Development of photosensitive liposomes for the controlled release of drugs

The facility to controlled triggered release from a “cage” system remains a key requirement for novel drug delivery. Earlier studies have shown that Bis-Azo PC based photosensitive liposomes are beneficial for drug delivery. Thus, the aim of this project was to develop photosensitive liposomes that can be used for the controlled release of drugs through UV irradiation, particularly therapeutic agents for the treatment of psoriasis. Bis-Azo PC was successfully synthesized and incorporated into a range of liposomal formulations, and these liposomes were applied for the controlled release of BSA-FITC. Bis-Azo PC sensitized liposomes were prepared via interdigitation fusion method. IFV containing optimum cholesterol amount in terms of protein loading, stability and photo-trigger release of protein was investigated. Further studies investigated the stability and triggered release of the HMT from IFV. Finally, permeation behavior of HMT and HMT-entrapped IFV through rat skin was examined using Franz cell. Results from protein study indicated that the stable entrapment of the model protein was feasible as shown through fluorescence spectroscopy and maximum of 84% protein release from IFV after 12 min of UV irradiation. Moreover, stability studies indicated that IFV were more stable at 4 0C as compared to 25 0C. Hence, DPPC:Chol:Bis-Azo PC (16:2:1) based IFV was chosen for the controlled release of HMT and these studies exhibited that photo-trigger release and stability data of HMT-entrapped IFV are in line with the protein results. Franz cell work inferred that HMT-entrapped IFV attributed to slower skin permeation as compared to HMT. CLSM also demonstrated that HMT can be used as a fluorescent label for the in vitro skin study. Overall, the work highlighted in this thesis has given useful insight into the potentials of Bis-Azo PC based IFV as a promising carrier for the treatment of psoriasis.

Studies of the stability and metabolism of tumour inhibitory tetrazinones

Temozolomide is an imidazotetrazinone with antineoplastic properties. It is structurally related to dacarbazine. Temozolomide was not metabolized in vitro by liver fractions. Chemical decomposition appears to play an important r^ole in its in vitro and in vivo disposition. In contrast, 3-methylbenzotriazinone, a structural analogue, was metabolized by hepatic microsomes to afford benzotriazinone and a hydrophilic metabolite. The cytotoxicity of temozolomide, dacarbazine, 5-[3-(hydroxy-methyl-3-methyl-triazen-1-yl]imidazole-5-carboxamide (HMMTIC) and 3-monomethyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) were investigated in TLX5 murine lymphoma cells. Unlike dacarbazine, which was not toxic, MTIC, HMMTIC and temozolomide were cytotoxic in the absence of microsomes. Decarbazine was only cytotoxic in the presence of microsomes. The formation of MTIC from dacarbazine, HMMTIC and temozolomide was determined by reversed phase high performance liquid chromatography in mixtures incubated under conditions identical to those described before. MTIC was generated chemically from temozolomide and HMMTIC metabolically from dacarbazine. Using [14C]temozolomide, it was found that, in mice, the major route of excretion of the drug is via the kidneys. An acidic metabolite (metabolite I) was found in the urine of mice which had received temozolomide but its identity has not been established. 1H NMR, UV and chemical analyses revealed that Metabolite I possesses an intact NNN linkage and the site of metabolism is at the N3 methyl group. A further acidic metabolite (metabolite II) was found in the urine of patients. Metabolite II was unambiguously identified as the 8-carboxylic acid derivative of temozolomide. In vitro cytotoxicity assay showed that ony metabolite II is cytotoxic but not metabolite I. Pharmacokinetic studies of temozolomide and MTIC in vivo were performed on mice bearing TLX5 tumour. Temozolomide was eliminated from the plasma monophasically with a t1/2 of 0.7hr. MTIC was identified as a product of decomposition. MTIC was eliminated rapidly with a t1/2 of 2min. Though temozolomide shares many biochemical and biological similarities with clinically used dacarbazine, the results obtained in this study show that it differs markedly in its pharmacokinetic properties from dacarbazine, as temozolomide produced relatively sustained plasma levels which were reflected by drug concentrations in the tumour.

Developmental stability and leadership effectiveness

Developmental stability is the degree to which we can withstand environmental or genetic stressors during development. Fluctuating asymmetry (FA), concerns the extent to which the right and left side of the body is asymmetrical and is one way to measure developmental stability. Two studies were carried out that examined both the predictive value of leader FA with leadership behaviors and its role in facilitating group performance. The first study examined the hypothesis that a leader's FA is correlated with scores on the Multifactor Leadership Questionnaire (MLQ). The results revealed individuals with a more asymmetrical morphology scored higher on the transformational, but not transactional, dimensions of leadership behavior. A second study examined the hypothesis that asymmetrical morphology and leadership effectiveness would share a positive relationship. In this study participants who led a business game exercise, revealed a positive relationship between FA and self-reported well-being and task satisfaction. Importantly, there was also a positive correlation between the leader's FA score and group performance. The role that developmental stability may play in leadership effectiveness is discussed in the wider context of evolutionary psychology.

Standing in front of bulldozers? Explaining policy stability in land use planning

Policy towards planning presents scholars of politics and public policy with a significant puzzle. Since 1947, there has been a surprising level of stability in the system used to plan the use of land. On the other hand, there has been growing evidence that insufficient land has been released for development. The paper considers the question why, in spite of the planning system demonstrably failing to allocate sufficient land, fundamental reform of the system has not been achieved. In answering the question, the paper considers in particular attempts at reform under the Labour governments from 1997 to 2010. It argues that there is an interplay of interests, ideas and institutions: public attitudes, the interests of certain sections of the population, and institutions which are responsive to these attitudes and interests combined to stymie policy reform. As a consequence, radical reform was not achieved, and the paper concludes that attempt to find a technical “fix” to the planning system are unlikely to succeed. A diagnosis recognising the political and distributive nature of the problem will be required.

Symptoms of hyperactivity and inattention can mediate deficits of postural stability in developmental dyslexia

DDevelopmental dyslexia is a reading disorder associated with impaired postural control. However, such deficits are also found in attention deficit hyperactivity disorder (ADHD), which is present in a substantial subset of dyslexia diagnoses. Very few studies of balance in dyslexia have assessed ADHD symptoms, thereby motivating the hypothesis that such measures can account for the group differences observed. In this study, we assessed adults with dyslexia and similarly aged controls on a battery of cognitive, literacy and attention measures, alongside tasks of postural stability. Displacements of centre of mass to perturbations of posture were measured in four experimental conditions using digital optical motion capture. The largest group differences were obtained in conditions where cues to the support surface were reduced. Between-group differences in postural sway and in sway variability were largely accounted for by co-varying hyperactivity and inattention ratings, however. These results therefore suggest that postural instability in dyslexia is more strongly associated with symptoms of ADHD than to those specific to reading impairment.

Synthesis and evaluation of novel ligands for quantum dot stabilisation and polymerisation studies

This thesis describes the design and synthesis of a variety of functionalised phosphine oxides and sulfides, based on the structure of trioctylphosphine oxide, synthesised for the purpose of surface modification of quantum dots. The ability of the ligands to modify the surface chemistry via displacement of the original hexadecylamine capping layer of quantum dots was evaluated. Finally the surface modified quantum dots were investigated for enhancement in their inherent properties and improved compatibility with the various applications for which they were initially designed. Upon the commencement of research involving quantum dots it became apparent that more information on their behaviour and interaction with the environment was required. The limits of the inherent stability of hexadecylamine capped quantum dots were investigated by exposure to a number of different environments. The effect upon the stability of the quantum dots was monitored by changes in the photoluminescence ability of their cores. Subtle differences between different batches of quantum dots were observed and the necessity to account for these in future applications noted. Lastly the displacement of the original hexadecylamine coating with the "designer" functionalised ligands was evaluated to produce a set of conditions that would result in the best possible surface modification. A general procedure was elucidated however it was discovered that each displacement still required slight adjustment by consideration of the other factors such as the difference in ligand structure and the individuality of the various batches of quantum dots. This thesis also describes a procedure for the addition of a protective layer to the surface of quantum dots by cross-linking the functionalised ligands bound to the surface via an acyclic diene metathesis polymerisation. A detailed description of the problems encountered in the analysis of these materials combined with the use of novel techniques such as diffusion ordered spectroscopy is provided as a means to overcome the limitations encountered. Finally a demonstration of the superior stability, upon exposure to a range of aggressive environments of these protected materials compared with those before cross-linking provided physical proof of the cross-linking process and the advantages of the cross-linking modification. Finally this thesis includes the presentation of initial work into the production of luminescent nanocrystal encoded resin beads for the specific use in solid phase combinatorial chemistry. Demonstration of the successful covalent incorporation of quantum dots into the polymeric matrices of non-functionalised and functionalised resin beads is described. Finally by preliminary work to address and overcome the possible limitations that may be encountered in the production and general employment of these materials in combinatorial techniques is given.

Spray drier simulation and air flow pattern studies

The literature pertaining to the key stages of spray drying has been reviewed in the context of the mathematical modelling of drier performance. A critical review is also presented of previous spray drying models. A new mathematical model has been developed for prediction of spray drier performance. This is applicable to slurries of rigid, porous crust-forming materials to predict trajectories and drying profiles for droplets with a distribution of sizes sprayed from a centrifugal pressure nozzle. The model has been validated by comparing model predictions to experimental data from a pilot-scale counter-current drier and from a full-scale co-current drier. For the latter, the computed product moisture content was within 2%, and the computed air exit temperature within 10oC of experimental data. Air flow patterns have been investigated in a 1.2m diameter transparent countercurrent spray tower by flow visualisation. Smoke was introduced into various zones within the tower to trace the direction, and gauge the intensity, of the air flow. By means of a set of variable-angle air inlet nozzles, a variety of air entry configurations was investigated. The existence of a core of high rotational and axial velocity channelling up the axis of the tower was confirmed. The stability of flow within the core was found to be strongly dependent upon the air entry arrangement. A probe was developed for the measurement of air temperature and humidity profiles. This was employed for studying evaporation of pure water drops in a 1.2m diameter pilot-scale counter-current drier. A rapid approach to the exit air properties was detected within a 1m distance from the air entry ports. Measured radial profiles were found to be virtually flat but, from the axial profiles, the existence of plug-flow, well-mixed-flow and some degree of air short-circuiting can be inferred. The model and conclusions should assist in the improved design and optimum operation of industrial spray driers.

Delivery and stability of antisense oligonucleotide conjugates in vitro

The efficacy of antisense oligonucleotide (ODN) therapy is dependent on four major parameters: delivery to cells, intracellular stability and localisation and efficient action at the target site.The aim of this project was to study the delivery of ODNs to macrophages and to assess the stability of two ODN conjugates, in vitro. The first conjugate aimed to improve uptake of ODNs via mannose receptor mediated delivery, the second investigated the improved delivery of ODN conjugates via non-specific lipophilic interaction with the cell membrane. A mono-mannose phosphoramidite derivative was designed and synthesised and a mono-mannose ODN conjugate synthesised by standard phosphoramidite chemistry. Delivery of this conjugate was enhanced to RAW264.7 and J774 macrophage cell lines via a mechanism of receptor mediated endocytosis. The delivery of three lipophilic ODN conjugates, cholesterol (cholhex), 16-carbon alkyl chain (C16) and hexa-ethylene glycol (HEG) moieties and an unconjugated ODN were assessed in RAW264.7 macrophages. All three conjugates increased the lipophilicity of the ODN as assessed from partition coefficient data. Both the cholhex and unconjugated ODNs were found to have higher degrees of cellular association than the C16 and HEG conjugates. Cellular uptake studies implicated internalisation of these ODNs by an adsorptive endocytosis mechanism. Following endocytosis, ODNs must remain stable during their residence in endosomal/lysosomal compartments prior to exiting and exerting their biological action in either the cytosol or nucleus. Assessment of in vitro stability in a lysosomal extract revealed the cholhex conjugate and unconjugated ODNs to have a longer half-life than the C16 and HEG conjugated ODNs, highlighting the influence of conjugate moieties on lysosomal stability. The effects of base composition and length on stability in a lysosomal extract revealed the longest half-life for homo-cytidine ODNs and ODNs over 20 nucleotides in length. These studies suggest that the above conjugates can enhance cellular association and delivery of antisense ODNs to cultured macrophages. This may lead to their use in treating disorders such as HIV infection, which affects this cell type.

The effect of certain hydrophilic polymers on suspension stability

The adsorption of nonionic surface active agents of polyoxyethylene glycol monoethers of n hexadecanols on polystyrene latex and nonionic cellulose polymers of hydroxyethyl cellulose, hydroxypropyl cellulose and hydroxypropyl methylcellulose on polystyrene latex and ibuprofen drug particles have been studied. The adsorbed layer thicknesses were determined by means of microelectrophoretic and viscometric methods. The conformation of the adsorbed molecules at the solid-liquid interface was deduced from the molecular areas and the adsorbed layer thicknesses. Comparison of the adsorption results obtained from polystyrene latex and ibuprofen particles was made to explain the conformation difference between these two adsorbates. Sedimentation volumes and redispersibility values were the main criteria used to evaluate suspension stability. At low concentrations of surface active agents, hard caked suspensions were found, probably due to the attraction between the uncoated areas or, the mutual adsorption of the adsorbed molecules on the bare surface of the particles in the sediment. At high concentrations of hydroxypropyl cellulose and hydroxypropyl methylcellulose, heavily caked sediments were attributed to network structure formation by the adsorbed molecules. An attempt was made to relate the characteristics of the suspensions to the potential energy of interaction curves. Generally, the agreement between theory and experiment was good, but for hydroxyethyl cellulose-ibuprofen systems discrepancies were found. Experimental studies showed that hydroxyethyl cellulose flocculated polystyrene latex over a rather wide range of concentrations; similarly, hydroxyethyl cellulose-ibuprofen suspensions were also flocculated. Therefore, it ls suggested that a term to account for flocculation energy of the polymer should be added to the total energy of interaction. A rheometric method was employed to study the flocculation energy of the polymer.

Characterisation and oxidative stability of speciality plant seed oils

The past decade has seen an influx of speciality plant seed oils arriving into the market place. The need to characterise these oils has become an important aspect of the oil industry. The characterisation of the oils allows for the physical and chemical properties of the oil to be determined. Speciality oils were characterised based on their lipid and fatty acid profiles and categorised as monounsaturated rich (oleic acid as the major acyl components e.g. Moringa and Marula oil), linoleic acid rich (Grape seed and Evening Primrose oil) or linolenic acid rich (Flaxseed and Kiwi oil). The quality of the oils was evaluated by determining the free fatty acid content, the peroxide value (that measures initial oxidation) and p-anisidine values (that determines secondary oxidation products containing the carbonyl function). A reference database was constructed for the oils in order to compare batches of oils for their overall quality including oxidative stability. For some of the speciality oils, the stereochemistry of the triacylglycerols was determined. Calophyllum, Coffee, Poppy and Sea Buckthorn oils stereochemistry was determined. The oils were enriched with saturated and/or a monounsaturated fatty acids at position sn-1 and sn-3. The sn-2 position of the four oils was esterified with a polyunsaturated and/or a monounsaturated fatty acid indicating that they follow a typical acylation pathway and no novel acylation activity was evident from these studies (e.g enrichment of saturates at the sn-2 position). The oxidative stability of the oils was evaluated at 18oC and 60oC and the effect of adding a-tocopherol at commercially used level i.e 750ppm was assessed. The addition of 750ppm of a-tocopherol at 18oC increased the oxidative stability of Brown flax, Moringa, Wheat germ and Yangu oils. At 60oC Brown Flax, Manketti and Pomegranate oil polymerised after 48 hours. The addition of 750ppm a-tocopherol delayed the onset of polymerisation by up to 48 hours in Brown Flax seed oil. Pomegranate oil showed a high resistance to oxidation, and was blended into other speciality oils at 1%. Pomegranate oil increased the oxidative stability of Yangu oil at 18oC. The addition of Pomegranate oil to Wheat germ oil at 60oC, decreased the peroxide content by 10%. In Manketti and Brown Flaxseed oil at elevated temperatures, Pomegranate oil delayed the onset of polymerisation. Preliminary studies of Pomegranate oil blending to Moringa and Borage oil showed it to be more effective than a-tocopherol for certain oils. The antioxidant effects observed following the addition of Pomegranate oil may be due to its conjugated linolenic acid fatty acid, punicic acid.

A comparative study of cationic liposome and niosome-based adjuvant systems for protein subunit vaccines:characterisation, environmental scanning electron microscopy and immunisation studies in mice

Vesicular adjuvant systems composing dimethyldioctadecylammonium (DDA) can promote both cell-mediated and humoral immune responses to the tuberculosis vaccine fusion protein in mice. However, these DDA preparations were found to be physically unstable, forming aggregates under ambient storage conditions. Therefore there is a need to improve the stability of such systems without undermining their potent adjuvanticity. To this end, the effect of incorporating non-ionic surfactants, such as 1-monopalmitoyl glycerol (MP), in addition to cholesterol (Chol) and trehalose 6,6′-dibehenate (TDB), on the stability and efficacy of these vaccine delivery systems was investigated. Differential scanning calorimetry revealed a reduction in the phase transition temperature (T c) of DDA-based vesicles by ∼12°C when MP and cholesterol (1:1 molar ratio) were incorporated into the DDA system. Transmission electron microscopy (TEM) revealed the addition of MP to DDA vesicles resulted in the formation of multi-lamellar vesicles. Environmental scanning electron microscopy (ESEM) of MP-Chol-DDA-TDB (16:16:4:0.5 μmol) indicated that incorporation of antigen led to increased stability of the vesicles, perhaps as a result of the antigen embedding within the vesicle bilayers. At 4°C DDA liposomes showed significant vesicle aggregation after 28 days, although addition of MP-Chol or TDB was shown to inhibit this instability. Alternatively, at 25°C only the MP-based systems retained their original size. The presence of MP within the vesicle formulation was also shown to promote a sustained release of antigen in-vitro. The adjuvant activity of various systems was tested in mice against three subunit antigens, including mycobacterial fusion protein Ag85b-ESAT-6, and two malarial antigens (Merozoite surface protein 1, MSP1, and the glutamate rich protein, GLURP). The MP- and DDA-based systems induced antibody responses at comparable levels whereas the DDA-based systems induced more powerful cell-mediated immune responses. © 2006 The Authors.

Radiolabelling of antigen and liposomes for vaccine biodistribution studies

A relatively simple and effective method to follow the movement of pharmaceutical preparations such as vaccines in biodistribution studies is to radiolabel the components. Whilst single radiolabelling is common practice, in vaccine systems containing adjuvants the ability to follow both the adjuvant and the antigen is favourable. To this end, we have devised a dual-radiolabelling method whereby the adjuvant (liposomes) is labelled with 3H and the antigen (a subunit protein) with 125I. This model is stable and reproducible; we have shown release of the radiolabels to be negligible over periods of up to 1 week in foetal calf serum at 37° C. In this paper we describe the techniques which enable the radiolabelling of various components, assessing stability and processing of samples which all for their application in biodistribution studies. Furthermore we provide examples derived from our studies using this model in tuberculosis vaccine biodistribution studies. © 2010 by the authors.

Tissue-specific selection of reference genes is required for expression studies in the mouse model of maternal protein undernutrition

Suboptimal maternal nutrition during gestation results in the establishment of long-term phenotypic changes and an increased disease risk in the offspring. To elucidate how such environmental sensitivity results in physiological outcomes, the molecular characterisation of these offspring has become the focus of many studies. However, the likely modification of key cellular processes such as metabolism in response to maternal undernutrition raises the question of whether the genes typically used as reference constants in gene expression studies are suitable controls. Using a mouse model of maternal protein undernutrition, we have investigated the stability of seven commonly used reference genes (18s, Hprt1, Pgk1, Ppib, Sdha, Tbp and Tuba1) in a variety of offspring tissues including liver, kidney, heart, retro-peritoneal and inter-scapular fat, extra-embryonic placenta and yolk sac, as well as in the preimplantation blastocyst and blastocyst-derived embryonic stem cells. We find that although the selected reference genes are all highly stable within this system, they show tissue, treatment and sex-specific variation. Furthermore, software-based selection approaches rank reference genes differently and do not always identify genes which differ between conditions. Therefore, we recommend that reference gene selection for gene expression studies should be thoroughly validated for each tissue of interest. © 2011 Elsevier Inc.

Stability analysis of tree structured decision functions

In multicriteria decision problems many values must be assigned, such as the importance of the different criteria and the values of the alternatives with respect to subjective criteria. Since these assignments are approximate, it is very important to analyze the sensitivity of results when small modifications of the assignments are made. When solving a multicriteria decision problem, it is desirable to choose a decision function that leads to a solution as stable as possible. We propose here a method based on genetic programming that produces better decision functions than the commonly used ones. The theoretical expectations are validated by case studies. © 2003 Elsevier B.V. All rights reserved.