Lipid bicontinuous cubic phases have attracted enormous interest as bio-compatible scaffolds for use in a wide range of applications including membrane protein crystallisation, drug delivery and biosensing. One of the major bottlenecks that has hindered exploitation of these structures is an inability to create targeted highly swollen bicontinuous cubic structures with large and tunable pore sizes. In contrast, cubic structures found in vivo have periodicities approaching the micron scale. We have been able to engineer and control highly swollen bicontinuous cubic phases of spacegroup Im3m containing only lipids by (a) increasing the bilayer stiffness by adding cholesterol and (b) inducing electrostatic repulsion across the water channels by addition of anionic lipids to monoolein. By controlling the composition of the ternary mixtures we have been able to achieve lattice parameters up to 470 Å, which is 5 times that observed in pure monoolein and nearly twice the size of any lipidic cubic phase reported previously. These lattice parameters significantly exceed the predicted maximum swelling for bicontinuous cubic lipid structures, which suggest that thermal fluctuations should destroy such phases for lattice parameters larger than 300 Å.

Bicontinuous cubic structures offer enormous potential in applications ranging from protein crystallisation to drug delivery systems and have been observed in cellular membrane structures. One of the current bottlenecks in understanding and exploiting these structures is that cubic scaffolds produced in vitro are considerably smaller in size than those observed in biological systems, differing by almost an order of magnitude in some cases. We have addressed this technological bottleneck and developed a methodology capable of manufacturing highly swollen bicontinuous cubic membranes with length scales approaching those seen in vivo. Crucially, these cubic systems do not require the presence of proteins. We have generated highly swollen Im3m symmetry bicontinuous cubic phases with lattice parameters of up to 480 Å, composed of ternary mixtures of monoolein, cholesterol and negatively charged lipid (DOPS or DOPG) and we have been able to tune their lattice parameters. The swollen cubic phases are highly sensitive to both temperature and pressure; these structural changes are likely to be controlled by a fine balance between lipid headgroup repulsions and lateral pressure in the hydrocarbon chain region.

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Lipids are ubiquitous in nature; together with proteins they constitute the plasma membranes of cells, and take part in a plethora of biological processes, ranging from cell signalling 1 and actin assembly 2 through to endo-and exo-cytosis 3 . More recently, lipids and other amphiphiles have been used as vehicles for drug delivery 4 . Understanding lipid polymorphism is the key to understanding biochemical control at membranes and to the development of intracellular delivery systems.When mixed with a polar solvent, amphiphiles self-assemble into fluid interfacial structures where polar headgroups at the interface shield hydrocarbon chains from contact with the solvent. Most biological amphiphiles form 'inverse' liquid-crystalline phases where the interface curves towards the polar region. Conversely, most simple surfactants form 'normal' phases, where the interface bends away from the polar solvent and towards the chain region. Those lipids with a tendency for curvature play important roles in regulating the dynamics and function of membrane proteins, and are essential for enabling lipid based drug/gene delivery systems to fuse with the cell membrane. Those lipids with the greatest tendency for inverse interfacial curvature form spherical structural elements, where a core of water is surrounded by a monolayer of lipid. In some cases such inverse micelles are found to self-assemble into a close packing of cubic symmetry. Although only one such packing, of space group Fd3m, has so far been identified 5 , we have sought to find different inverse micellar packings, of primitive, body-centred or facecentred cubic symmetry, mirroring the more diverse range of normal micellar cubic phases found in surfactant systems 6,7 . Here we report only the second liquid crystalline structure based on a close packing of inverse micelles: a 3-D hexagonal inverse micellar phase, of space group P6 3 /mmc.Seddon et al. 8 previously reported the formation of an Fd3m phase in excess water for a mixture of two typical lipids, dioleoylphosphatidylcholine (DOPC) and dioleoylglycerol (DOG). They also observed a different phase at lower water concentrations, but were unable to give an unambiguous phase determination, although electrical conductivity measurements and freeze-fracture electron microscopy suggested that this phase also consisted of packed inverse micelles. The Fd3m cubic phase was also found in a mixture of egg phosphatidylcholine, egg phosphatidylethanolamine, 1,2-diacylglycerol (derived from egg phosphatidylcholine) and cholesterol, at 1,2-diacylglycerol concentrations in excess of 40 mol% 9 . For the present study, we prepared ternary lipid mixtures comprising DOPC, DOG and cholesterol, of molar ratios 1:2:1 and 1:2:2, in excess water. Small-angle X-ray scattering (SAXS) experiments were carried out at the European Synchrotron Radiation Facility, Grenoble, France. Detailed information regarding the experimental procedure can be found in the Supporting Information.The phase behaviour of both ternary mixtures was found to be v...