Yasutaka Matsuo

Sub-100 nm Gold Nanoparticle Vesicles as a Drug Delivery Carrier enabling Rapid Drug Release upon Light Irradiation

Previously, we reported gold nanoparticles coated with semifluorinated ligands self-assembled into gold nanoparticle vesicles (AuNVs) with a sub-100 nm diameter in tetrahydrofuran (THF). (1) Although this size is potentially useful for in vivo use, the biomedical applications of AuNVs were limited, as the vesicular structure collapsed in water. In this paper, we demonstrate that the AuNVs can be dispersed in water by cross-linking each gold nanoparticle with thiol-terminated PEG so that the cross-linked vesicles can work as a drug delivery carrier enabling light-triggered release. Rhodamine dyes or anticancer drugs were encapsulated within the cross-linked vesicles by heating to 62.5 °C. At this temperature, the gaps between nanoparticles open, as confirmed by a blue shift in the plasmon peak and the more efficient encapsulation than that observed at room temperature. The cross-linked AuNVs released encapsulated drugs upon short-term laser irradiation (5 min, 532 nm) by again opening the nanogaps between each nanoparticle in the vesicle. On the contrary, when heating the solution to 70 °C, the release speed of encapsulated dyes was much lower (more than 2 h) than that triggered by laser irradiation, indicating that cross-linked AuNVs are highly responsive to light. The vesicles were efficiently internalized into cells compared to discrete gold nanoparticles and released anticancer drugs upon laser irradiation in cells. These results indicate that cross-linked AuNVs, sub-100 nm in size, could be a new type of light-responsive drug delivery carrier applicable to the biomedical field.

Gold Nanoparticle Arrangement on Viral Particles through Carbohydrate Recognition: A Non-Cross-Linking Approach to Optical Virus Detection

We propose a new approach to optical virus detection based on the spatial assembly of gold nanoparticles on the surface of viruses. Since JC virus-like particles (VLPs) comprise a repeating viral capsid protein that binds to sialic acid, the conjugation of sialic acid-linked Au particles with VLPs enables the spatial arrangement of Au particles on the VLP surface. This structure produced a red shift in the absorption spectrum due to plasmon coupling between adjacent Au particles, leading to the construction of an optical virus detection system. Our system depends not on the simple cross-linking of VLPs and Au particles, but on an ordered Au structure covering the entire surface of the VLPs and can be applied to various virus detection systems using the inherent ligand recognition of animal viruses.

Hydrophilic gold nanoparticles adaptable for hydrophobic solvents.

Surface ligand molecules enabling gold nanoparticles to disperse in both polar and nonpolar solvents through changes in conformation are presented. Gold nanoparticles coated with alkyl-head-capped PEG derivatives were initially well dispersed in water through exposure of the PEG residue (bent form). When chloroform was added to the aqueous solution of gold nanoparticles, the gold nanoparticles were transferred from an aqueous to a chloroform phase through exposure of the alkyl-head residue (straight form). The conformational change (bent to straight form) of immobilized ligands in response to the polarity of the solvents was supported by NMR analyses and water contact angles.

Biomimetic bi-functional silicon nanospike-array structures prepared by using self-organized honeycomb templates and reactive ion etching

We demonstrate here a creation of a novel biomimetic bi-functional surface by using dry etching of silicon with a self-organized porous polymer. The novel silicon structures with hierarchical nanospike-array structures covered with fluorocarbons were fabricated by dry etching of silicon through the porous polymer masks. The anti-reflection and superhydrophobic properties were realized due to their large surface areas and low surface energies. These masks and silicon substrates offer great advantages and are suitable for various practical applications, including high efficiency solar cells.

Oligosaccharide‐Mediated Nuclear Transport of Nanoparticles

The transport of nanoparticles into the cellular nucleus is a potentially important technique because it can open the way to a wide range of applications, including the sequence-specific detection of genomic DNA, efficient DNA transfection, and the specific entry of drugs into the nucleus. It has been reported that the nuclear import of proteins larger than 40 kD does not occur by passive diffusion. Similarly, the nuclear import of macromolecules or particles is strictly regulated. Therefore, the nuclear import of nanoparticles that contain gold nanoparticles and quantum dots has been achieved by coating the surface with classical nuclear localization signals (NLS), that is, short, highly positively charged peptides. However, the problem remains that positively charged particles can interact with serum protein, resulting in rapid clearance from the plasma compartment. Because the cationic NLS interacts with negatively charged DNA, NLS peptides do not work as efficient signals for transport of DNA into the nucleus; this implies that the use of peptide-based cationic NLS might be limited when using DNA-displaying nanoparticles. Monsigny et al. have shown that sugars can also work as nuclear localization signals. The neoglycoproteins, BSA (bovine serum albumin)–glucose, BSA–mannose and BSA–fucose are rapidly transported into the nucleus of HeLa cells, whereas BSA without chemical modifications is not. Because carbohydrates normally show high biocompatibility and water solubility, they are suitable for use in the modification of synthetic carriers and nanoparticles. Previously, however, only the application of these carbohydrate signals to the nuclear import of proteins was examined, and there are no reports on the effectiveness of carbohydrate signals for the nuclear import of artificial materials, such as nanoparticles and polymers. Previous reports that used BSA have focused only on monosaccharides as a signal. In this paper, we expand the varieties of carbohydrates tested from monosaccharides to oligosaccharides in the search for an efficient signal that is applicable to the nuclear import of nanoparticles. Herein, we present our unique finding that nanoparticles (quantum dots) that display oligo a-glucopyranoside on their surface are readily transported into the nucleus of digitonin-permeabilized HeLa cells. Semiconductor QDs have a diameter of several nanometers and their specific transport inside the cell can be readily achieved through the display of multiple ligands on their surface. As far as we know, this is the first report to describe the import of nanoparticles into the nucleus without the use of cationic NLS. Because BSA that has been substituted with a-glucopyranoside has been reported to be efficiently transported into the cell nucleus, we synthesized neoglycolipids that contain various carbohydrates comprised of different numbers of glucose units (Scheme 1). In addition to a-monoglucopyranoside–lipid 3, we synthesized maltose(Glca1-4Glc)–lipid 4, maltotriose(Glca1-4Glca1-4Glc)–lipid 5, and panose(Glca1-6Glca1-4Glc)–lipid 6. The cellotriose(Glcb1-4Glcb1-4Glc)–lipid 7 is a trisaccharide that is composed of only a b-linked glucose, thus lipid 7 was used as a control to a-linked glucose. Neoglycolipids were synthesized based on a previously described method 11] by starting from fully acetylated carbohydrates, hexa(ethyleneglycol) and 11-bromoundecene. CdTe nanocrystals that had been stabilized with mercaptopropionic acid (MPA) were prepared in water as described by Yang et al. Sugar-displaying CdTe QDs were synthesized by surface exchange from MPA to the neoglycolipid in water (Figure 1A), and then purified by using spin filtration. Ligand exchange occurring on the surface of the QDs was confirmed by MALDI-ToF mass spectrometry. Ligands immobilized on inorganic nanoparticles, such as QDs, were detached from the surface during the laser deposition process, and the mass corresponding to the molecular weight of neoglycolipids was clearly detected (Figure 1B). Furthermore, the display of sugars on the QDs was visually confirmed by trapping on a lectin-immobilized column. For example, maltotriose 5–QDs were specifically trapped on a ConA (concanavalin A; a-mannose and a-glucose specific) agarose column, whereas they were not trapped on WGA (wheat germ agglutinin; GlcNAc specific) or LCA (lens culinaris agglutinin; branched-fucose specific) agarose columns (data not shown). We explored the interactions between sugar-displaying CdTe QDs and digitonin-permeabilized HeLa cells. Digitonin treatment causes partial damage to the plasma membrane and increases the permeability of cells. Digitonin permeabilization has been used often in the study of biochemical processes that are related to the import and export of nuclear proteins. Although live cells become semi-intact upon digitonin treatment due to the leakage of cytoplasmic proteins through the plasma membrane, the nuclear membrane is left intact. Import buffer (pH 7.3, 20 mm HEPES, 110 mm KOAc, 5 mm NaOAc, 2 mm MgACHTUNGTRENNUNG(OAc)2, 0.5 mm EGTA) that contained the sugar-displaying CdTe QDs (0.6 mgmL , [a] Dr. K. Niikura, Dr. Y. Matsuo, Dr. K. Ijiro Research Institute for Electronic Science, Hokkaido University N21W10, Kita-ku, Sapporo 001-0021 (Japan) Fax: (+81)11-706-9361 E-mail : kniikura@poly.es.hokudai.ac.jp ijiro@poly.es.hokudai.ac.jp [b] S. Sekiguchi, T. Nishio Department of Chemistry, Hokkaido University N21W10, Kita-ku, Sapporo 001-0021 (Japan) [c] T. Masuda, Dr. H. Akita, Dr. H. Harashima Faculty of Pharmaceutical Science, Hokkaido University N12 W6, Kita-Ku, Sapporo 060-0812 (Japan) [d] Dr. K. Kogure Department of Biophysical Chemistry, Kyoto Pharmaceutical University Misasagi-Nakauchicho 5, Yamashinaku, Kyoto 607-84142 (Japan)

Accumulation of O-GlcNAc-Displaying CdTe Quantum Dots in Cells in the Presence of ATP

Lectins are carbohydrate-binding proteins that are responsible for trafficking, cellular adhesion, and signaling. Recently, it was found that several chaperone proteins also have lectin-like activity. For example, calnexin and calreticulin, which are chaperones in the endoplasmic reticulum (ER), bind to the terminal glucose residue in N-glycans of glycoproteins so as to induce correct protein folding. Another group of carbohydratebinding chaperones is the heat shock protein 70 (HSP70) family. S5ve and co-workers showed that the N-acetylglucosamine (GlcNAc)-specific protein has a molecular weight of 70 kDa, thus they named the protein carbohydrate-binding protein 70 (CBP70). 6] Minic et al. reported the first evidence that an HSP70-like protein specifically binds to the GlcNAc residue. Recently, Lefebvre and co-workers directly proved that the O-GlcNAc-specific protein is a member of the HSP70 family by using an MS-based proteomics approach. They also found that the GlcNAc-directed lectin activity of HSP70 can be modulated by glucose availability and utilization. O-GlcNAc modifications are ubiquitous in eukaryotes where they cause rapid cycling in response to various cellular signals. However, the function of O-GlcNAc is not fully understood. Therefore, Lefebvre’s finding gave new insights into the meaning of the OGlcNAc modification of proteins. We focused on this chaperone–carbohydrate interaction to monitor the behavior of cellular chaperones. As the concentration of chaperones is known to rise in response to various forms of cellular stress, a sugar-based chaperone probe would offer a novel and simple tool for cellular-stress imaging. In this paper, we present the synthesis of sugar-displaying semiconductor quantum dots (QDs) and discuss the behavior of the chaperone-related GlcNAc-displaying QDs in cells. Semiconductor QDs are an important tool for tracking various biomolecules in vitro due to their resistance to photobleaching. Apart from their emission properties, QDs offer two advantages when used as a scaffold: first, QDs provide an excellent platform for the display of multivalent carbohydrates as a mimic of naturally occurring proteins; second, the size of QDs is similar to that of conventional proteins, thus the sugar-displaying QDs would behave similarly to glycoproteins. 24] Several types of sugar-modified QDs have been reported. Chaikof et al. synthesized glycopolymer-coated QDs utilizing the strong binding of a biotin-terminated lactose-bearing polymer with avidin-coated QDs, while Penad5s et al. have explored the synthesis of various gold-based glyconanoparticles as bioactive probes. They also reported the one-step synthesis of “glyco-quantum dots” displaying Le or maltose on cadmium sulfide (CdS) nanoparticles. Here, we synthesized various ACHTUNGTRENNUNGneoglycolipid-coated CdTe QDs by using a surface-exchange method and investigated their behavior in digitonin-treated HeLa cells with or without ATP.

Dissecting the Few-Femtosecond Dephasing Time of Dipole and Quadrupole Modes in Gold Nanoparticles Using Polarized Photoemission Electron Microscopy

Dipole and quadrupole modes are the two lowest orders of localized surface plasmon resonance (LSPR) eigenmodes in metallic nanoparticles. Of these two modes, the quadrupole mode is forbidden for symmetric metallic nanoparticles excited by linearly polarized light at normal incidence. Here, we demonstrate excitation of the quadrupole mode in symmetrical gold (Au) nanoblocks shined with s-polarized light at oblique incidence. In particular, we probe the near-field LSPR in Au nanoblocks using photoemission electron microscopy (PEEM) and find that at oblique incidence, the dipole and quadrupole modes can be selectively excited, in terms of near-field enhancement, by manipulating the light polarization state. More importantly, by time-resolved PEEM measurements, we experimentally demonstrate that the quadrupole mode in symmetrical Au nanoblocks has longer dephasing time than that of the dipole mode.

Low pH-triggered model drug molecule release from virus-like particles.

The essential properties required for carriers in a drug-delivery system are not only active targeting and cell entry, but also retention and release into the target cells of the drug molecules. Many techniques for appending controllable release functions at low pH or in a reducing environment to synthetic drug carriers, such as liposomes, polymers, and nanogels, have been reported. Such synthetic vehicles, however, possess low targeting ability, and the external display of specific ligands for recognition by cell surface receptors is required. Virus-like particles (VLPs), which consist of viral proteins without viral nucleic acids, are ideal drug carriers due to their inherent targeting potential and efficient cellular uptake. The VLPs derived from JC virus can be transfected into mammalian cells with high efficiency by receptor-mediated endocytosis in the same manner as the native virus. Many studies have been carried out to use the VLPs as nanomaterials. However, the concept of “controlled drug release” has not yet been introduced to the VLPs. VLP transport proceeds according to the life cycle of the native virus; thus drugs cannot be released at a particular stage during intracellular transport, such as in the endosome or cytosol. Fabrication of a conceptual path to encapsulation of drug molecules and controlled release at the desired position in cells widens the possibility of using VLPs as drug carriers. Herein, we first present “drug-releasing VLPs” in which the release mechanism is triggered by changes in pH based on hexahistidine motif (His6) affinity (Figure 1 A). The drug-releasing VLPs we have developed are composed of two viral proteins (VPs), a major coat protein, VP1, and inner core protein, VP2. VP2 was used as an anchoring molecule to display His6 tags inside the VLPs. The His6 tag offers specific and reversible attachment of the drug molecules. Because the pKa of the histidine residue is around 6.5, the protonation of histidines at a lower pH reduces the His6 affinity; this leads to the pH-mediated release of the attached molecules. The JC VLPs have apertures of around 1 nm, through which small drug molecules can enter and escape. As a first step, we investigated the incorporation of proteins into the JC VLPs by using green fluorescence protein (GFP), which can be used as a reporter protein (Figure 1 B). GFP was fused to the N terminus of VP2 (GFP–VP2) and co-expressed with VP1 in Escherichia coli. The expressed VP1 and GFP–VP2 can associate with each other and form GFP-incorporated VLPs (VLP–GFPs) inside E. coli. The resultant VLP–GFPs were purified by density gradient centrifugation and analysed by SDS-PAGE and immunoblotting (see Supporting Information). On average, 1.5 L of E. coli culture provides 750 mg of crude protein, which consists of E. coli lysate, and a final yield of 2 mg of purified VLPs. The structures of purified VLP–GFPs were observed by liquid atomic force microscopy (AFM) and scanning transmission electron microscopy (STEM) (Figure 2). VLP–GFPs had a spherical shape of around 40–50 nm in diameter, similar to the VLPs consisting solely of VP1, though the surface of the VLP– GFPs looked flatter than that of VLPs (Figure 2 A and B). This subtle surface difference might be attributed to conformational changes of the VLPs that arise from VP2 binding. In the STEM images, the white areas are negatively stained, and the dark areas are unstained; this suggests the presence of a protein. Compared with VLPs, the interior of the VLP–GFPs appeared less stained (Figure 2 C and D). Though not conclusive, Figure 1. Schematic illustration of drug-releasing VLPs. A) The encapsulation and low pH-triggered release of drug molecules from the VLPs by using His6-tag affinity. B) Incorporation of a peptide/protein into the VLPs by using VP2 as an anchoring molecule. PDBID = 1SVA.

Water transport mechanism through open capillaries analyzed by direct surface modifications on biological surfaces

Some small animals only use water transport mechanisms passively driven by surface energies. However, little is known about passive water transport mechanisms because it is difficult to measure the wettability of microstructures in small areas and determine the chemistry of biological surfaces. Herein, we developed to directly analyse the structural effects of wettability of chemically modified biological surfaces by using a nanoliter volume water droplet and a hi-speed video system. The wharf roach Ligia exotica transports water only by using open capillaries in its legs containing hair- and paddle-like microstructures. The structural effects of legs chemically modified with a self-assembled monolayer were analysed, so that the wharf roach has a smart water transport system passively driven by differences of wettability between the microstructures. We anticipate that this passive water transport mechanism may inspire novel biomimetic fluid manipulations with or without a gravitational field.

DNA-templated plasmonic Ag/AgCl nanostructures for molecular selective photocatalysis and photocatalytic inactivation of cancer cells

Silver halide (AgX, X = Cl, Br, I)-based materials represent an emerging class of heterogeneous photocatalysts. Despite progress in the synthesis of carrier-separated AgX-based photocatalysts, a number of issues remain unaddressed, including complicated synthesis, unfavorably large size and therefore poor photocatalytic performance of the resultant structures. Here we show the one-step DNA-programmable synthesis of Ag/AgCl nanostructures that takes only approximately 1 min for photocatalytic application. The optimal DNA-encapsulated structures show DNA sequence-specific sizes down to less than 40 nm with a Ag/AgCl composition ratio of 2 : 1, affording a vastly increased surface area and higher photocatalytic activity than any Ag/AgX nanostructures reported previously by over two orders of magnitude. From a physical standpoint, importantly, the plasmonic nanostructured silver in Ag/AgCl accelerates the photocatalytic reaction in terms of fast electron injection to AgCl, leading to enhanced hole-electron separation and high-performance photocatalysis under visible light. To test the effect of DNA encapsulation on the Ag/AgCl nanostructures, both positively and negatively charged organic compounds serve as the model pollutants to assess their photocatalytic selectivity. Our results show that the photodecomposition of the positively charged compounds obeys a first-order rate law, whereas the negatively charged compound is decomposed with zero-order kinetics. This comparison offers a mechanistic insight into reaction kinetics on the DNA-encapsulated photocatalyst. We further find that the DNA-encapsulated Ag/AgCl photocatalysts are robust and can be recycled. To extend the applicability of the Ag/AgCl nanostructures, their use in the efficient photocatalytic inactivation of cancer cells is also demonstrated for the first time, opening up a new avenue to daylight-based theranostics.