Membrane fusion and inverted phases

Abstract

We have found a correlation between liposome fusion kinetics and lipid phase behavior for several inverted phase forming lipids. N-Methylated dioleoylphosphatidylethanolamine (DOPE-Me), or mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), will form an inverted hexagonal phase (HII) at high temperatures (above TH), a lamellar phase (L alpha) at low temperatures, and an isotropic/inverted cubic phase at intermediate temperatures, which is defined by the appearance of narrow isotropic 31P NMR resonances. The phase behavior has been verified by using high-sensitivity DSC, 31P NMR, freeze-fracture electron microscopy, and X-ray diffraction. The temperature range over which the narrow isotropic resonances occur is defined as delta TI, and the range ends at TH. Extruded liposomes (approximately 0.2 microns in diameter) composed of these lipids show fusion and leakage kinetics which are strongly correlated with the temperatures of these phase transitions. At temperatures below delta TI, where the lipid phase is L alpha, there is little or no fusion, i.e., mixing of aqueous contents, or leakage. However, as the temperature reaches delta TI, there is a rapid increase in both fusion and leakage rates. At temperatures above TH, the liposomes show aggregation-dependent lysis, as the rapid formation of HII phase precursors disrupts the membranes. We show that the correspondence between the fusion and leakage kinetics and the observed phase behavior is easily rationalized in terms of a recent kinetic theory of L alpha/inverted phase transitions. In particular, it is likely that membrane fusion and the L alpha/inverted cubic phase transition proceed via a common set of intermembrane intermediates.

On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids

“…Equimolar mixtures of DODAC with POPG and LBPA did not exhibit a well-defined T BH value, as narrow 'isotropic' 31 P NMR spectra were observed between −3°C and 22°C, with H II phase spectra at temperatures above 26°C (data not shown). Such isotropic spectra have been associated with nonbilayer structures such as cubic phases, 45 which basically consist of three-dimensional intercalated networks of lipid tubules or inverted micelles, 46 and, like the H II phase, have been associated with lipid intermediates occurring during membrane fusion. 47 …”

“…45 It would be expected that formation of DOPS-DOTAP ion pairs following addition of DOPS LUV would result in dissociation of the DOTAP from the plasmid. This was tested by assaying the accessibility of the plasmid in the complexes before and after addition of the DOPS LUV, employing the PicoGreen assay described in Materials and methods.…”

Photoinduced Fusion of Liposomes

“…Liposome fusion was correlated with the onset of the isotropic 31 P-NMR resonances which are known to indicate the formation of inverted cubic phases or inverted cubic phase precursors in a variety of lipid systems (Ellens et al, 1986b;Ellens et al, 1989). …”

“…The most common of these nonlamellar phases is the inverted hexagonal (Hn) phase, but various inverted cubic (Qn) phases can also be formed. The presence of nonlamellar lipids has been postulated to be important both for transient formation of non-bilayer structures during membrane fusion (Siegel, 1986b;Ellens et al, 1989), and to endow the membranes with other physical properties crucial to the survival of the cell 31 Gruner, 1985;Gruner, 1989). Nonlamellar lipid phases have been reviewed by a number of authors (Luzzati, 1968;Cullis et aI., 1985;Gruner et al, 1985;Mariani et aI., 1988;Larsson, 1989;Seddon, 1990;Tate et al, 1991 The various phases are usually designated by the nomenclature of Luzzati (Luzzati, 1968 The lipid region is of fixed and limited thickness, whereas the water region is not.…”

“…anionic liposomes in the presence of multivalent ions Ohki et al, 1982;Bentz & Duzgunes, 1985;CarmonaRibeiro et al, 1985;Bentz et al, 1988); liposomes containing polymorphic lipids, such as phosphatidylethanolamine (PE) 1, at temperatures near TH 82 (Ellens et al, 1985;Ellens et al, 1986a;Ellens et al, 1986b;Ellens et aI., 1987;Ellens et al, 1989;; neutralliposomes in the presence of dehydrating agents such as PEG (Parente & Lentz, 1986;Lentz et al, 1992;Massenburg & Lentz, 1993); so-called pH-sensitive liposomes at low pH or in the presence of multivalent cations (Connor et al, 1984;Ellens et al, 1984;Duzgunes et al, 1985;Nayar & Schroit, 1985;Straubinger et al, 1985;Leventis et al, 1987;Collins et al, 1989); formation of diacylglycerol (DAG) by reaction of phospholipase C with PC-containing liposomes (Nieva et aI., 1989;Luk et aI., 1993); and cationic liposomes in the presence of anionic liposomes (FeIgner et al, 1987;Stamatatos et al, 1988;Leventis & Silvius, 1990 In all cases, membrane destabilization most likely occurs only after interbilayer contact. The prerequisite of membrane contact to the destabilization of anionic membranes in the presence of divalent cations was demonstrated by a number of workers (Liao & Pestegard, 1980b;Liao & Pestegard, 1980a;Wilschut et al, 1980;Nir et al, 1983a).…”