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Development of tools and technologies for the characterisation of novel drug delivery systems

Riester, Pamela 2017. Development of tools and technologies for the characterisation of novel drug delivery systems. PhD Thesis, Cardiff University.
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Abstract

Biological macromolecules such as peptides have a great potential to act as therapeutic agents to cure disease. However, they are unable to translocate biological membranes and require drug delivery systems (DDSs) for intracellular delivery. In order to evaluate the ability of novel DDSs to deliver peptide cargo, it is crucial that we are able to assess the functional delivery of the peptide to its intracellular target. In this study a technique based on split green fluorescent protein (GFP) has been developed to address this. Split GFP consists of two non-fluorescent fragments, GFP1-10 and a small peptide fragment called M3. In the presented assay, GFP1-10 acts as a cytosolic target protein expressed in HeLa cells and M3 mimics the peptide cargo that is delivered by a DDS. Upon functional delivery of the M3 peptide by a DDS to the cytosol, M3 and GFP1-10 complement to form full length GFP and GFP fluorescence is rescued. In this thesis, the development of this split GFP based method is described. Furthermore, a stable cell line expressing GFP1-10 was generated and we were able to show that our model DDS, the cell penetrating peptide octaarginine (R8), functionally delivers M3 to its cytosolic GFP1-10 target in a concentration dependent manner. The addition of two Phenylalanine residues to R8-M3 (FFR8-M3) significantly increases M3 delivery. Furthermore it is shown that M3 delivery can be investigated in real time using a widefield microscope. Moreover, the split GFP complementation assay not only shows if and when the M3 peptide cargo is delivered to its intracellular target but also where it localises in the cell. It is demonstrated that the subcellular localisation of complemented GFP is dependent on the DDS utilised to deliver M3. A second system has been developed within this project where the fluorescent protein mCherry acts as an expression marker of GFP1-10 expression. This system has the potential to correlate complemented GFP fluorescence with the amount of expressed GFP1-10 in order to gain information how much M3 peptide has been delivered to the cytosol.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Biosciences
Date of First Compliant Deposit: 17 March 2018
Last Modified: 03 May 2019 02:34
URI: http://orca.cf.ac.uk/id/eprint/109953

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