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Interaction of Grepafloxacin with Large Unilamellar Liposomes: Partition and Fluorescence Studies Reveal the Importance of Charge Interactions Catarina Rodrigues,† Paula Gameiro,*,† Salette Reis,‡ J. L. F. C. Lima,‡ and Baltazar de Castro† CEQUP/Departamento de Quı´mica, Faculdade de Cieˆ ncias, Universidade do Porto, 4169-007 Porto, Portugal, and CEQUP/Laborato´ rio de Quı´mica-Fı´sica, Faculdade de Farma´ cia, Universidade do Porto, 4050-047 Porto, Portugal Received May 30, 2002. In Final Form: August 1, 2002 The partition and location of grepafloxacin, a “second-generation” fluoroquinolone with enhanced efficacy against Gram(+) bacteria, in bilayers of dimyristoyl-L-R-phosphatidylcholine and dimyristoyl-L-Rphosphatidylglycerol have been studied by spectrophotometric and fluorimetric methods, and the partition coefficients (Kp) have also been determined by conventional drug quantification after phase separation. The Kp values obtained by the different methods are identical within experimental error and were found to be larger than those reported for other fluoroquinolones, which is attributed to the less acidic character of grepafloxacin caused by the methyl substituents in the heterocyclic and the piperazine rings. Thus, at the physiological pH grepafloxacin exists 20% in the cationic form, in contrast to what happens to other fluoroquinolones for which only the zwitterionic and anionic forms exist to any appreciable extent at pH 7.4. The fluorescence studies have also revealed that grepafloxacin is not deeply buried inside the lipid bilayers, despite the presence of the two methyl groups, but is located near the phospholipid headgroups, as has been found with other fluoroquinolones. Furthermore, our data suggest that the enhanced Gram(+) activity of grepafloxacin can be due to the charge interaction that occurs between the cationic form of the drug and the negatively charged membrane surface of the Gram(+) bacteria at physiological pH.
Introduction Fluoroquinolones are widely used therapeutically to treat bacterial infections due to their highly potent, widespectrum antimicrobial activity, which is related to the inhibition of DNA synthesis.1,2 These drugs have a good tissue penetration, and the recent quest for novel fluoroquinolones with a broader spectrum activity has shown that it can be related to their penetration ability.3 However, the precise molecular mechanism and kinetics of fluoroquinolones’ entry into bacteria is not known. Several studies with different bacteria strains showed that the transport channel proteins may be important for the uptake of these antibiotics, but whether the entry of the antibiotic is through the lipid/protein interface or through the porin channels is still unknown. Nevertheless, the primary capture of the drug by the bacterial membrane seems to be biologically relevant.4 As drugs must partition to a well-defined, energetically favorable location, orientation, and conformation in the membrane bilayers before diffusing to an intrabilayer receptor binding site,5 insights about the mechanism of drug entry into cells can be obtained from the study of * Corresponding author. Mailing address: Prof. Paula Gameiro, CEQUP/Departamento de Quı´mica, Faculdade de Cieˆncias, Universidade do Porto, R. Campo Alegre, 4169-007 Porto, Portugal. Tel: +351 226082889. Fax: +351 226082959. E-mail: agsantos@ fc.up.pt. † CEQUP/Departamento de Quı´mica. ‡ CEQUP/Laborato ´ rio de Quı´mica-Fı´sica. (1) Beermann, D. Quinolone antibacterials. In Handbook of Experimental Pharmacology; Kuhlmann, J., Dalhiff, A., Zeiler, H. J., Eds.; Springer: London, 1998; Vol. 127. (2) Wiedemann, B.; Heisig, P. J. Antimicrob. Chemother. 1997, 40, 19. (3) Niwa, M.; Hotta, K.; Kanamori, Y.; Matsuno, H.; Kozawa, O.; Hirota, M.; Uematsu, T. Eur. J. Pharmacol. 2001, 428, 121. (4) Chevalier, J.; Malle´a, M.; Pa`ges, J. M. Biochem. J. 2000, 348, 223.
their partition and location in membrane models, such as liposomes, since they can mimic the chemical and structural anisotropic environment of cell membranes but lack the transport machinery. Modulating the physical or chemical characteristics of the liposomes can change substantially the partition and location of drugs and may provide clues to understand their pharmacokinetic and pharmacodynamic profiles and as a final goal to elucidate the role of the lipid environment in the transport of fluoroquinolones in bacterial strains.5,6 Drug partition in water/liposomes can be quantified by their partition coefficients (Kp) which normally are determined by drug quantification in each phase, after phase separation. However, the encumbrance of these methods, specifically incomplete drug separation, can be overcome by the use of spectroscopic measurements directly on the liposome suspensions. These latter methods have also been shown to be effective for the determination of partition coefficients of drugs with short-chain lipids that bear negatively charged polar heads, for which the separation methods fail. Notwithstanding their obvious advantages, they have yet to make significant inroads in biological applications, despite the known incapacity of the phase separation methods to quantify the partition of fluoroquinolones in dimyristoyl-L-R-phosphatidylglycerol or dipalmitoyl-L-R-phosphatidylglycerol liposomes.7-9 The combination of partition coefficients and acidity constants (5) Mason, R. P.; Rhodes, D. G.; Herbette, L. G. J. Med. Chem. 1991, 34, 869. (6) Efthymiopoulos, C. J. Antimicrob. Chemother. 1997, 40, 35. (7) Bedard, J.; Bryan, L. E. Antimicrob. Agents Chemother. 1989, 33, 1379. (8) Vazques, J. L.; Montero, M. T.; Merino, S.; Domenech, O.; Berlanga, M.; Vinas, M.; Hernandez-Borrell, J. Langmuir 2001, 17, 1009. (9) Rodrigues, C.; Gameiro, P.; Reis, S.; Lima, J. L. F. C.; Castro, B. Biophys. Chem. 2001, 94, 97.
10.1021/la0205093 CCC: $22.00 © 2002 American Chemical Society Published on Web 11/21/2002
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Langmuir, Vol. 18, No. 26, 2002 Chart 1
to assess possible electrostatic contributions to the interaction of drugs with liposomes is also a useful approach, but again one that is not widely used.9-11 On the other hand, fluorescence quenching and fluorescence anisotropy have been widely and successfully used not only to determine drug location and drug insertion into liposomes but also to gather information about membrane fluidity. An important piece of information that can stem from location studies of hydrophilic drugs relates to the assessment of possible contributions of hydrophobic components to control the extent or orientation of insertion that can enhance the electrostatic interaction. In this work, the partition and the location of grepafloxacin in two types of lipid bilayers with different hydrophobic/electrostatic characteristics, those of dimyristoyl-L-R-phosphatidylcholine (DMPC) and dimyristoyl-LR-phosphatidylglycerol (DMPG), has been studied. Grepafloxacin, for which the chemical structure is depicted in Chart 1, is one of the “second-generation” fluoroquinolones, which differ from their “first-generation” counterparts by having at least two methyl groups. Their effect on the partition and location of grepafloxacin, when compared with those of other fluoroquinolones,7,8,12 can provide clues to understand their mechanism of entry on membrane surfaces and specially to understand the role of the lipid environment in this transport. The application of spectroscopic methods enabled the determination of the partition coefficients of grepafloxacin in DMPC and DMPG liposomes, which is, to the best of our knowledge, the first successful such determination for fluoroquinolones in liposomes. The analysis of the obtained values in connection with the results of the location studies allowed us to estimate the extent of electrostatic interactions of grepafloxacin with the liposomes and to stress the effect that small changes in chemical structure can have on the interactions with the membrane surface. Experimental Section Reagents and Sample Preparation. Grepafloxacin was a gift from Glaxo-Wellcom (Brentford, U.K.); DMPC) and DMPG were from Avanti Polar Lipids. N-(2-Hydroxyethyl)piperazineN′-ethanesulfonic acid (HEPES), 1,6-diphenyl-1,3,5-hexatriene (DPH), and trimethylammonium-diphenylhexatriene (TMADPH) were from Sigma, and all other chemicals were from Merck (grade, pro analysi). All lipid suspensions were prepared with aqueous 10 mM HEPES buffer solution (I ) 0.1 M NaCl, pH 7.4). Extrusion of liposomes was performed on a Lipex Biomembranes extruder attached to a circulating water bath. (10) Papahadjopoulos, D.; Jacobson, K.; Poste, G.; Shepherd, G. Biochim. Biophys. Acta 1975, 394, 504. (11) Seelig, A.; Allegrini, P. R.; Seelig, J. Biochim. Biophys. Acta 1988, 939, 267. (12) Montero, M. T.; Hernandez-Borrell, J.; Keough, K. M. W. Langmuir 1998, 14, 2451.
Rodrigues et al. Phase separation was attempted using either an ultracentrifuge (Sorvall Du Pont) or a centrifugal filter unit (Centricon 30.000 units) in conjunction with a Kubota 6900 centrifuge. Physical Measurements. Absorption spectra were recorded with a UNICAM UV-300 spectrophotometer equipped with a constant-temperature cell holder. All spectra were recorded at 37 ( 0.1 °C in 1 cm cuvettes with a slit width of 2 nm, using a spectral window from 225 to 400 nm. Absorption spectra of sample solutions (suspensions) were measured against a reference solution of the same composition but without drug. Alternatively, sample and reference were both measured against a HEPES buffer, and the spectrum for the reference was subtracted from that of the sample. The final corrected absorption spectra obtained by either procedure were equal, within experimental error. Derivative spectra were calculated using the Savitzky-Golay method,13 in which a second-order polynomial convolution of 13 points was employed. Fluorescence measurements were performed at 37 ( 0.1 °C in 1 cm cuvettes using a Perkin-Elmer LS 50B spectrofluorimeter equipped with a thermostated cell holder, using a slit width of 3 nm. Beer’s law was found to be obeyed for the following drug concentration ranges in HEPES buffer solutions: from 5.5 to 27.6 µM for UV/vis spectrophotometry and from 1.0 to 9.6 µM for spectrofluorimetry. Potentiometric measurements were carried out with a Crison 2002 pH meter and 2031 buret controlled by a personal computer. The electrode assembly was made up of an Orion 900029/4 AgCl/ Ag reference electrode and a Russel SWL glass electrode. System calibration was performed by the Gran method14 in terms of hydrogen ion concentration, using a strong acid/strong base titration [HCl (1 mM)/NaOH (≈20 mM)] with solutions whose ionic strength was adjusted to 0.1 M with NaCl. Titrations were always carried out under an argon atmosphere at 25 °C in a double-walled glass cell. The size distribution of extruded DMPC and DMPG liposomes, with and without added grepafloxacin, was determined by quasielastic light scattering analysis using a Malvern Instruments Zeta Sizer 5000. Lipid concentrations in vesicle suspensions were determined by phosphate analysis, using a modified version of the Fiske and Subbarow method.15 Potentiometric Determinations. Acidity constants of grepafloxacin were obtained by titrating 20.00 mL of an acidified aqueous solution of the drug (≈1 mM, HCl ≈ 1 mM) with NaOH (≈20 mM). All titrations were performed at 25.0 ( 0.1 °C under argon, and for all solutions the ionic strength was adjusted to 0.1 M with NaCl. Calculations were performed with data obtained from at least six independent titrations using the program Hyperquad.16 Liposome Preparation. Liposomes were prepared by evaporation to dryness with a stream of argon of a lipid solution in chloroform (DMPC) or in chloroform/methanol (1:1) (DMPG). The films were left under vacuum overnight to remove all traces of the organic solvent. The resulting dried lipid films were dispersed with 10 mM HEPES buffer (0.1 M NaCl, pH 7.4), and the mixture was vortexed above the phase transition temperature (37 ( 0.1 °C) to produce multilamellar liposomes (MLV). Frozen and thawed MLVs were obtained by repeating five times the following cycle: freezing the vesicles in liquid nitrogen and thawing the sample in a water bath at 37 ( 0.1 °C. Lipid suspensions were equilibrated at 37 ( 0.1 °C for 30 min and extruded 10 times through polycarbonate filters (100 nm) to produce large unilamellar vesicles (LUVs). Drug-Liposome Preparation. Incubation Method. Samples were prepared by mixing a known volume of drug and a suitable aliquot of vesicle suspension in HEPES (LUVs), whereas the corresponding reference solutions were prepared identically, but without drug. All suspensions were then vortexed for 5 min and incubated at 37 ( 0.1 °C for 30 min, and typically two sets of 10 samples (1.5 mL) were used in each experiment. Encapsulation Method. Two identical lipid films were prepared by the method described above. One of them was dispersed with 3 mL of HEPES buffer solution which contained a known amount (13) Savitzky, A.; Golay, M. J. E. Anal. Chem. 1964, 36, 1611. (14) Gran, G. Analyst 1952, 77, 661. (15) Fiske, C. H.; Subbarow, Y. J. Biol. Chem. 1925, 66, 375. (16) Gans P.; Sabatini, A.; Vacca, A. Talanta 1996, 43, 1739.
Interaction of Grepafloxacin with Liposomes Table 1. Summary of UV/Visible, Fluorescence, and Acid/Base Properties of Grepafloxacin UV/vis spectra λmax (nm) 273 323 337
Langmuir, Vol. 18, No. 26, 2002 10233 Table 2. Paritition Coefficient of Grepafloxacin in DMPG and DMPCa DMPC
fluorescence spectra
acidity constants
λexc ) 330 nm λem ) 441 nm
pKa1 ) 6.78 ( 0.05 pKa2 ) 8.33 ( 0.10
of drug, while the other was dispersed with the same volume of HEPES buffer and used as a reference. LUVs were then obtained as described above. Quenching Experiments. The quencher, KI, was freshly prepared, and sodium thiosulfate (0.1 mM) was added to the KI stock solution to prevent I3- formation. Iodide was quantified by titration with a standard solution of KIO3 in a mixture of carbon tetrachloride and hydrochloric acid.17 Samples were prepared by mixing a known volume of drug (final concentration, 10 µM), a suitable aliquot of vesicle suspension (lipid concentration in the range from 0 to 700 µM), and KI (final concentration in the range from 0.001 to 0.2 M) in HEPES buffer. All suspensions were then vortexed for 5 min and incubated at 37 ( 0.1 °C for 30 min. In the quenching experiments, the excitation wavelength was 330 nm and fluorescence was monitored at 441 nm. Steady-State Anisotropy Experiments. Lipids were dissolved in the appropriate solvent in a round-bottom flask (25 mL) and mixed with stock solutions of DPH or TMA-DPH in the same solvent. The solvent was removed with a stream of argon, and the lipid films containing the fluorescent probes were dried overnight under vacuum. Dried lipid films were suspended in HEPES buffer and vortexed for 5 min. The corresponding LUVs were prepared by the method described above. Grepafloxacin solutions (in HEPES buffer) were added to liposome suspensions to obtain a final concentration of 25 µM in drug and 1 mM in lipid. The molar ratio DPH/lipid and TMA-DPH/lipid was 1:300. Reference solutions were prepared identically but without grepafloxacin. The anisotropy was recorded at several temperatures between 15 and 40 °C, with an accuracy of (0.1 °C. The excitation wavelength for DPH was set at 360 nm and the emission wavelength was 427 nm, whereas for TMA-DPH the corresponding values were 365 and 427 nm. Grepafloxacin fluorescence at any of these wavelengths is negligible.
Results and Discussion Grepafloxacin has two relevant ionizable functional groups, as do similar fluoroquinolones used as antibiotics: a basic piperazinyl group in the 7-position and a carboxylic acid group in the 3-position of the quinolone ring (Chart 1). The acid dissociation constants for grepafloxacin were obtained by potentiometric titrimetry using established methods.18,19 Despite the rough similarity observed between the values (Table 1) of grepafloxacin and those of other analogous fluoroquinolones, the existence of small differences has important consequences at the physiological pH 7.4.20,21 Whereas other fluoroquinolones (norfloxacin, ofloxacin, and ciprofloxacin) exist ≈90% in the zwitterionic and 10% in the anionic form, grepafloxacin exists only 75% in the zwitterionic form, with 20% in the cationic and 5% in the anionic form. As, to the best of our knowledge, there are no reported spectral data for grepafloxacin, relevant information from the UV/visible spectrum and fluorescence emission spectrum is included in Table 1. It must be stressed that the spectral properties (17) Vogel, A. I. A Text Book of Quantitative Analysis: Theory and Practice; Longmans and Green: London, 1948. (18) Castro, B.; Gameiro, P.; Lima, J. L. F. C. Anal. Chim. Acta 1993, 281, 53. (19) Castro, B.; Gameiro, P.; Guimara˜es, C.; Lima, J. L. F. C.; Reis, S. J. Pharm. Sci. 1998, 87, 356. (20) Ross, D. L.; Riley, C. M. J. Pharm. Biomed. Anal. 1994, 121, 1325. (21) Kawai, Y.; Matsubayashi, K.; Hakusui, H. Chem. Pharm. Bull. 1996, 44, 1425.
Kp (M-1)
DMPG
Kp (M-1)
second derivative 250 ( 16 second derivative 2690 ( 64 phase separation 252 ( 20 fluorescence method 2650 ( 140 a The mean particle size for liposomes with grepafloxacin (≈20 µM) was found to be 103 ( 4 nm for DMPC and 98 ( 2 nm for DMPG.
of grepafloxacin are very similar to those of other fluoroquinolones.22-25 Partition Coefficients. The partition coefficient26 of any compound between vesicle suspensions and aqueous solution is defined as
Kp )
(Cm/Ct)/[lipid] (Cw/Ct)/[water]
(1)
in which C is the drug molar concentration, the subscripts m and w stand for drugs in lipid and in aqueous media, and [lipid] and [water] represent lipid and water molar concentrations. In this work, two independent methods were used to calculate the drug concentrations and thus the partition coefficients, one with and the other without phase separation. Partition Coefficients Determined After Phase Separation of Drug/DMPC Liposome Suspensions. The concentrations can be determined from absorption spectra of the aqueous and of the lysed lipid ethanolic solutions, which are obtained after phase separation. This method has proved to be applicable to water/DMPC suspensions, but attempts to separate the lipid and aqueous phases, even by ultracentrifugation, were not successful for DMPG liposomes, probably a consequence of their small size and negative charge.9,27 For water/DMPC suspensions, all separations were performed with the centrifugal units described in the Experimental Section, and to ensure that drug recovery was complete, the amount of drug was always determined spectrophotometrically before and after phase separation. The liposomes were lysed with ethanol to release the drug, and in the spectrophotometric determinations a standard curve in ethanol was used to minimize experimental errors; all measurements were repeated at least twice.9,27 The Kp values obtained by direct application of eq 1 are listed in Table 2. Partition Coefficients Determined Without Phase Separation of Drug/Liposome Suspensions. The UV/vis spectra of grepafloxacin show a decrease in absorption intensities in the presence of increasing amounts of either DMPC or DMPG (Figure 1). Furthermore, the absorption spectra of grepafloxacin in DMPG, but not in DMPC, exhibit isosbestic points and a shift in λmax with increasing lipid concentration, an observation that provides a clear indication that the drug exists in two forms in solution. By using second-derivative spectrophotometry, the effect (22) Mera´s, I. D.; Pena, A. M.; Ca´ceres, M. I. R.; Lo´pez, F. S. Talanta 1998, 45, 899. (23) Drakopoulos, A. I.; Ioannou, P. C. Anal. Chim. Acta 1997, 354, 197. (24) Djurdjevic, P. T.; Jelikic-Stankov, M.; Stankov, D. Anal. Chim. Acta 1995, 300, 253. (25) Rieutord, A.; Vazquez, L.; Soursac, M.; Prognon, P.; Blais, J.; Bourget, Ph.; Mahuzier, G. Anal. Chim. Acta 1994, 290, 215. (26) The partition coefficient as defined by eq 1 is defined by IUPAC as the partition ratio; IUPAC does not recommend the use of the former definition. Its use throughout this work reflects the common practice in the biological literature. (27) Rodrigues, C.; Gameiro, P.; Reis S.; Lima, J. L. F. C.; Castro, B. Anal. Chem. Acta 2001, 428, 103.
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Figure 1. Absorption spectra (1) and second-derivative spectra (2) of grepafloxacin in DMPC (A) and DMPG (B) at different lipid concentrations (µM): (1) 0, (2) 175, (3) 250, (4) 375, (5) 500, and (6) 675.
of background signals is eliminated, and the resolution of overlapped signals is improved by sharpening them.28-30 The values of partition coefficients, without phase separation, were obtained by fitting eq 2 to experimental second-derivative spectrophotometric data (∆D versus [L]) for a given drug concentration, using a nonlinear leastsquares regression method:9,27,31
∆D )
KpECt[lipid] [water] + Kp[lipid]
(2)
where D ) d2A/d2λ, ∆D ) Dm - Dw, E ) d2/d2λ and ) m - w, and the other symbols have their usual meanings. The partition coefficients are listed in Table 2, and for DMPC they are identical, within experimental error, to that obtained by phase separation. Fluorescence spectra of grepafloxacin, on the other hand, exhibit an increase in fluorescence intensity with increasing concentration of DMPC or of DMPG vesicles, which reflects its partition into a more hydrophobic environment. Fluorescence data were analyzed using the equation32
∆I )
∆Imax Kp[lipid] [water] + Kp[lipid]
(3)
(28) Talsky, G.; Mayring, L.; Kreuzer, H. Angew. Chem., Int. Ed. 1978, 17, 785. (29) Sommer, L. Analytical Absorption Spectrophotometry in the Visible and Ultraviolet: The Principles; Studies in Analytical Chemistry No. 8; Elsevier: Amsterdam, 1989; pp 186-199. (30) Kitamura, K.; Imayoshi, N. Anal. Sci. 1992, 8, 497. (31) Kitamura, K.; Imayoshi, N.; Goto, T.; Shiro, H.; Mano, T.; Nakai, Y. Anal. Chem. Acta 1995, 304, 101. (32) Coutinho, A.; Prieto, M. Biophys. J. 1995, 69, 2541.
in which ∆I ) I - I0 stands for the difference between the fluorescence intensity of the drug measured in the presence (I) and the absence (I0) of lipid vesicles, and ∆Imax ) I∞ I0 is the maximum value of this difference, where I∞ stands for the limiting value of I. The measurable spectral changes due to drug/lipid interaction can be used to obtain the partition coefficient, as the background signals due to liposome light scattering do not interfere under the experimental conditions used. The Kp values obtained by fitting experimental data for DMPG and DMPC to eq 3 are identical, within experimental error, to those obtained by second-derivative spectrophotometry (Table 2) and for DMPC are also identical to that determined by phase separation. This observation lends additional support to the validity of the methodology used to determine partition coefficients of drugs in liposomes without phase separation.10,27 Analysis of the data in Table 2 reveals that grepafloxacin exhibits a measurable affinity for both lipid environments, even though the partition coefficient of grepafloxacin in DMPG is 10 times greater than that in DMPC liposomes. This observation suggests a larger affinity of grepafloxacin for negatively charged liposomes, which must be a consequence of an ionic interaction between the positive form of the drug, at physiological pH, and the phosphate groups of the negatively charged heads of DMPG. The partition coefficients of grepafloxacin are, to the best of our knowledge, the first to be reported for fluoroquinolones. Ciprofloxacin, one of the most studied fluoroquinolones, has a structure very similar to that of grepafloxacin, but the partition coefficients, although not reported, were claimed to be very small in a lipid environment.8,12 The larger partition coefficients of grepafloxacin must be associated with the presence of the
Interaction of Grepafloxacin with Liposomes
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Figure 2. Fluorescence intensities vs quencher concentration (I-) for a solution of grepafloxacin in the absence of liposomes (0) and in the presence of DMPC (9) and DMPG ([). Table 3. Stern-Volmer Quenching Constant (KSV) for Grepafloxacin in DMPG and DMPCa DMPC (µM)
KSV (M-1)
DMPG (µM)
KSV (M-1)
250 500 700
21.5 ( 0.3 18.2 ( 0.6 15.3 ( 0.3
250 500 700
10.93 ( 0.03 8.86 ( 0.05 6.61 ( 0.07
a For grepafloxacin in aqueous buffer (HEPES) solution, K SV ) 25.7 ( 0.2 M-1.
two methyl groups in its structure (Chart 1). These groups increase the pKa values, and at the physiological pH the drug exists not only in a zwitterionic form (like the other fluoroquinolones) but also in a cationic form that interacts more strongly with the negatively charged lipid heads. On the other hand, the presence of the methyl groups may require the compound to be inserted differently into the bilayers and thus enhance the electrostatic interactions. In such a situation, hydrophobic and electrostatic interactions may strengthen each other. Location Studies. Fluorescence quenching and fluorescent anisotropy, the latter using fluorescent probes, have been used to provide information about membrane fluidity, drug insertion, and drug location into liposomes.33 Quenching Studies. Insights about the location of grepafloxacin, a fluorescent molecule, can be obtained from its fluorescence quenching with iodide, a quencher that is water soluble and that is expected to affect the fluorescence properties of grepafloxacin only when the drug is not located deeply in the membrane. In Figure 2 are depicted the fluorescence intensities of grepafloxacin versus iodide concentration, in the absence and the presence of DMPC and DMPG liposomes. Quenching data were analyzed using the classic Stern-Volmer equation
F0 ) 1 + kSV[Q] F
(4)
where F0 and F are the fluorescence intensities in the absence and the presence of the quencher Q and kSV is the Stern-Volmer constant. The observed Stern-Volmer plots were linear, always with correlation coefficients higher than 0.999, and the values obtained for kSV are collected in Table 3. This linearity indicates the existence of only one type of quenching, and since the electronic spectra of grepafloxacin do not change in the presence of I-, the quenching (33) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum: New York, 1999.
must be collisional.33 The slight decrease in kSV values with increasing lipid concentration can be attributed to a small partition of the quencher in the lipid phase.34 The Stern-Volmer constants decrease in the order (grepafloxacin in buffer solution) < (grepafloxacin in DMPC) < (grepafloxacin in DMPG), in agreement with what was expected from the partition coefficient, as a larger partition coefficient makes the drug less accessible to iodide. The differences in the fluorescence intensity of grepafloxacin free or incorporated in liposomes reflect the different environments of the drug. For free grepafloxacin, almost all fluorophores are accessible to iodide, whereas when the fluoroquinolone is incorporated in liposomes only a fraction of total grepafloxacin molecules are accessible to iodide. The existence of an unquenched population confirms the interaction of some grepafloxacin molecules with the liposomes. Steady-State Anisotropy Studies. To complement the information provided by quenching data, fluorescence anisotropy with the probes DPH and TMA-DPH was used to study the location of grepafloxacin molecules in DMPC and DMPG liposomes and the influence of their incorporation on membrane fluidity. DPH is deeply buried, as expected from its hydrophobic nature, and packs well with fatty acyl chains, whereas TMA-DPH, with a cationic group attached to the DPH phenyl ring, is located more shallowly than DPH, and thus TMA-DPH is expected to monitor lipid order changes closer to the water/membrane interface than does DPH.35 To avoid competition between drug and probe molecules for placement in the hydrophobic core of the liposome vesicles,36 a 1:300 probe/lipid molar ratio was used throughout this work. The degree of fluorescence anisotropy (r) is defined by the following equation:
r)
IVV - IVHG IVV + 2IVHG
(5)
where IVV and IVH are the intensities measured in directions parallel and perpendicular to the excitation beam. The correction factor G is the ratio of the detection system sensitivity for vertically and horizontally polarized light, which is given by the ratio of vertical to horizontal components when the excitation light is polarized in the horizontal direction, G ) IHV/IHH.33 The temperature dependence of DPH and TMA-DPH anisotropy in neutral and negatively charged liposomes is shown in Figure 3. The anisotropy of DPH, in both liposomes, is insensitive to the presence of grepafloxacin, whereas that of TMA-DPH changes slightly when grepafloxacin is added to the liposome suspensions. Furthermore, the lipid transition temperature (Tm) in the presence of drug monitored by TMA-DPH anisotropy shows a small decrease, less than 1 °C, and an increase of anisotropy above the phase transition. The observation that the anisotropy of DPH is not affected by grepafloxacin whereas that of TMA-DPH is makes it clear that the interaction of grepafloxacin with these lipids must occur close to the water/membrane interface. (34) Chalpin, D. B.; Kleinfeld, A. M. Biochim. Biophys. Acta 1983, 731, 465. (35) Moya-Quiles, M. R.; Mun˜oz; Delgado, E.; Vidal, C. J. Chem. Phys. Lipids 1996, 79, 21. (36) Blatt, E.; Sawyer, W. H. Biochim. Biophys. Acta 1985, 822, 43.
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Figure 3. Temperature dependence of the anisotropy of DPH (A) and TMA-DPH (B) in DMPC (1) and DMPG (2) liposomes in the absence (9) and the presence (b) of grepafloxacin (20 µM).
Furthermore, the small change in transition temperature (Tm) in the presence of grepafloxacin and the increase in the anisotropy above the phase transition, taken together, suggest that grepafloxacin interacts electrostatically with the headgroup of the lipids, which causes changes in the orientation of the phosphoryl group, and does not penetrate deeply into the membrane.8 Concluding Remarks The interaction of grepafloxacin in liposomes occurs to a larger extent than that found for other fluoroquinolones, a consequence of its different acid/base properties and more hydrophobic structure. Indeed, when compared with ciprofloxacin, the most studied fluoroquinolone, grepafloxacin has two more methyl groups, one in the heterocyclic ring and the other in the piperazine ring, and our data show that their presence changes significantly the physicochemical characteristics of grepafloxacin, enhancing strongly the interaction of fluoroquinolones with liposomes and consequently modifying/improving the mechanism of entry of the drugs into cell membranes. The results of fluorescence quenching and fluorescence anisotropy studies are in total agreement with the data obtained for partition coefficients and also suggest that grepafloxacin must be located near the phospholipid headgroup, with a stronger interaction with the negatively charged head of phosphatidylglycerol. In view of these results, the mechanism by which grepafloxacin permeates through the phospholipid bilayer must include an electrostatic adsorption at the interface region and this association must be the first step that governs the mechanism of interaction of these drugs with bacterial natural membranes.
This work shows that a slightly difference in the chemical structure of an active substance can bring large changes in some of its physicochemical properties. Grepafloxacin is a second-generation fluoroquinolone that maintains broad Gram(-) activity and has its Gram(+) activity increased compared to that of the first-generation fluoroquinolones. This increase in Gram(+) activity has been claimed to be associated with the introduction of the methyl group in the piperazine ring.37 This work shows that the introduction of this methyl group decreases the acidity of the fluoroquinolone carboxylic group, which increases the concentration of the cationic species at physiological pH and consequently the partition of the fluoroquinolone, particularly into negatively charged surface membranes. Analyses of our data seem to suggest that the increase of the Gram(+) activity of grepafloxacin, when compared with that of first-generation fluoroquinolones, can be due to the charge interaction that must occur at the negatively charged membrane surface of the Gram(+) bacteria at physiological pH. Although grepafloxacin is not being commercialized at present, our results show the importance of the acid/base properties of drugs in their physiological activity and can help in the development of new drugs with analogous, but increased, therapeutic activity. Acknowledgment. Partial financial support for this work was provided by “Fundac¸ a˜o para a Cieˆncia e Tecnologia” (FCT, Lisboa) through Project POCTI/34308/ QUI/2000. C.R. thanks FCT for a fellowship. LA0205093 (37) Domagala, J. M. J. Antimicrob. Chemother. 1994, 33, 685.
