13834
J. Phys. Chem. B 2008, 112, 13834–13841
A Monolayer Study on Interactions of Docetaxel with Model Lipid Membranes Alfonso Ferna´ndez-Botello,*,† Francesc Comelles,‡ M. Asuncio´n Alsina,§ Pilar Cea,| and Francisca Reig† Peptides Department and Surfactant Department, Institute for Chemical and EnVironmental Research (IIQAB-CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain, Physicochemistry Department, Faculty of Pharmacy, UniVersity of Barcelona, AVda. Joan XXIII s/n, 08028, Barcelona, Spain, Department of Organic and Physical Chemistry (Faculty of Science) and Institute of Nanoscience of Arago´n (INA), UniVersity of Zaragoza, 50009 Zaragoza, Spain ReceiVed: July 21, 2008; ReVised Manuscript ReceiVed: September 3, 2008
Docetaxel (DCT) is an antineoplastic drug for the treatment of a wide spectrum of cancers. DCT surface properties as well as miscibility studies with L-R-dipalmitoyl phosphatidylcholine (DPPC), which constitutes the main component of biological membranes, are comprehensively described in this contribution. Penetration studies have revealed that when DCT is injected under DPPC monolayers compressed to different surface pressures, it penetrates into the lipid monolayer promoting an increase in the surface pressure. DCT is a surface active molecule able to decrease the surface tension of water and to form insoluble films when spread on aqueous subphases. The maximum surface pressure reached after compression of a DCT Langmuir film was 13 mN/m. Miscibility of DPPC and DCT in Langmuir films has been studied by means of thermodynamic properties as well as by Brewster angle microscopy (BAM) analysis of the mixed films at the air-water interface, concluding that DPPC and DCT are miscible and they form nonideally mixed monolayers at the air-water interface. Helmholtz energies of mixing revealed that no phase separation occurs. In addition, Helmholtz energies of mixing become more negative with decreasing areas per molecule, which suggests that the stability of the mixed monolayers increases as the monolayers become more condensed. Compressibility values together with BAM images indicate that DCT has a fluidizing effect on DPPC monolayers. Introduction Taxanes have antineoplastic activity in Vitro and in ViVo against a wide variety of cancer cells. Taxanes include paclitaxel and docetaxel. Paclitaxel was first found as a natural product from the Pacific yew tree Taxus breVifolia,1 while docetaxel is a second-generation taxane derived from the needles of the European yew tree Taxus baccata.2 Both compounds were approved by the U.S. Food and Drug Administration (FDA) for the treatment of several carcinomas including breast, advanced ovarian, non small cell lung, head and neck, colon, and AIDS-related Kaposi′s sarcoma.3 Docetaxel (DCT, molecular weight ) 807 g/mol, trademarked as Taxotere by Rhone Poulenc Rorer DCT) is a complex diterpenoid, which features a rigid taxane ring and a flexible side chain (Figure 1). Docetaxel differs from paclitaxel at two positions in its chemical structure. It has a hydroxyl functional group on carbon 10, whereas paclitaxel has an acetate ester and a tert-butyl substitution exists on the phenylpropionate side chain.4 By tumor type, differences in activity between docetaxel and paclitaxel are statistically insignificant, but docetaxel often has greater cytotoxic activity against human breast cancer cell lines than paclitaxel,5 and is nowadays an effective option in the treatment of patients with metastatic breast cancer after failure of prior chemotherapy.5-10 * Corresponding author. Phone: +34 934006100. E-mail: afbqpp@ iiqab.csic.es. † Peptides Department, Institute for Chemical and Environmental Research (IIQAB-CSIC). ‡ Surfactant Department, Institute for Chemical and Environmental Research (IIQAB-CSIC). § University of Barcelona. | University of Zaragoza.
Figure 1. Chemical structure of docetaxel (DCT).
Docetaxel induces apoptosis by interfering with microtubule dynamics, in particular by preventing tubulin depolymerization.11 Actively dividing cells are thus induced to undergo apoptosis, triggered by bcl-2 phosphorylation, involving the activation of the c-Raf-1/Ras or the p53/p21WAF1/CIP1 signaling pathway.12 DCT shows very low water solubility, and the only available formulation for clinical use consists of a solution (40 mg/mL) in a vehicle containing a high concentration of Tween 80. Unfortunately, this vehicle has been associated with several hypersensitivity reactions such as nephrotoxicity, neurotoxicity, and cardiotoxicity.13,14 In order to eliminate the Tween 80-based vehicle and in the attempt to increase the drug solubility, alternative dosage forms have been suggested, e.g., liposomal formulations for controlled and targeted delivery of the drug.15 In this context, a deep knowledge of the interactions between docetaxel and phospholipids within the lipid bilayer membrane could provide significant insight into the therapeutic performance
10.1021/jp806423k CCC: $40.75 2008 American Chemical Society Published on Web 10/10/2008
Interactions of Docetaxel with Model Lipid Membranes of docetaxel-liposome formulations and into mechanisms of action and cellular effects of DCT. In addition, molecular interactions between biological cell membranes and drugs have a decisive influence on the pharmacokinetic properties of the drugs.16-19 Thus, it is well-known that some anticancer compounds can modify the phase transition, organization of lipid domains, fluidity, and mobility of the cell membrane.20-22 Taken together, these observations highlight the need for exploration of interactions between DCT and L-R-dipalmitoyl phosphatidylcholine (DPPC, the main component of biological membranes) and the crucial role that these molecular interactions play in transport, distribution, accumulation, and efficacy of the drug. To the best of our knowledge, DPPC-DCT interactions have not been studied before. However, there is a large body of excellent published work on molecular interactions between paclitaxel and lipid monolayers at the air-water interface.23-25 Thus, although there is a gap between monolayers and actual lipid bilayers or cell membranes, studies between DCT and lipid monolayers at the air-water interface can give us some insights into the interactions between the drug and the lipids. In this context, Langmuir films represent an excellent tool with which to study drug molecules at interfaces,26-28 as surface pressure, area per molecule, chemical composition, and temperature can be precisely controlled.29-32 Thus, the main objective of this contribution is to comprehensively explore and describe the molecular interactions between DPPC and DCT. These interactions have been studied using different approaches following the methodology and theoretical interpretation of previous works with similar compounds,23-25 which include studies of surface activity, penetration of DCT in DPPC monolayers at different surface pressures, and compression isotherms of pure and mixed Langmuir films (DPPC/DCT) of different molar compositions. It is hoped that these studies will contribute to the design of further liposomal formulations. Experimental Section Materials. L-R-Dipalmitoyl phosphatidylcholine (DPPC) and docetaxel (DCT) were supplied by Sigma-Aldrich (St. Louis, MO) and stored at -4 °C. 8-Anilino-1-naphthalene sulfonic acid (ANS) was purchased from Sigma (Steinheim, Germany). Solvents and salts were provided by Probus analytical grade. Water was distilled twice over permanganate and passed trough a Milli-Q filtration system. Its resistivity was 18.2 MΩ · cm. Phosphate buffer saline (PBS) was prepared by dissolving 58 g of disodium hydrogen phosphate (12H2O), 5.2 g of sodium dihydrogen phosphate (2H2O), and 5.2 g of sodium chloride in 2 L of Milli-Q water. Aggregation of Docetaxel. Aggregation was studied by using the fluorescent probe ANS dissolved in 10-2 M PBS (pH 7.4). From this concentrated solution, a diluted one (10-5 M) was obtained and used for the experiments. Four quartz cells were prepared by adding 2.5 mL of PBS and 50 µL of diluted ANS solution, and were titrated with aliquots of a 10-3 M docetaxel stock solution in acetonitrile. The concentration of DCT in the cuvettes ranged between 10-7 and 10-4 M. ANS fluorescence spectra were recorded at 22 °C (λex ) 370 nm, λem ) 470 nm, bandpass 4 nm) in an Aminco-Bowman spectrophotometer. All experiments were carried out at room temperature (22 ( 1 °C). Surface Activity of Docetaxel. Surface pressure experiments were carried out on a Kru¨ss K12 tensiometer using the Wilhelmy hanging plate method. A platinum plate was half-dipped in the liquid solution contained in a cylindrical Teflon cuvette (5 cm in diameter and 1.1 cm depth), which was thermoregulated at 22 °C and provided with a lateral
J. Phys. Chem. B, Vol. 112, No. 44, 2008 13835 hole to allow the injection of DCT solutions into the aqueous subphase. Due to the low solubility of DCT in water, the use of an organic solvent was required, and consequently, it was essential to determine the possible effect of the solvent on the surface tension when injected in the aqueous media. To choose the more appropriate solvent, a preliminary study involving different solvents (ethanol, chloroform, acetonitrile, methanol, and dimethylsulfoxide) was performed. Investigations established that acetonitrile can be injected up to 100 µL into the subphase of the 20 mL container without significant modification (less than 1 mN/m) of the initial surface tension of a free aqueous surface. Increasing volumes of DCT solutions (7 mM in acetonitrile) were injected (through the lateral hole) in a stirred subphase (PBS solution), and the surface pressure increase was recorded in individual measurements performed every 30 s. The system was considered stable after five consecutive values which differed in less than 0.1 mN/m. Penetration of Docetaxel in Monolayers. These experiments were carried out using the cylindrical Teflon cuvette described above. A few drops of a DPPC chloroform solution were spread on the aqueous surface (PBS) in order to form lipid monomolecular films of different surface pressures. After each DPPC deposition, the spreading solvent was allowed to evaporate for 15 min before proceeding to the measurement of the initial surface pressure. As soon as the initial surface pressure was stabilized, 50 µL of a DCT solution in acetonitrile was injected into the subphase through the lateral hole and surface pressure changes were recorded. Compression Isotherms. The ability of DCT to spread on aqueous surfaces and to form monolayers was examined using a KSV 5000 Teflon trough with a surface area of 770 cm2 that was housed in a closed box to avoid dust deposition and water evaporation. A solution of DCT in chloroform was delivered from a syringe held very close to the surface, allowing the surface pressure to return to a value as close as possible to zero between each addition. The solvent was allowed to completely evaporate over a period of at least 15 min before two mechanically coupled barriers, compressed symmetrically the monolayer at constant sweeping speed of 10 mm/min. All of the experiments were carried out at room temperature (22 ( 1 °C). Miscibility Studies. Required volumes of DPPC and DCT stock solutions (1.36 mM) in chloroform were mixed in order to obtain the desired molar ratios of both components. Aliquots of the mixtures were spread on the aqueous surface and the π-A isotherms were recorded following the procedure described above. Brewster Angle Microscope. A mini-Brewster angle microscope (mini-BAM) from Nanofilm Technologie GmbH (Go¨ttingen, Germany) was employed for the direct visualization of the monolayers at the air-water interface. Results and Discussion Aggregation of Docetaxel. In order to rule out the presence of aggregates that could create some artifacts during measurements and induce misinterpretations, the hydrophobic fluorescent probe method was applied.33 This experiment is based on the fact that dyes that show larger dipole moments in the excited state than in the ground state yield emission spectra which depend on the polarity of the environment. ANS is one of the most frequently used hydrophobic fluorescent probes.34 Transference of ANS from a polar to a nonpolar environment results in a blue shift of the maximum fluorescence emission, together
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Γ)-
∂γ 1 · 2.303 · RT ∂ log c
(
)
p,T
(1)
where (∂γ/∂ log c) is the slope of the linear part of the graph below the surface tension stabilization. In addition, the area occupied per adsorbed molecule at the saturated interface, A, expressed in Å2, was calculated by applying the equation
A ) 1016/NAΓ Figure 2. Aggregation of DCT in aqueous media, represented by ANS fluorescence intensity versus DCT concentration. The inset graph shows for comparison purposes the fluorescence intensity of ANS samples to which acetonitrile was added (same amounts of volume as those used in the DCT titration).
Figure 3. Surface tension versus DCT concentration (logarithmic scale). The inset graph shows the surface pressure vs DCT concentration.
with a significant increase in the quantum yield. Therefore, the self-aggregation behavior of DCT at various concentrations in aqueous solution can be determined from the fluorescence emission spectra of 10-6 M ANS solution. Docetaxel dissolved in acetonitrile (10-3 M) was used to titrate an ANS solution and the fluorescence intensity recorded. The results are shown in Figure 2. The influence of acetonitrile in ANS fluorescence was also measured in parallel for comparison (see inset of Figure 2). The fluorescence signal of ANS in water (measured at λem ) 470 nm) is very low, but the signal increases gradually with the concentration of DCT in the media, reflecting the partitioning of ANS into the hydrophobic microdomains of self-aggregates. According to the results illustrated in Figure 2, we adjusted the DCT concentration in most of our experiments to a maximum of 20 µM. Adsorption of Docetaxel at the Air-Water Interface. The adsorption of DCT molecules at the aqueous interface was monitored by the decrease of surface tension, measured at constant area, as a function of DCT concentration. The dependence of the surface tension (γ) with the DCT concentration (logarithmic scale) is illustrated in Figure 3. A linear decrease of surface tension with increasing DCT concentration takes place until a concentration of ca. 20 µM is reached. At higher DCT concentrations, the surface tension remains constant (ca. 55 mN/m), suggesting the beginning of some type of aggregation. This result is in excellent agreement with the value determined using the hydrophobic fluorescent probe method described above (Figure 2). Docetaxel adsorption at the saturated interface, Γ, expressed in mol · cm-2 can be calculated using the Gibbs equation:35
(2)
where NA is Avogadroʼs number and Γ is the surface excess calculated from eq 1. From these calculations, the surface excess and area per molecule at the saturated interface are Γ ) 1.71 × 10-10 mol · cm-2 and A ) 97.3 Å2 · molecule-1. The interfacial adsorption can also be expressed in terms of the surface pressure, π, defined as the difference between the surface tension of the solvent, γ0, and the surface tension, γ, after the injection of the DCT solution, i.e., π ) γ0 - γ. The inset of Figure 3 shows the variation of surface pressure with DCT concentration. The values in the inset of Figure 3 are the media of three different experiments. From this graph, it can be concluded that the presence of DCT clearly promotes an increase in the surface pressure up to a maximum of ca. 18 mN/m, which remains fairly constant for DCT concentrations higher than 20 µM. This result indicates that a stable monolayer is formed at the interface, and the beginning of a micelization process occurs at concentrations of ca. 20 µM. Insertion of Docetaxel in DPPC Monolayers. DCT penetration from the subphase into DPPC monolayers at the air-water interface was investigated. Depending on the volume of the DPPC chloroform solutions spread on the aqueous surface, different initial surface pressures, πi, were obtained. To each of these DPPC monolayers, a fixed volume (50 µL) of a 15.12 mM DCT solution in acetonitrile was injected in the subphase, which resulted in a 37.78 µM DCT concentration in the cuvette. Although 20 µΜ is enough to attain the equilibrium of surface tension, this larger DCT concentration was used to ensure a constant concentration of DCT monomer able to interact with the DPPC monolayers. The interaction between the DCT monomer and the DPPC monolayers was recorded in terms of the increase of the surface pressure vs the initial surface pressure. These experiments were carried out for DPPC monolayers at six different initial surface pressures. Docetaxel insertion promotes an immediate increase in the surface pressure (∆π) of the system. The fast stabilization of the surface pressure values is especially worthy of note. Thus, measurements were carried out recording changes in surface pressure for 2 h (to analyze the kinetics of the insertion process). The results indicated that no more than 10 min were needed to attain the equilibrium values. This behavior was similar to that obtained above for surface pressure measurements (without lipid monolayer), where the drug adsorption at the air-water interface was completed in about 10 min. Moreover, these data are in agreement with the kinetic process described for paclitaxel.24 Another important parameter that can be derived from the penetration study is the so-called critical surface pressure (πcr). Figure 4 illustrates the increment of surface pressure (∆π) after DCT injection versus the initial DPPC surface pressure (πi). The critical surface pressure, ca. 47.5 mN/m, is a theoretical value, determined by the intersection of the extrapolated line (∆π f 0 mN/m) and the horizontal x-axis. No increase in the surface pressure beyond the critical surface pressure can be
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J. Phys. Chem. B, Vol. 112, No. 44, 2008 13837
Figure 4. Lineal plot of the increment in surface pressure after DCT injection in the subphase as a function of the initial DPPC surface pressure.
Figure 6. Surface pressure of DPPC/DCT mixed films versus mole fraction of DPPC at the indicated areas per molecule.
xDCT < 0.8 are nearly identical to that of pure DPPC. They are characterized by the LE-LC phase transition as well as by collapse surface pressures similar to that of the pure DPPC film, with the exception of mixed films of xDCT ) 0.8 which show a maximum surface pressure of ca. 35 mN/m under the experimental conditions used. The monolayer compressibility (Cs) has been calculated for pure and mixed films using the equation29
Figure 5. Surface pressure vs area per molecule isotherms of pure DPPC, pure DCT, and mixed films onto a PBS aqueous subphase at 22 °C at the indicated mole fractions of DCT. The inset graph shows the compressibility values for the pure and mixed films vs the surface pressure.
expected, no matter how high the initial drug concentration in the subphase or the initial surface pressure area are. Compression Isotherms of Docetaxel. Figure 5 shows the surface pressure vs area per molecule isotherm of DCT onto a PBS aqueous subphase. The pure DCT monolayer does not exhibit surface pressures >13 mN/m on compression, which is indicative of a low surface activity of DCT at the air-water interface. A similar behavior has been observed for paclitaxel monolayers at the air-water interface which show a maximum surface pressure of ca. 10 mN/m.23,36,37 DCT/DPPC Mixed Monolayers. Both DPPC and DCT form stable Langmuir films at the air-water interface. Therefore, the nature of molecular interactions and also the miscibility of DPPC and DCT can be examined by quantitative analysis of π-A isotherms of mixed monolayers at the air-water interface. Representative compression isotherms of DPPC/DCT at several molar ratios are illustrated in Figure 5. The π-A isotherm of pure DPPC monolayer is well-known, and the isotherm presented in this paper is in good agreement with those reported before.24,38 The π-A isotherm is clearly characterized by a phase transition from liquid expanded (LE) to liquid condensed (LC) state that appears around 17 mN/m. The area per molecule at which the takeoff in the mixed monolayers occurs increases gradually with increasing DCT mole fraction, while increasing percentages of DCT cause the isotherms to shift to smaller areas per molecule at high surface pressures. In addition, the shapes of π-A isotherms of the mixed DPPC/DCT monolayers with
Cs ) -
1 ∂A · A ∂π
( )
T
(3)
The compressibility values vs the surface pressure are illustrated in the inset of Figure 5. The incorporation of DCT molecules into the DPPC monolayers significantly increases the film compressibility at low surface pressures; i.e., a fluidizing effect on the film occurs. This phenomenon was observed before for the DPPC/paclitaxel system25,37,39 and interpreted in terms of changes in the packing of the acyl chains of DPPC induced by paclitaxel.25 Miscibility Analysis of DPPC/DCT Monolayers. The phase rule applied to a multicomponent surface film at the air-water interface, which was developed by Defay and Crisp,40 may help to discern the homogeneity and miscibility of the components in the film.29 Thus, at constant temperature, at constant pressure, and in the absence of any externally imposed electrical potentials, the number of degrees of freedom, f, for the system is given by the expression
f ) CB + CS - PB - PS + 1
(4)
where CB is the number of components in the bulk phase, CS is the number of components restricted to the surface, PB is the number of phases in equilibrium with each other, and PS is the number of surface phases present in equilibrium with each other. In a system of two miscible components forming a monomolecular film at the air water interface, eq 4 indicates that there is one degree of freedom in this system (f ) 1). Consequently, if DPPC and DCT were miscible in all proportions, the surface pressure should vary continuously with the composition. Figure 6 illustrates the variation of surface pressure with the DCT molar fraction at several constant areas per molecule. The variation
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πA,ideal ) XDPPC · (πDPPC)A + XDCT · (πDCT)A
(5)
where (πDPPC)A and (πDCT)A represent the surface pressures of the pure components at an area per molecule A. The interactions between the two components can be quantitatively studied by the surface pressure increment, ∆π, which is calculated as the difference between the ideal surface pressure, πA,ideal, and the experimental value, πA,exptl:
∆π ) πA,exptl - πA,ideal
Figure 7. Area per molecule versus DCT mole fraction for the indicated surface pressures. Solid lines are an eye guide joining the experimental data. Dashed lines represent the additivity rule, and dotted lines are an extrapolation for xDCT f 1 for those surface pressures at which the experimental data can not be obtained.
of the surface pressure for the DPPC/DCT mixed films with the mole fraction of DCT is consistent with the two components being miscible at the air-water interface. The nature of molecular interactions and also the miscibility of the two components can be examined by quantitative analysis of the deviations of the area per molecule in the mixed films with respect to the ideality (additivity rule).35,41,42 For completely immiscible or ideal mixed monolayers, the area per molecule follows the additivity rule (Amixed film ) xDPPC · ADPPC + xDCT · ADCT), i.e., the excess area is zero, while positive or negative deviations from the additivity rule are indicative of some degree of molecular interactions between the different molecular components. In particular, positive deviations from the additivity rule in the mixed system imply some type of repulsive interactions, with DPPC-DPPC or DCT-DCT interactions broken and weaker DPPC-DCT interactions formed. On the contrary, negative deviations from the additivity rule suggest increased attractive interactions between the two components in the mixed monolayer. Figure 7 shows the experimental values of the area per molecule versus the mole fraction of DCT at several surface pressures; dashed lines in Figure 7 represent the values for the additivity rule and dotted lines correspond to the linear extrapolation of the experimental values of the areas per molecule for xDCT f 1, which can not be experimentally determined for high surface pressures due to low surface activity of DCT. From the data in Figure 7, it can be concluded that low proportions of DCT are capable of breaking DPPC-DPPC interactions, with a maximum deviation from the additivity rule for a mole fraction of DCT of ca. 0.4. It should also be noted that an increase in the surface pressure leads to lower deviations from the additivity rule, with mixed monolayers showing negligible deviations from the additivity rule at surface pressures higher than 30 mN/m. The DDPC/DCT system is more sensitive to changes in the surface pressure than in the molecular area. Then, it may be more convenient to analyze the excess property by monitoring changes in the surface pressure at any given value of the molecular area. The ideal value of surface pressure of the mixed monolayer, πA,ideal, at any given molecular area, A, can be determined from those of the pure monolayers at the same given area using the equation
(6)
Figure 8 shows the surface pressure increment at five representative areas per molecule. These surface pressure increments are indicative again of nonideal miscibility of the two components. Slightly positive deviations at large areas per molecule indicate a pressure increase effect, which is equivalent to an area expansion effect. This result suggests a net repulsive interaction between the two components, which is consistent with the results obtained for the excess area deviations. In contrast, negative surface pressure increments were observed for all DCT mole fractions at small areas per molecule. Such negative pressure increments are indicative of a pressure reduction effect, which indicates a net attractive interaction between the two components. Figure 8 also indicates that deviation from ideality increases with increasing DCT mole fraction, showing the maximum deviations in the 0.4-0.6 DCT mole fraction range, with the precise position of the minimum in the ∆π vs xDCT plot depending on the area per molecule. Any deviation, either positive or negative, from ideality involves the generation of an energy known as excess free energy of mixing, ∆GEm, which represents the energy associated with the mixing process of the two pure components in the bidimensional phase and can be determined using the expression42,43 E ∆Gm )
∫ π0 Α12 dπ - x1 ∫ π0 Α1 dπ - x2 ∫ π0 Α2 dπ (7)
where x1 and x2 are the mole fractions of components 1 and 2, respectively, in the mixed monolayer; A1 and A2 represent the area per molecule of the pure monolayers at the same surface pressure. π1 and π2 are the surface pressures of components 1 and 2, at a given molecular area; A12 and π12 are the mean area
Figure 8. Surface pressure increments of the DPPC/DCT monolayers at the air-water interface plotted as a function of the mole fraction of DCT at the indicated areas per molecule.
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per molecule and surface pressure in the mixed films. However, the use of this expression is not convenient for the present case because the isotherms are more sensitive to the surface pressure than to the molecular area. This is the reason why the Helmholtz excess energy of mixing, ∆AEm, has been determined in this work, as it is permits a more accurate analysis of energy changes with varying surface pressure, instead of dealing with varying molecular areas.25 In addition, the Helmholtz energy of mixing, ∆Am, provides an indication of the interactions and stability of mixed films. For the process of mixing, the value of the Helmholtz energy of mixing, ∆Am, is the sum of the excess energy and the ideal free energy of mixing, ∆Aideal m . Thus, the Helmholtz excess energy and the Helmholtz energy of mixing have been calculated according to the equations35 E ∆Am )
∫ AA π12 dA - x1 ∫ AA π1 dA - x2 ∫ AA π2 dA 0
0
E ideal ∆Am ) ∆ Am + ∆ Am
ideal ∆ Am ) RT(x1 ln x1 + x2 ln x2)
0
(8) (9)
(10)
where A0 is the area where π gives the first increment from π ) 0 and A is the molecular area at which the Helmholtz energy is to be calculated. Figure 9a and b illustrates the variation of ∆AEm and ∆Am with DCT mole fraction at several representative areas per molecule. These plots indicate that a more stable system is obtained when the two materials are mixed. In addition, both ∆AEm and ∆Am become more negative with smaller molecular areas, which suggests that the stability of the mixed monolayers becomes higher as the monolayers become more condensed. Negative values of ∆Am and the presence of only one minimum suggest that no phase separation in the mixed films occurs.41,44 The foregoing properties have been calculated from the π-A data points, and therefore, other independent methods are necessary to provide additional information of the phase behavior in the mixed films. Thus, Brewster angle microscopy (BAM) investigations were made during the compression of the Langmuir films and gave further insight about the formation of DPPC, DCT, and mixed DPPC/DCT monolayers (Figure 10). BAM images are quite homogeneous with no apparent threedimensional aggregates, which is indicative of the formation of true monomolecular films for both the pure and the mixed films. In addition, BAM images for the mixed films resemble those of the pure films, with no evidence of phase separation within the micro-BAM resolution (in the micron range). DPPC monolayers show a clear increase in the brightness of the images with increasing surface pressure, which is indicative of a gradual tilt of the molecules and an increase in the film thickness. The presence of DCT, especially for xDCT g 0.4, yields BAM images with a lower brightness at high surface pressures which suggest that the presence of DCT induces a different arrangement of DPPC molecules in the film. This result is consistent with previous observations (FTIR and AFM analysis) of DPPC and paclitaxel which showed that the taxol derivative increases the mobility of the acyl chains of the lipid, resulting in a fluidizing effect on the monolayer as well as the extent of liquid condensed phases.37,39 Therefore, all of the techniques used to analyze the films described here indicate that the two molecules are miscible in the whole range of molar fractions, evidencing a nonideal mixing behavior. DPPC/DCT mixed films show a maximum stability
Figure 9. (a) Excess Helmholtz energy of mixing versus mole fraction of DCT for the indicated areas per molecule and (b) Helmholtz energy of mixing versus mole fraction of DCT for the indicated areas per molecule.
of the system for a DCT mole fraction of 0.6, in contrast with mixtures of DPPC and paclitaxel which show minimum values of the excess Helmholtz energy for the mixed monolayers for xDCT ) 0.05.25 Conclusions To the best of our knowledge, this is the first study that reports the interactions of docetaxel (DCT), an antineoplastic drug for the treatment of a wide spectrum of cancers, with dipalmitoyl phosphocholine (DPPC), the main component of biological membranes. These investigations have been centered on the measurement of DCT surface activity, penetration of DCT into DPPC monolayers at different surface pressures, and compression isotherms of pure and mixed Langmuir films (DPPC/DCT) containing different proportions of the two components. Adsorption of DCT at the air-water interface, when injected into the water subphase, takes place with a maximum surface coverage of 1.71 × 10-10 mol · cm-2. Penetration studies of DCT into a DPPC monolayer revealed that DCT promotes an immediate increase in the surface pressure of DPPC monolayers with fast kinetics and a stabilization time of ca. 10 min. This result indicates that DCT molecules are absorbed at the interface where they can penetrate into the phospholipid matrix. Mixed DPPC/ DCT films have been prepared by the Langmuir technique.
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Figure 10. BAM images of DPPC, DCT, and DPPC/DCT Langmuir films at the indicated surface pressures and compositions. The field of view along the x-axis for the BAM images is 2350 µm.
Miscibility of the two components in the Langmuir films has been analyzed by means of thermodynamic properties as well as by BAM analysis of the films at the air-water interface, concluding that DPPC and DCT are miscible and the mixing is nonideal. Nonideal mixing behavior was revealed by deviations of the area from the additivity rule as well as by the analysis of the pressure reduction effect. Positive deviations in the area from
the additivity rule at low surface pressures and a pressure increase effect at large areas per molecule indicate a net repulsive interaction between the two components in the gas and liquid expanded phases. In contrast, compression of the mixed monolayers results in negative surface pressure increments which indicate a net attractive interaction between the two components in the condensed monolayers. The stability of
Interactions of Docetaxel with Model Lipid Membranes the mixed monolayers was investigated by analyzing the Helmholtz energies of mixing which revealed that no phase separation in the mixed films occurs and the mixed films are more stable than the pure monolayers, with a maximum stability for xDCT ) 0.6. BAM images confirm the miscibility of the two materials and reveal that the presence of DCT induces a different arrangement of the DPPC molecules in the film. Acknowledgment. The competent technical assistance of Mr. Ignacio Sanchez and Mrs. Jessica Priego is gratefully acknowledged. This study was supported by the Ministerio de Industria y Energia as a Cenit Project. A.F.-B. is grateful for support received from the Departament d′Universitats, Recerca i Societat, Generalitat de Catalunya and Lipotec S. A. in the frame of Beatriu de Pino´s grant. P.C. is grateful for financial assistance from Ministerio de Educacio´n y Ciencia (MEC) from Spain and “fondos FEDER” in the framework of the project CTQ200605236 as well as to DGA for its support through the interdisciplinary project PM079/2006. References and Notes (1) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A. T. J. Am. Chem. Soc. 1971, 93, 2325. (2) Denis, J. N.; Correa, A.; Greene, A. E. J. Org. Chem. 1990, 55 (6), 1957. (3) Brown, D. T. In Taxus: The Genus Taxus; Itokawa, H., Lee, K.H., Eds.; Taylor and Francis: 2003; p 387. (4) Clarke, S. J.; Rivory, L. P. Clin. Pharmacokinet. 1999, 36 (2), 99. (5) Lyseng-Williamson, K. A.; Fenton, C. Drugs 2005, 65 (17), 2513. (6) Fulton, B.; Spencer, C. M. Drugs 1996, 51 (6), 1075. (7) Figitt, D. P.; Wiseman, L. R. Drugs 2000, 59 (3), 621. (8) Keam, S. J.; Scott, L. J.; Am, J. Cancer 2004, 5 (5), 325. (9) Lee, K. S.; Ro, J.; Nam, B. H.; Lee, E. S.; Kwon, Y.; Kwon, H. S.; Chung, K. W.; Kang, H. S.; Kim, E. A.; Kim, S. W.; Shin, K. H.; Kim, S. K. Breast Cancer Res. Treat. 2008, 109 (3), 481. (10) Yong, W. P.; Wang, L. Z.; Tham, L. S.; Wong, C. I.; Lee, S. C.; Soo, R.; Sukri, N.; Lee, H. S.; Goh, B. C. Cancer Chemother. Pharmacol. 2008, 62 (2), 243. (11) Horwitz, S. B.; Fant, J.; Shiff, P. B. Nature 1979, 277, 655. (12) Wang, L. G.; Liu, X. M.; Kreis, W.; Budman, D. R. Cancer Chemother. Pharmacol. 1999, 44 (5), 355. (13) Szebeni, J.; Muggia, F. M.; Alving, C. R. J. Natl. Cancer Inst. 1998, 90 (4), 300. (14) Argyriou, A. A.; Koltzenburg, M.; Polychronopoulos, P.; Papapetropoulos, S.; Kalofonos, H. P. Crit. ReV. Oncol. Hematol. 2008, 66 (3), 218. (15) Alexopoulos, A.; Karamouzis, M. V.; Stavrinides, H.; Ardavanis, A.; Kandilis, K.; Stavrakakis, J.; Georganta, C.; Rigatos, G. Ann. Oncol. 2004, 15 (6), 891.
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