Design and synthesis of novel hydrogels for biological applications

The aims of this project were:1) the synthesis of a range of new polyether-based vinylic monomers and their incorporation into poly(2-hydroxyethyl methacrylate) (poly(HEMA)) based hydrogel networks, of interest to the contact lens industry.2) the synthesis of a range of alkyltartronic acids, and their derivatives. These molecules may ultimately be used to produce functionalised poly(-hydroxy acids) of potential interest in either drug delivery or surgical suture applications. The novel syntheses of a range of both methoxy poly(ethylene glycol) acrylates (MPEGAs) and poly(ethylene glycol) acrylates (PEGAs) are described. Products were obtained in very good yields. These new polyether-based vinylic monomers were copolymerised with 2-hydroxyethyl methacrylate (HEMA) to produce a range of hydrogels. The equilibrium water contents (EWC) and surface properties of these copolymers containing linear polyethers were examined. It was found that the EWC was enhanced by the presence of the hydrophilic polyether chains.Results suggest that the polyether side chains express themselves at the polymer surface, thus dictating the surface properties of the gels. Consequentially, this leads to an advantageous reduction in the surface adhesion of biological species. A synthesis of a range of alkyltartronic acids is also described. The acids prepared were obtained in very good yields using a novel four-stage synthesis. These acids were modified to give potassium monoethyl alkyltartronates. Although no polyesterification is described in this thesis, these modified alkyltartronic acid derivatives are considered to be potentially excellent starting materials for poly (alkyltartronic acid) synthesis via anhydrocarboxylate or anhydrosulphite cyclic monomers.

Characterization and cytotoxicity studies of thiol-modified polystyrene microbeads doped with [(Mo6X8)(NO3)6]2- (X=Cl, Br, I)

Halide octahedral molybdenum clusters [(Mo6X8)L6]n- possess luminescence properties that are highly promising for biological applications. These properties are rather dependent on the nature of both the inner ligands X (i.e. Cl, Br, or I) and the apical organic or inorganic ligands L. Herein, the luminescence properties and the toxicity of thiol-modified polystyrene microbeads (PS-SH) doped with [(Mo6X8)(NO3)6]2- (X=Cl, Br, I) were studied and evaluated using human epidermoid larynx carcinoma (Hep2) cell cultures. According to our data, the photoluminescence quantum yield of (Mo6I8)@PS-SH is significantly higher (0.04) than that of (Mo6Cl8)@PS-SH (6Br8)@PS-SH (6X8)@PS-SH showed that all three types of doped microbeads had no significant effect on the viability and proliferation of the cells.

A simple, sensitive and selective quantum-dot-based western blot method for the simultaneous detection of multiple targets from cell lysates

Quantum dots (Qdots) are fluorescent nanoparticles that have great potential as detection agents in biological applications. Their optical properties, including photostability and narrow, symmetrical emission bands with large Stokes shifts, and the potential for multiplexing of many different colours, give them significant advantages over traditionally used fluorescent dyes. Here, we report the straightforward generation of stable, covalent quantum dot-protein A/G bioconjugates that will be able to bind to almost any IgG antibody, and therefore can be used in many applications. An additional advantage is that the requirement for a secondary antibody is removed, simplifying experimental design. To demonstrate their use, we show their application in multiplexed western blotting. The sensitivity of Qdot conjugates is found to be superior to fluorescent dyes, and comparable to, or potentially better than, enhanced chemiluminescence. We show a true biological validation using a four-colour multiplexed western blot against a complex cell lysate background, and have significantly improved previously reported non-specific binding of the Qdots to cellular proteins.

Interrogation of a sonogashira cross-coupling of 8-bromoguanosine with phenylacetylene on amberlite:evidence for Pd/Cu ion binding and propagation of Pd/Cu nanoparticles

The reactivity of Amberlite (IRA-67) base "heterogeneous" resin in Sonogashira cross-coupling of 8-bromoguanosine 1 with phenylacetylene 3 to give 2 has been examined. Both 1 and 2 coordinate to Pd and Cu ions, which explains why at equivalent catalyst loadings, the homogeneous reaction employing triethylamine base is poor yielding. X-ray photo-electron spectroscopy (XPS) has been used to probe and quantify the active nitrogen base sites of the Amberlite resin, and postreaction Pd and Cu species. The Pd2Cl3(PPh)2 precatalyst and CuI cocatalyst degrade to give Amberlite-supported metal nanoparticles (average size ∼2.7 nm). The guanosine product 2 formed using the Amberlite Pd/Cu catalyst system is of higher purity than reactions using a homogeneous Pd precatalyst, a prerequisite for use in biological applications. Copyright © Taylor and Francis Group, LLC.

Optical fibre sensors with applications in gas and biological sensing

This thesis describes the study of various grating based optical fibre sensors for applications in refractive index sensing. The sensitivity of these sensors has been studied and in some cases enhanced using novel techniques. The major areas of development are as follows. The sensitivity of long period gratings (LPGs) to surrounding medium refractive index (SRI) for various periods was investigated. The most sensitive period of LPG was found to be around 160 µm and this was due to the core mode coupling to a single cladding mode but phase matching at two wavelength locations, creating two attenuation peaks, close to the waveguide dispersion turning point. Large angle tilted fibre gratings (TFGs) have similar behaviour to LPGs, in that they couple to the co-propagating cladding modes. The tilted structure of the index modulation within the core of the fibre gives rise to a polarisation dependency, differing the large angle TFG from a LPG. Since the large angle TFG couple to the cladding mode they are SRI sensitive, the sensitivity to SRI can be further increased through cladding etching using HF acid. The thinning of the cladding layer caused a reordering of the cladding modes and shifted to more SRI sensitive cladding modes as the investigation discovered. In a SRI range of 1.36 to 1.40 a sensitivity of 506.9 nm/URI was achieved for the etched large angle TFG, which is greater than the dual resonance LPG. UV inscribed LPGs were coated with sol-gel materials with high RIs. The high RI of the coating caused an increase in cladding mode effective index which in turn caused an increase in the LPG sensitivity to SRI. LPGs of various periods of LPG were coated with sol-gel TiO2 and the optimal thickness was found to vary for each period. By coating of the already highly SRI sensitive 160µm period LPG (which is a dual resonance) with a sol-gel TiO2, the SRI sensitivity was further increased with a peak value of 1458 nm/URI, which was an almost 3 fold increase compared to the uncoated LPG. LPGs were also inscribed using a femtosecond laser which produced a highly focused index change which was no uniform throughout the core of the optical fibre. The inscription technique gave rise to a large polarisation sensitivity and the ability to couple to multiple azimuthal cladding mode sets, not seen with uniform UV inscribed gratings. Through coupling of the core mode to multiple sets of cladding modes, attenuation peaks with opposite wavelength shifts for increasing SRI was observed. Through combining this opposite wavelength shifts, a SRI sensitivity was achieved greater than any single observed attenuations peak. The maximum SRI achieved was 1680 nm/URI for a femtosecond inscribed LPG of period 400 µm. Three different types of surface plasmon resonance (SPR) sensors with a multilayer metal top coating were investigated in D shape optical fibre. The sensors could be separated into two types, utilized a pre UV inscribed tilted Bragg grating and the other employed a post UV exposure to generate surface relief grating structure. This surface perturbation aided the out coupling of light from the core but also changed the sensing mechanism from SPR to localised surface plasmon resonance (LSPR). This greatly increased the SRI sensitivity, compared to the SPR sensors; with the gold coated top layer surface relief sensor producing the largest SRI sensitivity of 2111.5nm/URI was achieved. While, the platinum and silver coated top layer surface relief sensors also gave high SRI sensitivities but also the ability to produce resonances in air (not previously seen with the SPR sensors). These properties were employed in two applications. The silver and platinum surface relief devices were used as gas sensors and were shown to be capable of detecting the minute RI change of different gases. The calculated maximum sensitivities produced were 1882.1dB/URI and 1493.5nm/URI for silver and platinum, respectively. Using a DFB laser and power meter a cheap alternative approach was investigated which showed the ability of the sensors to distinguish between different gases and flow rates of those gases. The gold surface relief sensor was coated in a with a bio compound called an aptamer and it was able to detect various concentrations of a biological compound called Thrombin, ranging from 1mM to as low as 10fM. A solution of 2M NaCl was found to give the best stripping results for Thrombin from the aptamer and showed the reusability of the sensor. The association and disassociation constants were calculated to be 1.0638×106Ms-1 and 0.2482s-1, respectively, showing the high affinity of the Aptamer to thrombin. This supports existing working stating that aptamers could be alternative to enzymes for chemical detection and also helps to explain the low detection limit of the gold surface relief sensor.

Transglutaminase 2 cross-linking of matrix proteins: biological significance and medical applications

This review summarises the functions of the enzyme tissue transglutaminase (TG2) in the extracellular matrix (ECM) both as a matrix stabiliser through its protein cross-linking activity and as an important cell adhesion protein involved in cell survival. The contribution of extracellular TG2 to the pathology of important diseases such as cancer and fibrosis are discussed with a view to the potential importance of TG2 as a therapeutic target. The medical applications of TG2 are further expanded by detailing the use of transglutaminase cross-linking in the development of novel biocompatible biomaterials for use in soft and hard tissue repair.

Physical and biological properties of a novel siloxane adhesive for soft tissue applications

The aim of this study was to investigate the adhesive properties of an in-house amino-propyltrimethoxysilane-methylenebisacrylamide (APTMS-MBA) siloxane system and compare them with a commercially available adhesive, n-butyl cyanoacrylate (nBCA). The ability of the material to perform as a soft tissue adhesive was established by measuring the physical (bond strength, curing time) and biological (cytotoxicity) properties of the adhesives on cartilage. Complementary physical techniques, X-ray photoelectron spectroscopy, Raman and infrared imaging, enabled the mode of action of the adhesive to the cartilage surface to be determined. Adhesion strength to cartilage was measured using a simple butt joint test after storage in phosphate-buffered saline solution at 37°C for periods up to 1 month. The adhesives were also characterised using two in vitro biological techniques. A live/dead stain assay enabled a measure of the viability of chondrocytes attached to the two adhesives to be made. A water-soluble tetrazolium assay was carried out using two different cell types, human dermal fibroblasts and ovine meniscal chondrocytes, in order to measure material cytotoxicity as a function of both supernatant concentration and time. IR imaging of the surface of cartilage treated with APTMS-MBA siloxane adhesive indicated that the adhesive penetrated the tissue surface marginally compared to nBCA which showed a greater depth of penetration. The curing time and adhesion strength values for APTMS-MBA siloxane and nBCA adhesives were measured to be 60 s/0.23 MPa and 38 min/0.62 MPa, respectively. These materials were found to be significantly stronger than either commercially available fibrin (0.02 MPa) or gelatin resorcinol formaldehyde (GRF) adhesives (0.1 MPa) (P <0.01). Cell culture experiments revealed that APTMS-MBA siloxane adhesive induced 2% cell death compared to 95% for the nBCA adhesive, which extended to a depth of approximately 100-150 μm into the cartilage surface. The WST-1 assay demonstrated that APTMS-MBA siloxane was significantly less cytotoxic than nBCA adhesive as an undiluted conditioned supernatant (P <0.001). These results suggest that the APTMS-MBA siloxane may be a useful adhesive for medical applications. © VSP 2005.

Writing and characterisation methods for UV-photosensitivity based devices and their applications

The underlying work to this thesis focused on the exploitation and investigation of photosensitivity mechanisms in optical fibres and planar waveguides for the fabrication of advanced integrated optical devices for telecoms and sensing applications. One major scope is the improvement of grating fabrication specifications by introducing new writing techniques and the use of advanced characterisation methods for grating testing. For the first time the polarisation control method for advanced grating fabrication has successfully been converted to apodised planar waveguide fabrication and the development of a holographic method for the inscription of chirped gratings at arbitrary wavelength is presented. The latter resulted in the fabrication of gratings for pulse-width suppression and wavelength selection in diode lasers. In co-operation with research partners a number of samples were tested using optical frequency domain and optical low coherence reflectometry for a better insight into the limitations of grating writing techniques. Using a variety of different fabrication methods, custom apodised and chirped fibre Bragg gratings were written for the use as filter elements for multiplexer-demultiplexer devices, as well as for short pulse generation and wavelength selection in telecommunication transmission systems. Long period grating based devices in standard, speciality and tapered fibres are presented, showing great potential for multi-parameter sensing. One particular scope is the development of vectorial curvature and refractive index sensors with potential for medical, chemical and biological sensing. In addition the design of an optically tunable Mach-Zehnder based multiwavelength filter is introduced. The discovery of a Type IA grating type through overexposure of hydrogen loaded standard and Boron-Germanium co-doped fibres strengthened the assumption of UV-photosensitivity being a highly non-linear process. Gratings of this type show a significantly lower thermal sensitivity compared to standard gratings, which makes them useful for sensing applications. An Oxford Lasers copper-vapour laser operating at 255 nm in pulsed mode was used for their inscription, in contrast to previous work using CW-Argon-Ion lasers and contributing to differences in the processes of the photorefractive index change

The biotechnological applications of transglutaminases

Transglutaminases catalyse a diverse range of reactions leading to the modification of proteins and peptides such that their physical, chemical and biological properties become changed. They are found in many different living organisms and as a consequence display subtle differences in their biochemical and physical properties. it is therefore not surprising that this group of enzymes have been exploited as applied biocatalysts in a wide range of commercial sectors varying from the textile industry to the highly lucrative cosmetic industry. in addition the pathophysiological importance of this group of enzymes has increased significantly over the last decade with their involvement noted in a number of human diseases. As a consequence their identification as therapeutic targets or as monitoring aids for a range of different diseases has caused significant interest from the diagnostics and pharmaceutical industries. This review describes some of the current applications of transglutaminases; together with their potential strategic importance and future uses.

Novel cationic polymers for use at biological interfaces

One of the main problems with the use of synthetic polymers as biomaterials is the invasion of micro-organisms causing infection. A study of the properties of polymeric antibacterial agents, in particular polyhexamethylene biguanide, has revealed that the essential components for the design of a novel polymeric antibacterial are a balance between hydrophilicity and hydrophobicity coupled with sites of cationicity. The effect of cation incorporation on the physical properties of hydrogels has been investigated. Hydrogel systems copolymerised with either N-vinyl imidazole or dimethylaminoethyl methacrylate have been characterised in terms of their water binding, mechanical and surface properties. It has been concluded that the incorporation of these monomers does not adversely affect the properties of such hydrogels and that these materials are potential candidates for further development for use in biomedical applications. It has been reported that hydro gels with ionic character may increase the deposition of biological material onto the hydrogel surface when it is in contact with body fluids. An investigation into the deposition characteristics of hydrogels containing the potentially cationic monomers has been carried out, using specific protein adsorption and in vitro spoilation techniques. The results suggest that at low levels of cationicity, the deposition of positively charged proteins is reduced without adversely affecting the uptake of the other proteins. The gross deposition characteristics were found to be comparable to some commercially available contact lens materials. A preliminary investigation into the development of novel antibacterial polymers has been completed and some novel methods of bacterial inhibition discussed. These methods include development of an hydrogel whose potential application is as a catheter coating.

The development and applications of the combined use of NMR and ultrasound

The objective of the research carried out in this report was to observe the first ever in-situ sonochemical reaction in the NMR Spectrometer in the megahertz region of ultrasound. Several reactions were investigated as potential systems for a sonochemical reaction followed by NMR spectroscopy. The primary problem to resolve when applying ultrasound to a chemical reaction is that of heating. Ultrasound causes the liquid to move and produces 'hot spots' resulting in an increase in sample temperature. The problem was confronted by producing a device that would counteract this effect and so remove the need to account for heating. However, the design of the device limited the length of time during which it would function. Longer reaction times were required to enable observations to be carried out in the NMR spectrometer. The fIrst and most obvious reactions attempted were those of the well-known ultrasonic dosimeter. Such a reaction would, theoretically, enable the author to simultaneously observe a reaction and determine the exact power entering the system for direct comparison of results. Unfortunately, in order to monitor the reactions in the NMR spectrometer the reactant concentrations had to be signifIcantly increased, which resulted in a notable increase in reaction time, making the experiment too lengthy to follow in the time allocated. The Diels-Alder Reaction is probably one of the most highly investigated reaction systems in the field of chemistry and it was this to which the author turned her attention. Previous authors have carried out ultrasonic investigations, with considerable success, for the reaction of anthracene with maleic anhydride. It was this reaction in particular that was next attempted. The first ever sonochemically enhanced reaction using a frequency of ultrasound in the megahertz (MHz) region was successfully carried out as bench experiments. Due to the complexity of the component reactants the product would precipitate from the solution and because the reaction could only be monitored by its formation, it was not possible to observe the reaction in the NMR spectrometer. The solvolysis of 2-chloro-2-methylpropane was examined in various solvent systems; the most suitable of which was determined to be aqueous 2-methylpropan-2-ol. The experiment was successfully enhanced by the application of ultrasound and monitored in-situ in the NMR spectrometer. The increase in product formation of an ultrasonic reaction over that of a traditional thermal reaction occurred. A range of 1.4 to 2.9 fold improvement was noted, dependent upon the reaction conditions investigated. An investigation into the effect of sonication upon a large biological molecule, in this case aqueous lysozyme, was carried out. An easily observed effect upon the sample was noted but no explanation for the observed effects could be established.

Cellular uptake and biological activity of synthetic hammerhead ribozymes

Glioblastoma Multiforme (GBM) is a highly malignant form of brain cancer for which there is no effective cure. The over-expression of a number of genes, including the epidermal growth factor receptor (EGFr), has been implicated as a causative factor of tumourigenesis. Ribozymes are a class of ribonucleic acid that possess enzymatic properties. They can inhibit gene-expression in a highly sequence specific manner by catalysing the trans-cleavage of target RNA. The potential use of synthetic hammerhead ribozymes as novel anti-brain tumour agents was investigated in this study. The successful use of synthetic, exogenously administered ribozymes for such applications will require chemical modifications that improve biological stability and a fundamental understanding of cellular uptake mechanisms. Chimeric 2'-O-methylated hammerhead ribozymes proved to be significantly more stable (>4000-fold) in serum than unmodified RNA ribozymes and exhibited high in vitro catalytic activity. The cellular association of an internally [32P]-labelled 2'-O-methylated chimeric ribozyme in U87-MG human glioma cells was temperature-, energy- and pH-dependent and involved an active process that could be competed with a variety of polyanions. Indications are that the predominant mechanism of uptake is by adsorptive and / or receptor mediated endocytosis. Twenty 2'-O-methylated chimeric ribozymes were designed to cleave various sites along the EGFr mRNA. In vitro, 18 ribozymes exhibited high activity in cleaving a complementary short substrate. Using LipofectAMINETM as a delivery agent, the efficacy of these ribozymes was evaluated in the A431 cell line, which expresses amplified levels of EGFr. Studies revealed that although the ribozymes were taken up by the cells and remained stable over a period of 4 days, no significant reduction in either EGFr expression or cell proliferation was evident. The presence of telomerase, a ribonucleoprotein responsible for telomere elongation, has been strongly associated with tumour progression. The biological activity of a 2'-O-methylated ribozyme targeted against the RNA component of telomerase was determined. The ribozyme exhibited specific dose-dependent inhibition of telomerase activity in U87-MG cell lysates with an IC50 of –4μM. When 4μM ribozyme was delivered to intact U87-MG cells, complexed to LipofectAMINETM, telomerase activity was significantly reduced to 74.5±4.17% of the untreated control. Free ribozyme showed no significant inhibitory effect demonstrating the importance of an appropriate delivery system for optimum delivery of exogenously administered ribozymes.

Proteoglycan analogues for ophthalmic and orthopaedic applications

The successful development of biomaterials is increasingly influenced by biomimesis essentially the use of biological structures as design templates. This approach has been used here in the design of injectable systems for the nucleus of the intervertebral disc, corneal inlays and intraocular lenses (IOLs). The strategy is based on the use of C-linked sulphonates to mimic the O-linked sulphate groups that are the hydration drivers in proteoglycans. The elastic modulus of the materials can be tailored for specific applications.

Decellularization of bovine corneas for tissue engineering applications

Scaffolds derived from processed tissues offer viable alternatives to synthetic polymers as biological scaffolds for regenerative medicine. Tissue-derived scaffolds provide an extracellular matrix (ECM) as the starting material for wound healing and the functional reconstruction of tissues, offering a potentially valuable approach for the replacement of damaged or missing tissues. Additionally, acellular tissue may provide a natural microenvironment for host-cell migration and the induction of stem cell differentiation to contribute to tissue regeneration. There are a number of processing methods that aim to stabilize and provide an immunologically inert tissue scaffold. Furthermore, these tissue-processing methods can often be applied to xenogenic transplants because the essential components of the ECM are often maintained between species. In this study, we applied several tissue-processing protocols to the cornea in order to obtain a decellularized cornea matrix that maintained the clarity and mechanical properties of the native tissue. Histology, mechanical testing and electron microscopy techniques were used to assess the cell extraction process and the organization of the remaining ECM. In vitro cell seeding experiments confirmed the processed corneas’ biocompatibility.

Solvability and asymptotics of the heat equation with mixed variable lateral conditions and applications in the opening of the exocytotic fusion pore in cells

We investigate a mixed problem with variable lateral conditions for the heat equation that arises in modelling exocytosis, i.e. the opening of a cell boundary in specific biological species for the release of certain molecules to the exterior of the cell. The Dirichlet condition is imposed on a surface patch of the boundary and this patch is occupying a larger part of the boundary as time increases modelling where the cell is opening (the fusion pore), and on the remaining part, a zero Neumann condition is imposed (no molecules can cross this boundary). Uniform concentration is assumed at the initial time. We introduce a weak formulation of this problem and show that there is a unique weak solution. Moreover, we give an asymptotic expansion for the behaviour of the solution near the opening point and for small values in time. We also give an integral equation for the numerical construction of the leading term in this expansion.