Polymers for the Neutralisation of Ricin Toxin

Abstract

This thesis employed the use of RAFT/MADIX polymerisation to prepare polymers for the neutralisation of ricin toxin. Two approaches are investigated: the first looks at the use of galactose-containing glycopolymers to inhibit ricin binding to cell surface receptors; the second involves the preparation of polymers for multivalent conjugation to anti-ricin antibody fragments. The first approach was examined briefly and showed some promise with flexibility of the linker appearing to be an important factor, presumably because a flexible linker enables the galactose moiety to achieve the right conformation for optimal binding. The second approach forms the major focus of this thesis and the biocompatible poly(N-vinyl pyrrolidone) (PVP) was the polymer of choice with the intention for use in therapeutic applications. A series of PVP star RAFT agents and their polymers were synthesised. Three different conjugation strategies are examined: amine-targeted conjugation via succinimide ester containing RAFT agent to produce PVP-NHS; thiol-targeted monovalent conjugation via aminolysis and divinyl sulfone modification of PVP-RAFT to give PVPDVS; and thiol-targeted bivalent conjugation via further modification of PVPDVS to contain a bis-sulfone based bivalent linker. End-functionalisation of PVP with these moieties was achieved for the first time therefore extending the usefulness and application of PVP polymers in bioconjugation. Conjugation specificities and strategies were confirmed with glutathione and somatostatin or BSA before being employed, with varying success, to conjugate with anti-ricin IgG Fab and rFab fragments. PVPNHS was also used to produce a seven-arm star polymer with a lysozyme core. The monovalent conjugation approaches were successfully used in linear polymer systems but have not been fully extended to multi-arm stars. Conjugation of proteins with the bivalent linker proved problematic and was further complicated by difficulties in selectively reducing disulfide bonds and/or removing reducing agent. In the case of Fab attachment it appears that size may be a factor with steric hindrance preventing such close conjugation of two separated monomeric Fab . Ultimately PVP end-functionalisation by NHS, DVS and bis-sulfone moieties was achieved and this extends the usefulness and application of PVP polymers in bioconjugation.