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C: Physical Processes in Nanomaterials and Nanostructures

In Silico Study of Gold Nanoparticle Uptake into Mammalian Cell: Interplay of Size, Shape, Surface Charge, and Aggregation Thodsaphon Lunnoo, Jirawat Assawakhajornsak, and Theerapong Puangmali J. Phys. Chem. C, Just Accepted Manuscript • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 7, 2019

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The Journal of Physical Chemistry

In Silico Study of Gold Nanoparticle Uptake into Mammalian Cell: Interplay of Size, Shape, Surface Charge, and Aggregation Thodsaphon Lunnoo,† Jirawat Assawakhajornsak,‡ and Theerapong Puangmali∗,†,¶ †Materials Science and Nanotechnology Program, Department of Physics, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand ‡Department of Physics, King’s College London, Strand, London, WC2R 2LS, United Kingdom ¶Department of Physics, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand E-mail: [email protected]

Abstract

tionship between size, shape, surface charge, and aggregation of Au nanostructures provides a clear view on the design of Au nanostructures for developing new diagnostic strategies and drug delivery.

The study of interactions between Au nanostructures and living cells is a fundamental aspect that can be applied for the promising applications in nanomedicine. In the present work, we performed coarse-grained molecular dynamics (MD) simulations to observe the internalization pathways of Au nanostructures (nanosphere, nanocage, nanorod, nanoplate, and nanohexapod) into an idealized mammalian plasma membrane at an unprecedented level of complexity. Compared with the simple lipid bilayer model consisting of two lipid species, the different cellular uptake pathways of the AuNP were found. We highlight that the complexity of the lipid bilayer models plays an important role in the uptake pathway of nanoparticles (NPs). The permeability of aggregated AuNPs was much less than the NP counterpart. Spherical AuNPs showed pronounced size and surface charge dependence in their translocation through the plasma membrane. The translocation rates of different Au nanostructures were also evaluated and we found that Au nanohexapod exhibited highest cellular uptake. Understanding the interrela-

INTRODUCTION Gold nanoparticles (AuNPs) have unique optical properties and are of particular interest for medical applications, e.g., biological imaging and near-infrared (NIR) photothermal therapies as they exhibit a strong localized surface plasmon resonance (LSPR). 1 AuNPs, in addition, can also be utilized for gene and drug delivery 2 and for developing new diagnostic strategies. 3 However, cellular mechanisms of the internalization of AuNPs across cell membrane are still ambiguous. This gap of knowledge hinders the design of AuNPs for their use in the aforementioned applications. Considering that membrane penetration is a key step to the successful implementation of AuNPs, promising new studies to understand fundamental aspects of the interactions between AuNPs and biological membranes are of critical importance.

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The ability of AuNPs to reach intracellular compartments depends upon their morphology and surface modification. Different sizes of nanoparticles (NPs) can be internalized into the cell via different pathways. The NP size also affects the rates of cellular uptake. Small NPs can cross the cell membrane through direct translocation process. In contrast, larger NPs can enter cells via endocytic pathways. 4 For the endocytosis, previous works, including experimental, 5 theoretical, 6,7 and computational studies, 8,9 have shown that the endocytic pathway is size-dependent and there may exist the optimal and minimum wrapping radius (Rmin ) for successful endocytosis. Although there are a great deal of simulation works about the size effect, the minimum size for successful endocytosis is still far from clear. Thus, a thorough understanding of the size-dependent endocytosis could provide a potential technique to explore the design of NPs in drug delivery. In addition to gold nanospheres, nonspherically shaped Au nanostructures are also widely used in therapeutics and biomedical diagnostics. 1 They are capable of generating sufficient heat to raise the local temperature and thus kill cancer cells. A wide variety of Au nanostructures, e.g., nanocage, nanorod, nanoplate, and nanohexapod, have been demonstrated for photothermal therapy with NIR light. These Au nanostructures exhibit a high photothermal efficiency. On account of their asymmetrical geometries, they show unique cellular uptake. 10 Understanding the effect of shapes on the cellular internalization could therefore suggest potential candidates as photothermal transducers for various theranostic applications. Besides morphology, surface chemistry also affects the internalization of AuNPs. 11 The use of charged ligands is often a requirement for biomedical applications as they prevent aggregation in aqueous solutions. NPs with different surface charges can be internalized by different pathways, i.e., via clathrin- (cation NPs) or dynamic-dependent (zwitterionic NPs) endocytosis. The screening effects of the counterions surrounding the charged NP and the electrostatic interactions between the NP and the lipid headgroups affect the permeation rates

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of charged NPs through the plasma membrane. Understanding of the interrelationship between size, shape, and surface charge of Au nanostructures is therefore crucial. NP aggregation, a ubiquitous phenomenon, could also influence the internalization mechanism. 25 Even though the functionalised AuNPs do not aggregate in the short term, numerous experiments have demonstrated the aggregation of AuNPs both in vitro 26 and in vivo. 27 It has been theoretically 28,29 and experimentally 25 studied that there exists a critical local NP density to trigger cellular uptake, which can be achieved through clustering of small NPs. The internalization pathways of multiple AuNPs are therefore affected by a cooperative process. 30 Furthermore, in comparison with single and monodisperse AuNPs, it was found that there was a 25% decrease in uptake of aggregated AuNPs with Hela and A459 cells. 31 In effect, AuNP aggregates might behave as larger particles, and thus being internalized via endocytic pathways. Nevertheless, the effect of aggregation on the NP internalization is still unclear and no simulation study has been reported to date. Membrane complexity is another factor that could affect the cellular uptake. Most of the computational studies carried out so far on the interaction of AuNPs with membranes involved very simple membrane models in which less than three lipid species were taken into account, as shown in Table 1. As a matter of fact, plasma membranes typically contain thousands of different lipid components, 32–34 as well as carbohydrates and proteins. This certainly affects membrane elastic, dynamic, and structural properties. In mammalian membranes, cholesterol is the most abundant sterol. 35 It produces a striking impact on the structural properties of phospholipid membranes. 36 It also plays a non-negligible role in the formation of lipid rafts. Many studies have reported that cholesterol imposes remarkable influences on the permeability of small molecules, such as organic molecules, 37 water, 38 and ions. 39 Additionally, most biological membranes feature a highly asymmetric composition of the two leaflets, maintained by active transport of lipids


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Table 1: The interactions of NPs with different lipid bilayer models which have been studied and published in the literature. The numbers in the parentheses are the ratios of each lipid species in the inner and outer leaflets. Note that, the lipid models are composed of the following lipid types: palmitoyloleoylphosphatidylcholine (POPC), dipalmitoylphosphatidylcholine (DPPC), cholesterol (CHOL), dipalmitoylphosphatidylglycerole (DPPG), palmitoyloleoyl phosphoserine (POPS), distearoyl phosphatidyl choline (DSPC), distearoyl phosphatidyl glycerol (DSPG), ceramide (CER), and free fatty acids (FFAs). Lipid type 1

2

3

Nanoparticle

Inner leaflet

Outer leaflet

AuNP 12,13 AuNP 14–17 Nanodimond 18 Fullerene 19 AuNP 20 AuNP 14 AuNP 14 AuNP 21 AuNP 10 AuNP 22 AuNP 23,24

POPC DPPC POPC POPC DPPC:CHOL(7:3) DPPC:DPPG(9:1) DPPC:DPPG(4:1) POPC DSPC:DSPG(3:1) DPPC:DPPG(3:1) CER:CHOL:FFAs(1:1:1)

POPC DPPC POPC POPC DPPC:CHOL(7:3) DPPC:DPPG(9:1) DPPC POPC:POPS(4:1) DSPC:DSPG(3:1) DPPC:DPPG(3:1) CER:CHOL:FFAs(1:1:1)

and crucial for the cell survival. As the membrane asymmetry affects all membrane properties, it is reasonable to conceive whether it affects the interaction with NPs via an asymmetric distribution of charge species. Inclusion of such a complexity into the study of membraneAuNP interactions holds the promise of reducing the gap between simulation and experiments. Taking all the aforementioned aspects into consideration, we can summarize some of the questions and challenges posted by recent computational studies and experiments on AuNPmembrane interactions as follows: (i) Does the complexity of a plasma membrane model affect the uptake pathways of AuNPs?, (ii) What is the minimum size for endocytosis?, (iii) Does the aggregation of AuNPs affect the permeability?, (iv) How does the surface charge affect the cellular uptake?, and (v) Which shape is a potential candidate for theranostic applications? To the extent of our knowledge, these questions remain unanswered. To gain insights into the cellular uptake mechanism of AuNPs in the realistic plasma membrane, we carry out coarsegrained molecular dynamic (CGMD) simulations. Never before has any other work been

computed to study the interactions between AuNPs and plasma membrane at this level of complexity. The transition regime of cellular uptake from direct translocation to endocytosis is evaluated. This is of great significance for the design of drug carrier and for avoiding cytotoxicity. The effect of aggregation on the endocytic pathway will be taken into account. Last but not least, the interplay between surface charge density and morphology of AuNPs on the cellular uptake will be also elucidated. These results may offer profound information about the design of AuNPs and give some guidelines to their applications in the field of nanomedicine.

METHODS Martini Force Field The Martini force field 40 developed by Marrink and co-workers was used throughout the present work. The model is based on a four-toone mapping, i.e., a single interaction centre, which is typically called bead, represents four heavy atoms plus associated hydrogens. Each bead has a number of subtypes, which allow an optimum between computational efficiency


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and chemical representability. Four real water molecules are mapped to a coarse-grained (CG) water bead. Ions are represented by a single CG bead, which represents both the ions and its first hydration shell.

in Figure 1. The charged species including phosphatidylinositol (PI), PI-phosphate, bisphosphate, and -trisphosphate (PIPs), phosphatidic acid (PA) and phosphatidylserine (PS) are placed in the inner leaflet. The glycolipids (GM) is in the outer leaflet. The zwitterionic lipids including sphingomyelin (SM), phosphatidylethanolamine (PE), and phosphatidylcholines (PC) are present in both leaflets, with PC and SM primarily in the outer leaflet (70%) and PE in the inner leaflet (80%). A few of the more prominent minor species were also included: ceramide (CER), diacylglycerol (DAG), lysophosphatidylcholine (LPC), with all the LPC in the inner leaflet and CER, and DAG primarily in the outer leaflet (6065%). The plasma membrane model consists of 63 different lipid species created by combining the lipid headgroups with various fatty acid tails, depending on their relative prevalence. The cholesterol concentration was 30 mol% in the PM. The PM model used in the current work was obtained from the Martini portal, http://cgmartini.nl. Details on simulation setup of the PM can be found in the literature. 34 Our simulation box is composed of ∼ 7000 lipids, ∼700000 CG water beads, ∼3000 Na+ , and ∼2000 Cl− , totalling over 712000 par-

Coarse-Grained Model of AuNP Our CG AuNP model is compatible with the Martini force field. The nanocrystalline structure of the AuNP is characterized by the facecentred cubic (FCC) lattice structure. The Au nanospheres were obtained by cutting the crystal out of a bulk gold face-centred-cube lattice. Five different sizes of AuNPs were studied in the present work including 2, 4, 6, 8, and 10 nm in diameter corresponding to the number of Au atoms of 249, 1985, 6699, 15707, and 30887 atoms, respectively. Other Au nanostructures, including nanocage, nanorod, nanoplate, and nanohexapod, were generated by our inhouse code. To coarse-grain the AuNP model, the atomistic Au nanotructure models were mapped into CG beads in accordance with the Martini mapping scheme. The force field parameters of the CG AuNP model followed the work of Lin and co-workers, 17 which was calibrated with the experimental data reproducing several structural and dynamic properties of AuNP in experiments. 41 Gold atoms were mapped into CG bead by 1:1 mapping and rigidly fixed in the new CG model. They were assigned to C5 interaction sites in the Martini CG force field. The nonbond interaction parameters for AuNP are as follows: σ = 0.47 nm and ϵ = 3.5 kJ/mol.

Idealized Mammalian Membrane

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Plasma Figure 1: Plasma membrane lipid and cholesterol distribution in the inner and outer leaflet. Cholesterols are coloured yellow and lipid head groups are coloured by types (PC, pink; SM, green; PE, cyan; PS, lime; GM, orange; PIPs, red; others, blue). The cholesterol distribution is asymmetric; 54% in the outer vs 46% in the inner leaflet.

An idealized mammalian plasma membrane (PM) model at near-atomic detail has been developed by the group of Marrink. 34 All major lipid headgroups which are in mammalian PMs were included. Their molar ratios and distribution were also set to closely mimic an idealized mammalian PM. 32,42–45 The comparison of the lipid compositions between the inner and outer leaflets is shown


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ticles in a box of 40×40×50 nm3 . All PM models used in the current work were equilibrated for 40 µs.

ns with a step size of 10 fs. The pressure was controlled at 1 atm by employing the ParrinelloRahman scheme, while the system temperature was set at 310 K using a Nose-Hoover thermostat. Lennard-Jones and short-ranged Coulombic interaction were reduced to zero gradually in the range of 0-1.2 nm using cut-off potentials. The weighted histogram analysis method (WHAM) 48 was applied to a set of umbrella sampling simulations to compute the PMF. Cell internalization of Au nanostructures can be considered by the transition state theory (TST). 10,49 The barrier of each translocation was calculated by taking the difference between the maximum and minimum energy points in the PMF curve. The rate constants of internalization were approximated by using the following equation [ ] ∆G‡ (z) kB T exp − (1) k= h kB T

Simulation Setup Au nanostructures were typically placed ∼ 9 nm above the upper membrane. After the insertion of the Au nanostructures, counterions (Na+ and Cl− ) were added to the simulation box to ensure electroneutrality. Periodic boundary conditions were applied and the temperature of the system was set at 310 K for all cases. To set the system in an NPT ensemble, Berendsen pressure coupling and Berendsen temperature coupling were used. The relative dielectric constant (ϵr ) was 15 for explicit screening, which is the default value of the force field. 40 The long-range electrostatic interactions were calculated by the particle mesh ewald (PME) method. van de Waals and shortranged electrostatic interactions are cut off at 1.2 and 1.4 nm, respectively. To gain insights into the internalization of Au nanostructures, pull simulations were performed over a maximum distance of ∼ 25 nm in the z direction by applying a constant force of 1000 kJ mol−1 nm−2 to the centre of mass (COM) of the Au nanostructures to make them pass through the plasma membrane. All simulations were performed by the GROMACS 5.0.7 package. 46 All visualizations have been done with Visual Molecular Dynamics (VMD). 47

where T is the temperature, kB is the Boltzmann constant, h is Planck’s constant, and ∆G‡ (z) is the free energy activation along the translocation coordinate z. Considering the translocation to be a first-order reaction, the half-life (t 1 ) of the internalization is 2

0.693 . (2) 2 k The half-life is related to the characteristic lifetime of internalization τ where τ = k1 . t1 =

RESULTS AND DISCUSSION

Umbrella Sampling To observe the uptake pathways of Au nanostructures, pull simulations were performed over a maximum distance of ∼ 25 nm in the z direction. The Au nanostructure was pulled slowly at a rate of 0.0002 nm/ps with a constant force of 1000 kJ mol−1 nm−2 to make it pass through the lipid bilayer. 23,24 To calculate the potentials of mean force (PMFs), a set of the geometries along the pull simulations was saved and used as windows for the calculation by umbrella sampling technique. NPT equilibration was performed for each window followed by a molecular dynamics (MD) production run of 1

Effect of Membrane Complexity The effect of the membrane model complexity on the interactions between AuNPs and lipid bilayer was initially studied. The cellular uptake of AuNPs in a simple negatively charged lipid membrane was compared with the AuNP uptake in the plasma membrane. The simple model was modelled using a 3:1 ratio of DSPC (neutral) and DSPG (negatively charged) lipid molecules self-assembled in the centre of a sim-


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centre of the lipid bilayers is at z = 0. Simulations of a neutral AuNP (2 nm in diameter) across two different lipid bilayer models show a significant difference in the free-energy activation barrier. Considering the DSPC/DSPG model, a AuNP has three free-energy barriers at z = −2.46, −7.06, and −8.88 nm, corresponding to the barrier heights of 43.47 ± 4.70, 28.61 ± 5.50, and 25.42 ± 7.03 kJ/mol, respectively. The PMF curve has a global minimum energy (m1) at z = 3.60 nm. The minimum energy is in accordance with the translocation behaviour of the AuNP at t = 23.85 ns shown in Figure 2b. The global maximum of the free energy (m2) happened at t = 51.60 ns as the AuNP reached the entrance of the membrane hole. For the plasma membrane model, the shape of free energy profile is significantly different from that of the DSPC/DSPG model. There only exists one obvious free energy barrier of 260.85 ± 6.04 kJ/mol at z = −14.87 nm. The energy profile firstly decreases (M1) as the AuNP came from bulk water to the plasma membrane, then it increases as approaching bilayer centre. Similar to the DSPC/DSPG model, the highest barrier was also found at the position where the AuNP was escaping from the outer leaflet (see Figure 2c at t = 39 ns). The barrier for the AuNP to translocate through a plasma membrane is much higher than that of the DSPC/DSPG model. It is worth noting that the symmetric PMF profiles are not found in the size range of AuNPs (2-10 nm) studied in the present work. We suggest that this is due to the deformation of the membrane. Smaller AuNP (d < 2 nm) can diffuse through the membrane by a simple diffusion without membrane deformation; thus, the symmetry of the PMF profile will be observed. Due to the wettability of the AuNP, it preferred to be located in the hydrophilic region of the DSPC/DSPG bilayer. Consequently, the local minima were observed at t = 23.85 (m1) and 52.30 ns (m3), as illustrated in Figure 2a. As expected, the maximum free energy barrier happened at the centre of DSPC/DSPG bilayer which is attributed to the transfer of weakly hydrophilic AuNP from the water to the hydrophobic region of bilayer. In contrast, the

Figure 2: (a) The PMFs of AuNP (2 nm in diameter) in two lipid bilayer models including DSPC/DSPG model (left), and plasma membrane model (right). The horizontal axis represents the distance between the COM of the AuNP and the centre of the bilayer in z direction. z = 0 is placed at the COM of the lipid bilayer at the equilibrium state (t = 0 ns). The errors of the calculated PMFs are represented by the transparent colours. (b) and (c) show representative snapshots of AuNPs, at the local maximum or minimum of PMFs, translocating through DSPC/DSPG model and plasma membrane model, respectively. For clarity, a cut through the membrane at the particle position is depicted and water molecules in both outer and inner cell are not shown.

ulation box. The plasma membrane model in the present work consists of 63 different lipid species, 34 combining 14 types of headgroups and 11 types of tails which are asymmetrically distributed across the two leaflets, closely mimicking an idealized plasma membrane. The free energy profiles of AuNPs translocating through two different lipid bilayer models are illustrated in Figure 2 a. The umbrella sampling windows of both models were densely distributed along the reaction coordinate (z) and well-overlapped histograms have been obtained, as shown in Figure S1 and S2 in the Supporting Information (SI). The free energy is set to be relative to the global minimum energy. The


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The Journal of Physical Chemistry 10 nm 0 ns

82 ns

100 ns

118 ns

122 ns

130 ns

0 ns

20 ns

30 ns

50 ns

60 ns

150 ns

0 ns

90 ns

130 ns

132 ns

137 ns

145 ns

(a)

(b)

(c)

Figure 3: Snapshots of internalizations of a neutral 10-nm-in-diameter AuNP, shown in yellow, in (a) the DSPC/DSPG bilayer, and (b) the plasma membrane. (c) The uptake of a positively charged AuNP with the surface charge density (σ = +0.4). Water molecules were removed for the purpose of clarity. Cholesterols are coloured yellow and lipid head groups are coloured by types (PC, pink; SM, green; PE, cyan; PS, lime; GM, orange; PIPs, red; others, blue).

PMF curve of the PM bilayer was different from that of the cholesterol-free model. This is attributed to the insertion of cholesterol in both inner and outer leaflets. Thus, the local minimum was not observed and the free energy barrier occurred once the AuNP was at the hydrophobic region of the PM bilayer. Recent study by Ingólfsson and co-workers suggests that the complex changes in composition of plasma membrane significantly affect overall bilayer properties, dynamics, and lipid organization of cellular membrane. 50 The underlying changes in bilayer properties are due primarily to the increased cholesterol concentration, which directly affects the membrane packing (the so-called condensing effect) and

the ordering of lipid hydrocarbon chains (the so-called ordering effect). 36,51–53 In addition, cholesterol also increases the thickness, and rigidity of the bilayers, preventing pore formation and restricting bilayer deformations. 54 We therefore conclude that the increase in the freeenergy activation barrier is due to the inclusion of cholesterols to a fluid phase bilayer, decreasing the permeability of the AuNP. According to the PMF curves of both lipid bilayer models, the AuNP uptake by cells is viewed as a sequence of two steps. The first step is the binding of NPs onto the cell surface while the PMF curves show the minimum values (m1 and M1 for DSPC/DSPG and plasma membrane models, respectively). The following step is the in-


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ternalization of NPs. At this stage, the PMF curves show the global maximum while NPs are escaping the lipid bilayers (see m2 and M3 in Figure 2). In addition to a huge difference in the freeenergy barrier, the complexity of the lipid bilayer also affects the uptake pathways of NPs. Membrane permeation can happen via direct translocation by simply diffusing across the lipid bilayer, or via endocytosis, in which the NP is surrounded by an area of plasma membrane, which then buds off inside the cell to form a membrane-bound vesicle. Small NPs (d < 10 nm) are likely to be internalized via direct translocation, while NPs cannot be endocytosed provided that their diameter is smaller than 10 nm. 4,55 We performed steered MD simulations to clarify which mechanisms drive the uptake process of sub-10 nm AuNPs. To examine the effects of model complexity on the cellular uptake pathway, the permeations of a 10nm-in-diameter AuNP in both DSPC/DSPG and plasma membrane models were compared, as illustrated in Figure 3. For the DSPC/DSPG model, a AuNP was placed above the outer leaflet of the membrane (Figure 3a). After the contact with the AuNP, the lower leaflet of the bilayer protruded downward to accommodate the penetration. Subsequently, the membrane started to bend around the AuNP until t = 118 ns when the bent membrane was in close proximity to the flat region separated only by a small neck. After that, a defect in the neck was created (t = 122 ns) resulting in the separation of the bent membrane from the flat one. Shortly after that, the membrane rupture was healed and the two membranes started to separate. The final snapshot at t = 130 ns depicts a fully internalized AuNP that is passivated by a small vesicle or endosome. The lipid composition of the vesicle (the ratio of DSPC:DSPG) is slightly different from that of the lipid bilayer (DSPC:DSPG=2.987:1). In contrast to the endocytic pathway of the AuNP in the DSPC/DSPG model, the direct translocation of a 10-nm-in-diameter AuNP was found in the plasma membrane, as shown in Figure 3b. The AuNP disrupted the bilayer packing and subsequently induced undu-

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lation in the bilayer. It also created vacancies inside the bilayer by pushing the contacting lipids towards the centre of the bilayer. Embedding of the AuNP into the bilayer also forced lipids and CHOL to reorganize. The AuNP was mostly surrounded by the CHOL molecules, as shown in Figure S3. Compared with the DSPC/DSPG model, the full wrapping of the AuNP cannot be observed in the plasma membrane model. The mechanism of the spontaneous encapsulation can be understood in terms of Helfrich theory of membrane elasticity. 56 A lower limit of the NP radius 55 below which √ the NP cannot be endocytosed 2kc /w, where kc is the bending is Rmin = rigidity of the membrane associated with the mean curvature and w is the attractive adhesion energy per unit area between the nanoparticle and the membrane. CHOL molecules play a key role in modulating the rigidity of cell membranes and controlling intracellular transport and signal transduction. 35,54,57,58 Thus, the bending rigidity of the PM model is more than that of the DSPC/DSPG model. Besides, the cellular uptakes of sub-10 nm AuNPs in the PM were also perforemed. As illustrated in Figure S4, each AuNP (2-8 nm) disrupted the plasma membrane and entered the interior of the bilayer via direct translocation process. Therefore, the minimum size (Rmin ) of a neutral AuNP that can be endocytosed in the PM membrane is larger than that of the CHOL-free bilayer model. To sum up, the complexity of the lipid bilayer significantly affects the internalization pathway of AuNPs. Besides the neutral AuNP, we also observed an internalisation of a positively charged AuNP (10 nm in diameter) into the plasma membrane. As illustrated in Figure 3c, the nanoparticle can be endocytosed spontaneously into the membrane and was coated by a small vesicle or endosome. However, the endocytosis of positively charged AuNPs smaller than 10 nm was not found, which is in accordance with the theoretical prediction. 55 This happens when the surface charge of the nanoparticle is positive and the strength of the electrostatic interaction between the positively charged AuNP and the negatively charged membrane are above a certain thresh-


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Figure 4: PMFs of permeation and representative snapshots of the MD trajectory of the penetration of (a) a 4-nm-in-diameter AuNP and (b) agglomerated AuNPs with an approximate size of 4 nm in diameter. The agglomerated AuNP is composed of five 2-nm-in-diameter AuNPs. Water molecules were removed for the purpose of clarity.

old. We conclude that in addition to the interaction between receptors and ligands on the surface of AuNPs, 56 the thermodynamic driving force for the endocytosis is also provided by the electrostatic interactions between nanoparticles and the membrane.

uptake and toxicity is related to the aggregation/agglomeration of nanomaterials. This matter, however, has been scarcely dealt with at the computational level, mainly due to the obvious difficulties involved in sampling the large length and time scales required. Here we evaluate the effect of aggregation on uptake kinetics of AuNPs in the idealized plasma membrane. To examine the effects of aggregation on the cellular uptake of AuNPs, the free-energy profile of a single 4-nm-in-diameter AuNP was compared with that of aggregated AuNPs, which comprised five 2-nm-in-diameter AuNPs. The aggregated AuNPs have a hydrodynamic size of ∼ 4 nm. Five neutral AuNPs were equilibrated in the water for 50 ns. This caused the van der Waals force (EvdW ) to drive a formation of aggregation. Then, pull simulations were performed over distances of 20 and 25 nm for single AuNP and aggregated AuNPs, respectively, by applying a constant force of 1000 kJ mol−1 nm−2 to the COM of the NPs to make them through the plasma membrane. The PMF curves and representative snapshots at the pronounced positions of single and aggregated AuNPs are shown in Figure 4. The free-energy activation barriers of the single and aggregated nanoparticles are 264.05±4.59 (at

Aggregation Effect When working with nanoparticles, it is always vital to quantify the size, shape, morphology, uniformity, and dispersity of the particles before subjecting them for various purposes. They may, however, aggregate in cell culture media due to exposure to proteins or ions which may cause unexpected results. The aggregation of NPs occurs once the van der Waals attractive forces between nanoparticles are greater than the electrostatic repulsive forces produced by the surface modification. High ion concentration in biological media can screen the repulsive force produced by the charged chemical groups on the nanoparticle surface. Apart from this, the high protein content can also cause a thermodynamically favoured replacement of surface-associated molecules with serum protein. These can presumably impede the targeting efficiency of nanoparticles to cells and affect the experiment reproducibility in biological applications. Furthermore, the degree of


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Table 2: Calculated barrier heights (kJ/mol) and rate constant of internalization (ki ) for spherical AuNPs with varying surface charge density σ (units/bead). Shape Nanosphere 2 nm

4 nm

Nanocage Nanorod Nanoplate Nanohexapod

σ

Barrier (kJ/mol)

−0.4 0.0 +0.4 −0.4 0.0 +0.4 0.0 0.0 0.0 0.0

213.28±4.57 260.85±6.04 48.62±4.98 699.25±5.30 264.05±4.59 231.52±2.88 443.97±40.34 310.56±8.91 321.16±5.81 80.25±5.75

z = −16.42 nm) and 709.07±3.12 (at z = −24.61 nm) kJ/mol, respectively. The free energy barrier, which is related to the permeability of AuNPs, of the aggregated AuNPs is much higher than that of the single particle. This is due to the interparticle interactions among AuNPs during translocation resulting in a decrease in permeability. The representative snapshots of the translocation process at certain positions are also shown in Figure 4. It is worth noticing that the barriers were found at the positions where AuNPs were escaping the plasma membrane (S3 and A4 in Figure 4a and 4b, respectively). Besides, we also performed simulations for larger aggregations. The aggregations of 10, 15 and 20 particles were also observed. The cellular uptake of these aggregates were “semi-endocytosis”, as can be seen in Figure S5-S7. They cannot be fully wrapped and a vesicle cannot be formed as the hydrodynamic sizes of the aggregates are probably smaller than Rmin . It is expected that the endosome could be formed provided that the effective size of aggregated particles is larger than 10 nm. Recently, Alexandre and Warren 31 developed a simple technique to produce transferin-coated AuNP aggregates of different sizes and characterized their uptake and toxicity in cell lines. While the aggregation did not elicit a unique toxic response, there was a 25% decrease in uptake of aggregated nanoparticles with Hela and

ki (s−1 )

t1 2 (s)

4.5×10−25 2.3×10−33 2.1×104 1.0×10−109 6.4×10−34 3.0×10−28 2.9×10−65 5.1×10−42 7.2×10−44 6.6×10−2

1.6×1024 3.0×1032 3.3×10−5 6.9×10108 1.1×1033 2.3×1027 2.4×1064 1.4×1041 9.6×1042 10.5

A549 cells in comparison to single and monodisperse nanoparticles. This is in good agreement with our simulations in which the aggregated AuNPs show much higher free-energy barrier than the single AuNP. It was recently reported by Wang and coworkers 59 that the introduction of a partner NP would significantly trigger the transmembrane penetration of a host spherical NP owing to the membrane-mediated cooperation between the NPs. A proper NP partner with a specific surface chemistry would greatly facilitate the NP permeability. For a host hydrophilic NP, a Janus-like NP would be most helpful. In contrast to the aforementioned work, partner NPs in our simulation are weakly hydrophilic NPs; therefore, cooperative penetration does not play an important role during the translocation process. The increased energy barrier associated with the transmembrane translocation of aggregated AuNPs originates from the interactions between AuNPs and the lipid tails.

Surface Charge Effect To examine the effects of surface charge on the permeability of AuNPs through the plasma membrane, we performed systematic simulations of 2- and 4-nm-in-diameter AuNPs featuring neutral, anionic, and cationic surface charge. The AuNP surface beads were given a Q0 particle type with a fractional neg-


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ative/positive charge per bead (σ) of ±0.4 units/bead, and the core was kept neutral as a C1 particle. The PMF curves of 2- and 4-nmin-diameter AuNPs with σ = −0.4, 0 and +0.4 are shown in Figure S8. The calculated barrier heights of these particles are summarized in Table 2. The cellular internalization of these AuNPs was quantified, providing a parametric evaluation of charge and size effects. Simulations of AuNPs with negatively charged surface (σ = −0.4) showed higher free-energy barrier than the positively charged AuNPs (σ = +0.4), indicating no preference for translocation across the lipid bilayer. Negatively charged AuNPs have high energy barriers in the PMF profiles because they were electrostatically repelled by the negatively charged lipid bilayer. For all cases, free-energy barriers increased with increasing AuNP size, which indicates the size-dependent internalization. Similar trends have been reported experimentally where negatively charged AuNPs showed low membrane adhesion and low translocation compared to their neutral and positively charged counterparts. Simulations also indicate the importance of AuNP adhesion to the cell membrane, which for negatively charged AuNPs is energetically less favourable. Our results are consistent with experimental observations of cellular uptake of sub-10 nm AuNPs. 11 As shown in Table 2, the translocation halflife, calculated by Eq. 2, provides a very useful estimate of the translocation efficacy. Spherical AuNPs (4 nm) show pronounced charge dependence in their translocations through the plasma membrane. Negatively charged AuNP interacts weakly with the cell membrane and has substantially long half-life compared with neutral and positively charged particles. The half-lifes of positively charged AuNPs (σ = +0.4) with d = 2 and 4 nm are 3.3 × 10−5 (instantaneous translocation) and 2.3 × 1027 s compared to t 1 = 1.6 × 1024 (d = 2 nm) and 2 6.9 × 10108 s (d = 4 nm) for negatively charged NPs. The half-life of AuNPs (in logarithmic scale) as a function of the surface charge density for AuNPs with 2 and 4 nm in diameter is shown in Figure 5. This demonstrates that size and surface charge interact in an interrelated

Figure 5: Half-life of nanoparticles (logarithmic scale) as a function of the surface charge densities (σ = +0.4, 0, and -0.4) for AuNPs with 2 and 4 nm in diameter.

fashion to modulate AuNP uptake into cells.

Shape Effect To study the effects of shapes on the internalization pathways of AuNPs, we have performed simulations for nanocage, nanorod, nanoplate, and nanohexapod. The effective size of each shape was chosen to be roughly equal to 4 nm. The COM of these AuNPs were initially placed at ∼ 10 nm above the plasma membrane. The representative snapshots of the cellular uptake pathways of each shape are shown in Figure 6. All Au nanostructures are internalized via direct translocation, which is a similar pathway to a spherical AuNP with the same size. It is worth noticing that all nanostructures reoriented themselves and used sharp tips/edges to disrupt the membrane before escaping into the inner cell. While escaping the inner leaflet, the highest free-energy barriers were observed. For example, as shown in Figure 6b, the nanorod had an orientation change during the uptake. Although the initial orientation of the nanorod’s main axis was parallel to the plasma membrane, it rotated during the uptake (as can be seen at t=21, 65, and 67 ns) and used a sharp end to disrupt and exit the plasma membrane. The nanorod


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Figure 6: Snapshots of different shapes of AuNP approaching the plasma membrane and undergoing reorientation during translocation. (a) nanocage, (b) nanorod, (c) nanoplate, and (d) nanohexapod, each with the longest characteristic length of 2 nm. Water molecules in both outer and inner cell are not shown for clarity.

was internalized with a nearly 90◦ entry angle. The uptake of nanorod into cells proceeds through a laying-down-then-standing-up sequence, consistent with recent CGMD simulations 60 wherein the membrane model consists of coarse-grained lipid agents stabilized in a twodimensional (2D) fluid surface in 3D space. The rotation of ellipsoidal nanoparticles during the translocation process was also reported. 61 It is worth noting that the initial orientation of particles plays a significant role in their rotation. Similarly, the same behaviour can be observed in nanocage, nanoplate, and nanohexapod, as one can see in Figure 6a, c, and d, respectively. It is thus expected that nanohexapods could be more effective in cellular uptake compared to other shapes, owing to the presence of six vertices. To assess the internalization of these nanotructures, we have evaluated the permeabil-

ity of Au nanostructures by comparing their translocation energy barriers. The barrier heights and half-lifes of Au nanostructures are summarized in Table 2. Our comparison studies of Au nanocage, nanorod, nanoplate, and nanohexapod indicate that Au nanohexapod exhibits the lowest free-energy barrier (∆G = 80.25 ± 5.75 kJ/mol), as illustrated in Figure 7. The free-energy barriers of nanocage, nanorod, and nanoplate are 443.97±40.34, 310.56 ±8.91, and 321.16 ± 5.81 kJ/mol, respectively. This indicates that Au nanohexapod shows higher cellular uptake than other shapes. Owing to the presence of sharp tips, Au nanohexapod could be more effective in cell uptake relative to those with smooth surfaces. These results are in good agreement with the experimental observations. Yucai and co-workers 1 assessed the potential use of Au nanohexpods as photothermal transducers by benchmarking against Au


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The Journal of Physical Chemistry (a) Nanocage

ever, a small increase was observed for neutrally charged particles. Mechanistic studies provide insight into the cellular uptake pathway of sub10 nm AuNPs. We found that direct translocation (passive diffusion) appears to be the predominant mechanism, as shown in Figure S4.

(b) Nanorod

ΔG

ΔG

(c) Nanoplate

ΔG

(d) Nanohexapod

CONCLUSIONS

ΔG

The study of interactions between Au nanostructures and mammalian plasma membrane, at an unprecedented level of complexity, was performed by coarse-grained molecular dynamics simulations to observe the effects of morphology and surface chemistry on the internalization pathways of Au nanostructures. The shapes of Au nanostructures that were studied in the present work are nanosphere, nanocage, nanorod, nanoplate, and nanohexapod. Different cellular uptake pathways of the AuNP were found in different lipid bilayer models. For a neutral 10-nm-in-diameter AuNP, it is internalized via endocytosis and direct translocation into the DSPC/DSPG bilayer and the plasma membrane models, respectively. We therefore highlight that the complexity of the lipid bilayer models plays a significant role in the uptake pathway of nanoparticles. The aggregation of AuNPs before cell uptake also affects the permeability. There was a decrease in uptake of aggregated nanoparticles due to the interparticle interactions. Shapes of Au nanostructures also affect the translocation rate and we found that nanohexapod shows higher permeability than other shapes and it is suitable for photothermal transducers. Our study demonstrates the interrelationship between the morphology and surface modification of Au nanomaterials that modulate nanoparticle uptake into cell, and provides a computational microscopy for designing nanostructures for specific biological applications.

Figure 7: The PMFs of (a) nanocage, (b) nanorod, (c) nanoplate and (d) nanohexapod. The horizontal axis represents the distance between the COMs of AuNP and bilayer in z direction.

nanorods and nanocages. They found that Au nanohexapods exhibited higher cell uptake, and lower cell cytotoxicity. Our simulations suggest that nanohexapods are potential candidates as photothermal transducers for various theranostic applications. By controlling the length of the arms, the LSPR peaks of the Au nanohexapods could be easily tuned from the visible to NIR region. 62

Size Effect Exploring the cellular uptake of AuNPs in plasma membrane along a single structural parametric axis, i.e., surface charge, aggregation, and shape, is discussed in the previous sections. However, interplay between structural factors is expected, e.g., size and surface charge of AuNPs. Based upon the simulation results presented in the aforementioned sections, we can correlate the cellular internalization efficiency of sub-10 nm AuNPs and observe a cellular uptake trend. As shown in Table 2, increasing particle sizes resulted in increasing translocation barrier height, which reflected the decreased cellular uptake. The increase of barrier height was obviously observed in negatively charged AuNPs in which the barrier was increased from 213.28 to 699.25 kJ/mol once the particle size was increased from 2 to 4 nm. How-


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ASSOCIATED CONTENT

Page 14 of 26

Drug Molecules. Adv. Drug Delivery Rev. 2013, 65, 663 – 676.

Supporting Information

(3) Mayer, K. M.; Hafner, J. H. Localized Surface Plasmon Resonance Sensors. Chem. Rev. 2011, 111, 3828–3857.

The Supporting Information is available free of charge on the ACS Publications website at DOI: xx.xxx/acs.jpcc.xxxxxx. Histograms, Cholesterol effect, Size effect, Aggregation effect, Potentials of mean force

(4) Rossi, G.; Monticelli, L. Gold Nanoparticles in Model Biological Membranes: A Computational Perspective. Biochim. Biophys. Acta 2016, 1858, 2380 – 2389.

AUTHOR INFORMATION

(5) Huang, K.; Ma, H.; Liu, J.; Huo, S.; Kumar, A.; Wei, T.; Zhang, X.; Jin, S.; Gan, Y.; Wang, P. C.; He, S.; Zhang, X.; Liang, X.-J. Size-Dependent Localization and Penetration of Ultrasmall Gold Nanoparticles in Cancer Cells, Multicellular Spheroids, and Tumors in Vivo. ACS Nano 2012, 6, 4483–4493.

Corresponding Author *E-mail: [email protected].

ORCID Thodsaphon Lunnoo: 0000-0002-3000-3486 Theerapong Puangmali: 0000-0003-2529-7528

(6) Deserno, M. Elastic Deformation of a Fluid Membrane upon Colloid Binding. Phys. Rev. E 2004, 69, 031903.

Notes The authors declare no competing financial interest.

(7) Li, X. Size and Shape Effects on ReceptorMediated Endocytosis of Nanoparticles. J. Appl. Phys. 2012, 111, 024702.

Acknowledgement This work was financially supported by The Thailand Research Fund (TRG5880016). We express our appreciation to the Bureau of Information Technology, Khon Kaen University, for the computational resource. T.L. is grateful to the Graduate School of Khon Kaen University for financial support.

(8) Ginzburg, V. V.; ; Balijepalli, S. Modeling the Thermodynamics of the Interaction of Nanoparticles with Cell Membranes. Nano Lett. 2007, 7, 3716–3722. (9) Ding, H.; Tian, W.; Ma, Y. Designing Nanoparticle Translocation through Membranes by Computer Simulations. ACS Nano 2012, 6, 1230–1238.

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Effect of size, shape, surface charge, and aggregation on the cellular uptake into mamalian cell of gold nanostructures. 82x44mm (299 x 299 DPI)


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(a) Nanocage 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

(b) Nanorod

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