To describe the action mechanisms of Bacteriochlorin a (BCA), a second generation photosensitizer, in phosphate buffer (PB) and in dimyristoyl phosphatidylcholine (DMPC) liposomes we carried out oxygen consumption and ESR measurements. In PB, where BCA was in a monomer-dimer equilibrium, our results suggested that the oxygen consumption was related to the BCA monomers concentration in solution. Incorporation of BCA in DMPC liposomes, by promoting the monomerization of BCA, increased 9-fold the oxygen consumption in comparison to the value in PB. The use of specific singlet oxygen quenchers (Azide and 9, 10-Anthracenedipropionic acid) in ESR and oxygen consumption experiments allowed us to assert that BCA was mainly a type II sensitizer when it was incorporated in DMPC. Finally, the cell survival of WiDr cells after a PDT treatment was measured for cells incubated with BCA in cell culture medium and cells incubated with BCA in DMPC. Irrespective of the dye concentration, the cell survival was lower when liposomes were used. This effect could be the result of a better BCA monomerization and / or a different BCA uptake in cells.

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.

Photodynamic therapy with bacteriochlorin a (BCA) as sensitizer induces damage to red blood cells in vivo. To assess the extent of the contributuion of reactive oxygen species (ROS) and to determine a possible reaction mechanism, competition experiments with assorted ROS quenching or/and enhancing agents were performed in human erythrocytes as model system and in phosphate buffer. In the erythrocyte experiments, a 2% suspension was incubated with BCA for 1 h, washed with phosphate-buffered saline, resuspended and subsequently illuminated with a diode laser using a fluence rate of 2.65 mW/cm2. Potassium leakage and hemolysis were light and BCA dose dependent. Adding tryptophan (3.3 mM), azide (1 mM) or histidine (10 mM) to the erythrocyte suspension before illumination delayed the onset of K-leakage and hemolysis suggesting a type II mechanism. The D2O did not affect K-leakage nor photohemolysis. Adding mannitol (13.3 mM) or glycerol (300 nM) also caused a delay in the onset of K-leakage and hemolysis, suggesting the involvement of radicals. In phosphate buffer experiments, it was shown using electron spin resonance (ESR) associated with spin-trapping techniques that BCA is able to generate O2-. and OH. radicals without production of aqueous electron. Visible or UV irradiation of the dye in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct DMPO-OH. Addition of ethanol or sodium formate produced supplementary hyperfine splittings due to the respective CH3CHOH. and CO2-. radical adducts, indicating the presence of free OH.. Production of DMPO-OH was partly inhibited by superoxide dismutase (SOD), catalase and desferrioxamine, suggesting that the iron-catalyzed decomposition of H2O2 was partly involved in the formation of one part of the observed OH.. The complementary inhibition of DMPO-OH production by azide and 9,10-anthracenedipropionic acid (ADPA) was consistent with 1O2 production by BCA followed by reaction of 1O2 with DMPO and decay of the intermediate complex to form DMPO-OH and free OH.. All our results seem to indicate that BCA is a 50%/50% type 1/type 2 sensitizer in buffered aqueous solutions and confirmed that the dye-induced hemolysis of erythrocytes was cell caused by a mixed type 1/type 2 mechanism.

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(4methylpyridinium)-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 concentrationdependent uptake by T. rubrum was established.

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.

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.

No abstract

Porphyrin Tri-P(4) may be a suitable photosensitizer for sterilization of RBCs. However, further exploration to optimize the method is necessary to reach clinically acceptable goals.