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Title: Intracellular delivery vehicles based on 2-(methacryloyloxy)ethyl phosphorylcholine.
Author: Warren, Nicholas John
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2011
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This thesis focuses on the use of both atom transfer radical polymerisation (A TRP) and reversible addition fragmentation chain transfer (RAFT) polymerisation for the synthesis of controlled-structure poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC}-based copolymers for biological applications. A TRP was used to prepare various fluorescent PMPC homopolymers, statistical copolymers and PMPC-poly(2-diisopropylamino)ethyl methacrylate) (PDPA) block copolymers. (Co)polymers were terminally labelled using a rhodamine 6G-functionalisecl ATRP initiator and statistical copolymers were prepared by copolymerising approximately one unit of either rhodamine 6G methacrylate, rhodamine B acrylate or the commercially available fluorescein-O-methacrylate per PMPC chain. Labelled copolymers prepared using the fluorescent methacrylic comonomers exhibited relatively low polydispersities for mean degrees of polymerisation up to 200. A post-polymerisation functionalisation technique was also used to attach commercially available thiol-reactive dyes to (co)polymers containing a central disulfide bond. Con focal microscopy studies revealed that efficient staining of human dermal fibroblast (HDF) cells was achieved within one hour incubation. Depending on the nature of the fluorescent label, organelle specificity was also observed: rhodamine labelled (co)polymers selectively stain the mitochondria, while fluorescein-labelled copolymers were distributed throughout the cellular matrix. MTT assays indicated negligible toxicity for all the (co)polymers after 24 h incubation at 37°C. Finally, uptake inhibition studies indicated that the internalisation pathway of the rhodamine-labelled polymers is energy-dependent and relies on recognition of the PC groups the (co)polymer chains by scavenger receptors on the cell surface. RAFT polymerisation was used to synthesise a series of PMPC macro-chain transfer agents (CTAs) using a range of RAFT CTAs. These syntheses were all well controlled with low polydispersity macro-CT As of varying molecular weights produced. After purification, selected PMPC macro-CTAs could be chain-extended with DPA to produce a series of pH-responsive amphiphilic diblock copolymers. By tailoring the relative block compositions and varying the preparation method, either spherical micelles, cylindrical (or worm-like) micelles or polymcrsomes could be produced. Using pH-induced self-assembly, it was only possible to form spherical micelles (PDPA DP:'S 59), polymersomes (PDPA DP ~ 77), or a mixture of the two phases (POP A DP = 67). Using both thin film rehydration and solvent-exchange techniques, it was also possible to form worm-like micelles. This was the major nano-structure observed when the PDPA DP was 67. Incubation of selected copolymers with HDF cells confirmed negligible cytotoxicity for all the copolymers, irrespective of end-group, DP or morphology. Moreover, it was also possible to deliver rhodamine B octadecyl ester to the HDF cells. These preliminary studies suggest that the internalisation kinetics may depend on the morphology of the nano-structures. Finally, the use of PMPC macro-CT As as steric stabilisers for the aqueous emulsion polymerisation of DPA was investigated. Using several PMPC25 macro-CTAs, a series of PMPC-PDP A nanolatexes were synthesised, for which the mean hydrodynamic diameter increased systematically with PDPA DP. The most stable formulations consisted of POP A DPs above 250. It was possible to encapsulate rhodamine B octadecyl ester during the synthesis of a PMPC2s-PDPA250 nanolatex and deliver it to HDF cells without causing significant toxicity (cell viability> 80 % after 24 h incubation at 37°C). Cross-linking such nanolatexes was achieved by addition of a small amount of dimethacrylate comonomer during the latex synthesis. This resulted in pH-responsive microgels that swelled on lowering the solution pH below approximately 6.5. Incorporation of a disulfide bond into the cross-linker made it possible to cleave the cross-links, causing the copolymer microgels to dissolve molecularly in acidic solution.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available