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Advisor(s)
Abstract(s)
Em Portugal estima-se que morrem 350 mulheres por ano com cancro do colo do útero, estando Portugal nos 20 países da Europa com maiores taxas de incidência e mortalidade. Este cancro resulta, geralmente, da infeção pelo vírus do papiloma humano e é a quarta causa de morte, nas mulheres, a nível mundial. A sua progressão resulta da expressão das oncoproteínas E6 e E7, sendo estas capazes de alterar o ciclo celular, interagindo com uma grande variedade de proteínas, nomeadamente com o supressor de tumor p53. Esta interação leva à marcação da p53 com a finalidade de ser degradada pelo proteossoma. Uma vez que a sua função é induzir a apoptose das células com danos no Àcido Desoxirribonucleico (ADN) ou que se encontram infetadas, quando esta proteína não é corretamente produzida ou é inibida, deixa de desempenhar as suas funções permitindo assim que ocorra uma proliferação descontrolada das células malignas, dando origem a tumores. Neste sentido, o desenvolvimento de estratégias terapêuticas como a terapia génica para a entrega de material genético utilizando vetores não virais de base polimérica, descritos como mais seguros e eficientes, surge como uma alternativa promissora para a terapia do cancro do colo do útero, tendo como objetivo a reposição dos níveis normais de p53 nas células cancerígenas, induzindo a apoptose das mesmas. Para além disto, e tendo em conta os avanços clínicos em terapia do cancro descritos na literatura, a conjugação da terapia génica com a quimioterapia traz inúmeros benefícios. Assim, o foco deste trabalho foi o desenvolvimento de um sistema de entrega capaz de incorporar em simultâneo um ADN plasmídico (pADN) e uma droga anti-cancerígena, metotrexato (MTX). O MTX é um fármaco que, não só permite conferir direcionamento e seletividade ao veículo de entrega pelas células cancerígenas, como também apresenta atividade quimioterapêutica, complementando assim o efeito terapêutico pretendido.
O polímero de polietilenimina (PEI) foi escolhido para a formulação dos sistemas que possam condensar e encapsular o pADN em poliplexos. Foi estudado o efeito do peso molecular e da arquitetura do polímero nas propriedades dos complexos PEI/pADN (nomeadamente a morfologia, tamanho, potencial zeta, taxa de encapsulação de pADN e citotoxicidade). Os resultados demonstram uma variação nas caraterísticas das nanopartículas com o peso molecular e arquitetura do polímero, mas principalmente com o rácio N/P utilizado para cada sistema. O estudo de screening realizado permitiu desenvolver poliplexos biocompatíveis, com morfologia, tamanho e carga superficial adequados para a incorporação de pADN e MTX, com vista à sua entrega direcionada a células tumorais do colo do útero. Os resultados obtidos foram promissores e incentivam à futura realização de testes in vitro de transfeção celular.
In Portugal, it is estimated that 350 women die each year with cervical cancer, being Portugal one of 20 countries of Europe with the highest incidence and mortality rates. This cancer is usually the result of human papilloma virus (HPV) infection and it is the fourth leading cause of death in women worldwide. Its progression results from the expression of HPV E6 and E7 oncoproteins, which are capable of altering the cell cycle, interacting with a wide variety of proteins, namely with the p53 tumor suppressor. This interaction leads to the labeling of p53 for the purpose of being degraded by the proteosome. Since its function is to induce infected cells or with DNA damage in apoptosis, it stops performing its function, leading to the uncontrolled proliferation of the malignant cells, causing tumors. In this sense, gene therapy, using of safe and efficient polymer-based non-viral delivery vectors of gene material, appears as a promising alternative for the therapy of cervical cancer with the aim of restoring normal levels of p53 in the target cells. Furthermore, considering the clinical advances in cancer therapy described in the literature, the combination of gene therapy with chemotherapy brings numerous benefits. Thus, the main goal of this work was the development of a delivery system capable of simultaneously incorporating plasmid DNA (pDNA) and an anti-cancer drug, methotrexate (MTX). MTX is a drug that not only allows to give targeting and selectivity to the delivery vehicle, but also has chemotherapeutic activity complementing the intended therapeutic effect. The use of polyethyleneimine (PEI) was the strategy chosen to condense and encapsulate the pDNA into polyplexes. The effect of molecular weight and polymer architecture on the properties (morphology, size, zeta potential, pDNA encapsulation rate and cytotoxicity) of the PEI/pDNA complexes, was studied. The results demonstrate a variation in the characteristics of the nanoparticles with the molecular weight and architecture of the polymer, but mainly with the N/P ratio used for each system. The screening study allowed the development of biocompatible polyplexes with morphology, size and surface loading suitable for the incorporation of pDNA and MTX, directed at its delivery to tumor cells of the cervix. The results obtained were promising and encouraged in vitro cell transfection tests.
In Portugal, it is estimated that 350 women die each year with cervical cancer, being Portugal one of 20 countries of Europe with the highest incidence and mortality rates. This cancer is usually the result of human papilloma virus (HPV) infection and it is the fourth leading cause of death in women worldwide. Its progression results from the expression of HPV E6 and E7 oncoproteins, which are capable of altering the cell cycle, interacting with a wide variety of proteins, namely with the p53 tumor suppressor. This interaction leads to the labeling of p53 for the purpose of being degraded by the proteosome. Since its function is to induce infected cells or with DNA damage in apoptosis, it stops performing its function, leading to the uncontrolled proliferation of the malignant cells, causing tumors. In this sense, gene therapy, using of safe and efficient polymer-based non-viral delivery vectors of gene material, appears as a promising alternative for the therapy of cervical cancer with the aim of restoring normal levels of p53 in the target cells. Furthermore, considering the clinical advances in cancer therapy described in the literature, the combination of gene therapy with chemotherapy brings numerous benefits. Thus, the main goal of this work was the development of a delivery system capable of simultaneously incorporating plasmid DNA (pDNA) and an anti-cancer drug, methotrexate (MTX). MTX is a drug that not only allows to give targeting and selectivity to the delivery vehicle, but also has chemotherapeutic activity complementing the intended therapeutic effect. The use of polyethyleneimine (PEI) was the strategy chosen to condense and encapsulate the pDNA into polyplexes. The effect of molecular weight and polymer architecture on the properties (morphology, size, zeta potential, pDNA encapsulation rate and cytotoxicity) of the PEI/pDNA complexes, was studied. The results demonstrate a variation in the characteristics of the nanoparticles with the molecular weight and architecture of the polymer, but mainly with the N/P ratio used for each system. The screening study allowed the development of biocompatible polyplexes with morphology, size and surface loading suitable for the incorporation of pDNA and MTX, directed at its delivery to tumor cells of the cervix. The results obtained were promising and encouraged in vitro cell transfection tests.
Description
Keywords
Mtx Padn Pei Poliplexos Terapia Génica