Hexagonal phase with ordered acyl chains formed by a short chain asymmetric ceramide

Highlights

• We examined the structured of an asymmetric ceramide.

• A sequence of SAXS diffraction peaks in ratios for hexagonal phase was found.

• The acyl chains according to WAXS and FTIR are in predominant all trans conformation.

• The topology corresponds to an inverted (water in oil) micelle.

• We speculate that the asymmetry of the acyl chains stabilize the structure.

Abstract

Ceramides constitute a group of lipids with usually high melting temperature that also favor negative curvature in membranes when mixed with other lipids. The short chain C10:0 ceramide is an asymmetric lipid which consists of an 18 carbon sphingosine base N-acylated with decanoic acid. According to high sensitivity differential scanning calorimetry, it shows a minor exothermic peak at 61 °C and a main endothermic transition at 75 °C. By small angle X-ray scattering and polarized light microscopy we found that, at temperatures below the main transition, the fully hydrated lipid dispersions are arranged in a tridimensional structure corresponding to an inverted hexagonal phase. Infrared spectroscopy and wide angle X-ray diffraction indicated that the acyl chains of ceramides exhibit a relatively high order in the hexagonal phase. As far as we know, this is the first report of a lipid hexagonal phase having highly ordered acyl chains. Molecular asymmetry due to the different length of the sphingosine and the N-acyl chains of C10:0 ceramide may explain why this novel phase is formed.

Introduction

Sphingolipids comprise a vast family of members composed by a long chain aminediol base N-linked to a fatty acid through an amide bond. The base most usually found is the 18 carbon sphingosine, with a trans double bond at position 4–5, (see Fig. 1), which is itself a bioactive lipid; however other variants with shorter or longer hydrocarbon chains, different hydroxylation level and saturation can be found [1], [2], [3], [4], [5], [6]. In addition, the N-acyl chain can also vary in length (ten in the present case, see Fig. 1), hydroxylation and unsaturation level, although long saturated fatty acyl residues are more often encountered in sphingolipids than in glycerol-based lipids; such variations transduce into largely different molecular packing and phase state [7].

In addition, a great variety of polar groups attached to hydroxyl group at position C1 give rise to the different families of sphingolipids: ceramides, with a single hydroxyl group; sphingomyelins, with phosphorylcholine; cerebrosides, with different sugars; gangliosides, with sugars and sialic acid residues [8]; among others.

The chemical variety of the sphingolipids is responsible for the large structural polymorphism observed due to the intrinsic natural curvature that the polar head group, in relation to the hydrocarbon moiety, imposes to the different lipid families [9]. In this regard, while gangliosides with a complex and charged head group self-assemble as micellar structures of different shapes and size [10], [11], sphingomyelins and cerebrosides form stable bilayers [3]. In the case of ceramides, it has been reported that natural extracts of bovine brain and N-palmitoyl-ceramide can form lamellar stackings [12], a feature also induced by other complex glycosphingolipids and some of their mixtures with phosphatidylcholine [13]. However, when mixed with inverted phase-forming lipids such as phosphatidylethanolamine, it was shown that ceramides N-acylated with fatty acids equal or longer than 8 carbons decreased the temperature of lamellar Lα to hexagonal II transition [14], suggesting that this ceramide stabilized the negative curvature of membranes. On the other hand, when mixed with dipalmitoyl phosphatidylcholine, an increase of the main transition temperature and reduction of bilayer thickness due to chain interdigitation is induced by egg-Ceramide [15].

Ceramides also display the highest melting transition temperatures described in lipid systems [16], [17], [18], owing to tight molecular packing, high Van der Waals interactions and subsequent hydrogen bonding [19]. In bilayer and monolayer membrane model systems, ceramides form highly ordered, solid phases [19], [20], [21] and, when mixed with either glycerophospholipids or sphingolipids, induce phase segregation of condensed domains and complex thermograms [18], [22], [23], [24]. However it should be pointed out that even the condensed C16:0 ceramide can undergo liquid-expanded to condensed transitions depending on temperature, besides other structural transitions in the condensed state [21]. Also, when the N-acyl chain length of ceramides is shortened to less than 14 carbons, expanded phase and expanded to condensed monolayer phase transition at room temperature have also been found in Langmuir monolayers [21].

In the present work, we studied the phase behavior of an asymmetric ceramide N-acylated with the 10 carbons long decanoic acid, C10:0 ceramide, when dispersed in excess water in bulk. Previous studies [21] have shown C10:0 ceramide is able to form stable monolayers with an expanded to condensed transition at 25 mN m⁻¹ at 24 °C. Combining differential scanning calorimetry (DSC), small angle X-ray scattering (SAXS), polarized light microscopy (PLM), wide angle X-ray scattering (WAXS) and Fourier transform infrared spectroscopy (FTIR) we could ascertain the presence of a novel lipid phase below the main transition that consists of an inverted hexagonal phase HII in which the acyl chains of C10:0 ceramide are highly ordered. As far as we know, this is the first report of a curved-tubular (hexagonal) phase with ordered acyl chains in pure lipid systems.