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New Insights on the Biophysical Interaction of Resveratrol with Biomembrane Models: Relevance for Its Biological Effects Ana Rute Neves, Cláudia Nunes, and Salette Reis J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b05419 • Publication Date (Web): 03 Aug 2015 Downloaded from http://pubs.acs.org on August 8, 2015

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The Journal of Physical Chemistry

New Insights on the Biophysical Interaction of Resveratrol with Biomembrane Models: Relevance for Its Biological Effects Ana Rute Neves1, Cláudia Nunes1, and Salette Reis1*

1

UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of

Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.

* Corresponding Author Salette Reis Department of Chemical Sciences, Faculty of Pharmacy of University of Porto Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal TEL: (+351)220428672 FAX: (+351)226093390 E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT

Resveratrol has been widely studied because of its pleiotropic effects in cancer therapy, neuroprotection and cardioprotection. It is believed that the interaction of resveratrol with biological membranes may play a key role in its therapeutic activity. The capacity of resveratrol to partition into lipid bilayers, its possible location within the membrane, and the influence of this compound on the membrane fluidity were investigated using membrane mimetic systems composed of egg L-α-phosphatidylcholine (EPC), cholesterol (CHOL), and sphingomyelin (SM). The results showed that resveratrol has greater affinity for the EPC bilayers than for EPC:CHOL [4:1] and EPC:CHOL:SM [1:1:1] membrane models. The increased difficulty in penetrating tight packed membranes is also demonstrated by fluorescence quenching of probes and by fluorescence anisotropy measurements. Resveratrol may be involved in the regulation of cell membrane fluidity, thereby contributing for cell homeostasis.

KEYWORDS: Resveratrol, liposomes, cholesterol, sphingomyelin, drug-membrane interactions.


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INTRODUCTION In the last decades, resveratrol has been widely studied because of its pleiotropic and promising effects in chemotherapy, cardioprotection, antioxidant and anti-inflammatory responses and neuroprotection 1,2. The mechanism by which resveratrol exerts its effects is still a matter of debate and it is thought to be through multiple pathways, including scavenging of free radicals

3,4

, suppression of cyclooxygenase (COX) activity 5,6, inhibition of cell proliferation

7,8

,

induction of apoptosis 7,9, and inhibition of enzymes, namely phospholipase C (PLC) and protein kinase C (PKC)

10

. Nevertheless, additional investigation is required to clarify such pathways

and the molecular mechanisms involved in the interaction of resveratrol with biological membranes may play a key role in its therapeutic activity, as suggested for other drugs which act through a membrane-lipid therapy

11-13

. In this regard, the present study aims to investigate the

effects of trans-resveratrol, which is the active form of the compound (Figure 1), on the biophysical properties of biomembranes in order to correlate these effects with the well documented pharmacological properties of this compound at physiological conditions (pH 7.4, I = 0.1M and 37 °C). Hence, the capacity of resveratrol to partition into the lipid bilayers, its possible location within the membrane, and the influence of this compound on the membrane fluidity were investigated by means of derivative spectrophotometry resolved spectrofluorimetry

15

14

, steady-state and time-

, and fluorescence anisotropy measurements

have shown the effect of resveratrol in membrane model systems

17-23

16

. Previous works

. However, the correct

location of resveratrol within the membrane and how the biophysical properties of the lipid bilayer are affected by resveratrol remain to be clarified, since there is some controversy in the literature results. While some studies showed that resveratrol interacts with the head group region of membranes

20,23

, other reported that resveratrol permeates into the intermembrane


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region of lipid bilayers

21,22

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. Likewise, resveratrol seems to increase membrane fluidity in some

studies [17, 19], and contrarily shows to induce stiffness on other liposomal models

22

. One of

the reasons for these contradictions may be due to the different membrane lipid compositions, namely the degree of unsaturation of fatty acids, the acyl chain length, the head group of the phospholipids, and the presence or absence of cholesterol and sphingomyelin lipids. Concerning this, different membrane model systems were used in the present study, simulating diverse phospholipid biological environments encountered by resveratrol. Membrane mimetic systems composed of phosphatidylcholine (PC), cholesterol (CHOL), and sphingomyelin (SM) with different ratios between the chosen lipids (Figure 1) leads to membranes with different fluidity, condensed domains and surface charge. For achieving this purpose, large unilamellar liposomes (LUVs) of pure EPC (egg L-α-phosphatidylcholine), EPC:CHOL in a molar ratio of 4:1, and EPC:CHOL:SM in a molar ratio of 1:1:1 were selected. EPC model system is a natural mixture of saturated and unsaturated lipids, conferring fluid properties to the lipid bilayer, which is recognized as the most common lipid arrangement in cells, being phosphatidylcholines the most abundant lipids in cell membranes

24

. Regarding EPC:CHOL model system, the presence of

cholesterol molecules makes the membrane more complex and organized, trying by this way to mimic cellular targets rich in cholesterol domains. Finally, EPC:CHOL:SM mimics quite well brain membranes bearing further complexity and structural organization that is typical of these cell membranes. In fact, brain is particularly enriched in cholesterol and sphingomyelin lipids, which are essential for neuronal development and survival, maintenance of membrane integrity in neurons, synapse maturation, and optimal synaptic activity 25-27. Furthermore, cholesterol and sphingomyelin can modulate the membrane fluidity and consequently its permeability 28, playing


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an important role in the maintenance of membrane organization and contributing for the physiology and function of the cell membrane 29,30.

Figure 1 – Chemical structures of the intervenient species in this study. (A) egg phosphatidylcholine (EPC) unsaturated predominant specie – 31% 18:1 PC; (B) sphingomyelin (SM) predominant specie – 33%; (C) cholesterol and (D) trans-resveratrol.

EXPERIMENTAL METHODS Materials trans-Resveratrol (> 99% purity) and EPC (> 99% purity) were obtained from Sigma Aldrich (St. Louis, MO, USA). Cholesterol (> 98% purity) and sphingomyelin (> 99% purity) were purchased from Avanti Polar Lipids, Inc. (Alabama, USA). The fluorescent probes 1,6diphenyl-1,3,5-hexatriene

(DPH)

and

1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-

hexatriene (TMA-DPH) were supplied by Molecular Probes (Invitrogen Corporation, Carlsbad,


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California, USA). Resveratrol solutions and lipid suspensions were prepared with phosphate buffer (pH 7.4), according to its water solubility (0.0688 mg/mL). For the preparation of phosphate buffer solutions, potassium phosphate monobasic was obtained from Sigma Aldrich and sodium hydroxide from Riedel-de Haën AG (Seelze, Germany). The buffers were prepared using double deionized water from arium water purification system (resistivity > 18 MΩ cm, Sartorius, Goettingen, Germany) and the ionic strength was adjusted to mimic physiological conditions with NaCl (I = 0.1 M).

Liposomes preparation Liposomes were prepared by the lipid film hydration method 31,32. The molar ratio mixture of the lipids (EPC, EPC:CHOL [4:1], or EPC:CHOL:SM [1:1:1]) was dissolved in chloroform/methanol (3:2, v/v). The organic solvents were then evaporated under a nitrogen stream to yield a dried lipid film. The lipid film was hydrated with phosphate buffer (pH 7.4) and vortexed at 40°C (well above the main phase transition temperature of the lipids). LUVs of 100 nm were prepared by extrusion of the multilamellar vesicles (MLVs) suspensions 10 times through a polycarbonate filter with a pore size of 100 nm, at 40°C (temperature well above the main phase transition of the lipids), using a LIPEX Extruder, Northern Lipids Inc. (Burnaby, Canada). The size of the LUVs formed was confirmed by Dynamic Light Scattering analysis in a Brookhaven Instrument (Holtsville, NY, USA). For fluorescence measurements, the probe (DPH or TMA-DPH) was co-dissolved with the lipids (EPC, EPC:CHOL or EPC:CHOL:SM) in the organic solvents mixture to give a probe/lipid molar ratio of 1:300.

Determination of partition coefficients by derivative spectrophotometry


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The partition coefficients (Kp) of resveratrol between LUVs suspensions of EPC, EPC:CHOL [4:1] or EPC:CHOL:SM [1:1:1] and the aqueous buffered solution were determined by derivative spectrophotometry. Resveratrol in phosphate buffer with a final concentration of 20 µM was added to LUVs suspensions with increasing concentrations of lipids (from 0 to 1000 µM) in a microplate, and incubated in the dark for 30 minutes and 37 °C, with agitation. The corresponding reference solutions were identically prepared in the absence of resveratrol. The absorption spectra (250–500 nm range) of samples and reference solutions were recorded at body temperature of 37 °C, in a multidetection microplate reader (Synergy HT; Bio-Tek Instruments), accordingly to a well-established protocol

14

. The mathematical treatment of the results was

performed using a developed routine, Kp Calculator

14

, which (i) subtracts each reference

spectrum from the correspondent sample spectrum to obtain corrected absorption spectra; (ii) determines the second and third derivative spectra in order to eliminate the spectral interferences due to light scattered by the lipid vesicles and to enhance the ability to detect minor spectral features and improve the resolution of bands; and (iii) calculates the Kp values by plotting the second or third derivative spectra values at wavelengths where the scattering is eliminated versus the lipids concentrations 14. After that, a non-linear least-squares regression method is applied by fitting the following equation to the plot, where Kp is the adjustable parameter:

= +

( − ) [] Ø 1 + [] Ø

(1)

In this equation, D is the second or third derivative intensities obtained from the absorbance values of resveratrol: DT refers to the total amount of resveratrol, Dw corresponds to resveratrol distributed in the aqueous phase, and Dm corresponds to resveratrol distributed on the


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lipid membrane phase; Kp is the partition coefficient of resveratrol in a specific liposome system; [L] is the molar concentration of the lipid; and VØ is the lipid molar volume of EPC (0.75 L mol1

), EPC:CHOL [4:1] (0.67 L mol-1), or EPC:CHOL:SM [1:1:1] (0.63 L mol-1).

Membrane location studies by fluorescence quenching The membrane location of resveratrol was assessed by steady-state fluorescence quenching and lifetime measurements using DPH and TMA-DPH probes with a known membrane position and depth

33-35

. The quenching studies were performed after incubation of increasing

concentrations of resveratrol (from 0 to 80 µM) with a fixed 500 µM concentration of labelled liposomes of EPC, EPC:CHOL [4:1] or EPC:CHOL:SM [1:1:1], in phosphate buffer (pH 7.4). Before fluorescence measurements, the samples were incubated in the dark for 30 minutes, at physiological temperature (37 °C), allowing resveratrol to reach the partition equilibrium between the lipid membranes and the aqueous medium. Measurements were carried out at 37 °C with excitation/emission wavelengths of 357/429 nm and 361/427 nm for DPH and TMA-DPH, respectively. The capacity of resveratrol to quench the fluorescence of DPH and TMA-DPH probes was evaluated by determination of the Stern–Volmer constant (KSV) from the slope of the Stern–Volmer plots obtained by steady-state fluorescence measurements (I0/I)

15

. In order to

study if the quenching mechanism was static or dynamic, the dynamic quenching constant (KD) was determined from the slope of the Stern-Volmer plots obtained by lifetime fluorescence measurements (τo/τ)

15

. In addition, it was also calculated the bimolecular quenching rate

constant (Kq=KSV/τ0, where τ0 is the fluorescence lifetime of the probes in the liposomes without resveratrol) which constituted a fundamental parameter to predict the location of resveratrol in


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the membrane, since the effect of the different microenvironment surrounding the different probes was eliminated 36.

Membrane fluidity studies by fluorescence anisotropy Fluorescence anisotropy with DPH and TMA-DPH probes was used to study the effect of resveratrol on the membrane fluidity. Different concentrations of resveratrol (0, 20 and 80 µM) were tested with a fixed concentration (500 µM) of labelled liposomes of EPC, EPC:CHOL [4:1] and EPC:CHOL:SM [1:1:1] in phosphate buffer (pH 7.4), after 30 minutes of incubation in the dark and 37 °C. Steady-state fluorescence anisotropy measurements (rs) were performed in a Jasco FP6500 spectrofluorimeter with polarizers. The excitation/emission wavelengths were set to 357/429 nm and 361/427 nm for DPH and TMA-DPH, respectively. Samples were excited with vertically polarized light and fluorescence intensities were recorded with the analyzing polarizer oriented parallel and perpendicular to the excitation polarizer. The anisotropy was recorded at several temperatures between 7°C and 37 °C, by intervals of 1 °C. The order parameter ( = / ) was calculated, where r0 is the fluorescence anisotropy in the absence of any rotational motion of the probe and r∞ reflects the restriction of probe motion in each particular membrane system

37,38

. r∞ can be calculated from rs values ( =

4 − 0.10), for 0.13 < rs < 0.28 39. 3

RESULTS AND DISCUSSION Determination of resveratrol partition coefficients The partition coefficient of resveratrol strongly influences its pharmacokinetic and pharmacodynamic properties, suggesting the distribution of the compound between the aqueous


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and the lipid environments and also the diffusion processes across the biological barriers inside the body

40-43

. In this work, derivative spectroscopic techniques combined with liposome/water

systems allowed the determination of resveratrol partition between the lipid and aqueous media 14

.

Figure 2 – Absorption spectra (A), first-derivative (B), and second-derivative (C) of resveratrol (20 µM) incubated in LUVs of EPC (black lines) and LUVs of EPC without resveratrol (gray lines) with increasing lipid concentrations (from 1 to 11) in phosphate buffer at physiological conditions (pH 7.4, 37 °C). The curve (D) represents the fitting curve to experimental secondderivative spectrophotometric data as a function of EPC concentration, using a nonlinear leastsquares regression method at wavelength 360 nm where the scattering is eliminated.


10

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In Figure 2A, 2B and 2C are presented, as an example, the absorption spectra, the firstderivative and the second-derivative of resveratrol with different concentrations of LUVs of EPC at pH 7.4 and 37 °C. The second-derivative spectra (Figure 2C) eliminate the background caused by light scattered of lipid vesicles and exhibit a shift in the λmax with increasing lipid concentrations, providing a clear indication that resveratrol partitions from the aqueous medium to the liposomes

14,44

. Figure 2D shows the best fit of equation 1 to the second-derivative

spectrophotometric data, collected at λ=360 nm, as a function of EPC concentration. Similar spectra were obtained for the other lipid model systems (Figures S1 for EPC:CHOL and Figure S2 for EPC:CHOL:SM) at the same physiological conditions - pH 7.4 and 37 °C. The values of Kp and log D obtained are listed in Table 1.

Table 1 – Partition coefficients (expressed as Kp and log D) of resveratrol in LUVs of EPC, EPC:CHOL [4:1] and EPC:CHOL:SM [1:1:1] at physiological conditions (pH 7.4 and 37 °C). System

Kp

log D

EPC

3384 ± 362

3.52 ± 0.09

EPC:CHOL

1386 ± 165

3.14 ± 0.05

EPC:CHOL:SM

939 ± 220

2.96 ± 0.10

Note: All values represent the mean ± standard deviation (n = 3).

The pKa values and octanol:water partition coefficients of resveratrol were also calculated using Marvin sketch calculator software from ChemaxonTM. From pKa values (8.49; 9.13 and 10.14 for positions C5; C4’ and C3, see Figure 1), it was possible to predict that around 92% of all resveratrol molecules were in the neutral form at pH 7.4, what explains the high values of resveratrol partition coefficients obtained experimentally (Table 1). The theoretical log Po/w calculated by ChemaxonTM was 3.40 which is very similar to the experimental log D obtained at


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pH 7.4 in the EPC membrane model (3.52 ± 0.09), but less comparable with the other two models (3.14 ± 0.05 for EPC:CHOL and 2.96 ± 0.10 for EPC:CHOL:SM). These findings highlight the advantage of using liposome/water systems to obtain the partition coefficients more accurately depending on the composition of the membrane. Consequently, resveratrol presents higher Kp values for LUVs of EPC, followed by EPC:CHOL [4:1], and finally EPC:CHOL:SM [1:1:1] which presents the lower partition coefficient, at the same physiological conditions (pH 7.4 and 37 °C). This might be attributed to the fact that the lipids are much more organized and packed in this latter brain model system in the presence of cholesterol and sphingomyelin molecules, making more difficult the diffusion of resveratrol into this lipid bilayer. Therefore, resveratrol partition into the more fluidic system is greater than in the more organized membranes. However, the capacity of resveratrol to partition even into the more packed system is probably due to its high lipophilicity and its planar structure (Figure 1) enabling the intercalation between the membrane phospholipids 17.

Resveratrol location studies The location of resveratrol inside the different membranes used in this study was tracked by the fluorescence quenching of two probes with a well-known membrane position. DPH is deeply buried in the hydrocarbon core of the lipid bilayer aligned parallel to the acyl chains 33,34 and TMA-DPH is anchored in the polar head region of phospholipids due to its charged group and therefore is located closer to the lipid/water interface 35. In the current study the steady-state fluorescence intensities and lifetimes were measured in labelled liposomes of EPC, EPC:CHOL [4:1] and EPC:CHOL:SM [1:1:1], at pH 7.4 and 37 °C. As an example, Figure 3 represents the excitation and emission spectra of DPH in LUVs of EPC, as well as the Stern-Volmer plots with


12

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increasing concentrations of resveratrol (quencher). Similar plots were obtained for the other membrane systems with both probes DPH and TMA-DPH, at the same physiological conditions.

Figure 3 – Excitation and emission spectra of fluorescence quenching (A) and Stern-Volmer plots (B) of the probe DPH in LUVs of EPC at physiological conditions (pH 7.4, 37 °C) by increasing concentration of resveratrol (from 1 to 7). Note: In B open symbols (○) represent Stern-Volmer plot obtained by steady-state fluorescence measurements (I0/I) and solid symbols (●) represent Stern-Volmer plot obtained by lifetime fluorescence measurements (τ0/τ).

Table 2 – Values of the Stern-Volmer constant (KSV), static quenching constant (KS), dynamic quenching constant (KD) and bimolecular quenching rate constant (Kq) obtained from measurements of fluorescence quenching of DPH and TMA-DPH by resveratrol in LUVs of EPC, EPC:CHOL [4:1] and EPC:CHOL:SM [1:1:1], at physiological conditions (pH 7.4, 37 °C). DPH

TMA-DPH

System

KSV / M-1

KS / M-1

KD / M-1

Kq / x 108 M-1 s-1

KSV / M-1

KS / M-1

KD / M-1

Kq / x 108 M-1 s-1

EPC

23.8 ± 3.3

22.8 ± 0.3

0.9 ± 0.1

31.0 ± 4.2

6.6 ± 0.3

5.0 ± 0.5

1.6 ± 0.1

23.8 ± 0.9

EPC:CHOL

11.0 ± 0.3

10.4 ± 1.5

0.6 ± 0.1

11.4 ± 0.4

3.7 ± 0.4

3.3 ± 0.3

0.4 ± 0.1

6.4 ± 0.7

4.3 ± 1.0

0.3 ± 0.1

4.5 ± 0.3

2.1 ± 0.2

1.9 ± 0.2

0.2 ± 0.1

3.2 ± 0.3

EPC:CHOL:SM 4.6 ± 0.3

Note: All values represent the mean ± standard deviation (n = 3).


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The quenching of fluorescence can be analyzed through the Stern-Volmer plots in Figure 4 and the Stern-Volmer constants (KSV) which are listed in Table 2 for all systems with both probes, at pH 7.4 and 37 °C. From the analysis of Table 2, it is possible to conclude that the quenching mainly occurs by the formation of a nonfluorescent ground-state complex between the probe and resveratrol (static quenching), since the static quenching constant (KS) values are much higher than the dynamic quenching constant (KD) values for both probes in all systems, being KSV ≈ KS. In addition, the bimolecular quenching rate constant (Kq) values can be used to predict the location of resveratrol in the membranes, by comparing the different probes and systems 36.

Figure 4 – Stern-Volmer plots of the probe DPH and TMA-DPH obtained by steady-state fluorescence measurements (I0/I) in LUVs of EPC (○), EPC:CHOL [4:1] (●), and EPC:CHOL:SM [1:1:1] (●) at physiological conditions (pH 7.4, 37 °C) by increasing concentration of resveratrol.

According to the results, resveratrol was able to quench both probes, DPH and TMA-DPH. However, the decrease of the probe fluorescence was more pronounced for DPH than for TMADPH in all the model systems, presenting higher values of Kq. This difference is mainly related to the lipophilicity and degree of ionization of the compound at pH 7.4. In fact, resveratrol is


14

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found mostly in its neutral form (92%) at physiological pH, enabling a higher accumulation in the deeper region of the membrane by stablishing hydrophobic interactions with the phospholipid tails, which is in agreement with other studies in the literature 17. Moreover, it is also possible to conclude that the quenching process is totally dependent on the organization level and the degree of lipid packing of the membrane, since the Kq values are higher in EPC, followed by EPC:CHOL and finally EPC:CHOL:SM for both probes. A tight packing in brain model systems hinders the diffusion of the compound into the membrane, as already seen in the last section, which translates into a smaller fluorescence deactivation of both probes in bilayers with cholesterol and sphingomyelin molecules.

Resveratrol effect on membrane fluidity Steady-state fluorescence anisotropy with DPH and TMA-DPH probes was used to study the effect of resveratrol on the membrane fluidity. The method is based on the adjustment of the probe rotational motion when the stiffness of the membranes is changed. The effect of temperature on the DPH and TMA-DPH fluorescence anisotropy in liposomes of EPC, EPC:CHOL [4:1] and EPC:CHOL:SM [1:1:1] without resveratrol is shown in Figure 5.


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Figure 5 – Steady-state anisotropy of DPH and TMA-DPH as a function of temperature in EPC (■), EPC:CHOL [4:1] (∆) and EPC:CHOL:SM [1:1:1] (○) liposomes, at pH 7.4.

It is possible to observe that the anisotropy value decreases much more with increasing temperature in the acyl chain region (given by the probe DPH) than in the region of the phospholipid heads (given by the TMA-DPH probe). This is because the area of the tails has much more freedom to move than the area of the heads, even at higher temperatures. At the same time, we cannot see the phase transition in none of these membrane model systems composed of EPC, because they are made of a fluid mixture of lipids whose Tm is definitely below the minimum temperature reached in this experiment. Moreover, from the interpretation of the Figure 5, we can easily find out that EPC model presents much lower anisotropy values for both probes than the other two systems. In fact, it is well documented that the presence of cholesterol and sphingomyelin molecules in the lipid bilayers promotes the formation and maintenance of specific domains in the liquid-ordered phase, making this a more rigid and organized membrane, and consequently with higher anisotropy values. 45,46.


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Figure 6 shows the influence of resveratrol on the DPH fluorescence anisotropy as a function of the temperature, in the three lipid model systems at pH 7.4. The analysis of Figure 6 reveals that the incorporation of resveratrol increases the anisotropy of EPC model system, especially for higher temperatures, resulting in a stiffening of the membrane. On the other hand, the effect of resveratrol is just the opposite for the other systems (EPC:CHOL and EPC:CHOL:SM), specially for lower temperatures, showing a fluidizing effect when the membranes are more rigid and organized. Similar results with milder effects were obtained for the other probe TMA-DPH (Figure S3). The order parameter (S) which reflects the restriction of probe motion was also calculated for DPH and TMA-DPH in all membrane model systems, at 37 °C as shown in Table 3.


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Figure 6 – Steady-state anisotropy of DPH as a function of temperature in the absence (■), and in the presence of resveratrol 20 µM (∆) and 80 µM (○) in EPC (A), EPC:CHOL [4:1] (B), and EPC:CHOL:SM [1:1:1] (C) liposomes, at pH 7.4.

Table 3 – Values of order (S) obtained for EPC, EPC:CHOL [4:1], and EPC:CHOL:SM [1:1:1] liposomes at physiological conditions (pH 7.4 and 37 °C) in the absence and in the presence of resveratrol, obtained by measuring steady-state anisotropy of DPH and TMA-DPH. S (order) System DPH

TMA-DPH

EPC

n/d

0.494 ± 0.002

EPC + RSV 20 µM

n/d

0.498 ± 0.003

EPC + RSV 80 µM

n/d

0.518 ± 0.004


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EPC:CHOL

0.418 ± 0.002

0.627 ± 0.001

EPC:CHOL + RSV 20 µM

0.417 ± 0.004

0.625 ± 0.002

EPC:CHOL + RSV 80 µM

0.417 ± 0.003

0.624 ± 0.004

EPC:CHOL:SM

0.469 ± 0.003

0.683 ± 0.002

EPC:CHOL:SM + RSV 20 µM

0.449 ± 0.001

0.681 ± 0.002

EPC:CHOL:SM + RSV 80 µM

0.428 ± 0.001

0.677 ± 0.003

Note: All values represent the mean ± standard deviation (n = 3). n/d: no data with the probe DPH in EPC due to the fluidity of the system at 37 °C.

The presence of resveratrol increases the order parameter at 37 °C in the EPC membrane, indicating a decrease of the rotational mobility of the probe TMA-DPH. The same parameter could not be calculated for the probe DPH in EPC system because the region of the hydrocarbon chains near the center of the bilayer is even more disordered than the head group region

47

.

Concurrently, the effect of resveratrol on the order parameter was exactly the antagonistic for the other more organized models (EPC:CHOL and EPC:CHOL:SM), showing a fluidizing effect which explains the reduction of the order parameter for this two systems. The slight reduction on the brain mimetic model membranes order in the presence of resveratrol may be a strong indication that resveratrol itself allows to increase its penetration into this membranes what can be in the origin of its neuroprotective effects. These results provide evidence that the effect of resveratrol depends on the organizational level and fluidity of the different membranes under study, producing opposite effects in an attempt to adjust the conditions of their own membrane fluidity.

CONCLUSIONS


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The results on the literature show some controversy regarding the interactions of resveratrol with membrane model systems and its subsequent effects

17-23

. The origin of this

controversy can be explained by the different size and degree of saturation of the phospholipids acyl chains and the presence or absence of cholesterol and sphingomyelin lipids in the membrane model systems used. Thus, the present study brings some clarification through the use of different systems that allow mimicking different membrane barriers within the body, namely EPC model system which mimics the most common fluid lipid arrangement in cells; EPC:CHOL bilayer that represents a more organized membrane cellular targets rich in cholesterol domains; and EPC:CHOL:SM that mimics quite well brain membranes bearing further complexity and structural organization to the brain. The results showed that resveratrol has greater affinity for the EPC bilayers than for EPC:CHOL and EPC:CHOL:SM membrane models, probably because EPC is more fluid and allows a greater interaction and penetration of the polyphenol compound. For this reason, resveratrol intercalates easier and deeper between the EPC phospholipids, therefore contributing to the increase of microviscosity and stiffness of the bilayer. On the other hand, the increased difficulty in penetrating tight packed brain membranes composed of sphingomyelin and cholesterol lipids was already expected, since it is well known that central nervous system barriers present greater complexity and selectivity to the passage of substances 48,49

. However, we have found out that resveratrol has the ability to penetrate these membranes,

although to a lesser extent, perhaps due to its lipophilic characteristics, leading to a fluidizing effect, which allows the adjustment of the membrane homeostasis of brain barriers and what can be in the origin of its neuroprotective effects. Therefore, the results showed that the action of resveratrol depends on the initial state of membrane fluidity and order. This finding suggests a dual effect similar to what happens in the presence of cholesterol, thereby adjusting and


20

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controlling the fluidity of membranes. Indeed, it has been shown that cholesterol interacts differently within the membrane, depending on its position in the bilayer. Cholesterol partitions into the hydrophobic core of the membrane and causes a dramatic decrease in lipid fluidity in this region of the membrane, while the effects of this compound on the lipid packing order diminish when we move to a more superficial region of the membrane

50,51

. Additionally, the

lipid pattern of the cell membranes plays a key role in several membrane processes, namely in the localization, function, and activity of a number of membrane proteins, regulating cell signaling

52

. Consequently, resveratrol may be involved in controlling the activity of

transmembrane proteins such as G proteins, PLC and PKC, thereby regulating signal transduction, membrane trafficking, cell proliferation and susceptibility to apoptosis, through the organization and regulation of the cell membrane fluidity and order 17,18. In conclusion, this work contributed to identify the effects of resveratrol on the biophysical properties of biological membranes, which may explain the different pharmacological activities described for this compound, during its transit through different environments encountered in the organism.

ACKNOWLEDGMENTS This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE and by National Funds through FCT - Foundation for Science and Technology under the Pest-C/EQB/LA0006/2013 and FCOMP-01-0124-FEDER3728. The work also received financial support from the European Union (FEDER funds) under the framework of QREN through Project NORTE-07-0162-FEDER-000088. To all financing


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Page 22 of 28

sources the authors are greatly indebted. ARN and CN also acknowledge the FCT for financial support

through

the

PhD

grant

SFRH/BD/73379/2010

and

Post-Doc

Grant

SFRH/BPD/81963/2011.

SUPPORTING INFORMATION AVAILABLE Figure S1 – Absorption spectra (A), first-derivative (B), and second-derivative (C) of resveratrol (20 µM) incubated in LUVs of EPC:CHOL [4:1] (black lines) and LUVs of EPC:CHOL [4:1] without resveratrol (gray lines) at increasing lipid concentrations (from 1 to 11) in phosphate buffer at physiological conditions (pH 7.4, 37 °C). The curve (D) represents the fitting curve to experimental second-derivative spectrophotometric data as a function of EPC:CHOL [4:1] concentration, using a nonlinear least-squares regression method at wavelength 360 nm where the scattering is eliminated.

Figure S2 – Absorption spectra (A), first-derivative (B), and second-derivative (C) of resveratrol (20 µM) incubated in LUVs of EPC:CHOL:SM [1:1:1] (black lines) and LUVs of EPC:CHOL:SM [1:1:1] without resveratrol (gray lines) at increasing lipid concentrations (from 1 to 11) in phosphate buffer at physiological conditions (pH 7.4, 37 °C). The curve (D) represents the fitting curve to experimental second-derivative spectrophotometric data as a function of EPC:CHOL:SM [1:1:1] concentration, using a nonlinear least-squares regression method at wavelength 360 nm where the scattering is eliminated.


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Figure S3 – Steady-state anisotropy of TMA-DPH as a function of temperature in the absence (■), and in the presence of resveratrol 20 µM (∆) and 80 µM (○) in EPC (A), EPC:CHOL [4:1] (B), and EPC:CHOL:SM [1:1:1] (C) liposomes, at pH 7.4.

This information is available free of charge via the Internet at http://pubs.acs.org COMPETING INTERESTS The authors declare no competing financial interest.

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