Mechanism of Action of Thymol on Cell Membranes Investigated

Article pubs.acs.org/Langmuir

Mechanism of Action of Thymol on Cell Membranes Investigated through Lipid Langmuir Monolayers at the Air−Water Interface and Molecular Simulation Joaõ Victor N. Ferreira, Tabata M. Capello, Leonardo J. A. Siqueira, Joaõ Henrique G. Lago, and Luciano Caseli* Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of São Paulo, Diadema, SP, Brazil ABSTRACT: A major challenge in the design of biocidal drugs is to identify compounds with potential action on microorganisms and to understand at the molecular level their mechanism of action. In this study, thymol, a monoterpenoid found in the oil of leaves of Lippia sidoides with possible action in biological surfaces, was incorporated in lipid monolayers at the air−water interface that represented cell membrane models. The interaction of thymol with dipalmitoylphosphatidylcholine (DPPC) at the air−water interface was investigated by means of surface pressure−area isotherms, Brewster angle microscopy (BAM), polarization-modulation reflection−absorption spectroscopy (PM-IRRAS), and molecular dynamics simulation. Thymol expands DPPC monolayers, decreases their surface elasticity, and changes the morphology of the lipid monolayer, which evidence the incorporation of this compound in the lipid Langmuir film. Such incorporation could be corroborated by PM-IRRAS since some specific bands for DPPC were changed upon thymol incorporation. Furthermore, potential of mean force obtained by molecular dynamics simulations indicates that the most stable position of the drug along the lipid film is near the hydrophobic regions of DPPC. These results may be useful to understand the interaction between thymol and cell membranes during biochemical phenomena, which may be associated with its pharmaceutical properties at the molecular level. interface can be investigated by a variety of techniques.15 Interactions of bioactive molecules with lipid Langmuir monolayers have been reported in the literature for synthetic drugs,16−18 natural products,19,20 and metallic nanoparticles.21 The fact that Langmuir monolayers of lipids may represent a model for half a membrane is extensively defended in the literature (e.g., see ref 14). The aqueous subphase serves as a model for the external phase of the cell membrane, which is composed by water and hydrophilic molecules. Given the relatively high surface tension of water, the air phase represents the hydrophobic core of the bilayer from a thermodynamic point of view. Studies on the interaction of thymol with lipid Langmuir monolayers are scarce in the literature, being so far reported its surface activity in soybean phosphatidylcholine monolayers,22,23 pure or mixed with a membrane protein (GABAA receptor). In these papers, thymol is reported to penetrate in phosphatidylcholine monolayers, even at high initial values of surface pressures, which indicated its high ability to incorporate in the lipid film. The insertion of thymol in phosphatidylcholine-based liposomes is also reported in the literature,24 where its antioxidant and antimicrobial activities are investigated. Other

1. INTRODUCTION Thymol (2-isopropyl-5-methylphenol) is a monoterpenoid found in the oil of thyme and extracted from Thymus vulgaris as well as from other kinds of plants.1 It is a substance of aromatic odor and with antiseptic properties that has been used in culinary purposes because of its characteristic flavor.2 Thymol is reported as a potent biocide, with antimicrobial properties when used alone or in combination with other compounds such as carvacrol. It acts reducing bacterial resistance to common drugs such as penicillin.3 Also, thymol has been reported as antifungal, acaricidal, antidiabetic, antiinflammatory, antioxidant, antileishmanial, antiallergic, and antitumoral.4−12 However, its molecular mechanism of action is not precisely known, although some evidence suggest that its biocidal properties are related to membrane disruption.6 In this sense, it is of interest the use of models to investigate the mechanisms related to the interactions between thymol and the surface of cell membranes, which may be useful to understand this process at the molecular level. Among these models, lipid Langmuir monolayer formed at the air−water interface seems to be a convenient approach. This technique is based on spreading amphiphilic molecules at the air−water interface, forming a monomolecular film.13 When this monolayer is constituted of membrane lipids, it can represent a model for half a membrane,14 and interactions between molecules at the © XXXX American Chemical Society

Received: February 16, 2016

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DOI: 10.1021/acs.langmuir.6b00600 Langmuir XXXX, XXX, XXX−XXX

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Langmuir kinds of models for cell membranes are related to the computational systems, which have allowed studies of complex systems, including biomembrane models. Molecular dynamics simulations of biomembrane models have been performed for lipid bilayers25−29 and monolayers,30,31 and some of them investigate the interaction of drugs with biomembranes. For example, the interaction of the local anesthetic benzocaine with membrane models was studied,32 being reported that this drug disorders the structure of the membrane. In this paper, thymol, isolated from essential oil from leaves of Lippia sidoides, was incorporated in Langmuir monolayers of dipalmitoylphosphatidylcholine (DPPC) and investigated with tensiometry, surface specific vibrational spectroscopy, Brewster angle microscopy (BAM), and molecular dynamics simulation. The main objective of this work was to obtain information at the molecular level on how this drug interacts with cell membranes.

Figure 1. Chemical structure of thymol. (δ/ppm, 75 MHz, CDCl3): 151.1 (C-1), 131.6 (C-2), 126.0 (C-3), 121.6 (C-4), 136.3 (C-5), 116.1 (C-6), 26.6 (C-7), 22.7 (C-8 and C9), 20.8 (C-10). 2.5. Langmuir Monolayers. The Langmuir monolayers were obtained by spreading a chloroform solution of DPPC (0.5 mg/mL) on the surface of an aqueous solution. For preliminary tests, thymol solutions, dissolved in chloroform at a concentration of 0.5 mg/mL, were also spread alone on the air−water interface in order to test the surface activity of this compound. For mixed thymol−lipid monolayers a previous solution of thymol were spread at the air−water interface a few millimeters below the preformed DPPC monolayer and allowed at least 30 min for homogenization. Final thymol concentration in the aqueous subphase varied between 20 and 60 ng/mL. Surface pressure−area (π−A) isotherms were obtained with a miniKSV Langmuir trough equipped with a surface pressure sensor (Wilhelmy method) and an interface compression rate of 5 Å2 molecule−1 min−1. After 20 min allowed for evaporation of chloroform, the monolayer was compressed until the collapse. For PM-IRRAS studies, the monolayer was compressed until the desired surface pressure (30 mN/m). The surface pressure was maintained at this surface pressure by moving the barriers, and the stabilization of the monolayer was monitored until no additional movement of the barriers was needed. The PM-IRRAS measurements were taken using a KSV PMI 550 instrument (KSV Instruments, Ltd., Helsinki, Finland) at a fixed incidence angle of 80°. Brewster angle microscopy (BAM) images were obtained with a microBAM from KSV-Instruments. All experiments were carried out at a controlled room temperature (300 K) and conducted at least three times to test the reproducibility. For each isotherm, one representative curve is shown. For each spectrum an average of at least 6000 scans is shown. 2.6. Molecular Dynamics Simulation. The MD simulations were performed with GROMACS package35−37 of a system constituted of 4000 water molecules forming an aqueous slab that was placed between two DPPC monolayers as depicted in Figure 2. Each DPPC

2. MATERIALS AND METHODS 2.1. General. DPPC were purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in chloroform (Synth, Diadema, Brazil) to a concentration of 0.5 mg/mL. The water employed as subphase was previously purified using a Milli-Q-Plus system (resistivity 18.2 MΩ· cm, pH 5.5). Silica gel (Merck, 230−400 mesh) was used for column chromatographic separation. GC chromatograms were obtained on a Shimadzu GC-2010 gas chromatograph equipped with an FID detector and an automatic injector (Shimadzu AOC-20i) using a RtX-5 (5% phenyl, 95% polydimethylsiloxane (Restek, Bellefonte, PA, 30 m × 0.32 mm × 0.25 m film thickness) capillary column. These analyses were performed by injecting 1.0 μL of a 1.0 mg/mL solution of sample material in CH2Cl2 in a split mode (1:10) employing helium as the carrier gas (1 mL/min) under the following conditions: injector and detector temperatures of 220 and 250 °C, respectively; oven programmed temperature from 40 to 240 °C at 3 °C/min, holding 5 min at 240 °C. GC/LREIMS analysis (70 V and an ion source temperature of 230 °C) was conducted on a Shimadzu GC-17A chromatograph interfaced with a MS-QP-5050A mass spectrometer using helium as the carrier gas. The identification of the each compound was performed by comparison of recorded mass spectra with those available in the system library. 1H and 13C NMR spectra were recorded at 300 and 75 MHz, respectively, on a Bruker Avance III spectrometer using CDCl3 as solvent. 2.2. Plant Material. Leaves of Lippia sidoides (Verbenaceae) were collected in Atlantic Forest Biomes in São Paulo State, Brazil, on March 15th, 2012. The specimen was identified by Prof. Euder G. A. Martins by comparison of the voucher with that deposited in the Herbarium of the University de São Paulo-SPF. 2.3. Essential Oil Extraction. Fresh leaves (approximately 500 g) of L. sidoides were subjected to hydrodistillation in a Clevenger type apparatus for 4 h. After extraction using CH2Cl2, the essential oil was dried over anhydrous Na2SO4 and filtered, and the solvent was evaporated under reduced pressure. 2.4. Fractionation of Crude Essential Oil. Crude essential oil (1 g) was subjected to column chromatography separation over SiO2 soaked with AgNO3 (15%). The column was eluted with n-pentane, npentane:CH2Cl2, and CH2Cl2 to afford 32 fractions (5 mL). Each fraction was analyzed by GC/FID, and those which displayed chromatographic similarities were pooled together33 This procedure gave eight groups of fractions (A−H) being group E (350 mg) composed bythymol (99% of purity determined by FID-GC), which structure was confirmed by NMR spectral analysis and comparison with data described in the literature.34 Thymol (2-isopropyl-5-methylphenol); see Figure 1 for its structure: Colorless amorphous solid, LREIMS (70 eV) m/z (int rel): 150 (25), 135 (100), 115 (12), 107 (10), 91 (17), 77 (12), 65 (8), 51 (7). 1H NMR (δ/ppm, 300 MHz, CDCl3): 1.66 (d, J = 7.0 Hz, H-8 and H-9), 2.65 (s, H-10), 3.64 (hept, J = 7.0 Hz, H-7), 6.94 (br s, H-6), 7.15 (br d, J = 7.6 Hz, H-4), 7.51 (d, J = 7.6 Hz, H-3). 13C NMR

Figure 2. Snapshot of DPPC monolayer obtained after the equilibration run at 300 K. monolayer contains 32 lipid molecules. The box dimensions of the equilibrated system without thymol are 3.97 × 3.97 × 23.34 nm. Along the z-axis there is a vacuum slab of 10 nm that keeps the DPPC monolayers distant from each other. Periodic boundary conditions have been applied in all directions. The starting configuration was generated with Packmol program,38 placing the two layers of 32 DPPC molecules side by side along z-axis of the water slab in the middle. The system was equilibrated at 300 K for 20 ns in the NVT ensemble, with B

DOI: 10.1021/acs.langmuir.6b00600 Langmuir XXXX, XXX, XXX−XXX

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Langmuir an area per molecule of 48.8 Å2, which resembles the area per molecule at 30 mN/m observed experimentally. One thymol molecule was placed in the vacuum slab, distant approximately 1 nm from the DPPC−vacuum interfaces of an equilibrated configuration of the system described above. With this configuration, thymol was pulled into the monolayer, that is, along the z-axis of the simulation box over 500 ps, using a spring constant of 1000 kJ mol−1 nm−2 and a pull rate of 0.01 nm ps−1. We have used a dummy atom (q = 0.00e and ε = 0.0 kJ mol−1) constrained at (2.0, 2.0, 3.0 nm) in the simulation box as reference atom to pull thymol. In the end of this pulling procedure, we encountered thymol in the middle of the water slab. In order to perform the so-called umbrella sampling, we selected configurations that placed thymol molecule distant from the previous position by approximately 0.1 nm, generating 40 windows. For each window, short equilibrations of 50 ps were carried out in the NPT ensemble and followed by 10 ns of MD runs, which were used for umbrella sampling. The weighted histogram analysis method (WHAM) was employed to analyze the results. It is worth mentioning that this procedure is similar to that used in Lemkul and Bevan’s work.39 The water molecules were modeled with the TIP3P force field, while DPPC molecules were modeled with a recently refined force field for phosphatidylcholine lipids.40 The thymol molecule was modeled with OPLS.41 Only aromatic and hydroxyl hydrogens were considered, while CH and CH3 groups were considered as single bodies by using a united atom model. The charges of thymol atoms/ groups are CHELPG charges obtained from its gas phase with the Gaussian 09 program.42 A time step of 2 fs was used in all the simulations. In order to compare with the experimental results, the simulations were performed at 300 K, controlled by the Nosé−Hoover thermostat with time constant of 0.5 ps. In the NPT simulations, the Parrinello− Rahman barostat, with the target pressure of 1.013 bar (semi-isotropic pressure coupling) and time constant of 10 ps, were employed. The equation of motion was integrated with the leapfrog algorithm, and the configurations stored every 100 ps. In order to treat the Lennard-Jones and short-range part of the Coulombic interactions, a cutoff distance of 1.0 nm was used. The long-range Coulombic interactions were dealt with the particle-mesh Ewald scheme. All bond lengths were constrained with the LINCS algorithm. The visualization of simulation snapshots were done with the VMD program.43

Figure 3. Surface pressure−area isotherms of monolayers of DPPC, pure or mixed with thymol (proportions in mol indicated in the inset).

that around the percentage of 1% of the drug there is a saturation point for its incorporation. However, increasing to 3% the amount of thymol, we observe a substantial expansion of the monolayer. This expansion must be associated with the formation of lipid−drug aggregates, as further detailed in this paper when we discuss the BAM images. Also, the plateau corresponding to the LE−LC transition is shifted to higher surface pressures and becomes less defined. Transitions with a variable pressure are typical of nonfirst order transitions, common for mixed systems. Also the surface elasticity decreases with the introduction of thymol as observed by the inclination of the curves, especially at high surface pressure values. Figure 4 compares the surface elasticity (E) of the monolayers, defined as −A(∂π/∂A)T (A: molecular area; π:

3. RESULTS AND DISCUSSION Thymol in the conditions employed in this paper presented low surface activity. It was spread on the air−water interface and compressed until the minimum area allowed by the Langmuir trough, and the surface pressure increased until values no higher than 5 mN/m. Figure 3 shows the surface pressure−area isotherms for DPPC monolayers. The pure DPPC monolayer presents a typical curve,44 with minimum area of 45 Å2/molecule and a typical plateau around 6 mN/m, representing the transition between the liquid-expanded (LE) and liquid-condensed (LC) phases. Introduction of thymol in the monolayer shifts the isotherm to higher molecular areas of DPPC, indicating the penetration of the drug in the lipid monolayer. For the surface pressure of 30 mN/m, which corresponds to the lateral pressure in natural membranes,45 the shift of area was from 45 to 47 Å2/molecule for 1% (and also for 2%) of thymol and from 45 to 52 Å2/molecule for 3% of thymol. For higher concentrations, the isotherm can no longer be shifted to higher areas. This indicates saturation in terms of incorporation of this compound in the lipid monolayer. Increasing the amount of thymol from 1 to 2%, we observe in low surface pressures a slight shift of the isotherm to higher areas. This shift is not observed in high surface pressures, suggesting a low capacity of penetration of the drug in this stage. This fact points to the fact

Figure 4. Surface elasticity−surface pressure isotherms of monolayers of DPPC, pure or mixed with thymol (proportions in mol indicated in the inset).

surface pressure; T: temperature). For the surface pressure of 30 mN/m the E values decreased from 230 mN/m, for pure DPPC, to values around 170 mN/m, for the mixed thymol− DPPC monolayers. This decrease is a consequence of a compressional gain of the mixed monolayer owing to the fact that the rigid structure of the well-packed phospholipid monolayer was smoothed by the presence of thymol. Decrease of values of E for lipid monolayers is reported in the literature for other drug−lipid films at the air−water interface.19,20 Figure 5 shows the PM-IRRAS spectra for DPPC monolayers with or without thymol at the surface pressure of 30 mN/m. Panel A shows the region corresponding to the main bands of C

DOI: 10.1021/acs.langmuir.6b00600 Langmuir XXXX, XXX, XXX−XXX

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mixed monolayer are related to the difference of reflectivity between the uncovered and covered monolayer, and they are attributed to surface water bends.47 The band in 1740−1770 cm−1 is attributed to CO stretches of DPPC, and the band in 1243 cm−1 can be attributed to phosphate stretches. With thymol, the phosphate band becomes less prominent, and the presence of the band centered in 1477 cm−1 may be attributed to aromatic ring vibrations of thymol. The band centered in 1749 cm−1 for the mixed monolayer in the region of CO stretches is more defined, which may be a consequence of the O−H bends of the thymol molecule. These results evidence the presence of thymol at the interface and show that the lipid polar groups were also affected. This may be related either to the presence of the drug close to the polar heads of the phospholipid or to an effect of the disorder of the monolayer provoked by the insertion of the drug close the alkyl chains of the lipid. Figure 6 shows the BAM images for DPPC monolayers without and with thymol. At the surface pressure of 30 mN/m, the image for pure DPPC is relatively homogeneous, which is in agreement with the literature. 48 This surface pressure corresponds to the region of to the liquid-condensed state, which must present no phase contrast. With the incorporation of thymol, brightly domains appear as a consequence of some DPPC molecules forming rich-in-thymol lipid domains. For lower concentrations of thymol (1 and 2%), there is homogeneous a pattern like that observed for pure DPPC, in contrast to images observed for 3% thymol. This fact can be related to the fact that from 2 to 3% a significant expansion of the monolayer is observed (Figure 3), suggesting that the ability of penetration of thymol into the DPPC films be related with a synergistic effect of association of the drug with the lipid that forms such domains. Thymol is a hydrophobic molecule with a high partition coefficient, log P = 3.3, which suggests that it has a high affinity by the apolar part of lipids. Given the fact that DPPC has a hydrophilic and a hydrophobic domain, it is worth investigating the preferential location of thymol in the DPPC monolayer. By means of molecular dynamics simulation, the preferential location of gases in ionic liquids was accomplished by the calculation of the potential of mean force (PMF) along the zaxis of the simulation box.49 In the same way, the PMF of thymol across the DPPC−water monolayer/film was calculated, that is, thymol was pulled from the vacuum phase up to water phase, along the z-axis of the simulation box. The upper panel of Figure 7 shows the PMF of thymol along the z-axis, considering as reference state the drug in the vacuum phase, PMF = 0.0 kJ mol−1. The bottom panel of Figure 7 depicts the

Figure 5. PM-IRRAS spectra for monolayers of DPPC, pure or mixed with thymol (3% in mol).

the alkyl tails of the phospholipid. For DPPC monolayers these bands are centered in 2918 and 2847 cm−1 and are attributed respectively to the antisymmetric and symmetric stretches of the CH2 groups from the alkyl chains of the phospholipid. With the addition of thymol, the antisymmetric band is shifted to 2914 cm−1. Also, the ratio between the intensities of the antisymmetric and symmetric bands varied from 1.82 to 1.34. As this ratio is usually described as an order parameter,46 this decrease suggests that the insertion of thymol in the lipid layer disorders the close-packed structure of the monolayer, which has been previously compressed to the surface pressure of 30 mN/m. This fact is in agreement with the decrease of surface elasticity observed in Figure 4. Panel B shows the region where some of the main bands for the hydrophilic regions of DPPC appear. The negative band to the baseline around 1680 cm−1 for pure DPPC monolayers and the positive band to the baseline in the same region for the

Figure 6. BAM for monolayers of DPPC, pure (left) or mixed with thymol 3% in mol (right). Images were obtained at 30 mN/m. Field of view: 3600 × 4000 μm. D

DOI: 10.1021/acs.langmuir.6b00600 Langmuir XXXX, XXX, XXX−XXX

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thymol reaches the water phase, the molecular area turns back to values around 48.8 Å2. The literature reports that thymol self-aggregates in water with a critical micellar concentration of 4 μM,22 above the concentration used in the aqueous subphase in our study (50− 100 nM), which indicates that thymol is not saturated at the surface in our case. It is also reported the ability of this compound to penetrate in monomolecular layers of soybean phosphatidylcholine (sPC) at the air−water interface, even at surface pressures above the equilibrium.23 These authors also reported that changes in the dipolar arrangement and in the molecular packing of a monolayer containing an intrinsic membrane protein. In this study, thymol was reported to bind to the receptor protein, changing the molecular organization microenvironment. Turina et al.23 tested the penetration ability of several monoterpenes, including thymol, showing that it is able to penetrate in DPPC monolayers at surfaces pressures as high as 61.5 mN/m for concentrations of 0.5 μg/mL of thymol. In our work, we tested concentrations considerably lower in order to achieve a smaller drug: DPPC molecular proportion. Despite that, a noticeable penetration could be inferred. The surface pressure−area isotherms showed that thymol, at relatively small concentrations, was able to incorporate in DPPC monolayers, expanding them, and decreasing their molecular order. These facts can be related to a disruption of the close-packed lipid monolayer, which can be associated with the change in the morphology of the Langmuir film since the insertion of the drug induced the formation of some domains in contrast to the homogeneous pattern observed for DPPC at 30 mN/m. Additionally, the potential of mean force obtained by molecular dynamics simulation pointed that the favorable position of thymol along the lipid monolayer is near the DPPC hydrophobic tails. Such results then suggest that thymol penetrates the DPPC monolayer, with area shift compatible with molecular simulation and tensiometry. Going back to the PM-IRRAS spectra in Figure 4, we observe that only with this technique we cannot be sure if the changes in the bands for vibrational transitions of phosphate upon incorporation if thymol are related to a direct interaction of this drug with phosphate or due to a change in the organization of the lipid molecules. However, as molecular simulation data suggest a position of thymol closer to the hydrophobic tails of the lipid, it is probable that the lack of nitidity of the band for phosphate must be associated with the increasing disorder of the monolayer. The literature shows some examples that report the properties of terpenes at the air−water interface, with or without lipids. Usually, terpenes with a higher molecular weight can be directly spread on the air−water interface, rather than interacting with lipids. In the literature, it is reported that lupane-type pentacyclic triterpenes (lupeol, betulin, and betulinic acid) form crystalline phases in their pure monolayers.50 These results definitively show that these compounds are able to reorganize at the interface when induced by a mechanical stress such as interfacial compression. When lipids are copresent at the interface, this stress is caused not only by the compression of the air−water interface but also by the condensation of the supramolecular structure formed by the lipids. The literature reports the interaction of other monoamphiphilic pentacyclic triterpenes with cardiolipins and phosphatidylglycerols extracted from mitochondrial and bacterial membranes.51 They were comparatively characterized in binary Langmuir monolayers, revealing that some of them

Figure 7. Potential of mean force calculated for thymol across the vacuum−DPPC and DPPC−water interfaces at 300 K, upper panel. Mass density of DPPC and water was calculated at 300 K.

mass density of DPPC and water, showing the vacuum−DPPC (z ∼ 5 nm) and DPPC−water (z ∼ 7.5 nm) interfaces. Around 5 nm, the PMF presents its minimum values, around −12.0 kJ mol−1, which corresponds to thymol at the vacuum−DPPC interface. Between 5.5 and 6.5 nm (apolar phase of DPPC) there is a plateau in the PMF around 12.0 kJ mol−1. Further pulling thymol into the DPPC monolayer, from 6.5 to 7.8 nm, the PMF increases linearly up to ∼78 kJ mol−1. In the DPPC− water interface and in the water phase, 8 < z < 9 nm, the PMF shows an oscillation around 60 kJ mol−1. Therefore, following the PMF profile, the preferential location of thymol in the monolayer model is at the vacuum−DPPC interface. Moreover, the free energy of partitioning thymol from vacuum to water is around 70 kJ mol−1. In accordance, the partition energy obtained when thymol is pulled from water to vacuum is around 70 kJ mol−1 as well. As discussed previously, the introduction of thymol into the DPPC monolayer shifted the isotherms to higher areas; for instance, with 1 and 2% of thymol, the shift was 2 Å2/molecule in comparison with monolayer without the terpene. The molecular area is obtained straightforwardly in the simulations. Figure 8 shows the molecular area obtained in the presence of thymol in different depths of DPPC monolayer. With thymol at the vacuum−DPPC interface the molecular area shifts ∼1 Å2. Higher shifts of ∼2 Å2 are observed with thymol inside the apolar phase of DPPC and at DPPC−water interface. When

Figure 8. Molecular area of DPPC along with thymol pulling into the monolayer film (upper panel) and mass density of DPPC and water calculated at 300 K (lower panel). E

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arrangement of the system, which is not straightforward by using classical pharmaceutical approaches.

are able to disintegrate the cardiolipin containing domains in mitochondrial and bacterial membranes, which was explained based on the phenomenon of chiral discrimination. These results were discussed in reference to the biological activity of the studied compounds, pointing out that the molecular mechanism of biological activity of these terpenes may be membrane-related and that the charge of the anionic phospholipids may be a key factor for such interaction. Also, grazing incidence diffraction studies for the interactions between ursane-type antimicrobial triterpenes and bacterial anionic phospholipids52 emphasized the significance of the interactions between the cyclopropane ring present in the hydrophobic part of the bacterial phospholipids and the terminal ring of the terpene structure. It was proposed that the significant differences between the systems with the triterpenes are connected with the formation of hydrogen bonds between the hydrophobic moieties. A sequential study of the same group53 showed that at a higher proportion of one of these compounds can lead to the disintegration of cardiolipinrich domains present in bacterial membrane. Another interesting work showed that monolayers composed of bacterial phospholipids were used as model membranes to study interactions of the naturally occurring phenolic compounds from plant essential oil compounds active against pathogenic microorganisms.54 Mixing these compounds with selected lipids indicated that they are able to modify the lipid monolayer structures by integrating into the monolayer, forming aggregates of antimicrobial−lipid complexes. Another interesting work with the compound hexadecaprenol, a potential bactericide, showed that it decreases the activation energy and increases membrane conductance and membrane permeability coefficient in Langmuir monolayers by modulating the molecular organization of the membrane.55 The authors suggested that hexadecaprenol modifies lipid membranes by the formation of fluid microdomains, which was speculated in terms of the fact that electrical transmembrane potential can accelerate the formation of pores in lipid bilayers modified by long chain polyprenols. General speaking, all these papers on related terpenes and similar compounds show that such drugs act in lipid Langmuir monolayer representing cell membranes by altering some physical chemical properties, such as reducing the packing effectiveness of the lipids, increasing the membrane fluidity, and altering the total dipole moment in the monolayer. For our case, the outcomes presented in this present study also indicate an intrinsic interaction between thymol and DPPC at the air−water interface, which could be related to the prospective use of this compound in pharmaceutical processes that involve interaction of thymol with cell membranes and other biointerfaces. Although the obvious limitation of the approach of using lipid Langmuir monolayers to understanding such complex systems, it is important to mention that interactions of such compounds may differ with interactions regarding bilayers, but provide a first clue on the molecular mechanism of the drug with the outmost layer of the cell. Therefore, we emphasize that our results may be valuable to analyze drug−membrane systems, taking into account the molecular structure of the drug and the physical−chemical characteristics of the models employed, such as membrane organization, partitioning, film expansion, surface rheological properties, and preferential localization of the drug. The effects observed in this paper emerge from the supramolecular

4. CONCLUSIONS In this present paper, the interaction of thymol with model membranes at the air−water interface was studied with tensiometry, infrared spectroscopy, microscopy, and dynamics simulation, focusing on surface pressure values that approximate natural membrane lateral pressures. Thymol was proved to incorporate into a simplified model of the cell membrane outer layer in a proof-of-concept experiment. This compound expands DPPC monolayers, decreases their surface elasticity, and therefore changes the physical−chemical properties of the films. A thermodynamic preferential location of the drug along the biomembrane model could be inferred to be near the hydrophobic tails of DPPC. We hope these results may have a significant impact on the understanding of the interaction between thymol and cell membrane surfaces, which may be useful to access molecular information on pharmaceutical properties of this drug.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (L.C.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Brazilian agencies FAPESP (grants 2013/10213-1 and 2015/23446-0) and CNPq. J.V.N. Ferreira was a CNPq fellow.

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REFERENCES

(1) Nabavi, S. M.; Marchese, A.; Izadi, M.; Curti, V.; Nabavi, S. F. Plants belonging to the genus Thymus as antibacterial agents: from farm to pharmacy. Food Chem. 2015, 173, 339−347. (2) Burt, S. Essential oils: their antibacterial properties and potential applications in foods − a review. Int. J. Food Microbiol. 2004, 94, 223− 253. (3) Palaniappan, K.; Holley, R. A. Use of natural antimicrobials to increase antibiotic susceptibility of drug resistant bacteria. Int. J. Food Microbiol. 2010, 140, 164−168. (4) Ahmad, A.; Khan, A.; Yousuf, S.; Khan, L. A.; Manzoor, N. Proton translocating ATPase mediated fungicidal activity of eugenol and thymol. Fitoterapia 2010, 81, 1157−1162. (5) Andersen, A. Final report on the safety assessment of sodium pchloro-m-cresol, p-chloro-m-cresol, chlorothymol, mixed cresols, mcresol, o-cresol, p-cresol, isopropyl cresols, thymol, o-cymen-5-ol, and carvacrol. Int. J. Toxicol. 2006, 25, 29−127. (6) Trombetta, D.; Castelli, F.; Sarpietro, M. G.; Venuti, V.; Cristani, M.; Daniele, C.; Saija, A.; Mazzanti, G.; Bisignano, G. Mechanisms of Antibacterial Action of Three Monoterpenes. Antimicrob. Agents Chemother. 2005, 49, 2474−2478. (7) Araújo, L. X.; Novato, T. P.; Zeringota, V.; Matos, R. S.; Senra, T. O.; maturano, R.; Prata, M. C.; Daemon, E.; Monteiro, C. M. Acaricidal activity of thymol against larvae of Rhipicephalus microplus (Acari: Ixodidae) under semi-natural conditions. Parasitol. Res. 2015, 114, 3271−3276. (8) Saravanan, S.; Pari, L. Role of thymol on hyperglycaemia and hyperlipidemia in High fat diet-induced type 2 diabetic C57BL/6J mice. Eur. J. Pharmacol. 2015, 761, 279−287. (9) Gholijani, N.; Gharagozllo, M.; Farjadian, S.; Amirghofran, Z. Modulatory effects of thymol and carvacrol on inflammatory transcription factors in lipopolysaccharide-treated macrophages. J. Immunotoxicol. 2016, 13, 157−164.

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DOI: 10.1021/acs.langmuir.6b00600 Langmuir XXXX, XXX, XXX−XXX

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Langmuir (10) Llana-Ruiz-Cabello, M.; Gutiérrez-Praena, D.; Puerto, M.; Pichardo, S.; Jos, Á .; Cameán, A. M. In vitro pro-oxidant/antioxidant role of carvacrol, thymol and their mixture in the intestinal Caco-2 cell line. Toxicol. In Vitro 2015, 29, 647−656. (11) de Morais, S. M.; Vila-Nova, N. S.; Bevilaqua, C. M.; Rondon, F. C.; Lobo, C. H.; de Alencar Araripe Noronha Moura, A.; Sales, A. D.; Rodrigues, A. P.; de Figuereido, J. R.; Campello, C. C.; Wilson, M. E.; de Andrade, H. F., Jr. Thymol and eugenol derivatives as potential antileishmanial agents. Bioorg. Med. Chem. 2014, 22, 6250−6255. (12) Zhou, E.; Fu, Y.; Wei, Z.; Yu, Y.; Zhang, X.; Yang, Z. Thymol attenuates allergic airway inflammation in ovalbumin (OVA)-induced mouse asthma. Fitoterapia 2014, 96, 131−137. (13) Langmuir, I. J. The constitution and fundamental properties of solids and liquids. J. Am. Chem. Soc. 1917, 39, 1848−1906. (14) Brockman, H. Lipid Monolayers: Why Use Half a Membrane to Characterize Protein-Membrane Interactions? Curr. Opin. Struct. Biol. 1999, 9, 438−444. (15) Dynarowicz-Łatka, P.; Dhanabalan, A.; Oliveira, O. N., Jr. Modern physicochemical research on Langmuir monolayers. Adv. Colloid Interface Sci. 2001, 91, 221−293. (16) Goto, T. E.; Caseli, L. The interaction of mefloquine hydrochloride with cell membrane models at the air−water interface is modulated by the monolayer lipid composition. J. Colloid Interface Sci. 2014, 431, 24−30. (17) Pacholati, C. P.; Lopera, E. P.; Pavinatto, F. J.; Caseli, L.; Nobre, T. M.; Zaniquelli, M. E. D.; Viitala, T.; D’Silva, C.; Oliveira, O. N., Jr. The interaction of an antiparasitic peptide active against African Sleeping Sickness with cell membrane models. Colloids Surf., B 2009, 74, 504−510. (18) Herculano, R. D.; Pavinatto, F. J.; Caseli, L.; D’Silva, C.; Oliveira, O. N., Jr. The lipid composition of a cell membrane modulates the interaction of an antiparasitic peptide at the air−water interface. Biochim. Biophys. Acta, Biomembr. 2011, 1808, 1907−1912. (19) Ferreira, J. V. N.; Grecco, S. S.; Lago, J. H. G.; Caseli, L. Ultrathin films of lipids to investigate the action of a flavonoid with cell membrane models. Mater. Sci. Eng., C 2015, 48, 112−117. (20) Gonçalves, G. E. G.; de Souza, F. S.; Lago, J. H. G.; Caseli, L. The interaction of eugenol with cell membrane models at the air− water interface is modulated by the lipid monolayer composition. Biophys. Chem. 2015, 207, 7−12. (21) Cancino, J.; Nobre, T. M.; Oliveira, O. N., Jr.; Machado, S. A. S.; Zucolotto, V. A new strategy to investigate the toxicity of nanomaterilas using Langmuir monolayers as membrane models. Nanotoxicology 2013, 7, 61−70. (22) Sánchez, M. E.; Turina, A. V.; García, D. A.; Nolan, M. V.; Perillo, M. A. Surface activity of thymol: implications for an eventual pharmacological activity. Colloids Surf., B 2004, 34, 77−86. (23) Turina, A. V.; Nolan, M. V.; Zygadlo, Z.; Perillo, M. A. Natural terpenes: Self-assembly and membrane partitioning. Biophys. Chem. 2006, 122, 101−113. (24) Liolos, C. C.; Gortzi, O.; Lalas, S.; Tsaknis, J.; Chinou, I. Liposomal incorporation of carvacrol and thymol isolated from the essential oil of Origanum dictamnus L. and in vitro antimicrobial activity. Food Chem. 2009, 112, 77−83. (25) Anezo, C.; de Vries, A. H.; Höltje, H.-D.; Tieleman, P. D.; Marrink, S. J. Methodological Issues in Lipid Bilayer Simulations. J. Phys. Chem. B 2003, 107, 9424−9433. (26) Leontiadou, H.; Mark, A. E.; Marrink, S. J. Antimicrobial peptides in action. J. Am. Chem. Soc. 2006, 128, 12156−12161. (27) Cerezo, H.; Zuniga, J.; Batista, A.; Requena, A.; Cerón-Carrasco, J. P. Conformational changes of β-carotene and zeaxanthin immersed in a model membrane through atomistic molecular dynamics simulations. Phys. Chem. Chem. Phys. 2013, 15, 6527−6538. (28) Bennett, W. F. D.; Tieleman, D. P. The Importance of Membrane DefectsLessons from Simulations. Acc. Chem. Res. 2014, 47, 2244−2251. (29) Filipe, H. A. L.; Moreno, M. J.; Rog, T.; Vattalainen, I.; Loura, L. M. S. J. Phys. Chem. B 2014, 118, 3572−3581.

(30) Baoukina, S.; Monticelli, L.; Marrink, S. J.; Tieleman, D. P. Pressure-Area Isotherm of a Lipid Monolayer from Molecular Dynamics Simulations. Langmuir 2007, 23, 12617−12623. (31) Laing, C.; Baoukina, D. P.; Tieleman, D. P. Molecular dynamics study of the effect of cholesterol on the properties of lipid monolayers at low surface tensions. Phys. Chem. Chem. Phys. 2009, 11, 1916−1922. (32) Porasso, R. D.; Bennett, W. F. D.; Oliveira-Costa, S. D.; Cascales, J. J. L. Study of the Benzocaine Transfer from Aqueous Solution to the Interior of a Biological Membrane. J. Phys. Chem. B 2009, 113, 9988−9994. (33) Lago, J. H. G.; Reis, A. A.; Roque, N. F. Chemical composition from volatile oil of the stem bark of Guarea macrophylla Vahl. ssp. tuberculata Vellozo (Meliaceae). Flavour Fragrance J. 2002, 17, 255− 257. (34) Diniz, S. P. S. S.; Specian, V.; Oliveira, R. C.; Romero, A. L. Activity of Thyme (Thymus vulgaris L.) Essential Oil Against Phytopathogenic Fungi. Cient., Ciênc. Biol. Saúde. 2009, 11, 15−18. (35) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 2008, 4, 435−447. (36) Van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. C. GROMACS: Fast, Flexible and Free. J. Comput. Chem. 2005, 26, 1701−1719. (37) Lindahl, E.; Hess, B.; van der Spoel, D. GROMACS 3.0: A package for molecular simulation and trajectory analysis. J. Mol. Model. 2001, 7, 306−317. (38) Martínez, L.; Andrade, R.; Birgin, E. G.; Martínez, J. M. Packmol: A package for building initial configurations for molecular dynamics simulations. J. Comput. Chem. 2009, 30, 2157−2164. (39) Lemkul, J. A.; Bevan, D. R. Assessing the Stability of Alzheimer’s Amyloid Protofibrils Using Molecular Dynamics. J. Phys. Chem. B 2010, 114, 1652−1660. (40) Jambeck, J. P. M.; Lyubartsev, A. P. Derivation and Systematic Validation of a Refined All-Atom Force Field for Phosphatidylcholine Lipids. J. Phys. Chem. B 2012, 116, 3164. (41) Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. Development and Testing of the OPLS All-Atom Force Field on Conformational Energetics and Properties of Organic Liquids. J. Am. Chem. Soc. 1996, 118, 11225−11236. (42) Gaussian 09, Revision C.03: Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A.; Peralta Jr., J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian, Inc.: Wallingford, CT, 2009. (43) Humphrey, W.; Dalke, A.; Schulten, K. VMD - Visual Molecular Dynamics. J. Mol. Graphics 1996, 14, 33−38. (44) Forsheimer, M.; Mohwald, H. Development of equilibrium domain shapes in phospholipid monolayers. Chem. Phys. Lipids 1989, 49, 231−241. (45) Blume, A. A comparative study of the phase transitions of phospholipid bilayers and monolayers. Biochim. Biophys. Acta, Biomembr. 1979, 557, 32−34. (46) Huang, C.; Lapides, J. R.; Levin, I. W. Phase-Transition Behavior of Saturated, Symmetric Chain Phospholipid Bilayer Dispersions Determined by Raman Spectroscopy: Correlation between Spectral and Thermodynamic Parameters. J. Am. Chem. Soc. 1982, 104, 5926−5930. G

DOI: 10.1021/acs.langmuir.6b00600 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir (47) Huo, Q.; Dziri, L.; Desbat, B.; Russel, K. C.; Leblanc, R. M. Polarization-modulated infrared reflection absorption spectroscopic studies of a hydrogen-bonding network at the air−water interface. J. Phys. Chem. B 1999, 103, 2929−2934. (48) McConglogue, C. W.; Vanderlick, T. K. A Close Look at Domain Formation in DPPC Monolayers. Langmuir 1997, 13, 7158− 7164. (49) Dang, L. X.; Wick, C. D. Anion Effects on Interfacial Absorption of Gases in Ionic Liquids. A Molecular Dynamics Study. J. Phys. Chem. B 2011, 115, 6964−6970. (50) Broniatowski, M.; Flasiński, M.; Wydro, P. Lupane-Type Pentacyclic Triterpenes in Langmuir Monolayers: A Synchrotron Radiation Scattering Study. Langmuir 2012, 28, 5201−5210. (51) Broniatowski, M.; Flasiński, M.; Zięba, K.; Miśkowiec, P. Langmuir monolayer studies of the interaction of monoamphiphilic pentacyclic triterpenes with anionic mitochondrial and bacterial membrane phospholipids  Searching for the most active terpene. Biochim. Biophys. Acta, Biomembr. 2014, 1838, 2460−2472. (52) Broniatowski, M.; Flasinski, M.; Wydrob, P.; Fontainec, P. Grazing incidence diffraction studies of the interactions between ursane-type antimicrobial triterpenes and bacterial anionic phospholipids. Colloids Surf., B 2015, 128, 561−567. (53) Broniatowski, M.; Mastalerz, P.; Flasiński, M. Studies of the interactions of ursane-type bioactive terpenes with the model of Escherichia coli inner membraneLangmuir monolayer approach. Biochim. Biophys. Acta, Biomembr. 2015, 1848, 469−476. (54) Nowotarska, S. W.; Nowotarski, K. J.; Situ, C. Effect of Structure on the Interactions between Five Natural Antimicrobial Compounds and Phospholipids of Bacterial Cell Membrane on Model Monolayers. Molecules 2014, 19, 7497−7515. (55) Janas, T.; Nowotarski, K.; Gruszecki, W. I.; Janas, T. The effect of hexadecaprenol on molecular organisation and transport properties of model membranes. Acta Biochim. Pol. 2000, 47, 661−673.

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DOI: 10.1021/acs.langmuir.6b00600 Langmuir XXXX, XXX, XXX−XXX