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Effects of Ciprofloxacin on Escherichia coli Lipid Bilayers: An Atomic Force Microscopy Study Sandra Merino,†,‡ O Å scar Dome`nech,†,‡ Ismael Dı´ez,‡,§ Fausto Sanz,‡,§ | Miquel Vin˜as, M. Teresa Montero,*,†,‡ and Jordi Herna´ndez-Borrell†,‡ Departament de Fisicoquı´mica, Departament de Quı´mica Fı´sica, and Centre de Bioelectro` nica i Nanobiocie` ncia (CBEN), Universitat de Barcelona, 08028 Barcelona, Spain, and Laboratori de Microbiologia, Campus de Bellvitge, Universitat de Barcelona, 08907 L’Hospitalet de Llobregat, Spain Received February 11, 2003. In Final Form: April 7, 2003 The interaction of 6-fluoroquinolones with phospholipids may account for their remarkable activity against a broad spectrum of microorganisms. In preceding papers, we have shown that ciprofloxacin (CPX) and its N-4-butylpiperazinyl derivative (BCPX) might be surface-active drugs that interact at the lipidwater interface where the headgroups reside. In this work atomic force microscopy (AFM) was applied to observe the effects of CPX and BCPX on surface planar bilayers (SPBs) formed with the phospholipid extract of membranes of Escherichia coli (E. coli). Two main features were observed when liposomes of E. coli preincubated with CPX were spread onto mica: (i) the degree of surface coverage by the liposomes was reduced in comparison with the degree covered by the liposomes nonincubated with the drug; and (ii) at high lipid-to-drug ratio patches of drug were observed which protrude from the surrounding lipid domains. BCPX, on the other hand, facilitates liposome extension onto mica. In situ experiments, carried out by injecting CPX or BCPX on SPBs, confirmed the observed behavior ex situ. CPX patches were interpreted as the result of drug self-association and the fact that this drug has a negligible partitioning into the bilayers. On the other hand BCPX, which is able to integrate into the bilayer, promotes its extension.
Introduction 6-Fluoroquinolones (6-FQs) are a group of antibiotics with a broad spectrum of activity against gram-positive and gram-negative microorganism and some mycobacterium species as well.1 It has been thought that this unusual activity could be attributed, at least in part, to their ability to diffuse through lipid bilayers,2 (hydrophobic via). However, the hydrophilic via (porin pathway)3 of entry seems to be the major entry route of 6-FQs into the cytoplasm. However, there is evidence that supported the fact that many drugs, including 6-FQs, are extruded out of the cytoplasm by efflux pumps. Their mechanism of function seems to be dependent on the surrounding lipid bilayer environment.4 Hence, that the ability of 6-FQ to interact with the lipid bilayers becomes of crucial interest in understanding the mechanism of entry and efflux as well. Pioneering work, by Bedard and Bryan,5 points to the involvement of ionic and hydrophobic forces in the binding of ciprofloxacin (CPX; Figure 1) with liposomes bearing negative surface charge. An electrostatic interaction was also evidenced in the fluorimetric titration of CPX in the presence of negatively charged liposomes.6 * To whom correspondence should be addressed at Departament de Fisicoquı´mica, Facultat de Farma`cia, Universitat de Barcelona. 08028 Barcelona, Spain. E-mail:
[email protected]. † Departament de Fisicoquı´mica. ‡ Centre de Bioelectro ` nica i Nanobiocie`ncia (CBEN). § Departament de Quı´mica Fı´sica . | Laboratori de Microbiologia. (1) Hooper, D. C., Wolfson, J. S., Eds. Quinolone Antimicrobial Agents, 2nd ed.; ASM: Washington, DC, 1995. (2) Nikaido, H.; Thanassi, D. G. Antimicrob. Agents Chemother. 1993, 37, 1393-1399. (3) Berlanga, M.; Ruiz, N.; Herna´ndez-Borrell, J.; Montero, M. T.; Vin˜as, M. Can. J. Microbiol. 2000, 46, 716-722. (4) Putman, M.; Van Veen, H. W.; Konings, W. N. Microb. Mol. Biol. Rev. 2000, 64, 672-693. (5) Bedard, J.; Bryan, L. E. Antimicrob. Agents Chemother. 1989, 33, 1379-1382.
Figure 1. Structure of CPX(RdH) and BCPX (CH3(CH2)3-). Partition coefficient values expressed as log P, between octanol and E. coli lipid extract and buffer pH 7.4 have been previously published.9,26
Other authors,7 working with enrofloxacin (another 6-FQ) and using fluorescence polarization, microcalorimetry, and monolayer techniques, came to the conclusion that the level of integration of the drug with neutral phospholipids was very low. In apparent concordance, it has been demonstrated that the amount of CPX associated with neutral liposomes is less than 3% of the total amount of drug added.8 In view of such a low affinity of CPX for neutral membranes, we synthesized its homologous series by the introduction of N-alkyl substituents (one to five C) at the 4 position of the piperazynil group of CPX9 (Figure 1). (6) Va´zquez, J. L.; Berlanga, M.; Merino, S.; Dome`nech, O Å .; Vin˜as, M.; Montero, M. T.; Herna´ndez-Borrell, J. Photochem. Photobiol. 2001, 73, 14-19. (7) Mestres, C.; Alsina, M. A.; Busquets, M. A.; Haro, I.; Reig, F. Langmuir 1994, 10, 767-772. (8) Montero, M. T.; Freixas, J.; Herna´ndez-Borrell, J. Int. J. Pharm. 1997, 149, 161-170.
10.1021/la034232y CCC: $25.00 © 2003 American Chemical Society Published on Web 07/12/2003
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Figure 2. (A) Escherichia coli supported phospholipid bilayer. (B) Height profile along the drawn line in A. The measured step height between a noncovered area (white star) and the lipid bilayer plateau (black star) was 4.4 nm.
Importantly, CPX and the butyl alkylated compound (BCPX) induced significant changes in the electrostatic surface potential, which was, as expected, dependent on the liposome composition.10,11 On the other hand, both CPX and BCPX produce a decrease in the fluorescence anisotropy values and small, sometimes negligible, variations in the transition temperature of the phospholipids.11,12 The bulk of these studies suggested that the driving force in the interaction between CPX and model membranes is far from simple, involving hydrophobic and electrostatic components and, possibly, hydrogen bonding with the phospholipid headgroups.11 The effect caused by CPX and BCPX, as observed by means of the epifluorescence of phospholipid monolayers13,14 provides unequivocal evidence for an interfacial location of the drugs. To gain further insight into the effects of CPX and BCPX on biological membranes, we applied atomic force microscopy (AFM) to observe the effect of both drugs on polar lipid extract from Escherichia coli (E. coli). The objectives of this work were to study (i) the layer formation of E. coli lipid vesicles spread onto mica surface and (ii) the effect of CPX and BCPX on lipid vesicle extension. Experimental Section Chemicals. Escherichia coli polar lipid extract (nominally 67% phosphatidylethanolamine, 23.2% phosphatidylglycerol, and 9.8% cardiolipin) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Basically, the polar lipid extract is the total lipid extract precipitated with acetone and then extracted with diethyl ether.15 Ciprofloxacin and butylciprofloxacin were obtained from Cenavisa (Reus, Spain). Their purity was assessed by IR and HPLC. Resuspension buffer solution (RB): 10 mM (9) Maurer, N.; Wong, K. F.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1998, 1374, 9-20. (10) Merino, S.; Va´zquez, J. L.; Dome`nech, O Å .; Berlanga, M.; Vin˜as, M.; Montero, M. T.; Herna´ndez-Borrell, J. Langmuir 2002, 18, 32883292. (11) Grancelli, A.; Morros, A.; Caban˜as, M. E.; Dome`nech, O Å .; Merino, S.; Va´zquez, J. L.; Montero, M. T.; Vin˜as, M.; Herna´ndez-Borrell, J. Langmuir 2002, 18, 9177-9182. (12) 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. (13) Montero, M.T.; Herna´ndez-Borrell, J.; Keough, K. M. W. Langmuir 1998, 14, 2451-2454. (14) Herna´ndez-Borrell, J.; Montero, M. T. Int. J. Pharm. 2003, 252, 149-157. (15) Newman, M. J.; Wilson, T. H. J. Biol. Chem. 1980, 255, 1058310586.
Hepes pH 7.40, 150 mM NaCl, 20 mM CaCl2; I ) 0.15 m. Stock solutions of CPX and BCPX (300 µM) were prepared in RB after a few cycles of vortexing and sonication bath. Solutions were always freshly prepared. Vesicle Preparation. Chloroform/methanol (50:50 (v/v)) stock solutions of E. coli lipids were evaporated to dryness in a conical tube using a rotavapor. The resulting thin lipid film was then kept under high vacuum overnight to ensure the absence of organic solvent traces. Multilamellar vesicles were obtained by hydration in an excess of RB. Thereafter suspensions were filtered through Nucleopore (Costar, Cambridge, MA, USA) polycarbonate filters (200 nm nominal diameter) using an Extruder (Lipex Biomembranes Inc.,) device.16 Liposomes were diluted in RB to a final concentration of 100 µM total phospholipid. The size and polydispersity of the liposomes were monitored by quasielastic light scattering (QELS) using an Autosizer IIc photon correlation spectrophotometer (Malvern Instruments). AFM Observations. All images were recorded with a commercial Digital Instruments (Santa Barbara, CA, USA) Nanoscope III AFM fitted with a 15 µm scanner (d-scanner). Standard Si3N4 tips, with a nominal force constant of 0.1 N/m (Digital Instruments), were used, and the acting forces, either lateral or along the z-axis, were minimized using the tapping mode. Before every sample, the AFM liquid cell was washed with ethanol and ultrapure water (Milli Q reverse osmosis system) and allowed to dry in a N2 stream. Mica disks (green mica) were cleaved with scotch and glued onto a Teflon disk by a water-insoluble epoxy. These Teflon disks were glued onto a steel disk and then mounted onto the piezoelectric scanner. All images were scanned under aqueous solution. Prior to imaging the sample, the tip was stabilized in buffer. Force plots were recorded for each sample to control the repulsion of the tip, and images were flattened with the Nanoscope III program. Samples were prepared for AFM images following two different procedures: (i) to study the effect of drugs on vesicles extension, large unilamellar liposomes were preincubated with CPX or BCPX at 40 °C for 30 min and cooled at room temperature before the observation. Then, aliquots of 50 µL of liposome samples, in the absence and presence of drug, were pipetted onto freshly cleaved mica. After allowing the vesicles to adsorb at room temperature for 20 min, the surface was gently washed with Ca2+ free buffer. For in situ experiments (ii) 50 µL of liposomes was deposited onto freshly cleaved mica and the tip was immediately immersed in the buffer. To minimize the thermal drift of the cantilever, the tip engaging time was considered the starting zero (t ) 0) for a sequential observation, which corresponds to injecting appropriate volumes of drug solution into the AFM fluid cell to increase the final drug concentration. (16) Hope, M. J.; Bally, M. B.; Welb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55-65.
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Results and Discussion It has been shown that E. coli lipid extracts form lipid vesicles with monomodal size distribution after extrusion.17 The size distribution curve of the liposomes by QELS exhibited a monomodal distribution with a diameter of about 198 ( 11 nm. The polydispersity index was in all cases lower than 0.1, indicating a homogeneous size distribution of the vesicles. These liposomes deposited onto mica spread and form supported planar bilayers (SPBs),18,19 as shown in Figure 2. According to the procedure described in Experimental Section, the spreading process was stopped after 20 min of liposome deposition by washing with the Ca2+ free buffer. It is well-known that the presence of Ca2+ is required for the transformation (adsorption, fusion, and rupture) of vesicles into single-bilayer disks, which then coalesce and form the SPBs.20 As expected, by retrieving the cation, an uncompleted coverage of the surface results (up to approximately 85% in Figure 2A). Defects and/or holes, dark in the images, allowed the analysis of the lipid domain thickness by the difference between the two levels observed (lipid and mica). Thus, the SPBs from the E. coli lipid extract shown (see Figure 2B) had a step height of 4.4 ( 0.4 nm (n ) 10). This value is in reasonable agreement with values reported for other SPBs21,22 of different composition. However, it is important to notice here that the height depends on the phospholipid composition, on the Ca2+ presence, and also on the force applied with the tip.23 In the presence of CPX (100 µM) lipid domains of different sizes are observed (Figure 3A). The image in Figure 3A shows a large lipid planar bilayer coexisting with single-bilayer disks of small size. When liposomes incubated with 200 µM CPX where spread onto mica, large lipid regions of almost round shape, such as the one shown in Figure 3B, are observed. The corresponding cross section (Figure 3C) shows the large plateau of the lipid domain giving in the edge the height of a bilayer (3.7 ( 0.5 nm, black arrow in Figure 3C). Clearly, incubation of liposomes with CPX results in a decrease of the surface covered by the bilayer. This could be interpreted qualitatively due to the increase of the line tension (defined as the work done to increase the perimeter of the membrane with a unit length)22 by the presence of CPX may be related with the reduction of the mean molecular average area obtained from the compression isotherms of mixed monolayers of DPPC and CPX.11 A height profile of the line section depicted in Figure 3B is shown in Figure 3C, which strongly suggests that CPX might be segregated and remain adsorbed onto the bilayer. Interestingly, few small patches (see Figure 3B) with a step average height of 11 ( 2 nm (n ) 5), which protrude from the surrounding lipid domains, are observed. Remarkably, those patches appear only at the highest concentration of CPX and have not ever been observed in the absence of drug (Figure 2) or at the lowest CPX concentration (see Figure 3A). Therefore, it seems highly likely that those patches are drug aggregates and could be also related with the enriched (17) White, G. F.; Racher, K. I.; Lipski, A.; Hallet, F. R.; Wood, J. M. Biochim. Biophys. Acta 2000, 1468, 175-186. (18) Leonenko, Z. V.; Carnini, A.; Cramb, D. T. Biochim. Biophys. Acta 2000, 1509, 131-147. (19) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806-1815. (20) Nollert, P.; Kiefer, H.; Ja¨hnig, F. Biophys. J. 1995, 69, 14471455. (21) Jass, J.; Tja¨rnhage, T.; Puu, G. Biophys. J. 2000, 79, 31533163. (22) Muresan, A. S.; Lee, K. Y. J. Phys. Chem. 2001, 105, 852-855. (23) Beckman, M.; Nollert, P.; Kolb, H. A. J. Membr. Biol. 1998, 161, 227-233.
Figure 3. E. coli lipid supported phospholipid bilayer incubated with (A) 100 µM and (B) 200 µM of CPX. (C) Height profile along the drawn line in B, showing aggregates (red arrows) and edge domain (black arrow) heights.
domains of 6-FQs earlier observed by epifluorescence microscopy11,13,24 of lipid monolayers. BCPX is a molecule with a higher lipophilicity than CPX9 (see Figure 1). Therefore it is able to penetrate deeper into the bilayer.11,25 Figure 4 illustrates the SPBs obtained after extension of liposomes incubated with 100 µM BCPX. This figure shows a surface even more covered, although qualitatively, than the lipid extract itself (Figure 2A). Holes, are still visible and allowed to measure a depth of 4.3 ( 0.2 (n ) 2), which agrees with the values obtained in Figure 2A. Complete coverage of the surface was obtained by depositing liposomes incubated with 200 µM BCPX, and no defects are observed (data not shown). The (24) Nag, K.; Keough, K. M. W.; Montero, M. T.; Trias, J.; Pons, M.; Hernandez-Borrell, J. J. Liposome Res. 1996, 6, 713-736. (25) Grancelli, A.; Cladera, J.; Villaverde, J.; Trias, J.; Morros, A. In Spectroscopy of Biological Molecules: Modern Trends; Carmona, P., Navarro, R., Herranz, A., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1997; pp 321-322.
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Figure 4. (A) E. coli supported phospholipid bilayer incubated with 100 µM BCPX. (B) Height profile along the drawn line in A.
Figure 5. Real-time series of E. coli SPBs (in fluid) on mica obtained by in situ injection of increasing amounts of CPX. Solution concentrations are indicated in each image. Time after onset of imaging: (A) 0, (B) 10, (C) 20, and (D) 30 min.
surface was in this case molecularly smooth, and no domains were observed, indicating a good mixing of both components. Contrarily to what we have observed with CPX, BCPX appears to facilitate liposome extension onto mica presumably by reduction of the line tension. In situ experiments were performed to monitor the effects produced by increasing amounts of drugs on the
two-dimensional growth of E. coli SPBs. In Figure 5, a series in real time corresponding to accumulative concentrations of CPX injected over the same area are shown. Small patches were observed immediately to the first injection of drug (Figure 5B), which grew in number upon increasing CPX concentration. Clearly, these patches should be attributed to CPX itself, an observation which
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Figure 6. Time-lapse series of E. coli SPBs (in fluid) obtained by injection in situ of increasing amounts of BCPX in mica. Concentrations reached are indicated on top of the corresponding image. Time elapsed after imaging onset: (A) 0, (B) 10, (C) 20, and (D) 30 min.
seems related to the property of this drug to self-associate inside liposomes at pH 4.9, forming stacks.8 Although our observations were carried out at neutral pH, it cannot be excluded that variations in the local pH at the membrane surface and/or the influence of the lipid composition could induce self-association and drug confinement onto the lipid surface. The series of images in Figure 5 show that CPX diffuses on the surface of the lipid domain and through the buffer as well. Indeed, CPX patches are not firmly adsorbed to the SPB because they are able to move under the influence of the jet stream produced by the injection of the drug. On the other hand, it has been shown that CPX has an almost negligible partitioning into lipid bilayers;8,11,26 therefore we conclude that the patches observed should be attributed to the non-entrapped drug, which is squeezed-out from the lipid environment. Similar conclusion was achieved from 31P-MNR measurements carried out with neutral liposomes.11 This time-lapse series evidenced that the maximum spreading is achieved in 30 min. The SPB then remains stable, and no more mica surface is covered after that (26) Va´zquez, J. L.; Merino, S.; Dome`nech, O.; Berlanga, M.; Vin˜as, M.; Montero, M. T.; Herna´ndez-Borrell, J. Int. J. Pharm. 2001, 220, 53-62.
time. Similarly to the previous experiments (Figure 3), an incomplete coverage results. Judging from the craggy edges (see the inset in Figure 5C,D) observed in the image, the CPX concentration reached at that time (Figure 5D, 200 µM) seems to produce a partial disruption and or solubilization of the SPB. A detergent-like effect could occur at this high concentration. The effect of increasing concentrations of BCPX on SPB formation is shown in Figure 6. As mentioned above, BCPX seems to enhance SPB formation by increasing the rate of spreading. To probe this, it is noteworthy that the imaging process of Figure 6 was started earlier than in Figure 5. Therefore, large planar bilayers still coexist with smaller domains of round shape. An almost total coverage of the surface was obtained in 30 min (i.e., compare Figures 5D and 6D). The results obtained suggested that BCPX promotes the extension of the lipid layer. This effect should be attributed to the ability of the alkyl derivative to be integrated into the bilayer, in concordance with previous works.10-13 This would affect in some way the time relaxation process of SPB formation.17 As expected from its partitioning behavior,21 neither patches nor irregularities on the surface were observed in this case.
Effects of Ciprofloxacin on E. coli Lipid Bilayers
The results presented in this work emphasize the fact that E. coli lipid membrane has an effect of confining CPX aggregates on its surface. Although this phenomenon is presently not fully understood, it could have important biological implications. Thus, from CPX patches, molecules could move along the surface increasing the probability of collision with absorbing surface domains of natural occurring membranes, receptors, or efflux pumps. In addition, the local accumulation achieved could play a role in the ability shown by CPX to form pores in black lipid bilayers.27 As an interstitial component integrated into the membrane, BCPX could be encapsulated with higher efficiency (27) Ruı´z, N. Dissertation Thesis, University of Barcelona, Barcelona, Spain, 2002. Vin˜as, M.; Benz, R. Unpublished observations.
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than CPX in liposomes and used as a drug delivery system. Otherwise the insertion of BCPX into the membrane seems to be related with the activity of an efflux pump.12 Further experiments are presently carried out in our laboratory that will enable us to understand the implications of CPX and BCPX membrane interaction. Acknowledgment. S.M. is the recipient of a fellowship of “Recerca i Doce`ncia de la U.B.”. This work was supported by Grants TIC 2002-04280-C03-01 and SAF 2002-00698 from Ministerio de Ciencia y Tecnologı´a, Spain. The authors thank the SCT of University of Barcelona for equipment availability. LA034232Y
