Size Segregation of Gold Nanoparticles into Bilayer-like Vesicular Assembly.

Size segregation of nanoparticles with different sizes into highly ordered, unique nanostructures is important for their practical applications. Herein, we demonstrate spontaneous self-assembly of the binary mixtures of small and large gold nanoparticles (GNPs; 5/15, 5/20, or 10/20 in diameter) in the presence of a tetra(ethylene glycol)-terminated octafluoro-4,4'-biphenol ligand, namely, TeOFBL, resulting in a size-segregated assembly. The outer single layer of large GNPs forming a gold nanoparticle vesicle (GNV) encapsulated the inner vesicle-like assembly composed of small GNPs, which is referred to as bilayer-like GNV and similar to the molecular bilayer structure of a liposome. The size segregation was driven by the solvophobic feature of the TeOFBLs on the surface of GNPs. A time-course study indicated that size segregation occurred instantaneously during the mixing stage of the self-organization process. The size-segregated precursors quickly fused with each other through the inner-inner and outer-outer layer fashion to form the bilayer-like GNV. This study provides a new approach to creating biomimetic bilayer capsules with different physical properties for potential applications such as surface-enhanced Raman scattering and drug delivery.

Visualization of DNA Replication in Single Chromosome by Stable Isotope Labeling.

Among the inheritance of cellular components during cell division, deoxyribonucleic acid (DNA) and its condensate (chromosome) are conventionally visualized using chemical tag-labeled nucleotide analogs. However, associated mutagenesis with nucleotide analogs in the visualization of chromosomes is cause for concern. This study investigated the efficiency of using stable isotope labels in visualizing the replicating cultured human cell-chromosomes, in the absence of analog labels, at a high spatial resolution of 100 nm. The distinct carbon isotope ratio between sister chromatids reflected the semi-conservative replication of individual DNA strands through cell cycles and suggested the renewal of histone molecules in daughter chromosomes. Thus, this study provides a new, powerful approach to trace and visualize cellular components with stable isotope labeling.Key words: stable isotope, chromosome replication, semi-conservative replication, imaging, mass spectrometry.

Vesicle Formation by the Self-Assembly of Gold Nanoparticles Covered with Fluorinated Oligo(ethylene glycol)-Terminated Ligands and Its Stability in Aqueous Solution.

Water-stable gold nanoparticle vesicles (GNVs) with hollow interiors have attracted attention due to their great potential for biological applications; however, their preparation through the self-assembly approaches has been restricted due to the limited understanding of their critical mechanistic issues. In this paper, we demonstrate that a fluorinated tetra (ethylene glycol) (FTEG)-terminated tetra (ethylene glycol) (EG4), namely, FTEG-EG4, ligand can self-assemble with gold nanoparticles (5 and 10 nm) into GNVs with a hollow structure in THF due to the solvophobic feature of the ligand. Time-dependent studies showed that the GNVs with a closely packed surface derived from the incomplete and irregular GNVs, but not through the fusion of the GNV precursors. After dialysis in water, the assemblies retained vesicular structures in water, even though GNVs aggregated together, which was initiated by the hydrophobic interactions between the FTEG heads of the surface ligands on GNVs. This study provides a new insight into the design of novel small surface ligands to produce water-stable GNVs for biological applications.

Intrahelical Interactions in an α-Helical Coiled Coil Determine the Structural Stability of Tropomyosin.

Tropomyosin (Tpm) is a two-stranded parallel α-helical coiled-coil protein, and studying its structure is crucial for understanding the nature of coiled coils. Previously, we found that the N-terminal half of the human skeletal muscle α-Tpm (α-Tpm 140) was less structurally stable in the presence of phosphate ions than the coiled-coil protein carrier (CCPC) 140 variant with 18 mutated residues, in which all amino acid residues located at the interface between the two α-helices were completely conserved. A classical hypothesis explains that interhelical interactions stabilize the coiled-coil structure. In this study, we tested the hypothesis that the structural stability of Tpm and its variant is governed by the binding of multivalent ions that form a bridge between charged side chains located at positions b, c, and f of the heptad repeat on a single α-helical chain. We found that the structural stability of α-Tpm 140 and CCPC 140 markedly increased upon addition of divalent cations and divalent anions, respectively. We also clarified that the structural stability of the α-Tpm 140/CCPC 140 heteromeric coiled-coil molecule was governed by the stability of a less stable α-helical chain. These results demonstrated that the entire structural stability of Tpm is determined by the stability of a single α-helix. Our findings provide new insights into the study of the structure of coiled-coil proteins.

Strategy for Finely Aligned Gold Nanorod Arrays Using Polymer Brushes as a Template.

The development of a strategy for the assembly of nanoscale building blocks, in particular, anisotropic nanoparticles, into desired structures is important for the construction of functional materials and devices. However, control over the orientation of rod-shaped nanoparticles on a substrate for integration into solid-state devices remains challenging. Here, we report a strategy for the fabrication of finely aligned gold nanorod (GNR) arrays using polymer (DNA) brushes as a nanoscale template. The gold nanorods modified with cationic surface ligands were electrostatically adsorbed onto the DNA brush substrates under various conditions. The orientational behavior of the GNRs was examined by spectral analyses and transmission electron microtomography (TEMT). As a result, we found several important factors, such as moderate interaction between GNRs and polymers and polymer densities on the substrate, related to the vertical alignment of GNRs on the substrates. We also developed a purification method to remove the undesired adsorption of GNRs onto the arrays. Finally, we have succeeded in the fabrication of extensive vertical GNR arrays of high quality via the easy bottom-up process.

Two-Step Assembly of Thermoresponsive Gold Nanorods Coated with a Single Kind of Ligand.

Gold nanorods (GNRs) coated with a single kind of ligand show thermoreponsive two-step assembly to provide a hierarchical structure. The GNRs (33 nm in length × 14 nm in diameter) coated with a hexa(ethylene glycol) (HEG) derivative form side-by-side assemblies at 30 °C (TA1 ) as a steady state through dehydration. By further heating to over 40 °C (TA2 ), larger assemblies, which are composed of the side-by-side assembled units, are formed as hierarchical structures. The dehydration temperature of the HEG derivative varies depending on the free volume of the HEG unit, which corresponds to the curvature of the GNRs. Upon heating, dehydration first occurs from the ligands on the side portions with a lower curvature, and then from the ligands on the edge portions with a higher curvature. The different sized GNRs (33 × 8 and 54 × 15 nm) also show two-step assembly. Both the TA1 and TA2 are dependent on the diameter of the GNRs, but independent of their length. This result supports that the dehydration is dependent on the free volume, which corresponds to the curvature. Anisotropic assembly focusing on differences in curvature provides new guidelines for the fabrication of hierarchical structures.

Self-Priming Enzymatic Fabrication of Multiply Modified DNA.

The self-priming synthesis of multiply modified DNA by the extension of repeating unit duplex "oligoseeds" provides a source of versatile DNA. Sterically-demanding nucleotides 5-Br-dUTP, 7-deaza-7-I-dATP, 6-S-dGTP, 5-I-dCTP as well as 5-(octadiynyl)-dCTP were incorporated into two extending oligoseeds; [GATC]5 /[GATC]5 and [A4 G]4 /[CT4 ]4 . The products contained modifications on one or both strands of DNA, demonstrating their recognition by the polymerase as both template (reading) and substrate (writing). Nucleobase modifications that lie in the major groove were reliably read and written by the polymerase during the extension reaction, even when bulky or in contiguous sequences. Repeat sequence DNA over 500 bp long, bearing four different modified units was produced by this method. The number, position and type of modification, as well as the overall length of the DNA can be controlled to yield designer DNA that offers sequence-determined sites for further chemical adaptations, targeted small molecule binding studies, or sensing and sequencing applications.

Size-Defined Cracked Vesicle Formation via Self-Assembly of Gold Nanoparticles Covered with Carboxylic Acid-Terminated Surface Ligands.

The self-assembly of gold nanoparticles (GNPs) into a defined structure, particularly hollow capsule structures, provides great potential for applications in materials science and medicine. However, the complexity of the parameters for the preparation of those structures through self-assembly has limited access to critical mechanistic questions. With this in mind, we have studied GNP vesicle (GNV) formation through self-assembly by the surface modification of GNPs with low-molecular-weight ligands. Here, we successfully prepared GNVs composed of GNPs with a diameter of 30 nm by surface modification with carboxylic acid-terminated fluorinated oligo(ethylene glycol) ligands (CFLs). As the carboxylic acid has two states (protonated and deprotonated), the balance of the attraction and repulsion between GNPs covered with CFLs is tunable. Sodium carboxylate-terminated fluorinated oligo(ethylene glycol) ligands (SCFLs) provided smaller GNVs than did CFLs at 0.8 × 1011 NPs/mL. Time-course study revealed that CFL-covered GNPs quickly form small aggregates and gradually grow to larger GNVs (ca. 200 nm), but no gradual growth was observed for SCFL-covered GNPs. This result indicated that the electrostatic repulsion inhibits fusion of the small GNVs. The size of the GNVs formed with the aid of CFLs was independent of the initial GNP concentration, but the extinction spectra were concentration-dependent. Electron microscopy imaging and simulations supported the defect formation in the assemblies. These results provided new insights into the vesicle formation mechanism.

pH-Responsive Coassembly of Oligo(ethylene glycol)-Coated Gold Nanoparticles with External Anionic Polymers via Hydrogen Bonding.

Stimuli-responsive assembly of gold nanoparticles (AuNPs) with precise control of the plasmonic properties, assembly size, and stimuli responsivity has shown potential benefits with regard to biosensing devices and drug-delivery systems. Here we present a new pH-responsive coassembly system of oligo(ethylene glycol) (OEG)-coated AuNPs with anionic polymers as an external mediator via hydrogen bonding in water. Hydrogen-bond-driven coassemblies of OEG-AuNPs with poly(acrylic acid) (PAA) were confirmed by the monitoring of plasmonic peaks and hydrodynamic diameters. In this system, the protonation of anionic polymers on change in pH triggered the formation of hydrogen bond between the OEG-AuNPs and polymers, providing sensitive pH responsivity. The plasmonic properties and assembly size are affected by both the ratio of PAA to AuNPs and the molecular weight of PAAs. In addition, the attachment of hydrophobic groups to the surface ligand or anionic polymer changed the responsive pH range. These results demonstrated that the coassembly with an external mediator via hydrogen bonding provides a stimuli-responsive assembly system with tunable plasmonic properties, assembly size, and stimuli responsivity.

Yolk/Shell Assembly of Gold Nanoparticles by Size Segregation in Solution.

We demonstrate that binary mixtures of small and large gold nanoparticles (GNPs) (5/15, 5/30, 10/30, and 15/30 nm in diameter) in the presence of a glucose-terminated fluorinated oligo(ethylene glycol) ligand can spontaneously form size-segregated assemblies. The outermost layer of the assembly is composed of a single layer of small-sized GNPs, while the larger-sized GNPs are located in the interior, forming what is referred to as a yolk/shell assembly. Time course study reveals that small and large GNPs aggregate together, and these kinetically trapped aggregations were transformed into a size-segregated structure by repeating fusions. A yolk/shell structure was directly visualized in solution by X-ray laser diffraction imaging, indicating that the structure was truly formed in solution, but not through a drying process.

Reverse Size Dependences of the Cellular Uptake of Triangular and Spherical Gold Nanoparticles.

Gold nanoparticles (GNPs) show promise as both drug and imaging carriers with applications in both diagnosis and therapy. For the safe and effective use of such gold nanomaterials in the biomedical field, it is crucial to understand how the size and shape of the nanomaterials affect their biological features, such as in vitro cellular uptake speed and accumulation as well as cytotoxicity. Herein, we focus on triangular gold nanoparticles (TNPs) of four different sizes (side length 46, 55, 72, and 94 nm; thickness 30 nm) and compare the cellular internalization efficiency with those of spherical nanoparticles (SNPs) of various diameters (22, 39, and 66 nm). Both surfaces were coated with anionic thiol ligands. Inductively coupled plasma-emission spectrometry (ICP-ES) data demonstrated that TNPs with longer sides showed higher levels of uptake into RAW264.7 and HeLa cells. On the other hand, in the case of SNPs, those with smaller diameters showed higher levels of uptake in both cells. Our results support the notion of a reverse size dependence of TNPs and SNPs in terms of cellular uptake. For HeLa cells, in particular, 20-fold more efficient internalization was observed for TNPs with longer sides (72 nm side length) compared to SNPs (66 nm) with a similar surface area. These results highlight the importance of the shape of nanomaterials on their interactions with cells and provide a useful guideline for the use of TNPs.

Noncationic Rigid and Anisotropic Coiled-Coil Proteins Exhibit Cell-Penetration Activity.

Numerous cationic peptides that penetrate cells have been studied intensively as drug delivery system carriers for cellular delivery. However, cationic molecules tend to be cytotoxic and cause inflammation, and their stability in the blood is usually low. We have previously demonstrated that a rigid and fibrous cationic coiled-coil protein exhibited cell-penetrating ability superior to that of previously reported cell-penetrating peptides. Making use of structural properties, here we describe the cell-penetrating activity of a rigid and fibrous coiled-coil protein with a noncationic surface. A fibrous coiled-coil protein of pI 6.5 penetrated 100% of the cells tested in vitro at a concentration of 500 nM, which is comparable to that of previously reported cell-penetrating peptides. We also investigated the effect of cell-strain dependency and short-term cytotoxicity.

Superior cell penetration by a rigid and anisotropic synthetic protein.

Molecules with structural anisotropy and rigidity, such as asbestos, demonstrate high cell-penetrating activity but also high toxicity. Here we synthesize a biodegradable, rigid, and fibrous artificial protein, CCPC 140, as a potential vehicle for cellular delivery. CCPC 140 penetrated 100% of cells tested in vitro, even at a concentration of 3.1 nM-superior to previously reported cell-penetrating peptides. The effects of cell-strain-dependency and aspect ratio on the cell-penetrating activity of CCPC 140 were also investigated.

Synthesis of Janus-like gold nanoparticles with hydrophilic/hydrophobic faces by surface ligand exchange and their self-assemblies in water.

This study aims at the synthesis of Janus gold nanoparticles (Janus GNPs) with hydrophilic/hydrophobic faces by a simple ligand exchange reaction in an homogeneous system and at the elucidation of the self-assembled structures of the Janus GNPs in water. As hydrophilic surface ligands, we synthesized hexaethylene glycol (E6)-terminated thiolate ligands with C3, C7, or C11 alkyl chains, referred to as E6C3, E6C7, and E6C11, respectively. As a hydrophobic ligand, a butyl-headed thiolate ligand C4-E6C11, in which a C4 alkyl was introduced on the E6C11 terminus, was synthesized. The degree of segregation between the two ligands on the GNPs (5 nm in diameter) was examined by matrix-assisted laser desorption/ionization time-of fright mass spectrometry (MALDI-TOF MS) analysis. We found that the choice of immobilization methods, one-step or two-step addition of the two ligands to the GNP solution, crucially affects the degree of segregation. The two-step addition of a hydrophilic ligand (E6C3) followed by a hydrophobic ligand (C4-E6C11) produced a large degree of segregation on the GNPs, providing Janus-like GNPs. When dispersed in water, these Janus-like GNPs formed assemblies of ∼160 nm in diameter, whereas Domain GNPs, in which the two ligands formed partial domains on the surface, were precipitated even when the molar ratio of the hydrophilic ligand and the hydrophobic ligand on the surface of the NPs was almost 1:1. The assembled structure of the Janus-like GNPs in water was directly observed by pulsed coherent X-ray solution scattering using an X-ray free-electron laser, revealing irregular spherical structures with uneven surfaces.

Spatially Uniform and Quantitative Surface-Enhanced Raman Scattering under Modal Ultrastrong Coupling Beyond Nanostructure Homogeneity Limits.

We developed a substrate that enables highly sensitive and spatially uniform surface-enhanced Raman scattering (SERS). This substrate comprises densely packed gold nanoparticles (d-AuNPs)/titanium dioxide/Au film (d-ATA). The d-ATA substrate demonstrates modal ultrastrong coupling between localized surface plasmon resonances (LSPRs) of AuNPs and Fabry-Pérot nanocavities. d-ATA exhibits a significant enhancement of the near-field intensity, resulting in a 78-fold increase in the SERS signal for crystal violet (CV) compared to that of d-AuNP/TiO2 substrates. Importantly, high sensitivity and a spatially uniform signal intensity can be obtained without precise control of the shape and arrangement of the nanoscale AuNPs, enabling quantitative SERS measurements. Additionally, SERS measurements of rhodamine 6G (R6G) on this substrate under ultralow adsorption conditions (0.6 R6G molecules/AuNP) show a spatial variation in the signal intensity within 3%. These findings suggest that the SERS signal under modal ultrastrong coupling originates from multiple plasmonic particles with quantum coherence.

Gold nanoparticles coated with semi-fluorinated oligo(ethylene glycol) produce sub-100 nm nanoparticle vesicles without templates.

Gold nanoparticles (NPs) with diameters of 5, 10, and 20 nm coated with semifluorinated oligo(ethylene glycol) ligands were formed into sub-100 nm hollow NP assemblies (NP vesicles) in THF without the use of a template. The NP vesicles maintained their structure even after the solvent was changed from THF to other solvents such as butanol or CH(2)Cl(2). NMR analyses indicated that the fluorinated ligands are bundled on the NPs and that the solvophobic feature of the fluorinated bundles is the driving force for NP assembly. The formed NP vesicles were surface-enhanced Raman scattering-active capsules.

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.

Hysteresis in the Thermo-Responsive Assembly of Hexa(ethylene glycol) Derivative-Modified Gold Nanodiscs as an Effect of Shape.

Anisotropic gold nanodiscs (AuNDs) possess unique properties, such as large flat surfaces and dipolar plasmon modes, which are ideal constituents for the fabrication of plasmonic assemblies for novel and emergent functions. In this report, we present the thermo-responsive assembly and thermo-dynamic behavior of AuNDs functionalized with methyl-hexa(ethylene glycol) undecane-thiol as a thermo-responsive ligand. Upon heating, the temperature stimulus caused a blue shift of the plasmon peak to form a face-to-face assembly of AuNDs due to the strong hydrophobic and van der Waals interactions between their large flat surfaces. Importantly, AuNDs allowed for the incorporation of the carboxylic acid-terminated ligand while maintaining their thermo-responsive assembly ability. With regard to their reversible assembly/disassembly behavior in the thermal cycling process, significant rate-independent hysteresis, which is related to their thermo-dynamics, was observed and was shown to be dependent on the carboxylic acid content of the surface ligands. As AuNDs have not only unique plasmonic properties but also high potential for attachment due to the fact of their flat surfaces, this study paves the way for the exploitation of AuNDs in the development of novel functional materials with a wide range of applications.

Improving in vivo hepatic transfection activity by controlling intracellular trafficking: the function of GALA and maltotriose.

The successful control of intracellular trafficking (i.e., endosomal escape and nuclear delivery) is prerequisite for the development of a gene delivery system. In the present study, we developed an in vivo hepatic gene delivery system using a plasmid DNA (pDNA)-encapsulating lipid envelope-type nanoparticle, to which we refer as a multifunctional envelope-type nanodevice (MEND). The critical structural elements of the MEND are a DNA/protamine condensed core coated with lipid bilayers including serum-resistant cationic lipids. Intravenous administration of bare MEND represents minimal transfection activity. For the surface modification of functional devices, hydrophobic moieties were chemically attached, which are shed in the spontaneous orientation outward from the MEND surface by anchoring to the lipid bilayers. Modification of the pH-dependent fusogenic peptide GALA as an endosome escape induced transfection activity by 1 and 2 orders of magnitude. In an attempt to induce the nuclear delivery of pDNA, maltotriose, a recently characterized nuclear localization signal, was additionally modified. As a result, transfection activity further enhanced by 1 order of magnitude, and it reached to the higher level obtained for a conventional lipoplex and an in vivo jetPEI-Gal, with less hepatic toxicity. The data show that the combination of GALA and maltotriose results in a highly potent functional device that shows an enhanced endosomal escape and nuclear delivery in vivo.

Self-repairing filamentous actin hydrogel with hierarchical structure.

A chemically cross-linked filamentous actin (F-actin) gel consisting of globular actin (G-actin) as repeating units was prepared. The F-actin gel was cross-linked by covalent bonds, and the main chain is represented by the self-assembly of G-actin with a high-ordered hierarchical structure. The gel exhibited good mechanical performance with a storage modulus >1 kPa and undergoes reversible sol-gel transitions in response to changes in the salt concentration (chemical-induced sol-gel transition) as well as to shear strain (mechanical-induced sol-gel transition). Therefore, the gel exhibits self-repairing ability through dynamic polymerization and depolymerization across the structure hierarchies under repeated shear stress.