PG Lipid

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Fluoroquinolone-Biomembrane Interaction at the DPPC/ PG Lipid-Bilayer Interface Sandra Merino,† Jose´ Luis Va´zquez,†,§ O Å scar Dome`nech,† Mercedes Berlanga,‡ ‡ Miquel Vin˜as, M. Teresa Montero,† and Jordi Herna´ndez-Borrell*,† 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 October 11, 2001. In Final Form: January 24, 2002 The interaction of two fluoroquinolones, ciprofloxacin (CPX) and its N-4-butylpiperazinyl derivative (BCPX), with liposomes bearing negative surface charge was studied by fluorescence methods. Binding of 1-anilino-8-naphthalenesulfonate to the liposome surface and diphenylhexatriene (DPH) and diphenylhexatriene-propionic (DPH-AP) acid fluorescence anisotropy measurements were differently affected by the two. On the basis of the variations of the surface potential and phase transition temperature (Tm) of the phospholipids studied, it was concluded that (i) both drugs interact electrostatically at the lipid membrane interface and (ii) the butyl side chain of BCPX led to a different interaction with the lipid headgroups. This behavior is then related to the activity of an efflux pump Serratia marcescens NIMA, which appear to be affected by the ability of CPX and BCPX to interact with the inner surface of the plasma membrane.

Introduction One of the most serious problems that has evolved from the extensive use and misuse of antibiotics is the emergence of resistant microorganisms. Among the different mechanisms that result in resistance, efflux pump systems1 have received close scrutiny. Bioenergetically, they depend on the release of energy either from ATP hydrolysis or from the gradient of protons, or other ions, which are coupled with the translocation of the drug. Much less is known about the structural requirement for their function and the molecular mechanism of their action. However, despite their nonspecific nature, the efflux mechanisms are far from a simple diffusion or permeation through a channel-like protein. Certain physicochemical features of the drugs such as lipophilicity or ionization seem to play a crucial role in the activity of the efflux system,2,3 It is likely that the efflux pumps require as a first step the insertion of the drugs into the inner leaflet of the plasma membrane.4 Therefore, for understanding the molecular mechanism of drug transport through biomembranes, it is crucial to investigate the effects of drugs on the lipid bilayer. Ciprofloxacin (CPX) is a 6-fluoroquinolone antibiotic currently under clinical use for which many resistances have been reported in a large number of microbial species. In this respect several physicochemical features of CPX should taken into account. Thus, CPX (i) is only slightly soluble in water and consequently it shows moderate †

Laboratori V de Fisicoquı´mica. Laboratori de Microbiologia. Present address: Howard Hughes Medical Institute, University of California Los Angeles, 5-748 Macdonald Building, Box 951662, Los Angeles, CA 90095-1662. * Corresponding author: e-mail [email protected]. ‡ §

(1) Van Veen, H. W.; Putman, M.; Margolles, A.; Sakamoto, K.; Konings, W. N. Biochim. Biophys. Acta 1999, 1461, 201-206. (2) Renau, T. E.; Sanchez, J. P.; Shapiro, M. A.; Dever, J. A.; Gracheck, S. J.; Domagala, J. M. J. Med. Chem. 1995, 38, 2947-2977. (3) Takenouchi, T.; Tabata., F.; Iwata, Y.; Hanzawa, H.; Sugawara, M.; Ohya, S. Antimicrob. Agents Chemother. 1996, 40, 1835-1842. (4) Putman, M.; van Veen, H. W.; Konings, W. N. Microb. Mol. Biol. Rev. 2000, 64, 672-693.

hydrophobicity, (ii) displays two proton binding sites that result in a microspeciation equilibrium (Figure 1), (iii) shows little tendency to bind to neutral5 liposomes but does bind with those with negatively surfaces charged,6 and (iv) exhibits a remarkable tendency to self-aggregate spontaneously in lipid monolayers.7 The biological importance of all of these properties, in relation to the mechanisms of entry into the cytoplasm and/or efflux from it, has remained until now unclear. Elsewhere, it has been claimed that 6-fluoroquinolones can be useful in the treatment of acid-alcohol resistant bacteria,8 which would clearly have important sanitary implications. CPX belongs to an amphoteric class of drugs characterized by the existence of many as two proton binding sites. In solution, an equilibrium is such that as many as four different microspecies can be found at a given pH (Figure 1). The combination of potentiometric and spectrophotometric techniques for the quantitation of each microspecies originally developed for amino acids has proven to be effective for quinolones.9 Basically, the method involves following the degree of protonation of the carboxylic function (RCOO-) by continuously monitoring the absorbance at 324 nm. With the value of RCOO- at a given pH and the macroscopic pKi values obtained by fluorimetric titration,10 the value of the microscopic constant11 k21 can be readily calculated. The remaining microconstants (pki,j) (5) Maurer, N.; Wong, K. F.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1998, 1374, 9-20. (6) Va´zquez, J. L.; Montero, M. T.; Merino, S.; Dome`nech, O.; Berlanga, M.; Vin˜as, M.; Herna´ndez-Borrell, J. Langmuir 2001, 17, 1009-1014. (7) Nag, K.; Keough, K. M. W.; Montero, M. T.; Trias, J.; Pons, M.; Herna´ndez-Borrell, J. J. Liposome Res. 1996, 6, 713-736. (8) Majumdar, S.; Flasher, D.; Friend, D. S.; Nassos, P.; Yajko, D.; Hadley, W. K.; Du¨zgu¨nes, N. Antimicrob. Agents Chemother. 1992, 36, 2808-2815. (9) Takacks-Novak, K.; Noszal, B.; Hermecz, I.; Kereszturi, G.; Podanyi, B.; Szasz, G. J. Pharm. Sci. 1990, 79, 1023-1028. (10) Va´zquez, J. L.; Berlanga, M.; Merino, S.; Dome`nech, O.; Vin˜as, M.; Montero, M. T.; Herna´ndez-Borrell, J. Photochem. Photobiol. 2001, 73, 14-19. (11) Va´zquez, J. L.; Montero, M. T.; Merino, S.; Dome`nech, O.; Berlanga, M.; Vin˜as, M.; Herna´ndez-Borrell, J. Int. J. Pharm. 2001, 220 (1-2), 53-62.

10.1021/la015627p CCC: $22.00 © 2002 American Chemical Society Published on Web 03/09/2002

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Figure 1. Microspeciation scheme of 6-fluoroquinolones (top), log P, and concentrations of microspecies at pH 7.4 (bottom). The mole fraction of each microspecies was calculated using the macroconstant (Ki) and microconstant (kij) values previously published.10-12

Chemicals. Dipalmitoylphosphadidylcholine (DPPC, >99%) and dipalmitoylphosphatidylglycerol (DPPG, >99%) were purchased from Avanti Polar Lipid Co. (Alabaster, Al). Phospholipid purity was assessed by thin-layer chromatography. 1-Cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid (CPX) was obtained from Cenavisa (Reus, E), and the BCPX was synthesized according to a procedure described

elsewhere.13 The purity of the compounds was assessed by IR and HPLC. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was purchased from Sigma (St. Louis, MO). 1-Anilino-8-naphthalenesulfonate (ANS), 1,6-diphenyl-1,3,5-hexatriene (DPH), and (2-carboxyethyl)-1,6-diphenyl-1,3,5-hexatriene (DPH-PA) were obtained from Molecular Probes (Eugene, OR). Buffers were Hepes pH 7.4, 50 mM; I ) 0.15 m and phosphate buffer saline (PBS) pH 7.4. Tripticase Soy Broth (TSB) and Tripticase Soy Agar (TSA) and Mueller Hinton broth (MH) were purchased from Scharlau (Barcelona, E). Vesicle Preparation. Chloroform/methanol (50:50, v/v) stock solutions of DPPC and DPPG were mixed to obtain the desired molar ratio (0.9:0.1, mol/mol) DPPC/DPPG. In anisotropy experiments, an aliquot of a similar organic solution of drugs was added to obtain the desired initial drug-lipid molar ratio. The organic solvent was evaporated to dryness in a conical tube by using a rotavapor, and the lipid film obtained was lyophilized for approximately 2 h to ensure absence of organic solvent traces. Multilamellar liposomes were obtained by hydration in water or buffer excess. The milky suspensions were then filtered through polycarbonate membranes (100 nm nominal diameter) using an Extruder device to obtain large unilamellar vesicles. Liposomes were then purified by passage through a molecular exclusion minicolumn of Sephadex G-50. Size and polydispersity of each preparation were monitored systematically by quasi-elastic lightscattering (QLS) using an Autosizer IIc photon correlation spectrophotometer (Malvern, Instruments, UK). Fluorescence Measurements. All were carried out using an SLM-Aminco 8100 spectrofluorometer provided with a jacketed cuvette holder. The temperature was controlled using a circulatory bath (Haake, Germany, sensitivity 0.1 °C). Slits of excitation and emission were set to 8 and 4 nm, respectively.

(12) Herna´ndez-Borrell, J.; Montero, M. T. J. Chem. Educ. 1996, 74, 1311-1314.

(13) Montero, M. T.; Freixas, J.; Herna´ndez-Borrell, J. Int. J. Pharm. 1997, 149, 161-170.

follow by means of the relationships existing between the micro- and macroconstants. It follows, then, that the fraction of each microspecies at pH 7.4 can be calculated.9 A detailed discussion about this method and its application to CPX has been published elsewhere.12 Since the ability to partition into membranes appears to be an important determinant for entry and/or efflux of antibiotics, we have synthesized the N-4-butylpiperazinyl derivative (BCPX), which has a substantially higher log P than CPX. We report here the effect of both drugs, CPX and BCPX, on liposomes mimicking the phospholipid electrical charge of the inner plasma membrane of the microorganisms. In the first part, binding experiments are performed to investigate the effect of fluoroquinolones on the lipid surface and estimate the variation in the surface potential. In the second part, the influence of the drugs on the fluorescence anisotropy of phospholipids is investigated. On the basis of these experimental results, both the accumulation and its effect on the efflux pump activity of a Serratia marcescens are discussed. Materials and Methods

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Calculation of the Variations in the Surface Membrane. Drugs (50 µM) were incubated with liposomes (50 µM total lipid concentration) at 50 °C for 30 min. Samples were kept at room temperature for 30 min before the experiments were performed. Then, increasing amounts of a stock solution 9 mM of ANS in methanol were added to the desired final concentration (0-125 µM). Corrections for the inner filter effects and scattering were introduced. The measurements were done using excitation and emission wavelengths of 380 and 500 nm, respectively. Before titration, the analytical signals were corrected for the background scattering by subtraction of the respective blank. The adsorption data of ANS binding on bilayers were fitted to a Langmuir isotherm

IF )

IF,max[ANS] Kapp + [ANS]

(I)

where Kapp is the apparent dissociation constant at a given surface potential, [ANS] is the concentration of the probe, and IF and IF,max are the fluorescence intensities at a given concentration and maximum binding of the probe to the phospholipids, respectively. For practical purposes, ANS bound concentration was considered proportional to the fluorescence intensities. As in previous studies,6 we assumed that intensities of fluorescence are proportional to the extent of ANS molecules bound to the liposomes. Using the values of the apparent constant in absence (Kapp) and presence (K′app) of different amounts of drugs, it is possible to calculate the variation of the surface membrane potential according to

( )

∆ψ ) RT/F ln

K′app Kapp

Figure 2. Fluorescence intensity as a function of ANS concentration for DPPC:DPPG, 0.9:0.1 mol/mol liposomes, in the presence of CPX (open circles) and BCPX (open diamonds). Each experiment is presented with its own reference (filled circles and filled diamonds, respectively). The drug/total lipid molar ratio was 1. Data are means ( SD for three experiments.

(II)

where R is the universal gas constant, T the temperature, and F the Faraday constant. This equation was used to estimate the modification of the absolute surface potential of the liposomes by the drugs. Steady-State Anisotropy Experiments. DPH in tetrahydrofuran and DPH-PA in methanol were incorporated in liposomes following an incubation of 3 µL of concentrated stock solutions of the probe in 1500 µL of liposome suspension during 30 min at 50 °C. The final lipid/fluorescent probe molar ratio was 637. The anisotropy was recorded in the range between 15 and 63 °C. Between 15-31 and 47-63 °C the anisotropy was recorded each 4 °C and between 31 and 47 °C each 2 °C. The absorption and fluorescence spectra of the probes were not independent of fluoroquinolone presence. Several assays were done to monitor the influence of the drug on the fluorescence properties of the probes. Hence, that excitation wavelength was at 381 nm to avoid the concomitant excitation of the drug.14,15 The fluorescence emission of the probes was collected at 426 nm. The vertical and horizontally polarized emission intensities were corrected for background scattering by subtraction of the corresponding polarized intensities of a blank containing unlabeled suspension. Steady-state anisotropy (rs) values were calculated according to

rs )

IVV - GIHV IVV + 2GIHV

(III)

where IVV and IHV are the intensities measured in directions parallel and perpendicular to the exciting beam and G is the grating correction factor equal to IHV/IHH. The polarization values reported in Figure 3 are the average of several measurements. All the individual points were within 5% of the reported values. Bacteria. Serratia marcescens NIMA16 was used in this work. Bacteria were incubated at 30 °C over 18 h and maintained on TSA slants and culture in TSB for experiments.

Figure 3. Steady-state polarization of DPH (A) and DPH-PA (B). In the absence (filled circles) and presence of CPX (open circles) and BCPX (triangle) for DPPC:DPPG, 0.9:0.1 mol/mol, as a function of temperature. The drug/total lipid molar ratio was 0.05. Each point is the mean value of three experiments, but the SD are not shown for sake of clarity. Data were fitted to sigmoid curves as it was previously reported.6

(14) Montero, M. T.; Herna´ndez-Borrell, J.; Nag, K.; Keough, K. M. W. Anal. Chim. Acta 1993, 290, 58-64. (15) Tiefenbacher, E. M.; Haen, E.; Przybilla, B.; Kurz, H. J. Pharm. Sci. 1994, 83, 463-467. (16) Williams, R. R.; Quadri, S. M. H. The pigment of Serratia. In The Genus Serratia; von Graevenitz, A., Rubin, S., Eds.; CRC Press: Boca Raton, FL, 1980; pp 31-75.

CPX and BCPX Accumulation and Efflux Assays. Isolates were harvested by centrifugation (9000g) at room temperature, washed, and concentrated 10-fold in phosphate buffer saline (PBS), pH 7.5. 6-Fluoroquinolones were added to 1 mL aliquots to a final concentration of 10 µg/mL. At convenient times, samples were centrifuged in a microfuge at 10 000 rpm, 4 °C, for 1 min.

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Table 1. Parameters Obtained from the Fitting of the ANS Binding Data on DPPC:DPPG, 0.9:0.1 mol/mol, Liposomes CPX or BCPX (µM) 0 50

IF,max CPX BCPX

CPX

351 303

28.21 33.51

417 379

Kapp BCPX 27.64 37.24

Table 2. Transition Temperature (Tm) and Cooperativity (B) of DPPC/DPPG, 0.9:0.1 mol/mol, Liposomes in Absence and Presence of CPX or BCPX DPH

∆ψ (mV) CPX BCPX +4.42

+7.66

Pellets were washed in 1 mL of chilled PBS at pH 7.5 and resuspended in 1 mL of 0.1 M glycine-HCl buffer, pH 3.0. Finally, they were incubated at room temperature overnight to allow bacterial lysis. Afterward, suspensions were centrifuged to remove bacterial debris. The concentration of CPX or BCPX in the supernatants was determined fluorimetrically. For the efflux assays cells were incubated 3 min with the antibiotic before the addition of the metabolic inhibitor CCCP at 100 µM final concentration. Samples were manipulated as in the accumulation assays.

ref CPX BCPX

DPH-PA

Tm (°C)

B

Tm (°C)

B

40.56 ( 0.09 40.77 ( 0.12 41.15 ( 0.10

24570 ( 1117 32990 ( 2628 46520 ( 4708

41.63 ( 0.10 40.00 ( 0.17 41.93 ( 0.10

54890 ( 5346 49960 ( 7542 62420 ( 5662

In a previous work,10 we have showed that the titration curves of CPX and BCPX in the presence of liposomes formed with acidic phospholipids exhibit a significant shift of both macroconstants of ionization (see Figure 1). The simplest hypothesis is that an electrostatic interaction exists between fluoroquinolones and the zwitterionic and acidic phospholipids of the bilayers. In a subsequent work,6 we showed that there is a competition between ANS (a fluorescent label used to study the hydrophilic phosphate moiety17) and CPX. In view of these results we applied here the same experiments to BCPX. The binding of CPX and BCPX to DPPC/DPPG liposomes (Figure 2) produced a decrease in the emission fluorescence. These results (Figure 2) are triplicates of experiments performed with aliquots of two different populations of liposomes (one for CPX and the other for BCPX). For comparative purposes a blank for each population is presented. The results are in agreement with a preferential interaction between these drugs and the acidic components of the membrane. Thus, probe affinity for the bilayer binding sites is affected similarly by both drugs. However, when the data were fitted to the isotherm (eq I), the values of I F,max and Kapp obtained indicated that CPX displaces more ANS molecules than BCPX does (Table 1). As can be seen, CPX and BCPX produce a decrease in I F,max of about 15 and 10%, respectively. Strikingly, the ∆ψ values show that the negative surface charge borne by DPPC/DPPG liposomes (∼-25 mV)10 becomes slightly more screened by BCPX than by CPX under these conditions. This phenomenon should be attributed to the existence of an electrostatic bonding between the microspecies bearing positive charges (Figure 1) and the negative groups present at the liposomes surface. In a previous work we suggested that BCPX might anchor into neutral phospholipid monolayers formed at the air-water interface.7 Similar behavior is expected to occur in bilayers. Hence, to investigate the localization and the effects of CPX and BCPX on DPPC:DPPG liposomes, we have measured the changes in the fluorescence anisotropy (rs) of DPH and DPH-PA. It is worth noting that DPH is preferentially located near the center of the bilayer while DPH-PA molecules are anchored with the polar groups at the aqueous interface. Figure 3 shows the temperature profile of rs for both probes in the absence and presence of CPX and BCPX. Neither of the drugs caused significant differences of DPH and DPH-PA fluorescence anisotropy values compared to DPPC:DPPG

liposomes in the absence of drugs (Figure 3). However, in the experiments with DPH (Figure 3A) a slight increase of the main phase transition temperature (Tm) is observed in the presence of both drugs. The presence of CPX and BCPX in DPPC:DPPG liposomes (Figure 3B) caused a small decrease of the DPH-PA fluorescence anisotropy below Tm. Conversely, both drugs increased slightly the rs values of DPH-PA above Tm. In Table 2 Tm and B (cooperativity) values, calculated from the slope and inflection point of the data to sigmoid curves, are given. As shown, while CPX decreases Tm in DPH-PA liposomes and has almost no effect in DPH liposomes, BCPX caused an increase in Tm in all cases. This suggest that, in contrast to CPX, BCPX may form hydrogen bonding with the headgroups of the phospholipids sited at the interface, presumably with the hydroxyl groups of DPPG. On the other hand, the cooperativity of the transition increased in the presence of BCPX, which suggests that the BCPX molecules are located deeper in the membrane than CPX. Actually, the overall anisotropy experiments suggest that BCPX may lie in an extended orientation in the bilayer with the butyl chain anchored and the carboxylic group at the phospholipid/water interface. In contrast, CPX might establish contact only with the polar head region at the phospholipid/water interface. Presumably, although both drugs are able to neutralize the surface charges, the insertion of BCPX into the membrane would lead to a different competition with the ANS molecules which may explain the quantitative differences in the surface potential values calculated (Table 1). However, it is important to note that the concentration of the negative microspecies for BCPX is approximately 4-fold that for CPX (Figure 1). From this consideration a repulsive effect exerted by the negative surface of the bilayers on the drugs could be substantially stronger for BCPX than for CPX in solution. In fact, this could be interpreted as a rate-limiting step in the capture of the drug by the inner leaflet of the membranes. Although the evidence supporting definite mechanisms of CPX and BCPX interaction with the species of phospholipids present is sketchy, the data presented may elucidate the antibacterial behavior of these drugs. Recently, we have described an efflux pump in Serratia marcescens.18 When the accumulation of both drugs, CPX and BCPX, was studied in the NIMA strain (see methods), no qualitative differences were observed (Figure 4). Thus, both molecules reach similar concentration under steadystate conditions. Remarkably, the collapse of the pH gradient by addition of CCCP revealed a slight but intriguing difference in the kinetics of the phenomena. Although the concentration reached in 12 min for both drugs after addition of the protonophore was similar, a slower efflux for BCPX than for CPX seems to operate. This difference could be physiologically significative and, in view of the binding and anisotropy experiments, can be attributed to a different capture mechanism of CPX and BCPX by the bilayer.

(17) Ma, J. Y. C.; Ma, J. K. H.; Weber, K. C. J. Lipid Res. 1985, 26, 735-744.

(18) Berlanga, M.; Va´zquez, J. L.; Herna´ndez-Borrell, J.; Montero, M. T.; Vin˜as, M. Microb. Drug Resist. 2000, 6, 111-117.

Results and Discussion

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Figure 4. CPX (circles) and BCPX (diamonds) (10 mg/L outer concentration) accumulation, with (open symbols) and without (filled symbols) the metabolic inhibitor CCCP (100 µM) by S. marcescens NIMA. Data are means ( SD of three individual experiments.

According to the most currently accepted models for drug transport through efflux pumps,1 a capture mechanism by the membrane occurs before the drug reaches its target protein. We therefore suggest that the drugs studied here could both be initially bound electrostatically on the inner monolayer of the membrane, only subsequently reaching the protein. Therefore, the relatively low perturbation of membrane lipid ordering by CPX and BCPX might be important for ensuring the mobility of the drug. On the other hand, the detailed analysis of the microspecies present in solution (Figure 1) allows for

Merino et al.

interpretation of the kinetics of the efflux process, which is slightly slower for BCPX, as deriving from an electrostatic repulsion exerted by the inner phospholipid monolayer on the drug. Some additional points should be also considered. First, it cannot be discounted that the cytoplasm protein membrane can be directly involved in the interaction with the drugs. As was shown previously,19 the negative charge of one or more amino acid residues could be involved in the phenomena negating the role of the phospholipids. Second, the different lipophilicities of the CPX and BCPX could also be relevant. Thus, it is reasonable to assume that because BCPX anchors deeper into the membrane, it follows a different mechanism than CPX to reach the protein. Third, given the differences observed in the antimicrobial activity, CPX appears to be better substrate than BCPX for our efflux pump. The microbiological evaluation of new compounds carrying alkyl chains of different length in the N-4 position of the piperazinyl group of CPX has recently been initiated in our laboratory.11 NMR and FTIR studies designed to elucidate further the molecular mechanism of interaction are currently in progress and will be released in a following paper. Acknowledgment. S.M. has been supported by 2001 Agustı´ Pedro Pons award. This work has been supported by DGICYT (Grant PM98-0189) and Generalitat de Catalunya (Grant 2000 SGR00017). We thank Cenavisa Laboratories for the generous gift of ciprofloxacin and to Sandra Hurle and Dr. Christopher Cramer for the critical reading of the manuscript. LA015627P (19) Edgar, R.; Bibi, E. EMBO J. 1999, 18, 822-832.