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.

Quil A-lipid powder formulations releasing ISCOMs and related colloidal stuctures upon hydration

The aim of the present study was to prepare solid Quil A-cholesterol-phospholid formulations (as powder mixtures or compressed to pellets) by physical mixing or by freeze-drying of aqueous dispersions of these components in ratios that allow spontaneous formation of ISCOMs and other colloidal stuctures upon hydration. The effect of addition of excess cholesterol to the lipid mixtures on the release of a model antigen (PE-FITC-OVA) from the pellets was also investigated. Physical properties were evaluated by X-ray powder diffractometry (XPRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and polarized light microscopy (PLM). Characterization of aqueous colloidal dispersions was performed by negative staining transmission electron microscopy (TEM). Physically mixed powders (with or without PE-FITC-OVA) and pellets prepared from the same powders did not spontaneously form ISCOM matrices and related colloidal structures such as worm-like micelles, ring-like micelles, lipidic/layered structures and lamellae (hexagonal array of ring-like micelles) upon hydration as expected from the pseudo-temary diagram for aqueous mixtures of Quil A, cholesterol and phospholipid. In contrast, spontaneous formation of the expected colloids was demonstrated for the freeze-dried lipid mixtures. Pellets prepared by compression of freeze-dried powders released PE-FITC-OVA slower than those prepared from physically mixed powders. TEM investigations revealed that the antigen was released in the form of colloidal particles (ISCOMs) from pellets prepared by compression of freeze-dried powders. The addition of excess cholesterol slowed down the release of antigen. The findings obtained in this study are important for the formulation of solid Quil A-containing lipid articles as controlled particulate adjuvant containing antigen delivery systems. (c) 2004 Elsevier B.V. All rights reserved.

Preparation of immuno-stimulating complexes (ISCOMs) by ether injection

preparation of liposomes, as a new, continuous and potentially scaleable method for the preparation of ISCOMs. Phosphatidylcholine (PC) and cholesterol (Chol) were dissolved in ether, which was injected into an aqueous solution, maintained at 55 degrees C, containing Quil A. The influences of the following variables on ISCOM formation were investigated: ratio of PC:Quil A:Chol used, pumping rate, total lipid mass and concentration of buffer salts and Quil A in the aqueous phase. All samples were characterized by negative stain transmission electron microscopy, photon correlation spectroscopy and sucrose ultracentrifugation gradient. It was demonstrated that ISCOMs could be produced by this method but the homogeneity of the preparation was influenced by the conditions used. Homogeneous ISCOM preparations were consistently produced only when the weight ratio of PC:Quil A:Chol was 5:3:2 with a total lipid mass of 20 mg, the Quil A dissolved in a 0.01 M phosphate buffer at a concentration of 6 mg in 4 ml, and the ether solution injected into the warmed buffer solution at a rate of 0.2 ml/min. Changing any of these variables resulted in more heterogeneous preparations in which ISCOMs typically co-existed with other colloidal structures such as worm-like and helical micelles, liposomes, lamellae and lipidic particles. (C) 2005 Elsevier B.V. All rights reserved.

Pluronic F127-modified liposome-containing tacrolimus-cyclodextrin inclusion complexes:improved solubility, cellular uptake and intestinal penetration

Objective The aim of this study was to investigate Pluronic F127-modified liposome-containing cyclodextrin (CD) inclusion complex (FLIC) for improving the solubility, cellular uptake and intestinal penetration of tacrolimus (FK 506) in the gastrointestinal (GI) tract. Methods Molecular modelling was performed to screen the optimal CD for the solubilization of FK 506. FLIC was prepared by thin-lipid film hydration with the inclusion complex solutions followed by membrane extrusion. Dilution tests were conducted in simulated gastric fluids and phosphate-buffered solution of sodium taurocholate to investigate the solubility improvement of FK506. The cellular uptake of nanocarriers was studied in Caco-2 cells, and intestinal mucous membrane penetration in the GI tract was evaluated in Sprague-Dawley rats. Key findings The results showed that β-CD had the strongest binding energy with the guest molecule among the CDs. The prepared FLIC has an average diameter of 180.8 ± 8.1 nm with a spherical shape. The solubility and cellular uptake of FK 506 was greatly improved by the incorporation of CD complexes in the Pluronic F127-modified liposomes. Intestinal mucous membrane penetration was also significantly improved by the preparation of FLIC. Conclusion With improved drug solubility and intestinal mucous membrane penetration, FLIC shows a promising oral delivery system for FK 506. © 2013 Royal Pharmaceutical Society.

Rapid quantification and validation of lipid concentrations within liposomes

Quantification of the lipid content in liposomal adjuvants for subunit vaccine formulation is of extreme importance, since this concentration impacts both efficacy and stability. In this paper, we outline a high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method that allows for the rapid and simultaneous quantification of lipid concentrations within liposomal systems prepared by three liposomal manufacturing techniques (lipid film hydration, high shear mixing, and microfluidics). The ELSD system was used to quantify four lipids: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, dimethyldioctadecylammonium (DDA) bromide, and D-(+)-trehalose 6,6′-dibehenate (TDB). The developed method offers rapidity, high sensitivity, direct linearity, and a good consistency on the responses (R2 > 0.993 for the four lipids tested). The corresponding limit of detection (LOD) and limit of quantification (LOQ) were 0.11 and 0.36 mg/mL (DMPC), 0.02 and 0.80 mg/mL (cholesterol), 0.06 and 0.20 mg/mL (DDA), and 0.05 and 0.16 mg/mL (TDB), respectively. HPLC-ELSD was shown to be a rapid and effective method for the quantification of lipids within liposome formulations without the need for lipid extraction processes.

Development of paclitaxel-loaded liposomal systems with anti-her2 antibody for targeted therapy

Purpose: To develop liposome formulations containing monoclonal antibody anti-HER2 (MabHer2), and Paclitaxel (PTX). Methods: Seven different liposomal systems containing PTX, or MabHer2 or a combination of PTX and MabHer2 were made using lipid film hydration technique and sonication. The effects of liposome preparation conditions and extraction methods on antibody structure were investigated by polyacrylamide gel electrophoresis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The characteristics of the liposomes were determined by a zetasizer, while drug-loading efficiency was evaluated by high-performance liquid chromatography. The cytotoxic effect of the liposome formulations was evaluated on MDA-MB-453 (HER2+) and MCF-7 (HER2-) breast cancer cell lines by MTT assay. Results: The antibody was not significantly affected by the stress conditions and the method of extraction. The particle size of liposomes was < 200 nm while the amount of incorporated PTX was 97.6 % for liposome without cationic agent and 98.2 % for those with cationic agent. Recovery of MabHer2 was 94.38 % after extraction. Combined PTX/MabHer2 liposome was more toxic on HER2 overexpressing positive MDA-MB-453 cell line than PTX-loaded liposomes and MabHer2. MabHer2 and combined PTX/MabHer2 liposomes showed no toxic effects on HER2 overexpressing negative MCF-7 cells relative to cationic PTX-loaded liposomes. Conclusions: This results obtained show that PTX can be encapsulated successfully into liposoma systems and that owing to Her2 specific antibody, these systems can be delivered directly to the target cell.

Influences of Hydration Force and Elastic Strain Energy on the Stability of Solid Film in a Very Thin Solid-on-Liquid Structure

Since hydration forces become very strong at short range and are particularly important for determining the magnitude of the adhesion between two surfaces or interaction energy, the influences of the hydration force and elastic strain energy due to hydration-induced layering of liquid molecules close to a solid film surface on the stability of a solid film in a solid-on-liquid (SOL) nanostructure are studied in this paper. The liquid of this thin SOL structure is a kind of water solution. Since the surface forces play an important role in the structure, the total free energy change of SOL structures consists of the changes in the bulk elastic energy within the solid film, the surface energy at the solid-liquid interface and the solid-air interface, and highly nonlinear volumetric component associated with interfacial forces. The critical wavelength of one-dimensional undulation, the critical thickness of the solid film, and the critical thickness of the liquid layer are studied, and the stability regions of the solid film have been determined. Emphasis is placed on calculation of critical values, which are the basis of analyzing the stability of the very thin solid film.

Contribution of different physical forces to the disjoining pressure of a thin water film being pressed by an oil droplet

With an objective to understand the nature of forces which contribute to the disjoining pressure of a thin water film on a steel substrate being pressed by an oil droplet, two independent sets of experiments were done. (i) A spherical silica probe approaches the three substrates; mica, PTFE and steel, in a 10 mM electrolyte solution at two different pHs (3 and 10). (ii) The silica probe with and without a smeared oil film approaches the same three substrates in water (pH = 6). The surface potential of the oil film/water was measured using a dynamic light scattering experiment. Assuming the capacity of a substrate for ion exchange the total interaction force for each experiment was estimated to include the Derjaguin-Landau-Verwey-Overbeek (DLVO) force, hydration repulsion, hydrophobic attraction and oil-capillary attraction. The best fit of these estimates to the force-displacement characteristics obtained from the two sets of experiment gives the appropriate surface potentials of the substrates. The procedure allows an assessment of the relevance of a specific physical interaction to an experimental configuration. Two of the principal observations of this work are: (i) The presence of a surface at constant charge, as in the presence of an oil film on the probe, significantly enhances the counterion density over what is achieved when both the surfaces allow ion exchange. This raises the corresponding repulsion barrier greatly. (ii) When the substrate surface is wettable by oil, oil-capillary attraction contributes substantially to the total interaction. If it is not wettable the oil film is deformed and squeezed out. (C) 2010 Elsevier Inc. All rights reserved.

Counterion exchange at the surface of polyelectrolyte multilayer film for wettability modulation

Counterions present at the surface of polyelectrolyte multilayers (PEMs) were utilized for modulation of surface wettability via ion exchange. The PEM film was dipped in aqueous solutions of different anions, respectively, and the water contact angle of the surface varied from about 10 degrees to 120 degrees, depending on the hydration characteristics of the anion. The ion exchange mechanism was verified by X-ray photoelectron spectroscopy. The process was rapid and reversible. Ionic strength of the polyelectrolyte solution used for preparing the PEMs was found to be crucial to the surface wetting properties and the reversibility and kinetics of the process, and the effects were correlated to the surface density of the excess charge and counterion. This work provides a general, facile and rapid approach of surface property modulation.

Polymer modified cement: hydration microstructure and diffusion properties

Investigations concentrated on the styrene butadiene rubber (SBR) latex and formulations included standard carboxylated and special carboxylated latexes. The aqueous component, containing the stabilisers and antifoaming agent but not the polymer solids, was also used. For comparison, limited investigations were carried out using other polymer types e.g. acrylic, ethylene-vinyl acetate (EVA), and redispersible powders rather than emulsions. The major findings were: 1) All latex systems investigated acted as retarders for cement hydration. The extent of retardation depends on the type of polymer. The mechanism for cement hydration may be changed, and excessive retardation influences properties. 2) Polymer modified cements exhibited either similar or coarser pore structures compared with unmodified cements. Results suggest that polymer mainly exists in a mixture of cement hydrates and polymer phase. Very little evidence was found for the formation of a distinct polymer film phase. 3) During the first few days of curing the polymer solids are removed from the pore solution and concentrations of OH-, Na+ and K+ are reduced. These observations are probably a result of polymer-cement surface interactions since there was no evidence of any chemical reactions or degradation of the polymer. 4) Improved diffusional resistance of modified cements depends on the ability to achieve adequate workability at low w/c ratio, rather than modification of matrix structure.