Noboru Ohta

Oriented Salts: Dimension‐Controlled Charge‐by‐Charge Assemblies from Planar Receptor–Anion Complexes

Salts, ionic compounds comprising cations (positive ions) and anions (negative ions), are essential materials for biotic activities. They are also utilized as inorganic minerals for industry. The appropriate arrangement of charged species through electrostatic interactions is a significant issue for constructing ordered nanoscale architectures in various states. For example, most inorganic, organic, and inorganic–organic hybrid salts use electrostatic interactions between ions to form organized three-dimensional (3D) crystal structures. The 3D structures defined herein include not only crystals of isomeric space groups in a cubic system but also non-isomeric crystals. Appropriate pairs of cations and anions yield ionic liquids, which are partially ordered but essentially nondimensional (0D) states. In ionic liquids, bulky geometries of both the cationic and the anionic species effectively prevent crystallization owing to significantly weaker ionic interactions. In contrast to crystals and liquids from ions, soft materials formed by electrostatic interactions between charged components have been reported as liquid crystals on the basis of ionic mesogens. 4] For example, Kato et al. reported various ionic liquid crystals comprising alkyl-substituted imidazolium salts, which afford columnar structures and have ionic conduction. Compared to such ionic mesophases, in which the locations of either cations or anions cannot be confirmed, more rigidly organized structures with a certain level of mobility in their building subunits are also useful for various applications such as ferroelectric materials. In contrast to bulky components, planar cationic and anionic molecules effectively interact with each other and form charge-by-charge assemblies composed of alternately stacking charged components. Aided by supplementary van der Waals interactions along with electrostatic and p–p interactions, dimension-controlled charge-by-charge assemblies will form not only crystals but also soft materials such as supramolecular gels, liquid crystals, and other organized structures. In comparison to p-conjugated cations, which are often based on sp-hybridized planar geometries, p-conjugated planar anionic species are required to delocalize their excess electrons, for example by depositing them in aromatic systems, to prevent them from suffering an electrophilic attack. Focusing on these perspectives, one of the strategies for forming planar anions is the complexation of electronically neutral p-conjugated anion receptors and spherical halide anions. As p-conjugated planes for associating with halide anions, BF2 complexes of 1,3-dipyrrolyl-1,3-propanediones efficiently bind spherical anions with inversion of pyrrole rings (1 and 2 ; Scheme 1 a). 8] Receptor 1 affords single crystals, which are composed of 1D columnar structures of alternately stacking chloride and bromide complexes and tetrapropylammonium (TPA) countercations, and are prepared from a hydrocarbon solvent. In contrast to these crystal states, an alkyl-substituted receptor 2 exhibits the formation of anion-responsive supramolecular octane gel, which is temporally transformed into a solution state by the addition of tetrabutylammonium (TBA) salts owing to the formation of soluble ion pairs comprising fairly aliphatic TBA cations and receptor–anion complexes. 9] Therefore, the introduction of planar cations in place of bulky TBA cations may form fine-tuned supramolecular organized structures as soft materials using p–p stacking and electrostatic interactions along with van der Waals forces. Herein, we present the [*] Y. Haketa, Prof. Dr. H. Maeda College of Pharmaceutical Sciences, Institute of Science and Engineering, Ritsumeikan University Kusatsu 525–8577 (Japan) Fax: (+ 81)77-561-2659 E-mail: maedahir@ph.ritsumei.ac.jp

Hydrophobic Molecules Infiltrating into the Poly(ethylene glycol) Domain of the Core/Shell Interface of a Polymeric Micelle: Evidence Obtained with Anomalous Small-Angle X-ray Scattering

Polymeric micelles have been extensively studied as nanoscale drug carriers. Knowing the inner structure of polymeric micelles that encapsulate hydrophobic drugs is important to design effective carriers. In our study, the hydrophobic compound tetrabromocathecol (TBC) was chosen as a drug-equivalent model molecule. The bromine atoms in TBC act as probes in anomalous small-angle X-ray scattering (ASAXS) allowing for its localization in the polymeric micelles whose shape and size were determined by normal small-angle X-ray scattering (SAXS). Light scattering measurements coupled with field flow fractionation were also carried out to determine the aggregation number of micelles. A core-corona spherical model was used to explain the shape of the micelles, while the distribution of bromine atoms was explained with a hard-sphere model. Interestingly, the radius of the spherical region populated with bromine atoms was larger than the one of the sphere corresponding to the hydrophobic core of the micelle. This result suggests that the TBC molecules infiltrate the PEG hydrophilic domain in the vicinity of the core/shell interface. The results of light scattering and SAXS indicate that the PEG chains at the shell region are densely packed, and thus the PEG domain close to the interface has enough hydrophobicity to tolerate the presence of hydrophobic compounds.

A possible regulation mechanism of water content in human stratum corneum via intercellular lipid matrix

We studied the water regulation mechanism in human stratum corneum which is composed of corneocytes and intercellular lipid matrix by the ex vivo small- and medium-angle X-ray diffraction. Under the normal condition water molecules are stored mainly in the corneocytes. When the water content increased, from the small-angle X-ray diffraction of the human stratum corneum we obtained the swelling behavior of the short lamellar lipid structure as a result of incorporating a very small amount of water into water layers between neighboring the lipid bilayers, and its diffraction peak width became narrow and turned to wide at the water content of 20-30wt%. In addition as evidence for uptake of water in the corneocytes, we observed the structural modification of soft keratins in the corneocytes from the medium-angle X-ray diffraction. Based upon these results we propose that the water content in the human stratum corneum is regulated to be at 20-30wt% so as to stabilize the short lamellar structure in the intercellular lipid matrix.

Novel method to observe subtle structural modulation of stratum corneum on applying chemical agents.

In the development of functional chemicals such as percutaneous penetration enhancers and cosmetics, the structural evidence at the molecular level in stratum corneum (SC) is highly desirable. We developed a method to observe a minute structural change of intercellular lipid matrix and corneocytes on applying the chemicals to the SC using synchrotron X-ray diffraction technique. The performance of the present method was demonstrated by applying typical chemicals, chloroform/methanol mixture, hydrophilic ethanol and hydrophobic d-limonene. From the small- and wide-angle X-ray diffraction we obtained the following results: on applying chloroform/methanol mixture the intercellular lipids were extracted markedly, on applying ethanol the intercellular lipid structure was slightly disrupted, ethanol molecules were taken into the corneocytes and in addition the pools of ethanol seem to be formed in the hydrophilic region of the intercellular lipid matrix in the SC, and on applying d-limonene the repeat distance of the long lamellar structure increased by incorporating d-limonene molecules, the intercellular lipid structure was slightly disrupted, and the pools of d-limonene were formed in the hydrophobic region of the intercellular lipid matrix in the SC.

Small-angle fibre diffraction studies of corneal matrix structure: a depth-profiled investigation of the human eye-bank cornea

In the cornea of the eye light transmission is facilitated by the regular arrangement and uniform diameter of collagen fibrils that constitute the bulk of the extracellular corneal matrix. Matrix architecture, in turn, is believed to be governed by interactions between collagen fibrils and proteoglycan molecules modified with sulfated glycosaminoglycan side chains. Here, we outline the contribution made by small-angle X-ray scattering studies of the cornea in understanding the role of sulfated glycosaminoglycans in the control of collagen architecture in cornea, and present new depth-profiled microbeam data from swollen human eye-bank corneas that indicate no significant change in collagen fibril diameter throughout the tissue, but a lower collagen interfibrillar spacing in the anterior-most stromal regions compared with the ultrastructure of the deeper cornea.

Evaluation of enamel crystallites in subsurface lesion by microbeam X-ray diffraction

Early caries lesion is a demineralization process that takes place in the top 0.1 mm layer of tooth enamel. In this study, X-ray microbeam diffraction was used to evaluate the hydroxyapatite crystallites in the subsurface lesion of a bovine enamel section and the results are compared with those obtained by transversal microradiography, a method commonly used for evaluation of tooth mineral. Synchrotron radiation from SPring-8 was used to obtain a microbeam with a diameter of 6 microm. Wide-angle X-ray diffraction reports the amount of hydroxyapatite crystals, and small-angle X-ray scattering reports that of voids in crystallites. All three methods showed a marked decrease in the enamel density in the subsurface region after demineralization. As these diffraction methods provide structural information in the nanometre range, they are useful for investigating the mechanism of the mineral loss in early caries lesion at a nanometre level.

Structure of amyloid fibrils of hen egg white lysozyme studied by microbeam X-ray diffraction

Structure of spherical aggregates formed by hen egg white lysozyme (HEWL) was studied with microbeam X-ray diffraction. Aggregates with a diameter of 50-100 microm were formed after incubation of HEWL at pH 1.6 and 60 degrees C up to 60 days. The scattering from the aggregate in solution showed a marked symmetry demonstrating it as a spherulite. A reflection at 1/0.46 nm(-1) along the fiber axis showed the presence of beta-sheets along the fiber. There were strong equatorial reflections at 1/2.4 and 1/1.2 nm(-1). The similarities to other amyloid fibers suggest that molecules are planar in the direction perpendicular to the fiber axis and beta-strands are making hydrogen bonds to neighboring molecules.

Kinetics of low pH-induced lamellar to bicontinuous cubic phase transition in dioleoylphosphatidylserine/monoolein

Recently, it has been well recognized that the modulation of electrostatic interactions due to surface charges can induce transitions between lamellar liquid-crystalline (L(α)) and inverse bicontinuous double-diamond cubic (Q(II)(D)) phases in biological lipids. To reveal their kinetic pathway and mechanism, we investigated the low pH-induced L(α) to Q(II)(D) phase transitions in 20%-dioleoylphosphatidylserine (DOPS)/80%-monoolein (MO) using time-resolved small-angle x-ray scattering and a rapid mixing method. At a final pH of 2.6-2.9, the L(α) phase was transformed completely into the hexagonal II (H(II)) phase within 2-10 s after mixing a low pH buffer with a suspension of multilamellar vesicles of 20%-DOPS∕80%-MO (the initial step). Subsequently, the H(II) phase slowly converted into the Q(II)(D) phase and completely disappeared within 15-30 min (the second step). The rate constants of the second step were obtained using the singular value decomposition analysis. On the basis of these data, we discuss the underlying mechanism of the kinetic pathway of the low pH-induced L(α) to Q(II)(D) phase transitions.

Structural analysis of cell membrane complex of a hair fibre by micro‐beam X‐ray diffraction

The cell membrane complex in the cuticle of a human hair fibre or a rat whisker is composed of three layers, that is, β, δ and β layers. The X-ray diffraction technique is a powerful tool to investigate the pathway of aqueous molecules and ions across the cuticle. Small-angle scattering experiments using a micro X-ray beam, which can be applied to a cuticle of 5 µm thickness, provide the structural information on the cell membrane complex without interference from other structures. Taking into account the variation of thickness in the δ and β layers, the overall features of the diffraction profile in a small-angle region can be explained satisfactorily. The method makes it possible to analyse the structure of β, δ and β layers without assuming an ambiguous background in the diffraction profile, and was used for the analysis of a human hair fibre and a rat whisker. In a rat whisker, the X-ray diffraction was stronger and the variation in the layer thickness smaller than in a human hair fibre. This may be due to the fact that the rat whisker had not been washed with soap or cosmetically treated, whereas the variation may depend on the lipids or the proteins that each species naturally has. It is proposed that the method represents convenient tool for quantitative analysis to estimate the thickness of δ and β layers in the cell membrane complex.

pH-Responsive and selective protein adsorption on an amino acid-based zwitterionic polymer surface

The synergistic interactions between the α-amine and the carboxylic acid in an amino acid have recently been studied as bio-based zwitterions. Here, we report a new amphiphilic polymer containing glutamic acid grafted to the end of a dodecyl polymer side chain, which contains the α-amine and the γ-carboxylic acid of the glutamic acid moiety. The polymer self-assembled into a multilayered structure in the thin film, and the glutamic acid moieties in the polymer side chains were exposed to the polymer film/water interface. Annealing the sample enhanced the formation of a well-oriented lamellar structure in the films. Due to the presence of the glutamic acid moieties at the interface, the surface charge was controllable by pH in buffer solutions, resulting in zwitterionic character at neutral pH. It has been widely accepted that zwitterionic surfaces can exhibit non-fouling for proteins. Interestingly enough, the polymer film showed charge-selective protein adsorption since the synergistic interaction between the α-amine and the γ-carboxylic acid was weaker than conventional amino acid-based zwitterionic systems. This is due to the separated state of the functional groups by a three carbon spacer.