Synthesis and characterisation of imprinted polymeric receptor mimics

Molecularly imprinted polymers (MIPs) are crosslinked polymers containing bespoke functionalised cavities arising from the inclusion of template molecules in the polymerisation mixture and their later extraction. When the polymers are prepared functional polymerisable monomers are included which become part of the polymer matrix and serve to decorate the cavities with functionality appropriate to the template molecules. Overall, binding sites are created which have a memory for the template both in terms of shape and matching functionality. Fluorescent molecularly imprinted polymers have the benefit of a fluorophore in their cavities that may respond to the presence of bound test compound by a change in their fluorescence output. The work presented falls into three main areas. A series of fluorescent MIPs was prepared with a view to generating material capable of mimicking the binding characteristics of the metabolically important cytochrome isoform CYP2D6. The MIPs re-bound their templates and various cross-reactivities were encountered for test compound/drug recognition. One MIP in particular exhibited a rational discrimination amongst the related synthetic templates and was reasonably successful in recognising CYP2D6 substrates from the drug set tested. In order to give some insights into binding modes in MIPs, attempts were made to produce functional monomers containing two or more fluorophores that could be interrogated independently. A model compound was prepared which fitted the dual-fluorophore criteria and which will be the basis for future incorporation into MIPs. A further strand to this thesis is the deliberate incorporation of hydrophobic moieties into fluorescent functional monomers so that the resulting imprinted cavities might be biomimetic in their impersonation of enzyme active sites. Thus the imprinted cavities had specific hydrophobic regions as well as the usual polar functionality with which to interact with binding test compounds.

Synthesis and evaluation of polystyrene based soluble polymeric supports for organic synthesis

Several copolymers of linear polystyrene were prepared for evaluation as soluble polymeric supports for organic synthesis. These polymers were utilized for the synthesis of ?2-isoxazoline compounds. The target compounds were synthesized via 1,3-dipolar cycloaddition reactions between polymer bound alkenes and nitrile oxides generated in situ from their corresponding aldoximes. The cleaved ?2-isoxazoline compounds were tested for biological activity against Mycobacterium fortuitum. To compare the success of these linear polystyrene copolymers, some of the ?2-isoxazoline compounds synthesized on soluble polymeric supports were also prepared via traditional crosslinked polymer supports. The polymer-bound ?2-isoxazolines were also tested for antimicrobial activity. In addition attempts were made to prepare polymers containing the ?2-isoxazolines but anchored by non-hydrolysable bonds. Although the copolymers of polystyrene gave good loading capacity in mmol/g, and being soluble in chlorinated solvents it was possible to monitor the reactions by 1H NMR spectroscopy, the cleavage of the polymer bound products proved to be quite troublesome. Product purification was not as straightforward as it was anticipated. Isolation of the cleaved target compounds proved to be time consuming and laborious when compared to the traditional organic synthesis and solid phase organic synthesis (SPOS). Polymer-bound ?2-isoxazolines close to the polymer backbone exhibited some biological activity against Staphylococcus aureus. Polymers with substitution at the para-position of the aryl substituent at position 3 of isoxazoline ring showed antimicrobial activity.

A study in radiopaque polymeric materials

The problems associated with x-ray-transparent denture base are defined and conventional approaches to their solution are assessed. Consideration of elemental absorption parameters leads to the postulation that atoms such as zinc, and bromine, may be effective radiopacifiers over at least part of the clinical x-ray spectrum. These elements had hitherto been considered too light to be effective. Investigation of copolymers of methylmethacrylate and p-bromostyrene revealed no deleterious effects arising from the aromatically brominated monomer (aliphatic bromination caused UV destabilisation). For effective x-ray absorption a higher level of bromination would be necessary, but the expense of suitable compounds made further study unjustifiable. Incorporation of zinc atoms into the polymer was accomplished by copolymerisation of zinc acrylate with methylmethacrylate in solution. At high zinc levels this produced a powder copolymer convenient for addition to dental polymers in the dough moulding process. The resulting mouldings showed increasing brittleness at high loadings of copolymer. Fracture was shown to be through the powder particles rather than around them, indicating the source of weakness to be in the internal structure of the copolymer. The copolymer was expected to be cross-linked through divalent zinc ions and its insolubility and infusibility supported this. Cleavage of the ionic cross links with formic acid produced a zinc-free linear copolymer of high molecular weight. Addition of low concentrations of acrylic acid to the dough moulding monomer appeared to 'labilise' the cross links producing a more homogeneous moulding with adequate wet strength. Toxicologically the zinc-containing materials are satisfactory and though zinc is extracted at a measurable rate in an aqueous system, this is very small and should be acceptable over the life of a denture. In other respects the composite is quite satisfactory and though a marketable product is not claimed the system is considered worthy of further study.

Cathodic protection of reinforced concrete:anodic processes in cements and related electrolytes

The nature and kinetics of electrode reactions and processes occurring for four lightweight anode systems which have been utilised in reinforced concrete cathodic protection systems have been studied. The anodes investigated were flame sprayed zinc, conductive paint and two activated titanium meshes. The electrochemical properties of each material were investigated in rapidly stirred de-oxygenated electrolytes using anodic potentiodynamic polarisation. Conductive coating electrodes were formed on glass microscope slides, whilst mesh strands were immersed directly. Oxygen evolution occurred preferentially for both mesh anodes in saturated Ca (OH)2/CaC12 solutions but was severely inhibited in less alkaline solutions and significant current only passed in chloride solutions. The main reactions for conductive paint was based on oxygen evolution in all electrolytes, although chlorides increased the electrical activity. Self-corrosion of zinc was controlled by electrolyte composition and the experimental set-up, chlorides increasing the electrical activity. Impressed current cathodic protection was applied to 25 externally exposed concrete slabs over a period of 18 months to investigate anode degradation mechanisms at normal and high current densities. Specimen chloride content, curing and reinforcement depth were also variables. Several destructive and non-destructive methods for assessing the performance of anodes were evaluated including a site instrument for quantitative "instant-off- potential measurements. The impact of cathodic protection on the concrete substrate was determined for a number of specimens using appropriate methods. Anodic degradation rates were primarily influenced by current density, followed by cemendtious alkalinity, chloride levels and by current distribution. Degradation of cementitious overlays and conductive paint substrates proceeded by sequential neutralisation of cement phases, with some evidence of paint binder oxidation. Sprayed zinc progressively formed an insulating layer of hydroxide complexes, which underwent pitting_ attack in the presence of sufficient chlorides, whilst substrate degradation was minimal. Adhesion of all anode systems decreased with increasing current density. The influence of anode material on the ionic gradients which can develop during cathodic protection was investigated. A constant current was passed through saturated cement paste prisms containing calcium chloride to central cathodes via anodes applied or embedded at each end. Pore solution was obtained from successive cut paste slices for anion and cation analyses. Various experimental errors reduced the value of the results. Characteristic S-shaped profiles were not observed and chloride ion profiles were ambiguous. Mesh anode specimens were significantly more durable than the conductive coatings in the high humidity environment. Limited results suggested zinc ion migration to the cathode region. Electrical data from each investigation clearly indicated a decreasing order of anode efficiency by specific anode material.

Polymeric films for buccal drug delivery

The aim of this research project is to evaluate whether or not pullulan films are suitable to buccal drug delivery of a phosphodiesterase5 (PDE5) inhibitor yonkenafil, which was discovered in our research group and currently is under phase II clinical trial for treatment of erectile dysfunction. Variable formulations of pullulan films were designed and the films were prepared. Mechanical properties of the films, in vitro drug release and polymer dissolution, in vitro drug penetration through porcine esophageal mucosa were investigated. The plasticization effects of solvents, polyols and acids to the films were studied by tensile test, and differential scanning calorimetry, thermogravimetric analysis, fourier transform-infrared, scanning electron microscopy, optical microscopy was applied to analyse the structure and chemical-bonding between pullulan and the additives within the films. Release mathematics models were used in the study of the mechanism of drug releases and polymer dissolutions. Ethanol, menthol, fatty acids, and sodium dodecyl sulphate were employed as penetration enhancers to pretreat the tissue. Various plasticizers and acids were applied into the films and the result showed polyethylene glycol 400 and 600 had the excellent plasticization effect on the drug-free pullulan films, while lactic acid was the best plasticizer for the drug-loaded films. Both PEG400 and lactic acid had a great effect on the drug release from the films in vitro, and all the results indicated that the hydroxyl and carboxyl groups of pullulan and the additives influenced the mechanical properties of the films significantly, and also altered drug release mechanisms. Ethanol shows the greatest enhancing ability on the drug permeation through the porcine esophageal mucosa. A possible mechanism for this is that ethanol interferes with the structure of the lipids in the mucosa, resulting in increased partitioning of the drug into the membrane.

Fabrication of magnetic drug-loaded polymeric composite nanofibres and their drug release characteristics

Magnetic polymer nanofibres intended for drug delivery have been designed and fabricated by electrospinning. Magnetite (Fe3O4) nanoparticles were successfully incorporated into electrospun nanofibre composites of two cellulose derivatives, dehydroxypropyl methyl cellulose phthalate (HPMCP) and cellulose acetate (CA), while indomethacin (IDN) and aspirin have been used as model drugs. The morphology of the neat and magnetic drug-loaded electrospun fibres and the release characteristics of the drugs in artificial intestinal juice were investigated. It was found that both types of electrospun composite nanofibres containing magnetite nanoparticles showed superparamagnetism at room temperature, and their saturation magnetisation and morphology depend on the Fe3O4 nanoparticle content. Furthermore, the presence of the magnetite nanoparticles did not affect the drug release profiles of the nanofibrous devices. The feasibility of controlled drug release to a target area of treatment under the guidance of an external magnetic field has also been demonstrated, showing the viability of the concept of magnetic drug-loaded polymeric composite nanofibres for magneto-chemotherapy.

Thiol-containing microspheres as polymeric ligands for the immobilisation of quantum dots

The first demonstration "polymeric ligands" for the immobilisation of quantum dots (QDs) is presented. Specifically, thiol-containing polystyrene microspheres were synthesised and used to incorporate QDs via a swelling/doping strategy. The resultant composite materials were shown to be highly stable against QD leaching in both apolar and polar solvents and retained an identical QD emission profile to non-immobilised QDs. This straightforward approach also allows easy access to controllable and reproducible multiple-QDcontaining microspheres.

Effects of gentamicin therapy on urinary excretion of retinol-binding protein, lysozyme and electrolytes

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A new 3-D polymeric spin transition compound: [tris(1,4-bis-(tetrazol-1-yl) butane-N1,N1′)iron(II)] bis(perchlorate)

A series of novel polymeric compounds of formula [M(btzb)3][ClO4]2 (Mll = Fe, Ni or Cu) with btzb = 1,4-bis-(tetrazol-1-yl)butane have been prepared and their physical properties investigated. The btzb ligand has been prepared and its crystal structure determined, together with a tentative crystal structure of the 3-D compound [Fe(btzb)3][ClO4]2. The model of the latter shows two symmetry-related, interpenetrating Fe-btzb networks in which the iron(II) ions approach each other as close as 8.3 and 9.1 Å. This supramolecular catenane undergoes a sharp thermal spin transition around 160 K with hysteresis (20 K) along with a pronounced thermochromic effect. The spin crossover behaviour has been followed by magnetic, DSC, optical spectroscopy and 57Fe Mössbauer spectroscopy measurements. Irradiation with green light at low temperature leads to population of the metastable high-spin state for the thermally active iron(ll) ions. The nature of the spin crossover behaviour has been discussed in detail.

Polymeric microspheres as protein transduction reagents

Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere-protein linkage was stable for ≥90 h at 37°C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake. © 2014 by The American Society for Biochemistry and Molecular Biology Inc.

Electrocoating of carbon fibres:a route for interface control in carbon fibre reinforced poly methylmethacrylate?

A simple method of creating defined PMMA and poly (MMA-co-Cz) electrocoatings on carbon fibres is described. The electrodeposition of poly methylmethacrylate (PMMA) onto unsized, unmodified carbon fibres was performed by simple constant current electrolyses of methylmethacrylate (MMA) monomer in dimethylformamide (DMF) solutions and the 'pur' liquid monomer using sodium nitrate and lithium perchlorate as supporting electrolytes. The presence of polymeric coatings successfully attached to the carbon fibres was verified by scanning electron microscopy and photoelectron spectroscopy (XPS). Performing the electrolysis in dilute MMA in DMF solutions ([MMA]

In vivo biocompatibility evaluation of composite polymeric materials for use in a novel artificial heart valve

A novel trileaflet polymer valve is a composite design of a biostable polymer poly(styrene-isobutylene-styrene) (SIBS) with a reinforcement polyethylene terephthalate (PET) fabric. Surface roughness and hydrophilicity vary with fabrication methods and influence leaflet biocompatibility. The purpose of this study was to investigate the biocompatibility of this composite material using both small animal (nonfunctional mode) and large animal (functional mode) models. Composite samples were manufactured using dip coating and solvent casting with different coating thickness (251μm and 50μm). Sample's surface was characterized through qualitative SEM observation and quantitative surface roughness analysis. A novel rat abdominal aorta model was developed to test the composite samples in a similar pulsatile flow condition as its intended use. The sample's tissue response was characterized by histological examination. Among the samples tested, the 25μm solvent-cast sample exhibited the smoothest surface and best biocompatibility in terms of tissue capsulation thickness, and was chosen as the method for fabrication of the SIBS valve. Phosphocholine was used to create a hydrophilic surface on selected composite samples, which resulted in improved blood compatibility. Four SIBS valves (two with phosphocholine modification) were implanted into sheep. Echocardiography, blood chemistry, and system pathology were conducted to evaluate the valve's performance and biocompatibility. No adverse response was identified following implantation. The average survival time was 76 days, and one sheep with the phosphocholine modified valve passed the FDA minimum requirement of 140 days with approximately 20 million cycles of valve activity. The explanted valves were observed under the aid of a dissection microscope, and evaluated via histology, SEM and X-ray. Surface cracks and calcified tissue deposition were found on the leaflets. In conclusion, we demonstrated the applicability of using a new rat abdominal aorta model for biocompatibility assessment of polymeric materials. A smooth and complete coating surface is essential for the biocompatibility of PET/SIBS composite, and surface modification using phosphocholine improves blood compatibility. Extrinsic calcification was identified on the leaflets and was associated with regions of surface cracks.