Interaction of Antibiotics with Lipid Vesicles on Thin Film Porous

Jul 11, 2013 - E-mail: [email protected]; Telephone: +61 8 8302 5508; Fax: +61 8 8302 ... Elena Tenenbaum , Nadav Ben-Dov , and Ester Segal...
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Interaction of Antibiotics with Lipid Vesicles on Thin Film Porous Silicon Using Reflectance Interferometric Fourier Transform Spectroscopy Taryn Guinan,† Cédric Godefroy,‡ Nicole Lautrédou,§ Stephanie Pace,† Pierre-Emmanuel Milhiet,‡,§ Nicolas Voelcker,*,† and Frédérique Cunin*,∥ †

Mawson Institute, University of South Australia, Adelaide, Australia Institut National de la Santé et de la Recherche Médicale, Unité 1054, 34090 Montpellier, France § Centre National de la Recherche Scientifique, UMS 3426/MRI, Centre de Biochimie Structurale, UMR5048 CNRS, U1054 INSERM, 34090 Montpellier, France ∥ Institut Charles Gerhardt Montpellier, UMR 5253 CNRS-ENSCM-UM2-UM1, Matériaux Avancés pour la Catalyse et la Santé, Ecole Nationale Supérieure de Chimie de Montpellier, 8 rue de l’Ecole Normale, 34296 Montpellier, France ‡

S Supporting Information *

ABSTRACT: The ability to observe interactions of drugs with cell membranes is an important area in pharmaceutical research. However, these processes are often difficult to understand due to the dynamic nature of cell membranes. Therefore, artificial systems composed of lipids have been used to study membrane properties and their interaction with drugs. Here, lipid vesicle adsorption, rupture, and formation of planar lipid bilayers induced by various antibiotics (surfactin, azithromycin, gramicidin, melittin and ciprofloxacin) and the detergent dodecyl-b-D-thiomaltoside (DOTM) was studied using reflective interferometric Fourier transform spectroscopy (RIFTS) on an oxidized porous silicon (pSi) surface as a transducer. The pSi transducer surfaces are prepared as thin films of 3 μm thickness with pore dimensions of a few nanometers in diameter by electrochemical etching of crystalline silicon followed by passivation with a thermal oxide layer. Furthermore, the sensitivity of RIFTS was investigated using three different concentrations of surfactin. Complementary techniques including atomic force microscopy, fluorescence recovery after photobleaching, and fluorescence microscopy were used to validate the RIFTS-based method and confirm adsorption and consequent rupture of vesicles to form a phospholipid bilayer upon the addition of antibiotics. The method provides a sensitive and real-time approach to monitor the antibiotic-induced transition of lipid vesicles to phospholipid bilayers.

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energy transfer (FRET), fluorescence correlation spectroscopy (FCS), etc.),2 usually combined with NMR spectroscopy, dynamic light scattering (DLS), X-ray or neutron reflection and diffraction methods. UV−vis spectroscopy, Fourier transform infrared (FTIR), and calorimetry methods are also commonly used to characterize changes in the lipids organization when exposed to membrane-active drugs. Other techniques are more suitable to probe SLB films, such as ellipsometry, X-ray photoelectron spectroscopy (XPS), surface plasmon resonance (SPR), quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM).1,3−6 In particular, AFM allows high resolution imaging of SLB under physiological conditions, and the recent development of highspeed AFM (HS-AFM) promises to offer real time imaging of

INTRODUCTION

The interaction of drugs with cell membranes is an important area in pharmaceutical research. Biological lipid bilayer membranes are highly dynamic molecular assemblies whose functions and activity can be disturbed when exposed to membrane-active molecules and drugs. On the other hand, the biological activity of drugs and their pharmacological properties are strongly influenced by interactions with cell membranes. Several artificial systems essentially composed of lipids have been developed to study membrane properties and their interactions with drugs.1 They are usually designed in the form of vesicles (large or giant unilamellar vesicles, LUV and GUV, respectively), or in the form of supported lipid bilayers (SLB) depending on the analytical techniques that are used to probe their structure, composition, and phase properties. Classical techniques to investigate the way drugs interact with membranes include fluorescence techniques (fluorescence lifetime imaging microscopy (FLIM), fluorescence resonance © 2013 American Chemical Society

Received: May 12, 2013 Revised: July 4, 2013 Published: July 11, 2013 10279

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model membrane features.7−9 In addition, SPR, in combination with AFM or not, was exploited as a tool to monitor in real time protein−membrane interactions, formation of SLB, as well as permeation event across membranes.3,10,11 The main advantage of label-free sensor methods such as SPR or QCM-D is the possibility of investigating the kinetics of interactions and the possibility for multiplexing. However, all of the above methods require very expensive instrumentation and are therefore not necessarily suitable for laboratories, which do not have access to these tools. Optical interferometry is an alternative technique that only requires inexpensive and readily available components including a thin film material such as pSi. pSi is a nanomaterial formed via electrochemical etching of crystalline silicon in hydrofluoric acid (HF), which is suitable as a substrate for supported lipid bilayer membranes since those membranes retain their fluidity and mimic biological membranes due to the access of fluid to both sides of the membrane.5,12,13 pSi also has suitable optical properties for optical interferometry. For example, fringe patterns are observed when analyzing pSi films by optical interferometry, caused by thin film Fabry− Perot interferences. The fringes maxima are described by the Fabry−Perot equation, mλ = 2 nL, (where m is an integer, L is the thickness of the film, n is the average refractive index of the film, and λ is the wavelength of incident light). By extracting the frequential information contained in the reflectance spectrum of the porous silicon film, the fast Fourier transform (FFT) directly yields the product 2 nL of thickness and refractive index of the reflecting layer as the single peak in a frequency spectrum. This method is known as reflective interferometric Fourier transform spectroscopy (RIFTS).14,15 Monitoring the variation of amplitude and position of the FFT peak from the reflecting film provides information on events and changes that occur at the interfaces of the film. In a previous study, we have shown that the RIFTS method could be used to characterize the evolution of lipid vesicle layers on the pSi surface.16 Vesicle deposition on the pSi surface resulted in a significant decrease in the amplitude of the reflected light, and of the amplitude of the Fourier transform of the reflected light, compared to bare pSi. This was due to the decrease of the refractive index contrast at the pSi surface upon adsorption of lipid vesicles. The subsequent formation of a lipid bilayer from spontaneous rupture and/or fusion of the adsorbed lipid vesicle layer led to an increase of the amplitude of the reflected light back to its initial value, and therefore was indistinguishable from the case of bare pSi in phosphate buffered saline (PBS). Here, the RIFTS method was employed to monitor the vesicle rupture and formation of planar lipid bilayers induced by various antibiotics on an oxidized pSi surface. We intended to demonstrate that RIFTS on pSi is an analytical platform suitable for investigation of the mechanism of antibiotic action on cell membranes. We used the antibiotics surfactin, azithromycin, gramicidin, melittin and ciprofloxacin and the detergent dodecyl-b-Dthiomaltoside (DOTM). Surfactin, a lipopeptide produced by Bascillus subtillus, is thought to solubilize lipids via a detergentlike action.7,17−22 The peptide melittin is extracted from honey bee venom and has been reported to cause the fusion of small and large unilamellar zwitterionic vesicles, including those made from dipalmitoylphosphatidlycholine (DPPC).18 Gramicidin is a pore forming linear polypeptide.19 Ciprofloxacin has been shown to strengthen acyl chains in DPPC vesicles below the transition temperature, while having no effect on the diameter

of the vesicles.23 Azithromycin causes a time-dependent erosion of DPPC domains in mixed dioleoylphosphatidylcholine (DOPC)/DPPC bilayers;7,21 however, the effect on vesicles is not well understood. DOTM, a detergent that causes the complete solubilization of vesicles, was used as a control. Zwitterionic (neutral) DPPC vesicles were adsorbed onto negatively charged oxidized pSi, where bilayer formation via vesicle rupture is not favorable, resulting in a stable layer of vesicles. The rupture of these vesicles was induced by addition of antibiotics and this was investigated in situ using RIFTS, by monitoring the changes in refractive indices contrast at the surface of pSi. Furthermore, complementary techniques such as DLS, AFM, fluorescence microscopy, and fluorescence recovery after photobleaching (FRAP), were used for validation purposes and to confirm the adsorption and solubilization/ rupture of vesicles to form a planar phospholipid bilayer on pSi.

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MATERIALS AND METHODS

Chemicals and Reagents. Ethanol (99.9%), HF (48%), surfactin, gramicidin, melittin, ciprofloxacin, chloroform, and methanol (MeOH, 99.9%) were purchased from Sigma-Aldrich (France). Phosphatebuffered saline (PBS, pH 7.4) was purchased from Invitrogen. Azithromycin was purchased from Appollo Scientific (France). DPPC, DOPC, dioleoyltrimethylammonium-propane (DOTAP), and dioleoyl phosphatidylethanolamine-rhodamine (DOPE-Rhod) lipids were purchased from Avanti polar lipids (USA). DOTM was purchased from Anatrace (USA) and mica from Goodfellow (France). pSi Preparation. pSi layers were fabricated via electrochemical etching of boron doped p++ monocrystalline silicon substrate (100), with a resistivity of 0.0008−0.0012 Ω cm (Siltronix, Inc.). pSi films were prepared in a solution containing 3:1 (volume ratio) 48% HF/ ethanol. The silicon wafers were etched at a constant current density of 30 mA/cm2 for 325 s. The samples were subsequently rinsed thoroughly with ethanol and dried in a stream of nitrogen. pSi Surface Oxidation. The predominately hydride-terminated porous layer was thermally oxidized using a muffle furnace in air (Nabertherm, B170, 30−3000 °C) at a temperature of 350−600 °C for 5 h. After oxidation, the substrates were stored in absolute ethanol to prevent contamination. Preparation of Drug Solutions. A stock solution of melittin (1 mM) was prepared in PBS and stored at −20 °C. Stock solutions of azithromycin (5 mM), surfactin (5 mM), ciprofloxacin (20 mM), and gramicidin (5 mM) were prepared in ethanol and stored at −20 °C. Further dilutions of the stock solutions were prepared freshly before each experiment using PBS. Preparation of Detergent Solutions. A 10 mM stock solution of DOTM was prepared by dissolving 5.76 mg DOTM into 1 mL of Milli-Q water. Preparation of LUVs. Multilamelar vesicle (MLVs) solutions were prepared from stock solutions of DPPC (10 mM), DOPE-Rhod (1 mM), and of DOPC (10 mM) in chloroform/MeOH (2:1 ratio). For experiments requiring the adsorption of LUVs onto pSi, the following mol % ratios were added to a glass vial, DPPC, 99% and DOPE-Rhod, 1%. In experiments requiring the fusion of vesicles to form a bilayer DOPC, 89%, DOTAP, 10%, and DOPE-Rhod, 1% were used. The solvent was evaporated under nitrogen at 35 °C. Subsequently, lipids were suspended in PBS at 70 °C. LUVs of 100 nm in size on average were formed at 70 °C by extrusion of MLVs through polycarbonate membranes (Avanti) and analyzed routinely using a Zetasizer Nano S (Malvern, France) DLS system. RIFTS Measurements. A pSi film was placed into a custom-made flow cell and sealed under a glass coverslip (24 mm × 30 mm) using grease. The reflectance spectra of the film was recorded using an Ocean Optics USB2000 CCD spectrometer coupled to a bifurcated optical fiber, in the range 600−1000 nm, in a back reflection configuration. A tungsten light source was focused onto the center of the pSi surface, and the reflected light was detected at a direction normal to the surface sample. A FFT was applied to the reflectance 10280

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spectrum (algorithm from the Wavemetrics Inc. (www.wavemetrics. com) IGOR program library (FFT)). Filtered PBS was injected into the flow cell using a peristaltic pump at 200 μL/min for 5 min until a stable baseline was obtained. LUVs (0.7 mM, 4 mL) were injected into the flow cell at 200 μL/min. The flow was halted, and vesicle interaction was measured over a period of at least 40 min until a stable baseline was obtained. PBS was reintroduced into the flow cell to rinse the LUV coated-pSi surface at 200 μL/min. Once a stable baseline was obtained, the drug was introduced into the flow cell at 200 μL/min. The interaction between vesicles and each drug was measured for at least 40 min, after which the surface was rinsed with PBS at 200 μL/ min. Afterward, these surfaces were analyzed using AFM and fluorescence microscopy to confirm the data observed. AFM Imaging. AFM imaging was performed in tapping mode using a Nanoscope IIIa AFM equipped with a fluid cell (Bruker, Nanosurfaces division), under ambient conditions, using a J scanner and MLCT silicon nitride tips (Bruker, Nanosurfaces division) on integral cantilevers with a nominal spring constant of 0.03 or 0.10 N/ m. DPPC + 1% DOPE-Rhod vesicles (35 μL, 0.7 mM) suspended in PBS were pipetted on the pSi film at room temperature. After 1 h, the pSi surfaces with adsorbed vesicles was rinsed a minimum of 20 times using Tris:NaCl buffer (pH 7.4) and imaged using AFM. Afterward, the drug was added to the surface and left to interact with the vesicles for 16 h at room temperature. The surface was then rinsed a further 20 times with Tris:NaCl buffer and imaged immediately. Confocal Fluorescence Microscopy. One percent of fluorescent lipids of DOPE-Rhod were incorporated in DPPC and DOPC/ DOTAP/DOPE-Rhod (0.89:0.1) lipid solutions. Fluorescence images were obtained using an upright Zeiss LSM 510 confocal microscope, with a X25NA0.85 immersion objective. DOPE-Rhod was excited at a wavelength of 543 nm and emission was collected in a 560−615 nm bandpass filter. For FRAP measurements, 15 scans of the entire field of view were made at prebleach intensity and then a circular region of interest (ROI) was bleached. 250 iterations of the Argon Laser 477, 488, and 514 nm at 75% intensity and 100% acousto-optic tunable filter (AOTF) were used for bleaching. For recovery of the fluorescence, 50 scans were collected after bleaching. Pictures were collected every 15 s. Images were recorded for 60 min with a 512 × 512 pixel picture size. We verified that the energy of the 543 nm laser used for the postbleaching added no bleaching effect by recording control regions outside the bleaching ROI (dashed circle in Figure 1).

addition of antibiotics and then after 16 h. A minimum of three replicate DLS measurements was performed for each sample.

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RESULTS AND DISCUSSION The pSi film etched at 30 mA/cm2 for 325 s was characterized by SEM and AFM. SEM micrographs of the pSi surfaces are depicted in Figure S1A and in Figure S1B (Supporting Information). They show that homogeneous mesopores with approximately 6 nm in diameter and pore depths of 3 μm were obtained using these electrochemical etching conditions. Average pore diameter of 6 nm was confirmed by nitrogen adsorption/desorption analysis (data not shown). Pore diameters of 6 nm were desirable so that adsorption of the vesicles (100 nm in diameter) was restricted to the surface of the film. AFM images show that the pSi has an root-meansquare (rms) roughness of approximately 0.5 nm. pSi was thermally oxidized at 600 °C to form a negatively charged stable hydrophilic surface that has been noted to be favorable for phospholipid vesicle deposition and bilayer formation.16 IR spectra of the freshly etched and thermally oxidized pSi films (Figure S2) confirmed the anticipated surface characteristics for each surface type. The freshly etched pSi (trace I) showed bands at 904 cm−1 and 2084 cm−1, attributed to Si−H2 scissor vibration and Si−H stretching vibrations, respectively. After the thermal treatment at 600 °C (trace II), these bands were no longer observed. Only a band centered at 1035 cm−1 was observed, corresponding to the stretching vibration of Si−O. Suspensions of LUVs with diameters of approximately 100 nm were generated using a mixture of DPPC doped with 1% DOPE-Rhod, and deposited on the oxidized pSi surface. Due to the phase transition temperature (Tm = 41 °C) of DPPC lipids, bilayer formation was not favorable at room temperature via lipid vesicle rupture and fusion. This hypothesis was confirmed using FRAP (Figure 1). Figure 1 displays the confocal fluorescence microscopy image and the FRAP acquisition of DPPC + DOPE-Rhod 1% vesicles supported on a thin film of pSi. The fluorescence recovery curve for the bleached area shows no recovery after 60 min. This was expected for intact vesicles adsorbed on the pSi as they do not possess lateral mobility, and as the mobility of lipids within a vesicle is restricted to the given vesicle. By contrast, rupturing of lipid vesicles into a continuous bilayer will result in efficient fluorescence recovery due to the long distance lateral mobility of the lipids within the bilayer. This is shown in Figure S3 where the fluorescence recovery for DOPC (89%) + DOTAP (10%) + DOPE-Rhod (1%) on mica was investigated as a control for the formation of a bilayer. Six minutes after bleaching, fluorescence recovery was observed. DOPC lipids enriched with positive DOTAP lipids were chosen because FRAP experiments could not be conducted using DPPC lipids on pSi or mica as a continuous phospholipid bilayer could not be formed. Since the laser spot size on the fluorescence microscope is approximately 1 μm in size and only small regions of a few hundred nanometers in size of bilayers were observed with the DPPC lipid system, the complete bilayer would be photobleached with minimal or no surrounding lipids for fluorescence recovery to occur. The FRAP experiment here clearly confirmed that intact vesicles of DPPC + DOPE-Rhod 1% were stably adsorbed to the surface of oxidized pSi. AFM imaging was also used to confirm the presence of vesicles on the oxidized pSi surface (Figure 3A).

Figure 1. Confocal fluorescence microscope image and FRAP acquisition trace of DPPC/DOPE-Rhod (0.99:0.01) lipid vesicles on thermally oxidized pSi. (Left) Image during FRAP experiment at the time of photobleaching. The FRAP area is represented by the plain circle. The dashed circles indicate the positions of the control areas. Spot size is 50 μm. (Right) Corresponding FRAP traces over 60 min (plain dots). Dashed dot curve is the control area. DLS Measurements. Particle size analysis of the LUVs was performed using a Zetasizer Nano S (Malvern, France) DLS system. Data was analyzed using DTS nano software, and LUVs with a diameter of 100 nm were routinely produced. For all measurements, the system had a laser manually set to attenuator7 and a dispersant viscosity set to 0.8872 cP at 25 °C. In order to follow the changes in vesicle structure, we measured the change in count rate 5 min after the 10281

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mM),17 and so it was expected that a similar pattern would be observed as for DOTM. For ciprofloxacin (cmc = 0.03 mM), a concentration of 0.1 mM was chosen, as no effect was observed at lower concentrations. However, higher concentrations could not be investigated without exceeding established ethanol concentrations. For melittin, which does not form micelles, a concentration of 0.0105 mM was chosen since this was the lowest concentration that caused a change in FFT amplitude for RIFTS. Gramicidin is a channel forming antibiotic, and it was unknown how this drug would interact with the LUV suspensions. However, a concentration of 0.005 mM was chosen because concentrations were limited by solubility in ethanol. For azithromycin, a concentration of 1 mM was used as previous studies by Berquand et al. used these concentration to study the effects on DPPC:DOPC bilayers.21 Only melittin caused a slight decrease in intensity immediately after introduction in the vesicle suspension, but following the same profile as the decrease in intensity in the control experiment. Melittin, ciprofloxacin, gramicidin, and azithromycin did not cause immediate decreases in the intensity values, but destabilization of the vesicles was observed after the 16 h incubation. To further investigate the effect of the antibiotics and of DOTM on the lipid vesicles, AFM imaging was conducted on vesicles adsorbed to an oxidized pSi surface and exposed to the antibiotics and to DOTM for 16 h. Representative tapping mode AFM images taken at room temperature with adsorbed vesicles are shown in Figure 3A. The rms value for pSi surfaces

DLS was used in order to investigate the changes in the dimensions of the DPPC LUVs vesicles upon addition of antibiotics. Detailed information about the vesicle diameter, size distribution, solubilization, and vesicle aggregation was derived from the count rate value (Figure 2). A decrease in

Figure 2. DLS comparison of count rates for DPPC vesicles, and for DPPC vesicles exposed to DOTM (2.15 mM), melittin (0.0105 mM), surfactin (0.05 mM), ciprofloxacin (0.1 mM), gramicidin (0.005 mM), and azithromycin (1 mM). Error bars are calculated from standard deviation for three replicates.

count rate would indicate the destabilization of vesicles, whereas an increase in count rate would suggest the formation of aggregates or multilamelar vesicles in solution.24 DLS studies conducted overnight were used to analyze the stability of DPPC LUVs in PBS without the presence of antibiotics. The count rate for DPPC LUVs overnight did not change significantly, suggesting good stability. The stability of vesicles in ethanol−water was also investigated (Figure S4). At 10% ethanol/PBS ratio the count rate decreased immediately within 5 min, showing that this ethanol concentration ruptures the vesicles. For concentrations below 5% ethanol/PBS, no change in count rate was observed after 16 h. Consequently, for all antibiotics dissolved in ethanol, dilutions were made to ensure that no more than 1% ethanol/PBS was present. Each antibiotic was added to a vesicle suspension and analyzed after 5 min and 16 h (Figure 2). DOTM, a detergent widely used in membrane biochemistry, which causes the destabilization of vesicles above a critical micelle concentration (cmc of 0.05 mM) was first tested as a reference membrane-active compound. A concentration of 2.15 mM, which exceeded the cmc, was chosen to ensure complete solubilization of lipids. A decrease in intensity was observed after 5 min, which decreased further after 16 h, suggesting that lipids have been solubilized. The same experiment was performed using the five antibiotics, melittin, surfactin, ciprofloxacin, gramicidin, and azithromycin. For surfactin and ciprofloxacin, concentrations were chosen so that they were equal or higher to their respective cmc described in the literature. Surfactin caused an instant significant increase in intensity after 5 min, which indicates that the mass of the vesicles increased, which could be due to the increase of the vesicles size, or to the formation of multilamelar vesicles in solution, or eventually to the interaction of surfactin with the vesicles surface. Interestingly, surfactin is known to exhibit a detergent-like activity toward membranes above its cmc (0.007

Figure 3. AFM images of (A) DPPC vesicles on pSi surface, and after addition of (B) surfactin (0.05 mM) and (C) DOTM (2.15 mM) after 16 h. The image was performed in tapping mode in Tris:NaCl buffer. 10282

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the FFT of the reflectivity peak. No change in the value 2 nL (Figure S6A) nor in the amplitude (Figure S6B) was observed for at least 7 h after the vesicles were adsorbed to the pSi surface confirming stability of the vesicle/pSi system, as also shown by AFM (Figure 3A). The vesicle-covered pSi films were then exposed to an aqueous solution of DOTM and to aqueous solutions of antibiotics (in PBS or in PBS/ethanol) in a customized flow cell, and reflectivity spectra were collected as a function of time, at room temperature. First, the effect of DOTM on the DPPC + DOPE-Rhod 1% vesicles/pSi system was investigated (Figure 4A−B). A reasonably high concentration of DOTM of 2.15

with immobilized DPPC + 1% DOPE-Rhod vesicles, measured over an area of 1 μm × 1 μm, was 11 nm (Figure 3A). In comparison, unaltered pSi surface had an rms of 0.5 nm over the same image area. Hence, the pSi surfaces with immobilized vesicles prepared in this study are significantly rougher in comparison to the unaltered pSi surface. This is due to the presence of vesicles on the pSi surface, and is likely to be affected by multilayers of vesicles on the surface and valleys between adjoining vesicles. AFM imaging was then conducted for the pSi surfaces with immobilized vesicles when exposed to DOTM (2.15 mM) and surfactin (0.05 mM) (Figure 3B,C). The AFM data confirmed that DOTM caused the complete solubilization of the vesicles (Figure 3C), consistent with the literature.25 The rms value for the DOTM treated vesicles, measured over an area of 1 μm × 1 μm, was 1.1 nm. This value is close to the rms observed for pSi in Tris buffer, and the difference is likely to be due to the presence of some unremoved lipids. In contrast to DOTM, introduction of surfactin did not result in complete solubilization of the lipids, but resulted in the formation of a bilayer with holes at the pSi surface (Figure 3B). A section analysis confirmed that the bilayer thickness was 5.5 nm, consistent with a DPPC bilayer.26 Surfactin here was observed to strongly destabilize the vesicles, which was not obvious from the DLS measurements. AFM imaging was also performed for the oxidized pSi surfaces with adsorbed vesicles after being exposed overnight to melittin, gramicidin, ciprofloxacin, and azithromycin, which, according to DLS, destabilized vesicles after an overnight incubation (Figure S5A−D, respectively). Patches of bilayers and multibilayers were formed onto the pSi surface, upon incubation with melittin, which is likely to be due to the destabilization of vesicles due to the insertion of melittin, in good agreement with the literature.27 The insertion of melittin may cause the remodeling of membranes resulting in stacked membrane patches (Figure S5A). Few vesicles could still be observed after treatment with azithromycin (Figure S5D), which confirmed the destabilization of the vesicles with azithromycin, observed in DLS after 16 h. However, stacked lipids and aggregates were observed for gramicidin, and vesicles or aggregates were still present at the pSi surface when treated for 16 h with ciprofloxacin, indicating that the vesicles were quite stable overnight after the treatment with these antibiotics. In summary, the main information that was obtained from DLS is that DOTM produced a more rapid destabilization of the vesicles than the antibiotics; moreover, AFM showed an important destabilization of the lipid vesicles after 16 h exposure to DOTM, surfactin, melittin, and azithromycin, resulting in the formation of lipid bilayers for vesicles exposed to surfactin and melittin. The RIFTS method was then employed to monitor the destabilization of the DPPC + DOPE-Rhod 1% vesicles at the pSi surface, induced by the antibiotics surfactin, azithromycin, gramicidin, melittin, and ciprofloxacin, and by DOTM.14,15,28 The RIFTS method makes it possible to monitor real time changes in the conformation of lipid vesicles adsorbed to the pSi surface by monitoring index contrast changes at the surface of pSi.15 We have monitored the amplitude and the position (2 nL) of the FFT of the reflectivity of the DPPC + DOPE-Rhod 1% lipid vesicles/pSi system. Prior to the exposure to the various drug molecules, the stability of DPPC + DOPE-Rhod 1% vesicles on pSi surfaces was investigated over a period of 7 h using RIFTS (Figure S6A). Data was presented as the percentage change in the value of 2 nL, and in amplitude of

Figure 4. Experimental RIFTS data for pSi film in PBS with adsorbed DPPC + 1% DOPE-Rhod vesicles and exposed to DOTM (2.15 mM). (A) Value of 2 nL as a function of time. (B) Percent change in the amplitude of the FFT peak as a function of time. (C) Possible situations during vesicle dissolution.

mM was used since that provoked complete dissolution of lipid vesicles in DLS and AFM experiments (Figure 2A and Figure 3D). Immediately after the introduction of DOTM, an abrupt increase in the amplitude of the FFT of the reflectivity peak was observed, which was attributed to the solubilization and disappearance of the vesicles.16 The schematic in Figure 4C illustrates this process. The reflectance spectrum for a vesicle coated pSi structure can be described by the equation: R(λ) = [ρa 2 + ρb 2 + ρc 2 ] + 2ρa ρb cos(2d pSi) + 2ρb ρc cos(2d ves) + 2ρa ρc cos(2(d pSi + d ves))

(1)

Where dpSi and dves are the phase relationships described by d pSi = 2πn pSiLpSi /λ

and

d ves = 2πn vesLves /λ

npSi, nves are the refractive indices of the pSi and of the lipid vesicle layers, and LpSi and Lves are the thickness (in nm) of the pSi and of the lipid vesicle layers, respectively. λ is the wavelength of incident light. 10283

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Figure 5. Experimental RIFTS data for a pSi film in PBS with adsorbed DPPC + 1% DOPE-Rhod lipid vesicles (0.7 mM) and exposed to solutions of surfactin of various concentrations. Value of 2 nL as a function of time for (A) surfactin (0.005 mM), (B) surfactin (0.01 mM), and (C) surfactin (0.05 mM). Percent change in the amplitude of the FFT peak as a function of time for (D) surfactin (0.005 mM), (E) surfactin (0.01 mM), and (F) surfactin (0.05 mM).

The terms ρa, ρb, and ρc represent the refractive index contrasts between layers, defined as:

decrease in the index contrast at the new interface, due to the higher refractive index of a lipid bilayer (1.45) compared to a vesicle monolayer (1.35).15 A decrease in the index contrast at the new interface should generate a decrease in the amplitude of the FFT peak of the reflectivity. Nevertheless, because of its low thickness (about 5 nm), the lipid bilayer could not be detected by the RIFTS method, which then provided the same response than for bare pSi surface. Interestingly, for surfactin (0.01 mM), a concentration just above the cmc, an increase in the amplitude of the FFT of the reflectivity peak was also observed (Figure 5E). However the conformation changes observed for the DPPC vesicles here were slower at this lower concentration of surfactin. The peak shifted to the longer incubation with a plateau of around 200 min (Figure 5E) instead of 50 min for 0.05 mM (Figure 5F), which could indicate that vesicles first underwent destabilization that led to a membrane remodeling resulting in the delayed rupture of the vesicles . The baseline did not appear to recover completely, which could indicate that lipid vesicles or lipid aggregates were still present at the pSi surface, after exposure of surfactin (0.01 mM). For surfactin concentrations (0.005 mM) below the cmc, no change in the amplitude of the FFT peak was observed (Figure 5D), suggesting that vesicles were still present on the pSi surface. Therefore, the effect of surfactin on the adsorbed vesicles as determined by RIFTS showed the expected concentration-dependence. Confocal fluorescence microscopy confirmed the RIFTS results for the interaction of DPPC + 1% DOPE-Rhod lipid vesicles absorbed on pSi and surfactin (0.05 mM) (Figure 6). No fluorescence was observed for pSi in PBS (Figure 6A). However, after the addition of the LUVs (Figure 6B), grainy fluorescence was observed across the pSi surface. However, some brightly fluorescing dots were also observed that may be due to the aggregation of some vesicles on the surface. After the addition of surfactin (0.05 mM) (Figure 6C), fluorescent domains were observed corresponding to the formation of a patchy bilayer, which was consistent with results obtained from both AFM and RIFTS experiments. These results suggest that RIFTS could also be coupled with fluorescence microscopy to distinguish between the formation of a lipid bilayer or complete solubilization and removal of lipid vesicles upon the interaction with antibiotics.

ρa = (nsoln − n ves)/(nsoln + n ves) ρb = (n ves − npSi)/(n ves + npSi) ρc = (n pSi − nSi)/(n pSi + nSi)

where nsoln and nSi are the refractive indices of the buffer solution, and of bulk Si, respectively. When the lipid vesicles are solubilized and disappear from the pSi surface, the pSi/vesicle and the vesicle/PBS interfaces disappear in favor of the pSi/PBS interface. The refractive index contrast at the pSi surface increases consequently causing the increase of the amplitude of the FFT of the reflectivity the signal.16 After rinsing with PBS, the amplitude of the FFT peak was observed to stabilize due to the removal of the free lipids from the solution. In comparison, no significant change in the 2 nL value was observed upon introduction of DOTM, indicating that the solubilization of the vesicles did not affect the refractive index in the pSi layer. The gradually decreasing baseline in the value 2 nL was attributed to the slow dissolution of pSi in PBS.29 A control experiment showed that addition of DOTM solution to pSi without vesicles did not generate changes in the 2 nL value nor in the amplitude of the FFT peak. The RIFTS method was then used with the DPPC + DOPERhod 1% vesicles/pSi system exposed to three different concentrations of surfactin, an antibiotic which showed interesting effects on DPPC vesicles with DLS and AFM (Figure 2 and 3B). Figure 5A−C shows corresponding 2 nL data for interactions occurring between DPPC + 1% DOPERhod vesicles/pSi surface and surfactin at 0.05, 0.01, and 0.005 mM, respectively. No change in the value 2 nL was observed at either of these surfactin concentrations. In contrast, after addition of 0.05 mM surfactin, a concentration well above the molecule’s cmc, the amplitude of the FFT peak of the reflectivity increased over a period of approximately 30 min and then stabilized slightly below the original FFT amplitude observed for pSi in PBS (Figure 5F). This result was consistent with the rupturing of vesicles and formation of a lipid bilayer, as observed with AFM (Figure 3B). In this case, the pSi/vesicle interface was replaced by a pSi/lipid bilayer interface with a 10284

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detected at the time scale of the present RIFTS experiment. Indeed, ciprofloxacin, gramicidin, and azithromycin showed only a long-term effect on the lipid vesicles in DLS (Figure 2) at the same concentrations as used in RIFTS. In addition, adsorbed lipid vesicles were still observed in AFM after exposure to gramicidin and ciprofloxacin. Two populations of active substances were identified with the RIFTS method: DOTM, surfactin, and melittin, which were shown to have a rapid effect on the lipid vesicles; and gramicidin, ciprofloxacin, and azithromycin, which were shown not to have an effect, or to have a slow effect on the lipid vesicles. Moreover, the RIFTS method was also informative on the kinetics of the changes (destabilization, remodeling, or dissolution) occurring on vesicles when exposed to the antibiotics.

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CONCLUSIONS The RIFTS method was used to investigate in real time the effect induced by various antibiotics (surfactin, azithromycin, gramicidin, melittin, and ciprofloxacin) and DOTM on lipid vesicles adsorbed on an oxidized porous silicon (pSi) surface. The RIFTS method applied in this paper appeared to be robust and is cost-effective in comparison with techniques such as AFM and DLS. Moreover, it correlated very well with the results obtained from DLS and from AFM, suggesting that it can constitute an interesting preliminary technique to monitor time and concentration dependent changes in membrane model systems, induced by antibiotics without the need for advanced technology. Furthermore, the RIFTS method can easily be coupled with fluorescence microscopy to confirm the presence of a lipid bilayer after an increase in FFT peak amplitude is observed.

Figure 6. Fluorescence images of (A) pSi films in PBS, (B) pSi film with adsorbed DPPC + 1% DOPE-Rhod vesicles in PBS, and (C) pSi film with adsorbed vesicles and exposed to surfactin (0.05 mM) in PBS.

The interaction of lipid vesicle suspension was investigated using RIFTS with melittin (0.0105 mM), gramicidin (0.005 mM), ciprofloxacin (0.1 mM) and azithromycin (1 mM) (Figure 7A−D, respectively). We have observed previously by DLS and AFM that melittin transformed lipid vesicles and induced lipid bilayer formation (Figure 2 and Figure 3C). Here, a significant increase in the amplitude of the FFT peak, was observed as a result of the addition of melittin (Figure 7A), which was consistent with the AFM and DLS control. For the three other antibiotics: gramicidin, ciprofloxacin, and azithromycin, no significant change in the amplitude of the FFT of the reflectivity peak was observed over the time scale of the experiment (Figure 7B−D). This indicated that ciprofloxacin, gramicidin, and azithromycin appear to have no effect on the lipid vesicles conformation at the concentrations used in the study, or that eventual effects were too slow and then were not

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ASSOCIATED CONTENT

S Supporting Information *

SEM and AFM micrographs of pSiO2 films; IR spectra of hydride terminated and thermally oxidized pSi films; FRAP of DOPC/DOTAP/DOPE-Rhod (0.89:0.10:0.01); DLS of vesicle suspension with ethanol; AFM images of vesicles on pSiO2 exposed to gramicidin, ciprofloxacin, and azithromycin; RIFTS

Figure 7. Percent change in the amplitude of the FFT peak as a function of time for pSi film in PBS with adsorbed DPPC + 1% DOPE-Rhodamine lipid vesicles (0.7 mM) and then exposed to (A) melittin (0.0105 mM), (B) gramicidin (0.005 mM), (C) ciprofloxacin (0.1 mM) and (D) azithromycin (1 mM). 10285

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data for pSiO2 exposed to vesicles; Comparison of the 2 nL value and FFT peak amplitude for pSiO2 exposed to DOTM; AFM image of vesicles on pSiO2 exposed to surfactin (0.005 mM) and the data for the 2 nL value for melittin, gramicidin, azithromycin, and ciprofloxacin. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Telephone: +33 4 67 16 34 44; Fax: +33 4 67 16 34 70 (F.C.). E-mail: nico.voelcker@ unisa.edu.au; Telephone: +61 8 8302 5508; Fax: +61 8 8302 5613 (N.V.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The authors acknowledge funding providing by the French Australian S&T FAST/EGIDE program for international travel and living expenses. Corine Gerardin is acknowledged for advice and expertise provided for DLS experiments.

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