1. Running head: GRAPHENE AND GRAPHENE OXIDE
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Graphene And Graphene Oxide
Gudyne Wafubwa
Pwani University

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Graphene Nanomaterial Toxicity in vitro
Introduction
Although carbon known as a natural chemical element found in the body, it can be a
source of toxicity. Carbon derivatives such as; GO and rGO still have to show a sufficient and
acceptable safety level before being widely used. Many in vitro and in vivo studies have shown
that carbon-based nonmaterial's including Graphene and Reduced Graphene oxide can be toxic
while others have shown they are safe for biomedicine application. Toxicity variations depend on
the physiological and chemical structure of the Graphene where it is functionalized, multilayer,
reduced, oxidized and exposure routes consideration (Bianco, 2013). In vitro toxicity showed a
blurred idea on the cytotoxicity of carbon-based nanomaterial and Graphene family (Nezakati et
al., 2014). Ali-Boucetta et al. (2012) have showed no noticeable cytotoxic effects for up to 100
µg/mL of GO on lung carcinoma A549 cell line after 24 hours incubations.
In a study aimed to establish the biocompatibility and antimicrobial effects of Graphene
oxide, Ruiz et al. (2011) concluded that in both bacteria and mammalian cells, Graphene oxide
does not have intrinsic antibacterial, bacteriostatic, and cytotoxic properties. Ruiz et al. (2011)
suggested that nanoparticles with low mammalian cell cytotoxic effect and high antimicrobial
activity are ideal for biomedical applications. More studies were done to bring a clear
interpretation of Graphene toxicity. The tests were done using cell viability based on the LDH
(lactate dehydroxylase enzyme) which is released from the cell when it is undergoing a stressed
cytotoxic level leading to necrosis. MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium
bromide) used to evaluate cell vitality based on normal mitochondrial function when converting

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MTT into formazan (Van Meerloo et al., 2011). Different human cell lines were tested in vitro
with those assays to understand the direct cellular effect of the carbon-based nanomaterial. They
include lung cancer cell line A549, red blood cells, and platelets.
Talking about the concentration of the Graphene, it is important to know that a direct
relationship is linking Graphene oxide concentration and its toxic effect. The effect is more than
10 μg/mL of Graphene oxide that can cause metabolic changes and increase the toxicity (Zhang
et al., 2010). Increased level of LDH was noticed when platelets are exposed to enormous layers
of Graphene oxide, and this is due to increase in cellular oxidative stress related to high level of
reactive oxygen species generation (Singh et al., 2011). A549 lung cancer cell line, as
mentioned above, was treated with diversity sizes of 160, 430, and 780 nm Graphene oxide.
Toxicity and cell stress increase as the size increases, showing a line graph (Bitounis et al.,
2013). Human Fibroblast cell lines or (HDF) were incubated with increased concentrations of
Graphene oxide and found that levels of 20 µg/ml and lower have no significant effects on cell
viability. Where 50 µg/ml and more induce cell toxicity and reduce cell viability that leads to
activation of programmed cell death. Not only concentration fluctuations would affect cell
viability. Other explanations such as incubation period can directly and negatively affect the cell
when increased (Wang et al., 2011). The hemolytic action was reported when the blood cells cell
line was in vitro treated with separately single layers of Graphene oxide, and dose dependent.
Safety studies of GO in Vivo
The body of the living things responds positively to GO as it does to other biomaterials that are
safe to it.

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In the figure above, mice are used to test the GOs biocompatibility. It is established that
the GO is better than the Graphene itself. By use of microvascular endothelial cell line and
primary maurine- derived mesenchymal stem cells, it is found that Graphene oxide and reduced
Graphene oxide are both toxic to cells. The toxicity is at higher concentration and less toxic at a
lower concentration of 1mg/ml. The effect is due to the low concentration that allows materials
to enter the body immune system and be cleared faster from the body (Dumé, 2015)
Safety Study of rGO in mammalian cell and E.coli
To determine the safety measures of rGO, the researchers developed an antibacterial paper that is
composed of GO and rGO; the paper was tested its interaction with E. coli and its
biocompatibility with the mammalian cell. It is established that the nanosheet attacks the cell
membrane of the E. coli preventing it further growth but the same results are not experienced in a
mammalian cell (Gelotte, 2010).
Safety Study for Graphene in vivo
Although Graphene has acquired a wide range of importance in the medicine field, it is important
to examine safety measures. The study which deals with this substance, therefore, it is vital that
one should use a small single Graphene sheet that can be readily internalized by the macrophages
in the body and be easily removed from the site of deposition. It is also important that one should
use water soluble, colloidal and stable dispersions of Graphene material. The approach will aid in
minimizing aggregation in vivo and finally, one should consider using excretable Graphene so as

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to be degraded easily. This safety measures are vital and can be applied to all Graphene-related
substances (Cyrill, Kostas, & Ali-Buocetta, 2015).
Toxicity Study of GO in Vitro
In a systematic study on the toxicity of GO at the cellular level, Chang et al. (2010) found
that GO did not have apparent toxicity to A549 cells. However, they found that GO can not only
induce a slight decline in the cell viability at high concentrations but has also produced cellular
oxidative stress even at low levels. Wang et al. (2013), studied cytotoxic effect for different
levels of GO from 1 to 100 µg/mL with human lung fibroblast cells. The result showed a dose-
dependent pattern. When a transition electron was used, it showed that GO cannot readily
penetrate the cell. However, their study suggests that it is the size of GO sheets that determines
the toxicity at a high concentration, and, therefore, larger sheets. In this plight, it is seen that GO
has got higher biocompatibility to cells and animals at a higher level. This is through the study of
GO toxicity to human fibroblast at a concentration above 50 µg/ml that was determined to be
toxic (Changa, et al., 2010).
Toxicity Study of Reduced Graphene Oxide in vivo and in-vitro
In order to study the biocompatibility of rGO, the researchers compared the
biocompatibility of GO and rGO by use of U87 andU118 giloma cell in Vitro model and in Vivo
model, the U87 tumors was cultured on the chicken embryo. For in vitro, the analysis of GO and
rGO was made, from the study of in vitro, it seen that GO and rGO enter the giloma cell with
varied cytotoxicity, rGO having higher toxicity value than GO. On the other hand, in vivo, the
mass of tumor reduced, with the rGO having more elevated apoptotic markers. It is therefore

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observed that rGO kills the cells through apoptosis and has high biocompatibility than GO
(Jaworski, et al., 2015).
Toxicity study of Graphene in vitro
Toxicological study of Graphene shows that it is non-cytotoxic on Hela cell line. This is
because of its inability to induce a significant amount of apoptosis and ROS in the Hela cells.
Also, Graphene is biocompatible to other cells such as mammalian cells and it does not induce
any observable abnormality, this ability of Graphene to be friendly compatible to cells has made
it be the most important way to transport biological species for future research (Gollavell &
Ling, 2012).
Biomedical Applications
Biomedical Imaging
The most significant and useful applications of GO and rGO in biomedicine is
biomedical imaging. The ability of graphene and graphene derivatives to be conjugated with
fluorophores to produce fluorescence, enabled researchers to diagnose certain cell types such as
cancer cells in near-infrared region using biomedical imaging (Peng et al., 2010). Also, Sun et al.
(2008) found that the GO nanoparticles were photoluminescent in the visible and infrared
regions and concluded that GO can be used for living cell imaging in the near-infrared region
due to its intrinsic photoluminescence properties. Even more precisely, cellular imaging using
fluorescently functionalized GO can detect the defects and presence of proteins, organelles, and
specific targeted DNA by using Raman spectroscopy which is helpful in identifying specific
DNA lesion and cancer cells. For instance, Liu et al. (2012) proved in their study the cellular

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uptake for GO with cervical cancer (Hela 229) cell line. They used GO nanoparticles for cellular
imaging by conjugating them with gold (Au) nanoparticles and incubated them with Hela 229
cells for 24 hours. GO facilitated cellular uptake of Au nanoparticles.
Raman spectroscopy was used to detect the presence of GO hybridized with Au
nanoparticles that were taken inside Hela 229 cells. Figure 5 illustrated Raman monochromatic
light scattering of Hela 229 cells with pristine GO and modified GO with Au nanoparticles (GO-
Au) after 24 hours incubation (Liu et al., 2012).
Figure 5: Microscopic images of Hela 229 cells (A,C,and E) and Raman spectroscopy images
(B,D,and F). 24 hours incubation of Hela 229 cells without pristine GO or GO-Au (A and B). 24

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hours incubation of Hela 229 cells with pristine GO (C and D). 24 hours incubation of Hela 229
cells with GO-Au (E and F) (Liu et al., 2012).
Besides, fluorescence imaging, magnetic resonance imaging is very powerful in non-invasive
imaging technique that is well used in clinical practices. Because of the ability to induce local
inhomogeneity of the magnetic field, the superparamagnetic are widely used as the T2 contrast
agent. To increase the biocompatibility, a coated amino-dextrans with Fe3O4 is always
immobilized into GO. The composite acquires proper physiological stability and a lower
cytotoxicity that is then well internalized by Hela cells. This composite has a more enhanced
MRI signal. Positron emission tomography has a better tissue penetration; this is because it is
conjugated with GO together with CD105 together with an antibody TRC105 and copper-64 that
is best used to target breast cancer (Yang, Asiri, Tang, Du, & Lin, 2013)
Drug Delivery
Besides, GO has recently been used as an essential component in drug delivery. The GO
used for drug purpose is approximately 1-3 layers and has the size ranging from fewer
nanometers to thousands of nanometers; it has a planar structure with sp2
hybridization that gives
the GO excellent biocompatibility and proper solubility thus enabling it to have a high capability
to load drugs. However, the conjugation of GO with different systems is allowed through the
COOH and OH groups. For instance, various studies on targeted delivery of anti-cancer drugs
have used the functionalized GO (Pan, Sahoo, & Li, 2012). In an Aldrich study, SN38, a
camptothecin derivative became a soluble drug in water and serum when conjugated with
Polyethylene glycol (PEG) functionalized GO (GO PEG SN38).‒ ‒

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Although irinotecan (CPT-11) is an FDA approved prodrug for reducing cell viability of
human colon cancer cell lines (HTC-116), this study showed that the (nGO PEG SN38) was‒ ‒
more efficient by three orders of magnitude at reducing the colon cancer cell viability. Moreover,
the effectiveness of nGO PEG SN38 was similar to SN38 in DMSO (Luedtke, 2012). It has‒ ‒
been established that the combined use of drugs in medical purpose has a broad range of
importance, for instance, in combating breast cancer this is aimed at reducing the resistance of
drug by cancer cells. In the effort of combining two more drugs with GO with folic acid ligands,
increases the fight against breast cancer as compared to when one single drug is used. Over the
recent years, it has been noted that the rise of GO-based drugs has expanded from treatment of
cancer to the treatment of non-cancer such as bacterial diseases. However, adjusting the pH value
controls drug release (Zhang, Liu, Zhang, & He, 2012). On the other hand, rGO are also applied
in drug delivery devices like microneedle arrays. With the introduction of rGO, it leads to
improvement of the mechanical property of chitosan ( graphene oxide composite films). When
rGO is added, the electrical conductivity of the chitosan increases and this gave away for the
nanocomposites electroporation to be used in drug delivery. The content of the rGO increased

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drug delivery ability because the drug was linked with rGO (Mater & Chem, 2014)

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The discovery of new drug delivery devices that utilizes graphene sheets is used to administer
two anticancer medications to the targeted cells separately (Shipman, 2015)
Hyperthermia Treatment
Hyperthermia refers to a form of cancer treatment. The hyperthermia treatment exposes
body tissues to high temperatures that range up to 113 degrees Celsius. Recent studies reveal that
such high temperatures can kill the cancerous cells without tampering with the body tissues.
Treating cancer with such methods is not widely used and studied in clinical trials. Graphene
Oxide (GO) is a form of hyperthermia treatment. GO has an infrared absorption ability that has a
two-dimensional small size shape. GO has a unique performance when compared to other types
of nanoparticles. However, during the induction process, great care should be taken. The
hyperthermia laser models employ on the destruction of cancer, and it tends to control the
damages that may arise due to damage caused by the GOs. The laser is also used to monitor the
temperature rise of the GOs, which comprises of the culture medium (Ooi, 2013).

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References
Changa, Y., Yang, S.-T., Liu, Y., Wang, H., Cao, A., Liu, J.-H., et al. (2010). In vitro toxicity
evaluation of graphene oxide on A549 cells. Elsevier.
Cyrill, B., Kostas, K., & Ali-Buocetta, H. (2015). Safety Considerations for Graphene: Lessons
Learnt from Carbon Nanotubes. SciFinder, np.
Dumé, B. (2015, April 16). Graphene oxide could make safe bioimplant material. Nanotechweb,
np.
Gelotte, S. (2010, September 10). Antibacterial Paper Could Extend Shelf Life. Retrieved
August 1, 2015, from foodqualityandsafety:
http://www.foodqualityandsafety.com/article/antibacterial-paper-could-extend-shelf-life/
Gollavell, G. i., & Ling, Y.-C. (2012). Multi-functional graphene as an in vitro and in vivo
imaging probe. Researchgate, 2544.
Jaworski, S., Sawosz, E., Kutwin, M., M, W., Hinzmann, M., Grodzik, M., et al. (2015). In vitro
and in vivo effects of graphene oxide and reduced graphene oxide on glioblastoma.
International Journal of Nanomedicine, np.
Liu, S., Zeng, T. H., Jiang, R., Kong, J., Chen, Y., Hofmann, M., et al. (2011). Antibacterial
Activity of Graphite, Graphite Oxide, Graphene Oxide, and Reduced Graphene Oxide:
Membrane and Oxidative Stress. AcsNano, 6975.

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Mater, J., & Chem, B. (2014). High and conductive chitosan–reduced graphene oxide
nanocomposites for transdermal drug delivery. Journal of Materials Chemistry B, 3707-
3898.
Ooi, S. F. (2013, March 1 ). Laser Induced Hyperthermia of Superficial Tumors: Thermal
Damage Model With Regeneration of Healthy Tissue. Retrieved August 1, 2015, from
unsw:
https://www.engineering.unsw.edu.au/sites/eng/files/u7/PDFs/seow_fern_ooi_poster.pdf
Pan, Y., Sahoo, N. G., & Li, L. (2012). The application of graphene oxide in drug delivery.
Informahealthcare, 1365-1376.
Shipman, M. (2015). New Drug Delivery Technique Uses Graphene to Deliver Anticancer
Drugs. Sciencetechdaily, np.
Yang, Y., Asiri, A. M., Tang, Z., Du, D., & Lin, Y. (2013). Graphene-based materials for
biomedical applications. Materialstoday, 365-373.
Zhang, L., Liu, M., Zhang, Z., & He, S. (2012). Biomedical Applications of Graphene.
Theranostics, 283-294.