Encapsulation and Release of Drug Molecule Pregabalin Based on

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C: Physical Processes in Nanomaterials and Nanostructures

Encapsulation and Release of Drug Molecule Pregabalin Based on Ultrashort Single-Walled Carbon Nanotubes Junlang Chen, Dangxin Mao, Xiaogang Wang, Guoquan Zhou, Songwei Zeng, Liang Chen, Chaoqing Dai, and Shangshen Feng J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.9b00675 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 15, 2019

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The Journal of Physical Chemistry

Encapsulation and Release of Drug Molecule Pregabalin Based on Ultrashort Single-Walled Carbon Nanotubes

Junlang Chen#, †, Dangxin Mao#, †, Xiaogang Wang†, Guoquan Zhou†, Songwei Zeng ‡, Liang Chen†, Chaoqinq Dai*, † Shangshen Feng*, †

†Department ‡School

of Optical Engineering, Zhejiang A&F University, Lin'an 311300, China

of Information and Industry, Zhejiang A&F University, Lin'an 311300, China

Email: [email protected], [email protected]

Abstract Carbon nanotubes (CNTs) have been regarded as one of the most hopeful candidates for transporting drugs to target cells because of their huge surface area, hollow structure and enhanced cellular uptake. The idea of using their hollow channels as containers to load and unload small drug molecules has been proposed for many years. However, the encapsulation of drug into CNT, the internalization of CNT-drug conjugates in the cell membrane and the successive drug release at atomic level remain unclear. In this work, we performed molecular dynamics simulations to investigate the potential application of CNT as a nanocarrier to transport and deliver drug molecules. Pregabalin (PRE) was selected as a model drug, as its size and polarity are suitable for transporting through CNT hollow channels. The simulation can be divided into three stages. First, PRE was encapsulated into the optimized CNT in the water solution and the PRE-CNT complex was formed, then this complex readily entered the lipid bilayer and finally PRE released one by one from CNT into the membrane. Compared with the direct insertion of PRE in the membrane, the PRE-CNT complex can reduce the energy barrier to enter the membrane and pass the bilayer center. The fast release of PRE from CNT benefits from its amphipathicity. 1 ACS Paragon Plus Environment

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The electrostatic interaction between its polar groups and lipid headgroups pull the PRE molecules out of the CNT. The results indicate that the loading and unloading of PRE based on CNT both are energetically favorable. CNTs exhibit great potential as nanovehicles to carry and deliver particular drug molecules.

Introduction Carbon nanotubes (CNTs) have shown prospective potential in the fields of biotechnology and biomedicine due to the unique atomic structure and mechanical, optical and chemical properties.1-3 For example, CNTs act as vehicles to transport drug molecules.4-7 Large surface area enables various functionalization for either covalent attachment or noncovalent adsorption with drug molecules.8-12 The formed CNT-drug conjugates show high cellular uptake via passive penetration or endocytosis mechanism depending on the high hydrophobicity as well as their needle like shape.13-15 They can accumulate in tumor tissues because of the enhanced permeability and retention effect.16 Moreover, modified CNTs or functionalized CNTs have higher solubility and are less cytotoxic than nonfunctionalized ones.17-19 Kam et al. covalently attached the protein streptavidin to the surface of single-walled carbon nanotubes (SWCNTs) and showed that the nanotubes can carry large drugs and the complex can be successfully transported into cells.8 The same research group also formed protein–SWCNT and DNA-SWCNT conjugates using a noncovalent adsorption scheme, where the proteins and oligonucleotides absorb spontaneously onto the sidewalls of acid-oxidized SWCNT.9 The proteins and oligonucleotides were found to be readily transported into living cells with nanotubes acting as the carrier via the energy-dependent endocytosis. In other studies, they transported short SWCNTs with various functionalizations of proteins and oligonucleotides into various mammalian cells.10 Singh et al. functionalized CNTs with ammonium and then associated with plasmid DNA. The Nanotube-DNA complexes were delivered to a mammalian cell line and exhibited upregulation of marker gene expression over naked DNA.20 Pantarotto et al. found that the gene 2 ACS Paragon Plus Environment

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expression levels of CNT-DNA complexes were up to ten times higher than those achieved with DNA alone.21 Complementary to experimental studies, molecular dynamics (MD) simulations have been extensively employed to study CNT loading and unloading drug molecules.22-35 For example, Kavyani et al. investigated the loading of protein ubiquitin and drug pyrene onto the drug carrier carbon nanotube−dendrimer, using coarse-grained MD simulations.32 They found that a stable complex can be formed by properly adjusting the weight ratio of polyamidoamine to protein. Li et al. studied the optimization of noncovalent loading of vinblastine on SWCNT.31 Taking temperature, chirality and functionalization into consideration, they demonstrated terminal esterification of carbon nanotubes strengthened the drug−carrier interactions of all systems. The functionalized CNTs of armchair type were suitable for drug delivery at both 277 and 300 K due to the strong drug−carrier interactions. The hollow channel can also be used to transport small molecules. In this regard, CNTs serve as nanocapsules to protect encapsulated drug from hydrolysis, degradation or reaction with healthy cells before the drug is delivered to the target site. The large inner volume can be filled with the desired chemical, ranging in size from small drug molecules to peptides and proteins. Our previous work have utilized MD simulations to study the transport properties of ultrashort CNTs when embedded in the lipid bilayer.36 The inserted CNTs exhibit selective transport between water, dimethyl sulfoxide and urea according to the CNT size and the polarity of these small molecules. Hilder and Hill theoretically investigated the encapsulation behavior of anticancer drug molecule cisplatin entering nanotubes.5 They determined the critical tube radius (about 0.48 nm) required for efficient encapsulation, and this radius that provide the maximum uptake of cisplatin. MD simulations performed by Liu and Wang showed that the peptide zadaxin can spontaneously insert the (14, 14) CNT and move around the center of the tube.24 As to unloading, drug molecule pyrazinamide initially encapsulated within CNT will be squeezed out and replaced by fullerene with the increasing temperature.29 Cisplatin can release from CNT when the complex is 3 ACS Paragon Plus Environment


positioned at the interface between water and lipid bilayer.27 Upon exposure to an external magnetic field, Panczyk group simulated the anticancer drug cisplatin and carmustine release from CNTs capped by magnetic nanoparticles.33, 34 However, the above simulations are only performed in the water solution without cell membranes. The passive penetration of drug-CNT complex into the membrane and the subsequent drug release have yet to be observed. In this work, we use classic MD simulations to investigate the encapsulation of small drug molecule pregabalin into an optimized CNT in the water solution and release of these drug molecules into the lipid membrane. We find PRE can readily enter the CNT and reside there steadily. Similarly, the PRE-CNT complex can spontaneously permeate into the lipid bilayer and then PRE molecules begin to release from CNT into the bilayer. Our simulations confirm the good suction and expulsion capacity of a particular nanotube ((7, 7) CNT) in association with a particular drug molecule (pregabalin). Free energy calculations demonstrate both the suction and expulsion are energetically favorable, indicating that utilizing CNT hollow channels to carry and deliver drug molecules is feasible.

Computational Methods The simulation system consisted of a fully hydrated lipid bilayer ， one pristine SWCNT and small drug molecules. The hydrated bilayer developed by Berendsen and Tieleman was composed of 128 dipalmitoylphosphatidylcholine (DPPC) lipids and about 4000 water molecules.37 We constructed armchair (6, 6), (7, 7) and (8, 8) CNTs with their radii ranged from 0.42 to 0.55 nm. Pregabalin (abbreviated as PRE) was adopted as a model drug molecule, as its size and polarity are suitable for transport through CNT hollow channels. The force field parameters for CNTs and DPPC lipids were taken from Hummer et al. and Berger et al., respectively.38-40 The force field parameters for PRE (see Supporting Information 1) were developed by Automated Topology Builder (ATB, https://atb.uq.edu.au/index.py),41 which is based on GROMOS force field and is compatible with those of DPPC lipids. Water was represented by the SPC model.42 The carbon atoms in CNTs were treated as 4 ACS Paragon Plus Environment

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uncharged Lennard-Jones (LJ) spheres with a cross section of σcc = 0.34 nm and a depth of the potential well of εcc = 0.36 kJ/mol.40 All simulations were performed under the isothermal-isobaric (NPT) ensemble by using the Gromacs package 4.5.6.43, 44 The vdW interactions were treated with smooth cutoff at a distance of 1 nm, whereas the particle-mesh Ewald method was used to calculate the long-range electrostatic interactions.45,

46

Periodic boundary conditions

were employed in all directions. The pressure was controlled semi-isotropically by a Berendsen barostat and the temperature was kept stable at 323K using the V-rescale thermostat.47, 48 Bond lengths within DPPC and water molecules were constrained by the LINCS and the SETTLE algorithms, which allows a integration step of 2 fs.49, 50 The free energy of PRE across the bilayer and CNT was computed from the potential of mean force (PMF) using umbrella sampling. First, Steered MD simulations were conducted to pull PRE from the aqueous phase to the center of mass (COM) of the membrane or CNT. Then, 35 configurations were generated along the z-axis direction (reaction coordinate). The z coordinates of COM distance between the molecule and membrane in each configuration differed by about 0.08 nm to ensure sufficient sampling. Each window was run for 10ns, and data in the last 5 ns was used for sampling. Eventually, the PMF profile was obtained by the Weighted Histogram Analysis Method (WHAM), implemented in the GROMACS package as 'g_wham'.51 When sampling through the CNTs, they were position-restrained by a spring of 1000 kJ mol-1 nm-2.

Results and Discussion 1 PRE translocation through the lipid membrane

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Figure 1. Trajectory of two PRE molecules in the membrane. (A) The initial configuration. (B) The final structure. (C) The z-coordinates of the COM of PRE as a function of simulation time, where the center of the bilayer is set as z=0 nm. For comparison purposes, we first investigated the translocation of PRE across the hydrated lipid bilayer. Initially, we put only one PRE in the aqueous phase and another one in the bilayer center (Figure 1A) to look for the preferable locations PRE stays. Figure 1C shows the vertical distance between the COM of PRE and lipid bilayer. The boundaries of the bilayer in z-direction are defined as the averaged z-coordinates of phosphorus atoms in the upper and lower leaflets, marked as the dashed lines at z= ± 1.8 nm in Figure 1C. We observed that the inner PRE went straight and quickly toward the lipid headgroups and remained at the transition location between lipid polar headgroups and non-polar tails for the rest of the simulation. The outer PRE diffused randomly in the water for the first 6 ns. However, once it reached the interface, the PRE was fast adsorbed into the membrane and stayed at the similar location till the end of simulation (see Figure 1B).

2 The distribution of PRE in the lipid bilayer


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Figure 2. The distribution of PRE in the lipid bilayer. (A) Initial structure, (B) final snapshot, (C) mass density profile of the system. (D) Potential of mean force (PMF) of PRE across the membrane. The labeled ΔGpen denotes the bilayer center penetration barrier. To further corroborate the locations that PRE prefers to stay, we increased the PRE concentration in the water solution, as illustrated in Figure 2A. Figure 2B shows the final structure of the system, in which all the PRE molecules moved into the membrane and remained there. Each PRE molecule presented the similar orientation with its polar groups interacting with the lipid headgroups and its methyl groups immersed in the lipid tails. Mass density profile of PRE demonstrated that they were distributed almost evenly in the two leaflets (see Figure 2C). The two symmetric peaks were at z=±1.1 nm, close to the lipid headgroups. The translocation of PRE can be more quantitatively elucidated by the PMF of PRE at different trans-membrane positions, as shown in Figure 2D. The PMF of PRE in the aqueous phase far away from the interface, where the interactions between PRE and lipid membrane can be neglected, was set as zero. We found that PRE enter the membrane should overcome an energy barrier, though it was not high (about 3.6 kJ/mol). Once overcoming this barrier, the PMF declined rapidly and reached the minimal (approximate -11.9 kJ/mol) at symmetric positions z=±1.1 nm. Such 7 ACS Paragon Plus Environment


positions were in good agreement with the mass density profile, further confirming the energetically favorable location of PRE in the membrane. In addition, there was a high energy barrier at z=0 nm, approximately 29.2 kJ/mol, preventing PRE from passing the bilayer center.

3 PRE encapsulation into the optimal CNT

Figure 3. PMF profiles of PRE across the (6, 6), (7, 7) and (8, 8) CNTs. The two dashed lines denote the positions of two CNT ends (The CNT center was fixed at z=0). The potential of PRE in the water far away from the CNT ends was set to zer0. We next began to study PRE encapsulation into the CNT at the aqueous phase. To find the optimal pore size of CNT to accommodate PRE molecules, we compared the PMF profiles of PRE in (6, 6), (7, 7) and (8, 8) CNTs with the same length of 2.4 nm (Figure 3), and found that PRE in the (7, 7) CNT owned the lowest free energy, indicating that PRE in the (7, 7) CNT was the most energetically favorable. Thus, we chose (7, 7) CNT to load PRE molecules. Figure 4 shows the dynamic process and stability of PRE encapsulation into (7, 7) CNT. It was found that once close to the entrance of CNT, the PRE was fast adsorbed into the hollow channel and then reside there for the rest of the simulation, as the COM distances in xy-plane and z-axis between PRE and CNT were never bigger than the radius (approximate 0.47 nm) and half length of CNT (about 2.4 nm long, see Figure 4C and 4D). PMF profile (Figure 3, red curve) shows that there is a huge fall (-72 kJ/mol) when PRE enters the CNT, compared with that in the water. Such huge fall makes the PRE strongly trapped in the CNT. It should be noted that another structure of PRE-CNT complex is that PRE is 8 ACS Paragon Plus Environment

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adhered to the outer CNT surface (see Supporting Information 2, Figure S1). However, this structure is not stable, but always appears in the simulation, as the PRE is easily trapped in these local minima.

Figure 4. The encapsulation of PRE into (7, 7) CNT from the water solution. (A) Initial configuration. (B) Final structure. Water molecules are not shown for clarity. (C) The COM distance between PRE and CNT in xy-plane. (D) The COM distance between PRE and CNT in z-direction.

4 The stability of 3 PREs in (7, 7) CNT

Figure 5. The stability of 3 PREs in the (7, 7) CNT. (A, C) Two orientations of three PREs in the CNT. (B, D) The COM distance between each PRE and CNT in CNT axis. We repeated the simulation of PRE in the CNT to test the stability of PRE-CNT complex in the water solution. The CNT was 2.4 nm in length and could load 3 PRE 9 ACS Paragon Plus Environment


molecules. Two separate simulations were conducted, in which three PRE molecules were put in the CNT with two different orientations, as shown in Figure 5A and 5C. As aforementioned, the free energy of PRE in the CNT was -72 kJ/mol. Therefore, the structure of PRE molecules in the CNT was highly stable. Only half of the PRE molecule at the end left the CNT for the 50 ns MD simulation. We changed the orientation of the three PRE molecules in the CNT, and compared the interaction energies of three patterns of two adjacent PRE molecules (labeled as S1, S2 and S3), as shown in Figure 6. Among the three structures, S2 presents the strongest interactions, as their polar groups interact with each other. Correspondingly, the electrostatic energy climbs to dramatically -53.6 kJ/mol. The interaction energy of S1 (-8.465 kJ/mol) is a little stronger that of S2 (-3.888 kJ/mol), since there is no strong electrostatic interaction between the two molecules.

Figure 6. The interactions of three patterns of two PRE molecules. (A) The three conformations of two adjacent PRE molecules in the CNT. CNT and water molecules are not shown to highlight these structures. (B-D) The electrostatic, vdW and total interaction energy of the three structures.

5 The permeation of PRE-CNT complex in the membrane


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Figure 7. The insertion of PRE-CNT complex in the membrane. (A) Snapshots at critical times. (B) The COM distance in bilayer normal and interaction energy between CNT and lipid membrane as functions of simulation time. The inset highlights the second energy fall when a lipid enters after two PRE molecules leave the CNT. We then began to study the passive penetration of PRE-CNT complex in the lipid membrane, and placed one such complex out of the membrane (see Figure 7A, snapshot at t=0 ns). After diffusing for a short while, the complex adjusted its orientation and entered the membrane quickly (see snapshot at t=6.3 ns). At t=6.7 ns, the whole body of complex had been already in the bilayer center. Similar to the graphene and fullerene, the fast insertion was driven by the hydrophobic interactions between CNT and lipid tails.52,

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To obtain a more quantitative picture of these

interactions, we calculated the interaction energy between CNT and lipid bilayer, as shown in Figure 7B. Here, the interaction energy was defined as the vdW interaction between CNT and membrane, as the carbon atoms on CNT were uncharged. The energy curve presented the same trend as that of COM distance. The energy difference of CNT in and out of the bilayer reached approximately 900 kJ/mol. It was clear that the huge fall of the energy made CNT fast adsorbed into the membrane. Once entered the membrane, CNT could not leave, just reside in the bilayer center with almost vertical orientation. Similarly, we calculated the PMF of CNT across the lipid bilayer (see Supporting Information 2, Figure S2). Because of its high 11 ACS Paragon Plus Environment


hydrophobility, CNT in the bilayer center reached the lowest free energy, approximately -100 kJ/mol. Compared with the direct insertion of PRE in the membrane, the PRE-CNT complex can reduce the energy barrier to enter the membrane and pass the bilayer center. On the other hand, the lipid bilayer was kept intact after the insertion, indicating that small and short CNTs are fairly biocompatible.

6 PRE release from CNT into the membrane

Figure 8. PRE release from CNT into the membrane. (A) Snapshots at critical times. (B) The COM distance between each PRE and lipid bilayer. After entering the membrane, the complex was kept stable for about 30 ns. Then, we observed the first release of PRE from CNT into the membrane began at t=38.4 ns and completed at 38.8 ns (see Figure 8, snapshots at t=38.4 and 38.8 ns). Interestingly, the second and third PRE molecules continued to climb towards the entrance of CNT. At t=42 ns, the second PRE molecule also was ejected from the CNT. The driving force originated from two factors: one was the interactions between PRE and DPPC lipids, mainly the electrostatic interactions between their polar groups, as PRE climb out of the CNT with the orientation of its polar groups towards the CNT ends. The evidence is that if the PRE-CNT complex was fixed in the bilayer center with the orientation of PRE’s hydrophobic groups outwards the CNT, PRE molecules are difficult to release (see Support Information 2, Figure S3). The other was the hydrophobic interactions between CNT and lipid tails. The release of PRE was accompanied by the insertion of lipid tails in the CNT (see the inset in Figure 7B) 12 ACS Paragon Plus Environment

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because of the strong van der Waals attractions and hydrophobic interactions between the lipid tails and CNT inner walls. That was why there was a sharp energy fall (approximately 300 kJ/mol) near t=40 ns in Figure 7B, indicating that the interactions between CNT and lipid tails were much stronger than those between CNT and PRE molecules.

Figure 9. (A) The most energetically favorable location of PRE, (B) PMF profile of PRE along the CNT axis. However, the last one remained in the CNT till the end of simulation (see Figure 7A, snapshots at t=200 ns). We calculated the PMF of PRE across the CNT embedded in the lipid bilayer, and found that the lowest PMF was located at z ≈ ±1.1 nm. Figure 9A shows the corresponding structure of the system. Half of PRE (polar groups) was out and the rest (hydrophobic methyl groups) was in the CNT. Such location as well as the orientation of PRE was the most energetically favorable. The free energy of PRE at this position is -94.8 kJ/mol, which is approximately 22 kJ/mol lower than that in the CNT in water solution. This is because the CNT is surrounded by lipid tails, which enhance the adsorption of PRE in the CNT. We repeated the above system with different orientations of three PRE molecules in the CNT and showed the similar results (see Supporting Information 2, Figure S4) that two out of three PRE molecules leaved the CNT, implying that the release of PRE from CNT after the complex entering the membrane was very robust. The shortcoming of this complex is that one PRE molecule cannot release from the CNT. To further confirm that one PRE molecule will be trapped in the CNT, we performed two separate simulations with CNT loading one or two drug molecules (see Supporting Information 2, Figure S5). When CNT loading one PRE molecule, it cannot break away from the CNT during 13 ACS Paragon Plus Environment


the whole simulation (Figure S5B, snapshot at 200 ns). Similarly, when CNT was filled with two drug molecules, one leaved and the other was still trapped in the CNT (see Figure S5D). The length of CNT (2.4 nm) was close to the thickness of lipid tails (2.8 nm), and they are both hydrophobic, therefore, CNT in the bilayer center was almost vertical. Such orientation was unfavorable to damp the last PRE molecule.

Figure 10. Shorter CNT loading and release two PRE molecules. (A) Snapshots at critical times. Water molecules are not shown for clarity. (B) The COM distance between each PRE and lipid bilayer. To improve the release rate of CNT, we construct a shorter CNT with the length of 1.6 nm and just carrying two PRE molecules (Figure 10A, snapshot at t=0 ns). Interestingly, it was found that no PRE remained in the CNT. At t=28.9 ns, two PRE molecules both leave the CNT, indicating that the release rate is dependent on the length of CNT. When CNT became shorter, it was prone to tilt in the bilayer center and drug molecules were easy to release. The (7, 7) CNT with the length of 1.6 nm was much suitable to load and unload PRE molecules.

Conclusions In summary, using MD simulations, we have investigated the encapsulation of small drug molecule PRE into CNT from the water solution, the insertion of PRE-CNT complex in the lipid bilayer and the release of PRE from the CNT into the lipid membrane. The results show that in the water solution, PRE prefers to reside in 14 ACS Paragon Plus Environment

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the CNT and owns the lowest free energy. The PRE-CNT complex can readily enter the lipid bilayer, due to the hydrophobic interactions between CNT and lipid tails. Then, the drug PRE molecules release one by one from CNT into the lipid membrane, which is driven mainly by the electrostatic interactions between lipid headgroups and PRE polar groups. The orientation of PRE at the ends with polar groups outwards is beneficial to release. CNTs exhibit prospective potential as nanovehicles to carry and deliver small drug molecules through their hollow channels.

Supporting Information Available The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. The topology and force field parameters of PRE. The adsorption of PRE on CNT surface. PMF profiles of PRE and CNT across the lipid bilayer. The robustness of PRE release from the CNT into the membrane.

Acknowledgements This work was partially supported by the National Natural Science Foundation of China (Grant No. 11875236, 61575178, 11574272, U1832150), the Zhejiang Provincial Natural Science Foundation of China (Grant No. LY16A040014, LY17F050011, LY18A040001) and Zhejiang Provincial Science and Technology Project (Grant No. LGN18C200017).

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