Development of New Nanocarriers for the Delivery of siRNA against RUNX1/ETO gene for the Treatment of Acute Myeloid Leukaemia

Abstract

A new platform for the delivery of short interfering RNA (siRNA) was investigated. Polymer-coated AuNPs were functionalised with phosphorothioate modified (PS) nucleic acids. The PS-modification allows their conjugation onto AuNPs though the formation of Au-S bonds. The conjugation of PS-single strand oligonucleotide (PSssODN) on polymer-coated AuNPs resulted in high loading efficiency. Particles prepared with PS-siRNA, which is a double-stranded molecule, did not show loading of siRNA. The addition of three PS-modifications on the siRNA (3PS-siRNA) did not improve the loading efficiency. These observations suggest that the conjugation of PS-ssODN onto AuNPs was not exclusively driven by the Au-S bond formation, but that the exposed bases within the single strand can also drive the conjugation onto Au, presumably through the formation of Au-N bonds. Work in this thesis also investigated the introduction of pH-sensitivity into a siRNA delivery platform. An important feature of any nanocarrier is its stimuli response towards an intracellular trigger. Polymers presenting pH-responsiveness are potentially useful in siRNA delivery as the endosomal acidic environment within the target cells can be harnessed to trigger siRNA release. The pH-sensitivity of model hydrazone and imine bonds was evaluated. The model hydrazones tested did not show the required pH-sensitivity, however, two imines were identified stable at pH 7 and hydrolyzed at pH 5, a suitable pH-sensitivity for siRNA applications. Time limits prevented the further development of the imine system, however, the model hydrazone was successfully appended onto polymer scaffolds and conjugated onto AuNPs. Their complexation with siRNA resulted in 60 % loading efficiency, however, the particles did not release siRNA after incubation in a buffer at pH 5.0. This observation confirms the poor pH-sensitivity of hydrazone bonds. Further studies must be developed to determine the pH-sensitivity of imines in polymer systems, and thus, evaluate its potential as candidates for the safe delivery of siRNA.