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.

Environmental scanning electron microscope imaging of vesicle systems

The structural characteristics of liposomes have been widely investigated and there is certainly a strong understanding of their morphological characteristics. Imaging of these systems, using techniques such as freeze-fracturing methods, transmission electron microscopy, and cryo-electron imaging, has allowed us to appreciate their bilayer structures and factors that influence this. However, there are a few methods that study these systems in their natural hydrated state; commonly, the liposomes are visualized after drying, staining and/or fixation of the vesicles. Environmental scanning electron microscopy (ESEM) offers the ability to image a liposome in its hydrated state without the need for prior sample preparation. We were the first to use ESEM to study the liposomes and niosomes, and have been able to dynamically follow the hydration of lipid films and changes in liposome suspensions as water condenses onto, or evaporates from, the sample in real-time. This provides an insight into the resistance of liposomes to coalescence during dehydration, thereby providing an alternative assay for liposome formulation and stability.

Environmental scanning electron microscope imaging of vesicle systems

The structural characteristics of liposomes have been widely investigated and there is certainly a strong understanding of their morphological characteristics. Imaging of these systems, using techniques such as freeze-fracturing methods, transmission electron microscopy, and cryo-electron imaging, has allowed us to appreciate their bilayer structures and factors which can influence this. However, there are few methods which all us to study these systems in their natural hydrated state; commonly the liposomes are visualized after drying, staining, and/or fixation of the vesicles. Environmental Scanning Electron Microscopy (ESEM) offers the ability to image a liposome in its hydrated state without the need for prior sample preparation. Within our studies we were the first to use ESEM to study liposomes and niosomes and we have been able to dynamically follow the hydration of lipid films and changes in liposome suspensions as water condenses on to, or evaporates from, the sample in real time. This provides insight into the resistance of liposomes to coalescence during dehydration, thereby providing an alternative assay of liposome formulation and stability.

Microscopy imaging of liposomes:from coverslips to environmental SEM

A plethora of techniques for the imaging of liposomes and other bilayer vesicles are available. However, sample preparation and the technique chosen should be carefully considered in conjunction with the information required. For example, larger vesicles such as multilamellar and giant unilamellar vesicles can be viewed using light microscopy and whilst vesicle confirmation and size prior to additional physical characterisations or more detailed microscopy can be undertaken, the technique is limited in terms of resolution. To consider the options available for visualising liposome-based systems, a wide range of microscopy techniques are described and discussed here: these include light, fluorescence and confocal microscopy and various electron microscopy techniques such as transmission, cryo, freeze fracture and environmental scanning electron microscopy. Their application, advantages and disadvantages are reviewed with regard to their use in analysis of lipid vesicles.

Caking behaviour of spray-dried milk powders:using scanning probe microscopy to study nanoscale surface properties and material composition

Spray drying is widely used to manufacture many powdered products, with the drying process parameters having significant influence over the final powder's surface properties and propensity for unwanted caking. In most cases caking experiments are performed on bulk powders, but especially in multi-component powders, it is often difficult to interpret these results, where interaction effects between particles can be complex. Here the technique of scanning probe microscopy is used to characterize the nanoscale properties of spray dried model milk powders in order to investigate the surface properties of the powders.

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.