Small cationic DDA:TDB liposomes as protein vaccine adjuvants obviate the need for TLR agonists in inducing cellular and humoral responses

Most subunit vaccines require adjuvants in order to induce protective immune responses to the targeted pathogen. However, many of the potent immunogenic adjuvants display unacceptable local or systemic reactogenicity. Liposomes are spherical vesicles consisting of single (unilamellar) or multiple (multilamellar) phospholipid bi-layers. The lipid membranes are interleaved with an aqueous buffer, which can be utilised to deliver hydrophilic vaccine components, such as protein antigens or ligands for immune receptors. Liposomes, in particular cationic DDA:TDB vesicles, have been shown in animal models to induce strong humoral responses to the associated antigen without increased reactogenicity, and are currently being tested in Phase I human clinical trials. We explored several modifications of DDA:TDB liposomes--including size, antigen association and addition of TLR agonists--to assess their immunogenic capacity as vaccine adjuvants, using Ovalbumin (OVA) protein as a model protein vaccine. Following triple homologous immunisation, small unilamellar vesicles (SUVs) with no TLR agonists showed a significantly higher capacity for inducing spleen CD8 IFN? responses against OVA in comparison with the larger multilamellar vesicles (MLVs). Antigen-specific antibody reponses were also higher with SUVs. Addition of the TLR3 and TLR9 agonists significantly increased the adjuvanting capacity of MLVs and OVA-encapsulating dehydration-rehydration vesicles (DRVs), but not of SUVs. Our findings lend further support to the use of liposomes as protein vaccine adjuvants. Importantly, the ability of DDA:TDB SUVs to induce potent CD8 T cell responses without the need for adding immunostimulators would avoid the potential safety risks associated with the clinical use of TLR agonists in vaccines adjuvanted with liposomes.

Preparation, characterisation and entrapment of a non-glycosidic threitol ceramide into liposomes for presentation to invariant natural killer T cells

Dendritic cells (DCs) are able to present glycolipids to invariant natural killer T (iNKT) cells in vivo. Very few compounds have been found to stimulate iNKT cells, and of these, the best characterised is the glycolipid a-galactosylceramide, which stimulates the production of large quantities of interferon-gamma (IFN-?) and interleukin-4 (IL-4). However, aGalCer leads to overstimulation of iNKT cells. It has been demonstrated that the aGalCer analogue, threitol ceramide (ThrCer 2), successfully activates iNKT cells and overcomes the problematic iNKT cell activation-induced anergy. In this study, ThrCer 2 has been inserted into the bilayers of liposomes composed of a neutral lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or dimethyldioctadecylammonium bromide (DDA), a cationic lipid. Incorporation efficiencies of ThrCer within the liposomes was 96% for DSPC liposomes and 80% for DDA liposomes, with the vesicle size (large multilamellar vs. small unilamellar vesicles) making no significant difference. Langmuir-Blodgett studies suggest that both DSPC and DDA stack within the monolayer co-operatively with the ThrCer molecules with no condensing effect. In terms of cellular responses, IFN-? secretion was higher for cells treated with small DDA liposomes compared with the other liposome formulations, suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.

Liposomes:Formulation and characterisation as contrast agents and as vaccine delivery systems

Liposome systems are well reported for their activity as vaccine adjuvants; however novel lipid-based microbubbles have also been reported to enhance the targeting of antigens into dendritic cells (DCs) in cancer immunotherapy (Suzuki et al 2009). This research initially focused on the formulation of gas-filled lipid coated microbubbles and their potential activation of macrophages using in vitro models. Further studies in the thesis concentrated on aqueous-filled liposomes as vaccine delivery systems. Initial work involved formulating and characterising four different methods of producing lipid-coated microbubbles (sometimes referred to as gas-filled liposomes), by homogenisation, sonication, a gas-releasing chemical reaction and agitation/pressurisation in terms of stability and physico-chemical characteristics. Two of the preparations were tested as pressure probes in MRI studies. The first preparation composed of a standard phospholipid (DSPC) filled with air or nitrogen (N2), whilst in the second method the microbubbles were composed of a fluorinated phospholipid (F-GPC) filled with a fluorocarbon saturated gas. The studies showed that whilst maintaining high sensitivity, a novel contrast agent which allows stable MRI measurements of fluid pressure over time, could be produced using lipid-coated microbubbles. The F-GPC microbubbles were found to withstand pressures up to 2.6 bar with minimal damage as opposed to the DSPC microbubbles, which were damaged at above 1.3 bar. However, it was also found that DSPC-filled with N2 microbubbles were also extremely robust to pressure and their performance was similar to that of F-GPC based microbubbles. Following on from the MRI studies, the DSPC-air and N2 filled lipid-based microbubbles were assessed for their potential activation of macrophages using in vitro models and compared to equivalent aqueous-filled liposomes. The microbubble formulations did not stimulate macrophage uptake, so studies thereafter focused on aqueous-filled liposomes. Further studies concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyldioctadecylammonium (DDA) and immunomodulatory trehalose dibehenate (TDB) with the addition of polyethylene glycol (PEG). One of the proposed hypotheses for the mechanism behind the immunostimulatory effect obtained with DDA:TDB is the ‘depot effect’ in which the liposomal carrier helps to retain the antigen at the injection site thereby increasing the time of vaccine exposure to the immune cells. The depot effect has been suggested to be primarily due to their cationic nature. Results reported within this thesis demonstrate that higher levels of PEG i.e. 25 % were able to significantly inhibit the formation of a liposome depot at the injection site and also severely limit the retention of antigen at the site. This therefore resulted in a faster drainage of the liposomes from the site of injection. The versatility of cationic liposomes based on DDA:TDB in combination with different immunostimulatory ligands including, polyinosinic-polycytidylic acid (poly (I:C), TLR 3 ligand), and CpG (TLR 9 ligand) either entrapped within the vesicles or adsorbed onto the liposome surface was investigated for immunogenic capacity as vaccine adjuvants. Small unilamellar (SUV) DDA:TDB vesicles (20-100 nm native size) with protein antigen adsorbed to the vesicle surface were the most potent in inducing both T cell (7-fold increase) and antibody (up to 2 log increase) antigen specific responses. The addition of TLR agonists poly(I:C) and CpG to SUV liposomes had small or no effect on their adjuvanticity. Finally, threitol ceramide (ThrCer), a new mmunostimulatory agent, was incorporated into the bilayers of liposomes composed of DDA or DSPC to investigate the uptake of ThrCer, by dendritic cells (DCs), and presentation on CD1d molecules to invariant natural killer T cells. These systems were prepared both as multilamellar vesicles (MLV) and Small unilamellar (SUV). It was demonstrated that the IFN-g secretion was higher for DDA SUV liposome formulation (p<0.05), suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.

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.

Design and development of cationic liposomes as DNA vaccine adjuvants

Cationic liposomes have been extensively explored for their efficacy in delivering nucleic acids, by offering the ability to protect plasmid DNA against degradation, promote gene expression and, in the case of DNA vaccines, induce both humoural and cellular immune responses. DNA vaccines may also offer advantages in terms of safety, but they are less effective and need an adjuvant to enhance their immunogenicity. Therefore, cationic liposomes can be utilised as delivery systems and/or adjuvants for DNA vaccines to stimulate stronger immune responses. To explore the role of liposomal systems within plasmid DNA delivery, parameters such as the effect of lipid composition, method of liposome preparation and presence of electrolytes in the formulation were investigated in characterisation studies, in vitro transfection studies and in vivo biodistribution and immunisation studies. Liposomes composed of 1,2-dioleoyl-sn-glycero 3-phosphoethanolamine (DOPE) in combination with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-stearoyl-3- trimethylammonium-propane (DSTAP) were prepared by the lipid hydration method and hydrated in aqueous media with or without presence of electrolytes. Whilst the in vitro transfection efficiency of all liposomes resulted to be higher than Lipofectin, DSTAP-based liposomes showed significantly higher transfection efficiency than DOTAP-based formulations. Furthermore, upon intramuscular injection of liposomal DNA vaccines, DSTAP-based liposomes showed a significantly stronger depot effect at the injection site. This could explain the result of heterologous immunisation studies, which revealed DSTAP-based liposomal vaccines induce stronger immune responses compared to DOTAP-based formulations. Previous studies have shown that having more liposomally associated antigen at the injection site would lead to more drainage of them into the local lymph nodes. Consequently, this would lead to more antigens being presented to antigen presenting cells, which are circulating in lymph nodes, and this would initiate a stronger immune response. Finally, in a comparative study, liposomes composed of dimethyldioctadecylammonium bromide (DDA) in combination with DOPE or immunostimulatory molecule of trehalose 6,6-dibehenate (TDB) were prepared and investigated in vitro and in vivo. Results showed that although DDA:TDB is not able to transfect the cells efficiently in vitro, this formulation induces stronger immunity compared to DDA:DOPE due to the immunostimulatory effects of TDB. This study demonstrated, while the presence of electrolytes did not improve immune responses, small unilamellar vesicle (SUV) liposomes induced stronger humoural immune responses compared to dehydration rehydration vesicle (DRV) liposomes. Moreover, lipid composition was shown to play a key role in in vitro and in vivo behaviour of the formulations, as saturated cationic lipids provided stronger immune responses compared to unsaturated lipids. Finally, heterologous prime/boost immunisation promoted significantly stronger immune responses compared to homologous vaccination of DNA vaccines, however, a single immunisation of subunit vaccine provoked comparable levels of immune response to the heterologous regimen, suggesting more immune efficiency for subunit vaccines compared to DNA vaccines.

Liposome-mediated DNA immunisation via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper, we have investigated the application of liposome-entrapped DNA and their cationic lipid composition on such potency after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into liposomes composed of 16 μmol egg phosphatidylcholine (PC), 8 μmoles dioleoyl phosphatidylethanolamine (DOPE) or cholesterol (Chol) and either the cationic lipid 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP) or cholesteryl 3-N-(dimethyl amino ethyl) carbamate (DC-Chol). This method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded incorporation values of 90-94% of the DNA used. Mixing or rehydration of preformed cationic liposomes with 100 μg plasmid DNA also led to similarly high complexation values (92-94%). In an attempt to establish differences in the nature of DNA association with these various liposome preparations their physico-chemical characteristics were investigated. Studies on vesicle size, zeta potential and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, formulation of liposomal DNA by the dehydration-rehydration generated submicron size liposomes incorporating most of the DNA in a manner that prevents DNA displacement through anion competition. The bilayer composition of these dehydration-rehydration vesicles (DRV(DNA)) can also further influence these physicochemical characteristics with the presence of DOPE within the liposome bilayer resulting in a reduced vesicle zeta potential. Subcutaneous liposome-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG1 and 1gG2a) engendered by the plasmid encoded NP were substantially higher after dosing twice, 28 days apart with 10 μg liposome-entrapped DNA compared to naked DNA. At all time points measured, mice immunised with naked DNA showed no greater immune response compared to the control, non-immunised group. In contrast, as early as day 49, responses were significantly higher in mice injected with DNA entrapped in DRV liposomes containing DOTAP compared to the control group and mice immunised with naked DNA. By day 56, all total IgG responses from mice immunised with both DRV formulations were significantly higher. Comparison between the DRV formulations revealed no significant difference in immune responses elicited except at day 114, where the humoural responses of the group injected with liposomal formulation containing DC-Chol dropped to significantly lower levels that those measured in mice which received the DOTAP formulation. Similar results were found when the IgG1 and IgG2a subclass responses were determined. These results suggest that, not only can DNA be effectively entrapped within liposomes using the DRV method but that such DRV liposomes containing DNA may be a useful system for subcutaneous delivery of DNA vaccines. © 2003 Taylor & Francis Ltd.

Manipulation of the surface pegylation in combination with reduced vesicle size of cationic liposomal adjuvants modifies their clearance kinetics from the injection site, and the rate and type of T cell response

The mechanism behind the immunostimulatory effect of the cationic liposomal vaccine adjuvant dimethyldioctadecylammonium and trehalose 6,6′- dibehenate (DDA:TDB) has been linked to the ability of these cationic vesicles to promote a depot after administration, with the liposomal adjuvant and the antigen both being retained at the injection site. This can be attributed to their cationic nature, since reduction in vesicle size does not influence their distribution profile yet neutral or anionic liposomes have more rapid clearance rates. Therefore the aim of this study was to investigate the impact of a combination of reduced vesicle size and surface pegylation on the biodistribution and adjuvanticity of the formulations, in a bid to further manipulate the pharmacokinetic profiles of these adjuvants. From the biodistribution studies, it was found that with small unilamellar vesicles (SUVs), 10% PEGylation of the formulation could influence liposome retention at the injection site after 4 days, whilst higher levels (25 mol%) of PEG blocked the formation of a depot and promote clearance to the draining lymph nodes. Interestingly, whilst the use of 10% PEG in the small unilamellar vesicles did not block the formation of a depot at the site of injection, it did result in earlier antibody response rates and switch the type of T cell responses from a Th1 to a Th2 bias suggesting that the presence of PEG in the formulation not only control the biodistribution of the vaccine, but also results in different types of interactions with innate immune cells. © 2012 Elsevier B.V.

Surfactant vesicle-mediated delivery of DNA vaccines via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper we have compared the potency of lipid-based and non-ionic surfactant based vesicle carrier systems for DNA vaccines after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into various vesicle formulations. The DRV method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded high DNA vaccine incorporation values (85-97% of the DNA used) in all formulations. Studies on vesicle size revealed lipid-based systems formed cationic submicron size vesicles whilst constructs containing a non-ionic surfactant had significantly large z-average diameters (>1500 nm). Subcutaneous vesicle-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG 1 and 1gG 2a) engendered by the plasmid encoded nucleoprotein were substantially higher after dosing twice, 28 days apart with 10 μg DRV-entrapped DNA compared to naked DNA. Comparison between the lipid and non-ionic based vesicle formulations revealed no significant difference in stimulated antibody production. These results suggest that, not only can DNA be effectively entrapped within a range of lipid and non-ionic based vesicle formulations using the DRV method but that such DRV vesicles containing DNA may be a useful system for subcutaneous delivery of DNA vaccines. © 2004 Elsevier B.V. All rights reserved.

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-entrapped plasmid DNA:characterisation studies

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.

Effect of vesicle size on tissue localization and immunogenicity of liposomal DNA vaccines

The formulation of plasmid DNA (pDNA) in cationic liposomes is a promising strategy to improve the potency of DNA vaccines. In this respect, physicochemical parameters such as liposome size may be important for their efficacy. The aim of the current study was to investigate the effect of vesicle size on the in vivo performance of liposomal pDNA vaccines after subcutaneous vaccination in mice. The tissue distribution of cationic liposomes of two sizes, 500 nm (PDI 0.6) and 140 nm (PDI 0.15), composed of egg PC, DOPE and DOTAP, with encapsulated OVA-encoding pDNA, was studied by using dual radiolabeled pDNA-liposomes. Their potency to elicit cellular and humoral immune responses was investigated upon application in a homologous and heterologous vaccination schedule with 3 week intervals. It was shown that encapsulation of pDNA into cationic lipsomes resulted in deposition at the site of injection, and strongest retention was observed at large vesicle size. The vaccination studies demonstrated a more robust induction of OVA-specific, functional CD8+ T-cells and higher antibody levels upon vaccination with small monodisperse pDNA-liposomes, as compared to large heterodisperse liposomes or naked pDNA. The introduction of a PEG-coating on the small cationic liposomes resulted in enhanced lymphatic drainage, but immune responses were not improved when compared to non-PEGylated liposomes. In conclusion, it was shown that the physicochemical properties of the liposomes are of crucial importance for their performance as pDNA vaccine carrier, and cationic charge and small size are favorable properties for subcutaneous DNA vaccination.

αα'-trehalose 6,6'-dibehenate in non-phospholipid-based liposomes enables direct interaction with trehalose, offering stability during freeze-drying

Trehalose is a well known protector of biostructures like liposomes and proteins during freeze-drying, but still today there is a big debate regarding its mechanism of action. In previous experiments we have shown that trehalose is able to protect a non-phospholipid-based liposomal adjuvant (designated CAF01) composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6-dibehenate (TDB) during freeze-drying [D. Christensen, C. Foged, I. Rosenkrands, H.M. Nielsen, P. Andersen, E.M. Agger, Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying, Biochim. Biophys. Acta, Biomembr. 1768 (2007) 2120-2129]. Furthermore it was seen that TDB is required for the stabilizing effect of trehalose. Herein, we show using the Langmuir-Blodgett technique that a high concentration of TDB present at the water-lipid interface results in a surface pressure around 67 mN/m as compared to that of pure DDA which is approximately 47 mN/m in the compressed state. This indicates that the attractive forces between the trehalose head group of TDB and water are greater than those between the quaternary ammonium head group of DDA and water. Furthermore, addition of trehalose to a DDA monolayer containing small amounts of TDB also increases the surface pressure, which is not observed in the absence of TDB. This suggests that even small amounts of trehalose groups on TDB present at the water-lipid interface associate free trehalose to the liposome surface, presumably by hydrogen bonding between the trehalose head groups of TDB and the free trehalose molecules. Hence, for CAF01 the TDB component not only stabilizes the cationic liposomes and enhances the immune response but also facilitates the cryo-/lyoprotection by trehalose through direct interaction with the head group of TDB. Furthermore the results indicate that direct interaction with liposome surfaces is necessary for trehalose to enable protection during freeze-drying.

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.

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.