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Onychomycosis is predominantly caused by the dermatophytes Trichophyton rubrum, Trichophyton mentagrophytes and Trichophyton tonsurans. The main treatment obstacle concerns low nail-plate drug permeability. In vitro antifungal photodynamic treatment (PDT) and nail penetration enhancing effectiveness have been proven for multifunctional photosensitizer 5,10,15-tris(4-N-methylpyridinium)-20-(4-(butyramido-methylcysteinyl)-hydroxyphenyl)-[21H,23H]-porphine trichloride (PORTHE). This study investigates single PORTHE green laser/LED PDT of varying degrees of ex vivo onychomycoses in a human nail model. T. mentagrophytes, T. rubrum, T. tonsurans onychomycoses were ex vivo induced on nail pieces at 28 °C (normal air) and 37 °C (6.4% CO2) during 3 to 35 days and PDTs applied to the 37 °C infections. All dermatophytes showed increasingly nail plate invasion at 37 °C between 7 and 35 days; arthroconidia were observed after 35 days for T. mentagrophytes and T. tonsurans. Using 81 J/cm² (532 nm) 7-day T. mentagrophytes onychomycoses were cured (92%) with 80 µM PORTHE (pH 8) after 24 h propylene glycol (PG, 40%) pre-treatment and 35-day onychomycoses (52%-67%) with 24 h PORTHE (40-80 µM)/40% PG treatment (pH 5). 28 J/cm² LED light (525 ± 37 nm) improved cure rates to 72%, 83% and 73% for, respectively, T. mentagrophytus, T. rubrum and T. tonsurans 35-day onychomycoses and to 100% after double PDT. Data indicate PDT relevance for onychomycosis.

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Novel multifunctional photosensitizers (MFPSs), 5,10,15-tris(4-N-methylpyridinium)-20-(4-phenylthio)-[21H,23H]-porphine trichloride (PORTH) and 5,10,15-tris(4-N-methylpyridinium)-20-(4-(butyramido-methylcysteinyl)-hydroxyphenyl)-[21H,23H]-porphine trichloride (PORTHE), derived from 5,10,15-Tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and designed for treatment of onychomycosis were characterized and their functionality evaluated. MFPSs should function as nail penetration enhancer and as photosensitizer for photodynamic treatment (PDT) of onychomycosis. Spectrophotometry was used to characterize MFPSs with and without 532 nm continuous-wave 5 mW cm(-2) laser light (± argon/mannitol/NaN3 ). Nail penetration enhancement was screened (pH 5, pH 8) using water uptake in nails and fluorescence microscopy. PDT efficacy was tested (pH 5, ± argon/mannitol/NaN3 ) in vitro with Trichophyton mentagrophytus microconida (532 nm, 5 mW cm(-2) ). A light-dependent absorbance decrease and fluorescence increase were found, PORTH being less photostable. Argon and mannitol increased PORTH and PORTHE photostability; NaN3 had no effect. PDT (0.6 J cm(-2) , 2 µm) showed 4.6 log kill for PORTH, 4.4 for Sylsens B and 3.2 for PORTHE (4.1 for 10 µm). Argon increased PORTHE, but decreased PORTH PDT efficacy; NaN3 increased PDT effect of both MFPSs whereas mannitol increased PDT effect of PORTHE only. Similar penetration enhancement effects were observed for PORTH (pH 5 and 8) and PORTHE (pH 8). PORTHE is more photostable, effective under low oxygen conditions and thus realistic candidate for onychomycosis PDT.

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Owing to the accessibility of skin to light, many applications of photodynamic treatment (PDT) have been developed within dermatology. The recent increase of dermatological antimicrobial PDT investigations is related to the growing problem of bacterial and fungal resistance to antibiotics. This review focuses on the susceptibility of dermatophytic fungi, in particular Trichophyton rubrum, to PDT and shows its potential usefulness in treatment of clinical dermatophytoses. There are no data indicating significant differences in PDT susceptibility between various dermatophytes and it is unlikely that treatment problems of especially T. rubrum with current antimycotics would occur in case of PDT. Red light 5-aminolevulinic acid-mediated PDT is after repeated sessions successful in in vivo treatment of onychomycosis (fungal nail infection) caused by various dermatophytes. Regarding skin dermatophytoses, UVA-1 PDT with cationic porphyrins appears to be safe and efficient. Most effective toward T. rubrum ex vivo is 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) when combined with UVA-1 radiation or red light; this creates the possibility of efficiently treating nail infections and remaining spores in hair follicles. If the promising in vitro and ex vivo results could be transferred to clinical practice, then PDT has a good prospect to become a worthy alternative to established antifungal drugs.

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Sunscreens are used to provide protection against adverse effects of ultraviolet (UV)B (290-320 nm) and UVA (320-400 nm) radiation. According to the United States Food and Drug Administration, the protection factor against UVA should be at least one-third of the overall sun protection factor. Titanium dioxide (TiO2) and zinc oxide (ZnO) minerals are frequently employed in sunscreens as inorganic physical sun blockers. As TiO2 is more effective in UVB and ZnO in the UVA range, the combination of these particles assures a broad-band UV protection. However, to solve the cosmetic drawback of these opaque sunscreens, microsized TiO2 and ZnO have been increasingly replaced by TiO2 and ZnO nanoparticles (NPs) (<100 nm). This review focuses on significant effects on the UV attenuation of sunscreens when microsized TiO2 and ZnO particles are replaced by NPs and evaluates physicochemical aspects that affect effectiveness and safety of NP sunscreens. With the use of TiO2 and ZnO NPs, the undesired opaqueness disappears but the required balance between UVA and UVB protection can be altered. Utilization of mixtures of micro- and nanosized ZnO dispersions and nanosized TiO2 particles may improve this situation. Skin exposure to NP-containing sunscreens leads to incorporation of TiO2 and ZnO NPs in the stratum corneum, which can alter specific NP attenuation properties due to particle-particle, particle-skin, and skin-particle-light physicochemical interactions. Both sunscreen NPs induce (photo)cyto- and genotoxicity and have been sporadically observed in viable skin layers especially in case of long-term exposures and ZnO. Photocatalytic effects, the highest for anatase TiO2, cannot be completely prevented by coating of the particles, but silica-based coatings are most effective. Caution should still be exercised when new sunscreens are developed and research that includes sunscreen NP stabilization, chronic exposures, and reduction of NPs' free-radical production should receive full attention.

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Today, various anthropogenic sources account for an increasing atmospheric nanoparticle (NP) concentration and thus increase of human exposure to NPs. The situation may become problematic since commercial applications of nanotechnology expand more rapidly than the scientific knowledge on NP exposure. This review focuses on skin as a route of exposure for NPs and the toxicological impact in skin with special attention to physicochemical properties of NPs and skin. We will review data published on NP skin penetration, toxicological issues and on physicochemical NP characterisation. NPs are reported to be localised mainly in hair follicle openings and on the stratum corneum surface. Some studies report the localisation of NPs in the deeper layers of the stratum corneum, the viable epidermis and deeper hair follicle parts. Sporadically, penetration into the dermis is reported for 4 to 5 nm sized quantum dots. NP interactions with epidermal and dermal cells may cause cytotoxicity and undesired immune responses, especially in damaged skin. NP characteristics promoting skin penetration are still unclear. For sunscreen NP substances there are indications for cytotoxicity (TiO2) and genotoxicity (ZnO). Significant data gaps comprise skin penetration and toxicological areas of (metal) particles smaller than 10 nm. The importance of skin barrier function in NP exposure is underlined by NP's skin cell damaging potential. Although NP skin studies display, increasingly, a multidisciplinary character (penetration, toxicity studies) the results are often contradicting. Standardisation of available test systems for NPs and focusing on the correlating physicochemical NP properties to penetration potential is recommended.

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Dermatophytes are fungi that cause infections of keratinized tissues. We have recently demonstrated the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment (PDT) with 5,10,15-Tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) in 5 mm citric acid/sodium citrate buffer (pH 5.2, formulation I). In this work, we examined the penetration of Sylsens B in healthy and with T. rubrum infected skin and we investigated the susceptibility of T. rubrum to PDT using formulation I and UVA-1 radiation (340-550 nm). Skin penetration studies were performed with formulations I and II (Sylsens B in PBS, pH 7.4) applied on dermatomed skin, human stratum corneum (SC), disrupted SC by T. rubrum growth and SC pretreated with a detergent. No penetration was observed in healthy skin. Disruption of SC by preceding fungal growth caused Sylsens B penetration at pH 7.4, but not at pH 5.2. However, chemically damaged SC allowed Sylsens B to penetrate also at pH 5.2. UVA-1 PDT was applied ex vivo during two fungal growth stages of two T. rubrum strains (CBS 304.60 and a clinical isolate). Both strains could be killed by UVA-1 alone (40 J/cm(2)). Combined with formulation I (1 and 10 microm Sylsens B for, respectively, CBS 304.60 and the clinical isolate), only 18 J/cm(2) UVA-1 was required for fungal kill. Therefore, PDT with 10 microm Sylsens B (formulation I) and 18 J/cm(2) UVA-1 could be considered as effective and safe. This offers the possibility to perform clinical studies in future.

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Treatment strategies for superficial mycosis caused by the dermatophyte Trichophyton rubrum consist of the use of topical or oral antifungal preparations. We have recently discovered that T. rubrum is susceptible to photodynamic treatment (PDT), with 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) as a photosensitizer. The susceptibility appeared to depend on the fungal growth stage, with PDT efficacy higher with microconidia when compared to mycelia. The aim of this study was to investigate, with the use of scanning electron microscopy, the morphological changes caused by a lethal PDT dose to T. rubrum when grown on isolated human stratum corneum. Corresponding dark treatment and light treatment without photosensitizer were used as controls. A sub-lethal PDT dose was also included in this investigation The morphologic changes were followed at various time points after the treatment of different fungal growth stages. Normal fungal growth was characterized by a fiber-like appearance of the surface of the hyphae and microconidia with the exception of the hyphal tips in full mycelia and the microconidia shortly after attachment to the stratum corneum. Here, densely packed globular structures were observed. The light dose (108 J/cm2) in the absence of Sylsens B, or the application of the photosensitizer in the absence of light, caused reversible fungal wall deformations and bulge formation. However, after a lethal PDT, a sequence of severe disruptions and deformations of both microconidia and the mycelium were observed leading to extrusion of cell material and emptied fungal elements. In case of a non-lethal PDT, fungal re-growth started on the remnants of the treated mycelium.

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BACKGROUND: Photodynamic treatment (PDT) refers to a treatment with light-activated agents (photosensitizers) in combination with visible light and molecular oxygen. Recently, we have demonstrated that the porphyrins, 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and deuteroporphyrin monomethylester (DP mme) are excellent photosensitizers to be used against Trichophyton rubrum both in vitro and ex vivo. OBJECTIVES AND METHODS: The objective of this study was to investigate the key factors involved in PDT efficacy of both photosensitizers in an ex vivo situation during different fungal growth stages using a recently developed ex vivo model. The study focused on the influence of pH and ion strength of incubation media, photochemical properties of the photosensitizers (spectra and singlet oxygen production), and the effect of several scavengers of reactive oxygen species (sodium azide, histidine, mannitol) and phenylmethylsulphonylfluoride (keratinase inhibitor) on the PDT efficacy. RESULTS AND CONCLUSIONS: The results show that an optimal pH and low concentrations of calcium are crucial for a selective binding of Sylsens B to the fungus, leading to an increased PDT efficacy. This selective binding to T. rubrum cannot be accomplished for DP mme. It can be concluded that the prerequisite for successful treatment is a use of a low molarity solution of pH 5, supplemented with a chelating agent and a keratinase activity-repressing agent. Under these conditions, PDT with Sylsens B inactivates, initially via singlet oxygen, effectively the fungus in different fungal growth stages.

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BACKGROUND: Dermatophytes are fungi that can cause infections of skin, hair and nails because of their ability to feed on keratin. Superficial mycoses are among the most prevalent infectious diseases worldwide. Two important restrictions of current therapeutic options are the recurrence of the infection and prolonged treatment. This is especially true for infections caused by Trichophyton rubrum, a widely distributed dermatophyte. The application of photosensitizers for treatment of fungal infections is, within the field of photodynamic treatment (PDT), relatively new. Recently, we demonstrated that the porphyrins 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and deuteroporphyrin monomethylester (DP mme) were excellent photosensitizers towards T. rubrum when using red light. OBJECTIVES AND METHODS: To evaluate the photodynamic effectiveness of the porphyrins in a situation that mimics the clinical situation, we developed an ex vivo model using human stratum corneum. This model offers the possibility of applying PDT at different time points during the germination and subsequent development of T. rubrum microconidia. The model was used for two different incubation media, Dulbecco's modified Eagle medium (DMEM) and distilled water. RESULTS AND CONCLUSIONS: We demonstrated that the PDT susceptibility of T. rubrum depended on the time of PDT application after spore inoculation. A decrease in susceptibility was observed with increasing time of PDT application for both photosensitizers in DMEM. Changing the incubation medium to distilled water resulted in an increased fungicidal effect for Sylsens B and in a decreased effect for DP mme. We conclude that T. rubrum is susceptible to PDT in a situation that mimics the clinical situation. The fungicidal effect of PDT on fungal spores is of particular importance.

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The application of photosensitizers for the treatment of fungal infections is a new and promising development within the field of photodynamic treatment (PDT). Dermatophytes, fungi that can cause infections of the skin, hair and nails, are able to feed on keratin. Superficial mycoses are probably the most prevalent of infectious diseases in all parts of the world. One of the most important restrictions of the current therapeutic options is the return of the infection and the duration of the treatment. This is especially true in the case of infections of the nail (tinea unguium) caused by Trichophyton rubrum, an anthropophilic dermatophyte with a worldwide distribution. Recently, we demonstrated that 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and deuteroporphyrin monomethylester were excellent photosensitizers toward T. rubrum when using broadband white light. This study demonstrates the photodynamic activity of these photosensitizers with red light toward both a suspension culture of T. rubrum and its isolated microconidia. The higher penetration depth of red light is important for the PDT of nail infections. In addition, we tested the photodynamic activity of a newly synthesized porphyrin, quinolino-[4,5,6,7-efg]-7-demethyl-8-deethylmesoporphyrin dimethylester, displaying a distinct peak in the red part of the spectrum. However, its photodynamic activity with red light toward a suspension culture of T. rubrum appeared to be only fungistatic. Sylsens B was the best photosensitizer toward both T. rubrum and its microconidia. A complete inactivation of the fungal spores and destruction of the fungal hyphae was found. In studies into the photostability, Sylsens B appeared to be photostable under the conditions used for fungal PDT. A promising result of this study is the demonstration of the complete degradation of the fungal hyphae in the time after the PDT and the inactivation of fungal spores, both with red light. These results offer the ingredients for a future treatment of fungal infections, including those of the nail.

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In the past few years, there has been an increase in the application of photosensitizers for medical purposes. A good standardized test system for the evaluation of the mutagenic potentials of photosensitizers is therefore an indispensable device. In the standard Ames test, white light itself was proven to be mutagenic and the result influenced by the light source. Lack of a reliable positive control is another problem in many genotoxicity test systems used for the evaluation of mutagenicity of photosensitizers. Based on the validated somatic mutation and recombination test, known as SMART, and using Drosophila melanogaster, we developed the Photo-SMART and demonstrated that methylene blue, known to induce photomutagenicity, can act as a positive control in the presented test system. The SMART scores for the loss of heterozygosity caused predominantly by homologous mitotic recombination. The Photo-SMART can be used to detect photogenotoxicity caused by short-lived photoproducts or by stable photoproducts or both. We demonstrated the Photo-SMART to be a good standardized test system for the evaluation of mutagenic potentials of the photosensitizer 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (TPP). We demonstrated that TPP was mutagenic using the Photo-SMART. For hematoporphyrin, the results of the Photo-SMART indicate the absence of mutagenicity.

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Dermatophytes are fungi that can cause infections (known as tinea) of the skin, hair and nails because of their ability to use keratin. Superficial mycoses are probably the most prevalent of infectious diseases worldwide. One of the most distinct limitations of the current therapeutic options is the recurrence of the infection and duration of treatment. The present study shows that Trichophyton rubrum in suspension culture is susceptible to photodynamic treatment (PDT), a completely new application in this area. T. rubrum could be effectively killed with the use of the light-activated porphyrins deuteroporphyrin monomethylester (DP mme) and 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B). The photodynamic efficacy was compared with that of some other photosensitizers that are well known in the field of PDT: the porphyrins deuteroporphyrin and hematoporphyrin, the drug Photofrin and several phthalocyanines. It was demonstrated that with the use of broadband white light, the phthalocyanines and Photofrin displayed a fungistatic effect for about 1 week, whereas all the porphyrins caused photodynamic killing of the dermatophyte. Sylsens B was the most effective sensitizer and showed no dark toxicity; therefore, in an appropriate formulation, it could be a promising candidate for the treatment of various forms of tinea. For Sylsens B and DP mme, which displayed the best results, a concentration-dependent uptake by T. rubrum was established.

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