Chianese, Valeria (2015) Effect of density and interaction of surface-bound complexes on substrate-mediated delivery. [Tesi di dottorato]

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Efficient and facile transfection of nucleic acids is a good tool for biological research and diagnostic applications. The crescent develop of gene transfer techniques for genomic studies, in particular has involved an increase of the investigations on gene transfer mediated by the substrate. This strategy is suitable for the manufacturing of screening platforms, such as microarrays, to in vivo and in vitro assays. Substrate-mediated transfection methods were described for delivering DNA in a slow release manner, but there is a critical need to modulate gene transfer process. In other words, a good gene delivery platform have to stably retain the gene vectors for prolonged periods and at same time mediate a efficient gene transfer when cells come in contact with it. To this address, we have investigated on different approaches of substrate-meditated delivery. In particular, have studied three kinds of interaction between gene vectors and substrate with the aim to find a compromise between the interaction strength and the effective gene transfer from substrate. In the first instance, the process through which DNA-vectors were stably tethered to a glass substrate result inefficient to mediate the transfection, despite PEI/DNA complexes were internalized by the cells seeded on this functionalized substrates. We hypothesize that a low surface density of complexes, therefore an ineffective immobilization capability affect the subsequent gene transfer. Another factor be taken into account is the high co-localization of the internalized complexes with lysosomal compartments which suggests the likely involvement of a wrong mechanism of internalization by the cells placed on modified substrate. Starting from this results, we have subsequently designed a substrate-mediate delivery with PEI/DNA complexes aspecifically adsorbed to glass slides. As expected, the successful transfection was probably affected by substrate release of the gene particles, as the quantification of the DNA/PEI complexes internalized by cells seeded on substrates in time indicates. In this case, in fact, there is not high lysosomal co-localization of the complexes inside cells on substrates. At least, we have conduct a preliminary study with the aim to test a specific adsorption of gene particles to the substrate. To address this purpose we have investigated the binding affinity of a linker peptide to fibronectin coated substrate. Preliminary results confirm the specificity of selected peptide for the protein, in this way it will be possible adsorbed PEI/DNA complexes in specific way to a coated substrate, that simultaneously promotes cellular adhesion. This strategy to built a substrate-mediate gene delivery platform can be implement with the spatial protein patterning such as adhesive/transfective islands on substrate

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