New Insights into the Stratum Corneum Lipid Organization by X-Ray Diffraction Analysis

The characteristic 13-nm lamellar phase that is formed by lipids in the outermost layer of the skin, the stratum corneum (SC), is very important for the barrier function of the skin. To gain more insight into the molecular organization of this lamellar phase, we performed small-angle x-ray diffraction (SAXD) using various lipid mixtures mimicking the lipid composition in SC. In the SAXD pattern of each mixture, at least seven diffraction orders were observed, attributed to the lamellar phase with a repeat distance ranging from 12.1 to 13.8 nm. Using the sampling method based on the variation in repeat distance, we selected phase angles for the first six diffraction orders. Using these phase angles for the lamellar phase, a high-resolution electron density distribution could be calculated. Subsequently, from SAXD patterns of isolated SC, the electron density distribution of the lamellar phase was also calculated and appeared to be very similar to that in the lipid mixtures. This demonstrates that the lipid mixtures serve as an excellent model for the lipid organization in SC, not only with respect to the repeat distance, but also in terms of the electron density distribution within the unit cell.     

Effect of Ceramide Tail Length on the Structure of Model Stratum Corneum Lipid Bilayers

Lipid bilayers composed of non-hydroxy sphingosine ceramide (CER NS), cholesterol (CHOL), and free fatty acids (FFAs), which are components of the human skin barrier, are studied via molecular dynamics simulations. Since mixtures of these lipids exist in dense gel phases with little molecular mobility at physiological conditions, care must be taken to ensure that the simulations become decorrelated from the initial conditions. Thus, we propose and validate an equilibration protocol based on simulated tempering, in which the simulation takes a random walk through temperature space, allowing the system to break out of metastable configurations and hence become decorrelated from its initial configuration. After validating the equilibration protocol, which we refer to as random-walk molecular dynamics, the effects of the lipid composition and ceramide tail length on bilayer properties are studied. Systems containing pure CER NS, CER NS + CHOL, and CER NS + CHOL + FFA, with the CER NS fatty acid tail length varied within each CER NS-CHOL-FFA composition, are simulated. The bilayer thickness is found to depend on the structure of the center of the bilayer, which arises as a result of the tail-length asymmetry between the lipids studied. The hydrogen bonding between the lipid headgroups and with water is found to change with the overall lipid composition, but is mostly independent of the CER fatty acid tail length. Subtle differences in the lateral packing of the lipid tails are also found as a function of CER tail length. Overall, these results provide insight into the experimentally observed trend of altered barrier properties in skin systems where there are more CERs with shorter tails present.     

Recovery of Skin Barrier After Stratum Corneum Removal by Microdermabrasion

Microdermabrasion is widely used as a non-invasive cosmetic technique that has recently been adapted to selectively remove stratum corneum to increase skin permeability for transdermal drug delivery. This study measured the kinetics of skin barrier recovery after stratum corneum removal using microdermabrasion in hairless guinea pigs. The skin was abraded at two sites on each animal, one of which was allowed to recover under occlusion while the other remained non-occluded. Histological measurements showed that skin barrier properties to sulforhodamine B largely recovered within 12 h, and the stratum corneum appeared largely reformed within 24 h for both occluded and non-occluded skin. Skin electrical resistance measurements showed significant recovery of the skin barrier within 24 h. We conclude that transdermal drug delivery may occur for up to 12 h after microdermabrasion in guinea pigs; however, humans will probably have a longer recovery time due to expected slower skin healing rates.     

Resistance to Water Diffusion in the Stratum Corneum Is Depth-Dependent

The stratum corneum (SC) provides a permeability barrier that limits the inflow and outflow of water. The permeability barrier is continuously and dynamically formed, maintained, and degraded along the depth, from the bottom to the top, of the SC. Naturally, its functioning and structure also change dynamically in a depth-dependent manner. While transepidermal water loss is typically used to assess the function of the SC barrier, it fails to provide any information about the dynamic mechanisms that are responsible for the depth-dependent characteristics of the permeability barrier. This paper aims to quantitatively characterize the depth-dependency of the permeability barrier using in vivo non-invasive measurement data for understanding the underlying mechanisms for barrier formation, maintenance, and degradation. As a framework to combine existing experimental data, we propose a mathematical model of the SC, consisting of multiple compartments, to explicitly address and investigate the depth-dependency of the SC permeability barrier. Using this mathematical model, we derive a measure of the water permeability barrier, i.e. resistance to water diffusion in the SC, from the measurement data on transepidermal water loss and water concentration profiles measured non-invasively by Raman spectroscopy. The derived resistance profiles effectively characterize the depth-dependency of the permeability barrier, with three distinct regions corresponding to formation, maintenance, and degradation of the barrier. Quantitative characterization of the obtained resistance profiles allows us to compare and evaluate the permeability barrier of skin with different morphology and physiology (infants vs adults, different skin sites, before and after application of oils) and elucidates differences in underlying mechanisms of processing barriers. The resistance profiles were further used to predict the spatial-temporal effects of skin treatments by in silico experiments, in terms of spatial-temporal dynamics of percutaneous water penetration.     

Characterization of Stratum Corneum Molecular Dynamics by Natural-Abundance 13C Solid-State NMR

Despite the enormous potential for pharmaceutical applications, there is still a lack of understanding of the molecular details that can contribute to increased permeability of the stratum corneum (SC). To investigate the influence of hydration and heating on the SC, we record the natural-abundance ¹³C signal of SC using polarization transfer solid-state NMR methods. Resonance lines from all major SC components are assigned. Comparison of the signal intensities obtained with the INEPT and CP pulse sequences gives information on the molecular dynamics of SC components. The majority of the lipids are rigid at 32°C, and those lipids co-exist with a small pool of mobile lipids. The ratio between mobile and rigid lipids increases with hydration. An abrupt change of keratin filament dynamics occurs at RH = 80–85%, from completely rigid to a structure with rigid backbone and mobile protruding terminals. Heating has a strong effect on the lipid mobility, but only a weak influence on the keratin filaments. The results provide novel molecular insight into how the SC constituents are affected by hydration and heating, and improve the understanding of enhanced SC permeability, which is associated with elevated temperatures and SC hydration.     

Basic Nanostructure of Stratum Corneum Lipid Matrices Based on Ceramides [EOS] and [AP]: A Neutron Diffraction Study

The goal of this study was to investigate the nanostructure of SC lipid model membranes comprising the most relevant SC lipids such as the unique-structured ω-acylceramide [EOS] in a near natural ratio with neutron diffraction. In models proposed recently the presence of ceramide [EOS] and FFA are necessary for the formation of one of the two existent crystalline lamellar phases of the SC lipids, the long-periodicity phase as well as for the normal barrier function of the SC. The focus of this study was placed on the influence of the FFA BA on the membrane structure and its localization within the membrane based on the ceramides [EOS] and [AP]. The internal nanostructure of such membranes was obtained by Fourier synthesis from the experimental diffraction patterns. The resulting neutron scattering length density profiles showed that the exceptionally long ceramide [EOS] is arranged in a short-periodicity phase created by ceramide [AP] by spanning through the whole bilayer and extending even further into the adjacent bilayer. Specifically deuterated BA allowed us to determine the exact position of this FFA inside this SC lipid model membrane. Furthermore, hydration experiments showed that the presented SC mimic system shows an extremely small intermembrane hydration of ∼1 Å, consequently the headgroups of the neighboring leaflets are positioned close to each other.     

A Model of Solute Transport through Stratum Corneum Using Solute Capture and Release

A one-dimensional model of solute transport through the stratum corneum is presented. Solute is assumed to diffuse through lipid bi-layers surrounding impermeable corneocytes. Transverse diffusion (perpendicular to the skin surface) through lipids separating adjacent corneocytes, is modeled in the usual way. Longitudinal diffusion (parallel to the skin surface) through lipids between corneocyte layers, is modeled as temporary trapping of solute, with subsequent release in the transverse direction. This leads to a linear equation for one-dimensional transport in the transverse direction. The model involves an arbitrary function whose precise form is uncertain. For a specific choice of this function, closed form expressions for the Laplace transform of solute out-flux at the inner boundary, and for the time lag are obtained in the case that a constant solute concentration is maintained at the outer skin surface, with the inner boundary of the stratum corneum kept at zero concentration, and with the stratum corneum initially free of solute.     

Diffusion Behaviors of Fluorescence Probe Molecules Through the Stratum Corneum Layer Under Physical Stress

This study experimentally demonstrates how application of an external physical stress onto the skin membrane affects the permeation of penetrating molecules. As a proxy of active compounds, in this study, a series of fluorescence probe molecules were utilized. We observed that skin permeation could be enhanced by imparting vertical strokes from a tapping head consisting of projections onto the skin. This was confirmed with consistency from in vitro and in vivo transdermal permeation studies. After an effective physical stress was applied to the skin, the permeation depth of probe molecules remarkably increased, which was comparable to the case of topical treatment. This seems to arise from temporal disordering of the stratum corneum layer in response to the applied physical stress.     

Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures

There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixtures using a powerful experimental platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This experimental approach is utilized to probe the domain-specific sequestering and oligomerization state of α_vβ₃ and α₅β₁ integrins in bilayers, which contain coexisting liquid-disordered/liquid-ordered (l_d/l_o) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains l_d phase). Comparison with previously reported integrin sequestering data in bilayer-spanning l_o-l_d phase separations demonstrates that bilayer asymmetry has a profound influence on α_vβ₃ and α₅β₁ sequestering behavior. For example, both integrins sequester preferentially to the l_o phase in asymmetric bilayers, but to the l_d phase in their symmetric counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering.     

Gross Cystic Disease Fluid Protein 15 in Stratum Corneum Is a Potential Marker of Decreased Eccrine Sweating for Atopic Dermatitis

It is well known that eccrine sweating is attenuated in patients with atopic dermatitis (AD). We have reported by using proteome analysis that gross cystic disease fluid protein 15 (GCDFP15), a substance secreted from eccrine sweat glands, is decreased in tape-stripped stratum corneum (SC) samples from AD patients. The aim of this study was to evaluate GCDFP15 production by eccrine glands with SC samples and to assess sweating in AD. SC samples were obtained from 51 healthy control (HC) and 51 AD individuals. Sweat samples were from 18 HC and 12 AD subjects. GCDFP15 was quantified by ELISA. By immunohistochemistry, the expression of GCDFP15 in eccrine glands was examined in normal and AD skin specimens. To identify GCDFP15-producing cells, double immunofluorescence staining for GCDFP15 and S100 protein was performed in frozen sections. To address the mechanism underlying the decreased eccrine sweating in AD patients, we examined the expression of cholinergic receptor M3 (CHRM3), a receptor for acetylcholine-induced sweating, in eccrine sweat glands. The amounts of GCDFP15 in the SC extracts were significantly lower in AD than HC (P < 0.0001). The sweat samples from AD patients also had lower levels of GCDFP15 concentration (P < 0.05). Immunohistochemistry showed positive GCDFP15 staining in the eccrine gland secretory cells and the ductal and acrosyringial lumen in normal skin, but AD lacked clear staining. Immunofluorescence staining revealed that GCDFP15 was co-expressed with S100 protein, suggesting that the clear cell of eccrine glands produces GCDFP15. Finally, we found that the expression of CHRM3 was depressed in AD, suggesting contribution to the low sweating. The SC of AD patients contains a low amount of GCDFP15 due to both low sweating and low GCDFP15 concentration in the sweat. GCDFP15 in SC is a potential marker for dysregulated sweating in AD.     

Prediction of Drug Solubility in Lipid Mixtures from the Individual Ingredients

The purpose of this research was to investigate the relationship of drug solubility in a complex lipid mixture to that of the individual ingredients with the goal of substantiating a quantitative equation that can be applied in formulation development of lipid dosage forms. To this end, the solubility of four drugs, which span a large range of physicochemical properties, was evaluated in 18 lipid ingredients that cover the major lipid classes. To assess the solubility relation in complex lipid mixtures in an unbiased manner, the experiments were created as an experimental design with the ability to detect cubic curvature in the solubility-lipid composition space. The results demonstrated that for all drugs, irrespective of their significantly distinct physicochemical properties, solubility in lipid mixtures can be readily estimated as a simple weighted average of the drug solubility in the individual ingredients. This result is of great value to formulators who can minimize a large number of solubility experiments once a basis set of solubility is determined in individual lipids.     

Changes in Cholesterol Level Alter Integrin Sequestration in Raft-Mimicking Lipid Mixtures

The influence of cholesterol (CHOL) level on integrin sequestration in raft-mimicking lipid mixtures forming coexisting liquid-ordered (l_o) and liquid-disordered (l_d) lipid domains is investigated using complementary, single-molecule-sensitive, confocal detection methods. Systematic analysis of membrane protein distribution in such a model membrane environment demonstrates that variation of CHOL level has a profound influence on l_o-l_d sequestration of integrins, thereby exhibiting overall l_d preference in the absence of ligands and l_o affinity upon vitronectin addition. Accompanying photon-counting histogram analysis of integrins in the different model membrane mixtures shows that the observed changes of integrin sequestration in response to variations of membrane CHOL level are not associated with altering integrin oligomerization states. Instead, our experiments suggest that the strong CHOL dependence of integrin sequestration can be attributed to CHOL-mediated changes of lipid packing and bilayer thickness in coexisting l_o and l_d domains, highlighting the significance of a biophysical mechanism of CHOL-mediated regulation of integrin sequestration. We envision that this model membrane study may help clarify the influence of CHOL in integrin functionality in plasma membranes, thus providing further insight into the role of lipid heterogeneities in membrane protein distribution and function in a cellular membrane environment.     

Lipid Interactions of a Ciliary Membrane TRP Channel: Simulation and Structural Studies of Polycystin-2

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Comparative Molecular Dynamics Simulation Studies of Protegrin-1 Monomer and Dimer in Two Different Lipid Bilayers

Antimicrobial peptides interact specifically with the membrane of a pathogen and kill the pathogen by releasing its cellular contents. Protegrin-1 (PG-1), a β-hairpin antimicrobial peptide, is known to exist as a transmembrane monomer in a 1,2-dilauroylphosphatidylcholine (DLPC) bilayer and shows concentration-dependent oligomerization in a 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer. To examine its structure, dynamics, orientation, and interaction in membranes, we performed comparative molecular dynamics simulations of PG-1 monomer and dimer in DLPC and POPC bilayers for a total of 840 ns. The PG-1 monomer exhibits larger tilting in DLPC than in POPC due to a hydrophobic mismatch. PG-1 tilting is dependent on its rotation angle. The specific orientation of PG-1 in membranes is governed by the interactions of its aromatic residues with lipid headgroups. The calculated ¹⁵N and ¹³CO chemical shifts of Val¹⁶ in DLPC reveal that there are different sets of tilt and rotation angles that satisfy the experimental values reasonably, suggesting that more experiments are needed to determine its orientation. The dimer simulations show that the dimer interface is better preserved in POPC than in DLPC because POPC's greater hydrophobic thickness causes reduced flexibility of the C-terminal strands. Both monomer and dimer simulations show membrane thinning around PG-1, largely due to arginine-lipid interactions.     

Membrane Protein Frustration: Protein Incorporation into Hydrophobic Mismatched Binary Lipid Mixtures

Bacteriophage M13 major coat protein was reconstituted in different nonmatching binary lipid mixtures composed of 14:1PC and 22:1PC lipid bilayers. Challenged by this lose-lose situation of hydrophobic mismatch, the protein-lipid interactions are monitored by CD and site-directed spin-label electron spin resonance spectroscopy of spin-labeled site-specific single cysteine mutants located in the C-terminal protein domain embedded in the hydrophobic core of the membrane (I39C) and at the lipid-water interface (T46C). The CD spectra indicate an overall α-helical conformation irrespective of the composition of the binary lipid mixture. Spin-labeled protein mutant I39C senses the phase transition in 22:1PC, in contrast to spin-labeled protein mutant T46C, which is not affected by the transition. The results of both CD and electron spin resonance spectroscopy clearly indicate that the protein preferentially partitions into the shorter 14:1PC both above and below the gel-to-liquid crystalline phase transition temperature of 22:1PC. This preference is related to the protein tilt angle and energy penalty the protein has to pay in the thicker 22:1PC. Given the fact that in Escherichia coli, which is the host for M13 bacteriophage, it is easier to find shorter 14 carbon acyl chains than longer 22 carbon acyl chains, the choice the M13 coat protein makes seems to be evolutionary justified.     

Ligand Binding Alters Dimerization and Sequestering of Urokinase Receptors in Raft-Mimicking Lipid Mixtures

Lipid heterogeneities, such as lipid rafts, are widely considered to be important for the sequestering of membrane proteins in plasma membranes, thereby influencing membrane protein functionality. However, the underlying mechanisms of such sequestration processes remain elusive, in part, due to the small size and often transient nature of these functional membrane heterogeneities in cellular membranes. To overcome these challenges, here we report the sequestration behavior of urokinase receptor (uPAR), a glycosylphosphatidylinositol-anchored protein, in a planar model membrane platform with raft-mimicking lipid mixtures of well-defined compositions using a powerful optical imaging platform consisting of confocal spectroscopy XY-scans, photon counting histogram, and fluorescence correlation spectroscopy analyses. This methodology provides parallel information about receptor sequestration, oligomerization state, and lateral mobility with single molecule sensitivity. Most notably, our experiments demonstrate that moderate changes in uPAR sequestration are not only associated with modifications in uPAR dimerization levels, but may also be linked to ligand-mediated allosteric changes of these membrane receptors. Our data show that these modifications in uPAR sequestration can be induced by exposure to specific ligands (urokinase plasminogen activator, vitronectin), but not via adjustment of the cholesterol level in the planar model membrane system. Good agreement of our key findings with published results on cell membranes confirms the validity of our model membrane approach. We hypothesize that the observed mechanism of receptor translocation in the presence of raft-mimicking lipid mixtures is also applicable to other glycosylphosphatidylinositol-anchored proteins.     

High-Melting Lipid Mixtures and the Origin of Detergent-Resistant Membranes Studied with Temperature-Solubilization Diagrams

The origin of resistance to detergent solubilization in certain membranes, or membrane components, is not clearly understood. We have studied the solubilization by Triton X-100 of binary mixtures composed of egg sphingomyelin (SM) and either ceramide, diacylglycerol, or cholesterol. Solubilization has been assayed in the 4–50°C range, and the results are summarized in a novel, to our knowledge, form of plots, that we have called temperature-solubilization diagrams. Despite using a large detergent excess (lipid/detergent 1:20 mol ratio) and extended solubilization times (24–48 h) certain mixtures were not amenable to Triton X-100 solubilization at one or more temperatures. DSC of all the lipid mixtures, and of all the lipid + detergent mixtures revealed that detergent resistance was associated with the presence of gel domains at the assay temperature. Once the system melted down, solubilization could occur. In general adding high-melting lipids limited the solubilization, whereas the addition of low-melting lipids promoted it. Lipidomic analysis of Madin-Darby canine kidney cell membranes and of the corresponding detergent-resistant fraction indicated a large enrichment of the nonsolubilized components in saturated diacylglycerol and ceramide. SM-cholesterol mixtures were special in that detergent solubilization was accompanied, for certain temperatures and compositions, by an independent phenomenon of reassembly of the partially solubilized lipid bilayers. The temperature at which lysis and reassembly prevailed was ∼25°C, thus for some SM-cholesterol mixtures solubilization occurred both above and below 25°C, but not at that temperature. These observations can be at the origin of the detergent resistance effects observed with cell membranes, and they also mean that cholesterol-containing detergent-resistant membrane remnants cannot correspond to structures existing in the native membrane before detergent addition.     

Ligand Binding Alters Dimerization and Sequestering of Urokinase Receptors in Raft-Mimicking Lipid Mixtures

Lipid heterogeneities, such as lipid rafts, are widely considered to be important for the sequestering of membrane proteins in plasma membranes, thereby influencing membrane protein functionality. However, the underlying mechanisms of such sequestration processes remain elusive, in part, due to the small size and often transient nature of these functional membrane heterogeneities in cellular membranes. To overcome these challenges, here we report the sequestration behavior of urokinase receptor (uPAR), a glycosylphosphatidylinositol-anchored protein, in a planar model membrane platform with raft-mimicking lipid mixtures of well-defined compositions using a powerful optical imaging platform consisting of confocal spectroscopy XY-scans, photon counting histogram, and fluorescence correlation spectroscopy analyses. This methodology provides parallel information about receptor sequestration, oligomerization state, and lateral mobility with single molecule sensitivity. Most notably, our experiments demonstrate that moderate changes in uPAR sequestration are not only associated with modifications in uPAR dimerization levels, but may also be linked to ligand-mediated allosteric changes of these membrane receptors. Our data show that these modifications in uPAR sequestration can be induced by exposure to specific ligands (urokinase plasminogen activator, vitronectin), but not via adjustment of the cholesterol level in the planar model membrane system. Good agreement of our key findings with published results on cell membranes confirms the validity of our model membrane approach. We hypothesize that the observed mechanism of receptor translocation in the presence of raft-mimicking lipid mixtures is also applicable to other glycosylphosphatidylinositol-anchored proteins.     

中药药浴联合卤米松乳膏对银屑病患者角质层神经酰胺及屏障功能的影响

摘要 目的：研究中药药浴联合卤米松乳膏对银屑病患者角质层神经酰胺及屏障功能的影响。方法：本研究招募2020年3月~2021年5月在我院皮肤门诊科就诊的80例银屑病患者,所有患者经临床病理确诊寻常型银屑病。其中男性患者52例,女性患者28例,患者年龄23~72岁,平均年龄48.29±6.33岁。实验分为两组：对照组（常规药物治疗）和观察组（中药药浴联合卤米松乳膏进行治疗）。观察患者病情康复情况,据PASI评分比较银屑病患者的总体严重程度,通过DLQI评分和VAS评估患者皮肤干燥和脱屑,通过电容检测和经表皮失水评估银屑病患者皮肤的水化程度和屏障功能,通过反相-液相色谱-质谱法分析患者角质层细胞中神经酰胺Cer和Cer（[NP]/[NS]）。通过蛋白印迹检测患者病变区域VEGFR-1和VEGFR-2的蛋白表达。结果：（1）根据PASI比较银屑病患者的总体严重程度,治疗前3周,两组患者PASI评分比较无差别（P>0.05）,第5周、8周和12周观察组较对照组的PASI评分降低（P<0.05）。（2）观察组较对照组DLQI和VAS评分降低（P<0.05）。（3）观察组较对照组病患者病变皮肤的电容增大,经表皮水分降低（P<0.05）。（4）观察组组较对照组Cer含量和Cer（[NP]/[NS]）升高（P<0.05）。（5）观察组较对照组VEGFR-1和VEGFR-2的蛋白表达降低。结论：中药药浴联合卤米松乳膏通过提高银屑病患者Cer的含量和Cer（[NP]/[NS]比率,维持角质层细胞的屏障功能,从而改善患者的皮肤干燥和瘙痒,角质层神经酰胺及屏障功能。