Membrane Insertion and Phospholipids Extraction by Graphyne

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Membrane Insertion and Phospholipids Extraction by Graphyne Nanosheets Zonglin Gu, Zaixing Yang, Binquan Luan, Xifa Zhou, Linbi Hong, Hong Zhou, Judong Luo, and Ruhong Zhou J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b10548 • Publication Date (Web): 12 Jan 2017 Downloaded from http://pubs.acs.org on January 23, 2017

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The Journal of Physical Chemistry

Membrane Insertion and Phospholipids Extraction by Graphyne Nanosheets Zonglin Gu1,‡, Zaixing Yang1,‡, Binquan Luan2‡, Xifa Zhou3, Linbi Hong2, Hong Zhou1, Judong Luo3,* & Ruhong Zhou1,2,4,*

1. School for Radiological and Interdisciplinary Sciences (RAD-X) and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, China, 215123 2. Computational Biological Center, IBM Thomas J. Watson Research Center, Yorktown Heights, NY 10598, USA 3. Department of Oncology, Changzhou Tumor Hospital, Soochow University, Changzhou, 213001, China 4. Department of Chemistry, Columbia University, New York, NY 10027, USA

‡These authors contribute equally * To whom correspondence should be addressed. E-mail: [email protected](J.D.L.); [email protected] (R. H. Z.)

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Abstract: The presence of both sp1- and sp2-hybridized carbon atoms in graphyne (GY), a nanomaterial resembling the 2D graphene, has led researchers to speculate possible outstanding properties of this material. Recent studies have also been successful in synthesizing derivatives of GY, such as graphdiyne (GY-2), which holds promises in various applications, notably biomedicine. In anticipation of successful synthesis and wide applications of GY as a novel 2D nanomaterial in the near future, we utilized in silico molecular modeling to examine and predict the material’s potential impacts while placed in a biological system, in particular its interactions with cell membranes. Intriguingly, we found that while in the vicinity, GY can be spontaneously inserted into POPC membrane and extract large amounts of phospholipids from it. When compared with graphene, GY shows a relatively weaker capability of lipid extraction though, which is also confirmed by free energy perturbation (FEP) calculations where the POPC lipid molecule shows a larger reduction in free energy when being extracted from the membrane to the graphene surface than to the graphyne surface. This difference in lipid-extraction capability was mainly due to the significantly different carbon-density in nanosheets.


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Introduction Carbon nanomaterials (CNMs), such as 0-dimensional (0-D) fullerene1, 1-D carbon nanotube (CNT)2 and 2-D graphene3, have drawn much attention from multiple fields due to their outstanding physical, chemical and mechanical properties4-6. These materials are deemed promising especially with respect to their potential usages as antitumor and antibacterial nanodrugs in biological systems7-13. Recently, researchers have begun to study another CNM, namely graphyne (GY)14 and its derivatives, particularly after the successful synthesis of graphdiyne (GY-2)15, due to its 1) similar allotrope structure to graphene, 2) the ethynyl group connecting each benzene ring, as well as 3) features assembling layers of sp- and sp2-hybridized carbon atoms. Many researchers have therefore speculated and proposed the material’s potential applications. Several recent studies indicated that the controllable, uniform and repeating triangular atomistic pores of GY enabled the material to be used as a superior separation device for H2 purification16-17. Recently, Fan et al. discovered that GY can be applied in water desalination, and that GY-3 and -4 not only have higher salt rejection but also possess the water permeability several orders of magnitude higher than conventional reverse osmosis membranes18. Moreover, metal-doped GYs have been shown to be potentially suitable for H2 storage due to their additional in-plane π states and enhanced binding energy to Ca19-20. Its special structure (low carbon density) endows it easier than graphene in terms of crumpling.21 GY was also reported to extract cholesterol from protein cluster, thus acting as a potential nanomedicine to remove extra cholesterol with its distinct porous structure and promising surface adhesion.22 Last but not least, the material has also been proven to exhibit high mobility and capacity of Li in its multilayer structure, suggesting a potential application for Li ion batteries23-24. However, despite of its similarity in structure with graphene and the many promising properties mentioned above, biomedical applications of GY have so far remained largely uncovered mostly due to its difficulty in experiments (GY has not yet been synthesized). In order to better understand the potential usage of this 2D



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nanomaterial in potential biological settings, we explored GY’s effect on a biological membrane using molecular dynamics (MD) simulation approaches. Our simulations revealed that GY displayed good membrane insertion and phospholipids extraction capability, despite that it was not as strong as that of graphene’s. This discovery thus indicates the potential application of GY as antibacterial nanodrug, while implying this material’s potential cytotoxicity at the same time.

Models and methods As a measure to thoroughly examine the potential effects of graphyne while in the vicinity of the membrane, we constructed three initial systems (i.e., simulation-1, simulation-2 and simulation-3) using POPC lipid bilayers. Herein, simulation-1 and simulation-2 shared similar initial configurations, with GY’s longer edge facing the membrane. In simulation-1, one carbon atom, situated at the sheet-corner and far away from the membrane was restrained (mimicking coating substrates such as stainless-steel25); while in simulation-2, there was no such positional restraint on any atom. In addition, the initial distances between the membrane and GY nanosheet were 1.8 nm and 1.0nm for simulation-1 and simulation-2, respectively. Such distances were deliberately chosen to accelerate the insertion process in the second simulation. Simulation-3 was a “docking” simulation with the GY entirely restrained, similar to our previous “docking” simulation for graphene11. This setting enables us to probe if the phospholipids extraction was due to kinetic effects or not (refer to Results section for further discussion). Additional “docking” simulations using the graphene nanosheet were performed to compare differences in extraction capability between two types of nanosheets. GY was constructed with the PyMol software package at a size of 3.929×6.236 nm2 and graphene was built using VDW software package at a size of 4.043×6.018 nm2.The materials’ parameters were attached in Supporting information.

POPC

membrane

was

constructed

from

the

link:

http://www.charmm-gui.org, with a total number of 188 lipids. Each system was solvated with water after initial simulation setting was configured, with water molecules in clash with lipid bilayersdeleted. This solvated complex was then


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simulated with molecular dynamics (MD) method that has been widely used in the studies of biomolecules26-30 and nanomaterials31-43. All MD simulations were carried out with software package GROMACS (version 4.6.6)44. We then utilized the VMD software45 to visualize and analyze the simulation results. The CHARMM 36 force field46-48 and TIP3P water model49 were adopted for POPC molecules and water molecules, respectively. The temperature and pressure were fixed at 300K and 1atm using v-rescale thermostat50 and Berendsen barostat (semi-isotropic pressure)51, respectively. Periodic boundary conditions were applied in all directions. The long-range electrostatic interactions were treated with PME method52-53, and the van der Waals (vdW) interactions were calculated with a cutoff distance of 1.0 nm. All solute bonds were maintained constant at their equilibrium values with the LINCS algorithm54, while water geometry was also constrained using the SETTLE algorithm55.

Results In our previous study11, we found that graphene nanosheets can penetrate into and extract large amounts of phospholipids from E. coli cell membranes because of the strong dispersion interactions between graphene and lipid molecules. This insertion and extraction result in membrane stress and subsequent cell death, thus indicating graphene’s potential cytotoxicity. While the structure of GY highly resembles the structure of graphene, the biological effects of GY remain largely unknown. We thereby follow the previous approach11 to examine the cytotoxicity of GY by exploring its interaction with cell membranes, with simulations carried out using GY nanosheet and POPC membrane. Firstly, we started with simulations of a system (named as simulation-1) containing one GY nanosheet and POPC membrane, as shown in Figure 1A. In this system, GY was initially placed 1.8 nm above the membrane, with a carbon atom at one corner (large vdW sphere) marked as the restrained atom in simulations. Figure 1 illustrates one typical trajectory with the interactions between GY and the membrane



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at various key time points throughout the simulation. At ~30 ns, GY began to touch the POPC surface (Figure 1B). The insertion of GY into POPC lasted ~10 ns, as shown in Figure 1C. At 48 ns, two phospholipids began to climb up to the surface of GY. From 48 ns to 50 ns, while the extraction continued, one phospholipid had obviously been entirely pulled up, with the entire membrane moved up slightly (moving towards GY; Figure 1D-E). Finally at 68 ns, the insertion of GY into the membrane was prominent enough, and the number of phospholipids extracted was no longer negligible, with the membrane shifted towards GY by a significant amount.

Figure 1. Time evolution of GY nanosheet insertion and lipid extraction of simulation-1. (A-F) Snap-shots of the simulation system at 0, 30, 40, 48, 50 and 68 ns, respectively. The GY nanosheet is shown as a yellow-bonded sheet with the restrained atom shown as the yellow vdW sphere. The nitrogen and phosphorus atoms of POPC membrane are displayed by blue and tan vdW spheres while the rest of the membrane structure is illustrated with lines. The key extracted lipids are presented with cyan (carbon) and red (oxygen) vdW spheres.


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Figure 2. Atom contact number, vdW energy (A) and center of mass distance (B) between POPC membrane and GY nanosheet as a function of simulation time. Green and red shaded areas indicate the periods of insertion and extraction, respectively.

In order to quantify the entire process, we calculated the atom contact number (ACN), vdW energy and center of mass (COM) distance between GY and membrane, as a function of the simulation time (Figure 2). Herein, the atom contact number was defined as the number of lipid heavy atoms that were within 0.6 nm from any GY surface atoms. As shown in Figure 2, from 0 to 30 ns, changes in ACN (ACNs of other two trajectories were shown in Figure S1) and vdW energy were almost negligible, whereas fluctuations of the COM distance were prominent due to the movement of GY in an environment filled with water. From 30 ns to 40 ns (namely the insertion course), ACN increased by a small percentage while vdW energy decreased, indicating an enhanced interaction between GY and the membrane, while the COM distance exhibited little change. Starting from 40 ns, GY underwent two periods, namely i) andante extraction period and ii) speedy insertion and extraction period. From 40 ns to 50 ns (andante extraction period), the increase in ACN was modest, while vdW energy and COM distance decreased slightly, suggesting the extraction was slow in the early stage. The slow-moving trend also implies that the phospholipids packed on the GY surface still exhibit relatively strong interactions with both the POPC membrane and the GY nanosheet. These phospholipids thus cannot migrate quickly and completely to the GY surface, yet cannot fall back to the membrane either. This situation would change drastically once one phospholipid was



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drawn almost entirely away from the membrane (as shown in Figure 1E), marking the start of the speedy insertion and extraction period. ACN increased almost exponentially while vdW energy decreased sharply. The increasing and decreasing tendencies of ACN and vdW were largely symmetrical, suggesting that vdW interaction is the main reason behind the insertion and extraction processes. Meanwhile, the COM distance exhibited rapid decline. These observations suggested that in the speedy insertion and extraction period, many phospholipids were extracted quickly from the membrane (Figure 1F), largely because of their strong vdW attractions with GY, while this strong interaction also caused the membrane to shift upward (Figure 1 B).

Figure 3. Analyses of simulation-2. Atom contact number and vdW interaction energy between POPC and GY nanosheet (A). The thickness of POPC membrane as a function of simulation time (B). Time evolution of GY nanosheet inserting into the POPC membrane (C). The color settings are same to Figure 1, but without one restrained carbon atom and key lipids. In this system, we just show the insertion process with GY nanosheet having no restraint.

In the above simulation, we applied a position restraint by fixing one carbon atom and discovered that GY nanosheet could insert into POPC membrane while extracting


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large amounts of phospholipids during the process. We continued with a second simulation without imposing any restraint (namely simulation-2), to mimic free GYs in solution (as opposed to GY or graphene coated on stainless steel etc25). The result indicated that GY nanosheet could penetrate into POPC membrane regardless. Figure 3 illustrates the evolution of interactions between GY and the membrane in one representative trajectory (two other ACN curves were shown in Figure S2). GY was placed 1.0 nm instead of 1.8nm above the membrane so as to accelerate the insertion process. In this simulation, GY started contacting with the membrane at 14 ns. From 14 ns to 28 ns, GY promptly penetrated into the membrane. ACN increased from ~0 to ~764 while vdW energy decreased from ~0 to ~-641.12 kcal/mol. The thickness of membrane increased from ~49.45 Å to ~64.02 Å and reached the maximum at ~28ns (Figure 3C). During this period, only a few numbers of phospholipids were attracted by GY. They climbed up to the GY surface (i.e., extraction) and formed an obvious “embossment” (layers of lipids). This membrane “embossment” was caused by the strong interaction of GY and membrane (including both vdW and hydrophobic interactions). At 40 ns, the insertion arrived at its equilibrium state, with GY largely “buried” into the membrane. The thickness of the membrane recovered from ~64.02 Å to ~58.96 Å. After 40 ns, ACN and vdW energy kept almost constant at ~1073 and ~-898.89 kcal/mol, respectively. The thickness of membrane also reduced only slightly after 40 ns. It is noted that when the GY size is larger than the one used in simulation, not all GY surface can be buried inside the membrane. Thus, the part of GY that is exposed to water can extract lipids from membrane, as shown in the simulation-1.



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Figure 4. Time evolution of fully docked and restrained GY nanosheet extracting the lipids (simulation-3). (A-D) Snap-shots of the simulation system at 0, 5.2, 14.4, 30.0 ns, respectively. (E-F) Front and back views of the same state shown in (D). Color settings are as Figure 1, but without one restrained carbon atom.

To further validate that lipid extraction is indeed possible and not caused by potential confounds such as kinetic effects, we performed an additional GY “docking” simulation following the similar approach in our previous work for graphene11, namely simulation-3. In this simulation, position restraints were applied such that all GY atoms were fixed during the entire simulation process. Figure 4 demonstrates the trajectories at key time points throughout the simulation. The GY nanosheet was placed above the POPC membrane with its orientation perpendicular to the membrane surface and its tail fully contacting the membrane (Figure 4A). Only after 5.2 ns, one lipid had already climbed up to the GY surface, as shown in Figure 4B. At 14.4 ns, nearly half of the GY surface was covered by phospholipids (Figure 4C). Merely 16 ns later, GY was already completely packed by phospholipids (Figure 4D). After


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examining the last few trajectory snapshots from different orientations (including Figure 4D, E and F), we discovered that GY was tightly carpeted with two layers of phospholipids, while our previous graphene docking simulation also showed phospholipids climbing up to the graphene surface11. One thing to note however, is that we used a mixed POPE/POPG lipid bilayer in previous work to mimic E. coli membrane, instead of the POPC lipid bilayer in the current study. In order to compensate for this difference, we carried out additional simulations with a comparable graphene nanosheet (namely simulation-4) to probe the differences between GY and graphene in terms of their lipid extraction capacities. Figure 5A illustrates the ACNs of six trajectories under both GY and graphene “docking” simulations. The ACN trajectories clearly show that GY extracts lipids slower than graphene, indicating a weaker extraction capability of GY. Figure 5B further shows the initial “adsorption rate” (i.e., derivatives of the lipid extraction at early fast extraction stage, as shown by the black bars) and final “adsorption capacity” (i.e., the total lipid adsorption amount per unit area once at equilibrium stage, as shown by the red bars) for each trajectory. Once again, both the adsorption rate and capacity imply that graphene has a relatively stronger membrane extraction capability than GY.

Figure 5. Comparison of “docking” simulations for GY and GRA (graphene) nanosheets. (A) Atom contact number (ACN) of six trajectories for two kinds of simulation systems. (B) The mean adsorption rate at quick extraction stage for every trajectory (black) and adsorption capacity (i.e., adsorption amount per unit area, red) for every trajectory from 80 ns to 100 ns.



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In order to better understand the mechanism behind observed differences in extraction capability of the two similar nanomaterials, we continued our analyses with the free energy perturbation (FEP) method53,54, which aims to calculate the free energy differences during processes of lipid’s extraction from the membrane to the nanomaterial’s surface. Figure 6 shows the thermal dynamic cycles used in our FEP method when calculating the free energy difference ∆∆G. According to this cycle, ∆∆ ∆ ∆ ,

(1)

where ∆∆G is the free energy difference for transferring a lipid from the membrane to the GRA/GY surface. To calculate ∆G1 and ∆G2 with the FEP method, a lipid on the GRA/GY surface and a lipid in the membrane are annihilated, as shown in Fig. 6a and 6b, respectively. Furthermore, ∆G1’ and ∆G1” refer to free energy changes for annihilating a lipid on the GY and GRA surfaces, respectively.


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Figure 6. An illustration of the thermodynamic cycle for computing ∆∆G in the process of transfer a lipid from the membrane to the graphyne surface (a) and to the graphene surface (b).

Theoretically, ∆G can be computed from the following ensemble average53,

∆- B ln〈

B

〉

(2)

where kB is the Boltzmann constant; T is the temperature; Vi and Vf are Hamiltonian at the initial (i) and the final (f) stages. In order to accurately capture the process, multiple intermediated stages (denoted by λ) are inserted, enabling a gradual annihilation procedure. λ equals to 0 and 1 for the initial (with a lipid equilibrated on its substrate) and the final (with a lipid annihilated on its substrate) states, respectively.

Figure 7. The cumulative free energy differences ∆G(λ) (=G(λ)-G(0)), averaged over ten independent simulations, for annihilating a lipid in the membrane (∆G2), on the graphene surface (∆G1”) and on the graphyne surface (∆G1’).

Figure 7 shows the cumulative free energy differences during a lipid annihilation process. When λ=1, ∆G1” > ∆G1’ > ∆G2. Thus, the free energy change is least when annihilating a lipid from the membrane, suggesting that it is energetically favorable to extract a lipid from the membrane to the GY/GRA surface (∆∆G

Membrane Insertion and Phospholipids Extraction by Graphyne

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