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<strong>AFM imaging of DOPC/DPPC bilayers incubated with ciprofloxacin and moxifloxacin</strong><br> To gain insight into ciprofloxacin- and moxifloxacin-membrane<sup></sup>interactions, supported lipid bilayers made of DOPC/DPPC were<sup></sup>prepared by fusion of unilamellar vesicles on mica. Time-lapse<sup></sup>AFM topographic images were recorded in solution, in the absence<sup></sup>and in the presence of fluoroquinolones.<sup></sup> <p> In the absence of drug, classical images previously published<sup></sup>(13<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#BIB13"><img src="http://www.biophysj.org/icons/fig-down.gif" alt="Go"></a>) were obtained. They displayed two discrete height levels<sup></sup>reflecting phase separation between gel phase DPPC and liquid-crystalline<sup></sup>phase DOPC. The DPPC gel domains were well defined and homogenous,<sup></sup>with a size ranging from 0.15 to 1.5 <em>µ</em>m. The height difference<sup></sup>between DPPC domains and the fluid DOPC matrix was 1.10 ±<sup></sup>0.05 nm.<sup></sup> </p> <p> When bilayers were incubated with either ciprofloxacin (<em>top<sup></sup>panels</em>) or moxifloxacin (<em>bottom panels</em>), we observed a decrease<sup></sup>of the size of DPPC domain with time (<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG2">Fig. 2</a>). The height differences<sup></sup>between gel phase DPPC and fluid phase DOPC remains constant<sup></sup>during the incubation. We note that for all incubation times<sup></sup>the bilayer surface was devoid of defects, i.e., holes in the<sup></sup>upper monolayer or in the bilayer were never observed. To evaluate<sup></sup>the kinetics of the erosion process, a plot of the average domain<sup></sup>areas was represented as a function of time. <a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG3">Fig. 3</a> shows that<sup></sup>within a few hours, the area of DPPC domains decreased from<sup></sup>114.1 to 75.6 <em>µ</em>m<sup>2</sup> and 83.3 to 42.3 <em>µ</em>m<sup>2</sup> for ciprofloxacin<sup></sup>and moxifloxacin, respectively. Thus, ciprofloxacin induced<sup></sup>a decrease of the surface occupied by the DPPC domain of 27%.<sup></sup>This erosion process was more marked with moxifloxacin since<sup></sup>it reached a value of 58%. The kinetic trend was also different<sup></sup>for ciprofloxacin compared to moxifloxacin, as reflected by<sup></sup>the linear and the exponential-like processes, respectively.<sup></sup>This suggests that, in contrast with ciprofloxacin, different<sup></sup>regimes of erosion had to be distinguished with moxifloxacin.<sup></sup>To assess whether such an alteration of DPPC domains could be<sup></sup>due to mechanical perturbation by the scanning tip, we performed<sup></sup>the same measurements on a control bilayer that was not incubated<sup></sup>with drugs. We obtained an area decrease of 3% indicating that<sup></sup>the time-dependent erosion of the DPPC gel domains is due to<sup></sup>the action of the antibiotics rather than to a simple scanning<sup></sup>effect. </p> <p> <strong>Partition of ciprofloxacin and moxifloxacin measured by phase transfer</strong><br> The differences in behavior of ciprofloxacin and moxifloxacin<sup></sup>observed by AFM experiments might be related to their ability<sup></sup>to partition between aqueous and hydrophobic environments. To<sup></sup>this end, we followed the transfer of the ciprofloxacin and<sup></sup>moxifloxacin from an aqueous to a lipidic phase, using egg yolk<sup></sup>phosphatidylcholine dissolved in chloroform (<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG4">Fig. 4</a>). Mostly,<sup></sup>egg yolk phosphatidylcholine is a mixture of C16 and C18 saturated<sup></sup>alkyl chains at C-1, and C18 unsaturated alkyl chain at C-2.<sup></sup>Its thickness and degree of hydration, as well as mean acyl-chain<sup></sup>area, are well known (25<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#BIB25"><img src="http://www.biophysj.org/icons/fig-down.gif" alt="Go"></a>,26<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#BIB26"><img src="http://www.biophysj.org/icons/fig-down.gif" alt="Go"></a>). Egg yolk phosphatidylcholine is<sup></sup>commonly used to mimic lipid membranes and shows close characteristics<sup></sup>of synthetic lipids used in this study. For example, the thickness<sup></sup>of egg yolk phosphatidylcholine bilayer was estimated to be<sup></sup>30 Å, a close value to the thickness of DPPC (29.3 Å)<sup></sup>and DOPC (30 Å; deduced from the thickness of DSPC, which<sup></sup>has the same carbon number in the alkyl chain as DOPC (26<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#BIB26"><img src="http://www.biophysj.org/icons/fig-down.gif" alt="Go"></a>)).<sup></sup>In the absence of lipid, more than 40% of ciprofloxacin was<sup></sup>detected in the aqueous phase, while only 5% of moxifloxacine<sup></sup>was found in these conditions. A huge amount of this latter<sup></sup>was found at the interface. The addition of lipids to the organic<sup></sup>phase, with a lipid/drug molar ratio up to 50:1 did not change<sup></sup>significantly the drug phase transfer. </p> <p> <strong>Transfer of fluoroquinolones from lipid monolayer to aqueous phase</strong><br> To get more insight on the partition of fluoroquinolones between<sup></sup>lipid and aqueous phases, we investigated the ability of fluoroquinolones<sup></sup>to be released from a lipid monolayer to an aqueous phase, by<sup></sup>using the Langmuir trough technique. The amount of fluoroquinolone<sup></sup>found in the subphase increased with the initial quantity of<sup></sup>fluoroquinolone in the monolayer (data not shown). For a DOPC/DPPC/fluoroquinolone<sup></sup>ratio 1:1:2, a plateau value was reached within 10 min for ciprofloxacin<sup></sup>and 15 min for moxifloxacin (<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG5">Fig. 5</a>). At this equilibrium state,<sup></sup>the percentage of fluoroquinolone detected in the subphase was<sup></sup>clearly higher for ciprofloxacin (<img src="http://www.biophysj.org/math/sim.gif" alt="~">70%) as compared to moxifloxacin<sup></sup>(at <img src="http://www.biophysj.org/math/sim.gif" alt="~">40%). In the experimental conditions used, no lipid was<sup></sup>detected by phospholipid assay in the buffer that supports the<sup></sup>lipid-fluoroquinolone monolayer. </p> <p> <strong>Conformational analysis of the interactions between fluoroquinolones and lipids</strong><br> In an attempt to correlate our experimental data with molecular<sup></sup>modeling, the interaction of ciprofloxacin and moxifloxacin<sup></sup>with a model membrane was calculated using the IMPALA method.<sup></sup>This procedure was used to study the membrane behavior of both<sup></sup>molecules when crossing the bilayer from the hydrophilic environment<sup></sup>to the hydrophobic.<sup></sup> </p> <p> <a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG6">Fig. 6 <em>A</em></a> shows the most stable position of each molecule into<sup></sup>the membrane. Both fluoroquinolones are clearly located at the<sup></sup>hydrophilic-hydrophobic interface. The molecules were embedded<sup></sup>into the membrane, with their mass center near the phospholipid<sup></sup>headgroup/acyl-chain interface (<img src="http://www.biophysj.org/math/sim.gif" alt="~">13 Å from the bilayer<sup></sup>center), as shown on the plot of the mass center position versus<sup></sup>the restraints (<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG6">Fig. 6 <em>B</em></a>). It should be noted that differences<sup></sup>were seen between ciprofloxacin and moxifloxacin. The interaction<sup></sup>of moxifloxacin notably appeared more favorable than that of<sup></sup>ciprofloxacin, since the restraint value of the most stable<sup></sup>position was 1.5 kcal/mol lower for moxifloxacin as compared<sup></sup>to ciprofloxacin. </p> <p> <strong>Effect of fluoroquinolones on lipid monolayer—surface-pressure isotherms</strong><br> To investigate the ability of fluoroquinolones to modify the<sup></sup>surface pressure versus area isotherms of DOPC/DPPC (1:1) monolayers,<sup></sup>we investigated the effect of increasing amounts of antibiotics<sup></sup>on these isotherms curves.<sup></sup> </p> <p> For the sake of accuracy, we took into account the release of<sup></sup>fluoroquinolones from the lipid to the aqueous phases in the<sup></sup>determination of the quantity of drug remaining in the monolayer<sup></sup>at the air-water interface, on the monolayers isotherms. We<sup></sup>therefore recalculated the monolayer compression isotherms using<sup></sup>the proportion of fluoroquinolones remaining in the monolayer<sup></sup>after 30 min. Results are illustrated in <a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG7">Fig. 7</a>. </p> <p> The curve corresponding to pure DOPC/DPPC (1:1) is in perfect<sup></sup>agreement with the one already reported (27<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#BIB27"><img src="http://www.biophysj.org/icons/fig-down.gif" alt="Go"></a>). As already evoked<sup></sup>by Montero et al. (28<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#BIB28"><img src="http://www.biophysj.org/icons/fig-down.gif" alt="Go"></a>), pure ciprofloxacin or moxifloxacin does<sup></sup>not form a film at the air-water interface. In presence of fluoroquinolones,<sup></sup>the isotherms were shifted toward the small molecular areas.<sup></sup>This effect is more pronounced with moxifloxacin (<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG7">Fig. 7 <em>B</em></a>)<sup></sup>than ciprofloxacin (<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG7">Fig. 7 <em>A</em></a>).<sup></sup> </p> <p> In addition, fluoroquinolones also affected the collapse pressure:<sup></sup>46.0 mN/m for DOPC/DPPC; 45.9 mN/m, 44.8 mN/m, and 37.7 mN/m,<sup></sup>for initial proportions of DOPC/DPPC/moxifloxacin of 1:1:0.4,<sup></sup>1:1:1, and 1:1:2, respectively; and 46.0 mN/m, 40.5 mN/m, and<sup></sup>37.8 mN/m, for initial proportions of DOPC/DPPC/ciprofloxacin<sup></sup>of 1:1:0.4, 1:1:1, and 1:1:2, respectively. This disruption<sup></sup>of the lipid monolayer stability at a high compression level<sup></sup>is more pronounced at a high level of fluoroquinolone in the<sup></sup>mixed monolayer.<sup></sup> </p> <p> <strong>Effect of fluoroquinolones on lipid conformation—ATR-FTIR</strong><br> Because the lower area occupied by lipids in presence of fluoroquinolones<sup></sup>might be partly due to a change in the orientation of lipid<sup></sup>at the interface by straightening up their fatty acid chains,<sup></sup>we used ATR-FTIR to investigate the effect of ciprofloxacin<sup></sup>and moxifloxacin on conformation and orientation of acyl chain<sup></sup>of lipids.<sup></sup> </p> <p> Nonpolarized ATR-FTIR spectra of supported layers of DPPC, drug<sup></sup>(ciprofloxacin or moxifloxacin), and DPPC/drug at a molar ratio<sup></sup>of 1:1 were recorded (<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG8">Fig. 8</a>, <em>top panel</em>). As the drug proportion<sup></sup>increased, the drug spectrum appeared in the DPPC/drug mixture<sup></sup>spectrum, notably at 1630 cm<sup>–1</sup>. Interestingly, in DPPC/drug<sup></sup>spectra, the DPPC <em><img src="http://www.biophysj.org/math/nu.gif" alt="{nu}"></em>(C=O) band at 1736 cm<sup>–1</sup> was modified<sup></sup>in terms of frequency and shape, suggesting a modification of<sup></sup>the interfacial lipid carbonyl groups. Analysis of the lipid<sup></sup>C-H wagging (<em><img src="http://www.biophysj.org/math/nu.gif" alt="{nu}"></em><sub>w</sub>(CH<sub>2</sub>)) allowed us to get information on lipid<sup></sup>chain conformation and proportion of the chains in the all-<em>trans</em><sup></sup>conformation (23<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#BIB23"><img src="http://www.biophysj.org/icons/fig-down.gif" alt="Go"></a>). Here the wagging band at 1200 cm<sup>–1</sup><sup></sup>was selected because it has little overlap with other lipid<sup></sup>or drug absorption. As shown in <a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG8">Fig. 8</a>, <em>bottom panel</em>, area evolution<sup></sup>of DPPC peak at 1206–1193 cm<sup>–1</sup> as function of increasing<sup></sup>amounts of fluoroquinolones decreased by up to 60 and 72% for<sup></sup>ciprofloxacin and moxifloxacin, respectively. These data indicated<sup></sup>a loss of all-<em>trans</em> conformation and the appearance of a kink<sup></sup>somewhere between C-2 and C-6 of the chain.<sup></sup> </p> <p> <a></a> </p> <p> </p> <p> <strong>Effect of fluoroquinolones on lipid orientation—ATR-FTIR</strong><br> To get information on molecular orientation in the absence or<sup></sup>in the presence of both fluoroquinolones, we took advantage<sup></sup>from the fact that, in an ordered membrane deposited on the<sup></sup>germanium crystal (oriented multilayers), all the molecules<sup></sup>have the same orientation with respect to a normal to the germanium<sup></sup>plate. Measuring the spectral intensity with two orientations<sup></sup>of the incident-light electric field obtained with a polarizer<sup></sup>allowed us to obtain information on several chemical groups<sup></sup>of the lipid molecule. The dichroic spectrum of pure DPPC and<sup></sup>DPPC/drug (molar ratio 1:1) mixture were obtained by subtracting<sup></sup>the spectrum recorded with perpendicular-polarized light from<sup></sup>that recorded with the parallel-polarized light using the lipid<sup></sup><em><img src="http://www.biophysj.org/math/nu.gif" alt="{nu}"></em>(C=O) band at 1780–1700 cm<sup>–1</sup> as a reference (<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG9">Fig. 9</a>)<sup></sup>(29<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#BIB29"><img src="http://www.biophysj.org/icons/fig-down.gif" alt="Go"></a>). Interestingly the dichroic spectra of DPPC/drug mixture<sup></sup>displayed strong dichroism for bands assigned to the drug, notably<sup></sup>at 1630 and 1465 cm<sup>–1</sup>, suggesting a well-organized, well-defined<sup></sup>orientation of the drug in the DPPC bilayer. The orientation<sup></sup>of the lipid acyl chain can be estimated from the wagging band<sup></sup>(<em><img src="http://www.biophysj.org/math/nu.gif" alt="{nu}"></em><sub>w</sub>(CH<sub>2</sub>)). The dipole of this transition is oriented parallel<sup></sup>to the all-<em>trans</em> chain (23<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#BIB23"><img src="http://www.biophysj.org/icons/fig-down.gif" alt="Go"></a>). In turn, positive deviations of<sup></sup>the dichroism spectrum demonstrate that the chains are mainly<sup></sup>perpendicular to the germanium surface, i.e., perpendicular<sup></sup>to the membrane plane since AFM recording demonstrated that<sup></sup>membranes orient themselves parallel to the germanium surface,<sup></sup>even when natural membranes are used (30<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#BIB30"><img src="http://www.biophysj.org/icons/fig-down.gif" alt="Go"></a>). In <a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG9">Fig. 9</a>, <em>bottom<sup></sup>panel</em>, we plotted the area evolution of the wagging peak integrated<sup></sup>between 1206 and 1193 cm<sup>–1</sup> as a function of the DPPC/drug<sup></sup>molar ratio. Both fluoroquinolones induced a marked and similar<sup></sup>decrease of the area when they were added at low concentration<sup></sup>(1:0.2 molar ratio). When the amounts of fluoroquinolones were<sup></sup>increased, the area decreased further in presence of ciprofloxacin<sup></sup>but remained almost stable for moxifloxacin. This observation<sup></sup>was similar for the four wagging peaks (indicated by <em>arrows</em>;<sup></sup><a href="http://www.biophysj.org/cgi/content/full/94/8/3035#FIG9">Fig. 9</a>, <em>top panel</em>) (1275–1261 cm<sup>–1</sup>, 1253–1240<sup></sup>cm<sup>–1</sup>, 1229–1216 cm<sup>–1</sup>, and 1206–1193<sup></sup>cm<sup>–1</sup>). </p> <p> To quantify the orientation of DPPC all-<em>trans</em> chains, we measured<sup></sup>the dichroic ratio for wagging band at 1200 cm<sup>–1</sup>. <em>R</em><sup>ATR</sup><sup></sup>(<em>A</em><sub>//</sub>/<em>A</em><sub><img src="http://www.biophysj.org/math/perp.gif" alt="{perp}"></sub>) was 6.8 with an isotropic dichroic ratio of 1.33 (calculated<sup></sup>from <em><img src="http://www.biophysj.org/math/nu.gif" alt="{nu}"></em>(C=O) band at 1755–1750 cm<sup>–1</sup> (29<a href="http://www.biophysj.org/cgi/content/full/94/8/3035#BIB29"><img src="http://www.biophysj.org/icons/fig-down.gif" alt="Go"></a>)). On the<sup></sup>basis of this determination, the angle between the acyl chains<sup></sup>of DPPC and the normal at the germanium surface was found to<sup></sup>be 21°. The same calculation was done in the presence of<sup></sup>fluoroquinolones at a lipid/drug ratio of 1:1. The angle was<sup></sup>27° and 20° in the presence of ciprofloxacin and moxifloxacin,<sup></sup>respectively. These data suggested that in contrast to ciprofloxacin,<sup></sup>moxifloxacin had no effect on the orientation of the acyl chains<sup></sup>and did not induce additional disorder. </p> <p> </p> <p> </p>
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