The intercellular lipid structure of the stratum corneum (SC) plays a key role in skin barrier function. A depth profile of the intercellular lipid conformation and the lipid lateral packing order ...were measured in vivo in the human SC using confocal Raman microscopy. The depth profiles of the 2880 cm(-1)/2850 cm(-1) peak ratio intensity, which represent the C-H stretching and lateral packing order of lipids, and the 1080 cm(-1)/(1130 cm(-1) + 1060 cm(-1)) peak ratio, which represents the C-C skeleton vibration and trans-gauche conformation order of lipids, were investigated. The influence of keratin on the lipid peaks at 2850 cm(-1) and 2880 cm(-1) was excluded by the developed mathematical algorithm. The results show that the trans-conformation and lateral packing order of the intercellular lipids reach their maximum value in the SC at 20-40% of its depth and then decrease towards the stratum granulosum. These results show that at a depth of 20-40% (normally corresponding to a depth of 4-8 μm) the SC exhibits the most ordered lipids and therefore the highest skin barrier function. The lateral packing of lipids is more disordered on the surface and in the deeper parts of the SC, which may be associated with a reduced skin barrier function.
The influence of the surface functionalization of silica particles on their colloidal stability in physiological media is studied and correlated with their uptake in cells. The surface of 55 ± 2 nm ...diameter silica particles is functionalized by amino acids or amino- or poly(ethylene glycol) (PEG)-terminated alkoxysilanes to adjust the zeta potential from highly negative to positive values in ethanol. A transfer of the particles into water, physiological buffers, and cell culture media reduces the absolute value of the zeta potential and changes the colloidal stability. Particles stabilized by L-arginine, L-lysine, and amino silanes with short alkyl chains are only moderately stable in water and partially in PBS or TRIS buffer, but aggregate in cell culture media. Nonfunctionalized, N-(6-aminohexyl)-3-aminopropyltrimethoxy silane (AHAPS), and PEG-functionalized particles are stable in all media under study. The high colloidal stability of positively charged AHAPS-functionalized particles scales with the ionic strength of the media, indicating a mainly electrostatical stabilization. PEG-functionalized particles show, independently from the ionic strength, no or only minor aggregation due to additional steric stabilization. AHAPS stabilized particles are readily taken up by HeLa cells, likely as the positive zeta potential enhances the association with the negatively charged cell membrane. Positively charged particles stabilized by short alkyl chain aminosilanes adsorb on the cell membrane, but are weakly taken up, since aggregation inhibits their transport. Nonfunctionalized particles are barely taken up and PEG-stabilized particles are not taken up at all into HeLa cells, despite their high colloidal stability. The results indicate that a high colloidal stability of nanoparticles combined with an initial charge-driven adsorption on the cell membrane is essential for efficient cellular uptake.
The secondary and tertiary structure of keratin and natural moisturizing factor (NMF) are of great importance regarding the water regulating functions in the stratum corneum (SC). In this in vivo ...study, the depth-dependent keratin conformation and its relationship to the hydrogen bonding states of water and its content in the SC, are investigated using confocal Raman microscopy. Based on the obtained depth-profiles for the β-sheet/α-helix ratio, the stability of disulphide bonds, the amount of cysteine forming disulphide bonds, the buried/exposed tyrosine and the folding/unfolding states of keratin, a "three layer model" of the SC, regarding the keratin-water-NMF interaction is proposed. At the uppermost layers (30-0% SC depth), the keratin filaments are highly folded, entailing limited water binding sites, and NMF is mostly responsible for binding water. At the intermediate layers (70-30% SC depth), the keratin filaments are unfolded, have the most water binding sites and are prone to swelling. At the bottom layers (100-80% SC depth), the water binding sites are already occupied with water and cannot swell substantially. The hydrogen bonding states of water molecules can only be explained by considering both, the molecular structure of keratin and the contribution of NMF as a holistic system.
In this study, the skin penetration and cellular uptake of amorphous silica particles with positive and negative surface charge and sizes ranging from 291 ± 9 to 42 ± 3 nm were investigated. Dynamic ...light scattering measurements and statistical analyses of transmission electron microscopy images were used to estimate the degree of particle aggregation, which was a key aspect to understanding the results of the in vitro cellular uptake experiments. Despite partial particle aggregation occurring after transfer in physiological media, particles were taken up by skin cells in a size-dependent manner. Functionalization of the particle surface with positively charged groups enhanced the in vitro cellular uptake. However, this positive effect was contrasted by the tendency of particles to form aggregates, leading to lower internalization ratios especially by primary skin cells. After topical application of nanoparticles on human skin explants with partially disrupted stratum corneum, only the 42 ± 3 nm particles were found to be associated with epidermal cells and especially dendritic cells, independent of their surface functionalization. Considering the wide use of nanomaterials in industries and the increasing interest for applications in pharmaceutics and cosmetics versus the large number of individuals with local or spread impairment of the skin barrier, e.g., patients with atopic dermatitis and chronic eczema, a careful dissection of nanoparticle-skin surface interactions is of high relevance to assess possible risks and potentials of intended and unintended particle exposure.
Mast cells (MCs) are multifunctional cells of the immune system and are found in skin and all major tissues of the body. They contribute to the pathology of several diseases including urticaria, ...psoriasis, atopic dermatitis and mastocytosis where they are increased at lesional sites. Histomorphometric analysis of skin biopsies serves as a routine method for the assessment of MC numbers and their activation status, which comes with major limitations. As of now, non-invasive techniques to study MCs in vivo are not available. Here, we describe a label-free imaging technique to visualize MCs and their activation status in the human papillary dermis in vivo. This technique uses two-photon excited fluorescence lifetime imaging (TPE-FLIM) signatures, which are different for MCs and other dermal components. TPE-FLIM allows for the visualization and quantification of dermal MCs in healthy subjects and patients with skin diseases. Moreover, TPE-FLIM can differentiate between two MC populations in the papillary dermis in vivo-resting and activated MCs with a sensitivity of 0.81 and 0.87 and a specificity of 0.85 and 0.84, respectively. Results obtained on healthy volunteers and allergy and mastocytosis patients indicate the existence of other MC subpopulations within known resting and activated MC populations. The developed method may become an important tool for non-invasive in vivo diagnostics and therapy control in dermatology and immunology, which will help to better understand pathomechanisms involving MC accumulation, activation and degranulation and to characterize the effects of therapies that target MCs.
•Intercellular lipids order profile measured by confocal Raman microscopy in vivo.•Topically applied oils influence the intercellular lipids order in stratum corneum.•Oils do not permeate through the ...stratum corneum in vivo.•Plant-derived oils influence deeper-located layers more than mineral-derived oils.
The intercellular lipids (ICL) of stratum corneum (SC) play an important role in maintaining the skin barrier function. The lateral and lamellar packing order of ICL in SC is not homogenous, but rather depth-dependent.
This study aimed to analyze the influence of the topically applied mineral-derived (paraffin and petrolatum) and plant-derived (almond oil and jojoba oil) oils on the depth-dependent ICL profile ordering of the SC in vivo.
Confocal Raman microscopy (CRM), a unique tool to analyze the depth profile of the ICL structure non-invasively, is employed to investigate the interaction between oils and human SC in vivo.
The results show that the response of SC to oils’ permeation varies in the depths. All oils remain in the upper layers of the SC (0–20% of SC thickness) and show predominated differences of ICL ordering from intact skin. In these depths, skin treated with plant-derived oils shows more disordered lateral and lamellar packing order of ICL than intact skin (p<0.05). In the intermediate layers of SC (30–50% of SC thickness), the oils do not influence the lateral packing order of SC ICL (p>0.1), except plant-derived oils at the depth 30% of SC thickness. In the deeper layers of the SC (60–100% of SC thickness), no difference between ICL lateral packing order of the oil-treated and intact skin can be observed, except that at the depths of 70–90% of the SC thickness, where slight changes with more disorder states are measured for plant-derived oil treated skin (p<0.1), which could be explained by the penetration of free fatty acid fractions in the deep-located SC areas.
Both oil types remain in the superficial layers of the SC (0–20% of the SC thickness). Skin treated with mineral- and plant-derived oils shows significantly higher disordered lateral and lamellar packing order of ICL in these layers of the SC compared to intact skin. Plant-derived oils significantly changed the ICL ordering in the depths of 30% and 70–90% of the SC thickness, which is likely due to the penetration of free fatty acids in the deeper layers of the SC.
Many modern cosmetic or sunscreen products contain nano-sized components. Nanoemulsions are transparent and have unique tactile and texture properties; nanocapsule, nanosome, noisome, or liposome ...formulations contain small vesicles (range: 50 to 5000 nm) consisting of traditional cosmetic materials that protect light-or oxygen-sensitive cosmetic ingredients. Transdermal delivery and cosmetic research suggests that vesicle materials may penetrate the stratum corneum (SC) of the human skin, but not into living skin. Depending on the physical/chemical properties of the ingredient and the formulation, nano-sized formulations may enhance or reduce skin penetration, albeit at a limited rate. Modern sunscreens contain insoluble titanium dioxide (TiO2) or zinc oxide (ZnO) nanoparticles (NP), which are colorless and reflect/scatter ultraviolet (UV) more efficiently than larger particles. Most available theoretical and experimental evidence suggests that insoluble NP do not penetrate into or through normal as well as compromised human skin. Oral and topical toxicity data suggest that TiO2 and ZnO NP have low systemic toxicity and are well tolerated on the skin. In vitro cytotoxicity, genotoxicity, and photogenotoxicity studies on TiO2 or other insoluble NP reporting uptake by cells, oxidative cell damage, or genotoxicity should be interpreted with caution, since such toxicities may be secondary to phagocytosis of mammalian cells exposed to high concentrations of insoluble particles. Caution needs to be exercised concerning topical exposure to other NP that either have characteristics enabling some skin penetration and/or have inherently toxic constituents. Studies on wear debris particles from surgical implants and other toxicity studies on insoluble particles support the traditional toxicology view that the hazard of small particles is mainly defined by the intrinsic toxicity of particles, as distinct from their particle size. There is little evidence supporting the principle that smaller particles have greater effects on the skin or other tissues or produce novel toxicities relative to micro-sized materials. Overall, the current weight of evidence suggests that nano-materials such as nano-sized vesicles or TiO2 and ZnO nanoparticles currently used in cosmetic preparations or sunscreens pose no risk to human skin or human health, although other NP may have properties that warrant safety evaluation on a case-by-case basis before human use.
The influence of a topically applied formulation containing components of natural moisturizing factor (NMF) on barrier-related parameters of the stratum corneum (SC) was investigated in vivo using ...confocal Raman microspectroscopy in a randomized, placebo-controlled double-blind study on 12 volunteers for 14 days. This method allowed for the elucidation of subtle differences between the verum and the placebo even though the components of the verum naturally occur in the SC. This differentiation is not possible non-invasively by conventional methods. In this study, we found that the applied verum and placebo formulations disrupted the equilibrium of water, NMF and lipids in the SC. The adverse effects of the formulation could be mitigated by incorporating it into a simplified supplementation of NMF molecules. As a long-term effect, the amount of strongly bound water increases at 30-40% SC depth (
< 0.05) and the amount of weakly bound water decreases at 30-40% SC depth (
< 0.05) for the verum. This supplement was also unexpectedly able to prevent intercellular lipids (ICL) disorganization in selected depths. In the long term, the verum treatment limited the lateral disorganization of the ICL to the upper 20% SC depth. Further research is required to elucidate the interplay of these factors in the SC, to better understand their contribution to the equilibrium and barrier function of the skin. This understanding of the interaction of these naturally occurring components could help in the future to develop and optimize topical treatments for diseases like psoriasis, atopic dermatitis, ichthyosis where the skin barrier is disrupted.
Atopic dermatitis (AD)/atopic eczema is a chronic relapsing inflammatory skin disease affecting nearly 14% of the adult population. An important pathogenetic pillar in AD is the disrupted skin ...barrier function (SBF). The atopic stratum corneum (SC) has been examined using several methods, including Raman microspectroscopy, yet so far, there is no depth-dependent analysis over the entire SC thickness. Therefore, we recruited 21 AD patients (9 female, 12 male) and compared the lesional (LAS) with non-lesional atopic skin (nLAS) in vivo with confocal Raman microspectroscopy. Our results demonstrated decreased total intercellular lipid and carotenoid concentrations, as well as a shift towards decreased orthorhombic lateral lipid organisation in LAS. Further, we observed a lower concentration of natural moisturising factor (NMF) and a trend towards increased strongly bound and decreased weakly bound water in LAS. Finally, LAS showed an altered secondary and tertiary keratin structure, demonstrating a more folded keratin state than nLAS. The obtained results are discussed in comparison with healthy skin and yield detailed insights into the atopic SC structure. LAS clearly shows molecular alterations at certain SC depths compared with nLAS which imply a reduced SBF. A thorough understanding of these alterations provides useful information on the aetiology of AD and for the development/control of targeted topical therapies.
Preclinical studies frequently lack predictive value for human conditions. Human cell-based disease models that reflect patient heterogeneity may reduce the high failure rates of preclinical ...research. Herein, we investigated the impact of primary cell age and body region on skin homeostasis, epidermal differentiation, and drug uptake. Fibroblasts derived from the breast skin of female 20- to 30-year-olds or 60- to 70-year-olds and fibroblasts from juvenile foreskin (<10 years old) were compared in cell monolayers and in reconstructed human skin (RHS). RHS containing aged fibroblasts differed from its juvenile and adult counterparts, especially in terms of the dermal extracellular matrix composition and interleukin-6 levels. The site from which the fibroblasts were derived appeared to alter fibroblast-keratinocyte crosstalk by affecting, among other things, the levels of granulocyte-macrophage colony-stimulating factor. Consequently, the epidermal expression of filaggrin and e-cadherin was increased in RHS containing breast skin fibroblasts, as were lipid levels in the stratum corneum. In conclusion, the region of the body from which fibroblasts are derived appears to affect the epidermal differentiation of RHS, while the age of the fibroblast donors determines the expression of proteins involved in wound healing. Emulating patient heterogeneity in preclinical studies might improve the treatment of age-related skin conditions.
PDF
