The interaction of mefloquine hydrochloride with cell membrane models at the air–water interface is modulated by the monolayer lipid composition

Journal of Colloid and Interface Science 431 (2014) 24–30

Contents lists available at ScienceDirect

Journal of Colloid and Interface Science www.elsevier.com/locate/jcis

The interaction of mefloquine hydrochloride with cell membrane models at the air–water interface is modulated by the monolayer lipid composition Thiago Eichi Goto, Luciano Caseli ⇑ Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of Sao Paulo, Diadema, SP, Brazil

a r t i c l e

i n f o

Article history: Received 23 April 2014 Accepted 23 May 2014 Available online 14 June 2014 Keywords: Mefloquine hydrochloride Malaria Langmuir monolayers DPPC DPPG DODAB Stearic acid PM-IRRAS Antiparasitic drug

a b s t r a c t The antiparasitic properties of antiparasitic drugs are believed to be associated with their interactions with the protozoan membrane, encouraging research on the identification of membrane sites capable of drug binding. In this study, we investigated the interaction of mefloquine hydrochloride, known to be effective against malaria, with cell membrane models represented by Langmuir monolayers of selected lipids. It is shown that even small amounts of the drug affect the surface pressure–area isotherms as well as surface vibrational spectra of some lipid monolayers, which points to a significant interaction. The effects on the latter depend on the electrical charge of the monolayer-forming molecules, with the drug activity being particularly distinctive for negatively charged lipids. Therefore, the lipid composition of the monolayer modulates the interaction with the lipophilic drug, which may have important implications in understanding how the drug acts on specific sites of the protozoan membrane. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Mefloquine hydrochloride (MFH), or 2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol monohydrochloride, is a synthetic compound used to prevent and treat certain forms of malaria [1,2]. Its molecular formula is C17H17ClF6N2O, being an analogue of quinine. MFH presents a positive charge at low pHs, but it is electrically neutral at physiological pHs. Moreover, it presents polarity due to the presence of fluorine, amine and hydroxyl groups. Studying alternative drugs for malaria treatment has received growing interest because malaria remains one of the most important infectious diseases in the world. The administration of a combination of drugs has been applied to kill the parasite that causes the disease. Many of these drugs currently employed are toxic, and for this reason, the search for alternative drugs is of particular interest. MFH has been reported as one of these alternative drugs with low toxicity [3,4], serving also for the treatment of diseases such as those caused by schistosomes [5]. Because of the drug’s microbicidal activity against protozoa, a possible molecular process that involves interaction with the membrane of the protozoa ⇑ Corresponding author. E-mail address: [email protected] (L. Caseli). http://dx.doi.org/10.1016/j.jcis.2014.05.050 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.

[6–10] has been reported, but the molecular mechanism thereof has not been fully elucidated. Thus, the use of cell membrane models able to reveal the mechanism of this drug at the molecular level can be useful. In this sense, the use of Langmuir monolayers of lipids may be an appropriate approach because these monolayers are able to mimic half of a membrane [11], and intrinsic molecular interactions between drugs and lipid surfaces can be accessed. Lipid Langmuir monolayers have been used to investigate the interaction of membranes with proteins [12], enzymes [13], polysaccharides [14], nonbiological polymers [15], metal nanoparticles [16] and drugs [17]. Furthermore, this technique allows for the control of several properties of the lipid monolayer with ease, such as surface packing, surface pressure and composition. Herein, we report on the interaction of the drug MFH with cell membrane models using the Langmuir technique. Tensiometry and Polarization-Modulation Infrared Reflection–Absorption Spectroscopy (PM-IRRAS) were employed to study monolayers formed with selected lipids. 2. Experimental The lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), stearic acid (HSt), 1,2-dipalmitoyl-sn-glycero-3-phospho-(10 -rac-

T.E. Goto, L. Caseli / Journal of Colloid and Interface Science 431 (2014) 24–30

glycerol) sodium salt (DPPG), dioctadecyldimethylammonium bromide (DODAB), and stearic acid (HSt) were purchased from Sigma–Aldrich (St. Louis, MO, USA) and dissolved in chloroform (Synth, Diadema, Brazil) to a concentration of 0.5 mg/mL. The monolayer subphase approximated physiological conditions and consisted of a 50 mM phosphate buffer and 150 mM NaCl at a pH of 7.4. The water employed was purified using a MilliQ-Plus system (resistivity 18.2 MX cm, pH 5.5). MFH was purchased from Sigma–Aldrich and was dissolved in chloroform to a concentration of 0.2 mg/mL. All the chemical structures of the materials employed in this work are show in Fig. 1. The Langmuir monolayers were then obtained by spreading a chloroform solution of one of the lipids on the surface of the aqueous buffer solution. For preliminary tests, MFH solutions were also spread alone at the air–water interface to test the surface activity of this compound. Surface pressure–area (p–A) isotherms were obtained in a mini-KSV Langmuir trough equipped with a surface pressure sensor (the Wilhelmy method), with an interface compression rate of 5 Å2 molecule1 min1. This value of compression rate was chosen because is low enough to ensure the reproducibility of the isotherms. For mixed MFH-lipid monolayers, lipids and drug were previously mixed in chloroform to obtain the proportion desired. After allowing the chloroform to evaporate for 20 min (the absence of this solvent was confirmed by vibrational spectroscopy), the monolayer was compressed until it collapsed. For PM-IRRAS studies, the monolayer was compressed until the desired surface pressure was achieved. The surface pressure was maintained at the desired value by moving the barriers, and the stabilization of the monolayer was monitored until no significant movement of the barriers was required. PM-IRRAS measurements were performed using a KSV PMI 550 instrument (KSV Instruments, Ltd., Helsinki, Finland) at a fixed incidence angle of 80°. Surface pressure–area isotherms were obtained to evaluate the manner in which the drug shifts the monolayer to large areas and to investigate the mixed monolayer in the 2-D states achievable by the monolayer from the expanded phase to collapse. Thus, the monolayer was expanded to the maximum area allowed by the Langmuir trough and then compressed until it collapsed. All experiments were carried out at a controlled room temperature (20 °C). Each surface pressure–area isotherm or spectrum was obtained at least three times to ensure the reproducibility of the experiments. The isotherms and spectra were highly reproducible and no significant variations from the first data obtained were observed.

25

3. Results e discussion First, it is important to note that when the drug was spread alone at the air–water interface, no considerable increase in surface pressure was observed, which means that no detectable surface pressure could be measured. Even after the compression of the interface that contained the drug, the surface pressure did not increase, which indicates the absence of the surface activity of MFH, which is probably caused by its low spreading coefficient. With the presence of the lipid, however, all surface pressure–area isotherms reported here showed some effect caused by the presence of the drug. This result indicates that the surface activity of MFH was induced by the presence of lipids at the air–water interface. Fig. 2 shows the surface pressure–area isotherms obtained for DPPC and the effect of the drug at different concentrations. This lipid was chosen primarily because it is considered a model lipid for mimicking cell membranes using Langmuir monolayers [18]. We observed an expansion of the monolayer because the isotherms were progressively shifted to larger molecular areas for DPPC. It is important to emphasize that the molecular area displayed in the isotherms refer always to the lipid because MFH, although insoluble in water, does not present surface activity when alone, and does not spread uniformly at the air–water interface. A drug concentration of 5% represents the limit for expansion because at higher relative amounts of MHF the curve is not shifted to areas larger than that for a 5% concentration of the drug. This result is indicative that an excess of the incorporating compound aggregates at the interface and cannot expand the monolayer. For example, referring to the shift in the isotherm at 30 mN/m, which is the surface pressure corresponding to the lateral pressure of a natural membrane [19], for pure DPPC, the molecular area is 44.0 Å2, and for the mixed monolayer, this area increases to 53.4 Å2. This increase is more than 20% higher than that expected considering (i) the molar proportion of MFH, (ii) the relative molecular size of MFH and (iii) the fact that all drug molecules penetrate the lipid monolayer. This higher area is probably caused by electrostatic repulsions between the polar groups of the drug and the lipids, which may expand the monolayer. Such electrostatic repulsions may be related to the choline group from DPPC, which contains a quaternary ammonium group, positively charged, and a phosphate group, negatively charged. These groups may interact with some polar groups of MFH such as hydroxyl, fluorine and amines, through ion–dipole interactions.

Fig. 1. Structures of MFH (A), DPPC (B), DODAB (C), DPPG (D), and HSt (E).

26

T.E. Goto, L. Caseli / Journal of Colloid and Interface Science 431 (2014) 24–30

Surface Pressure (mN/m)

80

A

70

DPPC DPPC + 1% MFH DPPC + 2% MFH DPPC + 5% MFH

60 50 40 30 20 10 0 20

40

60

80

100

Mean Molecular Area (Å2) DPPC DPPC + 1% MFH DPPC + 2% MFH DPPC + 5% MFH

B E (mN/m)

400

200

of impurities at the interface. The degenerated transition for DPPC– MFH mixed monolayer is therefore related to intrinsic interactions of the drug with the lipid at the interface. Another effect clearly observed is the decrease in the surface elasticity (E). This property is related to the degree of packing of the monolayer and is defined as the inverse of the surface compressibility of a film: E = A(op/oA)T, where p is the surface pressure, A is the molecular area and T is temperature. Fig. 2B shows that for a given surface pressure, E decreases as long as MFH is incorporated. For example, at a surface pressure of 30 mN/m, E decreases from 327 mN/m for pure DPPC to 163 mN/m for DPPC– MFH (5%). This decrease indicates that the drug alters the ability of the lipid to achieve higher states of surface packing, which is related to the difficulty of the lipid molecules to arrange in an ordered state of packing when a second component is present. Fig. 3 shows the PM-IRRAS spectra for DPPC without or with MFH incorporated. For the region related to C–H stretching vibrations (Panel A), two main bands appear: one centered at 2918 cm1, ascribed to asymmetric stretching for CH2, and the other centered at 2848 cm1, assigned to symmetric stretching. With MFH incorporation, the positions of the bands do not change noticeably; however, the enhancement of a shoulder at approximately 2870 cm1 is clearly observed, indicating the CAH stretching of CH3 groups. This shoulder is more distinct for a 2% concentration of MFH. The ratio of symmetric to asymmetric peak intensities also varies considerably: the ratio increases from 0.56 for pure DPPC to 0.70, 0.77 and 0.87 for DPPC + MFH (1%, 3% and 5%, respectively). This result may be related to the incorporation of the drug into the alkyl chains

0 0

20

40

Surface Pressure (mN/m) PM-IRRAS signal (arb.units)

In addition, the phase transition between the liquid-expanded and liquid-condensed states changes considerably. For pure DPPC, this transition is denoted by a plateau between the molecular areas of approximately 70 and 50 Å2, with the surface pressure remaining constant at approximately 5 mN/m. This result is in agreement with results reported in the literature for DPPC [20,21]. With increasing amounts of drug, this plateau not only appears to shift to higher values of surface pressure, but also becomes less defined. Increasing values of surface pressure for the plateau region is an indication that to achieve higher condensed states, more energy is needed to pack the molecules at the air–water interface, whereas the poor definition of the plateau indicates that there is no longer a first-order phase transition, which is thermodynamically expected for non-ideal mixtures. Then a degenerated phase transition between the liquid-expanded (LE) and liquid-condensed (LC) states of DPPC in the presence of MFH appears resulting that at higher MFH concentrations the LE to LC phase transition disappears. Thus, no plateau region is observed at 5% MFH in the mixture (Fig. 2A). The presence of a degenerated phase transition between LE and LC phases could indicate the presence of impurities at the interface or an artefact related to the monolayer compression rate. For that, both cases were checked. Lower compression rates were essayed and no change in the isotherms was observed. Also, the presence of impurities was evaluated for aqueous subphases, DPPC and MFH solutions. Since (i) no variation of surface tension for a bare interface even with compression was observed; (ii) isotherms for pure DPPC did not presented degenerated transitions; and (iii) MFH did not caused any increase in surface tension with compression, it is highly probable that there is no problem with the presence

2848

A

DPPC + 5% MFH

DPPC + 2% MFH DPPC + 1% MFH DPPC

3000

2950

2900

wavenumber

B

1523

2850

2800

(cm-1)

1363

1234

PM-IRRAS signal (arb.units)

Fig. 2. (A) Surface pressure–area and (B) surface elasticity–surface pressure isotherms for DPPC and DPPC–MFH (relative amounts in mol are shown in the inset).

2918

1084 DPPC + 5% MFH DPPC + 2% MFH DPPC + 1% MFH DPPC

1800

1600

1400

1200

1000

wavenumber (cm-1) Fig. 3. PM-IRRAS spectra for DPPC and DPPC–MFH at 30 mN/m (relative amounts in mol are shown in the inset).

T.E. Goto, L. Caseli / Journal of Colloid and Interface Science 431 (2014) 24–30

of the phospholipids, which changes the degree of packing and therefore alters the vibrational moment. Panel B shows the region where the absorption of the vibration of hydrophilic groups of DPPC appears. The negative band at 1680 cm1 is related to the in-plane bending of OH bonds in surface water molecules in a close-packing state [22]; the band at 1523 cm1 is related to the in-plane bending of OH bonds in water without hydrogen bonding. These bands are a result of the difference in reflectivity of the water interface covered with the monolayer and that not covered with the monolayer. So, although information on water vibration from vapor and aqueous subphase is removed in the PM-IRRAS spectra, the orientation of water molecules at the air–water interface could change upon monolayer spreading. That is the reason related to fact that in many PM-IRRAS spectra, a band centered at about 1680 cm1appears. The band at 1234 cm1 is attributed to the stretching of phosphate groups; and the band centered at 1084 cm1 is due to the stretching of CAOH bonds. Upon MFH incorporation, there were slight changes in these bands, except for the band centered at 1084 cm1, which became broader. Also, ester vibrations for DPPC appear in 1730–1750 cm1. With the incorporation of MFH, the band centered at 1363 cm1 appeared in the spectra. According to reports in the literature regarding the assignment of vibrational bands for this compound, this band may be assigned to C@C stretching in the quinoline ring [23]. This result is an indication that MFH was incorporated into the DPPC monolayer. To test the effect of MFH in lipids with negative net charges, monolayers were formed with DPPG. The surface pressure–area isotherm (Fig. 4) shows a typical curve [24], with an onset in areas close to 50 Å2, at which the monolayer goes from exhibiting

Surface Pressure (mN/m)

70

A DPPG DPPG + 1% MFH DPPG + 2% MFH DPPG + 5% MFH

60 50 40 30 20 10 0 20

40

60

80

Mean Molecular Area

120

(Å2)

DPPG DPPG + 1% MFH DPPG + 2% MFH DPPG + 5% MFH

B 400

E (mN/m)

100

300 200 100 0 0

10

20

30

40

50

Surface Pressure (mN/m) Fig. 4. (A) Surface pressure–area and (B) surface elasticity–surface pressure Isotherms for DPPG and DPPG–MFH (relative amounts in mol are shown in the inset).

27

negligible surface pressure values to a state of higher surface elasticity. With MFH, the profile of the isotherms changes considerably, showing a compressible state at higher areas. For instance, at a surface pressure of 30 mN/m, the surface elasticity is 213 mN/m for pure DPPG monolayers and decreases to values close to 80 mN/m for mixed MFH–DPPG monolayers. Furthermore, a small shoulder at surface pressure values of approximately 35 mN/m can be observed, which resembles a plateau in the phase transitions. This shoulder becomes more evident with higher relative amounts of MFH. Another effect observed is that the collapse of the monolayer occurred at lower surface pressure values when the drug was incorporated. For pure DPPC, the monolayer collapsed at 67 mN/ m, whereas for the MFH–DPPC mixed monolayer, the collapse occurred at 45 mN/m, which is related to the lower resistance of the monolayer to compression. Moreover, the shift to higher areas appeared to be more pronounced when the drug was incorporated into DPPG monolayers. For DPPC monolayers, the maximum area shift in the isotherm at 30 mN/m was 25%, whereas for DPPG monolayers, the maximum shift at the same surface pressure was 60%. Overall, these results are indicative of a more prominent effect of the drug on this negatively charged lipid. Also, from the surface pressure–area and elasticity-pressure isotherms (Fig. 4A and B, respectively), it seems that DPPG monolayer present a solid state, but mixtures of DPPG–MFH monolayers show a LE phase up to the monolayer collapse. Equilibrium surface pressure data for pure DPPG is PPG–MFH mixtures would give some insight of isotherms. As equilibrium surface pressure (value at which the monolayer or bilayer is in equilibrium with the bulk phase) for this phospholipid is lower than the values obtained with this isotherm [25], this phospholipid start to spread when chain melting occurs (transition from solid-liked to liquid-liked phase). This low values of equilibrium surface pressure is attributed to low van der Waals interactions between the hydrocarbon chains, which allow the crystals to spread. Also, if the lipid head is hydrated enough it can be spread uniformly at the interface, allowing for compression to higher states of packing reaching metastates close to the monolayer collapse. Also, as the monolayer relaxes faster in two dimensions than into the third dimension, the compression can be carried out to values of surface pressure above the equilibrium spreading pressure. For the mixed monolayer, however, the accommodation of the lipids is hampered by the presence of the drug, leading the monolayer to a state of more fluidity, as demonstrated in Fig. 4B. Fig. 5 shows the PM-IRRAS spectra for MFH–DPPG monolayers. The bands for symmetric and asymmetric CH stretching vibrations of CH2 appear centered at 2850 and 2915 cm1, respectively, for a pure DPPG monolayer. With MFH, the spectra change significantly. The asymmetric band has its maximum shifted to higher wavenumbers; the symmetric band becomes less evident; and new bands, probably related to CH3 vibrations, become more evident, such as the band centered at 2955 cm1. These results suggest a higher degree of disorganization caused by MFH in this negatively charged lipid membrane. Panel B in Fig. 5 shows a band centered at 1753 cm1, ascribed to carbonyl stretching vibrations of DPPG. The bands centered at 1578 and 1525 cm1 can be attributed water bends, and the one at 1469 cm1 may be ascribed to CAH bending. These bands appear to have been practically unaltered with MFH incorporation. Phosphate stretching mode appeared in a discrete band at 1156 cm1. The band centered at 1356 cm1 (for 5% MFH, and for other drug quantities, at wavenumbers close to this value) is related to C@C bonds in the quinoline ring and is a marker of drug incorporation. Although the negative charge of this lipid may play an important role in determining the effects of this drug on the physicochemical properties of the monolayer, this drug is also

T.E. Goto, L. Caseli / Journal of Colloid and Interface Science 431 (2014) 24–30 2915

A

2850

PM-IRRAS signal (arb.units)

2955

DPPG + 5% Mef DPPG + 2% Mef DPPG + 1% Mef

DPPG

3000

2950

2900

2850

2800

wavenumber (cm-1)

PM-IRRAS signal (arb.units)

B DPPG + 5% MFH DPPG + 2% MFH DPPG + 1% MFH DPPG

1800

1600

1400

1200

40 mN/m at a surface pressure of 30 mN/m. This value does not vary significantly with the introduction of the drug. Fig. 7 shows the PM-IRRAS spectra for DODAB. In the alkyl stretching mode region, the main bands centered at 2918 and 2848 cm1, attributed to asymmetric and asymmetric stretching vibrations for CAH in CH2 groups, respectively, do not vary to a great extent with MFH incorporation. Moreover, the main bands in the region 1800–1000 cm1 do not change considerably when MFH is incorporated into the DODAB monolayer. This finding also indicates that the drug has negligible surface activity in the presence of DODAB, which suggests a low molecular affinity for this positively charged lipid. The negligible surface activity of MFH in the presence of DODAB could also suggest that MFH is not spread at the air–water interface from the DODAB–MFH mixture. In order to check if the insolubility of DODAB–MFH mixed monolayer causes lose of MFH molecules from the interface due to their dissolution into the aqueous subphase, compression-cycles cycles were carried out and any hysteresis was observed. Consequently, a possible solubilization of the mixed monolayer should be discarded. To ensure that the main effects of this drug on the lipid monolayers were electrostatic in nature, with respect to the negatively charged polar heads in particular, surface pressure–area isotherms with a negatively charged fatty acid were obtained (Fig. 8). The isotherm for HSt presents a typical profile [26] with a liquidexpanded phase at 31 and 22 Å2/molecule and a liquid-condensed state that starts at surface pressures as low as 27 mN/m until collapse is attained at approximately 50 mN/m. The literature reports that the most frequent structure of stearic acid monolayer is

1000

2918

wavenumber (cm-1) Fig. 5. PM-IRRAS spectra for DPPG and DPPG–MFH at 30 mN/m (relative amounts in mol are shown in the inset).

observed to have a significant effect on the hydrophobic groups of the lipid. Fig. 6 shows the effect of MFH on a positively charged lipid: DODAB. Even with increasing amounts of MFH, the isotherms do not vary considerably. This finding suggests that little effect is observed when the drug is incorporated into the monolayer formed by this charged lipid. In addition, DODAB presents a compressible monolayer with a surface elasticity value of approximately

PM-IRRAS signal (arb.units)

28

2848

A

DODAB + 5% MFH

DODAB + 2% MFH DODAB + 1% MFH DODAB

3000

2950

2900

2850

2800

wavenumber (cm-1) 1523

DODAB DODAB + 1% MFH DODAB + 2% MFH DODAB + 5% MFH

40

PM-IRRAS signal (arb.units)

Surface Pressure (mN/m)

50

30 20 10 0

30

60

90

120

150

180

210

Mean Molecular Area (Å2) Fig. 6. Surface pressure–area isotherms for DODAB and DODAB–MFH (relative amounts in mol are shown in the inset).

B DODAB + 5% MFH DODAB + 2% MFH

DODAB + 1% MFH DODAB

1800

1600

1400

1200

1000

wavenumber (cm-1) Fig. 7. PM-IRRAS spectra for DODAB and DODAB–MFH at 30 mN/m (relative amounts in mol are shown in the inset).

29

T.E. Goto, L. Caseli / Journal of Colloid and Interface Science 431 (2014) 24–30

Surface Pressure (mN/m)

2916

HSt HSt + 1% Mef HSt + 2% Mef HSt + 5% Mef

40 30 20 10 0 10

15

20

25

30

35

40

45

50

55

60

PM-IRRAS signal (arb.units)

A

50

Mean Molecular Area (Å2)

2847

A

HSt + 5% MFH HSt + 2% MFH HSt + 1% MFH

HSt

3000

2950

2900

2850

2800

wavenumber (cm-1) HSt HSt + 1% Mef HSt + 2% Mef HSt + 5% Mef

B

E (mN/m)

400

200

0 0

10

20

30

40

50

Surface Pressure (mN/m) Fig. 8. (A) Surface pressure–area and (B) surface elasticity–surface pressure isotherms for HSt and HSt–MFH (relative amounts in mol are shown in the inset).

PM-IRRAS signal (arb.units)

B

1466

1378

1727

HSt + 5% MFH

HSt + 2% MFH HSt + 1% MFH HSt

1800

1600

1400

wavenumber

1200

1000

(cm-1)

Fig. 9. PM-IRRAS spectra for HSt and HSt-mefloquine at 30 mN/m (relative amounts in mol are shown in the inset).

liquid-condensed, one which becomes solid at higher surfaces pressures [26]. These structures are observed in Fig. 8A and conformed by data of elasticity (Fig. 8B). When the drug is incorporated, the surface pressure values increase substantially at higher molecular areas (in the range of 32 and 42 Å2/molecule) when compared with the isotherm for pure HSt monolayer, for which the surface pressure values are close to zero. For the pure lipid monolayer, a phase transition occurs at 27 mN/m, whereas for the MFH–HSt mixed monolayers, this transition occurs at higher values of surface pressure (28–43 mN/m). The surface pressure increases with increasing concentrations of MFH, and from a single inflection point for pure HSt, this transition features a shoulder when 5% (in mol) of the drug is incorporated into the monolayer. The collapse of the mixed monolayers, however, occurs at surface pressure values as high as those observed for the pure HSt monolayers. Overall, we can conclude that with MFH, the isotherm changes significantly, corroborating the impression that the major effects of this drug are exerted on negatively charged lipids. Surface elasticity values also appear to vary with the drug concentration; for pure HSt, these values are approximately 200 mN/m at a surface pressure of 30 mN/m (Panel B for Fig. 8), and with 5% MFH, this value decreases to approximately 75 mN/m. Fig. 9 shows the PM-IRRAS spectra for HSt. The bands centered at 2916 and 2847 cm1 correspond, respectively, to asymmetric and symmetric stretching vibrations for CH2. The shape of the spectra does not vary significantly with drug incorporation. However, it can be observed that the ratio between the maximum intensities of the peaks for the symmetric and asymmetric stretching vibrations varies to some extent: it increases from 0.56 for pure HSt to 0.74, 0.82 and 0.88 for HSt mixed with MFH (1%, 2% and 5%,

respectively). This result indicates that the monolayer changed its state of packing upon drug adsorption. The PM-IRRAS spectra in Panel B show a band centered at 1727 cm1 indicating the C@O stretching mode of the pure HSt monolayer. With drug incorporation, the maximum is slightly reduced to lower wavenumbers. The band centered at 1466 cm1 is related to the CAH bending mode and appears to have been unaltered with the introduction of MFH. The band centered at 1378 cm1 is attributed to C@C vibrations in the quinoline group of mefloquine and is an indicative of drug incorporation. Overall, the data show that MFH has also affinity for negatively charged lipids, as observed for DPPG and HSt. It is probable, therefore, that the surface activity of this drug in the presence of these lipids is induced by dipole–ion interactions, probably between the OH and NH groups of mefloquine and its respective cationic sites. Interactions between mefloquine and membranes (potassium channels) are reported to be related to the positive charge of the drug, and the results obtained in this study corroborate this finding. It is also relevant that this interaction affects the alkyl chains of the lipid monolayers. Although hydrophobic interactions are not the driving factor for the incorporation of MFH into the lipid monolayers (for instance, for DODAB, no effect was observed), the electrostatic interactions may either induce some disorder in the alkyl chains or to some extent lead the drug to be in close contact with the hydrophobic tails of the monolayers. Moreover, because the shift in the surface pressure–area isotherms leads to higher areas than expected for such small molar quantities of MFH, we can conclude that the monolayers expanded with MFH incorporation. This result suggests that the drug imposed

30

T.E. Goto, L. Caseli / Journal of Colloid and Interface Science 431 (2014) 24–30

a higher degree of lateral repulsion among the interfacial molecules present at the air–water interface. Although a negative charge appears to be a crucial factor for the drug to significantly alter the physicochemical properties of the monolayers, a relevant interaction was observed between the drug and DPPC, which presented some negatively charged groups, although electrically neutral (or zwitterionic). However, for DODAB, a positively charged lipid, a negligible effect was observed. The charge on the membrane of the protozoa that causes malaria is mainly negative [27], and it is probable that the drug’s effect on natural membranes could be somehow associated with ionic attraction. As MFH is neutral, such effects should be based therefore on ion–dipole interactions. However, the effects of MFH on DPPG monolayer are very distinct from those on HSt monolayer. The mixed monolayer either with DPPC or HSt partially retained the characteristics of the pure lipid at high surface pressures, for example, the final collapse surface pressure. Distinctly, the mixtures with the anionic DPPG show a different behavior, independently of the MFH concentration the collapse occurs at 45 mN/m. This particular behavior is possibly related with the negative charge of DPPG interacting with positive charge of the drug. In contrast, for DODAB, a positively charge lipid, negligible effects were observed. Also, for the zwitterionic lipid, DPPC, some effects were also observed. Comparing the drug/lipid isotherms for the same concentration of drug (1% MFH in relation to the lipid), the following observation can be emphasized: (i) the drug is retained in the DPPG monolayer until high surface pressures; (ii) the drug is retained in the mixture with DPPG, but the mixed monolayer collapses at 45 mN/m; (iii) the drug is completely excluded from the DODAB monolayer; (iv) the drug is excluded from the HSt monolayer at surface pressures higher than 20 mN/m. The findings indicate that the drug/lipid interaction is strongly dependent on the polar group of the lipids. It can be therefore suggested that the repulsive nature of the interaction points to a possible disruptive effect of the drug. Furthermore, the large changes in fluidity arising from intermolecular bonds and aggregation of the drug may affect the surface relaxation and surface elasticity of the membrane. Drug incorporation may decrease the ability of the membrane to compress and expand reversibly to respond to cell events such as ion transport, cell reproduction and cell signaling. One may speculate that this reduced surface flexibility of the protozoan membrane could be related to the drug’s parasite-killing property. 4. Conclusions We have shown that the antiparisitic MFH is able to affect Langmuir monolayers even at low concentrations at the air–water interface, thus indicating that interactive effects may occur. The effects induced on the monolayers depend on the charge of the monolayer as well as on the polar head of the lipid. For negatively

charged lipids, such as DPPG and HSt, the drug markedly affected the surface pressure–area isotherms as well as vibrational spectra, but presented some distinctive effects. The results may help explain the mechanism by which this drug acts on specific sites of the protozoan membrane, providing molecular-level detail on how the drug is incorporated into the membrane model. Possible biological implications of the finding reported here may be related to the strong effects caused by incorporating the antiparasitic drug into the membrane model mainly at values of surface pressure that approximate natural membrane surface pressures. Acknowledgments This work was supported by FAPESP – Brazil (2013/10213-1), CNPq – Brazil (470890/2012-6), rede nBionet: Films and Sensors (CAPES – Brazil) and INEO – Brazil (INCT-CNPq). T.E. Goto was a CAPES fellow. References [1] A.C. Boareto, J.C. Muller, E.L.B. Lourenço, N. Lombardi, A.C. Lourenço, I. Rabitto, R.N. de Morais, F.S. Rios, P.R. Dalsenter, Hum. Exp. Toxicol. 9 (2013) 930–941. [2] P. Schlagenhauf, M. Adamcova, L. Regep, M.T. Schaerer, H.G. Rhein, Malaria J. 9 (2010) 357. [3] K.J. Palmer, S.M. Holliday, R.N. Brogden, Drugs 45 (1993) 430–475. [4] S.A. Ward, E.J. Sevene, I.M. Hastings, F. Nosten, R. McGready, Lancet Infect. Dis. 7 (2007) 136–144. [5] S. Xiao, Parasitol. Res. 11 (2013) 3723–3740. [6] C.P. Wu, A. Klokouzas, S.B. Hladky, S.V. Ambudkar, M.A. Barrand, Biochem. Pharmacol. 14 (2005) 500–510. [7] R.C. San George, R.L. Nagel, M.E. Fabry, Biochim. Biophys. Acta 23 (1984) 174– 181. [8] M. Foley, L. Int, J. Parasitol. 27 (1997) 231–240. [9] R. Chevli, C.D. Antimicrob, Agents Chemother. 21 (1982) 581–586. [10] D. Caridha, D. Yourick, M. Cabezas, L. Wolf, T.H. Hudson, G.S. Dowl, Antimicrob. Agents. Chemother. 52 (2008) 684–693. [11] H. Brockman, Curr. Opin. Struct. Biol. 9 (1999) 438–443. [12] A. Garcia-Gonzalez, A.L. Flores-Vazquez, A.P.B. de la Rosa, Vazquez-E.A. Martinez, J. Ruiz-Garcia, J. Phys. Chem. B 117 (2013) 14046–14058. [13] T.F. Schmidt, L. Caseli, T.M. Nobre, M.E.D. Zaniquelli, O.N. Oliveira, Colloid Surf. A-Physicochem. Eng. Asp. 321 (2007) 206–210. [14] L. Caseli, F.J. Pavinatto, T.M. Nobre, M.E.D. Zaniquelli, T. Viitala, O.N. Oliveira, Langmuir 24 (2008) 4150–4156. [15] A. Sakai, S.H. Wang, L.O. Péres, L. Caseli, Synth. Met. 161 (2011) 1753–1759. [16] T.E. Goto, L. Caseli, Langmuir 29 (2013) 9063–9071. [17] N. Hussein, C.C. Lopes, P.C.A. Pernambuco Filho, B.R. Carneiro, J. Colloid Interface Sci. 402 (2013) 300–306. [18] B. Gzyl, M. Paluch, Colloid Polym. Sci. 126 (2004) 60–64. [19] A. Brume, Biochim. Biophys. Acta 557 (1979) 32–44. [20] K.J. Klopfer, T.K. Vanderlick, J. Colloid Interface Sci. 182 (1996) 220–229. [21] V.V. Tscharner, H.M. McConnell, Biophys. J. 36 (1981) 409–419. [22] J. Saccani, S. Castano, F. Beaurain, M. Laguerre, B. Desbat, Langmuir 20 (2004) 9190–9197. [23] J.A. Obaleye, M.R. Caira, A.C. Tella, Struct. Chem. 20 (2009) 859–868. [24] D. Vollhardt, V.B. Fainerman, S. Siegel, J. Phys. Chem. B 104 (2000) 4115–4121. [25] H. Mansour, G. Zografi, Langmuir 23 (2007) 3809–3819. [26] G.A. Overbeck, D. Mobius, J. Phys. Chem. 97 (1993) 7999–9004. [27] J. Kang, C. Xiao-Liang, L. Wang, D. Rampe, J. Pharmacol. Exp. Ther. 299 (2001) 290–296.