X-ray diffraction to probe the kinetics of ice recrystallization inhibition

Understanding the nucleation and growth of ice is crucial in fields ranging from infrastructure maintenance, to the environment, and to preserving biologics in the cold chain. Ice binding and antifreeze proteins are potent ice recrystallization inhibitors (IRI), and synthetic materials that mimic this function have emerged, which may find use in biotechnology. To evaluate IRI activity, optical microscopy tools are typically used to monitor ice grain size either by end-point measurements or as a function of time. However, these methods provide 2-dimensional information and image analysis is required to extract the data. Here we explore using wide angle X-ray scattering (WAXS/X-ray powder diffraction (XRD)) to interrogate 100's of ice crystals in 3-dimensions as a function of time. Due to the random organization of the ice crystals in the frozen sample, the number of orientations measured by XRD is proportional to the number of ice crystals, which can be measured as a function of time. This method was used to evaluate the activity for a panel of known IRI active compounds, and shows strong agreement with results obtained from cryo-microscopy, as well as being advantageous in that time-dependent ice growth is easily extracted. Diffraction analysis also confirmed, by comparing the obtained diffraction patterns of both ice binding and non-binding additives, that the observed hexagonal ice diffraction patterns obtained cannot be used to determine which crystal faces are being bound. This method may help in the discovery of new IRI active materials as well as enabling kinetic analysis of ice growth

Understanding natural and synthetic ice-active materials to aid in the development of new cryoprotective formulations

Ice formation and growth is of interest to many different fields, including food science, mechanical engineering, agriculture and cryobiology, however little is understood about the underlying mechanisms behind the nucleation and growth processes. The need to increase our understanding of ice and how it is affected by compounds with ‘antifreeze’ properties is fundamental to improving techniques for the storage of biologics. Nature has evolved to contend with a range of harsh climates; in particular, they produce cryoprotectants enabling them to survive sub-zero temperatures. Inspired by Nature’s ingenious response a range of synthetic protein mimics have been developed, which have ice growth inhibition activity. The scientific principles behind ice nucleation and growth, and the materials that affect them, as well as current techniques for analysis are detailed in Chapter 1. This thesis reports on ice-activity for a range of compounds, studying their micro- and macroscopic effects on ice, as well as any potential cryoprotective capabilities, with the view to further fundamental understanding of ice growth inhibition and aid in the development of future potent cryoprotectants. A diverse range of methods including microscopy, X-ray Diffraction (XRD) and solid state nuclear magnetic resonance (SSNMR) were used to aid in characterisation and analysis by monitoring structural changes as well as the antifreeze macromolecule:ice interface. Chapter 2 investigates cryostorage of a range of biological materials using an organic solvent-free formulation consisting of an ice growth inhibiting polymer and a secondary bulking agent. Chapter 3 details X-ray diffraction (XRD) as a new method for studying ice growth continuously as a function of time, confirming its potential as a supplementary tool to study ice growth. Chapter 4 builds upon results from microscopy and XRD-based methods by using solid state nuclear magnetic resonance (SSNMR) to enable the study of molecular-level details experimentally. SSNMR provides further evidence for the ‘turning on’ of ice recrystallisation activity (IRI) for poly(vinyl alcohol) and ice-binding for a variety of compounds. Chapter 5 focuses on ice nucleation specifically. A range of previously untested materials that feature design motifs associated with nucleators reported on in the literature were examined for ice nucleation effectiveness and IRI activity, finding none to inhibit ice growth, and that structure alone is not enough to infer nucleation effectiveness

Synthesis of anthracene conjugates of truncated antifreeze protein sequences : effect of the end group and photocontrolled dimerization on ice recrystallization inhibition activity

Biomacromolecular antifreezes distinguish ice from water, function by binding to specific planes of ice, and could have many applications from cryobiology to aerospace where ice is a problem. In biology, antifreeze protein (AFP) activity is regulated by protein expression levels via temperature and light-regulated expression systems, but in the laboratory (or applications), the antifreeze activity is “always on” without any spatial or temporal control, and hence methods to enable this switching represent an exciting synthetic challenge. Introduction of an abiotic functionality into short peptides (e.g., from solid-phase synthesis) to enable switching is also desirable rather than on full-length recombinant proteins. Here, truncated peptide sequences based on the consensus repeat sequence from type-I AFPs (TAANAAAAAAA) were conjugated to an anthracene unit to explore their photocontrolled dimerization. Optimization of the synthesis to ensure solubility of the hydrophobic peptide included the addition of a dilysine solubilizing linker. It was shown that UV-light exposure triggered reversible dimerization of the AFP sequence, leading to an increase in molecular weight. Assessment of the ice recrystallization inhibition activity of the peptides before and after dimerization revealed only small effects on activity. However, it is reported here for the first time that addition of the anthracene unit to a 22-amino-acid truncated peptide significantly enhanced ice recrystallization inhibition compared to the free peptide, suggesting an accessible synthetic route to allow AFP activity using shorter, synthetically accessible peptides with a photoreactive functionality

Ice recrystallisation inhibiting polymers prevent irreversible protein aggregation during solvent-free cryopreservation as additives and as covalent polymer-protein conjugates

Protein storage and transport is essential to deliver therapies (biologics), enzymes for biotechnological applications, and underpins fundamental structural and molecular biology. To enable proteins to be stored and transported it is often essential to freeze them, requiring cryoprotectants such as glycerol or trehalose. Here we explore the mechanisms by which poly(vinyl alcohol), PVA, a potent ice recrystallisation inhibitor protects proteins during freeze/thaw to enable solvent-free cryopreservation with a focus on comparing mixing, verses polymer-protein conjugation. A panel of poly(vinyl alcohol)s are investigated including commercial, well-defined (from RAFT), and PVA-protein conjugates, to map out PVA’s efficacy. Enzymatic activity recovery of lactate dehydrogenase was found to correlate with post-thaw aggregation state (less aggregated protein had greater activity), which was modulated by PVA’s ice recrystallisation inhibition activity. This macromolecular cryoprotectant matched the performance of glycerol, but at lower additive concentrations (as low as 1 mg.mL−1). It was also demonstrated that storage at −20 °C, rather than −80 °C was possible using PVA as a cryoprotectant, which is not possible with glycerol storage. A second protein, green-fluorescent protein (GFP), was used to enable screening of molecular weight effects and to obtain PVA-GFP bioconjugates. It was observed that covalent attachment of RAFT-derived PVA showed superior cryoprotectant activity compared to simple mixing of the polymer and protein. These results show that PVA is a real alternative to solvent-based protein storage with potential in biotechnology, food and therapeutics. PVA is already approved for many biomedical applications, is low cost and available on a large scale, making it an ideal cryoprotectant formulation enhancer

Ice recrystallization inhibiting polymers enable glycerol-free cryopreservation of microorganisms

All modern molecular biology and microbiology is underpinned by not only the tools to handle and manipulate microorganisms but also those to store, bank, and transport them. Glycerol is the current gold-standard cryoprotectant, but it is intrinsically toxic to most microorganisms: only a fraction of cells survive freezing and the presence of glycerol can impact downstream applications and assays. Extremophile organisms survive repeated freeze/thaw cycles by producing antifreeze proteins which are potent ice recrystallization inhibitors. Here we introduce a new concept for the storage/transport of microorganisms by using ice recrystallization inhibiting poly(vinyl alcohol) in tandem with poly(ethylene glycol). This cryopreserving formulation is shown to result in a 4-fold increase in E. coli yield post-thaw, compared to glycerol, utilizing lower concentrations, and successful cryopreservation shown as low as 1.1 wt % of additive. The mechanism of protection is demonstrated to be linked not only to inhibiting ice recrystallization (by comparison to a recombinant antifreeze protein) but also to the significantly lower toxicity of the polymers compared to glycerol. Optimized formulations are presented and shown to be broadly applicable to the cryopreservation of a panel of Gram-negative, Gram-positive, and mycobacteria strains. This represents a step-change in how microorganisms will be stored by the design of new macromolecular ice growth inhibitors; it should enable a transition from traditional solvent-based to macromolecular microbiology storage methods

Lifetime differences in the B⁰_s system and consequences for B⁰_s lifetime measurements

We show here a low molecular weight hydrogelator based on a functionalised-dipeptide which is stable down to temperatures of −12 °C despite being made from >99% water. This stabilty at low temperature can be extended to ∼−40 °C by gelling water : glycerol mixtures. The temperature range is wider than that of the glycerol : water mixtures alone. The rheological properties of the gels do not change at this low temperature compared to that of gels at 25 °C. This freezing point depression offers a potentially new method of transporting gels and offers the prospect of hydrogels being used at much lower working temperatures whilst retaining the desired rheological properties, this is useful for cryopreservation

The detailed statistics of YCRD.

<p>The detailed statistics of YCRD.</p

Facially Amphipathic Glycopolymers Inhibit Ice Recrystallization

Antifreeze glycoproteins (AFGPs) from polar fish are the most potent ice recrystallization (growth) inhibitors known, and synthetic mimics are required for low-temperature applications such as cell cryo­preservation. Here we introduce facially amphi­pathic glyco­polymers that mimic the three-dimensional structure of AFGPs. Glyco­polymers featuring segregated hydro­philic and hydro­phobic faces were prepared by ring-opening metathesis polymerization, and their rigid conformation was confirmed by small-angle neutron scattering. Ice recrystallization inhibition (IRI) activity was reduced when a hydro­philic oxo-ether was installed on the glycan-opposing face, but significant activity was restored by incorporating a hydrophobic dimethyl­fulvene residue. This biomimetic strategy demonstrates that segregated domains of distinct hydro­philicity/hydro­phobicity are a crucial motif to introduce IRI activity, which increases our understanding of the complex ice crystal inhibition processes