Interaction of Antiseptic Compounds with Intercellular Lipids of Stratum Corneum

The interaction between three kinds of antiseptic compounds and components of intercellular lipids in the stratum corneum was characterized in terms of thermodynamics at pH 7.5 and 25°C, and the different mechanisms used to penetrate the stratum corneum were clarified. Anionic surfactants such as benzalkonium chloride and benzethonium chloride mainly bound to cholesterol (CH) and cholesterol sulfate with high affinity (10⁵–10⁶ M⁻¹) to extract endogenous CH from the stratum corneum and penetrated through the intercellular route. Chlorhexidine gluconate also bound to CH and accumulated in the stratum corneum without removing endogenous CH. An amphoteric surfactant of dodecyldiaminoethylglycine hydrochloride seemed to be incorporated into the lipid bilayer and bound to ceramide with its polar end close to the lipid polar heads by hydrophobic interaction. This revised version was published online in August 2006 with corrections to the Cover Date.     

Interactions between a skin penetration enhancer and the main components of human stratum corneum lipids

The study employed isothermal titration calorimetry (ITC) technique to investigate the interactions between a skin penetration enhancer, laurocapram, and three representatives of the human stratum corneum (SC) intercellular lipids, i.e., ceramide-3, cholesterol and behenic acid. Experiments were performed using a TAM2277 (Thermometric AB, Sweden) calorimeter. Results showed that, firstly, two laurocapram molecules were bound to one ceramide-3 molecule in propylene glycol solution. The binding was enthalpy-driven and system entropy decreased after binding. Secondly, the binding ratio between cholesterol and laurocapram molecule was one to one. It was mainly an entropy-driven process, suggesting the ordered cholesterol molecules in SC lamella were disturbed by the introduction of laurocapram. Hydrogen bonding might be the main force of the binding. The three different interaction modes showed that the chemical enhancers selectively interacted with the components of SC lipids so that the SC can still retain its barrier function.     

FUNCTIONS OF LIPIDS ON HUMAN SKIN

In order to clarify the roles of lipids in the water-holding property of stratum corneum, the forearm skin of healthy male volunteers was treated with acetone/ether (1/1) or sodium dodecyl sulfate (5%) for 1-30 min. A prolonged treatment period of 5-30 min produced a chapped and scaly appearance of the stratum corneum without any inflammatory reactions. Under these conditions, there was a marked decrease in the water-holding capacity of the stratum corneum accompanied by a selective loss of stratum corneum lipids such as cholesterol, cholesterol esters, and sphingolipids. Two daily applications of the isolated stratum corneum lipids to experimentally induced dry skins caused a significant increase of conductance, accompanied by a marked improvement in the level of scaling. Meanwhile, the isolated sebaceous lipids exhibited no significant recovery in both the conductance value and the scaling. Out of chroma-tographically separated fractions of the stratum corneum lipids, topical applications of ceramide fraction induced the highest increase in the conductance value. Topical applications of synthesized pseudo-ceramides also showed a significant recovery of the water-retaining properties accompanied by an improvement in the scaling only when the polar group has an amide bond in the major linkage. Analysis of the alkyl chain structures has revealed that a structural requirement for the recovery of the water retaining capacity is the presence of saturated-straight alkyl chains, not unsaturated or branched alkyl chains. These structural characteristics required for water-retaining function also paralleled their capacity to form multiconcentric lamellae vesicles in vitro which is also capable of acquiring bound water as shown by DSC thermograms. The present study suggests that ceramides with relatively shorter alkyl chain length serve as a water modulator in the multi-lipid bilayers through the stratum corneum.     

The action modes of Lippia sidoides (Cham) essential oil as penetration enhancers on snake skin

The aim of this work was to study the effect of Lippia sidoides essential oil (LSEO) on stratum corneum lipids and the permeation of salicylic acid. DSC and FTIR spectroscopy were applied. LSEO 1% (v/v) significantly enhanced salicylic acid flux through snake skin. According to the DSC curves changes in the transition temperature of the lipids were observed indicating that LSEO can interact with stratum corneum. The IR spectrum of skin treated with LSEO showed a decrease in the peak intensity for CH₂ stretchings (2920–2850 cm^–1) however the peak positions did not alter suggesting the extraction of the lipids.     

Penetration Process of a Hydrated Deep Eutectic Solvent Through the Stratum Corneum and its Application as a Protein Penetration Enhancer

The penetration mechanism of choline chloride-glycerol deep eutectic solvent (DES) through the stratum corneum (SC) as a potential solvent for a novel enhancer of protein penetration into the skin was investigated in a wide and small angle X-ray diffraction study. We found that DES penetrated through intercellular lipids but not the corneocytes. DES seemed to extract a portion of lipids of the short lamellae in the SC. Hydrated DES with a DES to water weight ratio of 9 to 1 (9DES-1H₂O) showed the strongest interaction with the lipids in the SC compared with water, DES, and hydrated DESs with another weight ratio of DES to water (DES : water=8 : 2). In a skin penetration test with a fluorescently labelled lysozyme, 9DES-1H₂O increased the amount of penetration through the SC by two-fold compared with HEPES buffer.     

Penetration and growth of DPPC/DHPC bicelles inside the stratum corneum of the skin

The effect of dipalmitoyl phosphatidylcholine (DPPC)/dihexanoyl phosphatidylcholine (DHPC) bicelles on the microstructure of pig stratum corneum (SC) in vitro was evaluated. The physicochemical characterization of these nanoaggregates revealed small disks with diameters around 15 nm and a thickness of 5.4 nm. Upon dilution, the bicelles grow and transform into vesicles. Cryogenic scanning electron microscopy (cryo-SEM) images of the SC pieces treated with this system showed vesicles of about 200 nm and lamellar-like structures in the intercellular lipid areas. These vesicles probably resulted from the growth and molecular rearrangement of the DPPC/DHPC bicelles after penetrating the SC. The presence of lamellar-like structures is ascribed to the interaction of the lipids from bicelles with the SC lipids. The bicellar system used is suitable to penetrate the skin SC and to reinforce the intercellular lipid areas, constituting a promising tool for skin applications.     

WATER UPTAKE INTO STRATUM CORNEUM; PARTITION BETWEEN LIPIDS AND PROTEINS

Water uptake in natural and reaggregated stratum corneum was determined by weight difference after storage in an atmosphere of controlled relative humidity.

Interlayer spacing in separated lipids as a function of their water content was determined by low-angle X-ray diffractometry. These values were used as a calibration curve to determine the water content of the lipid bilayers in reaggregated stratum corneum.

The results revealed different behavior of the lipid models compared to natural lipids of the stratum corneum. The additional water taken up after reaggregation of equilibrated lipids and proteins, was equally partitioned between the protein and the natural lipid fraction, while the models gave a proportionally higher water uptake into the lipids at high relative humidity. It is obvious that the models, so far, do not mimic all the properties of the natural stratum corneum lipids.     

Influence of the level of cholesteryl sulfate in the solubilization of stratum corneum lipid liposomes by sodium dodecyl sulfate

 The role played by cholesteryl sulfate (Chol-sulf) in the solubilization of liposomes modeling the stratum corneum (SC) lipids by sodium dodecyl sulfate (SDS) was studied. We determined the surfactant-to-lipid molar ratios and the bilayer/aqueous phase surfactant partition coefficients of this interaction by varying the proportion of Chol-sulf, the relative proportions of the others lipids remaining constant. These parameters were determined by monitoring the changes in the static light scattering of the system during solubilization. The fact that the free surfactant concentration was always similar to its critical micelle concentration indicates that the liposome solubilization was mainly ruled by the formation of mixed micelles. The SDS ability to saturate and solubilize SC liposomes decreased as the proportion of Chol-sulf in the bilayers increased until a minimum was reached for a Chol-sulf proportion of about 15%. Inversely, the SDS partitioning into liposomes (or affinity with these bilayers) increased as the proportion of Chol-sulf increased until a maximum was reached at similar Chol-sulf proportions (10–15%). Hence, in these Chol-sulf proportions (similar to that existing in the intercellular lipids, which was 10%) the ability of SDS molecules to interact with liposomes exhibits a minimum despite their enhanced partitioning into liposomes. These effects may be related to the reported dependencies of the level of Chol-sulf on the abnormalities in the skin barrier function and on the SC intercellular cohesion. Received: 12 October 1999 Accepted: 20 January 2000     

Retaining Skin Barrier Function Properties of the Stratum Corneum with Components of the Natural Moisturizing Factor—A Randomized,Placebo-Controlled Double-Blind In Vivo Study

The influence of a topically applied formulation containing components of natural moisturizing factor (NMF) on barrier-related parameters of the stratum corneum (SC) was investigated in vivo using confocal Raman microspectroscopy in a randomized, placebo-controlled double-blind study on 12 volunteers for 14 days. This method allowed for the elucidation of subtle differences between the verum and the placebo even though the components of the verum naturally occur in the SC. This differentiation is not possible non-invasively by conventional methods. In this study, we found that the applied verum and placebo formulations disrupted the equilibrium of water, NMF and lipids in the SC. The adverse effects of the formulation could be mitigated by incorporating it into a simplified supplementation of NMF molecules. As a long-term effect, the amount of strongly bound water increases at 30–40% SC depth (p < 0.05) and the amount of weakly bound water decreases at 30–40% SC depth (p < 0.05) for the verum. This supplement was also unexpectedly able to prevent intercellular lipids (ICL) disorganization in selected depths. In the long term, the verum treatment limited the lateral disorganization of the ICL to the upper 20% SC depth. Further research is required to elucidate the interplay of these factors in the SC, to better understand their contribution to the equilibrium and barrier function of the skin. This understanding of the interaction of these naturally occurring components could help in the future to develop and optimize topical treatments for diseases like psoriasis, atopic dermatitis, ichthyosis where the skin barrier is disrupted.     

Fatty acids influence "solid" phase formation in models of stratum corneum intercellular membranes

Stacked intercellular lipid membranes in the uppermost epidermal layer, the stratum corneum (SC), are responsible for skin's barrier function. These membranes are unique in composition, the major lipids being ceramides (Cer), cholesterol, and free fatty acids (FFA) in approximately equimolar proportions. Notably, SC lipids include chains much longer than those of most biological membranes. Previously we showed that Cer's small hydrophilic headgroup enabled SC model membranes composed of bovine brain ceramide (BBCer), cholesterol, and palmitic acid in equimolar proportion to solidify at pH 5.2. In order to determine the influence of FFA chain length on the phase behavior of such membranes, we used 2H NMR and FT-IR to study BBCer/cholesterol/FFA dispersions containing linear saturated FFA 14-22 carbons long. Independent of chain length, the solid phase dominated the FFA spectrum at physiological temperature. Upon heating, each dispersion underwent phase transitions to a liquid crystalline phase (only weakly evident for the membrane containing FFA-C22) and then to an isotropic phase. The phase behavior, the lipid mixing properties, and the transition temperatures are shown to depend strongly on FFA chain length. A distribution of FFA chain lengths is found in the SC and could be required for the coexistence of a proportion of solid lipids with some more fluid domains, which is known to be necessary for normal skin barrier function.     

Dispersity of stratum corneum in the mixed aqueous solutions of binary mixtures between N,N-dimethyldodecylamine oxide and sodium dodecyl sulfate or two terpenes

Solubilization of cholesterol, differential scanning calorimetric (DSC), nuclear magnetic resonance (NMR) and dynamic light scattering (DLS) measurements were performed in order to reveal the dispersion mechanisms of stratum corneum (SC) into each intact corneocytes in the following systems: (1) in the aqueous mixed solutions of sodium dodecyl sulfate (SDS) and N,N-dimethyldodecylamine oxide (C₁₂DMAO); (2) in the aqueous micellar solutions of C₁₂DMAO containing solubilized α-terpineol (α-T); and (3) in the aqueous micellar solutions of C₁₂DMAO containing solubilized limonene. The intercellular lamellar structure of SC was revealed to be disrupted and/or removed in all these solutions. However, considering the micellar sizes and the interaction among molecules in micelle, the dispersion mechanisms in these three systems were different each other. The three dispersion mechanisms of SC were estimated and discussed on the basis of the results of solubilization, DSC, NMR and DLS, respectively.     

Label free non-invasive imaging of topically applied actives in reconstructed human epidermis by confocal Raman spectroscopy

Raman spectroscopy has become a versatile tool for the in vivo characterization of skin. Here we describe use of Raman spectroscopy for high resolution optical cross sectioning to resolve skin constituents and administered drugs at the cellular level. Percutaneous penetration is typically studied using permeation cells with biopsies of animals or human skin. Although this technique provides valuable clinical data, little insight is gained in the microstructure of drug penetration (intercellular or transcellular) or in the mode of action of applied vehicles or penetration enhancers. Therefore, a Raman microspectroscopic method was combined with a confocal scanning setup to image the microstructure of commercially available skin models (SkinEthic^®) and the spatial distribution of penetrated actives. The models’ microstructure was scanned without any special treatment or environment such as cutting, staining, freezing, or application of vacuum. The non-invasive Raman images reveal the layered structure of stratum corneum. This in particular for lipids while water tends to be more evenly distributed. When penetration of the hydrophilic active glycerol and the lipophilic octyl methoxycinnamate, OMC, was studied, a strong correlation between the local distribution of skin constituents and the hydrophilic/lipophilic character of the active was observed.     

Transport processes in responding lipid membranes: a possible mechanism for the pH gradient in the stratum corneum

The "acidic mantle" of the skin surface has been related to several essential functions of the skin, although the origin of the acidity is still obscure. In this paper, we investigate how different transport processes can influence the local proton concentration inside a membrane consisting of oriented lipid bilayers. This system is chosen as a simple model of the extracellular lipids in the upper layer of the skin, the stratum corneum. We present a theoretical model for diffusional transport over the membrane in the presence of an osmotic gradient and a gradient in CO(2), taking into account the influence of these gradients on the lipid structure and the local electrostatics. We are also discussing the complications in applying the concept of pH to the stratum corneum. From this, we make the following conclusions: (i) The definition of pH in the stratum corneum is ambiguous, and thus, all statements regarding pH should always be related to a clear definition. (ii) A natural definition of pH in the stratum corneum can be proposed which takes into account local heterogeneity, local charges, and the fact that the stratum corneum is not in thermodynamical equilibrium. (iii) Diffusive transport across an oriented bilayer stack in the presence of an osmotic gradient and/or a gradient in CO(2) can give rise to a substantial gradient in pH. (iv) The results from the simplified model can be correlated to experimental observations of pH in the stratum corneum.     

Dispersion of stratum corneum in an aqueous mixed solution of surfactants: the effect of a mixture of sodium dodecyl sulfate and N,N-dimethyldodecylamine oxide

Molecular interactions between sodium dodecyl sulfate (SDS) and N,N-dimethyldodecylamine oxide (C₁₂DMAO), whose mixtures were effective for dispersion of stratum corneum (SC) into intact corneocytes, were studied and found to be strongest at an SDS/C₁₂DMAO molar ratio of 1/3, when dispersion of SC was most effective and the sizes of the mixed micelles were largest.

This dispersion effect was confirmed as being caused by the stronger solubilizing power of molecular complexes formed between SDS and C₁₂DMAO in the binary mixed solutions by using ¹H NMR. The mechanisms for removing intercellular lipids such as ceramides, cholesterol and their derivatives, which play an important role as adhesives among the corneocytes, and for dispersing SC into intact cells were proposed on the basis of supporting data obtained from ¹H-NMR and light scattering measurements.     

ESI–MS method for in vitro investigation of skin penetration by copper–amino acid complexes: from an emulsion through a model membrane

Copper can be found in many cosmetic formulations, mainly as complexes with peptides, hydroxyacids or amino acids. The main reason that the usage of this element in this context is still increasing is its beneficial biochemical activity, although the mechanism that enables its complexes to permeate through skin barriers is largely unknown. The ability of copper complexes with amino acids to penetrate through the stratum corneum and participate in copper ion transport processes is key to their cosmetic and pharmaceutical activities. The penetration process was studied in vitro in a model system, a Franz diffusion cell with a liposome membrane, where a liquid crystalline system with physicochemical properties similar to those of the intercellular cement of stratum corneum was used to model the skin barrier. The influences of various ligands on the model membrane migration rate of copper ions was studied, and the results highlighted the crucial roles of metal ion complex structure and stability in this process.     

The effect of internal lipids on the water sorption kinetics of keratinised tissues

Water has a large influence on the properties of keratinised tissues. The water diffusion properties of keratinised tissues are known to be governed by the cell membrane complex, which is mainly composed of internal lipids. The main aim of this work was to characterise the differences in the water sorption and desorption behaviour of human hair and stratum corneum (SC) both with and without internal lipids. Absorption and desorption curves were obtained using a thermogravimetric balance equipped with a controlled humidity chamber. The results demonstrate that the role of the intercellular lipids in the SC is more marked than in hair, which is likely due to the greater amount of lipids present in its structure. Therefore, lipid structures in the SC are essential both to prevent changes in the water-holding capacity of the skin and to maintain the water permeability of the SC.     

EVALUATION OF MODEL FOR STRATUM CORNEUM LIPID BARRIER AGAINST STEROID DIFFUSION

The ability of three matrices to model the barrier properties of the lipid domain of stratum corneum (SC) against permeation of seven steroids was studied. Model matrices were water and oleic acid/oleate; a mixture of unsaturated and saturated fatty acids/soap; or a more complex matrix also containing phospholipids, sphingolipids, cholesterol and ceramides.

Permeability coefficients (K.) were similar in the three models, supporting the hypothesis that the barrier to steroid permeation is determined by the structural organization of the lipids, not by the chemical structure of individual substances. Parabolic relationships were found between K values and octanol/water partition coefficients (P_oct) of the steroids, with an optimum permeability at log P_oct, of 3·0. All three models showed good resistance to permeability by steroids. The effects of cationic, anionic and non-ionic surfactants on the permeability of hydrocortisone within the water oleic acid/oleate matrix were also investigated. Permeability increased with anionic surfactants, decreased with cationic surfactants and varied little with non-ionic surfactants. The matrices tested appeared able to model the effect of surfactants on the permeability of hydrocortisone through the SC     

Influence of the hydrophobic tail of alkyl glucosides on their ability to solubilize stratum corneum lipid liposomes

The lytic interactions of a series of alkyl glucosides (alkyl chain lengths ranging from C₈ to C₁₂) with liposomes formed by a mixture of lipids modeling the stratum corneum (SC) lipid composition were investigated. The surfactant-to-lipid molar ratios (Re) and the normalized bilayer/aqueous phase partition coefficients (K) were determined by monitoring the changes in the static light-scattering (SLS) of the system during solubilization. The fact that the free surfactant concentrations were always similar to their critical micelle concentrations indicates that the liposome solubilization was mainly ruled by the formation of mixed micelles. At the two interaction levels studied (100 and 0% SLS) the nonyl glucoside showed the highest ability to saturate and to solubilize liposomes (lowest Re values), whereas the dodecyl glucoside showed the highest degree of partitioning into liposomes or affinity with these structures (highest K values). Comparison of the data for octyl glucoside with that reported for the interaction of this surfactant with phosphatidylcholine (PC) liposomes shows that whereas the SC lipid liposomes were more resistant to the action of this surfactant (higher Re values), its degree of partitioning into SC bilayers was both in the saturation and solubilization of liposomes similar to that exhibited in PC vesicles (similar K values). Received: 27 November 2000/Accepted: 19 February 2001     

SKIN PERMEATION ENHANCEMENT BY FATTY ACIDS

The present study elucidates the skin permeation enhancement effects of a number of fatty acids, i.e. straight-chain saturated (SFA), monounsaturated (MUFA) and polyunsaturated acids (PUFA). The effects were studied using human stratum corneum (SC) and p-aminobenzoic acid (PABA) as a model permeant. The fatty acids in propylene glycol (FA/PG) were applied according to a pre-treatment/co-treatment protocol. SFA with 6 to 12 carbons exhibit a parabolic correlation between enhancement effect and chain-length, with a maximum at nonanoic-decanoic acids (with 9 and 10 carbons). All cis-6-, 9-, 11- or 13-octadecenoic acids (MUFA) enhance the permeation of PABA to the same extent. PUFA — linoleic (LA), α-linolenic (ALA) and arachidonic acids — enhance PABA permeation stronger than MUFA but additional double bonds do not further increase the degree of enhancement. The enhancement effects of fatty acids on the PABA penetration through SC are structure-dependent, associated with the existence of a balance between the permeability of pure fatty acids across SC and the interaction of the acids to skin lipids. Based on this and other studies, a set of mechanisms of action is proposed for fatty acids.