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Abstract(s)
A terapia génica tem tido um interesse crescente em estudos científicos devido às suas potencialidades no tratamento de doenças, como o cancro, infeções e particularmente doenças genéticas. A eficácia da terapia génica está dependente do desenvolvimento de sistemas de transporte e entrega de material genético, devendo estes promover uma entrega do material genético de forma eficiente e segura. Já foram estudados diferentes tipos de vetores, tanto virais como não virais, sendo sugerido que os vetores não virais são muito mais seguros, devido as suas diversas características, como a versatilidade, grande capacidade de empacotamento, capacidade de funcionalização química e baixa imunogenicidade. No conjunto de vetores não virais, as bases poliméricas se destacam como uns dos mais promissores. Portanto ao longo desse projeto foi proposto avaliar a utilização de polímeros sensíveis ao pH, que possam proteger e encapsular DNA plasmídico (pDNA) de uma maneira simples e eficaz, para futuramente serem usados em diversas aplicações terapêuticas. Destacam-se neste projeto os diblocos de copolímeros POEOMA-b-PDPA, que possuem uma responsividade ao pH, devido à incorporação do PDPA, que permite também a interação eletrostática desses copolímeros com os ácidos nucleicos em pH ácido, como resultado da protonação dos grupos amina terciária deste polímero em valores de pH abaixo de seu pKa (cerca de 6,2). Esses copolímeros foram utilizados em diversos ensaios visando a caracterização física e química das nanopartículas preparadas, consistindo na avaliação da carga superficial (potencial zeta), morfologia das nanopartículas formuladas em diferentes valores de pH, caracterizada por Microscopia Eletrónica de Varrimento (MEV), e a proteção do pDNA, na presença de DNaseI e meio DMEM. A viabilidade celular foi igualmente determinada por MTS, para verificar a segurança dessas nanopartículas. Os resultados baseados no copolímero POEOMA-b-PDPA, mostram que estes copolímeros têm uma boa capacidade de proteção do pDNA, um potencial zeta positivo e um tamanho que pode permitir que as nanopartículas se mantenham em circulação por bastante tempo, sem a presença de citotoxicidade. Globalmente, os resultados indicam um potencial interessante desses copolímeros para aplicações biomédicas. Assim, os resultados demonstram que foi possível desenvolver um método simples, eficiente e prático para a entrega de pDNA.
Gene therapy has had a growing interest in scientific studies due to its potential in the treatment of diseases such as cancer, infections, and specifically for genetic diseases. The effectiveness of gene therapy is linked to the development of delivery systems for the protection and transport of genetic material, which should promote a competent and safe delivery to target cells. Different types of vectors, both viral and non-viral, have already been studied, being recognized that non-viral vectors are much safer, due to their various characteristics, such as versatility, large packaging capacity, chemical functionalization capacity, and low immunogenicity. In the set of non-viral vectors, the polymeric materials stand out as one of the most promising. Therefore, throughout this project, it was proposed to evaluate pH-responsive polymers, which can protect and encapsulate plasmid DNA in a simple and effective way, to be used in future therapeutic applications. In this project the diblock copolymers POEOMA-bPDPA, which have a PDPA responsiveness to pH, have been used. The PDPA content is also responsible for the electrostatic interaction of these copolymers with nucleic acids at acidic pH, as a result of the protonation of the tertiary amine groups of this polymer at pH values below its pKa (around 6.2). These copolymers were used through experiments aiming at the physical and chemical characterization of the studied nanoparticles, consisting of the evaluation of the surface charge (zeta potential), the morphology of the nanoparticles, formulated at different pH values, characterized by Scanning Electron Microscopy (SEM), and pDNA protection, in the presence of DNaseI and DMEM medium. Cell viability was also determined by MTS, to verify the safety profile of these nanoparticles. The results based on the POEOMA-bPDPA copolymer, show that these copolymers promote a good pDNA protection capacity, present a positive zeta potential and a size that can allow the nanoparticles to remain in circulation for a long time, without the presence of cytotoxicity. Overall, this suggests the potential of these copolymers to be used in biomedical applications. Thus, the results demonstrate that it was possible to develop a simple, efficient, and practical method for pDNA delivery.
Gene therapy has had a growing interest in scientific studies due to its potential in the treatment of diseases such as cancer, infections, and specifically for genetic diseases. The effectiveness of gene therapy is linked to the development of delivery systems for the protection and transport of genetic material, which should promote a competent and safe delivery to target cells. Different types of vectors, both viral and non-viral, have already been studied, being recognized that non-viral vectors are much safer, due to their various characteristics, such as versatility, large packaging capacity, chemical functionalization capacity, and low immunogenicity. In the set of non-viral vectors, the polymeric materials stand out as one of the most promising. Therefore, throughout this project, it was proposed to evaluate pH-responsive polymers, which can protect and encapsulate plasmid DNA in a simple and effective way, to be used in future therapeutic applications. In this project the diblock copolymers POEOMA-bPDPA, which have a PDPA responsiveness to pH, have been used. The PDPA content is also responsible for the electrostatic interaction of these copolymers with nucleic acids at acidic pH, as a result of the protonation of the tertiary amine groups of this polymer at pH values below its pKa (around 6.2). These copolymers were used through experiments aiming at the physical and chemical characterization of the studied nanoparticles, consisting of the evaluation of the surface charge (zeta potential), the morphology of the nanoparticles, formulated at different pH values, characterized by Scanning Electron Microscopy (SEM), and pDNA protection, in the presence of DNaseI and DMEM medium. Cell viability was also determined by MTS, to verify the safety profile of these nanoparticles. The results based on the POEOMA-bPDPA copolymer, show that these copolymers promote a good pDNA protection capacity, present a positive zeta potential and a size that can allow the nanoparticles to remain in circulation for a long time, without the presence of cytotoxicity. Overall, this suggests the potential of these copolymers to be used in biomedical applications. Thus, the results demonstrate that it was possible to develop a simple, efficient, and practical method for pDNA delivery.
Description
Keywords
Copolímeros Dna Plasmídeo Encapsulamento Poeoma-B-Pdpa Terapia Génica