Model Skin Lipid Research Articles

Effects of imidazolium ionic liquids on skin barrier lipids – Perspectives for drug delivery

Ionic liquids (ILs) have great potential to facilitate transdermal and topical drug delivery. Here, we investigated the mechanism of action of amphiphilic ILs 1-methyl-3-octylimidazolium bromide (C8MIM) and 3-dodecyl-1-methylimidazolium bromide (C12MIM) in skin barrier lipid models in comparison to their complex effects in human skin. C8MIM incorporated in a skin lipid model was a better permeation enhancer than C12MIM for water and model drugs, theophylline and diclofenac. Solid state 2H NMR and X-ray diffraction indicated that both ILs prefer the cholesterol-rich regions in skin lipids without significantly perturbing their lamellar arrangement and that C8MIM induces the formation of an isotropic lipid phase to a greater extent compared to C12MIM. C12MIM applied topically to the lipid model or human skin as a pretreatment was more potent than C8MIM. When co-applied with the drugs to human skin, aqueous C12MIM was more potent than C8MIM in enhancing theophylline permeation, but neither IL affected (even decreased) diclofenac permeation. Thus, the IL’s ability to permeabilize skin lipid barrier is strongly modulated by its ability to reach the site of action and its interactions with drug and solvent. Such an interplay is far from trivial and requires detailed investigation to realize the full potential of ILs.

Effect of sphingosine and phytosphingosine ceramide ratio on lipid arrangement and barrier function in skin lipid models

The lipids in the uppermost layer of the skin, the stratum corneum (SC), play an important role in the skin barrier function. The three main subclasses in the SC lipid matrix are ceramides (CER), cholesterol, and free fatty acids. In inflammatory skin diseases, such as atopic dermatitis and psoriasis, the SC lipid composition is modulated compared to the composition in healthy SC. One of the main alterations is the molar ratio between the concentration of CER N-(tetracosanoyl)-sphingosine (CER NS) and CER N-(tetracosanoyl)-phytosphingosine (CER NP), which correlated with an impaired skin barrier function. In the present study, we investigated the impact of varying the CER NS:CER NP ratios on the lipid organization, lipid arrangement, and barrier functionality in SC lipid model systems. The results indicate that a higher CER NS:CER NP ratio as observed in diseased skin did not alter the lipid organization or lipid arrangement in the long periodicity phase encountered in SC. The trans-epidermal water loss, an indication of the barrier functionality, was significantly higher for the CER NS:CER NP 2:1 model (mimicking the ratio in inflammatory skin diseases) compared to the CER NS:CER NP 1:2 ratio (in healthy skin). These findings provide a more detailed insight into the lipid organization in both healthy and diseased skin and suggest that in vivo the molar ratio between CER NS:CER NP contributes to barrier impairment as well but might not be the main factor.

The intriguing molecular dynamics of Cer[EOS] in rigid skin barrier lipid layers requires improvement of the model

Omega-O-acyl ceramides such as 32-linoleoyloxydotriacontanoyl sphingosine (Cer[EOS]) are essential components of the lipid skin barrier, which protects our body from excessive water loss and the penetration of unwanted substances. These ceramides drive the lipid assembly to epidermal-specific long periodicity phase (LPP), structurally much different than conventional lipid bilayers. Here, we synthesized Cer[EOS] with selectively deuterated segments of the ultralong N-acyl chain or deuterated or 13C-labeled linoleic acid and studied their molecular behavior in a skin lipid model. Solid-state 2H NMR data revealed surprising molecular dynamics for the ultralong N-acyl chain of Cer[EOS] with increased isotropic motion toward the isotropic ester-bound linoleate. The sphingosine moiety of Cer[EOS] is also highly mobile at skin temperature, in stark contrast to the other LPP components, N-lignoceroyl sphingosine acyl, lignoceric acid, and cholesterol, which are predominantly rigid. The dynamics of the linoleic chain is quantitatively described by distributions of correlation times and using dynamic detector analysis. These NMR results along with neutron diffraction data suggest an LPP structure with alternating fluid (sphingosine chain-rich), rigid (acyl chain-rich), isotropic (linoleate-rich), rigid (acyl-chain rich), and fluid layers (sphingosine chain-rich). Such an arrangement of the skin barrier lipids with rigid layers separated with two different dynamic “fillings” i) agrees well with ultrastructural data, ii) satisfies the need for simultaneous rigidity (to ensure low permeability) and fluidity (to ensure elasticity, accommodate enzymes, or antimicrobial peptides), and iii) offers a straightforward way to remodel the lamellar body lipids into the final lipid barrier.

Phytosphingosine ceramide mainly localizes in the central layer of the unique lamellar phase of skin lipid model systems

Understanding the lipid arrangement within the skin’s outermost layer, the stratum corneum (SC), is important for advancing knowledge on the skin barrier function. The SC lipid matrix consists of ceramides (CERs), cholesterol, and free fatty acids, which form unique crystalline lamellar phases, referred to as the long periodicity phase (LPP) and short periodicity phases. As the SC lipid composition is complex, lipid model systems that mimic the properties of native SC are used to study the SC lipid organization and molecular arrangement. In previous studies, such lipid models were used to determine the molecular organization in the trilayer structure of the LPP unit cell. The aim of this study was to examine the location of CER N-(tetracosanoyl)-phytosphingosine (CER NP) in the unit cell of this lamellar phase and compare its position with CER N-(tetracosanoyl)-sphingosine (CER NS). We selected CER NP as it is the most prevalent CER subclass in the human SC, and its location in the LPP is not known. Our neutron diffraction results demonstrate that the acyl chain of CER NP was positioned in the central part of the trilayer structure, with a fraction also present in the outer layers, the same location as determined for the acyl chain of CER NS. In addition, our Fourier transformed infrared spectroscopy results are in agreement with this molecular arrangement, suggesting a linear arrangement for the CER NS and CER NP. These findings provide more detailed insight into the lipid organization in the SC lipid matrix.

The importance of ceramide headgroup for lipid localisation in skin lipid models

The stratum corneum's lipid matrix is a critical for the skin's barrier function and is primarily composed of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The lipids form a long periodicity phase (LPP), a unique trilayer unit cell structure. An enzyme driven pathway is implemented to synthesize these key lipids. If these enzymes are down- or upregulated as in inflammatory diseases, the final lipid composition is affected often altering the barrier function. In this study, we mimicked down regulation of enzymes involved in the synthesis of the sphingosine and CER amide bond. In a LPP lipid model, we substituted CER N-(tetracosanoyl)-sphingosine (CER NS) with either i) FFA C24 and free sphingosine, to simulate the loss of the CER amide bond, or ii) with FFA C24 and C18 to simulate the loss of the sphingosine headgroup. Our study shows the lipids in the LPP would not phase separate until at least 25% of the CER NS is substituted keeping the lateral packing and conformational ordering unaltered. Neutron diffraction studies showed that free sphingosine chains localized at the outer layers of the unit cell, while the remaining CER NS head group was concentrated in the inner headgroup layers. However, when FFA C18 was inserted, CER NS was dispersed throughout the LPP, resulting in an even distribution between the inner and outer water layers. The presented results highlight the importance of the CER NS headgroup structure and its interaction in combination with the carbon chain invariability for optimal lipid arrangement.

Effects of (R)- and (S)-α-Hydroxylation of Acyl Chains in Sphingosine, Dihydrosphingosine, and Phytosphingosine Ceramides on Phase Behavior and Permeability of Skin Lipid Models.

Ceramides (Cers) with α-hydroxylated acyl chains comprise about a third of all extractable skin Cers and are required for permeability barrier homeostasis. We have probed here the effects of Cer hydroxylation on their behavior in lipid models comprising the major SC lipids, Cer/free fatty acids (C 16-C 24)/cholesterol, and a minor component, cholesteryl sulfate. Namely, Cers with (R)-α-hydroxy lignoceroyl chains attached to sphingosine (Cer AS), dihydrosphingosine (Cer AdS), and phytosphingosine (Cer AP) were compared to their unnatural (S)-diastereomers and to Cers with non-hydroxylated lignoceroyl chains attached to sphingosine (Cer NS), dihydrosphingosine (Cer NdS), and phytosphingosine (Cer NP). By comparing several biophysical parameters (lamellar organization by X-ray diffraction, chain order, lateral packing, phase transitions, and lipid mixing by infrared spectroscopy using deuterated lipids) and the permeabilities of these models (water loss and two permeability markers), we conclude that there is no general or common consequence of Cer α-hydroxylation. Instead, we found a rich mix of effects, highly dependent on the sphingoid base chain, configuration at the α-carbon, and permeability marker used. We found that the model membranes with unnatural Cer (S)-AS have fewer orthorhombically packed lipid chains than those based on the (R)-diastereomer. In addition, physiological (R)-configuration decreases the permeability of membranes, with Cer (R)-AdS to theophylline, and increases the lipid chain order in model systems with natural Cer (R)-AP. Thus, each Cer subclass makes a distinct contribution to the structural organization and function of the skin lipid barrier.

High concentration of the ester-linked ω-hydroxy ceramide increases the permeability in skin lipid model membranes

The ester-linked ω-hydroxy acyl chain linked to a sphingosine base referred to as CER EOS is essential for the skin barrier lipid organization. While the majority of the skin lipids form a dense, crystalline structure, associated with low permeability, the unsaturated moiety of CER EOS, (either the linoleate or the oleate chain) exists in a liquid phase at the skin's physiological temperature. Thus, the relationship between CER EOS and barrier function is not entirely comprehended. We studied the permeability and lipid organization in skin lipid models, gradually increasing in CER EOS concentration, mixed with non-hydroxy sphingosine-based ceramide (CER NS) in an equimolar ratio of CERs, cholesterol, and free fatty acids (FFAs) mimicking the ratio in the native skin. A significant increase in the orthorhombic-hexagonal phase transition temperature was recorded when CER EOS concentration was raised to 70 mol% of the total CER content and higher, rendering a higher fraction of lipids in the orthorhombic phase at the expense of the hexagonal phase at physiological temperature. The model's permeability did not differ when CER EOS concentration ranged between 10 and 30% but increased significantly at 70% and higher. Using CER EOS with a perdeuterated oleate chain, it was shown that the fraction of lipids in a liquid phase increased with CER EOS concentration, while the neighboring CERs and FFAs remained in a crystalline state. The increased fraction of the liquid phase therefore, had a stronger effect on permeability than the increased fraction of lipids forming an orthorhombic phase.

Behavior of 1-Deoxy-, 3-Deoxy- and N-Methyl-Ceramides in Skin Barrier Lipid Models

Ceramides (Cer) are essential components of the skin permeability barrier. To probe the role of Cer polar head groups involved in the interfacial hydrogen bonding, the N-lignoceroyl sphingosine polar head was modified by removing the hydroxyls in C-1 (1-deoxy-Cer) or C-3 positions (3-deoxy-Cer) and by N-methylation of amide group (N-Me-Cer). Multilamellar skin lipid models were prepared as equimolar mixtures of Cer, lignoceric acid and cholesterol, with 5 wt% cholesteryl sulfate. In the 1-deoxy-Cer-based models, the lipid species were separated into highly ordered domains (as found by X-ray diffraction and infrared spectroscopy) resulting in similar water loss but 4–5-fold higher permeability to model substances compared to control with natural Cer. In contrast, 3-deoxy-Cer did not change lipid chain order but promoted the formation of a well-organized structure with a 10.8 nm repeat period. Yet both lipid models comprising deoxy-Cer had similar permeabilities to all markers. N-Methylation of Cer decreased lipid chain order, led to phase separation, and improved cholesterol miscibility in the lipid membranes, resulting in 3-fold increased water loss and 10-fold increased permeability to model compounds compared to control. Thus, the C-1 and C-3 hydroxyls and amide group, which are common to all Cer subclasses, considerably affect lipid miscibility and chain order, formation of periodical nanostructures, and permeability of the skin barrier lipid models.

Barrier Capability of Skin Lipid Models: Effect of Ceramides and Free Fatty Acid Composition.

The skin is an effective barrier that prevents the influx of harmful substances from the environment and the efflux of body fluid. This barrier function is ascribed to the intercellular lipids present in the outermost layer of the skin referred to as the stratum corneum (SC). These lipids are composed mainly of ceramides (CERs), cholesterol, and free fatty acids (FFAs). Alterations in the SC lipid composition and barrier function impairment occur in several skin diseases including atopic dermatitis (AD). As the etiology of AD is multifactorial, establishing the relationship between the changes in SC lipid composition and barrier function impairment in the patients remains a challenge. Here, we employed model membrane systems to investigate the contribution of various anomalies in the SC CER and FFA composition observed in AD patients' skin to the barrier dysfunction. Using ethyl-p-aminobenzoate permeation and transepidermal water loss values as markers for barrier function, we determined that the alterations in SC lipid composition contribute to the impaired barrier function in AD patients. By the use of biophysical techniques, we established that the largest reduction in barrier capability was observed in the model with an increased fraction of short-chain FFAs, evident by the decrease in chain packing density. Modulations in the CER subclass composition impacted the lamellar organization while having a smaller effect on the barrier function. These findings provide evidence that AD therapies normalizing the FFA composition are at least as important as normalizing CER composition.

New insight into phase behavior and permeability of skin lipid models based on sphingosine and phytosphingosine ceramides

The intercellular lipid matrix of the stratum corneum (SC), which consist mainly of ceramides (CERs), free fatty acids and cholesterol, is fundamental to the skin barrier function. These lipids assemble into two lamellar phases, known as the long and short periodicity phases (LPP and SPP respectively). The LPP is unique in the SC and is considered important for the skin barrier function. Alterations in CER composition, as well as impaired skin barrier function, are commonly observed in diseased skin, yet the understanding of this relationship remains insufficient. In this study, we have investigated the influence of non-hydroxy and α-hydroxy sphingosine-based CERs and their phytosphingosine counterparts on the permeability and lipid organization of model membranes, which were adjusted in composition to enhance formation of the LPP. The permeability was compared by diffusion studies using ethyl-p-aminobenzoate as a model drug, and the lipid organization was characterized by X-ray diffraction and infrared spectroscopy. Both the sphingosine- and phytosphingosine-based CER models formed the LPP, while the latter exhibited a longer LPP repeat distance. The ethyl-p-aminobenzoate flux across the sphingosine-based CER models was higher when compared to the phytosphingosine counterparts, contrary to the fact that the α-hydroxy phytosphingosine-based CER model had the lowest chain packing density. The unanticipated low permeability of the α-hydroxy phytosphingosine-based model is probably associated with a stronger headgroup hydrogen bonding network. Our findings indicate that the increased level of sphingosine-based CERs at the expense of phytosphingosine-based CERs, as observed in the diseased skin, may contribute to the barrier function impairment.

Probing the interactions among sphingosine and phytosphingosine ceramides with non- and alpha-hydroxylated acyl chains in skin lipid model membranes

Ceramides (Cers) are significant constituents of the stratum corneum (SC), the uppermost skin layer responsible for skin barrier properties. Cers are a heterogeneous group of lipids whose mutual interactions are still unclear. To better understand these interactions, we characterized model membranes containing stearic acid, cholesterol, cholesterol sulfate and one or more of the following ceramides: N-stearoyl-sphingosine (CerNS), N-stearoyl-phytosphingosine (CerNP) and N-(2-hydroxy)stearoyl-phytosphingosine (CerAP). Small angle X-ray scattering and FTIR spectroscopy were used to study lipid arrangement, phase separation and thermotropic behaviour. In the one-Cer systems, the membranes with CerNP showed strong hydrogen bonding and significant phase separation, even after phase transition, while the systems containing CerAP and CerNS had increased lipid miscibility. The multi-Cer systems exhibited different behaviour. In particular, the membrane containing all three Cers was a highly miscible system with narrow one-step phase transition, which, of all the studied samples, occurred at the lowest temperatures. Our results show that even a small variation in Cer structure results in substantially different phase behaviour, which is further affected by the presence of other Cer subclasses. Interestingly, the phase behaviour of the most complex three-Cer system was simpler than that of the others, highlighting the importance of lipid diversity in real SC.

Probing the role of ceramide hydroxylation in skin barrier lipid models by 2H solid-state NMR spectroscopy and X-ray powder diffraction

In this work, we studied model stratum corneum lipid mixtures composed of the hydroxylated skin ceramides N-lignoceroyl 6-hydroxysphingosine (Cer[NH]) and α-hydroxylignoceroyl phytosphingosine (Cer[AP]). Two model skin lipid mixtures of the composition Cer[NH] or Cer[AP], N-lignoceroyl sphingosine (Cer[NS]), lignoceric acid (C24:0) and cholesterol in a 0.5:0.5:1:1 molar ratio were compared. Model membranes were investigated by differential scanning calorimetry and 2H solid-state NMR spectroscopy at temperatures from 25 °C to 80 °C. Each component of the model mixture was specifically deuterated for selective detection by 2H NMR. Thus, the exact phase composition of the mixture at varying temperatures could be quantified. Moreover, using X-ray powder diffraction we investigated the lamellar phase formation. From the solid-state NMR and DSC studies, we found that both hydroxylated Cer[NH] and Cer[AP] exhibit a similar phase behavior. At physiological skin temperature of 32 °C, the lipids form a crystalline (orthorhombic) phase. With increasing temperature, most of the lipids become fluid and form a liquid-crystalline phase, which converts to the isotropic phase at higher temperatures (65–80 °C). Interestingly, lignoceric acid in the Cer[NH]-containing mixture has a tendency to form two types of fluid phases at 65 °C. This tendency was also observed in Cer[AP]-containing membranes at 80 °C. While Cer[AP]-containing lipid models formed a short periodicity phase featuring a repeat spacing of d = 5.4 nm, in the Cer[NH]-based model skin lipid membranes, the formation of unusual long periodicity phase with a repeat spacing of d = 10.7 nm was observed.

Effects of Ceramide and Dihydroceramide Stereochemistry at C-3 on the Phase Behavior and Permeability of Skin Lipid Membranes.

Ceramides (Cer) are key components of the skin permeability barrier. Sphingosine-based CerNS and dihydrosphingosine-based CerNdS (dihydroCer) have two chiral centers; however, the importance of the correct stereochemistry in the skin barrier Cer is unknown. We investigated the role of the configuration at C-3 of CerNS and CerNdS in the organization and permeability of model skin lipid membranes. Unnatural l-threo-CerNS and l-threo-CerNdS with 24-C acyl chains were synthesized and, along with their natural d-erythro-isomers, incorporated into membranes composed of major stratum corneum lipids (Cer, free fatty acids, cholesterol, and cholesteryl sulfate). The membrane microstructure was investigated by X-ray powder diffraction and infrared spectroscopy, including deuterated free fatty acids. Inversion of the C-3 configuration in CerNS and CerNdS increased phase transition temperatures, had no significant effects on lamellar phases, but also decreased the proportion of orthorhombic packing and decreased lipid mixing in the model membranes. These changes in membrane organization resulted in membrane permeabilities that ranged from unchanged to 5-fold higher (depending on the permeability markers, namely, water loss, electrical impedance, flux of theophylline, and flux of indomethacin) compared to membranes with natural CerNS/NdS isomers. Thus, the physiological d-erythro stereochemistry of skin Cer and dihydroCer appears to be essential for their correct barrier function.

Effect of Size and Surface Charge of Gold Nanoparticles on their Skin Permeability: A Molecular Dynamics Study

Molecular level understanding of permeation of nanoparticles through human skin establishes the basis for development of novel transdermal drug delivery systems and design and formulation of cosmetics. Recent experiments suggest that surface coated nano-sized gold nanoparticles (AuNPs) can penetrate the rat and human skin. However, the mechanisms by which these AuNPs penetrate are not well understood. In this study, we have carried out coarse grained molecular dynamics simulations to explore the permeation of dodecanethiol coated neutral hydrophobic AuNPs of different sizes (2–5 nm) and surface charges (cationic and anionic) through the model skin lipid membrane. The results indicate that the neutral hydrophobic AuNPs disrupted the bilayer and entered in it with in ~200 ns, while charged AuNPs were adsorbed on the bilayer headgroup. The permeation free energy calculation revealed that at the head group of the bilayer, a very small barrier existed for neutral hydrophobic AuNP while a free energy minimum was observed for charged AuNPs. The permeability was maximum for neutral 2 nm gold nanoparticle (AuNP) and minimum for 3 nm cationic AuNP. The obtained results are aligned with recent experimental findings. This study would be helpful in designing customized nanoparticles for cosmetic and transdermal drug delivery application.

Effects of 6-Hydroxyceramides on the Thermotropic Phase Behavior and Permeability of Model Skin Lipid Membranes.

Ceramides (Cer) based on 6-hydroxysphingosine are important components of the human skin barrier, the stratum corneum. Although diminished concentrations of 6-hydroxyCer have been detected in skin diseases such as atopic dermatitis, our knowledge on these unusual sphingolipids, which have only been found in the skin, is limited. In this work, we investigate the biophysical behavior of N-lignoceroyl-6-hydroxysphingosine (Cer NH) in multilamellar lipid membranes composed of Cer/free fatty acids (FFAs) (C16-C24)/cholesterol/cholesteryl sulfate. To probe the Cer structure-activity relationships, we compared Cer NH membranes with membranes containing Cer with sphingosine (Cer NS), dihydrosphingosine, and phytosphingosine (Cer NP), all with the same acyl chain length (C24). Compared with Cer NS, 6-hydroxylation of Cer not only increased membrane water loss and permeability in a lipophilic model compound but also dramatically increased the membrane opposition to electrical current, which is proportional to the flux of ions. Infrared spectroscopy revealed that Cer hydroxylation (in either Cer NH or Cer NP) increased the main transition temperature of the membrane but prevented good Cer mixing with FFAs. X-ray powder diffraction showed not only lamellar phases with shorter periodicity upon Cer hydroxylation but also the formation of an unusually long periodicity phase (d = 10.6 nm) in Cer NH-containing membranes. Thus, 6-hydroxyCer behaves differently from sphingosine- and phytosphingosine-based Cer. In particular, the ability to form a long-periodicity lamellar phase and highly limited permeability to ions indicate the manner in which 6-hydroxylated Cer contribute to the skin barrier function.

Penetration of Gold Nanoparticles through Human Skin: Unraveling Its Mechanisms at the Molecular Scale.

Recent experimental studies suggest that nanosized gold nanoparticles (AuNPs) are able to penetrate into the deeper layer (epidermis and dermis) of rat and human skin. However, the mechanisms by which these AuNPs penetrate and disrupt the skin's lipid matrix are not well understood. In this study, we have used computer simulations to explore the translocation and the permeation of AuNPs through the model skin lipid membrane using both unconstrained and constrained coarse-grained molecular dynamics simulations. Each AuNP (1-6 nm) disrupted the bilayer packing and entered the interior of the bilayer rapidly (within 100 ns). It created a hydrophobic vacancy in the bilayer, which was mostly filled by skin constituents. Bigger AuNPs induced changes in the bilayer structure, and undulations were observed in the bilayer. The bilayer exhibited self-healing properties; it retained its original form once the simulation was run further after the removal of the AuNPs. Constrained simulation results showed that there was a trade-off between the kinetics and thermodynamics of AuNP permeation at a molecular scale. The combined effect of both resulted in a high permeation of small-sized AuNPs. The molecular-level information obtained through our simulations offers a very convenient method to design novel drug delivery systems and effective cosmetics.

Free fatty acids chain length distribution affects the permeability of skin lipid model membranes

The lipid matrix in the stratum corneum (SC) plays an important role in the barrier function of the skin. The main lipid classes in this lipid matrix are ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The aim of this study was to determine whether a variation in CER subclass composition and chain length distribution of FFAs affect the permeability of this matrix. To examine this, we make use of lipid model membranes, referred to as stratum corneum substitute (SCS). We prepared SCS containing i) single CER subclass with either a single FFA or a mixture of FFAs and CHOL, or ii) a mixture of various CER subclasses with either a single FFA or a mixture of FFAs and CHOL. In vitro permeation studies were performed using ethyl-p-aminobenzoic acid (E-PABA) as a model drug. The flux of E-PABA across the SCS containing the mixture of FFAs was higher than that across the SCS containing a single FA with a chain length of 24 C atoms (FA C24), while the E-PABA flux was not effected by the CER composition. To select the underlying factors for the changes in permeability, the SCSs were examined by Fourier transform infrared spectroscopy (FTIR) and Small angle X-ray scattering (SAXS). All lipid models demonstrated a similar phase behavior. However, when focusing on the conformational ordering of the individual FFA chains, the shorter chain FFA (with a chain length of 16, 18 or 20 C atoms forming only 11m/m% of the total FFA level) had a higher conformational disordering, while the conformational ordering of the chains of the CER and FA C24 and FA C22 hardly did not change irrespective of the composition of the SCS. In conclusion, the conformational mobility of the short chain FFAs present only at low levels in the model SC lipid membranes has a great impact on the permeability of E-PABA.

Kinetic Mechanism of Phase Separation in Stratum Corneum Models by IR Spectroscopy

The primary barrier to permeability in mammalian skin resides within the ∼15 μm thick outermost layer, the stratum corneum (SC) which is formed from anucleated corneocytes embedded in a lamellar lipid matrix. Studies of lipid structural organization in human SC have begun to provide a basis for understanding barrier function with the potential for elucidating how skin organization is altered in disease states. In contrast, the kinetics and mechanisms of the development of lamellar structures, lipid packing motifs, and domain formation have not been widely probed. Yet, molecular level information concerning the mechanisms and kinetics of lipid domain formation would help define potential pathways for permeation of hydrophobic species as well as to possibly provide clues as to why a variety of ceramides and additional chemical species are present in the SC. In addition, understanding the formation and dissipation kinetics of domains formed from particular ceramide and fatty acid species would aid in defining biological processes such as epidermal desquamation. The current study demonstrates the feasibility of utilizing IR spectroscopy for studying lateral phase separation and the development of the early stages of lamellar structure in skin lipid models formed from two fairly standard ternary equimolar mixtures, namely ceramide[NS]/stearic acid/cholesterol and ceramide[AS]/stearic acid/cholesterol mixtures. The IR measurements show that the following sequence of events occurs in ceramide[NS]/stearic acid/cholesterol mixtures: (1) rearrangements of H-bonds in the ceramide component, (2) formation of small ceramide domains and orthorhombic phases, followed by (3) rearrangement of H-bonds in stearic acid, and (4) formation of relatively large and pure orthorhombic domains of stearic acid, probably indicative of the onset of lamellar phase formation. Notably different kinetic behavior is observed for the stearic acid component in the ceramide[AS] mixture.

Ceramides in the Skin Lipid Membranes: Length Matters

Ceramides are essential constituents of the skin barrier that allow humans to live on dry land. Reduced levels of ceramides have been associated with skin diseases, e.g., atopic dermatitis. However, the structural requirements and mechanisms of action of ceramides are not fully understood. Here, we report the effects of ceramide acyl chain length on the permeabilities and biophysics of lipid membranes composed of ceramides (or free sphingosine), fatty acids, cholesterol, and cholesterol sulfate. Short-chain ceramides increased the permeability of the lipid membranes compared to a long-chain ceramide with maxima at 4-6 carbons in the acyl. By a combination of differential scanning calorimetry, Fourier transform infrared spectroscopy, X-ray diffraction, Langmuir monolayers, and atomic force microscopy, we found that the reason for this effect in short ceramides was a lower proportion of tight orthorhombic packing and phase separation of continuous short ceramide-enriched domains with shorter lamellar periodicity compared to native long ceramides. Thus, long acyl chains in ceramides are essential for the formation of tightly packed impermeable lipid lamellae. Moreover, the model skin lipid membranes are a valuable tool to study the relationships between the lipid structure and composition, lipid organization, and the membrane permeability.

Investigating the barrier function of skin lipid models with varying compositions

The lipids in the uppermost layer of the skin, the stratum corneum (SC), play an important role in the barrier function. The main lipid classes in stratum corneum are ceramides, cholesterol, and free fatty acids. In previous publications, a lipid model was presented, referred to as the stratum corneum substitute (SCS), that closely mimics the SC lipid organization and SC barrier function. In the present study, we use the SCS to study the effect of changes in lipid organization on the lipid barrier function using benzoic acid as permeation compound. First, in the SCS, we increased the level of one of the three major lipid classes keeping the ratio between the other lipid classes constant. An increased cholesterol level resulted in an increase in phase-separated cholesterol and a reduction in the permeability. An increase in ceramide or free fatty acid level resulted in the formation of additional phases, but had no significant influence on the permeability. We also examined models that mimic selected changes in lipid composition reported for dry or diseased skin. The SCS that mimics the composition in recessive X-linked ichthyosis skin displayed a twofold increase in permeability. This increase is possibly related to the formation of an additional, less ordered phase in this model.