Molecular Perspective Mechanism for Drug Loading on Carbon

Subscriber access provided by TUFTS UNIV

B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Molecular Perspective Mechanism for Drug Loading on Carbon nanotube-Dendrimer: A Coarse grained Molecular Dynamics Study Sajjad Kavyani, Mitra Dadvar, Hamid Modarress, and Sepideh Amjad-Iranagh J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b04434 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Molecular Perspective Mechanism for Drug Loading on Carbon nanotube-Dendrimer: A Coarse grained Molecular Dynamics Study Sajjad Kavyani, Mitra Dadvar, Hamid Modarress*, Sepideh Amjad-Iranagh Department of Chemical Engineering, Amirkabir University of Technology * Corresponding Author Email: [email protected] Tel:

+982164543176 Fax: +982166405847 Abstract The loading mechanism of the protein ubiquitin, and the drug pyrene, as a representatives of large and small molecules, onto the drug carrier carbonnanotube-polyamidoamine(CNTPAMAM), was studied by using coarse grained (CG) molecular dynamics simulation. The results indicated that, the optimum and stable drug delivery system for protein loading can be obtained by inserting the molecules in the sequence of: i) PAMAM, ii) protein and iii) PAMAM. Also, it was found that, by properly adjusting the weight ratio of PAMAM to the protein, defined as Mw PAMAM/Mw protein (where Mw is molecular weight), can lead to achieve a stable system for loading the protein. However, for pyrene loading, it was found that the insertion sequence has no significant effect and only encapsulation of the pyrene molecules into PAMAM and adjustment of the weight ratio of PAMAM to pyrene (Mw PAMAM/Mw pyrene), can affect the stability of the drug delivery system.

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Introduction In recent years, carbon nanotubes (CNTs) have been proposed as an effective tool in various fields of applications due to their unique optical, electrical, chemical and mechanical properties.1–7 Drug delivery is one of these applications where the CNTs showed a significant potential8,9 by acting as a delivery system for large molecules such as gene and proteins and also small drug molecules such as pyrene.10–12 However, poor solubility of CNTs in aqueous and physiological solvents is one of the main drawbacks for their application in drug delivery systems.13,14 Recently, it has been reported that by functionalization of CNTs with dendritic polymers (covalently and non-covalently) such as dendrimers, the solubility of CNTs in aqueous solvent can be enhanced significantly.3,15–18 Poly-amidoamine (PAMAM) dendrimers, with their significance characteristics such as uniform structure and controlled size19, showed huge potential to be used for enhancing the solubility of CNTs.20,21 It is notable that as a stand-alone molecule, PAMAM dendrimers have been extensively used in drug delivery of molecules with various sizes and structures.21,22 The PAMAM dendrimers, not only are capable of enhancing the solubility of CNTs, but, by introducing their unique properties into the CNT, are able to modify various CNT’s structural parameters. For example, the CNT-PAMAMs were successfully utilized to immobilize a kind of lipase (BCL) for employing in biodiesel production.23 It has been reported that modifying single walled CNTs with PAMAM dendrimers can effectively reduce their cytotoxicity and increase their cellular uptake which introduce the CNT-PAMAM as an effective tool for utilization in gene delivery and cancer therapy.24 The composition of the PAMAM dendrimers with both single and multi-walled CNTs represent a promising gene delivery tool, in which by increasing


Page 2 of 47

Page 3 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the free amine groups on the dendrimer surface, the transfection efficiency of the CNT-PAMAM is effectively enhanced.25 The ability of PAMAM dendrimers in loading and encapsulating drugs or other agents can add some major aspects to the CNT-PAMAM applications. As the cavities inside the PAMAM in the CNT-PAMAM can host various molecules, they can be used to accommodate nanoparticles, up to few hundred atoms, such as gold nanoparticles, where the resulted CNT-PAMAM-Au can be utilized in drug delivery applications.26 Composition of CNT and PAMAM dendrimer with Pt nanoparticles, by encapsulating Pt into PAMAM, has been used as a glutamate biosensor.27,28 Molecular dynamics (MD) simulations is an effective tool for studying various phenomena from molecular perspective,

13,29–39

such as CNT-dendrimer drug carriers. The CNT-PAMAM drug

carrier has been studied by MD simulation for different generation of the PAMAM dendrimers where it has been shown that at higher generations, the interaction of the PAMAM with CNT increases. Also, the results show that for the same generation of PAMAM and poly(propyl ether imine) (PETIM) dendrimer, the PAMAM has stronger interactions with CNT, compared with PETIM.13,40 It was reported that two PAMAM-wrapped-CNTs have lesser binding affinity toward each other than two pristine CNTs. It has been found that for protonated PAMAM dendrimers the interaction of two CNT-PAMAMs is repulsive, and as a result, can act as effective dispersed agents compared to non-protonated PAMAMs30. Also it has been shown that by increasing the generation number of non-protonated PAMAM, a proper dispersion of CNTPAMAMs in water is obtained which can be utilized in drug delivery system.31 In this work, we intend to examine the mechanism for loading of small or large molecules onto the CNT-PAMAM as a carrier. The parameters affecting the loading mechanism such as the type



of chemical groups used in functionalization of the CNT-PAMAM as well as concentration of the loading molecule and the sequence and type of their insertion were investigated. Method GROMACS43–45 software package (version 5.1.5) was used to perform the MD simulations. The MARTINI force-field (MFF)37,46 was utilized for coarse graining the atoms in the simulation boxes, where in the MFF nearly every four atoms, excluding H, are lumped into a single bead. The simulations were performed with the time step of 20 fs. Considering the recommended cutoff radius of 1.2 nm, for MARTINI force field37,46, then, for the CNT with the length of 9 nm, the simulation box length would be obtained as: 2×1.2 + 9 = 11.4 nm, Therefore, the actual size of the simulation box, was chosen as: ~11.5×11.5×11.5 nm3, to keep the simulation time to a minimum level, for time saving. The MARTINI water model was used to fill the simulation boxes, where each box contained ~ 10000 water beads (40000 water molecules). The temperature and pressure of the simulation boxes were coupled to 315 K at 1 bar, respectively, by Berendsen coupling method.47 The periodic boundary conditions (PBC) were set in three directions of x, y and z. The cut-off radius of 1.2 nm was applied to calculate van der Waals interactions and, in the case of electrostatics interactions, the particle mesh Ewald (PME)48 method was used. To visualize the molecules, visual molecular dynamics (VMD) software49 was utilized. The PAMAM model represented by Lee et. al50,51 was used to simulate the PAMAM dendrimers (Figure 1a). Simulations consider the protonated state of molecules at pH=7, therefore, the primary amines of the dendrimers (terminal amines on the PAMAM surface, represented by “Qd” bead type) were protonated, but the tertiary amines (represented by “N0” bead type) were in neutral state.


Page 4 of 47

Page 5 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The graphene model represented by Titov et. al.34 was used to obtain the CNT structure where every benzene ring was coarse grained into three “SC4” beads (Figure 1b)35,36. However, it has been found that the “CNP” bead type represent the carbon materials more properly than “SC4” 35,36

, thus, the “SC4” bead type in the Titov’s model was replaced by “CNP”. For all of the

simulations, the CNT had a diameter of 1.7 nm with the length of 9 nm. For covalent functionalization of the CNT with the PAMAM dendrimer, the PAMAM branching chains were covalently attached to the CNT surface with the bond length of 0.47 nm and the force constant of 1250 kJ mol-1, where these represent the MARTINI standard bond parameters.37 It is noteworthy that in the CNT-PAMAM only 2 percent of CNT surface sites were functionalized to the PAMAM, and the number of covalent branching chain terminals of PAMAM which are functionalized to the CNT is nearly 7 times more than a non-covalent CNT-PAMAM structure with 64 terminals, since a non-covalent CNT-PAMAM structure, due to electrostatics repulsion forces between the PAMAM terminals in pH = 7 (by presence of positively charged primary amine groups on the surface), limited number of terminals can take part in functionalization with the CNT. During the course of the simulations, the CNT x, y and z components were kept constant. For simulating the pyrene molecules, a previously developed model was utilized41, where in this model, every benzene ring was represented with three SC4 beads (Figure 1c). Both bead types, “CNP” and “SC4” were examined for pyrene molecule simulation in our previous work41 and it was found that by using “SC4” bead type more accurate results can be obtained compared to experimental data42, especially when pyrene molecules are encapsulating in the PAMAM dendrimer. Application of this model led to evaluation of proper results for encapsulation of pyrene molecules into the PAMAM and PPI dendrimers.41 In order to investigate the loading



mechanism of large and small molecules into the CNT-PAMAM, the pyrene molecule as a representative of a small drug molecule and the protein ubiquitin as a representative of a large molecule (Rg ≈ 1.17 nm as we evaluated for this protein by MD simulation) were used. It is noteworthy that ubiquitin is a protein which can be found in almost all human tissues.52–56

Figure 1. Molecular and coarse grained representation of PAMAM, CNT and pyrene structure. In order to model ubiquitin, the martinize.py script57 was utilized for coarse graining its atomic structure, as obtained from protein data bank (1UBQ)58. Various representation models of ubiquitin protein are shown in Figure S1. In order to find the best approach of loading, different delivery systems were designed and simulated, based on the sequence of the protein and PAMAM insertion into the CNT system. It is noteworthy no structural change such as conformation or degradation has been considered for the protein, since the aim of this work was merely investigating the loading of the protein (as a large molecule) onto the CNT-PAMAM systems.


Page 6 of 47

Page 7 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Result and Discussion All of the delivery systems were simulated for the time length of 670 ns and the results were obtained in the last 200 ns. (a) Loading a large molecule (protein) in a delivery system The investigated simulation systems for protein loading are listed in Table 1. In this table, the abbreviation used to name each system represents: system number, functionalization types of PAMAM (nc for non-covalent, and c for covalent functionalization of the PAMAM to the CNT) followed by the sequence of insertion of the dendrimer (PAMAM) and its generation number, and the protein (Pr). For example, for the system #6, the abbreviation “CNTnc-PAMAM4-PrPAMAM4”, represent a delivery system containing a non-covalent functionalization of CNT to PAMAM with the insertion sequence of: i) PAMAM fourth generation, ii) protein and iii) PAMAM fourth generation. For the sake of obtaining realistic and accurate results, the insertion of each molecule into the CNT system (every step of the insertion sequence), was simulated for 670 ns. To show that the delivery systems have reached the equilibrium state in the course of the simulation, the distance between center of masses (COMs) of the last inserted PAMAM and the COM of the protein have been calculated and plotted versus time in Figure 2, as an example, for randomly selected delivery systems. Figure 2 shows that the COM distances have remained constant which indicates that the systems finally have reached their equilibrium states. The last snapshots of the simulation systems are represented in Figure 3.



Table 1. List of studied delivery systems.+ System Delivery number(#) System abbreviation #1 CNTc-PAMAM4-Pr #2 CNTnc-PAMAM4-Pr #3 CNTnc-Pr-PAMAM4 #4 CNTnc-PAMAM4-PAMAM4-Pr #5 CNTnc-Pr-PAMAM4-PAMAM4 #6 CNTnc-PAMAM4-Pr-PAMAM4 #7 CNTnc-PAMAM5-Pr-PAMAM5

Sequence of inserting molecules First Second Third PAMAM4* Protein PAMAM4 Protein Protein PAMAM4 PAMAM4 PAMAM4 Protein Protein PAMAM4 PAMAM4 PAMAM4 Protein PAMAM4 PAMAM5 Protein PAMAM5

+

It is notable that the c and nc in the systems’ names are referred to the covalent and non-covalent functionalization of PAMAM to the CNT. The number after the PAMAM refers to its generation size. * The numbers (4 or 5) after PAMAMs represent their generation.

Figure 2. Variation of the distance between the center of mass (COM) of the last inserted PAMAM dendrimer and the COM of protein as a function of time (ns)


Page 8 of 47

Page 9 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. The last snapshots of the delivery systems consisting of the carrier CNT-PAMAM and the loaded protein molecule. For each delivery system, the right snapshot is in the xy plane (front view) and the left one is in the yz plane (side view). Figure 3 shows that, the sequence of inserting the protein and the PAMAM dendrimer can significantly affects the loading mechanism of the protein into the CNT-PAMAM. In the case of just one PAMAM dendrimer in the solvent, for the system #2, CNTnc-PAMAM4-Pr, in Figure 3b, it is seen that the protein is located on the top of the PAMAM where in this case the PAMAM almost acts as the carrier for the protein. However, for the system #3, CNTnc-PrPAMAM4, the first inserted protein shows a great tendency to wrap around the CNT (Figure 3c) where the long branching chains of PAMAM, acts like a hood for the protein and cover both the protein and the CNT by wrapping around both. For the covalently functionalized CNT to the PAMAM in the system #1, CNTc-PAMAM4-Pr, it can be seen clearly in Figure 3a that, 9 ACS Paragon Plus Environment


although the protein tends to interact with the CNT, the thick layer of PAMAM branching chains prevent its penetration to get through and reach the CNT surface, therefore, only the PAMAM is interacting with the protein and as a result the protein is kept on the PAMAM surface, and in this way, in the #1, CNTc-PAMAM4-Pr system can act as the carrier for the protein. The radial probability (RP) of the protein and the PAMAM, referenced to the z-axis of the CNT, are plotted in Figure 4. This figure shows that for the system #1, CNTc-PAMAM4-Pr (Figure 4a), the PAMAM branching chains almost have covered the CNT surface, and therefore, the protein shows no peak around the CTN surface at the distance of ~1.3 nm from z-axis of the CNT, where it is the closest distance of the MARTINI beads from z-axis of the CNT. This distance is approximated as: (1.7/2 + 0.47) ≈ 1.3 nm, where 1.7 nm is the CNT diameter and the 0.47 nm is approximately the van der Waals radius of the MARTINI beads. Figure 4a also shows that in this system, the protein has no tendency to distribute into the water, and the multiple sharp RP peaks, at various distances indicate that the protein by forming a bulky and unstable structure is located on the PAMAM surface in the #1, CNTc-PAMAM4-Pr system. This unstable structure would increase the chance of early release of the protein from the system #1, CNTc-PAMAM4Pr system, therefore the system cannot be considered a proper and reliable carrier for the loaded protein. For #2, CNTnc-PAMAM4-Pr system (Figure 4b), the RP plot of the protein shows a high intensity peak at the CNT surface (~1.3 nm) which indicates that the protein has reached the CNT surface and is interacting with it. But, as there are multiple sharp RP peaks for the protein, the formation of a bulky structure for the protein is probable and therefore, the interaction of the protein and the CNT cannot be strong. This plot also shows that the RP of the PAMAM dendrimer is very sharp which indicates that it is loaded on the CNT surface and it has covered the CNT surface completely. However, considering Figure 4c, it can be seen the protein is


Page 10 of 47

Page 11 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


adsorbed on the CNT structure and its RP peaks at the CNT surface (~1.3 nm) is very sharp and then there is no evidence for formation of a bulky structure. Therefore, in this case, the protein tends to extend and distribute its structure and then with strong interactions with CNT wrap around the CNT which as a consequence the protein obtains a stable loading position on the CNT surface. Also, in Figure 4c, the RP peaks of the PAMAM dendrimer show a very sharp peak at the CNT surface (~1.3 nm), similar to the protein’s RP peak. Thus it is evident that the PAMAM has wrapped around the CNT strongly and has formed a stable system suitable to be used in drug delivery applications.



Figure 4. The radial probability (RP) of the PAMAM dendrimer, as a whole, and the protein referenced to the CNT z-axis for systems: (a) #1, CNTc-PAMAM4, (b) #2, CNTnc-PAMAM4Pr and (c) #3, CNTnc-Pr-PAMAM4. The RPs of the dendrimer core and the generation layers G0 to G4 for systems: (d) #1, CNTc-PAMAM4-Pr, (e) #2, CNTnc-PAMAM4-Pr and (f) #3, CNTnc-Pr-PAMAM4. It should be noted that the RP values in this figure for (a), (b) and (c) have been normalized to avoid curves overlapping and achieve a more clear representation.


Page 12 of 47

Page 13 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The RP plots for generation layers of the PAMAM has been calculated and are shown in Figure 4d to 5f. These figures indicate that by increasing the generation layer from G0 to G4, the intensity of the RP peaks has reduced, where the RP peak of the G4 layer represent the smallest intensity near the CNT surface. By comparing Figure 4a and Figure 4d, it can be seen that, the protein scarcely interacted with the G0 and G1 layers as it cannot penetrate into the thick layer of the covalently functionalized branching chains of the PAMAM. Then the layers G4, G3 are mostly interacting with the protein, and therefore, the protein remains uncovered and vulnerable to interact with the surrounding water medium. Comparison of Figure 4b and Figure 4e shows that for system #2, CNTnc-PAMAM4-Pr, most of the interior layers of the PAMAM are taking part in interaction with the protein and some part of the protein has penetrated into the PAMAM and interacted with the G0 and G1 layers. However, in the case of the system #3, CNTnc-PrPAMAM4, by comparing Figure 4c with Figure 4f, it is seen that all of the interior layers of the PAMAM, even the core of the dendrimer, have interacted with the protein, This indicates the presence of strong interaction between the PAMAM and the protein which can lead to formation of a stable structure. By increasing the concentration of PAMAM to twice as that of the CNT, to obtain the system of #4, CNTnc-PAMAM4-PAMAM4-Pr, it is seen in Figure 3d that, when the first PAMAM is inserted into the system, it adsorbs randomly on the CNT surface, but, by inserting the second PAMAM, since both PAMAMs have positive charge on their chain branching terminals, due to primary amine groups on their surface, they locate themselves at farthest possible distance from each other, on the CNT surface to minimize the repulsive electrostatics potential, as caused by positively charged chain branching terminals. Therefore, the CNT surface is almost completely covered after insertion of the second PAMAM.



Figure 5. Charge probability of branching chain terminals of the dendrimer and the counterions Cl- versus box length in (a) x and (b) z directions. In order to investigate what happens to the PAMAM dendrimers in presence of counterions the charge probability of the positive charges on the PAMAM surface and the counterions Cl- are calculated, along x and z directions of the simulation box, and are plotted in Figure 5. This figure shows that the positive charges on the PAMAM surface are sharply located on the dendrimer surface whereas the counterions Cl-, are widely distributed throughout the box length which indicates that the counterions cannot neutralize the positive charges at pH = 7. Therefore, due to 14 ACS Paragon Plus Environment

Page 14 of 47

Page 15 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


high probability of positive charges (presence of densely localized positive charges) on the PAMAM surface and repulsive interactions between the dendrimer terminals of PAMAM, the counterions cannot mediate agglomeration of PAMAM molecules. Since, the two PAMAMs have almost covered the entire CNT surface, then by inserting the protein into the system, it can only interact with the PAMAM. However, for more scrupulous interpretation of the behavior of the systems: #5, CNTnc-Pr-PAMAM4-PAMAM4 and #6, CNTnc-PAMAM4-Pr-PAMAM4, we recourse to consider the other structural factors which affect their behavior. For example, in the case of #5, CNTnc-Pr-PAMAM4-PAMAM4 system, it is expected that the two PAMAMs, similar to the system #3, CNTnc-Pr-PAMAM4, cover the protein as a hood, however, the results in Figure 3e indicates that by inserting the second PAMAM, the repulsive electrostatics interactions between the second and the first PAMAMs cause the first one to move further away to minimize the repulsive interaction potential and in this way a room is created for location of the second PAMAM on the CNT surface. Therefore, the so called hood, which has been created primarily by the first PAMAM, and could act as a coverage for the protein, would be pushed away and removed from the protein surface and as a result the loaded protein will be left bare and unprotected, exposed to the surrounding water medium. Figure 3e demonstrates that in the #5, CNTnc-Pr-PAMAM4-PAMAM4 system, although the CNT has kept the loaded protein molecule on its surface, the protein is not completely covered by the PAMAMs and therefore it can be affected by the water molecules. Whereas in the #6, CNTnc-PAMAM4-Pr-PAMAM4 system, as it is seen in Figure 3f, the protein is completely sandwiched between the two PAMAMs and in this way not only the repulsion between the two PAMAMs has been reduced to a minimum, but the PAMAMs have



remained fixed and stable at their positions on the CNT surface and as a result the protein has been covered and protected by the PAMAM. The RP plots of the PAMAM dendrimers and the protein, in the systems with two PAMAMs, have been calculated and are illustrated in Figure 6. This figure shows that for the #4, CNTncPAMAM4-PAMAM4-Pr system (Figure 6a), the RP of the two dendrimers are very similar, which indicates the presence of just a thin layer of PAMAM branching chains on the CNT surface. This behavior of PAMAM is due to the electrostatics repulsions between the PAMAMs which force them to reduce their repulsive potential to a minimum level, by adsorption on the CNT surface, in a very similar manner, at a further distance, from each other. As a result, the RP peak, in Figure 6a, of the protein (at ~1.3 nm) shows that the protein has slightly penetrating into the thin layer of PAMAM to reach the CNT surface and weakly interacted onto the CNT surface. Also, the protein’s RP plot for the #4, CNTnc-PAMAM4-PAMAM4-Pr system, in Figure 6a, represents multiple sharp peaks with strong intensity, which indicates that the protein has a bulky structure and does not tend to distribute its structure on the CNT surface. Since the protein has formed a bulky structure, as it is seen in Figure 6b, and its chain have not wrapped around the CNT-PAMAM structure, it can be released early if it be exposed to the water medium. However, Figure 6b for the #5, CNTnc-Pr-PAMAM4-PAMAM4 system, shows that the RP plot of the protein has only one sharp RP peak at the CNT surface (~1.3 nm), which indicates that the protein has interacted strongly with the CNT and wrapped up around it. Figure 6c for the #6, CNTnc-PAMAM4-Pr-PAMAM4 system, shows that the RP plot of the first PAMAM has small fluctuations, which is due to formation of a stable system. This figure has a high intensity RP peak for the protein at ~1.3 nm which indicates that the protein has interacted strongly with the CNT. Also, the presence of slight fluctuations in the RP plot (Figure 6c) with no sign of bulky


Page 16 of 47

Page 17 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


structure, confirms that the protein has obtained a very stable position in this system. However, RP plot of the second PAMAM in Figure 6c appears at ~1.6 nm. Although this peak is near the CNT surface, but its intensity gradually increases to a maximum intensity peak at ~4 nm, which indicates that the branching chains of the second PAMAM not only has interacted strongly with the protein structure but also covered the protein surface as a hood. By comparing the RP plots of the protein in Figure 4b and Figure 6c, it is evident that the second PAMAM has pushed and compressed the protein on the CNT surface and in this way, instead of forming a bulky structure, has stabilized its position by promoting its interaction with the CNT.



Figure 6. The radial probability (RP) of the PAMAM dendrimers, as a whole, and the protein for systems: (a) #4, CNTnc-PAMAM4-PAMAM4-Pr, (b) #5, CNTnc-Pr-PAMAM4-PAMAM4 and (c) #6, CNTnc-PAMAM4-Pr-PAMAM4. Note that: PAMAM* and PAMAM** are the first and second inserted PAMAMs, respectively 18 ACS Paragon Plus Environment

Page 18 of 47

Page 19 of 47


1 2 3 4 5 6 7 8 9 10 11 12 13 Table 2. The number of close contacts of the protein with: PAMAM dendrimer (as a whole and PAMAM’s core and generation layers G0 to G5), CNT, water 14 and CNT+PAMAMs.+ 15 system core G0 G1 G2 G3 G4 G5 PAMAM* PAMAM** CNT water CNT & PAMAMs 16 #1, CNTc-PAMAM4-Pr 0 0.3 2.3 1.6 3.6 15 23.2 0 96.5 23.2 17 #2, CNTnc-PAMAM4-Pr 0.1 1.9 2.6 4.2 6.3 14.2 29.3 6.5 92.3 35.9 18 #3, CNTnc-Pr-PAMAM4 1.5 4.1 5.1 7.4 10.1 15.1 43.5 73.4 82.1 117 19 0.1 0.7 2.8 5.8 9 13.7 32.3 0 1.3 91.7 33.6 20 #4, CNTnc-PAMAM4-PAMAM4-Pr 0 0 0.1 1.6 8.7 20.7 17.1 14.1 91.0 97.2 122.2 21 #5, CNTnc-Pr-PAMAM4-PAMAM4 2.3 6.4 8.8 14.1 17.1 25.4 31.1 43.1 19.3 72.2 93 22 #6, CNTnc-PAMAM4-Pr-PAMAM4 23 #7, CNTnc-PAMAM5-Pr-PAMAM5 0 0.3 2.1 5.4 9.2 15.3 22.3 22 33 0 70.1 55 24 + PAMAM* and PAMAM** are the first and second inserted PAMAMs respectively 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 19 44 ACS Paragon Plus Environment 45 46 47


Table 2 represents the number of close contacts of the beads in the protein with the beads in the

following named components in each system which includes: the PAMAM (core and G0 to G5 generation layers), the first and the second PAMAMs, the CNT, the surrounding water medium and both the (CNT+PAMAMs). The close contacts are defined as those, where the distance between the beads of the protein and: PAMAM, CNT or water is approximately 0.53 nm. This distance corresponds to the equilibrium distance (re) at the minimum of Lennard-Jones potential (re = 21/6σ)60. As for most of the beads37,46 in the MARTINI force field the size parameter is σ = 0.47 nm, the equilibrium distance (re) between the beads which are in close contact can be evaluated as; re = 21/6σ ≈ 0.53 nm. Table 2 indicates that in the #6, CNTnc-PAMAM4-PrPAMAM4 system, the second PAMAM dendrimer has a strong interaction with the protein. Table 2 also shows that the protein in this system has the minimum number of close contacts with

the water medium. This indicates that the protein is in a stable and safe location in this delivery system and it is well isolated from the water medium, since it is sandwiched between two PAMAMs. To investigate the effect of higher generation number of the PAMAM on the behavior of #6, CNTnc-PAMAM4-Pr-PAMAM4 systems, the fifth generation of the PAMAM dendrimer in the system #7, CNTnc-PAMAM5-Pr-PAMAM5 was considered, where for this system, Figure 7 represents clearly the occurrence of sandwiching of the protein between the two PAMAMs. Although, in this system, the number of close contact of the protein with water (Table 2) shows that the protein has been isolated from the water medium by the PAMAM, but the number of close contacts of the protein with the PAMAMs, as a whole, and with their inner layers (in Table 2) are significantly smaller than those in the #6, CNTnc-PAMAM4-Pr-PAMAM4 system. This table also illustrates that in the #7, CNTnc-PAMAM5-Pr-PAMAM5 system, the protein have no


Page 20 of 47

Page 21 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


close contact with the CNT surface. The reason for this behavior can be explained by inspecting Figure 7. This figure shows the snapshots for the system #7, CNTnc-PAMAM5-Pr-PAMAM5 during the course of the simulations.

Figure 7. The sandwiching of the protein between two PAMAM in the #7, CNTnc-PAMAM5Pr-PAMAM5 system, The snapshots are taken respectively at 20, 600, 620, 1200, 1220 and 1800 ns. The right snapshot is in the xy plane (front view) and the left one is in the yz plane (side view).



As it is seen in Figure 7, the branching chains of the fifth generation of PAMAM gradually in a progressive manner cover the CNT surface by a thick layer and in this way prevent the protein to reach the CNT surface and interact with it. Therefore the protein molecule are loosely positioned on the CNT surface and are highly susceptible to be released from the system by interaction with the surrounding water molecules.

Figure 8. The radial probability (RP) of the protein from COM of the CNT in the systems of #6, CNTnc-PAMAM4-Pr-PAMAM4 and #7, CNTnc-PAMAM5-Pr-PAMAM5. Considering the RP plots of the protein in the systems of #7, CNTnc-PAMAM5-Pr-PAMAM5 as represented in Figure 8, supports the above interpretations related to snapshots presented in Figure 7 in the #7, CNTnc-PAMAM5-Pr-PAMAM5 system. As it is seen in this figure, the protein has no RP peak at the surface of the CNT which shows that the protein has not reached the CNT surface to interact with it.. This figure also shows that the RP peaks of the protein in the 22 ACS Paragon Plus Environment

Page 22 of 47

Page 23 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


#6, CNTnc-PAMAM4-Pr-PAMAM4 system has very small fluctuations compared to the RP peaks of the #7, CNTnc-PAMAM5-Pr-PAMAM5 system. The comparatively large fluctuations of the protein’s RP in the #7, CNTnc-PAMAM5-Pr-PAMAM5 system, in Figure 8, indicates that there is no interaction between the CNT and the protein, and protein has formed a bulky structure on the CNT surface. To investigate the behavior of these two systems further, we consider the weight ratio parameter which is defined as Mw

PAMAM/Mw protein,

where Mw is the molecular

weight. The evaluated weight ratio for #6, CNTnc-PAMAM4-Pr-PAMAM4 system is: Mw PAMAM/Mw protein

protein

≈ 1.6 and for #7, CNTnc-PAMAM5-Pr-PAMAM5 system is: Mw

PAMAM/Mw

≈ 3.3. Therefore, it seems that by manipulating the weight ratio, the stability of the CNT-

dendrimer drug delivery system can be adjusted somehow to become appropriate for loading of a large molecule such as protein. The results, in Figure 7 and Figure 3f manifest that for the large values of the weight ratio, where the weight of the PAMAM is significantly larger than that of the protein, the PAMAMs would cover a larger portion of the CNT structure and as a result the protein cannot reach the CNT surface. It is notable that the size of the PAMAM dendrimer depends on molecular weight29, therefore, for PAMAM with higher molecular weight, the branching chains have longer length. But, at low values of the weight ratio (smaller PAMAM size or generation), the PAMAM coverage around the CNT can be adjusted so that some parts of the CNT remain uncovered to allows the protein to interact with the CNT surface (Figure 8). Thus, in the #7, CNTnc-PAMAM5-Pr-PAMAM5 system, although the protein has been sandwiched between PAMAMs and isolated from the water medium (Figure 7), but it has been prevented to reach the CNT surface to interact with it to form a firm and stable structure. However, for the #6, CNTnc-PAMAM4-Pr-PAMAM4 system which has smaller weight ratio,



not only the protein has been sandwiched and isolated from the water medium, but it has reached to the CNT surface to interact with it and to form a stable drug delivery system. Inspecting the results in Figure 6c and Table 2, it can be stated that the #6, CNTnc-PAMAM4-PrPAMAM4 system represents an optimum structure for loading of large molecule such as the protein ubiquitin, since the two PAMAM dendrimers have sandwiched the loaded protein and provided sufficient isolation, to keep it stable and safe to approach intact to the targeted cells and then released it due to interactions with the targeted cells. Therefore, this system can be proposed as a proper system for application as an effective CNT-dendrimer drug delivery system. (b) Loading a small molecule (pyrene) in a delivery system Pyrene was selected as a representative of small molecules, and was used to investigate the loading mechanism of small molecules into the CNT-PAMAM system as a drug carrier. In order to find the proper procedure to load the pyrene, we used a similar approach as explained in the previous section, where in each step, the pyrene and PAMAM were added to the CNT system in various sequences. The simulated delivery systems are listed in Table 3. The name of each studied system indicates the sequence of insertion, the number of PAMAM and pyrene, and how they were added to the designed delivery system. For example, for the system number #6, the “CNTnc-PAMAM4-20Py-PAMAM4” system represent an abbreviated name for a system in which the molecules were inserted in the following order: i) PAMAM fourth generation, ii) 20 pyrene molecules and iii) PAMAM fourth generation. It is notable that in the systems where the pyrene molecules were encapsulated into the PAMAM, prior to insertion into the CNT system, the system name is designated as: CNTnc-PAMAM4(20Py), which means that 20 pyrene molecules were encapsulated into the PAMAM dendrimer and then both the PAMAM and the encapsulated pyrene molecules were inserted into the CNT system, simultaneously. Every step 24 ACS Paragon Plus Environment

Page 24 of 47

Page 25 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


was simulated for 670 ns and the last 200 ns was used for analysis of the obtained results. Figure 9 represents the distance between the COM of the last inserted PAMAM and the COM of the CNT, as example, for some randomly selected delivery systems. This figure indicates that the delivery systems have reached the equilibrium state. Since with small fluctuations, the distance has remained constant about a mean value.



Page 26 of 47

1 2 3 Table 3.List of studied delivery systems.+ 4 Sequence of inserting molecules System Number 5 Delivery system 6 number (#) of pyrene First Second Third 7 #1 CNTc-PAMAM4-20Py 20 PAMAM Pyrene 8 #2 CNTnc-PAMAM4-20Py 20 PAMAM Pyrene 9 10 #3 CNTnc-20Py-PAMAM4 20 Pyrene PAMAM 11 #4 CNTnc-PAMAM4(20Py) 20 PAMAM(20Py) 12 #5 CNTnc-PAMAM4-PAMAM4-20Py 20 PAMAM PAMAM Pyrene 13 14 #6 CNTnc-PAMAM4-20Py-PAMAM4 20 PAMAM Pyrene PAMAM 15 #7 CNTnc-PAMAM4-PAMAM4(20Py) 20 PAMAM PAMAM(20Py) 16 #8 CNTnc-PAMAM4(4Py) 4 PAMAM(4Py) 17 18 + It is notable that the c and nc in the system names is referred to the covalent and non-covalent functionalization of PAMAM to CNT. The 19 number after each PAMAM refers to its generation size. Also, PAMAM(Py) stands for a PAMAM dendrimer structure with 20 21 encapsulated pyrene molecules, where the number before Py represents the number of pyrene molecules. 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Figure 9. Variation of the distance between COM of the last inserted PAMAM dendrimers and 50 the COM of the CNT versus time (ns), as example, for randomly selected delivery systems 51 52 53 54 55 56 57 58 26 59 ACS Paragon Plus Environment 60

Page 27 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 10. The last snapshots for the CNT-PAMAM drug carrier systems with the pyrene molecule as a loaded molecule. For each delivery system, the right snapshot is in the xy plane (front view) and the left one is in the yz plane (side view).



Figure 10 represents the last snapshots of the CNT-PAMAM delivery systems with the pyrene molecules as the loaded drug. This figure shows that for the #1, CNTc-PAMAM4-20Py system (Figure 10a), the pyrene molecules have penetrated through the branching chains of the PAMAM. Therefore, the lack of proper coverage of the absorbed pyrene molecules by PAMAM’s branching chains make the pyrene molecules vulnerable to interact with the water medium and facilitate their unwanted release from the CNT surface. Then, #2, CNTncPAMAM4-20Py cannot be considered as a proper drug delivery system. However, Figure 10c shows that by inserting the PAMAM dendrimer after the pyrene molecules (in the #3, CNTnc20Py-PAMAM4 delivery system), the PAMAM by wrapping around the CNT, covers the adsorbed pyrene molecules. But, as the pyrene molecules tend to distribute themselves on the entire CNT surface, the PAMAM is unable to cover the CNT surface completely. The RP plots of the pyrene molecules and PAMAM (including its core and G0 to G4 generation layers) for the #1, CNTc-PAMAM4-20Py, #2, CNTnc-PAMAM4-20Py and #3, CNTnc-20Py-PAMAM4 systems have been calculated referenced to the z-axis of CNT and are represented in Figure 11. Figure 11a for the #1, CNTc-PAMAM4-20Py system, shows that there is a high intensity peak due to presence of the pyrene molecules on the CNT surface (~1.3 nm). This indicates that, unlike the protein molecule (as shown in Figure 4a), the pyrene molecules, due to their small size, can penetrate into the PAMAM branching chains, to form a stable drug delivery system. Also, this figure for the same system, shows that the pyrene molecules are widely distributed from the CNT surface into the PAMAM branching chains. Whereas for the #2, CNTncPAMAM4-20Py system, the RP peaks (in Figure 11b) of the PAMAM and pyrene molecules with significantly high intensity are located near the CNT surface (~1.3 nm). The RP peaks in


Page 28 of 47

Page 29 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


this figure confirms that both pyrene and PAMAM are strongly interacting with the CNT surface.



Figure 11. The radial probability (RP) of the PAMAM dendrimer, as a whole, and the pyrene molecules referenced the CNT z-axis for: (a) #1, CNTc-PAMAM4-20Py, (b) #2, CNTnc-PAMAM4-20Py and (c) #3, CNTnc-20Py-PAMAM4. The RP of the dendrimer generation layers for: (d) #1, CNTc-PAMAM4-20Py, (e) #2, CNTncPAMAM4-20Py and (f) #3, CNTnc-20Py-PAMAM4. 30 ACS Paragon Plus Environment

Page 30 of 47

Page 31 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


However, as it is seen in Figure 11b and Figure 11c, the RP of PAMAM for the #3, CNTnc20Py-PAMAM4 system (Figure 11c) is located in the range of ~1.7 nm with higher intensity compared to that of #2, CNTnc-PAMAM4-20Py system in Figure 11b. This indicates that the pyrene molecules in this system are located under the branching chain of PAMAM isolated and therefore this system is suitable for drug delivery applications. But, the wide distribution of the RP peaks, as it is seen in Figure 11f, compared to Figure 11e, for the interior structure of the PAMAM as well as the high intensity of RP peaks at the range of ~1.7 nm, specially for the core and G0 layer, indicates the occurrence of some kind of structural change in the PAMAM which can be attributed to the presence of pyrene molecules beneath the PAMAM branching chains, on the CNT surface. Figure 10e shows that for the #5, CNTnc-PAMAM4-PAMAM4-20Py system, after inserting the second PAMAM into the system, the repulsive electrostatics interactions between the two PAMAMs, forced them to be adsorbed, at the furthest possible distance from each other on the CNT surface in order to minimize their repulsion interactions potential. By inserting the pyrene molecules into the #5, CNTnc-PAMAM4-PAMAM4-20Py system, as explained for the #5, CNTnc-PAMAM4-20Py system (Figure 10b), the pyrene molecules tend to be adsorbed into the uncovered CNT surface. This behavior results in an instability in the delivery system, since the pyrene molecules are not properly isolated and there is a high probability of their early release due to interactions with water molecules. However, in the #6, CNTnc-PAMAM4-20PyPAMAM4 system (in Figure 10f), by considering the facts that, the inserted first PAMAM dendrimer and the pyrene molecules (as it was discussed for the #2, CNTnc-PAMAM4-20Py system in Figure 10b), compete for adsorption into the CNT surface, and also, due to the repulsion between the PAMAMs, the second PAMAM should take adsorption position far from



the first PAMAM. Therefore, the only possible location for the second PAMAM for adsorption would be where the pyrene molecules were already adsorbed. By adsorption of the second PAMAM, its branching chains tend to wrap around the adsorbed pyrene molecules on the CNT surface. As a result, the pyrene molecules would be isolated and therefore the drug delivery system as obtained in this way will be safe and stable for delivering the pyrene molecules. In the case #7, CNTnc-PAMAM4-PAMAM4(20Py) system in Figure 10g, the second PAMAM has encapsulated all the pyrene molecules in its structure, where no pyrene molecule have been released from it. This behavior can be attributed to strong interactions between the second PAMAM and the encapsulated pyrene molecules. Figure 12, represents the RP plots of the PAMAMs and pyrene molecules for the systems containing two PAMAMs. In Figure 12c, for the system of #7, CNTnc-PAMAM4-PAMAM4(20Py), there is no RP peak near the CNT surface (~1.3 nm) neither for the second PAMAM nor for the pyrene molecules. This evidently indicate that these molecules have no interaction with the CNT surface. But, there is a very small overlap of the RP peaks for the first and second PAMAM which is due to their weak interaction with each other.


Page 32 of 47

Page 33 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 12. The radial probability (RP) of the PAMAM dendrimers, as a whole, and the pyrene molecules for: (a) #5, CNTnc-PAMAM4-PAMAM4-20Py, (b) #6, CNTnc-PAMAM4-20PyPAMAM4 and (c) #7, CNTnc- PAMAM4-PAMAM4(20py). Note that: PAMAM* and PAMAM** are the first and second inserted PAMAM, respectively. 33 ACS Paragon Plus Environment


For further investigation of the delivery systems containing the PAMAM with encapsulated pyrene molecules, we studied the systems: #4, CNTnc-PAMAM4(20Py) and #8, CNTncPAMAM4(4Py), containing only one PAMAM but two different number of pyrene molecules respectively 20 and 4. Although the pristine PAMAM tend to wrapped around the CNT surface as it is seen in Figure 10d clearly shows that in the #4, CNTnc-PAMAM4(20Py) system, the PAMAM with encapsulated pyrene molecules, do not tend to interacted with the CNT at all. Also, the encapsulated pyrene molecules have not been released from the PAMAM interior structure, and remained in their positions. These results show that the binding between the branching chains of the PAMAM and pyrene is so strong and the structure formed for PAMAM and encapsulated pyrene is so stable that cannot be affected by presence of CNT. On the other hand, Figure 10h shows that for the #8, CNTnc-PAMAM4(4Py) system, with less number of pyrene molecules, only the core and G0 layer of PAMAM are involved in interactions with the encapsulating pyrene molecules, and as a result the branching chains of the outer layers of PAMAM can interact with the CNT and wrap around it. As explained earlier, the weight ratio of the PAMAM to the encapsulated molecules inside the PAMAM determines the capability of the branching chains of PAMAM to interact with the CNT. The weight ratio which in this case is defined as: Mw PAMAM/Mw pyrene, is equal to 3.5 and 17.5 for the #4, CNTnc-PAMAM4(20Py) and #8, CNTnc-PAMAM4(4Py) systems, respectively. According to the above interpretation of the obtained results it is evident that in the #4, CNTnc-PAMAM4(20Py) system, with the weight ratio of 3.5, the pyrene and PAMAM maintain their structure. But, for the system of #8, CNTncPAMAM4(4Py), with the weight ratio of 17.5, the outer branching chains of the PAMAM change their structure to wrap around the CNT. These results again emphasize the importance of


Page 34 of 47

Page 35 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


weight ratio (dendrimer/drug) which can influence the structural behavior of CNT-dendrimer systems.

Figure 13. The radial probability (RP), referenced to the CNT z-axis of the PAMAM dendrimer, for #8, CNTnc-PAMAM(4Py) system: (a) PAMAM as a whole, and the pyrene molecules and (b) the core and the generation layers G0 to G4 of the PAMAM. Considering the results represented in Figure 13a, for the RP plots (referenced to the CNT z-axis) for PAMAM and pyrene molecules in the system of #8, CNTnc-PAMAM4(4Py), again confirms the effect of weight ratio in the studied systems. Figure 13a shows that PAMAM and the pyrene 35 ACS Paragon Plus Environment


molecules have a high intensity peak near the CNT surface (at ~1.3 nm). Whereas due to wrapping of the branching chains of the PAMAM around the CNT surface, it seems that somehow the pyrene molecules have penetrated into the PAMAM interior layers and interacted with the CNT surface. Inspecting the RP plots of the interior generation layers of the PAMAM in Figure 13b lends support to the above interpretation, since as this figure evidently demonstrates the core and the G0 layers of PAMAM have not interacted with the CNT surface, since their highest intensity RP peak is at ~1.8 nm which is far from the CNT surface (at ~1.3 nm). Whereas the encapsulated pyrene molecules are mostly located in the core and G0 layer41 of PAMAM, and the location of RP peaks for the core and G0 layer of PAMAM (in Figure 13b) indicates that they have maintained their structure, although they have the pyrene molecules encapsulated into their interior layers. Also, the interaction of PAMAM dendrimer with the CNT is sufficiently strong13 to form a stable CNT-PAMAM structure. Therefore, it can be stated that the for higher weight ratios (Mw PAMAM/Mw pyrene), not only the pyrene molecules can be loaded to and remain firmly stable on the CNT surface in the drug delivery system, but, the whole CNT-dendrimer drug delivery system would remain stable and intact as a drug carrier. To approve these interpretations further we proceed with calculating the number of close contacts, between the beads of PAMAM, CNT and pyrene, where the results of these calculations are presented in Table 4. According to the results presented in this table, the PAMAM dendrimer in the #8, CNTnc-PAMAM4(4Py) system has a strong interaction with the CNT, which indicates the stability of the CNT-PAMAM structure. Also, the results in this table confirms this point that by changing the weight ratio, the stability and loading capacity of the delivery system can be adjusted to a required level for drug delivery applications.


Page 36 of 47

Page 37 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 4. The number of close contacts of the PAMAM dendrimer with the CNT, and also, the weight ratio (Mw PAMAM/Mw pyrene) for #4, CNTnc-PAMAM4(20Py) and #8, CNTnc-PAMAM4(4Py) systems Mw PAMAM/Mw pyrene Delivery system Number of contacts #4, CNTnc-PAMAM4(20Py) 2.68 3.5 17.5 #8, CNTnc-PAMAM4(4Py) 84.14


The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Table 5. The number of close contacts of the pyrene molecules with: PAMAM dendrimer (as a whole and with PAMAM’s core generation layers G0 to G4), CNT, water.+ system core G0 G1 G2 G3 G4 PAMAM* PAMAM** CNT #1, CNTc-PAMAM4-20Py 6.04 17.81 40.10 63.12 42.23 169.33 94.59 #2, CNTnc-PAMAM4-20Py 0 0.09 2.98 8.69 17.57 18.92 48.27 144.06 #3, CNTnc-20Py-PAMAM4 0.19 3.49 11.01 27.47 37.44 23.85 103.48 31.21 #4, CNTnc-PAMAM4(20Py) 2.00 7.46 13.21 20.34 25.09 14.01 82.01 0.00 #5, CNTnc-PAMAM4-PAMAM4-20Py 1.39 8.46 13.39 24.58 34.88 28.53 14.86 60.51 135.76 #6, CNTnc-PAMAM4-20Py-PAMAM4 0.00 1.54 10.87 32.73 54.26 40.08 41.78 63.06 149.28 #7, CNTnc-PAMAM4-PAMAM4(20Py) 4.98 16.2 26.59 38.26 41.51 20.77 0.00 100.46 0.00 #8, CNTnc-PAMAM4(4Py) 1.15 3.92 5.11 7.27 8.82 4.02 30.06 12.25 +

PAMAM* and PAMAM** are the first and second inserted PAMAM respectively.


Page 38 of 47

and the water 43.33 96.20 71.59 30.67 65.43 45.61 27.31 6.28

Page 39 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The number of close contacts between the pyrene molecules and PAMAM, CNT and water molecules, in each delivery system, has been calculated and represented in Table 5. This table shows that for the delivery system #1, CNTc-PAMAM4-20Py, which has a large number of PAMAM branching chains, the number of close contacts with the pyrene molecules is the highest, and since the number of close contacts between the pyrene and the CNT surface is also significantly large, it can be stated that the pyrene molecules have penetrated into the PAMAM structure and interacted with the CNT. As it is seen in Table 5 for the #8, CNTncPAMAM4(4Py) system, the number of close contacts of the pyrene molecules with water molecules is very small which indicates that the pyrene molecules are well isolated from the surrounding water medium. On the other hand, the pyrene molecules have sufficient number of close contacts with the CNT (~13 close contacts) and the PAMAM (~30 close contacts) as it is reported in Table 5. This means that, the encapsulated pyrene molecules are attached by strong interaction to the main components of the system i.e. PAMAM and CNT, and as a result, the pyrene molecules as a loaded drug, possess a stable and firm positions in this drug delivery system. These interpretations as based on the close contact results in Table 5 are supported by considering the RP peaks in Figure 13 and the snapshots in Figure 10 (d, g and h), that the pyrene molecules have been strongly attached to the CNT-PAMAM system by being well encapsulated into the PAMAM. Therefore, it can be stated that among the designed systems for delivery of pyrene as small molecule, the #8, CNTnc-PAMAM4(4Py) system has the desired characteristics which indicates the strong interactions between PAMAM and the CNT, and between PAMAM and the pyrene molecules. Thus, in this system, the pyrene molecules are kept stable and safe from the effect of surrounding water molecules. Another advantage of this system is that; the loading capacity and the interaction of the PAMAM with the CNT can be adjusted by



varying the value of weight ratio (Mw PAMAM/Mw pyrene ). Considering these characteristics, the #8, CNTnc-PAMAM4(4Py) delivery system is proposed as the most stable structure for the safe delivery of pyrene and other small molecules. Conclusion In this work, the loading mechanism of the drug pyrene (as a representative of small molecules) and the protein ubiquitin (as a representative of large molecules) was investigated by utilizing coarse grained (CG) molecular dynamics simulation. The results showed that the loaded protein ubiquitin tends to interact with the CNT and can distribute its structure into the surroundings medium only in the presence CNT. In the #1, CNTc-PAMAM4-Pr system, it was found that the protein cannot penetrate through the thick layer of PAMAM branching chains, and then it would agglomerate into a bulky structure on the CNT-PAMAM surface resulting an unstable system. This behavior of PAMAM indicates that increasing the number of branching chain of PAMAM around the CNT may not necessarily enhance the capabilities of the CNT-PAMAM as a drug delivery system. However, by prior insertion of the protein and then non-covalent functionalization of PAMAM to the CNT, it was seen that the protein would interacts with the CNT and wrapped around it. On the other hand, the inserted PAMAM by wrapped around the adsorbed protein on the CNT surface, can cover the protein structure to form a stable delivery system. The results also indicate that by inserting the protein and PAMAM in the following sequence of i) PAMAM, ii) protein and iii) PAMAM, to a delivery system such as #6, CNTncPAMAM4-Pr-PAMAM4, the two PAMAM dendrimers can sandwich the protein structure and isolate it from the surrounding water medium. It was observed that, the generation number of the PAMAMs is a determining factor, where by manipulating the weight ratio parameter (Mw PAMAM/Mw protein)

the stability of the drug delivery systems can be adjusted. The results showed 40 ACS Paragon Plus Environment

Page 40 of 47

Page 41 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


that the delivery system: #6, CNTnc-PAMAM4-Pr-PAMAM4 with Mw PAMAM/Mw pyrene ≈ 1.6, is the most stable system which can keep the loaded protein in a safe position, isolated from the surrounding water medium, in the delivery system, and prevent its unwanted early release. For loading of small molecules pyrene into the studied delivery systems, the results indicated that the pyrene molecules have strong interaction with the CNT, therefore, they have tendency to be absorbed on the CNT surface and in a competitive absorption they seize the priority over PAMAM to occupy most of the adsorption sites on the CNT surface. But, in this case, the pyrene molecules remain uncovered and unprotected from the surrounding water molecules and hence constitute an unstable drug delivery system. Therefore in the CNTc-PAMAM system, since there are large number of functionalized PAMAM branching chains, the small pyrene molecule would penetrate into the PAMAM branching chains to interact with the CNT surface to form a stable delivery system. But, if the pyrene molecules be encapsulated preliminarily into the PAMAM prior to insertion into the system, the resulted delivery system #8, CNTnc-PAMAM4(4Py) would be the most stable, due to strong binding between the interior structure of PAMAM and pyrene molecules and this would prevent the unwanted early release of the pyrene molecules from the delivery system. The results also indicated that in the systems of #8, CNTnc-PAMAM4(4Py), by changing the weight ratio parameter (Mw PAMAM/Mw pyrene), the interaction of the PAMAM and the CNT as well as its loading capacity can be adjusted in order to obtain an effective drug delivery system. These results suggest an important possible loading mechanism of molecules onto CNT-PAMAM as a carrier, however, the simulation results always need to be confirmed by experiments. Supporting Information



Extra results as obtained in this work and cited in the manuscript are provided in the supporting information.


Page 42 of 47

Page 43 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


References (1)

Ouyang, M.; Huang, J.-L.; Lieber, C. M. Fundamental Electronic Properties and Applications of Single-Walled Carbon Nanotubes. Acc. Chem. Res. 2002, 35 (12), 1018– 1025.

(2)

Fagan, J. A.; Simpson, J. R.; Bauer, B. J.; Lacerda, S. H. D. P.; Becker, M. L.; Chun, J.; Migler, K. B.; Walker, A. R. H.; Hobbie, E. K. Length-Dependent Optical Effects in Single-Wall Carbon Nanotubes. J. Am. Chem. Soc. 2007, 129 (15), 10607–10612.

(3)

Tasis, D.; Tagmatarchis, N.; Bianco, A.; Prato, M. Chemistry of Carbon Nanotubes. Chem. Rev. 2006, 106 (3), 1105–1136.

(4)

Reich, S.; Thomsen, C.; Maultzsch, J. Carbon Nanotubes: Basic Concepts and Physical Properties; John Wiley & Sons: Weinheim, Germany, 2004.

(5)

Gu, L.; Luo, P. G.; Wang, H.; Meziani, M. J.; Lin, Y.; Veca, L. M.; Cao, L.; Lu, F.; Wang, X.; Quinn, R. A. Single-Walled Carbon Nanotube as a Unique Scaffold for the Multivalent Display of Sugars. Biomacromolecules 2008, 9 (9), 2408–2418.

(6)

Herrero, M. A.; Toma, F. M.; Al-Jamal, K. T.; Kostarelos, K.; Bianco, A.; Da Ros, T.; Bano, F.; Casalis, L.; Scoles, G.; Prato, M. Synthesis and Characterization of a Carbon Nanotube− Dendron Series for Efficient siRNA Delivery. J. Am. Chem. Soc. 2009, 131 (28), 9843–9848.

(7)

Giacalone, F.; Campisciano, V.; Calabrese, C.; Parola, L.; Syrgiannis, Z.; Prato, M.; Gruttadauria, M. Single-Walled Carbon Nanotube-Polyamidoamine Dendrimer Hybrids for Heterogeneous Catalysis. ACS Nano 2016, 10 (4), 4627–4636.

(8)

Liu, Z.; Sun, X.; Nakayama-ratchford, N.; Dai, H. Supramolecular Chemistry on WaterSoluble Carbon Nanotubes for Drug Loading and Delivery. ACS Nano 2007, 1 (1), 50–56.

(9)

Liu, P. Modification Strategies for Carbon Nanotubes as a Drug Delivery System. Ind. Eng. Chem. Res. 2013, 53 (28).

(10)

Wong, N.; Kam, S.; Dai, H.; V, S. U. Carbon Nanotubes as Intracellular Protein Transporters : Generality and Biological Functionality. J. Am. Chem. Soc. 2005, 6021– 6026.

(11)

Bianco, A.; Kostarelos, K.; Prato, M. Applications of Carbon Nanotubes in Drug Delivery. Curr. Opin. Chem. Biol. 2005, 9, 674–679.

(12)

Sheng, B.; Lee, S.; Jagusiak, A.; Panczyk, T.; Kiat, H.; Han, W.; Pastorin, G. Carbon Nanotubes for Delivery of Small Molecule Drugs. Adv. Drug. Deliv. Rev. 2015, 65 (15), 1964–2015.

(13)

Vasumathi, V.; Pramanik, D.; Sood, A. K.; Maiti, P. K. Structure of a Carbon Nanotube– dendrimer Composite. Soft Matter 2013, 9, 1372–1380.

(14)

Sun, J.; Hong, C.; Pan, C. Surface Modification of Carbon Nanotubes with Dendrimers or Hyperbranched. Polym. Chem. 2011, 2, 998–1007.

(15)

Chen, X.; Wu, X.; Zou, J.; Wan, J. A Convenient Route to Modified Multiwall Carbon Nanotubes with Liquid Crystal Molecules via Covalent Functionalization. J. Appl. Polym. Sci. 2012, 124 (4), 3399–3406.

(16)

Ogoshi, T.; Saito, T.; Yamagishi, T.; Nakamoto, Y. Solubilization of Single-Walled 43 ACS Paragon Plus Environment


Carbon Nanotubes by Entanglements between Them and Hyperbranched Phenolic Polymer. Carbon 2009, 47 (1), 117–123. (17)

Xu, L.; Ye, Z.; Cui, Q.; Gu, Z. Noncovalent Nonspecific Functionalization and Solubilization of Multi-Walled Carbon Nanotubes at High Concentrations with a Hyperbranched Polyethylene. Macromol. Chem. Phys. 2009, 210 (24), 2194–2202.

(18)

Ghosh, M.; Maiti, S.; Dutta, S.; Das, D.; Das, P. K. Covalently Functionalized SingleWalled Carbon Nanotubes at Reverse Micellar Interface: A Strategy to Improve Lipase Activity. Langmuir 2012, 28 (3), 1715–1724.

(19)

Naylor, A.; Goddard, W. A. Starburst Dendrimers. 5. Molecular Shape Control. J. Am. Chem. Soc. 1989, 111 (7786), 2339–2341.

(20)

Caminade, A.-M.; Majoral, J.-P. Dendrimers and Nanotubes: A Fruitful Association. Chem. Soc. Rev. 2010, 39 (6), 2034–2047.

(21) Astruc, D.; Boisselier, E.; Ornelas, C. Dendrimers Designed for Functions: From Physical, Photophysical, and Supramolecular Properties to Applications in Sensing, Catalysis, Molecular Electronics, Photonics, and Nanomedicine. Chem. Rev. 2010, 110 (4), 1857– 1959. (22)

Cells, M. S.; Santos, L.; Pandita, D.; Pe, A. P.; Granja, P. L.; Balian, G.; Toma, H. Receptor-Mediated Gene Delivery Using PAMAM Dendrimers Conjugated with Peptides Recognized by. Mol. Pharm. 2010, 7 (3), 763–774.

(23)

Fan, Y.; Su, F.; Li, K.; Ke, C.; Yan, Y. Carbon Nanotube Filled with Magnetic Iron Oxide and Modified with Polyamidoamine Dendrimers for Immobilizing Lipase toward Application in Biodiesel Production. Nat. Sci. Reports 2017, 2017 (March), 1–13.

(24)

Pan, B. F.; Cui, D. X.; Xu, P.; Huang, T.; Li, Q.; He, R.; Gao, F. Cellular Uptake Enhancement of Polyamidoamine Dendrimer Modified Single Walled Carbon Nanotubes. J. Biomed. Pharm. Eng. 2007, 1, 13–16.

(25)

Yang, K.; Qin, W.; Tang, H.; Tan, L.; Xie, Q.; Ma, M.; Zhang, Y.; Yao, S. Polyamidoamine Dendrimer-Functionalized Carbon Nanotubes-Mediated GFP Gene Transfection for HeLa Cells: Effects of Different Types of Carbon Nanotubes. J. Biomed. Mater. Res. A 2011, 99 (2), 231–239.

(26)

Herrero, M. A.; Guerra, J.; Myers, V. S.; Gomez, M. V.; Crooks, R. M.; Prato, M. Gold Dendrimer Encapsulated Nanoparticles as Labeling Agents for Multiwalled Carbon Nanotubes. ACS Nano 2010, 4 (2), 905–912.

(27)

Tang, L.; Zhu, Y.; Xu, L.; Yang, X.; Li, C. Amperometric Glutamate Biosensor Based on Self-Assembling Glutamate Dehydrogenase and Dendrimer-Encapsulated Platinum Nanoparticles onto Carbon Nanotubes. Talanta 2007, 73, 438–443.

(28)

Tang, L.; Zhu, Y.; Yang, X.; Li, C. An Enhanced Biosensor for Glutamate Based on SelfAssembled Carbon Nanotubes and Dendrimer-Encapsulated Platinum Nanobiocomposites-Doped Polypyrrole Film. Anal. Chim. Acta 2007, 597, 145–150.

(29)

Maiti, P. K.; Çaǧın, T.; Wang, Guofeng; Goddard, W. A. Structure of PAMAM Dendrimers : Generations 1 through 11. Macromolecules 2004, 37 (16), 6236–6254.

(30)

Pramanik, D.; Maiti, P. K. Dendrimer Assisted Dispersion of Carbon Nanotubes : A 44 ACS Paragon Plus Environment

Page 44 of 47

Page 45 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Molecular Dynamics Study. Soft Matter 2016, 12, 8512–8520. (31)

Pramanik, D.; Maiti, P. K. DNA Assisted Dispersion of Carbon Nanotubes and Comparison with Other Dispersing Agents. Appl. Mater. Interfaces 2017, 9 (40), 35287– 35296.

(32)

Liu, Y.; Chen, C.-Y.; Chen, H.-L.; Hong, K.; Shew, C.-Y.; Li, X.; Liu, L.; Melnichenko, Y. B.; Smith, G. S.; Herwig, K. W.; et al. Electrostatic Swelling and Conformational Variation Observed in High-Generation Polyelectrolyte Dendrimers. J. Phys. Chem. Lett. 2010, 1 (13), 2020–2024.

(33)

Liu, Y.; Bryantsev, V. S.; Diallo, M. S.; Goddard, W. A. PAMAM Dendrimers Undergo pH Responsive Conformational Changes without Swelling. J. Am. Chem. Soc. 2009, 131, 2798–2799.

(34)

Titov, A. V; Kral, P.; Pearson, R. Sandwiched Graphene - Membrane Superstructures. ACS Nano 2010, 4 (1), 229–234.

(35)

Baoukina, S.; Monticelli, L.; Tieleman, D. P. Interaction of Pristine and Functionalized Carbon Nanotubes with Lipid Membranes. J. Phys. Chem. B 2013, 117, 12113−12123.

(36)

Monticelli, L. On Atomistic and Coarse-Grained Models for C 60 Fullerene. J. Chem. Theory Comput. 2012, 8 (1), 1370–1378.

(37)

Marrink, S. J.; Risselada, H. J.; Yefimov, S.; Tieleman, D. P.; de Vries, A. H. The MARTINI Force Field: Coarse Grained Model for Biomolecular Simulations. J. Phys. Chem. B 2007, 111 (27), 7812–7824.

(38)

Tieleman, S. J. M. and D. P. Perspective on the Martini Model. Chem. Soc. Rev. 2013, 42 (16), 6801–6822.

(39)

Sajjad Kavyania, Mitra Dadvara, Hamid Modarressa, S. A.-I. A Coarse Grained Molecular Dynamics Simulation Study on the Structural Properties of CarbonnanotubeDendrimer. Soft Matter 2018, 14 (16), 3151–3163.

(40)

Jayamurugan, G.; Vasu, K. S.; Rajesh, Y. B. R. D.; Kumar, S.; Vasumathi, V.; Maiti, P. K.; Sood, A. K. Interaction of Single-Walled Carbon Nanotubes with Poly (Propyl Ether Imine) Dendrimers. J. Chem. Phys. 2012, 134, 104507.

(41)

Kavyani, S.; Amjad-iranagh, S.; Dadvar, M.; Modarress, H. Hybrid Dendrimers of PPI(core) − PAMAM(shell): A Molecular Dynamics Simulation Study. J. Phys. Chem. B 2016, 120, 9564–9575.

(42)

Kannaiyan, D.; Imae, T. pH-Dependent Encapsulation of Pyrene in PPI-core:PAMAMShell Dendrimers. Langmuir 2009, 25 (9), 5282–5285.

(43)

Van Der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. C. GROMACS: Fast, Flexible, and Free. J. Comput. Chem. 2005, 26 (16), 1701–1718.

(44)

Hess, B.; Kutzner, C. GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. J. Chem. Theory Comput. 2008, 4, 435–447.

(45)

Mark Abraham, Berk Hess, David van der Spoel, and E.; Lindahl. GROMACS User Manual; 2016.

(46)

Monticelli, L.; Kandasamy, S. K.; Periole, X.; Larson, R. G.; Tieleman, D. P.; Marrink, S. The MARTINI Coarse-Grained Force Field : Extension to Proteins. 2008, 819–834. 45 ACS Paragon Plus Environment


(47)

Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R. Molecular Dynamics with Coupling to an External Bath. J. Chem. Phys. 1984, 81 (8), 3684.

(48)

Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. A Smooth Particle Mesh Ewald Method. J. Chem. Phys. 1995, 103 (19), 8577.

(49)

Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual Molecular Dynamics. J. Mol. Graph. 1996, 14 (1), 33–38.

(50)

Lee, H.; Larson, R. G. Effects of PEGylation on the Size and Internal Structure of Dendrimers : Self-Penetration of Long PEG Chains into the Dendrimer Core. Macromolecules 2011, 44, 2291–2298.

(51)

Lee, H.; Choi, J. S.; Larson, R. G. Molecular Dynamics Studies of the Size and Internal Structure of the PAMAM Dendrimer Grafted with Arginine and Histidine. Macromolecules 2011, 44 (21), 8681–8686.

(52)

Goldstein, G.; Scheid, M.; Hammerling, U.; Boyse, E. A.; David, H.; Niall, H. D. Isolation of a Polypeptide That Has Lymphocyte-Differentiating Properties and Is Probably Represented Universally in Living Cells. Proc. Natl. Acad. Sci. U.S.A 1975, 72 (1), 11–15.

(53)

Elsasser, S.; Finley, D. Delivery of Ubiquitinated Substrates to Protein-Unfolding Machines. Nat. Cell Biol. 2005, 7 (8), 742–749.

(54)

Vitte, A.; Jalinot, P. Intracellular Delivery of Peptides via Association with Ubiquitin or SUMO-1 Coupled to Protein Transduction Domains. BMC Biotechnol. 2008, 11, 1–11.

(55)

Xu, Z.; Zhang, S.; Luan, B.; Zhou, R.; Li, J. Sequential Protein Unfolding through a Carbon Nanotube Pore. Nanoscale 2016, 8 (24), 12143–12151.

(56)

Firouzi, M. A. M.; Pishkenari, H. N.; Mahboobi, S. H.; Meghdari, A. Manipulation of Biomolecules : A Molecular Dynamics Study. Curr. Appl. Phys. 2014, 14 (9), 1216–1227.

(57)

Jong, D. H. De; Singh, G.; Bennett, W. F. D.; Arnarez, C.; Wassenaar, T. A.; Scha, L. V; Periole, X.; Tieleman, D. P.; Marrink, S. J. Improved Parameters for the Martini CoarseGrained Protein Force Field. J. Chem. Theory Comput. 2013, 9 (1), 687–697.

(58)

Vijay-kumar, S.; Bugg, C. E.; Cook, J. Structure of Ubiquitin Refined at 1.8 A Resolution. .I. Mol. Bid. 1987, 194, 531–544.

(59)

Margaret E. Baron. The Origins of Infinitesimal Calculus; 1969.

(60)

Graef, M. De; Mchenry, M. E. Structure of Materials: An Introduction to Crystallography, Diffraction and Symmetry; 2007.


Page 46 of 47

Page 47 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphical Abstract


Molecular Perspective Mechanism for Drug Loading on Carbon

Recommend Documents