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PEGylated graphene oxide elicits strong immunological responses despite surface passivation

Authors:
  • Suzhou Alphamab Co. Ltd

Abstract and Figures

Engineered nanomaterials promise to transform medicine at the bio–nano interface. However, it is important to elucidate how synthetic nanomaterials interact with critical biological systems before such products can be safely utilized in humans. Past evidence suggests that polyethylene glycol-functionalized (PEGylated) nanomaterials are largely biocompatible and elicit less dramatic immune responses than their pristine counterparts. We here report results that contradict these findings. We find that PEGylated graphene oxide nanosheets (nGO-PEGs) stimulate potent cytokine responses in peritoneal macrophages, despite not being internalized. Atomistic molecular dynamics simulations support a mechanism by which nGO-PEGs preferentially adsorb onto and/or partially insert into cell membranes, thereby amplifying interactions with stimulatory surface receptors. Further experiments demonstrate that nGO-PEG indeed provokes cytokine secretion by enhancing integrin β8-related signalling pathways. The present results inform that surface passivation does not always prevent immunological reactions to 2D nanomaterials but also suggest applications for PEGylated nanomaterials wherein immune stimulation is desired.
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ARTICLE
Received 13 May 2016 |Accepted 6 Jan 2017 |Published 24 Feb 2017
PEGylated graphene oxide elicits strong
immunological responses despite surface
passivation
Nana Luo1,2,*, Jeffrey K. Weber3,*, Shuang Wang1,2, Binquan Luan3, Hua Yue1, Xiaobo Xi1,2, Jing Du4,
Zaixing Yang5, Wei Wei1, Ruhong Zhou3,5,6 & Guanghui Ma1,2,7
Engineered nanomaterials promise to transform medicine at the bio–nano interface. However,
it is important to elucidate how synthetic nanomaterials interact with critical biological
systems before such products can be safely utilized in humans. Past evidence suggests that
polyethylene glycol-functionalized (PEGylated) nanomaterials are largely biocompatible
and elicit less dramatic immune responses than their pristine counterparts. We here report
results that contradict these findings. We find that PEGylated graphene oxide nanosheets
(nGO-PEGs) stimulate potent cytokine responses in peritoneal macrophages, despite not
being internalized. Atomistic molecular dynamics simulations support a mechanism by which
nGO-PEGs preferentially adsorb onto and/or partially insert into cell membranes, thereby
amplifying interactions with stimulatory surface receptors. Further experiments demonstrate
that nGO-PEG indeed provokes cytokine secretion by enhancing integrin b
8
-related signalling
pathways. The present results inform that surface passivation does not always prevent
immunological reactions to 2D nanomaterials but also suggest applications for PEGylated
nanomaterials wherein immune stimulation is desired.
DOI: 10.1038/ncomms14537 OPEN
1State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China. 2University of
Chinese Academy of Sciences, Beijing 100049, PR China. 3Computational Biology Center, IBM Thomas J. Watson Research Center, Yorktown Heights,
New York 10598, USA. 4Institute of Biomechanics and Medical Engineering, Department of Engineering Mechanics, Tsinghua University, Beijing 100084,
PR China. 5Institute of Quantitative Biology and Medicine, SRMP and RAD-X, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher
Education Institutions, Soochow University, Suzhou 215123, PR China. 6Department of Chemistry, Columbia University, New York, New York 10027, USA.
7Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing 211816, PR China. *These authors
contributed equally to this work. Correspondence and requests for materials should be addressed to W.W. (email: weiwei@ipe.ac.cn) or to R.Z.
(email: ruhongz@us.ibm.com) or to G.M. (email: ghma@ipe.ac.cn).
NATURE COMMUNICATIONS | 8:14537 | DOI: 10.1038/ncomms14537 | www.nature.com/naturecommunications 1
Bio–nano interfaces form when nanomaterials come into
contact with biomolecular assemblies, such as protein
complexes or lipid membranes1,2. Within a given medium,
the physicochemical properties of bio–nano interfaces are mainly
dictated by the diverse compositions, morphologies and surface
chemistries that engineered nanomaterials can possess3,4.
By tuning these characteristics, a myriad of nanomaterial
functionalities can be realized for biomedical applications in
biosensing, drug delivery, imaging and tissue engineering5,6.
Important prerequisites for such biomedical applications
involve establishing the in vivo stability and biocompatibility
of nanomaterials in question7,8. Accumulating evidence has
suggested that the intrinsic activities of nanomaterials are
often overridden by the adsorption of biomolecular coronae
from the biological milieu9,10. These coronal molecules bestow
nanomaterials with new properties that transform their
interactions at the bio–nano interface, interfering with both
designed nanomaterial properties and innate biomolecular
functions. To bypass these effects, nanomaterials can be coated
with antifouling, hydrophilic and charge-neutral moieties such as
polyethylene glycol (PEG) chains11,12. The resultant ‘passivated’
surfaces have been shown to discourage internalization by
macrophages, allowing engineered nanomaterials to elude the
body’s preliminary line of defense against intruding particles13–15.
Such characteristics, in principle, are thought to prevent
macrophage activation and subsequent immunological response,
thus ensuring the safe use of exogenous nanomaterials.
Two-dimensional (2D) nanomaterials have garnered particular
attention due to their biomedical applicability16–18. Graphene
derivatives, for example, possess large and specific surface areas
that yield excellent adsorption propensities for drug delivery and
have intrinsic photoluminescence properties that facilitate live
cell imaging. Previous research into nanoparticle passivation,
however, has largely concentrated on traditional spherical
materials, such as micelles, liposomes and artificial polymers19.
Accordingly, here we study the immunological impact of surface-
passivated nano-graphene oxide (nGO), a prototypical and widely
encountered 2D nanomaterial.
Intriguingly, we found that the macrophage response to
PEGylated nGO was more dramatic than might be hypothesized.
Despite our presumption of non-internalization being largely
true, nGO-PEG was still shown to activate macrophages by
promoting high levels of cytokine secretion. We discovered that
this macrophage excitation was triggered through physical
contact between nGO-PEG and cell membranes, interactions
that enhanced cell mobility and migration. Applying gene
chip analysis, we demonstrated that nGO-PEG stimuli were
transduced into chemical signals through the upregulation of the
integrin b
8
and activation of subsequent signalling pathways. Our
molecular dynamics (MD) simulations support the notion that
while PEGylated nanosheets are less likely to be internalized, they
are even more likely to adsorb onto/partially insert into the
membrane surface in face-on/edge-on configurations and thus
solicit integrin-mediated signalling pathways. We explicate all of
these results in depth below.
Results
Elevated cytokine response to nGO-PEG. Foreign bodies that
enter human serum are normally engulfed by macrophages,
which in turn alter physiological behaviours involving cytokine
secretion, inflammation and other related stress responses20,21.
PEG is commonly conjugated to nanomaterial surfaces to
avoid such internalization by immune cells22,23. Negligible
internalization of nGO-PEG by macrophages was indeed
observed in our experiments, as indicated by the absence of
intracellular fluorescence signal (purple) in the nGO-PEG
confocal image (Fig. 1a). Signal from internalized nanosheets,
however, is clearly present in cells exposed to pristine nGO.
In further contrast with pristine nGO, which caused substantial
nuclear damage to cells, the nuclear characteristics of nGO-PEG-
exposed macrophages (for example, shape, area, roundness
and intensity) remained consistent with those of normal cells
(Supplementary Fig. 1). Coupled with the results of other viability
tests (such as the CCK-8, Live-Dead and Annexin-V/PI assays)
that can be found in our past work13, our data suggest that
nGO-PEGs are highly biocompatible.
In agreement with past observations, internalization of pristine
nGOs stimulated the secretion of activation-associated cytokines
such as interleukin (IL)-6, monocyte chemotactic protein-1,
interferon-g, tumour necrosis factor-aand IL-12 (Fig. 1b) in
macrophages. But, contrary to our expectations, substantially
greater cytokine production (even higher than in the positive
control group of cells treated with lipopolysaccharides) was
observed in macrophages incubated with the supposedly inert
nGO-PEG (Fig. 1b). To explore these puzzling results, we first
measured cytokine levels as a function of time (Fig. 1c).
These data confirm that nGO-PEG indeed triggers the most
conspicuous cytokine response among the materials tested
(Fig. 1c). In a dosage-dependent assay (featuring nGO-PEG
concentrations ranging from 2.5 to 10 mgml1), the levels of all
activation-associated cytokines detected increased with increasing
nGO-PEG dosage; little change was observed in the secretion of
suppressive factor IL-10 (Fig. 1d). Neither free PEG nor a simple
mixture of nGO and PEG (featuring no covalent conjugation
between the two groups) aroused macrophage activity on the
scale seen with nGO-PEG (Supplementary Fig. 2). Moreover,
cytokine production levels were found to be positively correlated
with the density of conjugated PEG chains (Supplementary
Fig. 3). Chemical conjugation between PEG and nGO thus seems
to be important for the marked increase in cytokine secretion
observed with PEGylated GO.
Impact of nGO-PEG on macrophage membranes. We next
sought to explicitly measure the impact of nGO-PEG on cell
membrane parameters. After 24 h of incubation with nGO-PEG,
confocal images revealed profoundly extended filopodia (green)
intertwined with nGO-PEG (purple) on macrophage surfaces
(Fig. 2a). These filopodia were also visible in transmission
electron micrographs (Fig. 2b).
To gauge the impact of these nGO-PEG interactions on cell
membrane integrity, we performed a lactate dehydrogenase
leakage assay (Fig. 2c). Only very slight leakage was detected in
the nGO-PEG-treated group, indicating that the membranes of
nGO-PEG-exposed macrophages remain, in large part, intact and
able to maintain normal cellular functions. As membrane motility
is also highly correlated to membrane function, we labelled cell
membranes with DiO and employed the fluorescence recovery
after photobleaching technique to characterize membrane diffu-
sion properties (Fig. 2d, Supplementary Fig. 4). According to the
observed recovery kinetics, normal cells exhibited a recovery half
time (t
1/2
) of 12.21 s and a diffusion coefficient of 0.031 mm2s1.
By contrast, exposure to pristine nGO resulted in a recovery half
time (t
1/2
) of 7.51 s and an increased membrane diffusion
coefficient of 0.145 mm2s1. The slope of the fluorescence curve
for nGO-PEG-treated cells was even steeper, featuring a half time
of 5.5 s; the corresponding diffusion coefficient (0.166 mm2s1)
was determined to be higher than that of either normal or nGO-
exposed cells. This increased membrane mobility within the
nGO- and nGO-PEG-treated groups is likely attributable to
interactions between the nanosheets and cell membranes, a
phenomenon that is more pronounced with nGO-PEG exposure.
The precise mechanisms by which diffusive dynamics are
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14537
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accelerated remain to be elucidated. As discussed in the context of
our simulations, one expects direct contact with nGOs to freeze
local membrane segments, arresting diffusion through site-
specific interactions with lipids. Enhanced macrophage activation,
however, could very well result in increased diffusion rates via
some downstream process. Regardless of the underlying mechan-
ism, heightened lipid mobility should serve to improve the
transport properties of nGO-PEGs adsorbed onto the membrane,
perhaps further amplifying macrophage activity.
The observed increase in membrane mobility could, in part,
be associated with intensified macrophage migration induced by
the presence of nGO-PEG (Fig. 2e). Within 1 h of observation,
normal cells persisted in quiescent and inactive states, remaining
at their original locations. The addition of pristine nGOs caused
cells to migrate only a small distance farther. Cells exposed to
nGO-PEG, however, greatly extended their spheres of migration,
with some cells even moving outside of our field of vision over the
monitoring period (Supplementary Fig. 5). More quantitatively,
nGO-PEG prompted cell trajectories to approach the periphery of
a30mm spherical area, while untreated and nGO-treated cells
remained near the centre of that region (Supplementary Fig. 6).
The presence of non-internalized nGO-PEG thus is distinctly
associated with an increase in macrophage motility, movement
that is likely a result of their activation.
Ctrl nGO nGO-PEG
Ctrl
nGO
nGO-PEG
LPS
Ctrl
nGO
nGO-PEG
LPS
Ctrl
nGO
nGO-PEG
LPS
Ctrl
nGO
nGO-PEG
LPS
104
104
103
103
102
102
101
101
100
100
IL-6
IL-10
MCP-1
IFN-γ
TNF-α
IL-12
6,000
5,000
4,000
3,000
2,000
1,000
0
Cytokine levels (pg ml–1)
Cytokine levels (pg ml–1)
6 h
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24 h
48 h 2,000
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0
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0
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0246810
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024 6810
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0
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0
IL-6 IL-10 MCP-1
IFN-γTNF-αIL-12
IL-6
IL-10
MCP-1
IFN-γ
TNF-α
IL-12
nGO-PEG (μg ml–1)
PE-cy5
PE
a
b
cd
Figure 1 | Impact of nGO and nGO-PEG on macrophage behaviour. (a) Internalization of nGO and nGO-PEG observed by confocal imaging (purple dots,
marked with white arrows: nGO complexes). Scale bar: 5 mm. (b) Flow cytometric dotplots of cytokine stimulation induced by nGO over 24 h. (c) Histogram
of total cytokine concentrations as a function of time. Each cytokine concentration column is displayed as the mean value of three replicas. (d) Cytokine
secretion induced by nGO-PEG at different concentrations after 24h coincubation. Dosages in acwere fixed at 10 mgml1. Data are presented as
means±s.d., with n¼3.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14537 ARTICLE
NATURE COMMUNICATIONS | 8:14537 | DOI: 10.1038/ncomms14537 | www.nature.com/naturecommunications 3
Molecular basis of membrane-nGO-PEG interactions.
Considering the unique properties possessed by 2D nanomaterials,
one might posit that the planar structure of nGO-PEG could
define the interactions responsible for its macrophage activation.
As a rudimentary test of this hypothesis, we incubated macro-
phages in the presence of PEGylated carbon spheres (B200 nm)
and one-dimensional carbon nanotubes (B4mm length) (Fig. 3a).
Under similar constraints on surface area and dose (10 mgml1),
2D nGO-PEG induced, by far, the highest levels of cytokine
secretion, membrane diffusion and cell migration (Fig. 3b,
Supplementary Fig. 7). This trend in cytokine secretion is
conserved among pristine nanomaterials as well (Supplementary
Fig. 8). Based on simple physical arguments, 2D PEGylated
nanomaterials should indeed have the most pronounced
interactions with cell membranes. The nGO-PEG, for example,
might favour plane-to-plane interactions with cell membranes,
which should cover more area (and thus support stronger van der
Waals interactions) and persist longer than the point-to-plane
and line-to-plane ‘binding modes’ of PEGylated carbon spheres
and one-dimensional carbon nanotubes, respectively (Fig. 3c).
Such planar interaction characteristics, in turn, could translate
into high flux through activating receptors on the macrophage
surface, as we discuss later.
Seeking clearer insight into the microscopic interactions
between nGO-PEG and macrophage surfaces, we performed
extensive molecular dynamics simulations of GO nanosheets
120 ***
***
*
100
80
60
40
20
0
0
–20 –10 0 10 20
Bleaching duration = 7.68 s Bleaching duration = 7.65 s Bleaching duration = 7.92 s
Diffusion co-
efficient: 0.031 µm2 s–1
Diffusion co-
efficient: 0.145 µm2 s–1
T1/2=12.21 s T1/2=7.51 s T1/2=5.50 s
30
Time (s)
r=30 µm
Ctrl nGO nGO-PEG
r=30 µmr=30 µm
40 50 60 70 –20 –10 0 10 20 30
Time (s)
40 50 60 70 –20 –10 0 10 20 30
Time (s)
40 50 60 70
Normalized intensity (%)
20
40
60
80
100
ab c
0
20
40
60
80
100
0
20
40
60
80
100
P-ctrl
N-ctrl
nGO
nGO-PEG
Relative LDH leakage
d
e
Diffusion co-
efficient: 0.166 µm2 s–1
Figure 2 | Impact of nGO and nGO-PEG on cell migration and membrane integrity. (a) Confocal (top view; Scale bar: 1 mm) and (b) TEM images
(side view; scale bar: 200 nm) showing interactions between nGO-PEG and macrophage filopodia. (c) Membrane integrity analysis conducted through
lactate dehydrogenase leakage assays. The designations p-ctrl and n-ctrl represent positive and negative control, respectively. (d) Kinetics of macrophage
membrane fluorescence recovery after photobleaching in the absence/presence of nGO and nGO-PEG. (e) Trajectories of cells in the absence or presence
of nGO and nGO-PEG (n¼6 cells), where the graphical sphere radius is 30 mm (see Supplementary Movies 1 and 2 for more information). Data are
presented as means±s.d. with n¼3. *Po0.05, ***Po0.001.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14537
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(both pristine and PEGylated) in the presence of lipid
membranes. Notably, past experimental and simulation work
has demonstrated that pristine nGO has destructive effects
on phospholipid bilayers via direct incisive mechanisms and
further compromises membrane integrity through aggressive lipid
extraction24–28.
Corroborating this past evidence, both incisive and extractive
mechanisms were indeed observed in our simulations of pristine
nGO-membrane interactions (Fig. 4). Lipid extraction events
buffeted the insertion process at early times (for example,
at t¼30 ns), as recoil forces drew the nGO further toward the
membrane centre. As the simulation proceeded, a depletion of
lipid density became evident in membrane regions away from the
point of nGO interaction. After a few hundred nanoseconds, the
top of the nanosheet nearly disappeared below the membrane
surface, almost penetrating the opposite side of the bilayer.
Both this physical incision and the long-range density deficit
induced by lipid adsorption onto the GO surface translate
into destabilization of the membrane’s structure; these results
support past experimental observations of pristine nGO inflicting
damage on macrophage cell membranes13. The complete nGO
insertion observed here also suggests that nGO internalization by
6,000
4,000
Same
surface area
CS CNTnGO
a
b
c
nGO-PEG Planeplane
Pointplane
Lineplane
Cell membrane
CS-PEG
CNT-PEG
Shape
Dimension
10 μg ml–1
Cytokine levels (pg ml–1)
0
Ctrl
LPS
CS-PEG
CNT-PEG
nGO-PEG
CS-PEG
CNT-PEG
2,000
IL-6 IFN-γ
TNF-α
IL-12
IL-10
MCP-1
Figure 3 | Effects of PEGylated carbon nanomaterials on cytokine secretion. (a) Atomic force microscopic (AFM), scanning electron microscopic (SEM)
and TEM images of graphene oxide, carbon spheres and carbon nanotubes, respectively. Scale bars: 200 nm. (b) Inflammatory cytokines secreted by
macrophages when exposed to different PEGylated carbon nanomaterials, normalized by surface area and concentration. (c) Conjecture for interaction
modes between carbon nanomaterials and cell membranes resulting in different levels of cytokine secretion.
t = 0 ns
ab
t = 30 ns t = 100 ns Equilibrated
(t > 320 ns) Pegylated GO/membrane COM
PEG/GO COM
50
40
30
Separation (Å)
20
10
0 0.5
Time (μs)
1
2.5
5
7.5
10
12.5
GO/membrane COM
t = 0 ns
PEGylated
GO
t = 80 ns t = 120 ns Equilibrated
(t > 320 ns)
GO
Figure 4 | Simulation of nGO- and nGO-PEG-membrane interactions from edge-on configurations. (a) System snapshots relevant to the observed
membrane insertion processes, with GO carbons represented in grey and covalently linked PEG chains rendered in purple. (b) Centre-of-mass (COM)
displacement data recorded over the course of the simulation trajectories. The PEG/GO COM separation trace (which highlights the PEG extrusion process
during insertion) is presented on an alternative vertical scale.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14537 ARTICLE
NATURE COMMUNICATIONS | 8:14537 | DOI: 10.1038/ncomms14537 | www.nature.com/naturecommunications 5
macrophages might proceed via both passive and active means,
as reported elsewhere26,27,29.
Further MD simulations were performed to elucidate the
passivating effects of PEG conjugation with nGO. A PEGylated
GO nanosheet (with four PEG chains protruding from the nGO
edges and two from the nGO faces) was first simulated in the
absence of a membrane. Starting from fully extended polymer
configurations, the PEG chains quickly collapsed and adsorbed
onto the GO surface (Supplementary Fig. 9), as seen with protein
adsorbates30. The 2D nature of nGO thus should largely be
conserved in nGO-PEG. Placing an equilibrated nGO-PEG edge-
on above the membrane surface, we observed that surface-bound
PEG chains compete with lipid adsorption, impeding nanosheet
insertion and preventing complete membrane penetration.
Adsorbed serum proteins have been observed to mediate
similar obstructions of graphene insertion31. As the centre-of-
mass separation traces in Fig. 4b indicate, nGO-PEG insertion
proceeds more gradually than in the case of pristine nGO.
Interestingly, the bound PEG molecules resist desorption over the
course of interactions with the membrane: the adsorbed chains
are extruded upwards as lipids attach to the nGO surface and
draw the nanosheet downward. Once available area on the GO
surface has been exhausted, the insertion process terminates,
leaving the PEG-covered graphene surface partially exposed on
the membrane exterior. The damage incurred by the membrane,
accordingly, is moderate compared with that seen for pristine
nGO. Adsorbed PEG not only serves as a steric hindrance to
membrane penetration but also occupies surface area that would
otherwise attract lipids and further deplete the equilibrium
density of the bilayer. One expects these basic consequences of
PEG functionalization—that is, diminished membrane cutting
and lipid extraction capabilities—to apply to nGO-PEGs of
various sizes, explaining why PEGylated nanosheets are more
benign than their unprotected counterparts13.
The notion that nGO-PEG is less damaging to cell membranes
may provide the key to understanding its immunoactive
properties. Though macrophages should remain highly viable
upon exposure to nGO-PEG, that viability does not prevent
PEGylated nanosheets from adhering to cell membranes. Partially
inserted nGO-PEGs, similar to that shown in Fig. 4, are likely to
remain in that state for extended time periods and diffuse across
the cell surface. It is even more likely, however, that nGO-PEG
will also adsorb onto membranes in face-on configurations.
The simulations featured in Fig. 5 support this statement: face-on
nGOs and nGO-PEGs immediately attached to the membrane
surface and showed no signs of desorbing. Though estimating
quantitative binding kinetics from these simulation trajectories is
not possible, total interaction energies after face-on absorption
favour the nGO-PEG by an approximate factor of two (Fig. 5b).
An interesting phenomenon emerges that explains this surplus
nGO-PEG interaction energy: after initial face-on contact, loops
and termini of PEG chains desorb to form transient ‘anchors’
that bore into the lipid bilayer (Fig. 5c). One would not expect
these single-chain anchors to compromise membrane integrity;
however, the additional surface area and polar moieties made
available by protruding PEG molecules enhance both electrostatic
and van der Waals interactions with the membrane
(Supplementary Fig. 10). Indeed, compared with pristine nGO,
this augmented interaction energy is correlated with spatially
tighter binding between nGO-PEG and the membrane
(Supplementary Fig. 11).
Regardless of orientation, nGO-PEGs should thus be more
capable than pristine nGOs of attaching to and diffusing across
macrophage surfaces and activating cytokine-related receptors
embedded in membranes. Our simulation data suggest several
complementary reasons for the high activity of macrophages
exposed to nGO-PEG. First, nGO-PEGs are unlikely to be
internalized via direct/passive mechanisms, meaning adsorbed
nGO-PEGs should persist longer on macrophage surfaces and
elicit a correspondingly stronger cytokine response. Second,
nGO-PEGs bound face-on to membranes seem more likely to
remain in that state, as the substantially enhanced membrane
interaction energies of nGO-PEGs indicate. Third, macrophages
exposed to nGO-PEG are more likely to stay viable than those
treated with pristine nGO, implying that the entire macrophage
ensemble should be better able to transmit cytokine-related
messages in response to nGO-PEG adsorption. Finally,
adsorbed nGO-PEGs (present at a surplus compared with
pristine nGOs) might also recruit immunoactive membrane
proteins to nanosheet-binding sites by inducing changes in
membrane curvature and dynamics; such effects have been
proposed in previous work on graphenes and other classes of
nanoparticles29,32–34. Due to the high computational cost of
atomistic simulations, long-range curvature effects could not
be probed in the present work. Though enhanced diffusion was
observed upon nGO-PEG exposure at the cellular scale, one
t = 0 ns
ab
c
t = 6 ns t = 100 ns Time (ns)
400 600
t = 500 ns
Energy (kcal mol–1)
2000
0
–800
PEGylated GO/membrane
GO/membrane
–1,600
t = 0 ns t = 20 ns t = 100 ns
Figure 5 | Simulation of nGO- and nGO-PEG-membrane interactions from side-on configurations. (a) Representative snapshots of the membrane
adsorption process. (b) Total interaction energies between pristine and PEGylated nGOs and the lipid bilayer. (c) Illustration of PEG desorption events
that lead to the burial of PEG anchors in the membrane, events that further enhance nGO-PEG/membrane-binding energies.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14537
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expects an opposite effect in areas local to substrate/membrane
contact. Direct lipid–nanosheet interactions should arrest lipid
diffusion—via a process not unlike a glass transition—as our
calculations confirm (Supplementary Fig. 12). Whether or
not mesoscale diffusion and curvature effects emerge from
basic membrane physics represents an intriguing topic for
future study.
Mechanism of nGO-PEG-induced cytokine secretion. In order
to alter the cellular behaviour, the above interactions between
nGO-PEG and cell membranes must be converted into chemical
signals35,36. To investigate the mechanism by which this signal
transduction occurs, we performed a broad-spectrum gene
screening on macrophages exposed to nGO-PEG. Based on our
gene chip analysis, we noticed that a multitude of membrane
protein-related genes were significantly upregulated and
downregulated within the nGO-PEG group (Fig. 6a). Notable
among translations of upregulated genes, integrins are known
to be of particular importance for mediating cell adhesion,
migration and the activation of divergent signalling
pathways37,38. Using antibody-blocking experiments, we thus
IL-6 IFN-γ
TNF-α
IL-12
IL-10
MCP-1
β8 siRNA
Ctrl nGO-PEG
P
P
P
P
P
P
P
P
P
0 8.13 10.46
CK nGO-PEG Gene
ab c
d
f
eg
Itgb7 –2.16
–3.05
–9.29
–10.83
–3.68
19.27
8.51
3.95
3.01
1.69
2.01
4.57
14.19
7.74
3.54
3.31
2.01
3.72
6.71
2.32
26.52
Itgb3bp
Itga6
Itgb5
Fn1
Itgb8
Itga1
Itgal
Itga5
Itgb1
Itgb2
Cav1
Src
Zyx
Vasp
Flnb
Akt3
Birc3
Ccnd3
Rac2
Pdgfrb
Fold
change
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40 μg ml–1
Integrin β8
GAPDH
4
**
3
Integrin β8/GAPDH
2
1
0
FAK
P
Src
Vin
Ras
PI3k
PKC
Raf
Mek
Erk
Fos
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nGO-PEG
nGO-PEG
Ctrl
Ctrl nGO-PEG
nGO-PEG
anit-itg α1
anit-itg αL
anit-itg β1
anit-itg β2
anit-itg β8
20 μg ml–1 anit-itg β8
10 μg ml–1 anit-itg β8
100 μg ml–1 anit-itg αv
β8 siRNA
Cytokine levels (pg ml–1)
2,000
p-FAK
FAK
2.0
1.5
***
1.0
p-FAK/FAK
0.5
0.0
100
50
Fold change
3
2
1
0
Shc1
Hras1
Kras
Raf1
Map2k3
Map2k1
Mapk3
Mapk1
Fosl1
Fos
Pik3ca
Prkca
Prkcb
Nfkb1
Ctrl
0
P
Figure 6 | Mechanistic investigation of high cytokine secretion driven by nGO-PEG. (a) Heatmap of membrane-related genes in the control and
nGO-PEG groups. (b) Secreted inflammatory cytokine concentrations after the application of different integrin inhibitors or b
8
siRNA interference.
(c,d) Western blotting analysis of integrin b8 and p-FAK after 24 h. Full gel images are included in Supplementary Figs 19 and 20. (e) Colocalization of
integrin b
8
(green) and vinculin (red), with high-magnification insets included at the bottom. Scale bar: 5 mm.(f) Extent of gene expression change
(measured by quantigene detection) in the control/nGO-PEG/siRNA groups after 24 h. (g) Proposed activation pathways that could result in high levels of
cytokine secretion upon the introduction of nGO-PEG. Data are presented as means±s.d. with n¼3. **Po0.01 and ***Po0.001.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14537 ARTICLE
NATURE COMMUNICATIONS | 8:14537 | DOI: 10.1038/ncomms14537 | www.nature.com/naturecommunications 7
explored the signalling pathways of the integrins most affected by
nGO-PEG (Fig. 6b). Suppression of integrins a
1
/a
L
/b
1
/b
2
had
little effect on cytokine secretion upon exposure to nGO-PEG, but
blockage of the most highly upregulated integrin b
8
(Fig. 6a,c),
substantially reduced cytokine concentrations with near
uniformity. As integrin receptors are composed of aand b
subunits, we also interrogated the behaviour of a
v
, the sole
binding partner of b
8
. In a manner complementary to b
8
,a
substantial (though slightly less dramatic) decrease in cytokine
concentrations was also seen upon a
v
blockage (Supplementary
Fig. 13). This observation was conserved within the entire a
v
subgroup, a set of proteins consisting of four additional
heterodimers that were diluted to the effective concentration of
a
v
b
8
. A further short interfering RNA (siRNA) experiment
(Supplementary Figs 14 and 15) demonstrated that a deficiency in
b
8
gene expression diminished the effect of nGO-PEG on
cytokine secretion (Fig. 6b), again suggesting a key role for b
8
in nGO-PEG-induced signal transduction.
Early in integrin signalling pathways, macromolecular
complexes called focal adhesions serve as carriers that transmit
signals into the cell interior39. This signalling process
proceeds through the recruitment of focal adhesion kinase
(FAK) to integrin clusters, which is activated upon binding
via autophosphorylation40,41. Monitoring the expression of
phospho-FAK (p-FAK) over the course of 2 days by western
blotting (Fig. 6d, Supplementary Fig. 16), we found that
nGO-PEG gradually provoked the production of p-FAK with a
significant difference from normal cells (Po0.001). In addition to
p-FAK, the protein vinculin is also a key component in focal
adhesion complexes42. We thus imaged the distribution of
integrin and vinculin using confocal microscopy (Fig. 6e). In
contrast with untreated cells, the introduction of nGO-PEG not
only promoted the expression of integrin b
8
but also affected its
distribution around the cellular perimeter. Strong colocalization
of vinculin and integrin (shown in the high-magnification
inset of Fig. 6e) was also observed in response nGO-PEG
exposure, suggesting that a proliferation of focal adhesions and
concomitant signal transduction occurs as vinculin is recruited to
nGO-PEG-binding sites.
Considering the putative pathways indicated by gene chip
analysis (Supplementary Table 1), we made a further effort to
quantify the expression of relevant genes through quantigene
detection. As shown in Fig. 6f and Supplementary Figs 16 and 17,
the presumed physical interactions with nGO-PEG result in a
many fold upregulation of genes, such as Shc1, Kras, Map2k1 and
Fos. Similar to our antibody-blocking experiments, pretreatment
with integrin b
8
siRNA depressed the expression to levels more
comparable to those of the control group.
Considered together, our results suggest a clear mechanism by
which exposure to nGO-PEG stimulates cytokine secretion
(Fig. 6g). Integrin a
v
b
8
, residing on the membrane surface,
is activated by interactions with membrane-adsorbed or -inserted
nGO-PEG, resulting in vinculin recruitment and autophosphor-
ylation of FAK. P-FAK then arouses elevated expression of the
genes Ras, Raf, Mek and Erk, which in turn governs the enhanced
expression of the nuclear transcription factor Fos. The genes for
phosphoinositide-3 kinase and protein kinase C are also activated,
encouraging the translocation of nuclear factor-kB from the
cytoplasm into the nucleus (Supplementary Fig. 18). These events
act in concert to boost cytokine synthesis and secretion by
macrophages, potentially leading to further immunological
responses downstream.
In conclusion, surface passivation is widely considered to be
an effective dampener of crosstalk at the bio–nano interface,
improving nanomaterial stability and biocompatibility and
circumventing macrophage internalization and activation.
However, our studies have demonstrated that PEGylation of
2D nanomaterials is less passivizing than previously believed.
Although stable, biocompatible and non-internalized, nGO-PEG
was still able to activate macrophages by triggering a potent
release of cytokines. One suspects nGO-PEG’s high available
surface area for membrane interactions—representative of all 2D
nanomaterials—dictates stimulatory effects on the cells in
question. Distinct changes in membrane morphologies were
observed upon nGO-PEG exposure, alongside augmented
membrane mobilities and enhanced cell migration. Our simula-
tion work on nGO-PEG/membrane interactions suggested
specific molecular mechanisms (via edge- and face-on contact)
by which macrophage activation might be facilitated. Further
experiments established that the integrin a
v
b
8
plays a crucial
initiating role in signal transduction related to nGO-PEG/
membrane binding. Through the upregulation of integrin b
8
and the subsequent activation of FAK-related intracellular
signalling pathways, the external stimulus carried by nGO-PEG
is transduced into chemical signals that ultimately give rise to
macrophage activation.
Importantly, the present study indicates that surface
passivation might not always allow 2D nanomaterials to escape
immunological responses. We did note an occurrence of cytokine
downturn at the time of second stimulus (Supplementary Fig. 21),
an observation that suggests nGO-PEG-induced macrophage
activation might decay over time. Interestingly, very comparable
levels of activation were also observed with the PEGylation
of the non-carbon-based 2D nanomaterial MoS
2
(Supplementary
Fig. 22). Questions as to whether such activation by 2D
PEGylated nanomaterials would prime further inflammatory or
immunological responses, and to what extent, demand further
in vivo experimentation. In the meantime, it would be worthwhile
to explore the potential for nGO-PEG-induced activation in
related systems such as dendritic cells, which are critical antigen-
presenting cells in the immune system43. Indeed, our
observations also suggest that PEGylated nanomaterials may be
suitable for use in situations in which the immune system
requires stimulation44,45. The fact that nGO-PEG elicits a
vigorous immune response without causing conspicuous
damage to macrophages is perhaps promising from a
therapeutic perspective. Though more research is certainly
needed, targeted cytokine secretion induced by carefully
delivered nanomaterials such as nGO-PEG could serve as an
effective component of future immunotherapies. Further work on
the immunological implications of nGO-PEG exposure will not
only supplement our current knowledge of the effects of surface
functionalization but will also pave the way for safer and more
effective biomedical applications for these novel nanomaterials.
Methods
Cell cultures.Peritoneal macrophages (PMØs) were harvested from stimulated
C57BL/6 mice according to a typical protocol46 and cultured under standard
conditions. C57BL/6 mice were ordered from Charles River Laboratories (USA).
All animal experiments were performed in compliance with the institutional ethics
committee regulations and guidelines on animal welfare. The murine macrophage
cell line J774A.1 was supplied by the ATCC (American Type Culture Collection).
More details of cell culture and other methods can be found in Supplementary
Methods.
Nanomaterial synthesis and characterization.PEGylation of pristine nGO
(single layered, with B200 nm lateral size), as well as other carbon-based
nanomaterials, was performed based on previously established methods47. In brief,
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (20 mM) was introduced
into a pristine nGO suspension (B500 mgml1) and sonicated for 15 min, after
which mPEG-NH
2
was added and allowed to react overnight. The final products
were harvested by centrifugation at 70,000gafter repeated washing with deionised
water. Nanomaterial morphologies were imaged using an atomic force microscope
(Bruker), a scanning electron microscope and a transmission electron microscope
(JEOL). More detailed characterizations of nGO and nGO-PEG are included in
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14537
8NATURE COMMUNICATIONS | 8:14537 | DOI: 10.1038/ncomms14537 | www.nature.com/naturecommunications
Supplementary Figs 23 and 24; Supplementary Tables 2 and 3 and our previous
study13.
Cytokine secretion measurements.To measure cytokine secretion levels, PMØ
cells were exposed to various GO solutions over distinct time periods
(6, 12, 24 and 48 h) and at different dosages (10 and 40 mgml1). Cytokine
secretion (IL-6, IL-10, IL-12, tumour necrosis factor-a, monocyte chemotactic
protein-1, and interferon-g) was evaluated by flow cytometry (Beckman Coulter).
Cell imaging.For imaging applications, PMØs were seeded (1 105ml 1)ina
petri dish and incubated with nGO complexes at 10–40 mgml1for 24 h. NGO
complex imaging was performed using flow cytometry or confocal laser scanning
microscopy (Leica), invoking graphene’s intrinsic photoluminescence. The
cytoskeleton and nuclei were separately stained with rhodamine-phalloidin
(green pseudocolour in images, Invitrogen) and Hoechst. For characterization of
membrane morphologies, cells treated with nGO-PEG were stained and fixed, cut
on a Reichert Ultracut microtome (Leica) and imaged using transmission electron
microscope. Lactate dehydrogenase leakage assays and fluorescence recovery after
photobleaching experiments were conducted using standard procedures. Cell
trajectory videos were recorded inside an Ultraview (PerkinElmer) incubator in
bright field mode.
Gene chip analysis.In preparation for gene chip analysis, PMØ cells were
cultured and then exposed to 10 mgml1nGO-PEG for 12, 24 and 48 h,
respectively. RNA was extracted from each sample using 0.6 ml of trizol reagent
and sent to Shanghai Gene Corporation for further analysis. Pathway analysis was
performed using the KEGG database. Quantigene detection was carried out on
PMØ cells split into control, nGO-PEG and b
8
-gene-silenced nGO-PEG groups
after 12, 24 and 48 h of coincubation.
Antibody-blocking experiments.Anti-integrins b
1
,b
2
(Millipore), a
1
(Abcam),
a
L
,b
8
(Santa Cruz Biotechnology) and a
v
(Biolegend) were added at the indicated
concentrations and allowed to incubate 4 h; cells were subsequently exposed to
culture medium containing 10 mgml1nGO-PEG for 24 h. Cytokine levels were
then detected as described above. Transfection of mouse integrin b
8
siRNA into
PMØs was performed with an RNAimax reagent (Invitrogen) according to the
manufacturer’s instructions.
Western blotting.Primary antibodies for GADPH (1:1,000, Goodhere
Corporation), FAK (1:1,000, Santa Cruz), p-FAK (1:500, Cell Signaling
Technology), integrin b8 (1:500, Biolegend) and horseradish peroxidase-
conjugated anti-rabbit IgG (1:2,000, Cell Signaling Technology) were used for
immunoblotting analysis. Immunofluorescence experiments were conducted using
b
8
integrin (1:200) and vinculin (1:200, Sigma-Aldrich) for primary antibodies and
anti-rabbit IgG conjugated with Alexa Fluor 488 and anti-mouse IgG with Alexa
Fluor 647 dyes for confocal laser scanning microscopic imaging. Quantigene
detection was carried out on PMØ cells split into control, nGO-PEG and
b
8
-gene-silenced nGO-PEG groups after 12, 24 and 48 h of coincubation.
Simulation parameters and configuration.nGO sheet coordinates and
chemistries were first generated (using the VMD Nanotube Builder plugin48)to
comply with a slightly reduced version of the standard Lerf–Klinowski model49.
Starting PEG chain configurations, each consisting of 15 monomers, were then
created manually; PEG chains were covalently attached to nGO through amide
linkages similar to those used in our experiments. Force field parameters for PEG
monomers and termini were extracted directly from the CHARMM ether force
field50, while parameters related to GO functionalities and amide linkages were
adapted from similar motifs within the CHARMM27 force field51.
Simulation setup.The initial nGO-PEG configuration was minimized and
equilibrated in isolation using the NAMD simulation package52,applyingaLangevin
integrator (310 K; 1 atm), the CHARMM27 force field, the TIP3P water model, PME
electrostatics and normal SETTLE constraints. An equilibrated snapshot of our nGO-
PEG system is shown in Supplementary Fig. 10. The 1-palmitoyl-2-oleoyl-sn-glycero-
3-phosphocholine (POPC) membrane fragments featured in our simulations were
generated and equilibrated using a standard procedure.
Production simulations.For production simulations, an equilibrated nGO-PEG
(or pristine nGO) was initialized above the membrane in either a face- or edge-on
configuration; trajectory data were collected for several hundreds of nanoseconds
(in some cases, for a microsecond) using the same force field and simulation
parameters described above.
Data availability.Data supporting the findings of this study are available within
this article (and its Supplementary Information file) and from the corresponding
author upon reasonable request.
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Acknowledgements
We thank Ding Ma’s Group of Peking University for offering the use of GO and CNT
powders; we also thank Hua Zhang’s group of Nanyang Technological University for
the use of MoS
2
solution. This work was partly supported by National Science and
Technology Major Project (2014ZX09102045-004), 973 Program (2013CB531500) and
National Natural Science Foundation of China (21320102003, 11374221, 11574224,
31370018). This project was partially funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD). R.Z. acknowledges the
support from the IBM Blue Gene Science Program (W125859, W1464125, W1464164).
J.D. is also supported by Tsinghua University (2012Z02133).
Author contributions
G.M. and R.Z. conceived the study and designed all experiments. N.L., S.W., H.Y., X.X.
J.D. and W.W. performed and analysed all experiments. J.K.W., B.L., Z.Y. performed the
molecular dynamics simulations and computational analysis. N.L., J.K.W., W.W., R.Z.
and G.M. wrote the manuscript with support from all authors.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing financial interests: The authors declare no competing financial
interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Luo, N. et al. PEGylated graphene oxide elicits strong
immunological responses despite surface passivation. Nat. Commun. 8, 14537
doi: 10.1038/ncomms14537 (2017).
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14537
10 NATURE COMMUNICATIONS | 8:14537 | DOI: 10.1038/ncomms14537 | www.nature.com/naturecommunications
... Thus, PEG could potentially affect the binding affinity of ligands to their receptors. Furthermore, Luo et al. (Luo et al., 2017) found that PEGylated graphene oxide (GO) nanosheets provoked cytokine secretion in macrophages by enhancing integrin β 8 -related signalling. Indeed, computational modelling suggested that PEGylation, which is thought to "passivate" nanomaterials (Shi et al., 2022), may transform GO into a biologically relevant signalling entity . ...
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The history of brain drug delivery is reviewed beginning with the first demonstration, in 1914, that a drug for syphilis, salvarsan, did not enter the brain, due to the presence of a blood–brain barrier (BBB). Owing to restricted transport across the BBB, FDA-approved drugs for the CNS have been generally limited to lipid-soluble small molecules. Drugs that do not cross the BBB can be re-engineered for transport on endogenous BBB carrier-mediated transport and receptor-mediated transport systems, which were identified during the 1970s–1980s. By the 1990s, a multitude of brain drug delivery technologies emerged, including trans-cranial delivery, CSF delivery, BBB disruption, lipid carriers, prodrugs, stem cells, exosomes, nanoparticles, gene therapy, and biologics. The advantages and limitations of each of these brain drug delivery technologies are critically reviewed.
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Two-dimensional (2D) nanomaterials such as graphene are increasingly used in research and industry for various biomedical applications. Extensive experimental and theoretical studies have revealed that 2D nanomaterials are promising drug delivery vehicles, yet certain materials exhibit toxicity under biological conditions. So far, it is known that 2D nanomaterials possess strong adsorption propensities for biomolecules. To mitigate potential toxicity and retain favorable physical and chemical properties of 2D nanomaterials, it is necessary to explore the underlying mechanisms of interactions between biomolecules and nanomaterials for the subsequent design of biocompatible 2D nanomaterials for nanomedicine. The purpose of this review is to integrate experimental findings with theoretical observations and facilitate the study of 2D nanomaterial interaction with biomolecules at the molecular level. We discuss the current understanding and progress of 2D nanomaterial interaction with proteins, lipid membranes, and DNA based on molecular dynamics (MD) simulation. In this review, we focus on the 2D graphene nanosheet and briefly discuss other 2D nanomaterials. With the ever-growing computing power, we can image nanoscale processes using MD simulation that are otherwise not observable in experiment. We expect that molecular characterization of the complex behavior between 2D nanomaterials and biomolecules will help fulfill the goal of designing effective 2D nanomaterials as drug delivery platforms.
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Most research on the toxicology of nanomaterials has focused on the effects of nanoparticles that enter the body accidentally. There has been much less research on the toxicology of nanoparticles that are used for biomedical applications, such as drug delivery or imaging, in which the nanoparticles are deliberately placed in the body. Moreover, there are no harmonized standards for assessing the toxicity of nanoparticles to the immune system (immunotoxicity). Here we review recent research on immunotoxicity, along with data on a range of nanotechnology-based drugs that are at different stages in the approval process. Research shows that nanoparticles can stimulate and/or suppress the immune responses, and that their compatibility with the immune system is largely determined by their surface chemistry. Modifying these factors can signifi cantly reduce the immunotoxicity of nanoparticles and make them useful platforms for drug delivery. © 2010 Nature Publishing Group, a division of Macmillan Publishers Limited and published by World Scientific Publishing Co. under licence. All Rights Reserved.
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Nanoparticles (NPs) are in use to efficiently deliver drug molecules into diseased cells. The surfaces of NPs are usually grafted with polyethylene glycol (PEG) polymers, during so-called PEGylation, to improve water solubility, avoid aggregation, and prevent opsonization during blood circulation. The interplay between grafting density σp and grafted PEG polymerization degree N makes cellular uptake of PEGylated NPs distinct from that of bare NPs. To understand the role played by grafted PEG polymers, we study the endocytosis of 8 nm sized PEGylated NPs with different σp and N through large scale dissipative particle dynamics (DPD) simulations. The free energy change Fpolymer of grafted PEG polymers, before and after endocytosis, is identified to have an effect which is comparable to, or even larger than, the bending energy of the membrane during endocytosis. Based on self-consistent field theory Fpolymer is found to be dependent on both σp and N. By incorporating Fpolymer, the critical ligand-receptor binding strength for PEGylated NPs to be internalized can be correctly predicted by a simple analytical equation. Without considering Fpolymer, it turns out impossible to predict whether the PEGylated NPs will be delivered into the diseased cells. These simulation results and theoretical analysis not only provide new insights into the endocytosis process of PEGylated NPs, but also shed light on the underlying physical mechanisms, which can be utilized for designing efficient PEGylated NP-based therapeutic carriers with improved cellular targeting and uptake.
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Clarifying the mechanisms of cellular interactions of graphene family nanomaterials is an urgent issue to the development of guidelines for safer biomedical applications and to the evaluation of health and environment impacts. By combining large-scale computer simulations, theoretical analysis, and experimental discussions, here we present a systematic study on the interactions of graphene nanosheets having various oxidization degrees with a model lipid bilayer membrane. In the mesoscopic simulations, we investigate the detailed translocation pathways of these materials across a 56 × 56 nm2 membrane patch which allows us to fully consider the role of membrane perturbation during this process. A phase diagram regarding the transmembrane translocation mechanisms of graphene nanosheets is thereby obtained in the space of oxidization degree and particle size. Then, we propose a theoretical approach to analyze the effects of various initial equilibrium states of graphene nanosheets with membrane on their following cellular uptake process. Finally, we demonstrate that the simulation and theoretical results reproduce some important experimental findings towards the mechanisms of cytotoxicity and antibacterial activity of graphene materials. These results not only provide new insight into the cellular internalization mechanism of graphene-based nanomaterials but also offer fundamental understanding on their physicochemical properties which can be precisely tailored for safer biomedical and environment applications.