Cite this: RSC Advances, 2013, 3, 1660

Received 26th November 2012,

Accepted 28th November 2012
Graphene coatings for enhanced hemo-compatibility of
nitinol stents3
DOI: 10.1039/c2ra23073a
www.rsc.org/advances
Ramakrishna Podila,
ab
Thomas Moore,
c
Frank Alexis
c
and Apparao M. Rao
bd
In this study, we used graphene, a one-atom thick sheet of carbon
atoms, to modify the surfaces of existing implant materials to
enhance both bio- and hemo-compatibility. This novel effort
meets all functional criteria for a biomedical implant coating as it
is chemically inert, atomically smooth and highly durable, with
the potential for greatly enhancing the effectiveness of such
implants. Specifically, graphene coatings on nitinol, a widely used
implant and stent material, showed that graphene coated nitinol
(GrNiTi) supports excellent smooth muscle and endothelial cell
growth leading to better cell proliferation. The authors further
determined that the serum albumin adsorption on GrNiTi is
greater than that of fibrinogen, which is an important and well
understood criterion for promoting a lower thrombosis rate.
These hemo- and biocompatible properties, along with high
strength, chemical inertness and durability provide graphene with
an edge over most antithrombogenic coatings for biomedical
implants and devices.
Introduction
The past three decades have witnessed the discovery of novel
materials-based therapies and devices for disease treatment and
diagnosis. Novel alloy materials such as nitinol (NiTi) and stainless
steel are often used in biomedical implant manufacturing due to
their superior mechanical properties.
13
Nevertheless, difficulties
regarding the exogenous cytotoxicity, and bio- and hemo-compat-
ibility of the implant materials must be first resolved prior to the
safe and effective use of such devices. The metallic nature of these
alloys results in metal leaching, lack of cell adhesion, proliferation,
and thrombosis when they (e.g., catheters, blood vessel grafts,
vascular stents, artificial heart valves, etc.) come into contact with
flowing blood.
1,4,5
The interaction of proteins or living cells with
the implant surface can lead to a strong immunological response,
and the ensuing cascade of biochemical reactions can adversely
affect the device functionality. Therefore, it is necessary to control
the interactions between the biomedical implant and its
surrounding biological environment. Surface modification is often
employed to mitigate these adverse physiological responses to the
implant material. An ideal surface coating is expected to have a
high adhesion strength, chemical inertness, smoothness, and
hemo- and biocompatibility. Previously, numerous materials
including diamond-like carbon, SiC, TiN, TiO
2
and many polymer
materials have been tested as bio-compatible implant surface
coatings.
1,623
These materials, however, do not yet meet all the
functional criteria stipulated by the United States Food and Drug
Administration for implant coatings.
The discovery of graphene, a single atom thick layer of sp
2
carbon, has expanded the research in the development of novel
multifunctional materials. An ideal candidate for implant surface
coatings, graphene is chemically inert, atomically smooth and
highly durable. Furthermore, graphene has been used in many
biomedical applications including the growth of neuronal cells
and human osteoblasts on graphene.
12,13
In this investigation of
the viability of graphene as a surface coating for biomedical
implants, we show that graphene coated nitinol (GrNiTi) meets
all functional criteria, while also supporting excellent smooth
muscle and endothelial cell growth, leading to better cell
proliferation. We also show that the serum albumin adsorption
on GrNiTi is higher than fibrinogen, an important and well
known criterion for promoting a lower thrombosis rate. In
addition, (i) our detailed spectroscopic measurements confirmed
the lack of charge transfer between graphene and fibrinogen,
suggesting that graphene coatings inhibit platelet activation on
the surface of the implants; (ii) graphene coatings do not exhibit
any significant in vitro toxicity for endothelial and smooth muscle
cell lines, confirming their biocompatibility; and (iii) graphene
coatings are chemically inert, durable and impermeable in an
environment that is more severe compared to that of the blood
flow environment. These hemo- and biocompatible properties,
along with high strength, chemical inertness and durability,
a
Brody School of Medicine, East Carolina University, Greenville, NC, 27834, USA
b
Department of Physics and Astronomy, Clemson University, Clemson, SC, 29634,
USA. E-mail: arao@g.clemson.edu
c
Department of Bioengineering, Clemson University, Clemson, SC, 29634, USA.
E-mail: falexis@clemson.edu
d
Center for Optical Materials Science and Engineering Technologies, Clemson
University, Clemson, SC, 29634, USA
3
Electronic supplementary information (ESI) available. See DOI: 10.1039/c2ra23073a
RSC Advances
COMMUNICATION
1660 | RSC Adv., 2013, 3, 16601665 This journal is The Royal Society of Chemistry 2013
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provide graphene with an edge over most antithrombogenic
coatings for use in manufacturing biomedical implants and
devices.
Experimental
The graphene samples used in this study were grown on Cu
substrates using a chemical vapor deposition technique, and
subsequently transferred to 4.5 mm
2
NiTi substrates. Briefly, Cu
foils (1 cm 6 1 cm) were placed in a 1 inch quartz tube furnace
and heated to 1000 uC in the presence of 50 sccm of H
2
and 450
sccm of Ar. Next, CH
4
(1 and 4 sccm) was introduced into the
furnace at different flow rates for 2030 min. The samples were
finally cooled to room temperature under flowing H
2
, Ar and CH
4
.
Next, the Cu foils were spin-coated with poly(methyl methacrylate)
(PMMA diluted with 4% anisole) at 4000 rpm followed by a heat
treatment for 5 min at 150 uC. Graphene attached to the PMMA
layer was obtained by etching the Cu foil using Transene Inc. CE-
100 etchant, and subsequent rinsing with 10%HCl followed by de-
ionized water for 10 mins. The samples were transferred to NiTi
substrates (4.5 mm
2
) and annealed at 450 uC in Ar (300 sccm) and
H
2
(700 sccm) for 2 h to remove most of the PMMA. Finally, the
substrates were washed with acetone to dissolve the residual
PMMA to obtain the GrNiTi sample. A Dilor XY triple grating
monochromator was used for the micro-Raman characterization
(using the 1006 objective) of all GrNiTi samples with 514.5 nm
excitation from an Ar
+
ion laser.
For etching experiments, GrNiTi or graphene/Cu substrates
were exposed to 70% nitric acid until the NiTi/Cu was partially
etched away. The etch rate was measured by calculating the
surface area of copper substrates etched per minute.
Rat aortic endothelial cells (Cell application Inc.) were cultured
on a gelatin coated 8 chamber slide. For testing the cell growth,
pristine and GrNiTi substrates were placed in wells without any
gelatin coating. Scanning electron microscopy images were
obtained using a Hitachi S-4800 SEM. Additionally, rat aortic
smooth muscle cells were also grown in CellBind 96-well plates as
a control group (Corning) in Dulbeccos Modified Eagle Medium
(ATCC).
For testing cell viability, cells (both endothelial and
smooth muscle cells) were seeded at 10
4
cells/well in wells
containing pristine NiTi, 1 sccm or 4 sccm GrNiTi
substrates, in which the stated sccm corresponds to the
methane flow used in the CVD growth of graphene. Cells were
grown for the desired time period in an incubator at 37 uC
and 5% CO
2
, exchanging media every other day. For the MTT
assay, medium was removed and fresh media containing 0.5
mg ml
21
thiazolyl blue tetrazolium bromide (or MTT
obtained from Sigma) was added to each well. Cells were
then incubated for an additional 3 h. Next, media were gently
removed and 100 ml of dimethylsulfoxide (Sigma) was added
to each well. After allowing 10 min for the MTT crystals to
dissolve, the solution was transferred to another well plate.
Absorbance was read at 490 nm and the percent viability was
determined by normalizing the averaged absorbance to that
of the pristine NiTi sample. Five repeats were done for each
sample type. CellTiter 96 Aqueous One Solution Cell
Proliferation Assay (Promega, CA, USA) was used to assess
rat aortic endothelial cell viability. Cell culture supernatant
was removed and 100 ml of phenol red free Dulbeccos
Modified Eagles Medium (DMEM) with 20 ml of the MTS
reagent was added to each cell. The cells were incubated at 37
uC in 5% humidified CO
2
for 30 min. A Thermo Scientific
plate reader was used to measure the absorbance at 490 nm.
Cellular viability was calculated using the equation: %
Cellular Viability = 100 6 Test Sample Absorbance/Control
Absorbance.
For confocal imaging of rat aortic smooth muscle cells,
substrates were placed in an 8-chamber slide (Thermo
Scientific). The cells were seeded at 25 000 cells/chamber and
incubated for 3 days at 37 uC and 5% CO
2
. Cells were fixed on the
substrate with 4% formaldehyde in phosphate buffered saline,
and made permeable with 0.1% Triton-X. Actin was stained with
Alexa Fluor 488 phalloidin (Life Technologies) and nuclei were
stained by mounting in fluorescent mounting medium containing
DAPI (Vector Labs). Confocal images were collected using a Nikon
Confocal TI. A chamber seeded with cells without any substrate
was used as a control.
Substrate dimensions were measured with calipers before
starting the protein adsorption experiments. Three measurements
were taken for each side of the approximately square samples and
averaged to get the length and width. Each sample, pristine NiTi, 1
sccm and 4 sccm GrNiTi were incubated with 1 mg ml
21
albumin in phosphate buffered saline (PBS) or 1 mg ml
21
fibrinogen in PBS at room temperature for 3 h. Similar samples
were combined in a microcentrifuge tube with 200 ml of reducing
sample buffer and boiled for 5 min. Samples were then diluted in
a Tris/Glycine/SDS buffer (Bio-Rad) and run through a 415% Tris
polyacrylamide electrophoresis gel (Bio-Rad) at 90 V for 100 min.
Gels were then stained with SYPRO Red (Life Technologies) and
imaged using FluorChem SP imaging equipment. The fluores-
cence intensity from each sample was quantified using ImageJ
software, and normalized to the total area of substrate. Fibrinogen
adsorption was compared to albumin adsorption.
Western blotting was performed to analyze total actin in rat
aortic smooth muscle cells. Cells were seeded at 10
4
cells/substrate
in a 96-well plate and grown for three days before the media was
removed. The total protein was extracted using an RIPA buffer and
a standard BCA assay (Lamda) was performed to quantify the total
amount of protein. Samples were diluted to the identical RIPA
concentrations and then boiled in a reducing sample buffer for 5
min. Proteins were separated by a 415% Tris polyacrylamide gel
via electrophoresis at 90 V for 100 min, and a kaleidoscope protein
standard (Bio-Rad) was used to assess protein molecular weight.
Proteins were then transferred to a PVDF membrane and blocked
with a 1% non-fat dry milk (Bio-Rad) solution. Total actin was
tagged with a rabbit anti-rat actin antibody (Sigma), and a BM
chemiluminescent kit (Roche) was used to detect the primary
antibody. Membranes were imaged using FluorChem SP imaging
equipment and fluorescent intensity was measured using ImageJ
software. Fluorescent intensity was normalized relative to the
pristine NiTi sample.
This journal is The Royal Society of Chemistry 2013 RSC Adv., 2013, 3, 16601665 | 1661
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Results and discussion
Fig. 1a shows an optical image of a chemical vapor deposition
(CVD) grown polycrystalline graphene sample on a Cu foil (25 mm
thick) with evidence of crystal grains of Cu. Using micro-Raman
spectroscopy we confirmed that the as-grown film on the Cu foil is
either a monolayer or few-layer graphene when 1 sccm and 4 sccm
methane flow rates are used in our CVD experiments. Clearly, the
1 sccm (4 sccm) sample exhibits an intense (relatively weaker) G9
band indicative of monolayer (few-layer) graphene (Fig. 1b). Fig. 1c
shows an atomic force microscopy (AFM) image of a few-layer
graphene sheet that was transferred using well established
methods (see Methods for further details) onto NiTi substrates
to yield a smooth GrNiTi with a surface roughness R
q
= 5 nm. It is
well known that the surface topography strongly affects the cell
shape and cytoskeletal assembly in endothelial cells and smooth-
muscle cells. Here, the cell lines respond to the mechanical
stresses by changing their lipid-bilayer fluidity, which may
adversely affect protein translocation and the entry of activators
such as calcium into the cells. More importantly, an increase in
the cell membrane stress gradient can change both the
conformation and density of cell surface receptors. In order to
test the influence of graphene on the stress gradients of cells, we
used confocal optical microscopy to determine smooth muscle
and endothelial cell morphology.
As shown in Fig. 2b, a non-spherical smooth muscle cell (SMC)
morphology was observed on pristine NiTi when compared to its
morphology on a glass slide (Fig. 1a). Further, the cells are sparsely
spread indicating the weak adhesion of SMCs to pristine NiTi.
Conversely, SMCs on the GrNiTi (both 1 and 4 sccm) surfaces are
dense and spherical in shape, similar to the control. Thus it is
evident that the graphene coating, similar to that depicted in
Fig. 1, is capable of considerably reducing the stress gradients in
the cells and lead to a better cell morphology compared to non-
coated NiTi. In order to measure the cell viability and proliferation,
we performed an MTT assay on the SMCs grown on pristine and
GrNiTi substrates over durations of three and seven days
respectively. Here, the MTT dye (yellow color) was reduced into
formazan dye (purple color) by the active reductase enzymes, thus
permitting quantification of the healthy and proliferating cells (or
the materials cytotoxicity) via colorimetric measurements. The
data in Fig. 3a indicates little evidence of excess toxicity of GrNiTi
substrates over NiTi at the end of the three and seven day cycles
thus confirming that graphene, in addition to supporting cell
proliferation, does not induce excess toxicity compared to the
pristine NiTi substrates.
To further confirm the excess non-toxicity of graphene over
NiTi, we also performed detailed MTS 3-(4,5-dimethylthiazol-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay
(Fig. 3b) and electron microscopy studies (Fig. 4) using rat-aortic
endothelial cells (RAECs). The outcome of these studies was akin
to that discussed in Fig. 2 and 3a for SMCs. Here, the RAECs on
pristine NiTi were sparse and elongated but ellipsoidal and dense
on the GrNiTi, confirming the reduction in stress gradients
provided by both the graphene coating, and the absence of excess
toxicity from the graphene coating for RAECs. The rationale for
using the MTS assay (instead of MTT) lies in its greater
compatibility with the RAEC growth media and conditions.
Interestingly, both the 1 sccm and 4 sccm GrNiTi exhibited a
similar response to two different cell lines, implying the lack of
dependence of cell morphology and viability on the number of
graphene layers and the cell type.
Fig. 1 a) A typical optical image of a CVD grown polycrystalline graphene on a Cu foil (scale bar: 10 mm); b) Micro-Raman spectra of single- and few-layer graphene prepared
using 1 sccm and 4 sccm flow rates of methane, respectively. As expected, the G9 band intensity is greater in the micro-Raman spectrum of the single-layer graphene. c) An
AFM image of few-layer graphene transferred onto a NiTi substrate exhibits a surface roughness of y5 nm. Scale bar = 500 nm.
Fig. 2 Confocal optical microscopy images for SMCs grown on a) control glass slide,
b) pristine NiTi, c) 1 sccmGrNiTi and d) 4 sccmGrNiTi substrates (scale bar: 50 mm).
1662 | RSC Adv., 2013, 3, 16601665 This journal is The Royal Society of Chemistry 2013
Communication RSC Advances
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Blood clotting in the vicinity of implant materials has been a
major hindrance in the development of effective implant
technologies. As mentioned earlier, the implant material triggers
a coagulation cascade upon contact with blood. The interaction
between biomedical implants and blood begins with the adsorp-
tion of plasma proteins (serum albumin, fibrinogen, etc.) on its
surface. Initially, highly abundant proteins (serum albumin,
fibrinogen, and fibronectin) are adsorbed and subsequently
replaced by factor XII and high molecular weight kininogen. The
ratio of adsorbed fibrinogen to albumin is crucial in the
determination of the hemo-compatibility of the biomedical
implant. A low ratio of fibrinogen/albumin adsorbed on the
biomedical implant surface has been correlated with low platelet
adhesion and thrombus formation.
1
As shown in Fig. 5a, GrNiTi
exhibits a low fibrinogen/albumin ratio relative to pristine NiTi,
suggesting better hemo-compatibility arising from graphene. The
fibrinogen/albumin ratio was significantly lower for both 1 and 4
sccm GrNiTi indicating that the hemo-compatibility of graphene
is layer-independent.
We next address the underlying mechanism that is responsible
for the low fibrinogen/albumin ratio exhibited by 1 and 4 sccm
GrNiTi in Fig. 5a. It is well known that an electron transfer from
fibrinogen molecules to the implant material is responsible for the
formation of fibrin as a first step towards thrombus growth, and
subsequent failure of the implant.
1
As depicted in Fig. 5b,
fibrinogen exhibits a semi-conductor-like density of electronic
states with an energy gap of 1.8 eV. The Fermi levels (E
F
) of
fibrinogen and GrNiTi equilibrate at their interface. An electron
transfer from the fibrinogen molecule to the GrNiTi is only
possible from the occupied electronic states of the fibrinogen
molecule into empty electronic states of GrNiTi at the same
energy level. Both single- and few-layer graphene are semi-metals
at roomtemperature with a lowdensity of states r(E) in the vicinity
of E
F
.
24
Thus, the electron transfer from fibrinogen to graphene is
insignificant due to low values of r(E). This intrinsic property of
graphene coatings is crucial for inhibiting any charge transfer
from fibrinogen (and subsequent blood clotting).
We employed micro-Raman spectroscopy to confirm that the
charge transfer dynamics between fibrinogen and GrNiTi is
indeed insignificant. The Raman spectrum of graphene exhibits
several sharp features due to resonance effects. Notably, the
tangential band (G-band) arises from the planar vibration of
carbon atoms and was previously found to be highly sensitive to
charge transfer.
25
The G-band frequency is known to upshift
(downshift) when any acceptor (donor) species interacts with
Fig. 4 Scanning electron microscopy images for RAECs grown on a) pristine NiTi, b)
1 sccm GrNiTi, and c) 4 sccm GrNiTi substrates. These images indicate that the
graphene coating results in better cell morphology for RAECs. Scale bar = 10 mm.
Fig. 5 a) Fibrinogen/albumin ratio for pristine NiTi, GrNiTi (1 and 4 sccm samples);
b) electronic density schematic of states for fibrinogen and graphene showing the
equilibration of the Fermi level E
F
(dashed line) and the energetically unfavored
charge transfer from fibrinogen. Fig. 3 a) A MTT assay for SMCs showing the absence of graphene-induced excess
toxicity compared to pristine NiTi; b) The three-day cell viability results for RAECs in a
MTS assay.
This journal is The Royal Society of Chemistry 2013 RSC Adv., 2013, 3, 16601665 | 1663
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graphene via hole (electron) transfer. Importantly, the lineshape of
the G-band deviates from a symmetric Lorentzian to an
asymmetric BreitWignerFano (BWF) lineshape in the presence
of a charge transfer.
25
Upon fibrinogen adsorption on GrNiTi, no
shift in the G-band frequency of graphene was found (see
supplementary information Fig. S13), thus confirming the absence
of charge transfer between Gr-NiTi and fibrinogen. Such inhibition
of charge transfer and low fibrinogen/albumin ratio indicate good
hemo-compatibility of graphene coatings.
Lastly, we address the permeability and durability of graphene
coatings in an environment that is more severe compared to that
of the flowing blood environment. To this end, we refer to Fig. 6a
in which the graphene coated portion of the coin (y95% Cu)
remains protected from oxidation when exposed to H
2
O
2
while the
bare region of the coin is discolored upon contact with 5% H
2
O
2
(see the magnified optical microscope image in Fig. 6a).
Graphenes sp
2
honeycomb lattice serves as a natural diffusion
barrier and is expected to prevent metal ion leaching from the
implant material.
26,27
We also exposed GrNiTi substrates to 70%
nitric acid until the NiTi was partially etched away. Our in situ
Raman spectroscopy of GrNiTi immersed in HNO
3
showed no
change in the D- and G-band frequencies of graphene implying
that the graphene coating is extremely durable (Fig. 6b).
Furthermore, we find that the graphene coating in GrNiTi
reduces the etch rate of the underlying copper, as shown in Fig. 6c.
Conclusions
In summary, our detailed spectroscopic measurements confirmed
the lack of charge transfer between graphene and fibrinogen,
suggesting that graphene coatings inhibit platelet activation by
implants. Additionally, graphene coatings do not exhibit any
significant in vitro toxicity for SMCs and RAECs, confirming their
biocompatibility. Further, graphene coatings were found to be
chemically inert, durable and impermeable in an environment
that is more severe than the flowing blood environment. All of
these characteristics make graphene superior to other coatings for
biomedical implants and devices.
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1664 | RSC Adv., 2013, 3, 16601665 This journal is The Royal Society of Chemistry 2013
Communication RSC Advances
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RSC Advances Communication
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