Neil R. Cameron

Lectins: tools for the molecular understanding of the glycocode

Recent progress in glycobiology has revealed that cell surface oligosaccharides play an essential role in recognition events. More precisely, these saccharides may be complexed by lectins, carbohydrate-binding proteins other than enzymes and antibodies, able to recognise sugars in a highly specific manner. The ubiquity of lectin-carbohydrate interactions opens enormous potential for their exploitation in medicine. Therefore, extraordinary effort is made into the identification of new lectins as well as into the achievement of a deep understanding of their functions and of the precise mechanism of their association with specific ligands. In this review, a summary of the main features of lectins, particularly those found in legumes, will be presented with a focus on the mechanism of carbohydrate-binding. An overview of lectin-carbohydrate interactions will also be given, together with an insight into their energetics. In addition, therapeutic applications of lectins will be discussed.

High internal phase emulsions (HIPEs) — Structure, properties and use in polymer preparation

High internal phase emulsions (HIPEs) are concentrated systems possessing a large volume of internal, or dispersed phase. The volume fraction is above 0.74, resulting in deformation of the dispersed phase droplets into polyhedra, which are separated by thin films of continuous phase. Their structure, which is analogous to a conventional gas-liquid foam of low liquid content, gives rise to a number of peculiar and fascinating properties including high viscosities and viscoelastic rheological behaviour. Like dilute emulsions, HIPEs are both kinetically and thermodynamically unstable; nevertheless, it is possible to prepare metastable systems which show no change in properties or appearance over long periods of time.

Study of the formation of the open-cellular morphology of poly(styrene/divinylbenzene) polyHiPE materials by cryo-SEM

The formation of the interconnected morphology of open-cell styrene/divinylbenzene (DVB) PolyHIPE copolymers has been studied by scanning electron microscopy on frozen HIPE samples at different stages of polymerisation, a technique known as cryo-SEM. The transition from discrete emulsion droplets to interconnected cells was observed to occur around the gelpoint of the polymerising system. This would suggest that the formation of holes between adjacent cells is due to the contraction of the thin monomeric films on conversion of monomer to polymer, as a result of the higher density of the latter.

The relationship between the mechanical properties and cell behaviour on PLGA and PCL scaffolds for bladder tissue engineering

Previous work on 2D synthetic films showed growth of human bladder stromal cells was enhanced on materials with lower moduli that mimic the elastic properties of native tissue. This study developed 3D synthetic foam scaffolds for soft tissue engineering by emulsion freeze-drying. Foams of poly(lactide-co-glycolide) (PLGA) and poly(epsilon-caprolactone) (PCL) were extensively characterised using scanning electron microscopy, mercury porosimetry, dynamic mechanical analysis, degradation analysis, size exclusion chromatography and differential scanning calorimetry. Foams of 85-88% porosity and 35 microm pore diameter were selected for further study; the storage modulus of PCL foams was around half that of PLGA (2 MPa vs 4 MPa) and closer to the reported value for native bladder tissue. Urinary tract stromal cells showed a 4.4 and 2.4-fold higher attachment and rate of growth, respectively, on PCL scaffolds, as assessed by a modified 3-[4,5-dimethyl(thiazol-2yl)-3,5-diphery] tetrazolium bromide assay. A greater contractile force was exerted by cells seeded in PLGA than on PCL scaffolds, raising the possibility that the reduced rate of proliferation of cells on PLGA scaffolds may reflect differentiation into a contractile phenotype. This study has generated PCL foam scaffolds with properties that may be pertinent to the tissue engineering of the bladder and other soft tissues.

A spoonful of sugar: the application of glycopolymers in therapeutics

Glycopolymers, synthetic polymers displaying carbohydrate moieties, have been linked to many potential applications at the biology–chemistry interface. One area that holds particular promise is the employment of glycopolymers as vehicles for therapeutics or as therapeutics themselves. This review summarises some of the more prominent examples as well as those in the early stages of development.

Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge

Cultured cells are dramatically affected by the micro‐environment in which they are grown. In this study, we have investigated whether HepG2 liver cells grown in three dimensional (3‐D) cultures cope more effectively with the known cytotoxic agent, methotrexate, than their counterparts grown on traditional two dimensional (2‐D) flat plastic surfaces. To enable 3‐D growth of HepG2 cells in vitro, we cultured cells on 3‐D porous polystyrene scaffolds previously developed in our laboratories. HepG2 cells grown in 3‐D displayed excellent morphological characteristics and formed numerous bile canaliculi that were seldom seen in cultures grown on 2‐D surfaces. The function of liver cells grown on 3‐D supports was significantly enhanced compared to activity of cells grown on 2‐D standard plasticware. Unlike their 2‐D counterparts, 3‐D cultures were less susceptible to lower concentrations of methotrexate. Cells grown in 3‐D maintained their structural integrity, possessed greater viability, were less susceptible to cell death at higher levels of the cytotoxin compared to 2‐D cultures, and appeared to respond to the drug in a manner more comparable to its known activity in vivo. Our results suggest that hepatotoxicity testing using 3‐D cultures might be more likely to reflect true physiological responses to cytotoxic compounds than existing models that rely on 2‐D culture systems. This technology has potential applications for toxicity testing and drug screening.

Tailoring the morphology of emulsion-templated porous polymers

Methods with which to tailor the morphology of polystyrene-based emulsion-templated (PolyHIPE) materials are presented. Increasing the temperature of the aqueous phase used to prepare the parent emulsion leads to an increase in average void and interconnect size in the resulting porous material. Additionally, the presence in the aqueous phase of small quantities of organic additives that are capable of partitioning between the two emulsion phases also affects the morphology of the porous material obtained. The additives examined were tetrahydrofuran (THF), methanol and poly(ethylene glycol) (PEG), all of which were found to increase the average void and interconnect diameters. It is suggested that THF and, to a lesser extent, PEG enhance Ostwald ripening, resulting in emulsion coarsening over time. Evidence for this was gleaned from NMR experiments to determine the rates of water diffusion in each emulsion. However, methanol was shown not to affect the rate of water diffusion. An alternative mechanism by which methanol could affect the emulsion stability is by depleting surfactant from the interface. However, higher levels of surfactant in emulsions containing methanol did not have a significant effect on morphology. To explain this, we suggest that methanol may result in depletion of surfactant from the emulsion interface, however additional surfactant serves not only to replace this depleted surfactant but also to increase the number of w/o micelles in the continuous phase. These facilitate transport of water between droplets, thus negating the effect of replacing the surfactant lost from the interface.

Multicopy multivalent glycopolymer-stabilized gold nanoparticles as potential synthetic cancer vaccines.

Mucin-related carbohydrates are overexpressed on the surface of cancer cells, providing a disease-specific target for cancer immunotherapy. Here, we describe the design and construction of peptide-free multivalent glycosylated nanoscale constructs as potential synthetic cancer vaccines that generate significant titers of antibodies selective for aberrant mucin glycans. A polymerizable version of the Tn-antigen glycan was prepared and converted into well-defined glycopolymers by Reversible Addition–Fragmentation chain Transfer (RAFT) polymerization. The polymers were then conjugated to gold nanoparticles, yielding ‘multicopy-multivalent’ nanoscale glycoconjugates. Immunological studies indicated that these nanomaterials generated strong and long-lasting production of antibodies that are selective to the Tn-antigen glycan and cross-reactive toward mucin proteins displaying Tn. The results demonstrate proof-of-concept of a simple and modular approach toward synthetic anticancer vaccines based on multivalent glycosylated nanomaterials without the need for a typical vaccine protein component.

Emulsion-templated porous materials (PolyHIPEs) for selective ion and molecular recognition and transport: applications in electrochemical sensing

Functionalised emulsion-templated polymers (PolyHIPEs) are reported as new materials for electroanalytical applications. PolyHIPEs, which are prepared from high internal phase emulsions (HIPEs), were tailored by optimisation of polymerisation conditions to yield well-defined, tubular, porous membranes. The PolyHIPE membrane backbone was activated by incorporating ionophores, graphite particles, electron mediators and enzymes. The results show that a valinomycin ionophore impregnated, plasticised membrane shows a Nernstian response to K+ ions with improved detection limits and selectivity coefficients compared to traditional PVC membranes. The graphite/mediator/enzyme loaded membranes exhibit quasi-reversible redox behaviour with semi-infinite linear diffusion at fast scan rates tending to radial diffusion at slow scan rates. Additionally, composite, asymmetric membrane structures with a porous PolyHIPE membrane and a PVC membrane exclude proteins such as BSA (bovine serum albumin) and α1 acid glycoprotein (AAG). These preliminary results demonstrate that plasticised membranes with functionalised skeletons and with controllable porosity such as PolyHIPE membranes are very promising for the fabrication of sensors with integrated separation.

Ultra‐High Surface Area Functional Porous Polymers by Emulsion Templating and Hypercrosslinking: Efficient Nucleophilic Catalyst Supports

4-Dimethylaminopyridine (DMAP) is a highly efficient and important organocatalyst used for a variety of organic reactions, including the acylation and silylation of hindered alcohols, the Baylis–Hillman reaction and the ringopening polymerisation of lactide and other lactones. Heterogeneous versions supported on soluble and crosslinked polymers as well as inorganic nanoparticles have been described. Soluble catalysts and nanoparticles can show high activities but require extra isolation steps for catalyst recycling. On the other hand, catalysts supported on insoluble polymer beads, whilst being simple to recycle, tend to suffer from a drop in activity compared to the homogeneous catalyst. Furthermore, the performance of catalysts supported on gel-type polymer beads is limited by solvent-dependent access to the reactive sites inside the beads. The use of permanently porous (often referred to as macroporous ) polymer beads overcomes these solvent limitations, however reactions with these supports can be slow as mass transfer to the active sites on the internal surface of the porous bead occurs by diffusion only. The mass transport limitations of permanently porous beads can be overcome by using emulsion-templated porous polymers (polyHIPEs), which possess very large pores (1– 100 mm) permitting mass transfer by convection rather than diffusion. PolyHIPE materials are produced from high internal phase emulsions (HIPEs), where the dispersed phase occupies >74 % of the emulsion volume. PolyHIPEs have been prepared from either oil-in-water (o/w) or water-in-oil (w/o) emulsions and have been used in a flow through manner as scavengers, reagents, solid-phase synthesis supports and chromatography media. For polyHIPEs to function as a heterogeneous catalyst support, a high surface area is required. PolyHIPE materials with surface areas up to 690 m g 1 can be prepared by the addition of an organic porogen to the monomer phase together with careful choice of HIPE stabilising surfactant. However the resulting materials are rather weak mechanically. An alternative approach to introducing high surface areas is the hypercrosslinking method. Hypercrosslinked polymers contain a very high density of crosslinks together with molecular-sized pores (micropores), and exhibit important properties such as an ultra-high surface area (up to 2000 m g ) and the ability to sorb large amounts of both thermodynamically good and poor solvents, due to high matrix rigidity and a much reduced degree of chain entanglement. Hypercrosslinked polystyrene, in the form of beads, can be prepared by Lewis acid catalysed post polymerisation crosslinking of poly(4-vinylbenzyl chloride) (pVBC) (Scheme 1). High surface area polyHIPE materials for gas storage have recently been prepared by this method. Hypercrosslinked polyHIPE is a potentially attractive catalyst support material due to the combination in one material of an interconnected network of macropores, facilitating access of reagents to the surface, with an ultra-high surface area produced by the hypercrosslinking induced microporosity. Furthermore, hypercrosslinking VBC polyHIPEs to less than full conversion would leave residual chloromethyl functionality for the attachment of catalysts. In this work, we demonstrate proof of this concept; the hypercrosslinking of VBC polyHIPEs is controlled to leave unreacted benzyl chloride moieties with which to anchor DMAP, leading to a highly efficient, recyclable nucleophilic catalyst (Scheme 1). [a] J. Wall, Prof. N. R. Cameron Department of Chemistry and Biophysical Sciences Institute Durham University South Road, Durham, DH1 3LE (UK) Fax: (+44) 191-3844737 E-mail : n.r.cameron@durham.ac.uk [b] Dr. I. Pulko, Prof. P. Krajnc Faculty of Chemistry and Chemical Engineering University of Maribor Smetanova 17, 2000 Maribor (Slovenia) Fax: (+386)2-2527-774 E-mail : peter.krajnc@uni-mb.si [c] Dr. I. Pulko Polymer Technology College Pod gradom 4, 2380 Slovenj Gradec (Slovenia) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200903043.