Showing papers by "Joke A. Bouwstra published in 2013"

Ceramides in the skin lipid membranes: length matters

Abstract: Ceramides are essential constituents of the skin barrier that allow humans to live on dry land. Reduced levels of ceramides have been associated with skin diseases, e.g., atopic dermatitis. However, the structural requirements and mechanisms of action of ceramides are not fully understood. Here, we report the effects of ceramide acyl chain length on the permeabilities and biophysics of lipid membranes composed of ceramides (or free sphingosine), fatty acids, cholesterol, and cholesterol sulfate. Short-chain ceramides increased the permeability of the lipid membranes compared to a long-chain ceramide with maxima at 4-6 carbons in the acyl. By a combination of differential scanning calorimetry, Fourier transform infrared spectroscopy, X-ray diffraction, Langmuir monolayers, and atomic force microscopy, we found that the reason for this effect in short ceramides was a lower proportion of tight orthorhombic packing and phase separation of continuous short ceramide-enriched domains with shorter lamellar periodicity compared to native long ceramides. Thus, long acyl chains in ceramides are essential for the formation of tightly packed impermeable lipid lamellae. Moreover, the model skin lipid membranes are a valuable tool to study the relationships between the lipid structure and composition, lipid organization, and the membrane permeability.

Characterization of stratum corneum molecular dynamics by natural-abundance 13C solid-state NMR

Abstract: Despite the enormous potential for pharmaceutical applications, there is still a lack of understanding of the molecular details that can contribute to increased permeability of the stratum corneum (SC). To investigate the influence of hydration and heating on the SC, we record the natural-abundance (13)C signal of SC using polarization transfer solid-state NMR methods. Resonance lines from all major SC components are assigned. Comparison of the signal intensities obtained with the INEPT and CP pulse sequences gives information on the molecular dynamics of SC components. The majority of the lipids are rigid at 32°C, and those lipids co-exist with a small pool of mobile lipids. The ratio between mobile and rigid lipids increases with hydration. An abrupt change of keratin filament dynamics occurs at RH = 80-85%, from completely rigid to a structure with rigid backbone and mobile protruding terminals. Heating has a strong effect on the lipid mobility, but only a weak influence on the keratin filaments. The results provide novel molecular insight into how the SC constituents are affected by hydration and heating, and improve the understanding of enhanced SC permeability, which is associated with elevated temperatures and SC hydration. (Less)

Knock-down of filaggrin does not affect lipid organization and composition in stratum corneum of reconstructed human skin equivalents.

Abstract: Human skin mainly functions as an effective barrier against unwanted environmental influences. The barrier function strongly relies on the outermost layer of the skin, the stratum corneum (SC), which is composed of corneocytes embedded in an extracellular lipid matrix. The importance of a proper barrier function is shown in various skin disorders such as atopic dermatitis (AD), a complex human skin disorder strongly associated with filaggrin (FLG) null mutations, but their role in barrier function is yet unclear. To study the role of FLG in SC barrier properties in terms of SC lipid organization and lipid composition, we generated an N/TERT-based 3D-skin equivalent (NSE) after knock-down of FLG with shRNA. In these NSEs, we examined epidermal morphogenesis by evaluating the expression of differentiation markers keratin 10, FLG, loricrin and the proliferation marker ki67. Furthermore, the SC was extensively analysed for lipid organization, lipid composition and SC permeability. Our results demonstrate that FLG knock-down (FLG-KD) did not affect epidermal morphogenesis, SC lipid organization, lipid composition and SC permeability for a lipophilic compound in NSEs. Therefore, our findings indicate that FLG-KD alone does not necessarily affect the functionality of a proper barrier function.

Increased Presence of Monounsaturated Fatty Acids in the Stratum Corneum of Human Skin

Abstract: Previous results showed that our in-house human skin equivalents (HSEs) differ in their stratum corneum (SC) lipid organization compared with human SC. To elucidate the cause of the altered SC lipid organization in the HSEs, a recently developed liquid chromatography/mass spectrometry method was used to study the free fatty acid (FFA) and ceramide composition in detail. In addition, the SC lipid composition of the HSEs and human skin was examined quantitatively with high-performance thin-layer chromatography. Our results reveal that all our HSEs have an increased presence of monounsaturated FFAs compared with human SC. Moreover, the HSEs display the presence of ceramide species with a monounsaturated acyl chain, which are not detected in human SC. All HSEs also exhibit an altered expression of stearoyl-CoA desaturase, the enzyme that converts saturated FFAs to monounsaturated FFAs. Furthermore, the HSEs show the presence of 12 ceramide subclasses, similar to native human SC. However, the HSEs have increased levels of ceramides EOS and EOH and ceramide species with short total carbon chains and a reduced FFA level compared with human SC. The presence of unsaturated lipid chains in HSE offers new opportunities to mimic the lipid properties of human SC more closely.

Nanolayered chemical modification of silicon surfaces with ionizable surface groups for pH-triggered protein adsorption and release: application to microneedles

Abstract: The aim of this work was to develop a nanolayered pH sensitive coating method whereby proteins are coated at a suitable pH on the surface of chemically modified biomedical/bioanalytical microdevices and protein release is triggered by a pH-shift upon contact with the physiological environment. In this work such a coating was developed and was applied onto microneedles. First, the surface of microneedle arrays was modified with basic groups with a surface pKa below physiological pH. This modification was a multistep procedure: first the surface was hydroxylated in a piranha mixture, then 3-aminopropyl-triethoxysilane was coupled (yielding a “pH independent” surface with a positive charge over a broad pH range), next 4-pyridinecarboxaldehyde was coupled to the obtained surface amine groups and finally the imine bond was reduced by sodium cyanoborohydride. The obtained pH-sensitive pyridine-modified microneedles were coated with ovalbumin at surface pKa > pH > pI of the protein; thus the surface of the microneedles is positively charged and the protein is negatively charged. The coating efficiency of ovalbumin was 95% for the amine-modified (pH independent) and the pyridine-modified (pH sensitive) surfaces, whereas a non-modified surface had a coating efficiency of only 2%. After the protein-coated microneedle arrays were pierced into the skin, having a pH > surface pKa of the microneedle arrays, 70% of the protein was released within 1 minute, whereas the protein release from pH independent microneedle arrays was only 5%. In conclusion, we developed a procedure to efficiently coat microneedle arrays with proteins that are released upon piercing into human skin.

From the structure of the skin barrier and dermal formulations to in vitro transport models for skin absorption: skin research in the Netherlands and in Germany.

Abstract: This review presents an overview of German and Dutch research institutions and their studies in the field of skin drug delivery and adjacent topics. In the Netherlands, the involved research groups are mainly localized in Leiden, whereas in Germany the skin research institutions are spread over the whole country. The scientific studies in the Netherlands focus on the in-depth analysis of human skin composition and its individual components as well as on the development and characterization of dermal drug delivery systems ranging from liquid crystalline systems and vesicles up to microneedles with an emphasis on examining the interactions of these drug delivery systems with the human skin in vitro and in vivo. In Germany, the individual areas of research span from in-depth investigations on various drug delivery systems intended for skin application and the development of novel in vitro models for skin absorption testing up to in vivo studies focusing on the biological performance of topically applied actives. Furthermore, sophisticated analytical techniques are applied for the elucidation of skin assembly and transport processes. In addition, experimentally derived data are correlated with advanced computational modelling. Even though the individual research topics in the Netherlands and Germany are quite diverse, the exchange of knowledge and interdisciplinary collaborations between the two neighbouring countries were and are still frequently made. In this context, the review aims at highlighting crosslinks between the different institutions and individual persons to complete the picture. For each institution, the principal investigators and their studies are presented and the upcoming young scientists are introduced as an outlook for the field. This review does not claim completeness, but is rather intended to give a general overview of Dutch and German research in the field of skin drug delivery and adjacent topics.