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Cholesterol concentration effect on the bilayer properties and phase formation of niosome bilayers: A molecular dynamics simulation study
Journal of Molecular Liquids 256 (2018) 591–598
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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq
Cholesterol concentration effect on the bilayer properties and phase formation of niosome bilayers: A molecular dynamics simulation study Saowalak Somjid a, Sriprajak Krongsuk a,b,c,⁎, Jeffrey Roy Johns d a
Department of Physics, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand Integrated Nanotechnology Research Center, Khon Kaen University, Khon Kaen 40002, Thailand Nanotec-KKU Center of Excellence on Advanced Nanomaterials for Energy Production and Storage, Khon Kaen 40002, Thailand d Melatonin Research Group, Faculty of Pharmaceutical Science, Khon Kaen University, Khon Kaen 40002, Thailand b c
a r t i c l e
i n f o
Article history: Received 9 November 2017 Received in revised form 14 February 2018 Accepted 17 February 2018 Available online 21 February 2018 Keywords: Niosome bilayer Cholesterol concentration Condensation effect Compressibility Molecular dynamics
a b s t r a c t Niosome bilayers formed by the mixture of sorbitan monostearate (Span60) and cholesterol molecules with different cholesterol concentrations (0 to 70 mol% Chol) were studied by molecular dynamics simulations at the temperature of 298 K and the pressure of 1 bar. Structural properties and dynamics of niosome bilayers were characterized as a function of cholesterol concentration. We found that addition of cholesterol in the niosome bilayer significantly changes the molecular structure and stability. Increasing of cholesterol concentrations causes the area per molecule to increase and bilayer thickness to decrease, suggesting that the niosome bilayer is expanded from the gel to the liquid ordered phase. This contrasts with DOPC or DMPC lipid bilayers which show a condensation effect with increasing cholesterol content. The calculated isothermal area compressibility of the niosome bilayer shows a remarkable increase at moderate concentration (40–50 mol% Chol) and slight change at the higher concentrations. This suggests that the preparation of niosome with the higher cholesterol concentrations does not significantly improve the bilayer compressibility. With increasing cholesterol concentration the Span60 tails gradually adopt a more conformation while the orientation and dynamics of the Span60 groups are rather unaffected. The hydrogen bond interactions of the Span60/chol/water and Span60/water system increased with increasing cholesterol concentration, leading to improved bilayer stability. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Niosomes are novel vesicular drug delivery systems that have numerous advantages over conventional drug carriers because of their special characteristics, such as high entrapment and release rate, low toxicity and biodegradability [1–3]. Structurally, niosomes are very similar to liposomes, but they are composed of non-ionic surfactants e.g. sorbitans (Spans) or polysorbates (Tweens) usually in combination with cholesterol [4,5]. This characteristic makes them resistant to oxidation and hydrolysis, making formulation relative to liposomes much cheaper and simpler, and giving good long-term stability [6]. Additionally, these surfactants are biocompatible, very low toxicity and cheaply available at high purity [2]. On the other hand, liposomes composed of phospholipids that are not stable in air and rapidly undergo oxidation and hydrolysis, leading to poor long-term stability and making formulation very costly [7]. Furthermore, natural sources of phospholipids are difficult and costly to purify and the cost of synthetic phospholipids make them prohibitive for commercial applications. Therefore, many ⁎ Corresponding author at: Department of Physics, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail address: [email protected] (S. Krongsuk).
https://doi.org/10.1016/j.molliq.2018.02.077 0167-7322/© 2018 Elsevier B.V. All rights reserved.
studies from experimental and theoretical researchers have been carried out on the synthesis and characterization of niosomes to improve drug encapsulation [4,6–12]. It is important to understand the molecular level properties of the surfactant assembly, molecular structure, and dynamical behavior, as these give rise to the macromolecular properties of fluidity, elasticity, and shear strength that are critical for stability of nanoparticulate vesicles [3,5,13], their behavior within other fluids such as blood, and their transport across cell membranes [3]. Cholesterol (Chol) plays an important role in the physical properties of lipid membranes, such as the maintenance of proper fluidity, the reduction of passive permeability, and an increase in the mechanical strength of the membrane [14]. Addition of cholesterol into the lipid bilayers thus increases their stability [15] and changes the phase transition from gel to liquid ordered phase [7]. The influence of cholesterols on the physical properties and phase transition of lipid bilayers has been widely investigated by experimental and theoretical studies [6,9,11,12]. However, only a few have been addressed niosome bilayers [7,8,18–20]. These previous works provided insight into the bilayer structure, stability and conditions of the niosome formation. The study of Nasseri [4] showed that the niosome prepared from Span60 with 50 mol% cholesterol inclusion is the most stable over a wide range of temperatures. Recently, the simulation of the pure Span60 bilayer and
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the Span60 bilayer with 50 mol% cholesterol inclusion conducted by Ritwiset et al. [18] provided detailed information on the bilayer structures and dynamical properties which are consistent with experimental data [4]. Their study reveals that the niosome with 50 mol% cholesterol inclusion significantly changes bilayer properties and phase formation. To the best of our knowledge, a molecular dynamics simulation of the niosome bilayers formed by different composition ratios of Span60 and cholesterol has never been studied before. In addition, previous studies of lipid bilayers reported the existence of condensation effects related to the increasing of cholesterol in the bilayer [14,17,21]. Therefore, it is of interest to investigate the cholesterol influence on the structure and phase formation of the niosome bilayer. In this study, we focused on the bilayer structure and dynamic properties of the niosome bilayers with different cholesterol concentrations by varying from 10 mol% to 70 mol% cholesterol, which covers a full range of niosome preparation from the experiment of Nasseri [4]. We used molecular dynamics simulation techniques to investigate these systems. Structural and dynamical properties such as area per molecule, bilayer thickness, bilayer compressibility, order parameters, diffusion, and hydrogen bond analysis were calculated from the simulations. Such information is necessary for design and development of drug delivery systems based on niosome materials.
2. Materials and methods 2.1. Model and initial system setting Molecular models of niosome bilayers formed by the mixture of Span60 and cholesterol molecules were generated by using CELLmicrocosmos 2.2 software [22] with cholesterol concentrations of 0, 10, 20, 30, 40, 50, 60 and 70 mol%, respectively. The number of Span60 and cholesterol molecules for each system is given in Table 1. These molecules were placed randomly in a rectangular box of 6.00 × 6.00 × 14.00 nm3, with their hydrophilic parts in each layer pointing away from each other while their hydrophobic parts faced each other. To build the niosome bilayer with water solution, a water slab consisting of 3895 water molecules in a rectangular box of 6.00 × 6.00 × 3.30 nm in the x, y and z directions, respectively, was simulated at the temperature of 298 K and the pressure of 1 bar using the Gromacs 4.5.4 package [23,24]. After equilibration was reached, this water slab was taken to cap at the top and the bottom of the niosome bilayer, leading to a total number of 7790 water molecules. The same procedure was carried out for all niosome bilayer systems. The united atom description was applied to hydrocarbon sites for the Span60 and cholesterol molecules. The force field parameters for the Span60 and cholesterol molecules were adopted from the literature [9,18,25]. The SPC/E model was employed to describe water molecules. The validation of these force field parameters have been done in our previous works [9,18].
2.2. Simulation details Firstly, all niosome bilayers underwent energy minimization (EM) by using the steepest descent method to remove undesired overlaps between neighboring atoms. After that, all systems were subjected to molecular dynamics at the constant temperature of 298 K and pressure of 1 bar. It should be noted that in this study we performed the MD simulations of niosome bilayers at room temperature (25°C) which is below the main transition temperature of Span60 (56–58 °C) [26]. At room temperature, the pure Span60 bilayer is in the gel phase. However, the addition of cholesterol to the gel phase bilayers can disrupt local packing order, leading to phase change of the niosome formation. To maintain a constant pressure, a Berendsen barostat [27] with semi-isotropic coupling in the lateral direction (in xy- plane) and the normal direction (in z direction) were used with a time constant (τP) of 0.5 and a compressibility of 4.5 × 10−5 bar−1, respectively. The v-rescale thermostat with a coupling constant τT of 0.1 ps was employed to maintain a constant temperature in which the Span60, cholesterol, and water were coupled separately. Periodic boundary conditions were applied in all three directions. The bonds between atoms of the molecules were constrained by using the LINCS algorithm [28] with fourth order expansion. The equations of motion were integrated using the leap-frog algorithm with a time step (Δt) of 2 fs. The electrostatic interactions were calculated using the Fast Particle Mesh Ewald (PME) method [29] and fourth order (cubic) interpolation was used with a grid spacing of 0.15 nm. The coulomb and van der Waal interactions were cut off after a distance of 1.5 nm. The neighborlist was updated every 10 time step. All systems were simulated for 100 ns (60 ns initial equilibrium, 40 ns production run). The total energy, temperature, and area per molecule were monitored and the equilibrium time employed was to be sufficient to obtain reasonable convergence of the structural properties of our interest such as area per molecule (see Supporting Fig. S1-S3). Molecular structures and configurations of these systems were visualized using the VMD program [30]. 3. Results and discussion 3.1. Density profiles The mass density distributions of individual compositions of the niosome bilayer including the Span60, cholesterol, and water molecules for all systems were calculated as given in Fig. 1. The density distributions of the head and tail groups of Span60 molecules were separately plotted for clarify. Clearly, the density profile of Span60 head group is a well-defined structure having the two pronounced peaks that are overlapping the water density. This implies that the Span60 head group in each leaflet prefers locally to absorb at the water surface due to the strong hydrophilic interactions. The addition of cholesterol molecules in the bilayer causes a decrease in the density peaks of Span60
Table 1 Number of Span60 and cholesterol molecules for each niosome bilayer formed by the mixture of Span60 with different cholesterol concentrations. Structural properties including area per molecule (A0), bilayer thickness (d), isothermal area compressibility (KA), and tilt angle (α) were calculated during the final 40 ns of each simulation. Cholesterol concentration (mol% Chol)
Number of molecules Span60
Chol
Total
0
230 (175)a 230 206 182 154 130 (100)a 104 78
–
230 (175)a 256 258 260 256 260 (200)a 260 256
10 20 30 40 50 60 70 a
These data were taken from [18].
26 52 78 102 130 (100)a 156 178
A0 (Å2)
d (nm)
KA (mN·m−1)
Tilt angle (α°) Span60
Chol
22.70 ± 0.21 (23.60 ± 0.10)a 21.37 ± 0.22 22.07 ± 0.07 23.69 ± 0.09 25.06 ± 0.07 26.71 ± 0.07 (26.20 ± 0.10)a 28.32 ± 0.09 30.21 ± 0.08
4.63 ± 0.02 (3.001 ± 0.004)a 4.98 ± 0.02 4.88 ± 0.01 4.65 ± 0.02 4.45 ± 0.01 4.13 ± 0.01 (4.186 ± 0.001)a 4.01 ± 0.01 3.83 ± 0.02
208 ± 37
19.36 ± 1.02 (34.3 ± 0.5)a 17.57 ± 0.67 11.14 ± 0.70 11.35 ± 0.61 11.08 ± 0.66 15.43 ± 0.59 (34.3 ± 0.5)a 12.67 ± 0.57 18.16 ± 0.94
–
146 ± 17 266 ± 90 250 ± 59 316 ± 138 326 ± 135 300 ± 85 339 ± 105
22.20 ± 1.14 13.31 ± 0.95 11.05 ± 0.72 12.66 ± 0.57 11.35 ± 0.54 (15.4 ± 0.8)a 11.17 ± 0.48 11.83 ± 0.49
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Fig. 1. Mass density distributions for all compositions of niosome bilayer with different cholesterol content are presented for (a) 0 mol%, (b) 10 mol%, (c) 20 mol%, (d) 30 mol%, (e) 40 mol%, (f) 50 mol%, (g) 60 mol%, and (h) 70 mol%, respectively.
head group and the shifting of these two peaks closer together. Consequently, the bilayer thickness decreases with increasing cholesterol concentration as seen in Table 1. For the density of Span60 tail group, it is clearly seen at the lower concentrations (10–20 mol% Chol) that there are two broad peaks separated with a minimum point at the center of the bilayer. However, these peaks are combined to form a small peak located at the bilayer center for the higher cholesterol
concentrations (≥40 mol% Chol). This means that the tail groups of Span60 molecules in each leaflet were pushed closer together due to the increasing contribution of cholesterol-Span60 interactions as reported in our previous work [18]. The density peaks of cholesterol molecules are well organized and located near the bilayer center, suggesting that cholesterol molecules are less favored to interact with water molecules. However, the density of cholesterol molecules overlaps more
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with the water density as the cholesterol concentration increases. This indicates that there are more cholesterol molecules interacting with water molecules, leading to an increase of the hydrogen bond number of the cholesterol/water system as seen in Table 2. 3.2. Bilayer properties Table 1 shows structural properties of niosome bilayers including area per molecule (A0), bilayer thickness (d), isothermal area compressibility (KA) and tilt angle (α) as a function of cholesterol concentration (mol% Chol). The area per molecule is defined as
A0 ¼
Lx Ly A ¼ N NSpan60 þ NChol
ð1Þ
where A is the surface area in the xy-plane obtained from the product of cell size in x- (Lx) and y- (Ly) directions, respectively and N is the total number of the molecules in one layer which is the sum of the Span60 number (NSpan60) and cholesterol number (NChol). The bilayer thickness is defined as the distance between the two density peaks of the Span60 head groups as displayed in Fig. 1. It is clearly seen from Table 1 that the area per molecule increases whereas the bilayer thickness decreases with increasing cholesterol concentration. Furthermore, the area per molecule increases and the bilayer thickness decreases exhibiting a monotonic trend, as seen in Fig. 2. These results contrast with the case of phospholipid bilayers such as DOPC and DMPC lipids [14,17] for which addition of more cholesterol molecules in the bilayer causes decreasing area per molecule and increasing bilayer thickness. Such a characteristic can be explained in terms of the condensation effect [16] which involves conservation of chain volume during chain stretching. In fact, the areas per molecule for the pure Span60 and pure cholesterol are 21 Å2 [9] and 37 Å2 [8], respectively. The area per molecule for all cholesterol concentrations lies between these two values, approaching to the area per molecule of the pure cholesterol. This result indicates that niosome bilayers formed by pure Span60 are more condensed than those formed by mixtures of Span60 and cholesterol. According to the formation of pure Span60 bilayer in the gel phase at the room temperature [26], however, the addition of cholesterol to the gel phase bilayers can disrupt local packing order, increasing the molecular area and decreasing the thickness of the niosome bilayers. This result indicates that the properties of the niosome bilayer changes from the gel to the liquid ordered phase as cholesterol concentration increases. Our result is in good agreement with the study of Wilkhu [8] showing that the condensation effect does not exist in a mixture of MPG:Chol:DCP monolayers. The inclusion of cholesterol in the niosome formulations helps to prevent “rigid” molecular packing of Span60 and subsequently the area per molecule increases.
Fig. 2. Area per molecule (A0), bilayer thickness (d) and isothermal area compressibility (KA) calculated from the simulations of niosome bilayers as a function of the cholesterol concentration.
The isothermal area compressibility (KA) can be examined in terms of the area per molecule and its fluctuation as given in Eq. (2) [31], KA ¼
2kB bT NbA0 N ; N δA20
ð2Þ
where kB is the Boltzmann constant, bT N is the average temperature, bA0 N is the average area per molecule and δA20 is the variance associated to A0. It is clearly seen in Table 1 that the KA values are in the interval of 150–340 mN·m−1 which are good agreement with the experimental measurement of the POPC lipid [32]. Although no experimental measurement of KA for the niosome bilayers is available to date, this result shows the bilayer compressibility increasing with increasing cholesterol concentrations as depicted in the inset of Fig. 2. The KA values show a remarkable increase at moderate concentrations (40–50 mol% Chol) and slight change at higher concentrations. This suggests that the preparation of niosomes with higher cholesterol concentrations does not significantly improve the bilayer compressibility (elasticity). From the measurement of the shear modulus of niosomes [4], it was reported the highest value was at a cholesterol concentration of 47.5 mol%. Increasing cholesterol further resulted in decreasing shear modulus. 3.3. Molecular orientation The average orientation of Span60 tails can be characterized in terms of order parameter (Sz). This order parameter can vary between 1 (full
Table 2 Average hydrogen bond number per molecule (bnHBN) and average van der Waals interaction (bEvdWN) calculated from the simulations of niosome bilayers with different cholesterol concentrations. The hydrogen bonds formed by the Span60/Span60 (SS), the Span60/water (SW), the Span60/cholesterol (SC), and the cholesterol/water (CW) systems were investigated for this study. Cholesterol concentration (mol% Chol)
0 10 20 30 40 50 60 70 a
These data were taken from [18].
bnHBN
bEvdW N (kJ/mol)
SW
SS
SC
CW
SS
2.17 ± 0.05 (2.11 ± 0.06)a 2.10 ± 0.05 2.49 ± 0.06 3.04 ± 0.08 3.53 ± 0.09 3.97 ± 0.10 (3.82 ± 0.11)a 4.35 ± 0.12 4.88 ± 0.15
1.89 ± 0.03 (1.26 ± 0.04)a 1.23 ± 0.03 1.14 ± 0.03 1.13 ± 0.04 0.94 ± 0.04 0.89 ± 0.05 (0.67 ± 0.05)a 0.68 ± 0.06 0.44 ± 0.07
–
–
−234.01 ± 0.16
SC
CC
0.16 ± 0.01 0.33 ± 0.01 0.53 ± 0.02 0.72 ± 0.33 0.85 ± 0.04 (0.84 ± 0.04)a 1.00 ± 0.05 1.09 ± 0.05
0.00 0.22 ± 0.03 0.41 ± 0.04 0.56 ± 0.05 0.88 ± 0.05 (0.83 ± 0.05)a 1.16 ± 0.04 1.40 ± 0.05
−225.84 ± 0.13 −202.28 ± 0.03 −173.65 ± 0.12 −147.26 ± 0.12 −123.14 ± 0.18
−347.95 ± 0.23 −302.82 ± 0.24 −278.07 ± 0.22 −227.83 ± 0.24 −190.02 ± 0.11
−39.47 ± 0.09 −65.54 ± 0.07 −77.27 ± 0.06 −96.84 ± 0.05 −113.78 ± 0.07
−99.34 ± 0.27 −71.28 ± 0.38
−149.72 ± 0.07 −117.57 ± 0.26
−132.74 ± 0.13 −147.41 ± 0.14
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order along the interface normal) and −1/2 (full order perpendicular to the normal). In this study, we calculated the Sz which measures the relative orientation of a vector from Cn-1 to Cn+1 with respect to the bilayer normal (z-axis). The average Sz is defined as following equation [33] Sz ¼
3 1 b cos2 θz N− ; 2 2
ð3Þ
where θz is the angle between the a vector from Cn−1 to Cn+1 and the bilayer normal (z-axis). The angular bracket represents a time and ensemble average. For this study we employed a united atom for describing the hydrocarbon group (CH2 or CH3). The order parameters obtained from all bilayer systems as a function of the carbon atom of the Span60 are presented in Fig. 3. The orientations of the Span60 tail and that of the ring system of cholesterol were examined in terms of the average of the tilt angle (α) with respect to the bilayer normal. The tilt angle of Span60 was defined as the angle between the vector connecting from C1 to C18 (see Fig. 4a) and the bilayer normal vector (z-axis). The tilt angle of cholesterol was defined as the vector pointing from C20 to C5 (see Fig. 4b) and the bilayer normal vector. The calculated distributions of the tilt angles are presented in Fig. 5. It is clearly seen in Fig. 3 that in the presence of cholesterol the order parameters are higher and there is more variation for all carbon atom sites compared to the niosome bilayer without cholesterol inclusion. The Sz shows more variation with increasing cholesterol concentration, especially for the carbon atom sites ranging from C8 to C16 (definition of each carbon atom site is referred to in Fig. 4a). This reveals that the Span60 tail has high orientation mobility when cholesterol is incorporated into the niosome bilayer. In our previous study [18], we have reported the relationship between the order parameter and the phase formation of the niosome bilayer. The niosome bilayer without cholesterol inclusion exhibits small variation of order parameter and corresponds to the gel phase with a more tilted lipid chain (34°) [18]. Addition of cholesterol molecules in the bilayer influences the phase formation by inhibiting the tilt of the Span60 tails, causing more variation of the order parameter. As can be seen in Table 1, the ring system of cholesterol shows more tilt (~22°) at the lower concentrations, but for the rest of the concentrations the tilt angles of cholesterol are small and nearly the same (~11°). However, in the case of the Span60 tail groups the average tilt angle has more variation and lies between 11° and 18° with increasing concentration. This indicates that there is a phase change from gel to liquid ordered phase. Clear pictures of these molecular orderings and orientations of niosome bilayers at different concentrations is displayed in Fig. 6. A similar result reported by
Fig. 4. Molecular structures with atom numbering of (a) Span60 and (b) cholesterol, respectively.
de Mayer and Smit [17] reveals that the phase formation of a lipid bilayer strongly depends on cholesterol concentration and temperature. The lipid bilayers formed in the gel phase at lower cholesterol concentrations (b10 mol%), and formed liquid phase at higher cholesterol concentrations. Fig. 7 shows 2-dimensional radial distribution function (2D–rdf) plots for Span60/Span60 (SS), Span60/Chol (SC), and Chol/ Chol (CC) systems. Only lateral distributions (xy plane) were considered in order to clearly illustrate the impact of the variable cholesterol concentrations. The centers of mass of Span60 head group and cholesterol were employed for the 2D-rdf calculations. At the lower cholesterol concentrations (0–30 mol% Chol), the 2D-rdfs of SS and CC show higher structural order over a long range. There are several shape peaks, especially for the CC-rdf. This characteristic is similar to solid structure. With further cholesterol concentration increase the SS-rdf and CC-rdf show less structural order having one and two pronounced peaks in the short range (r b 1.5 nm), respectively. This is similar to liquid structure. Clearly, the CC-rdf exhibits sharper peaks than the SS-rdf implying that the cholesterol molecules have less mobility. 3.4. Hydrogen bond and energetic interaction analysis To investigate the influence of cholesterol on the molecular interactions between water and the head group of Span60 as well as among the Span60 themselves, the average hydrogen bond number per molecule and van der Waal interactions were calculated for all systems, as given in Table 2. It is clearly seen that the number of hydrogen bonds formed by Span60/Span60 (SS) decrease, but that formed by the Span60/water (SW), the Span60/cholesterol (SC), and the cholesterol/water (CW) increased as the cholesterol concentration increased. In addition, the hydrogen bond number of the Span60/water greatly increased. This result indicates that the niosome bilayers formed by Span60 themselves are more loosely packed when cholesterol molecules are added, leading to more contribution of water molecules to interact with the Span60 head group. According to the average van der Waal interactions obtained from each system, the interactions of the Span60/Span60 and Span60/cholesterol systems decreased in strength while the interaction of cholesterol/cholesterol system increased. This result indicates that insertion of cholesterol molecules helps to improve the bilayer stability via the hydrogen bond interactions. 3.5. Lateral diffusion Mobility of the Span60 and cholesterol molecules in the lateral surface can be also calculated from the mean square displacement (MSD) correlation functions defined as follows [30],
Fig. 3. Order parameters (Sz) of the Span60 tail group calculated from the simulations of niosome bilayers with different cholesterol concentrations.
MSD ¼
N D 1X 2 jr⃑ ðt Þ−r⃑ i ð0Þj i; N i¼1 i
ð4Þ
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Fig. 5. Tilt angle distributions of (a) Span60 tail group and (b) the ring plane of cholesterol as a function of cholesterol concentrations.
where, r⃑ i is the position vector of particle i and N is the number of particles. This equation describes the average of the mean square displacement of particle i calculated from all particle positions of the trajectory. The MSD plots of the cholesterol and Span60 movements in the lateral surface were presented in Fig. 8a and b, respectively. The lateral diffusion coefficient, DL, can be extracted from the slope of these plots and given in Table 3. It is clearly seen from these plots that the Span60 molecules exhibit higher movement in the lateral surface than the cholesterol molecules do as cholesterol concentrations increase. Addition of cholesterol in the niosome bilayers causes a reduction of the Span60 packing. Thus the Span60 molecules can more readily move laterally through the surface. However, the cholesterol diffusion is smaller and fluctuation larger because the cholesterol motion along the bilayer normal becomes more restricted with increasing concentrations. This is the result of increasing van der Waal interaction between cholesterol molecules, leading to lower cholesterol mobility in the bilayer plane. It should be noted that the calculated diffusion coefficient strongly
depends on the time and length scales used by the MD simulation technique. The lateral diffusion was calculated on the short and medium time scales, when the molecular movement of a lipid mainly comes from the conformational changes of the hydrocarbon chains [34]. 4. Conclusion In this study we employed molecular dynamics simulations to investigate the structural and dynamical properties of niosome bilayers formed by the mixture of Span60 and cholesterol molecules at different cholesterol concentrations varying from 0 mol% to 70 mol% Chol. We found that cholesterol significantly affects on the bilayer structure and its related properties such as area per molecule, thickness, and compressibility. Addition of cholesterol in the niosome bilayers causes increase in the area per molecule, leading to decreasing of the bilayer thickness due to phase change from gel to the liquid phase. The tail group of Span60 shows more ordering and mobility while cholesterol
Fig. 6. Final configurations of niosome bilayers (water not shown) obtained from MD simulations for all different cholesterol concentrations. Span60 and cholesterol molecules, represented with red and green colors, respectively.
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Fig. 7. Two dimensional radial distribution functions (2D-rdfs), calculated only the xy-plane for the Span60/Span60 (SS), Span60/cholesterol (SC), and cholesterol/cholesterol (CC) systems in different concentrations. The 2D-rdfs were calculated from Span60 head group to Span60 head group (black line) and to cholesterol (red line) as well as that calculated from cholesterol to cholesterol (blue line). The centers of mass of Span60 head group and cholesterol were used throughout this calculation.
molecules are more restricted with increasing cholesterol concentration. Insertion of cholesterol in the niosome bilayers can help to improve the mechanical strength of the bilayer with the higher compressibility modulus, leading to increased niosome stability. The hydrogen bonds of the Span60/water, Span60/cholesterol, and cholesterol/water
systems play an important role for the niosome stability. Finally, our study suggests that adding more cholesterol into niosome bilayers can increase their stability and rigidity and decrease the release rate of an encapsulated material, important in the formulation of niosomes for delivery of chemical entities.
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Fig. 8. Mean squared displacement (MSD) of lateral diffusion (a) for cholesterol and (b) for the Span60 molecules obtained from the simulations of niosome bilayers with different cholesterol concentrations.
Table 3 Lateral diffusion coefficients, DL, of the Span60, Span60 headgroup, Span60 tail group, and cholesterol for different cholesterol concentrations. Cholesterol concentration (mol% Chol)
0 10 20 30 40 50 60 70 a
Lateral diffusion coefficient, DL, [10−8 cm2/s] Span60
Span60-head
Span60-Tail
Cholesterol
0.36 ± 0.11 (0.33 ± 0.03)a 0.13 ± 0.09 0.22 ± 0.04 0.27 ± 0.03 0.64 ± 0.89 0.35 ± 0.16 (1.04 ± 0.04)a 0.43 ± 0.62 1.16 ± 0.15
0.46 ± 0.06 (0.60 ± 0.05)a 0.16 ± 0.04 0.37 ± 0.05 0.42 ± 0.09 0.95 ± 0.07 0.60 ± 0.23 (1.49 ± 0.04)a 0.58 ± 0.71 1.78 ± 0.43
0.27 ± 0.15 (0.12 ± 0.02)a 0.10 ± 0.13 0.10 ± 0.03 0.16 ± 0.01 0.38 ± 0.11 0.15 ± 0.11 (0.67 ± 0.06)a 0.32 ± 0.66 0.67 ± 0.07
–
These data were taken from [18].
Acknowledgment This work was supported by the National Research Council of Thailand (Grant No. 600079) and by the Research and Technology Transfer Affairs (KKUSyn60_001). S.S acknowledges the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0176/2558). The authors would like to acknowledge Dr. Jeffrey Roy Johns for generous discussion and proofreading of manuscript. The Bureau of Information Technology, Khon Kaen University is also acknowledged for high performance computing support. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.molliq.2018.02.077. References [1] [2] [3] [4] [5] [6] [7] [8] [9]
0.09 ± 0.13 0.13 ± 0.09 0.11 ± 0.02 0.31 ± 0.15 0.11 ± 0.05 (0.56 ± 0.06)a 0.11 ± 0.29 0.36 ± 0.30
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[10] T.-X. Xiang, B.D. Anderson, Adv. Drug Deliv. Rev. 58 (2006) 1357–1378. [11] G. Parthasarathi, N. Udupa, P. Umadevi, G. Pillai, J. Drug Target. 2 (1994) 173–182. [12] A. Manosroi, P. Wongtrakul, J. Manosroi, H. Sakai, F. Sugawara, M. Yuasa, M. Abe, Colloids Surf. B: Biointerfaces 30 (2003) 129–138. [13] C.T. Lo, A. Jahn, L.E. Locascio, W.N. Vreeland, Langmuir 26 (2010) 8559–8566. [14] T. Róg, M. Pasenkiewicz-Gierula, FEBS Lett. 502 (2001) 68–71. [15] R. Gupta, B. Rai, J. Phys. Chem. B 119 (2015) 11643–11655. [16] W.-C. Hung, M.-T. Lee, F.-Y. Chen, H.W. Huang, Biophys. J. 92 (2007) 3960–3967. [17] F. de Meyer, B. Smit, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 3654–3658. [18] A. Ritwiset, S. Krongsuk, J.R. Johns, Appl. Surf. Sci. 380 (2016) 23–31. [19] S. Han, Bull. Kor. Chem. Soc. 34 (2013) 946–948, https://doi.org/10.5012/bkcs.2013. 34.3.946. [20] Y. Myung, S. Yeom, S. Han, J. Liposome Res. 26 (2016) 336–344. [21] P. Jedlovszky, M. Mezei, J. Phys. Chem. B 107 (2003) 5322–5332. [22] B. Sommer, T. Dingersen, C. Gamroth, S.E. Schneider, S. Rubert, J. Krüger, K.-J. Dietz, J. Chem. Inf. Model. 51 (2011) 1165–1182. [23] B. Hess, C. Kutzner, D. van der Spoel, E. Lindahl, J. Chem. Theory Comput. 4 (2008) 435–447. [24] D. Van Der Spoel, E. Lindahl, B. Hess, G. Groenhof, A.E. Mark, H.J.C. Berendsen, J. Comput. Chem. 26 (2005) 1701–1718. [25] M. Höltje, T. Förster, B. Brandt, T. Engels, W. von Rybinski, H.-D. Höltje, Biochim. Biophys. Acta Biomembr. 1511 (2001) 156–167. [26] G.P. Kumar, P. Rajeshwarrao, Acta Pharm. Sin. B 1 (2011) 208–219. [27] H.J.C. Berendsen, J.P.M. Postma, W.F. van Gunsteren, A. DiNola, J.R. Haak, J. Chem. Phys. 81 (1984) 3684–3690. [28] A. Baranyai, D.J. Evans, Mol. Phys. 70 (1990) 53–63. [29] H.J.C. Berendsen, D. van der Spoel, R. van Drunen, Comput. Phys. Commun. 91 (1995) 43–56. [30] W. Humphrey, A. Dalke, K. Schulten, J. Mol. Graph. 14 (1996) 33–38. [31] D. Poger, A.E. Mark, J. Chem. Theory Comput. 6 (2010) 325–336. [32] X. He, O. Guvench, A.D. MacKerell, M.L. Klein, J. Phys. Chem. B 114 (2010) 9787–9794. [33] T.J. Piggot, J.R. Allison, R.B. Sessions, J.W. Essex, J. Chem. Theory Comput. 13 (2017) 5683–5696. [34] A.P. Lyubartsev, A.L. Rabinovich, Soft Matter 7 (2010) 25–39.
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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq
Cholesterol concentration effect on the bilayer properties and phase formation of niosome bilayers: A molecular dynamics simulation study Saowalak Somjid a, Sriprajak Krongsuk a,b,c,⁎, Jeffrey Roy Johns d a
Department of Physics, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand Integrated Nanotechnology Research Center, Khon Kaen University, Khon Kaen 40002, Thailand Nanotec-KKU Center of Excellence on Advanced Nanomaterials for Energy Production and Storage, Khon Kaen 40002, Thailand d Melatonin Research Group, Faculty of Pharmaceutical Science, Khon Kaen University, Khon Kaen 40002, Thailand b c
a r t i c l e
i n f o
Article history: Received 9 November 2017 Received in revised form 14 February 2018 Accepted 17 February 2018 Available online 21 February 2018 Keywords: Niosome bilayer Cholesterol concentration Condensation effect Compressibility Molecular dynamics
a b s t r a c t Niosome bilayers formed by the mixture of sorbitan monostearate (Span60) and cholesterol molecules with different cholesterol concentrations (0 to 70 mol% Chol) were studied by molecular dynamics simulations at the temperature of 298 K and the pressure of 1 bar. Structural properties and dynamics of niosome bilayers were characterized as a function of cholesterol concentration. We found that addition of cholesterol in the niosome bilayer significantly changes the molecular structure and stability. Increasing of cholesterol concentrations causes the area per molecule to increase and bilayer thickness to decrease, suggesting that the niosome bilayer is expanded from the gel to the liquid ordered phase. This contrasts with DOPC or DMPC lipid bilayers which show a condensation effect with increasing cholesterol content. The calculated isothermal area compressibility of the niosome bilayer shows a remarkable increase at moderate concentration (40–50 mol% Chol) and slight change at the higher concentrations. This suggests that the preparation of niosome with the higher cholesterol concentrations does not significantly improve the bilayer compressibility. With increasing cholesterol concentration the Span60 tails gradually adopt a more conformation while the orientation and dynamics of the Span60 groups are rather unaffected. The hydrogen bond interactions of the Span60/chol/water and Span60/water system increased with increasing cholesterol concentration, leading to improved bilayer stability. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Niosomes are novel vesicular drug delivery systems that have numerous advantages over conventional drug carriers because of their special characteristics, such as high entrapment and release rate, low toxicity and biodegradability [1–3]. Structurally, niosomes are very similar to liposomes, but they are composed of non-ionic surfactants e.g. sorbitans (Spans) or polysorbates (Tweens) usually in combination with cholesterol [4,5]. This characteristic makes them resistant to oxidation and hydrolysis, making formulation relative to liposomes much cheaper and simpler, and giving good long-term stability [6]. Additionally, these surfactants are biocompatible, very low toxicity and cheaply available at high purity [2]. On the other hand, liposomes composed of phospholipids that are not stable in air and rapidly undergo oxidation and hydrolysis, leading to poor long-term stability and making formulation very costly [7]. Furthermore, natural sources of phospholipids are difficult and costly to purify and the cost of synthetic phospholipids make them prohibitive for commercial applications. Therefore, many ⁎ Corresponding author at: Department of Physics, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail address: [email protected] (S. Krongsuk).
https://doi.org/10.1016/j.molliq.2018.02.077 0167-7322/© 2018 Elsevier B.V. All rights reserved.
studies from experimental and theoretical researchers have been carried out on the synthesis and characterization of niosomes to improve drug encapsulation [4,6–12]. It is important to understand the molecular level properties of the surfactant assembly, molecular structure, and dynamical behavior, as these give rise to the macromolecular properties of fluidity, elasticity, and shear strength that are critical for stability of nanoparticulate vesicles [3,5,13], their behavior within other fluids such as blood, and their transport across cell membranes [3]. Cholesterol (Chol) plays an important role in the physical properties of lipid membranes, such as the maintenance of proper fluidity, the reduction of passive permeability, and an increase in the mechanical strength of the membrane [14]. Addition of cholesterol into the lipid bilayers thus increases their stability [15] and changes the phase transition from gel to liquid ordered phase [7]. The influence of cholesterols on the physical properties and phase transition of lipid bilayers has been widely investigated by experimental and theoretical studies [6,9,11,12]. However, only a few have been addressed niosome bilayers [7,8,18–20]. These previous works provided insight into the bilayer structure, stability and conditions of the niosome formation. The study of Nasseri [4] showed that the niosome prepared from Span60 with 50 mol% cholesterol inclusion is the most stable over a wide range of temperatures. Recently, the simulation of the pure Span60 bilayer and
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the Span60 bilayer with 50 mol% cholesterol inclusion conducted by Ritwiset et al. [18] provided detailed information on the bilayer structures and dynamical properties which are consistent with experimental data [4]. Their study reveals that the niosome with 50 mol% cholesterol inclusion significantly changes bilayer properties and phase formation. To the best of our knowledge, a molecular dynamics simulation of the niosome bilayers formed by different composition ratios of Span60 and cholesterol has never been studied before. In addition, previous studies of lipid bilayers reported the existence of condensation effects related to the increasing of cholesterol in the bilayer [14,17,21]. Therefore, it is of interest to investigate the cholesterol influence on the structure and phase formation of the niosome bilayer. In this study, we focused on the bilayer structure and dynamic properties of the niosome bilayers with different cholesterol concentrations by varying from 10 mol% to 70 mol% cholesterol, which covers a full range of niosome preparation from the experiment of Nasseri [4]. We used molecular dynamics simulation techniques to investigate these systems. Structural and dynamical properties such as area per molecule, bilayer thickness, bilayer compressibility, order parameters, diffusion, and hydrogen bond analysis were calculated from the simulations. Such information is necessary for design and development of drug delivery systems based on niosome materials.
2. Materials and methods 2.1. Model and initial system setting Molecular models of niosome bilayers formed by the mixture of Span60 and cholesterol molecules were generated by using CELLmicrocosmos 2.2 software [22] with cholesterol concentrations of 0, 10, 20, 30, 40, 50, 60 and 70 mol%, respectively. The number of Span60 and cholesterol molecules for each system is given in Table 1. These molecules were placed randomly in a rectangular box of 6.00 × 6.00 × 14.00 nm3, with their hydrophilic parts in each layer pointing away from each other while their hydrophobic parts faced each other. To build the niosome bilayer with water solution, a water slab consisting of 3895 water molecules in a rectangular box of 6.00 × 6.00 × 3.30 nm in the x, y and z directions, respectively, was simulated at the temperature of 298 K and the pressure of 1 bar using the Gromacs 4.5.4 package [23,24]. After equilibration was reached, this water slab was taken to cap at the top and the bottom of the niosome bilayer, leading to a total number of 7790 water molecules. The same procedure was carried out for all niosome bilayer systems. The united atom description was applied to hydrocarbon sites for the Span60 and cholesterol molecules. The force field parameters for the Span60 and cholesterol molecules were adopted from the literature [9,18,25]. The SPC/E model was employed to describe water molecules. The validation of these force field parameters have been done in our previous works [9,18].
2.2. Simulation details Firstly, all niosome bilayers underwent energy minimization (EM) by using the steepest descent method to remove undesired overlaps between neighboring atoms. After that, all systems were subjected to molecular dynamics at the constant temperature of 298 K and pressure of 1 bar. It should be noted that in this study we performed the MD simulations of niosome bilayers at room temperature (25°C) which is below the main transition temperature of Span60 (56–58 °C) [26]. At room temperature, the pure Span60 bilayer is in the gel phase. However, the addition of cholesterol to the gel phase bilayers can disrupt local packing order, leading to phase change of the niosome formation. To maintain a constant pressure, a Berendsen barostat [27] with semi-isotropic coupling in the lateral direction (in xy- plane) and the normal direction (in z direction) were used with a time constant (τP) of 0.5 and a compressibility of 4.5 × 10−5 bar−1, respectively. The v-rescale thermostat with a coupling constant τT of 0.1 ps was employed to maintain a constant temperature in which the Span60, cholesterol, and water were coupled separately. Periodic boundary conditions were applied in all three directions. The bonds between atoms of the molecules were constrained by using the LINCS algorithm [28] with fourth order expansion. The equations of motion were integrated using the leap-frog algorithm with a time step (Δt) of 2 fs. The electrostatic interactions were calculated using the Fast Particle Mesh Ewald (PME) method [29] and fourth order (cubic) interpolation was used with a grid spacing of 0.15 nm. The coulomb and van der Waal interactions were cut off after a distance of 1.5 nm. The neighborlist was updated every 10 time step. All systems were simulated for 100 ns (60 ns initial equilibrium, 40 ns production run). The total energy, temperature, and area per molecule were monitored and the equilibrium time employed was to be sufficient to obtain reasonable convergence of the structural properties of our interest such as area per molecule (see Supporting Fig. S1-S3). Molecular structures and configurations of these systems were visualized using the VMD program [30]. 3. Results and discussion 3.1. Density profiles The mass density distributions of individual compositions of the niosome bilayer including the Span60, cholesterol, and water molecules for all systems were calculated as given in Fig. 1. The density distributions of the head and tail groups of Span60 molecules were separately plotted for clarify. Clearly, the density profile of Span60 head group is a well-defined structure having the two pronounced peaks that are overlapping the water density. This implies that the Span60 head group in each leaflet prefers locally to absorb at the water surface due to the strong hydrophilic interactions. The addition of cholesterol molecules in the bilayer causes a decrease in the density peaks of Span60
Table 1 Number of Span60 and cholesterol molecules for each niosome bilayer formed by the mixture of Span60 with different cholesterol concentrations. Structural properties including area per molecule (A0), bilayer thickness (d), isothermal area compressibility (KA), and tilt angle (α) were calculated during the final 40 ns of each simulation. Cholesterol concentration (mol% Chol)
Number of molecules Span60
Chol
Total
0
230 (175)a 230 206 182 154 130 (100)a 104 78
–
230 (175)a 256 258 260 256 260 (200)a 260 256
10 20 30 40 50 60 70 a
These data were taken from [18].
26 52 78 102 130 (100)a 156 178
A0 (Å2)
d (nm)
KA (mN·m−1)
Tilt angle (α°) Span60
Chol
22.70 ± 0.21 (23.60 ± 0.10)a 21.37 ± 0.22 22.07 ± 0.07 23.69 ± 0.09 25.06 ± 0.07 26.71 ± 0.07 (26.20 ± 0.10)a 28.32 ± 0.09 30.21 ± 0.08
4.63 ± 0.02 (3.001 ± 0.004)a 4.98 ± 0.02 4.88 ± 0.01 4.65 ± 0.02 4.45 ± 0.01 4.13 ± 0.01 (4.186 ± 0.001)a 4.01 ± 0.01 3.83 ± 0.02
208 ± 37
19.36 ± 1.02 (34.3 ± 0.5)a 17.57 ± 0.67 11.14 ± 0.70 11.35 ± 0.61 11.08 ± 0.66 15.43 ± 0.59 (34.3 ± 0.5)a 12.67 ± 0.57 18.16 ± 0.94
–
146 ± 17 266 ± 90 250 ± 59 316 ± 138 326 ± 135 300 ± 85 339 ± 105
22.20 ± 1.14 13.31 ± 0.95 11.05 ± 0.72 12.66 ± 0.57 11.35 ± 0.54 (15.4 ± 0.8)a 11.17 ± 0.48 11.83 ± 0.49
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Fig. 1. Mass density distributions for all compositions of niosome bilayer with different cholesterol content are presented for (a) 0 mol%, (b) 10 mol%, (c) 20 mol%, (d) 30 mol%, (e) 40 mol%, (f) 50 mol%, (g) 60 mol%, and (h) 70 mol%, respectively.
head group and the shifting of these two peaks closer together. Consequently, the bilayer thickness decreases with increasing cholesterol concentration as seen in Table 1. For the density of Span60 tail group, it is clearly seen at the lower concentrations (10–20 mol% Chol) that there are two broad peaks separated with a minimum point at the center of the bilayer. However, these peaks are combined to form a small peak located at the bilayer center for the higher cholesterol
concentrations (≥40 mol% Chol). This means that the tail groups of Span60 molecules in each leaflet were pushed closer together due to the increasing contribution of cholesterol-Span60 interactions as reported in our previous work [18]. The density peaks of cholesterol molecules are well organized and located near the bilayer center, suggesting that cholesterol molecules are less favored to interact with water molecules. However, the density of cholesterol molecules overlaps more
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with the water density as the cholesterol concentration increases. This indicates that there are more cholesterol molecules interacting with water molecules, leading to an increase of the hydrogen bond number of the cholesterol/water system as seen in Table 2. 3.2. Bilayer properties Table 1 shows structural properties of niosome bilayers including area per molecule (A0), bilayer thickness (d), isothermal area compressibility (KA) and tilt angle (α) as a function of cholesterol concentration (mol% Chol). The area per molecule is defined as
A0 ¼
Lx Ly A ¼ N NSpan60 þ NChol
ð1Þ
where A is the surface area in the xy-plane obtained from the product of cell size in x- (Lx) and y- (Ly) directions, respectively and N is the total number of the molecules in one layer which is the sum of the Span60 number (NSpan60) and cholesterol number (NChol). The bilayer thickness is defined as the distance between the two density peaks of the Span60 head groups as displayed in Fig. 1. It is clearly seen from Table 1 that the area per molecule increases whereas the bilayer thickness decreases with increasing cholesterol concentration. Furthermore, the area per molecule increases and the bilayer thickness decreases exhibiting a monotonic trend, as seen in Fig. 2. These results contrast with the case of phospholipid bilayers such as DOPC and DMPC lipids [14,17] for which addition of more cholesterol molecules in the bilayer causes decreasing area per molecule and increasing bilayer thickness. Such a characteristic can be explained in terms of the condensation effect [16] which involves conservation of chain volume during chain stretching. In fact, the areas per molecule for the pure Span60 and pure cholesterol are 21 Å2 [9] and 37 Å2 [8], respectively. The area per molecule for all cholesterol concentrations lies between these two values, approaching to the area per molecule of the pure cholesterol. This result indicates that niosome bilayers formed by pure Span60 are more condensed than those formed by mixtures of Span60 and cholesterol. According to the formation of pure Span60 bilayer in the gel phase at the room temperature [26], however, the addition of cholesterol to the gel phase bilayers can disrupt local packing order, increasing the molecular area and decreasing the thickness of the niosome bilayers. This result indicates that the properties of the niosome bilayer changes from the gel to the liquid ordered phase as cholesterol concentration increases. Our result is in good agreement with the study of Wilkhu [8] showing that the condensation effect does not exist in a mixture of MPG:Chol:DCP monolayers. The inclusion of cholesterol in the niosome formulations helps to prevent “rigid” molecular packing of Span60 and subsequently the area per molecule increases.
Fig. 2. Area per molecule (A0), bilayer thickness (d) and isothermal area compressibility (KA) calculated from the simulations of niosome bilayers as a function of the cholesterol concentration.
The isothermal area compressibility (KA) can be examined in terms of the area per molecule and its fluctuation as given in Eq. (2) [31], KA ¼
2kB bT NbA0 N ; N δA20
ð2Þ
where kB is the Boltzmann constant, bT N is the average temperature, bA0 N is the average area per molecule and δA20 is the variance associated to A0. It is clearly seen in Table 1 that the KA values are in the interval of 150–340 mN·m−1 which are good agreement with the experimental measurement of the POPC lipid [32]. Although no experimental measurement of KA for the niosome bilayers is available to date, this result shows the bilayer compressibility increasing with increasing cholesterol concentrations as depicted in the inset of Fig. 2. The KA values show a remarkable increase at moderate concentrations (40–50 mol% Chol) and slight change at higher concentrations. This suggests that the preparation of niosomes with higher cholesterol concentrations does not significantly improve the bilayer compressibility (elasticity). From the measurement of the shear modulus of niosomes [4], it was reported the highest value was at a cholesterol concentration of 47.5 mol%. Increasing cholesterol further resulted in decreasing shear modulus. 3.3. Molecular orientation The average orientation of Span60 tails can be characterized in terms of order parameter (Sz). This order parameter can vary between 1 (full
Table 2 Average hydrogen bond number per molecule (bnHBN) and average van der Waals interaction (bEvdWN) calculated from the simulations of niosome bilayers with different cholesterol concentrations. The hydrogen bonds formed by the Span60/Span60 (SS), the Span60/water (SW), the Span60/cholesterol (SC), and the cholesterol/water (CW) systems were investigated for this study. Cholesterol concentration (mol% Chol)
0 10 20 30 40 50 60 70 a
These data were taken from [18].
bnHBN
bEvdW N (kJ/mol)
SW
SS
SC
CW
SS
2.17 ± 0.05 (2.11 ± 0.06)a 2.10 ± 0.05 2.49 ± 0.06 3.04 ± 0.08 3.53 ± 0.09 3.97 ± 0.10 (3.82 ± 0.11)a 4.35 ± 0.12 4.88 ± 0.15
1.89 ± 0.03 (1.26 ± 0.04)a 1.23 ± 0.03 1.14 ± 0.03 1.13 ± 0.04 0.94 ± 0.04 0.89 ± 0.05 (0.67 ± 0.05)a 0.68 ± 0.06 0.44 ± 0.07
–
–
−234.01 ± 0.16
SC
CC
0.16 ± 0.01 0.33 ± 0.01 0.53 ± 0.02 0.72 ± 0.33 0.85 ± 0.04 (0.84 ± 0.04)a 1.00 ± 0.05 1.09 ± 0.05
0.00 0.22 ± 0.03 0.41 ± 0.04 0.56 ± 0.05 0.88 ± 0.05 (0.83 ± 0.05)a 1.16 ± 0.04 1.40 ± 0.05
−225.84 ± 0.13 −202.28 ± 0.03 −173.65 ± 0.12 −147.26 ± 0.12 −123.14 ± 0.18
−347.95 ± 0.23 −302.82 ± 0.24 −278.07 ± 0.22 −227.83 ± 0.24 −190.02 ± 0.11
−39.47 ± 0.09 −65.54 ± 0.07 −77.27 ± 0.06 −96.84 ± 0.05 −113.78 ± 0.07
−99.34 ± 0.27 −71.28 ± 0.38
−149.72 ± 0.07 −117.57 ± 0.26
−132.74 ± 0.13 −147.41 ± 0.14
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order along the interface normal) and −1/2 (full order perpendicular to the normal). In this study, we calculated the Sz which measures the relative orientation of a vector from Cn-1 to Cn+1 with respect to the bilayer normal (z-axis). The average Sz is defined as following equation [33] Sz ¼
3 1 b cos2 θz N− ; 2 2
ð3Þ
where θz is the angle between the a vector from Cn−1 to Cn+1 and the bilayer normal (z-axis). The angular bracket represents a time and ensemble average. For this study we employed a united atom for describing the hydrocarbon group (CH2 or CH3). The order parameters obtained from all bilayer systems as a function of the carbon atom of the Span60 are presented in Fig. 3. The orientations of the Span60 tail and that of the ring system of cholesterol were examined in terms of the average of the tilt angle (α) with respect to the bilayer normal. The tilt angle of Span60 was defined as the angle between the vector connecting from C1 to C18 (see Fig. 4a) and the bilayer normal vector (z-axis). The tilt angle of cholesterol was defined as the vector pointing from C20 to C5 (see Fig. 4b) and the bilayer normal vector. The calculated distributions of the tilt angles are presented in Fig. 5. It is clearly seen in Fig. 3 that in the presence of cholesterol the order parameters are higher and there is more variation for all carbon atom sites compared to the niosome bilayer without cholesterol inclusion. The Sz shows more variation with increasing cholesterol concentration, especially for the carbon atom sites ranging from C8 to C16 (definition of each carbon atom site is referred to in Fig. 4a). This reveals that the Span60 tail has high orientation mobility when cholesterol is incorporated into the niosome bilayer. In our previous study [18], we have reported the relationship between the order parameter and the phase formation of the niosome bilayer. The niosome bilayer without cholesterol inclusion exhibits small variation of order parameter and corresponds to the gel phase with a more tilted lipid chain (34°) [18]. Addition of cholesterol molecules in the bilayer influences the phase formation by inhibiting the tilt of the Span60 tails, causing more variation of the order parameter. As can be seen in Table 1, the ring system of cholesterol shows more tilt (~22°) at the lower concentrations, but for the rest of the concentrations the tilt angles of cholesterol are small and nearly the same (~11°). However, in the case of the Span60 tail groups the average tilt angle has more variation and lies between 11° and 18° with increasing concentration. This indicates that there is a phase change from gel to liquid ordered phase. Clear pictures of these molecular orderings and orientations of niosome bilayers at different concentrations is displayed in Fig. 6. A similar result reported by
Fig. 4. Molecular structures with atom numbering of (a) Span60 and (b) cholesterol, respectively.
de Mayer and Smit [17] reveals that the phase formation of a lipid bilayer strongly depends on cholesterol concentration and temperature. The lipid bilayers formed in the gel phase at lower cholesterol concentrations (b10 mol%), and formed liquid phase at higher cholesterol concentrations. Fig. 7 shows 2-dimensional radial distribution function (2D–rdf) plots for Span60/Span60 (SS), Span60/Chol (SC), and Chol/ Chol (CC) systems. Only lateral distributions (xy plane) were considered in order to clearly illustrate the impact of the variable cholesterol concentrations. The centers of mass of Span60 head group and cholesterol were employed for the 2D-rdf calculations. At the lower cholesterol concentrations (0–30 mol% Chol), the 2D-rdfs of SS and CC show higher structural order over a long range. There are several shape peaks, especially for the CC-rdf. This characteristic is similar to solid structure. With further cholesterol concentration increase the SS-rdf and CC-rdf show less structural order having one and two pronounced peaks in the short range (r b 1.5 nm), respectively. This is similar to liquid structure. Clearly, the CC-rdf exhibits sharper peaks than the SS-rdf implying that the cholesterol molecules have less mobility. 3.4. Hydrogen bond and energetic interaction analysis To investigate the influence of cholesterol on the molecular interactions between water and the head group of Span60 as well as among the Span60 themselves, the average hydrogen bond number per molecule and van der Waal interactions were calculated for all systems, as given in Table 2. It is clearly seen that the number of hydrogen bonds formed by Span60/Span60 (SS) decrease, but that formed by the Span60/water (SW), the Span60/cholesterol (SC), and the cholesterol/water (CW) increased as the cholesterol concentration increased. In addition, the hydrogen bond number of the Span60/water greatly increased. This result indicates that the niosome bilayers formed by Span60 themselves are more loosely packed when cholesterol molecules are added, leading to more contribution of water molecules to interact with the Span60 head group. According to the average van der Waal interactions obtained from each system, the interactions of the Span60/Span60 and Span60/cholesterol systems decreased in strength while the interaction of cholesterol/cholesterol system increased. This result indicates that insertion of cholesterol molecules helps to improve the bilayer stability via the hydrogen bond interactions. 3.5. Lateral diffusion Mobility of the Span60 and cholesterol molecules in the lateral surface can be also calculated from the mean square displacement (MSD) correlation functions defined as follows [30],
Fig. 3. Order parameters (Sz) of the Span60 tail group calculated from the simulations of niosome bilayers with different cholesterol concentrations.
MSD ¼
N D 1X 2 jr⃑ ðt Þ−r⃑ i ð0Þj i; N i¼1 i
ð4Þ
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Fig. 5. Tilt angle distributions of (a) Span60 tail group and (b) the ring plane of cholesterol as a function of cholesterol concentrations.
where, r⃑ i is the position vector of particle i and N is the number of particles. This equation describes the average of the mean square displacement of particle i calculated from all particle positions of the trajectory. The MSD plots of the cholesterol and Span60 movements in the lateral surface were presented in Fig. 8a and b, respectively. The lateral diffusion coefficient, DL, can be extracted from the slope of these plots and given in Table 3. It is clearly seen from these plots that the Span60 molecules exhibit higher movement in the lateral surface than the cholesterol molecules do as cholesterol concentrations increase. Addition of cholesterol in the niosome bilayers causes a reduction of the Span60 packing. Thus the Span60 molecules can more readily move laterally through the surface. However, the cholesterol diffusion is smaller and fluctuation larger because the cholesterol motion along the bilayer normal becomes more restricted with increasing concentrations. This is the result of increasing van der Waal interaction between cholesterol molecules, leading to lower cholesterol mobility in the bilayer plane. It should be noted that the calculated diffusion coefficient strongly
depends on the time and length scales used by the MD simulation technique. The lateral diffusion was calculated on the short and medium time scales, when the molecular movement of a lipid mainly comes from the conformational changes of the hydrocarbon chains [34]. 4. Conclusion In this study we employed molecular dynamics simulations to investigate the structural and dynamical properties of niosome bilayers formed by the mixture of Span60 and cholesterol molecules at different cholesterol concentrations varying from 0 mol% to 70 mol% Chol. We found that cholesterol significantly affects on the bilayer structure and its related properties such as area per molecule, thickness, and compressibility. Addition of cholesterol in the niosome bilayers causes increase in the area per molecule, leading to decreasing of the bilayer thickness due to phase change from gel to the liquid phase. The tail group of Span60 shows more ordering and mobility while cholesterol
Fig. 6. Final configurations of niosome bilayers (water not shown) obtained from MD simulations for all different cholesterol concentrations. Span60 and cholesterol molecules, represented with red and green colors, respectively.
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Fig. 7. Two dimensional radial distribution functions (2D-rdfs), calculated only the xy-plane for the Span60/Span60 (SS), Span60/cholesterol (SC), and cholesterol/cholesterol (CC) systems in different concentrations. The 2D-rdfs were calculated from Span60 head group to Span60 head group (black line) and to cholesterol (red line) as well as that calculated from cholesterol to cholesterol (blue line). The centers of mass of Span60 head group and cholesterol were used throughout this calculation.
molecules are more restricted with increasing cholesterol concentration. Insertion of cholesterol in the niosome bilayers can help to improve the mechanical strength of the bilayer with the higher compressibility modulus, leading to increased niosome stability. The hydrogen bonds of the Span60/water, Span60/cholesterol, and cholesterol/water
systems play an important role for the niosome stability. Finally, our study suggests that adding more cholesterol into niosome bilayers can increase their stability and rigidity and decrease the release rate of an encapsulated material, important in the formulation of niosomes for delivery of chemical entities.
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Fig. 8. Mean squared displacement (MSD) of lateral diffusion (a) for cholesterol and (b) for the Span60 molecules obtained from the simulations of niosome bilayers with different cholesterol concentrations.
Table 3 Lateral diffusion coefficients, DL, of the Span60, Span60 headgroup, Span60 tail group, and cholesterol for different cholesterol concentrations. Cholesterol concentration (mol% Chol)
0 10 20 30 40 50 60 70 a
Lateral diffusion coefficient, DL, [10−8 cm2/s] Span60
Span60-head
Span60-Tail
Cholesterol
0.36 ± 0.11 (0.33 ± 0.03)a 0.13 ± 0.09 0.22 ± 0.04 0.27 ± 0.03 0.64 ± 0.89 0.35 ± 0.16 (1.04 ± 0.04)a 0.43 ± 0.62 1.16 ± 0.15
0.46 ± 0.06 (0.60 ± 0.05)a 0.16 ± 0.04 0.37 ± 0.05 0.42 ± 0.09 0.95 ± 0.07 0.60 ± 0.23 (1.49 ± 0.04)a 0.58 ± 0.71 1.78 ± 0.43
0.27 ± 0.15 (0.12 ± 0.02)a 0.10 ± 0.13 0.10 ± 0.03 0.16 ± 0.01 0.38 ± 0.11 0.15 ± 0.11 (0.67 ± 0.06)a 0.32 ± 0.66 0.67 ± 0.07
–
These data were taken from [18].
Acknowledgment This work was supported by the National Research Council of Thailand (Grant No. 600079) and by the Research and Technology Transfer Affairs (KKUSyn60_001). S.S acknowledges the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0176/2558). The authors would like to acknowledge Dr. Jeffrey Roy Johns for generous discussion and proofreading of manuscript. The Bureau of Information Technology, Khon Kaen University is also acknowledged for high performance computing support. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.molliq.2018.02.077. References [1] [2] [3] [4] [5] [6] [7] [8] [9]
0.09 ± 0.13 0.13 ± 0.09 0.11 ± 0.02 0.31 ± 0.15 0.11 ± 0.05 (0.56 ± 0.06)a 0.11 ± 0.29 0.36 ± 0.30
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