Cubic lipid-water systems

Luzzati V, Tardieu A, Gulik-Krzywicki T, Rivas E and Reiss-Flusson F 1968 Structure of the cubic phases of lipid-water systems Nature 220 485-8... 

In this work we will focus on the use of the cubic phase as a delivery system for oligopeptides - Desmopressin, Lysine Vasopressin, Somatostatin and the Renin inhibitor H214/03. The amino acid sequences of these peptides are given in Table I. The work focuses on the cubic phase as a subcutaneous or intramuscular depot for extended release of peptide drugs, and as a vehicle for peptide uptake in the Gl-tract. Several examples of how the peptide drugs interact with this lipid-water system will be given in terms of phase behaviour, peptide self-diffusion, in vitro and in vivo release kinetics, and the ability of the cubic phase to protect peptides from enzymatic degradation in vitro. Part of this work has been described elsewhere (4-6). 

It should be pointed out that cubic phases, such as the one discussed in this work, frequently occur in lipid-water systems (77), and the concept of using cubic phases as drug vehicles is therefore not limited to the use of monoolein only. From a toxicological stand-point, it is tempting to try to use membrane lipids, such as phospholipids, instead of monoolein for parenteral depot preparations (18-20). 

Figure 6. Cubic structures in lipid-water systems based on space-filling polyhedra. The data from the monoglyceride-water cubic phases fit with the body-centered structure to the right.

Cubic lipid phases have a very much more complex architecture than lamellar and hexagonal phases. Their structural characteristics have been elucidated only very recently, and it has become clear that their subtleties are the key to a variety of biological problems. We will consider those subtleties in some detail. The three fundamental cubic minimal surfaces - the P-surface, the D-surface and the gyroid (or G-surface), introduced in Chapter 1, can all be foimd in cubic lipid-water phases. The lipid bilayer is centred on the surface with the polar heads pointing outwards. Water fills the labyrinth systems on each side of the surface. These cubic phases will be termed Cp, CD and CG/ respectively. It is likely that there are other more complex IPMS morphologies in cubic phases of lipid-water mixtures, as yet uncharacterised. 

Among the various lipids that form cubic phases, monoglycerides have been studied most. Phosphatidylcholines exhibit cubic phases only at very low water content, whereas phosphatidylethanolamines form cubic phases in similar regions of the lipid-water system as monoglycerides e.g. GMO). Lysophospholipids (single-chain lipids) exhibit cubic phases of quite different kinds, liiey will not be discussed here as they are not considered to be relevant in the formation of self-assembled structures of biological... 

When a bicontinuous cubic lipid-water phase is mechanically fragmented in the presence of a liposomal dispersion or of certain micellar solutions e.g. bile salt solution), a dispersion can be formed with high kinetic stability. In the polarising microscope it is sometimes possible to see an outer birefringent layer with radial symmetry (showing an extinction cross like that exhibited by a liposome). However, the core of these structures is isotropic. Such dispersions are formed in ternary systems, in a region where the cubic phase coexists in equilibrium with water and the L(x phase. The dispersion is due to a localisation of the La phase outside cubic particles. The structure has been confirmed by electron microscopy by Landh and Buchheim [15], and is shown in Fig. 5.4. It is natural to term these novel structures "cubosomes". They are an example of supra self-assembly. 

Several details, described by Bassot and coworkers [73, 76], such as the presence of a continuous membrane, and especially its bicontinuity, are consistent with our cubic membrane model. Indeed, similarities between the structure of cubic phases in lipid-water systems (c/. Chapters 4 and 5) and that of the photosome membrane system have been discussed by Bassot and coworkers [77]. These particularly well developed cubic membranes have been exhaustively studied surpassed only by studies of the PLB cubic membrane. 

Most cubic phases in lipid-water systems exhibit unit cell parameters not larger than 20 mn, while the imit cell of cubic membranes is usually larger than 100 nm. Some exceptioi have been apparently found [131, 132] although at this stage such findings should be treated with caution, as the determination of lattice parameters is dependent on the indexing of diffraction patterns, based only on a small niunber of reflections. Further, in lipid-protein-water, lipid-poloxamer-water and lipid-cationic surfactant-water systems, cubic phases with cell parameters of the order of 50 nm have been observed [56,127, 128]. Due to the small number of reports dealing with the... 

It is now well established that proteins can induce phase transitions in lipid membranes, resulting in new structures not found in pure lipid-water systems (c/. section 5.1). However, this property is not peculiar to proteins the same effect can be induced by virtually any amphiphilic molecule. Depending on the structure and nature of proteins, their interactions with lipid bilayers can be manifested in very different ways. We may further assume that the role of proteins in the biogenesis of cubic membranes is analogous to that in condensed systems, and lipids are necessary for the formation of a cubic membrane. This assumption is supported by studies of membrane oxidation, which induce a structure-less proteinaceous mass [113]. However, the existence of a lipid bilayer by itself does not guarantee the formation of a cubic membrane, as proteins may also play an essential role in setting the membrane curvature. In this context, note that the presence of chiral components e.g. proteins) may induce saddle-shaped structures characteristic of cubic membranes. (This feature of chiral packings has been discussed briefly in section 4.14)... 

Luzzati, V., Tardieu, A., Gulik-Krzywicki, T., Rivas, E. and Reiss-Husson, F. (1968) Structure of cubic phase of lipid-water systems. Nature, 220,485. 

The single-gyroid (SG) IMDS with 74i32 (No. 214) symmetry was first discovered in 1967 by Luzzati et al. as a cubic phase occurring in strontium soap surfactants and in pure lipid-water systems [12, 13]. In 1970, Schoen identified the minimal... 

This rod-system structure was considered to be the general structure for cubic lipid-water phases (Luzzati et al., 1968). Thus anhydrous strontium myristate, lecithin and galactolipids from maize chloroplasts with low water content were thought to have the structure shown in Fig. 8.10 with the polar groups arranged in two network systems of rods. The inverse structure, i.e. with hydrocarbon chains forming the rod systems and water forming the outer medium, was proposed to exist in aqueous systems of potassium soaps of lauric, myristic and palmitic acid and in aqueous systems of lauroyl- and palmitoyl-trimethylammonium bromide. 

In 1960, Luzzati et al. [3,4] found the so-called cubic phase between the hexagonal and lamellar phases in a lipid-water system. The cubic structure attributed to space group 1 3 consists of rods of finite length that join three by three to form two three-dimensional networks. 

The phase diagram of such curved surfaces in the amphiphilic system has been studied by Huse and Liebler [8] on the basis of the elastic energy of the surface. The phase behavior is determined by the balance of the surface tension, the bending rigidity and the saddle-splay modulus of the curved interface and the IPMS structure appears when the saddle-splay modulus increased to a some critical value. Mathematically, more than 30 species of curved surfaces have been reported as IPMS [9] and, experimentally, several types of IPMS structures have been found in cubic phases of lipid-water systems [10]. Concerning the formation mechanism of the cubic network, Ranpon and Charvolin [6] found the epitaxial relationships between reticular planes of these three phases in the... 

Another phase which has attracted recent interest is the gyroid phase, a bicontinuous ordered phase with cubic symmetry (space group Ia3d, cf. Fig. 2 (d) [10]). It consists of two interwoven but unconnected bicontinuous networks. The amphiphile sheets have a mean curvature which is close to constant and intermediate between that of the usually neighboring lamellar and hexagonal phases. The gyroid phase was first identified in lipid/ water mixtures [11], and has been found in many related systems since then, among other, in copolymer blends [12]. 

Hyde ST, Andersson S, Ericsson B, Larsson K. A cubic strucmre consisting of a lipid bilayer forming an infinite periodic minimum surface of the gyroid type in the glyceroknonooleate-water system. Zeitschrift fur Kristallographie 1984 168 213-219. 

Cubic phases are also unique in their ability to accommodate proteins as compared to other lipid-water phases. A wide range of globular proteins with molecular weights 5,000-150,000 are known to form cubic phases when mixed with lipids and water. So far few single ternary lipid-protein-water phase diagrams have been completely determined [7], [13] one system that has been looked at is that of monoolein-water-lysoz5one. Protein incorporation results in increased water swelling, and all three phases, Cp, Cd and CG/ occur. The protein molecules are located in the water channel systems and retain their native structure. This has been proved by thermal analysis of the phase, and measurements of enzymatic activity [7]. 

Besides the asymmetry between monolayers in cytomembranes, two of the more obvious differences between cubic phases and membranes are the unit cell size and the water activity. It has been argued that tire latter must control the topology of the cubic membranes [15], and hence tiiat the cubic membrane structures must be of the reversed type (in the accepted nomenclature of equilibrium phase behaviour discussed in Chapters 4 and 5 type II) rather than normal (type I). All known lipid-water and lipid-protein-water systems that exhibit phases in equilibrium with excess water are of the reversed type. Thus, water activity alone cannot determine the topology of cubic membranes. Cubic phases have recently been observed with very high water activity (75-90 wt.%), in mixtures of lipids [127], in lipid-protein systems [56], in lipid-poloxamer systems [128], and in lipid A and similar lipopolysaccharides [129,130]. 

A cubic phase of space group laid (which was the first cubic structure to be solved [161] and is among the most commonly observed [162]). The structure of laid belongs to a body-centered space group of rods (which are essentially a surfactant bilayer with a circular cross section) connected 3 x 3 to generate two interwoven but unconnected 3D networks [162]. A chiral cubic phase of space group PA Il has been observed so far in only one lipid-protein-water system [163]. Its proposed structure is similar to that of laid. It has one water-lipid network interwoven with one network of quasi-spherical inverse micelles that encloses the protein molecules. 

The effect of adding various lipids to monoolein was discussed above. These lipids are soluble in either water (bile salt) or oil (triglyceride) or hardly soluble at all (lecithin). If a substance that is soluble in both water and oil, e.g., propylene glycol, is added to the monoolein-water system, the cubic liquid crystal undergoes a transition to a sponge or L3 phase [13], as shown in Fig. 5. The structure of the sponge phase has been described as a melted bicontinuous cubic phase [14]. 

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