Efeito citotóxico da Terapia Fotodinâmica associando Photogem e LED azul e vermelho em cultura de células normais
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Data
2009-03-20
Autores
Ribeiro, Ana Paula Dias [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Para considerar a Terapia Fotodinâmica (PDT) como tratamento clínico da estomatite protética, é necessário conhecer tanto o potencial antifúngico, como efeito citotóxico desta terapia sobre células normais do indivíduo. Assim, o objetivo desse estudo in vitro foi avaliar a citotoxicidade da PDT antifúngica com o fotossensibilizador Photogem® associado ao LED azul e ao LED vermelho em cultura de fibroblastos L929 e células odontoblastóides MDPC-23. As células foram cultivadas (30.000 células/cm2) em placas de 24 compartimentos por 48 horas e ambos os tipos celulares foram incubados com Photogem® (0, 10, 25, 50, 100 ou 150 mg/L) e irradiados ou não pelo LED azul (460 ± 3 nm; 25,5 ou 37,5 J/cm2; 22 mW/cm2) ou LED vermelho (630 ± 3 nm; 70 ou 100 J/cm2; 25 mW/cm2) . O metabolismo celular foi determinado 0, 12 e 24 horas após a PDT utilizando o teste do metiltetrazolium (MTT), e a morfologia celular avaliada pela microscopia eletrônica de varredura (MEV). A técnica de citometria de fluxo foi utilizada para avaliar o tipo de morte celular (necrose ou apoptose) assim como estimar os níveis intracelulares das espécies reativas de oxigênio (EROs). Observou-se redução do metabolismo celular estatisticamente significante para todas as concentrações do Photogem® quando irradiadas em qualquer dose de luz, sendo essa redução de 90 a 97% tanto para as células L929 quanto MDPC-23 (ANOVA and Dunnet’s post hoc tests; p<0.05). Essa redução da atividade mitocondrial não foi dependente da concentração do fotossensibilizador e nem da dose de luz empregada. Também, foi demonstrado que a atividade mitocondrial das células submetidas a PDT não foi recuperada após 12 ou 24 horas, caracterizando um dano irreversível. A presença do Photogem® e da luz isoladamente não alterou estatisticamente a atividade mitocondrial de ambas as linhagens. As células submetidas...
In order to consider the photodynamic therapy (PDT) as a clinical treatment for candidosis, it is necessary to know its antifungal potential and its cytotoxicity effect on normal cells. Therefore, the purpose of this in vitro study was to evaluate the cytotoxicity of PDT with Photogem® associated to blue and red LED on L929 and MDPC-23 cell cultures. The cells (30.000 cells/cm2) were seeded in 24-well plates for 48 hours, incubated with Photogem® (10, 25, 50, 100 or 150 mg/L) and were irradiated or not with a blue LED source (460 ± 3 nm; 25.5 or 37.5 J/cm2; 22 mW/cm2) or with a red LED source (630 ± 3 nm; 75 or 100 J/cm2; 22 mW/cm2). Cell metabolism was evaluated by the MTT assay (ANOVA and Dunnet’s post hoc tests; p<0.05) and cell morphology was examined by scanning electron microscopy. Flow cytometry was employed to analyze the type of PDT-induced cell death (necrosis or apoptosis) as well as to estimate intracellular production of reactive oxygen species (ROS). There was a statistically significant decrease of mitochondrial activity for all Photogem® concentrations associated to blue or red LED regardless irradiation time; this reduction ranged from 90 to 97% for both cell lines. This reduction, however, was not dependent on the photosensitizer concentration. It was also demonstrated that the mitochondrial activity of the cells submitted to PDT was not recovered after 12 or 24 hours, characterizing irreversible cell damage. PDT-treated cells presented an altered morphology with ill-defined limits. In both cell lines, there was a predominance of necrotic cell death when in contact with the photosensitizer. The presence of Photogem® and exposure to blue and red LED increased the intracellular levels of ROS in both L929 and MDPC- 23 cells in a dose-dependent manner. The association of Photogem® and blue LED caused severe toxic effects on normal cell culture... (Complete abstract click electronic access below)
In order to consider the photodynamic therapy (PDT) as a clinical treatment for candidosis, it is necessary to know its antifungal potential and its cytotoxicity effect on normal cells. Therefore, the purpose of this in vitro study was to evaluate the cytotoxicity of PDT with Photogem® associated to blue and red LED on L929 and MDPC-23 cell cultures. The cells (30.000 cells/cm2) were seeded in 24-well plates for 48 hours, incubated with Photogem® (10, 25, 50, 100 or 150 mg/L) and were irradiated or not with a blue LED source (460 ± 3 nm; 25.5 or 37.5 J/cm2; 22 mW/cm2) or with a red LED source (630 ± 3 nm; 75 or 100 J/cm2; 22 mW/cm2). Cell metabolism was evaluated by the MTT assay (ANOVA and Dunnet’s post hoc tests; p<0.05) and cell morphology was examined by scanning electron microscopy. Flow cytometry was employed to analyze the type of PDT-induced cell death (necrosis or apoptosis) as well as to estimate intracellular production of reactive oxygen species (ROS). There was a statistically significant decrease of mitochondrial activity for all Photogem® concentrations associated to blue or red LED regardless irradiation time; this reduction ranged from 90 to 97% for both cell lines. This reduction, however, was not dependent on the photosensitizer concentration. It was also demonstrated that the mitochondrial activity of the cells submitted to PDT was not recovered after 12 or 24 hours, characterizing irreversible cell damage. PDT-treated cells presented an altered morphology with ill-defined limits. In both cell lines, there was a predominance of necrotic cell death when in contact with the photosensitizer. The presence of Photogem® and exposure to blue and red LED increased the intracellular levels of ROS in both L929 and MDPC- 23 cells in a dose-dependent manner. The association of Photogem® and blue LED caused severe toxic effects on normal cell culture... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Fotoquimioterapia, Fibroblastos, Odontoblastos, Photochemotherapy, Hematoporphyrin derivative, Toxicity, Fibroblasts, Odontoblasts
Como citar
RIBEIRO, Ana Paula Dias. Efeito citotóxico da Terapia Fotodinâmica associando Photogem e LED azul e vermelho em cultura de células normais. 2009. 194 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Odontologia de Araraquara, 2009.