Materials Today Communications 26 (2021) 102056

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Materials Today Communications

journal homepage: www.elsevier.com/locate/mtcomm

Adhesive properties of graphene oxide and its modification with RGD

peptide towards L929 cells
Joanna Jagiełło a, *, Marcin Kuśmierz b, Ewa Kijeńska-Gawrońska c, d,
Magdalena Winkowska-Struzik a, Wojciech Święszkowski d, Ludwika Lipińska a
a
Łukasiewicz Research Network- Institute of Microelectronics and Photonics, 133 Wólczyńska Str., 01-919 Warsaw, Poland
b
Maria Curie-Skłodowska University, Institute of Chemical Sciences, Faculty of Chemistry, 3 Maria Curie-Skłodowska Square, 20-031 Lublin, Poland
c
Warsaw University of Technology, Centre for Advanced Materials and Technologies CEZAMAT, 19 Poleczki Str., 02-822 Warsaw, Poland
d
Warsaw University of Technology, Faculty of Material Science and Engineering, 141 Wołoska Str., 02-507 Warsaw, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: Properly prepared substrate for cell culturing is a crucial factor to maintain good viability and proliferation of the
Graphene oxide cells. Graphene oxide (GO), due to its physicochemical properties, can be easily modified with biomolecules,
RGD peptide which enable the interaction with different types of cells. Moreover, highly hydrophilic GO flakes itself can
Biocompatibility
provide good conditions for cell culture. The aim of the study was to determine the adhesion ability of GO to­
L929 cells
Adhesion
wards L929 cells by comparing the morphology and viability with the results obtained for cell culturing on GO
modified with the Arg-Gly-Asp (RGD) peptide and standard tissue culture polystyrene well plates. This work
presents the routes of efficient modification of GO with RGD molecules. This peptide is present in proteins
building chondrocytes, fibroblasts, macrophages, and extra- and intracellular matrices and is responsible for
binding the cell integrins. Successful functionalization was conducted with the use of carbodiimide (EDC⋅HCl)
and N-Hydroxysuccinimide (NHS) that activated the GO surface for binding molecules (peptides) with amine
groups. Samples were characterized with SEM, Raman, FTIR and XPS spectroscopy. One step functionalization
revealed as a very effective method for peptide immobilization on the GO giving ca. 71 μg/cm2 of RGD attached
to the GO surface. On the basis of these studies, the materials for cellular research were selected. Morphology and
viability/proliferation of L929 cells incubated on the prepared graphene substrates were investigated. Biocom­
patibility of each of the tested materials was found on the basis of adhesion and viability tests. GO, because of
oxygen functional groups present on its surface, led to a better cellular response in comparison to control glass
slides and standard polystyrene well plates, making this material a competitive substrate for cell culturing. The
presence of RGD on the GO surface did not affect the viability of L929 cells, which leaded to the conclusion about
good adhesive properties of GO towards the cells. Based on the presented work, it can be concluded that GO is a
material that can be successfully used for cell culture and can be competitive with standard polystyrene substrate
due to the possibility of its modification according to specified needs.

1. Introduction amphiphilicity, honeycomb structure and surface chemistry) of GO

In the field of biomedicine, more and more attention is being paid to
graphene derivatives, such as graphene oxide and reduced graphene
oxide (rGO). Their defected structure with functional oxygen groups
attached to the surface leads to various properties suitable for many
medical applications [1,2]. Moreover, ease of the fabrication procedure
and the facility of their modification are huge advantages that make GO
and rGO more accessible. The physicochemical features (including
provide desired interactions with biomolecules and different cell types.
Graphene properties can be tuned more easily than other materials and,
for this reason, it gives better opportunities to mimic the cellular
microenvironment to maintain pluripotency, and to enable attachment
and proliferation of cells. Therefore, among the multitude of available
materials, oxidized graphene flakes have resulted in a strong interest in
the development of new biomaterials.
The distinct features of GO and rGO were found to enhance

* Corresponding author.
E-mail address: Joanna.Jagiello@imif.lukasiewicz.gov.pl (J. Jagiełło).

https://doi.org/10.1016/j.mtcomm.2021.102056
Received 10 August 2020; Received in revised form 14 December 2020; Accepted 8 January 2021
Available online 20 January 2021
2352-4928/© 2021 Elsevier Ltd. All rights reserved.
J. Jagiełło et al.
osteogenesis [3–9], neurogenesis [10–14], adipogenesis [15,16] and to
improve scaffolds for cardiac tissue engineering [17–19]. Among others,
this was due to good adhesion of the cells leading to their great
spreading without a morphological disorder. The adhesive properties
towards cells and biocompatibility can even be improved by binding
proteins, such as collagen [20,21] and fibronectin [22,23] or by binding
peptides. In this work, tripeptide Arg-Gly-Asp (RGD) built of arginine,
glycine and asparagine (amino acids) molecules was chosen to fabricate
new composites with GO as substrates for L929 cell cultures.
The RGD peptide was used because its sequence plays an essential
role in the binding of cell integrins [24]. Some of these integrins can
recognize the exact amino-acid sequences in protein ligand molecules.
RGD can be recognized by integrin cell receptors even in a synthetic
form. RGD occurs, for example, in fibronectin – a protein present on the
surface of chondrocytes, in fibroblasts, and in macrophages as well as in
the intracellular and extracellular matrices of connective tissues and
biological fluids.
The literature provides information about application of RGD pep­
tide in the composites of antitumor action as well as for cell growth
promotion. For example, CTAB-GNRs (hexadecyltrimethylammonium
bromide-gold nanorods) were functionalized by an RGD-HK (His-Lys
sequence) peptide to target the overexpressed αvβ3 integrin in
cancerous cells, increase the biocompatibility, and gain the ability of
gene/drug delivery. It was stated that the RGD peptide promoted
cellular uptake of the GNRs [25]. In another paper, to improve ECs
(human endothelial cells) adhesion and growth on chitosan and
chitosan-TPP (tripolyphosphate) films, cell adhesive peptide GRGD
(Gly–Arg–Gly–Asp) was photochemically grafted to its surface. The
adhesion and growth of ECs in chitosan and chitosan-TPP surfaces was
poor, while grafted GRGD on those surfaces did promote the growth of
ECs. Even though polystyrene culture well showed the highest relative
growth rate, GRGD grafted surfaces did enhance the rates by about 50 %
in comparison to the of ungrafted surfaces [26]. Similar work was
conducted to improve ECs adhesion and growth on the surface of
polyethylene glycol modified polyurethane (PU-PEG), by grafting cell
adhesive peptide GRGD to the surface. Human endothelial cells were
well adhered and grown on the PU-PEG-GRGD surface and the
enhancement efficacy for the growth of ECs was well correlated with the
surface concentration of GRGD [27].
Molecules of the RGD peptide are much smaller than fibronectin and
therefore its attachment to the GO layer can be conducted in a more
controlled way. In this work, GORGD was synthesized using different
routes. All of them included a step that involved the use of EDC and NHS
compounds as zero-length linkers which activate carboxyl groups for
covalent bonding of amine (I) groups. Activation commonly consists of
forming a reactive NHS-ester by a two-step reaction between the car­
boxylic acid and a carbodiimide. After EDC and NHS addition to car­
boxylic acids, the initial intermediate O-acylisourea is formed, followed
by a reaction between O-acylisourea and NHS to yield the activated
NHS-ester [28].
2. Materials and methods
2.1. Materials
The compounds used for GO synthesis were: graphite (carbon flakes
with an average size of 300–425 μm), sulphuric acid (POCH S.A. 96–98 %,
pure p. a.), orthophosphoric acid (Chempur, pure p.a.), potassium per­
manganate (Chempur, pure p.a.) and perhydrol (Chempur, pure p.a.). The
following compounds were purchased from Sigma Aldrich: chloroacetic
acid, sodium hydroxide, N-Hydroxysuccinimide (NHS), N-(3-Dimethyla­
minopropyl)-N′ -ethylcarbodiimide hydrochloride (EDC⋅HCl), and Arg-
Gly-Asp peptide (RGD), fluorescamine and dimethyl sulfoxide (DMSO).
The following were used for cell culture: Medium RPMI 1640, penicillin-
streptomycin solution (Gibco, Thermo Fischer Scientific, UK), heat-
inactivated fetal bovine serum (Biowest, Europe), Trypsin-EDTA
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Materials Today Communications 26 (2021) 102056
solution, glutaraldehyde (Sigma Aldrich, Poland), and MTS assay
(Promega, Poland). The L929 fibroblast cell line was purchased from the
ATCC company.
2.2. Methods
2.2.1. Characterization methods
The morphology of the materials was examined using SEM micro­
scopy (Auriga CrossBeam Workstation, Carl Zeiss) and AFM microscopy
(FastScan, Bruker). The chemical structure was studied with Raman
spectroscopy (Renishaw inVia, excitation laser source of 532 nm and
laser power below 1 mW), FTIR spectroscopy (VERTEX 80v, Bruker) and
XPS spectroscopy (UHV Multi-chamber XPS System, PREVAC). Immu­
nostaining analysis was carried out using a Laser Confocal Microscope
(DM 600, Leica, USA). Quantification of immobilized RGD peptide was
investigated with a multi-mode microplate reader (Synergy 2, BioTek)
and the viability and proliferation of L929 cells with a spectrophoto­
metric plate reader (FLUOstar Omega, BMG Labtech).
2.2.2. Graphene oxide (GO) preparation
GO was prepared by a modified Hummers method [29]. In brief:
graphite flakes were added gradually to the reactor containing
concentrated sulphuric acid (H2SO4) and orthophosphoric acid (H3PO4).
After that, potassium permanganate (KMnO4) was slowly added in
excess to graphite weight. The oxidation process was conducted for a
few hours and was stopped by the addition of deionized water followed
by a perhydrol (30 % H2O2). The water suspension of the obtained
graphite oxide was left to sediment. The purifying process was carried
out via centrifugation and dialysis. Due to specific shearing forces acting
on graphite oxide flakes during purification, exfoliation lead to gra­
phene oxide formation.
2.2.3. Graphene oxide functionalization with RGD peptide
Functionalization was carried out on thin layers of GO deposited on
15 mm diameter glass slides compatible with polystyrene (PS) 24-well
plates, as well as directly on PS 24-well plates (an area of one well is
1.9 cm2). To get uniformly covered glasses and wells, water from GO
suspensions was replaced with ethanol and the ethanol GO suspension
was drop-casted in the appropriate amount to obtain the concentration
10 μg per cm2. It was then left at RT until the ethanol evaporated. This
concentration was used based on previous studies [30], which showed
that such concentration was the best for cell cultures. Modification was
conducted according to two different routes: (a) two-step and (b)
one-step functionalization. The first route (a) involves a simultaneous
use of EDC and NHS followed by RGD addition. In the second route (b),
the mixture of EDC, NHS and RGD water solution was applied on the GO
layer.
2.2.3.1. Two-step functionalization. The experiment was carried out
according to the following procedure: 200 μl of EDC⋅HCl (0.3 μg/μl) and
NHS (0.25 μg/μl) water solution was applied on a pristine GO layer and
left for 24 h to react. Then the above solution was removed and the
modified GO layer was washed with ultrapure water. Afterwards, 200 μl
of 0.5 μg/μl of RGD water solution was applied on this layer and left for
next 24 h. After the specified time had elapsed, the RGD solution was
removed and functionalized GO layer was washed with ultrapure water.
The sample was named GO-EDC-RGD. The second sample was prepared
with 30-times higher concentration of EDC⋅HCl and NHS in comparison
to the first one, i.e. 10 μg/μl and 7.5 μg/μl, respectively, while concen­
tration of RGD was unchanged. The sample was named GO-EDC30-
RGD.
2.2.3.2. One-step functionalization. Based on FTIR measurements of the
materials produced according to the two-step functionalization, the
higher concentration of EDC⋅HCl and NHS was chosen for the simplified
J. Jagiełło et al.
one-step functionalization procedure (following GO-EDC30-RGD sample
concentrations): 200 μl of water solution of EDC⋅HCl, NHS and RGD
mixture was applied on GO layer and left to react for 24 h. Non-bonded
molecules was removed by washing the layers with ultrapure water.
Prepared sample was marked as GO-EDC30-RGDmix.
The additional experiment was carried out for 4-times higher con­
centration of RGD: 2.0 μg/μl following the same above procedure and
was labelled GO-EDC30-RGD4mix.
The schematic reaction of GO functionalization with RGD in the
presence of EDC and NHS compounds can be depicted as follows:
2.2.3.3. Quantification of immobilized RGD peptide on the GO surface.
For the GO-EDC30-RGD4mix sample, the mount of immobilized RGD
peptide was quantified with the use of fluorescamine assay [31]. Briefly,
50 μl of fluorescamine solution (4 mg/mL in DMSO) was added to the
mixture of NHS, EDC⋅HCl and RGD after it reacted with GO layer. After
short vortexing, fluorescence intensity was measured using the plate
reader (390 nm of excitation and 475 nm of emission wavelength). The
measured intensity corresponded to the amount of non-bonded RGD
peptide. The amount of peptide immobilized on GO surface was calcu­
lated from the difference between the amount of added and unbound
peptide. Standard calibration curve was obtained from the fluorescence
intensity of known concentrations of RGD peptide (0− 2 μg/μl).
2.2.4. Biological studies
2.2.4.1. Cell culture and seeding. L929 fibroblast cells were cultured in
RPMI 1640 media supplemented with 10 % FBS and 1% penicillin-
streptomycin solution in a 75 cm2 cell culture flask until sufficient
confluence was attained. Further, cells were detached from the flask
with a 0.05 % trypsin-EDTA solution and counted under an optical mi­
croscope using a hemocytometer. Cells were seeded on 24-well plates
and glasses with deposited materials previously prepared according to
the described methods. The cells were incubated for 24, 48 and 72 h at
37 ◦ C in an atmosphere of 5% CO2.
2.2.4.2. Cell morphology. The morphology of L929 fibroblast cells was
investigated with the use of SEM. After 72 h of culturing, the cells were
fixed with 3% glutaraldehyde (3 h at RT) and rinsed three times with
distilled water. Then the dehydration process was conducted with
increasing concentrations of ethanol (50, 70, 90 and 100 %, 15 min for
each rinse) and finally treated with HMDS (hexamethyldisilazane). The
samples were kept at RT overnight for drying. Prior to SEM observations,
the samples were sputter-coated with gold.
2.2.4.3. Cell viability/proliferation. The cell adhesion and viability/
proliferation studies were performed using colorimetric MTS assay ([3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo­
phenyl)-2H-tetrazolium] inner salt). The principle of this assay is a
3
Materials Today Communications 26 (2021) 102056
reduction of yellow tetrazolium salt and formation of purple for­
mazonum crystals, which is caused by mitochondrial enzyme dehydro­
genase secreted by metabolically active cells. The number of viable cells
is directly proportional to the absorbance at 490 nm. MTS assay tests
were conducted after 24, 48 and 72 h of cell culturing. Samples were
rinsed with PBS to remove unattached cells, followed by incubation with
20 % MTS reagent in a bovine serum-free media (RPMI 1640) for 3 h at
37 ◦ C. The absorbance of formed formazan dye was measured using a
spectrophotometric plate reader at a wavelength of 490 nm.
2.2.4.4. Immunostaining. In order to investigate the cells’ morphology,
immunostaining of actin cytoskeleton was performed. The samples were
washed three times with PBS, fixed with ice-cold methanol for 30 min at
20 ◦ C followed by permeation with 0.1 % TritonX-100 for 1 min.
Nonspecific labeling was blocked by incubating the samples in 2% wt.
BSA (bovine serum albumin) solution. Further, the samples were incu­
bated with Alexa Fluor Phalloidin (diluted at 1:40) for 2 h. Samples were
then rinsed three times with PBS (5 min each) and DRAQ5 (diluted at
1:500) was added for 15 min in order to stain cell nuclei. After washing
thrice with PBS (10 min each) samples were observed under a confocal
microscope (DM600, Leica, USA).
2.2.4.5. Statistical analysis. All the experiments were repeated at least
three times and the standard deviation (SD) is shown in the figures.
Statistical analysis was carried out using ANOVA followed by the Tukey
test. A value of p ≤ 0.05 was considered statistically significant.
3. Results
3.1. Physicochemical characterization of graphene materials
The analysis of the SEM image (Fig. 1 A) showed that the average
lateral size of the GO flakes was in the range of 20–80 μm. No agglom­
erates were visible in SEM observations. The thickness, and thus the
estimated number of GO layers, was examined using atomic force mi­
croscopy (AFM). The colors (shades of green, color online) of the map in
Fig. 1B () correspond to the thickness of the GO flakes – increased
thickness (lighter colors) is observed due to their overlapping and the
creation of wrinkles. The thickness of flakes ranges from 0.8 to 4.0 nm.
Knowing that the measured thickness of pure graphene (exfoliated or
CVD) is between 0.4 and 1.7 nm [32] and taking into account functional
groups on the GO surface which contribute to the total thickness, it can
be assumed that GO is of 1–4 layer thickness [33].
In Fig. 2 A Raman spectra is presented for natural graphite flakes.
The two most intense features are centered at ~1582 cm− 1 (G peak) and
~2700 cm− 1 (2D band). The G peak is always observed in graphite
samples and corresponds to stretching mode of C–C chains within
carbon structure. The G peak is due to doubly degenerate Brillouin zone
J. Jagiełło et al. Materials Today Communications 26 (2021) 102056

Fig. 1. SEM image of GO (graphene oxide) flakes deposited on Si substrate (A) and the thickness profiles of the GO flakes obtained from Atomic Force Microscopy
(AFM) measurements (B).

Fig. 2. Raman spectra of the graphite (A) and GO (graphene oxide) flakes (B).

Fig. 3. FTIR spectra of the graphite (A) and GO (graphene oxide) fakes (B).

4
J. Jagiełło et al.
center, while 2D mode is the second order of zone-boundary phonons. In
Fig. 2 A there is no feature around 1350 cm− 1, corresponding to first-
order zone-boundary phonons. Appearing of D peak is related to de­
fects in carbon chicken-wire structure and is visualized as breathing
mode of carbon aromatic rings. The 2D mode of bulk graphite consists of
two components 2D1 and 2D2, about 25 % and 50 % the height of G
peak, respectively [34]. The analysis of Raman spectra of GO sample
(Fig. 2 B) showed that in the contrary to graphite there is a D mode
appearing about 1355 cm− 1, which is related to defects in a carbon
structure. The intensity ratio of those two main features (D and G peaks)
ID/IG is about 0.91. The G band is blueshifted (1590 cm− 1) and asym­
metric in comparison to graphite which can be explain with
defect-related D’ mode appearance around 1610 cm− 1 [35,36]. The
visible background originates from GO fluorescence. The peak between
D and G (D**) at around 1490 cm− 1 can be assigned to sp3 hybridization,
amorphous structure or isolated epoxide groups [37,38].
FTIR measurements of the materials deposited on glass supports
were made using the ATR technique. The spectrum of GO is shown in
Fig. 3 B. The two bands presented at 1730 cm-1 and 1630 cm− 1 corre­
spond to C– – O, − COOH and C– – C stretching and bending vibrations,
respectively, while peaks at 1225 cm− 1 and 1060 cm− 1 originate from
(CC)>O (epoxides) and C–OH, respectively. CH3/CH2, as well as
adsorbed water, can be assigned to 1380 cm-1 band [39]. Because there
are no oxygen-related peaks on the FTIR spectrum of graphite surface
(Fig. 3 A), the FTIR results of GO indicates that the oxidation of graphite
flakes occurred during the reaction carried out according to the modi­
fied Hummers method.
FTIR measurements were carried out to verify the presence of RGD
peptide on the GO surface after chemical synthesis. In the FTIR spectra
Fig. 4. FTIR spectra of the GO (graphene oxide) modified with RGD (Arg-Gly-Asp) peptide according to three described routes (A) and comparison of the FTIR
spectra between GO modified with RGD peptide of two different concentrations (B), spectrum of non-modified RGD peptide (C).
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Materials Today Communications 26 (2021) 102056
shown in Fig. 4 A, there are bands at 1745, 1645 and 1064 cm− 1
attributed to C–– O, C–– C and (CC)>O, C–OH vibrations, respectively,
that are also characteristic for the non-modified GO sample (Fig. 4 A).
The amide I band (between 1600 and 1700 cm− 1) is mainly associated
with the C– – O stretching vibration and is related to the backbone
conformation. The most important difference appears at 1542 cm− 1,
where the band attributed to N–H bending vibration and C–N
stretching vibrations (in amide II) appears in the spectrum of the
modified GO [40,41]. These characteristic peaks are present in the
spectrum of pure RGD peptide sample shown in the Fig. 4 C (at
1672 cm-1 and 1548 cm− 1) what confirms the presence of RGD mole­
cules in RGD-modified GO samples and properly assigned bonds in their
spectra (Fig. 4 A and B). The amide A (about 3300 cm− 1) and amide B
(about 2900 cm− 1) bands originate from a Fermi resonance between the
first overtone of amide II and the N-H stretching vibration. The presence
of these chemical groups indicates the incorporation of RGD peptide
molecules on the GO surface. From FTIR studies it can be seen that the
most effective functionalization of GO occurs when EDC⋅HCl, NHS and
RGD are mixed together (one-step functionalization). This effect is due
to self-conjugation of RGD peptide molecules containing carboxyl and
amine groups. Therefore, the addition of EDC⋅HCl and NHS not only
leads to binding molecules with -NH2 groups to GO flakes, but also acts
as zero-length crosslinkers between peptide molecules. In consequence,
it gives a stronger response under FTIR measurements in the wave­
number range corresponding to amide vibrations. Moreover, electro­
static interactions between negatively charged GO and positively
charged self-conjugated RGD chains can promote the functionalization
of GO flakes.
FTIR spectra in the Fig. 4 B show that the intensity of C–
– O vibrations
J. Jagiełło et al. Materials Today Communications 26 (2021) 102056

Fig. 5. Deconvolution of XPS C1 s spectrum of the GO (A) GO-RGD (B) and deconvolution of XPS N1 s spectrum of the GO-RGD (C); atomic content obtained from the
survey spectra of GO and GO-RGD (D); GO – graphene oxide, GO-RGD – GO modified with peptide Arg-Gly-Asp.

(1730 cm− 1) for GO modified with RGD is very low, compared to non- concentrated RGD, peaks in the region between 3200 cm− 1 and
modified GO. It suggests that part of them were involved in the reac­ 3400 cm− 1 are sharper due to O–H and N–H stretching of amide A.
tion with RGD. Both GO-modified samples are characterized by a strong Based on the results obtained from FTIR measurements, the material
absorbance at 1542 cm− 1, related to amide II due to in-plane N–H vi­ with the highest content of the peptide (GO-EDC30-RGD4mix) was
brations and stretching C–N vibrations and by the band at 1655 cm− 1, further examined by XPS spectroscopy to investigate its chemical
typical for amide I due to carbonyl and C–N groups. When comparing structure more precisely. GO-EDC30-RGD4mix is hereinafter referred to
two different concentrations of RGD solutions used for GO functionali­ as GO-RGD.
zation, it can be seen that after functionalization with a higher XPS was used to determine the chemical composition of the samples.
Fig. 5 A shows the XPS C1 s spectrum of GO. It can be decomposed into
Table 1 components corresponding to the following functional groups: sp2-hy­
Information obtained from the deconvolution of the XPS C1 s spectrum of GO bridized carbon (C– – C), sp3-hybridized carbon (C–C), hydroxyl/phe­
and GO-RGD (GO – graphene oxide, GO-RGD – GO modified with peptide Arg- nols (C OH), epoxy ((CC)>O), ether (C–O–C), carbonyl (C–
– – O) and
Gly-Asp). carboxyl (O–C– – O) groups. Carbonate ions (CO2−3 ) and defective car­
Chemical Position At. % Position for At. % Literature bon structures can also be assigned. Table 1 summarizes the atomic
groups for GO for GO GO-RGD for GO- percentages of these groups found in the sample. The obtained results
(eV) (eV) RGD indicate that the epoxide groups are the most common groups in the GO
C = C sp2 284.46 10.22 284.50 13.13 [39,42,43,44, structure (~25 %). Hydroxyl (~13 %) and ether (~17 %) are also
45] among the major oxygen groups. Carboxyl groups are of 4% at. only,
C-H 284.91 8.98 284.96 11.15 [43,46] which corresponds to their occurrence mainly on the edges of GO flakes.
C-C sp3 285.38 5.09 285.49 11.74 [39,43,44,45]
After GO functionalization (Fig. 5 B, Table 1) the carboxyl groups’
C = C-O, C- 285.48 2.29 285.90 4.95 [42,46,47]
N
C-OH 286.34 12.69 286.20 12.03 [39,43,45]
(CC)>O 286.81 24.95 286.69 16.36 [39,43,44,45] Table 2
C-O-C 287.26 16.87 287.22 12.74 [39,43,44,45] Information obtained from the deconvolution of the XPS N1 s spectrum of GO-
C=O 287.82 5.22 287.82 7.97 [39,43,44,45] RGD (GO – graphene oxide, GO-RGD – GO modified with peptide Arg-Gly-Asp).
O-C = O 288.43 4.11 288.42 3.86 [39,32,33,34,
Chemical groups [34,38, Description Position At. %
35,36,37,38,39,
42] (eV)
40,41,42,43,44,
45] >C = N-R Imine with sp2 nitrogen 397.81 5.2
Carbonate 289.10 1.66 289.02 1.44 [48] N < C5 Pyridinic nitrogen 398.83 4.4
ions C-N < R1R2, -CN Amine, nitrile 399.42 66.8
283.08 0.63 283.29 0.24 [39,49] -CN < CC, N-C = O Graphitic/amide-like 400.28 9.1
Defective
283.57 2.77 283.67 0.92 [39,49] groups
structure
284.02 4.53 284.10 3.47 [39,49] =N+< Quaternary nitrogen 401.58 14.6

6
J. Jagiełło et al.
content remains at a similar level before and after functionalization
(~4%), although part of them present on the GO surface were involved
in the creation of amide bonds with amine groups from RGD. This can be
explained by the fact that RGD molecules also contain carboxyl groups,
which gives the signal in XPS measurement. However, the atomic % of
carbon involved in C–N bonds slightly increased (from ~2.3 % for GO
to ~5% for GO-RGD). Moreover, the decrease of epoxide (~16 %) and
ether (~13 %) groups is observed, which can also suggest that some of
these highly reactive groups in GO were involved in the reaction with
RGD molecules.
Fig. 5 D provides the data of elemental composition of GO and GO-
RGD obtained from the XPS survey spectra. The atomic percentage of
nitrogen content (N1 s region) in the GO-RGD sample, which was 11.3
%, is important. The graph for the N1 s area (Fig. 5 C, Table 2) shows
that the concentration of nitrogen atoms forming amide groups (bonds
with graphene material) [42,50] for GO-RGD was 9.1 %. It can be stated
that some nitrogen atoms were built into the graphene structure,
forming pyridine rings [46]. This is possible due to the presence of point
defects in the graphene lattice of GO (in the structure of the conjugated
C–– C double bonds), which creates the conditions for the nitrogen atoms
to build into and close the 6-atom ring. However, the highest content of
N atoms concerned amine/nitrile groups (66.8 %), which is in agree­
ment with the chemical structure of RGD molecules.
Based on the results obtained from FTIR and XPS spectra, it can be
concluded that the RGD peptide molecules were effectively built into the
structure of GO flakes in the GO-RGD sample.
Scanning microscopy was used to present the morphology of GO and
GO-RGD layers before L929 cells seeding. Presented pictures of GO and
GO-RGD layers deposited on the glass substrate for cell culturing do not
show significant differences (Fig. 6). These layers are uniform with
wrinkles created by thin flakes overlapping during the solvent (ethanol)
evaporation. Therefore we can suspect that the morphology of the layers
will not be a factor influencing any differences in the vital functions of
the L929 cells incubating on these substrates. It should be mentioned
that there was no possibility to depict GO and GO-RGD layers on PS
substrate (well plates) due to its inadequate geometry for SEM method.
The amount of immobilized RGD peptide on the GO film (GO-RGD
sample) was assessed by the fluorescamine assay as a difference between
added and unbound RGD amount.
After the functionalization occurred, from 400 μg (200 μl) of RGD
solution added, 135.8 ± 11.8 μg was immobilized on the GO layer (1.9
cm2, 10 μg/cm2), what implies 71.5 ± 6.2 μg/cm2. The mass and molar
ratio of RGD to GO was of ca. 3.8 and 0.4, respectively.
3.2. In vitro cell studies
For cellular studies, GO and GO-RGD were used. Based on the pre­
vious studies [30], it can be stated that GO is biocompatible towards
human cells. The aim of these studies was to examine the adhesive
properties of GO and its modified form with the RGD peptide with
respect to the cells. This peptide, as mentioned, is involved in the
Fig. 6. SEM images of the GO and GO-RGD layers deposited on glass substrate for L929 cells seeding.
7
Materials Today Communications 26 (2021) 102056
binding of adhesive proteins and the adhesion is an essential aspect
affecting cell viability and proliferation. This research answers the
question of whether GO creates proper conditions for cellular adhesion
or whether it can be improved, introducing the specific amino acid
sequence contained in the RGD peptide.
The study of the viability of L929 cells was conducted on graphene
materials deposited on slides (glass plates) and on polystyrene (PS) 24-
well plates. The polystyrene substrate led to better adhesion of these
materials.
The viability studies (Fig. 7) show that for each sample the number of
L929 cells increases with the increasing incubation time. Each of the
tested materials is therefore considered biocompatible and does not
exhibit cytotoxicity. In the case of tests conducted with glass plates after
72 h of culture, the number of cells on the graphene materials exceeds
their number on the glass control (Fig. 7 A). Functionalization with
peptide turned out not to have a significant influence on the cell’s
viability. The above study shows that GO and its modified forms
improve the viability and proliferation of L929 cells compared to glass
substrate used as a control material.
However, the results for GO and GO-RGD are affected by an error due
to poor adhesion of these layers to the slides in the presence of a culture
medium, which made the MTS staining procedure difficult.
Biocompatibility of the materials was confirmed by tests carried out
with the use of polystyrene substrate (Fig. 7 B). The polystyrene itself
turned out to be more cell-friendly when compared to a glass substrate,
resulting in better cell growth. Also, in this case, with increasing incu­
bation time, the number of L929 cells in all tested samples increased.
After 24 and 48 h of culture, cell viability and proliferation are very
similar. Differences appear after 72 h, when pure GO material turns out
to have the best effect on the cells.
The obtained results suggest that pure GO has a very good, sufficient
capacity for cellular adhesion, even better than the polystyrene substrate
commonly used as a control material for studies of the cells.
Adhesion is a key prerequisite for cell growth and is assessed on the
basis of cell morphology and surface area. Cell proliferation is a dynamic
process that involves the stretching and proliferation of cells in all di­
rections in order to examine the environment and thus control the length
of the filopodia.
Cells imaged by scanning electron microscope (Fig. 8) show normal
morphology, good adhesion and proper coating of the substrate. In the
case of GO and especially GO-RGD, even the formation of more than one
layer of the cells can be observed. The cells have a clearly outlined, long
filopodia. Paddle elongation facilitates cell adhesion and migration.
L929 cells after 72 h of culture on GO and GO-RGD were immuno­
stained to visualize actin cytoskeleton and nuclei of the cells. GO and
GO-RGD were deposited on the PS substrate due to great adhesion of
these materials to polystyrene even during the staining procedure. The
cytoskeleton was stained with fluorescently marked phalloidin, which
binds to the actin fibers and the nuclei was visualized by staining with
DRAQ5. The images (Fig. 9) obtained from the confocal microscope
show that the elongation of the cells is very good and similar on GO
J. Jagiełło et al. Materials Today Communications 26 (2021) 102056

Fig. 7. Viability of L929 cells incubated for 24, 48 and 72 h on two forms of flake graphene deposited on slides and incubated on non-modified slides (glass plates) as
a control (A). Viability of L929 cells incubated for 24, 48 and 72 h on four forms of flake graphene deposited on polystyrene (PS) well plates and incubated on non-
modified well plates (PS) as a control (B). Absorbance is proportional to the number of living cells. Statistical analysis: * for p < 0.05, ** for p < 0.01 was calculated
according to ANOVA, Tukey test. p ≤ 0.05 was considered statistically significant; GO – graphene oxide, GO-RGD – GO modified with peptide Arg-Gly-Asp.

Fig. 8. SEM images of L929 cells incubated on different substrates: GO (A), GO-RGD (B), slide-glass (C); incubation time - 72 h; GO – graphene oxide, GO-RGD – GO
modified with peptide Arg-Gly-Asp.

(Fig. 9 A) and GO-RGD (Fig. 9 B) substrate. The cells spread uniformly
on the entire available substrate and only the available space limits their
elongation. The obtained images indicate that both materials proved to
be a good substrate for L929 cell growth.
4. Discussion
Comparing the two routes of modification of GO with RGD peptide, it
can be stated that the one-step process, where the three compounds
(NHS, EDC⋅HCl and RGD molecules) were applied on the GO surface

8
J. Jagiełło et al.
Fig. 9. Confocal microscope images of L929 cells cultured on GO (A) and GO-RGD (B) for 72 h. Green and blue signals come from the stained actin and nuclei,
respectively; scale bars =100 μm; GO – graphene oxide, GO-RGD – GO modified with peptide Arg-Gly-Asp; materials were deposited on PS substrate (For inter­
pretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
together (mixed), was much more effective than the process in which
NHS and EDC⋅HCl were used to modify GO followed by the addition of
RDG. It was examined using FTIR spectroscopy (Fig. 4), where the bands
connected with amine groups (especially at 1542 cm− 1) were of higher
intensity for the material obtained through the one-step method. The
absorbance at 1542 cm-1 is related to amide II due to in-plane N–H
vibrations and stretching CN vibrations. The area of 1640− 60 cm− 1 is
typical for amide I due to carbonyl and C–N groups, but is also present
in the spectrum of GO and is attributed to C–
– C bonds. Therefore, mainly
the 1542 cm− 1 band can provide unambiguous information about
functionalization. The efficiency of the functionalization was investi­
gated with XPS measurements (Fig. 5). The total amount of N atoms was
found to be 11.3 % at. N 1s area) with the amine groups of 66 % at., and
nitrogen atoms forming amide groups (bonds with graphene material) of
9.1 % at. and forming pyridine rings (N atoms embedded in GO defected
structure and closing the 6-atom ring) of 4.4 % at.
The better efficiency of one-step GO functionalization with RGD
peptide, when compared to the two-step route, can be explained as
follows. The idea of using crosslinkers (NHS, EDC⋅HCl) is to create a
semistable ester with molecules containing amine groups (primary
amines) that are highly reactive towards molecules with carboxyl
chemical groups [51]. Therefore, in the presence of the crosslinkers,
RGD molecules containing carboxyl and amine groups can polymerize.
The addition of EDC⋅HCl and NHS directly to the RGD solution not only
leads to binding of molecules with -NH2 groups to GO flakes (with
− COOH groups), but also acts as zero-length crosslinkers between
peptide molecules. The functionalization was very effective, as the mass
of RGD immobilized on the GO layer was 71.5 ± 6.2 μg/cm2. This is the
amount of RGD molecules bonded to the GO layer with self-conjugated
chains of this peptide.
A biological evaluation of GO and GO-RGD substrates revealed the
high potential of these materials in cell culture (Fig. 7). It was deter­
mined that both these substrates have a similar effect on L929 cells’
viability and proliferation. That means that the adhesion of pure GO is at
a very high level and does not need to be improved. The addition of Arg-
Gly-Asp (amino acids sequence) that plays an essential role in the
binding of cell integrins did not influence GO adhesive properties in a
significant way. Moreover, the viability and proliferation of the cells
were found to be better when cultured on a GO substrate rather than on
a glass substrate or commercially used polystyrene well plates. Taking
into account these results and the possibilities of biochemical modifi­
cations of GO with molecules, e.g. enhancing cell differentiation, pro­
moting desired cell response [52] and antibacterial molecules [53], GO
substrates seem to be a good alternative for commonly used polystyrene
plates. It should also be emphasized that, among different forms of
9
Materials Today Communications 26 (2021) 102056
graphene, GO is the most biocompatible form, as shown in the previous
work [30]. Among others, this is due to its hydrophilicity and the
presence of oxygen groups on the surface. The production of reactive
oxygen species (ROS), which are responsible for cell stress and appear in
contact with other graphene forms, like reduced GO, does not take place
on GO substrates. This can be explained by the higher energy of ROS
generation when the concentration of oxygen groups is higher [54].
There are some articles concerning biological activity of GO modified
with similar peptides as a part of antitumor and growth promotion
composites. GO–PLL–SDGR (poly-L-lysine and Arg-Gly-Asp-Ser func­
tionalized graphene oxide), a GO-based carrier was prepared and used to
deliver VEGF-siRNA for cancer therapy. GO–PLL–SDGR could release
VEGF-siRNA slowly and last for over 200 h, which was critical for
enhancing the effects of gene silence. In addition, GO–PLL–SDGR could
deliver VEGF-siRNA into tumor cells successfully and downregulate the
expression of VEGF effectively in vitro [55]. In the another study,
nanofiber matrices composed of PLGA and M13 bacteriophage with
RGD peptide displayed on its surface (RGD-M13 phage) were prepared
via electrospinning. The cellular behaviors of C2C12 mouse myoblasts
on the RGD/PLGA nanofiber matrices were evaluated when GO was
added in the culture media to determine if a combination of GO and
matrices can be applied as a novel strategy for skeletal tissue regener­
ation. It was confirmed that when the matrices were used with GO, the
myogenic differentiation of C2C12 myoblasts was effectively stimulated
and accelerated [56]. Graphene oxide modified with hyaluronic acid
(HA) and RGD was designed as a dual receptor targeting drug delivery
system to enhance the specificity and efficiency of anticancer drug de­
livery. The nanocarrier displayed a high Dox loading efficiency,
pH-response and sustained drug release behavior. In vitro toxicity
studies showed that the GO-HA-RGD exhibited very low cytotoxicity,
but GO-HA-RGD/Dox possessed much higher cytotoxicity for SKOV-3
cells than that of the both GO-HA/Dox (single receptor) and GO/Dox
(without receptor) [57].
5. Conclusions
In this study, the GO in the form of flakes was effectively modified
with RGD peptide. The optimal methods of the binding of RGD mole­
cules to the GO surface were developed and described. One-step func­
tionalization was an effective method to immobilize RGD peptide on the
GO substrate with the RGD amount of 71.5 μg/cm2.
Cellular studies show that the substrate in the form of unmodified GO
has the most beneficial effect on the culture of L929 fibroblast cells. The
addition of the RGD peptide, which is responsible for better cell adhe­
sion, does not improve GO functionality. This is confirmed by the results
J. Jagiełło et al.
of vitality and morphology (SEM and confocal microscope). Oxygen
functional groups present on the GO surface seem to be crucial for cell
culture to provide an environment for proper morphology and viability
of the cells. Moreover, the good wettability of such surface gives a
positive effect.
Based on the work carried out, it can be concluded that GO is a
material that can be successfully used for cell culture and can be
competitive with standard polystyrene media. The advantage of GO is
that the surface can be modified according to needs, e.g. growth factors,
antibacterial compounds. Such modifications cannot be carried out on
polystyrene substrates.
Funding
This study was funded by the Ministry of Science and Higher Edu­
cation in Poland (Grant ID: 352570).
CRediT authorship contribution statement
Joanna Jagiełło: Conceptualization, Methodology, Writing - orig­
inal draft, Investigation, Formal analysis, Visualization. Marcin
Kuśmierz: Writing - review & editing, Formal analysis. Ewa Kijeńska-
Gawrońska: Writing - review & editing, Formal analysis. Magdalena
Winkowska-Struzik: Writing - review & editing, Visualization, Formal
analysis. Wojciech Święszkowski: Writing - review & editing. Ludwika
Lipińska: Writing - review & editing, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors would like to kindly acknowledge Violetta Cecuda-
Adamczewska, PhD for fluorescence studies to calculate the peptide
content on the GO layers (from Department of Pharmacy, Cosmetic
Chemistry and Biotechnology, Łukasiewicz-IChP, Warsaw, Poland).
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