The chemical modification of polymer blends by reactive processing

The primary objective of this research was to examine the concepts of the chemical modification of polymer blends by reactive processing using interlinking agents (multi-functional, activated vinyl compounds; trimethylolpropane triacrylates {TRIS} and divinylbenzene {DVD}) to target in-situ interpolymer formation between immiscible polymers in PS/EPDM blends via peroxide-initiated free radical reactions during melt mixing. From a comprehensive survey of previous studies of compatibility enhancement in polystyrene blends, it was recognised that reactive processing offers opportunities for technological success that have not yet been fully realised; learning from this study is expected to assist in the development and application of this potential. In an experimental-scale operation for the simultaneous melt blending and reactive processing of both polymers, involving manual injection of precise reactive agent/free radical initiator mixtures directly into molten polymer within an internal mixer, torque changes were distinct, quantifiable and rationalised by ongoing physical and chemical effects. EPDM content of PS/EPDM blends was the prime determinant of torque increases on addition of TRIS, itself liable to self-polymerisation at high additions, with little indication of PS reaction in initial reactively processed blends with TRIS, though blend compatibility, from visual assessment of morphology by SEM, was nevertheless improved. Suitable operating windows were defined for the optimisation of reactive blending, for use once routes to encourage PS reaction could be identified. The effectiveness of PS modification by reactive processing with interlinking agents was increased by the selection of process conditions to target specific reaction routes, assessed by spectroscopy (FT-IR and NMR) and thermal analysis (DSC) coupled dichloromethane extraction and fractionation of PS. Initiator concentration was crucial in balancing desired PS modification and interlinking agent self-polymerisation, most particularly with TRIS. Pre-addition of initiator to PS was beneficial in the enhancement of TRIS binding to PS and minimisation of modifier polymerisation; believed to arise from direct formation of polystyryl radicals for addition to active unsaturation in TRIS. DVB was found to be a "compatible" modifier for PS, but its efficacy was not quantified. Application of routes for PS reaction in PS/EPDM blends was successful for in-situ formation of interpolymer (shown by sequential solvent extraction combined with FT-IR and DSC analysis); the predominant outcome depending on the degree of reaction of each component, with optimum "between-phase" interpolymer formed under conditions selected for equalisation of differing component reactivities and avoidance of competitive processes. This was achieved for combined addition of TRIS+DVB at optimum initiator concentrations with initiator pre-addition to PS. Improvements in blend compatibility (by tensiles, SEM and thermal analysis) were shown in all cases with significant interpolymer formation, though physical benefits were not; morphology and other reactive effects were also important factors. Interpolymer from specific "between-phase" reaction of blend components and interlinking agent was vital for the realisation of positive performance on compatibilisation by the chemical modification of polymer blends by reactive processing.

The morphology of spray-dried particles

Samples of Various industrial or pilot plant spray-dried materials were obtained from manufacturers together with details of drying conditions and feed concentrations. The samples were subjected to qualitative and semi-quantitative examination to identify structural and morphological features. The results were related to measured bulk physical properties and to drying conditions. Single particles were produced in a convective drying process Analogous to spray drying, in which different solids or mixtures of solids were dried from solutions, slurries or pastes as single suspended droplets. The localized chemical and physical structures were analysed and in some cases the retention of volatiles monitored. The results were related to experimental conditions, viz.; air temperature, initial solids concentration and the degree of feed aeration. Three distinct categories of particle morphology were identified, i.e.; crystalline, skin-forming and agglomerate. Each category is evidence of a characteristic drying behaviour which is dependent on initial solids concentration. the degree of feed aeration, and drying temperature. Powder flow ability, particle and bulk density, particle-size, particle friability, and the retention of volatiles bear a direct relationship to morphological structure. Morphologies of multicomponent mixtures were complex, but the respective migration rates of the solutes were dependent on drying temperature. Gas-film heat and SDSS transfer coefficients of single pure liquid droplets were also measured over a temperature range of 50•C to 200•C under forced convection. Balanced transfer rates were obtained attributed to droplet instability or oscillation within the airflow, demonstrated in associated work with single free-flight droplets. The results are of relevance to drier optimisation and to the optimisation of product characteristics, e.g.; particle strength and essential volatiles-retention, in convective drying.

Mechanisms of antifatigue agents used in natural rubber

A large number of compounds containing quinonoid or hindered phenol functions were examined for their roles as antifatigue agents. Among the evaluated quinones and phenols expected to have macroalkyl radical scavenging ability, BQ, αTOC, γTOC and GM showed relatively good performance for fatigue resistance (although their performance was slightly less effective than the commercial aromatic amine antioxidants, IPPD and 6PPD). The compounds which were shown to have higher reactivity with alkyl radicals (via calculated reactivity indices) showed better fatigue resistance. This fact supports the suggestion that strong alkyl radical scavengers should be also effective antifatigue agents. Evidence produced based on calculation of reactivity indices suggests that the quinones examined react with alkyl radicals on the meta position of the quinone rings producing phenoxyl radicals. The phenoxyl radicals are expected either to disproportionate, to recombine with a further alkyl radical, or to abstract a hydrogen from another alkyl radical producing an olefine. The regeneration of quinones and formation of the corresponding phenols is expected to occur during the antifatigue activity. The phenol antioxidant, HBA is expected to produce a quinonoid compound and this is also expected to function in a similar way to other quinones. Another phenol, GM, which is also known to scavenge alkyl radicals showed good antifatigue performance. Tocopherols had effective antifatigue activity and are expected to have different antifatigue mechanisms from that of other quinones, hence αTOC was examined for its mechanisms during rubber fatiguing using HPLC analysis. Trimers of αTOC which were produced during vulcanisation are suggested to contribute to the fatigue activity observed. The evidence suggests that the trimers reproduce αTOC and a mechanism was proposed. Although antifatigue agents evaluated showed antifatigue activity, most of them had poor thermoxidative resistance, hence it was necessary to compensate for this by using a combination of antioxidants with the antifatigue agents. Reactive antioxidants which have the potential to graft on the polymer chains during reactive processing were used for this purpose. APMA was the most effective antioxidant among other evaluated reactive antioxidants. Although high ratio of grafting was achieved after optimisation of grafting conditions, it is suggested that this was achieved by long branches of APMA due to large extent of polymerisation. This is expected to cause maldistribution of APMA leading to reducing the effect of CB-D activity (while CB-A activity showed clear advantages for grafting). Further optimisation of grafting conditions is required in order to use APMA more effectively. Moreover, although synergistic effects between APMA and antifatigue agents were expected, none of the evaluated antifatigue agents, BQ, αTOC, γTOC and TMQ, showed significant synergism both in fatigue and thermoxidative resistance. They performed just as additives.

Automated mapping of environmental variables from a SEIS or SISE perspective

The INTAMAP FP6 project has developed an interoperable framework for real-time automatic mapping of critical environmental variables by extending spatial statistical methods and employing open, web-based, data exchange protocols and visualisation tools. This paper will give an overview of the underlying problem, of the project, and discuss which problems it has solved and which open problems seem to be most relevant to deal with next. The interpolation problem that INTAMAP solves is the generic problem of spatial interpolation of environmental variables without user interaction, based on measurements of e.g. PM10, rainfall or gamma dose rate, at arbitrary locations or over a regular grid covering the area of interest. It deals with problems of varying spatial resolution of measurements, the interpolation of averages over larger areas, and with providing information on the interpolation error to the end-user. In addition, monitoring network optimisation is addressed in a non-automatic context.

Evolutionary neural networks : models and applications

The scaling problems which afflict attempts to optimise neural networks (NNs) with genetic algorithms (GAs) are disclosed. A novel GA-NN hybrid is introduced, based on the bumptree, a little-used connectionist model. As well as being computationally efficient, the bumptree is shown to be more amenable to genetic coding lthan other NN models. A hierarchical genetic coding scheme is developed for the bumptree and shown to have low redundancy, as well as being complete and closed with respect to the search space. When applied to optimising bumptree architectures for classification problems the GA discovers bumptrees which significantly out-perform those constructed using a standard algorithm. The fields of artificial life, control and robotics are identified as likely application areas for the evolutionary optimisation of NNs. An artificial life case-study is presented and discussed. Experiments are reported which show that the GA-bumptree is able to learn simulated pole balancing and car parking tasks using only limited environmental feedback. A simple modification of the fitness function allows the GA-bumptree to learn mappings which are multi-modal, such as robot arm inverse kinematics. The dynamics of the 'geographic speciation' selection model used by the GA-bumptree are investigated empirically and the convergence profile is introduced as an analytical tool. The relationships between the rate of genetic convergence and the phenomena of speciation, genetic drift and punctuated equilibrium arc discussed. The importance of genetic linkage to GA design is discussed and two new recombination operators arc introduced. The first, linkage mapped crossover (LMX) is shown to be a generalisation of existing crossover operators. LMX provides a new framework for incorporating prior knowledge into GAs.Its adaptive form, ALMX, is shown to be able to infer linkage relationships automatically during genetic search.

DNA chip designed antisense oligodeoxynucleotides targeting EGFR MRNA for brain tumour therapy

Glioblastoma multiforme (GBM) is a malignant brain tumour for which there is currently no effective treatment regime. It is thought to develop due to the overexpression of a number of genes, including the epidermal growth factor receptor (EGFR), which is found in over 40% of GBM. Novel forms of treatment such as antisense therapy may allow for the specific inhibition of aberrant genes and thus they are optimistic therapies for future treatment of GBM. Oligodeoxynucleotides (ODNs) are small pieces of DNA that are often modified to increase their stability to nucleases and can be targeted to the aberrant gene in order to inhibit it and thus prevent its transcription into protein. By specifically binding to mRNA in an antisense manner, they can bring about its degradation by a variety of mechanisms including the activation of RNase H and thus have great potential as therapeutic agents. One of the main drawbacks to the utilisation of this therapy so far is the lack of techniques that can successfully predict accessible regions on the target mRNA that the ODNs can bind to. DNA chip technology has been utilised here to predict target sequences on the EGFR mRNA and these ODNs (AS 1 and AS2) have been tested in vitro for their stability, uptake into cells and their efficacy on cellular growth, EGFR protein and mRNA. Studies showed that phosphorothioate and 2'O-methyl ODNs were significantly more stable than phosphodiester ODNs both in serum and serum-free conditions and that the mechanism of uptake into A431 cells was temperature dependent and more efficient with the use of optimised lipofectin. Efficacy results show that AS 1 and AS2 phosphorothioate antisense ODNs were capable of inhibiting cell proliferation by 69% ±4% and 65% ±4.5% respectively at 500nM in conjunction with a non-toxic dose of lipofectinTM used to enhance cellular delivery. Furthermore, control ODN sequences, 2' O-methyl derivatives and a third ODN sequence, that was found not to be capable of binding efficiently to the EGFR mRNA by DNA chip technology, showed no significant effect on cell proliferation. AS 1 almost completely inhibited EGFR protein levels within 48 hours with two doses of 500nM AS 1 with no effect on other EGFR family member proteins or by control sequences. RNA analysis showed a decrease in mRNA levels of 32.4% ±0.8% but techniques require further optimisation to confirm this. As there are variations found between human glioblastoma in situ and those developed as xenografts, analysis of effect of AS 1 and AS2 was performed on primary tumour cell lines derived from glioma patients. ODN treatment showed a specific knockdown of cell growth compared to any of the controls used. Furthermore, combination therapies were tested on A431 cell growth to determine the advantage of combining different antisense approaches and that of conventional drugs. Results varied between the combination treatments but indicated that with optimisation of treatment regimes and delivery techniques that combination therapies utilising antisense therapies would be plausible.

Combinatorial approach to multi-substituted 1,4-Benzodiazepines as novel non-peptide CCK-antagonists

For the drug discovery process, a library of 168 multisubstituted 1,4-benzodiazepines were prepared by a 5-step solid phase combinatorial approach. Substituents were varied in the 3,5, 7 and 8-position on the benzodiazepine scaffold. The combinatorial library was evaluated in a CCK radiolabelled binding assay and CCKA (alimentary) and CCKB (brain) selective lead structures were discovered. The template of CCKA selective 1,4-benzodiazepin-2-ones bearing the tryptophan moiety was chemically modified by selective alkylation and acylation reactions. These studies provided a series of Asperlicin naturally analogues. The fully optimised Asperlicin related compound possessed a similar CCKA activity as the natural occuring compound. 3-Alkylated 1,4-benzodiazepines with selectivity towards the CCKB receptor subtype were optimised on A) the lipophilic side chain and B) the 2-aminophenyl-ketone moiety, together with some stereochemical changes. A C3 unit in the 3-position of 1,4-benzodiazepines possessed a CCKB activity within the nanomolar range. Further SAR optimisation on the N1-position by selective alkylation resulted in an improved CCKB binding with potentially decreased activity on the GABAA/benzodiazepine receptor complex. The in vivo studies revealed two N1-alkylated compounds containing unsaturated alkyl groups with anxiolytic properties. Alternative chemical approaches have been developed, including a route that is suitable for scale up of the desired target molecule in order to provide sufficient quantities for further in vivo evaluation.

The fabrication of biodegradable nanospheres from novel hydroxalkanoates for the delivery of actives of pharmaceutical and agricultural interest

This work describes the fabrication of nanospheres from a range of novel polyhydroxyalkanoates supplied by Monsanto, St Louis, Missouri, USA for the delivery of selected actives of both pharmaceutical and agricultural interest. Initial evaluation of established microsphere and nanosphere fabrication techniques resulted in the adoption and optimisation of a double sonication solvent evaporation method involving the synperonic surfactant F68. Nanospheres could be consistently generated with this method. Studies on the incorporation and release of the surrogate protein Bovine Serum Albumin V demonstrated that BSA could be loaded with between 10-40% w/w BSA without nanosphere destabilisation. BSA release from nanospheres into Hanks Balanced Salts Solution, pH 7.4, could be monitored for up to 28 days at 37°C. The incorporation and release of the Monsanto actives - the insecticide Admire® ({ 1-[(6-chloro-3-pyridinyl)methyIJ-N-nitro-2-imidazolidinimine}) and the plant growth hormone potassium salt Gibberellic acid (GA3K) from physico-chemically characterised polymer nanospheres was monitored for up to 37 days and 28 days respectively, at both 4°C and 23°C. Release data was subsequently fitted to established kinetic models to elaborate the possible mechanisms of release of actives from the nanospheres. The exposure of unloaded nanospheres to a range of physiological media and rural rainwater has been used to investigate the role polymer biodegradation by enzymatic and chemical means might play in the in vivo release of actives and agricultural applications. The potential environmental biodegradation of Monsanto polymers has been investigated using a composting study (International Standard ISO/FDIS 14855) in which the ultimate aerobic biodegradation of the polymers has been monitored by the analysis of evolved carbon dioxide. These studies demonstrated the potential of the polymers for use in the environment, for example as a pesticide delivery system.

The delivery of bioactive proteins using biodegradable microspheres

In this project, antigen-containing microspheres were produced using a range of biodegradable polymers by single and double emulsion solvent evaporation and spray drying techniques. The proteins used in this study were mainly BSA, tetanus toxoid, F1 and V, Y. pestis subunit vaccines and the cytokine, interferon-gamma. The polymer chosen for use in the vaccine preparation will directly determine the characteristics of the formulation. Full in vitro analysis of the preparations was carried out, including surface hydrophobicity and drug release profiles. The influence of the surfactants employed on microsphere surface hydrophobicity was demonstrated. Preparations produced with polyhydroxybutyrate and poly(DTH carbonate) polymers were also shown to be more hydrophobic than PLA microspheres, which may enhance particle uptake by antigen presenting cells and Peyer's patches. Systematic immunisation with microspheres with a range of properties showed differences in the time course and extent of the immune response generated, which would allow optimisation of the dosing schedule to provide maximal response in a single dose preparation. Both systematic and mucosal responses were induced following oral delivery of microencapsulated tetanus toxoid indicating that the encapsulation of the antigen into a microsphere preparation provides protection in the gut and allows targeting of the mucosal-associated lymphoid tissue. Co-encapsulation of adjuvants for further enhancement of immune response was also carried out and the effect on loading and release pattern assessed. Co-encapsulated F1 and interferon-gamma was administered i.p. and the immune responses compared with singly encapsulated and free subunit antigen.

Formulation and characterisation of an effective particulate delivery vehicle for the novel sub-unit vaccine antigen, Ag85B-ESAT-6

This research focused on the formation of particulate delivery systems for the sub-unit fusion protein, Ag85B-ESAT-6, a promising tuberculosis (TB) vaccine candidate. Initial work concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyl dioctadecyl ammonium (DDA). These studies demonstrated that addition of the immunomodulatory trehalose dibehenate (TDB) enhanced the physical stability of the system whilst also adding further adjuvanticity. Indeed, this formulation was effective in stimulating both a cell mediated and humoural immune response. In order to investigate an alternative to the DDA-TDB system, microspheres based on poly(DL-lactide-co-glycolide) (PLGA) incorporating the adjuvants DDA and TDB, either alone or in combination, were first optimised in terms of physico-chemical characteristics, followed by immunological analysis. The formulation incorporating PLGA and DDA emerged as the lead candidate, with promising protection data against TB. Subsequent optimisation of the lead microsphere formulation investigated the effect of several variables involved in the formulation process on physico-chemical and immunological characteristics of the particles produced. Further, freeze-drying studies were carried out with both sugar-based and amino acid-based cryoprotectants, in order to formulate a stable freexe-dried product. Finally, environmental scanning electron microscopy (ESEM) was investigated as a potential alternative to conventional SEM for the morphological investigation of microsphere formulations. Results revealed that the DDA-TDB liposome system proved to be the most immunologically efficient delivery vehicle studied, with high levels of antibody and cytokine production, particularly gamma-interferon (IFN-ϒ), considered the key cytokine marker for anti-mycobacterial immunity. Of the microsphere systems investigated, PLGA in combination with DDA showed the most promise, with an ability to initiate a broad spectrum of cytokine production, as well as antigen specific spleen cell proliferation comparable to that of the DDA-TDB formulation.