such as "Introduction", "Conclusion"..etc
In the absence of drug, classical images previously published(13) were obtained. They displayed two discrete height levelsreflecting phase separation between gel phase DPPC and liquid-crystallinephase DOPC. The DPPC gel domains were well defined and homogenous,with a size ranging from 0.15 to 1.5 µm. The height differencebetween DPPC domains and the fluid DOPC matrix was 1.10 ±0.05 nm.
When bilayers were incubated with either ciprofloxacin (toppanels) or moxifloxacin (bottom panels), we observed a decreaseof the size of DPPC domain with time (Fig. 2). The height differencesbetween gel phase DPPC and fluid phase DOPC remains constantduring the incubation. We note that for all incubation timesthe bilayer surface was devoid of defects, i.e., holes in theupper monolayer or in the bilayer were never observed. To evaluatethe kinetics of the erosion process, a plot of the average domainareas was represented as a function of time. Fig. 3 shows thatwithin a few hours, the area of DPPC domains decreased from114.1 to 75.6 µm2 and 83.3 to 42.3 µm2 for ciprofloxacinand moxifloxacin, respectively. Thus, ciprofloxacin induceda decrease of the surface occupied by the DPPC domain of 27%.This erosion process was more marked with moxifloxacin sinceit reached a value of 58%. The kinetic trend was also differentfor ciprofloxacin compared to moxifloxacin, as reflected bythe linear and the exponential-like processes, respectively.This suggests that, in contrast with ciprofloxacin, differentregimes of erosion had to be distinguished with moxifloxacin.To assess whether such an alteration of DPPC domains could bedue to mechanical perturbation by the scanning tip, we performedthe same measurements on a control bilayer that was not incubatedwith drugs. We obtained an area decrease of 3% indicating thatthe time-dependent erosion of the DPPC gel domains is due tothe action of the antibiotics rather than to a simple scanningeffect.
Partition of ciprofloxacin and moxifloxacin measured by phase transfer
The differences in behavior of ciprofloxacin and moxifloxacinobserved by AFM experiments might be related to their abilityto partition between aqueous and hydrophobic environments. Tothis end, we followed the transfer of the ciprofloxacin andmoxifloxacin from an aqueous to a lipidic phase, using egg yolkphosphatidylcholine dissolved in chloroform (Fig. 4). Mostly,egg yolk phosphatidylcholine is a mixture of C16 and C18 saturatedalkyl chains at C-1, and C18 unsaturated alkyl chain at C-2.Its thickness and degree of hydration, as well as mean acyl-chainarea, are well known (25,26). Egg yolk phosphatidylcholine iscommonly used to mimic lipid membranes and shows close characteristicsof synthetic lipids used in this study. For example, the thicknessof egg yolk phosphatidylcholine bilayer was estimated to be30 Å, a close value to the thickness of DPPC (29.3 Å)and DOPC (30 Å; deduced from the thickness of DSPC, whichhas the same carbon number in the alkyl chain as DOPC (26)).In the absence of lipid, more than 40% of ciprofloxacin wasdetected in the aqueous phase, while only 5% of moxifloxacinewas found in these conditions. A huge amount of this latterwas found at the interface. The addition of lipids to the organicphase, with a lipid/drug molar ratio up to 50:1 did not changesignificantly the drug phase transfer.
Transfer of fluoroquinolones from lipid monolayer to aqueous phase
To get more insight on the partition of fluoroquinolones betweenlipid and aqueous phases, we investigated the ability of fluoroquinolonesto be released from a lipid monolayer to an aqueous phase, byusing the Langmuir trough technique. The amount of fluoroquinolonefound in the subphase increased with the initial quantity offluoroquinolone in the monolayer (data not shown). For a DOPC/DPPC/fluoroquinoloneratio 1:1:2, a plateau value was reached within 10 min for ciprofloxacinand 15 min for moxifloxacin (Fig. 5). At this equilibrium state,the percentage of fluoroquinolone detected in the subphase wasclearly higher for ciprofloxacin (70%) as compared to moxifloxacin(at 40%). In the experimental conditions used, no lipid wasdetected by phospholipid assay in the buffer that supports thelipid-fluoroquinolone monolayer.
Conformational analysis of the interactions between fluoroquinolones and lipids
In an attempt to correlate our experimental data with molecularmodeling, the interaction of ciprofloxacin and moxifloxacinwith a model membrane was calculated using the IMPALA method.This procedure was used to study the membrane behavior of bothmolecules when crossing the bilayer from the hydrophilic environmentto the hydrophobic.
Fig. 6 A shows the most stable position of each molecule intothe membrane. Both fluoroquinolones are clearly located at thehydrophilic-hydrophobic interface. The molecules were embeddedinto the membrane, with their mass center near the phospholipidheadgroup/acyl-chain interface (13 Å from the bilayercenter), as shown on the plot of the mass center position versusthe restraints (Fig. 6 B). It should be noted that differenceswere seen between ciprofloxacin and moxifloxacin. The interactionof moxifloxacin notably appeared more favorable than that ofciprofloxacin, since the restraint value of the most stableposition was 1.5 kcal/mol lower for moxifloxacin as comparedto ciprofloxacin.
Effect of fluoroquinolones on lipid monolayer—surface-pressure isotherms
To investigate the ability of fluoroquinolones to modify thesurface pressure versus area isotherms of DOPC/DPPC (1:1) monolayers,we investigated the effect of increasing amounts of antibioticson these isotherms curves.
For the sake of accuracy, we took into account the release offluoroquinolones from the lipid to the aqueous phases in thedetermination of the quantity of drug remaining in the monolayerat the air-water interface, on the monolayers isotherms. Wetherefore recalculated the monolayer compression isotherms usingthe proportion of fluoroquinolones remaining in the monolayerafter 30 min. Results are illustrated in Fig. 7.
The curve corresponding to pure DOPC/DPPC (1:1) is in perfectagreement with the one already reported (27). As already evokedby Montero et al. (28), pure ciprofloxacin or moxifloxacin doesnot form a film at the air-water interface. In presence of fluoroquinolones,the isotherms were shifted toward the small molecular areas.This effect is more pronounced with moxifloxacin (Fig. 7 B)than ciprofloxacin (Fig. 7 A).
In addition, fluoroquinolones also affected the collapse pressure:46.0 mN/m for DOPC/DPPC; 45.9 mN/m, 44.8 mN/m, and 37.7 mN/m,for initial proportions of DOPC/DPPC/moxifloxacin of 1:1:0.4,1:1:1, and 1:1:2, respectively; and 46.0 mN/m, 40.5 mN/m, and37.8 mN/m, for initial proportions of DOPC/DPPC/ciprofloxacinof 1:1:0.4, 1:1:1, and 1:1:2, respectively. This disruptionof the lipid monolayer stability at a high compression levelis more pronounced at a high level of fluoroquinolone in themixed monolayer.
Effect of fluoroquinolones on lipid conformation—ATR-FTIR
Because the lower area occupied by lipids in presence of fluoroquinolonesmight be partly due to a change in the orientation of lipidat the interface by straightening up their fatty acid chains,we used ATR-FTIR to investigate the effect of ciprofloxacinand moxifloxacin on conformation and orientation of acyl chainof lipids.
Nonpolarized ATR-FTIR spectra of supported layers of DPPC, drug(ciprofloxacin or moxifloxacin), and DPPC/drug at a molar ratioof 1:1 were recorded (Fig. 8, top panel). As the drug proportionincreased, the drug spectrum appeared in the DPPC/drug mixturespectrum, notably at 1630 cm–1. Interestingly, in DPPC/drugspectra, the DPPC (C=O) band at 1736 cm–1 was modifiedin terms of frequency and shape, suggesting a modification ofthe interfacial lipid carbonyl groups. Analysis of the lipidC-H wagging (w(CH2)) allowed us to get information on lipidchain conformation and proportion of the chains in the all-transconformation (23). Here the wagging band at 1200 cm–1was selected because it has little overlap with other lipidor drug absorption. As shown in Fig. 8, bottom panel, area evolutionof DPPC peak at 1206–1193 cm–1 as function of increasingamounts of fluoroquinolones decreased by up to 60 and 72% forciprofloxacin and moxifloxacin, respectively. These data indicateda loss of all-trans conformation and the appearance of a kinksomewhere between C-2 and C-6 of the chain.
Effect of fluoroquinolones on lipid orientation—ATR-FTIR
To get information on molecular orientation in the absence orin the presence of both fluoroquinolones, we took advantagefrom the fact that, in an ordered membrane deposited on thegermanium crystal (oriented multilayers), all the moleculeshave the same orientation with respect to a normal to the germaniumplate. Measuring the spectral intensity with two orientationsof the incident-light electric field obtained with a polarizerallowed us to obtain information on several chemical groupsof the lipid molecule. The dichroic spectrum of pure DPPC andDPPC/drug (molar ratio 1:1) mixture were obtained by subtractingthe spectrum recorded with perpendicular-polarized light fromthat recorded with the parallel-polarized light using the lipid(C=O) band at 1780–1700 cm–1 as a reference (Fig. 9)(29). Interestingly the dichroic spectra of DPPC/drug mixturedisplayed strong dichroism for bands assigned to the drug, notablyat 1630 and 1465 cm–1, suggesting a well-organized, well-definedorientation of the drug in the DPPC bilayer. The orientationof the lipid acyl chain can be estimated from the wagging band(w(CH2)). The dipole of this transition is oriented parallelto the all-trans chain (23). In turn, positive deviations ofthe dichroism spectrum demonstrate that the chains are mainlyperpendicular to the germanium surface, i.e., perpendicularto the membrane plane since AFM recording demonstrated thatmembranes orient themselves parallel to the germanium surface,even when natural membranes are used (30). In Fig. 9, bottompanel, we plotted the area evolution of the wagging peak integratedbetween 1206 and 1193 cm–1 as a function of the DPPC/drugmolar ratio. Both fluoroquinolones induced a marked and similardecrease of the area when they were added at low concentration(1:0.2 molar ratio). When the amounts of fluoroquinolones wereincreased, the area decreased further in presence of ciprofloxacinbut remained almost stable for moxifloxacin. This observationwas similar for the four wagging peaks (indicated by arrows;Fig. 9, top panel) (1275–1261 cm–1, 1253–1240cm–1, 1229–1216 cm–1, and 1206–1193cm–1).
To quantify the orientation of DPPC all-trans chains, we measuredthe dichroic ratio for wagging band at 1200 cm–1. RATR(A///A) was 6.8 with an isotropic dichroic ratio of 1.33 (calculatedfrom (C=O) band at 1755–1750 cm–1 (29)). On thebasis of this determination, the angle between the acyl chainsof DPPC and the normal at the germanium surface was found tobe 21°. The same calculation was done in the presence offluoroquinolones at a lipid/drug ratio of 1:1. The angle was27° and 20° in the presence of ciprofloxacin and moxifloxacin,respectively. These data suggested that in contrast to ciprofloxacin,moxifloxacin had no effect on the orientation of the acyl chainsand did not induce additional disorder.
Enter the code exactly as it appears. All letters are case insensitive, there is no zero.