Reverse Micelles As Antioxidant Carriers: An Experimental and

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Reverse Micelles As Antioxidant Carriers: An Experimental and Molecular Dynamics Study Maria D. Chatzidaki,† Konstantinos D. Papavasileiou,*,†,‡ Manthos G. Papadopoulos,† and Aristotelis Xenakis*,† †

Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation, 116 35 Athens, Greece National Center for Scientific Research “Demokritos”, Institute of Nanoscience and Nanotechnology, Aghia Paraskevi Attikis, 153 10 Athens, Greece

‡

S Supporting Information *

ABSTRACT: Water-in-oil microemulsions with biocompatible components were formulated to be used as carriers of natural antioxidants, such as hydroxytyrosol (HT) and gallic acid (GA). The system was composed of a mixture of natural surfactants, lecithin and monoglycerides, medium chain triglycerides, and aqueous phase. A dual approach was undertaken to study the structure and dynamics of these complicated systems. First, experimental data were collected by using adequate techniques, such as dynamic light scattering (DLS) and electron paramagnetic resonance (EPR) spectroscopy. Following this, a coarse-grained molecular dynamics (CGMD) study based on the experimental composition using the MARTINI force field was conducted. The simulations revealed the spontaneous formation of reverse micelles (RMs) starting from completely random initial conformations, underlying their enhanced thermodynamic stability. The location of the bioactive molecules, as well as the structure of the RM, were in accordance with the experimental findings. Furthermore, GA molecules were found to be located inside the water core, in contrast to the HT ones, which seem to lie at the surfactant interfacial layer. The difference in the antioxidants’ molecular location was only revealed in detail from the computational analysis and explains the RM’s swelling observed by GA in DLS measurements.

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INTRODUCTION In nonpolar solutions, amphiphilic molecules with certain physicochemical characteristics aggregate with the polar head groups oriented at the interior, forming so-called “reverse micelles” (RMs).1−3 RMs have the ability to incorporate polar solventsmost commonly waterand hydrophilic molecules. Once the polar solvent is solubilized in the hydrophilic cores, the term “RM” is commonly (but incorrectly) used again.4 With the addition of larger amounts of aqueous solvent, some authors use the term “W/O microemulsion” to distinguish between small and larger amphiphilic aggregates.5 In most studies, “RMs” and “W/O microemulsions” are considered as synonyms and refer to swollen surfactant aggregates. These colloidal systems have been extensively studied for their applications in hosting molecules of different partition coefficients. Experimentally, W/O microemulsions are of significant interest because they resemble biological membranes © 2017 American Chemical Society

and can be considered as model drug-delivery systems with the ability to solubilize bioactive substances while retaining their activity.6−9 On the other hand, RMs have often been proposed in the literature as ideal microenvironments for hosting polar enzymes in their hydrophilic pools due to their large interfacial area.10 The complexity of the above systems as well as the chemistry at the interface has been discussed experimentally.11−13 From a theoretical point of view, much attention has been paid to the water behavior at hydrophobic surfaces.14−16 Recently, surfactantless micelles in the “pre-ouzo” region were characterized using molecular dynamics simulations.17−19 Simulation models for the continuous medium and the surfactant molecules could shed light on the structure, Received: January 20, 2017 Revised: April 12, 2017 Published: May 8, 2017 5077

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Langmuir Table 1. Compositions of the Model Systems Studied composition (molecules) system

MCT

lecithin

DMG

water

HT

GA

1 2 3

1860 (93.08% w/w) 1860 (93.03% w/w) 1860 (93.02% w/w)

40 (2.95% w/w) 40 (2.94% w/w) 40 (2.94% w/w)

80 (1.98% w/w) 80 (1.98% w/w) 80 (1.98% w/w)

1100 (1.99% w/w) 1100 (1.99% w/w) 1100 (1.99% w/w)

4 (0.06% w/w) 4 (0.07% w/w)

vegetable oil and two types of surfactants and AH were both experimentally formulated and computationally evaluated.

formulation, and dynamics of the soft self-assembles in the molecular level.20 Very few models of RMs have been proposed,21 mainly discussing the self-assembly of the wellknown ionic surfactant AOT, bis(2-ethylhexyl) sulfosuccinate sodium salt.22−26 Recently, molecular dynamic simulations have been proposed for the formation of RMs based on biocompatible surfactants such as phospholipids27,28 or monoglycerides.29 The use of a combination of surfactants with different morphologies and hydrophilic−lipophilic deviations (HLDs)30 shows numerous advantages regarding applications in the pharmaceutical, cosmetics, food, and other sectors. Although many experimental studies have dealt with such complex systems, modeling would shed light on the interactions and physicochemical characteristics in a molecular basis. Few cases of such complex nonbiocompatible31 or food-grade32 systems with mixtures of surfactants have been investigated theoretically so far. Generally, the determination of the nanostructure of W/ O microemulsions, when a mixture of surfactants and additives is used, is not trivial, neither theoretically nor experimentally. To this respect, only a few studies have compared the computational findings with experimental data on a single system leading to a more comprehensive approach.33 From an experimental point of view, much attention has been paid on the solubilization of hydrophilic molecules to the polar pools lacking theoretical support in a molecular basis. A few studies concerning enzyme folding and interactions at the interior of an RM in terms of molecular dynamics have been proposed.34 Phenolic antioxidants (AH)35 have been used as model encapsulated molecules in W/O microemulsions,36 mimicking a structure that resembles that of natural oils.37,38 Hydroxytyrosol (HT) is found in the leaves of the olive tree (Olea europaea), in extra virgin olive oil, and in olive mill wastewaters, while gallic acid (GA), an antioxidant with a lower partition coefficient, is found in extra virgin olive oil39 and green tea40 extracts. In the present study, a complex W/O microemulsion composed of medium-chain triglycerides (MCTs) as the oil phase, lecithin and monoglycerides as surfactants, and an aqueous phase was formulated. The system in the presence or absence of HT or GA was experimentally investigated for its structure, in terms of RM size, and membrane dynamics, using dynamic light scattering (DLS) and electron paramagnetic resonance (EPR) spectroscopy. Furthermore, a coarse-grained molecular dynamics study (CGMD) using the MARTINI force field was conducted to support experimental data. This investigation sheds light on the spontaneous formation mechanisms of RMs in the presence of water in a complex system containing two types of biocompatible surfactants in a triglyceride matrice. In addition, the molecular dynamic study was expanded for the same system in the presence of HT or GA, AH with different partitioning properties, in order to determine their localization within the microdomains and the possible induced RM conformational changes. To our knowledge, this is the first time that RM solutions consisting of

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EXPERIMENTAL SECTION

Materials. Medium-chain (caprylic/capric) triglycerides (Stelliesters MCT 6535) were from Stearinerie Dubois, France. Distilled monoglycerides of vegetable fatty acids (DMG 0295) were donated by Palsgaard, Denmark. Deoiled soy lecithin (Solac FS-B) was a kind gift from Solae (Switzerland). 5-Doxylstearic acid (5-DSA) and 16doxylstearic acid (16-DSA) spin probes, gallic acid (99.3%) 1,2 propanediol, and galvinoxyl free radical were obtained from Sigma− Aldrich (Germany). Hydroxytyrosol (98% purity) was from Extrasynthèse (France), and highly purified water was obtained from a Millipore Milli Q Plus device. All chemicals were used as received without any further purification. W/O Microemulsion Formulation and Antioxidant Encapsulation. Biodegradable RM solutions consisting of MCT as the oil phase, lecithin and DMG as surfactants, and 30% (w/w) propylene glycol (PG) in water as the aqueous phase were formulated as described by Chatzidaki et al.41 The aqueous phase was added gradually to obtain single-phase RM solutions with the following composition: 93.1% MCT, 4.9% of lecithin/DMG mixture (3:2 weight ratio), and 2% of aqueous phase (30% PG). The RM solutions were stored at 25 °C. For the formation of AH-loaded RM solutions, HT and GA were initially solubilized in ethanol at specific concentrations. Following this, the appropriate amounts of these solutions were transferred in test tubes and ethanol was evaporated. One mL of the RM solutions was then added to the tube where the antioxidant was previously deposited. The final concentration of antioxidants to the RM solutions was 0.7 mM. Dynamic Light Scattering Measurements. DLS measurements were performed using a Zetasizer Nano ZS (ZEN3600) from Malvern Instruments (U.K.) equipped with a He−Ne laser, using a noninvasive backscatter technology. The average hydrodynamic diameter of the RMs was measured at 25 °C at a detection angle of 173°. Aqueous and organic phases were filtered through 0.45 μm filters before mixing, and a quartz dustfree cuvette was used for the measurements. The autocorrelation functions were measured, and the average droplet size was calculated using the Stokes−Einstein equation Rh = kBT/(6πηD), where kB is the Boltzmann constant, T is the temperature in Kelvin, η is the viscosity of the solvent, and D is the diffusion coefficient. Analysis was obtained from the software supplied by the manufacturer using the cumulant method and the CONTIN routine. Electron Paramagnetic Resonance Spectroscopy. EPR spectra were recorded at constant room temperature (25 °C), using a Bruker EMX EPR spectrometer operating at the X-Band. Samples were contained in a WG-813-Q Wilmad (Buena, NJ) Suprasil flat cell. Typical instrument settings were as follows: center field, 348 mT; scan range, 10 mT; gain, 2.83 × 103; time constant, 163 ms; conversion time, 5 ms; modulation amplitude, 0.4 mT; frequency, 9.77 GHz. Data collection and analysis were performed using the Bruker WinEPR acquisition and processing program. Experimental results were analyzed in terms of rotational correlation time (τR) and order parameter (S) of the amphiphilic spin probes 5-DSA and 16-DSA as described elsewhere.42,43 Probes were initially solubilized in ethanol at specific concentrations. Following this, the appropriate amounts of these solutions were transferred in test tubes and ethanol was evaporated. One mL of the RM solutions was then added to the tube where the spin probe was previously deposited. The concentration of the spin probes was finally 10−4 M. 5078

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Figure 1. Fully atomistic structures of the RM solution molecules. The CG mapping scheme used is also illustrated, along with the bead types and masses (in g mol−1) employed. The aliphatic chain length is also depicted. Coarse-Grained Molecular Dynamics Simulation. To establish the spontaneous self-assembly of lecithin, DMG, and water into RM in MCT and elucidate on the behavior of the encapsulated HT and GA antioxidants, CGMD simulations were performed. Originally, representative fully atomistic configurations of the aforementioned molecules and their mixtures were prepared in conjunction with the experimental w/w compositions and are collected in Table 1. Initial conformations were obtained by means of the Scienomics MAPS software package,44 in which the constituent molecules were inserted into the cubic boxes using a modified configuration bias scheme.45 The resulting cubic unit cells with 12 nm edge lengths contained randomly distributed molecules described in a fully atomistic regime. These were in turn mapped to coarse-grained (CG) topologies in accordance to the MARTINI force field (version 2.0)46 by means of the Visual Molecular Dynamics (VMD) CG Builder utility.47 The initial fully atomistic description of the constituent molecules along with the mapping scheme used is illustrated in Figure 1. To ensure accurate kinetic description of the systems, realistic bead masses were specified. The MARTINI force field is based on a four-to-one mapping, i.e., four heavy atoms are represented by a bead or single interaction center. Oil and surfactant moieties were parametrized using the standard bead mapping for the MCT, lecithin, and DMG heads, along with combinations of the three-to-one mapping for some of their alkyl tail CH2 groups. HT and GA ring moieties were modeled according to the standard MARTINI two- or three-to-one mapping of ring atoms onto CG beads procedure.46 Water was mapped according to the standard four-to-one mapping. Also, in accordance to MARTINI, ∼7% of the water used was antifreeze particles (20 out of a total of 275 CG water beads) so as to ensure that freezing of the CG water will not occur.46 All CGMD simulations were performed by utilizing the NAMD 2.10 software,48 with periodic boundary conditions imposed in all directions. Prior to production runs and in order to nullify unphysical bad contacts between CG beads, all systems were subjected to a total of 40 000 conjugate gradient energy minimization cycles. Then, system temperature was gradually raised from 1 to 100 K in a 100 ps run with a 2 fs time step by performing Langevin dynamics, with a damping coefficient of 5 ps−1 in the canonical (NVT) ensemble. This was followed by 100 ps in the isothermal−isobaric (NPT) ensemble at the same temperature (100 K) and 0.33 atm pressure, maintained with a Nosé−Hoover Langevin piston,48,49 using a piston period of 2 ps and a

decay time of 1 ps. A cycle of interchanging 200 ps of NVT and NPT runs ensued, where T and P were raised from 100 to 300 K and from 0.33 to 1 atm, respectively. This practice allowed the avoidance of random crashes due to unphysical forces. All systems were then equilibrated for another 5 ns in the NPT ensemble, during which the time step was increased from 2 to 20 fs, which was maintained throughout the 85 μs NPT production runs. The key simulation parameters are summarized in Table 2.

Table 2. Summary of the Production CGMD Simulation Parameters simulated system

ensemble temperature pressure number of beads cubic unit cell edge length time step duration

1 (empty RM)

2 (HT loaded RM)

3 (GA loaded RM)

NPT 300 K 1 atm 19755 12 nm

NPT 300 K 1 atm 19771 12 nm

NPT 300 K 1 atm 19767 12 nm

20 fs 85 μs

20 fs 85 μs

20 fs 85 μs

Standard Lorentz−Berthelot combining rules50 were used for the well depth ε and the size parameter σ to describe nonbonded LennardJones interactions between sites of different type i and j according to the following expressions: εij =

εiεj and σij =

σi + σj 2

(1)

A cutoff of 1.2 nm was employed for the nonbonded interactions. The GROMACS switching function recommended defaults were applied for both Lennard-Jones (LJ) and Coulomb potentials for use of the MARTINI force field in NAMD. Specifically, the LJ potential was smoothly shifted to zero between 0.9 and 1.2 nm. A similar approach was employed for the electrostatic interactions, considering a Coulombic potential with a relative permittivity of 15 together with a shift function from 0 to 1.2 nm. The neighbor list was updated every 10 steps, with a 1.4 nm pair list cutoff. Analysis was performed by means of the GROMACS 4.6.7 software,51−55 on the last 40 ns of the 5079

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Langmuir produced trajectories, whereas radial distribution functions (RDFs) were calculated by means of the Visual Molecular Dynamics (VMD) software.47

probably due to interactions of GA with ethanol. Here, the rotational correlation time is identical with that of the empty state, indicating that GA is not interfering with the amphiphilic membrane as suggested from the swelling of the RM observed by DLS. Furthermore, 16-DSA, the free electron of which is located deeper in the hydrophobic tails, seems to move almost freely for both the empty and loaded cases. The identical profile of the spectra indicates that the bioactive molecules are much deeper inside the membrane and closer to the hydrophilic cores. MD Section. The spontaneous formation of small RMs was observed around the 1 μs timemark, and it was characterized by the aggregation of the lecithin, DMG, and water molecules into two individual clusters. These clusters merged into two larger formations after ∼16 μs that were stabilized and maintained for another 58 μs. It should be noted that this pattern is shared for the systems with HT or GA. After this point and by the end of the simulations, a single large RM had finally formed in all cases. Figure 2 shows snapshots from the simulations,

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RESULTS Experimental Section. Dynamic Light Scattering. Free and AH-loaded RM solutions were formulated as described previously by Chatzidaki et al.41 Systems were measured using DLS in the presence and absence of HT and GA in order to determine the hydrodynamic diameter of the reversed swollen micelles (Table 3). The mean size was 20 nm for both empty Table 3. Hydrodynamic Diameters of Free and AntioxidantLoaded Reverse Micelle (RM) Solutions (Antioxidants (AH): Hydroxytyrosol (HT) or Gallic Acid (GA)) system

average size, D (nm)

empty RM HT-loaded RM GA-loaded RM

20 ± 1 20 ± 1 30 ± 4

and HT-loaded RM solutions. The presence of HT shows no significant difference in the hydrodynamic radius, as seen previously in a 5-fold higher concentration.41 This is probably due to the amphiphilic character of HT as revealed from its partition coefficient (log Po/w = 1.1).56 On the other hand, the hydrodynamic diameter of GA-loaded systems was increased to 30 nm, indicating enhanced swelling of the RM, which indicates that GA is encapsulated at the hydrophilic core of the RM (log Po/w = −0.53).57 Figure S1 provides comparative information about the intensity/size profiles of the various samples. Electron Paramagnetic Resonance Spectroscopy. EPR measurements in the presence and absence of HT and GA were undertaken in order to investigate the possible changes of the interfacial properties of the surfactant layer using two different spin probes. 5-DSA contains a free electron attached to the fifth carbon of the stearic acid moiety, while 16-DSA contains a free electron attached to the 16th carbon. As shown in Table 4, the order parameter and the rotational correlation

Figure 2. Progression of the formation of the RM. Lecithin (gray), DMG (green), and water (blue) beads are illustrated. The unit cell is also shown, along with periodic images of the system. MCT molecules have been omitted for clarity.

Table 4. S and τR Parameters for Free and AntioxidantLoaded Microemulsions for 5- and 16-Spin Probes (Values Are Means ± SD (n = 3)) 5-DSA

indicating the progression from the initially amorphous system to the finally formed single RM, displaying the lecithin, DMG, and water molecule’s great potential for self-assembly inside the MCT phase, guided by both hydrophilic and hydrophobic effects. The complete evolution of the aggregation is shown in a movie file provided in the Supporting Information. The RM formed was then characterized on the basis of intermolecular distances by means of the g_clustsize tool in GROMACS.51−55 Lecithin, DMG, and water molecules that were located beyond 0.7 nm, a distance corresponding to the average location of their intermolecular RDF’s first minimum, were not considered as part of the RM (Figure 3). It should be noted that, as the local environment of the RM components is anisotropic, RDFs converge to zero and not unity at long distances.58 This is commonly encountered in systems in confinement or at interfaces.59−62 Typically, corrections are applied to normalize the density;63 however, as the RDF peak and minima positions are not affected, such a correction was not applied. The calculated molecules comprising the RMs according to clustering analysis are collected in Table 5. It can be deduced

16-DSA

spin probe

S

τR (ns)

S

τR (ns)

empty RM HT-loaded RM GA-loaded RM

0.13 ± 0.01 0.13 ± 0.00

2.19 ± 0.08 2.20 ± 0.08

0.04 ± 0.00 0.04 ± 0.00

0.47 ± 0.01 0.47 ± 0.02

0.13 ± 0.01

2.23 ± 0.08

0.04 ± 0.00

0.48 ± 0.01

time are not affected in the presence of encapsulated AH, meaning that the membrane’s rigidity remains unaltered. Following a previous study41 using the same system as carrier of HT, findings revealed a slight increase in the mobility of the spin probe in the presence of the bioactive molecule, indicating its partitioning at the oil and polar phases. In the present study, the concentration of HT is 5-fold less, leading to a decrease of the HT monomers in the medium. In a recent study,36 GA at the same concentration was encapsulated at a different microemulsion containing 50% w/w of surfactants and cosurfactants. Interestingly, in this study the addition of the GA increased the mobility of the spin probe, 5080

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Figure 3. Intermolecular RDFs of lecithin (gray), DMG (green), and water (blue). The dashed line indicates the distance criterion used for clustering analysis. The unit cell illustrating the reverse micelle in MCT is also shown.

This is also corroborated by clustering analysis, according to which only two HT molecules on average prefer to lie inside the RM’s water core and prefer to lie at the lecithin−DMG/ water interface (Figure 5). In contrast, all GA molecules are immersed inside the RM’s core.

Table 5. RM Composition Averages and Their Corresponding Fluctuations of the Model Systems Studied According to Clustering Analysis composition (molecules)

a

system

lecithin

DMG

watera

HT

1 2 3

40 40 40

52 (1) 53 (2) 52 (1)

215 (2) 218 (2) 213 (2)

2 (1)

GA

4

Including antifreeze water particles.

that the lecithin molecules constitute the RM’s building block, as they all participate in micelle formation. On the other hand, 52 DMG molecules participate on average in RM formation in all three model systems. This corresponds to ∼65% of their total population and illustrates that their role in RM formation is rather complementary. Therefore, the aggregation number, Nagg, i.e., the number of surfactant molecules forming the RM, is on average equal to 92 in all systems. The RM’s core comprises 215 water molecules on average, which accounts for roughly 78% of the total water content. These compositions correspond to an average waterto-surfactant molar ratio, W0 = [water]/[lecithin + DMG], value equal to 2.34 in all RMs. RDF analysis revealed that GA molecules prefer to lie inside the water core, in contrast to HT, which are less exposed to the RM’s water interior (Figure 4).

Figure 5. Clustered RMs. Lecithin (gray), DMG (green), and water (blue) beads are illustrated. Clipped RMs are also shown for bisect. HT (orange) and GA (red) antioxidant molecules are shown. HT lies at the water/surfactant interface, while GA is buried inside the water core.

RM formation is a dynamic process and the micelle’s size is not fixed but rather described as a distribution. The RM’s structural characteristics, either with or without the introduced antioxidants, were characterized by means of the radius of gyration, which was calculated by the g_gyrate module according to the following equation, Rg =

∑i ri 2mi ∑i mi

(2)

where mi is the mass of bead i and ri is the distance between each bead and the center of mass of each bead involved. Figure 6 shows the fluctuation of the radius of gyration of the entire micelle as a function of time, averaged over the last 40 ns of the production runs. It can be deduced that overall the micelle retains its stability, as portrayed by the constant fluctuation of Rg. Furthermore, to acquire a sense of its overall shape, the RM’s eccentricity, e, was calculated from the three time-averaged components of the

Figure 4. Intermolecular RDFs of HT (orange) and GA (red) with water (WAT). The different intensities highlight the exposure of each antioxidant in water. 5081

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Figure 6. Calculated radii of gyration of the entire lecithin/DMG/ water reverse micelle (RM) systems without antioxidants and including HT and GA molecules, respectively, over the last 40 ns of the simulation. The ensemble averages of Rg are also illustrated with black lines.

Figure 7. Calculated eccentricity values, e, of the entire lecithin/ DMG/water RMs with and without HT and GA molecules over the last 40 ns of the simulation. The ensemble averages of e are also illustrated with black lines.

principle moments of inertia, namely, Ix, Iy, and Iz. The eccentricity values were evaluated by the following expression,58,64 I e = 1 − min Iavg (3)

antioxidant concentrations used in the simulations are not able to induce changes in RM’s size. However, results from RDF (Figure 4) and density analysis provide insight on their location, which along with the subtle differences in Rs hint at the underlying cause of the swelling observed for the GAcontaining RMs. Specifically, the calculated total density of all systems is equal to 814 ± 1 kg m−3, a value lower by ∼14% from the experiment (i.e., 950 kg m−3); this is attributed to the CG description and particularly to the addition of water antifreeze particles.46 Since the RM sphericity was established, the radial density profiles with respect to the micelle’s center of mass (r = 0) were calculated and are illustrated in Figure 8. It can be deduced that the water core is protected from the MCT phase by the lecithin and DMG interface, occupying the interior of the RM, as shown by the broad region of nonzero density delimited at ±2.3 nm. As far as the HT and GA molecules are concerned, density profiles clearly illustrate their tendency to be less and more localized in the water core of the RM, respectively, in agreement with the aforementioned RDF analysis. This finding is supportive of the GA tendency to localize in the water core, hence explaining the swelling observed in the experiment.

where Imin is the moment of inertia along the x, y, or z axis with the smallest value and Iavg is the average value over all three axes. The moments of inertia were extracted from the radius of gyration analysis. In general, eccentricity values equal to zero are characteristic of perfect spherical shapes, while values close to unity are indicative of flat or needle-like shapes. From the eccentricity values presented in Table 6 and Figure 7, it is deduced that the RMs possess uniform, spherical characteristics. In this case, Rg relates to the Stokes or effective radius of the RMs, Rs, by the following expression,65

Rs =

5 ⟨R g⟩ 3

(4)

where the brackets denote the ensemble average. Rs describes the radius of an equivalent sphere with the same diffusion constant, rather than a specific physical length. In Table 6 the calculated Rs values of the entire micelles are reported, along with their corresponding Rg values. The system sizes examined by MD are smaller by an order of magnitude compared to their experimental ones, reaching ∼8 nm in effective diameter. Also, our simulations show that these dimensions remain essentially unchanged for all systems, as Rg computed values show minor discrepancies, falling within statistical uncertainty. This essentially means that the

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CONCLUSIONS Structural analysis of the formation of RM solutions using MCT as the oil phase, lecithin and DMG as surfactants, and aqueous phase was effectively performed. The formulated systems were able to solubilize hydrophilic antioxidants with different partition coefficients. The molecular dynamics of such a complex system was extensively studied to be compared with the experimental findings. The CGMD simulations revealed

Table 6. Ensemble Averages of the RM’s Aggregation Number, Nagg; Water-to-Surfactant Ratio, W0; Radius of Gyration of the Entire Micelle, Rg; Effective Radius of the Entire Micelle, Rs; Radius of Gyration of the Micelle’s Water Core, RWAT ; Principle g Moments of Inertia, I1, I2, and I3; and Eccentricity Values, e system

Nagg

W0

Rg (nm)

Rs (nm)

RWAT (nm) g

I1 (104amu nm2)

I2 (104amu nm2)

I3 (104amu nm2)

e

1 2 3

92 93 92

2.34 2.34 2.32

2.63 (0.02) 2.65 (0.02) 2.67 (0.02)

3.40 3.42 3.45

1.66 (0.03) 1.68 (0.03) 1.69 (0.03)

5.00 (0.14) 5.41 (0.15) 5.21 (0.14)

5.52 (0.13) 6.02 (0.16) 5.62 (0.15)

5.91 (0.18) 6.43 (0.18) 6.04 (0.18)

0.09 0.09 0.07

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Figure 8. Density profiles as a function of distance from the RM center, r, of the lecithin (LEC)/DMG/water (WAT) systems (a) without antioxidants, (b) including HT, and (c) including GA molecules. HT tendency to lie at the WAT/LCT/DMG interface is clearly illustrated. On the other hand, GA is located inside the RM’s water core.

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that RM formation is spontaneous, starting from completely amorphous initial mixture conformations, underlying their enhanced thermodynamic stability. This was the primary objective of our study, highlighting the agreement between the experimental and computational findings. Moreover, the dynamics of the systems in the molecular scale as well as the detailed characterization of the RM’s structure and antioxidants’ location were evaluated using a comprehensive computational work. Computations are again supportive of the experimental measurements. No significant structural changes are induced to the RMs upon the addition of antioxidant molecules at these small concentrations in CGMD simulations. However, density analysis showed that GA molecules are solely immersed in the RM’s water core, while HT is more likely to interfere with the interface, in accordance with the DLS data. Concerning the membrane dynamics, the data obtained by the EPR technique were not conclusive probably because of the small antioxidant concentrations. Nevertheless, the difference in the bioactive molecules location was revealed in detail from the computational analysis. Molecular dynamics of such complex systems is therefore important not only to support the structural characterization but also to shed light to the mechanisms of the spontaneous formation and interactions. In this work, we used a simplified computational model to get an insight of the experimental findings in a molecular basis. It would therefore be of interest to further investigate (i) the size effects on RM shape and structural characteristics; (ii) the gradual increase of the antioxidant’s concentration, in order to establish a relationship on the maximum water core loading; and (iii) the inclusion of propylene glycol, in order to observe and examine possible rearrangements of water and antioxidant molecules in the aqueous pools.

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AUTHOR INFORMATION

ORCID

Konstantinos D. Papavasileiou: 0000-0002-2322-7422 Aristotelis Xenakis: 0000-0002-5596-7433 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by computational time granted from the Greek Research & Technology Network (GRNET) in the National HPC facility, ARIS, under Project ID pr001017. Scienomics SARL is acknowledged for providing MAPS software used to generate initial structures of the systems simulated.

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REFERENCES

(1) Luisi, P. L.; Straub, B. Reverse Micelles: Biological and Technological Relevance of Amphiphilic Structures in Apolar Media; Plenum Pub Corp.: 1984. (2) De Gennes, P.; Taupin, C. Microemulsions and the flexibility of oil/water interfaces. J. Phys. Chem. 1982, 86 (13), 2294−2304. (3) Moulik, S.; Paul, B. Structure, dynamics and transport properties of microemulsions. Adv. Colloid Interface Sci. 1998, 78 (2), 99−195. (4) Chevalier, Y.; Zemb, T. The structure of micelles and microemulsions. Rep. Prog. Phys. 1990, 53 (3), 279. (5) Zulauf, M.; Eicke, H. F. Inverted micelles and microemulsions in the ternary system water/aerosol-OT/isooctane as studied by photon correlation spectroscopy. J. Phys. Chem. 1979, 83 (4), 480−486. (6) Constantinides, P. P. Lipid microemulsions for improving drug dissolution and oral absorption: physical and biopharmaceutical aspects. Pharm. Res. 1995, 12 (11), 1561−1572. (7) Lawrence, M. J.; Rees, G. D. Microemulsion-based media as novel drug delivery systems. Adv. Drug Delivery Rev. 2000, 45 (1), 89−121. (8) Kalaitzaki, A.; Poulopoulou, M.; Xenakis, A.; Papadimitriou, V. Surfactant-rich biocompatible microemulsions as effective carriers of methylxanthine drugs. Colloids and Surfaces A. Colloids Surf., A 2014, 442, 80−87. (9) Theochari, I.; Goulielmaki, M.; Danino, D.; Papadimitriou, V.; Pintzas, A.; Xenakis, A. Drug nanocarriers for cancer chemotherapy based on microemulsions: the case of Vemurafenib analog PLX4720. Colloids Surf., B 2017, 154, 350−356. (10) Stamatis, H.; Xenakis, A.; Kolisis, F. N. Bioorganic reactions in microemulsions: the case of lipases. Biotechnol. Adv. 1999, 17 (4), 293−318. (11) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed.; John Wiley: 1980.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b00213. DLS results of reverse micellar solutions (PDF) Movie files illustrating simulation progression toward self-assembly (AVI) 5083

DOI: 10.1021/acs.langmuir.7b00213 Langmuir 2017, 33, 5077−5085

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Langmuir (12) Maitra, A. Determination of size parameters of water-Aerosol OT-oil reverse micelles from their nuclear magnetic resonance data. J. Phys. Chem. 1984, 88 (21), 5122−5125. (13) Papadimitriou, V.; Pispas, S.; Syriou, S.; Pournara, A.; Zoumpanioti, M.; Sotiroudis, T. G.; Xenakis, A. Biocompatible microemulsions based on limonene: formulation, structure, and applications. Langmuir 2008, 24 (7), 3380−3386. (14) Geiger, A. Mechanisms of the molecular mobility of water. J. Mol. Liq. 2003, 106 (2), 131−146. (15) Lee, C. Y.; McCammon, J. A.; Rossky, P. The structure of liquid water at an extended hydrophobic surface. J. Chem. Phys. 1984, 80 (9), 4448−4455. (16) Harpham, M. R.; Ladanyi, B. M.; Levinger, N. E.; Herwig, K. W. Water motion in reverse micelles studied by quasielastic neutron scattering and molecular dynamics simulations. J. Chem. Phys. 2004, 121 (16), 7855−7868. (17) Schoettl, S.; Marcus, J.; Diat, O.; Touraud, D.; Kunz, W.; Zemb, T.; Horinek, D. Emergence of surfactant-free micelles from ternary solutions. Chemical Science 2014, 5 (8), 2949−2954. (18) Zemb, T. N.; Klossek, M.; Lopian, T.; Marcus, J.; Schöettl, S.; Horinek, D.; Prevost, S. F.; Touraud, D.; Diat, O.; Marčelja, S.; et al. How to explain microemulsions formed by solvent mixtures without conventional surfactants. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (16), 4260−4265. (19) Schöttl, S.; Horinek, D. Aggregation in detergent-free ternary mixtures with microemulsion-like properties. Curr. Opin. Colloid Interface Sci. 2016, 22, 8−13. (20) Mudzhikova, G.; Brodskaya, E. Computer simulation of reverse micelles and water-in-oil microemulsions. Colloid J. 2012, 74 (3), 269−279. (21) Brown, D.; Clarke, J. Molecular dynamics simulation of a model reverse micelle. J. Phys. Chem. 1988, 92 (10), 2881−2888. (22) Faeder, J.; Ladanyi, B. Molecular dynamics simulations of the interior of aqueous reverse micelles. J. Phys. Chem. B 2000, 104 (5), 1033−1046. (23) Linse, P.; Halle, B. Counterion NMR in heterogeneous aqueous systems: A molecular dynamics simulation study of the electric field gradient. Mol. Phys. 1989, 67 (3), 537−573. (24) Linse, P. Molecular dynamics study of the aqueous core of a reversed ionic micelle. J. Chem. Phys. 1989, 90 (9), 4992−5004. (25) Alaimo, M.; Kumosinski, T. Investigation of hydrophobic interactions in colloidal and biological systems by molecular dynamics simulations and NMR spectroscopy. Langmuir 1997, 13 (7), 2007− 2018. (26) Chowdhary, J.; Ladanyi, B. M. Molecular simulation study of water mobility in aerosol-OT reverse micelles. J. Phys. Chem. A 2011, 115 (23), 6306−6316. (27) Vierros, S.; Sammalkorpi, M. Phosphatidylcholine reverse micelles on the wrong track in molecular dynamics simulations of phospholipids in an organic solvent. J. Chem. Phys. 2015, 142 (9), 094902. (28) Abel, S. p.; Galamba, N.; Karakas, E.; Marchi, M.; Thompson, W. H.; Laage, D. On the Structural and Dynamical Properties of DOPC Reverse Micelles. Langmuir 2016, 32 (41), 10610−10620. (29) Bradley-Shaw, J. L.; Camp, P. J.; Dowding, P. J.; Lewtas, K. Glycerol Monooleate Reverse Micelles in Nonpolar Solvents: Computer Simulations and Small-Angle Neutron Scattering. J. Phys. Chem. B 2015, 119 (11), 4321−4331. (30) Kunz, W.; Testard, F.; Zemb, T. Correspondence between curvature, packing parameter, and hydrophilic− lipophilic deviation scales around the phase-inversion temperature. Langmuir 2009, 25 (1), 112−115. (31) Tobias, D. J.; Klein, M. L. Molecular dynamics simulations of a calcium carbonate/calcium sulfonate reverse micelle. J. Phys. Chem. 1996, 100 (16), 6637−6648. (32) Liu, J.; Wu, J.; Sun, J.; Wang, D.; Wang, Z. Investigation of the phase behavior of food-grade microemulsions by mesoscopic simulation. Colloids Surf., A 2015, 487, 75−83.

(33) Negro, E.; Latsuzbaia, R.; De Vries, A.; Koper, G. Experimental and molecular dynamics characterization of dense microemulsion systems: morphology, conductivity and SAXS. Soft Matter 2014, 10 (43), 8685−8697. (34) Martinez, A. V.; DeSensi, S. C.; Dominguez, L.; Rivera, E.; Straub, J. E. Protein folding in a reverse micelle environment: The role of confinement and dehydration. J. Chem. Phys. 2011, 134 (5), 055107. (35) Shahidi, F.; Janitha, P.; Wanasundara, P. Phenolic antioxidants. Crit. Rev. Food Sci. Nutr. 1992, 32 (1), 67−103. (36) Chatzidaki, M. D.; Mitsou, E.; Yaghmur, A.; Xenakis, A.; Papadimitriou, V. Formulation and characterization of food-grade microemulsions as carriers of natural phenolic antioxidants. Colloids Surf., A 2015, 483, 130−136. (37) Xenakis, A.; Papadimitriou, V.; Sotiroudis, T. G. Colloidal structures in natural oils. Curr. Opin. Colloid Interface Sci. 2010, 15 (1), 55−60. (38) Chen, B.; Han, A.; Laguerre, M.; McClements, D. J.; Decker, E. A. Role of reverse micelles on lipid oxidation in bulk oils: impact of phospholipids on antioxidant activity of α-tocopherol and Trolox. Food Funct. 2011, 2 (6), 302−309. (39) Boskou, D. Olive oil: Chemistry and technology; AOCS Press: Champaign, IL, 1996. (40) Wang, H.; Helliwell, K.; You, X. Isocratic elution system for the determination of catechins, caffeine and gallic acid in green tea using HPLC. Food Chem. 2000, 68 (1), 115−121. (41) Chatzidaki, M. D.; Arik, N.; Monteil, J.; Papadimitriou, V.; LealCalderon, F.; Xenakis, A. Microemulsion versus emulsion as effective carrier of hydroxytyrosol. Colloids Surf., B 2016, 137, 146−151. (42) Avramiotis, S.; Papadimitriou, V.; Hatzara, E.; Bekiari, V.; Lianos, P.; Xenakis, A. Lecithin organogels used as bioactive compounds carriers. A microdomain properties investigation. Langmuir 2007, 23 (8), 4438−4447. (43) Chatzidaki, M. D.; Papadimitriou, K.; Alexandraki, V.; Tsirvouli, E.; Chakim, Z.; Ghazal, A.; Mortensen, K.; Yaghmur, A.; Salentinig, S.; Papadimitriou, V.; et al. Microemulsions as Potential Carriers of Nisin: Effect of Composition on Structure and Efficacy. Langmuir 2016, 32 (35), 8988−8998. (44) Scienomics MAPS. http://www.scienomics.com/. (45) Ramos, J.; Peristeras, L. D.; Theodorou, D. N. Monte Carlo Simulation of Short Chain Branched Polyolefins in the Molten State. Macromolecules 2007, 40 (26), 9640−9650. (46) Marrink, S. J.; Risselada, H. J.; Yefimov, S.; Tieleman, D. P.; de Vries, A. H. The MARTINI force field: Coarse grained model for biomolecular simulations. J. Phys. Chem. B 2007, 111 (27), 7812− 7824. (47) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual molecular dynamics. J. Mol. Graphics 1996, 14 (1), 33−38. (48) Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.; Chipot, C.; Skeel, R. D.; Kale, L.; Schulten, K. Scalable molecular dynamics with NAMD. J. Comput. Chem. 2005, 26 (16), 1781−1802. (49) Feller, S. E.; Zhang, Y. H.; Pastor, R. W.; Brooks, B. R. Constant-Pressure Molecular-Dynamics Simulation - the Langevin Piston Method. J. Chem. Phys. 1995, 103 (11), 4613−4621. (50) Smith, F. T. Atomic Distortion and the Combining Rule for Repulsive Potentials. Phys. Rev. A: At., Mol., Opt. Phys. 1972, 5 (4), 1708−1713. (51) Lindahl, E.; Hess, B.; van der Spoel, D. GROMACS 3.0: a package for molecular simulation and trajectory analysis. J. Mol. Model. 2001, 7 (8), 306−317. (52) Van Der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. C. GROMACS: Fast, flexible, and free. J. Comput. Chem. 2005, 26 (16), 1701−1718. (53) Berendsen, H. J. C.; van der Spoel, D.; van Drunen, R. GROMACS: A message-passing parallel molecular dynamics implementation. Comput. Phys. Commun. 1995, 91 (1), 43−56. (54) Bekker, H.; Berendsen, H. J. C.; Dijkstra, E. J.; Achterop, S.; Vondrumen, R.; Vanderspoel, D.; Sijbers, A.; Keegstra, H.; Reitsma, B.; Renardus, M. K. R. GROMACSA Parallel Computer for 5084

DOI: 10.1021/acs.langmuir.7b00213 Langmuir 2017, 33, 5077−5085

Article

Langmuir Molecular-Dynamics Simulations. In 4th International Conference on Computational Physics (PC 92); DeGroot, R. A., Nadrchal, J., Eds.; World Scientific Publishing: 1993. (55) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 2008, 4 (3), 435−447. (56) Rietjens, S. J.; Bast, A.; de Vente, J.; Haenen, G. R. M. M. The olive oil antioxidant hydroxytyrosol efficiently protects against the oxidative stress-induced impairment of the NO• response of isolated rat aorta. American Journal of Physiology-Heart and Circulatory Physiology 2007, 292 (4), H1931−H1936. (57) Lu, Z.; Nie, G.; Belton, P. S.; Tang, H.; Zhao, B. Structure− activity relationship analysis of antioxidant ability and neuroprotective effect of gallic acid derivatives. Neurochem. Int. 2006, 48 (4), 263−274. (58) Salaniwal, S.; Cui, S. T.; Cochran, H. D.; Cummings, P. T. Molecular Simulation of a Dichain Surfactant/Water/Carbon Dioxide System. 1. Structural Properties of Aggregates. Langmuir 2001, 17 (5), 1773−1783. (59) Ashbaugh, H. S.; Pratt, L. R.; Paulaitis, M. E.; Clohecy, J.; Beck, T. L. Deblurred observation of the molecular structure of an oil− water interface. J. Am. Chem. Soc. 2005, 127 (9), 2808−2809. (60) Shen, Y.; He, X.; Hung, F. R. Structural and dynamical properties of a deep eutectic solvent confined inside a slit pore. J. Phys. Chem. C 2015, 119 (43), 24489−24500. (61) Zokaie, M.; Foroutan, M. Comparative study on confinement effects of graphene and graphene oxide on structure and dynamics of water. RSC Adv. 2015, 5 (49), 39330−39341. (62) Abbaspour, M.; Akbarzadeh, H.; Abroodi, M. A new and accurate expression for the radial distribution function of confined Lennard-Jones fluid in carbon nanotubes. RSC Adv. 2015, 5 (116), 95781−95787. (63) Soper, A. K.; Bruni, F.; Ricci, M. A. Water confined in Vycor glass. II. Excluded volume effects on the radial distribution functions. J. Chem. Phys. 1998, 109 (4), 1486−1494. (64) Marchi, M.; Abel, S. Modeling the Self-Aggregation of Small AOT Reverse Micelles from First-Principles. J. Phys. Chem. Lett. 2015, 6 (1), 170−174. (65) Dashevskaya, S.; Kopito, R. B.; Friedman, R.; Elbaum, M.; Epel, B. L. Diffusion of anionic and neutral GFP derivatives through plasmodesmata in epidermal cells of Nicotiana benthamiana. Protoplasma 2008, 234 (1), 13.

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