In Vitro Antimicrobial Photodynamic Therapy Against Trichophyton mentagrophytes Using New Methylene Blue as the Photosensitizer. / Terapia fotodinámica antimicrobiana in vitro aplicada sobre Trichophyton mentagrophytes con nuevo azul de metileno como fotosensibilizador.

INTRODUCTION AND OBJECTIVES: Antimicrobial photodynamic therapy combines the use of a photosensitizing drug with light and oxygen to eradicate pathogens. Trichophyton mentagrophytes is a dermatophytic fungus able to invade the skin and keratinized tissues. We have investigated the use of new methylene blue as the photosensitizing agent for antimicrobial photodynamic therapy to produce the in vitro inactivation of T mentagrophytes. MATERIAL AND METHODS: A full factorial design was employed to optimize the parameters for photoinactivation of the dermatophyte. The parameters studied were new methylene blue concentration, contact time between the photosensitizing agent and the fungus prior to light treatment, and the fluence of red light (wavelength, 620-645nm) applied. RESULTS: The minimum concentration of new methylene blue necessary to induce the death of all T. mentagrophytes cells in the initial suspension (approximate concentration, 106 colony forming units per milliliter) was 50µM for a fluence of 81J/cm2 after a contact time of 10minutes with the photosensitizing-agent. Increasing the concentration to 100µM allowed the fluence to be decreased to 9J/cm2. CONCLUSIONS: Comparison of our data with other published data shows that the susceptibility of T. mentagrophytes to antimicrobial photodynamic therapy with new methylene blue is strain-dependent. New methylene blue is a photosensitizing agent that should be considered for the treatment of fungal skin infections caused by this dermatophyte.

Photodynamic fungicidal efficacy of hypericin and dimethyl methylene blue against azole-resistant Candida albicans strains.

Antimicrobial photodynamic therapy (aPDT) is an emerging alternative to treat infections based on the use of photosensitisers (PSs) and visible light. To investigate the fungicidal effect of PDT against azole-resistant Candida albicans strains using two PSs with a different mechanism of action, hypericin (HYP) and 1,9-dimethyl methylene blue (DMMB), comparing their efficacy and the reactive oxygen species (ROS) species involved in their cytotoxicity. Azole-resistant and the azole-susceptible C. albicans strains were used. Solutions of 0.5 and 4 McFarland inoculum of each Candida strain were treated with different concentrations of each PS, and exposed to two light-emitting diode light fluences (18 and 37 J cm⁻²). Mechanistic insight was gained using several ROS quenchers. The minimal fungicidal concentration of HYP for ≥3 log10 CFU reduction (0.5 McFarland) was 0.62 µmol l⁻¹ for most strains, whereas for DMMB it ranged between 1.25 and 2.5 µmol l⁻¹. Increasing the fluence to 37 J cm⁻² allowed to reduce the DMMB concentration. Higher concentrations of both PSs were required to reach a 6 log10 reduction (4 McFarland). H2O2 was the main phototoxic species involved in the fungicidal effect of HYP-aPDT whereas ¹O2 was more important for DMMB-based treatments. aPDT with either HYP or DMMB is effective in killing of C. albicans strains independent of their azole resistance pattern. HYP was more efficient at low fungal concentration and DMMB at higher concentrations.

Singlet molecular oxygen quenching by the antioxidant dimethylmethoxy chromanol in solution and in ex vivo porcine skin.

Singlet-oxygen is a non-radical reactive oxygen species believed to play a major role in many photooxidation processes in connection with diverse photo-biological processes such as skin ageing or photocarcinogenesis. Dimethylmethoxy chromanol (3,4-dihydro-6-hydroxy-2,2-dimethyl-7-methoxy-1(2H)-benzopyran) is a potent antioxidant used in cosmetic and pharmaceutical formulations. We have assessed the singlet oxygen quenching ability of dimethylmethoxy chromanol, by monitoring the near-IR phosphorescence of singlet-oxygen in solution and in ex vivo porcine skin samples. Dimethylmethoxy chromanol quenches singlet oxygen with a rate constant of (1.3 ± 0.1) × 108 M⁻¹ s⁻¹ in solution. Consistent with this, a clear reduction in the singlet oxygen lifetime and emission intensity was observed when ex vivo porcine skin samples were treated with dimethylmethoxy chromanol.

Riboflavin applications to grapevine leaves and berries blue-light post-harvest treatments modifies grape anthocyanins and amino acids contents.

Light is an energy source and key environmental factor for plants. Out of the different light wavelengths, blue-light is one of the most relevant spectral regions because of its relation to anthocyanins biosynthesis. Among the compounds present in grapes, anthocyanins determine their main organoleptic and healthy properties; while a minimum concentration of ammonium and amino acids is necessary for a desirable development of the alcoholic fermentation. Moreover, amino acids are precursors of several volatile compounds synthetized during the fermentation. The aim of this study was to assess the influence of riboflavin (vitamin B2) applications, at harvest and one week later,to grapevine leaves in combination with post-harvest blue-light irradiation on Tempranillo (Vitis vinifera L.) grape anthocyanins and amino acidscomposition. The combination of blue-light irradiation and two riboflavin doses as well as theseindividual factors affected both grape anthocyanins and amino acidsconcentrations. After one week of storage, anthocyanins concentration diminished when clusters were irradiated with blue-light; while for amino acids content, the trend to increase or decrease is dependent on the riboflavin dose applied in vines and the storage time.

Porphycenes: facts and prospects in photodynamic therapy of cancer.

The photodynamic process induces cell damage and death by the combined effect of a photosensitizer (PS), visible light, and molecular oxygen, which generate singlet oxygen ((1)O(2)) and other reactive oxygen species that are responsible for cytotoxicity. The most important application of this process with increasing biomedical interest is the photodynamic therapy (PDT) of cancer. In addition to hematoporphyrin-based drugs, 2nd generation PSs with better photochemical properties are now studied using cell cultures, experimental tumors and clinical trials. Porphycene is a structural isomer of porphyrin and constitutes an interesting new class of PS. Porphycene derivatives show higher absorption than porphyrins in the red spectral region (lambda > 600 nm, epsilon > 50000 M-(1)cm(-1)) owing to the lower molecular symmetry. Photophysical and photobiological properties of porphycenes make them excellent candidates as PSs, showing fast uptake and diverse subcellular localizations (mainly membranous organelles). Several tetraalkylporphycenes and the tetraphenyl derivative (TPPo) induce photodamage and cell death in vitro. Photodynamic treatments of cultured tumor cells with TPPo and its palladium(II) complex induce cytoskeletal changes, mitotic blockage, and dose-dependent apoptotic or necrotic cell death. Some pharmacokinetic and phototherapeutic studies on experimental tumors after intravenous or topical application of lipophilic alkyl-substituted porphycene derivatives are known. Taking into account all these features, porphycene PSs should be very useful for PDT of cancer and other biomedical applications.

Tuning the local solvent composition at a drug carrier surface: the effect of dimethyl sulfoxide/water mixture on the photofunctional properties of hypericin-ß-lactoglobulin complexes.

Aggregation is a major problem for the anti-microbial photodynamic applications of hydrophobic photosensitizers since it strongly reduces the amount of singlet oxygen generated in aqueous solutions. Binding of hypericin (Hyp) to the milk whey protein ß-lactoglobulin (ßLG), occurring at the two hydrophobic cavities located at the interface of the protein homodimer, can be exploited to confer water-solubility and biocompatibility to the photosensitizer. The introduction of a small amount of the organic cosolvent dimethyl sulfoxide (DMSO) leads to a remarkable improvement of the photophysical properties of the complex Hyp-ßLG by increasing its fluorescence emission and singlet oxygen photosensitization quantum yields. Surprisingly, the ability of the complex to photo-inactivate bacteria of the strain Staphylococcus aureus is strongly reduced in the presence of DMSO, despite the higher yield of photosensitization. The reasons for this apparently contradictory behavior are investigated, providing new insights into the use of carrier systems for hydrophobic photosensitizers.

Necrotic cell death induced by photodynamic treatment of human lung adenocarcinoma A-549 cells with palladium(II)-tetraphenylporphycene.

In this study we describe photodamaging and photokilling effects of palladium(II)-tetraphenylporphycene (PdTPPo) (previously incorporated into dipalmitoylphosphatidylcholine liposomes) on the human lung adenocarcinoma A-549 cell line. No dark cytotoxicity was found when the drug was applied at 10(-6) M or 5 x 10(-7) M for 1 or 18 h, respectively. After 1-h treatment with 10(-7) M or 5 x 10(-7) M PdTPPo followed by red light irradiation for variable times, dose-dependent lethal effects were observed in A-549 cells. Apoptosis was not found after the above photodynamic treatments or under even milder sublethal conditions. In contrast to HeLa cells subjected to PdTPPo photosensitization where either apoptosis or necrosis were induced, morphological analysis and electrophoretical DNA pattern of A-549 cells always revealed a clearly necrotic death mechanism. However, A-549 cells died by apoptosis after serum and L-glutamine deprivation, indicating that only the photodynamically induced apoptosis was inhibited. Immunofluorescent labeling revealed that microtubules and actin microfilaments were immediately and strongly damaged by photodynamic treatments with PdTPPo. No metaphase arrest and/or mitotic alterations were observed after phototreatments. Present results show that the cell type plays a fundamental role in relation to the apoptotic or necrotic response to photosensitization, and that cytoskeletal components are important targets implicated in cell death processes.

Quantum yield of production of singlet molecular oxygen (1 delta g) in aqueous dispersions of small unilamellar lipid vesicles. A time-resolved near-IR phosphorescence study.

The quantum yield of formation and kinetic behaviour of O2(1 delta g) in D2O dispersions of small unilamellar vesicles (SUVs) of dipalmitoyl phosphatidylcholine were studied by time-resolved detection of near-IR phosphorescence. At a SUV concentration of 26 nM, O2(1 delta g) is not quenched by the vesicles. It diffuses quickly through the lipid bilayer and a partition equilibrium of O2(1 delta g) between the lipid bilayer and the buffer is attained before decay occurs. In this equilibrium situation O2(1 delta g) is mostly located in the buffer phase, which permits the determination of absolute quantum yields for O2(1 delta g) production, phi delta, by comparison of the luminescence in the dispersions with that in neat D2O. The maximal phi delta values for the sensitizers incorporated in the SUV bilayer were 0.47 +/- 0.09 for the dipyridyl complex of zinc(II) phthalocyanine (ZnPc), 0.35 +/- 0.08 for porphycene, and 0.36 +/- 0.08 for 2,7,12,17-tetra-n-propylporphycene. These values are equal to those in neat organic solvents but lower than those previously obtained in SUVs by using chemical trapping agents. The high degree of organization of the environment around the sensitizers does not influence their efficiency of producing O2(1 delta g). While no concentration dependence is observed for ZnPc (at least up to a local concentration of 20 mM in the bilayer), phi delta for both porphycenes significantly decreases above a local concentration of 4 mM in the bilayer. This result is expected in view of previous observations on the concentration dependence of other photophysical parameters of the porphycenes in such microheterogeneous media.

Photophysical properties of neutral and cationic tetrapyridinoporphyrazines.

We describe the synthesis and photophysical properties of a series of neutral and cationic 3,4-tetrapyridinoporphyrazines, potential lead photosensitizers for photodynamic inactivation of bacteria. Tetracationic TPyPzs exist essentially as monomers in aqueous systems, but the presence of trialkylated compounds due to incomplete quaternization of the outer nitrogen atoms induces severe aggregation. The absorption, fluorescence, triplet, and singlet oxygen quantum yields for both the neutral and cationic compounds are comparable to those of the related phthalocyanines.

Incorporation of hydrophobic porphyrins into liposomes: characterization and structural requirements.

The ability of photosensitisers to give reactive oxygenated products is considered decisive for photodynamic applications, but the hydrophobic nature of many porphyrins makes necessary to obtain suitable pharmaceutical formulations. This paper reports the structural photosensitiser features that allow the preparation of stable liposomal formulations. Metallated and non-metallated TPPs and TPyPs and different lipid/porphyrin ratios were considered in order to procure liposomal preparations containing porphyrin concentrations adequate to necessary doses. The results show that the incorporation of porphyrins into liposomes can be related with their ability to form aggregates in a watery media. Thus, ZnTPP, which structural properties avoid the formation of aggregates, was efficiently incorporated into stable liposomes. Moreover, the efficient generation of singlet oxygen by ZnTPP liposomal suspensions has been shown. Because of this, the synthesis of hydrophobic porphyrin derived structures or other sensitisers, which do not aggregate in a watery media and with Q-bands shifted to higher lambda values than ZnTPP, will be efficiently incorporated into liposomes and useful for clinical applications.

An artificial neural network model for predicting the subcellular localization of photosensitisers for photodynamic therapy of solid tumours.

Photodynamic therapy (PDT) is a promising modality for the treatment of tumours based on the combined action of a photosensitiser (PS), visible light and molecular oxygen, which generates a local oxidative damage that leads to cell death. The site where the primary photodynamic effect takes place depends on the subcellular localization of the PS and affects the mode of action and efficacy of PDT. It is therefore of prime interest to develop structure-subcellular localization prediction models for a PS from its molecular structure and physicochemical properties. Here we describe such a prediction method for the localization of macrocyclic PSs into cell organelles based on a wide set of physicochemical properties and processed through an artificial neural network (ANN). 128 2D-molecular descriptors related to lipophilicity/hydrophilicity, charge and structural features were calculated, then reduced to 76 by using Pearson's correlation coefficient, and finally to 5 using Guyon and Elisseeff's algorithm. The localization of 61 PSs was compiled from literature and distributed into 3 possible cell structures (mitochondria, lysosomes and "other organelles"). A non-linear ANN algorithm was used to process the information as a decision tree in order to solve PS-organelle assignment: first to identify PSs with mitochondrial and/or lysosomal localization from the rest, and to classify them in a second stage. This sequential ANN classification method has permitted to distinguish PSs located into two of the most important cell targets: lysosomes and mitochondria. The absence of false negatives in this assignation, combined with the rate of success in predicting PS localization in these organelles, permits the use of this ANN method to perform virtual screenings of drug candidates for PDT.

Terapia fotodinámica antimicrobiana in vitro aplicada sobre Trichophyton mentagrophytes con nuevo azul de metileno como fotosensibilizador / In vitro antimicrobial photodynamic therapy against Trichophyton mentagrophytes using new methylene blue as the photosensitizer

INTRODUCCIÓN Y OBJETIVOS: La terapia fotodinámica antimicrobiana combina el uso de un fármaco fotosensibilizante, la luz y el oxígeno para erradicar microorganismos patógenos. Trichophyton mentagrophytes es un hongo dermatofito capaz de invadir la piel y tejidos queratinizados. El objetivo de este trabajo es aplicar la terapia fotodinámica antimicrobiana para la inactivación in vitro de T. mentagrophytes utilizando el nuevo azul de metileno como agente fotosensibilizador. MATERIAL Y MÉTODOS: Se aplica un diseño factorial completo para optimizar los parámetros que permiten la fotoinactivación del dermatofito. Se tiene en cuenta la concentración del nuevo azul de metileno, el tiempo de contacto entre el fotosensibilizador y el hongo antes del tratamiento con luz y la fluencia de luz roja aplicada entre 620 y 645 nm. RESULTADOS: La mínima concentración de nuevo azul de metileno que produce una mortalidad de todas las células de T. mentagrophytes de la suspensión inicial (concentración∼106 ufc/ml) es 50 M para una fluencia de 81 J/cm-2 y un tiempo previo de contacto hongo-fotosensibilizador de 10 min. Si se aumenta la concentración a 100 M la fluencia que se necesita disminuye a 9 J/cm-2. CONCLUSIONES: La comparación de nuestros datos con otros publicados muestra que la susceptibilidad de T. mentagrophytes a la terapia fotodinámica antimicrobiana con nuevo azul de metileno es cepa-dependiente. El nuevo azul de metileno es un fotosensibilizador a tener en cuenta para el tratamiento de las micosis cutáneas causadas por este dermatofito

INTRODUCTION AND OBJECTIVES: Antimicrobial photodynamic therapy combines the use of a photosensitizing drug with light and oxygen to eradicate pathogens. Trichophyton mentagrophytes is a dermatophytic fungus able to invade the skin and keratinized tissues. We have investigated the use of new methylene blue as the photosensitizing agent for antimicrobial photodynamic therapy to produce the in vitro inactivation of T mentagrophytes. MATERIAL AND METHODS: A full factorial design was employed to optimize the parameters for photoinactivation of the dermatophyte. The parameters studied were new methylene blue concentration, contact time between the photosensitizing agent and the fungus prior to light treatment, and the fluence of red light (wavelength, 620---645 nm) applied. RESULTS: The minimum concentration of new methylene blue necessary to induce the death of all T. mentagrophytes cells in the initial suspension (approximate concentration, 106 colony forming units per milliliter) was 50 M for a fluence of 81 J/cm2 after a contact time of 10 minutes with the photosensitizing-agent. Increasing the concentration to 100 M allowed the fluence to be decreased to 9 J/cm2. CONCLUSIONS: Comparison of our data with other published data shows that the susceptibility of T. mentagrophytes to antimicrobial photodynamic therapy with new methylene blue is straindependent. New methylene blue is a photosensitizing agent that should be considered for the treatment of fungal skin infections caused by this dermatophyte

Photodamaging effects of tetraphenylporphycene in a human carcinoma cell line.

The photosensitizing effects of tetraphenylporphycene (TPPo) and light on HeLa cells, with emphasis on cell viability and the microtubular network, have been investigated. The survival of the cells incubated with TPPo was dependent on both drug concentration and light dose. The integrity of microtubules (MTs) was evaluated by immunofluorescence staining of alpha-tubulin. Interphasic and mitotic MTs were altered after 30 min of incubation with 5 microM TPPo followed by light irradiation. The degree of damage depended on light exposure time: 3 or 15 min corresponded to survival rates of approximately 50% or < 5%, respectively. Sublethal treatment led to the gradual accumulation of cells in metaphase, which caused an increase in the mitotic index (MI), with a maximum being found 6 h later. The number of cells in metaphase, as well as the MI, were within control values 24 h after sublethal photodynamic treatment. Lethal treatment provoked irreversible damage of interphasic and mitotic MTs. In addition, cell surface modifications such as bleb projections of the plasma membrane were also observed immediately after lethal treatment. It is concluded that both cellular structures, plasma membrane and MTs, constitute important targets for the phototoxic action of TPPo.

Photosensitizing properties of palladium-tetraphenylporphycene on cultured tumour cells.

In this study we describe photokilling properties and effects on the mitotic index (MI) of cultured HeLa cells induced by palladium(II)-tetraphenylporphycene (PdTPP(0)). The drug was synthesized by refluxing tetraphenylporphycene (TPP(0)) and PdCl2 in dimethylformamide, followed by evaporation and purification by chromatography. Cells were treated with different concentrations of PdTPPo incorporated into dipalmitoylphosphatidylcholine liposomes, and red light irradiation (lambda > 600 nm) was performed at 21 mW/cm2. No dark toxicity was found when the drug was applied under our experimental conditions. Using lethal (LD(100)) treatments, cells showed the immediate occurrence of bubbles on the plasma membrane, whereas homogeneous nuclear condensation and loss of cytoplasm appeared 3-24 h later. An increased MI was found 6-8 h after sublethal LD(25) and LD(40) treatments as well as a high proportion of abnormal metaphases with altered spindle microtubules. Chromatin condensation and fragmentation were observed 8 h after LD(75) treatments. These results show that in comparison with TPP(0), the new sensitizer PdTPPo has more efficient photokilling properties which could be very valuable for the photodynamic therapy of cancer.

Uptake of tetraphenylporphycene and its photoeffects on actin and cytokeratin elements of HeLa cells.

In the present work we have continued our studies in the photobiological properties of the 2,7,12,17-tetraphenylporphycene (TPPo). In particular, the uptake, the subcellular localization and the photoeffects on two cytoskeletal elements (actin, microfilaments and cytokeratin intermediate filaments) of HeLa cells have been analyzed. The uptake kinetics of TPPo, determined by fluorescence spectroscopy, was initially very rapid, reaching saturation at approximately 6 h of incubation. This porphycene tends to be accumulated mainly in rounded particles distributed throughout the cytoplasm. The morphological comparison of the localization pattern of TPPo and those of acridine orange and rhodamine 123, which are fluorescence markers for lysosomes and mitochondria respectively, allowed us to confirm that this porphycene is mainly accumulated in lysosomal organelles. The results obtained after treatment with TPPo and red light indicated that this compound is very effective in mediating the photodestruction of lysosomes. The photosensitizing effects on the cytoskeletal elements studied depended on both the irradiation time and the elapsed time after treatment. The implications of damage to lysosomes and actin and cytokeratin filaments on the process of cell death is discussed.