Luminescent dual sensors reveal extracellular pH-gradients and hypoxia on chronic wounds that disrupt epidermal repair.

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.

Penetration of normal, damaged and diseased skin--an in vitro study on dendritic core-multishell nanotransporters.

A growing intended or accidental exposure to nanoparticles asks for the elucidation of potential toxicity linked to the penetration of normal and lesional skin. We studied the skin penetration of dye-tagged dendritic core-multishell (CMS) nanotransporters and of Nile red loaded CMS nanotransporters using fluorescence microscopy. Normal and stripped human skin ex vivo as well as normal reconstructed human skin and in vitro skin disease models served as test platforms. Nile red was delivered rapidly into the viable epidermis and dermis of normal skin, whereas the highly flexible CMS nanotransporters remained solely in the stratum corneum after 6h but penetrated into deeper skin layers after 24h exposure. Fluorescence lifetime imaging microscopy proved a stable dye-tag and revealed striking nanotransporter-skin interactions. The viable layers of stripped skin were penetrated more efficiently by dye-tagged CMS nanotransporters and the cargo compared to normal skin. Normal reconstructed human skin reflected the penetration of Nile red and CMS nanotransporters in human skin and both, the non-hyperkeratotic non-melanoma skin cancer and hyperkeratotic peeling skin disease models come along with altered absorption in the skin diseases.

Increased cutaneous absorption reflects impaired barrier function of reconstructed skin models mimicking keratinisation disorders.

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.

Filaggrin deficiency leads to impaired lipid profile and altered acidification pathways in a 3D skin construct.

Mutations in the filaggrin (FLG) gene are strongly associated with common dermatological disorders such as atopic dermatitis. However, the exact underlying pathomechanism is still ambiguous. Here, we investigated the impact of FLG on skin lipid composition, organization, and skin acidification using a FLG knockdown (FLG-) skin construct. Initially, sodium/hydrogen antiporter (NHE-1) activity was sufficient to maintain the acidic pH (5.5) of the reconstructed skin. At day 7, the FLG degradation products urocanic (UCA) and pyrrolidone-5-carboxylic acid (PCA) were significantly decreased in FLG- constructs, but the skin surface pH was still physiological owing to an upregulation of NHE-1. At day 14, secretory phospholipase A2 (sPLA2) IIA, which converts phospholipids to fatty acids, was significantly more activated in FLG- than in FLG+. Although NHE-1 and sPLA2 were able to compensate the FLG deficiency, maintain the skin surface pH, and ensured ceramide processing (no differences detected), an accumulation of free fatty acids (2-fold increase) led to less ordered intercellular lipid lamellae and higher permeability of the FLG- constructs. The interplay of the UCA/PCA and the sPLA2/NHE-1 acidification pathways of the skin and the impact of FLG insufficiency on skin lipid composition and organization in reconstructed skin are described.

Cytoprotective effects of opioids on irradiated oral epithelial cells.

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.

Reconstructed skin models as emerging tools for drug absorption studies.

INTRODUCTION: 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. AREAS COVERED: 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. EXPERT OPINION: 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.

Formulation development of lyophilized, long-term stable siRNA/oligoaminoamide polyplexes.

Polyplexes based on precise oligoaminoamides exhibited promising results in non-viral siRNA delivery. However, one serious limitation is insufficient stability of polyplexes in liquid, which raises the demand for lyophilized, long-term stable formulations. Two different siRNA/oligoaminoamide polyplexes were prepared. Freeze-thaw experiments were performed, in order to test various formulations containing sucrose, trehalose, lactosucrose, and hydroxypropyl-ß-cyclodextrin for their cryoprotective potential and to investigate the influence of the oligoaminoamide structure on particle stability. Selected formulations were lyophilized and tested for storage stability up to 6 months. Moreover, reconstitution of the lyophilisates in reduced volume as a technique to prepare higher concentration formulations was studied. Samples were analyzed for particle size, gene silencing, cytotoxicity, turbidity, subvisible particles, osmolarity, residual moisture content, glass transition temperature, and morphology. Depending on the oligoaminoamide, siRNA polyplexes maintained particle size and gene silencing efficiency in the absence or presence of low amounts (7%) of stabilizers after freeze-thawing, lyophilization, and reconstitution. Particle stability was highly dependent on the oligoaminoamide used, but independent of the presence of cysteines that form intra-particular disulfide bridges. In contrast to all other excipients, hydroxypropyl-ß-cyclodextrin did not provide sufficient stability. For lyophilized 5%/10% sucrose and 7% lactosucrose formulations, long-term stability was demonstrated at 40 °C with retained particle size, retained gene silencing activity, unchanged turbidity values, low numbers of subvisible particles, low residual moisture level, and sufficiently high glass transition temperature. Hence, this work is a promising approach in order to provide long-term stable siRNA polyplex formulations that are ready to use after a simple reconstitution step.

Targeting inflammation and wound healing by opioids.

Opioid receptors are expressed on peripheral sensory nerve endings, cutaneous cells, and immune cells; and local application of opioids is used for the treatment of inflammatory pain in arthritis, burns, skin grafts, and chronic wounds. However, peripherally active opioids can also directly modulate the inflammatory process and wound healing. Here, we discuss the underlying mechanisms of opioid action and the conceivable therapeutic approaches for opioid treatment, as investigated in experimental and clinical studies. A large number of in vitro experiments and animal model investigations have produced evidence that peripherally active opioids can reduce plasma extravasation, vasodilation, proinflammatory neuropeptides, immune mediators, and tissue destruction. In contrast to currently available anti-inflammatory agents, opioids have not demonstrated organ toxicity, thus making them interesting candidates for drug development. Few clinical studies have tapped into this potential to date.

Polyglycerol coatings of glass vials for protein resistance.

Proteins are surface active molecules which undergo non-specific adsorption when getting in contact with surfaces such as the primary packaging material. This process is critical as it may cause a loss of protein content or protein aggregation. To prevent unspecific adsorption, protein repellent coatings are of high interest. We describe the coating of industrial relevant borosilicate glass vials with linear methoxylated polyglycerol, hyperbranched polyglycerol, and hyperbranched methoxylated polyglycerol. All coatings provide excellent protein repellent effects. The hyperbranched, non-methoxylated coating performed best. The protein repellent properties were maintained also after applying industrial relevant sterilization methods (≥200 °C). Marginal differences in antibody stability between formulations stored in bare glass vials and coated vials were detected after 3 months storage; the protein repellent effect remained largely stable. Here, we describe a new material suitable for the coating of primary packaging material of proteins which significantly reduces the protein adsorption and thus could present an interesting new possibility for biomedical applications.

A thermosensitive morphine-containing hydrogel for the treatment of large-scale skin wounds.

PURPOSE: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Surfactant free preparation of biodegradable dendritic polyglycerol nanogels by inverse nanoprecipitation for encapsulation and release of pharmaceutical biomacromolecules.

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.

Comparison of four different particle sizing methods for siRNA polyplex characterization.

The ability to reliably determine the size of siRNA polyplexes is the key for the rational design of particles and their formulation, as well as, their safe application in vivo. At the moment, no standard technique for size measurements is available. Each method has different underlying principles and hence may give different results. Here, four different analytical methods were evaluated for their suitability to analyze the characteristics of homogeneous and heterogeneous siRNA polyplexes: dynamic light scattering (DLS), atomic force microscopy (AFM), nanoparticle trafficking analysis (NTA), and fluorescence correlation spectroscopy (FCS). Three different siRNA polyplex compositions generated with different, precise, and hydrophobically modified oligoaminoamides were used in this study. All of the evaluated methods were suitable for analysis of medium sized, homogeneous siRNA polyplexes (~120 nm). Small particles (<40 nm) could not be tracked with NTA, but with the other three methods. Heterogeneous polyplexes were generally difficult to analyze. Only by visualization with AFM, the heterogeneity of those polyplexes was observable. FCS was the only method suitable for measuring polyplex stability in 90% fetal bovine serum. Physico-chemical characteristics of polyplexes are important quality criterions for successful in vivo application and future formulation development. Therefore, a comprehensive analysis by more than one method is of particular importance.

Lyophilization of synthetic gene carriers.

Lyophilization, also known as freeze-drying, is a widely used method for stabilization, improvement of long-term storage stability, and simplification of the handling of drugs and/or carrier systems. Lyophilization is time- and energy-consuming; hence, optimized processes are required to avoid time loss and higher costs without compromising product stability. Since the last decade nonviral, synthetic carriers for gene delivery are of increasing interest. However, these systems suffer from poor physical stability in aqueous solution or suspension. Hence, to ensure long-term storage stability lyophilization of the gene carrier systems is favored. Though, lyophilized products retrieving original carrier size and transfection efficiency after reconstitution are mandatory. This chapter gives an overview of the basic steps and troubleshooting for successful lyophilization of synthetic gene carriers. Furthermore the required excipients and their mechanism of action are summarized.

Morphine stimulates cell migration of oral epithelial cells by delta-opioid receptor activation.

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.

Non-analgesic effects of opioids: peripheral opioid effects on inflammation and wound healing.

Opioids are the gold standard for pain treatment but systemic opioid use is accompanied by central and intestinal side effects. As opioid receptors are expressed on peripheral sensory nerve endings, cutaneous and immune cells, local opioid application is being used for pain reduction in patients with inflammatory lesions such as burns, skin grafts, arthritis, acute or chronic wounds. In addition, peripherally active opioids have anti-inflammatory effects and can modulate wound healing. This review will cover anatomical, physiological and pathophysiological characteristics of opioid receptors and their ligands in peripheral tissues. We will then focus on mechanisms and the functional role of peripheral opioids in inflammation and wounds in experimental and clinical studies. Controversial results, methodological issues, implications for pharmacology, and therapeutic prospects for inflammatory diseases and wound healing will be discussed.

Hallmarks of atopic skin mimicked in vitro by means of a skin disease model based on FLG knock-down.

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.

3D-wound healing model: influence of morphine and solid lipid nanoparticles.

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 180 nm), and tested improvement of wound closure in a new human-based 3D-wound healing model. Standardised wounds were induced by CO(2)-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.

SLN for topical application in skin diseases--characterization of drug-carrier and carrier-target interactions.

The modes of drug-particle interactions considerably influence drug delivery by nanoparticulate carrier systems and drug penetration into the skin. The exact mechanism of the drug loading and its release are still ambiguous. Therefore, the loading process, the interaction of the agent and the lipid matrix of solid lipid nanoparticles (SLNs) as well as the uptake of the loaded agent by skin lipids were analysed by electron spin resonance (ESR) and parelectric spectroscopy (PS) using spin probes (TEMPO, TEMPOL, and CAT-1) as model drugs differing in their lipophilicity. The spin probes were closely attached to the particles lipid surface (TEMPO) or located in the layers of the surfactant (CAT-1), respectively. Furthermore, two distinct sub-compartments on the SLN were found. To simulate the processes at the phase boundary SLN dispersion/skin, skin lipid mixtures were prepared and the transfer process of the spin labels was followed by ESR tomography. Transfer rates were related to the lipophilicity of the spin probe, the lipid mixture and the applied pharmaceutical formulation, SLN dispersion and aqueous solution, respectively. In particular, SLN accelerated in particular the distribution of the lipophilic agents.

Influence of nanocarrier type and size on skin delivery of hydrophilic agents.

The nanoparticulate carrier systems solid lipid nanoparticles (SLN) and dendritic core-multishell (CMS) nanotransporters gained interest for the topical treatment of skin diseases as they facilitate the skin penetration of loaded lipophilic drugs. Here, we studied if these carrier systems are also suitable drug delivery systems for more hydrophilic agents using the dye rhodamin B as model compound. Furthermore, the influence of the particle size on the skin penetration was investigated. Loading rhodamin B onto SLN (250-340 nm) and CMS nanotransporters (20-30 nm), the dye amount increased significantly in viable epidermis and dermis as compared to a conventional cream. CMS nanotransporters were most efficient. Creating nanoparticles of 50-200 nm demonstrated only marginal size effect for the skin penetration. Therefore, the superiority of the CMS nanotransporters seems to be attributed to the character of the nanoparticles and not to its smaller size.

Influences of opioids and nanoparticles on in vitro wound healing models.

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-beta1 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.