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Understanding loading, diffusion and releasing of Doxorubicin and Paclitaxel dual delivery in graphene and graphene oxide carriers as highly efficient drug delivery systems
Journal Pre-proofs Full Length Article Understanding Loading, Diffusion and Releasing of Doxorubicin and Paclitaxel Dual Delivery in Graphene and Graphene Oxide Carriers as Highly Efficient Drug Delivery Systems Hassan Hashemzadeh, Heidar Raissi PII: DOI: Reference:
S0169-4332(19)33036-3 https://doi.org/10.1016/j.apsusc.2019.144220 APSUSC 144220
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Applied Surface Science
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9 May 2019 27 August 2019 28 September 2019
Please cite this article as: H. Hashemzadeh, H. Raissi, Understanding Loading, Diffusion and Releasing of Doxorubicin and Paclitaxel Dual Delivery in Graphene and Graphene Oxide Carriers as Highly Efficient Drug Delivery Systems, Applied Surface Science (2019), doi: https://doi.org/10.1016/j.apsusc.2019.144220
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Understanding Loading, Diffusion and Releasing of Doxorubicin and Paclitaxel Dual Delivery in Graphene and Graphene Oxide Carriers as Highly Efficient Drug Delivery Systems Hassan Hashemzadeh1 and Heidar Raissi2* 1Department
of Chemistry, University of Birjand, Birjand, Iran
Tel: +985632502064, Email: [email protected] 2Department
of Chemistry, University of Birjand, Birjand, Iran
Tel: +985632502064, Email: [email protected]
*
Corresponding E-mail: [email protected]
Abstract: The adsorption mechanism of Doxorubicin (DOX) and Paclitaxel (PTX) mixture (1:1) on graphene (GRA) and graphene oxide (GOX) is determined using the molecular dynamics (MD) simulation and free energy calculation. The results indicate that the drug molecules spontaneously move toward the carriers. In the GRA system, the drug molecules form strong π-π interactions with the graphene surface, while the formation of intermolecular hydrogen bonds between the drug molecules and carrier expects in the GOX system due to different surface chemistry. The range of the drug-carrier intermolecular distances is around 2.5-4 A°. It is found that the binding energy of PTX (-487.67 Kj/mol) with the graphene is higher than DOX (1
373.53 Kj/mol). In the GOX system, the oxygen-containing functional groups lead to a decrease in the binding of PTX (-414.79 Kj/mol) and DOX (-121.12 Kj/mol) to the carrier. Moreover, the study of drug release in acidic pH shows that some drug molecules can be desorbed from the carrier due to strong electrostatic repulsion. Finally, the interaction of the drug delivery systems (DDSs) and membrane cell is investigated. It is found that the graphene-based DDS cannot spontaneously diffuse into the membrane cell, while the GOX-based DDS easily penetrate in the membrane cell. Keywords: Dual Delivery, pH effect, Lipid bilayer, Molecular dynamics simulation, Free energy calculation.
1. Introduction: In recent years, nanocarriers are employed as efficient drug delivery systems (DDSs) in nanomedicine and chemotherapy that can deliver drug molecules specifically into cancer cells and control the drug release rate. Nanomaterials often have excellent biocompatibility and a large surface area[1–3] that these properties make it possible to load more drug amount and design a smart DDS by using surface modification. Generally, nanomaterials are employed to improve the efficacy of conventional chemotherapy drugs [4–7]. In the last decade, graphene-based materials have been of interest in a wide range of applications such as solar cells[8–10] and drug delivery[11–13]. Graphene (GRA) is constructed from a monolayer of carbon atoms which is tightly packed into a twodimensional honeycomb lattice. This nanovector can form strong π-π interaction with aromatic drug molecules due to the large hydrophobic surface. GRA widely is 2
used to transport numerous biomolecules such as chemotherapy drugs [14–16]. Graphene oxide (GOX) is a chemically modified GRA that its surface is decorated with oxygen-containing functional groups such as carbonyl, hydroxyl, carboxyl and epoxy groups. The oxygen-containing functional groups of GOX caused that this carrier easily dispersed in the water environment. This carrier can form hydrogen bonds and electrostatic interactions in addition to π-π interaction. There are several studies focused on GRA and GOX for the biomedical applications [17,18]. Picaud et al.[19] performed density functional theory (DFT) calculation and molecular dynamics (MD) simulation to study the transfer of Zn Phthalocyanine (ZnPC) in the biological environment with GRA carrier. They indicated the adsorption of ZnPC on GRA does not affect the optical properties of the drug. Nouranian and coworkers[20] employed MD simulation to investigate the behavior of GRA and GOX in the loading, diffusion, and release of doxorubicin (DOX). Hasanzade and Raeisi[21] study the adsorption properties of paclitaxel (PTX) drug molecule on GRA, GOX, and chitosan functionalized GRA by using MD simulation. Their results revealed that the adsorption pattern of PTX on these carriers is different due to the difference in surface chemistry. PTX is a potent hydrophobic drug molecule with low solubility in water that is used in the treatment of a wide range of cancers. This drug is a strong inhibitor of cell proliferation, which has high severe side effects[22,23]. It is found that PTX in conjunction with the drug carriers and/or other chemotherapeutic drugs shows more therapeutic efficiency and lower toxicity[24]. DOX is another conventional chemotherapy drug which is classified in the anthracycline drugs family. DOX intercalates in the DNA double helix and thereby prevents DNA replication and cell division process[25]. It is shown that the penetration of the DOX molecule into the cell membrane is faced with some problems[26]. Recently, it is found that the 3
combination of two drugs (dual delivery) could be enhanced drugs efficiency compared to free drugs. Several studies have been reported, indicating the dual delivery strategy has the great potential to be more effective in compression with traditional DDS[1,27,28]. It is well known that normal pH level in the human body is ~7.4, while in near of a cancerous tumor is about 5-5.5. This difference in pH level is an interesting factor which drug molecule can respond to it. [11,15,20]. Maleki et al.[29] performed MD simulation to study the pH-sensitive co-adsorption of DOX and PTX on different carbon-based (i.e., fullerene, CNT, and GOX) carriers in conjugation with Nisopropyl acrylamide (PIN). The obtained results indicated that CNT has a stronger interaction with drugs than GOX and fullerene. Moreover, they found that in acidic pH, CNT has an excellent release property. In other work, Sgarlata et al.[30] studied the adsorption and hydration behavior of gemcitabine–poly (acrylic acid)–GOX complex at the range of pH values. They showed at the acidic condition the interaction of polymer and GOX be stronger, whereas the drug-polymer interaction is weaker. The obtained results of the computational studies can provide a fundamental knowledge for the development of DDS and improve their properties. There are a few theoretical studies that have assessed the co-loading systems; therefore, further studies are needed to develop and design better-performing systems. On the other hand, the interaction of co-loading systems with the cell membrane can also provide insight into the action mechanism of the graphene-based DDSs. In this study, MD simulation is performed to provide comprehensive information from the loading, diffusion, and release properties of the dual delivery of PTX and DOX drug molecules by GRA and GOX. The adsorption of these drugs on the 4
graphene and graphene oxide by using all-atom MD simulation and binding free energy has been investigated. Also, the release of drug from the carriers by protonation of the drug molecules has been examined. Finally, the diffusion of drugGRA and drug-GOX DDSs into a membrane cell is evaluated.
2. The Strategy of Calculations: 2.1 Molecular Models and Initial Structures: The initial structure of graphene 40×40 Å2 (containing 678 carbon atoms) is constructed by using the Nanotube Modeler Package. The dangling bonds at the edges are saturated with hydrogen atoms. To create a GOX model, the graphene surface based on Chen et al. work[31] is decorated with hydroxyl and epoxy functional groups. It should be noted that the hydrogen atoms at the edge of GOX are replaced with the carboxylic group. By adding these groups to graphene, the final carbon and oxygen (C:O) ratio in the GOX system is around 7:2. DOX and PTX molecules are given from the PubChem database[32]. 2.2 Force fields and simulation boxes: For all of the components, the force fields parameters are taken from the CHARMM force field[33]. The force field parameters for the drug molecules are generated by using the SwissParam web server[34]. In order to obtain the partial charge of the drug molecules, the density functional theory calculation at M06-2X[35] functional level along with 6-31G** basis set is performed and partial charges are extracted from the NBO analysis. For the study drug "loading" two systems (i.e., GRA and GOX) are designed. In each of the GRA and GOX system, eight drug molecules 5
(four DOX and four PTX) are located approximately 2 nm away from the graphene and graphene oxide surface (see Figure 1). In order to study the drug "release" process at the acidic pH, we build two other systems (i.e., pGRA and pGOX). To create these two systems, the final orientation of the drug molecules on the carrier surfaces are taken from the GRA and GOX systems, and then the relevant amine group of the drug molecules is protonated. Finally, the "diffusion" of the drug delivery systems based on GRA and GOX carriers into membrane cell is investigated. It is found that cholesterol (CHL) is an important molecule which presence in the majority of animal cell membranes and can be affected the chemicalphysical properties of a cell membrane [36,37]. The Presence of CHL in the membrane significantly changes its property. It is observed that by adding CHL to lipid bilayers the area per lipid is decreased, lipid tail order as well as bilayer thickness are increased [38]. Therefore, a cell membrane, including mixed the 1-
palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and CHL with identical leaflets, is selected to simulate a cell environment. In this study, each of leaflet contains 200 POPC and 50 CHL (membrane containing 20% cholesterol) is provided. All of simulation boxes are solvated with the water molecules which explicitly treated by using the TIP3P [39] model. Moreover, NaCl ions are replaced with the water to neutralize the system and reproduce a correct biological environment. It should be noted that the membrane is progressively equilibrated using MD simulations during 20 ns. Detail of the simulation boxes is provided in Table 1. 2.3 MD simulation: All of MD simulations in the present work are performed using the GROMACS 5.1.4 package[40]. The temperature and pressure are kept in 310 K and 1 bar using 6
V-rescale and Berendsen algorithm, respectively[41,42]. It should be noted that Nose-Hoover thermostat[43] and Parrinello-Rahman barostat[44] are used in the systems containing the membrane. Also, semi-isotropic scaling behavior, which treating the z-axis (normal to the bilayer) differently from the x and y-axis is applied for barostat. All of production MD runs under periodic boundary conditions is performed for 60 ns in each of the systems. LINCS algorithm is employed to constrain all bonds at their equilibrium length. Particle-mesh Ewald (PME) method is used to treat long-range electrostatic interactions, and the nonbonded interactions are calculated with a 1.2 nm range cutoff. For molecular visualization, visual molecular dynamics (VMD) package[45] is utilized. 2.4 Free energy calculation: In order to close inspection of the drug loading process, the binding free energy calculation is performed using Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method. The binding free energy for adsorbing the drug molecules on the carrier surfaces is carried out using the g-mmpbsa script introduced by Rashmi Kumari[46]. This code includes all the required subroutines from the GROMACS and the APBS packages to calculate the enthalpic components of the MM-PBSA interaction.
3. Results and discussions: The co-loading of DOX and PTX mixture (1:1) on GRA and GOX nanocarriers is investigated using MD simulation and free energy calculations. Then, the release process of the drug molecules from the carriers is examined. Finally, the diffusion of the DDSs into a membrane cell is evaluated. 7
Several snapshots from the
simulation boxes are provided, and different analyses are performed to quantify the adsorption process. 3.1 PTX and DOX adsorption on Graphene and graphene oxide: In this part, the adsorption behavior of PTX and DOX drug molecules on GRA and GOX nanosheets is studied. Different snapshots of the GRA and GOX systems as a function of time are depicted in Figure S1. As can be seen in the final snapshot, the drug molecules spontaneously move toward the carriers, indicating the drug molecules have a good tendency to form a stable complex with the graphene and graphene oxide surface. In the GRA system, all of PTX and DOX molecules adsorbed on the carrier surface, while in the GOX system, one DOX molecule and one PTX molecule overlap on the other drug molecules through π-π stacking and hydrogen bonds (HBs) and the other drug molecules are quite stable on the GOX throughout the simulation. The snapshots of the closet adsorbed drugs to GRA and GOX surfaces are represented in Figure 2. It is found that PTX molecule through its three benzene rings enables to form strong π-π stacking interactions with graphene surface. Furthermore, the X-H…π (X:C and O) intermolecular interactions between PTX molecule and the carrier surface are also observed. In our previous work, we found that two types of π-π stacking interactions (i.e., T-shaped and parallel π-π stacking) can be formed in adsorption of PTX on CNT [47]. As can be seen in Figure 2, in similar with CNT behavior, PTX able to form T-shaped and parallel π-π stacking interactions with graphene and range of intermolecular interaction is around of 2.5 to 4.0 A°. While the C-H…π and O-H…π intermolecular interactions between PTX and graphene occur at a distance of about 2.5-3.5 A°. The DOX is absorbed on the surface of graphene approximately in a similar manner to the PTX molecule. Close inspection of Figure 2 panel (a) shows that DOX molecule 8
interacts with graphene surface through formation of “parallel” π-π stacking and XH…π (X: X, N, and O) interactions. In these interactions, intermolecular distances are varied from 3.0 to 4.0 A° that for both of the drug molecules are comparable with those obtained for CNT carrier[27,47]. In the GOX system, the drug molecules have different adsorption behavior in comparison to the GRA system due to the presence of functional groups on graphene oxide surface. In this system, the oxygencontaining functional groups lead to the formation of several intermolecular HBs between GOX and the drug molecules while the number of π-π stacking interaction is reduced compared to the GRA system (see Figure 2). The intermolecular interactions between PTX/DOX and graphene oxide are in the range of 2.5-3.5 A°. As can be seen in the final snapshot of the GOX system, some of the drug molecules prefer to overlap with the other drug molecules due to reducing the hydrophobic surface of the carrier. This overlap occurs with the formation of the π-π stacking and the X-H…π interactions between the drug molecules. In order to quantify of adsorption behavior, the binding and decomposing energies (vdW and electrostatic) which obtained from free energy calculation are evaluated. All of the energy values for GRA and GOX systems are tabulated in Table 2. The binding energy for DOX and PTX molecules in investigated systems is negative, which indicates the adsorption process is spontaneous. In the GRA system, all of the energy values (i.e., vdW, elec, and binding energies) for PTX are higher than those for the DOX molecule. It reveals that the PTX adsorption on the graphene surface is preferable than DOX. This may be related to the formation of stronger and more hydrophobic interactions (i.e., π-π stacking and X-H…π) between PTX and GRA during the adsorption process. This obtained results in line with Karnati and Wang [27] study, indicating the adsorption of PTX on the CNT surface is stronger than the DOX molecule. 9
Furthermore, the decomposition of the binding energy showed that the vdW energy has more contribution than elec term in the adsorption process of drug molecules. This result can be attributed to the hydrophobic nature of the carrier and the drug molecules. As can be seen in Table 2, the vdW interaction between the drug molecules and GOX surface is decreased with adding oxygen-containing functional groups to the carrier surface, while the role of electrostatic interaction in adsorption process is increased. On the other hand, it is noted that the strength of binding energy between the drug molecules and the carrier is reduced. This behavior is already observed for PTX and DOX molecules in other works. Hasanzade and Raissi[21] studied the adsorption of PTX drug on the GRA and GOX using molecular dynamics simulation. Their obtained results indicated that the interaction of PTX with GRA is stronger than its interaction with GOX. Sanyal et al.[48] investigated the binding characterization of DOX with graphene and graphene oxide by using DFT calculation and fluorescence spectroscopy. According to DFT calculation and fluorescence measurements, they concluded that GRA has a better binding behavior in DOX adsorption in comparison to GOX. This observation can be attributed to the hydrophobic nature of PTX and DOX molecules, which by reduction of the hydrophobic area in the GOX system, the interaction of the drug molecules with the carrier is decreased. Furthermore, it is observed that there is no significant competition between the two drugs during the co-loading of PTX and DOX into graphene and graphene oxide surface. As can be seen in Table 2, PTX-DOX interaction energy in the GOX system is higher than the GRA system. This fact can be attributed to the overlap of PTX and DOX drugs with other drugs which caused π-π stacking, X-H…π, and HB interactions between drugs molecules form.
10
To examine the distribution of drug molecules around of GRA and GOX surfaces, the radial distribution function (RDF) between the drug molecules and the carrier calculated and results represented in Figure 3. Due to the strong repulsive forces between the carriers and the drug molecules, RDF is zero in short distances. Interestingly, as can be seen in Figure 3, the nonzero values of function g(r) for PTX started in the longer distances in comparison with DOX. It may be related to bigger molecular size and more steric effect of PTX molecule, which caused the repulsive force to be stronger. On the other hand, the steric effect in the GOX system is more prominent due to present the oxygen atoms on the carrier surface. Therefore, it can be expected the nonzero values of g(r) function for GOX system started from longer distances. As can be seen in Figure 3 panel (a), the drug molecules at a range about 0.5-2.0 nm are distributed around of graphene surface. The probability of finding the PTX drug around graphene surface is more than DOX due to stronger interaction energy (see Figure 3 and Table 2). The location of RDF peaks has a good correlation with the reported π–π interaction distances in the other conjugated systems and the quantum mechanics calculation (0.3 < nm)[2,49]. The GOX system has different drug adsorption behavior in comparison with the GRA system. As expected, due to the presence of oxygen on the surface of the carrier, the HBs and HB-likes are formed between the drug molecules and graphene oxide. As can be seen in Figure 3 panel (b), the sharp RDF peak between GOX and DOX is located at 0.7 nm which can be related to HB interaction, while this peak is not observed for PTX. The second peak of DOX and the first peak of PTX are located at 1.0 and 0.9 nm, respectively. These peaks can be attributed to HB-like interactions between the drugs and GOX. Furthermore, it is observed that the RDF peak intensity for PTX is higher than DOX due to the formation of more HB-like interaction. The π-π interaction between drug molecules and GOX surface is formed in longer distance compared with GRA. The observed small peaks around 1.5 to 2 nm for the drugs in the GOX 11
system may be related to the overlap of two drug molecules on the other drugs. Because of the fewer number of drug molecules at this distance, the intensity of these peaks is lower than the main peak. Different quantities are evaluated to gain better insight into the diffusion properties of drug molecules and kinetics of the adsorption process. Figure S2 shows the interaction energy (i.e., vdW + elec) of the drug molecules with the carriers as a function of time. In the investigated systems, the drug molecules spontaneously diffused toward the GRA and GOX surfaces and adsorbed approximately after 0.5 ns on the carrier surface. It can be evidenced by a noticeable decrease in the interaction energies after this time. In the GRA system, PTX and DOX molecules almost have the same diffusion behavior and their diffusion is faster than GOX system. The mean-square displacement (MSD) is calculated based on the following equation: 𝑀𝑆𝐷(∆𝑡) = < (𝑟𝑖(∆𝑡) ― 𝑟𝑖(0))2 > = < ∆𝑟𝑖(∆𝑡)2 >
(1)
Where 𝑟𝑖(𝑡) ― 𝑟𝑖(0) is the distance traveled by center of mass (COM) of the particle i over some time interval of length ∆𝑡. The self-diffusion coefficient Di can be obtained from the long-time limit of MSD with using Einstein relation: 𝐷𝑖 =
1 lim 𝑀𝑆𝐷(∆𝑡) 6∆t→∞
(2)
Figure S3 indicates the MSDs of the drug molecules in two investigated systems. The obtained results reveal that the order of Di (Table 3) for the drug molecules on the carrier surfaces is as follows: DOXGRA > DOXGOX > PTXGRA > PTXGOX, where the subscript stands for GRA-graphene system and GOX-graphene oxide system. 12
The diffusion coefficient of the drug molecule can be affected by three factors: i) the interaction energy between drug and carrier ii) the molecular weight of drug molecule and iii) the surface chemistry of the carrier In the investigated systems, the diffusion coefficient for PTX molecule is lower than the DOX molecule due to more molecular weight and stronger interaction energy. Furthermore, in the GOX system, the diffusion coefficients are significantly lower than the corresponding values in the GRA system. This observation can be attributed to the blocking effect of the functional groups on the GOX surface. According to the obtained results, it is found that the surface chemistry of the carrier has the most impact on the mobility of the drug molecule. The obtained results indicate that functionalization of the carrier surface with proper groups leads to a controllable drug release. 3.2 Protonated drug molecules: It is well known that in the neighborhood of a cancerous tumor, the local pH is about 5-5.5. Therefore, for studying the anticancer drug release from carrier near the cancer cells we consider the acidic pH level. For investigating the effect of acidic pH on releasing of the drug molecules two systems, including protonated drug molecules, are studied. As mentioned in “The Strategy of Calculations:” section, the final orientation of drugs on the carriers in natural systems are used for constructing protonated cases. The obtained results from the protonated systems (i.e., pGRA and pGOX systems) reveal that some of the protonated drug molecules tend to release from the carrier surfaces. The calculated interaction energies between the drug molecules and GRA and GOX in the protonated systems are given in Table S1. It is found that the vdW and elec energies in pGRA and pGOX systems are weaker than those in the natural cases. To gain a deeper insight into these systems, the distribution of the protonated drug molecules around the carriers is examined. The RDF plots of 13
DOX and PTX with the carriers in the protonated systems are displayed in Figure 4. It is observed that the probability of finding drug molecules around the GRA and GOX is decreased in these systems. Close inspection of Figure 4 shows that the RDF peaks intensity related to intermolecular HBs of DOX molecule with GOX surface has reduced significantly. Furthermore, the location of the π-π stacking peaks in protonates systems almost places at the further distance in comparison with the natural systems. These observations confirm that the protonated drug molecules tend to release from graphene and graphene oxide surfaces. Also, it is found that the protonation of drugs due to strong electrostatic repulsion mainly affects HB interactions, which also observed in similar systems[20,27]. The decomposition of interaction energy indicates that the electrostatic contribution has the main role in drug release from the carrier surfaces (see Table S1). 3.3 Drug delivery system close to membrane cells: To evaluate the interaction of the investigated DDSs with a membrane cell two different simulation systems are designed. The final orientations of the drug molecules on the carrier surfaces (i.e., graphene and graphene oxide) extracted from the last time of trajectory of natural systems and then the amine groups of drug molecules protonated. These DDSs are located close to the membrane cell (detail of the membrane cell provided in the “The Strategy of Calculations:” section) at a distance about 2 nm above the membrane. In order to evaluate the validity of the membrane model, the area per lipid for the lipid bilayers and density profile are analyzed. The calculated area per lipid for the bilayer is 52.24 Å2/lipid which has a good agreement with reported similar membranes (53.0 Å2/lipid) [50]. Furthermore, investigation of the density profiles of the membrane components and water during 20 ns (see Figure S4) confirms that 14
the bilayer properly equilibrated. When the Drugs-GRA systems placed near the membrane cell, it is randomly moved into the water-lipid interface. During simulation time, the DDS moves away from the membrane and diffuses into the bulk water phase. It is observed that one of the DOX molecules desorb from the carrier surface and penetrate to membrane cell. The obtained energies are provided in Table S2 and the GRA-POPC elec energy (as an example of the energy variation during simulation time) as a function of time is given in Figure S5 panel (a). The averaged GRA-POPC and GRA-CHL interaction energies during simulation time are almost negligible. The time variation of GRA-POPC elec energy indicates that energy values fluctuate around zero at the first 25 ns, and after that become flat and the energy value is zero. It can be concluded that the carrier for penetrating into membrane cell reorient in the initial times of simulation, but probably this process cannot spontaneously occur due to high barrier energy. On the other hands, in the previous works [19,51] were indicated that there is a competition between the lipid membrane and the water environment for interaction with DDS. Here, graphenebased DDS tends to interact with water rather than the bilayer. This fact can be related to formation HB between water molecules and the drug molecules which located on the carrier surface. In should be mentioned that the energy plot becomes zero after 25 ns which related to getting out on the cut-off radius. This phenomenon confirmed by calculating the distance of COM between the carrier and POPC_CHL, indicating the carrier is moved away from the water-membrane interface and is placed in the bulk water phase (Figure S5 panel (b). The obtained results emphasized that drug delivery systems based on graphene nanosheet cannot spontaneously diffuse into the membrane cell. Furthermore, the drug molecules adsorbed on the graphene surface (except one of the DOX molecules) due to strong interaction energy between the drug molecules. 15
The interaction energies (vdW and elec) between DOX molecules and POPC are given in Figure S5 panel (c). As can be seen in this Figure, the drug molecule penetrates to the membrane and interacts with head groups after 5 ns (decreasing in interaction energies). After that, the drug molecule moves among the head groups (electrostatic interaction fluctuates around -200 kJ/mol). Also, as the drug approaches to the membrane hydrophobic tails, the vdW energy is decreased as a function of time. Our obtained results are in line with the Picaud et. al.[26] outcomes and indicate the DDS based on graphene, unlike the isolated graphene[26,52] cannot spontaneously diffuse into the membrane cell. This behavior can be attributed to the presence of the drug molecules on the surface of graphene, which can form HBs with water molecules. In other words, when drug molecules are adsorbed on the graphene surface, the DDS tends to diffuse into the water bulk phase due to the formation of HBs between the drugs and water molecules. This conclusion can be confirmed by calculating the number of HB. The number of HB between the water molecules and the drug molecules is calculated based on cutoffs of 3.5 Å for the distance donoracceptor and results are presented in Figure S6. The number of HBs between water and DDS is decreased from 0 to 10 ns due to the presence of the DDS in near of the cell membrane and the desorption of the one drug molecule from the carrier surface. From 10-60 ns, by moving the DDS toward the water phase, the number of hydrogen bonds is raised and is reached to an equilibrium value. The behavior of graphene oxide in the presence of the membrane cell is completely different from the graphene. The initial and final snapshots from the simulation box are depicted in Figure 5. The interaction energies between various components are given in Table S2. The variation of elec energy between GOX and POPC as a function of simulation time is provided in Figure S7. The contribution of vdW and 16
elec energies in the interaction of GOX with POPC are -252.29 and -607.21 kJ/mol, respectively. The obtained energies and the final snapshot indicate that the DDS based on graphene oxide spontaneously and rapidly penetrates to the membrane cell and consequently the elec energy between POPC and graphene oxide decreases (Figure S7).It should be noted that the same behavior is observed for vdW energy between POPC and GOX (data are not shown). Furthermore, as can be seen in Table S2, the interaction between cholesterol of membrane and each of the DDS components (i.e., GOX, PTX, and DOX) is negligible. It is found that cholesterol does not considerably affect the membrane-DDS interactions during the DDS diffusion process. Close inspection of the final snapshot shows that one DOX molecule and one PTX molecule desorbed from the carrier surface during diffusion of DDS into the membrane cell. Due to weak interaction, these drugs easily desorbed from the carrier and located on top of the membrane. Picaud and co-workers[26] showed that the DOX molecule in isolated form could not diffuse into the membrane cell due to strong repulsion. Here, the desorbed PTX and DOX molecules from the carrier surface have remained within the water-lipid interface and cannot diffuse into the membrane cell. 4. Conclusion: In this study, co-loading of the DOX and PTX molecules on GRA and GOX surface is investigated using MD simulation and free energy calculations to the development and design of new drug delivery systems for biomedical purposes. The obtained results indicated that the drug molecules spontaneously adsorbed on GRA and GOX surface. In the GRA system, the drug molecules by their aromatic rings prefer to create strong π-π interaction with the carrier surface, which supplemented by X-π interactions. In GOX systems, in addition to these interactions, the carrier can form 17
HB interaction with the drug molecules. The adsorption of the drug molecules on GOX is weaker than the GRA surface due to the difference in its surface chemistry. Moreover, PTX and DOX molecules can be released from the carrier surfaces in response to low pH condition. It is observed that strong charge-charge repulsion between the drug molecules and the carrier surface has the main role in the drugreleasing process. Furthermore, the interaction between the drug delivery systems (DDSs) and membrane cell is investigated. It is found that the graphene-based DDS cannot spontaneously diffuse into the membrane cell, while the GOX-based DDS quickly penetrates in the membrane cell.
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25
Figure 1. (a)/(c) top view and (b)/(d) side view of the initial structure the GRA/GOX system. Color code: PTX molecule: blue and DOX molecule: red. The water and ions molecules are not shown for clarity.
26
Figure 2. Close snapshot of DOX/PTX interacting with GRA (A)/(B) and GOX (C)/(D) surface.
27
Figure 3. Radial distribution function (RDF) of the drug molecules with respect to (A) GRA and (B) GOX surface.
Figure 4. Radial distribution function (RDF) of the drug molecules with respect to the carrier surface in (A) pGRA and (B) pGOX systems. 28
Figure 5. The initial (A) and final (B) snapshot from GOX-MEM simulation box. Color code PTX: blue, DOX: red GOX: black POPC: cyan CHL: purple.
29
System natural protonate membrane
Carrier PTX DOX POPC CHL Box size (nm) GRA graphene 4 4 8.00×8.00×8.00 GOX graphene oxide 4 4 8.00×8.00×8.00 pGRA graphene 4 4 8.00×8.00×8.00 pGOX graphene oxide 4 4 8.00×8.00×8.00 GRA-MEM graphene 4 4 400 100 11.44×11.44×22.00 GOX-MEM graphene oxide 4 4 400 100 11.44×11.44×22.00 Table 1. Detail of the simulation boxes which used in this work.
Table 2. The van der Walls (vdW), electrostatic (elec) and binding energies between different components (all in kJ/mol). The values in parenthesis refer to the standard deviations of the data. System GRA
GOX
vdW
elec
Binding
GRA-DOX
-392.23 (+/- 0.26)
-14.62 (+/- 0.23)
-373.53 (+/- 0.32)
GRA-PTX
-458.36 (+/- 0.42)
-83.07 (+/- 0.21)
-487.67 (+/- 0.67)
DOX-PTX
-140.13 (+/- 7.70)
-65.1 (+/- 8.90)
-
GOX-DOX
-152.77 (+/- 2.25)
-50.39 (+/- 2.90)
-121.12 (+/- 8.95)
GOX-PTX
-380.11 (+/- 3.50)
-234.28 (+/- 3.69)
-414.79 (+/- 4.06)
DOX-PTX
-147.87 (+/- 6.42)
-81.68 (+/- 7.52)
-
Table 3. Average diffusion coefficient (Di) of DOX and PTX in GRA and GOX systems. System
Drug
D1(10-5 cm2/s)
GRA
DOX
6.07 ± 0.007
PTX
4.11 ± 0.003
DOX
5.39 ± 0.005
PTX
2.48 ± 0.001
GOX
30
GA
31
highlights
The drug molecules form strong π-π interactions with the graphene surface. The interaction of drug molecules with graphene is stronger than graphene oxide The bonding energy of Paclitaxel with graphene is higher than Doxorubicin. The graphene-based DDS cannot spontaneously diffuse into the membrane cell The graphene oxide-based DDS easily penetrate in the membrane cell.
32
S0169-4332(19)33036-3 https://doi.org/10.1016/j.apsusc.2019.144220 APSUSC 144220
To appear in:
Applied Surface Science
Received Date: Revised Date: Accepted Date:
9 May 2019 27 August 2019 28 September 2019
Please cite this article as: H. Hashemzadeh, H. Raissi, Understanding Loading, Diffusion and Releasing of Doxorubicin and Paclitaxel Dual Delivery in Graphene and Graphene Oxide Carriers as Highly Efficient Drug Delivery Systems, Applied Surface Science (2019), doi: https://doi.org/10.1016/j.apsusc.2019.144220
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© 2019 Published by Elsevier B.V.
Understanding Loading, Diffusion and Releasing of Doxorubicin and Paclitaxel Dual Delivery in Graphene and Graphene Oxide Carriers as Highly Efficient Drug Delivery Systems Hassan Hashemzadeh1 and Heidar Raissi2* 1Department
of Chemistry, University of Birjand, Birjand, Iran
Tel: +985632502064, Email: [email protected] 2Department
of Chemistry, University of Birjand, Birjand, Iran
Tel: +985632502064, Email: [email protected]
*
Corresponding E-mail: [email protected]
Abstract: The adsorption mechanism of Doxorubicin (DOX) and Paclitaxel (PTX) mixture (1:1) on graphene (GRA) and graphene oxide (GOX) is determined using the molecular dynamics (MD) simulation and free energy calculation. The results indicate that the drug molecules spontaneously move toward the carriers. In the GRA system, the drug molecules form strong π-π interactions with the graphene surface, while the formation of intermolecular hydrogen bonds between the drug molecules and carrier expects in the GOX system due to different surface chemistry. The range of the drug-carrier intermolecular distances is around 2.5-4 A°. It is found that the binding energy of PTX (-487.67 Kj/mol) with the graphene is higher than DOX (1
373.53 Kj/mol). In the GOX system, the oxygen-containing functional groups lead to a decrease in the binding of PTX (-414.79 Kj/mol) and DOX (-121.12 Kj/mol) to the carrier. Moreover, the study of drug release in acidic pH shows that some drug molecules can be desorbed from the carrier due to strong electrostatic repulsion. Finally, the interaction of the drug delivery systems (DDSs) and membrane cell is investigated. It is found that the graphene-based DDS cannot spontaneously diffuse into the membrane cell, while the GOX-based DDS easily penetrate in the membrane cell. Keywords: Dual Delivery, pH effect, Lipid bilayer, Molecular dynamics simulation, Free energy calculation.
1. Introduction: In recent years, nanocarriers are employed as efficient drug delivery systems (DDSs) in nanomedicine and chemotherapy that can deliver drug molecules specifically into cancer cells and control the drug release rate. Nanomaterials often have excellent biocompatibility and a large surface area[1–3] that these properties make it possible to load more drug amount and design a smart DDS by using surface modification. Generally, nanomaterials are employed to improve the efficacy of conventional chemotherapy drugs [4–7]. In the last decade, graphene-based materials have been of interest in a wide range of applications such as solar cells[8–10] and drug delivery[11–13]. Graphene (GRA) is constructed from a monolayer of carbon atoms which is tightly packed into a twodimensional honeycomb lattice. This nanovector can form strong π-π interaction with aromatic drug molecules due to the large hydrophobic surface. GRA widely is 2
used to transport numerous biomolecules such as chemotherapy drugs [14–16]. Graphene oxide (GOX) is a chemically modified GRA that its surface is decorated with oxygen-containing functional groups such as carbonyl, hydroxyl, carboxyl and epoxy groups. The oxygen-containing functional groups of GOX caused that this carrier easily dispersed in the water environment. This carrier can form hydrogen bonds and electrostatic interactions in addition to π-π interaction. There are several studies focused on GRA and GOX for the biomedical applications [17,18]. Picaud et al.[19] performed density functional theory (DFT) calculation and molecular dynamics (MD) simulation to study the transfer of Zn Phthalocyanine (ZnPC) in the biological environment with GRA carrier. They indicated the adsorption of ZnPC on GRA does not affect the optical properties of the drug. Nouranian and coworkers[20] employed MD simulation to investigate the behavior of GRA and GOX in the loading, diffusion, and release of doxorubicin (DOX). Hasanzade and Raeisi[21] study the adsorption properties of paclitaxel (PTX) drug molecule on GRA, GOX, and chitosan functionalized GRA by using MD simulation. Their results revealed that the adsorption pattern of PTX on these carriers is different due to the difference in surface chemistry. PTX is a potent hydrophobic drug molecule with low solubility in water that is used in the treatment of a wide range of cancers. This drug is a strong inhibitor of cell proliferation, which has high severe side effects[22,23]. It is found that PTX in conjunction with the drug carriers and/or other chemotherapeutic drugs shows more therapeutic efficiency and lower toxicity[24]. DOX is another conventional chemotherapy drug which is classified in the anthracycline drugs family. DOX intercalates in the DNA double helix and thereby prevents DNA replication and cell division process[25]. It is shown that the penetration of the DOX molecule into the cell membrane is faced with some problems[26]. Recently, it is found that the 3
combination of two drugs (dual delivery) could be enhanced drugs efficiency compared to free drugs. Several studies have been reported, indicating the dual delivery strategy has the great potential to be more effective in compression with traditional DDS[1,27,28]. It is well known that normal pH level in the human body is ~7.4, while in near of a cancerous tumor is about 5-5.5. This difference in pH level is an interesting factor which drug molecule can respond to it. [11,15,20]. Maleki et al.[29] performed MD simulation to study the pH-sensitive co-adsorption of DOX and PTX on different carbon-based (i.e., fullerene, CNT, and GOX) carriers in conjugation with Nisopropyl acrylamide (PIN). The obtained results indicated that CNT has a stronger interaction with drugs than GOX and fullerene. Moreover, they found that in acidic pH, CNT has an excellent release property. In other work, Sgarlata et al.[30] studied the adsorption and hydration behavior of gemcitabine–poly (acrylic acid)–GOX complex at the range of pH values. They showed at the acidic condition the interaction of polymer and GOX be stronger, whereas the drug-polymer interaction is weaker. The obtained results of the computational studies can provide a fundamental knowledge for the development of DDS and improve their properties. There are a few theoretical studies that have assessed the co-loading systems; therefore, further studies are needed to develop and design better-performing systems. On the other hand, the interaction of co-loading systems with the cell membrane can also provide insight into the action mechanism of the graphene-based DDSs. In this study, MD simulation is performed to provide comprehensive information from the loading, diffusion, and release properties of the dual delivery of PTX and DOX drug molecules by GRA and GOX. The adsorption of these drugs on the 4
graphene and graphene oxide by using all-atom MD simulation and binding free energy has been investigated. Also, the release of drug from the carriers by protonation of the drug molecules has been examined. Finally, the diffusion of drugGRA and drug-GOX DDSs into a membrane cell is evaluated.
2. The Strategy of Calculations: 2.1 Molecular Models and Initial Structures: The initial structure of graphene 40×40 Å2 (containing 678 carbon atoms) is constructed by using the Nanotube Modeler Package. The dangling bonds at the edges are saturated with hydrogen atoms. To create a GOX model, the graphene surface based on Chen et al. work[31] is decorated with hydroxyl and epoxy functional groups. It should be noted that the hydrogen atoms at the edge of GOX are replaced with the carboxylic group. By adding these groups to graphene, the final carbon and oxygen (C:O) ratio in the GOX system is around 7:2. DOX and PTX molecules are given from the PubChem database[32]. 2.2 Force fields and simulation boxes: For all of the components, the force fields parameters are taken from the CHARMM force field[33]. The force field parameters for the drug molecules are generated by using the SwissParam web server[34]. In order to obtain the partial charge of the drug molecules, the density functional theory calculation at M06-2X[35] functional level along with 6-31G** basis set is performed and partial charges are extracted from the NBO analysis. For the study drug "loading" two systems (i.e., GRA and GOX) are designed. In each of the GRA and GOX system, eight drug molecules 5
(four DOX and four PTX) are located approximately 2 nm away from the graphene and graphene oxide surface (see Figure 1). In order to study the drug "release" process at the acidic pH, we build two other systems (i.e., pGRA and pGOX). To create these two systems, the final orientation of the drug molecules on the carrier surfaces are taken from the GRA and GOX systems, and then the relevant amine group of the drug molecules is protonated. Finally, the "diffusion" of the drug delivery systems based on GRA and GOX carriers into membrane cell is investigated. It is found that cholesterol (CHL) is an important molecule which presence in the majority of animal cell membranes and can be affected the chemicalphysical properties of a cell membrane [36,37]. The Presence of CHL in the membrane significantly changes its property. It is observed that by adding CHL to lipid bilayers the area per lipid is decreased, lipid tail order as well as bilayer thickness are increased [38]. Therefore, a cell membrane, including mixed the 1-
palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and CHL with identical leaflets, is selected to simulate a cell environment. In this study, each of leaflet contains 200 POPC and 50 CHL (membrane containing 20% cholesterol) is provided. All of simulation boxes are solvated with the water molecules which explicitly treated by using the TIP3P [39] model. Moreover, NaCl ions are replaced with the water to neutralize the system and reproduce a correct biological environment. It should be noted that the membrane is progressively equilibrated using MD simulations during 20 ns. Detail of the simulation boxes is provided in Table 1. 2.3 MD simulation: All of MD simulations in the present work are performed using the GROMACS 5.1.4 package[40]. The temperature and pressure are kept in 310 K and 1 bar using 6
V-rescale and Berendsen algorithm, respectively[41,42]. It should be noted that Nose-Hoover thermostat[43] and Parrinello-Rahman barostat[44] are used in the systems containing the membrane. Also, semi-isotropic scaling behavior, which treating the z-axis (normal to the bilayer) differently from the x and y-axis is applied for barostat. All of production MD runs under periodic boundary conditions is performed for 60 ns in each of the systems. LINCS algorithm is employed to constrain all bonds at their equilibrium length. Particle-mesh Ewald (PME) method is used to treat long-range electrostatic interactions, and the nonbonded interactions are calculated with a 1.2 nm range cutoff. For molecular visualization, visual molecular dynamics (VMD) package[45] is utilized. 2.4 Free energy calculation: In order to close inspection of the drug loading process, the binding free energy calculation is performed using Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method. The binding free energy for adsorbing the drug molecules on the carrier surfaces is carried out using the g-mmpbsa script introduced by Rashmi Kumari[46]. This code includes all the required subroutines from the GROMACS and the APBS packages to calculate the enthalpic components of the MM-PBSA interaction.
3. Results and discussions: The co-loading of DOX and PTX mixture (1:1) on GRA and GOX nanocarriers is investigated using MD simulation and free energy calculations. Then, the release process of the drug molecules from the carriers is examined. Finally, the diffusion of the DDSs into a membrane cell is evaluated. 7
Several snapshots from the
simulation boxes are provided, and different analyses are performed to quantify the adsorption process. 3.1 PTX and DOX adsorption on Graphene and graphene oxide: In this part, the adsorption behavior of PTX and DOX drug molecules on GRA and GOX nanosheets is studied. Different snapshots of the GRA and GOX systems as a function of time are depicted in Figure S1. As can be seen in the final snapshot, the drug molecules spontaneously move toward the carriers, indicating the drug molecules have a good tendency to form a stable complex with the graphene and graphene oxide surface. In the GRA system, all of PTX and DOX molecules adsorbed on the carrier surface, while in the GOX system, one DOX molecule and one PTX molecule overlap on the other drug molecules through π-π stacking and hydrogen bonds (HBs) and the other drug molecules are quite stable on the GOX throughout the simulation. The snapshots of the closet adsorbed drugs to GRA and GOX surfaces are represented in Figure 2. It is found that PTX molecule through its three benzene rings enables to form strong π-π stacking interactions with graphene surface. Furthermore, the X-H…π (X:C and O) intermolecular interactions between PTX molecule and the carrier surface are also observed. In our previous work, we found that two types of π-π stacking interactions (i.e., T-shaped and parallel π-π stacking) can be formed in adsorption of PTX on CNT [47]. As can be seen in Figure 2, in similar with CNT behavior, PTX able to form T-shaped and parallel π-π stacking interactions with graphene and range of intermolecular interaction is around of 2.5 to 4.0 A°. While the C-H…π and O-H…π intermolecular interactions between PTX and graphene occur at a distance of about 2.5-3.5 A°. The DOX is absorbed on the surface of graphene approximately in a similar manner to the PTX molecule. Close inspection of Figure 2 panel (a) shows that DOX molecule 8
interacts with graphene surface through formation of “parallel” π-π stacking and XH…π (X: X, N, and O) interactions. In these interactions, intermolecular distances are varied from 3.0 to 4.0 A° that for both of the drug molecules are comparable with those obtained for CNT carrier[27,47]. In the GOX system, the drug molecules have different adsorption behavior in comparison to the GRA system due to the presence of functional groups on graphene oxide surface. In this system, the oxygencontaining functional groups lead to the formation of several intermolecular HBs between GOX and the drug molecules while the number of π-π stacking interaction is reduced compared to the GRA system (see Figure 2). The intermolecular interactions between PTX/DOX and graphene oxide are in the range of 2.5-3.5 A°. As can be seen in the final snapshot of the GOX system, some of the drug molecules prefer to overlap with the other drug molecules due to reducing the hydrophobic surface of the carrier. This overlap occurs with the formation of the π-π stacking and the X-H…π interactions between the drug molecules. In order to quantify of adsorption behavior, the binding and decomposing energies (vdW and electrostatic) which obtained from free energy calculation are evaluated. All of the energy values for GRA and GOX systems are tabulated in Table 2. The binding energy for DOX and PTX molecules in investigated systems is negative, which indicates the adsorption process is spontaneous. In the GRA system, all of the energy values (i.e., vdW, elec, and binding energies) for PTX are higher than those for the DOX molecule. It reveals that the PTX adsorption on the graphene surface is preferable than DOX. This may be related to the formation of stronger and more hydrophobic interactions (i.e., π-π stacking and X-H…π) between PTX and GRA during the adsorption process. This obtained results in line with Karnati and Wang [27] study, indicating the adsorption of PTX on the CNT surface is stronger than the DOX molecule. 9
Furthermore, the decomposition of the binding energy showed that the vdW energy has more contribution than elec term in the adsorption process of drug molecules. This result can be attributed to the hydrophobic nature of the carrier and the drug molecules. As can be seen in Table 2, the vdW interaction between the drug molecules and GOX surface is decreased with adding oxygen-containing functional groups to the carrier surface, while the role of electrostatic interaction in adsorption process is increased. On the other hand, it is noted that the strength of binding energy between the drug molecules and the carrier is reduced. This behavior is already observed for PTX and DOX molecules in other works. Hasanzade and Raissi[21] studied the adsorption of PTX drug on the GRA and GOX using molecular dynamics simulation. Their obtained results indicated that the interaction of PTX with GRA is stronger than its interaction with GOX. Sanyal et al.[48] investigated the binding characterization of DOX with graphene and graphene oxide by using DFT calculation and fluorescence spectroscopy. According to DFT calculation and fluorescence measurements, they concluded that GRA has a better binding behavior in DOX adsorption in comparison to GOX. This observation can be attributed to the hydrophobic nature of PTX and DOX molecules, which by reduction of the hydrophobic area in the GOX system, the interaction of the drug molecules with the carrier is decreased. Furthermore, it is observed that there is no significant competition between the two drugs during the co-loading of PTX and DOX into graphene and graphene oxide surface. As can be seen in Table 2, PTX-DOX interaction energy in the GOX system is higher than the GRA system. This fact can be attributed to the overlap of PTX and DOX drugs with other drugs which caused π-π stacking, X-H…π, and HB interactions between drugs molecules form.
10
To examine the distribution of drug molecules around of GRA and GOX surfaces, the radial distribution function (RDF) between the drug molecules and the carrier calculated and results represented in Figure 3. Due to the strong repulsive forces between the carriers and the drug molecules, RDF is zero in short distances. Interestingly, as can be seen in Figure 3, the nonzero values of function g(r) for PTX started in the longer distances in comparison with DOX. It may be related to bigger molecular size and more steric effect of PTX molecule, which caused the repulsive force to be stronger. On the other hand, the steric effect in the GOX system is more prominent due to present the oxygen atoms on the carrier surface. Therefore, it can be expected the nonzero values of g(r) function for GOX system started from longer distances. As can be seen in Figure 3 panel (a), the drug molecules at a range about 0.5-2.0 nm are distributed around of graphene surface. The probability of finding the PTX drug around graphene surface is more than DOX due to stronger interaction energy (see Figure 3 and Table 2). The location of RDF peaks has a good correlation with the reported π–π interaction distances in the other conjugated systems and the quantum mechanics calculation (0.3 < nm)[2,49]. The GOX system has different drug adsorption behavior in comparison with the GRA system. As expected, due to the presence of oxygen on the surface of the carrier, the HBs and HB-likes are formed between the drug molecules and graphene oxide. As can be seen in Figure 3 panel (b), the sharp RDF peak between GOX and DOX is located at 0.7 nm which can be related to HB interaction, while this peak is not observed for PTX. The second peak of DOX and the first peak of PTX are located at 1.0 and 0.9 nm, respectively. These peaks can be attributed to HB-like interactions between the drugs and GOX. Furthermore, it is observed that the RDF peak intensity for PTX is higher than DOX due to the formation of more HB-like interaction. The π-π interaction between drug molecules and GOX surface is formed in longer distance compared with GRA. The observed small peaks around 1.5 to 2 nm for the drugs in the GOX 11
system may be related to the overlap of two drug molecules on the other drugs. Because of the fewer number of drug molecules at this distance, the intensity of these peaks is lower than the main peak. Different quantities are evaluated to gain better insight into the diffusion properties of drug molecules and kinetics of the adsorption process. Figure S2 shows the interaction energy (i.e., vdW + elec) of the drug molecules with the carriers as a function of time. In the investigated systems, the drug molecules spontaneously diffused toward the GRA and GOX surfaces and adsorbed approximately after 0.5 ns on the carrier surface. It can be evidenced by a noticeable decrease in the interaction energies after this time. In the GRA system, PTX and DOX molecules almost have the same diffusion behavior and their diffusion is faster than GOX system. The mean-square displacement (MSD) is calculated based on the following equation: 𝑀𝑆𝐷(∆𝑡) = < (𝑟𝑖(∆𝑡) ― 𝑟𝑖(0))2 > = < ∆𝑟𝑖(∆𝑡)2 >
(1)
Where 𝑟𝑖(𝑡) ― 𝑟𝑖(0) is the distance traveled by center of mass (COM) of the particle i over some time interval of length ∆𝑡. The self-diffusion coefficient Di can be obtained from the long-time limit of MSD with using Einstein relation: 𝐷𝑖 =
1 lim 𝑀𝑆𝐷(∆𝑡) 6∆t→∞
(2)
Figure S3 indicates the MSDs of the drug molecules in two investigated systems. The obtained results reveal that the order of Di (Table 3) for the drug molecules on the carrier surfaces is as follows: DOXGRA > DOXGOX > PTXGRA > PTXGOX, where the subscript stands for GRA-graphene system and GOX-graphene oxide system. 12
The diffusion coefficient of the drug molecule can be affected by three factors: i) the interaction energy between drug and carrier ii) the molecular weight of drug molecule and iii) the surface chemistry of the carrier In the investigated systems, the diffusion coefficient for PTX molecule is lower than the DOX molecule due to more molecular weight and stronger interaction energy. Furthermore, in the GOX system, the diffusion coefficients are significantly lower than the corresponding values in the GRA system. This observation can be attributed to the blocking effect of the functional groups on the GOX surface. According to the obtained results, it is found that the surface chemistry of the carrier has the most impact on the mobility of the drug molecule. The obtained results indicate that functionalization of the carrier surface with proper groups leads to a controllable drug release. 3.2 Protonated drug molecules: It is well known that in the neighborhood of a cancerous tumor, the local pH is about 5-5.5. Therefore, for studying the anticancer drug release from carrier near the cancer cells we consider the acidic pH level. For investigating the effect of acidic pH on releasing of the drug molecules two systems, including protonated drug molecules, are studied. As mentioned in “The Strategy of Calculations:” section, the final orientation of drugs on the carriers in natural systems are used for constructing protonated cases. The obtained results from the protonated systems (i.e., pGRA and pGOX systems) reveal that some of the protonated drug molecules tend to release from the carrier surfaces. The calculated interaction energies between the drug molecules and GRA and GOX in the protonated systems are given in Table S1. It is found that the vdW and elec energies in pGRA and pGOX systems are weaker than those in the natural cases. To gain a deeper insight into these systems, the distribution of the protonated drug molecules around the carriers is examined. The RDF plots of 13
DOX and PTX with the carriers in the protonated systems are displayed in Figure 4. It is observed that the probability of finding drug molecules around the GRA and GOX is decreased in these systems. Close inspection of Figure 4 shows that the RDF peaks intensity related to intermolecular HBs of DOX molecule with GOX surface has reduced significantly. Furthermore, the location of the π-π stacking peaks in protonates systems almost places at the further distance in comparison with the natural systems. These observations confirm that the protonated drug molecules tend to release from graphene and graphene oxide surfaces. Also, it is found that the protonation of drugs due to strong electrostatic repulsion mainly affects HB interactions, which also observed in similar systems[20,27]. The decomposition of interaction energy indicates that the electrostatic contribution has the main role in drug release from the carrier surfaces (see Table S1). 3.3 Drug delivery system close to membrane cells: To evaluate the interaction of the investigated DDSs with a membrane cell two different simulation systems are designed. The final orientations of the drug molecules on the carrier surfaces (i.e., graphene and graphene oxide) extracted from the last time of trajectory of natural systems and then the amine groups of drug molecules protonated. These DDSs are located close to the membrane cell (detail of the membrane cell provided in the “The Strategy of Calculations:” section) at a distance about 2 nm above the membrane. In order to evaluate the validity of the membrane model, the area per lipid for the lipid bilayers and density profile are analyzed. The calculated area per lipid for the bilayer is 52.24 Å2/lipid which has a good agreement with reported similar membranes (53.0 Å2/lipid) [50]. Furthermore, investigation of the density profiles of the membrane components and water during 20 ns (see Figure S4) confirms that 14
the bilayer properly equilibrated. When the Drugs-GRA systems placed near the membrane cell, it is randomly moved into the water-lipid interface. During simulation time, the DDS moves away from the membrane and diffuses into the bulk water phase. It is observed that one of the DOX molecules desorb from the carrier surface and penetrate to membrane cell. The obtained energies are provided in Table S2 and the GRA-POPC elec energy (as an example of the energy variation during simulation time) as a function of time is given in Figure S5 panel (a). The averaged GRA-POPC and GRA-CHL interaction energies during simulation time are almost negligible. The time variation of GRA-POPC elec energy indicates that energy values fluctuate around zero at the first 25 ns, and after that become flat and the energy value is zero. It can be concluded that the carrier for penetrating into membrane cell reorient in the initial times of simulation, but probably this process cannot spontaneously occur due to high barrier energy. On the other hands, in the previous works [19,51] were indicated that there is a competition between the lipid membrane and the water environment for interaction with DDS. Here, graphenebased DDS tends to interact with water rather than the bilayer. This fact can be related to formation HB between water molecules and the drug molecules which located on the carrier surface. In should be mentioned that the energy plot becomes zero after 25 ns which related to getting out on the cut-off radius. This phenomenon confirmed by calculating the distance of COM between the carrier and POPC_CHL, indicating the carrier is moved away from the water-membrane interface and is placed in the bulk water phase (Figure S5 panel (b). The obtained results emphasized that drug delivery systems based on graphene nanosheet cannot spontaneously diffuse into the membrane cell. Furthermore, the drug molecules adsorbed on the graphene surface (except one of the DOX molecules) due to strong interaction energy between the drug molecules. 15
The interaction energies (vdW and elec) between DOX molecules and POPC are given in Figure S5 panel (c). As can be seen in this Figure, the drug molecule penetrates to the membrane and interacts with head groups after 5 ns (decreasing in interaction energies). After that, the drug molecule moves among the head groups (electrostatic interaction fluctuates around -200 kJ/mol). Also, as the drug approaches to the membrane hydrophobic tails, the vdW energy is decreased as a function of time. Our obtained results are in line with the Picaud et. al.[26] outcomes and indicate the DDS based on graphene, unlike the isolated graphene[26,52] cannot spontaneously diffuse into the membrane cell. This behavior can be attributed to the presence of the drug molecules on the surface of graphene, which can form HBs with water molecules. In other words, when drug molecules are adsorbed on the graphene surface, the DDS tends to diffuse into the water bulk phase due to the formation of HBs between the drugs and water molecules. This conclusion can be confirmed by calculating the number of HB. The number of HB between the water molecules and the drug molecules is calculated based on cutoffs of 3.5 Å for the distance donoracceptor and results are presented in Figure S6. The number of HBs between water and DDS is decreased from 0 to 10 ns due to the presence of the DDS in near of the cell membrane and the desorption of the one drug molecule from the carrier surface. From 10-60 ns, by moving the DDS toward the water phase, the number of hydrogen bonds is raised and is reached to an equilibrium value. The behavior of graphene oxide in the presence of the membrane cell is completely different from the graphene. The initial and final snapshots from the simulation box are depicted in Figure 5. The interaction energies between various components are given in Table S2. The variation of elec energy between GOX and POPC as a function of simulation time is provided in Figure S7. The contribution of vdW and 16
elec energies in the interaction of GOX with POPC are -252.29 and -607.21 kJ/mol, respectively. The obtained energies and the final snapshot indicate that the DDS based on graphene oxide spontaneously and rapidly penetrates to the membrane cell and consequently the elec energy between POPC and graphene oxide decreases (Figure S7).It should be noted that the same behavior is observed for vdW energy between POPC and GOX (data are not shown). Furthermore, as can be seen in Table S2, the interaction between cholesterol of membrane and each of the DDS components (i.e., GOX, PTX, and DOX) is negligible. It is found that cholesterol does not considerably affect the membrane-DDS interactions during the DDS diffusion process. Close inspection of the final snapshot shows that one DOX molecule and one PTX molecule desorbed from the carrier surface during diffusion of DDS into the membrane cell. Due to weak interaction, these drugs easily desorbed from the carrier and located on top of the membrane. Picaud and co-workers[26] showed that the DOX molecule in isolated form could not diffuse into the membrane cell due to strong repulsion. Here, the desorbed PTX and DOX molecules from the carrier surface have remained within the water-lipid interface and cannot diffuse into the membrane cell. 4. Conclusion: In this study, co-loading of the DOX and PTX molecules on GRA and GOX surface is investigated using MD simulation and free energy calculations to the development and design of new drug delivery systems for biomedical purposes. The obtained results indicated that the drug molecules spontaneously adsorbed on GRA and GOX surface. In the GRA system, the drug molecules by their aromatic rings prefer to create strong π-π interaction with the carrier surface, which supplemented by X-π interactions. In GOX systems, in addition to these interactions, the carrier can form 17
HB interaction with the drug molecules. The adsorption of the drug molecules on GOX is weaker than the GRA surface due to the difference in its surface chemistry. Moreover, PTX and DOX molecules can be released from the carrier surfaces in response to low pH condition. It is observed that strong charge-charge repulsion between the drug molecules and the carrier surface has the main role in the drugreleasing process. Furthermore, the interaction between the drug delivery systems (DDSs) and membrane cell is investigated. It is found that the graphene-based DDS cannot spontaneously diffuse into the membrane cell, while the GOX-based DDS quickly penetrates in the membrane cell.
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25
Figure 1. (a)/(c) top view and (b)/(d) side view of the initial structure the GRA/GOX system. Color code: PTX molecule: blue and DOX molecule: red. The water and ions molecules are not shown for clarity.
26
Figure 2. Close snapshot of DOX/PTX interacting with GRA (A)/(B) and GOX (C)/(D) surface.
27
Figure 3. Radial distribution function (RDF) of the drug molecules with respect to (A) GRA and (B) GOX surface.
Figure 4. Radial distribution function (RDF) of the drug molecules with respect to the carrier surface in (A) pGRA and (B) pGOX systems. 28
Figure 5. The initial (A) and final (B) snapshot from GOX-MEM simulation box. Color code PTX: blue, DOX: red GOX: black POPC: cyan CHL: purple.
29
System natural protonate membrane
Carrier PTX DOX POPC CHL Box size (nm) GRA graphene 4 4 8.00×8.00×8.00 GOX graphene oxide 4 4 8.00×8.00×8.00 pGRA graphene 4 4 8.00×8.00×8.00 pGOX graphene oxide 4 4 8.00×8.00×8.00 GRA-MEM graphene 4 4 400 100 11.44×11.44×22.00 GOX-MEM graphene oxide 4 4 400 100 11.44×11.44×22.00 Table 1. Detail of the simulation boxes which used in this work.
Table 2. The van der Walls (vdW), electrostatic (elec) and binding energies between different components (all in kJ/mol). The values in parenthesis refer to the standard deviations of the data. System GRA
GOX
vdW
elec
Binding
GRA-DOX
-392.23 (+/- 0.26)
-14.62 (+/- 0.23)
-373.53 (+/- 0.32)
GRA-PTX
-458.36 (+/- 0.42)
-83.07 (+/- 0.21)
-487.67 (+/- 0.67)
DOX-PTX
-140.13 (+/- 7.70)
-65.1 (+/- 8.90)
-
GOX-DOX
-152.77 (+/- 2.25)
-50.39 (+/- 2.90)
-121.12 (+/- 8.95)
GOX-PTX
-380.11 (+/- 3.50)
-234.28 (+/- 3.69)
-414.79 (+/- 4.06)
DOX-PTX
-147.87 (+/- 6.42)
-81.68 (+/- 7.52)
-
Table 3. Average diffusion coefficient (Di) of DOX and PTX in GRA and GOX systems. System
Drug
D1(10-5 cm2/s)
GRA
DOX
6.07 ± 0.007
PTX
4.11 ± 0.003
DOX
5.39 ± 0.005
PTX
2.48 ± 0.001
GOX
30
GA
31
highlights
The drug molecules form strong π-π interactions with the graphene surface. The interaction of drug molecules with graphene is stronger than graphene oxide The bonding energy of Paclitaxel with graphene is higher than Doxorubicin. The graphene-based DDS cannot spontaneously diffuse into the membrane cell The graphene oxide-based DDS easily penetrate in the membrane cell.
32