Low amounts of PEG-lipid induce cubic phase in phosphatidylethanolamine dispersions

AbstractBy using time-resolved X-ray diffraction we demonstrate that low amounts (5–10 mol%) of a phospholipid with two saturated hydrocarbon acyl chains 14 carbon atoms long and PEG550 chain covalently attached to its phosphoethanolamine polar head group, DMPE(PEG550), induce spontaneous formation of a cubic phase with lattice constant 20.5 nm (cubic aspect #8, space group Im3m) in aqueous dispersions of dielaidoylphosphatidylethanolamine (DEPE). This phase displays a highly resolved X-ray diffraction pattern with 17 low-angle reflections. The cubic phase was found to intrude in the temperature range between the lamellar liquid crystalline (Lα) phase and the inverted hexagonal phase (HII) known to form in pure DEPE/water dispersions. A higher DMPE(PEG550) amount of 20 mol% was found to eliminate the non-lamellar phases in the temperature scale up to 100°C. DMPE grafted with PEG5000 only shifts the Lα-HII transition of DEPE to higher temperatures but does not promote formation of cubic phase. These findings indicate that, consistent with their bulky head groups, the PEG-lipids decrease the tendency for negative interfacial mean curvature of the DEPE bilayers

New phases induced by sucrose in saturated phosphatidylethanolamines: an expanded lamellar gel phase and a cubic phase

AbstractA new lamellar gel phase (Lβ) with expanded lamellar period was found at low temperatures in dihexadecylphosphatidylethanolamine (DHPE) and dipalmitoylphosphatidylethanolamine (DPPE) dispersions in concentrated sucrose solutions (1–2.4 M). It forms via a cooperative, relatively broad transition upon cooling of the Lβ gel phase of these lipids. According to the X-ray data, the transformation between Lβ and Lβ is reversible, with a temperature hysteresis of 6–10°C and a transition width of about 10°C. No specific volume changes and a very small heat absorption of about 0.05 kcal/mol accompany this transition. The Lβ−Lβ transition temperature strongly depends on the disaccharide concentration. From a value of about 10°C below the melting transition of DHPE, it drops by 25°C with decrease of sucrose concentration from 2.4 M to 1 M. The low-temperature gel phase Lβ has a repeat spacing by 8–10 Å larger than that of the Lβ gel phase and a single symmetric 4.2 Å wide-angle peak. It has been observed in 1. 1.25, 1.5 and 2.4 M solutions of sucrose, but not in 0.5 M of sucrose. The data clearly indicate that the expanded lamellar period of the Lβ phase results from a cooperative, reversible with the temperature, increase of the interlamellar space of the Lβ gel phase. Other sugars (trehalose, maltose, fructose, glucose) induce similar expanded low-temperature gel phases in DHPE and DPPE. The Lβ phase is osmotically insensitive. Its lamellar period does not depend on the sucrose concentration, while the lattice spacings of the Lα, Lβ and HH phases decrease linearly with increase of sucrose concentration. Another notable sugar effect is the induction of a cubic phase in these lipids. It forms during the reverse HH−Lα phase transition and coexists with the Lα phase in the whole temperature range between the HH and Lβ phases. The cubic phase has only been observed at sucrose concentrations of 1 M and above. In accordance with previous data, sucrose suppresses the Lα phase in both lipids and brings about a direct Lβ−HH phase transition in DHPE. A rapid, reversible gel-subgel transformation takes place at about 17°C in both DPPE and DHPE. Its properties do not depend on the sucrose concentration. The observed new effects of disaccharides on the properties of lipid dispersions might be relevant to their action as natural protectants

Calorimetric monitoring data of the evolution of the lamellar-inverted hexagonal phase transition in phosphatidylethanolamine dispersions upon temperature cycling

The data presented in this article are related to the research article entitled ''Cubic phases in phosphatidylethanolamine dispersions: formation, stability and phase transitions'' (Tenchov and Koynova, 2017) [1]. This article presents thermodynamic data obtained by differential scanning calorimetry following the evolution of the Lα - HII endotherm upon temperature cycling during the lamellar to cubic phase conversion

Cationic O-ethylphosphatidylcholines and their lipoplexes: phase behavior aspects, structural organization and morphology

AbstractEthylphosphatidylcholines, positively charged membrane lipid derivatives in which the anionic charge of the phosphate oxygen has been eliminated by ethylation, are promising nonviral, metabolizable transfection agents. We studied in detail the phase behavior, structural organization and morphology of the ethylphosphatidylcholines and their lipoplexes. Unlike the other phospholipids, dehydration does not change the melting transition temperature of O-ethyl-dipalmitoylphosphatidylcholine (EDPPC). Neither does an isoelectric amount of DNA, when added to the EDPPC aqueous dispersion. This is ascribed to the inability of EDPPC to form hydrogen bonds because of its headgroup modification. Similarly to its parent lipid DPPC, EDPPC displays a subtransition at 15 °C in its differential scanning calorimetry (DSC) heating scans after prolonged low-temperature incubation. The cooling behavior of the O-ethylphosphatidylcholines is sensitive to the thermal prehistory and the ionic strength. Different aggregate morphologies in the solid and the liquid-crystalline phases—respectively lamellar sheets and vesicles, as documented by light microscopy—are considered responsible for the cooling pattern. The interconversion between these morphologies is slow or even kinetically hindered, however, increasing the ionic strength to physiological values facilitates the conversion. The interdigitated chain arrangement of EDPPC gel phase tolerates incorporation of DNA between the bilayers. The minimum observed separation between the DNA strands is ∼30–32 Å, at DNA/lipid molar ratio ≥1. Formation of lipoplexes with DNA ordered in a 1-D lattice sandwiched between interdigitated lipid bilayers is reported for the first time

Mixing behavior of saturated short-chain phosphatidylcholines and fatty acids

Phase diagrams of the hydrated pseudo-binary mixtures dilauroylphosphatidylcholine (DLPC)/lauric acid (LA) and dimyristoylphosphatidylcholine (DMPC)/myristic acid (MA) have been constructed using high-sensitivity DSC and time-resolved X-ray diffraction. They are of a different type compared to those of the longer chain phosphatidylcholine (PC)/fatty acid (FA) mixtures, the latter being of maximum azeotropic point type. Eutectic points were distinguished in the phase diagrams, at 75 mol% MA and 49°C for the DMPC/MA mixture, and at ca. 67 mol% LA and 29°C for the DLPC/LA mixture. Regions of liquid–liquid and solid–solid phase separation have been located according to the shape of the phase diagrams and observed by X-ray diffraction. Limited regions (2–4°C) of liquid–liquid phase immiscibility exist at compositions with slightly prevailing fatty acid molar content. For instance, at 67 mol% MA, a phase separation between Lα phase enriched in DMPC and HII phase enriched in MA takes place in the temperature range 51–55°C. Solid phase immiscibility is detected between 60 and 90 mol% fatty acid. The studied PC/FA mixtures form compound subgel polymorphic phases (one in the DMPC/MA mixture, with DMPC/MA 1:2 molar stoichiometry, and two in the DLPC/LA mixture, with about 40 and 60 mol% LA, respectively) upon low-temperature equilibration. In the liquid crystalline phase region, non-lamellar phases dominate the phase diagrams, especially in their fatty acid-rich part. With increasing FA content, the nonlamellar phases arrange in the sequence: bicontinuous cubic phases (Ia3d, Pn3m, Im3m)→hexagonal phase (HII)→micellar cubic phase (Fd3m)→isotropic phase (I). With the eutectic composition (75 mol% MA), only HII phase is detected above the melting transition in the DMPC/MA mixture. At higher FA content (85 mol%), micellar cubic phase of space group Fd3m form in the two PC/FA mixtures. At lower FA content (<75 mol%), at least three cubic phases (Ia3d, Pn3m, Im3m) form. The order of their appearance with increasing temperature varies with the PC/FA ratio. They exist either as single phases or concurrently to the HII phase

An ordered metastable phase in hydrated phosphatidylethanolamine: the Y-transition

By using time-resolved X-ray diffraction, differential scanning calorimetry and scanning densitometry, we observed rapid formation at low temperature of a metastable ordered phase, termed LR1 phase, in fully hydrated dihexadecylphosphatidylethanolamine (DHPE). The LR1 phase has the same lamellar repeat period as the gel Lβ phase but differs from the latter in its more ordered, orthorhombic hydrocarbon chain arrangement. It forms at about 12°C upon cooling and manifests itself as splitting of the sharp, symmetric wide-angle X-ray peak of the DHPE gel phase into two reflections. This transition, designated the ‘Y-transition’, is readily reversible and proceeds with almost no hysteresis between cooling and heating scans. Calorimetrically, the LR1→Lβ transition is recorded as a low-enthalpy (0.2 kcal/mol) endothermic event. The formation of the LR1 phase from the gel phase is associated with a small, about 2 μl/g, decrease of the lipid partial specific volume recorded by scanning densitometry, in agreement with a volume calculation based on the X-ray data. The formation of the equilibrium Lc phase was found to take place from within the LR1 phase. This appears to be the only observable pathway for crystallisation of DHPE upon low-temperature incubation. Once formed, the Lc phase of this lipid converts directly into Lβ phase at 50°C, skipping the LR1 phase. Thus, the LR1 phase of DHPE can only be entered by cooling of the gel Lβ phase. The data disclose certain similarities between the low-temperature polymorphism of DHPE and that of long-chain normal alkanes

Time-resolved x-ray diffraction and calorimetric studies at low scan rates: II. On the fine structure of the phase transitions in hydrated dipalmitoylphosphatidylethanolamine

The phase transitions of dipalmitoylphosphatidylethanolamine (DPPE) in excess water have been examined by low-angle time-resolved x-ray diffraction and calorimetry at low scan rates. The lamellar subgel/lamellar liquid-crystalline (L(c) → L(α)), lamellar gel/lamellar liquid-crystalline (L(β) → L(α)), and lamellar liquid-crystalline/lamellar gel (L(α) → L(β)) phase transitions proceed via coexistence of the initial and final phases with no detectable intermediates at scan rates 0.1 and 0.5°C/min. At constant temperature within the region of the L(β) → L(α) transition the ratio of the two coexisting phases was found to be stable for over 30 min. The state of stable phase coexistence was preceded by a 150-s relaxation taking place at constant temperature after termination of the heating scan in the transition region. While no intermediate structures were present in the coexistence region, a well reproducible multipeak pattern, with at least four prominent heat capacity peaks separated in temperature by 0.4-0.5°C, has been observed in the cooling transition (L(α) → L(β)) by calorimetry. The multipeak pattern became distinct with an increase of incubation time in the liquid-crystalline phase. It was also clearly resolved in the x-ray diffraction intensity versus temperature plots recorded at slow cooling rates. These data suggest that the equilibrium state of the L(α) phase of hydrated DPPE is represented by a mixture of domains that differ in thermal behavior, but cannot be distinguished structurally by x-ray scattering

Transfection Activity of Binary Mixtures of Cationic O-Substituted Phosphatidylcholine Derivatives: The Hydrophobic Core Strongly Modulates Physical Properties and DNA Delivery Efficacy

A combination of two cationic lipid derivatives having the same headgroup but tails of different chain lengths has been shown to have considerably different transfection activity than do the separate molecules. Such findings point to the importance of investigating the hydrophobic portions of cationic amphiphiles. Hence, we have synthesized a variety of cationic phosphatidylcholines with unusual hydrophobic moieties and have evaluated their transfection activity and that of their mixtures with the original molecule of this class, dioleoyl-O-ethylphosphatidylcholine (EDOPC). Four distinct relationships between transfection activity and composition of the mixture (plotted as percent of the new compound added to EDOPC) were found, namely: with a maximum or minimum; with a proportional change; or with essentially no change. Relevant physical properties of the lipoplexes were also examined; specifically, membrane fusion (by fluorescence resonance energy transfer between cationic and anionic lipids) and DNA unbinding (measured as accessibility of DNA to ethidium bromide by electrophoresis and by fluorescence resonance energy transfer between DNA and cationic lipid), both after the addition of negatively charged membrane lipids. Fusibility increased with increasing content of second cationic lipid, regardless of the transfection pattern. However, the extent of DNA unbinding after addition of negatively charged membrane lipids did correlate with extent of transfection. The phase behavior of cationic lipids per se as well as that of their mixtures with membrane lipids revealed structural differences that may account for and support the hypothesis that a membrane lipid-triggered, lamellar→nonlamellar phase transition that facilitates DNA release is critical to efficient transfection by cationic lipids