The archaebacterial phospholipids and glycolipids are structurally different from those of bacterial and eukaryotic membranes, being diphytanyl glycerol diether compounds [1]–[4] in which isopranoid chain alcohols are linked to glycerol by ether bonds. In some archaeal microorganisms the phytanyl chains can combine and form biphytanyl chains which link to two glycerols (or to one glycerol and one nonitol) forming tetraether bipolar (bolaform amphiphilic) lipids. While diether lipids assemble in bilayers, tetraether bipolar lipids constitute monolayer membranes. Figure 1 illustrates two basic types of archaeal lipids: diphytanyl glycerol or archaeol and so called caldarchaeol. The ether bond and the almost complete absence of unsaturation in the hydrophobic tail of lipids of archaebacteria are considered adaptive traits of microorganisms which are able to thrive in harsh or extreme environments, such as saturated salt solutions [5], anoxic [5] or highly oxidized [6] conditions and hot waters [5], [7]. A different set of life-fundamental enzymes are involved in archaebacterial lipid biosynthesis [8]. Also, different chirality of the glycerol phosphate moiety [3] protects them against hydrolysis by phospholipases secreted by other organisms [9]. These properties could be of advantage in using archaebacterial lipids in human and veterinary medicine and are therefore a subject of increasing interest.

The role of archaebacterial lipids as vaccine adjuvants [10]–[12] and the possibility to use liposomes constituted of archaebacterial lipids as delivery system of drugs, genes and proteins [7], [13] provides an incentive to study interactions between archaebacterial and eukaryotic lipids, as well as to study the effect of different biologically important molecules on the mediated interactions between membranes composed of different lipid species.

Liposomes prepared from the lipid extracts of archaebacteria (archaeosomes), constituted of mixtures of various polar lipids, have been used in reconstitution studies [14], [15], in the study of the characteristics of membrane permeability [16] and as a delivery system in the immune response to specific antigens [10], [13], [17]. It has been shown that archaeosomes can endure extreme temperatures [18], [19] and resist extreme alkaline-baso-acidic and non-archeal enzyme degradation [19].

Giant lipid vesicles with the dimensions of living cells (GVs) are appropriate for study of properties and interactions of lipids. The advantage of using GVs is that they are large enough to be observed in real time under the phase contrast microscope or fluorescent optical microscope. GVs made of standard lipids of bacterial and eukaryotic membranes (also named in the following as non-archaebacterial lipids) were thoroughly studied experimentally [20]–[29] and theoretically [30]. In studying complex interactions between membranes constituted of standard phospholipids and proteins, it was found that certain plasma proteins mediate attractive interaction between membranes thereby causing close contact between GVs [31], [32], which is an essential step in some biologically important processes such as fusion and fission of vesicles with the mother membrane.

Until now studies involving GVs composed of archaebacterial lipids have mainly considered tetraether bipolar lipids in monolayers. Bagatolli et al. [25] have reported that GVs composed of archaeal tetraether lipids can be formed by the electroformation method, while Cavagnetto et al. [33] studied GVs composed of lipid fractions extracted from the thermophilic archaeobacterium Sulfolobus solfataricus mixed with eukaryotic lipids.

It is of interest to further study GVs composed of archaebacterial lipids and their interactions with the molecules in the surrounding solution. In the present work we have studied the GVs constituted of bilayer-forming archaeal diether phospholipids and glycolipids extracted from halophilic microorganisms inhabiting hypersaline environments, such as coastal salterns and continental salt lakes. We describe the shape and size of GVs constituted of different pure archaebacterial diether phospholipids and glycolipids and of mixtures of archaebacterial and non-archaebacterial lipids, by changing the proportions of membrane constituents. Besides assessing the population of GVs for average size and yield, GVs were used to study the mediating effect of two biologically important molecule species that act as anticoagulants and were previously studied in non-archaebacterial GV systems. These molecules are beta 2 glycoprotein I (β2-GPI), which is commonly found in the pheripheral blood of vertebrates and acts as a cofactor in binding certain antibodies to negatively charged lipids [34], [35], and heparin which is also known for its anti-tumour progression effect [36], [37].

Using the electroformation method we created GVs composed of pure archaebacterial lipids and of different mixtures of archaebacterial and non-archaebacterial lipids.

Previous studies considered lipid extracts composed of all lipid components of the membrane, of phospholipids and phosphoglycolipids (especially bipolar tetraether lipids) [9], [15], [16] and also mixtures of tetraether lipids with standard lipids [33]. Recently, it has been shown that liposomes consisting of diether lipids isolated from hyperthermophilic archaea Aeropyrum pernix have many physicochemical properties similar to those composed of tetraether lipids [48]. Novelty of our work is that the vesicles are giant and that they have been prepared by mixtures of non commercial individual archaeal lipids isolated and purified in our laboratory, including pure negatively charged diether lipids.

It is of interest to compare archaeal and non-archaeal (standard) lipids, when possible. In general, the archaeal analog of standard lipids (archaeal PC versus standard PC (POPC for example)) have the same headgroups but different chains; other examples are archaeal PG versus standard PG and archaeal BPG versus standard BPG (i.e. cardiolipin). Pure glycolipid GVs or glycolipid rich GVs might be important in clinical applications because of their adjuvant properties [10].

We studied the influence of lipid composition on the size and yield of these GVs.

In general, phosphatidyl choline GVs were the largest and most abundant while addition of other species decreased the size and the yield of GVs both, archaeal and non-archaeal systems. Vesicles constituted of aPC were smaller than vesicles constituted of POPC. As the polar heads of aPC and POPC are identical, the effect can be attributed to the lipid tails. It seems that branched chains increase the curvature. The proportions of the areas of headgroups and chains determine the prefered curvature [49]. Larger area pertaining to headgroups with respect to the area pertaining to the chains implies larger curvature, so it is indicated that branched chains are more compactly packed in the bilayer. Low yield of GVs composed of a lipid mixture similar to that present in natural membranes of microorganisms of Halorubrum genus could be a consequence of the absence of salts in the medium, since ions in the medium are important in stabilizing vesicles composed of negatively charged lipids.

BPG was unfavourable for creation of GVs within the given electroformation method. This is in agreement with our attempt to create GVs of cardiolipin within the same electroformation method, which had been unsuccessful previously [50]. We found only singular GVs in the sample obtained from BPG while many crystal-like particles were observed. The question remains whether these singular GVs were actually composed of remnants of other lipids on the electroformation electrodes. According to Israelachvili [49], lipid self-assembly results from the balance of interaction free energy, entropy and molecular geometry which determines local and global shape of a vesicle. The concept of Israelachvili was further elaborated by including orientational ordering of membrane constituents [51], [52]. The flexibility and reorientational mobility of cardiolipin is impaired which favours highly anisotropic membrane curvature and enhances the propensity of cardiolipin to form strongly curved non-lamellar phases [53] (such as inverted hexagonal and cubic phases which can also be present in other lipid systems [54], [55]).

Bagatolli et al. [25] have previously used archaeal tetraether lipids to prepare GVs for the study of configuration of fluorescence probe Laurdan in the membrane composed of the polar lipid fraction E (PLFE) from the thermoacidophilic archaebacteria Sulfolobus acidocaldarius. They have observed spherical vesicles while it was previously found that the majority (95%) of GVs created by this method are unilamellar [20]. In our samples, albeit prepared with the same method, there was a considerable proportion of nonspherical or multilamellar vesicles, some of them enclosing obvious internal structures. Also in our samples GVs appear spherical immediately after the formation. However, we performed experiments the next day. Until used, the suspension with vesicles was left for sedimentation in the gravity field to increase the concentration of GVs at the bottom of the tube. As almost all vesicles have attached remnants of the nanotubular network which is formed in electroformation and torn when GVs are rinsed from the observation chamber, with time, the difference between the areas of the outer and the inner membrane layer decreases and attached nanotubes become thicker and shorter. Eventually, they are integrated in the mother vesicle which becomes flaccid and subject to stronger flickering. As the process continues, invaginations appear and are internalized by the mother vesicle to finally yield globular vesicles with numerous internal structures. This process is common in GVs created by electroformation [56]. As many GVs composed of archaebacterial lipids had internal structures, it is indicated that after the formation they are presented with a large pool of membraneous nanostructures attached to GVs. Also, the portion of GVs with obvious internal structures was on the average larger for smaller GVs and for GVs with larger PC content (not shown).

The mixture of lipids in solvent was applied to the electroformation electrodes manually, which may have resulted in an uneven distribution of lipid in different areas. Further, lipid segregation can occur during the electroformation as lipids of a certain species have preference for a given curvature and respond differently to the AC electric field. A nonuniform lateral distribution of lipid species may contribute to the heterogeneity of GVs in the population.

In determining the GV yield, both, the GVs’ size and their number have to be taken into consideration. Also, there are several layers of GVs, while the focus of the microscope is on the cross section revealing a single layer. Another method to assess the GV yield would be the use of flow cytometry for counting GVs [57].

Different lipids in mixtures used for creation of GVs have different (temperature dependent) solubility properties in the organic solvents used for lipid storage. The electroformation method was performed at room temperature for all the lipid mixtures. To attain a greater yield of GVs composed of lipids with a higher gel to liquid crystalline phase transition temperature, different electroformation temperatures should be considered, however, it should be taken into account that polar lipid membranes of archaea are assumed to be in the liquid crystalline phase over a wide temperature range 0–100°C.

GVs were examined by applying the fluorescent marker NAO which was considered to bind selectively to cardiolipin, but was recently shown to also bind to other archaebacterial lipids [58]. Our observations confirm that binding of NAO to lipids is not cardiolipin-specific.

We have used the differential scanning calorimetry to determine the transition temperature of lipid vesicles prepared from pure aPC and from its non-archeal structural analogue DPPC. DSC was performed on MLVs, since we were not able to produce a sufficient amount of GVs to reach the the required lipid concentration for measurement with DSC. However, the curvature of most membranes in MLVs as well as in GVs can be considered very small and therefore both systems can be considered as equivalent in this respect. The DSC scan of MLVs composed of aPC showed one relativetly low and wide (barely recognisable) peak while the DSC scan of MLVs composed from DPPC showed two well defined peaks corresponding to the main phase transitions and the pretransition (Fig. 8). The observed minuteness of the single peak in aPC may contribute to higher yield of aPC GVs comparing to DPPC by electroformation at the room temperature (data not shown).

The attractive interaction between archaebacterial and non-archaebacterial membranes can be mediated by the same molecules as in eukaryotic membranes so the archaeosomes could approach the cell membrane very closely which is a prerequisite for the interaction and uptake of archaeosomes by the cell to take place. We have studied the effect of two relevant molecules (beta 2 glycoprotein I and heparin) and found that they both act similarly in mediating attractive interaction between archaeal and standard membranes. Since mediated interaction mechanisms are non-specific [32] (they derive from the charge distribution in the membrane surface, the shape of mediating molecules and charge distribution within them [59]–[61] and on the preferential orientation of water molecules near the membrane [62]), we assumed and finally have shown that the mediated interaction between the GVs composed of archaebacterial lipids takes place. The mechanism of the interaction is based on the minimization of the collective free energy of the membrane and of the adjacent solution which depends on the orientational ordering of molecules with internally distributed charge (e.g., protein and water molecules) in spatially varying electric field. In archaeal membranes effects similar to the ones observed in non-archaeal membranes were expected since the headgroups which essentially determine the electric field in the vicinity of the membrane are similar in archaeal phospholipids and in non-archaeal phospholipids. As the fusion of vesicles with target cells is a possible mechanism of drug delivery into the cells, it is of relevance to show that blood proteins can promote adhesion or fusion between vesicles constituted of archaebacterial lipids and of mixtures of archaebacterial and standard lipids. This is especially important since it was found that archaeal liposomes do not fuse easily in conditions which are physiological in vertebrates [63]. Archaeal lipids are in some respect different from non-archaeal ones while in other respects they are similar. This ambivalence could be an advantage, as archaeosomes should be more resistant to enzymes and still be able to interact with the host membrane and perform the delivery. Without the latter, high resistance of archaeosomes would be of no benefit.

Adhesion of phospholipid membranes has been a subject of thorough experimental and theoretical research as it represents an essential step for biologically important processes such as endo- and exo-cytosis and fusion of cells [64], [65]. In particular, these processes are important for the efficiency of drug delivery by liposomes. Experimental studies on lamellar membrane stacks by osmotic stress method, microscopy studies and micropipette aspiration (reviewed in [64], [65]) yielded the adhesion constants between 10⁻³ and 10⁻⁴ J/m², which is much larger than what we have obtained by the interaction mediated by β2-GPI (of the order of 10⁻⁸ J/m²). In contrast, a method which is based on a minimization of the contact area theoretical description of adhesion [66],(7)where 1/R is the contact curvature, yields values in the same range as our results; in these experiments the contact curvatures obtained from micrographs were of the order of 1/10 µm [67], [68]. It is evident that there is large scattering of data on γ obtained by different methods, mostly due to different experimental techniques used, which implies also different experimental systems. In our opinion, systems of lamellar stacks and interactions with non-membranous materials reveal important physical properties of membranes and adhesion, however, they are further from realistic to be used for drug-delivery systems comparing to systems consisting of populations of adhering GVs observed live by the optical microscopy. Further, studying mediated interaction between GVs includes suggesting models of mediated interaction which are basic for manipulating adhesion. Based on the structure of β2 glycoprotein I and its binding to phospholipid membranes [69]–[71], a bridging interaction was suggested as a mechanism of β2 glycoprotein I-mediated adhesion between GVs [59]. The hydrophobic part of the molecule is inserted into the membrane while the other part with a positively charged region is sticking out and can form a bridge with the membrane of another negatively charged vesicle [59]. In the case of heparin, the relevant mechanism is suggested to be orientational ordering of heparin which bears spatially distributed charge [60]–[62]. This interaction is nonspecific and it does not involve chemical binding of molecules. It could be described as an entropic effect. The membranes do not touch, but are driven very closely together. The distance is smaller than a nanometer, and the area of the contact is maximized, so the effect is interpreted as an adhesion. Both, archaeal and standard lipid GVs satisfy the conditions in which such interaction takes place (they form surfaces between which the mediating molecules are confined) and additionally, both may have charged headgroups, or spatially distributed charge on the headgroups, so they are similar in this respect.

We found statistically significant (p = 0.018) correlation (r = 0.598) between the measured adhesion constant γ and the measured average effective angle of contact Y which justifies the use of the effective angle of contact Y in analysis of the effect of mediating molecules on GV adhesion. Namely, the effective angle of contact Y is considerably less time consuming and also more convenient for assessment of a large number of contacts.