Langmuir 1997, 13, 6533-6539
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Interaction of Chlorpromazine with Phospholipid Membranes: A Monolayer and a Microelectrophoresis Approach M. Bennouna,† J. Ferreira-Marques,‡ S. Banerjee,‡ J. Caspers,*,‡ and J. M. Ruysschaert‡ Universite´ Abdelmalek Esaadi, Faculte´ des Sciences de Tetouan, BP 2121 Tetouan, Morocco, and Universite´ Libre de Bruxelles, Laboratoire de Chimie Physique des Macromole´ cules aux Interfaces, CP 206/2, Bd du Triomphe, B-1050 Bruxelles, Belgium Received February 25, 1997. In Final Form: September 2, 1997X Chlorpromazine penetration into the lipid core of the membrane was demonstrated through measurements on lipid monolayers (surface pressure and surface potential). The surface pressure measurements allow us to calculate the intrinsic binding constant (partition coefficient) for the lipid-Chlorpromazine interaction. This latter value is in correct agreement with the obtained results by electrophoretic mobilities measurements on liposomes.
Introduction Chlorpromazine (Cpz) is a phenotiazine with neuroleptic activity, showing a large capacity to interact with biological membranes and sometimes be used as a local anesthetic.1-9 Cpz has an amino group and because of its pKa value,10-12 exists essentially in its charged form at physiological pH (with a small amount of neutral form). In some earlier works, the local anesthetic-lipid interaction has been interpreted in terms of a “Langmuir” adsorption9,13-16 and, in others, in terms of a partition coefficient.17,18 The partition coefficient was found to be concentration and pH dependent.18-20 Therefore, this partition coefficient should be considered as apparent since the Cpz concentration in the bulk and near the interface were considered to be identical. Moreover, in the vicinity of the pKa, the †
Universite´ Abdelmalek Esaadi. Universite´ Libre de Bruxelles. X Abstract published in Advance ACS Abstracts, November 1, 1997. ‡
(1) Lee, A. G. Mol. Pharmacol. 1977, 13, 474-487. (2) Kwant, W. O.; Seeman, P. Bochim. Biophys. Acta 1969, 183, 530543. (3) Frenzel, F.; Arnold, K.; Nuhn, P. Biochim. Biophys. Acta 1978, 507, 185-197. (4) Ro¨mer, J.; Bickel, M. H. Biochem. Pharmacol. 1979, 28, 799805. (5) Cater, B. R.; Chapman, S. M.; Hawes; Saville, J. Biochim. Biophys. Acta 1974, 363, 54-69. (6) Leterrier, F.; Mendyk, A.; Viret, J. Biochem. Pharmacol. 1976, 25, 2469-2474. (7) Seeman, P.; Weinstein, J. Biochem. Pharmacol. 1966, 15, 17371752. (8) Seeman, P. Pharmacol. Rev. 1972, 24, 583-655. (9) Anteneodo, C.; Bisch, P. M.; Ferreira-Marques, J. Eur. Biophys. J. 1955, 23, 447-452. (10) Yamagushi, T.; Watanabe, S.; Kimoto, E. Biochim. Biophys. Acta 1985, 820, 157-164. (11) Chatten, L. G.; Harris, L. E. Anal. Chem. 1963, 34, 1495-1501. (12) Wajnberg, E.; Tabak, M.; Nussenzveig, P. A.; Lopes, C. M.; Louro, R. W. Biochim. Biophys. Acta 1988, 944, 185-190. (13) Rooney, E. K.; Lee, A. G. Biochim. Biophys. Acta 1983, 732, 428-440. (14) Zachowski, A.; Durand, Ph. Biochim. Biophys. Acta 1988, 937, 411-416. (15) Maher, P.; Singer, S. J. Biochemistry 1984, 23, 232-240. (16) Rosso, J.; Zachowski, A.; Devaux, P. R. F. Biochim. Biophys. Acta 1988, 942, 271-279. (17) Luxnat, M.; Galla, H. J. Biochim. Biophys. Acta 1986, 856, 274282. (18) Luxnat, M.; Mu¨ller, H. J.; Galla, H. J. Biochem. J. 1984, 224, 1023-1026. (19) Welti, R.; Mullikin, L. J.; Yoshimura, T.; Helmkamp, G. M. Biochemistry 1984, 23, 6086-6091. (20) Roth, S.; Seeman, P. Biochim. Biophys. Acta 1972, 255, 207219.
S0743-7463(97)00203-5 CCC: $14.00
simultanous presence of the protonated and unprotonated form of Cpz was not taken into account. Furthermore, one must take into account the critical micellar concentration (CMC) of Cpz in the interpretation of the interaction mechanism between the drug and the lipidic structure, since Cpz beyond a given concentration can behave as a detergent, and this can explain the increasing value of the apparent partition coefficient observed in basic media.12,19 In order to obtain a more unified view, the Cpzphospholipid interaction was studied in the present work by considering the nature of the lipid material (neutral or charged lipids, having different fluidity) and the pH effects, on different model membranes. The drug penetration into the lipidic material was verified by measurements on monolayers (surface pressure and surface potential). In addition, we made electrophoretic mobility measurements of multilamellar vesicles. The studies were performed at concentrations lower than the CMC.12 It was found that a simple partition coefficient is able to describe the Cpz-phospholipid interaction, for both anionic and neutral lipids and that the protonated and unprotonated forms of Cpz are able to penetrate into the lipidic layer. The intrinsic association constants for Cpzlipids (partition coefficients) were determined separately for the protonated and unprotonated forms of Cpz. Materials and Methods Cardiolipin (sodium salt, from bovine heart) (CL), DL-Rdipalmitoylphosphatidic acid (sodium salt) (DPPA), DL-R-dipalmitoylphosphatidylcholine (DPPC), phosphatidic acid from egg yolk (PA), egg phosphatidylcholine (PC), and chlorpromazine hydrochloride [2-chloro-10-(3-(dimethylamino)propyl)phenotiazine hydrochloride] (Cpz) were from Sigma Chemical Co. For monolayer measurements, the lipid was dissolved in chloroform or chloroform-methanol (3:1 V:V) mixture and spread with an Agla microsyringe on buffered subphases made of sodium acetate-HCl (10-3 M, pH 4.5) or phosphate (10-3 M, pH 8). Cpz was added in the subphase. Surface potential (∆V) was measured using the vibrating plate method.21-25 The surface pressure measurements were performed using a LAUDA-FILM balance. (21) Noblet, A.; Ridelaire, H.; Sylin, G. J. Phys. E.: Sci. Instrum. 1984, 17, 233-236. (22) Caspers, J.; Landuyt-Caufriez, M.; Deleers, M.; Ruysschaert, J. M. Biochim. Biophys. Acta 1979, 554, 23-38. (23) Goormaghtigh, E.; Chatelain, P.; Caspers, J.; Ruysschaert, J. M. Biochim. Biophys. Acta 1980, 597, 1-14. (24) Guilmin, T.; Goormaghtigh, E.; Brasseur, R.; Caspers, J.; Ruysschaert, J.M. Biochim. Biophys. Acta 1982, 685, 169-176.
© 1997 American Chemical Society
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Multilamellar vesicles (MLV) were obtained by dissolution of lipids in chloroform or chloroform-methanol mixtures (3:1, V:V) followed by evaporation of the solvent (first under N2 and then under vacuum overnight). The lipid films were vortexed in sodium acetate-HCl (10-3 M, pH 4.5) or in phosphate (10-3 M, pH 8) buffer mixtures above the transition temperature of the lipids. Cpz was added after MLV formation. The electrophoretic mobility of MLV was measured in a Rank Brothers Mark II apparatus. All the measurements were carried out in the stationary level26 of the cylindrical capillary cell (length 8 cm) with an applied voltage of approximatively 40 V (dc) at 25 °C. The migration rate of the particles was determined at least 10 times in each direction. (a) Surface Potential. The surface potential (∆V) is the potential variation resulting from the spreading of the lipid monolayer on the water surface. This potential change can be written as the sum of two terms: a dipolar contribution (p is the average vertical contribution to the total dipole associated to the lipid and the drug in the monolayer) and ψ0, the electrostatic potential at the interface:27
∆V )
p + ψ0 Ar0
Here, δ refers to the “free absorbed” DH+. An additional parameter δ′ refers to the absorbed DH(+)-Cl(-) species, resulting DHCl /nL). This from the Cl- adsorption on the absorbed drug (nabs (+) (-) 13 DH -Cl interaction is taken into account through a KCl equilibrium constant:
KCl )
KNa )
(2)
where A0 is the initial area of the lipid layer (A0 ) ALNL), AL (0.65 nm2) and AD are the lipid and drug areas, and NL and ND are the number of lipid and drug molecules in the monolayer after drug penetration. Measurements of ∆A at constant surface pressure will allow the determination of the drug-lipid association constant, as described as follows. At pH 4.5, when the drug exists only in its charged form (DH+), the surface charge density (σ) for anionic lipids is given by
-N*e -(z - δ)NLe ) A A0 + ∆A
(3)
where e is the absolute value of the electronic charge, N* is the resulting number of charged sites at the interface at equilibrium, z is the mole fraction of free lipidic anionic sites (n(-) L /nL), and δ is the number of moles of absorbed drugs (DH+) divided by the (+) /nL). nL is the total number of moles of lipids in the layer (nabs number of moles of lipids in the monolayer. For neutral lipids (z ) 0) at the same pH, the surface charge density is given by
σ)
δNLe N*e ) A A0 + ∆A
1-z z(Na+)0
(6)
(Cl-)0 and (Na+)0 are the equilibrium concentrations of Cl- and Na+ at the interface. One can write, from ∆A ) ADND and A0 ) ALNL (in eq 2):
(1)
A is the area of the monolayer, r is the relative permittivity of the medium, and 0 is the permittivity of vacuum. From the ∆V values determined experimentally and the ψ0 values calculated from the electrophoretic mobility measurements (see below), the dipolar contribution can be estimated. A strong penetration of the drug into the lipid layer will significantly affect the resulting p value. Some details about the determination of the dipolar contribution are presented in the Results and Discussion. (b) Surface Pressure. If the drug penetrates the lipid layer, an increase of the area (∆A) of the film maintained at constant surface pressure (20 m Nm-1) will be observed with increasing amounts of Cpz in the subphase. At low drug concentrations in the subphase, one can accept the additivity of the areas:28,29
σ)
(5)
Such a Cl- adsorption is not taken into account for Cpz interacting with anionic lipids. For anionic lipids, the Na+ adsorption on the negative lipidic sites is taken into account through a KNa equilibrium constant:
δ + δ′ )
A0 + ∆A ) ALNL + ADND
δ′ δ(Cl-)0
ND ∆AAL ) NL A0AD
Assuming that AL and AD are not very different,29 that relation can be simplified to δ + δ′ ≈ ∆A/A0. Thus, the experimental determination of ∆A and A0 at constant pressure allows us to estimate δ + δ′ (δ′ ) 0 with anionic lipids). Relation 5 or 6 (and the knowledge of KCl and KNa) allows us to determine δ and z, and relation 3 or 4 yields the charge density. The surface electrostatic potential at the interface, ψ027,30 is given by the Gouy-Chapman relation:
ψ0 )
2RT -1 σ sh F (8RTr0C)1/2
(8)
R is the gas constant, F is the Faraday constant, T is the absolute temperature, and C is the ionic concentration (10-1 or 10-2 M NaCl in the present work). The concentrations of other charged species present in the solution were neglected as compared with the NaCl concentration. The equilibrium concentration of each charged species (Cl-, Na+, DH+) at the interface,26 (X)0, is related to the bulk equilibrium concentration ((X)∞) by the Boltzmann factor:
(X)0 ) (X)∞e-ZFψ0/RT
(9)
Z is the valence of the ion. The initial DH+ bulk concentration ((DH+)∞,i) is the sum of the equilibrium bulk concentration ((DH+)∞) and the concentration of the absorbed drug:
(DH+)∞,i ) (DH+)∞ + (δ + δ′)CL
(10)
CL is the number of moles of lipid divided by the volume of the aqueous phase. By combination of the previous relations (and for given KCl and KNa values), δ, δ′, and (DH+)0, the equilibrium DH+ concentration in water near the interface, can be determined. The partition coefficient KDH can thus be calculated:26
(4)
(25) Caspers, J.; Goormaghtigh, E.; Ferreira, J.; Brasseur, R.; Vandenbranden, M.; Ruysschaert, J. M. J. Colloid Interface Sci. 1983, 91, 546-554. (26) Banerjee, S.; Caspers, J.; Bennouna, M.; Sautereau, A. M.; Tocanne, J. F.; Ruysschaert, J. M. Langmuir 1995, 11, 1134-1137. (27) Aveyard, R.; Haydon, D. A. An Introduction to the Principles of Surface Chemistry; Cambridge University Press: Cambridge, U.K., 1973; pp 1-57. (28) Seelig, A. Biochim. Biophys. Acta 1987, 899, 196-204. (29) Seelig, A.; Allegrini, P. R.; Seelig, J. Biochim. Biophys. Acta 1988, 939, 267-276.
(7)
KDH )
(+) Xabs
(DH+)0
(11)
KDH is equal to K′DHvw, where vw is the molar volume of water and (+) (+) (+) K′DH ) Xabs /X(+) 0 (Xabs and X0 are respectively the drug concentrations (in mole fraction) in the lipid phase and in water, near (+) the interface), Xabs is equal to δ(δ + 1)-1 for anionic lipids and -1 to δ(δ + δ′ + 1) for neutral lipids. We assume that the charge (30) McLaughlin, S. Current Topics in Membrane and Transport; Academic Press: New York, 1977; Vol. 9, pp 71-144.
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of the absorbed drug is localized in the vicinity of the lipid head group and modifies the surface charge density.28,29,31,32 At pH 8, where both DH+ and D, the protonated and unprotonated form of Cpz (pKa ) 8.6),12 must exist10-12 in the bulk and at the interface (depending on the surface pH), the relative proportions of the two forms of Cpz in the bulk can be determined using the acidic constant of the drug in water (Ka($)):
Ka($) )
(D)∞(H+)∞ (DH+)∞
(12)
(H+)0 can be related to (H+)∞ by relation 9. An additional partition coefficient KD can be defined for the unprotonated form of the drug:
KD )
D Xabs
(D)∞
(13)
D where Xabs is given by δ1(1 + δ1 + δ2 + δ′)-1. In relation 11, (+) Xabs is now given by δ2(1 + δ1 + δ2 + δ′)-1. Here δ1, δ2, and δ′ D (+) DHCl /nL, nabs /nL, and nabs /nL (δ′ ) 0 are respectively given by nabs with anionic lipids). The surface charge density for anionic and neutral lipids are given by relations 3 (with δ2 in the numerator) and 4 (with δ1 in the numerator), respectively. In the case of phosphatidic acid it is suitable to consider the second ionization of the acidic head group in the phospholipid. The surface charge density is given by
(z1 + z2 - δ2)NLe N*e σ))A A0 + ∆A
(14)
where z1 and z2 are respectively the fraction of free negatively charged sites 1 and 2 in the monolayer (see Appendix). Cl- adsorption on absorbed DH+ (for neutral lipid) and Na+ adsorption on negatively charged sites are taken into account through appropriate equilibrium constants (see before). The initial bulk drug concentration is the sum of the DH+ and D concentrations in the bulk plus the concentration of the absorbed drug:
(Dtot)∞,i ) (DH+)∞ + (D)∞ + (δ1 + δ2 + δ′)CL
(15)
By combination of the previous relations and using the KDH value determined at pH 4.5, the parameters δ1, δ2, δ′, (DH+)0, and (D)∞ can be determined. Thus, the KD partition coefficient (relation 13) can be calculated. (c) Electrophoretic Mobility. Under our experimental conditions,26 where the Debye length is lower than 3 nm and the diameter of the liposomes is larger than 100 nm, the electrophoretic mobility µ can be related to the ξ potential through the Helmholtz-Smoluchowski equation:33-36
ξ)
µη 0r
(16)
ξ is the potential at the hydrodynamic plane of shear and η is the viscosity of water. According to several authors35,37,38 the hydrodynamic plane of shear can be localized at 0.2 nm from the charged surface plane (31) Kuchinka, E.; Seelig, J. Biochemistry 1989, 28, 4216-4221. (32) Seelig, A.; McDonald, P. Biochemistry 1989, 28, 2490-2496. (33) James, A. M. In Surface and Colloid Science; Good, R. J., Stromberg, R. S., Eds.; Plenum Press: New York, 1978; Vol. II, pp 121-185. (34) Sherbet, G. V. The Biological Characterization of the Cell Surface; Academic Press: New York, 1978; pp 36-53. (35) Eisenberg, M.; Gresfaldi, T.; Riccio, T.; McLaughlin, S. Biochemistry 1979, 18, 5213-5223. (36) Wiersma, P. H.; Loebb, A. L.; Overbeek, J. Th. G. J. Colloid Interface Sci. 1966, 22, 78-99. (37) McLaughlin, A.; Eng, W. K.; Vaio, G.; Wilson, T.; McLaughlin, S. J. Membr. Biol. 1983, 76, 183-193. (38) Winiski, A. P.; Eisenberg, M.; Langner, M.; McLaughlin, S. Biochemistry 1988, 27, 386-392.
Figure 1. Schematic procedure to calculate the partition coefficient KDH at pH 4.5 in the case of anionic lipids: (a) by microelectrophoretic measurements on liposomes; (b) by ∆A measurements at constant surface pressure on monolayers. The encircled symbols are the experimental starting parameters. (DH+)∞,i ) initial bulk drug concentration; CL ) lipid concentration. of the vesicles. The ψ0 value in the surface plane is related to the ξ potential through the Gouy-Chapman theory of the electrical double layer (for more details, see Winiski et al. and McLaughlin30,38). That electrostatic surface potential is related to the surface charge density (σ) and to the concentration of monovalent electrolyte in the bulk through relation 8. In relations 3, 4, and 14, the ratio NL(A0 + ∆A)-1 can be replaced by AL-1(1 + δt)-1 where δt is the total amount of the absorbed drug (at pH 4.5 δt is given by δ and δ + δ′, respectively, for anionic and neutral lipids; at pH 8 δt is given by δ1 + δ2 and δ1 + δ2 + δ′ respectively for anionic and neutral lipids). In relations 10 and 15, CL is now the average number of moles of lipids in the outer layer of the MLV divided by the volume of the aqueous phase. Combination of these relations with the relations given in the previous section (5 or 6, 8, and 9) and with relation 16 allow us to determine KDH (at pH 4.5) and KD (at pH 8) (relations 11-13). Conversely, these relations allow to draw the theoretical evolution of µ as a function of the drug concentration (or its logarithm) for given values of KDH and KD both for anionic and neutral lipids and to compare that calculated curve to the experimental data. In Figure 1 a comparison of the two methods (surface pressure and electrophoretic mobility) used in the present work to determine the partition coefficients is shown.
Results and Discussion (a) Electrophoretic Mobility. The electrophoretic mobility of the neutral lipid vesicles becomes more and more positive with increasing drug concentration. At pH 4.5 (Figure 2a,b), the drug is protonated (DH+) and the charge density is directly related to the drug absorption at the surface of the vesicles. At pH 8 (Figure 3a,b) the electrophoretic mobility is systematically lower than that at pH 4.5 (Figure 2). At this pH the protonated (DH+) and unprotonated (D) forms of Cpz are simultaneously present in the medium. The electrophoretic mobility of vesicles made of anionic lipids becomes less and less negative with increasing Cpz concentration (Figure 4a,b). However, at a defined concentration in Cpz, the vesicles are immobilized and a further increase in drug concentration changes the direction of migration of the vesicles in the electric field, as a consequence of the fact that the net surface charge is positive (Figure 4a).
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Figure 2. Measured electrophoretic mobility (filled symbols) of egg PC MLV at pH 4.5 as a function of the logarithm of the Cpz concentration (T ) 25 °C; CL ) 10-5 M; (a) 10-1 M NaCl, (b) 10-2 M NaCl). The theoretical curves (solid lines) were calculated as follows: (upper curve) KDH ) 104 M-1; (lower curve) KDH ) 6 × 103 M-1; (average value) KDH ) 8 × 103 M-1.
Figure 3. Measured electrophoretic mobility (filled symbols) of egg PC MLV at pH 8 as a function of the logarithm of the Cpz concentration (T ) 25 °C; CL ) 10-5 M; (a) 10-1 M NaCl, (b) 10-2 M NaCl). The theoretical curves (solid lines) were calculated as follows: (curve A) KD ) 0.1KDH ) 8 × 102 M-1; (curve B) KD ) KDH ) 8 × 103 M-1; (curve C) KD ) 3KDH ) 2.4 × 104 M-1; (curve D) KD ) 10KDH ) 8 × 104 M-1.
At pH 4.5 (Figure 2), the Cpz-egg PC interaction can be satisfactorily described in terms of the “partition” model described before. In the range of studied drug concentrations, the KDH value near 8 × 103 M-1 fits the experimental data at the two studied ionic strengths. The calculations were performed by taking into account the adsorption of Cl- on the absorbed DH+ (KCl ) 0.5 M-1). In the case of the egg PA-Cpz interaction at pH 4.5 (Figure 4a) a “partition” model, with approximately the same numerical value for the partition coefficient (6 × 103 M-1) as that obtained for the egg PC-Cpz interaction, fits the electrophoretic mobility experimental data. A value of KNa ) 0.5 M-1 was taken into account for the Na+ adsorption on lipid anionic sites.26 At pH 8, the DH+ and D concentrations of Cpz must be calculated using the acidic dissociation constant of Cpz in water (pKa(w) ) 8.6) as described in the Appendix. Therefore, two association constants must be considered: KD and KDH. It is assumed that the DH+ absorption was identical at pH 4.5 and pH 8.
For neutral lipids, several values of KD were tested, as illustrated in Figure 3. It was found that the experimental results were satisfactorily described when KD and KDH values were considered of the same magnitude (KD ) 2 × 104 M-1 and KDH ) 0.8 × 104 M-1 for egg PC). For phosphatidic acid, the second ionization of the phosphate group was taken into account. As observed in the Cpz-neutral lipid interaction, there was a best agreement between the experimental electrophoretic mobilities and the calculated values (Figure 4b) when KD was not very different from KDH (KD ) 1.2 × 104 M-1 and KDH ) 0.6 × 104 M-1 for egg PA). The realtively similar values of KD and KDH show that the pKa shift13 of Cpz between the membrane (absorbed drug) and the bulk (free drug) is probably lower than the value previously reported.13 Our results suggest a ∆pKa value lower than 0.5, allowing us to consider that the basicity of Cpz will be slightly lower at the interface than in a purely aqueous medium.
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Figure 4. Measured electrophoretic mobility (filled symbols) of egg PA MLV at pH 4.5 (a) and 8 (b) as a function of the logarithm of the Cpz concentration (T ) 25 °C, CL ) 10-5 M, 10-1 M NaCl). The theoretical curves (solid lines) were calculated as follows: (a) KDH ) 6 × 103 M-1; (b) KDH ) 2KDH ) 1.2 × 104 M-1. Table 1. K and ∆G°298 Values (∆G° ) - RT ln 55.5 K) for the Cpz-Phospholipid Interactionsa K
(mol-1)
DH+
∆G° (KJ‚mol-1) a
D DH+ D
egg PC
DPCC
egg PA
DPPA
CL
8× 2 × 104 -32.2 -34.5
2× 4 × 103 -28.8 -30.5
6× 1.2 × 104 -31.5 -33.2
2× 4 × 103 -28.8 -30.5
103 2 × 103 -27.0 -28.8
103
103
103
103
These values allow the best fitting of the experimental curves µ ) f(log(Cpz)∞,i).
It must be mentioned that at pH 8, microscope observation revealed that the number of vesicles increases at a Cpz concentration higher than 10-4 M. Above this value, the suspension becomes turbid. Above 5 × 10-3 M Cpz, the vesicles were no more visible under the microscope, suggesting the formation of Cpz-lipid mixed micelles not detectable under the microscope. Taking this fact into account and the CMC of Cpz12 the upper part of the curves at pH 8 were presented with a dotted line (Figures 3 and 4b). Table 1 summarizes the results obtained with all the phospholipids used in the present work and show that the membrane fluidity (compare egg PC with DPCC and egg PA with DPPA) plays a relatively minor role in the Cpzlipid interaction. (b) Surface Potential. Measurements were performed at a surface pressure of 20 mN m-1. Addition of Cpz molecules into the subphase will increase the surface potential as a result of the surface charge modification. The decrease of the surface potential observed at Cpz concentrations beyond 10-5 M (Figure 5) means that the total dipole moment has been modified (relation 1). For a monolayer made with neutral lipids (ψ0 ) 0), the ∆V initial value is ∆Vdip (where ∆Vdip ) pf/Ar0). The f f subscript f refers to free lipids (drug not added). In the presence of an interacting cationic drug (∆Vd), one can write ∆Vd ) ∆Vdip d + ψ0, and
∆(∆V)dip ) ∆(∆V) - ψ0
(17)
- ∆Vdip where ∆(∆V)dip (given by ∆Vdip d f ) is the total dipolar variation resulting from the binding of the drug molecules to the lipid layer. ∆(∆V) is an experimental value (given by ∆Vd - ∆Vf). For anionic lipids, one can write in the absence of drug: + ψ0,f, and the in presence of drug: ∆Vd ) ∆Vf ) ∆Vdip f
∆Vdip d + ψ0,d. Thus
∆(∆V)dip ) ∆(∆V) - ∆ψ0
(18)
where ∆ψ0 ) ψ0,d - ψ0,f. Thus, from the experimental ∆(∆V) values and the calculated ψ0 contributions for different Cpz concentrations (using the KD and KDHvalues determined by microelectrophoresis), the ∆(∆V)dip contributions were calculated at both pH 4.5 and pH 8 (Figure 5). In most previous works, the drug dipolar contribution was considered as a constant24,25 and therefore ∆Vdip d ∆(∆V)dip showed a linear evolution in terms of the amount of the associated drug. No linear evolution is observed in the present work. The uncharged form (D) appears more efficient than the charged one (DH+) in modifying the surface properties of the lipid monolayer. Figure 5 illustrates the obtained results with egg PA. The dipolar variation (∆(∆V)dip) observed at both pH 4.5 and 8 suggests a penetration of the drug into the monolayer and confirms the hypothesis of a "partition" model describing the Cpzlipid interaction. Identical data were obtained for the neutral lipids (results not shown). (c) Surface Pressure. Figure 6 (parts a and b) shows the evolution of ∆A/A0 in terms of the logarithm of the Cpz concentration at pH 4.5 and 8 for egg PC and egg PA monolayers at constant surface pressure (20 mN m-1). It is shown that a very low Cpz concentration is sufficient to induce a detectable ∆A variation. An important drug penetration into the hydrophobic region of the lipid layer can be suggested, in agreement with the “partition” model. At pH 4.5 the calculated KDH “average value” for Cpz concentration between 3 × 10-7 and 10-5 M, is 2 × 104 M-1 for the egg PC-Cpz interaction and 2 × 103 M-1 for the egg PA-Cpz interaction. These values are comparable to the results obtained by electrophoretic mobility measurements.
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Figure 6. Measured evolution of the relative area variation (circle and square symbols) of egg PC (a) (filled symbols) and egg PA (b) (open symbols) monolayers as a function of the logarithm of the Cpz concentration (T ) 25 °C; 10-1 M NaCl; surface pressure ) 20 mN m-1). The theoretical curves (solid and dotted lines) were calculated as follows: (curve 1) pH 8, (a) (egg PC) KD ) 2.5KDH ) 2 × 104 M-1 and AD/AL ) 1, (b) (egg PA) KD ) 2KDH ) 1.2 × 104 M-1 and AD/AL ) 1; (curve 2) pH 4.5, (a) (egg PC) KDH ) 8 × 103 M-1 and AD/AL ) 1, (b) (egg PA) KDH ) 6 × 103 M-1 and AD/AL ) 1; (curve 3) pH 8, (a) (egg PC) same conditions as for curve 1 except that AD/AL ) 1.15, (b) (egg PA) same conditions as for curve 1 except that AD/AL ) 0.85; (curve 4) pH 4.5, (a) (egg PC) same conditions as for curve 2 except that AD/AL ) 1.15, (b) (egg PA) same conditions as for curve 2 except that AD/AL ) 0.85.
Figure 5. Surface potential measurement (filled symbols) of egg PA monolayers at pH 4.5 (a) and 8 (b) as a function of the logarithm of the Cpz concentration (T ) 25 °C; 10-1 M NaCl; surface pressure ) 20 mN m-1): (1) experimental curve; (2) calculated evolution of ∆ψ0 (see text); (3) deduced evolution of ∆(∆V)dip (see text).
Conversely, these last values of KDH and KD were used to calculate a theoretical curve ∆A/A0 ) f(log(Dtot)∞,i). (Dtot)∞,i is the initial Cpz concentration in the bulk ((DH+)∞,i + (Dt)∞,i). Figure 6 (parts a and b) compares the evolution of such calculated curves with the experimental results (using the KDH and KD values reported in table 1) at pH 4.5 and 8 for egg PC and egg PA. The agreement is generally better at low than at high drug concentrations, especially if the hypothesis AD/AL ) 1 (in relation 7) is replaced by AD/AL < 1 (we tested the a value of 0.85) in the case of the Cpz-egg PA interaction or by AD/AL > 1 (we tested a value of 1.15) for the Cpz-egg PC interaction. However, the discrepancy between the calculated and
experimental ∆A/A0 values became more important in the case of the Cpz-egg PA interaction for Cpz concentrations higher than 3 × 10-6 mol/L. That will probably be correlated with the nonadditivity of the areas (relation 2) at high drug concentrations, for that mixture (chargecharge interactions). An interesting point to emphasize, concerning the comparison between the two techniques used here to determine the partition coefficient (microelectrophoretic mobilities measurements on liposomes and ∆A measurements at constant surface pressure on monolayers), is illustrated by Figure 1. In the first method (microelectrophoretic mobility, µ), the δ parameter is deduced from ψ0 (obtained from µ and the ζ potential), which is dependent on the area variation through an sh-1x/A relation (eqs 8 and 3 (or 4)). A relative error of 10% on the denominator does not significantly affect the δ determination in that case. In contrast, in the second method, the δ parameter is directly correlated to the experimental ∆A determination (eq 7) at constant surface pressure. In such conditions, the δ parameter is very more sensitive to the ∆A accuracy in the second method than in the first one. This factor must be taken into account to explain the differences observed here and for other studies using these techniques.
Interaction of Chlorpromazine with Phospholipids
Conclusions Microelectrophoresis was used here for the evaluation of the lipid-drug intrinsic association constant. It was found that the lipid-drug interaction can be satisfactorily described in terms of a simple “partition” model. The best fitting was for the partition coefficient, values between 6 × 103 and 2 × 104 M-1 for charged and uncharged Cpz in the case of the Cpz-egg PA and-egg PC interactions. The values were found between 2 × 103 and 4 × 103 M-1 for Cpz-DPPA and Cpz-DPPC and near 103 M-1 in the case of the Cpz-cardiolipin interaction (Table 1). Our values are, when earlier results exist, in agreement17 or lower13 than those determined by other authors. The lipidic fluidity was found to play a minor role in these interactions. The surface charge density plays a key role, increasing or decreasing the concentration of the charged form of Cpz near the surface, in relation with the sign of the resulting surface potential in relation 8. Similar intrinsic partition coefficient values were obtained both for the neutral lipids and the “corresponding” charged ones (compare egg PC with egg PA). The same observation was reported by Ohki39 for the interaction of other anesthetics with phospholipids. However, the interaction of Cpz with phospholipids is not purely electrostatic in nature. The unprotonated form of Cpz interacts with phospholipids with, at least, the same affinity. Surface pressure measurements on monolayers allow us to confirm the drug penetration into the lipid layer and the magnitude of the partition coefficient values obtained by microelectrophoretic mobility measurments on liposomes. Surface potential measurements show that the penetration of Cpz into the lipid layer induces an alteration of the dipolar contribution of the lipids, which would have as a consequence a modification of the permeability of lipids to cations. Since the high positive polarization potential (∆Vdip) of phospholipids can perhaps be correlated to the low permeability of lipid bilayers to cations,40 the decrease of ∆Vdip induced by Cpz could be responsible for a modification of the membrane permeability. In prelimiary experiments, we observed that Cpz led to a 4-fold increase in conductance of the asolectin membrane.
Langmuir, Vol. 13, No. 24, 1997 6539
Ka(w) ) (D)∞(H+)∞/(DH+)∞ ) 2.5 × 10-9 (pKa ) 8.6) (A1) (H+)0 ) (H+)∞e-ZFψ0/RT
(39) Ohki, S. Biochim. Biophys. Acta 1984, 777, 56-66. (40) Haydon, D. A.; Myers, V. B. Biochim. Biophys. Acta 1973, 307, 429-443.
+
(A2) (A3)
D In this model, Xabs (in relation 13)
(a) Neutral Lipids. (+) (in relation 11) are respectively given by δ1(1 + and Xabs δ1 + δ2 + δ′)-1 and δ2(1 + δ1 + δ2 + δ′)-1. The surface charge density (obtained from relations 16 and 8 for a given µ value) is equal to eδ2AL-1(1 + δ1 + δ2 + δ′)-1 (from relation 4). (Cl-)0 was calculated from relation 9. KCl is given by relation 5 (where δ ) δ2). A combination of these relations (including (A1)-(A3) allows us to calculate (DH+)∞ and (D)∞. Finally relation 15 allows us to calculate (Dtot)∞,i. D (+) and Xabs are (b) Cardiolipin. With this lipid, Xabs -1 respectively equal to δ1(1 + δ1 + δ2) and δ2(1 + δ1 + δ2)-1. The charge density is equal to -e(z - δ2)AL-1(1 + δ1 + δ2)-1 (from relation 3). AL is the area of one acidic site of cardiolipin (the lipid area divided by 2) and the two acidic sites of each cardiolipin molecule are considered to have the same pKa (fully ionized at pH 8). (Na+)0 was calculated from relation; 9 KNa is given by relation 6. A combination of these relations (including (A1)-(A3)) allows us to calculate (DH+)∞ and (D)∞. Finally, relation 15 (with δ′ ) 0) allows us to calculate (Dtot)∞,i. (c) Phosphatidic Acid (Egg PA and DPPA). In this case, two distinct acidic sites will be considered for each lipid in the liposome. The two “site constants” are defined41 as follows:
z1(H+)0 Ka(1) ) 1 - z1 - γ1 Ka(2) )
z2(H+)0 1 - z2 - γ2
where zi and γi are respectively the fraction of free negatively charged sites i and the fraction of negatively charged sites i associated with Na+ in the liposome. The first pKa was taken equal to 1.5 and the second equal to 6.42 For the association with Na+, we defined the following two constants:
KNa(1) )
Appendix We describe in this appendix the calculations allowing us to obtain the theoretical isotherm µ ) f log(Dtot)∞,i at pH 8 for given values of KD and KDH. (Dtot)∞,i is the total Chlorpromazine concentration in the aqueous phase. KNa and KCl were taken equal to 0.5 M-1. At this pH, the following equilibria relations will be taken into account (DH+(w) h D(w) + H+(w), where the subscript w refers to the drug dissociation in water):12
-ZFψ0/RT
(DH )0 ) (DH )∞e +
KNa(2) )
1 - z1 z1(Na+)0 1 - z2 z2(Na+)0
For simplicity, the value of KNa(2) was taken equal to KNa(1) ) 0.5 M-1. (Na+)0 was calculated from relation 9.The charge density is equal to -e(z1 + z2 - δ2)AL-1(1 + δ1 + δ2)-1. A combination of these relations (including (A1)-(A3)) allows us to calculate (DH+)∞ and (D)∞. Finally, relation 15 (with δ′ ) 0) allows us to calculate (Dtot) ∞,i. LA970203+ (41) Tanford, Ch. In Physical Chemistry of Macromolecules; J. Wiley: New York, 1967; pp 526-586. (42) Blume, A.; Tuchtenhagen, J. Biochemistry 1992, 31, 4636-4642.
