Georgette Madelmont

Differential scanning calorimetry studies of hydration forces with phospholipid multilamellar systems

Abstract True differential calorimetry using a DSC device was used to study the thermal behavior of two hydrated phospholipids: DPPC or dipalmitoyl phosphatidyl choline and PI phosphatidylinositolmonophosphate. The amount of water varied between 15% (w/w) and 60% (w/w). The relevance of ice-melting thermograms to the surface forces emerging from the lipid bilayers and affecting the structural water molecules which constitute the aqueous separation (between bilayers) is demonstrated. The melting of the frozen structural water is not cooperative. For fully hydrated multibilayers, the corresponding molar enthalpy of fusion L is less than 50% of the molar enthalpy for bulk ice melting. In the present report, the difference in the aspects of the thermograms obtained with either the fluid PI or the rigid DPPC bilayers is assigned to a significant thermal expansion of the fluid PI bilayers.

Cooperativity of an acid phospholipid reaction with basic hydrophobic polyelectrolytes. III: Phospholipid-polyion electrostatic interaction and mesophase hydration

Abstract DSC thermograms have been established for two fully hydrated (40 and 50% water w/w) mixtures of a hydrophobic polyelectrolyte and respectively either one acidic or one amphoteric phospholipid. The temperature range of the heating scans, −65 to 50°C, allowed the study of ice melting and of phospholipids gel to liquid-crystal transitions. The phospholipids are wheat phosphatidylinositolmonophosphate PI and synthetic DPPC dipalmitoylphosphatidylcholine. The macromolecular constituent is the polysoap poly(2-methyl-5-vinylhexyl-pyridinium bromide) or PVPC6. Increasing amounts of PVPC6 affect only slightly the transition temperature for both phospholipids although PI is acidic while DPPC is amphoteric. This temperature is 22 to 23°C below 0°C for PI. The effects of PVPC6 on the enthalpy of transition ΔHt for PI and for DPPC are different. For PI, ΔHt decreases sharply in the range of phospholipid mole fraction 0.5-0.6 (cooperativity of PI binding, L. Ter-Minassian-Saraga,J. Colloid Interface Sci.70, 245 (1979 reference 16). For DPPC, it is minimum at x = 0.5. The analysis of ice melting thermograms is original. It provides three classes of water molecules which vary with the ratio PVPC6/phospholipid: bulk water melting at 0°C, supercooled water melting below 0°C, and nonmelting (non-freezing) water of hydration. The last class of water molecules is considerably reduced (dehydration) by the electrostatic interaction between acidic PI and basic PVPC6. It is considerably enhanced by the interpenetration between the amphoteric DPPC and PVPC6 molecules. Pure PI and DPPC do not display significant specificity toward water binding. An interpretation is proposed for the shape of the ice-melting peaks obtained for lamellar systems. It is based on the hypothesis that the free energy of water inside the aqueous separation between the bilayers is lower than in bulk water on account of molecular interactions water-bilayer. The interpretation is satisfactory for the amphoteric DPPC pure or mixed with PVPC6. The relevance of the present results to “condensation” in mixed monolayers and to biological systems is discussed.

Cholesterol-induced modulation of membrane hydration studies by thermal analysis

Incorporation of cholesterol Ch into dipalmitoylphosphatidyl choline DPPC multibilayers induces disorder in the low temperature gel state and order in the liquid crystalline state as shown by IR spectroscopy [ 11. Differential scanning calorimetry DSC [24] and X-ray diffraction studies [ 1,5] of fullyhydrated, mixed DPPC t Ch bilayers show that their behaviour is complex at low Ch content. At low Ch content and temperature the system is biphasic [5]. One of the phases is DPPC in the gel state. The second displays a 3 5 mn thick aqueous spacing broader than the 1.9 mn thick aqueous spacing of the DPPChydrated gel phase. Ch and DPPC were purchased from Fluka and Merck, respectively and were used as received. Samples were prepared from stock solutions in chloroform/ methanol, 9/l (v/v). The solvents were of spectroscopic grade purity. Ch and DPPC solutions were deposited inside the DSC cups and the solvent evaporated in vacuum and dark. The dry sample composition is expressed as mol fraction of DPPC, xDPPC. It weighed -0.5 mg + 0.005 mg. An equal amount of water was added. The cup was sealed and incubated at 60°C for 32 h. Leakage of water out of the cups was controlled by weighing. The samples were stocked at -18°C. We have used thermal analysis [6,7] (DSC differential scanning calorimetry) to measure directly the amount of water forming the interbilayer aqueous spacing in a multibilayer system. We apply this technique to the systems studied by other techniques such as the X-ray diffraction technique [5] and various dynamic techniques [8]. The agreement between our results and those in [5,8] are better than qualitative . Cholesterol modulates membrane antigenity expression by affecting the membrane microviscosity [9] and may be involved in modulating the ionic channels in reconstituted acetyl choline receptors vesicles [lo]. We have shown [7] that bilayer fluidity and hydration modulation are correlated properties. Furthermore, membrane-membrane interaction, adhesion and fusion may be modulated by its state of hydration. We wonder whether membrane antigenity is not related to both membrane fluidity and to state of hydration, which are correlated membrane properties. The thermograms (fig.1) were obtained with a 990-910 Du Pont de Nemours Thermal Analyzer equipped with a mechanical cooling accessory. Heating-cooling cycles were performed at 2°C min-’ between -65 and 60°C. In general the sensitivities used were 48 wal . s-l. cm-’ or 96 peal . s-l. cm-‘. The areas of the peaks were measured witha planimeter and converted into heat amounts. The values for the molar enthalpy for the gel-liquid crystal transition of DPCC were obtained by dividing the heat of the high temperature endothermic transition (fig.1 b) by the amount of DPPC only. The temperatures at the peaks maxima are shown in fig.2 for the various systems. The endothermic low temperature (<O”C) peaks and the ‘exothermic’ peaks at 0°C (fig.la) correspond to frozen water melting inside the sample and inside the reference cups, respectively. A reference thermogram [6,7] (not shown) established with pure water in the reference cup and an empty cup on the sample holder calibrates the apparatus. The interpretation of these peaks has been given elsewhere [6,7]. It is based

Vinblastine and vincristine action on gel-fluid transition of hydrated DPPC

Abstract The effect of two anti-cancer agents, vinblastine sulphate (VLBS) and vincristine sulphate (VCRS), on the gel-liquid crystal transition of fully hydrated dipalmitoylphosphatidylcholine (DPPC) has been studied by differential scanning calorimetry (DSC). DSC diagrams were established for various mixtures of DPPC + agent and a fixed (50%) amount of water. It is concluded that VLBS perturbs the hydrated DPPC structure more strongly than VCRS. This conclusion confirms the idea proposed by Hort-Legrand and Metral [5] that these anti-mitotic drugs may also affect the functioning of cell membranes.

Interaction of vinblastine sulfate with artificial phospholipid membranes: A study by differential scanning calorimetry and spin labeling

The effect of the antimitotic drug vinblastine sulfate has been studied on fully hydrated dipalmitoylphosphatidylcholine (DPPC) liposomes in the temperature range 0 degrees to 60 degrees using differential scanning calorimetry and electron spin resonance spectroscopy with two fatty acid spin labels. In the gel phase, vinblastine interacts essentially with the DPPC polar heads and induces an important disorganization of the phospholipidic bilayer. The co-operativity of the main thermal transition is decreased. In the crystal-liquid phase, the drug penetrates inside the artificial membrane and induces the formation of domains which increased thermal stability. These effects are opposite to those observed with the drug isaxonine which is used to reduce the axonal degenerating effects due to vinblastine.

The binding of the radioprotective agent cysteamine with the phospholipidic membrane headgroup-interface region

The interaction of the aminothiol radioprotector cysteamine (beta-mercaptoethylamine) (CYST) with dipalmitoylphosphatidylcholine (DPPC) artificial membranes has been studied by differential scanning calorimetry (DSC), turbidimetry and spin labeling. This hydrophilic molecule displays a biphasic, concentration-dependent binding to the phospholipidic head groups at neutral pH. In the CYST/DPPC molar ratio 1:160-1:2 (mole/mole) an increasing ordering effect is observed. At high concentrations (over 3:1 ratio), this ordering effect decreases. With the symmetric disulfide dimer cystamine, the biphasic effect is not shown and the membrane rigidity decrease is obtained only at concentration ratio higher than 1:1. The charge repartition of the cysteamine molecule has been shown to be disymmetric, +0.52 e on the NH3 group and +0.19 e on the SH extremity, [38] whereas the cystamine molecule is electrostatically symmetrical. These properties could be related to their membrane effects. With cysteamine, at a low concentration, an electrostatic bridging between the negatively charged phosphate groups of the polar heads induces the increase in membrane stability: the molecules behave like a divalent cation. At high concentrations a displacement of the slightly charged SH extremity by the amine disrupts the bridges and induces the decrease in rigidity: the drug behaves like a monovalent cation. Due to its symmetric charge and its double length, such an effect is not observed with cystamine. This study could bring further information about the interactions between cysteamine and polyelectrolytic structures (ADN for example) and about the radioprotective properties of this drug.

Isaxonine base is a strong perturber of phospholipid bilayer order and fluidity—A differential scanning calorimetry and spin labeling study

The effects of the neurotropic drug isaxonine on fully hydrated dipalmitoyl-phosphatidyl-choline (DPPC) bilayers has been studied in the temperature range 0 degree-60 degrees, using differential scanning calorimetry and electron spin resonance spectroscopy, with two stearic acid spin labels. At low concentration (1% mol/mol), isaxonine is trapped in the polar interface and enhances the phospholipid multibilayers organization in the gel state. In contrast, at high concentration (30% mol/mol), the drug disorganizes the phospholipidic structures and may induce domain formation by phase separation. The strong interactions of isaxonine at the lipid-water interface change the ionization state of the stearic acid spin labels which become totally ionized. Then isaxonine acts as a modifier of the surface pH of the bilayer. The strong membrane effects of isaxonine may explain in part its pharmacological properties in vivo.