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This study describes the formulation of immuno-stimulating complexes (ISCOMs) utilising the ethanol injection technique. Cholesterol and phosphatidylcholine were dissolved in ethanol and the resulting solution was rapidly injected into a stirred, aqueous solution of the triterpene-saponin mixture Quil-A. The reversed experiment was also carried out by adding the aqueous Quil-A solution to a solution of cholesterol/phosphatidylcholine dissolved in ethanol. This was done by either rapid injection or dropwise addition of the aqueous Quil-A solution. The colloidal dispersions obtained by ethanol injection and reversed addition were compared with formulations obtained by the dialysis and lipid-film hydration methods. In a further experiment, the preparation of ISCOMs from liposomes as precursor structures was investigated. Transmission electron microscopy was used to analyse the resulting colloidal dispersions. Samples were also compared with respect to homogeneity of obtained particle species. The ethanol injection technique led to formation of ISCOMs in high numbers within 2 h post formulation. The reversed rapid injection resulted in a similar colloidal dispersion, differing from the former mainly due to the presence of some helical micellar structures. The reversed, dropwise addition led to the formation of helices as the predominant colloidal structure. Of the three previously established methods, only dialysis led to the formation of ISCOMs within 48 h. The lipid-film hydration method and the approach using liposomes as precursor structures did not produce ISCOMs under the conditions and within the time periods investigated. However, it is known that dispersions prepared by the hydration method equilibrate towards ISCOMs after longer storage. Ethanol injection and reversed rapid injection are simple, cost-effective and quick methods to produce ISCOMs.

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