RESUMEN
The topical photodynamic therapy (PDT) is mainly used in the treatment of dermato-oncological diseases. The distribution and functionality of the photosensitizer Tetrahydroporphyrin-Tetratosylat (THPTS) was investigated using microscopic and spectroscopic methods after topical application to excised porcine skin followed by irradiation. The distribution of THPTS was determined by two-photon tomography combined with fluorescence lifetime imaging (TPT/FLIM) and confocal Raman microspectroscopy (CRM). The radicals were quantified and characterized by electron paramagnetic resonance (EPR) spectroscopy. Results show a penetration depth of THPTS into the skin down to around 12 ± 5 µm. A penetration of THPTS through the stratum corneum was not clearly observable after 1 h penetration time, but cannot be excluded. The irradiation within the phototherapeutic window (spectral range of visible and near infrared light in the range ≈ 650-850 nm) is needed to activate THPTS. An incubation time of 10 min showed the highest radical production. A longer incubation time affected the functionality of THPTS, whereby significant less radicals were detectable. During PDT mainly reactive oxygen species (ROS) and lipid oxygen species (LOS) are produced. Overall, the irradiation dose per se influences the radical types formed in skin. While ROS are always prominent at low doses, LOS increase at high doses, independent of previous skin treatment and the irradiation wavelength used.
Asunto(s)
Fármacos Fotosensibilizantes/farmacocinética , Porfirinas/farmacocinética , Especies Reactivas de Oxígeno/metabolismo , Piel/metabolismo , Administración Cutánea , Animales , Rayos Infrarrojos , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/administración & dosificación , Porfirinas/administración & dosificación , Especies Reactivas de Oxígeno/análisis , Análisis Espacio-Temporal , Porcinos , Factores de Tiempo , Distribución Tisular/efectos de la radiaciónRESUMEN
The sun protection factor (SPF) is related to the selected UV filters. The objective of this study was to evaluate and compare the rheological behavior and texture profile of two sunscreen formulations and to correlate these data with the obtained SPF values. Two formulations (F1 and F2) were developed with the same type and amount of UV filters - whereby one of them also contained ethoxylated lanolin as additional film former (F2). Their rheological behavior, texture profile and in vitro and in vivo SPF were analyzed. The film-forming properties were evaluated by skin profilometry and diffuse reflectance spectroscopy. The structures of the formulations were examined by two-photon tomography combined with fluorescence lifetime imaging, and the penetration profile into the stratum corneum was investigated by tape stripping. The formulation with lanolin presented lower and constant values for physical-mechanical parameters, with a higher and better reproducible SPF. Both formulations did not penetrate the viable epidermis. In conclusion, formulations with better surface deposition on the skin surface can influence the film formation and, consequently, improve the SPF. These findings are important to improve the efficacy of sunscreen formulations and reduce the addition of UV filters.
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Factor de Protección Solar , Protectores Solares , Piel , Análisis Espectral , Rayos UltravioletaRESUMEN
The exposure to sun radiation is indispensable to our health; however, a long-term and high exposure could lead to cell damage, erythema, premature skin aging, and promotion of skin tumors. An underlying pathomechanism is the formation of free radicals which may induce oxidative stress at elevated concentrations. Different skin models, such as porcine-, murine-, human- ex vivo skin, reconstructed human skin (RHS) and human skin in vivo, were investigated during and after irradiation using X- and L-band EPR spectroscopy within different spectral regions (UVC to NIR). The amount of radical formation was quantified with the spin probe PCA and the radical types were measured ex vivo with the spin trap DMPO. The radiation dose influences the types of radicals formed in the skin. While reactive oxygen species (ROS) are always pronounced at low doses, there is an increase in lipid oxygen species (LOS) at high doses. Furthermore, the radical types arise independent from the irradiation wavelength, whereas the general amount of radical formation differs with the irradiation wavelength. Heat pre-stressed porcine skin already starts with higher LOS values. Thus, the radical type ratio might be an indicator of stress and the reversal of ROS/LOS constitutes the point where positive stress turns into negative stress.Compared to light skin types, darker types produce less radicals in the ultraviolet, similar amounts in the visible and higher ones in the infrared spectral region, rendering skin type-specific sun protection a necessity.
RESUMEN
Ultraviolet (UV) radiation leads to the formation of free radicals, which may cause immunological modulations, skin aging or skin cancer. Sunlight exposure in the UVA region according to CIE 85 promotes almost 46% of radical formation in skin. A critical radical concentration characterized by the inversion of the domination of primary ROS (reactive oxygen species) to an excess of secondary LOS (lipid oxygen species) is proven for the spectral regions UV and or VIS light and is intended to be a marker for an imbalance in the redox system, which can no longer compensate harmful effects. To investigate whether this transition point is also universally valid for one spectral region, the radical formation during and after targeted UVA in situ-irradiation at 365 ± 5 nm and three different irradiances (31, 94 and 244 mW/cm2) was investigated in ex vivo porcine skin using x-band electron paramagnetic resonance (EPR) spectroscopy. The quantification was performed with the spin probe 3-(carboxy)-2,2,5,5-tetramethylpyrrolidin-1-oxyl (PCA), the spin trap 5,5-Dimethyl-1-Pyrroline-N-Oxide (DMPO) was used to characterize the radical species. Furthermore, the viability of the skin cells after irradiation was controlled by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, skin integrity was examined by histological analysis. A significant dose dependence in the radical formation is given at higher irradiance. The transition point was detected in the range of 0.5 MED after irradiation with the highest irradiance. From this point on the proportion of LOS increases with increasing dose and the proportion of ROS decreases. After switching off the UVA irradiation no further quantitative changes were detected, but rapid changes in the radical pattern were observed demonstrating the importance of in situ irradiation during the use of spin traps. Heat-pre-stressed skin showed more LOS than ROS already at the beginning of the irradiation, leading to the assumption that the transition point to the distress-level has already been reached. In summary, a postulated transition point could be verified for the UVA spectral region using only one spin trap combined with in-situ irradiation. A certain degree of stress is necessary to detect an inversion of the ratio of ROS to LOS. This reversal indicates an imbalance in the redox status. However, at low intensities no changes at all in radical pattern appeared over time (dose), probably it can be compensated by adaptation processes of the skin.
Asunto(s)
Estrés Oxidativo , Rayos Ultravioleta , Animales , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Especies Reactivas de Oxígeno , Porcinos , Rayos Ultravioleta/efectos adversosRESUMEN
BACKGROUND AND OBJECTIVES: In the oral cavity, the mucosal tissues may develop a number of different pathological conditions, such as inflammatory diseases (gingivitis, periodontitis) and autoimmune disorders (eg, oral lichen planus) that require therapy. The application of topical drugs is one common therapeutic approach. However, their efficacy is limited. Dilution effects due to saliva hinder the adherence and the penetration of drug formulations. Therefore, the bioavailability of oral topical drugs is insufficient, and patients may suffer from disease over years, if not life-long. MATERIAL AND METHODS: In the present study, we characterized core-multishell (CMS) nanocarriers for their potential use as drug delivery systems at oral mucosal tissues. For this purpose, we prepared porcine masticatory as well as buccal mucosa and performed Franz cell diffusion experiments. Penetration of fluorescently labeled CMS nanocarriers into the mucosal tissue was analyzed using confocal laser scanning microscopy. Upon exposure to CMS nanocarriers, the metabolic and proliferative activity of gingival epithelial cells was determined by MTT and sulforhodamine B assays, respectively. RESULTS: Here, we could show that the carriers penetrate into both mucosal tissues, while particles penetrate deeper into the masticatory mucosa. Electron paramagnetic resonance spectroscopy revealed that the 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy-labeled glucocorticoid dexamethasone loaded on to the CMS nanocarriers was released from the carriers in both mucosal tissues but with a higher efficiency in the buccal mucosa. The release from the nanocarriers is in both cases superior compared to the release from a conventional cream, which is normally used for the treatment of inflammatory conditions in the oral cavity. The CMS nanocarriers exhibited neither cytotoxic nor proliferative effects in vitro. CONCLUSION: These findings suggested that CMS nanocarriers might be an innovative approach for topical drug delivery in the treatment of oral inflammatory diseases.
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Dexametasona/administración & dosificación , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Glucocorticoides/administración & dosificación , Mucosa Bucal/efectos de los fármacos , Nanopartículas , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dexametasona/farmacocinética , Células Epiteliales/efectos de los fármacos , Encía/citología , Glucocorticoides/farmacocinética , Espectroscopía de Resonancia Magnética , Microscopía ConfocalRESUMEN
Highly hydrophilic, responsive nanogels are attractive as potential systems for the topical delivery of bioactives encapsulated in their three-dimensional polymeric scaffold. Yet, these drug carrier systems suffer from drawbacks for efficient delivery of hydrophobic drugs. Addressing this, ß-cyclodextrin (ßCD) could be successfully introduced into the drug carrier systems by exploiting its unique affinity toward dexamethasone (DXM) as well as its role as topical penetration enhancer. The properties of ßCD could be combined with those of thermoresponsive nanogels (tNGs) based on dendritic polyglycerol (dPG) as a crosslinker and linear thermoresponsive polyglycerol (tPG) inducing responsiveness to temperature changes. Electron paramagnetic resonance (EPR) studies localized the drug within the hydrophobic cavity of ßCD by differences in its mobility and environmental polarity. In fact, the fabricated carriers combining a particulate delivery system with a conventional penetration enhancer, resulted in an efficient delivery of DXM to the epidermis and the dermis of human skin ex vivo (enhancement compared to commercial DXM cream: â¼2.5 fold in epidermis, â¼30 fold in dermis). Furthermore, DXM encapsulated in ßCD tNGs applied to skin equivalents downregulated the expression of proinflammatory thymic stromal lymphopoietin (TSLP) and outperformed a commercially available DXM cream.
RESUMEN
Exogenous factors can cause an imbalance in the redox state of biological systems, promoting the development of oxidative stress, especially reactive oxygen species (ROS). To monitor the intensity of ROS production in secondary keratinocytes (HaCaT) by diesel exhaust particles and thermoresponsive nanogels (tNG), electron paramagnetic resonance (EPR) spectroscopy after 1 and 24 h of incubation, respectively, was applied. Their cytotoxicity was analyzed by a cell viability assay (XTT). For tNG an increase in the cell viability and ROS production of 10% was visible after 24 h, whereas 1 h showed no effect. A ten times lower concentration of diesel exhaust particles exhibited no significant toxic effects on HaCaT cells for both incubation times, thus normal adult human keratinocytes (NHK) were additionally analyzed by XTT and EPR spectroscopy. Here, after 24 h a slight increase of 18% in metabolic activity was observed. However, this effect could not be explained by the ROS formation. A slight increase in the ROS production was only visible after 1 h of incubation time for HaCaT (9%) and NHK (14%).
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Espectroscopía de Resonancia por Spin del Electrón , Nanopartículas/toxicidad , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/análisis , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Nanopartículas/química , Especies Reactivas de Oxígeno/metabolismo , Emisiones de Vehículos/toxicidadRESUMEN
BACKGROUND: A nutrition rich in fruit and vegetables and a healthy lifestyle become more and more important in the industrial countries to counteract oxidative stress and promote health. For many years, it has been possible to control human cutaneous carotenoids noninvasively by resonance Raman spectroscopic systems and by spatially resolved reflectance spectroscopy. METHODS: Ten volunteers took a commercially available fruit and vegetable extract daily for a time period of 5 weeks. A second group served as control group and did not take any supplements (10 volunteers). To monitor the status of the cutaneous carotenoids noninvasively, an optical sensor based on multiple spatially resolved reflectance spectroscopy was applied once a week. RESULTS: The study could demonstrate that the intake of the supplement significantly increase the cutaneous carotenoid values of the young adults by 50%. The control group without any supplementation showed also significantly increased values, ie, by 10%, which might be due to the fact that their lifestyle was controlled. CONCLUSION: The results illustrate that a biofeedback by measuring the skin carotenoids could improve the lifestyle of young adults and that a regular consumption of fruit and vegetables directly or as a drink can increase the concentration of cutaneous carotenoids significantly.
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Carotenoides/metabolismo , Piel/química , Suplementos Dietéticos , Femenino , Frutas , Humanos , Masculino , Extractos Vegetales/administración & dosificación , Espectrometría Raman , Verduras , Adulto JovenRESUMEN
The skin and especially the stratum corneum (SC) act as a barrier and protect epidermal cells and thus the whole body against xenobiotica of the external environment. Topical skin treatment requires an efficient drug delivery system (DDS). Polymer-based nanocarriers represent novel transport vehicles for dermal application of drugs. In this study dendritic core-multishell (CMS) nanoparticles were investigated as promising candidates. CMS nanoparticles were loaded with a drug (analogue) and were applied to penetration studies of skin. We determined by dual-frequency electron paramagnetic resonance (EPR) how dexamethasone (Dx) labelled with 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA) is associated with the CMS. The micro-environment of the drug loaded to CMS nanoparticles was investigated by pulsed high-field EPR at cryogenic temperature, making use of the fact that magnetic parameters (g-, A-matrices, and spin-lattice relaxation time) represent specific probes for the micro-environment. Additionally, the rotational correlation time of spin-labelled Dx was probed by continuous wave EPR at ambient temperature, which provides independent information on the drug environment. Furthermore, the penetration depth of Dx into the stratum corneum of porcine skin after different topical applications was investigated. The location of Dx in the CMS nanoparticles is revealed and the function of CMS as penetration enhancers for topical application is shown.
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Dexametasona/química , Dexametasona/metabolismo , Nanopartículas/química , Nanopartículas/metabolismo , Piel/metabolismo , Administración Cutánea , Animales , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Polímeros/química , Polímeros/metabolismo , Absorción Cutánea/efectos de los fármacos , Marcadores de Spin , PorcinosRESUMEN
Dendritic core-multi shell (CMS) particles are polymer based systems consisting of a dendritic polar polyglycerol polymer core surrounded by a two-layer shell of nonpolar C18 alkyl chains and hydrophilic polyethylene glycol. Belonging to nanotransport systems (NTS) they allow the transport and storage of molecules with different chemical characters. Their amphipihilic character CMS-NTS permits good solubility in aqueous and organic solutions. We showed by multifrequency electron paramagnetic resonance (EPR) spectroscopy that spin-labeled 5-doxyl stearic acid (5DSA) can be loaded into the CMS-NTS. Furthermore, the release of 5DSA from the carrier into the stratum corneum of porcine skin was monitored ex vivo by EPR spectroscopy. Additionally, the penetration of the CMS-NTS into the skin was analyzed by fluorescence microscopy using indocarbocyanine (ICC) covalently bound to the nanocarrier. Thereby, no transport into the viable skin was observed, whereas the CMS-NTS had penetrated into the hair follicles down to a depth of 340 µm ± 82 µm. Thus, it could be shown that the combined application of fluorescence microscopy and multi-frequency EPR spectroscopy can be an efficient tool for investigating the loading of spin labeled drugs to nanocarrier systems, drug release and penetration into the skin as well as the localization of the NTS in the skin.