Self-assembly of Palm Kernel Oil Wax Esters in Aqueous Media : A Molecular Dynamics Study

Experimentally determined phase diagram of palm kernel oil-based wax esters (PKOEs) nano-emulsion was selected to explore an atomic insight into the self-assembly of PKOEs nano-emulsion using molecular dynamics (MD) simulation technique. A model system including PKOEs molecules and nonionic Tween80 surfactant in aqueous solution was prepared. We used MD to explore a detail atomic insight into the self-assembly process of and the shape of the micelle formed. MD simulation was performed for 20 ns. MD results involving the radius of gyration, the solvent accessible surface areas, the number of clusters formed, and the moment of inertia analysis revealed that the model prepared showed a stable prolate ellipsoid shape.


Introduction
Inflammatory problems are related to skin disorders which can be controlled using non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen and diclofenac (Consola et al., 2007).NSAIDs are widely used as pain killers but they can be toxic if they consumed in high dosages.Oral therapy of NSAIDs, e.g.ibuprofen is limited due to the irritation and the ulceration of the gastro-intestinal mucosa (Al-Saidan, 2004).Therefore, they have been formulated in different topical and transdermal emulsions in order to reduce their side effects (Cho & Choi, 1998;Adrian et al., 2004;Cevc & Vierl, 2010).The nano size of nano-emulsions helps significantly solve the penetration problem of drug at stratum corneum (Swarnalatha et al., 2008).Nano-emulsions are dispersion of nano size droplets (20-200 nm) (Solans et al., 2005).Nano-emulsions can't form spontaneously because of the surface tension at oil/water interface, thus an extra energy is usually required to break the bigger droplets to smaller ones (Mason et al., 2006).Currently, unsaturated palm oil-based wax esters (PKOEs) are widely used in the formulation of nano-emulsions for cosmetic and pharmaceutical applications (Salim et al., 2011(Salim et al., , 2012)).Palm oil-based wax esters are synthesized using enzymatic reactions (Sulaiman et al., 2005).PKOEs are a mixture of different esters which illustrate both lipophilic and hydrophilic properties.
Additionally, theoretical studies of nano-emulsion of palmitate ester have illustrated the potential application of palm oil esters for transdermal delivery of NSAIDs (AbediKarjiban et al., 2012).They selected two different compositions of palmitate ester and Tween80 surfactant molecules in water with and without diclofenac acid from an experimentally determined phase diagram.They found a prolate-like shape for both systems, while adding the hydrophobic diclofenac produced a more compact structure.Molecular dynamics (MD) simulation technique is used to predict the structure and dynamics of the self-assembled structures for many years.MD can effectively simulate the molecular systems at atomic details using selected force fields based on Newton's second law of motion (Van Gunsteren et al., 2008).In MD, atoms and molecules can interact for a selected period of time and produce an atomic detail of the dynamics of atoms involved.The traditional MD simulation calculations have been extensively carried out to explore the self-assembly process in surfactant molecules such as octyl glucoside (Bogusz et al., 2000(Bogusz et al., , 2001)), C 12 E 4 in β-cyclodextrin (Cunha-Silva & Teixeira-Dias, 2002), sodium dodecyl sulfate (SDS) (Bruce et al., 2002), and sodium octanoate (DeMoura & Freitas, 2004, 2005).
Abdul Rahman et al. (2009) reported the MD simulation calculations of palm oil-based esters (POEs) nano-emulsions according to experimental results.They selected a composition of water/palm oil ester/span20 with a ratio of 75:5:20 and carried out MD calculations.They got a spherical micelle in which the palm oil ester molecules were surrounded by nonionic Span20 surfactants.In another study by Abdul Rahman et al. (2010), a model consisted of oleyl oleate ester with a mixture of nonionic Span20 and Tween80 molecules was simulated in aqueous solution to find the structural properties of the aggregated structures formed.Their MD simulation results showed a cylindrical shape in which the oleyl oleate/Tween80 system was more compact compared to the oleyl oleate/Span20 model.Both Span20 and Tween80 are among very commonly used nonionic surfactants in nano-emulsion preparation.Here, a model system containing palm kernel oil-based wax ester and Tween80 has been explored using all-atom level MD simulations calculations based on our previously reported experimental results (Salim et al., 2012).MD was utilized to give a detail insight into palm kernel oil-based wax esters self-assembly and the shape of the aggregates formed.
All molecular structures were geometry optimized using WinGAMESS (Schmidt et al., 1993) by applying Density Functional Theory (DFT) based on Becke's three-parameter hybrid functional method (B3LYP) followed by optimization of the molecules at 6-31G level of theory.MD simulations were performed using GROMACS version 3.3.2(Lindahl et al., 2001;Van der Spoel et al., 2005) using Optimized Potential for Liquid Simulation-All Atom (OPLS-AA) force field (Jorgensen et al., 1996).The randomly placed starting structure was prepared using Packmol (Martinez & Martinez, 2003).Later on, the model obtained was placed into a 1000 nm 3 cubic simulation box using SPC water model.
Energy minimization was performed using the steepest descent and conjugate gradient techniques and it continued until the energy converged to the maximum force smaller than 50 kJ.mol - .nm - .The minimized model was then underwent 1 ns of pre-equilibration using isobaric-isothermal ensemble (NPT) followed by a 20 ns of MD production simulation in a canonical ensemble (NVT).The integrating timestep was set at 2 fs and periodic boundary condition was applied.Temperature kept constant at 300K using Berendsen coupling while the pressure turned off during production simulation (Berendsen et al., 1984).Particle Mesh Ewald (PME) method was used to control the electrostatics interactions with a cut-off value of 0.9 nm (Essman et al., 1995).The cut-off value for van der Waals (VDW) short range forces was also set at 0.9 nm in order to correlate with electrostatic interactions calculations computed during neighbour searching.The coupling time was 0.1 ps and all-bonds were constraints using LINCS algorithm (Hess et al., 1998).

Results and Discussion
Emulsions exhibit a size distribution rather than a fixed size at equilibrium (Maillet et al., 1999;Konidala et al., 2006).The aggregation of the Tween80 and PKOEs molecules can be further explained using radius of gyration (R g ) results to find the compactness of the aggregates obtained.Figure 1 shows the fluctuation of R g as a function of simulation time.From Figure 1, at 0 ns, the total R g of the system was 3.92±0.05nm and it reduced to 3.08±0.10nm at the end of simulation.However, the biggest compactness was detected from 16 to 17 ns with an average value of 2.49±0.05nm.The big fluctuations from 10 ns onward until the end of simulation could be as a result of periodic boundary condition.
We calculated the size of the aggregate formed by computing the average diameter of the aggregate at the end of the simulation using VEGAZZ program (Pedretti et al., 2004).The average diameter of the aggregate formed was 5.78±0.05nm compared to which it was approximately consistent with experimentally measured value of 6.03±0.05nm (Salim et al., 2012).The snapshot pictures of PKOEs and Tween80 surfactant molecules in the simulation box are shown in Figure 2 Figure 4 shows the solvent accessible surface area (SASA) fluctuation as a function of time.The SASA analysis was done by using g_sas tool in GROMACS version 3.3.2which computes hydrophobic, hydrophilic and total solvent accessible surface area.The average initial values of the hydrophobic, hydrophilic and the total SASA were 427.30±3.80nm 2 , 64.20±1.20 nm 2 , and 491.40±3.80nm 2 , respectively.A drastic decrease was observed for the hydrophobic and the total SASA until 3 ns and then it remained more or less constant until the end of simulation.The minimum value of SASA was detected around ~18 ns with an average value of 171.00±1.20 nm 2 for the hydrophobic part and 202.40±1.20 nm 2 for the total accessibility where the most compact aggregate was detected.Our SASA results may propose that the self-assembly of the PKOEs esters and Tween80 surfactant molecules could be mainly due to the hydrophobic interaction resulting in from the amphiphilic characteristics of both esters and nonionic surfactant molecules.

Conclusion
Palm kernel oil-based nano-emulsion formulation can be a suitable alternative for transdermal delivery of active ingredients such as NSAIDs.These kinds of formulations can be used as carriers for the delivery of NSAIDs by removing the adverse effects of drug due to the oral therapy showed experimentally (Salim et al., 2011(Salim et al., , 2012)).We performed all-atom level MD simulations of the model Water, PKOEs, and Tween80 provided by our experimental groups to explore how can the self-assembly of nano-emulsions containing palm kernel oil wax esters and nonionic Tween80 surfactants proceed in aqueous solution and what is the shape of aggregate formed.Our structural analysis showed a stable prolate ellipsoid shape at the end of MD simulation.From our results, it can clearly be seen that the self-assembly of the esters and surfactants might be mainly due to the hydrophobic interaction due to the amphiphilic characteristics of both esters and surfactant molecules.