Physicochemical and biological characterisation of novel immunomodulators
Native phosphatidylinositol mannosides (PIMs) isolated from the cell wall of mycobacteria, and synthetic analogues, have been reported to have the ability to both suppress and stimulate the immune system. These immunomodulating properties have resulted in PIMs being utilised for the treatment of atopic diseases and as adjuvants to improve the efficacy of vaccines. While numerous studies have examined the biological activity of these molecules, little data is available on the behaviour of these compounds at a molecular level. The aim of this thesis was to assess the physicochemical properties of a series of synthetic PIM analogues and to attempt to correlate these characteristics with immunological activity. To accomplish this, the flexibility and lipophilicity of synthetic PIM2 was varied by changing the polar head group (inositol versus glycerol) and the length of the fatty acid residues (C0, C10, C16 and C18). A series of six phosphatidylinositol dimannoside (PIM2) and phosphatidylglycerol dimannoside (PGM2) compounds was synthesised and characterised. Examination of their behaviour at an air/water interface using a Langmuir trough technique, showed that surface pressure-area (π-A) isotherms were significantly influenced by the length of lipid acyl chains and by the nature of the head group. Stronger hydrophobic attractive forces between the longer fatty acid chains enabled a closer packing in the pure monolayers. Furthermore, the more flexible glycerol head group led to more tightly arranged molecules at the air/water interface. In aqueous solution acylated PIM2 and PGM2 were observed to self-assemble into spherical aggregates, as confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The length of the fatty acid chains affected the size of the formed aggregates. In a mouse model of allergic asthma all compounds showed modest immunosuppressive activity. Replacement of the inositol core with the three carbon glycerol unit maintained biological activity. The deacylated PGM2, which did not show self-organisation, had no effect on the eosinophil numbers but did impact on the expansion of OVA-specific CD4+ T cells. In order to investigate adjuvant activities, the phosphoglycolipids were incorporated into liposome-based delivery systems prepared using phosphatidylcholine (PC). To obtain insight into bilayer interactions and incorporation of the phosphoglycolipids into PC bilayers, binary Langmuir monolayers were investigated. In mixed films the phosphoglycolipids were found to be miscible with PC based on evaluation of collapse pressures and deviations of experimental molecular areas from calculated ideal values. ConA agglutination assays confirmed the surface display of the mannosyl residues. However, using murine bone marrow-derived dendritic cells (BMDC), no significant adjuvant activity or effect on vaccine uptake was detected in vitro. Investigations of the immunostimulatory activities in vivo revealed that modified liposomes were unable to elicit an improved antigen-specific humoral or cellular immune response, as compared to unmodified PC liposomes. The fact that inclusion of mannosylated phosphoglycolipids into liposomal bilayers did not enhance immune responses may reflect the complexity of mannose recognition and signalling. The findings may indicate that the conformation of the mannose moieties when presented in the prepared liposomes was not optimal for receptor binding. In conclusion, physicochemical properties of the investigated compounds were determined and data gained may help to elucidate the requirements for receptor interactions of synthetic PIM2 and PGM2. With regards to biological effects, the impact of structural variations was less pronounced. Varied lipophilicity did not necessarily correlate with immunological activities and no conclusive structure-function relationship could be established.
Advisor: Hook, Sarah; Rades, Thomas; Larsen, David
Degree Name: Doctor of Philosophy
Degree Discipline: School of Pharmacy
Publisher: University of Otago
Keywords: Phosphatidylinositol dimannoside; PIM analogues; Langmuir trough study; Immunomodulation; Modified liposomes
Research Type: Thesis