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Structural and calorimetrical studies of the effect of different aminoglycosides on DPPC liposomes
Colloids and Surfaces B: Biointerfaces 69 (2009) 116–121
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Structural and calorimetrical studies of the effect of different aminoglycosides on DPPC liposomes Ágnes Oszlánczi a , Attila Bóta b,∗,1 , Gábor Czabai a , Erwin Klumpp c a b c
Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, H-1521 Budapest, Hungary Chemical Research Centre – Hungarian Academy of Sciences, Institute of Nanochemistry and Catalysis, HU-1025 Budapest, Pusztaszeri út 59-67., Hungary Agrosphere Institute, Research Centre, D-52425 Jülich, Germany
a r t i c l e
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Article history: Received 30 June 2008 Received in revised form 17 November 2008 Accepted 18 November 2008 Available online 25 November 2008 Keywords: Aminoglycosides DPPC liposomes DSC Small- and wide-angle X-ray scattering (SWAXS) Unbinding
a b s t r a c t The effects of tobramycin, spectinomycin and streptomycin on 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC)/water vesicle system were studied by using differential scanning calorimetry (DSC) and simultaneous small- and wide-angle X-ray scattering (SWAXS) in the 0–1 lipid/aminoglycoside molar range. The changes in enthalpy between the thermally adjacent phases are decreased, but the pace of decrease is totally different for the three investigated AGs. The alterations in the lamellar arrangement and the chain packing are rather tendentious and are extended by increased AG concentrations depending on the type of the AG. In the case of tobramycin and streptomycin, still sharp Bragg peaks of SAXS curves shift to smaller values of the scattering variable, while spectinomycin results in an entire loss of multilayer correlation representing an increased amount of unbound bilayers. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The members of the aminoglycoside class of antibiotics have a wide spectrum of antimicrobial activity [1]. Most of the clinically important Gram-negative [1,2], and as well some of the Gram-positive [1] bacterial infections are treated by these powerful drugs. The major problems associated with these antibiotics are the oto- and nephrotoxicity in humans [2,3], and the drug-resistance of the pathogens that have aminoglycoside-modifying enzymes [1]. In human beings these molecules are taken up by proximal tubular cells and are accumulated in lysosomes [3,4]. Several researchers used negatively charged liposomes in a low pH environment to model lysosomal circumstances and examine their interaction with aminoglycosides. They have found a specific interaction between phosphatidylinositol diphosphate (PIP2) – a negatively charged lipid – and aminoglycosides (AGs) [4–6]. In vitro [7], and in vivo [8] studies have shown that AGs bind to PIP2 molecules in the bilayer of both the biological and artificial membranes, and that the AG-PIP2 complexes inhibit the activities of lysosomal phospholipases [4]. The inhibition of the breakdown of different phospholipids causes
∗ Corresponding author. E-mail address: [email protected] (A. Bóta). 1 Leave from Budapest University of Technology and Economics, Dep. of Phys. Chem. and Mat. Sci. 0927-7765/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2008.11.010
phospholipidosis which is considered to be the first phase of toxicity [4,9]. The stability of the AG-PIP2 complex and its consequence, the toxic effect are different, depending on the type of the AG [4–6]. The stability of the complex is related to electrostatic interactions, but hydrophobic interactions may also exist between the drug and the lipid molecules [6,9,10]. Although extensive work has been done by different research groups on the lipid–AG interactions, several conflicting results have been published. As mentioned before, electrostatic interactions were suggested between AGs and an anionic lipid molecule, the PIP2. These findings were based on gel filtration technique, conformational calculations [4,5], and NMR spectroscopy [6]. Structural analysis (freeze-fracture method) has also been used to examine the toxic effect of these antibiotics [11]. Some authors have detected integrity changes in some multilamellar liposome systems by leakage of entrapped molecules [9,10]. The disintegration of the liposomes may be explained by the interaction of the AGs and the hydrophobic domain of the bilayer. Comprehensive research has been carried out to clear up the effect of AGs on model membranes, and only a very weak and slowly proceeding interaction has been found between the AGs and the lipid molecules [12]. In this study the effect induced by some clinically important AGs on the structural and thermotropical changes of a human model membrane has been presented by differential scanning calorimetry (DSC), and simultaneous small- and wide-angle X-ray scattering (SWAXS) measurements. The characteristic features of the
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1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/water multilamellar system was thoroughly examined and reported widely in scientific literature [13–15]. Depending on temperature, different multilamellar structures can be observed in the fully hydrated DPPC liposomes. These are the crystalline (Lc ), the gel (Lˇ ), the rippledgel (Pˇ ), and the liquid crystalline (L˛ ) phases, with of 59, 64, 70.5 and 67 Å characteristic periodicities, respectively [16–18]. Different chain packings result in different subcells (hybrid in the gel phase and hexagonal in the rippled-gel phase). Transitional points occur at 18 ◦ C (sub-transition), 33 ◦ C (pretransition), and 41 ◦ C (main transition), with characteristic endotherm enthalpy changes [16,18]. Formation of defect structures is favoured, especially, during the pretransition, because of the strong fluctuations in the whole system, and the effect of the foreign molecules become stronger than at other transitions [19]. The DPPC molecule, as one of the most common lipids in biomembranes, has proved to be a suitable model membrane constituent in the investigation of the effect of toxic molecules [20–26]. 2. Materials and methods Synthetic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, purity > 99%) was purchased from Avanti Polar Lipids, Inc. (Alabaster, Ala, USA). The different aminoglycosides (AGs): spectinomycin, streptomycin and tobramycin were obtained from Sigma–Aldrich (Steinheim, Germany; purity >99%). Substances were used without further purification. The dried lipids were dissolved in sodium phosphate buffer (10 mM, pH 7.4), prepared from deionized, triple-quartz-distilled water to obtain a lipid concentration of 20 wt.%. The sample was heated above the phase transition (50 ◦ C) for 15 min, cooled to 4 ◦ C, reheated and kept above 50 ◦ C again. After each heating period the samples were stirred intensively. On average 10 repetitions of this process was needed to achieve homogeneous dispersions. For the preparation of systems containing antibiotics the concentrations of AGs were determined at the molar ratios of 10−2 , 10−1 , and 1 (AG/lipids). In the case of the treated systems the AG powders and the pure DPPC were mixed, then dissolved and temperaturetreated. The DSC measurements were performed with a DSC 2920 instrument (TA Instrument, US) operated at a heating rate of 1 ◦ C/min in the temperature range from 25 to 50 ◦ C. The DSC curves were recorded in the heating direction. The reference pan was empty. The calibration of the calorimeter was carried out by using a pure indium sample (Tonset = 156.6 ◦ C). The transition points were characterized by the temperature at which the heat-flow curve exhibited a minimal value.
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SAXS and WAXS measurements were performed using a modified Kratky Compact camera (Anton Paar, Graz, Austria) supplied with two position sensitive detectors (M.Braun, Garching, Germany). Simultaneous detection in the small- and wide-angle region was performed with this SWAXS camera. The scattering of Ni-filtered Cu K␣ radiation ( = 1.542 Å) was recorded in the smallangle range from 10−3 to 10−1 Å−1 and in the wide-angle range from 0.2 to 0.3 Å−1 of the scattering variable, defined as s = (2/) sin , where 2 is the scattering angle (s = 1/d, d is a characteristic periodicity in the sample). For the X-ray measurements the samples were transferred into thin-walled capillaries (Hilderberg, Germany) with a diameter of 1 mm. After centrifugation to remove air bubbles the capillaries were sealed with a two-component synthetic resin and transferred into metal capillary holders that were placed into an aluminium block. This block was positioned directly into the beam line and was used as a thermal incubator for controlled annealing at characteristic temperatures of the lipid systems. After a 10 min incubation of the samples at the respective temperatures the exposure took 600 s for the detection in the small- and wide-angle X-ray scattering regions. 3. Results and discussion 3.1. Thermometric results The pure DPPC multilamellar system exhibits two endothermic phase transitions at 35.3 and at 40.7 ◦ C, corresponding to the pretransition between the gel (Lˇ ) and rippled-gel (Pˇ ) phases, and to the main transition (chain-melting) between the rippledgel and liquid crystalline phases (L˛ ), respectively. The influence of the three different aminoglycosides (AGs: streptomycin, STR; tobramycin, TOB; spectinomycin, SPE, see their chemical structure in Fig. 1) on the phase transition behaviour of the DPPC/water systems are characterized by DSC experiments in the range of AG/lipid molar ratios from 10−2 to 1 (Fig. 2). Transition temperature changes of the antibiotic-doped systems appear only at the pretransition. The apexes of the first dips on the thermograms shift to lower or higher temperature values, but they do so significantly only at 1 AG/lipid molar ratio, indicating that this transition is affected by the chemical structures of the AGs. Structurally similar aminoglycosides like streptomycin and tobramycin increase the pretransitional temperature to 36.4 ◦ C, while spectinomycin, a non-aminoglycoside aminocyclitol still ranked as an AG, decreases it to 33.7 ◦ C. The chain-melting temperature of the system is not affected considerably by the drug molecules (the results remain within the standard deviation of the DSC measurement: ±0.1 ◦ C) as shown in Fig. 2 as well as in Fig. 3A.
Fig. 1. Chemical structure of the three investigated aminoglycosides (streptomycin, STR; tobramycin, TOB; spectinomycin, SPE).
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around 1/4.2 Å−1 both in the gel and the rippled-gel phases, which results from the hybrid and the hexagonal chain packing arrangement in the bilayers, respectively. At 45 ◦ C, after the chain-melting process, the peak disappears entirely.
Fig. 2. DSC curves of the DPPC/water systems at different AG concentrations. Molar ratios and the type of AG (streptomycin, STR; tobramycin, TOB; spectinomycin, SPE) are marked on each curve.
The changes in enthalpy of the pre- and main transition of the DPPC/water systems were thoroughly investigated and the values are in accordance with our previous data (6.55 ± 0.6 and 45.03 ± 4.5 kJ/mol, respectively) [16]. Contrary to the temperature changes, pretransitional enthalpy is not influenced by any of the examined AGs. The areas of the dips representing the changes in enthalpy (Fig. 3B) vary only at the main transitions and are mostly dependent on the concentration and to a lesser extent on the type of the AGs. At 10−2 AG/lipid molar ratio the change in enthalpy of the melting process is strongly decreasing (tobramycin: 30.6 kJ/mol; streptomycin: 32.2 kJ/mol; spectinomycin: 33.3 kJ/mol). At 10−1 and 1 molar ratio the values of streptomycin and spectinomycin treated systems are nearer to that of the pure one, but they still exhibit smaller enthalpy values. At 10−1 TOB/lipid ratio 38.42 kJ/mol, while at 1 molar ratio more significant decrease in enthalpy, 29.76 kJ/mol can be observed. 3.2. Structural study by X-ray diffraction According to the thermotropic behaviour of the systems, the structural studies were carried out at 26, 37, and at 45 ◦ C by simultaneous small- and wide-angle X-ray scattering (SWAXS), corresponding to the gel (Lˇ ), rippled-gel (Pˇ ), and to the liquid crystalline (L˛ ) phases, respectively. The SWAXS patterns of the pure lipid system show the characteristic features of the phases (at every temperature four curves with increasing AG/lipid molar ratios – from bottom to up: 0, 10−2 , 10−1 , 1 – are shown, Figs. 4–6). In the beginning range of the SAXS curves an intense scattering can be observed as a consequence of inhomogeneities with a size-range of several hundreds Å. The first Bragg reflection peaks on the scattering curves, which are typical of the multilamellar systems, appear at 1/64 Å−1 (Lˇ ), 1/70.5 Å−1 (Pˇ ), and 1/67 Å−1 (L˛ ). In the WAXS profiles the maximum of the broadened Bragg reflections are located
Fig. 3. (A) Thermodynamic parameters of the pre- and main transitions of the AG/DPPC model systems: transition temperatures. The type of AG (streptomycin, STR; tobramycin, TOB; spectinomycin, SPE) is marked on each curve. (B) Thermodynamic parameters of the pre- and main transitions of the AG/DPPC model systems: transition enthalpies. The type of AG (streptomycin, STR; tobramycin, TOB; spectinomycin, SPE) is marked on each curve.
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Fig. 4. (A–B) SAXS and WAXS diffractograms, at the denoted temperatures of the pure DPPC and the TOB/DPPC systems, in the range of 10−2 to 1 molar ratio.
The effect of the concentration (in the range of 10−2 to 1 AG/lipid molar ratio) of the chosen antibiotics on the structural changes of the model membranes can be seen in Figs. 4–6. The three examined antibiotics show some similarities in their influence, but significant divergences can also be observed in the layer arrangements of the doped systems. The gel phase (at 26 ◦ C) is not affected at 10−2 molar ratio of any of the used AGs (Figs. 4–6). The intensity and the location of the peaks are the same as in the case of the pure liposomes. At 10−1 SPE/lipid molar ratio the location and the broadening of the peak remains as in the DPPC/water system, but the intensity of the peak diminishes significantly (Fig. 5). Tobramycin and streptomycin added systems result in the same patterns at this molar value (Figs. 4 and 6, respectively). At the equal molar amount of the AGs and the lipid molecules, spectinomycin causes the strongest effect: the SAXS peak disappears almost entirely. With the other two antibiotics the maximum of the peaks shift to higher values
Fig. 5. (A–B) SAXS and WAXS diffractograms, at the denoted temperatures of the pure DPPC and the SPE/DPPC systems, in the range of 10−2 to 1 molar ratio.
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Fig. 6. (A–B) SAXS and WAXS diffractograms, at the denoted temperatures of the pure DPPC and the STR/DPPC systems, in the range of 10−2 to 1 molar ratio.
of the scattering variable, that is from 1/64 to 1/62.5 Å−1 , meaning that the distance between the lipid layers become smaller. As we expected (see Section 1), the rippled-gel phase (37 ◦ C) was the most sensitive to the presence of the AGs. The SAXS patterns of the TOB/lipid and SPE/lipid systems show gradual broadening and the intensity of the peaks start to decrease continuously from 10−2 molar ratio upwards. At 1 molar ratio of the tobramycin the location of the peak jumps to a higher scattering variable, while with spectinomycin the Bragg peak ceases to exist. In the presence of streptomycin the intensity and the broadening of the peaks do not change with the different concentrations of the drug, but the maximum of the peaks shift from 1/70.5 to 1/72.5 Å−1 at 10−1 and to 1/69.5 Å−1 at 1 STR/lipid molar ratio. At 45 ◦ C (liquid crystalline phase) strong fluctuations in the layer arrangement of all antibiotic-treated system can be observed. The peak maximums on the SAXS patterns alternate within a wide range of scattering variable depending on the concentration of AG-molecules. At 10−2 TOB/lipid molar ratio the distance of the layers decreases as the position of its characteristic peak moves from 1/66.5 to 1/64.5 Å−1 – than at 10−1 molar ratio from 1/66.5 to 1/65 Å−1 . At the equimolar ratio of the tobramycin the layer distance is reduced strongly as the peak appears at 1/61 Å−1 . The spectinomycin show the same tendency depending on concentration, but at the highest examined SPE/lipid ratio, the peak shift to smaller value than that of the pure system (1/69 Å−1 ). By increasing the ratio of the drug, the intensity of the peaks strongly decreases. At 1 SPE/lipid molar ratio the intensity of the first Bragg reflection is reduced extremely. On the SAXS patterns of the streptomycindoped systems at higher STR/lipid ratios only the locations of the peaks vary. At 10−1 molar ratio the peak shifts to lower scattering variables (from 1/66 to 1/67 Å−1 ), while the peak moves to 64 Å−1 at 1 molar ratio. With the subcell formation changes the same tendency can be examined (Figs. 4–6, B panel), thus the WAXS results can be summarized without focusing on any of the used drugs. At the gel phase, as the ratio of the antibiotics increases, the maximum of the WAXS peaks shift to lower values of the scattering variable, indicating the increase of the distance of the neighbouring lipid molecules by intercalation of AG molecules. Taking into account the chemical characters of the AGs, it can be supposed that the location of these molecules is close to the head groups. The intensity of the peaks
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decrease at 10−1 , and the subcells of the chain packing disappear at 1 molar ratio. WAXS pictures of the rippled-gel phase show the loss of the regular lipid arrangement at higher antibiotic ratios. As in the pure system, subcells cease to exist in all doped samples above the chain-melting temperature. The fact that signal of the subcells vanishes, relates to non-specific interactions between the AG and the lipid molecules as no characteristic subcell is formed by the presence of AGs. Moreover, the relatively minor changes in enthalpy also indicate that those interactions which determine the calorimetrical features are not influenced in the gel phases of the doped systems. 3.3. Interpretation of the SAXS patterns The studied AGs influenced the lamellar arrangement of the vesicles differently. Basically, two phenomena (1) shifting of the still sharp Bragg peaks of SAXS curves to smaller values of the scattering variable or (2) drastic broadening in the Bragg reflections can be observed. The shift is a consequence of the reduction of the thickness of each layer caused by embedding AG molecules. The sharp reflections indicate that the presence of foreign molecules does not destroy the regularity of the multilamellar arrangement, which is typical of DPPC/water system. This effect appears in the case of streptomycin in all investigated temperature domains. Moreover, a significant shift can be observed in the tobramycindoped system (Fig. 4, at 45 ◦ C, the periodicity of the pure system was decreased in the presence of equimolar AG from 67 to 61 Å). In this system the main transition undergoes a significantly low change in enthalpy indicating also the increased presence of the AG molecules, whereby the characteristic interactions, connecting to the chain-melting, are reduced; therefore the affected interactions are hydrophobic. The broadening in the Bragg reflections is characteristic for the spectinomycin-doped systems indicating a severe loss in multilamellar correlation. Generally, the broadening and the final disappearance of the Bragg reflections, caused by any foreign molecules, can be explained in several cases by the paracrystalline theory considering only the average fluctuations of the one-dimensional layer arrangement [27–29]. To obtain direct visual information in the real space we have executed freeze-fracture procedure on AG-vesicle systems. In the electronmicrographs the aggregation of liposomes is typical, especially in the case of higher spectinomycin concentration as it was also observed in other lipid systems and published in the earlier [11]. The characteristic, well ordered lamellar arrangement of the pure lipid–water system, however, is present even at the presence of spectinomycin. In Fig. 7, the surface morphology of the fractured gel phase of DPPC/water system is shown to represent the existence of the closely packed stacks
Fig. 7. Freeze-fracture morphology of the gel phase of DPPC/water multilamellar vesicles.
Fig. 8. Computed and measured SAXS curves. The domains are assumed to have only one and two layers (bottom curve), strongly different layer numbers (middle curve) and a measured, typical broadened scattering curve (Fig. 5A, 28 ◦ C), (upper curve).
of curved-layers. Based on this picturesque information we can suppose that the vesicles are composed of quasi-concentric stacks having different layer numbers. In these “domains” the lamellar arrangement is regular while the water shells between them are extremely extended so the correlation between the domains are hindered, resulting the broadening of the Bragg reflections. By increasing the concentration of spectinomycin, the layer number of the coherent domains is reduced while that of the extended water shells between them is increased. Considering this feature, we have calculated SAXS profiles using the centrosymmetrical shell model [19]. In Fig. 8, two computed SAXS curves and a measured, typical broadened scattering curve (Fig. 5A, 26 ◦ C, upper curve) can be observed. If the domains are assumed to be extremely small (having only one and two layers) the shape of the scattering curve is similar to the form factor (bottom curve). Supposing strongly different layer number in domains, the appearance of a number of small peaks can be observed (middle curve). The third curve presented is a measured one, under higher magnification (upper curve). In the latter we can reveal also a complex pattern with several small peaks sitting on an extremely broad hump, indicating the existence of an inhomogeneous radial domain size inside the vesicles. 4. Conclusion In spite of the fact that spectinomycin ranked to AGs (a nonaminoglycoside, as it was already mentioned) induces a severe loss in the multilamellar correlation of DPPC/water model membrane systems, the other two investigated true AGs (tobramycin, streptomycin) do not show this effect. The loss of the multilamellar correlation is the consequence of the increased amount of the unbound phospholipids bilayers, known as unbinding and described several years ago [30–32]. This phenomenon possesses high biological importance (for example: fission and fusion of membranes). Recently, the changes in the electrostatic interactions and the surface modulation were considered as the reason for this effect in the presence of bivalent copper ions [33]. Although, during this effect the water shells are extremely extended, we can suppose that the location of spectinomycin is still close to the head groups (they are not placed in the whole water shells) because the wide-angle reflections characteristic for the chain packing of the double layers are strongly and tendentiously effected. The slight differences in the chemical character of spectinomycin may cause the alterations in molecular interactions which lead to the unbinding effect. Spec-
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troscopic studies are required to reveal the details of this significant effect on the layer correlation, which is strongly connected to the antimicrobial mechanisms of the different AGs. Acknowledgements This work was supported by the Hungarian Scientific Funds OTKA (Bóta, T 43055) and a German-Hungarian Research Project. We wish to thank Cs. Novák for giving the opportunity for the calorimetric measurements and Mrs. E. Tóth for the technical assistance at DSC measurements. References [1] [2] [3] [4] [5] [6]
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Structural and calorimetrical studies of the effect of different aminoglycosides on DPPC liposomes Ágnes Oszlánczi a , Attila Bóta b,∗,1 , Gábor Czabai a , Erwin Klumpp c a b c
Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, H-1521 Budapest, Hungary Chemical Research Centre – Hungarian Academy of Sciences, Institute of Nanochemistry and Catalysis, HU-1025 Budapest, Pusztaszeri út 59-67., Hungary Agrosphere Institute, Research Centre, D-52425 Jülich, Germany
a r t i c l e
i n f o
Article history: Received 30 June 2008 Received in revised form 17 November 2008 Accepted 18 November 2008 Available online 25 November 2008 Keywords: Aminoglycosides DPPC liposomes DSC Small- and wide-angle X-ray scattering (SWAXS) Unbinding
a b s t r a c t The effects of tobramycin, spectinomycin and streptomycin on 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC)/water vesicle system were studied by using differential scanning calorimetry (DSC) and simultaneous small- and wide-angle X-ray scattering (SWAXS) in the 0–1 lipid/aminoglycoside molar range. The changes in enthalpy between the thermally adjacent phases are decreased, but the pace of decrease is totally different for the three investigated AGs. The alterations in the lamellar arrangement and the chain packing are rather tendentious and are extended by increased AG concentrations depending on the type of the AG. In the case of tobramycin and streptomycin, still sharp Bragg peaks of SAXS curves shift to smaller values of the scattering variable, while spectinomycin results in an entire loss of multilayer correlation representing an increased amount of unbound bilayers. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The members of the aminoglycoside class of antibiotics have a wide spectrum of antimicrobial activity [1]. Most of the clinically important Gram-negative [1,2], and as well some of the Gram-positive [1] bacterial infections are treated by these powerful drugs. The major problems associated with these antibiotics are the oto- and nephrotoxicity in humans [2,3], and the drug-resistance of the pathogens that have aminoglycoside-modifying enzymes [1]. In human beings these molecules are taken up by proximal tubular cells and are accumulated in lysosomes [3,4]. Several researchers used negatively charged liposomes in a low pH environment to model lysosomal circumstances and examine their interaction with aminoglycosides. They have found a specific interaction between phosphatidylinositol diphosphate (PIP2) – a negatively charged lipid – and aminoglycosides (AGs) [4–6]. In vitro [7], and in vivo [8] studies have shown that AGs bind to PIP2 molecules in the bilayer of both the biological and artificial membranes, and that the AG-PIP2 complexes inhibit the activities of lysosomal phospholipases [4]. The inhibition of the breakdown of different phospholipids causes
∗ Corresponding author. E-mail address: [email protected] (A. Bóta). 1 Leave from Budapest University of Technology and Economics, Dep. of Phys. Chem. and Mat. Sci. 0927-7765/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2008.11.010
phospholipidosis which is considered to be the first phase of toxicity [4,9]. The stability of the AG-PIP2 complex and its consequence, the toxic effect are different, depending on the type of the AG [4–6]. The stability of the complex is related to electrostatic interactions, but hydrophobic interactions may also exist between the drug and the lipid molecules [6,9,10]. Although extensive work has been done by different research groups on the lipid–AG interactions, several conflicting results have been published. As mentioned before, electrostatic interactions were suggested between AGs and an anionic lipid molecule, the PIP2. These findings were based on gel filtration technique, conformational calculations [4,5], and NMR spectroscopy [6]. Structural analysis (freeze-fracture method) has also been used to examine the toxic effect of these antibiotics [11]. Some authors have detected integrity changes in some multilamellar liposome systems by leakage of entrapped molecules [9,10]. The disintegration of the liposomes may be explained by the interaction of the AGs and the hydrophobic domain of the bilayer. Comprehensive research has been carried out to clear up the effect of AGs on model membranes, and only a very weak and slowly proceeding interaction has been found between the AGs and the lipid molecules [12]. In this study the effect induced by some clinically important AGs on the structural and thermotropical changes of a human model membrane has been presented by differential scanning calorimetry (DSC), and simultaneous small- and wide-angle X-ray scattering (SWAXS) measurements. The characteristic features of the
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1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/water multilamellar system was thoroughly examined and reported widely in scientific literature [13–15]. Depending on temperature, different multilamellar structures can be observed in the fully hydrated DPPC liposomes. These are the crystalline (Lc ), the gel (Lˇ ), the rippledgel (Pˇ ), and the liquid crystalline (L˛ ) phases, with of 59, 64, 70.5 and 67 Å characteristic periodicities, respectively [16–18]. Different chain packings result in different subcells (hybrid in the gel phase and hexagonal in the rippled-gel phase). Transitional points occur at 18 ◦ C (sub-transition), 33 ◦ C (pretransition), and 41 ◦ C (main transition), with characteristic endotherm enthalpy changes [16,18]. Formation of defect structures is favoured, especially, during the pretransition, because of the strong fluctuations in the whole system, and the effect of the foreign molecules become stronger than at other transitions [19]. The DPPC molecule, as one of the most common lipids in biomembranes, has proved to be a suitable model membrane constituent in the investigation of the effect of toxic molecules [20–26]. 2. Materials and methods Synthetic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, purity > 99%) was purchased from Avanti Polar Lipids, Inc. (Alabaster, Ala, USA). The different aminoglycosides (AGs): spectinomycin, streptomycin and tobramycin were obtained from Sigma–Aldrich (Steinheim, Germany; purity >99%). Substances were used without further purification. The dried lipids were dissolved in sodium phosphate buffer (10 mM, pH 7.4), prepared from deionized, triple-quartz-distilled water to obtain a lipid concentration of 20 wt.%. The sample was heated above the phase transition (50 ◦ C) for 15 min, cooled to 4 ◦ C, reheated and kept above 50 ◦ C again. After each heating period the samples were stirred intensively. On average 10 repetitions of this process was needed to achieve homogeneous dispersions. For the preparation of systems containing antibiotics the concentrations of AGs were determined at the molar ratios of 10−2 , 10−1 , and 1 (AG/lipids). In the case of the treated systems the AG powders and the pure DPPC were mixed, then dissolved and temperaturetreated. The DSC measurements were performed with a DSC 2920 instrument (TA Instrument, US) operated at a heating rate of 1 ◦ C/min in the temperature range from 25 to 50 ◦ C. The DSC curves were recorded in the heating direction. The reference pan was empty. The calibration of the calorimeter was carried out by using a pure indium sample (Tonset = 156.6 ◦ C). The transition points were characterized by the temperature at which the heat-flow curve exhibited a minimal value.
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SAXS and WAXS measurements were performed using a modified Kratky Compact camera (Anton Paar, Graz, Austria) supplied with two position sensitive detectors (M.Braun, Garching, Germany). Simultaneous detection in the small- and wide-angle region was performed with this SWAXS camera. The scattering of Ni-filtered Cu K␣ radiation ( = 1.542 Å) was recorded in the smallangle range from 10−3 to 10−1 Å−1 and in the wide-angle range from 0.2 to 0.3 Å−1 of the scattering variable, defined as s = (2/) sin , where 2 is the scattering angle (s = 1/d, d is a characteristic periodicity in the sample). For the X-ray measurements the samples were transferred into thin-walled capillaries (Hilderberg, Germany) with a diameter of 1 mm. After centrifugation to remove air bubbles the capillaries were sealed with a two-component synthetic resin and transferred into metal capillary holders that were placed into an aluminium block. This block was positioned directly into the beam line and was used as a thermal incubator for controlled annealing at characteristic temperatures of the lipid systems. After a 10 min incubation of the samples at the respective temperatures the exposure took 600 s for the detection in the small- and wide-angle X-ray scattering regions. 3. Results and discussion 3.1. Thermometric results The pure DPPC multilamellar system exhibits two endothermic phase transitions at 35.3 and at 40.7 ◦ C, corresponding to the pretransition between the gel (Lˇ ) and rippled-gel (Pˇ ) phases, and to the main transition (chain-melting) between the rippledgel and liquid crystalline phases (L˛ ), respectively. The influence of the three different aminoglycosides (AGs: streptomycin, STR; tobramycin, TOB; spectinomycin, SPE, see their chemical structure in Fig. 1) on the phase transition behaviour of the DPPC/water systems are characterized by DSC experiments in the range of AG/lipid molar ratios from 10−2 to 1 (Fig. 2). Transition temperature changes of the antibiotic-doped systems appear only at the pretransition. The apexes of the first dips on the thermograms shift to lower or higher temperature values, but they do so significantly only at 1 AG/lipid molar ratio, indicating that this transition is affected by the chemical structures of the AGs. Structurally similar aminoglycosides like streptomycin and tobramycin increase the pretransitional temperature to 36.4 ◦ C, while spectinomycin, a non-aminoglycoside aminocyclitol still ranked as an AG, decreases it to 33.7 ◦ C. The chain-melting temperature of the system is not affected considerably by the drug molecules (the results remain within the standard deviation of the DSC measurement: ±0.1 ◦ C) as shown in Fig. 2 as well as in Fig. 3A.
Fig. 1. Chemical structure of the three investigated aminoglycosides (streptomycin, STR; tobramycin, TOB; spectinomycin, SPE).
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around 1/4.2 Å−1 both in the gel and the rippled-gel phases, which results from the hybrid and the hexagonal chain packing arrangement in the bilayers, respectively. At 45 ◦ C, after the chain-melting process, the peak disappears entirely.
Fig. 2. DSC curves of the DPPC/water systems at different AG concentrations. Molar ratios and the type of AG (streptomycin, STR; tobramycin, TOB; spectinomycin, SPE) are marked on each curve.
The changes in enthalpy of the pre- and main transition of the DPPC/water systems were thoroughly investigated and the values are in accordance with our previous data (6.55 ± 0.6 and 45.03 ± 4.5 kJ/mol, respectively) [16]. Contrary to the temperature changes, pretransitional enthalpy is not influenced by any of the examined AGs. The areas of the dips representing the changes in enthalpy (Fig. 3B) vary only at the main transitions and are mostly dependent on the concentration and to a lesser extent on the type of the AGs. At 10−2 AG/lipid molar ratio the change in enthalpy of the melting process is strongly decreasing (tobramycin: 30.6 kJ/mol; streptomycin: 32.2 kJ/mol; spectinomycin: 33.3 kJ/mol). At 10−1 and 1 molar ratio the values of streptomycin and spectinomycin treated systems are nearer to that of the pure one, but they still exhibit smaller enthalpy values. At 10−1 TOB/lipid ratio 38.42 kJ/mol, while at 1 molar ratio more significant decrease in enthalpy, 29.76 kJ/mol can be observed. 3.2. Structural study by X-ray diffraction According to the thermotropic behaviour of the systems, the structural studies were carried out at 26, 37, and at 45 ◦ C by simultaneous small- and wide-angle X-ray scattering (SWAXS), corresponding to the gel (Lˇ ), rippled-gel (Pˇ ), and to the liquid crystalline (L˛ ) phases, respectively. The SWAXS patterns of the pure lipid system show the characteristic features of the phases (at every temperature four curves with increasing AG/lipid molar ratios – from bottom to up: 0, 10−2 , 10−1 , 1 – are shown, Figs. 4–6). In the beginning range of the SAXS curves an intense scattering can be observed as a consequence of inhomogeneities with a size-range of several hundreds Å. The first Bragg reflection peaks on the scattering curves, which are typical of the multilamellar systems, appear at 1/64 Å−1 (Lˇ ), 1/70.5 Å−1 (Pˇ ), and 1/67 Å−1 (L˛ ). In the WAXS profiles the maximum of the broadened Bragg reflections are located
Fig. 3. (A) Thermodynamic parameters of the pre- and main transitions of the AG/DPPC model systems: transition temperatures. The type of AG (streptomycin, STR; tobramycin, TOB; spectinomycin, SPE) is marked on each curve. (B) Thermodynamic parameters of the pre- and main transitions of the AG/DPPC model systems: transition enthalpies. The type of AG (streptomycin, STR; tobramycin, TOB; spectinomycin, SPE) is marked on each curve.
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Fig. 4. (A–B) SAXS and WAXS diffractograms, at the denoted temperatures of the pure DPPC and the TOB/DPPC systems, in the range of 10−2 to 1 molar ratio.
The effect of the concentration (in the range of 10−2 to 1 AG/lipid molar ratio) of the chosen antibiotics on the structural changes of the model membranes can be seen in Figs. 4–6. The three examined antibiotics show some similarities in their influence, but significant divergences can also be observed in the layer arrangements of the doped systems. The gel phase (at 26 ◦ C) is not affected at 10−2 molar ratio of any of the used AGs (Figs. 4–6). The intensity and the location of the peaks are the same as in the case of the pure liposomes. At 10−1 SPE/lipid molar ratio the location and the broadening of the peak remains as in the DPPC/water system, but the intensity of the peak diminishes significantly (Fig. 5). Tobramycin and streptomycin added systems result in the same patterns at this molar value (Figs. 4 and 6, respectively). At the equal molar amount of the AGs and the lipid molecules, spectinomycin causes the strongest effect: the SAXS peak disappears almost entirely. With the other two antibiotics the maximum of the peaks shift to higher values
Fig. 5. (A–B) SAXS and WAXS diffractograms, at the denoted temperatures of the pure DPPC and the SPE/DPPC systems, in the range of 10−2 to 1 molar ratio.
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Fig. 6. (A–B) SAXS and WAXS diffractograms, at the denoted temperatures of the pure DPPC and the STR/DPPC systems, in the range of 10−2 to 1 molar ratio.
of the scattering variable, that is from 1/64 to 1/62.5 Å−1 , meaning that the distance between the lipid layers become smaller. As we expected (see Section 1), the rippled-gel phase (37 ◦ C) was the most sensitive to the presence of the AGs. The SAXS patterns of the TOB/lipid and SPE/lipid systems show gradual broadening and the intensity of the peaks start to decrease continuously from 10−2 molar ratio upwards. At 1 molar ratio of the tobramycin the location of the peak jumps to a higher scattering variable, while with spectinomycin the Bragg peak ceases to exist. In the presence of streptomycin the intensity and the broadening of the peaks do not change with the different concentrations of the drug, but the maximum of the peaks shift from 1/70.5 to 1/72.5 Å−1 at 10−1 and to 1/69.5 Å−1 at 1 STR/lipid molar ratio. At 45 ◦ C (liquid crystalline phase) strong fluctuations in the layer arrangement of all antibiotic-treated system can be observed. The peak maximums on the SAXS patterns alternate within a wide range of scattering variable depending on the concentration of AG-molecules. At 10−2 TOB/lipid molar ratio the distance of the layers decreases as the position of its characteristic peak moves from 1/66.5 to 1/64.5 Å−1 – than at 10−1 molar ratio from 1/66.5 to 1/65 Å−1 . At the equimolar ratio of the tobramycin the layer distance is reduced strongly as the peak appears at 1/61 Å−1 . The spectinomycin show the same tendency depending on concentration, but at the highest examined SPE/lipid ratio, the peak shift to smaller value than that of the pure system (1/69 Å−1 ). By increasing the ratio of the drug, the intensity of the peaks strongly decreases. At 1 SPE/lipid molar ratio the intensity of the first Bragg reflection is reduced extremely. On the SAXS patterns of the streptomycindoped systems at higher STR/lipid ratios only the locations of the peaks vary. At 10−1 molar ratio the peak shifts to lower scattering variables (from 1/66 to 1/67 Å−1 ), while the peak moves to 64 Å−1 at 1 molar ratio. With the subcell formation changes the same tendency can be examined (Figs. 4–6, B panel), thus the WAXS results can be summarized without focusing on any of the used drugs. At the gel phase, as the ratio of the antibiotics increases, the maximum of the WAXS peaks shift to lower values of the scattering variable, indicating the increase of the distance of the neighbouring lipid molecules by intercalation of AG molecules. Taking into account the chemical characters of the AGs, it can be supposed that the location of these molecules is close to the head groups. The intensity of the peaks
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decrease at 10−1 , and the subcells of the chain packing disappear at 1 molar ratio. WAXS pictures of the rippled-gel phase show the loss of the regular lipid arrangement at higher antibiotic ratios. As in the pure system, subcells cease to exist in all doped samples above the chain-melting temperature. The fact that signal of the subcells vanishes, relates to non-specific interactions between the AG and the lipid molecules as no characteristic subcell is formed by the presence of AGs. Moreover, the relatively minor changes in enthalpy also indicate that those interactions which determine the calorimetrical features are not influenced in the gel phases of the doped systems. 3.3. Interpretation of the SAXS patterns The studied AGs influenced the lamellar arrangement of the vesicles differently. Basically, two phenomena (1) shifting of the still sharp Bragg peaks of SAXS curves to smaller values of the scattering variable or (2) drastic broadening in the Bragg reflections can be observed. The shift is a consequence of the reduction of the thickness of each layer caused by embedding AG molecules. The sharp reflections indicate that the presence of foreign molecules does not destroy the regularity of the multilamellar arrangement, which is typical of DPPC/water system. This effect appears in the case of streptomycin in all investigated temperature domains. Moreover, a significant shift can be observed in the tobramycindoped system (Fig. 4, at 45 ◦ C, the periodicity of the pure system was decreased in the presence of equimolar AG from 67 to 61 Å). In this system the main transition undergoes a significantly low change in enthalpy indicating also the increased presence of the AG molecules, whereby the characteristic interactions, connecting to the chain-melting, are reduced; therefore the affected interactions are hydrophobic. The broadening in the Bragg reflections is characteristic for the spectinomycin-doped systems indicating a severe loss in multilamellar correlation. Generally, the broadening and the final disappearance of the Bragg reflections, caused by any foreign molecules, can be explained in several cases by the paracrystalline theory considering only the average fluctuations of the one-dimensional layer arrangement [27–29]. To obtain direct visual information in the real space we have executed freeze-fracture procedure on AG-vesicle systems. In the electronmicrographs the aggregation of liposomes is typical, especially in the case of higher spectinomycin concentration as it was also observed in other lipid systems and published in the earlier [11]. The characteristic, well ordered lamellar arrangement of the pure lipid–water system, however, is present even at the presence of spectinomycin. In Fig. 7, the surface morphology of the fractured gel phase of DPPC/water system is shown to represent the existence of the closely packed stacks
Fig. 7. Freeze-fracture morphology of the gel phase of DPPC/water multilamellar vesicles.
Fig. 8. Computed and measured SAXS curves. The domains are assumed to have only one and two layers (bottom curve), strongly different layer numbers (middle curve) and a measured, typical broadened scattering curve (Fig. 5A, 28 ◦ C), (upper curve).
of curved-layers. Based on this picturesque information we can suppose that the vesicles are composed of quasi-concentric stacks having different layer numbers. In these “domains” the lamellar arrangement is regular while the water shells between them are extremely extended so the correlation between the domains are hindered, resulting the broadening of the Bragg reflections. By increasing the concentration of spectinomycin, the layer number of the coherent domains is reduced while that of the extended water shells between them is increased. Considering this feature, we have calculated SAXS profiles using the centrosymmetrical shell model [19]. In Fig. 8, two computed SAXS curves and a measured, typical broadened scattering curve (Fig. 5A, 26 ◦ C, upper curve) can be observed. If the domains are assumed to be extremely small (having only one and two layers) the shape of the scattering curve is similar to the form factor (bottom curve). Supposing strongly different layer number in domains, the appearance of a number of small peaks can be observed (middle curve). The third curve presented is a measured one, under higher magnification (upper curve). In the latter we can reveal also a complex pattern with several small peaks sitting on an extremely broad hump, indicating the existence of an inhomogeneous radial domain size inside the vesicles. 4. Conclusion In spite of the fact that spectinomycin ranked to AGs (a nonaminoglycoside, as it was already mentioned) induces a severe loss in the multilamellar correlation of DPPC/water model membrane systems, the other two investigated true AGs (tobramycin, streptomycin) do not show this effect. The loss of the multilamellar correlation is the consequence of the increased amount of the unbound phospholipids bilayers, known as unbinding and described several years ago [30–32]. This phenomenon possesses high biological importance (for example: fission and fusion of membranes). Recently, the changes in the electrostatic interactions and the surface modulation were considered as the reason for this effect in the presence of bivalent copper ions [33]. Although, during this effect the water shells are extremely extended, we can suppose that the location of spectinomycin is still close to the head groups (they are not placed in the whole water shells) because the wide-angle reflections characteristic for the chain packing of the double layers are strongly and tendentiously effected. The slight differences in the chemical character of spectinomycin may cause the alterations in molecular interactions which lead to the unbinding effect. Spec-
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troscopic studies are required to reveal the details of this significant effect on the layer correlation, which is strongly connected to the antimicrobial mechanisms of the different AGs. Acknowledgements This work was supported by the Hungarian Scientific Funds OTKA (Bóta, T 43055) and a German-Hungarian Research Project. We wish to thank Cs. Novák for giving the opportunity for the calorimetric measurements and Mrs. E. Tóth for the technical assistance at DSC measurements. References [1] [2] [3] [4] [5] [6]
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