Langmuir 1996,11, 1134-1137
1134
Evaluation of Drug-Lipid Association Constants from Microelectrophoretic Mobility Measurements S. Banerjee,*lt J. Caspers,?M. Bennouna,*A. M. Sautereau,! J. F. Tocanne,! and J. M. Ruysschaertt Laboratoire de Chimie Physique des Macromolicules aux Interfaces, CP 20612, Universiti Libre de Bruxelles, Bd du Triomphe, B-1050 Bruxelles, Belgium, Dipartement de Biologie, Laboratoire de Biochimie et de Physiologie, Universitk Abdelmalek Essabdi, Faculti des Sciences de Tktouan, B.P. 2121, Titouan, Morocco, and Dkpartement “Glycoconjuguis et Biomembranes”, Laboratoire de Pharmacologie et de Toxicologie Fondamentale du CNRS, et Universiti Paul Sabatier, 118 Route de Narbonne, F-31062 Toulouse, France Received August 30, 1994. In Final Form: December 29, 1994@ Electrophoretic mobilities of negatively charged lipid vesicles were measured after incubation with cationic drugs (adriamycin,celiptium,ethidium bromide, tetracaine) at different drug concentrations. We show here that comparison of the measured electrophoretic mobility with adsorption isotherms allows calculation of the drug-bindingconstants over a large range of drug concentrations,whatever the structure of the drug, its topology into the lipid bilayer, or its aggregation state.
Introduction Model membranes have been extensively used to describe drug-lipid interactions in monolayer and in bilayer systems.’-16 Binding of cationic drugs to anionic lipids modifies the negative surface charge density. This change of the electrical potential profile in the vicinity of the membrane-water interface is accompanied by a modification of the ionic species surface concentrations (including H+)and of several membrane properties (ionic permeability, enzymatic activity).l7J8 Several cationic drugs as adriamycin, celiptium, ethidium bromide, and tetracai’ne have been reported to interact with anionic p h o s p h o l i p i d ~ . ~ - ~Although J~J~ this process is mainly electrostatic, it was recently suggested that binding of several charged drugs, including adriamycin and celipt Universite Libre de Bruxelles.
* Universite Abdelmalek Essaldi.
Universite Paul Sabatier. Abstract published inAdvanceACSAbstracts, March 15,1995. (1)El Mashak, E. M.; Tocanne, J. F. Eur. J . Biochem. 1980,105, 593-601. (2)Goormaghtigh, E.; Chatelain, P.; Caspers, J.;Ruysschaert, J.-M. Biochim. Biophys. Acta 1980,597,1-14. (3)Goormaghtigh, E.; Chatelain, P.; Caspers, J.;Ruysschaert, J.-M. Biochim. Pharmacol. 1980,29,3003-3010. (4)Goormaghtigh, E.; Caspers, J.; Ruysschaert, J.-M. J . Colloid Interface Sci. 1981,80,163-170. ( 5 ) Goormaghtigh, E.; Brasseur, R.; Vandenbranden, M.; Caspers, J.; Ruysschaert, J.-M. Bioelectrochem. Bioenerg. 1982,9,489-498. (6)Guilmin, T.; Goormaghtigh, E.; Brasseur, R.; Caspers, J.; Ruysschaert, J.-M. Biochim. Biophys. Acta 1982,685,169-176. (7)Terce, F.; Tocanne, J. F.; Laneelle, G. Biochem. Pharmacol. 1985, 32,2189-2194. (8)Terce, F.; Tocanne, J. F.; Laneelle, G. Eur. J.Biochim. 1983,133, 349-354. ( 9 ) Nicolay, K.; Timmers, R. J. M.; Spoelstra, E.; Van der Neut, R.; Fok, J. J.; Huigen, Y. M.; Verkleij, A. J.;de Kruijff, B. Biochim. Biophys. Acta 1984,778,359-371. (10)Sautereau, A. M.; Cros, S.;Tocanne, J. F. Biopharm.DrugDispos. 1986,7,310-325. (11)Sautereau, A. M.;Betermier, M.; Altibelli, A.; Tocanne, J. F. Biochim. Biophys. Acta 1989,978,276-282. (12)de Wolf, F. A.; Maliepaard, M.; Van Dorsten, F.; Berghuis, I.; Nicolay, K.; de Kruijff, B. Biochim. Biophys. Acta 1991,1096,67-80. (13)Ohki, S.Biochim. Biophys. Acta 1984,777,56-66. (14)Lee, A. G. Biochim. Biophys. Acta 1978,514,95-104. (15)Fernandez, M. S. Biochim. Biophys. Acta 1981,646,27-30. (16)Westmans, J.; Boulanger, Y.; Ehrenberg, A,; Smith, I. C. P. Biochim. Biophys. Acta 1982,685,315-328. (17)McLaughlin, S.In Current Topics in Membrane a n d Transport; Academic Press: New York, 1977;Vol. 9,pp 71-144. (18)Botte, P.; Symons, M.; Swysen, C.; Sybesma, C.; Lannoye, R.; Hurwitz, H.D.J.Electroanal. Chem. 1977,214,407-425. 5
@
tium, to lipidsl1J2 cannot be described satisfactorily in terms of a classical Langmuir adsorption isotherm mainly because of the cooperative nature of the drug-lipid interaction, related to the capacity of the drug to selfassociate or because of the penetration of the drug into the lipid bilayer.11J9s20 We describe here a theoretical approach which allows from a comparison of the electrophoretic mobility with adsorption models to calculate the drug-lipid association constant whatever the degree of drug-drug self-association or the mode of penetration of the drug into the lipid bilayer (in the vicinity ofthe lipid-water interface or inside the hydrophobic lipid core).
Material and Methods Cardiolipin (sodium salt, from bovine heart) and egg lecithin were purchased from Sigma Chemical Co. (St. Louis, MO). Adriamycin (doxorubicin hydrochloride) was provided by the National Cancer Institute. Ethidium bromide was from Merck. Celiptium was from Sanofi and tetracaine (hydrochloride) from Sigma. The purity of the phospholipids was checked by TLC on Silica gel plates (Merck, Darmstadt, Germany) using CHClfleOW7 M NH3 (230:90:15) (v/v/v) as solvent. No impurities could be detected using this procedure. Lipids were dissolved in chloroform and the solvent was removed, first under NZand then under vacuum. Multilamellar vesicles (MLV)were formed after buffer addition M, (MOPS, 3-(N-morpholino)propanesulfonicacid) pH 7.4; 10-1 M NaC1) to the lipid film and mechanical vortexing. The drug was added to the lipid film after liposome formation. Electrophoretic mobilities of MLV were measured in a Rank Brothers Mark I1 apparatus. Measurements were carried out in the stationary l e ~ e l ~ in l - a~ capillary ~ cylindric cell (length 8 cm) with an applied voltage of (19)Garnier-Suillerot, A.; Gattegno, L. Biochim. Biophys.Acta 1988, 936,50-60. (20)de Wolf, F. A. Biosci. Rep. 1991,11, 275-284. (21)James, A. M. In Surface a n d Colloid Science; Good, R. J., Stromberg, R. S., Eds.; Plenum Press: New York, 1978;Vol. 11, pp 121- 185. (22)Sherbet, G. V. In The Biological Characterization of the Cell Surface; Academic Press: New York, 1978;pp 36-53. (23)Eisenberg, M.; Gresfaldi, T.; Riccio, T.; McLaughlin, S. Biochemistry 1979,18,5213-5223.
0743-746319512411-1134$09.00/0 0 1995 American Chemical Society
Cationic Drug-Anionic Lipid Association Constants
Langmuir, Vol. 11, No. 4, 1995 1135
approximately 40 V (dc) at 25 “C. The sign of the applied electric field was changed alternatively and the migration rate of the particles was determined at least 10 times in each direction. The size of the MLV was determined by photon correlation spectroscopy (ionized Ar Laser, green region of the spectrum, intensity adjusted t o 3 x lo3photons s-l) in the Department of Colloid Chemistry (ULB, Brussels). The mean diameter of cardiolipin MLV was in the 450500 nm range (in lo-’ M NaCl solutions). As in our experimental conditions the Debye length is inferior to 3 nm and the diameter of the liposomes larger than 100 nm, the electrophoreticmobilityp can be related to the zeta potential ( E ) through the Helmholtz-Smoluchowski e q u a t i ~ n ~ l - ~ ~
(D+)..and (Na+)..are the equilibrium bulk concentrations of these species. If the drug-lipid interaction is considered as an ideal and localized a d s o r p t i ~ n , ~ a ~“Langmuir-Stern” ~~* (LS) isotherm describes the progressive covering of the lipid surface by the adsorbate.”,29 Lipid-Na+ ( K N ~ and ) lipiddrug (KD)“Langmuir-Stern” association constants can be written
and KD(LS) = P/.(D+)o
(7)
y is the fraction of accessible anionic lipid sites bound to Na+, /3 the fraction of accessible anionic lipid sites associated with D+,and z the fraction of free anionic lipid 77 is the viscosity of the aqueous phase, 5 the electrical sites (z = 1 - y in saline medium in the absence of drug zeta potential qJ at the hydrodynamic plane of shear, EO or other adsorbate, and z = 1 - y - /3 when D+ and Na+ the permittivity ofvacuum, and erthe relative permittivity are simultaneouslypresent in the medium). In this model, of the medium. N*/NL’(relation 3) is equal toz, the fraction of free anionic According to several a ~ t h o r and s ~in~ the ~ ~ experi~ ~ ~ ~ lipid head group at equilibrium. KNJLS) was calculated mental conditionshere described,the hydrodynamicplane from relations 1, 2, 3, 5, and 6 with y = 1 - z (drug not of shear is located at a distance of 0.2 nm from the charged added). In our experiments, Na+ is in large excess, surface of the vesicles. The +O value in the surface plane whereas for D+,the mass conservation implies: is related to the zeta potential through the GouyChapman theory (for more details, see McLaughlin17and (8) Winiski et a1.26). On the other hand, +O is related to the surface charge X is the mole fraction of anionic head groups in the den~ity’~,~~ liposomes, CLthe total concentration of accessible lipid head groups, and (D+)..,,ithe initial bulk concentration of the drug. From eqs 1to 8, KD(LS)can be calculated using the p experimental values corresponding to several (D+)+ concentrations. Conversely,for a value of&, the previous R is the gas constant, F the Faraday, T the absolute equations enable the theoretical curve p = f(log(D+),,i)to temperature, and C the ionic concentration. cr, the surface be calculated. charge density, is given by: However, it is an oversimplification to consider that drugs adsorb mainly at the air-water interfacb; most (3) drugs are amphiphilic and are inserted into the core of the lipid bilayer. If the drug penetrates into the hydrophobic core of the MLV, a partition equilibrium can be e is the absolute value of the electron charge, N* the defined resulting number of charged sites on the surface of the MLV at equilibrium, X the number of anionic lipid head groups in the outer layer (N’L)divided by the total number of lipid head groups in the outer layer (NL),and AL the D+water defines the drug at the lipid-water interface area of one of these head groups (0.65nm2). We assumed the drug within the hydrophobic core of the and D+MLV that drug adsorption does not modify significantly the AL bilayer. value. From eqs 2 and 3, N*/NL’ can be calculated for a In this model,the charge of the absorbed drug is localized given ~$0 value. The q0is calculated from the measured in the vicinity of the polar lipid head groups and modifies p value, for different bulk drug concentrations (D+)+ the surface charge density of the MLV.13,30-33An asThe D+ and Na+ equilibrium concentrations at the sociation constant can be defined interface, (D+)oand (Na+Io,are given byl1sz3
6 = PV/eoer
(1)
(4) and
RD(partition)= XDMLV/(~+),
(9)
K’Dis equal to KDU,, where uw is the molar volume of water andKD =%‘MLv/~Do;PMLv a n d P o are respectively the drug concentration (in mole fraction) in the MLV and in water, in the vicinity ofthe interface. (D+)ois evaluated
(24)Wiersema, P. H.; Loebb, A. L.; Overbeek, J. Th.G . J. Colloid Interface Sci. 1966,22,78-99. (25)McLaughlin,A.;Eng, W. K.; Vaio, G.; Wilson, T.; McLaughlin, S. J. Membr. Biol. 1983,76, 183-193. (26)Winiski, A. P.; Eisenberg, M.; Langner, M.; McLaughlin, S. Biochemistry 1988,27,386-392. (27)Aveyard, R.;Haydon, D. A. In A n Introduction to the Principles of Surface Chemistry; Cambridge University Press: Cambridge, 1973; pp 1-57.
(28)Habib, M. A.;Bockris, J. 0. M. In Comprehensive Treatise of Electrochemistry 1. The Double Layer; Bockris, J. 0. M., Conway, B. E., Yeager, E., Eds.; Plenum Press: New York, 1980;pp 135-219. (29)McLaughlin,S.;Harary, H. Biochemistry 1976,15,1941-1947. (30)Seelig, A.Biochim. Biophys. Acta 1987,899,196-204. (31)Kuchinka, E.; Seelig, J. Biochemistry 1989,28,4216-4221. (32)Seelig, A.;Allegrini, P. R.; Seelig, J. Biochim. Biophys. Acta 1988,939,267-276. (33)Seelig, A.;McDonald, P. Biochemistry 1989,28,2490-2496.
1136 Langmuir, Vol. 11, No. 4, 1995
Banerjee et al.
1
L
x.1
0-
x.
0.5
x:o.2
-5
I -5
-6
loglD+IO,i
-4
Figure 1. Measured electrophoreticmobilities (filledsymbols) of cardiolipin MLV (X= 1)and of cardiolipin-lecithin MLV (X = 0.5 and 0.2) as a function of the logarithm of the adriamycin M concentration. C = 10-I.M NaC1, T = 25 "C, CL= 5 x in MOPS M. The electrophoretic mobility (solid lines) was calculated by combining the Fowler-Stern adsorption isotherm and the partition model.
+
as described in eq 4. PMLV is equal to 6(6 11-l (6 is defined as nDMLV/nL,the number of moles of absorbed drugs divided by the number of moles of accessible lipid). Since more than one drug molecule can absorb per accessible lipid (6 > 11, electrophoretic mobilities can be positive. However, drug absorption may modify the liposome area,A.32 This contribution should be taken into account in relation 3. Assuming thatAL and&, the areas occupied per lipid and drug molecules, are not significantly different32
From relations 3 and 10 it becomes
o=---N*e -
A
-N*e ALNL(1 6)
+
(11)
-5
-6
loglD*lo,,l
-4
Figure 2. Measured electrophoreticmobilities(filledsymbols) of cardiolipin MLV (X= 1)and of cardiolipin-lecithin MLV (X = 0.2) as a function of the logarithm of the celiptium M concentration. C = 10-l M NaC1, T = 25 "C; CL= 5 x M. The electrophoretic mobility (solidline)was in MOPS calculatedby combiningthe Fowler-Stern adsorptionisotherm and the partition model.
X - 0.5
x = 0.2 -- 55
-1
Figure 3. Measured electrophoretic mobilities of cardiolipin MLV (X= 1)and of cardiolipin-lecithin MLV (filled symbols) as a function of the logarithm of the ethidium bromide M concentration. C = 10-1M NaC1; T = 25 "C, CL = 5 x in MOPS M. The electrophoretic mobility was calculated (solid line) from the Langmuir-Stern model.
On the other hand, N*/NLis equal to the difference between the molar ratios of free anionic head groups and absorbed cationic drugs. One can write
N*INL = ZX - 6
121
(12) /I
The Na+ adsorption is taken into account through relation 6. For D+, the mass conservation implies
From the relations 1, 2, 4, 5 , 6, 9, 11, 12, and 13 and a defined K'D(partition1 value, the theoretical curve p = fl(D+),,J can be calculated.
Results At high drug concentrations, the mobility of the anionic cardiolipin MLV becomes positive in the presence of adriamycin and celiptium (Figures 1and 2). Such positive values were not measured for ethidium bromide and tetracaine (Figures 3 and 4). Moreover, a drastic increase of electrophoretic mobility around M revealed ahighly cooperative binding of adriamycin and celiptium to cardiolipin. At low adriamycin and celiptium concentration, the electrophoretic mobility of liposomes made of cardiolipin and lecithin decreases (in absolute value) with the surface
-L
-3
/ I l l
-2
log10'1,,
Figure 4. Measured electrophoreticmobilities(filledsymbols) of cardiolipin MLV as a function of the logarithm of the tetracalne concentration. C = lo-' M NaC1, T = 25 "C, CL = 5x M in MOPS M. Comparison with the calculated curves: curve 1, Langmuir-Stern model KD(LS)= lo2 M-l; curve 1'; Langmuir-Stern model KD(LS)= lo3 M-l; curve 2, partition model K'D = 70 M-I. charge density in agreement with the Gouy-Chapman theory. The sequence is opposite at high drug concentrations (Figures 1and 2) and cannot be interpreted in terms of a classical Langmuir adsorption. This discrepancy between values of electrophoretic mobility at low and high drug concentration was not detected €or ethidium bromide (Figure 3) at least in the concentration range studied.
Cationic Drug-Anionic Lipid Association Constants These data clearly put forward that, except at low drug concentrations, a “Langmuir-Stern” adsorption model does not describe satisfactorily the adsorption of adriamycin and celiptium on anionic lipids. The KD(LS) (relations 1 to 8) adriamycin-cardiolipin association constants calculated from the p = fllog(D+),,i(Figure 1, X = 1)experimental curve were lo4M-l and 2 x lo5M-’ between (D+),,i = 3 x M and 3 x M. A valuable model should describe both the existence of positive electrophoretic mobilities of anionic lipids and the drastic increase of mobility observed at high drug concentrations (Figures 1 and 2, for X = 1). Positive mobilities mean that more than one positively charged drug binds to one lipid head group. These positive values can be interpreted in terms of an unspecific drug penetration into the lipid bilayer through a partition coefficient (relation 9). Abrupt increase of mobility around M drug concentration was mainly observed for drugs which selfassociate in solution around this concentration.2 The “Fowler-Guggenheim” model introduces in the Langmuir isotherm an exponential term characterizing the interaction between adsorbate^.^^ From the equilibrium drug concentration (D+)oat the interface, a “Fowler-Stern” (FS) association constant can be defined:
Langmuir, Vol. 11, No. 4,1995 1137 (KD(LS)= 2 x lo3M-l) (Figure 3). For tetracaine (Figure 4),6J3-1ethe “Langmuir-Stern” model fits the experimental data for drug concentrations lower than 3 x M. (The KD(LS) association constant (lo3 M-l) is in agreement with preceding monolayer evaluations6)but a partition model with a RD(partition) of 70 M-’ (Figure 4) describesthe experimental electrophoreticmobility curve over a larger range of concentration than the “LangmuirStern” model. This 70 M-l association constant is in agreement with previous determinati011.l~
Conclusion
The present work provides evidence that the interaction of cationic drugs that penetrates into the lipid bilayer (tetracaine) or adsorbs at the lipid-water interface (ethidium bromide) can be described in terms of a partition model or a “Langmuir-Stern” model. The environment oftetracaine in a lipid bilayer6has been shown to be mainly hydrophobic(dielectricconstant equal to 12). For ethidium bromide, a dielectric constant higher than 50 was rep ~ r t e d indicating ,~ that the drug binds mainly to the periphery of the vesicles, at the lipid-water interface.37 A “Fowler-Stern” model combined with a partition contribution describes quite satisfactorily the adsorption of drugs (adriamycin, celiptium) that s e l f - a s ~ o c i a t e . ~ ~ - ~ ~ Evaluation of a drug-lipid association constant at a unique drug concentrationcan be misleading and explains discrepancies mentioned in the literature. The evaluation U is the interaction energy between adsorbates and c the number of nearest neighbors in the adsorbed l a ~ e r . 2 ~ of the adriamycin-cardiolipin association constant illustrates this point quite convincingly. Most of the Through relation 4, relation 14 becomes reported association constant^^,^ were calculated from the Langmuir isotherm for an unique drug concentration and did not consider drug-drug interaction. We provided evidence that a “Langmuir-Stern” isotherm did not fit all the p = fl(D+).+) curve (data not shown) and therefore FVo is the electrostatic contribution and CUP describes that the association constant values will depend on the nonelectrostatic effects. drug concentration. We proposed here a strategy that A FS adsorption isotherm combined with the partition allows a satisfactory description of the cationic drugcoefficient model was shown to describe satisfactorily the anionic lipid interaction whatever the drug-drug selfadriamycin-cardiolipin interaction (KD’= 2 x lo3 M-l association, the structure and location of the drug into and KD(FS) = 2 x lo3 M-l, and cU/RT = -5 (cU= -12.5 the lipid bilayer (in the vicinity of the lipid-water interface kJlmol)) (Figure 1,X = 1)and the celiptium-cardiolipin or inside the lipid core) over a large range of drug interaction KD‘ = lo5 M-’ and KD(FS)= lo4 M-’ (CURT concentrations. Work is in progress in our laboratory to = -5.2) (Figure 2, X = 1) in the 10-6-10-4 M-l drug extend this approach to the analysis of peptide-lipid concentrations range. The competitive adsorption of Na+ interactions. was considered through a binding constant KN,(LS)of 0.5 M-1.23,34-36
In mixed MLV made of cardiolipin and lecithin (X = 0.5)and incubated with adriamycin,KD’andKD(FS)values equal to 2 x lo3M-’ andcUIRT = -3 (Figure 1)allow the experimental data to fit. For X = 0.2, C U R Tis equal to zero (Figure 1). We assumed that in this last case each anionic lipid is surrounded with neutral lipids. The “drug self-association”in the adsorbed layer is therefore strongly dependent upon the nature of the interface and, in the present case, upon the initial surface charge density. Comparison with the adsorption models indicates that drug-drug interactions are not significantly involved in the ethidium bromide and tetracaihe-cationic lipids interactions. A “Langmuir-Stern” model (relations 2 to 8) was shown to describe quite satisfactorilythe measured electropheretic mobility of cardiolipin MLV incubated with ethidium bromide in the 10-6-10-4 concentration range (34)Kurland, R.; Newton, A.; Nir, S.; Papahadjopoulos, D. Biochim. Biophys. Acta 1979,555,137-147. (35)Lakhdar-Ghazal,F.;Tocanne,J. F. Biochim.Biophys.Acta 1988, 945. ._ _ 19-27. -(36)Ermakov, Y.A. Biochim. Biophys. Acta 1990,1023,91-97. I
Acknowledgment. We thank Dr. F. Dumont and B. Meere from the Department of Colloid Chemistry (ULB) for the photon correlation spectroscopymeasurements and interpretation, Drs. A. Lopez (Department “Glycoconjugu6s et Biomembranes”,CNRS-Toulouse)and F. A. de Wolf (Institute of Molecular Biology and Medical Biotechnology, State University-Utrecht) for helpful discussions. We thank Ph. Duquenoy for the preparation of the manuscript. LA940681t (37)Shinitzky, M. Isr. J. Chem. 1974,12,879-890. (38)Huart, P.; Brasseur, R.; Goormaghtigh, E.; Ruysschaert, J. M. Biochim. Biophys. Acta 1984,799, 199-201. (39)Brasseur, R.; Ruysschaert, J. M. Biochim. Biophys. Acta 1986, 238, 1-11. (40)Barthelemy-Clavey, V.; Maurizot, J. C.; Dimicoli, J. L.; Sicard, P. FEES Lett. 1974,46, 5-10. (41)Menozzi, L.; Valentini, M.; Vannini, E.; Arcamone, F. J.Pharm. Sci. 1984,73,766-770. (42)Chaires, J. B.; Dattagupta, N.; Crothers, D. M. Biochemistry 1982,21,3927-3931.
