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Computational Study of Superparamagnetic Nanocapsules Crossing the Blood-Brain Barrier: A Robotics Approach
Mustapha Hamdi and Antoine Ferreira

AbstractThis work proposes molecular dynamics simulation results that are used to derive guidelines for the design of the superparamagnetic nanocapsules (spherical-like or tubular-like) dedicated to cross the brain-blood barrier by magnetic forces. The computational studies focus mainly that on the physical processes occurring during the nanocapsule-targeting and endocytosis process in the endothelial barrier of brain capillaries. We focus our study on capillaries that have opened fenestrations when the integrity of the endothelial barrier is perturbed by tumors. We identied three different categories of governing parameters: geometrical (radius and length of nanocapsules); biophysical (ligand-to-receptor surface density ratio; non-specic interaction parameter) and biological (ligandreceptor binding afnity). It was demonstrated from MD simulations that the optimized size was for CNTs with dimensions greater than for 20 to 200 nm. Finally, the simulation results show that the superparamagnetic nanorobotic capsules were able to cross the endothelial barrier by using magnetic gradients forces compatible with medical constraints. Index TermsNanorobotics, robotics design, molecular dynamics, drug delivery system, magnetic steering.

Fig. 1. Concept of targeted therapy contributing to the local recruitment, via magnetic guidance, of functionalized superparamagnetic nanocapsules on the dysfunctional endothelium.

I. INTRODUCTION The major problem in drug delivery to the brain is the presence of the blood-brain barrier (BBB) which limits drug penetration even if in certain pathological situations the BBB is partly disrupted. Under normal conditions the BBB acts as a barrier to toxic agents due to the brain endothelial cells that form the vessels walls. It displays important morphological characteristics such as the presence of tight junctions between the cells, the absence of fenestrations that together restrict the passage of compounds from the blood into the brain. Nevertheless, several disorders and diseases can affect the brain leading to some loss in BBB integrity [3]. The diffusion of drugs from the blood into the brain depends mainly upon the ability of the biologically active to traverse the membrane. The use of magnetic nanocapsules (MNPs) to deliver drugs to the brain across the blood-brain barrier (BBB) may provide a signicant advantage to current strategies [15][18]. The chemotherapy magnetically controlled under MRI, is a novel technique that was proposed by Martel [12],[10]. This novel technique relies on magnetically propelling a therapeutic magnetic microcarrier through an MRI system. This therapeutic device can be coupled to or encapsulated within the specialized drug molecules. Moreover, the surface of these capsules is bio-conjugated with specic antibodies so that they are not detected by the immune system. The passage through the BBB
This work was supported by European Unions 7th Framework Program and its research area ICT-2007.3.6 Micro/nanosystems under the project NANOMA (Nano-Actuactors and Nano-Sensors for Medical Applications). M. Hamdi and A. Ferreira are with the Laboratoire PRISME, Ecole Nationale Sup rieure dIng nieurs de Bourges, 88 Bd Lahitolle, 18000, e e Bourges, France antoine.ferreira@ensi-bourges.fr

may be due to the recognition of these nanocapsules by the receptor at the surface of the endothelial cells of the BBB [11]. These new targeted drug delivery systems are mainly tested by trial and error. Since this is a very expensive approach, computational modeling methods are currently developed in order to reduce the risk and uncertainty inherent in the nanocapsule-development process [6][5]. This paper deals with the computational simulation of superparamagnetic nanocapsules crossing the blood-brain barrier via an hybrid approach: mechanical steering driven by magnetic gradients and chemical functionalization (Fig.1). The goal is to contribute to the local recruitment, via magnetic guidance, of magnetoliposomes on the dysfunctional endothelium of the BBB. This study extends previous studies on navigation modeling [2],[4] domain. The use of molecular dynamics (MD) models helps to identify the physical parameters that signicantly affect the efciency of the penetration process. This information will be used to determine design criteria - the size, charge and shape of various nanocapsules - that optimize their penetration into the BBB. The computational study is mainly based on current MD packages (NAMD2 software [13] with CHARMM27 force eld [16]) to simulate the interaction with the endothelial barrier. To improve the endocytosis process of the nanocapsules by the brain endothelial cells, we demonstrate computationally that the accurate control of magnetic gradient forces steers the nanocapsules in order to generate forces compatible with the endothelial barrier. The remainder of the paper is as follows. Section 2 presents the robotic design of different types of magnetic nanocapsules. Based on these designs, Section 3 and 4 present simulation

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Fig. 2. Biological functionalization of nanocapsules: (a) spherical nanocapsule composed of shell-cross-linked assemblies of amphiphilic block copolymers (b) spherical-shaped model, (c) template-synthesized nanotube coated with amino acids derivatives (NH3+ peptides) and (d) carbon nanotube-shaped model.

Fig. 3. Magnetic functionalization using superparamagnetic nanocapules (MNPs).(a) Biologically functionalized carbon nanotube encapsulating MNPs. Magnetic eld distribution for different non-symmetric (b) and symmetric (c) nanocapsules using Comsol multiphysics simulation.

results of the molecular recognition and mediated endocytosis of nanocapsules when interacting with endothelial cells. Then, simulation results are presented when considering natural and magnetically controlled endocytosis. Finally, Section 5 presents a discussion and conclusion. II. NANOCAPSULE ROBOTIC D ESIGN This section presents the robotic design of superparamagnetic nanocapsules functionalized with biological ligands. These ligands may be used as molecular Trojan horses to ferry large-molecule drugs, e.g. doxorubicin (DOX), across the BBB. A. Biological functionalization The transport of the nanocapsule through the BBB is mediated by specialized ligand-specic receptor systems coated on the nanoparticle surface, including the insulin receptor (IR) or the transferrin receptor (TfR), which are highly expressed on the capillary endothelium of brain. In order to probe the nanocapsule shape, size, physical and biological properties, various spherical and cylindrical nanocapsules coated externally with biological lipid-soluble peptides (Fig.2) have been tested. As molecular recognition peptides, we use PEGMA-b-AEMA diblock copolymers coatings. The peptides are covalently bound to the nanotube and/or nanoparticle via atomistic linkers, i.e. carbon-carbon (C-C) or carbonhydrogen (C-N) bonds, and computationally-devised amino acid linkers. To demonstrate the stability in water of the anchoring polypeptide/CNT interaction, we took into consideration the charge distributions on CNT in order to optimize the energy distribution. The peptide residues were parameterized through Quantum Dynamics (QD) simulation (Spartan software) in terms of bond length, dihedral angles and Coulomb

charges at around the equilibrium state. The presence of pHresponsive DEAEMA moieties in the HEGMAx-b-DEAEMAy micelles renders these polypeptides capable of responding to pH changes occurring in the surrounding environment. We modeled cleavable covalent bonds as a promising strategy for pH-dependent DOX release. For the PEGMA-b-AEMA diblock copolymers coatings, we used semi-empirical PM3 or Hartree-Fock molecular orbital models [17]. B. Magnetic functionalization The magnetic properties are provided by the encapsulation of superparamagnetic iron oxide nanoparticles. A general view of the magnetically and biologically functionalized microrobot is illustrated in Figure 3(a). Superparamagnetism is a special form of magnetism with characteristics from both ferromagnetism and paramagnetism. Two microrobot congurations were tested, e.g., cylindrical-type and sphericaltype. As shown in the nite element modeling simulations in (Fig.3)(b) and (Fig.3)(c), the magnetic signatures are orientation dependent. As expected the eld is large (red) at the front and back of the nanocapsule, and small (blue) on the top and the bottom of the cylinder. The effect of the magnetic eld is simulated through the modied velocity Verlet algorithm [1] coupled to NAMD2. In the rest of the paper, we use F e3 O4 nanoparticles which has magnetization equal to 1.83 MA/m. C. Drug encapsulation The presence of pH-responsive DEAEMA moieties in the HEGMAx-b-DEAEMAy micelles renders these systems capable of responding to pH changes occurring in the surrounding environment. In particular, the DEAEMA units are hydrophobic (non-ionizable) above a certain pH value (the pKa value of the DEAEMA) whereas below this value they become

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ionizable and hydrophilic. We modeled cleavable covalent bonds as a promising strategy for pH-dependent drug release (doxorobucin). Specic short peptide sequences have been developed that are enzymatically cleaved near the target. III. M OLECULAR R ECOGNITION BY THE E NDOTHELIAL BARRIER A. Preliminary steps The main program of the computational is NAMD2 [5], a fast, scalable program which implements the popular CHARMM27 force eld [6] for molecular dynamics. The absolute speed of NAMD2 is an essential ingredient of a responsive interactive system. It runs on a parallel cluster composed of 12 quadri-core Pentium IV-4.1GHz personal computers. All the simulations were carried out at 303.15 K and 1 atm . The van der Waals interactions were truncated at 15 , whereas the real part of the electrostatic interactions was truncated at 18.7 A, and a multiple time-step integrator scheme was used to optimize the simulations. The molecular species in each simulation were distributed as follows: The simulation of a f -CNT above a lipid bilayer was composed of the tube, 256 CG DMPC lipids, and 4,263 W sites (representing 12,789 water molecules). The simulations of CNT and f-CNT embedded in a bilayer consisted of 256 CG DMPC lipids, 2,192 W sites (representing 6,576 water molecules). All other simulations mentioned in the article are geometrical variations of these basic simulations. B. Adhesion forces analysis We validated by molecular dynamics simulation the surface binding afnity of PEGMA-b-AEMA diblock copolymers coating nanocapsules prototypes (spherical-shaped model and carbon nanotube-shaped model). More specically, we studied the SWCNT/FexOy/PEGMAx-b-AEMAy hybrid materials as binding receptors. Due to their large structure size, intensive calculations of energy minimization were required. That is why we restricted the study to the terminal group of the diblock copolymer, namely NH3 groups. A lipid bilayer model composed of 256 coarse-grained (CG) DMPC lipids is used in the following molecular dynamics simulations. In the CG model, each DMPC lipid molecule consists of 13 interaction sites, eight of which are hydrophobic (four for each alkanoyl tail) and ve of which are hydrophilic, three for the glycerol moiety and one each for the charged choline and phosphate. To further characterize the interaction of cylindrical CNT with a brain endothelial cell, we analyzed the forces balance in Fig.5 between specic interactions formed by the formation of molecular bonds (ligand/receptor) denoted by Fl/r and the non-specic interactions arising at the cell/particle interface denoted by short-range repulsive (van der Waals: Fvdw , steric: Fsteric ) and attractive (electrostatic: Fe ). The hydrodynamic force is denoted F . These forces are dened as follow: 1) van der Waals and electrostatic forces: When the nanocapsule and the wall are not in contact, they interact each other through van der Waals and electrostatic forces. These two interaction forces have different dominant regimes. In fact, when the capsule is close to the wall, Van der Waals force is (a)

Fig. 4. Lipid bilayer model composed of 256 coarse-grain (CG) DMPC lipids used in molecular dynamics simulations.

(b)
Fig. 5. Forces balance analysis: (a) Forces balance between the functionalised CNT and the cell surface. MD simulations of (b) non-specic energies (Fsteric +Fvdw -Fe ) and (c) specic energies (Fl/r ) when the nanocapsule is in close proximity of the biomembrane.

dominant. As the capsule move away from the wall, the Van der Waals force rapidly decrease and the electrostatic force becomes dominant. The van der Waals potential between the spherical capsule and the wall is given by [8]: Vvdw = Ah 6 1 1 H + + ln H 2+H 2+H n (1)

where Ah is the Hamaker constant and H = h/r is normalized separation h between the capsule and the wall. Then, the attractive Van der Waals interaction force is given by differenciating (1): Fvdw = Vvdw (2)

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Fig. 6. Snapshots of the molecular PEGMA-b-AEMA diblock copolymers and cell recognition of a FeYOy-decorated CNT (termed SWCNT/FexOy/PEGMAx-b-AEMAy) nanocapsule for computational time t = 0 ns to t = 8 ns.

The electrostatic force between the capsule and the wall considered as an uncharged surface is given by [7]: Fe = q2 n 4 0 (R + h)2 (3)

Fig. 7. 2-D displacement of the CNT position during cell binding. TEM images showing a multi-walled carbon nanotube crossing the cell membrane (in cooperation with Pr. K. Kostarelos from the School of Pharmacy, University of London).

with q the capsules charge, the relative permittivity of the medium in which the interaction occurs and 0 the vacuum permittivity. This force could be attractive or repulsive depending on the charge sign of the endothelial cells (negative) and of the capsule (negative or positive). In our study, the capsule is assumed to be charged negatively. Thus, the electrostatic force is a repulsive force. 2) Steric force: The therapeutic capsules, used for drug delivery, are generally functionalized via polymer chain grafting. The presence of these chains affects the interaction of the capsule with the endothelial glycocalyx consisting of a negatively charged, organized mesh of membranous glycoproteins, proteoglycans, glycosaminoglycans (GAGs) and associated plasma proteins. The repulsive steric force using the Derjaguin approximation can be expressed as: Fsr = RWsr = 0.36 Ref f (h/RG ) 2 kb T e RG (4)

Fig. 8. Root mean square values characterizing the adhesion of ligand/receptor for different values of N1 =5-to-14.

with Ref f the effective capsules radius including the contribution of the polymer chains, kb is the Boltzmann constant, T is the absolute temperature, h is the distance of the capsule from the endothelium and RG is the unperturbed radius of gyration. C. Simulation and Analysis To understand the molecular kinetics forces (Fsteric ,Fvdw ,Fe ) of molecular recognition, Fig.6 shows sequence of cell recognition of the nanocapsule in close proximity to the biomembrane surface. We reported the 3-D position of the nanocapsules termini during cellular binding in Fig.7. The nanocapsule is coated with specic recognition ligands (PEGMAx-b-AEMA micelles) that bounds to the cell surface in order to perform a site selective delivery. When the nanocapsule is in close proximity to the biomembrane surface (few tens of nanometer), the non-specic interactions are dominant. These forces are related to the balance between attractive and repulsive short range forces. It explains why

between t=2 ns and t=8 ns, the CNT rotates due to the strong specic interactions that are governed by the formation of molecular bonds among ligand peptides distributed over the particle surface and receptors expressed at the cell membrane. We tested the adhesion of the coated ligands connected to the outer walls of the CNT when it adheres rmly for various dislodging hydrodynamic forces (Fig.8). The internalization behavior can be signicantly affected also by the nonuniform distribution of ligand molecules on the particle surface. If sufciently large ligand densities are generated at rims with small curvature, the particle could be internalized regardless of its orientation with respect to the cell membrane. We functionalized the outer wall of the carbon nanotube with an uniform surface density of ligands m1 = (N1 / 2RL) with R, L and N1 representing the radius and the length of the CNT and the number of ligands. Figure 8 illustrates steric force for different surface coverage . It shows that the higher the coverage of the surface is, the higher the repulsion becomes. To estimate the adhesion properties of the molecular recognition for a given hydrodynamic force, we simulated a R=20nm and L=180 nm with the number of ligands ranging from N1 =5 to N1 =15. As shown in Fig.8, the root mean square (RMSD) values valid the assumption that the rm steady-state adhesion depends strongly on the density m1

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Fig. 9. f -CNT-based nanocapsule spontaneously adsorbs onto the membrane while purely hydrophobic CNT placed in the water subphase results in a slow insertion process.

of ligands. The hydrodynamic dislodging forces (F =50 pN) exerted on CNT adhering at the cell are optimal for values up to N1 =8. We will keep this surface density value of ligands in the future MD simulations. IV. NATURAL M ECHANISM OF R ECEPTOR -M EDIATED E NDOCYTOSIS A. Receptor-Mediated Endocytosis Once nanocapsule are adhered to the cell surface, it may be internalized into the cell through thermodynamic driving force. When considering amphiphilic carbon nanotubes, there is mounting evidence that f -CNT are capable of efcient cellular uptake by a mechanism that has not yet been clearly identied [9], [14]. However, the nature of the functional group at the CNT surface seems to play a determinant role in the mechanism of interaction with cells. Our MD simulations demonstrate that a generic CNT coated with PEGMAx-bAEMAy-diblock copolymers spontaneously wraps and inserts across a lipid bilayer. Dynamic simulation studies show clearly that when considering CNTs, the nanocapsule is not uptaken into the cell and stays at the surfaces bilayer with a random thermal uctuation (Fig.9(b)). On contrary, the f-CNT spontaneously adsorbs onto the membrane, and is gradually embedded by deforming the membrane (see Fig.9(a)). Partial immersion subsequently takes place with the f -CNT long axis remaining essentially parallel to the membrane plane and then tilt to be perpendicular to the lipid bilayer. This behavior have been identied experimentally by the authors in [9] where the f -CNT can migrate through lipid bilayers of plasma membrane via a similar mechanism. The mechanism of uptake of this type of f -CNT appears to be passive and endocytosis-independent. To explain this phenomenon, the electrostatic potential map (hydrophobic energy) are depicted during nanocapsule-lipid bilayer interaction (see Fig.10). The potentials are depicted by blue color (positive potential) and red color (negative potential). In Fig.10(a), the electrostatic potential map represents random distributed energies leading to small attractive forces. The interaction between f -CNTlipids are not strongly pronounced in order to demonstrate attraction. The hydrophobic energy presented by the PEGMAx-

Fig. 10. The electrostatic potential map during nanocapsule-lipid bilayer interaction: (a) CNT without functionalization and (b) CNT with functionalization.

b-AEMAy diblock copolymers (shown in Fig.10(b)) features a high attractive energy between f -CNT and lipids that facilitate the CNT nanopenetration into the cell membrane. B. Inuence of Size and Shape The objective of this study is to analyze the effect of nanocapsule shape and size on receptor-mediated endocytosis. We evaluated quantitatively through MD simulations how shape of nanocapsules impacted its uptake. To study the effect, we used the robotic designs of spherical-shaped and rodshaped carbon nanocapsules presented in Fig.2. We observed from these preliminary results that a large difference in the uptake of the different size and shape exists (see Fig.11). The efciency of cellular uptake is evaluated for a time-scale of 60ns: (5) = (dmax d60 ) 100% dmax where dmax and d60 represents the maximum penetration distance (in nm) of the nanocapsule at its nal state, and at the computation time T =60 ns, respectively. The MD simulations show that the uptake of rod-shaped nanocapsule (CNTs) is lower than their spherical counterpart without (Fig.11(a)) and with (Fig.11(b)) functionalization. We conrmed some design rules. The uptake efciency of the nanocapsule is independent of the shape and size when the nanocapsule is uncoated. The random distribution of thermodynamic driving force referring to the amount of free energy required to drive the cell is not sufcient even for larger computational time. The difference in the density of surface chemistries (from the stabilizing ligands coatings) between the spherical and rod-shaped structures may be one of the reasons for the

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Fig. 11. The effect of shape and size of the nanocapsules on cellular uptake. Comparison of uptake of rod-shaped nanoparticles (CNTs with aspect ration 1:5, 1:7 and 1:9) and spherical-shaped nanoparticles: (a)not functionalized CNT and(b)functionalized f-CNT.

Fig. 12. The variation of the wrapping time (ns) as a function of the aspect ratio G.

difference of uptake. The CNT possess a rod-shaped (1:3 1:5 1:7 and 1:9) hat induces a larger contact area with the cell membrane receptors than the spherical-shaped nanocapsule (1:1) when the longitudinal axis of the CNT interacts with the receptors. It explains why cellular uptake of rod-shaped structures with lower aspect-ratio (1:5) is greater than higher aspect ratio (1:9). It is suggested that spherical nanoparticles that were 55 nm would have the fastest wrapping time and the receptor-ligand interaction can produce enough free energy to drive the NPs into the cell (see Fig.12). It should be noticed that for larger computation times (100ns), higher aspect ratio (1:9) CNTs completed their internalization.

Fig. 13. Snapshots of the f -CNT/cell interaction mechanism simulations corresponding to the sub-states molecular. The f -CNT molecule spontaneously adsorbs onto the membrane. Partial immersion subsequently takes place with the f -CNT long axis remaining essentially parallel to the contant magnetic eld B0 . During this process a few lipids form salt bridges with the hydrophilic termini of the CNT. Next, random thermal uctuations drive one end of the f-CNT toward the core of the membrane. At this point, the long axis of the CNT aligns perpendicular to the bilayer plane and is steered by the magnetic gradient eld in z-direction.

C. Receptor-Mediated Endocytosis using Magnetic Gradients After minimizing the potential energy in its initial state, we conducted MD simulations with a magnetic eld of B0 =1.5 T directed along the z-axis direction. Note that B0 is sufcient to ensure a saturation magnetization Msat . The simulations are carried out using NAMD2 software based on the modied velocity Verlet algorithm [9]. The effect of the constant magnetic eld manifests itself in the structural change of the f -CNT orientation and the lipids chains unfolds into a linear form with a noticeable orientation parallel to the z-axis.

Fig. 14. The mean square displacement of the centre of mass of carbon nanotube in the absence (dashed line) and presence (solid line) of magnetic gradient force steering.

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To improve the endocytosis process, we applied a magnetic gradient force Fm along the z-axis to steer the superparamagnetic f -CNT. The following relation Fm = V ( M . ) B (6)
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was inserted in NAMD2 software. The parameters V , M and B are the nanocapsules total volume, the magnetization of the material and the magnetic eld gradient (uniform on the working simulation box) respectively. Our overall success rate was 80 percent for ten simulations ranging in simulation raging from 0 to 40 ns. When the insertion did occur, it could be rapid (around 30 ns after the bilayer formation). During the random starting point simulations, bilayer formation was rapid and reproducible (around 50ns), but correct insertion of the f -CNT did not always occurs within the simulation time-scales as shown in Fig.13. The f -CNT insertion into the DPPC bilayer did not cause signicant perturbations judging from the bilayer thickness which had an average value of 41A, compared to 40A for simulations of pure bilayer of identical lipids. The mean square displacement of the center of mass of carbon nanotube is shown in Fig.14. The difference between < | r(t)|2 > with and without magnetic gradient B is distinct. Without magnetic gradient, the plot of < | r(t)|2 > is somehow similar to the natural endocytosis process, the slope of < | r(t)|2 > is low hence the translational mobility of carbon nanotube within the lodgment is very weak. The value of < | r(t)|2 > and its slope spectacularly increases when the magnetic gradient is applied. The increase of < | r(t)|2 > reects a simple fact that the magnetic gradient force steers the f -CNT in the lipid bilayer. V. CONCLUSION Through molecular dynamics simulations we predicted the recognition, the adhesive and the endocytotic performances of functionalized hydrophobic robotic nanocapsules w.r.t different size and shape structures. Spherical-shaped (micellar nanoparticle) and rod-shaped (single wall carbon nanotube) have been simulated into a biomembrane. The mechanism involves a two-step process where the functionalized CNT is rst attracted by the cell membrane through molecular recognition ligands decorating on the nanocapsule then, it reorients to adopt a transmembrane conguration. We identied three different categories of governing parameters: geometrical (radius and length of nanocapsules); biophysical (ligand-to-receptor surface density ratio; non-specic interaction parameter) and biological (ligandreceptor binding afnity). Furthermore, the preliminary results on applying magnetic gradients demonstrates that the endocytosis process can be favorized offering the possibility to cross the blood-brain barrier. R EFERENCES
[1] M. Al-Haik and Y. Hussaini, Molecular dynamics simulation of magnetic eld induced orientation of nanotube-polymer composite, Japanese Journal of Applied Physics, vol. 45, p. 89848987, 2006. [2] L. Arcese, M. Fruchard, and A. Ferreira, Endovascular magneticallyguided robots : Navigation modeling and optimization, IEEETransactions on Biomedical Engineering, vol. 59, no. 4, pp. 236243, 2012.

Preprint submitted to 2012 IEEE/RSJ International Conference on Intelligent Robots and Systems. Received March 10, 2012.