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The photodynamic inactivation mediated by 1,3,5,7-tetramethyl-8-[4-(N,N,N-trimethylamino)phenyl]-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene 3 and 8-[4-(3-(N,N,N-trimethylamino)propoxy)phenyl]-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene 4 was investigated on Staphylococcus aureus, Escherichia coli and Candida albicans. In vitro experiments indicated that BODIPYs 3 and 4 were rapidly bound to microbial cells at short incubation periods. Also, fluorescence microscopy images showed green emission of BODIPYs bound to microbial cells. Photosensitized inactivation improved with an increase of the irradiation time. Similar photoinactivation activities were found for both BODIPYs in bacteria. The photoinactivation induced by these BODIPYs was effective for both bacteria. However, the Gram-positive bacterium was inactivated sooner and with a lower concentration of a photosensitizer than the Gram-negative bacterium. After 15 min irradiation, the complete eradication of S. aureus was obtained with 1 μM photosensitizer. A reduction of 4.5 log in the E. coli viability was found when using 5 μM photosensitizer and 30 min irradiation. Also, the last conditions produced a decrease of 4.5 log in C. albicans cells treated with BODIPY 3, while 4 was poorly effective. On the other hand, the effect of the addition of KI on photoinactivation at different irradiation periods and salt concentrations was investigated. A smaller effect was observed in S. aureus because the photosensitizers alone were already very effective. In E. coli, photokilling potentiation was mainly found at longer irradiation periods. Moreover, the photoinactivation of C. albicans mediated by these BODIPYs was increased in the presence of KI. In solution, an increase in the formation of the BODIPY triplet states was observed with the addition of the salt, due to the effect of external heavy atoms. The greater intersystem crossing together with the formation of reactive iodine species induced by BODIPYs may be contributing to enhance the inactivation of microorganisms. Therefore, these BODIPYs represent interesting photosensitizers to inactivate microorganisms. In particular, BODIPY 3 in combination with KI was highly effective as a broad spectrum antimicrobial photosensitizer.

The photodynamic activity of 5,10,15,20-tetra(4-N-methylpyridyl)porphyrin (TMPyP) was evaluated in vitro on Candida albicans cells under different experimental conditions. This tetracationic porphyrin binds rapidly to C. albicans cells, reaching a value of ∼1.7 nmol 10(-6) cells when the cellular suspensions (10(6) CFU mL(-1) ) were incubated with 5 μM sensitizer. The amount of cell-bound sensitizer is not appreciably changed when cultures are incubating for longer times (>15 min) but it diminishes with the number of washing steps. Photosensitized inactivation of C. albicans cellular suspensions increases with both sensitizer concentration and irradiation time, causing a ∼5 log decrease of cell survival when the cultures are treated with 5 μM TMPyP and irradiated for 30 min. However, the photocytotoxicity decreases after one washing step, with the decrease in cell-bound sensitizer. The growth of C. albicans cells was arrested when the cultures were exposed to 5 μM TMPyP and visible light. On agar surfaces, the phototoxic effect of this sensitizer, which caused an inactivation of C. albicans cells, remained high. No growth was observed in areas containing TMPyP and irradiated. Moreover, in small C. albicans colonies, C. albicans cells were completely inactivated. These studies indicate that TMPyP is an effective sensitizer for photodynamic inactivation of yeasts in both liquid suspensions or localized on a surface.

The photodynamic mechanism of action induced by 5-(4-trifluorophenyl)-10,15,20-tris(4-N,N,N-trimethylammoniumphenyl)porphyrin (TFAP(3+)), 5,10,15,20-tetrakis(4-N,N,N-trimethylammoniumphenyl)porphyrin (TMAP(4+)) and 5,10,15,20-tetrakis(4-N-methylpyridyl)porphyrin (TMPyP(4+)) was investigated on Candida albicans cells. These cationic porphyrins are effective photosensitizers, producing a ~5 log decrease of cell survival when the cultures are incubated with 5 μM photosensitizer and irradiated for 30 min with visible light. Studies under anoxic conditions indicated that oxygen is necessary for the mechanism of action of photodynamic inactivation of this yeast. Furthermore, photoinactivation of C. albicans cells was negligible in the presence of 100 mM azide ion, whereas the photocytotoxicity induced by these porphyrins increased in D(2)O. In contrast, the addition of 100 mM mannitol produced a negligible effect on the cellular phototoxicity. On the other hand, in vitro direct observation of singlet molecular oxygen, O(2)((1)Δ(g)) phosphorescence at 1270 nm was analyzed using C. albicans in D(2)O. A shorter lifetime of O(2)((1)Δ(g)) was found in yeast cellular suspensions. These cationic porphyrins bind strongly to C. albicans cells and the O(2)((1)Δ(g)) generated inside the cells is rapidly quenched by the biomolecules of the cellular microenvironment. Therefore, the results indicate that these cationic porphyrins appear to act as photosensitizers mainly via the intermediacy of O(2)((1)Δ(g)).

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Mechanistic aspects in the photodynamic inactivation of Candida albicans sensitized by a dimethylaminopropoxy porphyrin and its equivalent with cationic intrinsic charges,