Temperature-Dependent Lipid Extraction from Membranes by Boron

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Temperature-Dependent Lipid Extraction from Membranes by Boron Nitride Nanosheets Zhen Li, Yonghui Zhang, Chun Chan, Chunyi Zhi, Xiaolin Cheng, and Jun Fan ACS Nano, Just Accepted Manuscript • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

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Temperature-Dependent Lipid Extraction from Membranes by Boron Nitride Nanosheets Zhen Li,† Yonghui Zhang,† Chun Chan,† Chunyi Zhi,†* Xiaolin Cheng,‡* and Jun Fan†¶┴*

†

Department of Materials Science and Engineering, City University of Hong Kong, Hong Kong,

China ‡

Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State

University, Columbus, Ohio, United States of America ¶

City University of Hong Kong Shenzhen Research Institute, Shenzhen 518057, China

┴

Center for Advanced Nuclear Safety and Sustainable Development, City University of Hong

Kong, Hong Kong, China

*Corresponding Author E-mails: [email protected] (Chunyi Zhi), [email protected] (Xiaolin Cheng), and [email protected] (Jun Fan)

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ABSTRACT

Two-dimensional (2D) materials can mechanically insert into cell membranes and extract lipids out, thus leading to the destruction of cell integrity. On the one hand, the cytotoxicity of 2D materials can be harnessed in surface engineering to resist biofouling; on the other hand, it causes great concern on in vivo biomedical applications ranging from drug delivery to nanoimaging. To understand the nature of this cytotoxic behavior and find strategies to control it, we performed molecular dynamics (MD) simulations on the lipid extraction of hexagonal boron nitride (BN) nanosheets from lipid membranes. Interestingly, we observed that the lipid extraction behavior suddenly disappears as temperature decreases. Structural analyses revealed that this temperature dependence is related to the lipid membrane phase transition, which was confirmed by an additional membrane model with phase state regulated by cholesterol. The potential of mean force (PMF) calculation was adopted to clarify the thermodynamic origin of these results, which also indicates directions to adjust the lipid extraction behavior of nanomaterials. Overall, this work suggests that the cytotoxic mechanical interactions between 2D materials and cell membranes can be controlled by temperature and other factors which can induce phase transitions of lipid membranes, and that the thermodynamic threshold of the lipid extraction varies for surfaces with different curvature. This work clarifies the thermodynamics in the lipid extraction phenomenon of 2D materials, and indicates possible strategies to adjust the antibacterial performance or cytotoxicity of 2D materials.

KEYWORDS 2D materials, boron nitride, cytotoxicity, lipid membrane, lipid extraction, phase transition, molecular dynamics simulations


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Two-dimensional (2D) materials are playing an increasingly important role in science and industry due to their structural feature as well as extraordinary electrical, thermal, mechanical properties. In nanomedicine, 2D materials provide versatile platforms for biofunctionalization, yielding striking applications in nano-imaging, nano-sensing, drug delivery, etc.1-4 In particular, boron nitride (BN) nanosheet has attracted exceptional interests because of its distinctive chemical stability compared with carbon-based nanomaterials.5-7 Despite promising perspective, many problems are still challenging the in vivo applications of BN nanosheets and other 2D materials, one of which is cytotoxicity.8-11 For example, when exposed to graphene nanosheets, the metabolic activity of neuronal PC12 cells can dramatically decrease, and the cell morphology can be damaged.12 However, the cytotoxicity of 2D materials is a double-edged sword; it can also be harnessed in materials engineering to obtain antibacterial surfaces.13-15 For specific applications, we must be able to control the cytotoxicity of 2D materials; and understanding the nature of it is a prerequisite. There have been a large number of studies on the cytotoxicity of nanomaterials, which indicated that the biocompatibility of nanomaterials greatly depends on their geometry (shape, size),16, 17 concentration,12, 18 surface properties,19-21 and so on. However, the mechanisms are elusive, and many conclusions in the literatures are at odds. Graphene oxide was found toxic in some studies,22, 23 but, was also reported to be nontoxic and even capable of enhancing cellar growth.24 Polyethylene glycol (PEG) chains can be used to modify nanomaterials for better biocompatibility,25 but strong immunological responses were recently observed for PEGylated graphene oxide.26 The study on the cytotoxicity of BN nanomaterials is very preliminary, and few existing studies also sparked controversy. Chen et al. reported that BN nanotubes are


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nontoxic to human embryonic kidney 293 cells;27 however, Horváth et al. found that BN nanotubes are even more cytotoxic than carbon nanotubes to the same kind of cells.28 The cytotoxicity of 2D materials results from chemical, physical and mechanical interactions between 2D materials and soft biological structures.8 To understand the cytotoxicity of 2D materials in greater depth and resolve existing debates, molecular dynamics (MD) simulations have been employed to reveal molecular insights into the cytotoxic interactions between 2D materials and biological components including proteins,29,

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DNA,31 and cell

membranes.23, 26, 32-37 Especially, the cell membrane is the initial part of a cell to interplay with 2D materials, potentially leading to the destruction of cell integrity and dysfunctions. Using MD simulations, the interaction between cell membranes and graphene nanosheets with different size,32, 33, 35 shape,33, 36 and oxidization degree34, 35 have been studied, which uncovered some cellular internalization pathways and cytotoxicity mechanisms. Of particular interest is that 2D materials were found to be able to penetrate and extract lipids from cell membranes; and in this way, generating pores and leading to the destruction of cell integrity.23, 38, 39 The lipid extraction behavior is resulted from short-range van der Waals attractions and strong hydrophobic interactions between lipid tails and 2D materials;23 when piercing cell membranes, 2D materials robustly extract lipids to decrease the water/nanosheet interface energy. Therefore, the lipid extraction should be a basic motion of 2D materials when interplaying with cells membranes. If the lipid extraction process can be prevented, the cytotoxicity of 2D materials should be mitigated. To some extent, controlling the lipid extraction behavior of 2D materials means controlling their cytotoxicity. Motivated by this, we aimed to reveal the molecular dynamic details and the thermodynamic origin of the lipid extraction


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process and find possible strategies to control the lipid extraction behavior and cytotoxicity of 2D materials. Thinking from a molecular view, lipids in the vicinity of 2D materials should be under a “tug-of-war” between the nanosheet and the membrane. To stop the lipid extraction, definitely we can weaken the strength of the nanosheet through controlling the properties of 2D materials (hydrophobicity, surface modification, geometry, etc.); for example, surface modification with proteins or polymers has proven effective to reduce the cytotoxicity of 2D materials.19-21 In addition, enhancing the strength of the membrane should also work. The strength of membranes in the tug-of-war may be affected by membrane components, electrical field, ion concentration, pH value, temperature, and so forth; they are all possible factors to adjust the lipid extraction (mechanical cell-destruction) behavior of 2D materials. However, these factors have been scarcely studied although they may explain experimental results and controversies. Here, we report a temperature effect on the lipid extraction from lipid membranes by 2D materials. With MD simulations, we found that the lipid extraction behavior of BN nanosheets is greatly temperature-sensitive. Molecular insights and mechanisms for this characteristic were clarified in this work. The tug-of-war between the nanosheet and the membrane was quantified with potential of mean force (PMF) calculations. These results are important for future studies on adjusting the cytotoxicity or antibacterial performance of 2D materials.


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RESULTS AND DISCUSSION

BN Nanosheets Can Extract Lipids from Membranes. To simulate the interaction between BN nanosheets and lipid membranes, a piece of rectangular BN nanosheet was horizontally placed above pre-equilibrated lipid membranes at a distance (Figure 1, at 0 ns). An atom at one corner was restrained in position, as the models reported by Tu et al.23 to mimic the “blades” role of nanosheets22 in experiments. We firstly performed simulations with POPC (1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine) lipid membranes, and found that BN nanosheets can insert into membranes and extract lipids out (Figure 1 and the movie in Supporting Information) as graphene nanosheets do. As shown in Figure 1, the BN nanosheet first randomly moves away from the initial configuration. Gradually, the edge of the BN nanosheet binds to the lipid membrane (30 ns), and lipids climb onto the BN nanosheets (70 ns). As the lipid extraction process goes on, the BN nanosheet penetrates the lipid membrane (200 ns). The adsorption of lipids on the surface of BN nanosheets was further confirmed in experiments (Figure S1 and section SI-1 in Supporting Information).

Figure 1. The lipid extraction process of the BN nanosheet from the POPC lipid membrane at 290 K. The BN nanosheet (yellow) was horizontally place above the membrane, with an atom at one corner (yellow sphere) restrained in position. Water is shown in transparent surface, lipids in membrane are shown in lines, and extracted lipids are shown in spherical atoms.


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Temperature-Dependent Lipid Extraction. The model of the POPC bilayer was further simulated at 300 K and 310 K, and the POPC lipid extractions were all observed (Figure S2 in Supporting Information). Then, we performed simulations with DMPC (1,2-dimyristoyl-snglycero-3-phosphocholine) membranes at the three temperatures. At 300 K (Figure 2A) and 310 K (Figure S2 in Supporting Information), similar to POPC membranes, BN nanosheets also extract lipids from DMPC membranes. However, the DMPC lipid extraction behavior suddenly disappears at 290 K (Figure 2C). To uncover more detailed dynamic information, we tracked the z positions of lipids and the bottom of the BN nanosheet, as shown in Figure 2B and 2D. At 300 K, after touching the DMPC membrane, the BN nanosheet extracts lipids towards positive z-axis and gradually inserts into the DMPC membrane (Figure 2B). However, at 290 K, although the BN nanosheet binds to the DMPC membrane quickly (at ~20 ns), it stays there and no lipid can be extracted from the membrane (Figure 2D). In short, the lipid extraction was observed at 290, 300, and 310 K for POPC membranes; however, for DMPC membranes, the lipid extraction happens at 300 and 310 K but disappears at 290 K. Thus, we speculated that the lipid extraction should be a temperature-dependent behavior. We also performed simulations with larger fully restrained BN nanosheets, which show the temperature-dependent lipid extraction more clearly (see Figure S3 in Supporting Information). The elimination of DMPC lipid adsorption on BN surfaces at low temperature was also observed in experiments (Figure S4 in Supporting Information). This temperature dependence is very interesting and meaningful. It suggests that the mechanical interactions between cell membranes and 2D materials could be controlled by temperature; thus, temperature may affect the antibacterial performance and cytotoxicity of 2D materials. Song et al. reported their experimental result that elevated temperature can enhance


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the disinfection effects of graphene oxide treatment,40 which may be explained by this temperature-dependent lipid extraction phenomenon.

Figure 2. Temperature-dependent lipid extraction from DMPC membranes. (A) The BN nanosheet extracts lipid from the DMPC membrane at 300 K, but (C) the lipid extraction disappears at 290 K. (B) and (D) show the z-position evolution of the phosphorus atom in lipids and the bottom of the BN nanosheet at 300 K and 290 K, respectively. To confirm the universality of the temperature-dependence of the lipid extraction, the membranes of Escherichia coli were also simulated. In the work of Tu et al.,23 graphene and graphene oxide nanosheets can extract lipids from the inner and outer membranes of Escherichia coli at 310 K, but the temperature dependence was not studied. We built the models according to the work of Tu et al.,23 with the outer membrane modelled by pure POPE (1-palmitoyl-2-oleoylsn-glycero-3-phosphoethanolamine) (lipopolysaccharides (LPS) were omitted for simplicity,


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please see section SI-5 in Supporting Information for detail) and the inner membrane modelled by 3:1 mixed POPE and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol))). Here, we found that as the temperature decreases to 290 K, the lipid extraction by BN nanosheets disappears for the pure POPE membrane (the outer membrane of Escherichia coli); for the POPE-POPG membrane (the inner membrane of Escherichia coli), the lipid extraction disappears when decreasing temperature to 280 K (see Figure S5 in Supporting Information). Taken together, the lipid extraction behavior is generally temperature-dependent for different lipid membranes, although the critical temperature varies. For the POPC membrane, the lipid extraction happens at 290, 300, and 310 K, but a much lower temperature should also stop it. The critical temperature to switch on/off the lipid extraction varies for different lipid membranes, which may be a reason for that the cytotoxicity of 2D materials changes for different kinds of cells.41

The Temperature Dependence is Related to Phase Transition. The temperature dependence of the lipid extraction is interesting and it hints a possible strategy to adjust the antibacterial performance or cytotoxicity of 2D materials; but what is the reason? We projected the averaged distribution of lipid head-to-tail vectors onto the x-y plane, and found that the orientation of DMPC is well ordered at 290 K (Figure 3A). However, the orientation of DMPC at 300 K is disordered (Figure 3B). DMPC at 310 K and POPC at 290, 300, and 310 K are all disordered (Figure S6). That is to say, the lipid extraction cannot happen for ordered lipid membranes, and vice versa. The splay angle distribution of DMPC at 290 K is quite narrow compared with other cases (Figure 3C and Figure S7 in Supporting Information). The bending rigidity, KC, for the DMPC membrane at 290 K is extremely larger than other cases (Figure 3D). Obviously, as temperature decreases to 290 K, the DMPC membrane transforms from liquid phase into gel


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phase. Therefore, the temperature-dependent lipid extraction should be related to such a disorderorder phase transition of lipid membranes.

Figure 3. The averaged distribution of lipid head-to-tail (from the phosphorus atom to the two terminal carbon atoms in tails) vectors projected onto the membrane plane for the DMPC membrane at (A) 290 K and (B) 300 K. (C) Normalized probability density of finding a pair of lipids at an angle α with respect to each other. (D) The bending modulus of lipid bilayers. The disordered DMPC bilayer transforms into ordered phase and the lipid extraction disappears when decreasing temperature from 300 K to 290 K, which is consistent with the phase transition temperature (Tm) of the DMPC bilayer at about 297 K in experiments.42 The Tm of POPE is about 298 K;42 therefore, decreasing temperature from 300 K to 290 K also stops the lipid extraction from the POPE membrane in our simulations (Figure S5 in Supporting Information). POPG is a lipid with much lower Tm (271 K);42 therefore, mixing POPG with POPE could lower the Tm of POPE, and the critical temperature for the lipid extraction decreases (Figure S5 in Supporting Information). An extremely lower temperature is needed to stop the lipid extraction from POPC membranes because the Tm of POPC is about 271 K,42 which is why we observed lipid extraction even at 290 K (Figure 1).


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Figure 4. (A) The addition of 30 mol% cholesterol (Chol.) into the DMPC membrane could dramatically enhance its bending modulus, KC. (B) The addition of cholesterol greatly prevents the lipid extraction, and only two DMPC molecules were extracted out at 300 K (in comparison with pure DMPC at 300 K as shown in Figure 2A). (C) The z-evolution of the BN nanosheet and DMPC lipids. The cholesterol molecules in (B) are shown in red.

In addition to temperature, factors including electrostatic effects43 and the addition of cholesterol44 also affect the phase state of lipid membranes. To further confirm the relationship between the lipid extraction and the phase state of lipid membranes, the ordering effect of cholesterol was also studied. Specifically, 30 mol % cholesterol molecules were added to the disordered membrane of DMPC at 300 K. The addition of cholesterol induces the ordering of DMPC lipids and thus dramatically increases the bending rigidity of the lipid membrane (Figure 4A). With this mixed membrane, the lipid extraction of the BN nanosheet was studied. As shown in Figure 4B, although there are also lipids extracted, the amount is extremely limited (only two


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at the end of the simulation). With tracking the z-position of the BN nanosheet and DMPC lipids (Figure 4C), we can find that the BN nanosheet can hardly extract lipids although it binds to the surface of the membrane at very early stage. Comparing the DMPC membrane with and without cholesterol at 300 K (Figure 2AB and Figure 4), we confirmed that the lipid extraction is closely related to the phase state of lipid membranes. Thermodynamics in the Lipid Extraction “Tug-of-War”. As mentioned earlier, lipids in the vicinity of 2D materials are under a “tug-of-war” between the nanosheet and the lipid membrane. When the strength of the nanosheet dominates the tug-of-war, it extracts lipids from the membrane. The phase transition induced by temperature or cholesterol may enhance the strength the membrane, thus hindering the lipid extraction process. The strength of the nanosheet and the membrane in the tug-of-war is a reflection of the thermodynamic preference of lipids. To quantify the thermodynamic preference, we performed PMF calculations for pulling a lipid molecule away from the membrane (Figure 5A1 and 5C1) or from the BN surface (Figure 5A2 and 5C2). While lipids moving away from their initial positions, the PMF values firstly increase and finally reach a plateau, at which distance the steered lipid and the membrane (or the BN nanosheet) no longer interact with each other. For POPC at 290, 300, and 310 K, the effect of temperature on the PMF of pulling a lipid away from the membrane and from the BN surface is negligible (Figure 5A). Moreover, the free energy barrier of pulling a lipid away from BN surface (∆GPOPC/BN) is obviously larger than that from the lipid membrane (∆GPOPC/membrane), demonstrating that staying on the surface of the BN nanosheet is a lower free energy state than staying in the membrane. That explains why POPC lipids can be extracted by the BN nanosheet from the membrane (Figure 1 and Figure S2 in Supporting Information).


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For each kind of 2D material, the ∆G of pulling a kind of lipid from its surface (∆Glipid/nanosheet, as the ∆GPOPC/BN in Figure 5A2) should be almost a temperature-independent constant which is determined by the surface properties of the 2D material. This constant can be taken as a “thermodynamic threshold” for the lipid extraction; the lipid extraction is theoretically possible when the threshold (∆Glipid/nanosheet) is larger than ∆Glipid/membrane. We plot such a relationship into a thermodynamic diagram as shown in Figure 5B, the thermodynamic threshold splits the diagram into two regions (red and blue). The ∆GPOPC/membrane at 290, 300, and 310 K are all located in the red area (extraction area), where the lipid extraction is a thermodynamically favorable behavior.

Figure 5. (A) Potential of mean force (PMF) for pulling a POPC molecule away from (A1) the membrane and from (A2) the surface of BN nanosheet. (B) Thermodynamic diagram for POPC/BN systems which contains free energy barrier for pulling a POPC molecule away from the membrane (rhombus dots) and from the surface of BN nanosheet (the solid line on the


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boundary of the red area and the blue area). (C) and (D) are results for DMPC corresponding to POPC results in (A) and (B), respectively. The thermodynamics for DMPC models were also calculated and analyzed (Figure 5C and 5D). The PMF of pulling a lipid from the pure DMPC membrane at 280 K and 295 K was also studied (the dash lines in Figure 5C1) to show the phase transition more clearly. We have learnt that the DMPC membrane transforms from disordered phase into ordered phase when decreasing temperature from 300 K to 290 K (Figure 3). Accordingly, the ∆GDMPC/membrane dramatically changes and it jumps from the red area into the blue area (Figure 5D). That is why the DMPC lipid extraction disappears at 290 K (Figure 2). Moreover, at 300 K, the addition of cholesterol could greatly increase the ∆GDMPC/membrane (the green line in Figure 5C1), shifting the thermodynamic point towards the blue area (the green dot in Figure 5D). Because of this thermodynamic shifting effect, the lipid extraction process can be slowed down by the addition of cholesterol (Figure 4). Based on these thermodynamic results, we conclude that the lipid extraction is a thermodynamically selected result. Decreasing temperature and adding cholesterols could increase the preference of lipids staying in membranes (∆Glipid/membrane), thus inhibiting the lipid extraction process. The value of ∆Glipid/membrane should be different for different cell types, which could explain why the cytotoxicity of 2D materials varies for different cells.41 To adjust the antibacterial performance or cytotoxicity of 2D materials, we may control the ambient environment of cells such as temperature and other factors that affect the value of ∆Glipid/membrane.

Tuning the Thermodynamic Threshold by Curving Surfaces. Based on the thermodynamic diagram (Figure 5B and 5D), mitigating the cytotoxic lipid extraction behavior means shifting the value of ∆Glipid/membrane from the red area towards the blue area. Decreasing temperature and


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adding cholesterol are effective as demonstrated above; in addition, we can shift the thermodynamic threshold (∆Glipid/nanosheet) to change the boundary of the blue and red regions. That is another part to adjust the cytotoxic interactions between 2D materials and cell membranes: controlling the strength of 2D materials in the “tug-of-war”.

Figure 6. (A) Potential of mean force (PMF) for pulling a DMPC molecule away from convex, flat, and concave BN surfaces. (B) The thermodynamic diagram for the system of DMPC membrane and convex/flat/concave BN surface. ∆Glipid/nanosheet should be decreased by weakening the interactions between 2D materials and membranes, such as surface modification with proteins and polymers. Besides, surface curvature also affects the interaction between nanomaterials and biomolecules.45-48 We compared the thermodynamic threshold of convex, flat, and concave BN nanosheets for DMPC (Figure 6), using a half (10, 10) BN nanotube to model the convex and concave surfaces. The atom number of the flat BN flake here is the same as that of the convex and the concave BN flakes, to eliminate the size effect. As shown in Figure 6A, the free energy barrier (thermodynamic threshold) of pulling a DMPC away from the flat BN surface is ~25 kcal·mol-1 which is almost


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identical as calculated with the infinite flat BN surface (Figure 5C2). The PMF profile is dramatically affected by curvature, with ∆GDMPC/BN decreased by the convex surface and increased by the concave surface. These changes greatly affect the thermodynamic diagram (Figure 6B). With the convex BN surface, the thermodynamic threshold (∆GDMPC/BN) decreases to the lower limit of ∆GDMPC/membrane; thus DMPC lipids cannot be extracted by the convex BN surface even at 300 K and 310 K. With the concave BN surface, all the ∆GDMPC/membrane values are located within the extraction area (the red area), demonstrating its enhanced lipid extraction ability.

Figure 7. The side view (left) and the top view (right) of the DMPC extraction by a curved BN nanosheet at 300 K. The BN nanosheet is constructed by a half of a (10, 10) BN nanotube.


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Based on these PMF calculation results, the surface curvature of 2D materials may be used to tune the lipid extraction behavior. For example, according to Figure 6B, at 300 K, the flat BN nanosheet can extract DMPC from membrane; the concave BN surface can promote the extraction; however, the convex BN surface should not be able to extract DMPC from the membrane. To confirm the PMF results, we performed an MD simulation at 300 K with a half (10, 10) BN nanotube and DMPC membrane (Figure 7). It is observed that the curved BN nanosheet inserts into membrane, which is followed by the DMPC lipid extraction. Interestingly, DMPC lipids only climb onto the concave side of the BN nanotube (Figure 7), verifying the above thermodynamic analyses.

CONCLUSION The lipid extraction behavior of 2D materials is like a “tug-of-war” between 2D materials and cell membranes. If the strength of 2D materials dominates, the lipid extraction happens, and vice versa. We observed that decreasing temperature can stop the lipid extraction, and this temperature dependence is related to the phase transition of lipid membranes. Potential of mean force (PMF) calculations quantified the tug-of-war, showing that the lipid extraction is determined by the free energy barrier of pulling a lipid away from the nanosheet surface (∆Glipid/nanosheet, thermodynamic threshold) and from the membrane (∆Glipid/membrane). A thermodynamic diagram associates these free energy barriers and the lipid extraction, clearly explaining the temperature dependence and the effect of cholesterol. Moreover, the thermodynamic threshold can be changed by the surface curvature of nanosheets, which switches on/off the lipid extraction.


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The lipid extraction is a basic behavior in mechanical interactions between 2D materials and cell membranes. With adjusting the lipid extraction, we can control the insertion of 2D materials into cell membranes, and the destruction of cell membranes may be controlled. The thermodynamic diagram presented in this work indicates directions: shifting ∆Glipid/membrane or ∆Glipid/nanosheet. The value of ∆Glipid/membrane can be changed by temperature and other factors that induce phase transitions of cell membranes; the value of ∆Glipid/nanosheet can be tuned by curvature, surface modification, etc. The thermodynamic framework in this work can quantify lipid-nano interactions and relate them to the cytotoxicity of nanomaterials, which could facilitate future studies in nanotoxicology and nanomedicine. Decreasing temperature for mitigating the cytotoxicity of 2D materials for in vivo applications may be still impractical at present; but according to the physics uncovered in this work, the cytotoxic lipid extraction is tightly related to the phase behavior of lipids which not only depends on temperature but also can be affected by membrane component, protein behaviors, metabolic activities, etc.49 Thus, this work is also expected to trigger future studies on biological activities involving nanomaterials. In addition, controlling temperature is meaningful and practical for the in vitro application of 2D materials in engineering (e.g., antibacterial) surfaces. The lipid bilayer models studied in this work are still far away from real cell membranes which are mixtures of lipids, proteins, carbohydrates, and so forth. For example, the outer membrane model of Escherichia coli (pure POPE lipids) neglects LPS which should also affect the interaction between nanomaterials and lipids. Highly accurate models have been developed in recent years;50-53 more sophisticated cases should be taken into account using these models in future studies.


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METHODS Models. Three basic building blocks were used to assemble our initial models: pre-relaxed lipid bilayers, BN nanosheets, and water. To obtain pre-relaxed lipid bilayers, membranes containing 64 lipids (32 in each upper leaflet and 32 in each bottom leaflet) were generated using CHARMM-GUI Membrane Builder54-59. Then, the lipid membranes were simulated at different temperatures for 300 ns in the NPT ensemble, to obtain fully relaxed lipid bilayer (RLB) building blocks. Four kinds of BN building blocks were used, including the square BN nanosheet with the dimension of ~ 45 × 45 Å2 (type A), the infinite periodic BN nanosheet (type B), original or flattened half (10, 10) BN nanotubes (type C), and the rectangular BN nanosheets with the dimension of ~ 50 × 70 Å2 (type D). The TIP3P water model was adopted in all simulations. Models for lipid extraction simulations were constructed with 2 × 2 extended RLB building blocks (128 lipids in the upper leaflet and 128 lipids in the bottom leaflet), water molecules, and type A or type D BN nanosheets. The type D BN nanosheet was only used in docking simulations (Figure S3 in Supporting Information), in which the type D BN nanosheet was fully restrained in space and initially placed perpendicular to lipid membranes. In other models, the type A BN nanosheet with only an atom at one corner restrained was used, and its plane was initially parallel to lipid membranes as shown in Figure 1. These two BN models are further discussed in Supporting Information (section SI-7). Models for membrane phase analyses and PMF calculations were assembled by RLBs that contain 64 lipids or type B BN nanosheets and water molecules. Type C BN nanosheets, the RLB of DMPC at 300 K, and water molecules were used when studying the effect of curvature (Figure 6 and 7).


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Simulation

Setup.

MD

simulations

were

performed

with

GROMACS

5.0.660

(www.gromacs.org) package. The CHARMM 36 force field61 was employed for lipids, TIP3P water model62 was used, and BN nanosheets were modeled with the non-bonded force field parameters developed by Won and Aluru.63, 64 The bonds, angles, and dihedrals of the type A BN nanosheet were described with the universal force field (UFF).65 The periodic boundary condition was applied along all the three dimensions. All the lipid extraction models with the type A BN nanosheet were simulated in the NPT ensemble for 200 ns with a time step of 2 fs. The pressure of the models was maintained at 1 bar with the Parrinello-Rahman barostat,66 and temperature was kept by the V-rescale thermostat.67 We treated the electrostatic interactions with the particle mesh Ewald (PME) method,68 and a cutoff distance of 12 Å was used for van der Waals interaction calculations. Potential of Mean Force (PMF) Calculation. The PMF profiles were calculated with the umbrella sampling method69-71 and the g_wham tool in GROMACS. The interval of umbrella sampling windows is 1.2 Å. In each window, the distance between the phosphorus atom of the pulled lipid and its starting position along the z-axis was restrained with a harmonic force constant of 1000 kJ·mol-1·nm-2. Each window was simulated for 25 ns, and the PMF results reached convergence for the last 15 ns (see Figure S8, S9 and S10 in Supporting Information) which was used for sampling. Analysis. The z position evolution of lipids and nanosheets, the projection of lipid head-to-tail vectors, the splay angle distribution and bending modulus of lipid membranes were derived using locally written scripts. For the z position evolution of lipids and BN nanosheets, the simulation boxes were divided into slices along the z-axis with 1 Å bin width, the percentage of phosphorus atoms in each slice was calculated for each frame of the simulation trajectory and presented by a


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green-red-blue color gradient. To calculate the projection of lipid head-to-tail vectors, the x-y plane of simulation boxes was divided into 2 × 2 Å2 grids. Each lipid was assigned to a grid that contains the phosphorus atom of the lipid, and this process was repeated for all the frames (2000 frames) in the last 20 ns. The head points (phosphorus atoms) and corresponding vectors to tail points (terminal carbon atoms of the tails) of all the lipids contained in each grid were averaged. The bending rigidity (KC) of lipid membranes were calculated according to the method developed by Khelashvili et al.72 Model images were generated and rendered by the Visual Molecular Dynamics (VMD)73 software.


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ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21403182), Research Grants Council of Hong Kong (CityU 21300014), CityU grants (7004387 & 9680136), NSFC/RGC Joint Research Scheme, under Project N_CityU123/15 and 5151101197, Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (the second phase) under Grant No. U1501501.

ASSOCIATED CONTENT The authors declare no competing financial interest. Supporting Information The following files are available free of charge. Experiments on the lipid adsorption on BN surfaces, lipid extraction MD results and membrane phase state analyses for POPC and DMPC at additional temperatures, temperature-dependent lipid extraction results for the outer (pure POPE) and inner (3:1 mixed POPE and POPG) membranes of Escherichia coli, the evolvement and convergence test of PMF calculations (PDF) Lipid extraction process of the BN nanosheet (POPC membrane at 290 K) (MPG) AUTHOR INFORMATION Corresponding Author Chunyi Zhi*

Email: [email protected]

Xiaolin Cheng*


Jun Fan*



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