Influence of Parabens on Bacteria and Fungi Cellular Membranes

Article Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX

pubs.acs.org/JPCB

Influence of Parabens on Bacteria and Fungi Cellular Membranes: Studies in Model Two-Dimensional Lipid Systems Michał Flasiński,*,† Sara Kowal,† Marcin Broniatowski,† and Paweł Wydro‡ †

Department of Environmental Chemistry, Faculty of Chemistry and ‡Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Kraków, Poland S Supporting Information *

ABSTRACT: Langmuir monolayers were used to study the influence of four commercially applied parabens on multicomponent systems composed of lipid species characteristic of the cellular membrane of microorganisms found in carbohydrates and proteins reaching products, including food and cosmetics. The aim of the undertaken studies was to shed new light on the problem of parabens’ interactions with membrane lipids and their affinity for monolayers differing with regard to the composition, mutual lipid ratios, and physicochemical properties. The discussion is based on the π−A isotherm characteristics, surface morphology observation performed with BAM, and analysis of the diffraction data collected for the periodically ordered lipid domains present in the investigated multicomponent films. Our studies revealed that the selected parabens are capable of surface film modification and that the magnitude of this effect increases with the number of methylene groups in the ester part of paraben molecules. We found that the strongest destructive effect was observed for model 1 (Staphylococcus aureus), a lower effect was observed for model 2 (Pseudomonas aeruginosa), and the lowest effect was observed for model 3 (Candida albicans). It was inferred that such a trend appears due to the composition of the artificial membranes, i.e., above all, in the presence or lack of sterol molecules and the content of negatively charged lipids.

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INTRODUCTION Parabens (PBs) are esters of p-hydroxybenzoic acid which are widely known from their broad antimicrobial activity. Over the last 50 years, these compounds have been applied as preservative agents in processed food and beverages, cosmetics, and toiletries as well as in pharmaceuticals.1 The main advantages of PB application result from the low costs of their synthesis, chemical and pH stability, nonperceptible taste and odor, and no influence on the consistency and coloration of commercial products.2 Thanks to these properties, parabens have often been considered to be the perfect additives, revealing broad preserving properties. From a practical point of view, the most useful are water-soluble PBs, having in their molecular structure short alkyl substituents, usually from methyl to butyl (Scheme 1).1,3 On the other hand, the elongation of the alkyl chain increases the strength of the antimicrobial properties considerably. For

example, BuPB has 4-fold higher antimicrobial potential than EtPB; however, the water solubility of BuPB is 12 times lower than that of EtPB.4 For that reason, parabens are frequently applied as mixtures in order to equalize good antimicrobial properties and sufficient water solubility.5−7 As far as the preserving potency of parabens is concerned, it is known that these compounds can be applied to a wide spectrum of microbes that are known to develop in carbohydrates and protein reaching products, including food and cosmetics, i.e., both Gram-negative and Gram-positive bacterial strains as well as fungi, represented presumably by molds and yeasts. Results from in vitro studies concerning the influence of parabens on Candida albicans suggested that the dose-dependent effect of these antimicrobials is not solely related to the inhibition of biofilm growth but rather is based on the direct influence on the cells.8 However, as emphasized by many authors, the exact mechanism of antibacterial activity demonstrated by parabens has not yet been elucidated.9,10 It was postulated that these compounds are capable of DNA, RNA, and some vital enzyme synthesis inhibition.10,11 Other studies pointed out that parabens may interfere with the respiration of microorganisms;10,12 however, most often it was postulated that the mode

Scheme 1. Chemical Structure of the Studied Parabens

Received: October 13, 2017 Revised: January 10, 2018 Published: February 12, 2018 © XXXX American Chemical Society

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DOI: 10.1021/acs.jpcb.7b10152 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol), sodium salt (POPG), and 1′,3′-bis[1,2-dimyristoyl-sn-glycero-3phospho]-sn-glycerol, sodium salt (TMCl). They were synthetic products (≥99%) purchased from Avanti Polar Lipids, Inc. Ergosterol (≥95%) used in the experiments was purchased from Sigma-Aldrich. Lipid solutions with a concentration in the range of 1.5−2.5 mg/mL were prepared in a chloroform/methanol 9/1 (v/v) mixture. The following applied solvents were provided by Sigma-Aldrich: chloroform of spectroscopic purity (99.9% stabilized by ethanol) and methanol (99%). The following investigated parabens (PBs) were purchased from Sigma-Aldrich: MePB (methyl phydroxybenzoate), EtPB (ethyl p-hydroxybenzoate), PrPB (propyl p-hydroxybenzoate), and BuPB (butyl p-hydroxybenzoate) of ≥99.0% purity. In the experiments, PB solutions in ultrapure water with a resistivity of ≥18.2 MΩ·cm obtained from a Milli-Q system (Merck Millipore synergy system) were used as subphases in the Langmuir trough. The concentrations of the applied PB solutions were in the range of 10−6−10−3 M. Methods. Langmuir Technique. The experiments were performed with the KSV-NIMA double barrier trough with a nominal area of 273 cm2. Surface pressure was measured to an accuracy of ±0.1 mN/m using a Wilhelmy plate made of filter paper connected to an electrobalance. The required amounts of lipid solutions were spread on a pure water surface or alternatively on the subphase of a paraben solution of a given concentration with a Hamilton microsyringe precise to 1 μL. In each experiment, the monolayer was left to equilibrate for at least 10 min before monolayer compression was initiated with a barrier speed of 10 cm2/min. This procedure guaranteed the reproducibility of the recorded isotherms; however, a longer monolayer equilibration prior to compression was also tested. In the case of experiments in the presence of PBs in the water subphase, the lipid monolayers were spread directly on the surface of a prepared solution of the desired concentration filling the trough. The temperature during the experiments (20 °C) was controlled thermostatically with the circulating water system (Julabo). During the penetration experiments, a mixed lipid monolayer was compressed to the desired surface pressure (15 or 30 mN/m), and afterward 500 μL of a concentrated ethanolic solution of PB was injected into the subphase via the injection port. The volume of the alcoholic solution was applied so that the paraben concentration in the subphase was 1 × 10−4 M. In order to ensure that the solvent does not influence the results, as the blank test we also applied the same volume of pure ethanol without the studied paraben. Moreover, there was no influence of the injected paraben solution on the surface tension measured without the spread monolayer. The results from the penetration experiment are presented here as bar graphs showing changes in the surface pressure after an identical period of time (ca. 1 h, π1h) and the initial surface pressure after a short relaxation of the compressed monolayer (5 min, π0): Δπ = π1h − π0. The magnetic stirrer was applied to ensure efficient mixing of the introduced solution. In order to ensure the reproducibility of the results, all of the isotherms were measured at least three times. Brewster Angle Microscopy (BAM). Brewster angle microscopy experiments were performed with an UltraBAM instrument (Accurion GmbH, Göttingen, Germany) equipped with a 50 mW laser emitting light of p polarization at a wavelength of 658 nm, a 10× magnification objective, a polarizer, an analyzer, and a CCD camera. The spatial resolution of the BAM was 2 μm.

of action of aliphatic parabens was based on their ability to disrupt a plasma membrane by the modification of its integrity and permeability,13,14 the induction of a potassium efflux,15 or the alteration of the transmembrane potential.10 Interestingly, Bredin and co-workers reported that parabens are able to destabilize the bacterial membrane by mimicking the activity of pore-forming proteins or polymyxins, which cause similar kinetics of potassium release from Escherichia coli cells.15 However, the connection between the susceptibility of the cell toward paraben activity and the composition, architecture, and main physicochemical properties of the cellular membrane is still not known. In some review articles, one can also find the assumption that the antimicrobial activity of parabens is higher toward fungi than toward bacteria and that these compounds are more effective in the fight against Gram-positive rather than Gram-negative bacterial strains.1,16 However, other results demonstrated that the susceptibility of bacteria is opposite, namely, that the sensitivity to the paraben antimicrobial effect was greater for Gram-negative E. coli than for Staphylococcus aureus.10 The aim of this study was to discuss the influence of parabens on model microbial plasma membranes in light of the interactions between paraben molecules and mixed lipid systems in a two-dimensional environment. In our investigation, we were looking for the answers to the following questions: What is the relationship between the lipid composition of the studied model membrane and the efficiency of the PB activity? What is the difference between the activity of the four tested parabens in the model lipid systems? What is the influence of PBs on model membrane condensation? Which of the investigated artificial membrane reveals the highest susceptible to paraben action? In searching for answers, we applied the Langmuir monolayer technique in order to build model lipid systems containing compounds found in the cellular membrane of selected bacteria and fungi. We prepared three multicomponent models having a composition and mutual proportion of lipid species characteristic of both Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria as well as the yeast representative (Candida albicans). These pathogenic organisms are often found in contaminated cosmetics and food, causing allergies and poisoning.17 The main advantage of such model studies is the possibility to reduce the number of variables that can have an influence on the paraben−lipid interactions. The properties of the prepared lipid surface films were tested in a series of complementary experiments, starting from the surface pressure (π)−mean molecular area (A) isotherm registration and stability measurements carried out at a constant mean molecular area connected to penetration experiments through microscopic observations with the BAM technique and finally investigations of the crystalline 2D domain characteristics performed with grazing incidence X-ray diffraction (GIXD). The combination of such a broad technique arsenal enabled us to obtain a comprehensive view of paraben behavior in twodimensional lipid systems, which is of great importance in the context of their antimicrobial activity.

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EXPERIMENTAL SECTION Materials. The following phospholipids were applied in the preparation of model membranes: 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine, sodium salt (POPS), 1B


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The Journal of Physical Chemistry B Table 1. Composition of the Investigated Model Membranes lipid

model 1 (Staphylococcus aureus)19

model 2 (Pseudomonas aeruginosa)20

POPG TMCl POPE POPC POPS ergosterol

0.580 0.420

0.228 0.119 0.652

model 3 (Candida albicans)21

0.200 0.254 0.150 0.396

Figure 1. Excess areas of mixing values calculated for the multicomponent monolayers of model membranes of Staphylococcus aureus, model 1 (a), Pseudomonas aeruginosa, model 2 (b), and Candida albicans, model 3 (c) investigated at π = 15 and 30 mN/m on the surface of pure water and on subphases containing paraben solutions of 1 × 10−4 M concentration.

Grazing Incidence X-ray Diffraction (GIXD). GIXD experiments were performed at the SOLEIL synchrotron (Paris, France) on the liquid surface diffractometer (SIRIUS beamline). At the beamline, the dedicated Langmuir trough placed in a gastight canister was mounted on the goniometer of the diffractometer. Before each experiment, the canister was sealed and flushed with helium to reduce the oxygen level. Such a procedure guaranteed a reduction of the scattering background and minimized the beam damage during the experiment. After at least 30 min, a monolayer was compressed to the target surface pressure, which afterward was held constant during the entire experiment. The surface pressure was measured with a Wilhelmy balance (R&K, Germany) equipped with a filter paper stripe as a π sensor. The detailed construction of the diffractometer working at the SIRIUS beamline and the parameters of the synchrotron beam applied in the GIXD experiments are described on the SOLEIL Web site (www.synchrotron-soleil.fr) as well as in the previous paper.18 Model Membranes. Mixed multicomponent solutions of desirable compositions were prepared from the respective lipid stock solutions and deposited onto the water subphase or PB solutions with the Hamilton microsyringe (±1.0 μL). The composition of the studied mixed monolayers (model membranes) was estimated on the basis of the literature data and gathered in Table 1. The presented values indicate the molar fractions of the respective lipids in the mixtures. Data Analysis. Information concerning the monolayer state was obtained with the application of traditional approach proposed by Riedel and Davies.22 According to the definition, the compression modulus, expressed as CS−1 = −A

lipid molecules in the mixed monolayers as well as on the mutual interactions between film-forming components, the excess area per molecule was calculated for the investigated multicomponent monolayers according to the equation Aexc = A1,2,...,i − (A1X1 + A2X2 +...+ AiXi), where A1,2,...,i is the mean area per lipid molecule in the multicomponent monolayers and A1, A2, and Ai are the areas occupied by molecules in the respective one-component films. X1, X2, and Xi are the mole fractions of components 1, 2, and i in the mixed monolayers. For the clarity of data presentation, the π−A curves recorded for the mixed surface films were presented in the Supporting Information (Figures S1−S3).

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RESULTS In Figure 1, the excess areas of mixing values calculated on the basis of the registered π−A isotherms for the multicomponent monolayers made from lipid species characteristic of the plasma membranes of different composition were gathered. As can be seen in Figure 1, the investigated parabens reveal different influences on the studied model systems. In the case of model 1, there is the largest difference with regard to the impact of paraben molecules differing in the length of the ester hydrophobic moiety. For the monolayer spread on the surface of ultrapure water, one can find the negative values of Aexc, indicating the miscibility of the components and favorable interactions between molecules forming mixed films, as compared to the interactions in pure monolayers of the respective components. The situation is different for the monolayer compressed on the subphase containing paraben solutions. In the presence of MePB, Aexc increases; however, it still remains negative, whereas in the case of EtPB the excess area is close to zero. On the other hand, for both PrPB and BuPB, positive values of the excess area were found, which

( ddAπ ), was

calculated from the recorded π−A isotherms. With respect to the influence of PBs on the mean molecular area occupied by C


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Figure 2. Increase in the excess area of mixing values for the investigated model membranes compressed to surface pressures of 15 mN/m (a) and 30 mN/m (b) on the surface of pure water and paraben solutions of 1 × 10−4 M concentration.

Figure 3. Maximal values of the compression modulus calculated for three model lipid systems constructed from lipid classes characteristic of the plasma membrane of Staphylococcus aureus, model 1 (a), Pseudomonas aeruginosa, model 2 (b), and Candida albicans, model 3 (c). The monolayers were spread on a water surface and aqueous solutions of parabens.

Moreover, in the case of MePB and EtPB, Aexc values are very close to that obtained for the mixed monolayer and the surface of pure water. Surprisingly, even in the case of the monolayer compressed on the surface of BuPB solution to a surface pressure of 30 mN/m, the excess area of mixing is negative, which means that the favorable interactions between lipid molecules are retained in the presence of paraben molecules. In order to compare the effect revealed by the four studied parabens on the characteristics of the model lipid membranes, the differences between the excess area of mixing calculated for mixed monolayers spread on the surface of PB solutions as well as on the surface of pure water were calculated (ΔAexc) and presented in Figure 2. As can be noticed in Figure 2, regardless of the studied model system and the applied surface pressure, the increase in Aexc was larger in the case of a paraben molecule having a longer hydrophobic fragment (MePB < EtPB < PrPB < BuPB). It is

means that due to the incorporation of paraben molecules into the monolayer and its fluidization the lipid−lipid interactions become thermodynamically unfavorable. The general trend observed in the case of model 2 looks quite similar; however, the quantitative effect is different as compared to model 1. The introduction of MePB does not lead to significant changes, whereas for EtPB and PrPB the increase in Aexc is pronounced and quantitatively comparable in both cases. For BuPB, the observed effect is the largest regardless of the surface pressure. It should be mentioned here that for models 1 and 2 the excess areas of mixing values are notably larger for π = 15 mN/m, as compared to 30 mN/m. This means that paraben molecules strongly affect the monolayer being in a state of low condensation, where molecules are packed less densely than at higher surface pressure. In contrast to models 1 and 2, for model 3, the excess area of mixing was negative in all experiments with the exception of the model membrane compressed on the surface of BuPB solution to π = 15 mN/m. D


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Figure 4. BAM images recorded during compression of the mixed monolayer constructed from lipid characteristic for the Staphylococcus aureus cell membrane (model 1) on a water surface (top row), a 1 × 10−4 M MePB solution (middle row), and a 1 × 10−4 M BuPB solution (bottom row).

also apparent that ΔAexc is larger for lower surface pressure and grows in order of model 3 < model 2 < model 1. The next parameter investigated in the context of the paraben influence on the model lipid membrane was the compression modulus calculated directly on the basis of the recorded π−A isotherms. The maximal values of CS−1 were plotted in the functional paraben solution concentrations in Figure 3. One can find that all of the investigated multicomponent monolayers reach the liquid condensed state since the maximal values of the compression modulus are 214, 239, and 229 mN/ m, for models 1−3, respectively. The presence of paraben molecules in the subphase during monolayer compression caused the CS−1 values to become smaller. The observed effect increases with the paraben solution concentration and is the largest for BuPB. In the case of a paraben concentration of 10−3 M, there is an evident trend with regard to the correlation of the fluidizing effect revealed by parabens and the size of their ester (hydrophobic) fragment. Interestingly, for model 3 there is the largest difference between the influence of studied parabens on the condensation of model membranes. It can also be observed that for models 1 and 2 the decrease in the monolayer condensation was similar in the cases of PrPB and BuPB at concentrations of 10−5 and 10−4 M, respectively. The discussion of the fluidizing effect revealed by the studied paraben molecules was complemented by the conclusions drawn from the morphological studies. Therefore, in our studies Brewster angle microscopy was applied in order to visualize the surface domains in situ. The collected images were gathered in Figure 4 and Figure S4. For model 3, the observed monolayer was homogeneous in the broad range of surface pressure (data not shown). BAM images recorded for the mixed monolayer of model 1 spread on the surface of pure water as well as on MePB and BuPB solutions with a concentration of 1 × 10−4 M were compared for three surface pressures as indicated in the Figure

4 caption. Generally, an analysis of the images confirms the conclusions drawn from compression modulus calculations. One can find that in the presence of paraben solution in the subphase both the size and the surface density of the condensed LC domains decrease. For example, at a surface pressure of 10 mN/m, in the case of an ultrapure water subphase, these domains possess a characteristic, well-developed dendritic shape. Such a structural profile is typical for phospholipid monolayers during the LE−LC phase transition.23 On the other hand, in the presence of MePB molecules, the condensed domains are significantly smaller and have a worse-evolved contour. The incorporation of BuPB molecules into the studied surface film results in an even larger diminishing of the LC domain shape. They resemble small bright spots dispersed in the predominant phase of low condensation. It can be clearly seen that for images gathered in the rows, from top to bottom, the ratio of the LC to LE phase decreased. This can lead to the conclusion that the condensation of the monolayer compressed on the subphase containing paraben molecules decreased. In the case of a surface pressure of 30 mN/m, the observed effect is not as pronounced; however, the decrease in the domain size can be undoubtedly noticed. For the mixed monolayer compressed on the surface of pure water and MePB solution to the highest surface pressure tested (40 mN/m), the condensed LC domains are densely packed on the surface and only a very small area is covered by the dark regions characteristic of the expanded monolayer. Interestingly, in the case of BuPB, in the investigated surface film apart from the condensed domains, very bright spots of the three-dimensional (collapsed) phase can be noticed. In the next part of our experiments, the evolution of the surface pressure after paraben solution injection into the water subphase was measured. The obtained π vs time curves were presented in the Supporting Information, Figure S5, whereas the ultimate changes in the surface pressures were plotted in Figure 5. E


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by the investigated lipid 2D systems, we applied a grazing incidence X-ray diffraction technique (GIXD). The obtained results were compared with the diffraction measurements performed for the studied monolayers spread on the surface of ultrapure water. Diffraction signals which are characteristic of the surface films containing periodically ordered domains were registered for all model systems. In the case of the monolayers corresponding to models 1 and 2, compressed on pure water to a surface pressure of 30 mN/m, single Bragg peaks were registered with their maxima at 1.487 and 1.480 Å−1, respectively (Figure 6.). These values are typical for the diffraction signals obtained for Langmuir monolayers of amphiphilic molecules possessing saturated or monounsaturated hydrocarbon chains.24 A single, symmetrical Bragg peak localized in the horizon, i.e., at qz = 0 Å−1, is indicative of the hexagonal phase, in which lipid molecules with their hydrophobic parts are oriented along the surface normal.25 In the presence of BuPB molecules in the subphase, the location of the maximum changed only slightly (Table 2), which indicates

Figure 5. Results of penetration experiments expressed as the surface pressure changes after the injection of paraben solution into the subphase.

As can be seen in Figure 5, the influence of the investigated parabens injected beneath the model lipid membranes leads in most cases to a significant increase in the surface pressure, which is connected to the penetration of paraben molecules into the mixed lipid monolayers. This effect is notably stronger in the case of lower initial surface pressure (10 mN/m), as compared to 30 mN/m. In the latter case for MePB (model 2) and both MePB and EtPB (model 3), the decrease in the initial π was observed, which is connected to the desorption of the monolayer material into the bulk phase. As expected, the incorporation of parabens into the surface film was the strongest for more bulky PrPB and BuPB molecules. In the case of these two compounds, the magnitude of the observed effect increased in order of model 2 < model 1 < model 3, whereas for MePB and EtPB, a different trend was found: model 1 < model 2 < model 3. In order to shed light onto the impact of BuPB, which revealed the strongest influence in the preceding experiments, on molecular organization of the condensed domains formed

Table 2. Structural Parameters Calculated for the Investigated Model Membranes Based on GIXD Data

model 1

model 2

model 3

Bragg peak Qxy [Å−1]

lattice parameters [Å, Å, deg]

water

1.487

BuPB

1.484

water

1.480

BuPB

1.482

water

1.487 1.480

BuPB

1.483

a = 4.879 γ = 120° a = 4.889 γ = 120° a = 4.902 γ = 120° a = 4.895 γ = 120° a = 4.872 b = 8.491 γ = 90° a = 4.892 γ = 120°

area [Å2]

Lxy [Å]

20.615

266

20.700

285

20.810

212

20.751

309

41.368 (20.684)

160

20.725

276

Figure 6. Diffraction signals (Bragg peaks) registered for the model lipid systems constructed from lipid classes characteristic of the plasma membrane of Staphylococcus aureus, model 1 (a), Pseudomonas aeruginosa, model 2 (b), and Candida albicans, model 3 (c) on a pure water surface as well as on the surface of BuPB solution at π = 30 mN/m. The registered Bragg peaks were fitted using a Lorentzian function and were offset for clarity of data presentation. F


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parabens, the revealed effect is significantly lower: it becomes weaker together with decreasing values of the octanol/water coefficient in the following order: PrPB > EtPB > MePB. From the viewpoint of model membrane compositions, we found that the largest disturbing effect revealed by PBs was demonstrated for model 1 mimicking Staphylococcus aureus, it was lower for model 2, and it was the lowest for model 3. The susceptibility of bacterial model membranes on the action of parabens is in agreement with the biological studies performed on living bacterial strains. Namely, in the literature, one can find the results suggesting that parabens are more active toward Grampositive than toward Gram-negative bacteria.1 On the other hand, the same results indicate that fungi are more susceptible to PBs’ destructive influence than they are to bacteria. The opposite tendency was observed in our studies concerning the characteristics of model membranes spread on the preformed paraben solutions. In this case, the influence of MePB and EtPB on model 3 was very weak as compared to that on the remaining model systems. This is most likely connected to the lipid composition of the investigated membranes. First, in a model Candida albicans membrane a relatively high content of ergosterol is present. Sterol molecules are known to display both condensing and ordering effects,28 which are responsible for the tight packing of lipid molecules in two-dimensional systems. It is also known that sterols form highly ordered, condensed domains with the membrane lipids having long, saturated acyl chains, especially with phosphatidylcholines and sphingomyelins.29,30 For the mentioned reasons, the ΔAexc value for the monolayer studied on a pure water surface is negative. Moreover, the mentioned effects positively influence the integrity of lipid monolayers, hindering paraben effects. On the other hand, in the mixed monolayer made from lipid representatives characteristic of a membrane of Staphylococcus aureus, solely two (negatively charged) lipids are present: POPG and cardiolipin. These molecules are able to form intermolecular hydrogen bonds; however, the electrostatic repulsions between charged polar headgroups hinders this fact.31,32 This in turn may lead to easier access of paraben molecules to the surface region. The intermediate susceptibility of model 2 can be explained by the presence of a large amount of POPE in this mixed film, which together with POPG molecules forms a densely packed monolayer due to the formation of the hydrogen bonds.32,33 It is also worth noticing here that from our previous studies performed in onecomponent lipid monolayers it turned out that parabens interact more strongly with a lipid of mammalian membranes (saturated zwitterionic PCs) than with bacteria (negatively charged PG and cardiolipin).27 Interestingly, the results obtained from molecular dynamics simulations showed that MePB, PrPB, and BuPB bind to the phosphatidylcholine bilayer and are able to penetrate the polar headgroup region but not the hydrophobic core of the membrane.34 Another effect revealed by parabens concerns the decrease in the lipid monolayers’ condensation. It can be found that this influence is the largest for the mixed film of model 2, which initially possesses the largest condensation on the surface of pure water. Interestingly, in the case of both models 1 and 2, the fluidizing effect demonstrated by parabens is very similar for PrBP and BuPB solutions of the same concentration. Generally, the decrease in condensation is the smallest for model 3, which is connected to the presence of sterol molecules in this model membrane. Similarly, the high resistivity of the model membrane rich in sterols was also observed for water-soluble

a rather minor influence of this paraben on the characteristics of the well-organized periodically ordered domains. The situation is different in the case of model 3 on the free water surface, and for this monolayer, two diffraction signals were recorded. The first one is localized to the horizon with the maximum at qxy = 1.480 Å−1, and the second one is beyond the horizon at qxy = 1.487 Å−1. Such a diffraction pattern is characteristic of the twodimensional centered rectangular lattice with a molecular tilt toward the nearest neighbor (NN).25,26 Interestingly, the incorporation of BuPB molecules into the surface region leads to the modification of the periodically ordered domains’ organization. In place of two Bragg peaks, only one can be observed with its maximum at qxy = 1.483 Å−1. This also means that the molecular tilt is lifted due to the lipid−paraben interactions. The incorporation of BuPB molecules into the mixed lipid monolayers caused a slight modification of the crystallographic area for the two-dimensional unit cells (Table 2). For models 1 and 3, this area expands, while in the case of model 2, it becomes smaller. It can also be found that in all cases the range of crystallinity in the model membranes increases in the presence of BuPB in the subphase. This finding can be interpreted as the increase in molecular organization in the film, occurring due to the paraben−lipid headgroup interactions. Moreover, the intensity of Bragg peaks registered for monolayers investigated on the BuPB subphase is lower than for surface films spread on pure water, which means that less film-forming material is implicated in the formation of the periodically ordered fraction. In other words, the surface coverage of the LC phase is lower, which is in agreement with the BAM observation regarding the diminishing of the surface density of LC domains.

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DISCUSSION The affinity of paraben molecules for the lipid phase and the proven activity of these compounds at the level of natural lipid bilayers encouraged us to perform comprehensive studies focused on the interactions of four broadly applied as food and cosmetic preservative compounds (parabens) with lipid components of microorganism membranes. From our previous studies performed with the application of one-component monolayers formed by mammalian lipids, we find that both the class of lipids and the size of the hydrophobic paraben fragment and the solution concentration have significant impacts on lipid−paraben interactions.27 In the present work, our intention was to widen the investigations for the multicomponent mixed lipid systems containing phospholipids and the representative of sterol found in natural bilayers of bacteria and fungi cells. The aim of the studies was to analyze the influence of parabens on the simplified models, namely, Langmuir monolayers made of lipid species typical of the microorganism plasma membranes, in order to shed new light on paraben behavior in the membrane lipid environment. From our analysis based on the excess area of mixing (ΔAexc), it can be inferred that the most unfavorable interactions in the mixed lipid systems occur as the result of PB molecules’ incorporation into the surface film and are the largest in the presence of BuPB in the subphase. This result is connected to the fact that among the investigated parabens, BuPB possesses the largest hydrophobic (ester) fragment and therefore the affinity of this compound for the lipid environment is the highest. In the case of the remaining G


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was found for model 1 mimicking Staphylococcus aureus, lower for model 2 (Pseudomonas aeruginosa), and the lowest for model 3 (Candida albicans). The origin of this trend is directly connected to the composition of the artificial membranes, that is, above all, the presence or lack of a sterol component and the content of negatively charged lipids (cardiolipin and POPG). The other factor determining the parabens’ ability to disrupt studied model membranes is their condensation, which decides the potential of PB molecules to modify the properties of the lipid films. Moreover, the performed experiments demonstrated that at the higher surface pressure BuPB, in contrast to parabens having smaller ester substituents, is able to initialize the formation of three-dimensional crystallites. On the other hand, it is also interesting that the presence of PB molecules in the surroundings of periodically ordered lipid domains causes an increase in the range of periodic order and the removal of the molecular tilt. The undertaken model studies proved that the antimicrobial effectiveness of paraben molecules is strongly connected to the composition of lipid membranes and their physical properties as well as to the chemical structure of PBs.

plant hormones incorporated beneath the studied monolayers.35 The mentioned fluidization of the lipid film was proven on the basis of the microscopic observations. This effect was the largest for the monolayers being in the liquid expanded (LE) state, and it is manifested as the inhibition of LC domain formation occurring as a result of the presence of paraben molecules in the surface region. Additional evidence of such behavior was inferred from the penetration experiment, in which the significantly larger influence of paraben molecules was noticed for the monolayers investigated at a surface pressure of 10 mN/m rather than 30 mN/m. This is an interesting finding since parabens are not the surface-active compounds; however, their molecules are able to be accommodated at the surface as a result of interactions with lipid polar headgroups. The increase in the surface pressure during the penetration experiments proved also that paraben molecules possessing larger acyl fragments (PrPB and BuPB) are more effective as far as incorporation onto surface films is concerned. Upon compression of the surface film toward the higher pressure range, water-soluble molecules of PBs tend to reposition into the bulk aqueous phase since the distances between film-forming lipid molecules are decreased. However, in the case of BuPB having, among the investigated parabens, the largest affinity for the lipid phase, the formation of threedimensional crystallites can be observed. Therefore, on the basis of the presented results, it can be concluded that in the studied systems two alternative mechanisms of the artificial membrane’s disruption may be possible, depending on the ordering of lipid films with respect to fluidization and crystallization. Bearing in mind the above-mentioned rationalization, it is also interesting to observe what happens with the monolayer at the molecular level. In order to obtain information regarding the properties and organization of the condensed, periodically ordered region, we performed GIXD studies. We found that the presence of paraben molecules in the surface region reveals a minor influence on the well-ordered monolayers which is manifested as an increase in the 2D crystalline domain size, connected to the lipid−paraben interactions. Moreover, in the case of model 3, the removal of molecular tilt was observed. These two findings show that for the highly ordered domains within the monolayers an increase in order takes place, which is in contradiction to the abovementioned global effect of the surface film fluidization. From the diffraction experiments, it was also inferred that the larger the fraction of periodically ordered domains in the monolayer (more intense Bragg peak), the smaller the influence of PB molecules. These also explain why parabens interact more effectively with the lipid monolayers of lower condensation, in which the contribution of the two-dimensional crystalline domains is small.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b10152. Surface pressure (π)−mean molecular area (A) isotherms registered for the mixed monolayers of the investigated model membranes. BAM images recorded during compression of the mixed monolayer made from lipid classes typical of the Pseudomonas aeruginosa model membrane (model 2) and evolution of the surface pressure after the injection of paraben solution into the water subphase beneath the mixed monolayers of the studied model systems. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: fl[email protected]. Phone: +48 0-12-686-2568. ORCID

Michał Flasiński: 0000-0002-8330-5701 Marcin Broniatowski: 0000-0003-0292-1826 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge SOLEIL for the provision of synchrotron radiation facilities, and we thank Dr. Philippe Fontaine for assistance in using the SIRIUS beamline. M.F. acknowledges financial support from the Foundation of Habilitation Projects in the Department of Chemistry, Jagiellonian University in Krakow (scientific project no. 406.3115.31.2016: Application of the model lipid systems in studies on the ecotoxicity of selected xenobiotics: plant growth regulators and food and consmetic preservatives).

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CONCLUSIONS Our studies performed in the model two-dimensional systems revealed that the representatives of parabens broadly applied in the cosmetics and food industries cause cause a significant alteration of the lipid monolayer characteristics. This modification of the artificial plasma membrane properties is responsible for the paraben destructive activity at the level of microorganism membranes, as shown also in biological tests. The results of the performed investigation proved that the effectivity of parabens’ action is correlated with the size of the ester substituent in the homologues series from methyl to butyl. We observed that the largest disturbing effect revealed by PBs

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