Loss-of-function mutations in the filaggrin gene (FLG) are a strong predisposing factor for atopic dermatitis, although their relevance to the disease pathomechanism needs further elucidation. The ...generation of an in vitro model of atopic skin would not only permit further evaluation of the underlying pathogenetic mechanisms and the testing of new treatment options, but would also allow toxicological studies to be performed in a simple, rapid and inexpensive manner. In this study, we have knocked down FLG expression in human keratinocytes and created three-dimensional skin models, which we used to investigate the impact of FLG on epidermal maturation and on skin absorption and its response to irritation. Histopathological evaluation of the skin models showed impaired epidermal differentiation in the FLG knock-down model. In addition, skin irritation induced by an application of sodium dodecyl sulphate resulted in significantly higher lactate dehydrogenase leakage, and interleukin (IL)-6 and IL-8 levels, than in the control model. To assess the effect of filaggrin deficiency on skin absorption of topically applied agents, we quantified the percutaneous absorption of lipophilic and hydrophilic model drugs, finding clinical relevance only for lipophilic drugs. This study clearly demonstrates that important clinical characteristics of atopic skin can be mimicked by using in vitro skin models. The FLG knock-down construct is the first step toward an in vitro model that allows clinical and toxicological studies of atopic-like skin.
For efficient pain reduction in severe skin wounds, topical opioids may be a new option – given that wound healing is not impaired and the vehicle allows for slow opioid release, since long intervals ...of painful wound dressing changes are intended. We investigated the influence of opioids on the wound healing process via in vitro models, migration assay and scratch test. In fact, morphine, hydromorphone, fentanyl and buprenorphine increased the number of migrated HaCaT cells (spontaneously transformed keratinocytes) twofold. In the scratch test, morphine accelerated the closure of a monolayer wound (scratch). As possible slow release application forms are nanoparticulate systems like solid lipid nanoparticles (SLN) and dendritic core-multishell (CMS) nanotransporters, we evaluated the effect of unloaded nanoparticles on HaCaT cell migration, too. CMS nanotransporters did not inhibit migration, SLN even enhanced it (twofold). Applying morphine plus unloaded nanoparticles reduced morphine effects possibly due to uptake into CMS nanotransporters and adsorption to the surface of SLN. In contrast to SLN, TGF-β1 was taken up by CMS nanotransporters, too. Both nanoparticles are tolerable by skin and eye as derived from Episkin-SM
TM skin irritation test and HET-CAM assay. No acute toxic effects were observed either. In conclusion, opioids as well as the investigated nanoparticulate carriers conform the essential conditions for topical pain reduction.
Oral mucositis is one of the most common side effects of chemoradiation regimens and manifestation can be dose-limiting for the therapy, can impair the patient's nutritional condition and quality of ...life due to severe pain. The therapeutic options are limited; often only an alleviation of the symptoms such as pain reduction by using systemic opioids is possible. Stimulating opioid receptors on peripheral neurons and dermal tissue, potent analgesic effects are induced e.g. in skin grafted patients. Advantageous effects on the cell migration and, thus, on the wound healing process are described, too. In this study, we investigated whether opioid receptors are also expressed on oral epithelial cells and if morphine can modulate their cell migration behavior. The expression of the opioid receptors MOR, DOR and KOR on primary human oral epithelial cells was verified. Furthermore, a significantly accelerated cell migration was observed following incubation with morphine. The effect even slightly exceeded the cell migration stimulating effect of TGF-ß: After 14 h of morphine treatment about 86% of the wound area was closed, whereas TGF-ß application resulted in a closed wound area of 80%. With respect to morphine stimulated cell migration we demonstrate that DOR plays a key role and we show the involvement of the MAPK members Erk 1/2 and p38 using Western blot analysis.Further studies in more complex systems in vitro and in vivo are required. Nevertheless, these findings might open up a new therapeutic option for the treatment of oral mucositis.
In this paper we report a novel approach to generate biodegradable polyglycerol nanogels on different length scales. We developed a mild, surfactant free inverse nanoprecipitation process to template ...hydrophilic polyglycerol nanoparticles. In situ crosslinking of the precipitated nanoparticles by bioorthogonal copper catalyzed click chemistry allows us to obtain size defined polyglycerol nanogels (100–1000nm). Biodegradability was achieved by the introduction of benzacetal bonds into the net points of the nanogel. Interestingly, the polyglycerol nanogels quickly degraded into low molecular weight fragments at acidic pH values, which are present in inflamed and tumor tissues as well as intracellular organelles, and they remained stable at physiological pH values for a long time. This mild approach to biodegradable polyglycerol nanogels allows us to encapsulate labile biomacromolecules such as proteins, including the therapeutic relevant enzyme asparaginase, into the protein resistant polyglycerol network. Enzymes were encapsulated with an efficacy of 100% and after drug release, full enzyme activity and structural integrity were retained. This new inverse nanoprecipitation procedure allows the efficient encapsulation and release of various biomacromolecules including proteins and could find many applications in polymer therapeutics and nanomedicine.
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(Per)-cutaneous morphine absorption using the finite dose approach over a period of 6h.
Topically applied opioids are an option to induce efficient analgesia in patients with severe skin wounds. For ...ongoing pain reduction, the vehicle should provide sustained drug release in order to increase the intervals during the regular wound dressing changes. In addition, the formulation should not impair wound healing. Hydrogels provide a moist wound environment, which is known to facilitate the healing process.
Investigating poloxamer hydrogels as a carrier system for morphine in terms of release behavior and (per-)cutaneous absorption, poloxamer 407 25wt.% hydrogel sustained morphine release up to 24h. The drug release rate decreased with increasing concentration of the gel forming triblock copolymer. Poloxamer 407 25wt.% hydrogel retarded morphine uptake into reconstructed human skin and percutaneous drug absorption compared to a hydroxyethyl cellulose reference gel.
The results of our in vitro study indicate that the thermosensitive poloxamer 407 25wt.% hydrogel is an appropriate carrier system for the topical application of morphine with regard to sustained drug release and ongoing analgesia.
As humans can come into contact with xenobiotics intentionally or accidentally, knowledge about the skin absorption of these substances is crucial and requires reliable models and test procedures. ...Animal experiments should be avoided whenever possible, instead of making the use of in vitro systems. Furthermore, due to limited availability of normal and especially diseased human skin, alternative test systems such as reconstructed skin models are urgently required.
This article discusses the advantages and limitations of excised human skin, animal skin and reconstructed skin models for absorption testing in vitro. Furthermore, the authors also describe the standard procedure for skin absorption testing and give an excursion to the applicability of artificial membranes. Finally, the article highlights the progress in the development of reconstructed disease models and provides an extensive overview about past and ongoing research in this field.
The development and validation of in vitro systems for skin absorption testing is inevitable. More research efforts are required for the development of reconstructed disease models. Reconstructed skin models need to be improved, especially in terms of complexity to mimic the in vivo situation better. It should not, however, be the main goal to imitate the in vivo situation exactly, but to establish reliable systems that ensure predictive and reliable data.
For efficient pain reduction in severe skin wounds, topically applied opioids may be a new option. Moreover, by stimulating keratinocyte migration opioids may also accelerate wound healing. Yet, ...conventional formulations failed to consistently provide sufficient pain control in patients which may be due to local drug degradation or insufficient concentrations at the target site. After having excluded major morphine glucuronidation by keratinocytes and fibroblasts, we next aimed for an optimised formulation. Since long intervals for painful wound dressing changes are intended, the formulations should allow for prolonged opioid release and should not impair the healing process. We developed morphine-loaded solid lipid nanoparticles (SLN, mean size about 180nm), and tested improvement of wound closure in a new human-based 3D-wound healing model. Standardised wounds were induced by CO2-laser irradiation of reconstructed human full-thickness skin equivalents (EpiDermFT™). Morphine, morphine-loaded and unloaded SLN accelerated reepithelialization. Keratinocytes almost completely covered the dermis equivalent after 4 days, which was not the case when applying the vehicle. In conclusion, acceleration of wound closure, low cytotoxicity and irritation as well as possible prolonged morphine release make SLN an interesting approach for innovative wound management.
Wound repair is a quiescent mechanism to restore barriers in multicellular organisms upon injury. In chronic wounds, however, this program prematurely stalls. It is known that patterns of ...extracellular signals within the wound fluid are crucial to healing. Extracellular pH (pHe) is precisely regulated and potentially important in signaling within wounds due to its diverse cellular effects. Additionally, sufficient oxygenation is a prerequisite for cell proliferation and protein synthesis during tissue repair. It was, however, impossible to study these parameters in vivo due to the lack of imaging tools. Here, we present luminescent biocompatible sensor foils for dual imaging of pHe and oxygenation in vivo. To visualize pHe and oxygen, we used time-domain dual lifetime referencing (tdDLR) and luminescence lifetime imaging (LLI), respectively. With these dual sensors, we discovered centripetally increasing pHe-gradients on human chronic wound surfaces. In a therapeutic approach, we identify pHe-gradients as pivotal governors of cell proliferation and migration, and show that these pHe-gradients disrupt epidermal barrier repair, thus wound closure. Parallel oxygen imaging also revealed marked hypoxia, albeit with no correlating oxygen partial pressure (pO2)-gradient. This highlights the distinct role of pHe-gradients in perturbed healing. We also found that pHe-gradients on chronic wounds of humans are predominantly generated via centrifugally increasing pHe-regulatory Na+/H+-exchanger-1 (NHE1)-expression. We show that the modification of pHe on chronic wound surfaces poses a promising strategy to improve healing. The study has broad implications for cell science where spatial pHe-variations play key roles, e.g. in tumor growth. Furthermore, the novel dual sensors presented herein can be used to visualize pHe and oxygenation in various biomedical fields.
Oral mucositis is a common side effect of chemotherapy and radiation therapy accompanied with acute inflammation and ulceration of the oral mucosa. Opioids can improve the wound healing of dermal and ...oral tissue when applied locally. The aim of this study was to investigate if morphine exhibits cytoprotective effects on oral epithelial cells postirradiation. Hence, oral epithelial cells were exposed to increasing doses (3–30 Gy) of ionization radiation. We assessed the effects of the radiation on cell viability, proinflammatory cytokine release (interleukin IL‐1α, IL‐6), and matrix metalloproteinase (MMP‐1, ‐8, and ‐9) expression. As expected, radiation significantly impaired cell viability and morphology and resulted in enhanced IL release. However, morphine‐treated cells consistently showed higher cell viability postirradiation: 9.19 ± 1.16% after 24 hours and 7.45 ± 0.93% after 48 hours compared with the control. In terms of proinflammatory cytokines, the release of IL‐1α and IL‐6 was significantly reduced, too, being most pronounced at 48 hours postradiation. Additionally, we observed a significant reduction of MMP‐1 and especially MMP‐9 expression in morphine‐treated cells. The results clearly indicate anti‐inflammatory as well as cytoprotective effects of morphine on irradiated oral epithelial cells. Interestingly, the protective effects of morphine are not related to a decrease in cell apoptosis or necrosis. Before final conclusions can be drawn, further studies in more complex systems in vitro and in vivo are required. Nevertheless, these findings further underline the high potential of opioids for the treatment of topical wounds and inflammatory conditions.
The aim of this study was to assess a recently established 3D model of congenital ichthyosis, representing severe epidermal barrier function defects, for skin penetration and permeation. We have ...generated disease models by knock‐down of either TGM1 or ALOXE3 in primary human keratinocytes, and using keratinocytes and fibroblasts from patients with congenital ichthyosis. The results indicate disturbed barrier function as demonstrated by increased permeation of testosterone and caffeine particularly in TGM1 knock‐down models compared to control models. In addition, enhanced penetration of the model dye nile red incorporated into solid lipid nanoparticles and core‐multishell nanotransporters, respectively, was evident in disease models. Thus, in vitro skin disease models reproduce differences in barrier permeability and function seen in congenital ichthyosis and pave the way to personalised disease models. Furthermore, our findings indicate that nanocarriers may be useful in new, topical therapeutic approaches for the currently very limited treatment of congenital ichthyosis.
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