Pierre Tancrède

The effect of aggregation state of amphotericin-B on its interactions with cholesterol- or ergosterol-containing phosphatidylcholine monolayers

Amphotericin B (AmB) is the most effective antibiotic used in the treatment of systemic fungal infections. It is generally thought that the activity of this drug results from its interaction with ergosterol, the main sterol of fungi membranes. However, AmB also interacts with cholesterol, the major sterol of mammal membranes, thus limiting the usefulness of this drug due to its relatively high toxicity. The aim of the present work is to study the molecular basis of the interactions of AmB with these sterols contained in a DOPC film by using the monolayer technique. Two different concentrations of the sterols in the film (13 and 30%) at an initial surface pressure of 30 mN/m were studied, which correspond to conditions found in various biological membranes. Four concentrations of AmB in the subphase, ranging from a molecularly dispersed to a highly aggregated state of the drug were studied. Our results show that the monomeric form of AmB interacts with the ergosterol containing film solely. On the other hand, when AmB is dispersed as a pre-micellar or as a highly aggregated state in the subphase, a very significant selectivity of its interactions between the two sterols is observed which is shown in our experimental results by a difference of 8 mN/m in the surface pressure when AmB interacts with ergosterol as compared to cholesterol. We show that the activity of AmB is most likely related to the micellar form of the antibiotic. In addition, we observe that upon increasing the amount of ergosterol in the film, the insertion of AmB is largely promoted, results that are discussed in terms of the molecular organization of the sterols within the monolayer film. We show that these results provide a better understanding of the action of AmB (activity/toxicity) at the membrane level.

The structuring effects of amphotericin B on pure and ergosterol- or cholesterol-containing dipalmitoylphosphatidylcholine bilayers: a differential scanning calorimetry study

Amphotericin B (AmB) is the most widely used polyene antibiotic to treat systemic fungal infections which affect an increasing number of immunocompromised patients. It is generally thought that AmB forms pores within the fungi membranes by interacting with ergosterol, the main sterol of fungi. However, it also interacts with the cholesterol contained in mammalian cells, hence its toxicity. In order to have a better understanding of the interactions prevailing between AmB and sterols, differential scanning calorimetry was used to study various mixtures incorporating from 6.5 to 25 mol% of AmB in pure dipalmitoylphosphatidylcholine (DPPC) vesicles and in ergosterol- or cholesterol-containing DPPC vesicles. The sterol concentration was kept constant at 12.5 mol% with respect to the phospholipid. Our results show that three phases co-exist when AmB is dispersed in the pure phospholipid. One corresponds to the phospholipid phase alone. The two others are characterised by a broad transition at temperatures higher than the main transition temperature of the pure phospholipid, corresponding to the drug in interaction with the aliphatic chains of the lipid. The fact that the transition temperatures of these additional components are higher than that of the pure phospholipid suggests that AmB interacts strongly with the aliphatic chains of the lipid, consistent with the idea prevailing in the literature that AmB by itself may form pores in a lipid matrix. When AmB interacts with cholesterol-containing bilayers the thermograms also present three components. Upon increasing the concentration of AmB, though, an important broadening of these components is observed which is explained in terms of destabilisation of the organisation of the aliphatic chains. The situation is strikingly different if ergosterol is present in the lipid matrix. The thermograms remain unmodified as the concentration of AmB is increased and a broad transition, now involving only two components when the thermograms are decomposed, is observed. An analysis of the results shows that various interacting units, e.g. AmB+DPPC and (AmB+ergosterol)+DPPC, are present within the membrane. These units involve the phospholipid and hence contribute to its structurisation. The important differences between the thermograms obtained with the ergosterol- as compared to the cholesterol-containing bilayers, in spite of the structural similarity of these two sterols, provides strong evidence for the selectivity of interaction of AmB with ergosterol as compared to cholesterol. It is thus clear that the action of AmB on cholesterol- as compared to ergosterol-containing membranes results from different mechanisms. Finally, UV-visible spectra of AmB in pure as well as sterol-containing DPPC vesicles show the presence of absorption bands that give support to the interpretation derived from the calorimetric data.

The effect of surfactants on the aggregation state of amphotericin B

We have studied the effect of two surfactants, one non-ionic, lauryl sucrose (LS) and the other ionic, sodium deoxycholate (DOC), on the aggregation state of amphotericin B (AmB) and its selectivity towards ergosterol and cholesterol. It is shown that the addition of these surfactants has very similar effects on the AmB micelles. Below the critical micellar concentration of the surfactants, mixed micelles with AmB are first formed as a result of the penetration of the surfactant molecules into the AmB micelles. At higher concentrations of the surfactant molecules, the micellar structure is completely destroyed and AmB is found as monomers in solution. When the concentration of the surfactant is further increased, micelles of the surfactant molecules are built up, AmB remaining in monomeric form. However, the critical micellar concentration of LS is modified by the presence of AmB in solution, while that of DOC is not affected, thereby indicating that the interactions of AmB with LS are stronger than those of DOC with AmB. We also show that both surfactants enhance the selectivity of the AmB binding to sterols at exactly the concentrations of the surfactants which induce the monomerization of the antibiotic. It is observed that the maximal selectivity is found at a concentration of the surfactants corresponding to their particular CMC in presence of the antibiotic.

The effects of amphotericin B on pure and ergosterol- or cholesterol-containing dipalmitoylphosphatidylcholine bilayers as viewed by 2H NMR.

Amphotericin B (AmB) is a widely used polyene antibiotic to treat systemic fungal infections. This drug is known to be lethal to fungal cells but it has also side effect toxicity on mammalian cells. The mechanism of action of AmB is thought to be related to the difference of the main sterol present in the mammalian and the fungal cells, namely cholesterol and ergosterol, respectively. The effect of AmB has been investigated on pure dipalmitoylphosphatidylcholine (DPPC) and on cholesterol- and ergosterol-containing DPPC bilayers by 2H NMR spectroscopy. The 2H NMR results first confirm that AmB forms a complex with sterol-free DPPC bilayers, the interaction causing the structurization of the lipids and the increase of the gel-to-lamellar fluid DPPC phase transition temperature with increasing concentration of the antibiotic. The results also show that the effects of AmB on cholesterol- and ergosterol-containing DPPC bilayers are remarkably different. On one hand, the drug causes an increase of the orientational order of the lipid acyl chains in cholesterol-containing membranes, mostly in high cholesterol content membranes. On the other hand, the addition of AmB disorders the DPPC acyl chains when ergosterol is present. This is thought to be due to the direct complexation of the ergosterol by AmB, causing the sterol ordering effect to be weaker on the lipids.

The interactions of amphotericin B with various sterols in relation to its possible use in anticancer therapy

Amphotericin B (AmB) is still the most common anti-fungal agent used to treat systemic fungal infections. It is known that this antibiotic acts by forming pores with the ergosterol contained in the membranes of fungi, but it also interacts with the cholesterol contained in the membranes of eukaryotic cells, hence its toxicity. AmB may also interact with the most common oxidation products of cholesterol found in vivo, together with interacting with biosynthetic precursors of cholesterol, namely, lanosterol and 7-dehydrocholesterol (7-DHC). The purpose of the present work was to study the interactions in solution between AmB and these various sterols, the techniques used being UV-Vis spectroscopy and differential scanning calorimetry. The results are globally interpreted in terms of the structural differences between the sterols. We show that AmB selectively interacts with 7-DHC which, according to a recent hypothesis proposed in the literature, has been identified in connexion with a therapeutic strategy against hepatocellular carcinomas. We find that the affinity of AmB towards 7-DHC is even greater than the affinity of the antibiotic towards ergosterol. We also find that AmB selectively interacts with the principal oxidation product of cholesterol, 7-ketocholesterol, a situation that has to be taken into account when AmB is administered.

A kinetic study of the oxidation effects of amphotericin B on human low‐density lipoproteins

The UV‐visible results of this kinetic study show that amphothericin B as Fungizone is a much stronger oxidant than CuSO4, itself a powerful oxidant of low‐density lipoprotein (LDL). Amphotericin B as AmBisome alone has no oxidizing effect on LDL while a mixture of both AmBisome and CuSO4 induces an important potentialization of the LDL oxidation. These results allow us to believe that the high toxicity of amphotericin B is related to its capacity to modify and to weaken the structure of LDL. In addition, differential scanning calorimetry experiments show that the oxidative modifications of LDL by CuSO4 or by amphotericin B proceed through different mechanisms.

Interactions in mixed monolayers between dioleoyl-l-phosphatidylcholine and all-trans retinal

Abstract The surface pressure isotherms of all- trans retinal and dioleoyl- l -phosphatidylcholine at the nitrogen-water interface were studied at 20.5°C. The extrapolated limiting areas per molecule are 49 and 83 A 2 molecule −1 , respectively. The properties of seven mixtures of all- trans retinal and dioleoyl- l -phosphatidylcholine, covering the whole range of molar fractions were also measured. The results show that an almost constant collapse pressure is observed at 44 ± 1 mN m −1 for all the mixtures studied. Furthermore, an apparent collapse pressure is also observed at 16, 18, 22, 26, 32, and 35 mN m −1 at a molar fraction of dioleoyl- l -phosphatidylcholine of 0.100, 0.200, 0.400, 0.500, 0.600, and 0.800, respectively. An analysis of the results in terms of the surface phase rule shows that below the apparent collapse pressure, the components are completely miscible. Negative deviations from ideality were observed in the region of miscibility for all the systems, as shown by the negative deviations to the additivity rule and the negative excess free energies of mixing calculated for all the systems at 5, 7, 10, and 13 mN m −1 . Furthermore, it is shown that above the apparent collapse pressure, all-trans retinal is rejected from the monolayer, the rejection being total at the collapse pressure.

Excess free energies of interaction of chlorophyll a with monogalactosyldiacylglycerol and phytol a mixed monolayer study

Abstract The surface pressure isotherms of chlorophyll a , monogalactosyldiacylglycerol and phytol at the air-water interface were studied on a Langmuir trough at 20.0±0.5° C . The subphase was a phosphate buffer, 10 −3 M at pH 8.0. The extrapolated limiting areas per molecule are 115, 82 and 38 A 2 /molecule, respectively. The isotherms of eight mixtures of chlorophyll a with monogalactosyldiacylglycerol and eight mixtures of chlorophyll a with phytol, covering in both cases the whole range of molar fractions have also been measured. The results for the mixed monolayers were analysed in terms of the additivity rule. They show that a small negative deviation with respect to ideality is observed upon mixing chlorophyll a with monogalactosyldiacylglycerol. However, chlorophyll a forms an ideal two-dimensional solution when mixed with phytol. The excess free energies of mixing of chlorophyll a with monogalactosyldiacylglycerol as a function of concentration were calculated from the surface pressure isotherms at 10, 15, 20 and 25 mN·m −1 . The values are negative, reflecting the interaction prevailing between these components in the monolayers. For the four surface pressures studied, the excess free energies are symmetrical with respect to the mode fraction. The values for an equimolar mixture range from −300 to −540 J·mol −1 at 10 and 25 mN·m −1 , respectively. A comparison between the thermodynamics of mixing of chlorophyll a with monogalactosyldiacylglycerol and phytol suggests that the polar head of monogalactosyldiacylglycerol together with the polar groups of chlorophyll a are probably involved in the interaction. However, this does not completely rule out the possibility that structural effects due to a different packing of chlorophyll a with monogalactosyldiacylglycerol and phytol may also be involved. Furthermore it is shown that the small interactions between these constituents are not inconsistent with the specific coupling existing between the apoprotein of the chlorophyll a -protein complexes and chlorophyll a .

General method for the purification of lipids for surface pressure studies : Application to monogalatosyldiglyceride

Abstract In order to prepare lipids free of surface-active contaminants ad suitable for the measurement of surface pressure isotherms as general purification method has been developed involving preparative thin-laye chromatography. A detailed description of the procedure and blank experiments carried out with a monolayer trough are presented. The method provides a rapid purification of various classes of lipids, the whole procedure taking less than 1 day. It gives highly reproducible results, better than 2 A 2 per molecule at any surface pressure. As an example of an application, the results obtained with a commercial sample of monogalactosyldiglyceride, a major plant lipid, are reported.

Formation of asymmetrical planar lipid bilayer membranes from characterized monolayers

A device allowing the formation of lipid bilayers by apposition of characterized monolayers has been designed and constructed. It is essentially composed of two solid Teflon Langmuir troughs, pressed along one another. The troughs are both equipped with a movable barrier for the compression of the monolayers and a float-type torsion balance for the detection of the surface pressures. The lipid bilayers are formed across a tiny hole (0.225 mm diameter) punched through a polymer film clamped in between the two troughs and are characterized by their electrical properties (specific resistance and capacitance). Experimental results have been obtained on asymmetrical membranes of phosphatidylethanolamine and phosphatidylserine formed from monolayers and from lipid films many molecular dimensions in thickness. Two polymer supports, Teflon and polypropylene, were used and coated with either squalene or petroleum jelly. The results show that the stability of the bilayer, particularly when formed from monolayers, is a direct function of the interactions of the lipids with the coated support. The most stable membranes are obtained when Teflon is used as support and squalene as coating. The most important advantage of the technique presented here is found when the bilayers are formed from mixtures of components deposited at the interface. Indeed, our system allows a characterization of the miscibility of the components in the monolayers prior to form the membrane, as opposed to a complete ignorance of the molecular organization when the bilayers are formed by apposing lipid films.