Langmuir 2001, 17, 1009-1014
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Location and Nature of the Surface Membrane Binding Site of Ciprofloxacin: A Fluorescence Study Jose´ Luis Va´zquez,†,§ M. Teresa Montero,† Sandra Merino,† O Å scar Dome`nech,† Mercedes Berlanga,‡ Miquel Vin˜as,‡ 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 June 19, 2000. In Final Form: October 18, 2000 In this work, the interaction between the antibiotic ciprofloxacin (CPX) and liposomes formed with zwitterionic and acidic phospholipids was studied using fluorescence methods. Binding of 1-anilino-8naphthalene sulfonate to the liposome surface was dependent on the presence of CPX and the lipid composition. The data were fitted to a Freundlich-like isotherm. The binding constant (K), maximum concentration bound to liposomes (Cm), and cooperativity (b) were obtained. K values, in the presence and absence of CPX, were used to calculate the variation in the surface potential (∆Ψ) of the liposomes. Fluorescence quenching and anisotropy measurements suggest that the drug interacts with the headgroups of the phospholipids and does not penetrate deeper in the bilayer. No significant changes were observed in the cooperativity of the phospholipid transition. Hydrogen bonding with dipalmitoylphosphatidylethanolamine and electrostatic interactions with dipalmitoylphosphatidylglycerol and zwitterionic phospholipids such as dipalmitoylphosphatidylcholine appear to be involved in the interaction occurring in natural membranes.
Introduction The broad spectrum of action shown by the new fluoroquinolones against Gram-positive and Gram-negative bacteria has launched new research in almost all fields, including the synthesis of new molecules, clinical studies, and the development of new strategies for their delivery. Noteworthy is the possible use of fluoroquinolones in the treatment of emerging tuberculosis and of the secondary infections suffered by AIDS patients. The unusual activities observed against acid-alcohol resistant bacteria, which are characterized by an external cell surface of complex architecture,1 seem to derive from some of the particular physicochemical properties of fluoroquinolones. It has been demonstrated that this effectiveness against such species can be attributed neither to differences in lipophilicity2 nor to differences in susceptibilities for their intracellular target, the DNA gyrase.3 However, a nonspecific ability of fluoroquinolones to penetrate bacterial envelopes would explain their wide range of antimicrobial activity. Thus, the study of the mechanism of action of fluoroquinolones may fall in part in the domain of membranology, particularly in the investigation of the nature of the interaction between these drugs and phospholipids. Several routes have been described for the transport of drugs, mainly antibiotics, through bacterial envelopes: (i) a hydrophilic pathway through porins governed by * Corresponding author. Departament de Fisicoquı´mica, Facultat de Farma`cia, U.B. 08028-Barcelona, Spain. E-mail: jhernan@ farmacia.far.ub.es. † Laboratori V de Fisicoquı´mica, Facultat de Farma ` cia. ‡ Laboratori de Microbiologia, Campus de Bellvitge. § Present address: Howard Hughes Medical Institute, University of California Los Angeles, 5-748 Macdonald Building, Box 951662, Los Angeles, CA 90095-1662. (1) Nikaido, H.; Kim, S. H.; Rosenberg, E. Y. Mol. Microbiol. 1993, 8, 1025-1030. (2) Renau, T. E.; Sa´nchez, J. P.; Shapiro, M. A.; Dever, J. A.; Gracheck, S. J.; Domagala, J. M. J. Med. Chem. 1995, 38, 2974-2977. (3) Shen, L.; Mitscher, L. A.; Sharma, P. N.; O’Donnell, T. J. Biochemistry 1989, 28, 3886-3893.
Fick’s law;4 (ii) a “self-promoted” mechanism which causes the disruption of the outer bacterial membrane used by aminoglicosides;5 (iii) a hydrophobic pathway, based on the equilibrium between the noncharged microspecies across membranes, proposed for amphoteric antibiotics;6 (iv) a pore-forming action, described for the lantibiotic nisin.7 In view of their physicochemical properties, some of these mechanisms can be imagined for fluoroquinolones and would include a preliminary binding step on the membrane surface. Importantly, some resistance mechanisms related to the specific membrane functions have been developed by bacteria and result in a decreased uptake8 or an increased efflux of the antibiotics.9 Because of this, a hypothetical mechanism has been proposed that recognizes the existence of a primary capture step of the drug by the inner lipid monolayer.10 Although the whole mechanism of accumulation of the drug is not simple,6 because of the coexistence of influx and efflux mechanisms, the involvement of a binding step to the inner or outer monolayer membrane seems to be biologically relevant.11,12 With respect to the binding and/or translocation through the natural membranes, the role of the lipid environment in the accumulation of fluoroquinolones remains unsettled. In the present work, we investigated the interaction of ciprofloxacin, a 6-fluoroquinolone currently in clinical use, on neutral and charged liposomes at neutral pH. The (4) De´che`ne, M. H.; Leying, H.; Cullman, W. Chemotherapy 1990, 36, 13-23. (5) Hancock, R. E. W.; Raffle, J.; Nicas, T. I. Antimicrob. Agents Chemother. 1981, 19, 777-785. (6) Nikaido, H.; Tanassi, D. G. Antimicrob. Agents Chemother. 1993, 37, 1393-1399. (7) Garcera´, M. J. G.; Elferink, M. G. L.; Driessen, A. J. M.; Konings, W. N. Eur. J. Biochem. 1993, 212, 417-422. (8) Hirai, K. H.; Aoyama, T.; Irikura, S.; Iyobe, S.; Mitsuhashi, S. Antimicrob. Agents Chemother. 1986, 29, 535-538. (9) Xian-Zhi, L.; Livermore, D. M.; Nikaido, H. Antimicrob. Agents Chemother. 1994, 38, 1732-1741 (10) Nikaido, H. J. Bacteriol. 1996, 178, 5853-5859. (11) Bedard, J.; Bryan, L. E. Antimicrob. Agents Chemother. 1989, 33, 1379-1382. (12) Furet, Y. X.; Deshusses, J.; Pechere, J. C. Antimicrob. Agents Chemother. 1992, 36, 2506-2511.
10.1021/la0008582 CCC: $20.00 © 2001 American Chemical Society Published on Web 01/24/2001
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general purpose of our experiments was to better understand the phospholipid-fluoroquinolone interactions.13 Specifically, the objectives were to investigate (i) the molecular membrane surface requirements for drug recognition, (ii) the level of spontaneous integration of ciprofloxacin into the bilayer, and (iii) the role of liposome surface charge. Experimental Section Chemicals. Dipalmitoylphosphatidylcholine (DPPC, >99%), dipalmitoylphosphatidylethanolamine (DPPE, >99%), dipalmitoylphosphatidylglycerol (DPPG, >99%), and Escherichia coli acetone/ether washed phospholipids (nominally 57.5% PE, 15.1% PG, 9.8% Cardiolipin, and 17.6% others) were purchased from Avanti Polar Lipids (Alabaster, AL). Phospholipid purity was assessed by thin-layer chromatography. Ciprofloxacin (CPX) was obtained from Cenavisa (Reus, Spain). The purity of the compound was assessed by IR and HPLC. The probes 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluene sulfonate (TMA-DPH) and 1-anilino-8-naphthalene sulfonate (ANS) were obtained from Molecular Probes (Eugene, OR). Quenchers were purchased from Sigma (St. Louis, MO). The buffer solution was HEPES, pH 7.40, 50 mM, I ) 0.15 molal. Solubility Determinations and Preparation of Stock Solutions of CPX. The solubility of ciprofloxacin was determined following a method described elsewhere14 with minor modifications. Saturated solutions of CPX were prepared in the HEPES buffer and stirred overnight at room temperature. The samples were filtered, and concentration was measured spectrophotometrically by UV absorption (λmax ) 278 nm). The working solutions (50-150 µM) were prepared by weighing and dissolving the appropriate amount of CPX in 5 mL volumetric flasks. The complete dissolution of CPX becomes apparent after a few cycles of vigorous vortexing and small periods of sonication in a bath. Vesicle Preparation. Chloroform/methanol (50:50, v/v) stock solutions of DPPC, DPPE, and DPPG were mixed to obtain the desired molar ratio DPPC/DPPE or DPPC/DPPG (0.9:0.1 mol/ mol). Except for the binding experiments, an aliquot of a similar organic solution of CPX was added to obtain the desired drug/ total lipid ratio (1:10, mol/mol). The organic solvent was evaporated to dryness in a conical tube by using a rotavapor, and the lipid film obtained was lyophilized15 for approximately 2 h to ensure the absence of organic solvent traces. Multilamellar liposomes were obtained by hydration in excess of buffer. Thereafter, the suspensions 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 quasielastic light-scattering (QLS) using an Autosizer IIc photon correlation spectrophotometer (Malvern, Instruments, U.K.). In fluorescence quenching experiments, an additional step of exclusion gel chromatography using Sephadex G-50 was needed in order to remove the noninteracting drug. 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 4 and 4 nm and 8 and 8 nm, respectively. Binding Experiments. These studies consisted of the incubation of increasing amounts of drug (0, 50, 100, and 150 µM) with different types of liposomes at 50 °C for 60 min while maintaining the total lipid concentration constant (50 µM). Thereafter, samples were stored at room temperature and immediately titrated with a concentrated stock solution of ANS in ethanol. Under continuous stirring, the fluorescence emission of ANS was measured at 500 nm and the excitation wavelength was set at 380 nm. The adsorption data of ANS binding on bilayers (13) Montero, M. T.; Herna´ndez-Borrell, J.; Keough, K. M. W. Langmuir 1998, 14, 2451-2454. (14) Ross, D. L.; Riley, C. M. Int. J. Pharm. 1990, 63, 237-250. (15) Liposome Methodology; Leserman, L. D., Barbet, J., Eds.; E Ä ditions INSERM: Paris, 1982.
Va´ zquez et al. were fitted to an equation derived from the Freundlich isotherm:16
(K[ANS]∞)b [ANS]B ) Cmax 1 + (K[ANS]∞)b
(I)
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 concentrations, respectively. In practice, ANS bound concentration was considered to be proportional to the fluorescence intensity. Equation I can be used to calculate the variations in the electrostatic surface potential according to
∆Ψ )
( )
Kfq RT ln F K0
(II)
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. Quenching Experiments. We have followed the same methodology used in previous work.17 The quenchers, KI and acrylamide, were freshly prepared in HEPES buffer. Sodium thiosulfate (0.1 mM) was added to the KI stock solution to prevent I3- formation. Iodobenzene and iododecanoic acid were prepared before use in dimethyl sulfoxide (2 × 10-2 M). The intensities were corrected for the dilution and the inner filter effect. CPX was excited at 278 nm, and fluorescence was monitored at 420 nm for quenching experiments. The experiments were carried out at 25 and 50 °C, and the total lipid concentration was 1 mM. The CPX concentration was 1 µM. The data were analyzed according to the Stern-Volmer equation for dynamic and static quenching.18
F0/F ) (1 + Ksv[Q])(eV[Q])
(III)
where F0 and F are the fluorescence intensities in the absence and presence of the quencher, Ksv and V are the dynamic and static quencher constants, respectively, and Q is the concentration of the quencher. Steady-State Anisotropy Experiments. The probe, TMADPH, was incorporated in liposomes following the incubation of 3 µL of concentrated stock solution of the probe in methanol in 1500 µL of liposome suspension for 30 min at 50 °C. The final lipid/fluorescent probe ratio was 1000:1.57 mol/mol. The anisotropy was recorded in the range between 15 and 63 °C. Between 15 and 31 °C and between 47 and 63 °C, the anisotropy was recorded at 4° intervals, and between 31 and 47 °C 2° intervals were used. The absorption and fluorescence spectra of TMADPH were not independent of the presence of fluoroquinolones. Therefore, several assays were done to monitor the influence of the drug on the fluorescence properties of the probes. The excitation wavelength was 381 nm, and emission was observed at 426 nm. The vertically and horizontally polarized emission intensities were corrected for the background scattering by subtraction of the corresponding polarized intensities of a blank containing the unlabeled suspension. Steady-state anisotropy (rs) values were calculated according to
rs )
IVV - GIHV IVV + 2GIHV
(IV)
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. The individual points were within 5% of the reported values. The parameters B and Tm are the cooperativity and temperature (16) Serra, R.; Mas, F.; Dı´az-Cruz, J.; Arin˜o, C.; Estevan, M. Electroanalysis 2000, 12, 60-65. (17) Va´zquez, J. L.; Montero, M. T.; Trias, J.; Herna´ndez-Borrell, J. Int. J. Pharm. 1998, 171, 75-86. (18) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983.
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of the gel-liquid-crystal phase transition of the lipid. They were calculated from the slope and inflection point of the data fitted to sigmoid curves.19
Results and Discussion The CPX shows a moderate hydrophilicity (log Poc/w ) -0.987)13 near neutral pH that would correlate with a moderate affinity of CPX for lipid environments. Nevertheless, it has been reported recently20 that CPX shows (i) an almost null tendency to bind to DPPC (
