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

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

Stratum corneum lipid phase transitions and water barrier properties

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

Interactions of elastic and rigid vesicles with human skin in vitro: electron microscopy and two-photon excitation microscopy.

Interactions between vesicle formulations and human skin were studied, in vitro, in relation to their composition and elasticity. The skin ultrastructure was investigated using transmission electron microscopy (TEM), freeze-fracture electron microscopy (FFEM) and two-photon fluorescence microscopy (TPE). The main difference between the vesicle formulations was their elasticity. Elastic vesicle formulations contained bilayer forming surfactants/lipids and single-chain surfactant octaoxyethylenelaurate-ester (PEG-8-L), whereas rigid vesicles contained bilayer surfactants in combination with cholesterol. TEM results showed three types of interactions after non-occlusive application of elastic PEG-8-L containing vesicle formulations on human skin: (1) the presence of spherical lipid structures containing or surrounded by electron dense spots; (2) oligolamellar vesicles were observed between the corneocytes in the upper part of the stratum corneum; and (3) large areas containing lipids, surfactants and electron dense spots were observed deeper down into the stratum corneum. Furthermore, after treatment with vesicles containing PEG-8-L and a saturated C12-chain surfactant, small stacks of bilayers were found in intercellular spaces of the stratum corneum. Rigid vesicles affected only the most apical corneocytes to some extent. FFEM observations supported the TEM findings. Major morphological changes in the intercellular lipid bilayer structure were only observed after treatment with PEG-8-L containing elastic vesicles. TPE showed a distinct difference in penetration pathways after non-occlusive application of elastic or rigid vesicles. After treatment with elastic vesicles, thread-like channels were formed within the entire stratum corneum and the polygonal cell shape of corneocytes could not be distinguished. Fluorescent label incorporated in rigid vesicles was confined to the intercellular spaces of the upper 2-5 micrometer of the stratum corneum and the cell contours could still be distinguished.     

Preparation,Characterization, and <Emphasis Type="Italic">In Vitro</Emphasis> Permeation Study of Terbinafine HCl in Poloxamer 407<Emphasis Type="Bold">-</Emphasis>Based Thermogelling Formulation for Topical Application

Upon topical administration, a high penetration rate of antifungal drug into the infected site is desirable to reduce treatment length and systemic side effects which occur especially after a prolonged peroral administration. Thermogelling formulations composed of poloxamer 407, medium chain triglycerides, isopropyl alcohol, dimethyl isosorbide, and water for topical application were developed, and a lipophilic drug terbinafine HCl (TBF) was incorporated. Previously, a remarkable high permeation rate of a hydrophilic drug 5-aminolevulinic acid from this vehicle was evident compared to different creams from German Pharmacopoeia. By varying the composition of vehicle constituents, a broad range of consistencies and appearances was obtained. Up to 4% TBF could be solubilized in the vehicle. TBF fluxes at steady state across human stratum corneum from these formulations were higher than those from the German Pharmacopoeia Basiscreme Deutscher Arzneimittel Codex and a marketed product at similar concentration of 1%. TBF fluxes increased along with a higher content of TBF in the formulation. The amount of TBF retained in stratum corneum was higher compared to those from both standards of comparison (p < 0.01). The thermodynamic activity of TBF in the thermogelling formulation was lower compared to those in other formulations. Therefore, the nature of the vehicle and its interaction with TBF are suggested to play a significant role in explaining higher fluxes along with higher TBF content. Differential scanning calorimetry measurements revealed comparable T2 and T3 endothermic shifts from all examined formulations suggesting equal influences to the skin lipids.     

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

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

Effect of Hydroxyl Groups and Rigid Structure in 1,4-Cyclohexanediol on Percutaneous Absorption of Metronidazole

In a previous study, a synergistic retardation effect of 1,4-cyclohexanediol and 1,2-hexanediol on percutaneous absorption and penetration of metronidazole (MTZ) was discovered. A complex formation between 1,4-cyclohexanediol and 1,2-hexanediol was proposed to be responsible for the observed effect. The objective of this study was to investigate the necessity of hydroxyl group and the ring structure in 1,4-cyclohexanediol on percutaneous absorption and penetration of MTZ. Eleven formulations were studied in an in vitro porcine skin model using glass vertical Frans Diffusion Cell. 1,4-Cyclohexanediol was changed into 1,4-cyclohexanedicarboxylic acid, trans (and cis)-1,2-cyclohexanediol and 1,6-hexanediol, respectively, to study if H-bonding or ring structure would influence the retardation effect. MTZ was applied at infinite dose (100 mg), which corresponded to 750 μg of MTZ. Based on modifier ratios (MR) calculated by the flux values, the retardation effect on percutaneous absorption and penetration of MTZ was found in the formulations containing 1,4-cyclohexanedicarboxylic acid or cis-1,2-cyclohexanediol (MR values were 0.47 for which only contains 1,4-cyclohexanedicarboxylic acid, 0.74 for the formulation containing both 1,4-cyclohexanedicarboxylic acid and 1,2-hexanediol, and 0.90 for the formulation containing cis-1,2-cyclohexanediol and 1,2-hexanediol, respectively). The results showed that the hydroxyl group and structure of 1,4-cyclohexanediol played a significant role in retardation effects and provided valuable insight on the mechanisms of retardation effect through structure–activity relationships.     

Quantitative analysis of histidine and cis and trans isomers of urocanic acid by high-performance liquid chromatography: a new assay method and its application

To elucidate the factors involved in dry skin and the skin damage caused by UV light, it is necessary to analyze small amounts of stratum corneum to determine amino acid contents. A new assay method for this purpose is described. Dabsylated amino acids including histidine and the cis and trans isomers of urocanic acid were analyzed quantitatively by high-performance liquid chromatography (HPLC), using a reversed-phase column. Histidine and the isomers of urocanic acid were separated from 36 other amino acids thought to be present in the extract of stratum corneum. In the presence of the 36 amino acids, standard calibration curves were obtained from 0.25 to 2.5 pmol/μl, for histidine and for both isomers of urocanic acid. The coefficients of variation for the reproducibility of the analysis at 1.0 pmol/μl were 3.8%, 2.9% and 2.5% for the cis and trans isomers of urocanic acid and for histidine, respectively. Amounts of 2 to 50 pmol of cis and trans isomers of urocanic acid and histidine in the stratum corneum were detected. The ratio of the cis to the trans isomer of urocanic acid in sunburned stratum corneum was more than three times that in normal stratum corneum. This method appears to be useful for the determination of small amounts of histidine and of the cis and trans isomers of urocanic acid in the stratum corneum.     

Identification of two opaque2 modifier loci in Quality Protein Maize

Genetic modifiers of opaque2 convert the soft, starchy endosperm of opaque2 maize mutants to a hard, vitreous phenotype, while maintaining the enhanced lysine content of the grain. Genetic analysis of F2 segregating seeds from crosses of opaque2 by modified opaque2 genotypes indicated that the modifiers are complex traits that act codominantly. We developed two different segregating F2 populations and mapped the modifier loci by restriction fragment length polymorphism (RFLP) analysis. A relationship was found between formation of vitreous endosperm and the locus encoding the gamma-zein storage protein, which maps near the centromere of chromosome 7. Endosperm modification was consistently associated with the presence of two rather than one gamma-zein gene at this locus. A second modifier locus was mapped near the telomere of chromosome 7L.     

Impact of optical clearing on ex vivo human skin optical properties characterized by spatially resolved multimodal spectroscopy

A spatially resolved multimodal spectroscopic device was used on a two-layered “hybrid” model made of ex vivo skin and fluorescent gel to investigate the effect of skin optical clearing on the depth sensitivity of optical spectroscopy. Time kinetics of fluorescence and diffuse reflectance spectra were acquired in four experimental conditions: with optical clearing agent (OCA) 1 made of polyethylene glycol 400 (PEG-400), propylene glycol and sucrose; with OCA 2 made of PEG-400 and dimethyl sulfoxide (DMSO); with saline solution as control and a “dry” condition. An increase in the gel fluorescence back reflected intensity was measured after optical clearing. Effect of OCA 2 turned out to be stronger than that of OCA 1, possibly due to DMSO impact on the stratum corneum keratin conformation. Complementary experimental results showed increased light transmittance through the skin and confirmed that the improvement in the depth sensitivity of the multimodal spectroscopic approach is related not only to the dehydration and refractive indices matching due to optical clearing, but also to the mechanical compression of tissues caused by the application of the spectroscopic probe.     

Physical and chemical perturbations of the supramolecular organization of the stratum corneum lipids: In vitro to ex vivo study

Background: FTIR spectroscopy is classically used to study the supramolecular organization of the stratum corneum lipids. Exposure to UV_A is responsible for a small decrease in packing observed on cutaneous lipid films. Methods: Lipid films and human skin biopsies were either exposed to UV_A irradiation of 120 J/cm², UV_B irradiation of 0.15 J/cm² or put in contact with ethanol. Using FTIR in vitro and IR microspectroscopy ex vivo provided information on: i) the precise localisation of the stratum corneum in the skin, ii) its thickness, and iii) the organization of its constituted lipids. Results: Different action modes were observed for UV irradiation and the contact with ethanol with a certain destabilisation of the lipidic layer. Ethanol was also found to be responsible for the creation of pores. The destabilisation of the lipid cement was mainly observed ex vivo. Conclusion: The barrier function of the skin is affected by the action of physical and chemical external agents at the molecular level. The increased laxity of the lipid packing could enable the percutaneous penetration velocity of actives.     

Interactions Between non-ionic Surfactant Vesicles and human stratum corneum in vitro.

Abstract

The effects of non-ionic surfactant vesicles (NSVs) on human skin in vitro were studied in relation to the physico-chemical properties of the vesicles. The interactions between NSVs and skin were visualized using both freeze fracture electron microscopy and confocal laser scanning microscopy. The physico-chemical properties of the NSVs were varied in a systematic way, using a broad series of polyoxyethylene monoalkyl ether type surfactants (C_nEO_m). The number of oxyethylene units (m) was varied between 3, 7 and 10, and the number of carbon atoms (n) was either 12 or 18. Both the effects of liquid state vesicles composed of ^C12^Eo3,7 ^and C9=9^EO10 surfactants and gel state vesicles (C18EO37) were investigated. After the application of the NS V suspension on the stratum corneum surface two essentially different types of vesicle-skin interactions were visualized. Firstly, an interfacial interaction, involving the adsorption of vesicles, and the deposition of bilayer sheets on the outermost layers of the stratum corneum observed for all NSV formulations tested in this study. Secondly, effects on the ultrastructure of the stratum corneum were observed: the appearance of water pools observed for the liquid state vesicles only, and ultrastructural changes of the intercellular lipid domains are induced only observed after treatment with C₁₂EO₃ NSVs. Neither changes in the ultrastructure of the viable epidermis, nor changes in deeper skin layers were observed.     

Individual variation of human plantar stratum corneum lipids, determined by automated multiple development of high-performance thin-layer chromatography plates

The stratum corneum lipids are unique in composition and have been used frequently as a model system of the skin's lipid barrier. Automated multiple development (AMD) of high-performance thin-layer chromatography plates in combination with a 25-step gradient, based on methanol, diethyl ether and n-hexane separated the six major human plantar stratum corneum lipids. Post-chromatographic staining of these lipids with a solution of MnCl₂H₂SO₄ at 130°C or a solution of CuSO₄H₃PO₄ at 140°C allowed visualization of the lipids and quantification. The MnCl₂H₂SO₄ solution stained saturated fatty acids less intensity. Therefore, the CuSO₄H₃PO₄ solution was used for quantification and we found, on average, 2.06% (w/w) cholesterol 3-sulphate, 20.16% (w/w) free fatty acids, 20.25% (w/w) ceramides, 43.53% (w/w) non-esterified sterols, 4.56% (w/w) triacylglycerols and 9.4% (w/w) sterolesters in the human plantar stratum corneum extracts. The concentration of phospholipids was less than 1% (w/w). In addition, the lipid composition of twenty different human plantar stratum corneum extracts was determined. Statistics revealed a correlation between the ratio of free fatty acids and non-esterified sterols (r=0.832, p<0.01, n=20). Several control experiments proved that this correlation is not due to the extraction method, the post-chromatographic staining procedure or bacterial contamination of the stratum corneum.     

Covalently bound ω-hydroxyacylsphingosine in the stratum corneum

Pig epidermis was heat separated, and the stratum corneum was isolated after trypsinization. Exhaustive extraction of the stratum corneum fraction with chloroform/methanol mixtures yielded 14.7% lipid on a dry weight basis. After mild saponification of the extracted residue, additional lipid could be extracted which accounted for 2.1% of the stratum corneum weight. This bound lipid proved to consist mainly (91.9%) of N-(ω-hydroxyacyl)sphingosines in which the amide-linked ω-hydroxyacids were 28 to 34 carbon atoms in length. The release of this lipid by mild alkaline hydrolysis indicates that it is bound through an ester linkage. Half of the hydroxyceramide molecules reacted in situ with acidic acetone, suggesting that half of these molecules are attached to the stratum corneum through the ω-hydroxyl function, while the other half may be linked through one of the hydroxyl groups of the sphingosine.     

Estradiol distribution and penetration in rat skin after topical application,studied by high resolution autoradiography

Summary Transdermal pathways and targets in the skin for estradiol were investigated using dry-mount autoradiography.³H-estradiol-17 was applied at doses of 30.1 pmol, 120.4 pmol and 301 pmol/cm² to shaved rat skin in the dorsal neck region. Vehicles were DMSO, ethylene glycol or sesame oil. After 2 h of topical treatment with 30.1 pmol³H-estradiol×cm^–2 dissolved in DMSO a distinct cellular distribution was apparent. Target cells with concentrations of radioactivity were found in epidermis, sebaceous glands, dermal papillae of hair and fibroblasts. After treatment with 120.4 and 301 pmol/cm², a penetration gradient of radioactivity was recognizable however it masked specific cellular and subcellular uptake. The stratum corneum accumulated and retained radioactivity, apparently forming a depot for the hormone. Strong concentration and retention of the hormone was conspicious in sebaceous glands for more than 24 h, suggesting that sebaceous glands serve as a second storage site for the hormone. In all autoradiograms two penetration pathways to the dermis were visible: one through the stratum corneum and epidermis, the other through the hair canals and hair sheaths.This work was supported by US PHS grant NS09914     

Animal Experiments—An Essential Component for the Development of Liposomal Anticancer Agents

The poor selectivity of anticancer drugs often leads to their multiplicate dose-limiting toxicities in humans, which severely restricts their clinical application. In this study, a novel liposomal formulation of zedoary turmeric oil (ZTO) targeting the insulin receptor (IR) was prepared by covalently conjugating insulin to the terminal of the polyethylene glycol (PEG) chain of sterically stabilized liposomes. In vitro assays indicated that a higher uptake of insulin-modified sterically stabilized liposomes (ISSLs) was observed in SMMC-7721 hepatocarcinoma cells overexpressing insulin receptors. IC₅₀ values of ISSLs, NTLs (nontargeted liposomes), and ZTO injection (free ZTO) against SMMC-7721, determined by MTT assays, were 157.2, 256.7, and 43.3?μg·ml^?1, respectively. Plasma-clearance profiles of ZTO in the liposomal formulations were then compared with that of ZTO injection. The liposomal formulations showed much longer terminal half-lives (11.24 and 14.73 hours for ISSLs and NTLs, respectively) than that of ZTO injection (1.45 hours). All results above indicated the ISSLs were potentially useful for the treatment of IR (+) tumors and are worthy of further investigation.     

Conformations of peptides containing a chiral cyclic α, α‐disubstituted α‐amino acid within the sequence of Aib residues

A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac₅c^dOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and ¹H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac₅c^dOM]‐(Aib)₄‐OEt ( 1) and Cbz‐(Aib)₄‐[(S,S)‐Ac₅c^dOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 3₁₀‐helical structure was present in 1 and that a right‐handed (P) 3₁₀‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Estradiol distribution and penetration in rat skin after topical application, studied by high resolution autoradiography

Transdermal pathways and targets in the skin for estradiol were investigated using dry-mount autoradiography. 3H-estradiol-17 beta was applied at doses of 30.1 pmol, 120.4 pmol and 301 pmol/cm2 to shaved rat skin in the dorsal neck region. Vehicles were DMSO, ethylene glycol or sesame oil. After 2 h of topical treatment with 30.1 pmol 3H-estradiol x cm-2 dissolved in DMSO a distinct cellular distribution was apparent. Target cells with concentrations of radioactivity were found in epidermis, sebaceous glands, dermal papillae of hair and fibroblasts. After treatment with 120.4 and 301 pmol/cm2, a penetration gradient of radioactivity was recognizable however it masked specific cellular and subcellular uptake. The stratum corneum accumulated and retained radioactivity, apparently forming a depot for the hormone. Strong concentration and retention of the hormone was conspicuous in sebaceous glands for more than 24 h, suggesting that sebaceous glands serve as a second storage site for the hormone. In all autoradiograms two penetration pathways to the dermis were visible: one through the stratum corneum and epidermis, the other through the hair canals and hair sheaths.     

Development and Evaluation of Ethyl Cellulose-Based Transdermal Films of Furosemide for Improved In Vitro Skin Permeation

Transdermal films of the furosemide were developed employing ethyl cellulose and hydroxypropyl methylcellulose as film formers. The effect of binary mixture of polymers and penetration enhancers on physicochemical parameters including thickness, moisture content, moisture uptake, drug content, drug–polymer interaction, and in vitro permeation was evaluated. In vitro permeation study was conducted using human cadaver skin as penetration barrier in modified Keshary–Chein diffusion cell. In vitro skin permeation study showed that binary mixture, ethyl cellulose (EC)/hydroxypropyl methylcellulose (HPMC), at 8.5:1.5 ratio provided highest flux and also penetration enhancers further enhanced the permeation of drug, while propylene glycol showing higher enhancing effect compared to dimethyl sulfoxide and isopropyl myristate. Different kinetic models, used to interpret the release kinetics and mechanism, indicated that release from all formulations followed apparent zero-order kinetics and non-Fickian diffusion transport except formulation without HPMC which followed Fickian diffusion transport. Stability studies conducted as per International Conference on Harmonization guidelines did not show any degradation of drug. Based on the above observations, it can be reasonably concluded that blend of EC–HPMC polymers and propylene glycol are better suited for the development of transdermal delivery system of furosemide.     

Experimental penetration of Trichophyton mentagrophytes into human stratum corneum

We present confirmation of the experimental penetration of Trichophyton mentagrophytes into human stratum corneum under designated conditions of temperature and humidity. When stratum corneum, obtained from healthy human heel region, was incubated at 100% humidity, mycelium was observed in the corneum layer on day 2 at 35 °C and 27 °C, and on day 4 at 15 °C. At 90% humidity, the hyphae penetrated into the stratum corneum on day 4 at 35 °C, and on day 6 at 27 °C. Whereas, at 80% humidity, no fungal elements were observed in the stratum corneum at both 27 °C and 35 °C for up to 7 day. These data suggested that humidity was a more important environmental factor for penetration than temperature, and that at least 90% humidity is necessary for dermatophytes to penetrate into the stratum corneum within a few days. Mean humidity in the interdigital space between the fourth and fifth toes was found to be approximately 98%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in structure of ventral epidermis of Rana ridibunda during metamorphosis

Summary The organisation of the ventral epidermis organisation was followed throughout ontogenesis in Rana ridibunda. Epidermis of tadpoles with 2–3 limbs was organised into two layers: a stratum germinativum consisting of elongated columnar cells, and an outer stratum corneum consisting of two types of cuboid cells. Two types of cells can be distinguished; they are a light (clear) cell and a dark (dense) cell. In the 4-legged tadpoles the stratum corneum cells start to flatten and a replacement layer appeared underneath. A well-defined stratum germinativum is found and within it, epidermal glands. Moulting took place for the first time in tadpoles just before metamorphosis, and a well-organised stratum granulosum was formed still containing the two main types of epidermal glands. The flask cells appear in the juveniles for the first time, greatly increasing in numbers in the adult epidermis.