Drug Interactions in Liposomes Studied by Rheological

Nov 1, 1996 - (6) Boury, F.; Ivanova, Tz.; Panaiotov, I.; Proust, J. E.; Bois, A.; Richou,. J. J. Colloid Interface Sci. 1995, 169, 380. Figure 1. Str...
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Langmuir 1996, 12, 6098-6103

Phospholipid/Drug Interactions in Liposomes Studied by Rheological Properties of Monolayers A. Doisy,†,‡ J. E. Proust,*,† Tz. Ivanova,†,§ I. Panaiotov,†,§ and J. L. Dubois‡ Biophysique Pharmaceutique, Faculte´ de Pharmacie, 16 boulevard Daviers, 49100 Angers, France, and Roussel-Uclaf, De´ partement Gale´ nique, 102 route de Noisy, 92230 Romainville, France Received June 26, 1995. In Final Form: July 16, 1996X In order to obtain a good knowledge of the pharmaceutical form, we sought to localize the antiacneic drug RU 58841 in liposomes. The mixed drug/phospholipid monolayer obtained by spreading phospholipids at the air/aqueous surface of drug solutions was studied using the film balance technique to clarify the organization of the film respect to its composition and state of compression. A rheological approach based on Maxwell’s model was found to be useful to discriminate between the influence of each constituent in the monolayer. From the study of the isotherms and the rheological behavior of mixed monolayers at low surface pressures it was deduced that the drug could be localized between the phospholipids polar headgroups. In the opposite case, at high surface pressures, the drug molecule was believed to be trapped in the hydrophobic part of the phospholipidic layer and behave like an insoluble one. The surface film formation kinetics of drug-loaded liposomes is faster than the corresponding kinetics of unloaded liposomes.

Introduction Liposomes are spherical vesicles with one or several phospholipid bilayers. When they are dispersed in aqueous media, hydrophilic substances can be entrapped in the inner spheres whereas lipophilic drugs may be concentrated in the phospholipidic bilayers. Thus liposomes are of great interest to carry and introduce drugs into cells. A good appreciation of the drug action-mode requires a knowledge of the interactions between drug and phospholipid. The aim of this study was to evaluate the tendency of drugs to accumulate either in the hydrophilic or hydrophobic areas of liposomes by correlating the variation of mixed monolayer compositions with the compressional state of the film obtained by a Langmuir balance. The drug, RU 58841 (Figure 1), is a potent antiacneic agent which is poorly soluble in water (0.47 mg/mL (J. L. Dubois, Roussel-Uclaf, personal communication)). This molecule is characterized by hydrophilic and hydrophobic areas (its partition coefficient between octanol and water is about 1.8 (J. L. Dubois, Roussel-Uclaf, personal communication)) and as a consequence tends to accumulate between the polar headgroups and the hydrophobic chains of the phospholipids. Since two monolayers make up the lipid bilayer forming the liposome membrane, the monolayer model system has been applied extensively to investigate the interfacial behavior of phospholipids and lipid/drug interactions.1 Experiments with monolayers have the advantage that the arrangement of molecules can be easily controlled by changing the molecular area and the surface pressure of the monolayer. Two approaches have proven successful for investigating lipid/drug interactions in monolayers. In one approach, the lipid-drug mixture is spread out at the surface,1,2 and the surface pressure/area isotherms of the insoluble mixed monolayers are determined. In the other approach, †

Biophysique Pharmaceutique. Roussel-Uclaf. § Permanent Address: Biophysical Chemistry Laboratory, University of Sofia, J. Bourchier 1 str., 1126 Sofia, Bulgaria. X Abstract published in Advance ACS Abstracts, November 1, 1996. ‡

(1) Dhathathreyan, A. Colloids Surf. 1993, 81, 269. (2) Wiedmann, T. S.; Jordan, K. R. Langmuir 1991, 7, 318.

S0743-7463(95)00516-6 CCC: $12.00

Figure 1. Structure of RU 58841.

the monolayer interactions between the pharmacologically active substance and the lipids are investigated by injecting the drug into the subphase beneath a lipid film at a constant surface area.3-5 In this case, the penetration of the drug can be estimated by the surface pressure increase of the lipidic film. In the present study, we try to obtain information about the phospholipid/RU-55841 interactions and the location of the drug in the lipid in an aqueous environment. The drug however forms partially soluble monolayers, thus to investigate the properties of the mixed drug/phospholipids monolayers obtained by adsorption of drug and soreading of phospholipids we used two approaches: a large compression/expansion far away from the equilibrium and a small linear monolayer deformation, by employing recently developed methodology.6 Likewise, the spreading and the destabilization of phospholipid liposomes containing RU at the air-water interface was studied. Materials and Methods The phospholipid mixture (L) Lipoid E100-35 (average mol wt 777) was supplied by Lipoid KG (Ludwigshafen). This mixture, composed of 94% egg phosphatidylcholine, is described in Table 1. The drug (RU) RU-58841 (mol wt 369.4) was synthesized by Roussel Uclaf and was used without firther purification (Figure 1). Analytical grade chloroform, obtained from Prolabo, was used for spreading monolayers. The aqueous subphase consisted of a 0.05 M phosphate buffer solution at pH 7. Water was ultrapure and obtained from a Milli Q Plus system (Millipore, France). Both the surface pressure and surface tension were measured (3) Sicre, P.; Cordoba, J. J. Colloid Interface Sci. 1989, 132, 94. (4) Birdi, K. S. J. Colloid Interface Sci. 1976, 57, 228. (5) Bourhim, N.; Elkebbaj, M. S.; Gargouri, Y.; Giraud, P.; Rietschoten, J.; Olivier, C.; Verger, R. Colloids Surf. 1993, 1, 203. (6) Boury, F.; Ivanova, Tz.; Panaiotov, I.; Proust, J. E.; Bois, A.; Richou, J. J. Colloid Interface Sci. 1995, 169, 380.

© 1996 American Chemical Society

Phospholipid/Drug Interactions in Monolayers

Langmuir, Vol. 12, No. 25, 1996 6099

Table 1. Chemical Compounds of Lipoid E100-35 composition of lipoid E100-35 phosphatidylcholines dipalmytoyl, DPPC distearyl, DSPC dioleyl, DOPC lysophosphatidylcholine sphingomyeline cholesterol apolar lipids R-tocopherol

% (p/p) 94 31-33 20-26 29-35 1 3 0.5 1.0 0.1

at 25 °C. Liposomes (obtained using a Microfluidiseur at 60 °C) were purchased from Roussel-Uclaf. The total phospholipid concentration was 49.6 g/L for the placebo and 107.6 g/L for liposomes loaded with RU-58841. Liposome size measurements were performed with a Coulter model N4MD submicrometer particle analyzer (Coulter, Margency). The operating temperature was 25 °C and the detection angle was 90°. The mean particle diameter was 44 nm for placebo liposomes and 85 nm for RU-58841 liposomes, with a monopeak dispersion. The phospholipid / RU-58841 molar ratio was about 10/1. Phospholipid Monolayers. Lipoid E100-35 was dissolved in chloroform (2 mg/mL) and spread on the aqueous subphase (26 µL) over the maximum available area (927 cm2) of a Langmuir film balance (Lauda FW2, Lauda-Ko¨nighshafen, Germany) by means of an Exmire microsyringe (Prolabo, France), after the water surface was cleaned by suction. Ten minutes was allowed for solvent evaporation before the start of the compression. The value of the surface pressure after spreading was less than 0.1 mN/m. Dynamic isotherms and hysteresis cycles were performed with deformation rates of 10, 150, or 370 cm2/min, and the variation of the surface presure versus molecular area was directly recorded on the Lauda computer. RU 58841 Monolayers. Solutions of RU 58841 in phosphate buffer were prepared with concentrations between 0.2 and 0.001 mg/mL. The corresponding equilibrium surface tensions, γRU were measured with an accuracy of 0.02 mN/m by the Whilhelmy plate method using an electronic balance (Analytic AC 210S001V1, Sartorius, Go¨ttingen, Germany). Lipoid E100-35/RU 58841 Monolayers. The lipoid solution was spread out on adsorbed monolayers of RU with bulk concentrations between 0.2 and 0.005 mg/mL. After the equilibrium time of drug adsorption, lipoid was spread on the subphase. A spreading quantity of 26 µL had no effect on the initial surface pressure, which was determined from the variation of the surface tension measured by the Whilhelmy plate method. A large compression of the mixed film (adsorbed RU and spread lipoid) at a rate of 150 cm2/min was performed, and the variation of the excess surface pressure was recorded. This measured surface pressure is in fact the excess pressure in the compressionnal compartment of the film balance (imposed by the compression of the mixed film) with respect to the initial equilibrium surface pressure ΠRU (ΠRU ) γ0 - γRU) due to the adsorption of the drug. Rheological Measurements at the Air/Water Interface. The rheological properties of pure lipid, pure drug, and mixed lipid/drug monolayers were tested using a recently developed approach.6 Small monolayer compressions or expansions were applied at a given surface pressure with different velocities using the mobile barrier of the film balance. Surface pressure was measured as a function of time during compressions or expansions and during the relaxation process which followed the perturbation. A Sartorius balance fitted with a Wilhelmy plate and connected to a computer allowed us to measure the variations of the surface pressure versus time with an accuracy of 0.02 mN/m. The fastest acquisition rate was approximately five measurements per second. Liposomes. Variable volumes (4.4, 9.8, or 41 µL) of liposomal suspensions were spread at a constant surface area (10.7 cm2) by means of an Exmire microsyringe. The subphase was phosphate buffer and the temperature was 25 °C. Variations of the surface tension with time were recorded. Zero time was taken at the beginning of the spreading procedure. Prior to spreading the water surface was cleaned by suction.

Figure 2. (a) Surface tension of RU 58841 in phosphate buffer solutions as a function of concentration (in log scale) at 25 °C. (b) Adsorption isotherm of RU 58841 from curve a and Gibbs equation.

Figure 3. Compression isotherms of mixed Lipoid E100-35/ RU 58841 monolayers (150 cm2/min) at 25 °C. RU 58841 subphase concentrations are as follows: 0; 0.005 mg/mL; 0.01 mg/mL; 0.03 mg/mL; 0.05 mg/mL; 0.1 mg/mL; 0.2 mg/mL. The pressure of the mixed film is described as a function of the molecular area of the spread lipoid. For all pressure and for all concentration of the drug in the subphase, ∆A shows the increase of the molecular area.

Results and Discussion (1) Surface Pressure/Area Isotherms. (a) Equilibrium Isotherm of Drug Monolayers. Plots of the surface tension (γ) versus RU log concentrations are shown in Figure 2a. RU exhibited a decrease in γ with increasing concentration up to the appearance of discontinuity at the concentration corresponding to the critical micellar concentration (cmc) at about 0.4 mg/mL. From this figure, it can be concluded that the highest surface pressure reached after adsorption equilibrium is about 20 mN/m, corresponding to a surface tension of 52 mN/m. The equilibrium Gibbs isotherm (Figure 2b) of the drug is obtained from the variation of the surface tension versus the logarithm of the drug concentration. The variation of the surface excess value Γ was calculated graphically according to the Gibbs equation [Γ ) -(1/RT)(dγ/d ln c)]: for each surface pressure the values of the molecular area reported in Figure 2b correspond to the mean slopes (dγ/d ln c) of curve a. These calculations give values of 70 Å2 per adsorbing RU molecule corresponding to the highest concentrations of RU. Computer-simulated models suggest that this area is consistent with molecules lying flat on the surface. (b) Isotherm of Lipoid Monolayer. The dynamic Π/A isotherm of lipoid, performed at 25 °C, is shown in Figure 3. The maximum surface pressure was 67 mN/m and the evaluated molecular area at the collapse level was 50 Å2. Like the DOPC isotherm,7 this curve does not exhibit a LE-LC transition, but as with DPPC monolayers this lipid film can be compressed at high surface pressures (7) Beitinger, H.; Vogel, V.; Mobius, D.; Rahmann, H. Biochim. Biophys. Acta 1989, 984, 293. (8) Reinhardt-Schlegel, H.; Kawamura, Y.; Furuno, T.; Sasabe, H. J. Colloid Interface Sci. 1991, 147, 295.

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Figure 4. Increase of the molecular area of lipoid due to the presence of the drug in the monolayer as a function of the surface pressure. (∆A is deduced from Figure 3.) (‚‚-‚‚) Equilibrium isotherm of the drug deduced from adsorption measurements (Figure 2b).

without collapse.8 Thus, one can suppose that the monolayer is in a somewhat expanded state. The compressionexpansion cycle gave the same Π/A relationship, and no hysteresis cycle was found. When compression experiments were performed at lower or higher rates of compression, the isotherms recorded were practically coincident. Consequently, one can assume that the phospholipid monolayer is quite stable. Thus, the obtained dynamic Π/A curves are the true equilibrium isotherms. (c) Mixed Lipoid/RU Monolayers. When compression experiments were performed at lower or higher rates of compression, the isotherms registered were practically coincident. Consequently, one can assume that the mixed monolayer is quite stable. The dependence of the dynamical surface pressure Π as a function of the apparent area A per lipid molecule is shown in Figure 3. It can be noted that these isotherms are characterized by a plateau, or a shoulder, at the lower RU 58841 subphase concentrations, at about 20 mN/m. We can see on this figure that the increase of the apparent area ∆A, which is due to the presence of the drug in the mixed monolayer, depends on the surface pressure and on the concentration of the drug in the subphase. In Figure 4, the surface pressure is plotted as a function of the increase of the molecular area ∆A (obtained from Figure 3) for each drug concentration in the subphase. This increase in the molecular area, which must be attributed to the drug molecules in the mixed film, can be compared to the molecular area of the drug at the same surface pressure in Gibbs equilibrium conditions (Figure 2b). For all subphases and small compressions, the area of the drug is greater in the mixed film than in the pure monolayer. In these regions, the interactions between lipoid and drug seems to be repulsives and it is probable that the interactions occur in the vicinity of the polar heads. On the contrary, for higher compression levels, the apparent area of the drug is smaller in the mixed film than in the pure monolayer. In these regions the interactions between drug and lipoid appears to be attractive and it is probable that the molecule displaces more of the polar region than the hydrophobic one, thus allowing the drug to penetrate between the hydrocarbon chains of the lipidic layer. It is also probable that this condensation effect can be attributed to a reorientation of drug molecules toward the right. However another explanation could be the progressive expulsion of the drug into the subphase, and to corroborate our interpretation based on the retention of the drug in the hydrophobic part of the lipidic layer, rheological studies were performed.

Doisy et al.

Figure 5. (a) Rheological model of the monolayer. (b) Surface pressure change ∆Π during the time T of compression (c) at a constant velocity Ub, followed by a relaxation (r).

(2) Rheological Studies. (a) Theoretical Approach. (The derivations of the used expressions are given in much more detail in ref 6.) The rheological dilatational properties involving dilatational mechanical stress of a surface film were studied using a theoretical approach based on two-dimensional rheology.9 In order to describe the surface pressure change, ∆Π ) Π(t) - Πi (Figure 5a), during the time T of the compression at a constant velocity Ub followed by relaxation, we supposed that at any one moment the total surface pressure change ∆Π ) Π(t) - Πi can be written as a sum of equilibrium ∆Πe and nonequilibrium ∆Πne contributions

∆Π ) ∆Πe + ∆Πne

(1)

The equilibrium part ∆Πe depends on the equilibrium surface dilatational elasticity Ee

∆Πe ) Ee

Ubt Ai

(2)

where Ai is the initial surface before the mechanical stress and

Ubt ∆A ≡ Ai Ai is the corresponding strain . This elastic behavior is represented by the upper branch of the mechanical model in Figure 5a. The nonequilibrium part ∆Πne of the total surface pressure change correlates to the accumulation of elastic energy during the compression. Dissipation of this accumulated energy occurs on compression as well as relaxation and can be interpreted as a molecular reorganization in the monolayer. This viscoelastic behavior can be described using Maxwell’s equation

d∆Πne ∆Πne Ub + ) Ene dt τ Ai

(3)

where ∆Πne is the applied stress f, Ene is the nonequilibrium surface dilatational elasticity, and τ is the specific time of relaxation. The viscoelastic behavior is represented by the lower branch of the mechanical model in Figure 5a. The two branches of the mechanical model are coupled in parallel according to eq 1 corresponding to the additivity of stresses. From the solution of (3) using the initial conditions ∆Πne ) 0 at t ) 0, (1) and (2), we obtained the following equation to describe the viscoelastic behavior of the monolayer (9) van Voorst Vader, F.; Erkens, J. F.; van den Tempel, M. Trans Faraday Soc. 1964, 60, 1170.

Phospholipid/Drug Interactions in Monolayers

∆Π τ A ) Ee + Ene (1 - e-t/τ) Ubt i t

Langmuir, Vol. 12, No. 25, 1996 6101

(4)

Using the experimental values found for ∆Π(t) along with eq 4, it is possible to determine the nonequilibrium part (Ene) and the equilibrium part (Ee) of the dilatational elasticity. The specific time of relaxation τ could be easily determined from experiments where the time of compression T was much smaller than the time of the relaxation process τ. Subsequently the following expression for the relative relaxation

∆Π(t) - ∆Π∞ ∆Π0 - ∆Π∞



Figure 6. Dynamical elasticity as a function of surface pressure for Lipoid E100-35 spread monolayer (values calculated from Figure 3).

Π(t) - Π∞ Π0 - Π∞

is obtained

ln

Πt - Π∞ t )Π0 - Π∞ τ

(5)

(b) Results. The insoluble lipid monolayer behaves as an elastic two-dimensional body without any relaxation process. From the equilibrium isotherm (Figure 3), the elasticities of the monolayer E ) -A(dΠ/dA) were calculated and plotted against the surface pressure (Figure 6). Figure 7 shows the typical results obtained for a mixed (L/RU) monolayer corresponding to the most concentrated RU subphase; the initial surface pressure Πi was about 46 mN/m. The variation of the excess surface pressure ∆Π with time for this mixed film is shown in Figure 7a. This variation was obtained by performing a small compression or expansion at a rate of 10 or 370 cm2/min, followed by relaxation after removal of the stress had ceased. From these results, we applied the theoretical approach to determine the relaxation time τ and the equilibrium and nonequilibrium parts of dilatational elasticity. Firstly, the relaxation time was deduced from the relaxation effect obtained with the higher relative rate of compression. The plots of the logarithm of the relative relaxations against time are shown in Figure 7b. It can be seen that the experimental points are very close to straight line. The characteristic time τ was calculated from the slope of this line. Subsequently, the experimental data obtained when applying the low compression rate was found to fit eq 4 well. Typical results of this linearization are shown in Figure 7c. The slope of the curve corresponds to the nonequilibrium elasticity while the equilibrium elasticity occurs at the origin. The results obtained for different mixed monolayers at various surface pressures are summarized in Table 2. The obtained values of τ, Ee, and Ene are presented. The characteristic time is of the order of magnitude of 10 s. The equilibrium elasticity increases with the increasing of the surface pressure. From the last column it is apparent that, for all surface pressures, the equilibrium elasticity of mixed monolayers is always lower than the elasticity of the pure phospholipidic film. This decrease in elasticity can be correlated to the presence of RU in the mixed film at any surface pressure. For higher surface pressures, a nonequilibrium elasticity, Ene, appears. If we attribute this trend to the desorption of the RU molecules in the subphase, nonequilibrium elasticity should have been observed at any pressure and not just for the higher pressures as can be seen in Table 2. This behavior is most probably due to the rearrangement of RU molecules in the monolayer at pressures above 20

Figure 7. Typical results of rheological comportment and parameter calculation for mixed monolayer: concentration of RU in the subphase, 0.2 mg/mL; initial surface pressure, 46 mN/m. (a) Variation with time of the surface pressure excess ∆Π. The rates of compression or expansion were 370 cm2/min (C1;E1) and 10 cm2/min (C2;E2). (b) Variation of ln(Π(t) - Π∞)/(Π0 - Π∞) versus time from C1 curve. Equation 5 gives the relaxation time τ. (c) Variation of (∆Π/Ubt)A0 versus (τ/t)(1 - e-t/τ). Equation 4 gives the Ee and Ene values. Table 2. Rheological Parameters Obtained for Different RU 58841 Subphase Concentrations at Various Surface Pressuresa RU 58841 concentration Πi Π0 - Π∞ (mg/mL) (mN/m) (mN/m) 0.005 0.01 0.03 0.05 0.1 0.2

13 29 41.8 14.7 21.8 31.4 29 39 16 21 35 19.5 22.5 35 19 38.5 46

0.4 0.8 0.1 0.4 0.45 0 0.3 0.5 0.05 0.6 1.3

τ (s) 14.5 10.8 10.5 4.9 10.1 9 9.7 15.6 8.7 10.3

Ene Ee Elip - Emix (mN/m) (mN/m) (mN/m)

2.5 5.6 4.8 4 5

7.5 12

7.5 12.2

10 14.5

0.9 8.7

15 61.3 85.3 30.5 47.6 73 78 91 19 25.5 81 16.6 15.4 62.4 15.6 80.9 92.9

49 69 29 47 52 58 53 24 61 65 41 75 89 60 77 34 22

a The values of the last column were calculated using the data from Figure 10.

mN/m. This consideration would therefore corroborate the hypothesis of the expulsion of RU from the polar region of the layer toward the hydrophobic chains of the phospholipids. (3) Liposomes. (a) Theoretical Approach. According to previously published data,10 the behavior of liposomes spread at the air/water interface can generally be described using two simultaneous competitive processes (Figure 8): (10) Ivanova, Tz.; Raneva, V.; Panaiotov, I.; Verger, R. Colloid Polym. Sci. 1993, 271, 290.

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Doisy et al.

Figure 8. Behavior of liposomes spread at the air/water interface: (a) spreading layer of liposome suspension; (b) mechanism of the film formation from a liposome suspension.

Irreversible liposome diffusion from the interface to the bulk phase, liposomes are perfectly closed vesicles which do not influence surface pressure. Ιrreversible liposome adsorption at the interface and transformation into superficial mesophases, the liposomes are destroyed and form open tensioactive structures. The diffusion process prevails when small amounts of a liposomal solution are spread whereas the surface transformation process is predominant with a large amount of liposomes. These two phenomena may be considered separately. The diffusion controlled process has been described mathematically,10,11 by assuming that the liposomal suspension initially spread forms a layer of constant thickness l and by resolving Fick’s equation. The irreversible surface transformation process can be described by a Langmuir-type equation neglecting the desorption term

(

n* dn* ) KdC0d 1 dt n∞*

)

(6)

where C0 is the concentration of liposomes in the first subsurface layer able to be transformed, C0d is the same quantity related per unit surface of destroyable vesicles present in the subsurface layer, n* is the number of destroyed vesicles at the interface present per unit area at time t, n*∞ is the maximal number of destroyed liposomes present in a close-packed layer, (1 - (n*/n∞*)) is the available free surface area at time t, and K is the surface transformation rate constant of the interfacial reorganization process. In the general case, where both transformation and diffusion processes are involved, the following expression has been deduced10

Figure 9. Variation of ln(1 - (Γ(t)/Γ∞)) versus xt1/2 for placebo (a) and loaded liposomes (b) to determine the kinetic constant of the liposome transformation (eqs 7 and 8) (a) Variation with time of surface pressure after spreading placebo liposomes: 4.4 µL (a); 9.8 µL (b); 41 µL (c). (b) Variation with time of surface pressure after spreading RU 58841 loaded liposomes: 4.4 µL (a); 9.8 µL (b); 41 µL (c).

with the corresponding theoretical predictions described by eq 7 and by assuming that the surface density Γ(t) directly depends upon the number of destroyed liposomes n*(t), we can obtain the following expression

Γ(t) n*(t) ) Γ∞ C0d

(8)

Thus, from eqs 7 and 8, by plotting ln(1 - (Γ(t)/Γ∞)) as

a function of xt1/2, the rate of liposome transformation K (Figure 9) was determined to be 1.4 × 10-3 s-1 for placebo liposomes whereas K is 2.5 × 10-3 s-1 for RU 58841/ liposomes (the calculated diffusion coefficients D were about 9.7 × 10-8 cm2/s for placebo and 5.05 × 10-8 cm2/s for RU liposomes). Thus it can be seen that RU accelerates the transformation of closed vesicles into destroyed ones. Conclusion

(b) Results. The variation of surface pressure with time at a constant surface area after spreading different volumes of placebo (a) or RU (b) liposomal solution is shown in Figure 9. It is immediately apparent that the increase in surface pressure is earlier and more pronounced in the case of RU liposome spreading. In agreement with ref 10 it is clear that the transformation controlled process predominates for a large initial amount of liposomes. As a first approximation, the superficial phospholipidic film formed from liposomes can be considered as a monolayer. From the phospholipid isotherm it is possible to obtain Π ) f(1/Γ) where Γ is the phospholipidic superficial density. By comparing Π ) f(1/Γ) and Π ) f(t) relationships, it is possible to plot 1/Γ as a function of time. Thus we can determine Γ∞ and then Γ(t)/Γ∞, describing the evolution of the relative superficial density. By comparing experimental results of surface pressure

From the Lipoid E100-35 surface pressure/area relationships, the phospholipid film appears to be in an expanded state. The dynamical isotherms of mixed Lipoid E100-35/RU 58841 monolayers are characterized by a plateau at a surface pressure of about 20 mN/m which corresponds to a possible modification of the monolayer structure. In order to gain a better appreciation of this transition, it would be interesting to study this structure by electron microscopy or atomic force microscopy. These methods have already been used to investigate the structure of bipolar molecular films.12 From dynamic isotherm measurements, we have deduced that at low pressures, the drug could lie between the polar headgroups of phospholipids. For higher pressures, a behavioral difference can be noted and the drug molecules could be “squeezed out” of the polar region toward the hydrophobic chains of the phospholipids. To corroborate this hypothesis, we have investigated the rheological properties of these monolayers under dilational deformation. The methodology used is based upon a linear approximation and has already proved successful in gaining new information on the structure and behavior of polymer monolayers.6 From the data collected it can be seen that while the lipid film behaves as a two-dimensional elastic body, the mixed monolayers exhibit viscoelastic properties specially at high surface pressures. It is also clear that the equilibrium elasticity is always lower for mixed films than for phospholipidic

(11) Ivanova, Tz.; Georgiev, G.; Panaiotov, I.; Ivanova, M. Prog. Colloid Polym. Sci. 1989, 79, 24.

(12) Gulik, A.; Tchoreloff, P.; Proust, J. E. Chem. Phys. Lipids 1990, 53, 341.

(

ln 1 -

)

n* 2Kl xt )C0d xDπ

(7)

Phospholipid/Drug Interactions in Monolayers

monolayer. This decrease confirms that penetration of RU into the phospholipids may effectively take place at any pressure. Furthermore, a nonequilibrium elasticity appears only for higher surface pressures and can be correlated to a reorganization of the lipid/drug monolayer such as an expulsion of the RU molecules toward the hydrophobic chains of the phospholipids. This conclusion seems reasonable considering that the Lipoid monolayer is in an expanded state even at high surface pressures, (13) Marcelja, S. Biochim. Biophys. Acta 1974, 367, 165.

Langmuir, Vol. 12, No. 25, 1996 6103

thus allowing drug molecules to concentrate between the phospholipids. Furthermore, since the lateral pressure in the liposome membrane is found to be higher than 20 mN/m, according to Marcelja.13 RU molecules could tend to accumulate between the hydrocarbon chains of phospholipids. Finally, from a liposome spreading study, the destruction of loaded liposomes appears to occur earlier and more important than for placebo liposomes. LA950516+