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Bioconjugate Chem. 1991, 2,398-402
Interaction of Doxorubicin with Lipid Systems Jordi Hernlndez, Antoni Mart!, and Joan Estelrich' Unitat de Fisicoquimica, Facultat de Farmticia, Universitat de Barcelona, 08028-Barcelona, Catalonia, Spain. Received May 15, 1991
The action of doxorubicin, a cancer chemotherapeutic agent, on liposomes of dipalmitoylphosphatidylcholine obtained by extrusion, with or without cholesterol, and on monolayers of the same composition, was determined by photon correlation spectroscopy, penetration kinetics, and adsorption isotherms. Doxorubicin produced a wider vesicle distribution in both of liposomes, but liposomes containing cholesterol underwent an increase of mean diameter as well as polydispersity. In contrast, films containing cholesterol showed least interaction with the drug. Since both lipids are neutral, any interaction must be primarily hydrophobic.
INTRODUCTION Doxorubicin hydrochloride (DXR) is a cancer chemotherapeutic agent with an anthracycline structure, which consists of an aglycon, adriamycinone, combined with an amino sugar, daunosamine. A pharmaceutical form which contains lactose as an excipient, is manufactured by Farmitalia (Milan, Italy) and sold under the trademark of Adriamycin. Doxorubicin interacts widely with some membrane models such as lipid bilayers and liposomes (1-12). The affinity of DXR of liposomal membranes which bear no net charge is moderate. This interaction is believed to be dominated by forcesother than ionic ones, since the affinity is only slightly dependent on the concentration of metal ions in the solution (2). In any case, an electrostatic interaction is observed between the ammonium group of DXR and the phosphate group of neutral phospholipids (8). In contrast, DXR interacts strongly with positively or negatively charged liposomes. However, binding of DXR to charged vesicles is not strictly dependent on electrostatic interactions, because the binding characteristics of a DXR derivative, daunorubicin, which presents a comparable basicity, were totally different from those of DXR (10). Therefore, DXR binding to the lipid bilayers appears to be dominated by hydrophobic interactions, although it is not clear where DXR is localized in the lipid bilayer. In energy-transfer experiments it has been observed that anthracyclines interact preferentially with the hydrophobic core of lipid bilayers, while from measurements of circular dichroism, two different binding sites of DXR with lipids have been described. In the first one, the dihydroanthraquinone moiety lies outside the lipid bilayer, and in the second, it is embedded in the bilayer. The localization of DXR in membranes depends on the molecular packing of the lipid. In this way, DXR can penetrate a monolayer at low surface pressure but is squeezed out from the monolayer above a surface pressure of 23 "am-1. For this reason, DXR does not readily penetrate natural membranes, since they present a tight lipid molecular packing. The lateral surface pressure of red-cell membrane ranges from 31 to 35 mN-m-' and this corresponds to a lateral pressure of a monolayer higher than 25 "am-1 (11). On the basis of NMR studies, other authors have suggested that phospholipid bilayers become more fluid
* Author to whom correspondence should be addressed.
upon interaction with DXR and that the latter binds with the same affinity to liposomes of various compositions (6). The aim of this study is to determine the influence of DXR on a planar membrane model, namely lipid monolayers, formed by neutral lipids and to compare this effect with the action of DXR on liposomes of the same composition as a model of curved bilayer. This is an aspect that can contribute to a better understanding of the drugmembrane interactions. EXPERIMENTAL PROCEDURE'
Chemicals. L-a-Dipalmitoylphosphatidylcholine (DPPC), specified as 99% pure, and cholesterol (CHOL), type 99+ % pure, were purchased from Sigma. Doxorubicin hydrochloride used in monolayers was purchased from Sigma and that used for liposome preparation was a gift of Farmitalia-Carlo Erba (Barcelona,Spain). Organic solvents (chloroform, n-hexane, and ethanol) obtained from Merck were without surface-active impurities. Water was double-distilled, the last time in presence of potassium permanganate, in borosilicate apparatus, and then purified through a Mill-& system (Millipore). Preparation of Liposomes. Liposomes of DPPC (or DPPC and CHOL at equal molar ratio) were obtained by the extrusion method (13). Briefly, 40 pmol of DPPC or DPPC and CHOL were dissolved in chloroform. The solution was dried under a stream of dry nitrogen and then lyophilized. Lipids were rehydrated in 7 mL of 145 mM NaCl, 10mM Tris (pH 7.4) buffer solution containing DXR at 2 mM concentration. Hence, the molar ratio between lipid and DXR was 2.88. The multilamellar vesiclesproduced above were extruded seven times through polycarbonate membrane filters of variable pore size (Nucleopore) using nitrogen pressures of up 55 X 105 N*m-*. Unbound DXR was separated from bound DXR to liposomes by means of gel-filtration chromatography. Aliquots of 0.5 mL were applied on a column (30 X 0.9 cm i.d.) filled with Sephadex G-50 (Pharmacia LKB). The elution was carried out with the Tris buffer at a flow rate of 27.72 mL/cm2.h. Monolayer Technique. Films of DPPC or DPPC: CHOL (l:l, molar ratio) dissolved in n-hexane-ethanol were spread over the subphase of a Teflon trough filled Abbreviations used: CHOL, cholesterol; DPPC, L-a-dipalmitoylphosphatidylcholine;DXR, doxorubicin hydrochloride; NMR, nuclear magnetic resonance; SUVs, small unilamellar vesicles;
T,, transition temperature.
1Q43-1802/91/29Q2-Q398$02.5Q~Q 0 1991 American Chemical Society
Interactlon of Doxorubicin with Lipid Systems
Table I. Influence of Doxorubicin on Liposome Size liposome filter pore mean diameter, poly-. composition size, nm nm f SD dispersity 200 161.5 f 41.9 0.064 DPPC DPPC 100 112.9 f 23.1 0.041 DPPC 50 77.4 f 22.2 0.077 DPPC + DXR 200 166.9 f 83.0 0.201 DPPC + DXR 100 115.4f 58.7 0.207 DPPC + DXR 50 84.3f 39.1 0.179 DPPC:CHOL 200 184.5f 66.4 0.116 DPPC:CHOL 100 120.5f 30.7 0.082 DPPC:CHOL 50 111.2f 46.0 0.147 DPPC:CHOL + DXR 200 585.1 f 744.0 0.679 DPPC:CHOL + DXR 100 169.8f 114.3 0.318 DPPC:CHOL + DXR 50 128.0f 57.4 0.169
with 10 mM Tris and 145 mM NaCl buffer (pH 7.4) solution. Compression isotherms were measured using an electromicrobalance (Sartorius A - 1 2 0 3 based on the Wilhelmy method and coupled to a chart recorder to give a continuous reading of force on the dipping plate. The dimensions of the Teflon trough were 28.4 X 17.45 X 0.625 cm. Compression started at least 30 min after spreading and the compression rate was of 5 A2/molecule per min. For each experiment, buffer surface tension was first measured. DXR in the above solution was injected into the aqueous subphase beneath the lipid film and the subphase solution was stirred well by a magnetic stirrer (1 min). The final concentration of DXR in the bulk was 10 or 20 nM. Penetration kinetics were performed by spreading the necessary amount of DPPC to obtain monolayers of an initial pressure of 5 or 10 mN-m-l. The area of the Teflon trough was 124 cm2. Different volumes of DXR solution were injected into the subphase with a Hamilton syringe to attain DXR concentrations in the range of 10-40 nM. In kinetic assays, agitation in the subphase was maintained by two magnetic Teflon stirrer bars turning at 250 rpm. All data reported here are the average of three measurements. The temperature was kept at 24 f 1 "C. The accuracy of the surface tension measurements was f0.25 "am-'. Photon Correlation Spectroscopy (PCS). Fluctuations in scattered light intensity generated by the diffusion of vesicles in solution were analyzed. Analysis of the intensity fluctuations using a multichannel digital correlator allowed the mean diameter of vesicles to be obtained. PCS was performed in an Autosizer IIc photon correlation spectrometer (Malvern Instruments) consisting of a 5-mW, 632.8-nm, helium-neon laser irradiating the scattering cell placed inside a temperature-regulated enclosure. Data acquisition was via a Malvern 7032-N, 72-channel multibit correlator. Experimental conditions were temperature, 25.0 "C; reference angle, 90"; viscosity, 0.899 X Pa-s; refractive index, 1.330. Two principle methods can be used for data analysis. Assuming a log-normal size distribution of particles, the method of cumulant analysis (14) is available, while for broad monomodal distributions (polydisperse systems) the model independent analysis, which does not assume any particular form of the distribution, is used.
RESULTS AND DISCUSSION Influence of DXR on Size of Liposomes. Size and polydispersity of liposomes obtained by extrusion and the influence of DXR on these parameters are shown in Table I. Mean diameters with their standard deviation and polydispersity were determined by PCS using the cumulants method and Gaussian analysis for mean diameter and standard deviation determinations, respectively. Polydispersity values are within the range of 0-1. Values of
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polydispersity 10.100 correspond to monodisperse population. As can be observed, DXR barely altered the mean diameter of DPPC liposomes, although it enhanced the standard deviation and the polydispersity. So, we can conclude that the presence of DXR in such liposomes results in wider vesicle distribution. On the contrary, the action on DXR on mixed liposomes produced an increase both in size and polydispersity. It is clear that the largest liposomes were those most affected, while liposomes extruded through filters with 50-nm pores underwent a variation like that observed in DPPC liposomes. These variations in size and polydispersity can be explained by means of aggregation and fusion processes, although the main mechanism is the fusion, since determinations of size by photon correlation spectroscopy at 50 "C (temperature above the T, of DPPC) afford us values similar to those obtained at 25 "C. As in SUVs, the T, is always lower than in pure lipid; we have determined the variations in size of DPPC liposomes at temperatures ranging from 20 to 50 "C and we have checked that DPPC liposomes extruded through a 0.1-pm porous membrane show the minimal size and polydispersity a t 33-34 "C; at this temperature, size parameters remained high. In the absence of DXR, a homogeneous vesicle population is obtained, but when DXR is present in the rehydratation buffer, the thermodynamically metastable vesicle suspension becomes less stable and the aggregation processes are now more likely to be due to the DXR charge. Aggregates of vesicles may fuse to form larger vesicles and small micelles. Liposomes which contains CHOL show, indeed, a larger average size. The presence of CHOL in DPPC liposomes produces a slight increase in z average diameter when it is present at equal molar ratio, but the increase is bigger if the ratio is 1.6:l (DPPC:CHOL). (Liposomes of this composition and extruded by a 100-nm membrane filter have a mean diameter of 146 f 54.) As the packing constraints of bilayer vesicles are determined by both the entropy of the system and the molecular geometry and as the latter is measured by a critical packing parameter that relates the optimal cross-sectional molecular area, the hydrocarbon chain volume and the maximal hydrophobic length (15),the insertion of DXR in the bilayer will modify these parameters and, thus, the vesicles will be of different sizes. Increase in bilayer fluidity (6)or aggregation and fusion of small unilamellar liposomes of DPPC has been observed elsewhere (16). Given that mixing the freeze-dried lipid and DXR can be close to an equilibrium because the lipid molecules will incorporate DXR molecules rather easily when they are forming liposomes, we mixed a liposome and a DXR solution and checked the vesicle size variation at several time intervals in order to rule out the localization of DXR being a consequence of the codispersion of DXR and lipids. Under these conditions, DXR also produced the modification of both polydispersity and z average diameter within vesicles. The aggregation process is fast (30 s after the last extrusion the size increase can be monitored by photon correlation spectroscopy) for the first 30 min and it becomes slower. Table I1 exhibits the variation in size 30 min after the mixing. Measurementsof PenetrationKinetics. Initially we determined the adsorption kinetics of DXR to the airwater interface in the absence of the lipid monolayer. DXR concentrations ranging from 10 to 100 nM were injected into the subphase. Above 90 nM the increase in surface pressure presented a plateau (steady state), but anyway,
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Table 11. Influence of Incubation of DXR on Size and Polydispersity of Liposomes liposome compositiono membrane DPPC DPPCCHOL pore,nm DPPC +DXR DPPC:CHOL +DXR 50
92.4 (0.100) 116.1 (0.061) 188.0 (0.089)
100 200
100.4 (0.356) 117.4 (0.189) 251.3 (0.386)
91.7 (0.099) 126.5 (0.102) 168.8 (0.072)
69
'
113.6 (0.319) 203.9 (0.617) 221.0 (0.636)
Values show the z average diameter (nm) and the polydispersity (in brackets) 30 min after the incubation.
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Figure 3. Variation in surface pressure for the interaction of doxorubicin at four concentrations with dipalmitoylphosphatidylcholine cholesterol monolayer. The initial surface pressure of the film was 5 mN.m-1 (for conditions, see caption of Figure
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Figure 1. Variation in surface pressure for the interaction of doxorubicin at four concentrations @,lo nM; A, 20 nM; x, 30 nM; and W, 40 nM) with dipalmitoylphosphatidylcholinemonolayer. Subphase buffer: 145 mM NaCl, 10 mM Tris-HC1 (pH 7.4). The initial surface pressure of the film was 5 mN-m-l.
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Figure 4. Variation in surface pressure for the interaction of doxorubicin at four concentrations with dipalmitoylphosphatidylcholine cholesterol monolayer. The initial surface pressure of the film was 10 mN-m-l (for conditions, see caption of Figure
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Figure 2. Variation in surface pressure for the interaction of doxorubicin a t four concentrations with dipalmitoylphosphatidylcholine monolayer. The initial surface pressure of the film was 10 mN.m-1 (for conditions, see caption of Figure 1). the increase never exceeded 0.96 mN-m-'. This plateau was reached in 15-20 min. In Figures 1-4the registered increase in surface pressure of lipid monolayers produced by solutions of DXR at 1020 nM concentration range are drawn as a function of time. Previously, it has been checked that a DPPC or DPPC/CHOL monolayer alone does not exhibit any change in its surface pressure over the period of 40 min. Figures 1 and 2refer to DPPC monolayers at initial surface ) 5 and 10 mN-m-', respectively. As in the pressure ( ~ i of surface activity experiments, an increase in surface pressure (AT) of a lipid monolayer is taken to indicate an interaction of the substance present in the subphase with the lipid film. It can be established from these plots that the greater the concentration, the greater the interaction obtained. But a t identical drug concentration, the increase obtained was significantly greater when a more expanded monolayer was employed. So, a 6.77"em-' increase in surface pressure was observed at ~i = 5 mN-m-', whereas
at = 10 mN-m-' it was 2.49. Thus, the drug-lipid interaction is highly dependent on the initial pressure of the monolayer being lower for more compressed monolayers. At surface pressures from 20 to 40 "am-', this increase is progressively abolished and can be accounted for by a partial exclusion of DXR from monolayer at high pressures. Figures 3 and 4 show the effect of DXR on DPPC:CHOL monolayers. I t can be observed that the interaction with such lipids layers followed a similar pattern, although the curves were closer to each other than in DPPC monolayers and they seem to lead to constant values. An important difference between the two kinds of lipids layers was the time necessary to reach the steady state. Thus, monolayers of DPPC presented a plateau about 35 min after the injection of DXR into the subphase, while DPPC: CHOL monolayers needed much longer to uptake DXR. Thus, saturation values AT for DPPCCHOL may be higher than the DPPC ones. Unfortunately, incubation times longer than 35 min alter monolayer properties and it was not possible to check the DXR incorporation after this time. Measurements of Compression Isotherms. Measurements of the surface pressure-area ( F A ) of DPPC films are shown in Figure 5. The compression isotherms were characteristic of lipids in the liquid-expanded state. From this figure it is clear that the adsorption of DXR to the lipid film produced a less condensed monolayer. This finding, which is a consequence of the drug-lipid interaction, was more evident at a surface pressure of 10
Bioconjugate Chem., Vol. 2, No. 6, 1991 401
Interaction of Doxorubicin with LipM Systems 50 U
40th 40
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Area (A' /molecule) Figure 5. Surface pressure-molecular area isotherms for adsorbed films of doxorubicin a t the dipalmitoylphosphatidylcholinewater (Tris buffer, pH 7.4) interface. Concentration of , 20. doxorubicin (nM): 0,0; X, 10; .
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Area (A' /molecule) Figure 6. Surface pressure-molecular area isotherms for adsorbed films of doxorubicin at the dipalmitoylphosphatidylcho1ine:cholesterol-water (Tris buffer, pH 7.4) interface (for conditions, see caption of Figure 5 ) .
mN*m-l. At this pressure, which corresponds to the expanded-ondensed liquid-phase transition of ure DPPC monolayers, the area/molecule was nearly 77 while in the presence of 10 and 20 nM DXR it rose to 95 and 99 A2,respectively. However, the three curves tended to collapse a t similar A (45mN-m-l), corresponding to the limiting molecular area of 44 f 1A2/molecule. Such values are in good agreement with those described elsewhere (17). In contrast, the effect of DXR on mixed films was almost insignificant (Figure 6). In this case, isotherms were parallel from 5 to 20 mN-m-l surface pressure, and at higher surface pressure the isotherms were superimposable. So, the amount of DXR incorporated appears to be small. Nevertheless, it must be borne in mind that DPPC:CHOL membranes are much harder than DPPC membranes and, for this reason, it is logical to suppose that the uptake of the molecules from the water phase is considerably slow. The stiffness of the DPPC:CHOL is evident from Figures 5 and 6;the onset of the A-A curve for DPPC:CHOL is very steep. Moreover, with such mixed films it is not possible to observe the effect of the interaction at the surface pressure of the phase transition, because it is eliminated when the cholesterol mole fraction in a mixed film is higher than 0.23 (18). To sum up, we may conclude that both methods, monolayer technique and photon correlation spectroscopy, show that DXR interacts with the lipid systems used. So, the effect of DXR on liposomesinvolvesthe vesicle distribution becoming broader in DPPC liposomes. With DPPC: CHOL liposomes, not only was the distribution wider but
12,
the average diameter also increases. DXR is believed to induce fusion and aggregation. It is also important to notice that the extension of this effect varies with the liposome curvature. On the other hand, results from penetration kinetics and compression isotherms also indicate a interaction between DXR and monolayers. The binding of watersoluble substances, such as DXR, to a lipid monolayer may be due to hydrophobic interactions for noncharged and partially hydrophobic chains of the molecule, and to electrostatic interactions for polar or ionizable groups. DXR in the hydrochloride form contains a charged tertiary amine and can establish electrostatic interactions, but due to its structure it can also behave as a hydrophobic substance and penetrate into the hydrophobic core of the lipid layer. In this way, a weak insertion of the planar anthracenic part of anthracycline into the lipidic leaflet has been observed (10). This insertion into the hydrocarbon region explains that the apparent area occupied is higher in the presence of DXR, as well as the enhancement of liposome size. Furthermore, as both lipids DPPC and CHOL, are neutral, the main kind of interaction between DXR and such lipids must be primarily hydrophobic. Finally, it is important to remark on the apparently opposite effect of CHOL in liposomes in comparison to monolayers. So, while the interaction was greater in liposomes containing CHOL, mixed monolayers experienced the minimum disturbance in their ordered configuration. Bearing in mind that the interaction between lipid and DXR has been only observed in monolayers at low initial surface pressures and, on the other hand, at room temperature, liposomal lipids are in the gel state and, consequently, the lateral surface pressure is high, it is difficult to make a comparison between the two lipids systems as far as the DXR interaction is concerned. ACKNOWLEDGMENT
This research was supported in part by the CICYT (No. 86-0484)(Spain). We thank Robyn Rycroft for his help in translation to the English version. LITERATURE CITED ( 1 ) Goldman, R., Facchinetti, T., Bach, D., Raz, A., and Shinitzky, M. (1978)A differential interaction of daunomycin, adriamycin and their derivatives with human erythrocytes and phospholipid bilayers. Biochim. Biophys. Acta 512, 254269. ( 2 ) Karczmar, G. S., and Tritton, T. R (1979) The interaction of adryamicin with small unilamellar vesicle liposomes. A fluorescence study. Biochim. Biophys. Acta 557, 306-319. (3) Crommelin, D. J. A., Slaats, N., and van Blois, L. (1983) Preparation and characterization of doxorubicin-containing liposomes: I. Influence of liposome charge and pH of hydration medium on loading capacity and particle size. Znt. J.Pharm. 16,79-92. (4) Burke, T. G., and Tritton, T. R. (1984)Ligand self-association at the surface of liposomes: a complication during equilibriumbinding studies. Anal. Biochem. 143, 135-140. (5) Burke, T. G., and Tritton, T. R. (1985) Structural basis of anthracycline selectivity for unilamellar phosphatidylcholine vesicles: an equilibrium binding study. Biochemistry24,17681770. (6) Burke,T. G.,andTritton,T. R. (1985)Locationanddynamics of anthracyclines bound to unilamellar phosphatidylcholine vesicles. Biochemistry 24, 5972-5980. (7) Griffin, E., Vanderkooi, J., Maniara, G., and Erecinska, M. (1986) Anthracycline binding to synthetic and natural membranes. A study using resonance energy transfer. Biochemistry 25, 7875-7880.
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(8) Giuliani, A. M., Boicelli, C. A., De-Angelis, L., Giomini, M., Giustini, M., and Trotta, E. (1988)Doxorubicin interactions with neutral phospholipids: An infrared and thermal analysis study. Stud. Biophys. 123, 45-52. (9) Nicolay, K., Sautereau, A. M., Tocanne, J. F., Brasseur, R., Huart, P., Ruysschaert, J. M., and de Kruijff, B. (1988)A comparative model membrane study on estructural effects of membrane-active positively charged anti-tumor drugs. Biochim. Biophys. Acta 940, 197-208. (10)Henry-Toulme, N. Stefanska;B., Borowski, E., and Bolard, J. (1988)Structural basis for the binding of antitumor anthracycline antibiotics to model membrane: Circular dichroism studies. Mol. Pharmacol. 33,574-579. (11) Dupou-CBzanne, L.,Sautereau, A. M., and Tocanne, J. F. (1989) Localization of adryamicin in model and natural membranes. Eur. J . Biochem. 18, 695-702. (12) Wolf, F.A. de, Maliepaard, M., van Dorsten, F., Berghuis, I., Nocolay, K., and de Kruijff, B. (1991)Comparable interaction of doxorubicin with various acidic phospholipids results in changes of lipid order and dynamics. Biochim. Biophys. Acta 1096, 67-80.
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(13) Mayer, L. D., Hope, M. J., and Cullis, P. R. (1986)Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim. Biophys. Acta 858,161-168. (14) Koppel, D. E. (1972)Analysis of macromolecular polydispersity in intensity correlation spectroscopy: The method of cumulants. J . Chem. Phys. 57,4814-4820. (15) Maggio, B., Albert, J.,and Yu, R. K. (1988)Thermodynamic geometric correlations for the morphology of self-assembled structures of glycosphingolipids and their mixtures with dipalmitoylphosphatidylcholine. Biochim. Biophys. Acta 945, 145-160. (16) Constantinides, P.P.,Tritton, T. T., and Sartorelli, A. C. (1988)Interaction of adryamicin with single and multibilayer dipalmitoylphosphatidylcholine vesicles: spin-labeling and calorimetric study. J. Liposome Res. 1, 35-62. (17) von Tscharner, V., and McConnell, H. M. (1981) An alternative view of phospholipid phase behavior at the airwater interface. Biophys. J. 36, 409-419. (18) Cadenhead, D. A., and Kellner, M. J. (1976)The miscibility of dipalmitoylphosphatidylcholineand cholesterol in monolayers. J. Colloid Interface Sci. 57, 224-227.
