The chain conformational order of ergosterol- or cholesterol-containing DPPC bilayers as modulated by Amphotericin B: a FTIR study

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Chemistry and Physics of Lipids 151 (2008) 41–50

The chain conformational order of ergosterol- or cholesterol-containing DPPC bilayers as modulated by Amphotericin B: a FTIR study Isabelle Fournier a , Joanna Barwicz a , Mich`ele Auger b , Pierre Tancr`ede a,∗ a

b

Universit´e du Qu´ebec a` Trois-Rivi`eres, D´epartement de Chimie-Biologie, BP 500, Trois-Rivi`eres, QC G9A 5H7, Canada Universit´e Laval, D´epartement de Chimie, Centre de recherche en Sciences et Ing´enierie des Macromol´ecules, Qu´ebec, QC G1K 7P4, Canada Received 22 May 2007; received in revised form 8 August 2007; accepted 20 September 2007 Available online 25 September 2007

Abstract Amphotericin B (AmB) is the most widely used antibiotic to treat systemic fungal infections. However, the molecular mechanism of its activity is still not completely understood. In the present work we have used FTIR spectroscopy to investigate the conformational state of the aliphatic chains of DPPC liposomes using the 2850 cm−1 band, associated with the methylene symmetric stretching mode. The liposomes were either binary mixtures of the lipid with AmB, cholesterol or ergosterol, or ternary systems of these constituents. The two sterols contribute to an ordering of the aliphatic chains of the lipid, this ordering being slightly more important with ergosterol. In the gel state, AmB does not change the conformational order of DPPC even at high concentration. In the fluid phase, however, the drug clearly structures its lipid environment. Our results show that AmB can initiate a redistribution of the ergosterol in the plane of the membrane, but not of the cholesterol molecules, which might constitute an additional mechanism to explain the activity of the antibiotic. Crown Copyright © 2007 Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Amphotericin B; FTIR; Ergosterol; Cholesterol; MLV; DPPC

1. Introduction In the modern pharmacopia used to treat systemic fungal infection Amphotericin-B (AmB) remains, more than 50 years after its introduction, the drug with the largest spectrum of action. It is still the most widely used in spite of the documented toxic effects associated with its administration. To circumvent these side effects, numerous new formulations of the drug have been proposed (Hartsel and Bolard, 1997; Hann and Prentice, 2001; Lemke et al., 2005; Soni and Wagstaff, 2005). However these new formulations have not upheld all their promises with the exception of AmBisome® , a lipid based preparation of the antibiotic that is of limited use, however, owing to its high cost. As a consequence, the original dispersion of AmB in deoxycholate micelles, known as Fungizone® , is still widely used. This situation is certainly related to the fact that the molecular mechanism of action of AmB is still not completely understood. Thus, a better understanding of how AmB behaves within bio-

∗

Corresponding author. Tel.: +1 819 376 5011x3397. E-mail address: [email protected] (P. Tancr`ede).

logical membranes, the site of its action, would be important to reduce the toxicity of the drug and improve its therapeutic index. It is now generally accepted that the in vivo activity of AmB is related to its interactions with the ergosterol contained in fungal membranes, but AmB also interacts with the cholesterol contained in mammalian cells, hence its toxicity. The prevailing model explaining the mode of action of AmB implies the formation of pores in the membrane involving the antibiotic and the sterols, thus yielding to cellular death (Bolard, 1986; Andreoli, 1974; de Kruijff and Demel, 1974). In recent years we have been more specifically interested in understanding the selectivity of interaction of AmB with ergosterol as compared to cholesterol when these components are dispersed in a lipid matrix. We have shown, through differential scanning calorimetry (DSC) studies (Fournier et al., 1998), that the drug could structure around itself the lipid environment, an idea consistent with the finding that AmB alone could form pores within the lipid matrix (Baginski et al., 1997). The thermograms of DPPC containing AmB and either ergosterol or cholesterol also presented important differences in spite of the great structural similarities of these sterols. These differences were related to the selectivity of interaction of AmB

0009-3084/$ – see front matter. Crown Copyright © 2007 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2007.09.006

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with ergosterol as compared to cholesterol in the lipid bilayer, thus suggesting that these interactions resulted from different mechanisms, as confirmed by a 2 H NMR study on the same lipid systems (Paquet et al., 2002). In this context, it appeared to us that the structuration of the lipid environment by either AmB itself, or when sterols are also involved, was sufficiently important to deserve further investigation. In this respect, Fourier-transform infrared spectroscopy (FTIR) clearly stands as one among the most useful techniques to get further knowledge on such a structuration. Indeed, FTIR is inherently well-suited to study conformational order in phospholipid acyl chains (Mantsch, 1984; Mendelsohn et al., 1991; Zhang et al., 1992; Mendelsohn and Senak, 1993; O’Leary, 1993; Mendelsohn and Moore, 1998; Los and Murata, 2004). It provides valuable informations complementary to those obtained by 2 H NMR, namely because the time scale of the two techniques are different. More specifically, the quadrupolar interactions giving rise to a signal in 2 H NMR spread over ∼100 kHz, and therefore the time scale associated with the measurement is approximately 10−5 s (Mendelsohn and Senak, 1993). The quadrupolar interaction is thus averaged by trans/gauche isomerization since the lifetime of a conformer is evaluated to be ∼10−10 s. FTIR, on the other hand, is sensitive to such time scales. Among the various spectral regions rich in information in the FTIR spectrum of DPPC (Mendelsohn and Senak, 1993; O’Leary, 1993), we have been mainly interested in the region centered at 2850 cm−1 , corresponding to the methylene symmetric stretching mode. Two reasons were behind this choice. First, AmB presents no absorption in this region (Schwartzman et al., 1978; Espuelas et al., 1997), therefore the absorption at 2850 cm−1 will solely reflect the absorption of the lipid CH2 groups. Second, it has been shown in the literature (Mantsch, 1984; Zhang et al., 1992; Mendelsohn and Senak, 1993; O’Leary, 1993; Mendelsohn and Moore, 1998; Los and Murata, 2004) that the absorption at 2850 cm−1 is closely related to the conformational order of the lipid chain, which is known to reflect the instantaneous state of organization of the membranes from which all other, longer time scale properties, follow (Mendelsohn et al., 1991). Thus, the information gathered by FTIR will complement the informations on the organization of the lipid environment by AmB previously obtained by DSC (Fournier et al., 1998) and 2 H NMR (Paquet et al., 2002). In the present work we have thus used FTIR spectroscopy to investigate the conformational state of the aliphatic chains of DPPC liposomes. The liposomes were either binary mixtures of DPPC with AmB or cholesterol or ergosterol, or ternary systems incorporating DPPC, AmB and either ergosterol or cholesterol. To establish comparisons between the results, the composition of the various lipid mixtures was chosen to be the same as that used in our previous work (Fournier et al., 1998; Paquet et al., 2002). The FTIR spectra were recorded as a function of temperature from below to above the phase transition of the lipid. The results provide evidence that ergosterol and cholesterol have a similar effect on the conformational order of the lipid chains. Indeed, both contribute to an ordering of the aliphatic chains of the lipid, this ordering being slightly more important with ergos-

terol. We show that in the presence of AmB, differences between ergosterol and cholesterol become manifest, which suggest that a redistribution of the ergosterol molecules is occurring in the plane of the membrane. 2. Materials and methods 2.1. Materials AmB, cholesterol, ergosterol and dimethyl sulfoxide (DMSO) were purchased from Sigma Co. (St-Louis, MO). DPPC or DPPC deuterated on the sn-2 acyl chain (DPPC-d31 ) were purchased from Avanti Polar Lipids (Alabaster, AL). Chloroform was obtained from Anachemia (spectroscopic grade, Montr´eal, QC). The water used to prepare all the solutions was distilled and demineralized on a Sybron-Barnstead system (Fisher Scientific Co., Montr´eal, QC). Phosphate buffer (PBS) 0.02 M at pH 7.0 was used for the liposome preparation. 2.2. Preparation of the liposome solutions DPPC, ergosterol and cholesterol are first dissolved in chloroform while AmB is solubilized in the smallest volume of DMSO possible. The exact quantities necessary to prepare a solution at the final concentration required are taken from these solutions. The solvent is then evaporated (Rotovapor R 110, Buechi-Brinkman, Germany) to dryness and the lipid film is resuspended in the buffer. It was verified by FTIR spectroscopy that at the concentrations used, DMSO was completely removed from the suspension during the evaporation. Multilamellar vesicles (MLV) are prepared from this lipid suspension through four freeze–thaw–vortex cycles, the high temperature of the cycle being at 50 ◦ C, i.e. higher than the main transition temperature of DPPC. All the solutions prepared have the same final lipid concentration, 4 mg/ml (5.45 × 10−3 M), while the concentration of the sterol was 12.5 and 28 mol% with respect to the phospholipid. Three different concentrations of AmB were used: 6.25, 12.5 and 25 mol% with respect to the lipid. UV–vis spectra, taken from the supernatant solution when the vesicles are centrifuged, indicate that more than 98% of the AmB is indeed incorporated in the vesicles. 2.3. Fourier transform infrared spectroscopy The FTIR spectra of the various mixtures were recorded against the pure PBS buffer on a Perkin-Elmer Spectrum 2000 spectrometer using a mercury cadmium telluride detector cooled with liquid nitrogen. The temperature was programmed using an Omega microprocessor-based temperature and process controller. The lipid sample was deposited between CaF2 windows separated by a 50 ␮m cell spacer. For each sample, interferograms were averaged on 100 scans at 2 cm−1 resolution. The baseline was corrected by using GRAMS/32 software program (Galactic Industries Corporation, Salem, MA). The methylene symmetric stretching frequency was obtained from the center of gravity calculated at the top 10% of the band (Cameron et al., 1982).

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Fig. 1. FTIR spectra recorded at 30 ◦ C. A: pure DPPC; B: DPPC + 28 mol% cholesterol; C: DPPC + 28 mol% ergosterol.

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Fig. 3. Temperature dependence of the frequency of the methylene symmetric stretching mode for the pure DPPC-d31 and its binary mixtures incorporating 28 mol% cholesterol or ergosterol.

3. Results and discussion 3.1. Effect of sterols on the DPPC bilayers Fig. 1 displays an example of the FTIR spectra recorded for the pure DPPC bilayers (curve A) in the gel state (30 ◦ C), as well as for DPPC incorporating either 28% cholesterol (curve B) or 28% ergosterol (curve C). The spectra show two bands, the methylene symmetric stretching mode at 2850 cm−1 and the methylene antisymmetric stretching mode at 2920 cm−1 . The analysis presented below will only concern the 2850 cm−1 band because, as discussed in the introduction, this band is more sensitive to trans-gauche isomerization within the chain (Kodati and Lafleur, 1993; Kodati et al., 1994). The spectra of Fig. 1 show that upon addition of cholesterol, and even more so for ergosterol, the maximum absorption at 2850 cm−1 is slightly shifted towards lower wavenumber values. This shift is most clearly seen in Fig. 2, which shows how the wavenumber of the methylene symmetric stretching mode varies as a function of temperature for the DPPC bilayers that contain either 12.5% or 28% of cholesterol or ergosterol. It is observed that at low temperatures, i.e. in the gel phase of the lipid, the presence of cholesterol or ergosterol in the lipid

Fig. 2. Temperature dependence of the frequency of the methylene symmetric stretching mode for the pure DPPC and its binary mixtures with 12.5 and 28 mol% cholesterol or ergosterol.

bilayer induces a decrease in the wavenumber associated with the methylene symmetric stretching mode. This effect is more important for ergosterol than for cholesterol and gets larger as the concentration of the sterols is increased. A decrease of the wavenumber of the 2850 cm−1 band is normally associated with an increase of the conformational order of the aliphatic chains of the lipid, resulting from an increase in the number of trans conformers with respect to gauche conformers (Kodati and Lafleur, 1993; Kodati et al., 1994). This highly reproducible result is surprising owing to the fact that it is generally admitted that in the gel phase, cholesterol fluidifies the bilayer. So, to verify that this decrease was not due to a possible interference of the sterol molecules on the methylene symmetric stretching mode of the lipid, we have recorded the FTIR spectra of deuterated DPPC-d31 , either pure, or incorporating 28% of cholesterol or ergosterol. The results, presented in Fig. 3, clearly indicate that the two sterols decrease the wavenumber associated to the CD2 symmetric stretching mode, even below the lipid main phase transition temperature. It thus appears that the sterols could increase the chain conformational order of DPPC in the gel phase. Interestingly, such a decrease in the wavenumber of the methylene group in the gel phase has also been observed by various authors (Senak et al., 1992; McMullen et al., 1994; Severcan et al., 1995; Dicko et al., 1998) but no rationale was offered. The following tentative explanations may be proposed. It is important to note that below the gel-to-fluid phase transition temperature, the lipid aliphatic chains are not completely in an all-trans conformation. Yellin and Levin (1977) have indeed found that the onset of the hydrocarbon chain-gauche isomerization in the gel state of DPPC is approximately −40 ◦ C. Thus, at 30 ◦ C for instance, i.e. under the gel-to-fluid phase transition temperature, the isomerization process is well under way. More quantitatively, Mendelsohn et al. (1991) have shown by FTIR spectroscopy that about 11% of gauche conformers are present in DPPC at 34 ◦ C for the fourth carbon of the acyl chain while this fraction is decreased to about 2% in the presence of 33 mol% of cholesterol. The overall conformational order of DPPC thus seems to be increased in the presence of cholesterol, a result consistent with our finding.

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Similarly, Weisz et al. (1992) have shown, from 2 H NMR of DMPC/cholesterol bilayers, that even below the phase transition, the rates measured for the trans-gauche isomerization are lower for the sterol containing bilayer than those for the pure lipid. Again, this is consistent with our finding that cholesterol does not seem to disorder the aliphatic chains in the gel state but, contrarily, seems to order those chains. Finally, it has also been suggested that a decrease of the acyl chain tilt angle also induces a decrease in the frequency of the methylene symmetric and antisymmetric stretching modes (Dicko et al., 1998) due to an increase in the intermolecular coupling or an increase of the van der Waals interactions (Kodati et al., 1994). All these ideas certainly make it evident that the final story of the binary lipid/cholesterol mixture has not yet been written. Fig. 2 also shows the gel-to-fluid phase transition. For DPPC, the transition is sharp, and located at 41.5 ◦ C, a value identical to the literature data obtained by FTIR (Inoue et al., 2001; Toyran and Servecan, 2003), and by DSC (Rolland et al., 1991; Fournier et al., 1998; Tahir et al., 1999). However, for the sterolcontaining bilayers, the transition is broadened, corresponding to a decrease in the cooperativity of the transition. This broadening has also been put in evidence by DSC (Vist and Davis, 1990; McMullen et al., 1993; McMullen and McElhaney, 1995; Fournier et al., 1998). As expected, this decrease in the cooperativity of the transition is more important as the concentration of the sterols is increased. In the fluid phase, it is clear that the wavenumber of the methylene symmetric stretching mode is decreased as compared to that of DPPC, thus corresponding to an increase in the number of trans conformers. This conformational ordering of the DPPC chains by the sterols in the fluid phase has been well documented for cholesterol in the literature (Mantsch and McElhaney, 1991; Senak et al., 1992; Zhang et al., 1992; Mendelsohn and Senak, 1993; O’Leary, 1993; Smondryev and Berkowitz, 1999; Chiu et al., 2002; Endress et al., 2002). It also corresponds to what has been recorded by DSC for the same systems (McMullen et al., 1994; Fournier et al., 1998). It is also consistent with the molecular dynamics investigation of the cholesterol effects in a DPPC bilayer as published by Tu et al. (1998). These authors have shown that cholesterol has a significant effect on the subnanosecond time scale lipid chain dynamics (slowing down of the reorientational motion of the methylene groups along the entire length of the hydrocarbon chains), a time scale comparable to the time scale probed by FTIR spectroscopy. More recently, this ordering effect on DPPC has also been evidenced for ergosterol and lanosterol, in addition to cholesterol, through similar molecular dynamics simulation studies (Cournia et al., 2007). Finally, Fig. 2 shows that the ordering effect increases as the sterol concentration is increased and is more important for ergosterol than for cholesterol, at least for the highest concentration used in the present study. This latter effect has to be correlated to the molecular structure of the sterols used. Although these two sterols have similar structures, ergosterol bears an additional double bond on its steroid nucleus and both an additional methyl group and double bond on its side chain. These features are such that ergosterol offers a more rigid, planar structure, than cholesterol, allowing stronger van der Waals interactions

Fig. 4. FTIR spectra recorded at 30 ◦ C. A: pure DPPC; B to D: DPPC + 6.25, 12.5 and 25 mol% AmB, respectively.

to be established with the lipid chains (Charbonneau et al., 2001; Urbina et al., 1995, 1998), with the concomitant result of a higher condensing effect of ergosterol in the liquid phase of either DPPC (Cournia et al., 2007) or DMPC (Czub and Baginski, 2006). 3.2. Effect of AmB on the DPPC bilayers Fig. 4 presents a typical spectrum obtained for DPPC in the gel phase at 30 ◦ C incorporating three different concentrations of AmB (Fig. 4, spectra B to D). The spectra are compared to that of the pure lipid (Fig. 4, spectrum A) at the same temperature. Contrary to what was observed with the sterols (Fig. 1), Fig. 4 shows that the position of the methylene symmetric stretching mode recorded at 2850 cm−1 does not change when AmB is within the bilayer in the gel state. Fig. 5A displays how the wavenumber of the methylene symmetric stretching mode varies as a function of temperature for the three concentrations of AmB used. As stated above, below the transition temperature, the position of the band does not change with respect to that of the pure DPPC as the concentration of AmB is increased. This implies that the conformational order of DPPC does not change even in the presence of 25 mol% of AmB. In this context, it might be important to recall that AmB is an amphiphilic molecule with a very rigid structure. As a consequence, Sternal et al. (2004) have recently argued that AmB cannot be present as monomers in a saturated lipid bilayer, their work involving DMPC, though, rather than DPPC bilayers. The antibiotic is rather present as aggregates of molecules that spontaneously form hydrophilic pores, which are thought to be the functional units in biological membranes. Thus, the AmB molecules, contrary to the sterols, are not dispersed molecularly in the plane of the membrane, but aggregated as pores perpendicular to the plane of the membrane surface. Their perturbing effect on the lipid molecules would be less and therefore the conformational order of the lipid chain would, on average, stay almost the same as that of the pure lipid. This aggregation of AmB within the bilayer was also manifest in the thermograms that we had recorded for the same mixtures (Fournier et al., 1998). Fig. 5B presents the thermogram of the pure DPPC, as well as that of AmB at 12.5 mol% in DPPC.

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Fig. 6. Temperature dependence of the frequency of the methylene symmetric stretching mode for the pure DPPC, its binary mixtures with 28 mol% cholesterol and its ternary mixtures with 28 mol% cholesterol and, in addition, 6.25, 12.5 and 25 mol% AmB.

laboratory by UV–vis spectroscopy (to be submitted) do suggest that above the transition temperature, the pores themselves are aggregated. Fig. 5. (A) Temperature dependence of the frequency of the methylene symmetric stretching mode for the pure DPPC and its binary mixtures with 6.25, 12.5 and 25 mol% AmB; (B) thermograms recorded for DPPC and its binary mixture with 12.5 mol% AmB.

The thermogram of the mixture shows that the pre-transition of the pure DPPC is still present while it is abolished at very low sterol concentrations (McMullen and McElhaney, 1995). The main phase transition associated with the pure DPPC is also clearly displayed. In fact, surprisingly, it is still observed even at 25 mol% of AmB (Fournier et al., 1998). This strongly suggests that a lateral phase separation of the components is occurring in the plane of the membrane, one of the phases being composed of pure DPPC (Tm = 41.5 ◦ C), while the other, at higher temperatures, involves lipid molecules structured by the pores of AmB. Fig. 5A also displays the main phase transition of the pure lipid at 41.5 ◦ C. As stated above, the transition is very cooperative for the pure DPPC but less so when AmB is incorporated in the bilayer. Fig. 5A and B show that the temperature range involved in the completion of the transition is the same either when recorded through both FTIR spectroscopy or DSC, which implies that the results obtained by these two techniques are consistent. Also, when one compares the FTIR data presented in Fig. 5A for AmB and Fig. 2 for either cholesterol or ergosterol, it becomes obvious that for similar concentrations, the gel-tofluid phase transition is much more cooperative when AmB is present in the bilayer. This implies, as noted above, that AmB is not dispersed molecularly in the bilayer, but rather present in its aggregated form (pores). As the temperature is further increased, i.e. within the fluid phase, one also notes that AmB contributes to increase the conformational order of the lipid chains, but to a lesser extent than the sterols. This is again consistent with the presence of pores of AmB. In fact, recent results obtained in our

3.3. Effect of AmB on cholesterol containing DPPC bilayers We have also recorded the FTIR spectra of DPPC containing both cholesterol and AmB. The results appear in Fig. 6 for DPPC containing 28 mol% cholesterol and, in addition, either 6.25, 12.5 or 25 mol% AmB. We have also recorded the spectra for DPPC containing 12.5 mol% cholesterol and the same concentrations of AmB (results not shown) and the trend in the results to be discussed below was essentially the same for these two sterol concentrations. To compare the data, Fig. 6 also presents the melting curves for the pure DPPC and for DPPC containing 28 mol% cholesterol. The first noticeable effect of adding AmB to a bilayer containing 28 mol% cholesterol is the large broadening of the gel-to-fluid phase transition. It is however quite remarkable to note that a phase transition is still apparent in the melting curve even when the bilayer contains as much as 28 mol% cholesterol and 25 mol% AmB. This is consistent with our DSC data which showed, in the same conditions, that thermotropic transitions were also recorded. The second noticeable result to be remarked is that the ordering effect of cholesterol in the gel phase is not observed even at the lowest AmB concentration used. Thus, the particular molecular arrangement yielding to the ordering effect of cholesterol in its binary mixtures with DPPC, as shown in Fig. 2, may not be present in the ternary mixture. This may not be completely surprising considering the fact that the three components (DPPC, AmB and cholesterol) are present all together when the liposomes are prepared. Matsuoka and Murata (2002) have also invoked such an argument to rationalize the differences they observed in the permability of cholesterol-containing PC liposomes when AmB was present either as a ternary constituent when the bilayer was prepared or when it was added to the binary cholesterol-PC mixture from the aqueous subphase. In

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addition, it is known that the cholesterol-rich domains in the bilayer are of short lifetimes (10−9 to 10−7 s) (Subczy´nski et al., 1994; Schroeder et al., 1995). Therefore, it may be speculated that AmB, even at low concentrations, could affect these lifetimes and thus change the population of the trans-gauche conformers to which FTIR is so sensitive as it probes such timescales. Finally, above the transition temperature, we observe that the wavenumber values for the ternary mixtures gradually tend toward values recorded for the binary cholesterol–DPPC mixtures, all the values being identical at 60 ◦ C. This behaviour is consistent with the aggregation of the pores formed by AmB within the membrane at high temperatures as referred to above, the aggregation resulting from the important difference between the lipid fluidity as compared to the rigidity of the AmB molecules. More importantly, the fact that the fluid bilayer now behaves almost as a membrane only containing cholesterol suggests that this sterol is not associated with the pores formed by AmB and thus, that there is no specific interactions between AmB and cholesterol, as suggested in our previous work (Fournier et al., 1998; Paquet et al., 2002). 3.4. Effect of AmB on ergosterol containing DPPC bilayers The same type of study was performed on DPPC bilayers containing both 28 mol% ergosterol and 6.25, 12.5 or 25 mol% AmB. The melting curves are presented in Fig. 7. Once again, it is observed, in agreement with our DSC results (Fournier et al., 1998), that the main phase transition of DPPC is still observed, albeit considerably broadened, even in the presence of both AmB and ergosterol at a total concentration of more than 50% per mole of DPPC. However, it is clear that, below the phase transition, the behaviour of AmB towards ergosterol, as compared to that of cholesterol in the DPPC bilayer, is strikingly different. It is indeed observed that when ergosterol is present in the bilayer, the wavenumber gradually increases towards the value recorded either for the pure DPPC bilayer or to that of the DPPC bilayers containing AmB only. The antibiotic thus gradually decreases the effect normally induced on the DPPC chains by the presence of 28 mol % of ergosterol in the bilayer. When put in the context of our previous studies, either by UV–vis spectroscopy (Fournier et al., 1998) or through the use of the monolayer

Fig. 7. Temperature dependence of the frequency of the methylene symmetric stretching mode for the pure DPPC, its binary mixtures with 28 mol% ergosterol and its ternary mixtures with 28 mol% ergosterol and, in addition, 6.25, 12.5 and 25 mol% AmB.

technique (Barwicz and Tancr`ede, 1997), and when also put in the context of a further discussion of our calorimetric data presented in the next section, the present FTIR results on the ternary DPPC/AmB/ergosterol mixtures are consistent with a direct interaction between AmB and ergosterol within the DPPC membrane. In addition, the effect observed is the same with the two highest concentrations of AmB used. The same general trend also prevails in the fluid phase, which shows that these AmBergosterol associations seem to prevail even up to 60 ◦ C. It is thus clear that AmB seems to induce a redistribution of the ergosterol molecules within the bilayer. 3.5. Comparative analysis with the calorimetric data In the context of the analysis of the FTIR results presented so far, we have been led to revisit the calorimetric data of our previous work (Fournier et al., 1998). Table 1 presents a new set of data on the thermal effect associated to the gel to fluid phase transition of DPPC in the various mixtures. The transitions recorded for the various systems are presented in increasing order of temperature. It is observed that the main transition of the pure DPPC, recorded at T3 = 41.5 ◦ C, is associated to a heat effect equal to 93 mJ. This corresponds to 34.2 kJ/mol on a molar basis, in agreement with the data reported by McMullen and McElhaney (1995). For the bilayers containing either 12.5 mol%

Table 1 Heat effecta (mJ) associated with the phase transitions of the decomposed thermograms for the various systems studied

DPPC DPPC + cho DPPC + ergo DPPC + AmB DPPC + cho + AmB DPPC + ergo + AmB

H1 (T1 = 40.5 ◦ C)

H2 (T2 = 41.5 ◦ C)

H3 (T3 = 41.5 ◦ C)

H4 (T4 = 42.7 ◦ C)

H5 (T5 = 43.5 ◦ C)

H6 (T6 = 45.3 ◦ C)

– 35.8 42.4 – 1.2 21.4

– 20.3 26.8 – – –

93.0 – – 5.0 – –

– – – 36.5 19.4 –

– – – – – 33.4

– – – 14.5 4.4 –

The relative concentrations of either sterol and AmB are 12.5 and 25%, respectively. a In all our DSC experiments, the cell was filled with exactly the same amount of lipid (from a solution containing 4 mg/ml DPPC), which allows a comparison of the heat effects for all the mixtures studied. For DPPC, the heat effect was obtained from the area corresponding to the peak associated with the main gel-to-fluid phase transition and, for the mixtures, from the areas corresponding to the various components of the decomposed thermograms.

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cholesterol or ergosterol, two transitions are recorded, at the same temperatures for the two sterols, corresponding to the melting of the sterol-poor (T1 = 40.5 ◦ C) and sterol-rich components (T2 = 41.5 ◦ C). Also, for both sterols, the heat effect associated with the poor-sterol region is larger than that associated with the sterol-rich regions. On the other hand, when the bilayer contains 25 mol% AmB, Table 1 shows that, as noted in our previous work (Fournier et al., 1998), three transitions are recorded: one, at T3 = 41.5 ◦ C, corresponding to the main transition of the pure DPPC, the other two, at higher temperatures (T4 = 42.7 ◦ C and T6 = 45.3 ◦ C), corresponding to the lipids structured by the antibiotic. The fact that the thermogram clearly presents a component corresponding to the pure DPPC, which represents about 10% of the total heat effect for that mixture, is a clear indication that a phase separation is prevailing within the bilayer. In addition, one observes that the overall heat of transition is reduced by only about 40% with respect to that of the pure lipid even if the membrane contains as much as 25 mol% of AmB. This decrease of the overall heat on one hand, and the existence of transitions at higher temperatures implying an increase of the conformational order of the lipid chains on the other hand, are indications that monomeric molecules of AmB may also be present within the plane of the bilayer, at equilibrium with the AmB aggregated as pores. In this sense the AmB monomers would act as classical “impurities” in the membrane and contribute to the perturbation of the lipid molecules, somehow like sterols or other small organites do. When the DPPC bilayer includes both cholesterol and AmB, three transitions are recorded, the first one at T1 = 40.5 ◦ C, corresponding to the cholesterol-poor region. The other two, at T4 = 42.7 ◦ C and T6 = 45.3 ◦ C correspond exactly to those recorded when AmB alone is present in the DPPC bilayer. In the ternary AmB–cholesterol–DPPC mixture AmB is thus interacting with the lipid as if it was by itself in the bilayer, indicating that no specific interactions with cholesterol occur. However, the perturbing effect of cholesterol on the lipid is manifest through the fact that the total heat effect associated with the three transitions is about two times smaller than that of the binary AmB–DPPC mixture. Thus, these results do confirm the FTIR results presented previously for this ternary system (Fig. 6). The results of the decomposition process for the thermograms of the DPPC bilayers containing both ergosterol and AmB are strikingly different. First, the total heat effect associated with all the transitions observed here is about twice that recorded for the ternary DPPC mixture containing both cholesterol and AmB (54.8 mJ versus 25.0 mJ). Thus, the presence of both AmB and ergosterol in the DPPC bilayer is less perturbing of the lipid organization than the concomitant presence of AmB and cholesterol in the lipid. Second, as noted above for either AmB alone or AmB and cholesterol in the DPPC bilayer, a clear phase separation occurs in the ternary mixture containing AmB and ergosterol. One of the phases is centered at T1 = 40.5 ◦ C, corresponding to the ergosterol-poor domains. More interestingly, the main component, a broad transition centered at around at T5 = 43.5 ◦ C, is a new phase that was not observed neither for the binary DPPC/AmB nor for the binary DPPC/ergosterol mixtures. This transition is a clear indication that a direct interaction

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between AmB and ergosterol occurs within the DPPC bilayer and the transition at 43.5 ◦ C is thus associated with the structuration of the lipid by the antibiotic specifically interacting with ergosterol. Again, this is in general agreement with the analysis of the FTIR results of Fig. 7 presented above. This is also consistent with our previous finding, obtained through UV–vis spectra, that a direct interaction prevails between AmB and ergosterol in the DPPC bilayer (Fournier et al., 1998). In this sense, therefore, the DSC data bring support and complement the interpretation of the FTIR results presented above for the ternary systems. These findings on the various organization states of DPPC in binary mixtures with AmB or in its ternary mixtures with AmB and cholesterol or ergosterol can be summarized very schematically as shown in Fig. 8. Thus, Fig. 8A corresponds to the binary DPPC + AmB mixture and shows the three lipid species associated with the three phase transitions recorded by DSC and referred to above in Table 1: one is found at T3 = 41.5 ◦ C and corresponds to the pure DPPC, while the other two, at T4 = 42.7 ◦ C and T6 = 45.3 ◦ C are associated with the surrounding lipid molecules structured by AmB or trapped between the pores of AmB associated as aggregates. These latter lipids are somehow similar to those trapped between proteins at high protein levels, as suggested by Mendelsohn and Senak (1993). The same lipid species are also found when cholesterol is present in addition to AmB in the bilayer (Fig. 8B), but the lipid undergoing a transition at T1 = 40.5 ◦ C now corresponds to the lipid under the influence of cholesterol. Finally, Fig. 8C shows the representation for the mixture containing both AmB and ergosterol within the DPPC matrix. For this mixture, two lipid phases are present, one at T1 = 40.5 ◦ C corresponding to DPPC under the influence of ergosterol and the other one at T5 = 43.5 ◦ C being associated with the lipid structured by the AmB-ergosterol units or by AmB itself. Owing to the fact that this latter transition is rather broad, we cannot exclude that some of the lipids associated to those trapped within the aggregates of AmB and transiting at 45.3 ◦ C, are still present (Fig. 8C, black circles). Although ergosterol and cholesterol are structurally very similar, the only difference being for ergosterol, as noted above, an additional double bond on the steroid nucleus and an additional methyl and double bond on its side chain, it is clear that when either of these sterols are present in a DPPC bilayer already containing AmB, the bilayer presents strikingly different thermotropic transitions. In this context, Bagi´nski et al. (2002) recall that the small structural differences between these two sterols may have important consequences because these differences are ultimately additive. So, when numerous molecules are involved, these differences will yield to significant influence on the stability of the pores or of their aggregates. More specifically, Bagi´nski et al. (2002) have shown though molecular dynamics simulation that AmB is slightly more rigid in contact with ergosterol, and vice-versa, in the AmB/ergosterol channel as compared to AmB in a AmB/cholesterol channel. This mutual increase of stability allows for stronger van der Waals interactions to be established between AmB and ergosterol as compared to those between cholesterol and AmB. In this latter context, the cholesterol molecules are more free to move independently from the antibiotic molecules. The AmB channel

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I. Fournier et al. / Chemistry and Physics of Lipids 151 (2008) 41–50

Fig. 8. Schematic representation of: A: DPPC + AmB; B: DPPC + AmB + Chol; C: DPPC + AmB + Ergo. DPPC at T1 = 40.5 ◦ C or T3 = 41.5 ◦ C (); DPPC at ); Chol (⊕); Ergo ( ). T4 = 42.7 ◦ C or at T5 = 43.5 ◦ C ( ); DPPC at T6 = 45.3 ◦ C (䊉); AmB (

surrounded by ergosterol molecules is thus both more rigid and more stable. This is consistent with the finding that the lifetime of the AmB channel in a lipid matrix incorporating ergosterol is 100 times longer than when the lipid incorporates cholesterol (Cotero et al., 1998). One may add, in addition, that the minute differences between ergosterol and cholesterol also explain how AmB will differentially adsorb to DPPC monolayers (Barwicz and Tancr`ede, 1997; Gagos et al., 2005) or bilayers (Lopes and Castanho, 2002) containing either sterol. In this sense, therefore, the ideas presented here thus give overall support to the schematic molecular representation of the various membranes of Fig. 8. It is clear, from the analysis presented so far, that the overall structure of the DPPC matrix incorporating both AmB and sterols is the reflection of the fine balance of the interactions between the various components present. We have focused, in the preceding paragraph, on the interactions between AmB and the sterols, but the interactions of the sterol molecules with DPPC are also themselves of the utmost importance in the understanding of how the constituents will be organized in the lipid matrix. In this respect, H˛ac-Wydro et al. (2005) have shown, from studies on DPPC monolayers incorporating either cholesterol or ergosterol, that the excess free energies of interaction between DPPC and either cholesterol or ergosterol are negative, but five times larger for cholesterol. This, coupled with the information given above that the interactions between AmB and ergosterol are stronger than those between AmB and cholesterol, is a good indication that the ergosterol molecules in the

plane of the membrane are most likely to be distributed in the vicinity of the AmB molecules as depicted above in Fig. 8C. Again, it is clear that the minute differences in the structure of the sterols are responsible for the two additive effects discussed so far, i.e. the more favorable, specific, interactions of ergosterol with AmB and the less favorable interactions of ergosterol with DPPC, as compared to the equivalent interactions when ergosterol is substituted for cholesterol in the bilayer. In conclusion, we have shown that when AmB is incorporated into a bilayer containing either sterol, the minute differences in the structure of these sterols lead to important differences in the organization of the various constituents within the bilayer that could hardly have been predicted. Indeed, the final organization of the membrane reflects the affinity of each constituent – lipid, sterol and AmB – for the others. We have thus been able to show that AmB could initiate a redistribution of the ergosterol, but not the cholesterol molecules, in the plane of the membrane. This might constitute an additional mechanism, besides the formation of pores by the antibiotic, to explain the activity of the antibiotic. This redistribution of the ergosterol molecules around the AmB pores would deprive in sterol the rest of the membrane the consequences being, for example, an alteration of the enzymatic activity of membrane proteins. Acknowledgements This work was supported by a grant from the Natural Sciences and Engineering Council of Canada, to which we are grateful.

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