In vivo methods for the analysis of the penetration of topically applied substances in and through the skin barrier

The efficacy of a drug is characterized by its action mechanism and its ability to pass the skin barrier. In this article, different methods are discussed, which permit this penetration process to be analysed non‐invasively. Providing qualitative and quantitative information, tape stripping is one of the oldest procedures for penetration studies. Although single cell layers of corneocytes are removed from the skin surface, this procedure is considered as non‐invasive and is applicable exclusively to the stratum corneum. Recently, optical and spectroscopic methods have been used to investigate the penetration process. Fluorescence‐labelled drugs can be easily detected in the skin by laser scanning microscopy. This method has the disadvantage that the dye labelling changes the molecular structures of the drug and consequently might influence the penetration properties. The penetration process of non‐fluorescent substances can be analysed by Raman spectroscopy, electron paramagnetic resonance, CARS and multiphoton microscopic measurements. Using these methods, the concentration of the topically applied formulations in different depths of the stratum corneum can be detected by moving the laser focus from the skin surface deeper into the stratum corneum. The advantages and disadvantages of these methods will be discussed in this article.     

Study of surfactant-skin interactions by skin impedance measurements

The stratum corneum (SC) plays a very critical physiological role as skin barrier in regulating water loss through the skin and protects the body from a wide range of physical and chemical exogenous insults. Surfactant-containing formulations can induce skin damage and irritation owing to surfactant absorption and penetration. It is generally accepted that reduction in skin barrier properties occurs only after surfactants have penetrated/permeated into the skin barrier. To mitigate the harshness of surfactant-based cleansing products, penetration/permeation of surfactants should be reduced. Skin impedance measurements have been taken in vitro on porcine skin using vertical Franz diffusion cells to investigate the impact of surfactants, temperature and pH on skin barrier integrity. These skin impedance results demonstrate excellent correlation with other published methods for assessing skin damage and irritation from different surfactant chemistry, concentration, pH, time of exposure and temperature. This study demonstrates that skin impedance can be utilized as a routine approach to screen surfactant-containing formulations for their propensity to compromise the skin barrier and hence likely lead to skin irritation.     

J. Cosmet. Sci., 59, 59–69 (January/February 2008)Biological activities of selected peptides: Skin penetration ability of copper complexes with peptides

This study concerning the permeability through skin barriers of copper complexes with peptides is an important part of the research on their biological activity. The transport of copper complexes through the skin is essential in treatment of dermatological dysfunctions connected to the deficiency of these elements in the skin. During the last several years, a special interest in transepidermal copper delivery has been observed. This is the reason why copper compounds have been used as active compounds in care cosmetics. Yet, the transport process of copper complexes with tripeptides, glycyl-histidyl-lysine GHK, or γ-glutamylcysteinyl-glycine GSH through the stratum corneum has received very little attention in the literature so far. The penetration ability of GHK-Cu and GSH-Cu through the stratum corneum and the influence of the complexes with tripeptide on the copper ion transport process is the key factor in their cosmetic and pharmaceutical activity. The in vitro penetration process was studied in the model system, a Franz diffusion cell with a liposome membrane, where liquid crystalline systems of physicochemical properties similar to the ones of the intercellular cement of stratum corneum were used as a standard model of a skin barrier. The results obtained demonstrated that copper complexes permeate through the membranes modeling the horny lipid layer and showed the influence of peptides on the dynamics of copper ion diffusion.     

Targeted delivery of salicylic acid from acne treatment products into and through skin: role of solution and ingredient properties and relationships to irritation

Salicylic acid (SA) is a beta‐hydroxy acid and has multifunctional uses in the treatment of various diseases in skin such as acne, psoriasis, and photoaging. One problem often cited as associated with salicylic acid is that it can be quite irritating at pH 3–4, where it exhibits the highest activity in the treatment of skin diseases. We have identified strategies to control the irritation potential of salicylic acid formulations and have focused on hydroalcoholic solutions used in acne wipes. One strategy is to control the penetration of SA into the skin. Penetration of the drug into various layers of skin, i.e. epidermis, dermis, and receptor fluid, was measured using a modified Franz in vitro diffusion method after various exposure times up to 24 h. A polyurethane polymer (polyolprepolymer‐15) was found to be an effective agent in controlling delivery of SA. In a dose‐dependent fashion it targeted delivery of more SA to the epidermis as compared to penetration through the skin into the receptor fluid. It also reduced the rapid rate of permeation of a large dose of SA through the skin in the first few hours of exposure. A second strategy that proved successful was incorporation of known mild nonionic surfactants like isoceteth‐20. These surfactants cleanse the skin, yet due to their inherent mildness (because of their reduced critical micelle concentration and monomer concentration), keep the barrier intact. Also, they reduce the rate of salicylic acid penetration, presumably through micellar entrapment (either in solution or on the skin surface after the alcohol evaporates). Cumulative irritation studies showed that targeting delivery of SA to the epidermis and reducing the rapid early rate of penetration of large amounts of drug through the skin resulted in a reduced irritation potential. In vivo irritation studies also showed that the surfactant system is the most important factor controlling irritancy. SA delivery is secondary, as formulations with less SA content reduced the rate of delivery to the receptor and yet were some of the most irritating formulations tested, presumably due to the action of the specific anionic surfactant on the barrier. Alcohol content also did not appreciably affect irritation and SA delivery; formulations with considerably low alcohol content but containing anionic versus nonionic surfactant systems exhibited considerably higher irritancy. Thus the surfactant type was again the predominant factor in those studies, although arguably alcohol plays some role (solubilization of SA). Results showed that both polymers and mild surfactants work in concert to provide the optimal formulation benefits of targeted delivery and reduced irritation. Synergistic relationships among hydroalcoholic formulation components will be discussed along with the mechanisms likely involved in controlling delivery of SA to skin.     

The state of nano-sized titanium dioxide (TiO2) may affect sunscreen performance

In the past several years, there has been a trend in the sunscreen/cosmetics industry to replace micron-sized titanium dioxide (TiO(2)) particles with nanoscale materials. The increased use of nanoscale TiO(2) has resulted in questions about these and other nanoproducts. This study examines the effects of using nanoscale TiO(2) on ultraviolet (UV) attenuation in simple to complex sunscreen formulations. UV light attenuation, product stability, and potential damage to the skin barrier were examined with both nanoscale and microscale TiO(2) particles. Results indicate that none of the formulations decreased the barrier function of the skin and the best UV attenuation occurs when the TiO(2) particles are stabilized with a coating and evenly distributed such as with non-agglomerated coated nanoscale materials. This indicates that nanoscale TiO(2) may have better efficacy while lacking toxicity.     

Methodology to improve epidermal barrier homeostasis: How to accelerate the barrier recovery?

Good water-impermeable barrier function is vital for healthy skin. Abnormality of the barrier function is observed in a variety of skin diseases, such as atopic dermatitis, psoriasis and contact dermatitis. Moreover, repeated barrier disruption induces epidermal hyperplasia and inflammation. On the other hand, acceleration of the barrier recovery prevents epidermal hyperplasia induced by barrier disruption in a dry environment. Thus, methods to improve the barrier function are very important for clinical dermatology. Recently, we have been searching for new reagents and/or new materials to improve barrier homeostasis. In this review, I will describe our recent findings and show how they provide the basis for a new perspective for clinical dermatology.     

Skin barrier structure, function and formation - learning from cryo-electron microscopy of vitreous, fully hydrated native human epidermis

The hydration level of the stratum corneum largely depends on the physical state and molecular organization of the stratum corneum intercellular lipid matrix. A better understanding of stratum corneum lipid organization may thus aid the development of more rational cosmetic formulations. Several experimental and theoretical problems of a fundamental character remain, however, unresolved. These are, e.g. that precise quantitative skin barrier compositional data are difficult to obtain and that in vitro experimental skin barrier models usually are of limited value because of the prevailing non-equilibrium conditions in vivo. However, new experimental methods have recently been developed, which may help to overcome some of these limitations. These are, e.g. direct electrospray ionization-mass spectrometry (ESI-MS) of intact long-chain skin ceramides and direct high-resolution cryo-transmission electron microscopy (cryo-TEM) of vitreous sections of native, fully hydrated epidermis. Here, we show that cryo-transmission electron micrographs of vitreous normal human skin often dramatically differ from those obtained by conventional electron microscopy of resin-embedded skin. Our epidermal cryo-TEM data are subsequently discussed in relation to central problems of present conceptions of skin barrier structure, function and formation.     

Transdermal delivery of two antioxidants from different cosmetic formulations

The efficacy of any cosmetic product containing a functional ingredient is determined by the skin delivery of the active molecule, which is influenced by the type of the vehicle and the molecule itself. This study was designed to compare the percutaneous absorption habits of the antioxidants carcinine and lipoic acid out of various formulations by means of the porcine skin model. Initial evaluation of the in vitro porcine skin model has demonstrated its feasibility for various substances and formulations [1, 2]. Increasing legal requirements for risk assessment in the cosmetic industry have led to the development of this alternative test method. The penetration properties are determined by the OECD Guideline TG 428: Skin Absorption: in vitro Method [3, 4], which allows the use of porcine skin for penetration studies. Porcine skin is used because of its similarity to human skin in terms of its morphology and the essential permeation characteristics [5]. The mass balances for each tested formulation type of the antioxidants show individual penetration behaviours with significant differences. The presented data plainly demonstrate that the lipophilic lipoic acid has a distinct higher penetration potential than the hydrophilic carcinine. The chosen vehicle can enhance or reduce the transdermal delivery of both tested antioxidants. Modern effective cosmetic formulations will work only, if the active ingredients penetrate into the epidermis. In conclusion, the correct selection of a suitable formulation plays an important role during product development.     

Prediction of the percutaneous penetration of ultra-violet filters used in sunscreen formulations

The application of a mathematical model to estimate the extent of transdermal absorption of UV-filters commonly used in sunscreen formulations is described. Percutaneous penetration is not a factor that has been properly addressed in the literature and the penetration/time profiles generated here suggest that significant amounts of certain of these compounds may penetrate the skin and enter the systemic circulation. The results presented indicate that further research in this area is necessary and the authors suggest that in vitro experiments with human skin are conducted with both current and new UV filters to quantify the degree of dermal penetration of these substances.     

Skin permeability to aromatic carboxylic acids: an in vitro comparison in pig ear, white mouse and brown mouse

A comparative study in vitro was undertaken for pig ear, white mouse, and brown mouse in which the percutaneous absorption of the following compounds, labelled with ¹⁴C-carboxyl, were studied: benzoic acid, 2-methylbenzoic acid, and 4-methylbenzoic acid. The effects of temperature on the penetration through various skin membranes have been evaluated. The possible influences of chemical structure on transport across the skin were discussed. The results indicated that brown mouse skin was the most permeable membrane to all acids. Introduction of the methyl group to the aromatic ring, in the ortho and in the para positions, increased the diffusion coefficients for the benzoic acid.     

Assessment of the variation of skin barrier function with anatomic site,age, gender and ethnicity

The skin is the largest organ of the human body and its functions include protection, thermoregulation sensation and secretion. Significant advances in our understanding of how the morphology and physiology of the skin contribute to the skin’s barrier role have been achieved in recent years. The aim of this review is to summarize the principal approaches which have been used to assess variation in skin barrier function with anatomic site, age, gender, and ethnicity. The methods discussed include trans‐epidermal water loss (TEWL) measurement, assessment of corneocyte size, response to vasoactive compounds and attenuated total reflectance Fourier transform infrared (ATR‐FTIR) interrogation of skin. The utility of the various methods is considered and the most important findings in the literature to date are highlighted.     

Predicting skin penetration of actives from complex cosmetic formulations: an evaluation of inter formulation and inter active effects during formulation optimization for transdermal delivery

Twenty products, containing a radiolabelled form of each active in typical cosmetic formulations, were made and applied to female human epidermal membranes mounted in Franz diffusion cells for 48 h under ‘in use’ conditions. The products consisted of combinations of five formulations (a hydro‐alcoholic gel, an oil in water emulsion, a water in oil emulsion, a microemulsion and an oil) with four model drug actives (testosterone, hydrocortisone, 5‐fluorouracil and ketoconazole). Steady‐state flux appeared to be reached by 8 h and maintained for all products, other than for the microemulsions, consistent with the actives being present in the residual formulation on the skin at saturation. The recovery for each active at the end of the 48‐h study (from a series of stratum corneum tape strips, the remaining skin, cumulative amount penetrating into the receptor solution, product washed from the skin and on the donor chamber cap) ranged from 86.5% to 100.6%. The rank order of the fluxes for the actives from the hydro‐alcoholic gel is consistent with the known active molecular size and polarity determinants for maximum epidermal flux. Actives with similar steady‐state (maximum) fluxes from a range of formulations had retention in the stratum corneum and similar transport rate constants through the stratum corneum. The microemulsion formulation significantly enhanced both the stratum corneum steady‐state flux and transport rate constant for 5‐fluorouracil, hydrocortisone and testosterone. The penetration flux of each active could be related to its size and polarity and appeared maximal when the actives in the different cosmetic formulations applied to the skin under ‘in use’ conditions were likely to remain in the residual product on the skin as a saturated solution after solvent evaporation. Enhanced penetration fluxes can be achieved by formulation selection and an appropriate choice/mix of emollients/adjuvants. The principles described here provide a framework for understanding the delivery of cosmetic ingredients from various formulations.     

Skin penetration and stabilization of formulations containing microfine titanium dioxide as physical UV filter

Microfine titanium dioxide (TiO(2)) has become a frequently used physical UV filter in sunscreen formulations. Penetration of microfine TiO(2) into human skin seems to be possible because of the mean particle size of 20 nm. The small particle size results in a high surface activity of the primary particles and causes a formation of agglomerates in the formulation. The aim of this study was to investigate the in vivo and in vitro penetration behaviour of the physical UV filter into human skin. Furthermore, a stable sunscreen formulation with microfine TiO(2) which does not penetrate into the skin should be developed. According to our experiments, microfine TiO(2) penetrates deeper into human skin from an oily dispersion than from an aqueous one. Therefore, an o/w emulsion containing the dispersed micropigment in the aqueous phase was manufactured. Microfine TiO(2) cannot penetrate into human skin from this emulsion, but the storage stability of the formulation is very low at different temperatures. The encapsulation of the micropigment into liposomes does not result in a better stability but it causes a higher penetration depth of the particles into the skin.     

Characterization of natural polymers as functional barriers for cellulose-based packaging materials

Cellulose-based packaging materials are currently the most commonly used food packaging materials due to their light weight, stability and affordable price. However, the use of recycled paper and board adds to the risk that undesirable substances migrate into the packed goods, since contaminants are not completely removed during the recycling process and can accumulate in the final product. The only available fast and practical solution that can be used to reduce the migration of these substances is the application of functional barriers in the packaging. The applied barriers are currently mostly synthetic, which either serve only a moderate barrier function and/or have the disadvantage that it is often more complex and expensive to recycle the resulting packaging material. The aim of this project is to evaluate different bio-based or biodegradable polymers with regards to their barrier properties. Due to the fact that the transport phenomena are mainly driven by (gas phase) migration, methods based on gas chromatography (GC), including GC coupled with mass spectrometry (GC-MS) and flame ionization detection (GC-FID), GC-FID coupled online with high pressure liquid chromatography (HPLC-GC-FID), and comprehensive GCxGC-MS were used to qualify and quantify the migrated substances. This use of a wide range of different methods and instruments yielded excellent results, allowing us to comprehensively characterize the biopolymers and their barrier function.     

The effect of cream and ointment bases on the steady state penetration of permeants through intact skin: the reciprocal of the onset time of a pharmacodynamic effect as parameter of response

To characterize cream or ointment bases for cosmetic or pharmaceutical purposes with regard to their effect on permeant penetration through intact healthy skin, the measurement of the pharmacodynamic response of a suitable model drug incorporated in these bases has been shown to be a promising approach. In general, it may be distinguished between thermodynamic vehicle effects owing to different permeant escaping tendencies from the vehicles and penetration-enhancing vehicle effects resulting from a change of the stratum corneum structure, which manifests itself in an increase of the permeant diffusion coefficient and/or its solubility in this barrier. As the latency time of onset of a pharmacodynamic effect, usually used as reciprocal value, represents a suitable response parameter under certain circumstances, this study was done to further evaluate this parameter with regard to the determination of relative bioavailability and penetration enhancement data obtained from simulated dose- and activity-response curves assuming infinite dose conditions, i.e. zero order penetration kinetics and considering varying lag times of drug penetration. The results indicate that bioavailability and enhancement factors may be determined accurately from the horizontal distance between dose- or activity-response curves of a standard and a test preparation as long as the curves are parallel to each other, as it is the case with uniform lag times of permeant penetration. Non-parallel curves observed with varying lag times indicate an influence of the vehicles on the permeant diffusion coefficient in the barrier. Enhancement factors from these curves may be obtained after determination of the lag times from the plateau region of the curves, subsequent subtraction of these values from the measured latency time data, and finally plotting of the reciprocal data as a function of the drug activity. Enhancement factors then correspond to the inverse logarithm of the horizontal distances between the resulting parallel curves.     

Lipid nanoparticles (SLN & NLC) for delivery of vitamin E: a comprehensive review

The antioxidative and photoprotective properties of vitamin E have caused it to be included as an active agent in various pharmaceutical and cosmetic products. However, its lipophilicity, chemical instability and poor skin penetration have limited the effectiveness of these formulations. For that reason, many attempts to include it in different drug delivery systems have been made. In recent decades, lipid nanoparticles have received special attention due to their advantages of compatibility with the skin, ability to enhance penetration of drugs in the stratum corneum, protection of the encapsulated substance against degradation induced by the external medium and control of drug release. This work reviews the current status of the encapsulation of vitamin E in lipid nanoparticles. We describe the most important methods for obtaining and characterizing lipid nanoparticles containing vitamin E (LNP‐VE), various techniques for the evaluation of vitamin E's properties after encapsulation, the main in vitro and in vivo studies of the potential effectiveness or toxicity of LNP‐VE, the formulations and stability studies of this delivery system, the commercial products based on LNP‐VE and the regulatory aspects related to lipid nanoparticles. Finally, we discuss the most relevant advantages of encapsulating vitamin E in such particles and critical aspects that still demand attention to enhance the potential of solid lipid nanoparticles to deliver vitamin E.     

Superior effect of isostearyl isostearate on improvement in stratum corneum water permeability barrier function as examined by the plastic occlusion stress test

Dry skin is a major dermatological problem and consumer research indicates that although current moisturizers are effective they are not completely meeting consumer expectation. Several technological approaches have been taken but influencing stratum corneum (SC) lipid phase behaviour as a novel water permeability barrier‐enhancing and moisturizing mechanism has only been started to be investigated recently. Both the long periodicity SC lipid lamellar phase and the orthorhombic lipid packing state have been proposed to define optimal SC water permeability barrier properties. Several lipophillic moisturizers have been tested for their ability to modify SC lipid lateral packing namely glyceryl monoisostearate (GMIS), isopropyl isostearate (IPIS) and isostearyl isostearate (ISIS) of which IPIS and ISIS are reported to induce the orthorhombic phase. Despite the improvements in the lateral packing of SC lipids, these ingredients have been shown not to improve transepidermal water loss. However, using a novel skin surface water loss method we have observed for the first time significant improvements in SC water permeability barrier function for ISIS compared with IPIS, GMIS and petrolatum. However, using synthetic membranes and measuring water vapour transport rates we showed that the isostearyl esters were not occlusive like petrolatum. As the effects of ISIS were not because of what would be considered as true occlusion, we propose that the differences in the SC water permeability barrier properties from use of ISIS to the other ingredients tested are because of its reported effects on SC lipid phase behaviour. Further studies probably using spectroscopic approaches, however, will be needed to specifically test this hypothesis in vivo.     

肠道黏液层降解菌Akkermansia muciniphlia和肠道屏障功能关系研究进展

肠道屏障功能异常与肥胖等一系列慢性代谢性疾病相关。Akkermansia muciniphlia是一种肠道黏液层降解菌,与肠道屏障功能关系密切,其丰度在部分代谢性疾病临床病例和动物模型中变化显著,提示该菌可能参与代谢,但具体作用机制有待明确和归纳。本文从A. muciniphlia与肠道黏液层、代谢疾病以及肠道免疫关系这3 个角度,综述了近年来相关研究进展,试图探讨该菌对肠道屏障功能的影响和作用机制,为通过饮食预防慢性代谢疾病提供新的思路。     

Ethnic skin types: are there differences in skin structure and function?1

People of skin of colour comprise the majority of the world's population and Asian subjects comprise more than half of the total population of the earth. Even so, the literature on the characteristics of the subjects with skin of colour is limited. Several groups over the past decades have attempted to decipher the underlying differences in skin structure and function in different ethnic skin types. However, most of these studies have been of small scale and in some studies interindividual differences in skin quality overwhelm any racial differences. There has been a recent call for more studies to address genetic together with phenotypic differences among different racial groups and in this respect several large-scale studies have been conducted recently. The most obvious ethnic skin difference relates to skin colour which is dominated by the presence of melanin. The photoprotection derived from this polymer influences the rate of the skin aging changes between the different racial groups. However, all racial groups are eventually subjected to the photoaging process. Generally Caucasians have an earlier onset and greater skin wrinkling and sagging signs than other skin types and in general increased pigmentary problems are seen in skin of colour although one large study reported that East Asians living in the U.S.A. had the least pigment spots. Induction of a hyperpigmentary response is thought to be through signaling by the protease-activated receptor-2 which together with its activating protease is increased in the epidermis of subjects with skin of colour. Changes in skin biophysical properties with age demonstrate that the more darkly pigmented subjects retaining younger skin properties compared with the more lightly pigmented groups. However, despite having a more compact stratum corneum (SC) there are conflicting reports on barrier function in these subjects. Nevertheless, upon a chemical or mechanical challenge the SC barrier function is reported to be stronger in subjects with darker skin despite having the reported lowest ceramide levels. One has to remember that barrier function relates to the total architecture of the SC and not just its lipid levels. Asian skin is reported to possess a similar basal transepidermal water loss (TEWL) to Caucasian skin and similar ceramide levels but upon mechanical challenge it has the weakest barrier function. Differences in intercellular cohesion are obviously apparent. In contrast reduced SC natural moisturizing factor levels have been reported compared with Caucasian and African American skin. These differences will contribute to differences in desquamation but few data are available. One recent study has shown reduced epidermal Cathepsin L2 levels in darker skin types which if also occurs in the SC could contribute to the known skin ashing problems these subjects experience. In very general terms as the desquamatory enzymes are extruded with the lamellar granules subjects with lowered SC lipid levels are expected to have lowered desquamatory enzyme levels. Increased pores size, sebum secretion and skin surface microflora occur in Negroid subjects. Equally increased mast cell granule size occurs in these subjects. The frequency of skin sensitivity is quite similar across different racial groups but the stimuli for its induction shows subtle differences. Nevertheless, several studies indicate that Asian skin maybe more sensitive to exogenous chemicals probably due to a thinner SC and higher eccrine gland density. In conclusion, we know more of the biophysical and somatosensory characteristics of ethnic skin types but clearly, there is still more to learn and especially about the inherent underlying biological differences in ethnic skin types.