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

Abstract

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 4 degrees C) 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.

In Vitro Cutaneous Application of ISCOMs on Human Skin Enhances Delivery of Hydrophobic Model Compounds Through the Stratum Corneum

“…Quil A micelles (Fig. 3, white arrow) were also observed in the ISCOM preparations as described in the literature (19,20).…”

Cationic cage-like complexes formed by DC-cholesterol, Quil-A, and phospholipid

“…This interaction may lower the rigidity of the ring-like subunits, enabling their aggregation to spherical particles in absence of a phospholipid. cholesterol) this was also discussed by Demana et al 28 Moreover, the function of the phospholipid as a fluidiser may become redundant in the cationic cage-like structure as discussed above.…”

“…12,28 This method offers the advantage that, unlike in the widely used dialysis procedure, no losses of components are expected, 29 which simplifies interpretation of colloidal structures obtained. It was hypothesized that, if ISCOMs are prepared according to the dialysis procedure, a residual amount of surfactant might at least partially take over the role of the phospholipid, 10 and a recent publication by our group, in which colloidal structures prepared by either the lipid-film hydration method or the dialysis procedure were compared, supported this interpretation.…”

“…It was hypothesized that, if ISCOMs are prepared according to the dialysis procedure, a residual amount of surfactant might at least partially take over the role of the phospholipid, 10 and a recent publication by our group, in which colloidal structures prepared by either the lipid-film hydration method or the dialysis procedure were compared, supported this interpretation. 28 ISCOM matrix formation was indeed obtained as a minor colloidal structure on the binary cholesterol:Quil-A axis if samples were dialysed, but not if the lipid-film hydration method was used. However it appears that the binary interaction between DC-CHOL and Quil-A leads to disc/ring-micelles with a lower rigidity compared to colloids formed by cholesterol and Quil-A at comparable molar ratios, as these aggregated to spherical cage-like particles in the absence of phospholipid.…”

Saponins from Quillaja saponaria Molina: Isolation, Characterization and Ability to Form Immuno Stimulatory Complexes (ISCOMs)

“…In contrast to Morein's earlier studies [16], Dalsgaard et al reported that both Quadri-1 and Quadri-2 formed homogeneous ISCOM matrices when prepared by dialysis. The main difference between the two studies was that Dalsgaard et al included phospholipid in their ISCOM formulation, a component which has recently been shown to be critical for ISCOM formation when crude Quil A is used as the saponin [18]. In another study, the ability of three RP-HPLC purified saponin fractions, designated QH-A, QH-B and QH-C, to form ISCOMs was investigated [19].…”