Delivery of proteins and nucleic acids into cells is a major challenge to the development of biological therapeutics. Cell penetrating peptides (CPPs) and cationic liposomes have been shown to internalise short interfering RNA (siRNA) to achieve gene silencing, but no standard reagent exists which can safely deliver macromolecules both in vitro and in vivo. Small Molecule Carriers (SMoCs) are amphipathic α-helix mimetics displaying guanidine groups in order to mimic the structure of the CPP penetratin. Previously, SMoCs were shown to effectively deliver active proteins into cells. It is hypothesized that SMoCs may also be applied to the delivery of siRNA into cells in order to knockdown target genes. In addition, since cell surface binding is thought to be a crucial step in CPP internalisation, a new SMoC which optimises binding to proteoglycans may be more efficiently taken up. The aims of this thesis are to optimise the synthesis of the SMoCs in order to increase the quantity of product; to demonstrate that SMoCs may be used as siRNA delivery agents; and to design and synthesize a new SMoC which maximises siRNA uptake. The synthesis of SMoCs has been significantly enhanced, with the development of new reagents to improve the yields and cost of production. The electrostatic interactions of SMoCs with siRNA have been characterised, using NMR to examine a π-cation interaction which may contribute to anion binding, as well as determination of binding affinities using ITC and gel shift assays. Initial experiments using SMoC-siRNA complexes show significant mRNA knowdown which demonstrates the potential of SMoCs as siRNA delivery vectors. Finally, a new SMoC has been designed and synthesized which represents the first in a new class of dendritic SMoCs which are designed to maximise binding to the cell surface. This SMoC is also capable of delivering siRNA into cells, and may also be expanded by the addition of targeting peptides.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:565496 |
| Date | January 2011 |
| Creators | Gooding, M. J. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1333223/ |
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