Preparation, characterisation and entrapment of a non-glycosidic threitol ceramide into liposomes for presentation to invariant natural killer T cells

Abstract

Dendritic cells (DCs) are able to present glycolipids to invariant natural killer T (iNKT) cells in vivo. Very few compounds have been found to stimulate iNKT cells, and of these, the best characterised is the glycolipid α-galactosylceramide, which stimulates the production of large quantities of interferon-gamma (IFN-γ) and interleukin-4 (IL-4). However, αGalCer leads to overstimulation of iNKT cells. It has been demonstrated that the αGalCer analogue, threitol ceramide (ThrCer 2), successfully activates iNKT cells and overcomes the problematic iNKT cell activation-induced anergy. In this study, ThrCer 2 has been inserted into the bilayers of liposomes composed of a neutral lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or dimethyldioctadecylammonium bromide (DDA), a cationic lipid. Incorporation efficiencies of ThrCer within the liposomes was 96% for DSPC liposomes and 80% for DDA liposomes, with the vesicle size (large multilamellar vs. small unilamellar vesicles) making no significant difference. Langmuir-Blodgett studies suggest that both DSPC and DDA stack within the monolayer co-operatively with the ThrCer molecules with no condensing effect. In terms of cellular responses, IFN-γ secretion was higher for cells treated with small DDA liposomes compared with the other liposome formulations, suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.

Figure 1

CD1d agonists αGalCer 1 and ThrCer 2.

Figure 2

Vesicle size (nm) of MLV (a) and SUV (b) of DSPC and DDA liposomes with and without the inclusion of ThrCer represented by storage at 25 °C. Size were measured in Tris buffer (1 mM) using a Brookhaven ZetaPlus instrument. Results represent mean ± SD of triplicate experiments.

Figure 2

Vesicle size (nm) of MLV (a) and SUV (b) of DSPC and DDA liposomes with and without the inclusion of ThrCer represented by storage at 25 °C. Size were measured in Tris buffer (1 mM) using a Brookhaven ZetaPlus instrument. Results represent mean ± SD of triplicate experiments.

Figure 3

Entrapped ThrCer release profile of DSPC and DDA MLV (a) and SUV (b) from liposomes when stored in Tris and under simulated in vivo conditions (50% FCS, 37 °C).

Figure 3

Entrapped ThrCer release profile of DSPC and DDA MLV (a) and SUV (b) from liposomes when stored in Tris and under simulated in vivo conditions (50% FCS, 37 °C).

Figure 4

The surface pressure-area isotherms of mixed and pure monolayers at the air/water interface. The mixed and pure monolayers were of (a) DSPC, (b) DDA and ThrCer. The air/water interface was at 20 °C. Results denote mean ± SD, from 3 independent batches.

Figure 4

The surface pressure-area isotherms of mixed and pure monolayers at the air/water interface. The mixed and pure monolayers were of (a) DSPC, (b) DDA and ThrCer. The air/water interface was at 20 °C. Results denote mean ± SD, from 3 independent batches.

Figure 5

IFNγ release from iNKT cells co-cultured with DCs pulsed with liposomes