Production of reactive oxygen species after photodynamic therapy by porphyrin sensitizers.

The objectives of this study was to investigate the production of reactive oxygen species (ROS) after photodynamic therapy (PDT) in vitro. We examined second generation sensitizers, porphyrines (TPPS4, ZnTPPS4 and PdTPPS4) and compared their effectivity on ROS generation in G361 cell line. Used porphyrines are very efficient water-soluble aromatic dyes with potential to use in photomedicine and have a high propensity to accumulate in the membranes of intracellular organelles like lysosomes and mitochondria. Interaction between the triplet excited state of the sensitizer and molecular oxygen leads to produce singlet oxygen and other ROS to induce cell death. Production of ROS was verificated by molecular probe CM-H2DCFDA and viability of cells was determined by MTT assay. Our results demonstrated that ZnTPPS4 induces the highest ROS production in cell line compared to TPPS4 and PdTPPS4 at each used concentration and light dose. These results consist with a fact that photodynamic effect depends on sensitizer type, its concentration and light dose.

Comparison of sensitizers by detecting reactive oxygen species after photodynamic reaction in vitro.

The production of reactive oxygen species (ROS) has a crucial effect on the result of photodynamic therapy (PDT). Because of this fact, we examined the ROS formation by means of three porphyrin sensitizers (TPPS(4), ZnTPPS(4) and PdTPPS(4)) and compared their effectivity for induction of cell death in the G361 (human melanoma) cell line. The porphyrins used are very efficient water-soluble aromatic dyes with a potential application in photomedicine and have a high tendency to accumulate in the membranes of intracellular organelles such as lysosomes and mitochondria. Interaction between the triplet excited state of the sensitizer and molecular oxygen leads to the production singlet oxygen and other reactive oxygen species to induce cell death. Production of ROS was investigated by molecular probe CM-H(2)DCFDA. Our results demonstrated that ZnTPPS(4) induces the highest ROS production in the cell line compared to TPPS(4) and PdTPPS(4) at concentrations of 1, 10, and 100 microM and light dose of 1 J cm(-2). We also observed a consequence between ROS production and cell survival. In conclusion, these results demonstrate that photodynamic effect depends on sensitizer type, its concentration and light dose.

In vitro photodynamic therapy on melanoma cell lines with phthalocyanine.

Photodynamic therapy (PDT) is a new treatment modality of tumours. The photochemical interactions of sensitizer, light, and molecular oxygen produce singlet oxygen and other forms of active oxygen, such as peroxide, hydroxyl radical and superoxid anion. Phthalocyanine ClAlPcS(2), belonging among the promising second generation of sensitizers, was tested as an inducer of photodamage. We report the production of reactive oxygen species (ROS) and the phototoxicity of ClAlPcS(2) assessed using G361 melanoma cells. A semiconductor laser (lambda=675nm, output power 21mW) was used as a source for evocation of the photodynamic effect. ROS generation and H(2)O(2) release after PDT on G361 cells were detected using probe CM-H(2)DCFDA and recorded by luminescence spectrometer. Viability studies show, that the optimum phototoxic effect tested on G361 melanoma cells was determined in the combination of laser dose of 25Jcm(-2) and phthalocyanine ClAlPcS(2) concentration of 5microg/ml. This combination of phthalocyanine concentration and corresponding radiation dose was lethal for melanoma cells.

Photodynamic therapy with zinc-tetra(p-sulfophenyl)porphyrin bound to cyclodextrin induces single strand breaks of cellular DNA in G361 melanoma cells.

The basis of photodynamic therapy (PDT) is the phototoxicity resulting from co-action of light, sensitizer and oxygen. In this study we demonstrate in vitro phototoxicity measurement on G361 cell lines using ZnTPPS(4) sensitizer bound to cyclodextrin hpbetaCD. We have proved its photodamage effect on cancer cell lines in the visible region of spectrum. We used the halogen lamp (24V/250W) as a source of radiation. After 24h incubation of cell cultures with 10 microM ZnTPPS(4) and 1mM cyclodextrine hpbetaCD, the cells were irradiated for 7.5 min at the total irradiation dose of 12.5 Jcm(-2). Analysis of DNA damage in the cell line after PDT was proved by comet assay and using inversion fluorescent microscope with image analysis. This treatment method gave rise to DNA damage. The used radiation dose of visible light in the absence of sensitizers does not induce DNA breaks in tumour cells. In conclusion, binding of ZnTPPS(4) sensitizer to cyclodextrin hpbetaCD may improve the efficacy of PDT for the treatment of malign melanoma.