Effect of antibiotic amphotericin B on structural and dynamic properties of lipid membranes formed with egg yolk phosphatidylcholine

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1 Chemistry and Physics of Lipids 147 (2007) Effect of antibiotic amphotericin B on structural and dynamic properties of lipid membranes formed with egg yolk phosphatidylcholine Monika Hereć a,b, Akhmed Islamov c, Alexander Kuklin c, Mariusz Gagoś a,d, Wiesław I. Gruszecki a, a Department of Biophysics, Institute of Physics, Maria Curie-Skłodowska University, Lublin, Poland b Department of Theoretical Physics, The John Paul II Catholic University of Lublin, Lublin, Poland c Frank Laboratory of Neutron Physics, Joined Institute of Nuclear Research, Dubna, Russia d Department of Physics, Agricultural University, Lublin, Poland Received 14 July 2006; received in revised form 21 March 2007; accepted 24 March 2007 Available online 31 March 2007 Abstract Amphotericin B (AmB) is a popular antibiotic applied in treatment of deep-seated mycotic infections. The mode of action of AmB is based upon interactions with biomembranes but exact binding properties of the antibiotic to the lipid membranes still remain obscure. Effect of incorporation of AmB into egg yolk phosphatidylcholine membranes in the concentration range from 0.01 to 5 mol% on structural and dynamic properties of lipid bilayers was studied with application of small-angle neutron scattering, X-ray diffractometry and Fourier-transform infrared spectroscopy (FTIR). The results of the experiments show that AmB is located predominantly in the headgroup region of the membranes at concentrations below 1 mol%. The process of AmB aggregation, at concentrations above 1 mol%, is associated with ordering effect within the acyl chain region and therefore indicates incorporation of AmB into the hydrophobic membrane core Elsevier Ireland Ltd. All rights reserved. Keywords: Amphotericin B; Biomembranes; Polyene antibiotics; Lipid membranes 1. Introduction Antifungal polyene macrolide antibiotic amphotericin B (AmB; Fig. 1) is a representative of the group of chemotherapeutics which mechanism of action consists in change of membrane cell permeability (Aracava et al., 1981; Brajtburg et al., 1990; Gallis et al., 1990; Ellis, 2002; Baginski et al., 2006). Destroying natural selective Corresponding author. Tel.: ; fax: address: wieslaw.gruszecki@umcs.lublin.pl (W.I. Gruszecki). membrane permeability is an effect of incorporation of amphiphilic molecules of amphotericin B into the lipid phase and their interaction with lipid molecules (Hartsel et al., 1991; Wolf and Hartsel, 1995). Rod-shaped structure of AmB molecule, with the polar head mycosamine, the hydroxyl groups on the one side of the macrolide ring and the polyene fragment on the opposite side, makes it possible to interact both with the polar part of the lipid membrane and acyl chains. The consequence of such a structure of AmB is also a formation of molecular aggregates (De Kruijff et al., 1974; De Kruijff and Demel, 1974; Bolard et al., 1991; Barwicz et al., 1993; Caillet et al., 1995; Fujii et al., 1997; Gruszecki et al., 2002) /$ see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi: /j.chemphyslip

2 M. Hereć et al. / Chemistry and Physics of Lipids 147 (2007) Fig. 1. Chemical structure of amphotericin B. that play an important role in AmB mode of therapeutic action. It has been postulated that not only formation of porous molecular structures by AmB, but also modification of the physical properties of the lipid bilayers modulates membrane permeability to ions (Barwicz and Tancrede, 1997; Fournier et al., 1998; Wojtowicz et al., 1998; Charbonneau et al., 2001; Gagos et al., 2001; Milhaud et al., 2002; Paquet et al., 2002; Zumbuehl et al., 2004; Gagos et al., 2005; Baginski et al., 2006; Gabrielska et al., 2006). Such mechanisms can be particularly effective at low concentrations of AmB, at which formation of aggregated molecular structures of the drug within the lipid phase cannot be expected (Gagos et al., 2001; Gruszecki et al., 2003a,b). On the other hand, the recent findings show that AmB increases the barrier for transmembrane ion transport, while incorporated to the lipid membranes at low concentrations instead of acting as an ionophore (Herec et al., 2005). In the present work, we address the problem of molecular interactions between AmB and lipids in the membranes containing very low and relatively high concentrations of the drug, in order to explore further, the molecular mechanisms responsible for action of this antibiotic with respect to biomembranes. 2. Materials and methods Amphotericin B was purchased from Sigma Chem. Co. (St. Louis, USA). AmB was dissolved in and recrystallized from 2-propanol water (4:6, v/v) and purified by means of HPLC directly before use. A Supelco PKB- 100 column was applied (length 25 cm, internal diameter 4.6 mm) and the solvent mixture 2-propanol water (4:6, v/v) was used as a mobile phase. The final concentration of AmB was calculated from the absorption spectra. The molar extinction coefficient in the absorption maximum at 408 nm was M 1 cm 1. Owing to the low concentration of AmB eluted from the column (absorbance level below 0.2 at the optical path-length 1 cm) and owing to the fact that absorbance level decreased linearly upon dilution we considered the solution as composed mostly of monomeric AmB and the determination based on absorbance measurement accurate. Egg yolk phosphatidylcholine (EYPC) was obtained from Sigma Chem. Co. and used without further purification. The AmB-containing EYPC membranes were investigated in the form of either unilamellar liposomes (SANS, electronic absorption) or multibilayers (X-ray, FTIR). The multibilayers composed of 40 bilayers were formed on glass support or on ZnSe crystal with EYPC and AmB at molar concentration from 0.01 to 5 mol% according to the procedure described in detail previously (Gruszecki and Sielewiesiuk, 1990, 1991). Briefly, the lipid multibilayers were deposited to a solid support by means of evaporation from ethanol. After deposition the lipid films were transferred to a vacuum, for 30 min, in order to remove possible residuals of organic solvent, and then exposed for 30 min to relative humidity 80% in order to hydrate the lipid multibilayers. The unilamellar liposomes were prepared in 100 mm Tricine buffer (ph 7.6) by extrusion technique, with application of liposome extruder (Avestin Inc., Canada) using filters with 50 nm pores, as described in detail previously (Herec et al., 2005). The small-angle neutron scattering (SANS) with twodetector system measurements were performed at the small-angle time-of-flight axially symmetric neutron scattering spectrometer MURN at the IBR-2 fast pulsed reactor of the Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research in Dubna. For complete description of technical details, see Kuklin et al. (2005). The samples were poured into quartz cells (Hellma, Müllheim, Germany) to provide the 2 mm sample thickness. The sample temperature was set and controlled electronically at 20.0 ± 0.1 C. The sample in quartz cell

3 80 M. Hereć et al. / Chemistry and Physics of Lipids 147 (2007) was equilibrated minimally for 1 h at this temperature before the measurements. The scattering patterns were corrected for background effect. The coherent scattering intensity was obtained by using a vanadium standard scatterer. Further details of measurements and data interpretation were described previously (Uhrikova et al., 2000, 2003). The θ 2θ X-ray diffractometric measurements were performed with Dron-HZG 4 apparatus equipped with a copper anode (λ = 1.54 Å) and a nickel filter. The methodology of measurements and data interpretation were described previously (Klose et al., 1996). Electronic absorption spectra of liposome suspension was recorded with a double-beam UV Vis spectrophotometer Cary 300 Bio from Varian equipped with a thermostated cuvette holder. Infrared absorption spectra were reordered with Fourier-transform infrared spectrometer (FTIR) from Bruker Optik, Germany, model Vector 33 supplied with an attenuated total reflection (ATR) accessory. Before (30 min) and during measurements the instrument was purged with argon. Lipid multilayers were deposited to a ZnSe crystal support as described above. Typically 100 interferograms were collected, Fourier transformed and averaged. Absorption spectra in the region between 4000 and 600 cm 1, at a resolution of one data point every 0.6 cm 1, were obtained using a clean crystal as the background. Spectral analysis was performed with Grams 32 software from Galactic Industries (USA). All measurements were done at 20 C. 3. Results and discussion Fig. 2 presents the X-ray diffractograms of the EYPC multibilayers containing incorporated different amount of AmB. The multibilayer periodicity, representing the Fig. 3. AmB concentration dependence of X-ray determined EYPC multibilayer periodicity. Mean from three to six determinations ± S.D. lipid bilayer thickness and the layer of tightly bound water between the lipid bilayers (Gruszecki et al., 1992), determined on the basis of the Bragg s law for the samples containing different amount of AmB are presented in Fig. 3. As can be seen, incorporation of the drug does not influence the multibilayer periodicity parameter, in the concentration range between 0.1 and 3 mol%. Fig. 4 presents the electron density profiles across the EYPC bilayer and the bilayer containing incorporated AmB, calculated on the basis of the X-ray diffractograms. The thickness of the lipid bilayer, calculated as a distance between the maxima of the profile (minimum of the second derivative of the profile), was 39.3 Å in the case of pure EYPC, 39.2 Å in the case of 0.2 mol% AmB and Fig. 2. θ 2θ X-ray diffractograms recorded from the multilayers formed with EYPC containing 0.01 and 1 mol% amphotericin B, indicated. Fig. 4. Electron density profiles from X-ray studies obtained by Fourier reconstruction as a function of distance z along the normal to the bilayer with the center of bilayer at z = 0. Bilayers were formed with pure EYPC and EYPC containing 0.2 mol% amphotericin B (indicated).

4 M. Hereć et al. / Chemistry and Physics of Lipids 147 (2007) Å in the case of the membrane containing 1.0 mol% AmB (not shown). The X-ray analysis shows therefore that AmB does not affect essentially the membrane thickness. Accordingly, no very pronounced effect had been observed on the shape of the electron density profiles. Slight changes in the profile corresponding to the polar headgroup region of AmB-containing membranes (z spacing +20 and 20 Å; Fig. 4) may be associated with possible rearrangement of phosphate groups, responsible predominantly for the X-ray scattering, owing to the interactions of these groups with the drug. Such an effect suggests the polar membrane zone as a place of localization of AmB in the lipid bilayer. On the other hand, certain alterations in the electron density profiles due to the presence of AmB can be also observed at z spacing values ±6 and ±12 Å, corresponding to the hydrophobic core of the membrane. It is possible that such an effect represents a fraction of antibiotic molecules that penetrate the membrane but an indirect effect of AmB bound to the polar headgroup region on the membrane interior may not be excluded. The X-ray diffractometry applied to the lipid multibilayer showed no effect of AmB on the periodicity parameter of the system. Effect of the antibiotic of thickness of a single bilayer lipid membrane can be analysed with application of small-angle neutron scattering technique applied to unilamellar liposome suspension (see Fig. 5). The calculated liposomal membrane thickness values are 37.6 ± 0.6 Å in the case of pure EYPC, 37.6 ± 0.6 Å in the case of EYPC containing 0.2 mol% AmB and 36.6 ± 0.6 Å in the case of Fig. 5. The dependence of SANS intensity I(Q) on the scattering vector Q for extruded unilamellar EYPC liposomes (A), and liposomes containing 0.2 mol% AmB (B) and 1 mol% AmB (C). The calculated liposomal membrane thickness values are 37.6 ± 0.6 Å in the case of pure EYPC, 37.6 ± 0.6 Å in the case of EYPC containing 0.2 mol% AmB and 36.6 ± 0.6 Å in the case of EYPC containing 1 mol% AmB. EYPC containing 1 mol% AmB. The lipid membrane thickness values of liposomes, determined on the basis of the neutron scattering data are slightly lower than the thickness of the lipid bilayer in the hydrated multilayer systems. The difference may be directly related to different model membrane systems examined (multilayers versus liposomes) or to differences in the approach applied to determine the lipid bilayer thickness (distance of the maxima of electron density profiles versus determination based on the radius of gyration of liposomes; Uhrikova et al., 2000, 2003). In both model systems AmB did not influence the thickness of the membrane. Interestingly, incorporation of comparable amount of carotenoid pigments, characterized by rigid, rod-like polyene chain as in the case of AmB, increases the lipid membrane thickness, owing to the van der Waals interactions between the pigments and alkyl lipid chains, that promote their extended conformation (Gruszecki, 1999, 2004; Gruszecki and Strzalka, 2005). The lack of such an effect in the case of AmB suggests that either the additive is not located in the hydrophobic core of the membrane or remains in the aggregated form that restricts its influence on the structural and dynamic properties of lipid membranes owing to the inhomogeneous distribution. The localization of AmB in the polar headgroup region of the lipid bilayer has also been concluded on the basis of the UV Vis linear dichroism (Gruszecki et al., 2003a,b) and FTIR linear dichroism (Gagos et al., 2005) studies. The results of the linear dichroism measurements are consistent with a presence of a certain fraction of amphotericin B parallel to the plane of the membrane and owing to the amphiphilic structure of the antibiotic and to the energetic reasons a localization of this fraction outside the hydrophobic core of the lipid bilayer has been concluded (Gruszecki et al., 2003a,b; Gagos et al., 2005). A localization of other polyene antibiotic, Nystatin, in the membrane polar headgroup region has been concluded on the basis of fluorescence quenching measurements (Moribe et al., 2000) and fluorescence decay kinetics analysis (Coutinho et al., 2004). Fig. 6 presents the IR absorption spectra in the fingerprint region of hydrated lipid multibilayers formed on ZnSe crystal with pure EYPC and multibilayers containing different amount of AmB. Several bands visible in this spectral region represent vibrations of the groups located in the polar headgroup region of the membrane: antisymmetric N + (CH 3 ) 3 stretching vibrations of choline (969 cm 1 ), symmetric (1090 cm 1 ) and antisymmetric (1249 cm 1 ) stretching vibrations of PO 2 group, C O stretching of the ester group (1169 cm 1 ) and stretching vibrations of the C O P O C fragment (1066 cm 1 )(Lewis and Mcelhaney, 1996). The fre-

5 82 M. Hereć et al. / Chemistry and Physics of Lipids 147 (2007) Fig. 7. Dependence of the position of the absorption band corresponding to the ν as PO 2 vibrations in IR absorption spectra of EYPC multilayers on AmB concentration. Fig. 6. Infrared absorption spectra of multilayers formed with pure EYPC and EYPC containing different concentrations of amphotericin B (indicated) in cm 1 spectral range. Multilayers were deposited on ZnSe crystal. Dashed lines indicate position of selected absorption bands in the lipid sample without AmB. in the case of the C O P O C stretching vibrations (Fig. 8), that can be also interpreted in terms of hydrogen bonding to this group. Interestingly, the shape of the dependencies corresponds well to the concentrationdependent aggregation of AmB in the system (Fig. 9). This can be seen from the analysis of the electronic absorption spectra of the EYPC liposome suspension containing different amount of AmB (Fig. 9). The absorption spectra of AmB in liposomes display relatively complex structure owing to the fact that they are quency of antisymmetric stretching (ν a )ofpo 2 is exceptionally sensitive to hydrogen bonding (Barth and Zscherp, 2002). As can be seen, the position of this band is sensitive to AmB presence in the system. Fig. 7 presents the AmB concentration dependence of the ν a PO 2 band position. The band becomes shifted towards lower frequencies upon incorporation of AmB to the membranes. This effect can be readily interpreted in terms of hydrogen bonding to PO 2 group, either AmB or water molecules, influenced by the presence of the drug in the system. The dependency presented in Fig. 7 is clearly biphasic: a strong concentration dependence below 1 mol% AmB and practically unchanged band position in the concentration range above 1 mol%. Similar, biphasic concentration dependency can be observed Fig. 8. Dependence of the position of the absorption band corresponding to the stretching vibrations of the C O P O-C fragment in IR absorption spectra of EYPC multilayers on AmB concentration.

6 M. Hereć et al. / Chemistry and Physics of Lipids 147 (2007) Fig. 9. Electronic absorption spectra recorded from EYPC liposomes containing incorporated different amount of AmB (indicated). The inset shows AmB concentration dependency of the absorbance ratio recorded at 408 and 325 nm, corresponding to the absorption maxima that represent monomeric and aggregated form of AmB, respectively. composed of the spectra of various organization forms of the drug, including monomers (absorption maximum at 408 nm), dimers (absorption maximum at 350 nm) and larger aggregates (absorption maximum at 325 nm) (Barwicz et al., 1993; Gaboriau et al., 1997; Gagos et al., 2001; Gruszecki et al., 2003a,b; Baginski et al., 2006). The dependency of the absorbance ratio recorded at 408 and 325 nm, presented in the inset to Fig. 9, shows that AmB remains largely in the aggregated form at concentrations above 1 mol% with respect to lipid. This particular concentration can be referred to as the aggregation threshold of AmB in the lipid system applied. The symmetric stretching vibrations of the N + (CH 3 ) 3 groups are also sensitive to the AmB presence in the membranes (Fig. 10). Interestingly, the corresponding spectral band, that peaks at 926 cm 1, becomes shifted to 910 cm 1 upon AmB incorporation but the maximum of this band can be seen again at 926 cm 1 at the relatively high concentration of the drug (5 mol%). According to the analysis presented in Fig. 9, AmB remains largely aggregated at such a high concentration. Apparently, aggregated drug has limited influence on the membrane region characterized by location of the choline groups. This effect is most probably associated with inhomogeneous distribution of aggregated molecules of AmB with respect to the lipid phase. Aggregation of AmB in the lipid membrane system is considered as a principal molecular mechanism that enables penetration of the drug into the hydrophobic core of the lipid bilayer owing to the amphiphilic character of AmB molecule Fig. 10. Infrared absorption spectra of multilayers formed with pure lipid EYPC and EYPC containing different concentrations of AmB, in cm 1 spectral range. Multilayers were deposited on ZnSe crystal. Dashed lines indicate position of the absorption bands assigned to the ν s N + (CH 3 ) 3 vibrations. (see Fig. 1) (De Kruijff et al., 1974; Fujii et al., 1997; Gruszecki et al., 2002, 2003a,b; Matsuoka and Murata, 2003; Baginski et al., 2006). Fig. 11 presents the IR absorption spectra in the region characteristic of the symmetric C H stretching vibrations of methylene groups of lipid alkyl chains that form a hydrophobic core of the lipid bilayer. As can be seen from the analysis of the position of this band (Fig. 12), the incorporation of AmB at low concentrations, results in the increase in the frequency of CH 2 vibrations. This spectral effect is indicative of the increase in the motional freedom of alkyl chains (decreased order parameter). It is highly probable that AmB molecules bound to the polar headgroup region increase distance between the lipid molecules and provide more freedom for gauche-trans isomerization of alkyl chains within the hydrophobic membrane core. The process of formation of molecular aggregates of AmB, at concentrations higher than 1 mol%, is associated with a shift of the CH 2 vibration frequencies towards lower values. Such a shift is diag-

7 84 M. Hereć et al. / Chemistry and Physics of Lipids 147 (2007) Fig. 12. Dependence of the position of the infrared absorption band corresponding to the symmetric stretching vibrations of the methylene groups of alkyl chains of EYPC multilayers on AmB concentration. The position of the band in the EYPC sample without AmB indicated with dashed line. Note that all experimental points are located above this line in the case of AmB concentrations lower than 1 mol% and below this line in the case of AmB concentrations exceeding 1 mol%. Fig. 11. Infrared absorption spectra of multilayers formed with pure lipid EYPC and EYPC containing different concentrations of AmB, in cm 1 spectral range. Multilayers were deposited on ZnSe crystal. Dashed lines indicate position of the absorption band assigned to the ν s CH 2 vibrations. nostic of alkyl chain ordering (Lewis and Mcelhaney, 1996) and therefore reflects binding of AmB molecules into the hydrophobic core of the membrane. Such a binding results in van der Waals interactions between alkyl chains of the lipid molecules and rigid polyene chains of AmB, that restrict gauche-trans isomerization of alkyl chains. An ordering effect of AmB with respect to lipid membranes has been also demonstrated with application of differential scanning calorimetry (Fournier et al., 1998) and 2 H NMR technique (Paquet et al., 2002). Another aspect of such a binding is formation of porous aggregated structures, characterized by the internal diameter of 0.6 nm (Gruszecki et al., 2002). The results of the experiments presented in this work show that the polyene antibiotic amphotericin B in a monomeric form binds preferentially to the phospho- lipid membrane polar headgroup region. This fraction of the antibiotic molecules is most probably responsible for the sealing effect manifested by an increase of the barrier for transmembrane ion transport (Herec et al., 2005). At concentrations exceeding the aggregation threshold (1 mol% in EYPC membranes at room temperature) AmB forms molecular structures that penetrate the hydrophobic core of the lipid bilayer. Acknowledgement This research was financed by the Ministry of Science and Higher Education of Poland from the budget funds for science in the years within the research project 2P05F References Aracava, Y., Schreier, S., Phadke, R., Deslauriers, R., Smith, I.C.P., Effect of amphotericin B on membrane permeability-kinetics of spin probe reduction. Biophys. Chem. 14, Baginski, M., Cybulska, B., Gruszecki, W.I., Interaction of macrolide antibiotics with lipid membranes. In: Ottova-Liu, A. (Ed.), Advances in Planar Lipid Bilayers and Liposomes. Elsevier Science, Amsterdam, pp Barth, A., Zscherp, C., What vibrations tell us about proteins. Q. Rev. Biophys. 35, Barwicz, J., Gruszecki, W.I., Gruda, I., Spontaneous organization of amphotericin B in aqueous medium. J. Colloid Interface Sci. 158,

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