Tension-Induced Translocation of Ultra-Short Carbon Nanotube

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Tension-Induced Translocation of Ultra-Short Carbon Nanotube through a Phospholipid Bilayer Yachong Guo, Marco Werner, Ralf Seemann, Vladimir A Baulin, and Jean-Baptiste Fleury ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b04657 • Publication Date (Web): 19 Nov 2018 Downloaded from http://pubs.acs.org on November 20, 2018

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Tension-Induced Translocation of Ultra-Short Carbon Nanotube through a Phospholipid Bilayer Yachong Guo,†,‡ Marco Werner,¶ Ralf Seemann,§ Vladimir A. Baulin,∗,‡ and Jean Baptiste Fleury∗,§ †National Laboratory of Solid State Microstructure, Department of Physics, Nanjing University, Nanjing 210093, China ‡Departament d’Enginyeria Quimica, Universitat Rovira i Virgili 26 Av. dels Paisos Catalans, 43007 Tarragona, Spain ¶Departament d’Enginyeria Quimica, Universitat Rovira i Virgili 26 Av. dels Pa¨ısos Catalans, 43007 Tarragona, Spain §Universitat des Saarlandes, Experimental Physics and Center for Biophysics, 66123 Saarbruecken, Germany E-mail: [email protected]; [email protected]

Abstract Increasing awareness of bioeffects and toxicity of nanomaterials interacting with cells puts in focus the mechanisms by which nanomaterials can cross lipid membranes. Apart from well-discussed energy-dependent endocytosis for large objects and passive diffusion through membranes by solute molecules, there can exist other translocation mechanisms based on physical principles. We show the importance of membrane tension on the translocation through lipid bilayers of ultra-short carbon nanotubes (USCNTs). By using a combination of a microfluidic setup and self-consistent mean field theory we observed that under membrane tension, USCNT inserted into a lipid bilayer

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may spontaneously nucleate an unstable local pore, allowing it to escape from the bilayer. We demonstrated that stretching of the membrane is essential for triggering this mechanism of translocation, and no translocation is observed at low membrane tension. For this purpose, a quantitative analysis of the kinetic pathway associated with USCNT translocation induced by tension was performed in a specially designed microfluidic device, simultaneously combining optical fluorescence microscopy and electrophysiological measurements. Important outcome of these findings is the identification of the way to control the nanomaterials translocation through lipid bilayer by membrane tension that can be useful in many practical applications.

Keywords Lipid bilayers, Carbon nanotubes, Microfluidics, Translocation dynamics, Tension

Carbon nanotubes (CNT) are promising materials for a wide range of biotechnological applications such as biosensors, electrochemical probes for cells, scanning probe microscopy, cell manipulation, heat transmitters, photo-voltaic applications, and nanoscale electric conductors. 1,2 The surface of carbon nanotubes can be readily functionalized with ligands 3 and biomolecules 4,5 (peptides, RNA) that makes them a useful tool for single cell biosensing. 2,6 The ability of carbon nanotubes to penetrate into living cells is a subject of intensive debates over the last decades. Carbon nanotubes are found inside cells 7–11 but the exact translocation mechanism 12,13 either by means of direct piercing through a membrane or by an endocytotic pathway could not be always identified. However, due to a large energy barrier of order 100 kT, 12 direct piercing is questionable and endocytosis pathway is probably enabled in most practical situations. In turn, endocytosis pathway rely on energy-dependent nonspecific interactions between CNTs and cell membranes; endocytosis is difficult to control

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or reproduce in artificial membranes, while targeting to a specific cell type, or cell organelle, is almost impossible. 14 Insertion and consequent translocation of CNTs through a lipid bilayer by passive diffusion is not a general phenomenon and may be observed only for very small, less than 10 nm, objects with a balanced hydrophobicity, adjusted to lipid bilayer properties. It was suggested that alternating hydrophobic and hydrophilic stripes along the CNTs may significantly decrease the translocation barrier, 15 or that heterogeneous surface properties could be responsible for direct penetration of CNTs in plant cells. 16,17 Moreover, the size of CNTs dramatically affects their possibility to translocate: translocation time increases exponentially with the diameter and the length of the CNT, 12 while hydrophobicity of CNTs promotes their permanent trapping into the core of the bilayer. 18,19 Recent experimental studies demonstrated that lipid-coated single-walled ultra-short carbon nanotubes (USCNTs) of length ≈ 10 nm can easily be inserted into model lipid bilayers with a perpendicular orientation 20 and behave as synthetic ion channels, enhancing the transport of small solute molecules across the membrane. In addition, inserted USCNTs enable transport of large linear macromolecules across the membrane, such as single-strained DNA, which can find applications in gene delivery and sequencing. Depending on their size and chemical composition, embedded USCNTs present a tunable ion selectivity similar to natural membrane ion channel. 21 Apart from interest in biomedical applications, these properties can find applications in physical transport through structures on the nano-scale. Beyond biotechnological advantages, CNTs can provoke risks, in particular, toxicity issues for cells and tissues, 11,22,23 which may limit biomedical applications of CNTs. Toxicity of a nano-objects depends on different factors including functionalization, aggregation state, size, length, diameter and surface chemistry. In this respect, the possibility of spontaneous translocation of CNTs through lipid bilayers and other biological barriers, such as blood brain barrier and kidney filtration barrier may be a leading factor in the toxicity of CNTs. In this article we present a theoretical and experimental study of conditions when ultra-

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Figure 1: Suggested translocation mechanism of hydrophobic USCNT through a stretched lipid bilayer. DOPC-capped USCNT (diameter 0.8 nm) after being wrapped by a lipid layer. The lipid-covered USCNT destabilize the bilayer by generating pores and pass through the bilayer by taking away non-fluorescent and fluorescent lipids.

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short nanotubes (USCNTs) can spontaneously translocate through a lipid bilayer if the bilayer tension raises to sufficient levels by stretching (See Fig.1). First we present a theoretical basis for the mechanism and conditions for USCNTs to translocate spontaneously through lipid bilayer. Next, with an experimental setup, we present a direct experimental proof of USCNTs translocation induced by bilayer stretching. The corresponding translocation kinetic pathway is characterized with electrophysiological and simultaneous fluorescent measurements.

Theoretical Model of Spontaneous Translocation under Stretching We use the Single Chain Mean Field (SCMF) theory 12 to study the insertion and translocation of lipid covered USCNT through a phospholipid bilayer (see Materials and Methods), which was previously used to study energy barrier of insertion of CNTs 12 into lipid bilayers and for translocation of lipid-covered hydrophobic nanoparticles through lipid bilayer. 24 We model a short USCNT, length 10 nm, inserted perpendicular to the bilayer, which is the lowest energy orientation. 25 Since for a long CNT, length 1 µm, the energy barrier is significantly higher, it will be in with a parallel orientation to the bilayer, and hence, not included in the modelling. Within this theory, USCNT is modeled as a hydrophobic cylinder with a diameter 0.8 nm. The hydrophobic character of USCNT is modeled by a negative interaction parameter between lipid tails and the surface of the cylinder, . From previous works, 12,24 we found that the crossover between wetting and non-wetting by lipids of CNT is between −6.0 kT to −6.5 kT, i.e. we observe spontaneous wetting of CNTs by lipids for = −6.5 kT and non-wetting or ”naked” CNTs for > −6 kT. The free energy cost per lipid of USCNT insertion is defined as the energy difference between USCNT placed in the center of the bilayer in a perpendicular orientation and the free energy of USCNT in the solution (reference state). The insertion of a hydrophobic 5


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A)

Figure 2: Interaction regimes of lipid-covered USCNT and lipid bilayer A) without stretching and B) with stretching. Strong interaction with lipid tails = −6.5 kT and stretching above (A − A0 )/A0 = 5% are prerequisites for pore formation and translocation (Escape regime). The dashed lines in the figures below correspond to solution without stretching (curves above).

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USCNT into a lipid bilayer is a multi-step process accompanied by the exchange of lipids between the USCNT and the bilayer, which is reflected in coexistence of several solutions in the SCMF theory. First regime is the insertion of USCNT with lipid bilayer, where USCNT interacts with the head region of the bilayer for large distances from the bilayer center. Upon further insertion in to the bilayer, USCNT can spontaneously embed into the core of the bilayer through a first-order transition corresponding to breaking the bilayer and rearrangement of heads layer in the embedding regime. Under no stretching conditions corresponding to tensionless membranes we observe the following regimes presented in Figure 2A. When USCNT with = −6.0 kT (corresponds to no lipid wetting) interact with the lipid bilayer, the USCNT inserts into the core. This process leads to a considerable decrease of the free energy to ∼ −70 kT into embedding regime, where USCNT is trapped in the minimal energy state. In this state the tails of the lipids are in direct contact with the USCNT, but the interaction is not strong enough to cover the surface of the USCNT with lipids. For stronger interactions, e.g. = −6.5 kT (lipid wetting), lipids cover the surface of USCNT, but the interaction between the USCNT and the lipid tails is still not strong enough to qualitatively change the bilayer structure and USCNT is thus stay trapped in the bilayer with embedding regime being the minimal energy state. However, a qualitative difference in the free energy landscapes can be observed under stretching of the lipid bilayer at (A − A0 )/A0 ≈ 5% of area per lipid A with respect to equilibrium area A0 . As in tensionless membrane case, for the less hydrophobic USCNT, = −6.0 kT, we observe only two solutions: insertion and embedding, whereas in case of the strong interaction energy, corresponding to wetting of USCNT by lipids, = −6.5 kT, we observe a new regime, ”escape”, where USCNT is covered with lipids and surrounded by spontaneously formed hydrophilic pore with heads of lipids turned inside the pore (Fig. 2B). In such configuration the lipids forming the pore around USCNT and lipids covering USCNT are not well connected and thus USCNT can quit the bilayer with almost no energy

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Figure 3: A) Microfluidic setup: a water channel is separated with a lipid bilayer. The USCNTs (not fluorescent, non-visible) are added on one side, a USCNT becomes visible after the contact with the lipid bilayer (after being wrapped by fluorescent lipids). B) A sequence of fluorescent images is showing a single USCNT translocating through a fluorescent lipid bilayer. The position of the USCNT moving out from the bilayer is marked by a dashed circle. cost and is accompanied by lipid extraction from the bilayer.

Direct Observation of a Single USCNT Translocation Event The microfluidic setup (Fig. 3A) consist of two buffer droplets separated by a lipid bilayer composed of 98% DOPC and 2% of fluorescent lipid Rho-DOPE. One droplet contains only a buffer solution while the other droplet contains a dispersion of DOPC-coated CNTs. 20 Fluorescent lipids are initially present only in the lipid bilayer, while the dispersed CNTs are coated with non-fluorescent DOPC, and are thus invisible in fluorescent microscopy. We use CNTs of two lengths: ”long” CNTs of ≈ 1µm and ”ultra short” CNTs ≈ 10 nm (USCNTs).

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For both nanotubes, the diameter is fixed to 0.8 nm (see Materials and Methods). For the following experiments we employ only long CNTs, and we use a CNTs concentrations in the range from 1 to 400 ng/ml. Since the mass concentration of CNTs is 100 times higher than that of USCNTs, for experiments with short USCNTs, we use a concentration range from 0.01 to 4 ng/ml in order to match the number of nanotubes. Adding lipid-coated CNTs (1µm length) in one buffer droplet and waiting 20-30 min for the bilayer formation, we detect no change of the current intensity through the bilayer for applied voltage (between −50mV and 50mV ). if the CNTs were inserted perpendicularly to the bilayer, a characteristic (current-voltage) signal should be measured as the CNTs allows the transport of charges across the bilayer. 20 This implies that either lipid-coated CNTs (1µm length) are not embedded into the bilayer core, or they are inserted in the bilayer core with a parallel orientation. Conducting the same experiments with lipid-coated USCNTs (10nm length) a characteristic current-voltage signal across the lipid bilayer is measured, which increases proportionally to the USCNT concentration Fig. 4A. This demonstrates successful insertion of the USCNTs to the bilayer core in perpendicular orientation, while inserted USCNTs behave as synthetic ionic channels. Additionally, measured ion transport signals are in good agreement with the reported values for similar systems. 20,21,26 Furthermore, at low concentrations 0.01 ng/ml, our experimental setup allows for detection and characterization of a single translocation event across a bilayer. 24 We have not observed any translocation events for long CNTs, whatever the tested bilayer tension. However, we found that USCNTs, inserted perpendicularly in the lipid bilayer, can spontaneously quit the bilayer if this bilayer is stretched above a tension of 4mN/m. Thus, two conditions should be fulfilled for translocation event: (i) USCNT is oriented perpendicularly in the bilayer; (ii) tension above critical threshold should be applied. The bilayer tension could be control by changing the chemical composition of the oily phase at the edges of the bilayer. Hence, we could gently tune the bilayer tension in the range of 1mN/m to 8mN/m (see Ma-

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Pore

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0

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Figure 4: A) Current-voltage characteristics (I-V curves) measured for a lipid bilayer without nanotube (purple squares), a lipid bilayer containing a concentration 1 ng/ml USCNTs (blue circles), a concentration 2 ng/ml (orange triangles), a concentration 4 ng/ml (pink inverted triangles); B) (top) Conductance trace of a single translocation event observed in a DOPC lipid bilayer under the tension 4 mN/m for a USCNT of 0.8 nm; (bottom) Control conductance of a lipid bilayer without USCNT and without tension (purple) and without USCNT but under tension 4 mN/m (blue); C) Frequency time of the measured translocating events for a DOPC bilayer under the tension of 4 mN/m in the presence of USCNT (with a concentration of 0.01 ng/ml). terials and Methods). We found that translocation events of USCNTs can only be observed for bilayer tension above a critical threshold of 4mN/m. An example of direct observation of a single USCNTs translocating through a bilayer is present in Fig. 3.B. Lipid-covered USCNTs are not visible before the contact with the bilayer since DOPC lipids initially covering the USCNTs are not fluorescent, while observation of fluorescent object on the other side of the bilayer, where initially only the buffer was placed, indicates that the USCNTs were in contact with the bilayer and extracted fluorescent lipids from the bilayer. It also demonstrate that some of the USCNTs crossed the bilayer on the other side. Apart from optical detection of single translocation event, patch-clamp measurements permit to get a detailed information about the translocation pathway, timescales (Fig 4.B). Starting at t = 0 ms, the conductance is increasing to G = 0.57nS for a period of ≈ 4 ms. This small conductance jump suggests a creation of a gap between USCNT and the bilayer, which may correspond to a hydrophilic pore around USCNT in escape regime in 10


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our theoretical model, Fig. 2.B. Since this pore created by USCNT represents a truncated cylinder, one can estimate the corresponding gap size dg between the nanotube and the bilayer as: dg ≈

Gl ≈ 0.16 nm 2πkRU SCN T

(1)

where l ≈ 4nm is the bilayer thickness, and the radius of a USCNT RU SCN T coated with a DOPC monolayer is ≈ 2nm, k = 1.15 S/m is the bulk electrolyte conductivity (estimated for 100 mM NaCl at 30◦ C). The size of the gap dg is large enough to allow USCNTs to escape due to thermal motion, 12,24 while the gap lifetime is ∆t ≈ 4ms. We assume, that the gap is not uniformly formed during a pore lifetime since the process of lipid extraction and reorganization of the bilayer may not be instant and takes a few ms, while a thin lipid bridge may maintain the USCNT connected to the bilayer. After this time, the conductance jumps back to G = 4.2nS for ∆t ≈ 3.4 ms. Based on our theoretical model, this conductance increase could be the consequence of the lipid-covered USCNTs that is quitting the bilayer and form a pore. We can confirm this assumption by calculating the radius of such pore Rpore with a cylindrical shape, from the corresponding conductance value, r Rpore =

Gl ≈ 2.17 nm. kπ

(2)

This value is in good agreement with the pore radius generated by a 0.8nm USCNTs coated with a lipid DOPC monolayer. which is consistent with our theoretical understanding. The lifetime of the pore is of a few milliseconds, after this time the conductance drops abruptly control value corresponding to an intact lipid bilayer indicating the closing of a transient pore and confirming that the USCNT has left the bilayer within a few milliseconds (Fig. 4.B-C). This scenario is only observed for tensions above critical threshold of 4mN/m, while below this value, although some rare events of USCNT escape could be observed by fluorescence microscopy, no convincing electrophysiological signal could be measured, as the bilayer conductance stays at the control value corresponding to an intact lipid bilayer.

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Conclusion Using a combination of SCMF theory and a microfluidic technique, we characterize the insertion and the spontaneous translocation of ultra-short carbon nanotubes (USCNTs) through a lipid bilayer. We demonstrate that high membrane tension is a mandatory prerequisite for such translocation, as the discussed translocation mechanism that is unlikely to occur in tensionless membranes. The detailed kinetic pathway associated to a translocation event is predicted by using SCMF theory. For sufficiently strong hydrophobic attraction between the lipid and the nanotube, it is predicted that the lipid molecules reorganize and flip their tails toward the USCNT, and subsequently completely wrap the USCNT. Under tension, trans-membrane USCNTs eventually act as nucleation points for the spontaneous opening of instable pores in the membrane for partial tension release, while the USCNTs remain wrapped by lipids. In case the resulting hydrophilic pore circumvents the USCNT, the nanotube may escape from the membrane environment by diffusion. The predicted translocation pathway has been tested by microfluidic experiments, which allowed to characterize the kinetic pathway of single translocation events by simultaneous optical and electrophysiological measurements. Under low tension, no translocation events have been observed experimentally, while above a threshold tension of ≈ 4mN/m signatures of individual translocation events have been found in conductance traces in agreement with fluorescence microscopy. According to our theoretical understanding, bilayer stretching reduces the local number of lipids interacting with the surface of USCNT, and facilitates the pore formation necessary to release the USCNT from the bilayer. Fluorescence measurements demonstrate an exchange of lipids during translocation between USCNT and bilayer membrane, which likely leads to a change of the USCNT chemical potential in solution. SCMF results, on the other hand, highlight that throughout the whole translocation pathway the free energy is decreased. An open question therefore is if USCNTs 12


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that escaped from the membrane would be able to reinsert into the membrane. The suggested translocation mechanism will allow for specific targeting or permanent localization of USCNTs in cells and organelles as function of their elastic properties and tension. Even if the tension values required to trigger translocation seem particularly high for biological cells, these conditions might be relevant during cell division. 27 Moreover, our study reveals important conditions to use nanotube porins, embedded into a lipid bilayer, as high-flux nanofilters for purifying water at astonishingly high rates, which might be one of the solutions against possible global water threat. 21 Those applications rely on the fact that USCNTs stay in the membrane for a long time, and their escape has to be avoided. On the other hand, tension induced escape offers the possibility for controlled USCNT removal from the bilayers by stretching, which can used in many practical applications along with the use of USCNTs as synthetic ion channels. Fine-tuned coatings of USCNTs may open a perspective to deliver objects such as RNA, ligands, or short biopolymers through the membrane or into specific membranes.

Materials and Methods Microfluidics and Lipids Microchannels with rectangular cross section were fabricated using typical soft lithography protocols. Channel dimensions were 300 µm in width and 140 µm in height. The device was molded with a SU-8 photoresist on a silicon wafer using Sylgard 184 (Dow Corning, USA). The surface of the Sylgard 184 devices was exposed to nytrogen plasma (Diener electronic GmbH, Germany) and sealed with a plasma treated glass cover slide. The sealed device was rendered hydrophobic by heating it to 135◦ C over night. The liquids were dispensed from syringes (Hamilton Bonaduz AG, Switzerland), which were connected to the microfluidic device by Teflon tubing. Computer controlled syringe pumps were used to control the injection of the water and oil phases, respectively. DOPC (1,2-dimyristoyl-sn-glycero13


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3-phosphocholine), fluorescent Rho-DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), were purchased from Avanti Polar Lipids. Experiments are performed at 30◦ C to have the lipids phospholipids molecules in a fluid phase. A variant of the Droplet Interface Bilayer (DiB) 28 technique was used to produce a freestanding lipid membrane in a microfluidic chip. 24,29 Using a volume controlled system with syringe pumps, two water droplets were injected face-to-face into microchannels with a crossgeometry which was previously filled with a fluorescent lipid solution. After a few seconds, the water-oil interface of each fingers is covered with a monolayer of DOPC molecules. Once the two liquid fingers are brought in contact, the two lipid monolayers interact and form a lipid bilayer within a short time. 24,28,29 The system is then stable and can be analyzed, simultaneously, with fluorescence microscopy and by electrophysiological inspection.

Carbon Nanotubes Single-walled carbon nanotubes (long CNTs) with diameter ≈ 0.8nm and an average length around 1 µm were purchased at Sigma-Aldrich. Ultra-short carbon nanotubes (USCNTs) of length ≈ 10nm were produced from long CNTs in a two-step process following standard methods. 20,26,30 One hundred milligrams of the purified USCNTs were suspended in 24ml mixture of concentrated H2 SO4 /HNO3 (3/1) in a 250ml conical flask and sonicated in a water bath for 48h at 35 − 40◦ C. The suspension was then diluted with 200ml of water and neutralized with 6 M NaOH solution. The relatively long CNTs were collected on a 100nm pore size filter membrane (PTFE; Millipore) and the USCNTs were collected in the filtrate. The solution was taken for centrifugal ultrafiltration for 40 min and then MilliQ water was added to dilute the solution for further centrifugal ultrafiltration. The washing and ultrafiltration were repeated three times before the water was completely removed. It results that USCNTs were produced with a diameter of 0.8nm with a length around ≈ 10 nm. 26,30 Stable dispersion of CNTs in water is made possible by coating them with a lipid DOPC monolayer following standard procedure. 31 14


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Figure 5: A) Temporal evolution of the surface tension of a DOPC monolayer at a oil/buffer interface measured using the pendant droplet technique. B) The angle θ gives the bilayer contact angle of a lipid bilayer with a 100 µm diameter.

Bilayer Tension To tune the membrane tension, we added between 5 and 0.1% of Silicone oil AR20 (Sigma) to the squalene phase. This silicone oil has the advantage to not insert into the bilayer core, like the squalene oil, 32 but it is able to reduce the water/oil interfacial energy which is also reducing the bilayer tension. As a result, the bilayer tension could be tuned between 8mN/m (for pure squalene) to 4mN/m (corresponding to 0.1% of silicone oil AR20) and 1.5mN/m (corresponding to 5% of silicone oil AR20). Surface tensions of the various lipid monolayers at oil/water interfaces were measured with the pendant drop method using a commercial measurement device (OCA 20, DataPhysics Instruments GmbH, Filderstadt, Germany). Oil solution with a concentration of 5 mg/ml DOPC were produced by introducing droplet from a steel needle into the surrounding oil phase. As oils, squalene oil are used as function of different silicone AR20 concentration. The shape of all droplets were fitted with the Young-Laplace equation to obtain their interfacial tension. After the initial formation of a droplet, the DOPC lipids adsorb to the interface leading to a reduction in interfacial tension. This decrease was recorded over several minutes until a plateau was reached (see fig. 5.A). From the values of the surface tension and the bilayer contact angle θ, which were obtained from pendant drop measurements or optical micrographs (see fig5.B), respectively, the bilayer tension can be calculated using Youngs

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equation 33,34 Γ = 2γcosθ

(3)

It results that the bilayer tension Γ is equal to 8mN/m for pure squalene, 3 to 4mN/m for 0.1 vol% of silicone oil AR20 and from 3mN/m to 1.5mN/m for 1 vol% to 5 vol% of silicone oil AR20.

Patch Clamp Ag/AgCl electrodes were prepared by inserting a electrode in a borosilicate glass pipette (outer diameter 1.5 mm, inner diameter 0.86 mm, Vendor) containing an electrolyte agarose solution. The electrodes are carefully introduced into the buffer compartment of the Sylgard 184 device using a micromanipulator. The lipid membrane conductance was measured using the standard function provided by the patch clamp amplifier EPC 10 USB (HekaElectronics). A 10 mV sinusoidal wave with a frequency of 20 kHz was used as an excitation signal.

Single Chain Mean Field Theory Single Chain Mean Field (SCMF) theory 12,25,35–37 provides a detailed description of the mechanical and equilibrium properties of lipid bilayers at the molecular level. The phospholipids are modeled at a coarse-grained level: 37 the DOPC lipid molecule is modeled as three freely joint spherical beads of equal radius 4.05 ˚ A and connected by the stiff bond of 10 ˚ A. The beads interact through square well potentials: between two hydrophobic beads, tail ”T”, εT T = −2.1 kT with the interaction range r = 12.15 ˚ A between one hydrophilic bead, head ”H” and implicit solvent ”S”, εHS = −0.15 kT with interaction range r = 12.15 ˚ A. The solvent molecule in our model is considered of the same radius as the spherical beads. This model accurately describe the equilibrium and mechanical properties of DMPC lipid bilayers. 37 USCNT is modeled as a rigid cylinder with the height 10 nm and the diameter

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0.8 nm. This cylinder is inaccessible for lipids and solvent, thus representing exclusion zone. However, tails of the lipids can interact with the surface of the cylinder through interaction parameter . We assume no interaction at the top and the bottom ends of the USCNT, which is consistent with the process of fabrication of USCNTs, where the caps of the USCNTs remain hydrophilic. The interaction parameter between tails and surface of USCNT varies between −6.0 kT (when lipids do not wrap the USCNT) and −6.5 kT (lipid wrapping). Since a cylinder inserted perpendicularly into a bilayer has an axial symmetry, the simulation box of size 200 × 200 × 300 ˚ A is discretized into 2D cylindrical geometry oriented around the z-axis in the center of simulation box. The sampling of the molecule conformational space is 5 000 000 conformations which gives enough precision of energy calculation. 37 As a result of the calculations, the SCMF theory gives directly the equilibrium volume fraction of lipids and the free energy of the system for a given position of the cylinder with respect to the center of the bilayer. In order to calculate the free energy we assume that the simulation box represents a part of an extensive system with a USCNT located inside the simulation box, where the rest of the extensive system is a continuous repetition of the USCNT perturbed membrane, used as a reference state for the free energy. This allows to calculate the free energy F of a large system from the calculation of the simulation box. It can be written as a sum of the free energy of the simulation box Fbox and the free energy of the equilibrium system out of the box Fout . If we assume there is no USCNT inside the simulation box as a reference state, F can be denoted through the total volume V and the total number of lipids N of the large system, the free energy per lipid of the bilayer, fA , and the free energy of pure solvent, fs = (φ0 /Vsol ) ln(φ0 /Vsol ), where Vs is the volume of the solvent and φ0 is the bulk solvent volume fraction. F = Fbox + Fout = V fs + N fA

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The free energy difference due to the insertion of a USCNT (Fig. 2) yields the form

∆F = Fbox − N fA − (Vbox − Vobj )fs

(5)

where N is the equilibrium number of lipids in the box, Vbox is the volume of the box, and Vobj in is the part of the USCNT inside the box. Thus, the reference state for free energy difference is chosen to be the energy of unperturbed bilayer (zero energy in Fig. 2). Experimental results in the main text correspond to DOPC lipid bilayer. However, the same translocation effect has been observed for DMPC bilayer when the membrane tension is above 4mN/m (for same USCNT concentration and temperature), and this translocation effect is not observed for membrane with a lower tension. See Supporting Information.

Supporting Information Supporting Information Available: The results for the interaction between a DMPC lipid bilayer and a single USCNT. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information Corresponding Authors Vladimir A. Baulin and Jean-Baptiste Fleury

Acknowledgement VB, MW and YG acknowledge funding from Marie Curie Actions under EU FP7 Initial Training Network SNAL 608184. RS and JBF acknowledge funding from SFB 1027. YG acknowledge funding from NSFC 11804151 and FRFCU 14380108 18


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References 1. Bekyarova, E.; Ni, Y.; Malarkey, E. B.; Montana, V.; McWilliams, J. L.; Haddon, R. C.; Parpura, V. Applications of Carbon Nanotubes in Biotechnology and Biomedicine. J. Biomed. Nanotechnol. 2005, 1, 3–17. 2. Klumpp, C.; Kostarelos, K.; Prato, M.; Bianco, A. Functionalized Carbon Nanotubes as Emerging Nanovectors for the Delivery of Therapeutics. Biochim. Biophys. Acta, Biomembr. 2006, 1758, 404–412. 3. Sun, Y. P.; Fu, K.; Lin, Y.; Huang, W. Functionalized Carbon Nanotubes: Properties and Applications. Acc. Chem. Res. 2002, 35, 1096–1104. 4. Pantarotto, D.; Singh, R.; McCarthy, D.; Erhardt, M.; Briand, J.-P.; Prato, M.; Kostarelos, K.; Bianco, A. Functionalized Carbon Nanotubes for Plasmid DNA Gene Delivery. Angew. Chem. Int. Ed. 2004, 43, 5242–5246. 5. Lu, Q.; Moore, J. M.; Huang, G.; Mount, A. S.; Rao, A. M.; Larcom, L. L.; Ke, P. C. RNA Polymer Translocation with Single-Walled Carbon Nanotubes. Nano Lett. 2004, 4, 2473–2477. 6. Kouklin, N. A.; Kim, W. E.; Lazareck, A. D.; Xu, J. M. Carbon Nanotube Probes for Single-Cell Experimentation and Assays. Appl. Phys. Lett. 2005, 87, 173901. 7. Porter, A. E.; Gass, M.; Muller, K.; Skepper, J. N.; Midgley, P. A.; Welland, M. Direct Imaging of Single-Walled Carbon Nanotubes in Cells. Nat. Nanotechnol. 2007, 2, 713– 717. 8. Porter, A. E.; Gass, M.; Bendall, J. S.; Muller, K.; Goode, A.; Skepper, J. N.; Midgley, P. A.; Welland, M. Uptake of Noncytotoxic Acid-Treated Single-Walled Carbon Nanotubes into the Cytoplasm of Human Macrophage Cells. ACS Nano 2009, 3, 1485– 1492. 19


ACS Nano 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

9. Kam, N. W. S.; Jessop, T. C.; Wender, P. A.; Dai, H. Nanotube Molecular Transporters: Internalization of Carbon Nanotube-Protein Conjugates into Mammalian Cells. J. Am. Chem. Soc. 2004, 126, 6850–6851. 10. Kam, N. W. S.; O’Connell, M.; Wisdom, J. A.; Dai, H. Carbon Nanotubes As Multifunctional Biological Transporters and Near-Infrared Agents for Selective Cancer Cell Destruction. Proc. of Nat. Acad. Sci. 2005, 102, 11600–11605. 11. Cheng, C.; M¨ uller, K. H.; Koziol, K. K. K.; Skepper, J. N.; Midgley, P. A.; Welland, M. E.; Porter, A. E. Toxicity and Imaging of Multi-Walled Carbon Nanotubes in Human Macrophage Cells. Biomaterials 2009, 30, 4152–4160. 12. Pogodin, S.; Baulin, V. A. Can a Carbon Nanotube Pierce through a Phospholipid Bilayer? ACS Nano 2010, 4, 5293–5300. 13. Raffa, V.; Ciofani, G.; Nitodas, S.; Karachalios, T.; DAlessandro, D.; Masinie, M.; Cuschieri, A. Can the Properties of Carbon Nanotubes Influence Their Internalization by Living Cells? Carbon 2008, 46, 1600–1610. 14. Miaczynska, M.; Stenmark, H. Mechanisms and Functions of Endocytosis. J. Cell Biol. 2008, 180, 7–11. 15. Pogodin, S.; Slater, N. K. H.; Baulin, V. A. Surface Patterning of Carbon Nanotubes Can Enhance Their Penetration through a Phospholipid Bilayer. ACS Nano 2011, 5, 1141–1146. 16. Liu, Q.; Chen, B.; Wang, Q.; Shi, X.; Xiao, Z.; Lin, J.; Fang, X. Carbon Nanotubes as Molecular Transporters for Walled Plant Cells. Nano Lett. 2009, 9, 1007–1010. 17. Khodakovskaya, M.; Dervishi, E.; Mahmood, M.; Xu, Y.; Li, Z.; Watanabe, F.; Biris, A. S. Carbon Nanotubes are Able to Penetrate Plant Seed Coat and Dramatically Affect Seed Germination and Plant Growth. Acs Nano 2009, 3, 3221–3227. 20


Page 20 of 24

Page 21 of 24 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

ACS Nano

18. Yue, T.; Xu, Y.; Li, S.; Luo, Z.; Zhang, X.; Huang, F. Surface Patterning of Single-walled Carbon Nanotubes Enhances Their Perturbation on a Pulmonary Surfactant Monolayer: Frustrated Translocation and Bilayer Vesiculation. RSC Adv. 2017, 7, 20851–20864. 19. Gangupomu, V. K.; Capaldi, F. M. Interactions of Carbon Nanotube with Lipid Bilayer Membranes. J. Nanomater. 2011, 2011, 1–6. 20. Geng, J., Kim, K., Zhang, J., Escalada, A., Tunuguntla, R., Comolli, L.R., Allen, F.I., Shnyrova, A.V., Cho, K.R., Munoz, D., Wang, Y.M., Grigoropoulos, C.P., Ajo-Franklin, C.M., Frolov, V.A., Noy, A. Stochastic Transport through Carbon Nanotubes in Lipid Bilayers and Live Cell Membranes. Nature 2014, 514, 612–615. 21. Tunuguntla, R. H.; Henley, R. Y.; Yao, Y. C.; Pham, T. A.; Wanunu, M.; Noy, A. Enhanced Water Permeability and Tunable Ion Selectivity in Subnanometer Carbon Nanotube Porins. Science 2017, 357, 792–796. 22. Liu, J.; Hopfinger, A. J. Identification of Possible Sources of Nanotoxicity from Carbon Nanotubes Inserted into Membrane Bilayers Using Membrane Interaction Quantitative Structure-Activity Relationship Analysis. Chem. Res. Toxicol. 2008, 21, 459–466. 23. Davoren, M.; Herzog, E.; Casey, A.; Cottineau, B.; Chambers, G.; Byrne, H. J.; Lyng, F. M. In Vitro Toxicity Evaluation of Single Walled Carbon Nanotubes on Human A549 Lung Cells. Toxicol. In. Vitro 2007, 21, 438–448. 24. Guo, Y.; Terazzi, E.; Seemann, R.; Fleury, J. B.; Baulin, V. A. Direct Proof of Spontaneous Translocation of Lipid-Covered Hydrophobic Nanoparticles through a Phospholipid Bilayer. Sci. Adv. 2016, 2, e1600261. 25. Pogodin, S.; Baulin, V. Equilibrium Insertion of Nanoscale Objects into Phospholipid Bilayers. Curr. Nanosci. 2011, 7, 721–726.

21


ACS Nano 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

26. Liu, L.; Yang, C.; Zhao, K.; Li, J.; Wu, H. C. Ultrashort Single-Walled Carbon Nanotubes in a Lipid Bilayer As a New Nanopore Sensor. Nat. Commun. 2013, 4, 2989. 27. Fischer-Friedrich, E.; Hyman, A. A.; Jlicher, F.; Mller, D. J.; Helenius, J. Quantification of Surface Tension and Internal Pressure Generated by Single Mitotic Cells. Sci. Rep. 2015, 4, 6213. 28. Bayley, H.; Cronin, B.; Heron, A.; Holden, M. A.; Hwang, W. L.; Syeda, R.; Thompson, J.; Wallace, M. Droplet Interface Bilayers. Mol. BioSyst. 2008, 4, 1191–1208. 29. Vargas, J. N.; Seemann, R.; Fleury, J. B. Fast Membrane Hemifusion via Dewetting between Lipid Bilayers. Soft Matter 2014, 10, 9293–9299. 30. Liu, J.; Rinzler, A.G.; Dai, H.; Hafner, J.H.; Bradley, R.K.; Boul, P.J.; Lu, A.; Iverson, T.; Shelimov, K.; Huffman, C.B.; Rodriguez-Macias, F.; Shon, Y.-S.; Lee, T.R.; Colbert, D.T.; Smalley, R.E. Fullerene Pipes. Science 1998, 280, 1253–1256. 31. Douroumis, D.; Fatouros, D. G.; Bouropoulos, N.; Papagelis, K.; Tasis, D. Colloidal Stability of Carbon Nanotubes in an Aqueous Dispersion of Phospholipid. Int. J. Nanomed. 2007, 2, 761. 32. Taylor, G. J.; Venkatesan, G. A.; Collier, C. P.; Sarles, S. A. Direct In Situ Measurement of Specific Capacitance, Monolayer Tension, and Bilayer Tension in a Droplet Interface Bilayer. Soft Matter 2015, 11, 7592–7605. 33. Needham, D.; Haydon, D. Tensions and Free Energies of Formation of “Solventless” Lipid Bilayers. Measurement of High Contact Angles. Biophys. J. 1983, 41, 251–257. 34. Bibette, J.; Calderon, F. L.; Poulin, P. Emulsions: Basic Principles. Rep. Prog. Phys. 1999, 62, 969. 35. Ben-Shaul, A.; Szleifer, I.; Gelbart, W. M. Chain Organization and Thermodynamics in Micelles and Bilayers. I. Theory. J. Chem. Phys. 1985, 83, 3597–3611. 22


Page 22 of 24

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36. Szleifer, I.; Ben-Shaul, A.; Gelbart, W. Chain Statistics in Micelles and Bilayers: Effects of Surface Roughness and Internal Energy. J. Chem. Phys. 1986, 85, 5345–5359. 37. Pogodin, S.; Baulin, V. A. Coarse-Grained Models of Phospholipid Membranes within the Single Chain Mean Field Theory. Soft Matter 2010, 6, 2216–2226.

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Tension-Induced Translocation of Ultra-Short Carbon Nanotube

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