Incorporation of ovalbumin into ISCOMs and related colloidal particles prepared by the lipid film hydration method

The ann of this study was to investigate the incorporation of a model antigen, fluorescently labelled ovalbumin (FITC-OVA), into various colloidal particles including immune stimulating complexes (ISCOMs), liposomes, ring and worm-like micelles, lamellae and lipidic/layered structures that are formed from various combinations of the triterpene saponin Quil A, cholesterol and phosphatidylethanolamine (PE) following hydration of PE/cholesterol lipid films with aqueous Solutions of Quil A. Colloidal dispersions of these three components were also prepared by the dialysis method for comparison. FITC-OVA was conjugated with palmitic acid (P) and PE to produce P-FITC-OVA and PE-FITC-OVA, respectively. Both P-FITC-OVA and PE-FITC-OVA could be incorporated in all colloidal structures whereas FITC-OVA was incorporated only into liposomes. The incorporation of PE-FITC-OVA into all colloidal structures was significantly higher than P-FITC-OVA (P < 0.05). The degree of incorporation of protein was in the order: ring and worm-like micelles < liposomes and lipidic/layered structures < ISCOMs and lamellae. The incorporation of protein into the various particles prepared by the lipid film hydration method was similar to those for colloidal particles prepared by the dialysis method (provided both methods lead to the formation of the same colloidal structures). In the case of different colloidal structures arising due to the preparation method, differences in encapsulation efficiency were found (P < 0.05) for formulations with the same polar lipid composition. This study demonstrates that the various colloidal particles formed as a result of hydrating PE/cholesterol lipid films with different amounts of Quil A are capable of incorporating antigen, provided it is amphipathic. Some of these colloidal particles may be used as effective vaccine delivery systems. (C) 2004 Elsevier B.V. All rights reserved.

Pseudo-ternary phase diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by the lipid-film hydration method

Pseudo-ternary phase diagrams of the polar lipids Quil A, cholesterol (Chol) and phosphatidylcholine (PC) in aqueous mixtures prepared by the lipid film hydration method (where dried lipid film of phospholipids and cholesterol are hydrated by an aqueous solution of Quil A) were investigated in terms of the types of particulate structures formed therein. Negative staining transmission electron microscopy and polarized light microscopy were used to characterize the colloidal and coarse dispersed particles present in the systems. Pseudo-ternary phase diagrams were established for lipid mixtures hydrated in water and in Tris buffer (pH 7.4). The effect of equilibration time was also studied with respect to systems hydrated in water where the samples were stored for 2 months at 4degreesC. Depending on the mass ratio of Quil A, Chol and PC in the systems, various colloidal particles including ISCOM matrices, liposomes, ring-like micelles and worm-like micelles were observed. Other colloidal particles were also observed as minor structures in the presence of these predominant colloids including helices, layered structures and lamellae (hexagonal pattern of ring-like micelles). In terms of the conditions which appeared to promote the formation of ISCOM matrices, the area of the phase diagrams associated with systems containing these structures increased in the order: hydrated in water/short equilibration period < hydrated in buffer/short equilibration period < hydrated in water/prolonged equilibration period. ISCOM matrices appeared to form over time from samples, which initially contained a high concentration of ring-like micelles suggesting that these colloidal structures may be precursors to ISCOM matrix formation. Helices were also frequently found in samples containing ISCOM matrices as a minor colloidal structure. Equilibration time and presence of buffer salts also promoted the formation of liposomes in systems not containing Quil A. These parameters however, did not appear to significantly affect the occurrence and predominance of other structures present in the pseudo-binary systems containing Quil A. Pseudo-ternary phase diagrams of PC, Chol and Quil A are important to identify combinations which will produce different colloidal structures, particularly ISCOM matrices, by the method of lipid film hydration. Colloidal structures comprising these three components are readily prepared by hydration of dried lipid films and may have application in vaccine delivery where the functionality of ISCOMs has clearly been demonstrated. (C) 2003 Elsevier B.V. All rights reserved.

A comparison of pseudo-ternary diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by lipid-film hydration and dialysis

Pseudo-ternary diagrams for Quil A, phospholipid (phosphatidylcholine (PC) or phosphatidylethanolamine (PE)) and cholesterol were established in order to identify combinations that result in the formation of immune-stimulating complex (ISCOM) matrices and other colloidal structures produced by these three components in aqueous systems following lipid-film hydration or dialysis (methods that can be used to produce ISCOMs). In addition, the effect of equilibration time (1 month at 4degreesC) on the structures formed by the various combinations of the three components was investigated. Depending on the ratio of Quil A, cholesterol and phospholipid, different colloidal particles, including ISCOM matrices, liposomes and ring-like micelles, were found irrespective of the preparation method used. In contrast, worm-like micelles were only observed in systems prepared by lipid-film hydration. For samples prepared by dialysis, ISCOM matrices were predominantly found near the Quil A apex of the pseudo-ternary diagram (> 50% Quil A). On the other hand, for samples prepared by lipid-film hydration, ISCOM matrices were predominantly found near the phospholipid apex of the pseudo-ternary diagram (> 50% phospholipid). The regions in the pseudo-ternary diagrams in which ISCOM matrices were observed increased following an extended equilibration time, particularly for samples prepared by lipid-film hydration. Differences were also observed between pseudoternary diagrams prepared using either PE or PC as phospholipids.

Formation of mesostructured titania thin films using iospropoxide precursors

Mesostructured titania thin films were prepared by an evaporation-induced self-assembly process. The highly acidic sot precursors contained titanium(IV) tetraisopropoxide (TTIP) as a titanium source, a tri-block copolymer Pluronic P123 as a template, and acetylacetonate and HCl as hydrolysis inhibitors. Characteristics of the resultant titania thin films were studied using X-ray diffraction (XRD) analysis, N-2-adsorption/desorption analysis, and transmission electron microscopy (TEM). XRD and TEM investigations on the as-synthesised films revealed the appearance of cubic-like, pseudohexagonal, and lamellar mesophases; depending on the amount of water in the sols of film precursors. Template removal by a calcination process yields high surface area (320-360 m(2)/g) mesoporous materials with crystalline anatase frameworks. Water content also influences the degree of anatase crystallinity of the calcined films. Higher water content resulted in improved anatase crystallinity. These nanostructured materials are of interest for photocatalysts, pbotoelectrochemical solar cells and other photonic devices. (C) 2003 Elsevier B.V. All rights reserved.