Growth process and molecular packing of a self-assembled lipid nanotube: phase-contrast transmission electron microscopy and XRD analyses.

Phase-contrast transmission electron microscopy (PC-TEM) and quick freezing method have been combined to study the initial growing process of a self-assembled lipid nanotube in water. The PC-TEM enabled us to detect thin lamellar edge structure and the very fast growth of the newborn edge to a thin tube with high contrast. The thin tube acts as a core structure for further growth into thick complete lipid nanotube. The initially formed nanotube structure is denoted as a "core tube". The core tube has uniform wall structure that consists of five lamellar layers and the inner and outer diameters of the core tube are 130 and 180 nm, respectively. The evaluated lamellar spacing of 4.6 nm is well compatible with that measured by X-ray diffraction. We also discussed the molecular packing of the nanotube from the pitch angle determined by the PC-TEM images, X-ray diffraction pattern in wide-angle region, and IR spectroscopy. The subcell structure of the nanotube is assigned to an orthorhombic type. The twisting angle between the neighboring lipid molecules is determined as ca. 0.26 degrees for the first time; it is a crucial parameter for the formation of a lipid nanotube in chiral packing but has not been elucidated before.

Formation of self-assembled glycolipid nanotubes with bilayer sheets.

Rolled-up morphology of bilayer sheets in a self-assembled glycolipid nanotube (LNT) in water was carefully examined by using a cryogenic transmission electron microscope (cryo-TEM) with a rapid-freezing specimen-preparation technique. The LNTs were obtained under a series of self-assembly conditions: boiling of an aqueous dispersion of glycolipid N-(11-cis-octadecenoyl)-beta-D-glucopyranosylamine, subsequent gradual cooling, and incubation at room temperature for several days. Cryo-TEM images revealed that the LNT walls consist of a multilayer structure with interlayer distance of about 4.7 nm. These layers correspond to constituent lipid bilayers. From the result of precise cryo-TEM observations and analyses, we confirmed the rolled-up morphology of the lipid bilayer sheets in a complete self-assembled glycolipid nanotube.

Alignment of glycolipid nanotubes on a planar glass substrate using a two-step microextrusion technique.

We have developed a two-step microextrusion technique to align lipid nanotubes of 200 nm in diameter in parallel on planar glass substrates. This technique is useful to align self-assembled molecular nanofibers or nanotubes with diameters ranging from 100 to 300 nm. In the first step, we applied relatively large air pressure (approximately 40 hPa) onto a microcapillary filled with aqueous dispersion of lipid nanotubes to push them out. An aqueous droplet with 60 microm diameter was then extruded from the tip of the microcapillary. After one end of the lipid nanotube moved out, we changed the air pressure to be smaller, approximately 20 hPa to reduce the flow rate of the dispersion. The decrease in size of the droplet allowed us to fix the exposed end of the lipid nanotube onto the planar substrate. By dragging the microcapillary along the planar surface, we were able to align the whole nanotube onto the substrate. Using this technique, we have achieved the parallel alignment of the lipid nanotubes on the glass substrate.

FT-IR study of the interlamellar water confined in glycolipid nanotube walls.

The local hydrogen-bonding environment of water confined in glycolipid nanotubes (LNTs) was investigated by Fourier transform infrared (FT-IR) spectroscopy. Using X-ray diffraction (XRD), we estimated the thickness of an interlamellar water layer, which was confined between the bilayer membranes constructing the walls of the LNTs, to be 1.3 +/- 0.3 nm. FT-IR spectroscopic measurement of the confined water showed an obvious reduction in IR absorption in both the low-energy (around 3000 cm(-1)) and high-energy regions (around 3600 cm(-1)) of the OH stretching band as compared to bulk water. The reduction around 3000 cm(-1) indicated a decrease in the relative proportion of the water molecules with a long-range network structure due to a geometrical restriction. This agrees with the results obtained for other multilamellar systems. On the other hand, the remarkable reduction around 3600 cm(-1), which was not observed in the other systems, indicated the absence of weakly hydrogen-bonded water aggregates due to the effect of sugar headgroups.

Molecular structure of glucopyranosylamide lipid and nanotube morphology.

A series of glucopyranosylamide lipids, N-(X-octadecenoyl)-beta-D-glucopyranosylamine [X = 13-cis (1), 11-cis (2), 9-cis (3), 6-cis (4), and 9-cis,12-cis (5)] and their saturated homologue N-octadecanoyl-beta-d-glucopyranosylamine (6), which differ in the position of a cis double bond in the C18 hydrocarbon chains, have been synthesized. The effect of the cis double bond position on the chiral self-assembly of each glycolipid has been examined by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, UV, and circular dichroism (CD). The 11-cis derivative 2 was observed to self-assemble in water to form a uniform hollow cylinder structure with about 200-nm outer diameters in >98% yields. The obtained nanotubes from 2 showed the narrowest distribution of outer diameters and also gave a negative CD band around 234-236 nm, showing the largest CD intensity among the glycolipids investigated. Thus, we found that the position of a cis double bond significantly influences the homogeneity of the outer diameters as well as growth behavior of the self-assembled nanotube structures. Chiral molecular packing driven by a possible bending structure of the unsaturated glycolipids is playing a critical role in determining tubular morphology through molecular self-assembly.

Self-assembly and subsequent accumulation of lipid nanotubes at oil/water interfaces.

We utilized oil/water interfaces as a new field to produce lipid nanotubes (LNTs), which are formed by the self-assembly of lipid molecules, and possess hollow nanometer-wide cylindrical structures. Compared to the self-assembling field in bulk water, oil/water interfaces produced shorter lipids nanotubes less than 10 microm long more efficiently. In addition, we found that the oil/water interface accumulates lipid nanotubes spontaneously. This methodology is favorable to fabricate LNTs as new nano-fluidic devices, or sensors that require accumulation and alignment in two dimensions.

Confined organization of Au nanocrystals in glycolipid nanotube hollow cylinders.

Mild fabrication of anisotropic metal-lipid nanotube (LNT) nanocomposites, in which Au nanoparticles of 3-10 nm wide are organized in a glycolipid nanotube hollow cylinder, has been achieved by filling the internal channel of the LNT with HAuCl(4) aqueous solution by capillary force and subsequent photochemical reduction of [AuCl(4)](-).

A quartz crystal microbalance method for rapid detection and differentiation of shiga toxins by applying a monoalkyl globobioside as the toxin ligand.

A simple globobiosyl (Gb2) ceramide mimic carrying a monoalkyl chain (C18) was applied for a monolayer Langmuir-Blodgett (L-B) technique to detect Shiga toxins (Stxs) by a quartz crystal microbalance (QCM) method. The artificial glycolipid, synthesized from penta-O-acetyl-D-galactopyranose via a conventional glycosidation pathway, was developed at the air-water surface for the formation of the monolayer film. Then, the film was transferred onto a QCM cell surface modified with alkanethiols. Upon the addition of each of Stx-1 and Stx-2, the decrease of frequency reached saturation within 45 min at a few nanogram order per quartz cell. Binding constants (Ka) estimated for each of Stx-1 and Stx-2 showed little difference between the two toxins. On the other hand, in the presence of an artificial acrylamido Gb2 copolymer as a competitive inhibitor, the two toxins showed a large difference in the binding behavior to the L-B monolayer.