Nanotoxicity of Boron Nitride Nanosheet to Bacterial Membranes

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Nanotoxicity of Boron Nitride Nanosheet to Bacterial Membranes Yonghui Zhang, Chun Chan, Zhen Li, Jiale Ma, Qiangqiang Meng, Chunyi Zhi, Hongyan Sun, and Jun Fan Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00025 • Publication Date (Web): 08 Apr 2019 Downloaded from http://pubs.acs.org on April 9, 2019

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Nanotoxicity of Boron Nitride Nanosheet to Bacterial Membranes Yonghui Zhang†,‡, Chun Chan†, Zhen Li†, Jiale Ma†, Qiangqiang Meng†, Chunyi Zhi†, Hongyan Sun§ & Jun Fan*,†,¶ †

Department of Materials Science and Engineering, City University of Hong Kong, 83

Tat Chee Avenue, Hong Kong, China ‡

School of Materials and Energy, Guangdong University of Technology, No. 100

Waihuan Xi Road, Guangzhou Higher Education Mega Center, Panyu District, Guangzhou, China ¶ Center

for Advanced Nuclear Safety and Sustainable Development, City University of

Hong Kong, 83 Tat Chee Avenue, Hong Kong, China §

Department of Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Hong

Kong, China

* To whom correspondence should be addressed. E-mail address: [email protected]

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ABSTRACT

Boron nitride nanosheet is a novel material with great potential in biomedical applications. A deep understanding of the basic interaction mechanisms between biosystems and foreign boron nitride nanosheets can help to better clarify the potential risks of this nanomaterials and provide guidance on its safe design. In this paper, we show that boron nitride nanosheets can cause degradation of bacterial cell membranes via experimental and simulation-based approaches. Our extensive molecular dynamics simulations results reveal that boron nitride nanosheets cause the toxicity to both the bacterial outer and inner membrane in which hydrophobic effect plays an important role. The spontaneous lipid extraction by boron nitride nanosheet is agreed with free energy calculations. A liquid to gel phase transition is induced by boron nitride nanosheet in the outer model membrane of bacteria, indicating that boron nitride nanosheet may cause higher toxicity to the outer membrane than the inner membrane. Our findings may offer new insights into the molecular basis of boron nitride’s cytotoxicity and antibacterial activity.

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INTRODUCTION In recent years, manufactured nanomaterials have received increasing attention throughout a diverse range of biomedical fields. Promising applications of nanomaterials in areas such as bio-sensing, bio-imaging,1 gene delivery,2 antibiofouling and tumor diagnosis and therapy3 foretell a preponderance of nanomaterial-based therapies in human medicine. Among readily synthesized nanomaterials, boron nitride (BN) has gained particular interests due to its extraordinary physical, morphological, thermal and electrical conductivity properties. However, the potential for widespread human exposure raises safety concerns about BN. Consequently, the emerging field of nanotoxicology is of paramount importance for the advancement of nanotherapeutics. For safe applications of the nanoscale products, it is essential that thorough safety assessments are conducted in order to protect human health and the environment.4 Preliminary work has been performed to investigate boron nitride nanotube (BNNT) structures and their compatibility with living cells. Thomas et al. used computational approaches to analyse the stability of BNNTs in lipid bilayers and to investigate the process through which the nanotubes enter the lipid bilayer.5 Highly biocompatible and concentrated dispersions of BNNTs were tested on human neuroblastoma cells by Ciofani et al..6 Horvath et al. investigated the effects of BNNTs on the viability and metabolic status of different cell types and found that BNNTs were cytotoxic for all cell types studied,7 while Chen et al. found that highly pure BNNTs were not cytotoxic.8 Although there are many studies on BNNTs, few studies focus on the cytotoxicity of boron nitride nanosheet (BNNS). Gram-negative bacteria are kinds of bacteria which can be detected by staining techniques.9 They are attracting more and more interests due to their utility in many biotechnological processes and their increasing antibiotic resistance. More and more attention is payed to bacterial diseases as gram-negative microorganisms keep on acquiring resistance to the accessible range of antibiotic drugs,10-12 causing persistent chronic infections and adding to heightening healthcare cost around the world.13 The most ordinarily known gram-negative bacteria is Escherichia coli (E. coli), discovered

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normally in human stomach related framework and broadly utilized in biomedical research and industry. The interaction of bacteria with their surrounding environment is mediated, directly or indirectly, through the membranes that constitute the cell envelopes. The cell wall of gram-negative bacteria consists of two lipid membranes, the outer membrane and the inner membrane.14 As the membrane presents an additional barrier to antibiotics entering gram-negative bacteria, biophysical and structural studies are of significant interest.15 Understanding the molecular structure of the bacterial cell membrane and how the membrane interacts with foreign materials such as antibiotics are therefore important in combating bacterial resistance and development of the cellular delivery. In our previous study, bio-membranes are simply modeled as homogeneous bilayers composed of single type of phospholipid,16 which may not be able to represent the complicated bio-membranes precisely. In this study, experiments show that BNNSs cause the degradation of E. coli membranes. Extensive molecular dynamics (MD) simulations reveal the interactions between BNNS and bilayer membranes models that incorporate the heterogeneity of the non-protein components. A simplified single-component bilayer composed of palmitoyloleoylglycerophosphatidylethanolamine (POPE) and a doublecomponent bilayer composed of POPE and palmitoyloleoylglycerophosphatidylglycerol (POPG) lipids in a 3:1 ratio are investigated, which are frequently used to mimic bacterial outer and inner membranes,17-21 respectively. Results show that BNNS can firstly extract phospholipids from the bilayer membrane and finally surrounded by the bilayer in all simulations. The dynamic lipid extraction process is analyzed. It’s found that the wetting of BNNS with lipids and wetting of lipids with solvents contribute most in the lipid extraction process, while separation of lipid tail group and dewetting of BNNS are the main resistance interactions. Free energy calculations further confirm the spontaneous single lipid extraction. After the BNNS is inserted into the bilayer, the bending rigidity of the membrane increases, and the diffusivity of lipid molecules is restricted due to the local lipid molecules become more order. Furthermore, a liquid to gel phase transition is observed in the outer model membrane with BNNS system. This study provides a molecular-level understanding of the biocompatibility of BNNS and offers new perspectives for the design of nanocarrier, antibiotics and other biomedical applications. 4


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EXPERIMENTAL SECTION Preparation of BNNSs. h-BN powder was purchased from Zibo Jonye Ceramic Technologies Co., Ltd, Shandong province, China. BNNSs were fabricated by liquid phase exfoliation with the assistance of ultrasonic bath KX-1740Q1 at 120 W. In a typical run, 1.0 g of pristine h-BN micro-powder was added into 200 ml ethanol and then sonicated for 1 h. After the sonication, the whitish suspension was allowed to stay for 15 min and then the supernatant was collected through centrifuging at 2500 RPM for 10 min while the sediment was disposed. The BNNSs product was collected by filtration and then dried at 60⁰C prior to future use. Bacterial culture. E. coli DH5α, as model gram-negative bacteria, were grown overnight in LB medium (Luria-Bertani broth, Lennox modification) at 37⁰C and harvested at the exponential growth phase via centrifugation. The E. coli DH5α cells were washed twice to remove residual growth-medium constituents and resuspended in sterile saline solution (0.9% NaCl). The E. coli cells were quantified by assessing the optical absorption at 600 nm (OD600). The bacterial suspensions used for all experiments contained ~1×107 colony forming units (CFUs) per milliliter. Transmission electron microscopy. The 1×107 CFU/ml E. coli cells were suspended in BNNSs (100 mg/ml) solution and cultured at 37⁰C for 2.5 h. The E. coli cells were then fixed with 2.5% glutaraldehyde for 30 min. Cells were washed with 0.9% NaCl, fixed with 1% aqueous OsO4 (Fluka) for 1 h, and washed again twice with 0.9% NaCl. Cells were then dehydrated via ethanol series (70%, 90% and 100% for 15 min, respectively) and embedded in Epon/Araldite resin (polymerization at 65⁰C for 24 h). Thin sections (70 nm) containing cells were placed on the grids and stained for 1 min each with 4% uranyl acetate (1:1 acetone/water) and 0.2% Raynolds lead citrate (water), air-dried, and examined under the transmission electron microscopy (TEM) (Joel JEM-2010). Molecular dynamics simulations. Two simplified bacterial membranes were investigated in our simulations: the outer and inner membrane of gram-negative bacteria. The outer membrane was modeled with pure POPE lipid molecules (lipopolysaccharides in the outer membrane were omitted for simplicity), and the inner membrane was modeled with 3:1 mixed POPE-POPG lipids (summarized in Table 1), according to 5


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previous studies.17-21 The bilayer membranes, each contained 256 lipids (128 lipids per layer), were first generated by the Membrane Builder22-24 plugin of CHARMM-GUI.25 All-atom MD simulations were performed for these bilayer systems with/ without BNNS. For the simulations of bilayer systems, each bilayer membrane was solvated in a large enough water box with periodic boundary conditions. 64 potassium ions were randomly added to neutralize the electric charges of the inner bilayer membrane. The systems containing the bilayer membrane and solvents (ions) were minimized until maximal force were less than 100 kJ·mol-1·nm-1. The minimized systems were then heated up to 310 K. Standard membrane builder equilibration runs were performed followed by 300 ns production runs. Convergence of the inner membrane simulation was estimated by computing the time evolution of radial distribution function (RDF) of lipid molecules (show in Figure S1). For the simulations of bilayer with BNNS systems, a 4.2 nm×4.1 nm BNNS containing 576 atoms was firstly generated by VMD.26 The equilibrated bilayer membrane was placed in the x-y plane with the nanosheet plane oriented vertically above it. The model was then put in a rectangular box with periodic boundary conditions and solvated in water. Standard membrane builder minimization and equilibration runs were performed prior to the production run. In the docking simulations, all atoms in the nanosheet were restrained in space to study the interaction between lipid molecules and BNNS. In this case the kinetic effect from the moving nanosheet was restricted. In order to improve statistical accuracy, three independent simulations (150 ns) were performed to study the lipid extraction process in membranes with BNNS systems. After the BNNS was surrounded by the bilayer membrane and the systems reached equilibrium state (700 ns docking simulation), the restrains on BNNS were then removed. 300 ns final production runs were performed to study the change of membrane properties for each system. All the simulations were carried out with the GROMACS27-28 package version 5.0.6. The CHARMM3629-30 force field was adopted for lipid molecules. The force field for hBN was derived from previous studies,31-32 with the boron and nitride atoms treated as uncharged Lennard-Jones particles. The cross-sections of boron and nitride were set to be

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 BB  3.453 Å and  NN  3.365 Å ,

respectively. While the depth of the potential wells of boron and

nitride were set to be

 BB  0.3971 k J/ mol and  NN  0.606 k J/ mol ,

respectively. Water was

represented by the CHARMM TIPS3P model.33-35 Periodic boundary conditions were applied in all directions. Production simulations were run with a time step of 2 fs in the NPT ensemble, using a semi-isotropic Parrinello-Rahman pressure coupling scheme36-37 to control the pressure at 1 atm. Temperature was controlled at 310 K by the NoseHoover thermostat38-39 with a coupling coefficient of 1 ps. The particle-mesh Ewald method40 was used to calculate the long-range electrostatic interactions, whereas the van der Waals interactions were treated with a cutoff distance of 12 Å. The length of all bonds involving hydrogens was kept constant at their equilibrium values with the LINCS algorithm.41 VMD26 was used to visualize the simulation results. Analysis was performed with GROMACS and VMD for the last 50 ns data of all systems unless otherwise stated. Free energy perturbation calculations. Free energy calculations of lipid extraction were performed with GROMACS 5.0.627-28 as detailed in the results section, using a nonphysical thermodynamic path. The ligand (POPE/POPG lipid) van der Waals interactions were recoupled, and the charges were reappeared using a linear alchemical pathway with ∆λ=0.05 for the van der Waals and ∆λ=0.05 for the coulombic transformations, respectively. A total of 21 windows for the lipid-membrane complex simulations and 21 windows for the lipid-BNNS simulations were therefore employed for one ∆∆G calculation. For each window, energy minimization was first carried out using steepest descent algorithm. Subsequently, the system was simulated for 25 ps in canonical ensemble, with harmonic position restraints applied to the solute (lipid) heavy atoms in which the force constant was set to be 1000 kJ∙mol-1∙nm-2. Temperature was coupled to 310 K using Langevin dynamics.42 300 ps simulations with position restrained were performed in the isothermal–isobaric ensemble, using the Berendsen weak coupling algorithm.43 Finally, 15 ns unrestrained production runs were performed using Hamiltonian-exchange Langevin dynamics with a 2 fs time-step in the NPT ensemble with the Parrinello–Rahman pressure coupling scheme.44 A soft-core potential was employed for the van der Waals interactions transformed.45 Convergence of the free energy calculation were estimated in Figure S2, and free energy was calculated with the last 10 ns production data. 7


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RESULTS AND DISCUSSION Membrane degradation cause by BNNSs. Graphene has emerged as a novel antibacterial material.46-47 Recently, it is reported that graphene nanosheet can damage the E. coli membranes.48 However, little about the antibacterial property of its analogue, hexagonal BNNS, is known so far. Figure 1 presents the TEM images of the cell morphology of E. coli incubated with 100 mg/ml BNNSs at 37⁰C. Among them, three typical morphologies can be identified. The E. coli cells incubated without the presence of BNNSs show an intact membrane with uniform contrast (Figure 1a). After being incubated with BNNSs, some E. coli cell membranes are partially damaged (Figure 1b-e), displaying relatively lower surface phospholipid density, that is, sparser lipids, but not obvious destroyed yet. Moreover, some E. coli cells completely lose their cellular integrity (Figure 1f). Their membranes are severely damaged. Some E. coli cells are even missing their cytoplasm entirely, exhibiting almost empty nest structures. The broken membrane of E. coli cells is thought to be induced by interaction between BNNSs and phospholipids and the degree of membrane fragmentation is attributed to different exposure of cells to BNNSs and the diversity of each individual cell. The dynamic process at the cellular level for this BNNSs-induced degradation of E. coli cell membranes is thus interesting as it is responsible for the observed phenomena in our experiments and the following simulations. Simulations reveal molecular detail. To understand how BNNS interacts with lipid bilayers of bacteria at a molecular level, all-atom MD simulations were used to examine the interaction between BNNS and modeled bacterial membranes. Two types of phospholipids, POPE and POPG, which are commonly found in gram-negative bacteria, namely E. coli, were adopted to mimic the bacterial cell membrane. As the cell wall of gram-negative bacteria consists two lipid bilayers (the outer and inner layer), the outer layer was modeled with pure POPE lipid molecules, while the inner layer was modeled with POPE-POPG lipids with a ratio of 3:1, which was adopted in previous studies.17-21 We refer to the above two model systems as Outer_GramN, Inner_GramN for short. Similar to our previous simulation setup,16 the BN nanosheet was placed perpendicular to the plane of the bilayer and restrained to its original position in space, with its lower side barely reaching the lipid molecules (left wing of Figure 2a, b ). 8


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After the simulation started, the lipid membrane adopted some adaptive motions to adjust to the existence of BNNS. Soon the phospholipid molecules near the BNNS displayed larger fluctuations due to the van der Waals attraction between them and adjacent BN molecules. As the adjacent phospholipid molecules got closer and closer to the nanosheet, some of the phospholipids head group atoms started touching the lower side of BNNS. A few nanoseconds later, one phospholipid was extracted onto the BNNS as a consequence of the attractions. Within a few nanoseconds, many other phospholipids were cooperatively dragged out of the bilayer and adsorbed onto the BNNS surface (middle wing of Figure 2a, b). The main driving force still stems from the van der Waals attractions between the BNNS and the lipid molecules. Once a lipid is extracted, the lipid hydrophobic tails tend to spread out, mainly in the hydrophobic regions of the nanosheet, while the hydrophilic head groups prefer to contact the polar groups of the solvents via favorable electrostatic and van der Waals interactions. This adsorption of phospholipid molecules can be observed on both side of the BNNS, as expected. More interestingly, multilayer climbing of phospholipid molecules was also observed, mainly due to the hydrophobic interactions between the tails of lipid molecules. The disruptive extraction of phospholipid molecules led not only to a sparser lipid bilayer but also to a deformation of the membrane due to strong dragging forces from the BNNS, thus resulting in the loss of cell membrane integrity (middle wing of Figure 2a, b). Within a few hundreds of nanoseconds, serious membrane deformations occurred, with the bilayer membrane moving upward. Finally, the whole bilayer membrane was dragging towards the BNNS, with the BNNS swallowed inside (right wing of Figure 2a, b). Our findings is consistent with what Chen et al., Tu et al. and Zhang et al. found that similar lipid extractions were caused by graphene and graphene oxide nanosheets (The dispersion coefficient is compared in table 2).48-50 To further analyze this dynamics extraction process, the interaction energy between the BNNS and the lipid bilayer was compared with other computational results. As illustrated in Figure 3a, the interaction profiles between lipids and BNNS start from near zero, indicating that the nanosheet has little interaction with the membrane at the beginning of the simulations. As the bilayer starts to interact with the BNNS, the energy between the bilayer and BNNS drops quickly. The interaction is so strong that it 9


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overcomes the attraction between lipid molecules in the same layer due to the hydrophobic effects.51 Finally, the energy profiles between BNNS and the bilayer stay at relatively stable values, indicating that the systems reach their equilibrium state. Besides the energy profile, the movement of the center of mass (COM) of the adjacent lipid molecules towards the nanosheet was computed and compared (Figure 3b). Similarly, three distinguishable stages can be observed from the profile. Initial at around 3.1 nm, the COM distance profiles stay around their initial value indicating the adjustment of the lipid molecules in bulk water. The drop of the distance profiles corresponds to the adsorption of the lipid molecules onto the BNNS. The subsequent gradual decrease in COM distance corresponds to further enhancements in interaction from the BNNS’s continuous dragging on the membrane and direct extraction of lipid molecules. Figure 3c shows the temporal evolution of the number of lipid molecules extracted onto the BNNS. The number of lipid molecules on BNNS starts from zero at the beginning and gradually increases to around twelve. Since the area of the BNNS is constant, the number of lipid molecules on the BNNS is negatively correlated with the acyl chain length of the lipids. The final number of lipid molecules on BNNS is similar, mainly because the tail lengths of the phospholipids we investigated (POPE, POPG) are the same. Furthermore, the area coverage of the BNNS by lipids was calculated as a function of time in Figure 3d, showing how BNNS was covered by the lipids. All these results showed that the quick extraction process took place within tens of nanoseconds. To identify the driving force of the lipid extraction process, we calculated the interaction energy between each of the following groups of molecules: BNNS, lipid head group, lipid tail group and solvent (Figure 4a shows representative results calculated from Sim Outer_3, results calculated from other simulation sets are show in Figure S3) and found that hydrophobic interactions played a dominant role through nanoscale dewetting. It’s found that the first energy profile drop came from the tail-solvent interactions, or the wetting process of lipid tail groups. Meanwhile the resisting interaction came from the tail-tail interaction, which may attribute to the adjust movement of lipids. As the head groups were disorganized by the BNNS, the ΔEHead-Head raised a little bit, while the ΔEBNNS-Head decreased as these two parts were getting closer. As the extraction process took place, the ΔEBNNS-Tail experienced a sharp drop, at the same time since BNNS was 10


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dewetting, ΔEBNNS-Sol raised a lot. The energy change clearly shows that the wetting of BNNS with lipids and wetting of lipids with solvents contribute most for this process, while the separation of lipid tail-tail interactions and dewetting of BNNS are the main resistance interactions. The time evolution of total energy of the representative systems shows the lipid extraction process is energetically driven (Figure 4b). It’s been argued that the hydrophobic interaction is originating from the nonpolar solute which disrupt the hydrogen bonds between molecules of liquid water.52 These water molecules rearrange themselves around the non-hydrogen-bonding surface, so as to minimize the number of disrupted hydrogen bonds. We further analyzed the solvent-accessible surface area of BNNS together with the bilayer and found that the lipid extraction and BNNS insertion process is an event that minimize such a surface (Figure 4c) and thus is entropically favored. To further confirm the above spontaneous lipid extraction caused by BNNS, we performed additional stochastic dynamics simulations using the free energy perturbation (FEP) method. In order to calculate the free energy differences for a single lipid extracted from the membrane to the surface of BNNS, a thermal dynamic cycle is constructed (Figure 5a). According to this cycle, ΔΔG = ΔG2 – ΔG1 , where ΔΔG is the free energy difference of transferring a lipid molecule from the bilayer to the surface of BNNS, ΔG1 represents the free energy change of coupling a lipid molecule to the bilayer while ΔG2 refers to free energy difference of recoupling a lipid onto the BNNS surface. To calculate ΔG2 and ΔG1 with the FEP method, a lipid on the BNNS surface and a lipid in the membrane are reappeared, as shown in Figure 5a. Theoretically, ΔG can be computed from the following ensemble average, ΔG (A→B) = − kBT ln A , where kB is the Boltzmann constant; T is the temperature; EA and EB are Hamiltonian at the initial A and the final B stages. In order to accurately capture the process, multiple intermediated stages (denoted by λ) are inserted, enabling a gradual reappearance procedure. λ equals to 0 and 1 for the initial (with a lipid annihilated on its substrate) and the final (with a lipid equilibrated on its substrate) states, respectively. Figure 5b-d show the cumulative free energy differences during a lipid reappearance process (convergence assessment is show in Figure S2). For the transferring of a POPE from the Outer_GramN and the POPG from the Inner_GramN to the BNNS surface process, when λ = 1, ΔG1 > 11


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ΔG2. In other words, the free energy change is lesser when coupling a lipid into the membrane. As ΔΔG < 0, it is energetically favorable to transfer lipids from the membrane to the BNNS surface (shown in Table 3), which confirms the spontaneous lipids extraction by BNNS in terms of free energy calculations. While for the POPE in the Inner_GramN system, ΔG1 ≈ ΔG2 suggests the free energy difference for POPE binds to membrane and binds to BNNS surface is very small. As the whole lipid extraction is a dynamic process results from the BNNS-lipid, lipid-lipid, BNNS-solvent, lipid-solvent and solvent-solvent interaction, our FEP calculation in a way explains the ideal single lipid extraction phenomenon. Membrane properties affected by BNNS. As the BNNS was finally enveloped by the bilayer, we further removed the restrains on BNNS to allow it interacts with the membrane freely. To study how BNNS affects the bilayer as it inserts into the membrane, the bending moduli was first calculated using the method developed by Khelashvili et al..53 We found that the bending moduli of Outer_GramN increased from 26.4 (+1.89/4.08) kBT to 99.75 (+27.92/-17.39) kBT, and that of Inner_GramN increased from 44.03 (+3.88/-11.69) kBT to 93.35 (+25.79/-14.51) kBT, after the insertion of BNNS (Figure 6a). This shows the bilayers are more resist bending when affected by BNNS. Besides bending modulus, the lateral diffusion constants of lipid molecules were calculated and compared (Figure 6b,c). For the Outer_GramN system, diffusivity decrease from 7.77±0.38×10-8 cm2/s to 0.65±0.18×10-8 cm2/s for POPE molecules. When considering the Inner_GramN, diffusivity decrease from 8.69±2.73×10-8 cm2/s to 3.32±0.83×10-8 cm2/s for POPE molecules, while for POPG molecules it decreases from 8.45±3.13×10-8 cm2/s to 2.47±3.32×10-8 cm2/s. The mobility of lipid molecules is restricted after the insertion of the BNNS. We also computed the fraction of gauche conformation of lipid tails (dihedral angle between ±120˚) (Figure 6a) before and after the insertion of BNNS. The gauche conformation friction decreases from 0.31 to 0.24 for Outer_GramN. For the Inner_GramN, the gauche fraction decreases from 0.31 to 0.28. It’s obvious that all the gauche fraction values decrease after the insertion of BNNS, indicating that the BNNS increases the order of the lipid molecules.

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The sharp decrease of the diffusivity, the increase of bending moduli and the change of gauche conformation fraction (Figure 6) show that the Outer_GramN membrane is seriously affected by the inserted BNNS. We further projected our results along two dimensions (x-y plane) for detail analysis. To gain insights of the detail arrangement of lipid molecules around BNNS, an examination of average lipid density in the plane of the Outer_GramN with/without BNNS was performed (Figure 7a). The blue region in the center of each figure represents the excluded volume due to the existence of BNNS, as we look down from the top of a bilayer. 2D density of lipid molecules shows that for the Outer_GramN system, certain high-density region is found not only around BNNS, but spread over the ~9 nm bilayer, displaying the adsorption of lipid molecules whose motions are highly restricted. This indicates that the lipid molecules in bilayer are closed packed with limited mobility. However, certain ordered pattern can not be observed in the system without BNNS, indicating the arrangement of lipid molecules is induced by the BNNS. As the melting temperature of POPE is 298 K (Table 1), we further performed a control simulation for the Outer_GramN membrane in 295 K, in which lipid should be in the gel phase, and found that lipid molecules formed similar pattern as that in 310 K with BNNS (Figure 7a). To further exam the ordering of the above system, the order parameter (SCD, defined in SI-3) of lipid tails was calculated and compared (Figure 7b). The SCD results clearly show that both the Outer_GramN membrane in 295 K and that in 310 K with BNNS are much order than the membrane in 310 K without BNNS. So far, a liquid to gel phase transition is identified in the Outer_GramN system with BNNS. As the melting temperature of POPE is 298 K, which is lower than the temperature in the simulation, suggesting that the transition is induced by the interaction between the BNNS and POPE lipids.

CONCLUSIONS In summary, by using both comprehensive experimental and theoretical approaches, we demonstrated that lipid molecules could be spontaneously attracted to the surface of BNNS from both bacterial outer and inner membrane. First found in our TEM images, the direct extraction of phospholipids from lipid membranes was validated by our MD simulations. Cooperative movements of extracted lipid molecules were also observed and

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finally lead to the insertion of BNNS into the bilayer. Both the destructive lipid extraction and the insertion of nanosheet suggest that BNNS can induce serious membrane stress. Further analysis implies that the hydrophobic effect which minimize the non-hydrogenbonding surface also plays an important role in the extraction process. The spontaneous single lipid extraction was further confirmed by free energy calculations. The insertion of BNNS was found to affect the structure and properties of the bilayers. The change in gauche fraction and order parameter of lipids suggest that the lipid tails become more ordered. A liquid to gel phase transition was observed in the Outer_GramN system with BNNS, in which the properties of bilayer was dramatically affected. Thus, our current findings may aid in the understanding of how BNNSs affect the structure and properties of bacterial membranes, providing molecular-level guidance for the development of novel nano-carriers, antibiotics as well as other biomedical and clinical applications. Our study might also facilitate the cytotoxicity studies of other 2D materials that might alter the structure and properties of certain biological systems.

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Figure 1. Morphology of E. coli cells exposed to BNNSs. TEM images show E. coli undergoing changes in morphology after incubation with 100 μg/ml BNNSs at 37 ⁰C for 2.5 h. Two typical morphologies can be identified: (a) Initial morphology of E. coli showing an unbroken phospholipids layer. (b-f) Cell membranes damaged by BNNSs. Some bacteria exhibit a lower density of surface phospholipids and some show the complete loss of membrane integrity. The degraded membrane of E. coli cells is caused by interaction between BNNSs and phospholipids while the degree of membrane fragmentation is due to different exposure of cells to BNNSs and the diversity of each individual cell. Scale bars in (a), (c), (e) and (f) are 500 nm while those in (b) and (d) are 1μm.

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Figure 2. Lipid extraction confirmed in MD simulations. Snapshots of Outer_GramNBNNS (a), Inner_GramN-BNNS (b) systems at 0 ns, 50 ns and 500 ns, respectively. The BNNS is shown in gray. The lipid head group atoms are shown as yellow and red spheres. The backbone of lipid tails is shown as green and blue sticks for POPE and POPG, respectively. Both simulation results indicate that lipid extraction may occur in both simplified outer and inner model membranes. The continuous extraction of phospholipids from the bilayer causes the lipid bilayer becomes sparser and induces serious deformation of the membrane, which further results in the loss of membrane integrity.

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Figure 3. Adsorption dynamics of lipid molecules. Time evolution of interaction energy between membrane and BNNS (a), center of mass distance (vertical) between BNNS and surrounding lipid molecules (b), number of lipids extracted onto the BNNS (c) and area percentage of the BNNS covered by lipid molecules (d). All figures with a step up/down behavior describe that the spontaneous lipid extraction and nanosheet insertion processes take place within tens of nanoseconds.

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Figure 4. Driven force of the lipid extraction process. (a) Time evolution of the change in interaction energy. Interactions with lipid are decomposed into interactions with lipid head groups and interactions with lipid tail groups. The interaction energy of each pair at 0 ns is set to be the reference energy (dashed line). (b) Time evolution of the system total energy. (c) Time evolution of the solvent-accessible surface area of BNNS together with bilayer membrane.

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Figure 5. Free energy calculations account for lipid extraction. (a) Thermodynamic cycle for computing ΔΔG of transferring one lipid molecule from the bilayer membrane to the BNNS surface. (b) Free energy differences for coupling a POPE lipid to the Outer_GramN membrane and the BNNS surface systems. (c, d) Free energy differences for coupling a POPE (c)/ POPG (d) lipid to the Inner_GramN membrane and the BNNS surface systems, respectively. The negative ΔΔG indicates that the ideal single lipid extraction/ transfer from the bilayer to BNNS is energetically favorable and spontaneous.

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Figure 6. Property of bilayer membranes with/ without BNNS. (a) Bending moduli (KC) of bilayers with/without BNNS. (b) Fraction of gauche dihedral angles in the above systems. All torsional angles around C-C bonds in lipid tails are counted. (c) Lateral diffusivity of lipid molecules in the above systems.

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Figure 7. Phase transition induced by BNNS in the Outer_GramN membrane. (a) 2D number density of lipid molecules in Outer_GramN (295 K), Outer_GramN (310 K) and Outer_GramN-BNNS (310 K). (b) Comparison of SCD in the above corresponding systems.

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Table 1. Phospholipids in two bilayer membrane systems Charge (e)

sn1

sn2

Tm (°C)

Chemical formula

POPE

0

16:0

18:1

25

C39H76NO8P

POPG

-1

16:0

18:1

-2

C40H76O10P

Table 2. Comparison of the dipole-dipole dispersion coefficient (C6,ij) between different atoms (i) and atoms in BN/graphene nanosheet (j). j

C6,ij (Å6∙kJ∙mol-1)

i

B

N

C

H

56.5

60.8

57.4

C

3189.4

3652.7

2968.6

N

3404.7

3887.4

3182.4

O

2070.0

2355.8

1943.8

P

9202.8

9202.8

8532.3

Table 3. Free energy of transferring one lipid molecule from Inner/Outer membrane to the BNNS surface. ΔΔG (kJ∙mol-1)

Inner→BNNS

Outer→BNNS

POPE

1±3

-20 ± 4

POPG

-61 ± 6

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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGEMENT The author J.F. thanks the projects supported by the Research Grants Council of Hong Kong (CityU 21300014 & CityU 11306517), and CityU grants (7004387 & 9680136). This research was also supported by NSFC/RGC Joint Research Scheme, under Project N_CityU123/15 and 5151101197 to C.Z..

REFERENCES 1. Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S., Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics. Science 2005, 307 (5709), 538. 2. Rosi, N. L.; Giljohann, D. A.; Thaxton, C. S.; Lytton-Jean, A. K.; Han, M. S.; Mirkin, C. A., Oligonucleotide-modified gold nanoparticles for intracellular gene regulation. Science 2006, 312 (5776), 1027-30. 3. Grossman, J. H.; Mcneil, S. E., Nanotechnology in Cancer Medicine. Physics Today 2012, 65 (8), 38-42. 4. Oberdörster, G.; Oberdörster, E.; Oberdörster, J., Nanotoxicology: An Emerging Discipline Evolving from Studies of Ultrafine Particles. Environmental Health Perspectives 2005, 113 (7), 823-839. 5. Thomas, M.; Enciso, M.; Hilder, T. A., Insertion mechanism and stability of boron nitride nanotubes in lipid bilayers. Journal of Physical Chemistry B 2015, 119 (15), 4929-36. 6. Ciofani, G.; Danti, S.; D’Alessandro, D.; Moscato, S.; Menciassi, A., Assessing cytotoxicity of boron nitride nanotubes: Interference with the MTT assay. Biochemical & Biophysical Research Communications 2010, 394 (2), 405. 7. Horváth, L.; Magrez, A.; Golberg, D.; Zhi, C.; Bando, Y.; Smajda, R.; Horváth, E.; Forró, L.; Schwaller, B., In vitro investigation of the cellular toxicity of boron nitride nanotubes. Acs Nano 2011, 5 (5), 3800-10. 8. Chen, X.; Wu, P.; Rousseas, M.; Okawa, D.; Gartner, Z.; Zettl, A.; Bertozzi, C. R., Boron Nitride Nanotubes Are Noncytotoxic and Can Be Functionalized for Interaction with Proteins and Cells. Journal of the American Chemical Society 2009, 131 (3), 890-1. 9. Baron, S., Epidemiology--Medical Microbiology. University of Texas Medical Branch at Galveston: 1996.

23


Langmuir 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

10. Mah, T.-F.; Pitts, B.; Pellock, B.; Walker, G. C.; Stewart, P. S.; O'toole, G. A., A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance. Nature 2003, 426 (6964), 306310. 11. Allen, H. K.; Donato, J.; Wang, H. H.; Cloud-Hansen, K. A.; Davies, J.; Handelsman, J., Call of the wild: antibiotic resistance genes in natural environments. Nature Reviews Microbiology 2010, 8 (4), 251-259. 12. Fisher, J. F.; Meroueh, S. O.; Mobashery, S., Bacterial resistance to β-lactam antibiotics: compelling opportunism, compelling opportunity. Chemical reviews 2005, 105 (2), 395-424. 13. Roberts, R. R.; R Douglas Scott, I.; Hota, B.; Kampe, L. M.; Abbasi, F.; Schabowski, S.; Ahmad, I.; Ciavarella, G. G.; Cordell, R.; Solomon, S. L., Costs attributable to healthcare-acquired infection in hospitalized adults and a comparison of economic methods. Medical care 2010, 48 (11), 1026-1035. 14. Gupta, R. S., What are archaebacteria: life's third domain or monoderm prokaryotes related to gram ‐ positive bacteria? a new proposal for the classification of prokaryotic organisms. Molecular microbiology 1998, 29 (3), 695-707. 15. Tenover, F. C., Mechanisms of antimicrobial resistance in bacteria. The American journal of medicine 2006, 119 (6), S3-S10. 16. Zhang, Y.; Li, Z.; Chan, C.; Ma, J.; Zhi, C.; Cheng, X.; Fan, J., Ordering of lipid membranes altered by boron nitride nanosheets. Physical Chemistry Chemical Physics 2018. 17. Hong, C.; Tieleman, D. P.; Wang, Y., Microsecond molecular dynamics simulations of lipid mixing. Langmuir 2014, 30 (40), 11993-12001. 18. Pasenkiewicz-Gierula, M.; Murzyn, K.; Róg, T.; Czaplewski, C., Molecular dynamics simulation studies of lipid bilayer systems. ACTA BIOCHIMICA POLONICA-ENGLISH EDITION2000, 47 (3), 601-612. 19. Hallock, K. J.; Lee, D.-K.; Omnaas, J.; Mosberg, H. I.; Ramamoorthy, A., Membrane composition determines pardaxin's mechanism of lipid bilayer disruption. Biophysical journal 2002, 83 (2), 1004-1013. 20. Lee, S.-Y.; MacKinnon, R., A membrane-access mechanism of ion channel inhibition by voltage sensor toxins from spider venom. Nature 2004, 430 (6996), 232-235. 21. Murzyn, K.; Róg, T.; Pasenkiewicz-Gierula, M., Phosphatidylethanolaminephosphatidylglycerol bilayer as a model of the inner bacterial membrane. Biophysical journal 2005, 88 (2), 1091-1103. 22. Jo, S.; Kim, T.; Im, W., Automated builder and database of protein/membrane complexes for molecular dynamics simulations. PloS one 2007, 2 (9), e880. 23. Jo, S.; Lim, J. B.; Klauda, J. B.; Im, W., CHARMM-GUI Membrane Builder for mixed bilayers and its application to yeast membranes. Biophys J 2009, 97 (1), 50-8. 24. Wu, E. L.; Cheng, X.; Jo, S.; Rui, H.; Song, K. C.; Davila-Contreras, E. M.; Qi, Y.; Lee, J.; Monje-Galvan, V.; Venable, R. M.; Klauda, J. B.; Im, W., CHARMM-GUI Membrane Builder toward realistic biological membrane simulations. Journal of computational chemistry 2014, 35 (27), 1997-2004. 25. Jo, S.; Kim, T.; Iyer, V. G.; Im, W., CHARMM-GUI: a web-based graphical user interface for CHARMM. Journal of computational chemistry 2008, 29 (11), 1859-65. 26. Humphrey, W.; Dalke, A.; Schulten, K., VMD: visual molecular dynamics. Journal of molecular graphics 1996, 14 (1), 33-8. 27. Berendsen, H. J. C.; Vanderspoel, D.; Vandrunen, R., Gromacs - a Message-Passing Parallel Molecular-Dynamics Implementation. Comput Phys Commun 1995, 91 (1-3), 43-56.

24


Page 24 of 27

Page 25 of 27 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

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28. Pronk, S.; Pall, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M. R.; Smith, J. C.; Kasson, P. M.; van der Spoel, D.; Hess, B.; Lindahl, E., GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 2013, 29 (7), 845-54. 29. Klauda, J. B.; Venable, R. M.; Freites, J. A.; O'Connor, J. W.; Tobias, D. J.; MondragonRamirez, C.; Vorobyov, I.; MacKerell, A. D., Jr.; Pastor, R. W., Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. The journal of physical chemistry. B 2010, 114 (23), 7830-43. 30. Vanommeslaeghe, K.; Hatcher, E.; Acharya, C.; Kundu, S.; Zhong, S.; Shim, J.; Darian, E.; Guvench, O.; Lopes, P.; Vorobyov, I.; Mackerell, A. D., Jr., CHARMM general force field: A force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. Journal of computational chemistry 2010, 31 (4), 671-90. 31. Won, C. Y.; Aluru, N. R., Water permeation through a subnanometer boron nitride nanotube. J Am Chem Soc 2007, 129 (10), 2748-9. 32. Won, C. Y.; Aluru, N. R., Structure and dynamics of water confined in a boron nitride nanotube. J Phys Chem C 2008, 112 (6), 1812-1818. 33. Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L., Comparison Of Simple Potential Functions for Simulating Liquid Water. J Chem Phys 1983, 79 (2), 926-935. 34. Neria, E.; Fischer, S.; Karplus, M., Simulation of activation free energies in molecular systems. The Journal of chemical physics 1996, 105 (5), 1902-1921. 35. Durell, S. R.; Brooks, B. R.; Ben-Naim, A., Solvent-induced forces between two hydrophilic groups. The Journal of Physical Chemistry 1994, 98 (8), 2198-2202. 36. Martyna, G. J.; Tobias, D. J.; Klein, M. L., Constant pressure molecular dynamics algorithms. The Journal of Chemical Physics 1994, 101 (5), 4177-4189. 37. Parrinello, M.; Rahman, A., Polymorphic Transitions In Single-Crystals - a New Molecular-Dynamics Method. J Appl Phys 1981, 52 (12), 7182-7190. 38. Nosé, S., A molecular dynamics method for simulations in the canonical ensemble. Molecular physics 1984, 52 (2), 255-268. 39. Hoover, W. G., Canonical dynamics: Equilibrium phase-space distributions. Physical review. A, General physics 1985, 31 (3), 1695-1697. 40. Tom, D.; Darrin, Y.; Lee, P., Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems. J. Chem. Phys. 1993, 98 (12), 10089-10092. 41. Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M., LINCS: A linear constraint solver for molecular simulations. Journal of computational chemistry 1997, 18 (12), 1463-1472. 42. van Gunsteren, W. F.; Berendsen, H. J., Computer simulation of molecular dynamics: Methodology, applications, and perspectives in chemistry. Angewandte Chemie International Edition in English 1990, 29 (9), 992-1023. 43. Berendsen, H. J.; Postma, J. v.; van Gunsteren, W. F.; DiNola, A.; Haak, J., Molecular dynamics with coupling to an external bath. The Journal of chemical physics 1984, 81 (8), 36843690. 44. Parrinello, M.; Rahman, A., Polymorphic transitions in single crystals: A new molecular dynamics method. J Appl Phys 1981, 52 (12), 7182-7190. 45. Beutler, T. C.; Mark, A. E.; van Schaik, R. C.; Gerber, P. R.; Van Gunsteren, W. F., Avoiding singularities and numerical instabilities in free energy calculations based on molecular simulations. Chemical physics letters 1994, 222 (6), 529-539. 46. Liu, S.; Zeng, T. H.; Hofmann, M.; Burcombe, E.; Wei, J.; Jiang, R.; Kong, J.; Chen, Y., Antibacterial activity of graphite, graphite oxide, graphene oxide, and reduced graphene oxide: membrane and oxidative stress. ACS nano 2011, 5 (9), 6971-6980. 25


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47. Ji, H.; Sun, H.; Qu, X., Antibacterial applications of graphene-based nanomaterials: Recent achievements and challenges. Advanced drug delivery reviews 2016, 105, 176-189. 48. Tu, Y.; Lv, M.; Xiu, P.; Huynh, T.; Zhang, M.; Castelli, M.; Liu, Z.; Huang, Q.; Fan, C.; Fang, H., Destructive extraction of phospholipids from Escherichia coli membranes by graphene nanosheets. Nature nanotechnology 2013, 8 (8), nnano. 2013.125. 49. Chen, J.; Zhou, G.; Chen, L.; Wang, Y.; Wang, X.; Zeng, S., Interaction of graphene and its oxide with lipid membrane: a molecular dynamics simulation study. The Journal of Physical Chemistry C 2016, 120 (11), 6225-6231. 50. Zhang, L.; Xu, B.; Wang, X., Cholesterol extraction from cell membrane by graphene nanosheets: a computational study. The Journal of Physical Chemistry B 2016, 120 (5), 957-964. 51. Tu, Y.; Lv, M.; Xiu, P.; Huynh, T.; Zhang, M.; Castelli, M.; Liu, Z.; Huang, Q.; Fan, C.; Fang, H.; Zhou, R., Destructive extraction of phospholipids from Escherichia coli membranes by graphene nanosheets. Nat Nanotechnol 2013, 8 (8), 594-601. 52. Silverstein, T. P., The real reason why oil and water don't mix. Journal of chemical education 1998, 75 (1), 116. 53. Khelashvili, G.; Kollmitzer, B.; Heftberger, P.; Pabst, G.; Harries, D., Calculating the Bending Modulus for Multicomponent Lipid Membranes in Different Thermodynamic Phases. Journal of chemical theory and computation 2013, 9 (9), 3866-3871.

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TOC Figure

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Nanotoxicity of Boron Nitride Nanosheet to Bacterial Membranes

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