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Sistema de entrega génica de pBMP-2 baseado em Carbon Dots/Polietilenimina para diferenciação osteogénica de hMSCs
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Resumo(s)
A aplicação da técnica de manipulação génica a células estaminais, tornando possível o
controle da diferenciação celular, tem aberto novos caminhos à medicina regenerativa.
Neste contexto, uma vez que as células mesenquimais humanas (hMSCs) são difíceis de
transfetar, é importante desenvolver estratégias capazes de facilitar esse processo. O
objetivo principal desta dissertação foi, pois, desenvolver um sistema, à base de pontos
de carbono (carbon dots, CDs) e polietilenimina (PEI) para a entrega do gene da proteína
morfogénica do osso-2 (BMP-2) em hMSCs, com a finalidade de induzir a sua
diferenciação osteogénica. Este sistema apresenta a vantagem de associar os CDs
(nanopartículas fluorescentes que apresentam pequeno tamanho, biocompatibilidade e
facilidade de funcionalização) ao PEI (um polímero catiónico amplamente utilizado como
sistema de entrega de genes devido à sua capacidade de compactar o DNA e facilitar a
sua entrada nas células), tirando partido das qualidades dos dois componentes. Os
nanoveículos CDs/PEI formados a diferentes proporções mássicas (1CDs/1PEI,
1CDs/2PEI e 1CDs/4PEI) mostraram capacidade de complexar e neutralizar o DNA
plasmídico (pDNA). Os nanoplexos assim formados mostraram, ainda, capacidade de
transfetar o pDNA codificante da proteína verde fluorescente e da luciferase (pEGFPLuc),
a diferentes razões N/P, tanto em células HEK293T, como nas hMSCs. Adicionalmente,
estes nanoplexos foram aplicados ao transporte do gene da BMP-2 em hMSCs e, nas
razões N/P10 e N/P15, foi estudada a citotoxicidade e diferenciação osteogénica. Estes
estudos mostraram que se obteve transfeção do pBMP-2 com os nanoplexos CDs/PEI em
estudo onde, houve um aumento da atividade metabólica ao longo do tempo, propondo
a ocorrência da diferenciação celular. Aparentemente, ocorreu uma expressão inicial da
BMP-2, suficiente para aumentar a atividade metabólica celular e expressar a ALP no
período inicial da diferenciação (7 dias), não sendo claramente evidente, a formação de
depósitos de cálcio/fosfato, mesmo após os 21 dias.
The application of gene manipulation techniques to stem cells, making it possible to control cell differentiation, has opened up new avenues for regenerative medicine. In this context, since human mesenchymal stem cells (hMSCs) are difficult to transfect, it is important to develop strategies capable of facilitating this process. The main aim of this dissertation was therefore to develop a system based on carbon dots (CDs) and polyethylenimine (PEI) for the delivery of the bone morphogenic protein-2 (BMP2) gene into hMSCs, with the aim of inducing their osteogenic differentiation. This system has the advantage of combining CDs (fluorescent nanoparticles that are small in size, biocompatible and easy to functionalize) with PEI (a cationic polymer widely used as a gene delivery system due to its ability to compact DNA and facilitate its entry into cells), taking advantage of the qualities of both components. The CDs/PEI nanovehicles formed at different mass ratios (1CDs/1PEI, 1CDs/2PEI and 1CDs/4PEI) showed the ability to complex and neutralize plasmid DNA (pDNA). The nanoplexes thus formed also showed the ability to transfect the pDNA coding for green fluorescent protein and luciferase (pEGFPLuc), at different N/P ratios, in both HEK293T cells and hMSCs. In addition, these nanoplexes were applied to the transport of the BMP-2 gene in hMSCs and, at N/P10 and N/P15 ratios, cytotoxicity and osteogenic differentiation were studied. These studies showed that pBMP2 was transfected with the CDs/PEI nanoplexes under study, where there was an increase in metabolic activity over time, suggesting that cell differentiation was occurring. Apparently, there was an initial expression of BMP-2, sufficient to increase cellular metabolic activity and expressing ALP in the initial period of differentiation (7 days), and the formation of calcium/phosphate deposits was not clearly evident, even after 21 days.
The application of gene manipulation techniques to stem cells, making it possible to control cell differentiation, has opened up new avenues for regenerative medicine. In this context, since human mesenchymal stem cells (hMSCs) are difficult to transfect, it is important to develop strategies capable of facilitating this process. The main aim of this dissertation was therefore to develop a system based on carbon dots (CDs) and polyethylenimine (PEI) for the delivery of the bone morphogenic protein-2 (BMP2) gene into hMSCs, with the aim of inducing their osteogenic differentiation. This system has the advantage of combining CDs (fluorescent nanoparticles that are small in size, biocompatible and easy to functionalize) with PEI (a cationic polymer widely used as a gene delivery system due to its ability to compact DNA and facilitate its entry into cells), taking advantage of the qualities of both components. The CDs/PEI nanovehicles formed at different mass ratios (1CDs/1PEI, 1CDs/2PEI and 1CDs/4PEI) showed the ability to complex and neutralize plasmid DNA (pDNA). The nanoplexes thus formed also showed the ability to transfect the pDNA coding for green fluorescent protein and luciferase (pEGFPLuc), at different N/P ratios, in both HEK293T cells and hMSCs. In addition, these nanoplexes were applied to the transport of the BMP-2 gene in hMSCs and, at N/P10 and N/P15 ratios, cytotoxicity and osteogenic differentiation were studied. These studies showed that pBMP2 was transfected with the CDs/PEI nanoplexes under study, where there was an increase in metabolic activity over time, suggesting that cell differentiation was occurring. Apparently, there was an initial expression of BMP-2, sufficient to increase cellular metabolic activity and expressing ALP in the initial period of differentiation (7 days), and the formation of calcium/phosphate deposits was not clearly evident, even after 21 days.
Descrição
Palavras-chave
Manipulação génica Células estaminais mesenquimais humanas (hMSCs) Pontos de carbono (CDs) Polietilenimina (PEI) BMP-2 (proteína morfogenética óssea) Diferenciação osteogénica Gene manipulation Human Mesenchymal Stem Cells (hMSCs) Carbon dots (CDs) Polyethylenimine (PEI) BMP-2 (Bone Morphogenetic Protein 2) Osteogenic differentiation Bioquímica Aplicada . Faculdade de Ciências Exatas e da Engenharia