Carbon Nanotube Review

Perspective
pubs.acs.org/jmc
Carbon Nanotubes in Biomedical Applications: Factors, Mechanisms,

and Remedies of Toxicity
Miniperspective
Reem Alshehri, Asad Muhammad Ilyas, Anwarul Hasan,*,,, Adnan Arnaout, Farid Ahmed,
and Adnan Memic*,
Center of Nanotechnology, King Abdulaziz University, Jeddah, 21589, Saudi Arabia

Center of Excellence in Genomic Medical Research, King Abdulaziz University, Jeddah, 21589, Saudi Arabia
Department of Mechanical and Industrial Engineering, College of Engineering, Qatar University, Doha 2713, Qatar
Biomedical Engineering and Department of Mechanical Engineering, Faculty of Engineering and Architecture, American University
of Beirut, Beirut 1107 2020, Lebanon
Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Womens
Hospital, Harvard Medical School, Boston Massachusetts 02115, United States
ABSTRACT: Carbon nanotubes (CNTs) represent one of the most

studied allotropes of carbon. The unique physicochemical properties of
CNTs make them among prime candidates for numerous applications in
biomedical elds including drug delivery, gene therapy, biosensors, and
tissue engineering applications. However, toxicity of CNTs has been a
major concern for their use in biomedical applications. In this review,
we present an overview of carbon nanotubes in biomedical applications;
we particularly focus on various factors and mechanisms aecting their
toxicity. We have discussed various parameters including the size, length,
agglomeration, and impurities of CNTs that may cause oxidative stress,
which is often the main mechanism of CNTs toxicity. Other toxic pathways
are also examined, and possible ways to overcome these challenges have
been discussed.
such as nanosensors and nanorobots,2 and tissue engineering
applications.
However, in order for the CNTs to be more broadly used and
eventually end up in a clinical settings, their biological properties,
behavior, and performance need to be thoroughly understood.5,6
Furthermore, when produced at large scales, CNTs should also
have well characterized biological, environmental, and safety
proles. However, CNTs unlike many of their chemical agent
counterparts do not always come with well-dened structure and
purity. CNTs can signicantly vary in size, morphology, structure,
and purity based on the preparation, purication, and functionalization method used to synthesize them.5,6 Therefore, the
interaction between CNTs and the biological environment is very
complex and sometimes unpredictable. Similarly, researchers have
found that CNTs can show dierent levels of toxicity depending
on their manufacturing method, surface-to-volume ratio, shape,
concentration, aspect ratio, extent of oxidation, composition,
functional group(s), and the applied dosage.710 In addition,
CNTs are highly hydrophobic, which plays a major role in
reducing their biocompatibility.11 They have the ability to damage
1. INTRODUCTION
Merging of nanotechnology with medicine presents an unprecedented opportunity for developing novel materials that can signicantly improve treatment and diagnosis of diseases. It is also
expected that the ability to make and use nanomaterials at
industrial scales will be the driving force for the emerging new
economies and industries. Carbon nanotubes (CNTs) are novel
nanomaterials that have unique properties and potential to be
developed into useful products at industrial scales, as they are
attractive for a variety of applications in various elds. More
specically, CNTs have the potential to revolutionize biomedical
research due to their important electrical, chemical, thermal,
mechanical, and structural properties which have made them an
area of great research interest.1 They have the ability to display
metallic, semiconducting, and superconducting electron transport
properties. They are also known for their high elastic modulus
compared to all other materials.1 In the past decade, numerous
studies have been conducted on the application of CNTs in the
biomedical eld.2 It has been shown that CNTs can be used in
numerous applications including diagnostic tools and devices in
radiation oncology, biosensors, probes and quantum dots,2
nanouidic systems, biopharmaceutical applications such as drug
delivery and drug discovery,3,4 implantable biomedical devices,
XXXX American Chemical Society
Received: November 12, 2015
DOI: 10.1021/acs.jmedchem.5b01770
J. Med. Chem. XXXX, XXX, XXXXXX
Journal of Medicinal Chemistry
Perspective
and an array of functional group and biomedical applications4,24

(Figure 2B).
the DNA and the cell membrane as well. They can also cause
toxicity through oxidative stress, mitochondrial activities modication, protein synthesis, and altered intracellular metabolic
routes.10 The most common CNT mechanisms that lead to
cytotoxicity also include necrosis and apoptosis.10
In this review, we discuss the critical roles of CNTs in
biomedical applications. We state the synthesis methods of
CNTs, and we summarize the recent advances in application of
CNTs in drug delivery, bioimaging, biosensing, and tissue
engineering applications. The paper also summarizes how
physicochemical properties of CNTs such as size, shape, surface
chemistry, and the route of synthesis can aect their toxicity.
Finally, the mechanisms responsible for CNTs toxicity and the
potential remedies to overcome their pitfalls have also been
presented.
3. CNTs IN BIOMEDICAL APPLICATIONS

CNTs possess numerous unique features that make them a
promising material for an array of biomedical applications. Some
of their unique features include their unique structure giving rise
to an extraordinary combination of mechanical, electrical, and
optical properties.25 Although CNTs are hydrophobic in nature,
they can be functionalized to suit particular applications. Several
of these functionalization techniques have useful biomedical
applications. Functionalization moieties can be introduced to
CNTs such that they specically interact with cell surface
receptors that can guide their internalization. These receptormediated targeting strategies can facilitate specic cell loading,
thereby lowering the quantity of drugs needed in disease
treatment. In addition, these strategies can help in minimizing
systemic toxicity and inammation.26
3.1. Drug and Gene Delivery. Many drug delivery systems
have been designed using CNTs for treatment of a variety of
diseases. In fact, CNT-based anticancer drugs have attracted
much attention, and many depend on two strategies: rst,
selective targeting, which is accomplished through functionalization with specic tumor receptors, and second, the controlled
release of the drugs usually present in tumor environment such as
lower pH.2630 In fact, this enables CNTs to deliver small amounts
of drugs to the specic tumor site by which minimization of
systemic toxicity and reduction of the undesirable side eects of
normal anticancer drugs is achieved. Cheng et al., 2011, developed
a CNT-based anticancer drug that overcame the problem of
multidrug resistant cancer cells and at the same time was eective
on sensitive cancer cells without aecting cell proliferation and cell
cycle.31 In addition, using a noncovalent approach, they developed
peptide-modied SWCNTs that could be loaded with the anticancer drug, tamoxifen. This system when tested in vitro and in
vivo showed high antitumor eect and ecient tumor targeting.32
There are many other important types of CNT-based drugs, for
example, using CNTs as carrier for immunization against some
antigens.9 In fact, induction of immune response against tumors
can be achieved through CNT based vaccination as in Villa et al.s
2011 work, which used SWCNTs as a tool for successfully delivering tumor antigens.33 Other types of substances such as acetylcholine (Ach) that cannot be transported to the brain by
traditional ways in which normal drugs are transported can be
successfully transported using SWCNTs.34 The lack of Ach is
associated with Alzheimers disease where neurons may be unable
to synthesize it.35 Yang et al. showed the capability of SWCNTs to
deliver Ach into the brain of mice.34 Moreover, cell-selective
nuclear targeting of anticancer drugs has been achieved using
functionalized MWCNTs.36
Other advantages with functionalization of CNTs have been
reported in many studies using covalent conjugation strategies.
For example, Khazaei et al. found that covalent functionalization
of SWCNT with amide and ester bonds facilitates the slow
administration of drug for longer periods and improves the
solubility in aqueous and organic media.37 In another study,
covalently attached liposomes were found to enable the delivery
of large doses of a drug.38 Baligelli et al., 2013, found that
targeting intracellular organelles like mitochondria with
covalently functionalized CNTs for the delivery of therapeutics
could be one eective strategy to better understand dierent
genetic disorders.39 Also, dierent types of chemically functionalized MWNTs have been used to study their interaction with
2. CARBON NANOTUBES SYNTHESIS

CNTs have been extensively studied in many disciplines such as
engineering, medicine, biology, and chemistry due to their
unique properties and promising applications.12,13 Their
diameter is approximately 1 nm and their length varies from
1 to 100 m. The carbon atoms in CNTs are arranged to form a
cylinder of graphite layers.14 There are mainly two types of
CNTs: single-walled nanotubes (SWNTs) which are formed of
one layer of graphene and multiwalled nanotubes (MWNTs),
which are formed of multiple layers of graphene, as shown in
Figure 1. Other types of carbon based nanomaterials include
carbon nanohorns, fullerenes, carbon nanotorous, carbon
nanopeapods, and carbon nanobuds.2,15 CNTs are produced
using three dierent techniques: the carbon arc-discharge
technique;2,16 the laser-ablation technique; and the chemical
vapor deposition (CVD) technique, shown in Figure 2. Arcdischarge technique was rst used by Ijima in 1991 to develop the
MWNTs.16 Shortly after that, Ijima and Ichihashi17 and Bethune
et al.18 used metal catalyst in the same technique to produce
the rst SWNTs. Later, Thess et al.19 used the laser-ablation
technique to synthesize bundles of aligned SWNTs. Yacaman
et al.20 was the rst team to propose catalytic growth of MWNTs
by CVD. One synthesis method used carbon y ash as a catalyst
for the CNTs growth.21 Yasui et al. adopted the use of coal y ash
which contains relatively small amount of carbon.22 This method
is very promising, as it is very cost-eective and can extend
the applications of CNTs.21 In addition, cobalt (Co) catalyzed
MWCNTs lms have been produced using low pressure chemical
vapor deposition on silicon oxide grown silicon substrate.23 These
Co MWCNTs can be used as sensors for detecting carbon monoxide (CO) gas.23
Pristine CNTs often lack the necessary solubility that impose
great limitations for their biomedical applications. As-produced
CNTs are almost completely insoluble in any aqueous solution
or organic solvent except by sonication that usually only leads to
CNT dispersions. Therefore, it is critical to functionalize CNTs
not only to make them more soluble but to allow their
integration into many organic, inorganic, and biological systems
and applications.4,24 There are three main approaches for CNT
modication, namely, (a) the covalent functionalization of the
-conjugated skeleton of CNT using various chemical groups and
reactions; (b) the noncovalent adsorption of numerous functional
bio/molecules; and (c) the endohedral lling of their inner
empty cavity24 (Figures 1a and 2D). For example, 1,3-dipolar
cycloaddition of azomethine ylides to CNTs is one of the simple,
powerful, and highly utilized functionalization approaches
that lead to soluble CNTs that can be used for further reactions
B
Perspective
Figure 1. Structures of carbon nanotubes and their eld of use: (a) schematic representation showing the structures of SWCNTs and MWCNTs;162
(b) schematic illustration showing the dierent applications of carbon nanotubes such as drug delivery, diagnostics, biosensors, biomedical imaging, as
well as tissue engineering and regenerative medicine.
can be easily attached by strong noncovalent bonds.41 Noncovalent functionalizations of CNTs have been used in many
medical applications including many successful models for CNTbased drugs.42,43 Many successful drug models were developed
neural tissue cells and such studies could help in developing

eective gene and drug delivery systems and future use of CNTs
in CNS applications.40 On the other hand, some molecules that
cannot form covalent linkages (such as aromatic compounds)
C
Perspective
Figure 2. Schematic illustration showing dierent synthesis methods of carbon nanotubes. (A) Chemical vapor deposition (CVD) method used for
carbon nanotubes synthesis. (B) Laser-ablation technique used in the synthesis of carbon nanotubes. (C) Carbon arc-discharge technique performed to
synthesize carbon nanotubes.163 (D) Dierent functionalization strategies of CNTs showing (i) cycloaddition with in situ generated dichlorocarbene,
(ii) photoinduced generation of reactive nitrenes, (iii) functionalization by nitrenes, (iv) 1,3 dipolar cycloaddition of azomethine ylides, (v) reaction
scheme for uorination of nanotubes, defunctionalization, and further derivatization; (vi) 1,3 dipolar cycloaddition of nitrile amines; (vii) reaction
pathway for obtaining water-soluble ammonium-modied nanotubes. The last can be used for the delivery of biomolecules.
This feature can make them an eective tool in gene silencing

and gene delivery. Eorts have been made for developing
ecient and specic nonviral gene delivery systems using CNTs.
For instance, functionalizing CNTs with poly(lactic-co-glycolic)
(PLFA) was shown to deliver proapoptotic protein caspase-3
(CP3) into osteocarcinoma cells.45 CNTs have also been used to
deliver the GFP gene into cultured cell lines.46,47 On the other
using noncovalent interactions allowing for selective targeting and

proper release of the drug in tumor environments at reduced pH
and the eective killing of cancer cells.2729 Further noncovalent
functionalization of SWCNT with modied polyethylenemines has
been shown to be a successful gene delivery vector system in vivo.44
Another great advantage of CNTs is that it can be loaded
with a variety of biomolecules like siRNAs, genes, and DNA.
D
Perspective
made them a powerful tool to obtain fast biological species

detection at low concentration, which made CNT-based
biosensors important in the eld of ultrasensitive biosensing
applications (Figure 3).63
CNT-based biosensors have many advantages over commercially available, silicon based, or other material-based sensors.
These advantages include (i) high sensitivity, due to their high
surface-to-volume ratio and hollow tubular structure, where
CNTs can oer high biological activity because of their ability to
be used to immobilize enzymes;64 (ii) fast response time, where
CNTs can promote electron-transfer reactions due to their high
ability to mediate fast electron-transfer kinetics, i.e., NADH and
hydrogen peroxide reaction; (iii) less surface fouling eects and
low potential of redox reaction; (iv) long life span and high
stability.63
As mentioned earlier, CNTs are known for their high surface
area to volume ratio which can be a contribution to the
biomolecular conjugation, and it is being taken into consideration in constructing enzyme biosensors. For this reason,
enzyme biosensors are one of the most commonly used type of
biosensors.65 As an example, Vicentini et al. was able to develop a
tyrosinase biosensor which is glassy carbon electrode based,
adjusted with functionalized MWCNT, 1-butyl-3-methylimidazolium chloride (IL), and tyrosinase (Tyr) within a dihexadecyl
phosphate (DHP) lm.66 This combination of the electrocatalytic activity of MWCNTs and the biocompatibility and the
conductivity of IL has enhanced the biosensors response signal;
this biosensor has proven its durability and stability.63 Another
enzyme biosensor was developed to detect androsterone by
using 3-hydroxysteroid dehydrogenase immobilized onto a
CNTs/IL/NAD+ composite electrode.67 The most unique
characteristic of CNTs taken into consideration while developing
an enzyme biosensor is their large surface area, which contributes
to the biomolecular conjugation.65 Enzyme biosensors can act as
glucose detectors in the blood. Glucose detection is one of the
most important application of currently used enzyme biosensors.68
The other well-known CNT based biosensors are the DNA
biosensors. They have been heavily used for medical diagnostics,
forensic science, and many other applications.63 DNA, whether it
is single stranded (ssDNA) or double stranded (dsDNA), is the
main sensing element in the DNA biosensors (Figure 3).63
Researchers have found ssDNA to be highly adsorptive to CNTs,
unlike dsDNA which cannot bind to CNTs.69 Researchers have
taken advantage of this unique property of DNAs and used it to
build CNT-DNA biocomplexes for biosensing technologies to
target various molecules.63 Tang et al. were able to fabricate a
SWCNT-FET-based electronic DNA biosensor; these biosensors can be used in systems-on-chip applications.70 DNA biosensors are simpler in their setup and chemistry, compared
to other commercial biosensors such as electrochemical and
optical DNA biosensors.63 In addition, DNA methyltransferase
(DNA MTase) control and inhibition are now possible due to
the ultrasensitive DNA biosensor that was developed using the
uorescence polarization detection and MWCNT signal
amplication.71 Furthermore, a glassy carbon based sensitive
DNA biosensor was recently designed by Zhang et al.; it is
modied with MWCNTs, polydopamine (PDA), and gold
nanoparticles and used as a DNA sequencing detector.72 This
innovative sensing approach has proven its sensitivity and
selectivity; it is being used as human serum samples in experiments for targeting DNA complements and have shown
promising results so far.63 Several types of the CNT biosensors are illustrated in Figure 3. The unique characteristics
hand, gene silencing was also successfully performed on many

cells lines.48,49 In vivo, gene silencing was achieved in mice model
without any signs of toxicity or induction of immune response.50
Huang et al. showed that functionalizing MWCNTs with polyethylenimine was able to deliver siRNA successfully into the
HeLa-S3 cells.51 In addition, DNA molecules were delivered
using SWCNTs that not only transported them but also
protected them from digestion by nucleases present in the
cytoplasm.52
3.2. Biomedical Imaging. Biomedical imaging is a eld that
is being heavily studied and that incorporates dierent
approaches from dierent elds of science. It is an emerging
tool that can oer high resolution imaging of the behavior of cells,
tissues, organs, or complete body. CNTs, having unique physical
properties, can be manipulated to be subjected to dierent
methods of biomedical imaging in order to analyze and improve
their functionalities and response to their environment.53
One method of biomedical imaging is uorescence emission in
the near-infrared region, being a label-free technique that can be
done at high speed, giving access to monitor the nanotubes in live
cells.54 Photoacoustic imaging (which allows deeper tissues to be
imaged, using dierent contrast agents to target specic sites55)
and magnetic resonance imaging are two additional methods that
can be performed on CNTs, due to their high absorbance and the
impurities in the form of metallic nanoparticles they contain.
In fact, results of in vivo experiments done by Vittorio et al. have
shown that CNTs can be guided by an external magnetic source
toward a specic organ.56 In addition, a method used is scanning
gate imaging of two coupled quantum dots, done by electron
transport through the wall.57
Modication of CNTs and addition of elements to their
structure can bring new perspectives of analysis of their behavior.
One technology is assembling gold nanostructures, which has
proven to enhance uorescence intensity and has achieved
ferritin receptor-mediated targeting and biomedical imaging
both in vitro and in vivo, proving itself as an important
biomedical imaging agent. Eective multifunctional platforms
can be made out of the nanotubes, thus enhancing their
properties and the width of their application, as is for the
eectiveness of the imaging. This can be done by adding the
dierent types of nanoparticles to CNTs that include among
others gold nanoparticles, quantum dots, iron oxide nanoparticles,
upconversion nanoparticles, PET imaging nanoprobes.58 Some of
these nanoparticles may contribute, in some cases, to the toxicity
of the CNTs,58 which makes the platform unsuitable for cellular
life. However, this can be avoided by not exceeding the toxic
amount of the element to be used. In fact, two published papers so
far have used ligands to target tumors in vivo, and it has been proven
ecient by modifying the ligands used and their concentration.59
This technology is proving itself as a new, powerful technique to
treat cancer.
Biomedical imaging is proving itself as a watershed in nanotechnology and biomedical engineering, providing easy-to-control
platforms in living cells and allowing the emergence of new
solutions and perspectives of analysis in dierent elds.
3.3. Carbon Nanotubes in Biosensors. Biosensors are one
of the most important application of CNTs, where they combine
their biological recognition with a chemical/physical transduction in order to detect biomolecules.60 CNTs have excellent
mechanical, electrical, and electrochemical properties, high surface
area, and high exposure sensitivity to various biomolecules; this
has made CNTs highly eective sensing elements for biosensors.16,61,62 The high surface area-to-volume ratio of CNTs has
E
Perspective
Figure 3. Dierent types of carbon nanotubes based biosensors. (a) PEGylated SWNTs with RGD conjugation and radiolabeling used for in vivo tumor
targeting. Reproduced with permission from Nature Nanotechnology.164Copyright 2006 Nature Publishing Group. (b) Scheme of SWNTs with three
dierent isotope compositions (13C-SWNT, 12C12/13C-SWNT, 12C-SWNT), conjugated with dierent targeting ligands.165 (c) Experimental setup
of the two coupled single wall carbon nanotube quantum dots in a multiple quantum dot system.166 (d) Schematic illustration showing dierent types of
CNT-biosensors, enzymatic CNT biosensors, and DNA-functionalized CNT biosensors, as well as the combination of nanoparticles with CNTs and
enzymes to form specic type of biocomposites for biomedical applications. (e) CNT electrode array used for sensitive detection of DNA hybridization
in the presence of Ru(bpy)32+ mediated with guanine oxidation Gox.167 (f) Schematic of a SWCNT eld-eect transistor (FET). SWCNTs are being
coated to a biotin layer which allows streptavidin to bind and thereby change the characteristics of FET.168 (g) Schematic representation of the CNT
electrically contacted glucose oxidase electrode where GOx is constituted on the FAD units. Reproduced with permission from Angewandte Chemie,
International Edition.169 Copyright 2004 Wiley-VCH.
approaches, cells are seeded or encapsulated in a suitable

biomaterial for growing engineered tissues.73,74,78 Ensuring proper
mechanical, electrical, and biological properties of biomaterials is a
challenge in the eld of tissue engineering73,75 where CNTs can
play important roles. Recently, CNTs have been used in tissue
engineering in varieties of applications including improving the
mechanical and electrical properties of scaolds, sensing the cell
of CNTs have made them extremely important for biosensing

applications.
3.4. Tissue Engineering and Regenerative Medicine.
Tissue engineering and regenerative medicine are new approaches
in medicine intended for developing engineered articial tissues
for applications in replacement grafts, and tissue models for in vitro
disease studies and drug discovery.7378 Usually, in these
F
Perspective
the bers have enhanced the myotube contraction amplitude, as

well as myotubes maturation.86 Thus, CNTs are also emerging as
a potential useful component for various biomaterials in tissue
engineering applications.
microenvironments, tracking of cells, and delivery of suitable

chemical and biological agents.79 The success of some studies in
using CNTs as scaolds for nerve generation in vitro has opened a
new horizon for neural tissue engineering. Use of CNT-based
scaolds has been reported to improve neural growth,80 bone
growth,81 cardiac tissue growth.82 While in neural and cardiac
tissue growth the CNTs are generally used as enhancer of electrical
properties of the scaolds, the bone growth can be achieved by
functionalization of CNTs with groups attracting calcium
cations.83 Recently, MWCNTs functionalized with broblast
growth factors also displayed ability to be used as scaolds for bone
formation.84 Another potential use of CNTs in tissue engineering
is to engineer biohybrid tissue actuators.85 Inspired by muscle
tissues, biological machines, sensing, and adapting to the environment, biohybrid tissue actuators can be built; these devices and
actuators can be used to handle numerous challenges in biorobotics and drug screening.85 Furthermore, electrospun gelatin
MWCNTs bers were used as scaolds for myoblasts (C2C12)
growth; MWCNTs method has enhanced the mechanical
properties of the resulting bers.86 Under electrical stimulation,
4. TOXICITY OF CNT
Numerous toxicological studies of CNTs have been reported in
literature, both in vitro (Table 1) and in vivo (Table 2), some of
which are contradictory to each other due to the variabilities in
parameters including the type of functionalization, method of
preparation, and the doses of CNTs. In addition to these
variables, other simple laboratory variables such as dierent
types of cell-viable indicator dyes can contribute to dierent
cytotoxicity proles. The most common indicator dyes include
commassie blue, alamar blue, neutral red, MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and
WST-1 (a water-soluble tetrazolium salt). Hence, it is important
to mention this simple observation before assessing parameters
that can lead to CNT cytotoxicity results and their variability.87
Table 1. Comparison of CNT Toxicity in Vitro Using Biological Model Systems

model system
unmodied SWCNTs
pristine SWCNTs
pristine SWCNTs
pristine SWCNTs
SWCNTs
0.5 DMSO pristine SWCNT
SWCNTs
functionalized SWCNTs
acid-functionalized single-walled carbon
nanotubes (AF-SWCNTs)
2-methacryloyloxyethylphosphorylcholine
SWCNTs and phosphorylcholine grafted
SWCNTs
uorescein-labeled poly(ethylene glycol)modied single-walled carbon nanotubes
RNA-polymer SWCNT conjugate
FITC-SWCNT
streptavidin-SWCNT
6-aminohexanoic acid-derivatized SWCNTs
unmodied MWCNTs
pristine-multiwall carbon nanotubes
pristine MWCNTs
pristine MWCNTs
pristine MWCNTs
MWCNTs
MWCNTs
functionalized MWCNTs
carboxylated-MWCNTs
four types of functionalized multiwalled CNTs
plasmid DNA-MWCNT
MWCNTs-polyglycerol (PG)
MWCNTs functionalized by carboxylation
surface-functionalized multiwalled carbon
nanotubes
human vein endothelial cells

(HUVEC)
RAW264.7 cells
mesothelioma cell line MSTO-211H
HeLA cells
human embryo kidney (HEK 293) cells
A549 lung epithelial cells
dosage and outcome
ref
550 g/mL. They observed damage caused by cell ber contact and oxidative
stress probably due to contaminant metals
At concentration of 100 g/mL they induced apoptosis through mitochondrial
dysfunction and ER stress
7.5 g/mL water, 10% decrease in cell proliferation and activity
100 g/mL, no eect on growth rate
25 g/mL, G1 cell arrest and apoptosis
1.56800 g/mL, cytotoxic at 400 and 800 g/mL
173
174
118
175
176
177
1 mg/mL signicantly lowered the ability of these cell lines to present galactosylceramide antigen
5, 10, 20, 40, and 80 g/mL had greatly reduced but cannot completely
eliminated cytotoxicity
178
HeLa, KB-31, and KB-85 cells
exhibited low toxicity at concentration of 0.001100 mg/L
180
MCF-7 breast cancer

HeLa cell lines
HL60 and Jurkat cells
human epidermal HEK keratinocytes
1 mg/mL, no signicant cell damage

510 mg/mL, 50% survival of HeLa cells
0.025 mg/mL, no adverse eects
0.000000050.05 mg/mL, dose dependent decrease in viability
181
182
183
184
human alveolar (A549) epithelial cells

and normal bronchial (BEAS-2B)
cells
A549 human lung epithelial cells
1100 g/mL induced membrane damage, particularly in BEAS-2B cells
185
At doses of 12.5100 g/mL, pristine-MWCNT caused a time- and dosedependent ROS increase and higher levels of lipid hydroperoxides compared
to the controls
1 mg/mL, toxicity observed
40 g/mL, low toxicity was observed using the concentrations
100 g/mL, no toxicity was observed
toxicity observed on the cells using the MWCNTs
186
187
188
189
92
520 g/mL for up to 24 h incubation showed cell surface changes
190
At 100 g/mL, three of the tested nanotubes activated immune-related

pathways in THP1 but not in Jurkat cells
10 mg/mL 50% survival of HeLa cells
1 mg/mL, no toxicity observed
191
functionalization completely eliminated inammasome activationat

concentrations from 6.25 to 50 g/mL
The order of toxicity to BEAS2B cells was determined to be COOH > O > NH2
> pristine MWCNTs at 5200 mg/L concentration.
157
three cell lines, LA4, MHS, and JAWSII

RAW264.7, HUVEC and L929 cells
murine alveolar macrophages (MH-S)

T lymphocytes
human platelets
human macrophage cells
human pulmonary epithelial cells
(A549)
Jurkat and THP1 cells
HeLa cell lines in vitro
HT1080 cell line (human
Fibrosarcoma)
THP-1 cells and primary alveolar
macrophages from C57BL/6 mice
human bronchial epithelial cells,
BEAS2B cells
G
179
192
193
194
Perspective
Table 2. Comparison of CNT Toxicity in Vivo Using Biological Model Systems

unmodied SWCNTs
pristine SWCNTs
pristine SWCNTs
pristine SWCNT
pristine SWCNT
oxidized single-wall carbon nanotubes
DTPA-SWCNT
DOTA-SWCNT
SWCNT functionalized
using phospholipids
conjugated to hyaluronan
unmodied MWCNTs
pristine MWCNTS
MWCNTS
puried MWCNTs
MWCNTs
MWCNTs
functionalized
MWCNTs
several functionalized
MWCNTs
carboxyl group functionalized MWCNT
glucosamine-MWCNT
DTPA-MWCNT
14
C-taurine-MWCNTs
model
dosage and outcome
ref
male ICR mice

development of the mouse
embryo
intravenous injection, systemic, rabbit in vivo
intravenous injection, systemic, mice in vivo
SWCNT, mice model
At concentration of 100 g/mL they induced acute inammatory response in the lungs of mice.
10 ng to 30 g/mouse. At concentrations above 100 ng/mouse reactive oxygen species (ROS) were detected in
placentas of malformed but not of normally developed fetuses
7.5 mL of 20 g/kg body mass. They observed little to no toxicity.
174
195
Single 600 g injection showed no observable toxicity.
197
0.064 mg/kg oral administration, genotoxic eect
198
Swiss-Webster mice
development of the mouse
embryo
with female BALB/c mice
in vivo
mouse model
C57BL/6 mice
0.25, 0.5, and 0.75 mg/kg, enhanced ROS including increased DNA damage and decreased the mitotic index
10 ng to 30 g/mouse. At concentrations above 100 ng/mouse cytotoxic eects to the mouse development were
observed.
20 g/L PBS, no acute toxicity after single 200 L dose
199
195
male Wistar rats

lungs of SpragueDawley
rats in vivo and macrophages in vitro
subcutaneous tissue of rats
with MWCNTs with an
average length of 220 and
825
Swiss albino mice model
mouse model
C57Bl/6 mice
male Swiss-Webster mice
intraperitoneally into female

Kunming mice
intravenous injection, systemic, female BALB/c
mice
intravenous administration
PEGylated MWCNTs
196
200
0.012 mg, low toxicity

100 L at 1 mg/kg was intravenously injected into the tail vein of C57BL/6 mice and did not alter the total
number of leukocytes or increased liver enzyme release
201
202
0.20 or 0.55 mg via intratracheal instillationwere not cleared from lungs of rats following 364 day follow-up
suggesting potential toxicity
observable toxicity
203
204
low toxicity
96
60 and 100 mg/kg, liver toxicity observed

40 g, acute lung toxicity
205
206
surface charge in determining the pulmonary brogenic eects of MWCNTs at a with nal dose at 2 mg/kg using
pulmonary aspiration
C-MWCNTS were administered at doses of 0.250.75 mg kg1 day1 for 5 days, body-weight gain of the mice
decreased, induced reactive oxygen species (ROS), and enhanced the activities of serum amino-transferases
(ALT/AST), alkaline phosphatases (ALP,) and concentration of lipid hydroperoxide compared to control.
300 L single dose, suspension concentration unknown had good biocompatibility
207
208
20 g/L PBS, no acute toxicity after single 200 L dose
200
10 g, low level acute toxicity

10 and 60 mg/kg, induce toxicity in liver
210
211
4.1. What Makes CNTs Cytotoxic? Factors that contribute

to cytotoxicity of CNTs include the amount and kind of metal
impurities, length and type of carbon nanotubes, the presence of
surface functionalization and its type, and the existence of
dispersant or surfactant in dispersant solution41 (Tables 1 and 2).
Below, we discuss the factors contributing to toxicity and the
plausible mechanisms by which CNTs exert their toxicological
eects (Figure 4).
4.2. Eect of Metal Catalyst Impurities. Several studies
have demonstrated that metal impurities introduced during the
synthesis of CNTs could have a signicant impact on toxicity.
Pulskamp et al., in one of their studies, have shown the toxicological eect of dierent puried and commercial SWCNTs and
MWCNTs.88 What they observed is that three commercial
CNTs samples substantially increased the ROS that contributed
to oxidative stress and decreased the mitochondrial membrane
potential. However, when compared to acid-puried SWCNTs,
with low metal content, they observed less toxic eects, similar to
that of the negative controls, exhibiting little or no cytotoxicity.
For all four CNTs cases, IL-8 and TNF- levels in A549 epithelial
209
cells or rat macrophages were not increased by their presence.

However, another study showed this observation does not
stretch across all cell lines. For example, loss of cell viability in
human neuroblastoma cells (SH-SY5Y) was observed with
MWCNTs treatment. Several MWCNT preparations were
assessed ranging from of 97% purity with or without acid
treatment; in comparison to MWCNTs of 99% purity, all CNT
preparations showed no development of oxidative stress during
prolonged cells culture. Only when the concentration of
MWCNTs was increased did the eect of metal impurities or
other contaminant became evident.89 Other reports that looked
at in vivo toxicity showed a correlated increase in CD8+ levels and
CD4+/CD8+ ratio of peripheral T-cell in subcutaneous mice
implanted with iron-contaminated MWCNTs. In addition, other
cytokine levels were increased in comparison to MWCNTs
puried at 1800 C and even more pronouncedly with those
MWCNT puried by 2800 C heat treatment. The group
proposed that the release of water-soluble ions from iron or other
metallic impurities induced the generation of ROS.90 It has also
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unpuried MWCNTs signicantly altered the level of expression

of the DJ-1, a protein that may be involved in protection against
oxidative damage.91 In general, from these studies and others
similar to it we can observe that the metal impurities may have
impact on the toxicity of CNTs. However, the amount, concentration, method of administration, and the assay used to assess
the impurity contribution toward toxicity should be carefully
accounted for.
4.3. Impact of CNT Length on Toxicity. Length of CNTs
has been shown to aect the toxicity of CNTs mainly due to
failure of their cellular internalization, which in part has been
reported to be greatly aected by CNTs length, showing that the
smaller the MWCNTs (sub 1 m), the easier the penetration to
cell membrane.93 In fact, long CNTs can cause biopersistence94
or CNTs retention. Results of Murphy et al., 2011, clearly
demonstrated this association.95 Their study found that short
CNTs instilled into pleural cavity were cleared, while long CNTs
did not. Retention of long CNTs can lead to severe inammation
which causes progressive brosis.95 Similarly, longer CNTs with
825 nm length have been shown to induce more inammation
than 220 nm CNTs. A proposed illustration to these results is the
poor macrophage engulfment with increase of length.96
In addition, long MWCNTs (515 m and diameter 2060
nm) were found to be more genotoxic in human alveolar
carcinoma epithelial cells (A549) compared to the shorter ones
(length 12 m and diameter 60100 nm). Furthermore,
the study also showed that long MWCNTs with the same length
(515 m) but with dierent diameters may exhibit dierent
level of toxicity, suggesting that smaller diameter of <2 nm
signicantly reduces DNA damage and thereby the toxicity.97
It has also been reported that the absorption of long CNTs in
rat small intestine is lower than that for short ones.98 On the
other hand, one study involving MWCNT indicated that smaller
nanoparticles are more cytotoxic than larger nanoparticles.99 An
in vitro model by Simon-Deckers et al., 2008, suggested no
association.100 This emphasizes the involvement of other factors
contributing to CNTs toxicity.
4.4. Eects of the Type of CNTs: Single Wall versus
Multiwall. Single and multiwalled are two dierent structural
characteristics of carbon nanotubes. They dier mainly in
structure, size, and chemical surface state. SWCNTs have one
cylindrical graphene layer with diameter range 0.62.4 nm,
whereas MWCNTs have many concentric cylinders of graphitic
shells. MWCNTs have larger diameter ranging from 2.5 to
100 nm.9 SWCNTs have large surface area with strong van der
Waals forces. This may lead to the tendency of forming bundles
as well as reduced eciency of their surface area. On the other
hand, MWCNTs have lower tendency for bundle formation due
to relatively lower surface area and the presence of many active
defects in their side walls.87,101 However, functionalization of CNTs
can help in the reduction of aggregation state and therefore reduce
their toxicity.102
Results of Fraczek et al., 2008, showed that SWCNTs form
smaller aggregates compared with MWCNTs aggregates.103
The author linked the dierences among CNTs toxicity in vivo to
surface structure and size. SWCNTs in the study contained
hydroxyl OH and carboxyl COOH groups that made their
surface more hydrophilic compared to MWCNTs. This
hydrophobicity enables them to be uniformly dispersed inside
tissues, forming small aggregates ranging from 5 to 30 m that
can be phagocytosed. In contrast, MWCNTs form larger aggregates 300 m that cannot be phagocytosed.103 The smaller size
and length of SWCNTs make them less aggregated in vivo
Figure 4. Factors aecting carbon nanotubes toxicity on blood cells.

(a) Illustration showing how the structure of CNTs can aect phagocytosis
by macrophages and tissue clearing. Low aspect ratio MWCNTs can be
engulfed by macrophages, while it is not the case with high aspect ratio
MWCNTs. Reproduced with permission from Nature Biotechnology.170
Copyright 2008 Nature Publishing Group. (b) Besides their dimensions,
other considerations should be taken into consideration when it comes
to safety of CNTs, such as preventing their aggregation and increasing
their solubility, which makes excretion of urine easier and thereby
preventing accumulation of tissue. Reproduced with permission from
Nature Biotechnology.170 Copyright 2008 Nature Publishing Group.
(c) Schematic illustration of the factors aecting CNT toxicity. It shows
how these factors such as functionalization, impurities, size, and shape
have the ability to mediate CNT toxicity.171
been reported by Haniu et al., 2010, that impurities are involved

in the inhibition of cell proliferation.91
Those that argue against these studies point to a report by
Cheng et al., 2009, performed on human macrophage cells
instead of rat cells.92 The group did not nd dierences in cell
death between puried MWNTs via heat and unpuried tubes.
Another piece of evidence that supports this hypothesis or the
lack of it is the absence of toxicity in Fe2O3 treated tubes.
Additionally, the Haniu et al. 2010 study found that puried and
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and facilitate their uptake by macrophages.102 A study of

Di Giorgio et al., 2011, showed the mechanism of toxicity by the
brous surface of MWCNTs and SWCNTs, though the
mechanisms are dierent. In the case of MWCNTs, the plasma
membrane damage and aberrant phagocytosis caused the toxicity
due to the brous shape of MWCNTs. In the case of SWCNTs,
impurities may play the primary role in the induction of oxidative
damage to cells.104 Results of a Walker et al. 2009 study
conducted in vitro indicate that both of the puried SWCNT and
MWCNTs induce similar eects with no sign of toxicity at
low concentrations (0.040.4 g/mL).105 As a result, we can
conclude that dierences among SWCNT and MWCNTs in
their ability to form aggregates and in variability in size and
surface state can be expressed as variability in toxicity among
CNTs types.
4.5. Toxicity Eects of Solubilizing Agents. The
attractive interaction between individual CNTs and their
tendency to form bundles106 require an agent to help their
dispersion in aqueous media. Results of a comparison between
SWCNTs solubilized in natural dispersant (gum arabic, amylose,
Suwannee River natural organic matter) and synthetic ones
(polyvinylpyrrolidone, Triton X-100) on both prokaryotic
(E. coli) and eukaryotic (WB-F344 rat liver epithelial cells)
cells clearly indicated that the toxicity of solubilizing agent
increases SWCNTs toxicity.107 Dispersion of MWCNTs has
been successfully obtained using DNA. Such dispersions involve
noncovalent interaction between DNA strands and surface
of MWCNTs resulting in high solubility and stability without
loss of electrochemical properties.108 In contrast, use of sodium
dodecyl sulfate (SDS) as solubilizing agent for SWCNTs induced
growth inhibition in normal rat kidney epithelial cells (NRK-52E)
at relatively low concentration whereas higher concentrations of
SDS solubilized SWCNTs were found to cause apoptosis and
genotoxicity.109
4.6. Eect of CNT Functionalization on Toxicity.
Functionalization of CNTs involves the modication of their
surface by introducing functional groups that may result in
enhanced solubility in aqueous media.110 The process may be
either a covalent modication or a noncovalent one111 and can
help in achieving greater compatibility with physiological systems
by resulting in higher solubility in aqueous media.25,112,113 Study
performed by Coccini et al., 2010, correlated the reduction of
cytotoxicity of CNTs with chemical functionalization that
increase the solubility, improve the dispersibility, and reduce
the CNTs agglomeration.110
Another advantage of functionalization is the ability to
conjugate CNTs with various groups that help in cell specic
targeting25 and cellular processing and elimination.114 In the past
few years, many promising studies demonstrating the reduction
of toxicity by functionalization have been reported. Use of biological molecules such as proteins and antibodies in conjugation
might help with the selective binding of CNTs to target
biomolecules and reduce the amount of the material needed.115
Functionalized MWCNTs were capable of crossing cell membrane in RAW 264.7 murine macrophages and nonphagocytic
A459 human lung carcinoma cells. Additionally, functionalization
with 220 kDa lectin protein was shown to reduce toxicity and
apoptosis in J774A macrophage when compared with unpuried
CNTs, puried CNTs, and uorescein isothiocyanate functionalized CNTs, suggesting the ability of the functional group
(220 kDa lectin) to interact with receptors present on macrophages.116 On the other hand, functionalization (COOH SWCNT)
with carboxylic acid was shown to induce higher toxicity compared
to nonfunctionalized SWCNTs in the HUVEC cell line.117 This

proves the role of type of functionalizing group in the induction
or reduction of toxicity and the importance of the right selection
of functionalizing group for the intended application.
4.7. Eect of Aggregation State. There is a direct
relationship between the aggregation state of CNTs and their
relative toxicity. The higher the agglomeration state of CNTs, the
more toxic they are. The reason is higher agglomeration state
results in bigger, stier, and more solid CNTs agglomerates.118
The same concentration of SWCNTs with dierent agglomeration states was reported to have dierent impacts on toxicity in
vitro, showing that toxicity increases with increase in the
agglomeration state of SWCNTs (Figure 5).119 In vivo, a high
degree of agglomeration of MWCNTs was shown to result in
increased accumulation of CNTs in vital organs like such as
the lungs and livers, subsequently initiating an inammatory
response.120
We can thus conclude that a number of factors aect the
toxicity of CNTs including their physicochemical properties such
as length, type, and degree of agglomeration, the amount of
metallic impurities, the type of solubilizing agent, and the type of
functionalizing group on their surface. Modifying these factors
would have a strong inuence on the production of biocompatible and less toxic CNTs. Further, functionalization with
specic favorable groups can be used in reduction of toxicity of
CNTs.
5. MECHANISMS OF CNT TOXICITY

The type and physical properties of CNTs mentioned in the
section above have a great inuence on the type and degree of
toxicity. The literature reporting the mechanisms of toxicity
presents three possible mechanisms of CNTs cytotoxicity that
include oxidative stress, cell membrane injury, and genotoxicity
as discussed below.
5.1. Oxidative Stress. Oxidative stress is dened as
imbalance between the production of reactive oxygen species
(ROS) or levels of oxygen free radicals that overcome the
antioxidant defense system.121 This imbalance can be expressed
as diminished levels of antioxidants, such as GSH or increase in
the production of reactive oxygen species. The resultant ROS can
easily oxidize proteins, nucleic acids, and fats, resulting in loss of
function of these molecules that end with disturbed homeostasis.102,122 In vitro studies show both decrease in GSH levels
and increase in ROS production as a function of increased
SWCNT concentration.123 Similarly, decrease in GSH was
proposed to be involved in E. coli loss of viability induced by
SWCNT.124 Similar pattern of cytotoxicity was observed for
MWCNT in dose-dependent manner for human embryonic
kidney cells (HEKs).99 Jiang et al., 2013, showed recently that
surface functionalization of MWCNTs is a determining factor for
ROS cytotoxicity inside the cell.125 In vivo, it has been shown that
intravenously injected SWCNTs in mice showed low toxicity.126
On the other hand, pretreatment of neuronal PC12 cells with
dierent concentrations of vitamin E (VE) has been shown to
protect against SWCNTs induced toxicity in a dose-dependent
manner by increasing cell viability, reducing cell apoptosis, and
reducing ROS formation.127 The induction of oxidative stress by
CNT has been shown to be inuenced greatly by their physical
and chemical properties104 and by the presence of reactive
species and transition metals.88,128 In fact, accumulation of
nanomaterials inside the cell or at higher concentrations can by
itself participate in oxidative stress induction or cell death.129
Oxidative stress was shown to interfere with cellular signaling
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Figure 5. Pulmonary toxicity of single-wall carbon nanotubes in mice 90 days after intratracheal instillation with 0.5 mg of a test material per mouse.
(a) Serum control. (b) Carbon black. (c) CNT. Abnormal appearances were present in parts of the lung that were exposed to NT. (d) PNT, even
distribution of particles. (e) RNT. Granulomas may result due to the presence of clusters of black pigment. (f) RNT. Dorsal view shows some necrotic
changes. (g) Carbon black. (h) Quartz, showing lymphocytes gathered around the area where macrophages, containing quartz particles, are present.
(i) CNT. Granulomas contained black particles. (j) RNT. Granulomas at low magnication. (k) RNT. Granulomas at high magnication. (l) PNT.
Necrosis of a large granuloma. Parts al are reproduced with permission from Toxicological Sciences.172 Copyright 2004 Oxford University Press.
(m) Schematic gure showing the dierent eects of CNTs addition to various tissues. It causes some modulations in the immune system which may
lead to cancer. They also aect platelets formation as well as destruction of the red blood cells membrane. In some cases, they can cause cell death.
also reported in association with reduced GSH levels after

MWCNTs treatment.99
5.2. Membrane Injury. Many reports have considered
membrane damage as another possible way in which CNTs can
induce cytotoxicity. Among these reports, the study of Hirano
et al., 2008, demonstrate the role of membrane injury and not
oxidative stress in cytotoxicity when exposing highly puried
MWCNTs (67 nm) to mouse macrophages (J774.1).138 These
results were conrmed by loss of both oxidative stress signal
transduction and absence of the eect antioxidant N-acetylcysteine
(NAC) as well as glutathione synthesis inhibitor, buthionine
sulfoximine BSO on cytotoxicity. In addition, both of the apoptotic
and MAP kinases pathways were not activated signicantly by
the treatment. The study proposes that membrane extension in the
course of MWCNTs engulfment results in membrane injury.
In another study, dendritic cells (DCs) were able to phagocytose
MWCNTs and show no cytotoxicity or immune activity.139 In fact,
macrophages extension along the CNTs ber to engulf particles
may cause lysosomes damage and induce cell death by necrosis. In
addition, ROS can be produced as a result of membrane injury.104
Another report shows that double wall CNTs (DWCNTs) aect
membrane integrity through induction of potassium eux that
leads to Nlrp3 inammasome activation, with no role of ROS in
cytotoxicity.140
5.3. Genotoxicity of CNT. Genotoxicity represents any
chromosomal or DNA damage which includes gene mutations
and chromosome breaks and rearrangements.141 There are
many factors that may assist CNTs interaction with genetic
material and development of genotoxicity, including its ability to
penetrate nuclear membrane,104 the similarity in size to those to
which in turn may exert apoptosis. In addition, oxidative DNA

damage, cytotoxicity, and necrosis were reported as consequences of CNTs induced oxidative stress.104 Gene expression
analyses of lung and skin cells treated with MWCNTs have been
linked with serious retardation of some of the major cell
functions. These aects range from reduction of cellular growth
and metabolism at low doses to reduced proliferation and
induction of innate immune responses at higher ones. Apoptosis,
necrosis, and a possible G2/M cell cycle block all may be
involved in the reduction of proliferation.130 Furthermore,
Cheng et al., 2011, reported that SWCNTs delay the cell cycle
phase and inhibit cell growth that could result in loss of cell
proliferation.123 Pathways dependent on NADPH-oxidase
activity have been primarily implicated in plumanory responses
to SWCNT oxidative stress and lung toxicity, most likely caused
by mitochondrial dysfunction.131 Others have also shown that
SWCNT induced hydroxyl and oxygen radical generation can
activate several molecular pathways that include MAPK, AP-1,
NF-B, and Akt signaling.132134 Finally, oxidative biodegradation pathways such as enzyme generated hypochlorite and
myeloperoxidase reactive intermediates might play a role in ROS
response to SWCNT.135,136 Oxidative stress induced by MWCNTs
in rat lung epithelial cells (LE) resulted in apoptosis and immense
DNA breakage by inhibiting cell viability and increased level of
tumor suppressor proteins p53, p21 and bax proteins. NF-B
activity increased with signicant reduction of IB. Additionally,
ATP and ADP/ATP ratio shows marked decline in cellular ATP
content and cytochrome c release.137 Increased lipid peroxidation,
which is one of the major manifestations of oxidative damage, was
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microtubules,142 and high anity of SWCNTs to G-C rich region

in DNA sequences.143 Lindberg et al. showed that SWCNTs and
MWCNTs induced DNA damage in mesothelial cells in vitro.144
Genotoxicity of MWCNTs was reported not only to human and
mammalian cells but also on plant cells (Allium cepa) and plasmid
DNA (pBR322).145,146 Carbon nanomaterials may contribute
directly (primary genotoxicity) or indirectly to genotoxicity. The
direct way can take place when nanomaterials interact with DNA
or mitotic apparatus, and the indirect way may result from oxidative
stress and inammatory responses.147
5.3.1. Primary Genotoxicity of CNTs. Primary genotoxicity of
CNTs ensued by the direct interaction of particles with the DNA.
Muller et al., 2008, demonstrate the direct induction of genotoxicity by MWCNTs through aneugenic and clastogenic
eects.148 The aneugenic eect or chromosome loss can be
explained by physical interaction of CNTs with components of
the mitotic spindle during cell division or the interaction with
proteins that are directly or indirectly involved in chromosome
segregation like tubulin and actin resulting in genetic instability,
in the form of micronuclei chromosomal imbalances or aneuploidy
in daughter cells. On the other hand, the clastogenic eect involves
the formation of adducts or chromosomal aberrations. Additionally, physical association between SWCNTs and DNA, microtubule
and centrosome fragments were reported to cause multipolar
mitotic spindles and aneupoidy.149
5.3.2. Secondary Genotoxicity of CNTs. CNTs indirect
genotoxicity arises by the generation of ROS by inammatory
cells, when they interact with dierent cell components that
might result in clastogenic eect of CNTs.129 In fact, the
generated ROS are able to interact with DNA, causing oxidation
of DNA, DNA breakage, or lipid peroxidation mediated DNA
adducts.150 The link between oxidative stress and genotoxicity
has been described in many reports.104,129,151,152 Interestingly,
purity and dispersion properties exert nongenotoxic eect in
SWCNTs, suggesting that minimizing factors associated with
ROS production such as purity of CNT may reduce the chance of
genotoxicity.153
5.4. Interaction with the Immune System. It is interesting
that CNTs can be recognized by vertebrate innate immune
system. The recognition takes place via the complement system,
which is composed of over 35 proteins and glycoproteins present
in cell surface body uids.154 Salvador Morales et al. in 2006 were
the rst to identify the pathway of activation of human serum
complement system, which may generate the inammatory
peptides C3a, C4a, and C5a(c).155 Although nonfunctionalized
SWCNTs, DWCNTs, and MWCNTs and most functionalized
CNTs in vitro can activate complement systems, there are other
ways in which some types of CNTs can activate the complement
including the lectin pathway. Functionalization of CNTs does
not prevent complement activation, but it can minimize it.156
Additionally, exposure of CNTs in some cell types has been
associated with inammation. High concentration of MWCNTs
has been shown to induce an overexpression of genes related to
immune response including innate immune response and
inammation in human skin broblast.130 HEK293 cells exposed
to MWCNTs for 48 h cause a time-dependent signicant
increase in IL-8 level.99
In summary, physical properties of CNT (length, type) metal
catalyst impurities, solubilizing agent, functionalization of CNT,
cell type, and aggregation state play an important role in the
biological responses to CNTs. The toxicity mechanisms may
involve oxidative stress; some studies propose membrane injury.
Genotoxicity may also result.
6. CONCLUSIONS
Carbon nanotubes (CNTs) are a novel class of nanomaterials
that have enormous potential for applications in biomedical
systems including delivery of therapeutics, biomedical imaging,
and biosensors and as scaolds for tissue engineering. With their
unique physicochemical properties, CNTs can be functionalized
with various biomolecules either covalently or noncovalently
to increase their biocompatibility and decrease the cytotoxicity.
The strategy of functionalization has been shown to be eective
in various studies for drug delivery and gene delivery systems.
However, the cytotoxicity has still remained as the limiting factor
for use of CNTs in biological systems. As short-walled or multiwalled nanotubes are developed, numerous studies have been
conducted to understand the relation between their physical
properties and cytotoxicity. Bioactivity of MWCNTs has been
shown to be aected by their diameter, length, and functionalization in vitro and in vivo.157 Similarly, SWCNTs toxicity is
shown to be aected by their size and length. These parameters
are found to result in cytotoxicity by causing changes in the ROS
generation.158 The method of CNT synthesis inuences the
viability and behavior of cells and the interaction of CNTs with
cells. The presence of metal impurities in CNTs has substantial
role on response of cells, and it has been shown that metal
impurity such as iron can induce cytotoxic and genotoxic
eects.159 Understanding the factors responsible for the CNT
toxicity is required for further development of safe CNTs based
applications. Dierent ways in which CNTs cause toxicity
have been recognized, but the mechanisms involved are still in
investigational stages. Recently, genotoxic eect of CNT by
direct contact with DNA was shown to induce mutations in
DNA.160 Also, genome wide gene expression study has been
performed in vivo in animal models to understand the molecular
mechanism of CNTs toxicity at the organism level.161 Although
several studies have been performed employing new approaches
for toxicological characterization of CNTs in biological systems,
complete understanding of the cellular uptake, internalization,
and changes in gene expression associated with the CNT toxicity
has still remained elusive. Such understanding will be required for
future development of CNTs for biomedical applications.
AUTHOR INFORMATION
Corresponding Authors
*A.H.: e-mail, ahasan@qu.edu.qa and mh211@aub.edu.lb;

phone, +97470569169.
*A.M.: e-mail, amemic@kau.edu.sa; phone, +966564975404.
Notes
The authors declare no competing nancial interest.

Biographies
Reem Alshehri received her B.Sc. and M.Sc. in Biochemistry from King
Abdulaziz University, Jeddah, Saudi Arabia. Her research area
concentrated on the mechanisms of mercuric compound toxicity and
their role in initiating of oxidative stress, autoimmunity, and response of
autistics to organic forms of mercury. Her research interests include
molecular mechanisms contributing to autism and neuroscience and
using nanotechnology tools in the treatment and diagnosis of
neurodegenerative diseases.
Asad Muhammad Ilyas graduated with a B.Sc. in Biology. He received
his Masters degree from University of Skovde, Sweden, with a major in
Molecular Biology in 2010. He completed his Ph.D. in Biochemistry
with a major in Molecular Biology from King Abdulaziz University in
2016. His research focuses on targeting multiple signaling pathways for
the treatment of acute myeloid leukemia, including interventions using
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Perspective
interdisciplinary research award from American University of

Beirut, and the CNRS grant from National Council for Scientic
Research, Lebanon.
nanotechnology. In addition, his research focuses on studying the

mechanism of cancer related genes in the development of leukemic
hematopoiesis.
Anwarul Hasan, Ph.D., P.Eng., is an Assistant Professor of Biomedical

Engineering and Department of Mechanical Engineering at the
American University of Beirut, Lebanon, and the Department of
Mechanical and Industrial Engineering at Qatar University, Qatar. He is
also a visiting Assistant Professor at the Harvard Medical School (HMS)
in Cambridge, MA, U.S. Earlier, Dr. Hasan worked in industry during
20102011 and was a National Science and Engineering Research
Council of Canada (NSERC) Fellow at Harvard and Massachusetts
Institute of Technology during 20122013. Dr. Hasan obtained his
Ph.D. from University of Alberta, Canada, in 2010. His current research
interests are in the areas of biomaterials, biomechanics, and tissue
engineering, particularly for cardiovascular, bone, and neural applications.
ABBREVIATIONS USED
Ach, acetylcholine; ADP, adenosine diphosphate; ATP,
adenosine triphosphate; BAX, BCL2-associated X protein;
BSO, buthionine sulfoximine; CD, cluster of dierentiation;
CNS, central nervous system; CNT, carbon nanotube; CP3,
proapoptotic protein caspase-3; CVD, chemical vapor deposition; DC, dendritic cell; DHP, dihexadecyl phosphate; DWCNT,
double wall carbon nanotube; dsDNA, double stranded
deoxyribose nucleic acid; FET, uoroethyl-L-tyrosine; GSH,
glutathione; HUVEC, human umbilical vein endothelial cell;
IL, interleukin; MAPK, mitogen-activated protein kinase;
DNA Mtase, DNA methyltransferase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide); MWCNT, multiwalled nanotube; NAD, nicotinamide adenine dinucleotide;
NADP, nicotinamide adenine dinucleotide phosphate; NF-B,
nuclear factor -light-chain-enhancer of activated B cells; PDA,
polydopamine; PEG, polyethylene glycol; PET, positron
emission tomography; PLFA, poly(lactic-co-glycolic); ROS,
reactive oxygen species; SDS, sodium dodesyl sulfate; ssDNA,
single stranded deoxyribose nucleic acid; SWCNT, single-walled
nanotube; TNF, tumor necrosis factor; WST-1, water-soluble
tetrazolium salt
Adnan Arnaout is a fourth year mechanical engineering student at the

American University of Beirut, minoring in biomedical engineering, with
special interest in tissue engineering. He is an undergraduate researcher
at the Nano Micro-Technologies and Tissue Engineering Lab at the
American University of Beirut. His research focuses on developing
hydrogels for tissue engineering, as well as microuidics.
Farid Ahmed completed his Ph.D. in Human Biology from the Ludwig
Maximilians University, Munich, Germany, in 2006. He performed his
postdoctoral studies at the Clinical Co-Operative Group-Leukemia in
Munich. Dr. Ahmeds current research interests are studying the
molecular regulation of normal and malignant hematopoiesis and HSC
self-renewal with the hope to identify underlying processes that lead to
leukemic transformation and progression. Projects in Dr. Ahmeds
group focus on identication and characterization of candidate leukemic
stem cell (LSC) in hematopoietic malignancies and therapeutic
targeting of the leukemic cells that drive human leukemia. Dr. Ahmed
is currently a member of several scientic societies such as International
Society for Stem Cell Research (ISSCR), European Hematology
Association (EHA), and ISEHSociety for Hematology and Stem
Cells.
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Adnan Memic graduated summa cum laude with a B.Sc. in Chemistry.

He received his Ph.D. in Chemistry/Biochemistry from Wayne State
University, Detroit, MI, U.S., with Mark Spaller. He was a postdoctoral
fellow in Chicago with Brian Kay at the University of Illinois. He was
previously a Visiting Assistant Professor of Toxicology and Pharmacology at Dartmouths Geisel Medical School. He joined King Abdulaziz
University in 2010, was promoted to Associate Professor of Nanotechnology, and is concurrently a part-time Lecturer on Medicine at
Harvard Medical School. His research focuses on bioactive molecule
discovery and development including generation of biomaterials, carbon
nanomaterials, chemical and peptide analog libraries, protein and
antibody engineering toward solving challenges in targeted drug
delivery, biosensing, and tissue engineering and regenerative medicine
applications.
ACKNOWLEDGMENTS
This project was funded by the National Plan for Science,
Technology and Innovation (MAARIFAH), King Abdulaziz City
for Science and Technology, the Kingdom of Saudi Arabia,
Award 12-MED3096-3. The authors also acknowledge with
thanks Science and Technology Unit, King Abdulaziz University.
A.H. acknowledges Grant NPRP9-144-3-021 from Qatar
Foundation, Grants QUUG-CENG-MIE-15/16-7 and QUSTCENG-FALL-15/16-20 from Qatar University, the startup grant,
the University Research Board grant, and the Farouk Jabre
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Carbon Nanotubes in Biomedical Applications: Factors, Mechanisms,

Center of Nanotechnology, King Abdulaziz University, Jeddah, 21589, Saudi Arabia

ABSTRACT: Carbon nanotubes (CNTs) represent one of the most

Received: November 12, 2015

Journal of Medicinal Chemistry

and an array of functional group and biomedical applications4,24

3. CNTs IN BIOMEDICAL APPLICATIONS

2. CARBON NANOTUBES SYNTHESIS

Journal of Medicinal Chemistry

neural tissue cells and such studies could help in developing

Journal of Medicinal Chemistry

This feature can make them an eective tool in gene silencing

using noncovalent interactions allowing for selective targeting and

Journal of Medicinal Chemistry

made them a powerful tool to obtain fast biological species

hand, gene silencing was also successfully performed on many

Journal of Medicinal Chemistry

approaches, cells are seeded or encapsulated in a suitable

of CNTs have made them extremely important for biosensing

Journal of Medicinal Chemistry

the bers have enhanced the myotube contraction amplitude, as

microenvironments, tracking of cells, and delivery of suitable

Table 1. Comparison of CNT Toxicity in Vitro Using Biological Model Systems

human vein endothelial cells

dosage and outcome

HeLa, KB-31, and KB-85 cells

exhibited low toxicity at concentration of 0.001100 mg/L

MCF-7 breast cancer

1 mg/mL, no signicant cell damage

human alveolar (A549) epithelial cells

1100 g/mL induced membrane damage, particularly in BEAS-2B cells

520 g/mL for up to 24 h incubation showed cell surface changes

At 100 g/mL, three of the tested nanotubes activated immune-related

functionalization completely eliminated inammasome activationat

three cell lines, LA4, MHS, and JAWSII

murine alveolar macrophages (MH-S)

Journal of Medicinal Chemistry

Table 2. Comparison of CNT Toxicity in Vivo Using Biological Model Systems

dosage and outcome

male ICR mice

Single 600 g injection showed no observable toxicity.

0.064 mg/kg oral administration, genotoxic eect

male Wistar rats

intraperitoneally into female

0.012 mg, low toxicity

60 and 100 mg/kg, liver toxicity observed

20 g/L PBS, no acute toxicity after single 200 L dose

10 g, low level acute toxicity

4.1. What Makes CNTs Cytotoxic? Factors that contribute

cells or rat macrophages were not increased by their presence.

Journal of Medicinal Chemistry

unpuried MWCNTs signicantly altered the level of expression

Figure 4. Factors aecting carbon nanotubes toxicity on blood cells.

been reported by Haniu et al., 2010, that impurities are involved

Journal of Medicinal Chemistry

and facilitate their uptake by macrophages.102 A study of

to nonfunctionalized SWCNTs in the HUVEC cell line.117 This

5. MECHANISMS OF CNT TOXICITY