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Authors
Abstract(s)
O cancro é uma doença que continua a proliferar em todo o mundo, sendo uma das
principais causas de mortalidade, porém a probabilidade de encontrar a cura tem vindo a
aumentar em resultado dos vários estudos desenvolvidos ao longo dos últimos anos. O
cancro do colo do útero é a doença mais relevante associada à infeção pelo vírus do papiloma
humano (HPV), principalmente quando não é detetado precocemente, evoluindo para
formas invasivas. A sobreexpressão das oncoproteínas HPV E6 e E7 interfere na regulação
e proliferação do ciclo celular através do comprometimento das proteínas supressoras de
tumor p53 e pRb, respetivamente. Embora a vacinação profilática contra o HPV seja
atualmente a melhor estratégia para prevenir o cancro do colo do útero, esse tipo de vacina
não é eficaz no tratamento de infeções pré-existentes. Para preencher essa lacuna, vários
tipos de vacinas terapêuticas contra o HPV têm sido estudadas. As vacinas de DNA têm
assumido particular importância devido à sua capacidade de gerar respostas imunes
celulares e humorais, a partir do uso de sequências de material genético do patogénio que
se pretende combater. O DNA minicircular (mcDNA) é uma molécula de DNA inovadora e
promissora, uma vez que a ausência de sequências procarióticas supera algumas das
principais limitações do DNA plasmídeo tradicional.
O sucesso das vacinas de DNA está fortemente dependente do desenvolvimento de sistemas
eficientes de entrega de genes que devem ser capazes de condensar e proteger o DNA,
ligando-se à membrana e internalizando-se nas células eucarióticas, superando todos os
obstáculos intracelulares e extracelulares. Os vetores virais oferecem alta eficiência de
transfecção para células eucarióticas. No entanto, apresentam desvantagens significativas,
como antigenicidade, potenciais efeitos oncogénicos, possível recombinação do vírus ou
dificuldade de produção e armazenamento em larga escala. Para superar essas limitações,
várias tentativas têm sido feitas para desenvolver sistemas de entrega de genes não virais
baseados em lipossomas, polímeros catiónicos sintéticos ou naturais e péptidos de
penetração celular (CPPs).
A polietilenimina (PEI) é o polímero catiónico sintético mais utilizado, existindo em
diversos pesos moleculares e estruturas lineares ou ramificadas. A abundância de aminas
favorece a formulação de sistemas de entrega PEI/DNA com elevada carga superficial, o que
melhora a eficiência da transfecção, mas também aumenta a toxicidade. A conjugação de
PEI com outras moléculas é uma abordagem possível para reduzir a toxicidade. Para
aumentar a eficiência das vacinas de DNA, várias estratégias têm-se concentrado no
direcionamento de células apresentadoras de antigénios (APCs). O desenvolvimento de
novas formulações contendo ligandos específicos para direcionar e entregar vacinas de DNA em APCs tem sido explorado. Neste contexto, os ligandos de manose são frequentemente
usados para ligar a recetores de manose que são altamente expressos em superfícies de DC
e macrófagos.
Este trabalho teve como objetivo o desenvolvimento de novos sistemas binários e ternários,
baseados no polímero PEI, no péptido R8 e em ligandos de manose, para entrega
direcionada de uma vacina de mcDNA às APCs. Os sistemas foram formulados com vários
rácios N/P (relação entre grupos amina e grupos fosfato) e as suas propriedades estruturais,
físico-químicas e de estabilidade foram estudadas, a fim de identificar o sistema de entrega
mais eficiente. Para avaliar a captação e internalização celular bem como a eficiência de
transfeção destes sistemas, foram realizados ensaios de transfeção in vitro com diferentes
linhas celulares.
Os resultados obtidos neste trabalho revelam que a elevada densidade de carga e capacidade
de condensação do PEI e R8 aumentam a interação com o mcDNA, levando à formação de
partículas menores. A adição do polímero PEI ao sistema binário R8-manose/mcDNA não
só reduz o tamanho como também aumenta o potencial zeta e a estabilidade do sistema. Os
estudos de microscopia de fluorescência confocal confirmaram a localização intracelular de
sistemas de direcionamento, resultando na captação sustentada de mcDNA. Além disso, a
eficiência da transfecção in vitro pode ser influenciada pela presença de R8-manose, com
grandes implicações para a expressão génica. Os sistemas ternários R8-
manose/PEI/mcDNA podem ser considerados ferramentas valiosas para instigar novas
pesquisas, visando avanços no campo das vacinas de DNA.
Cancer is a disease that continues to spread worldwide and is a major cause of mortality, but the likelihood of finding a cure has increased due to the various studies that have been done in recent years. Cervical cancer is the most relevant disease associated with human papillomavirus (HPV) infection, especially when it is not detected early and develops into invasive forms. Overexpression of HPV oncoproteins E6 and E7 disrupts cell cycle regulation and proliferation through the involvement of tumor suppressor proteins p53 and pRb, respectively. Although prophylactic HPV vaccination is currently the best strategy to prevent cervical cancer, this type of vaccine is not effective in treating pre-existing infections. To address this gap, several types of therapeutic vaccines against HPV have been investigated. DNA vaccines have gained particular importance due to their ability to generate cellular and humoral immune responses by using sequences of the genetic material of the pathogen to be targeted. Mini-circular DNA (mcDNA) is an innovative and promising DNA molecule, as the absence of prokaryotic sequences overcomes some of the major limitations of traditional plasmid DNA. The success of DNA vaccines depends heavily on the development of efficient gene delivery systems that must be able to condense and protect DNA, bind to the membrane, and internalize into eukaryotic cells, overcoming all intracellular and extracellular barriers. Viral vectors provide high transfection efficiency for eukaryotic cells. However, they have significant drawbacks, such as antigenicity, potential oncogenic effects, possible recombination of the virus, or difficulties in large-scale production and storage. To overcome these limitations, several attempts have been made to develop delivery systems for non-viral genes based on liposomes, synthetic or natural cationic polymers and cellpenetrating peptides (CPPs). Polyethylenimine (PEI) is the most commonly used synthetic cationic polymer, , existing in various molecular weights and linear or branched structures. The abundance of amines favors the formulation of PEI/DNA deliver systems with a high surface loading, which improves the efficiency of transfection, but also increases toxicity. Conjugation of PEI with other molecules is a possible approach to reduce toxicity. To increase the efficiency of DNA vaccines, several strategies have focused on targeting APCs. The development of new formulations containing specific ligands to target DNA vaccines to APCs has been explored. In this context, mannose ligands that bind to mannose receptors, highly expressed on DC surfaces and macrophages, are commonly used. The aim of this work was to develop new binary and ternary systems based on the polymer PEI, R8 and mannose ligands for the targeted delivery of an mcDNA vaccine to APCs. The systems were formulated with different N/P ratios and their structural, physicochemical and stability properties were studied to identify the most efficient delivery system. To evaluate the cell uptake and internalization as well as the transfection efficiency of these systems, in vitro transfection assays were performed using different cell lines. The results obtained in this work show that the high charge density and condensation ability of PEI and R8 increase the interaction with mcDNA, leading to the formation of smaller particles. The addition of the PEI polymer to the binary R8-mannose / mcDNA system not only reduces the size, but also increases the zeta potential and stability of the system. Fluorescence Confocal microscopy studies confirmed the intracellular location of the targeting systems, resulting in sustained mcDNA uptake. Moreover, the efficiency of in vitro transfection can be affected by the presence of R8-mannose, which has significant implications for gene expression. The ternary R8-mannose / PEI / mcDNA systems can be considered as valuable tools to initiate new research aimed at advancing the field of DNA vaccines.
Cancer is a disease that continues to spread worldwide and is a major cause of mortality, but the likelihood of finding a cure has increased due to the various studies that have been done in recent years. Cervical cancer is the most relevant disease associated with human papillomavirus (HPV) infection, especially when it is not detected early and develops into invasive forms. Overexpression of HPV oncoproteins E6 and E7 disrupts cell cycle regulation and proliferation through the involvement of tumor suppressor proteins p53 and pRb, respectively. Although prophylactic HPV vaccination is currently the best strategy to prevent cervical cancer, this type of vaccine is not effective in treating pre-existing infections. To address this gap, several types of therapeutic vaccines against HPV have been investigated. DNA vaccines have gained particular importance due to their ability to generate cellular and humoral immune responses by using sequences of the genetic material of the pathogen to be targeted. Mini-circular DNA (mcDNA) is an innovative and promising DNA molecule, as the absence of prokaryotic sequences overcomes some of the major limitations of traditional plasmid DNA. The success of DNA vaccines depends heavily on the development of efficient gene delivery systems that must be able to condense and protect DNA, bind to the membrane, and internalize into eukaryotic cells, overcoming all intracellular and extracellular barriers. Viral vectors provide high transfection efficiency for eukaryotic cells. However, they have significant drawbacks, such as antigenicity, potential oncogenic effects, possible recombination of the virus, or difficulties in large-scale production and storage. To overcome these limitations, several attempts have been made to develop delivery systems for non-viral genes based on liposomes, synthetic or natural cationic polymers and cellpenetrating peptides (CPPs). Polyethylenimine (PEI) is the most commonly used synthetic cationic polymer, , existing in various molecular weights and linear or branched structures. The abundance of amines favors the formulation of PEI/DNA deliver systems with a high surface loading, which improves the efficiency of transfection, but also increases toxicity. Conjugation of PEI with other molecules is a possible approach to reduce toxicity. To increase the efficiency of DNA vaccines, several strategies have focused on targeting APCs. The development of new formulations containing specific ligands to target DNA vaccines to APCs has been explored. In this context, mannose ligands that bind to mannose receptors, highly expressed on DC surfaces and macrophages, are commonly used. The aim of this work was to develop new binary and ternary systems based on the polymer PEI, R8 and mannose ligands for the targeted delivery of an mcDNA vaccine to APCs. The systems were formulated with different N/P ratios and their structural, physicochemical and stability properties were studied to identify the most efficient delivery system. To evaluate the cell uptake and internalization as well as the transfection efficiency of these systems, in vitro transfection assays were performed using different cell lines. The results obtained in this work show that the high charge density and condensation ability of PEI and R8 increase the interaction with mcDNA, leading to the formation of smaller particles. The addition of the PEI polymer to the binary R8-mannose / mcDNA system not only reduces the size, but also increases the zeta potential and stability of the system. Fluorescence Confocal microscopy studies confirmed the intracellular location of the targeting systems, resulting in sustained mcDNA uptake. Moreover, the efficiency of in vitro transfection can be affected by the presence of R8-mannose, which has significant implications for gene expression. The ternary R8-mannose / PEI / mcDNA systems can be considered as valuable tools to initiate new research aimed at advancing the field of DNA vaccines.
Description
Keywords
Cancro do Colo do Útero Manose Péptido R8 Polietilenimina Vacinas de Dna Minicircular