RuCl3(dppb)(VPy) - American Chemical Society

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Correlation of [RuCl3(dppb)(VPy)] Cytotoxicity with its Effects on the Cell Membranes: An Investigation Using Langmuir Monolayers as Membrane Models B. Sandrino,*,† T. T. Tominaga,‡ T. M. Nobre,§ L. Scorsin,† E. C. Wrobel,† B. C. Fiorin,† M. P. de Araujo,∥ L. Caseli,⊥ O. N. Oliveira, Jr.,§ and K. Wohnrath*,† †

Departamento de Química, Universidade Estadual de Ponta Grossa, 84035-900, Ponta Grossa, PR, Brasil Departamento de Física, Universidade Estadual do Centro Oeste, 85040-080, Guarapuava, PR, Brasil § Instituto de Física de São Carlos, Universidade São Paulo, 13560-970, São Carlos, SP, Brasil ∥ Departamento de Química, Universidade Federal do Paraná, 81531-980, Curitiba, PR, Brasil ⊥ Departamento de Ciências Exatas e da Terra, Universidade Federal de São Paulo, 09972-270, Diadema, SP, Brasil ‡

S Supporting Information *

ABSTRACT: One of the major challenges in drug design is to identify compounds with potential toxicity toward target cells, preferably with molecular-level understanding of their mode of action. In this study, the antitumor property of a ruthenium complex, mer-[RuCl3(dppb)(VPy)] (dppb =1,4-bis(diphenylphosphine)butane and VPy = 4-vinylpyridine) (RuVPy), was analyzed. Results showed that this compound led to a mortality rate of 50% of HEp-2 cell with 120 ± 10 μmol L−1, indicating its high toxicity. Then, to prove if its mode of action is associated with its interaction with cell membranes, Langmuir monolayers were used as a membrane model. RuVPy had a strong effect on the surface pressure isotherms, especially on the elastic properties of both the zwitterionic dipalmitoylphosphatidylcholine (DPPC) and the negatively charged dipalmitoylphosphatidylglycerol (DPPG) phospholipids. These data were confirmed by polarization-modulated infrared reflection−absorption spectroscopy (PM-IRRAS). In addition, interactions between the positive group from RuVPy and the phosphate group from the phospholipids were corroborated by density functional theory (DFT) calculations, allowing the determination of the Ru complex orientation at the air−water interface. Although possible contributions from receptors or other cell components cannot be discarded, the results reported here represent evidence for significant effects on the cell membranes which are probably associated with the high toxicity of RuVPy.

1. INTRODUCTION The biological activity of Ru complexes as antitumor agents was first described by Dwyner and co-workers, who attributed the effect to the ligand 1,10-phenanthroline or related bases.1,2 Ru complexes interact with proteins and lipids located on cell surfaces through noncovalent interactions, but the most relevant parameters for such activity are coordination and state of oxidation.3,4 The antitumor activity of Ru complexes containing heterocyclic nitrogenous ligands are probably due to noncovalent interaction between aromatic cyclic ligands and biomolecules (e.g., for DNA intercalation).5 The interest in Ru compounds has decreased after the breakthrough with the discovery of antitumor activity of Pt complexes (cisplatin), which is ascribed to their ability to interact with DNA molecules after entering the cells.6 Nevertheless, Pt complexes have been found to exhibit low specificity toward cancer cells, being highly toxic also for healthy cells. This has brought further motivation to revisit the possible use of Ru compounds, whose ability to coordinate with different ligands was explored © 2014 American Chemical Society

to obtain antitumor agents with the same efficiency of cisplatin, but with lower toxicity and antimetastatic activity.7 This low cytotoxicity can be achieved because ruthenium mimics iron in binding biological molecules such as serum proteins.8 The antimetastatic activity has not yet been elucidated, but it has been suggested that the low level of oxygen in the tumor environment could transform Ru III into the more reactive Ru II, which can bind to various biomolecules, including DNA.9 It seems that the mechanism of action for Ru complexes differs from those well established for Pt complexes,10−16 involving four contributions:17 (i) electrostatic binding, (ii) external binding, (iii) groove binding, and (iv) intercalative binding. Current interest in the design of new ruthenium complexes as therapeutic agents focuses on the role that phosphine ligands play in determining the chemical properties, and hence Received: June 6, 2014 Revised: August 16, 2014 Published: August 18, 2014 10653

dx.doi.org/10.1021/jp505657x | J. Phys. Chem. B 2014, 118, 10653−10661

The Journal of Physical Chemistry B

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concentrations of RuVPy (5−250 μg mL−1) dissolved in DMSO and diluted using the culture medium with a final DMSO concentration of 1%. Controls were always treated with the same amount of DMSO as used in the corresponding experiments. After the incubation period, the cells were washed with PBS and fresh medium was added. Following this treatment, the cells were kept at 37 °C, 5% CO2, and 95% air for an additional 48 h. Cell viability was assessed using a colorimetric assay, in the presence of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma), based on the cellular conversion of a tetrazolium salt into formazan.30 50 μL of 1 mg mL−1 solution of MTT in IMDM medium were added to each well, followed by 3 h incubation at 37 °C, 5% CO2, 95% air. The MTT solution was removed, and 50 μL of ethanol and 150 μL of a solution containing PBS and isopropanol (1:1) were added to each well in order to solubilize the crystals formed. The absorbance of each well was read on a microplate reader (Versamax, Molecular Device) at 550 nm, being proportional to the number of living cells. Experiments were performed in sextuplicate for each concentration of RuVPy and for the control cells. The results were expressed as the mean ± standard deviation and the survival index (Supporting Information) was calculated as follows: Survival ratio (%) = (Adrug/Acontrol) × 100%, where A = absorbance.31 The mean inhibitory concentration, IC50, was taken as the concentration that caused 50% inhibition of cell viability, being calculated using Calcusyn,32 the multiple drug effect analysis software. 2.3. Computational Simulations. The quantum chemical calculation for the RuVPy molecule, whose structure is shown in Figure 3, was carried out using density functional theory (DFT) with B3LYP/lanl2dz implemented on GAUSSIAN package.33 The optimized geometrical structure of RuVPy complex was used to infer the conformation that RuVPy should adopt at the air−water interface, based on the area per molecule for a condensed monolayer from π−A isotherms. In general, this area was associated with the geometrical triangle area among atoms H67, H55 and C75 (Figure 3A). The electrostatic potential map was visualized using the Molekel 5.3 program,34 which allowed one to identify the higher density electronics groups. 2.4. Langmuir Monolayers. The complex RuVPy and lipid molecules were cospread at the air−water interface. Langmuir monolayers were produced by spreading chloroform solutions, for RuVPy and DPPC, or 4:1 (v:v) chloroform:methanol solutions for DPPG, onto the surface of a PBS buffer (pH 7.4, 150 mM NaCl) in a Mini KSV Langmuir trough (KSV, Finland) equipped with a Wilhelmy plate as surface pressure sensor. Mixed (RuVPy/DPPC and RuVPy/DPPG) monolayers were prepared by mixing different volumes of RuVPy and phospholipid stock solutions at 1 mM of concentration. Appropriate volumes were mixed to render the concentrations of RuVPy 10, 30, 50, 70 and 90% in mol. Aliquots of 40 μL of these mixed solutions were then spread onto the PBS buffer surface with a micrometric syringe. The solvent was allowed to evaporate for ca. 10 min before monolayer compression. The surface pressure versus area per molecule (π-A) isotherms were recorded with symmetrical compression of the monolayers with two barriers at a constant speed of 10 mm min−1 (∼5 Å2 molecule−1 min−1). The experiments were performed at 20 ± 1 °C and the isotherms represent the average of at least two measurements for each concentration of the Ru complex. The cospreading method was used here because RuVPy is not

biological activity of these complexes.18,19 Phosphine complexes of various transition metals (Au, Ag, Cu, Ru, Rh, Pt, Pd) have been evaluated as potential antitumor agents in various human tumor cell lines.20−25 For Au complexes, for instance, activity has been attributed to the presence of the phosphine ligands, since similar complexes without them had a very low activity.26 trans-Phosphine amine complexes investigated by NavarroRanninger et al. were found to induce apoptosis in both cisplatin sensitive and resistant cell lines, representing a promising means of treating cancers where cisplatin is inactive.27 One of the earliest works of biological activity with biphosphine ligand coordinated to ruthenium complexes was performed with fac-[RuCl3(NO)(dppf)] (dppf =1,1′-bis(diphenylphosphino)ferrocene), which yielded a highly potent compound with IC50 = 10 ± 3 μmol L−1 in MDA-MB-231 cell line, a value that is lower than those observed for the RuCl3NO· 2H2O precursor, free dppf ligand and cisplatin.20 The cytotoxic activity of mer-[RuCl 3 (dppb)H 2 O] (dppb =1,4 bis(diphenylphospine)butane) against MCF-7, TK-10, and UACC-62 human tumors was of the same order of magnitude as that of the clinical drugs etoposide and colchicines.21 Therefore, coordination to biphosphine ligands to ruthenium complexes with oxidation state +2 or +3 improved the cytotoxic activity. In this study, we investigated the interaction of mer[RuCl3(dppb)(VPy)] (VPy = 4-vinylpyridine), RuVPy, with human laryngeal carcinoma cells as well as artificial membranes. For the latter, molecular-level information could be obtained with RuVPy in Langmuir monolayers simulating the membrane environment. The interaction between RuVPy and phospholipid monolayers was also explored in terms of change in morphology using Brewster angle microscopy (BAM) and in the spectroscopic properties with polarization modulatedinfrared reflection absorption spectroscopy (PM-IRRAS).

2. MATERIALS AND METHODS 2.1. Materials. All chemicals used were of reagent grade or comparable purity. All solvents were purchased from Merck. mer-[RuCl3(dppb)(VPy)] was synthesized through a wellestablished route for similar complexes.28,29 The ligands 1,4bis(diphenylphosphino)butane (dppb) and 4-vinylpyridine were used as received from Aldrich. The phospholipid with chirality levogero L-dipalmitoylphosphatidylcholine (referred to as DPPC hereafter) and a racemic mixture of the racdipalmitoylphosphatidylglycerol (DPPG hereafter) were acquired from Avanti Polar Lipids and used without further purification. The electronic absorption spectra for compounds solution (0,5 mmol L−1 ) were recorded in a Varian spectrophotometer model Cary 50 Bio using a quartz cell, light path 5 mm. 2.2. Cell Viability Assays. The biological activity was tested using human laryngeal carcinoma HEp-2 cell (ATCC CCL 23) and VERO (African green monkey kidney) control cell lines. The latter were cultured in Iscove’s modified Dulbecco’s medium (IMDM, Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum, 10000 IU mL−1 penicillin and 10 mg mL−1 streptomycin in a humidified environment with 5% CO2 at 37 °C. Cells were maintained at 37 °C in a moist environment as a sub confluent monolayer and were routinely sub cultured twice a week. For the assays, 96-well plates were used, and in each well 1 × 105 cells were incubated for 24 h at 37 °C in 5% CO2. Then, the medium was removed and the cells were incubated for 24 h with different 10654



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soluble in water. Furthermore, the amount of drug molecules interacting with the phospholipid is known, which would not be the case if RuVPy was adsorbed from the subphase. Brewster Angle Microscopy. A BAM 2 Brewster angle microscope from Nanofilm Technology, equipped with a 30 mW laser emitting p-polarized light at 690 nm, allowed imaging the surface with a 2 μm resolution. The reflected light at the Brewster angle (52.16°) was detected by means of a CCD camera and recorded with image processing software to improve contrast. The principles of detection in a BAM microscope are described in ref 35. When a Langmuir film is formed, the refractive index at the air/water interface is slightly changed, yielding reflected light toward the camera. An image of the interfacial film structure is formed by the contrast of the regions with lower molecular concentration of the film (dark regions, without reflection) and spots where the water surface is covered with higher molecular concentration of the film (bright regions, reflection). Polarization Modulation Infrared Reflection Absorption Spectroscopy (PM-IRRAS). PM-IRRAS measurements were taken with a KSV PMI 550 instrument (KSV instrument Ltd., Helsinki, Finland). The experimental setup used was similar to that described by Geraldo and co-workers.36 The Langmuir trough is placed in a way that the light beam reaches the monolayer at a fixed incidence angle of 80°, at which the intensity is maximum with a low level of noise. The incoming light is continuously modulated between s- and p-polarization at a high frequency, which allows for the simultaneous measurement of the spectra for the two polarizations. The difference between the spectra provides surface-specific information, and the sum gives the reference spectrum. The effect from water vapor is reduced with the simultaneous measurements. The same equipment and conditions were used for characterizing Langmuir−Blodgett (LB) films deposited onto Si substrates, obviously with no PBS background. 2.5. Langmuir−Blodgett Deposition. LB films were transferred from Langmuir monolayers at a pressure of 30 mN m−1, which is close to the biomembrane pressure.37 Z-type LB films with 3 layers were deposited onto Si substrates. Transfer ratios close to 1.0 were obtained by varying the dipping speed from 0.5 mm/min (RuVPy monolayer) to 5 mm/min (pure phospholipid and mixed monolayers).

Figure 1. Effect of RuVPy on the survival index of HEp-2. Cells were treated for different RuVPy concentrations for 24 h. Each column represents the mean ± SD for two experiments, each performed in sextuplicate.

being therefore able to interact with target molecules efficiently.38,39 In a cell membrane the complex of RuVPy could be reduced to Ru(II), but for our model membrane we found that this does not occur. This conclusion was based on UV−vis spectra taken for RuVPy mixed with phospholipids, which are shown in Figure S1 of the Supporting Information. The interaction with the cell membranes should also be crucial because metal complexes are known to induce abnormalities such as membrane blebbing40 and mitochondrial damage, which depend on the plasma membranes. Furthermore, the ability of metal complexes to diffuse into the cell is determined by their interaction with the membrane lipids.41 Indeed, on these interactions depends the amount of metal to be administered because the ruthenium complex is lodged in the lipophilic layer.42 The lipophilic character of Ru complexes has been proven important for the cytotoxicity in a comparison between a complex containing a pyrimidine deoxyribo or ribonucleoside ligand and another with a trans[RuCl4(DMSO)2]− Na+ precursor.42 3.2. Interaction with Cell Membrane Models. The high toxicity of RuVPy for both human laryngeal carcinoma cell lines, HEp-2cells, and VERO line (control cells) points to a mode of action that may depend strongly on the interaction with the core of cell membranes, for specific receptors in the membrane would not be required. Because mimicking the whole cell membranes is not feasible when one wishes to obtain precise molecular-level information, here we used phospholipid Langmuir monolayers to represent the membrane. In order to take into account the importance of surface charge, use was made of a zwitterionic (DPPC) and an anionic (DPPG) phospholipids, and the effects from incorporating RuVPy were studied with several methods. We evaluated and systematically compared the monolayer behavior of DPPC and DPPG individually as a function of complex concentration by measuring surface pressure−area (π−A) isotherms. 3.2.1. Surface Pressure Isotherms. The surface pressure isotherm for RuVPy monolayer over PBS buffer subphase is presented in Figure 2. There is no coexistence of liquidexpanded (LE) and liquid-condensed (LC) phases, and the limiting area, taken from extrapolating the curve at the condensed state to zero surface pressure, is ∼55 Å2. The inset brings the compressional modulus, Cs−1, defined as Cs−1 = −A(∂π/∂A)43 at constant temperature. According to the

3. RESULTS AND DISCUSSION 3.1. Toxicity of RuVPy. The cytotoxic activity of RuVPy against human laryngeal carcinoma cell lines, HEp-2, and control cell line VERO, was examined over a period of 24 h of incubation. The MTT colorimetric assay was used to analyzed cell viability and expressed as IC50. Figure 1 shows the Hep-2 survival curves for different concentrations of RuVPy, which was effective against laryngeal cancer cells with an IC50 value of 120 ± 10 μmol L−1. It also exhibited cytotoxic activity against normal VERO cells, with IC50 = 67 ± 7 μmol L−1 indicating that this complex is not specific for tumor cells. In comparison, a much higher dose (306.9 μmol L−1) was necessary for reaching a high cell mortality of human carcinoma cells when mer-[RuCl3(dppb)H2O], a ruthenium complex with similar structure to RuVPy, was used.21 A possible mechanism to explain the cytotoxicity of RuVPy is the one reported for Ru(III) complexes, referred to as prodrugs. Within this mechanism, the anticancer activity is ascribed to the complex reduction to Ru(II) species, which are more reactive and likely to be involved in ligand exchange reactions, 10655



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Figure 2. π−A isotherms for (A) RuVPy/DPPC and (B) RuVPy/DPPG mixed monolayers in different molar ratios (indicated in the inset) spread on PBS buffer at 20 °C. Inset: Compressibility modulus (Cs−1) versus surface pressure for the pure compounds and mixtures.

definition of Davies and Rideal,44 the RuVPy monolayer is mostly in the liquid-condensed phase, which means Cs−1 values are between 100 and 250 mN m−1. The π−A isotherms for pure DPPC and DPPG are consistent with the literature.45 RuVPy was incorporated in DPPC or DPPG monolayers by cospreading, with concentrations ranging from 10 to 90% in mol, which means 100 to 900 μmol L−1. These values were chosen to match those employed in the cytotoxic study. The incorporation of RuVPy drastically affected the DPPC and DPPG monolayers, especially their elasticity (Figure 2). The effect on the plateau region, correspondent to the coexistence of LE−LC phases depends on the RuVPy concentration. For 10% of RuVPy in DPPC monolayers, the plateau (LE to LC) is still observed, but at 7 mN m−1, with the limiting area per molecule being shifted to 62 Å2, higher than for pure DPPC. With RuVPy concentrations from 30 to 70% the plateau was no longer observed. Also worth mentioning is the decrease in Cs−1 for these monolayers, especially at 25−30 mN m−1 (pressure corresponding to the lipid packing in a cell membrane). Cs−1 decreased with increasing concentration of RuVPy, i.e., the film became more fluid. For 90:10% in mol of RuVPy:DPPC, the surface pressure isotherm is similar to that of pure RuVPy, as one should expect, while Cs−1 was higher than for pure RuVPy. The overall behavior departs from the additive rule for a binary mixture,46 which means that some molecular-level interaction takes place between RuVPy and DPPC. The thermodynamic parameters of mixed RuVPy/DPPC monolayers at the air−liquid interface, such as excess area (ΔAE), excess free energy (ΔGE) and free energy of mixing (ΔGM), were calculated for distinct surface pressures (π = 1, 3, 5, 10, 15, 20, 25, and 30 mN m−1) to better explore miscibility (Figure S2), using eqs 1−4 ΔAE = Aexp − A12 = Aexp − (X1A1 + X 2A 2 )

ΔG E =

∫0

π

Aexp − A12 dπ =

∫0

gas universal constant, and T is the absolute temperature.46−48 Figure S2a−e in the Supporting Information shows the effects from incorporation of RuVPy. For low π (1−15 mN m−1), ΔGE is positive suggesting predominant repulsive interactions,49 except for the 90:10 RuVPy:DPPC ratio, for which ΔGE was always negative. At higher surface pressures, ΔGE becomes negative and therefore attractive interactions between molecules of the distinct components predominate. In fact, ΔGE was increasingly negative as the surface pressure increased, indicating that the degree of molecular interaction was enhanced by rearranging the molecules at the interface.46 This same behavior was observed in the thermodynamic study of mixed monolayers with DPPC with dioleoyltrimethylammonium propane (DoTAP) at the air−water interface, pointing to associative interactions especially at higher mole fractions of DoTAP.49 The free energy of mixing, ΔGM, was negative under all conditions studied for mixed RuVPy/DPPC monolayers, as shown in Figure S2e in the Supporting Information. Therefore, the mixed films were more stable than the monolayers of neat compounds, and the maximum stability was obtained for 50 and 70% of RuVPy at π = 30 mN m−1, for which ΔGM = −3.49 and −3.61 kJ mol−1, respectively. These observations are consistent with data in the literature. In the mixed system formed by DPPC and a terpenoid molecule, trans-dehydrocrotonin (t-DCTN), ΔGE was positive and ΔGM was negative. Therefore, the insertion of t-DCTN into the phospholipid monolayer resulted in stable films. Since the same behavior was observed for RuVPy, we may infer that a stable complex of saturated phospholipids and RuVPy is formed at the air/water interface.50 The surface pressure isotherm for pure DPPG in Figure 2B is in agreement with those reported in the literature for a PBS subphase,45 with a plateau at 8 mN m−1. The limiting area was ∼55 Å2/molecule and the collapse pressure was ∼70 mN m−1. The isotherm was considerably affected by addition of RuVPy, as depicted in Figure S3a,b in the Supporting Information. Upon adding RuVPy the LE-LC coexistence phase was extinguished and, for π values higher than 27 mN m−1, the area per molecule was smaller than for pure DPPG. According to the Cs−1 values, the mixed monolayers are less compressible than their components separately. Figure S3d shows positive ΔGE for π between 5 and 25 mN m−1, except for 90% of RuVPy, thus indicating repulsive interactions between the molecules. At π = 30 mN m−1, the excess free energy was negative, probably because the degree of molecular interaction was enhanced by packing the molecules and this led to

(1)

π

dπ

Aexp − (X1A1 + X 2A 2 ) (2)

ΔG M = ΔG E + ΔG ID

(3)

ΔG ID = RT (X1 ln X1 + X 2 ln X 2)

(4)

where A1 and A2 denote the mean molecular area (mma) of each single (pure) system of species 1 and 2, respectively, A12 is the observed mma for their mixture, and the molar fractions of the compounds 1 and 2 are represented as X1 and X2; R is the 10656



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Figure 3. (A) Structure and (B) electrostatic potential map of RuVPy in space (isovalue 0.05). Headline area between the H67, H55, and C75 atoms, which corresponds to the extrapolated area (43 Å2) for the condensed monolayer of neat RuVPy. The electrostatic potential is represented with a color scale from red (−0.18 au) to blue (0.17 au).

attractive interactions.48 Similarly to the mixed films with DPPC, the free energy of mixing ΔGM was negative, except for 10% RuVPy, and therefore the RuVPy/DPPG films are more stable than the corresponding monolayers of the neat compounds. The maximum stability was measured for 90% of RuVPy at π = 30 mN m−1, with ΔGM = −3.01 mol−1 kJ (Figure S3e. The thermodynamic parameter of ΔGM shows that the stability of RuVPy/phospholipid monolayers depends upon the type of polar groups in the phospholipid, and ΔGM was found to be more negative for the binary system with DPPC than for DPPG. These observations are consistent with the literature, being related to the charge of DPPG polar headgroup, which, even at high concentration of RuVPy, appear to affect the miscibility of the binary system.51 Figure 3 shows the result of DFT calculations for RuVPy in vacuum, which was used to infer the conformation of RuVPy at the air−water interface, assuming this conformation to be related to the minimum area per molecule in the π−A isotherms. The extrapolated area for the condensed monolayer of neat RuVPy was ca. 43 Å2, corresponding to the projected area onto the surface established by the atoms H67, H55 and C75. From this conformation and from the electrostatic potential map for a neutral molecule of RuVPy in Figure 3B obtained with DFT calculations, we could determine the groups in contact with the aqueous surface for a condensed monolayer. 3.2.2. Morphological Analysis. Brewster angle microscopy provided morphological information about the effect of RuVPy over phospholipid monolayers. Figure 4A for RuVPy shows no domain formation. At 0 mN m−1, the monolayer is not uniform, while upon compression, condensed regions coallesce resulting in a very bright image. At 55 mN m−1 grooves appear, denoting the collapse of the monolayer, consistent with the surface pressure isotherm. Similar images were reported for bimetallic ruthenium tris-bipyridine complexes [Ru(bpy)3Ph4Ru(bpy)L2](PF6)4 (bpy = 2,2′-bipyridine, Ph = phenyl, L= 4,4′-dinon-1-

enyl-2,2′-bipyridine).52 For pure DPPC and DPPG monolayers, the images are consistent with the literature,53,54 and typical domains for DPPC and DPPG monolayers over PBS buffer are observed. The incorporation of RuVPy affects the morphology of a DPPC monolayer, as indicated in Figure 5A. Domains started to appear later in RuVPy/DPPC monolayer, with RuVPy (10%), at 7 mN m−1. However, differences in the shape of the domains are more evident, since at similar surface pressures domains are smaller, with less defined chirality. For the mixed RuVPy/DPPG monolayer in Figure 5B, the domains observed were also smaller. Hence, RuVPy prevents formation of large domains of both DPPC and DPPG, probably owing to the repulsive interactions between RuVPy and the phospholipids, as inferred from the positive ΔGE in the thermodynamic analysis (except for the film containing 90% of RuVPy). 3.2.3. Spectroscopic Data. Figure 6 shows the PM-IRRAS spectra for monolayers at 30 mN/m, which corresponds to the pressure of a cell membrane,37 of neat RuVPy, DPPC, DPPG, and the mixtures containing 10% of RuVPy (RuVPy/DPPC or RuVPy/DPPG). A broad band at ca. 1675 cm−1, assigned to the deformation of the vibrational mode of the liquid water at the interface55 or restructuring of water molecules56 was observed in all films. This band was upward for neat DPPC and mixed RuVPy/DPPG monolayers, but downward for neat RuVPy, neat DPPG, and mixed RuVPy/DPPC monolayers. For RuVPy, bands appeared at 1190 and 1275 cm−1 assigned to the P− C6H5 stretching and CH2 scissors deformation of the dppb ligand, respectively.57 Two strong bands appeared at ca. 2920 and 2850 cm−1, assigned to the asymmetric and symmetric C− H stretches for CH2, respectively, coming from dppb and VPy ligands57 and from the phospholipids DPPC or DPPG.58 The ratio between the peak intensity of the symmetric C−H stretches (Is, ca. 2850 cm−1) and the asymmetric C−H stretches (Ias, ca. 2920 cm−1) increases when the order in the monolayer 10657



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Figure 5. π−A isotherms and BAM images for monolayers obtained with the mixture of (A) RuVPy/DPPC and (B) RuVPy/DPPG, with RuVPy (10%), spread on PBS buffer at 20 °C.

interaction between RuVPy and the phospholipids were noted in the spectra of parts A and B of Figure 6, which show bands at ca. 1505 and ca. 1530 cm−1, assigned to C−N vibration and CC stretching of the pyridine ligand, respectively.57 Since these bands were absent in the RuVPy monolayer, their presence points to interaction between RuVPy and DPPC or DPPG.56,60 The band due to CO stretching in neat DPPC appears at 1749 cm−1, being shifted to 1763 cm−1 for the mixed RuVPy/DPPC monolayer. RuVPy also affected the CO stretching band from DPPG, but the shift was opposite to the case of DPPC, from 1759 cm−1 for neat DPPG to 1738 cm−1 in the mixed RuVPy/DPPG monolayer. In summary, RuVPy induced strong changes in the headgroup region of both DPPC and DPPG monolayers, particularly in the PO group owing to change in hydration and H-bonding with water molecules.36 There was practically no penetration of RuVPy into the hydrophobic tails of the phospholipids, even though the overall chain ordering decreased. Scheme 1 depicts this interaction, where the geometric arrangement was based on previous work with reflectivity measurements on DPPC monolayers.61 The assumed length for the RuVPy molecule close to 10 Å corresponds to the arrangement of H67, H55, and C75 atoms at the air/water interface. Particularly important was the interaction with the negatively charged PO group of the phospholipids, which should be expected from the computational simulations using DFT, for the electrostatic potential map in Figure 3B indicates positive potential sites in the pyridine group of RuVPy. In order to confirm the adsorption of the complex by polar heads of DPPC or DPPG molecules, LB films were transferred from Langmuir monolayers at a pressure of 30 mN m−1. Similar PM-IRRAS results were obtained for the LB films of neat phospholipids, RuVPy and mixtures (Figure S4 a-d) in the Supporting Information, which confirm the

Figure 4. π−A isotherms and BAM images for monolayers of (A) RuVPy, (B) DPPC, and (C) DPPG spread on PBS buffer at 20 °C.

chains is decreased.59 Therefore, the incorporation of RuVPy induced disorder since Is/Ias increased, probably because RuVPy molecules affected the packing of DPPC and DPPG molecules, consistent with more fluid monolayers as inferred from the compressional modulus data. However, penetration of RuVPy into the hydrophobic tail region must be limited since the signal assigned to δ(CH2) vibrations for DPPC (1461 cm−1) and DPPG (1466 cm−1) was not strongly affected by RuVPy, at 1461 cm−1 for RuVPy/DPPC and RuVPy/DPPG. The strongest effects from incorporation of RuVPy occurred for the headgroups of the phospholipids, particularly with the PO2− group. For neat DPPC monolayers the bands assigned to the asymmetric stretching (νas-PO2−) are observed at 1275 and 1240 cm−1, while the bands assigned to the symmetric stretching (νs-PO2−) appear at 1083 and 1030 cm−1. For the RuVPy/DPPC mixed monolayer those bands were shifted to 1250 and 1222 cm−1 for νas-PO2− and are completely vanished in the case of νs-PO2−. For DPPG the bands for νas-PO2− appeared at 1250 and 1216 cm−1, but vanished in the mixed RuVPy/DPPG monolayer. The bands due to νs-PO2− were observed at 1100 and 1042 cm−1, and were shifted to 1093 and 1051 cm−1 for the mixed film. Further changes induced by 10658



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Figure 6. PM-IRRAS spectra for monolayers at 30 mN m−1 of the pure compounds and mixed RuVPy (10%) with DPPC (A) and DPPG (B). Baseline is the line of the zero level of the PM-IRRAS signal.

on the interaction with the cell membrane. We tested the latter hypothesisin a very simplified wayby using Langmuir monolayers as model membranes. The strong interaction was confirmed by the significant changes in the packing and morphology of both zwitterionic DPPC and anionic DPPG monolayers. Furthermore, using PM-IRRAS we were able to identify the interacting groups in the phospholipids and in RuVPy, which was consistent with the electronic properties obtained with DFT for RuVPy. Even though RuVPy is not able to penetrate into the hydrophobic tail regions of the phospholipid monolayers, the elastic properties of the latter were changed considerably. If real membranes become more fluid upon interacting with RuVPy as the Langmuir monolayers did, this is the likely mechanism for cell death, though we cannot discard the importance of other cell components which were not tested in our study.

Scheme 1. Idealized Organization of RuVPy and DPPC Molecules at the Air/PBS Buffer Interfacea

a

The configuration and length of RuVPy inserted in the DPPC monolayer were obtained from the DFT calculations. The portion of the DPPC molecule immersed into the aqueous subphase was taken from reflectivity measurements by G. Fragneto et al.61.

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ASSOCIATED CONTENT

S Supporting Information *

Spectroscopic data in the UV−vis region for RuVPy complex and phospholipids/RuVPy mixtures, area per molecule plotted against the relative concentration of RuVPy at various pressures, and excess areas (ΔAE), excess free energies (ΔGE), and free energy of mixing (ΔGM) for the mixtures, along with the PM-IRRAS spectrum for LB films deposited on a Si substrate. This material is available free of charge via the Internet at http://pubs.acs.org.

existence of molecular-level interactions between RuVPy and the phospholipids.

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CONCLUSIONS Toxicity assays have indicated that the RuVPy complex is highly toxic for both cancer and control cells, thus indicating a lack of specificity that could be related to a mode of action depending 10659



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AUTHOR INFORMATION

Corresponding Authors

*Telephone: + 55 42 32203731. Fax: + 55 16 33739825. Email: [email protected] (B.S.). *E-mail: [email protected] (K.W.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by FAPESP, CNPq, CAPES, and nBioNet (Brazil). REFERENCES

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