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Lipid—drug electrostatic interactions in model membranes
Lipid-Drug Electrostatic Interactions in Model Membranes E. GOORMAGHTIGH, J. CASPERS, AND J. M. RUYSSCHAERT Laboratoire de Chimie Physique des MacromolOcules aux Interfaces, Universitd Libre de Bruxelles, Campus Plaine, C.P. 206/2, Bruxelles 1050, Belgium Received June 9, 1980; accepted August 6, 1980 Model membranes have been used to describe the interaction between charged drugs (acridine orange, ethidium bromide) and lipids. A new method of evaluation of the surface charge density is described. It allows, from surface potential measurements, the evaluation of the drug-lipid association constant. The low perturbation of the monolayer dipole orientation after complexation is correlated to the low penetration of the drug into the lipid bilayer of liposomes as shown by a fluorescence titration technique. INTRODUCTION
lipid molecules was associated with a hydrophobic contribution due to the drug penetration into the bilayer. Fluorescence spectra of the drug in the presence of liposomes of different composition allowed confirmation of this statement.
Most drugs encounter biological membranes at some points in their interaction with cells, prior to reaching their target. Consequently, it is essential to understand the mechanism of the drug-membrane interaction. However, the complex composition of membranes does not allow a simple analysis of this interaction. A possible way to approach this problem is to use model membranes for testing more specifically the lipid contribution in the drug-membrane interaction (1). Drug adsorption on lipid layers can be expected to have two effects. First, it will modify the lipid matrix fluidity. Second, it will change the membrane charge (2, 3). It is precisely the purpose of the present work to propose a new method of evaluation of the surface charge density in model membranes. The lipid was spread at the air-water interface and the drugs injected into the aqueous subphase. Ethidium bromide and acridine orange were chosen as water-soluble drugs because of their effects on membrane sites (4-6). Surface potential measurements allowed an immediate determination of the drug-lipid association constant. A possible change in the dipole moment orientation of
MATERIALS AND METHODS
DL-t~-Dipalmitoyl phosphatidylcholine (DPPC), egg phosphatidylcholine (Egg PC), and cardiolipin (CL) were purchased from Sigma Chemical Company. Acridine orange and ethidium bromide were Merck products. All chemicals were of analytical grade and water was tridistilled. Buffered solutions (Tris-HC1 10-4 M, pH 7.4, NaC1 10-3 M) were used to prepare the subphase. Phospholipids were spread at the air-water interface from a chloroform solution using an Agla Microlitre syringe. All experiments were carried out at 25°C. To prepare small unilamellar liposomes, lipids were dissolved in CHC13 in a spherical flask. The solution was evaporated to dryness and further dried under vacuum. Multilamellar liposomes were obtained by mechanical stirring (vortex mixer) of a lipid film in buffer. The temperature was maintained above the corresponding lipid phase 163 0021-9797/81/030163-08502.00/0
Journal of Colloid and Interface Science, Vol. 80, No. 1, March 1981
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
164
GOORMAGHTIGH, CASPERS, AND RUYSSCHAERT 6(,'@) (mY)
n=20
reaction can be written: D + + P - ~ DP
n=lO n=8
[1]
with an association constant (2):
n=6 6=4
[DP]
K -
-
cr0 - o"
1
o"
[D+]s
[D+]s[P -] n=2
0
I 4.1
I &3
I I 12 16 x lO-3(chorge/~ 2 )
FIG. 1. Theoretical evolution of the surface potential increase (A(At#))as a function of the charge density owhen the concentration of monovalent salt increases from C1 = I0-a M to C2 = n x C1. n values are reported on each curve.
transition t e m p e r a t u r e Tc (7). Small unilamellar liposomes were obtained by sonication of the multilamellar liposome dispersion (Branson Sonifier B12). The t e m p e r a t u r e was kept a b o v e Tc during sonication. The vibration electrode technique was used to measure the surface potential (8, 9). Before injection of drug or salt solutions into the subphase, an equivalent a m o u n t of buffer was withdrawn f r o m the subphase to maintain constant the distance b e t w e e n the vibrating plate and the monolayer. Surface pressure m e a s u r e m e n t s were made on a Cahn R.G. electrobalance using the Wilhelmy method (10). A platinum plate was used. F l u o r e s c e n c e spectra were recorded with a differential spectrofluorimeter F O C C I MK1. The theoretical curves (Figs. 1, 2, 6, 7) have been obtained on a CDC 6600 computer.
[2]
where or and tr0 are, respectively, the surface charge density after and before complexation and [D+]s is the m o l a r concentration o f D ÷ at the interface. [D+]~ is related to the bulk concentration through a Boltzmann distribution: [D+]s = [D+]o~ exp(-e~/kT)
[3]
where e is the electronic charge, k the Boltzmann constant, and ¢ the surface potential after complexation. F r o m a general point of view, the electrostatic potential tk (mV) associated to the lipid m o n o l a y e r is described by the G o u y - C h a p m a n theory. At 25°C (10, 11): 134 o= 50.4 s h - ' - -
[4]
6112
where o- is the surface charge density in charge/,&, 2 and C is the molar salt concentration in the subphase (monovalent ions). Classically, the lipid m o n o l a y e r is spread at the a i r - w a t e r interface. After injection of the drug into the subphase, the evolution of the surface potential is followed until
z~(A¢) I (mY)
1) C1 =10 -4 M/L 2) C1 =10~3 3) C 1 =10 -2 4) C1=510 -2
RESULTS
Surface Potential Measurements The knowledge of tr, the surface charge density after complexation and of o'0, the surface charge density before complexation allows evaluation of the c o m p l e x a t i o n constant b e t w e e n a lipidic anionic site (P-) and a positively charged drug (D+). The Journal of Colloid and Interface Science, Vol. 80, N o . 1, M a r c h 1981
OI ! 0
4!1
8!3
112
3 cr ll6 ~10- (chorgel~, 2)
Fie. 2. Theoretical evolution of the surface potential incrase (A(At#)) as a function of the charge density tr when the concentration of monovalent salt is increased 10 times from various initial salt concentrations C1.
INTERACTIONS
equilibrium is reached. The increase in surface potential AV (mV) can be described by: AV = A4' +
127rA/zz
A
[51
where A• is the change in the electrostatic potentialexpressedin mV, A~zis the change of the vertical componentof the total dipole momentexpressed in mDebye, and A is the area occupied per molecule in A2/molecule. To eliminate the problem of the evaluation of the change in the dipolar contribution after the drug-lipid complex formation, we developed a new technique based on the Gouy-Chapman theory allowing the determination of 0.. It consists in varying the salt concentration by injecting a saturated solution of NaNO3 in the subphase. Since evidence has been reported that no modification of the dipolar orientation of the monolayer will occur (11, 12), the increase of the surface potential A(A~) after salt injection can be described from the Gouy-Chapman theory. At 25°C, A(Atb) = 50.4 x (sh_l 134o-
(C2),/2
sh_l 1340-)
(C1---~,I-~
165
IN MODEL MEMBRANES
[61
where C1 and C2 are, respectively, the concentration in monovalent ions in the subphase (M) before and after NaNOz injection and 0- is the surface charge density (charge/A2). Figures 1 and 2 illustrate relation [6] for (1) different factors n of salt concentration increase (C2 = n .C,). C1 was chosen equal to 10-3 M; and (2) different C, values, n was chosen equal to 10. It can be seen that the choice of the A(A~b) value, C, and n will be of prime importance for the precision obtained on 0-. For reasons discussed in the appendix, we have chosen A ( A ~ ) e x t = 25 mV, n -- 10, and C~ = 10-3 M. The validity of relation [6] was tested in charged lipid monolayers. Monolayers of well-defined charge density 0-o were obtained by spreading mixtures of a neutral lipid (DPPC) and of a negatively charged one
TABLEI C o m p a r i s o n b e t w e e n T h e o r e t i c a l ( E q . [6]) a n d E x p e r i m e n t a l I n c r e a s e o f S u r f a c e P o t e n t i a l A(Aq0
as a Function of the Surface Charge Density o- W h e n the N a N O 3 Concentration Increases from 10-3 M to 10 -2 M a Proportion CL/DPPC (w/w)
o- 0 (charge//~)
1:0 4:1 3:2 2:3 1:4 3:17 1:9 1:19 1:32 1:99 0:1
1.6 1.3 1.0 6.7 3.3 2.5 1.6 8.3 5.0 1.6
z × × x × × × × )< × 0
10 -2 10 -z 10 -2 10 -3 10 -3 10 -3 10 -3 10 -4 10 -4 10 -4
A(A~)th~or (mY) 58.0 58.0 58.0 57.9 57.5 56.5 55.9 51.0 43.7 22.0 0
A(Aq~)e~ (mV)
53 +_ 5 53 -+ 5 53 -+ 5 53 +- 5 53 -+ 5 53 -+ 5 52 -+ 5 48 +_ 5 40 -+ 5 25 -+ 5 0-+ 5
Subphase was a buffered solution (Tris-HC1, p H = 7.4, 10 -4 M). T = 25°C. T h e s u r f a c e p r e s s u r e o f the m o n o l a y e r w a s 25 m N / m .
(cardiolipin) in different proportions. The surface pressure was kept equal to 25 mN/m. Seventy microliters of a saturated NaNO3 solution was injected in 80 ml of buffered subphase ([NaNO3] = 10-3 M) at 25°C under stirring to reach a 10-2 M NaNO3 concentration. Surface potential increase was recorded as a function of time. Surface potential values at equilibrium are reported in Table I and compared with the theoretical values calculated from Eq. [6]. The good agreement between experimental and theoretical values demonstrates that the surface potential change observed after the salt concentration increase can be described in terms of an electrostatic interaction (Gouy-Chapman theory) without any contribution of the monolayer dipole orientation. This conclusion allows us to present this method as a tool to calculate an unknown charge density after complexation. We have then applied this method to calculate the association constant between positively charged drugs (acridine orange, ethidium bromide) and negatively charged Journal of Colloid and Interface Science, Vol. 80, No. 1, March 1981
166 t, (~¢) (mY) ~0
GOORMAGHTIGH,
//
CASPERS, ~(chorge/A2) ×1~4
3~
17 08 0 q
1,G
aoxlO"2 chorge/~ 2
FIG. 3. Increase of the surface potential A(Atk) of the complexed monolayer after injection of NaNO3 in the subphase as a function of the surface charge density before complexation tr0. The NaNOa concentration was modified from I0 -a to 10-z M. The right ordinate gives the surface charge density tr equivalent to the increase of the surface potential (Eq. [6]). Subphase was a buffered solution (Tris-HCl, pH 7.4, 10-4 M, NaNO3 10-3 M, T = 25°C. The surface pressure was 25 mN/m.
lipids (cardiolipin). Monolayers of welldefined charge density o-0 were obtained by spreading mixture of DPPC and cardiolipin in different proportions (see Table I). Adsorption experiments on a pure DPPC monolayer were carried out for each drug at the same concentration as in Fig. 3. Surface potential and surface pressure measurements demonstrated that no adsorption occurred (AV = 0 --- 5 mV; A I I = 0 -+ 0.5 mN/m). For this reason, DPPC was used as an inert lipid. For each o-0 value, i.e., for each monolayer composition, a known amount of the drug was injected into the subphase. At equilibrium, the salt concentration of the subphase was modified from 10-a to 10-2 M by injection of NaNOa. The increase of the
AND
RUYSSCHAERT
surface potential is reported for each o-0value in Fig. 3. The left ordinate gives the experimental data A(Ato) and the right ordinate the charge density o- obtained from A(Ato) by Eq. [6]. For reasons discussed in the appendix, for each curve, o-0 was determined at A(Ato)ext = 25 mV correspond ing to cr = 1.7 10-4 charge/]k ~. From o- and o-0, tO and [D+]3, the association constant K can be obtained (Eqs. [2] to [4]). K values are reported in Table II. Comparison between AV (Eq. [5]) and AtO (Table III) allows an evaluation of the dipolar term change. AtO is calculated from Eq. [7]: AtO = 50.4 × (sh_ a 134 tr (CO ~12
sh_ 1 134 ~__01 (C1)1/2 1.
[7]
Generally, a low or near zero A/~z value is associated with a low penetration of the drug into the lipophilic region of the model membrane. In the next part of this work, we will try to confirm this conclusion by fluorescence measurements. Fluorescence Measurements The maximum emission wavelength of the is strongly dependent on the dielectric constant of the medium surrounding the dye (Fig. 4). Consequently, fluorescence spectra can be used as a probe of the dielectric constant of the medium in which the dye is embedded (13). In fact, the dielectric constant profile in a phospholipidic bilayer has been evaluated by Shinitzky (14). The position of the maximum emission has f l u o r e s c e n c e hma x
TABLE II Association Constants of the Cardiolipin-Positively Charged Drug Complexa
Acridine orange Ethidium bromide
ED+]~
[D+],
o'o
o"
K (Eq. [21)
(mole/liter)
(mole/liter)
(charge/A s)
(charge/A s)
(liter/mole)
10 -5 2 x 10 -5
2 . 6 × 10 -5 5 . 2 x 10 -5
1.6 x 10 -2 1.4 x 10 -2
1.7 x 10 -4 1.7 x 10 -4
4 + 2 x l0 g 2 + I x 10 6
Subphase was a buffered solution (Tris-HC1, pH = 7.4, 10-4 M, NaNO3 10-8 M). T = 25°C. The surface pressure was 25 mN/m. Journal of Colloid and b~terface Science, Vol. 80, No. 1, March 1981
INTERACTIONS IN MODEL MEMBRANES T A B L E III
XMAX
Increase of the Surface Potential of the CardiolipinDPPC Monolayer after Injection of the Drug into the Aqueous Subphase a
Acridine orange Ethidium bromide
167
( AV)exl)erimental
( Al~)theo~tteal
(mY)
(mY)
230 _+ 30 200 _+ 13
203 203
ACRIDINE ORANGE BgS f
590
. . . .
#ord~ot,#,,n
EGG PC DPPC
Do
E~THI DIUM BROMIDE a Comparison between experimental values and theoretical predictions calculated from equation 7. Subphase was a buffered solution (Tris-HC1, pH = 7.4, 10 -4 M, NaNO3 10 -3 M). T = 25°C. The surface pressure of the monolayer was 25 mN/m.
been measured as a function of the dielectric constant. Media of different dielectric constants were used: buffer (• = 80), buffermethanol 2/1 v/v (e = 66), buffer-methanol 1/1 v/v (• = 58), methanol (• = 33), ethanol (~ = 24) (15). Figure 5 shows the position of the maximum emission wavelength as a function of the liposomes concentration. If Xmax does not depend on the liposome concentration, one can conclude that no more free drug is present in the solution. At this
)'max Eth. br
mo
EGGPC
ao -- c,c DPPcC,C,
so
cardiotipin 515
0
I 0.1
L 02
I 03
] I 04 [LIPIDS] rnglml
FIG. 5. Fluorescence titration of and ethidium bromide 5 × 10 -6 M liposomes of DPPC, egg PC, and buffered solution (Tris-HC1, pH NaNO3 10 -3 M). T = 25°C.
acridine orange by unilamellar cardiolipin in a = 7.4, 1 0 - 4 M ,
point • is deduced from hmax. The values of • are reported in Table IV. It must be concluded that these two dyes certainly do not penetrate into the hydrophobic region of the bilayer. This result is in good agreement with the surface potential predictions and with Massari's work (16, 17).
kmQx
Acr.or.
~
cridine orQnge
610 ---~,
605
520 ~
520
-4¢--
6
0
DISCUSSION
The surface potential approach is particularly well adapted to the study of the electrostatic interactions between soluble drugs and lipids. However, the value of the surface charge density before and after the drug injection cannot be immediately calculated.
0
T A B L E IV
515 ~
595
Dielectric Constants of the Lipidic Medium Surrounding Each Drug in Unilamellar L i p o s o m e s a Dielectric constant
Ethidium bromide SlO
20
40
60
590 80
E,
FIG. 4. Xmax (nm) of acridine orange and ethidium bromide as a function of the dielectric constant of the solvent. The concentration of dye was 10 -6 M. T = 25°C.
Ethidium bromide Acridine orange
Cardiolipin
DPPC
Egg PC
75 70
80 80
80 80
Buffered solution (Tris-HC1, pH = 7.4, 10 -4 M, NaNO8 10 -a M). T = 25°C. Journal of Colloid and Interface Science, Vol. 80, No. 1, March 1981
168
GOORMAGHTIGH,
CASPERS, AND RUYSSCHAERT
Indeed, surface potential measurements depend both on the surface charge density change (described by the Gouy-Chapman theory) and on the total dipole orientation modification (in fact, its vertical projection). In order to overcome this difficulty, two possible ways have been described. The most classical way is to record the surface potential increase due to the drug injection as a function of its bulk concentration. This method allows us to define a bulk concentration corresponding to a half maximum effect. From this concentration, an association constant can be calculated. This method is however imprecise since the concentration is often difficult to define, particularly when the water solubility of the drug is a limiting factor. Moreover, one assumes that the charge and dipole effects do not depend on the drug concentration. From a molecular point of view, it seems difficult to accept this statement since each drug molecule will modify the state of the lipid layer (surface charge density, packing, accessibility to the surface). In a preceding paper, we proposed another method to dissociate the electrostatic term from the dipolar term (2). It was based on surface radioactivity measurements (18) allowing determination of the equilibrium drug surface concentration. However, the availability and the stability of radioactive drugs are limiting factors. In the present paper, we proposed a simple technique to evaluate independently the charge and the dipolar effect. It is based on a modification of the subphase ionic concentration. The first part of this work consisted of the verification of the theoretical predictions. The good agreement between theoretical and experimental data demonstrated that the surface charge density of a lipid monolayer can be evaluated from the surface potential increase. In fact, the Gouy-Chapman theory is based on the hypothesis that the charge is uniformly spread over the surface and that the counterions behave as point charges. Deviations from the theoretical valJournal of Colloid and Interface Science, Vol. 80, No. 1, March 1981
ues of the surface potential appear mainly for fully ionized films (1 charge/60 A 2) and high salt concentration (up to 0.1 M) (19). With the method proposed here, the measurements are carried out at (r = 1.7 × 10-4 charge/Ae (or 1 charge/6000 A 2) and at 10-2 M salt concentration. This method has been applied to evaluate the surface charge density of monolayers after complexation by acridine orange and ethidium bromide. Our results demonstrated the existence of a high drug-lipid electrostatic association constant and a low penetration of the drugs into the lipid monolayers. These results are in good agreement with our fluorescence measurements and with Massari's work (16, 17). From a general point of view, we must however point out some limitations of the proposed method: (1) The drug-monolayer equilibrium must not be modified by the ionic concentration increase. This point could with difficulty be assumed for low association constants. However, with most of the biologically specific drugs, the high association constant allows elimination of this eventuality. (2) Some reorganization of the interface structure (including the drug) could occur due to the increase in salt concentration. Indeed, hydrophobic effects can be sensitive to salt addition. This eventuality would be detected from the shape of the A(A~) = f(o'0) curve. Indeed, an additional dipolar term will affect the A(A~k) value at the plateau (o-> 1.7 × 10-3) (Table 1). Moreover, the plateau will deviate from horizontality since the additional term depends on the number of drug molecules complexed at the interface and is therefore a function of o'. (3) Another difficulty could arise from the choice of the inert lipid. Lipids with highly saturated long chains seem however to prevent any nonspecific drug adsorption. To conclude, the new technique presented here allows a direct estimation of the electrostatic interactions between drugs and lipids but remains restricted to specific inter-
169
I N T E R A C T I O N S IN M O D E L M E M B R A N E S
actions. A precise evaluation o f the association constants of antimitotic-lipid-specific complex is now in progress in our laboratory.
60
APPENDIX
The purpose of this appendix is to define the experimental parameters n, C1, and ~(h0)~x t in order to obtain the best precision for or. However, other conditions such as the nonperturbation of the equilibrium by the increase of salt concentration will be taken into account. Figures 6 and 7 give in the ordinate the relative error on the extrapolated o- value as a function of these three parameters, if we consider an absolute error of 5 mV on the experimental data (A(&~b)). F r o m Fig. 6, giving the error as a function of C~ and n, it appears that the initial salt concentration has only a very weak effect on the precision, whereas the best precision is obtained with high n values. F r o m another Point of view, n and C~ must be chosen low enough in order to avoid a massive introduction Of salt into the subphase since this could perturbate the equilibrium described in Eq. [1]. For each experiment, we have controlled that equilibrium was maintained after salt injection. Surface potential and surface pressure were recorded simultaneously. Indeed, drug adsorption on the lipid monolayer will induce an increase of the surface pressure whereas an eventual desorption of the drug will de-
GO
-
-/
•'~/~/ ' '
/
'
I O0
4020
~ ...........
40
~000
10
20
40
60 n
FIG. 7. Calculated relative error for ~r as a function of the salt concentration increase term n and of the A(At0)ext value. An absolute error of 5 mV has been considered for A(AtO). The initial salt concentration has been chosen equal to i0 -~ M. If n = I0 and A(St0)ext = 25 mV, the percentage of error on or is 28.1%.
crease the surface pressure. For acridine orange and ethidium bromide, no modification of the surface pressure was observed after salt injection. This means that the equilibrium conditions were maintained during the experiments. For these reasons, we have chosen the following experimental conditions:C~ = 10-z M andn = 10. F r o m Fig. 7, giving the relative error on cr as a function of n and A(AqJ)ext, it appears that the error will be minimum if h(AqJ)ext = 25 mV when n - - 1 0 a n d C l = 10- s M . ACKNOWLEDGMENT One of us (E.G.) thanks I.R.S.I.A. (Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture) for financial assistance.
ERROR o n o
40
(*
40 20
•
REFERENCES
20
'
-7 1
-3
5
l o g C1
FIG. 6. Calculated relative error for cr as a function of the salt concentration increase term n and of the initial salt concentration ~C!- o- is obtained at A(~q~)ext = 25 mV. If C~ = 10-3 M and n = 10, the percentage of error on cr is 28.1%.
1. Tritton, T. R., Murphree, S. A., and Sartorelli, A. C., Biochem. Pharmacol. 26, 2319 (1977). 2. Goormaghtigh, E., Chatelain, P., Caspers, J., and Ruysschaert, J. M., Biochim. Biophys. Acta 597, 1 (1980). 3. Lee, A. G., Biochim. Biophys. Acta 514, 95 (1978). 4. Gittler, G., Rubalcava, B., and Coswell, A., Biochim. Biophys. Acta 197, 419 (1969). 5. Azzi, A., and Santato, M., Biochem. Biophys. Res. Commun. 44, 211 (1971). 6. Fico, R. M., Chen, T. K., and Canellakis, E. S., Science 198, 53 (1977). Journal of Colloid and Interface Science,
Vol. 80, N o . 1, M a r c h 1981
170
GOORMAGHTIGH, CASPERS, AND RUYSSCHAERT
7. Ruysschaert, J. M., Tenenbaum, A., Berliner, C., and Delmelle, M., FEBS Lett. 81, 406 (1977). 8. Caspers, J., Landuyt-Caufriez, M., Deleers, M., and Ruysschaert, J. M., Biochim. Biophys. Acta 554, 23 (1979). 9. Caspers, J., Berliner, C., Ruysschaert, J. M., and Jaff6, J.,J. Colloid lnterface Sci. 49, 433 (1974). 10. Gaines, G. L., in "Insoluble Monolayers at LiquidGas Interfaces." Wiley-Interscience, New York, 1966. 11. Davies, J. T., and Rideal, E. K., in "Interracial Phenomena," 2nd ed., p. 75. Academic Press, New York, 1963. 12. MacDonald, R. C., and Bangham, A. D.,J. Membrane Biol. 7, 29 (1972). 13. Goldman, R., Fachinetti, T., Bach, D., Raz, A.,
Journal of Colloid and Interface Science, Vol.80, No. 1, March1981
14. 15. 16. 17. 18.
19.
and Shinitzky, M., Biochim. Biophys. Acta 512, 254 (1978). Shinitzky, M., lsr. J. Chem. 12, 879 (1974). Turner, J., and Brand, P., Biochemistry 7, 512 (1968). Massari, S., and Pascolini, D., Biochemistry 16, 1189 (1977). Massari, S., Pascolini, D., and Gradenigo, G., Biochemistry 17, 4465 (1978). Kummer, J., Ruysschaert, J. M., and Jaff6, J., Berichte yon VI. Internationalen Kongress fiir Grenzfl~ichenaktive Stoffe, Band II, 1,285. Carl Hauser Verlag, Miinchen, 1973. Hauser, H., Drake, A., and Phillips, M. C., Eur. J. Biochem. 62, 335 (1976).
lipid molecules was associated with a hydrophobic contribution due to the drug penetration into the bilayer. Fluorescence spectra of the drug in the presence of liposomes of different composition allowed confirmation of this statement.
Most drugs encounter biological membranes at some points in their interaction with cells, prior to reaching their target. Consequently, it is essential to understand the mechanism of the drug-membrane interaction. However, the complex composition of membranes does not allow a simple analysis of this interaction. A possible way to approach this problem is to use model membranes for testing more specifically the lipid contribution in the drug-membrane interaction (1). Drug adsorption on lipid layers can be expected to have two effects. First, it will modify the lipid matrix fluidity. Second, it will change the membrane charge (2, 3). It is precisely the purpose of the present work to propose a new method of evaluation of the surface charge density in model membranes. The lipid was spread at the air-water interface and the drugs injected into the aqueous subphase. Ethidium bromide and acridine orange were chosen as water-soluble drugs because of their effects on membrane sites (4-6). Surface potential measurements allowed an immediate determination of the drug-lipid association constant. A possible change in the dipole moment orientation of
MATERIALS AND METHODS
DL-t~-Dipalmitoyl phosphatidylcholine (DPPC), egg phosphatidylcholine (Egg PC), and cardiolipin (CL) were purchased from Sigma Chemical Company. Acridine orange and ethidium bromide were Merck products. All chemicals were of analytical grade and water was tridistilled. Buffered solutions (Tris-HC1 10-4 M, pH 7.4, NaC1 10-3 M) were used to prepare the subphase. Phospholipids were spread at the air-water interface from a chloroform solution using an Agla Microlitre syringe. All experiments were carried out at 25°C. To prepare small unilamellar liposomes, lipids were dissolved in CHC13 in a spherical flask. The solution was evaporated to dryness and further dried under vacuum. Multilamellar liposomes were obtained by mechanical stirring (vortex mixer) of a lipid film in buffer. The temperature was maintained above the corresponding lipid phase 163 0021-9797/81/030163-08502.00/0
Journal of Colloid and Interface Science, Vol. 80, No. 1, March 1981
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
164
GOORMAGHTIGH, CASPERS, AND RUYSSCHAERT 6(,'@) (mY)
n=20
reaction can be written: D + + P - ~ DP
n=lO n=8
[1]
with an association constant (2):
n=6 6=4
[DP]
K -
-
cr0 - o"
1
o"
[D+]s
[D+]s[P -] n=2
0
I 4.1
I &3
I I 12 16 x lO-3(chorge/~ 2 )
FIG. 1. Theoretical evolution of the surface potential increase (A(At#))as a function of the charge density owhen the concentration of monovalent salt increases from C1 = I0-a M to C2 = n x C1. n values are reported on each curve.
transition t e m p e r a t u r e Tc (7). Small unilamellar liposomes were obtained by sonication of the multilamellar liposome dispersion (Branson Sonifier B12). The t e m p e r a t u r e was kept a b o v e Tc during sonication. The vibration electrode technique was used to measure the surface potential (8, 9). Before injection of drug or salt solutions into the subphase, an equivalent a m o u n t of buffer was withdrawn f r o m the subphase to maintain constant the distance b e t w e e n the vibrating plate and the monolayer. Surface pressure m e a s u r e m e n t s were made on a Cahn R.G. electrobalance using the Wilhelmy method (10). A platinum plate was used. F l u o r e s c e n c e spectra were recorded with a differential spectrofluorimeter F O C C I MK1. The theoretical curves (Figs. 1, 2, 6, 7) have been obtained on a CDC 6600 computer.
[2]
where or and tr0 are, respectively, the surface charge density after and before complexation and [D+]s is the m o l a r concentration o f D ÷ at the interface. [D+]~ is related to the bulk concentration through a Boltzmann distribution: [D+]s = [D+]o~ exp(-e~/kT)
[3]
where e is the electronic charge, k the Boltzmann constant, and ¢ the surface potential after complexation. F r o m a general point of view, the electrostatic potential tk (mV) associated to the lipid m o n o l a y e r is described by the G o u y - C h a p m a n theory. At 25°C (10, 11): 134 o= 50.4 s h - ' - -
[4]
6112
where o- is the surface charge density in charge/,&, 2 and C is the molar salt concentration in the subphase (monovalent ions). Classically, the lipid m o n o l a y e r is spread at the a i r - w a t e r interface. After injection of the drug into the subphase, the evolution of the surface potential is followed until
z~(A¢) I (mY)
1) C1 =10 -4 M/L 2) C1 =10~3 3) C 1 =10 -2 4) C1=510 -2
RESULTS
Surface Potential Measurements The knowledge of tr, the surface charge density after complexation and of o'0, the surface charge density before complexation allows evaluation of the c o m p l e x a t i o n constant b e t w e e n a lipidic anionic site (P-) and a positively charged drug (D+). The Journal of Colloid and Interface Science, Vol. 80, N o . 1, M a r c h 1981
OI ! 0
4!1
8!3
112
3 cr ll6 ~10- (chorgel~, 2)
Fie. 2. Theoretical evolution of the surface potential incrase (A(At#)) as a function of the charge density tr when the concentration of monovalent salt is increased 10 times from various initial salt concentrations C1.
INTERACTIONS
equilibrium is reached. The increase in surface potential AV (mV) can be described by: AV = A4' +
127rA/zz
A
[51
where A• is the change in the electrostatic potentialexpressedin mV, A~zis the change of the vertical componentof the total dipole momentexpressed in mDebye, and A is the area occupied per molecule in A2/molecule. To eliminate the problem of the evaluation of the change in the dipolar contribution after the drug-lipid complex formation, we developed a new technique based on the Gouy-Chapman theory allowing the determination of 0.. It consists in varying the salt concentration by injecting a saturated solution of NaNO3 in the subphase. Since evidence has been reported that no modification of the dipolar orientation of the monolayer will occur (11, 12), the increase of the surface potential A(A~) after salt injection can be described from the Gouy-Chapman theory. At 25°C, A(Atb) = 50.4 x (sh_l 134o-
(C2),/2
sh_l 1340-)
(C1---~,I-~
165
IN MODEL MEMBRANES
[61
where C1 and C2 are, respectively, the concentration in monovalent ions in the subphase (M) before and after NaNOz injection and 0- is the surface charge density (charge/A2). Figures 1 and 2 illustrate relation [6] for (1) different factors n of salt concentration increase (C2 = n .C,). C1 was chosen equal to 10-3 M; and (2) different C, values, n was chosen equal to 10. It can be seen that the choice of the A(A~b) value, C, and n will be of prime importance for the precision obtained on 0-. For reasons discussed in the appendix, we have chosen A ( A ~ ) e x t = 25 mV, n -- 10, and C~ = 10-3 M. The validity of relation [6] was tested in charged lipid monolayers. Monolayers of well-defined charge density 0-o were obtained by spreading mixtures of a neutral lipid (DPPC) and of a negatively charged one
TABLEI C o m p a r i s o n b e t w e e n T h e o r e t i c a l ( E q . [6]) a n d E x p e r i m e n t a l I n c r e a s e o f S u r f a c e P o t e n t i a l A(Aq0
as a Function of the Surface Charge Density o- W h e n the N a N O 3 Concentration Increases from 10-3 M to 10 -2 M a Proportion CL/DPPC (w/w)
o- 0 (charge//~)
1:0 4:1 3:2 2:3 1:4 3:17 1:9 1:19 1:32 1:99 0:1
1.6 1.3 1.0 6.7 3.3 2.5 1.6 8.3 5.0 1.6
z × × x × × × × )< × 0
10 -2 10 -z 10 -2 10 -3 10 -3 10 -3 10 -3 10 -4 10 -4 10 -4
A(A~)th~or (mY) 58.0 58.0 58.0 57.9 57.5 56.5 55.9 51.0 43.7 22.0 0
A(Aq~)e~ (mV)
53 +_ 5 53 -+ 5 53 -+ 5 53 +- 5 53 -+ 5 53 -+ 5 52 -+ 5 48 +_ 5 40 -+ 5 25 -+ 5 0-+ 5
Subphase was a buffered solution (Tris-HC1, p H = 7.4, 10 -4 M). T = 25°C. T h e s u r f a c e p r e s s u r e o f the m o n o l a y e r w a s 25 m N / m .
(cardiolipin) in different proportions. The surface pressure was kept equal to 25 mN/m. Seventy microliters of a saturated NaNO3 solution was injected in 80 ml of buffered subphase ([NaNO3] = 10-3 M) at 25°C under stirring to reach a 10-2 M NaNO3 concentration. Surface potential increase was recorded as a function of time. Surface potential values at equilibrium are reported in Table I and compared with the theoretical values calculated from Eq. [6]. The good agreement between experimental and theoretical values demonstrates that the surface potential change observed after the salt concentration increase can be described in terms of an electrostatic interaction (Gouy-Chapman theory) without any contribution of the monolayer dipole orientation. This conclusion allows us to present this method as a tool to calculate an unknown charge density after complexation. We have then applied this method to calculate the association constant between positively charged drugs (acridine orange, ethidium bromide) and negatively charged Journal of Colloid and Interface Science, Vol. 80, No. 1, March 1981
166 t, (~¢) (mY) ~0
GOORMAGHTIGH,
//
CASPERS, ~(chorge/A2) ×1~4
3~
17 08 0 q
1,G
aoxlO"2 chorge/~ 2
FIG. 3. Increase of the surface potential A(Atk) of the complexed monolayer after injection of NaNO3 in the subphase as a function of the surface charge density before complexation tr0. The NaNOa concentration was modified from I0 -a to 10-z M. The right ordinate gives the surface charge density tr equivalent to the increase of the surface potential (Eq. [6]). Subphase was a buffered solution (Tris-HCl, pH 7.4, 10-4 M, NaNO3 10-3 M, T = 25°C. The surface pressure was 25 mN/m.
lipids (cardiolipin). Monolayers of welldefined charge density o-0 were obtained by spreading mixture of DPPC and cardiolipin in different proportions (see Table I). Adsorption experiments on a pure DPPC monolayer were carried out for each drug at the same concentration as in Fig. 3. Surface potential and surface pressure measurements demonstrated that no adsorption occurred (AV = 0 --- 5 mV; A I I = 0 -+ 0.5 mN/m). For this reason, DPPC was used as an inert lipid. For each o-0 value, i.e., for each monolayer composition, a known amount of the drug was injected into the subphase. At equilibrium, the salt concentration of the subphase was modified from 10-a to 10-2 M by injection of NaNOa. The increase of the
AND
RUYSSCHAERT
surface potential is reported for each o-0value in Fig. 3. The left ordinate gives the experimental data A(Ato) and the right ordinate the charge density o- obtained from A(Ato) by Eq. [6]. For reasons discussed in the appendix, for each curve, o-0 was determined at A(Ato)ext = 25 mV correspond ing to cr = 1.7 10-4 charge/]k ~. From o- and o-0, tO and [D+]3, the association constant K can be obtained (Eqs. [2] to [4]). K values are reported in Table II. Comparison between AV (Eq. [5]) and AtO (Table III) allows an evaluation of the dipolar term change. AtO is calculated from Eq. [7]: AtO = 50.4 × (sh_ a 134 tr (CO ~12
sh_ 1 134 ~__01 (C1)1/2 1.
[7]
Generally, a low or near zero A/~z value is associated with a low penetration of the drug into the lipophilic region of the model membrane. In the next part of this work, we will try to confirm this conclusion by fluorescence measurements. Fluorescence Measurements The maximum emission wavelength of the is strongly dependent on the dielectric constant of the medium surrounding the dye (Fig. 4). Consequently, fluorescence spectra can be used as a probe of the dielectric constant of the medium in which the dye is embedded (13). In fact, the dielectric constant profile in a phospholipidic bilayer has been evaluated by Shinitzky (14). The position of the maximum emission has f l u o r e s c e n c e hma x
TABLE II Association Constants of the Cardiolipin-Positively Charged Drug Complexa
Acridine orange Ethidium bromide
ED+]~
[D+],
o'o
o"
K (Eq. [21)
(mole/liter)
(mole/liter)
(charge/A s)
(charge/A s)
(liter/mole)
10 -5 2 x 10 -5
2 . 6 × 10 -5 5 . 2 x 10 -5
1.6 x 10 -2 1.4 x 10 -2
1.7 x 10 -4 1.7 x 10 -4
4 + 2 x l0 g 2 + I x 10 6
Subphase was a buffered solution (Tris-HC1, pH = 7.4, 10-4 M, NaNO3 10-8 M). T = 25°C. The surface pressure was 25 mN/m. Journal of Colloid and b~terface Science, Vol. 80, No. 1, March 1981
INTERACTIONS IN MODEL MEMBRANES T A B L E III
XMAX
Increase of the Surface Potential of the CardiolipinDPPC Monolayer after Injection of the Drug into the Aqueous Subphase a
Acridine orange Ethidium bromide
167
( AV)exl)erimental
( Al~)theo~tteal
(mY)
(mY)
230 _+ 30 200 _+ 13
203 203
ACRIDINE ORANGE BgS f
590
. . . .
#ord~ot,#,,n
EGG PC DPPC
Do
E~THI DIUM BROMIDE a Comparison between experimental values and theoretical predictions calculated from equation 7. Subphase was a buffered solution (Tris-HC1, pH = 7.4, 10 -4 M, NaNO3 10 -3 M). T = 25°C. The surface pressure of the monolayer was 25 mN/m.
been measured as a function of the dielectric constant. Media of different dielectric constants were used: buffer (• = 80), buffermethanol 2/1 v/v (e = 66), buffer-methanol 1/1 v/v (• = 58), methanol (• = 33), ethanol (~ = 24) (15). Figure 5 shows the position of the maximum emission wavelength as a function of the liposomes concentration. If Xmax does not depend on the liposome concentration, one can conclude that no more free drug is present in the solution. At this
)'max Eth. br
mo
EGGPC
ao -- c,c DPPcC,C,
so
cardiotipin 515
0
I 0.1
L 02
I 03
] I 04 [LIPIDS] rnglml
FIG. 5. Fluorescence titration of and ethidium bromide 5 × 10 -6 M liposomes of DPPC, egg PC, and buffered solution (Tris-HC1, pH NaNO3 10 -3 M). T = 25°C.
acridine orange by unilamellar cardiolipin in a = 7.4, 1 0 - 4 M ,
point • is deduced from hmax. The values of • are reported in Table IV. It must be concluded that these two dyes certainly do not penetrate into the hydrophobic region of the bilayer. This result is in good agreement with the surface potential predictions and with Massari's work (16, 17).
kmQx
Acr.or.
~
cridine orQnge
610 ---~,
605
520 ~
520
-4¢--
6
0
DISCUSSION
The surface potential approach is particularly well adapted to the study of the electrostatic interactions between soluble drugs and lipids. However, the value of the surface charge density before and after the drug injection cannot be immediately calculated.
0
T A B L E IV
515 ~
595
Dielectric Constants of the Lipidic Medium Surrounding Each Drug in Unilamellar L i p o s o m e s a Dielectric constant
Ethidium bromide SlO
20
40
60
590 80
E,
FIG. 4. Xmax (nm) of acridine orange and ethidium bromide as a function of the dielectric constant of the solvent. The concentration of dye was 10 -6 M. T = 25°C.
Ethidium bromide Acridine orange
Cardiolipin
DPPC
Egg PC
75 70
80 80
80 80
Buffered solution (Tris-HC1, pH = 7.4, 10 -4 M, NaNO8 10 -a M). T = 25°C. Journal of Colloid and Interface Science, Vol. 80, No. 1, March 1981
168
GOORMAGHTIGH,
CASPERS, AND RUYSSCHAERT
Indeed, surface potential measurements depend both on the surface charge density change (described by the Gouy-Chapman theory) and on the total dipole orientation modification (in fact, its vertical projection). In order to overcome this difficulty, two possible ways have been described. The most classical way is to record the surface potential increase due to the drug injection as a function of its bulk concentration. This method allows us to define a bulk concentration corresponding to a half maximum effect. From this concentration, an association constant can be calculated. This method is however imprecise since the concentration is often difficult to define, particularly when the water solubility of the drug is a limiting factor. Moreover, one assumes that the charge and dipole effects do not depend on the drug concentration. From a molecular point of view, it seems difficult to accept this statement since each drug molecule will modify the state of the lipid layer (surface charge density, packing, accessibility to the surface). In a preceding paper, we proposed another method to dissociate the electrostatic term from the dipolar term (2). It was based on surface radioactivity measurements (18) allowing determination of the equilibrium drug surface concentration. However, the availability and the stability of radioactive drugs are limiting factors. In the present paper, we proposed a simple technique to evaluate independently the charge and the dipolar effect. It is based on a modification of the subphase ionic concentration. The first part of this work consisted of the verification of the theoretical predictions. The good agreement between theoretical and experimental data demonstrated that the surface charge density of a lipid monolayer can be evaluated from the surface potential increase. In fact, the Gouy-Chapman theory is based on the hypothesis that the charge is uniformly spread over the surface and that the counterions behave as point charges. Deviations from the theoretical valJournal of Colloid and Interface Science, Vol. 80, No. 1, March 1981
ues of the surface potential appear mainly for fully ionized films (1 charge/60 A 2) and high salt concentration (up to 0.1 M) (19). With the method proposed here, the measurements are carried out at (r = 1.7 × 10-4 charge/Ae (or 1 charge/6000 A 2) and at 10-2 M salt concentration. This method has been applied to evaluate the surface charge density of monolayers after complexation by acridine orange and ethidium bromide. Our results demonstrated the existence of a high drug-lipid electrostatic association constant and a low penetration of the drugs into the lipid monolayers. These results are in good agreement with our fluorescence measurements and with Massari's work (16, 17). From a general point of view, we must however point out some limitations of the proposed method: (1) The drug-monolayer equilibrium must not be modified by the ionic concentration increase. This point could with difficulty be assumed for low association constants. However, with most of the biologically specific drugs, the high association constant allows elimination of this eventuality. (2) Some reorganization of the interface structure (including the drug) could occur due to the increase in salt concentration. Indeed, hydrophobic effects can be sensitive to salt addition. This eventuality would be detected from the shape of the A(A~) = f(o'0) curve. Indeed, an additional dipolar term will affect the A(A~k) value at the plateau (o-> 1.7 × 10-3) (Table 1). Moreover, the plateau will deviate from horizontality since the additional term depends on the number of drug molecules complexed at the interface and is therefore a function of o'. (3) Another difficulty could arise from the choice of the inert lipid. Lipids with highly saturated long chains seem however to prevent any nonspecific drug adsorption. To conclude, the new technique presented here allows a direct estimation of the electrostatic interactions between drugs and lipids but remains restricted to specific inter-
169
I N T E R A C T I O N S IN M O D E L M E M B R A N E S
actions. A precise evaluation o f the association constants of antimitotic-lipid-specific complex is now in progress in our laboratory.
60
APPENDIX
The purpose of this appendix is to define the experimental parameters n, C1, and ~(h0)~x t in order to obtain the best precision for or. However, other conditions such as the nonperturbation of the equilibrium by the increase of salt concentration will be taken into account. Figures 6 and 7 give in the ordinate the relative error on the extrapolated o- value as a function of these three parameters, if we consider an absolute error of 5 mV on the experimental data (A(&~b)). F r o m Fig. 6, giving the error as a function of C~ and n, it appears that the initial salt concentration has only a very weak effect on the precision, whereas the best precision is obtained with high n values. F r o m another Point of view, n and C~ must be chosen low enough in order to avoid a massive introduction Of salt into the subphase since this could perturbate the equilibrium described in Eq. [1]. For each experiment, we have controlled that equilibrium was maintained after salt injection. Surface potential and surface pressure were recorded simultaneously. Indeed, drug adsorption on the lipid monolayer will induce an increase of the surface pressure whereas an eventual desorption of the drug will de-
GO
-
-/
•'~/~/ ' '
/
'
I O0
4020
~ ...........
40
~000
10
20
40
60 n
FIG. 7. Calculated relative error for ~r as a function of the salt concentration increase term n and of the A(At0)ext value. An absolute error of 5 mV has been considered for A(AtO). The initial salt concentration has been chosen equal to i0 -~ M. If n = I0 and A(St0)ext = 25 mV, the percentage of error on or is 28.1%.
crease the surface pressure. For acridine orange and ethidium bromide, no modification of the surface pressure was observed after salt injection. This means that the equilibrium conditions were maintained during the experiments. For these reasons, we have chosen the following experimental conditions:C~ = 10-z M andn = 10. F r o m Fig. 7, giving the relative error on cr as a function of n and A(AqJ)ext, it appears that the error will be minimum if h(AqJ)ext = 25 mV when n - - 1 0 a n d C l = 10- s M . ACKNOWLEDGMENT One of us (E.G.) thanks I.R.S.I.A. (Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture) for financial assistance.
ERROR o n o
40
(*
40 20
•
REFERENCES
20
'
-7 1
-3
5
l o g C1
FIG. 6. Calculated relative error for cr as a function of the salt concentration increase term n and of the initial salt concentration ~C!- o- is obtained at A(~q~)ext = 25 mV. If C~ = 10-3 M and n = 10, the percentage of error on cr is 28.1%.
1. Tritton, T. R., Murphree, S. A., and Sartorelli, A. C., Biochem. Pharmacol. 26, 2319 (1977). 2. Goormaghtigh, E., Chatelain, P., Caspers, J., and Ruysschaert, J. M., Biochim. Biophys. Acta 597, 1 (1980). 3. Lee, A. G., Biochim. Biophys. Acta 514, 95 (1978). 4. Gittler, G., Rubalcava, B., and Coswell, A., Biochim. Biophys. Acta 197, 419 (1969). 5. Azzi, A., and Santato, M., Biochem. Biophys. Res. Commun. 44, 211 (1971). 6. Fico, R. M., Chen, T. K., and Canellakis, E. S., Science 198, 53 (1977). Journal of Colloid and Interface Science,
Vol. 80, N o . 1, M a r c h 1981
170
GOORMAGHTIGH, CASPERS, AND RUYSSCHAERT
7. Ruysschaert, J. M., Tenenbaum, A., Berliner, C., and Delmelle, M., FEBS Lett. 81, 406 (1977). 8. Caspers, J., Landuyt-Caufriez, M., Deleers, M., and Ruysschaert, J. M., Biochim. Biophys. Acta 554, 23 (1979). 9. Caspers, J., Berliner, C., Ruysschaert, J. M., and Jaff6, J.,J. Colloid lnterface Sci. 49, 433 (1974). 10. Gaines, G. L., in "Insoluble Monolayers at LiquidGas Interfaces." Wiley-Interscience, New York, 1966. 11. Davies, J. T., and Rideal, E. K., in "Interracial Phenomena," 2nd ed., p. 75. Academic Press, New York, 1963. 12. MacDonald, R. C., and Bangham, A. D.,J. Membrane Biol. 7, 29 (1972). 13. Goldman, R., Fachinetti, T., Bach, D., Raz, A.,
Journal of Colloid and Interface Science, Vol.80, No. 1, March1981
14. 15. 16. 17. 18.
19.
and Shinitzky, M., Biochim. Biophys. Acta 512, 254 (1978). Shinitzky, M., lsr. J. Chem. 12, 879 (1974). Turner, J., and Brand, P., Biochemistry 7, 512 (1968). Massari, S., and Pascolini, D., Biochemistry 16, 1189 (1977). Massari, S., Pascolini, D., and Gradenigo, G., Biochemistry 17, 4465 (1978). Kummer, J., Ruysschaert, J. M., and Jaff6, J., Berichte yon VI. Internationalen Kongress fiir Grenzfl~ichenaktive Stoffe, Band II, 1,285. Carl Hauser Verlag, Miinchen, 1973. Hauser, H., Drake, A., and Phillips, M. C., Eur. J. Biochem. 62, 335 (1976).