SERR Study of the Interaction of Anthracyclines with Mono - American

Sep 1, 1997 - The interaction of positively charged anthracyclines, pirarubicin, and ... of the screen formed by pirarubicin adsorbed on DPPA polar he...
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Langmuir 1997, 13, 5634-5643

SERR Study of the Interaction of Anthracyclines with Mono- and Bilayers of Charged Phospholipids C. Heywang, M. Saint-Pierre-Chazalet,* M. Masson, and J. Bolard Laboratoire de Physicochimie Biomole´ culaire et Cellulaire, CNRS URA 2056, Universite´ Paris 6, Case 138, 4 place Jussieu, 75252 Paris cedex 05, France Received December 18, 1996. In Final Form: March 26, 1997X The interaction of positively charged anthracyclines, pirarubicin, and adriamycin with planar monolayers and bilayers of zwitterionic and anionic phospholipids (POPC and DPPA) is reported, by using a surface pressure technique and surface-enhanced resonance Raman scattering (SERRS). The results are compared with those of a previous study concerning the pirarubicin-POPC interaction. The surface pressure technique enabled us to determine the speed of penetration and the amount of anthracycline trapped in phospholipid monolayers. The behavior of both anthracyclines was different, depending on their hydrophilic-lipophilic balance and the charges of phospholipids. We observed, in particular, that the presence of anionic DPPA in the monolayer does not enhance the percentage of pirarubicin at the interface as compared to the adsorption in a pure POPC monolayer. This could be due to the formation of a screen by pirarubicin adsorbed on the polar head groups of anionic phospholipids. These molecules would prevent the other molecules of pirarubicin dissolved in the bulk from penetrating more deeply into the phospholipids. These monolayers were transferred by a Langmuir-Blodgett technique onto silver-coated prisms, in order to study them by SERRS and have information on the orientation of the anthracycline in the phospholipid bilayer thus obtained. The spectrum of a phospholipid bilayer containing anionic DPPA (POPC-DPPA 80-20 mol %), put in contact with an aqueous solution of pirarubicin showed that this anthracycline is able to cross the bilayer and is lying parallel to silver after crossing. This result confirms the hypothesis of the screen formed by pirarubicin adsorbed on DPPA polar head groups, proposed after the surface pressure study. Adriamycin in contact with a POPC bilayer is able to cross this bilayer too. It does not remain in the bilayer, probably because of its weak hydrophobicity, and is lying on silver after crossing. In the presence of anionic DPPA in the bilayer, adriamycin remains adsorbed on the polar head groups of the second monolayer (in contact with water) and does not penetrate into the bilayer.

1. Introduction Surface-enhanced Raman scattering (SERS) is a powerful technique for the study of biological molecules1 and lipid bilayers used as a simplified membrane model, because of its short range of detection.2-4 An original setup built in our laboratory and already described has been used to study phospholipid planar bilayers and the interaction between an anthracycline, pirarubicin, and zwitterionic phospholipid planar bilayers in contact with water.5,6 Anthracyclines are molecules used in the treatment of some cancers. Although DNA is considered to be their main target, it has been shown that the interaction between the antibiotic and the cellular membrane could play a role in the efficiency of this molecule.7 Moreover a resistance to the drug appears during treatment:8 one way to overcome this problem is to increase the amount of these antibiotics incorporated into the membrane, in order to enhance the level taken into the cells. A better understanding of the interaction between the anthracy* Correspondence should be addressed to M. Saint-Pierre Chazalet. Telephone: 33 1 44 27 42 40. Fax: 33 1 44 27 75 60. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, September 1, 1997. (1) Cotton, T. M. In Spectroscopy of Surfaces; Clark, R. J. H., Hester, R. E., Eds.; John Wiley and Sons Ltd: New York, 1988; Chapter 3. (2) Kovacs, G. J.; Loutfy, R. O.; Vincett, P. S.; Jennings, C.; Aroca, R. Langmuir 1986, 6, 689. (3) Cotton, T. M.; Uphaus, R. A.; Mo¨bius, D. J. Phys. Chem. 1986, 90, 6071. (4) Aroca, R.; Guhathakurta-Ghosh, U. J. Am. Chem. Soc. 1989, 111, 7681. (5) Saint-Pierre-Chazalet, M.; Masson, M.; Bousquet, C.; Bolbach, G.; Ridente, Y.; Bolard, J. Thin Solid Films 1994, 244, 852. (6) Heywang, C.; Saint-Pierre Chazalet, M.; Masson, M.; GarnierSuillerot, A.; Bolard, J. Langmuir 1996, 12, 6459. (7) Tritton, T.; Yee, G. Science 1982, 217, 248. (8) Bradley, G.; Juranka, P. F.; Ling, V. Biochim. Biophys. Acta 1988, 948, 87.

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cline molecules and phospholipid membranes will facilitate the rational design of more efficient derivatives. Studies have already been realized on the anthracycline-membrane interaction with anionic phospholipidcontaining monolayers, vesicles, and cellular membranes. Indeed anthracyclines are amphiphilic molecules positively charged, and their interaction with anionic phospholipids plays an important role in the penetration into the cells. Circular dichroism has shown that adriamycin binds to negatively charged phospholipid vesicles, containing egg phosphatidic acid (EPA) or cardiolipin.10 Two different patterns are obtained depending on the lipid/ adriamycin ratio: this result suggests that there are two types of binding sites. In the first type, there is an electrostatic interaction between the adriamycin positive charge and the negative polar head group. In the second type, the adriamycin anthraquinone group is, in addition, embedded in the lipid bilayer. This suggests an hydrophobic interaction too. Other studies using surface pressure techniques, conformational analysis or fluorescence transfer confirm the existence of these interactions.11,12 Speelmans et al. have studied the permeability and the crossing of adriamycin through the phospholipid bilayer too, by observing the fluorescence of the anthracycline in contact with DNA-containing large unilamellar vesicles (LUVs) of dioleoylphosphatidylserine (phospholipids with anionic phosphatidylserine being present in the inner layer of the erythrocyte membrane).13 They showed that, in (9) Dupou-Ce´zanne, L.; Sautereau, A. M.; Tocanne, J. F. Eur. J. Biochem. 1989, 181, 695. (10) Henry, N.; Fantine, E. O.; Bolard, J.; Garnier-Suillerot, A. Biochemistry 1985, 24, 7085. (11) Nicolay, K.; Sautereau, A. M.; Tocanne, J. F.; Brasseur, R.; Huart, P.; Ruysschaert, J. M.; de Kruijff, B. Biochim. Biophys. Acta 1988, 940, 197. (12) Ferrer-Montiel, A. V.; Gonzalez-Ros, J. M.; Ferragut, J. A. Biochim. Biophys. Acta 1988, 937, 379.

© 1997 American Chemical Society

Interaction of Anthracyclines with Phospholipids

the presence of DOPS in LUVs, the permeability coefficient of adriamycin decreased as compared to the one observed in zwitterionic vesicles. Some hypotheses were proposed: the concentration of adriamycin free and transportable could be decreased because of the higher interaction between the anthracycline and DOPS. Another possibility would be that the packing of phospholipids would be different in the presence of anionic phospholipids and could limit the crossing of the bilayer by adriamycin. Finally, Speelmans et al. suggested that adriamycin forms complexes with the anionic phospholipids of the inner layer, these complexes tightening the polar head groups at the interface. Under these conditions, there would be an accumulation of adriamycin on the inner layer, due to a lower penetration. Lastly, de Wolf et al. studied the consequences of the interaction of adriamycin with anionic phospholipids by using fluorescence, nuclear magnetic resonance (NMR), and small-angle X-ray scattering and proposed a model of interaction depending on the molar adriamycin/phospholipid ratio.14 At low ratio (0.01-0.02), the amino function of adriamycin would be in interaction with the polar head groups, whereas the chromophore would be embedded in lipids. If the ratio increases, drug-drug interaction would occur and the chromophores would be stacked at the surface of the polar head groups. This last type of interaction would create aggregates, limiting a deeper penetration of adriamycin in the bilayer. The fluorescence also enabled us to determine a mean dielectric constant of the chromophore environment: de Wolf et al. showed that this constant was between 40 and 60 and suggested that the chromophore would thus be close to the membrane surface. In our previous paper, we studied the interaction of an anthracycline, pirarubicin, with monolayers and bilayers of palmitoyloleoylphosphatidylcholine (POPC), a zwitterionic phospholipid.6 We have shown that electrostatic and hydrophobic interactions are important and have determined the orientation of the anthracycline in planar POPC bilayers. Here, in order to improve the previous results, we introduced in POPC 20% of a negatively charged phospholipid, dipalmitoylphosphatidic acid (DPPA): this phospholipid enables us to modify the hydrophilic-lipophilic balance and thus the anthracycline-phospholipid interactions and to simulate the inner layer of a biological membrane, the composition of the membrane being asymmetric.15 Indeed the inner layer contains anionic phospholipids like phosphatidylserine,16 and the interaction of anthracycline with the inner layer could be important for the equilibrium between anthracycline molecules inside and outside the cell. This interaction could have an influence on the efficiency of the molecule. We studied here what influence the presence of the anionic phospholipid has on the penetration speed and the incorporation level of two positively charged anthracyclines, pirarubicin and adriamycin. We used first a surface pressure technique to compare the interaction between the two anthracyclines (pirarubicin or adriamycin) and POPC or POPC-DPPA (80-20 mol %) monolayers. Then we transferred the anthracycline/phospholipid monolayers by a Langmuir-Blodgett technique onto planar supports and studied the bilayers thus obtained by surface-enhanced Raman scattering (SERS) to have information on the orientation of the two (13) Speelmans, G.; Staffhorst, R.; de Kruijff, B.; de Wolf, F. A. Biochemistry 1994, 33, 13761. (14) de Wolf, F. A.; Maliepaard, M.; van Dorsten, F.; Berghuis, I.; Nicolay, K.; de Kruijff, B. Biochim. Biophys. Acta 1991, 1096, 67. (15) Devaux, P. F. Biochemistry 1991, 30, 1163. (16) Op den Kamp, J. A. F. Annu. Rev. Biochem. 1979, 48, 47.

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Figure 1. Chemical formulas of (a) doxorubicinone and (b) adriamycin and pirarubicin. (c) Geometrical model for pirarubicin proposed after the modeling study.

anthracyclines in the phospholipid bilayers. Next, the crossing of a “preformed” bilayer (a pure phospholipid bilayer put in contact with an aqueous solution of anthracycline) was studied and compared for both anthracyclines. 2. Materials and Methods 2.1. Materials. Palmitoyloleoylphosphatidylcholine (POPC, a zwitterionic phospholipid) and dipalmitoylphosphatidic acid (DPPA, an anionic phospholipid with one negative charge at pH ) 5.5) were purchased from Sigma (St. Louis, MO) and were 99% pure. These phospholipids were dissolved in ethanol/chloroform 1/1 (v/v), at a concentration of 10-3 M. Doxorubicin (or adriamycin, Figure 1b) and 4′-O-(tetrahydropyranyl)doxorubicin or pirarubicin (Figure 1b) were kindly provided respectively by Farmacia Laboratory (Italy) and Laboratoire Roger Bellon (France). They were dissolved in Millipore water (pH ) 5.5) or in an organic solvent (dimethyl sulfoxide, DMSO) at an initial concentration of 10-3 M. These two anthracyclines differ by the presence of a hydrophobic THP group in pirarubicin. Both adriamycin and pirarubicin have an amino function positively charged at pH ) 5.5. 2.2. Methods. 2.2.1. Monolayers. Monolayers were prepared using a laboratory-built Langmuir trough. The subphase was Millipore water at pH ) 5.5 (R ) 18 MΩ). The surface pressure was measured with a displacement force transducer (Kaman Sciences Corporation, Colorado Springs, CO), and an electronic device enabled us to keep constant the monolayer pressure by monitoring the barrier displacement. This system was used during penetration experiments and layer transfers. All experiments were performed at 21(1 °C. Two methods were used to study the anthracyclinephospholipid interaction: (i) Adsorption and/or Penetration of the Anthracycline into a Phospholipid Monolayer. A phospholipid monolayer was spread at the air-water interface and compressed to 2 mN/m. This pressure was then kept constant. An aqueous solution of anthracycline was then injected in the subphase, at a final concentration of 0.4, 1, or 1.6 µM.6

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At equilibrium the anthracycline/phospholipid monolayer was compressed. We will call this kind of monolayers “adsorbed” monolayers in the following paragraphs. (ii) Spreading of a Mixed Anthracycline/Phospholipid Monolayer. We spread at the interface various mixed solutions of POPC or POPC-DPPA (80-20 mol %)/ anthracycline (into organic solvents) and compressed them. This kind of monolayers will be called “spread” monolayers in the following paragraphs. 2.2.2. Preparation Technique of Planar Bilayers; SERR Study. We used the same setup as in our previous study to transfer anthracycline/phospholipid bilayers from the air-water interface to the prism.5,6 The technique of transfer was the following. The first monolayer was transferred according to a classical Langmuir-Blodgett technique: molecules at the interface were compressed at a pressure of 25 mN/m and at a speed of 5 mm‚min-1 onto a high-index rutile prism. Previously, a thin layer of silver had been deposited onto a face of the prism, this face being perpendicular to the air-water interface during the first transfer. The silver layer is indispensable for the following SERR experiments. To transfer the second layer, the prism was first rotated by 90°, the silver coating being thus parallel to the air-water interface, and carefully brought in contact with the monolayer spread at the interface. After 2 min, the prism was dipped into water and fixed in a little Teflon box in the bottom of the trough. Under these conditions, the polar head groups of the second layer remained in contact with water. It was possible to remove the prism with the bilayer out of the subphase without destroying the bilayer. Three kinds of planar bilayers were realized for the SERR study: (i) “Adsorbed” Bilayers. They were obtained after adsorption of an aqueous solution of anthracycline into a POPC or a POPC-DPPA (80-20 mol %) monolayer, compression to 25 mN/m, and transfer. (ii) “Spread” Bilayers. They were obtained after spreading and compression to 25 mN/m of an anthracycline/ phospholipid mixture (we chose a solution containing initially 66.6% antibiotic to stay under the same conditions as in our preceding study: in the case of pirarubicin, this ratio enabled us to keep a high level of antibiotic at the interface without spreading too many molecules). (iii) “Preformed” Bilayers. We transferred a phospholipid bilayer onto the prism and then put it in contact with an aqueous solution of the anthracycline at a concentration of 0.4 or 2 µM. This last model is the closest to a cellular membrane in contact with an exogenous molecule. The face coated with the monolayer or the bilayer was then illuminated with an Ar+ laser beam at 488 nm, under an angle of incidence greater than the limit angle of reflection, enabling the formation of an evanescent wave. The thickness of the silver layer being 15 nm, the metal thus deposited forms islands, which enable us to enhance the Raman signal by a maximum factor of 106 (SERS effect).17 Two phenomena are responsible for this effect: a modification of the polarizability tensor of the molecules and an amplification of the electrical field near the metal. Under these conditions, it is impossible to realize polarization experiments, since the wave of excitation is completely depolarized. However it is possible to obtain information about the orientation of molecules on silver, since the range of SERS is short (a few nanometers): only the bands corresponding to the molecules or the parts of the molecules which are close to silver are enhanced. In the presence of phospholipids, the molecules observed are really in contact with the monolayer or the bilayer. (17) Bousquet, C.; Masson, M.; Harrand, M. J. Raman Spectrosc. 1995, 26, 273.

Heywang et al.

Figure 2. Penetration kinetics of pirarubicin in (a) a POPC monolayer and (b) a POPC-DPPA (80-20 mol %) monolayer. The pressure of the phospholipid monolayer during the penetration was kept constant at 2 mN/m (pirarubicin concentration in the subphase ) 1.6 µM, pH ) 5.5, T ) 21 ( 1 °C).

The wavelength used in this study being in the pirarubicin or adriamycin absorption band, the signal was still increased (surface-enhanced resonance Raman scattering or SERRS). It was then collected in a direction perpendicular to the surface sample and analyzed with a Dilor (Lille, France) triple monochromator, associated with a data acquisition system. 3. Surface Pressure Study 3.1. Results. 3.1.1. Study of the Pirarubicin-Phospholipid Interaction. Our main goal was to study on one hand the penetration speed of pirarubicin into the phospholipid monolayer and on the other hand the level incorporated. We compared the previous results obtained with monolayers of pure zwitterionic POPC6 with those obtained with monolayers composed of a POPC-DPPA (80-20 mol %) mixture. We studied the antibiotic-phospholipid interaction by using two techniques: “adsorption and/or penetration” and “spreading” of pirarubicin. (i) Adsorption and/or Penetration of an Aqueous Solution of Pirarubicin into a Phospholipid Monolayer: “Adsorbed” Monolayers. (a) Penetration Speed. Figure 2 shows the penetration kinetics of pirarubicin in a POPC or a POPC-DPPA (80-20 mol %) monolayer at a surface pressure of 2 mN/m. We observe that in the presence of DPPA, the duration necessary for the penetration is about 1 h (5 or 6 times faster than in the presence of pure POPC). Pirarubicin being positively charged under our experimental conditions, the presence of negative molecules at the interface favors the adsorption of pirarubicin: an electrostatic interaction occurs in this adsorption. (b) Antibiotic Level at the Interface. When equilibrium was reached after adsorption and/or penetration, we compressed the molecules at the interface and obtained an isotherm shifted to greater areas as compared to the those for phospholipid isotherm. We can use the difference between the two isotherms (POPC-DPPA and POPCDPPA/pirarubicin) to estimate the pirarubicin level staying at the interface, by taking into account the molecular area of pirarubicin estimated thanks to a modeling study (Figure 1c).6 This study performed in our laboratory was realized with the Hyperchem Windows sofware (Autodesk), which gave the structure of pirarubicin in vacuum after an energy minimization. Two possibilities of orientation at the interface (anthraquinone part perpendicular or parallel to the interface) could be considered. In both cases the mean molecular area was estimated to 90 Å2. We observed, first, that the saturation of the phospholipid monolayer occurs at about 1 µM for both POPC and

Interaction of Anthracyclines with Phospholipids

Figure 3. Mean molecular area at 25 mN/m versus the initial percentage of pirarubicin in the spread solution (pH ) 5.5, T ) 21 ( 1 °C) of pirarubicin/POPC-DPPA (80-20 mol %) mixtures. The straight line gives the theoretical mean molecular area obtained if there was no interaction between antibiotic and phospholipid.

POPC/DPPA monolayers (data not shown). Second, the maximum percentages of pirarubicin incorporated in POPC and POPC-DPPA monolayers after penetration at 2 mN/m and compression to 25 mN/m are respectively 11% and 13%. Thus the presence of the anionic phospholipid does not greatly enhance the penetration level of pirarubicin in the monolayer, whereas the penetration speed is drastically increased. (ii) Spreading of Mixed Phospholipid/Pirarubicin Solutions: “Spread” Monolayers. From the isotherms of the mixed solutions of POPC-DPPA (80-20 mol %)/pirarubicin (results not shown), we have determined the amount of pirarubicin remaining at the interface after compression and have reported (Figure 3) the variation of the mean molecular area of each mixture at 25 mN/m versus the initial mole percentage of pirarubicin in the spread solution, in the presence of POPC or POPC-DPPA (8020 mol %). Pirarubicin alone (“mixture” containing 100% antibiotic) is able to remain at the interface after spreading.6 However its isotherm does not reach 25 mN/m; thus, we considered that the mean molecular area corresponding to an initial mole-percentage of 100% is equal to zero on Figure 3. We can define an additivity straight line passing through the two extreme points (0 and 100% pirarubicin). We observe that all experimental points are above it (mean molecular area greater than the area of pure phospholipids): phospholipids being insoluble in the subphase, we can conclude that the presence of a POPC or a POPCDPPA (80-20 mol %) monolayer enables us to keep pirarubicin at the interface. According to these results, we can estimate the percentage of pirarubicin staying at the interface at 25 mN/m versus the initial percentage of antibiotic in the spread mixture (Figure 4). As previously, the percentages are estimated by taking into account a molecular area of 90 Å2 for the pirarubicin molecule. We observe that the percentage of pirarubicin staying in POPC-DPPA monolayers versus the total number of molecules in the monolayers is always 10%, whatever the initial level of pirarubicin is. In the presence of pure POPC this percentage increases regularly until 38%. This result seems surprising, since pirarubicin is positive and POPC is zwitterionic, whereas DPPA is anionic. The presence of DPPA would have been expected to increase the level of pirarubicin at the interface as compared to the case for pure POPC. 3.1.2. Study of the Adriamycin/Phospholipid Interaction. We compare now the results obtained with adriamycin (Figure 1b). This molecule is positively charged

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Figure 4. Percentage of pirarubicin remaining at the interface after compression at 25 mN/m of (a) POPC/pirarubicin mixtures and (b) POPC-DPPA (80-20 mol %)/pirarubicin mixtures versus the initial percentage of pirarubicin in the spread mixture (pH ) 5.5, T ) 21 ( 1 °C).

but is devoid of the hydrophobic THP group: adriamycin is less hydrophobic than pirarubicin. (i) “Adsorbed” Monolayers. As previously, we compared the penetration of adriamycin into a POPC or POPCDPPA monolayer kept at 2 mN/m. However this penetration was so slow in POPC monolayers that we decided to use only a strong concentration (1.6 µM) and to limit the penetration time to 15 min. We observed first that the presence of DPPA in spread phospholipids enhanced the penetration speed about twice as compared to that for pure POPC. As previously we determined the percentage of adriamycin incorporated into phospholipids thanks to an estimation of the mean molecular area of adriamycin. This mean molecular area was considered in two extreme cases, the long axis of the anthraquinone part being parallel or perpendicular to the air-water interface, as for pirarubicin. But, contrary to the case for pirarubicin, this area was different according to these two possible orientations, and an average of 75 Å2 per molecule was used for the calculations. Thus after compression to 25 mN/m, the level of adriamycin incorporated into the monolayer versus the total number of molecules at the interface was 4% in the case of POPC, whereas it was 11% in the case of the POPC-DPPA (80-20 mol %) mixture. (ii) “Spread” Monolayers. As in the case of pirarubicin, we compared the amount of adriamycin staying at the air-water interface at 25 mN/m in POPC and POPCDPPA (80-20 mol %) spread monolayers. We observed that the maximum percentages of adriamycin incorporated into the phospholipids were respectively about 9 and 10%. Thus the amount of adriamycin at the interface is not quite different in the presence or in the absence of DPPA in the spread phospholipids. 3.2. Discussion. Surface pressure measurements are thus powerful for studying the interaction of anthracyclines with phospholipids at the air-water interface. The main points are the following. (i) Case of Pirarubicin. The results show that pirarubicin is able to adsorb at the interface more quickly into POPC-DPPA monolayers than into pure POPC monolayers. It is not surprising, since DPPA is anionic and pirarubicin positively charged under our experimental conditions, that an electrostatic interaction increases the speed of adsorption. However, we observe that (a) the percentage of pirarubicin remaining at the interface is slightly more important in the “adsorbed” POPC-DPPA monolayers than in pure POPC monolayers; and (b) all the negative charges of DPPA are not neutralized. These results are surprising since other studies showed that anthracyclines bind preferentially to membrane models containing anionic phospholipids and neutralize the negative charges

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of these lipids.18 In our case, we observe a maximum percentage of pirarubicin of about 10% at the interface in the presence of 20% DPPA (or 20% negative charges in the mixture). Thus half of these charges are not neutralized. However we can suppose that the POPC-DPPA bilayers are not perfectly homogenous. Clusters concentrated with DPPA could form among POPC, one chain being different in the two phospholipids. Pirarubicin would be preferentially bound to these clusters by electrostatic interaction, as compared to the case for POPC, and locally concentrated. This local concentration could create a steric hindrance, which would limit the access of pirarubicin to DPPA: only half the DPPA molecules could be neutralized because of the limited access and the cohesion of molecules at 25 mN/m. This hypothesis is in agreement with the size of the polar head group of DPPA (its mean molecular area being around 40 Å2) as compared to the molecular area of pirarubicin estimated to 90 Å2. This difference implies a steric hindrance at the level of adsorbed pirarubicin molecules. At last, these molecules of pirarubicin adsorbed onto the polar head groups would form a screen which would hinder other molecules from penetrating more deeply into the monolayer. The percentage of pirarubicin in contact with phospholipids would be thus limited. (ii) Case of Adriamycin. The percentages kept at the interface after the adsorption and/or penetration are different according to the nature of the phospholipid monolayer, contrary to the case for pirarubicin. They are more important in the presence of POPC-DPPA monolayers than in the presence of pure POPC monolayers. This difference of behavior can be explained by a different hydrophilic-lipophilic balance, which would favor the electrostatic interaction in the case of adriamycin. However the percentages kept at the interface are always lower than those observed with pirarubicin. We suppose that this difference can be explained by the presence or the absence of the hydrophobic THP group. According to the higher percentage observed in the case of pirarubicin, it seems that the hydrophobic component of the interaction favors the insertion of pirarubicin into the phospholipid monolayer. Because adriamycin does not have the THP group, its hydrophilic-lipophilic balance is modified compared to the balance of pirarubicin. This parameter plays thus a very important role into the anthracycline-membrane interaction, as shown by Goormaghtigh et al.18 In order to complete these results, a SERR study has been performed to compare the orientations of pirarubicin and adriamycin in the presence of POPC or POPC-DPPA. Indeed we saw that a hypothesis explaining the surface pressure results obtained with pirarubicin is a different insertion of this molecule into POPC and POPC-DPPA monolayers. The insertion of adriamycin could be different too, according to its lower hydrophobicity. Under these conditions, SERRS is powerful for checking these hypotheses. 4. SERR Study 4.1. Results. Our goal was to study the orientation of the two anthracyclines in POPC-DPPA (80-20 mol %) planar bilayers and to compare it with the results obtained with pure POPC bilayers. In order to compare the spectra more easily, the spectrum of graphitized carbon (resulting from a photochemical reaction between CO2 present in air or water and the metal coating) was always subtracted from the (18) Goormaghtigh, E.; Chatelain, P.; Caspers, J.; Ruysschaert, J. M. Biochim. Biophys. Acta 1980, 597, 1.

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solution and bilayer SERR spectra. This graphitized carbon spectrum gives two broad bands at 1375 and 1590 cm-1.19 This last region is thus difficult to use for the interpretation of results. 4.1.1. Orientation of Pirarubicin and Adriamycin Deposited from an Aqueous Solution onto the Silver Coating. We studied, in our previous work, the orientation of pirarubicin deposited from an aqueous solution onto the silver coating.6 Its SERR spectrum is presented in Figure 5a. We were particularly interested in the bands at 980 cm-1, corresponding to the chromophore breathing, the bands at 1206 and 1240 cm-1, assigned to the δ(CO-H) vibration, and the bands at 1260 and 1400 cm-1, assigned to the ring stretch.20-22 Another band at 1450 cm-1 was studied too. This band (present in the THP spectrum recorded in our previous study6) can be only partly assigned to the δ(CH2) of the THP group (Table 1), since this band appears in the adriamycin spectrum too, whereas this molecule does not have the THP group. In this case, it is assigned to the ring stretch.20 By comparing the relative intensities of these bands in the resonance Raman (in the absence of silver) and the SERR spectra of pirarubicin and doxorubicinone (the anthraquinone part “cleared” from the sugar and the THP group, Figure 1a), we proposed the following orientation of pirarubicin on silver: its chromophore and sugar (OH) function would be close to silver, the anthraquinone part being more or less perpendicular to the silver coating, whereas the hydrophobic THP group would be far from it. In the same study we proposed that doxorubicinone would be lying on the silver coating, the vibrations of the rings being enhanced, particularly at 1260 and 1400 cm-1 (Figure 5c, Table 1). Here, we studied the orientation of adriamycin under the same conditions as those for pirarubicin. Its SERR spectrum is shown in Figure 5b. On the whole, it is closer to the doxorubicinone spectrum than to the pirarubicin spectrum: the band at 980 cm-1 has about the same intensity as that in the doxorubicinone spectrum, if we compare the relative intensities versus the intensity of the band at 1240 cm-1, which is less variable in every spectrum. The band at 1206 cm-1 is slightly more intense, but it is weaker than that in the pirarubicin spectrum. The peak at 1260 cm-1 appears like a shoulder, as in the pirarubicin spectrum, but it is more intense. This spectrum suggests that adriamycin would have the same type of orientation as doxorubicinone and would be more or less lying on silver. 4.1.2. SERR Study of the Pirarubicin/Phospholipid Mixed Bilayers. We describe here the results obtained with the pirarubicin/POPC-DPPA (80-20 mol %) bilayers. The results concerning the pirarubicin/POPC bilayers were obtained in our previous work.6 In all cases, the integrity of the bilayer was checked by observing the 2750-3050 cm-1 region: the presence of an intense band at 2960 cm-1 (assigned to the asymmetric stretching vibration of methyl groups) shows that the structure of the bilayer is maintained and that the presence of the anthracycline does not destroy it.5 (i) Pirarubicin in “Adsorbed” and “Spread” Bilayers. (a) “Adsorbed” Bilayers. We estimated, thanks to the surface pressure study, that the percentage of pirarubicin (19) Nabiev, I. R.; Efremov, R. G.; Chumanov, D. G. Sov. Phys. Usp. 1988, 31, 241. (20) Nonaka, Y.; Nakamoto, K. J. Raman Spectrosc. 1990, 21, 133. (21) Smulevich, G.; Feis, A.; Mantini, A. R.; Marzocchi, M. P. Ind. J. Pure Appl. Phys. 1988, 26, 207. (22) Nabiev, I.; Chourpa, I.; Manfait, M. J. Phys. Chem. 1994, 98, 1344.

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ν (cm-1) Figure 5. SERR spectrum of (a) pirarubicin in aqueous solution (C ) 1 µM, pH ) 5.5), (b) adriamycin in aqueous solution (C ) 1 µM, pH ) 5.5), and (c) doxorubicinone (powder). Table 1. Principal Assignments of the Vibrational Modes of SERR Spectra of Pirarubicin, Adriamycin, Doxorubicinone, and the Different Phospholipid-Antibiotic Bilayers band frequency (cm-1) assignment pirarubicin (SERRS) doxorubicinone (SERRS) “adsorbed” POPC-DPPA/pirarubicin bilayer “spread” POPC-DPPA/pirarubicin bilayer preformed POPC-DPPA bilayer put in contact with an aqueous solution of pirarubicin (0.4 µM) adriamycin (SERRS) “adsorbed” POPC/adriamycin bilayer “adsorbed” POPC-DPPA/adriamycin bilayer preformed POPC bilayer put in contact with an aqueous solution of adriamycin (2 µM)

980

1206

1240

chromophore breathing

δ(C-O-H)

δ(C-O-H)

+++a + +++ ?c ++

+++ + +++ ++ +++

+++ ++ +++ +++ +++

++ ++

+++ ++

++ ++

++ ++

+++ broad band maximum 1240 cm-1 +++ broad band

1260

1400-1410

1450

shb +++ +++ sh+ sh+++

+ +++ +++ +++ +++

δ(CH2) (THP group) ring stretch + sh sh++ sh+ ++

sh broad band

+++ +++

+ sh

+++ +++

sh sh

ring stretch

+++

ring stretch

a +++/++/+/- symbols indicate whether the band is very intense, intense, visible, or invisible. b The letters “sh” indicate that the band appears like a shoulder. c The question mark (?) indicates the ambiguous cases.

in the POPC-DPPA monolayer was about 13% (see paragraph 3.1.1 (i)). The spectrum of the “adsorbed” bilayers (Figure 6a) presents the bands previously described: the band at 980 cm-1 has about the same intensity as that in the doxorubicinone spectrum; the intensity of the band at 1206 cm-1 is weaker than that in the pirarubicin spectrum (Figure 5a) but stronger than that in the doxorubicinone spectrum, whereas the shoulder at 1260 cm-1 is enhanced as compared to that in the pirarubicin spectrum; the band at 1240 cm-1 is always intense.

On the whole, this spectrum is thus closer to the doxorubicinone one, this result suggesting that pirarubicin in POPC-DPPA (80-20 mol %) “adsorbed” bilayers is rather lying on silver. (ii) “Spread” Bilayers. We estimated the percentage of pirarubicin in the POPC-DPPA (80-20 mol %) monolayer to 10% (see paragraph 3.1.1 (ii) and Figure 4). The spectrum of the “spread” bilayer is reported in Figure 6b: we cannot observe a band at 980 cm-1, and the band at 1206 cm-1 is less intense than previously. The shoulder at 1260 cm-1 has about the same intensity as

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ν (cm-1) Figure 6. SERR spectrum of (a) an “adsorbed” POPC-DPPA (80-20 mol %)/pirarubicin bilayer, after penetration in a POPCDPPA monolayer of an aqueous solution of pirarubicin (C ) 1.6 µM, pH ) 5.5, T ) 21 ( 1 °C, the surface pressure being kept at 2 mN/m during penetration and the penetration time limited to 15 min) and after compression to 25 mN/m; (b) a “spread” POPCDPPA (80-20 mol %)/pirarubicin bilayer, after spreading of a mixed POPC-DPPA/pirarubicin solution (66.6% pirarubicin in the mixture) and after compression to 25 mN/m; and (c) a preformed POPC-DPPA (80-20 mol %) bilayer put in contact with an aqueous solution of pirarubicin, (C ) 0.4 µM, pH ) 5.5).

that in the “adsorbed” bilayer spectrum and is thus more intense than that in the spectrum of the pirarubicin solution deposited on silver. Finally this spectrum is closer to the doxorubicinone one than to the pirarubicin solution one: this suggests that pirarubicin in “spread” bilayers is rather lying on the silver coating than perpendicular to it. If we now compare these results with those obtained for pirarubicin/POPC bilayers (in the absence of anionic DPPA), we observe that the position of pirarubicin is different in “adsorbed” POPC and POPC-DPPA bilayers. In our previous work,6 it was concluded that pirarubicin is embedded in the “adsorbed” POPC bilayers and perpendicular to the silver coating. Here we observe that at least a part of the antibiotic would be lying on silver. However, the orientation of pirarubicin is similar in the “spread” POPC and POPC-DPPA bilayers, since we observed in the two cases pirarubicin lying on silver. (ii) Pirarubicin in Contact with a “Preformed” Phospholipid Bilayer. We chose a weak concentration for the pirarubicin solution (0.4 µM) to avoid saturation of the phospholipid bilayer and, thus, to keep an intact structure. Although the pirarubicin solution was initially separated from silver by the POPC-DPPA bilayer, we observe the antibiotic spectrum (Figure 6c). Since SERRS has a short range,1-4 the signal decreases rapidly when the

distance between the molecule and the silver coating increases. Thus the pirarubicin spectrum that we observe corresponds to molecules which are very close to the silver coating. Since these molecules were initially separated from silver by the phospholipid bilayer, it means that pirarubicin has crossed the bilayer to come close to the silver coating. The spectrum is very similar to the “adsorbed” bilayer spectrum (Figure 6a), even if the band at 1206 cm-1 is more intense. We can conclude that pirarubicin in the preformed POPC-DPPA 80-20 mol % bilayer is, in the majority, lying on silver. 4.1.3. SERR Study of the Adriamycin/Phospholipid Mixed Bilayers. (i) Adriamycin in “Adsorbed” and “Spread” Bilayers. We studied the orientation of this second antibiotic in pure POPC and POPC-DPPA 80-20 mol % bilayers. The same types of bilayers as for pirarubicin were prepared. (a) “Adsorbed” POPC or POPC-DPPA (80-20 mol %)/ Adriamycin Bilayers. From the surface pressure study, the percentages of incorporated adriamycin are estimated respectively at 4% and 11% (see paragraph 3.1.2 (i)). The spectra of the “adsorbed” POPC and POPC-DPPA/ adriamycin bilayers (Figure 7a and b) are very similar and look like those of the adriamycin solution and doxorubicinone. In both cases, adriamycin would be more or less lying on the silver coating.

Interaction of Anthracyclines with Phospholipids

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ν (cm-1) Figure 7. SERR spectrum of (a) an “adsorbed” POPC/adriamycin bilayer, after penetration in a POPC monolayer of an aqueous solution of adriamycin (C ) 1.6 µM, pH ) 5.5, T ) 21 ( 1 °C, the surface pressure being kept to 2 mN/m during penetration and the penetration time limited to 15 min) and after compression to 25 mN/m; (b) an “adsorbed” POPC-DPPA (80-20 mol %)/ adriamycin bilayer (same conditions as above); and (c) a POPC preformed bilayer put in contact with an aqueous solution of adriamycin, (C ) 2 µM, pH ) 5.5).

(b) “Spread” POPC or POPC-DPPA (80-20 mol %)/ Adriamycin Bilayers. Their spectra between 900 and 1800 cm-1 are unusable (data not shown). In the best case we can observe a very broad band between 1200 and 1300 cm-1. This observation is surprising because we estimated that the adriamycin percentages in the POPC and POPCDPPA (80-20 mol %) monolayers were respectively about 9 and 10%. (ii) Adriamycin in Contact with a “Preformed” POPC Bilayer. The surface pressure showed that this antibiotic penetration was slow and weak. So we used a concentrated adriamycin solution: 2 µM. We can observe the adriamycin spectrum (Figure 7c). Thus this spectrum shows that adriamycin crossed the bilayer like pirarubicin, even if it is less hydrophobic than pirarubicin. The spectrum is very similar to the doxorubicinone one, as is the case for pirarubicin under the same conditions: after crossing, adriamycin lies on silver. This result confirms that, under our experimental conditions, adriamycin does not remain in the bilayer because of its weak hydrophobicity. The same experiment was realized with a preformed bilayer of POPC-DPPA (80-20 mol %), the concentration of the aqueous solution of adriamycin being the same (2 µM). Its SERR spectrum presented only the two broad bands of graphitized carbon at 1375 and 1590 cm-1 (data not shown). No band corresponding to adriamycin was visible.

4.2. Discussion. This SERR study enables us to improve the hypotheses that we proposed after the Surface Pressure study. Table 2 summarizes the orientations of pirarubicin and adriamycin found in the different phospholipid bilayers. (i) Case of Pirarubicin. The orientation of the molecule is different according to the mode of preparation and the charge of the phospholipids present in the bilayer. In the presence of DPPA, we observed that pirarubicin is always lying on silver and thus adsorbed on the polar head groups of phospholipids, in all the bilayers (“adsorbed”, “spread”, or “preformed” bilayers). These results confirm the importance of the electrostatic interaction between the polar head groups of DPPA and the positive charge of pirarubicin, and they are in agreement with the hypothesis proposed after the surface pressure study: pirarubicin adsorbed on the polar head groups forms a screen, which hinders other molecules from penetrating into the monolayer and limits the percentage of pirarubicin remaining at the interface. Moreover this hypothesis is in agreement with the results of de Wolf et al., obtained with adriamycin.14 They proposed that adriamycin would be out of the phospholipid bilayer if the ratio adriamycin/phospholipid is greater than 0.02. The percentage of pirarubicin in the POPC-DPPA monolayers being about 10-12%, pirarubicin is thus expected to be rather out of the bilayer and perhaps stacked with other molecules.

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Table 2. Summary of the Orientation of Pirarubicin and Adriamycin in POPC and POPC-DPPA Bilayers Observed in SERRS orientation of the anthraquinone part on the silver coating

type of bilayer observed in SERRS

anthraquinone part perpendicular or tilted on silver w anthracycline embedded into phospholipids

“adsorbed” POPC/pirarubicin bilayers6 “adsorbed” POPC-DPPA/pirarubicin bilayers “spread” POPC/pirarubicin bilayers6 “spread” POPC-DPPA/pirarubicin bilayers “preformed” POPC bilayers in contact with an aqueous solution of pirarubicin (0.4 µM)6 “preformed” POPC-DPPA bilayers in contact with an aqueous solution of pirarubicin (0.4 µM) “adsorbed” POPC/adriamycin bilayers “adsorbed” POPC-DPPA/adriamycin bilayers “spread” POPC/adriamycin bilayers “spread” POPC-DPPA/adriamycin bilayers “preformed” POPC bilayers in contact with an aqueous solution of adriamycin (2 µM) “preformed” POPC-DPPA bilayers in contact with an aqueous solution of adriamycin (2 µM)

anthraquinone part lying on silver w anthracycline adsorbed on the polar head groups of phospholipids

+a + + + + +

?c ?

+ + ? ? +

-b

-b

a The symbol “+” indicates the major orientation observed in each case. b The symbol “-” indicates that no orientation could be determined. Adriamycin does not penetrate into the bilayer and is likely adsorbed on the polar head groups of the second monolayer (far from the silver coating). c The question mark (?) indicates the cases where no spectrum was obtained.

In the case of the “preformed” POPC-DPPA bilayers, we observed that pirarubicin is able to cross the bilayer and is lying on silver after the crossing, contrary to the case for pirarubicin under the same conditions of concentration (0.4 µM) in contact with a “preformed” bilayer of pure POPC. In this last case, the antibiotic is embedded into phospholipids and more or less perpendicular to the silver layer.6 This difference of orientation of pirarubicin in the absence or the presence of DPPA confirms once again the importance of the electrostatic interaction between pirarubicin and negative polar head groups and confirms the hypothesis of a screen formed by adsorbed pirarubicin. (ii) Case of Adriamycin. The SERR spectra of “adsorbed” POPC or POPC-DPPA bilayers and “preformed” POPC bilayers show that adriamycin is rather out of the bilayers. This similar orientation of adriamycin in the presence or the absence of DPPA confirms the weaker hydrophobicity of adriamycin, which would be unable to penetrate deeply into the bilayer and would stay adsorbed onto the polar head groups. Contrary to the previous cases, the “spread” POPC and POPC-DPPA/adriamycin bilayers did not give usable spectra, although the percentages of adriamycin were repectively 9 and 10%. Several hypotheses are possible: First adriamycin could be so deep into the bilayer that it would not be illuminated and we could not observe it. This hypothesis is inconsistent with the weak hydrophobicity of adriamycin shown in the Surface Pressure study. Second, adriamycin would not remain in the bilayer after its formation. The affinity of adriamycin for the subphase would be greater than the affinity for the bilayer. This last hypothesis is more likely, since adriamycin is not very hydrophobic, but disagrees with the fact that we observe the adriamycin spectrum in the case of the “adsorbed” bilayers. A third possibility would be the following: the molecules of adriamycin in interaction with phospholipids in the “spread” monolayer would not remain in the layer during the transfer onto the support. The electrostatic interaction would be too weak to maintain adriamycin in contact with lipids. This fact would be enhanced by the very weak concentration of adriamycin in the subphase with this method (this concentration being estimated to be around 10-7 M). In the case of the “adsorbed” bilayers, the concentration of adriamycin in the subphase is more

important (around 1.6 µM) and could help to maintain adriamycin in contact with the polar head groups during the transfer. This kind of problem was not observed in the case of pirarubicin, probably because of the presence of hydrophobic interactions between the THP group and phospholipids, which increases the strength of the binding. At last the adriamycin spectrum was not obtained with POPC-DPPA “preformed” bilayers. The absence of the adriamycin bands means that adriamycin did not cross the bilayer. This result is very interesting for two major reasons. (i) This result is in agreement with the hypothesis of a screen formed by the anthracycline molecules on the polar head groups of DPPA. Adriamycin is likely adsorbed on the negative polar head groups of the second monolayer (in contact with the solution). As it is less hydrophobic than pirarubicin, it is unable to cross the bilayer and remains adsorbed, hindering a deeper penetration by other molecules. (ii) It confirms the very weak range of detection of SERRS under our experimental conditions, since we did not observe adriamycin in aqueous solution above the phospholipid bilayer, its thickness being estimated to be about 50-60 Å. It confirms that the spectrum of the aqueous solution is not superimposed. This hypothesis agrees with the fast decrease of the Raman signal intensity with distance (the intensity being expected to follow a (a/r)12 rule, where a is the radius of the silver island and r is the distance of the observed point to the center of the island).1 Another possibility is that the level of adriamycin, which has crossed the bilayer, is too weak to be detectable. However the enhancement of the Raman signal is so important that we should detect a spectrum, even if it is bad. The results obtained with adriamycin are partly in agreement with the models proposed by Speelmans et al. on the one hand and de Wolf et al. on the other hand: they suggested that adriamycin is adsorbed on the polar head groups in the presence of anionic phospholipids, at least for a sufficient adriamycin/phospholipid ratio. Under our experimental conditions, the SERR spectra of the different adriamycin/phospholipid bilayers show that adriamycin either would not penetrate into the bilayer or would be rather adsorbed on the polar head groups (Table 2).

Interaction of Anthracyclines with Phospholipids

However this last position is observed even in the absence of anionic phospholipids. It can be explained by the weak hydrophobicity of adriamycin, which is thus unable to penetrate and/or to remain in the bilayer. This weak hydrophobicity is confirmed by the weak octanol/buffer partition coefficient of adriamycin (1.1) determined by Goldman et al.23 Lastly, Garnier-Suillerot at al. studied the interactions of numerous derivatives of adriamycin, daunorubicin, and idarubicin with large unilamellar vesicles (LUVs) of phosphatidylcholine, with various amounts of phosphatidic acid and cholesterol, and concluded that the presence of anionic phospholipids does not enhance the percentage of anthracycline in their model membrane. They proposed that roughly all the tested anthracyclines would be located at the level of the polar head groups of phospholipids.24 5. Conclusion Our goal was to study the role of negative charges of phospholipids in the interaction of two anthracyclines, pirarubicin and adriamycin, with planar phospholipid bilayers (containing DPPA, a negatively charged phospholipid), after having studied the interaction of pirarubicin with POPC, a zwitterionic phospholipid, in a previous (23) Goldman, R.; Facchinetti, T.; Bach, D.; Raz, A.; Shinitzky, M. Biochim. Biophys. Acta 1978, 512, 254. (24) Gallois, L.; Fiallo, M.; Laigle, A.; Priebe, W.; Garnier-Suillerot, A. Eur. J. Biochem. 1996, 241, 879.

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work.6 A preliminary surface pressure study enabled us to determine the type of interaction and the level of antibiotic incorporated into phospholipids (or adsorbed on the polar head groups, this second possibility being rather observed in this work), whereas SERRS gave us information about the orientation of the anthracyclines in the planar phospholipid bilayers. It has been shown that there is an accumulation of pirarubicin on the polar head groups in the presence of anionic DPPA in the phospholipid bilayers, this accumulation creating a screen which hinders the penetration of other molecules. As the composition of biological membrane is asymmetric (the inner layer presenting a higher percentage of anionic phospholipids), we can suppose that the passive diffusion of pirarubicin is different, when it is interacting with the inner or the outer layer of the membrane. Pirarubicin adsorbed onto the polar head groups of the inner layer could hinder the passive diffusion of other molecules, as previously proposed by Speelmans et al.13 In the case of adriamycin, the interaction with zwitterionic or anionic phospholipids is less important, and adriamycin is unable to penetrate deeply in the bilayer, or is ejected out of it, these properties being likely bound to the weaker hydrophobicity of the molecule as compared to that of pirarubicin. LA962113V