Development and Characterization of a 96-Well Exposure System for Safety Assessment of Nanomaterials.

In this study, a 96-well exposure system for safety assessment of nanomaterials is developed and characterized using an air-liquid interface lung epithelial model. This system is designed for sequential nebulization. Distribution studies verify the reproducible distribution over all 96 wells, with lower insert-to-insert variability compared to non-sequential application. With a first set of chemicals (TritonX), drugs (Bortezomib), and nanomaterials (silver nanoparticles and (non-)fluorescent crystalline nanocellulose), sequential exposure studies are performed with human lung epithelial cells followed by quantification of the deposited mass and of cell viability. The developed exposure system offers for the first time the possibility of exposing an air-liquid interface model in a 96-well format, resulting in high-throughput rates, combined with the feature for sequential dosing. This exposure system allows the possibility of creating dose-response curves resulting in the generation of more reliable cell-based assay data for many types of applications, such as safety analysis. In addition to chemicals and drugs, nanomaterials with spherical shapes, but also morphologically more complex nanostructures can be exposed sequentially with high efficiency. This allows new perspectives on in vivo-like and animal-free approaches for chemical and pharmaceutical safety assessment, in line with the 3R principle of replacing and reducing animal experiments.

Characterization of Classical Flexural and Nummular Forms of Atopic Dermatitis in Childhood with Regard to Anamnestic, Clinical and Epidermal Barrier Aspects.

Nummular (coin-shaped) and classical (flexural) atopic dermatitis differ morphologically, but no other distinguishing features are known. The aim of this study was to determine differences and similarities of both variants in children. Detailed interviews, clinical examinations, biophysical measurements and electron microscopic analyses were performed on 10 children with nummular atopic dermatitis, 14 with classical atopic dermatitis and 10 healthy controls. Nummular atopic dermatitis affected more boys than girls and manifested less frequently within the first year of life than classical atopic dermatitis. Localization, distribution and morphology of the eczema varied more over time, and expression of keratosis pilaris was more severe in children with nummular atopic dermatitis. Both disease groups showed reduced hydration, increased transepidermal water loss and reduced intercellular lipid lamellae in lesional skin areas compared with non-lesional areas. These findings underline the separate classification of both variants. Further research is necessary to investigate the potential of diverging therapeutic approaches.

Comparison of Lipid Foam Cream and Basic Cream on Epidermal Reconstruction in Mild Atopic Eczema.

INTRODUCTION: Basic therapy is of central importance in the treatment of atopic eczema. Using electron microscopic images, the morphology of epidermal skin barrier and its lipids was investigated after application of a lipid foam cream and basic cream. METHODS: Patients with two contralateral comparable atopic eczema (local SCORAD 1-10) on the forearms were tested. Eczema was treated with a lipid foam cream or basic cream twice daily for 28 days. At the beginning, after 14 days, and at the end of application, the local SCORAD, trans-epidermal water loss (TEWL), skin hydration, intercellular lipid length in the intercellular space of the stratum corneum (SC), and skin lipids were determined. RESULTS: After application of the foam cream, the epidermal skin barrier could be completely restored and corresponded to healthy skin, while the epidermal skin barrier could not reach this state after care with the basic cream. The content of lipids in the SC increases significantly by 31% after basic cream treatment, whereas they are significantly increased by 85% after application of the lipid foam cream. The local SCORAD improved for both treatments to about the same extent, and no significant results could be shown for TEWL and skin hydration. CONCLUSION: In subjects with mild atopic eczema, the lipid foam cream leads to a measurable recovery of the skin barrier which is much more pronounced in comparison to the basic cream.

Epidermal Regeneration Induced by Comfrey Extract: A Study by Light and Electron Microscopy.

INTRODUCTION: An accelerated healing of superficial wounds was demonstrated in clinical trials with a topical comfrey preparation (Symphytum × uplandicum Nyman). The effect has previously not been examined in skin models. METHODS: An established in vitro model of epidermal cells with the typical strata was used for the observation of effects of applied substances on skin regeneration. Damage corresponding to a typical abrasion was created on day 1 by punching an opening into the epidermal fine structure down to the stratum basale. Samples were either untreated (controls) or exposed to comfrey cream on days 2, 3, 5, and 6. Tissue samples were taken for light and electron microscopy on days 1, 4, and 7. RESULTS AND CONCLUSIONS: Application of comfrey cream led to a quicker regeneration of skin cells and to an earlier differentiation of the cells towards a normal fine structure with a visible distinction of epidermal strata, keratin, and corneocyte formation within 4-7 days. The study covered the early days of skin regeneration and confirms the benefits observed in published clinical trials and non-interventional studies in patients with abrasions.

Effects of pimecrolimus compared with triamcinolone acetonide cream on skin barrier structure in atopic dermatitis: a randomized, double-blind, right-left arm trial.

Patients with atopic dermatitis (AD) have an epidermal barrier dysfunction, which allows invasion of allergens to occur. Stratum corneum skin barrier is formed by corneocytes and extracellular lipids extruded from the epidermal lamellar bodies. In a controlled, randomized, double-blinded, right-left comparison study we investigated the effect of pimecrolimus (PIM) cream compared with triamcinolone acetonide cream (TA) on the skin barrier in 15 patients with symmetrical elbow lesions of AD. In punch biopsies, before and after treatment, skin lipid bilayer and lamellar body structure were examined by transmission electron microscopy (TEM). Partial Eczema Area and Severity Index (pEASi), stratum corneum hydration, and transepidermal water loss (TEWL) were monitored on days 1, 8 and 22. The pEASi was significantly more improved with TA compared with PIM, whereas stratum corneum hydration was slightly more improved after treatment with PIM. The TEM revealed a strong reduction in lamellar bodies in lesional skin of AD; only 32% of the lamellar bodies were normal. A significantly higher number of normal lamellar bodies was found after 3 weeks of treatment with PIM (58%; p < 0.005). An increase in lamellar bodies also occurred with TA treatment (46%; p < 0.05); however, significantly less than with PIM (p < 0.05). Clinical score and TEWL were more improved after treatment with TA, whereas the lamellar bodies were more normal after treatment with PIM.

Comparison of effects of tacrolimus ointment and mometasone furoate cream on the epidermal barrier of patients with atopic dermatitis.

BACKGROUND: The skin barrier plays a crucial role in the pathophysiology of atopic dermatitis. The quality of the skin barrier can be assessed using a new semi-quantitative method to measure intercellular lipid lamellae. This procedure was used to evaluate the influence of the topical application of the calcineurin inhibitor tacrolimus 0.1% ointment (Protopic®) versus mometasone furoate cream (Ecural®) on the quality of the skin barrier. PATIENTS AND METHODS: 20 adult patients with active atopic dermatitis (SCORAD 10-63) were included in an open, non-interventional study. Lesions on their forearms were treated twice daily over 10 days with either tacrolimus 0.1% ointment or mometasone furoate cream. At the beginning and the end of the treatment period, SCORAD, TEWL and skin hydration were determined and the intercellular lipids were measured using transmission electron microscopy. RESULTS: The SCORAD improved in both groups nearly to the same extent, whereas TEWL and skin hydration improved significantly only in the tacrolimus group. Using the semi-quantitative analysis of intercellular lipid length per 1,000 nm(2) intercellular space, a twofold increase for mometasone furoate cream and a fourfold increase for tacrolimus 0.1% ointment were determined. CONCLUSIONS: In addition to its known antiinflammatory effect, tacrolimus 0.1% ointment leads also to a measurable increase of the lipids of the skin barrier in patients with atopic dermatitis, exceeding the effect of mometasone furoate cream.

Fluorescence Labeling of Cellulose Nanocrystals-A Facile and Green Synthesis Route.

Efficient chemical modification of cellulose nanocrystals (CNCs) by grafting commonly involves aprotic solvents, toxic reactants, harsh reaction conditions, or catalysts, which have negative effects on the particle character, reduced dispersibility and requires further purification, if products are intended for biomedical applications. This work, in contrast, presents a robust, facile, and green synthesis protocol for the grafting of an amino-reactive fluorophore like fluorescein isothiocyanate (FITC) on aqueous CNCs, combining and modifying existent approaches in a two-step procedure. Comparably high grafting yields were achieved, which were confirmed by thermogravimetry, FTIR, and photometry. The dispersive properties were confirmed by DLS, AF4-MALS, and TEM studies. The presented route is highly suitable for the introduction of silane-bound organic groups and offers a versatile platform for further modification routes of cellulose-based substrates.

Size Matters! Issues and Challenges with Nanoparticulate UV Filters.

Preparations containing pigments have been used since ancient times to protect against negative effects of solar radiation. Since the 1950s, sunscreen products containing micronized TiO2 and ZnO have been marketed. These products were soon regarded as cosmetically unattrac-tive due to their property of remaining as a white paste on the skin, a result of particle sizes. In order to eliminate these unfavourable properties, particle size distribution was lowered into a range below 100 nm, a size threshold for decreasing the particle's optical property to reflect visible light. After 2000, new nanoparticulate organic filters were developed. Effects of both the inorganic and organic nanoparticulate substances - alone or in combination - with non-particulate UV filters were well documented and had shown great effectiveness. At the time, nanotechnology fuelled great hope in the progress of science and technology, including the health sector and cosmetics industry. Instead, influenced by images from the science fiction literature of self-replicating nanorobots destroying all living matter or health and environmental disasters caused by asbestos, fear of this new unknown amongst the general population has hindered acceptance and progress of nano-enabled products. Consumers have started to suspect that the particles permeate through skin, are absorbed by the blood and are distributed throughout the body, causing disease. Not least because of public pressure, cosmetics - which include sunscreen products - became the first product segment in which appropriately manufactured substances were subject to stringent rules. Despite advanced regulation and rigorous approval procedures for nanoparticulate UV filters, widespread reservations remain. Possible reasons could be a lack of knowledge of current legislation and unclear ideas about nature and behaviour of nanoparticles. Against this background, we discuss the nature and behaviour of nanoparticulate UV filters within finished products, on the skin and potentially in the skin, and the regulatory framework that ensures that nanoparticulate UV filters and the sunscreen products containing them are safe to use.

Comparing the effects of proactive treatment with tacrolimus ointment and mometasone furoate on the epidermal barrier structure and ceramide levels of patients with atopic dermatitis.

BACKGROUND: The epidermal skin barrier and lipids that are integral to its structure are impaired in atopic dermatitis (AD). Current treatment guidelines include proactive therapy. OBJECTIVE: This study assessed the effect of 12 weeks of proactive treatment with tacrolimus ointment 0.1% (TAC) compared with mometasone furoate cream (MF) on specific skin barrier lipids in patients with AD who previously received 10 days of reactive treatment with either agent. METHODS: This was an open-label, non-interventional study. In the reactive phase, forearm lesions in 20 patients were treated with either TAC or MF twice daily for 10 days. In the subsequent proactive phase, patients applied TAC or MF twice weekly for 12 weeks (n = 16 patients). RESULTS: Over the 12-week proactive treatment period, the mean local SCORAD significantly decreased in the TAC and MF treatment group. Levels of total and individual ceramides increased in both groups. Normalized intercellular lipid lamellae values were significantly higher with proactive TAC treatment than MF and undistinguishable from healthy skin. CONCLUSION: The results show that proactive treatment with TAC is superior in restoring the skin barrier.

Influence of Physicochemical Characteristics and Stability of Gold and Silver Nanoparticles on Biological Effects and Translocation across an Intestinal Barrier-A Case Study from In Vitro to In Silico.

A better understanding of their interaction with cell-based tissue is a fundamental prerequisite towards the safe production and application of engineered nanomaterials. Quantitative experimental data on the correlation between physicochemical characteristics and the interaction and transport of engineered nanomaterials across biological barriers, in particular, is still scarce, thus hampering the development of effective predictive non-testing strategies. Against this background, the presented study investigated the translocation of gold and silver nanoparticles across the gastrointestinal barrier along with related biological effects using an in vitro 3D-triple co-culture cell model. Standardized in vitro assays and quantitative polymerase chain reaction showed no significant influence of the applied nanoparticles on both cell viability and generation of reactive oxygen species. Transmission electron microscopy indicated an intact cell barrier during the translocation study. Single particle ICP-MS revealed a time-dependent increase of translocated nanoparticles independent of their size, shape, surface charge, and stability in cell culture medium. This quantitative data provided the experimental basis for the successful mathematical description of the nanoparticle transport kinetics using a non-linear mixed effects modeling approach. The results of this study may serve as a basis for the development of predictive tools for improved risk assessment of engineered nanomaterials in the future.

Comparison of Epidermal Barrier Integrity in Adults with Classic Atopic Dermatitis, Atopic Prurigo and Non-Atopic Prurigo Nodularis.

A deficient epidermal barrier is a key feature of atopic dermatitis (AD) and comprises altered lipid and protein content and composition of the stratum corneum resulting in disturbed water balance. Clinically, eczematous lesions on dry skin and pruritus develop. Pruritic nodules occur in prurigo nodularis (PN), another chronic skin disease, which can be associated with atopy. We aimed at comparing the three clinical pictures, classic AD, atopic prurigo (AP), and non-atopic PN, to healthy controls regarding the epidermal barrier. We determined clinical parameters and performed biophysical measurements, histology/immunohistochemistry, electron microscopy, and molecular biological analysis. We found distinctively elevated clinical scores, reduced hydration and increased transepidermal water loss, epidermal hyperplasia and inflammation reduced filaggrin and increased loricrin and involucrin expression, as well as reduced intercellular lipid lamellae in all three disease groups. These findings show a severe disruption in epidermal barrier structure and function in all three disorders so that epidermal barrier impairment is now proven not only for AD but also for PN.

Synthesis and in vitro evaluation of cyclodextrin hyaluronic acid conjugates as a new candidate for intestinal drug carrier for steroid hormones.

Steroid hormones became increasingly interesting as active pharmaceutical ingredients for the treatment of endocrine disorders. However, medical applications of many steroidal drugs are inhibited by their very low aqueous solubilities giving rise to low bioavailabilities. Therefore, the prioritized oral administration of steroidal drugs remains problematic. Cyclodextrins are promising candidates for the development of drug delivery systems for oral route applications, since they solubilize hydrophobic steroids and increase their rate of transport in aqueous environments. In this study, the synthesis and characterization of polymeric ß-cyclodextrin derivates is described, which result from the attachment of a hydrophilic ß-CD-thioether to hyaluronic acid. Host-guest complexes of the synthesized ß-cyclodextrin hyaluronic acid conjugates were formed with two poorly soluble model steroids (ß-estradiol, dexamethasone) and compared to monomeric ß-cyclodextrin derivates regarding solubilization and complexation efficiency. The ß-cyclodextrin-drug (host-guest) complexes were evaluated in vitro for their suitability (cytotoxicity and transport rate) as intestinal drug carriers for steroid hormones. In case of ß-estradiol, higher solubilities could be achieved by complexation with both synthesized ß-cyclodextrin derivates, leading to significantly higher intestinal transport rates in vitro. However, this success could not be shown for dexamethasone, which namely solubilized better, but could not enhance the transport rate significantly. Thus, this study demonstrates the biocompatibility of the synthesized and characterized ß-cyclodextrin derivates and shows their potential as new candidate for intestinal drug carrier for steroid hormones like ß-estradiol.

Claudin-1 decrease impacts epidermal barrier function in atopic dermatitis lesions dose-dependently.

The transmembrane protein claudin-1 is a major component of epidermal tight junctions (TJs), which create a dynamic paracellular barrier in the epidermis. Claudin-1 downregulation has been linked to atopic dermatitis (AD) pathogenesis but variable levels of claudin-1 have also been observed in healthy skin. To elucidate the impact of different levels of claudin-1 in healthy and diseased skin we determined claudin-1 levels in AD patients and controls and correlated them to TJ and skin barrier function. We observed a strikingly broad range of claudin-1 levels with stable TJ and overall skin barrier function in healthy and non-lesional skin. However, a significant decrease in TJ barrier function was detected in lesional AD skin where claudin-1 levels were further reduced. Investigations on reconstructed human epidermis expressing different levels of claudin-1 revealed that claudin-1 levels correlated with inside-out and outside-in barrier function, with a higher coherence for smaller molecular tracers. Claudin-1 decrease induced keratinocyte-autonomous IL-1ß expression and fostered inflammatory epidermal responses to non-pathogenic Staphylococci. In conclusion, claudin-1 decrease beyond a threshold level results in TJ and epidermal barrier function impairment and induces inflammation in human epidermis. Increasing claudin-1 levels might improve barrier function and decrease inflammation and therefore be a target for AD treatment.

Skin acidification with a water-in-oil emulsion (pH 4) restores disrupted epidermal barrier and improves structure of lipid lamellae in the elderly.

The pH of the skin surface increases with age and thus reduces epidermal barrier function. Aged skin needs appropriate skin care to counterbalance age-related pH increase and improve barrier function. This confirmatory randomized study investigated the efficacy of water-in-oil (w/o) emulsions with either pH 4 or pH 5.8 in 20 elderly subjects after 4 weeks of treatment. After the treatment, the skin was challenged with a sodium dodecyl sulphate (SDS) solution in order to analyze barrier protection properties of both formulations. The pH 4 w/o emulsion resulted in a significantly lower skin pH compared with the pH 5.8 w/o emulsion and an improved skin hydration after 4-week treatment. Further, the pH 4 emulsion led to more pronounced improvements in length of intercellular lipid lamellae, lamellar organization as well as lipid levels than the pH 5.8 emulsion. Following SDS-induced barrier damage to the skin, the pH of all test areas increased, but the area treated with the pH 4 emulsion showed the lowest increase compared with baseline. In addition, even after the SDS challenge the skin area treated with the pH 4 emulsion still maintained a significantly increased length of intercellular lipid lamellae compared with the beginning of the study. This study provides evidence that topical application of a w/o emulsion with pH 4 reacidifies the skin in elderly and has beneficial effects on skin moisturization, regeneration of lipid lamellae and lipid content. Application of a pH 4 emulsion can improve the epidermal barrier as well as the stratum corneum organization in aged skin.

Influence of Topical Formulations: Lipid Lamella Organization and Lipid Composition of Stratum Corneum as a Surrogate Marker for Barrier Integrity.

Skin barrier repair therapies often involve the use of medicated and non-medicated topical preparations. To measure the effect of topical preparations, clinical (scoring systems, for example, Score of Atopic Dermatitis, Dermatology Quality of Life Index) and biophysical procedures (e.g., trans-epidermal water loss, skin hydration) are widely used. However, the results of these procedures describe the condition of the barrier indirectly. A direct assessment of skin barrier integrity is primarily possible by electron-microscopic examination, visualization and morphometric analysis of the lipid lamellae in the intercellular space of the stratum corneum (SC) and by quantitatively characterizing the composition of key SC lipids. Recently, the combination of a non-invasive lipid barrier visualization (Lipbarvis®) technique (SC sampling and morphometric analysis) and SC lipid composition analysis (chromatographic analysis) has been proposed to directly characterize the skin barrier integrity. Initial experience demonstrates that morphometric analysis of the lipid lamellae organization in the intercellular space of the SC as well as the characterization of the composition of key SC lipids may serve as surrogate marker to study the influence of topical non-medicated preparations including pH-lowered preparations.

Echinacea purpurea-derived alkylamides exhibit potent anti-inflammatory effects and alleviate clinical symptoms of atopic eczema.

BACKGROUND: Atopic eczema (AE) is a chronic inflammatory and pruritic skin disease. There is still an unmet need for topical anti-inflammatory and anti-pruritic substances exhibiting an excellent safety profile. The endocannabinoid system is known to regulate various aspects of cutaneous barrier and immune functions, thus targeting it may be a valid approach for alleviating the symptoms of AE. OBJECTIVE: To assess the putative efficacy of Echinacea purpurea-derived alkylamides (Ec. extract) activating cannabinoid (CB)-2 receptors in exerting anti-inflammatory effects and alleviating symptoms of AE. METHODS: In vitro anti-inflammatory efficiency was investigated by monitoring the effects of Ec. extract on poly-(I:C)-induced pro-inflammatory cytokine expression (Q-PCR) and release (ELISA) of HaCaT keratinocytes. Irritancy and sensitization potential (assessed by Human Repeat Insult Patch Test; Clinical trial 1); clinical efficiency in alleviating symptoms of AE (Clinical trial 2) as well as effects on human skin structure and lipid content (Clinical trial 3 followed by transmission electron microscopy and HPTLC) were investigated in randomized double blind clinical trials. RESULTS: Ec. extract significantly reduced mRNA expression as well as release of poly-(I:C)-induced pro-inflammatory cytokines (IL-6 and IL-8) in keratinocytes. Thus, not surprisingly, the well-tolerated (Clinical trial 1) Ec. extract-based cream reduced local SCORAD statistically significantly, not only compared to baseline, but also compared to the comparator (Clinical trial 2). Of great importance, besides the in vitro anti-inflammatory effects, administration of the Ec. extract-based cream also resulted in significantly higher levels of overall epidermal lipids, ceramide EOS (ω-esterified fatty acid+sphingosine sphingoid base), and cholesterol at Day 15 compared to baseline as well as significantly greater numbers of intercellular lipid lamellae in the intercellular space (Clinical trial 3). CONCLUSION: The investigated Ec. extract shows great potential in alleviating cutaneous symptoms of AE, and by exerting remarkable anti-inflammatory actions and restoring the epidermal lipid barrier, it will be very likely a well-tolerated, powerful novel ingredient for the adjuvant therapy of AE.

A new topical panthenol-containing emollient: Results from two randomized controlled studies assessing its skin moisturization and barrier restoration potential, and the effect on skin microflora.

PURPOSE: Two randomized, intra-individual comparison studies were performed in healthy subjects to evaluate the skin moisturization and barrier restoration potential of a new topical panthenol-containing emollient (NTP-CE) (Study 1), and its effect on skin microflora (Study 2). METHODS: In Study 1 (N = 23), two skin areas, one challenged with 0.5% sodium dodecyl sulfate (SDS) solution and one unchallenged, were treated with NTP-CE for 3 weeks. Transepidermal water loss (TEWL), skin hydration, and intercellular lipid lamellae (ICLL) organization were measured at regular intervals during the study. In Study 2 (N = 20), quantitative bacterial cultures were obtained over 6 h from a skin area undergoing wash stress with 10% SDS with subsequent single application of NTP-CE. RESULTS: In Study 1, mean AUC for TEWL reduction from baseline was more pronounced with NTP-CE compared with control (-168.36 vs. -123.38 g/m2/h, p = 0.023). NTP-CE use was also associated with statistically significant improvements in stratum corneum hydration and an increase in mean ICLL length from baseline (day 22: 120.61 vs. 35.85 nm/1000 nm2, p < 0.001). In Study 2, NTP-CE use had no negative impact on bacterial viability. CONCLUSIONS: NTP-CE use has favorable and lasting effects on barrier function and repair as well as skin hydration without negatively influencing bacterial viability.