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Abstract(s)
Na última década, vários investigadores têm realizado diversos estudos com o intuito de projetar e desenvolver sistemas de entrega de material genético que pudessem ser biocompatíveis e competentes para internalizar em células alvo. Este enorme contributo para o sucesso das terapias génicas foi complementado com a aplicação de sistemas de co-entrega que permitem entregar simultaneamente genes e drogas anticancerígenas. O efeito sinergético demonstrado por estes agentes trouxe um enorme progresso na eficácia terapêutica para o tratamento de diversos cancros.
O cancro do colo do útero é um dos cancros com maior incidência nas mulheres em todo o mundo, especialmente em mulheres jovens. O aparecimento desta doença advém da infeção pelo vírus do papiloma humano (HPV), sendo os genótipos de elevado risco responsáveis por 99,7% dos casos. Em particular, os genótipos do tipo 16 e 18 são responsáveis por mais de 70% das ocorrências. A infeção por HPV é responsável pela integração do genoma viral nas células do colo do útero levando à interrupção da sequência da proteína E2 que é repressora das oncoproteínas E6 e E7. A oncoproteína E6 induz a degradação do supressor de tumor p53, responsável por regular a apoptose das células. No caso da proteína E7, inibe a atuação da proteína do retinoblastoma (pRb) levando a uma desregulação do ciclo celular e consequente crescimento descontrolado das células.
O objetivo deste trabalho prende-se por repor os níveis da p53 no interior das células cancerígenas do colo do útero através da entrega de um plasmídeo (pDNA) que codifica para este supressor de tumor, e em simultâneo entregar também um fármaco anticancerígeno com o objetivo final de induzir a apoptose destas células de forma mais eficiente. Para este fim, foi desenvolvido um sistema de co-entrega baseado no polímero catiónico polietilenimina (PEI) que permitiu encapsular o DNA plasmídico que contém o gene da p53. Adicionalmente, o fármaco anticancerígeno metotrexato (MTX) foi também encapsulado nestes vetores poliméricos. A utilização da droga MTX na formulação destes sistemas deve-se, não só ao seu efeito terapêutico já comprovado em diversos tipos de cancro, mas também ao facto de ser reconhecido pelos recetores do ácido fólico, abundantemente presentes nas células tumorais, conferindo assim um direcionamento dos sistemas para as células alvo.
Os resultados obtidos neste trabalho revelam que os dois sistemas formulados (PEI/pDNA e PEI/pDNA/MTX) produziram nanotransportadores com características apropriadas em termos de morfologia, tamanho e carga à superfície para a captação e internalização celular dos dois agentes terapêuticos. As células HeLa foram transfetadas com sucesso pelos sistemas que contêm o gene responsável pela produção da proteína supressora de tumor p53. O efeito terapêutico dos dois sistemas foi avaliado, comprovando-se que os poliplexos que continham os dois bioativos inibiram com maior eficácia o crescimento das células tumorais em comparação com o sistema PEI/pDNA. O efeito sinergético evidenciado nas células cancerígenas com a utilização desta co-entrega de um agente genético e de um fármaco anticancerígeno é promissor para estudos adicionais in vivo e futura aplicação terapêutica. Além disso, a utilização combinada de quimioterapia e terapia génica nas células alvo representa um avanço incrível no desenvolvimento de intervenções terapêuticas inovadoras, podendo assim contribuir grandemente para a evolução do tratamento de vários tipos de cancro.
In the last decade, several researchers have conducted diverse studies with the aim of designing and developing delivery systems for genetic material that could be biocompatible and competent to internalize in target cells. This enormous contribution to the success of gene therapies was been complemented by the application of co-delivery systems, which allow to simultaneously deliver genes and anticancer drugs. The synergistic effect demonstrated by these agents has brought tremendous progress in therapeutic efficacy for the treatment of various cancers. Cervical cancer is one of the cancers with the highest incidence in women worldwide, especially in young women. The cause of this disease is due to infection by the human papillomavirus (HPV), with high risk genotypes accounting for 99.7% of the cases. Specifically, genotypes of type 16 and 18 are responsible for more than 70% of occurrences. HPV infection is responsible for integrating the viral genome into the cells of the cervix, leading to the disruption of the E2 protein sequence – repressor of E6 and E7 oncoproteins. The E6 oncoprotein induces degradation of the p53 tumor suppressor, responsible for regulating cell apoptosis while E7 protein inhibits the action of the retinoblastoma protein (pRb), leading to a dysregulation of the cell cycle and consequent uncontrolled growth of cells. The aim of this work is to restore the p53 levels within cervical cancer cells by delivering a plasmid encoding this tumor suppressor gene and, simultaneously, delivering an anticancer drug with the ultimate goal of inducing apoptosis of these cells more efficiently. To this end, a co-delivery system based on the cationic polymer polyethyleneimine (PEI) was developed, which allowed to encapsulate the plasmid DNA containing the p53 gene. In addition, the anticancer drug methotrexate (MTX) was also encapsulated into these polymeric vectors. The use of the MTX drug in the formulation of these systems is due not only to its already proven therapeutic effect in several types of cancer but also to the fact that it’s recognized by folic acid receptors, abundantly present in tumor cells, thus conferring cancer cells targeting effect. The results obtained in this work show that the two formulated systems (PEI/pDNA and PEI/pDNA/MTX) produced nanotransporters with appropriate characteristics in terms of morphology, size and surface charge, gene and drug loading/encapsulation efficiencies suitable for both the cellular uptake and internalization of the two therapeutic agents. HeLa cells were successfully transfected by systems containing the gene responsible for the production of p53 tumor suppressor protein. The therapeutic effect of the two systems was evaluated, proving that the polypeptides containing the two therapeutic agents were more effective to inhibit tumor cell growth, when compared to the PEI/pDNA system. The synergistic effect evidenced in cancer cells with the use of this co-delivery of a gene agent and an anticancer drug is promising for additional in vivo studies and future therapeutic application. In addition, the combined use of chemotherapy and gene therapy in target cells represents an incredible advance in the development of innovative therapeutic interventions and can thus greatly contribute to the evolution of the treatment of various types of cancer.
In the last decade, several researchers have conducted diverse studies with the aim of designing and developing delivery systems for genetic material that could be biocompatible and competent to internalize in target cells. This enormous contribution to the success of gene therapies was been complemented by the application of co-delivery systems, which allow to simultaneously deliver genes and anticancer drugs. The synergistic effect demonstrated by these agents has brought tremendous progress in therapeutic efficacy for the treatment of various cancers. Cervical cancer is one of the cancers with the highest incidence in women worldwide, especially in young women. The cause of this disease is due to infection by the human papillomavirus (HPV), with high risk genotypes accounting for 99.7% of the cases. Specifically, genotypes of type 16 and 18 are responsible for more than 70% of occurrences. HPV infection is responsible for integrating the viral genome into the cells of the cervix, leading to the disruption of the E2 protein sequence – repressor of E6 and E7 oncoproteins. The E6 oncoprotein induces degradation of the p53 tumor suppressor, responsible for regulating cell apoptosis while E7 protein inhibits the action of the retinoblastoma protein (pRb), leading to a dysregulation of the cell cycle and consequent uncontrolled growth of cells. The aim of this work is to restore the p53 levels within cervical cancer cells by delivering a plasmid encoding this tumor suppressor gene and, simultaneously, delivering an anticancer drug with the ultimate goal of inducing apoptosis of these cells more efficiently. To this end, a co-delivery system based on the cationic polymer polyethyleneimine (PEI) was developed, which allowed to encapsulate the plasmid DNA containing the p53 gene. In addition, the anticancer drug methotrexate (MTX) was also encapsulated into these polymeric vectors. The use of the MTX drug in the formulation of these systems is due not only to its already proven therapeutic effect in several types of cancer but also to the fact that it’s recognized by folic acid receptors, abundantly present in tumor cells, thus conferring cancer cells targeting effect. The results obtained in this work show that the two formulated systems (PEI/pDNA and PEI/pDNA/MTX) produced nanotransporters with appropriate characteristics in terms of morphology, size and surface charge, gene and drug loading/encapsulation efficiencies suitable for both the cellular uptake and internalization of the two therapeutic agents. HeLa cells were successfully transfected by systems containing the gene responsible for the production of p53 tumor suppressor protein. The therapeutic effect of the two systems was evaluated, proving that the polypeptides containing the two therapeutic agents were more effective to inhibit tumor cell growth, when compared to the PEI/pDNA system. The synergistic effect evidenced in cancer cells with the use of this co-delivery of a gene agent and an anticancer drug is promising for additional in vivo studies and future therapeutic application. In addition, the combined use of chemotherapy and gene therapy in target cells represents an incredible advance in the development of innovative therapeutic interventions and can thus greatly contribute to the evolution of the treatment of various types of cancer.
Description
Keywords
Cancro do Colo do Útero Metotrexato Polietilenimina Sistemas de Co-Entrega Vírus do Papiloma Humano