Molecular Organization of Nalidixate Conjugated Calixarenes in

Article pubs.acs.org/JPCB

Molecular Organization of Nalidixate Conjugated Calixarenes in Bacterial Model Membranes Probed by Molecular Dynamics Simulation and Langmuir Monolayer Studies Beata Korchowiec,*,† Jacek Korchowiec,|| Marcelina Gorczyca,|| Jean-Bernard Regnouf de Vains,‡ and Ewa Rogalska*,‡ †

Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian University, ul. R. Ingardena 3, 30-060 Krakow, Poland || Department of Theoretical Chemistry, Faculty of Chemistry, Jagiellonian University, ul. R. Ingardena 3, 30-060 Krakow, Poland ‡ Structure et Réactivité des Systèmes Moléculaires Complexes, BP 239, CNRS/Université de Lorraine, 54506 Vandoeuvre-lès-Nancy Cedex, France S Supporting Information *

ABSTRACT: Two p-tert-butylcalix[4]arene derivatives bearing one or two nalidixic acid groups connected to the lower rim of ptert-butylcalix[4]arene through the propylenic spacer were studied upon interaction with model bacterial membranes. Indeed, these derivatives were developed recently as new macrocyclic antibiotic carriers for antibacterial therapy. To obtain molecular level information about the interaction between the calixarene conjugates and a membrane lipid, atomistic molecular dynamics simulation, as well as surface pressure, surface potential, polarization modulation infrared reflection−absorption spectroscopy, and Brewster angle microscopy studies of 1,2-dimyristoyl-snglycero-3-phosphoethanolamine (DMPE)−calixarene derivative films were performed. The results obtained indicate that the interaction between the calixarene derivatives and DMPE occurs via the phosphate and carbonyl groups present in the lipid. Although both calixarene derivatives increase the chain tilt and conformational disordering of the DMPE molecules, these effects are more important in the case of the monosubstituted derivative. Importantly, the two derivatives have an opposite impact on hydration of the phosphoglyceride polar head.

1. INTRODUCTION Calixarenes, a class of polyphenolic macrocycles, have both fundamental and practical importance.1,2 Different calix[n]arene derivatives can be obtained synthetically by attaching various moieties on the aromatic crown. Some calixarene derivatives have interesting therapeutic properties.3,4 Recently, an anti-infectious activity was demonstrated with some derivatives.5−8 In our group, different derivatives bearing antibiotic moieties were conceived as possible drug carriers, releasing the antibiotic upon hydrolysis.9 An oral administration of the of p-tert-butylcalix[4]arenebased derivatives would lead to the release of the soluble antibacterial agent in the intestinal compartment; the insoluble calixarene molecule could be thus easily eliminated from the organism.10−13 The calixarenes used in the present study (Figure 1) have one14 or two10 nalidixic arms linked to the lower rim of the of p-tert-butylcalix[4]arene platform via a carboxylate function present in the quinolone moiety. Their interfacial and complexation properties were studied before.14−16 © XXXX American Chemical Society

Indeed, quinolones have aroused much scientific and clinical interest since their discovery in the early 1960s. Nalidixic acid is an inhibitor of the bacterial DNA gyrase, an enzyme essential for DNA replication and transcription. It is effective against both Gram-positive and Gram-negative bacteria. Nalidixic acid was the first quinolone available for clinical use.17 It was proposed in the literature that the incorporation of quinolones into cells occurs via simple diffusion mechanism.18 However, the quinolones containing piperidine or pyrazine rings are zwitterionic, and passive diffusion mechanism may not fully explain their high intestinal absorption, selective tissue distribution, and selective excretion because amphiphilicity plays a crucial role in passive diffusion of molecules across the membranes. Recently, involvement of membrane transporters in a high membrane permeability of the quinolone agents in various tissues was proposed.19 It was demonstrated that, first, the conformational changes occurring in a bacterial multidrug Received: July 17, 2014 Revised: November 12, 2014

A

DOI: 10.1021/jp507151r J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

Gaussian 09.27 A limited conformational analysis was done for calix I and calix II by performing rotation along single bonds in propyl linkages. In the DMPE molecule, all trans conformations in the hydrophobic chains were assumed. The simulation setup included a water slab situated between two vacuum slabs. Two monolayers, each composed of 100 surface active molecules, were placed at the vacuum/water interfaces using a symmetric model. Pure DMPE, calix I, and calix II monolayers, as well as mixed DMPE/calix I and DMPE/calix II monolayers were investigated. Calixarene mole fractions xcalix = 0.25, 0.50, and 0.75, the same at both interfaces, were taken into account. The calixarene derivatives and DMPE molecules were randomly distributed in the monolayers. The simulation was performed under constant number of particles (N), constant temperature (T), constant normal pressure (pn), and constant surface tension (γ): NpnγT ensemble.28 Simulations were run at 293.15 K. Temperature was controlled by Langevin dynamics with damping parameter set to 5 ps. The z-pressure value was set to 1.01325 bar. The surface tension was set to 48.8 mN m−1. The Nosé−Hoover Langevin piston was used (LangevinPistonPeriod = 200 fs, LangevinPistonDecay = 100 fs). All simulations were run with periodic boundary conditions. The cutoff of 12 Å was employed for van der Waals interactions. The particle mesh Ewald scheme29 was used for computing the long-range electrostatic energy. The initial configuration of all systems was obtained after 1 ns NVT simulation. The NpnγT simulations with 2 fs time steps (rigid bonds with hydrogen atoms) were carried out for 20 ns to let the system reach equilibrium. After equilibration, a production run of 4 ns was performed. The data were stored after every 4 ps. The analysis was performed by averaging the information acquired from 1000 computational frames. Compression Isotherms and Brewster Angle Microscopy. The Π−A isotherms were carried out with a KSV 2000 Langmuir balance (KSV Instruments, Helsinki). The isotherms were measured for pure lipid monolayers, as well as their various mixtures. The surface pressure was monitored using a platinum Wilhelmy plate. Surface potential (ΔV) was measured with a KSV Spot 1 (KSV Instruments, Helsinki) using a vibrating plate and a stainless steel counter electrode. The thermostated Teflon trough of the effective film area of 765 cm2 was equipped with two hydrophilic Delrin barriers (symmetric compression). Before each measurement, the subphase surface was cleaned by sweeping and suction. The stability of the surface potential signal was checked before each experiment, after cleaning the water surface. After the ΔV signal had reached the constant value, it was zeroed, and the film was spread on the subphase. The lipid solutions were spread with a Hamilton syringe on the free surface of water and left for 15 min to allow solvent evaporation and to reach an equilibrium state of the monolayer. All isotherms were recorded upon symmetric compression of the monolayer at a constant barrier speed of 2.5 Å2 molecule−1 min−1. In the case of binary mixtures, the surface pressure and surface potential were plotted against the mean molecular area, obtained by dividing the total surface area by the number of molecules spread on the surface. For each monolayer composition, measurements were repeated at least three times. The Π−A and ΔV−A isotherms were recorded at 20 ± 0.1 °C. The standard deviation obtained from compression isotherms was ± 0.5 Å2 on molecular area (A), ± 0.2 mN m−1 on surface pressure and ± 0.01 V on surface potential.

transporter LmrP are decisive for transport activity and, second, that these changes are phosphatidylethanolamine-dependent.20 Indeed, LmrP reconstituted into proteoliposomes lacking phosphatidylethanolamine did not show any transport activity. The calixarene derivatives studied in this work are more hydrophobic compared to quinolones used presently as drugs; the increased hydrophobicity is due to the presence of the calixarene moiety and to the absence of the carboxylic group. Although below pH 4 the two derivatives would bear a positive charge at the heterocyclic nitrogen atom present in the heterocycle,21,22 the net charge would be zero at pH above this value. It can be expected that these features of the two derivatives would facilitate diffusion across the membrane. Here, the membrane-related characteristics of calixarene derivatives were studied in lipid environment using Langmuir film technique. This technique was used before for studying new calixarene derivatives with biologically relevant properties upon interaction with model membranes.23 The fact that the two derivatives used in this work differ by the number of the grafted nalidixic acid moieties permits estimating the contribution of the pendant arms to the properties of the mixed calixarene-lipid films. DMPE was used as a model membrane lipid because, first, it plays a particular role in the transport of quinolones across membranes and, second, it is useful for Brewster angle microscopy (BAM) studies because it forms characteristic domains in the liquid expanded-liquid condensed phase transition region. Surface pressure−molecular area (Π−A) isotherms, surface potential−molecular area (ΔV−A) isotherms, BAM and polarization-modulation infrared reflection−absorption spectroscopy (PM-IRRAS) were used to characterize the monolayers. The data obtained from the isotherms were used to calculate the thermodynamic functions of the films with the aim to better understand film properties and the calixarene− DMPE interaction. On the other hand, molecular modeling of the calixarene derivatives allowed a more reliable interpretation of the experimental results in terms of conformational rearrangement of these molecules upon interaction with DMPE. The overall results may be useful when designing new synthetic calixarenebased drugs and for understanding of how these molecules interact with cell membranes. This study prepares the ground for further research on the biological activity of calixarene− antibiotic conjugates.

2. EXPERIMENTAL SECTION Materials. Calix I: p-tert-butylcalix[4]arene-mono-propylnalidixate and calix II: p-tert-butylcalix[4]arene-bis-propylnalidixate were synthesized and characterized as described previously.10,14 Synthetic DMPE (∼99% pure), chloroform, and methanol (both 99.9% pure) were from Sigma-Aldrich. Calix I, calix II, and DMPE were dissolved in chloroform/methanol mixture (3:1 v/v) to achieve a final concentration of 0.5 mg mL−1. The stock solutions of calix I, calix II, and DMPE were used for preparing 0.25, 0.5, and 0.75 calix I or calix II mole fraction mixtures. The solutions were stored at 4 °C. Milli-Q water (Millipore) with resistivity of 18 MΩ cm and surface tension of 72.8 mN m−1 at 20 °C was used in all experiments. Methods. Molecular Dynamics Simulations. Molecular dynamics (MD) calculations were carried out using NAMD2 package24 and CHARMM27 force field25 including a rigid TIP3 water molecule.26 Initial geometries of DMPE, calix I, and calix II were obtained at B3LYP/6-31G(d) level of theory using B


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Figure 1. Structures of (A) p-tert-butylcalix[4]arene-mono-propylnalidixate (calix I), (B) 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, and (C) p-tert-butylcalix[4]arene-bis-propylnalidixate (calix II).

the difference providing surface specific information, and the sum providing the reference spectrum. As the spectra are measured simultaneously, the effect of water vapor is largely reduced. The PM-IRRAS spectra of the film-covered surface, S(f), as well as that of the pure water, S(w), were measured and the normalized difference ΔS/S = [S(f) − S(w)]/S(w) is reported. In total, 6000 interferogram scans (10 scans per second) have been acquired for each spectrum. In the mid-IR region, the wavenumber at which the half-wave retardation takes place can be freely selected. Here, the maximum of PEM efficiency was set either to 1500 or to 2900 cm−1 for analyzing the carbonyl stretching or methylene stretching regions of the spectra, respectively. The spectral range of the device is 800− 4000 cm−1, and the resolution is 8 cm−1.

The compression isotherms allowed determining the compressibility modulus, CS−1,30 as:

CS−1 = −A(∂Π/∂A)T

(1)

The Gibbs energy of mixing, ΔGmix, was calculated from Π− A isotherms using the following formula: ΔGmix =

∫0

Π

[A12 − (x1A1 + x 2A 2 )]dΠ

(2)

where A12 is the mean molecular area in the mixed monolayer at a given surface pressure, A1 and A2 are the mean molecular areas of the pure components 1 and 2 at the same surface pressure, and x1 and x2 are the mole fractions of the two lipid components in the mixed film.31,32 We want to mention that thermodynamic terms used here were defined in our previous papers.23,33 Namely, in accordance with the definition of Gibbs energy of mixing, ΔGmix = G − (x1G1 + x2G2), eq 2 gives ΔGmix instead of ΔGexc given by Goodrich31 and Bacon et al.32 The morphology of the films was imaged with a computerinterfaced KSV 2000 Langmuir balance combined with a Brewster angle microscope (KSV Optrel BAM 300, Helsinki). The Teflon trough dimensions were 6.5 cm × 58 cm × 1 cm; other experimental conditions were as described above. Polarization-Modulation Infrared Reflection−Absorption Spectroscopy (PM-IRRAS). The PM-IRRAS spectra34 of DMPE and all mixed DMPE/calixarene monolayers spread on pure water subphase were acquired at 20 °C. The Teflon trough dimensions were 36.5 cm × 7.5 cm × 0.5 cm; other experimental conditions were as described in the preceding paragraph. The PM-IRRAS measurements were performed using a KSV PMI 550 instrument (KSV Instruments Ltd., Helsinki, Finland). The PMI 550 contains a compact Fourier Transform IR-spectrometer equipped with a polarizationmodulation (PM) unit on one arm of a goniometer and a MCT-detector on the other arm. The incident angle of the light beam can be freely chosen between 40° and 90°; here, the incident angle was 79°. The spectrometer and the PM-unit operate at different frequencies, allowing separation of the two signals at the detector. The PM unit consists of a photoelastic modulator, which is an IR-transparent, ZnSe piezoelectric lens. The incoming light is continuously modulated between s- and p- polarization at a frequency of 74 kHz. This allows simultaneous measurement of spectra for the two polarizations,

3. RESULTS AND DISCUSSION Molecular Dynamics of the Pure and Mixed DMPE/ Calixarene Monolayers. The structures of the molecules studied in this work are presented in Figure 1. The snapshots of the equilibrated upper monolayers (see the Molecular Dynamics Simulations section) at the water−vacuum interface are collected in Figures 2 and 3. The section along xz surface with depth y containing one monolayer of the surface active molecules is plotted. Water molecules within 10 Å from DMPE and calixarene derivatives are depicted. The pictures were obtained using a vmd graphical package.35 Figure 2A−C corresponds to pure calix I, calix II, and DMPE monolayers, respectively. The snapshots of DMPE/calix I and DMPE/calix II mixed systems obtained with xcalix = 0.25, 0.50, and 0.75 are shown in Figure 3A, D, Figure 3B, E, and Figure 3C, F, repectively. It can be observed that ordering (i.e., all-trans conformation in the DMPE chains seen in the pure phospholipid film) decreases in mixed monolayers (Figure 3 vs Figure 2C snapshots). Indeed, the size of the calixarene aromatic crown is too small to stabilize the trans conformers in z-direction. On the other hand, the aromatic crown increases spacing between the DMPE molecules and favors conformational movements. The disordering of the monolayer increases with xcalix for both calixarene derivatives. Partial density profiles, ρY = ρY(z) (g cm−3), of pure monolayers (Figure 4) show the composition of the system along the normal (z) to the interface (xy surface), where Y corresponds to water (black lines) or selected functional C


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CH3 groups in the mixed films are broader compared to the pure film and the maximum density corresponding to the terminal CH3 groups in DMPE is shifted toward the water phase. These results indicate an increased conformational mobility and disordering of the hydrocarbon chains in the mixed films compared to pure DMPE films. The latter effect can be seen as well in the snapshots displayed in Figure 3 and in the tilt angle distribution plots (Figure 5A, B). An increase of the DMPE hydrophobic chain mobility with increasing xcalix can be seen in the tilt angle distribution of the hydrocarbon chain present in the sn-1 position (Figure 5A). The tilt vector in DMPE is defined by the first CH2 and the terminal CH3 carbon atoms in the hydrocarbon chains. The tilt angle is formed by the tilt vector and the normal to the surface. Because behavior of both chains is similar, the results obtained with the sn-2 chain are shown in the Supporting Information. In the pure DMPE monolayer, the tilt angle probability distribution (Figure 5A, black curve) is asymmetric with the maximum (i.e., the most probable value) situated at around 10 degrees; the mean value of the tilt angle is 15 degrees. This indicates a predominantly trans conformation in the hydrocarbon chains. The width of the tilt angle distribution increases upon introduction of calix I or calix II to the film (Figure 5A; solid and dashed lines, respectively). On the other hand, an increased number of maxima is observed. These effects, which are connected to an increasing rotational mobility around the C−C bonds, can be seen in Figure 3 snapshots. Ordering of hydrophobic chains in DMPE can be quantified using the average trans conformation parameter, S: S = |3⟨cos2 θ ⟩ − 1| /2

where θ refers to the dihedral angle formed with the C−C bonds in the hydrocarbon chain. The total order in the chain corresponds to 1.0, whereas disorder corresponds to 0.0. The computed values of S are plotted in Figure 5B. It can be observed that S values are significantly higher in the case of pure DMPE monolayer compared to mixed monolayers, indicating a preference for trans conformation in the DMPE acyl chains. In the mixed films, the S values are slightly higher for xcalix = 0.25 compared to xcalix = 0.50 and 0.75. These results show that calix I and calix II introduce disordering of the DMPE acyl chains and favor their conformational mobility. Moreover, to quantify ordering of the pure DMPE monolayer, the rotational order parameter, g, was computed:

Figure 2. Cross-section of pure calix I (A), calix II (B), and DMPE (C) monolayers in xz surface with width (Δy) containing one molecular layer. The air−water interface is indicated by water molecules separated by no more than 10 Å from the DMPE, calix I, or calix II molecules.

groups. The density of amine, phosphate, and carbonyl groups as well as methyl terminal groups of hydrocarbon chains were taken into account in the case of DMPE molecule. For both calixarene derivatives, the hydroxyl and carbonyl functional groups linked to the lower rim of the molecule and methyl groups from the tert-butyl substituents were considered. In the pure DMPE monolayer (Figure 2C), the hydrophilic headgroups and hydrophobic tails are oriented toward water and the vapor phase, respectively. The density profiles (Figure 4A) show that water penetrates into the polar headgroup region, whereas the space occupied by the acyl chains is dehydrated. In the case of calix I and calix II monolayers (Figure 4B, C), water molecules penetrate partially into the hydrophobic zone. Distribution of the CH3 groups in both derivatives is broad and asymmetric. In the case of the calix II monolayer, clearly distinguished maxima are observed at the interface indicating that some calix II molecules penetrate into and stay completely immersed in the water phase. A more important penetration of calix II than calix I molecules into the water subphase is clearly seen in Figure 2. In the mixed films, the density plots of DMPE and calixarenes are similar to those obtained for the pure films (data not shown). However, the density profiles of the DMPE

g = ⟨2cos2(φ) − 1⟩

where φ is the azimuthal angle of the tilt vector; g = ± 1 and g = 0 indicate order and disorder, respectively. As the g values do not exceed 0.1, it can be concluded that there is rotational (conformational) disordering in the DMPE molecules in pure monolayers. The ordering of the calix I and calix II molecules in the monolayer was analyzed, as was the case with DMPE. In Figure 6 is shown the tilt angle distribution for the calixarene cone (Figure 6A) defined by its normal and for the nalidixic acid groups (Figure 6B). In the case of calix II, an average value for both nalidixic acid groups is taken into account. It can be observed that the tilt angle distribution of the cone is lower in the case of calix II (black solid line) compared to calix I (red solid line). This observation is true both for pure calixarene monolayers (solid black and red lines) and for mixed D


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Figure 3. Cross-section of mixed DMPE/calix I (A−C) and DMPE/calix II (D−F) monolayers in xz surface with width (Δy) containing one molecular layer for xcalix = 0.25 (A, D), 0.50 (B, E), and 0.75 (C, F). The air−water interface is indicated by water molecules separated by no more than 10 Å from the DMPE, calix I, or calix II molecules.

presented in Figure 8. The Π−A isotherm (Figure 8, solid black line) of pure DMPE show the liquid expanded−liquid condensed phase transition (LE−LC) at a surface pressure of approximately 6 mN m−1 and collapse pressure at 55 mN m−1, which is consistent with the published data.36 The films formed with calix I (Figure 8A) and calix II (Figure 8B) have liquid expanded or liquid condensed character, respectively (CS−1 equal to 82 or 134 mN m−1, respectively).14 The Π−A and ΔV−A isotherms provide information concerning molecule organization in the films. In the mixed films, an increase of the molecular area values was observed at all pressures and potentials compared to the pure lipid. However, the calixarene induced expansion of the film is lower with calix I compared to calix II, which may be due to a smaller area occupied by this derivative in the film (Figure 8). Inspection of the ΔV−A isotherms37 (Figure 8, dashed lines) permits detection of gas−liquid expanded (G−LE) phase transitions, which are not visible in the Π−A isotherms. In the pure calix I and DMPE/calix I films, the onset of the G−LE phase transition is observed at around 150, 190, 220, and 260 Å2 for xcalix = 0.25, 0.50, 0.75 or pure calix I, respectively. In the case of DMPE/calix II, the onset of this phase transition can be seen at around 190 and 280 Å2 for xcalix = 0.50 and 0.75. The LE−LC phase transition shown by the discontinuities in the corresponding Π−A isotherms can be also visualized as minima in the compressibility modulus−surface pressure (CS−1−Π) plots (Figure 9). The LE−LC transition was not

monolayers corresponding to xcalix = 0.75 (dashed black and red lines). The fact that the tilt angle is smaller for calix II compared to calix I indicates that the nalidixate moiety in calix I adopts several different orientations due to the internal rotation within the propylene linker. In the pure calix II monolayer, the internal rotation is limited due to the intermolecular stabilization between the two nalidixate moieties (Figure 1C). The radial pair distribution function, G(r), shown in Figure 7 gives additional information concerning the DMPE headgroups. It can be observed that oxygens present in the phosphate groups (Figure 7A−C) form hydrogen bonds with water molecules (the first maximum at around 4 Å). The fact that the first peak for pure DMPE film is situated between the DMPE/calix I (red curve) and DMPE/calix II (green curve) films indicates that hydration of the phosphate group in the mixed DMPE/calix I is higher compared to pure DMPE or mixed DMPE/calix II films. The propensity of the carboxylic ester oxygens (Figure 7D−F) to form hydrogen bonds is lower compared to phosphate groups, as indicated by a lower value of G(r) at the first maximum. These findings indicate that the DMPE headgroup hydration in the films increases in the order DMPE/calix II < pure DMPE < DMPE/calix I. Compression Isotherms and Brewster Angle Microscopy. The effect of calixarene derivatives on the model lipid membrane was probed experimentally using Langmuir film technique. The obtained Π−A and ΔV−A isotherms of pure DMPE, calix I or calix II, and mixed DMPE/calixarene films are E


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The surface pressure of the LE−LC phase transition varies significantly with the composition of the film, namely, it increases with calixarene concentration (insets of Figure 9). It is also clearly visible that DMPE/calix II mixed monolayers undergo the transition to the condensed phase at lower surface pressures (9.3, 12.7, and 20.4 mN m−1 for xcalix = 0.25, 0.5, and 0.75, respectively) compared to DMPE/calix I (10.6 and 13.9, mN m−1 for xcalix = 0.25 and 0.5, respectively). These results confirm that both derivatives induce a more liquid like character of the films; this effect is more pronounced in the case of calix I. At the lowest calixarene content (xcalix = 0.25), the LE−LC phase transition shows clearly in the compression isotherms of the mixed films formed with calix I as well as with calix II. BAM micrographs taken at the LE−LC phase transition of pure phospholipid and mixed phospholipid/calixarene films are presented in Figure 10. The snapshots presented in the first and the second column correspond to the beginning of the LE−LC phase transition and to a surface pressures higher by 1 mN m−1, respectively. It can be seen that in the case of the mixed films the domains are smaller and more numerous; the domains observed in the DMPE/calix I are smaller compared to DMPE/calix II film. Overall, BAM results are in accordance with compression isotherms indicating a more liquid like character of the film containing calix I compared to calix II. At mole fractions above 0.25, no domains could be observed; the uniformly black BAM images up to the collapse point indicated that the films were isotropic. Interaction between Monolayer Components. To get a quantitative insight in the intermolecular interaction between 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine and calixarene derivatives in mixed Langmuir films, mean molecular area (MMA), mean surface potential (MSP), as well as ΔGmix (eq 2) analyses were performed at an arbitrarily chosen surface pressure of 24 mN m−1. The additive values of pure compounds are used as reference in the plots of MMA, MSP, and ΔGmix versus xcalix (Figure 11).23,33 A comparison between the MMA of additive and experimental mixing as a function of the xcalix mole fraction at an arbitrary chosen surface pressures of 24 mN m−1 is shown in Figure 11A. The experimental MMA values of DMPE/calix I films are close to the calculated additive values. However, the latter films have lower CS−1 values (Figure 9), which indicates a less ordered character compared to the

Figure 4. Partial density profiles, ρY = ρY(z), of pure DMPE (A), calix I (B), and calix II (C) monolayers, where Y in ρY corresponds to water or functional groups as indicated in panels A−C.

observed in the case of DMPE/calix I mixture at xcalix = 0.75 (Figure 9A, blue line). At this concentration, the Π−A isotherm of the mixed film resembles that of the pure calix I.

Figure 5. Tilt angle probability distribution function (A) and ordering of the hydrocarbon chain (B) in position sn-1 for pure DMPE (black lines), mixed DMPE/calix I (solid lines), and DMPE/calix II (dashed lines) monolayers. Red, blue, and green lines correspond to xcalix = 0.25, 0.50, and 0.75, respectively. (A) The horizontal axis corresponds to angles formed between the vector representing the hydrocarbon chains in extended conformation (vector originating on C2 and going to C14) and the normal to the surface; (B) numbers on the x axis represent the consecutive C−C bonds, where 1 is the bond between C2 and C3. The results obtained with the sn-2 chains were close to sn-1 (see Supporting Information). F


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Figure 6. Tilt angle probability distribution function of the calixarene cone (A) and the nalixidic acid moieties (B). Solid or broken lines correspond to pure or mixed calix I (red) and calix II (black) monolayers, respectively. Mixed monolayers were prepared with xcalix = 0.75.

films containing calix II. Another difference between the effect of calix I and calix II in mixed films can be seen in the MSP− xcalix plots presented in Figure 11B. Here, the higher values of MSP measured with DMPE/calix II films show a better ordering of molecules at the air−water interface. Based on modeling, this result was not unexpected. Indeed, due to the molecule conformation, the two pendant arms present in calix II would favor a more vertical orientation of the molecules at the interface. The energy of film formation, ΔGmix, is presented in function of xcalix in Figure 11C. The fact that the ΔGmix curves are symmetric indicates that interactions DMPE−DMPE, calix I− calix I (or calix II−calix II), and DMPE-calix I (or DMPE−calix II) are comparable. Consequently, it can be expected that distribution of DMPE and calix I or calix II in the films is random. It can be noticed that the energy of film formation is approximately 2 times higher in the case of DMPE/calix II. This difference of the ΔGmix values may be explained by different hydration of DMPE in the two mixtures. Indeed, taking into account the fact that ΔG of hydration of the DMPE molecules is negative,38 dehydrating of DMPE upon mixing with calix II increases the positive ΔGmix, while hydrating of DMPE by calix I lowers it. It should be noted that the effect of more important hydration of the DMPE polar head upon interaction with calix I compared to pure DMPE and less important in the case of calix II was demonstrated with molecular modeling and PM-IRRAS.

Figure 7. Radial pair distribution function, G(r), of water oxygen atoms close to the phosphate group (A, B, C) and carbonyl oxygens (D, E, F) in the DMPE headgroup. Monolayers containing 0.25 (A, D), 0.50 (B, E), and 0.75 (C, F) mole fraction of DMPE. Black, red, and green curves correspond to pure DMPE, DMPE/calix I, and DMPE/calix II monolayers, respectively.

Figure 8. Π−A (solid lines) and ΔV−A (dashed lines) isotherms of binary mixtures DMPE/calix I (A) and DMPE/calix II (B) spread on water at 20 °C. Black curves: pure DMPE film; red curves: xcalix = 0.25; green curves xcalix = 0.50; blue curves: xcalix = 0.75, cyan curve: pure calix I and magenta curve: pure calix II film. G


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Figure 9. CS−1−Π dependency for DMPE/calix I (A) and DMPE/calix II (B) monolayers. Black curves: pure DMPE film; red curves: xcalix = 0.25; green curves xcalix = 0.50; blue curves: xcalix = 0.75, cyan curve: pure calix I and magenta curve: pure calix II film.

Figure 10. BAM micrographs of pure and mixed DMPE/calixarene monolayers. The mixed films contained 0.25 mole fraction of calixarene. Pure DMPE (A, B); DMPE/calix I (C, D); DMPE/calix II (E, F). The images were taken at: Π = 6.3 (A), 7.3 (B), 10.0 (C), 11.0 (D), 9.0 (E) and 10.0 (F) mN m−1. Scale: the width of the snapshots corresponds to 150 μm.

PM-IRRAS Spectroscopy of the Monomolecular Films. Although the interfacial properties and mechanism of action of molecules such as phosphoglycerides and proteins have been intensively studied in Langmuir films using PM-IRRAS over the last years,39,40 this approach was rarely used with molecules resulting from organic synthesis,14,41−43 such as macrocycles.14,43 Here, PM-IRRAS was used to explore the effect of the structure of the calixarene derivatives on the molecular organization of phospholipid films. PM-IRRAS spectra of Langmuir films were recorded on a pure water subphase at Π = 24 mN m−1. Plots of the variation in the lipid characteristic wavenumbers with the calixarene content are shown in Figure 12. The wavenumbers of the CH2 symmetric and antisymmetric modes can be used to monitor the degree of conformational order of alkyl chains in different molecules. When the hydrocarbon chain is highly ordered (trans-zigzag conformation), the bands due to CH2 symmetric and antisymmetric modes appear at around 2850 and 2920 cm−1, respectively. If gauche conformers (conformational disorder) are induced in the alkyl chains, these bands shift to higher frequencies.44−47 The methylene stretching region of the pure DMPE film revealed the symmetric and antisymmetric stretching vibrations

Figure 11. Thermodynamic analysis of DMPE/calix I (○) and DMPE/calix II (●) films at Π = 24 mN m−1, T = 20 °C. Dotted lines: additive mixing.

at 2851 and 2920 cm−1, as shown in Figure 12A and B, respectively. The wavenumber values obtained indicate that in a pure phospholipid monolayer the alkyl chains are well packed and highly orderedthat is, the conformation is all-trans. The presence of calix I or calix II in the phospholipid layer induces a disorder by increasing the number of gauche conformations. The latter shows as a shift of the asymmetric CH2 stretching frequency to 2924 and 2922 cm−1 for DMPE/calix I and DMPE/calix II system, respectively. The same tendency was observed for the symmetric CH2 stretching. In the case of the asymmetric CH2 stretching, the shift was observed with the H


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band is observed in the mixed film containing calix I, whereas a blueshift is seen in the case of calix II. These results suggest an increase of the carbonyl group hydration in the presence of calix I, and dehydration in the presence of calix II. The latter is in accordance with molecular modeling The PM-IRRAS data corresponding to the asymmetric PO2− stretching vibrations43 in DMPE are presented in Figure 12D. The frequency of this signal in the pure DMPE monolayer is centered at 1233 cm−1. The νas(PO2−) signals measured in the DMPE/calix I and DMPE/calix II mixed films, for xcalix = 0.25 are centered at 1237 and 1221 cm−1, respectively. It can be concluded that the PO2− groups are hydrated in the presence of calix I, although calix II would rather lead to dehydration of the PO2− moiety. It can be observed that hydration of the PO2− groups in the presence of calix I or calix II follows the same tendency as that observed in the case of CO; the PM-IRRAS results obtained with the two moieties converge with those obtained with molecular modeling.

4. CONCLUSIONS The results obtained show that mixed films formed with DMPE and calix I or calix II have a more liquid-like character compared to pure DMPE. This effect, which is more pronounced in the case of calix I, is due to a higher conformational and orientational mobility of molecules in the mixed films formed with the latter compared to calix II. Obviously, the second nalidixic moiety present in calix II permits interactions with DMPE, which do not exist in the case of the DMPE/calix I films. This proposal is supported by the fact that hydration of the DMPE polar heads is higher in the DMPE/calix I films compared to DMPE/calix II. The DMPE− calix II interactions probably occur via the phosphate and carbonyl groups, as shift of stretching vibrations to the higher frequencies was observed using PM-IRRAS; the latter effect is often associated with dehydration of these moieties. Importantly, both calixarene derivatives and DMPE are randomly distributed in the monolayers. Because mixed DMPE/calix I monolayers have a more liquid-like character compared to DMPE/calix II, diffusion of calix I in the monolayer would be easier compared to calix II. Consequently, it can be supposed that translocation across cell membranes would be easier in the case of calix I. This observation, as well as the effect of phospholipid hydration in the presence of calix I, can be useful for developing new calixarene-based drug carriers.

Figure 12. Characteristic vibrational wavenumbers of DMPE/calix I (○) and DMPE/calix II (●) mixed films obtained from PM-IRRAS spectra as a function of calixarene mole fraction. Surface pressure: 24 mN m−1.

increase of xcalix, whereas the symmetric CH2 stretching appears to be less dependent on the latter. The highest frequencies of the methylene stretching, indicating a higher number of gauche conformers and larger changes in the acyl chain packing, were observed with calix I compared to calix II. Overall, the PMIRRAS results show an increasing conformational disorder of DMPE acyl chains upon interaction with calixarene derivatives. These results are in accordance with the molecular dynamics simulation indicating that hydrophobic acyl chains become more tilted and flexible upon interaction with calix I and calix II. These results are also consistent with the compression isotherm analysis, showing a more fluid-like character of the mixed films compared to pure DMPE. As described in the literature, for groups involved in Hbonding to water, the stretching mode absorption bands shift to lower frequency, whereas bending mode absorption bands shift to higher frequency, as H-bonding increases and entropy decreases.48,49 The CO stretching region (Figure 12C) in mixed films reveals vibrations from the DMPE polar head and from nalidixic acid groups in calixarene derivatives; the latter is absent in the pure lipid film. As shown in Figure 12C, the carbonyl stretching vibration in DMPE monolayer appears at 1733 cm−1. For mixed films with the highest DMPE content (xcalix = 0.25), the DMPE ν(CO) signal values are close to those observed in the pure lipid film. The MD simulations show that the DMPE ν(CO) values in the pure lipid and in mixed DMPE/calixarene films were close (Figure 7D), as a result of a comparable hydration of the carbonyl groups. The presence of calix I or calix II at xcalix = 0.50 and xcalix = 0.75 affects the position of the CO stretching bands more significantly. Moreover, a redshift of the ν(CO) stretching

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ASSOCIATED CONTENT

S Supporting Information *

Figures showing tilt angle probability distribution function and ordering of the hydrocarbon chain in position sn-2 for pure DMPE, mixed DMPE/calix I, and DMPE/calix II monolayers. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +48 (12) 663 22 51. Fax: +48 (12) 634 05 15. *E-mail: [email protected]. Tel.: +33 3 83 68 43 45. Fax: +33 3 83 68 43 22. Notes

The authors declare no competing financial interest. I


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ACKNOWLEDGMENTS This work was supported by the Polish National Science Centre, Project No. 2012/07/B/ST5/00890. M.G. acknowledges the financial support from an Interdisciplinary Ph.D. Studies project entitled “Molecular sciences for medicine” cofinanced by the European Social Fund within the Human Capital Operational Programme. The assistance of Adel Ben Salem in organic synthesis is gratefully acknowledged. Calculations were performed at Faculty of Chemistry of Jagiellonian University on the computer cluster purchased with the financial support from the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (Contract No. POIG.02.01.0012-023/08) and on PL-Grid infrastructure at ACK CYFRONET with the support of the “HPC Infrastructure for Grand Challenges of Science and Engineering” Project.

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Molecular Organization of Nalidixate Conjugated Calixarenes in

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