This thesis describes the development of novel pH responsive hyperbranched polymers via the Reversible Addition Fragmentation chain Transfer (RAFT) polymerisation technique, for the targeted drug delivery of cancer therapeutics via folate mediated endocytosis.
This thesis consists of four chapters, described as such: Chapter one introduces the common topics within the subject area, with a focus on the issues surrounding current drug efficacy and efficiency within cancer therapies. A broad review of the current state of the disease in the 21st century and current therapeutic developments regarding nanotechnology and targeted drug delivery are discussed. With an insight into a multitude of polymerisation and bioconjugation techniques including Free Radical Polymerisation (FRP), Atom Transfer Radical Polymerisation (ATRP), Nitroxide Mediated Polymerisation (NMP) and RAFT polymerisation, highlighting the advantages and disadvantages of each and the ability of “controlled/living” polymerisation techniques to synthesise materials with complex topology. Furthermore, an introduction into the world of “smart” materials and hyperbranched polymers is discussed, emphasising the advantages of stimuli responsive materials in drug delivery and beyond due to their ability to respond sharply to changes in the external environment facilitating actions such as drug release, gelation, changes in hydrophilic/hydrophobic character and self-assembly characteristics of the macromolecule.
Chapter two discusses the analytical methodologies adapted in detail with a background of the techniques and the methodology and hardware behind them. Additionally, contained within this chapter are all the calibration details and the details of standards run within an experimental section.
Chapter three focuses on the design and synthesis of novel hyperbranched polymers and the experimental and analytical methodology adopted discussed. Three monomers were chosen as the building blocks for RAFT co-polymerisation: 2-propyl acrylic acid (PAA) 2-(dimethylamino) ethyl methacrylate (DMAEMA) and disulfanebis(ethane-2,1-diyl) (DSDA) using 4-cyano-4-(((dodecylthio)carbonothioyl)thio)pentanoic acid as RAFT agent. The polymerisation of this macromolecule was investigated at different monomer reaction feed ratio compositions to deduce the “sweet spot” for control and optimisation. Further work then investigated the control of molecular weight and dispersity via further experiments increasing RAFT agent ratio into reaction feeds. Characterisation was performed via Nuclear Magnetic Resonance (NMR), Size Exclusion Chromatography (SEC) and Dynamic Light Scattering (DLS). This is the foundation of the project, with further bio conjugation reactions via Steiglich esterification to modify the hyperbranched structures and biological studies assessing the design of the structure discussed in chapter three.
Chapter four, as previously stated focuses on the behaviour of the material post modification, with the synthesis Poly(ethylene glycol) (PEG) based linkers used to exploit high functionality associated with hyperbranched structures to covalently bond ligands or interest onto the structure. The experimental and analytical methodology are also discussed in this chapter. Folic acid was chosen as the targeting ligand, as to exploit folate mediated endocytosis, whilst the commonly used chemotherapeutic drug gemcitabine was used for drug efficacy and efficiency studies via the MTT cytotoxicity study. Characterisation of conjugates was performed via Fourier Transform Infra-Red (FTIR) Ultra Violet Visible Spectroscopy (UV-VIS), NMR and DLS. For biological investigations the HeLa cell line was chosen as the model cell line, for both cytotoxicity and cell uptake studies, which were characterised via UV-VIS and confocal microscopy respectively.
Chapter five acts as a summation of the results presented in this thesis and aims to draw rational conclusions from the data. The initial design is critiqued against alternatives, such as: alternative monomeric building block and alternative targeting moieties, whilst possible enhancements to the structure are discussed, as a basis for future work concerning this structure. Finally, the project aims, and project vision are evaluated against the results provided.