Antifreeze proteins (AFPs) are produced by polar fish in order to survive in sub-zero conditions in their habitat. These AFPs adhere to ice and inhibit the growth of larger crystals which can be difficult to reverse and fatal to the organism. The application of AFPs for use in human cell and tissue cryopreservation has been hypothesised to improve cell recover and reduce the need for organic solvent based cryoprotectants. Modulation of the ice recrystallisation inhibition (IRI) activity has been previously achieved by the fusion of type III AFP to larger maltose-binding protein, and through engineering a larger ice-binding site. In particular, these larger AFP fusions showed more thermal hysteresis activity: a phenomenon wherein the freezing point of ice is depressed below its melting point. An increase in IRI activity upon the linking of AFP type III to a dendrimer scaffold has also been reported. This scaffold both increased the size of the molecule, and allowed the displaying of multiple ice binding sites. Herein we employ a polymer-coated gold nanoparticle scaffold to enable the assembly of dense antifreeze protein layers on the nanoparticle surface. A mixture of chemical and biological ligations methods are used to enable tuning of the surface and the resulting materials show synergistic enhancements in the observed ice growth inhibition activity. The polymers in this study were prepared by controlled RAFT polymerisation, terminally functionalised by post-polymerisation modification to afford a functional group with the capacity to interact with a protein tag. These functional polymers were attached to small (&lt;5 nm) AuNPs by a simple mixing procedure, any required post-polymerisation modification was applied and the proteins were attached. Uniformity of AuNP shape/size and successful conjugation, were characterised at each step. IRI activity was assessed by the ‘SPLAT’ assay, wherein ice crystal recrystallisation size is determined by cryo-light microscopy. A library of multimeric AFP-AuNP hybrid materials was synthesized, and their IRI activity relative to free (monomeric) AFP was determined. This procedure showed the potential in using protein-AuNP materials to probe protein activity in other contexts. Further investigation is required to fully ascertain the mechanisms behind this activity enhancement, and the nature of the interaction with the ice surface, as well as potential usage in human cells.

Hybrid multivalent antifreeze protein/polymer materials

256th National Meeting (2018)

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