| Summary: | Poly (N-isopropylacrylamide) (PNIPAM) polymers are thermo-responsive and change conformation above their lower critical solution temperature (LCST). However, there is evidence that highly branched PNIPAM can be driven through a conformational change from open chain to globule at temperatures below the LCST by interaction of groups placed on the chain ends with a compatible molecule/ligand. It is less clear how the linear analogue would behave and our hypothesis is that it would collapse but with the chain ends shielded within the globule. In this study we explored the behaviour of a highly branched PNIPAM and a linear analogue that had the antibiotic vancomycin placed at the chain ends. Vancomycin binds to residues in the cell wall of Gram-positive bacteria so binding to bacteria should result in the highly branched PNIPAM undergoing a conformational change. The aim of the work reported in this thesis, therefore, was to compare the behaviour of linear and highly branched PNIPAM functionalised with vancomycin when interacting with Staphylococcus aureus with a view to better understand the role of conformation in polymer responsiveness. The reporting systems employed were the ability of the polymers to cause aggregation of bacteria and the response of the fluorescent solvatochromic dye, nile red. The highly branched PNIPAM (HB-PNIPAM) was synthesised via self-condensing vinyl polymerisation (SCVP)-reversible addition-fragmentation chain transfer (RAFT) polymerisation using 4-vinylbenzyl-1-pyrrolecarbodithioate as the chain transfer monomer. The polymer chain ends were functionalised with vancomycin via activation of carboxylic acids to succinimidyl derivative. A linear analogue of the polymer was synthesised using vinyl benzoic acid as comonomer to provide the same fraction of repeating units and aryl groups, because HB-PNIPAM consists of aryl branching points. The coil-to-globule conformational transition of the two polymers was studied by micro differential scanning calorimetry (microDSC) and turbidimetry. It was found that the LCSTs of highly branched polymers with pyrrole and carboxylic acid chain ends could be measured by turbidimetry (cloud point) and by microDSC. A cloud point for vancomycin-ended PNIPAM could only be detected for the linear version, not the HB-PNIPAM-van. However, the LCST of both polymers could be measured by microDSC. In aggregation tests with bacteria in aqueous suspension, the L-PNIPAM-van and HB-PNIPAM-van behaved very differently. Even though both polymers contained the same amount of vancomycin, only the HB-PNIPAM-van caused aggregation of S. aureus. These observations support a concept that the HB-PNIPAM-van has a core-shell structure above its LCST, with the densely packed core originating from desolvated PNIPAM and the outer shell stemming from solvated polymer that is swollen because of the presence of vancomycin. In contrast, when the L-PNIPAM-van initially bound to S .aureus and desolvation occurred, most of the vancomycin residues become shielded within the globule so are not available to stabilise the collapse and consequently bind more bacteria. It is suggested that the outer shell swelling is stabilised by electrostatic repulsion of adjacent vancomycin residues. However, a further complexity was identified which was the degree of hydrophobicity and charge of the bacterial cells. We also sought to confirm that vancomycin end groups of both linear and highly branched polymers were still functional and able to bind to their targets. The data presented here indicate that as the vancomycin residues on the chain ends of the polymers bind to their targets D-Ala-D-Ala and the two polymers did not differ in this respect. To probe the solvation state of the polymers when interacting with the target for vancomycin, two approaches were taken. First in the presence of the D-Ala-D-Ala peptide, hydration was disrupted by binding to vancomycin so that less energy was required to drive the polymer through its LCST. Second, a solvatochromic dye, nile red, provided information on the environmental polarity of the polymer. The hypothesis was that nile red could provide information on phase transition because loss of water should shift the emission wavelength or fluorescence intensity. It was found that in the presence of 108cfu/ml of S. aureus and HB-PNIPAM-van nile red fluorescence increased with the number of bacteria almost in a dose dependent manner, whereas there was relatively little change in fluorescence with the L-PNIPAM-van. This supports a model of change in the solvation of only the microenvironment around the chain ends of HB-PNIPAM-van, rather than of the whole polymer segment. Finally, we have shown that highly purified vancomycin functionalised L-PNIPAM and HB-PNIPAM, which bind to the cell surface of S. aureus without killing activity. We conclude, therefore, that the binding interaction primarily involves surface-located D-Ala-D-Ala on the bacterial cell wall.
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