The Generation of Novel Aptamers for Glioma and their Pharmacokinetic Profile In Vitro

Manangazira, Tendai Desmond (2013) The Generation of Novel Aptamers for Glioma and their Pharmacokinetic Profile In Vitro. Masters thesis, University of Central Lancashire.

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Over the past fourteen years, the survival rate of glioma patients with the use of current therapeutic strategies has not significantly improved. This has been due to the difficulty in selectively targeting the highly infiltrative glioma cells for surgical removal, radiotherapy and chemotherapeutic treatment, which make tumour recurrence and eventual mortality inevitable. The use of aptamers has been postulated as a targeting mechanism for the selective treatment of cancer.

Aptamers are single stranded, structured nucleic acid ligands with the capacity to specifically bind to target molecules. Aptamers were obtained through SELEX (the Systematic Evolution of Ligands by EXponential Enrichment), an evolutionary selection process that isolates aptamers with a high affinity for the target molecules (low Kd) from a pool of 1014 to 1015 oligonucleotide sequences.

In this study, SELEX was optimised for the selection of aptamers against the grade IV glioma cell line U87MG with counter selection carried out against the non-cancerous foetal astrocyte cell line SVGp12. Optimisation of the SELEX protocol involved the adjustment of PCR parameters, selection cell numbers and identification of ideal cloning plasmid. Eight novel aptamers were isolated, purified and sequenced utilising the optimised method of selection.

The pharmacokinetic profile of U87TDM1 and GL44 was established utilising a fluorescence quenching assay. In utilising aptamers for the targeted therapy of glioma it is important to understand aptamer availability in serum therefore the flourescence quenching assay was carried out which illustrated aptamer intaraction with serum proteins.

Further investigations are required to characterise the affinity and specifity pharmacokinetic behaviour and stability of U87TDM1 and GL44. At present, both aptamers show potential for selective targeting of grade IV GBM cell line U87MG.

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