Deeper Layers Of Stratum Corneum Research Articles

Investigation of the cutaneous penetration behavior of dexamethasone loaded to nano-sized lipid particles by EPR spectroscopy, and confocal Raman and laser scanning microscopy

An improvement of the penetration efficiency combined with the controlled release of actives in the skin can facilitate the medical treatment of skin diseases immensely. Dexamethasone (Dx), a synthetic glucocorticoid, is frequently used for the treatment of inflammatory skin diseases. To investigate the penetration of nano-sized lipid particles (NLP) loaded with Dx in comparison to a commercially available base cream, different techniques were applied. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the penetration of Dx, which was covalently labeled with the spin probe 3-(Carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA). The penetration into hair follicles was studied using confocal laser scanning microscopy (CLSM) with curcumin-loaded NLP. The penetration of the vehicle was followed by confocal Raman microscopy (CRM). Penetration studies using excised porcine skin revealed a more than twofold higher penetration efficiency for DxPCA into the stratum corneum (SC) after 24h incubation compared to 4h incubation when loaded to the NLP, whereas when applied in the base cream, almost no further penetration was observed beyond 4h. The distribution of DxPCA within the SC was investigated by consecutive tape stripping. The release of DxPCA from the base cream after 24h in deeper SC layers and the viable epidermis was shown by EPR. For NLP, no release from the carrier was observed, although DxPCA was detectable in the skin after the complete SC was removed. This phenomenon can be explained by the penetration of the NLP into the hair follicles. However, penetration profiles measured by CRM indicate that NLP did not penetrate as deeply into the SC as the base cream formulation.In conclusion, NLP can improve the accumulation of Dx in the skin and provide a reservoir within the SC and in the follicular infundibula.

In vivo penetration of bare and lipid-coated silica nanoparticles across the human stratum corneum

Skin penetration of silica nanoparticles (NP) currently used in pharmaceutical and cosmetic products is a topic of interest not only to evaluate their possible toxicity, but also to understand their behaviour upon contact with the skin and to exploit their potential positive effects in drug or cosmetic delivery field. Therefore, the present work aimed to elucidate the in vivo mechanism by which amorphous hydrophilic silica NP enter human stratum corneum (SC) through the evaluation of the role played by the nanoparticle surface polarity and the human hair follicle density. Two silica samples, bare hydrophilic silica (B-silica, 162±51nm in size) and hydrophobic lipid-coated silica (LC-silica, 363±74nm in size) were applied on both the volar and dorsal side of volunteer forearms. Twelve repetitive stripped tapes were removed from the human skin and evaluated for elemental composition by Energy Dispersive X-ray (EDX) analysis and for silicon content by Inductively Coupled Plasma quadrupole Mass Spectrometry (ICP-MS). All the stripped tapes revealed nanosized structures generally located in the broad spaces between corneocytes and characterized by the same elemental composition (relative weight percentage of silicon and silicon to oxygen weight ratio) than that of the applied samples. However, only about 10% B-silica permeated until the deepest SC layers considered in the study indicating a silica retention in the upper layers of SC, regardless of the hair follicle density. Otherwise, the exposure to LC-silica led to a greater silica skin penetration extent into the deeper SC layers (about 42% and 18% silica following volar and dorsal forearm application, respectively) indicating that the NP surface polarity played a predominant role on that of their size in determining the route and the extent of penetration.

Accessing Raman spectral variability in human stratum corneum for quantitative in vitro depth profiling

The opportunity of label‐free and non‐destructive detection of substances inside human skin by confocal Raman microscopy represents a novel approach for investigating drug penetration and permeation. Several studies already introduced confocal Raman microscopy for depth profiling in skin; however, the reported results show high deviations. Thus, analysis of the spectral variability of human skin itself is a necessary prerequisite for systematic quantitative investigations of drug penetration and permeation by confocal Raman microscopy.In our work, we acquired Raman depth profiles from excised human skin samples after abdominal plastic surgery and investigated the absolute intensity fluctuation of four major skin derived Raman peaks. The results prove the expected high variability in spectral intensity, but we could not detect dissimilarities between different skin donors. A detailed analysis of the major endogenous skin components revealed differences in spatial distribution which consequently affects their individual applicability as reference peaks for relative depth profiling. Furthermore, we discovered an increase in signal variability in deeper stratum corneum layers, which has to be considered in future substance depth profiling investigations. In addition, we discovered an exponential decay of the Raman signal for all major skin components accounting for signal attenuation inside biological tissue. Based on this mathematical description, quantitative follow‐up of substances in human skin can be realized. All in all, the results of this study elucidate the necessity of substantial understanding of endogenous spectral characteristics inside human skin as essential prerequisite for rational depth profiling of substances in human skin. Copyright © 2013 John Wiley & Sons, Ltd.

Encapsulation of the UV Filters Ethylhexyl Methoxycinnamate and Butyl Methoxydibenzoylmethane in Lipid Microparticles: Effect on in vivo Human Skin Permeation

Background: Lipid microparticles loaded with the UVB filter ethylhexyl methoxycinnamate (EHMC) and the UVA filter butyl methoxydibenzoylmethane (BMDBM) were evaluated for their effect on the sunscreen agent’s percutaneous penetration. Methods: Microparticles loaded with EHMC or BMDBM were prepared by the melt emulsification technique using stearic acid or glyceryl behenate as lipidic material, respectively, and hydrogenate phosphatidylcholine as the surfactant. Nonencapsulated BMDBM and EHMC in conjunction with blank microparticles or equivalent amounts of the 2 UV filters loaded in the lipid microparticles were introduced into oil-in-water emulsions and applied to human volunteers. Skin penetration was investigated in vivo by the tape-stripping technique. Results: For the cream with the nonencapsulated sunscreen agents, the percentages of the applied dose diffused into the stratum corneum were 32.4 ± 4.1% and 30.3 ± 3.3% for EHMC and BMDBM, respectively. A statistically significant reduction in the in vivo skin penetration to 25.3 ± 5.5% for EHMC and 22.7 ± 5.4% for BMDBM was achieved by the cream containing the microencapsulated UV filters. The inhibiting effect on permeation attained by the lipid microparticles was more marked (45–56.3% reduction) in the deeper stratum corneum layers. Conclusions: The reduced percutaneous penetration of BMDBM and EHMC achieved by the lipid microparticles should preserve the UV filter efficacy and limit potential toxicological risks.

Quantitative Assessment of the Transport of Elastic and Rigid Vesicle Components and a Model Drug from these Vesicle Formulations into Human Skin In Vivo

The aim of this study was to quantitatively assess the distribution profiles of elastic and rigid vesicle material in human skin in vivo. Furthermore, the distribution profiles of the model drug ketorolac applied in these vesicle formulations was investigated. A deuterium-labelled phospholipid was incorporated into these vesicles to serve as a marker for the vesicle material. The vesicles were loaded with ketorolac at saturated concentrations. Vesicle solutions were applied non-occlusively onto the skin and the treated site was sequentially tape-stripped. Tape-strips were analyzed for vesicle material using attenuated total reflectance-Fourier transform infrared spectroscopy and for ketorolac by extraction of the tape-strips followed by high pressure liquid chromatography. Distribution profiles in the stratum corneum (SC) were obtained for the elastic and rigid vesicle material and for the ketorolac. These profiles have suggested that elastic vesicle material can rapidly enter the deeper layers of the SC and can reach almost the SC-viable epidermal junction. Rigid vesicle material, however, did not penetrate deep into the SC. Furthermore, the elastic vesicles were better than the rigid vesicles in the enhancement of ketorolac transport into human SC. The distribution profile of ketorolac in the deeper SC layers was, however, different from that of the vesicle material. This suggests that once the elastic vesicles partition into the SC, the ketorolac is released from the vesicles. The elastic vesicles are superior to the rigid vesicles both in terms of vesicular transport into the SC and in terms of therapeutic potential as a skin delivery vehicle.

Insights in the biochemical composition of skin as investigated by micro infrared spectroscopic imaging

The conventional methods for biochemical and histophatological tissue characterisation necessitate complex and “time consuming” sample manipulations and the results are rarely quantifiable. Micro infrared imaging is a relatively new approach for the examination of tissue sections. In contrast to standard light microscopy based procedures, the IR approach requires neither sample staining nor complicated fixation. The contrast in infrared images is solely derived from differences in the spectral and spatial heterogeneity of the biochemical components. In this study the morphological organisation and biochemical composition of “Gottingen” pig skin is analysed. The upper layer of the skin, the so called stratum corneum, is composed of corneocytes (terminally differentiated cells) imbedded in a lipid matrix. Based on the integral intensity of specific bio-marker absorption bands for the lipid as well as for the protein fraction (cells), infrared images were generated (functional group imaging). These infrared images represent the biochemical distribution of the analysed fraction within the sample. The characteristic bio-marker bands for the lipids are: the asymmetric (∼2920 cm−1) and symmetric methylene (∼2851 cm−1) stretching absorptions, and the carbonyl (∼1740 cm−1) vibration. The protein fraction is imaged by using the integral intensity of the asymmetric methyl vibration (∼2957 cm−1) as well as by the amide I (∼1655 cm−1), amide II (∼1545 cm−1) and amide B (∼3080 cm−1) absorptions. Evidence is given, that the upper stratum corneum is characterised by large areas of lipids and aggregated corneocytes. Phase separation is also observed for the deeper stratum corneum layers. However, the detected domains are much smaller. This demonstrates that the stratum corneum is not so regularly organised as described in the simplified “bricks and mortar” model. The different layers of the skin (stratum corneum, epidermis and dermis) are easily made visible by using the presented imaging technique and are fairly different concerning its biochemical composition. Micro infrared imaging is also used for analysing the penetration and distribution of a sun cream lotion in the skin. Depending on the used formulation, the distribution in the tissue can be quite different. The presented example demonstrates how this technique can be used for the development of topical applied formulations. The results in this report suggest that micro infrared spectroscopic imaging may become a new and promising investigation tool in bio-medicinal research and will provide a wealth of information on tissues that is not readily accessible by other physical and biochemical techniques.

Distribution of sunscreens on skin

The effectiveness of sunscreens was originally achieved by incorporation of soluble organic UV absorbers such as cinnamates and others into cosmetic formulations. Determinations of the sun protection factor (SPF) of emulsions containing different organic UV absorbers clearly indicate that the efficacy depends on the absorption characteristics of each single UV filter substance. Nowadays, micronised pigments such as titanium dioxide or zinc oxide have also been found to be protective against harmful UV rays. Our investigations using optical and electron microscopy proved that neither surface characteristics, particle size nor shape of the micronised pigments result in any dermal absorption of this substance. Micronised titanium dioxide is solely deposited on the outermost surface of the stratum corneum and cannot be detected in deeper stratum corneum layers, the human epidermis and dermis.

The Human Stratum corneum Layer: An Effective Barrier against Dermal Uptake of Different Forms of Topically Applied Micronised Titanium Dioxide

Electron microscopy visualisation and light microscopic investigations of three different application forms of titanium dioxide proved that neither surface characteristics, particle size nor shape of the micronised titanium dioxide result in any dermal absorption of this substance: Micronised titanium dioxide is solely deposited on the outermost surface of the stratum corneum and cannot be detected in deeper stratum corneum layers, the human epidermis and dermis.