ISCOMs and related colloidal particles prepared by the lipid film hydration method for antigen delivery
Demana, Patrick Hulisani
Purposes: The aims of this thesis were to physicochemically investigate the formation of ISCOM matrices and other types of colloidal particles formed in aqueous dispersion as a function of different mass ratios of Quil A, cholesterol and phospholipid (phosphatidylcholine (PC) or phosphatidylethanolamine (PE)) prepared by the hydration method and to investigate the delivery of subunit vaccines to antigen presenting cells using these colloids. Methods: The hydration method, recently developed for the preparation of ISCOMs or ISCOM matrices, was used to produce these colloids and other related structures. Factors such as effects of buffer salts, equilibration time and type of phospholipid on the formation of ISCOM matrices and other colloidal particles prepared were investigated. The standard dialysis method for the preparation of ISCOMs was also used for comparison to prepare various colloidal particles. Colloidal particles were characterized by negative staining transmission electron microscopy (TEM). Polarized light microscopy (PLM) was used to identify samples containing cholesterol crystals. Incorporation of a model antigen (modified ovalbumin) into various colloidal particles was investigated by fluorescence spectroscopy following analytical sucrose density gradient ultracentrifugation. Physical properties of solid Quil A-cholesterol-phospholipid formulations (as powder mixtures or compressed to pellets) with or without model antigen were characterized by X-ray powder diffractometry (XRPD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and PLM. Release of model antigen from compressed pellets was investigated in vitro. Flow cytometric analysis (FACS) was used to investigate the in vitro delivery of antigen incorporated into various colloidal structures to murine bone-marrow derived dendritic cells (BMDC). Results: Pseudo-ternary diagrams revealed that depending on the mass ratio of Quil A, cholesterol and phospholipid, various colloidal particles including ISCOM matrices, liposomes, lipidic/layered structures, ring-like micelles, and worm-like micelles could be identified in the different regions of the diagrams. In the presence of these predominant colloids, helices and lamellae (hexagonal pattern of ring-like micelles) structures were also formed as minor structures. Buffer salts and equilibration time were important factors for the formation of ISCOM matrices and liposomes. The type of phospholipid affected the morphology of ISCOM matrices and lamellae. ISCOM matrices were predominantly found near the phospholipid apex of the pseudo-ternary diagram following sample preparation by the hydration method. On the other hand, samples prepared by the dialysis method produced ISCOM matrices that were predominantly found near the Quil A apex of the pseudo-ternary diagram. No ISCOM matrices could be formed in any binary mixtures prepared by the hydration method in contrast to the dialysis method. Worm-like micelles could only be formed if samples were prepared by the hydration method. An incorporation study demonstrated that the various colloidal particles formed as a result of hydrating phospholipid/cholesterol lipid films with different amounts of Quil A are capable of incorporating antigen, provided it is amphipathic. Freeze-dried lipid powder mixtures were found to contain a lower degree of crystalline cholesterol compared to physically mixed powders. Consequently, physically mixed powders (with or without model antigen) and pellets prepared from the same powders did not spontaneously form ISCOM matrices and related colloidal structures upon hydration as expected from the pseudo-ternary diagram. Release of antigen incorporated into ISCOM particles was relatively slower from the pellets made using freeze-dried powders in contrast to pellets prepared from the physically mixed powders. Using ISCOMs, liposomes and ring-like micelles, it was demonstrated that the model antigen incorporated into these particles could be delivered to dendritic cells leading to activation and proliferation of transgenic T cells. Conclusions: Depending on the mass ratio of Quil A, cholesterol and phospholipid, ISCOM matrices and other types of colloidal structures such as liposomes, lipidic/layered structures, ring-like micelles, lamellae (hexagonal array of ring-like micelles) and wormlike micelles prepared by the hydration method could be identified in the different regions of pseudo-ternary diagram. All the colloids containing Quil A were capable of incorporating an antigen, provided it is amphipathic. Delivery of antigen to DC and immunestimulatory effects of various colloidal particles could be demonstrated.
Advisor: Rades, Thomas; Davies, Nigel; Hook, Sarah
Degree Name: Doctor of Philosophy
Degree Discipline: Pharmacy
Publisher: University of Otago
Research Type: Thesis