Photosensitization in membrane models and in neuronal cells

• Ano de defesa: 2021
• Autor(a) principal: Gomes, Raphael de Avila Narciso
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: Células neuronais; Complexos de rutênio; DMMB; Fotossensibilização; Lipid membranes; Membranas; Neuronal cells; Photodynamic therapy; Photosensitization; Ruthenium complexes; Terapia fotodinâmica
• Link de acesso: https://www.teses.usp.br/teses/disponiveis/46/46136/tde-26042022-083934/

Photodynamic therapy (PDT) is being extensively studied and tested to treat different types of diseases, such as cancer, leishmaniosis, malaria, among others. PDT usually comprises the use of photosensitizers, oxygen and light to increase the oxidative stress in cells and tissues. Preferred photosensitizers display: i) high absorption coefficient in the visible range of the light spectra; ii) high efficiency of triplet excited state generation with energy suitable to allow efficient energy transfer to form singlet oxygen; iii) physical-chemical and biochemical properties that allow interaction with important biological targets. Usually organic compounds, such as porphyrin or phenothiazinium derivatives, are used as photosensitizers (PS), but ruthenium complexes are also promising compounds to function as PDT PS. In this work the photodynamic effects of ruthenium photoactive complexes were evaluated in membrane small unilamellar vesicles (SUVs) and giant unilamellar vesicles (GUVs) and in a cellular model (Mouse Hippocampal Neuronal Cell Line - HT22). The phototoxicity of the complexes in HT22 cells were analysed, and the photochemical damage in the cell and mitochondria was observed by flow cytometry and microscopy. Experiments were also designed to compare its efficiency with an efficient phenothiazinium (dimethyl-methylene blue DMMB). The ruthenium complexes were able to damage the membranes of GUVs and SUVs and the damage efficiency does not seem to be related to the efficiency of singlet oxygen generation, but to membrane bonding. In HT22 cells Ru complexes were able to damage mitochondria. Damage could not be reverted by general anti-oxidant agents. The LD50 were calculated to be from 17 to 25 M from 8000 to 25000 cells, which implicates in intracellular concentrations of ~80pM/cell. DMMB also causes severe damage in mitochondria as indicated by mitochondria morphology, mitoSox staining, and lipid peroxidation. The LD50 were calculated to be 30 to 35 and 40 nM for incubations with 5000, 6000 and 8000 cells, respectively, which implicates in intracellular concentrations of ~2pM/cell. It is clear that DMMB is more efficient (~40 times) in terms of causing cell death (compared PS intracellular concentrations at the LD50), probably because for its higher tendency to bind more efficiently to membranes. We expect to have higher efficiency in Ru complexes with more lipophilic ligands.