ORGANIZATION OF ANTIBIOTIC AMPHOTERICIN B IN MODEL LIPID

CELLULAR & MOLECULAR BIOLOGY LETTERS

Volume 8, (2003) pp 161 – 170 http://www.cmbl.org.pl Received 30 September 2002 Accepted 13 February 2003

ORGANIZATION OF ANTIBIOTIC AMPHOTERICIN B IN MODEL LIPID MEMBRANES. A MINI REVIEW WIESŁAW I. GRUSZECKI1*, MARIUSZ GAGOŚ2, MONIKA HEREĆ1 and PETER KERNEN3 1 Department of Biophysics, Institute of Physics, Maria Curie-Skłodowska University, 20-031 Lublin, Poland, 2Department of Physics, Agricultural University, Lublin, Poland, 3Zyomyx, Inc., 26101 Research Road, Hayward, CA 94545, USA Abstract: Amphotericin B (AmB) is a polyene antibiotic frequently applied in the treatment of fungal infections. According to the general understanding, the mode of action of AmB is directly related to the molecular organization of the drug in the lipid environment, in particular to the formation of pore-like molecular aggregates. Electronic absorption and fluorescence techniques were applied to investigate formation of molecular aggregates of AmB in the lipid environment of liposomes and monomolecular layers formed at the argon-water interface. It appears that AmB dimers, stabilized by van der Waals interactions, are present in the membrane environment along with the aggregates formed by a greater number of molecules. Linear dichroism measurements reveal that AmB is distributed between two fractions of molecules, differently oriented with respect to the bilayer. Molecules in one fraction remain parallel to the plane of the membrane and molecules in the other one are perpendicular. Scanning Force Microscopy imaging of the surface topography of the monolayers formed with AmB in the presence of lipids reveals formation of pore-like structures characterized by the external diameter close to 17 Å and the internal diameter close to 6 Å. All the findings are discussed in terms of importance of the molecular organization of AmB in the pharmacological action, as well as of the toxic side effects of the drug. Key Words: Amphotericin B, Molecular Aggregates, Lipid Membranes

*Corresponding author, Fax + (4881) 537 61 91, E-mail: [email protected] Abbreviations used: AmB - amphotericin B; SFM - scanning force microscopy; DPPC dipalmitoylphosphatidylcholine

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INTRODUCTION

Fig. 1. Chemical structure of amphotericin B

Amphotericin B (AmB) is a polyene antibiotic frequently used in medical treatment of systemic fungal infections. According to the general understanding, the pharmacological action of the drug is directly associated with the formation by its molecules of pore structures across biomembranes, which considerably affects physiological ion transport [1, 2]. The ability of AmB to form hydrophilic pores results directly from the amphiphilic molecular structure of the drug (see Fig. 1). Selectivity of AmB toward lipid membranes of fungi is most probably related to the presence of ergosterol (instead of cholesterol), the sterol supposed to participate in the formation of membrane pores [1, 2]. On the other hand, the ionic channel activity has also been observed in the lipid membrane system containing AmB without any sterols [3]. Aggregation of AmB and pore formation in the ergosterol-free membranes is most probably a direct cause of the toxic side effects associated with medical treatment with the application of preparations based on AmB [4]. It has been proposed that not only formation of porous molecular structures, but also affection of the physical properties of the lipid bilayers brings about increased membrane permeability to ions [5]. This effect is expected to be particularly pronounced at low concentrations of AmB, promoting monomeric organization of the drug within the lipid phase. In this work we present our recent research, carried out with the application of electronic spectroscopic techniques, aimed to elucidate the molecular organization of AmB in the lipid membrane system. Understanding of molecular mechanisms that govern the organization of AmB in the lipid environment seems to be important not only for the understanding of various biological effects, but also for minimizing severe side effects of the drug. SPECTROSCOPY OF AMPHOTERICIN B IN LIPID MEMBRANES The electronic absorption spectrum of AmB present as a monomer in the 2propanol:water mixture and embedded in lipid membranes is shown in Figure 2. A very distinct hypsochromic shift of the spectrum is indicative of the formation of molecular aggregates, the so-called “card pack” or H-type structures. Such a spectral effect may be interpreted as a manifestation of the formation of


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Fig. 2. Absorption spectra of AmB: in monomeric form in solution of 2-propanol:water (4:6, v:v) - solid line, in aggregate form in suspension of DPPC liposomes containing 5 mol% AmB - dashed line.

molecular pores: hydrophobic [6] or hydrophilic [7], depending on the polarity of the environment. On the other hand, a very similar spectral shift can be reproduced in the monomolecular layers formed with AmB [8]. The molecular organization of AmB in this system gave rise to the hypsochromic spectral shift, but an analysis of the sample topography, carried out with the application of Scanning Force Microscopy (SFM), revealed semi homogeneous distribution of molecules [8]. Interestingly, the presence of lipids in the monolayer system, at a concentration as low as 10 mol%, has been found to promote formation of pore structures by AmB molecules, characterized by the internal diameter of 6 Å and the external diameter of 17 Å [8]. The SFM image of such a structure is presented in Figure 3. The distance between the chromophores of the molecules involved in the formation of the hydrophilic pore (a possible model presented in Fig. 4) is comparable with the distance between the chromophores in the linear aggregate, and therefore both molecular structures, in principle, may give rise to very similar spectroscopic effects. The spectral shift accompanying the aggregation can be described by the following formula [9]:  mπ  ν m = ν mon + 2 β cos   N +1

(1)

where νm is the position of the m-th excitation state in the spectra of the aggregated form, νmon is the position of the electronic transition in the spectrum

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Fig. 3. Scanning Force Microscopy image of a two-component AmB-DPPC monolayer (9:1) deposited to mica. The 3D-view is shown with scan sizes of 2nm x 2nm and is plotted with z-range of 0.1 nm. A pore-like structure marked with a white circle.

cylindrical aggregate

chromophore

polar groups

AmB Fig. 4. Schematic representation of the cylindrical aggregate structure of AmB


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Fig. 5. Temperature dependences of absorption spectra of DPPC liposomes containing 1 mol% AmB

of monomeric chromophores, N is the number of all exciton states, and m is the number of the exciton state considered. In the case of a maximum hypsochromic shift m=1. β is a dipole-dipole coupling matrix element [9] and is expressed as: β=

µ mon

2

4πε oη 2 R 3

(cosθ − 3 cos α ) 2

(2)

In the above formula µmon is the dipole transition moment of a monomer, εo is the dielectric permittivity of free space, η is the refractive index of the medium, and R is the distance between the centers of the transition dipoles of the nearest neighbors in the aggregate. The angle between monomeric transition moment vectors θ, and the angle between the transition moment vector and the axis connecting the centers of the transition moment vectors of neighboring molecules α, were assumed to be 0 deg. and 90 deg. in the case of H-aggregates of AmB. Figure 5 presents temperature dependencies of the electronic absorption spectra of AmB embedded in dipalmitoylphosphatidylcholine (DPPC) liposomes at a concentration of 1 mol%. The two spectral forms, corresponding to monomeric and aggregated AmB, can be distinguished in the temperature range both below and above the main phase transition (Pβ’→Lα). The short-wavelength absorption bands of AmB, indicative of aggregated molecular structures, which are particularly pronounced in the ordered phase of DPPC, decrease in the intensity at the temperatures above the phase transition.

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As may be seen, the short-wavelength absorption maximum shifts also from ca. 350 nm to 332 nm in this temperature range. Analyzing the formation of molecular structures manifested by the hypsochromic shift from 408 nm (monomeric AmB) to 332 nm one arrives, for example, at a model of a linear aggregate formed with 6 molecules or at a model of a cylindrical aggregate formed with 8 AmB molecules, in which polar groups are located inside a structure (see Fig. 4). In the first case the neighboring chromophores are spaced by R=4.9 Å and in the second by R=4.7 Å. A value of 11.3 Debye for the transition dipole of monomeric AmB was taken for calculations, as evaluated based on the integration of the absorption spectra in 2-propanol:water (see Fig. 2), and η was taken as 1.40, corresponding to the hydrophobic core of the DPPC membrane [7]. Other combinations of the number of molecules (between 6 and 10) and the distance of chromophores are also possible to reproduce the spectroscopic effects observed in the AmB-containing DPPC liposomes [7]. Interestingly, linear dichroism measurements carried out in the AmB-containing oriented multibilayers formed with DPPC reveal that the average dipole transition moment of monomeric AmB forms an angle close to 40 deg. with respect to the axis normal to the plane of the lipid membrane. Such a result can be understood as an expression of the presence of AmB in two, orthogonal pools of AmB, one oriented in the plane of the membrane (ca. 38 % molecules) and one perpendicular to it (ca. 62 % molecules). Figure 6A presents the electronic absorption spectra of AmB embedded in oriented DPPC multibilayers, recorded with the probing light beam polarized in the plane of incidence, and with a tilt angle of 0 deg. and 45 deg. The difference between these spectra (Fig. 6B) shows a distinct band in the short-wavelength region, thus indicating that aggregated molecular forms are primarily present in the fraction oriented in the plane of the membrane. Very likely, this fraction represents linear aggregates, not involved in the formation of pores across the membrane. On the other hand, one should not exclude the effects of horizontally oriented AmB molecules on the physical properties of lipid membranes. The chromophores of those molecules are also located in the hydrocarbon zone of the membrane (most probably in the interface region), but polar groups are very likely to form hydrogen bonds with head groups of lipids. Currently we are working on a method to distinguish the effects of these different pools of AmB on physical properties of lipid membranes, in particular directly related to passive ion transport. Despite the exact model of the aggregated forms of AmB that give rise to the hypsochromic shift, discussed above, this spectral effect may be applied to follow aggregation level of the drug in the lipid membrane environment. Recent studies carried out in our laboratory revealed that also dimeric forms of AmB have a specific spectroscopic signature [10]. A relatively high fluorescence quantum yield of the dimers of AmB (Q=4, excitation at 352 nm), as compared to the fluorescence quantum yield of the monomers (Q=0.06, excitation at 408 nm), enables discrimination of these two spectral forms.


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Fig. 6. Electronic absorption spectra of an oriented multilayer formed with 200 bilayers of DPPC containing 2 mol% AmB, recorded with polarized light in which the electric vector was oriented parallel with respect to the plane of incidence and the axis normal to the plane of the sample was tilted by 0 and 45 deg. with respect to the direction of the measuring beam (panel A) and the difference of these spectra (panel B). Fluorescence Intensity [a.u.]

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Fig. 7. Temperature dependences of the fluorescence signal corresponding to the dimeric (Ex 352 nm, Em 471 nm) and monomeric (Ex 408 nm, Em 606 nm) form of AmB embedded in DPPC liposomes at 5 mol% concentration and the ratio of fluorescence intensities, representing changes of monomers and dimers. Lines were fit in the temperature range below 35oC and above 45oC. The line cross-section points at the temperature of the main phase transition of lipid membranes formed with DPPC (Tm ).


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Fig. 8. Temperature dependences of the ratio of Gaussian components representing the 0-0 transition of the monomeric structure (centered at 408 nm) and the aggregated structure (centered at 334 nm) produced on the basis of the Gaussian analysis of absorption spectra of DPPC liposomes containing 1 mol% AmB (upper panel) and temperature dependences of the ratio of fluorescence intensities, representing changes of proportion of monomers (Ex 408 nm, Em 606 nm) and dimers (Ex 352 nm, Em 471) of AmB embedded in DPPC liposomes at 1 mol%.

Figure 7 presents the temperature dependencies of fluorescence signals corresponding to the dimeric and monomeric forms of AmB embedded in DPPC liposomes. The ratio of these traces displays a distinct transition that corresponds to the main phase transition of DPPC. As may be seen, the rise in the temperature of liposomes results in a relative increase in the population of monomers. A very similar effect was observed in the case of AmB aggregated structures, as demonstrated by means of the Gaussian analysis of the electronic absorption spectra of AmB-containing DPPC liposomes (see Fig. 8). Figure 9 presents concentration profiles of the dimer to monomer and aggregate to monomer ratios, based on fluorescence and absorption measurements, respectively. Apparently, the maximum that appears at 1 mol% AmB in DPPC in the dimer concentration, corresponds to the minimum in the concentration of the aggregated structures. Such an apparent paradox may be understood in terms of composition of molecular structures of AmB at the expense of dimers. The


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Fig. 9. Concentration profile of the AmB dimerization (panel A) and aggregation (panel B) level expressed as the ID/IM and IA/IM ratio, respectively, corresponding to 35 oC. The level of dimers and aggregates relative to monomers was determined on the basis of fluorescence and absorption measurements, as explained in the caption to Fig. 8.

appearance of the minimum in the aggregation level corresponding to a certain concentration of AmB, itself, seems to be particularly interesting and may suggest different (opposite?) biological effects of the drug, depending on its actual local concentration in the lipid phase. Acknowledgements. This work was supported by the Polish Committee for Scientific Research (KBN) Grant No. 3P04A 076 22. REFERENCES 1. De Kruijff, B. and Demel, R.A. Polyene antibiotic-sterol interaction in membranes of Acholeplasma Laidlawii cells and lecithin liposomes; III Molecular structure of the polyene antibiotic-cholesterol complex. Biochim. Biophys. Acta. 339 (1974) 57-70.

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2. Baginski, M., Resat, H. and McCammon, J.A. Molecular properties of amphotericin B membrane channel: a molecular dynamics simulation. Mol. Pharmacol. 52 (1997) 560-570. 3. Cotero, B.V., Rebolledo-Antunez, S. and Ortega-Blake, I. On the role of sterol in the formation of the amphotericin B channel. Biochim. Biophys. Acta 1375 (1998) 43-51. 4. Hartsel, S. and Bolard, J. Amphotericin B: new life for an old drug. TiPS 17 (1996) 445-449. 5. Wójtowicz, K., Gruszecki, W. I., Walicka, M. and Barwicz, J. Effect of amphotericin B on dipalmitoylphosphatidylcholine membranes: calorimetry, ultrasound absorption and monolayer technique studies. Biochim. Biophys. Acta 1373 (1998) 220-226. 6. Barwicz, J., Gruszecki, W.I. and Gruda I. Spontaneous Organization of Amphotericin B in Aqueous Medium. J. Colloid Interf. Sci. 158 (1993) 7176. 7. Gagoś, M., Koper, R. and Gruszecki, W.I. Spectrophotometric analysis of organisation of dipalmitoylphosphatidylcholine bilayers containing the polyene antibiotic amphotericin B. Biochim. Biophys. Acta 1511 (2001) 90-98. 8. Gruszecki, W.I., Gagoś, M. and Kernen, P. Polyene Antibiotic Amphotericin B in Monomolecular Layers: Spectrophotometric and Scanning Force Microscopic Analysis. FEBS Lett. 524 (2002) 92-96. 9. Parkash, J., Robblee, J. H., Agnew, J., Gibbs, E., Collings, P., Pasternack, R. F. and de Paula, J. C. Depolarized Resonance Light Scattering by Porphyrin and Chlorophyll a Aggregates. Biophys. J. 74 (1998) 2089-2099. 10. Gruszecki, W.I., Gagoś, M. and Hereć, M. Dimers of polyene antibiotic amphotericin B detected by means of fluorescence spectroscopy: molecular organization in solution and in lipid membranes. J. Photochem. Photobiol. B: Biol. 69 (2003) 49-57.