Self-Association of Amphotericin B: Spontaneous Formation of

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Self-Association of Amphotericin B: Spontaneous Formation of Molecular Structures Responsible for the Toxic Side Effects of the Antibiotic Joanna Starzyk,† Marcin Gruszecki,‡ Krzysztof Tutaj,† Rafal Luchowski,† Radoslaw Szlazak,† Piotr Wasko,† Wojciech Grudzinski,† Jacek Czub,§ and Wieslaw I. Gruszecki*,† †

Department of Biophysics, Institute of Physics, Maria Curie-Skłodowska University, Lublin, Poland Department of Informatics and Statistics, Medical University of Gdansk, Gdansk, Poland § Department of Physical Chemistry, Gdansk University of Technology, Gdansk, Poland ‡

S Supporting Information *

ABSTRACT: Amphotericin B (AmB) is a lifesaving antibiotic used to treat deep-seated mycotic infections. Both the pharmaceutical activity and highly toxic side effects of the drug rely on its interaction with biomembranes, which is governed by the molecular organization of AmB. In the present work, we present a detailed analysis of self-assembly of AmB molecules in different environments, interesting from the physiological standpoint, based on molecular spectroscopy techniques: electronic absorption, circular dichroism, steady state and time-resolved fluorescence and molecular dynamic calculations. The results show that, in the water medium, AmB self-associates to dimeric structures, referred to as “parallel” and “antiparallel”. AmB dimers can further assemble into tetramers which can play a role of transmembrane ion channels, affecting electrophysiological homeostasis of a living cell. Understanding structural determinants of self-assembly of AmB opens a way to engineering preparations of the drug which retain pharmaceutical effectiveness under reduced toxicity.

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INTRODUCTION Amphotericin B (AmB, Figure 1) is a life-saving polyene antibiotic used as a gold standard to treat systemic fungal infections.1 The drug has been in use for over 50 years due to its exceptional effectiveness, regardless of numerous severe side effects which can be lethal to patients.2 Despite almost half of a century of research on the mode of action of AmB, this issue seems to still be open and is a subject of intensive studies carried out in several laboratories.3−6 According to a generally accepted model, AmB self-associates in the lipid membrane environment into a pore-like structure (actually, two compatible and aligned structures, one in each lipid monolayer forming the bilayer lipid membrane).7 Such structures can act as transmembrane channels affecting physiological ion transport which lead to cell death. Recently, the results of a combined spectroscopic study and molecular modeling have shown that a single molecular organization form of AmB, a tetramer, can span a lipid bilayer and act as a transmembrane ion channel.8 The results of the studies carried out with model lipid membrane systems show that, opposite to higher drug concentration, AmB at relatively low molar fractions (below 3 mol %), with respect to lipids, does not enter the membrane interior9,10 and seals the membranes rather than induces ion leakage.11 In this respect, the selective interaction of AmB to ergosterol, the sterol in fungi, is of great significance.3,12−14 Very interestingly, it has been recently reported that AmB can © 2014 American Chemical Society

exist in the form of large, sponge-like extramembraneous aggregates which are able to extract ergosterol from lipid bilayers, leading to lethal destabilization of biomembranes.4 All the results referred to above point to the importance of molecular organization of AmB in both desirable pharmacological effects and toxic side effects of the drug. Spectra of different organization forms of AmB can be recorded by various molecular spectroscopy techniques, including electronic absorption,15−17 CD,15,18,19 fluorescence,8,20,21 FTIR,6,14 and Raman scattering.22 On the other hand, in most cases, the analyses enabled distinguishing of monomeric AmB from the structures formed by a large number of molecules, without insight into the problem of formation of small aggregates. Moreover, it appeared that the spectra typically assigned to AmB monomers are in general more complex and hide components of small aggregates of the drug.8 In the present work, we address the problem of spectral signatures of individual molecular organization forms of AmB, important in biological activity of the drug. Comparative spectroscopic analyses based on electronic absorption, CD, steady-state, and time-resolved fluorescence techniques enabled elaboration of “spectral tools” applicable in analysis of the molecular Received: October 10, 2014 Revised: November 7, 2014 Published: November 10, 2014 13821

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Figure 1. Chemical structure (upper panel) and atomic model (lower panel) of the AmB molecule. In the lower panel, the AmB molecule is divided into four segments which were used for the analysis of interactions in dimeric AmB structures. These four segments (I−IV) have been arbitrarily selected in order to estimate involvement of different groups of AmB in the formation and stabilization of a dimer (see also Figure 9).

ultrasonic processor (Sonics Inc., USA) for 15 cycles of 3 s with 100% amplitude with a titanium probe. AmB-containing lipid multibilayers were prepared by evaporation of a suspension of AmB-containing liposomes at the surface of a quartz slide. Before measurements, lipid layers were transferred to a vacuum for 30 min and afterward left to hydrate for 1 h in air. Such a procedure yielded formation of a lipid multilayer consisting of ca. 100 bilayers. Spectroscopic Measurements. Absorption spectra were recorded with a Cary 50 UV−vis spectrophotometer (Varian, Australia). Fluorescence excitation and fluorescence emission spectra were recorded with a Cary Eclipse fluorescence spectrophotometer (Varian, Australia). Fluorescence excitation spectra were recorded in the range 300−480 nm with correction for the lamp characteristics and with application of the long-wavelength-pass 495 nm filter in front of the detector. Fluorescence emission spectra were recorded with application of the long-wavelength-pass 400 nm filter placed in front of the detector. During recording of fluorescence excitation and emission spectra, both the excitation and emission slits were set to 5 nm bandwidth. CD spectra were recorded with application of a Chirascan Plus Spectrometer (Applied Photophysics, U.K.). Spectra were recorded in the range 220−600 nm, at room temperature, with a 1 nm resolution. The scan rate was 60 nm/min. Recorded spectra were analyzed with application of the Grams/AI software from Thermo Scientific (USA). Lifetime measurements were conducted by time-correlated single photon counting (TCSPC) experiments, using a FluoTime 300 fluorescence lifetime spectrometer from PicoQuant (Germany). Samples were excited at 402.3 nm (with a solid state laser LDH−P−C−405 with a pulse width of 70 ps). Fluorescence lifetime was recorded with the application of long-wavelength-pass 400 and 420 nm filters placed in front of the detector in order to eliminate scattered and reflected light. The laser excitation repetition rate was fixed to 20 MHz

organization of AmB in pharmacological preparations and biological systems.

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EXPERIMENTAL SECTION Materials. Amphotericin B (AmB) 80% grade from Streptomyces, dipalmitoylphosphatidylcholine (DPPC), dimethyl sulfoxide (DMSO), and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich Chem. Co. (USA). Potassium hydroxide (KOH) was obtained from POCH (Poland). As described previously,8 crystalline AmB was suspended in chloroform−water (1:1, v/v) and vortexed for 30 min, and then collected from the interphase and evaporated under nitrogen. Dry AmB was dissolved in 2-propanol−water (4:6, v/v) and centrifuged for 5 min at 8765 × g, in order to remove microcrystals of the drug still remaining in the sample, and was purified by means of HPLC. Water used for experiments was purified by a Milli-Q system from Merck Millipore (France, specific resistivity 18.2 MΩ cm). Sample Preparation. In order to transfer AmB to different solvents (DMSO, PBS, pH 12 water alkalized with KOH), AmB dissolved in HPLC mobile phase (2-propanol:water, 4:6, by volume) was dried under a vacuum and transferred to the required solvent. The final concentration of AmB in each solvent was 9.2 × 10−6 M. In order to prepare AmB-containing liposomes, DPPC was dissolved in ethanol and then admixed with pure AmB dissolved in 2-propanol−water (4:6, v:v). The mixture was dried under gaseous nitrogen. The dried sample was further kept under a vacuum for 30 min in order to remove traces of organic solvents. Liposomes were formed in water. Large multilamellar vesicles (MLVs) were prepared by rehydration with Milli-Q water and incubation in a circulating water bath for 5 min at 50 °C (a temperature above the main phase transition of DPPC) with brief vortex mixing for 10 min. This process was repeated three times. Small unilamellar vesicles (SUVs) were prepared by sonication with a VCX-130 13822

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and detection performed with a microchannel plate photomultiplier tube (MCP- PMT, Hamamatsu, Japan). Time decays were fitted using an exponential model implemented in the FluoFit Pro software package (v 4.5.3.0) and expressed by the relation I (t ) =

∑ αi exp(−t /τi) i

(1)

where τi are the decay times and αi are the pre-exponential factors (amplitudes) of the individual components (∑αi = 1). Quartz cuvettes with a 1 cm optical path were applied for all measurements. Fluorescence Lifetime Imaging Microscopy (FLIM). FLIM measurements were performed on a confocal MicroTime 200 (PicoQuant, GmbH, Germany) system coupled to an OLYMPUS IX71 microscope. The samples were excited at a 405 nm pulsed laser, with a 20 MHz repetition rate. The fwhm of a pulse response function was 52 ps (measured by PicoQuant, Inc.). The time resolution was kept at 4 ps. Photons were collected with a 60× water immersed objective (NA 1.2, OLYMPUS UPlanAPO). Therefore, the restricted effective confocal volume was 0.21 fL. The scattered light was removed by a ZT 405RDC band-pass interference filter (Chroma Technology), and the observation was made through an additional 430 long-wavelength-pass filter. Fluorescence photons were collected with the single photon sensitive avalanche photodiode (τ-SPAD) with processing accomplished by the HydraHarp400 time-correlated single photon counting (TCSPC) module. Data analysis was performed using the SymPhoTime 64 software package (v. 1.6). Molecular Dynamics Simulations. We performed a series of molecular dynamics simulations of AmB dimer in an aqueous environment. All energy minimizations and molecular dynamics simulations were performed using NAMD.23 For the antibiotic molecule, the same parameter set was used as in the number of previous studies.24−26 The preformed dimers (see below) were placed in a 5.3 × 5.3 × 5.3 nm3 box containing 14540 TIP3P water molecules and 14 K+ and 14 Cl− ions, respectively, to obtain a physiological concentration of 0.15 mol/dm3. The pressure was kept constant at 1 bar using the Langevin piston method.27 The temperature was maintained at 300 K according to Langevin dynamics. The particle-mesh Ewald method28 was used to calculate long-range electrostatic interactions with a mesh size of ∼1.0 Å and cubic interpolation. The LennardJones potential and forces were smoothly switched off between 10 and 12 Å. The SHAKE algorithm29 was used to constrain covalent bonds involving hydrogen atoms, except for water, for which the SETTLE algorithm30 was applied. This allowed for a time step of Δt = 2 fs to be used to integrate the equations of motion in the velocity Verlet algorithm. The initial coordinates for the AmB monomers were obtained from the most recent and accurate crystallographic data.31 We considered two different possible dimer topologies: parallel and antiparallel (see Figure S1, Supporting Information). Their initial structures were obtained as the low-energy configurations from a systematic search procedure described in detail in Neumann et al.26

Figure 2. Absorption and circular dichroism spectra of AmB dissolved in DMSO (a), water alkalized to pH 12 (b), and PBS (c). AmB is expected to remain in a monomeric form in DMSO and in a water phase at pH 12 but in the aggregated form in PBS. Optical path length 1 cm.

between 350 and 450 nm represents the strongly allowed electronic transition from the ground energy state (S0, 11Ag−) to the S2 excited state (11Bu+). Very distinct vibrational substructure of the absorption band, which can be observed in the monomeric form, is not pronounced in the case of the aggregated AmB (Figure 2c). The CD spectrum recorded in DMSO (Figure 2a) can be assigned to monomeric AmB with two distinct bands: the positive band representing the S0 → S2 (11Ag− → 11Bu+) transition and the negative band representing the 11Ag− → 11Ag+ transition. The observation that both transitions give rise to the opposite sign CD bands can be understood on the basis of the fact that the dipole moments of these transitions make a large angle in polyenes.33 In the previous studies, the CD activity was solely attributed to the aggregated forms of AmB.15,34,35 As can be seen from Figure 2a, a very distinct CD spectrum of monomeric AmB can also be recorded, despite the fact that the ellipticity values are clearly not as high as in the case of the aggregated AmB forms (Figure 2c). On the other hand, the fact that monomeric and aggregated structures of AmB give rise to very different and specific CD spectra can be applied to monitor very precisely the molecular organization processes of the drug. Both the electronic absorption and CD spectra of AmB in the water environment (Figure 2c) display spectral features typical for aggregated molecules.35 Despite apparent similarities in the absorption spectra of AmB dissolved in DMSO and in the water medium alkalized to pH 12 (both systems believed to

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RESULTS Molecular Spectroscopy. Figure 2 presents the absorption and CD spectra of AmB, recorded in the solvent systems in which the drug appears as a monomer (panels a and b) and as an aggregate (panel c).16,17,21,32 The main absorption band 13823

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toward shorter wavelengths and the second one with clearly resolved vibrational substructure, shifted toward longer wavelengths. Such a behavior can be interpreted as demonstration of the presence of at least two different molecular assemblies in the sample. Interestingly, the analysis of CD spectra shows the time evolution of samples dissolved in water alkalized to pH 12 (Figure S2, Supporting Information). The analysis of the spectra recorded immediately after the sample preparation and after prolonged incubation shows appearance of unknown spectral forms of AmB at the expense of monomers. The fact that this new spectral form consists of the bands shifted toward both lower and higher wavelengths suggests that monomers associate into molecular structures which give rise to excitonic interactions. Depending on the mutual orientation of monomers involved in formation of a supramolecular structure, low-energy-shifted or high-energy-shifted bands may appear more or less intensive or even observed exclusively. A subfraction of organized molecular structures of AmB can also be detected in solution, by means of conventional UV−vis absorption spectroscopy, under decreased temperature of the sample (at 77 K, Figure 3). Enhanced spectral resolution enables detection of the bands additional to those representing monomeric AmB. Analysis of the spectrum shows, clearly resolved, the two sets of spectra (Figure 3), the one set corresponding to monomeric AmB (marked with dots) and the second one shifted toward higher energies by 1245 cm−1 (marked with asterisks). The spectral shift observed can be assigned to excitonic interactions. The fact that the shifted spectrum retains its vibrational substructure suggests a low number of molecules involved in formation of an aggregate (most probably a dimer). The splitting of the 0−1 and 0−2 vibrational bands, observed, can also be interpreted in terms of contribution of the ν1 and ν2 vibrational modes of a polyene (CC and CC, respectively). On the other hand, such a splitting is not observed in the 0−0 band, which makes the interpretation based on a hypsochromic spectral shift more

Figure 3. Absorption spectra of AmB dissolved in the glycerol− ethanol mixture (1:1, v:v) at room temperature (a) and at liquid nitrogen temperature (b). The spectra were normalized at the maximum.

ensure monomeric organization of the drug), the CD spectra recorded from the same samples display considerable differences (Figure 2a and b). Two different spectral bands in the CD spectrum of AmB in the water medium at pH 12 can be resolved, one similar to the aggregated sample but less shifted

Figure 4. Diagram of localization of the energy levels of AmB in different molecular organization forms. Shaded areas represent excitonic bands. The dipole−dipole interaction energies for excitonic interactions (β) for each structure are shown, based on the spectroscopic data, along with the interchromophore distance, calculated on the basis of the exciton splitting theory.8 See the text for more information. 13824

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Figure 5. Fluorescence emission and fluorescence lifetime-associated spectra of AmB. The fluorescence emission spectrum is the average of the spectra recorded with excitation set at 395, 400, and 405 nm. The solvent spectrum (water pH 12) was subtracted from each spectrum in order to correct for the Raman scattering effect. Each point in the lifetime-associated spectra was calculated following the formula: (cps × %)/(1 − T), where cps - counts per second, % - fraction of a lifetime component, T - transmission of the sample at excitation wavelength. Presented results are the average of seven global analyses of AmB fluorescence kinetics in different solvents.

Table 1. Fluorescence Kinetics Parameters Corresponding to the AmB Dimers Referred to as “Parallel” (τ = 1.8 ns) and “Antiparallel” (τ = 6.8 ns) Determined in Different Solventsa solvent type

solvent

average lifetime τav (ns)

6.8 ns lifetime component fraction (%)

1.8 ns lifetime component fraction (%)

fraction 1.8 ns/6.8 ns ratio

polar protic

water ethanol 2-propanol dimethyl sulfoxide tetrahydrofuran chloroform

0.561 0.449 0.502 2.653 1.157 3.060

2.8 0.9 1.5 31.5 10.5 36.5

6.3 3.7 3.0 20.3 9.3 26.8

2.3 4.1 2.0 0.6 0.9 0.7

polar aprotic nonpolar a

The intensity-averaged lifetime (τav) determined in each system, based on four-exponential deconvolution, is also presented.

in the CD spectra recorded from the sample composed of aggregated AmB (Figure 2c) can be observed at 335 nm. This means that the structures formed in the water medium alkalized to pH 12 are not as large as in the case of the aggregated sample or that the distance between chromophores in such a structure is greater. Interestingly, the position of the energy level corresponding to this band (340 nm, see Figure 4) has its counterpart at the energy level corresponding to 510 nm, in the AmB fluorescence emission spectrum (Figure 5c): these energy levels are localized on the energy scale symmetrically with respect to the S2 energy level of monomer (Figure 4). Such an agreement goes along with the interpretation that both the energy levels originate from the exciton-shifted S2 state, toward lower and higher energies. The molecular distance in such a structure can be calculated as R = 4.2 Å, assuming a dimeric organization of the structure. Such a distance is close to the dimeric structure of AmB referred to as “parallel”, concluded to be formed in the water medium, on the basis of the results of the molecular dynamics calculations (5.07 ± 0.14 Å, see the section below). The fluorescence emission spectrum of AmB, presented in Figure 5, is superimposed on the fluorescence lifetime-associated spectra (the examples of the original AmB

justified. Predominantly, the hypsochromically shifted bands are observed, with minor contribution from the bathochromically shifted ones, which suggests that the aggregated structures formed are close to H-type structures. On the other hand, the fact that the CD bands are clearly visible implies that molecules in the structures are not rigorously parallel.36 The red-shifted CD spectral component represents most probably the same dimer which gives rise to the hypsochromic spectral shift of 1245 cm−1. Localization of the energy levels of such a dimeric structure is proposed in the scheme presented in Figure 4. Application of the exciton splitting theory, based on the assumptions and methodology described previously,8 leads to the conclusion that the chromophore distance in such a structure is R = 6.6 Å. Such a distance corresponds very well to the dimeric structure of AmB referred to as “antiparallel”, concluded to be formed in the water medium, based on results from molecular dynamics calculations (7.59 ± 1.12 Å, see the section below). Let us call this AmB structure “dimer_AP”. A more pronounced (blue) spectral shift is associated with appearance of the second band in the CD spectrum (Figure 2b). The center of the doublet (of the negative and positive bands) is located at 340 nm. The center of the doublet visible 13825

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Figure 7. Atomic models of parallel and antiparallel AmB dimers in a water environment. The figure presents final geometries obtained from the performed simulations (t = 200 ns).

monomeric form (6.8 ns vs 3 ns). Such a phenomenon can be understood in terms of a very small energetic separation between the two excitonic levels, in the range of molecular vibration energy (see Figure 4). Such a small energy gap between the excitonic levels allows multiple circulation between those energetic states, driven by thermal excitations, on the one hand, which are followed by spontaneous relaxation acts, on the other hand. It is possible that such a mechanism is responsible for a relatively long fluorescence lifetime observed, owing to the fact that fluorescence originates from the lowest excitonic band. Importantly, the fluorescence lifetime values, characteristic for the two types of dimers, are distinctly different, which makes analysis of the appearance of these two molecular organization forms of AmB convenient and precise. The fact that the dimer_P structures are more readily formed in polar environments (see Table 1) led to the conclusion that it has a more polar character as compared to the dimer_AP. This is also supported by molecular dynamics calculations (see the section below), where dimer_P creates more water binding than dimer_AP. As can be seen, an additional spectral component can be resolved, characterized by the fluorescence lifetime, 0.350 ns (Figure 5e) and 80 ps (Figure 5f), in the longwavelength region, which suggests that this spectrum represents another AmB aggregated structure. Comparison of the fluorescence excitation spectrum with the one-minus-transmission spectrum of AmB in the aggregated state (see Figure S4, Supporting Information) shows a very large mismatch, proving the relatively low fluorescence quantum yield of AmB in the aggregated state. It is therefore possible that the 0.35 ns and 80 ps lifetime components represent fluorescence of another type of small molecular assembly, which could be a tetramer (a result of association of two dimers), but certainly not large molecular aggregates which do not give rise to the fluorescence signal. In principle, two types of tetramers could

Figure 6. Fluorescence autocorrelation curves of AmB in two different solutions: in DMSO (a) and in water alkalized to pH 12 for the fresh sample (b) and 24 h after the preparation (c). Diffusion coefficients D determined for the fluorescence emitting AmB structures are presented in each panel.

fluorescence decay kinetics are presented in Figure S3, Supporting Information). The localization on the energy scale and the fluorescence lifetime values determined for monomeric AmB agree very well with those found previously.8 The comparison of the lifetime-associated spectra (Figure 5) with localization of the energy levels (Figure 4) led to the assignment of a lifetime of τ = 6.8 ns to the dimer_AP and τ = 1.8 ns to the structure referred to as a dimer parallel (dimer_P). Interestingly, the fluorescence lifetime of the dimer_AP structure is relatively long, as compared to the 13826

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Figure 8. Time evolution of the distance (top) and angle (bottom) between two AmB monomers forming a dimer. The distance was measured between the centers of mass of hydrophobic AmB chromophores (Figure 1 - from C20 to C33). The angle was measured between two longest principal axes of the two AmB molecules forming a dimer (see right panel). The values of 0 and 180° correspond to the parallel and antiparallel geometry, respectively. Different line colors correspond to two spatial geometries of the AmB dimer illustrated in Figure S1 (Supporting Information).

Figure 10. Distribution of the number of hydrogen bonds formed between AmB and water molecules for AmB dimer (top) and AmB monomer (bottom).

Figure 9. Electrostatic (top) and van der Waals (bottom) dimerization energies decomposed into the contributions between the selected structural elements of AmB molecules (as illustrated in Figure 1). Error bars illustrate the standard error value for estimated energies.

this form, as presented in Figure 4, results from both the lifetime associated spectrum (low-energy level, the spectral origin at 523 nm, Figure 5) and the CD spectrum (high-energy level, center of the doublet at 335 nm, Figure 2c). Moreover, both of the energy levels are located ideally symmetrically with respect to the S2 energy level. Assuming that this energy gap (Figure 4) corresponds to a tetramer (3.2 × β), the calculated chromophore distance is 4.8 Å, which suggests that this tetrameric structure is formed out of the parallel dimers, present predominantly in a water system. The conclusion on formation of dimeric structures of AmB in the water phase (alkalized to pH 12) and further association of dimers into the larger structures has very strong support from the fluorescence correlation spectroscopy (FCS) analysis (Figure 6). The presence of a single spectral form of AmB dissolved in DMSO, corresponding to monomers, is represented by a single diffusion coefficient of D = 99 μm2/s. Two distinctly resolved

Table 2. Total Electrostatic (Eel) and van der Waals (EvdW) Energy for Two Considered Geometries geometry

Eel (kcal/mol)

EvdW (kcal/mol)

antiparallel parallel

−5.81 −15.34

−24.57 −30.34

represent molecular assembly of two types of dimers, namely, dimers_P and dimers_AP. As can be seen from the comparison of the fluorescence spectrum and the lifetime-associated spectrum, the emission component characterized by the 0.35 ns lifetime reproduces very well part of the fluorescence emission spectrum. This suggests that the molecular organization form of AmB, associated with this spectrum, is ubiquitous in the AmB preparation in water. It has to be emphasized that localization of the excitonic energy levels of 13827

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exposed to interaction with water molecules. A prolonged incubation of such a sample results in formation of the structures characterized by the diffusion coefficient D2 = 1.8 μm2/s, in expense of the molecular organization forms assigned to dimers. Such AmB forms can be assigned to tetramers. Molecular Dynamics of AmB Dimers. To study structural and dynamical properties of putative AmB dimers in aqueous environment, we performed a series of molecular dynamics simulations. We considered two different initial geometries of a dimer: parallel and antiparallel. Figure 7 illustrates the final structures of the AmB dimers in the parallel and antiparallel orientation. To characterize the structural properties of the simulated complexes and to determine their stability over time, we first calculated the evolution of the distance between the heptane chromophores of two AmB molecules forming a dimer (Figure 8, top panel). It is clearly visible that, although the distances fluctuate to a certain extent, they are quite stable on a 200 ns time scale, showing that the dimers did not dissociate during the simulation which also supports the experiment (see section above). The average distance between AmB molecules was equal to 7.59 ± 1.12 Å for antiparallel geometry and 5.07 ± 0.14 Å for parallel geometry. The average distances determined based on the molecular dynamics simulations are in good agreement with values found based on the spectroscopic analyses (Figure 4). Additionally, smaller fluctuations of the distance observed for the parallel dimer (standard deviation of 0.86 Å) compared to the antiparallel dimer (2.0 Å) may indicate a higher stability of the former. The bottom panel of Figure 8 demonstrates the time evolution of the angle between the two longest principal axes of the two AmB molecules forming a dimer. It can be seen that the fluctuation rate for the antiparallel geometry is again higher than that for the parallel one. For example, at t ≈ 130 ns for the antiparallel geometry, the angle changes by about 90°. To investigate molecular determinants of possible differences in the stability of parallel and antiparallel dimers, we decomposed the dimerization energies into interactions between individual elements of the AmB structure (Figure 9). Symmetry of the interaction energies between corresponding elements (see, e.g., I−III and III−I or I−II and II−I) suggests a symmetry in the dimer structure (see also Figure 7). The total electrostatic and van der Waals contributions to the dimerization energy (Table 2) show that, for both geometries, AmB dimers are stabilized especially by van der Waals interactions (see also Table S1, Supporting Information). This suggests a hydrophobic nature of the dimerization process, as the majority of the AmB polar groups are involved in the interaction with water molecules rather than in the dimerstabilizing contacts. As can also be seen from Table 2, in the parallel orientation, AmB molecules interact with each other more favorably, suggesting an explanation for the possible higher stability of this particular geometry of the dimer. Figure 7 further indicates that this higher stability is due to the stronger van der Waals interactions (larger contact surface) between the polyol chain of one molecule and the chromophore of the other, and between the chromophores. Additional stabilization results from the interaction between the polar heads with each other and between them and the polyol and chromophore chains. In the case of the antiparallel geometry, the dimer is stabilized mostly by the large contact surface between polyol chains and the electrostatic interaction between the polyol chains and the tail hydroxyl group (OH−

Figure 11. Results of the fluorescence lifetime analysis of AmB incorporated into small unilamellar vesicles formed with DPPC. Relative amplitudes of four fluorescence lifetime components are presented versus the molar percentage of the drug with respect to lipid. Note the relatively high data scattering in the sample containing AmB at very low concentration.

Figure 12. Results of the determination of the mean orientation of the transition dipole of AmB (which is close to the long axis of the AmB molecule) with respect to the axis normal to the plane of the lipid multibilayer formed with DPPC. The dichroic ratio, to calculate the AmB orientation, was determined at 350 nm, in order to take into account all the molecular organization forms. The level corresponding to the magic angle is shown with the dashed line.

diffusion coefficients can be resolved in the AmB samples in the water medium alkalized to pH 12, D1 = 115 μm2/s and D2 = 4.7 μm2/s. The diffusion coefficients D1 and D2 can be assigned to monomeric and dimeric structures, respectively, owing to the fact that determination of diffusion coefficients with application of the FCS technique is based on fluorescence signal and that analysis of the steady-state and time-resolved fluorescence characteristics of the same sample lead to the conclusion that monomeric and dimeric structures are present. The relatively large difference between the D1 and D2 values can be interpreted in terms of a substantially greater hydrodynamic radius of the dimeric structure, in which the hydrophobic polyene chains are hidden inside and all the polar groups are 13828

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Figure 13. FLIM images of the areas of the glass slides covered with polylysine on which deposited was AmB from the water solution at pH 7 (left panel) or the single AmB-containing multilamellar vesicle formed with DPPC (1 mol % AmB with respect to lipid). The results of the FLIM analysis (fluorescence lifetime distribution) are presented below the images along with the results of fluorescence lifetime analysis based on the components: 0.350, 1.8, 3, 6.8 ns (the amplitudes presented in the form of a histogram). The upper panel presents the distribution of the fluorescence lifetime fraction assigned to AmB monomers, and the middle panel presents the fractions assigned to dimers_P, dimers_AP, and tetramers (indicated).

fluorescence lifetime amplitudes of AmB incorporated into liposomes formed with DPPC, in dependence of the molar concentration of the drug with respect to the lipid. UV−vis absorption spectra of the samples are presented in Figure S5 (Supporting Information). As can be seen from Figure 11, fluorescence lifetime analysis reveals the presence of four spectral forms, characterized by the fluorescence lifetime components which can be assigned to the tetramers formed out of the dimers_P (0.35 ns), the dimers_P (1.8 ns), and the dimers_AP (6.8 ns) and the tetramers formed out of dimer_AP (80 ps). It is possible that the more hydrophobic structures (dimers_AP) are incorporated into the lipid bilayer core, by spanning the membranes. On the other hand, it is possible that more hydrophilic structures (dimers_P) are localized in the membrane periphery, anchored to the polar headgroup regions. In the case of tetramers, both localizations with respect to the lipid bilayer can be predicted. The linear-dichroism-based analysis of the orientation of AmB chromophore with respect to the axis normal to the plane of the membrane (Figure 12) shows that the mean orientation angle determined is relatively large at low AmB concentrations, suggesting that a certain fraction of the drug can be oriented within the plane of the membrane.9,20 In the case of 1 mol % AmB, the mean orientation angle of 58° is consistent with the fractions of 28% oriented vertically with respect to the lipid bilayer and 72% in the membrane plane. A vertically oriented AmB fraction increases along with the increase in AmB concentration (the

C35 in Figure 1), in agreement with the previous computational study.37 Above, we concluded that polar groups are involved in interaction with water molecules. To check this directly, we calculated the distribution of a number of hydrogen bonds between AmB molecules and water (Figure 10). The criterion used to detect hydrogen bonding was strictly geometric; namely, the D−A distance was supposed to be less than 3.5 Å, and the value of the D−H−A angle had to be less than 40°, where D, A, and H are the donor, acceptor, and hydrogen atoms, respectively. From the obtained results, we see that in the parallel geometry of the dimer AmB molecules are more hydrated than in the antiparallel one. The average numbers of bonds between AmB and water for the antiparallel and parallel geometry are 28 and 30, respectively. Additionally, to determine if the dimer formation is accompanied by dehydration of AmB molecules, we obtain a similar distribution of the number of hydrogen bonds for the isolated AmB monomer. We found (Figure 10, bottom panel) that the number of hydrogen bonds created by each AmB molecule in the dimer is only slightly smaller than that in their monomeric state. This suggests a lack of dehydration during dimerization and a hydrophobic nature of dimer stabilization. Application to Lipid Membranes. Owing to the fact that the site of pharmacological action of AmB is biomembranes, molecular organization of the drug was also studied in model lipid membranes. Figure 11 presents the distribution of relative 13829

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Figure 14. Absorption and CD spectra of DPPC liposome suspensions containing incorporated AmB at different molar concentrations, indicated.

are most probably distributed between the membrane core and the polar headgroup regions. Interestingly, the considerable fraction of the AmB tetramers (τ = 350 ps) is proved to appear in the lipid bilayer system, which can span a membrane and act as a transmembrane pore able to transmit ions. Molecular organization of AmB in the DPPC membranes was also studied with application of the CD technique (Figure 14). As can be seen from the analysis of the CD spectra of AmB present in the lipid membrane system, a certain fraction of the drug remains in the monomeric form, even at relatively high concentrations. This fraction can be monitored by the negative CD band in the short wavelength region. It can be predicted that monomeric AmB is bound to the polar headgroup membrane region. The analysis also shows that the spectral bands assigned to dimeric structures are present at all the AmB concentrations, in contrast to the tetrameric structures, which are particularly observed at 5 and 7 mol % AmB with respect to the lipid.

orientation angle decreases), suggesting that molecular assemblies of AmB can better adopt a trans-membranous localization as compared to monomers. A similar conclusion has been made on the basis of the recent EPR studies of AmB in model lipid membranes.38 Interestingly, the character of changes of the fluorescence lifetime relative amplitudes (Figure 11) shows that the presence of AmB in the membranes at higher concentrations is associated with formation of tetramers (τ = 80 ps), in expense of hydrophobic dimers (dimers_AP, τ = 6.8 ns). On the other hand, at all the molar concentrations, very high are relative fractions of tetramers formed out of the dimers_P, present massively in the water phase. Such tetramers have been postulated to play a role of transmembrane ion channels.8 We applied fluorescence lifetime imaging microscopy (FLIM) to address the question regarding distribution of the molecular organization forms of AmB in a liposome. Figure 13 presents the FLIM analysis of a single, multilamellar lipid vesicle, binding 1 mol % AmB. As can be seen, the four major lifetime components have been resolved: 3, 1.8, 6.8, and 0.35 ns, corresponding to the monomers, dimers_P, dimers_AP, and the tetramers formed by association of dimers_P, respectively. The 80 ps lifetime component was not resolved in the microscopic experiments due to the time response of the FLIM detector limitation (∼300 ps). Such a result shows that, in the membrane system, a large fraction of the AmB molecules remain in the form of organized supramolecular structures. Both types of dimers, more hydrophobic and more hydrophilic,

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DISCUSSION In the present work, we applied molecular spectroscopy techniques combined with the molecular dynamics calculations to discern the organization of the polyene antibiotic AmB in the environments interesting from the physiological standpoint. This knowledge seems to be not only intriguing but also important, owing to the fact that molecular organization of the drug is recognized as being directly responsible for both the pharmacological effect and the toxic side-effects. All the results 13830

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Notes

point at an extremely high capacity of AmB for self-assembly, leading to formation of the two types of dimers which can further self-assemble yielding the tetrameric structures. The concept on association of AmB molecules into dimeric structures follows directly from the results presented above. In particular, such structures give rise to new excitonically shifted spectra (electronic absorption, fluorescence, and CD) which retain their vibrational substructure. The distance between the chromophores, calculated on the basis of the spectral shifts observed, agrees with the molecular distance determined by the molecular dynamics calculations, for the selfassociated AmB dimers. The molecular spectroscopy measurements (in particular time evolution of CD spectra of AmB solution) show that AmB dimers can further associate to tetramers. AmB tetramer formation, out of dimers, follows also from the fluorescence lifetime analysis and from the results of the FCS experiments. Fluorescence lifetime analysis and the FLIM technique show that tetramers of AmB are ubiquitous molecular organization forms not only in a water medium but also in the lipid membranes. Such a finding seems particularly important, taking into consideration the fact that, according to the structural analysis, tetrameric structures of AmB, located in the lipid bilayer, can function as transmembrane pores, enabling leakage of small ions across the membrane and therefore disturbing the physiological ion transport and equilibrium.8 Such a mechanism has to be considered as a potential cause of severe toxic side effects of AmB in treatment of deep-seated mycotic infections. According to the recent reports, the main molecular mechanism responsible for pharmacological effectiveness of AmB is a sequestration of ergosterol from fungal membranes, which makes them physiologically nonfunctional and leads to cell death.4,5 Interestingly, AmB has been shown to form extramembraneous aggregates, attached to the membrane surface, which extract and bind ergosterol from the yeast membranes.4 On the other hand, AmB can also bind to other lipid membranes, including biomembranes of human cells. Understanding of the molecular mechanisms responsible for toxic activity of the drug seems critical in elaborating a formula of the drug which retains its pharmacological potential but, at the same time, will not be able to form structures responsible for high toxicity in patients. In light of the results of the present study, such structures are the AmB dimers, which are able to associate into tetramers, being able to play the role of transmembrane pores.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research has been performed within the framework of the project “Molecular Spectroscopy for BioMedical Studies” financed by the Foundation for Polish Science within the TEAM program (TEAM/2011-7/2). M.G. and J.C. thank Academic Computer Centre TASK (Gdansk, Poland) and Cyfronet (Krakow, Poland) for granting CPU time. M.G.’s work was supported by Regional Specialist Hospital in Biala Podlaska during his employment from 2012 to 2013 under framework agreement “Molecular studies in biomedicine”. The research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Development of Eastern Poland Operational Programme. The authors would like to thank Karol Sowinski for critical reading of the manuscript.

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ABBREVIATIONS AmB, amphotericin B; DPPC, dipalmitoylphosphatidylcholine; PBS, phosphate buffered saline; CD, circular dichroism; FLIM, fluorescence lifetime imaging microscopy; FCS, fluorescence correlation spectroscopy

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ASSOCIATED CONTENT

S Supporting Information *

Atomic models of AmB dimer in water, time evolution of absorption and circular dichroism spectra of AmB dissolved in water alkalized to pH 12, fluorescence decay kinetics of AmB in different environments, comparison of the absorption and fluorescence excitation spectra of AmB dissolved in PBS, absorption spectra of AmB incorporated into DPPC small unilamellar vesicles at different molar concentrations with respect to lipid, and a table with listed electrostatic and van der Waals energies between groups of AmB in a dimer. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

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*Phone: + (48 81) 537 62 52. Fax: + (48 81) 537 61 91. Email: [email protected]. 13831

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