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Human immune protein C1q selectively disaggregates carbon nanotubes Maximilien Saint Cricq, Jesús Carrete, Christine Gaboriaud, Edmond Gravel, Eric Doris, Nicole Thielens, Natalio Mingo, and Wai Li Ling Nano Lett., Just Accepted Manuscript • Publication Date (Web): 22 May 2017 Downloaded from http://pubs.acs.org on May 23, 2017
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Human immune protein C1q selectively disaggregates carbon nanotubes M. Saint Cricqa, J. Carretea, C. Gaboriaudb, E. Gravelc, E. Dorisc, N. Thielensb , N. Mingoa,*, and W. L. Lingb,* a
b
c
Univ. Grenoble Alpes, CEA LITEN, F-38000 Grenoble, France
Univ. Grenoble Alpes, CEA, CNRS, IBS, F-38000 Grenoble, France
Service de Chimie Bioorganique et de Marquage (SCBM), CEA, Université Paris-Saclay, 91191 Gif-sur-Yvette, France
† Corresponding author,
[email protected] * Corresponding author,
[email protected].
KEYWORDS: Carbon nanotubes. C1q. Toxicity. Molecular dynamics. Free energy. Solvation.
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ABSTRACT
We atomistically compute the change in free energy upon binding of the globular domain of the complement protein C1q to carbon nanotubes (CNTs) and graphene in solution. Our modeling results imply that C1q is able to disaggregate and disperse bundles of large diameter multi-walled CNTs but not those of thin single-walled CNTs, and we validate this prediction with experimental observations. The results support the view of a strong binding, with potential implications for the understanding of the immune response and biomedical applications of graphitic nanomaterials.
The protein C1q is the initiator of the classical complement pathway, a proteolytic cascade involved in innate immunity. Moreover, C1q is a multi-functional protein: besides activating the complement cascade by direct binding with intruders and by recognizing bound antibodies in adaptive immunity, it plays an important role in processes such as brain maturation, pregnancy, auto-immunity, and cancer control 1. Whether and how C1q binds to nanoparticle surfaces is therefore a critical piece of information, since this binding can interfere with the many important physiological activities that involve the protein, and inappropriate activation of the complement can trigger a potentially severe or even lethal immune response 2. Of particular concern is the case of carbon nanotubes (CNTs), which are becoming increasingly widespread in technological applications. An extensive literature exists on C1q binding to various systems, including CNTs1,3,4. Nonetheless, interaction of C1q with CNTs has only been investigated experimentally, and no quantitative measurement or modeling of the energetics of this interaction has previously been reported. A quantification of the CNT-C1q interaction at the atomic level is thus of utmost importance to determine how C1q binds to CNTs, and to quantify the strength of the interaction.
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Furthermore, we show here that this interaction relates to the important problem of dispersing CNTs. Carbon nanotube agglomeration has indeed been suggested to influence toxicity 5,6. Whereas experimental aspects of the interaction between CNTs and C1q have been addressed in connection with complement activation 7 and organized binding 8, dispersion of CNT bundles by C1q had never been previously investigated theoretically. We have performed such energetic modeling. The answer to these questions has allowed us to predict that C1q can selectively disaggregate CNT bundles depending on the individual CNT diameter, in agreement with our experimental observations. Disaggregating (or dispersing) nanotube bundles is an important and widespread scientific and technological problem in general 9. This problem is closely related to the issue of CNT functionalization by adsorbed molecules, and numerous investigations have been carried out to determine the ability of surfactants to disperse CNTs 10–16. In contrast, reports on diameterspecific solubilisation are wanting 17. An exception is Ref. [18], which used cyclic peptides of selected sequence length to promote selective enrichment of small-diameter CNTs dispersed in solution. However the phenomenon reported there is radically different from the one we identify here: the sorting in that case was produced by polymers wrapping around sufficiently small CNTs and leading to the smaller diameters being singled out. In contrast, here we report on the selection of the larger diameters, resulting from a very different mechanism. Regarding modeling, one previous study with molecular mechanics exists on the dispersion of CNTs by a small molecule sodium dodecyl sulphate, which wraps around CNTs 12. However, this work did not explicitly model the solvent and it did not address diameter-selectivity. In the following, we present the results of molecular dynamics (MD) simulations of the interaction between the C1q globular region (gC1q) - the domain previously shown to bind
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CNTs, and a CNT or a graphene sheet in aqueous solution. By taking statistics of the forces, we are able to quantify the free energy of the system as a function of the distance between the gC1q and the nanotube, showing the presence of an attractive interaction between them. We show that this interaction depends on factors like nanotube diameter and orientation. Afterwards we use the MD results to propose a model of nanotube bundle disaggregation from which a coherent picture emerges, relating the relative ability of gC1q to disaggregate CNT bundles depending on the radius of the CNTs. We then provide experimental evidence, based on transmission electron microscopy (TEM), of this diameter dependence. Possible toxicological implications are also discussed. The C1q protein has a flower shaped structure comprising six collagen stems, held together by disulphide bonds at one end and branched out on the other end to terminate in a globular domain about 5 nm in diameter (gC1q). Figure 1 shows a model of how the gC1q domains, whose crystal structure has been solved 19, are positioned in the full flexible C1q molecule. The depicted collagen-like moiety is a model that has taken into account the sequence-related geometrical parameters observed in other collagen-like molecules 20. A Ca2+ ion sits at the apex of each gC1q opposite to its site of attachment to the collagen arm. Specific C1q binding to appropriate targets via gC1q is known to trigger complement activation19. Some models have been proposed on how
Figure 1: Model of full C1q showing the six globular recognition domains, gC1q. Each gC1q is 5 nm in diameter.
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attachment of the gC1q can induce conformational changes in C1q to initiate the complement cascade 20. For the purpose of determining whether CNTs can bind C1q, we restrict ourselves to investigating a single gC1q unit interacting with a CNT in aqueous solution at room temperature. Just one gC1q molecule contains 6153 atoms, and including the CNT and the water molecules in a 1000 nm3 simulation box brings the total number of atoms to over 118,000. This large amount of atoms prevents the use of ab-initio approaches, and makes empirical force field MD the method of choice. Determining whether there is binding is far from trivial, because large fluctuations take place in a solvated system. Even with careful initial equilibration, the forces on the gC1q center of mass oscillate wildly over time, and its distance to the nanotube will not consistently decrease or increase over time, but will display a complex, multi-scale time dependence due to thermal motion, preventing us from drawing a clear conclusion about the character of the interaction based only on the trajectory. Instead, the quantity to be calculated is the change in free energy, ∆A(x), as a function of the gC1q-nanotube distance, x. If there is binding, ∆A will present a minimum at the adsorption distance, and the depth and extent of the curve will provide crucial information about the character of the average force between the two systems in solution. Various methods exist for the calculation of free energy differences from MD 21–23. When only liquids and solids are involved as is the case here, we can work with the Helmholtz free energy and consider constant temperature (T) and volume (V) conditions. For the problem in hand, it is best to obtain the free energy directly from the averaged forces. This is done using the identity (see eq. 34 in Ref. [22], or eq. 15.11 in Ref. [24]):
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( , , ) = ,
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(1)
where x is a macroscopic parameter (in this case the distance measured from the gC1q centre of mass to the nanotube), and − ( , , )/ is the ensemble averaged force associated with x (i.e. the average of minus the derivative of the total energy with respect to x). In the context of MD, ensemble averages are approximated by trajectory averages. This type of approach is often employed to study the free energy of adsorption of large molecules on solid-liquid interfaces 25. In order to investigate the difference between small and large diameter nanotubes, we have considered the interaction of gC1q with two different systems: 1) a (15,15) nanotube (roughly 2 nm in diameter) to represent thin CNTs; and 2) a graphene sheet as a representative of large diameter CNTs. Simulations were carried out with GROMACS 4.6 26 using the OPLS-AA force field for all interactions, including the modifications by A. Minoia to deal with nanotubes 27. Water was modeled explicitly, using a Simple Point Charge (SPC) model. A cubic simulation box with 10 nm side and periodic boundary conditions was employed. The systems were relaxed, solvated, and equilibrated to room temperature, prior to the production MD runs. We used GROMACS’s “pulling” method to generate initial configurations at many different distances x between the gC1q and nanotube/graphene centers of mass. For each distance we then ran MD simulations through 1 ns to obtain the total force versus time, Fx(t), that needs to be applied on the gC1q center of mass in order to keep the distance x unchanged. The time average of this force, () = lim → ( ) , yields the derivative of the free energy with respect to the
center of mass distance. By averaging over 1 ns, we obtain a reasonable estimate of the average
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force at each particular distance x. The change in free energy between two distances x1 and x2 can then be obtained numerically by integrating () .
The results for the interaction with a thin CNT and with graphene are compared in Figure 2. In both cases, there is a net attractive force at longer distances, which turns repulsive at shorter separations. Inspection of the atomic configuration at short distance shows that when the force becomes repulsive the molecule begins to deform. The change in free energy as a function of distance is also shown in Figure 2. Remarkably, the change in free energy is one order of
Figure 2. Computed average forces and free energies in aqueous solution at 300K for interactions between (a) graphenegC1q and (b) (15,15) CNT-gC1q. Configuration snapshots at different distances between the molecules’ centers of mass: (c) CNT-gC1q, 42.1nm; (d) CNT-gC1q, 36.6 nm; (e) graphene-gC1q, 49.8 nm; (f) graphene-gC1q, 20.9 nm. The Ca2+ ion is highlighted in red. For better clarity the water molecules are not shown.
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magnitude smaller for adsorption on the thin CNT than on graphene. This is due to the reduced interacting surface available on the thin CNT, since the CNT’s diameter is 2.5 times smaller than that of gC1q. The adsorption free energy is about 1 eV on the 2 nm diameter CNT, and ~10 eV on graphene. In both cases, |∆A|>>kBT, meaning that gC1q binds strongly to CNT as a result of the large number of atoms at the interface. The orientation of the gC1q plays a role in the strength of the interaction. We found a strong attractive interaction when the vector between the protein center and the Ca2+ ion is roughly parallel to the nanotube surface. In contrast, weaker or no adsorption was found when the Ca2+ is facing the graphene or CNT, nor when it is placed completely opposite to it (see supplementary material). Since the opposite end from the Ca2+ site on gC1q is where gC1q joins the collagen domain, this later finding assures us that the presence of the collagen domain in the whole C1q molecule likely will not invalidate the results we get from our simplified system of representing C1q by gC1q. It has been suggested that the Ca2+ ion is involved in the specific bindings of complement activators 20,28. Our finding that CNTs do not bind at the Ca2+ site would suggest that CNT binding is not specific to complement activation, in agreement with our previous experimental results that C1q binds CNTs but does not activate complement 8. Carbon nanotubes in aqueous environment are typically bundled together and entangled. Our previous experimental observations have shown that adding micromolar of C1q molecules to the medium results in the disaggregation and total dispersion of the ~25 nm wide multi-walled CNTs (MWCNTs) produced by arc-discharge 8. Imaging by transmission electron microscopy (TEM) shows that a layer of closely packed protein molecules coats the straight MWCNTs. In contrast, bundles of much thinner (~1 nm diameter) single-walled CNTs (SWCNTs) are not dispersed by the addition of C1q.
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We can understand this behavior by using our calculated free energy results and a simple model of nanotube adhesion. For a hexagonal packing of the CNTs in a bundle, the adhesion energy per nanotube is 3 times the adhesion energy between two nanotubes (i.e. the adhesion energy of the bundle is six times the number of tubes divided by two -to avoid double counting-). Since CNTs bind to each other by short-ranged, Van der Waals forces, one can estimate the interaction energy in terms of an effective contact surface (see Figure 3). Assuming that the interaction vanishes beyond a certain cutoff distance d, and is constant at closer distances, the effective contact surface is l·a, where l is the contacting length of the nanotubes, and a is defined in Figure 3. For r>>d it is easy to show that ≈ 2#$%&
(2)
(This same dependence of the adhesion energy on the square root of the nanotube radius was numerically verified in Ref. [29] using empirical potentials.) The energy required to split a
nanotube off the bundle, ()* ∝ 3 - ∙ , has to be compared with the energy gained by coating
the nanotube with gC1q, /0( . The latter is proportional to the number of gC1q molecules that can be accommodated on the surface on the nanotube, i.e.
/0( ≈ ()1 ($%& )-
22$%&
3 $4%5
(3)
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Here 3 $4%5 gives an idea of the CNT surface area that can be covered by one gC1q, where B
is a dimensionless number of order 1, and ()1 ($%& ) is the free energy of adsorption, which
depends on the nanotube radius, as discussed in the previous section. Thus, Ecoat grows faster than Eadh with the nanotube radius, meaning that the thicker the nanotubes the easier it is to disaggregate the bundle. An additional factor comes to enhance this effect: when the radius of the CNT becomes comparable to or smaller than the radius of the gC1q, the adsorption free energy is smaller than for adsorption on large diameter CNT, due to a reduced interaction
surface, as discussed earlier. Thus, for $%& ≫ $4%5 one has /0( ∝ $%& , but for $%& ~
