Naohiro Kameta

Soft Nanotube Hydrogels Functioning As Artificial Chaperones

Self-assembly of rationally designed asymmetric amphiphilic monomers in water produced nanotube hydrogels in the presence of chemically denatured proteins (green fluorescent protein, carbonic anhydrase, and citrate synthase) at room temperature, which were able to encapsulate the proteins in the one-dimensional channel of the nanotube consisting of a monolayer membrane. Decreasing the concentrations of the denaturants induced refolding of part of the encapsulated proteins in the nanotube channel. Changing the pH dramatically reduced electrostatic attraction between the inner surface mainly covered with amino groups of the nanotube channel and the encapsulated proteins. As a result, the refolded proteins were smoothly released into the bulk solution without specific additive agents. This recovery procedure also transformed the encapsulated proteins from an intermediately refolding state to a completely refolded state. Thus, the nanotube hydrogels assisted the refolding of the denatured proteins and acted as artificial chaperones. Introduction of hydrophobic sites such as a benzyloxycarbony group and a tert-butoxycarbonyl group onto the inner surface of the nanotube channels remarkably enhanced the encapsulation and refolding efficiencies based on the hydrophobic interactions between the groups and the surface-exposed hydrophobic amino acid residues of the intermediates in the refolding process. Refolding was strongly dependent on the inner diameters of the nanotube channels. Supramolecular nanotechnology allowed us to not only precisely control the diameters of the nanotube channels but also functionalize their surfaces, enabling us to fine-tune the biocompatibility. Hence, these nanotube hydrogel systems should be widely applicable to various target proteins of different molecular weights, charges, and conformations.

Confinement Effect of Organic Nanotubes Toward Green Fluorescent Protein (GFP) Depending on the Inner Diameter Size

Transportation, release behavior, and stability of a green fluorescent protein (GFP, 3x4 nm) in self-assembled organic nanotubes with three different inner diameters (10, 20, and 80 nm) have been studied in terms of novel nanocontainers. Selective immobilization of a fluorescent acceptor dye on the inner surface enabled us to not only visualize the transportation of GFP in the nanochannels but to also detect release of the encapsulated GFP to the bulk solution in real time, based on fluorescence resonance energy transfer (FRET). Obtained diffusion constants and release rates of GFP markedly decreased as the inner diameter of the nanotubes was decreased. An endo-sensing procedure also clarified the dependence of the thermal and chemical stabilities of the GFP on the inner diameters. The GFP encapsulated in the 10 nm nanochannel showed strong resistance to heat and to a denaturant. On the other hand, the 20 nm nanochannel accelerated the denaturation of the encapsulated GFP compared with the rate of denaturation of the free GFP in bulk and the encapsulated GFP in the 80 nm nanochannels. The confinement effect based on rational fitting of the inner diameter to the size of GFP allowed us to store it stably and without denaturation under high temperatures and high denaturant concentrations.

Controllable biomolecule release from self-assembled organic nanotubes with asymmetric surfaces: pH and temperature dependence

The release behavior of fluorescent dyes, oligo DNAs and spherical proteins from self-assembled organic nanotubes having 7-9 nm inner diameters has been studied in terms of novel nanocontainers with high-axial ratios. Both much smaller inner diameters and asymmetric inner and outer surfaces are characteristic of the nanotubes. The acid-dissociation constant (pKa) of the amino groups located at the inner surface and the thermal phase transition temperature (Tg-l) of the nanotube were evaluated based on the pH titration and variable-temperature circular dichroism (CD) spectroscopic experiments, respectively. Each guest was slowly released from both open ends of the nanotube under weak alkaline conditions (pH 8.5), as a result of the decrease in electrostatic attraction between the inner surface and the guests. Elevated temperatures above the obtained Tg-l converted the monolayer membrane of the nanotube from a solid state to a fluid one, promoting the remarkably fast release of the guests. The unique release properties of the nanotube as a nanocontainer with two terminal open ends were compared with those of liposomes that posses a closed hollow space covered with fluid bilayer membranes.

Functionalized organic nanotubes as tubular nonviral gene transfer vector

Tubular nanomaterials are expected to represent a novel nonviral gene transfer vectors due to the unique morphology and potential biological functionalities. Here we rationally constructed functionalized organic nanotubes (ONTs) for gene delivery through the co-assembly of bipolar glycolipid, arginine-lipid and PEG-lipid. The arginine- and PEG-functionalized ONTs efficiently formed complexes with plasmid DNA without aggregation, and protect DNA from enzymatic degradation; while the arginine-functionalized ONTs aggregated with DNA as large bundles. Long ONTs exceeding 1μm in length was rarely taken up into the cells, while those with a length of 400-800nm could effectively deliver plasmid DNA into cells and induce high transgene expression of green fluorescense protein. This study demonstrated the usefulness of functionalized ONT in gene delivery, and the functionalized ONT represents a novel type of tubular nonviral gene transfer vector.

Biologically responsive, sustainable release from metallo-drug coordinated 1D nanostructures

A multistep self-assembly process produced one-dimensional nanostructures that consisted of a monolayer membrane functionalized with a ligand that acted as a coordination site for an anticancer Pt complex. Control of the mode of the networks of intermolecular hydrogen bonds within the monolayer membrane of the nanostructures completely determined the morphologies of the one-dimensional nanostructures to be nanotapes having widths of 20-40 nm and nanotubes having widths of 16 nm (8 nm inner diameter and 4 nm membrane thickness). Various spectroscopic measurements and microscopic observations revealed that the ligand in a nanotape was located on the surface, whereas the ligand in a nanotube was selectively located on the inner surface of the nanochannel. We calculated the stability constants of the nanotape and nanotube with an anticancer Pt complex to be 107.81 and 106.53, respectively. The nanotape and nanotube were able to not only stably coordinate the anticancer Pt complex in Milli-Q water but also release it in phosphate-buffered saline through a ligand exchange reaction. With respect to sustainable, slow release of the drug, the nanotube, which has a nanochannel to store the drug, was superior to the nanotape.

Cisplatin-encapsulated organic nanotubes by endo-complexation in the hollow cylinder.

A bipolar glycolipid self-assembles into organic nanotubes upon its chelation with an anticancer drug cis-dichlorodiamineplatinum(II) (CDDP). The facile synthesis of glycolipid, chelation-assisted formation of the nanotubes, and efficient loading and prolonged release of CDDP demonstrate a new approach to high-axial supramolecular drug nanocarriers.

Electric moulding of dispersed lipid nanotubes into a nanofluidic device.

Hydrophilic nanotubes formed by lipid molecules have potential applications as platforms for chemical or biological events occurring in an attolitre volume inside a hollow cylinder. Here, we have integrated the lipid nanotubes (LNTs) by applying an AC electric field via plug-in electrode needles placed above a substrate. The off-chip assembly method has the on-demand adjustability of an electrode configuration, enabling the dispersed LNT to be electrically moulded into a separate film of parallel LNT arrays in one-step. The fluorescence resonance energy transfer technique as well as the digital microscopy visualised the overall filling of gold nanoparticles up to the inner capacity of an LNT film by capillary action, thereby showing the potential of this flexible film for use as a high-throughput nanofluidic device where not only is the endo-signalling and product in each LNT multiplied but also the encapsulated objects are efficiently transported and reacted.

Self-assembled organic nanotubes embedding hydrophobic molecules within solid bilayer membranes

We have carried out co-assembly of N-(11-cis-octadecenoyl)-β-D-glucopyranosylamine 1 with hydrophobic molecules, 8-anilinonaphthalene-1-sulfonate (1,8-ANS) 2 or Zn-phthalocyanine 3 in a mixed solvent of organic solvents and water to form nanotubes embedding the hydrophobic molecules. Transmission electron microscopic (TEM), fluorescence microscopic observations, and X-ray diffraction (XRD) analysis revealed the formation of nanotubes where the interdigitated bilayer membranes of 1 function as an embedded matrix for the hydrophobic molecules. We have compared the release behavior of 2 embedded in the bilayer membranes with that of 2 encapsulated inside the nanotube hollow cylinder of 60-nm inner diameter. Although the encapsulated molecules 2 proved to be slowly released from both open ends of the hollow cylinder at room temperature, the embedded ones kept staying in the bilayer membranes. Similarly, the embedded molecules 3 kept staying in the interdigitated bilayer membranes at temperatures below a thermal phase transition temperature (Tg-l = 59 °C) of the nanotubes. However, when the solid-state bilayer membranes of the nanotubes converted into a fluid bilayer membrane at temperatures above the Tg-l, the molecules 3 was instantly released. Such self-assembled nanotubes embedding hydrophobic molecules are applicable to medical diagnosis systems containing deliveries of drugs, photosensitizers, fluorescence- and spin-probes.

Chiral sensing for amino acid derivative based on a [2]rotaxane composed of an asymmetric rotor and an asymmetric axle.

A racemic [2]rotaxane, composed of an asymmetric rotor and an asymmetric axle, formed a diastereomer with an amino acid derivative, and showed an optical response for the chiral recognition.

Lipid Nanotube Encapsulating Method in Low-Energy Scanning Transmission Electron Microscopy Analyses

The lipid nanotube (LNT) encapsulating method is a rational sample fixation method that can be used to mount samples for transmission electron microscopy analyses. By employing the LNT encapsulating method in 30 kV low-voltage scanning transmission electron microscopy (LV-STEM), it is possible to record multiangle images of ferritin without using the negative staining method. We have also recorded a tilted series of high-contrast LV-STEM images and reconstructed three-dimensional images. These results show that LNTs have sufficient durability for LV electron beam, and indicate the potential of the LNT encapsulating method as a sample fixation method of LV electron microscopy.