Purification of DNA vaccine to prevent or treat cervical cancer

• Autor(a) principal: Simões, Ana Rita Santos
• Data de Publicação: 2016
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/6271
• topic: Cromatografia de Afinidade; Hpv; Mgco3; Monolito de Arginina Com Braço Espaçador; Nanopartículas; Plasmídeo Superenrolado; Vacina de Dna Plasmídico.; Domínio/Área Científica::Ciências Médicas::Ciências da Saúde

Human Papillomavirus (HPV) is worldwide sexually transmitted and associated with 99.7% of cervical cancer. The cancer progression is due to the expression of the oncoproteins E6 and E7, which can alter the cell cycle and are responsible for the viral replication and transformation of host cells. The vaccines available are only preventive ones, it being necessary to develop therapeutic ones, to prevent and treat a pre-existent infection. Deoxyribonucleic acid (DNA) vaccination along with the use of plasmid DNA (pDNA) as a non-viral vector arises as a good strategy that can activate both humoral and cellular immune responses, allowing the prevention and treatment of HPV infections. The combination of the amino-acid affinity chromatography (AC) with the innovative monolithic supports appears as a promising approach to obtain highly purified supercoiled (sc) pDNA – the active biological conformation – with high purity and recovery. This allows the selective interaction of specific ligands to the target biomolecule adding to the higher capacity of monoliths when compared to conventional chromatographic supports. Monoliths also allow the use of high flow rates, which allows a fast purification procedure and decreases the retention time of the target biomolecule, avoiding its degradation. In the present work, different elution strategies (manipulation of sodium chloride (NaCl) concentrations and/or pH and competition) were explored, in order to purify the supercoiled HPV-16 E6/E7MUT pDNA, by using the arginine monolith with spacer arm. The best elution strategy applied on both laboratorial and preparative scales allowed the removal of impurities within the regulatory agency recommendations, with 93.3% and 98.5% of purity degree, respectively. This reinforces the applicability of this monolith for the sc pDNA purification. Moreover, only one in thousands naked plasmids presented to the cells reach the nucleus and are expressed. The use of nanoparticles is a valuable strategy that permits the protection of the pDNA by avoiding the enzymatic degradation and facilitates the specific delivery, enhancing the cellular transfection. Thus, different magnesium carbonate (MgCO3) systems were characterized regarding its encapsulation efficiency (around 87%), morphology (round shape), size (99.7-237.4 nm) and zeta potential (positive). These data suggest that the developed nanoparticles are suitable for cellular uptake and thus appropriate for therapeutic applications. Additionally, in vitro studies accompanied with confocal microscopy were performed, which revealed that all the formulated systems are able to transfect eukaryotic cells.