‘Click’ functionalised polymer resins : a new approach to the synthesis of surface attached bipyridinium and naphthalene diimide [2]rotaxanes

Herein we describe the design and synthesis of a series of solid-tethered [2]rotaxanes utilising crown ether-naphthalene diimide or crown ether- bipyridinium host guest interactions. TentaGel polystyrene resins were initially modified in a two-stage procedure to azide functionalised beads before the target supramolecular architectures were attached using a copper catalysed “click” procedure. The final assembly was examined using IR spectroscopy and gel-phase 1H High Resolution Magic Angle Spinning (HR MAS) NMR spectroscopy. The HR MAS technique enabled a direct comparison between the solid-tethered architectures and the synthesis and characterisation of analogous solution-based [2]rotaxanes to be made.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix

Adipose tissue forms when basement membrane extract (Matrigel™) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 μL collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and β-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, β-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.

The use of phospholipid fatty acid analysis to measure impact of acid rock drainage on microbial communities in sediments

The impact of acid rock drainage (ARD) and eutrophication on microbial communities in stream sediments above and below an abandoned mine site in the Adelaide Hills, South Australia, was quantified by PLFA analysis. Multivariate analysis of water quality parameters, including anions, soluble heavy metals, pH, and conductivity, as well as total extractable metal concentrations in sediments, produced clustering of sample sites into three distinct groups. These groups corresponded with levels of nutrient enrichment and/or concentration of pollutants associated with ARD. Total PLFA concentration, which is indicative of microbial biomass, was reduced by >70% at sites along the stream between the mine site and as far as 18 km downstream. Further downstream, however, recovery of the microbial abundance was apparent, possibly reflecting dilution effect by downstream tributaries. Total PLFA was >40% higher at, and immediately below, the mine site (0-0.1 km), compared with sites further downstream (2.5-18 km), even after accounting for differences in specific surface area of different sediment samples. The increased microbial population in the proximity of the mine source may be associated with the presence of a thriving iron-oxidizing bacteria community as a consequence of optimal conditions for these organisms while the lower microbial population further downstream corresponded with greater sediments' metal concentrations. PCA of relative abundance revealed a number of PLFAs which were most influential in discriminating between ARD-polluted sites and the rest of the sites. These PLFA included the hydroxy fatty acids: 2OH12:0, 3OH12:0, 2OH16:0; the fungal marker: 18:2ω6; the sulfate-reducing bacteria marker 10Me16:1ω7; and the saturated fatty acids 12:0, 16:0, 18:0. Partial constrained ordination revealed that the environmental parameters with the greatest bearing on the PLFA profiles included pH, soluble aluminum, total extractable iron, and zinc. The study demonstrated the successful application of PLFA analysis to rapidly assess the toxicity of ARD-affected waters and sediments and to differentiate this response from the effects of other pollutants, such as increased nutrients and salinity.

Delivery of therapeutic molecules using electrosprayed polymeric particles for applications in tissue engineering

This thesis has developed an innovative technology, electrospraying, that allows biodegradable microparticles to deliver pharmaceuticals that aid bone regeneration. The establishment, characterisation and optimisation of the technique are a step forward in developing an affordable and safe alternative to the products used currently in the clinical setting for the treatment of musculoskeletal disorders. The researcher has also investigated electrospraying as a coating technique on biodegradable structures that are used to replace damaged tissues, in order to provide localised and efficient drug delivery in the site of the defect to help tissue reconstruction.

Experimental and numerical study of polymeric foam efficacy in portable water filled barriers

Portable, water filled road safety barriers are used to provide protection and reduce the potential hazard due to errant vehicles in areas where the road conditions change frequently (e.g. near road work sites). As part of an effort to reduce excessive working widths typical of these systems, a study was conducted to assess the effectiveness of introducing polymeric foam filled panels into the design. Surrogate impact tests of a design typical of such as barrier system were conducted utilising a pneumatically powered horizontal impact testing machine up to impact energies of 7.40 kJ. Results of these tests are utilised to examine the barrier behaviour, in addition to being used to validate a couple FE/SPH model of the barrier system. Once validated, the FE/SPH model it utilised as the basis for a parametric study into the efficacy and effects of the inclusion of polymeric foam filled panels on the performance of portable water filled road safety barriers. It was found that extruded polystyrene foam functioned well, with a greater thickness of the foam panel significantly reducing the impacting body velocity as the barrier began to translate.

Pathological excretion patterns of urinary proteins in miners highly exposed to dinitrotoluene

A cohort of 161 underground miners who had been highly exposed to dinitrotoluene (DNT) in the copper-mining industry of the former German Democratic Republic was reinvestigated for signs of subclinical renal damage. The study included a screening of urinary proteins excreted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and quantitations of the specific urinary proteins α 1-microglobulin and glutathione-S-transferase α (GST α) as biomarkers for damage of the proximal tubule and glutathione-S-transferase π (GST π) for damage of the distal tubule. The exposures were categorized semiquantitatively (low, medium, high, and very high), according to the type and duration of professional contact with DNT. A straight dose-dependence of pathological protein excretion patterns with the semiquantitative ranking of DNT exposure was seen. Most of the previously reported cancer cases of the urinary tract, especially those in the higher exposed groups, were confined to pathological urinary protein excretion patterns. The damage from DNT was directed toward the tubular system. In many cases, the appearance of Tamm-Horsfall protein, a 105-kD protein marker, was noted. Data on the biomarkers α 1-microglobulin, GST α, and GST π consistently demonstrated a dose-dependent increase in tubular damage, which confirmed the results of screening by SDS-PAGE and clearly indicated a nephrotoxic effect of DNT under the given conditions of exposure. Within the cluster of cancer patients observed among the DNT-exposed workers, only in exceptional cases were normal biomarker excretions found.

Melatonin synthesis : a possible indicator of intolerance to shiftwork

Hypothesis Melatonin synthesis, which is directly controlled by the central circadian pacemaker indicates the circadian phase better than rectal temperature. Methods: Thirty four men (16-32 years, 7 morning, 13 neither, 14 evening types) performed a constant routine (24-26-hr bedrest, < 30 lux, 18-20°C, hourly isocaloric diet). Salivary melatonin level was determined hourly and rectal temperature was continuously recorded. Results: The nadir of rectal temperature occurred 1.5 hr (P = 0.017), the onset of melatonin synthesis 3 hr earlier (P < 0.0001) in morning than in evening types. Morningness was not related to the quantitative but significantly to the temporal parameters, closer to those of melatonin than of rectal temperature. Conclusions: The melatonin onset is a more reliable indicator of the diurnal type than the nadir of rectal temperature. As morningness has been associated with intolerance to shiftwork, melatonin profiling provides a suitable basis for the establishment of directed preventive measures.

Colloidal drug probe : Method development and validation for adhesion force measurement using Atomic Force Microscopy

This study demonstrates a novel technique of preparing drug colloid probes to determine the adhesion force between a model drug salbutamol sulphate (SS) and the surfaces of polymer microparticles to be used as carriers for the dispersion of drug particles from dry powder inhaler (DPI) formulations. Model silica probes of approximately 4 lm size, similar to a drug particle used in DPI formulations, were coated with a saturated SS solution with the aid of capillary forces acting between the silica probe and the drug solution. The developed method of ensuring a smooth and uniform layer of SS on the silica probe was validated using X-ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Using the same technique, silica microspheres pre-attached on the AFM cantilever were coated with SS. The adhesion forces between the silica probe and drug coated silica (drug probe) and polymer surfaces (hydrophilic and hydrophobic) were determined. Our experimental results showed that the technique for preparing the drug probe was robust and can be used to determine the adhesion force between hydrophilic/ hydrophobic drug probe and carrier surfaces to gain a better understanding on drug carrier adhesion forces in DPI formulations.

Association of the SNP rs2623047 in the HSPG modification enzyme SULF1 with an Australian Caucasian Breast Cancer Cohort

Breast cancer is the second most common cancer worldwide and the most common cancer reported in women. This malignant tumour is characterised by a number of specific features including uncontrolled cell proliferation. It ranks fifth in the world as a cause of cancer death in women. Early diagnosis increases 5 year survival rates up to 95%. Heparan sulfate proteoglycans (HSPGs) are complex proteins composed of a core protein to which a number of highly sulfated side chains are synthesised by a highly co-ordinated process resulting in distinct sulfation patterns, which determine specific interations with cell-signaling partners including growth factors, their receptors, ligands and morphogens. The enzymes responsible for chain initiation, elongation and sulfation are critical for creating HS chain variability conferring biological functionality. This study investigated single nucleotide polymorphism in SULF1, the enzyme responsible for the 6-0 desulfation of heparan sulfate side chains. We investigated this SNP in an Australian Caucasian case-control breast cancer population and found a significant association between SULF1 and breast cancer at both the allelic and genotypic level (allele, p=0.016; genotype, p=0.032). Our results suggest the res2623047 SNP in SULF1 may impact breast cancer susceptibility. Specifically, the T allele of rs2623047 in SULF1 is associated with a increased risk of developing breast cancer in our cohort. The identification of markers including SULF1 may improve detection of this disease at its earliest stages improving patient treatment and prognosis.

Process considerations related to the microencapsulation of plasmid DNA via ultrasonic atomization

An effective means of facilitating DNA vaccine delivery to antigen presenting cells is through biodegradable microspheres. Microspheres offer distinct advantages over other delivery technologies by providing release of DNA vaccine in its bioactive form in a controlled fashion. In this study, biodegradable poly(D,L-lactide-coglycolide) (PLGA) microspheres containing polyethylenimine (PEI) condensed plasmid DNA (pDNA) were prepared using a 40 kHz ultrasonic atomization system. Process synthesis parameters, which are important to the scale-up of microspheres that are suitable for nasal delivery (i.e., less than 20 μm), were studied. These parameters include polymer concentration; feed flowrate; volumetric ratio of polymer and pDNA-PEI (plasmid DNA-polyethylenimine) complexes; and nitrogen to phosphorous (N/P) ratio. PDNA encapsulation efficiencies were predominantly in the range 82-96%, and the mean sizes of the particle were between 6 and 15 μm. The ultrasonic synthesis method was shown to have excellent reproducibility. PEI affected morphology of the microspheres, as it induced the formation of porous particles that accelerate the release rate of pDNA. The PLGA microspheres displayed an in vitro release of pDNA of 95-99% within 30 days and demonstrated zero order release kinetics without an initial spike of pDNA. Agarose electrophoresis confirmed conservation of the supercoiled form of pDNA throughout the synthesis and in vitro release stages. It was concluded that ultrasonic atomization is an efficient technique to overcome the key obstacles in scaling-up the manufacture of encapsulated vaccine for clinical trials and ultimately, commercial applications.

Towards the design of a scalable and commercially viable technique for plasmid purification using a methacrylate monolithic stationary phase

A monolithic stationary phase was prepared via free radical co-polymerization of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with pore diameter tailored specifically for plasmid binding, retention and elution. The polymer was functionalized. with 2-chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) for anion-exchange purification of plasmid DNA (pDNA) from clarified lysate obtained from E. coli DH5α-pUC19 culture in a ribonuclease/ protease-free environment. Characterization of the monolithic resin showed a porous material, with 68% of the pores existing in the matrix having diameters above 300 nm. The final product isolated from a single-stage 5 min anion-exchange purification was a pure and homogeneous supercoiled (SC) pDNA with no gDNA, RNA and protein contamination as confirmed by ethidium bromide agarose gel electrophoresis (EtBr-AGE), enzyme restriction analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This non-toxic technique is cGMP compatible and highly scalable for production of pDNA on a commercial level.

Creation of protein loaded biodegradable microparticles via ultrasonic atomization suitable for nasal delivery

Improved biopharmaceutical delivery may be achieved via the use of biodegradable microspheres as delivery vehicles. Biodegradable microspheres offer the advantages of maintaining sustained protein release over time whilst simultaneously protecting the biopharmaceutical from degradation. Particle samples produced by ultrasonic atomization were studied in order to determine a feed stock capable of producing protein loaded poly-ε-caprolactone (PCL) particles suitable for nasal delivery (i.e., less than 20 μm). A 40 kHz atomization system was used with a 6 mm full wave atomization probe. The effect of solids percent, feed flow rate, volumetric ratio of the polymer stock to the protein stock, and protein concentration in the protein stock on particle size characteristics were determined. It was shown that feed stocks containing 100 parts of 0.5 or 1.0% w/v PCL in acetone with one part 100 mg ml -1 BSA and 15 mg ml -1 PVA produced particles with a mass moment diameter (D[4,3]) of 13.17 μm and 9.10 μm, respectively in addition to displaying high protein encapsulation efficiencies of 93 and 95%, respectively. The biodegradable PCL particles were shown to be able to deliver encapsulated protein in vitro under physiological conditions.

Straightforward biodegradable nanoparticle generation through megahertz-order ultrasonic atomization

Simple and reliable formation of biodegradable nanoparticles formed from poly-ε-caprolactone was achieved using 1.645 MHz piston atomization of a source fluid of 0.5% w/v of the polymer dissolved in acetone; the particles were allowed to descend under gravity in air 8 cm into a 1 mM solution of sodium dodecyl sulfate. After centrifugation to remove surface agglomerations, a symmetric monodisperse distribution of particles φ 186 nm (SD=5.7, n=6) was obtained with a yield of 65.2%. © 2006 American Institute of Physics.

Synthesis of novel interlocked architectures in solution and solid phase

Mechanically interlocked molecules, such as catenanes and rotaxanes, are fascinating due to their unique sensing and catalytic properties and their potential to act as molecular motors or switches. Traditionally their synthesis has been laborious and expensive, however this research project endeavoured to overcome this challenge by exploring novel ways of preparing mechanically interlocked molecules both in solution and on surfaces. A series of disulfide-linked macrocycles, [2]catenanes and [2]rotaxanes were synthesised in solution using reversible dynamic covalent chemistry. Subsequently, the interlocked architectures were adapted into solid-tethered systems via attachment to swelling polystyrene resins.