Interaction of 6-Fluoroquinolones with Dipalmitoylphosphatidylcholine

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Interaction of 6-Fluoroquinolones with Dipalmitoylphosphatidylcholine Monolayers and Liposomes Anna Grancelli,† Antoni Morros,† Miquel E. Caban˜as,‡ O Å scar Dome`nech,§ § §,| § Sandra Merino, Jose´ Luis Va´zquez, M. Teresa Montero, Miguel Vin˜as,⊥ and Jordi Herna´ndez-Borrell*,§ Unitat de Biofı´sica, Departament de Bioquı´mica i Biologia Molecular, Facultat de Medicina, U.A.B. 08193-Bellaterra, Spain; Laboratori V de Fisicoquı´mica, Facultat de Farma` cia, U.B. 08028-Barcelona, Spain; and Laboratori de Microbiologia, Campus de Bellvitge, U.B. 08907-L’Hospitalet de Llobregat, Spain Received April 16, 2002. In Final Form: September 9, 2002 The interaction of 6-fluoroquinolones at the lipid-water interface is the primary step for the activity of membrane transporters involved in the generation of drug resistance. In this work the interaction of the antibiotic ciprofloxacin (CPX) and its N-4-butylpiperazinyl derivative (BCPX) with dipalmitoylphosphatidylcholine (DPPC) as a model membrane is described. BCPX forms a stable film at the air-water interface and induces a condensing effect of the DPPC monolayer. A basic thermodynamic analysis was performed which suggests a possible segregation of the drug at the lowest proportion studied. The temperature dependence of 3-(4-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid fluorescence anisotropy measurements shows that the incorporation of CPX or BCPX into bilayers does not greatly modify the DPPC lamellar gel state. In the liquid-crystalline phase, BCPX lowered the anisotropy values and both drugs lowered the main transition temperature (Tm) of pure DPPC by approximately 1 °C. On the other hand, significant changes in the cooperativity of the phospholipid transition were found only in the presence of BCPX. In 31P-NMR experiments the presence of BCPX induced, both below and above the main transition, a strong line shape narrowing of the DPPC spectra that suggests an increase in the local mobility of the phosphate groups; that is, the interactions between DPPC headgroups are altered by the presence of BCPX. Although the incorporation of CPX also promotes a remarkable line shape narrowing in the fluid phase, it only slightly modifies the spectral parameters of DPPC in the gel phase. This suggests that CPX could be “squeezed out” or segregated from the lipid-water interface when the phospholipids are in this tight packed phase. The segregation is likely to occur to defects on the gel phase. Values of 1-anilino-8naphthalenesulfonate binding to the liposome surface were fitted to a Freundlich-like isotherm. The binding constant (K) and maximum concentration bound to liposomes (Cm) both are dependent on the structure of the drugs, which indicates a definite effect due to the drug hydrophobicity. K values in the presence and absence of drug were used to calculate the variation in the surface potential (∆Ψ) of the liposomes. All the results are consistent with an electrostatic interaction of 6-fluoroquinolones at the lipid-water interface. This interaction is favored by the presence of the N-4-butyl chain and could have important implications in the efflux of this drug from bacteria.

Introduction 6-Fluoroquinolones are a group of synthetic antibiotics structurally related to nalidixic acid, which were introduced more than 10 years ago and are used for a wide range of infections.1 The extensive use and misuse of these compounds in both human and veterinary medicine led to the emergence and spread of resistant clones. These appear mainly by mutations in the structural genes encoding their intracellular targets (DNA gyrase and topoisomerase IV) but also in combination with modifications in membrane permeability (decrease in influx and/ or increased efflux).2,3 There is evidence that one of the mechanisms involved in the development of resistance is the extrusion of drugs * Corresponding author. E-mail: [email protected]. † Unitat de Biofı´sica. ‡ Servei de Ressona ` ncia Magne`tica Nuclear (SeRMN). § Laboratori V de Fisicoquı´mica. | Present address: Howard Hughes Medical Institute, University of California Los Angeles, 5-748 Macdonald Building, Box 951662, Los Angeles, CA 90095-1662. ⊥ Laboratori de Microbiologia. (1) Hooper, D. C.; Wolfson, J. S. In Quinolone Antimicrobial Agents, 2nd ed.; American Society for Microbiology; Washington, DC, 1995.

by efflux pumps.4 It is noteworthy that their mechanism of action is (i) unspecific, due to the ability to extrude chemically unrelated compounds, and (ii) dependent on the surrounding lipid bilayer environment. It is believed4 that substrates are transported first from the lipid bilayer to the transporter protein by a still unclear mechanism.5 Therefore, it seems that a capture mechanism of the drug by the inner leaflet of the cytoplasmic membrane is required. Hence, the understanding of the lipid surfacedrug interaction becomes of biological relevance. Ciprofloxacin (CPX) is currently used as a paradigm for 6-fluoroquinolones. In a previous work6 we reported a preferential interaction between CPX and the negatively charged group present at the lipid bilayer. Another work7 (2) Hirai, K.; Aoyama, H.; Irikura, T.; Iyobe, S.; Mitsuhashi, S. Antimicrob. Agents Chemother. 1986, 30, 248-253. (3) Charvalos, E.; Tselentis, Y.; Hamzehpour, M. M.; Ko¨hler, T.; Peche`re, J. C. Antimicrob. Agents Chemother. 1995, 39, 2019-2022. (4) Van Bambeke, F.; Balzi, E.; Tulkens, P. M. Biochem. Pharmacol. 2000, 60, 457-470. (5) Putman, M.; Van Veen, H. W.; Konings, W. N. Microb. Mol. Biol. Rev. 2000, 64, 672-693. (6) Va´zquez, J. L.; Montero, M. T.; Merino, S.; Dome`nech, O Å .; Berlanga, M.; Vin˜as, M.; Herna´ndez-Borrell, J. Langmuir 2001, 17, 1009-1014.

10.1021/la025837h CCC: $22.00 © 2002 American Chemical Society Published on Web 11/02/2002

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Figure 1. Structure of CPX (R ) H) and BCPX (R ) CH3(CH2)3-).

suggested that introduction of a butyl chain in the N-4position of the piperazinyl groups of CPX (to give BCPX) led to a different mechanism of interaction with liposomes. Zwitterionic and neutral microspecies predominate (approximately 85%) under the present experimental conditions.8 We have also shown that CPX and BCPX (Figure 1) have different modes of interaction with liposomes, which could be related to the activity of a microbial efflux pump system in Serratia marcescens. Hence, to gain insight into the molecular level phenomena, we have extended here our work by means of 31P-NMR spectroscopy in combination with 1-anilino-8-naphthalenesulfonate (ANS) binding and fluorescence anisotropy experiments in bilayers and mixing measurements in monolayers. The objectives of this study were (i) to investigate the nature of the forces that determine the interaction of CPX and BCPX at the lipid-bilayer interface; (ii) to ascertain the effect of each drug on the headgroup region; and (iii) to provide information about the capture mechanism exerted by membranes on 6-fluoroquinolones. Experimental Section Chemicals. Dipalmitoylphosphatidylcholine (DPPC > 99%) was purchased from Avanti Polar Lipid Co. (Alabaster, AL). 1-Cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid (CPX) was obtained from Cenavisa (Reus, E), and the BCPX was synthesized according to a procedure elsewhere described.9 The purity of the compounds was assessed by IR and HPLC. 1-Anilino-8-naphthalenesulfonate (ANS) and 3-(4-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (PA-DPH) were obtained from Molecular Probes (Eugene, OR). Buffers were 20 mM Tris, pH 7.4, I ) 0.15 m, for monolayers and 31P-NMR experiments and 50 mM Hepes, pH 7.4, I ) 0.15 m, for fluorescence experiments. Monolayer Studies. Stock solutions of the lipid and 6-fluoroquinolones were made up in chloroform/methanol (50:50, v/v) to known concentrations of about 1 mM. To examine how the alkylation of the N-4-piperazinyl group of CPX affected the mean molecular areas in mixed fluoroquinolone/DPPC monolayers, force-area isotherms of the monolayers at two different compositions (0.7:0.3 and 0.5:0.5, DPPC/fluoroquinolone) were obtained. Monolayers were spread by depositing a volume of about 50 µL of the stock solution onto the buffer solution subphase (22 ( 2 °C). After 10 min for solvent evaporation, the monolayers were compressed at a constant rate of 20 mm2/s. The compression speed and the time allowed before compression were maintained constant in all experiments. The equipment used in this work was a KSV LB 5000 system (Helsinki) with a 15 cm × 54 cm trough. The interaction between two components (i.e. DPPC and BCPX) in a mixed monolayer can be evaluated from the calculation of the excess Gibbs energy (GE),10,11 which is given by (7) Va´zquez, J. L.; Montero, M. T.; Merino, S.; Dome`nech, O Å .; Berlanga M.; Vin˜as, M.; Herna´ndez-Borrell, J. Photochem. Photobiol. 2001, 73, 14-19. (8) Va´zquez, J. L.; Montero, M. T.; Merino, S.; Dome`nech, O Å .; Berlanga, M.; Vin˜as, M.; Herna´ndez-Borrell, J. Langmuir 2002, 18, 3288-3292. (9) Montero, M. T.; Freixas, J.; Herna´ndez-Borrell, J. Int. J. Pharm. 1997, 149, 161-170. (10) Pagano, R. E.; Gershfeld, J. J. Phys. Chem. 1972, 76, 12381243.

∫ [A π

0

12

- (X1A1 + X2A2)] dπ

(I)

where A12 is the average molecular area in the binary mixture, A1 and A2 are the molecular areas of pure monolayers of components 1 and 2, respectively, and X1 and X2 are their corresponding mole fractions in the mixed monolayers. In this work, GE was evaluated by integration of the π-A isotherms using the software package Origin. The Gibbs energy of mixing is given by

∆mixG ) ∆mixGid + GE

(II)

where the first term, the ideal Gibbs energy of mixing (∆mixGid), can be calculated from the equation

∆mixGid ) RT(X1 ln X1 + X2 ln X2)

(III)

where R is the gas constant and T is the temperature. Vesicle Preparation. Chloroform/methanol (50:50, v/v) solutions containing the appropriate amounts of DPPC and CPX or BCPX were mixed to give the desired final lipid/drug molar ratio. The solutions were dried under a stream of oxygen-free N2 in a conical tube, and the thin film obtained was kept under high vacuum for approximately 3 h to remove organic solvent traces. Unsonicated multilamellar vesicles (MLVs) for the 31P-NMR experiments were obtained by resuspending the film with 10 mL of buffer by vortex mixing and keeping the samples at 50 °C until a homogeneous suspension was obtained. The final suspension was then frozen by using liquid nitrogen. The freeze and thaw cycle was repeated two more times. The resulting multilamellar vesicles were then pelleted by ultracentrifugation at 115000g for 1 h at 20 °C. The hydrated pellet was then resuspended in 300 µL of supernatant and placed in a conventional 5 mm NMR tube. A capillary tube containing D2O was added for fieldfrequency stabilization. For fluorescence studies, the suspensions obtained after resuspension of the film were filtered through polycarbonate membranes (100 nm nominal diameter) using an Extruder device obtained from Lipex Biomembranes (Vancouver, BC, Canada) to obtain large unilamellar vesicles. The size and polydispersity of each preparation were monitored systematically by quasi-elastic light scattering using an Autosizer IIc photon correlation spectrophotometer (Malvern, Instruments, U.K.). Fluorescence Measurements. All measurements were carried out using an SLM-Aminco 8100 spectrofluorometer provided with a jacketed cuvette holder. The temperature was controlled to within 0.1 °C using a circulating water bath (Haake, Germany). The excitation and emission slits were 8 and 8 nm and 4 and 4 nm, respectively. Steady-State Anisotropy Experiments. Experimental details for these experiments have been previously reported.6-8 Briefly, steady-state anisotropy (rs) values were calculated according to

rs )

IVV - GIVH IVV + 2GIVH

(IV)

where IVV and IVH are the polarized intensities measured in directions parallel and perpendicular to the exciting beam and G is the grating correction factor equal to IHV/IHH. The parameters B and Tm, calculated from the slope and the inflection point of the data fitted to sigmoid curves, are the cooperativity and temperature of the gel-to-liquid-crystal phase transition of the lipid, respectively. The following equation was fitted to the anisotropy versus temperature data:

rs ) rs1 + p1T +

rs2 - rs1 + p2T - p1T 1 + 10B(1/T-1/Tm)

(V)

where T is the absolute temperature, Tm is the midpoint of the phase transition, B is a measure of the cooperativity of the (11) Mill, I.; Cvitas, T.; Homan, K.; Kallay, N.; Kutchitsu, K. Quantities, Units and Symbols in Physical Chemistry; Blackwell Scientific: Oxford, 1988; p 46.

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transition, p1 and p2 correspond to the slopes of the straight lines at the beginning and at the end of the plot, and r1 and r2 are the anisotropy intercepting values at the y axis. From rs values the limiting anisotropy (r∞) was determined using the following relationship12

The chemical shift anisotropy (CSA) of the phosphorus atoms of phospholipids is a parameter frequently used to characterize the 31P-NMR spectra and is obtained as the chemical shift difference between the high field peak and the low field shoulder.18 Since the 31P-NMR spectra of mixed liposomes do not always show a well-defined shoulder at low field, the determination of CSA directly from the powder pattern is not very accurate.19 We used instead the second moment (M2) of the powder pattern spectra,20 which can be accurately evaluated and can be used to make objective comparisons among experimental spectra.21 M2 values report on the square of the headgroup order parameter22 and so reflect the changes occurring in the structure and dynamics of the phosphate headgroups.23

r∞ ) 4/3rs - 0.10

for

0.13 < rs < 0.28

(VI)

r∞ reflects restriction of probe motion and can be converted to an order parameter (S)

S2 ) r∞/r0

(VII)

where r0 is the fluorescence anisotropy in the absence of any rotational motion of the probe. Binding Experiments. Experimental details for these experiments have been previously reported.6-8 Basically, the adsorption data of ANS binding to bilayers were fitted to an equation derived from the Freundlich isotherm13

(K[ANS]∞)b [ANS]B ) Cmax 1 + (K[ANS]∞)b

(VIII)

where K is the binding constant, Cmax is the maximum concentration bound to liposomes, b is interpreted as the cooperativity of the binding process, and the B and ∞ subscripts refer to the bound and free ANS concentration, respectively. Equation VIII can be used to calculate the variations in the electrostatic surface potential according to

RT Kfq ∆Ψ ) ln F K0

(IX)

where R and F are the gas and Faraday constants, respectively, and Kfq and K0 are the binding constants for ANS in the presence and absence of fluoroquinolones, respectively. 31P-Nuclear Magnetic Resonance. 31P-NMR spectra were recorded on a Bruker ARX-400 spectrometer (Bruker Espan˜ola, S.A., Madrid) operating at 161.98 MHz using a single 90° pulse sequence, with proton-decoupling during signal sampling by means of a Waltz-16 composite pulse sequence.14,15 The single pulse sequence was used instead of the phase-cycled Hahn echo pulse sequence16 to obtain spectra with higher signal-to-noise ratios.17 Each spectrum was the result of accumulating 14 000 scans sampled using 4096 complex data points, with the 90° pulse width in the range 12-14 µs (Beff ) 21-18 kHz), and a spectral width of 20 000 Hz. An interpulse delay of 1.2 s was used to further improve the signal-to-noise ratio, which resulted in moderate saturation of the narrow peak at high field. Before Fourier transformation, signals were zero-filled to 16 384 points and an exponential multiplication resulting in a line broadening of 5 Hz was applied. Twelve spectra were recorded from each sample at temperatures ranging from 25 to 50 °C. The sample temperature was set and monitored ((0.1 °C) with the aid of a Bruker BVT-2000 variable temperature unit, and the sample temperature was allowed to stabilize prior to acquisition. Spectra were processed on a SGI Indy workstation running the XWINNMR software. All chemical shift values are quoted in parts per million (ppm) with reference to pure lysophosphatidylcholine micelles (0 ppm). The real part of each phased spectra was smoothed using the Grams 32 (Galactic Corporation) software. The second moment was calculated from the smoothed spectra by using the Jandel Sigma Plot (Jandel Corporation) software. (12) van Blitterswijk, W. J.; van Hoeven, R. P.; van der Meer, D. Biochim. Biophys. Acta 1981, 644, 323-332. (13) Serra, R.; Mas, F.; Dı´az-Cruz, J.; Arin˜o, C.; Estevan, M. Electroanalysis 2000, 12, 60-65. (14) Killian, J. A.; Fabrie, C. H. J. P.; Baart, W.; Morein, S.; de Kruijff, B. Biochim. Biophys. Acta 1992, 1105, 253-262. (15) Go´mez-Ferna´ndez, J. C.; Llamas, M. A.; Aranda, F. J. Eur. J. Biochem. 1999, 259, 739-746. (16) Rance, M.; Byrd, R. A. J. Magn. Reson. 1983, 52, 221-240. (17) Lewis, R. N. A. H.; Sykes, B. D.; McElhaney, R. N. Biochemistry 1998, 27, 880-887.

∑(w - M ) I ∑I 2

M2 )

i

1

i

(X)

i

where wi and Ii are the frequency and intensity of the ith data point, respectively, and M1 is the first moment, wich corresponds to the isotropic chemical shift of the sample. The short recycling delay used in these experiments (TR ) 1.2 s) results in small differential saturation effects, that in turn translate into moderately overestimated M2 values with respect to those calculated from fully relaxed spectra (TR > 3 s). This fact does not hinder the qualitative interpretation of the changes induced in M2 by the presence of CPX/BCPX.

Results and Discussion Monolayer Compression Isotherms. It is known from previous studies24 that (i) pure CPX does not form a film at the air-water interface and (ii) there is a shift of the isotherms toward lower area per molecule for DPPC/ CPX mixed monolayers. The surface pressure-area per molecule isotherms for DPPC/BCPX monolayers at various molar ratios were shown in Figure 2. The isotherm for DPPC including the liquid-expanded to liquid-condensed phase transition and the collapse pressure value (65.7 mN/m) was consistent with others in the literature.24-26 As expected from its log P (partition coefficient) value,9 BCPX forms a stable film at the air-water interface (Figure 2). Incorporation of BCPX into a DPPC monolayer induces a condensing effect and modifies its structure. The collapse pressure for the monolayers with XBCPX ) 0.3 and XBCPX ) 0.5 were 69.8 and 59.7 mN/m, respectively. The values of the excess Gibbs energy (GE) for the monolayer with the highest proportion of BCPX were all negative (see Table 1). However, this monolayer becomes even more condensed than the pure drug; therefore, these values should be taken with caution.27 On the other hand, the features for the monolayer with the lowest proportion of BCPX, particularly the positive values of GE, are usually interpreted as a result of a small tendency toward lateral phase segregation of the drug from the phospholipid environment. This phenomenon was also observed for (18) Cullis, P. R.; de Kruijff, B. Biochim. Biophys. Acta 1978, 507, 207-218. (19) Beyer, K. Biochim. Biophys. Acta 1986, 855, 365-374. (20) Herreros, B.; Metz, A. W.; Harbison, G. S. Solid State Nucl. Magn. Reson. 2000, 16, 141-150. (21) Davis, J. H. Biochim.Biophys. Acta 1983, 737, 117-171. (22) Le´onard, A.; Dufourc, E. J. Biochimie 1991, 73, 1295-1302. (23) Gaillard, S.; Renou, J. P.; Bonnet, M.; Vignon, X.; Dufourc, E. J. Eur. Biophys. J. 1991, 19, 265-274. (24) Montero, M. T.; Herna´ndez-Borrell, J.; Keough, K. M. W. Langmuir 1998, 14, 2451-2454. (25) Notter, R. H.; Tabak, S. A.; Mavis, R. D. J. Lipid Res. 1980, 21, 10-22. (26) Min˜ones, J.; Rodriguez Patino, J. M.; Conde, O.; Carrera, C.; Seoane, R. Colloids Surf. 2002, 203, 273-286. (27) The absence of drugs in the aqueous subphase by possible desorption from the monolayer was demonstrated by fluorimetry.

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Figure 2. Surface pressure-area per molecule isotherms of DPPC (1), BCPX (4), and mixed DPPC/BCPX monolayers at different molar ratios: 0.7/0.3 (2); 0.5/0.5 (3). (GE)

Table 1. Excess Gibbs Energy of Mixing and Gibbs Energy of Mixing (∆mixG) (J/mol) at Different Surface Pressures for the Two Mixed Monolayers Studied 0.5:0.5 DPPC/BCPX π (mN/m)

GE

40 30 20 10

-579.17 -246.45 -204.55 -189.72

Figure 3. Steady-state polarization of PA-DPH in the absence (triangles) and presence of CPX (open circles) and BCPX (filled circles) for DPPC liposomes as a function of temperature. Each point is the mean value of three experiments, but SD is not shown for the sake of clarity; continuous lines are the fitted curves. The total lipid/drug molar ratio was 20. Table 2. Transition Temperature (Tm) and Cooperativity (B) of DPPC Liposomes in the Absence and Presence of CPX or BCPX Calculated from the Inflection Point of PA-DPH Anisotropy Curves (Figure 2)a

∆mixG

GE

-2274.29 -1941.57 -1899.67 -1884.85

757.93 686.62 458.35 129.16

∆mixG -740.87 -812.18 -1040.45 -1369.64

other 6-fluoroquinolones.28 In addition, increasing surface pressure results in larger values of the excess Gibbs energy indicating greater phase segregation at higher surface pressures. The values of Gibbs energy of mixing for the mixed monolayers studied here were negative. Fluorescence Polarization. PA-DPH is a fluorescent probe that is currently used to obtain information of the lipid bilayer-water interface.29 The temperature dependence of PA-DPH fluorescence anisotropy for DPPC liposomes in the presence and absence of CPX and BCPX is shown in Figure 3. For pure DPPC, rs decreased slightly with increasing temperature, with a sharp drop occurring at 42.9 °C, the Tm temperature. This result is consistent with previous anisotropy experiments6 and earlier work using differential scanning calorimetry (DSC).24 As can be seen, the incorporation of CPX or BCPX does not greatly modify the DPPC lamellar gel state. At temperatures below Tm, both drugs caused an increase in rs values. A decrease above Tm is produced by BCPX but not by CPX. The variations of Tm and the cooperativity values in the presence and absence of both drugs, as observed by PADPH fluorescence anisotropy, are reported in Table 2. Inspection of the table reveals that CPX and BCPX both (28) Nag, K.; Keough, K. M. W.; Montero, M. T.; Trias, J.; Pons, M.; Hernandez, J. J. Liposome Res. 1996, 6, 713-736. (29) Marsh, D. Handbook of Lipid Bilayers; CRC Press: Boca Raton, FL, 1990; pp 135-156.

31P-NMR

PA-DPH

0.7:0.3 DPPC/BCPX DPPC CPX BCPX

Tm (°C)

B

rs

S

Tm (°C)

42.9 ( 0.1 41.8 ( 0.2 41.6 ( 0.1

26 270 ( 1412 27 210 ( 1169 34 060 ( 2148

0.2528 0.2336 0.2129

0.792 0.748 0.697

41.2 ( 0.1 40.4 ( 0.1 39.4 ( 0.1

a The anisotropy (r ) and order parameter (S) were calculated s from eq VII at 43 °C, and Tm was calculated from the inflection points of Figure 4B.

lowered by approximately 1 °C the Tm of the pure DPPC. These findings are in agreement with an electrostatic interaction between the drugs and the headgroup of the phospholipids previously reported.6,8 However, these marginal decreases suggest that the driving force is not exclusively the electrostatic interaction but rather the hydrophobic interaction.7,24 Interestingly, whereas the cooperativity (B) of the transition was not substantially affected by CPX, values increased in the presence of BCPX. These data confirm that the effect of BCPX is on the headgroup region and also at a certain level of the hydrophobic core of the bilayer. Furthermore, the presence of 6-fluoroquinolones decreased the anisotropy and order parameter of PA-DPH, indicating an increased rotational mobility of the probe (Table 2). These values were calculated at 43 °C according to eqs IV and VII, for the phase coexistence range, the one that allows the application of eq VII. The present data are consistent with DSC data24 and support the idea that the alkylated piperazinyl derivatives of CPX would intercalate between phospholipid molecules. Thus, the addition of the four-carbon chain group would place the drug somewhere in the bilayer, probably in the vicinity of the first four carbons of the acyl chain, with its carboxylate group being directed to the interface. In this

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Figure 5. Temperature dependence of the second moment (M2) calculated from spectra of pure DPPC (triangles), DPPC/ CPX (open circles), and DPPC/BCPX (filled circles). The total lipid/drug molar ratio was 6. Data are means ( SD for two experiments.

Figure 4. Solid-state 31P-NMR spectra at 25 and 50 °C corresponding to pure DPPC (A), DPPC/CPX (B), and DPPC/ BCPX (C). All spectra were normalized to the same height. The total lipid/drug molar ratio was 6.

way, its carboxyl group could to some extent participate in an electrostatic interaction with the phospholipid headgroup. Therefore, to investigate the molecular level of interaction, 31P-NMR was applied. 31 P-Nuclear Magnetic Resonance. Figure 4 shows the 31P-NMR powder pattern spectra of DPPC in the presence and absence of CPX or BCPX, at two different temperatures below (25 °C) and above (50 °C) the gelto-liquid crystal phase transition. Both 31P-NMR spectra, below and above Tm, show the typical features for DPPC arranged as a bilayer16 structure. The incorporation of CPX in the DPPC lamellar gel state (Figure 4B, left) did not induce significant change in the spectrum when compared to that of the pure phospholipid. Notably, the presence of BCPX results in the narrowing of the gel phase DPPC spectra (Figure 4C, left). Remarkably, in the liquidcrystalline phase, however (Figure 4B and C), both drugs produced a similar effect that led to a considerably narrower spectrum, as compared with that of pure DPPC. Figure 5 shows the temperature dependence of the second spectral moment (M2) for DPPC in the presence or absence of both fluroquinolones. For pure DPPC a temperature increase leads to a monotonic decrease in M2 (Figure 5) followed by a drastic decrease corresponding to the main transition temperature of the lipid bilayer which can be attributed to an increase in the phosphate heads’ local motions.30 As can be seen, the presence of BCPX induces a very strong decrease in the values of M2, as compared with those for pure DPPC, both below and above the main transition. It is striking to note that whereas CPX does not modify the thermal variations in M2 below Tm, it promotes a remarkable decrease of M2 as compared with that for pure DPPC above Tm. Table 2 shows Tm values calculated from fitting the data of Figure 5 to sigmoid curves in the temperature (30) Montenez, J. P.; van Bambeke, F.; Piret, J.; Schanck, A.; Brasseur, R.; Tulkens, P. M.; Mingeot-Leclercq, M. P. Eur. J. Pharmacol. 1996, 314, 215-227.

range encompassing the main transition.31,32 A value of 41.2 °C, lower than that obtained in the anisotropy experiments, resulted in pure DPPC. However, both values fell in the range of values commonly found in the literature.33 Importantly, in the presence of CPX or BCPX, the Tm values of DPPC underwent decreases of about 1 and 2 °C, respectively. A decrease in CSA or in line width of the 31P-NMR powder pattern spectra could result from a change in the size of the multilamellar liposomes17 that in this case could be induced by the presence of fluoroquinolones. We ruled out such a possibility by monitoring the size and distribution of the vesicles, in the presence and absence of drugs, by light scattering. Therefore, the decrease in M2 values observed in the presence of CPX or BCPX should indicate either an increase in the local mobility of the phosphate groups or a change in their average orientation with respect to the bilayer normal.34 The binding of BCPX to the lipid-water interface could disrupt interheadgroup electrostatic interactions, which restrict the axial and lateral motions of the phospholipid molecules. That is, the insertion of BCPX molecules between neighboring headgroups could result in removal of phosphate-choline interactions. A similar effect could be proposed for CPX but only in the liquid-crystalline phase, as indicated by the M2 decrease. As mentioned above, an alternative explanation could be a reorientation of the phosphate moiety of DPPC headgroups due to the presence of the drug. ANS Binding Experiments. ANS is an anionic probe that fluoresces only when it binds electrostatically with the head region of the phospholipids. As can be seen in Figure 6, the effect of the incorporation of either CPX or BCPX results in a competence mechanism with ANS, as demonstrated by comparison with fluorescence values obtained with pure DPPC. The values obtained by fitting the isotherm (eq VII) to the ANS binding data and the calculated surface potential values (eq VIII) are shown in Table 3. As expected, Cmax and K in the presence of BCPX were lower than the values when CPX was present. On the other hand, the cooperativity values obtained were similar in all cases. Remarkably, the variations in the (31) De Boeck, H.; Zidovetzki, R. Biochim. Biophys. Acta 1988, 507, 244-252. (32) Browning, J. L.; Seelig, J. Biochemistry 1980, 19, 1262-1270. (33) Chen, S. C.; Sturtevant, J. M.; Gaffney, B. J. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 5060-5063. (34) Girault, L.; Lemaire, P.; Boudou, A.; Debouzy, J. C.; Dufourc, E. Eur. Biophys. J. 1996, 24, 413-421.

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Table 3. Parameters Obtained from ANS Binding Data on DPPC Liposomesa Cmax [CPX or BCPX] (µM) 0 50

CPX

Kapp (10-2)

B BCPX

24.03 ( 1.37 18.53 ( 0.45 17.05 ( 0.54

CPX

BCPX

0.83 ( 0.08 1.08 ( 0.07 0.94 ( 0.05

CPX

BCPX

0.19 ( 0.03 0.25 ( 0.02 0.17 ( 0.01

Ψ (mV) CPX

BCPX

+7.04

-2.85

a

Cmax is the maximum fluorescence intensity (related with the maximum concentration bound), B is the cooperativity, Kapp is the binding constant, and Ψ is the variation in the surface potential of liposomes.

Conclusions

Figure 6. Fluorescence intensity as a function of ANS concentration for DPPC liposomes in the absence of drugs (triangles) and in the presence of CPX (open circles) and BCPX (filled circles) at 25 °C. The total lipid/drug molar ratio was 1. Data are means ( SD for three experiments.

electrostatic surface potential reveal an opposite effect caused by each drug. Although this is a reproducible effect,6,8 it is important to notice that the DPPC/drug molar ratio was very high (1:1). Therefore, the relative values calculated should be taken only as indicative of the competence mechanism, different for each drug, occurring at the headgroup region. It was not possible with this technique to obtain significative variations of the electrostatic surface potential values with lower proportions of drug.

The monolayer, binding, and anisotropy experiments reported in the present work emphasize the fact6-8 that CPX and BCPX might be surface active drugs that interact at the lipid-bilayer interface where the headgroups are located. 31P-NMR experiments provide evidence that fluoroquinolones have an effect on the headgroup ordering and/or average orientation of the DPPC headgroups, which appears to depend on the phospholipid phase. Thus, whereas in the fluid phase CPX and BCPX both enhance the mobility of the headgroups of DPPC, in the gel phase CPX has no effect on the phosphate group ordering. It is possible that the tight packing of the gel phase does not allow the molecule of CPX to insert itself among the headgroups. Previous monolayer experiments24 and those in the present study point to the possibility that CPX could be segregated or “squeezed out” from the phospholipid environment below Tm and that eventually the drug is incorporated in structural defects of the gel phase. This will explain why the insertion on both drugs in the bilayer results in a slow efflux activity for some bacteria.8 Nevertheless, it remains to be demonstrated if such a capability makes the BCPX a better or worse substrate for the efflux pumps of other bacteria. This is why biophysical (FTIR with liposomes and fluorescence with spheroplasts) in combination with microbiological studies8 are currently in progress in our laboratory. Acknowledgment. S.M. is the recipient of a fellowship of “Recerca i Doce`ncia de la UB”. This work was supported by Grants PB93-0809 and PM98-0189 from DGICYT and 2000 SGR 0017. To the reviewers we offer thanks for the valuable comments. We acknowledge Dr. Teodor Parella (SeRMN, UAB) for technical help and Montserrat Vila for her participation in the initial stage of this work. Special thanks are extended to Sandra Hurle for correcting the English. LA025837H