Structural characterization of cationic DODAB bilayers containing C24:1 β-glucosylceramide

Abstract

The effect of 5 mol%, 9 mol%, and 16 mol% of C24:1 β-glucosylceramide (βGlcCer) on the structure of cationic DODAB bilayers was investigated by means of differential scanning calorimetry (DSC), electron spin resonance (ESR) spectroscopy and fluorescence microscopy. βGlcCer is completely miscible with DODAB at all fractions tested, since no domains were observed in fluorescence microscopy or ESR spectra. The latter showed that βGlcCer destabilized the gel phase of DODAB bilayers by decreasing the gel phase packing. As a consequence, βGlcCer induced a decrease in the phase transition temperature and cooperativity of DODAB bilayers, as seen in DSC thermograms. ESR spectra also showed that βGlcCer induced an increase in DODAB fluid phase order and/or rigidity. Despite their different structures, a similar effect of loosening the gel phase packing and turning the fluid phase more rigid/organized has also been observed when low molar fractions of cholesterol were incorporated in DODAB bilayers. The structural characterization of mixed membranes made of cationic 2 lipids and glucosylceramides may be important for developing novel immunotherapeutic tools such as vaccine adjuvants.

Thermotropic Behavior and Structural Organization of C24:1 Sulfatide Dispersions and Its Mixtures with Cationic Bilayers

“…The single heating and cooling peaks at approximately 27.5 °C observed for pure C24:1 SF (Figure ) contrast with the complex thermotropic behavior of C24:1 beta-glucosylceramide (βGlcCer), which presents several peaks upon heating and a single hysteretic peak upon cooling . Considering that SF and βGlcCer have the same C24:1 ceramide moiety, the differences in thermotropic behavior could be attributed to the negative charge of the SF headgroup, whose electrostatic repulsion might hinder the formation of metastable phases and of high-temperature melting events observed for the neutral βGlcCer, which has a main phase-transition temperature around 66 °C when heated. , Electrostatic repulsion is an important factor in lipid-phase organization and was shown to weaken intermolecular hydrogen bonding in SFs when compared to neutral cerebrosides …”

“…Despite their different thermotropic behaviors, pure SF and βGlcCer show similar structural organization in EPR spectroscopy assays: both form lamellar structures that are not interdigitated since a flexibility gradient is present at all temperatures (Figure ). Even though the C4- trans bond of the sphingosine base would facilitate the compact packing of the ceramide chains, , the 15- cis bond of the C24:1 acyl chain would increase the lateral space requirements (Figure ), resulting in a decreased ceramide chain packing. , Accordingly, EPR experiments with 16-doxylstearate show that this cis double bond prevents any form of interdigitation in SFs …”

“…This V-shaped conformation would result in a large lateral area requirement that would decrease the packing of the DODAB gel phase, as observed in Figure . The V-shaped conformation would also explain the increase in DODAB fluid-phase rigidity: the larger S eff and smaller h –1 / h 0 values in Figure indicate that the acyl chains of DODAB are more ordered and have less freedom of movement in the presence of SF, as they do in the presence of βGlcCer and αGalCer. , Paramagnetic-labeled galactosylceramides were also shown to increase the rigidity of membranes in the fluid phase …”

“…Indeed, a steep increase in fluidity can be observed between 25 and 30 °C, which is consistent with the broad thermal event around 27.5 °C observed in Figure 2. No coexistence of phases is observed in the 25−30 °C temperature range (Figure 7), which contrasts with the coexistence of rigid and fluid EPR signals around the gel−fluid transition of DODAB 39,40 (see the Supporting Information).…”

Cationic Nanostructures for Vaccines Design

“…In order to improve fusion of cell membranes with DODAB bilayers, which are in the rigid gel state at room temperature [ 75 ], DODAB bilayer fluidity had to be increased by using DODAB combinations with other lipids and surfactants such as the glycolipid TDB [ 15 , 76 ], the surfactant monoolein (1-monooleoyl-rac-glycerol) [ 77 ], the C24:1 β-glucosylceramide [ 78 ] or the glycolipid de-O-acylated lipooligosaccharide (dLOS) as a booster vaccine against tuberculosis [ 79 ]. In particular, intranasal immunization with DODAB/TDB combined with influenza antigen A (H3N2) induced superior humoral and cell-mediated responses; there was an effective facilitation of uptake by DC, DC maturation in vitro, increased mucosal IgA production, increased IgG, IgG1, and IgG2b antibody titers in comparison with other formulations using cationic lipids after intranasal administration in vivo [ 80 ].…”