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Authors
Abstract(s)
Ao longo dos últimos anos, algumas terapias, como a quimioterapia, braquiterapia,
imunoterapia ou radioterapia, tornaram-se o tratamento convencional para certas
doenças devido à sua eficácia, no entanto, mostraram ser muito invasivas, citotóxicas e
pouco seletivas. Mesmo com a exposição conjunta e continuada destes tratamentos, os
mesmos não demonstram eficácia suficiente para curar tais doenças, sendo usados de
forma paliativa.
Com o desenvolvimento da Ciência dos materiais e rápido progresso da
nanotecnologia, a utilização de materiais nanométricos na entrega de genes por
aplicações de terapia génica mudaram o atual paradigma da terapêutica, uma vez que
esta terapia possibilita a inserção de material genético em células e tecidos alvo de um
indivíduo, de forma mais direcionada e menos citotóxica.
Neste sentido, o desenvolvimento de vetores de entrega de material genético
seguros, seletivos, biocompatíveis e fáceis de produzir surge como uma estratégia
adequada para fins terapêuticos. Entre os vetores virais e sintéticos, estes últimos,
nomeadamente os de base polimérica, apresentam características promissoras para
serem explorados com vista à sua aplicação em contexto clínico.
O núcleo e a mitocôndria das células eucarióticas animais possuem genoma
próprio e o funcionamento de ambos os organelos está interligado. Diversas doenças
humanas estão associadas a mutações que ocorrem num ou em ambos os organelos.
A terapia génica nuclear tem sido amplamente investigada nas últimas décadas e
evoluiu consideravelmente com vários ensaios clínicos em curso. Contrariamente, a
terapia génica mitocondrial é uma área, até ao momento, pouco estudada com apenas
alguns resultados publicados internacionalmente. Estes trabalhos têm demonstrado o
potencial efeito terapêutico da criação de vetores adequados à entrega de ADN
mitocondrial a mitocôndrias.
Assim, neste trabalho foram desenvolvidos sistemas de transporte de ADN plasmídico
(pADN), com capacidade de direcionamento a organelos celulares específicos, tendo
como objetivo final restaurar o normal funcionamento celular. Para tal, fez-se a
complexação de pADN com o polímero polietilenamina (PEI) e PEI associado ao agente
espaçador poli (etileno glicol) (PEG) e modificado com o composto trifenilfosfónio (TPP)
que possui afinidade mitocondrial (PEI-PEG-TPP) a fim de formular nanopartículas biocompatíveis, com propriedades de tamanho, carga superficial e morfologia adequadas
aos processos de captação e internalização celular.
Deste modo, pretendeu-se desenvolver vetores com capacidade de direcionamento
ao núcleo e mitocôndria das células, respetivamente. O seu potencial efeito terapêutico
foi investigado, foi feita a otimização das propriedades destes vetores a fim de melhorar
a sua performance de transfeção celular in vitro e assim promover avanços significativos
no desenvolvimento de sistemas de entrega direcionada de pADN com efeito terapêutico.
Também foi avaliada a citotoxicidade dos sistemas.
Os resultados demonstram uma variação nas caraterísticas das nanopartículas
formuladas, quer ao nível do tamanho, carga à superfície ou citotoxicidade induzida,
consoante o sistema formulado. Foi possível a formulação de poliplexos biocompatíveis,
com boa capacidade de incorporação de pADN e entrega eficaz às células.
Throughout the last years, some therapies, such as chemotherapy, brachytherapy, immunotherapy or radiotherapy, have become the conventional treatment for certain diseases due to its effectiveness. However, the exposure to these treatments has shown to be invasive, cytotoxic and not very selective. Even the combined and continuous exposure of these therapies, show to be ineffective enough to cure. With the development of material sciences and the fast progress in nanotechnology, the use of nanometric materials in gene delivery by gene therapy applications has changed the current paradigm of therapy, since it allows the insertion of genetic material in targeted cells and tissues, in a more targeted and less cytotoxic way. In this regard, the development of safe, selective, biocompatible and easy to produce delivery vectors based on genetic material appears as a suitable strategy for therapeutic purposes. Among the viral and synthetic vectors, the latter, namely the polymeric-based ones, present promising features to their application in a clinical context. The nucleus and mitochondria of animal eukaryotic cells have their own genome, and their functioning is connected between them. Several human diseases are associated with mutations occurring in one or both of these organelles. Nuclear genetic therapy has been widely studied on the last decades and has evolved significantly with multiple ongoing clinical trials. On the other hand, mitochondrial genetic therapy has been less studied. The few studies that have been published show the potential therapeutic effect of the creation of appropriate vectors for mitochondrial DNA delivery to the mitochondria. Thus, in this work, plasmid DNA (pDNA) transport systems were developed and directed to specific cellular organelles in order to restore normal cellular functioning. To do so, pDNA was complexed with the polyethyleneamine polymer (PEI) and modified PEI associated with the polyethylene glycol (PEG) spacer and triphenylphosphonium compound (TPP) with mitochondrial affinity (PEI-PEG-TPP), in order to formulate biocompatible nanoparticles, with the suitable properties concerning size, surface charge and morphology required to the uptake and cellular internalization. In this way, the aim was to develop vectors targeting the nucleus and mitochondria, respectively. Their potential therapeutic effect was investigated and the properties of these vectors were optimized in order to improve their in vitro cell transfection rate and thus promote the development of targeted delivery systems with therapeutic effect. The cytotoxicity of the systems was also evaluated. Depending on the formulated system, the results have shown a variation in the characteristics of the formulated nanoparticles, either in terms of size, surface charge or induced cytotoxicity. It was possible achieve biocompatible polyplexes, with good pDNA incorporation and effective delivery to cells.
Throughout the last years, some therapies, such as chemotherapy, brachytherapy, immunotherapy or radiotherapy, have become the conventional treatment for certain diseases due to its effectiveness. However, the exposure to these treatments has shown to be invasive, cytotoxic and not very selective. Even the combined and continuous exposure of these therapies, show to be ineffective enough to cure. With the development of material sciences and the fast progress in nanotechnology, the use of nanometric materials in gene delivery by gene therapy applications has changed the current paradigm of therapy, since it allows the insertion of genetic material in targeted cells and tissues, in a more targeted and less cytotoxic way. In this regard, the development of safe, selective, biocompatible and easy to produce delivery vectors based on genetic material appears as a suitable strategy for therapeutic purposes. Among the viral and synthetic vectors, the latter, namely the polymeric-based ones, present promising features to their application in a clinical context. The nucleus and mitochondria of animal eukaryotic cells have their own genome, and their functioning is connected between them. Several human diseases are associated with mutations occurring in one or both of these organelles. Nuclear genetic therapy has been widely studied on the last decades and has evolved significantly with multiple ongoing clinical trials. On the other hand, mitochondrial genetic therapy has been less studied. The few studies that have been published show the potential therapeutic effect of the creation of appropriate vectors for mitochondrial DNA delivery to the mitochondria. Thus, in this work, plasmid DNA (pDNA) transport systems were developed and directed to specific cellular organelles in order to restore normal cellular functioning. To do so, pDNA was complexed with the polyethyleneamine polymer (PEI) and modified PEI associated with the polyethylene glycol (PEG) spacer and triphenylphosphonium compound (TPP) with mitochondrial affinity (PEI-PEG-TPP), in order to formulate biocompatible nanoparticles, with the suitable properties concerning size, surface charge and morphology required to the uptake and cellular internalization. In this way, the aim was to develop vectors targeting the nucleus and mitochondria, respectively. Their potential therapeutic effect was investigated and the properties of these vectors were optimized in order to improve their in vitro cell transfection rate and thus promote the development of targeted delivery systems with therapeutic effect. The cytotoxicity of the systems was also evaluated. Depending on the formulated system, the results have shown a variation in the characteristics of the formulated nanoparticles, either in terms of size, surface charge or induced cytotoxicity. It was possible achieve biocompatible polyplexes, with good pDNA incorporation and effective delivery to cells.
Description
Keywords
Adn Plasmídico Polietilenimina Polímeros Catiónicos Sistemas de Entrega Terapia Génica Transfeção Celular