RESUMO
Gene transfer into muscle cells is a key issue in biomedical research. Indeed, it is important for the development of new therapy for many genetic disorders affecting this tissue and for the use of muscle tissue as a secretion platform of therapeutic proteins. Electrotransfer is a promising method to achieve gene expression in muscles. However, this method can lead to some tissue damage especially on pathologic muscles. Therefore there is a need for the development of new and less deleterious methods. Triblock copolymers as pluronic L64 are starting to be used to improve gene transfer mediated by several agents into muscle tissue. Their mechanism of action is still under investigation. The combination of electrotransfer and triblock copolymers, in allowing softening electric field conditions leading to efficient DNA transfection, could potentially represent a milder and more secure transfection method. In the present study, we addressed the possible synergy that could be obtained by combining the copolymer triblock L64 and electroporation. We have found that a pre-treatment of cells with L64 could improve the transfection efficiency. This pre-treatment was shown to increase cell viability and this is partly responsible for the improvement of transfection efficiency. We have then labelled the plasmid DNA and the pluronic L64 in order to gain some insights into the mechanism of transfection of the combined physical and chemical methods. These experiences allowed us to exclude an action of L64 either on membrane permeabilization or on DNA/membrane interaction. Using plasmids containing or not binding sequences for NF-κB and an inhibitor of NF-κB pathway activation we have shown that this beneficial effect was rather related to the NF-κB signalling pathway, as it is described for other pluronics. Finally we address here some mechanistic issues on electrically mediated transfection, L64 mediated membrane permeabilization and the combination of both for gene transfer.