Destructive extraction of phospholipids from Escherichia coli membranes by graphene nanosheets

Destructive extraction of phospholipids fromEscherichia coli membranes by graphenenanosheetsYusong Tu1,2, Min Lv2, Peng Xiu3, Tien Huynh4, Meng Zhang2, Matteo Castelli4, Zengrong Liu1,

Qing Huang2*, Chunhai Fan2, Haiping Fang2 and Ruhong Zhou3,4,5*

Understanding how nanomaterials interact with cell membranes is related to how they cause cytotoxicity and is thereforecritical for designing safer biomedical applications. Recently, graphene (a two-dimensional nanomaterial) was shown tohave antibacterial activity on Escherichia coli, but its underlying molecular mechanisms remain unknown. Here we showexperimentally and theoretically that pristine graphene and graphene oxide nanosheets can induce the degradation of theinner and outer cell membranes of Escherichia coli, and reduce their viability. Transmission electron microscopy showsthree rough stages, and molecular dynamics simulations reveal the atomic details of the process. Graphene nanosheetscan penetrate into and extract large amounts of phospholipids from the cell membranes because of the strong dispersioninteractions between graphene and lipid molecules. This destructive extraction offers a novel mechanism for the molecularbasis of graphene’s cytotoxicity and antibacterial activity.

The widespread use of nanomaterials in biomedicine (forexample, in gene delivery1, cellular imaging2 and tumourtherapy3) has been accompanied by an increasing interest in

understanding their interactions with tissues, cells and biomole-cules4, as well as how they might affect the integrity of cell mem-branes5,6 and proteins7,8. Recent studies on the interaction ofgraphitic nanomaterials such as zero-dimensional fullerenes9,10

and one-dimensional carbon nanotubes11,12 with cell membranesshow that these materials can enter cells either through direct pen-etration13,14 (usually the case for small-sized nanoparticles) or viaendocytosis11,15.

The interaction between graphene and cells has not yet beenstudied to the same extent, probably because of the intrinsicallyhigher complexity of such interactions. Graphene—a two-dimen-sional single-atom-thick nanomaterial with unique structural,mechanical and electronic properties—has many potential biomedi-cal applications16–18. Its high specific surface area allows high-density biofunctionalization for nanotechnology-based drugdelivery17–19. Furthermore, its smooth, contiguous topography andbiopersistence play a unique role in foreign-body-induced carcino-genesis and tumour progression20,21. Moreover, because graphenedemonstrates ultrahigh in vivo tumour uptake in mice, it is poten-tially effective for photothermal ablation of tumours22.Meanwhile, recent studies have shown that graphene and grapheneoxide have strong antibacterial activity23–26 towards Escherichia coli(E. coli)23–25. This cytotoxicity is thought to arise from physicaldamage resulting from direct interactions between the grapheneand the bacteria cell membranes23–25. The graphene-induced cyto-toxicity is largely reduced when the nanosheets are surrounded byproteins27, such as serum proteins. Therefore, graphene is less athreat to humans or other mammalians but is lethal to bacteria,

which means it has the potential to be effective as a novel antibiotic.However, the detailed dynamical process and underlying molecularmechanism of how graphene induces degradation of the bacterialcell membrane remain unclear.

Here, we show experimentally and theoretically that graphenenanosheets can insert/cut through the cell membranes of E. coliand vigorously extract large amounts of phospholipids fromthe membranes. Our experiments show that this process causesdegradation of E. coli membranes and reduces bacteria viability.Molecular dynamics simulations reveal atomic details on howgraphene and graphene oxide interact with the inner and outermembranes of E. coli. To the best of our knowledge, such adestructive extraction of phospholipids by graphene has notbeen reported previously, and the findings may offer insightsfor the better design of graphene-based antibiotics or otherbiomedical applications.

Three rough stages of membrane degradationGraphene oxide was used (for water solubility) in the transmissionelectron microscopy (TEM) experiment, and then both grapheneand graphene oxide in molecular dynamics simulations, to enablecomparison with our own experiments as well as those of others.Figure 1 presents TEM images of the cell morphology of E. coli incu-bated with 100 mg ml21 graphene oxide nanosheets at 37 8C. Thegraphene nanosheets were oxidized using the modified Hummersmethod28 to generate water-dispersible graphene oxide nanosheets.Roughly three stages (stages I, II and III) of E. coli cell morphologywere observed during this 2.5 h incubation process. The E. coli cellscan initially tolerate graphene oxide nanosheets for a short period oftime, particularly under low concentrations. Figure 1a represents theinitial morphology of the E. coli (control run, Stage I). In Stage II,

1Institute of Systems Biology, Shanghai University, Shanghai 200444, China, 2Division of Interfacial Water and Division of Physical Biology, ShanghaiInstitute of Applied Physics, Key Laboratory of Interfacial Physics and Technology, Chinese Academy of Sciences, PO Box 800-204, Shanghai 201800,China, 3Department of Engineering Mechanics and Soft Matter Research Center, Zhejiang University, Hangzhou 310027, China, 4IBM Thomas J. WatsonResearch Center, Yorktown Heights, New York 10598, USA, 5Department of Chemistry, Columbia University, New York 10027, USA.

*e-mail: [email protected]; [email protected]

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the E. coli cell membranes are partially damaged, with some display-ing a lower surface phospholipid density, that is, sparser lipids butno obvious cuts yet (see the cells marked with arrows inFig. 1b,c). In the final stage (Stage III), the E. coli cells lose their cel-lular integrity; their membranes are severely damaged, and some areeven missing their cytoplasm entirely (‘empty nests’, see markedcells in Fig. 1d–f). It should be noted that the exact timing ofeach stage cannot be determined easily due to the resolution limitand the diversity within each individual cell. Nevertheless, theserough but representative stages help us understand, to somedegree, the dynamic process at the cellular level for this grapheneoxide-induced degradation of E. coli cell membranes.

Simulations reveal two types of interaction mechanismTo understand the underlying molecular mechanism behind thisstrong antibacterial activity, we performed molecular dynamicssimulations to study the detailed interactions of both graphene andgraphene oxide nanosheets with E. coli membranes. Both the outerand inner membranes of E. coli were simulated with all-atom lipidmodels in explicit solvent (see Methods for more details). We firststarted with graphene nanosheets to model an earlier experimentby Akhavan and Ghaderi24, where graphene nanosheets weredeposited on stainless-steel substrates by electrophoretic deposition.They showed with more direct evidence that the sharpened edgesof these nanosheets may act like ‘blades’, which can insert and cutbacteria cell membranes24, similar to those in our Stage III TEMimages. One way to model this (that is, mimicking the stainless-steel substrate) in computer simulations is to position/restrain

some carbon atoms on one edge of the graphene nanosheet (seeschematic view in Supplementary Fig. S1, showing the nanosheet‘suspended’ above the membrane). The initial suspending distancefrom the membrane surface is found not to be critical. Multiplesimulations have shown that a vertical distance of �3.5–4.7 nmfrom the membrane surface (0.7–0.9 times the graphene nanosheetlength) can always lead to the spontaneous insertion of a graphenenanosheet into both outer and inner membranes.

Two representative trajectories for graphene nanosheet insertionare shown in Fig. 2, one for the outer membrane (Fig. 2a,Supplementary Movie S1) and one for the inner (Fig. 2b,Supplementary Movie S2). Our unbiased simulations clearly showthat the graphene nanosheet ‘suspended’ above the membranescan insert into both the outer and inner E. coli membranes withina few tens to hundreds of nanoseconds. Three distinguishablemodes were observed during this spontaneous entrance of thegraphene nanosheet into both membranes (see SupplementaryMovies S1 and S2 for more details and the slight differencesbetween them): (i) the swing mode, (ii) the insertion mode and(iii) the extraction mode. In the swing mode, the graphenenanosheet, with its initially unbiased orientation, undergoes aswing motion, back and forth, around the restrained atom, fortens to hundreds of nanoseconds. During this swing mode, thetail end of the graphene nanosheet touches the membrane surfacemultiple times (before swinging back into the bulk solution). Inthe insertion mode, the tail end of the graphene nanosheet is even-tually trapped by the membranes due to strong van der Waals attrac-tions from the membrane lipids and hydrophobic interactions.

Type A

0.5 µm

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Type B

Type B

Type B

Type A

a b c

d e f

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Figure 1 | Morphology of E. coli exposed to graphene oxide nanosheets. TEM images showing E. coli undergoing changes in morphology after incubation

with 100 mg ml21 graphene oxide nanosheets at 37 8C for 2.5 h. Three stages of destruction can be seen. a, Initial morphology of E. coli (control or Stage 1;

two individual TEM images (inset and main image) are shown, the scale bar applies to both). b,c, Partial damage of cell membranes, with some bacteria

showing a lower density of surface phospholipids (Stage II). Arrows indicate a Type B mechanism, where graphene nanosheets extract phospholipids from

the cell membrane. d–f, Three representative images showing the complete loss of membrane integrity, with some showing ‘empty nests’ and missing

cytoplasm (Stage III). d and f are representative images showing a Type A mechanism, where graphene nanosheets cut off large areas of membrane

surfaces. In e, both Type A and Type B mechanisms are shown.

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Once the tail end starts to enter, it takes only a few nanoseconds forthe graphene nanosheet to cut into the lipid membranes. This directinsertion/cutting supports the earlier experiment by Akhavan andGhaderi24, as well as our current TEM experiment, and is referredto as the ‘Type A’ mechanism hereafter (see cells marked ‘Type A’in Fig. 1). In the extraction mode, surprisingly, the graphenenanosheet starts to vigorously extract the phospholipid moleculesfrom the lipid bilayers onto its own surfaces (Fig. 2). The disruptiveextraction of phospholipid molecules leads not only to a sparserlipid bilayer but also to a deformation of the membrane due tostrong dragging forces from the graphene nanosheet, thus resultingin the loss of cell membrane integrity. This surprising lipid extrac-tion is named the ‘Type B’ mechanism. It was first revealed in ourmolecular dynamics simulations, and then validated by carefulexamination of our staged TEM images (see cells marked ‘Type B’in Fig. 1). This strong extraction-induced deformation might alsohelp to explain the membrane wrapping in endocytosis29 ofvarious nanoparticles15,30.

Interestingly, if we released the position restraint after insertion,to mimic experiments without the graphene-holding substrates suchas our own TEM experiment (Fig. 1), the phospholipids were foundto be extracted from cell membranes even faster for both the outerand inner membranes, with several lipids extracted within a fewnanoseconds (Supplementary Fig. S2). More simulations were per-formed later with fully restrained graphene sheets (graphene‘docking’ simulations), which is presumably the hardest scenariofor lipid extraction to occur, but again, to our surprise, turbulentlipid extractions were observed (more below).

To better understand the insertion process, we further analysedthe physical interactions between graphene and lipid membranes.Figure 3a,b shows interaction energy profiles between the graphenenanosheet and the two E. coli membranes, together with movementsof the centre of mass (COM) of these two membranes towards thenanosheet. Again, three distinguishable modes can be seen fromthese energy profiles. Initial high-energy plateaux describe theswing mode of the graphene nanosheet in bulk water. Then, sharp

13.2 ns 142.0 ns

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187.0 ns 500.0 ns

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Figure 2 | Graphene nanosheet insertion and lipid extraction. a,b, Representative simulated trajectories of graphene nanosheet insertion and lipid extraction

in the outer membrane (pure POPE) and inner membrane (3:1 mixed POPE–POPG) of E. coli (the snapshot times are shown in the top left corners). Water is

shown in violet and the phospholipids in tan lines with hydrophilic charged atoms as coloured spheres (hydrogen, white; oxygen, red; nitrogen, dark blue;

carbon, cyan; phosphorus, orange). The graphene sheet is shown as a yellow-bonded sheet with a large sphere marked at one corner as the restrained atom

in simulations. Extracted phospholipids are shown as larger spheres.

ARTICLES NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2013.125





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Figure 3 | Interaction energy profiles. a,b, Time evolution of the interaction energy between the graphene nanosheet and the membrane (red), the COM

of the membrane along the z-direction towards the nanosheet (black) for the representative trajectories shown in Fig. 2 (a, outer membrane; b, inner

membrane). c,d, Time evolution of bilayer thickness (red) and area per lipid (black) of membranes in the ‘extracted region’ (c, outer membrane; d, inner

membrane). Blue vertical regions indicate the insertion time of the graphene nanosheet in both membranes. Black and green shaded areas indicate the states

before and after graphene insertion, respectively.

a0.0 ns b 68.0 ns

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Figure 4 | Lipid extraction by graphene in docking simulations. a–f, A representative trajectory of a fully restrained graphene nanosheet docked at the

surface of the outer membrane (pure POPE). The simulation time is indicated in each snapshot, with the last snapshot shown in more view angles

(e,f, rotated anticlockwise by angle from its previous view to obtain the current view). Colour settings are as in Fig. 2, but with water hidden for clarity.






energy collapses correspond to the insertion mode. The subsequentgradual decrease in energy corresponds to further enhancements ininteraction from graphene’s continuous dragging on the membraneand direct extraction of lipid molecules. This strong attractionbetween graphene and membrane lipids largely arises from gra-phene’s unique two-dimensional structure with all sp2 carbons,which facilitates exceptionally strong dispersion interactionsbetween graphene and lipid molecules, so strong that it can over-come the self-attraction among lipid molecules within the mem-brane. Meanwhile, we also estimated the effects of grapheneinsertion and lipid extraction on the overall properties of themembrane bilayers. Figure 3c,d shows the time evolution ofthe local bilayer thickness and area per lipid for both inner andouter membranes near the insertion and extraction region, that is,the ‘extracted region’ (see Supplementary PS1 for the exactdefinition). Again, sharp changes in both the thickness (increase)and area per lipid (decrease) correspond to the insertion mode,with both membranes displaying similar but significant defor-mations overall, consistent with the above COM movement analysis.The phospholipids’ tail order parameters, on the other hand, do notshow significant changes (Supplementary Fig. S3), indicating thatthe acyl chain orientations are not affected much, as onewould expect.

Lipid extraction by graphene nanosheets is robustAs mentioned, a novel Type B mechanism was discovered in oursimulations, where destructive extractions of lipid molecules by gra-phene nanosheets are seen, which can result in sparser lipid den-sities and eventual loss of cell membrane integrity. To furtherverify that this lipid extraction is real, and not just due to somekinetic effects, an additional graphene docking simulation was per-formed using the outer membrane (pure palmitoyloleoylphosphati-dylethanolamine, POPE) as an example (a larger 7.62 × 3.69 nm2

graphene nanosheet was used to demonstrate this effect moreclearly; see Methods for more details of the setup). As shown inFig. 4 (Supplementary Movie S3), the entire graphene nanosheetwas restrained in space, with its plane oriented perpendicular tothe membrane surface and its tail barely touching it, which is pre-sumably the hardest scenario under which lipid extraction canoccur, because there is no help available from the moving graphenenanosheet (kinetic effects). For the first �60 ns, the lipid membraneexperienced some adaptive motions to adjust to the graphenedocking. Shortly after that, nearby phospholipid molecules dis-played larger fluctuations, perturbing the seemingly smooth mem-brane surface. Some phospholipids then started to climb up alongthe graphene nanosheet as a consequence of the strong attractionsfrom the graphene (Fig. 4b). Within a few tens of nanoseconds,many other phospholipids also joined in this ‘climbing’.Interestingly, this phospholipid climbing seems to be highly coop-erative due to the collective movements of the hydrophobic tailsat the water/graphene interface (Fig. 4c). More interestingly, multi-layer climbing of phospholipid molecules was also observed �30 nsafter the first extraction event (Fig. 4d–f). In addition, the climbingof phospholipid molecules was also seen on the other side of the gra-phene nanosheet (Fig. 4d–f), indicating that this process can occursimultaneously along both sides (a total of �17 lipids were extractedby the end). Within a few hundreds of nanoseconds, serious mem-brane deformations occurred, with the COM moving up by�1.5 nm. As shown in the last two subfigures (Fig. 4e,f ), the hydro-phobic tails of these extracted phospholipids tend to spread outevenly onto the entire graphene nanosheet to maximize contactwith the hydrophobic graphene surface, while their hydrophilichead groups prefer to be solvated in bulk water (SupplementaryFig. S4 shows their orientation distributions). These results indicatethat water plays a significant role in the lipids’ structure and orien-tation at the graphene/water interface.

It is interesting to note that all these complicated and collectivemovements of phospholipids on the graphene nanosheet firststarted from seemingly short-range van der Waals attractionsbetween the graphene and lipid molecules. Once extracted, thestrong hydrophobic interactions between the graphene and thelipid tails played another important role through nanoscale ‘dewet-ting’ (that is, expelling water from the graphene surface). This stronghydrophobic packing is similar to the hydrophobic collapses inmany biomolecular self-assemblies, such as cell membrane for-mation and protein folding; many recent studies31–33 have shownthat nanoscale dewetting (or drying) can provide significantdriving forces for the collapse speed and system stability. It shouldbe noted that this potential Type B mechanism was initially over-looked in our TEM images due to the limited resolution. Onlyafter analysing the results from our computer simulations did werealize that there is a potentially novel molecular mechanism forthis graphene-induced degradation of bacteria cell membranes.This demonstrates again the importance of the synergy betweenthe complementary experimental and theoretical approaches.

Another way to show the robustness of this lipid extraction bygraphene nanosheets is to artificially adjust the well depth of thevan der Waals potential, 1CC, of graphene carbon atoms.Following the same procedure described above, fully restrained gra-phene nanosheets were docked at the E. coli outer membranesurface, but with different depths of potential well 1′CC (1/8, 1/6,1/4, 1/2, 1, 2 and 41CC). All the docking simulations were run for250 ns, with the last 50 ns used for the calculation of average inter-action energies. Figure 5 presents the average interaction energybetween the nanosheet and the lipid membrane (pure POPE) atdifferent 1

′CC values. A fast decrease in interaction energy was

observed when phospholipid molecules were extracted out of themembrane and absorbed onto the graphene nanosheet. In contrast,a relatively high constant value was found when no extractionoccurred. This indicates quantitatively that the graphene nanosheetcan, in principle, extract phospholipids from the membrane when1′CC . 1

41CC, that is, a very robust extraction capability undernormal conditions, largely due to its exceptionally large flatsurface area full of sp2 carbons. It is interesting to note that it hasbeen found recently that graphene can also induce cytotoxicity to

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Figure 5 | Robustness of lipid extraction by graphene. The average

interaction energy between the lipid membrane and the fully restrained

graphene nanosheet docked at the membrane surface with different depths

of potential well 1’CC. The two different background patterns indicate two

distinguished regions, with 1’CC ≤ 1/41CC and 1’CC . 1/41CC. It shows that

the graphene nanosheet can, in principle, extract phospholipids from the

membrane when 1’CC . 1/41CC (that is, a very robust extraction capability

under normal conditions).






neuronal PC12 cells, partially due to its aggregation/agglomerationon cell membranes13. This robust lipid extraction capability of gra-phene may also help explain its toxicity to other cells such as PC12.

As mentioned above, our insertion simulations with two differ-ent-sized graphene nanosheets, as well as the docking simulationswith an even larger size, indicate that larger graphene nanosheetsare more disruptive to bacteria cell membranes, either because ofstronger insertion and cutting (Type A) or a greater number oflipid extractions (Type B). To further confirm this, we performedadditional E. coli antibacterial activity experiments by increasingthe lateral sizes and concentrations of the graphene oxide. Withrepeated oxidation processes using the Hummers’ method (seeMethods and Supplementary PS6 for more details), we generatedvarious graphene oxide nanosheets with different sizes (seeGO1, GO2 and GO3 in Supplementary Fig. S5, with lateral sizesof �500, �200 and �50 nm, respectively). It is clear that after2.5 h incubation, larger graphene oxide nanosheets display amuch stronger antibacterial activity than smaller ones (90.9%,51.8% and 40.1% for GO1, GO2 and GO3, respectively, underthe same 100 mg ml21 concentration, Supplementary Fig. S6a).The increase in GO1 concentration also results in a persistent

increase in antibacterial activity (54.3%, 71.4%, 90.9% for25, 50 and 100 mg ml21, respectively, Supplementary Fig. S6b),because more graphene oxide nanosheets are now activelyengaged in the membrane cutting and lipid extraction. Thesefindings are also consistent with another very recent experimentby Liu et al34.

Simulations with graphene oxide confirm lipid extractionFinally, we simulated the graphene oxide nanosheet to furtherdemonstrate the robustness of this lipid extraction. As mentionedabove, in our TEM experiments we had to use graphene oxideinstead of pure graphene in order to obtain high water dispersabil-ity. For a more direct comparison with our TEM experiments, wethen simulated the interaction between graphene oxide and mem-branes using the Lerf–Klinowski graphene oxide structural modelwith a molecular formula of C10O1(OH)1(COOH)0.5, which rep-resents typical outcomes from the standard oxidation process35–37.Figure 6 shows representative configurations for graphene oxideinteracting with both the outer and inner E. coli membranes inthe same docking simulations. Again, both POPE and palmitoylo-leoylphosphatidylglycerol (POPG) phospholipids were extracted

a

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Figure 6 | Lipid extraction by graphene oxide nanosheets. a,b, Representative configurations of a fully restrained graphene oxide nanosheet docked at both

the outer (a) and inner (b) membrane surfaces, with initial conformations shown in side view and final ones shown in top view (both sides). The simulation

time is indicated in the top left corner of each snapshot. Colour settings are as in Fig. 2, but with water hidden for a clearer view.






from the membranes by the graphene oxide nanosheets. The maindriving force still stems from the strong van der Waals attractionsbetween the graphene oxide nanosheet and the membrane lipids.Once extracted, hydrophobic interactions once again play a domi-nant role through nanoscale dewetting. The lipid hydrophobictails tend to spread out, mainly in the unoxidized hydrophobicregions, while the hydrophilic head groups prefer to contact polaroxide groups via favourable electrostatic interactions.

It was recently proposed that large unoxidized residual graphene-like regions (sp2 domain) can exist on graphene oxidenanosheets35,38,39. Gomez-Navarro et al. found that, upon oxidation,isolated highly oxidized areas (a few nanometres in size) are formed,while at least �60% of the surface remains undisturbed38. In fact, theexistence of large unoxidized regions has been utilized experimen-tally to achieve the oxidative cutting and unravelling of carbonnanotubes40. Our own UV–vis spectra data also show that themaximum absorption peak of graphene, resulted from the sp2

domain of carbon atoms, displays an obvious blueshift uponrepeated heavy oxidation (see GO3 curve in SupplementaryFig. S7) due to the presence of oxygen and the increased numberof sp3 bonds. With less oxidation (and larger lateral size), this blue-shift is weakened (see GO1 curve in Supplementary Fig. S7), indicat-ing that there are larger sp2 domains with an increase in grapheneoxide lateral size. To further illustrate this point, we performedquantum mechanics calculations on the graphene oxidation path-ways (see Supplementary PS7 for detailed computation methodsand analyses). Our quantum mechanics calculations indicate thatfurther oxidations near the previously oxidized sites can signifi-cantly lower the energy barrier (by up to 14.5 kcal mol21) andsmooth the oxidation reaction pathways (increasing the reactionrate by 1011-fold) on graphene (Supplementary Figs S8 and S9).These quantum mechanics calculations present a consistentpicture regarding the possibility of condensed oxidation sites on gra-phene, thus resulting in potentially large sp2 domains on grapheneoxide nanosheets. It should be noted that our simulated grapheneoxide nanosheets (7.62 × 3.69 nm2) have sizes only �0.01% of theGO1 used in our TEM experiment (�500 × 500 nm2), so it isvery likely that sp2 domains with similar sizes to our current gra-phene nanosheets can exist on GO1. Furthermore, in reality, pureunoxidized graphene nanosheets do exist that can display evenmore power in extracting lipid molecules from membranes.

ConclusionsIn summary, using both experimental and theoretical approaches,we show that there are two types of molecular mechanism for thegraphene-induced degradation of E. coli cell membranes: one bysevere insertion and cutting and the other by destructive extractionof lipid molecules. This direct extraction of phospholipids from lipidmembranes was first observed in our computer simulations, andthen validated by our TEM images. This strong attraction betweengraphene and membrane lipids is largely derived from graphene’sunique two-dimensional structure with all sp2 carbons, which facili-tates exceptionally strong dispersion interactions between grapheneand lipid molecules. Cooperative movements of extracted lipidmolecules were also observed on the graphene two-dimensionalsurface due to the redistribution of the hydrophobic tails tomaximize hydrophobic interactions with the graphene surface.This behaviour was found for both outer and inner membranes ofE. coli. The destructive lipid extraction mediated by graphenenanosheets is also found to be very robust.

Both the severe graphene insertion and destructive lipid extrac-tion suggest that graphene nanosheets can induce serious mem-brane stress, and thus significantly reduce cell viability. Theresulting antibacterial activity also increases with increasing gra-phene lateral size and concentration. Even though these findingsare from E. coli, we believe similar mechanisms also apply to

other types of bacteria24,26. Thus, our current findings might haveimplications in the design of novel antibiotics and other clinicalapplications. In particular, we envisage that graphene mightbecome a new type of ‘green’ antibacterial material for everydayuse, with little bacterial resistance due to its ‘physical damage’-based bacterial killing mechanism41. It might also stimulate andfacilitate the cytotoxicity studies of other nanomaterials in thisemerging field of nanotoxicology.

MethodsPreparation of graphene oxide. Graphene oxide nanosheets were prepared fromnormal graphite powder by a modified Hummers’ method, as in our previouswork23,27,28. The lateral size of the nanosheets was �205 nm, and the thickness was�1.0 nm. Graphene oxides with other lateral sizes (from �50 nm to �500 nm) werealso prepared for further validations (see Supplementary PS6).

Bacterial culture. E. coli DH5a, as model Gram-negative bacteria, were grownovernight in LB medium (Luria-Bertani broth, Lennox modification) at 37 8C andharvested at the exponential growth phase via centrifugation. The E. coli DH5a cellswere washed twice to remove residual growth-medium constituents and resuspendedin sterile saline solution (0.9% NaCl). The E. coli cells were quantified by assessingthe optical absorption at 600 nm (OD600). The bacterial suspensions used for allexperiments contained �1 × 107 colony forming units (CFUs) per millilitre.

Transmission electron microscopy. The �1 × 107 CFU ml21 E. coli cells weresuspended in graphene oxide nanosheets (100 mg ml21) and cultured at 37 8C for2.5 h. The E. coli cells were then fixed with 2.5% glutaraldehyde for 30 min. Cellswere washed with 0.9% NaCl, fixed with 1% aqueous OsO4 (Fluka) for 1 h, andwashed 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/Aralditeresin (polymerization at 65 8C for 15 h). Thin sections (90 nm) containing cells wereplaced on the grids and stained for 1 min each with 4% uranyl acetate (1:1acetone/water) and 0.2% Raynolds lead citrate (water), air-dried, and examinedunder the TEM (Joel JEM-1230).

Computer simulation methods. We carried out molecular dynamics simulationsfor graphene nanosheets interacting with E. coli outer and inner membranes. Twocommonly found phospholipids in Gram-negative bacteria (for example, E. coli42)were used in our simulations, with the outer membrane modelled by pure POPE andthe inner membrane by a mixture of POPE and POPG (3:1 ratio)43–45. The Bergerlipid force field, one of the most commonly used and extensively validatedmembrane force fields46–48, was adopted in our simulations. Two sizes of graphenenanosheets, 4.92 nm × 1.99 nm (with 256 lipids) and 5.41 nm × 3.69 nm (with�340 lipids), interacting with both outer and inner membranes, were simulated(Supplementary Fig. S1). More details of the molecular dynamics simulationmethods are provided in Supplementary PS1.

For the graphene ‘docking’ simulations, all carbon atoms in the graphenenanosheet were restrained in space, with the nanosheet plane oriented vertically tothe membrane surface (x–y plane), which is presumably the hardest condition underwhich lipid extraction can occur because no kinetic effect is available from themoving graphene nanosheet. The lowest carbon atoms were initially 2 nm above themembrane surface (Fig. 4). After 20 ns of pre-equilibration, the z-position restraintson the graphene nanosheet were released, and the nanosheet was pulled along thez-direction to the membrane surface with a velocity of 0.05 Å ps21. When thegraphene nanosheet reached the membrane surface, all carbon z-positions wererestrained again to study the potential lipid extraction by the dockedgraphene nanosheet.

We also simulated the graphene oxide nanosheets, which were constructedbased on the Lerf–Klinowski structural model with a molecular formula ofC10O1(OH)1(COOH)0.5 (that is, two epoxy, two hydroxyl on both sides of thegraphene basal plane, and one carboxyl group on the edges of the graphene, per 20carbon atoms), which represents typical outcomes from standard oxidationprocesses35–37. These epoxy and hydroxyl groups are grafted on both sides of thegraphene basal plane, randomly, but localized with high correlations betweenoxidization loci, and the carboxyl groups are attached to the graphene edges38,39,49.The high correlation in oxidation sites resulted from the fact that a carbon atom onsome broken p-bond may be oxidized with much higher probability (once itsneighbouring atom has been oxidized) than its counterpart with an intact p bond.Our quantum mechanics calculations also confirm these experimental findings(see more detailed computation and analysis in Supplementary Figs S8 and S9).

Received 11 November 2012; accepted 28 May 2013;published online 7 July 2013; corrected after print 26 November 2013

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AcknowledgementsThe authors thank B. Berne, Yuliang Zhao, Guosheng Shi, Huan Zhang, Jingyuan Li,Seung-gu Kang, Zhen Xia and P. Das for discussions. This work was partially supported bythe National Natural Science Foundation of China (grant nos 11290164, 11204269,11172158 and 11105088), the National Basic Research Program of China (2012CB932400,2013CB933800 and 2012CB932600) and the First-class Discipline of Universities inShanghai. The authors acknowledge the IBM Blue Gene supercomputer and ShanghaiSupercomputer Center for computational resources. R.Z. acknowledges support from theIBM Blue Gene Science Program.

Author contributionsR.H.Z., Q.H., H.P.F. and Y.S.T. conceived and designed the experiments and simulations.Y.S.T., P.X., T.H. and M.Z. performed the simulations. M.L. performed the experiments.Y.S.T., P.X., H.P.F., R.H.Z., Q.H., C.H.F. and Z.R.L. analysed the data. Y.S.T., R.H.Z., H.P.F.,P.X., M.C., Q.H. and C.H.F. co-wrote the paper. All authors discussed the results andcommented on the manuscript.

Additional informationSupplementary information is available in the online version of the paper. Reprints andpermissions information is available online at www.nature.com/reprints. Correspondence andrequests for materials should be addressed to Q.H. and R.Z.

Competing financial interestsThe authors declare no competing financial interests.





www.nature.com/reprints



In the version of this Article originally published, it was not made clear that the two Escherichia coli cells in the bottom left of Fig. 1a are from a different TEM image to the others. The figure and caption have now been corrected in the PDF and HTML versions of the Article.

Destructive extraction of phospholipids from Escherichia coli membranes by graphene nanosheets Yusong Tu, Min Lv, Peng Xiu, Tien Huynh, Meng Zhang, Matteo Castelli, Zengrong Liu, Qing Huang, Chunhai Fan, Haiping Fang and Ruhong Zhou

Nature Nanotechnology 8, 594–601 (2013); published online 7 July 2013; corrected after print 26 November 2013.

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Destructive extraction of phospholipids from Escherichia coli membranes by graphene nanosheets

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