Langmuir 1998, 14, 2451-2454
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Fluoroquinolone-Biomembrane Interactions: Monolayer and Calorimetric Studies M. T. Montero,*,† J. Herna`ndez-Borrell,† and K. M. W. Keough‡ Departament de Fisicoquı´mica, Facultat de Farma` cia, Universitat de Barcelona, 08028-Barcelona, Spain, and Department of Biochemistry and Discipline of Pediatrics, Memorial University of Newfoundland, St. John’s, Newfoundland, A1B 3X9 Canada Received June 27, 1997. In Final Form: February 11, 1998
The interaction of two 6-fluoroquinolones, ciprofloxacin and its pentyl derivative, with model membranes was studied as a function of drug hydrophobicity as reflected in the pentyl side chain at position 4 in the piperazinyl group, by epifluorescence surface balance technique and differencial scanning calorimetry. In the first part of this study drug incorporation in dipalmitoylphosphatidylcholine monolayers was investigated using epifluorescence microscopy. In the second part, the influence of the presence of the hydrophobic chain and fluoroquinolone content on the thermotropic behavior of liposomes was investigated. Both monolayer and calorimetric studies demonstrated that hydrophobicity of the drug had a role in determining the nature of drug-membrane interaction as seen in greater perturbations of the lipid packing by pentyl ciprofloxacin.
Introduction Fluoroquinolones are a group of antibiotics which are currently the focus of attention of many research groups. They appear to be efficient against multidrug resistant tuberculosis (MDRTB) and particularly beneficial in improving the conditions of AIDS patients secondarily affected by Mycobacterium tuberculosis and M. aviumintracellulare complex.1 Their unusual activity against acid alcohol resistant bacteria, which show a strong barrier of complex architecture2 composed basically of mycolic acids and lipids, suggests that a physicochemical feature of those drugs plays an essential role in drug action in addition to their specificity against DNA gyrase. With the currently avalaible information it seems that a prerequisite for activity in the 6-fluoroquinolone series of antibiotics is an adequate hydrophilic-lipophilic molecular balance. Moreover, lipophilicity seems to play a role in development of resistance in bacteria.3 Three means have been proposed for the entry of fluoroquinolones into the cytoplasm: via the porin pathway,4 by a “self-promoted” route similar to that used by cationic compounds,5 and through a hydrophobic pathway.6 Although the porin pathway might be the most important, the hydrophobic route of permeation is likely also involved to some extent7 as might be expected from the moderate hydrophobicity of the drugs.8 To better understand the mechanisms of entry of the drug into cells, * To whom correspondence should be addressed. † Universitat de Barcelona. ‡ Memorial University of Newfoundland. (1) Hooper, D. C.; Wolfson, J. S. In Quinolone Antimicrobial Agents 2nd ed; A. S. M.: Washington, DC, 1995. (2) Nikaido, H.; Kim, S. H.; Rosenberg, E. Y. Mol. Microb. 1993, 8 (6), 1025-1030. (3) Nikaido, H. J. Bacteriol. 1996, 178 (20), 5853-5859. (4) Dechene, M.; Leying, H.; Cullmann, W. Chemotherapy 1990, 36, 13-23. (5) Hancock, R. E. W.; Rafle, V. J.; Nicas, T. I. Antimicrob. Agents Chemother. 1981, 19, 777-785. (6) Nikaido, H.; Thanassi, D. G. Antimicrob. Agents Chemother. 1993, 37, 1393-1399. (7) Georgopapadokou, N. H. In Drug transport in antimicrobial and anticancer chemotherapy; Georgopapadokou, N. H., Ed.; Marcel Dekker: New York, 1995; pp 145-162.
useful information can come from the study of drug interaction with membrane models such as liposomes and monolayers. The objective of this study was to analyze the interaction between fluoroquinolones and phospholipids in model membranes using differential scanning calorimetry (DSC) and monolayers using epifluorescence microscopic surface balance techniques to gain insight into the incorporation of drugs in the bilayer with the view to understanding the proposed hydrophobic pathway of entry into the bacterial cytoplasm. We investigated the effects of variations on the structure of the fluoroquinolone ciprofloxacin on the thermotropic phase behavior of DPPC bilayers and characteristics of DPPC monolayers. The structures of ciprofloxacin (CIP) and its pentyl derivative (P-CIP) are shown in Figure 1. Materials and Methods Materials. L-R-1,2-Dipalmitoyl-sn-glycerophosphocholine (DPPC, >99%) was obtained from Sigma, St. Louis, MO. The fluorescent probe 1-palmitoyl-2-[12- (nitro-2-1,3-benzoxadiaol4-yl)amino}dodecanoyl]phosphatidylcholine (NBD-PC), was obtained from Avanti Polar Lipids. DPPC and NBD-PC were pure as judged by thin-layer chromatography. Ciprofloxacin and its derivative were gifts from Cenavisa Labs (Reus, Tarragona, Spain). Chloroform and methanol of HPLC grade were from Fisher Scientific. The water was permanganate double-distilled before use. Methods. Monolayer Studies. Stock solutions of the lipid and fluoroquinolones were made up in chloroform/methanol (3: 1) to known concentrations of about 1 mM. DPPC and probe were mixed in chloroform/methanol (3:1) mixtures, typically in a molar ratio of (25:1), and spread on the surface of the subphase. The subphase was 5 mM Tris-HCl buffer, pH 7.40, containing 0.15 M NaCl. To examine how the presence of the hydrocarbon chain of the fluoroquinolone derivatives affected the mean molecular areas in mixed fluoroquinolone/DPPC monolayers, force-area isotherms of the monolayers at three different compositions (0.7:0.3, 0.5:0.5, and, 0.3:0.7, DPPC: fluoroquinolone) were obtained. The surface balance used in these studies (8) Montero, M. T.; Freixas, J.; Herna`ndez-Borrell, J. Int. J. Pharm. 1997, 149, 161-170.
S0743-7463(97)00688-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/04/1998
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Figure 1. Structure of ciprofloxacin and its pentyl derivative. is described elsewhere.9 About 20 nmol of the probe and DPPC mixtures were spread from a Hamilton syringe and at least 10 min was allowed for solvent evaporation before compression and visual observation. The probe, NBD-PC, was included at a concentration of 1 mol % in the lipid. The monolayers were formed over a buffer solution subphase and observed at ambient temperature (22 ( 2 °C). The monolayer was compressed at a barrier speed of 20 mm2/s in 20 increments. Compression speed and the time allowed before compression were mantained constant in all experiments. DSC Studies. Preparation of Multilamellar Liposomes. Chloroform/methanol (3:1) stock solutions of DPPC and fluoroquinolone, 1 mM, were mixed to obtain different ratios of DPPC/ fluoroquinolone (0.7:0.3 and 0.3:0.7). The solvents were removed under a nitrogen flow, and the resulting film was kept overnight in a vacuum to remove the residual solvents. Liposomes were obtained by adding 5 mM Tris buffer (pH 7.40) at 0.15 M NaCl, heating at a temperature of 50 °C (above that of the gel-liquid crystalline phase transition) for 1 h, and vortexing frequently. The samples and the buffer were degassed under vacuum for 15 min. A Micro-Cal, Inc., MC-2 scanning calorimeter was used for all measurements. Three cycles were used which consisted of initial heating, cooling, and reheating scans between 20 and 70 °C at a rate of 30 °C h-1. The Tris buffer solution was used as a reference.
Results and Discussion Monolayer Compression Isotherms. The surface pressure versus area per molecule isotherms in Figure 2 show that the average area per molecule increases with pentylciprofloxacin but decreases with ciprofloxacin with respect to the DPPC isotherm. The isotherm for DPPC was consistent with others in the literature.9,10 In the presence of the NBD-PC probe the isotherm was perturbed minimally, as has been seen by other investigators using the epifluorescence technique.9,11,12 The shift of the isotherms toward lower area per molecule with the ciprofloxacin is likely due to the drug dissolving in the subphase to some degree instead of it all of remaining in the monolayer at the air-water interface. The expansion of the π-A isotherms as a result of the lipid-drug interaction could be interpreted as a penetration of pentylciprofloxacin molecules into the DPPC monolayer. Incorporation of this derivative takes place at all the molar ratios of DPPC/fluoroquinolone studied (0.7:0.3, 0.5:0.5 (not shown in the figure), and 0.3:0.7). Pure ciprofloxacin does not form a film at the air-water interface but pentylciprofloxacin forms a stable film as shown in Figure 2. (9) Nag, K.; Boland, C.; Rich, N. H.; Keough, K. M. W. Rev. Sci. Instrum. 1990, 61, 3425-3430. (10) Beuer, G.; Galla, H.-J. Eur. Biophys J. 1987, 14, 403-408. (11) Mohwald, M. In Phospholipids Handbook; Cevc, G., Ed.; Marcel Dekker: New York, 1993. (12) Perez-Gil, J.; Nag, K.; Taneva, S.; Keough, K. M. W. Biophys. J. 1992, 63 (1), 197-204.
Figure 2. Surface pressure versus area per molecule of DPPC isotherms of mixed films of DPPC and fluoroquinolone at different molar ratios: (a) DPPC/ciprofloxacin (squares, DPPC; circles, 0.7:0.3; triangles, 0.3:0.7); (b) DPPC/pentylciprofloxacin (squares, DPPC; circles, 0.7:0.3; triangles, 0.3:0.7; ×, pentylciprofloxacin alone).
The compressibilities (Table 1) and the visual appearance of films at low surface pressures and high areas per molecule indicated that only the liquid expanded phase existed. The pentylciprofloxacin modifies the liquid expanded-liquid condensed coexistence regions more than the ciprofloxacin. In liquid condensed regions ciprofloxacin has minimal effect in comparison to pentylciprofloxacin which perturbs the packing somewhat in this region. The plot of the average area per molecule of the mixed films of fluoroquinolone and DPPC versus percentage of fluoroquinolone at three different surface pressures (5, 10, and 20 mN/m) is shown in Figure 3. These pressures are in the liquid expanded zone (5 mN/m), liquid expanded-liquid condensed coexistence regions (10 mN/ m), and liquid condensed phases of the pure DPPC monolayer. A straight line between the two pure components in these plots corresponds to ideal mixing where there is no excess interaction between the components in mixed films. A positive deviation of the observed curve from ideality implies interaction between the components of the mixture likely in such a way the drug perturbs the regular packing of DPPC. These plot the greater perturbing effect of pentylciprofloxacin in comparison to that of ciprofloxacin. Figure 4 shows typical images obtained by epifluorescence microscopy of monolayers of DPPC in the presence of an equal amount of either drug. DPPC films at 10 mN/m showed the well-known kidney-bean-shaped liquid condensed domains in a liquid expanded matrix in the mixed phase region. Notably, not only does pentylciprofloxacin form a monolayer (see Figure 2b), but at a surface pressure of 10 mN/m it appears to be in a mixed phase with probe-excluding solid domains in a probe-including fluid phase. The presence of 50 mol % of ciprofloxacin caused a small perturbation in DPPC packing, producing a reduction in size of the liquid-condensed domains and a loss of the kidney-bean shape. Pentylciprofloxacin, on the other hand, caused the appearance of many more but much smaller domains of liquid-condensed phase. This
Fluoroquinolone-Biomembrane Interactions
Langmuir, Vol. 14, No. 9, 1998 2453
Table 1. Values of Cs* (10-3 m/mN) in Presence of NBD-PC Π (mN/m)
DPPC
DPPC:CIP (0.7:0.3)
DPPC:CIP (0.3:0.7)
DPPC:P-CIP (0.7:0.3)
DPPC:P-CIP (0.3:0.7)
P-CIP
2 5 10 20
36.82 490.00 11.42 5.82
37.55 42.32 43.90 13.90
29.44 32.30 652.00 14.43
34.00 58.37 37.00 10.58
26.00 18.87 20.83 9.63
64.80 56.17 47.00 34.00
a
area/molecule (Å2/molecule)
DPPC
DPPC:CIP (0.7:0.3)
DPPC:CIP (0.3:0.7)
95 70 50
37.46 122.00 6.13
38.84 40.70 8.38
30.33 65.23 15.00
area/molecule (Å2/molecule)
DPPC
DPPC:P-CIP (0.7:0.3)
DPPC:P-CIP (0.3:0.7)
95 80 50
37.46 595.00 6.13
35.32 63.77 9.97
27.00 21.60 24.12
All the Cs values are negative.
Figure 4. Typical images obtained at 9-10 mN/m from monolayers of (a) DPPC, (b) DPPC containing 50 mol % ciprofloxacin, (c) DPPC containing 50 mol % pentylciprofloxacin, and (d) pentylciprofloxacin alone. Each monolayer contained 1 mol % NBD-PC. The dark probe excluding regions correspond to liquid-condensed domain in the pure DPPC. Table 2. Differential Scanning Calorimetric Data for Mixtures of DPPC and Fluoroquinolonesa
Figure 3. Plot of the average area per molecule of the mixed films of DPPC and fluoroquinolone versus the percentage of fluoroquinolone at three different surface pressures: (a) DPPC/ ciprofloxacin (squares, 5 mN/m; circles, 10 mN/m; triangles, 20 mN/m); (b) DPPC/pentyl ciprofloxacin (squares, 5 mN/m; circles, 10 mN/m; triangles, 20 mN/m).
is consistent with the greater solubility of pentylciprofloxacin in the monolayer. This type of change is typically seen when materials are mixed in the monolayer and interfering with the regular packing of the phospholipid.12 Calorimetric Analysis of Liposomes. As can be seen in Table 2 fluoroquinolone containing an alkyl side chain of five carbon atoms in a DPPC bilayer produce decreases in the transition temperature (Tm) of the gel liquid-crystal phase transition of the lipid indicating the incorporation of fluoroquinolone molecules into the bilayer structure. The effect of fluoroquinolone incorporation on the cooperativity of the transition of DPPC bilayers is shown in Figure 5. Increases in the total amount of fluoroquinolone incorporated in the bilayer increase the ∆T1/2 (width of the
sample DPPC DPPC:CIP 0.7:0.3 0.3:0.7 DPPC:P-CIP 0.7:0.3 0.3:0.7
∆Hcal (kcal/mol)
Tm (°C)
∆HVH (kcal/mol)
cooperative units
7.78
42.02
41.40
5.32
8.87 8.06
41.80 41.75
39.26 28.60
4.42 3.54
9.54 7.81
40.90 40.58
12.47 9.67
1.30 1.23
a T , transition temperature; ∆H m CAL, calorimetric enthalpy change of the transition; ∆HVH, van’t Hoff enthalpy; cooperative unit defined as ∆HVH/∆HCAL.
gel to liquid crystalline phase transition measured at DSC endotherm half-height). The values of ∆T1/2 are inversely related to the cooperativity of the phase transition. The increase in ∆T1/2 is dependent on the number of -CH2 groups in the side chains of the ciprofloxacin structure and on the proportion of drug to DPPC. Ciprofloxacin, without any hydrophobic side chain, has only a very small influence on the transition width, reflecting, presumably, its relatively high solubility in buffer as opposed to the
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The small values of ∆HVH found for pentylciprofloxacin/ DPPC and the broader thermal transitions are likely due to changes in intermolecular lipid chain interactions caused by the presence of fluoroquinolone molecule, resulting in a decrease in the cooperative unit size. These observations show that pentylciprofloxacin is much more soluble in membranes than its parent form ciprofloxacin, and that it resides in and perturbs the packing of the lipid. Its hydrophobicity and membrane solubility as opposed to those of ciprofloxacin would enhance the cellular uptake of pentylciprofloxacin by the hydrophobic pathway in comparison to the porin route. Its membrane solubility could lead to a reservoir of pentylciprofloxacin which might have some effect on pharmacokinetics of the drugs in the cells. The overall efficiency of the drug will result from the balance of membrane uptake and storage and transfer of the drug to its target, DNA gyrase. Figure 5. ∆T1/2 as a function of molar ratio DPPC/fluoroquinolone.
bilayer phase. The aliphatic chain of pentylciprofloxacin confers much greater solubility in the membrane to it. The pentyl derivative shows ∆T1/2 values as much as 2.8 times the values observed in ciprofloxacin (for a ratio 0.3:0.7 of DPPC/fluoroquinolone).
Acknowledgment. This work was supported by grants from DGICYT (PB93-0809) of Spain and the Medical Resarch Council of Canada. We thank Dr. K. Nag and Ms L. A. Worthman for their instruction and help with epifluorescence microscopic surface balance and Mrs. J. Stewart for assistance with calorimetric analysis. LA9706882
