Composite cell support membranes based on collagen and polycaprolactone for tissue engineering of skin

The preparation and characterisation of collagen:PCL composites for manufacture of tissue engineered skin substitutes and models are reported. Films having collagen:PCL (w/w) ratios of 1:4, 1:8 and 1:20 were prepared by impregnation of lyophilised collagen mats by PCL solutions followed by solvent evaporation. In vitro assays of collagen release and residual collagen content revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the composite that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. DSC analysis revealed the characteristic melting point of PCL at around 60°C and a tendency for the collagen component, at high loading, to impede crystallinity development within the PCL phase. The preparation of fibroblast/composite constructs was investigated using cell culture as a first stage in mimicking the dermal/epidermal structure of skin. Fibroblasts were found to attach and proliferate on all the composites investigated reaching a maximum of 2×105/cm2 on 1:20 collagen:PCL materials at day 8 with cell numbers declining thereafter. Keratinocyte growth rates were similar on all types of collagen:PCL materials investigated reaching a maximum of 6.6×104/cm2 at day 6. The results revealed that composite films of collagen and PCL are favourable substrates for growth of fibroblasts and keratinocytes and may find utility for skin repair. © 2003 Elsevier Ltd. All rights reserved.

Combinatorial solid phase synthesis of multiply substituted 1,4-benzodiazepines and affinity studies on the CCK2 receptor (Part 1)

One hundred sixty-eight multiply substituted 1,4-benzodiazepines have been prepared by a five-step solid-phase combinatorial approach using syn-phase crowns as a solid support and a hydroxymethyl-phenoxy-acetamido linkage (Wang linker). The substituents of the 1,4-benzodiazepine scaffold have been varied in the -3, -5, -7, and 8-positions and the combinatorial library was evaluated in a cholecystokinin (CCK) radioligand binding assay. 3-Alkylated 1,4-benzodiazepines with selectivity towards the CCK-B (CCK2) receptor have been optimized on the lipophilic side chain, the ketone moiety, and the stereochemistry at the 3-position. Various novel 3-alkylated compounds were synthesized and [S]3-propyl-5-phenyl-1,4-benzodiazepin-2-one, [S]NV-A, has shown a CCK-B selective binding at about 180 nM. Fifty-eight compounds of this combinatorial library were purified by preparative TLC and 25 compounds were isolated and fully characterized by TLC, IR, APCI-MS, and 1H/13C-NMR spectroscopy.

Solid-supported boronic acid conjugates for sugar and glycopeptide recognition

The Fmoc synthetic strategy was employed to synthesise two identical combinatorial peptide libraries on a hydrophilic PEG-PS resin. One library was appended with boronic acid moieties at two positionally-fixed locations. Successful inclusion of the boronic acid units was confirmed using a novel UV fluorescent colorimetric assay employing carminic acid as the dye compound. A study of the effect had by the resin-bound peptides bearing boronic acid groups on the binding characteristics of vancomycin, a medically relevant antibiotic glycoprotein, was conducted. In all, 132 library compounds were tested for their binding affinity with vancomycin, via immobilisation of the glycopeptide onto the solid support through hydrogen bonding or complexation with the boronic acid moieties. Subsequent cleavage via acidolysis afforded vancomycin containing solutions which were quantified by growth inhibition of methicillin susceptible Staphylococcus aureus. Comparison of the diameters of the resultant zones of inhibition and those produced by vancomycin of known concentrations afforded a means of calculating the vancomycin concentration of the cleavage solutions, and thereby determining the binding affinity of vancomycin to each peptide sequence. Five peptide sequences and twenty one of the peptidyl-boronic acid sequences showed zones of inhibition, demonstrating their reversible affinity for vancomycin. Three peptide sequences showed zones of inhibition in both libraries. The presence of boronic acid was therefore shown to impart, enhance, detract and remove the affinity of vancomycin to a range of resin-bound peptide sequences.

Solid phase synthesis of a metronidazole oligonucleotide conjugate

Direct, solid phase synthesis of an oligonucleotide conjugate of the antibiotic drug metronidazole was accomplished by the phosphoramidite method. Removal of protecting groups and cleavage from the controlled pore glass (CPG) solid support was successful using mild conditions (20% EtN in pyridine, then conc. NH (aq) at rt for 30 min) whereas standard conditions (conc. NH (aq) at 55°C for 16 h) cleaved the drug. © 2006 by MDPI.

Alumina-grafted SBA-15 as a high performance support for Pd-catalysed cinnamyl alcohol selective oxidation

Ultrathin alumina monolayers grafted onto an ordered mesoporous SBA-15 silica framework afford a composite catalyst support with unique structural properties and surface chemistry. Palladium nanoparticles deposited onto Al-SBA-15 via wet impregnation exhibit the high dispersion and surface oxidation characteristic of pure aluminas, in conjunction with the high active site densities characteristic of thermally stable, high-area mesoporous silicas. This combination confers significant rate enhancements in the aerobic selective oxidation (selox) of cinnamyl alcohol over Pd/Al-SBA-15 compared to mesoporous alumina or silica supports. Operando, liquid-phase XAS highlights the interplay between dissolved oxygen and the oxidation state of palladium nanoparticles dispersed over Al-SBA-15 towards on-stream reduction: ambient pressures of flowing oxygen are sufficient to hinder palladium oxide reduction to metal, enabling a high selox activity to be maintained, whereas rapid PdO reduction and concomitant catalyst deactivation occurs under static oxygen. Selectivity to the desired cinnamaldehyde product mirrors these trends in activity, with flowing oxygen minimising CO cleavage of the cinnamyl alcohol reactant to trans-β-methylstyrene, and of cinnamaldehyde decarbonylation to styrene. © 2013 Elsevier B.V.

LacI-mediated sequence-specific affinity purification of plasmid DNA for therapeutic applications

Affinity purification of plasmid DNA is an attractive option for the biomanufacture of therapeutic plasmids, which are strictly controlled for levels of host protein, DNA, RNA, and endotoxin. Plasmid vectors are considered to be a safer alternative than viruses for gene therapy, but milligram quantities of DNA are required per dose. Previous affinity approaches have involved triplex DNA formation and a sequence-specific zinc finger protein. We present a more generically applicable protein-based approach, which exploits the lac operator, present in a wide diversity of plasmids, as a target sequence. We used a GFP/His-tagged Lacl protein, which is precomplexed with the plasmid, and the resulting complex was immobilized on a solid support (TALON resin). Ensuing elution gives plasmid DNA, in good yield (>80% based on recovered starting material, 35-50% overall process), free from detectable RNA and protein and with minimal genomic DNA contamination. Such an affinity-based process should enhance plasmid purity and ultimately, after appropriate development, may simplify the biomanufacturing process of therapeutic plasmids.

Nucleoside and oligonucleotide approaches towards potential HIV chemotherapies

The use of oligonucleotides directed against the mRNA of HIV promises site-specific inhibition of viral replication. In this work, the effect of aralkyl substituents on oligonucleotide duplex stability was studied using model oligonucleotide sequences in an attempt to improve targeting of oligonucleotides to viral mRNA. Arakyl-substituted oligonucleotides were made by solid phase synthesis using either the appropriate aralkyl-substituted phosphoramidite or by post-synthetic substitution of a pentafluorophenoxy substituent by N-methylphenethylamine. The presence of phenethyl or benzoyl substituents invariably resulted in thermodynamic destabilisation of all duplexes studied. The methods which were developed for the synthesis of nucleoside intermediates for oligonucleotide applications were also used to prepare a series of nucleoside analogues derived from uridine, 2'-deoxyuridine and AZT. Crystal structures of six compounds were successfully determined. Anti-HIV activity was observed for most compounds in the series although none were without cytotoxicity. The most active compound of the series was the ribose nucleoside; 1-β-D-erythro-pentofuranosyl-4-pentafluorophenoxy-pyrimidine-2(1H)-one 95, derived directly from uridine. The same series of compounds also displayed very modest anti-cancer activity. To enable synthesis of prooligonucleotides and analogues for possible antisense applications, the properties of a new Silyl-Linked Controlled Pore Glass solid support were investigated. Synthesis of the sequences d(Tp)7T, d(Tps)7T and the base-sensitive d(Tp)3(CBzp)2(Tp)2T was achieved using the silyl-linked solid support in a fluoride-induced cleavage/deprotection strategy.

Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker

The sequence-specific affinity chromatographic isolation of plasmid DNA from crude lysates of E. coli DH5α fermentations is addressed. A zinc finger-GST fusion protein that binds a synthetic oligonucleotide cassette containing the appropriate DNA recognition sequence is described. This cassette was inserted into the Smal site of pUC19 to enable the affinity isolation of the plasmid. It is shown that zinc finger-GST fusion proteins can bind both their DNA recognition sequence and a glutathione-derivatized solid support simultaneously. Furthermore, a simple procedure for the isolation of such plasmids from clarified cell lysates is demonstrated. Cell lysates were clarified by cross-flow Dean vortex microfiltration, and the permeate was incubated with zinc finger-GST fusion protein. The resulting complex was adsorbed directly onto glutathione-Sepharose. Analysis of the glutathione-eluted complex showed that plasmid DNA had been recovered, largely free from contamination by genomic DNA or bacterial cell proteins. © 2002 Wiley Periodicals, Inc.

The influence of ageing on fatigue crack growth in SiC-particulate reinforced 8090

A study of the influence of SiC-particulate reinforcement on ageing and subsequent fatigue crack growth resistance in a powder metallurgy 8090 aluminium alloy-SiC composite has been made. Macroscopic hardness measurements revealed that ageing at 170°C in the composite is accelerated with respect to the unreinforced alloy, though TEM studies indicate that this is not due to the enhanced precipitation of S′. Fatigue crack growth rates in the naturally aged condition of the composite and unreinforced matrix are similar at low to medium values of ΔK, but diverge above ≈ 8 MPa√m owing to the lower fracture toughness of the composite. As a result of the presence of the reinforcement, planar slip in the composite is suppressed and facetted crack growth is not observed. Ageing at or above 170°C has a deleterious effect on fatigue crack growth. Increased ageing time decreases the roughness of the fracture path at higher growth rates. These effect are though to be due to microstructural changes occurring at or near to the SiC/matrix interfaces, providing sites for static mode failure mechanisms to operate. This suggestion is supported by the observation that as ΔK increases, crack growth rates become Kmax dependent, implying the crack growth rate is strongly influenced by static modes.

High speed DSC (hyper-DSC) as a tool to measure the solubility of a drug within a solid or semi-solid matrix

Conventional differential scanning calorimetry (DSC) techniques are commonly used to quantify the solubility of drugs within polymeric-controlled delivery systems. However, the nature of the DSC experiment, and in particular the relatively slow heating rates employed, limit its use to the measurement of drug solubility at the drug's melting temperature. Here, we describe the application of hyper-DSC (HDSC), a variant of DSC involving extremely rapid heating rates, to the calculation of the solubility of a model drug, metronidazole, in silicone elastomer, and demonstrate that the faster heating rates permit the solubility to be calculated under non-equilibrium conditions such that the solubility better approximates that at the temperature of use. At a heating rate of 400°C/min (HDSC), metronidazole solubility was calculated to be 2.16 mg/g compared with 6.16 mg/g at 20°C/min. © 2005 Elsevier B.V. All rights reserved.

Solid-phase combinatorial chemistry and scintillation proximity assays

The Scintillation Proximity Assay (SPA) is a method that is frequently used to detect and quantify the strength of intermolecular interactions between a biological receptor and ligand molecule in aqueous media. This thesis describes the synthesis of scintillant-tagged-compounds for application in a novel cell-based SPA. A series of 4-functianlised-2,5-diphenyloxazole molecules were synthesised. These 4-functionalised-2,5-diphenyloxazoles were evaluated by Sense Proteomic Ltd. Accordingly, the molecules were evaluated for the ability to scintillate in the presence of ionising radiation. In addition, the molecules were incorporated into liposomal preparations which were subsequently evaluated for the ability to scintillate in the presence of ionising radiation. The optimal liposomal preparation was introduced into the membrane of HeLa cells that were used successfully in a cell-based SPA to detect and quantify the uptake of [14C]methionine. This thesis also describes the synthesis and subsequent polymerisation of novel poly(oxyethylene glycol)-based monomers to form a series of new polymer supports. These Poly(oxyethylene glycol)-polymer (POP) supports were evaluated for the ability to swell and mass-uptake in a variety of solvents, demonstrating that POP-supports exhibit enhanced solvent compatibilities over several commercial resins. The utility of POP-supports in solid-phase synthesis was also demonstrated successfully. The incorporation of (4’-vinyl)-4-benzyl-2,5-diphenyloxazole in varying mole percentage into the monomer composition resulted in the production of chemically functionalised scintillant-containing poly(oxyethylene glycol) polymer (POP-Sc) supports. These materials are compatible with both aqueous and organic solvents and scintillate efficiently in the presence of ionising radiation. The utility of POP-Sc supports in solid-phase synthesis and subsequent in-situ SPA to detect and quantify, in real-time, the kinetic progress of a solid-phase reaction was exemplified successfully.In addition, POP-Sc supports were used successfully both in solid-phase combinatorial synthesis of a peptide nucleic acid (PNA)-library and subsequent screening of this library for the ability to hybridise with DNA, which was labelled with a suitable radio-isotape. This data was used to identify the dependence of the number and position of complimentary codon pairs upon the extent of hybridisation. Finally, a further SPA was used to demonstrate the excellent compatibility of POP-Sc supports for use in the detection and quantification of enzyme assays conducted within the matrix of the POP-Sc support.

A structural investigation of the alkali metal site distribution within bioactive glass using neutron diffraction and multinuclear solid state NMR

The atomic-scale structure of Bioglass and the effect of substituting lithium for sodium within these glasses have been investigated using neutron diffraction and solid state magic angle spinning (MAS) NMR. Applying an effective isomorphic substitution difference function to the neutron diffraction data has enabled the Na-O and Li-O nearest-neighbour correlations to be isolated from the overlapping Ca-O, O-(P)-O and O-(Si)-O correlations. These results reveal that Na and Li behave in a similar manner within the glassy matrix and do not disrupt the short range order of the network former. Residual differences are attributed solely to the variation in ionic radius between the two species. Successful simplification of the 2

Effect of thermal residual stresses on fatigue crack opening and propagation behavior in an Al/SiCp metal matrix composite

The effects of a thermal residual stress field on fatigue crack growth in a silicon carbide particle-reinforced aluminum alloy have been measured. Stress fields were introduced into plates of material by means of a quench from a solution heat-treatment temperature. Measurements using neutron diffraction have shown that this introduces an approximately parabolic stress field into the plates, varying from compressive at the surfaces to tensile in the center. Long fatigue cracks were grown in specimens cut from as-quenched plates and in specimens which were given a stress-relieving overaging heat treatment prior to testing. Crack closure levels for these cracks were determined as a function of the position of the crack tip in the residual stress field, and these are shown to differ between as-quenched and stress-relieved samples. By monitoring the compliance of the specimens during fatigue cycling, the degree to which the residual stresses close the crack has been evaluated. © 1995 The Minerals, Metals & Material Society.

Effect of thermal and mechanical processing on tensile properties of powder formed 2124 aluminium and 2124 Al-SiC p metal matrix composite

The aging responses of 2124 Al-SiC p metal matrix composite (MMC) and unreinforced matrix alloy are studied and related to variations in tensile properties. The MMC is aged from Wo starting conditions: (i) stretched and naturally aged and (ii) re-solution treated. Accelerated aging occurs in both MMC conditions compared with unreinforced alloy. Tensile strengths and elastic moduli are improved in the MMC compared with the alloy, but ductility is reduced. Stretched MMC exhibits higher strength but lower ductility and modulus than re-solutioned MMC. The re-solutioned MMC fails by microvoid coalescence in low aging conditions, and by void nucleation and shear in high aging conditions. Failure of the stretched MMC initiates at the surface at specimen shoulders, illustrating the increased notch sensitivity of this condition, and propagates via a zigzag shear fracture mode. Zigzag facet size increases on gross aging. Particle fracture occurs during tensile failure, but also before testing as a result of the manufacturing process. © 1995 The Institute of Materials.