Fabrication of an aptamer-functionalised silica nanoparticle construct and its separation by magnetic capture-hybridisation

Nanoparticles produced with surfaces functionalised by highly specific molecular tags are able to target aberrant cells and detect or eliminate them without causing damage to surrounding healthy tissues. Single-stranded DNA (ssDNA) and RNA which fold to form secondary or tertiary structures, termed aptamers, represent a new class of such molecular tags. The nanoparticles, in turn, may carry therapeutic payload or luminescent entities which enable elimination or visualisation of targeted cells respectively.This project presents fabrication and isolation of a surface-functionalised nanoparticle construct, namely aptamer-tagged silica nanoparticles. DNA aptamers were chosen with the intention to make them useful for clinical or diagnostic applications of targeting neoplastic cells. Indeed, the ssDNA applied here is known to bind mucin-1 which in turn is a biomarker found on the surface of metastatic breast cancer cells. The separation of the construct was made possible by the inclusion of oligonucleotide-bound superparamagnetic particles in the construct; these enabled separation by magnetic capture.This project investigates two approaches to fabrication of the construct. In the first approach, aptamers, oligonucleotides and magnetic particles are mixed in solution. In the second, silica nanoparticles are functionalised with aptamers, oligonucleotides are bound to magnetic particles and the resulting two parts are hybridised together. The first approach gives higher yields. This may suggest that binding of silica nanoparticles to aptamers may hinder aptamer hybridisation to oligonucleotide fragments, thus resulting in lower construct synthesis yields. However, it is not known yet how the yield changes upon addition of silica nanoparticles into the solution. Therefore, the second experimental approach provides a starting point for fabrication and purification of an anti-cancer drug targeting platform in a simple bench-top setting.In addition, this thesis discusses the fabrication of silica nanoparticles which were intended to constitute an element of the construct. The work on nanoparticle fabrication aimed to develop a quick and repeatable synthesis method which would result in monodisperse entities. Despite trying various experimental approaches, suitable particles could not be reproducibly obtained. Agglomeration was identified as a major obstacle in the silica nanoparticle production process.Finally, this project assesses whether the chosen aptamers bind to the metastatic breast cancer cells, which would be necessary if they were to be used for diagnosis or therapy. FACS analysis indeed indicate that ssDNA aptamers attach to the MCF7 cell line, but the optimum conditions for that attachment remain to be determined.