Glioblastoma are aggressive brain tumours with a median survival of 15 months even with the best currently available treatment options. microRNAs (miRNA) are ~23 nucleotide natural silencing RNAs that have great potentials to improve cancer treatment outcomes. Lack of a safe, stable and efficient delivery system has, however, hindered the use of miRNAs in clinical applications. The aim is therefore to develop a miRNA delivery system adapted to glioblastoma using linear chain cationic polyamidoamine (PAA) polymers. The first part involved the development of luciferase assay that combined the measurement of gene-knockdown efficiency and cytotoxicity of miRNA nanoparticles. The simple two-step procedure was more effective and sensitive compared to the conventional protein-based normalization method. The second part was focused on the development of miRNA nanoparticles. In the initial phase, conditions required for maximum miRNA-polymer binding was achieved, however, the newly developed miRNA-PAA-nanoparticles did not produce significant functional gene-knockdown after cell treatment. The second stage was focused on the optimization of nanoparticle formulation as a function of stability in physiological ionic concentration. Stable PAA-nanoparticles displaying moderate cellular uptake and gene-knockdown were obtained. The final stage of development was focused on PAA-nanoparticle tagging with biotin, which improved their cellular uptake. This work developed simple and informative luciferase assay; the stability of miRNA-PAA-nanoparticles was improved by thiol-crosslinking and the cellular uptake was enhanced by a simple but smart method of ligand tagging. Further optimizations are needed to increase the functional performance of these potential and clinically relevant thiol-stabilized RNAi vectors.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:719440 |
| Date | January 2017 |
| Creators | Anthiya Ramamoorthi Gopalram, Shubaash |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/39449/ |
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