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.

Adjuvant properties of a simplified C32 monomycolyl glycerol analogue

A simplified C32 monomycolyl glycerol (MMG) analogue demonstrated enhanced immunostimulatory activity in a dioctadecyl ammonium bromide (DDA)/Ag85B-ESAT-6 formulation. Elevated levels of IFN-gamma and IL-6 were produced in spleen cells from mice immunised with a C32 MMG analogue comparable activity to the potent Th1 adjuvant, trehalose 6,6'-di-behenate (TDB).

Lipoplexes formulation and optimisation:in vitro transfection studies reveal no correlation with in vivo vaccination studies

The aim of these studies was to compare the effect of liposome composition on physico-chemical characteristics and transfection efficacy of cationic liposomes both in vitro and in vivo. Comparison between 4 popularly used cationic lipids, showed 3b-N-(dimethylaminoethyl)carbamate (DC-Chol) to promote the highest transfect levels in cells in vitro with levels being at least 6 times higher than those of 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA). 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and dimethyldioctadecylammonium (DDA) and approximately twice as efficient as dipalmitoyl-trimethylammonium-propane (DPTAP). To establish the role of the helper lipid, DC-Chol liposomes were formulated in combination with either 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol (Chol) (1:1 molar ratio) with and without the addition of phosphatidyl choline. The choice of helper lipid incorporated within the bilayer was found to influence the formation of complexes, their resultant structure and their transfection efficiency in vitro, with SUV-DNA complexes containing optimum levels of DOPE giving higher transfection than those containing cholesterol. The inclusion of PC within the formulation also reduced transfection efficiency in vitro. However, when administered in vivo, SUV-DNA complexes composed of PC:Chol:DC-Chol at a molar ratio of 16:8:4 micromole/ml were the most effective at inducing splenocyte proliferation upon exposure to antigen in comparison to control spleens. These results demonstrate that there is no in vitro/in vivo correlation between the transfection efficacy of these liposome formulations and in vitro transfection in the above cell model cannot be taken as a reliable indicator for in vivo efficacy of DNA vaccines.

Liposomes act as stronger sub-unit vaccine adjuvants when compared to microspheres

The ability of liposomes and microspheres to enhance the efficacy of a sub-unit antigen was investigated. Microspheres were optimised by testing a range of surfactants employed in the external aqueous phase of a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation process for the preparation of microspherescomposed of poly(d,l-lactide-co-glycolide) and the immunological adjuvant dimethyl dioctadecyl ammonium bromide (DDA)and then investigated with regard to the physico-chemical and immunological characteristics of the particles produced. The results demonstrate that this parameter can affect the physico-chemical characteristics of these systems and subsequently, has a substantial bearing on the level of immune response achieved, both humoural and cell mediated, when employed for the delivery of the sub-unit tuberculosis vaccine antigen Ag85B-ESAT-6. Moreover, the microsphere preparations investigated failed to initiate immune responses at the levels achieved with an adjuvant DDA-based liposome formulation (DDA-TDB), further substantiating the superior ability of liposomes as vaccine delivery systems.

Pegylation of DDA:TDB liposomal adjuvants reduces the vaccine depot effect and alters the Th1/Th2 immune responses

The adjuvant efficacy of cationic liposomes composed of dimethyldioctadecylammonium bromide and trehalose dibehenate (DDA:TDB) is well established. Whilst the mechanism behind its immunostimulatory action is not fully understood, the ability of the formulation to promote a 'depot effect' is a consideration. The depot effect has been suggested to be primarily due to their cationic nature which results in electrostatic adsorption of the antigen and aggregation of the vesicles at the site of injection. The aim of the study was to further test this hypothesis by investigating whether sterically stabilising DDA:TDB with polyethylene glycol (PEG) reduces aggregation, and subsequently influences the formation of a depot at the site of injection. Results reported demonstrate that high (25%) levels of PEG was able to significantly inhibit the formation of a liposome depot and also severely limit the retention of antigen at the site, resulting in a faster drainage of the liposomes from the site of injection. This change in biodistribution profile was reflected in the immunisation response, where lower levels of IgG2b antibody and IFN-? and higher level of IL-5 cytokine were found. Furthermore entrapping antigen within DDA:TDB liposomes did not improve antigen retention at the injection site compared surface adsorbed antigen. © 2011 Elsevier B.V. All rights reserved.

Vaccine adjuvant systems: enhancing the efficacy of sub-unit protein antigens

Vaccination remains a key tool in the protection and eradication of diseases. However, the development of new safe and effective vaccines is not easy. Various live organism based vaccines currently licensed, exhibit high efficacy; however, this benefit is associated with risk, due to the adverse reactions found with these vaccines. Therefore, in the development of vaccines, the associated risk-benefit issues need to be addressed. Sub-unit proteins offer a much safer alternative; however, their efficacy is low. The use of adjuvanted systems have proven to enhance the immunogenicity of these sub-unit vaccines through protection (i.e. preventing degradation of the antigen in vivo) and enhanced targeting of these antigens to professional antigen-presenting cells. Understanding of the immunological implications of the related disease will enable validation for the design and development of potential adjuvant systems. Novel adjuvant research involves the combination of both pharmaceutical analysis accompanied by detailed immunological investigations, whereby, pharmaceutically designed adjuvants are driven by an increased understanding of mechanisms of adjuvant activity, largely facilitated by description of highly specific innate immune recognition of components usually associated with the presence of invading bacteria or virus. The majority of pharmaceutical based adjuvants currently being investigated are particulate based delivery systems, such as liposome formulations. As an adjuvant, liposomes have been shown to enhance immunity against the associated disease particularly when a cationic lipid is used within the formulation. In addition, the inclusion of components such as immunomodulators, further enhance immunity. Within this review, the use and application of effective adjuvants is investigated, with particular emphasis on liposomal-based systems. The mechanisms of adjuvant activity, analysis of complex immunological characteristics and formulation and delivery of these vaccines are considered.

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.

Gene delivery using cationic liposomes

The development of cationic liposomes for gene delivery has been ongoing for almost 20 years; however, despite extensive efforts to develop a successful therapeutic agent, there has been limited progress towards generating an effective pharmaceutical product. Since the introduction of N-(1-[2,3-dioley-loxy]propyl)-N,N,N-trimethylammonium chloride, an immense number of different cationic lipids have been synthesised and used to formulate cationic liposome - DNA complexes. Structural modification of the cationic lipids and the addition of components within the delivery system that can facilitate the fusion, cellular uptake and targeting of liposome - DNA complexes have all been used in a bid to enhance their transfection efficiency. Unfortunately, the overall impact of these improvements is still nominal, with the vast majority of clinical trials (∼ 85%) continuing to rely on more potent viral delivery of DNA despite their associated toxicity issues. Key characteristics of the most effective cationic liposomes for the delivery of plasmid DNA (from a consensus of the literature) is identified here and the problems of converting these attributes into an effective pharmaceutical product are outlined. © 2006 Informa UK Ltd.

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.

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.

Formulation and characterisation of lyophilised rapid disintegrating tablets using amino acids as matrix forming agents

Despite recent advances in the formulation of lyophilised rapid disintegrating tablets (RDTs), the inclusion of matrix supporting/disintegration enhancing agents has been limited to the use of saccharides and polyols. In this study, the feasibility of using amino acids as matrix forming agents in lyophilised RDTs was investigated. Twelve amino acids were chosen (alanine, arginine, threonine, glycine, cysteine, serine, histidine, lysine, valine, asparagine, glutamine and proline), and the suitability for freeze drying, mechanical properties and disintegration time after inclusion of the amino acids at varied concentration were studied. In addition, the porosity of the RDTs and wettability profile of the amino acids were investigated to understand the mechanisms of disintegration. The results suggest the suitability of these amino acids for the lyophilisation regime, as they displayed satisfactory safety margin between the glass transition and shelf temperature (-40 degrees C), except proline-based formulations. Moreover, the crystallisation behavior of alanine, glycine, cysteine and serine at high concentration increased the stability of the formulation. The characterisation of the RDTs suggests that high concentration of the amino acids is required to enhance the mechanical properties, whereas only optimum concentrations promote the disintegration. Moreover, wetting time of the amino acid and porosity of the tablet are the two factors that control the disintegration of RDTs.

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.

Advanced characterisation techniques

Editorial

Liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a CMI response

With respect to liposomes as delivery vehicles and adjuvants for vaccine antigens, the role of vesicle surface charge remains disputed. In the present study we investigate the influence of liposome surface charge and antigen-liposome interaction on the antigen depot effect at the site of injection (SOI). The presence of liposome and antigen in tissue at the SOI as well as the draining lymphatic tissue was quantified to analyse the lymphatic draining of the vaccine components. Furthermore investigations detailing cytokine production and T-cell antigen specificity were undertaken to investigate the relationship between depot effect and the ability of the vaccine to induce an immune response. Our results suggest that cationic charge is an important factor for the retention of the liposomal component at the SOI, and a moderate to high (>50%) level of antigen adsorption to the cationic vesicle surface was required for efficient antigen retention in the same tissue. Furthermore, neutral liposomes expressing poor levels of antigen retention were limited in their ability to mediate long term (14 days) antigen presentation to circulating antigen specific T-cells and to induce the Th1 and Th17 arms of the immune system, as compared to antigen adsorbing cationic liposomes. The neutral liposomes did however induce the production of IL-5 at levels comparable to those induced by cationic liposomes, indicating that neutral liposomes can induce a weak Th2 response.

Dissolution rate enhancement, in vitro evaluation and investigation of drug release kinetics of chloramphenicol and sulphamethoxazole solid dispersions

Formulation of solid dispersions is one of the effective methods to increase the rate of solubilization and dissolution of poorly soluble drugs. Solid dispersions of chloramphenicol (CP) and sulphamethoxazole (SX) as model drugs were prepared by melt fusion method using polyethylene glycol 8000 (PEG 8000) as an inert carrier. The dissolution rate of CP and SX were rapid from solid dispersions with low drug and high polymer content. Characterization was performed using fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). FTIR analysis for the solid dispersions of CP and SX showed that there was no interaction between PEG 8000 and the drugs. Hyper-DSC studies revealed that CP and SX were converted into an amorphous form when formulated as solid dispersion in PEG 8000. Mathematical analysis of the release kinetics demonstrated that drug release from the various formulations followed different mechanisms. Permeability studies demonstrated that both CP and SX when formulated as solid dispersions showed enhanced permeability across Caco-2 cells and CP can be classified as well-absorbed compound when formulated as solid dispersions. © 2013 Informa Healthcare USA, Inc.