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.     

Interactions of amphotericin B derivative of low toxicity with biological membrane components--the Langmuir monolayer approach

Amphotericin B (AmB)--a polyene macrolide antibiotic--exhibits strong antifungal activity, however, is known to be very toxic to mammalian cells. In order to decrease AmB toxicity, a number of its derivatives have been synthesized. Basing on in vitro and in vivo research, it was evidenced that one of AmB derivatives, namely N-methyl-N-D-fructopyranosylamphotericin B methyl ester (in short MF-AME) retained most of the antifungal activity of the parent antibiotic, however, exhibited dramatically lower animal toxicity. Therefore, MF-AME seems to be a very promising modification product of AmB. However, further development of this derivative as potential new antifungal drug requires the elucidation of its molecular mechanism of reduced toxicity, which was the aim of the present investigations. Our studies were based on examining the binding energies by determining the strength of interaction between MF-AME and membrane sterols (ergosterol-fungi sterol, and cholesterol-mammalian sterol) and DPPC (model membrane phospholipid) using the Langmuir monolayer technique, which serves as a model of cellular membrane. Our results revealed that at low concentration the affinity of MF-AME to ergosterol is considerably stronger as compared to cholesterol, which correlates with the improved selective toxicity of this drug. It is of importance that the presence of phospholipids is essential since--due to very strong interactions between MF-AME and DPPC--the antibiotic used in higher concentration is "immobilized" by DPPC molecules, which reduces the concentration of free antibiotic, thus enabling it to selectively interact with both sterols.     

Interactions of amphotericin B derivatives with lipid membranes—A molecular dynamics study

Amphotericin B (AmB) is a well-known polyene macrolide antibiotic used to treat systemic fungal infections. AmB targets more efficiently fungal than animal membranes. However, there are only minor differences in the mode of action of AmB against both types of membranes, which is a source of AmB toxicity. In this work, we analyzed interactions of two low toxic derivatives of AmB (SAmE and PAmE), synthesized in our laboratory, with lipid membranes. Molecular dynamics simulations of the lipid bilayers containing ergosterol (fungal cells) or cholesterol (animal cells) and the studied antibiotic molecules were performed to compare the structural and dynamic properties of AmB derivatives and the parent drug inside the membrane. A number of differences was found for AmB and its derivatives' behavior in cholesterol- and ergosterol-containing membranes. We found that PAmE and SAmE can penetrate deeper into the hydrophobic region of the membrane compared to AmB. Modification of the amino and carboxyl group of AmB also resulted in the conformational transition within the antibiotic's polar head. Wobbling dynamics differentiation, depending on the sterol present, was discovered for the AmB derivatives. These differences may be interpreted as molecular factors responsible for the improved selectivity observed macroscopically for the studied AmB derivatives.     

Interactions of amphotericin B derivatives with lipid membranes--a molecular dynamics study

Amphotericin B (AmB) is a well-known polyene macrolide antibiotic used to treat systemic fungal infections. AmB targets more efficiently fungal than animal membranes. However, there are only minor differences in the mode of action of AmB against both types of membranes, which is a source of AmB toxicity. In this work, we analyzed interactions of two low toxic derivatives of AmB (SAmE and PAmE), synthesized in our laboratory, with lipid membranes. Molecular dynamics simulations of the lipid bilayers containing ergosterol (fungal cells) or cholesterol (animal cells) and the studied antibiotic molecules were performed to compare the structural and dynamic properties of AmB derivatives and the parent drug inside the membrane. A number of differences was found for AmB and its derivatives' behavior in cholesterol- and ergosterol-containing membranes. We found that PAmE and SAmE can penetrate deeper into the hydrophobic region of the membrane compared to AmB. Modification of the amino and carboxyl group of AmB also resulted in the conformational transition within the antibiotic's polar head. Wobbling dynamics differentiation, depending on the sterol present, was discovered for the AmB derivatives. These differences may be interpreted as molecular factors responsible for the improved selectivity observed macroscopically for the studied AmB derivatives.     

Interaction between nystatin and natural membrane lipids in Langmuir monolayers--the role of a phospholipid in the mechanism of polyenes mode of action

Nystatin (NYS), a polyene antifungal antibiotic, has been investigated in Langmuir monolayers alone and in mixtures with mammalian and fungi membrane sterols (cholesterol and ergosterol, respectively) as well as with a model phospholipid (DPPC). The interactions between film molecules have been examined both in a qualitative and quantitative way with the excess area per molecule (AExc), excess free energy of mixing (DeltaGExc) and the interaction parameter (alpha). The obtained results have been compared with those previously reported for another polyene antimycotic: amphotericin B (AmB) mixed with lipids. Higher affinity of NYS has been observed for ergosterol vs. cholesterol, however, the strongest attractions were found for its mixtures with DPPC. The obtained results have been verified with biological studies reported previously for both antibiotics (NYS and AmB). A thorough analysis of the Langmuir experiment results performed for both polyenes enabled us to conclude that the presence of DPPC can be considered as a key factor affecting their antifungal activity as well as their toxicity towards host cells.     

Comparative molecular dynamics simulations of amphotericin B-cholesterol/ergosterol membrane channels

Amphotericin B (AmB) is a very effective anti-fungal polyene macrolide antibiotic whose usage is limited by its toxicity. Lack of a complete understanding of AmB's molecular mechanism has impeded attempts to design less toxic AmB derivatives. The antibiotic is known to interact with sterols present in the cell membrane to form ion channels that disrupt membrane function. The slightly higher affinity of AmB toward ergosterol (dominant sterol in fungal cells) than cholesterol (mammalian sterol) is regarded as the most essential factor on which antifungal chemotherapy is based. To study these differences at the molecular level, two realistic model membrane channels containing molecules of AmB, sterol (cholesterol or ergosterol), phospholipid, and water were studied by molecular dynamics (MD) simulations. Comparative analysis of the simulation data revealed that the sterol type has noticeable effect on the properties of AmB membrane channels. In addition to having a larger size, the AmB channel in the ergosterol-containing membrane has a more pronounced pattern of intermolecular hydrogen bonds. The interaction between the antibiotic and ergosterol is more specific than between the antibiotic and cholesterol. These observed differences suggest that the channel in the ergosterol-containing membrane is more stable and, due to its larger size, would have a higher ion conductance. These observations are in agreement with experiments.     

Effect of potassium ions at the different concentration on the interaction between AmB and the lipid monolayer containing cholesterol or ergosterol

AmB is an antifungal drug of polyene. Although it is prone to nephrotoxicity, it is still the gold standard in the clinical treatment of fungal infection. Sterol plays a decisive role in the drug activity of AmB. The antifungal activity of AmB depends on ergosterol in fungal membranes, and its toxicity is related to cholesterol in mammalian membranes. At the same time, AmB interacts with biofilms, leading to a significant loss of potassium ions and affecting the transport of potassium ions across membranes. Meanwhile, metal cation may also affect AmB molecules’ aggregation on the membrane. This paper mainly studied the effects of different concentrations of potassium ions on the interactions between AmB and lipid monolayers containing cholesterol or ergosterol and explored the differences in the impact of varying potassium ions on the drug activity of AmB on monolayers rich in these two kinds of sterols. The results show that potassium ions caused the collapse of lipid monolayer and lipid-AmB monolayer to disappear. The limiting molecular area of these monolayers also increased due to potassium ions. The limiting molecular area of the monolayer in the presence of ergosterol has a great difference in the different concentration of potassium ions, which is different from that in the presence of cholesterol. The presence of potassium ions, regardless of the intensity of K⁺ ions, increased the maximum elastic modulus of the lipid/sterol monolayer with and without AmB. The presence of potassium ions reduced the influence of AmB on the stability of the lipid monolayer containing cholesterol. The impact of AmB on the stability of the lipid monolayer containing ergosterol was related to the concentration of potassium ions. The potassium ions increased the area of the ordered “island” region on the lipid-AmB monolayer containing cholesterol, and the boundary of the microregion produced different degrees of curvature. However, on the lipid/ergosterol monolayer, 5 mM and 10 mM potassium ions made the holes caused by AmB more denser, and the diameter of holes become larger. These results can help to improve the effect of potassium ions on the transmembrane transport of substances affected by AmB. The results will provide a basis for further exploration of the effect mechanism of metal ions on the antifungal activity of polyene drugs.     

Membrane permeabilizing activity of amphotericin B is affected by chain length of phosphatidylcholine added as minor constituent

The effect of acyl-chain length of phospholipid on the membrane permeabilizing activity of amphotericin B (AmB) was examined using egg phosphatidylcholine (eggPC) liposomes containing 5% or 20% phosphatidylcholine with various lengths of fatty acyl chains from C(10) to C(18); 1,2-dicapryloyl-sn-glycero-3-phosphocholine (DCPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The membrane activity of AmB was evaluated by two methods; the drug was added to a liposome suspension (added-via-aqua), or mixed with lipids prior to liposome preparation (mixed-with-lipid). In both cases, K(+) influx by AmB was measured as pH change inside liposomes by 31P-NMR. The C(10) and C(12) acyl phospholipids markedly enhanced the activity of AmB, the C(14) and C(16) lipids virtually showed no effect, and the C(18) lipid was inhibitory to the AmB's action. Clear distinction between the C(12) and C(14) lipids, which differ only in acyl chains by two carbons, implies that molecular interaction between phospholipid and AmB is partly due to the matching of their hydrophobic length.     

Binding of antibiotic amphotericin B to lipid membranes: a 1H NMR study

The (1)H NMR technique was applied to study binding of AmB, an antifungal drug, to lipid membranes formed with egg yolk phosphatidylcholine. The analysis of (1)H NMR spectra of liposomes, containing also cholesterol and ergosterol (at 40 mol%), shows that AmB binds preferentially to the polar headgroups. Such a binding restricts molecular motion of the choline fragment in the hydrophilic region at the surface of liposomes but increases the segmental motional freedom in the hydrophobic core. The same effects are also observed in the sterol-containing membranes, except that the effect on the hydrophobic core was exclusively observed in the membranes containing ergosterol.     

Interaction of the polyene antibiotic amphotericin B with model membranes: differences between small and large unilamellar vesicles

The interaction of the polyene antibiotic amphotericin B (AmB) (Fig. 1) with large unilamellar vesicles (LUV) was monitored by circular dichroism (CD) and carboxyfluorescein (CF) release. LUV afford a far better model for biological membranes than small unilamellar vesicles (SUV) which have been used until now. With dimyristoyl phosphatidyl choline (DMPC) LUV (i.e., containing saturated acyl chains), a strong and not saturable binding for AmB/lipid ratios up to 0.5 was observed both above and below the phase transition temperature. Incorporation of cholesterol into the vesicles did not significantly change the interaction. With egg PC (EPC) LUV (i.e., containing unsaturated acyl chains), quite a different picture emerged: the binding reached saturation for AmB/lipid ratios of about 5 x 10(-3), a result not observed with EPC SUV. When sterols were introduced into membranes, the CD spectral features obtained in the presence of ergosterol were different from those obtained in the presence of cholesterol. Such a different behavior was not observed with SUV. We suggest that species whose CD spectrum was observed after 15 min in the presence of ergosterol-containing EPC LUV is the particular one which forms wide channels and induces a Ca2+ release. (H. Ramos, A. Attias, B.E. Cohen and J. Bolard, submitted for publication). The CF release from EPC LUV induced by AmB was very low, even at very high concentrations of the antibiotic (3 x 10(-4)M). In contrast, an important release of the fluorescent dye was observed with DMPC LUV at concentrations of approximately 10(-5)M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectrophotometric analysis of organisation of dipalmitoylphosphatidylcholine bilayers containing the polyene antibiotic amphotericin B

Amphotericin B (AmB) is a polyene antibiotic widely used in the treatment of deep-seated fungal infections. The mode of action of AmB is directly related to the effect of the drug on the lipid phase of biomembranes. In the present work the effect of AmB on the properties of lipid bilayers formed with dipalmitoylphosphatidylcholine (DPPC) and the effect of the lipid phase on the molecular organisation of AmB were studied with application of spectrophotometry in the UV-Vis region. The absorption spectra of AmB in lipid membranes display a complex structure with hypsochromically and bathochromically shifted bands indicative of formation of molecular aggregates of the drug. Formation of molecular aggregates was analysed at different concentrations of the drug in the lipid phase in the range 0.05--5 mol% and at different temperatures in the range 5--55 degrees C. The aggregation level of AmB in the ordered phase of DPPC displayed a minimum corresponding to a concentration of 1 mol% with respect to the lipid. An increase in the aggregation level was observed in the temperature region corresponding to the main phase transition. The structure of molecular aggregates of AmB is analysed on the basis of spectroscopic effects in terms of the exciton splitting model. Analysis of the position of the absorption maximum of AmB in the lipid phase of DPPC in terms of the theory of solvatochromc effects makes it possible to ascribe the refractive indices n=1.40 and n=1.49 to the hydrophobic core of the membrane in the L(alpha) and the P(beta)' phase respectively. Analysis of the aggregation of AmB in the lipid phase in relation to the physical state of the membrane reveals that the temperature range of the main phase transition of a lipid cluster in the immediate vicinity of AmB depends on its concentration. The termination of the phase transition temperature, as read from the AmB aggregation, varies between 42 degrees C at 1 mol% AmB in DPPC and 49 degrees C at 5 mol% AmB in DPPC. The exciton splitting theory applied to the analysis of the spectroscopic data makes it possible to calculate the diameter of the AmB pore as 2.8 A in the gel phase and 3.6 A in the fluid phase of the DPPC membrane, on the assumption that the pore is formed by nine AmB molecules.     

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.     

Comparative molecular dynamics study of lipid membranes containing cholesterol and ergosterol

Sterol molecules are essential for maintaining the proper structure and function of eukaryotic cell membranes. The influence of cholesterol (the principal sterol of higher animals) on the lipid bilayer properties was extensively studied by both experimental and simulation methods. In contrast, the effect of ergosterol (the principal fungal sterol) on the membrane structure and dynamics is much less recognized. This work presents the results of comparative molecular dynamics simulation of the hydrated dimyristoylphosphatidylcholine bilayer containing approximately 25 mol % of cholesterol or ergosterol. A detailed analysis of the molecular properties (e.g., bilayer thickness, lipid order, diffusion, intermolecular interactions, etc.) of both sterol-induced liquid-ordered membrane phases is presented. Presence of sterols in the membrane significantly changes its property, especially fluidity and molecular packing. Moreover, in accordance with the experiments, our calculations show that, compared to cholesterol, ergosterol has higher ordering effect on the phospholipid acyl chains. This different influence on the properties of the lipid bilayer stems from differences in conformational freedom of sterol side chains. Additionally, obtained models of lipid membranes containing human and fungal sterols, constituting the result of our work, can be also utilized in other chemotherapeutic studies on interaction of selected ligands (e.g., antifungal compounds) with membranes.     

Interaction of fusidic acid with lipid membranes: Implications to the mechanism of antibiotic activity

We have studied the effects of cholesterol and steroid-based antibiotic fusidic acid (FA) on the behavior of lipid bilayers using a variety of experimental techniques together with atomic-scale molecular dynamics simulations. Capillary electrophoretic measurements showed that FA was incorporated into fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes. Differential scanning calorimetry in turn showed that FA only slightly altered the thermodynamic properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, whereas cholesterol abolished all endotherms when the mole fraction of cholesterol (X(chol)) was >0.20. Fluorescence spectroscopy was then used to further characterize the influence of these two steroids on DPPC large unilamellar vesicles. In the case of FA, our result strongly suggested that FA was organized into lateral microdomains with increased water penetration into the membrane. For cholesterol/DPPC mixtures, fluorescence spectroscopy results were compatible with the formation of the liquid-ordered phase. A comparison of FA and cholesterol-induced effects on DPPC bilayers through atomistic molecular dynamics simulations showed that both FA and cholesterol tend to order neighboring lipid chains. However, the ordering effect of FA was slightly weaker than that of cholesterol, and especially for deprotonated FA the difference was significant. Summarizing, our results show that FA is readily incorporated into the lipid bilayer where it is likely to be enriched into lateral microdomains. These domains could facilitate the association of elongation factor-G into lipid rafts in living bacteria, enhancing markedly the antibiotic efficacy of FA.     

Cholesterol and ergosterol influence nystatin surface aggregation: relation to pore formation

Nystatin interaction with liposomes mimicking fungal and mammalian membranes (ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles, respectively) was studied by fluorescence spectroscopy. The activity of this antibiotic was also measured using a pyranine fluorescence detected K+/H+ exchange assay. Nystatin mean fluorescence lifetime varied with the antibiotic concentration and ergosterol content (0-30 mol%) of the lipid vesicles. It sharply increased from 5 to 37 ns upon reaching 100 molecules per liposome, reporting nystatin oligomerization in the membrane. Concomitantly, spectral alterations typical of excitonic coupling were detected and there was a pronounced increase in the initial rate of pore formation by nystatin. These findings suggest that nystatin exerts its antibiotic activity via a two-stage mechanism: at low antibiotic concentrations, surface-adsorbed monomeric antibiotic molecules perturb the lipid packing, changing the permeability properties of the ergosterol-rich liposomes. Upon reaching a critical threshold, nystatin mode of action switches to the classical model of transmembrane aqueous channel formation. In the presence of cholesterol-containing POPC liposomes, neither nystatin spectroscopic properties, nor the kinetics of K+ efflux varied with the antibiotic concentration suggesting that in this case the first stage of antibiotic mode of action always prevails or the assemblies formed by nystatin and cholesterol are very loose.     

Mechanism of Action of Amphotericin B at the Cellular Level. Its Modulation by Delivery Systems

Abstract

Amphotericin B (AmB) is the drug of choice for the treatment of systemic fungal infections, but its use is hampered by its severe side-effects. A better understanding of its mechanisms of action is needed to develop new AmB formulations with an optimal selectivity between fungal and mammalian cells. Interactions between AmB and cells depend on the concentration of the drug. Stimulatory effects, modulation of the activity of immunocompetent cells and inhibition of yeast adherence are early events that precede the actual cellular toxicity. If membrane permeability alterations are considered to be the first toxic step, cell death results not only from osmotic imbalances, but also from additional mechanisms, such as lipid peroxidation, inhibition of membrane enzymes and blockade of endocytosis. The selectivity between fungal and mammalian cells takes its origin from the difference in the nature of the membrane sterol: ergosterol in fungi, cholesterol in mammalian cells. Transmembrane pores result from different mechanisms according to the sterol: ergosterol-AmB complexes are formed from monomelic AmB in solution, which is the only form present in aqueous medium at low AmB concentrations, whereas pores in the cholesterol containing membrane result from the adsorption onto the membrane surface of aqueous self-associated AmB, that appears in medium when AmB concentration increases. The liposomes seem to sequester AmB in a manner which makes it unavailable for mammalian cells, but maintains its access to fungal cells. The transfer of AmB by progressive diffusion of free AmB through the aqueous phase could explain the enhancement of the therapeutic index of the drug by liposomes, since the induction of pore formation needs a higher threshold of drug for host cell than for fungal cell membranes. The closed structure of the vehicle is not required to enhance the selectivity of the drug: esters of sucrose or high concentration of sodium deoxycholate afford a protective effect as well. Macrophages, after phagocytosis of liposomal AmB, may be considered as a reservoir of AmB, from which the drug is progressively released. Finally, the strong binding of AmB to the delivery system reduces the amount of drug bound to serum components and thus the endocytosis of AmB through the LDL receptor, resulting in lower toxicity.     

Susceptibilities of phospholipid vesicles containing different sterols to amphotericin B-loaded lysophosphatidylcholine micelles

To investigate the susceptibilities of fungal and mammalian cells to amphotericin B (AmB), AmB-loaded lysophosphatidylcholine (LPC)micelles as drug delivery vehicles were incubated at 37 degrees C with phosphatidylcholine vesicles containing different sterols as model systems for fungal and mammalian cells. The binding and kinetics of AmB to sterols in the membranes were judged by UV-visible spectroscopy. In the 91% monomeric form, AmB interacted rapidly with ergosterol and slowly with 7-dehydrocholesterol (7-DHC), while it did not interact with cholesterol. In the 50% monomeric form, AmB formed complexes more rapidly with ergosterol or 7-DHC than in the monomeric form, whereas it did not still interact with cholesterol. The interaction was also characterized by resonance energy transfer between the fluorescent probe trimethylammonium diphenylhexatriene (TMA-DPH) and AmB. In the 91% monomeric form, AmB caused initial fluorescence quenching in bilayer membranes containing any sterol as well as sterol-free bilayer membranes due to the release of AmB and its incorporation within the membranes. However, a second phase of increasing fluorescence was found in the case of ergosterol alone. On the other hand, in the 47% monomeric form, AmB gave a biphasic intensity profile in membranes containing any sterol as well as sterol-free membranes. However, the extent of the second phase of increasing fluorescence intensity was markedly dependent upon sterol composition. Studies using sterol-containing vesicles provide important insights into the role of the aggregation state of AmB in its effects on cells.     

Molecular mechanism for pore-formation in lipid membranes by the hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata.

The pore-forming activity of CEL-III, a Gal/GalNAc specific lectin from the Holothuroidea Cucumaria echinata, was examined using artificial lipid membranes as a model system of erythrocyte membrane. The carboxyfluorescein (CF)-leakage studies clearly indicated that CEL-III induced the formation of pores in the dipalmitoyl phosphatidyl choline (DPPC)-lactosyl ceramide (LacCer) liposomes effectively but not in the DPPC-glucosyl ceramide (GlcCer) liposomes or DPPC liposomes. Such a leakage of CF was strongly inhibited by lactose, a potent inhibitor of CEL-III, suggesting that the leakage is mediated through the specific binding of CEL-III to the carbohydrate chains on the surface of the liposomes. The leakage of CF from the DPPC-lactosyl ceramide liposomes was pH-dependent, and it increased with increasing pH. The immunoblotting analysis and circular dichroism data indicated that upon interaction with liposomes, CEL-III associated to form an oligomer concomitantly with a marked conformational change. Furthermore, channel measurements showed that CEL-III has an ability to form small ion channels in the planar lipid bilayers consisting of diphytanoylphosphatidylcholine and human globoside (Gb4Cer)/LacCer.     

Interactions of ciprofloxacin with DPPC and DPPG: fluorescence anisotropy, ATR-FTIR and 31P NMR spectroscopies and conformational analysis

The interactions between a drug and lipids may be critical for the pharmacological activity. We previously showed that the ability of a fluoroquinolone antibiotic, ciprofloxacin, to induce disorder and modify the orientation of the acyl chains is related to its propensity to be expelled from a monolayer upon compression [1]. Here, we compared the binding of ciprofloxacin on DPPC and DPPG liposomes (or mixtures of phospholipids [DOPC:DPPC], and [DOPC:DPPG]) using quasi-elastic light scattering and steady-state fluorescence anisotropy. We also investigated ciprofloxacin effects on the transition temperature (T(m)) of lipids and on the mobility of phosphate head groups using Attenuated Total Reflection Fourier Transform Infrared-Red Spectroscopy (ATR-FTIR) and (31)P Nuclear Magnetic Resonance (NMR) respectively. In the presence of ciprofloxacin we observed a dose-dependent increase of the size of the DPPG liposomes whereas no effect was evidenced for DPPC liposomes. The binding constants K(app) were in the order of 10(5) M(-1) and the affinity appeared dependent on the negative charge of liposomes: DPPG>DOPC:DPPG (1:1; M:M)>DPPC>DOPC:DPPC (1:1; M:M). As compared to the control samples, the chemical shift anisotropy (Deltasigma) values determined by (31)P NMR showed an increase of 5 and 9 ppm for DPPC:CIP (1:1; M:M) and DPPG:CIP (1:1; M:M) respectively. ATR-FTIR experiments showed that ciprofloxacin had no effect on the T(m) of DPPC but increased the order of the acyl chains both below and above this temperature. In contrast, with DPPG, ciprofloxacin induced a marked broadening effect on the transition with a decrease of the acyl chain order below its T(m) and an increase above this temperature. Altogether with the results from the conformational analysis, these data demonstrated that the interactions of ciprofloxacin with lipids depend markedly on the nature of their phosphate head groups and that ciprofloxacin interacts preferentially with anionic lipid compounds, like phosphatidylglycerol, present at a high content in these membranes.