network in

the fields of Nano-Bio-Medicine and (2) to foster excellent young generation researchers in

these fields based on this network. We take it especially important to construct an Asian

network and invite several speakers in the related fields from these countries. The

keywords of the Symposium are cancer, molecular imaging, drug delivery, regenerative

medicine, neurodegenerative disease and other nanomedicine topics.

I would like to thank again all of the participants for their attendance. I hope that this

Symposium will not only be fruitful to all of the participants but also provide utmost

importance for the improvement in the quality of our life and various state-of-the-art

technologies. Thank you very much.

Chairmen of the Symposium

Shigeori TAKENAKA, Professor Kyushu Institute of Technology

Tsuneo URISU, Professor Nagoya University

3

Table of Contents

WELCOME-7th ISNM2013 3

Location of Kyushu Inst. Tech. 5

Programme 8

Plenary Talk 23

Key Note Speech 25

Special Lecturers 29

Invited Speakers Brain nanomedicine Special Session

38

Invited Speakers Japan-Korea Special Session for Bionano-sensing Technology

46

Invited Speakers 52

Poster Presentations 78

List of Authors 129

4

Location of Kyushu Inst. Tech.

Address : 1-1 Sensui-cho, Tobata-ku, Kitakyushu-shi, Fukuoka, 804-8550, JAPAN

Phone : +81-(0)93-884-3322

General Fax : +81-(0)93-884-3322

The Kyushu Institute of Technology is located in Fukuoka Prefecture on the island of Kyushu.

The nearest airport is Kitakyushu Airport and the second nearest is Fukuoka Airport. For

flights from Asian countries, these two airports provide convenient access to Kyutech.

When arriving at Narita International Airport or Kansai International Airport, transfer to

domestic flight bound for either Kitakyushu or Fukuoka airport is required. Or taking bullet

train (Shinkansen) from Tokyo or Osaka is an alternative option.

5

Access to Tobata campus in Kyushu Inst. Tech.

from Fukuoka City From Fukuoka Airport to Kokuraekimae Bus Stop by Nishitetsu Limited Express Bus for

Kokura (1 h 40 min.)

From Kokura Station to Kyushukodaimae Station by train (6 min.)

From Kyushukodaimae Station to Kyutech on foot (6 min.)

from Tokyo From Tokyo Station to Kokura Station by Shinkansen (4 h 40 min.)

From Kokura Station to Kyushukodaimae Station by train (6 min.)

From Kyushukodaimae Station to Kyutech on foot (6 min.)

from Osaka From Kansai Airport to Shin-Osaka Station by express train (50 min.)

From Shin-Osaka Station to Kokura Station by Shinkansen (2 h 20min.)

From Kokura Station to Kyushukodaimae Station by train (6 min.)

From Kyushukodaimae Station to Kyutech on foot (6 min.)

6

Tobata campus map Nakamura Centenary Hall

Main gate

Nakamura Centenary Hall

7

Programme

8

7th ISNM 2013 Time Table 2013.11.6 2013.11.7 2013.11.8 2013.11.9 2013.11.10

8:30 Registration Registration Registration

Shigeori TAKENAKA Opening remarks

Taek Dong CHUNG(SNU, Korea)Sihyun HAM

(Sookmyung Women's Univ., Korea)

Min-Ho LEE(KETI, Korea)Yoon-Bo SHIM (PNU, Korea)Haesik YANG(PNU, Korea)

Tsuneo URISU Closing remarks

Shigeori TAKENAKA(Kyushu Inst.Tech., Japan)

Tour ofTakenaka Lab.

CB

Masahiro TOMITA (Mie Univ., Japan)

Ayumi HIRANO-IWATA(Tohoku Univ., Japan)

Yoshihiro UTO (The Univ. Tokushima, Japan)

Lunch

Hideaki YOSHIMURA(The Univ.Tokyo, Japan)

Dinner Party(only invited speaker)

CB

Peilin CHEN(Academia Sinica, Taiwan)

BanquetCafé de Rouge Blanc

CB

Chie HOSOKAWA(AIST, Japan)

Kenjiro IWANAGA(Kyushu Dental Univ., Japan)

Koji HARANO(The Univ. Tokyo, Japan)

Madoka SUZUKI(Waseda Univ., Japan)

Moving to Dinner Place by bus.

Hideo HIGUCHI(The Univ. Tokyo, Japan)

Li-jun WAN (Chinese Academy of Science,

China)

CB

Lunch

Ikuo FUJII (Osaka Pref. Univ., Japan)

Tomohiro KONNO(The Univ. Tokyo, Japan)

Shinji SUEDA(Kyushu Inst. Tech., Japan)

Steven BOXER(Stanford Univ., USA)

Hiroyuki ASANUMA(Nagoya Univ., Japan)

Kanta TSUMOTO (Mie Univ., Japan)

Tsuneo URISU(Nagoya Univ., Japan)

Shigeru NISHIZAWA(Univ. Occupational & Environmental Health, Japan)

Alberto BIANCO (CNRS, France)

Masako YUDASAKA(AIST,Japan)

Yuko ICHIYANAGI(Yokohama Nation. Univ., Japan)

CB

Akio OJIDA(Kyushu Univ., Japan)

Yoko MATSUMOTO (Sojo Univ., Japan)

Poster

13:00

14:00

Chung-Shi YANG(Nation. Health Res. Inst.,

Taiwan)

Yuki MORI (Osaka Univ., Japan)

11:00

12:00

Ning GU(Southeast Univ., China)

Hikaru SATO (KEK, Japan)

Kosuke GONDA(Tohoku Univ., Japan)Michihiro NAKAMURA

(The Univ. Tokushima, Japan)

Tour ofKyushu Inst.

Tech.

9:00

10:00

Morio MATSUNAGA Opening remarks

Fuyuhiko TAMANOI(UCLA, USA)

Yasuhisa FUJITA(Shimane Univ., Japan)

Manabu TOKESHI(Hokkaido Univ., Japan)

Yoshinobu BABA(Nagoya Univ., Japan)

Kazuhiko ISHIHARA(The Univ. Tokyo, Japan)

20:00

17:00

18:00

19:00

15:00

Registration

16:00

CB

Lunch

Tomomi NEMOTO(Hokkaido Univ., Japan)

Takeharu NAGAI(Osaka Univ., Japan)

Nobutaka HANAGATA(NIMS,Hokkaido Univ., Japan)

Yoshiyuki KONISHI(Univ. Fukui, Japan)

CB

Kazuya UEZU (The Univ. Kitakyushu, Japan)

Shinsuke SANDO (Kyushu Univ.,Japan)

Miwa SUGIURA(Ehime Univ., Japan)

Takefumi YAMASHITA (The Univ. Tokyo, Japan)

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Thursday, 7th November, 2013 9:00 Opening

remarks Morio MATSUNAGA, President of Kyushu Inst. Tech., Japan

Chair: Shigeori TAKENAKA

9:10 P1-1 Fuyuhiko TAMANOI, UCLA, USA Discoveries at the Interface of Nanomaterials and Biology

Chair: Kosuke GONDA

9:40 K1-1 Hideo HIGUCHI, The Univ. Tokyo, Japan

Noninvasive in vivo imaging of neutrophil and tumor in mouse auricles

10:10 K1-2 Yoshinobu BABA, Nagoya Univ., Japan

Nano- and Quantum-Biodevices for Cancer Diagnosis, Cancer Therapy, and iPS Cell Based Regenerative Medicine

10:40 Break Chair: Yuko ICHIYANAGI

11:00 I1-1 Kosuke GONDA, Tohoku Univ., Japan High Accuracy Imaging of Cancer and Peripheral Artery Disease with X-ray CT and Fluorescence

11:20 I1-2 Michihiro NAKAMURA, The Univ. Tokushima, Japan

Organosilica Nanoparticles for Multimodal imaging

11:40 I1-3 Shinji SUEDA, Kyushu Inst. Tech., Japan

Development of bioassays using biotin ligase modified with a luminescent Tb3+ complex

12:00 Lunch Chair: Kanta TSUMOTO

13:00 S1-1 Li-jun WAN, Chinese Academy of Science, China

Surface Molecular Patterns: Method and Possible Application for Bioanalysis

13:30 S1-2 Alberto BIANCO, CNRS, France Chemical functionalization tailors the properties of carbon nanonotubes for biomedical applications

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14:00 I1-4 Masako YUDASAKA, AIST, Japan

Graphene-based biosensors

14:20 I1-5 Yuko ICHIYANAGI, Yokohama Nation. Univ., Japan

Optimization of Self-Heating Properties of Multifunctional Magnetic Nanoparticles

14:40 Break Chair: Yasuhisa FUJITA

14:55 S1-3 Ning GU, Southeast Univ., China

The enzyme-like activities of iron-based nanoparticles 15:25 I1-6 Akio OJIDA, Kyushu Univ., Japan

Nanotechnology for the detection and prognosis of disease stage: Protein quantification for clinical approach

15:45 I1-7 Kanta TSUMOTO, Mie Univ., Japan

Cell-Sized Liposomes: Basic Structures for Mimicking Cell Systems

16:05 I1-8 Yoko MATSUMOTO, Sojo Univ., Japan Anti-tumor Effects of Cationic Liposomes Against Tumor Cells Along With Apoptosis

16:25 I1-9 Hikaru SATO, KEK, Japan Introduction for the medical particle accelerator

16:45 Break Chair: Shinobu SATO

17:00 S1-4 Peilin CHEN, Academia Sinica, Taiwan Multifunctional Organic Platform for On Chip Cell Sensing, Sorting and Manipulation

17:30 I1-10 Kenjiro IWANAGA, Kyushu Dental Univ., Japan

Local delivery system of cytotoxic agents to tumors by focused sonoporation

17:50 I1-11 Koji HARANO, The Univ. Tokyo, Japan

Lung-Specific siRNA Delivery Using Water-Soluble Amino-Fullerene

18:10 I1-12 Madoka SUZUKI, Waseda Univ., Japan Single cell-scale thermometry by fluorescent temperature reporters

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Friday, 8th November, 2013 Chair: Masako YUDASAKA

9:00 S2-1 Kazuhiko ISHIHARA, The Univ. Tokyo, Japan Cell membrane permeable and cytocompatible phospholipid polymer nanoprobes conjugated with molecular beacons

9:30 S2-2 Takeharu NAGAI, Osaka Univ.,J apan

Genetically-encoded functional probes applicable in conjunction with photo-manipulation technologies

10:00 Break Chair: Takeharu NAGAI

10:10 K2-1 Steven BOXER, Stanford Univ., USA

Reactions, Interactions and Dynamics in Biological Membranes: Vesicles to Brains

10:40 I2-1 Hiroyuki ASANUMA, Nagoya Univ., Japan Functional siRNA for fluorescent monitoring of RISC with improved activity and strand selectivity

11:00 B2-1 Tomomi NEMOTO, Hokkaido Univ., Japan

Improvement of Penetration Depth and Lateral Spatial Resolution of Two-photon Microscopy with Novel Lasers

11:20 I2-2 Ikuo FUJII, Osaka Pref. Univ., Japan

Post Antibody Medicines: Generation of Molecular-targeting Peptides by Directed Evolution

11:40 I2-3 Tomohiro KONNO, The Univ. Tokyo, Japan

Cytocompatible phospholipid polymer hydrogels for cell engineering

12:00 Lunch

13:00 Poster presentation

Chair: Peilin CHEN

15:00 S2-3 Chung-Shi YANG, Nation. Health Res. Inst., Taiwan

Nanoparticles In Vivo and the Biomedical Applications 15:30 I2-4 Miwa SUGIURA, Ehime Univ., Japan

Efficient Photosynthetic Electron Transfer and Water Oxidation in Photosystem II

12

15:50 I2-5 Takefumi YAMASHITA, The Univ. Tokyo, Japan

Toward a better understanding of the molecular recognition mechanism: A molecular dynamics study

16:10 I2-6 Kazuya UEZU, The Univ. Kitakyushu, Japan

Computational Structural Analysis of β-1,3-D-Glucans as a Drug-Delivery Carrier

16:30 Break Chair: Shinsuke SANDO

16:45 B2-2 Shigeru NISHIZAWA, Univ. Occupational & Environmental

Health, Japan

Production of therapeutic medicine for cerebral vasospasm after subarachnoid hemorrhage base on basic research

17:05 B2-3 Shinsuke SANDO, Kyushu Univ., Japan Cell-Cell Adhesion Control by Nucleic Acid Aptamers

17:25 B2-4 Yuki MORI, Osaka Univ., Japan

In vivo MRI monitoring of immune cell migration in mouse brain using superparamagnetic nanoparticles

17:45 B2-5 Yoshiyuki KONISHI, Univ. Fukui, Japan Local regulation of molecular reactions in maintaining axonal morphology

18:05 B2-6 Chie HOSOKAWA, AIST, Japan

Laser Manipulation of Living Neuronal Network 18:25 B2-7 Tsuneo URISU, Nagoya Univ., Japan

Development of homogeneous neural network and multichannel incubation-type planar patch clamp -aiming high performance disease model chip-

18:45 Break

19:00 Banquet

13

Saturday, 9th November, 2013 Chair: Miwa SUGIURA

9:00 I3-1 Shigeori TAKENAKA, Kyushu Inst. Tech., Japan Ferrocenylnaphthalene diimide (FND)-based electrochemical telomerase assay (ECTA) for diagnosis of premalignant oral lesions

9:20 I3-2 Nobutaka HANAGATA, NIMS, Hokkaido Univ., Japan

Action of Immunostimulatory DNA Drug Can Be Changed by Nanoparticle

9:40 I3-3 Yasuhisa FUJITA, Shimane Univ., Japan

Investigation of functional Zinc Oxide nanoparticles for biomedical applications

10:00 I3-4 Manabu TOKESHI, Hokkaido Univ.,Japan

Easy-to-Use Detection of Biomarkers and Fabrication of Nanoparticles for Gene Therapy Using Microfluidic Devices

10:20 Break Chair: Shinji SUEDA

10:30 I3-5 Hideaki YOSHIMURA, The Univ. Tokyo, Japan

Analysis of RNA dynamics in living cells based on single molecule imaging

10:50 S3-1 Masahiro TOMITA, Mie Univ., Japan New Hybridoma Technology for Selective Production of Stereospecific Monoclonal Antibodies

11:20 I3-6 Ayumi HIRANO-IWATA, Tohoku Univ., Japan

Microfabricated Silicon Chips for Recording hERG Channel Activities

11:40 I3-7 Yoshihiro UTO, The Univ. Tokushima, Japan

Development of Immunomodulatory Cancer Therapy Based on Gc protein-derived Macrophage Activating Factor (GcMAF)

12:00 Lunch

14

13:00 Opening

remarks

The Sixth Japan-Korea Joint Symposium on Bio-microsensing Technology

Shigeori TAKENAKA, Director of RCBT, Kyushu Inst. Tech., Japan

Chair: Yoshihiro UTO

13:10 T3-1 Taek Dong CHUNG, SNU,Korea

Ionics and Gold Microshells for Bioanalysis 13:30 T3-2 Sihyun HAM, Sookmyung Women's Univ., Korea

To be Soluble, or not to be, that is a question 13:50 T3-3 Min-Ho LEE, KETI, Korea

Metalloid polymer nanoparticle functionalized graphene oxide working electrode for durable glucose sensing

14:10 T3-4 Yoon-Bo SHIM, PNU, Korea

Applications of Potential Modulated Microfluidic Channel System to Electrochemical Analysis

14:30 T3-5 Haesik YANG, PNU, Korea

Glucose-Oxidase Label-Based Redox Cycling for an Incubation Period-Free Electrochemical Immunosensor

14:50 Closing

remarks Tsuneo URISU, Nagoya Univ., Japan

15

Poster session (13:00-15:00 on 8th November, 2013) Poster Presentations Odd numbers: 13:00-14:00, Even numbers: 14:00-15:00 All posters will be displayed in the forum of Nakamura Centenary Hall

P01 Micro-tissue assembly by live-fabrication technique

using near-infrared-responsive gel A. Shirakihara1, H. Koga1, T. Sada2, T. Fujigaya2, N. Nakashima2, K. Nakazawa1

(1The University of Kitakyushu, 2Kyushu University) P02 Synthesis of FasL mimetic peptide modified

dendrimer and investigation of apoptosis inducing effect A. Minohoshi1, T. Yamamoto2, T. Mori1, A. Kishimura1, Y. Katayama1 (1Kyushu

University, 2Information Technologies and Nanotechnologies) P03 Embryoid body culture of mouse iPS cell using

microwell chips D.Miyamoto, K.Nakazawa (The University of Kitakyushu)

P04 Immunosuppressive DNA Nanomedicine against Autoimmune Disorders D. Bheema1,2, N. Hanagata1,2 (1Hokkaido University, 2National Institute for

Materials Science) P05 A rational design strategy to design latent ratiometric

fluorescent pH probes based on self-assembled SNARF derivatives E. Nakata, T. Morii (Kyoto University)

P06 Anti-inflammatory effect of IL-1 receptor antagonist gene transfection into synovial cells using sonoporation F. Nagao1, T. Tsuzisawa2, K. Iwanaga3, M. Habu3, I. Yoshioka1, T. Nishihara4, K.

Tominaga3 (1Division of Oral Medicine, 2School of Oral Health Science, 3Division

of Oral and Maxillofacial Surgery, 4Division of Infections and Molecular Biology,

Kyushu Dental University)

16

P07 Immobilization of antibodies on the solid support using Protein A fused to a substrate protein for biotin ligase H. Miyao1, A. Shiraishi1, Y. Ikeda1, S. Sueda1,2 (1Department of Bioscience and

Bioinformatics, 2RCBT, Kyushu Institute of Technology) P08 Fabrication of Micro Valve with Ultra-precision

Machining Method H. Sato1, T. Hirose1, H. Uno2, M. Suzui3, T. Urisu2, M. Yabuya1 (1Nachi-Fujikoshi

Corp, 2Nagoya University First Research Center for Advanced Nanobiodevices, 3Institute for Molecular Science)

P09 Calcium Mitigates the Toxicity of Zinc Oxide Nanoparticles to Human Lung Epithelial Cells H. Morita1, N. Hanagata1,2 (1National Institute for Materials Science, 2Hokkaido

University)

P10 Influence of the pattern size of three-dimensional micropatterned scaffolds on cell functions H. Sunami1,2, I. Yokota1,2, Y. Igarashi2 (1PRESTO (JST), 2Hokkaido University

Faculty of Advanced Science) P11 Regulation of DNAzyme activity by metal ion-directed

sequence edition of the oligonucleotide template carrying terpyridine units H.Ohura,1 C. Sirahama,1 T. Furuzono,1 Y. Kitamura1, 2, T. Ihara1,2 (1Kumamoto

University, 2CREST (JST)) P12 Ultra-efficient photothermal therapy for prostate

cancers using aptamer-modified gold nanostars H. Jo, S. Lee, C. Ban (Pohang University of Science and Technology)

P13 Influence of temperature on the neuronal oscillation in rat hippocampal slices I. Kageyama1, K. Natsume1,2 (1Grad. Sch. of Life Sci. and Sys. Eng., 2RCBT,

Kyushu Institute of Technology) P14 Stabilization of poly-L-lysine-based cancer-specific

vectors via disulfide bond formation K. LI1, G. Zhao1,T. Mori1,2,3, A. Kishimura1,2,3, Y. Katayama1,2,3, (1Department of

Applied Chemistry, 2The Center for Future Chemistry, 3International Research

Center for Molecular Systems, Kyushu University)

17

P15 High Resolution Imaging of Angiogenesis in Hind Limb Ischemic Model Mouse with Nanoparticles K. Kawamura1, K. Gonda1,2, Y. Hamada1, Y. Kubota1, N. Ohuchi1, 2 (1Department

of Nano-Medical Science, 2Department of Medical Physics, 3Department of

Surgical Oncology, Tohoku University)

P16 Synthesis of the PEI coated cobalt ferrite nanoparticles for magnetic hyperthermia K.Mori, K.Onuma, T.Kondo, K.Miike, T.Miyasaka, M.Hachisu, Y.Kikuchi,

Y.Ichiyanagi (Yokohama National University) P17 Magnetic Properties and MRI Contrast Effect on

CoxFe3-xO4 Nanoparticles K. Miike1, N. Sakai1, Y. Ichiyanagi1, Y. Hosokai2, A. Usui2, Y. Machida2, H. Saito2

(1Yokohama National University, 2Tohoku University) P18 Preparation of hydrophobic carrier for medical agents

using cross-linked polysaccharide K. Maeda1, S. Mochizuki1, Y. Sanada1,2, K. Sakurai1,2 (1University of Kitakyushu, 2JST-CREST)

P19 Synthesis of α-Arylcarboxylic Acid Amides from Enol Silyl Ether via Migratory-Amidation with 2-Azido-1,3-dimethylimidazolinium Hexafluorophosphate K. Murakami, M. Kitamura (Kyushu Institute of Technology)

P20 Bottom-up Brain Model for Nano-Brain Toxicology Assay K. Fujioka1, S. Hanada2, Y. Inoue3, F. Kanaya2, K. Shiraishi1, K. Ikeda1, K.

Yamamoto2, Y. Manome1 (1The Jikei University School of Medicine, 2National

Center for Global Health and Medicine, 3Toho University) P21 Influence of alkyl chain length of multivalent cationic

lipids with calix[4]arene backbone for transfection efficiency K. Nishina1, S. Mochizuki1, K. Sakurai1,2 (1The University of Kitakyushu, 2JST-CREST)

P22 QCM Detection of GPCR-ligand Binding Using Cell-derived Liposomes M. Yamanaka, S. Sueda, T. Yasuda (Kyushu Institute of Technology)

18

P23 A new electron transfer mediator for a NAD-glucose dehydrogenase based glucose sensor D.-M. Kim, M.-Y. Kim, S. S. Reddy, Y.-B. Shim (Pusan National University)

P24 Cyclic naphthalene diimide aiming at tetraplex DNA specific ligand M. M. Islam, S. Sato, S. Takenaka (Kyushu Institute of Technology)

P25 Development of silicon nanowires (SiNWs)/gold nanoparticles (AuNPs)-modified electrode for oligonucleotide sequence of dengue virus detection J. I. A. Rashid1,2, N. A. Yusof1,3, J. Abdullah1,3 (1Institute of Advanced

Technology, Universiti Putra Malaysia, 2National Defense University of

Malaysia, 3Department of Chemistry, Universiti Putra Malaysia) P26 Adjuvant delivery by novel complex made

frompolysaccharide SPG and CpG-ODN N. Miyamoto1, S.Mochizuki1,2, K. Sakurai1,2 (1The University of Kitakyushu, 2JST-CREST)

P27 Rhodium-catalyzed Reaction of Diazonaphthoquinones and Enol Ethers: Synthesis of Dihydronaphthofuran Derivatives and α-Naphthyl Esters N. Fukuzumi, K. Araki, H. Matsuzaki, M. Kitamura (Kyushu Institute of

Technology) P28 Distribution and toxicity evaluation of ZnO

nanoparticles in single intravenously exposed mice R. Ohno1,2, M. Tongu1, H. Hashimoto3, Y. Fujita4, J. Fujihara2 (1Department of

Experimental Animals, Interdisciplinary Center for Science Research,

Organization for Research, 2Department of Legal Medicine Shimane University

Faculty of Medicine, 3Center for the Promotion of Project Research,

4Interdisciplinary Graduate School of Science and Engineering, Shimane

University) P29 Production of Polymer-Encapsulated Particles using

Supercritical Anti-solvent Method R. Yunus1, M. Kalani2, G.H. Chong3 (1Institute of Advanced Technology, 2Department of Chemical and Environmental Eng., 3Faculty of Food Science

and Technology, Universiti Putra Malaysia)

19

P30 Electrochemical property of the unique HFBI protein on an electrode R. Yamasaki1, Y. Takatsuji1, M. Lienemann2, M. Linder2, T. Haruyama1 (1Kyushu

Institute of Technology, 2VTT Biotechnology) P31 Nano metal-suppoted ZnO spherical particle as

photo-catalyst S. Teraoka, S. Ikeno, T. Haruyama (Kyushu Institute of Technology)

P32 Colorimetric aptasensor towards Plasmodium lactate dehydrogenase for the rapid diagnosis of malaria S. Lee, H. Jo, C. Ban (Pohang University of Science and Technology)

P33 Aptasensor for cardiac biomarker Troponin I using Ferrocene (Fc) – modified silica nanoparticles S. Kim, S. Lee, H. Jo, C. Ban (Pohang University of Science and Technology)

P34 Novel cancer vaccine using CpG-DNA/β-1,3-glucan complex S. Mochizuki1, H. Morishita1, K. Sakurai1,2 (1The University of Kitakyushu, 2JST-CREST)

P35 Peptide-functionalized gold nanoparticle sensor for high throughput ligand assay of Estrogen Receptor S. Ikeno1, M. Cauchi2, Y. Takatsuji1, K. Sano1, C. Bessant2, T. Haruyama1

(1Kyushu Institute of Technology, 2Cranfield University) P36 Biocompatible Super Adhesives Using Nano Contact

S. Kinugawa1, R. Hayasaka1,2, S. Wang1,2, D. Kaneko1,2, A. Tsuge1

(1Department of Applied Chemistry, 2Frontier Research Academy for Young

Researchers, Kyushu Institute of Technology) P37 Highly Selective DNA Detection Using

Toehold-mediated Strand Exchange on Graphene Oxide T. Miyahata1, A. Futamura1, Y. Kitamura1,2, T. Ihara1,2 (1Kumamoto University, 2JST-CREST)

P38 Identification of A Polyhydroxyalkanoate Synthase Gene and Active Site from Marine Bacteria, Halomonas sp. O-1 and Halomonas elongata DSM 2581 T. Kihara1, M. Ilham2, K. Mizuno1, T Tsuge2 (1Kitakyushu National College of

Technology, 2Tokyo Institute of Technology)

20

P39 Autofluorescence Lifetime Imaging Microscopy T. Nakabayashi, N. Ohta (Hokkaido University)

P40 Effects of culture condition on micropatterned culture of HepG2 spheroids T. Morikyo, K. Nakazawa (The University of Kitakyushu)

P41 Magnetic and self heating properties of Mn-Zn ferrite nanoparticles as agents of hyperthermia treatment T. Kondo, K. Onuma, K. Miike, T. Miyasaka, M. Hachisu, K. Mori, Y. Ichiyanagi

(Yokohama National University) P42 Effect of fiber length on cellular uptake of

antigen-loaded peptide nanofiber T. Waku, K. Kawabata, S. Nishigaki, S. Kunugi, N. Tanaka (Kyoto Institute

Technology) P43 In Vivo Dual Imaging of Lymph Node Using X-ray CT

and Fluorescence Y. Kubota1, K. Gonda1,2, Y. Kobayashi3, T. Kamei4, T. Nakagawa4, H. Matsudo3,

K. Shibuya3, N. Ohuchi1,5 (1Department of Nano-Medical Science, 2Department

of Medical Physics, Tohoku University, 3Department of Biomolecular Functional

Engineering, Ibaraki University, 4Advanced Surgical Science and Technology, 5Department of Surgical Oncology, Tohoku University)

P44 Preparation of nano-carrier which is fused with cholesterol modified polysaccharide and β1,3-Gulucan Y. Maegawa1, S. Mochizuki1,2, Y. Sanada1,2, K. Akiyoshi3, K. Sakurai1,2 (1The

University of Kitakyusyu, 2JST-CREST, 3Kyoto University) P45 Swing molecular layer for high-sensitive affinity

sensor based on photo-excited current Y. Takatsuji, R. Wakabayashi, T. Haruyama (Kyushu Institute of Technology)

P46 Investigation of enhancement of DNA detection sensitivity with zinc oxide and silica nanoparticles Y. Hiwatashi1, T. Higashi1, Y. Fukui2, J. Fujihara2, H. Takeshita2, H. Hashimoto3,

Y. Fujita1 (1Interdisciplinary Graduate School of Science and Engineering,

Shimane University 2Department of Legal Medicine, Shimane University School

of Medicine, 3Center for the Promotion of Project Research, Shimane

University)

21

P47 A powerless separation method with glass balloons Y. Okamoto1, H. Yukawa1, M. Tokeshi1,3, Y. Baba1,2 (1FIRST Research Center for

Innovative Nanobiodevices, 2Department of Applied Chemistry, Nagoya

University, 3Division of Biotechnology and Macromolecular Chemistry,

Hokkaido University)

P48 Synthesis of peptide-DNA conjugates as imaging reagent for biological sodium ion Y. Imaichi1, H. Koga3, K. Nakazawa3, S. Sato1,2, S. Takenaka1,2 (1Department of

Applied Chemistry, 2RCBT, Kyushu Institute of Technology, 3The University of

Kitakyushu) P49 Enhanced Tumor Targeting by Nitric Oxide

Encapsulated Stealth Liposomes Y. Tahara1, Z. Hosain1, T. Mori1, T. Yamamoto2, A. Kishimura1, Y. Katayama1

(1Kyushu University, 2Information Technologies and Nanotechnologies) P50 Electrochemical periodontal disease assay using

ferrocenyl peptide immobilized-electrode S. Nagata1, J. Shimamoto1, T. Ohshima1, S. Sato1, T. Okinaga2, M. Usui2, W.

Ariyoshi2, K. Nakashima2, M. Nagayoshi2, C. Kitamura2, T. Nishihara2, S.

Takenaka1 (1Kyushu Institute of Technology, 2Kyushu Dental University)

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Plenary talk Plenary lecture will take place in Tobata Campus,

Multipurpose hall of Nakamura Centenary Hall

23

Discoveries at the Interface of Nanomaterials and Biology

Fuyuhiko Tamanoi

Dept. of Microbio., Immunol. & Molec. Genet. Jonsson Comprehensive Cancer Center University of California, Los Angeles

Nanomedicine is driven by the development of novel nanomaterials or modification of existing nanomaterials. Often times, when they were used in biological systems, unexpected features of the nanomaterials are uncovered. Thus, many of the new discoveries are made at the interface of Nanomaterials and Biology. In this talk, I will discuss our experience developing mechanized nanoparticles for targeted delivery and controlled release of anticancer drugs. Our effort started several years ago. We focused on using mesoporous silica nanoparticles (MSNs) that have a diameter of approximately 130 nm and that contain thousands of pores. Established chemical methods can be employed to modify these nanoparticles. We have developed a variety of mechanized nanoparticles including nanoimpeller that responds to light, nanovalve-equipped MSNs as well as iron oxide core MSNs. When our MSNs that have negatively charged surface were examined in biological systems, we observed a number of interesting results. First, we were surprised to see that MSNs taken up by cancer cells were exocytosed from the cells via mechanism involving lysosomal exocytosis. Second, by injecting in mice, we showed that more than 90% of MSNs was excreted into urine and feces. This raises an interesting issue about the route they use to get out of the body. Because of these features, we call MSNs “in-and-out nanoparticles”. Third, we found that proteins are bound to MSNs when they are exocytosed from cancer cells. By using mass spectrometry, we have identified a number of proteins that are bound to MSNs. Identity of these proteins are currently examined. This study raises the possibility that MSNs can be used as a probe to investigate their surroundings. References: [1] Yanes RE, Tarn D, Hwang AA, Ferris DP, Sherman SP, Thomas CR, Lu J, Pyle AD, Zink JI, Tamanoi F. (2013) Involvement of lysosomal exocytosis in the excretion of mesoporous silica nanoparticles and enhancement of the drug delivery effect by exocytosis inhibition. Small 9, 697.

[2] Lu J, Li Z, Zink JI, Tamanoi F. (2012) In vivo tumor suppression efficacy of mesoporous silica nanoparticles-based drug-delivery system: enhanced efficacy by folate modification. Nanomedicine. 8, 212-220. [3] Lu J, Liong M, Li Z, Zink J, Tamanoi F. (2010) Biocompatibility, biodistribution, and drug-delivery efficiency of mesoporous silica nanoparticles for cancer therapy in animals. Small. 16,1794-805.

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Key Note Speech Plenary lecture will take place in Tobata Campus,

Multipurpose hall of Nakamura Centenary Hall

25

Noninvasive in vivo imaging of neutrophil and tumor in mouse auricles

Hideo Higuchi, Kenji Kikushima and Sayaka Kita Department of Physics, Graduate School of Science, the University of Tokyo

7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

Tumor was exposed by dissecting the skin and a epicedium, so far. Many biological systems especially the immune system were activated by the dissection. To avoid the dissection, we developed the method of non-invasive imaging. The auricle was selected for non-invasive imaging because of thin and short hair. The auricle was illuminated by high power laser for short time (Fig1). We investigated the motility of vesicle in neutrophil, a kind of white blood cell, in mice. Neutrophil is activated by the inflammation of TPA and goes out of blood vessel to cure the inflammation. 1 day after the inflammation, many of neutrophil go out of the blood vessel. We observed clearly the movement of vesicle containing antibody-quantum dot (Fig.2). The shape of neutrophil becomes long and front domain may pull the cell. The velocity of the vesicle was changed very much from 0.3 to 3.1 um/s. Surprisingly, the velocity of 3 um/s is about three times of velocity of dynein and kinesin. The MSD plots indicate that the “stop and go” fashion.

Noninvasive imaging method was applied to visualize GFP in tumor cells in xenograft model mice. We prepared a novel xenograft model in the auricle of SCID mice with breast cancer (MDA-MB-231-GFP-tubulin) and glioma cells. Tumors composed of these cells were successfully formed in mouse auricle by improving injection method. The structures of tumor, cells and tumor vessel were visualized by tissue staining. The MDA-MB-231-GFP-tubulin cells were observed by fluorescence of GFP in separated tumors (ex vivo), separation of skin around tumor (invasive in vivo) and without injuring (noninvasive in vivo). We successfully performed real time observation of GFP within breast cancer cells under noninvasive condition.

Fig 1 Imaging method Fig. 2 Moving neutrophil in mouse auricle

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Nano- and Quantum-Biodevices for Cancer Diagnosis, Cancer Therapy, and iPS Cell Based Regenerative Medicine

Yoshinobu Baba1,2

1 Department of Applied Chemistry, School of Engineering, FIRST Research Center for Innovative Nanobiodevices,

Synchrotron Radiation Research Center, Department of Advanced Medical Science, School of Medicine, Nagoya University, Nagoya, Japan

2 National Institute of Advanced Industrial Science and Technology (AIST)

Nano-/quantum-biodevice is a piece of contrivance, equipment, machine, or component, which is created by the overlapping multidisciplinary activities associated with nano-/quantum-technology and biotechnology, intended for biological, medical, and clinical purposes. During the past decade, nano-/quantum-biodevice has progressively begun to focus on the establishment of main four fields of biomedical applications of nanotechnology, including 1) diagnostic devices, 2) molecular imaging, 3) regenerative medicine, and 4) drug delivery systems.

In this lecture, I will describe the development of nano-/quantum-biodevices for biomedical applications, including single cancer cell diagnosis for cancer metastasis, circulating tumor cell (CTC) detection by microfluidic devices, nanopillar devices for ultrafast analysis of genomic DNA and microRNA, nanopore devices for single DNA and microRNA sequencing, nanowire devices for exosome analysis, single-molecular epigenetic analysis, quantum switching in vivo imaging of iPS cells and stem cells, and quantum technology-based cancer theranostics [1-5].

Euglena-based “biomimetic mechanical system” enables us to develop reliable circulating tumor cell (CTC) separation and detection technique for cancer metastasis diagnosis. Immunopillar devices realized the fast and low invasive “from blood to analysis” type biomarker detection of cancer with fM detection sensitivity within 2 min. Additionally, nanopillar devices give us ultrafast separation of DNA and microRNA within 60 µs and nanopillar-nanopore integrated nanobiodevice enables us ultarafast single molecular DNA sequencing. Nanowire devices coupled with super-resolution optical microscopy are extremely useful to analyze exosomes from cancer cells and exosomal microRNA analysis. Quantum dots are applied to develop quantum-biodevices for single cancer cell diagnosis, single molecular epigenetic analysis, quantum switching in vivo imaging for iPS cell based regenerative medicine, and theranostic devices for cancer diagnosis/therapy. [1] M. Tabuchi, Y. Baba, et al., Nature Biotech., 22, 337 (2004). [2] T. Yasui, Y. Baba, et al., ACS Nano, 5, 7775 (2011). [3] M.F. Serag, Y. Baba, et al., Nano Lett., 12, 6145 (2012). [4] T. Yasui, Y. Baba, et al., ACS Nano, 7, 3029 (2013). [5] K. Hirano, Y. Baba, et al., Nano Lett., in press (2013).

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Reactions, Interactions and Dynamics in Biological Membranes: Vesicles to Brains

Steven G. Boxer

Department of Chemistry, Stanford University, Stanford, CA, USA, 94305-5012

Our lab has developed a wide range of methods for patterning lipid bilayers on solid supports [1]. These

2D fluids are interesting as a model for biological membranes, as a physical system with unusual properties, and as a step towards the creation of controlled interfaces between biological and non-biological surfaces. Methods have been developed for controlling the composition of patterned membrane corrals by variations on microcontact printing and microfluidics. Charged components can be moved around within these fluid surfaces by a form of 2D electrophoresis. Although this is a model membrane system, it provides an excellent platform for the development of advanced imaging and analysis methods, and components displayed in the supported bilayer model membrane can interact with and affect the function of native cell membranes.

Fluid planar lipid bilayers can be used as a platform to tether small vesicles by short complementary DNA sequences added as lipid head groups. Once tethered, vesicles are laterally mobile in the plane of the supported bilayer, so individual vesicle-vesicle interactions, including vesicle fusion, mediated by different components on the vesicle surface or in solution, can be observed directly. Because this is a completely synthetic system and the DNA sequence, length, spacer length and the nature of the membrane anchors can be controlled, this is an attractive system for systematic investigation of the requirements for vesicle docking and fusion. Alternatively, large patches of planar bilayers can be assembled near solid supports using the same DNA-tethering strategy: these DNA tethers can either be fixed on the surface or displayed on a supported lipid bilayer, in which case the DNA hybrids are mobile and interesting topological domains are observed when different length hybrids are present. The planar geometry of the supported bilayer and tethered membranes are well suited for surface sensitive imaging methods including interferometry and imaging mass spectrometry. The DNA-lipid conjugates can be used either in the 5’-5’ orientation to tether vesicles to bilayers or bilayers to surfaces (Figure 1; magenta) or in the 3’-5’ orientation for programmed membrane fusion as illustrated for an incoming vesicle and a tethered lipid patch in Figure 1 [2]. The synthetic DNA-based membrane fusion machinery can also be use for programmed transfer of dye-labeled lipids to cells in complex tissues such as fixed brain slices for staining individual neurons.

Figure 1: Schematic diagram of vesicle docking, hemi-fusion and fusion mediated by DNA-lipid conjugates where the DNA is anchored on the 3’ end on the incoming vesicle and on the 5’ end on the target. This design simulates an artificial synapse and the same mechanism can be used to target single cells for the transfer of membrane or content components [1]. [1] http://www.stanford.edu/group/boxer/membr_pubs.html [2] “Individual Vesicle Fusion Events Mediated by Lipid-anchored DNA,” B. van Lengerich, R.J. Rawle, P. M.

Bendix, and S G. Boxer, Biophysical Journal, 105 (2013) 409.

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Special Lecturers Plenary lecture will take place in Tobata Campus,

Multipurpose hall of Nakamura Centenary Hall

29

Surface Molecular Patterns: Method and Possible Application for Bioanalysis

Li-Jun Wan

CAS Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry, Chinese Academy of Sciences (CAS), Beijing 100190, China

E-mail: wanlijun@iccas.ac.cn As a bottom-up technique, self-assembly is considered to be a powerful method in surface molecular engineering. The precise control of nanostructure and arrangement of molecule is now becoming possible in a molecular assembly. In this presentation, the methodology for preparing molecular nanostructures on different substrates such as Au(111), Cu(111) and HOPG are reported. The main methods include non-induced formation and induced deposition. In induced deposition, external stimulations of thermal/electrode potential/ light irradiation, molecular template and local chemical condition are well employed. The assembly with the individual molecules could also be organized and polymerized into molecular nanowires, nanonetworks and various patterns. The structures are investigated by scanning tunneling microscopy (STM) in ambient and electrolyte solution. The figure in the left side is the coordinated assembly of a macrocycle molecule and a rectangular molecule on HOPG surface, including (a-c) STM images and (d) structural model. The rectangular molecule is positioning on the top layer of macrocycle molecule.The arrows in the figure show the molecular defects in the second layer. On the other hand, biomolecules can be also deposited on solid surface. For instance, amino acid molecules formed a (4 x 4) monolayer on Cu(111) surface. With the so-prepared molecular nanopatterns, biomolecules such as peptides can be deposited in the cavity of the network and mono-dispersed on solid surface, forming well-defined molecular nanostructure with different symmetry and molecular ratio. The patterned and modified surfaces are also sensitive to special chemicals, showing potentials in biomolecular analysis and biochemical sensor fabrication. At the last part a carbon-organic composite nanomaterial for drug dilivery was introduced. References: [1] Li-Jun Wan, Acc. Chem. Res., 39(2006)334. [2] R. Wen, G. B. Pan, Li-Jun Wan, J. Am. Chem. Soc., 130(2008)12123. [3] Chen Ting, Ge-Bo Pan, Li-Jun Wan et al., J. Am. Chem. Soc., 132(2010)1328. [4] Jia Liu, Xin Deng, Dong Wang, Li-Jun Wan, J. Am. Chem. Soc., 133(2011)21010.

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Chemical functionalization tailors the properties of carbon nanonotubes for biomedical applications

Alberto Bianco

CNRS, Institut de Biologie Moléculaire et Cellulaire, Laboratoire d’Immunopathologie et Chimie Thérapeutique, 15, Rue

René Descartes, 67084 Strasbourg, France. E-mail: a.bianco@ibmc-cnrs.unistra.fr The combination of the mechanical, thermal, chemical and electronic properties makes carbon nanotubes unique materials in nanoscience and nanotechnology. In addition, they are considered as an innovative material for a wide range of applications in the biomedical domain [1]. One major concern related to the extreme difficulty to manipulate carbon nanotubes, due to their insolubility in all types of solvents, particularly in aqueous solutions, has been solved. In this presentation we will describe the functionalization and biomedical applications of carbon nanotubes loaded with different therapeutic agents. In particular we will describe the possibility to use them as carriers for siRNA to alleviate brain related diseases [2]. Recently, we have devised the echogenic potential of carbon nanotubes [3]. Their use as novel contrast agents will be also presented. In a broader health context, we will finally address the critical issue of their potential toxic effects and how to alleviate them [4,5].

Figure 1. The asbestos-like pathogenicity observed for long, pristine nanotubes can be completely alleviated if their effective length is reduced as a result of chemical functionalization, using for example the 1,3-dipolar cycloaddition reaction.

[1] A. Battigelli, C. Ménard-Moyon, T. Da Ros, M. Prato and A. Bianco, Adv. Drug Deliv. Rev. 2013,

DOI:10.1016/j.addr.2013.07.006 [2] K. T. Al-Jamal, L. Gherardini, G. Bardi, A. Nunes, C. Guo, M. A. Herrero, A. Bianco, M. Prato, K.

Kostarelos and T. Pizzorusso, Proc. Natl. Acad. Sci. USA, 108 (2011) 10952. [3] L. G. Delogu, G. Vidili, E. Venturelli, C. Ménard-Moyon, M. A. Zoroddu, G. Pilo, P. Nicolussi, C. Ligios, D.

Bedognetti, F. Sgarrella, R. Manetti and A. Bianco, Proc. Natl. Acad. Sci. USA, 109 (2012) 16612. [4] J. Russier, C.Ménard-Moyon, E. Venturelli, E. Gravel, G. Marcolongo, M. Meneghetti, E. Doris and A.

Bianco, Nanoscale, 3 (2011) 893. [5] H. Ali-Boucetta, A. Nunes, R. Sainz, M. A. Herrero, B. Tian, M. Prato, A. Bianco and K. Kostarelos,

Angew. Chem. Int. Ed., 52 (2013) 2274.

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The enzyme-like activities of iron-based nanoparticles

N.Gu1,2, Y. Zhang, F.Yang, M.Ma, Z.W.Chen, X. Q. Zhang, S.L.Hu, Y.H.Wu, W. Zhang and LN.Song

Jiangsu Key Laboratory for Biomaterials and Devices, Nanjing, 210009

State Key Laboratory of Bioelectronics, Nanjing, 210096 School of Biological Science and Medical Engineering

Southeast University, Nanjing, P.R.China Due to the tremendous progress in nanotechnology and especially nanomaterials, nanobiotechnology has been shown various applications in medicine and health care. The research on action of nanomaterials on biological objects is the need for laying foundations, on which several kinds of artificial materials or structures have been found to exhibit unexpected enzyme-like activities, which will be promised wide applications in many fields. As an attractive example, Prof. Yan and her co-workers first reported the intrinsic peroxidase-like activity of ferromagnetic nanoparticles and its potential applications in decomposing phenol, immunosorbent assay for detection of hydrogen peroxide in acid rain and etc[1,3]. Since this finding, a variety of iron-based nanoparticles, such as Fe3O4@Pt, prussian blue(PB) and PB modified iron oxide nanoparticles had been investigated as such enzyme-like nanomaterials or so-called nanozymes[4,5]. In this presentation, I will introduce our work on the multifactor dependent enzyme-like activities of several iron-based nanozymes, and their potential applications in immunoassays and smart drug/contrast agent, such as a hydrogen peroxide-responsive O2 nanogenerator for ultrasound and magnetic resonance dual modality imaging[6,9]. 1 Presenter: Ning Gu

2 Corresponding author: Ning Gu; E-mail: guning@seu.edu.cn

References: [1] L. Gao, J. Zhuang, L. Nie, J.B. Zhang, Y. Zhang, N. Gu, T.H. Wang, J. Feng, D.L. Yang, S. Perrett, X.Y.

Yan., Nature Nanotechnology, 2 (9) (2007)577 [2] J. Zhang, J. Zhuang, L.Z. Gao, Y Zhang, N. Gu, J. Feng, D. Yang, J. Zhu, X.Y. Yan, Chemosphere, 73

(9)(2008)1524 [3] J. Zhuang, J. Zhang, L.Z. Gao, Y. Zhang, N. Gu, J. Feng, D. Yang, X.Y. Yan, Materials Letters, 62

(24)(2008)3972 [4] X.Q. Zhang, S.Wen, Y. Gong, Y. Zhang, T. Yang, C.Y. Wang and N. Gu, J. Mater. Chem., 20( 2010)5110 [5] M. Ma, J. Xie, Y. Zhang, Z.P. Chen, and N. Gu, Materials Letters, 105(2013)36. [6] Z.W. Chen, J.J. Yin, Y.T. Zhou, Y. Zhang, L.N.Song, M.J.Song, S.L. Hu, and N. Gu, ACS Nano,

6(5)(2012)4001 [7] Y.H. Wu, M.J. Song, Z. Xin, X.Q. Zhang, Y. Zhang, C.Y. Wang, S.Y. Li and N. Gu, Nanotechnology, 22

(2011) 225703 [8] F. Yang, S.L. Hu, Y, Zhang, X.W. Cai, Y. Huang, F. Wang, S. Wen, G.J. Teng, N. Gu, Adv. Mater.,

24(38)(2012)5205 [9] W. Zhang, Y. Zhang, Y.H. Chen, S. Y. Li, N Gu, S.L.Hu, Y. Sun, X. Chen and Q. Li, J. Nanosci. Nanotech.,

13(2013)60

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Multifunctional Organic Platform for On Chip Cell Sensing, Sorting and

Manipulation

Peilin Chen

Research Center for Applied Sciences, Academia Sinica, Taipei, Taiwan 11529

An optically transparent poly(3,4-ethylenedioxythiophene) (PEDOT) organic

electrode based electrical cell-substrate impedance sensing (ECIS) device has been developed to investigate the behavior of human mesenchymal stem cell (hMSC). We first conducted a control experiment on ECIS device by culturing the hMSC on the chip. According to our result, the impedance increased reflected the hMSC proliferation, attachment and motility during the first 16 hours of cell culture. It has been shown that different differentiation types of mesenchymal stem cells (for example, osteoblast and adipocyte lineages) have distinct dielectric properties, therefore featuring different impedance profiles.

In order to control the differentiation of human mesenchymal stem cell (hMSC), we also developed all-solution-processed multifunctional organic devices, comprising reduced graphene oxide (rGO) and dexamethasone 21-phosphate disodium salt (DEX) drug loaded poly(3,4-ethylenedioxythiophene) (PEDOT) microelectrode arrays on indium tin oxide glass, that can be used to manipulate differentiation. In our devices, the rGO micropatterns were used as the adhesive coating to attract the adhesion of hMSC cells whereas PLL-g-PEG coated PEDOT electrodes served as the anti-adhesive coating where no hMSC cells can attach. In addition, the PEDOT electrodes also work as drug releasing components where control DEX release from PEDOT matrix can be achieved via cyclic potential stimulation (CPS).

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Cell membrane permeable and cytocompatible phospholipid polymer nanoprobes conjugated with molecular beacons

Kazuhiko ISHIHARA1,2, Xiaojie LIN1, Tomohiro KONNO2

1Department of Materials Engineering, School of Engineering, The University of Tokyo

2Department of Bioengineering, School of Engineering, The University of Tokyo 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

To comprehensive understanding the distribution and dynamic of intracellular biomolecules and mechanism of intracellular bio-reactions, we proposed to prepare a specific functional nanoprobe through the combination of well-designed cytocompatible phospholipid polymer and molecular beacons (MBs). A water-soluble amphiphilic phospholipid polymers, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-N-succinimidyloxycarbonyl tetra(ethylene glycol) methacrylate] (PMBS), were synthesized to conjugate with MBs to form the nanoprobe through the chemical reaction between N-hydroxysuccinimide ester group and amine group of the MBs. Surface tension measurement indicated that the polymeric nanoprobes showed different conformations in their aqueous solutions, especially at a concentration of 1.0 mg/mL, the PMBS with large hydrophobic BMA unit composition would form polymer aggregate. The model cancerous cells almost remained 100 % viability after incubation with our fluorescent polymer or PMBS-MBs probe. One of the polymeric nanoprobes not only demonstrated higher specificity to target, resistance to the nonspecific adsorption of protein compared with the original MBs, but also could penetrate into the cell cytoplasm to image the intracellular message ribonucleic acid in living cells (Fig. 1). We conclude that the MPC polymer-MBs nanoprobes have great application potential and will promote the development of noninvasive monitoring intracellular biomolecules and bio-reaction. References [1] S. Tyagi, F. R. Kramer, Nat. Biotechnol.,14(3), 303-308 (1996). [2] K. Ishihara, T. Ueda, N. Nakabayashi, Polym. J., 22(5), 355-360 (1990). [3] T. Goda, Y. Goto, K. Ishihara, Biomaterials, 31(8), 2380-2387 (2010).

Fig. 1. Laser scanning confocal microscopy images of the Hela cells after cultivation with different probes: (A) PMPC/MBs; (B) PMBS721-MBs; (C) PMBS361-MBs. Scale bar: 10 μm.

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Genetically-encoded functional probes applicable in conjunction with photo-manipulation technologies

Takeharu Nagai1,2

1The Institute of Scientific and Industrial Research, Osaka University, Suita, Osaka, Japan

2PRESTO, Japan Science and Technology Agency, Tokyo, Japan Optogenetic tools including ChR2, LOV domain, and CALI (chromophore-assisted light inactivation) allows us to operate biomolecule function upon light irradiation, by which we are now able to investigate protein of interest in terms of functional difference according to the existing area inside cells and expression stage with real time resolution. To understand what‘s going on after the light-stimulation of those optogenetic tools, many researcher may want to visualize some other molecular dynamics and function by fluorescence imaging. However, excitation light for the imaging should mis-activate the optogenetic tools so that we cannot apply the fluorescence imaging in conjunction with the optogenetic technology. To overcome this problem, we have developed genetically-encoded luminescent probes which are based on hybrid between a mutated chemiluminescent protein and a fluorescent protein variant. In the symposium, I will introduce our recent achievement such as development of luminescent functional indicators for Ca2+, ATP and cAMP as well as genetically-encoded CALI methods, all of which would be useful for quantitative understanding of biological systems.

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Nanoparticles In Vivo and the Biomedical Applications

Chung-Shi Yang

Institute of Biomedical Engineering and Center for Nanomedcine, National Health Research Institutes, Zhunan, Taiwan

Efficient delivery of the nanoparticles (NPs) and the NPs-based formulating agents to target organs, as

well as their optimal retention in the cells, play critical roles of their biomedical efficacy. Although the

tissue- and cell-specific targeting of the NS have been intensively studied, there has been no efficient

ways to control the fate of NPs once they enter into the cells. Most of the existing approaches to

manipulate the intracellular retention of NPs are mostly “passive” and particle size-dependent. The

adverse effect of particle size may limit the utility of nanodelivery systems. Therefore, the development

of tunable “active” NP intracellular retention systems with fixed particle sizes remains a considerable

challenge. By replacing the synergistic anions of transferrin (Tf) immobilized on quantum dots (Tf-QDs,

ca. 25 nm), we have examined the feasibility of this concept. Substitution of synergistic anions of Tf

from carbonate (holo-Tf) to oxalate (oxa-Tf) significantly increased the intracellular accumulation of the

oxa-Tf-QDs as a result of (i) a delay in cellular removal triggered by oxalate (oxa-Tf)-inducedendosomal

Tf iron-release retardation and (ii) enhanced recycling of Tf-QD/TfR (Tf receptor) complexes from early

endosomes to the plasma membrane. This accumulation extended the intracellular NP retention interval.

The half- maximum fluorescence intensity of the oxa-Tf-QDs in vivo was 4 times higher than that of the

holo-Tf-QDs. Programming of the intracellular NP retention time was accomplished through

manipulation of the ratio of holo- and oxa-Tfs on the surfaces of the QDs. The preferential residence

and accumulation of the oxa-Tf-QDs in animals bearing tumors was also observed. Using this simple

and efficient approach, it was possible to readily achieve a desirable intracellular retention interval for the

NPs, which may serve as one of the efficient ways for therapeutic nanoparticles to be delivered and

accumulated in target tissues.

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New Hybridoma Technology for Selective Production of Stereospecific Monoclonal Antibodies

M. Tomita and K. Tsumoto

Division of Chemistry for Materials, Graduate School of Engineering, Mie University

1577 Kurima-Machiya-cho, Tsu, Mie 514-8507 Japan Email:tomita@chem.mie-u.ac.jp

Recently, monoclonal antibodies have attracted attention as possible new medicines because of their strong

and specific interactions with target antigens. Hybridoma technology, which was originally established by Köhler and Milstein [1], is one of the privileged technologies to raise monoclonal antibodies against antigens of interest. It enables the generation of hybridoma cells secreting monoclonal antibodies by fusing antigen-sensitized B lymphocytes with cancerous myeloma cells.

Already a number of hybridoma approaches such as poly(ethylene glycol) (PEG), Sendai virus (HVJ), pearl-chain formation, laser radiation and so on, have been reported. One example, the PEG-mediated method is still being used by many researchers. However, these methods lack control of cell fusion, resulting in non-specific fusion events. To address this problem, Lo et al [2] developed an advanced hybridoma technology termed the pulsed electric field (PEF) method, which is also known as B-cell targeting. The point for the PEF method is preselection of sensitized B lymphocytes by the desired antigen followed by selective fusion of B lymphocyte-myeloma cell complexes by electrical pulses. At least 10-times higher fusion efficiency was observed over original methods. However, even though the PEF method allows efficient production of monoclonal antibodies, most of them only feature specificity to primary structures of antigens.

Monoclonal antibodies specific to conformational structures of antigens are of particular interest now. They are considered as a next generation of therapeutic antibodies, especially when receptors are the target antigens. However, no practical protocols have been generally available. Therefore we have established a new hybridoma technology to selectively generate stereospecific monoclonal antibodies [3, 4]. There are two critical points for this. One is that DNA immunization is employed to immunize a mouse with an antigen. Antigens expressed in the mouse with this approach may be recognized with their structures intact by the immune system. The other is that sensitized B lymphocytes are selected by antigen-expressing myeloma cells by harnessing strong and specific interactions between antigens and antibodies, as shown in Fig. 1. The antigens expressed on the myeloma cells must have their intact structures maintained. Only thus can the B lymphocytes selected by antigen-expressing myeloma cells produce stereospecific antibodies. Finally, the B lymphocyte-myeloma cell complexes are selectively fused by electrical pulses [2]. The characteristic of electric fusion is that it only can fuse conjugated or attached cells, but cannot fuse unconjugated nor free cells. Theoretically, all hybridoma cells produced by this technology generate the aimed for stereospecific monoclonal antibodies.

We have investigated the production of specific monoclonal antibodies against thyroid-stimulating hormone receptor (TSHR), which belongs to of the family of G protein-coupled receptors (GPCRs), applying this new technology. As a result, monoclonal antibodies competing with TSH, an authentic ligand for TSHR, were obtained. Furthermore, monoclonal antibodies directed against EphA2 showed specific binding to MDA-MB-231 cancer cells which abundantly express EphA2 on their cell surfaces, but did not cross-react with recombinant EphA2. These results suggest that the new hybridoma technology may be applicable to selective production of stereospecific monoclonal antibodies against target antigens. This technology may also contribute for elucidation of innate functions of soluble antigens. [1] G. Köhler and C. Milstein, Nature, 256 (1975) 495. [2] M.M.S. Lo, T.Y. Tsong, M.K. Conrad, S.M. Strittmatter, L.D. Hester and S.H. Snyder, Nature, 310 (1984)

792. [3] M. Tomita and K. Tsumoto, Immunotherapy, 3 (2011) 371. [4] H. Yagami, H. Kato, K. Tsumoto and M. Tomita, Pharm. Pat. Analyst, 2 (2013) 249.

Fig. 1 Stereospecific targeting (SST) technique

Antigen-expressing myeloma cells

Sensitized B cells

B cell- myeloma cell complex

Electrical pulses

Hybridoma cell

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Invited Speakers Brain nanomedicine Special Session

Plenary lecture will take place in Tobata Campus,

Multipurpose hall of Nakamura Centenary Hall

38

Improvement of Penetration Depth and Lateral Spatial Resolution of Two-photon Microscopy with Novel Lasers

T. Nemoto1, 2, R. Kawakami1, 2, T. Hibi1, 2, S. Ipponjima1, 3, K. Sawada1, 3, and K. Iijima1

1 Laboratory of molecular and cellular biophysics, Research Institute for Electronic Science, Hokkaido University, Sapporo,

Japan 2 JST, CREST, Japan

3 Laboratory of functional neuroimaging, Graduate school of information science and technology, Hokkaido University, Sapporo, Japan

Fluorescence imaging by confocal and two-photon microscopy is widely used for observing brain tissue. To elucidate the molecular mechanism underlying neurotransmission, imaging dendritic spines at high resolution within deeper layers of thick brain slices, while keeping neural circuits nearly intact, is required. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption in organs. In this sense, two-photon microscopy has revealed vital information on neural activity for brain function, even in light of its limitation in imaging events at depths greater than a several hundred micrometers from the brain surface. For this study, we have developed a novel semiconductor-laser-base 1030 nm light source, which can generate optical pulses of 5 ps duration at 20 MHz repetition rate, and 2 W averaged output power. We examined the performance of this laser for two-photon imaging employing a laser scanning microscope (Olympus/FV1000), by evaluating the penetration depth in the imaging of the living H-Line mouse brain; and we compared the result with that of using a different laser source (Tsunami Ti:Sapphire laser operating at a wavelength of 910 nm). In addition, we developed a method of retain the mouse head under upright microscope stage. This retainer had a horizontal adjustment mechanism that reduce aberration arise from the objective lens and cover slip on the skull were not parallel. As a result, the newly developed laser has successfully demonstrated cortex layer V pyramidal neurons spreading to all of the cortex layers at a superior S/N ratio. Surprisingly, in young adult mouse (4 week-old), it also enabled to visualize hippocampal CA1 pyramidal neurons lying beneath the cortex. Thus, in vivo two-photon microscopy with this newly developed laser might give us insights on activities of neurons in the cortex or the hippocampus. In addition, we have recently demonstrated that the lateral spatial resolution of confocal laser scanning microscopy (CLSM) is dramatically improved by a vector beam [2]. Vector beams have distribution in intensity, the polarization, and the phase in the single beam. First, we developed a pair of liquid crystal devices (LCDs) that convert a linearly polarized (LP) Gaussian beam into a kind of vector beams, a radially polarized beam with six concentric rings, which can be referred to as a “higher-order radially polarized” (HRP) beam. These LCDs were inserted in front of an objective lens using a hand-made adaptor. In CLSM, we have introduced the LCDs, and investigated the role of the confocal aperture (CA) for the tight focusing of an HRP beam by measuring point-spread-functions (PSFs) using tiny fluorescent beads of which diameter is under the diffraction limit. The side-lobes of an HRP beam can be effectively suppressed by a practical size of the CA used in conventional CLSM. Under optimized conditions, each of the aggregated 0.17 µm beads could be distinguished individually, whereas in conventional CLSM they could not. We also visualized the finer structures of networks of filamentous cytoskeleton microtubule fluorescently-labeled in the COS-7, and primary culture of mouse neurons. Moreover, by taking an advantage of the LCDs that can utilize various wavelengths including near-infrared, we could employ an HRP beam for improving multi-photon microscopy [3]. An HRP beam visualized fine intracellular structures not only in fixed cells stained with various dyes, but also in living cells expressing a fluorescent protein. Moreover, HRP beams have extremely extended depth of field compared with an LP beam [3], which facilitates obtaining in-focus images, especially in time-lapse observations of living cells. Here, we will discuss these improvements and future application on the basis of our recent data. [1] R. Kawakami, et al., Sci. Rep., 3(2013) 1014. [2] Y. Kozawa, et al., Opt. Express, 19(2011)15947. [3] S. Ipponjima , et al., Microscopy , in press [4] We thank Prof. Shunichi Sato, Prof. Hiroyuki Yokoyama, Dr. Yuichi Kozawa, and Dr. Yuta Kusama in

Tohoku University for collaboration. This study was supported by Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Japan, by JSPS KAKENHI Grants of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and also by the Nano-macro Materials, Device and Research Alliance Project, and the Network Joint Research Center for Advanced Materials and Devices (MEXT).

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Production of therapeutic medicine for cerebral vasospasm after subarachnoid hemorrhage base on basic research

Shigeru Nishizawa

Department of Neurosurgery

University of Occupational and Environmental Health Cerebral vasospasm (CV) after subarachnoid hemorrhage (SAH) is major problem in the management of patient of SAH. Cerebral vasospasm is very characteristic, and it occurs only in cerebral arteries after SAH. Major cerebral arteries start to constrict 3-4 days later after the event, and continue for 2 weeks. Under such situation, serious cerebral ischemia happens and patients are suffered from severe neurological deficits even the initial treatment is successful. The main causes of CV have been investigated, but they still remain unclear. I have been focusing on the role of protein kinase C (PKC), which plays a significant role in the signal transduction across the cell membrane, in the pathophysiological mechanism of CV. Using canine SAH model and canine basilar artery, I measured PKC activity of vascular smooth muscle cells. The PLC activity was significantly enhanced after SAH, and time course of activation of PKC was correlated well with that of CV. According those results, I concluded PKC plays a significant role in the mechanism of CV. PKC has 11 isoforms, then, we investigated which PKC isoforms are involved in CV. Based on Western blotting analysis, I found PKCδ and PKCα are important in CV. Especially, PKCδ plays a role in the development of CV on day 4, and PKCα in the maintenance of CV. From these investigation, I tried to produce therapeutic medicine for CV. Using high through-put screening method, I searched chemicals to be able to inhibit both PKCδ and PKCα. One chemical was found for this purpose, which is M-62885. Molecular weight of this chemical is 188.6Da, and IC50 value on PKCδ is 0.482m mol/L and 0.345 m mol/L on PKCα, respectively. The IC50 values on both PKC isoforms are very close, and was considered as an idealistic agent to inhibit the both. I tried this chemical for canine SAH model, and confirmed angiographic cerebral vasospasm was completely inhibited through the course. In Western blotting analysis, the activations of both PKCδ and PKCα were suppressed. These experimental results support my hypothesis. As the next step, I tried to make therapeutic agent using M 62885 with collaboration of some pharmaceutical company. Unfortunately, we could not succeed it yet. There are many problems such as technology, financial problem, so on. It has to be recognized that it is really not easy to bring basic research results in clinical medicine.

References 1. Nishizawa S, Nezu N, Uemura K: Direct evidence for a key role of protein kinase C in the development of

vasospasm after subarachnoid hemorrhage. J Neurosurgery 76:635-639, 1992 2. Nishizawa S, Peterson JW, Shimoyama I, Uemura K: Relation between protein kinase C and calmodulin systems in cerebrovascular contraction: Investigation of the pathogenesis of vasospasm after subarachnoid hemorrhage. Neurosurgery 31:711-716, 1992 3. Nishizawa S, Yamamoto S, Yokoyama T, Ryu H, Uemura K: Chronological changes of arterial diameter,

cGMP, and protein kinase C in the development of vasospasm. Stroke 26:1916-1921, 1995 4. Nishizawa S, Obara K, Nakayama K, Koide M, Yokoyama T, Yokota N, Ohta S: Protein kinase C d and a are

involved in the development of vasospasm after subarachnoid hemorrhage. Eur J Pharmacol 398:113-119, 2000

5. Nishizawa S, Obara K, Koide M, Nakayama K, Ohta S, Yokoyama T: Attenuation of canine cerebral vasospasm after subarachnoid hemorrhage by protein kinase C inhibitors despite augmented phosphorylation of myosin light chain. J Vasc Res 40:168-179, 2003

6. Nishizawa S: Roles of signal transduction mechanisms in cerebral vasospasm following subarachnoid hemorrhage: Overview. Acta Neurochir (Suppl) 110 (Pt 1): 27-30, 2011

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Cell-Cell Adhesion Control by Nucleic Acid Aptamers

S. Sando1,2

1 INAMORI Frontier Research Center, Kyushu University 2 CREST, JST

Nucleic acid aptamers are functional nuclei acids, selected using SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique, that bind to target molecules in biological environments. Because of high affinity and specificity, an attention has been paid to their application to biosensors. To date, various aptamer-based biosensors have been developed. In addition, new SELEX techniques such as cell SELEX have enabled selecting aptamers that bind specifically to biomolecules and biomarkers on cell membranes. There has been current attention of using aptamers as cell-recognition and cell function controlling bio-materials.

We have focused on aptamer-based analysis of cell surface biomolecules.1,2 Recently, we succeeded in demonstrating on-cell sensing of adenine compounds using fluorescent aptamer sensors anchored on cell membrane. In the study, we synthesized the fluorescent aptamer sensor labeled with lipophilic tag molecule tocopherol at its terminus and this aptamer-lipid conjugate was anchored directly on cell membranes to improve the spatiotemporal resolution of fluorescence imaging. The fluorescent aptamer sensor, anchored in cell surface, actually realized real-time fluorescence imaging of adenine compounds, released from rat astrocyte cells upon mechanical stimulation.1 In addition, it was revealed that the aptamer anchoring efficiency and retention are dependent on lipophilic tag molecules used.2

In this research, we tried to control cell-cell adhesion by the cell surface-immobilized aptamers.3,4 Cell-cell

adhesion is an important biological event, especially to induce immune cytotoxicity. Immune effector cells take roles to kill the target tumor cells. The immune system utilizes several pathways, including perforin/granzyme and Fas/FasL, to induce the immune cytotoxicity and these target cell killings require the close contacts between effector and target cells. Thus, cell-cell adhesion is indispensable to induce an effective cytotoxicity.

We designed and synthesized various aptamer-lipophilic tag conjugates, and evaluated them in the point of

anchoring and retention efficiency on cell surface to find applicable lipophilic tags for this purpose. Then, we applied these aptamer-modified cells for cell-cell adhesion. To evaluate the induction efficacy of cell-cell adhesion by on-cell aptamer, the aptamer-anchored cell and the target cell, stained with different fluorophores, were mixed and were analyzed by flow cytometry and confocal microscopy. It was clearly shown that the observed cell-cell adhesion was improved by anchoring a matched aptamer on cell membrane. Details will be discussed in this presentation. [1] T. Tokunaga, S. Namiki, K. Yamada, T. Imaishi, H. Nonaka, K. Hirose, S. Sando, J. Am. Chem. Soc., 134

(2012) 9561-9564. [2] T. Tokunaga, K. Kuwahata, S. Sando, Chem. Lett., 42 (2013) 127-129. [3] Á. M. Cuesta, N. Sainz-Pastor, J. Bonet, B. Oliva, L. Álvarez-Vallina, Trends Biotechnol., 28, (2010)

355-362. [4] X. Xiong, H. Liu, Z. Zhao, M. B. Altman, D. Lopez-Colon, C. J. Yang, L. –J. Chang, C. Liu, W. Tan, Angew.

Chem. Int. Ed., 52 (2013) 1472-1476.

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In vivo MRI monitoring of immune cell migration in mouse brain using superparamagnetic nanoparticles

Y. Mori1,2, T. Chen1, K. Ohno3, S. Yoshida4, Y. Tago4, M. Fukunaga1,2 and Y. Yoshioka1,2

1 Biofunctional Imaging, WPI Immunology Frontier Research Center (IFReC), Osaka University

2 Center for Information and Neural Networks (CiNet), National Institute of Information and Communications Technology and Osaka University

3 Institute for Chemical Research, Kyoto University 4 Frontier Biomedical Research Center, KANEKA Corporation

Immune cells play an important role in inflammatory diseases of the central nervous system (CNS). The

dynamical behavior of immune cells in intact/injured CNS has not been well characterized with in-vivo imaging technique. Noninvasive monitoring of immune cells before/after neuroinflammatory conditions may lead to a greater understanding of the mechanistic underpinning of both CNS injury and repair. Superparamagnetic nanoparticles of iron oxide (SPIO) contrast agents primarily affect T2 and T2

* of proton by virtue of their iron oxide crystals, which have a strong magnetic moment, thereby we can find SPIO-labeled cells as black dots on T2- and T2

*-weighted magnetic resonance images. In addition, we have attempted to improve the spatial resolution of MRI for single cell-level tracking. Recently, endogenous labeling of immune cells via intravenous injection of SPIO might provide a noninvasive MRI cellular tracking method [1]. Here, we use this technique to track cells in the CNS. We used endogenous labeling method, in vivo assays, and an 11.7 T high-field MRI to examine immune cell trafficking in the lipopolysaccharides (LPS)-induced inflammatory and healthy mouse brain.

We observed several black spots not only in LPS but also in healthy brains at 24 and 48 h post-SPIO administration. We confirmed histologically and attributed these black spots to SPIO-labeled endogenous phagocytes. Most of these spots disappeared within 1 week after the administration in both groups. We confirm the integrity of blood-brain barrier (BBB), and any leakage was observed neither in normal, nor in LPS-induced inflammatory brains throughout the MRI measurements. BBB prevents the passage of many substances into the CNS, including ionized water-soluble molecules with molecular weights greater than 500 Da [2]. Although the BBB prevents the passage of SPIO itself into the CNS, SPIO traversed the BBB by incorporating into peripheral phagocytic cells that naturally traverse the BBB. We also examined peripheral macrophage depletion methods, which revealed that few SPIO-labeled cells existed within the brain. In addition, we synthesized SPIO-type hybrid nanoparticles that have a novel polymer-brush surface structure [3]. These particles are not taken into phagocytes due to their stealth effects. Although these particles themselves have enough contrast effect, any signal change was not found within the brain after intravenous administration. These phenomena indicated that black spots in the MRI affected by infiltration of peripheral phagocytes but was not by the leak of SPIO.

We conclude that SPIO-enhanced MRI can detect and trace the endogenous immune cells’ migration from peripheral blood or tissues toward the mouse brain noninvasively. Surprisingly, we first visualized the immune cells’ migration across the BBB even under the physiological condition as well as during the inflammation in the in-vivo whole brain. Simple intravenous administration of nanoparticles and in-vivo MRI may offer a novel method for examining in situ immune cell dynamics in the CNS.

[1] Y. Mori, M. Umeda, M. Fukunaga, K. Ogasawara and Y. Yoshioka, Magn Reson Med Sci, 10 (2011) 219. [2] W. M. Pardridge, Neuron, 36 (2002) 555. [3] K. Ohno, T. Akashi, Y. Tsujii, M. Yamamoto and Y. Tabata, Biomacromolecules, 13 (2012) 927.

Figure 1. Representative MR images of control group (top row) and LPS-induced inflammation group

(bottom). The black dots in each slice of brain represent the distributions of phagocytic cells that incorporate SPIOs. Peripheral phagocytes infiltrated into the brain even in the normal condition as well as inflammatory condition. All images were acquired with a spatial resolution of 59 µm × 59 µm and 300 µm thick.

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Local regulation of molecular reactions in maintaining axonal morphology

Y. Konishi 1,2, T. Seno1, K. Kubota1 N. Sakae1 and H. Takada1 1 Graduate School of Engineering, University of Fukui

2 Research and Education Program for Life Science, University of Fukui

The establishment and maintenance of characteristic cellular morphology is especially important in

the nervous system, in which neurons make connection with specific targets, thereby enable to process the information. Recent studies have identified variety of extracellular molecules, such as axon guidance molecules, control neuronal morphology. On the other hand, isolated neurons in culture are capable to extend the axon and establish matured neuronal morphology. Molecular systems that control this cell-autonomous process remain to be elucidated. We have been studied on the intracellular molecular mechanisms that control axonal morphology, and identified signaling pathways that control growth of axons1. However, the mechanisms by which neurons locally control cellular nanostructures, such as F-actin/microtubules remained unsolved. Our aim is to demonstrate molecular systems by which neurons process spatial information and regulate cellular structure at right position at right time to establish and maintain the axonal morphology. We focused on the axonal branch morphology and tested the possibility that axonal transport might play roles to regulate the axonal branch pattern. Previous studies have revealed that the motor domain of kinesin (K5H) is accumulated in axon in hippocampal neurons2, 3. When K5H-GFP was introduced in cerebellar granule neurons, it accumulated in axonal tip but not in dendrites as observed in hippocampal neurons. We next analyzed the signal intensity of K5H-GFP in each branched axonal terminal to test if K5H-GFP is randomly sorted to each branch terminal. Quantitative analysis revealed that frequency of relative signal intensity does not fit to a normal distribution, indicating that there is the active system to deriver K5H-GFP into the particular axonal branches. Furthermore, we found that there is a positive correlation between signal intensity of K5H-GFP and axonal branch length, suggesting the possibility that axonal branch pattern is regulated via axonal transport (Fig.1). The long-term time lapse imaging of cultured neurons identified that the retraction of axonal branches in which relatively high amount of K5H is delivered is significantly smaller than other branches. These results indicate the existence of novel system that maintain branched axonal pattern via controlling axonal transport. There are several factors reported that regulate the kinesin transport. The degree of post-translational modifications on tubulin that consist microtubules is different between dendrites and axons, and are involved in the polarized transport of kinesin4,5. We found that the degree of microtubule modification is varied among axonal branches as well. Further study of molecular mechanism at high resolution is expected to clarify the detailed molecular system in maintaining axonal morphology.

[1] Y. Konishi, J. Stegmüller, T. Matsuda, S. Bonni and A. Bonni, Science, 303 (2004) 1026 [2] T. Nakata and N. Hirokawa, J. Cell Biol., 162 (2003) 1045 [3] C. Jacobson, B. Schnapp and G.A. Banker, Neuron 49 (2006) 797 [4] Y. Konishi, and M. Setou, Nat. Neurosci., 12 (2009) 559 [5] D.B. Arnold, Sci Signal., 2 (2009) pe49

Fig.1 Distribution of K5H-GFP in the axon of cerebellar granule neurons. A: K5H-GFP (green) accumulated in the axonal terminals. B: Frequency of relative K5H-GFP level calculated from each pairs of terminal axonal branches. C: Correlation between K5H-GFP signal level and axonal branch length.

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Fig. 1 Fluorescence images of NCAMs labeled with Q-dots in a neuron before (a, b), immediately after (c), 0.05 s after (d), and 0.15 s after the trapping laser irradiation (e). The gray and black lines indicate the focal spot of the laser and the position of Q-dots attached to NCAMs.

10 µm 1 µm

(a) (b) (c) (d) (e)

Laser Manipulation of Living Neuronal Network

C. Hosokawa1, N. Takeda1,2, Y. Ueda1,2, S. N. Kudoh2 and T. Taguchi3 1 Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka,

Ikeda, Osaka 563-8577, Japan 2 Graduate School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan

3 CiNet, National Institute of Information and Communication Technology, 1-4 Yamadaoka, Suita, Osaka 565-0871, Japan Neuronal networks are widely studied both experimentally and theoretically to elucidate the basis for brain systems referred to as learning and memory. Neurons in brain systems communicate through synaptic connections. The molecular dynamics including neurotransmitter receptors and a variety of scaffolding and signaling proteins at presynaptic and postsynaptic terminals are essential for synaptic plasticity and subsequent modulation of living neuronal network. For aiming artificial control of the synaptic transmission in living neuronal network, we propose and demonstrate novel approaches using laser manipulation techniques. As one approach, we have recently demonstrated local modulation of living neuronal network with direct cutting of neurons, micro-channel fabrication for arrangement of neurons, and laser-induced stimulation with a focused femtosecond laser. Here we demonstrate laser manipulation of synaptic vesicles and neural cell adhesion molecules (NCAMs) in neurons with optical tweezers toward modulating the synaptic transmission in a neuronal network. The optical trapping and assembling dynamics of molecules in hippocampal neuronal cells was evaluated by fluorescence imaging and fluorescence correlation spectroscopy (FCS). Primary cultures of dissociated hippocampal neurons were prepared from Wistar rats on embryonic day 18. When a 1064-nm laser beam for optical tweezers was focused on the synaptic vesicles labeled with a fluorescent dye FM1-43 in a neuronal cell, the fluorescence intensity at the focal spot increased gradually with the laser irradiation time, suggesting that optical trapping force causes vesicles assembly at the focus. The decay time of fluorescence autocorrelation curves obtained from FCS increased with the trapping laser power and the laser irradiation time, indicating that vesicle motion was restrained at the focus. This is caused because the vesicles form clusters in synapses, and these clusters are effectively trapped at the focal spot because of strong trapping force. Furthermore, optical trapping suppressed releasing synaptic vesicles after high K+ stimulation, suggesting that our method has a potential to manipulate synaptic transmission at single synapse level. To achieve laser manipulation of smaller molecules in neuronal cell, we applied optical tweezers to manipulate neural cell adhesion molecules (NCAMs) labeled with quantum dots (Q-dots) in neurons. The NCAM whose diameter is ~10 nm labeled with a few tens of nm-sized Q-dot can be trapped slight easily as compared to trapping of the single molecule. After immunofluorescence staining, the fluctuation of fluorescent puncta corresponding to Q-dots attached to NCAM was observed. After focusing a trapping laser on NCAMs labeled with Q-dots of a neuron, the fluorescence intensity of Q-dots gradually increased at the focal spot (Fig. 1), suggesting that Q-dots attached to NCAM around the laser spot were optically trapped and assembled. The assembling dynamics of NCAMs labeled with Q-dots in an optical trap was revealed by FCS that the particle motion of Q-dots attached to NCAM was constrained at the laser focus due to optical trapping force. Our method can be applied to laser-induced perturbation of living neuronal cells, which will achieve the realization of drug-free optical control of synaptic transmission in living neuronal network.

[1] C. Hosokawa, S. N. Kudoh, M. Suzuki, A. Kiyohara, and T. Taguchi, NeuroReport, 19 (2008) 771. [2] C. Hosokawa, S. N. Kudoh, M. Suzuki, A. Kiyohara, and T. Taguchi, Appl. Phys. Lett., 98 (2011) 163705. [3] C. Hosokawa, Y. Sakamoto, S. N. Kudoh, Y. Hosokawa, and T. Taguchi, Appl. Phys. A, 110 (2013) 607.

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Development of homogeneous neural network and multichannel incubation-type planar patch clamp

-aiming high performance disease model chip-

Tsuneo Urisu

Nagoya University, FIRST Research Center for Innovative Nanobiodevices,

Neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) are intractable diseases, for which

neither of cause and treatment methods are known in spite of more than 100 years of researches. The biggest

reason is that the patient’s neurons cannot be sampled in their lifetime. Recently mutated genes of patients are

being identified. By transfecting these mutated genes into animals, cloning of disease animal, the model animals,

was succeeded．Using these model animals, investigation of origin of the disease and the development of

medicines have been actively carried out. However, any successful results have not been obtained. Concerning

medicines, although it was effective in the model animals, its effect was small in the human patients. I think that

the essential difficulty is contained in the model animal method, where causative genes are excessively

transfected into animals which originally have not such a disease. Revolutionary change has been brought into

this field by the invention of iPS technology. We can make the neuron or neural networks by the differentiation

of iPS cells induced from the skin or blood cells of patients. By this method, we can make the in vitro neural

network of the patient him/her self, that is disease model chip, which expresses the disease characteristics. .

We have been, in recent several years, developing noble technology of neural network formation, technology of

multichannel ionchannel recording using incubation type planar patch clamp and several imaging technologies

such as Ca2+ imaging. And now, we are considering to combining these technologies and iPS technology to

originate the disease model chip of ALS. We are now developing several technologies of, chip fabricaton, cell

seeding for noble neural network, integrated microfluidic device of multi-ionchannel recordings, data analysis

and differentiation of iPS cells in parallel. I will introduce in the meeting about the present stage of our

developments. This work is the results of collaborations of all members of my group (Z-H Wang, H. Uno, Y.

Nagaoka, K. Kobayashi, S.Miho, M.Murakami, Y. Suzuki).

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Invited Speakers Japan-Korea Special Session

for Bionano-sensing Technology Plenary lecture will take place in Tobata Campus,

Multipurpose hall of Nakamura Centenary Hall

46

Ionics and Gold Microshells for Bioanalysis

T. D. Chung

School of Chemistry, Seoul National University, Seoul, Korea Handling ions in microfluidic systems is a strong way to create unprecedented opportunities at the interface between informational technology and biotechnology. This presentation will be dealing with how a simple and well known polyelectrolyte can play creative roles in introducing electrochemical methods to microfluidic chip systems. A few novel chip systems on the basis of polyelectrolytic gel electrodes(PGE), including iontronic diodes/logic gates, complete blood cell counter, ultra-fast ion-depletion micromixer, and efficient preconcentrator. Conductance across a pair of PGEs proportionally responds to the volume of micrometer scale objects that pass along the microchannel, e.g. the human blood cells and the functionalized microbeads. The same structure can lead to a microfluidic preconcentrator that makes very low concentration of ionic species and proteins and even nanoparticles dispersed in the solution. This is what resulted from concentration polarization near the PGEs, being easily and systematically driven by external electric control. Not only electrochemical principle but Raman spectroscopy can be met each other to create innovative chip devices. Sophisticatedly decorated microbeads allows high sensitive surface enhanced Raman scattering(SERS) that may amplify the analytical power of microfluidic system. In this presentation, a few examples of SERS-combined chips will be introduced to suggest innovative advances in chemistry as well as bioanalytical devices. [1] B. J. Kim, D.-J. Kim, Y.-R. Kim, S. Y. Lim, J. H. Bae, K.-B. Kim, T. D. Chung Adv. Mater., 24 (2012) 421. [2] C. M. Kang, S. Joo, J. H. Bae, Y.-R. Kim, Y. Kim, T. D. Chung Anal. Chem. 84 (2012) 901. [3] C. S. Jeon, I. Hwang, T. D. Chung Adv. Funct. Mater., 23 (2013) 1484, front cover. [4] H. Choi, K. B. Kim, C. S. Jeon, I. Hwang, S. Lee, H. K. Kim, H. C. Kim, T. D. Chung Lab on a Chip,

13 (2013) 970. [5] S. Kim, L. Piao, D. Han, B. J. Kim, T. D. Chung Adv. Mater., 25 (2013) 2056.

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To be Soluble, or not to be, that is a question

Sihyun Ham

Department of Chemistry, Sookmyung Women’s University Hyochangwon-gil 52, Yongsan-gu, Seoul 140-742, Korea

A number of human diseases such as Alzheimer’s, Parkinson’s, and type II diabetes are associated with the formation of toxic protein aggregates. Protein aggregation also represents a major bottleneck in the biotechnological production of polypeptide-based drugs and antibody-based reagents. Understanding the molecular determinants of the relative propensity for proteins to aggregate in a cellular environment has therefore been a central issue in attacking protein-aggregation diseases and in the development of human therapeutics. Despite the prevailing expectation that the protein aggregation can largely be attributed to the direct protein-protein interactions within an aggregate or in solution, we here unveil a crucial role of water in ruling the aggregation propensity of proteins both in vitro and in vivo. The protein overall hydrophobicity, defined solely by the hydration free energy of a protein in its monomeric state sampling its equilibrium structures, was shown to predominantly dictate the protein aggregation propensity in aqueous solutions. This is demonstrated for in total 61 protein systems of largely varying aggregation propensity, ranging from intrinsically disordered to natively folded forms. We also find striking and unexpected discrimination of positively and negatively charged residues by surrounding layered water in regulating the solubility of a protein. Guided by such observations, we provide novel protein design principles to control protein solubility, which impacts strategies for the biotechnological generation of aggregation-resistant proteins as biotherapeutics. [1] S.-H. Chong, C. Lee, G. Kang, M. Park, S. Ham, J. Am. Chem. Soc. 133, 7075, (2011). [2] C. Lee, S. Ham, J. Comp. Chem. 32, 349, (2011). [3] S.-H. Chong, M. Park, S. Ham, J. Chem. Theo. Comp., 8, 724, (2012). [4] S.-H. Chong, S. Ham, Proc. Natl. Acad. Sci. USA, 109, 7636, (2012). [5] S.-H. Chong, J. Yim, S. Ham, J. Phys. Chem. B, 117, 5503, (2013).

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Metalloid polymer nanoparticle functionalized graphene oxide working electrode for durable glucose sensing

Min-Ho Lee‡

‡Korea Electronics Technology Institute, Medical IT Technology, Gyeonggi-DO 463-816, Republic of Korea

ABSTRACT

A new class of functionalized graphene oxide (FGO) nanosheet based amperometric glucose biosensor platform has been fabricated. FGO nanosheet comprises of chemically bound

metalloid polymer hybrid (MPH) nanoparticles (average size of 12.5 ±2 nm) on the surface of a graphene oxide (GO) nanosheet. Spectroscopic characterization indicated that MPHs are well distributed, with a strong binding affinity between the GO nanosheets. The synergistic features of the metalloid polymer and the GO resulted in a unique three-dimensional nano-architecture on a gold-printed circuit board electrode (Au-PCB). The electrocatalytic response against a glucose sample is predominant, with a characteristic response time of 7s, correlation co-efficient of 0.9981 and a wide linear range of up to 55.5 mM. The stability of the nano-architecture modified on the electrode substrate is suitably durable for long-term application. The practical applicability of the fabricated electrode system was evaluated using a hyperglycemic clinical samples, and was compared with a commercial glucose biosensor. The obtained amperometric results were in good agreement with those of the commercial biosensor, and are promising for further clinical applications.

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Applications of Potential Modulated Microfluidic Channel System to Electrochemical Analysis

Yoon-Bo Shim, Hui-Bog Noh, You Jeong Kim

Department of Chemistry and Institute of BioPhysio Sensor Technology, Pusan National University, Busan 609-735,

Korea, ybshim@pusan.ac.kr, http//chemlab.pusan.ac.kr/echem

I will introduce electrochemical potential modulated microfluidic channels coupled with sensors to analyze the biological and chemical target species[1,2]. The sensor probes and channel walls can be modified with biomaterials and chemicals, such as antibodies, lipids, enzymes, and metalloproteins, where the sensing probe surfaces are composed of nanoparticles and/or conductive polymer composites. The electrochemical microfluidic channels operating by the the AC potential modulation methods performed preconcentration and separation of analytes from the complicate samples. For example, the electrochemical potential modulation microfluidic channel devise was examined for the separation and the detection of heavy metal ions and phenolic endocrine disruptors in the real samples. In addition, the electrochemical microfluidic channel immobilized with enzymes, glucose oxidase and hydrazine functionalized-conducting polymer layers were examined as a model enzyme-loaded biofuel cell device using glucose, whole blood, and H2O2 as fuels. [1] H.B. Noh, P. Chandra, Y.J.Kim, and Y.-B. Shim, Anal. Chem., 84 (2013) 9738. [2] M. J. A. Shiddiky, Y. -B., Shim, Microfluidic Devices in Nanotechnology, C. S. Kumar(Eds.), John Wiley

and Sons, (2010), 213.

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Glucose-Oxidase Label-Based Redox Cycling for an Incubation Period-Free Electrochemical Immunosensor

Haesik Yang

Department of Chemistry, Pusan National University

Catalytic reactions of enzyme labels in enzyme-linked immunosorbent assays require a long incubation period to obtain high signal amplification. We present herein a simple immunosensing scheme in which the incubation period is minimized without a large increase in the detection limit. This scheme is based on electrochemical-enzymatic (EN) redox cycling using glucose oxidase (GOx) as an enzyme label, Ru(NH3)6

3+ as a redox mediator, and glucose as an enzyme substrate. Fast electron mediation of Ru(NH3)6

3+ between the electrode and the GOx label attached to the electrode allows high signal amplification. The acquisition of chronocoulometric charges at a potential in the mass transfer-controlled region excludes the influence of the kinetics of Ru(NH3)6

2+ electrooxidation and also facilitates high signal-to-background ratios. The reaction between reduced GOx and Ru(NH3)6

3+ is rapid even in air-saturated Tris buffer, where the faster competitive reaction between reduced GOx and dissolved oxygen also occurs. The direct electrooxidation of glucose at the electrode and the direct electron transfer between glucose and Ru(NH3)6

3+ that undesirably increase background levels occur relatively slowly. The detection limit for the EN redox cycling-based detection of cancer antigen 125 (CA-125) in human serum is slightly higher than 0.1 U/mL for the incubation period of 0 min, and the detection limits for the incubation periods of 5 and 10 min are slightly lower than 0.1 U/mL, indicating that the detection limits are almost similar irrespective of the incubation period and that the immunosensor is highly sensitive.

glucose Gluconicacid

2Ru(NH3)62+2Ru(NH3)6

3+

2e–

GOx

ITO

CA-125

avidin

IgG

IgG

Figure 1. Schematic of electrochemical immunosensor using GOx label-based EN redox cycling.

[1] A. Singh, S. Park and H. Yang, Anal. Chem., 85 (2013) 4863.

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Invited Speakers Plenary lecture will take place in Tobata Campus,

Multipurpose hall of Nakamura Centenary Hall

52

High Accuracy Imaging of Cancer and Peripheral Artery Disease with X-ray CT and Fluorescence

K. Gonda1,2, Y. Kobayashi3, H. Tada4 and N. Ohuchi2,4

1 Department of Medical Physics, Graduate School of Medicine, Tohoku University

2 Department of Nano-Medical Science, Graduate School of Medicine, Tohoku University 3 Department of Biomolecular Functional Engineering, College of Engineering, Ibaraki University

4 Department of Surgical Oncology, Graduate School of Medicine, Tohoku University

Cancer and arterial sclerosis are main cause of the death in Japan. The effective development of diagnosis in early stage and treatment methods for those diseases is greatly expected. Imaging technology is very important for the development for the diagnosis and treatment. We have been developing the technologies for X-ray computed tomography (CT) imaging or fluorescence imaging to clarify the mechanism for cancer and angiogenesis in peripheral artery disease and develop the diagnostic methods for these diseases with high accuracy and highly-quantitative sensitivity [1-5].

We newly prepared gold nanoparticles as contrast agents for X-ray CT imaging. The gold nanoparticles is coated with silica-layer or PEG chains. Gold is superior to conventional iodine-base compounds contrast agent in term of X-ray absorption coefficients. As the resolution of X-ray CT imaging is around several tens of micrometers, the CT imaging can visualize at levels ranging from small tissues to whole body by high penetrative power of X-rays. Quantum dots (QDs) is one of recently-developed fluorescence nanoparticles. The brightness and photostability of QDs is much greater than those of conventional fluorescent dyes or proteins. To perform various biomedical fluorescence imaging, we are using modified QDs and own in vivo imaging system with spatial accuracy of 9 nm. Fluorescence imaging generally has the resolution with hundreds of nanometer and high quantitative sensitivity because the fluorescence signal intensity is proportional to the intensity of the photon excitation energy. However, as the fluorescence imaging was affected by optical scatter and absorption in cells or tissues, tissue permeability of fluorescence is not so good. Therefore, fluorescence imaging is suitable to visualize at levels ranging from molecular to small tissues. The technology integration of both advantages for X-ray CT and fluorescence imaging is thought to greatly contribute to development of medical imaging with high accuracy and highly-quantitative sensitivity at levels ranging from molecular to whole body. Here we introduce high accuracy imaging of cancer and peripheral artery disease with X-ray CT or fluorescence and discuss the technology integration of both imaging.

[1] Gonda K, Watanabe TM, Ohuchi N, Higuchi H. In vivo nano-imaging of membrane dynamics in metastatic tumor cells using quantum dots. Journal of Biological Chemistry 285:2750-2757 (2010).

[2] Hikage M, Gonda K, Takeda M, Kamei T, Kobayashi M, Kumasaka M, Watanabe M, Satomi S, Ohuchi N. Nano-imaging of the lymph network structure with quantum dots. Nanotechnology 21: 185103 (2010).

[3] Kobayashi Y, Inose H, Nakagawa T, Gonda K, Takeda M, Ohuchi N, Kasuya A. Control of shell thickness in silica-coating of Au nanoparticles and their X-ray imaging properties. Journal of Colloid & Interface Science 358: 329-333 (2011).

[4] Hamada Y, Gonda K, Takeda M, Sato A, Watanabe M, Yambe T, Satomi S, Ohuchi N. In vivo imaging of the molecular distribution of the VEGF receptor during angiogenesis in a mouse model of ischemia. Blood 118: e93-e100 (2011).

[5] Gonda K, Miyashita M, Watanabe M, Takahashi Y, Goda H, Okada H, Nakano Y, Tada H, Amari M, Ohuchi N. Development of a quantitative diagnostic method of estrogen receptor expression levels by immunohistochemistry using organic fluorescent material-assembled nanoparticles. Biochemical and Biophysical Research Communications 426: 409-414 (2012)

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53

Organosilica Nanoparticles for Multimodal imaging

M. Nakamura1, K. Hayashi1, H. Kubo2, M. Harada3, K. Izumi4, K. Ishimura1

1Department of Anatomy and Cell Biology, Institute of Health Biosciences, The University of Tokushima Graduate School 2Advanced Clinical Research Center, Fukushima Medical University

3Department of Radiology, Institute of Health Biosciences, The University of Tokushima Graduate School 4Department of Molecular and Environmental Pathology, Institute of Health Biosciences,

The University of Tokushima Graduate School

Organosilica nanoparticles made from a single organosilicate are new nanomaterials for bioassay, imaging, and nanomedicine [1, 2]. Organosilica nanoparticles are both structurally and functionally different from typical silica nanoparticles (inorganosilica nanoparticles) prepared from tetraethoxyorthosilicate because the organosilica particles contain both interior and exterior functionalities such as mercaptopropyl residue (Fig. 1). The organosilica nanoparticles allow for facile surface and internal functionalization, offering new opportunities to create multifunctionalized nanoparticles. Various sizes of fluorescent organosilica nanoparticle containing various types of fluorescent dye can be prepared using a one-pot synthesis. [1] Functional fusions of organosilica nanoparticles and other functional nanoparticles such as quantum dots, gold nanoparticles, and iron oxides are possible based on organosilica particles technology [2] (Fig. 2). Multifunctionalized organosilica nanoparticles have high potential to create novel imaging systems and therapeutic applications [3, 4].

Multifunctionalized organosilica nanoparticles were applied to multimodal imaging. The multimodal thiol-organosilica nanoparticles containing magnet nanoparticles and fluorescent dye showed unique magnetic resonance imaging (MRI) signals and high fluorescence intensity. We injected multimodal thiol-organosilica nanoparticles into normal mice, rats, and Long−Evans Cinnamon (LEC) rat intravenously. Injected mice revealed alteration of signal of livers on MRI and unique fluorescent pattern of liver on mesoscopic fluorescent imaging using multipurpose zoom fluorescence microscopy. The LEC rat revealed tumors in liver clearly on MRI, and showed pathologic fluorescent histological pattern of liver on mesoscopic fluorescent imaging. We performed a correlation analysis between MRI, mesoscopic fluorescent imaging and histological findings of liver. We demonstrated new approach to evaluate pathological imaging and advantages of multimodal imaging using MRI and mesoscopic fluorescence imaging. [1] M. Nakamura and K. Ishimura, J. Phys. Chem. C, 111 (2007) 18892. M. Nakamura and K. Ishimura,

Langmuir, 24 (2008) 5099. M. Nakamura and K. Ishimura, Langmuir, 24 (2008) 12228. K. Hayashi, M. Nakamura and K. Ishimura, Chem. Comm., 47 (2011) 1518. M. Nakamura, A. Awaad, K. Hayashi, K. Ochiai and K. Ishimura, Chem. Mater. 24 (2012) 3772.

[2] M. Nakamura, S. Ozaki, M. Abe, T. Matsumoto and K. Ishimura, J. Mater. Chem., 21 (2011) 4689. [3] A. Awaad, M. Nakamura and K. Ishimura, Nanomedicine: NBM, 8 (2012) 627. A. Awaad, M. Nakamura and

K. Ishimura, Int. J. Nanomed., 7 (2012) 1423. M. Nakamura, K. Miyamoto, K. Hayashi, A. Awaad, M. Ochiai and K. Ishimura, Nanomedicine: NBM, 9 (2013) 274.

[4] M. Nakamura, Nanostructured Oxides. Wiley-VCH Verlag GmbH & Co. KGaA, (2009) 109. M. Nakamura, Nanotechnol. Rev. 1 (2012) 469.

Fig. 1. Schematic structure of inorganosilica nanoparticles and organosilica nanoparticles

Fig. 2. Schematic structure of multimodal organosilica nanoparticles

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Development of bioassays using biotin ligase modified with a luminescent Tb3+ complex

S. Sueda1,2, T. Ikeda1, H. Hayashi1, Y. Shinboku1, and T. Kusaba1

1 Department of Bioscience and Bioinformatics, Kyushu Institute of Technology 2 Research Center for Bio-microsensing Technology, Kyushu Institute of Technology

Complexes of lanthanide ions, particularly Tb3+ and Eu3+, are very powerful tools for analyzing biological samples because of their luminescence properties such as long emission lifetime, narrow emission bands, and large stokes shift. Their long-lived luminescence enables the time-resolved measurement to remove background emission. When lanthanide complexes are used as donors in resonance energy transfer system in combination with short-lived fluorescence acceptor, the signal from luminescence resonance energy transfer (LRET) allows for highly sensitive detection of molecular interactions such as protein-protein interactions. In this paper, we will present bioassays using biotin protein ligase (BPL) modified with a luminescent Tb3+ complex. BPL catalyzes a covalent attachment of biotin to a specific lysine residue of biotin carboxyl carrier protein (BCCP). We found that biotinylation from archaeon Sulfolobus tokodaii has a unique property that the enzyme BPL forms a stable complex with its product, biotinylated BCCP [1]. By employing this unique enzyme reaction, we developed a fluorescent labeling system of proteins, where the target proteins fused to BCCP were labeled through biotinylation with BPLs carrying fluorophores [2]. In the present work, a mutant of BPL with an additional cysteine residue was modified on their cysteine residues with a luminescent Tb3+ complex carrying a maleimide group (Fig. 1), and two types of bioassays were constructed by using the modified BPL; one is a tyrosine kinase assay and another is an assay for determination of biotin and ATP. In the tyrosine kinase assay, a Src homology 2 (SH2) domain from human lymphocyte-specific tyrosine kinase was genetically fused to BCCP, and the resulting fusion protein was labeled with Tb3+ complex-modified BPL [3]. The labeled SH2 fusion proteins were employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide was produced by phosphorylation to the substrate peptide by Src tyrosine kinase. Our assay allows for a reliable determination of the activity of Src kinase lower than 10 pg/µL, and the detection limit was estimated to be about 2 and 1 pg /µL for 2 and 6 h reactions, respectively.

In the S. tokodaii biotinylation, a stable complex of BPL with BCCP is formed based on the reaction with biotin and ATP as cosubstrates. Thus, the determination of biotin and ATP is enabled by monitoring the complexation between BPL and BCCP. Here, LRET was used for monitoring the complexation. To construct the assay based on LRET, BCCP was labeled with fluorescein which can act as an acceptor for the donor Tb3+ complex. The emission spectra of the reaction solution containing the Tb3+ complex-modified BPL and fluorescein-labeled BCCP were recorded by time-resolved measurement. As expected, the sensitized emission of fluorescein based on LRET was observed through the excitation of Tb3+ complex. To examine the sensitivity of this assay, the reactions were conducted with various concentration of biotin and ATP. From these experiments, the detection limit was estimated to be 1 pM and 0.1 nM for biotin and ATP, respectively. This detection limit for biotin is classified into the highest sensitivity reported so far, and this is the first example of the highly sensitive biotin assay in a homogeneous format.

Fig. 1 (a) A model structure of S. tokodaii BPL complexed with BCCP. The structure on the right was generated by rotating the left by 90°. (b) Chemical structure of the Tb3+ complex used in this study. [1] S. Sueda, Y.-Q. Li, H. Kondo, and Y. Kawarabayasi, Biochem. Biophys. Res. Commun., 344 (2006) 155. [2] S. Sueda, S. Yoneda, and H. Hayashi, ChemBioChem, 12 (2011)1367. [3] S. Sueda, Y. Shinboku, and T. Kusaba, Anal. Sci., 29 (2013) 491.

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Toxicity Mechanism of Nanocarbon

M. Yang1, M. Zhang1, Y. Tahara1, S. Iijima1 ,2, M. Yudaska1

1 National Institute of Advanced Industrial Science and Technology (AIST), Nanotube Research Center, Tsukuba, 305-8565, Japan

2 Faculty of Science and Technology, Meijo University, Shiogamaguchi, Nagoya, 468-8502, Japan

Various types of nanocarbons have been found, and many of them have high potential in medical applications. We have been demonstrating that carbon nanohorns (CNHs), a type of nanocarbons having tubule structure with diameters of 2-4 nm and length of 40-50 nm [1], can store anticancer drugs in their inner nano-spaces and the released drugs have higher therapeutic effects in cell and animal tests [2]. CNHs can absorb near infrared light and emit the thermal energy, therefore has high effect of photo-hyperthermia therapy [3]. Multiple functionalizations of CNHs are easy, which is useful for the biocompatibility control of CNHs [3] and in providing tumor targeting property. The CNH forms spherical robust-aggregate with sizes of 100 nm [2], and the aggregates can be separated into smaller aggregates or individual CNHs by using oxidative acids [4]. Their sizes are in the criterion of EPR effective, therefore the passive targeting to tumors may be possible. For the future medcal applications, the toxicity of CNHs should be studied carefully now. The biodistribution of CNHs in mice has been studied quantitatively by using the unique labels of Gd2O3 nanopatricles that are embedded within CNHs [5]. The CNHs in the organs were quantified by measuring the Gd quantities by ICP. The CNHs biodistribution depended on the size and surface functionalization of CNHs [6-7], however, those remained in the body are always accumulated in macrophages or endothelial cells. Thus we have studied the cytotoxicity by using murine macrophage cell line, RAW264.7. The cytotoxicity appeared at the high-uptake condition, where the cell apoptosis and necrosis were found [8]. Although the cells died, the cytokines releases were not significant in the quantities [8]. Also in the mice experiments, the cytokine release was not increased by the intravenous injection of CNHs, and indeed the blood test did not show any abnormality [7]. It is reported that uptake of nanocarbons generates ROS in vitro, and the ROS induces inflammatory responses and cell death. However, how the nanocarbons induce ROS generation is not clear. Thus we have studied the ROS generation mechanism and its relation to the cell apoptosis. The RAW264.7 cells were incubated with CNHs that were treated with light-assisted oxidation with H2O2 to afford plenty of oxygenated groups. The obtained CNHs were highly dispersible in culture medium. The CNH quantities engulfed by RAW264.7 were quantified by the optical absorption intensity of cell lysate at 700 nm. The CNHs were accumulated in lysosomes and lysosome membranes permeabilization (LMP) occurred, coinciding with the previous results [8]. After LMP, ROS was generated, and mitochondrial membrane dysfunction proceeded, activating the caspase 3/7. When the cathepsins (lysosome enzymes) inhibitors were used, ROS generation was suppressed and the mitochondrial membrane dysfunction was reduced. This suggests that LMP caused by CNHs was the first events, and the lysosome enzymes released in cytosols could induce the ROS generation. Supporting this conclusion, the ROS inhibitors did not suppress the LMP. We further studied the behavior of CNHs in lysosomes with transmission electron microscopy observation. Some of the CNHs in the lysosomes attached to the lysosome membranes, where the membrane structures were disordered. We tentatively believe that this is the major reason of LMP caused by CNHs. [1] Iijima et al. Chem. Phys. Lett. 1999, 309, 165–70. [2] Ajima et al. ACS nano 2008, 2, 2057-2064. [3] Zhang et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 14773-14778. [4] Zhang et al. Small, 2012, 8, 2524-31. [5] Miyawaki, Matsumura, et al. ACSNano, 2009, 3, 1399-1406. [6] Tahara, et al. Nanotechnology, 2011, 22, 265106(6pp) [7]Zhang, et al. Nanomedicine, 2013, 9, 657-64. [8]Tahara et al. Biomaterials, 2012, 33, 2762-2769.

Contact: M. Yudasaka (m-yudasaka@aist.go.jp)

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Optimization of Self-Heating Properties of Multifunctional Magnetic Nanoparticles

Yuko Ichiyanagi

Department of Physics, Graduate School of Engineering, Yokohama National University

Fe3-xCoxO4 (x = 0, 0.3, 0.5) magnetic particles were prepared by our novel wet chemical method. In this sense, cobalt ions were doped into magnetite (Fe3O4) particles. Particle size was controlled between 6 and 22 nm. Functionalization by amino group, carboxyl group and thiol group was already established. Then cancer cell selective magnetic particles were further developed by conjugating with folic acid [1]. In order to realize magnetic hyperthermia treatment, optimization of magnetic parameters for nanoparticles was performed. If the system follows Néel relaxation, AC magnetic susceptibility causes power loss [2]. AC magnetic susceptibility was measured and imaginary part χ” was analyzed for heat dissipation ability. The largest magnetization value was observed at a composition where x = 0.3 of Fe3-xCoxO4 system. Then we have examined particle size dependence of magnetization for this composition. Fig. 1 shows the temperature dependence of imaginary part of magnetic susceptibility χ” of the sample of x=0.3 for various particle sizes. The peak shifted higher temperature side as the particle size increased. The particle size of 7.5 nm was highly expected for heating effect by external field at room temperature. The temperature rise of this sample upon an alternative field was measured, and it was found that the temperature increased by about 20 K under AC frequency of 100 Hz and field of 1 Oe. From the temperature increase rate, heat dissipation was evaluated quantitatively according to Néel relaxation theory as shown in Fig.2. Temperature increase depending on the magnetic field strength and frequency supported that the sample with composition of x=0.3 with diameter of 7.5 nm was appropriate for use as an agent in hyperthermia treatment. Drastic effect of magnetic hyperthermia was observed in vitro experiment. On the other hand, it was found that Fe3-xCoxO4 nanoparticles were effective as an agent for MRI imaging [3]. Our multifunctional magnetic nanoparticles would be expected as agents of future biomedical applications. [1] S. Taira, S. Moritake, Y. Kai, T. Hatanaka, Y. Ichiyanagi and M. Setou, eJ. Surf. Sci. Nanotech., 5 (2007) 23. [2] D. Shigeoka, H. Katayanagi, Y. Moro, S. Kimura, T. Mashino, T. Hiroki and Y. Ichiyanagi, J. Phys .Conf.

Ser. 200 (2010) 122002. [3] N. Sakai, L. Zhu, A. Kurokawa, H. Takeuchi, S. Yano, T. Yanoh, N. Wada, S. Taira, Y. Hosokai, A. Usui, Y.

Machida, H. Saito and Y. Ichiyanagi, J. Phys .Conf. Ser.s 352 (2012) 012008

150 200 250 300 350 4000.0

0.5

1.0

1.5

2.0

2.5 f = 100 HzH = 1 Oe

Sample : Fe2.7Co0.3O4

Imag

inary

part

of

AC su

scep

tibili

ty , c

" / 10

-3 em

u g-1 O

e-1

Temperature / K

6.8 nm 7.5 nm 9.8 nm 13.2 nm 18.0 nm

Fig.1 Temperature dependence of imaginary part of magnetic susceptibility χ“ of the sample of Fe2.7Co0.3O4 MNPs for various particle sizes.

5 10 15 20 250.0

0.1

0.2

0.3

0.4

0.5

Heat

dis

sipa

tion /

W g

-1

Particle diameter / nm

measured value estimated value

Fig.2 Optimization of heat dissipation depending on particle size of the sample of Fe2.7Co0.3O4 MNPs.

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Developments of the Metal-ion Containing Fluorescent Probes

Akio Ojida

1 Graduate School of Pharmaceutical Sciences, Kyushu University Fluorescence sensing with small molecular chemosensors is a versatile technique for elucidation of function of various biological substances. I first present a new fluorescent chemosensor for nucleoside polyphosphates such as ATP using metal-anion coordination chemistry. The chemosensor 1-2Zn(II) is comprised of the two sites of 2,2’-dipicolylamine (Dpa)-Zn(II) as the binding motifs and xanthene as a fluorescent sensing unit for nucleoside polyphosphates. The chemosensor 1-2Zn(II) selectively senses nucleoside polyphosphates with a large fluorescence enhancement (F/Fo > 15) and strong binding affinity (Kapp ≈ 1 x 106 M-1), whereas no detectable fluorescence change was induced by monophosphate species and various other anions. The ‘turn-on,’ fluorescence of 1-2Zn(II) is based on a new mechanism, which involves the binding-induced recovery of the conjugated form of the xanthene ring, from its non-fluorescent de-conjugated state which was formed by an unprecedented nucleophilic attack of zinc-bound water. The selective and highly sensitive ability of 1-2Zn(II) to detect nucleoside polyphosphates enables its bio-analytical applications in fluorescence visualization of ATP particulate stores in living cells, demonstrating the potential utility of 1-2Zn(II). As the second topic, I also present the design of the dual emission fluorescent probes for metal ions based on a unique fluorescence sensing mechanism. This mechanism involves the non-coordination contact between an aromatic ring of a fluorophore and a metal ion, which causes a large fluorescence emission shift of the fluorophore. Screening assay revealed that the fluorescent probes displayed a large emission red-shift upon binding to a variety of metal ions, such as CdII AgI, HgII, and AuIII. The unique structures of the fluorescent CdII and AgI complexes such as 1-Cd(II) and 1-Ag(II) were solved by X-ray crystallography. These studies revealed that the metal ions are positioned in close proximity of the fluorophores and form an arene–metal ion contact (AM-contact). The electronic structure calculations based on time-dependent density functional theory (TDDFT), in which the metal ions, such as CdII and AgI, electrostatically modulate the energy levels of the molecular orbitals of the fluorophore, provide theoretical support for this sensing mechanism. The fluorescent probe incorporating this sensing mechanism was successfully applied not only to visualize toxic CdII ion trafficking in living cells, but also to sense bioactive hydrogen sulfide (H2S) with a ratiometric signal, both of which highlight the usefulness of this sensing mechanism in biological fluorescence analysis.

[1] Y. Kurishita, K. Kohira, A. Ojida, I. Hamachi, J. Am. Chem. Soc., 134, 18779 (2012). [2] Y. Kurishita, K. Kohira, A. Ojida, I. Hamachi, J. Am. Chem. Soc., 132, 13290 (2010). [3] A. Ojida, I. Takashima, T. Kohira, H. Nonaka, I. Hamachi, J. Am. Chem. Soc., 130, 12095 (2008).

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Cell-Sized Liposomes: Basic Structures for Mimicking Cell Systems

K. Tsumoto1, M. Nishigami1, T. Mori1 and M. Tomita1

1 Division of Chemistry for Materials, Graduate School of Engineering, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie

514-8507, Japan Artificial membranes have been utilized for research on structure and functions of membrane proteins, biomembrane micro-domains, etc. In special, phospholipid vesicles, that is, liposomes are often employed for these purposes. Generally, liposomes are classified into several types according to the diameter (small (~50 nm), large (~100 - 1000 nm), and giant vesicles (~1 - 100 μm)) and lamellarity (unilamellar and multilamellar vesicles). Giant liposomes, which are usually called giant unilamellar vesicles (GUVs), have diameters similar to actual living cells. For several decades, such cell-sized liposomes are considered to be basic platform structures for construction of artificial cell systems to mimic cellular functions. In the present talk, we report our improved simple methods for preparation of cell-sized unilamellar liposomes, observation of their morphological changes, and reconstitution of membrane proteins on the liposome membranes. Although vesicular structures consisting of phospholipids are stable in aqueous conditions, cell-sized vesicles are sometimes difficult to form especially in the presence of salt when the chief component of the phospholipid is phosphatidylcholine (PC), which is electrostatically neutral. Since PC is dominant phospholipid in biomembranes, it would be very useful to efficiently generate giant liposomes of PC. Two hydration methods, electroformation and gentle hydration (natural swelling), are often used for preparation of giant liposomes; the electroformation are very popular, but a special apparatus is required. Instead, the gentle hydration method does not need such apparatus, and the dry phospholipid films are subjected to simple hydration with aqueous solutions; however, the efficiency of the hydrations may strongly depend on the composition of the dry films and hydration solutions. When the films contain none or a small amount of acidic phospholipids such as phosphatidylglycerol (PG), it is difficult to obtain spherical giant unilamellar liposomes. Recently, we found that solutes, sugar (hexose) [1] and sodium iodide (NaI) [2], entrapped in advance within interlayer spaces of lamellar dry films could efficiently promote formation of spherical cell-sized liposomes only consisting of dioleoylphosphatidylcholine (DOPC) even in the presence of salt (up to ~100 mM NaCl). Because the solutes are soluble in not only water but also methanol, it is possible to dissolve DOPC together with the solutes in a mixed solvent (chloroform/methanol). After evaporating the solutions, solute-sandwiched phospholipid films are formed: Upon bathing the dry films, interlayer osmotic pressure can be induced immediately and increase each interlayer distance of lamellar films through unbinding transition, resulting in generation of cell-sized liposomes. This protocol is very simple and moderate enough to easily encapsulate biological molecules such as proteins without degradation of the function. Second, we introduce agarose gel-embedded cell-sized liposomes [3]. The gel-embedded giant liposomes are so intact that the membranes can serve as a barrier against hydrophilic reagents. Interestingly, probably because entrapment inside gels might reduce a rate of deformation of lipid membranes, we successfully observed morphological changes of giant liposomes using a confocal laser scanning microscope in real time: Destabilization of lipid membranes were induced by addition of the surfactant Triton X-100 and the deformation was monitored with permeation and collapse distinguishable. An intermediate of invagination of liposome membranes could be also captured very clearly. Finally, we report our novel method to reconstitute recombinant membrane proteins such as hormone receptors and channels on cell-sized liposomes through membrane fusion between the liposomes and recombinant baculovirus budded virus enveloped particles that display the recombinant proteins [4, 5]. The reconstituted membrane proteins could be visualized on individual proteins through specific binding with fluorescently labeled ligands, suggesting that the structure and function of the membrane protein could be kept intact. We believe that such techniques with cell-sized liposomes could be useful in research on nanomedicine related to properties of biomembranes. [1] K. Tsumoto, H. Matsuo, M. Tomita, and T. Yoshimura, Colloid. Surf. B: Biointerfaces, 68 (2009) 98. [2] N.L. Yamada, M. Hishida, H. Seto, K. Tsumoto, and T. Yoshimura, EPL (Europhys. Lett.), 80 (2007) 48002. [3] K. Tsumoto, M. Oohashi, and M. Tomita, Colloid Polym. Sci., 289 (2011) 1337. [4] K. Tsumoto, and T. Yoshimura, Methods Enzymol. 465 (2009) 95. [5] K. Kamiya, J. Kobayashi, T. Yoshimura, and K. Tsumoto, Biochim. Biophys. Acta-Biomembranes, 1798

(2010) 1625.

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Anti-tumor Effects of Cationic Liposomes Against Tumor Cells Along With Apoptosis

Y. Matsumoto

Department of Applied Life Sciences, Graduate School of Engineering, Sojo University

Tumor cell membranes show changes in the composition, and anionic lipids (phosphatidylserine (PS)) in the outer plasma membrane of tumor cells increase as compared with the normal cells. Negatively charged characteristics of tumor cell membranes are important as a target for novel chemotherapy. To aim at selective chemotherapy against carcinoma having anionic rich cell membranes, three-component cationic liposomes (CL) composed of dimyristoylphosphatidylcholine (DMPC), polyoxyethylene(21)dodecyl ether (C12(EO)21) and O,O’-ditetradecanoyl-N-(α-trimethylammonio acetyl) diethanolamine chloride (2C14ECl) were produced and the inhibitory effects of CL on the growth of tumor cells in vitro and in vivo were investigated. The physical properties of CL were examined on the basis of a dynamic light scattering measurement and electron microscopy [1]. The mean hydrodynamic diameter of cationic CL were 100 nm, which remained stable for more than one month at 37. It is worthy to note that CL having 100 nm in diameter could avoid the reticularendothelial system. The IC50 values of CL against human renal cell carcinoma (OS-RC-2) cells were much larger than those of normal human renal proximal tubule epithelial (RPTE) cells. CL having positive charge could fuse and accumulate in negatively charged tumor cell membranes through electrostatic interaction between membranes of CL and tumor cells. Induction of apoptosis by CL for OS-RC-2 cells was examined on the basis of flow cytometric analysis and TUNEL method. Fairly high apoptotic DNA rates were obtained after the treatment with CL and OS-RC-2 cells were dyed in green, indicating that CL induced apoptosis for OS-RC-2 cells. Induction of apoptosis by CL for colon carcinoma was also obtained [2]. The activity of caspase-3, -8, and -9 in OS-RC-2 cells drastically increased after the treatment with CL for 4 h, indicating that CL could execute apoptosis through caspase-3, -8, and -9 in the caspase cascade [3]. Mitochondrial transmembrane potential was decreased after the treatment with CL, suggesting that the mitochondrial pathway should be also implicated in apoptosis induced by CL. The induction of apoptosis by CL in mice model of renal carcinoma using TUNEL method in vivo was examined. A large number of apoptotic cells were observed in kidney (tumor cells) sections of mouse model of renal carcinoma after the treatment with CL using an optical microscope [4]. Moreover, we examined the therapeutic effects of CL using a mouse model of renal carcinoma after the inoculation of OS-RC-2 cells in vivo. The median survival time was 15 days in the control group. It is noteworthy that significantly increased survival rates (by 130%) were obtained in mice after the treatment with CL. A statistically significant difference (p<0.01) was confirmed in the prolonged survival rate of mice treated with CL in comparison with that of controls. These results indicate that CL prolongs survival of mice in these models of renal carcinoma. Inhibitory effects of CL without any drug on the growth of renal cell carcinoma in vitro and in vivo along with apoptosis were observed. The results obtained in this study might be applied in clinical applications for patients with renal carcinoma. Fig.1 Schematic representation of anti-tumor effects of CL against tumor cells. [1] M. Umebayashi et al., Biol. Pharm. Bull., 31, (2008) 1816. [2] M. Hino et al., Biol. Pharm. Bull., 35, (2012) 2097. [3] Y. Matsumoto et al., Int. J. Cancer, 115, (2005) 377. [4] M. Umebayashi et al., Anticancer Res., 30, (2010) 327.

+

+ +

++

+++

Phosphatidylserine

Cationic Liposome

Tumor cell

++

+

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++ +

+

+

Electrostatic interaction

Apoptotic signal

+ CL

Fragmentation of DNA

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Introduction for the medical particle accelerator

H. SATO

High Energy Accelerator Research Organization (KEK) At the age of Sir Rutherford’s famous nuclear experiment, every nuclear scientist deeply desired the particle accelerators, even if it is very low energy and low intensity [1]. More than one hundred years after that, accelerators have been developed historically to provide higher beam energy and beam intensity, and extended to collider machines. Accelerated beam is extracted to use not only for elementary particle and nuclear physics experiment but also another several fields such as material science and medical treatment [2]. In recent years, particle beams have often been used in the production of isotopes for nuclear medicine, cancer therapy, ion doping and industrial processing using the nanotechnology. Many medical accelerator facilities have been constructed and planned in the world [3]. More than 20 facilities are operated, under construction, and planned in Japan [4]. The preferred energy of a proton beam should be about 300 MeV. The synchrotron type of accelerator and cyclotron are suitable for this requirement. Not only the accelerated particle but also the synchrotron radiation light is useful for the high-technological imaging and medical treatment. Neutron is also effective for the high-technological imaging and medical treatment. For example, figure shows the fluoroscopy of Beetle by neutron flux. In this talk, the author will present a review of particle accelerator history and introduce the recent status of medical accelerator facilities. . Figure; Fluoroscopy of Beetle by Neutron Flux

References [1] “The Making of the Atomic Bomb” written by Richard Rhodes, first published by Simon and Schuster in

1987. [2] Joint Accelerator Conference Website, http://www.jacow.org/ [3] “Status of the Accelerator Industry in North America”, J.E. Clayton et al., Proceedings of IPAC’10, May 2010, Kyoto, Japan. [4] http://www.nirs.go.jp/ENG/index.shtml

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Local delivery system of cytotoxic agents to tumors by focused sonoporation

K, Iwanaga1, T, Nishihara2, and K, Tominaga1

1Department of Science of Physical Function, Division of Maxillofacial Surgery, Kyushu Dental Univercity. 2Department of Health Promotion, Divison of Infections and Molecular Biology, Kyushu Dental Univercity.

Recently, ultrasound-targeting microbubble destruction has been employed in molecular gene therapy, and a new potent nonviral gene transfer method known as ‘sonoporation’ has been developed. We investigated the efficiency of sonoporation toward growth inhibition of human gingival squamous carcinoma cell line, Ca9-22, in vitro and in vivo. We found that the delivery of bleomycin (BLM) by sonoporation induced cytotoxic effect toward Ca9-22 cells in vitro. Our in vivo results showed that tumors nearly disappeared in Ca9-22 cell-implanted nude mice treated with a low dose of BLM followed by sonoporation. Thus, we used sonoporation to transfect a cdtB-expressing plasmid into Ca9-22 cells and examined cell viability in vitro and in vivo. We found that an administration of cdtB-expressing plasmid followed by sonoporation induced marked growth inhibition of Ca9-22 cells and apoptotic cells were also observed in vitro and in vivo[1]. In addition, we developed a specific drug delivery system for squamous cell carcinoma that uses sonoporation with the anti-epidermal growth factor receptor (EGFR) antibody. Administration of a low dose of BLM by sonoporation with the anti-EGFR antibody produced a marked growth inhibition of Ca9-22 cells in vitro. Interestingly, the population of apoptotic cells was remarkably increased when a low dose of BLM was delivered using sonoporation with the Fab fragment of the anti-EGFR antibody[2]. However, microbubbles are unstable and their targeting ability is insufficient for clinical use. To circumvent these problems, we developed nobel polyethyleneglycol (PEG) modified liposomes (Nano bubble liposome) containing perfluoropropane, which is an ultrasound imaging gas. Polyethylene glicolylated (PEG) liposome avoids uptake by the reticuloendothelial system, thus improving drug delivery to tumor while decreasing systemic toxicity. PEG liposomes containing doxorubicin, Doxil®, have been used to treat Koposi’s sarcoma and ovarian cancer in clinics. Doxil® circulates around the body at a stable state and accumulates in tumor by passive targeting. Although it is difficult to release doxorubicin after accumulation into the tumor, we made a new bubble liposomes from Doxil® and investigated the effect on cytotoxicity by combination with this new Doxil® bubble liposome , encapsulated doxorubicin bubble liposomes(EDBL), and ultrasound in Ca9-22 cells. The combination of EDBL and ultrasound was found to enhance the cytotoxity of Ca9-22 cells treated by doxorubicin. These findings suggest that local administration of cytotoxic agents with sonoporation is a useful method for molecular cancer therapy. [1] K Iwanaga, K Tominaga, K Yamamoto, M Habu, H Maeda, S Akifusa, T Tsujisawa, T Okinaga, J Fukuda,

T Nishihara. Cancer Gene Therapy, 14(2007)354-363. [2] H Maeda, K Tominaga, K Iwanaga, F Nagao, M Habu, T Tsujisawa, Y Seta, K Toyoshima, J Fukuda, T

Nishihara. J Oral Pathol Med, 38(2009)572-579.

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Lung-Specific siRNA Delivery Using Water-Soluble Amino-Fullerene

K. Harano1, K. Minami1, K. Okamoto2, K. Doi2, E. Noiri2, and E. Nakamura1

1 Department of Chemistry, The University of Tokyo, Tokyo, Japan 2 Department of Hemodialysis and Apheresis, University Hospital, The University of Tokyo, Tokyo, Japan

Therapeutic application of RNA interference (RNAi) has been considered as an alternative treatment

of chemotherapy [1]. Versatile non-viral vehicles, which can guide small interfering RNA (siRNA) to the target cells and tissues, have been developed for in vitro as well as in vivo delivery. For in vivo delivery, those vehicles are mainly accumulated in reticuloendothelial systems. Among other tissues lung is an important target tissue for chemotherapy, but the efficient strategies of the lung-specific drug delivery for therapeutic applications are limited. Here we show the efficient strategy that a water-soluble cationic amino-fullerene, (tetrapiperazino)fullerene epoxide (TPFE, Figure a), deliver siRNA selectively to the lung by an agglutination-induced accumulation into the lung [2]. We found that the size of submicrometer-sized particles of TPFE-siRNA complexes can be controlled to micrometer-sized globules by agglutination with plasma proteins under coincubation with serum. The TPFE-siRNA complex spontaneously agglutinated in bloodstream, accumulated and remained in the narrow lung capillaries, and deliver siRNA into the lung cells to achieve the lung-specific knockdown (Figure b).

TPFE was dissolved into acidic buffer and mixed with siRNA to form siRNA-TPFE complex as slightly cationic submicrometer-sized globules, which were characterized by dynamic light scattering, AFM and STEM. The complexation is mediated by the electrostatic interaction between the cationic amines and the anionic phosphate RNA backbone as well as hydrophobic effect of fullerene core. The siRNA-TPFE complex further interacts with plasma proteins in mouse serum to agglutinate into micrometer-sized particles. The siRNA-TPFE complex showed significant resistance against degradation of siRNA under a simulated biological condition. In vitro experiment showed that TPFE delivers not only chemically stabilized siRNA (Stealth RNA, Invitrogen) but also free siRNA more effectively into cultured cells than commercially-available transfection reagent Lipofectamine2000.

We injected siRNA-TPFE complexes intravenously into mice and determined the knockdown efficiency in tissue specimens. The siRNA-TPFE complex showed significant RNAi activity only in the lung (Figure c). The siRNA-TPFE-protein ternaly complex formed immediately in bloodstream and stacked in the narrow lung capillaries. Furthermore, we applied our delivery systems for in vivo animal model of sepsis. Delivery of toll-like receptor 4 (TLR4) siRNA to lipopolysaccharide (LPS)-treated mice suppressed TLR4 expression levels as well as neutrophil accumulation levels in lung. Our results suggest the potential of siRNA delivery using a water-soluble fullerene for clinical application as well as the utility of the strategy of lung-specific delivery via the agglutination-induced accumulation.

Figure. Water-soluble amino-fullerene for siRNA delivery in vivo. (a) Structure of (tetrapiperazino)fullerene epoxide (TPFE). (b) Schematic illustration of lung specific delivery of siRNA by TPFE through hierarchical assembly of TPFE, siRNA and serum proteins to form micrometer-sized particles. (c) Biodistribution of injected siRNA in each organ at 24 h after the injection. mRNA expression of EGFP was evaluated by real-time PCR. Black and white bars indicate the EGFP-targeting siRNA and scrambled siRNA as a negative control, respectively. ** P < 0.005 versus scrambled siRNA group. [1] A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, Driver, S. E.; Mello, C. C. Nature 391 (1998) 806–811. [2] (a) R. Maeda-Mamiya, E. Noiri, H. Isobe, K. Okamoto, K. Doi, T. Sugaya, T. Izumi, T. Homma, E. Nakamura, Proc. Natl. Acad. Sci. U.S.A., 107 (2010) 5339–5344. (b) H. Isobe, W. Nakanishi, N. Tomita, S. Jinno, H. Okayama, E. Nakamura, Chem.–Asian J. 1–2 (2006) 167–175.

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Single cell-scale thermometry by fluorescent temperature reporters

M. Suzuki1,2

1 Waseda Bioscience Research Institute in Singapore, Waseda University, 11 Biopolis Way #05-01/02 Helios, Singapore, 138667 Singapore

2 Organization for University Research Initiatives, Waseda University, #304, Block 120-4, 513 Wasedatsurumaki-cho, Shinjuku-ku, Tokyo, 162-0041 Japan

Thermocouples are widely used to measure our body temperature or the room temperature at homes and workplaces. Temperature distribution of objects as large as or larger than human bodies can conveniently be imaged by infrared cameras. At the scale of single cell (< ~101 µm), especially in aqueous solution, fluorescent temperature reporters have advantages in spatial resolution and in compatibility with live samples. We have previously shown that the heat pulse, the short-lived localized temperature distribution, applied externally modulates the intracellular protein activities non-invasively, thereby causing Ca2+ dynamics in single HeLa cells [1] and Ca2+-independent contraction in single cardiomyocytes [2]. In pursuit of the physiological significance of theses cellular responses to heat pulses and the endogenous thermogenesis possibly generating heat pulses inside the cell, we are developing fluorescent "small" thermometers. I will present the initial development of our methods using glass micro needles filled with temperature sensitive fluorophores for the thermometry at the scale of single cells [3] and its application [4]. In this probe, glass wall is enough transparent for heat to transfer, but it intercepts any objects to reach the fluorophores that are sensitive to intracellular parameters such as pH and ionic strength. Then I will report new nanothermometers composed of fluorescent dyes embedded in polymer nanoparticles [5]. These nanothermometers are sensitive selectively to the temperature but not to other intracellular parameters. I will show the characterization of these materials and new findings obtained in HeLa cells by the temperature measurement in real-time under optical microscope. [1] V. Tseeb, M. Suzuki, K. Oyama, K. Iwai and S. Ishiwata, HFSP J., 3 (2009) 117-123. [2] K. Oyama, A. Mizuno, S.A. Shintani, H. Itoh, T. Serizawa, N. Fukuda, M. Suzuki and S. Ishiwata, Biochem.

Biophys. Res. Commun., 417 (2012) 607-612. [3] V. Zeeb, M. Suzuki and S. Ishiwata, J. Neurosci. Methods, 139 (2004) 69-77. [4] M. Suzuki, V. Tseeb, K. Oyama and S. Ishiwata, Biophys. J., 92 (2007) L46-48. [5] K. Oyama, M. Takabayashi, Y. Takei, S. Arai, S. Takeoka, S. Ishiwata, and M. Suzuki, Lab Chip, 12

(2012) 1591-1593.

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Functional siRNA for fluorescent monitoring of RISC with improved activity and strand selectivity

H. Asanuma1, A. Ito1, J. Takai1, M. Urushihara1, H. Ito1, T. Fujii1, X.G. Liang1,2, and Y. Kamiya1,3

1 Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 Japan

2 School of Food Science and Technology, Ocean University of China, Shinan-qu, Yushan Road No. 5, Qingdao 266003, China

3 Ecotopia Science Institute, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 Japan

Small interference RNA (siRNA) is now commonly used as a useful tool for knockdown of target gene due to its powerful silencing effect. A standard siRNA typically has a symmetric structure consisting of around 20 bp RNA duplex. The antisense (guide) strand of siRNA is incorporated into an RNAi machinery as the specificity determinants of the RNAi pathway. We assumed that development and application of siRNA-based molecular probe would provide information on details of RNAi system. To design such molecular probes, it is necessary to overcome several issues including improvement of selective uptake of antisense strand for making RISC, because misincorporation of sense (passenger) strand into RISC induces unexpected results. Previously, we have developed a methodology for modification of nucleic acids by use of base-surrogates with D-threoninol as scaffold for functional molecules [1-3]. In this method, a functional molecule can be introduced at any position of the sequence in any desired numbers. Using this methodology in conjunction with biochemical assays, we optimized a design of siRNA used as a molecular probe and, then attempted to visualize the siRNAs in the cells to investigate the mechanisms of RNAi.

Firstly, to determine the suitable position for chemical modification on siRNA without any disturbance of RNAi activity, we synthesized siRNAs having an azobenzene moiety through the D-threoninol at various positions on either the sense or the antisense strand of siRNA. For analyses of the strand selectivity and RNAi activity simultaneously, we constructed two reporter plasmids in which target sequence (SENSE) or its complementary sequence (ANTISENSE) was attached at the 3’-UTR region of the firefly luciferase expression vector pGL3 in the forward direction. By analyzing the activities of luciferase expressed by pGL3s in siRNA-transfected cells, both RNAi activity and strand selectivity were evaluated. Our data indicated that azobenzene modification near the 5’-end of sense strand increased the RNAi activity and the strand selectivity [4]. This effect was also observed even when siRNA was modified with other functional molecules on both sense and antisense strand. Based on this information, we designed a siRNA with Thiazole orange as a fluorophore on the antisense strand and Methyl red as a quencher on the sense strand for monitoring the siRNA in cell. During chasing in Hela cells by using confocal fluorescence microscopy, the fluorescence foci of Thiazole orange were gradually increased. These fluorescence foci were colocalized with AGO2 which is essential component of RISC (Figure 1). In conclusion, we propose a novel design of siRNA-based molecular probe with improvement of the strand selectivity in RISC assembly and without suppression of their activities [5]. Using the siRNA introducing the quencher-fluorophore pair, we successfully selective monitoring of mature RISC which embedded the antisense strand in cells. [1] H. Ito, X.G. Liang, H. Nishioka, and H. Asanuma, Org. Biomol. Chem. 8 (2010), 5519.. [2] M.G. Zhou, X.G. Liang, T. Mochizuki, and H. Asanuma, Angew. Chem. Int. Ed. 49 (2010), 2167. [3] T. Fujii, M. Urushihara, H. Kashida, H. Ito, X.G. Liang, M. Yagi-Utsumi, K. Kato, and H. Asanuma, Chem.

Eur. J. 18 (2012), 13304. [4] H. Ito, M. Urushihara, X.G. Liang, and H. Asanuma, ChemBioChem, 13 (2012) 311. [5] Y. Kamiya, A. Ito, H. Ito, M. Urushihara, J. Takai, T. Fujii, X.G. Liang, H. Kashida, and H. Asanuma, Chem.

Sci, 4 (2013) 4016.

anti AGO2S5Mr-A5To Merge

Quenching Light Up !

5’

3’

3’

5’

Anti-sense

Sense5’3’

Figure 1. Selective monitoring of RISC involving anti-sense strand with Thiazole orange upon dissociation of sense strand with Methyl red.

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Post Antibody Medicines: Generation of Molecular-targeting Peptides by Directed Evolution

Ikuo Fujii

Department of Biological Science, Graduate School of Science,

Osaka Prefecture University

The aim of our study is to investigate molecular design relying on evolutionary processes, called as “directed evolution”, to generate a novel class of biofunctional molecules. In our previous work, we have succeeded to develop a directed evolutionary process for improving catalytic efficiency and binding affinity of catalytic antibodies [1, 2]. Therefore, now, we apply our technology and know-how to directed evolution of antibody-like targeting peptides

At present, antibodies are indisputably the most successful reagents in molecular targeting therapy. However, use of antibodies has been limited due to the biophysical properties and the cost to manufacture. To enable new applications where antibodies show some limitations, we have developed an alternative-binding molecule with non-immunoglobulin domain. The molecule is a helix-loop-helix peptide, which is stable against natural enzymes in vivo and is too small to be non-immunogenic [3]. Since the molecule folds by virtue of the interactions between the amino acid residues positioned inside the helix-loop-helix, the solvent-exposed, outside residues would be randomized to give a library of helix-loop-helix peptides [4].

We constructed a phage-displayed library of the structured peptides and screened the library for G-CSF receptor. Finally, the screened peptides was cyclized by introduction of a disulfide-bond linkage into the N- and C-termini. The cyclic peptide showed strong binding affinity (Kd of 4 nM) to the receptor and a long half life (15 days) in mouse sera, proving an enzyme-resistant property. Futhermore, imminization of the peptide to mice elicited non-immunigenecity. The peptide is named “microAntibodes” due to haveing the same properties as those of antibodies. We have applied our peptide library for VEGF, hIgG/Fc, cytokines (IL-4R, IL-5R, IL-6R) and kinases (Aurora A and B) to get their microAntibodies [5]. This semi-rational strategy, which combines phage-displayed libraries with de novo designed peptides, provides a new way to generate structured functional peptides for useful tools in the field of chemical biology as well as altanatives to antibody medicines.

[1] I. Fujii, S. Fukuyama, Y. Iwabuchi & R. Tanimura, Nature, Biotechnology, 16, 463-467 (1998). [2] N. Takahashi, H. Kakinuma, L. Liu, Y. Nishi, and I. Fujii, Nature Biotechnology, 19, 563-567 (2001). [3] N. Suzuki and I. Fujii, Teterahedron Lett., 40, 6013-6017 (1999). [4] I. Fujii, Y. Takaoka, K. Suzuki and T. Tanaka, Tetrahedron Lett., 42, 3323-3325 (2001). [5] D. Fujiwara, Z. Ye, M. Gouda, K. Yokota, T. Tsumuraya, and I. Fujii Bioorg. Med. Chem. Lett., 20, 1776-1778 (2010).

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Cytocompatible phospholipid polymer hydrogels for cell engineering

Tomohiro Konno

Department of Bioengineering, School of Engineering, The University of Tokyo A cytocompatible phospholipid polymer hydrogel was prepared using with phospholipid polymer bearing phenylboronic acid and poly(vinyl alcohol) (PVA). A water-soluble phospholipid polymer, poly (2-methacryloyloxyethyl phosphorylchoine (MPC) -co- n-butyl methacrylate -co- p-vinyl phenylboronic acid (PMBV), was synthesized. The phenylboronic acid moiety in the PMBV can reversibly bind to cis-diol groups in the PVA chain under a mild condition (room temperature, normal pressure, and neutral condition)(1). The PMBV/PVA hydrogel can reversibly encapsulate cells without any reduction of their bioactivity during the encapsulate period. The cell functions in the PMBV/PVA hydrogels were investigated. Functional cells including stem cells were suspended in the PMBV solution. The PMBV/PVA hydrogels were prepared by mixing with PMBV and PVA solutions. The encapsulated cells in the PMBV/PVA hydrogels were stored in an incubator. To dissociate the PMBV/PVA hydrogels, D-sorbitol solution was added in the hydrogels. After dissociation of the hydrogels, the encapsulated cells were recovered by centrifuge. The recovered cells were plated under the conventional culture condition, and the cell functions were evaluated. The PMBV/PVA hydrogels were formed within 20 seconds after mixing the polymer solutions (Fig. 1). The PMBV/PVA hydrogel can be stable in the cell culture medium during preservation period of cell immobilization. The expression level of house keeping genes of immobilized cells was as same as normal cell culture condition (Fig. 2). This result indicated that the PMBV/PVA hydrogel could provide suitable circumstance for immobilized cells. The encapsulated cells were observed by phase contrast microscopy, and the cells were spherical shape, and the cells were not excessively proliferated in the hydrogels. When the cells were encapsulated in the PMBV/PVA hydrogel (storage modulus = 1100 Pa), the cell cycle of encapsulated cells was converged to G1 phase after 72 hour (Fig. 3). This result indicated that the solute permeability was affected to not only cell proliferation but also cell cycle character of encapsulated cells. It was concluded that the cytocompatible phospholipid polymer hydrogel is a promising platform material to provide a high reliable cell source for cell engineering. References 1. T. Konno and K. Ishihara,Biomaterials, 28 (2007)

1770. 2. T. Aikawa, T. Konno, K. Ishihara, Soft Matter, 9

(2013) 4628. 3. H. Oda, T. Konno, K. Ishihara, Biomaterials, 34

(2013) 5891.

Fig. 1. Gelation and dissociation mechanism of PMBV/PVA hydrogels

Fig. 2. Amount of gene expression of immobilized cells in the PMBV/PVA hydrogels for 1 day.

Fig. 3. Incubation time dependency of cell cycle of immobilized cells at G1 phase in various storage modulus of the PMBV/PVA hydrogels.

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Efficient Photosynthetic Electron Transfer and Water Oxidation in Photosystem II

M. Sugiura

Proteo-Science Research Center, Ehime University / JST-PRESTO

miwa.sugiura@ehime-u.ac.jp In the photosynthetic primary process of plants, algae and cyanobacteria, Photosystem II (PSII)

cooperates in series to convert photon energy into chemical energy through light-induced charge separation and

subsequent electron transfers. The light-driven water oxidation by PSII enzyme is responsible for the

production of O2 on Earth, and is at the origin of the synthesis of most of the biomass. PSII is a supracomplex

that consists with 17 membrane-spanning proteins and 3 extrinsic proteins involving over 80 cofactors such as

chlorophylls, ß-carotenes, non-heme irons, plastoquinones, lipids, and so on [1]. The reaction centre proteins,

D1 and D2, bind all the redox-active cofactors involved in the

energy conversion process and the following sequence of

reactions occurs. As shown in Fig. 1, firstly, a special form of

Chla, P680, acts as an exciton trap and converted to a strong

reducing agent after excitation (P680*). Within a few

picoseconds, P680* reduces a pheophytin (Pheo) to form the

radical par state P680+•Pheo-•, then Pheo-• reduces a primary

plastoquineone QA to produce P680+•Pheo-•. The P680

+•, which

has a very high redox potential (> 1.2 V), oxidises a TyrZ to

form TyrZ+•P680PheoQA

- on a nanosecond. On a millisecond scale, QA- reduces a second plastoqunone QB to

form TyrZ+•P680PheoQAQB

-. The oxidised TyrZ+• extracts an electron and proton from a Mn4CaO5 cluster. A

second photochemical turnover reduces QB- to QB

2-. Two further photochemical turnovers provide the

Mn4CaO5 cluster with a total of four oxidising equivalents.

The main cofactors involved in the function of PSII are borne by the D1 and D2 proteins. In

cyanobacteria, the D1 protein may be encoded by several psbA genes which are known to be possibly expressed

differentially depending on the environmental conditions. In the cyanobacterium Thermosynechococcus

elongatus there are 3 different psbA genes coding for D1. Among the 344 residues constituting D1 there are 21

differences between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2 and 27 between PsbA2 and PsbA3. By

those substitutions functions of PSII are modified especially in water oxidation rate and redox potentials of

cofactors[1-6]. In this symposium, we will present the relationship between the difference of molecular

structure of D1 and functions, especially focused on charge separation, electron transfer rates and water

oxidation rate.

References: [1] Y. Umena, K. Kawakami, J.-R. Shen, and N. Kamiya, Nature, 473 (2011) 55-60. [2] M. Sugiura, Y. Kato, R. Takahashi, H. Suzuki, T. Watanabe, T. Noguchi, F. Rappaport, and A. Boussac, Biochim. Biophys.

Acta., 1797 (2010) 1491-1499. [3] S. Ogami, A. Boussac, and M. Sugiura, Biochim. Biophys. Acta., 1817 (2012) 1322-1330. [4] M. Sugiura, S. Ogami, M. Kusumi, S. Un, F. Rappaport, A. Boussac, J. Biol. Chem., 287 (2012) 13336-13347. [5] Y.Kato, M. Sugiura, A. Oda, T. Watanabe, Proc. Natl. Acad. Sci. U.S.A., 106 (2009) 17365-17370. [6] Y. Kato, T. Shibamoto, S. Yamamoto, T. Watanabe, N. Ishida, M. Sugiura, F. Rappaport, A. Boussac, Biochim. Biophys.

Acta, 1817 (2012) 1998-2004.

Fig. 1. Electron transport and water oxidation in PSII.

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Toward a better understanding of the molecular recognition mechanism: A molecular dynamics study

Takefumi Yamashita1 1 Laboratory for Systems Biology and Medicine (LSBM), Research Center for Advanced Science and Technology (RCAST),

the University of Tokyo Due to recent advances of computer power, algorithms, software, and force fields, many molecular dynamics (MD) studies have demonstrated that it is now possible to solve various molecular biology problems. For example, Shaw and his co-workers utilized a special-purpose supercomputer for MD simulations called Anton and simulated the protein folding [1]. It is noteworthy that the force field is fully physics-based and not only proteins but also water are explicitly represented at the atomic level. Thus, the simulations provide atomic-level insight into the folding process. In Japan, K computer, which reaches the rate of 10 PF, is now available for scientific studies including large-scale MD simulations. Considering these facts, we believe that MD simulations with high-performance computers are helpful tools for drug design today. From the physicochemical viewpoint, one of the most important concepts of the drug design is “molecular recognition”. A drug recognizes its target protein specifically, which means that it binds strongly its target protein but not others. If the drug interacted with many proteins, it would trigger serious adverse effects. Because the binding free energy is a property that can describe the binding affinity of the target protein for the ligand, it is essential to evaluate the binding free energy in the drug discovery process. Recently, Fujitani et al. developed an accurate binding free energy calculation protocol named MP-CAFEE [2]. In the case of FKBP, the error of the free energy prediction was smaller than 1 kcal/mol. Since K computer was officially released in September 2012, we have been conducting several applications of MP-CAFEE to target proteins in order to discover candidates of the cancer drug [3]. In the drug design, we sometimes observe that small modification can decrease the binding affinity considerably. In some cases, even a single methylation affects the binding affinity. Although there are some empirical interpretations of such a methylation effect, it is still unclear why it can lead to the significant affinity reduction. In my presentation, I’ll be discussing the reason based on the all-atom MD simulation and providing atomistic insight into the molecular recognition [4]. Also, I plan to talk about the effect of a single mutation on the antigen-antibody interaction. Note that antibodies can recognize their targets specifically. Although antibodies are expected utilized in cancer therapy, the mechanism of the molecular recognition of the antibody is not yet clear at least at the molecular level [5].

(a) (b)

Figure (a) A drug trapped by its target protein: (b) An antibody (Fv) binding its antigen

References [1] D. E. Shaw et al., Science, 330 (2010) 341 [2] H. Fujitani et al., J. Chem. Phys., 123 (2005) 084108. [3] H. Fujitani et al., J. Phys.: Conf. Ser. 454 (2013) 012018 [4] T. Yamashita (in preparition) [5] T. Nakayama et al. (submitted)

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Computational Structural Analysis of β-1,3-D-Glucans as a Drug-Delivery Carrier

K. Uezu 1, T. Okobira 2 and K. Sakurai 1

1 Life and Environment Engineering, The University of Kitakyushu, Kitakyushu, Fukuoka 808-0135, Japan

2 Chemical Science and Engineering, Ariake National College of Technology, Omuta, Fukuoka 836-8585, Japan Introduction The β-1,3-D-glucan family is one of the abundant polysaccharides on the earth. Curdlan known as the simplest β-1,3-D-glucan takes a right-handed 61 triple helix with a 17.6 Å pitch. The triple helix can be dissociated to a single chain by dissolving in dimethylsulfoxide (DMSO). When water is added to the DMSO solution, the helical structure is partially retrieved (renature process). We found that when the single chain of curdlan is renatured together with specific polynucleotides such as poly (cytidylic acid) : poly(C), they form a stoichiometric complex, instead of retrieving the curdlan triple helix [1]. One of the novel features is that the complexation occurs in highly stoichiometrical manner and the stoichiometric number indicates that the complex consists of the two glucose chains and one poly(C) chain to form a new triple helix. This unique property has made it possible to utilize these polysaccharides as a potential drug-delivery carrier for various functional polynucleotides. Miyoshi extensively studied how hydrogen bonds stabilize the β-1,3-D-glucan triple helix, using a semi-empirical quantum-mechanics calculation (MOPAC) and ab initio calculations [2], and Okobira simulated the conformation of three kinds of β-1,3-D-glucan in aqueous solution at room temperature by molecular dynamics method [3]. Furthermore, our spectroscopic study [1] showed that the hydrogen bonding interaction is essentially important for the curdlan/poly(C) complexation. The present paper constructs a structural model for the curdlan/poly(C) complex and, using a computational approach, explores interactions in the complex in the molecular level. Methods Before performing the MOPAC calculation, we carried out pre-optimization for the above obtained model with Molecular Mechanics (MM) method using MM3 in CAChe 5.0 version (Fujitsu Ltd. Japan). For the resultant complex, the MOPAC calculation was done with the MOPAC2002 in CAChe 5.0 version. In order to avoid unfavorable repulsions between the phosphate anions and to simulate electric shielding effects by salts in the experiment, we added the same number of oxonium cations as that of the phosphate anions. All the atoms were allowed to move during the calculation. To simulate the molecules in water, we used the conductor-like screening model (COSMO), setting a dielectric constant 78.4 at 298 K. Results and discussion Fig. 1 presents the geometry-optimized structure of the curdlan/poly(C) complex (total 15 residues, 2.5 pitches). The MOPAC caluculation evaluated the pitch to be 18.0 Å, which is 0.4 Å longer than the curdlan triple helix and 0.6 Å shorter than the poly(C) single helix. It seems that the calculation compensates both initial pitches to provide a middle value, that is, the curdlan helix extends and the poly(C) helix compresses to fit with each other. Both chains (curdlan and poly(C)) maintain the 61 helix structure. The original curdlan helix has the intermolecular hydrogen bond array [2]. The remaining two glucoses formed hydrogen bond between O2-O2’ and the O2-O2’ distance between them is changed from 3.33 to 2.85 Å, that is, the curdlan helix comes close each other. This situation can make more room to accommodate the poly(C) chain and the shorter distance cause the more favorable situation to form the hydrogen bond. The poly(C) deforms in large extent upon the complexation. The diameter of the helix is 11.0 Å in the original chain, while it is 15.3 Å in the complex. Furthermore, as described above, the pitch is decreased by 0.8 Å. We already demonstrated that the circular dichroism (CD) spectrum was not changed by the complexation [1]. This means that spatial arrangement of the cytosine is not drastically altered upon the complexation, and poly(C) keeps the 61 helix. In calculation result, the 61 helix is not also changed and this fact is consistent with the experimental result. [1] K. Sakurai, K. Uezu, M. Numata, T. Hasegawa, C. Li, K. Kaneko and S.

Shinkai, Chem. Comm., 35 (2005) 4383. [2] K. Miyoshi, K. Uezu, K. Sakurai and S. Shinkai: Chem. & Biodivers., 1 (2004) 916. [3] T. Okobira, K. Miyoshi, K. Uezu, K. Sakurai and S. Shinkai: Biomacromolecules, 9 (2008) 783.

Fig. 1 Structure of the curdlan/poly(C) complex

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Ferrocenylnaphthalene diimide (FND)-based electrochemical telomerase assay (ECTA) for diagnosis of premalignant oral lesions

Shinobu Sato,1 Kumiko Mori,2 Mana Hayakawa,2 Masaaki Kodama,2 Manabu Habu,2 Osamu

Takahashi,3 Tatsuji Nishihara,3 Kazuhiro Tominaga,2 and Shigeori Takenaka1 1Department of Applied Chemistry and Research Center for Bio-microsensing Technology, Kyushu Institute of Technology:

Kitakyushu, Fukuoka 804-8550 2Department of Oral and Maxillofacial Surgery, Division of Maxillofacial Diagnostic and Surgical Science,

3Department of Health Promotion, Division of Infections and Molecular biology, Kyushu Dental University: Kitakyushu, Fukuoka 803-8580

Cancer is completely cured by early detection and cancer screening methods have been reported using its several makers. However, present cancer markers are not enough in terms of sensitivity and specificity. On the other hand, telomerase as a ribonucleoprotein is expected to excellent cancer marker because of the specific expression only in cancer cell [1].Telomerase repeat amplification protocol (TRAP) assay known as telomerase detection method is required tedious and time-consuming steps [2] and more simple and rapid screening method of telomerase has been required. We have been developing an electrochemical telomerase assay (ECTA) using ferrocenylnaphthalene diimide (FND) as tetraplex binder [3]. This method based on the elongation reaction of telomere DNA repeat for telomerase substrate (TS) primer-immobilized electrode by telomerase and subsequently formation of tetraplex DAN structure formation in the presence of potassium ion resulting in the concentration of FND on the electrode and current increase based on the concentrated FND.

Thus far, we evaluated telomerase activities of four types of culture cells connected with oral cancer of HSC-2 cells, human oral squamous cell carcinoma cell lines, and HSC-3 cells, human tongue squamous cell carcinoma cell lines, Ca9-22 cells, human gingival squamous cell carcinoma cell lines, and SAS cells, and human tongue squamous cell carcinoma cells using TRAP and ECTA methods. Distinct telomerase activity was observed over 100 cells by TRAP method and the activity was observed over 10 cells by ECTA one where current increasing ratio based on lysates form the four cultured cells shows the good correlation with expression of mRNA from hTERT gene as telomerase catalytic factor. This shows the current increasing ration of ECTA is correlated with the expression of hTERT gene.

As a next step, exfoliate buccal cells, which have a high risk of developing cancer, and tissue from oral cancer patients and healthy volunteers were collected and telomerase activities were evaluated for their lysates with ECTA and TRAP methods. Correct diagnostic rates were 50% and 10% for tissue and exfoliate buccal cells, respectively, by TRAP method. On the other hand, they were 85% and 90% by ECTA method. Since current increasing ratios were 41% and 13% as an average for cancer and healthy tissues, respectively, blind test for 56 samples of exfoliate buccal cells was carried out using threshold of 30% current increase ratio in ECTA method and diagnosis was archived with 84% correct decision. Telomerase activities of leukoplakia and oral lichen planus as benign adenoma were tested by ECTA method. Current increasing ratios were 31%, 26%, and 15% for cancer, benign adenoma, and healthy exfoliate buccal cells, respectively and the current increasing ratio of benign adenoma was located between cancer and healthy ones. These results show that early diagnosis of oral cancer is realized by the screening of exfoliate buccal cells using ECTA method regularly.

[1] L. Harrington, Cancer Letter, 194 (2003) 139. [2] N. W. Kim et al., Science, 266 (1994) 2011. [3] K. Mori et al., Clinical Chemistry, 59 (2013) 289-295.

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Action of Immunostimulatory DNA Drug Can Be Changed by Nanoparticle

N. Hanagata1,2

1 Nanotechnology Innovation Station, National Institute for Materials Science 2 Graduate School of Life Science, Hokkaido University

Oligodeoxynucleotides (ODNs) that contain unmethylated cytosine-phosphate-guanine (CpG) motifs have the potential to stimulate the immune system via interaction with the pattern-recognition receptor, Toll-like receptor 9 (TLR9). In humans, TLR9 is expressed in B cells and plasmacytoid dendritic cells (pDC). TLR9 activation in B cells induces cytokines—including interleukin-6 (IL-6), IL-10, and IL-12—through the signal-transduction pathways involved in nuclear factor-κB. Meanwhile, TLR9 activation in pDC induces type-I interferons, tissue necrosis factor-α (TNFα), IL-6, IL-12, and IFNγ-inducible 10-kDa protein. The potential of CpG ODNs to stimulate the immune system via activation of TLR9 can be applied to infectious diseases treatment, cancer therapy, and allergy treatment CpG ODNs are divided into 4 classes according to their sequence properties. Class-A and class-B CpG ODNs have been well characterized. Class-A CpG ODNs have a palindromic structure, consisting of a phosphodiester backbone in the center of the sequence, and poly-G motifs with a phosphorothioate backbone at each end. Class-B CpG ODNs have a linear structure, consisting entirely of a phosphorothioate backbone. IFN-α is induced by class-A, but not by class-B CpG ODNs. By contrast, class-B CpG ODNs are able to proliferate and activate B cells, leading to IL-6 production. Meanwhile, class-A CpG ODNs have a lower potential to induce IL-6.

Under certain physiological conditions, class-A CpG ODNs form nanometer-sized multimers by self-assembly, because of the palindromic and poly-G sequences. By contrast, class-B CpG ODNs lack the potential to form such higher-order structured multimer. IFN-α induction by class-A CpG ODNs is thought to result from the formation of multimers, the average size of which has been demonstrated to be ~100 nm in length. Interestingly, IFN-α production was observed when class-B CpG ODNs were loaded onto 180-nm polystyrene nanoparticles, suggesting that nanoparticles are able to alter the cytokine induction mediated by interaction between class-B CpG ODNs and TLR9. However, the mechanism by which class-B CpG ODNs acquires the potential to induce IFN-α remains unclear. In the present study, we aimed to clarify this mechanism. For this purposes, we prepared silicon nanoparticles (Si-NPs) with an average diameter of 3.4 nm, because silicon has a low inherent toxicity. The Si-NP possessed a substantial photoluminescence quantum yield in the visible region. Generally, polycations such as polyethyleneimine and poly-L-lysine are used for modifying nanoparticles to electrostatically bind negatively charged DNA. However, polycations have been reported to result in nonspecific binding of negatively charged molecules, and also to promote the formation of nanoparticles aggregates, which are thought to cause side effects. It is likely that the toxicity of polycations may affect cytokine induction in the immune system, and also the interaction between CpG ODN and TLR9. Thus, we modified the surface of Si-NP with allylamine, to ensure a positive charge, and electrostatically bound to CpG ODN molecules. Furthermore, alternative surface modification of Si-NP was performed by introducing maleimide into allylamine amino group to covalently crosslinked CpG ODN molecules onto Si-NP.

Here, we present the binding mode dependent bifurcation of cytokine induction (Figure 1) and discuss the possible mechanism of bifurcated cytokine induction by interaction between class-B CpG ODN molecules and TLR9. Our discoveries also provide that nanoparticles play roles in not only delivery of CpG ODN but also control of CpG ODN action.

Figure 1. Bifurcation of TLR9 signaling by different binding mode of CpG ODN onto silica nanoparticle

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Investigation of functional Zinc Oxide nanoparticles for biomedical applications

Yasuhisa Fujita1, Hideki Hashimoto2, Miki Tongu3, Takaya Yamada3, Kohji Nishimura4 Keisuke Yoshikiyo5, Hideo Akiyoshi5, Tatsuyuki. Yamamoto5, Akihiro Matsumoto6, Masatoshi Fujii6, Junko Fujihara6, Tomoko Toga6,

Haruo Takeshita6 and Takeshi Isobe6

1Interdisciplinary Graduate School of Science and Engineering, Shimane University, Matsue 690-8504, Japan

2 Center for the Promotion of Project Research, Shimane University, Matsue 690-8504, Japan 3 Dept. of Experimental Animals, Interdisciplinary Center for Science Research, Shimane University, Izumo, 693-8501,

Japan 4 Dept. of Molecular and Functional Genomics, Interdisciplinary Center for Science Research, Shimane University, Matsue

690-8504, Japan 5 Faculty of Life and Environmental Science, Shimane University, Matsue 690-8504, Japan

6School of Medicine, Shimane University, Izumo, 693-8501, Japan

The increasing cost of health care due to advanced medical technologies is a serious problem facing localities with an aging population. The development of nano-materials that are economical, simple, and practical in use is important in this issue. We have been developing unique simple medical nanotechnologies that are safe, offer peace of mind under the concept of co-creation in nanotechnology collaboration project.

Zinc oxide (ZnO) is a low-cost and abundant material and has unique features for the use of biomedical applications. ZnO is a semiconductor with bandgap energy of 3.37 eV at room temperature and a large exciton binding energy of 60 meV, which result in bright room-temperature near UV emission. In this work, nanophotonics of ZnO particles and their application to biomedical technologies are studied.

We have synthesized ZnO nanoparticles by using a gas evaporation method [1]. The particles show strong excitonic emission at the wavelength of 380 nm. Visible emission is also obtained by controlling the defect such as oxygen vacancies, interstitial zinc, excess oxygen etc. Because ZnO has anisotropic crystal structure, it has nonlinear optical properties that enable the second harmonic generation using near IR laser irradiation [2]. These nanoparticles also have a photocatalytic feature that is applicable for photo-therapeutic agents. These properties are controlled by defect formation and surface treatments.

The decrease of fluorescence intensity of ZnO nanoparticles is observed in the solution by photo quenching. This quenching is prevented by core-shell structure by silica coating [3]. Silica coated ZnO has a good fluorescence property and applicable for medical bio-imaging because the core-shell structure inhibit the photo-quenching and also photocatalytic properties of semiconductor materials. In addition, we are developing novel drug delivery devices with fluorescence property such as Cyclodextrin appended ZnO nanoparticles.

By using these technologies, photo-diagnostic and photo- therapeutic agents will be realized. We also study the safety, or toxicity of nanomaterials on organisms [4].

References [1] K. Senthilkumar, O. Senthilkumar, K. Yamauchi, M. Sato, S. Morito, T. Ohba, M. Nakamura, and Y. Fujita, Phys. Status Solidi B., 246, No.4, (2009) 885-888. [2] B. E. Urban, J. Lin, O Senthilumar, K. Senthilkumar, Y. Fujita, and A. Neogi, Optical Materials Express, 1, No.4, (2011) 658-669. [3] M. Nakamura, S. Ozaki, M. Abe, T. Matsumoto and K. Ishimura, J. Mater. Chem., 21 (2011) 4689-4695. [4] R. Ohno et al., Arch Toxicol submitted.

Fig.1 Scanning Electron Microscope image of Silica coated ZnO nanoparticles.

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Easy-to-Use Detection of Biomarkers and Fabrication of Nanoparticles for Gene Therapy Using Microfluidic Devices

M. Tokeshi1-3

1 Division of Biotechnology and Macromolecular Chemistry, Faculty of Engineering, Hokkaido University, Japan

2 FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Japan 3 The Priority Research Project, “The Knowledge Hub” of Aichi, Japan

Microfluidic devices have great potential for medical and life science applications. Recent progresses in micro- and nano-fabrication technologies have allowed us to fabricate smaller and more precise artificial nanostructures for manipulating biomolecules such as DNA, RNA, and proteins, and to form highly functional micro- and nano-particles such as liposome, quantum dot, gel particle.

In this presentation, I will present about several topics: easy-to-use detection of biomarkers [1,2], fabrication of functional nanoparticles using the microfluidic devices [3-5] and so forth. Moreover, future challenges and potentials of micro- and nano-fluidic devices for biotechnology, medicine, and clinical diagnostics will be also discussed.

The immunodetection of multiple biomarkers in a single sample is very important for many applications including clinical diagnosis, drug discovery, and so on. However, the current methods, such as planer arrays and beads-based suspension arrays, require a long reaction time or relatively large sample volume, or troublesome procedures. From viewpoint of practical use, it requires a rapid, small sample volume, easy-to-use and inexpensive. We developed a new rapid and easy-to-use multiplex immunoassay chip, “Immuno-Pillar Chip”, in which hydrogel pillars including many antibodies immobilized onto 1 μm-diameter polystyrene beads are fabricated inside a microchannel [1,2]. To evaluate the chip performance, we demonstrated a multiplex assay measuring three biomarkers: α-fetoprotein (AFP), C-reactive protein (CRP), and prostate-specific antigen (PSA). The assay is completed within 10 min, and the limit of detections (LODs) were about 100 pg/ml for three biomarkers.

The fabrication of functional micro- and nano-particles, such as liposome, quantum dot, gel particle, etc., using microfluidic devices has several advantages over conventional batch methods. For example, the size controlled fabrication of particles can be easily realized by controlling the experimental conditions. We have developed several types of fabrication method of the multifunctional envelope-type of nanodevices (MEND) using microfluidic devices [3-5].. Recently, we report the reliable fabrication of MEND using a microfluidic device that had an array of a staggered chaotic mixer with bas-relief structures on the floor of its reaction channel. The chaotic mixer array allowed homogeneous precursor mixing and that led to reliable results in fabricating MEND. Using this device, we realized that the fabrication of very small-sized MENDs (less than 100 nm). Such small-sized MENDs enable efficient delivery to a cancer tissue. Acknowledgements This research was partially supported by a Grant-in-Aid from the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST), JSPS KAKENHI Grant Number 24655054, and the Asahi Glass Foundation. References [1] M. Ikami, A. Kawakami, M. Kakuta, Y. Okamoto, N. Kaji, M. Tokeshi and Y. Baba, Lab Chip, 10 (2010)

3335. [2] W. Jin, K. Yamada, M. Ikami, N. Kaji, M. Tokeshi, Y. Atsumi, M. Mizutani, A. Murai, A. Okamoto, T.

Namikawa, Y. Baba and M. Ohta, J. Microbiol. Methods, 92 (2013) 323. [3] H. Kuramoto, Y. -S. Park, N. Kaji, M. Tokeshi, K. Kogure, Y. Shinohara, H. Harashima and Y. Baba, Anal.

Bioanal. Chem., 391 (2008) 2729. [4] K. Kitazoe, J. Wang, N. Kaji, Y. Okamoto, M. Tokeshi, K. Kogure, H. Harashima and Y. Baba, Lab Chip, 11

(2011) 3256 [5] K. Kitazoe, Y. -S. Park, N. Kaji, Y. Okamoto, M. Tokeshi, K. Kogure, H. Harashima and Y. Baba, PLoS

ONE, 7 (2012) e39057.

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Analysis of RNA dynamics in living cells based on single molecule imaging

Hideaki Yoshimura1, Toshimichi Yamada1, Hiroki Segawa1, Asumi Inaguma2 and Takeaki Ozawa1

1 Department of Chemistry, School of Science, The University of Tokyo 2 Institute of Molecular Science,

Live cell imaging, especially single-molecule imaging, is a powerful approach to analyze intracellular

molecular dynamics. Recent studies revealed that messenger RNA (mRNA) shows subcellular localizations, which related with various cellular functions. In addition, variety of non-coding RNAs (ncRNAs) are recently reported to be implicated in various biological events. Thus RNAs are now considered an important targets for bioimaging studies. Live-cell RNA imaging techniques, however, have not sufficiently developed, because of the lack of RNA probes for live-cell fluorescence imaging. An innovation of a probe that selectively labels and visualizes target RNAs in living cells will promote the investigation about relation between RNA dynamics and its physiological functions.

Here we developed RNA probes for live-cell imaging using an RNA binding domain, pumilio homology domain (PUM-HD) of human pumilio1. PUM-HD specifically binds to 8-base RNA sequence, and can be designed to alter the specificity to bind another specific sequence of 8-base RNA. We prepared PUM-HD mutants that bind specifically to the target RNA and fused to split fragments of enhanced green fluorescent protein (EGFP). Upon binding of the PUM-HD mutants to target RNAs, EGFP fragment reconstitution occurs and recover fluorescence. The developed probe was expressed in living cultured cells and subjected to total internal reflection fluorescence (TIRF) microscopy to capture images of the target RNA dynamics in living cells.

We first observed β-actin mRNA using the present probe. As the results of the TIRF microscopy observation, many fluorescent spots, which represent single probe molecules binding to β-actin mRNAs, were observed in the cytosol. Some of the spots were localized on microtubules and moved directionally. These results indicate that the present probe has an ability to visualize the localization and single-molecule motion of endogenous β-actin mRNA in living cells. In addition to β-actin mRNA, we also observed telomere repeat-containing RNA, TERRA. TERRA is an ncRNA that localize in the nucleus and implicated to formation of telomere complexes at the terminus of chromosome DNAs. We performed simultaneous observation of telomere DNA maker proteins and TERRA using the PUM-HD-based probe. As the result, TERRA showed a free diffusion motion in the nucleoplasm and a confined motion around the telomere DNA. This result revealed that TERRA motion is restricted by telomere complexes, suggesting implication between function of telomere complexes and TERRA dynamics.

The present study showed that the RNA probes has an ability to visualize single molecule dynamics of RNAs in living cells. The present principle of RNA visualization will produce a useful approach to analysis dynamics of various RNA spices in living cells. [1] H. Yoshimura, A Inaguma, T. Yamada and T. Ozawa, ACS Cnem. Biol., 7 (2012) 999. [2] T. Yamada, H Yoshimura, A Inaguma and T. ozawa, Anal. Chem., 83 (2011) 5708.

Fig. 1 ． Outline of β-actin mRNA imaging using a Pumilio-based probe. The probe consists of two mutated PUM-HDs and reconstituted EGFP. Subcellular localization and single molecule motion of the mRNA was visualized by the present probe using a TIRF microscope.

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Microfabricated Silicon Chips for Recording hERG Channel Activities

A. Hirano-Iwata1, Y. Ishinari

1, Y. Kimura

2 and M. Niwano

1,2

1 Graduate School of Biomedical Engineering, Tohoku University, Sendai 980-8579, Japan

2 Laboratory for Nanoelectronics and Spintronics, Research Institute of Electrical Communication,

Tohoku University, Sendai, 980-8577, Japan.

Ion-channel proteins are of crucial physiological importance and are major targets for drug design.

Recording ion-channel activities by measuring ion currents after reconstitution of ion-channels in artificial

bilayer lipid membranes (BLMs) represents a simple method to investigate channel function and to screen the

effect of drug candidates. However, the mechanical instability of BLMs prevents them from being widely used.

Recently, we succeeded in the formation of mechanically stable BLMs by preparing membranes in

microfabricated silicon chips [1]. The key feature that stabilized the BLMs is probably the tapered shape of the

pore edge, which allows reducing the stress on the bilayer at the pore edge. In this presentation, we report on

an application of this stable BLM device to measure ion currents of the human ether-a-go-go-related gene

(hERG) channel, which has been found to be related to serious arrhythmic side effects following drug treatment

[2].

The basis of the device was a silicon wafer

covered with a silicon nitride (Si3N4) layer.

Micropores were fabricated in the Si3N4 layer using

wet chemical etching. The surface of the device

was further coated with thermal oxide and

Teflon-AF [3]. After the device was treated with a

silane-coupling reagent, artificial BLMs were

prepared in the micopores by the monolayer folding

method. Owing to the use of SiO2/Teflon

dielectric layer, the current noise level of the BLM

was as small as 1-2 pA in peak-to-peak after

low-pass filtered at 1 kHz. This noise level

enabled observation of single-channel activities of

hERG channels. Average hERG single-channel

conductance was 11 ± 1.2 pS, which is similar to a

reported value (12 pS) by the patch-clamp method.

Then we examined effects of typical blockers on

hERG channel activities. Fig. 1 shows the effects

of E-4031 and astemizole on hERG currents at the single-channel level. E-4031 is a specific channel blocker

of hERG, and astemizole is an antihistamine that has been withdrawn from the market in most countries due to

its side effect on the hERG channel. Before the addition of the drugs (control), stepwise currents were

observed with a single-channel conductance of ~13 pS. The addition of E-4031 completely blocked the current,

confirming that the control currents stemmed from the permeation of ions through the hERG channel. After a

thorough washout of E-4031, the channel activities were recovered. Finally, the addition of astemizole again

completely blocked the channel current, confirming its inhibitory effect on hERG channels reconstituted in a

BLM. Thus, the sensitivity of the hERG channel to most typical drugs has been successfully reproduced in the

present BLM reconstitution system. The maximum membrane lifetime was 65 h with incorporated hERG

channels. The BLM containing hERG channel was tolerant to repetitive (17 times) solution exchanges.

Compatibility of the BLMs to solution exchanges can be useful for increasing the experimental throughput from

single BLMs. The next step is to extend this stable BLM device to a multisite array format. Since we

previously reported on the simultaneous and automated formation of stable BLMs [4], in future studies, we plan

to improve the throughput by combining the channel-containing BLMs with the array format. The realization

of an ion channel array will open a variety of applications, including high-throughput drug screening procedures

that can be a complement to the patch-clamp method.

[1] A. Hirano-Iwata, K. Aoto, A. Oshima, T. Taira, R. Yamaguchi, Y. Kimura and M. Niwano, Langmuir, 26

(2010) 1949.

[2] A. Oshima, A. Hirano-Iwata, H. Mozumi, Y. Ishinari, Y. Kimura and M. Niwano, Anal. Chem., 85 (2013)

4363.

[3] A. Oshima, A. Hirano-Iwata, T. Nasu,

Y. Kimura and M. Niwano, Micro and Nanosystems, 4 (2012) 2.

[4] A. Hirano-Iwata, T. Nasu, A. Oshima, Y. Kimura and M. Niwano, Appl. Phys. Lett., 101 (2012) 023702.

Fig. 1 Blocking of a single hERG channel by E-4031 and

astemizole.

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Development of Immunomodulatory Cancer Therapy Based on Gc protein-derived Macrophage Activating Factor (GcMAF)

Y. Uto1, R. Takeuchi2, Y. Nakagawa2, K. Hirota3, H. Terada3, S. Onizuka4, K. Kubo5, D. Kuchiike1, 5,

M. Mette5, T. Inui1, 5, 6, Y. Endo7 and H. Hori1

1 Department of Life System, Institute of Technology and Science, The University of Tokushima, Tokushima, Japan 2 Morita Pharmaceutical Ind. Ltd., Chuo-ku, Tokyo, Japan

3 Faculty of Pharmaceutical Sciences & Center for Drug Delivery Research, Tokyo University of Science, Chiba, Japan 4 Nagasaki Municipal Hospital, Nagasaki, Japan

5 Saisei Mirai Cell Processing Center, Osaka, Japan 6 Kobe Saisei Mirai Clinic, Kobe & Inui Immunotherapy Clinic, Osaka, Japan

7 Central Research Resource Branch, Cancer Research Institute, Kanazawa University, Kanazawa, Japan The group-specific component (Gc) protein known as a vitamin D-binding protein (DBP), Gc globulin, is a 53-kDa human plasma protein. There are six major subtypes, the homodimers and heterodimers of Gc1f, Gc1s, and Gc2. The 1f1f subtype of the Gc protein (Gc1f1f protein) contains a branched trisaccharide with a galactose and a sialic acid moiety bound to N-acetylgalactosamine attached to the core protein. In an inflammatory response, the terminal Gal and SA of the Gc1f1f protein were hydrolyzed by membrane-bound β-galactosidase on an activated B-cell and sialidase on a T-cell, respectively, to produce Gc protein-derived macrophage activating factor (GcMAF)1. We hypothesized that preGc1f1fMAF, the Gc1f1f protein lacking galactose moiety alone, could be converted to GcMAF in vivo because sialic acid was cleaved by residual sialidase. We identified the sugar moiety of our prepared preGcMAF with a Western blot using peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA) lectin. We, furthermore, found that preGc1f1fMAF significantly enhanced phagocytic activity in mouse peritoneal macrophages but only in the presence of the mouse peritoneal fluid; its level of phagocytic activity was the same as that observed for GcMAF2. We also found that preGc1s1sMAF and Gc22MAF significantly enhanced the phagocytic activity of mouse peritoneal macrophages in both cases of the presence and absence of mouse peritoneal fluid. Antitumor activity of preGcMAF was observed regardless of its subtype. The injection route of preGcMAF strongly affected its antitumor activity, with subcutaneous and intramuscular administration being more favorable than the intraperitoneal or intravenous route. We have demonstrated that three major subtypes of β-galactosidase-treated human serum Gc protein can be used as the biological response modifier based on the macrophage activation. Now we are planning our clinical study of preGcMAF for some patients with stage IV pancreatic or liver cancers in the Nagasaki Municipal Hospital. Besides, a major problem for clinical use of GcMAF was in the purification procedure. In previous studies, GcMAF was purified from human serum using an affinity column modified with 25-hydroxyvitamin D3. The column should be cleaned for repeated use because of its decontamination. To overcome the problems associated with GcMAF purification, we prepared degalactosylated/desialylated human serum, which we coined serum GcMAF or the next-generation GcMAF3, using autologous serum. We detected GcMAF in the degalactosylated/desialylated human serum by western blotting using an anti-human Gc globulin antibody, and HPA lectin. We also found that serum GcMAF significantly enhanced the phagocytic activity of mouse peritoneal macrophages and extended the survival time of mice bearing Ehrlich ascites tumors4. We, in this symposium, would like to introduce our development of the novel cancer immunotherapy with preGcMAF or serum GcMAF as a glycoprotein-type nanomedicine. [1] N. Yamamoto and R. Kumashiro, J. Immunol, 151 (1993) 2794. [2] Y. Uto, S. Yamamoto, R. Takeuchi, Y. Nakagawa, K. Hirota, H. Terada, S. Onizuka, E. Nakata and H. Hori,

Anticancer Res., 31 (2011) 2489. [3] Y. Uto, H. Hori, K. Kubo, M. Ichihashi, N. Sakamoto, M. Mette and T. Inui, Nature, 485 (2012) Sponsor

Feature. [4] D. Kuchiike, Y. Uto, H. Mukai, N. Ishiyama, C.Abe, D. Tanaka, T. Kawai, K. Kubo, M. Mette, T. Inui, Y.

Endo and H. Hori, Anticancer Res., 33 (2013) 2881.

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Poster Presentations All posters will be displayed in the forum of

Nakamura Centenary Hall

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Micro-tissue assembly by live-fabrication technique using near-infrared-responsive gel

A. Shirakihara1, H. Koga1, T. Sada2, T. Fujigaya2, N. Nakashima2 and K. Nakazawa1

1 Department of Life and Environment Engineering, The University of Kitakyushu

2 Department of Applied Chemistry, Kyushu University Introduction: The formation of micro-tissue, which is formed following the rearrangement and compaction of cell aggregates, is a promising technique for tissue engineering and regenerative medicine, pharmacological and toxicological studies, and fundamental studies in cell biology. Recently, some useful techniques, such as cell-sheet technique and molding cell bead method, have been established for creating micro-tissues [1, 2]. However, in these techniques, the shape of micro-tissue is dependent on the mold structure of scaffold. Therefore, the change of micro-tissue shape and/or the stepwise formation of micro-tissues during the culture period are limited. In this study, we developed a live-fabrication technique of microscaffold using a near-infrared (NIR)- responsive gel. Furthermore, we demonstrated the stepwise formation of micro-tissue by our live-fabrication technique. Materials and Methods: As a NIR-responsive gel, we designed a composite gel containing agarose and carbon nanotubes (CNTs). The concept of our live-fabrication is that NIR irradiation is absorbed by CNTs in the composite gel to cause local heating, which leads to local solation of the agarose gel and consequent formation of a selective micro-scaffold on a culture plate (Fig. 1). NIR irradiation to the gel was performed by microscope system that included an NIR laser device (1064 nm), and the location of NIR irradiation was controlled by the electric stage of the microscope system. Furthermore, the formation of micro-tissue composed with 3T3 cells and Hepa1-6 cell was demonstrated by the live-fabrication.

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Results and Discussion: The agarose gel only (control condition) showed no response to NIR irradiation. In contrast, upon NIR irradiation, the temperature of the CNT/agarose gel quickly reached the solation temperature and the selective micro-scaffold was generated. Furthermore, the size of micro-scaffolds (solation area) was controlled by NIR power, CNT concentration, and thickness of the gel, and the magnification of the objective lens.

To demonstrate the stepwise formation of micro-tissue, primary micro-scaffolds were created by NIR irradiation, and then 3T3 spheroids were formed in the scaffolds. After that, secondary micro-scaffolds were created by NIR irradiation again, and the seeded Hepa1-6 cells formed spheroids in the secondary micro-scaffolds. The both spheroids fused with increase of culture period, and consequently the micro-tissues composed with 3T3 and Hepa1-6 spheroids was formed (Fig. 2). This technique is promising for the assembly of stepwise and/or complex micro-tissues.

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[1] T. Shimizu, M. Yamamoto, A. Kikuchi, and T. Okano, Biomaterials 24(2003) 2309-2316. [2] Yukiko T.Matsunaga, Yuya Morimoro, and Shoji Takeuchi, Adv.Mater 23(2011) 90-94

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Synthesis of FasL mimetic peptide modified dendrimer and investigation of apoptosis inducing effect

Ayako MINOHOSHI1, Tatsuhiro YAMAMOTO2, Takeshi MORI1,

Akihiro KISHIMURA1, Yoshiki KATAYAMA1

1Department of Applied Chemistry, Graduate School of Engineering, Kyushu University 2Institute of Systems, Information Technologies and Nanotechnologies

A method for controlling apoptosis of target cells is highly important from both points of view of cancer

treatment and regenerative medicine. A Fas ligand (FasL), a cytokine inducing apoptosis, is type-II transmembrane protein consisting of 281 amino acids and belongs to tumor necrosis factor family. FasL exists on activated T cells and natural killer cells, and induces apoptosis to target cells that express Fas receptors. This induction of apoptosis is started from trimerization of Fas receptors triggered by trimer complex of FasL. Furthermore, it was reported that four-amino-acid sequence, WEWT, was designed by computational analysis of FasL-Fas receptor interaction, and the WEWT peptide possessed apoptosis inducing property [1]. The purpose of this study is to develop polymeric nanoparticle platform for controlling apoptosis inducing effect by using FasL mimetic peptide containing WEWT sequence.

First, we synthesized WEWT peptide by solid-phase synthesis. The peptide was purified by high-performance liquid chromatography. The molecular weight was confirmed by mass spectrometry (MALD-TOF). The WEWT peptide was modified on dendritic poly(L-lysine) of fourth generation (KG4) by varying reaction conditions for controlling modification rate. The synthesized KG4 dendrimer modified with WEWT peptides was purified by gel permeation chromatography. We determined the modification rate of WEWT peptide to KG4 from absorption measurement of tryptophan by UV-Vis spectroscopy. For the apoptosis evaluation, we chose the KG4 modified with WEWT peptide whose modification rate was 26% (Fig. 1).

In this presentation, I report the results of apoptosis-inducing activity evaluation in Jurkat cells treated with (WEWT)8-KG4 dendrimer by confocal microscopy and flow cytometry (Fig. 2). [1] Yoshimori, R. Takasawa, A. Hayakawa, M. Mizuno, J. Yoshida and S. Tanuma, Apoptosis, 10 (2005) 323.

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Embryoid body culture of mouse iPS cell using microwell chips

D.Miyamoto and K.Nakazawa

Department of Life and Environment Engineering, The University of Kitakyushu 1. Introduction Induced pluripotent stem (iPS) cells and embryonic stem (ES) cells possess self-renewal and pluripotency for differentiation into derivatives of all 3 germ layers (endoderm, mesoderm, and ectoderm). Therefore, they are a promising cell source for pharmacological and toxicological studies, cell-based therapies, and fundamental studies. The formation of cell aggregates called embryoid body (EB) is an initial step in stem cell differentiation, and EB culture has been widely used as a useful method for in vitro differentiation of stem cells. Among the various methods to generate EBs, microwell chips represent a promising technique for controlling EB size and producing a large number of homogenous EBs, and we have succeeded in the EB culture of mouse ES cell using the microwell chip [1, 2]. In this study, we investigated the effects of microwell chip on the EB culture of mouse iPS cells. 2. Material and Method The microwell chip consisted of 195 microwells (1000 μm in diameter, and 1000 μm in depth) in a triangular arrangement (1100 μm in pitch) on a polymethylmethacrylate plate with the surface modified by polyethylene glycol (PEG) to render it nonadhesive (Fig. 1). Hanging drop (HD) culture, the standard method for generating EBs, was used as a control condition. 3. Results and discussion

The iPS cells in each microwell began to aggregate within 1 d of seeding, and then spontaneously formed a single EB in each microwell within 3 d of culture. The EBs remained within the microwells and their size increased by cell proliferation. However, the EB growth rate of microwell chip culture was lower than that of HD culture.

The expression of typical differentiated-cell markers was evaluated at 10 d of culture. The expression level of hepatic markers (TTR and AFP) in the microwell chip culture was higher than that in the HD culture (Fig.2a and b). In contrast, the expression level of vascular formation markers (Flk1 and PDGFRβ) in the HD culture was higher than that in the microwell chip (Fig.2c and d).

These results indicated that the differentiation properties of iPS cells are regulated by the EB culture condition, and the microwell chip culture promotes hepatic differentiation rather than vascular differentiation.

Fig.2. Expression levels of differentiated-cell markers [1] Y. Sakai, Y. Yoshiura, and K. Nakazawa, Journal of Bioscience and Bioengineering, 111 (2011), 85-91. [2] K. Nakazawa, Y. Yoshiura, H.Koga, and Y. Sakai, Journal of Bioscience and Bioengineering, 2013 in press.

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Immunosuppressive DNA Nanomedicine against Autoimmune Disorders D. Bheema1,2 and N. Hanagata1,2

1Graduate School of Life Sciences, Hokkaido University, N10W8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan 2National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047, Japan

Innate immune responses represent the first line of defense against pathogens such as bacteria, virus, fungi and parasites. First step to induce innate immunity is the recognition of pathogens by “pattern recognition receptors (PRR)”. Cytosolic DNA sensor proteins such as DAI, IFI16 and RNA polymerase III, which are PRR, sense pathogen’s DNA followed by a signaling cascade in the cells that leads to the production of immune-mediator cytokines such as interleukin-6 (IL-6) and interferon-β (IFN- β). However, recently it is reported that the DNA sensor proteins also sense own DNA from dying cells, leading to the production of these cytokines, which results in autoimmune disorder [1]. The purpose of this study is to find a way for suppression of cytokines induced by an interaction of self-DNA with DNA sensor proteins.

We observed that fibroblast cells L929 induced IL-6 and IFN-β by the stimulation of B-form DNA (B-DNA) that is a model of own DNA. Our strategy is to suppress these immune-mediator cytokines using synthetic oligodeoxynucleotides (ODN) that has a higher affinity to DNA sensor protein than B-DNA. To find ODN that has a potential to suppress the cytokine induced by B-DNA, we synthesized three ODNs with different base sequences (CpG ODN, GpC ODN and CpG free ODN) and examined the suppression effect of these ODNs. Consequently, we observed slight suppression effect on both IL-6 and IFN-β in naked single-strand (ss) CpG ODN that contains cytosine-guanine (CG) dinucleotide sequence. To enhance the suppression effect, we prepared ODN complex with cationic liposome (lipofectamine), because it is well known that cationic lipids enhance internalization of DNA [2]. Indeed, cationic liposome improved the suppression effect in ssCpG ODN (Figure 1). Furthermore, double-strand (ds) CpG ODN complexed with cationic liposome showed more suppression effect than ssCpG ODN complex (Figure 1). We tested suppression effect of complementary ss ODN sequence of ssCpG ODN, but no significant enhancement of suppression effect was observed. This implies that the greater suppression effect of cytokines induced by B-DNA in dsCpG ODN complex is not caused by the complementary ssODN. Although we observed suppression effect of cytokines induced by B-DNA even in dsGpC ODN and dsCpG-free ODN complexes, the suppression effect was less than that of dsCpG ODN complex. To further improve the suppression effect in dsCpG ODN, we tested another cationic nanoparticle, silica nanoparticle (SiO2 NP) whose surface was modified with polycation. However, no significant suppression effect in dsCpG ODN complexed with cationic SiO2 NP was observed (Figure 1). In addition, alternate binding method of dsCpG ODN on to the surface of SiO2 NP also showed no suppression effect (Figure 1). This suggests that nanoparticle affect the interaction between dsCpG ODN and DNA sensor protein.

In conclusion, CpG ODN with CG dinucleotide had a considerable suppression effect of cytokines induced by B-DNA. Double-strand CpG ODN had more suppression effect compared to ssCpG ODN. In addition, dsODN complexed with cationic liposome exhibits improvement of suppression effect, while dsCpG ODN complexed with SiO2 NPs had no potential to suppress cytokines induced by B-DNA. This suggests that nanoparticles play a critical role in the interaction of dsCpG ODN with sensor protein.

References:

[1] G.N. Barber, Current Opinion in Immunology 23 (2011) 10 [2] P. Hawley-Nelson, and V. Ciccarone, Current Protocols in Cell Biology 20 (2003) 6

Figure 1. Immunosuppressive effect of CpG ODN complexes. Single and double strand CpG ODN consisting of 72 bases was bound onto cationic liposome, polycation-modified SiO2 nanoparticle, and streptavidin-modified SiO2 nanoparticle. These dsCpG ODN complexes were applied to fibroblast L929 cells and then the cells were stimulated by B-DNA. After 24h, IFN-β level was measured by ELISA.

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A rational design strategy to design latent ratiometric fluorescent pH probes based on self-assembled SNARF derivatives

Eiji Nakata, Takashi Morii

Institute of advanced Energy, Kyoto University, Kyoto, Japan

Gokasho, Uji, Kyoto, 611-0011, Japan

E-mail: nakata@iae.kyoto-u.ac.jp

Fluorescence imaging is one of the powerful techniques for continuous observation of dynamic

intracellular processes of living cells. Fluorescent probes bearing a fluorescence switching property

associated with a specific recognition or reaction of target biomolecule are therefore important for

fluorescence imaging. Thus, fluorescent probes continue to be developed to support various

approaches with different design strategies. When compared to simple intensity-changing fluorescent

probes, ratiometric fluorescent probes typically offer the advantage of less sensitivity to errors

associated with probe concentration, photobleaching, instrument sensitivity, and environmental effect.

For intracellular usage, ratiometric fluorescent probes based on small-molecules must be loaded into

the cells. Thus, probes having intrinsic fluorescence may obscure a change of intracellular signal if the

background fluorescence of remaining extracellular probes was high. To overcome the disadvantages

of using ratiometric fluorescent probes that are based on small-molecule fluorophores, it is necessary

to minimize extracellular background fluorescence.

Since tracking the dynamics of intracellular pH (pHi) is crucial to understanding the

mechanisms by which many physiological functions of cell and tissue activities are regulated, various

types of fluorescent pHi probes have been developed. Among these, SNARF (seminaphthorhodafluor)

are most commonly used ratiometric fluorescent pH probes, because of their unique fluorescent

characteristics: visible light excitation, dual-emission properties, and a neutral pKa region. However,

almost SNARF derivatives in which the phenolic substituent is protected to increase the cell

permeability still have strong fluorescence. Interestingly, a certain kind of SNARF derivative shows

unique fluorescent quenching properties, which causes it self-assemble in an aqueous condition.[1]

Based on such the unique characteristics, we developed a sophisticated ratiometric fluorescent pH

probe (latent ratiometric fluorescent pH probe) that minimizes background extracellular fluorescence,

and activates in the intracellular environment to effectively monitor pHi in a ratiometric manner.

References

[1] a) E. Nakata, et al., Bioorg. Med. Chem., 17, 6952 (2009). b) E. Nakata, et al., Chem. Comm., 46,

3526 (2010). c) E. Nakata, et al., Chem. Lett., 39, 734 (2010) d) E. Nakata, et al., Bioorg. Med. Chem.

Lett., 21, 1663 (2011). e) E. Nakata, et al., submitted.

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Anti-inflammatory effect of IL-1 receptor antagonist gene transfection into synovial cells using sonoporation

F. Nagao1, T. Tsuzisawa2, K. Iwanaga3, M. Habu3, I. Yoshioka1, T. Nishihara4 and K. Tominaga3

1 Department of Science of Physical Functions, Division of Oral Medicine,

2 Department of Functional Oral Rehabilitation, School of Oral Health Science, 3 Department of Science of Physical Functions, Division of Oral and Maxillofacial Surgery,

4 Division of Infections and Molecular Biology, Department of Oral Health Promotion, Science of Health Improvement, Kyushu Dental University

Objective: We had confirmed the increased of interleukin-1beta (IL-1 beta) level and the decreased interleukin-1 receptor antagonist (IL-1ra) level in the synovial fluid from the patients with temporomandibular joint arthritis [1]. Acceleration of suppressive mechanism of inflammation would be mandatory in management of arthritic conditions. In the present study, we tried to establish the sonoporation method as a new gene therapy. Materials and Methods: HIG-82 cells, a synovial cell line derived from a rabbit knee, were used in this experiment. pVIVO1-GFP/LacZ and pCI-neo-IL-1ra were transfected into HIG-82 cells by sonoporation[2]. To enhance the transfection efficiency, we used SonoVueTM as a micro bubble. The transfection efficiency of pVIVO-1-GFP/LacZ was examined by X-gal staining. The appearance of IL1-ra mRNA was confirmed by RT-PCR, and the expression of IL-1ra was examined by immunocytochemical analysis. IL-1ra-transfected HIG-82 cells were stimulated with lipopolysaccharide (LPS)[3], and the amounts of IL-1beta, IL-1ra and prostaglandin E2 (PGE2) were examined by ELISA. Result: Human IL-1ra mRNA was remarkably expressed in IL-1ra-transfected cells. In immunocytochemical analysis, the expression of hIL-1ra was detected in HIG-82 cells. The treatment of LPS enhanced the production of IL-1beta in HIG-82 cells and IL-1ra-transfected HIG-82 cells. IL-1ra-transfected HIG-82 cells spontaneously release IL-1ra in culture supernatant, and its level increased when the cells were cultured with LPS for 48 h. Interestingly, when IL-1ra-transfected HIG-82 cells were cultured with LPS for 60 h, a significant decrease of PGE2 release was observed. Conclusion: We showed that in vitro sonoporation-mediated transfer of IL-1ra plasmid had a remarkable effect on the regulation of inflammatory mediators. These findings suggest the possible application of sonoporation gene therapy using IL-1ra for the treatment of the inflammation in arthritis. [1] Tominaga K.., Habu M., Sukedai M., et al. Arch. Oral Biol, 49 (2004) 493. [2] Iwanaga K., Tominaga K., Yamamoto K., et al. Cancer Gene Ther, 14 (2007) 354. [3] Nishihara T., Maki E., Ishihara Y., et al. J. Periodontal Res, 24 (1989) 165.

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Immobilization of antibodies on the solid support using Protein A fused to a substrate protein for biotin ligase

H. Miyao1, A. Shiraishi1, Y. Ikeda1, S. Sueda1,2

1 Department of Bioscience and Bioinformatics, Kyushu Institute of Technology 2 Research Center for Bio-microsensing Technology, Kyushu Institute of Technology

[Introduction] The binding of antibodies to their specific antigens is utilized for various biological applications owing to their high specific interaction. In the most of these applications, antibodies need to be immobilized on the solid supports and here the immobilization are usually conducted via non-specific binding of antibodies to the supports. In these cases, the orientation of antibodies to the supports cannot be controlled, so that some of antibodies do not participate in the interaction with their antigens. Thus, the oriented immobilization of antibodies allows for the improvement of sensitivity of assays based on the antibodies. In the present work, we have attempted to develop the method for the oriented immobilization of antibody on the solid support using a biotinylation reaction from Sulfolobus tokodaii. [Methods] In the biotinylation reaction, biotin protein ligase (BPL) catalyzes a covalent attachment of biotin to a specific lysine residue of its substrate protein, biotin carboxyl carrier protein (BCCP). The biotinylation reaction from S. tokodaii has a unique property that BPL forms a stable complex with its product, biotinylated BCCP [1, 2]. In the present work, we have attempted to construct an immobilization method of antibodies by combining the S. tokodaii biotinylation with the specific binding ability of Protein A to the antibodies (Fig. 1). Specifically, Protein A fused to BCCP was immobilized through the biotinylation reaction on the solid support modified with BPL, and then the antibody was captured through the interaction between Protein A and the antibody. After that, the antigen was added to the support and the interaction between the antibody and antigen was investigated. Here, the sensor chip of quartz crystal microbalance (QCM) was used as a solid support to monitor the antibody-antigen interaction directly. For this purpose, the mutants of BPL, BPL-Cys1 and BPL-Cys2, were constructed, which have one or two cysteine residues located just in the opposite side of binding interface with BCCP; these mutated BPLs were fixed on the gold surface on QCM sensor chip through Au-S interaction. Also, two types of fusion proteins of Protein A and BCCP, Protein A(x1)-BCCP and Protein A(x2)-BCCP, were constructed by attaching one or two molecules of Protein A to N-terminus of BCCP. [Results] From the experiments with two types of BPL mutants, BPL-Cys2 carrying two cysteine residues was found to capture the Protein A-BCCP fusion protein more effectively than BPL-Cys1 carrying single cysteine residue. Thus, BPL-Cys2 was used for the subsequent experiments. Two types of Protein A-BCCP fusion proteins were fixed on the sensor chips through biotinylation and then anti-GFP antibody was captured on the respective sensor chips. As a result, the antibody in nanomolar range was effectively captured on the sensor chips in both systems, and the change in response was observed by addition of its antigen, GFP, in nanomolar range. From the several control experiments with different antigen and antibody, it was confirmed that the observed change in response was derived from the specific antigen-antibody interaction. Comparing the results with two types of Protein A-BCCP fusion proteins, Protein A(x2)-BCCP carrying two Protein A molecules was found to capture the antibody more effectively than Protein A(x1)-BCCP carrying single Protein A molecule. To evaluate our immobilization method of antibodies, a control experiment was conducted, in which anti-GFP antibody was fixed directly on the sensor chip and the change in response on addition of GFP was monitored. As a result, it was found that the change in response observed in our system was considerably larger than that in the control experiment.

Fig. 1 Immobilization of antibody on the solid support based on the biotinylation reaction from S. tokodaii [1] S. Sueda, Y.-Q. Li, H. Kondo, and Y. Kawarabayasi, Biochem. Biophys. Res. Commun., 344 (2006) 155. [2] S. Sueda, H. Tanaka, and M. Yamagishi, Anal. Biochem., 393 (2009) 189.

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Fabrication of Micro Valve with Ultra-precision Machining Method

Hideki Sato1, Tomohiro Hirose1, Hidetaka Uno2, Mitsukazu Suzui3, Tsuneo Urisu2, Makoto Yabuya1

1Nachi-Fujikoshi Corp. 2Nagoya University First Research Center for Advanced Nanobiodevices

3Institute for Molecular Science

Presently, microfluidic devices play important roles in the pharmaceutical, medical and life science fields. There is a need for fabrication of a microfluidic device comprised of mutiple fluidic channels to make diagnostic and research activities more efficient. To achieve the microfludic device comprised of multiple channels, a micro valve is essential as it allows the fluidic conveyance and discharge to all channels, closure of channels and electric resistance in some cases.

The micro valves developed by Studer and others are comprised of the channels with rounded cross sections and the actuation channels with membranes that connect directly with channels to deform with compressed air [1]. The actuation channel’s cross section is made into a rectangular shape while the cross section of the fluidic channel mold is made into a shape of an arc using the soft lithography which allows the reflowing of the photoresist. A high cutoff function of a fluid and electric resistance are expected with this method. However, the fluidic channel’s cross section becomes uniform across the entire area, causing it to be narrow in the parts where the valve function is not required. Thus, a high fluidic pressure is necessary for obtaining a sufficient flow. Another issue is that the electric resistance is high when the valve is opened, making it difficult to obtain a higher on/off ratio at the time when the electric characteristics are required.

Therefore, we have fabricated the fluidic channel mold with 3D geometry with the ultra-precision machining. Figure 1 shows the channel mold. The channel width is 0.2mm and the area crossed with the valve is a thin cylindrical surface of 20µm high. Other areas are box-shape with a height of 0.1mm, connecting seamlessly with the cylindrical area. Oxygen-free copper is used for the mold material. The surface roughness of the cylinder where the valve membrane contacts with is crucial; so this area in the mold is finished with theoretical roughness of 5nm. Nano Groover AMG62P for the optical-waveguide mold machining manufactured by Nachi-Fujikoshi and single-crystal diamond tool of R0.1mm are used for finish-machining. The 3D-geometry microfluidic channel is made by molding polydimethylsiloxane (PDMS) and the actuation channel is made by again pouring the PDMS into the photoresist-coated mold. These two channels are bonded to fabricate the micro valve that has the 3D-geometry fluidic channels. For comparison, the mold for a fluidic channel with a rectangular cross section using the lithography is fabricated for the same type of the micro valve.

These micro valves with two different fluidic channel geometries were tested by filling a physiological salt solution in the channels and each electric resistance was measured. The results are shown in Figure 2. The micro valve of the channels with rectangular cross sections exhibited a small amount of variation in electric resistance and sufficient electric resistance was not obtained. It was assumed that a gap remained between the membranes and fluidic channel walls even though the valve remained closed. On the other hand, the micro valve of 3D-geometry fluidic channels exhibited a drastic increase in electric resistance in proportion to the increase of controlled air pressure, performing the conspicuous valve functionality.

Reference [1] Vincent Studer, Giao Hang, Anna Pandolfi, Michael Ortiz et al., J. Appl. Phys. 95 (2004) 393.

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Calcium Mitigates the Toxicity of Zinc Oxide Nanoparticles to Human Lung Epithelial Cells

H. Morita1 and N. Hanagata1,2

1 Nanotechnology Innovation Station, National Institute for Materials Science, Japan

2 Graduate School of Life Science, Hokkaido University, Japan The sensitivity of human lung epithelial cells to the toxicity of zinc oxide (ZnO) nanoparticles (NPs) was found to vary according to differences in the dispersion medium, the physiological state in the cell growth stage, and the culture medium composition. This toxicity was not influenced by the Zn ions that were released from these particles, but rather by the particles themselves when they were taken up by cells. The origin of the toxicity of ZnO NPs was found to be the retardation of cell growth rates. Furthermore, at low calcium (Ca) concentrations, the toxicity was much higher than that at high Ca concentrations. As such, we demonstrate that ZnO NP toxicity can be mitigated by adding Ca to the culture medium; this is because the addition of Ca enhances functions that contribute to the cell cycle.

Zinc (Zn) is an essential trace element in biological processes, but an excess of Zn leads to undesirable effects. Therefore, Zn homeostasis is strictly controlled within cells. Due to their electronic and optoelectronic properties, Zn oxide (ZnO) nanoparticles (NPs) are used in biosensors and pigments, in rubber manufacture, and in the field of electronic materials. They also absorb ultraviolet (UV) light and have therefore been used in sunscreens and cosmetics. In recent years, climate change has resulted in increased UV exposure and a growing demand for sunscreen products. ZnO NPs also exhibit antibacterial activity and are therefore expected to find applications in prophylactic agents against bacterial infection. The growing demand for ZnO NPs has led to an increased risk of ZnO NP inhalation. When ZnO NPs are inhaled, they reach the alveoli and cause metal fume fever by inducing proinflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin-6 (IL-6). In guinea pigs, the inhalation of ZnO NPs has been reported to cause severe damage to pulmonary function.

It has been suggested that NPs with the same chemical composition can exhibit different cell toxicity effects depending on their size, surface area, and surface properties. It is reported that the toxicity of ZnO NPs in immune cells increases with decreasing particle size. In contrast, in mouse neural stem cells, the toxicity of ZnO NPs is reported to be dose dependent and not size dependent. Furthermore, in human lung epithelial cells, it is demonstrated that equal concentrations of ZnO NPs with particle sizes of 70 nm and 420 nm exert similar levels of toxicity. However, for the same surface area, the toxicity of 420-nm particles was greater than that of 70-nm particles. We also reported that the cell viability of human lung epithelial cells exposed to ZnO NPs with a particle size of 20 nm did not greatly differ from that for a particle size of 60 nm. Although there have been many reports on the cytotoxicity of ZnO NPs, other reports have found no cytotoxicity due to ZnO NPs in human dermal fibroblasts and T-cells. The cytotoxicity level of ZnO NPs with the same primary size depends on the concentration and aggregate size but is also thought to depend on the type of cell.

It has also been suggested that the toxicity of ZnO NPs is due to reactive oxygen species (ROS). This has been hypothesized to involve a continuous oxidation-reduction reaction taking place on the surface of ZnO NPs due to the redox property of amino acids-Zn2+/Zn+, such as glycine-Zn2+/Zn+. Furthermore, it has been suggested that the toxicity of silicon dioxide (SiO2) NPs to RAW264.7 cells originates from an increase in the intracellular concentration of Ca2+ as well as from the generation of ROS. This increase in the intracellular Ca2+ concentration has also been observed in human bronchial epithelial cells (BEAS-2B) exposed to ZnO NPs and in cells exposed to Ag NPs. The toxicity of ZnO NPs to BEAS-2B cells is reduced by the antioxidant N-acetylcysteine (NAC), but since NAC also blocks the influx of Ca2+ ions into the cell, it appears that the increase in ROS generation and intracellular Ca2+ concentration jointly contribute to toxicity. However, in this study, the cytotoxicity of ZnO NPs was mitigated by increasing the Ca concentration in the culture medium (Figure 1). This result seems to suggest that the increase in the intracellular concentration of Ca2+ ions in conjunction with the increase in ROS does not contribute to toxicity but is a defense mechanism to counteract the toxicity.

Figure 1. Effect of CaCl2 on the viability of cells exposed to ZnO NPs. The cells were incubated with DMEM + 10% FBS for 4 h, and the medium was then replaced with ZnO NPs containing 70% DMEM + 10% FBS with different concentrations of CaCl2. Cell viability was assayed after culture for another 24 h with ZnO NPs. The 70% DMEM contained 1.26 mM CaCl2.

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3 µm 6 µm 8 µm

10 µm 15 µm 20 µm

Bar: 20 µm

BA

1 cm

10

15

209

8

7

6

5

4

3

2

Length of each side of the triangle in each area [μm]

3D pattern-scaffold

20 µm3 µm

Cell Cell Cell

8 µm

Strong F-actinSmall and round

Weak F-actin Large extend

Slow growth

Cell properties similar to that on soft scaffold

Slow migration

Range: Subcellular size (3 – 20 µm, 50% pore ratio) Intensity of mechanical stress ?

Influence of the pattern size of three-dimensional micropatterned scaffolds on cell functions

H. Sunami1,2, I. Yokota1,2 and Y. Igarashi2

1 PRESTO, Japan Science and Technology Agency (JST)

2 Frontier Research Center for Post-genome Science and Technology, Hokkaido University Faculty of Advanced Science

To determine how the three-dimensional (3D) shape of scaffolds influences cell functions, 3D micropatterned scaffolds of various sizes were fabricated on a silicon substrate. The micropatterns were equilateral triangular pores with 3–20 µm long sides, and all had the same pore ratio (total pore area per unit area) and depth. The patterns only differed in terms of their 2D size (Fig. 1). Such scaffolds have not been previously generated, and thus the effects of pattern size on cell functions have not been addressed. NIH-3T3 cells were cultured on these micropatterned scaffolds, and their morphology, proliferation rate, migration rate, and level of F-actin expression were assessed. Cells became more rounded and F-actin expression decreased as the pattern size of the scaffold decreased. Relationships were also demonstrated between pattern size and cell proliferation and migration. These results suggest that the pattern size of 3D micropatterned scaffolds affects the level of mechanical stress that cells experience, and thereby influences F-actin expression, cell morphology, cell proliferation and cell migration (Fig. 2). Fig. 1. Properties of 3D patterned scaffolds of variously sized equilateral triangular pores generated by photolithography. (A) Overview image of the patterned silicon substrate taken with a digital camera. (B) Colour laser 3D microscopy images of the triangular pores of various sizes arranged in a scale pattern on the silicon substrate. Adjoining pores are not connected. The lengths of the sides of the triangles are indicated. Fig. 2. Schematic illustration of the level of mechanical stress that 3D micropatterned scaffolds exert on cells as a function of the unit size of the pattern. The level of mechanical stress that a substrate exerts on cells may decrease as the pattern size decreases. Scale bars represent 20 µm.

The migration and proliferation rates of NIH-3T3 cells cultured on these scaffolds tend to decrease as the pattern size decreases. Thus, the pattern size of 3D patterned scaffolds affects cell functions. Furthermore, the level of F-actin expression changes in response to mechanical stress, and this likely contributes to the relationship between pattern size and cell proliferation and migration. Although only patterns on the micrometre scale were used in this study, we expect that pattern size will affect cell functions on most 3D patterned scaffolds.

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Regulation of DNAzyme activity by metal ion-directed sequence edition of the oligonucleotide template carrying terpyridine units

Hiroyuki Ohura,1 Chisato Sirahama,1 Tomohiro Furuzono,1 Yusuke Kitamura1, 2 and Toshihiro Ihara1, 2

1 Department of Applied Chemistry and Biochemistry, Graduate School of Science and Technology,

Kumamoto University, Japan 2 CREST, Japan Science and Technology Agency, Japan

E-mail: toshi@chem.kumamoto-u.ac.jp

Two amidite reagents of terpyridine (terpy) derivatized

at their ortho or meta positions were synthesized. We can

build terpy units into any positions in the DNA backbone

with these amidite reagents using an automated DNA

synthesizer. Terpy was reported to form a stable complex

with Fe2+ and some other divalent transition metal ions

(Fig. 1a). The whole conformation (static structure) and

stiffness (dynamic structure) of terpy change with metal

binding (Fig. 1b). Therefore, the function of the

functional DNAs carrying terpy units in their backbone

would be regulated by reversible interaction with the

metal ions through concomitant change in the shape of

their backbone.

The formation of terpy complex was assessed by the

melting temperature change before and after the addition

of metal ions. Only meta-derivatized terpy-DNA

(m-terpy2DNA) showed the increase of the melting

temperature by the addition of an equivalent amount of

Fe2+ and Ni2+ ions. It shows that m-terpy2DNA forms

Ω-shaped structure with metal complexation. The

ortho-derivatized terpy-DNA, o-terpy2DNA, did not

form a 2:1 complex with metal ion because of the steric

hindrance of linker moieties.

It is well-known that some G-quadruplexes work as a

DNAzyme which shows peroxidase-like enzymatic

activity by forming a complex with the hemin. Here, we

demonstrated the regulation of the enzymatic activity of

G-quadruplex by Fe2+ or Ni2+-directed sequence edition

of the m-terpy2DNA. The sequences of the split G-quadruplex were designed to form the mature structure only

in the presence of Ω-conformer of m-terpy2DNA. That is, the Ω-conformer might serve as a template for

reconstructing the integrated DNAzyme. The formation of G-quadruplex structure was confirmed by the

oxidation of ABTS which was a substrate for the DNAzyme. As shown in Fig. 2, ABTS oxidation reaction was

activated only in the presence of metal ions.

Fig. 1. (a) Complexation of terpyridine with a metal ion. (b) Sequence edition by the formation of Ω-shaped duplex through the complexation between terpy-DNA and a metal ion.

Fig. 2. Time profiles of the catalytic reactions by the split DNAzyme in the presence (solid curve: Fe2+, dashed curve: Ni2+) and absence (dotted curve) of metal ions. The absorption change was monitored at 414 nm derived from the reaction product, ABTS•+.

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Ultra-efficient photothermal therapy for prostate cancers using aptamer-modified gold nanostars

H. Jo1, S. Lee1, and C. Ban1*

1 Department of Chemistry, Pohang University of Science and Technology, Pohang, Gyeongbuk, 790-784, Republic of Korea In recent years, noninvasive photothermal therapy using the near-infrared (NIR) light has received great attentions to effectively kill cancer cells and to reduce unwanted damages for other cells in contrast to other methods such as chemotherapy and radiation therapy [1]. Various photothermal agents including gold nanorods, gold nanoparticles, silica-cored gold nanoshells, carbon nanotubes, porphysomes, and polymeric nanoparticles have been developed for selective thermal therapy [2]. Among them, gold nanostars (AuNS) have shown numerous advantages compared with other materials, such as tunable plasmon bands in NIR, visualization under multiphoton microscopy, a high absorption-to-scattering ratio in the NIR, and high photothermolysis ability [3]. In this work, we designed an ultra-efficient photothermal complex that simultaneously targeted prostate-specific membrane antigen (PSMA) (+) and (–) prostate cancers. To improve selectivity and sensitivity, we introduced two aptamers that had been issued as a powerful biological ligand because of its high selectivity and binding strength to the target molecules. Dual aptamer-modified AuNS exhibited efficient intracellular delivery and effective photothermolysis. This new aptamer-based AuNS can be applied as an active photothermal agent in vivo.

[1] C. Dees, J. Harkins, M.G. Petersen, W.G. Fisher and E.A. Wachter, Photochem. Photobiol., 75 (2002) 296. [2] K.T. Nguyen, J. Bioengineer & Biomedical Sci., 2 (2012), 1000e112. [3] H. Yuan, C.G. Khoury, H. Hwang, C.M. Wilson, G.A. Grant and T. Vo-Dinh, Nanotechnology, 23 (2012)

075102.

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Influence of temperature on the neuronal oscillation in rat hippocampal slices

Itsuki Kageyama1 and Kiyohisa Natsume1, 2

1 Dept. of Brain Sci. and Eng., Grad. Sch. of Life Sci. and Sys. Eng., 2Research Center for Bio-Microsensing Technology

(RCBT), Kyushu Institute of Technology Neurons in a brain are sensitive and vulnerable to changes in brain temperature. The effects of the temperature on the excitatory postsynaptic potential, memory and behaviors are previously reported [1]. Theta and beta waves observed in rat hippocampus are related to memory and learning process [2]. What effect a change of the temperature has on these brain waves and brain function remains unknown. Theta ( > 12 Hz), β (12 -30 Hz) and γ oscillations( < 35 Hz) similar to the in vivo rhythms can be induced in hippocampal slices with the application of a cholinergic agent carbachol [3]. It is suggested that temperature affects theta rhythm. In a temperature window, the rhythm is induced in hippocampal slices. However, affects of temperature on the other neuronal oscillations have not been clarified so far. In the present study, we examined the impact of temperature on the carbachol-induced oscillations in hippocampal slices.

Hippocampal slices were prepared from male Wistar rats (50 – 160 g). A rat was anesthetized with isoflurane. Hippocampal formation was removed and slices were cut out with a slicer. Hippocampal slices (450 μm thick), were preserved in a chamber (27 oC) for an hour, and were transferred to the recording chamber which was set at one of the temperatures between 27 and 39 oC, and then carbachol was applied to the slice. The oscillation was recorded in the stratum pyramidal of CA3 region using a glass electrode. Carbachol-induced oscillation was measured using glass microelectrode. The temperature of the slices were continuously measured using a needle-thermocouple probe and a thermocouple thermometer. The probe was located in dentate gyrus of a slice. When carbachol could induce the oscillation, four parameters (frequency, amplitude, inter-burst interval (IBI), and duration) were measured.

Figure: Schematic diagram of a recording system.

In results, carbachol - induced neuronal oscillations were observed at the temperature between 27 and 35 oC. The oscillations were induced intermittently like bursts. The frequency of the oscillation in a burst ranged from 9.8 to 13.7 Hz at 27 oC. The frequency covered from θ to β frequency. The frequency increased with the temperature. It ranged from 12.7 to 17.6 Hz from 29 to 35 oC. The frequency was in the β range only. The frequency was significantly increased with the temperature. It was correlated with the temperature. Above 35 oC, the oscillation was not observed. Instead, at 39 oC, the epileptic discharges occurred, like febrile seizures epileptic discharges.

These results suggest that temperature should impact on the induction of hippocampal rhythm. Depending on the temperature, different brain rhythms will be induced, and different information processing will be done. The temperature may affect information processing through the control of inducing the neuronal oscillations. When the above the physiological temperature range, febrile seizures could be induced and information processing might be deteriorated. Recently it is reported that nanothermometer is developed [4]. In future, we would like to develop the system for suppressing febrile seizures using the nanothermometer. [1] T. Deboer and I. Tobler, Brain Res., 670 (1) (1995) 153–156. [2] B. H. Bland, Progress in Neurobiol., 26 (1) (1986) 1–54. [3] M. B. MacIver et al., Proc. Western Pharmacol. Soc., 29 (1986) 159–161. [4] G. Kucsko et al. Nature, 500 (2013) 54–59.

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Stabilization of poly-L-lysine-based cancer-specific vectors via disulfide bond formation

Kai LI1, Guoxi ZHAO1 ,Takeshi MORI1,2,3, Akihiro Kishimura 1,2,3,Yoshiki KATAYAMA1,2,3,

1Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 2The Center for Future

Chemistry, Kyushu University, 3International Research Center for Molecular Systems, Kyushu University

Introduction We have proposed a novel gene targeting strategy, called “drug or gene delivery system responding to cellular signals (D-RECS)”. The systems can activate transgene in response to malfunctions of signal transduction occurring inside diseased cells. In the precious study we succeed in activating gene expression in response to several protein kinase1). However, the polyplex formed from polymeric carrier and plasmid DNA unfavorably dissociates in blood circulation. A promising approach to stabilize the polyplex will be reversibly cross-linking of the polyplex with disulfide bonds. Here a new gene carrier, poly-l-lysine (PLL) is modified with

substrate peptide that can be phosphorylated by PKCα and is also modified with 15-Carboxy-1-pentadecanethiol (15-CPDT) that can form disulfide bond to increase the stability of polyplex in blood circulation.

Experiment PLL (Mw.15000-30000) was simply modified by peptide and 15-CPDT using HOBt and HBTU. After that, the protection group was removed by TFA. Four kinds of polymers with constant peptide modification ratio (5 mol%) and different 15-CPDT modification ratios (0, 5, 10 and 20 mol%) were prepred. The stability of complex that polymer and plasmid DNA formed (N/P=10) was evaluated by gel retardation analysis.

Result and Discussion The release of pDNA from the by oxidation of disulfide bond was examined.

Figure 1 shows the gel retardation assay of polyplexes fromed from each polymer. Free pDNA become weaker with the increase of 15-CDPT contents. On the contrary, the free pDNA bands were observed for reductive condition containing 20 mM DTT (lane 7-9). These results indicated that the disulfide cross-linking can stabilize the polyplexes and the polyplex can be weakened at the reductive condition 1) Riki Toita, et al, J. Am. Chem. Soc., 134, 1540-15417(2012)

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High Resolution Imaging of Angiogenesis in Hind Limb Ischemic Model Mouse with Nanoparticles

K. Kawamura1, K. Gonda1,2, Y. Hamada1, Y. Kubota1 and N. Ohuchi1, 2

1Department of Nano-Medical Science, Graduate School of Medicine, Tohoku University

2Department of Medical Physics, Graduate School of Medicine, Tohoku University 3Department of Surgical Oncology, Graduate School of Medicine, Tohoku University

Recently, the mechanisms of angiogenesis have been used for the treatment of ischemic heart disease and peripheral artery disease (PAD). Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis and has been applied to regenerative medicine. In our previous study, to induce angiogenesis at a selected site without the effects of inflammation and edema on angiogenesis, we developed new hind limb ischemic model mice (PAD model mouse) [1]. To understand the in vivo vascular remodeling and molecular dynamics of angiogenic factors, such as VEGF, we performed micro X-ray computed tomography (m-CT) imaging with polyethylene glycol-supported Au nanoparticles (Au/PEG) and fluorescence molecular imaging with VEGF-conjugated Quantum dots (VEGF-QD) using ischemic model mice. Here we report high resolution in vivo imaging of angiogenesis with these nanoparticles in the model mice.

Ischemic animal models have been used for the development of new angiogenesis therapy. In the previous animal studies, the efficacy of angiogenesis was primarily evaluated using angiography, laser doppler imaging, and the determination of histologic capillary density. Among these methods, the angiography by m-CT is thought to be the most effective to image in vivo capillary vessels generated by angiogenesis due to the resolution of in vivo imaging. Contrast agents are often used to enhance contrast of CT imaging in hospital and thus improve diagnostic accuracy. However, blood retention of previous contrast agents for CT imaging is not good because the agents are low molecules (< 5nm) and absorbed rapidly by the kidney after injection of that. Therefore, it was very difficult that angiogenesis in ischemic animal model is clearly visualized with previous contrast agents. In our previous study, Au/PEG was newly developed. We injected the Au/PEG into the tumor-bearing mice and examined blood retention of Au/PEG in mice with m-CT. The results showed that the more than 50% of Au-PEG retains in blood after 12h and thereby the Au/PEG was well-localized to tumor.

In this study, to investigate the mechanisms of angiogenesis in vivo, we injected the Au-PEG nanoparticles into the hind limb ischemic model mice and performed m-CT imaging and analyzed the three-dimensional architecture of hind limb microvascular network. In addition, to understand the detailed distribution of VEGF and the VEGF-receptor (VEGF-R), we made a VEGF-QD, injected into the model mice, and performed in vivo fluorescence imaging of gastrocnemius in where we induce selective ischemia.

The imaging data with m-CT is shown in Fig. 1. We succeeded in imaging distinctive vascular structure of angiogenesis. The m-CT imaging data showed typical corkscrew-like collateral vessels in ischemic leg. The corkscrew-like collateral vessels was not seen in normal leg. Therefore, It was believed that the development of these corkscrew-like collateral vessels is concerned with vascular remodeling induced by ischemia. In vivo fluorescence molecular imaging, VEGF-QDs bound to the VEGF-R on the membrane of endothelial cells in angiogenesis. The imaging data showed that only a 3-fold difference of VEGF-R distribution is involved in the formation of branched vasculature in angiogenesis, although previous ex vivo data showed 10 to 20-fold difference in its distribution, indicating that a method inducing a several-fold local increase of VEGF-R concentration may be effective in generating site-specific angiogenesis in ischemic disease [1]. We discuss an application of the angiogenesis mechanisms to the diagnosis and treatment of PAD. [1] Y. Hamada, K. Gonda, and N. Ohuchi et al., Blood, 118(13), e93-e100 (2011).

Fig. 1 m-CT imaging with Au/PEG in the ischemic model mice

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Synthesis of the PEI coated cobalt ferrite nanoparticles

for magnetic hyperthermia

K.Mori1, K.Onuma

1, T.Kondo

1, K.Miike

1, T.Miyasaka

1, M.Hachisu

1, Y.Kikuchi

1, Y.Ichiyanagi

1

1Graduate school of Engineering , Yokohama National University , Yokohama 240-8501

Magnetic hyperthermia is a new idea of a cancertherapy using the heat generated by the magnetic

nanoparticles in alternating magnetic field. In our laboratory, we have prepared various kind of magnetic

nanoparticles and reported magnetic properties and functionalization method for biomedical applications.

It is important to control particle size and improve dispersibility for drug delivery system (DDS) or

hyperthermia treatment.

In this study, in order to improve the dispersibility, Polyethyleneimine(PEI) was coated on the cobalt

ferrite nanoparticles.The sample was manufactured with our laboratory original wet process mixing

method. I precipitated precursor by adding Na2SiO3 and NaOH to a metal chloride. And I added PEI there

and stirred it at 363K.The provided sediment isolated it by centrifugal separation and I dried and annealed

it.

The sample was examined by XRD and modification of PEI was confirmed by FT-IR spectrum and

TG-DTA measurement. In addition, I roughly estimated the quantity that PEI was coated by TG-DTA.

The particle size was evaluated by a particle size distribution meter. Finally, we have measured generation

of heat of cobalt ferrite nanopariticles upon application of AC.

I can have successfully synthesized dispersive magnetic fine nanoparticles, and they would be utilized in

biomedical field, such as DDS or hyperthermia treatment.

0 100 200 300 400 500 600 700 800

-30

-20

-10

0

-80

-60

-40

-20

0

20

40

DT

A /

V

Temperature / K

TG

DTA

Mass of PEITG

/ %

Initial mass : 9.91mg

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Magnetic Properties and MRI Contrast Effect on CoxFe3-xO4 Nanoparticles Kazunari Miike 1, Naoki Sakai1, Yuko Ichiyanagi1, Yoshiyuki Hosokai 2, Akihito Usui2,

Yoshio Machida2, Haruo Saito2

1 Graduate School of Engineering, Yokohama National University 79-5 Tokiwadai, Hodogaya, Yokohama, Kanagawa, 240-8501, Japan

2 Graduate School of Medicine, Tohoku University 2-1 Seiryocho, Aoba, Sendai, Miyagi 980-8575, Japan

*yuko@ynu.ac.jp

In the diagnosis by Magnetic Resonance Imaging (MRI), the MRI agents are used for shortening the time and enhancement of the detection. Contrast image is expressed as the difference nuclear magnetic relaxation rate of protons in vivo. MRI contrast agent enhances the contrast of the bright and dark areas by reducing the magnetic relaxation of protons. Currently, main components of γ-Fe2O3 and Fe3O4 main have been used as MRI contrast agents to emphasize the relaxation T2 in the clinical scene. However, the relevance of these MRI relaxivity and physical properties have not been revealed. However, we have suggested so far there is correlation between corecivity Hc and MRI relaxivity R2 of CoFe2O4 nanoparticles controlled on various paricle size. In this study, we investigated correlation between the magnetic properties of nanoparticles and magnetic relaxivity in MRI contrast furthermore.

Based on the previous result, CoxFe3-xO4(x = 0.2, 0.4, 0.6, 0.8) nanoparticles were synthesized by our original wet chemical method. XRD measurement showed these nanoparticles were single-phase CoxFe3-xO4 encapsulated amorphous SiO2. Particle size was controlled by the annealing temperature. Magnetization curves were mesured by SQUID magnetometer at 300K. These samples had coercivity Hc from 301 to 631 Oe, and saturated magnetization Ms from 28.6 to 40.3 emu/g at 300K. MRI relaxation rate T2 and relaxivity R2 (=1/T2) was measured at room temperature under 0.3 T MRI system. Samples were dispersed in an agarose gel to prevent agglomeration of nanoparticles by the magnetic field.

We discovered CoxFe3-xO4 encapsulated amorphous SiO2 had larger R2 compared with material of commercial agents (Fig.1). We have found that this value would be affected by Ms value of nanoparticles (Fig.2). Local magnetic field around the protons would become large, as a result relaxation time T2 would have been shortened by increasing saturated magnetization.

We have concluded that the saturation magnetization magnetization Ms strongly improved imaging contrast, on the other hand, corecivity Hc would not affect the relaxation rate R2 so far as Hc would be small enough bellow about 600 Oe. It would be considered that the lager magnetization in particle causes interaction between magnetic spins and protons.

x = 0.2x = 0.4

x = 0.6x = 0.8

CoFe2O4 Fe3

O4γ-Fe2O3

Agarose0

10

20

30

40

50

60

Relax

zatio

n Ra

te , R

2 / m

s-1

Already in use

250 300 350 400 450 500 550 600 65044

46

48

50

52

54

56

28 30 32 34 36 38 40 42

T =300KRelax

zatio

n Ra

te , R

2 / m

s-1

Coresive force, Hc / Oe

Hc vs R2

Satulated magnetization, MS / emu g-1

Ms vs R2

[1] D.-H. Kim, et al., J. Magn. Magn. Mater., 321 (2009) 3899-3904 [2] L. E. W. LaConte, et al., J. Magn. Reson. Imaging, 26(2007)1634-1641 [3] Y. Ichiyanagi, et al., J. Therm. Anal. Cal., 99 (2010) 83-86 [4] N Sakai, Y Ichiyanagi, et al ., J. Phys. Conference Series 352 (2012) 012008

Fig.1 Relaxivity R2 for various composition of CoxFe3-xO4

Fig.2 Correlation between magnetic properties and R2 of CoxFe3-xO4 nanoparticles

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(b)

(a)

Preparation of hydrophobic carrier for medical agents using cross-linked polysaccharide

Kazuya Maeda1, Shinichi Mochizuki1, Yusuke Sanada1,2, and Kazuo Sakurai1,2

1 Graduate School of Engineering, University of Kitakyushu, 2 JST-CREST Introduction

Most of medical agents for cancer therapy are low molecular compounds, but there are some problems such as low retention time in blood and no cell specificity. When they are administered to a cancer patient, they are taken up by not only cancer cell but normal cells, disappeared quickly from blood stream and induce side effects. For the efficient cancer treatment, the development of drug delivery carriers is important. One of the drug delivery carriers is cholesterol-bearing pullulan (CHP) nanogel. In particular, cholesteryl-bearing pullulans form physically cross-linked nanogels by self-assembly. Since the nanogels trap hydrophobic molecules (antitumor drugs), proteins (enzymes, insulin, antigenic protein), and nucleic acids (DNA plasmids), it can be used as polymeric nanocarriers in cancer chemotherapy, protein delivery, and artificial vaccine [1,2]. We thought that carrier having ability recognized by specific cells could be prepared by using this technique. We have studied SPG as a delivery carrier to antigen presenting cells [3]. In this study, we modified SPG with hydrophobic molecules and prepared nanogel. Experimental Methods

Synthesis scheme for NSPG is shown Fig.1. We determined the modification rate of naphthyl ethyl isocyanate (RNEI) from UV absorbance and aggregation size of NSPG from DLS. We subsequently compared molecular morphology of SPG and NSPG from GPC. The ability to include DOX was examined with two kinds of methods. After dialyzing with water to cut out free DOX, we estimated the inclusion ratio of DOX into NPG nanogel with UV absorbance. We also estimated it from FFF. Results and Discussion

We could obtain some NSPGs with different modification rate of naphthalene. The aggregation sizes depended on RNEI. The size drastically increased at RNEI > 2.0%. It has been reported that SPG takes rod conformation [4]. But the radius gyration against the molecular weight for NSPG was small than that for SPG, indicating that the molecular morphology of NSPG takes hard sphere by cross-linking. From UV absorbance and FFF chromatogram, the inclusion ratio of DOX into NSPG was 1wt% (Fig. 2). This value can be said to be low, but NSPG has ability to be recognized by antigen presentation cells. Therefore we consider that it can be efficient carrier. Conclusion

We modified SPG with naphthalene (NSPG) and made dozens nano meter order particle at RNEI < 5%. NSPG aggregates in aqueous solution, and takes hard sphere. The inclusion ratio of DOX was 1wt%.

References [1] K.Akiyoshi et al., Macromolecules., 1993, 26.,3062-3068 [2] K. Akiyoshi et al., American Chemical Society., 2006 [3] N. Shimada, Bioconjugate Chem., 2006, 17 (5), pp 1136-1140 [4]T. Yanaki et al., American. Chemical. Society., 1980

Fig.1 Synthesis scheme

Fig.2 Amount of DOX included into the nanogel (a) FFF and Light scattering, (b) FFF and UV absorbance (495nm)

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Synthesis of α-Arylcarboxylic Acid Amides from Enol Silyl Ether via Migratory-Amidation with 2-Azido-1,3-dimethylimidazolinium

Hexafluorophosphate

Kento Murakami1 and Mitsuru Kitamura1

1 Department of Applied Chemistry, Kyushu Institute of Technology 1-1 Sensuicho, Tobata, Kitakyushu 804-8550, Japan

The reaction between electrophiles and enolates is one of the fundamental reactions in organic synthesis.

Various α-substituted carbonyl compounds could be thus synthesized by using alkyl halides/equivalents as the electrophiles. However, α-aryl carbonyl compounds are difficult to prepare by the simple nucleophilic substitution reaction of enolates to aryl halides, except when using strong electrophile like nitro-substituted aryl halide, or via a benzyne intermediate. Recently, the metal-catalyzed coupling of aryl halides and carbonyl compound derivatives was developed, and it is becoming one of the most efficient methods for synthesizing α-aryl carbonyl compounds.[1] Previously we reported an alternative method for introducing the aryl group at the α-position of carbonyl compounds on the basis of the internal migration of aryl groups;[2] i.e., by treating 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (ADMP, 1)[3,4] with benzyl aryl ketones 2a (R = aryl group) in the presence of Et3N, the aryl group (Ar1) in 2a can be migrated to the α-position of the carbonyl group to produce migratory-amidated compounds 3a (Scheme 1, path a). Various α,α-diaryl carboxylic acid amide derivatives 3a could be thus synthesized; however, the corresponding α-mono-aryl compounds 3b were not synthesized from alkyl aryl ketones 2b. Our ongoing investigations of the reaction of imidazolinium salt 1 and alkyl aryl ketone derivatives have demonstrated that α-mono-aryl compounds 3b could be synthesized by the reaction of enol silyl ethers of alkyl aryl ketone 4 with 1 (path b).[5] In the presentation, we report the results of these investigations in detail.

Scheme 1. Reaction of ADMP 1 with aryl ketone derivatives.

[1] For a review, see: C. C. C. Johansson, T. J. Colacot, Angew. Chem. Int. Ed., 49 (2010) 676. [2] M. Kitamura, S. Miyagawa, T. Okauchi, Tetrahedron Lett., 52 (2011) 3158. [3] Our recent study on the reaction of 2-azido-1,3-dimethylimidazolinium salt, see: a) M. Kitamura, T.

Koga, M. Yano, T. Okauchi, Synlett, (2012) 1335. b) M. Kitamura, N. Tashiro, S. Miyagawa, T. Okauchi, Synthesis, (2011) 1037. c) M. Kitamura, M. Yano, N. Tashiro, S. Miyagawa, M. Sando, T. Okauchi, Eur. J. Org. Chem., (2011) 458. d) M. Kitamura, N. Tashiro, R. Sakata, T. Okauchi, Synlett, (2010), 2503. e) M. Kitamura, N. Tashiro, Y. Takamoto, T. Okauchi, Chem. Lett., 39 (2010) 732. f) M. Kitamura, N. Tashiro, T. Okauchi, Synlett, (2009) 2943.

[4] ADMP 1 is a crystalline reagent having high thermal stability and low explosibility5b,c and is now commercially available from TCI (cat. No. A2457).

[5] M. Kitamura, K. Murakami, Y. Shiratake, T. Okauchi, Chem. Lett., 42 (2013) 691.

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Bottom-up Brain Model for Nano-Brain Toxicology Assay

K. Fujioka1, S. Hanada2, Y. Inoue3, F. Kanaya2, K. Shiraishi1, K. Ikeda1, K. Yamamoto2, and Y. Manome1

1 The Jikei University School of Medicine

2 National Center for Global Health and Medicine 3 Toho University

Recent technical innovations made it possible to mass-produce various nano materials. While they are currently used for articles of daily use such as foods and cosmetics and improves their quality, the safety of these materials is still under investigation. Several nanoparticles have ability to reach the mouse brain when these nanoparticles are inhaled, intravenously/ intraperitoneally administered, or applied on the skin. For example, Yamashita et al. reported that 70-nm silica particles reached to the brain of murine fetuses when the particles were intravenously administered to pregnant mice (K. Yamashita et al., Nat. Nanotech., 2011). Moreover, Kato et al. showed that quantum dots coated with captopril distributed area-specifically in brain tissue, when the dots were intraperitoneally administered (S. Kato et al., Nanotechnology, 2010). However, it is unknown whether penetrated nanoparticles affect neuronal activity, although nanoparticles' distributions in the brain tissue were reported. It may be difficult to understand the neuronal toxicological effects and mechanisms of nanoparticles in vivo assays directly, because most particles distribute in other organs, and penetrated particles localize in small tissue area into the brain. Therefore, we propose bottom-up brain model which combined with cellular assays for understanding the toxicological effects. Our bottom-up model includes blood-brain-barrier (BBB) model, which indicate apparent permeability coefficient (Papp) [1] and cellular assays (Figure 1). In our concepts, nanoparticles which have penetration ability were selected by means of BBB model and possible toxicological effects and mechanisms were assessed with integration of the cellular toxicological results using the penetrated particles. These selection and integrated toxicological studies will be useful method for predicting the in vivo effects, reducing animal experiments, and time for assessing. In this presentation, we report brief summary of silica particles examinations (Papp from the BBB model and threshold of toxicological concentration against neuron and neural stem cells etc.) with the bottom-up model.

Figure 1. Scheme of bottom-up brain model for assessing nanomaterial’s effects Acknowledgment This study was supported by Health and Labour Sciences Research Grants from the Ministry of Health, Labour and Welfare of Japan (H22-chemical-young-009). [1] S. Hanada, K. Fujoka, Y. Inoue, F. Kanaya, Y. Manome, K. Yamamoto, J. Phys. Conf. Ser., 429 (2013)

012028.

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：C3 ：C6 ：C9 ：C12 ：C15 O O

OOR RR R

N N N NNN

NN

NN

NN

NH2 NH2NH2 NH2

C3H7 C6H13 C9H19 C12H25 C15H31

R

Fig.1 The chemical structure of calix[4]- arene lipids

Influence of alkyl chain length of multivalent cationic lipids with calix[4]arene backbone for transfection efficiency

Koichi NISHINA1, Shinichi MOCHIZUKI2 and Kazuo SAKURAI3

1 The University of Kitakyushu 2 CREST, JST Co., Japan

Introduction Drug Delivery System (DDS) is expected to be useful to cure diseases like gene disorders. There are two types of DDS carriers. They are virus carrier and non-virus carrier. Recently non-virus carrier was studied because virus carrier has infection disease. Cationic lipid (CL) is one of non-virus carriers. A variety of gene carriers using cationic lipids has been studied[1,2]. Among them, multivalent cationic lipids are known to show better gene expression efficiency[3]. We synthesized calix[4]arene -based cationic lipids with different alkyl chain length (C3, 6, 9, 12, 15, Fig.1) and evaluated relationship between the alkyl chain length and the transfection efficiency. Experiment

We made DNA-CL complex to add the plasmid DNA encoding luciferase to CL at N/Pratio = 3. The size and structure of the complex were measured by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). A549 cells were transfected using the complex, and we measured luciferase expression. To check the process of gene expression, we measured cell cytotoxicity, cellular uptake, and release of plasmid DNA from a mimicked endosome. Result and Discussion

SAXS measurements showed lamella structures for all complexes.C6-lipid complexes showed the highest transfection efficiency among all complexes. C9-lipid and C12-lipid complexes showed the second and third efficiency, respectively. All complexes showed low cytotoxicity, and same cellular uptake. Importantly, release of plasmid DNA from a mimicked endosome was dependent on alkyl chain length. We found that the transfection efficiency is strongly influenced by the ability to release DNA from the complex.

Reference [1] Defu Zhi, and Defeng Zhao, Bioconjugate Chem., 2013, 24 (4) [2] Charles H Jones, and Blaine A. Pfeifer, Mol. Pharmaceutics [3] Valentina Bagnacani, and Rocco Ungaro, Bioconjugate Chem., 2012, 23 (5)

Fig.2 The relationship between the alkyl chain length and the transfection efficiency

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QCM Detection of GPCR-ligand Binding Using Cell-derived Liposomes

Makoto Yamanaka, Shinji Sueda and Takashi Yasuda

Kyushu Institute of Technology We developed a novel technique to produce cell-derived liposomes which have specific GPCRs (G protein-coupled receptors), and applied those liposomes to a QCM (quartz crystal microbalance) sensor for detecting interactions between GPCRs and their ligands. In our technique, the liposomes can be derived directly from cell membranes by chemical stimulation of human lymphocyte cells as we previously reported [1, 2]. Therefore, by preliminarily transfecting the cells with DNA encoding genes of specific membrane proteins, we can produce liposomes having those membrane proteins with complex structures such as GPCRs. Note that such membrane proteins on liposomes retain their original structures and activities. In this respect, our technique is much more advantageous than the conventional methods that produce liposomes from a phospholipid solution and invite a heavy risk of disruption of membrane proteins. In this research, we obtained cell-derived liposomes having BB2R (bradykinin B2 receptor) which is a GPCR for bradykinin, and applied the liposomes to a novel biosensor based on a QCM [3] which detects the binding of bradykinin molecules to BB2Rs on the liposomes (Fig. 1). Bradykinin is an oligopeptide having the molecular weight of approximately 1,060 Da and acts as a hormone to play significant roles in blood pressure reduction, pain generation, etc. The sensor was prepared in the following procedure. First, BB2Rs were expressed in human B lymphocytes, Ramos, using gene transfer of plasmid DNA encoding BB2R gene. Second, liposomes having BB2Rs were derived from the cells by chemical stimulation with sodium butyrate. Third, the liposomes were separated from the cells by filtration and micronized into tiny vesicles smaller than 1 μm in diameter by means of sonication treatment. Moreover, a SAM (self-assembled monolayer) with cysteamine and cell-anchoring molecules was formed on the surface of an Au electrode of a QCM sensor chip (Nihon Dempa Kogyo, Nominal frequency: 31.0 MHz). Finally, when the micronized liposomes were seeded on the electrode, they were immobilized on the electrode because the hydrophobic oleyl groups of cell-anchoring molecules bound to the liposome membranes. In this way, the QCM sensor having BB2R as a sensor probe was prepared for detection of bradykinin. To evaluate the detectability of the fabricated sensor, the sensor chip was equipped with a microchannel, and a PBS solution including bradykinin was injected into the microchannel. As a result, the significant reduction of resonant frequency of the sensor was obtained in real time. Figure 2 shows the frequency reduction obtained in response to a bradykinin solution of 1 mg/ml. The mass of bradykinin molecules which bound to the BB2Rs was calculated to be about 58 pg/mm2. In conclusion, we succeeded in detecting bradykinin using the QCM sensor where the liposomes having BB2Rs were immobilized. ACKNOWLEDGMENT This work was partly supported by the Grant-in-Aid for Challenging Exploratory Research and the Grant-in-Aid for Research Activity Start-up of MEXT (The Ministry of Education, Culture, Sports, Science and Technology), Japan.

Liposome having BB2Rs

QCM sensor chip

Au

Chemical stimulationand liposome derivation

BB2R

Transfection and expression ofbradykinin B2 receptor (BB2R) gene

DNA

Seeding and immobilization ofliposomes on a sensor chip Time [sec]

Freq

uenc

y sh

ift [H

z]

0 200 400 600 800 1000-15

-10

-5

0

Injection of bradykinin solution1 mg/ml

binding amounts : ∆m = 58 pg/mm2

∆f = -12.5 Hz

Figure 1. Schematic of cell-derived liposomes having BB2Rs and their application to a QCM sensor.

Figure 2. Frequency reduction in response to injection of a bradykinin solution.

[1] M. Yamanaka et al., Proc. μTAS 2012, (2012) 380. [2] M. Yamanaka et al., Cytotechnology, 62 (2010) 287. [3] M. Yamanaka et al., IEEJ Transactions on Sensors and Micromachines, 133 (2013) 155.

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A new electron transfer mediator for a NAD-glucose dehydrogenase based glucose sensor

Dong-Min Kim, Min-Yeong Kim, Sanapalli S. Reddy, and Yoon-Bo Shim

aDepartment of Chemistry and Institute of Biophysio Sensor Technology (IBST), Pusan National University, Busan 609-735, South Korea

A new novel electron transfer mediator, 5-(2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl)-1,10-phenanthroline Iron (III) chloride (Fe-PhenTPy) oriented to the nicotinamide adenine dinucleotide dependent glucose dehydrogenase (NAD-GDH) was synthesized through the Paal-Knorr condensation reaction[1]. FT-IR, 1H-, 13C-NMR, and mass spectroscopy were used to analyze the synthesized mediator. The electron transfer characteristics of Fe-PhenTPy were evaluated using voltammetry and impedance spectroscopy. A disposable amperometric glucose sensor was constructed on a screen printed carbon electrode (SPCE) employing Fe-PhenTPy & reduce grapheme oxide (RGO) as an electron transfer mediator, and NAD-GDH as a catalytic subunit. The experimental parameters to affect the performance and the stability of the sensor were extensively investigated. Interference effects by acetaminophen, ascorbic acid, dopamine, uric acid, caffeine and other mono-saccharides (fructose, lactose, mannose, and xylose) were completely avoided through coating the sensor surface with the Nafion film containing lead (IV) acetate. The proposed glucose sensor exhibited a dynamic range between 30 mg/dL and 600 mg/dL. The reliability of proposed glucose sensor was evaluated by the determination of glucose in artificial blood and human whole blood samples.

[1] J.Y. Hwang, J.I. Son, and Y.-B. Shim, Solar Energy Materials & Solar Cells, 94 (2010)1286.

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Cyclic naphthalene diimide aiming at tetraplex DNA specific ligand

Md. Monirul Islam, Shinobu Sato, and Shigeori Takenaka Research Center for Bio-microsensing Technology; Department of Applied Chemistry; Kyushu Institute of Technology; 1-1

Sensui-cho, Tobata-ku, Kitakyushu-shi, Fukuoka 804-8550 Japan Human telomere DNA sequence is known to form unique G-quadruplex structures by the different folding ways depending on the environmental conditions such as different metal cation. Such G-quadruplex has been known to form in a cell during cell cycles [1] and ligands having high preference for G-quadruplex structure are expected to stabilize strongly G-quadruplex structure of telomere parts of chromosome in the cell and are good candidate of new anti-cancer drug due to inhibit the access of telomerase contributing a development of cancer cell. Under the circumstances, many researchers focused at the development of ligands carrying preference for G-quadruplex DNA. Ligands carrying naphthalene diimide skeleton also discovered as a potential G-quadruplex specific one [2-4]. We found out that ferrocenylnaphtahlene diimide derivative bound to G-quadruplex structural DNA and applied to telomerase assay as an oral cancer diagnosis [5].

Here, we designed and synthesized cyclic napthalene diimide, cNDI (Fig. 1A). As its chemical structure, one of aromatic plane of naphtalene diimide of cyclic naphthalene diimide is expected to be covered by linker chain resulting in disturbing of duplex DNA binding, whereas this ligand is expected to bind to G-quadruplex DNA through the stacking between G-tetramer and one of aromatic plane of cyclic naphthalene diimide as shown in Fig. 1B.

Spectroscopic titration of cNDI with 5’-AGGG(TTAAGGG)3-3’ (A-core) which is part of human telomere sequence was carried out in 10 mM MES (pH 6.25) containing 1 mM EDTA and 100 mM KCl. Absorption maximum at 383 nm showed hypochromic effect with small red shift suggesting its binding to tetraplex DNA. Scatchard analysis using the spectra change of cNDI upon addition of A-core showed that the binding constant of cNDI with A-core was estimated as 4.1×106 M-1 with site size of 2.0. However, absorption maximum at 383 nm showed the small decreasing without spectra shift upon addition of calf thymus DNA. The precise binding constant of cNDI with calf thymus DNA could not be estimated using this spectra change, but this estimated tentatively as 1.4×105 M-1 suggesting its 31 times higher affinity for tetraplex DNA than double stranded DNA. Computer modeling of docking complex of cNDI with hybrid type tetraplex DNA structure suggested that naphthalene diimide plane is effectively stacked with plane formed by G-quadruplex and hydrogen bonding with nucleic bases and amino moieties of piperazine parts of cNDI. In this presentation, we will discuss the binding behavior of cyclic napthalene diimide with G-quadruplex structural or double stranded DNAs.

References [1] G. Biffi et al., Nature Chemistry, 5 (2013) 182-186. [2] S. M. Hampel et al., Bioorganic & Medicinal Chemistry Letters, 20 (2010), 6459-6463. [3] A. Peduto et al., Bioorganic & Medicinal Chemistry 19 (2011) 6419-6429. [4] I. Czerwinska et al., Bioorganic & Medicinal Chemistry 21 (2012) 6416-6422. [5] K. Mori et al., Clinical Chemistry, 59 (2013) 289-295.

N

N

O O

OO

N

N

HN O

HNO

Fig. 1. Chemical structure of cyclic naphthalene diimide (cNDI) (A) and the conceptual complex of cyclic naphthalene diimide with G-quadruplex structural DNA (B).

(A) (B)

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Development of silicon nanowires (SiNWs)/gold nanoparticles (AuNPs)-modified electrode for oligonucleotide sequence of dengue virus

detection

Jahwarhar Izuan Abdul Rashid1,2, Nor Azah Yusof 1,3*, Jaafar Abdullah1,3

1Institute of Advanced Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 2Department of Chemistry and Biology, Centre for Defense Foundation Studies, National Defense University of Malaysia,

Sungai Besi Camp, 57000, Kuala Lumpur, Malaysia. 3Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

There is an urgent need to develop rapid, simple, cost-effective medical devices for screening medical diseases for example dengue for early medical diagnosis. Such a system will also be useful in physician offices or for personal use at home. Initial Dengue virus infection symptoms are very similar to those of influenza, measles, malaria, typhus, yellow fever, and other virus infections, which make the diagnosis based on presenting symptoms problematic. ELISA assays for the detection of IgG and IgM antibodies to Dengue virus are available but unfortunately they are compromised by cross-reactivity with other flaviviruses and require at least 5 days post infection to mount a sufficient immune response to produce detectable antibodies in a patient. This might be too late for patients with severe dengue infection as complication e.g. bleeding tendency and plasma leakage occur at the same time. Late this year, researchers began to utilize silicon nanowire (SiNWs), semiconductors as promising nanomaterial in construction of chemical and biological sensing devices [1-5]. This is attributing to the SiNWs unique properties including excellent electronic/mechanical properties, favourable biocompability, stability at the atmosphere condition and large surface to volume ratio. These properties have been demonstrated in previous studies by many researchers as ultrasensitive SiNWS sensor for label and direct detection of nucleic acid. In this paper we presented preliminary studies on the development of silicon nanowires (SiNWs)/gold nanoparticles (AuNPs)-modified electrode DNA biosensor for oligonucleotide sequence of dengue virus detection as an alternative of ELISA assay. The fabrication and characterization of novel electrochemical DNA detection based on hybrid nanomaterial of SiNWs/AuNPs-modified ITO and screen printed gold electrode (SPGe) were demonstrated. Firstly, SiNWs/AuNPs-modified-electrode was developed by the dispersion of SiNWs in 3 aminoprophythoysilane (APTES) on both of electrode surface. This was followed by the decoration of Au nanoparticles on SiNWs surface using self-assembly monolayer (SAM) of APTES and dithiopropionic acid (DTPA) for DNA probe immobilization and hybridization. It was shown that the modification of ITO and SPG electrode with these hybrid nanomaterials can enhance the electrode surface area around 14 times and 9.3 times, respectively than their bare electrode. The electrochemical responses of methylene blue (MB) as redox indicator towards DNA immobilization and hybridization using both of modified electrodes are performed by cyclic voltammetry (CV). The results showed that the peak current of MB for both of modified electrode decreased upon hybridization of DNA probe with the target.

[1] L. Yang, H. Lin, Z. Zhang, L. Cheng, S. Ye, M. Shao. Sensors and Actuators B: Chemical 177 (2013) 260-264.

[2] Oh, Y., J., Jang, H.J., Cho, W.J. Islam and M.S. 2012. Sensor and Actuators. 171–172: 238-243. [3] Zhang, G.J., Huang, M.J., Ang, J.J., Liu, E.T. and K.V. Desai. Biosensors and Bioelectronics 26 (7) (2011)

3233-3239. [4] Kulkarnia, A., Xu, Y., Ahn, C., Amin, R., Park, S.H., Kima, T., M. Leed. Journal of Biotechnology 160

(2012) 91– 96. [5] He, Y.; Fan, C. and S.T. Lee. Nano Today 5 (2010): 282-295.

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Adjuvant delivery by novel complex made from polysaccharide SPG and CpG-ODN

N. Miyamoto1, S.Mochizuki1,2, and K. Sakurai1,2

1 The University of Kitakyushu of N.miyamoto, S. Mochizuki, and K. Sakurai

2 JST-CREST of K. Sakurai INTRODUCTION

To induce high immune response for vaccine, the administration of not only antigen but adjuvant is needed. CpG-DNA is expected as a novel adjuvant since the antigen-presenting cells of mammal are known to activate a Th1 immune response by recognizing a non-methyl CpG. Toll-like receptor 9 (TLR9) exists in endosome and recognizes a non-methyl CpG. Therefore the activation of cell using a CpG needs drug delivery system. We have studied interaction between SPG and nucleic acid. After being dissolved in alkaline solution (>0.25 N NaOHaq), a triple helix of SPG dissociates and becomes the random coiled single chain. The triple helix is retrieved when the solvent was neutralized in a neutral solution. We found that the DNA/SPG complex which one of triple helix chains was replaced by a nucleic acid is formed when poly(dA) and poly(C) existed in this process (Figure 1a). SPG is recognized by β-1,3-glucan receptor on the antigen-presenting cells (Figure 1b). This DNA/SPG complex is a potency as adjuvant delivery, but is still not be enough to obtain effective immuno response. Therefore adjuvant carrier with further advanced function is needed. EXPERIMENTAL METHODS

Design of carrier: We used two kinds of nucleic acids for complexatioin. One is CpG with dA40, and the other is complementary CpG (cCpG) with dA40. Crosslink complex is formed by mixing CpG/SPG and cCpG/SPG complex.

Cytokine IL-6 production: The sizes for CpG, complex and crosslink complex are evaluated with poly acrylamide and agarose gel electrophoresis. After splenocytes were treated with CpG, complex, crosslinked complex, we measured IL-6 production in the supernatant with enzyme-linked immune sorbent assay (ELISA) for IL-6. RESULTS AND DISCUSSION Gel electrophoresis: The bands for complex and crosslinked complex were observed at higher molecular weight position than nucleic acid. The band for crosslinked complex was observed at higher position than complex. (Figure 2a) IL-6 response: Crosslinked complex induced highest IL-6 secretion among all samples. CONCLUSION Crosslinked complex can make by use of nucleic hybridization between complexes. The cells treated with crossslinked complex induced highest cytokine production. Crosslink is expected as a carrier of adjuvant with high ability to induce a stronger immune response than complex.

Figure2.(a)Molecular size (b) immune response

Figure1．(a)SPG makes a complex with dA. (b)The complex is recognized by antigen presenting cell.

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Rhodium-catalyzed Reaction of Diazonaphthoquinones and Enol Ethers: Synthesis of Dihydronaphthofuran Derivatives and α-Naphthyl Esters

Noriyuki Fukuzumi,1 Keiichiro Araki,1 Hiroki Matsuzaki,1 Mitsuru Kitamura1

1 Department of Applied Chemistry, Kyushu Institute of Technology

1-1 Sensuicho, Tobata, Kitakyushu 804-8550, Japan

1,2-diazonaphthoquinones (1-oxo-2-diazonaphthoquinones and 2-oxo-1-diazonaphthoquinones) are unique cyclic α-diazocarbonyl compounds and are exclusively used as photoresists, e.g., the novolak-diazonaphthoquinone resist. The Wolff rearrangement of 1,2-diazonaphthoquinones, which is the key reaction of the photoresists, has been widely investigated. However, the development of other reactions has been partially limited because of the difficult synthesis of 1,2-diazonaphthoquinones. Recently, we developed an efficient synthetic method for 1,2-diazonaphthoquinones from naphthol by diazo-transfer with an azide imidazolinium salt (Scheme 1).[1] In this approach, 1-naphthols react with azide imidazolinium chloride (ADMC, 3) to give the corresponding 1-oxo-2-diazonaphthoquinones (2-diazonaphthoquinones) 1, regioselectively, while 2-oxo-1-diazonaphthoquinones (1-diazonaphthoquinones) 2 can be obtained from 2-naphthols.

Scheme 1

The naphthofuran skeleton is found in various natural products, and a number of their natural and synthetic derivatives show interesting biological activity. Various naphthofuran syntheses have been reported based on metal-mediated, photo-induced, thermal reactions. Kraus et al. reported the thermal-intermolecular cycloaddition reaction of enol ethers and 1.2-benzoquinones (135 °C in sealed tube) to afford dihydrobenzofurans (max 56% yield), which were transformed into benzofurans after treatment with p-toluenesulfonic acid in benzene. In the cycloaddition reaction, only dichloro-substituted benzoquinones were used to suppress the competing Wolff rearrangement. In the thermal reaction of diazo compounds, reactive carbene intermediates are formed, while a similar reaction was accomplished via metal carbene intermediates under milder conditions. We realized that dihydronaphthofuran derivatives could be synthesized by the metal-catalyzed intermolecular cycloaddition reaction of diazonaphthoquinones with enol ethers. Furthermore, we expected that this methodology could be extended for the synthesis of α-aryl carbonyl compounds, α-hydroxynaphthyl carbonyl compounds, by the transformation of the obtained cycloadducts. In this presentation, we describe the outcome of this investigation (Scheme 2).

Scheme 2

[1] M. Kitamura, N. Tashiro, R. Sakata, T. Okauchi, Synlett, (2010) 2503.

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Distribution and toxicity evaluation of ZnO nanoparticles in single intravenously exposed mice

R. Ohno1,2, M. Tongu1, H. Hashimoto3

, Y. Fujita4 and J. Fujihara2

1 Department of Experimental Animals, Interdisciplinary Center for Science Research,

Organization for Research, Shimane University 2 Department of Legal Medicine Shimane University Faculty of Medicine

3 Center for the Promotion of Project Research, Shimane University 4 Interdisciplinary Graduate School of Science and Engineering, Shimane University

Background

ZnO is suitable for bio-imaging applications because it has a wide band gap with a large excitation binding energy and ensures efficient UV-blue emission at room temperature [1]. Application of ZnO NPs is expected to apply to cancer diagnosis and therapy. To our knowledge, no information is available on ZnO NP toxicity following intravenous administration. In the present study, the lethal dose 50 (LD50) of intravenously administered ZnO NPs was calculated in mice and blood kinetics and tissue distribution of a toxic dose of ZnO NPs (0.2 mg/kg, 0.05mg/kg) were investigated after intravenous exposure [2].

Materials

ZnO dispersion was purchased from Sigma-Aldrich. Before intravenous administration, the dispersion was diluted with distilled water.

Methods - LD50 –

LD50 estimation was carried out using the Litchfield-Wilcoxon method.

- Kinetic study – Based on LD50, a dose of 0.2 mg/kg of ZnO NPs was used in this study. Six mice were used for each

time point (5, 15, 30, and 60 min), and an additional group received an equivalent volume of distilled water as a control. These mice received a single intravenous dose via tail vein and blood and tissues (lung, liver, kidney, and spleen) were collected. - Tissue accumulation study -

For this study, 6 mice were used for each time point (1, 3, and 6 days), and an additional group received an equivalent volume of distilled water as a control. These mice received a single intravenous dose (0.05, 0.2 mg/kg) via the tail vein and blood and tissues (lung, liver, kidney, and spleen) were collected - Pathological examination-

Tissue samples (liver, kidney, lung, and spleen) were fixed in 10% formalin for routine histologic processing. Paraffin sections were stained with hematoxylin and eosin for pathological examination. - Assay -

The concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine samples were measured using a competitive immunochromatography automatic analyzer. Zn analysis was performed by Metallo Assay Zinc LS.

Results and Discussions

Following the injection, ZnO NPs were rapidly removed from the blood and distributed to organs. Pulmonary emphysema was observed pathologically study in mice at 3 days after the 0.2 mg/kg dose and at 6 days after the 0.05 mg/kg dose. ZnO NPs were mainly accumulated in the lung and spleen within 60 min. From the long-term tissue distribution study, the liver showed peak concentration at 6 days, and spleen peaked at 1 day. The lungs kept high levels until 6 days. Tissue distribution and pathological study showed that the spleen, liver, and lungs are target organs for ZnO NPs. Accumulation in the liver and spleen may be due to the phagocytosis by macrophages. A dose-dependent increase in 8-OHdG was observed in mice treated with ZnO NPs. This study is the first to show information on kinetics and target organs following intravenous ZnO injection.

References [1] K. Senthilkumar, et al., Phys. Status Solidi B., 246, No.4, 885- 888 (2009). [2] R. Ohno et al., Arch Toxicol submitted.

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Production of Polymer-Encapsulated Particles using Supercritical Anti-solvent Method

R. Yunus1, M. Kalani2 and G.H. Chong3

1 Institute of Advanced Technology, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia 2 Department of Chemical and Environmental Eng., Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

3 Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia The best drug delivery method should be safe, inert, and comfortable to the patients. It should also be biocompatible, easily administrable or removable, with high drug loading and easy fabrication/sterilizing ability. Using biodegradable polymers for drug encapsulation is one of the best ways to achieve this [1, 2]. The usage of supercritical fluid (SCF) as an anti solvent is an effective and clean method in production of encapsulated particles [3]. This technique modifies particle size, its distribution and morphology. In this paper, a model system using paracetamol particles as a host particle and L poly lactic acid as a coating material was applied. The effect of pressure, temperature and polymer concentration on the mean particle size and morphology were investigated and the model system was optimized as well. The high diffusivity and low solubility of the anti solvent carbon dioxide in the semi crystalline polymer (LPLA) results in high mass transfer rate causing spherical nanoencapsulated particles to form with mean particle size of 300 nm as revealed in SEM and TEM images. The positions of FTIR spectra peaks of nanoencapsulated particles are similar to the pure polymer due to the high ratio of polymer respect to the drug. In addition, both the X- ray crystallography and DSC revealed to transform of crystalline drug to the amorphous phase due to the very fast mass transfer..

[1] M.M.J Cocero,F. Mattea and S. Varona, J. of Supercritical Fluids , 47 (2009) 546 [2] E. Reverchon and R. Adamia, J. of Supercritical Fluids, 37 (2006) 1. [3] M. Kalani and R. Yunus, Journal of Nanomedicine, 6 (2011) 1101.

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Electrochemical property of the unique HFBI protein on an electrode

R. Yamasaki1, Y. Takatsuji1, M. Lienemann2, M. Linder2, and T. Haruyama1*

¹Kyushu Institute of Technology, Department of Biological Functions and Engineering, Kitakyushu Science and Research Park, Kitakyushu, Fukuoka, 808-0196, Japan

*E-mail: haruyama@life.kyutech.ac.jp ²VTT Biotechnology, Espoo, Finland

1. Introduction

In this study, HFBI protein (derived from Trichoderma sp.) had been employed in order to perform high-through-put preparation of ordered nanostructure of protein molecules as a molecular interface. HFBI is originally from the cell wall of fungi and is a small amphiphilic protein [1]. The characteristic property of HFBI is formed self-organized monolayer on both air/water interface and water/solid interface in accurate two-dimensional ordered structure [2-3]. We found that the HFBI can be employed as molecular-carrier to form nano-structured self-organized membrane on solid surface (solid/liquid interface), because HFBI can be tagged with protein of interest by a way of genetic engineering [1-4]. In other words, HFBI will be able to act as “modifier” of surficial properties of solid materials. Such the functionalization of solid material surface is a concrete way for applied technologies. Moreover, physical property of HFBI molecule can be designed by genetic mutation.

In this study, self-organized, nano-structured, HFBI molecular layer was constructed on Highly oriented pyrolytic graphite (HOPG) electrode surface. Using the HFBI self-organized membrane coated electrode, its electrochemical properties have been investigated. In the experiments, wild type of HFBI was employed. In parallel, two different types of genetically mutated HFBI were prepared and were employed. Wild type HFBI has neutral charged. But two mutants have either negative or positive charge respectively. 2. Results and Discussion

In electrochemical impedance (IMP) measurement of HFBI self-organized membrane coated HOPG used [Fe(CN)6]3-/4- as a redox molecules, decreasing of charge-transfer resistance was observed in case of mutated to positive HFBI protein layer coater electrode. The observation shows that engineered HFBI (with positive charge) can be employed as molecular carrier to prepare molecular layer on an electrode when a negative charged redox probe is used. In the case of using positive charge redox molecules, the electrochemical impedance spectrum shows opposite result. In addition to this measurement, we investigated mutated HFBI can form similar self-organized membrane structure with wild type HFBI, or not. HFBI and mutated HFBI self-organized membrane were analyzed by Atomic Force Microscope (AFM) in solution. These were forming densely packed and just about same structural monolayer. In these results, it is clearly suggest that the self-organized protein layer can form nano-structure without suppression of electron transfer through the molecular layer. Therefore, it also suggest that HFBI molecule can employ as a precise molecular career. 3. Conclusion

Three different charged HFBI coated electrode showed different electrochemical characteristics respectively. This finding clearly suggests that the electrochemical function of protein molecular electronic devices can be designed through a nano-design of molecular interface. Due to our AFM observations, the present way of molecular interface preparation, accurate ordered nano-structure can be prepared through high-through-put self-organization process. This fact clearly shows that the HFBI is a potential molecule as molecular carrier to prepare nano-structured membrane with protein molecules on a sold surface. References [1] A. Iwanaga, H. Asakawa, T. Fukuma, M. Nakamichi, S. Shigematsua, M. B. Linder and T. Haruyama,

Colloids and Surfaces B, 84(2), 395-396 (2011) [2] S. Ikeno, G. R. Szilvay, M. Linder, H. Aritomi, M. Ajimi, and T.Haruyama, Sensors and Materials,

16(8), 413-420 (2004) [3] Y. Takatsuji, R. Yamasaki, A. Iwanaga, M. Lienemann, M. B. Linder and T. Haruyama, Colloids and

Surfaces B: Biointerfaces, 112, 186-191 (2013) [4] H. Asakawa, S. Tahara, M. Nakamichi, K. Takehara, S. Ikeno, M. Linder, and T. Haruyama, Langmuir,

25(16), 8841-8844 (2009)

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Nano metal-suppoted ZnO spherical particle as photo-catalyst

Seiya Teraoka, Shinya Ikeno, and Tetsuya Haruyama* Kyushu Institute of Technology, Department of Biological Functions and Engineering,

Kitakyushu Science and Research Park, Kitakyushu, Fukuoka, 808-0196, Japan

Photo-catalyst is a photo-excited material. In that time, excited electron (donor) and hole (acceptor) are formed in same time and in same crystal. Then it may act as either electron donor or acceptor. In this study, we have employed zinc oxide (ZnO) 5μm particle as a photo-catalyst. ZnO has been well known as an efficient photo-catalyst. However, ZnO has a problem of self-dissolution through photo-oxidation in solution [1]. Due to this agenda, ZnO photo-catalysts have not been well employed. In our present study, we have successfully suppressed the self-dissolution by optimized sintering process. We also modified the ZnO particle surface with precious nano-particles of precious metals, which is act as promoter nano-metal in photo-catalytic reaction. The sintered and precious metal-supported ZnO crystal shows a good photo-catalytic activity in an aqueous solution, without auto-dissolution. Introduction

Photo-catalyst shows oxidation or reduction reaction and super hydrophyric property by light irradiation. They are useful if they can use in the medical field. Therefore, we need studying of photo-catalysts to use in the medical field.

Photo-catalysts which assume light driving force are widely used for various purposes. ZnO is difficult to use as photo-catalyst because it has property that easy to self-dissolute through photo-oxidation [1]. However, (001) crystal face of ZnO have property that not easy to self-dissolute [2]. Therefore, using ZnO as photo-catalyst needs to control of crystal face. In addition improvement of photo-catalytic activity is important to apply ZnO as a photo-catalyst. The synthesis of the ZnO controlled a crystal face and support of promoter nano-metal to ZnO were required for improvement photo-catalytic activity. Results and discussion

ZnO spherical crystals were fabricated by the homogeneous precipitation method. The spherical partice size is 5μm, all the surface of this particle is (001) crystal face [3].

Photo-catalytic activity of ZnO was estimated by using decoloration of methylene blue solution. ZnO was supported by using Au and Ag particles as a promoter nano-metal. Photo-catalytic activity is largely enhanced by the sintered and supported nano-metal. We are considering that improvement of the electronic switching efficiency could be suppressed the self-dissolution by the sintered and supported nano-metal. Conclusion

In summary, photo-catalytic activity of ZnO spherical crystal improved substantially by support of promoter nano-metal. The sintered and precious metal modified ZnO crystals were showed a good photo-catalytic activity and stability in an aqueous solution. After the materialize investigation ZnO own, we will study Sterilization of the microbe. Reference [1] Kuveshni Govender, David S. Boyle, Peter B. Kenway and Paul O’Brien, J Mater. Chem 14, (2004) 2575-94 [2] McLaren A, Valdes-Solis T, Li G, Tsang SC. J Am Chem Soc, 131(35), (2009) 12540-1 [3] Kenji Matsumoto, Noriko Saito, Toshitsugu Mitate, Junichi Hojo, Miki Inada, and Hajime Haneda, crystal

growth design, 9, (2009) 5014-16

Figure 1. Decoloration of methylene blue by photo-catalytic activity of metal-supported ZnO

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Colorimetric aptasensor towards Plasmodium lactate dehydrogenase for the rapid diagnosis of malaria

S. Lee1, H. Jo1 and C. Ban1

1 Department of Chemistry, Pohang University of Science and Technology, Pohang, Gyeongbuk, 790-784, Republic of Korea Finding a rapid diagnostic technique for malaria has challenged scientists for the last century. In the present study, aptamer-based label-free colorimetric sensing of Plasmodium vivax lactate dehydrogenase (PvLDH) and Plasmodium falciparum LDH (PfLDH), biomarkers for malaria, was performed with the pL1 aptamer [1], unmodified gold nanoparticles (AuNPs) [2] and hexadecyltrimethylammonium bromide (CTAB) [3]. CTAB was considered an efficient material to aggregate AuNPs. The useful property of CTAB is that, it not only aggregates the AuNPs, but also controls their aggregation via its competitive binding to aptamers. The proposed biosensor offers simple, rapid, and sensitive detection of Plasmodium LDH (pLDH) proteins. In comparison with other pLDH sensors, the present method can rapidly detect low concentrations of pLDH through observation with the naked eye and UV-visible absorption spectroscopy. Both PvLDH and PfLDH were selectively detected using the aptasensor and showed low detection limits of 1.25 pM for PvLDH and 2.94 pM for PfLDH, whereas the aptasensor did not work for other proteins, such as BSA, lysozyme, and TEV protease. In addition, clinical evaluation trials were performed using UV-visible spectroscopy and transmission electron microscopy (TEM) imaging. Native pLDH in both the P. vivax and P. falciparum-infected blood samples was successfully detected with low detection limits. The detection limits of both P. vivax and P. falciparum were calculated using the linear fit, and the values were 42.25 parasites/μL and 57.53 parasites/μL, respectively. Therefore, the proposed aptasensor for pLDH can be utilized for the accurate and sensitive diagnosis of malaria.

[1] S. Lee, K. Song, W. Jeon, H. Jo, Y. Shim and C. Ban, Biosens. Bioelectron, 35 (2012) 291. [2] W. Jeon, S. Lee, M. DH and C. Ban, Anal. Biochem., 439 (2013) 11. [3] Y. Wu, L. Liu, S. Zhan, F. Wang and P. Zhou, The Analyst, 137 (2012) 4171.

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Aptasensor for cardiac biomarker Troponin I using Ferrocene (Fc) – modified silica nanoparticles

S. Kim1, S. Lee1, H. Jo1 and C. Ban1

1 Department of Chemistry, Pohang University of Science and Technology, Pohang, Gyeongbuk, 790-784, Republic of Korea Cardiac troponin I (cTnI) is well known as biomarker for early diagnosis of acute myocardial infarction (AMI). Using the systematic evolution of ligands by exponential enrichment (SELEX) method, we found ssDNA aptamers against cTnI. The aptamers were selectively bound to both the cTnI and cardiac troponin complexes. Using surface plasma resonance (SPR), the binding affinities of the cTnI aptamers were measured; the cTnI aptamers revealed a lower Kd value (270 pM) than the cTnI antibody (20.8 nM). Moreover, we report a new approach toward designing an electrochemical, label-free aptamer sensor using ferrocene (Fc)-modified silica nanoparticles as the probe. The developed cTnI aptasensor demonstrated an excellent analytical performance with a 2.3 pM detection limit (55.2 pg/mL; S/N = 3) and a wide linear detection range (1 – 10,000 pM) in buffer solution. The specificity of the aptamers was also evaluated using the proposed method; there was no interference attributed to non-specific binding proteins. Additionally, the cTnI was successfully detected in a human serum albumin (HSA) solution, and calibration plots were obtained. The cTnI concentrations in mimic patient samples were determined using a calibration plot. The obtained values were well correlated with the amounts actually added into the serum. Therefore, the cTnI aptamers and proposed the aptasensor might be applied instead of an antibody to detect cTnI, providing a new method for diagnosing AMI.

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Novel cancer vaccine using CpG-DNA/β-1,3-glucan complex

S. Mochizuki1, H. Morishita1 and K. Sakurai1,2

1 The University of Kitakyushu 2 JST, CREST

We have studied schizophyllan (SPG; Figure 1), a member of β-1,3-glucans, as a delivery carrier of oligo nucleotides since SPG can complex with particular homo nucleotides such as poly(C) or poly(dA) via combination of hydrogen bonding and hydrophobic interactions[1,2]. Therefore it is expected that the SPG can specifically deliver the bound oligonucleotides to antigen presenting cells (APCs) such as macrophages and dendritic cells. We have attached dA40 to antisense ODN (AS-ODN) or short interference RNA sequence to render ability to make complex with SPG and achieved efficient gene silencing in animal models of fulminant hepatitis and bowel disease[3-5]. SPG complexed with ODN can be incorporated into the peritoneal macrophages from fluorescent microscopic observation and flow cytometric analysis. These results phenomenologically suggested that the complex was incorporated into the cells through dectin-1. However we do not know whether the complex maintains the dectin-1 affinity in the molecular level and how strongly the complex binds to dectin-1 comparing with SPG. In this study, we demonstrated the affinity between the complex consisting of SPG and ODNs using the constructed soluble form of dectin-1 with quartz crystal microbalance (QCM) and elucidated the binding mechanism. We also prepared CpG-DNA/SPG complex and evaluated immune responses in vitro and in vivo.

The prepared dectin-1 has high specificity for not only SPG but DNA/SPG complex. When the splenocytes were stimulated with CpG-DNA/SPG complex, the cells induced a robust immune response even at low concentrations. The mice treated with CpG-DNA/SPG complex induced antigen specific immunogloblin and antigen specific cytotoxic T lymphocytes.

These findings indicate that SPG offers potential in biotechnology and CpG-DNA complex may be expected to be next cancer and influenza vaccine adjuvant.

[1] K. Sakurai, S. Shinkai. J. Am. Chem. Soc. 122 (2000) 4520. [2] K. Sakurai, M. Mizu, S. Shinkai. Biomacromolecules 2 (2001) 641. [3] S. Mochizuki, K. Sakurai. J. Control. Release, 151 (2011) 155. [4] H. Takedatsu, K. Mitsuyama, S. Mochizuki, T. Kobayashi, K. Sakurai, H. Takeda, Y. Fujiyama, Y.

Koyama, J. Nishihira, T. Sata. Mol. Ther., 20, (2012) 1234. [5] S. Mochizuki, H. Morishita, K. Sakurai. Bioorg. Med. Chem., 21 (2013) 2535.

O

HOOH

CH2OHO

HOOH

CH2O

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CH2OH

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HO

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OOO

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O

HOOH

CH2OHO

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CH2O

HOOH

CH2OH

O

HO

OH

CH2OH

OHO

OOO

nFigure 1. Structure of SPG

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Peptide-functionalized gold nanoparticle sensor for high throughput ligand assay of Estrogen Receptor

S. Ikeno1, M. Cauchi2, Y. Takatsuji1, K. Sano1, C. Bessant2, and T. Haruyama1

1 Department of Biological Functions and Engineering, Kyushu Institute of Technology, Japan

2 Bioinformatics Group, Cranfield Health, Cranfield University, United Kingdom

The development of high throughput analysis (HTA) with specific, responsive, rapid and low-cost method is required for identification of lead-substances in the drug discovery. Nuclear receptors (NR) play a key role in embryonic development, differentiation, reproduction and metabolic homeostasis. Therefore, NR attract attention as a target of drug development in human disease [1-2]. For example, estrogen receptor (ER) and androgen receptor (AR) are strongly involved in breast cancer and prostate cancer, respectively. The NR regulates the gene expression to form the complex with ligand and coactivator protein on the specific sequences of DNA. This complex form can be applied to the affinity sensor to examine the characteristics of ligand on NR.

In this study, authors have developed peptide-functionalized gold nanoparticles (GNPs) for HTA of ligands in agonistic/antagonistic actions on ER. The spectrum analysis of peptide-functional GNPs was performed by multivariate data analysis methods.

The synthetic peptide containing LXXLL motif of steroid-receptor coactivator-1 (SRC-1) can be used as a molecular-recognition element of ligand-activated ER. The peptide-functionalized gold nanoparticles (SRC1-GNPs) were constructed to modify the SRC-1 peptide on GNPs surface [3]. The absorbance spectrum of solution is changed by decrease of the colloidal stability in the solvent, when ligand-activated ER forms complexes with the SRC1-GNPs (Fig.1). The aggregations of SRC1-GNPs trigger the spectrum red shifts which provide character of ligand and quantitative information of agonist ligand with good sensitivity and selectivity through very simple procedure. The character of ligand was examined to analyze the spectrum by multivariate data analysis methods [4].

When ER and 17β estradiol (E2) were added into the

SRC1-GNPs solution, the nanoparticle sensor can recognize agonistic activity of ligand based on the spectrum red shifts of solution. The spectrum shifts was significant increased with concentration of additional E2. By using analyses combined with multivariate data analysis methods as principal components analysis (PCA) and hierarchical cluster analysis (HCA), this sensor can identify the ligand types that are strong agonistic, weak agonistic and antagonistic actions within 30 min.

This nanoparticle-based sensor combined with multivariate data analysis methods will be utilized as HTA in

drug discovery on the base of NR function. References [1] A. Aranda, A. Pascual, Phys. Rev 81 (2001) 1269. [2] A. Chawla, J.J Repa, R.M. Evans, D.J. Mangelsdorf, Science 294 (2001) 1866. [3] Y. Takatsuj, S. Ikeno and T. Haruyama, Sensors 12 (2012) 4952. [4] S. Wold, K. Esbensen and P. Geladi, Chemometrics and Intelligent Laboratory Systems 2 (1987) 37.

Fig.1 Schematic illustration of SRC1-GNPs based sensor for analysis of agonistic/antagonistic actions on ER

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Biocompatible Super Adhesives Using Nano Contact

Shougo Kinugawa1,*, Rina Hayasaka1,2, Siqian Wang1,2, Daisaku Kaneko1,2, Akihiko Tsuge1 1Department of Applied Chemistry, Faculty of Engineering, Kyushu Institute of Technology

2Frontier Research Academy for Young Researchers, Kyushu Institute of Technology Abstract

Wondering adhesive protein produced by mussels fascinates many researchers because of its special property for adhesion which can achieve strong adhesion onto both organic and inorganic surfaces even in seawater. The adhesive protein contains high concentration of amino acid 3,4-dihydroxy-L-phenylalanine (DOPA). The DOPA containing catechol group plays an important role of Mussels adhesion. We measured molecular level adhesion strength of plant-derived material containing catechol group by the same methods of Messersmith group [1], because we synthesize Mussel-mimicking polyester adhesives from polymer from plant-derived materials that also contain catechol groups in their main chains. In our presentation, we will report about molecular level adhesion force measurement of material containing catechol group and synthesizing method, characteristics of our Mussel-mimicking adhesives. We will also mention possible application for biomedical fields of our adhesives.

Experiment

-Measurement of molecular level adhesive strength We modified silicon nitride (Si3N4) cantilevers with 3-aminopropyltriethoxysilane and polyethylene glycol (PEG) molecular chain and finally reacted with caffeic acid containing catechol group to the end of PEG chain. Then we measured adhesive strength by using Atomic Force Microscope (AFM) with the cantilever.

-Synthesis of Mussel-mimicking adhesive

The monomers composing adhesives were selected under a conception of safeness to environment and human body. Based on this conception, 3,4-Dihydroxyhydrocinnamic acid (DHHCA) and 3-(3-Hydroxyphenyl)propionic acid (3HPPA) which can be respectively derived from cinnamon and berries were selected. The copolymer of poly(DHHCA-co-3HPPA)s were prepared in the presence of catalyst of hydroxyapatite. The reaction mixture was treated by acetic anhydride at 150°C and was polymerized for 180°C in vacuo for 20h. The excess acetic anhydride was evaporated during polymerization, and gradually increased its viscosity to finally solidify and not allow further agitation. After cooling down to room temperature, the solid was taken out from flask and milled to powder state of adhesives.

Results

-Measurement of molecular level adhesive strength Figure 1 shows molecular level interaction force between caffeic acid and glass in water. Figure 2 shows molecular level interaction force between only PEG and glass in water. Adhesive strength of caffeic acid with glass is estimated about 300pN. This value is stronger than two hydrogen bond force (tens of pN).

- Mussel-mimicking adhesive poly(DHHCA-co-3HPPA) Adhesion force between poly(DHHCA-co-3HPPA) and glass shows very strong value over 25MPa, and between the polymer and carbon is over 30MPa. Furthermore, from the results of MTT assay and ALP assay indicate that our adhesive have high biocompatibility. And our adhesives are also expected to help bone formation.

Acknowledgement This work was supported by JSPS KAKENHI Grant Number24710088. [1] Lee H, Scherer NF and Messersmith PB, PNAS, 103(2006) 12999

Fig. 2 Molecular level adhesive strength between PEG and glass in water.

Fig. 1 Molecular level adhesive strength between caffeic acid and glass in water.

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Highly Selective DNA Detection Using Toehold-mediated Strand Exchange on Graphene Oxide

T. Miyahata1, A. Futamura1, Y. Kitamura1,2, and T. Ihara1,2

1 Department of Applied Chemistry and Biochemistry, Graduate School of Science and Technology,

Kumamoto University, Japan 2 CREST, Japan Science and Technology Agency, Japan

Graphene oxide (GO), a partially oxidized graphene adsorbs single-stranded DNA (ssDNA) through π−π stacking and hydrophobic interactions with exposed nucleobases. Meanwhile the affinity for double-stranded DNA (dsDNA) is much lower because their nucleobases are put inside the hydrophilic coat of double helical backbones. This ss/dsDNA selectivity has been harnessed for GO-based DNA sensors, in which another unique property of GO as a super quencher of fluorescence has been used. In most of the sensors, quenched emission of the dye-labeled ssDNA probe adsorbed on GO is recovered by duplex formation with the complementary target accompanied by the release from GO (Fig. 1a).

However, it is reported that the conventional methods have intrinsic points of weakness such as the non-specific displacement of probes and adsorption of targets on the vacant sites appeared after the probe desorption. To solve the problems described above, we propose a new strategy for a fluorogenic DNA sensor based on toehold-mediated strand exchange on GO. As shown in Fig. 1b, the probes are indirectly immobilized on GO through the capture DNA. The capture DNA consists of an A20 tail and a partial sequence of the target DNA serving as an anchoring tail to GO and capturing moiety for the probe, respectively.

As shown in Fig. 2, the fluorescence intensity of the FAM-labeled probe was decreased with the increasing amount of GO. To minimize the loss of sensitivity due to non-specific binding, the GO surface were fully saturated by the probes for the use in the following experiments. By the addition of the target to this system, the probe was successfully released from GO surface through the strand exchange reaction. The single-stranded region appeared in the capture DNA after releasing the probe could cover the GO surface, preventing the non-specific adsorption of targets and probes. The emission recovery of the present system was higher than that of conventional one. One base difference in the targets were well-discriminated with higher contrast (Fig. 3).

Fig. 1. Schematic illustrations of the DNA analysis based on the adsorption and the desorption of the probe DNA on GO. (a) Conventional method. (b) Indirect adsorption of the probe DNA using a capture DNA and the sequence specific desorption through the strand exchange reaction.

Fig. 2. Fluorescence titration of the probe (a) and the probe/capture DNA duplex (b) (50 nM for each) with GO (0 – 9.6 µg/mL). The excitation wavelength was 490 nm.

Fig. 3. Time dependencies of the probe dissociation from GO. The emission intensities for the solutions containing the probe/GO (a) and the probe/capture/GO (b) were monitored after the addition of fully-matched DNA (solid curve) and one-base mismatched DNA (dotted curve).

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Identification of A Polyhydroxyalkanoate Synthase Gene and Active Site from Marine Bacteria,

Halomonas sp. O-1 and Halomonas elongata DSM 2581

Takahiro Kihara1, Mulyana Ilham2, Kouhei Mizuno*1, and Takeharu Tsuge2

1 Department of Materials Science and Chemical Engineering, Kitakyushu National College of Technology, 5-20-1 Shii, Kokuraminami-ku, Kitakyushu 802-0985, Japan

2 Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan

Polyhydroxyalkanoates (PHAs) are bacterial

intracellular biopolyesters that have wide potential applications for medical, agricultural, and biofuel industries. It is known that marine microorganisms include uncharacterized bacteria which are expected to have potential characters such as utilizing marine carbon sources and so on. Here, the PHA synthase genes from Halomonas sp. O-1 and Halomonas elongata DSM 2581 were firstly cloned and studied focusing on the potential active center called “lipase box”-like sequence [S Y C V G]. The site-directed mutagenesis was performed on the cysteine and serine in both Halomonas strains. Each of the wild type and mutated synthase genes was ligated with pGEM”ABex (containing phbARe and phbBRe) to build a complete set of genes required for PHA accumulation in the recombinant Escherichia coli JM109. The wild type synthases (phaCHO1 and phaCHe) accumulated P(3HB) up to 39.5 ± 4.5 and 28.4 ± 0.4 wt% of the dry cell weight, respectively. The substitution of the cysteine by alanine resulted in a complete loss of P(3HB) accumulation in both mutated synthases (phaCHO1 C329A and phaCHe C331A). The substitution of the serine by glycine (phaCHO1 S327G and phaCHe S329G) to put the typical lipase box-like sequence [G x C x G] in the synthases gave rise to P(3HB) accumulation up to 27.1 ± 0.2 and 37.3 ± 8.7 wt% of the dry cell weight, respectively. The Pha operons from H. sp. O-1 and H. elongata DSM 2581 consist of three genes, phaP1, -P2, -C, of which these two phaP1 and -P2 were investigated by using pGEM”ABex. Additionally, another potential PHA synthase gene referred as to phaC2 found in both Halomonas strains studied was also cloned and co-expressed with phbARe and phbBRe in the recombinant E. coli JM109, but no PHA accumulation occurred, indicating that the phaC2 alone could not work as a synthase.

Figure1. Site-directed mutation on the Halomonas lipase box.

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Autofluorescence Lifetime Imaging Microscopy

T. Nakabayashi and N. Ohta

Research Institute for Electronic Science, Hokkaido University, Japan Imaging of fluorescence from endogenous chromophores has received much attention because cellular environments can be maintained without exogenous probes, and diagnostic tests can be performed without staining time. Cells contain endogenous chromophores exhibiting fluorescence called autofluorescence, and each autofluorescent chromophore in cells is distinguished when the excitation and detection wavelengths are properly selected. In the present study, we have shown that autofluorescence lifetime imaging (AFLIM) can be applied to evaluate intracellular pH in a cell without exogenous probes. Fluorescence lifetime imaging is one of the fluorescence microscopic techniques and is advantageous over intensity measurements for quantitative analyses because fluorescence lifetime is independent of photobleaching and optical conditions. AFLIM is thus a promising tool for understanding intracellular environments without dye labeling [1–3].

We have investigated the sensing of intracellular pH using the autofluorescence lifetime of NADH that is one of the well-known autofluorescent chromophores [1]. Bulk measurements of the fluorescence decay profiles of NADH in buffer solution and in HeLa cells were first carried out with pH of 5–9. HeLa cells were incubated with medium containing ionophore that equalizes intracellular pH to extracellular one, and the intracellular pH can thus be evaluated by measuring the extracellular pH. The fluorescence lifetime of NADH remained almost unchanged with pH in buffer solution; however, NADH in HeLa cells exhibits the significant decrease in the fluorescence lifetime with increasing intracellular pH. Furthermore, the fluorescence lifetime in HeLa cells was ~ 5 times larger than that in buffer solution, indicating that the fluorescence lifetime of NADH is strongly affected by the interaction with proteins in cells. Fluorescence lifetime images of NADH in HeLa cells at different intracellular pH were measured, and the obtained lifetime images are shown in Fig. 1, together with the corresponding intensity images. It is shown that the fluorescence lifetime in the image becomes shorter as the intracellular pH increases, indicating that intracellular pH of a cell can be evaluated by AFLIM of NADH. The value of the fluorescence lifetime was not uniform in a cell and the fluorescence lifetime was smaller in nuclei than in other areas at each pH, and the magnitude of the pH-induced change was larger in nuclei than in other areas (Fig. 1). The small magnitude of the pH-induced change in the fluorescence lifetime in buffer indicates that the interaction between NADH and each protein site at which it is bound depends on intracellular pH, which results in the pH-induced change in the non-radiative decay rate of NADH.

The effect of pH on the fluorescence lifetime of FAD, that is a significant cofactor exhibiting autofluorescence, has also been investigated in buffer solution and in cells [2]. The fluorescence lifetime of FAD remained unchanged with pH in buffer solution. However, the fluorescence lifetime in HeLa cells was found to decrease with increasing intracellular pH. FAD is regarded to exist in protein-bound and free forms in living systems, and the interaction between FAD and the enzyme to which it is bound depends on intracellular pH, which induces the pH-induced change in the non-radiative decay rate of FAD in cells. AFLIM of FAD also exhibited the decrease in the fluorescence lifetime as the intracellular pH increases, leading us to a conclusion that intracellular pH of a cell can be evaluated by AFLIM of FAD. We believe that AFLIM can be applied to quantitatively measure changes in intracellular pH with physiological processes.

[1] S. Ogikubo, T. Nakabayashi, T. Adachi, Md. S. Islam, T. Yoshizawa, M. Kinjo, and N. Ohta, J. Phys. Chem. B, 115 (2011) 10385. [2] Md. S. Islam, M. Honma, T. Nakabayashi, M. Kinjo, and N. Ohta, Int. J. Mol. Sci., 14 (2013) 1952. [3] T. Nakabayashi and N. Ohta, Seibutsubutsuri, 53 (2013) 166.

pH 5 6 7 8 9 10

Intensity image

Lifetime image

1 ns 1.5 ns 2 ns 2.5 ns 3 ns Fig. 1. Fluorescence intensity images (upper) and fluorescence lifetime images (lower) of NADH in HeLa cells at the intracellular pH indicated at the top. Plots of the fluorescence lifetime as a function of intracellular pH using the values obtained at mitochondria (circles) and at nuclei (triangles) are also shown in the left.

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Effects of culture condition on micropatterned culture of HepG2 spheroids

T. Morikyo and K. Nakazawa

Department of Life and Environment Engineering, The University of Kitakyushu 【Introduction】 Recently, hepatocyte spheroids (three-dimensional multicellular aggregates) have attracted attention as a useful culture technique for tissue engineering, drug discovery screening technology, and fundamental studies in cell biology, because they have a tissue-like structure and can maintain the expression of high cell functions. Among various techniques, a microwell chip culture is one of useful techniques for generating spheroids. It can control the size of spheroids and array the spheroids on the chip. Furthermore, it is known that the properties of spheroids are influenced by the microwell conditions, such as diameter, pitch, and number of microwell [1, 2]. In this study, we evaluated the effect of the depth of the microwell on the spheroids properties. 【Materials and Methods】 The microwell chip comprised of 19 circular microwells (600 μm in diameter) in a triangular arrangement (4000 μm in pitch) on a poly-(methyl methacrylate) plate. The bottom of each microwell had a cell adhesion area (300 μm in diameter) modified with collagen, and the remaining part of each microwell was modified with polyethylene glycol to create the non-adhesive area (Fig. 1). Four similar chips with different depths of 0 μm (aspect ratio (diameter/depth) = 0, AR0-chip), 300 μm (AR0.5-chip), 600 μm (AR1.0-chip), and 900 μm (AR1.5-chip) were designed to investigate the effects of microwell depth on spheroids properties. 【Results and Discussion】 HepG2 cells adhered on a collagen area in the microwells and gradually formed spheroids by cell proliferation under all chip conditions. Fig. 2 shows a cross-section view of spheroids at 10day of culture. The spheroid size depended on the microwell depth and was smallest in the AR1.5-chip. In addition, the expression of liver specific function (albumin secretion activity) was highest in the AR0 chip. To understand these phenomena, we evaluated the oxygenation of spheroids in each chip. The glucose consumption (Fig. 3a) and lactate secretion rates (Fig. 3b) of spheroids was the highest in the AR1.5-chip. These results suggested that hypoxia condition occurs when spheroids are cultured in deeper microwells. From these findings, the depth of microwell affects to spheroids properties, and it is one of important factor for designing the microwell chips

[1] Y. Sakai and K. Nakazawa, Acta Biomaterialia, 3 (2007) 1033-1040. [2] Y. Sakai, S. Yoshida, Y. Yoshiura, R. Mori, T. Tamura, K. Yahiro, H. Mori, Y. Kanemura, M. Yamasaki and K.

Nakazawa, Journal of Bioscience and Bioengineering, 110 (2010), 223-229.

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600μm

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Magnetic and self heating properties of Mn-Zn ferrite nanoparticles as agents of

hyperthermia treatment

T. Kondo1 , K. Onuma

1 , K. Miike

1 , T. Miyasaka

1 , M. Hachisu

1 , K. Mori

1 and Y. Ichiyanagi

1*

1 Department of Physics, Graduate School of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya, Yokohama,

Kanagawa, 240-8501, Japan *yuko@ynu.ac.jp

Magnetic hyperthermia treatment is a new attempt to replace conventional hyperthermia treatment. We aim to kill the

cancer cells utilizing our magnetic nanoparticles, because they yields thermal energy upon the application of alternating

magnetic field. If we can prepare cell selective magnetic nanoparticles, cancer cells could be selectively destroyed by

magnetic hyperthermia treatment.

We have reported various ferrite nanoparticles surrounded by amorphous SiO2, and evaluated magnetic properties and

heating properties in the alternating magnetic field so far. In this report, in order to improve magnetization value, non

magnetic Zn ions were doped into MnFe2O4, and magnetic properties and self heating properties of Mn1-xZnxFe2O4 (x =

0, 0,1 0,2 0,3 0,5 0,7 0,9, 1) nanoparticles were studied. Interestingly, it was confirmed that the magnetization where x =

0.2 showed the largest value. So we prepared Mn0.8Zn0.2Fe2O4 (i.e. x=0.2) by wet chemical method and controlled

particle size. Obtained samples were confirmed as single-phase spinel structure by X-ray diffraction measurements.

Then the magnetization was measured for the sample between 13 ~ 30 nm by using SQUID magnetometer, in particular,

imaginary part of AC magnetic susceptibility (") was investigated. Because the imaginary part of AC manetic

susceptibility is important parameter to influence the amount of heating value under the constant frequency and magnetic field. Heating properties were measured by setting the sample in the alternating magnetic field. As the result,

the sample of 18 nm showed the highest temperature increase as expected by the AC magnetic susceptility

measurements. This phenomenon supported that self heating was caused by a mechanism of magnetic relaxation loss.

Finally, magnetic hyperthermia treatment was performed for prostate cancer cells, and the effective results of magnetic

hyperthermia treatment was confirmed.

[1]Y. Ichiyanagi, T. Uehashi, S. Yamada, Phys. stat. sol., 12 (2004) 3485-3488

[2] D. Shigeoka, H. Katayanagi, Y. Moro, S. Kimura, T. Mashino and Y. Ichiyanagi, J. Phys. Conf. Ser., 200 (2010)

122002

[3] Y.Ichiyanagi, D. Shigeoka, T. Hiroki, T. Mashino, S. Kimura, A. Tomitaka, K. Ueda, Y.Takemura, Thermochimica

Acta, 532 (2012) 123-126

0 50 100 150 200 250 3000

2

4

6

8

10

12

14

Time / s

Tem

per

ature

ris

e /

K

Mn(1-x)

ZnxFe

2O

4 (x=0.2) f = 15 kHz, H = 151 Oe

12.9 nm

15.3 nm

18 nm

23 nm

30 nm

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Effect of fiber length on cellular uptake of antigen-loaded peptide nanofiber

Tomonori Waku, Kazufumi Kawabata, Saki Nishigaki, Shigeru Kunugi, Naoki Tanaka

Department of Biomolecular Engineering, Kyoto Institute Technology

Peptide-based vaccines have attracted considerable attention as the next generation of prophylactic and

remedial immunotherapy. Antigen delivery to antigen-presenting cells is a key issue in developing effective vaccines. Nanoparticulate systems are one of the most promising approaches to efficiently deliver antigenic peptides. Recently, in addition to spherical nanoparticles, one-dimensional particles, such as nanorods, nanotube, and nanofiber, have attracted much attention as drug delivery carrier, since the particle shape may have a profound effect on their biological properties. In particular, the interactions of carbon nanotube with living system have been vigorously investigated. However, there is no consensus regarding the mechanism for cell uptake and cellular fate of carbon nanotube. Understanding the intracellular uptake and subcellular fate of one-dimensional materials is crucial for the design of delivery carriers.

Our goals are to understand the interactions of β-sheet peptide nanofibers, which are one of the one-dimensional materials that are promising as drug delivery carrier due to their morphological regularity and ease of chemical modification, with living cells and to develop the efficient delivery system of antigenic peptides by using the nanofibers as carriers. In this presentation, we report the preparation of antigenic peptide-loaded nanofibers by self-assembly of β-sheet peptide containing T-cell epitope and hydrophilic block, and their cellular uptake.

Two kinds of peptide conjugates consisting of four distinctive segments were designed as a simple model for the construction of antigenic peptide-loaded nanofibers. They were composed of a β-sheet forming block (FVIFLD; β), an antigen block (257SIINFEKL264; OVA49-57), a flexible-linker block (GSG), and a hydrophilic block (RKKRRQRRR; Tat49-57 or oligo(ethylene glycol) 12mer ; oligo(EG)12) (Fig. 1a). The β-OVA-TAT and

β-OVA-EG12 solution in PBS was incubated at 60 oC for 24 hours, nanofibers with a length of several hundreds of nanometers were formed. To evaluate the uptake of β-OVA-TAT and β-OVA-EG12

nanofibers (NFs) by cells, RAW264 cells, murine macrophage-like cell line, were incubated with fluorescence-labeled nanofibers for 30 min at 37 oC. Interestingly, β-OVA-EG12 NFs were more efficiently internalized by cells than β-OVA-TAT NFs. These results are contrary to our expectations, because pegylation is applied in order to reduce the non-specific phagocytosis. Next, in order to understand the cellular uptake behavior of β-OVA-EG12 NFs, we investigated the structures of the nanofibers in detail. XRD and FT-IR studies showed β-OVA-EG12 peptides partially adopted β-sheet structures in the state of nanofibers. TEM, AFM, and SAXS studies revealed the morphology of the β-OVA-EG12 NFs was rectangular solid rather than cylinder solid. On the basis of these results, we propose a model of β-OVA-EG12 NFs as shown in Fig. 1b, which show that nanofibers possess heterogeneous surface with a hydrophobic side and hydrophilic one. Thus, efficiently cellular uptake of β-OVA-EG12 NFs appears to be due to their characteristic amphiphilic structures. We will discuss the effect of fiber length on their cellular uptake of β-OVA-EG12 nanofibers.

References [1] T. Waku, Y. Kitagawa, K. Kawabata, S. Nishigaki, S. Kunugi, and N. Tanaka, Chem. Lett., in press

Figure 1. (a) Schematic illustration and sequence of the building block peptides for the construction of antigen-loaded nanofibers. (b) Proposed model of β-OVA-EG12 nanofibers.

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In Vivo Dual Imaging of Lymph Node Using X-ray CT and Fluorescence

Y. Kubota1, K. Gonda1,2 , Y. Kobayashi3, T. Kamei4, T. Nakagawa4, H. Matsudo3, K. Shibuya3 and N. Ohuchi1,5

1 Department of Nano-Medical Science, Graduate School of Medicine, Tohoku University

2 Department of Medical Physics, Graduate School of Medicine, Tohoku University 3 Department of Biomolecular Functional Engineering, College of Engineering, Ibaraki University

4Advanced Surgical Science and Technology, Graduate School of Medicine, Tohoku University 5 Department of Surgical Oncology, Graduate School of Medicine, Tohoku University

The technologies for X-ray computed tomography (CT) or fluorescence imaging has been intensively

developed in recent years. The X-ray CT imaging with gold nanoparticles as contrast agents has reported in many articles. Gold is superior to conventional iodine-base compounds contrast agent in term of X-ray absorption coefficients [1]. As the resolution of X-ray CT imaging is around several tens of micrometers, the CT imaging can visualize at levels ranging from small tissues to whole body by high penetrative power of X-rays. Quantum dots (QDs) is one of recently-developed fluorescence nanoparticles and used for various biological imaging. The brightness and photostability of QDs is much greater than those of conventional fluorescent dyes or proteins. Fluorescence imaging has the resolution with hundreds of nanometer and high quantitative sensitivity because the fluorescence signal intensity is proportional to the intensity of the photon excitation energy. However, as the fluorescence imaging was affected by optical scatter and absorption in cells or tissues, tissue permeability of fluorescence is not well. Therefore, fluorescence imaging is suitable to visualize at levels ranging from molecular to small tissues. It is thought that the technology integration of both advantages for X-ray CT and fluorescence imaging greatly contribute to development of medical imaging with high accuracy and highly-quantitative sensitivity at levels ranging from molecular to whole body. Here we developed silica-coated gold nanoparticles and silica-coated QD nanoparticles and report dual imaging of lymph node with these nanopartiles.

To make silica-coated gold nanoparticles and silica-coated quantum dots, silica coating was performed using a modified Stöber method based on the sol-gel method [2]. Gold nanoparticles or QDs were mixed with tetraethylorthosilicate (TEOS)/ethanol. Then a sodium hydroxide solution (a catalyst for the sol-gel reaction of TEOS) was mixed with the solution containing gold nanoparticles and TEOS or QDs and TEOS. As silica is relatively biocompatible material, it is expected that the silica-shell prevent aggregation and allergy of core in vivo. Silica-coated gold nanoparticles (Fig. 1) and silica-coated QDs nanoparticles (Fig. 2) was mixed before administration to a mice.

To evaluate CT contrast ability and fluorescent imaging ability of these silica-coated nanoparticles, we injected these nanoparticles mixture into lower legs of mice. Then both imaging was performed to examine distribution of these nanoparticles at subcutaneous of lower legs. The results showed that both nanoparticles were localized to lymph node via lymph vessels. Distribution of both agents was almost correspond to each other. Recently, lymph node diagnosis for cancer surgery is growing in importance because cancer cells metastasize to other parts of the body via lymph vessels or blood vessels. Therefore, dual imaging technology with silica-coated gold nanoparticles and silica-coated quantum dots was useful for development of lymph node diagnosis with high accuracy and highly-quantitative sensitivity.

[1]Hainfeld J F Br. J. Radiol 79, 248-53 (2006). [2]Kobayashi Y, Inose H, Nakagawa T, Gonda K, Takeda M, Ohuchi N and Kasuya J. Colloid Interface Sci. 358, 329-33 (2011).

Fig. 1 Fig. 2

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Preparation of nano-carrier which is fused with cholesterol modified

polysaccharide and β1,3-Gulucan

Yoshiya MAEGAWA1, Shinichi MOCHIZUKI

1,2, Yusuke SANADA

1,2, Kazunari AKIYOSHI

3

Kazuo SAKURAI1,2

1 The University of Kitakyusyu, 2 JST-CREST, 3 Kyoto University

Introduction

To protect the drug from uptake into non-targeting cells and degradation in blood stream, the drug needs a cell

specific delivery carrier. Akiyoshi et al have studied an antigenic protein delivery using nanogel consisting of

cholesterol modified hydrophilicity polysaccharide (CHCDex)[1]

. CHCDex forms a nanogel through its

hydrophobic interaction. In addition, this nanogel is famous for refolding the heatdenatured enzyme effectively

according to a similar mechanism such as molecular chaperone.

We have studied CpG-DNA delivery using β-(1,3)-D-glucan schizophyllan (SPG), which can make a complex

with nucleic acid[2]

. SPG is known to exist as a right-handed triple helix in neutral water and as a single chain in

alkaline solution (>0.25 N NaOHaq) [3]

. When the alkaline solution is neutralized to PH=7-8, the single chain

retrieves its original triple helix owing to the hydrophobic and hydrogen bonding interactions. β-(1,3)-D-glucan

is also known to be recognized by dectin-1 on antigen presenting cells such as macrophage and dendritic cell. If

CHCDex and SPG including antigenic protein and CpG-DNA, respectively, can be delivered to the antigen

presenting cells simultaneously, the induction of Th-1 immune response is expected. In this study, As a

preliminary step to achive this strategy, We prepared nano-carrier which is fused with cholesterol modified

polysaccharide and β1,3-glucan, and we attempted to specific protein delivery to antigen presenting cells.

Experiment

Preparation of a novel Polysaccharide nanogel

We obtained the fusion nanogel by neutralizing CHCDex sonicated in NaH2PO4 (330mM) and FITC-labeled

SPG in NaOHaq (>0.25 N). We measured it using Field Flow Fractionation (FFF) coupled to UV-vis 490nm.

Inclusion of BSA using by fusion nano-carrier

After obtaining fusion nanogel, we added RITC-labeled BSA to solution and incubated overnight. We

measured it using FFF coupled to UV-vis 550nm.

Uptake of the protein into the cells

d-raw cells were plated in 96 well pate at 2.0×104cells/dish and

cultured for 24 h. After the culture medium was removed, 100μl fusion

nano-carrier complex solution with RITC-BSA (10μg/ml) was added to

cells. After incubation for 4 h, cells were washed with PBS and

measured fluorescence from the RITC-BSA per cells.

Result and Discussion

We qualitatively confirmed the fusion nanogel used by FFF (Fig. 1).

A peak of FITC-SPG decreases, and a new peak is observed at

approximately eighteen minutes, indicating that the molecular weight of

FITC-SPG increased by adding nanogel. These results suggest that

the hydrophobic groups of CHCDex were embedded in the helix of

SPG since SPG is known to introduce hydrophobic materials at

renaturation process.

Uptake of the BSA was increased by using fusion nano-carrier (Fig.

2; SPG-CHCDex-R-BSA). It’s indicated that the carrier was

recognized by antigen presenting cells in the presence of SPG..

[1] Y. Ozawa, S. Sawada, N. Morimoto, K. Akiyoshi,

Macromolecular bioscience, 9(2009) 694.

[2] K. Sakurai, S. Shinkai, Journal of the American Chemical Society,

122(2000) 4520.

[3] T. L. Bluhm, Y. Deslandes, R. H. Marchessault, S. Perez, and M.

Rinaudo, Carbohydr. Res., 100(1982)117.

1.5x10-2

1.0

0.5

0.0

3025201510

UV

ab

sorb

an

ce (

49

0 n

m)

Elution time / min

Figure1 . Preparation of a fusion nanogelmeasured by FFF-UV-vis 490nm

0

1000

2000

3000

4000

5000

6000

Flu

ore

scen

ce i

nte

nsi

ty /

m

g p

rote

in *

* P < 0.05

Figure 2. fluorescence intensity of RITC-BSA taken into d-raw cells

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Swing molecular layer for high-sensitive affinity sensor based on photo-excited current

Y. Takatsuji, R. Wakabayashi and T. Haruyama

Kyushu Institute of Technology, Department of Biological Functions and Engineering,

Kitakyushu Science and Research Park, Kitakyushu, Fukuoka, 808-0196, Japan Abstract

The authors have developed the system which is a smart affinity detection method based on photo-excited current. In the system, it is important that the molecular layer is constructed and is possible to swing immobilized molecule on semi-conductor (FTO). Fluorescent pigment labeled affinity peptide was immobilized through the EC tag (electrochemical-tag) method [1-3]. This method is possible that a well-ordered molecule-solid juncture can be constructed electrochemically. In this study, we insisted the relation between swing molecular layer and photo-excited current and applied for nuclear receptor affinity sensor. Introduction

Biological affinity biomolecules, e.g., anti-bodies, nuclear receptors, play important roles in the functions of a living system. Such affinity molecules can be employed in the detection of material by sensor devices. In such applications, the key technology involves how to transduce molecular affinity binding into detectable signals. In our research, we employed photo-excited current for affinity sensing, which is performed on semi-conductor materials. It is important to prepare a molecular interface with immobilization of bio-affinity molecules (e.g., peptides or/and proteins) on semi-conductive material surface. In our research group, technologies and foundation study of molecular interface, including bonding molecules onto solid materials, have been developed as technical breakthrough [4]. EC tag method is a one of our achievements. The EC tag method is based on the electrochemical deposition of a coordinated metal ion in acyclic peptide ligand [1-3]. In this study, ECtag method were shown that it is possible to construct swing molecular layer and realized high-sensitive affinity sensor based on photo-excited current. Result and Discussion

Amount of immobilization sensor molecular by ECtag method can be controlled dependent on immobilization time. From the result, electron transfer rate were changed by amount of immobilization. It is shown that the molecular layer need to swing on a semi-conductor for efficient electron transfer. For application of affinity sensor, we designed a sensor molecular and constructed molecular layer. Therefore, we obtain decrease of sensor response which is an affinity of ligand activated nuclear receptor by photo-excited current (Fig.1). The sensor response was proportional to number of molecules which is ligand activated nuclear receptor in assay solution. Conclusion

For detection of molecular affinity, it is need to construct of swing molecular layer on semi-conductor surface. EC tag method permits smart molecular immobilization to prepare ordered molecular interface which may provide superior efficiency. This fact indicates that design and functionalization of molecular interfaces is critical in the development of molecular electronics devices. Especially in the case of molecular based devise fabrications, such as enzymes, receptors and antibodies, have inimitable functions. Reference [1] H. Sakamoto and T. Haruyama, Colloids and Surfaces B, 79, 83-87 (2010) [2] H. Sakamoto, S. Ikeno, T. Kato, N. Nishino,T. Haruyama, , Analytica chimica acta, 604, 76–80 (2007) [3] T. Haruyama, T. Sakai, K. Matsuno, Biomaterials, 26, 4944–4947 (2005) [4] T. Haruyama, Electrochemistry, 78(11), 888-895 (2010)

Fig.1 Photo-excited current measurement at a FTO electrode (dashed line), functional peptide/FTO electrode (gray line) and functional peptide/FTO electrode reacted ligand activated nuclear receptor (black line).

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02468

1012141618

Investigation of enhancement of DNA detection sensitivity with zinc oxide and silica nanoparticles

Y. Hiwatashi1, T. Higashi1, Y. Fukui2, J. Fujihara2, H. Takeshita2, H. Hashimoto3, and Y. Fujita1

1 Interdisciplinary Graduate School of Science and Engineering, Shimane University

2 Department of Legal Medicine, Shimane University School of Medicine 3 Center for the Promotion of Project Research, Shimane University

Introduction: Agarose gel electrophoresis is routinely used for a gene analysis, and microchip electrophoresis has been attracted recently. Microchip electrophoresis is fast and sensitive, results in high resolution, and allows for the easy separation of protein and DNA. Previously, Fujihara et al, have developed a quick method for a Deoxyribonuclease I activity assay using microchip electrophoresis. [1]. The previous research on microchip electrophoresis reported that the addition of ZnO nanoparticles in buffer solution provides the best enhancement factor of approximately 17 [2]. Also, ZnO nanoparticle size-dependency for the enhancement of DNA detection sensitivity has been reported [3]. However, it is difficult to examine the size-dependency of ZnO nanoparticles because the crystals of ZnO have a hexagonal-prism shape and show various aspect ratios. On the other hand, silica nanoparticles prepared by chemical methods have a spherical shape and are monodispersed. In this study, we tried to investigate the mechanism of the enhancement of DNA detection sensitivity by adding ZnO or silica nanoparticles to DNA-dye solutions. Methods: Silica nanoparticles were prepared by hydrolysis and polycondensation of tetraethoxysilane. ZnO nanoparticles were prepared by using a gas evaporation method. A Zn metal was melted and evaporated by the arc discharge between a carbon cathode and a Zn metal anode, and the ZnO nanoparticles were formed by the reaction of evaporated zinc and oxygen in the dry air.[4]. The particle size distributions of ZnO and silica nanoparticles were measured by using a dynamic light scattering particle size analyzer. ZnO and silica suspension were prepared by dispersing each of nanoparticles in pure water by using an ultrasonic homogenizer. The compositions of the samples are shown in Tabel 1. In the preparation of each sample, we first mixed the DNA (7.0 × 10ˉ⁶ [mol/l]) and 0.01% SYBRGold (a fluorescent dye for DNA)-water solution used as the PCR. Next, two samples were prepared by adding ZnO suspension or silica suspension to the DNA-SYBRGold solution. Another sample without nanoparticles was prepared as a control. The Photoluminescence (PL) quantum yields of three samples were measured by using a spectorofluorometer equipped with an integrating sphere with irradiating 480 nm light from a xenon lamp. Results and Discussion: Table 1: Sample composition The median sizes of ZnO and silica nanoparticles are around 430 nm and 410 nm, respectively. The PL quantum yields of three samples are shown in Figure 1. The quantum yields of the sample adding ZnO solution was about 2.0 times higher than that of the sample without nanoparticle while that of the sample adding silica solution was about 4.6 times higher than that of the sample without nanoparticles. It was considered that the enhancements were caused by Whispering gallery mode resonance (WGM) with the surface inside of silica nanoparticles or ZnO nanoparticles, and the difference in the quantum yield of the sample between adding silica solution and ZnO solution was caused by the difference of the shape of each nanoparticle. [1] Fujihara J, Tabuchi M, Yasuda T, Fujita Y, Takeshita H.

Rapid measurement of deoxyribonuclease I activity with the use of microchip electrophoresis based on DNA degradation.Anal.Biochem.(2011)413:78-79.Y. Fujita, M. Tabuchi, Japan Patent No. 4839449.

[2] Y. Hiwatashi et al., 6th International Symposium on Nanomedicine Abstract Book (2012) P-30.

[3] O. Senthilkumar, K. Yamauchi, K. Senthilkumar, T. Yamamae, Y. Fujita, and N. Nishimoto, J. Korean Phys. Soc. 53 (2008) 46-49.

Figure 1: Comparison of quantum yields

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A powerless separation method with glass balloons

*Yukihiro Okamoto1, Hiroshi Yukawa1, Manabu Tokeshi1,3, and Yoshinobu Baba1,2

1 FIRST Research Center for Innovative Nanobiodevices, Nagoya University 2 Department of Applied Chemistry, Graduate School of Engineering, Nagoya University

3 Division of Biotechnology and Macromolecular Chemistry, Hokkaido University [INTRODUCTION]

For in situ bioanalysis in developing countries, the attainment of powerless separation methods have been demanded because there is sometimes no available for electricity. Furthermore, in a production, the development of environmental friendly separation methods have been attempted e.g., no use of toxic chemicals, energy-saving separation etc. However, present separation methods require huge amount of energy, and cannot be applied for in situ bioanalysis and the production. In this paper, to overcome these problems and to attain powerless separation, we focused on hollow glass beads (HGBs), and modified their surface for separation. Surface modified hollow glass beads (sm-HGBs) could successfully separate biomolecules and cells in powerless manner and in large amount of samples. Therefore, sm-HGBs are expected to be applied for in situ bioanlysis and the production.

[PRINCIPLES] The principle of the separation with HGBs is shown in Figure 1. Sm-HGBs can sink by being stirred. Immediately after incubation, sm-HGBs start to float. During sinking and floating, analytes and sm-HGBs can effectively bind. After many sinking and flotation, sm-HGBs can be easily retrieved from the upper of the solution. [EXPERIMENTS AND RESULTS] DNA extraction was attempted with HGBs. Detail investigations indicate that HGBs could extract long (48 kbp) and short chain (10 bp) DNA in a short time (Fig 2). In addition, HGBs could extract DNA from blood. Thus, the powerless separation can be attained with HGBs. Furthermore, the cell separation with sm-HGB was attempted.

HGB’s surface was modified with antibody via carbodiimide method [1]. Table 1 indicates that sm-HGBs can attain high collection rate as well as high purity. In addition, sm-HGBs could retrieve tumor cells from bloods in large volume. Thus, sm-HGBs can permit powerless cell separation in easy manner. From these results, we can show that HGB overcomes the problems related to present separation methods and enables powerless separation in easy manner. Therefore, HGB is expected to be applied for in situ bioanalysis and the production. [REFERENCE] [1] Greg T. Hermanson, Bioconjugate Techniques, Academic Press, 1996.

Fig.2 Extraction efficiency of short and long chain DNA with HGB

Fig.1. Principle of separation with HGB

Table 1. Collection rate of H1975 cells (N=5) Sample Only H1975 Mixture of

H1975 and H1299

H1975 suspended in to

blood Collection rate

(%) 84 (SD 6.2) 72(SD 9.5) 62 (SD 5.4)

Sample volume was 4 ml in the case of only H1975 and mixture of H1975 andH1299, while 12 ml in the H1975 suspended into blood

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Synthesis of peptide-DNA conjugates as imaging reagent for biological sodium ion

Yuuki Imaichi,1 Haruka Koga,3 Koji Nakazawa,3 Shinobu Sato,1,2 Shigeori Takenaka1,2

1Department of Applied Chemistry,

2Research Center for Bio-microsensing Technology, Kyushu Institute of Technology, Kitakyushu 804-8550; 3Department of Life and Environment Engineering, The University of Kitakyushu, Kitakyushu, Fukuoka 808-0135, Japan

Intracellular sodium ion (Na+) is contributed with regulation of membrane potential in conjunction with potassium ion (K+) and operates as part of homeostasis. Regulation of membrane potential also contributes to signal transduction in our taste bud or nerve system. Therefore, imaging reagent of Na+ or K+ into or out of the cell is important to elucidate detailed mechanism in life phenomenon.

We have been developing Potassium Sensing Oligonucleotide, PSO, aiming at a fluorometric imaging of K+ ion in living cell based on G-rich oligonucleotide carrying two chromophores at both termini.1 When the oligonucleotide shows a preference for K+ by tetraplex structure formation and two chromophores are possible combination of fluorescence resonance energy transfer (FRET), two terminal chromophores of PSO close each other to form tetraplex structure with K+ and FRET signal should produce. On the other hand, one can estimate K+ concentration change from FRET signal change of PSO. Here we synthesized SSO-1 to develop Sodium Sensing oligonucleotide, SSO, to monitor Na+ in living cell as a first trial. SSO-1 is FAM chromophore modified oligonucleotide of the part of human telomere sequence, 5’-TAG GGT TAG GGT TAG GGT TAG GG-3’, as a sensing unit attached to peptide linker carrying TAMRA chromophore and biotin (Fig. 1A). This oligonucleotide has higher affinity for K+ rather than Na+, but its tetraplex structure with K+ is hybrid-1 form where two chromophores are located farther away position than that of basket from induced by Na+ (Fig. 1B). This expectation leads to effective FRET for Na+.

Fluorescence titration of 2 μM SSO-1 with NaCl or KCl was carried out in 20 mM Tris-HCl buffer (pH 7.4) at 25 ˚C. Fluorescence intensity at 518 nm derived from FAM decreased and at the same time fluorescence intensity at 585 nm derived from TAMRA increased upon addition of NaCl or KCl. This behavior comes from tetraplex structure formation of SSO-1 and subsequently increasing FRET efficiency in the presence of Na+ or K+. Fluoresce ratio (ΔRatio) obtained from F.I. at 585 nm divided by F.I. at 518 nm under 200 mM NaCl was larger than that under 200 mM KCl as we expected. Dissociation constant was estimated by the fitting of the salt depending ΔRatio change with the theoretical equation expecting the 1:2 binding. The obtained dissociation constants of SSO-1 with Na+ and K+ were 47.8±0.4 mM and 4.03±0.1 mM, respectively. Circular dichroism spectra of SSO-1 upon addition of NaCl showed positive Cotton effect at 245 and 295 nm suggesting the chair type tetraplex structure of SSO-1, whereas positive Cotton effect at 245 and 290 nm with was observed in the presence of KCl as typical aspect of hybrid type one. According to circular dichroism spectra change, similar dissociation constants were observed under NaCl and KCl.

SSO-1 shows the higher FRET change in Na+ than that in K+. However, dissociation constraint for Na+ was over 10-times larger than that for K+. When one can find out the oligonucleotide sequence carrying preference for Na+ rather than K+, true SSO will be realized to permit a monitoring of Na+ under excess amounts of K+ like an outside cell.

[1] K. Ohtsuka et al., Chem. Comm., 2012, 48, 4740-4742.

Fig. 1. (A) Chemical structure of SSO-1 and (B) the expected tetraplex structure of SSO-1 in the presence of Na+ or K+.

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Enhanced Tumor Targeting by Nitric Oxide Encapsulated Stealth Liposomes

Y. Tahara1, Z. Hosain, T. Mori1, T. Yamamoto2, A. Kishimura1 and Y. Katayama1

1 Department of Applied Chemistry, Faculty of Engineering, Kyushu University

2 Institute of Systems, Information Technologies and Nanotechnologies

A stealth liposome, which is a liposome covered with PEG chains, is an efficient carrier of drugs and imaging reagents to tumor due to the effective accumulation to tumor based on enhanced permeability and retention (EPR) effect. Nitric oxide (NO) is known as a diatomic free radical that induces the vascular smooth muscle relaxation to increase the blood flow. Here we hypothesized that the NO-encapsulated liposome will enhance the tumor accumulation due to the relaxation effect of the vascular smooth muscle (Figure 1). We used NOC18 as a NO donor because of its long half life of 21 hours under physiological condition1.

Liposome was prepared by a thin-film hydration method. The molar ratio of the lipid components was DPPC:cholesterol:PEG-DSPC = 55:40:5, including DiD as fluorophore 1 mol% (vs. total lipids). Then the NOC18 was mixed with liposome in 100 mM AMPSO buffer (pH 9) and the resulting dispersions were homogenized with an extruder at 55 through 200 nm polycarbonate filters. The obtained liposome was injected intravenously to CT-26-tumor-bearing mice (male ddY, n = 5). As negative controls, empty liposome dispersed in the AMPSO buffer containing or not containing NOC18 were injected similarly. After injection, in vivo biodistribution and tumor targeting characteristics of the liposomes were monitored using IVIS® Imaging System. A B

Figure 1. (A) Enhancement of EPR effect of stealth liposome-containing NOC18. (B) Structure of NOC18-conganing liposome prepared here.

The average size of the liposomes was 130 nm and average z-potential was -2 mV determined by DLS irrespective of the incorporation of NOC18. The size was suitable to expect the EPR effect. Figure 2 shows the fluoresence images of mice resulting from the liposomes. The NOC18-containing liposomes showed the highest accumulation in the tumor. This result showed that NO released from the liposome induced intravascular relaxation to improve the EPR effect of the stealth liposomes. Interestingly, the accumulation of the liposomes in the spleen was minimized in the NOC18-conganing liposomes. The mechanism is yet unclear, but the characteristics is quite important as the tumor targeting of the liposomes. Figure 2. The fluoresence imaging of mice injected intravenously with NOC17-containing liposome(A), empty liposome dissolved in AMPSO buffer containing (B) and not containing NOC17 (C). [1] J. A. Hrabie and J. R. Klose J. Org. Chem., 58(1993) 1472-1476. [2] Y. C. Barenholz J. Control. Release 160 (2012) 117–134

NO Liposome

Tumor

(A) (B) (C)

PEG2000-DSPE

DPPC

NOC18 (NO donor) DiD (Fluorophore)

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Electrochemical periodontal disease assay using ferrocenyl peptide immobilized-electrode

Shinichiro Nagata1, Junpei Shimamoto1, Takeshi Ohshima1, Shinobu Sato1, Toshinori Okinaga2,

Michihiko Usui2, Wataru Ariyoshi2, Keisuke Nakashima2, Masato Nagayoshi2, Chiaki Kitamura2, Tatsuji Nishihara2, and Shigeori Takenaka1

1Department of Applied Chemistry, Kyushu Institute of Technology, Kitakyushu 804-8550, Japan, 2Kyushu Dental University, Kitakyushu, Fukuoka 803-8580, Japan

In recent years several studies have clearly demonstrated a link between periodontal disease and systemic illnesses. In relation to this, it is important to develop a tool that will specifically diagnose this disease at the earliest possible stage. Basically, periodontal disease is connected with Gingipains (gp) generated by P. gingivalis. Gingipain as specific protease has two types of gps, the Arg-gingipain (Rgp) and Lys-gingipain (Kgp), which are categorized based on substrate specificity. In this study, we developed an electrochemical detection method1 for gps with electrodes carrying two ferrocenylpeptides, FRG or FKG. When these electrodes are treated with a sample solution containing either Rgp or Kgp, the current signals are expected to decrease in the matched combination of enzyme and substrate. As a result of this system, electrochemical assay of periodontal disease was specifically obtained by current signal change based on gp activity.

Ferrocenylpeptide, FRG, was synthesized and were immobilized on the gold electrode through Au-S linkage. Square wave voltammograms of these electrodes were measured before and after treatment of sample solution containing Rpg or culture of P. gingivalis.

Redox peak of FRG-immobilized electrode was observed at 0.30 V based on ferrocene moiety. When an FRG immobilized electrode was treated with 1.0 μg/mL Rgp, 83% of peak current was decreased, whereas no peak current decrease was observed in the presence of Rgp inhibitor, KTY-1. Use of FRG immobilized electrode gave a quantitative analysis of Rgp from ca. 3.0×105 cells of P. gingivalis as a detection limit. When this system is applied to saliva sample from patient with periodontal disease, large current decrease was observed.

Electrochemical assay of periodontal disease was first achieved by the method described here. This system is very interesting from the viewpoint of simultaneously detecting enzymatic activities of Rgp and Kgp.

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List of Authors

A Abdullah, Jaafar 103 Akiyoshi, Hideo 73 Akiyoshi, Kazunari 121 Araki, Keiichiro 105 Ariyoshi, Wataru 128 Asanuma, H. 65

B Baba, Yoshinobu 27, 125 Ban, C. 90, 110, 111 Bessant, C. 113 Bheema, D. 81 Bianco, Alberto 31 Boxer, Steven G. 28

C Cauchi, M. 113 Chen, Peilin 33 Chen, T. 42 Chen, Z.W. 32 Chong, G.H. 107 Chung, T. D. 47

D Doi, K. 63

E

Endo, Y. 77

F Fujigaya, T. 79 Fujihara, J. 106, 124 Fujihara, Junko 73 Fujii, Ikuo 66 Fujii, Masatoshi 73 Fujii, T. 65 Fujioka, K. 98 Fujita, Y. 106, 124 Fujita, Yasuhisa 73 Fukui, Y. 124 Fukunaga, M. 42 Fukuzumi, Noriyuki 105 Furuzono, Tomohiro 89 Futamura, A. 115

G Gonda, K. 53, 93, 121 Gu, N. 32

H Habu, M. 84 Habu, Manabu 71 Hachisu, M. 94, 119 Ham, Sihyun 48 Hamada, Y. 93 Hanada, S. 98 Hanagata, N. 72, 82, 87

Harada, M. 54 Harano, K. 63 Haruyama, T. 108, 113, 123 Haruyama, Tetsuya 109 Hashimoto, H. 106, 124 Hashimoto, Hideki 73 Hayakawa, Mana 71 Hayasaka, Rina 114 Hayashi, H. 55 Hayashi, K. 54 Hibi, T. 39 Higashi, T. 124 Higuchi, Hideo 26 Hirano-Iwata, A. 76 Hirose, Tomohiro 86 Hirota, K. 77 Hiwatashi, Y. 124 Hori, H. 77 Hosain, Z. 127 Hosokai, Yoshiyuki 95 Hosokawa, C. 44 Hu, S.L. 32

I Ichiyanagi, Y. 94, 119 Ichiyanagi, Yuko 57, 95 Igarashi, Y. 88 Ihara, T. 115 Ihara, Toshihiro 89 Iijima, K. 39

129

Iijima, S. 56 Ikeda, K. 98 Ikeda, T. 55 Ikeda, Y. 85 Ikeno, S. 113 Ikeno, Shinya 109 Ilham, Mulyana 116 Imaichi, Yuuki 126 Inaguma, Asumi 75 Inoue, Y. 98 Inui, T. 77 Ipponjima, S. 39 Ishihara, Kazuhiko 34 Ishimura, K. 54 Ishinari, Y. 76 Islam, Md. Monirul 102 Isobe, Takeshi 73 Ito, A. 65 Ito, H. 65 Iwanaga, K. 62, 84 Izumi, K. 54

J Jo, H. 90, 110, 111

K Kageyama, Itsuki 91 Kalani, M. 107 Kamei, T. 121 Kamiya, Y. 65 Kanaya, F. 98 Kaneko, Daisaku 114 Katayama, Y. 127 Katayama, Yoshiki 80,

92 Kawabata, Kazufumi 120 Kawakami, R. 39 Kawamura, K. 93 Kihara, Takahiro 116 Kikuchi, Y. 94 Kikushima, Kenji 26 Kim, Dong-Min 101 Kim, Min-Yeong 101 Kim, S. 111 Kim, You Jeong 50 Kimura, Y. 76 Kinugawa, Shougo 114 Kishimura, A. 127 Kishimura, Akihiro 80, 92 Kita, Sayaka 26 Kitamura, Chiaki 128 Kitamura, Mitsuru 97, 105 Kitamura, Y. 115 Kitamura, Yusuke 89 Kobayashi, Y. 53, 121 Kodama, Masaaki 71 Koga, H. 79 Koga, Haruka 126 Kondo, T. 119 Kondo, T. 94 Konishi, Y. 43 Konno, Tomohiro 34, 67 Kubo, H. 54 Kubo, K. 77 Kubota, K. 43 Kubota, Y. 93, 121

Kuchiike, D. 77 Kudoh, S. N. 44 Kunugi, Shigeru 120 Kusaba, T. 55

L Li, Kai 91 Lee, Min-Ho 49 Lee, S. 90, 110, 111 Liang, X.G. 65 Lienemann, M. 108 Lin, Xiaojie 34 Linder, M. 108

M Ma, M. 32 Machida, Yoshio 95 Maeda, Kazuya 96 Maegawa, Yoshiya 121 Manome, Y. 98 Matsudo, H. 121 Matsumoto, Akihiro 73 Matsumoto, Y. 60 Matsuzaki, Hiroki 105 Mette, M. 77 Miike, K. 94, 119 Miike, Kazunari 95 Minami, K. 63 Minohoshi, Ayako 80 Miyahata, T. 115 Miyamoto, D. 81 Miyamoto, N. 104 Miyao, H. 85 Miyasaka, T. 94, 119

130

Mizuno, Kouhei 116 Mochizuki, S. 104, 112 Mochizuki, Shinichi 96, 99, 122 Mori, K. 94, 119 Mori, Kumiko 71 Mori, T. 59, 127 Mori, Takeshi 80, 92 Mori, Y. 42 Morii, Takashi 83 Morikyo, T. 118 Morishita, H. 112 Morita, H. 87 Murakami, Kento 97

N Nagai, Takeharu 35 Nagao, F. 84 Nagata, Shinichiro 128 Nagayoshi, Masato 128 Nakabayashi, T. 117 Nakagawa, T. 121 Nakagawa, Y. 77 Nakamura, E. 63 Nakamura, M. 54 Nakashima, Keisuke 128 Nakashima, N. 79 Nakata, Eiji 83 Nakazawa, K. 79, 81, 118 Nakazawa, Koji 126 Natsume, Kiyohisa 91 Nemoto, T. 39 Nishigaki, Saki 120

Nishigami, M. 59 Nishihara, T. 62, 84 Nishihara, Tatsuji 71, 128 Nishimura, Kohji 73 Nishina, Koichi 99 Nishizawa, Shigeru 40 Niwano, M. 76 Noh, Hui-Bog 50 Noiri, E. 63

O Ohno, K. 42 Ohno, R. 106 Ohshima, Takeshi 128 Ohta, N. 117 Ohuchi, N. 53, 93, 121 Ohura, Hiroyuki 89 Ojida, Akio 58 Okamoto, K. 63 Okamoto, Yukihiro 125 Okinaga, Toshinori 128 Okobira, T. 70 Onizuka, S. 77 Onuma, K. 94, 119 Ozawa, Takeaki 75

R Rashid, Jahwarhar Izuan Abdul 103 Reddy, Sanapalli S. 101

S

Sada, T. 79 Saito, Haruo 95 Sakae, N. 43 Sakai, Naoki 95 Sakurai, K. 70, 104, 112 Sakurai, Kazuo 96, 99, 122 Sanada, Yusuke 96, 122 Sando, S. 41 Sano, K. 113 Sato, H. 61 Sato, Hideki 86 Sato, Shinobu 71, 102, 126, 128 Sawada, K. 39 Segawa, Hiroki 75 Seno, T. 43 Shibuya, K. 121 Shim, Yoon-Bo 50, 101 Shimamoto, Junpei 128 Shinboku, Y. 55 Shiraishi, A. 85 Shiraishi, K. 98 Shirakihara, A. 79 Sirahama, Chisato 89 Song, LN. 32 Sueda, S. 55, 85 Sueda, Shinji 100 Sugiura, M. 68 Sunami, H. 88 Suzui, Mitsukazu 86 Suzuki, M. 64

131

T Tada, H. 53 Tago, Y. 42 Taguchi, T. 44 Tahara, Y. 56, 127 Takada, H. 43 Takahashi, Osamu 71 Takai, J. 65 Takatsuji, Y. 108, 113, 123 Takeda, N. 44 Takenaka, Shigeori 71, 102, 126, 128 Takeshita, H. 124 Takeshita, Haruo 73 Takeuchi, R. 77 Tamanoi, Fuyuhiko 24 Tanaka, Naoki 120 Terada, H. 77 Teraoka, Seiya 109 Toga, Tomoko 73 Tokeshi, M. 74 Tokeshi, Manabu 125 Tominaga, K. 62, 84 Tominaga, Kazuhiro 71 Tomita, M. 37, 59 Tongu, M. 106 Tongu, Miki 73 Tsuge, Akihiko 114 Tsuge, Takeharu 116 Tsumoto, K. 37, 59 Tsuzisawa, T. 84

U

Ueda, Y. 44 Uezu, K. 70 Uno, Hidetaka 86 Urisu, Tsuneo 45, 86 Urushihara, M. 65 Usui, Akihito 95 Usui, Michihiko 128 Uto, Y. 77

W Wakabayashi, R. 123 Waku, Tomonori 120 Wan, Li-Jun 30 Wang, Siqian 114 Wu, Y.H. 32

Y Yabuya, Makoto 86 Yamada, Takaya 73 Yamada, Toshimichi 75 Yamamoto, K. 98 Yamamoto, T. 127 Yamamoto, Tatsuhiro 80 Yamamoto, Tatsuyuki. 73 Yamanaka, Makoto 100 Yamasaki, R. 108 Yamashita, Takefumi 69 Yang, Chung-Shi 36 Yang, F. 32 Yang, Haesik 51 Yang, M. 56

Yasuda, Takashi 100 Yokota, I. 88 Yoshida, S. 42 Yoshikiyo, Keisuke 73 Yoshimura, Hideaki 75 Yoshioka, I. 84 Yoshioka, Y. 42 Yudaska, M. 56 Yukawa, Hiroshi 125 Yunus, R. 107 Yusof, Nor Azah 103

Z Zhang, M. 56 Zhang, W. 32 Zhang, X. Q. 32 Zhang, Y. 32 Zhao, Guoxi 92

132