Langmuir 1994,10, 767-772
767
Interaction of Enrofloxacin with Phospholipid Mono- and Bilayers C.Mestres,? M. A. Alsina,*lt M. A. Busquets,t I. Haro,* and F. Reig* Unitat de Fisico Qutmica, Departament de Farmhcia, Facultat de Farmhcia, Raga Joan XXIII sln, 08028 Barcelona, Spain, and Departament de Paptids, CID-CSIC, Jordi Girona Salgado 18-26, 08034 Barcelona, Spain Received March 26,1993. I n Final Form: September 29,1995 In the present paper interactions between enrofloxacin and phosphatidylcholine, dipalmitoylphoaphatidylcholine, and mixtures of these lipids with cholesterol were studied by different techniques. Hydrophobicinteractionswere determinedby differentialscanningcalorimetry,carboxyfluoresceinleakage from liposomes, and compression isotherms of mixed monolayers. The penetration kinetics as well ae the polarization changes of the anilinonaphthalenesulfonate probe inserted into liposomes were used to study the effect of the drug in the lipid polar head motion. The drug was unable to form monolayers and compression isothermsshowed a low level of drug interaction with phospholipids. This was also confirmed by differential scanning calorimetry measurementsin which no change in the main transition temperature was observed but a slight broadening of the peaks. However, a strong influence in the fluidity of the polar part of the bilayers after incubation of liposomes with enrofloxacin was found.
Introduction Enrofloxacin belongs to the new quinolone carboxylic acid group of antibiotics and presents a broad antibacterial spectrum.lI2 This quinolone affects the bacterial cells by interfering with a wide variety of DNA related processes. The main target of enrofloxacin seems to be the DNAgyrase, nevertheless, the exact mechanism of action is not yet clearly understood.314 Enrofloxacin is under investigation as to its usefulness encapsulated in liposomes. Experiments carried out in our laboratory have shown substantial changes in the pharmacokinetic profile of this drug when administered entrapped in liposome^.^ The enrofloxacin molecule has very peculiar solubility characteristics. It is highly soluble in strong acid or basic media, but solubility is minimal at neutral pH. Apparently, the incorporation of this drug into liposomesshould be favored by neutral pH, but the encapsulation values achieved working in these conditions were lower than for acidic pH. Trying to understand the interactions between enrofloxacin and phospholipids at the molecular level, we have studied in detail the behavior of PC and DPPC/enrofloxacin mixtures in mono- and bilayers. Hydrophobic interactions were determined by differential scanning calorimetry (DSC), carboxyfluorescein (CF) leakage, and compression isotherms of mixed monolayers. The interactions between the phospholipid polar heads and the drug were studied by the penetration kinetics and the polarization of the hydrophobic fluorescent label anilinonaphthalene sulfonate (ANS).
Materials and Methods Chemicals. Enrofloxacin was kindly supplied by Infavet (Spain). Egg phosphatidylcholine (PC),dipalmitoylphosphati* To whom correspondenceshould be sent. t Unitat de Ffsico Qufmica, Departament de FarmAcia, Facultat
de FarmBcia. Departament de PBptids, CID-CSIC.
*
AbstractpubliehedinAduanceACSAbstmcts,February1,1994. (1)Sherr, M.Vet. Med. Reo. 1 9 8 7 , 2 , M . (2)Baudtiz, R. University of Veterinary Science, Budapeat: 1990, Uniphanna Co., Ltd.,pp 21-26. (3) Wolfson, J. 5.;Hooper, D. Antimicrob. Agents Chemother. 1985, 28,581-586. (4)Furet, Y. X.;Pechere, J. C. J. Antimicrob. Chemother. 1990,26, 7-15. (5) Cabanes, A.;Haro, I.; Rabanal,F.;Garcla Anthn, J. M.; Reig, F.In Liposomes in Drug Delivery: 21 years on; London, 1990.
dylcholine (DPPC), cholesterol (Chol), and aniliionaphthalenesulfonate (ANS) were purchased from Sigma. Carboxyfluorescein was from Eastman Kodak and was purified by column chromatography as described by Weinstein et d.6 All lipids were pure as judged by thin-layer chromatography. The lipids, enrofloxacin, and mixtures of both components of varying molar compositions were diesolved in chloroform and spread on subphasescomposed of NaClO.9%, pH 5.5, NaClO.9%, pH 7.4, and phosphate buffer saline (PBS) pH, 7.4. The presence of enrofloxacin at concentrationsranging from l(r to lod M does not change the pH of the water solutions. Water for the Langmuir f i i balance was prepared by distillation of deionized water over potassium permanganate in an all-glassapparatus. The pH of water was 5.5-6 and was freehly prepared every day. Methods. Compression isotherms were performed on a Langmuir film balance equipped with Wilhelmy platinum plate as described by Verger and de Haas.7 The output of the pressure pickup (Sartoriusmicrobalance) was calibrated by recording the well-known isotherm of stearic acid. This isotherm is characterized by a sharp phase transition at 25 mN/m on pure water at 20 "C. The Teflon trough (surfacearea 495 cm2,volume 310 mL) was regularly cleaned with hot chromicacid and rinsed with double distilledwater. Films were spread on the aqueous surface from a microsyringe and at least 10 min was allowed for solvent evaporation. Films were compressed at a rate of 4.2 cm/min. A change in the compression rate did not alter the shape of the isotherms. All isothermswere run at least4 timesin the direction of increasing pressurewith freshly prepared fibs. The accuracy of the system under the conditione in which the bulk of the reported measurementswere made was i0.5 mN/m for the surface pressure. All the measurements were made at a surface temperature of 21 i 1 "C, being the subphase constantly stirred to avoid inhomogenities. Penetration Studies. The penetration ability of enrofloxacin into monolayers was measured following the description given by Reig et al.8 Starting from lipid eolutione of 1mg/mL in CH Cla, the lipid was added dropwise and the surface pressure recorded until obtaining 5,10,or 20 mN/m. Once the desired (6)Weinstein, J. N.;Ralaton, E.; Leserman, L. D.; Klauener, R. D.; D r g t e n , P.; Henkart, P.; Blumenthal, R. In Liposome Technology; Gregoriadie,G., Ed.; CRC Preee Inc.: Boca Raton, FL, 19W,Vol. 3,p 183. (7)Verger, R.;de Haas, G. H. Chem. Phys. Lipids 1971,20,127-136. (8)Rei! F.;Buepuete, M.A.; Haro, I.; Rabanal, F.;M i a , M.A. J. Pharm. Scr. 1992,81 (6),546-560.
0743-746319412410-0767$04.50/0 0 1994 American Chemical Society
768 Langmuir, Vol. 10, No.3, 1994
Mestres et al.
fl 8e-11 6e-11 4e-11
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Figure 1. Dependencechangein the enrofloxacinsurfaceexcess on the initial concentration of this molecule in the subphase. 1s '1
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FC
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Figure 3. Surface pressure increases measured after injection of a 1W M enrofloxacin solution under different monolayers spread at 20 mN/m.
1
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CONCENTRATION (M)
Figure 2. Increase of surface pressure of different enrofloxacin solutions on monolayers of DPPC spread at an initial pressure of 5,10,and 20 mN/m. Chart 1. Shape of Enrofloxacin Molecule Approximated by Using the Hyperchem (Autodesk SA) Program
Area (nm2 molec-'1
.,
Figure 4. Compression isotherma of DPPC/enrofloxacinmonoDPPC; layers spread on subphases containing PBS, pH 7.4: 0 , DPPC/E (0.8/0.2); DPPC/E (0.6/0.4); 0 , DPPC/E (0.4/ 0.6); 0,DPPC/E (0.2/0.8); *, E.
*,
initial surface pressure was stabilized, increasing amounts of a concentrated drug solution were successively injected into the subphase and the experimental increases of surface pressures were recorded. The penetration process was achieved in 5 min but the successive additions were made at intervals of 30 min in order to be sure that the system had reached its equilibrium after each addition. The values reported are averagesof triplicate runs. Reproducibility was h0.5 mN/m for surface pressure. Calon'metricAnalysis. DPPC/enrofloxacinmixtures prepared for DSC studies were hydrated with 150 p L water and heated at 60 "C for 1 h. Calorimetric analyses were performed with a differential scanning calorimeter (Perkin-Elmer DSC-2 with intracooler). Weighed amounts of the liposomal samples were sealed in stainless steel pans. For each sample several scans were performed in both heating and cooling modes between 0 and 50 "C with heating rates of 5 "C/min. Indium was used as the calibration standard. Permeability Studies. Smallunilamellarvesicles (SW)were prepared by probe sonicationof multillamelarvesicles (MLV)in a CF 100mM solution. SUV were submitted to gel filtration and dialyzed overnight to obtain samples of high latency values.
Liposomal compositions were PC, PC/Chol (l/l),DPPC, and DPPC/Chol (l/l). Incubations were carried out as described in ref 8. Polarization Studies. These studies were carried out by incubating S W composed of PC or DPPC with ANS until saturation. The samples were then incubated with increasing amountsof enrofloxacinand fluorescencepolarizationcalculated, applying eq I. All these experiments were done at least in triplicate.
11and I 1 are fluorescence intensities recorded by analysis of polarizerparalleland perpendicular,respectively,to the exitation polarizer.
Results and Discussion The size and shape of enrofloxacin were approximated by using the Hyperchem (Autodesk SA) program. The
-
Langmuir, Vol. 10, No. 3,1994 769
Interaction of Enrofloxacin with Phospholipids
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molecule appears to have an elongated shape, the maximum distance among atoms being 1.28 nm as shown in Chart 1. Compression Isotherms. To study the influence of enrofloxacin on phospholipidmonolayers, the compression isotherms of DPPC were carried out, being this quinolone disolved in the aqueous subphase. As in previous studies: we found that the efficiency of encapsulation of this drug into liposomes was highly dependent on the pH and composition of the aqueous medium. These experiences were done on three subphases: PBS, pH 7.4; 0.9 % NaC1, pH 5.5 and 7.4. The concentration of the quinolone in the subphase was 10-6 and le5M and the relationship 35/1 DPPC/enrofloxacin. The presence of the drug under the monolayer did not modify either the compressibility or the area/molecule values. A t very low surface pressures, a slight expansion of the isotherm could be appreciated but up to 5 mN/m this effect disappeared. This behavior was similar for the three subphases assayed and suggests a low level of interaction. Penetration Kinetics. The surface activity of enrofloxacin was determined by injecting increasing amounts of this molecule into phosphate buffer. Two methods were used for this purpose: the additive process described by
Figure 6. Area/molecule values corresponding to the same amount of DPPC spread as a pure component or in a DPPC/ enrofloxacin mixture.
Reig et aL9and the conventionalprocess, injecting different amounts of drug solution in the reservoir. The values of surface pressure determined once the equilibrium was reached showed no differences between both methods. For this reason and to simplify, the following determinations were carried out in the additive way. By application of the Gibbs equation (11),the surface excess of these molecules was calculated and the relationship between this parameter and the subphase concentration is given in Figure 1
being R = 8.31 X lo7erg/(K mol), T = 294 K, AP = pressure increase, and a the concentration of enrofloxacin in the subphase. There is a clear tendency toward saturation at high concentrations of the enrofloxacin in the subphase, thus suggesting the existence of an equilibrium between the drug molecules in solution and a fraction of them in the surface. Nevertheless, this molecule is not able to form monolayers. The surface activity of enrofloxacin was also studied by its penetration on monolayers of PC, DPPC, PC/Chol (1/ l),and DPPC/Chol (l/l), being this molecule injected into a PBS subphase. The penetration process was very fast. The maximum pressure increases were achieved after 5 min and were dependent on the concentration of the drug in the subphase. Moreover, the higher the initial surface pressure of lipid monolayer, the lower the penetration level of enrofloxacin into the monolayer. This behavior is illustrated in Figure 2 for DPPC/enrofloxacin. As far as the influence of the compositionof the monolayer in the penetration is concerned, one can appreciate that the presence of cholesterol has a negative influence in this process. The values corresponding to monolayers at 20 mN/m are given in Figure 3. (9) Reig, F.; Busquets,M. A.; Garcfa AnMn, J. M.; Valencia, G.; Alsina,
M.A. Znt. J . Pharm. 1988,44,257-260.
Mestres et al.
770 Langmuir, Val. 10, No. 3,1994 PBS pH: 7.1
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Figure 7. Area/molecule calculated from the compression isothermsof DPPC/CHOL/enrofloxacin spread on three different subphases: NaClO.9%, pH 7.4 and 5.5; PBS, pH 7.4. Pressure in mN/m: 0, 5; tss, 10; X, 15; /tuo, 20;m, 30; 0,40. The effect is more acute in the case of DPPC. This behavior is related to the well-known rigidifying activity of cholesterol on phospholipid monolayers already described (Reig),lothat is also reflected in compressibility values for DPPC/Chol monolayers, lower than those of the PC/Chol or pure DPPC. These results seem to be in contradiction with the lack of interaction detected when working with compression isotherms of DPPC monolayers. Nevertheless, due to the design of the Langmuir balance used for this study, it is more accurate to measure pressure increases at constant area than changes in the area/ molecule at varying pressures. Only in the case of highly hydrophobic big molecules are the changes in areal molecule high enough to be measured. Miscibility of Phosphatidylcholine/Enrofloxacin. Although enrofloxacin molecules are not able to form (IO)b i g , F.;Meetree, C.; Haro,I.; Valencia, G.; Garck Ant& J. M.; Alsina, M.A. Colloid Polym. Sci. 1989, 267, 139-144.
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Figure 8. Area/molecule calculated from the compression isotherms of PC/enrofloracin spread on three different subphases: NaClO.9%;pH 7.4 and 5.5;PBS, pH 7.4. Pressure in mN/m: 0,5; +, 10; X, 15; A, 20;m, 30; 0,40. monolayers when spread into an aqueous subphase, their high insolubility allowed us to prepare mixed monolayers of this molecule with DPPC, PC, or DPPC/Chol (l/l).In these experiments, equimolecular organic solutions of lipids and enrofloxacin were mixed in different volumes so as to obtain different molar ratios. These mixtures were spread and compression isotherms recorded. The results obtained showed that when working with DPPC/enrofloxacin mixtures, no real collapse points could be observed. This behavior suggests that enrofloxacin alters the packing of DPPC molecules as will be seen also from the values of area/molecule described in the next paragraphs. Moreover, this fact was not dependent on the subphase composition. But on the contrary, the phase change of pure DPPC monolayers was highly influenced by the ionic content of the subphase
Langmuir, Vol. 10, No. 3, 1994 771
Interaction of Enrofloxacin with Phospholipids
TE
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(8.8mN/m, NaC1, pH 5.5; 6.94 mN/m, NaC1, pH 7.4; 11.38 mN/m, PBS, pH 7.4). The compressibility of the compression isotherms corresponding to DPPC/enrofloxacin mixtures decreases as the drug content increases (Figure 4). The area/molecule values calculated from the compression isotherms are represented in Figure 5. The last point corresponding to monolayers of pure enrofloxacin is lacking due to the inexistence of a real monolayer. Deviations from ideality are very small, although AGE^ cannot be quantified because one of the components does not form a monolayer. Nevertheless, the presence of PBS at pH 7.4 in the subphase has a positive effect on stabilizing the mixed monolayers and allowingthem to be compressed at higher surface pressures than for the other subphases. Probably, this effect is mainly due to a stronger incorporation of enrofloxacin in the monolayer, being its solubility is minimal at neutral pH. It is well known that in some cases the shape and area/ molecule values in isotherms can vary according to the amount of lipid spread on the subphase. To be sure that the differences in area/molecule found in the mixed monolayers are really due to the presence of enrofloxacin and not only the result of the presence of a lower amount of phospholipid on the surface, monolayers of pure DPPC were prepared that contain the same number of DPPC molecules as in the mixtures DPPC/enrofloxacin. The area/molecule values of DPPC calculated from the pure isotherms were smaller than those values of the mixed ones. This result is indicative of the presence of antibiotic
molecules as a stable component of the monolayer. The histogram given in Figure 6 represents these pairs of values when the subphase was PBS, pH 7.4, measured at 5 and 20 mN/m. I t seems as if the phospholipids and enrofloxacin dissolved in organic solvents could form some type of complexesor ion pairs that behave more hydrophobic than the parent components. The miscibility patterns of DPPC/Ch/enrofloxacin and PC/enrofloxacin present similar characteristics than those already described for DPPC. The diagrams of area/ molecule for both lipid compositionsat the three subphases are given in Figures 7 and 8. Calorimetric Studies. The DSC curves of mixtures DPPC/enrofloxacin prepared a t two pH values (5.5 and 7.4) combined the characteristic features of the thermograms of each component (Figure 9). The presence of increasing quantities of enrofloxacin results in a small but progressive decrease in the cooperativity of the DPPC main transition and a reduction in its AH while transition temperature (T,)remains unchanged. As expected enrofloxacin progressively reduces the temperature and AH of the DPPC pretransition. When the molar fraction of enrofloxacin is 0.60, the pretransition is completely abolished. As the shape of thermotropic curves broadens in mixtures, one can say that the interactions between both types of molecules do not affect strongly the intermolecular forces among molecules of the same type. One can
772 Langmuir, Vol. 10, No.3, 1994 Table 1. EffMt of Enrofloxacin in Membrane Fluidity.
ANS
PC fluorescence 149.7 polarization (P) 0.169 fluidity (UP) 5.91
PC/E DPPC DPPC/E DPPCb DPPC/Eb 180.7 218.5 239.8 98.7 96.1 0.198 0.177 0.213 0.128 0.228 4.69 7.81 4.38 5.05 5.64
a Fluorescence, polarization, and fluidity parameters measured after incubation of enrofloxacin with small unilamelar vesicles of different lipidic composition labeled with ANS.b 50 "C.
hypothesize the formation of clusters. In this case, from a quantitative point of view, the number of DPPC/ enrofloxacin molecules interacting each other will represent a small fraction of the total number present in the media. Moreover, if the type of interactions were mainly at the level of the polar heads, this will explain also the stability of T,independently of the phospholipid molar fraction.
Effect of Enrofloxacin on Phospholipid Bilayers. The incubation of liposomes containing CF with different amounts of enrofloxacin showed no leakage of this fluorescent marker. These results show that enrofloxacin remains at the surface of liposomes interacting mainly by electrostatic forces with its polar heads. The experiments carried out with SUV liposomes composed of PC (21 "C) and DPPC (21 and 50 "C) (Table l ) , using ANS as a fluidity marker, showed that the addition of enrofloxacin increases in all cases the polarization values of the samples inducing in consequence a decrease in the fluidity at the membrane surface. The changes observed in the polarization of ANS are not due to interactions between this marker and enrofloxacin as was previously determined by mixing different amounts of the quinolone and ANS. The effect of enrofloxacin in the fluidity of DPPC bilayers is highly dependent on the temperature. At temperatures above the T,the rigidifying effect is stronger than when lipids are in the gel phase. This result is not easily understood because this transition reflects changes in the mobility of the alkyl chains of phospholipids and the changes measured using ANS polarization are mainly related to the polar heads of phospholipids. Moreover, considering the results obtained by Namll for tumor promoters which are molecules having a similar
Mestres et al. shape to enrofloxacin, one can appreciate that this antibiotic produces a stronger disturbance in the fluorescence and polarization parameters. Having in mind the results obtained in the calorimetric scans and in the CF release measurements, one must conclude that there is no significant interaction between enrofloxacin and the membrane a t the level of the alkyl chains. Following the suggestion given by Nam" in his paper, the high differences in fluidity observed after incubation of liposomes with enrofloxacin can be due to changes in the orientation of the phosphorylcholine group from parallel to perpendicular to the membrane surface. This event could be induced by the presence of enrofloxacin that could stand stacked between the phosphorylcholine groups forming some kind of neutral ionic pairs. This new disposition would explain the increase in rigidity of the bilayer and also increases in pressure when working at constant area. The observed increase in the fluorescence quantum yield of the ANS would be due to the electrostatic neutralization followed after the interaction between PC and enrofloxacin and would be in agreement with the apparent increase in hydrophobicity of phospholipid/ enrofloxacin pairs compared to the behavior of both isolated molecules as already mentioned. Assuming that the molecule would probably locate with ita main axis parallel to the alkyl chains of the phospholipid and, the carboxyl end facing the aqueous subphase, the areas occupied per molecule would be approximately of 0.3 nm2. This value correlates well with the differences in area/molecule found between monolayers of pure DPPC and mixed monolayers of DPPC/enrofloxacin. For this reason it seems logical to accept that enrofloxacin has a similar effect in monolayers than cholesterol. Moreover, as the total length of the enrofloxacin molecule is 1.28 nm and the thickness of a phospholipid monolayer is around 2 nm, the disturbance created by this molecule affects mainly the polar part of the lipids.
Acknowledgment. Thiswork was supported by agrant (No. B1092-0982-C02-02) from CICYT (Spain). The technical assistance of Ms. Maria Osuna and Mr. Emilio Nogu6s is gratefully acknowledged. (11) Nam,K.Y.;Kimura, S.;Imanishi, Y.;Fujiki, H.Chem. 1989,34, 43-45.
