Graphene Oxide Touches Blood: In Vivo Interactions of Bio-Coronated 2D Materials

Abstract

Graphene oxide is the hot topic of biomedical and pharmaceutical research of this decade. However, its complex interactions with human blood components complicate a transition of the promising in vitro results to clinical settings. Even if graphene oxide is made with same atoms our organs, tissues, and cells, its bi-dimensional nature causes unique interactions with blood proteins and biological membranes and can lead to severe effects like thrombogenicity and immune cells activation. In this review, we will describe the journey of graphene oxide after injection in the bloodstream, from the initial interactions with plasma proteins to the formation of the “biomolecular corona”, and biodistribution. We will consider the link between the chemical properties of graphene oxide (and its functionalized/reduced derivatives), protein binding and in vivo response. We will also summarize data on biodistribution and toxicity in view of current knowledge of biomolecular corona influence on these processes. Our aim is to shed light on unsolved problems of graphene oxide corona literature to build the groundwork for future drug delivery technology development.

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Cite this: DOI: 10.1039/c8nh00318a
Graphene oxide touches blood: in vivo
interactions of bio-coronated 2D materials
V. Palmieri, *
ab
G. Perini,
a
M. De Spirito
a
and M. Papi
a
Graphene oxide is the hot topic in biomedical and pharmaceutical research of the current decade.
However, its complex interactions with human blood components complicate the transition from the
promising in vitro results to clinical settings. Even though graphene oxide is made with the same atoms
as our organs, tissues and cells, its bi-dimensional nature causes unique interactions with blood proteins
and biological membranes and can lead to severe effects like thrombogenicity and immune cell
activation. In this review, we will describe the journey of graphene oxide after injection into the
bloodstream, from the initial interactions with plasma proteins to the formation of the ‘‘biomolecular
corona’’, and biodistribution. We will consider the link between the chemical properties of graphene
oxide (and its functionalized/reduced derivatives), protein binding and in vivo response.Wewillalso
summarize data on biodistribution and toxicity in view of the current knowledge of the influence of the
biomolecular corona on these processes. Our aim is to shed light on the unsolved problems regarding the
graphene oxide corona to build the groundwork for the future development of drug delivery technology.
1. Introduction
Recent years have seen the explosion of biomedical research
on graphene-based materials (GBM), owing to the captivating
physical and chemical properties of this family of nano-
materials.
1
Thousands of scientific papers have been published
since 2010, the year Geim and Novoselov received the Nobel
Prize for research on graphene; it was pointed out recently that
as the number of works continues to grow, ‘‘the graphene
community could be easily overwhelmed by the collection of
this vast knowledge’’.
2
The nomenclature given to GBM as well
as the various methods of synthesis represent a source of
confusion since in some papers, what authors describe as
graphene/functionalized graphene is another member of this
ultrathin carbon family.
3
This leads to (sometimes apparent)
contradictions in the results ascribed to the same GBM.
In this review, we will shed light on these inconsistencies
and sum up the current knowledge about the in vivo bio-
interface of freely suspended monolayers of graphene oxide
(GO). We focus on GO since the low-cost production and
hydrophilic nature of this material still make it preferable
to other carbon materials.
4
The effects of GO on biological
systems are often compared to its reduced form, reduced
graphene oxide (rGO). Like GO, rGO can be obtained with
several protocols and can be characterized by variable C/O
atom ratios. In some works, GO has been compared to GBMs
that are different from rGO. In this review, we will stick to the
nomenclature used in the original papers, and we will also
describe the size data available to facilitate the readers in making
comparisons.
The in vivo fate of nanomaterials is generally influenced by
several factors, including the route of administration, nano-
material chemistry and physiological environment.
5
Numerous
physicochemical characteristics including lateral size, shape,
dose, exposure time, number of layers, chemical composition,
surface charge, stability, purity, and surface functionality can
influence the fate after injection, when materials are exposed
to the rich milieu of blood proteins.
6
The proteins in the
bloodstream cause an immediate and dramatic change in the
biological ‘‘identity’’ of nanomaterials. The result is the develop-
ment of a new interface, consisting of a dynamic shell of blood
macromolecules. This layer, given the protein enrichment, is
usually referred to as the protein corona or the biomolecular
corona (BC).
7
The BC determines the interactions with cells,
uptake and clearance and therefore affects the biodistribution
and delivery to the intended target sites.
8
The BC of GO is still poorly explored and few works have
considered the influence of this layer on in vitro and in vivo
effects. Here, we will first discuss the surface features of GO
and their links to amino acids and blood protein binding.
Then, we will discuss the GO BC composition and how the
BC can influence interactions with blood cells. Finally, we will
highlight biodistribution and biosafety concerns as well as
a
Fondazione Policlinico A. Gemelli IRCSS-Universita
`Cattolica Sacro Cuore, Largo
Francesco Vito 1, 00168, Roma, Italy. E-mail: valentina.palmieri@unicatt.it
b
Istituto dei Sistemi Complessi, Consiglio Nazionale delle Ricerche (ISC-CNR),
Via dei Taurini 19, 00185 Roma, Italy
Received 21st September 2018,
Accepted 17th October 2018
DOI: 10.1039/c8nh00318a
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future challenges related to the development of intravenously
injected GO-based pharmaceutical systems. The comprehension
of these aspects is involved in andwillimprovethefuturedesign
of injectable biocompatible GO.
2. The GO and rGO chemistry
GO and rGO are often defined as bidimensional materials but
only pristine graphene (the freely suspended single-atom-thick
sheet of hexagonally arranged, sp
2
-bonded carbon atoms
3
) can
be considered a true 2D material.
9
GO is a chemically modified
graphene derived from the oxidation and exfoliation of graphite
and rGO is produced from GO with several possible reduction
protocols in order to obtain the closest material to pristine
graphene.
3
rGO can be obtained by methods like thermal
reduction at high temperature (4900 1C),
4
chemical reduction
by reducing agents (like borohydrides, aluminum hydride,
hydrohalic acid and sulphur-containing reducing agents),
2
hydrothermal, electrochemical and bacteria-mediated reduction.
10
Since residual functional groups and defects remain on the basal
plane, rGO is not analogous to pristine graphene.
GO is primarily composed of carbon, oxygen and hydrogen
atoms with a C/O ratio between B1.5 and 2.5. Several possible
GO structures have been proposed.
2
Based on the widely
accepted Lerf–Klinowski model (see Fig. 1a adapted from
ref. 11), the GO basal-plane is highly populated with hydroxyls
and epoxides while the edges mainly consist of carboxyl and
carbonyl groups. Further, two regions in the GO plane can be
distinguished: one region made up of lightly functionalized
carbons, predominantly sp
2
-hybridized carbon (graphene-like)
atoms, and a second region of highly oxygenated, predomi-
nantly sp
3
-hybridized carbon atoms.
12
Due to the distribution
of functional groups, the edges of GO sheets are hydrophilic,
whereas the basal plane is mostly hydrophobic, and the result
is a giant amphiphilic sheet-like molecule.
12
Interestingly,
thermal annealing procedures at 80 1C for 1–9 days can modify
the distribution of the surface groups. During annealing,
Fig. 1 (a) The Lerf–Klinowski model of the GO structure, adapted
11
with permission from The Royal Society of Chemistry. (b) Analysis of protein residue
content and the correlation with the protein adsorption capacity on GO, rGO and single-walled carbon nanotubes (SWCNT). (b) The positive correlations
between the protein adsorption capacity and protein molecular weight (A), the number of hydrophobic amino acids (B) and the number of Tyr (C), Phe
(D), and Trp (E) residues, adapted with permission from ref. 25, American Chemical Society, Copyright (2015). (c) The binding of albumin, fibrinogen and
globulin depends on the lateral size and concentration of GO, as shown in this figure adapted
6
with permission from The Royal Society of Chemistry.
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clusters of oxygen functionalities are created on the GO surface
without reduction to rGO (B30% of oxygen content).
13
GO abundant surface oxygen groups provide plenty of reac-
tion sites for linking external species like proteins, enzymes,
peptides, bacteria, cells, and nucleic acids.
12,14
GO conversion to rGO produces variations in the physical and
surface properties and consequently alters the stability in solution
and interactions with biological systems.
11,15,16
rGO is indeed
more unstable and hydrophobic and tends to form aggregates in
solution.
17
Given the variety of reduction approaches and, conse-
quently, the possible surface features produced, it is not surprising
that contrasting results have been reported on the biological
systems.
4,18,19
The groups on the GO surface provide unique opportunities for
chemical modification via covalent bonds to obtain functionalized
GO. The functionalization of GO can be divided into two cate-
gories: edge functionalization (of carboxyl groups) and basal plane
functionalization (of hydroxyl and epoxide groups). Reactive
intermediate functionalization of rGO to directly functionalize
the sp
2
-hybridized basal plane is also possible; further details
can be found in other reviews.
11
Often, GO functionalization is
used to build GO-based polymer composites to enhance the
thermal and mechanical stabilityoftheoriginalpolymer.These
composites can be produced by covalent modification of GO
functional groups or via non-covalent interactions, taking advan-
tage of hydrogen bonding and van der Waals forces between the
polymer and GO.
11,20
Also,theproteinadsorptiononGOandrGO
can occur via covalent or non-covalent interactions. Covalent
binding is based on chemical reactions between the side groups
of amino acids and functional groups available on the GO surface.
In blood, non-covalent adsorption occurs through weak van der
Waals forces, hydrophobic, electrostatic, and p–pstacking
interactions.
12,21
The sp
2
hybridized honeycomb carbon lattice of
rGO and GO is hydrophobic and, therefore, interacts with the
hydrophobic regions of proteins, according to the protein
geometry.
9,22
The basal plane of the GO is also enriched with p
electrons, making p–pstacking interactions possible. At the same
time the oxygen groups of GO, whose composition is strictly
dependent on preparation and storing conditions
2
, allow further
hydrogen bonds and electrostatic bonds.
12
These electrostatic
bonds are strongly influenced by the charge on the proteins and
therefore by the pH and the ionic strength of the buffer. Bonding
on GO can also be mediated by van der Waals interactions.
23
However, while the electrostatic interactions are more pronounced
on GO, both van der Waals and electrostatic interactions play a
major role in the adsorption of proteins on rGO due to the increase
in the non-functionalized area on the surface.
24
In the following
sections, we will show how functionalization of the GO surface
alters protein adsorption and consequently BC properties.
3. Blood protein interactions with GO
and rGO
Several methods have been used to study the interactions
between GO/rGO and proteins. Intrinsic protein fluorescence
or light adsorption can be used to monitor the amount of
protein bound to nanomaterials.
25
Alternatively, the bicinchoninic
acid assay can be used to quantify the total unbound protein
quantities.
26
When using these methods, one should bear in mind
that the centrifugation step should be adapted to the size of GO
flakes used, to allow separation from unbound proteins. As an
example, a centrifugation speed of 10 000 rpm can isolate flakes
having an average lateral size of 120 nm, while ultracentrifugation
at 30 000 rpm is necessary for an average flake’s lateral size of
75 nm.
27
Fluorescence quenching spectroscopy is a convenient tech-
nique for investigating protein binding since GO is known to be
a universal quencher of fluorescent dyes and aromatic residues
(tryptophan (Trp), tyrosine (Tyr)), peptides and proteins, due to
strong p–pinteractions with the molecules.
6,28,29
From the
fluorescence quenching data, the quenching efficiency, asso-
ciation and dissociation constants, and binding cooperativity
can be estimated.
6,30
The quenching mechanism of GO has
been recently investigated with a technique based on a tunable
silica spacer to adjust the distance between GO and fluoro-
phores.
31
It was demonstrated that the quenching mechanism
of GO depends on the distance from the fluorescent molecule.
The exchange of electrons occurs at very short donor–acceptor
distances (Dexter transfer), while when the distance is
increased, the main mechanism of quenching is the Fo
¨rster
transfer (FRET), which is efficient even at distances greater
than 10 nm in the case of GO as the acceptor.
31
The quenching
of blood proteins has selective concentration-dependent
effects.
32
Although the fluorescence of blood plasma proteins
depends on three aromatic residues, i.e., tryptophan (Trp),
tyrosine (Tyr), and phenylalanine (Phe), Trp represents the
dominant source of absorption and intrinsic emission. The
Trp fluorescence is strongly influenced and highly sensitive to
its local microenvironment; fluorescence is high when Trp is in
the hydrophobic core of the protein, and weak when it is
exposed to a hydrophilic solvent. Generally, GO quenches Trp
of the three plasma proteins, namely, albumin, globulin, and
fibrinogen. However, it displays a selective amplification of
fibrinogen fluorescence at concentrations below 3 mgmL
1
.
As explained in the study by Kenry and colleagues, the small
sizes of albumin and globulin allow efficient quenching due to
the physical wrapping by the GO nanosheets and energy
transfer between GO and plasma proteins caused by the p–p
stacking and interaction.
32
Conversely, the large fibrinogen size
does not allow GO wrapping at low concentrations. This causes
a slight increase in the intrinsic fluorescence of fibrinogen,
thanks to the GO-induced aggregation of this protein. This
study highlights how, for GO and GBM nanomaterials, a careful
choice of methods and evaluation of concentration-dependent
effects should be made by researchers.
33
Protein binding constants on GO can be obtained by the
surface plasmon resonance technique
25
and the mechanism of
binding can be derived by isothermal titration calorimetry and
by performing quenching experiments at varying pH values
(to study the influence of electrostatic interactions).
30
After
labeling with convenient probes, the protein interaction with
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GO can be analyzed by using fluorescence correlation spectro-
scopy and fluorescence lifetime imaging microscopy.
34
Protein
conformational changes can be analyzed via circular dichroism and
also by the red shifting of the fluorescence peak that occurs with
Trp residues when exposed to a more hydrophilic environment.
30
Experimental data on GO interaction with amino acids and
proteinsaregiveninTable1,wherethephysiochemicalfeatures
of GO or GBM reported in each paper are also summarized. When
GO and graphene, having a thickness of 5–6 layers (hence a
few-layer graphene (FLG) according to literature
3
), were incubated
withaminoacids,bothinteractedstronglywithTrp,Tyr,andPhe.
GO showed significantly higher adsorption for Trp and Tyr,
possibly through hydrogen bonds, compared to FLG, which
exhibited p–pinteractions. Phe showed similar adsorption on
FLG and GO.
35
Also, molecular dynamics (MD) studies indicated
that GO nanoflakes and amino acid complexes are stabilized
by hydrogen bonding interactions, whereas graphene nanoflake
complexes are stabilized by p–pinteractions, leading to enhanced
binding energies for GO nanoflake complexes.
36
As shown in Fig. 1b, Chong et al. demonstrated positive
correlations between the adsorption capacity of GO, rGO and
single-wall carbon nanotubes (SWCNT), and protein molecular
weight, hydrophobic residues and Trp, Tyr, Phe residue
content.
25
The data indicate that surface atoms, as well as buried
residues, are involved in the phenomenon once the protein
partially unfolds and adsorbs on the carbonaceous surface.
25
Basic residues, such as arginine, were observed to play an equally
crucial role in the adsorption of blood proteins.
37,38
Experiments on the interaction between GO and purified
blood proteins have been focused on the most abundant
components in human plasma: albumin, fibrinogen and
g-globulin.
6
As shown in Fig. 1c, GO samples with variable
lateral sizes (LS) have been used to study the influence of the
amount of available surface on protein adsorption and the
behavior of these proteins on GO was largely different.
6
In this
work, albumin, fibrinogen and g-globulin proteins were incu-
bated with ‘small GO sheets’, having LS of B100 nm, obtained
after 240 minutes of sonication, to progressively larger sheets
up to B1000 nm LS of unsonicated ‘large GO sheets’. Albumin,
a small transport protein and the most abundant protein in
human plasma, has maximal adsorption on large GO sheets
and its secondary structure is minimally perturbed following
the adsorption.
6
Nevertheless, the functionality of albumin is
reduced when it is adsorbed to GO.
30
Indeed, epoxy groups on
GO crosslink with the surface of albumin, probably masking
binding sites and impeding bilirubin binding.
30
in contrast,
in GO-COOH, obtained through the oxidization of epoxy and
hydroxyl groups on the GO surface to carboxyl groups by
chemical modification with sodium chloroacetate, the addition
of carboxyl groups to the GO surface masks epoxy groups and
consequently the albumin functionality is preserved.
39
g-Globulin,
the second most abundant plasma protein, remains stable after
interaction with GO but is poorly affected by the size of GO sheets
to which it interacts in the range between B100 nm and
B1000 nm. Only at high concentration is g-globulin better
adsorbed by larger GO sheets (B1000 nm), while at concentra-
tions up to 5 mg mL
1
globulin saturates binding sites and is not
visible in the supernatant.
6
Fibrinogen, a large protein of the
coagulation system, is denatured on GO and is better adsorbed on
smaller GO sheets.
6
Chong and colleagues reported that for both GO and rGO,
the order of adsorption is fibrinogen 4immunoglobulin 4
transferrin (the iron-binding blood plasma glycoproteins that
control the free iron level in biological fluids) 4albumin.
25
Table 1 Amino acids and blood protein adsorption on GO
Material Main conclusions Ref.
Amino
acids
Few-layer graphene (FLG), B5–6 layers, (chemical exfoliation
of graphite)
Strong binding of aromatic residues: GO 4FLG adsorption of
Trp and Tyr, possibly through hydrogen bonds. Phe shows
similar adsorption on FLG and GO
35
GO (Hummers’ method) B6–8 nm thickness, avg. area
B25 mm
2
GO (lateral dimension 0.5–3 mm, thickness 0.8 nm) Positive correlations between adsorption capacity and protein
molecular weight, hydrophobic and Trp, Tyr, Phe residue content
25
rGO (lateral dimension 0.5–3 mm, 1–10 layers) purchased from
Chengdu Organic Chemical Company, Chinese Academy of
Science
Blood
proteins
GO (Hummers’ method) with variable lateral size from B100 nm
(small GO) up to B1000 nm (large GO), thickness 1.3 nm
Albumin is better adsorbed by large GO. g-Globulin is com-
pletely adsorbed up to 5 mg mL
1
, and at higher concentra-
tions is better adsorbed by large GO. Fibrinogen is completely
adsorbed up to 10 mg mL
1
and at higher concentrations is
better adsorbed on small GO
6
GO and rGO see above Adsorption order: fibrinogen 4immunoglobulin 4
transferring 4albumin
25
GO solution from Sigma-Aldrich (B2mm and thickness 1 nm) Adsorption order: complement factor H 4IgG 4albumin 26
rGO obtained with hydrazine monohydrate reduction protocol
(aggregated)
Albumin better adsorbed on GO, probably larger surface area
available
2D graphene nanoplatelets (GNP) purchased from Cheap Tubes,
Inc., Cambridgeport, VT, USA, thickness 10 nm, size of several mm
(from atomic force microscopy (AFM) images)
Albumin better adsorbed on GNP 40
Porous GO (PGO) prepared by the sequential acid and base
treatments of GO synthesized through the Hummers’ method
(Fig. 2e), thickness 2–4 nm, size of several mm (from AFM
images)
Fibrinogen and g-globulin better adsorbed on PGO
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Similar to Chong, in the study of Belling on GO and rGO, the
order of protein adsorption is as follows: complement factor
H4IgG 4albumin.
26
These data are in agreement with the
Kenry study, since albumin appeared in the supernatant even at
very low concentrations (0.5 mg mL
1
) followed by globulin
and fibrinogen, and was visible in the supernatants at 5 and
10 mg mL
1
, respectively (Fig. 1b and ref. 6).
The direct comparison of rGO and GO protein binding
capacity is difficult because (i) rGO is less stable and its exposed
surface varies, and (ii) the rGO synthesis procedure varies
among different studies. Chong utilized GO and rGO with lateral
dimension between 0.5 and 3 mmfromacommercialsource,and
although adsorption onto rGO was somewhat diminished when
juxtaposed with GO (Fig. 2a and b), especially for fibrinogen, the
authors concluded that significant differences between these two
materials were not visible.
25
Belling, who synthesized rGO by
chemical reduction, indicated a higher albumin loading of GO
caused by the less available binding surface of partially aggregated
rGO.
26
This effect was less visible for IgG and complement factor
H;howeverGOseemedtobetteradsorbproteiningeneral
(Fig. 2d). It should be noted that in both studies, the rGO was
unstable and tended to form multilayers or aggregate. Chong
considered this effect and normalized the milligrams of protein
adsorbed to the available surface, and surprisingly rGO was more
efficient in protein loading (Fig. 2c), which is consistent with the
idea that p–pand hydrophobic interactions are the primary
driving forces during protein binding (Chong, Ge et al., 2015).
25
To analyze the influence of oxygen functionalities using materials
with similar solubility, Kenry and his group selected carboxylic-
functionalized multi-wall nanotubes CNT (CNT-COOH), 2D
graphene nanoplatelets (GNPs) and porous graphene oxide (PGO)
(Fig. 2e). Results from the work are reported in Fig. 2f (albumin
adsorption), Fig. 2g (globulin adsorption) and Fig. 2h (fibrinogen
adsorption). The highest loading capacity for albumin was dis-
played by GNP, followed by CNT-COOH and PGO, suggesting that
the associations might be strongly dependent on the hydrophobic
interactions via p–pstacking between the aromatic rings of hydro-
phobic amino acids and the carbon nanomaterial surface. A reverse
trend was observed for globulin and fibrinogen, where PGO
displayed the highest capacity loading for both proteins, while
those of CNT-COOH and GNP were similar. The lower adsorption
of fibrinogen on CNT-COOH and GNP confirms Chong data and
might be due to the larger size of fibrinogen.
25
As such, fibrinogen
adsorption might require a higher surface area. However, globulin
is also poorly adsorbed on CNT-COOH and GNP, so the authors
ascribed the difference in adsorption to the amount of oxygen
functional groups on the surface.
40
In summary, the adsorption capacity of GO is influenced by
the aromatic residue content, protein size and hydrophobicity.
Fibrinogen is better adsorbed than albumin by GO, while GNP,
which is less oxygenated, better adsorbs albumin. The hydrophobic
interactions between protein amino acids and poorly oxygenated
GBM are less-favoured due to the instability of these nanomaterials.
Further systematic studies are needed to clarify the influence of
surface groups and nanomaterial dispersibility on protein adsorption.
4. The composition of the GO
biomolecular corona
In the journey of GO after injection in our body, the first
process that occurs is undoubtedly the binding of plasma
Fig. 2 Milligrams of proteins (bovine serum albumin (BSA), transferrin (Tf), immunoglobulin (Ig) and bovine fibrinogen (BFG)) adsorbed on GO (a) and
rGO (b); adapted with permission from the American Chemical Society, Copyright (2015).
25
Data for adsorption corrected for available surface indicate a
higher capacity of rGO in respect to other materials (c). Comparison between GO and rGO loading of immunoglobulin g (IgG), BSA, human serum
albumin (HSA) and factor H; reproduced with permission from ref. 26. (d) Protein adsorbed on three materials, namely carboxylic-functionalized
multi-walled CNT (CNT-COOH), 2D graphene nanoplatelets (GNPs) and porous graphene oxide (PGO) illustrated in (e), are reported in (f) albumin,
(g) globulin and (h) fibrinogen; adapted with permission from ref. 40.
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proteins on its surface (see Fig. 3-1). Intravenously injected
nanoparticles encounter multiple lines of defense intended to
neutralize the invaders. The first and most critical defense
line is the blood protein adsorption and formation of the
biomolecular corona (BC).
41
Of the thousands of proteins
present in our body, 10 to 50 may take part in a nanomaterial
BC.
42
The subset of blood proteins that have been identified in
at least one nanomaterial BC has been named ‘‘adsorbome’’.
43
Adsorbome is made of a total of 125 proteins divided into
highly and poorly abundant components by a threshold of 10%
of the total protein mass.
43
The BC appears to follow a general
structure: up to six proteins are adsorbed at high abundance,
and many more are adsorbed in low quantity. BC is species-
specific and there are only a few proteins in common among
human and mouse BC.
42
Studies on the BC composition of GO and GBM are limited
(Table 2). We will first consider the major issue in the charac-
terization of the BC of these nanomaterials: their instability
in solution, which differs from ultrapure water. rGO, pristine
graphene and, to a lesser extent, GO are prone to aggregation in
saline solution, phosphate buffered saline (PBS) and of course
plasma and serum.
17,33,44
To overcome this problem, the BC of
pristine graphene (G-BC) has been analyzed on solid substrates
in polystyrene wells or after direct exfoliation of graphite in
human serum. On graphene substrates (G-substrates), obtained
by chemical vapour deposition, the authors tested human
plasma concentrations from 2% up to 70%
45
and found that
plasma concentration influenced BC composition. Albumin
was the only attached protein on G-substrates at plasma
concentrations below 5%. With increasing concentrations of
plasma, first prothrombin and alpha-fetoprotein were added to
the BC, and then vitamin D binding protein and fibrinogen
beta chain enriched the BC. At the highest plasma concen-
tration the proteins adsorbed were inter-alpha-trypsin inhibitor
heavy chain, thrombospondin-1, complement component C7,
prothrombin, serum albumin, alpha-fetoprotein, fibrinogen
alpha chain, C4b-binding protein alpha chain, kininogen-1,
vitamin D-binding protein, fibrinogen beta chain, and cyto-
Fig. 3 Main results of GO interaction with blood components are sum-
marized in this illustration of the injection of GO flakes in the bloodstream.
The formation of the BC (1) prevents the hemolysis of red blood cells (2a).
Thrombosis (2b) and interaction with complement proteins (2c) are
ascribed to GO. In (2d) some of the possible fates after macrophage
encounters are shown: extracellular blocking or intracellular uptake. The
release of cytokines occurs when macrophages uptake GO. Aggregates of
GO in macrophage cytoplasm induce the production of pro-inflammatory
cytokines. Dendritic cells fail to present antigens to lymphocytes when
they uptake GO (2e). Lymphocyte activity is not inhibited, and BC protects
lymphocytes from apoptosis (2f).
Table 2 Pristine graphene and GO BC in human plasma/serum
Material Main components of the biomolecular corona Ref.
Pristine
graphene
Pristine graphene substrates on polystyrene obtained by
chemical vapor deposition
Inter-alpha-trypsin inhibitor heavy chain, thrombospondin-1,
complement component C7, prothrombin, serum albumin,
alpha-fetoprotein, fibrinogen alpha chain, C4b-binding protein
alpha chain, kininogen-1, vitamin D-binding protein, fibrinogen
beta chain, and cytochrome (70% plasma concentration)
45
Graphene (direct exfoliation of graphite in serum), lateral
size 200–300 nm, thickness 25 nm (protein layer attached)
Albumin, lipoproteins (mainly apolipoprotein A-1 and
apolipoprotein E), and vitronectin
46
Graphene
oxide
GO purchased from Graphene Supermarket (USA), flake
size 0.5–5 mm, thickness 1.1 nm
Transport, immune response protein, complement factors,
apolipoprotein E, inter-alpha-trypsin inhibitor heavy chain H1,
H2 and hyaluronan-binding protein 2 (in serum)
49
GO purchased from Nanjing XFNANO Materials Tech.
Co., Ltd, Nanjing, China lateral size 0.5–5 mm and
thickness of 0.7 nm
95% consists of organic molecules, ornithine, octadecenoic acid,
dodecanol, talose, and tetradecanoic acid show selective accu-
mulation (14 days in plasma)
54
GO (lateral size few mm) and nGO-PEG (10–50 nm) nGO-PEG has reduced protein binding and selectivity toward six
proteins: four immune-related factors (C3a/C3a (des-Arg),
clusterin, histidine-rich glycoprotein, vitronectin) in serum
55
GO, GO-NH
2
, GO-PAM, GO-PAA and GO-PEG Protein adsorption GO 4GO-PAM 4GO-NH
2
4GO-PAA 4
GO-PEG
56
Size of 100–500 nm, thickness depends on functionalization
(PAM, PAA or PEG became higher than GO with 2.9, 2.6 and
4.1 nm thickness, vs. 1.1 nm, respectively)
IgG highly adsorbed GO4GO-NH
2
4GO-PAM4GO-PAA4
GO-PEG
Influenced by surface modification include thrombospondin 1,
gelsolin and hemoglobin (less abundant in GO) in serum
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chrome.
45
Interestingly,mostoftheseproteinsarenotpartofthe
‘‘highabundance’’adsorbomedefinedbyWalkeyandChan.
43
Direct exfoliation of graphite in human serum is a recently
established ultra-sonication protocol to analyze the BC of
pristine graphene.
46
This method was developed by Castagnola
and colleagues to avoid the usage of dispersants that form an
adsorbed layer on the graphene surface and affect BC composition.
The method consists of 1 to 4 hours of ultrasonication of 10 w/v%
of natural flake graphite dispersed in a solution of serum at
different concentrations using a bath sonicator.
In this paper, the authors found that G-BC is composed of
albumin, lipoproteins (mainly apolipoprotein A-1 (ApoA-1) and
apolipoprotein E (ApoE)), and vitronectin.
46
The serum concen-
tration seems to slightly affect the G-BC composition in this
work. The available epitopes exposed include abundant ApoA-I,
which is known to be involved in early biological interactions,
both at the cell and organ level.
46,47
Despite the different
available surfaces and other physiochemical properties that
are known to vary between graphene in solid or soluble
form, the markedly different compositions of the corona might
arise from the protein source used in the experiments,
i.e. plasma and serum.
45,46
Indeed, while incubation of nano-
materials in serum is known to create a BC mainly formed
by apolipoproteins, in plasma, coagulation and complement
factors may also participate.
48
In a direct comparison of serum BC of GO and other carbon-
based nanomaterials, i.e., carbon black (CB) and multi-walled
carbon nanotubes (MWCNT), GO has the lowest affinity for
albumin because of its lower hydrophobicity, and a higher
adsorption capacity for low-abundant proteins of serum.
49
These data agree with the single protein studies of Kenry,
discussed in the previous section.
6,40
In another work, BC
formed in foetal bovine serum (FBS) shows that the adsorption
of protein is reduced when surface groups are reduced (rGO),
and that high material concentration also induces lower
adsorption efficacy.
50
With respect to CB or MWCNT, greater
amounts of proteins, especially complement components and
blood coagulation proteins, are adsorbed on GO corona. The
authors explained this higher protein concentration with the
low surface curvature of GO, combined with negatively charged
functional groups and high surface area availability. These
features result in several possible interactions, i.e., hydrogen
bonding and electrostatic interactions, in addition to hydrophobic
and van der Waals interactions of other nanomaterials.
49,51
The proteins identified in GO-BC are mainly involved in transport
and immune response.
49
All investigated nanomaterials possessed
a BC enriched with complement factors and apolipoproteins
(involved in targeting and translocation through the blood–brain
barrier) but apolipoprotein-E is selectively adsorbed by GO.
A particularly high number of complement factors was found in
GO-BC, together with proteins involved in hemostasis and tissue
structuring (hyaluronan-binding proteins, namely inter-alpha-
trypsin inhibitor heavy chain H1, H2 and hyaluronan-binding
protein 2).
49
Qualitative protein analysis shows that BC of GO is
influenced by the health conditions of blood donors, as demon-
strated for other nanoparticles.
52,53
Indeed, GO sheets incubated
with plasma from patients with different diseases/conditions,
including hypofibrinogenemia, blood cancer, thalassemia major,
thalassemia minor, rheumatism, fauvism, hypercholesterolemia,
diabetes, and pregnancy, and analysisbygelelectrophoresis
showed significant variations in the compositions of the BC. These
differences can be attributed to alterations in the plasma protein
compositions/content, the protein conformation, and/or the pro-
tein solubility.
52
When GO is submerged in human plasma for the long-term
(14 days) a biotransformation and reduction to rGO occurs,
mediated by free radicals.
54
During this period, the BC formed
is principally made by small organic molecules that account
for 95% of the corona composition. Some molecules, such
as ornithine, octadecenoic acid, dodecanol, talose, and tetra-
decanoic acid show a selective accumulation on GO.
54
Surface modification is a commonly adopted approach
to improve the biocompatibility of GO and to control the
formation of the BC.
Tan and colleagues compared the interactions of serum
proteins with GO and nGO-PEG, a nanometric GO functionalized
by a 10 kDa amine-terminated six-arm-branched polyethylene
glycol (PEG) via amide formation.
55
Unlike GO, which adsorbs
a significant amount of serum proteins without specificity,
nGO-PEG exhibits reduced protein binding and selectivity
toward six proteins: four immune-related factors (C3a/C3a
(des-Arg), clusterin, histidine-rich glycoprotein, vitronectin)
and two coagulation factors (contained platelet factor 4 and
thrombin). However, the association of thrombin and platelet
factor 4 might be a pseudo effect, since their circulation levels
are extremely low, but increase by more than 3 orders of
magnitude during the clotting protocol in serum preparation.
55
Further, given the size difference between nGO-PEG (nanometric
flakes) and GO (micrometric flakes), the potential size effect on the
above interactions should be investigated.
Xu and colleagues measured the effect of GO surface modi-
fication on the composition of GO-BC in mouse serum (Fig. 4).
GO was chemically modified to obtain aminated GO (GO-NH
2
),
prepared with GO dispersion in ammonia with hydrazine
hydrate as a reducing agent, GO-polyacrylamide (GO-PAM),
GO-polyacrylic acid (GO-PAA) and GO-PEG.
GO (100%) and GO-PAM (90.2%) showed higher protein
adsorption with respect to GO-NH
2
(54.3%), GO-PAA (39.3%)
and GO-PEG (43.9%), presumably due to their different surface
charges and hydrophobicities. The relative amount of IgG in the
BC was much greater than other proteins, and a big difference was
observed among GO (62.2%), GO-NH
2
(58.3%), GO-PAM (52.3%),
GO-PAA (36.8%) and GO-PEG (30.2%).
56
Other proteins influenced
by surface modification include thrombospondin 1, gelsolin and
haemoglobin (less abundant in GO).
56
Another functionalization strategy to modify GO-BC foresees
the spontaneous self-assembly of chitosan (CS) and GO via
electrostatic interactions to form CS-GO, which has a reduced
BSA and lysozyme adsorption capacity, and, in FBS, a reduction
of serum proteins uptake.
57
In summary, the composition of G-BC is affected both by the
nanomaterial state and the protein source. We can speculate that
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the work of Castagnola and colleagues
46
defines, to some extent,
the configuration of the BC of injected pristine graphene and that
the enrichment in apolipoproteins might be useful for targeted
delivery applications (as discussed in the conclusions). However,
unfunctionalized pristine graphene instability remains the
Achilles’ heel of this nanomaterial. As we will see in Section 6,
when GO is injected in blood, aggregates form, but in a size and
dose-dependent fashion.
58
ThismeansthatGOismorestableand
is suitable for an injected delivery system and consequently, its BC
should be precisely controlled. GO adsorbs a large amount of
proteins thanks to the highly available surface,
49
and this is
generally looked at as a disadvantageous feature in vivo,since
themoreproteins‘‘mark’’theforeign nanomaterial, the better it is
attacked by our immune system.
59
Many functionalization strate-
gies are therefore exploited to improve GO stealth properties.
However, this protein enrichment gives GO some advantages over
other nanomaterials in diagnostics and pharmaceutical applica-
tions. Indeed, the higher protein adsorption in the BC can be
exploited to select and enrich poorly concentrated biomarkers in
patients’ blood
52
and develop diagnostic tools based on the BC.
7
Secondly, the list of proteins found in GO-BC includes ApoE,
vitronectin and clusterin, which are important blood–brain barrier
(BBB)-directing molecules (ApoE) as well as targets of therapies
(Table 2).
60–62
rGO, whose BC, to the best of our knowledge, still
has to be fully characterized, can enter the brain thanks to a
transitory decrease in the BBB paracellular tightness.
63
Future
studies could be focused on delivery applications based on
GO/rGO selectively adsorbed proteins.
5. Effects of bio-coronated GO
materials on blood components
BC composition directly influences interactions with other
blood components (Fig. 3-2). For example, the presence of
antibodies, complement and clotting factors in the nano-
particle BC may activate clotting and coagulation cascades.
Further, the BC coating can promote phagocytosis and elimi-
nation from the circulation.
41
We will first consider data on the GO interaction with the red
blood cells (RBCs), given in Table 3. An intravenously injected
nanomaterial is likely to interact first with RBCs rather than other
cells, due to their abundance in blood. Hemolysis represents the
damage to RBCs that leads to the leakage of hemoglobin into
the bloodstream. After hemolysis, the nanomaterial may adsorb
released hemoglobin and/or adhere to cell debris, which can
increase its likelihood of elimination by macrophages.
8
Although
the literature is contradictory regarding GO effects on RBC, when
BC is introduced into the framework the results become clearer.
Due to the sharp edges of GO and rGO, hemolytic effects might
be expected in vivo, possibly caused by nanomaterial blades
disrupting cell membranes, as reported for GO interactions with
bacteria.
19
Feng and colleagues discovered RBC morphological altera-
tions and aggregation above 100 mgmL
1
and hemolytic effects
above 10 mgmL
1
reaching 96% at 500 mgmL
1
.
64
Lower hemo-
lytic concentrations have been reported by other groups.
65
Small GO flakes (few hundreds of nm) seem to be more
destructive.
66–68
The aggregation and hemolysis might be driven
by the hydrophobic interaction between the GO surface and
the lipid bilayer of RBCs, or other nonspecific interactions such
as hydrogen bonding between the glycocalyx and hydroxyls and
carboxyls of GO could also take place.
64
Alternatively, binding
between charged groups on the GO surface and positively charged
phosphatidylcholine of the RBC membrane could explain this inter-
action, but aminated rGO (G-NH
2
) does not induce hemolysis.
65,66
Even the stability in solution is known to play a role in the GO
interaction with living systems.
33
rGO and pristine graphene, with
lower oxygen content, also have lower hemolytic activity but are also
more prone to aggregate, yielding fewer cell contacts.
66,68
Fig. 4 Protein corona analysis of GO, GO-NH2, GO-PAM, GO-PAA and GO-PEG formed in mouse serum at 37 1C for 1 h. (a) SDS-PAGE analysis of
protein corona. (b) Normalized OD values of each protein corona, indicating the amount of protein adsorbed on GO. Asterisks (*) denote po0.05
compared to pristine GO. (c) Eleven highly abundant components identified by mass spectrometry. Reproduced with permission from ref. 56.
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As explained above, in vivo injection implies the formation
of the BC around GO and rGO (Fig. 3-1) and regardless of the
BC composition, several groups have demonstrated the physical
hindrance of BC between GO and eukaryotic cells (Fig. 3-2a).
The presence of a BC around GO flakes can completely inhibit
the interaction and therefore RBC hemolysis.
67
GO hemolysis is
also prevented by chitosan, dextran and curcumin coating or by
functionalization of the GO surface.
66,69,70
An interesting study
proposed the use of mussel-inspired dopamine (DA) for both the
GO reduction and functionalization.
71
DAhasmanyproperties:
(i) it adheres to solid surfaces in water solution without surface
pretreatment; (ii) once on the surface, DA can anchor a secondary
functional biopolymer via the thiol, imino and amine groups;
(iii) the catechol groups of DA can convert GO into chemically
reduced rGO. Cheng and colleagues exploited these features to
obtain heparin-grafted polyDA-rGO (Hep-gpRGO) and BSA-grafted
polyDA-rGO (BSA-g-pRGO) that greatly suppressed hemolysis ratios
(lowerthan1.8%,evenwithahighconcentrationof200mgmL
1
).
These contrasting results are not unique in the literature on
GO and cells, but they are explainable by the experimental
conditions. In most of the literature, the cytotoxicity of GO and
rGO against many cell lines has been demonstrated to be
caused by cellular membrane penetration and/or oxidative
stress induction.
6,72
However as for RBC, the physical damage
to the cell membrane is largely attenuated when GO is incu-
bated with BSA or FBS, due to the extremely high protein
adsorption ability of GO.
14,25,39,73,74
In cell culture medium
supplemented with FBS, GO is enriched with a BC of albumin
and IgG, irrespective of the lateral size GO.
75
In summary, the
presence of proteins in the cell culture medium influences the
results on cytotoxicity and we could consider that GO and rGO
are not hemolytic in vivo where abundant protective BC form on
their surfaces (Fig. 3-2a).
Hemostasis cascade prevents blood loss from injured tissue
and maintains blood fluidity. The final hemostasis is driven by
platelets, which form the clot, a mixture of red blood cells,
aggregated platelets, fibrin and other cellular elements (Fig. 3-2b).
If the clot forms abnormally, it can induce thrombosis.
Thrombogenicity is an important feature evaluated in nano-
material design for in vivo delivery and represents the propen-
sity to induce blood clotting and induce occlusion of a blood
vessel by a thrombus.
8
Nanoparticle thrombogenic properties are largely determined
by physicochemical properties andbyinteractionandmodulation
of the activity of various components of the coagulation system
such as platelets and plasma coagulation factors.
76
Furthermore,
nanoparticles engineered to have longer systemic circulation
times increase the likelihood of contact with blood components
including the coagulation system, with thrombogenicity risks.
8
In vitro studies on pristine graphene and GO provided
evidence that concentrations up to 75 mgmL
1
, do not interfere
with platelet function or the pathways of plasma coagulation
68
and that GO, up to 50 mgmL
1
, does not interfere with platelet
aggregation or fibrinogen polymerization.
64
GO completely blocks
clotting factors only at high concentrations (i.e. B500 mgmL
1
).
64
Contrasting results have been reported by Singh and
colleagues that described few-layer GO sheet (size between
0.2 and 5 mm) induction of platelet activation and thrombo-
embolism in vivo. At very low concentrations (2 mgmL
1
), GO
caused in vitro platelet aggregation through the intracellular
release of calcium from cytosolic stores, activation of
nonreceptor protein tyrosine kinases of the Src family and
enhancement of platelet integrin–fibrinogen interactions.
When administered in vivo (250 mgkg
1
body weight), 48%
of lung vessels were partially occluded after 15 minutes.
77
Interestingly, the thromboembolisms can be avoided if rGO
or GO are covered by a heparin corona.
22,29
This in vivo impact
on the coagulation cascade can be caused by an aggregation
of the nanomaterial after injection.
78
However, rGO is less
stable than GO and this hypothesis should be discarded since
thromboembolism of rGO is lower, with only 8% of vessels
occluded.
77
Another explanation could be that the adsorption
of coagulation factors onto the GO corona might cause their
contact activation.
76
Changing GO surface groups and creating
positively charged amine-modified rGO (N-GH
2
) at the same
concentration (250 mgkg
1
body weight) the thrombogenicity is
suppressed might influence both the interaction with blood
cells and the composition of BC.
56,65
The group of proteins of the complement system (Fig. 3-2c)
that promotes antigen phagocytosis and recruitment of neutro-
phils and macrophages to the site of inflammation is highly
abundant in the GO corona.
49
The in vitro GO complement-
activation test provided evidence that this material signifi-
cantly triggers complement activation by the increase in the
Table 3 Haemolytic effects of GO
Material Hemolysis BC effects Ref.
GO (Chengdu Organic Chemistry Co., Chinese Academy of
Science) average hydrodynamic diameter B500 nm
Starts from 10 mgmL
1
and reaches 96%
at 500 mgmL
1
—64
GO (graphite oxidation) and G-NH
2
(thermal annealing and
amination) few layers and 2 mm size
Starts from 2 mgmL
1
and reaches 60%
at 10 mgmL
1
—65
G-NH
2
not hemolytic
GO (Hummers’ methods) with size between 300 and 800 nm
and rGO from dehydration of GO (size of several mm)
GO = between 60% and 80% (psheet
lateral size) at 200 mgmL
1
—66
Size data refer to the hydrodynamic diameter in water rGO = 15% at 200 mgmL
1
Pristine graphene and GO thickness of 0.8 nm Not hemolytic (o10% at 75 mgmL
1
)— 68
GO (Graphene A, Cambridge, USA) hydrodynamic radius
from 660 nm to 100 nm (sonication) thickness of 0.8 nm
Between 60% and 80% (psheet
lateral size) at 200 mgmL
1
Inhibition of hemolysis if GO is
surrounded by a protein corona
67
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concentration of fragments C3a (proportional to GO concen-
tration) and C5a.
Authors have claimed that this activation might be driven
by both hydroxyl groups and the hydrophobic surface of the
GO skeleton.
64
A detailed study of the relation between GO
surface oxidation and complement activation confirmed this
hypothesis.
79
In this work, GO was synthesized through the
modified Hummers’ method and was mildly reduced to obtain
three nanomaterials having different oxygen content (i.e. 36%,
29%, 24% atomic percentage) and size of a few mm (from AFM
characterization). It was found that the decrease in oxygen
content reduced complement activation, as reflected in the
lower levels of both C5a and SC5b-9. This could be explained
by the instability of the flakes and the diminished exposure of
the GO surface in less oxidized GO, which is more prone to
forming irreversible flocculates in solution. On the other hand,
this phenomenon may be attributed to a combined effect of
oxygen-type functionality and topological change arising from
a wrinkling of the GO surface that may modulate the affinity
for recognized molecules or interaction with complement
regulators.
79
Since C5a can substantially potentiate IL-6 pro-
duction in lipopolysaccharide (LPS)-stimulated peripheral
human blood leukocytes, the effect of GO on IL-6 release in
human whole blood was tested in the same study. At concen-
trations below the GO-mediated complement activation, the
LPS-induced responses were inhibited by GO, probably through
a direct interaction of GO with LPS or GO and LPS binding
protein. This effect disappeared when the GO concentration
was above the complement activation threshold and the IL-6
cytokine release was induced.
79
Pre-coating of GO or rGO with a
corona of albumin or complement H factor, obtained after
incubation of the nanomaterials at 37 1C for 2 hours with these
proteins, can reduce complement activation by 40% and 90%,
respectively.
26
This interesting effect is mediated by both
the steric blocking of the interaction with complement compo-
nents and, for the complement H factor corona, by regulation
of the complement cascade.
26
In summary, GO activates the
complement system but the reduction of oxygen content and
the precoating with BC might prevent this effect.
Complement activation may result in altered biodistribution
caused by rapid clearance from the bloodstream via phago-
cytosis by mononuclear cells. In addition, complement
activation can support cell-mediated immunity through the
enhancement of B-cell responses and promotion of dendritic
cells (DC) and T-cell activation.
8
Understanding the interaction
of GO with immune cells is crucial for the development of
biomedical technologies and some recent interesting reviews
have focused on the mechanisms occurring when GBM interact
with immune systems.
18,72,80,81
The results on immune activa-
tion are contrasting but we can certainly assume that there is a
strict connection between GO size, oxidation and functionaliza-
tion and effects on immune cells.
18
For example, human
peripheral blood mononuclear cells (PBMC), which include
lymphocytes, monocytes, and dendritic cells, did not show
significant stimulation (proliferation)/immunosuppression
(cytotoxicity) below 75 mgmL
1
after incubation by either
pristine graphene or GO.
68
However, proinflammatory cyto-
kine expression quantification showed that PBMCs treated
with pristine graphene expressed relatively higher levels of
IL-8 and IL-6 compared to GO samples, thus indicating the
inflammatory potential of the former.
68
A recent study demonstrated that GO induced proinflam-
matory cytokine expression in a size-dependent fashion, with
smaller sized GO (o1mm) being more effective than larger ones
(1–10 mm).
18,82
Macrophages (Fig. 3-2d), the professional phagocytes of
nanomaterials, seem to better uptake GO compared to pristine
graphene, since the latter remains blocked on the cell surface,
probably because it is poorly dispersed in water.
68,72
It was
reported that at 50 mgmL
1
, pristine graphene induces macro-
phage apoptosis, while below this concentration it triggers
cytokine release and impairs macrophage function.
68,83,84
Ingested GO sheets form aggregates in macrophage cytoplasm
80,85
and induce the production of proinflammatory cytokines.
72,75,80
Macrophage phagocytosis depends on GO lateral size; large sheets
can align with the membranes of cells and develop a ‘‘masking’’
effect isolating cells from the environment.
86
This effect is
similar to the ‘‘wrapping’’ effect on bacterial cells that causes
their isolation and consequent growth inhibition.
19
Large
GO sheets (750–1330 nm), in comparison with their smaller
counterparts (50–350 nm), can induce M1 macrophage polari-
zation, NF-kB activation and the production of proinflammatory
cytokines.
87
A direct comparison of the effect of materials with similar
dispersibility but different oxidation states (GO and rGO nano-
platelets with size o100 nm) was conducted for monocytes and
macrophage precursors (Fig. 5). GO nanoparticles have been
obtained with the modified Hummers’ method and sonication,
while rGO was obtained via UV photoreduction of the former.
88
rGO is better ingested in respect to GO but induces differential
expression patterns of antioxidative enzymes. The effects of
exposed THP-1 cells could also pass to THP-1a as shown in
Fig. 5, reproduced from ref. 88 GO nanoparticles (GONPs)
demonstrated a stronger inhibition of THP-1a phagocytosis
towards E. coli as compared to rGO nanoparticles (rGONPs)
and both GONPs and rGONPs impaired the phagocytosis and
endocytosis abilities of THP-1a.
Concerning the BC effect, phagocytosis by macrophages
seems to be mediated by the Fcgreceptor that recognizes GO
opsonized by IgG.
75
Surface modification of GO with PEG
reduces IgG enrichment in the GO corona, and consequently
inhibits macrophage phagocytosis and improves biocompatibility.
However, it should also be kept in mind that other proteins (such
as complement C4, serotransferrin or unknown proteins) might
also cause specific ligand–receptor interactions and affect the
circulation time of functionalized GO.
56
Other important phagocytic cells are dendritic cells (DC)
that activate antigen-specific T cells, after (i) antigen capture,
(ii) intracellular processing and (iii) the presentation of the
antigen within the MHC complex on the cell surface.
The maturation of DC is induced significantly by GO in a
dose-dependent fashion, as reflected by the up-regulation of
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surface receptor phenotypes.
85,89
However, the GO-treated DC
failed to correctly present antigens to lymphocytes (Fig. 3-2e).
The uptake of antigens is not altered but GO interacts directly
with the subunit LMP7 of the immunoproteasome responsible
for antigen processing.
89
Below 50 mgmL
1
, T lymphocyte vitality is not affected
by GO or GO-COOH, a GO enriched with carboxyl.
39
Above
this concentration, vitality is reduced. Apoptosis seems to be
induced by an external mechanism, without internalization, via
ROS-dependent/independent signaling for GO-COOH and GO
respectively. Despite apoptosis induction, both nanomaterials
do not interfere with lymphocyte immune response ability.
It should be noted that when cells are treated with GO or
GO-COOH and the cell medium does not contain FBS, the
cytotoxicity is dramatically higher, indicating the formation
of a protecting BC around the nanomaterials.
39
T lymphocyte
apoptosis after treatment with GO was reported to be higher
(B70% at 100 mgmL
1
) by Zhi and colleagues.
85
A series of safety guidelines for the development of
graphene-based materials for in vivo applications like the use
of small, well-dispersed sheets that macrophages can efficiently
phagocytize, and the use of degradable GBM have been
proposed.
90
On the other hand, GO immunostimulatory activity
and high surface area for antigen adsorption have encouraged
studies on GO usage as an adjuvant in vaccine therapy.
91,92
Meng and colleagues explored the potential of ovalbumin and
carnosine functionalized GO to promote specific antibody
response and increase lymphocyte proliferation efficiency and
T-cell activation.
91
GO functionalized with both PEG and poly-
ethyleneimine (PEI), GO-PEG-PEI, can represent an innovative
method for the delivery of urease B antigen, specific for
Helicobacter pylori to dendritic cells. GO-PEG-PEI significantly
enhances the maturation of DCs that release interleukin 12
(IL-12) through the activation of multiple Toll-like receptors
(TLRs).
92
6. Biodistribution and biosafety of GO:
future challenges
The focus of this review is the GO interaction with blood
components and BC in light of the future design of GO
pharmaceutical delivery systems. Intravenously injected drug
delivery systems (DDS) developed so far include PEGylated
nanographene sheets for tumor passive targeting,
93
rGO func-
tionalized with chitosan and iron oxide magnetic nanoparticles
for the delivery of doxorubicin
94
and epidermal growth factor
receptor antibody-conjugated PEGylated nanographene oxide
for epirubicin delivery in tumors
55
(for a comprehensive
outlook of DDS based on graphene see ref. 95). Nanoparticles
intended for drug delivery applications are being engineered
to reduce their clearance and extend systemic circulation
times and thus increase the opportunity for targeted delivery.
However, the disadvantage of prolonged circulation times is
the greater chance of interaction with blood components and
activation of adverse effects.
Before any nanomaterial translation into clinical therapy,
there are biosafety concerns that need to be addressed. We have
seen how GO interacts with blood system components and
how BC can influence these interactions, but what is the
biodistribution and the toxicity when GO is administered
intravenously (i.v.)?
Studies have reported inconsistencies in GO effects in vivo,
as summarized in recent reviews.
90,96–98
The early study of Zhang and colleagues determined the
distribution and biocompatibility of i.v. injected GO in mice.
99
The half-life of GO in blood is much longer than in other
carbon nanomaterials (B5 hours). Within 48 hours after i.v.
injection, GO is cleared from the bloodstream and distributed
throughout various organs with preferred accumulation in the
lungs, liver, and spleen. The lack of pathological changes was
reported after 14 days of treatment at a low dose (1 mg kg
1
),
Fig. 5 Illustration of the short-term effects of GONPs and rGONPs on THP-1 cells, and the long-term effects on THP-1a differentiation from THP-1
cells. GONPs and rGONPs could have induced ROS formation and activated the NF-kB pathway in THP-1 cells. rGONPs could not fully transcript
proinflammatory genes due to lack of additional transcription factors. Reproduced with permission from ref. 72.
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but at a higher dose (10 mg kg
1
), granulomatous lesions,
pulmonary edema, inflammatory cell infiltration, and fibrosis
throughout the lung were observed.
99
Many studies confirmed
that the primary site of GO accumulation and toxicity in vivo is
the lungs.
97
It seems that the pathological effects on the lungs
are proportional to the degree of dispersion and oxidation of
GO. When directly injected into the lungs, GO induces severe
long-term (21 days) lung injury, while graphene flakes, either
in a dispersed or aggregated state, do not increase apoptosis
in lung macrophages.
100
The biodistribution of GO is size-
dependent. In the report by Zhang, the size of GO ranged from
10 to 800 nm and that this caused a distinctive clearance
behavior: particles with small size were quickly eliminated
through the renal route within 12 h post injection, while large
particles were intercepted by the lungs.
99
A systematic study on
GO size, dose and dosing frequency was conducted by Liu and
colleagues.
58
Liu intravenously administered two types of GO:
small GO flakes (s-GO, average hydrodynamic diameter of
B250 nm) and large GO flakes (l-GO, average hydrodynamic
diameter of B900 nm) at a single high dose (2.1 mg kg
1
)or
seven repeated low doses (0.3 mg kg
1
); irrespective of size, the
single high-dose administration of GO induced lung damage
and infiltration of inflammatory cells. In the lungs, GO accu-
mulated in the macrophages but not in the lymphocytes, which
were recruited but were not able to trap GO. In this study, the
authors claimed that although oxidative stress is a widely
existent phenomenon in cells exposed in vitro to GO, the
protective effect of proteins forming a BC around GO should
be considered in vivo.
Interesting size-dependent results were reported for multiple-
dose exposure. The s-GO did not induce renal damage or
accumulate in the kidneys since it was quickly eliminated
through the glomeruli. Conversely, l-GO failed to be cleared
through kidneys and induced damage. The lungs were damaged
only after multiple doses of l-GO. This effect depends on the
aggregation of GO with proteins that induce the blockage of
large GO-complexes in the lungs. The hypothesis relies on the
formation of multiple complexes of l-GO and proteins that enter
the capillaries and create multiple injury points and inflamma-
tory cell recruitment. s-GO could instead pass through lungs
capillaries after each low-dose administration. The kidneys and
lungs were more damaged by l-GO, while the s-GO preferentially
accumulated in the liver with toxic effects.
At a high single dose, s-GO can also damage the lungs since
at high concentration it forms large complexes that reach a
similar size to the l-GO protein complex.
44
In summary, s-GO is safer than l-GO, but at a single high
dose the aggregation with blood proteins, and therefore the BC,
induces the formation of toxic large complexes. These results
are in accordance with the study by Qu and colleagues, in which
GO (30–1000 nm) aggregation was suppressed with the addition
of Tween-80 surfactant.
101
While GO aggregated and accumulated
in the lung macrophages, Tween-80 GO was more stable and
accumulated in the liver inside Kuppfer cells (macrophages).
101
Interestingly, rGO seems to have reduced thrombogenicity in vivo,
improved biocompatibility and brain tissue targeting.
63,77,102
The intravenous administration of rGO (with a lateral size of
342 nm administered at 7 mg kg
1
in a single dose) led to
minor signs of toxicity in the blood, liver and kidneys and a lack
of inflammation after 7 days.
It is known that the adsorption of BC is a key factor affecting
the biodistribution of nanoparticles. The BC can act in two
ways: (i) promoting/avoiding the ingestion of phagocytic cells
and influencing circulating time; (ii) regulating the size of
nanomaterials. Size regulates distribution, and particles with
size smaller than capillaries are phagocytized mainly in the
liver, spleen and bone marrow; conversely, large particles are
trapped in the lungs.
44
GO therefore follows the rules of size distribution as other
nanomaterials. However, the BC also influences the uptake by
the immune cells both in the bloodstream (by monocytes,
platelets, leukocytes, and dendritic cells) and in tissues by
resident phagocytes (e.g., Kupffer cells in the liver, DC in lymph
nodes, macrophages and B cells in the spleen). IgG and comple-
ment proteins in the protein corona help to reorganize nano-
particles in immune cells and reticuloendothelial system organs.
59
Conversely, the macrophage uptake can be reduced by maintaining
the particle size at B150 nm and by conferring the nanomaterial
with hydrophilic molecules or albumin that reduce the opsonin
interactions
59,98
and non-specific protein adsorption.
103
The addition of polymer coatings such as PEG to the
nanomaterial surface is a tool for avoiding recognition by the
immune cells. PEG is known to prolong particle circulation in
the blood and significantly decrease uptake by the spleen and
liver-resident phagocytes. It has been hypothesized that PEG
creates a steric shield around the coated particle, effectively
preventing plasma proteins from adhering to the particle
surface and thus avoiding subsequent uptake by mononuclear
phagocytes. PEGylated GO sheets (10–30 nm) were reported
to be mainly distributed in the liver and spleen and did not
exhibit toxicity at high doses (20 mg kg
1
) within 90 days.
74,104
This was likely due to both the small size and PEGylation,
which stabilized the nanomaterial and prevented the aggrega-
tion as well as reduced the BC formation.
Luo and colleagues demonstrated that PEGylation of
GO does not prevent macrophage activation.
105
nGO-PEG,
a functionalized GO with a lateral size of B200 nm, prevents
macrophages uptake, but through physical contact with their
cell membranes, boosts the release of cytokines, potentially
leading to further immunological responses downstream.
Like PEGylation, dextran coating is a well-known strategy to
reduce the adsorption of proteins on nanomaterials and
improve biocompatibility.
106,107
Dextran-GO, obtained with a
protocol of covalent conjugation, showed rapid clearance from
the blood, accumulation in the liver and spleen and elimination
from the bodies of the mice after a week.
106
As we have seen in Section 3, the surface functionalization of
GO can modify its protein corona. The GO-PAA, can reduce the
organ impairments in the liver and lung with respect to GO-,
GO-NH
2
, GO-PAM and GO-PEG-treated mice.
56
Finally, the degradation of injected GO is an important
biosafety concern. Long-term interaction (14 days) of GO with
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plasma causes reduction and biodegradation with hole formation
caused by the action of hydroxyl radicals.
54
Once internalized
by the immune cells, biodegradable particles are digested
and cleared from the body, while non-biodegradable particles
accumulate in cells for extended periods.
A recent study investigated the degradation of large GO
(10 mm) and small GO (100 nm) by neutrophils myeloperoxidase
(MPO).
108
Both kinds of GO sheets were degraded within
12 hours by recombinant human MPO as well as neutrophils
after degranulation. Furthermore, the products of the reaction
were tested for toxicity on a human bronchial epithelial cell
line and demonstrated to be safe. It seems, however, that MPO
degradation is highly dependent on GO stability and that
aggregated GO sheets fail to undergo degradation. Further
studies should confirm neutrophil efficacy in GO disposal
in vivo.
109
Others reported that macrophages are instead
primarily involved in the degradation of carboxylated graphene
in vivo.
110
Although PEGylation reduces GO toxicity, it can hinder the
GO degradation. Both PEG or BSA coated GO/rGO are resistant
to enzyme horseradish peroxidase (HRP) digestion. In the same
study, Li and colleagues conjugated PEG to GO via a cleavable
disulfide bond (GO-SS-PEG) and improved the degradability
of nanoparticles that after reaching the designated tissues
may undergo cleavage of disulfide bonds by glutathione in
the cell cytoplasm/the myeloperoxidase in the phagosomes of
phagocytes.
74
Despite the great scientific advances, future studies for
in vivo application should focus on some weaknesses in gra-
phene research. First, graphene materials should be designed
to have more than just a stable small size for rapid excretion,
and degradable composition to limit toxicity. Detailed physico-
chemical characterization, including size, surface area, charge,
purity, oxygen content, stability in body fluids and the compo-
sition of the BC, should be described in papers aimed at the
pharmacological administration of graphene.
In 2017, Reina and colleagues defined guidelines for the
development of a clearer picture of the toxicity of graphene-
based materials to accelerate the transition of scientific results
into clinics.
111
These guidelines can be summarized as follows:
– use of the established nomenclature for the material
– provide physicochemical characterization (C/O ratio,
surface modification/functionalization, metal traces, size, the
number of layers, surface area, surface charge etc.) and manu-
facturing information
– use the recommended processing methods
– perform biocompatibility tests, cytotoxicity, genotoxicity,
biodegradation, distribution and accumulation into organs,
metabolism.
The BC forms in cell culture medium when FBS is present
and this influences the in vitro results. This and the peculiar
graphene optical properties might influence standard biological
assays like the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide) assay for cell metabolism and should be
considered when GO is tested in vitro. It is also possible that GO,
thanks to its high-protein binding properties, can block some
reagents used in the biological assays.
8
Dedicated protocols
should be established by the scientific community.
112
7. Conclusions
In the last decade, the design of injectable nanoparticles has
affected the control of the BC formation, the interface seen by
cells and tissues, in vivo. Combining all the experimental
results presented in this review, we can conclude that the BC
significantly affects several interactions of GO. The BC inhibits
the hemolytic effects of GO, regulates complement activation,
and mediates immune response activity and biodistribution.
Given the difficulty of precisely controlling the in vivo
interaction with proteins, most of the strategies designed to
modulate the BC are based on functionalization with anti-
fouling polymeric residues that suppress protein adsorption,
altogether lowering the targeting efficiency.
43
In the case of GO,
pre-coating with chitosan to reduce non-specific protein
adsorption, functionalization with dextran or PEG to improve
elimination from the body or modification of surface func-
tional groups by amination and carboxylation are examples of
approaches to modulating the BC.
One can imagine that unique nanomaterial BC properties
might be useful for specific cell targeting application. The BC
compositions of GO and rGO have just started to be studied,
but the first data are encouraging. As an example, ApoE residue
enrichment of the graphene corona can be useful for over-
coming the blood–brain barrier and targeting the cerebrovas-
cular endothelium for neurological disease treatment.
113
GO
complement activation can, in turn, be exploited for enhanced
antigen presentation in vaccines when administered via other
routes, e.g., subcutaneously or intradermally.
8
GO accumula-
tion in lungs can be exploited for the passive pulmonary
delivery of pharmaceuticals.
114
Furthermore, GO-BC displays unique features when submerged
in plasma with patients having diseases like diabetes, and can be
exploitable to develop BC-based diagnostic methods.
52,115
Future
studies should precisely describe the relationship between GO
oxidation, size and dispersibility and BC properties obtained after
incubation in serum or plasma. Another important unaddressed
question is how the corona might influence the biocompatibility of
the graphene-based bioengineering solid implants, considering
the variability of BC composition when scaffolds are implanted in
patients.
116
Based on the great advances in GO research, we can foresee
that new opportunities in nanomedical applications will be
available. We hope that the arguments presented in this review
will help researchers in moving forward and pushing the limits
of GO toward clinical applications.
List of abbreviations
AFM Atomic force microscopy
ApoA-1 Apolipoprotein A-1
ApoE Apolipoprotein E
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BBB Blood–brain barrier
BC Biomolecular corona
BFG Bovine fibrinogen
BSA Bovine serum protein
BSA-g-RGO BSA grafted polydopamine rGO
CB Carbon black
CNT-COOH Carboxylic-functionalized multi-walled CNT
CS Chitosan
DA Dopamine
DC Dendritic cells
DDS Drug delivery systems
FBS Foetal bovine serum
FLG Few-layer graphene
G-BC Biomolecular corona of graphene
GBM Graphene-based materials
G-NH
2
Aminated rGO
GNPs Graphene nanoplatelets
GO Graphene oxide
GONPs GO nanoparticles
GO-PAA GO-polyacrylic acid
GO-PAM GO-polyacrylamide
GO-PEG GO-polyethylene glycol
GO-PEG-PEI GO-polyethylene glycol-polyethyleneimine
GO-SS-PEG PEG conjugated to GO via a cleavable disulfide
bond
Hep-gpRGO Heparin-grafted polydopamine rGO
HRP Horseradish peroxidase
HSA Human serum albumin
Ig Immunoglobulin
IgG Immunoglobulin g
i.v. Intravenously
LPS Lipopolysaccharide
LS Lateral size
MD Molecular dynamics
MPO Myeloperoxidase
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide
MWCNT Multi-walled carbon nanotubes
PBS Phosphate buffered saline
PBMC Peripheral blood mononuclear cells
PEG Polyethylene glycol
PEI Polyethyleneimine
PGO Porous graphene oxide
Phe Phenylalanine
polyDA-rGO rGO functionalized with DA
RBCs Red blood cells
rGO Reduced graphene oxide
rGONPs rGO nanoparticles
SWCNT Single-walled carbon nanotubes
Tf Transferrin
Trp Tryptophan
Tyr Tyrosine
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by Fondazione Umberto Veronesi
(Postdoctoral Fellowships Grant 2018 to V. Palmieri). We are
thankful to Giulia Peragallo for her advice for Fig. 3 design.
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