Síntese, caraterização espetroscópica de novos corantes esquarílicos e avaliação in vitro do seu potencial fototerapêutico
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2018-12-17
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As cianinas esquarílicas são corantes polimetínicos que contêm na sua estrutura um anel central de quatro membros deficiente em eletrões e dois grupos terminais doadores de eletrões. Estes corantes podem ser utilizados no combate ao cancro, uma das doenças mais mortais a nível mundial definida como um conjunto de patologias caraterizadas pela proliferação celular descontrolada, através da sua aplicação como agentes fotossensibilizadores em terapia fotodinâmica. Neste trabalho foram sintetizadas e caraterizadas cianinas esquarílicas, tioesquarílicas e aminoesquarílicas simétricas derivadas da indolenina, que foram posteriormente avaliadas relativamente ao seu potencial fototerapêutico in vitro em linhas celulares tumorais e não- -tumorais. Estes compostos foram ainda avaliados relativamente à sua capacidade de formação de oxigénio singleto, uma ROS altamente citotóxica, à sua fotoestabilidade e à sua localização quando em contacto com as células. A presente dissertação de mestrado é composta por cinco capítulos. No primeiro capítulo é apresentada uma revisão bibliográfica sobre terapia fotodinâmica, bem como as propriedades que tornam as cianinas esquarílicas potenciais moléculas fotossensibilizadoras. No segundo capítulo são apresentados e discutidos os resultados relativos à síntese, caraterização espetroscópica e avaliação da capacidade de formação de oxigénio singleto e fotoestabilidade dos corantes esquarílicos. No terceiro capítulo são evidenciados e analisados os resultados obtidos nos estudos de citotoxicidade, de ensaios de MTT e de citometria de fluxo, de forma a avaliar o potencial fototerapêutico de corantes esquarílicos a várias concentrações e energias de irradiação, bem como no estudo de localização celular de algumas cianinas. No quarto capítulo são apresentados os métodos experimentais utilizados para a realização do presente trabalho, e no quinto capítulo são evidenciadas as referências bibliográficas mencionadas ao longo da dissertação. Face aos resultados observados relativamente à síntese e caraterização dos compostos conclui-se que foram obtidos corantes esquarílicos com elevado grau de pureza e que, dado absorverem na “janela fototerapêutica”, são potenciais fotossensibilizadores. Os resultados obtidos nos ensaios in vitro permitem concluir que, na generalidade, as cianinas esquarílicas não-substituídas no anel central de quatro membros, a derivada do ácido barbitúrico e a monotioesquarílica, foram as que revelaram ter efeitos mais promissores. Relativamente à sua localização celular, conclui-se que as cianinas não-substituídas e a derivada do ácido barbitúrico difundem para o meio intracelular, que não atravessam a membrana nuclear, nem se localizam preferencialmente nas mitocôndrias.
Squaraine cyanines are polymethynic dyes which contain in their structure an electrondeficient four membered central ring and two electron donor terminal groups. These dyes can be used in some therapeutic strategies against cancer, one of the deadliest diseases worldwide, defined as a set of pathologies characterized by uncontrolled cell proliferation, through its application as photosensitizing agents in photodynamic therapy. In this work, symmetrical squaraine, thiosquaraine and aminosquaraine dyes derived from indolenine were synthesized and their in vitro phototherapeutic potential in tumor and non-tumor cell lines was evaluated. These compounds were further studied for their ability to generate singlet oxygen, a highly cytotoxic oxygen reactive species, their photostability and their location when in contact with the cells. This dissertation is composed by five chapters. In the first chapter, a bibliographic review of photodynamic therapy, as well as the properties that make squaraine cyanine dyes potential photosensitizing molecules, are presented. In the second chapter, the results regarding synthesis, spectroscopic characterization and evaluation of the singlet oxygen generation capacity and photostability of squaraine dyes are presented and discussed. In the third chapter, the results obtained in the cytotoxicity studies, involving MTT and flow cytometry assays, were analyzed in order to evaluate the phototherapeutic potential of squaraines at various concentrations and irradiation energies, as well as the cell localization study of some dyes. In the fourth chapter we present the experimental methods used to carry out the present work, and the fifth chapter the bibliographical references mentioned throughout the dissertation are evidenced. The analysis of the results obtained with the synthesis and characterization of the compounds allows us to conclude that the squaraines were obtained with a high degree of purity and that, absorbing in the “phototherapeutic window”, are potential photosensitizers. The results obtained in the in vitro tests allow us to conclude that, in general, the most promising effects were observed with the unsubstituted squaraines in the four-membered central ring, the barbituric acid derivative and monothiosquaraine. Regarding its cellular location, it was concluded that the unsubstituted squaraines and the barbituric acid derivative diffuse into the intracellular environment, but neither cross the nuclear membrane, nor are preferentially located in the mitochondria
Squaraine cyanines are polymethynic dyes which contain in their structure an electrondeficient four membered central ring and two electron donor terminal groups. These dyes can be used in some therapeutic strategies against cancer, one of the deadliest diseases worldwide, defined as a set of pathologies characterized by uncontrolled cell proliferation, through its application as photosensitizing agents in photodynamic therapy. In this work, symmetrical squaraine, thiosquaraine and aminosquaraine dyes derived from indolenine were synthesized and their in vitro phototherapeutic potential in tumor and non-tumor cell lines was evaluated. These compounds were further studied for their ability to generate singlet oxygen, a highly cytotoxic oxygen reactive species, their photostability and their location when in contact with the cells. This dissertation is composed by five chapters. In the first chapter, a bibliographic review of photodynamic therapy, as well as the properties that make squaraine cyanine dyes potential photosensitizing molecules, are presented. In the second chapter, the results regarding synthesis, spectroscopic characterization and evaluation of the singlet oxygen generation capacity and photostability of squaraine dyes are presented and discussed. In the third chapter, the results obtained in the cytotoxicity studies, involving MTT and flow cytometry assays, were analyzed in order to evaluate the phototherapeutic potential of squaraines at various concentrations and irradiation energies, as well as the cell localization study of some dyes. In the fourth chapter we present the experimental methods used to carry out the present work, and the fifth chapter the bibliographical references mentioned throughout the dissertation are evidenced. The analysis of the results obtained with the synthesis and characterization of the compounds allows us to conclude that the squaraines were obtained with a high degree of purity and that, absorbing in the “phototherapeutic window”, are potential photosensitizers. The results obtained in the in vitro tests allow us to conclude that, in general, the most promising effects were observed with the unsubstituted squaraines in the four-membered central ring, the barbituric acid derivative and monothiosquaraine. Regarding its cellular location, it was concluded that the unsubstituted squaraines and the barbituric acid derivative diffuse into the intracellular environment, but neither cross the nuclear membrane, nor are preferentially located in the mitochondria
Descrição
Dissertação de Mestrado em Bioquímica apresentada à Universidade de Trás-os-Montes e Alto Douro
Palavras-chave
cianinas esquarílicas , terapia fotodinâmica , irradiação com LEDs , potencial fototerapêutico in vitro