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     

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.     

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.     

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.     

Interaction of Stratum Corneum Components With Benzalkonium Chloride. Isothermal titration calorimetric study

The interactions of benzalkonium chloride (BC)with components of stratum corneum, a model system of intercellular lipids in human stratum corneum and homogenized rat stratum corneum were characterized in terms of thermodynamics at pH 7.5 and 37°C. BC was strongly bound to cholesterol and cholesterol sulfate with higher affinities (10⁵~10⁶ M^-1) than to any other components of the stratum corneum by hydrophobic interaction and ionic interaction, respectively. BC binding to the model system of intercellular lipids significantly decreased only in the absence of cholesterol. It is indicated that cholesterol and its derivatives play an important role in the penetration and/or accumulation of BC in stratum corneum. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

POLYGLYCERYL OLEATE SURFACTANTS AND THE STRATUM CORNEUM LIPID

Polyglyceryl oleates with 3,6,10 glycerol groups formed lamellar liquid crystal with water. The penetration of water into amphiphilic bilayer was reduced with increased the number of glycerol groups. Polyglyceryl oleate entered into a liquid crystalline model of the stratum corneum lipids reduced the penetration of water and enhanced the stability towards low water content.     

Thermal Analysis Studies on Human Skin and Skin Barrier Modulation by Fatty Acids and Propylene Glycol

The thermal behaviour of human stratum corneum (SC) with various hydration levels was studied using differential thermal analysis DSC within the temperature range of –130 to 120°C. SC containing 20% water, resembling the intact condition, shows thermal transitions at around –20°C (representing water in skin), –10, 40, 70°C (representing skin lipids), 85°C (representing protein-associated lipids) and 100°C (representing skin protein). Dehydration of SC causes the transitions at –20 and 100°C to be invisible. Lipid extraction followed by dehydration eliminates all transitions. Further hydration produces a transition of water at around 0°C with a huge change in enthalpy. The perturbation effects of penetration enhancers fatty acids (FA) and propylene glycol (PG) were studied using DTA on SC after pretreatment with PG alone and FA/PG. The application of PG alone shifted the transitions at 70 and 85°C to lower temperatures. Additionally, the application to dehydrated stratum corneum removes the transitions at –10°C. Saturated fatty acids, e.g. nonanoic and decanoic acids, exert barely noticeable effects on the thermal behaviour of SC suggesting that they easily mix with the skin lipids. Thermal analysis also revealed that the cis-9- and 13-isomers of octadecenoic acid (monounsaturated fatty acids) form a separate domain containing mostly the pure fatty acids within the SC lipids and suppress the lipid transitions at 70/80°C. Polyunsaturated fatty acids linoleic and -linolenic acids — form separate domains but do not completely suppress the SC lipid transitions at 70/80°C as monounsaturated acids do. This study suggests different ways of perturbation by various fatty acids.This revised version was published online in November 2005 with corrections to the Cover Date.     

Reducing Variability in Pollution Data: The Use of Lipid Classes for Normalization of Pollution Data in Marine Biota

Abstract

Organochlorine residues (PCBs and pesticides) are usually normalized on lipid content, gravimetrically quantified after extraction in organic solvents. In this study, the lipid content of different biotic groups (e.g. phytoplankton, shrimp, bivalves, fish), obtained through different types of extraction (total extraction versus specific extraction of apolar lipids) and quantification techniques (total gravimetric determinations versus specific determinations of lipid classes (latroscan)) were compared. The different lipid quantification methods were interrelated. An overestimation of lipid content due to non-lipid co-extracts was shown to interfere with gravimetric lipid quantifications. The relative contribution of these co-extracts depended on the type of sample studied. Differences in pollution levels (individual PCB congeners) between different biotic groups (phytoplankton, shrimp, cockles, fish) and between different fish tissues are discussed in relation to lipid content, determined by the different methods. An important reduction in the variability of pollution levels was noted when data were normalized on total neutral lipid content (latroscan). Highly significant linear regression parameters were obtained between the pollutant and the total neutral lipid content of marine biotic samples from the same water mass. Water mass specific bio-lipid contamination levels, characteristic for the whole biotic community (excluding seabirds and sea mammals), were determined for the different PCB congeners, normalized on total neutral lipid content. These bio-lipid contamination levels were related to the K_ow of the congeners. This functional relationship could be described by a second order polynomial regression.     

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.     

TRANSMISSION OF HUMAN EPIDERMIS AND STRATUM CORNEUM AS A FUNCTION OF THICKNESS IN THE ULTRAVIOLET AND VISIBLE WAVELENGTHS

Abstract The dependence of radiation transmission on sample thickness was studied in isolated samples of human stratum corneum and full-thickness epidermis. The investigation also included samples of skin repeatedly exposed to UV-B. Transmission was measured in the ultraviolet and in the visible from 248–546 nm. Two methods, one microscopic and the other mechanical, were used to measure thickness. There was a good correlation between the results.
The dependence of transmission on thickness in these samples could be described satisfactorily by an exponential function, implying that the Lambert-Beer law is approximately valid. Thus, a single parameter, such as the half-value layer ( d _½), is sufficient to characterize absorption in the skin samples.
Water content of the isolated stratum corneum was influenced by maintenance conditions: samples floating on water containing a small amount of NaCl were more hydrated than samples floating on a more concentrated salt solution, or stored in air. Changes in water content of the samples resulted in changes of thickness and, to a lesser extent, of transmission. Approximate in vivo values of d _½ were computed after estimating the in vivo water content of stratum corneum.
Differences found in the shape of the transmission spectra of stratum corneum and full-thickness epidermis may reflect differences in chemical composition. The influence of wetting of the skin on its sensitivity to sunlight is explained in a new way.     

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.     

INTERACTION OF A MODEL EPIDERMAL LIPID WITH A VEGETABLE OIL ADDUCT

A study of the interaction between a model epidermal lipid and 2(alkoyloxy)-1-[(alkoyloxy)methyl]-ethyl-7-(4 heptyl-5,6-dicarboxyl-2-cyclo-hexene-1-yl)heptanoate, (G2) was made by use of small angle X-ray diffraction. The small angle X-ray diffraction pattern of the epidermal lipid model was first compared to the X-ray pattern obtained from human stratum corneum. Increase in the water content of the model epidermal lipid from 32% to 35% caused a shift in the broad diffraction peak from 50-80Å to 50-70Å. No additional shift was found at further addition of water. The addition of G2 to the 32% water model also caused a shift toward smaller interlayer spacing. The effect of G2 was most dramatic for the model epidermal lipid at the lowest water content.     

Analysis of electron spin resonance spectra of alkyl spin labels in excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes

The electron spin resonance (ESR) spectra of alkyl spin labels were observed in the excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes. The spectrum of 5-doxylstearic acid (5-NS) in the stratum corneum and order parameter obtained from the spectrum, indicated that the spin label was present in highly ordered lipid lamella. On the other hand, the spectrum of methyl ester of 5-NS (5-NMS) and its apparent rotational correlation time calculated from the spectrum, showed only a weakly immobilized component in the stratum corneum as well as in the whole excised skin. The ester spin label seemed to be scarcely present in the rigid lipid lamella, but mainly in the relatively fluid environment. On the other hand, cationic alkyl spin labels showed quite different spectra depending on their alkyl chain lengths. Long-chain 4-(N,N-dimethyl-N,-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-15) seemed to be present in the protein region of the stratum corneum as we recently reported, whereas hydrophilic quaternary ammonium spin label 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-1) seemed to be present in the bulk water of the skin, even in delipidized skin. These findings indicated that the different interaction and different localization of the alkyl spin labels depended on their electronic charge as well as their alkyl chain lengths.     

Proton spin lattice relaxation times in biological tissues Water and lipid role

Abstract

The multiphase model of water present in biological tissues derives mainly from the N.M.R. observation of a fraction of non-freezing water in biological tissues which is attributable to the water bound to macromolecules. Studies of this problem rule out completely the lipids as the source of the NMR signal. Our studies on nuclear spin lattice relaxation times of human and animal tissues have been made to understand if other contributions to the N.M.R. signal are present in addition to that coming from the water protons. We have measured directly by the N.M.R. method the relative water and lipid content and the relaxation time T ₁ as a function of the water content which was varied by controlled dehydration. The results show clearly that lipids contribute actively to the N.M.R. signal and the fast relaxation time T ₁ which is of the order of 100 ms in all biological tissues is related to the lipids. In view of these experimental observations we think that it is opportune to reconsider critically all the determinations of the ‘bound water’ made by the freezing procedure with the N.M.R. technique, and dedicate more attention to the lipids of biological membranes.     

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.     

An experimental approach to study the binding properties of vitamin E (alpha-tocopherol) during hairless mouse skin permeation.

An experimental approach to study the binding properties of vitamin E has been developed. Total vitamin E solubility in the skin was determined by a partition study, followed by in vitro skin permeation studies with whole skin and stripped skin. The amount of freely diffusable vitamin E in the diffusion process was determined from the permeation profiles of whole skin and stripped skin by employing a bi-layer model. The concentrations of vitamin E in the stratum corneum and viable dermis were determined separately. By subtracting this amount from the total concentration of vitamin E in the skin, as determined by the solubility study, the amount of bound vitamin E was determined. After skin permeation reached a steady state, the donor solution was removed and the permeation study continued (desorption study). During the entire period of the desorption experiment, the amount of vitamin E in the receptor solution hardly increased and remained constant. After the desorption experiment, vitamin E still remaining in the skin was determined by extracting with tissue solubilizer, SOLABLE, and is considered as the amount of vitamin E strongly bound in the skin. The concentrations of bound vitamin E determined by permeation and desorption studies coincided relatively well. To further investigate skin binding of vitamin E, a differential scanning calorimetry study was performed. Vitamin E-treated stratum corneum showed phase transitions at 76 and 85 degrees C, associated with lipid transitions. The thermal transitions associated with the lipid transition suggested interactions of vitamin E with lipid components of the skin. During skin permeation, vitamin E forms a very strong reservoir in the skin tissue and this amount of vitamin E, about 30%, exists as a bound-form with the lipid components of the stratum corneum.     

Influence of Amylose-Amylopectin Ratio on Properties of Extruded Starch Plastic Sheets

Abstract

Starch plastic sheets were prepared by extrusion processing of mixtures of granular high-amylopectin and high-amylose starches in the presence of glycerol and water as plasticizers. Amylose content varied between 0 and 70% (w/w). Structural characterization and determination of the mechanical properties of the sheets were performed after aging the materials between 40–65% relative humidity for 2 and 35 weeks and at 90% relative humidity for two weeks. The materials were semicrystalline and viscoelastic. The materials were described as complex heterogeneous multiphase materials. They consisted of amorphous and crystalline phases of amylose and amylopectin as well as granular structures and domains of amylose, amylopectin and amylose-amylopectin helices. Single-helical type crystallinity was formed solely by amylose directly after processing while B-type crystallinity was rapidly formed in amylose-rich materials and slowly during aging of amylopectin-rich materials.

The stress-strain and stress-relaxation properties were related to differences in amylose content, degree of crystallization and water content. The amorphous amylopectin rich materials were flexible and soft but showed an increase in stiffness and a decrease in elongation due to crystallization. Amylopectin-rich materials showed unfavorable relaxation, shrinkage and cracking during aging. The materials rich in amylopectin were sensitive to water content while the amylose-rich materials were not sensitive to water in the range of 9–13% (w/w). Stress-strain relaxation behaviors of the materials were dependent on starch structure and on experimental conditions such as strain rate and extension by which the ratio of elastic and viscous response were varied. An increase in relaxation times was found with increasing amylose content and water content for the materials with solely amylose crystallinity.     

Destabilization of whole skin lipid bio-liposomes induced by skin penetration enhancers and FT-IR/ATR (Fourier transform infrared/attenuated total reflection) analysis of stratum corneum lipids.

Whole skin lipid bio-liposomes (skin bio-liposomes), in size ranging from 2 to 8 microns, were prepared by a reverse phase evaporation technique using rat full thickness skin. Leakage of an encapsulated fluorescence probe, ANTS (delta-amino-1,3,6-naphthalene-trisulfonate), was measured by adding transdermal penetration enhancers (penetrants) into the medium where the skin bio-liposomes were present. Oleylamine induced a fast release of ANTS from the liposomes compared to lauryl-amine which showed a weak action. With these penetrants, the degree of ANTS release from the prepared bio-liposomes was found to correlate well with the results of frequency changes in the CH-asymmetric stretching band near 2920 cm-1 in the rat stratum corneum. The penetrant which caused relatively strong leakage of ANTS induced the significantly large shift of the peak toward the higher wave-numbers due to the perturbation in the structure of lipids of the stratum corneum. The skin bio-liposomes prepared from the rat full thickness skin could be useful in evaluating the penetrants.     

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