Infrared spectroscopy studies of mixtures prepared with synthetic ceramides varying in head group architecture: Coexistence of liquid and crystalline phases

The barrier function of the skin is provided by the stratum corneum (SC), the outermost layer of the skin. Ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) are present in SC and form highly ordered crystalline lipid lamellae. These lamellae are crucial for a proper skin barrier function. In the present study, Fourier transform infrared spectroscopy was used to examine the lipid organization of mixtures prepared from synthetic CERs with CHOL and FFAs. The conformational ordering and lateral packing of these mixtures showed great similarities to the lipid organization in SC and lipid mixtures prepared with native CERs. Therefore, mixtures with synthetic CERs serve as an excellent tool for studying the effect of molecular architecture of CER subclasses on the lipid phase behavior. In SC the number of OH-groups in the head groups of CER subclasses varies. Furthermore, acylCERs with a linoleic acid chemically bound to a long acyl chain are also identified. The present study revealed that CER head group architecture affects the lateral packing and conformational ordering of the CER:CHOL:FFA mixtures. Furthermore, while the majority of the lipids form a crystalline packing, the linoleate moiety of the acylCERs participates in a “pseudo fluid” phase.     

The role of ceramide chain length distribution on the barrier properties of the skin lipid membranes

The skin barrier function is provided by the stratum corneum (SC). The lipids in the SC are composed of three lipid classes: ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) which form two crystalline lamellar structures. In the present study, we investigate the effect of CER chain length distribution on the barrier properties of model lipid membranes mimicking the lipid composition and organization of SC. The membranes were prepared with either isolated pig CERs (PCERs) or synthetic CERs. While PCERs have a wide chain length distribution, the synthetic CERs are quite uniform in chain length. The barrier properties were examined by means of permeation studies using hydrocortisone as a model drug. Our studies revealed a reduced barrier in lipid membranes prepared with PCERs compared to synthetic CERs. Additional studies revealed that a wider chain length distribution of PCERs results in an enhanced hexagonal packing and increased conformational disordering of the lipid tails compared to synthetic CERs, while the lamellar phases did not change. This demonstrates that the chain length distribution affects the lipid barrier by reducing the lipid ordering and density within the lipid lamellae. In subsequent studies, the effect of increased levels of FFAs or CERs with a long acyl chain in the PCERs membranes was also studied. These changes in lipid composition enhanced the level of orthorhombic packing, reduced the conformational disordering and increased the barrier of the lipid membranes. In conclusion, the CER chain length distribution is an important key factor for maintaining a proper barrier.     

The effect of the chain length distribution of free fatty acids on the mixing properties of stratum corneum model membranes

The stratum corneum (SC) plays a fundamental role in the barrier function of the skin. The SC consists of corneocytes embedded in a lipid matrix. The main lipid classes in the lipid matrix are ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The aim of this study was to examine the effect of the chain length of FFAs on the thermotropic phase behavior and mixing properties of SC lipids. Fourier transform infrared spectroscopy and Raman imaging spectroscopy were used to study the mixing properties using either protonated or deuterated FFAs. We selected SC model lipid mixtures containing only a single CER, CHOL and either a single FFA or a mixture of FFAs mimicking the FFA SC composition. The single CER consists of a sphingoid base with 18 carbon atoms and an acyl chain with a chain length of 24 carbon atoms. When using lignoceric acid (24 carbon atoms) or a mixture of FFAs, the CER and FFAs participated in mixed crystals, but hydration of the mixtures induced a slight phase separation between CER and FFA. The mixed crystalline structures did not phase separate during storage even up to a time period of 3 months. When using palmitic acid (16 carbon atoms), a slight phase separation was observed between FFA and CER. This phase separation was clearly enhanced during hydration and storage. In conclusion, the thermotropic phase behavior and the mixing properties of the SC lipid mixtures were shown to strongly depend on the chain length and chain length distribution of FFAs, while hydration enhanced the phase separation.     

LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. In human SC, the literature currently reports 11 CER subclasses that have been identified. In this paper, a novel quick and robust LC/MS method is presented that allows the separation and analysis of all known human SC CER subclasses using only limited sample preparation. Besides all 11 known and identified subclasses, a 3D multi-mass chromatogram shows the presence of other lipid subclasses. Using LC/MS/MS with an ion trap (IT) system, a Fourier transform-ion cyclotron resonance system, and a triple quadrupole system, we were able to identify one of these lipid subclasses as a new CER subclass: the ester-linked ω-hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identification of a new CER subclass, this paper also describes the applicability and robustness of the developed LC/MS method by analyzing three (biological) SC samples: SC from human dermatomed skin, human SC obtained by tape stripping, and SC from full-thickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profile.     

Lipid organization in human and porcine stratum corneum differs widely, while lipid mixtures with porcine ceramides model human stratum corneum lipid organization very closely

The conformational disordering and lateral packing of lipids in porcine and human isolated stratum corneum (SC) was compared using Fourier transform infrared spectroscopy (FTIR). It was shown that SC of both species differ markedly, porcine SC lipids being arranged predominantly in a hexagonal lattice while lipids in human SC are predominantly packed in the denser orthorhombic lattice. However, the lipid organization of equimolar ceramide:cholesterol:free fatty acid (CER:CHOL:FFA) mixtures prepared with isolated porcine CER or human CER is very similar, only the transition temperatures differed being slightly lower in mixtures with porcine CER. Therefore, the difference in lateral packing between human and porcine stratum corneum is not due to the difference in CER composition. Furthermore, it is possible to use more readily available porcine CER in model lipid mixtures to mimic lipid organization in human SC. As the equimolar porcine CER:CHOL:FFA mixtures closely mimic the lipid organization in human SC, both human SC and this mixture were selected to examine the effect of glycerol on the lipid phase behaviour. It was found that high concentrations of glycerol change the lamellar organization slightly, while domains with an orthorhombic lateral packing are still observed.     

Lipid organization in human and porcine stratum corneum differs widely, while lipid mixtures with porcine ceramides model human stratum corneum lipid organization very closely

The conformational disordering and lateral packing of lipids in porcine and human isolated stratum corneum (SC) was compared using Fourier transform infrared spectroscopy (FTIR). It was shown that SC of both species differ markedly, porcine SC lipids being arranged predominantly in a hexagonal lattice while lipids in human SC are predominantly packed in the denser orthorhombic lattice. However, the lipid organization of equimolar ceramide:cholesterol:free fatty acid (CER:CHOL:FFA) mixtures prepared with isolated porcine CER or human CER is very similar, only the transition temperatures differed being slightly lower in mixtures with porcine CER. Therefore, the difference in lateral packing between human and porcine stratum corneum is not due to the difference in CER composition. Furthermore, it is possible to use more readily available porcine CER in model lipid mixtures to mimic lipid organization in human SC. As the equimolar porcine CER:CHOL:FFA mixtures closely mimic the lipid organization in human SC, both human SC and this mixture were selected to examine the effect of glycerol on the lipid phase behaviour. It was found that high concentrations of glycerol change the lamellar organization slightly, while domains with an orthorhombic lateral packing are still observed.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: Lipid composition and thermal phase behavior of the stratum corneum

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while α-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified ω-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T₂) at a slightly lower temperature (68 °C) than human skin (74 °C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Two new methods for preparing a unique stratum corneum substitute

Stratum corneum lipids play an important role in the barrier function of the skin. An in vitro permeation model consisting of synthetic lipids has previously been developed to replace human stratum corneum (SC) in permeation studies. This model is referred to as the stratum corneum substitute (SCS). In order to improve its reproducibility and to increase the efficiency in preparing the SCS, two new preparation methods are developed. Subsequently the properties of the SCS prepared by the various methods, i.e. the manual airbrush method, the rotor airbrush method and the linomat method, are investigated. The results show that the SCS prepared with the various methods share the properties of a uniform lipid composition and lipid distribution. Furthermore, irrespective of the preparation method, the lipids form crystalline lamellar phases, mimicking the lipid organization and orientation in human SC. As a result, permeation profiles of benzoic acid through SCS are very similar to human SC. The rotor method increases the efficiency and reproducibility of the manual airbrush method, while the linomat method reduces the lipid loss during preparation and results in SCS with a more uniform membrane thickness. In conclusion, the linomat method was chosen as the preferred method for preparing the substitute.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: lipid composition and thermal phase behavior of the stratum corneum.

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while alpha-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified omega-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T2) at a slightly lower temperature (68 degrees C) than human skin (74 degrees C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Characterization of overall ceramide species in human stratum corneum

Ceramides (CERs) in human stratum corneum (SC) play physicochemical roles in determining barrier and water-holding functions of the skin, and specific species might be closely related to the regulation of keratinization, together with other CER-related lipids. Structures of those diverse CER species, however, have not been comprehensively revealed. The aim of this study was to characterize overall CER species in the SC. First, we constructed 3D multi-mass chromatograms of the overall CER species, based on normal-phase liquid chromatography (NPLC) connected to electrospray ionization-mass spectrometry (ESI-MS) using a gradient elution system and a postcolumn addition of a volatile salt-containing polar solvent. The CERs targeted from the 3D chromatograms were structurally analyzed using NPLC-ESI-tandem mass spectrometry (MS/MS), which resulted in the identification of 342 CER species in the inner forearm SC. This led to the discovery of a new CER class consisting of alpha-hydroxy fatty acid and dihydrosphingosine moieties, in addition to the 10 classes generally known. The results also revealed that those CERs contain long-chain (more than C(18))-containing sphingoids and a great number of isobaric species. These novel results will contribute not only to physiochemical research on CERs in the SC but also to lipidomics approaches to CERs in the skin.     

The Lipid Organisation of the Skin Barrier: Liquid and Crystalline Domains Coexist in Lamellar Phases

The superficial layer of the skin, the stratum corneum, is the main barrier for diffusion of substances across the skin. The stratum corneum is composed of corneocytes embedded in lipid lamellae. In previous studies two lamellar phases have been identified with periodicities of 6.4 and 13.4 nm of which the 13.4 nm phase (long periodicity phase = LPP) is considered to be very important for the skin banier function. The main lipid classes in stratumcorneum are ceramides, free fatty acids and cholesterol. Until now 8 subclassesof ceramides are identified in human stratum corneum referred to as ceramide 1 to 8. Studies with mixtures prepared with isolated human ceramides revealed that cholesterol and ceramides are very important for the formation of the lamellar phases. After addition of free fatty acids the lipids are organised in an orthorhombic packing with a small proportion of lipids in a liquid phase. Our most recent results show that the presence of ceramide 1 and the formation of a liquid phase are crucial elements for the formation of the LPP. These observations and the broad-narrowbroad sequence of lipid layers in the LPP led us to propose a molecular model for this phase. This consists of one narrow central lipid layer with fluid domains with on both sides a broad layer with a crystalline structure. This model is referred to as `the sandwich model'.     

The skin barrier in healthy and diseased state

The primary function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid regions. As most drugs applied onto the skin permeate along the lipid domains, the lipid organization is considered to be very important for the skin barrier function. It is for this reason that the lipid organization has been investigated quite extensively. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid organization is different from that of other biological membranes. In stratum corneum, two lamellar phases are present with repeat distances of approximately 6 and 13 nm. Moreover the lipids in the lamellar phases form predominantly crystalline lateral phases, but most probably a subpopulation of lipids forms a liquid phase. Diseased skin is often characterized by a reduced barrier function and an altered lipid composition and organization. In order to understand the aberrant lipid organization in diseased skin, information on the relation between lipid composition and organization is crucial. However, due to its complexity and inter-individual variability, the use of native stratum corneum does not allow detailed systematic studies. To circumvent this problem, mixtures prepared with stratum corneum lipids can be used. In this paper first the lipid organization in stratum corneum of normal and diseased skin is described. Then the role the various lipid classes play in stratum corneum lipid organization and barrier function has been discussed. Finally, the information on the role various lipid classes play in lipid phase behavior has been used to interpret the changes in lipid organization and barrier properties of diseased skin.     

The skin barrier in healthy and diseased state

The primary function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid regions. As most drugs applied onto the skin permeate along the lipid domains, the lipid organization is considered to be very important for the skin barrier function. It is for this reason that the lipid organization has been investigated quite extensively. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid organization is different from that of other biological membranes. In stratum corneum, two lamellar phases are present with repeat distances of approximately 6 and 13 nm. Moreover the lipids in the lamellar phases form predominantly crystalline lateral phases, but most probably a subpopulation of lipids forms a liquid phase. Diseased skin is often characterized by a reduced barrier function and an altered lipid composition and organization. In order to understand the aberrant lipid organization in diseased skin, information on the relation between lipid composition and organization is crucial. However, due to its complexity and inter-individual variability, the use of native stratum corneum does not allow detailed systematic studies. To circumvent this problem, mixtures prepared with stratum corneum lipids can be used. In this paper first the lipid organization in stratum corneum of normal and diseased skin is described. Then the role the various lipid classes play in stratum corneum lipid organization and barrier function has been discussed. Finally, the information on the role various lipid classes play in lipid phase behavior has been used to interpret the changes in lipid organization and barrier properties of diseased skin.     

The nanoscopic molecular pathway through human skin

BackgroundKnowledge regarding the barrier properties of human skin is important for understanding skin pathology, developing of transdermal drug delivery systems and computational skin absorption models; however, the molecular pathways through human skin remains to be fully investigated on a nanoscopic level. In particular the nanoscopic pathway of molecules passing the intercellular lipid bilayers separating the corneocytes in the stratum corneum (SC) is not fully elucidated.MethodsUsing stimulated emission depletion microscopy (STED) and Förster resonance energy transfer (FRET) the molecular pathways through the SC, the main barrier of the skin, are determined for lipophilic and water-soluble molecules at a nanoscopic resolution.ResultsUsing STED and confocal microscopy, water-soluble dyes, were observed to be present in both the corneocytes and in the intercellular lipid matrix, whereas the lipophilic dyes were predominately in the intercellular lipid bilayers. FRET was observed in the SC between the lipophilic and water-soluble dyes, the existence of a minimum possible distance between acceptor and donor molecules of 4.0 ± 0.1 nm was found.ConclusionsThe results indicate that lipophilic molecules penetrate the stratum corneum via the intercellular lipids bilayers separating the corneocytes in the SC, while the more water-soluble molecules penetrate the stratum corneum via the transcellular route through the corneocytes and intercellular lipid bilayers via the polar head groups of lipid molecules in the bilayers.General significanceKnowledge of the nanoscopic molecular pathways through human skin will help understand the skin barrier function and will be of use for computational skin absorption models and transdermal drug delivery strategies.     

Preparation and characterization of a stratum corneum substitute for in vitro percutaneous penetration studies

The intercellular stratum corneum (SC) lipids form the main barrier for diffusion of substances through the skin. A porous substrate covered with synthetic SC lipids would be an attractive model to study percutaneous penetration, hereby replacing native human SC. Prerequisite is that this stratum corneum substitute (SCS) is prepared with a uniform lipid composition and layer thickness. Furthermore, the lipid organization and orientation should resemble that in SC. The objective of this study was to investigate the utility of an airbrush spraying device to prepare a SCS composed of cholesterol, ceramides and free fatty acids on a polycarbonate filter. The results demonstrate that a proper choice of solvent mixture and lipid concentration is crucial to achieve a uniform distribution of the applied lipids over the filter surface. A smooth and tightly packed lipid layer is only obtained when the equilibration conditions are appropriately chosen. The SCS possesses two crystalline lamellar phases with periodicities similar to those present in native SC. The orientation of these lamellae is mainly parallel to the surface of the polycarbonate filter, which resembles the orientation of the intercellular SC lipids. In conclusion, the airbrush technique enables generation of a homogeneous SCS, which ultimately may function as a predictive in vitro percutaneous penetration model.     

Preparation and characterization of a stratum corneum substitute for in vitro percutaneous penetration studies

The intercellular stratum corneum (SC) lipids form the main barrier for diffusion of substances through the skin. A porous substrate covered with synthetic SC lipids would be an attractive model to study percutaneous penetration, hereby replacing native human SC. Prerequisite is that this stratum corneum substitute (SCS) is prepared with a uniform lipid composition and layer thickness. Furthermore, the lipid organization and orientation should resemble that in SC. The objective of this study was to investigate the utility of an airbrush spraying device to prepare a SCS composed of cholesterol, ceramides and free fatty acids on a polycarbonate filter. The results demonstrate that a proper choice of solvent mixture and lipid concentration is crucial to achieve a uniform distribution of the applied lipids over the filter surface. A smooth and tightly packed lipid layer is only obtained when the equilibration conditions are appropriately chosen. The SCS possesses two crystalline lamellar phases with periodicities similar to those present in native SC. The orientation of these lamellae is mainly parallel to the surface of the polycarbonate filter, which resembles the orientation of the intercellular SC lipids. In conclusion, the airbrush technique enables generation of a homogeneous SCS, which ultimately may function as a predictive in vitro percutaneous penetration model.     

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 lipid phase transitions and water barrier properties

In mammals, the outer skin layer, the stratum corneum, is the ultimate barrier to water loss. In order to relate barrier function to stratum corneum structure, samples from porcine skin were investigated by using differential scanning calorimetry (DSC), infrared (IR) spectroscopy, and water permeability techniques. Results of DSC and IR studies show that stratum corneum lipids undergo thermal transitions between 60 and 80 degrees C similar to lipid thermotropic transitions seen in a variety of synthetic and biological membranes. Results of water flux experiments performed under conditions similar to those of the DSC and IR studies show an abrupt change in permeability at about 70 degrees C. At low temperatures, water flux values are similar to those obtained for human skin in vivo, yielding an activation energy of 17 kcal/mol, in excellent agreement with values obtained for water flux through a variety of lipid biomembranes. In contrast, at temperatures above about 70 degrees C, water flux is characterized by an activation energy only slightly higher than that of free diffusion, suggesting that the stratum corneum offers little diffusional resistance under these conditions. These combined results suggest that increased disorder in stratum corneum lipid structure, brought about by thermotropic transitions, results in dramatically altered diffusional resistance of this tissue to water flux. Thus, as found for numerous biological membranes, water flux and lipid order in porcine stratum corneum are inversely related.     

Comprehensive quantification of ceramide species in human stratum corneum

One of the key challenges in lipidomics is to quantify lipidomes of interest, as it is practically impossible to collect all authentic materials covering the targeted lipidomes. For diverse ceramides (CER) in human stratum corneum (SC) that play important physicochemical roles in the skin, we developed a novel method for quantification of the overall CER species by improving our previously reported profiling technique using normal-phase liquid chromatog­raphy-electrospray ionization-mass spectrometry (NPLC-ESI-MS). The use of simultaneous selected ion monitoring measurement of as many as 182 kinds of molecular-related ions enables the highly sensitive detection of the overall CER species, as they can be analyzed in only one SC-stripped tape as small as 5 mm × 10 mm. To comprehensively quantify CERs, including those not available as authentic species, we designed a procedure to estimate their levels using relative responses of representative authentic species covering the species targeted, considering the systematic error based on intra-/inter-day analyses. The CER levels obtained by this method were comparable to those determined by conventional thin-layer chromatography (TLC), which guarantees the validity of this method. This method opens lipidomics approaches for CERs in the SC.