Preparation of poly (alkylcyanoacrylate) nanoparticles by polymerization of water-free microemulsions

Phase diagrams of the pseudoternary systems ethyloleate, polyoxyethylene 20 sorbitan mono-oleate/sorbitan monolaurate and propylene glycol with and without butanol as a co-surfactant were prepared. Areas containing optically isotropic, one-phase systems were identified and samples therein designated as droplet, bicontinuous or solution type microemulsions using conductivity, viscosity and self-diffusion NMR. Nanoparticles were prepared by polymerization of selected microemulsions with ethyl-2-cyanoacrylate and the morphology of the particles was investigated. Addition of monomer to all types of microemulsions led to the formation of nanoparticles, which had an average size of 244 +/- 25 nm, an average polydispersity index of 0.15 +/- 0.04 and a zeta-potential of -17 +/- 3 mV. The formation of particles from water-free microemulsions of different types is surprising, particularly considering that polymerization is expected to occur at a water-oil interface by base-catalysed polymerization. It would appear that propylene glycol is sufficiently nucleophilic to initiate the polymerization. The use of water-free microemulsions as templates for the preparation of poly (alkylcyanoacrylate) nanoparticles opens up interesting opportunities for the encapsulation of bioactives which do not have suitable properties for encapsulation on the basis of water-containing microemulsions.

Tailoring surface properties to build colloidal diagnostic devices: Controlling interparticle associations

The ability to control the surface properties and subsequent colloidal stability of dispersed particles has widespread applicability in many fields. Sub-micrometer fluorescent silica particles (reporters) can be used to actively encode the combinatorial synthesis of peptide libraries through interparticle association. To achieve these associations, the surface chemistry of the small fluorescent silica reporters is tailored to encourage robust adhesion to large silica microparticles onto which the peptides are synthesized. The interparticle association must withstand a harsh solvent environment multiple synthetic and washing procedures, and biological screening buffers. The encoded support beads were exposed to different solvents used for peptide synthesis, and different solutions used for biological screening including phosphate buffered saline (PBS), 2-[N-morpholino]ethane sulfonic acid (VIES) and a mixture of MES and N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide (EDC). The number of reporters remaining adhered to the support bead was quantified after each step. The nature of the associations were explored and tested to optimize the efficiency of these phenomena. Results presented illustrate the influence of the surface functionality and polyelectrolyte modification of the reporters. These parameters were investigated through zeta potential and X-ray photoelectron spectroscopy.

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.

Hydrophobicity and surface electrostatic charge of conidia of the mycoparasite Coniothyrium minitans

The effect of increasing culture age on cell surface hydrophobicity (CSH) and cell surface electrostatic charge (measured as zeta potential) of conidia from five isolates of Coniothyrium minitans representing three different morphological types was examined. Conidial CSH of three isolates (A2 960/1, CH1 and CH2) decreased with culture age, whereas CSH of two others (B 1300/2 and IMI 134523) remained high for the whole 42 day experimental period. In contrast, cell surface electrostatic charge decreased uniformly in conidia of all five isolates for the first 34 d and then rose slightly at 42 d. The variation in cell surface electrostatic charge (spectrum width) of the sampled conidia decreased with age for all five isolates. In all five isolates cell surface electrostatic charge of conidia became increasingly negative as the pH of the buffer used to suspend conidia was increased from pH 3.0 to 9.0. No relationship between colony morphology of C. minitans and conidial CSH and cell surface electrostatic charge was found.

Characterisation of a harvesting step for monoclonal antibodies from mammalian cell culture

The focus of this research was defined by a poorly characterised filtration train employed to clarify culture broth containing monoclonal antibodies secreted by GS-NSO cells: the filtration train blinded unpredictably and the ability of the positively charged filters to adsorb DNA from process material was unknown. To direct the development of an assay to quantify the ability of depth filters to adsorb DNA, the molecular weight of DNA from a large-scale, fed-batch, mammalian cell culture vessel was evaluated as process material passed through the initial stages of the purification scheme. High molecular weight DNA was substantially cleared from the broth after passage through a disc stack centrifuge and the remaining low molecular weight DNA was largely unaffected by passage through a series of depth filters and a sterilising grade membrane. Removal of high molecular weight DNA was shown to be coupled with clarification of the process stream. The DNA from cell culture supernatant showed a pattern of internucleosomal cleavage of chromatin when fractionated by electrophoresis but the presence of both necrotic and apoptotic cells throughout the fermentation meant that the origin of the fragmented DNA could not be unequivocally determined. An intercalating fluorochrome, PicoGreen, was elected for development of a suitable DNA assay because of its ability to respond to low molecular weight DNA. It was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing pertinent monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 89.0 % of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples. Application of the fluorescence based assay resulted in characterisation of the physical parameters governing adsorption of DNA by various positively charged depth filters and membranes in test solutions and the DNA adsorption profile of the manufacturing scale filtration train. Buffers that reduced or neutralised the depth filter or membrane charge, and those that impeded hydrophobic interactions were shown to affect their operational capacity, demonstrating that DNA was adsorbed by a combination of electrostatic and hydrophobic interactions. Production-scale centrifugation of harvest broth containing therapeutic protein resulted in the reduction of total DNA in the process stream from 79.8 μg m1-1 to 9.3 μg m1-1 whereas the concentration of DNA in the supernatant of pre-and post-filtration samples had only marginally reduced DNA content: from 6.3 to 6.0 μg m1-1 respectively. Hence the filtration train was shown to ineffective in DNA removal. Historically, blinding of the depth filters had been unpredictable with data such as numbers of viable cells, non-viable cells, product titre, or process shape (batch, fed-batch, or draw and fill) failing to inform on the durability of depth filters in the harvest step. To investigate this, key fouling contaminants were identified by challenging depth filters with the same mass of one of the following: viable healthy cells, cells that had died by the process of apoptosis, and cells that had died through the process of necrosis. The pressure increase across a Cuno Zeta Plus 10SP depth filter was 2.8 and 16.5 times more sensitive to debris from apoptotic and necrotic cells respectively, when compared to viable cells. The condition of DNA released into the culture broth was assessed. Necrotic cells released predominantly high molecular weight DNA in contrast to apoptotic cells which released chiefly low molecular weight DNA. The blinding of the filters was found to be largely unaffected by variations in the particle size distribution of material in, and viscosity of, solutions with which they were challenged. The exceptional response of the depth filters to necrotic cells may suggest the cause of previously noted unpredictable filter blinding whereby a number of necrotic cells have a more significant impact on the life of a depth filter than a similar number of viable or apoptotic cells. In a final set of experiments the pressure drop caused by non-viable necrotic culture broths which had been treated with DNase I or benzonase was found to be smaller when compared to untreated broths: the abilities of the enzyme treated cultures to foul the depth filter were reduced by 70.4% and 75.4% respectively indicating the importance of DNA in the blinding of the depth filter studied.

Liposomes as carriers for polymyxins in the treatment of cystic fibrosis lung infections

Cystic fibrosis (CF) is the most common autosomal recessive disorder affecting Caucasian populations. The pathophysiology of this disorder predisposes the lungs of affected patients to chronic infection, typically by Pseudomonas aeruginosa, which is the main cause of morbidity and mortality. Recently, attention has focused on aerosolised polymyxins, which are given prophylactically in an effort to limit infection and subsequent lung damage. This class of antimicrobial compounds is highly active against P. aeruginosa and possess the advantage that resistance rarely develops. However, the rapid lung clearance of antibiotics is a well documented phenomenon and it was postulated that polymyxin treatment could be further improved by liposomal encapsulation. As part of the development of liposomal polymyxin B, analytical methodology (radiolabelling, HPLC and protein assay) applicable to liposomal formulations was established. Liposomes were prepared by the dehydration-rehydration method and encapsulation efficiencies were determined for a number of phospholipid compositions. Vesicles were characterised with respect to size, zeta potential, morphology and release characteristics. The surface hydrophobicity of vesicles was quantified by hydrophobic interaction chromatography and it was found that this method produced comparable results to techniques conventionally used to assess this property. In vivo testing of liposomal polymyxins demonstrated that encapsulation successfully prevented the rapid pulmonary clearance of PXB. Antimicrobial activity of liposomal formulations was quantified and found to be dependent on both the vesicle surface characteristics and their release profile. Investigation of the interaction of PXB with lipopolysaccharide was undertaken and results demonstrated that PXB caused significant structural distortion of the lipid A region. This may be sufficient to abrogate the potentiating action of LPS in the inflammatory cascade.

Electrodeposition of cermets

Modern engineering requirements are frequently near the limits of application of conventional materials. For many purposes, particularly tribological, the most satisfactory solution is frequently the application of a resistant coating to the surface of a common metal. Electrodeposited cermet coatings have proved very satisfactory: some of the factors underlying the cernet electrodeposition process have been investigated. A ceramic particle in contact with an electrolyte solution will carry a charge which may affect the kinetics of the suspended particle under electroplating conditions. Measurerment has been made of this charge on particles of silicon carbide, chrornium diboride and quartz, in contiact with solutions of copper sulphate/ sulphuric acid in terms of the electrokinetic (zeta) potential and also as surface charge density. The methocl used was that of streaming potential and streaming current measurement

Ionic and molecular diffusion in cementitious materials

The work described in this thesis is an attempt to provide improved understanding of the effects of several factors affecting diffusion in hydrated cement pastes and to aid the prediction of ionic diffusion processes in cement-based materials. Effect of pore structure on diffusion was examined by means of comparative diffusion studies of quaternary ammonium ions with different ionic radii. Diffusivities of these ions in hydrated pastes of ordinary portland cement with or without addition of fly ash were determined by a quasi-steady state technique. The restriction of the pore geometry on diffusion was evaluated from the change of diffusivity in response to the change of ionic radius. The pastes were prepared at three water-cement ratios, 0.35, 0.50 and 0.65. Attempts were made to study the effect of surface charge or the electrochemical double layer at the pore/solution interface on ionic diffusion. An approach was to evaluate the zeta potentials of hydrated cement pastes through streaming potential measurements. Another approach was the comparative studies of the diffusion kinetics of chloride and dissolved oxygen in hydrated pastes of ordinary portland cement with addition of 0 and 20% fly ash. An electrochemical technique for the determination of oxygen diffusivity was also developed. Non-steady state diffusion of sodium potassium, chloride and hydroxyl ions in hydrated ordinary portland cement paste of water-cement ratio 0.5 was studied with the aid of computer-modelling. The kinetics of both diffusion and ionic binding were considered for the characterization of the concentration profiles by Fick's first and second laws. The effect of the electrostatic interactions between ions on the overall diffusion rates was also considered. A general model concerning the prediction of ionic diffusion processes in cement-based materials has been proposed.

Influence of pretreatment on corrosion behaviour of duplex zinc/polymer coatings on steel substrates

An investigation has been undertaken to determine the major factors influencing the corrosion resistance of duplex-zinc coatings on steel substrates.Premature failure of these systems has been attributed to the presence of defects such as craters and pinholes in the polymer film and debonding of the polymer film from the zinc substrate.Defects found on commercially produced samples have been carefully characterised using metallographic and scanning electron microscopy techniques. The influence of zinc substrate surface roughness, polymer film thickness and degassing of conversion coatings films on the incidence of defects has been determined.Pretreatments of the chromate, chromate-phosphate, non chromate, and alkali-oxide types were applied and the conversion coatings produced characterised with respect to their nature and composition. The effect of degassing on the properties of the films was also investigated. Electrochemical investigations were carried out to determine the effect of the presence of the eta or zeta phase as the outermost layer of the galvanized coating.Flow characteristics of polyester on zinc electroplated hot-dip continuous and batch galvanized and zinc sprayed samples were investigated using hot-stage microscopy. The effects of different pretreatments and degassing after conversion coating formation on flow characteristics were determined.Duplex coatings were subjected to the acetic acid salt spray test. The effect on adhesion was determined using an indentation debonding test and the results compared with those obtained using cross-cut/peel and pull-off tests. The locus of failure was determined using scanning electron microscopy and X-ray photoelectron spectroscopy techniques.

Formulation and the characterisation of cationic liposomal adjuvants for the delivery of a promising subunit tuberculosis vaccine

Cationic liposomes of dimethyldioctadecylammonium bromide (DDA) incorporating the glycolipid trehalose 6,6-dibehenate (TDB) forms a promising liposomal vaccine adjuvant. To be exploited as effective subunit vaccine delivery systems, the physicochemical characteristics of liposomes were studied in detail and correlated with their effectiveness in vivo, in an attempt to elucidate key aspects controlling their efficacy. This research took the previously optimised DDA-TDB system as a foundation for a range of formulations incorporating additional lipids of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), by incrementally replacing the cationic content within DDA-TDB or reducing the total DDA-TDB dose upon its substitution, to ascertain the role of DDA and the effect of DDA-TDB concentration in influencing the resultant immunological performance upon delivery of the novel subunit TB vaccine, Ag85B–ESAT-6-Rv2660c (H56 vaccine). With the aim of using the DPPC based systems for pulmonary vaccine delivery and the DSPC systems for application via the intramuscular route, initial work focused on physicochemical characterisation of the systems with incorporation of DPPC or DSPC displaying comparable physical stability, morphological structure and levels of antigen retention to that of DDA-TDB. Thermodynamic analysis was also conducted to detect main phase transition temperatures and subsequent in vitro cell culture studies demonstrated a favourable reduction in cytotoxicity, stimulation of phagocytic activity and macrophage activation in response to the proposed liposomal immunoadjuvants. Immunisation of mice with H56 vaccine via the proposed liposomal adjuvants showed that DDA was an important factor in mediating resultant immune responses, with partial replacement or substitution of DDA-TDB stimulating Th1 type cellular immunity characterised by elevated levels of IgG2b antibodies and IFN-? and IL-2 cytokines, essential for providing protective efficacy against TB. Upon increased DSPC content within the formulation, either by DDA replacement or reduction of DDA and TDB, responses were skewed towards Th2 type immunity with reduced IgG2b antibody levels and elevated IL-5 and IL-10 cytokine production, as resultant immunological responses were independent of liposomal zeta potential. The role of the cationic DDA lipid and the effect of DDA-TDB concentration were appreciated as the proposed liposomal formulations elicited antigen specific antibody and cellular immune responses, demonstrating the potential of cationic liposomes to be utilised as adjuvants for subunit vaccine delivery. Furthermore, the promising capability of the novel H56 vaccine candidate in eliciting protection against TB was apparent in a mouse model.

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.

Lyophilisation and sterilisation of liposomal vaccines to produce stable and sterile products

The advantages of liposomes as delivery systems for peptide, protein and DNA vaccines is well-recognised, unfortunately their application has been stinted by their instability during storage and their limited shelf-life. Further, sterilisation of these systems has been problematic, with degradation of the liposomes being reported after sterilisation using the various techniques available. Work form our laboratory has investigated techniques that can be applied to particulate liposomal vaccines such that they can be prepared in a freeze-dried and sterile format. In this article, we describe techniques for the lyophilisation, cryoprotection and sterilisation of liposomal vaccines. Applying these methods allows for the retention of both the chemical integrity of the lipids and the key physico-chemical characteristics of the liposomes (e.g., particle size, zeta potential, and dynamic viscosity), thus supporting the enhanced transition of liposomal vaccines from the bench to the clinic. © 2006 Elsevier Inc. All rights reserved.

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.

Th1 immune responses can be modulated by varying dimethyldioctadecylammonium and distearoyl-sn-glycero-3-phosphocholine content in liposomal adjuvants

Objectives - Cationic liposomes of dimethyldioctadecylammonium bromide (DDA) combined with trehalose 6,6'-dibehenate (TDB) elicit strong cell-mediated and antibody immune responses; DDA facilitates antigen adsorption and presentation while TDB potentiates the immune response. To further investigate the role of DDA, DDA was replaced with the neutral lipid of distearoyl-sn-glycero-3-phosphocholine (DSPC) over a series of concentrations and these systems investigated as adjuvants for the delivery of Ag85B–ESAT-6-Rv2660c, a multistage tuberculosis vaccine. Methods - Liposomal were prepared at a 5?:?1 DDA–TDB weight ratio and DDA content incrementally replaced with DSPC. The physicochemical characteristics were assessed (vesicle size, zeta potential and antigen loading), and the ability of these systems to act as adjuvants was considered. Key findings - As DDA was replaced with DSPC within the liposomal formulation, the cationic nature of the vesicles decreases as does electrostatically binding of the anionic H56 antigen (Hybrid56; Ag85B-ESAT6-Rv2660c); however, only when DDA was completed replaced with DSPC did vesicle size increase significantly. T-helper 1 (Th1)-type cell-mediated immune responses reduced. This reduction in responses was attributed to the replacement of DDA with DSPC rather than the reduction in DDA dose concentration within the formulation. Conclusion - These results suggest Th1 responses can be controlled by tailoring the DDA/DSPC ratio within the liposomal adjuvant system.

Th1 immune responses can be modulated by varying dimethyldioctadecylammonium and distearoyl-sn-glycero-3-phosphocholine content in liposomal adjuvants

Objectives Cationic liposomes of dimethyldioctadecylammonium bromide (DDA) combined with trehalose 6,6′-dibehenate (TDB) elicit strong cell-mediated and antibody immune responses; DDA facilitates antigen adsorption and presentation while TDB potentiates the immune response. To further investigate the role of DDA, DDA was replaced with the neutral lipid of distearoyl-sn- glycero-3-phosphocholine (DSPC) over a series of concentrations and these systems investigated as adjuvants for the delivery of Ag85B-ESAT-6-Rv2660c, a multistage tuberculosis vaccine. Methods Liposomal were prepared at a 5: 1 DDA-TDB weight ratio and DDA content incrementally replaced with DSPC. The physicochemical characteristics were assessed (vesicle size, zeta potential and antigen loading), and the ability of these systems to act as adjuvants was considered. Key findings As DDA was replaced with DSPC within the liposomal formulation, the cationic nature of the vesicles decreases as does electrostatically binding of the anionic H56 antigen (Hybrid56; Ag85B-ESAT6-Rv2660c); however, only when DDA was completed replaced with DSPC did vesicle size increase significantly. T-helper 1 (Th1)-type cell-mediated immune responses reduced. This reduction in responses was attributed to the replacement of DDA with DSPC rather than the reduction in DDA dose concentration within the formulation. Conclusion These results suggest Th1 responses can be controlled by tailoring the DDA/DSPC ratio within the liposomal adjuvant system. © 2013 Royal Pharmaceutical Society.