International Journal of Cosmetic Science, 2014, 36, 505–515
doi: 10.1111/ics.12162
Review Article
The physical chemistry of the stratum corneum lipids M. Boncheva Corporate R&D Division, Firmenich SA, PO Box 239, Route des Jeunes 1, Geneva CH-1211, Switzerland
Received 25 July 2014, Accepted 11 September 2014
Keywords: infrared spectroscopy, molecular organization, skin barrier lipids, skin permeability, stratum corneum, X-ray diffraction
Synopsis The extracellular matrix of stratum corneum (SC) participates actively in the defensive performance of human skin. Understanding its physicochemical properties is essential for understanding the skin homoeostasis, for the development of efficient therapies for skin disorders and diseases and for ensuring the safety of products designed for topical application. This article summarizes the current knowledge of the composition, self-assembly and molecular organization of the SC lipids, reviews the evidence connecting these parameters and the barrier properties of human skin, and outlines the immediate issues in the field of SC lipid research. La matrice extracellulaire du stratum corneum (SC) parRESUM E: ticipe activement a la performance defensive de la peau humaine. Comprendre ses proprietes physico-chimiques est essentielle pour comprendre l’homeostasie de la peau, pour le developpement de therapies efficaces pour les troubles et les maladies de la peau, et pour assurer la securite des produits concßus pour une application topique. Cet article resume les connaissances actuelles sur la composition, l’auto-assemblage, et l’organisation moleculaire des lipides SC, examine les preuves reliant ces parametres et les proprietes de barriere de la peau humaine, et decrit les problemes immediats dans le domaine de la recherche sur les lipides SC. Introduction The field of skin research is replete with opportunities for those intent on working on scientifically interesting topics while making a noticeable, positive difference in people’s lives [1]. The range of important problems that the field can help solving is broad. It comprises (i) reducing infant mortality, especially in the developing countries where defective skin barrier is the second major cause of preterm mortality [2–6]; (ii) improving the skin health of some 20– 30% of the population in the developed countries which suffer from some sort of skin barrier dysfunction [7, 8]; (iii) developing efficient strategies for the dermal and transdermal delivery of drugs and vaccines [9]; (iv) increasing the national and personal security by developing methods to retard or prevent the skin penetration of chemical warfare agents, toxic spills and pesticides [10, 11]; (v) enhancing the safety of topical products (e.g. fragrances, cosmetics, cleansing agents, sunscreens and insect repellents) [12]. Thus, the Correspondence: Mila Boncheva, Corporate R&D Division, Firmenich SA, PO Box 239, Route des Jeunes 1, CH-1211 Geneva 8, Switzerland. Tel.: (+41) 22 780 3027; fax: (+41) 22 780 3334; e-mail: mila.boncheva@ firmenich.com
targets in skin research cover the full spectrum from counter-acting leaky skin and preventing its penetration by chemicals to pushing chemicals through it. One common major problem underlies these diverse topics: our insufficient understanding of the factors that determine skin permeability. Understanding the permeability of human skin to a large extent means understanding the extracellular lipid matrix of the stratum corneum (SC), the topmost skin layer which constitutes the main barrier to transdermal fluxes [13–19]. As the lipid matrix forms an uninterrupted pathway from the skin surface to the viable skin tissues, it provides the principle path of entry for many chemicals [20]. At first sight, backed by more than 100 years of excellent and extensive studies on the physical chemistry and biophysics of cell membrane lipids, we should have a fairly good grip on the properties of the SC lipids as well [21, 22]. Should we rely on this assumption, however? Can we apply directly the knowledge of the physical chemistry of the biomembrane lipids to rationalize the SC permeability, or do we need to consider the SC lipids as species apart? To answer these questions, this review compares the composition, local environment, self-assembly and molecular organization of the SC lipids to those of the lipids in the cell membrane. Composition and local environment of the SC lipid matrix In terms of composition, the SC and the cellular lipids differ drastically. Instead of the phospholipids, glycolipids and cholesterol common for cell membranes, the SC lipid matrix contains approximately equimolar amounts of ceramides, free fatty acids and cholesterol [23–25] (Fig. 1). The ceramide molecules consist of fatty acids amide linked to sphingoid bases. Fifteen ceramide subclasses have been identified so far in the extracellular space of SC [28–30]. The ceramide molecules have different headgroup architectures, all of them considerably smaller and less polar than those of the lipids in a typical phospholipid membrane. The different classes of ceramides molecules can participate in different inter- and intramolecular interactions with the other lipid molecules and thus make distinct contributions to the organization and the structural integrity of the lipid matrix [31–35]. Both the ceramides and the fatty acids of the SC have unusually long hydrocarbon chains with a broad distribution of lengths [29, 30, 35–37]. The variety of chain lengths and headgroup structures results in the existence of over 400 different ceramide species [29, 30, 38]. Most common for the ceramides are lengths of 24–26 carbon atoms for the fatty acid hydrocarbon chain and 18–22 carbon atoms for the sphingoid
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Figure 1 Chemical structures of the lipids of human SC (modified from [26]). The nomenclature of the most prevalent 12 ceramide classes is shown using the classification of Motta et al. [27] (in capital letters) and using the earlier classification based on chromatographic mobility (in parentheses). Each ceramide molecule contains one sphingoid and one fatty acid residue, amide-linked as shown above the table for Cer [NP]. The number ranges indicate the prevalent lengths of the hydrocarbon chains in ceramides and free fatty acids. The structures of the three most recently identified ceramides and a suggestion for an alternative ceramides nomenclature can be found in [28].
hydrocarbon chain [37], whereas most common for the fatty acids are lengths of 22–26 carbon atoms [39]. Contrary to the cell membrane lipids, the lipid hydrocarbon chains of both ceramides and fatty acids are predominantly saturated. The only SC lipids that contain non-saturated fatty acid residues (esterified to particularly long fatty acid chains of 30–32 carbon atoms) are the x-hydroxy (EO) ceramides; most of them exist covalently linked to proteins of the corneocyte envelope [25, 40, 41]. The conformation that the ceramides molecules adopt in vivo – splayed, hairpin, or V-shape – is still under debate [42, 43]. The SC matrix contains also minor (in mass but not in importance) ingredients, including cholesterol sulphate and cholesteryl esters [44, 45]. The local environment of the SC and cell lipids also differs. In a living tissue, the cell lipids exist in a water-rich environment; the headgroup hydration in a typical phospholipid membrane easily reaches 7–16 molecules per headgroup [22]. In contrast, the water content within the SC lipid lamellae is extremely low and they contain no free, unbound water. Infrared (IR) spectroscopy has shown that the phase transition temperature of the SC lipids decreases only slightly upon massive hydration [46, 47]. Both conventional and cryo-electron microscopy have shown that the lipid lamellae do not swell upon hydration [48]. More recently, calculations based on neutron diffraction demonstrated that the packing of the lipid molecules within the lamellae leaves no space for water molecules [49, 50]. Thus, the water content of the lamellae in their
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normal, air-dried state and at relative air humidity of up to 90% is estimated to be only about 1–2 molecules bound per headgroup, or ten times lower than a typical phospholipid membrane. The pH to which the lipids of living cells are exposed is neutral; it can be measured easily using pH electrodes. Applying the same strategy to measure the pH within the SC lipid matrix, however, can be very misleading. The often-quoted profiles of pH within the SC obtained using flat pH electrodes and tape-stripping [51, 52] – uniformly increasing with depth from acidic at the surface to neutral in the deep SC layers – carry no information about the lipid matrix. As these electrodes contact the skin surface through a small water droplet, what they measure in fact is the average distribution of water-soluble species over the SC surface. In principle, several other strategies can be used to estimate the pH within the SC lipid lamellae. Two-photon fluorescence lifetime imaging has shown that throughout the SC depth the extracellular space contains acidic microdomains of variable length, with an average pH value around 6 [53]. The number of these domains changes with the depth of the SC. The application of this strategy, however, necessitates making assumptions for the refractive index of the lipid matrix and the pKa values of the fluorescent probes within the lipid lamellae; furthermore, the Henderson–Hasselbalch correlation used to calculate the pH is valid only for dilute water solutions, a situation far from the reality within the SC lipids matrix. Another approach for calculating the proton concentrations
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 36, 505–515
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within the lipid bilayers of the SC matrix is based on simplified molecular models [54]. In principle, the calculation of the proton activity in non-aqueous medium such as the lipid lamellae can be based also on calculations of its Gibbs free energy of solvation approximating the lipids to a homogeneous oil phase [55]. It is not yet clear which of these approaches is best suited to describe the acidity of the lipid matrix. It is important to note that, strictly speaking, applying the conventional notion of pH to a system such as the lipid matrix is not correct: as there is no free water in the lamellae, talking about the activity of hydrated protons (i.e. pH by definition) makes no sense. In summary, both the composition and the local environment of the lipids within the SC and the cell membranes differ considerably. These differences, however, cannot explain the difference of three orders of magnitude between the observed permeation coefficients for water measured in the two systems (equal to approximately 103 cms1 in phospholipid membranes and approximately 103 cmh1 in the SC lipid matrix). The excellent works of Fettiplace on phospholipid bilayers have shown that differences in their composition can result in not more than an order of magnitude difference in their permeability [56, 57]. Clearly, additional factors related to differences in the structures of the two lipid systems are at play. Formation of the lipid lamellae The formation of the SC lipid lamellae differs considerably from all we know about the formation of a phospholipid bilayer [28, 58– 60]. Contrary to cell membranes, the process is independent on the presence of proteins. It starts with a mixture of polar lipid precursors, namely sphingomyelins, glucosylceramides, phospholipids and cholesterol [44] (Fig. 2). These precursors, synthesized (or imported) in the keratinocytes (for a review, see [28]), are transported in the trans-Golgi network where they self-assemble into short stacks within ovoid or tubular structures, the lamellar bodies (LB) [61–63]. The molecular organization of the precursors in the LB is not well understood. The details of following stages – secretion of the LB content into the extracellular space, processing of the precursors to the true SC lipids of lower polarity and self-assembly of the stacks into extended 3D lamellae – are even less clear. It
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is not known if the processing of all lipid types occurs at the same time or sequentially, how the short stacks fuse into lamellae, or to what extent their molecular organization changes upon their fusion. Essentially, the process of formation of the lipid lamellae is one of hierarchical, templated self-assembly involving two distinct stages: the first one comprises the organization of the precursor molecules into bilayer stacks within the LB, and the second one – the assembly of these stacks into lamellae within the extracellular space of the SC. The two stages of the self-assembly process differ considerably; only the first one bears some similarities with the formation of a phospholipids bilayer. 1 Components. The self-assembling components in the first stage are the polar precursor molecules, and in the second stage – the short bilayer stacks of the LB. 2 Temperature/agitation. For self-assembly to occur, the assembling components need to interact via balancing repulsive and attractive forces, or they must be able to move and adjust their relative positions [64]. The thermal energy which moves around the components of the first stage (i.e. the precursor molecules) is not enough to move nano-sized objects (i.e. the lipid bilayer stacks), so the energy for ‘mixing’ and movement in the second stage is probably provided by the lipid processing. 3 Interactions. In both stages, the interactions between the components are weak and non-covalent. Whereas attractive van der Waals forces between the long hydrocarbon chains are present in each stage, the patterns of hydrogen bonds between the more polar precursors and the less polar processed molecules are not necessarily the same [65–67]. Because of the different environment in which the self-assembly proceeds, the hydrophobic interactions involved in the two stages probably also differ: in the first one, they can be expected to arise from expelling water, similar to the formation of a lipid bilayer [68], whereas in the second one, they might be due to restructuring bound water molecules, in analogy with the situation observed in the processes of ligandreceptor binding [69]. 4 Templating. Both self-assembly stages appear to be assisted by external templating, in analogy with many other processes of biological self-assembly [70]. In the first one, it is provided by the
Figure 2 A scheme illustrating the self-assembly of the lipid lamellae. The process starts with a mixture of polar precursor molecules (sphingomyelins, glucosylceramides, cholesterol and phospholipids, shown on the left) which are synthesized or imported in the keratinocytes and subsequently transported in the trans-Golgi network (step 1). There, they self-assemble into short stacks within ovoid or tubular structures, the lamellar bodies (LB, step 2). The details and the exact sequence of following stages – secretion of the LB content into the extracellular space (step 3), processing of the precursors to the true SC lipids of lower polarity (ceramides, cholesterol and free fatty acids, shown on the right), and self-assembly of the stacks into extended 3D lamellae (step 4) – are not well understood. See the text for details.
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 36, 505–515
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restricting volume of the membrane-limited space within the LB and possibly by caveolins [58, 71] and in the second one – by the lipids covalently linked to the corneocyte envelope [35, 72]. Clearly, the energetics of this two-stage hierarchical, templated process can be expected to differ from the one-step self-assembly of phospholipid molecules into bilayers. The experimental evidence collected so far indicates that the SC lipid lamellae represent a static and not a dynamic self-assembled structure. By definition, this distinction is based on the final structure and not on the process itself [73]. Thus, static self-assembled structures are formed by minimization of the free energy of the system and exist at a global or local energy minimum. In the case of the self-assembly of the SC lipid lamellae, the system seems to be driven from one equilibrium state to another by non-equilibrium agitation, for example enzyme delivery and secretion of the bilayer stacks in the extracellular space. In contrast, dynamic selfassembled structures are non-equilibrium, energy dissipating ones, maintained in a steady state by constant supply of energy; if this supply stops, the systems fall apart and lose their order [74]. This situation clearly does not correspond to what we know about the SC lipid matrix: it maintains its molecular organization in isolated SC despite being detached from its source of energy, the living body. The fact that the lipid matrix is a static and not a dynamic selfassembled structure has an important practical implication: in principle, it should be possible to reproduce the energetically minimized structure of the extended lipid lamellae starting from the processed lipid molecules. The energetics of such one-step self-assembly process will be different from the one of the two-step process found in nature, but the final result should be identical. In other words, just as with model phospholipid membranes, we have the invaluable opportunity to work with model systems with systematically varied, simplified composition instead of treating the full complexity of the molecules in the lipid matrix [19]. As these things go, however, this blessing hides also a danger: depending on the composition and the self-assembly conditions, the resulting aggregates can have drastically different structures. Thus, for example, it is well known from studies with phospholipids that the chain length influences the type of chain packing: all other parameters being the same (e.g. headgroup architecture, sample preparation method, equilibration temperature, etc.), short- and long-chain lipids form phases of different packing at room temperature (i.e. hexagonal and orthorhombic, respectively) [75]. In vivo and in vitro studies of diseased and healthy SC have also demonstrated a direct correlation between the lipid chain length and the packing density and repeat distance in the resulting lipid phases; the relative content of the different ceramides classes also influences the molecular organization of the lipid lamellae [76–80]. As another example, the geometry and content of the lipid phases in samples of identical composition prepared in the exact same way except for a difference in the equilibration temperature of only 10°C are radically different [81]. Similar differences in the phase structure can be induced also by changing the deposition method and the solvent used [81, 82]. Thus, simplified model systems are a precious tool with which to study the physical chemistry of the SC lipids, but only provided a careful and judicious choice of their composition and protocol for preparation. Because the self-assembly of the lipid lamellae is a more complex process than the one of formation of phospholipid bilayers, these two parameters seem to be even more crucial in SC than in phospholipid model membranes.
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Molecular organization of the SC lipids and its relevance for the SC permeability There is a general consensus that the lipid molecules of the SC matrix form extended lamellae in the extracellular space surrounding the corneocytes [26]. In such three-dimensional structures, we distinguish between lateral organization (i.e. the molecular packing in the plane of the lamellae, roughly parallel to the SC surface) and lamellar organization (i.e. the symmetry and the repeat distance in the direction normal to the SC surface) (Fig. 3). Lateral organization In general, in lamellar lipid bilayers such as those found in the SC matrix, long-chain lipids can adopt three types of packing arrangements which differ in their rotational and translational mobilities (Fig. 4). In the most densely packed, orthorhombic (OR) phase, the lipid chains adopt an all-trans conformation and are organized in a rectangular crystalline lattice with no rotational or translational mobility. In the hexagonal (HEX) phase, the all-trans lipid chains have some rotational mobility along their long axes, but their translational mobility is restricted. In the liquid-crystalline (LIQ) phase, the chains have a high degree of gauche isomerization and their lateral organization is completely lost; the chains have both high rotational and high translational mobility in the plane of the membrane. Data from FTIR, WAXD, 2H NMR and electron diffraction have shown that all three phases are present in healthy human SC, with a notable prevalence of the OR phases [83–87], contrary to an earlier model of a single gel-phase matrix [88]. Thus, the average packing density in the SC lipid lamellae is higher than the one
Figure 3 A scheme illustrating the three-dimensional structure of the lipid lamellae. The ovals indicate the lateral organization (i.e. the molecular packing in the plane of the lamellae, roughly parallel to the SC surface) and the lamellar organization (i.e. the symmetry and the repeat distance in the direction normal to the SC surface) of the lipids.
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 36, 505–515
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Figure 4 Lateral molecular organization of the SC lipids. The schemes show the lateral chain packing (top row) and the chain conformation (bottom row) in the orthorhombic (OR), hexagonal (HEX) and liquid-crystalline (LIQ) phases formed by the lipids.
observed in the plasma membranes of living cells which contain predominantly LIQ phases [22, 89, 90]. The relative content of the phases varies within the SC thickness; in healthy adult skin, it does not correlate with age or gender [91]. The lipid molecules are inhomogeneously distributed throughout the different lipid phases, similarly to the cell membranes. Depending on their chain lengths and headgroup architectures, the ceramides and the free fatty acids may participate in the same or in separate OR lattices [34]. Reduction of the content of free fatty acids, shortening the chain lengths of both ceramides and fatty acids and mismatch between the lipid chain lengths inhibit the formation of OR [19, 33, 76, 92, 93]. Ceramide EOS was found to stabilize the OR phases [94]. The location and the mechanism of action of cholesterol in vivo, just like in phospholipids membranes, are still not fully understood [95–97]. It is not clear how the cholesterol molecules are distributed between the phases. Several studies with isolated SC, model lipid mixtures and human skin equivalents have shown presence of phase-separated, pure cholesterol crystals [49, 98]; little is known about them. The solid–solid phase transition (between OR and HEX) occurs at physiologically relevant temperatures, around 35–40°C [66, 99]. The solid–liquid transition (between OR or HEX and LIQ) occurs at non-physiological temperatures, around 70°C, but it can be caused by changes in the phase composition, for example by topical application of chemicals [85]. Thus, the design of effectors of the molecular organization has to consider also the timescale and reversibility of phase reformation following structural perturbation. The lateral lipid organization within the lamellae strongly influences the SC permeability for water. In analogy to phospholipids membranes, conformationally-disordered SC lipids are more permeable than ordered ones. At temperatures above the main phase transition of the SC lipids, around 70°C, the activation energy of the transport of water is lower than the one observed below the transition temperature [100]. What is different from phospholipids membranes, however, is that the flux of water across human skin, the trans-epidermal water loss (TEWL), closely correlates with the
type of solid phases present in the SC: the higher the OR content, the lower the flux of water [87]. These data confirm the observation that there is a strong contribution to the water transport from a non-polar pathway, through the lipid matrix [101]. The lateral lipid organization within the lipid matrix, however, is not the only factor that determines the SC permeability. Several studies have given indications that the lipid organization in the direction normal to the skin surface (i.e. the lamellar organization) also plays an important role in regulating the barrier efficiency [76, 80, 102]. Lamellar organization Whereas all evidence points towards the existence of regularly repeating units within the SC lamellae, there is still no general consensus on the symmetry and the dimensions of the unit cell of the lamellae (Fig. 5). Several models based on a combination of electron microscopy, SAXD, neutron diffraction of isolated SC, human skin equivalents and model lipid mixtures describe the lamellae as layered, centrosymmetric structures of long periodicity (or long-periodicity phases, LPP) with a repeat distance d 13 nm (Fig. 5A, [103–107]). Usually, they coexist with structures of shorter periodicity (or shortperiodicity phases, SPP) with d 6 nm. In SC, the LPP is found between the large, flat surfaces of adjacent corneocytes and the SPP – close to their edges [105]. In electron micrographs, the LPP lamellae appear as centrosymmetric stacks of broad-narrow-broad bands [25]. The existence and the structure of the LPP are extremely sensitive to the lipid composition (for a review, see [19]): (i) the presence of EO ceramides is crucial for the formation of this phase [108, 109]; (ii) the lamellae have to contain just the right fraction of EO ceramides to enable the formation of just the right amount of LIQ phases: if their EO content is high, the formation of SPP is impeded; if their EO content is too low, the formation of LPP is reduced [32]; (iii) the formation of the LPP also necessitates a broad distribution of chain lengths and a variety of headgroup
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(a)
(c)
(b)
Figure 5 Schematic drawings of the current models proposed for the lamellar molecular organization of the SC lipids. (a) Coexisting centrosymmetric phases of long (LPP) and short (SPP) periodicity with repeat distances of approximately 13 and 6 nm, respectively. (b) Phases of short periodicity with a repeat distance of approximately 4.2–4.8 nm. (c) Asymmetric phases of long periodicity with a repeat distance of approximately 11 nm. The solid lines indicate the borders of one lamella (unit cell), the dashed arrows indicate the characteristic periodic repeat distances of the phases, and the dotted lines delimit the individual lipid bilayers. See the text for a discussion of the main features of the models.
architectures [34, 49]; (iv) ceramides and cholesterol can form an LPP in absence of free fatty acids or in presence of short-chain free fatty acids, but it lacks domains with OR organization [92]. Another model based on X-ray and neutron diffraction studies of synthetic lipid mixtures describes the lipid lamellae as containing only phases of short periodicity with d 4.2–4.8 nm (Fig. 5B, [43, 49, 110, 111]). Finally, there is a model in which the unit cell is asymmetrical with d 11 nm (Fig. 5C, [48]). This model is based exclusively on cryo-electron microscopy of vitrified sections (CEMOVIS) and simulations of a lipid mixture of one particular composition which explicitly excludes the EO ceramides. The model does not specify the positions of the crystalline and gel phases within the lamellae. All these different models describe the lipid lamellae as seen through the prism of the particular samples and experimental techniques that the investigators used. The advantages and limitations of these techniques are inevitably reflected in the resulting molecular models. Thus, the majority of the experimental techniques for structural determination cannot be applied in vivo; it is, in
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principle, not excluded that the procedures of sample preparation (e.g. dehydration, fixation, or cutting) may have modified the sample morphology. The resolution of the experimental techniques keeps evolving, and often it is not wise to use as a reference results obtained from earlier versions of the techniques. In conventional electron microscopy, the contrast depends exclusively upon the specificity of stain binding; any, however, small amount of non-specifically bound stain might, in principle, modify the interpretation of the results. In samples of native SC which contain a limited number of repeats, it is often difficult to resolve the diffraction peaks and the samples have to be additionally re-crystallized. The images obtained from CEMOVIS are of exceptionally high quality, but at present it is not clear if their interpretation might change when comparing them with similar images collected from physical samples of known composition instead of images resulting from computer simulation. Most importantly, the various models of the lamellar lipid organization have been based either on SC samples of different origin and different preparation history, or on model systems of drastically different composition that have been formed
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under completely different conditions and have been studied by different experimental techniques; not surprisingly, the conclusions reached in these investigations also differ considerably. Under these circumstances, the models developed using several types of samples and investigated by several techniques seem most convincing. Thus, the coexistence of LPP and SPP and the geometry of the 13-nm LPP have been demonstrated by conventional electron microscopy and SAXD in a huge variety of substrates (hydrated full thickness skin, dried isolated SC, thermally treated SC, human skin equivalents and synthetic lipid mixtures); neutron diffraction and electron density calculations have indicated that the phase is symmetrical. At present, it is not clear why these observations differ from those made by CEMOVIS. I believe that to solve the current uncertainty regarding the lamellar molecular organization of the SC lipids, the most logical, straightforward and ultimately most useful next step is to perform a comparative study of physically identical samples (isolated SC and model lipid mixtures prepared following reproducible and standardized protocols) using several experimental techniques. Only this way, we can hope to develop a model which reflects the whole and not only parts of the picture. To what extent does the lamellar organization of the SC lipids correlate with the SC permeability? We are still in the early stages of our understanding. There are, however, strong indications that the LPP which is present in the SC of all species investigated to date plays an important role in SC permeability [86]. In the SC of patients with several diseases characterized by abnormally high skin permeability (atopic dermatitis, lamellar ichthyosis, psoriasis, Netherton syndrome and Chanarin–Dorfman disease), the periodicity of the LPP is decreased and its general appearance is significantly altered; concomitantly, the content of Cer [EO], the ceramides crucial for the formation of the LPP, is reduced (reviewed in [19, 86]). Studies on the penetration of ethyl para-amino benzoic acid and benzoic acid across model SC membranes have shown that the fluxes of these chemicals increase in absence of the LPP [102, 112]. Of course, a systematic investigation using molecules of different physicochemical properties will be needed to generalize the conclusions regarding the role of the LPP. There are several practical issues that may complicate such studies. In general, it is very difficult to disentangle the relative importance of the lateral and the lamellar organization for the penetration of molecules: without changing the sample composition, it is usually not possible to change the one without influencing the other. Furthermore, it cannot be excluded that the permeants influence the molecular organization during their passage through the SC; for experimental convenience, however, the permeability and the lipid molecular organization have usually been measured using two different samples. Conclusions and outlook The experimental evidence collected thus far indicates unambiguously that the SC lipids are in a class of their own. The principal characteristics of the SC lipid matrix – composition, pH, hydration, self-assembly, phase content and molecular organization – differ considerably from those of all other lipids found in biological structures. Since apparently the exceptional 3D organization of the SC lipids is crucial for the observed exceptional permeability of the SC, rationalizing the permeability of mammalian skin will be possible only if we take into account the specific particularities of the SC lipids and manage to evaluate their relative contribution to the
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parameters relevant for permeability, that is partition coefficient, diffusion coefficient and diffusion path length [113]. Such evaluation, however, necessitates to further develop and complete our understanding of the molecular details of the SC lipid matrix. The issues regarding the composition of the lipid matrix (e.g. if the EO ceramides are present in the interior of the lamellae or if they exist only covalently bound to the corneocyte envelope, what is the full spectrum of ceramides present in healthy and diseased SC, etc.) and the local environment within the lamellae (e.g. pH) are largely issues of methodology. Even though not trivial, these problems can certainly be resolved in the near future. The self-assembly of the lipid lamellae represents a considerably more complicated problem. The exact sequence of events is not well understood, and even their nature and the identity of all participants is not fully known. To understand the forces responsible for the formation and the stability of the extended lamellae, we need to take into account the fact that these structures are formed in a hierarchical, templated process which involves the precursor molecules and the molecular aggregates found in the LB rather than the processed lipids found in the mature lamellae. We have a very strong indication from the studies of molecular recognition that one can ignore the role of water and the interplay between enthalpy and entropy in biological systems only at one’s own risk and peril [114]. There are also many unknowns regarding the 3D molecular organization within the lipid lamellae. What are the limits within which the composition and 3D organization of the lipids in vivo can change while still providing a functional barrier? We are just beginning to recognize the role of the individual lipid classes for the structure of the lipid assemblies; a deeper understanding and further experimental evidence will be required before we can identify with certainty the structural and functional importance of each of them. The lipid organization around potential sites of structural defects in the lamellar structure, for example the cell borders, the SC enzymes and the phase boundaries, is also not well understood or proven. The reasons behind the presence of phase-separated cholesterol are not clear. I believe that wisely designed, model systems based on welldefined lipid mixtures will be invaluable to address these questions; indeed, numerous studies have already demonstrated that the skin barrier properties of healthy and diseased skin can be successfully reproduced and investigated in detail in synthetic lipid models (reviewed in [19]). Paying scrupulous attention to the minute aspects of composition, sample deposition, self-assembly conditions and measurements of performance can only improve the quality of these models – that is, the degree to which they resemble the original. The aphorism often ascribed to Einstein ‘Everything should be made as simple as possible – but not simpler’ provides a good practical guidance for designing useful models while avoiding the pitfall of tempting but harmful oversimplification. Our understanding of the structure and the properties of the SC lipid matrix will advance enormously by comparative studies performed on the same physical samples by different experimental methods. Acknowledgements I thank sincerely Joke A. Bouwstra, David J. Moore, George M. Whitesides, Phil W. Wertz and my colleagues from Firmenich R&D for many insightful discussions on the physical chemistry of lipids, and the anonymous reviewers of IJCS for their valuable comments. This work was financed by Firmenich SA.
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 36, 505–515
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References 1. Robert, M.S. and Adelaide, S.A., eds. Skin delivery – to Scheuplein and beyond. Skin Pharmacol. Physiol. 26. (2013). 2. WHO, Department of Child and Adolescent Health and Development. Epidemiology and management of common skin diseases in children in developing countries. Report WHO/FCH/CAH/05.12.(2005). Available at: http://www.who.int/maternal_child_ adolescent/documents/fch_cah_05_12/en/. Last accessed September 2014 3. Yasmin, S., Osrin, D., Paul, E. and Costello, A. Neonatal mortality of low-birth-weight infants in Bangladesh. Bull. World Health Organ. 79, 608–614 (2001). 4. Bang, A.T., Bang, R.A., Baitule, S.B., Reddy, H.M. and Deshmukh, M.D. Low birth weight and preterm neonates: can they be managed at home by mother and a trained village health worker? J. Perinatol. 25(Suppl. 1), S72–S81 (2005). 5. Darmstadt, G.L., Bhutta, Z.A., Cousens, S. et al. Evidence-based, cost-effective interventions: how many newborn babies can we save? Lancet 365, 977–988 (2005). 6. WHO, March of Dimes, The Partnership for Maternal, Newborn, and Child health. Save the Children. Born too soon: the global action report on preterm birth (2012). Available at: http://www.who.int/maternal_child_adolescent/documents/born_too_ soon/en/. Last accessed September 2014 7. Williams, H., Svensson, A., Diepgen, T. et al. (European Dermato-Epidemiology Network, EDEN). Epidemiology of skin diseases in Europe. Eur. J. Dermatol. 16, 212– 218 (2008). 8. Kunjumoideen, K. Allergy statistics. Available at: http://medicineworld.org/medicine/ allergy/allergy-statistics.html. Last accessed September 2014 9. Prausnitz, M.R. and Langer, R. Transdermal drug delivery. Nature Biotech. 26, 1261–1268 (2008). 10. Brain, K.R., Watkinson, A.C. and Walters, K.A. Reduction of the skin penetration of xenobiotics using chemical penetration retarders. In: NBC Risks: Current Capabilities and Future Perspectives for Protection (Sohns, T. and Voicu, V.A., eds.), pp. 271– 277. Kluwer Academic Publishers, Dordrecht, The Netherlands (1999). 11. Braue E.H., Jr., Doxzon, B.F., Lumpkin, H.L., Hanssen, K.A., Stevenson, R.S., Deckert, R.R. and Graham, J.S. Military perspectives in chemical penetration retardation. In: Percutaneous Penetration Enhancers (Smith, E.W. and Maibach, H.I., eds), pp.
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doi: 10.1111/ics.12162
Review Article
The physical chemistry of the stratum corneum lipids M. Boncheva Corporate R&D Division, Firmenich SA, PO Box 239, Route des Jeunes 1, Geneva CH-1211, Switzerland
Received 25 July 2014, Accepted 11 September 2014
Keywords: infrared spectroscopy, molecular organization, skin barrier lipids, skin permeability, stratum corneum, X-ray diffraction
Synopsis The extracellular matrix of stratum corneum (SC) participates actively in the defensive performance of human skin. Understanding its physicochemical properties is essential for understanding the skin homoeostasis, for the development of efficient therapies for skin disorders and diseases and for ensuring the safety of products designed for topical application. This article summarizes the current knowledge of the composition, self-assembly and molecular organization of the SC lipids, reviews the evidence connecting these parameters and the barrier properties of human skin, and outlines the immediate issues in the field of SC lipid research. La matrice extracellulaire du stratum corneum (SC) parRESUM E: ticipe activement a la performance defensive de la peau humaine. Comprendre ses proprietes physico-chimiques est essentielle pour comprendre l’homeostasie de la peau, pour le developpement de therapies efficaces pour les troubles et les maladies de la peau, et pour assurer la securite des produits concßus pour une application topique. Cet article resume les connaissances actuelles sur la composition, l’auto-assemblage, et l’organisation moleculaire des lipides SC, examine les preuves reliant ces parametres et les proprietes de barriere de la peau humaine, et decrit les problemes immediats dans le domaine de la recherche sur les lipides SC. Introduction The field of skin research is replete with opportunities for those intent on working on scientifically interesting topics while making a noticeable, positive difference in people’s lives [1]. The range of important problems that the field can help solving is broad. It comprises (i) reducing infant mortality, especially in the developing countries where defective skin barrier is the second major cause of preterm mortality [2–6]; (ii) improving the skin health of some 20– 30% of the population in the developed countries which suffer from some sort of skin barrier dysfunction [7, 8]; (iii) developing efficient strategies for the dermal and transdermal delivery of drugs and vaccines [9]; (iv) increasing the national and personal security by developing methods to retard or prevent the skin penetration of chemical warfare agents, toxic spills and pesticides [10, 11]; (v) enhancing the safety of topical products (e.g. fragrances, cosmetics, cleansing agents, sunscreens and insect repellents) [12]. Thus, the Correspondence: Mila Boncheva, Corporate R&D Division, Firmenich SA, PO Box 239, Route des Jeunes 1, CH-1211 Geneva 8, Switzerland. Tel.: (+41) 22 780 3027; fax: (+41) 22 780 3334; e-mail: mila.boncheva@ firmenich.com
targets in skin research cover the full spectrum from counter-acting leaky skin and preventing its penetration by chemicals to pushing chemicals through it. One common major problem underlies these diverse topics: our insufficient understanding of the factors that determine skin permeability. Understanding the permeability of human skin to a large extent means understanding the extracellular lipid matrix of the stratum corneum (SC), the topmost skin layer which constitutes the main barrier to transdermal fluxes [13–19]. As the lipid matrix forms an uninterrupted pathway from the skin surface to the viable skin tissues, it provides the principle path of entry for many chemicals [20]. At first sight, backed by more than 100 years of excellent and extensive studies on the physical chemistry and biophysics of cell membrane lipids, we should have a fairly good grip on the properties of the SC lipids as well [21, 22]. Should we rely on this assumption, however? Can we apply directly the knowledge of the physical chemistry of the biomembrane lipids to rationalize the SC permeability, or do we need to consider the SC lipids as species apart? To answer these questions, this review compares the composition, local environment, self-assembly and molecular organization of the SC lipids to those of the lipids in the cell membrane. Composition and local environment of the SC lipid matrix In terms of composition, the SC and the cellular lipids differ drastically. Instead of the phospholipids, glycolipids and cholesterol common for cell membranes, the SC lipid matrix contains approximately equimolar amounts of ceramides, free fatty acids and cholesterol [23–25] (Fig. 1). The ceramide molecules consist of fatty acids amide linked to sphingoid bases. Fifteen ceramide subclasses have been identified so far in the extracellular space of SC [28–30]. The ceramide molecules have different headgroup architectures, all of them considerably smaller and less polar than those of the lipids in a typical phospholipid membrane. The different classes of ceramides molecules can participate in different inter- and intramolecular interactions with the other lipid molecules and thus make distinct contributions to the organization and the structural integrity of the lipid matrix [31–35]. Both the ceramides and the fatty acids of the SC have unusually long hydrocarbon chains with a broad distribution of lengths [29, 30, 35–37]. The variety of chain lengths and headgroup structures results in the existence of over 400 different ceramide species [29, 30, 38]. Most common for the ceramides are lengths of 24–26 carbon atoms for the fatty acid hydrocarbon chain and 18–22 carbon atoms for the sphingoid
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Figure 1 Chemical structures of the lipids of human SC (modified from [26]). The nomenclature of the most prevalent 12 ceramide classes is shown using the classification of Motta et al. [27] (in capital letters) and using the earlier classification based on chromatographic mobility (in parentheses). Each ceramide molecule contains one sphingoid and one fatty acid residue, amide-linked as shown above the table for Cer [NP]. The number ranges indicate the prevalent lengths of the hydrocarbon chains in ceramides and free fatty acids. The structures of the three most recently identified ceramides and a suggestion for an alternative ceramides nomenclature can be found in [28].
hydrocarbon chain [37], whereas most common for the fatty acids are lengths of 22–26 carbon atoms [39]. Contrary to the cell membrane lipids, the lipid hydrocarbon chains of both ceramides and fatty acids are predominantly saturated. The only SC lipids that contain non-saturated fatty acid residues (esterified to particularly long fatty acid chains of 30–32 carbon atoms) are the x-hydroxy (EO) ceramides; most of them exist covalently linked to proteins of the corneocyte envelope [25, 40, 41]. The conformation that the ceramides molecules adopt in vivo – splayed, hairpin, or V-shape – is still under debate [42, 43]. The SC matrix contains also minor (in mass but not in importance) ingredients, including cholesterol sulphate and cholesteryl esters [44, 45]. The local environment of the SC and cell lipids also differs. In a living tissue, the cell lipids exist in a water-rich environment; the headgroup hydration in a typical phospholipid membrane easily reaches 7–16 molecules per headgroup [22]. In contrast, the water content within the SC lipid lamellae is extremely low and they contain no free, unbound water. Infrared (IR) spectroscopy has shown that the phase transition temperature of the SC lipids decreases only slightly upon massive hydration [46, 47]. Both conventional and cryo-electron microscopy have shown that the lipid lamellae do not swell upon hydration [48]. More recently, calculations based on neutron diffraction demonstrated that the packing of the lipid molecules within the lamellae leaves no space for water molecules [49, 50]. Thus, the water content of the lamellae in their
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normal, air-dried state and at relative air humidity of up to 90% is estimated to be only about 1–2 molecules bound per headgroup, or ten times lower than a typical phospholipid membrane. The pH to which the lipids of living cells are exposed is neutral; it can be measured easily using pH electrodes. Applying the same strategy to measure the pH within the SC lipid matrix, however, can be very misleading. The often-quoted profiles of pH within the SC obtained using flat pH electrodes and tape-stripping [51, 52] – uniformly increasing with depth from acidic at the surface to neutral in the deep SC layers – carry no information about the lipid matrix. As these electrodes contact the skin surface through a small water droplet, what they measure in fact is the average distribution of water-soluble species over the SC surface. In principle, several other strategies can be used to estimate the pH within the SC lipid lamellae. Two-photon fluorescence lifetime imaging has shown that throughout the SC depth the extracellular space contains acidic microdomains of variable length, with an average pH value around 6 [53]. The number of these domains changes with the depth of the SC. The application of this strategy, however, necessitates making assumptions for the refractive index of the lipid matrix and the pKa values of the fluorescent probes within the lipid lamellae; furthermore, the Henderson–Hasselbalch correlation used to calculate the pH is valid only for dilute water solutions, a situation far from the reality within the SC lipids matrix. Another approach for calculating the proton concentrations
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within the lipid bilayers of the SC matrix is based on simplified molecular models [54]. In principle, the calculation of the proton activity in non-aqueous medium such as the lipid lamellae can be based also on calculations of its Gibbs free energy of solvation approximating the lipids to a homogeneous oil phase [55]. It is not yet clear which of these approaches is best suited to describe the acidity of the lipid matrix. It is important to note that, strictly speaking, applying the conventional notion of pH to a system such as the lipid matrix is not correct: as there is no free water in the lamellae, talking about the activity of hydrated protons (i.e. pH by definition) makes no sense. In summary, both the composition and the local environment of the lipids within the SC and the cell membranes differ considerably. These differences, however, cannot explain the difference of three orders of magnitude between the observed permeation coefficients for water measured in the two systems (equal to approximately 103 cms1 in phospholipid membranes and approximately 103 cmh1 in the SC lipid matrix). The excellent works of Fettiplace on phospholipid bilayers have shown that differences in their composition can result in not more than an order of magnitude difference in their permeability [56, 57]. Clearly, additional factors related to differences in the structures of the two lipid systems are at play. Formation of the lipid lamellae The formation of the SC lipid lamellae differs considerably from all we know about the formation of a phospholipid bilayer [28, 58– 60]. Contrary to cell membranes, the process is independent on the presence of proteins. It starts with a mixture of polar lipid precursors, namely sphingomyelins, glucosylceramides, phospholipids and cholesterol [44] (Fig. 2). These precursors, synthesized (or imported) in the keratinocytes (for a review, see [28]), are transported in the trans-Golgi network where they self-assemble into short stacks within ovoid or tubular structures, the lamellar bodies (LB) [61–63]. The molecular organization of the precursors in the LB is not well understood. The details of following stages – secretion of the LB content into the extracellular space, processing of the precursors to the true SC lipids of lower polarity and self-assembly of the stacks into extended 3D lamellae – are even less clear. It
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is not known if the processing of all lipid types occurs at the same time or sequentially, how the short stacks fuse into lamellae, or to what extent their molecular organization changes upon their fusion. Essentially, the process of formation of the lipid lamellae is one of hierarchical, templated self-assembly involving two distinct stages: the first one comprises the organization of the precursor molecules into bilayer stacks within the LB, and the second one – the assembly of these stacks into lamellae within the extracellular space of the SC. The two stages of the self-assembly process differ considerably; only the first one bears some similarities with the formation of a phospholipids bilayer. 1 Components. The self-assembling components in the first stage are the polar precursor molecules, and in the second stage – the short bilayer stacks of the LB. 2 Temperature/agitation. For self-assembly to occur, the assembling components need to interact via balancing repulsive and attractive forces, or they must be able to move and adjust their relative positions [64]. The thermal energy which moves around the components of the first stage (i.e. the precursor molecules) is not enough to move nano-sized objects (i.e. the lipid bilayer stacks), so the energy for ‘mixing’ and movement in the second stage is probably provided by the lipid processing. 3 Interactions. In both stages, the interactions between the components are weak and non-covalent. Whereas attractive van der Waals forces between the long hydrocarbon chains are present in each stage, the patterns of hydrogen bonds between the more polar precursors and the less polar processed molecules are not necessarily the same [65–67]. Because of the different environment in which the self-assembly proceeds, the hydrophobic interactions involved in the two stages probably also differ: in the first one, they can be expected to arise from expelling water, similar to the formation of a lipid bilayer [68], whereas in the second one, they might be due to restructuring bound water molecules, in analogy with the situation observed in the processes of ligandreceptor binding [69]. 4 Templating. Both self-assembly stages appear to be assisted by external templating, in analogy with many other processes of biological self-assembly [70]. In the first one, it is provided by the
Figure 2 A scheme illustrating the self-assembly of the lipid lamellae. The process starts with a mixture of polar precursor molecules (sphingomyelins, glucosylceramides, cholesterol and phospholipids, shown on the left) which are synthesized or imported in the keratinocytes and subsequently transported in the trans-Golgi network (step 1). There, they self-assemble into short stacks within ovoid or tubular structures, the lamellar bodies (LB, step 2). The details and the exact sequence of following stages – secretion of the LB content into the extracellular space (step 3), processing of the precursors to the true SC lipids of lower polarity (ceramides, cholesterol and free fatty acids, shown on the right), and self-assembly of the stacks into extended 3D lamellae (step 4) – are not well understood. See the text for details.
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restricting volume of the membrane-limited space within the LB and possibly by caveolins [58, 71] and in the second one – by the lipids covalently linked to the corneocyte envelope [35, 72]. Clearly, the energetics of this two-stage hierarchical, templated process can be expected to differ from the one-step self-assembly of phospholipid molecules into bilayers. The experimental evidence collected so far indicates that the SC lipid lamellae represent a static and not a dynamic self-assembled structure. By definition, this distinction is based on the final structure and not on the process itself [73]. Thus, static self-assembled structures are formed by minimization of the free energy of the system and exist at a global or local energy minimum. In the case of the self-assembly of the SC lipid lamellae, the system seems to be driven from one equilibrium state to another by non-equilibrium agitation, for example enzyme delivery and secretion of the bilayer stacks in the extracellular space. In contrast, dynamic selfassembled structures are non-equilibrium, energy dissipating ones, maintained in a steady state by constant supply of energy; if this supply stops, the systems fall apart and lose their order [74]. This situation clearly does not correspond to what we know about the SC lipid matrix: it maintains its molecular organization in isolated SC despite being detached from its source of energy, the living body. The fact that the lipid matrix is a static and not a dynamic selfassembled structure has an important practical implication: in principle, it should be possible to reproduce the energetically minimized structure of the extended lipid lamellae starting from the processed lipid molecules. The energetics of such one-step self-assembly process will be different from the one of the two-step process found in nature, but the final result should be identical. In other words, just as with model phospholipid membranes, we have the invaluable opportunity to work with model systems with systematically varied, simplified composition instead of treating the full complexity of the molecules in the lipid matrix [19]. As these things go, however, this blessing hides also a danger: depending on the composition and the self-assembly conditions, the resulting aggregates can have drastically different structures. Thus, for example, it is well known from studies with phospholipids that the chain length influences the type of chain packing: all other parameters being the same (e.g. headgroup architecture, sample preparation method, equilibration temperature, etc.), short- and long-chain lipids form phases of different packing at room temperature (i.e. hexagonal and orthorhombic, respectively) [75]. In vivo and in vitro studies of diseased and healthy SC have also demonstrated a direct correlation between the lipid chain length and the packing density and repeat distance in the resulting lipid phases; the relative content of the different ceramides classes also influences the molecular organization of the lipid lamellae [76–80]. As another example, the geometry and content of the lipid phases in samples of identical composition prepared in the exact same way except for a difference in the equilibration temperature of only 10°C are radically different [81]. Similar differences in the phase structure can be induced also by changing the deposition method and the solvent used [81, 82]. Thus, simplified model systems are a precious tool with which to study the physical chemistry of the SC lipids, but only provided a careful and judicious choice of their composition and protocol for preparation. Because the self-assembly of the lipid lamellae is a more complex process than the one of formation of phospholipid bilayers, these two parameters seem to be even more crucial in SC than in phospholipid model membranes.
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Molecular organization of the SC lipids and its relevance for the SC permeability There is a general consensus that the lipid molecules of the SC matrix form extended lamellae in the extracellular space surrounding the corneocytes [26]. In such three-dimensional structures, we distinguish between lateral organization (i.e. the molecular packing in the plane of the lamellae, roughly parallel to the SC surface) and lamellar organization (i.e. the symmetry and the repeat distance in the direction normal to the SC surface) (Fig. 3). Lateral organization In general, in lamellar lipid bilayers such as those found in the SC matrix, long-chain lipids can adopt three types of packing arrangements which differ in their rotational and translational mobilities (Fig. 4). In the most densely packed, orthorhombic (OR) phase, the lipid chains adopt an all-trans conformation and are organized in a rectangular crystalline lattice with no rotational or translational mobility. In the hexagonal (HEX) phase, the all-trans lipid chains have some rotational mobility along their long axes, but their translational mobility is restricted. In the liquid-crystalline (LIQ) phase, the chains have a high degree of gauche isomerization and their lateral organization is completely lost; the chains have both high rotational and high translational mobility in the plane of the membrane. Data from FTIR, WAXD, 2H NMR and electron diffraction have shown that all three phases are present in healthy human SC, with a notable prevalence of the OR phases [83–87], contrary to an earlier model of a single gel-phase matrix [88]. Thus, the average packing density in the SC lipid lamellae is higher than the one
Figure 3 A scheme illustrating the three-dimensional structure of the lipid lamellae. The ovals indicate the lateral organization (i.e. the molecular packing in the plane of the lamellae, roughly parallel to the SC surface) and the lamellar organization (i.e. the symmetry and the repeat distance in the direction normal to the SC surface) of the lipids.
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Figure 4 Lateral molecular organization of the SC lipids. The schemes show the lateral chain packing (top row) and the chain conformation (bottom row) in the orthorhombic (OR), hexagonal (HEX) and liquid-crystalline (LIQ) phases formed by the lipids.
observed in the plasma membranes of living cells which contain predominantly LIQ phases [22, 89, 90]. The relative content of the phases varies within the SC thickness; in healthy adult skin, it does not correlate with age or gender [91]. The lipid molecules are inhomogeneously distributed throughout the different lipid phases, similarly to the cell membranes. Depending on their chain lengths and headgroup architectures, the ceramides and the free fatty acids may participate in the same or in separate OR lattices [34]. Reduction of the content of free fatty acids, shortening the chain lengths of both ceramides and fatty acids and mismatch between the lipid chain lengths inhibit the formation of OR [19, 33, 76, 92, 93]. Ceramide EOS was found to stabilize the OR phases [94]. The location and the mechanism of action of cholesterol in vivo, just like in phospholipids membranes, are still not fully understood [95–97]. It is not clear how the cholesterol molecules are distributed between the phases. Several studies with isolated SC, model lipid mixtures and human skin equivalents have shown presence of phase-separated, pure cholesterol crystals [49, 98]; little is known about them. The solid–solid phase transition (between OR and HEX) occurs at physiologically relevant temperatures, around 35–40°C [66, 99]. The solid–liquid transition (between OR or HEX and LIQ) occurs at non-physiological temperatures, around 70°C, but it can be caused by changes in the phase composition, for example by topical application of chemicals [85]. Thus, the design of effectors of the molecular organization has to consider also the timescale and reversibility of phase reformation following structural perturbation. The lateral lipid organization within the lamellae strongly influences the SC permeability for water. In analogy to phospholipids membranes, conformationally-disordered SC lipids are more permeable than ordered ones. At temperatures above the main phase transition of the SC lipids, around 70°C, the activation energy of the transport of water is lower than the one observed below the transition temperature [100]. What is different from phospholipids membranes, however, is that the flux of water across human skin, the trans-epidermal water loss (TEWL), closely correlates with the
type of solid phases present in the SC: the higher the OR content, the lower the flux of water [87]. These data confirm the observation that there is a strong contribution to the water transport from a non-polar pathway, through the lipid matrix [101]. The lateral lipid organization within the lipid matrix, however, is not the only factor that determines the SC permeability. Several studies have given indications that the lipid organization in the direction normal to the skin surface (i.e. the lamellar organization) also plays an important role in regulating the barrier efficiency [76, 80, 102]. Lamellar organization Whereas all evidence points towards the existence of regularly repeating units within the SC lamellae, there is still no general consensus on the symmetry and the dimensions of the unit cell of the lamellae (Fig. 5). Several models based on a combination of electron microscopy, SAXD, neutron diffraction of isolated SC, human skin equivalents and model lipid mixtures describe the lamellae as layered, centrosymmetric structures of long periodicity (or long-periodicity phases, LPP) with a repeat distance d 13 nm (Fig. 5A, [103–107]). Usually, they coexist with structures of shorter periodicity (or shortperiodicity phases, SPP) with d 6 nm. In SC, the LPP is found between the large, flat surfaces of adjacent corneocytes and the SPP – close to their edges [105]. In electron micrographs, the LPP lamellae appear as centrosymmetric stacks of broad-narrow-broad bands [25]. The existence and the structure of the LPP are extremely sensitive to the lipid composition (for a review, see [19]): (i) the presence of EO ceramides is crucial for the formation of this phase [108, 109]; (ii) the lamellae have to contain just the right fraction of EO ceramides to enable the formation of just the right amount of LIQ phases: if their EO content is high, the formation of SPP is impeded; if their EO content is too low, the formation of LPP is reduced [32]; (iii) the formation of the LPP also necessitates a broad distribution of chain lengths and a variety of headgroup
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(a)
(c)
(b)
Figure 5 Schematic drawings of the current models proposed for the lamellar molecular organization of the SC lipids. (a) Coexisting centrosymmetric phases of long (LPP) and short (SPP) periodicity with repeat distances of approximately 13 and 6 nm, respectively. (b) Phases of short periodicity with a repeat distance of approximately 4.2–4.8 nm. (c) Asymmetric phases of long periodicity with a repeat distance of approximately 11 nm. The solid lines indicate the borders of one lamella (unit cell), the dashed arrows indicate the characteristic periodic repeat distances of the phases, and the dotted lines delimit the individual lipid bilayers. See the text for a discussion of the main features of the models.
architectures [34, 49]; (iv) ceramides and cholesterol can form an LPP in absence of free fatty acids or in presence of short-chain free fatty acids, but it lacks domains with OR organization [92]. Another model based on X-ray and neutron diffraction studies of synthetic lipid mixtures describes the lipid lamellae as containing only phases of short periodicity with d 4.2–4.8 nm (Fig. 5B, [43, 49, 110, 111]). Finally, there is a model in which the unit cell is asymmetrical with d 11 nm (Fig. 5C, [48]). This model is based exclusively on cryo-electron microscopy of vitrified sections (CEMOVIS) and simulations of a lipid mixture of one particular composition which explicitly excludes the EO ceramides. The model does not specify the positions of the crystalline and gel phases within the lamellae. All these different models describe the lipid lamellae as seen through the prism of the particular samples and experimental techniques that the investigators used. The advantages and limitations of these techniques are inevitably reflected in the resulting molecular models. Thus, the majority of the experimental techniques for structural determination cannot be applied in vivo; it is, in
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principle, not excluded that the procedures of sample preparation (e.g. dehydration, fixation, or cutting) may have modified the sample morphology. The resolution of the experimental techniques keeps evolving, and often it is not wise to use as a reference results obtained from earlier versions of the techniques. In conventional electron microscopy, the contrast depends exclusively upon the specificity of stain binding; any, however, small amount of non-specifically bound stain might, in principle, modify the interpretation of the results. In samples of native SC which contain a limited number of repeats, it is often difficult to resolve the diffraction peaks and the samples have to be additionally re-crystallized. The images obtained from CEMOVIS are of exceptionally high quality, but at present it is not clear if their interpretation might change when comparing them with similar images collected from physical samples of known composition instead of images resulting from computer simulation. Most importantly, the various models of the lamellar lipid organization have been based either on SC samples of different origin and different preparation history, or on model systems of drastically different composition that have been formed
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under completely different conditions and have been studied by different experimental techniques; not surprisingly, the conclusions reached in these investigations also differ considerably. Under these circumstances, the models developed using several types of samples and investigated by several techniques seem most convincing. Thus, the coexistence of LPP and SPP and the geometry of the 13-nm LPP have been demonstrated by conventional electron microscopy and SAXD in a huge variety of substrates (hydrated full thickness skin, dried isolated SC, thermally treated SC, human skin equivalents and synthetic lipid mixtures); neutron diffraction and electron density calculations have indicated that the phase is symmetrical. At present, it is not clear why these observations differ from those made by CEMOVIS. I believe that to solve the current uncertainty regarding the lamellar molecular organization of the SC lipids, the most logical, straightforward and ultimately most useful next step is to perform a comparative study of physically identical samples (isolated SC and model lipid mixtures prepared following reproducible and standardized protocols) using several experimental techniques. Only this way, we can hope to develop a model which reflects the whole and not only parts of the picture. To what extent does the lamellar organization of the SC lipids correlate with the SC permeability? We are still in the early stages of our understanding. There are, however, strong indications that the LPP which is present in the SC of all species investigated to date plays an important role in SC permeability [86]. In the SC of patients with several diseases characterized by abnormally high skin permeability (atopic dermatitis, lamellar ichthyosis, psoriasis, Netherton syndrome and Chanarin–Dorfman disease), the periodicity of the LPP is decreased and its general appearance is significantly altered; concomitantly, the content of Cer [EO], the ceramides crucial for the formation of the LPP, is reduced (reviewed in [19, 86]). Studies on the penetration of ethyl para-amino benzoic acid and benzoic acid across model SC membranes have shown that the fluxes of these chemicals increase in absence of the LPP [102, 112]. Of course, a systematic investigation using molecules of different physicochemical properties will be needed to generalize the conclusions regarding the role of the LPP. There are several practical issues that may complicate such studies. In general, it is very difficult to disentangle the relative importance of the lateral and the lamellar organization for the penetration of molecules: without changing the sample composition, it is usually not possible to change the one without influencing the other. Furthermore, it cannot be excluded that the permeants influence the molecular organization during their passage through the SC; for experimental convenience, however, the permeability and the lipid molecular organization have usually been measured using two different samples. Conclusions and outlook The experimental evidence collected thus far indicates unambiguously that the SC lipids are in a class of their own. The principal characteristics of the SC lipid matrix – composition, pH, hydration, self-assembly, phase content and molecular organization – differ considerably from those of all other lipids found in biological structures. Since apparently the exceptional 3D organization of the SC lipids is crucial for the observed exceptional permeability of the SC, rationalizing the permeability of mammalian skin will be possible only if we take into account the specific particularities of the SC lipids and manage to evaluate their relative contribution to the
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parameters relevant for permeability, that is partition coefficient, diffusion coefficient and diffusion path length [113]. Such evaluation, however, necessitates to further develop and complete our understanding of the molecular details of the SC lipid matrix. The issues regarding the composition of the lipid matrix (e.g. if the EO ceramides are present in the interior of the lamellae or if they exist only covalently bound to the corneocyte envelope, what is the full spectrum of ceramides present in healthy and diseased SC, etc.) and the local environment within the lamellae (e.g. pH) are largely issues of methodology. Even though not trivial, these problems can certainly be resolved in the near future. The self-assembly of the lipid lamellae represents a considerably more complicated problem. The exact sequence of events is not well understood, and even their nature and the identity of all participants is not fully known. To understand the forces responsible for the formation and the stability of the extended lamellae, we need to take into account the fact that these structures are formed in a hierarchical, templated process which involves the precursor molecules and the molecular aggregates found in the LB rather than the processed lipids found in the mature lamellae. We have a very strong indication from the studies of molecular recognition that one can ignore the role of water and the interplay between enthalpy and entropy in biological systems only at one’s own risk and peril [114]. There are also many unknowns regarding the 3D molecular organization within the lipid lamellae. What are the limits within which the composition and 3D organization of the lipids in vivo can change while still providing a functional barrier? We are just beginning to recognize the role of the individual lipid classes for the structure of the lipid assemblies; a deeper understanding and further experimental evidence will be required before we can identify with certainty the structural and functional importance of each of them. The lipid organization around potential sites of structural defects in the lamellar structure, for example the cell borders, the SC enzymes and the phase boundaries, is also not well understood or proven. The reasons behind the presence of phase-separated cholesterol are not clear. I believe that wisely designed, model systems based on welldefined lipid mixtures will be invaluable to address these questions; indeed, numerous studies have already demonstrated that the skin barrier properties of healthy and diseased skin can be successfully reproduced and investigated in detail in synthetic lipid models (reviewed in [19]). Paying scrupulous attention to the minute aspects of composition, sample deposition, self-assembly conditions and measurements of performance can only improve the quality of these models – that is, the degree to which they resemble the original. The aphorism often ascribed to Einstein ‘Everything should be made as simple as possible – but not simpler’ provides a good practical guidance for designing useful models while avoiding the pitfall of tempting but harmful oversimplification. Our understanding of the structure and the properties of the SC lipid matrix will advance enormously by comparative studies performed on the same physical samples by different experimental methods. Acknowledgements I thank sincerely Joke A. Bouwstra, David J. Moore, George M. Whitesides, Phil W. Wertz and my colleagues from Firmenich R&D for many insightful discussions on the physical chemistry of lipids, and the anonymous reviewers of IJCS for their valuable comments. This work was financed by Firmenich SA.
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