Study of curcumin behavior in two different lipid bilayer models of liposomal curcumin using molecular dynamics simulation.

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Study of curcumin behavior in two different lipid bilayer models of liposomal curcumin using molecular dynamics simulation Seifollah Jalili

ab

a

& Marzieh Saeedi

a

Department of Chemistry, K. N. Toosi University of Technology, P. O. Box 15875-4416, Tehran, Iran b

Computational Physical Sciences Research Laboratory, School of Nano-Science, Institute for Research in Fundamental Sciences (IPM), P.O. Box 19395-5531, Tehran, Iran Accepted author version posted online: 26 Mar 2015.

Click for updates To cite this article: Seifollah Jalili & Marzieh Saeedi (2015): Study of curcumin behavior in two different lipid bilayer models of liposomal curcumin using molecular dynamics simulation , Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2015.1030692 To link to this article: http://dx.doi.org/10.1080/07391102.2015.1030692

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Publisher: Taylor & Francis Journal: Journal of Biomolecular Structure and Dynamics DOI: http://dx.doi.org/10.1080/07391102.2015.1030692

Study of curcumin behavior in two different lipid bilayer models of liposomal curcumin using molecular dynamics simulation

Downloaded by [Rutgers University] at 23:27 08 April 2015

Seifollah Jalilia,b,*and Marzieh Saeedia a

Department of Chemistry, K. N. Toosi University of Technology, P. O. Box 15875-4416, Tehran, Iran;

b

Computational Physical Sciences Research Laboratory, School of Nano-Science, Institute for

Research in Fundamental Sciences (IPM), P.O. Box 19395-5531, Tehran, Iran E-mails: [email protected], [email protected]

*

Corresponding author. Email: [email protected]

1

Abstract Liposomal formulation of curcumin is an important therapeutic agent for the treatment of various cancers. Despite extensive studies on the biological effects of this formulation in cancer treatment, much remains unknown about curcuminliposome interactions. Understanding how different lipid bilayers respond to curcumin molecule may help us to design more effective liposomal curcumin. Here, we used molecular dynamics simulation method to investigate the behavior of curcumin in two lipid bilayers commonly used in preparation of liposomal curcumin, namely DPPC and DMPG. First, the free energy barriers for translocation of one curcumin molecule from water to the lipid bilayer were determined by using the potential of mean force Downloaded by [Rutgers University] at 23:27 08 April 2015

(PMF). The computed free energy profile exhibits a global minimum at the solventheadgroup interface (LH region) for both lipid membranes. We also evaluated the free energy difference between the equilibrium position of curcumin in the lipid bilayer and bulk water as the excess chemical potential. Our results show that curcumin has the higher affinity in DMPG compared to DPPC lipid bilayer (−8.39 vs. −1.69 kBT) and this is related to more hydrogen bond possibility for curcumin in DMPG lipid membrane. Next, using an unconstrained molecular dynamic simulation with curcumin initially positioned at the center of lipid bilayer, we studied various properties of each lipid bilayer system in the presence of curcumin molecule that was in full agreement with PMF and experimental data. The results of these simulation studies suggest that membrane composition could have a large effect on interaction of curcumin- lipid bilayer. Keywords: liposomal curcumin; potential of mean force; molecular dynamics simulation; DPPC membrane; DMPG membrane. 1. Introduction Curcumin, which is found in traditional herbal remedy Curcuma longa, is primarily utilized as a natural yellow pigment. The curcumin structure (Figure1) is a linear diarylheptanoid which consists of two aromatic rings (aryl groups) joined by a seven-carbon chain (heptane) with hydroxyl and carbonyl functional groups. This diphenolic compound has a variety of biological activities and pharmacological properties such as anticancer, anti-inflammatory and anti-oxidant (Shang et al., 2010). An extensive review of literature has been specified curcumin as an excellent candidate for cancer treatment (Goel, Kunnumakkara, &Aggarwal, 2008).

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Despite the potential effectiveness of curcumin, there are several problems that have prevented its marketing as a drug. Major obstacles are the poor aqueous solubility, intense staining color, and extremely low oral bioavailability (Anand, Kunnumakkara, Newman, & Aggarwal, 2008). Fortunately, the advent of efficient drug delivery systems based on nanoscience and nanotechnology can improve curcumin’s bioavailability, protect it from degradation, and enhance its targeting capacity toward cancer tumors. Several types of nanoparticles such as phospholipid vesicles or liposomes (Li, Ahmed, Mehta, & Kurzrock, 2007;Narayanan, Nargi, Randolph, & Narayanan, 2009; Wang et al., 2008), micelles (Song et al., 2011), nanoemulsions (Ganta, Devalapally, & Amiji, 2010), solid-lipid nanoparticles (Kakkar& Kaur, 2011), polymeric nanoparticles (Yallapu, Gupta, Jaggi,& Chauhan, 2010), and cyclodextrins (Yadav et al., 2010) have been examined for delivery of curcumin to tumors. The hydrophobicity of curcumin provides an effective strategy to incorporate it in the hydrophobic domain of liposome nanoparticles. Liposomes, artificial vesicles consisting of an aqueous core enclosed in one or more phospholipid layers, are used to convey vaccines, drugs, enzymes or other substances to target cells and can protect them from external stimuli. Phospholipids and cholesterol are the major structural components of liposome. The most common phospholipid is phosphatidylcholine (PC) molecule which is not soluble in water and in aqueous media. The lipid molecules align themselves closely in planner bilayer sheets in order to minimize the unfavourable action between the bulk aqueous phase and long hydrocarbon fatty chain. The Glycerol containing phospholipids are the most common components used in liposome formulation and represent more than 50% of the weight of lipid in biological membranes. Several types of phospholipids such as dimyristoylphosphatidylcholine, DMPC (Li, Braiteh, & Kurzrock, 2005), DMPC/dimyristoylphosphatidylglycerol, DMPG (Wang et al., 2008; Li, Ahmed, Mehta, & Kurzrock, 2007), Egg yolk phosphatidylcholine, PC (Kunwar, Barik, Pandey, & Priyadarsini, 2006), dipalmitoylphosphatidylcholine, DPPC (Thangapazham et al., 2008), and DPPC/DMPG (Dhule et al., 2012) have been examined for preparation of liposomal curcumin. So far, most of studies on liposome-encapsulated curcumin have been focused on the biological effect of this formulation in cancer treatment (Aggarawal, Kurzrock, Li, & Mehta, 2004; Li, Braiteh, & Kurzrock, 2005; Narayanan et al., 2009), but not much attention has been given to the curcumin–liposome interactions (Barry et al., 2009; Karewicz et al., 2011). In addition, the lipid compositions as well as the liposome properties strongly influence the binding stability of curcumin in the liposomal structure. Thangapazham et al. (2008) have showed that curcumin selectively partitions with high efficiency into DMPC liposomes as compared to DPPC or Egg PC. They have observed that DMPC-based liposomal curcumin possesses greater encapsulation efficiency as compared with liposomes prepared with DPPC and PC. Sou et al. (2008) observed that liposomes consisting of DPPC and cholesterol could solubilize much less curcumin in the bilayer membrane than DMPC vesicles. This observation suggested that membranes containing cholesterol were unsuitable for stable embedding of curcumin. Therefore, the investigation of curcumin permeation into liposome as well as the interaction between different types of liposomal structure and curcumin plays an important role in designing liposomal composition for curcumin. In order to study the interaction of curcumin with liposome, the interaction with a phospholipid bilayer as a model for liposome can be examined. To investigate how curcumin interacts with a membrane, it is important to understand how it percolates through the membrane and localizes in a lipid bilayer. The curcumin 3


may be located on the surface of membrane at the hydrophilic/hydrophobic interface or within the hydrophobic domain. Experimental evidence suggests that curcumin is located in the hydrophobic domain of the lipid bilayer membrane (Karewicz et al., 2011; Sou, Inenaga, Takeoka, & Tsuchida, 2008). Julia el al. (2013) used the Caco-2 cells method to specify the percolation of curcumin through the membrane. Nevertheless, full understanding the interaction of curcumin with a lipid bilayer and the effects of drug on various physical properties of membrane at the molecular level is not yet achieved. Molecular dynamics (MD) computer simulation of lipids is a powerful technique to obtain detailed information about the structure and dynamics of different types of lipid assemblies that are concealed from the most experimental method (Feller, 2000). The benefit of MD simulations over traditional experiments is that the contributions from the different regions of the lipid bilayer can be studied at a molecular level, whereas experimental models can only approximate the membrane as a uniform barrier slab. In addition, MD simulations may provide valuable complementary information to experiment data about details of interactions between the drug molecule and different lipid bilayers (Bennett, MacCallum, Hinner, Marrink, & Tieleman, 2009). Numerous MD simulations have been performed concerning the molecular transport in membranes (Bemporad & Essex, 2004; Marrink & Berendsen, 1996). For example, the partitioning behavior of different amino acids and peptides in a model membrane host has been studied (Aliste & Tieleman, 2005; MacCallum, Bennett, & Tieleman, 2008). Bemporad, Luttmann, and Essex (2005) investigated the behavior of drugs and small organic compounds inside the membrane with atomistic details. A unique advantage of MD simulation is its ability to shed light on the free energy of translocation of a drug through the membrane. One such study scanned the free energy profile (or the potential of mean force, PMF) of drug as it travelled through the membrane. The PMF tells us the amount of work needed for a drug to percolate from the bulk water to a given depth in the membrane. Boggara and Krishnamoorti (2010) have studied partitioning of two non-steroid drugs (aspirin and ibuprofen) in a lipid membrane using PMF calculations. They found that both drugs had higher partition coefficients in the lipid bilayer than in water. The main goal of the present work is to perform a detailed study on the curcumin interaction with two different lipid bilayers (DPPC and DMPG) at the atomistic level using molecular dynamics simulations. Based on the study from Thangapazham et al. (2008) and Dhule et al. (2012), we have chosen two common lipid bilayers consisting of one zwitterionic phosphatiylcholine, DPPC and one negatively charged phosphatidylglycerol, DMPG. Although most of the liposomal curcumin have been prepared from mixtures of zwitterionic and anionic phospholipids, a detailed insight into the structural behavior of curcumin with these homogeneous one-component membranes is essential, before advancing to more complex liposomal curcumin. We applied two independent approaches to study the interaction between curcumin and each model of lipid bilayers. First, we used an umbrella sampling molecular dynamics protocol (Torrie & Valleau, 1977) to generate biased distributions of curcumin positions along the lipid bilayer. This method was used to derive a free energy profile that indicates energy barriers along the pathway. Next, we ran an unconstrained MD simulation with the curcumin initially located in the center of the lipid bilayer (z = 0). This simulation was analyzed to understand where curcumin

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resides in the lipid bilayer and how the curcumin molecule affects the bilayer properties of each membrane. To the best of our knowledge, there are no published results on molecular dynamics simulation of curcumin in a lipid bilayer and this study is the first one to provide detailed picture of effects of the lipid composition (specially the headgroup of PC versus PG phospholipid) on the curcumin-bilayer interaction using the MD simulation. Understanding the detailed behavior of curcumin in different types of lipid bilayers would enable us to develop the liposomal curcumin based on these properties. 2. Methods


2.1. System setup The chemical structure of the DPPC, DMPG, and curcumin molecules along with partial charges for the curcumin molecule are shown in Figure 1. DMPG molecule consists of two 13-carbon chains and has 2 carbons less than DPPC molecule in each chain. Moreover, the choline moiety of DPPC molecule is replaced with the glycerol group in DMPG molecule. In fact, the particular interest in this article is to investigate the effect of the chemical structure, specially the headgroup of lipid membrane, on the curcumin-bilayer interactions. Initial coordinates of a 128-lipid DPPC bilayer hydrated with 3655 water molecules based on 2 ns equilibration (Tieleman & Berendsen, 1996) have been obtained from P. Tielman’s Web site (http://moose.bio.ucalgary.ca). The initial coordinates of a 128-lipid DMPG bilayer neutralized with 128 Na+ counterions and hydrated with 3527 water molecules (Piggot, Holdbrook, & Khalid, 2011) have been obtained from Lipidbook Web site (Domański, Stansfeld, Sansom, & Beckstein, 2010). The racemic DMPG lipid bilayer was composed of equal numbers of L-DMPG and D-DMPG. Additional 3 nm layers of pure water were added to the DPPC and DMPG lipid bilayers to provide enough aqueous phases for curcumin positioning above the membrane. 2.2. Molecular dynamics simulations We employed MD simulation to study the interaction between curcumin and two different lipid membranes (DPPC and DMPG). DPPC lipid bilayers were modeled using GROMOS-96 force field with a modified version of the 43A1 parameter set (Chiu, Pandit, & Jakobsson, 2009). Force field parameters for the DMPG lipid bilayer were also taken from the GROMOS-53A6 parameter set (Oostenbrink, Villa, Mark, & Van Gunsteren, 2004).These parameters have previously been used by Piggot et al. (2011). It implies that both hydroxyl groups of the DMPG bear the same charges, in agreement with Pedersen et al. (2006). The Dundee PRODRG server (Schuettelkopf & Aalten, 2004), which is based on the GROMOS force field, was employed to prepare coordinate and topology files for curcumin. Partial charges on curcumin (Figure 1(c)) were found using the Hartree-Fock method and a 6-31G* basis set, solved with the Gaussian03 program (Frisch et al., 2004). The water molecules were explicitly represented by the simple point charge (SPC) model (Berendsen, Postma, van Gunsteren, & Hermans, 1981).

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The translocation of curcumin in the lipid bilayer is difficult to capture with the current accessible time scale of MD simulation. We chose two strategies to overcome this obstacle: first, by restraining the curcumin at different depths of each lipid bilayer, we computed the free energy profile of the system which is discussed later. Second, we placed the curcumin inside of each lipid bilayer to study the response of bilayer to the curcumin. The curcumin molecule was inserted into the center of a pre-equilibrated DPPC lipid bilayer using a series of scaling and energy minimization steps, as described by Kandt et al. (2007). The GROMACS program g_membed (Wolf el al., 2010) was also used for the curcumin insertion in the center of DMPG lipid bilayer. The initial orientation for the curcumin molecule in these simulations was parallel to the bilayer normal (z-axis). In addition to the above sets of simulations which all consisted of a single curucmin interacting with the membrane, we attempted to study multiple curcumins in the lipid bilayer to explore how would the interaction between curcumins affect the preferred location and orientation of drug in the lipid bilayer. We placed three curcumin molecules at random positions and orientations in the bilayer interior and simulated the system for 100 ns. One of the three curcumin molecules spontaneously partitioned into the solvent-headgroup interface after 1.5 ns. The two other curucmins did so after 30 ns. In this simulation, three curucmin molecules adopt an orientation and location remarkably similar to the results we obtained for one curumin. Therefore, we focus on the case of a single curucmin with each lipid bilayer to obtain a clear understanding of how lipid components affect the free energy and precise location of curcumin in the lipid membrane. In all simulations, the cut off distance for the Coulomb and van der Waals interactions were set to 1.2 nm. Coulomb interactions were calculated by the Particle Mesh Ewald (PME) method (Darden, York, & Pedersen, 1993). The temperature was maintained at 323K, well above the gelliquid transition temperature of 314 K for DPPC and 296 for DMPG bilayers, by means of NoseHoover thermostat (Hoover, 1985; Nosé, 1984) with a time constant of 0.5 ps. The reference pressure was set at 1 bar and controlled semi-isotropically using the Parinello-Rahman barostat (Parrinello& Rahman, 1981) with a time constant of 5 ps. All bond lengths were constrained using the LINCS algorithm (Hess, Bekker, Berendsen, & Fraaije, 1997), allowing an integration time step of 2 fs. All the MD simulations were performed using the GROMACS simulation package, version 4.5.4(van der Spoel et al., 2008). 2.3. Potential of mean force calculations To study the effect of lipid composition on the penetration of curcumin into two model lipid membranes, the free energy of transferring of one curcumin across the DPPC and DMPG bilayers was evaluated using the potential of mean force (PMF) calculations. The curcumin molecule was initially put in the bulk water. Then, it was pulled into each bilayer center along the z-axis using umbrella sampling method. A harmonic restraint of 1500 kJ mol−1nm−2 and a pulling rate of 0.01 nm/ps were applied to distance between the center of masses of curcumin and each lipid bilayer. Then 60 and 50 configurations were selected at different z locations in DPPC and DMPG bilayers, respectively. Each configuration was simulated for 20 ns. During the simulation, the curcumin molecule was constrained at the corresponding z locations, but was allowed to move unconstrained in the xy plane. The last 5 ns of each MD run were used to evaluate the free energy of curucmin in the lipid bilayers using the weighted histogram analysis method (Hub, de Groot, & van der Spoel, 6

2010). 3. Results and discussion


3.1. Potential of mean force (PMF) The free energy profiles of curcumin translocation in DPPC and DMPG lipid bilayers are shown in Figure 2. Calculations have been performed on one side of the membrane, and assumed to be symmetric with respect to the bilayer center. Bayesian bootstrap analysis (Hub et al., 2010) with100 bootstraps for the estimation of statistical errors showed that the largest errors for curcumin are 1.97 and 1.03 kBT in DPPC and DMPG bilayers, respectively, which are moderate values as compared with the other PMF results (Kyrychenko, Sevriukov, Syzova, Ladokhin, & Doroshenko, 2011; Meng & Xu, 2013). As in prior publications (Marrink & Berendsen, 1994), each individual leaflet of DPPC bilayer was partitioned into four regions and the bulk water was demonstrated as the SOL region. So, five resulting regions for DPPC bilayer are as follows (the distances are from the center of bilayer): low tail density (LT, 0 to 0.6 nm), high tail density (HT, 0.6 to 1.3 nm), high headgroup density (HH, 1.3 to 2.0 nm), low headgroup density (LH, 2.0 to 2.7 nm), and water solvent (SOL, more than 2.7 nm). The snapshot of the five regions lipid bilayer is depicted in Figure 2. Since the only significant difference between DMPG and DPPC membranes is the slightly shorter acyl chain (2carbon atoms for each chain); we shifted each region 0.2 nm closer to center of the bilayer for DMPG membrane. It is noted that the free energy is set to zero when the entire of drug is in the bulk water (SOL region). This region is considered as the reference point for the calculation of the change in the free energy (∆G). As can be seen from Figure 2, the general trend in the curcumin free energy profile is similar for DPPC and DMPG lipid bilayers. As curcumin molecule travels closer to the membrane, one part of the drug approaches to the headgroup region and the free energy is slightly declined from zero. When the curcumin reaches to the LH region, the PMF characteristically decreases. This decrease in the free energy is attributed to the transfer of the hydrophobic drug from water to an increasingly hydrophobic environment, as well as the electrostatic attraction of curcumin polar groups to charged lipid headgroups. In this region, the energy profiles show a global minimum at z = 2.36 and 2.32 nm in DPPC and DMPG bilayer, respectively. The corresponding free energy values for these global minimums are −1.69 and −8.39 kBT. The well depth in the PMF corresponds to the equilibrium position of the curcumin in the lipid bilayer. Sun, Lee, and Hung (2008) demonstrated two binding states for curcumin in the lipid bilayer. They reported that, in low and high concentrations of curcumin, it binds to the solvent-headgroup interface and hydrocarbon regions, respectively. In our study of the translocation of one curcumin in DPPC and DMPG lipid bilayers, the global minimum occurs in LH region. It can be concluded that curcumin tends to reside in the LH region, which it is in agreement with the experimental work (Sun et al., 2008). Although curcumin has a low solubility in water, this diphenolic compound has two hydroxyl groups, one at each end of the molecule, that interact favourably with water oxygen and lipid headgeoup oxygen atoms. Therefore, going deeper into the center of lipid bilayer is less favourable and the free energy of curcumin increases dramatically when it travels to HH region. Also, further penetration into the membrane requires an increase in free energy due to the steric hindrance from the lipid acyl chain. 7


The free energy profile of curcumin in DPPC bilayer also shows local minimum in HH region. The free energy change of curcumin translocation from global to local minimum is more than difference between water and global minimum because of reducing the proper interactions for polar groups of curcumin and increasing the steric barrier of the acyl chain. When curcumin approaches to the acyl chains of the bilayer, the free energy profile rapidly increases. The maximum in the free energy profile occurs in the LT region. The free energy profile of cucurmin in these two different lipid bilayers is similar to those obtained by Yacoub et al. (2011) for partitioning of doxorubicin in the DPPC/Cholesterol lipid bilayers. The free energy of curcumin desorption defines as the excess chemical potential of curcumin in the equilibrium position compared to water and it allows one to compare the relative affinity of curcumin in different bilayers. Zhang el al. (2008) and Bennett el al. (2009) have used this approach to describe cholesterol affinity in different lipid bilayers. Our results show that curcumin has a higher affinity in DMPG bilayer. It may be attributed to the decent interactions between curcumin molecule and hydroxyl groups of glycerol in DMPG lipid bilayer. So, it can be concluded that cucumin affinity in the lipid bilayers is strongly dependent on the composition and structure of the lipid bilayer. The main potential barrier in the free energy profile, determined by the difference between the absolute free energy minimum and the free energy in the bilayer center, is 20.8 and 30.75 kBT for curcumin in DPPC and DMPG bilayers, respectively, which are much larger than the values for doxorubicin, atenolol and ibuprofen in DPPC bilayer (Meng & Xu, 2013). The height of potential barrier for curcumin in DPPC bilayer is comparable to those for cholesterol in this lipid bilayer (Bennett, MacCallum, Hinner, Marrink, & Tieleman, 2009). Because of the long structure of curcumin, it is interesting to know how it is oriented when it penetrates through the lipid bilayer. The orientation of curcumin with respect to the DPPC bilayer in five regions is shown in Figure 3 with a series of snapshots taken from the umbrella sampling simulations. From the Figure, it can be seen that, moving from the water phase towards the LH region, the curcumin molecule tends to lie perpendicular to the bilayer normal. The reason for this orientation seems to be the feasibility for the curcumin’s polar groups to be close to the hydrophilic groups of DPPC, in order to form a larger number of hydrogen bonds (H-bonds) with oxygen atoms of DPPC. In addition, the curcumin tends to be aligned parallel to the lipid tails as it moves towards the bilayer interior (in the HT region), because the lipid molecules are tightly packed and the free space for curcumin is parallel to the bilayer normal. This kind of drug behavior in the HT region of lipid bilayer is also reported in both experimental (Frenzel, Arnold, & Nuhn, 1978) and simulation (Alper & Stouch, 1995; Bemporad et al., 2005) works. In the LT region, the lower part of the lipid chains, the curcumin prefers to tilt in order to form an intermolecular hydrogen bond and create a 6members ring. So, it can be concluded that the orientation of curcumin in the lipid bilayer can be affected by the hydrogen bonding opportunities with water and lipid molecules. Similar orientations were also observed for curucmin in DMPG lipid membrane. As we mentioned above, hydrogen bonding capability of the curcumin molecule is important because it is often correlated with the orientation of drug in the lipid bilayer. We have determined the number of hydrogen bonds formed to the pulled curucmin as it is transferred from water into the center of DPPC bilayer and is plotted for curcumin-lipid and curcumin-water couples in Figure 4. The presence of hydrogen bonds has been examined by two criteria: the distance between the donor and acceptor and the angle between the donor, hydrogen, and acceptor atoms. The cut-off values used for these parameters were 0.35 nm and135◦, respectively and the errors were estimated 8


using the block averaging method. As Figure 4 shows, the number of H-bond between curcumin and DPPC is less than 0.1 at all distances from the bilayer center. Therefore, hydrogen bonding between curcumin and DPPC does not have significant effects on the overall number of H-bonds and we can only consider the H-bonds between curcumin and water molecules. The maximum number of Hbonds between curcumin and water molecules is when the curucmin is in the bulk water (SOL region). Transferring the curucmin from water into its equilibrium position, there is a decrease in the number of hydrogen bonds. The number of hydrogen bonds decreases as it is moved into the bilayer interior, but doesn't reach to zero. Our analyses show that water molecules are able to penetrate into the bilayer interior to keep the hydrophilic group of the curcumin solvated. We observed at least one water molecule inside the bilayer interior when the curcumin is restrained at HT and LT regions, but we didn’t see any water defect formation which was reported for acetic acid in the lipid bilayer (Bemporad, Luttmann, & Essex, 2005). A similar trend of hydrogen bonding with water molecules is also observed for curcumin in DMPG lipid bilayer. In addition, a significant number of Hbonds between curcumin and glycerol group of DMPG lipid molecules are formed. Both experimental and simulation observations have also proved that the hydroxyl group of a PG lipid has the potential to form both intra- and intermolecular hydrogen bonds in the membrane (Elmore, 2006; Dicko, Bourque, & Pézolet, 1998). The contributions of key functional groups of curcumin in Hbond formation with water molecules have been averaged in each region along the pathway and are shown in Table 1. The average numbers of H-bond between curcumin and hydroxyl groups of glycerol in DMPG bilayer are shown in parentheses. H-bonds have been calculated with respect to the interior keto oxygen and enol hydroxyl, as well as the functional groups in one of the phenyl rings. Although the number of H-bonds with water molecules is very small in the HT and LT regions, hydration occurs at all depths, showing that water permeation to each bilayer core is taking place. Most of H-bonds on the curcumin involve the keto oxygen and methoxy oxygen groups, while the – OH groups form very few H-bonds. Moreover, the hydroxyl group of glycerol in DMPG bilayer form H-bonds with curcumin when it is in the LH and HH regions (the numbers of H-bond are shown in parentheses). As can be seen, the number of H-bonds between the key functional groups of curucmin and glycerol group is more in the LH region than in the HH region (0.97 vs. 0.79). So, it can be concluded that, the more hydrogen bond possibility for curcumin in DMPG lipid bilayer is evident to the high affinity of curcumin in equilibrium position of this lipid bilayer. These H-bonding results for curucmin in DPPC and DMPG lipid bilayers provide useful molecular level insights into experimental observations. The nonbonded energy contribution of curcumin with lipid, water, and ion in each region along the pathway of translocation were averaged and are shown in Table 2. As can be seen, the Lennard-Jones energy between the curcumin and water is about zero at z=0 nm and decreases with increasing the distance from the bilayer center. Moreover, the Lennard-Jones energy between curcumin and lipid increases from the center of bilayer (-197.84 kJ/mol for DPPC, and -226.09 kJ/mol for DMPG) and approaches to about zero in bulk water. These two terms cross each other in the LH region (at about z= 2.4 nm), which corresponds to the curcumin equilibrium position. Compared to the Lennard-Jones terms, the Coulomb energy contributes more to the total interaction energy. The Coulomb energy between curcumin and water in lipid bilayers decrease as the distance from the center of bilayer increase, and the smallest values are -284.41 and -276.19 kJ/mol in SOL region for DPPC and DMPG lipid bilayers, respectively. Curcumin interacts electrostatically with water

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molecules through its hydroxyl, ketone and methoxy groups. We can see that the variation sequence of the four selected interaction energies of curcmin in both lipid bilayers is similar. It is noted that in DMPG bilayer, in addition of Coulomb energy between curcumin and water, another Coulomb energy exists between curcumin and Na+ ions. As can be seen from Table 2, The Coulomb energy between curcumin and Na+ ions have the maximum value in HH region (−197.2 kJ/mol). So, we predicted that a great number of Na + ions are located in the HH region of DMPG membrane


3.2. Positioning of Curcumin in two different Bilayers To more explicate where curucmin localizes in two different lipid bilayers (DPPC and DMPG) and to understand how the curcumin molecule would affect the membrane properties of respective bilayers, we performed two unconstrained MD simulations. These simulations are complementary to simulations of previous section in which the drug restrained at different depths of the lipid bilayer. For the initial configuration, the drug was placed in the center of bilayer (z = 0), and it is roughly parallel to the membrane normal. These systems were simulated for 60 ns. For the purpose of comparison, two complementary simulations of DPPC and DMPG lipid bilayers without curcumin molecule (references systems) were also conducted for 60 ns. To analyze the impact of curcumin on two different bilayer properties we compared several parameters: the area per lipid, bilayer thickness and deuterium order parameters. All the parameters were averaged throughout the last 20 ns of the trajectory. In this work, the average area per lipid molecules was calculated by multiplying the XY dimensions of the simulation box and dividing the results by the number of lipid molecules present in one leaflet of the bilayer. Results showed no drift in evolution of the area per lipid for different simulated systems, and therefore, the systems were considered to be in equilibrium. The lipid thickness was calculated as the distance between the phosphate peaks in the electron density profile which was determined using the g_density program from the GROMACS package. The deuterium order parameter, SCD, is a common quantity to characterize the order in the lipid bilayers and is defined as:

S CD = 1 2 3 cos 2 θ i − 1 , Where θi is the angle between the molecular axis given by the carbon atoms Ci−1 and Ci+1 and the lipid bilayer normal. SCD was calculated in GROMACS by using g_order program. The average area per lipid and thickness for different simulation systems are presented in Table 3. The deuterium order parameter profile for sn1 hydrocarbon chain are shown in figure 5 (calculation for the sn2 chain show similar result), which compare the order of the lipid tails between the curcumin-free bilayer and the curcumin-bound bilayer. The areas per lipid and thickness values estimated from our simulations of reference systems (without curcumin molecule) are within the range of experimental values and the values obtained in previous simulations (Kučerka, Nieh & Katsaras, 2011; Marra, 1986; Nagle, 1993). From the result, it can be seen that the area per lipid for each bilayer was increased in the presence of curcumin. This can be explained by the fact that the curcumin molecule occupies some spaces between the lipid molecules and therefore increases the average area per lipid. The increased area of lipid bilayer in the presence of curcumin is coupled to a slight decrease in calculated thickness, in good agreement with the result for curcumin in DOPC/DOPE (Sun, Lee, & 10


Hung, 2008). As can be seen from figure 5, the curcumin also induces the ordering of hydrocarbon chain of two different bilayers. The increased order parameter of lipid bilayer tail in the presence of curcumin is qualitatively similar to cholesterol (Urbina, Pekerar, Patterson, Montez, & Oldfield, 1995). The ordering effect of the curcumin on the acyl chain of bilayer is in compliant with the experimental date (Barry et al., 2009). The time evolution of curucmin movement in the lipid membrane was shown with a series of snapshots in Figure 6. The curucmin molecule, which was initially placed in the center of the bilayer (Figure 6a), moves spontaneously away from the center of bilayer to the region near the carbonyl group of lipid molecules (Figure 7b). It moves within the first 2 and 5 ns in the DPPC and DMPG simulations, respectively. In this movement, curcumin orients itself perpendicular to the bilayer normal to maximize favourable contacts between polar groups of drug and the surroundings. Such localization of cucrcumin in the lipid bilayer is observed experimentally by fluorescence quenching (Karewicz at al., 2010). The curcumin molecule remains there for about 30 ns in both lipid membranes and then it shifts about 1 nm to the solvent-headgroup interface in the DPPC and DMPG simulations. This location corresponds to the equilibrium position of curcumin which was observed in the PMF curves (Figure 6c). Figure 6 also shows a close-up of hydrogen bonding and distances between the key functional groups of curcumin and water molecules for the last configuration in two different lipid bilayers. The minimum distances occur between water molecules and keto and two methoxy oxygen atoms. It is an evidence for more hydrogen bond formation between these key functional groups of curcumin (keto oxygen and methoxy oxygen) and water molecules compared to other functional groups which was also observed in Table 1. Moreover, curcumin molecule can form H-bond with hydroxyl group of glycerol in DMPG lipid membrane. To provide a better description of molecular dynamic behavior of curcumin in the lipid bilayer, we have analyzed the time evolution of the curucmin in two different lipid membranes. For this purpose, the z coordinate (along the bilayer normal) of the center of mass of the curcumin molecule as a function of the simulation time are depicted in Figure 7. As mentioned above, the curcumin molecule, which was placed in the center of the bilayer, moves gradually away from the center of the bilayer to the polar region of lipid head groups. Therefore, curcumin doesn’t show any tendency to stay in the center of the lipid bilayer. Because we used only one curcumin molecule in each of the simulated systems, this molecule distributes on the one of bilayer leaflets (top leaflet for both DPPC and DMPG bilayers). From the results shown in Figure 7, it is evident that fluctuation of curcumin in the DMPG bilayer are less than in the DPPC membrane, it is probably related to the high affinity of curcumin in the DMPG membrane. The center of mass positions of the choline or glycerol, phosphate, carbonyl, and terminal methyl groups of lipid molecules are also shown in Figure 7. As can be seen, the average location of the mentioned groups in the lipid bilayer is very stable. In order to have a better insight into the hydration and probability distribution of water molecules around the different groups of lipid bilayer, and to examine the effect of curcumin in permeation of water molecules into the lipid bilayer, the radial distribution function (RDF) analysis has been utilized. Figure 8 displays the RDF of oxygen atoms in water molecules around the oxygen atom of phosphate, carbonyl, and glycerol (in DMPG bilayer) groups. In the DPPC lipid bilayer, we observed the distinct first peak at 0.266 nm for phosphate and 0.272 nm for carbonyl groups which was consistent to 0.278, 0.272, and 0.276 nm for phosphate, carbonyl, and hydroxyl groups in DMPG 11

lipid membrane. From this figure, it can be seen that the systems containing curcumin have somewhat higher RDF compared to the reference systems. It's due to the orientation of curcumin in the lipid bilayer that creates more space in the lipid bilayer and allows water molecules to enter into the lipid bilayers. Analysis of electron density distribution is important to obtain the precise location of curcumin in two different lipid bilayers. Figure 9 show the electron density distribution for different groups in the simulation box for the last 20 ns of the simulations in DPPC and DMPG lipid membranes, in which all molecules have stable structures. For comparison, the electron density of reference systems are added by dashed lines. As shown in Figure 9, the presence of one curcumin molecule does not disturb the density distribution of the bilayer atoms. In both lipid membranes, curcumin molecule spends most of times in the head group regions with a broad peak observed at ~1 to 2.8 nm, which is close to the phosphate, choline or glycerol group’s peak.


4. Conclusions We present the results of atomistic MD simulations performed in an attempt to understand the nature of interaction between curcumin and two different types of lipid membranes (one zwitterionic phosphatiylcholine, DPPC and one negatively charged phosphatidylglycerol, DMPG). The umbrella sampling technique was used to calculate the potential of mean force (PMF) for transferring of curcumin across the lipid bilayers. Our analysis showed extra stabilization for curcumin in the solvent-headgroup interface and a perpendicular orientation of curcumin with respect to the bilayer normal to maximize favorable contacts with polar groups of lipid and water molecules. We also found that the composition of the bilayer and therefore the structure of the bilayer had a large effect on the affinity of curcumin in the lipid membrane. Curcumin had higher affinity in DMPG compared to DPPC lipid bilayer that can be related to hydrogen bonding between cucrumin and glycerol group of DMPG lipid bilayer. The electron density profiles extracted from the 60 ns unconstrained simulation supported the behavior deduced from the PMF curve. The simulation started with the curcumin molecule located in the center of bilayer and curcumin spontaneously diffused to the solvent-headgroup interface. The results of MD simulations justify that area per lipid and ordering of lipid bilayers increased in the presence of curcumin molecule. RDF of oxygen atoms in water around different oxygen atoms of lipid molecules shows somewhat higher value, compared to the reference systems, that shows more water penetration into the lipid bilayer in the presence of curcumin molecule. In summary, the results of this study provide valuable molecular level insight into understanding the mechanism of translocation of curcumin across the lipid bilayer and considering how membrane composition can affect the interaction of curcumin with lipid bilayer membranes. So, this khowledge may help us to design new liposomal cucumin. References Aggarawal, B. B., Kurzrock, R., Li, L., & Mehta, K. (2004).Liposomal curcumin for treatment of cancer.WO2004080396A2, Google Patents.

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Table 1. Average number of hydrogen bonds formed between the key functional groups of curcumin and water molecules in five regions along the bilayer normal. Functional groups are: −OH (enol hydroxyl), −O (keto oxygen), −OH (phenyl) and −OCH3 (methoxy oxygen). The number in parentheses shows the average number of hydrogen bonds between curcumin and hydroxyl groups of glycerol in DMPG lipid membrane. Lipid region

OH inner

keto Oxygen inner

OH phenyl

keto Oxygen phenyl

SOL

0.625

2.323

0.882

1.559

LH

0.459

2.212

0.683

1.453

HH

0.086

1.443

0.269

1.114

HT

0.02

1.026

0.008

0.908

LT

0.014

1.018

0.024

0.692

SOL

0.604

2.356

0.891

1.614

LH

0.525(0)

2.327 (0.609)

0.643 (0.04)

0.936 (0.317)

HH

0.584(0)

2.089 (0.634)

0.337 (0.02)

0.891 (0.144)

HT

0.059

0

0.333

1.139

LT

0.059

0.096

0.287

0.931


DPPC

DMPG

19

Table 2. Average nonbonded energy contributions (Lennard-Jones and Coulomb terms in kJ/mol) of curcumin with water, lipid, and ion in each region along the pathway of translocation. LJ

Coul

LJ

Coul

Coul

(CUR+SOL)

(CUR+SOL)

(CUR+Lipid)

(CUR+Lipid)

(CUR+Na)

SOL

-118.21

-284.41

-4.01

-2.18

-

LH

-99.74

-259.08

-49.04

-19.57

-

HH

-48.32

-172.31

-152.09

-40.92

-

HT

-4.67

-52.68

-195.6

-4.01

-

LH

-1.9

-2.68

-197.84

-0.06

-

SOL

-116.85

-276.19

-3.47

-3.11

-11.67

LH

-89.31

-217.63

-75.29

-30.89

-56.53

HH

-65.28

-108.31

-140.78

-18.92

-197.18

HT

-12.89

-51.08

-213.66

-14.69

-23.48

LH

-1.62

-4.92

-226.09

-4.24

-1.82


DPPC

DMPG

20

Table 3. Important quantitative properties of the simulated bilayer systems. Bilayer property

DPPC

DPPC+CUR

DMPG

DMPG+CUR

Area per lipid (nm )

0.623 ± 0.01

0.638 ± 0.02

0.608 ± 0.06

0.611 ± 0.02

Thickness (nm)

3.961± 0.03

3.808 ± 0.05

3.693 ± 0.02

3.582 ± 0.03

2


Note: The standard errors in the average values were estimated using block averaging method.

21


Figure Captions

Figure 1. Molecular structures of DPPC (a), DMPG (b), and Curcumin molecule (c) with partial atom charges.

22


Figure 2. Snapshot of the lipid bilayer system used (a). The vertical lines indicate the boundaries between the different regions (see text for definitions). Red and white tubes are water; red spherescholine; purple spheres-phosphate; green spheres-carbonyl; yellow spheres-terminal methyl; thin orange lines-lipid tails. Curcumin free energy profile estimated for half of the DPPC (b) and DMPG (c)

23

lipid bilayer. The five important regions described in the text are also shown in the plot. The


standard deviation values are shown with error bars.

24


Figure 3. Snapshots showing the curcumin orientation with respect to the DPPC bilayer from umbrella sampling simulations in SOL (a), LH (b), HH (c), HT (d), and LT (e) regions. The curcumin is shown as CPK and is colored in yellow. For DPPC, the nitrogen atoms are shown in blue, the oxygen atoms in red, the carbon atoms in cyan, and the phosphorous atoms in brown. Water molecules are not shown for clarity.

25


Figure 4. The average number of hydrogen bonds between curcumin and DPPC or water molecules along the bilayer normal.

26


Figure 5. Deuterium order parameter (SCD) profiles calculated for the sn1 fatty acid chain with presence of curcumin (solid line) and without curcumin molecule (dotted lines) in DPPC (a) and DMPG (b) lipid bilayers.

27


Figure 6. Snapshots illustrating the positioning of a curcumin molecule in the DPPC bilayer during a 60 ns unconstrained simulation (top). For clarity, acyl chains of DPPC have been omitted. Curcumin is shown in CPK representation and is colored in yellow. Water molecules are shown in red points. The 28

Close-ups illustrating the hydrogen bonds in final configuration between curcumin and water molecules or oxygen atoms of lipid bilayer in DPPC (a) and DMPG (b). The minimum distances


between them (in Å) are also shown.

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Figure 7. The z coordinates of the center of mass of curcumin molecule as a function of simulation time in DPPC (a) and DMPG (b) lipid bilayer. The center of mass positions of choline (glycerol in DMPG), phosphate, carbonyl, and terminal methyl groups of lipid molecules are also shown.

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Figure 8. Radial distribution functions of water oxygen around various oxygen atoms in phosphate, carbonyl, and hydroxyl groups of DPPC (a) and DMPG (b) lipid bilayer.

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Figure 9. Electron density profiles of various groups along the DPPC (a) and DMPG (b) lipid bilayers. The dashed curves show the electron density distribution of lipid bilayers without curcumin molecule. The density profiles were averaged over the last 20 ns of the simulations.

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Liposomal curcumin and its application in cancer.

Molecular dynamics simulation of the partitioning of benzocaine and phenytoin into a lipid bilayer.

Molecular dynamics simulation of interlayer water embedded in phospholipid bilayer.

Modulation of raft domains in a lipid bilayer by boundary-active curcumin.

Safety, tolerability and pharmacokinetics of liposomal curcumin in healthy humans.

Effect of cholesterol on behavior of 5-fluorouracil (5-FU) in a DMPC lipid bilayer, a molecular dynamics study.

One short cysteine-rich sequence pattern - two different disulfide-bonded structures - a molecular dynamics simulation study.

Curcumin specifically binds to the human calcium-calmodulin-dependent protein kinase IV: fluorescence and molecular dynamics simulation studies.

Mechanical properties of lipid bilayers from molecular dynamics simulation.

Simulation of lipid bilayer self-assembly using all-atom lipid force fields.

Molecular dynamics simulation study of methanesulfonic acid.

Comparative study of curcumin and curcumin formulated in a solid dispersion: Evaluation of their antigenotoxic effects.

Nanotechnology-applied curcumin for different diseases therapy.

Effect of nanostructured lipid vehicles on percutaneous absorption of curcumin.

Detecting Allosteric Networks Using Molecular Dynamics Simulation.

Phase-transition properties of glycerol-dipalmitate lipid bilayers investigated using molecular dynamics simulation.

Curcumin.

Proteomics screening of molecular targets of curcumin in mouse brain.

Understanding thermal phases in atomic detail by all-atom molecular-dynamics simulation of a phospholipid bilayer.

Anti-Inflammatory Effects of Novel Standardized Solid Lipid Curcumin Formulations.

Enhanced photocytotoxicity of curcumin delivered by solid lipid nanoparticles.

Lipid Models for United-Atom Molecular Dynamics Simulations of Proteins.

Comparative Neuroprotective Effects of Dietary Curcumin and Solid Lipid Curcumin Particles in Cultured Mouse Neuroblastoma Cells after Exposure to Aβ42.

Molecular chaperone dysfunction in neurodegenerative diseases and effects of curcumin.