ArticlePDF AvailableLiterature Review

Abstract and Figures

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.
Content may be subject to copyright.
This journal is ©The Royal Society of Chemistry 201 8 Nanoscale Horiz.
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
rsc.li/nanoscale-horizons
Nanoscale
Horizons
REVIEW
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
View Journal
Nanoscale Horiz. This journal is ©The Royal Society of Chemistry 20 18
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.
Review Nanoscale Horizons
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
This journal is ©The Royal Society of Chemistry 201 8 Nanoscale Horiz.
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 ppstacking
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 ppstacking 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 ppinteractions 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 pp
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
Nanoscale Horizons Review
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
Nanoscale Horiz. This journal is ©The Royal Society of Chemistry 20 18
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 ppinteractions. 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 ppinteractions, 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
Review Nanoscale Horizons
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
This journal is ©The Royal Society of Chemistry 201 8 Nanoscale Horiz.
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 ppand 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 ppstacking 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.
Nanoscale Horizons Review
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
Nanoscale Horiz. This journal is ©The Royal Society of Chemistry 20 18
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
Review Nanoscale Horizons
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
This journal is ©The Royal Society of Chemistry 201 8 Nanoscale Horiz.
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
Nanoscale Horizons Review
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
Nanoscale Horiz. This journal is ©The Royal Society of Chemistry 20 18
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.
Review Nanoscale Horizons
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
This journal is ©The Royal Society of Chemistry 201 8 Nanoscale Horiz.
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
Nanoscale Horizons Review
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
Nanoscale Horiz. This journal is ©The Royal Society of Chemistry 20 18
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
Review Nanoscale Horizons
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
This journal is ©The Royal Society of Chemistry 201 8 Nanoscale Horiz.
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.
Nanoscale Horizons Review
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
Nanoscale Horiz. This journal is ©The Royal Society of Chemistry 20 18
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
Review Nanoscale Horizons
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
This journal is ©The Royal Society of Chemistry 201 8 Nanoscale Horiz.
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
Nanoscale Horizons Review
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
Nanoscale Horiz. This journal is ©The Royal Society of Chemistry 20 18
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.
References
1 A. N. Banerjee, Graphene and its derivatives as biomedical
materials, future prospects and challenges, Interface Focus,
2018, 8(3), 20170056.
2 C. K. Chua and M. Pumera, Chemical reduction of gra-
phene oxide, a synthetic chemistry viewpoint, Chem. Soc.
Rev., 2014, 43(1), 291–312.
3 A. Bianco, H. Cheng, T. Enoki, Y. Gogotsi, R. H. Hurt,
N. Koratkar, T. Kyotani, M. Monthioux, C. R. Park,
J. M. D. Tascon and J. Zhang, All in the graphene family – a
recommended nomenclature for two-dimensional carbon
materials, Carbon, 2013, 65,16.
4 S. Pei and H. M. Cheng, The reduction of graphene oxide,
Carbon, 2012, 50(9), 3210–3228.
5 J. P. M. Almeida, A. L. Chen, A. Foster and R. Drezek,
In vivo biodistribution of nanoparticles, Nanomedicine,
2011, 6(5), 815–835.
6 Kenry, K. P. Loh and C. T. Lim, Molecular interactions of
graphene oxide with human blood plasma proteins, Nano-
scale, 2016, 8(17), 9425–9441.
7 D. Caputo, M. Papi, R. Coppola, S. Palchetti, L. Digiacomo,
G.CaraccioloandD.Pozzi,Aprotein corona-enabled blood
test for early cancer detection, Nanoscale, 2017, 9(1), 349–354.
8 M. A. Dobrovolskaia, P. Aggarwal, J. B. Hall and S. E.
McNeil, Preclinical studies to understand nanoparticle
interaction with the immune system and its potential
effects on nanoparticle biodistribution, Mol. Pharmaceutics,
2008, 5(4), 487–495.
9 Y. Zhang, C. Wu, S. Guo and J. Zhang, Interactions of
graphene and graphene oxide with proteins and peptides,
Nanotechnol. Rev., 2013, 2(1), 27–45.
10 D. F. Ba
´ez, H. Pardo, I. Laborda, J. F. Marco, C. Ya
´n
˜ez and
S. Bollo, Reduced graphene oxides, Influence of the
reduction method on the electrocatalytic effect towards
nucleic acid oxidation, Nanomaterials, 2017, 7(7), 168, DOI:
10.3390/nano7070168.
11 D. R. Dreyer, A. D. Todd and C. W. Bielawski, Harnessing
the chemistry of graphene oxide, Chem. Soc. Rev., 2014,
43(15), 5288–5301.
12 M. Simsikova and T. Sikola, Interaction of Graphene Oxide
with Proteins and Applications of their Conjugates,
J. Nanomed. Res., 2017, 5(2), 00109.
13 J. Yang, K. Y. Hsieh, P. V. Kumar, S. Cheng, Y. Lin, Y. Shen
and G. Chen, Enhanced Osteogenic Differentiation of Stem
Cells on Phase-Engineered Graphene Oxide, ACS Appl.
Mater. Interfaces, 2018, 10(15), 12497–12503.
14 W. Hu, C. Peng, M. Lv, X. Li, Y. Zhang, N. Chen, C. Fan and
Q. Huang, Protein corona-mediated mitigation of cytotoxi-
city of graphene oxide, ACS Nano, 2011, 5(5), 3693–3700.
Review Nanoscale Horizons
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
This journal is ©The Royal Society of Chemistry 201 8 Nanoscale Horiz.
15 J. Zhang, H. Yang, G. Shen, P. Cheng, J. Zhang and S. Guo,
Reduction of graphene oxide via L-ascorbic acid, Chem.
Commun., 2010, 46(7), 1112–1114.
16 A. G. Marrani, A. C. Coico, D. Giacco, R. Zanoni, F. A.
Scaramuzzo, R. Schrebler, D. Dinia, M. Bonomoa and
E. A. Dalchieled, Integration of graphene onto silicon
through electrochemical reduction of graphene oxide
layers in non-aqueous medium, Appl. Surf. Sci., 2018,
445, 404–414.
17 I. Chowdhury, N. D. Mansukhani, L. M. Guiney, M. C.
Hersam and D. Bouchard, Aggregation and stability of
reduced graphene oxide, complex roles of divalent cations,
pH, and natural organic matter, Environ. Sci. Technol.,
2015, 49(18), 10886–10893.
18M.Orecchioni,C.Me
´nard-Moyon,L.G.Deloguand
A. Bianco, Graphene and the immune system, challenges
and potentiality, Adv. Drug Delivery Rev., 2016, 105, 163–175.
19 V. Palmieri, M. C. Lauriola, G. Ciasca, C. Conti, M. De
Spirito and M. Papi, The graphene oxide contradictory
effects against human pathogens, Nanotechnology, 2017,
15, 152001, DOI: 10.1088/1361-6528/aa6150.
20 M. Papi, V. Palmieri, F. Bugli, M. De Spirito, M. Sanguinetti,
C.Ciancico,M.C.Braidotti,S.Gentilini,L.Angelaniand
C. Conti, Biomimetic antimicrobial cloak by graphene-oxide
agar hydrogel, Sci. Rep., 2016, 6(1), 12, DOI: 10.1038/s41598-
016-0010-7.
21 D. Li, W. Zhang, X. Yu, Z. Wang, Z. Su and G. Wei, When
biomolecules meet graphene from molecular level interac-
tions to material design and applications, Nanoscale, 2016,
8(47), 19491–19509.
22 D. Y. Lee, Z. Khatun, J. Lee, Y. Lee and I. In, Blood
compatible graphene/heparin conjugate through noncova-
lent chemistry, Biomacromolecules, 2011, 12(2), 336–341.
23 M. Hassan, M. Walter and M. Moseler, Interactions of
polymers with reduced graphene oxide, van der Waals
binding energies of benzene on graphene with defects,
Phys. Chem. Chem. Phys., 2014, 16(1), 33–37.
24 L.Baweja,K.Balamurugan,V.SubramanianandA.Dhawan,
Eectofgrapheneoxideontheconformational transitions of
amyloid beta peptide, A molecular dynamics simulation
study, J. Mol. Graphics Modell., 2015, 61, 175–185.
25 Y. Chong, C. Ge, Z. Yang, J. A. Garate, Z. Gu, J. K. Weber,
L. Jiajia and Z. Ruhong, Reduced cytotoxicity of graphene
nanosheets mediated by blood-protein coating, ACS Nano,
2015, 9(6), 5713–5724.
26 J. N. Belling, J. A. Jackman, S. Yorulmaz Avsar, J. H. Park,
Y. Wang, M. G. Potroz, A. R. Ferhan, P. S. Weiss and
N. J. Cho, Stealth immune properties of graphene oxide
enabled by surface-bound complement factor H, ACS
Nano, 2016, 10(11), 10161–10172.
27 H. Sun, A. Varzi, V. Pellegrini, D. Dinh, R. Raccichini,
A. Del Rio-Castillo, M. Prato, M. Colombo, R. Cingolani,
B. Scrosati, S. Passerini and F. Bonaccorso, How much
does size really matter? Exploring the limits of graphene as
Li ion battery anode material, Solid State Commun., 2017,
251, 88–93.
28 S. Li, A. N. Aphale, I. G. Macwan, P. K. Patra, W. G.
Gonzalez, J. Miksovska and R. M. Leblanc, Graphene oxide
as a quencher for fluorescent assay of amino acids,
peptides, and proteins, ACS Appl. Mater. Interfaces, 2012,
4(12), 7069–7075.
29 Kenry, Understanding the hemotoxicity of graphene nano-
materials through their interactions with blood proteins
and cells, J. Mater. Res., 2018, 33(1), 44–57.
30 Z. Ding, H. Ma and Y. Chen, Interaction of graphene oxide
with human serum albumin and its mechanism, RSC Adv.,
2014, 4(98), 55290–55295.
31 X. Wu, Y. Xing, K. Zeng, K. Huber and J. X. Zhao, Study of
Fluorescence Quenching Ability of Graphene Oxide with a
Layer of Rigid and Tunable Silica Spacer, Langmuir, 2018,
34(2), 603–611.
32 Kenry, K. P. Loh and C. T. Lim, Selective concentration-
dependent manipulation of intrinsic fluorescence of
plasma proteins by graphene oxide nanosheets, RSC Adv.,
2016, 6(52), 46558–46566.
33 V. Palmieri, F. Bugli, M. C. Lauriola, M. Cacaci, R. Torelli,
G. Ciasca, C. Conti, M. Sanguinetti, M. Papi and M. De
Spirito, Bacteria Meet Graphene, Modulation of Graphene
Oxide Nanosheet Interaction with Human Pathogens for
Effective Antimicrobial Therapy, ACS Biomater. Sci. Eng.,
2017, 3(4), 619–627.
34 J. Kuchlyan, N. Kundu, D. Banik, A. Roy and N. Sarkar,
Spectroscopy and fluorescence lifetime imaging micro-
scopy to probe the interaction of bovine serum albumin
with graphene oxide, Langmuir, 2015, 31(51), 13793–13801.
35 S. S. K. Mallineni, J. Shannahan, A. J. Raghavendra, A. M.
Rao, J. M. Brown and R. Podila, Biomolecular Interactions
and Biological Responses of Emerging Two-Dimensional
Materials and Aromatic Amino Acid Complexes, ACS Appl.
Mater. Interfaces, 2016, 8(26), 16604–16611.
36 H. Vovusha, S. Sanyal and B. Sanyal, Interaction of nucleo-
bases and aromatic amino acids with graphene oxide and
graphene flakes, J. Phys. Chem. Lett., 2013, 4(21), 3710–3718.
37Z.Gu,Z.Yang,L.Wang,H.Zhou,C.A.Jimenez-Cruzand
R. Zhou, The role of basic residues in the adsorption of blood
proteins onto the graphene surface, Sci. Rep., 2015, 5, 10873.
38 L. Baweja, K. Balamurugan, V. Subramanian and
A. Dhawan, Hydration patterns of graphene-based nano-
materials (GBNMs) play a major role in the stability of a
helical protein, a molecular dynamics simulation study,
Langmuir, 2013, 29(46), 14230–14238.
39 Z. Ding, Z. Zhang, H. Ma and Y. Chen, In vitro hemo-
compatibility and toxic mechanism of graphene oxide
on human peripheral blood T lymphocytes and serum
albumin, ACS Appl. Mater. Interfaces, 2014, 6(22),
19797–19807.
40 Kenry, A. Geldert, Y. Liu, K. P. Loh and C. T. Lim, Nano–bio
interactions between carbon nanomaterials and blood
plasma proteins, why oxygen functionality matters, NPG
Asia Mater., 2017, 9(8), e422.
41 P. P. Karmali and D. Simberg, Interactions of nano-
particles with plasma proteins, implication on clearance
Nanoscale Horizons Review
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
Nanoscale Horiz. This journal is ©The Royal Society of Chemistry 20 18
and toxicity of drug delivery systems, Expert Opin. Drug
Delivery, 2011, 8(3), 343–357.
42 V. Pareek, A. Bhargava, V. Bhanot, R. Gupta, N. Jain and
J. Panwar, Formation and Characterization of Protein
Corona Around Nanoparticles, A Review, Nanosci. Nano-
technol., 2018, 18, 6653–6670.
43 C. D. Walkey and W. C. Chan, Understanding and control-
ling the interaction of nanomaterials with proteins in a
physiological environment, Chem. Soc. Rev., 2012, 41(7),
2780–2799.
44 J. Liu, S. Yang, H. Wang, Y. Chang, A. Cao and Y. Liu, Effect
of size and dose on the biodistribution of graphene oxide
in mice, Nanomedicine, 2012, 7(12), 1801–1812.
45 H. Mao, W. Chen, S. Laurent, C. Thirifays, C. Burtea,
F. Rezaee and M. Mahmoudi, Hard corona composition
and cellular toxicities of the graphene sheets, Colloids
Surf., B, 2013, 109, 212–218.
46 V. Castagnola, W. Zhao, L. Boselli, M. L. Giudice, F. Meder,
E. Polo, K. R. Paton, C. Backes, J. N. Coleman and K. A.
Dawson, Biological recognition of graphene nanoflakes,
Nat. Commun., 2018, 9(1), 1577.
47 L.Digiacomo,F.Cardarelli,D.Pozzi,S.Palchetti,M.Digman,
E. Gratton, A. L. Capriotti, M. Mahmoudi and G. Caracciolo,
An apolipoprotein-enriched biomolecular corona switches
the cellular uptake mechanism and trafficking pathway of
lipid nanoparticles, Nanoscale, 2017, 9(44), 17254–17262.
48 V. Mirshafiee, R. Kim, M. Mahmoudi and M. L. Kraft, The
importance of selecting a proper biological milieu for
protein corona analysis in vitro, Human plasma versus
human serum, Int. J. Biochem. Cell Biol., 2016, 75, 188–195.
49 M. Sopotnik, A. Leonardi, I. Krizˇaj, P. Dus
ˇak, D. Makovec,
T. Mesaric
ˇ, N. P. Ulrih, I. Junkar, K. Sepc
ˇic
´and D. Drobne,
Comparative study of serum protein binding to three
different carbon-based nanomaterials, Carbon, 2015, 95,
560–572.
50 X. Wei, L. Hao, X. Shao, Q. Zhang, X. Jia, Z. R. Zhang,
Y. F. Lin and Q. Peng, Insight into the interaction of
graphene oxide with serum proteins and the impact of
the degree of reduction and concentration, ACS Appl.
Mater. Interfaces, 2015, 7(24), 13367–13374.
51 Z. Gu, Z. Yang, Y. Chong, C. Ge, J. K. Weber, D. R. Belland
and R. Zhou, Surface curvature relation to protein adsorp-
tion for carbon-based nanomaterials, Sci. Rep., 2015,
5, 10886.
52M.J.Hajipour,J.Raheb,O.Akhavan,S.Arjmand,
O. Mashinchian, M. Rahman, M. Abdolahad, V. Serpooshan,
S. Laurent and M. Mahmoudi, Personalized disease-specific
protein corona influences the therapeutic impact of graphene
oxide, Nanoscale, 2015, 7(19), 8978–8994.
53 G. Caracciolo, O. C. Farokhzad and M. Mahmoudi, Biolo-
gical identity of nanoparticles in vivo, clinical implications
of the protein corona, Trends Biotechnol., 2017, 35(3),
257–264.
54 X. Hu, D. Li and L. Mu, Biotransformation of graphene oxide
nanosheets in blood plasma affects their interactions with
cells, Environ. Sci.: Nano, 2017, 4(7), 1569–1578.
55 X. Tan, L. Feng, J. Zhang, K. Yang, S. Zhang, Z. Liu and
R. Peng, Functionalization of graphene oxide generates a
unique interface for selective serum protein interactions,
ACS Appl. Mater. Interfaces, 2013, 5(4), 1370–1377.
56 M. Xu, J. Zhu, F. Wang, Y. Xiong, Y. Wu, Q. Wang, J. Weng,
Z. Zhang, W. Chen and S. Liu, Improved in vitro and in vivo
biocompatibility of graphene oxide through surface
modification, poly(acrylic acid)-functionalization is super-
ior to PEGylation, ACS Nano, 2016, 10(3), 3267–3281.
57 T. Yan, H. Zhang, D. Huang, S. Feng, M. Fujita and
X. D. Gao, Chitosan-functionalized graphene oxide as a
potential immunoadjuvant, Nanomaterials, 2017, 7(3), 59.
58 J. H. Liu, T. Wang, H. Wang, Y. Gu, Y. Xu, H. Tang, G. Jiae
and Y. Liu, Biocompatibility of graphene oxide intra-
venously administrated in mice—effects of dose, size and
exposure protocols, Toxicol. Res., 2015, 4(1), 83–91.
59 P. Aggarwal, J. B. Hall, C. B. McLeland, M. A. Dobrovolskaia
and S. E. McNeil, Nanoparticle interaction with plasma
proteins as it relates to particle biodistribution, biocom-
patibility and therapeutic efficacy, Adv. Drug Delivery Rev.,
2009, 61(6), 428–437.
60 A. Moore, R. Weissleder and J. A. Bogdanov, Uptake of
dextran-coated monocrystalline iron oxides in tumor cells
and macrophages, J. Magn. Reson. Imaging, 1997, 7(6),
1140–1145.
61 A. Zoubeidi, K. Chi and M. Gleave, Targeting the cytopro-
tective chaperone, clusterin, for treatment of advanced
cancer, Clin. Cancer Res., 2010, 16(4), 1088–1093.
62 C. H. Stuart, K. R. Riley, O. Boyacioglu, D. M. Herpai,
W. Debinski, S. Qasem, F. C. Marini, C. L. Colyer and
W. H. Gmeiner, Selection of a Novel Aptamer Against
Vitronectin Using Capillary Electrophoresis and Next Gen-
eration Sequencing, Mol. Ther.–Nucleic Acids, 2016,
5(11), e386.
63 M. C. P. Mendonça, E. S. Soares, M. B. de Jesus, H. J.
Ceragioli, M. S. Ferreira, R. R. Catharino and M. A. da
Cruz-Ho
¨fling, Reduced graphene oxide induces transient
blood–brain barrier opening, an in vivo study,
J. Nanobiotechnol., 2015, 13(1), 78.
64 R. Feng, Y. Yu, C. Shen, Y. Jiao and C. Zhou, Impact of
graphene oxide on the structure and function of important
multiple blood components by a dose-dependent pattern,
J. Biomed. Mater. Res., Part A, 2015, 103(6), 2006–2014.
65 S. K. Singh, M. K. Singh, P. P. Kulkarni, V. K. Sonkar,
J. J. Gra
´cio and D. Dash, Amine-modified graphene,
thrombo-protective safer alternative to graphene oxide
for biomedical applications, ACS Nano, 2012, 6(3),
2731–2740.
66 K. H. Liao, Y. S. Lin, C. W. Macosko and C. L. Haynes,
Cytotoxicity of graphene oxide and graphene in human
erythrocytes and skin fibroblasts, ACS Appl. Mater. Interfaces,
2011, 3(7), 2607–2615.
67 M. Papi, M. Lauriola, V. Palmieri, G. Ciasca, G. Maulucci
and D. Spirito, Plasma protein corona reduces the haemo-
lytic activity of graphene oxide nano and micro flakes, RSC
Adv., 2015, 5(99), 81638–81641.
Review Nanoscale Horizons
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
This journal is ©The Royal Society of Chemistry 201 8 Nanoscale Horiz.
68 A. Sasidharan, L. S. Panchakarla, A. R. Sadanandan,
A. Ashokan, P. Chandran, C. M. Girish, D. Menon,
S. V. Nair, C. N. Rao and M. Koyakutty, Hemocompatibility
and macrophage response of pristine and functionalized
graphene, Small, 2012, 8(8), 1251–1263.
69 S. M. Chowdhury, S. Kanakia, J. D. Toussaint, M. D. Frame,
A. M. Dewar, K. R. Shroyer, W. Moore and B. Sitharaman,
In vitro hematological and in vivo vasoactivity assessment
of dextran functionalized graphene, Sci. Rep., 2013,
3, 2584.
70 F. Bugli, M. Cacaci, V. Palmieri, R. Di Santo, R. Torelli,
G. Ciasca, M. Di Vito, A. Vitali, C. Conti, M. Sanguinetti,
M. De Spirito and M. Papi, Curcumin-loaded graphene
oxide flakes as an effective antibacterial system against
methicillin-resistant Staphylococcus aureus,Interface Focus,
2018, 8(3), 20170059.
71 C. Cheng, S. Nie, S. Li, H. Peng, H. Yang, L. Ma, S. Sun and
C. Zhao, Biopolymer functionalized reduced graphene
oxide with enhanced biocompatibility via mussel inspired
coatings/anchors, J. Mater. Chem. B, 2013, 1(3), 265–275.
72 B. Zhang, P. Wei, Z. Zhou and T. Wei, Interactions of graphene
with mammalian cells, molecular mechanisms and bio-
medical insights, Adv. Drug Delivery Rev., 2016, 105, 145–162.
73 G. Duan, S. G. Kang, X. Tian, J. A. Garate, L. Zhao and
C. Ge, C Protein corona mitigates the cytotoxicity of
graphene oxide by reducing its physical interaction with
cell membrane, Nanoscale, 2015, 7(37), 15214–15224.
74 Y. Li, L. Feng, X. Shi, X. Wang, Y. Yang and K. Yang,
Surface Coating-Dependent Cytotoxicity and Degradation
of Graphene Derivatives, Towards the Design of Non-Toxic,
Degradable Nano-Graphene, Small, 2014, 10(8), 1544–1554.
75 H. Yue, W. Wei, Z. Yue, B. Wang, N. Luo and Y. Gao, et al.,
The role of the lateral dimension of graphene oxide in the
regulation of cellular responses, Biomaterials, 2012, 33(16),
4013–4021.
76 A. N. Ilinskaya and M. A. Dobrovolskaia, Nanoparticles and
the blood coagulation system. Part II, safety concerns,
Nanomedicine, 2013, 8(6), 969–981.
77 S. K. Singh, M. K. Singh, M. K. Nayak, S. Kumari,
S. Shrivastava and J. J. Gra
´cio, Thrombus inducing prop-
erty of atomically thin graphene oxide sheets, ACS Nano,
2011, 5(6), 4987–4996.
78 N. Kurantowicz, B. Strojny, E. Sawosz, S. Jaworski,
M. Kutwin and M. Grodzik, Biodistribution of a high dose
of diamond, graphite, and graphene oxide nanoparticles
after multiple intraperitoneal injections in rats, Nanoscale
Res. Lett., 2015, 10(1), 398.
79 P. P. Wibroe, S. V. Petersen, N. Bovet, B. W. Laursen and
S. M. Moghimi, Soluble and immobilized graphene oxide
activates complement system differently dependent on
surface oxidation state, Biomaterials, 2016, 78, 20–26.
80 S. P. Mukherjee, M. Bottini and B. Fadeel, Graphene and
the immune system, A romance of many dimensions,
Front. Immunol., 2017, 8, 673.
81 I. Dudek, M. Skoda, A. Jarosz and D. Szukiewicz, The
molecular influence of graphene and graphene oxide on
the immune system under in vitro and in vivo conditions,
Arch. Immunol. Ther. Exp., 2016, 64(3), 195–215.
82 M.Orecchioni,D.A.Jasim,M.Pescatori,R.Manetti,C.Fozza,
F. Sgarrella, D. Bedognetti, A. Bianco, K. Kostarelos and
L. G. Delogu, Molecular and genomic impact of large and
small lateral dimension graphene oxide sheets on human
immune cells from healthy donors, Adv. Healthcare Mater.,
2016, 5(2), 276–287.
83 Y. Li, Y. Liu, Y. Fu, T. Wei, L. Le Guyader, G. Gao, R. S. Liu,
Y. Z. Chang and C. Chen, The triggering of apoptosis in
macrophages by pristine graphene through the MAPK and
TGF-beta signaling pathways, Biomaterials, 2012, 33(2),
402–411.
84 H. Zhou, K. Zhao, W. Li, N. Yang, Y. Liu, C. Chen and
T. Wei, The interactions between pristine graphene and
macrophages and the production of cytokines/chemokines
via TLR-and NF-kB-related signaling pathways, Biomaterials,
2012, 33(29), 6933–6942.
85 X. Zhi, H. Fang, C. Bao, G. Shen, J. Zhang, K. Wang, S. Guo,
T. Wan and D. Cui, The immunotoxicity of graphene
oxides and the effect of PVP-coating, Biomaterials, 2013,
34(21), 5254–5261.
86 J. Russier, E. Treossi, A. Scarsi, F. Perrozzi, H. Dumortier,
L. Ottaviano, M. Meneghetti, V. Palermo and A. Bianco,
Evidencing the mask effect of graphene oxide, a compara-
tive study on primary human and murine phagocytic cells,
Nanoscale, 2013, 5(22), 11234–11247.
87 J. Ma, R. Liu, X. Wang, Q. Liu, Y. Chen, R. P. Valle,
Y. Y. Zuo, T. Xia and S. Liu, Crucial role of lateral size for
graphene oxide in activating macrophages and stimulating
pro-inflammatory responses in cells and animals, ACS
Nano, 2015, 9(10), 10498–10515.
88 J. Yan, L. Chen, C. C. Huang, S. C. Lung, L. Yang,
W. C. Wang, P. H. Lin, G. Suo and C. H. Lin, Consecutive
evaluation of graphene oxide and reduced graphene oxide
nanoplatelets immunotoxicity on monocytes, Colloids
Surf., B, 2017, 153, 300–309.
89 A. V. Tkach, N. Yanamala, S. Stanley, M. R. Shurin,
G. V. Shurin, E. R. Kisin, A. R. Murray, S. Pareso,
T. Khaliullin, G. P. Kotchey, V. Castranova, S. Mathur,
B.Fadeel,A.Star,V.E.KaganandA.A.Shvedova,Graphene
oxide, but not fullerenes, targets immunoproteasomes and
suppresses antigen presentation by dendritic cells, Small,
2013, 9(9–10), 1686–1690.
90 C. Bussy, H. Ali-Boucetta and K. Kostarelos, Safety
considerations for graphene, lessons learnt from carbon
nanotubes, Acc. Chem. Res., 2012, 46(3), 692–701.
91 C. Meng, X. Zhi, C. Li, C. Li, Z. Chen, X. Qiu, C. Ding, L. Ma,
H. Lu, D. Chen, G. Liu and D. Cui, Graphene oxides
decorated with carnosine as an adjuvant to modulate
innate immune and improve adaptive immunity in vivo,
ACS Nano, 2016, 10(2), 2203–2213.
92 L. Xu, J. Xiang, Y. Liu, J. Xu, Y. Luo, L. Feng, Z. Liu and
R. Peng, Functionalized graphene oxide serves as a novel
vaccine nano-adjuvant for robust stimulation of cellular
immunity, Nanoscale, 2016, 8(6), 3785–3795.
Nanoscale Horizons Review
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
Nanoscale Horiz. This journal is ©The Royal Society of Chemistry 20 18
93 K.Yang,S.Zhang,G.Zhang,X.Sun,S.T.LeeandZ.Liu,
Graphene in mice, ultrahigh in vivo tumor uptake and efficient
photothermal therapy, Nano Lett., 2010, 10(9), 3318–3323.
94 C. Wang, S. Ravi, U. S. Garapati, M. Das, M. Howell,
J. Mallela, S. Alwarapan, S. S. Mohapatra and S. Mohapatra,
Multifunctional chitosan magnetic-graphene (CMG) nano-
particles, a theranostic platform for tumor-targeted
co-delivery of drugs, genes and MRI contrast agents,
J. Mater. Chem. B, 2013, 1(35), 4396–4405.
95 D. Iannazzo, A. Pistone, I. Ziccarelli and S. Galvagno,
Graphene-based materials for application in pharmaceu-
tical nanotechnology, Fullerens, Graphenes and Nanotubes,
Elsevier, 2018, pp. 297–329.
96 T. P. D. Shareena, D. McShan, A. K. Dasmahapatra and
P. B. Tchounwou, A Review on Graphene-Based Nano-
materials in Biomedical Applications and Risks in Environ-
ment and Health, Nano-Micro Lett., 2018, 10(3), 53.
97 M. Ema, M. Gamo and K. Honda, A review of toxicity
studies on graphene-based nanomaterials in laboratory
animals, Regul. Toxicol. Pharmacol., 2017, 85, 7–24.
98 S. F. Kiew, L. V. Kiew, H. B. Lee, T. Imae and L. Y. Chung,
Assessing biocompatibility of graphene oxide-based nano-
carriers, a review, J. Controlled Release, 2016, 226, 217–228.
99 X. Zhang, J. Yin, C. Peng, W. Hu, Z. Zhu, W. Li, C. Fan and
Q. Huang, Distribution and biocompatibility studies of
graphene oxide in mice after intravenous administration,
Carbon, 2011, 49(3), 986–995.
100 M. C. Duch, G. S. Budinger, Y. T. Liang, S. Soberanes,
D. Urich, S. E. Chiarella, L. A. Campochiaro, A. Gonzalez,
N. S. Chandel, M. C. Hersam and G. M. Mutlu, Minimizing
oxidation and stable nanoscale dispersion improves the
biocompatibility of graphene in the lung, Nano Lett., 2011,
11(12), 5201–5207.
101 J. Zhu, M. Xu, M. Gao, Z. Zhang, Y. Xu, T. Xia and S. Liu,
Graphene oxide induced perturbation to plasma membrane
and cytoskeletal meshwork sensitize cancer cells to chemo-
therapeutic agents, ACS Nano, 2017, 11(3), 2637–2651.
102 M. C. P. Mendonça, E. S. Soares, M. B. de Jesus, H. J.
Ceragioli, S. P. Irazusta, A. G. Batista, M. A. Vinolo,
M. R. Maro
´stica Ju
´nior and M. A. da Cruz-Ho
¨fling,
Reduced graphene oxide, nanotoxicological profile in rats,
J. Nanobiotechnol., 2016, 14(1), 53.
103 M. Xie, H. Lei, Y. Zhang, Y. Xu, S. Shen, Y. Ge, H. Li and
J. Xie, Non-covalent modification of graphene oxide nano-
composites with chitosan/dextran and its application in
drug delivery, RSC Adv., 2016, 6(11), 9328–9337.
104 K. Yang, J. Wan, S. Zhang, Y. Zhang, S. T. Lee and Z. Liu,
In vivo pharmacokinetics, long-term biodistribution, and
toxicology of PEGylated graphene in mice, ACS Nano, 2010,
5(1), 516–522.
105 N. Luo, J. K. Weber, S. Wang, B. Luan, H. Yue, X. Xi, J. Du,
Z. Yang, W. Wei, R. Zhou and G. Ma, PEGylated graphene
oxide elicits strong immunological responses despite
surface passivation, Nat. Commun., 2017, 8, 14537.
106 S. Zhang, K. Yang, L. Feng and Z. Liu, In vitro and in vivo
behaviors of dextran functionalized graphene, Carbon,
2011, 49(12), 4040–4049.
107 H. H. Gustafson, D. Holt-Casper, D. W. Grainger and
H. Ghandehari, Nanoparticle uptake, the phagocyte
problem, Nano Today, 2015, 10(4), 487–510.
108 S. P. Mukherjee, A. R. Gliga, B. Lazzaretto, B. Brandner,
M. Fielden, C. Vogt, L. Newman, A. F. Rodrigues, W. Shao,
P. M. Fournier, M. S. Toprak, A. Star, K. Kostarelos,
K. Bhattacharya and B. Fadeel, Graphene oxide is degraded
by neutrophils and the degradation products are non-
genotoxic, Nanoscale, 2018, 10(3), 1180–1188.
109 R. Kurapati, J. Russier, M. A. Squillaci, E. Treossi,
C. Me
´nard-Moyon, A. E. Del Rio-Castillo, E. Vazquez,
P. Samorı
`, V. Palermo and A. Bianco, Dispersibility-
Dependent Biodegradation of Graphene Oxide by Myelo-
peroxidase, Small, 2015, 11(32), 3985–3994.
110 C. M. Girish, A. Sasidharan, G. S. Gowd, S. Nair and
M. Koyakutty, Confocal Raman imaging study showing
macrophage mediated biodegradation of graphene
in vivo,Adv. Healthcare Mater., 2013, 2(11), 1489–1500.
111 G. Reina, J. M. Gonza
´lez-Domı
´
nguez,A.Criado,E.Va
´zquez,
A. Bianco and M. Prato, Promises, facts and challenges for
graphene in biomedical applications, Chem.Soc.Rev., 2017,
46(15), 4400–4416.
112 L. Ou, B. Song, H. Liang, J. Liu, X. Feng, B. Deng, T. Sun
and L. Shao, Toxicity of graphene-family nanoparticles, a
general review of the origins and mechanisms, Part. Fibre
Toxicol., 2016, 13(1), 57.
113 R.DalMagro,B.Albertini,S.Beretta,R.Rigolio,E.Donzelli,
A. Chiorazzi, M. Ricci, P. Blasi and G. Sancini, Artificial
apolipoprotein corona enables nanoparticle brain targeting,
Nanomedicine, 2018, 14(2), 429–438.
114 Y. Wei and L. Zhao, Passive lung-targeted drug delivery
systems via intravenous administration, Pharm. Dev. Technol.,
2014, 19(2), 129–136.
115 M. Papi and G. Caracciolo, Principal component analysis
of personalized biomolecular corona data for early disease
detection, Nano Today, 2018, 21, 14–17.
116 V. Serpooshan, M. Mahmoudi, M. Zhao, K. Wei,
S. Sivanesan, K. Motamedchaboki, A. V. Malkovskiy, A. B.
Gladstone, J. E. Cohen, P. C. Yang, J. Rajadas, D. Bernstein,
Y. J. Woo and P. Ruiz-Lozano, Protein corona influences
cell–biomaterial interactions in nanostructured tissue
engineering scaffolds, Adv. Funct. Mater., 2015, 25(28),
4379–4389.
Review Nanoscale Horizons
Published on 17 October 2018. Downloaded on 11/2/2018 10:09:04 AM.
View Article Online
... The protein corona is one of the first factors to be addressed, which causes the rapid change of GO in the bloodstream [164]. This protein coating induces the size change of GO that substantially influences the interactions with the cells [34]. ...
... The body's non-specific immune defense relies on phagocytosis of foreign molecules via macrophages, providing a significant barrier to intravenous injections of GO nanocarriers. In addition, GO nanocarriers have a high probability of being removed by macrophages before reaching their destination and may initiate an inflammatory reaction [24,164]. In particular, when in the bloodstream, larger nanoparticles (>200 nm) have a higher risk of being recognized and sequestered [44,53,123,137] by the phagocytosing cells of the immune system (i.e., macrophages, dendritic cells, neutrophils, and B lymphocytes), which are responsible for recognizing the foreigner and destroying it via enzymatic digestion, as explained previously [31]. ...
Article
Full-text available
Functionalized graphene oxide (GO) nanoparticles are being increasingly employed for designing modern drug delivery systems because of their high degree of functionalization, high surface area with exceptional loading capacity, and tunable dimensions. With intelligent controlled release and gene silencing capability, GO is an effective nanocarrier that permits the targeted delivery of small drug molecules, antibodies, nucleic acids, and peptides to the liquid or solid tumor sites. However, the toxicity and biocompatibility of GO-based formulations should be evaluated, as these nanomaterials may introduce aggregations or may accumulate in normal tissues while targeting tumors or malignant cells. These side effects may potentially be impacted by the dosage, exposure time, flake size, shape, functional groups, and surface charges. In this review, the strategies to deliver the nucleic acid via the functionalization of GO flakes are summarized to describe the specific targeting of liquid and solid breast tumors. In addition, we describe the current approaches aimed at optimizing the controlled release towards a reduction in GO accumulation in non-specific tissues in terms of the cytotoxicity while maximizing the drug efficacy. Finally, the challenges and future research perspectives are briefly discussed.
... However, these materials are known to exert different degrees of toxicity in different animal and cell models following different administration routes (Ou et al. 2016). Known toxic effects of these materials include physical damage to cell membranes, oxidative stress, DNA damage, inflammatory response, apoptosis, autophagy and necrosis, which are determined by various factors such as the material's lateral size, surface structure, number of layers, functionalization, charge, impurities, synthetic method, aggregation, protein corona effect, dose, time of exposure and administration route (Seabra et al. 2014;Ou et al. 2016;Palmieri et al. 2019). ...
... Another issue with the use of colloidal materials, which may affect colloidal ultrasound contrast agents, is the formation of a hard protein corona upon lengthy exposure to systemic circulation. Adsorption of circulating proteins onto colloidal materials can alter their physicochemical properties (size, surface charge, surface composition and functionality), giving them a new biological identity that determines their physiological response, including agglomeration, cellular uptake, circulation time, signaling, kinetics, transport, accumulation and toxicity (Rahman et al. 2013;Palmieri et al. 2019;Breznica et al. 2020;Kopac 2021). These phenomena may result in the particles' displaying low response to stimuli, as well as failure to reach, concentrate and exert their action on target sites. ...
Article
Full-text available
The current work features process parameters for the ultrasound (25 kHz)-assisted fabrication of polydopamine-shelled perfluorocarbon (PDA/PFC) emulsion droplets with bimodal (modes at 100–600 nm and 1–6 µm) and unimodal (200–600 nm) size distributions. Initial screening of these materials revealed that only PDA/PFC emulsion droplets with bimodal distributions showed photoacoustic signal enhancement due to large size of their optically absorbing PDA shells. Performance of this particular type of emulsion droplets as photoacoustic agents were evaluated in Intralipid®–India ink media, mimicking the optical scattering and absorbance of various tissue types. From these measurements, it was observed that PDA/PFC droplets with bimodal size distributions can enhance the photoacoustic signal of blood-mimicking phantom by up to five folds in various tissue-mimicking phantoms with absorption coefficients from 0.1 to 1.0 cm⁻¹. Furthermore, using the information from enhanced photoacoustic images at 750 nm, the ultimate imaging depth was explored for polydopamine-shelled, perfluorohexane (PDA/PFH) emulsion droplets by photon trajectory simulations in 3D using a Monte-Carlo approach. Based on these simulations, maximal tissue imaging depths for PDA/PFH emulsion droplets range from 10 to 40 mm, depending on the tissue type. These results demonstrate for the first time that ultrasonically fabricated PDA/PFC emulsion droplets have great potential as photoacoustic imaging agents, which can be complemented with other reported characteristics of PDA/PFC emulsion droplets for extended applications in theranostics and other imaging modalities.
... De Paoli et al. (2014) reported that an IgG corona causes thrombocyte fragmentation, leading to induction of thrombocyte aggregation and release of platelet membrane microparticles. Palmieri et al. (2019) also extensively reviewed the adverse effects that protein corona coated graphene oxide has on blood integrity. Barbalinardo et al. (2018) reported that the cytotoxic activity of citrate-coated silver NPs toward NIH-3T3 cells was increased after protein corona formation, contrary to what was observed with CNTs (Gu et al. 2015) and graphene oxide (Hu et al. 2011). ...
Article
The surfaces of nanoparticles become covered by biomolecules in biological fluids. This protein ‘corona’ modifies materials’ characteristics and biological activity. The composition of the protein corona is dynamic, abundant biomolecules that bind first are subsequently replaced by less abundant but more tightly bound ones. Here, we explore the formation of the silver nanoparticle protein corona on exposure to cell culture media containing 10% fetal bovine serum supplemented Dulbecco's Modified Eagle's medium. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry analysis were used to monitor how different parameters such as incubation time, heating duration, cell culture medium, incubation temperature, and the number of washes affect the nanoparticle–protein corona complex. silver nanoparticles with and without bound proteins were characterized by electron microscopy, dynamic light scattering, and ultraviolet-visible-near-IR spectroscopy. The tetrazolium-based MTT assay was used to determine viability of A549 human lung adenocarcinoma cells treated with silver nanoparticles. Characterization of the nanoparticles before and after protein binding provided insights into their changing morphology on corona formation. Our results confirmed that the physiological environment directly affects protein corona formation on nanoparticle surfaces. In particular, incubation condition-dependent differences in the amount of bound proteins were observed. This work highlights the importance of environmental drivers of protein adsorption, which should be considered when predicting and/or controlling protein targets of silver nanoparticles.
... On their ''journey" through the body following i.v. injection, 2D materials will inevitably encounter and bind plasma proteins on their surface [130]. Growing evidence has been reported that pre-coating of nanomaterials with host proteins could enable regulatory control of nanomaterial-induced immune response, either preventing their rapid clearance by the mononuclear phagocyte system, for instance, by masking the complement-activating properties or boosting the immune response by coating with specific antibodies. ...
Article
Full-text available
Two-dimensional (2D) materials such as the graphene-based materials, transition metal dichalcogenides, transition metal carbides and nitrides (MXenes), black phosphorus, hexagonal boron nitride, and others have attracted considerable attention due to their unique physicochemical properties. This is true not least in the field of medicine. Understanding the interactions between 2D materials and the immune system is therefore of paramount importance. Furthermore, emerging evidence suggests that 2D materials may interact with microorganisms – pathogens as well as commensal bacteria that dwell in and on our body. We discuss the interplay between 2D materials, the immune system, and the microbial world in order to bring a systems perspective to bear on the biological interactions of 2D materials. The use of 2D materials as vectors for drug delivery and as immune adjuvants in tumor vaccines, and 2D materials to counteract inflammation and promote tissue regeneration, are explored. The bio-corona formation on and biodegradation of 2D materials, and the reciprocal interactions between 2D materials and microorganisms, are also highlighted. Finally, we consider the future challenges pertaining to the biomedical applications of various classes of 2D materials.
... In spite of the large number of scientific studies on CNMs, often, it is difficult to anticipate which type is the best performer for specific applications, and even more so in the case of biological uses where there is a high degree of biomolecular complexity [27][28][29][30]. Interactions between CNMs and biomolecules, such as proteins [31] and DNA [32], are very relevant in this regard for their key roles in the determination of the dynamic corona on their surface [33], which, in turn, affects their fate in vivo [34], spanning their biodistribution [35] to immune response [36,37] and biodegradation [38,39]. ...
Article
Full-text available
Carbon nanomaterials have attracted great interest for their unique physico-chemical properties for various applications, including medicine and, in particular, drug delivery, to solve the most challenging unmet clinical needs. Graphitization is a process that has become very popular for their production or modification. However, traditional conditions are energy-demanding; thus, recent efforts have been devoted to the development of greener routes that require lower temperatures or that use waste or byproducts as a carbon source in order to be more sustainable. In this concise review, we analyze the progress made in the last five years in this area, as well as in their development as drug delivery agents, focusing on active targeting, and conclude with a perspective on the future of the field.
... The identified substances are known for their bio-incompatibility by adversely affecting blood circulation, and vascular endothelium, contributing to the formation of thrombi, as well as increasing the risk of hemorrhaging. Nanoparticles such as graphene, regarded themselves as adjuvants (Xu et al., 2016;Cao, 2020;Chung et al., 2020), are known to induce pro-inflammatory cytokine expression -consider Blaylock's (2021) "cytokine storm" (also see Fleming, 2021) -and in combination with PEG supposedly to improve elimination from the body (Palmieri et al., 2019). However, the DiCoReTh-injections are actually designed to disperse the LNPs and their payloads throughout the body (Kostoff et al., 2021). ...
Article
Full-text available
The engineered spike protein of SARS-COV-2, and the corresponding infectious disease COVID-19 attributed to it, hold in their grip a large portion of humanity. The global race for a counter strategy quickly turned into a search for a vaccine as the preferred means to contain the virus. An unusually rapid development of different and completely new classes of experimental therapies that would widely be referred to as “vaccines” raised questions about safety, especially with regard to emergency use approval (EUA) being granted with unprecedented urgency and hardly any critical scrutiny. At present, independent researchers, even some former proponents and insiders, of the currently ongoing global experiment represented as a “vaccination” campaign point primarily to the lack of public safety studies based on empirical datasets that should be obtainable for the tens of millions, even hundreds of millions, of doses of mRNA and DNA vector therapeutics being distributed as “vaccines”. Studies regarding efficacy and “side effects” (sometimes fatalities or permanent iatrogenic injuries) of these experimental therapies have been by-passed in favor of short-term field data from real patients which inevitably raises scientific and ethical questions particularly in view of the fact that the persons and entities responsible for public safety hold deep financial and other vested interests in speeding along the distribution of the experimental pharmaceutical products. The lack of an open discussion about the experimental therapies for COVID-19 now being applied across all age groups, even children hardly impacted by COVID-19, is worrying. The core principle of open debate without pre-conceptions or vested interests in outcomes has been and continues to be utterly ignored. We hope to engage scientific discussion that will help decision-makers, the general public, and the media alike to consider the subject-matter of what is at stake in a context of reason rather than panic.
... Un'indagine simile che ha utilizzato allo stesso modo l'elettrone ambientale in microscopia (ESEM) per studiare la composizione dell'mRNA e dei "vaccini" vettoriali ha rivelato che queste iniezioni TeReCoMa contengono componenti che non sono menzionati nel foglio di istruzioni (TSC, 2021 Le sostanze identificate sono note per la loro bio-incompatibilità in quanto influiscono negativamente sulla circolazione sanguigna e sull'endotelio vascolare, contribuendo alla formazione di trombi, oltre ad aumentare il rischio di emorragia. È noto che le nanoparticelle come il grafene sono adiuvanti (Xu et al., 2016;Cao, 2020;Chung et al., 2020) che servono a indurre l'espressione di citochine pro-infiammatorie -si consideri la "tempesta di citochine" di Blaylock (2021) (vedi anche Fleming, 2021) -in combinazione con PEG presumibilmente per migliorare l'eliminazione dal corpo (Palmieri et al., 2019). Tuttavia, le iniezioni di TeReCoMa sono effettivamente progettate per disperdere gli LNP e i loro carichi utili in tutto il corpo (Kostoff et al., 2021). ...
Article
Full-text available
La proteina ingegnerizzata spike della SARS-COV-2 e la corrispondente malattia infettiva COVID-19 attribuita ad essa tengono in pugno una gran parte dell'umanità. La corsa globale per una strategia di contrasto si è rapidamente trasformata nella ricerca di un vaccino come mezzo preferenziale per contenere il virus. Uno sviluppo insolitamente rapido di diverse classi completamente nuove di terapie sperimentali diffuse come "vaccini", ha sollevato interrogativi sulla sicurezza, in particolare per quanto riguarda l'approvazione dell'uso di emergenza (EUA) che è stata concessa con un'urgenza senza precedenti e priva di qualsiasi esame critico contrario. Attualmente, ricercatori indipendenti, come anche alcuni ex proponenti e addetti ai lavori dell'esperimento globale attualmente in corso e rappresentato come una campagna di "vaccinazione", sottolineano soprattutto la mancanza di studi sulla sicurezza della campagna vaccinale che ha finito invece per strutturarsi su set di dati empirici che verranno ottenuti attraverso decine di milioni, anche centinaia di milioni, di dosi di mRNA e terapie vettoriali del DNA distribuite col nome di "vaccini". Gli studi riguardanti l'efficacia e gli "effetti collaterali" (talvolta fatalità o lesioni iatrogene permanenti) di queste terapie sperimentali sono stati omessi a favore di dati a breve termine presi sul campo su pazienti reali. Questa evidenza solleva inevitabilmente questioni scientifiche ed etiche, in particolare in considerazione del fatto che le persone e gli enti responsabili per la sicurezza pubblica hanno vasti interessi finanziari e di altro tipo che li portano ad accelerare la distribuzione di questi prodotti farmaceutici sperimentali. La mancanza di una discussione aperta sulle terapie sperimentali per il COVID-19 ora applicate su tutte le fasce di età, anche i bambini, che difficilmente sono colpiti dal COVID-19, è preoccupante. Il principio fondamentale del dibattito aperto senza preconcetti o sugli interessi nei risultati è stato e continua ad essere completamente ignorato. Speriamo di impegnare una discussione scientifica al fine di aiutare chi deve decidere, l'opinione pubblica e i media a considerare l'oggetto di ciò che è in gioco in un contesto di ragione piuttosto che di panico.
... These functional groups enhance the hydrophilicity of the GO surface and enable functionalization of GO with bio-active molecules [29] and interaction with a series of proteins. In vivo, these proteins may originate from blood serum [30], whereas in biotechnological approaches, distinct proteins might be chosen to generate bio-artificial or bio-mimetic surfaces of implants [31]. GO surfaces offer a broad spectrum for surface modification [32]. ...
Article
Graphene oxide (GO) is a promising material for bone tissue engineering, but the validation of its molecular biological effects, especially in the context of clinically applied materials, is still limited. In this study, we compare the effects of graphene oxide framework structures (F-GO) and reduced graphene oxide-based framework structures (F-rGO) as scaffold material with a special focus on vascularization associated processes and mechanisms in the bone. Highly porous networks of zinc oxide tetrapods serving as sacrificial templates were used to create F-GO and F-rGO with porosities >99% consisting of hollow interconnected microtubes. Framework materials were seeded with human mesenchymal stem cells (MSC), and the cell response was evaluated by confocal laser scanning microscopy (CLSM), deoxyribonucleic acid (DNA) quantification, real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and alkaline phosphatase activity (ALP) to define their impact on cellular adhesion, osteogenic differentiation, and secretion of vascular growth factors. F-GO based scaffolds improved adhesion and growth of MSC as indicated by CLSM and DNA quantification. Further, F-GO showed a better vascular endothelial growth factor (VEGF) binding capacity and improved cell growth as well as the formation of microvascular capillary-like structures in co-cultures with outgrowth endothelial cells (OEC). These results clearly favored non-reduced graphene oxide in the form of F-GO for bone regeneration applications. To study GO in the context of a clinically used implant material, we coated a commercially available xenograft (Bio-Oss® block) with GO and compared the growth of MSC in monoculture and in coculture with OEC to the native scaffold. We observed a significantly improved growth of MSC and formation of prevascular structures on coated Bio-Oss®, again associated with a higher VEGF binding capacity. We conclude that graphene oxide coating of this clinically used, but highly debiologized bone graft improves MSC cell adhesion and vascularization.
Article
Ultrasound has important applications, predominantly in the field of diagnostic imaging. Presently, colloidal systems such as microbubbles, phase-change emulsion droplets and particle systems with acoustic properties and multiresponsiveness are being developed to address typical issues faced when using commercial ultrasound contrast agents, and to extend the utility of such systems to targeted drug delivery and multimodal imaging. Current technologies and increasing research data on the chemistry, physics and materials science of new colloidal systems are also leading to the development of more complex, novel and application-specific colloidal assemblies with ultrasound contrast enhancement and other properties, which could be beneficial for multiple biomedical applications, especially imaging-guided treatments. In this article, we review recent developments in new colloids with applications that use ultrasound contrast enhancement. This work also highlights the emergence of colloidal materials fabricated from or modified with biologically derived and bio-inspired materials, particularly in the form of biopolymers and biomembranes. Challenges, limitations, potential developments and future directions of these next-generation colloidal systems are also presented and discussed.
Article
Protein corona refers to the structure composed of biomolecules adsorbed on the surface of nanomaterials. The study on the effect of the interaction between protein and nanoparticles can provide an important guide for the application of nanodrug delivery. To provide a reference for the research on fullerene (C60) nanocomplex drug delivery systems, this work studied the interaction between C60 nanocomplex and a variety of plasma proteins. Research showed that the protein binding with C60 nanocomplex did not change the charge properties of protein. The proteins induced the aggregation of C60 nanocomplex. The circular dichroism spectra showed that the secondary structure of the proteins changed after binding to C60 nanocomplex. The ultraviolet–visible spectra showed that the effect of C60 nanocomplex on proteins was concentration-dependent. The fluorescence spectra showed that C60 nanocomplex could intrinsic fluorescence alteration of proteins. The adsorption capacity of C60 nanocomplex to proteins was changed at 0 h and 4 h. The interaction between nanocomplex and proteins might affect the morphological characteristics of nanocomplex and the conformation of proteins. This work could provide a reference for the research and development of C60 nanocomplex and other carbon-based nanocomplex as nanoparticulate drug delivery systems.
Article
Full-text available
Graphene-based nanomaterials (GBNs) have attracted increasing interests of the scientific community due to their unique physicochemical properties and their applications in biotechnology, biomedicine, bioengineering, disease diagnosis and therapy. Although a large amount of researches have been conducted on these novel nanomaterials, limited comprehensive reviews are published on their biomedical applications and potential environmental and human health effects. The present research aimed at addressing this knowledge gap by examining and discussing: (1) the history, synthesis, structural properties and recent developments of GBNs for biomedical applications; (2) GBNs uses as therapeutics, drug/gene delivery and antibacterial materials; (3) GBNs applications in tissue engineering and in research as biosensors and bioimaging materials; and (4) GBNs potential environmental effects and human health risks. It also discussed the perspectives and challenges associated with the biomedical applications of GBNs. Open image in new window
Article
Full-text available
The systematic study of nanoparticle-biological interactions requires particles to be reproducibly dispersed in relevant fluids along with further development in the identification of biologically relevant structural details at the materials-biology interface. Here, we develop a biocompatible long-term colloidally stable water dispersion of few-layered graphene nanoflakes in the biological exposure medium in which it will be studied. We also report the study of the orientation and functionality of key proteins of interest in the biolayer (corona) that are believed to mediate most of the early biological interactions. The evidence accumulated shows that graphene nanoflakes are rich in effective apolipoprotein A-I presentation, and we are able to map specific functional epitopes located in the C-terminal portion that are known to mediate the binding of high-density lipoprotein to binding sites in receptors that are abundant in the liver. This could suggest a way of connecting the materials' properties to the biological outcomes.
Article
Full-text available
Graphene oxide (GO) has attracted significant interest as a template material for multiple applications due to its two-dimensional nature and established functionalization chemistries. However, for applications toward stem cell culture and differentiation, GO is often reduced to form reduced graphene oxide, resulting in a loss of oxygen content. Here, we induce a phase transformation in GO and demonstrate its benefits for enhanced stem cell culture and differentiation, while conserving the oxygen content. The transformation results in clustering of oxygen atoms on the GO surface, which greatly improves its ability toward substance adherence and results in enhanced differentiation of human mesenchymal stem cell (hMSCs) towards the osteogenic lineage. Moreover, the conjugating ability of modified GO strengthened, which was examined by auxiliary osteogenic growth peptide conjugation. Overall, our work demonstrates GO’s potential for stem cell applications, while maintaining its oxygen content, which could be enable further functionalization and fabrication of novel nano-bio interfaces.
Article
Graphene and its derivatives possess some intriguing properties, which generates tremendous interests in various fields, including biomedicine. The biomedical applications of graphene-based nanomaterials have attracted great interests over the last decade, and several groups have started working on this field around the globe. Because of the excellent biocompatibility, solubility and selectivity, graphene and its derivatives have shown great potential as biosensing and bio-imaging materials. Also, due to some unique physicochemical properties of graphene and its derivatives, such as large surface area, high purity, good bio-functionalizability, easy solubility, high drug loading capacity, capability of easy cell membrane penetration, etc., graphene-based nanomaterials become promising candidates for bio-delivery carriers. Besides, graphene and its derivatives have also shown interesting applications in the fields of cell-culture, cell-growth and tissue engineering. In this article, a comprehensive review on the applications of graphene and its derivatives as biomedical materials has been presented. The unique properties of graphene and its derivatives (such as graphene oxide, reduced graphene oxide, graphane, graphone, graphyne, graphdiyne, fluorographene and their doped versions) have been discussed, followed by discussions on the recent efforts on the applications of graphene and its derivatives in biosensing, bio-imaging, drug delivery and therapy, cell culture, tissue engineering and cell growth. Also, the challenges involved in the use of graphene and its derivatives as biomedical materials are discussed briefly, followed by the future perspectives of the use of graphene-based nanomaterials in bioapplications. The review will provide an outlook to the applications of graphene and its derivatives, and may open up new horizons to inspire broader interests across various disciplines.
Article
Recent years have witnessed unprecedented increase in the use of nanoparticles in various sectors viz. electronics, catalysis, agriculture, textile, cosmetics, bio-medicine, packaging, house-holds and food-associated consumer products. This has led to the inevitable release of nanoparticles into the environment, which can have negative impact on living beings. Humans can also be exposed to these nanoparticles either intentionally or accidently. Nanoparticles can enter in the human body through food chain, inhalation, open wounds, drugs and intravenous injections etc. In majority of these cases, the nanoparticles may pass through the various cell layers, cell sap and finally enter into the blood. Upon interaction with biological fluid, nanoparticles come in close proximity particularly to the proteins present in the fluid. The assembly of proteins surrounding the nanoparticle's surface is called as protein corona and their complex is known as protein-nanoparticle complex. Formation of protein corona is a vibrant and driving process, which plays a pivotal role in the functioning of nanoparticles in biological systems. Moreover, due to interaction of proteins with nanoparticles, conformational changes may occur in the native structure of protein, which may lead to change in the functioning of proteins towards its cellular interaction. The present review provides in-depth knowledge about the formation of protein corona around nanoparticles and the factors regulating this process. Further, it discusses various techniques that can be used for the protein corona analysis and obtaining information about molecular consequences upon nanoparticle's exposure. Finally, the functional aspects of protein-nanoparticle complex have been discussed in detail. In-depth understanding of protein-nanoparticles complex can be instrumental to generate well-suited nanoparticles with desired surface characteristics in the way to biological application.
Article
Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for serious hospital infections worldwide and represents a global public health problem. Curcumin, the major constituent of turmeric, is effective against MRSA but only at cytotoxic concentrations or in combination with antibiotics. The major issue in curcumin-based therapies is the poor solubility of this hydrophobic compound and the cytotoxicity at high doses. In this paper, we describe the efficacy of a composite nanoparticle made of curcumin (CU) and graphene oxide (GO), hereafter GOCU, in MRSA infection treatment. GO is a nanomaterial with a large surface area and high drug-loading capacity. GO has also antibacterial properties due mainly to a mechanical cutting of the bacterial membranes. For this physical mechanism of action, microorganisms are unlikely to develop resistance against this nanomaterial. In this work, we report the capacity of GO to support and stabilize curcumin molecules in a water environment and we demonstrate the efficacy of GOCU against MRSA at a concentration below 2 µg ml-1. Further, GOCU displays low toxicity on fibroblasts cells and avoids haemolysis of red blood cells. Our results indicate that GOCU is a promising nanomaterial against antibiotic-resistant MRSA.
Article
Wafer-scale integration of reduced graphene oxide with H-terminated Si(1 1 1) surfaces has been accomplished by electrochemical reduction of a thin film of graphene oxide deposited onto Si by drop casting. Two reduction methods have been assayed and carried out in an acetonitrile solution. The initial deposit was subjected either to potential cycling in a 0.1 M TBAPF6/CH3CN solution at scan rates values of 20 mV s⁻¹ and 50 mV s⁻¹, or to a potentiostatic polarization at Eλ,c = −3 V for 450 s. The resulting interface has been characterized in its surface composition, morphology and electrochemical behavior by X-ray photoelectron spectroscopy, Raman spectroscopy, atomic force microscopy and electrochemical measurements. The results evidence that few-layer graphene deposits on H-Si(1 1 1) were obtained after reduction, and use of organic instead of aqueous medium led to a very limited surface oxidation of the Si substrate and a very low oxygen-to-carbon ratio. The described approach is fast, simple, economic, scalable and straightforward, as one reduction cycle is already effective in promoting the establishment of a graphene-Si interface. It avoids thermal treatments at high temperatures, use of aggressive chemicals and the presence of metal contaminants, and enables preservation of Si(1 1 1) surface from oxidation.