Liposome (Lipodine™)-mediated DNA vaccination by the oral route

Plasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen (HBsAg) was incorporated by the dehydration-rehydration method into Lipodine™ liposomes composed of 16 μmoles phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC), 8 μmoles of (dioleoyl phosphatidylethanolamine (DOPE) or cholesterol and 4 μmoles of the cationic lipid 1,2-dioleoyl-3-(trimethylammonium propane (DOTAP) (molar ratios 1:0.5:0.25). Incorporation efficiency was high (89-93% of the amount of DNA used) in all four formulations tested and incorporated DNA was shown to be resistant to displacement in the presence of the competing anionic sodium dodecyl sulphate molecules. This is consistent with the notion that most of the DNA is incorporated within the multilamellar vesicles structure rather than being vesicle surface-complexed. Stability studies performed in simulated intestinal media also demonstrated that dehydration-rehydration vesicles (DRV) incorporating DNA (DRV(DNA)) were able to retain significantly more of their DNA content compared to DNA complexed with preformed small unilamellar vesicles (SUV-DNA) of the same composition. Moreover, after 4h incubation in the media, DNA loss for DSPC DRV(DNA) was only minimal, suggesting this to be the most stable formulation. Oral (intragastric) liposome-mediated DNA immunisation studies employing a variety of DRV(DNA) formulations as well as naked DNA revealed that secreted IgA responses against the encoded HBsAg were (as early as three weeks after the first dose) substantially higher after dosing with 100 μg liposome-entrapped DNA compared to naked DNA. Throughout the fourteen week investigation, IgA responses in mice were consistently higher with the DSPC DRV(DNA) liposomes compared to naked DNA and correlated well with their improved DNA retention when exposed to model intestinal fluids. To investigate gene expression after oral (intragastric) administration, mice were given 100 μg of naked or DSPC DRV liposome-entrapped plasmid DNA expressing the enhanced green fluorescent protein (pCMV.EGFP). Expression of the gene, in terms of fluorescence intensity in the draining mesenteric lymph nodes, was much greater in mice dosed with liposomal DNA than in animals dosed with the naked DNA. These results suggest that DSPC DRV liposomes containing DNA (Lipodine™) may be a useful system for the oral delivery of DNA vaccines.

Liposome formulation of poorly water soluble drugs:optimisation of drug loading and ESEM analysis of stability

Liposomes due to their biphasic characteristic and diversity in design, composition and construction, offer a dynamic and adaptable technology for enhancing drug solubility. Starting with equimolar egg-phosphatidylcholine (PC)/cholesterol liposomes, the influence of the liposomal composition and surface charge on the incorporation and retention of a model poorly water soluble drug, ibuprofen was investigated. Both the incorporation and the release of ibuprofen were influenced by the lipid composition of the multi-lamellar vesicles (MLV) with inclusion of the long alkyl chain lipid (dilignoceroyl phosphatidylcholine (C 24PC)) resulting in enhanced ibuprofen incorporation efficiency and retention. The cholesterol content of the liposome bilayer was also shown to influence ibuprofen incorporation with maximum ibuprofen incorporation efficiency achieved when 4 μmol of cholesterol was present in the MLV formulation. Addition of anionic lipid dicetylphosphate (DCP) reduced ibuprofen drug loading presumably due to electrostatic repulsive forces between the carboxyl group of ibuprofen and the anionic head-group of DCP. In contrast, the addition of 2 μmol of the cationic lipid stearylamine (SA) to the liposome formulation (PC:Chol - 16 μmol:4 μmol) increased ibuprofen incorporation efficiency by approximately 8%. However further increases of the SA content to 4 μmol and above reduced incorporation by almost 50% compared to liposome formulations excluding the cationic lipid. Environmental scanning electron microscopy (ESEM) was used to dynamically follow the changes in liposome morphology during dehydration to provide an alternative assay of liposome stability. ESEM analysis clearly demonstrated that ibuprofen incorporation improved the stability of PC:Chol liposomes as evidenced by an increased resistance to coalescence during dehydration. These finding suggest a positive interaction between amphiphilic ibuprofen molecules and the bilayer structure of the liposome. © 2004 Elsevier B.V. All rights reserved.

Vaccine entrapment in liposomes

The use of liposomes as carriers of peptide, protein, and DNA vaccines requires simple, easy-to-scale-up technology capable of high-yield vaccine entrapment. Work from this laboratory has led to the development of techniques that can generate liposomes of various sizes, containing soluble antigens such as proteins and particulate antigens (e.g., killed or attenuated bacteria or viruses), as well as antigen-encoding DNA vaccines. Entrapment of vaccines is carried out by the dehydration-rehydration procedure which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free vaccines. On rehydration, the large multilamellar vesicles formed incorporate up to 90% or more of the vaccine used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped vaccine still associated with the vesicles. A similar technique applied for the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 microm in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 30% or more of the spores used are associated with generated giant liposomes of similar mean size.

Three-dimensional fluid pressure mapping in porous media using magnetic resonance imaging with gas-filled liposomes

This paper presents and demonstrates a method for using magnetic resonance imaging to measure local pressure of a fluid saturating a porous medium. The method is tested both in a static system of packed silica gel and in saturated sintered glass cylinders experiencing fluid flow. The fluid used contains 3% gas in the form of 3-μm average diameter gas filled 1,2-distearoyl-sn-glycero-3-phosphocholine (C18:0, MW: 790.16) liposomes suspended in 5% glycerol and 0.5% Methyl cellulose with water. Preliminary studies at 2.35 T demonstrate relative magnetic resonance signal changes of 20% per bar in bulk fluid for an echo time TE=40 ms, and 6-10% in consolidated porous media for TE=10 ms, over the range 0.8-1.8 bar for a spatial resolution of 0.1 mm3 and a temporal resolution of 30 s. The stability of this solution with relation to applied pressure and methods for improving sensitivity are discussed. © 2007 Elsevier Inc. All rights reserved.

Exploring value-added benefits to enhance wider adoption of chipless RFID system

Radio-frequency identification technology (RFID) is a popular modern technology proven to deliver a range of value-added benefits to achieve system and operational efficiency, as well as cost-effectiveness. The operational characteristics of RFID outperform barcodes in many aspects. Despite its well-perceived benefits, a definite rationale for larger scale adoption is still not so promising. One of the key reasons is high implementation cost, especially the cost of tags for applications involving item-level tagging. This has resulted in the development of chipless RFID tags which cost much less than conventional chip-based tags. Despite the much lower tag cost, the uptake of chipless RFID system in the market is still not as widespread as predicted by RFID experts. This chapter explores the value-added applications of chipless RFID system to promote wider adoption. The chipless technology's technical and operational characteristics, benefits, limitations and current uses will also be examined. The merit of this chapter is to contribute fresh propositions to the promising applications of chipless RFID to increase its adoption in the industries that are currently not (or less popular in) utilising it, such as retail, logistics, manufacturing, healthcare, and service sectors. © 2013, IGI Global.

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.

Stock repurchases and treasury share sales:do they stabilize price and enhance liquidity?

Can companies reduce the volatility and increase the liquidity of their stocks by trading them? In the context of the Italian stock market, where companies have far more leeway to sell as well as buy their own stocks than in the U.S., the answer is yes. We examine the effects of trading (open-market share repurchases and treasury shares sales) on liquidity (bid–ask spread) and volatility (return variance). Further, we examine the impact of shareholder approvals of repurchase programs on liquidity and volatility. We find clear evidence that trading increases liquidity and reduces volatility. These results are consistent with our analysis of the motives Italian companies give for making share repurchases.

Effect of incorporating cholesterol into DDA:TDB liposomal adjuvants on Bilayer properties, biodistribution, and immune responses

Cholesterol is an abundant component of mammalian cell membranes and has been extensively studied as an artificial membrane stabilizer in a wide range of phospholipid liposome systems. In this study, the aim was to investigate the role of cholesterol in cationic liposomal adjuvant system based on dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) which has been shown as a strong adjuvant system for vaccines against a wide range of diseases. Packaging of cholesterol within DDA:TDB liposomes was investigated using differential scanning calorimetery and surface pressure-area isotherms of lipid monolayers; incorporation of cholesterol into liposomal membranes promoted the formation of a liquid-condensed monolayer and removed the main phase transition temperature of the system, resulting in an increased bilayer fluidity and reduced antigen retention in vitro. In vivo biodistribution studies found that this increase in membrane fluidity did not alter deposition of liposomes and antigen at the site of injection. In terms of immune responses, early (12 days after immunization) IgG responses were reduced by inclusion of cholesterol; thereafter there were no differences in antibody (IgG, IgG1, IgG2b) responses promoted by DDA:TDB liposomes with and without cholesterol. However, significantly higher levels of IFN-gamma were induced by DDA:TDB liposomes, and liposome uptake by macrophages in vitro was also shown to be higher for DDA:TDB liposomes compared to their cholesterol-containing counterparts, suggesting that small changes in bilayer mechanics can impact both cellular interactions and immune responses. © 2013 American Chemical Society.

Learning to reflect through technology, to enhance employability

"Reflection, if managed in an ordered way, can provide great opportunities for learning, understanding and clarifying thought, both in one's personal life and in learning and professional development." Moon, J (1999). Aston Business School's (ABS) 12cmonth professional placement programme has a number of espoused learning objectives, including helping students: 1. To benefit from the integration of university study and work experience in ways which facilitate critical reflection on each ofthese aspects. 2. To build a personal awareness of their own interests, competencies, values and potential. These objectives focus students' development to self-reflect critically, to make sense of their experiences/learning whilst undertaking their placements. Students complete a placement year Reflective Learning Journal, supported by the workplace supervisor, through regular meetings where objectives are agreed and reviewed. As well as reflection providing opportunities for students to make sense of their learning, it is also challenging! ABS is undertaking a pilot in 2008/9 to encourage students to engage with reflective practice, but employer feedback indicates an ability to think and analyse is often missing from the skill sets of graduates. Business students are using the PebblePad e-portfolio system as a tool to record their learning, reflect on their experiences in the workplace and to create their journals.

Professionalism and quality in European business courses:utilising research to enhance student learning and promote employability - a pan-European study

This..paper provides a comparative analysis of Quality Management and standards in four European countries, (the UK, Austria, Slovenia and Romania) and in doing so addresses the gap in academic knowledge about how the introduction and implementation of Quality Management Strategies can both facilitate and enhance student learning within Universities.

Employing DDBPSK in optical burst switched systems to enhance throughput

We demonstrate that doubly differential decoding can demodulate phase shift keyed data much faster after the switching event of a tunable laser than usual mth power single differential decoding. This technique can significantly improve throughput of optical burst switched networks.

High-throughput manufacturing of size-tuned liposomes by a new microfluidics method using enhanced statistical tools for characterization

Microfluidics has recently emerged as a new method of manufacturing liposomes, which allows for reproducible mixing in miliseconds on the nanoliter scale. Here we investigate microfluidics-based manufacturing of liposomes. The aim of these studies was to assess the parameters in a microfluidic process by varying the total flow rate (TFR) and the flow rate ratio (FRR) of the solvent and aqueous phases. Design of experiment and multivariate data analysis were used for increased process understanding and development of predictive and correlative models. High FRR lead to the bottom-up synthesis of liposomes, with a strong correlation with vesicle size, demonstrating the ability to in-process control liposomes size; the resulting liposome size correlated with the FRR in the microfluidics process, with liposomes of 50 nm being reproducibly manufactured. Furthermore, we demonstrate the potential of a high throughput manufacturing of liposomes using microfluidics with a four-fold increase in the volumetric flow rate, maintaining liposome characteristics. The efficacy of these liposomes was demonstrated in transfection studies and was modelled using predictive modeling. Mathematical modelling identified FRR as the key variable in the microfluidic process, with the highest impact on liposome size, polydispersity and transfection efficiency. This study demonstrates microfluidics as a robust and high-throughput method for the scalable and highly reproducible manufacture of size-controlled liposomes. Furthermore, the application of statistically based process control increases understanding and allows for the generation of a design-space for controlled particle characteristics.