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«Tatsuyuki Yoshii 2014 Preface and Acknowledgements The studies presented in this dissertation have been carried out under the direction of Professor ...»

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Development of functional biomaterials by self-assembled nanostructures.

Tatsuyuki Yoshii

2014

Preface and Acknowledgements

The studies presented in this dissertation have been carried out under the direction of Professor

Itaru Hamachi at the Department of Synthetic Chemistry and Biological Chemistry, Graduate

School of Engineering, Kyoto University, from April 2005 to September 2014. The study is focused

on the development of functional biomaterials by self-assembled nanostructures.

My heartfelt appreciation goes to Prof. Itaru Hamachi whose comments, suggestions, and encouragement were of inestimable value for my study. I am also indebt to Associate Prof. Masato Ikeda for his valuable and helpful advice, discussions and encouragement. I extend my sincere gratitude to Prof. Akio Ojida, Associate Prof. Shigeki Kiyonaka, Associate Prof. Shinya Tsukiji, Assistant Prof. Yousuke Takaoka, for their helpful suggestions.

I wish to acknowledge Prof. Kenji Urayama for technical help for rheological experiments. I also wish to acknowledge Prof. Kenji Matsuda and Assistant Prof. Takashi Hirose for their help with DLS analysis. I also wish to acknowledge Prof. Kazunari Akiyoshi, Dr. Sadaatsu Mukai, Prof.

Shiyoshi Yokoyama and Dr. Hiroyuki Aoki for technical help for CLSM imaging.

I thank the past and present members of Hamachi laboratory for their suggestions and cooperation, and with whom I shared enjoyable time. In particular, I express my appreciation to Assistant Prof. Hiroshi Tsutsumi, Assistant Prof. Takashi Sakamoto, Assistant Prof. Hiroshi Nonaka. I am also grateful to Dr. Takahiro Kohira, Dr. Yoshiyuki Ishida, Dr. Hangxiang Wang, Dr.

Harunobu Komatsu, Dr. Shohei Fujishima, Dr. Assistant Prof. Daishiro Minato, Dr. Keigo Mizusawa, Dr. Takahiro Hayashi, Dr. Rui Kamada, Dr. Rika Ochi, Dr. Hajime Shigemitsu, Dr.

Tomonori Tamura, Dr. Shohei Uchinomiya, Dr. Kazuya Matsuo, Dr. Yasutaka Kurishita, Mr.

Keishi Kiminami and Mr. Ryosuke Yasui.

I also wish to express mu gratitude to Ms. Ikuyo Miyamae for their help with official business, and I would like to thank JSPS (Japan Society for the Promotion of Science) Young Scientist Fellowship for financial support.

Finally, I wish to express mu deepest gratitude for my parents, Yasuyuki Yoshii and Kisako Yoshii who have supported mu education and encouraged me affectionally.

September 2014 Tatsuyuki Yoshii Table of contents General Introduction 1 Chapter 1 Montmorillonite-Supramolecular Hydrogel Hybrid for Fluorocolorimetric Sensing of Polyamines 25 Chapter 2 Two-Photon-Responsive Supramolecular Hydrogel for Controlling Materials Motion in Micrometer Space 56 Chapter 3 Reversible Assembly/Disassembly of Nanoprobes for Turn-on Fluorescent Imaging of Endogenous Proteins in Live Cells 90 List of Publications

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Introduction Molecular self-assembly relies on a spontaneous association of small molecules driven by non-covalent interactions, such as hydrogen bond, π-π interaction, hydrophobic interaction, and electrostatic interaction1. Through molecular self-assembly, one can construct various well-ordered nano- and micro-structures including micelles, vesicles, nanofibers, and nanotubes by designing the molecular components (Figure 1). Self-assembled nanostructure have various unique features. (1) Spectroscopic Change: Some designed π-conjugated molecules change their spectroscopic properties depending on self-assembled state.2 There are several types of spectral change such as fluorescence quenching or increase and color change. (2) Nano-space: Self-assembled nanostructures provide a unique nanospace different from solvent environment. Especially in water, the inner space of the nanostructure is generally hydrophobic and is protected from water or other hydrophilic molecules. (3) Reversible Formation and deformation: Since the driving forces for the formation of self-assembled nanostructure are non-covalent interactions, the structure can be formed reversibly. Thus, the formation of nanostructure can be controlled by the external stimuli.3 (4) Multivalent effect: The molecules are highly condensed in the nanospace through molecular self-assembly. Thus the nanostructure shows strong multivalent effects. Owing to these four features, the supramolecular materials based on molecular self-assembly hold great promise for a wide range of biological applications such as tissue engineering, biomolecular detection/imaging, and controlled drug release. However, there still remain significant challenges to create and functionalize the supramolecular nanostructure, due to the difficulty to control the self-assembly in aqueous media.

In this introduction section, I summarize the recent progress in the self-assembled nanostructure

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of small-molecules, which have emerged as new functional biomaterials for a variety of potential applications.

Figure 1. Schematic illustration of aggregate formation by amphiphilic small molecules in aqueous solution.

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Outstanding Researches to Explore Chemistry and Biology Using Self-assembled Nanostructure Biomolecular Sensing Biomolecules such as proteins, nucleic acids, lipids, sugars and other organic and inorganic molecules have distinct functions to maintain their homeostasis. The concentration and subcellular distribution of these biomolecules are precisely controlled in biological systems. To understand the biological system, the sensing and imaging of the biomolecules is essential. Several researchers have utilized the self-assembled nanostructures to detect come of the important biomolecules.





In biomolecular sensing, the high-throughput technology is attractive. By using the properties of supramolecular hydrogel,4 Hamachi et al. developed a semi-wet sensor-chip for biomolecules.5 The supramolecular hydrogel can immobilize proteins, enzymes, or artificial receptors without loss of their activities (Figure 2A). Furthermore, they can be equipped with unique fluorescent read-out systems such as environmentally-sensitive fluorescence enhancement in supramolecular hydrogel.

For example, a substrate for a protease bearing an environmentally sensitive dye was embedded in a supramolecular hydrogel. When the protease cleaves the peptide bond of the substrate, the resultant dye translocates from aqueous phase to the hydrophobic domain of the nanofiber, resulting in the increase of the fluorescence intensity (Figure 2B, C). A fluorescence resonance energy transfer (FRET)-based read out system can be also constructed in the supramolecular hydrogel by embedding the FRET acceptor in the nanofiber, which allowed us to estimate the enzyme activity more precisely by taking advantage of ratiometric detection.

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Figure 2. (A) Immobilization of various functional molecules to supramolecular hydrogel.

(B) Fluorescence spectral change of supramolecular gel containing enzymatic substrate upon addition of chymotrypsin. (C) Schematic illustration of the fluorescence dye redistribution upon enzymatic cleavage.

A group of fluorogenic molecules show strong fluorescence in self-assembled state but non-emissive in monomer state.2b,6 Such a phenomenon was called aggregation-induced emission (AIE). The main reason for the fluorescence increase is believed to be the restriction of intramolecular bond rotations. Kikuchi et al. reported an AIE based probe for detection of Sirt1 activity (Figure 3).7 The enzymatic deacetylation of K(Ac)PS-TPE triggers the electrostatic interaction between the anionic sulphonate and cationic lysine and automatically leads to fluorescence enhancement. Since the fluorescence increase of the probe was restrained in the

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presence of an HDAC inhibitor, they can also perform inhibitor assays by the probe. Thus, their prob may be valuable to the field of epigenetics and drug discovery.

Figure 3. (A) Enzymatic deacetylation of K(Ac)PS-TPE to form KPS-TPE with HDAC.

(B) Schematic representation of the aggregation-induced fluorescence enhancement of K(Ac)PS-TPE by HDAC reaction.

Fluorescence Imaging Fluorescence imaging is a powerful tool for visualizing biomolecules in a real-time manner with high spatial resolution. To achieve the selective imaging, switching of fluorescence in response to a reaction or binding of target molecule is needed.8 Exploitation of AIE described above is one of the promising strategies for the development of Turn-ON fluorescence imaging probes. Liu et al. reported the caspase-3/-7 probe via conjugation of a hydrophilic DEVD peptide sequence and a hydrophobic AIE fluorogen (Ac-DEVDK-TPE, Figure 4A).9 The probe is soluble in water and nonfluorescent. The specific cleavage of DEVD by caspase-3/-7 induces aggregation of the hydrophobic AIE residues and then the fluorescence turns on. This probe is capable of detecting caspase-3/-7 activities even in living cells (Figure 4B), and useful for real-time apoptosis imaging and in situ apoptosis-related drug screening.

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Figure 4. (A) Schematic illustration of monitoring of cell apoptosis by AIE-based caspase probe.

(B) Real-time fluorescence images showing staurosporine-induced apoptotic process of MCF-7 cells with Ac-DEVDK-TPE.

Contrary to AIE, many fluorescence dyes shows quenched fluorescence in the self-assembled state. Hamachi et al reported Turn-ON fluorescence probes for protein detection by using fluorescence quenching in the self-assembled state and fluorescence enhancement after disassembly.10 Amphiphilic molecules containing hydrophobic fluorescence dye and hydrophilic protein ligand assemble into the nanosphere in water. In the self-assembled state, the fluorescence of the dye was significantly quenched. In contrast, in the presence of target protein, the probe disassembles to enhance the drastic fluorescence change (Figure 5A). Furthermore, they extend this strategy for the cell-surface protein imaging by converting fluorophores to the more hydrophilic

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ones such as fluorescein or rhodamine.11 In this case, by introducing a hydrophobic module near the fluorophore, a new disassembly-driven turn-on nanoprobe was developed. They succeeded in imaging cancer-specific biomarkers such as the folate receptor (FR) and transmembrane-type carbonic anhydrases (CAs) under live cell conditions (Figure 5B). Furthermore, a cell-based inhibitor screening system for CAs under hypoxic live cell conditions was successfully demonstrated. An advantage of the probe is that the strategy can be applied to detection of the non-enzymatic proteins.

Figure 5. (A) Specific protein detection with disassembly-driven Turn-On fluorescent probes.

(B) Cell-surface protein imaging with disassembly-driven Turn-On fluorescent probes.

MRI Imaging Magnetic resonance imaging (MRI) is a promising technique for the visualization of biomolecules because of the noninvasive manner and utility for the deep tissue. The most widely used one is 1H-MRI. However, 1H-MRI has the limitation derived from low contrast-to-noise ratio because of the large background signals from water protons. By contrast, because 19F-MRI is highly

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sensitive and fluoride-containing molecules are not found in animal body, it has been paid much attention. Hamachi et al. reported aunique strategy to detect specific proteins with Turn-ON type

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The basis of the idea is that the 19F NMR signal is broadened and attenuated in the self-assembled state, but sharpens and recovers on their disassembly upon recognition by proteins (Figure 6B). As the signal response is derived from specific protein–ligand interactions, this strategy is applicable to the detection of both enzymes and non-enzymatic proteins.

Figure 6. (A) Schematic representation of the specific protein detection with disassembly-driven Turn-On 19F NMR probes.

(B) Turn-On 19F NMR signal of disassembly-driven Turn-On 19F NMR probe in the presence or absence of human carbonic anhydrase (hCA).

Controlled Release Controlled drug release has gained much attention because of the enhanced efficacy and economical standpoint. Supramolecular nanostructure can entrap drugs in the hydrophobic space.

The entrapment is effective for protecting from the nonspecific enzymatic degradation and avoiding undesired side-effect. The stimuli-responsive properties of the nanostructures are suitable to create a functional career for controlled release.

Thayumanavan et al. reported that dendritic amphiphilic nano-containers can be disassembled

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in stimuli-responsive manner.13 As shown in Figure 7A, the nanostructure formed by biotin-incorporated dendrimer disassembled upon the addition of the avidin through ligand-receptor interaction due to the change of the hydrophilic-lipophilic balance.13a Consequently, the guest molecules are released (Figure 7B). By altering the structure of amphiphile, they created the nano-container responsive to other stimuli such as photo-irradiation and enzymatic cleavage. 13b, c Figure 7. (A) Schematic illustration of protein-ligand binding-induced disassembly of dendritic micellar assemblies and resultant guest release. (B) Protein-induced release of the guest molecules from the dendritic micellar nano-containers.

Several researchers have reported covalent modifications of drugs to form nanostructures for controlled release. Cui et al. reported peptide nanofiber modified with anticancer drug such as camptothecin and taxol (Figure 8).14 The disulfide linker of the drug-peptide conjugate cleaves in the presence of glutathione, a reducing agent that exist with high concentration in cytosol. This strategy has the strong advantage for high drug loading compared to the non-covalent encapsulation.

Xu et al reported that the drug containing gelator shows unexpected isozyme selectivity. The conjugation of D-amino acids to naproxen, a nonsteroidal anti-inflammatory drug, afforded the supramolecular hydrogel. In addition, the obtained molecule showed high selectivity toward cyclooxygenase-2 (COX-2) than COX-1.15

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Figure 8. Schematic illustration of the degradation of the Drug Amphiphile (DA).



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