Sebastian Björklund

A water gradient can be used to regulate drug transport across skin

At normal conditions there is a substantial water gradient over the skin as it separates the water-rich inside of the body from the dry outside. This leads to a variation in the degree of hydration from the inside to the outside of skin and changes in this gradient may affect its structure and function. In this study we raise the question: How do changes in the water gradient across skin affect its permeability? We approach this problem in novel diffusion experiments that permit strict control of the gradient in the chemical potential of water and hence well-defined boundary conditions. The results demonstrate that a water gradient can be used to regulate transport of drugs with different lipophilic characteristics across the skin barrier. It is shown that the transport of metronidazole (log P(o/w)=0.0) and methyl salicylate (log P(o/w)=2.5) across skin increases abruptly at low water gradients, corresponding to high degrees of skin hydration, and that this effect is reversible. This phenomenon is highly relevant to drug delivery applications due to its potential of temporarily open the skin barrier for transdermal drug delivery and subsequently close the barrier after treatment. Further, the results contribute to the understanding of the occlusion effect and indicate the boundary conditions of the water gradient needed to make use of this effect.

Glycerol and urea can be used to increase skin permeability in reduced hydration conditions.

The natural moisturizing factor (NMF) is a group of hygroscopic molecules that is naturally present in skin and protects from severe drying. Glycerol and urea are two examples of NMF components that are also used in skin care applications. In the present study, we investigate the influence of glycerol and urea on the permeability of a model drug (metronidazole, Mz) across excised pig skin membranes at different hydrating conditions. The degree of skin hydration is regulated by the gradient in water activity across the membrane, which in turn depends on the water activity of the formulation in contact with the skin membrane. Here, we determine the water activity of all formulations employed using an isothermal calorimetric method. Thus, the gradient in water activity is controlled by a novel experimental set-up with well-defined boundary conditions on both sides of the skin membrane. The results demonstrate that glycerol and urea can retain high steady state flux of Mz across skin membranes at dehydrating conditions, which otherwise would decrease the permeability due to dehydration. X-ray diffraction measurements are performed to give insight into the effects of glycerol and urea on SC molecular organization. The novel steady state flux results can be related to the observation that water, glycerol, and urea all affect the structural features of the SC molecular components in a similar manner.

Characterization of Stratum Corneum Molecular Dynamics by Natural-Abundance 13C Solid-State NMR

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 13C 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.

Effects of water gradients and use of urea on skin ultrastructure evaluated by confocal Raman microspectroscopy.

The rather thin outermost layer of the mammalian skin, stratum corneum (SC), is a complex biomembrane which separates the water rich inside of the body from the dry outside. The skin surface can be exposed to rather extreme variations in ambient conditions (e.g. water activity, temperature and pH), with potential effects on the barrier function. Increased understanding of how the barrier is affected by such changes is highly relevant for regulation of transdermal uptake of exogenous chemicals. In the present study we investigate the effect of hydration and the use of a well-known humectant, urea, on skin barrier ultrastructure by means of confocal Raman microspectroscopy. We also perform dynamic vapor sorption (DVS) microbalance measurements to examine the water uptake capacity of SC pretreated with urea. Based on novel Raman images, constructed from 2D spectral maps, we can distinguish large water inclusions within the skin membrane exceeding the size of fully hydrated corneocytes. We show that these inclusions contain water with spectral properties similar to that of bulk water. The results furthermore show that the ambient water activity has an important impact on the formation of these water inclusions as well as on the hydration profile across the membrane. Urea significantly increases the water uptake when present in skin, as compared to skin without urea, and it promotes formation of larger water inclusions in the tissue. The results confirm that urea can be used as a humectant to increase skin hydration.

Chemical penetration enhancers in stratum corneum - Relation between molecular effects and barrier function.

Skin is attractive for drug therapy because it offers an easily accessible route without first-pass metabolism. Transdermal drug delivery is also associated with high patient compliance and through the site of application, the drug delivery can be locally directed. However, to succeed with transdermal drug delivery it is often required to overcome the low permeability of the upper layer of the skin, the stratum corneum (SC). One common strategy is to employ so-called penetration enhancers that supposedly act to increase the drug passage across SC. Still, there is a lack of understanding of the molecular effects of so-called penetration enhancers on the skin barrier membrane, the SC. In this study, we provide a molecular characterization of how different classes of compounds, suggested as penetration enhancers, influence lipid and protein components in SC. The compounds investigated include monoterpenes, fatty acids, osmolytes, surfactant, and Azone. We employ natural abundance (13)C polarization transfer solid-state nuclear magnetic resonance (NMR) on intact porcine SC. With this method it is possible to detect small changes in the mobility of the minor fluid lipid and protein SC components, and simultaneously obtain information on the major fraction of solid SC components. The balance between fluid and solid components in the SC is essential to determine macroscopic material properties of the SC, including barrier and mechanical properties. We study SC at different hydration levels corresponding to SC in ambient air and under occlusion. The NMR studies are complemented with diffusion cell experiments that provide quantitative data on skin permeability when treated with different compounds. By correlating the effects on SC molecular components and SC barrier function, we aim at deepened understanding of diffusional transport in SC, and how this can be controlled, which can be utilized for optimal design of transdermal drug delivery formulations.

Stratum corneum molecular mobility in the presence of natural moisturizers

The outermost layer of the skin, the stratum corneum (SC), is a lipid-protein membrane that experiences considerable osmotic stress from a dry and cold climate. The natural moisturizing factor (NMF) comprises small and polar substances, which like osmolytes can protect living systems from osmotic stress. NMF is commonly claimed to increase the water content in the SC and thereby protect the skin from dryness. In this work we challenge this proposed mechanism, and explore the influence of NMF on the lipid and protein components in the SC. We employ natural-abundance (13)C solid-state NMR methods to investigate how the SC molecular components are influenced by urea, glycerol, pyrrolidone carboxylic acid (PCA), and urocanic acid (UCA), all of which are naturally present in the SC as NMF compounds. Experiments are performed with intact SC, isolated corneocytes and model lipids. The combination of NMR experiments provides molecularly resolved qualitative information on the dynamics of different SC lipid and protein components. We obtain completely novel molecular information on the interaction of these NMF compounds with the SC lipids and proteins. We show that urea and glycerol, which are also common ingredients in skin care products, increase the molecular mobility of both SC lipids and proteins at moderate relative humidity where the SC components are considerably more rigid in the absence of these compounds. This effect cannot be attributed to increased SC water content. PCA has no detectable effect on SC molecular mobility under the conditions investigated. It is finally shown that the more apolar compound, UCA, specifically influences the mobility of the SC lipid regions. The present results show that the NMF components act to retain the fluidity of the SC molecular components under dehydrating conditions in such a way that the SC properties remain largely unchanged as compared to more hydrated SC. These findings provide a new molecular insight into how small polar molecules in NMF and skin care products act to protect the human skin from drying.

The effects of polar excipients transcutol and dexpanthenol on molecular mobility, permeability, and electrical impedance of the skin barrier

In the development of transdermal and topical products it is important to understand how formulation ingredients interact with the molecular components of the upper layer of the skin, the stratum corneum (SC), and thereby influence its macroscopic barrier properties. The aim here was to investigate the effect of two commonly used excipients, transcutol and dexpanthenol, on the molecular as well as the macroscopic properties of the skin membrane. Polarization transfer solid-state NMR methods were combined with steady-state flux and impedance spectroscopy measurements to investigate how these common excipients influence the molecular components of SC and its barrier function at strictly controlled hydration conditions in vitro with excised porcine skin. The NMR results provide completely new molecular insight into how transcutol and dexpanthenol affect specific molecular segments of both SC lipids and proteins. The presence of transcutol or dexpanthenol in the formulation at fixed water activity results in increased effective skin permeability of the model drug metronidazole. Finally, impedance spectroscopy data show clear changes of the effective skin capacitance after treatment with transcutol or dexpanthenol. Based on the complementary data, we are able to draw direct links between effects on the molecular properties and on the macroscopic barrier function of the skin barrier under treatment with formulations containing transcutol or dexpanthenol.

Humidity scanning quartz crystal microbalance with dissipation monitoring setup for determination of sorption-desorption isotherms and rheological changes

A new method to determine water sorption-desorption isotherms with high resolution in the complete range of water activities (relative humidities) is presented. The method is based on quartz crystal microbalance with dissipation monitoring (QCM-D). The QCM-D is equipped with a humidity module in which the sample film is kept in air with controlled humidity. The experimental setup allows for continuous scanning of the relative humidity from either dry to humid conditions or vice versa. The amount of water sorbed or desorbed from the sample is determined from the resonance frequencies of the coated quartz sensor, via analysis of the overtone dependence. In addition, the method allows for characterization of hydration induced changes of the rheological properties from the dissipation data, which is closely connected to the viscoelasticity of the film. The accuracy of the humidity scanning setup is confirmed in control experiments. Sorption-desorption isotherms of pig gastric mucin and lysozyme, obtained by the new method, show good agreement with previous results. Finally, we show that the deposition technique used to coat the quartz sensor influences the QCM-D data and how this issue can be used to obtain further information on the effect of hydration. In particular, we demonstrate that spin-coating represents an attractive alternative to obtain sorption-desorption isotherms, while drop-coating provides additional information on changes of the rheological properties during hydration.

A calorimetric method to determine water activity

A calorimetric method to determine water activity covering the full range of the water activity scale is presented. A dry stream of nitrogen gas is passed either over the solution whose activity should be determined or left dry before it is saturated by bubbling through water in an isothermal calorimeter. The unknown activity is in principle determined by comparing the thermal power of vaporization related to the gas stream with unknown activity to that with zero activity. Except for three minor corrections (for pressure drop, non-perfect humidification, and evaporative cooling) the unknown water activity is calculated solely based on the water activity end-points zero and unity. Thus, there is no need for calibration with references with known water activities. The method has been evaluated at 30 °C by measuring the water activity of seven aqueous sodium chloride solutions ranging from 0.1 mol kg(-1) to 3 mol kg(-1) and seven saturated aqueous salt solutions (LiCl, MgCl(2), NaBr, NaCl, KCl, KNO(3), and K(2)SO(4)) with known water activities. The performance of the method was adequate over the complete water activity scale. At high water activities the performance was excellent, which is encouraging as many other methods used for water activity determination have limited performance at high water activities.

Electrochemical monitoring of native catalase activity in skin using skin covered oxygen electrode

A skin covered oxygen electrode, SCOE, was constructed with the aim to study the enzyme catalase, which is part of the biological antioxidative system present in skin. The electrode was exposed to different concentrations of H2O2 and the amperometric current response was recorded. The observed current is due to H2O2 penetration through the outermost skin barrier (referred to as the stratum corneum, SC) and subsequent catalytic generation of O2 by catalase present in the underlying viable epidermis and dermis. By tape-stripping the outermost skin layers we demonstrate that SC is a considerable diffusion barrier for H2O2 penetration. Our experiments also indicate that skin contains a substantial amount of catalase, which is sufficient to detoxify H2O2 that reaches the viable epidermis after exposure of skin to high concentrations of peroxide (0.5-1mM H2O2). Further, we demonstrate that the catalase activity is reduced at acidic pH, as compared with the activity at pH 7.4. Finally, experiments with often used penetration enhancer thymol shows that this compound interferes with the catalase reaction. Health aspect of this is briefly discussed. Summarizing, the results of this work show that the SCOE can be utilized to study a broad spectrum of issues involving the function of skin catalase in particular, and the native biological antioxidative system in skin in general.