Low-flux electron diffraction study on body site dependence of stratum corneum structures in human skin.

For analysis of the structure of human skin stratum corneum (SC), we introduced low-flux electron diffraction (ED) and developed a new statistical analysis method for obtained ED intensity profiles. By use of this method we compared the differences in the intercellular lipid organization on the SC corneocytes collected at human forehead, cheek, and forearm by the grid-stripping method. As a result, we found a significant regional difference in the distribution of lipid hydrocarbon chain packing domains in the SC; the ring-type ED pattern with orthorhombic symmetry was more often observed in the forearm SC than in the forehead and cheek SCs. We also found that the dependence of the background electron diffraction intensity on the modulus of the scattering vector differed significantly among them. The present method for the analysis of a large number of ED patterns of noninvasively obtained SC samples could be a powerful tool to scrutinize the structural difference between the SCs under various experimental conditions.

Stratum Corneum Function: A Structural Study with Dynamic Synchrotron X-ray Diffraction Experiments.

Studies on the effectiveness of substances such as drugs and cosmetics that act on the skin require structural evidence at the molecular level in the stratum corneum to clarify their interaction with intercellular lipid and soft keratin. For this purpose, when applying the substances to the stratum corneum X-ray diffraction experiment is one of the powerful tools. To detect minute structural changes in a stratum corneum sample, using a "solution cell", dynamic synchrotron X-ray diffraction measurements were performed when applying aqueous solution of the substances to the stratum corneum: (1) It was found that a surfactant, sodium dodecyl sulfate, significantly disrupted the long-period lamellar structure. (2) To study the effects of water, structural modifications of the short-period lamellar structure and the soft keratin in corneocytes were measured as a function of time. At the initial water content of 15 wt%, the spacings of the short-period lamellar structure and the soft keratin increased toward those at the water content of 25 wt%, that is a key water content in the stratum corneum. (3) Nanoparticles composed of assembly of amphiphilic molecules are one of the leading pharmaceutical formulations. When the nanoparticles were applied, a new assembly of amphiphilic molecules originated from the nanoparticle appeared. This phenomenon suggests that the formation of the new assembly at the surface of skin is concerned with the release of the drug from the nanoparticles. (4) When ethanol was applied to the stratum corneum, only the liquid state in the intercellular lipid matrix was dissolved. After the removal of ethanol from this stratum corneum, the ordered hydrocarbon-chain packing structures appeared. From this fact we would propose that the liquid state region is the main pathway for hydrophobic drugs with a small molecular weight in connection with the so-called 500 Da rule. Here, not only the technique but also the background to these studies and the characteristic results obtained from these studies are explained.

Raman Study on Lipid Droplets in Hepatic Cells Co-Cultured with Fatty Acids.

The purpose of the present study was to investigate molecular compositions of lipid droplets changing in live hepatic cells stimulated with major fatty acids in the human body, i.e., palmitic, stearic, oleic, and linoleic acids. HepG2 cells were used as the model hepatic cells. Morphological changes of lipid droplets were observed by optical microscopy and transmission electron microscopy (TEM) during co-cultivation with fatty acids up to 5 days. The compositional changes in the fatty chains included in the lipid droplets were analyzed via Raman spectroscopy and chemometrics. The growth curves of the cells indicated that palmitic, stearic, and linoleic acids induced cell death in HepG2 cells, but oleic acid did not. Microscopic observations suggested that the rates of fat accumulation were high for oleic and linoleic acids, but low for palmitic and stearic acids. Raman analysis indicated that linoleic fatty chains taken into the cells are modified into oleic fatty chains. These results suggest that the signaling pathway of cell death is independent of fat stimulations. Moreover, these results suggest that hepatic cells have a high affinity for linoleic acid, but linoleic acid induces cell death in these cells. This may be one of the causes of inflammation in nonalcoholic fatty liver disease (NAFLD).

Simultaneous Measurements of Structure and Water Permeability in an Isolated Human Skin Stratum Corneum Sheet.

Stratum corneum (SC), the outermost layer of human skin, acts as an intelligent physicochemical interface between the inside and the outside of our body. To make clear the relationship between structure and physical barrier properties of SC, we developed a method that enables us to simultaneously acquire X-ray diffraction (XD) patterns and transepidermal water loss (TEWL) values using a spread SC sheet isolated from human skin. The synchrotron X-ray was incident on the SC sheet surface at an angle of 45° to avoid interference between the two kinds of measurements. Detailed comparison between XD and TEWL data suggested that the thermal behavior of water permeability is closely related to the thermal expansion of the lattice spacings of the hexagonal phases above 40 °C and to the existence ratio of the orthorhombic phase below 40 °C. Thus, the new method we developed can give useful information on the mechanism of water permeation in SC without ambiguity caused by separate measurements of structure and water permeability with different samples.

A Possible Percutaneous Penetration Pathway That Should Be Considered.

The intercellular lipids in the stratum corneum form structures composed of ordered phases with orthorhombic and hexagonal hydrocarbon-chain packing structures and, in addition, a structure composed of a disordered fluid phase. Although the fluid phase plays an important role in percutaneous penetration, little attention has been paid to it in the literature thus far. Recently, a method to estimate the proportion of the fluid phase within the lipids of the stratum corneum was proposed and it was shown to reach about 80%. However, since that study assumed uniform extraction of the intercellular lipids from the stratum corneum, the analysis might give rise to an overestimation of the proportion of the lipids in the fluid phase. We developed a way to investigate the proportion of the lipids in the fluid phase by treating with ethanol, into which the lipids in the fluid phase might be dominantly dissolved. From the experiment we pointed out the possibility that the proportion of the lipids in the fluid phase reached more than 50% of the whole intercellular lipids. Therefore, the fluid-phase region in the intercellular lipid matrix should be taken into account when considering the percutaneous penetration mechanism.

Effect of Solvent Dielectric Constant on the Formation of Large Flat Bilayer Stacks in a Lecithin/Hexadecanol Hydrogel.

We investigated the effect of dielectric properties of the aqueous medium on the novel type of hydrogel composed of a crude lecithin mixture (PC70) and hexadecanol (HD), in which charged sheet-like bilayers are kept far apart due to interbilayer repulsive interaction. We used dipropylene glycol (DPG) as a modifier of the dielectric properties and examined its effect on the hydrogel by synchrotron X-ray diffraction, differential scanning calorimetry (DSC), polarized optical microscopy, and freeze-fracture electron microscopy. We found that at a DPG weight fraction in the aqueous medium WDPG ≈ 0.4, the bilayer organization is transformed into unusually large flat bilayer stacks with a regular lamellar spacing of 6.25 nm and consequently disintegration of the hydrogel takes place. Semiquantitative calculation of the interbilayer interaction energy based on the Deyaguin-Landau-Verwey-Overbeek (DLVO) theory suggested that the reduction of the aqueous medium dielectric constant ε by DPG may lower the energy barrier preventing flat bilayers from coming closer together. We inferred that the size of the bilayer sheet increases because the reduction of ε promotes protonation of acidic lipids that work as edge-capping molecules.

Thermal phase transition behavior of lipid layers on a single human corneocyte cell.

We have improved the selected area electron diffraction method to analyze the dynamic structural change in a single corneocyte cell non-invasively stripped off from human skin surface. The improved method made it possible to obtain reliable diffraction images to trace the structural change in the intercellular lipid layers on a single corneocyte cell during heating from 24°C to 100°C. Comparison of the results with those of synchrotron X-ray diffraction experiments on human stratum corneum sheets revealed that the intercellular lipid layers on a corneocyte cell exhibit essentially the same thermal phase transitions as those in a stratum corneum sheet. These results suggest that the structural features of the lipid layers are well preserved after the mechanical stripping of the corneocyte cell. Moreover, electron diffraction analyses of the thermal phase transition behaviors of the corneocyte cells that had the lipid layers with different distributions of orthorhombic and hexagonal domains at 24°C suggested that small orthorhombic domains interconnected with surrounding hexagonal domains transforms in a continuous manner into new hexagonal domains.

Low-flux electron diffraction study for the intercellular lipid organization on a human corneocyte.

Human skin stratum corneum (SC) structures were investigated by electron diffraction (ED) with a very low-flux electron beam with the help of high-sensitivity detectors, the imaging plate and the CCD camera. This low-flux electron diffraction (LFED) method made it possible to minimize the unfavorable effect of electron beam damage and to give a reliable diffraction pattern from a small selected area (0.2µm(2)) on a corneocyte. Dependence of the 2-dimensional ED pattern on the size of the selected area showed that orientational correlation between lipid packing domains can persist over the area much larger than their domain size. The LFED method also allowed us to trace the detailed structural change induced by the electron beam damage. The ED diffraction peak for the lattice constant of about 4.1nm decayed in three steps. The detailed analysis of these three steps suggested that a different type of orthorhombic structure exists interacted with the well-described hexagonal and orthorhombic structures, in the process of decay resulting from electron beam damage.

Mechanism of gelation in the hydrogenated soybean lecithin (PC70)/hexadecanol/water system.

The crude phospholipid mixture (PC70) forms a homogeneous gel with hexadecanol (HD) in water, whereas the purified lipid does not. The fact that the crude material PC70 can be utilized for homogeneous gel preparation suits the cosmetic industry very well from the viewpoint of cost performance. In order to clarify the mechanism of the gelation, we investigated the structures and physicochemical properties of the PC70/HD/water system by rheometry, freeze-fracture electron microscopy, differential scanning calorimetry (DSC) and synchrotron X-ray diffraction. Our results suggested that the gelation is induced by change in bilayer morphology from closed vesicles to sheet-like structures with open edges covered by minor lipid components that are stiffened due to intercalation of HD molecules between phospholipids. The morphological change may give rise to homogeneous distribution of the bilayer sheets throughout the solution and formation of water continuum that may work as a network in the gel.

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.