The sustained delivery of antisense oligodeoxynucleotides using biodegradable polymer microspheres

Antisense technology is a novel drug discovery method, which provides an essential tool for directly using gene sequence information to rationally design specific inhibitions of mRNA, to treat a wide range of diseases. The efficacy of naked oligodeoxynucleotides (ODNs) is relatively short lived due to rapid degradation in vivo. The entrapment of ODNs within biodegradable sustained-release delivery systems may improve ODN stability and reduce dose required for efficacy. Biodegradable polymer microspheres were evaluated as delivery devices for ODNs and ribozymes. Poly(lactide-co-glycolide) polymers were used due to their biocompatibility and non toxic degradation products. Microspheres were prepared using a double emulsion-deposition method and the formulations characterised. In vitro release profiles were characterised by an initial burst effect during the first 48 hours of release followed by a more sustained release. The release profiles were influenced by microsphere size, copolymer molecular weight, copolymer ratio, ODN loading, ODN length, and ODN chemistry. The serum stability of ODNs was significantly improved when entrapped within polymer microspheres. The cellular association of ODNs entrapped within small spheres (1-2μm) was improved by approximately 20-fold in A431 carcinoma cells compared with free ODNs. Fluorescence microscopy studies showed a more diffuse subcellular distribution when delivered as a microsphere formulation compared with free ODNs, which exhibited the characteristic punctate periplasmic distribution. For in vivo evaluation, polymer microspheres containing fluorescently-labelled ODNs were stereo-taxically administered to the neostriatum of the rat brain. Free ODN resulted in a punctate cellular distribution after 24 hours. In comparison ODN delivered using polymer microspheres were intensely visible in cells 48 hours post administration, and fluorescence appeared to be diffuse covering both cytosolic and nuclear regions. Whole-body autoradiography was also used to evaluate the biodistribution of free tritium labelled ODN and ODN entrapped microspheres, following subcutaneous administration to Balb-C mice. Polymer entrapped ODN gave a similar biodistribution to free ODN. Free ODN was distributed within 24 hours, whereas polymer released ODN was observed still presented in organs and at the site of administration seven days post administration.

Biodegradable polymers for the sustained release of antisense nucleic acids

Antisense oligodeoxynucleotides can selectively inhibit individual gene expression provided they remain stable at the target site for a sufficient period of time. Thus, the efficacy of antisense oligodeoxynucleotides may be improved by employing a sustained release delivery system which would protect from degradation by nucleases whilst delivering the nucleic acid in a controlled manner to the site of action. Biodegradable polymer films and micro spheres were evaluated as delivery devices for the oligodeoxynucleotides and ribozymes. Polymers such as polylactide, polyglycolide, polyhydroxybutyrate and polyhydroxyvalerate were used due to their biocompatability and non toxic degradation products. Release profiles of antisense nucleic acids from films over 28 days was biphasic, characterised by an initial burst release during the first 48 hours followed by a more sustained release. Release from films of longer antisense nucleic acids was slower compared to shorter nucleic acids. Backbone type also affected release, although to a lesser extent than length. Total release of the nucleic acids is dependent upon polymer degradation, no degradation of the polymer films was evident over the 28 day period, due to the high molecular weight and crystallinity of the polymers required to make solvent cast films. Backbone length and type did not affect release from microspheres, release was generally faster than from films, due to the increased surface area, and low molecular weight polymers which showed signs of degradation over the release period, resulting in a triphasic release profile. An increase in release was observed when sphere size and polymer molecular weight were decreased. The polymer entrapped phosphodiester oligodeoxynucleotides and ribozymes had enhanced stability compared to free oligodeoxynucleotides and ribozymes when incubated in serum. The released nucleic acids were still capable of hybridising to their target sequence, indicating that the fabrication processes did not adversely effect the properties of the antisense nucleic acids. Oligodeoxynucleotides loaded in 2μm spheres had a 10 fold increase in macrophage association compared to free oligodeoxynucleotides. Fluorescent microscopy indicates that the polymer entrapped oligodeoxynucleotide is concentrated inside the cell, whereas free oligodeoxynucleotides are concentrated at the cell membrane. Biodegradable polymers can reduce the limitations of antisense therapy and thus offer a potential therapeutic advantage.

Studies on the thermal decomposition behaviour, kinetics and electrical conductivity of the non-isothermal decomposition of pyridine mono carboxylic acids and some of their transition metal complexes

The thesis is divided into four chapters. They are: introduction, experimental, results and discussion about the free ligands and results and discussion about the complexes. The First Chapter, the introductory chapter, is a general introduction to the study of solid state reactions. The Second Chapter is devoted to the materials and experimental methods that have been used for carrying out tile experiments. TIle Third Chapter is concerned with the characterisations of free ligands (Picolinic acid, nicotinic acid, and isonicotinic acid) by using elemental analysis, IR spectra, X-ray diffraction, and mass spectra. Additionally, the thermal behaviour of free ligands in air has been studied by means of thermogravimetry (TG), derivative thermogravimetry (DTG), and differential scanning calorimetry (DSC) measurements. The behaviour of thermal decomposition of the three free ligands was not identical Finally, a computer program has been used for kinetic evaluation of non-isothermal differential scanning calorimetry data according to a composite and single heating rate methods in comparison with the methods due to Ozawa and Kissinger methods. The most probable reaction mechanism for the free ligands was the Avrami-Erofeev equation (A) that described the solid-state nucleation-growth mechanism. The activation parameters of the decomposition reaction for free ligands were calculated and the results of different methods of data analysis were compared and discussed. The Fourth Chapter, the final chapter, deals with the preparation of cobalt, nickel, and copper with mono-pyridine carboxylic acids in aqueous solution. The prepared complexes have been characterised by analyses, IR spectra, X-ray diffraction, magnetic moments, and electronic spectra. The stoichiometry of these compounds was ML2x(H20), (where M = metal ion, L = organic ligand and x = water molecule). The environments of cobalt, nickel, and copper nicotinates and the environments of cobalt and nickel picolinates were octahedral, whereas the environment of copper picolinate [Cu(PA)2] was tetragonal. However, the environments of cobalt, nickel, and copper isonicotinates were polymeric octahedral structures. The morphological changes that occurred throughout the decomposition were followed by SEM observation. TG, DTG, and DSC measurements have studied the thermal behaviour of the prepared complexes in air. During the degradation processes of the hydrated complexes, the crystallisation water molecules were lost in one or two steps. This was also followed by loss of organic ligands and the metal oxides remained. Comparison between the DTG temperatures of the first and second steps of the dehydration suggested that the water of crystallisation was more strongly bonded with anion in Ni(II) complexes than in the complexes of Co(II) and Cu(II). The intermediate products of decomposition were not identified. The most probable reaction mechanism for the prepared complexes was also Avrami-Erofeev equation (A) characteristic of solid-state nucleation-growth mechanism. The tempemture dependence of conductivity using direct current was determined for cobalt, nickel, Cl.nd copper isonicotinates. An activation energy (ΔΕ), the activation energy (ΔΕ ) were calculated.The ternperature and frequency dependence of conductivity, the frequency dependence of dielectric constant, and the dielectric loss for nickel isonicotinate were determined by using altemating current. The value of s paralneter and the value of'density of state [N(Ef)] were calculated. Keyword Thermal decomposition, kinetic, electrical conduclion, pyridine rnono~ carboxylic acid, cOlnplex, transition metal compJex.

The effect of blending on selected physical properties of crude oils and their products

A study was made of the effect of blending practice upon selected physical properties of crude oils, and of various base oils and petroleum products, using a range of binary mixtures. The crudes comprised light, medium and heavy Kuwait crude oils. The properties included kinematic viscosity, pour point, boiling point and Reid vapour pressure. The literature related to the prediction of these properties, and the changes reported to occur on blending, was critically reviewed as a preliminary to the study. The kinematic viscosity of petroleum oils in general exhibited non-ideal behaviour upon blending. A mechanism was proposed for this behaviour which took into account the effect of asphaltenes content. A correlation was developed, as a modification of Grunberg's equation, to predict the viscosities of binary mixtures of petroleum oils. A correlation was also developed to predict the viscosities of ternary mixtures. This correlation showed better agreement with experimental data (< 6% deviation for crude oils and 2.0% for base oils) than currently-used methods, i.e. ASTM and Refutas methods. An investigation was made of the effect of temperature on the viscosities of crude oils and petroleum products at atmospheric pressure. The effect of pressure on the viscosity of crude oil was also studied. A correlation was developed to predict the viscosity at high pressures (up to 8000 psi), which gave significantly better agreement with the experimental data than the current method due to Kouzel (5.2% and 6.0% deviation for the binary and ternary mixtures respectively). Eyring's theory of viscous flow was critically investigated, and a modification was proposed which extends its application to petroleum oils. The effect of blending on the pour points of selected petroleum oils was studied together with the effect of wax formation and asphaltenes content. Depression of the pour point was always obtained with crude oil binary mixtures. A mechanism was proposed to explain the pour point behaviour of the different binary mixtures. The effects of blending on the boiling point ranges and Reid vapour pressures of binary mixtures of petroleum oils were investigated. The boiling point range exhibited ideal behaviour but the R.V.P. showed negative deviations from it in all cases. Molecular weights of these mixtures were ideal, but the densities and molar volumes were not. The stability of the various crude oil binary mixtures, in terms of viscosity, was studied over a temperature range of 1oC - 30oC for up to 12 weeks. Good stability was found in most cases.

Formulation of topical non-steroidal anti-inflammatory agents

Reversed-pahse high-performance liquid chromatographic (HPLC) methods were developed for the assay of indomethacin, its decomposition products, ibuprofen and its (tetrahydro-2-furanyl)methyl-, (tetrahydro-2-(2H)pyranyl)methyl- and cyclohexylmethyl esters. The development and application of these HPLC systems were studied. A number of physico-chemical parameters that affect percutaneous absorption were investigated. The pKa values of indomethacin and ibuprofen were determined using the solubility method. Potentiometric titration and the Taft equation were also used for ibuprofen. The incorporation of ethanol or propylene glycol in the solvent resulted in an improvement in the aqueous solubility of these compounds. The partition coefficients were evaluated in order to establish the affinity of these drugs towards the stratum corneum. The stability of indomethacin and of ibuprofen esters were investigated and the effect of temperature and pH on the decomposition rates were studied. The effect of cetyltrimethylammonium bromide on the alkaline degradation of indomethacin was also followed. In the presence of alcohol, indomethacin alcoholysis was observed and the kinetics of decomposition were subjected to non-linear regression analysis and the rate constants for the various pathways were quantified. The non-isothermal, sufactant non-isoconcentration and non-isopH degradation of indomethacin were investigated. The analysis of the data was undertaken using NONISO, a BASIC computer program. The degradation profiles obtained from both non-iso and iso-kinetic studies show that there is close concordance in the results. The metabolic biotransformation of ibuprofen esters was followed using esterases from hog liver and rat skin homogenates. The results showed that the esters were very labile under these conditions. The presence of propylene glycol affected the rates of enzymic hydrolysis of the ester. The hydrolysis is modelled using an equation involving the dielectric constant of the medium. The percutaneous absorption of indomethacin and of ibuprofen and its esters was followed from solutions using an in vitro excised human skin model. The absorption profiles followed first order kinetics. The diffusion process was related to their solubility and to the human skin/solvent partition coefficient. The percutaneous absorption of two ibuprofen esters from suspensions in 20% propylene glycol-water were also followed through rat skin with only ibuprofen being detected in the receiver phase. The sensitivity of ibuprofen esters to enzymic hydrolysis compared to the chemical hydrolysis may prove valuable in the formulation of topical delivery systems.

Developing a forest data portal to support multi-scale decision making

Forests play a pivotal role in timber production, maintenance and development of biodiversity and in carbon sequestration and storage in the context of the Kyoto Protocol. Policy makers and forest experts therefore require reliable information on forest extent, type and change for management, planning and modeling purposes. It is becoming increasingly clear that such forest information is frequently inconsistent and unharmonised between countries and continents. This research paper presents a forest information portal that has been developed in line with the GEOSS and INSPIRE frameworks. The web portal provides access to forest resources data at a variety of spatial scales, from global through to regional and local, as well as providing analytical capabilities for monitoring and validating forest change. The system also allows for the utilisation of forest data and processing services within other thematic areas. The web portal has been developed using open standards to facilitate accessibility, interoperability and data transfer.

General and multiplicative non-parametric corporate performance models with interval ratio data

The increasing intensity of global competition has led organizations to utilize various types of performance measurement tools for improving the quality of their products and services. Data envelopment analysis (DEA) is a methodology for evaluating and measuring the relative efficiencies of a set of decision making units (DMUs) that use multiple inputs to produce multiple outputs. All the data in the conventional DEA with input and/or output ratios assumes the form of crisp numbers. However, the observed values of data in real-world problems are sometimes expressed as interval ratios. In this paper, we propose two new models: general and multiplicative non-parametric ratio models for DEA problems with interval data. The contributions of this paper are fourfold: (1) we consider input and output data expressed as interval ratios in DEA; (2) we address the gap in DEA literature for problems not suitable or difficult to model with crisp values; (3) we propose two new DEA models for evaluating the relative efficiencies of DMUs with interval ratios, and (4) we present a case study involving 20 banks with three interval ratios to demonstrate the applicability and efficacy of the proposed models where the traditional indicators are mostly financial ratios. © 2011 Elsevier Inc.

Textbook (non-)adoption motives, legitimizing strategies and academic field configuration

Textbooks are an integral part of structured syllabus coverage in higher education. The argument advanced in this article is that textbooks are not simply products of inscription and embodied scholarly labour for pedagogical purposes, but embedded institutional artefacts that configure entire academic subject fields. Empirically, this article shows the various ways that motives of the (non-) adoption of textbooks have field institutional configuration effects. The research contribution of our study is threefold. First, we re-theorise the textbook as an artefact that is part of the institutional work and epistemic culture of academia. Second, we empirically show that the vocabularies of motive of textbook (non-) adoption and rhetorical strategies form the basis for social action and configuration across micro, meso and macro field levels. Our final contribution is a conceptualization of the ways that textbook (non-) adoption motives ascribe meaning to the legitimating processes in the configuration of whole subject fields.

A CFD study of biomass pyrolysis in a downer reactor equipped with a novel gas-solid separator-II thermochemical performance and products

A Eulerian-Eulerian CFD model was used to investigate the fast pyrolysis of biomass in a downer reactor equipped with a novel gas-solid separation mechanism. The highly endothermic pyrolysis reaction was assumed to be entirely driven by an inert solid heat carrier (sand). A one-step global pyrolysis reaction, along with the equations describing the biomass drying and heat transfer, was implemented in the hydrodynamic model presented in part I of this study (Fuel Processing Technology, V126, 366-382). The predictions of the gas-solid separation efficiency, temperature distribution, residence time and the pyrolysis product yield are presented and discussed. For the operating conditions considered, the devolatilisation efficiency was found to be above 60% and the yield composition in mass fraction was 56.85% bio-oil, 37.87% bio-char and 5.28% non-condensable gas (NCG). This has been found to agree reasonably well with recent relevant published experimental data. The novel gas-solid separation mechanism allowed achieving greater than 99.9% separation efficiency and < 2 s pyrolysis gas residence time. The model has been found to be robust and fast in terms of computational time, thus has the great potential to aid in future design and optimisation of the biomass fast pyrolysis process.

Power in distribution channels - Supplier assortment strategy for balancing power

This paper focuses on the move from buyer dominance toward interdependence between buyers and suppliers in a distribution channel. The paper introduces a case study collected through in-depth interviews and participative observations. It examines the relationships between a timber supplier and its customers in the builders' merchants sector. We stress the relevance of considering actions intended to change the power balance, rather than focusing only on trust. The power balance in a dyadic relationship is dynamic, and power positions need to be constantly re-evaluated. An important power resource is information asymmetry, manifested in the supplier's information about: products, regional and local demand, and the usage of the products. For practitioners, we highlight the possibility of exerting a non-coercive power resource, such as information asymmetry, in order to increase the relative power. Furthermore, being open about the power position between a buyer and a seller can foster a more efficient collaboration.

Effects of advanced glycation end-products (AGEs) on retinal pigment epithelial (RPE) cells lysosomal function:implications for age-related macular degeneration (AMD)

Purpose: RPE lysosomal dysfunction is a major contributor to AMD pathogenesis. Controlled activity of a major class of RPE proteinases, the cathepsins, is crucial in maintaining correct lysosomal function. Advanced glycation end-products (AGEs) accumulate in the Bruch’s membrane (BM) with age, impacting critical RPE functions and in turn, contributing to the development of AMD. The aim of this study was to assess the effect of AGEs on lysosomal function by analysing the expression, processing and activity of the cysteine proteinases cathepsins B, L and S, and the aspartic proteinase cathepsin D. Methods: ARPE-19 cells were cultured on AGE-containing BM mimics (matrigel) for 14 days and compared to untreated substrate. Expression levels and intracellular processing of cathepsins B, D, L and S, were assessed by qPCR and immunoblotting of cell lysates. Lysosomal activity was investigated using multiple activity assays specific to each of the analysed cathepsins. Statistical analysis was performed using the Student’s independent T-test. Results: AGE exposure produced a 36% decrease in cathepsin L activity when compared to non-treated controls (p=0.02, n= 3) although no significant changes were observed in protein expression/processing under these conditions. Both the pro and active forms of cathepsin S decreased by 40% (p=0.04) and 74% (p=0.004), respectively (n=3). In contrast, the active form of the cathepsin D increased by 125% (p=0.005, n= 4). However, no changes were observed in the activity levels of both cathepsins S and D. In addition, cathepsin B expression, processing and activity also remained unaltered following AGE exposure. Conclusions: AGEs accumulation in the extracellular matrix, a phenomenon associated with the natural aging process of the BM, attenuates the expression, intracellular processing and activity of specific lysosomal effectors. Altered enzymatic function may impair important lysosomal processes such as endocytosis, autophagy and phagocytosis of photoreceptor outer segments, each of which may influence the age-related dysfunction of the RPE and subsequently, AMD pathogenesis.