Review Skin Pharmacol Physiol 2008;21:58–74 DOI: 10.1159/000112956
Received: April 13, 2007 Accepted after revision: July 23, 2007 Published online: January 11, 2008
Properties of Ceramides and Their Impact on the Stratum Corneum Structure: A Review Part 2: Stratum Corneum Lipid Model Systems
D. Kessner a A. Ruettinger a M.A. Kiselev c S. Wartewig b R.H.H. Neubert a Institutes of a Pharmacy and b Applied Dermatopharmacy, Martin Luther University Halle-Wittenberg, Halle-Saale, Germany; c Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Dubna, Russia
Key Words Stratum corneum Ceramides SC lipid model systems Neutron diffraction
Abstract The stratum corneum (SC) represents the outermost layer of the mammalian skin, exhibits the main skin barrier and plays an important role in the water penetration pathway through the SC. Knowing the structure and properties of the SC at the molecular level is essential for studying drug penetration through the SC and for the development of new dermal drug delivery systems. Therefore, research interest is focused on the SC lipid matrix and on water diffusion through it. Thus, the ultimate aim is to design a lipid mixture that mimics the barrier properties of the human SC to a high extent and that can substitute the SC in drug delivery systems. This review summarizes various studies performed on either isolated animal or human ceramide based SC model systems, coming to the result that using synthetic lipids with a well-defined architecture allows good extrapolation to the in vivo situation. This review is the continuation of part 1 that is focused on a detailed description of the thermotropic and/or lyotropic phase behaviour of single ceramide types obtained by various experimental techniques. The objective of part 2 is to reflect the numerous studies on SC lipid model systems, namely binary, ternary and multicomponent systems, during the last decade. In this context, neutron diffraction as a
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prospective tool for analyzing the internal membrane structure is addressed in particular. Based on these new insights, current SC models are presented, whose validations are still under discussion. A profound knowledge about SC lipid organization at the molecular level is still missing. Copyright © 2008 S. Karger AG, Basel
Introduction
The lipid organization in the superficial layer of the skin, the stratum corneum (SC), is important for skin barrier function. The intercellular matrix of multilamellar organized lipids is mainly formed by ceramides (CER), cholesterol (CHOL) and long-chain free fatty acids (FFA) [1–3]. To date, 9 different classes of CER, representing the main constituent, are identified [4, 5]. They are classified as shown in figure 1. CER are known to play a key role in structuring and maintaining the SC barrier function [6, 7], although the function of each subclass has not been elucidated until now. Therefore, a detailed knowledge about the molecular arrangement of lipids in the SC is essential for a deeper understanding of the SC barrier properties and for a more rational design of dermal drug delivery systems. CER consist of a long-chain fatty acid bound to the amino group of a long-chain di- or trihydroxy sphingoid base (sphingosine, phytosphingosine, and 6-hydroxyProf. Dr. Dr. R.H.H. Neubert Institute of Pharmacy, Martin Luther University Halle-Wittenberg Wolfgang-Langenbeck-Strasse 4, DE–06120 Halle-Saale (Germany) Tel. +49 345 552 5000, Fax +49 345 552 7292 E-Mail
[email protected]
O O
O HN
OH OH
Ceramide 1 [EOS] O HN
O HN
OH OH
Ceramide 2 [NS]
OH
Ceramide 3 [NP]
OH OH
O O
O HN
OH OH
Ceramide 4 [EOH] OH OH
O
O HN
OH OH
Ceramide 5 [AS]
OH OH O
O
HN OH
HN
OH OH
OH
Ceramide 8 [AH]
OH OH
O
Fig. 1. Chemical structure and nomencla-
ture of CER of the SC. A = -Hydroxy fatty acid, EO = ester-linked -hydroxy acid, N = nonhydroxy fatty acid, P = phytosphingosine, S = sphingosine, H = 6-hydrosphingosine.
OH OH
Ceramide 6 [NH] OH
Ceramide 7 [AP]
HN
O
O HN
Ceramide 9 [EOP]
OH OH
OH
sphinganine). The acyl residue of CER can be hydroxylated at the -position to the carbonyl oxygen or at the end of the hydrocarbon chain (position). The first studies on the human SC tried to elucidate the lipid organization at a healthy state and made attempts to compare it to changes in diseased skin. Based on these studies, most of the prominent theoretic SC lipid models concerning the lipid assembly were proposed, which are still debated vigorously [8–16]. In the first studies, the samples used contained no well-defined lipids. They were characterized by varying head groups and chain length distribution. This fact made it complex to find a correlation between lipid structure and lipid organization and thereby validate each of the proposed models. Nowadays, a new possibility arises, using well-defined synthetic lipids, especially CER with a defined acyl chain and head group architecture. SC lipid model systems, prepared from the lipids in a fixed composition ratio, improve the knowledge about the structural and the phase
behaviour of the mixture as well as of the single components [17–20]. Further on, the impact of single lipid species and the interactions between the lipids as well as the influence by external parameters such as temperature, humidity and enhancer molecules can be examined on a high level. Finally, having well-defined lipid model systems allows a better extrapolation to the in vivo situation including the possibility to develop a suitable dermal drug delivery system. Therefore, the ultimate aim is to design a lipid mixture that mimics the barrier properties of the human skin to a high extent and that can substitute the SC in drug delivery systems. This review is the continuation of part 1, which summarizes the physical properties of various single CER types. The objective of part 2 is to review the numerous studies on SC lipid mixtures in the binary, ternary, quaternary or multicomponent organization in the form of bulk samples, vesicles and oriented layers. Prior to a detailed state-of-the-art report on this subject, we will especially
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address neutron diffraction because this method offers new possibilities to investigate the molecular architecture of SC lipid systems. Current SC lipid models of the SC barrier structure are presented. The arguments concerning the validation of each of these models are reviewed further on. Finally, the controversial discussions about the molecular arrangement inside the lipid matrix are summarized.
Properties of Lipid Systems as Revealed by Neutron Diffraction Experiments
Most of the applied experimental methods as X-ray diffraction, vibrational spectroscopy and differential scanning calorimetry (DSC) have been discussed in part 1. Neutron scattering is recommended as a suitable tool for studying the internal membrane arrangement of bilayer structures [20–24], although disadvantages to X-ray scattering exist, such as the weakness of the neutron beam and small availability of neutron sources [25, 26]. Whilst X-rays are scattered by the electrons surrounding the atomic nuclei, neutrons are scattered by the nucleus itself. The fact that the coherent neutron scattering lengths bcoh for hydrogen (bcoh = –3.741 fm) and deuterium (bcoh = 6.671 fm) are significantly different offers possibilities which are not available for X-ray scattering. Thus, and unlike X-rays, not only can neutrons ‘see’ hydrogen isotopes, but they can differentiate between them. Most of the lipid membranes are studied in partly or fully hydrated states. Varying the hydrogen to deuterium ratio in water, the contrast variation of the neutron scattering between lipid bilayer and water can be achieved without sufficient changes in the membrane structure. For example, in an aqueous dispersion with a concentration of 8% (w/w) D2O in H2O, the neutron scattering length density of the dispersion medium is zero and the neutron beam detects only the dispersed substance. This is the first important advantage of neutron scattering in comparison to X-rays. Variation of the D2O/H2O ratio in the surrounding medium (water or water vapour in the air) allows easily calculating the signs of the structure factors, a necessity to evaluate the neutron scattering length density across the unit cell of the multilamellar membrane by Fourier synthesis. In contrast, the determination of the structure factor signs in X-ray diffraction experiments is a problem which can only be solved on the base of the Shannon sampling theorem [27].
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The second important advantage of neutrons is their small absorption in lipids and water. This fact has a special importance for the investigation of diffraction in the reflection mode. The correction of the absorption could be omitted in neutron diffraction experiments on multilamellar membranes in case of a thickness of the lipid film of about 7–8 m [20]. In similar X-ray experiments special attention should be placed on the preparation of homogenous films and on the correction of the scattering intensities due to the absorption of X-rays in the multilamellar lipid membrane. The third advantage of neutron relative to X-ray scattering concerns the possibility of deuterium labelling. Specific deuteration of parts of the lipid, such as a selected methylen group, supplies precise information about the location of individual molecular groups. In most neutron scattering experiments on soft matter, the fully or partly deuterated lipids are applied as a unique tool to determine the exact position of molecular groups. When the neutron diffraction pattern from an oriented lipid bilayer system shows at least 3 diffraction orders, the neutron scattering length density profile across the bilayer can be reconstructed by Fourier synthesis as shown in equation (1): Ss ( x ) =
2Qhx ¬ 2 hmax Fh cos d ® d h =1
(1)
where Fh is the structure factor of the diffraction peak of the order h, and d is the lamellar repeat distance calculated from the peak position. The absolute value of the structure factors | Fh | = hIh
is given by the integrated intensity of the hth diffraction peak Ih. The sign of the structure factor (phase angle) having only the values +1 or –1 for centrosymmetric bilayers can be determined by H2O/D2O exchange [28, 29]. The achieved space resolutions of the Fourier synthesis %x = 0.6
d hmax
(2)
depend on the values of measured diffraction order hmax, as described in details in Kiselev et al. [20]. However, only the benefit of expensive specific deuteration does not allow neutron diffraction to overcome X-ray diffraction. In combination, both can extend the knowledge about the SC lipid structure and should be regarded as powerful tools for studies on lipid bilayer structures [30].
Kessner /Ruettinger /Kiselev /Wartewig / Neubert
SC Lipid Mixtures 90
Binary Systems Based on DSC and Raman spectroscopic data Neubert’s [31] group constructed the phase diagrams for the mixtures of CER together with different lipids also found in the SC. For these studies bovine brain CER type IV was used, which resembles CER[AS], therefore it will further be referred to as CER[AS]. In the first investigation the phase behaviour of CER[AS] was studied in mixtures with a saturated fatty acid [31]. The DSC heating curves for mixtures of CER[AS] with stearic acid (anhydrous) show 2 endothermic transitions that are typical of eutectic behaviour. The first transition is associated with the melting of the eutectic mixture and the second arises from the melting of the purely residual component. The corresponding phase diagram is presented in figure 2. The Raman spectroscopic data demonstrated that the hydrocarbon chains of both lipids are arranged in a highly ordered structure in the solid state. It appears that CER[AS] and stearic acid are immiscible. The addition of stearic acid to CER[AS] decreases the melting temperature of CER[AS] from 89 to 63.5 ° C. However, for the eutectic temperature a high amount of stearic acid is required to reach this low temperature. Therefore, stearic acid is not efficient enough to increase the fluidity of the CER molecule. Likewise, the mixtures of CER[AS] and CHOL both in the anhydrous and hydrated states show a eutectic behaviour indicating immiscibility of the lipids. The hydration of the CER[AS]/CHOL mixtures shifts the eutectic melting to a temperature of 55 ° C [32]. Complementarily, the phase behaviour of CER[AS] in a mixture with oleic acid in various molar ratios was studied [33], as the presence of free unsaturated fatty acids in the SC is established and the degree of unsaturation is said to have a profound effect on the membrane fluidity [34– 36]. Both lipids are also immiscible in the solid state. The phase diagram of both the anhydrous and fully hydrated systems is of a monotectic type because it was not possible to experimentally determine the eutectic point. In these binary mixtures oleic acid melts at about 12 ° C and forms fluid and separated domains above this temperature. Thus, it turns out that oleic acid does not ‘fluidize’ the structure of CER[AS] because of the total immiscibility of both components. These findings are contradictory to the results presented by Walker and Hadgraft [37]. Binary systems containing CER[NS] (structure see fig. 1) were applied in the following studies. Considering
the thermotropic response of the methylene stretching and scissoring infrared (IR) bands of hydrated equimolar binary mixtures, Chen et al. [38] deduce that CER[NS] and CHOL are well miscible at physiological temperature, whereas stearic acid was miscible with CHOL only at relatively high temperatures, where the fatty acid is disordered. On the other hand, a complex interaction between CER[NS] and stearic acid was observed. A separate fatty-acid-rich phase persisted until at least 50 ° C, while at higher temperatures the 2 components appear to be quite miscible and a preferential association of the fatty acid occurs with the base chain of CER[NS]. In another attempt [39] the membrane function was studied with a simplified model like Langmuir-Blodgett monolayers, whereby the 2-dimensional phase separation and phase transition was investigated by atomic force microscopy. In such studies, even small-scale irregularities and structures of the lipid matrix can be resolved. The influence of the CER acyl chain length in mixtures with FFA also of different acid chain length was investigated. These results illustrate that the short-acyl-chain CER showed a higher tendency to mix with the shortchain palmitic acid, while the long-acyl-chain CER mixed preferentially with the long-chain lignoceric acid, but both CER only show a partial miscibility with either fatty acid. It was concluded that a matching in chain length promotes the miscibility of the lipids investigated. In the same study both CER were investigated in mixtures with CHOL, where short-chain CER shows a higher miscibility with CHOL.
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Temperature (ºC)
85 80 75 70 65 60 0
20
60 40 Stearic acid (mol %)
80
100
Fig. 2. Phase diagram for mixtures of CER[AS] and stearic acid. From Neubert et al. [31] with permission from Elsevier.
61
The phase behaviour of SC lipids as a function of pH was elucidated by Kitson et al. [40] in a 2H nuclear magnetic resonance (NMR) study as the ionization state of charged lipids can certainly influence the molecular packing properties. For this study, bovine brain CER type III was used, which resembles CER[NS]. Therefore, it will be further referred to as CER[NS]. The mixtures of CER[NS] and palmitic acid showed that a more acidic pH appears to promote the development of both solid phases at lower temperatures and the formation of a non-bilayer phase at higher temperatures. On the other hand, the increase to pH = 7.4 resulted in a transition to a hexagonal phase at higher temperatures, which suggests that the polymorphic preference is regulated by the extent of protonation of the carboxyl group of the fatty acid. These findings support the hypothesis that the SC intercellular membranes contain domains of crystalline lipids. CER have also been identified as a central second messenger in cellular signalling cascades for differentiation, apoptosis and senescence. The thermal phase behaviour and structure of fully hydrated binary membranes composed of phospholipid and CER have been investigated by using DSC, small angle X-ray diffraction (SAXD) and small angle neutron scattering (SANS) [21, 41, 42]. As a general feature, it was found that the incorporation of CER molecules (CER[NS], CER[AP], CER[NP]) into dimyristoylphosphatidylcholine membranes causes an increase of the lamellar repeat distance. Ternary Systems Ternary mixtures containing CER, FFA and CHOL are widely used for physicochemical characterization of the SC barrier in most cases by Fourier transform infrared (FTIR) spectroscopy [43–49]. In the case of the hydrated equimolar mixture bovine brain CER[AS]/perdeuterated palmitic acid/CHOL the thermotropic response of the IR data reveals that CER[AS] and palmitic acid pack in separate highly ordered orthorhombic domains [44]. The fatty acid domains are stable up to 50 ° C and collapse at this temperature. Below 50 ° C, there is a strong hydrogen bonded carbonyl group in the head group. An abrupt change in the acid head group bonding occurs with the collapse of the crystalline chain packing. On the other hand, CER[AS] undergoes a broad transition that begins at 50 ° C and ends at 70 ° C. The collapse of the crystalline domains of both palmitic acid and CER[AS] at the same temperature demonstrates that distorted chain packing increases their susceptibility by the melting of the palmitic acid chains, although the CER[AS] chains are conformationally ordered. The band positions 62
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of the methylene stretching modes indicate that palmitic acid and CER[AS] are in a highly ordered conformation. The strong hydrogen bonding of the CER[AS] head group is not affected by changes in the inter- and intramolecular organization of lipid chains in the model system and is maintained even after the chains have been conformationally disordered. In the same study, the hydrated system of bovine brain CER[NS]/perdeuterated palmitic acid/CHOL shows a slightly different behaviour. Likewise, CER[NS] and palmitic acid are packed in separate domains with orthorhombic subcell structure. Otherwise, palmitic acid exhibits a broad melting transition with onset, midpoint and completion temperatures of 42, 50 and 60 ° C, respectively, and a less strong hydrogen bonding of the carbonyl group above 45 ° C. The chain melting of CER[NS] begins at 52 ° C and ends at 73 ° C with a Tm of –65 ° C. A strong hydrogen bonding between CER[NS] head groups persists up to 60 ° C and is not disrupted until the onset of the intramolecular conformational disorder of the chains. Taken all together, the results are in accordance with the domain mosaic model [14] because the systems investigated clearly show an organization of ordered orthorhombic lipid domains in all physiological temperature ranges. It is to emphasize that analogous ternary mixtures based on synthetic phytosphingosine-type CERs, CER[AP] and CER[NP], show a profoundly different behaviour [49]. The hydrated samples CER[AP]/stearic acid-d35/CHOL and CER[NP]/stearic acid-d35/CHOL appear to form intimate, homogeneous mixtures in which separate domains are not observed. Both systems undergo gradual and largely coordinated chain disordering processes over large temperature ranges. It has been observed that hydration is necessary to achieve these effects. The major findings in comparing the sphingosineand phytosphingosine-type CER based ternary lipid systems are summarized in table 1. It has been established in this study that the CER component drives the behaviour of the ternary system investigated, which means that the CER have the same chain packing in the ternary lipid systems as they do when hydrated individually. Therefore, if the phytosphingosine CER packs in a hexagonal subcell, the stearic acid also adopts the same packing. The results presented here are again consistent with the domain mosaic model proposed by Forslind [14]. Thereby, the sphingosine-type CER form distinct domains, in which the permeation occurs. In contrast, the phytosphingosine-type CER decrease the permeation through interaction with other lipid components making the mosaic more coherent. This result appears to explain the Kessner /Ruettinger /Kiselev /Wartewig / Neubert
Table 1. CER and fatty acid chain behaviour in ternary models
Attribute
CER[NS]
CER[NP]
CER[AP]
CER[AS]
Stearic acid chain packing Change in stearic acid chain packing? Stearic acid chain disordering Tm CER chain packing Change in CER chain packing? CER chain disordering Tm Stearic acid/CER chain disordering Tm Stearic acid CO frequency CER amide I frequency
orthorhombic no decreased orthorhombic no decreased separate similar similar
hexagonal yes increased hexagonal no decreased similar increased increased
hexagonal yes increased hexagonal no increased similar increased increased
orthorhombic no decreased orthorhombic no decreased similar increased similar
Summary of biophysical behaviour of phytosphingosine and sphingosine CER hydrated, ternary lipid systems containing d35-stearic acid and CHOL. Comparative statements are against the single species, i.e. either pure CER or fatty acid. From Rerek et al. [49] with permission from Elsevier.
higher levels of sphingosine-type CER compared to phytosphingosine CER in SC because a higher-level phytosphingosine-type CER could result in an SC with lower penetration rates. Elucidating the phase behaviour of mixtures of SC lipids in a monolayer with atomic force microscopy might also provide an insight into the role of CER and CHOL in the permeability barrier function of the SC. In this context, ten Grotenhuis et al. [50] investigated synthetic phytosphingosine-type CER (CER[NP]) with well-defined fatty acid chain length in monolayers mixed with CHOL and either palmitic acid or lignoceric acid. Short-acylchain CER showed a higher tendency to mix with CHOL and short-chain FFA than the long-acyl-chain CER. The latter demonstrated a preferred mixing with the longerchained FFA (lignoceric acid) and phase separation with CHOL. Thus, a bimodal distribution of CER amidelinked fatty acid chain length is required for the phase separation in a monolayer. Further, this separation can be correlated with the findings in bulk mixtures at moderate CHOL concentrations. Therefore, it is likely that in bulk mixture the short- and long-acyl-chain CER do not mix in the bilayer but form separated sublattices. The bovine brain CER[NS]/perdeuterated palmitic acid/CHOL system has also been investigated as monolayers at the air/water interface at room temperature by means of IR reflection absorption spectroscopy and Brewster angle microscopy (BAM) [45]. The BAM images of pure CER[NS] monolayers revealed that CER alone does not form a continuous ordered film but rather discrete domains of highly ordered molecules, which upon addition of palmitic acid and CHOL resulted in less
tightly packed fluid-like phases. Thus, the BAM images of the ternary lipid SC model demonstrated a more fluid homogeneous monolayer, consistent with a more plastic structure. This fluid structure visualized at the micrometre level by BAM is composed, however, of conformationally ordered lipids as detected at the molecular level by IR reflection absorption spectroscopy. Both the chain packing and the head group bonding are very different in the monolayers than in the bulk multilamellar system. In monolayers the chain packing is hexagonal, in contrast to the orthorhombic packing in multilayers. A splitting of the head group amide IR band, observed in multilamellar samples [51], is not found. This absence of amide mode splitting supports the assumption that in CER[NS] bulk samples there is a transversal hydrogen bonding between opposite head groups in adjacent bilayers; this arrangement is impossible for the monolayer. Velkova and Lafleur [47] systematically examined the thermotropic behaviour of several hydrated mixtures composed of bovine brain CER[NS], CHOL and perdeuterated palmitic acid with various compositions using FTIR spectroscopy. The results prove that below 40 ° C the lipid species exist in phase-separated crystalline domains and show very limited miscibility. Between 40 and 50 ° C, a transition from the crystalline to a liquid-ordered phase occurs, which involves CER[NS], CHOL and palmitic acid. In the case of a high CHOL content in the mixture, this liquid-ordered phase is stable up to 75 ° C. For low CHOL content, the mixtures undergo a second transition toward a more disordered phase which is likely not lamellar. The creation of theses phases is critically dependent on the lipid composition. They proposed that CHOL in
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64
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Time after quenching (spectra every 15 min)
Absorbance
high concentrations (60 mol%) supports the formation of a liquid-ordered phase and that in the mixtures investigated CHOL acts as a promoter of the lipid miscibility. These findings indicate the basic role of CHOL in the formation of the fluid-ordered phases that involve poorly hydrated lipids. The lipid distribution in the hydrated, equimolar mixture of bovine brain CER[NS], CHOL and perdeuterated palmitic acid has also been examined by Raman microscopy [52]. Applying the mapping mode of operation with a spatial resolution of the order of 1 m, the authors could detect distinct phase separations and domains enriched in each component of the ternary mixture with a size of a few tens of square micrometres. Fenske et al. [53] investigated ternary SC lipid mixtures composed of bovine brain CER[NS]/CHOL/perdeuterated palmitic acid in an equimolar ratio using 2H NMR. The cellular matrix study of the phase behaviour of oriented samples seems to be a suitable model as membranes of the SC are macroscopically ‘oriented’. The used of 2H NMR proofed to be a powerful tool for studying membrane structures and dynamics because it offers the possibility to study the different components separately. In the oriented samples an indication was found that the axis of motional averaging is the normal to the plane of the membrane and for the presence of liquid-ordered membranes, which is consistent with the finding by Abraham and Downing. [54]. It was stated that such a simple model provides general agreement with many features of the intact SC. In a first attempt, Moore et al. [55] recently studied the kinetics of membrane raft formation in 4 equimolar SC lipid mixtures, namely, CER[NS]/CHOL/stearic acid-d35, CER[AS]/CHOL/stearic acid-d35, CER[NS]/CHOL/palmitic acid-d31 and CER[NS]/CHOL/palmitic acid-d31; the term ‘lipid raft’ designates a non-randomly mixed cluster of ordered lipids containing closely packed acyl chains. To monitor the kinetics of formation of regions rich in fatty acids, the authors utilized a variant of a method to study spontaneous demixing in crystalline binary n-alkane mixtures. This approach is based on the fact that the shape of the methylene scissoring and rocking bands in the IR spectra of a binary mixture of same-length hydrogenated and deuterated n-alkanes is highly dependent on component concentration and that this dependence is quantitatively independent of chain length for chains longer than about 20 CH2 groups. The relevant experiments were performed as follows: the hydrated lipid mixtures were sandwiched between AgCl IR windows and placed in a temperature-controlled transmission cell.
1,070
1,075
1,080
1,085
1,090
1,095
1,100
Wave (n · cm–1)
Fig. 3. Time evolution of the CD2 IR scissoring contours of the
fatty acid component in the ternary model SC system CER[NS]/ CHOL/stearic acid-d35 (equimolar). From Moore et al. [55] with permission from the American Chemical Society.
The experiments were initiated by heating samples to 90 ° C and then cooling rapidly to the desired annealing temperature of 25, 30 or 35 ° C. When the desired temperature was achieved, the acquisition of the IR spectrum commenced and continued for a period of at least 9 h with a spectrum being registered every 15 min. In this manner the kinetics of the domain formation of the fatty acid component were monitored through the time evolution of the line widths and the onset of the splitting of the CD2 scissoring contour (for example see fig. 3) The findings qualitatively displayed interesting variation in the time evolution of domains between mixtures and between temperatures. The composition of stearic acid-d35 reached a limiting value (X = –0.8) which was significantly higher than that approached by palmitic acid-d31 (X = –0.55) at the end of a 9-hour period. In all cases, the time for domain formation was more rapid at 35 ° C than at lower temperatures, while the segregation of stearic acid-d35 enriched domains is much more rapid for a given CER than palmitic acid-d31 enriched domains. Kessner /Ruettinger /Kiselev /Wartewig / Neubert
In view of the varying roles postulated for different lipid classes in the structure of SC, a determination of the kinetics of lipid domain formation is of significance for understanding the details of the barrier structure. In addition, kinetic studies may have important physiological relevance, for example, dermal and transdermal drug delivery often requires (temporary) disruption of the SC lipid barrier. Rowat et al. [56] elucidated the influence of the penetration enhancer oleic acid on multilamellar dispersions composed of bovine brain CER[NS], CHOL-d6 and palmitic acid-d31 by means of solid state deuterium NMR. The results show that below 40 ° C, oleic acid-d2 is in an ‘isotropic’ phase, indicating that it is not incorporated into the lamellar membrane phase. Above the membrane’s crystalline to liquid crystalline melting temperature, Tm = 40–42 ° C, oleic acid interacts with lamellar membranes with a slight dependence on the content of oleic acid. The 2H NMR spectra of both the palmitic acid and CHOL component display an isotropic peak that grows with increasing temperature. This finding points out that oleic acid extracts a fraction of the endogenous membrane components, promoting phase separation in the system, which is consistent with the finding received by Francoeur et al. [57] and Ongpipattanakul et al. [58, 59]. Multicomponent Systems The first studies on the SC lipid organization by freeze fracture electron microscopy revealed a lamellar organization of the lipids in the SC intercellular spaces [60]. Only after introducing RuO4 as a post-fixation agent to visualize saturated SC lipids did electron microscopy studies show an unusual trilamellar repeat unit arrangement with a broad-narrow-broad appearance and an overall dimension of 13 nm, called the long-periodicity phase (LPP) [61]. White et al. [62] also detected the 13-nm periodicity in murine SC. By means of X-ray scattering techniques, multicomponent systems composed of CER, FFA, CHOL and CHOL sulphate were investigated in order to study the lipid organization in the SC on the molecular level. The first studies were undertaken on CERs isolated from pig SC, which was readily available [2, 9, 10, 63, 64]. According to McIntosh et al. [63], X-ray studies on a hydrated mixture of pig CER:CHOL:palmitic acid (molar ratio 2:1:1) revealed a single repeat unit of 13 nm, whose existence appears to be linked with the presence of the acylceramide CER[EOS], which is only found in the epidermis. The substitution of the skin CER with CER from
the bovine brain revealed no reflection that corresponds to the 13-nm structure, indicating that the lamellar structure of the SC depends on the presence of specific CER as well as on an appropriate ratio of CHOL and FFA. In a continuation of this study [64], the calculation of electron density profile was done after inducing swelling of the 13-nm repeating unit. The major finding was a repeating unit containing 2 asymmetric bilayers in which CHOL is asymmetrically distributed. Complementarily, Bouwstra’s [8] group studied the behaviour of isolated pig CER, either in a mixture containing CHOL or CHOL and long-chain FFA. The diffraction pattern revealed the presence of 2 lamellar units with periodicities of approximately 12 and 5 nm [shortperiodicity phase (SPP)], respectively. The behaviour of the CHOL/pig CER/FFA mixtures closely mimics that of the intact SC only in the presence of long-chain fatty acids. The detection of the broad-narrow-broad sequence in the LPP and the key role of CER[EOS] led to the proposal of the sandwich model [65]. Already in the early 1990s, the group of Abraham et al. [54] employed 2H NMR to explore SC model membranes prepared from isolated SC CER in mixtures with CHOL and perdeuterated palmitic acid (PA-d31) and cholesterol sulfate (ChS). Their study revealed that such mixtures undergo thermotropic transitions which lead to the formation of a hexagonal phase as well as isotropic phases at higher temperatures. They found that a decreased amount of CHOL in the mixture lowers the energy barrier for a transition to a hexagonal phase. In the absences of CHOL, a significant increase in disorder in the hexagonal phase resulted. The thermotropic behaviour also gives information about the interaction of the SC lipids with each other in conjunction with temperature-induced structural alternation. Therefore, Ongpipattanakul et al. [66] performed FTIR spectroscopy studies to analyze the behaviour of SC lipids. A complex polymorphism of isolated porcine SC as well as extracted porcine SC lipids was observed. Their results suggested that lipid segregation produces multiple transitions in intact SC. In contrast, the isolated SC lipids display only a single transition due to mixing of the extracted lipids. Another outcome of their investigation was that lipids coexist in solid and fluid phases at all temperatures well below chain melting temperature. Further on, studies focused on the effect of varying the CER composition on the SC lipid organization in model systems because it is known that there is a broad range of the CER composition in the SC depending on the sources and state of skin health [9]. Thus, experiments used
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mixtures containing pig CER1, 2, 3, 4, 5 and 6, respectively. It was shown that the lipid organization is insensitive towards changes in the CER composition for an equimolar CHOL:CER ratio as well as an equimolar mixture composed of CHOL:CER:FFA [9]. On the other hand, this influence increases for the case of a reduced CHOL/ CER molar ratio. A correlated effect on the in vivo situation is given. Additionally, other techniques were applied on extracted porcine SC. By using transmission electron microscopy, Kuempel et al. [10] determined the conditions under which the broad-narrow-broad pattern (typical of the 13-nm repeating unit) can be reconstituted from SC lipids in vitro. Thereby, heating in water induced the formation of many lamellae with repeating units in the range of 5–6 nm and no broad-narrow-broad pattern. The LPP only resulted after storage under desiccating conditions. A better approach is to use human CER as these have a larger relevance to the in vivo situation of human skin. In the study performed by Bouwstra et al. [11], this assumption is underlined by a comparison of pig CER and human CER. Both reveal the existence of the LPP as well as of the SPP, whose formation is dominated when CER[EOS] is absent. The differences occur by adding FFA. For the mixture of human CER/CHOL/FFA, a formation of the SPP was promoted. Additionally, in mixtures with pig CER/CHOL/FFA, no liquid phase was observed. On the other hand, by substituting pig CER with human CER, a liquid lateral packing could be detected, which is probably caused by a larger amount of the linoleic acid moiety in human CER compared to pig CER. Further, the amount of CHOL which is incorporated into the lipid lamellae is increased, which was attributed to an increased solubility of CHOL upon addition of FFA. In the same study, the influence of ChS was investigated. In mixtures containing human CER and ChS, the formation of SPP was enhanced, contrary to the findings with mixtures based on pig CER, where the formation of LPP was promoted. Up to this date, the function of each CER class is not known. However, the more lipophilic CER with -esterified fatty acids, and among them predominantly CER[EOS], are reported to be of considerable importance [12]. It is stated in the study of Bouwstra et al. [67] that human CER[EOS] with its unsaturated acyl chain is needed for the formation of the LPP as well as for the formation of a liquid phase. The latter induces a certain elasticity of the lipid lamellae and allows molecules to permeate along the lipid lamellae. If the fraction of lipids form66
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ing this fluid phase is too high, a reduction in barrier function could occur. During the last years, more emphasis has been placed on establishing mixtures containing synthetic CER with a well-defined acyl chain length and head group architecture. They are regarded as a useful tool to elucidate the role of the molecular structure of individual CER for structural assembly of the SC lipids. This development was necessary because of the difficulty to obtain information about the internal structure of the SC by using either native SC or isolated SC lipid mixtures. The advantage of synthetic CER in contrast to the isolated ones is obvious, namely undesirable effects such as variability of the native lipids can be overcome. Furthermore, the laborious isolation and separation of the SC lipids is not necessary anymore. De Jager et al. [17] used an equimolar CHOL/CER/ FFA mixture, whose synthetic CER fraction consisted of CER[EOS]/CER[NP]/bovine brain CER type IV in a 1:7:2 ratio. By choosing appropriate preparation methods, this mixture resembles the SC lipid organization. Further on, the influence of the CER type on the lipid formation was examined by introducing different synthetic CER mixtures, consisting of CER[EOS], CER[NS], CER[NP]C24, CER[AS], CER[NP]C16, CER[AP] and CER[EOP] in varying ratios. It was found that the exclusion of several CER classes does not affect the lipid organization [19]. However, by focusing on the role of the acylceramides CER[EOS] and CER[EOP], differences occur. The formation of the LPP was always observed when an acylceramide was present, but insensitive to a stepwise increase in CER[EOS] level, partial replacement of CER[EOS] by CER[EOP] shows no influence on the phase behaviour. Nevertheless, in case of a complete substitution of CER[EOS] fraction for CER[EOP], the formation of the LPP was reduced and phase-separated CER[EOP] resulted. This different behaviour is obviously linked with the more hydrophilic character of CER[EOP] caused by the additional OH group of the phytosphingosine backbone [19]. Beside X-ray scattering methods, other diffraction techniques became more prominent during the past years. In previous studies it was shown that X-ray and neutron scattering are very powerful tools to investigate the structural properties of the SC as well as of SC model membranes [25]. Small-angle neutron scattering experiments were undertaken by Charalambopoulou et al. [23]. Recent studies applied neutron diffraction on quaternary SC lipid model matrices for investigating their internal membrane structure [20]. Kessner /Ruettinger /Kiselev /Wartewig / Neubert
s(x) (AU)
3
2
Polar head groups 1 Cholesterol
Fig. 4. The neutron scattering length density s(x) of the CER6/Ch/PA/ChS membrane with composition 55/25/15/5 at 60% humidity, T = 32 ° C, and 8% D2O content (dots) and fitting curve (solid line). Arrows mark the 4 components: CH3 groups, CH2 chains, CHOL and polar head groups. From Kiselev et al. [20] with permission from Springer.
0 –25
–20
–15
–10
–5
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In the work of Kiselev et al. [20], the structure and hydration of an SC lipid model membrane composed of CER[AP]/CHOL/palmitic acid/Chs with a weight ratio of 55/25/15/5% was characterized. Determining the sign of the structure factor by substituting D2O by H2O allows one to calculate the neutron scattering length density profile [28], which is presented in figure 4. The major finding was the low hydration of the intermembrane space, which was concluded by the same value of 4.56 nm for the lamellar repeat distance and the membrane thickness at 60% humidity and T = 32 ° C. Additionally, a decrease of the CHOL content increases the membrane thickness. In this study, it could be shown that water diffusion through this model matrix is a slow process. At 97% humidity and T = 81 ° C, the membrane separates into 2 phases with repeat distances of 4.58 and 4.05 nm, respectively. It was stated that the SC lipid model membrane prepared on a quartz substrate allows one to study the influence of other CER and fatty acids on the structure and properties of SC model membranes. The application of deuterated lipids will be of particular interest for this type of method applied. In a continuation, there are current studies on the influence of various CER as well as FFA on the molecular arrangement of the SC lipid model matrix by neutron diffraction experiments. Thereby, using the benefit of spe-
A better insight into the SC lipid matrix is a prerequisite for understanding the skin barrier properties. Knowing the internal structure and hydration behaviour on the molecular level is essential for studying drug penetration through the SC and for a more rational design of transdermal drug delivery systems. During the last decades, various molecular skin barrier models have been developed, such as the stacked monolayer model [13], the domain mosaic model [14], the sandwich model [15] and the single gel phase model
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cific deuteration obviously provides interesting new insights into this organization [68, 69]. Using the advantage of quaternary systems with a well-defined composition ratio, Hatfield and Fung [70] investigated large unilamellar vesicles composed of bovine brain, either CER[NS] or CER[AS]/CHOL/FA/ChS, by electron paramagnetic resonance. To monitor the local dynamic properties of lipid molecules in the bilayer, spin labels were used. It could be demonstrated that through the whole temperature range investigated the molecular motion near the head group is more restricted than in the alkyl chain region of the bilayer.
Theoretical Models of SC Lipid Matrix
67
O
O
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Fig. 5. Stacked monolayer model. From Swartzendruber et al. [13] with permission from the Society of Investi-
gative Dermatology.
[16]. A short review for each is presented in the following. The stacked monolayer model describes a lamellar molecular arrangement for the intercellular lipid matrix of the SC, characterized by interdigitating of the alkyl chains of the CER in the stretched splayed chain conformation and by CHOL specifically distributed between different layers. This lipid organization could contribute to the intercellular broad-narrow-broad arrangement of lucent bands reported in classical transmission micrographs [13]. The proposed arrangement is displayed in figure 5. Forslind [14] formulated the domain mosaic model, where the intercellular lipid matrix is treated as a multilamellar 2-phase system with a discontinuous lamellar crystalline domain embedded in a continuous liquid crystalline domain (fig. 6). These structures are encircled by ‘grain borders’, formed by lipids in the fluid crystalline state. Diffusion of hydrophilic and hydrophobic substances through the barrier is allowed because of the fluid character of the bordering areas. Unlike the side-by-side arrangement of the crystalline and liquid structures in 1 layer, depicted in the domain mosaic model, the sandwich model suggested another lipid organization [15]. Thus, the domains are located in separate layers in a trilayer arrangement, according to the broad-narrow-broad sequence of the LPP. The liquid sub68
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Liquid crystalline grain border
Gel domain
Fig. 6. Domain mosaic model. From Norlén [16] with permission
from the Society of Investigative Dermatology.
lattice is located in the central layer, formed mainly by the unsaturated linoleic moiety of the acylceramides, namely CER[EOS], CER[EOP] and CER[EOH], and CHOL. In the 2 neighbouring sublattices, the crystallinity increases gradually due to the presence of less mobile long-satuKessner /Ruettinger /Kiselev /Wartewig / Neubert
rated hydrocarbon chains. Another feature is the small fraction of lipids constituting a fluid phase, which indicates that the central layer is a discontinuous phase. Substances penetrating through the SC have to pass the crystalline lipid lamellar region and partly diffuse through the less densely packed lipid regions. This effect on diffusion processes assumes to maintain the barrier properties [65]. In figure 7, this theoretical model is presented.
The single gel phase model, demonstrated in figure 8, differs significantly from the previously mentioned models and proposes that the skin barrier is formed by a single coherent lamellar gel structure in the intercellular space of the SC [16]. The barrier structure shows no phase separation, neither between liquid crystalline and gel phases nor between different crystalline phases with hexagonal and orthorhombic chain packing, respectively. The proposed single lipid structure exhibits small water content, a low degree of mobility and a low water permeability because of a dense packing of the constituent lipids. Contrary to the domain mosaic and the sandwich models, which propose that the CER are organized entirely in a hairpin conformation (i.e. with the 2 hydrocarbon chains pointing in the same direction), the single gel phase model supposes both hairpin and splayed chain conformations (chains point away from a central polar head group in the opposite directions) of CER in the SC lipid matrix [72]. There are controversial discussions about the unique molecular arrangement in the SC lipid matrix. The arguments are separated into 2 categories. Firstly, discussions which concentrate on the comparison of the current SC lipid models will be surveyed. Secondly, the molecular arrangement of the lipids in the LPP is controversially reflected. Swartzendruber et al. [13] and Bouwstra et al. [12] proposed molecular models of the SC lipid matrix which are based on the broad-narrow-broad electron density regions observed by transmission electron microscopy experiments and the long spacing of SAXD data (approximately 6 and 13 nm). They differ in the proposed alignment of the CER and CHOL moieties. Both models suggest an equal interfacial area of CER and CHOL, which show a planar alignment according to Swartzendruber et al. [13]. In contrast, Bouwstra’s [12] group favours a tail-to-tail arrangement. This assumption is underlined by the achieved similar values for the interfacial area of CHOL and CERs in the tail-to-tail arrangement, taken from the study of Dahlén and Pascher [73]. These authors also suggested possible conformations of CER, namely the hairpin and the splayed conformation, shortly characterized above. Especially, the sandwich model does not take these conformational possibilities [16] into account. On the other hand, those possible conformations as well as a postulated single coherent lamellar gel phase with an unperturbed barrier structure are considered in the single gel phase model proposed by Norlén [16]. It differs significantly from other suggestions about the lipid
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12.2 nm
Cholesterol Ceramide 1 Ceramides 2, 3, 4 and 6 Ceramide 5
Fig. 7. Sandwich model. The CERs are labelled according to the old nomenclature, therefore according to Motta et al. [71] the CER are named as follows: CER1 = CER[EOS], CER2 = CER[NS], CER3 = CER[NP], CER4 = CER[AS](C24), CER5 = CER[AS](C16), CER6 = [AP]. See also figure 1. From Bouwstra et al. [12] with permission from the American Society for Biochemistry and Molecular Biology.
One single and coherent lamellar gel phase
Fig. 8. Single gel phase model. From Norlén [16] with permission from the Society of Investigative Dermatology.
69
a
organization mentioned earlier. In comparison to the domain mosaic model by Forslind [14], it is stated that there is no phase separation between liquid crystalline and gel phases. Compared to the sandwich model proposed by Bouwstra et al. [15, 74] the absence of phase separation between different crystalline phases with hexagonal and orthorhombic lattices is postulated by the author himself [16]. On the contrary, the single gel phase model is often attacked by claiming the missing experimental data that could validate this structure [75]. Testing the homogeneity of the proposed single and coherent lamellar gel structure by applying time-resolved synchrotron X-ray diffraction on lower SC layers acquired by the stripping method is suggested by Norlén [16]. However, there is still a lack of confirming experimental data at the moment. Thus, Bouwstra and Ponec [75] refer to the confirmation of the sandwich model and the single gel phase model in respect of the absence of any grain boundaries. Nevertheless, the coexistence of hexagonal and orthorhombic chain packing, even in lower SC regions, cannot be verified by experimental data. Bouwstra’s [75, 76] group collected wide angle X-ray scattering (WAXS) data elucidating that there are no hexagonal phases in lower SC regions and that there is a transition from hexagonal to orthorhombic lattice by adding long-chain FFA. In summary, besides the lacking experimental data, the single gel phase model does not take into account the influence of individual lipids, especially the -acylceramides such as CER[EOS], which makes it difficult to prove the model. The last of the most prominent models which are under debate is the domain mosaic model proposed by Forslind [14]. It is consistent with the sandwich model in the point of claiming the presence of a liquid phase in the SC lipid matrix. The difference appears for the continuousness of the liquid phase from the superficial layer of the SC down to the viable epidermis, as it is described in the domain mosaic model. Here again, the criticism concentrates on the missing experimental data corroborating the suggestion [75]. 70
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Water
SC matrix. Transformation of SC membrane from partly dehydrated state (a) to fully hydrated by water excess (b). Kindly supplied by Mikhail A. Kiselev.
Water
Water
Fig. 9. Armature reinforcement model of
b
Altogether, the theoretical models described above have 1 disadvantage. They cannot explain the structural alteration of the SC lipid matrix under hydration by water excess. The armature reinforcement model of the SC lipid matrix founded on the hairpin conformation of short chain CER was proposed recently [20, 77]. The phenomena of the chain flip transition of the CER[AP] molecules from the hairpin conformation to the fully extended conformation was described in Kiselev et al. [20] and further developed in Kiselev [77] for the explanation of 2 experimental facts: (i) the disappearance of the first diffraction order and stability of the second diffraction order under hydration in water excess of the SC isolated from the porcine skin [24], and (ii) the transformation of the LPP to the SPP for the case of SC extracted from human skin under hydration by water excess [78]. The main principals of the armature reinforcement model are presented in figure 9. The steric contact of the bilayer leaflets is created by the hairpin conformation of the CER molecules as shown in figure 9a. The intermembrane space appears after hydration in water excess as seen in figure 9b. This space is hydrated by water. The appearance of the intermembrane space is a result of the transformation of the CER molecules from fully extended to the hairpin conformation, so-called chain flip transition. Finally, it can be stated that several molecular models of the SC lipid organization exist and are controversially discussed. Key features distinguishing between these models are the question of the existence of the lipid phase separation and the question of the presence of liquid lipid structures [72]. Summarizing, the validation of the theoretical models of the SC lipid matrix is hampered by experimental difficulties such as mimicking the environmental conditions and by substrate problems due to the variability of the native tissue and laborious isolation and separation of SC lipids. Difficulties related to the applied technical methods (including comparison of the results obtained by different methods and mimicking the in vivo situation) still exist. The problems concerning the variability of native tissue or of isolated CER have already been overKessner /Ruettinger /Kiselev /Wartewig / Neubert
come. The use of SC lipid mixtures containing synthetic CER with a well-defined acyl chain length and head group architecture can resemble the SC lipid matrix to a high extent [17–19, 67]. However, for none of the most prominent SC lipid models do studies exist which validate whether the proposed model correctly represents the lipid organization in the SC. The existence of the long-periodicity spacing is well accepted. Its formation requests the presence of the -acylceramide CER[EOS]. There is a strong debate about the molecular arrangement in the LPP. Using ruthenium tetroxide (RuO4) as a post-fixation agent in sample preparation techniques for transmission electron microscopy allows one to visualize the saturated SC lipids [61]. Multiple stacked lamellae in the intercellular spaces were revealed, among them 3-, 6- or 9-lamella patterns were most frequently observed [79]. In a first molecular model [13], it was described that the framework of the 3-band pattern is based on covalently bound -hydroxyceramides. It was suggested that the hydroxyacyl part of the molecules composed the broad lamellae and that the sphingosine tails interdigitated in the central narrow layer. The observed 6-band pattern was formed by covalently bound hydroxyceramides. It was proposed that some CER assumed a splayed conformation with 1 chain in one of the bilayers in the centre of the intercellular space and the other one in the narrow interdigitated layer. Furthermore, CER appeared to have an extended conformation to effectively link the bilayers. Summarizing, no special attention was paid to the function of each CER class and the special relevance of some of them. An important role was attributed to linoleate-containing acylceramides in a later published revision of that model [80]. The acylceramide molecules were assumed to have an extended conformation with the -hydroxyacylsphingosine residue in the broad lamellae and the linoleate tails inserted into the narrow layers. A similar model, the previously mentioned sandwich model, was published independently by Bouwstra et al. [15, 67]. All these ideas of the molecular arrangement in the LPP are based on a trilamellar repeating unit with a broad-narrow-broad appearance in the dimension of 13 nm. In contrast, transmission electron microscopy studies on pig SC prepared by using RuO4 were performed by Hill and Wertz [81]. The observed electron lucent bands revealed a uniform thickness of the trilamellar repeating units, which is in disagreement with the 5-3-5 nm dimen-
sion of the broad-narrow-broad arrangement. Moreover, the authors confirmed preceding suggestions that the linoleate chains are present only in the central lamella. This finding results in a higher ruthenium reduction in the central LPP unit, which consequently seems to be narrower than the adjacent ones. These outcomes are in agreement with studies relying on cryo-electron microscopy of vitreous human skin, which reported that the trilamellar conformation could not be observed [82]. This discrepancy could be caused by morphological changes due to ruthenium tetroxide fixation or dehydration in conventional sample preparation for electron microscopy. This finding supports the argument that the LPP phase (13-nm repeat unit) could be an artefact due to sample preparation. In line with this, several studies have reported that chemical fixation by ruthenium tetroxide led to severe changes in skin ultrastructure [83], which has to be taken into account for any interpretation. Complementarily, other investigations exist which discuss the arrangement inside the LPP. McIntosh [64] studied a mixture consisting of CHOL/pig CER/palmitic acid by X-ray diffraction. The electron density profiles were calculated after inducing swelling of the LPP by adding ChS and by influencing the pH. A repeating unit containing 2 asymmetric bilayers was revealed. This finding is not in line with the trilayer arrangement. It has to be mentioned that the lipid composition and the experimental conditions do not correlate with the in vivo situation. Therefore, more studies have to be performed in order to determine the correct lamellar arrangement in the SC lipid matrix. There is a general consensus about the unique role of -acylceramides in constituting the SC lipid barrier function [8, 9, 12, 18, 19, 63, 67]. Small angle X-ray scattering (SAXS) experiments performed by Bouwstra et al. [9, 12] on equimolar CHOL/human CER mixtures lacking CER[EOS] demonstrated that LPP was only weakly present. Furthermore, the influence of the fatty acid moiety of the -hydroxy acid of CER[EOS] was elucidated by using synthetic CER[EOS] derivatives containing either saturated or unsaturated fatty acid residues. The results revealed a necessity of an unsaturated fatty acid moiety forming a liquid phase for the formation of the LPP [67]. Subsequently, the focus was put on mixtures containing synthetic CERs as described earlier [17–19]. Abstracting the findings, it could be shown that exclusion of CER[EOS] reveals a reduced barrier function, indicating the importance of CER[EOS] for skin lipid behaviour as well as for skin barrier properties [84].
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In contrast, Hatta et al. [85] performed X-ray diffraction studies on normal and damaged mouse SC. The diffraction peak at 13 nm only appears for the normal SC. Then, the damaged SC was immersed in a suspension containing CER[NP], CHOL and FFA. Despite the absence of CER[EOS] in the suspension applied, the diffraction peak indicating the LPP re-appeared. However, this finding cannot disprove the predominant role of CER[EOS] for the formation of the LPP because it is not known whether CER[EOS] molecules remaining in the damaged SC trigger it forming the ordered lamellar structure.
Conclusion
During the past years a lot of new insights into the structural properties of the SC lipid model systems were acquired, especially by investigating the important role of the most prominent constituents, the CER. Several theoretical models proclaiming a possible structural organization of the SC lipid matrix were developed. Among them, the most important ones are the stacked mono-
layer model, the domain mosaic model, the sandwich model and the single gel phase model, which have been briefly discussed in the present review. However, the validation of the real SC lipid organization is still lacking. Therefore, an already known, promising strategy becomes widely accepted. Examining mixtures of well-defined SC lipids, with the anticipation of characterizing their thermotropic phase behaviour as well as the molecular interactions between the components selected, is a suitable approach to expand the knowledge about the lipid organization in the SC. For that purpose, a wide range of experimental methods should be applied, including the employ of neutron diffraction. Thereby, new aspects of SC lipid organization have already been obtained by the armature reinforcement model, based on neutron diffraction studies. Thereby, the ultimate objective is to learn how the interactions between the SC lipids can be influenced with respect to dermal and transdermal drug delivery, respectively. Concerning this, essential information is still missing. Consequently, there is a strong request to explore this research field more extensively.
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