Liposome-based cationic adjuvant formulations (CAF):past, present, and future

The use of liposomes as vaccine adjuvants has been investigated extensively over the last few decades. In particular, cationic liposomal adjuvants have drawn attention, with dimethyldioctadecylammonium (DDA) liposomes as a prominent candidate. However, cationic liposomes are, in general, not sufficiently immunostimulatory, which is why the combination of liposomes with immunostimulators has arisen as a strategy in the development of novel adjuvant systems in recent years. One such adjuvant system is CAF01. In this review, we summarize the immunological properties making CAF01 a promising versatile adjuvant system, which was developed to mediate protection against tuberculosis (TB) but, in addition, has shown promising protective efficacy against other infectious diseases requiring different immunological profiles. Further, we describe the stabilization properties that make CAF01 suitable in vaccine formulation for the developing world, which in addition to vaccine efficacy, are important prerequisites for any novel TB vaccine to reach global implementation. The encouraging nonclinical data led to a preclinical vaccine toxicology study of the TB model vaccine, Ag85B-ESAT-6/CAF01, that concluded that CAF01 has a satisfactory safety profile to advance the vaccine into phase I clinical trials, which are scheduled to start in 2009.

On the thermal stabilization of carbon-supported SiO2 catalysts by phosphorus:evaluation in the oxidative dehydrogenation of ethylbenzene to styrene and a comparison with relevant catalysts

A strategy to enhance the thermal stability of C/SiO2 hybrids for the O2-based oxidative dehydrogenation of ethylbenzene to styrene (ST) by P addition is proposed. The preparation consists of the polymerization of furfuryl alcohol (FA) on a mesoporous precipitated SiO2. The polymerization is catalyzed by oxalic acid (OA) at 160 °C (FA:OA = 250). Phosphorous was added as H3PO4 after the polymerization and before the pyrolysis that was carried out at 700 °C and will extend the overall activation procedure. Estimation of the apparent activation energies reveals that P enhances the thermal stability under air oxidation, which is a good indication for the ODH tests. Catalytic tests show that the P/C/SiO2 hybrids are readily active, selective and indeed stable in the applied reactions conditions for 60 h time on stream. Coke build-up during the reaction attributed to the P-based acidity is substantial, leading to a reduction of the surface area and pore volume. The comparison with a conventional MWCNT evidences that the P/C/SiO2 hybrids are more active and selective at high temperatures (450–475 °C) while the difference becomes negligible at lower temperature. However, the comparison with reference P/SiO2 counterparts shows a very similar yield than the hybrids but more selective to ST. The benefit of the P/C/SiO2 hybrid is the lack of stabilization period, which is observed for the P/SiO2 to create an active coke overlayer. For long term operation, P/SiO2 appears to be a better choice in terms of selectivity, which is crucial for commercialization.

Targeted delivery of a novel group of site-directed transglutaminase inhibitors to the liver using liposomes:a new approach for the potential treatment of liver fibrosis

Liver fibrosis and its end-stage disease cirrhosis are a main cause of mortality and morbidity worldwide. Thus far, there is no efficient pharmaceutical intervention for the treatment of liver fibrosis. Liver fibrosis is characterized by excessive accumulation of the extracellular matrix (ECM) proteins. Transglutaminase (TG)-mediated covalent cross-linking has been implicated in the stabilization and accumulation of ECM in a number of fibrotic diseases. Thus, the use of tissue TG2 inhibitors has potential in the treatment of liver fibrosis. Recently, we introduced a novel group of site-directed irreversible specific inhibitors of TGs. Here, we describe the development of a liposome-based drug-delivery system for the site-specific delivery of these TG inhibitors into the liver. By using anionic or neutral-based DSPC liposomes, the TG inhibitor can be successfully incorporated into these liposomes and delivered specifically to the liver. Liposomes can therefore be used as a potential carrier system for site-specific delivery of the TG2 inhibitors into the liver, opening up a potential new avenue for the treatment of liver fibrosis and its end-stage disease cirrhosis.

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.

Enzymatic stabilization of gelatin-based scaffolds

The definitive goal of this research is to develop protein-based scaffolds for use in soft tissue regeneration, particularly in the field of dermal healing. The premise of this investigation was to characterize the mechanical properties of gelatin cross-linked with microbial transglutaminase (mTGase) and to investigate the cytocompatibility of mTGase cross-linked gelatin. Dynamic rheological analysis revealed a significant increase in the storage modulus and thermal stability of gelatin after cross-linking with mTGase. Static, unconfined compression tests showed an increase in Young's modulus of gelatin gels after mTGase cross-linking. A comparable increase in gel strength was observed with 0.03% mTGase and 0.25% glutaraldehyde cross-linked gelatin gels. In vitro studies using 3T3 fibroblasts indicated cytotoxicity at a concentration of 0.05% mTGase after 72 h. However, no significant inhibition of cell proliferation was seen with cells grown on lower concentrations of mTGase cross-linked gelatin substrates. The mechanical improvement and cytocompatibility of mTGase cross-linked gelatin suggests mTGase has potential for use in stabilizing gelatin gels for tissue-engineering applications.

Increased potential of a cationic liposome-based delivery system:enhancing stability and sustained immunological activity in pre-clinical development

The combination of dimethyl dioctadecyl ammonium bromide (DDA) and the synthetic cord factor trehalose dibehenate (TDB) with Ag85B-ESAT-6 (H1 fusion protein) has been found to promote strong protective immune responses against Mycobacterium tuberculosis. The development of a vaccine formulation that is able to facilitate the requirements of sterility, stability and generation of a vaccine product with acceptable composition, shelf-life and safety profile may necessitate selected alterations in vaccine formulation. This study describes the implementation of a sterilisation protocol and the use of selected lyoprotective agents in order to fulfil these requirements. Concomitantly, close analysis of any alteration in physico-chemical characteristics and parameters of immunogenicity have been examined for this promising DDA liposome-based tuberculosis vaccine. The study addresses the extensive guidelines on parameters for non-clinical assessment, suitable for liposomal vaccines and other vaccine delivery systems issued by the World Health Organisation (WHO) and the European Medicines Agency (EMEA). Physical and chemical stability was observed following alteration in formulations to include novel cryoprotectants and radiation sterilisation. Immunogenicity was maintained following these alterations and even improved by modification with lysine as the cryoprotective agent for sterilised formulations. Taken together, these results outline the successful alteration to a liposomal vaccine, representing improved formulations by rational modification, whilst maintaining biological activity.

Solubility enhancement of poorly water soluble drugs using liposome technology

The aim of this work is to investigate the various parameters that could control the encapsulation of lipophilic drugs and investigate the influence of the physical properties of poorly water-soluble drugs on bilayer loading. Initial work investigated on the solubilisation of ibuprofen, a model insoluble drug. Drug loading was assessed using HPLC and UV spectrophotometric analysis. Preliminary studies focused on the influence of bilayer composition on drug loading to obtain an optimum cholesterol concentration. This was followed up by studies investigating the effect of longer alkyl chain lipids, unsaturated alkyl chain lipids and charged lipids. The studies also focused on the effects of pH of the hydration medium and addition of the single chain surfactant a-tocopherol. The work was followed up by investigation of a range of insoluble drugs including flurbiprofen, indomethacin, sulindac, mefenamic acid, lignocaine and progesterone to investigate the influence of drugs properties and functional group on liposomal loading. The results show that no defined trend could be obtained linking the drug loading to the different drug properties including molecular weight, log P and other drug specific characteristics. However, the presence of the oppositely charged lipids improved the encapsulation of all the drugs investigated with a similar effect obtained with the substitution of the longer chain lipids. The addition of the single chain surfactant a-tocopherol resulted in enhancement of drug loading and possibly is governed by the log P of the drug candidate. Environmental scanning-electron microscopy (ESEM) was used to dynamically follow the changes in liposome morphology in real time during dehydration thereby providing a alternative assay of liposome formulation and stability. The ESEM analysis clearly demonstrated ibuprofen incorporation enhanced the stability of PC:Chol liposomes.

Steric stabilization and dissolution characteristics of aqueous suspensions of pharmaceutical interest

The adsorption of two qroups of nonionic surface active agents and a series of hiqh molecular weiqht hydrophilic polymer fractions onto a polystyrene latex and a drug substance diloxanide furoate B.P. has been investigated. The presence of pores within the drug surface has been demonstrated and this is shown to increase the adsorption of low molecular weight polymer species. Differences in the maximum amount of polymer adsorbed at both solid-solution interfaces have been ascribed to the different hydrophobicities of the surface as determined by contact angle measurements. Adsorbed layer thicknesses of polymer on polystyrene latex have been determined by three techniques: microelectrophoresis, intensity fluctuation spectroscopy and by viscometric means. These results, in combination with adsorption data, were used to interpret the configuration of the adsorbed polymer molecules at the interface. The type of druq suspension produced on adsorbing the different polymers in the absence of electrostatic stabilization was correlated with theoretical prediuctions of suspension characteristics deduced from potential energy diagrams, The agreement was good for the adsorption of short chain length surfactants, but for the polyvinylalcohols, discrepancies were found between experiment and theory. This was attributed to the inappropriate use of a mean segment density approximation within the adsorbed layer to calculate attractive potentials between particles. A maximum in the redispersibility values for suspensions coated with adsorbed nonylphenylethoxylates was attributed to "partial static stabilization" of the particles in conjunction with the attractive forces operating in the sediment between bare surface patches on neighbouring particles. No significant change in the dissolution of the drug was observed when nonylphenylethoxylates were adsorbed due to desorption upon contact with the dissolution medium. Pluronic F68 and all the polyvinylalcohol fractions caused a reduction in the dissolution rate which is explained by the decreased diffusion of drug' through the adsorbed polymer layer.

Local and systemic delivery of a novel group of inhibitors of transglutaminase enzyme: A potential approach for treating of catheter-related complications and liver fibrosis

The present thesis investigates targeted (locally and systemically) delivery of a novel group of inhibitors of enzyme transglutaminases (TGs). TGs are a widely distributed group of enzymes that catalyse the formation of isopeptide bonds between the y-carboxamide group of protein-bound glutamines and the a-amino group of protein-bound lysines or polyamines. The first group of the novel inhibitors tested were the tluorescently labelled inhibitors of Factor XIIIa (FXIIIa). These small, non-toxic inhibitors have the potential to prevent stabilisation of thrombi by FXIIIa and consequently increase the natural rate of thrombolysis, in addition it reduces staphylococcal colonisation of catheters by inhibiting their FXIIIa¬mediated cross-linking to blood clot proteins on the central venous catheter (CVCs) surface. The aim of this work was to incorporate the FXIIIa inhibitor either within coating of polyurethane (PU) catheters or to integrate it into silicone catheters, so as to reduce the incidence of thrombotic occlusion and associated bacterial infection in CVCs. The initial work focused on the incorporation of FXIIIa inhibitors within polymeric coatings of PU catheters. After defining the key characteristics desired for an effective polymeric-coating, polyvinylpyrrolidone (PVP), poly(lactic-co-glycolic acid) (PLGA) or their combination were studies as polymers of choice for coating of the catheters_ The coating was conducted by dip-coating method in a polymer solution containing the inhibitor. Upon incubation of the inhibitor-and polymer-coated strips in buffer, PVP was dissolved instantly, generating fast and significant drug release, whilst PLGA did not dissolve, yielding a slow and an insufficient amount of drug release. Nevertheless, the drug release profile was enhanced upon employing a blend solution of PVP and PLGA. The second part of the study was to incorporate the FXIIIa inhibitor into a silicone elastomer; results demonstrated that FXIIIa inhibitor can be incorporated and released from silicone by using citric acid (CA) and sodium bicarbonate (SB) as additives and the drug release rate can be controlled by the amount of incorporated additives in the silicone matrix. Furthermore, it was deemed that the inhibitor was still biologically active subsequent to being released from the silicone elastomer strips. Morphological analysis confirmed the formation of channels and cracks inside the specimens upon the addition of CA and SB. Nevertheless, the tensile strength, in addition to Young's modulus of silicone elastomer strips, decreased constantly with an increasing amount of amalgamated CA/ SB in the formulations. According to our results, incorporation of FXIIIa inhibitor into catheters and other medical implant devices could offer new perspectives in preventing bio-material associated infections and thrombosis. The use of tissue transglutaminase (T02) inhibitor for treating of liver fibrosis was also investigated. Liver fibrosis is characterized by increased synthesis and decreased degradation of the extracellular matrix (ECM). Transglutaminase-mediated covalent cross-linking is involved in the stabilization of ECM in human liver fibrosis. Thus, TG2 inhibitors may be used to counteract the decreased degradation of the ECM. The potential of a liposome based drug delivery system for site specific delivery of the fluorescent TG2 inhibitor into the liver was investigated; results indicated that the TG2 inhibitor can be successfully integrated into liposomes and delivered to the liver, therefore demonstrating that liposomes can be employed for site-specific delivery of TG2 inhibitors into the liver and TG2 inhibitor incorporating liposomes could offer a new approach in treating liver fibrosis and its end stage disease cirrhosis.

Trigger release liposome systems:local and remote controlled delivery?

Target-specific delivery has become an integral area of research in order to increase bioavailability and reduce the toxic effects of drugs. As a drug-delivery option, trigger-release liposomes offer sophisticated targeting and greater control-release capabilities. These are broadly divided into two categories; those that utilise the local environment of the target site where there may be an upregulation in certain enzymes or a change in pH and those liposomes that are triggered by an external physical stimulus such as heat, ultrasound or light. These release mechanisms offer a greater degree of control over when and where the drug is released; furthermore, targeting of diseased tissue is enhanced by incorporation of target-specific components such as antibodies. This review aims to show the development of such trigger release liposome systems and the current research in this field.

Comparison of the depot effect and immunogenicity of liposomes based on dimethyldioctadecylammonium (DDA), 3β-[N-(N',N'-Dimethylaminoethane)carbomyl] cholesterol (DC-Chol), and 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP):prolonged liposome retention mediates stronger Th1 responses

The immunostimulatory capacities of cationic liposomes are well-documented and are attributed both to inherent immunogenicity of the cationic lipid and more physical capacities such as the formation of antigen depots and antigen delivery. Very few studies have however been conducted comparing the immunostimulatory capacities of different cationic lipids. In the present study we therefore chose to investigate three of the most well-known cationic liposome-forming lipids as potential adjuvants for protein subunit vaccines. The ability of 3ß-[N-(N',N'-dimethylaminoethane)carbomyl] cholesterol (DC-Chol), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and dimethyldioctadecylammonium (DDA) liposomes incorporating immunomodulating trehalose dibehenate (TDB) to form an antigen depot at the site of injection (SOI) and to induce immunological recall responses against coadministered tuberculosis vaccine antigen Ag85B-ESAT-6 are reported. Furthermore, physical characterization of the liposomes is presented. Our results suggest that liposome composition plays an important role in vaccine retention at the SOI and the ability to enable the immune system to induce a vaccine specific recall response. While all three cationic liposomes facilitated increased antigen presentation by antigen presenting cells, the monocyte infiltration to the SOI and the production of IFN-? upon antigen recall was markedly higher for DDA and DC-Chol based liposomes which exhibited a longer retention profile at the SOI. A long-term retention and slow release of liposome and vaccine antigen from the injection site hence appears to favor a stronger Th1 immune response.

Placenta growth factor-1 exerts time-dependent stabilization of adherens junctions following VEGF-induced vascular permeability

Increased vascular permeability is an early event characteristic of tissue ischemia and angiogenesis. Although VEGF family members are potent promoters of endothelial permeability the role of placental growth factor (PlGF) is hotly debated. Here we investigated PlGF isoforms 1 and 2 and present in vitro and in vivo evidence that PlGF-1, but not PlGF-2, can inhibit VEGF-induced permeability but only during a critical window post-VEGF exposure. PlGF-1 promotes VE-cadherin expression via the trans-activating Sp1 and Sp3 interaction with the VE-cadherin promoter and subsequently stabilizes transendothelial junctions, but only after activation of endothelial cells by VEGF. PlGF-1 regulates vascular permeability associated with the rapid localization of VE-cadherin to the plasma membrane and dephosphorylation of tyrosine residues that precedes changes observed in claudin 5 tyrosine phosphorylation and membrane localization. The critical window during which PlGF-1 exerts its effect on VEGF-induced permeability highlights the importance of the translational significance of this work in that PLGF-1 likely serves as an endogenous anti-permeability factor whose effectiveness is limited to a precise time point following vascular injury. Clinical approaches that would pattern nature's approach would thus limit treatments to precise intervals following injury and bring attention to use of agents only during therapeutic windows.

Stabilization of self-coherent OFDM with injection locked laser

With the rebirth of coherent detection, various algorithms have come forth to alleviate phase noise, one of the main impairments for coherent receivers. These algorithms provide stable compensation, however they limit the DSP. With this key issue in mind, Fabry Perot filter based self coherent optical OFDM was analyzed which does not require phase noise compensation reducing the complexity in DSP at low OSNR. However, the performance of such a receiver is limited due to ASE noise at the carrier wavelength, especially since an optical amplifier is typically employed with the filter to ensure sufficient carrier power. Subsequently, the use of an injection-locked laser (ILL) to retrieve the frequency and phase information from the extracted carrier without the use of an amplifier was recently proposed. In ILL based system, an optical carrier is sent along with the OFDM signal in the transmitter. At the receiver, the carrier is extracted from the OFDM signal using a Fabry-Perot tunable filter and an ILL is used to significantly amplify the carrier and reduce intensity and phase noise. In contrast to CO-OFDM, such a system supports low-cost broad linewidth lasers and benefits with lower complexity in the DSP as no carrier frequency estimation and correction along with phase noise compensation is required.