Environmental scanning electron microscopy offers real-time morphological analysis of liposomes and niosomes

Liposomes have been imaged using a plethora of techniques. However, few of these methods offer the ability to study these systems in their natural hydrated state without the requirement of drying, staining, and fixation of the vesicles. However, the ability to image a liposome in its hydrated state is the ideal scenario for visualization of these dynamic lipid structures and environmental scanning electron microscopy (ESEM), with its ability to image wet systems without prior sample preparation, offers potential advantages to the above methods. In our studies, we have used ESEM to not only investigate the morphology of liposomes and niosomes but also to dynamically follow the changes in structure of lipid films and liposome suspensions as water condenses on to or evaporates from the sample. In particular, changes in liposome morphology were studied using ESEM in real time to investigate the resistance of liposomes to coalescence during dehydration thereby providing an alternative assay of liposome formulation and stability. Based on this protocol, we have also studied niosome-based systems and cationic liposome/DNA complexes. Copyright © Informa Healthcare.

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.

On chlorosulphonation and the electron microscopy of polyethylene

It is shown that chlorosulphonation is a major aid to the electron microscopy of polyethylene for various samples which had mostly been crystallized at high pressures and included at least a proportion of the so-called chain-extended form. It is confirmed that sheets of excess electron density are produced at lamellar surfaces, but also including lateral surfaces. This is due primarily to the incorporation of chlorine and sulphur rather than to added uranium. The time to achieve an overall reaction varies sensitively with morphology, decreasing as the number of diffusion channels increases. Crystallinity is gradually lost, but sufficient crystals remain when a sample has become uniform, and in their initial orientations, for diffraction studies to be possible. The technique has been used to demonstrate that, during melt crystallization, the thickness of one lamella changes in response to altered growth conditions. This is direct confirmation that lamellar thickness is determined by secondary nucleation at the growth front. The tapered profile of a growing lamella previously observed in thick crystals of various polymers has been observed for chain-folded polyethylene lamellae, providing further evidence that this is a general feature of melt growth. © 1977 Chapman and Hall Ltd.

In situ aberration corrected-transmission electron microscopy of magnesium oxide nanocatalysts for biodiesels

Biofuels are promising renewable energy sources and can be derived from vegetable oil feedstocks. Although solid catalysts show great promise in plant oil triglyceride transesterification to biodiesel, the identification of active sites and operating surface nanostructures created during their processing is essential for the development of efficient heterogeneous catalysts. Systematic, direct observations of dynamic MgO nanocatalysts from a magnesium hydroxide-methoxide precursor were performed under controlled calcination conditions using novel in situ aberration corrected-transmission electron microscopy at the 0.1 nm level and quantified with catalytic reactivity and physico-chemical studies. Surface structural modifications and the evolution of extended atomic scale glide defects implicate coplanar anion vacancies in active sites in the transesterification of triglycerides to biodiesel. The linear correlation between surface defect density (and therefore polarisability) and activity affords a simple means to fine tune new, energy efficient nanocatalysts for biofuel synthesis. © 2009 Springer Science+Business Media, LLC.

Systems biology approach to study permeability of paracetamol and its solid dispersion

Physiological changes that take place at cellular level are usually reflective of their level of gene expression. Different formulation excipients have an impact on physiological behavior of the exposed cells and in turn affect transporter genes, enterocyte-mediated metabolism and toxicity biomarkers. The aim of this study was to prepare solid dispersion of paracetamol and evaluate genetic changes that occur in Caco-2 cell lines during the permeability of paracetamol alone and paracetamol solid dispersion formulations. Paracetamol-PEG 8000 solid dispersion was prepared by melt fusion method and the formulation was characterised using differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Formulation of solid dispersion resulted in the conversion of crystalline drug into an amorphous form. Permeability studies showed that paracetamol absorption was higher from the solid dispersion formulation. DNA microarrays analysis was carried out in order to investigate the involvement of any efflux/uptake transporters in paracetamol or its solid dispersion permeability. Neither transporter carriers nor efflux proteins were found to be involved in the absorption of paracetamol or its PEG solid dispersion. Gene expression analysis established that paracetamol toxicity was potentially reduced upon formulation into solid dispersion when ATP binding cassette (ABC) and solute carrier transporter (SLC) genes were analyzed.

The mechanical and wear behaviour of B(SiC) fibre-reinforced composite materials

The mechanical properties and wear behaviour of B(SiC) fibre-reinforced metal matrix composites (MMCs) and aluminium alloy (2014) produced by metal infiltration technique were determined. Tensile tests were peliormed at different conditions on both the alloy matrix and its composite, and the tensile fracture surfaces were also examined by Scanning Electron Microscopy (SEM). Dry wear of the composite materials sliding on hardened steel was studied using a pin-on-disc type machine. The effect of fibre orientation on wear rate was studied to provide wear resistance engineering data on the MMCs. Tests were carried out with the wear surface sliding direction set normal, parallel and anti-parallel to the fibre axis. Experiments were perfonned for sliding speeds of 0.6, 1.0 and 1.6 m/s for a load range from 12 N to 60 N. A number of sensitive techniques were used to examine worn surface and debris, i.e: Scanning Electron Microscopy (SEM), Backscattered Electron Microscopy (BSEM) and X-ray Photoelectron Spectroscopy (XPS). Finally, the effect of fibre orientation on the wear rate of the Borsic-reinforced plastic matrix composites (PMCs) produced by hot pressing technique was also investigated under identical test conditions. It was found that the composite had a markedly increased tensile strength compared with the matrix. The wear results also showed that the composite exhibited extremely low wear rates compared to the matrix material and the wear rate increased with increasing sliding speed and normal load. The effect of fibre orientation was marked, the lowest wear rates were obtained by arranging the fibre perpendicular to the sliding surface, while the highest wear was obtained for the parallel orientation. The coefficient of friction was found to be lowest in the parallel orientation than the others. Wear of PMCs were influenced to the greatest extent by these test parameters although similar findings were obtained for both composites. Based on the results of analyses using SEM, BSED and XPS, possible wear mechanisms are suggested to explain the wear of these materials.

Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6′- dibehenate) - A novel adjuvant inducing both strong CMI and antibody responses:a novel adjuvant inducing both strong CMI and antibody responses

Incorporation of the glycolipid trehalose 6,6′-dibehenate (TDB) into cationic liposomes composed of the quaternary ammonium compound dimethyldioctadecylammonium (DDA) produce an adjuvant system which induces a powerful cell-mediated immune response and a strong antibody response, desirable for a high number of disease targets. We have used differential scanning calorimetry (DSC) to investigate the effect of TDB on the gel-fluid phase transition of DDA liposomes and to demonstrate that TDB is incorporated into DDA liposome bilayers. Transmission Electron Microscopy (TEM) and cryo-TEM confirmed that liposomes were formed when a lipid film of DDA containing small amounts of TDB was hydrated in an aqueous buffer solution at physiological pH. Furthermore, time development of particle size and zeta potential of DDA liposomes incorporating TDB during storage at 4°C and 25°C, indicates that TDB effectively stabilizes the DDA liposomes. Immunization of mice with the mycobacterial fusion protein Ag85B-ESAT-6 in DDA-TDB liposomes induced a strong, specific Th1 type immune response characterized by substantial production of the interferon-γ cytokine and high levels of IgG2b isotype antibodies. The lymphocyte subset releasing the interferon-γ was identified as CD4 T cells.

The design of gas chromatography ionisation detectors:a study of the electron capture detector and the home ionisation detector

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Microemulsions as topical delivery vehicles for the anti-melanoma prodrug, temozolomide hexyl ester (TMZA-HE)

A prodrug, temozolomide acid hexyl ester (TMZA-HE), was identified as a skin-deliverable congener for temozolomide (TMZ) to treat skin cancers. Poor solubility and instability of TMZA-HE rendered a serious challenge for formulation of a topical preparation. Microemulsions (ME) were chosen as a potential vehicle for TMZA-HE topical preparations. ME systems were constructed with either oleic acid (OA) or isopropyl myristate (IPM) as the oil phase and tocopheryl (vitamin E) polyethylene glycol 1000 succinate (VE-TPGS) as a surfactant. Topical formulations of OA and IPM ME systems demonstrated beneficial solubilising ability and provided a stable environment for the prodrug, TMZA-HE. Significant differences between the microstructures of OA and IPM ME systems were revealed by freeze fracture electron microscopy (FFEM) and different loading abilities and permeation potencies between the two systems were also identified. In permeation studies, IPM ME systems, with inclusion of isopropyl alcohol (IPA) as a co-surfactant, significantly increased TMZA-HE permeation through silicon membranes and rat skin resulting in less drug retention within the skin, while OA ME systems demonstrated higher solubilising ability and a higher concentration of TMZA-HE retained within the skin. Therefore IPM ME systems are promising for transdermal delivery of TMZA-HE and OA ME systems may be a suitable choice for a topical formulation of TMZA-HE. © 2007 The Authors.

The visualisation of vitreous using surface modified poly(lactic-co-glycolic acid) microparticles

AIMS To demonstrate the potential use of in vitro poly(lactic-co-glycolic acid) (PLGA) microparticles in comparison with triamcinolone suspension to aid visualisation of vitreous during anterior and posterior vitrectomy. METHODS PLGA microparticles (diameter 10-60 microm) were fabricated using single and/or double emulsion technique(s) and used untreated or following the surface adsorption of a protein (transglutaminase). Particle size, shape, morphology and surface topography were assessed using scanning electron microscopy (SEM) and compared with a standard triamcinolone suspension. The efficacy of these microparticles to enhance visualisation of vitreous against the triamcinolone suspension was assessed using an in vitro set-up exploiting porcine vitreous. RESULTS Unmodified PLGA microparticles failed to adequately adhere to porcine vitreous and were readily washed out by irrigation. In contrast, modified transglutaminase-coated PLGA microparticles demonstrated a significant improvement in adhesiveness and were comparable to a triamcinolone suspension in their ability to enhance the visualisation of vitreous. This adhesive behaviour also demonstrated selectivity by not binding to the corneal endothelium. CONCLUSION The use of transglutaminase-modified biodegradable PLGA microparticles represents a novel method of visualising vitreous and aiding vitrectomy. This method may provide a distinct alternative for the visualisation of vitreous whilst eliminating the pharmacological effects of triamcinolone acetonide suspension.

Abrasion resistance of concrete

This thesis describes an experimental study of the abrasion resistance of concrete at both the macro and micro levels. This is preceded by a review related to friction and wear, methods of test for assessing abrasion resistance, and factors influencing the abrasion resistance of concrete. A versatile test apparatus was developed to assess the abrasion resistance of concrete. This could be operated in three modes and a standardised procedure was established for all tests. A laboratory programme was undertaken to investigate the influence, on abrasion resistance, of three major factors - finishing techniques, curing regimes and surface treatments. The results clearly show that abrasion resistance was significantly affected by these factors, and tentative mechanisms were postulated to explain these observations. To substantiate these mechanisms, the concrete specimens from the macro-study were subjected to micro-structural investigation, using such techniques as 'Mercury Intrusion Forosimetry, Microhardness, Scanning Electron Microscopy, Petrography and Differential Thermal Analysis. The results of this programme clearly demonstrated that the abrasion resistance of concrete is primarily dependent on the microstructure of the concrete nearest to the surface. The viability of indirectly assessing the abrasion resistance was investigated using three non-destructive techniques - Ultrasonic Pulse Velocity, Schmidt Rebound Hardness, and the Initial Surface Absorption Test. The Initial Surface Absorption was found to be most sensitive to factors which were shown to have influenced the abrasion resistance of concrete. An extensive field investigation was also undertaken. The results were used to compare site and laboratorypractices, and the performance in the accelerated abrasion test with the service wear. From this study, criteria were developed for assessing the quality of concrete floor slabs in terms of abrasion resistance.

Influence of impact angles on penetration rates of CRAs exposed to a high velocity multiphase flow

A combined flow loop - jet impingement pilot plant has been used to determine mass loss rates in a mixed gas - saltwater - sand multiphase flow at impact velocities up to 70 m/s. Artificial brine with a salt content of 27 g/1 was used as liquid phase. Sand content, with grain size below 150 µ, was 2.7 g/l brine. CO at a pressure of 15 bar was used as gas phase. The impact angle between jet stream (nozzle) and sample surface was varied between 30 and 90°. Rectangular stainless steel disc samples with a size of 20 × 15 × 5 mm were used. They were mechanically ground and polished prior to testing. Damaged surfaces of specimens exposed to the high velocity multiphase flow were investigated by stereo microscopy, scanning electron microscopy (SEM) and an optical device for 3D surface measurements. Furthermore, samples were investigated by applying atomic force microscopy (AFM), magnetic force microscopy (MFM) and nanoindentation. Influence of impact velocity and impact angle on penetration rates (mass loss rates) of two CRAs (UNS S30400 and N08028) are presented. Moreover effects of chemical composition and mechanical properties are critically discussed. © 2008 by NACE International.

A co-cultured skin model based on cell support membranes

Tissue engineering of skin based on collagen:PCL biocomposites using a designed co-culture system is reported. The collagen:PCL biocomposites having collagen:PCL (w/w) ratios of 1:4, 1:8, and 1:20 have been proven to be biocompatible materials to support both adult normal human epidermal Keratinocyte (NHEK) and mouse 3T3 fibroblast growth in cell culture, respectively, by Dai, Coombes, et al. in 2004. Films of collagen:PCL biocomposites were prepared using non-crosslinking method by impregnation of lyophilized collagen mats with PCL/dichloromethane solutions followed by solvent evaporation. To mimic the dermal/epidermal structure of skin, the 1:20 collagen:PCL biocomposites were selected for a feasibility study of a designed co-culture technique that would subsequently be used for preparing fibroblast/biocomposite/keratinocyte skin models. A 55.3% increase in cell number was measured in the designed co-culture system when fibroblasts were seeded on both sides of a biocomposite film compared with cell culture on one surface of the biocomposite in the feasibility study. The co-culture of human keratinocytes and 3T3 fibroblasts on each side of the membrane was therefore studied using the same co-culture system by growing keratinocytes on the top surface of membrane for 3 days and 3T3 fibroblasts underneath the membrane for 6 days. Scanning electron microscopy (SEM) and immunohistochemistry assay revealed good cell attachment and proliferation of both human keratinocytes and 3T3 fibroblasts with these two types of cells isolated well on each side of the membrane. Using a modified co-culture technique, a co-cultured skin model presenting a confluent epidermal sheet on one side of the biocomposite film and fibroblasts populated on the other side of the film was developed successfully in co-culture system for 28 days under investigations by SEM and immunohistochemistry assay. Thus, the design of a co-culture system based on 1:20 (w/w) collagen:PCL biocomposite membranes for preparation of a bi-layered skin model with differentiated epidermal sheet was proven in principle. The approach to skin modeling reported here may find application in tissue engineering and screening of new pharmaceuticals. © 2005 Elsevier Inc. All rights reserved.

Ion irradiation effects in nanocrystalline TiN coatings

Nitride materials and coatings have attracted extensive research interests for various applications in advanced nuclear reactors due to their unique combination of physical properties, including high temperature stability, excellent corrosion resistance, superior mechanical property and good thermal conductivity. In this paper, the ion irradiation effects in nanocrystalline TiN coatings as a function of grain size are reported. TiN thin films (thickness of 100 nm) with various grain sizes (8-100 nm) were prepared on Si substrates by a pulsed laser deposition technique. All the samples were irradiated with He ions to high fluences at room temperature. Transmission electron microscopy (TEM) and high resolution TEM on the ion-irradiated samples show that damage accumulation in the TiN films reduces as the grain size reduces. Electrical resistivity of the ion-irradiated films increases slightly compared with the as-deposited ones. These observations demonstrate a good radiation-tolerance property of nanocrystalline TiN films. © 2007 Elsevier B.V. All rights reserved.

Heavy ion irradiation effects in zirconium nitride

Polycrystalline zirconium nitride (ZrN) samples were irradiated with He +, Kr ++, and Xe ++ ions to high (>1·10 16 ions/cm 2) fluences at ∼100 K. Following ion irradiation, transmission electron microscopy (TEM) and grazing incidence X-ray diffraction (GIXRD) were used to analyze the microstructure and crystal structure of the post-irradiated material. For ion doses equivalent to approximately 200 displacements per atom (dpa), ZrN was found to resist any amorphization transformation, based on TEM observations. At very high displacement damage doses, GIXRD measurements revealed tetragonal splitting of some of the diffraction maxima (maxima which are associated with cubic ZrN prior to irradiation). In addition to TEM and GIXRD, mechanical property changes were characterized using nanoindentation. Nanoindentation revealed no change in elastic modulus of ZrN with increasing ion dose, while the hardness of the irradiated ZrN was found to increase significantly with ion dose. Finally, He + ion implanted ZrN samples were annealed to examine He gas retention properties of ZrN as a function of annealing temperature. He gas release was measured using a residual gas analysis (RGA) spectrometer. RGA measurements were performed on He-implanted ZrN samples and on ZrN samples that had also been irradiated with Xe ++ ions, in order to introduce high levels of displacive radiation damage into the matrix. He evolution studies revealed that ZrN samples with high levels of displacement damage due to Xe implantation, show a lower temperature threshold for He release than do pristine ZrN samples.