Nanomateriais hidrofílicos de carbono para o tratamento da doença de Alzheimer: uma nova estratégia terapêutica
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2016
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A doença de Alzheimer é a doença neurodegenerativa progressiva mais comum que afeta milhões de pessoas em todo o mundo. É caracterizada por um processo neurodegenerativo que promove a perda de sinapses e neurónios, principalmente em áreas associadas com a memória e a aprendizagem. Dados recentes suportam a hipótese de que a doença de Alzheimer emerge de um desequilíbrio bioenergético associado ao stresse oxidativo, a um perfil lipídico mitocondrial anormal e a disfunções mitocondriais, detetado, principalmente, pela perda de funcionalidade do complexo I mitocondrial. Assim sendo, a modelação da funcionalidade mitocondrial e do potencial redox das células cerebrais por agentes externos não-tóxicos apresenta-se como uma estratégia terapêutica atrativa para prevenir (ou interromper) o fenótipo patológico dependente da idade da doença de Alzheimer. Neste contexto, o principal objetivo deste trabalho consistiu no desenvolvimento de uma nova estratégia terapêutica baseada na utilização de nanomateriais hidrofílicos de carbono (NHC) com propriedades redox modeláveis. Os nanomateriais foram sintetizados por um novo método eletroquímico desenvolvido recentemente pelo nosso grupo.
Na primeira parte deste trabalho foi demonstrado que as propriedades redox do material hidrofílico podem ser seletivamente modeladas pela simples modificação da natureza do eletrólito utilizado na síntese eletroquímica. Assim, foram preparados dois tipos de nanomateriais, um que apresenta uma elevada capacidade para doar eletrões (NHC-o) e outro que apresenta, simultaneamente, a capacidade de doar e de receber eletrões (NHC-i). Com base na técnica de voltametria cíclica foram avaliadas as propriedades redox destes nanomateriais, a sua estabilidade química, a reversibilidade das reações de transferência de eletrões, os potenciais de eléctrodo e a sua dependência do pH. Os nanomateriais foram caracterizados quanto ao seu tamanho, forma, espessura, estrutura cristalina, natureza dos grupos funcionais e propriedades óticas utilizando as técnicas de TEM, HRTEM, AFM, XPS, UV-Vis e fluorescência.
Relativamente às propriedades redox dos NHC-i, concluiu-se que estes exibem um comportamento redox quase-reversível, que ocorre num intervalo de potenciais onde a cadeia respiratória mitocondrial opera. Esta característica sugere que os NHC-i poderão ser utilizados para promover, na fase aquosa da membrana interna mitocondrial, a oxidação do NADH e transferir os eletrões diretamente para o complexo III da cadeia respiratória mitocondrial, ultrapassando o défice de eletrões no complexo I. Por outro lado, foi demonstrado por testes antioxidantes (e.g. DPPH, ABTS, Folin-Ciocalteau) que os NHC-o apresentam uma elevada atividade de sequestro de radicais, comparável ao trolox. Tendo em conta estes resultados, a solução de NHC-o foi escolhida para prosseguir os estudos. Posteriormente, foi avaliada a capacidade dos NHC-o em ajustar o estado redox dos sistemas biológicos em condições de stresse. Os resultados revelaram que os NHC-o têm a capacidade de evitar a peroxidação lipídica e a oxidação dos grupos tiol celulares (proteínas e GSH) induzida por agentes oxidantes externos (e.g. tert-butil-hidroperóxido, par ascorbato/Fe+2).
Ensaios in vitro em que se utilizaram duas linhas tumorais humanas, AGS proveniente de carcinoma gástrico e SH-SY5Y proveniente de neuroblastoma, demonstraram a não-toxicidade dos NHC-o. Para além disso, mostrou-se que os NHC-o apresentam uma elevada capacidade para proteger as células contra a toxicidade do tert-butil-hidroperóxido, um indutor de morte celular associado a stresse oxidativo.
Em suma, concluiu-se que os NHC podem preservar ou recuperar o estado redox funcional das células e evitar a oxidação de biomoléculas (DNA, proteínas e lípidos), abrindo a possibilidade de serem aplicados como agentes terapêuticos em terapias upstream e downstream para prevenir/atenuar o fenótipo dependente da idade da doença de Alzheimer.
Alzheimer´s disease is the most common progressive neurodegenerative disease affecting millions of people worldwide. The Alzheimer’s brain is marked by neurodegeneration, comprising loss of synapses and neurons particularly in areas associated with memory and learning. Recent data support the idea that Alzheimer´s disease emerges as consequence of bioenergetics metabolism unbalance associated with oxidative stress, abnormal mitochondrial lipid profile and mitochondrial dysfunctions, mainly detected by the decline of complex I activity. Considering this framework, the modulation of both mitochondrial functionality and redox potential of brain cells by non-toxic external agents emerges as an attractive approach to prevent (or decrease) the age-dependent pathological phenotype of Alzheimer´s disease. In this context, the main motivation of this work was the development of a new therapeutic strategy based on the use of hydrophilic carbon nanomaterials (NHC) with tunable redox properties. The nanomaterials were prepared via a new electrochemical route recently developed in the group. In a first stage of this work, it was demonstrated that the redox ability of the hydrophilic material could be selectively tuned by simply modifying the nature of the electrolyte used in the electrochemical synthesis. In this way, it was prepared two pools of nanomaterials, one that can display a very high electron-donating ability (NHC-o) and another that displays simultaneously electron-accepting and electron-donating ability (NHC-i). Evaluation of the redox properties, their long-term stability, reversibility of the electron-transfer reactions, pH dependence, as well as electrode potentials, was provided by cyclic voltammetry technique. Characterization of their size, shape, thickness, crystallinity, surface functional groups and optical properties were accomplished by TEM, HRTEM, AFM, XPS and UV-vis, PL techniques. Considering the redox properties of the synthetized NHC-i, it was concluded that they exhibit a quasi-reversible redox behaviour within a narrow range of redox potentials, where the mitochondrial respiratory chain operates. This feature suggests that they might be used to promote the oxidation of NADH in aqueous interface of inner-mitochondrial membrane and deliver the electrons to complex III, overcoming deficiencies at complex I. On the other hand, it was demonstrated by antioxidant tests (e.g. DPPH, ABTS, Folin-Ciocalteau) that NHC-o exhibit a very high radical scavenging activity, comparable to trolox. In view of these results, the NHC-o solution was chosen to pursue the studies. Subsequently, it was evaluated the ability of NHC-o to modulate the redox state of biological systems under stress conditions. The results revealed that the NHC-o have the capacity to prevent the lipid peroxidation and cellular thiol groups oxidation (protein and GSH) induced by external oxidant agents (e.g. tert-butyl-hydroperoxide and ascorbate/ Fe+2). In vitro assays, performed with two human tumor cell lines, AGS cells from gastric carcinoma and SH-SY5Y cells from neuroblastoma, demonstrated the non-cytotoxicity of NHC-o. Moreover, it was also shown that the NHC-o exhibit high capacity of protection of cells against the toxicity of tert-butyl-hydroperoxide, known as a cell death inducer associated with oxidative stress. Summing up, it was concluded that hydrophilic carbon nanomaterials can preserve or restore the functional redox state of cells and avoiding the oxidation of biomolecules (DNA, protein and lipids), opening the way to be employed as therapeutics agents in upstream and downstream therapy to prevent/attenuate the age-dependent phenotype of Alzheimer´s disease.
Alzheimer´s disease is the most common progressive neurodegenerative disease affecting millions of people worldwide. The Alzheimer’s brain is marked by neurodegeneration, comprising loss of synapses and neurons particularly in areas associated with memory and learning. Recent data support the idea that Alzheimer´s disease emerges as consequence of bioenergetics metabolism unbalance associated with oxidative stress, abnormal mitochondrial lipid profile and mitochondrial dysfunctions, mainly detected by the decline of complex I activity. Considering this framework, the modulation of both mitochondrial functionality and redox potential of brain cells by non-toxic external agents emerges as an attractive approach to prevent (or decrease) the age-dependent pathological phenotype of Alzheimer´s disease. In this context, the main motivation of this work was the development of a new therapeutic strategy based on the use of hydrophilic carbon nanomaterials (NHC) with tunable redox properties. The nanomaterials were prepared via a new electrochemical route recently developed in the group. In a first stage of this work, it was demonstrated that the redox ability of the hydrophilic material could be selectively tuned by simply modifying the nature of the electrolyte used in the electrochemical synthesis. In this way, it was prepared two pools of nanomaterials, one that can display a very high electron-donating ability (NHC-o) and another that displays simultaneously electron-accepting and electron-donating ability (NHC-i). Evaluation of the redox properties, their long-term stability, reversibility of the electron-transfer reactions, pH dependence, as well as electrode potentials, was provided by cyclic voltammetry technique. Characterization of their size, shape, thickness, crystallinity, surface functional groups and optical properties were accomplished by TEM, HRTEM, AFM, XPS and UV-vis, PL techniques. Considering the redox properties of the synthetized NHC-i, it was concluded that they exhibit a quasi-reversible redox behaviour within a narrow range of redox potentials, where the mitochondrial respiratory chain operates. This feature suggests that they might be used to promote the oxidation of NADH in aqueous interface of inner-mitochondrial membrane and deliver the electrons to complex III, overcoming deficiencies at complex I. On the other hand, it was demonstrated by antioxidant tests (e.g. DPPH, ABTS, Folin-Ciocalteau) that NHC-o exhibit a very high radical scavenging activity, comparable to trolox. In view of these results, the NHC-o solution was chosen to pursue the studies. Subsequently, it was evaluated the ability of NHC-o to modulate the redox state of biological systems under stress conditions. The results revealed that the NHC-o have the capacity to prevent the lipid peroxidation and cellular thiol groups oxidation (protein and GSH) induced by external oxidant agents (e.g. tert-butyl-hydroperoxide and ascorbate/ Fe+2). In vitro assays, performed with two human tumor cell lines, AGS cells from gastric carcinoma and SH-SY5Y cells from neuroblastoma, demonstrated the non-cytotoxicity of NHC-o. Moreover, it was also shown that the NHC-o exhibit high capacity of protection of cells against the toxicity of tert-butyl-hydroperoxide, known as a cell death inducer associated with oxidative stress. Summing up, it was concluded that hydrophilic carbon nanomaterials can preserve or restore the functional redox state of cells and avoiding the oxidation of biomolecules (DNA, protein and lipids), opening the way to be employed as therapeutics agents in upstream and downstream therapy to prevent/attenuate the age-dependent phenotype of Alzheimer´s disease.
Descrição
Dissertação de Mestrado em Bioquímica
Palavras-chave
Bioquímica , Toxicidade , Stresse oxidativo , Doença de Alzheimer , Nanomateriais hidrofílicos de carbono , Propriedades Redox , Atividade antioxidante