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Review Article Open Access
Nanomedicine & Nanotechnology
Jain et al., J Nanomed Nanotechol 2012, 3:5
http://dx.doi.org/10.4172/2157-7439.1000140
Volume 3 • Issue 5 • 1000140
J Nanomed Nanotechol
ISSN:2157-7439 JNMNT an open access journal
Keywords: Carbon nanotubes; Toxicity; Biomedical applications
Introduction
Nanotechnology is the study of materials ranging in size from 0.1-
100 nm. Nanoscale materials have been categorized in dierent ways
by many popular ocial sources. For example, the National Academies
categorized nanoscale materials into four groups: (1) the metal
oxides (2) nanoclays (3) nanotubes and (4) quantum dots [1]. e
US Environmental protection agency (EPA) has classied them as (1)
carbon–based materials (2) metal based materials (3) dendrimers (4)
composites including nanoclays. Among these nanomaterials, CNTs
have generated signicant interest since Iijima [2] discovered CNTs
while synthesizing fullerenes in 1991. ese unique structures possess
tremendous strength, very high aspect ratio and excellent thermal and
electrical conductivity. us CNTs have found commercial applications
in electronics [3,4] optics [5], materials science [6,7] and architecture
[8]. More recently, CNTs have been used in biomedical engineering,
tissue engineering, drug delivery, gene therapy and biosensors.
However, the successful commercial application of CNTs in medicine
may depend largely upon their toxicity for humans, animals and the
environment. Chemical modication of CNTs in order to make them
more biocompatible is one of the major areas of current research
[9]. e intent of this review is to highlight various toxicity issues,
including the toxicity of CNTs at the cellular, genetic and systemic
level. e paper critically evaluates the ways to overcome toxicity in
order to exploit the complete potential of CNTs as an accessory tool for
various biomedical applications and allied elds. It also discusses the
regulatory concerns, risk of accidental exposure and the future market
potential of CNTs.
Classication and general properties of CNTs
CNTs are long chains of carbon held together with each carbon atom
bonded to three neighbouring carbon atoms forming sp2 hybridized
carbon. Since sp2 hybridization is stronger than the sp3 hybridization
found in diamond, CNTs have unique strength. From an atomic point
of view, a nanotube can be divided into two parts; the side-wall and the
end cap. e end caps can be considered as hemispherical fullerenes,
curved in 2D and the side wall contains less distorted carbon atoms and
is curved in 1D. CNTs are mainly classied into two types depending
upon the structure: (1) Single Walled Carbon Nanotubes (SWCNTs)
(2) Multiwalled Carbon Nanotubes (MWCNTs). SWCNTs are formed
when just one graphene sheet rolls up to form a tubular structure,
while more than one concentric graphene sheets creates MWCNTs
[10]. e width of CNTs ranges from a few to tens of nanometers, but
their length ranges from less than a micrometer to few millimeters.
CNTs can be classied as Carbon Nanohorns (CNHs), nanobuds and
nanotorus, depending upon their shape [11] .
Applications of CNTs
CNTs have been exploited for applications in drug delivery, gene
therapy, cancer therapy, vaccine delivery, imaging and diagnostics. Out
of the several applications, a few are discussed in more detail below.
CNTs in controlled and targeted drug delivery: CNTs have been
used to improve the pharmacological and therapeutic prole of a drug
molecule. e ability of functionalized CNT to penetrate into cells has
been exploited for the delivery of small drug molecules in a controlled
manner [12,13]. CNTs can be simultaneously functionalized with
moieties for targeting, imaging as well as therapy [14]. e outer surface
of the CNTs is modied to improve its solubility and biocompatibility
and the inner hollow core is used for insertion of the drugs [15]. A
multi-functionalization strategy was used to functionalize CNTs with
a uorescent probe for tracking aer cellular uptake and an antibiotic
moiety, amphotericin B was covalently linked for its controlled delivery
without any toxic eects [16,17]. e use of CNTs is widely accepted
for the treatment of cancer as they are capable of targeting malignant
cells because of better uptake by a specic population of malignant
cells without aecting the healthy cells and rapid elimination from
the body following systemic administration (Figure 1). Prato and
group also suggested that the most important factors governing the
fate of CNTs inside the body are their shape and size. Since they are
nanosize, they easily escape cellular opsonization or phagocytosis
*Corresponding author: Shreekumar Pillai, Department of Math and Science, Alabama
State University, P.O. Box 271, 915 S Jackson Street, Montgomery, AL 36101-0271,
USA, Tel: 334-229-7501; E-mail: spillai@alasu.edu
Received May 08, 2012; Accepted May 25, 2012; Published June 01, 2012
Citation: Jain S, Singh SR, Pillai S (2012) Toxicity Issues Related to
Biomedical Applications of Carbon Nanotubes. J Nanomed Nanotechol 3:140.
doi:10.4172/2157-7439.1000140
Copyright: © 2012 Jain S, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Toxicity Issues Related to Biomedical Applications of Carbon Nanotubes
Sapna Jain1, Shree R. Singh1 and Shreekumar Pillai2*
1Center for NanoBiotechnology Research, Alabama State University, Montgomery, Al-36104, USA
2Department of Math and Science, Alabama State University, P.O. Box 271, 915 S Jackson Street, Montgomery, AL 36101-0271, USA
Abstract
Carbon Nanotubes (CNTs) have emerged as a potential candidate for application in targeted drug delivery,
cancer treatment, gene therapy and diagnostics. This review critically evaluates the biomedical potential of CNTs,
and possible toxicological implications. Success of CNTs in other elds has not yet been translated to the biomedical
eld due to the lack of consistent data on toxicity, variation of toxicity due to characteristics such as shape and size
along with the method of preparation, and limited control over functionalized CNTs behavior. Thus there is a lack of a
predictable toxicity pattern of CNTs. This review summarizes the key ndings on the toxicity of CNTs and the role of
functionalization with hydrophilic moieties to reduce their toxicity, and increase cell penetrability for drug delivery and
gene therapy applications. Caution is urged when handling this ‘wonder material’ but the immense potential for its
commercial utilization and clinical trials infuses hope for future biomedical applications.
Citation: Jain S, Singh SR, Pillai S (2012) Toxicity Issues Related to Biomedical Applications of Carbon Nanotubes. J Nanomed Nanotechol 3:140.
doi:10.4172/2157-7439.1000140
Page 2 of 15
Volume 3 • Issue 5 • 1000140
J Nanomed Nanotechol
ISSN:2157-7439 JNMNT an open access journal
as a harmful foreign intruder [18]. Delivery of drug to tumor site is
through Enhanced Permeability and Retention (EPR) eect [19]. In
healthy tissues, the pore size of blood vessels is much smaller (2-6 nm)
as compared to tumor tissues (100-800nm) [20]. us, nanoparticles
ranging from 100-700nm cannot penetrate the healthy blood vessels
but easily penetrate inside the tumor blood vessels and accumulate at
the tumor site. Poly Ethylene Glycol (PEG) functionalized CNTs can
be conjugated with a cancer chemotherapy drug, Paclitaxel (PTX)
via the amide bond formation between the PEG chains and paclitaxel
and eectively inhibit cancer growth in mice [21]. PEG was used to
functionalize SWCNTs as the SWCNT-PEG complex did not cause any
toxicity in mice over several months aer intravenous injection. e
SWCNT-PTX conjugated were retained in blood for longer duration
(81.4 ± 7.4 min) than taxol (clinical drug formulation of PTX) (18.8 ±
1.5 min) and PTX coated with PEG (22.8 ± 1.0 min). us enhanced
EPR eect, prolonged circulation, better delivery ecacy and tumor
inhibition of the drug is achieved by SWCNT-PTX ( 5 mg/kg PTX) for
the 4T1 tumor model which is known to be resistant to PTX treatments.
CNTs as a non viral gene delivery system: Ammonium
functionalized CNTs are exploited for their application as a
delivery vector for gene-encoding nucleic acids. Stable complexes
can be formed from cationic CNTs and plasmid DNA, which are
demonstrated to enhance gene therapeutic capacity in comparison to
DNA alone [18] (Figure 2). e gene expression was upregulated 10
times higher than those achieved with DNA alone [22]. Ammonium
functionalized SWCNTs have been used to mediate the delivery of
Telomerase Reverse Transcriptase (TERT) siRNA into tumor cells. e
release of siRNA silences the targeted TERT gene that is critical for
the development and growth of tumors. Administering the SWCNT-
TERT-siRNA complex in mice bearing the Lewis carcinoma tumor,
resulted in suppression of the cancer cell growth and reduction of
average tumor weight [23]. Ammonium functionalized SWCNTs
were complexed with double stranded DNA and multifunctionalized
with phospholipid-PEG containing a tumor targeting moiety (folic
acid) [20]. Cationically functionalized CNTs with polyethyleneimine
and pyridinium moieties (CNT-PEI and CNT-pyridinium) have been
studied for siRNA delivery. Both functionalized CNTs complexed
with siRNA showed reduced toxicity and increased silencing activity
[24,25]. e ammonium functionalized SWCNT have been employed
for the delivery of temolerase reverse ranscriptase (TERT) siRNA into
tumor cells. ese functionalized CNTs released the siRNA to silence
the targeted TERT gene, which is critical for the development and
growth of the tumors. It was observed that the treatment cancer cells
with SWCNT-TERT-siRNA complexes resulted in suppression of the
cancer cells growth. It was concluded that functionalization of CNTs
might act as a key factor in obtaining an ecient nontoxic CNT-based
delivery system [21].
CNTs in diagnostics: CNTs can be useful in determining the
cause of disease (pathogen detection), level of disease progression
and pathological condition by acting as articial smart devices as
nanosensors and nanorobots [11]. CNT–based biosensor devices have
applications in detecting biological disorders such as autoimmune
diseases by exploiting the fact that it is able to detect antigens associated
with such diseases. ese sensors have also been used during space
missions as these nano-devices can be administered through skin
[26,27]. CNT based piezoelectric pressure sensor has the capability of
detecting the changes in resistance in SWCNT as the pressure is applied.
is can nd applications in blood pressure monitoring [28,29].
SWCNTs were used to modify glassy carbon electrodes for detection
of DNA from Salmonella enterica serover Typhimurium using
electrochemical impedimetric sensing technique. Electrode surface was
coated with single stranded DNA (ssDNA) functionalized SWCNTs.
e target DNA sensing was accomplished by measuring the change
in the impedance and charge transfer resistance before and aer the
complementary ssDNA binding takes place [30]. e immunological
sensor based on functionalized gold electrode allowed rabies antigen
detection [31]. e specic rabies antibodies were immobilized onto the
functionalized gold microelectrode and the interaction of the antibody
with specic antigen was measured with low limit detection with a
good reproducibility with impedance spectroscopy. Electrochemical
immunosensors using SWCNTs conjugated with antibody for highly
sensitive detection of a cancer biomarker in serum and tissue lysate
have been reported [32]. Amplied sensitivity was achieved by
Drug
Normal
drug delivery
drug loaded
inside the hollow
CNTs
Tumor targeting
ligards
(specific for the
target cells)
Targeted
drug delivery
Healthy cells
Receptor
nucleus
Healthy cells
target cell
(cancer cell)
target cell
(cancer cell)
nucleus
Figure 1: Efcient targeted drug delivery using CNTs versus inefcient non-
targeted drug delivery.
DNA
(a)
(d) (e)
(f)
(h)
(g)
(c)
(b)
DNA
wrapped
around
CNT
DNA
covalently
attached
to CNT
or
CNT-DNA
complex
enter
No entry
Traveling
gene expression
Golgi body
mitochondria protein
lysosomes
Hydrolytic
enzymes
Escaping
endo-lysosomal
pathway
DNA released
through cytoplasm
release to enter
nucleus
NH3
+
NH3
+
NH3
+
NH3
+
nucleus
endosomes
Figure 2: Gene delivery by CNTs (a) Plasmid DNA (DNA) by itself is unable
to penetrate the cell membrane (b) DNA is either covalently or non-covalently
attached to CNTs (c) DNA conjugated CNTs gain entry inside the cell (d)
DNA released in the cytoplasm (e) DNA travels through cytoplasm to enter
the nucleus (f) gene expressed (g) DNA-CNT complex escapes from endo-
lysosomal pathway and subsequent degradation by hydrolytic enzymes and (h)
nally goes to mitochondria.
Citation: Jain S, Singh SR, Pillai S (2012) Toxicity Issues Related to Biomedical Applications of Carbon Nanotubes. J Nanomed Nanotechol 3:140.
doi:10.4172/2157-7439.1000140
Page 3 of 15
Volume 3 • Issue 5 • 1000140
J Nanomed Nanotechol
ISSN:2157-7439 JNMNT an open access journal
hydroxyl (-OH) groups are more toxic than their pristine counterparts
[51]. e toxic eects of CNTs are negligible at low concentrations (5
µg-10 µg) but in order to use higher concentrations (50 µg-500 µg),
highest purity CNTs have to be used which minimizes the toxic eects
of residual catalysts or surface oxidation [52].
Physical attributes of CNTs responsible for toxicity: e physical
attributes of CNTs, like ber shape, length and the aggregation
status can also inuence the immunological responses and their
local deposition in tissues [53]. e shape and length of CNTs can
determine the internalization of CNT by macrophages and hence the
immune response. Shorter CNTs were found to be less toxic than the
longer CNTs. Shorter length CNTs when injected subcutaneously in
rat were found in the cytosol of the macrophages aer 4 weeks, but
longer CNTs were found to be free oating and causing inammation
[54] conrmed that the toxicity of MWCNTs was length dependent
and comparable with that of asbestos toxicity. Abdominal cavities of
rats were injected with MWCNTs and asbestos bers. Aer 24 h of
exposure increased immune response was observed, and aer 7 days
of exposure, granuloma formation was observed in both cases. ey
named the increased immune response due to MWCNTs exposure as
“frustrated phagocytosis” wherein macrophages were unable to engulf
long CNTs mainly because of their length.
Surface area and surface chemistry also inuence toxicity of a
nanoparticle. In one study, ve carbon based materials, graphite,
SWCNTs, MWCNTs, active carbon, and carbon black were tested for
their toxicity on broblast cells [55,56]. SWCNTs were found to be
most toxic because they had the lowest surface area. It was found that
hydrophobic SWCNTs with low surface area induce enhanced toxic
eects. CNTs inuenced extracellular matrix protein signaling that
resulted in deformation of cell membranes, and displacement of cell
organelles nally leading to cell death. When two nanoparticles have
comparable surface area, toxicity depends on their surface chemistry
[57]. It was observed that unrened SWCNTs were more toxic than
the rened SWCNTs. is was because the unrened SWCNTs
agglomerated and their surface area decreased, and lower surface
area resulted in higher toxicity. Whereas rened SWCNTs remained
dispersed and thus were less toxic [58].
Mechanism of uptake of CNTs
Several groups have worked towards establishing the uptake
mechanism of CNTs inside the lipid membrane bound cell and
their inuence in causing toxicity. Many studies have shown that
functionalized CNTs are able to cross the cell membrane [59,60].
ere are ve methods of internalization of CNTs inside the cells,
phagocytosis, macro-pinocytosis, clathrin-mediated endocytosis,
caveolin mediated path-ways, and clathrin/caveolin independent
pathways [61]. So far, the two most widely accepted mechanisms of
CNTs internalization proposed are (i) endocytosis/phagocytosis and
(ii) nanopenetration.
Active internalization of CNTs via endocytosis
Several studies have shown that the mechanism of internalization
of CNTs is by endocytosis [60,62]. It was observed that uorescently
labeled SWCNTs conjugated with protein or DNA were able to enter
HL60 cells and HeLa cells aer 1 h of incubation at 37ºC. By contrast,
protein and DNA alone were not located inside the cells under the
same experimental conditions [63]. Low energy, low temperature or
depletion of ATP (adenosine triphosphate) inhibits the endocytosis
process. Cellular uptake of CNTs was reduced at lower temperature
conjugation of SWCNTs with horse radish peroxidase (HRP) labels
and secondary antibodies (Ab(2)) at a high HRP/Ab (2) ratio. is way,
detection of low limit (4pg/ml) of prostate specic antigen (PSA) in
10 μl of undiluted calf serum was achieved. is immunosensor holds
promise for clinical screening of cancer biomarker and point-of-care
diagnostics.
Current toxicological knowledge: e broad and increasing range
of the biological applications of CNTs and other nanoparticles has
exposed humans, animals and the environment to these nanoparticles
[32,33]. us it has become imperative to evaluate the biocompatibility
and toxicity of CNTs on both human health and the environment. As
compared to bulk materials, nanomaterials could have a range of eects
due to their increased surface area to size ratio, which makes them
more reactive and potentially more harmful [34-37]. For example,
they may not be detected by the normal phagocytic defenses allowing
them to gain access to the blood or the nervous system. CNTs could
act like haptens to modify protein structures altering their function
or rendering them antigenic, raising the potential for autoimmune
eects [11,38-40]. Several studies [41,42] on the toxic eects of CNTs
suggest dierent mechanisms as to how nanoparticles induce toxic
eects. Some of the proposed mechanisms include: (i) Reduction in
size leads to increase in their relative surface area, which allows for
a greater contact area with cellular membranes, as well as a greater
capacity for absorption and transportation of toxic substances (ii)
Dierent electronic properties of nanoparticles as compared to their
bulk form could be a potential contributing factor [40]. Reduction of
size to the nano scale creates discontinuous crystal planes increasing
the number of structural defects and disrupting the well-structured
electronic conguration and altering their electronic properties [43-
45] (iii) Dierent surface chemistry of nanoparticles which make them
hydrophobic or hydrophilic, lyophilic or lyophobic, catalytically active
or inactive [43-45] (iv)e electron donor or acceptor active sites on
the surface of the nanoparticles can react with molecular dioxygen,
forming super oxide radicals (O2
-), which through dismutation or
fenton chemistry can generate additional reactive oxygen species (ROS)
[46] (v) Specic transport properties are due to dierences in shape,
aggregation, surface functionalization, and solubility of nanoparticles
[47,48]. Most of these properties can be controlled by adopting dierent
manufacturing processes, or altering physical conditions of the process
such as temperature, pressure, catalyst, dispersion techniques, or
choice of the dispersant to name a few.
Toxicity at the level of synthesis: One of the major causes of CNTs
toxicity arises from the catalyst residues le aer their production.
Synthesis of CNTs involves the use of metal catalysts like Fe, Ni, Co,
As, Mo etc. [49] that are quite toxic by themselves. ese elements if
not removed during the purication step, tend to catalyze oxidative
processes by free radical generation. e ROS thus generated cause
oxidative damage to cells and membranes. Catalysts also act by
interfering with the immune system at the cellular level. When CNTs
are engulfed by macrophages, Nicotinamide adenine dinucleotide
phosphate–oxidase (NADPH-oxidase) inside the cell produces
superoxides (O2
-). Fe based catalysts react with these superoxides to
form hydroxyl ions, resulting in oxidative stress and damage at the
molecular level [39].
Post fabrication treatment induced toxicity: It has been noted
that purication steps like oxidation by chemical etching greatly aect
CNTs toxicity. MWCNTs subjected to oxidation with nitric acid were
more toxic to T-lymphocytes than their impure counterparts [50]. It was
noted that CNTs with added carbonyl (CO), carboxylic (COOH), and
Citation: Jain S, Singh SR, Pillai S (2012) Toxicity Issues Related to Biomedical Applications of Carbon Nanotubes. J Nanomed Nanotechol 3:140.
doi:10.4172/2157-7439.1000140
Page 4 of 15
Volume 3 • Issue 5 • 1000140
J Nanomed Nanotechol
ISSN:2157-7439 JNMNT an open access journal
(4ºC) in the cells pre-incubated with ATP inhibitors like sodium azide
[64]. CNTs can also be internalized by clathrin mediated endocytosis.
When CNTs come in contact with cells, clathrin-coated pits are formed
under the plasma membrane and each pit then forms a vesicle inside
the cell [62]. Length of the CNTs inuences the internalization process.
Shorter CNTs are able to internalize more readily than the larger ones
[65]. Cellular uptake of CNTs was greatly reduced when cells were
grown in media rich in K+ and sucrose, that are known to inhibit the
formation of clathrin-coated vesicles in the cell membrane [66,67].
Passive internalization of CNTs via nanopenetration
Nanopenetration is an energy independent process, where
nanoparticles diuse passively through cellular membranes. Studies
have indicated that CNTs more likely penetrate inside the cell
passively just as a nano-needle would diuse across the cell membrane
[68,69]. Functionalized CNTs can cross cellular barriers by altering
their interaction with cells and alteration of intracellular transport
kinetics of the functionalized CNTs. To study the mechanism of
uptake, SWCNTs and MWCNTs were functionalized by several
moities including ammonium groups, antibodies, uorescent tags,
anticancer drugs and antibiotics. All these functionalized CNTs were
internalized even in the presence of endocytosis- inhibitors [70]. FITC-
labeled CNTs were also tracked in human T36 cells and murine 3T3
broblasts, showing that the internalization of CNTs was not hindered
by lowering the temperature or by the presence of sodium azide which
inhibits endocytosis [71].
e diusion mechanism of functionalized CNTs across the
membrane is not completely understood. According to one view
[72-74] the perpendicular orientation of CNTs to cell membranes
leads them to behave like nano-syringes and diuse inside the cells.
A two-step process of diusion has been suggested. CNTs are rst
‘accommodated’ onto the cell membrane and then oriented in a way
to achieve a ‘transmembrane conguration’. Hydrophilic and static
charge interactions between the CNTs and the lipid bilayer plays a lead
role in internalization of the CNTs [75] .
So far no conclusions can be drawn in support of just one
mechanism that best describes the internalization of CNTs inside the
cells. In fact, the arguments for both pathways look quite convincing.
Nevertheless several factors like type of functionalization, density and
the charge of the ligand, shape, size, agglomeration and hydrophobic
and hydrophilic interactions between cell surfaces and CNTs will
all play an important role in determining the CNTs internalization
mechanism. CNTs surface is hydrophobic but by functionalization
with hydrophilic groups, they can be made hydrophilic. us, their
interaction with the hydrophilic regions of the cell membrane will
greatly inuence the cellular uptake of CNT [56].
Mechanism of CNTs toxicity
CNT exposed cells undergo oxidative stress because of induction
of oxidants and toxic enzymes. Higher level of oxidative stress leads
to inammation and cytotoxicity. Protein kinase and nuclear factor-
Kappa B (NF-ƙB) signaling pathways regulate proinammatory
cytokines and apoptosis in response to oxidative stress. e decrease
in cell viability and elevated levels of the proinammatory cytokines
Interleukin-8(IL-8) and IL-1β indicates that MWCNTs can initiate an
inammatory response in Human Keratinocytes (HEKs) at 0.4mg/ml
dose [76]. Apoptosis could result from mitochondrial disruption and
release of pro-apoptotic factors [77]. Several mechanisms may underlie
the toxicity of CNTs as outlined below.
Free radical formation
e main cause of toxicity is related to oxidative stress by free radical
formation. ese excess free radicals oxidize lipids, protein, and DNA.
Oxidative stress may upregulate redox sensitive transcription factors,
activator protein-1 and kinases that cause inammatory responses.
Slow clearance caused due to agglomeration or accumulation of these
nanoparticles may produce free radicals, in the organs of Reticulo
Endothelial System (RES) making organs like the spleen, kidneys and
lungs, so targets for this oxidative stress [78].
Reactive Oxygen Species (ROS)
ROS are chemically reactive oxygen containing molecules that are
formed as byproducts of the normal metabolism of oxygen. However,
the level of ROS may increase due to environmental stress such as
exposure to radiation, foreign particles etc. ROS may lead to harmful
eects in cells like apoptosis, DNA damage, amino acid oxidation and
inactivity of enzymes [79]. Studies have shown that SWCNTs damage
DNA, cause changes in the cell cycle and generated apoptotic signals
by generating ROS. Most cells grown on media containing CNTs
altered the G1 phase of their cell cycle [80]. Wang et al. [78] have
determined that apoptosis of PC12 cells was induced by 4 to 5 fold
higher concentrations of ROS in cells exposed to SWCNTs (200 µg/
ml). MWCNTs have been shown to induce ROS in Human Umbilical
Vein Endothelial Cells (HUVEC) at 20 µg/ml concentration [81,82].
Increased inammatory responses
Poland and group [54] compared inammatory responses
produced by exposing mice to carbon black, asbestos and MWCNTs.
While carbon black initiated a normal foreign body response, where the
immune system recognized and destroyed foreign particles, MWCNTs
and asbestos exposure increased the release of polymorphonuclear
leukocytes and protein exudation, indicating an increased inammatory
response. e same group also showed the dierence in toxicity levels
between short and long MWCNTs. ey attributed the increased
inammatory response of long MWCNTs and asbestos to “frustrated
phagocytosis” in which the macropghages are unable to engulf the long
needle shaped CNTs. By contrast, in another study done on a mouse
macrophage RAW 264.7 cell line, CNTs induced ROS related necrosis,
apoptosis and chrosomal damage, but do not induce an inammatory
response [83].
Granuloma formation
Granuloma is a small nodule or a tiny collection of immune
cells formed when the immune system attempts to wall o foreign
substances, but is not able to eliminate them. CNTs as well as asbestos
both were found to cause granulomas in mice exposed for 7 days [54].
Intratracheal instillation of SWCNTs (diameter ranging from 0.7-0-1.5
nm) in the lungs of rats lead to blockage of the large airways as a result
of the formation of granulomas, with 15 % mortality within one day
[84] .
Apoptosis
SWCNTs caused the maximum apoptosis of ve carbon-based
nanomaterials that were tested including SWCNTs and MWCNTs for
toxicity on human broblast cells [85]. ese researchers hypothesized
that dispersed, hydrophobic materials with small surface area displayed
increased toxicity. ey proposed that the mechanism of toxicity due
to CNTs was due to extracellular matrix protein signaling resulting
in changes to the cell skeleton and the subsequent displacement of
organelles, resulting in membrane deformation and nally apoptosis.
Citation: Jain S, Singh SR, Pillai S (2012) Toxicity Issues Related to Biomedical Applications of Carbon Nanotubes. J Nanomed Nanotechol 3:140.
doi:10.4172/2157-7439.1000140
Page 5 of 15
Volume 3 • Issue 5 • 1000140
J Nanomed Nanotechol
ISSN:2157-7439 JNMNT an open access journal
ere is evidence showing that CNTs caused apoptosis in several cell
types including T- lymphocytes and HEK293 cells. Genes associated
with apoptosis (p16, bax, hrk, bak1, p53, p57FGFR2, TGF beta
receptor1 (TGFbetaR1) and TNFAIP2) were up-regulated by CNTs
[85]. Other studies also indicated the upregulation of genes responsible
for apoptosis [86]. e unfunctionalized (UP-CNTs), puried
(P-CNT), and FITC functionalized (FITC-CNTs) all caused decreased
cell viability and increased apoptosis [83] .
Functionalization approaches
Pristine CNTs have smooth surfaces without any hanging bonds,
which makes them chemically inert and insoluble in most organic and
inorganic solvents. us incompatibility of CNTs with most solvents
is one of the major drawbacks for their biomedical applications. ere
are 2 main methods to increase the solubility of CNTs: one is covalent
functionalization (nicknamed as ‘defect functionalization’), where
CNTs are covalently modied with functional groups on the surface, by
chemically decorating their side walls and tips via oxidation to generate
carboxylic groups or carboxylated fractions, which can be further
chemically modied via amidation or esterication [87-89]. e other
is a non-covalent functionalization approach which involves physical
absorption of small molecules or polymers onto the surface of CNTs
through hydrophobic interactions, π- π interactions or molecular
inclusions [68]. CNT functionalization schemes with the moieties of
biological interests that reduce toxicity are shown in the Table 1.
Covalent modication of CNTs
e covalent functionalization strategy can be further divided
into two main approaches (i) oxidation of CNTs followed by carboxyl
based couplings (ii) addition reactions to the sidewalls and tips of
CNTs. e oxidative functionalization approach involves the use of
strong acid treatment, which opens the tube cap and results in the
formation of holes in the side-walls of CNTs [90]. e carboxylic
group introduced into the cap and the side-walls enhances the
solubility of CNTs in aqueous solution. Further these carboxylated
CNTs can be modied via amidation or esterication to link a wide
variety of biomolecules like peptides, proteins, nucleic acids and other
therapeutic agents. Addition reactions can also be used for the covalent
modications of CNTs. Prato and colleagues developed 1,3–dipolar
addition of SWCNTs with azomethine ylides [91]. is strategy was
initially used to modify Buckminsterfullerene (C60) SWCNTs were
suspended in Dimethylformamide (DMF) with an aldehyde and
N-substituted glycine derivative, at 30ºC, resulting in the formation of
substituted pyrrolidine moieties on the SWCNTs surface. Streptavidin
functionalized nanotubes were obtained with improved solubility and
were used mainly to visualize the interaction of nanotubes with HL60
cells for 1 hour at 37ºC [60]. Apart from functionalization at defect
sites, various other methods have been adopted to improve solubility
of CNTs such as the addition of azomethine ylides, diazonium salts,
carbenes, nitrenes, and radicals to SWCNTs. One such approach
of direct covalent modication is Diels-Alder cycloaddition using
transition metals at enhanced pressure. In order to detect and quantify
covalent functionalization, the ratio of intensities of the D-band
to the G-band (ID/IG) in the Raman spectra was used as a probe.
D-band is increased in the functionalized SWCNTs as an indicator
of signicant conversion of sp2 to sp3 hybridization of carbon atoms
in CNTs [92,93]. e benets of addition reaction modications are
that they are systematic, and predictable chemistry can be achieved by
generating pyrrolidine rings on the side walls of CNT. Covalent bonds
are strong bonds and easy to control, and they oer the possibility for
multi-functionalization of CNTs, for possible use as drugs and other
therapeutics carrier.
Non-Covalent modication of CNTs
is refers to physical adsorption or wrapping of molecules or
polymers onto the surface of CNTs without any chemical bonding. e
advantage of this type of modication is that it preserves the structure
and desired properties of CNTs and at the same time improves its
solubility for further applications. ere are two main approaches for
non-covalent functionalization of CNTs. One is wrapping biomolecules
like DNA, RNA around the CNT walls and the other is through π-π
stacking interaction between aromatic rings of the loaded material
and π electrons of CNTs [68,94-96]. Various aromatic molecules like
pyrenes, porphyrins, conjugated polymers, peptides, saccharides, DNA
and other biomolecules and surfactants interact with the side walls of
CNTs by means of π-π stacking interactions [90]. Dierent approaches
for functionalization could be used based on the application of CNTs.
Covalent chemical reactions allows stable functionalization, improved
solubility and biocompatibility of CNT, but CNT properties could
be changed as damage is caused to the side walls thereby decreasing
the Raman scattering, near-infrared photoluminescence and optical
absorbance. us, the covalent functionalization approach could be
used for the delivery of drugs, genes and other bioactive molecules
inside the cells. However, covalent functionalization may not be an
ideal approach for sensing and imaging applications. On the other
hand, non-covalent functionalization maintains the structural and
optical properties of CNTs (Figure 3) and could be eectively used for
sensing and imaging purposes.
Functionalization of CNTs for improved biocompatibility
For biomedical applications, raw hydrophobic CNTs must be
functionalized in order to improve solubility and biocompatibility
(Table 2). e uptake of SWCNTs in biological systems in vitro
(cellular uptake) and in vivo (blood circulation and biodistribution)
largely depends on their surface chemistry [13,97,98]. Functionalized
CNTs with improved solubility and biocompatibility are able to cross
cell membranes and successfully transport bioactive molecules like
DNA, RNA, drugs and proteins into the targeted cells [99]. In fact
in vivo studies in mice have indicated that well functionalized CNTs
accumulate in the reticuloendothelial system and are slowly excreted
through bile without causing side eects to the mice. Chitosan wrapped
CNTs were found to improve dispersion and biocompatibility of CNTs
through non-covalent interaction [100]. Non-covalently functionalized
SWCNTs with amphiphilic phospholipid-poly ethylene glycol (PL-
PEG) displayed excellent solubility, biocompatibility and stability in
the aqueous phase. e hydrophobic lipid chains of PL-PEG strongly
adsorb on to the CNTs surface, whereas the hydrophilic PEG chain
improved solubility and biocompatibility of the entire functionalized
SWCNT unit [65,101]. In another study, carboxylated SWCNTs
were covalently conjugated with anti-HER2 chicken IgY antibody for
detection and selective destruction of breast cancer cells using NIR
radiations. e complex could be potentially used for detection and
selective photothermal ablation as well as targeting breast cancer cells
without the need of internalization by the cells. us this new strategy
utilizes unique intrinsic properties of HER2 IgY-SWCNT complexes
for both cancer detection and therapy [102].
An investigation has been conducted on how structure and type of
functionalization of SWCNTs and MWCNTs aects immunotoxicity
in Oncorhynchus mykiss, an aquatic vertebrate model [44]. Seven
dierent types of CNTs were used for exposure at a minimum of
three concentrations: SWCNTs, MWCNTs, SWCNHs suspended
Citation: Jain S, Singh SR, Pillai S (2012) Toxicity Issues Related to Biomedical Applications of Carbon Nanotubes. J Nanomed Nanotechol 3:140.
doi:10.4172/2157-7439.1000140
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in sodium deoxycholate, MWCNTs suspended using sodium
deoxycholate, water soluble MWCNTs with sulfonate group (anionic
groups), water soluble SWCNTs with PEG (neutral group), covalently
functionalized, water soluble SWCNTs with m-polyaminobenzene
sulfonic acid (PABS) (zwitterionic group). SWCNTs and MWCNTs
that were functionalized to be water soluble were more stimulatory
to IL-1β in macrophage cells compared with unfunctionalized
counter parts. Almost all the nanomaterials, mentioned above
were stimulatory at some concentration. SWCNHs and MWCNT
suspensions in sodium deoxycholate were stimulatory from 1 to 10 μg/
ml concentration. SWCNTs and MWCNTs that were functionalized to
be water-soluble were stimulatory only at highest concentrations (5-
Table 1: CNT functionalized with different groups and bio-molecules leading to the reduction of cell toxicity.
MATERIAL METHOD CONCENTRATON RESULTS AUTHOR
Polyamidoamine-MWCNT-
plasmid DNA Hela cells 50 µg/ml Decreased cytotoxicity
by 38% [153]
MWCNT-COOH;MWCNT-NH2
Human astrocyte D384
And lung A549 cells 1 µg/ml High solubility and dispersibility,
Reduced toxicity [154]
MWCNT-COOH,
MWCNT- PVA
MWCNT-Apatite
(PVA: Poly Vinyl alcohol)
MG-63 human osteoblastic
cells
MWCNT-Apatite shows higher cell viability than MWCNT-COOH
and MWCNT-PVA
[155]
pEGFPplasmid DNA-SWCNT Mouse B-cells
cortical neurons 0.1pM/10mL Only 10% non-viable cells [156]
Glycopolymer-SWCNT-
plasmid
(pEGFP)
HeLa cells 0.1 mg/ml No toxicity [157]
(SWCNT-NH3
+;MWCNT-
NH3
+)lysine
Functionalized with plasmid
DNA
A549 cells 150 µg/ml Good gene delivery vector [158]
Plasmid DNA-SWCNT and
Plasmid DNA-MWCNT HeLa cell line 10mg/ml Exhibit low toxicity, improved gene expression up to 10 folds. [22]
FITC-SWCT and
biotin-SA-SWCNT
HL60,3T3,Chinese hamster
ovary cell 0.05mg/ml Both the f-CNTs were internalized
inside the cells without toxicity by endocytosis. [63]
f-CNT prepared by 1,3
cycloaddition and oxidation/
amidation
B&T lymphocytes and
macropghages 1-10µg/ml
f-CNTs were uptaken by cells,
highly soluble and preserved the
activity of immunoregulatory cells
[159]
SWCNT-phenyl-SO3H,
SWCNT-phenyl- SO3Na,
SWCNT-phenyl-COOH,
SWCNT in pluronic F108
Cultured human
Dermal broblast 3µg/ml-30µg/ml
Reduced in toxicity observed with
Increased of degree of functionalization. f-CNT were less toxic
than surfactant stabilized SWCNT.
[160]
Pluronic F127(PF127)
surfactant
coated MWCT
Mouse cerebral cortex,
Cultured human
blastoma cells
0.01%,
5-10mµg/ml
No degeneration of neurons, no
Induction of apoptosis,
Presence of MWCNT reduced toxicity of PF127
[52, 161, 162]
MWCNT- NH2Fibroblast L929 cell line 1-50 µg/ml Non-toxic but reduced cell growth observed [163]
NH2-MWCNT-DNA
HUVEC cell & A375(human
melanoma cell line) 60µg/ml No cell toxicity [164]
SWCNT-AHA
(AHA: 6-aminohexanoic acid)
HEK cells(human
epidermal keratinocytes)
0.00000005-
0.05mg/ml Maintains cell viability [165]
PEI-MWCNT-DNA HEpG2 and COS7 cells 1mg/ml Low toxicity, improved gene expression up to 10 folds. [166]
Non covalently functionalized
with PEG
(PEG-SWCNT)
Oxidized, covalently
functionalized PEG
(PEG- O- SWCNT)
(PEG: Poly ethylene glycol)
mice 100µl PEG-SWCNT less toxic than PEG O SWCNT [167]
MWCNT-COOH,
MWCNT-Amide and
MWCNT- Benezimidazole
Fibroblast cells Functionalized CNTs show more than 50 % cell viability [168]
Citation: Jain S, Singh SR, Pillai S (2012) Toxicity Issues Related to Biomedical Applications of Carbon Nanotubes. J Nanomed Nanotechol 3:140.
doi:10.4172/2157-7439.1000140
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10 μg/ml) similar to positive control LPS. In general, functionalized
SWCNTs and MWCNTs were more stimulatory as compared to their
unfunctionalized counterparts.
Cytotoxicity
Treating cells with CNTs can result in a variety of cell fates. ere
have been reports that CNTs cause necrosis, where cells loose cell
Table 2: Functionalization strategies for improved solubility and biocompatibility of CNTs.
Functionalized
CNT
Modication
approach
Biocompatibility/
Solubility
Reference
Glucosamine-MWCNT Oxidative covalent Good biocompatibility [169]
CS-MWCNT
(CS: chitosan)
Oxidative covalent Reduced aggregation, high dispersibility
and long term stability in 2% acetic acid.
[170]
CS-MWCNT-Cu2+ Covalent Improved electrical conductivity and stability [19]
CS-MWCNT Wet-grinding assisted ultrasonication Improved wettability and homogeneous dispersion [171]
Galactose-MWCNT Oxidative covalent Improved dispersibility in aqueous solvents. [172]
PVP-MWCNT Polymerization and blending Remarkable solubility in water,
ethanol and dimethylformamide (DMF)
[173]
PPy-MWCNT
(PPy: Poly propylene)
Oxidative covalent Improved solubility and conductivity [174]
α-Pyrene-MWCNT Reversible addition –
fragmentation chain transfer
polymerization
Increased solubility with the surface coverage of the polymer. [175]
Pyrene-MWCNT Covalent Improved solubility [176]
MWCNT-COOH Acid oxidation Decreased length, increased solubility
and colloidal stability in polar solvents like
water and ethanol
[177]
MWCNT-Amine
(Amine: dicyandiamide and
phenylbiguamide)
Oxidative covalent Uniform dispersion, good interfacial binding,
better compatibility
[178]
SWCNT-OH
(hydroxyl nanotubes)
Fluorine displacement Stable suspension in water, ethanol and dimethyl formamide.
Improved compatibility with biomolecules
[88]
SWCNT-tetrauoric acetic
group
Covalent Improved dispersion in water, ethanol and DMF [179]
SWCNT-2-propanol-2-yl Covalent attachment under UV-light Bundle dissociation, improved solubility in organic solvent,
retained electronic structure
[180]
Cellulose-SWCNT Covalent Easily dispersed and stable for 7 days at the content of 10 mg/ml [181]
CNT-SDS
(SDS: sodium dodecylsufate;
Anionic surfactant)
Non-covalent Decreased aggregation,
Dispersion in both aqueous and organic solvents
[182, 183]
CNT-NaDDBS
(NaDDBS: Sodium dodecylbenzene
sulfonate)
Triton-100
SWCNT-pyrene-Protein Non-covalent Increased dispersion [184]
SWCNT-PEG-FITC Non-covalent
(π - π stacking of aromatic FITC)
Solubilized CNTs
MWCNT-PEI-DNA Non-covalent Improved dispersion [166]
Citation: Jain S, Singh SR, Pillai S (2012) Toxicity Issues Related to Biomedical Applications of Carbon Nanotubes. J Nanomed Nanotechol 3:140.
doi:10.4172/2157-7439.1000140
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membranes and lyse rapidly [78]. ere have been reports that cells
stop growing or dividing actively thereby losing their viability on
treatment with CNTs. Several authors have reported that cells can
activate a genetic program of controlled cell death (apoptosis) [83].
It has been reported that the probable mechanism of photothermal
killing of cancer cells with CNTs and grapheme involved necrotic as
well as apoptotic cell death characterized by caspase activation/DNA
fragmentation and cell membrane damage [103]. Apoptosis-associated
genes were up-regulated and tyrosine kinase activities were decreased,
with down – regulation of the expression of the related genes [85]. e
mechanism of SWCNTs cytotoxicity has been evaluated in terms of
induced changes on cytoskeletons and cell morphology. It is known
that certain proteins like Focal Adhesion Kinase (FAK) cadherin
collagen and bronectin, play an important role in cell adhesion. CNTs
insert into cell membranes and disturb the surface protein receptors.
FAK is related to reduced cell proliferation and adhesion. SWCNTs
disturb the distribution of FAK in a human cell line (HEK293) along
with a decrease in cell adhesion [85]. SWCNTs can indirectly cause
toxicity by reacting with culture media and altering their composition
and thereby reducing the availability of medium components to the
cells. When A549 cells were grown on media, which had previously
contained SWCNTs, signicant cytotoxicity was observed [104].
Alamar blue assay and clonogenic assay indicated the reduction of
proliferative activity of the cells rather than viability.
Dermal toxicity
Skin is at a high potential for both occupational and environmental
exposure to nanoparticles. Additionally, once the nanoparticles
are logded inside the avascular epidermis, they escape removal by
phagocytosis [105]. Currently, there is a lack of information on whether
nanomaterials can actually be absorbed across the skin’s stratum
corneum and can accumulate in dermal tissues. e products composed
of SWCNTs and MWCNTs were tested for their acute dermal toxicity
and acute eye irritability using rabbits and skin sensitization using
guinea pigs. None of the SWCNTs and MWCNTs caused toxic skin
sensitization eects. Only one of the products containing MWCNTs
caused slight eye irritation [106]. In other studies, unpuried SWCNTs
(containing iron as impurity) tested positive for their dermal toxicity on
murine epidermal cells (JB6 P+), EpiDerm FT engineered skin cells and
immune competent hairless SKH-1 mice [107]. e large surface area of
the skin and small size of nanoparticles makes it dicult to determine
their location in the skin and within the systemic circulation. If they
are systemically absorbed, CNTs would be diluted throughout the
entire body or may be deposited in major organs, thereby making their
detection and quantization dicult. Further, CNTs cannot be expected
Skin Cross-Section
Effects
Effects
Cutaneous irritaon
Membrane protein down
regulaon
Dermal cell cycle arre st
Carbon
Nanotubes
(CNTs)
epidermis
dermis
CNTs accumulates
around hair root
subcutaneous
tissue
Penetrates upper skin layer
hair shaft
Stratum
conneum
sweat pore
melanocytes
sweat gland
blood vessels
CNTs
Nose
Offactory nerves
Trachea
Lymph
nodes
CNT travel down
the respiratory tract
and invade alveoli
CNT crosses
air - blood tissue
barrier and reaches
body’s circulatory
system
1 .) 2.)
Blood vessels
Increased
Inflammatory
response
Granuloma
formation
Intersteial
fibrosis
Oxidative
Stress
CNT
Inhaled
Inhaled Alveoli
Affactory
nerves
Brain
entry into blood
circulatory system
entry into spleen,
kidney & organs
Lungs
Alveoli
Brain
CNTs
sebaceous
gland
Pacinian corpuscle
Figure 4: Entry of CNTs inside the human body via (a) indirect uptake by skin
pores by crossing the rst layer of protection (stratum corneum) and localization
around the root of the hair follicles (b) inhalation or respiratory system; where
CNTs enters alveoli and deposit on the epithelium from where they cross air-
blood tissue barrier to enter blood circulatory system and reach other tissues
and organs.
Non- Covalent
SP hybridization
2
SP hybridization
2SP hybridization
3
SP hybridization
2
Less stable
Preserves CNT
structure
More stable
Leads to defects
sites
Covalent
Ligand / Biomolecule
Covalent Bond
Functional groups
Figure 3: Covalent modication degrades CNT properties by converting SP2
hybridized carbon to SP3 type.
Citation: Jain S, Singh SR, Pillai S (2012) Toxicity Issues Related to Biomedical Applications of Carbon Nanotubes. J Nanomed Nanotechol 3:140.
doi:10.4172/2157-7439.1000140
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to be absorbed by the skin, similar to chemical absorption as they vary
widely in their shape, size and physiochemical properties which could
aect their dermotoxic potential and their penetration abilities across
the skin. Standardization becomes even more challenging for surface
modied CNTs [75,105,108,109]. MWCNTs penetrate through the
stratum corneum barrier, localize and initiate an irritation response in
dermal cells [110]. Proteomics studies were conducted to test the eect
of MWCNTs on human epidermal keratinocytes. An increase and
decrease in the expression of several proteins was observed in the CNTs
treated cells as compared to controls. ese altered protein expression
patterns on CNT treatment suggest cell cycle inhibition, deregulation
of intermediate lament expression, altered vesicular tracking and
membrane protein down-regulation [76].
Pulmonary toxicity
e release of ne CNTs nanoparticles in the atmosphere is a highly
energy intensive process and current dosages and processes do not
release signicant quantities of airborne CNTs into the atmosphere, but
there is a possibile health hazard due to cumulative eects, especially
when CNTs are handled in bulk. Nanoparticles may enter a living
organism by inhalation, ingestion, cutaneous absorption or through the
circulatory system. e respiratory system is one of the most important
systems identied for quick absorption and deposition of nanoparticles
in the body. Due to their small size, CNTs can easily disperse in air
and be inhaled into the lungs. erefore pulmonary toxicity is of prime
importance. Several in vitro [9,83,111] and in vivo [58,112,113] studies
have been conducted in the past to assess pulmonary toxicity due to
exposure to SWCNTs and MWCNTs. Instillation of SWCNTs into the
lungs of rats at a dose of 1or 5 mg/kg resulted in granuloma formation
[84]. Pulmonary toxicity is evaluated by studying lung index, alkaline
phosphate activity measurement (ALP) and oxidative stress pathway
evaluation [114]. Type II alveolar cells are responsible for secretion
of a hydrolase enzyme, (ALP, alkaline phosphatase) responsible for
removal of phosphate groups from dierent molecules. us, increase
of ALP activity in serum is an indicator of toxicity to Type II alveolar
cells. Lactate Dehydrogenase (LDH) is another measure for toxicity in
lungs. It is a cytoplasmic enzyme that catalyses the conversion of lactate
to pyruvate in the liver, lung, kidneys etc. e activity of LDH in these
tissues is much higher than that in the serum. Increased level of LDH
in the serum acts as an indicator of alveolar macrophage injury, which
leads to necrosis in the tissues.
Oxidative stress is yet another reliable measure for evaluation of
the toxic response in general. When nanoparticles enter into the lungs,
oxidative stress leads to increased production of ROS, which then
trigger lipid oxidation, and formation of Malondialdehyde (MDA),
and also Glutathione (GSH) an antioxidant that helps protect cells and
tissues from ROS. When lung damage occurs, there is an increase in
MDA level and decrease in GSH level [115,116].
ere are several factors that control the distribution of
nanomaterials within dierent parts of the respiratory tract, such
as their chemical reactivity with body proteins, the size or surface
characteristics [40]. Unlike ne particles, CNTs can escape clearance
by macrophages and travel from the alveoli to the blood circulation
followed by possible migration to other organs of the body. Physical
characteristics of nanoparticles like size, surface modications etc.
play a crucial role in extra-pulmonary translocation of nanoparticles
[107]. Once the nanoparticles are deposited, they are translocated to
extrapulmonary sites, from where they reach the circulatory system via
transcytosis (Figure 4). Removal of these deposited particles may take
place via physical as well as chemical translocation processes [112]. e
soluble components of nanoparticles either in lipids or in intracellular
uid undergo absorption into protein or subcellular components, or
into extracellular uids [117]. Several studies on biodistribution of
nanoparticles reveal that signicant amounts of carbon-based particles
(80-180 µg/m3) were found in the liver of rats on inhalation exposure
[118]. In another study, inhaled carbon particles reached the central
nervous system [119]. Alveolar macrophages are the rst line of
defense against invading nanoparticles in the lung. Instillation of 0.5mg
SWCNTs into mice induced macrophage activation and subsequent
pulmonary injury [120]. A dose dependent increase in inammatory
response was observed on treatment of mice with SWCNTs. e
aggregated CNTs were responsible for granulomaous inammation,
including discrete granulomas with hypertrophic epithelial cells in the
lungs aer 60 days of exposure [121].
Most of the inhaled nanoparticles are eliminated from the body
system by macrophages, through phagocytosis. e particles that are
not removed by phagocytosis, enter epithelial cells or nd their way
into interstitial spaces. ey may nally enter the systemic circulation
and the lymphatic system [40]. CNTs have been observed in liver
tissues of rats following inhalation [122]. Studies utilizing intratracheal
instillation of nanoparticles revealed the agglomerative nature of CNTs
in aqueous solutions [123-125] . Similar to this, pharyngeal aspiration
have shown evidence of agglomeration of CNTs in the proximal
alveolar regions of lungs.
Genotoxicity
Genotoxicity can be described as the deleterious eect of
nanoparticles on a cell’s genetic material. CNTs have the anity to
interact with DNA, thus rendering them potentially mutagenic or
carcinogenic. Owing to their cohesive nature, CNTs tend to form stable
aggregates inside organisms, causing inammatory as well as oxidative
stress at the sites of their deposition. ese eects, over the course
of time might lead to tissue/organ destruction and increase the risk
•Size/Shape
•Surface area
•Type
•Defect sites
•Surface
Chemistry
•Catalyst residues
•Agglomeraon
•Surface
funconalizaon
Factors Responsible
for Toxicity
•Uptake mechanism
•Route of exposure
•Inflammaon and
oxidave stress
•Biodestribuon
•Metabolism
•Accumulaon
•Cytotoxicity end
points
determinaons
•Effecve
concentraon
•Toxic limits
•Safe systemic dose
Biocompability /
Toxicity Assessment &
Evaluaon of Risk
Factors
•Human health risk
•Skin irritaon and
sensisaon
•Pulmonary toxicity
•DNA damage due
to oxidave stress
•Carcinogenic
•Developmental
effects
•Environmental
impact and risk
•Bioaccumulaon
and magnificaon
•Anbacterial
•Hatching delays
•Survival rates
•Germinaon
Known Hazards/Effects
Figure 5: Concept map explaining the various toxicity issues surrounding
CNTs and the risk assessment of accidental exposure on human health and
environment.
Citation: Jain S, Singh SR, Pillai S (2012) Toxicity Issues Related to Biomedical Applications of Carbon Nanotubes. J Nanomed Nanotechol 3:140.
doi:10.4172/2157-7439.1000140
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of cancer. Since the rst alarm raised by the Royal Society and Royal
Academy of Engineering in 2004, an enormous amount of research has
been conducted on genotoxicity issues of nanomaterials [126-131].
CNTs are able to induce cancer and mesothelioma in a similar
manner as asbestos. Within biological systems, they remain stable
and form aggregates in the micrometer size range. Animal studies
have indicated that MWCNTs and SWCNTs can induce stress related
inammatory responses, reactive oxygen and nitrogen species and
genotoxic eects associated with these eects [132]. Carcinogenic
properties of CNTs are associated with long term genotoxic stress.
ere are two ways CNTs can interact to cause genotoxicity, one is
direct interaction with DNA or the mitotic apparatus or indirectly
via oxidative stress and the inammatory response. Eukaryotic cells
develop several repair mechanisms in response to DNA damage,
to protect genomic integrity [133]. For instance, in the presence of
damaged DNA, the p53 protein is activated by protein phosphorylation
as a master guardian that arrests the cell cycle to provide time to repair
DNA damage. If the DNA damage goes beyond repair, enhanced
expression of p53 may trigger cell death by apoptosis. Under normal
conditions, when DNA is not damaged, p53 is expressed at lower levels.
Since the activation of p53 could be related to DNA damage, it could be
used as a molecular marker for assessing genotoxicity of MWCNTs to
mouse embryonic stem cells.
MWCNTs have long and thin morphology and produce asbestos
like toxic responses. ere have been interesting studies conducted on
intra-abdominal injection of MWCNTs with mesothelioma inducing
eects [134]. e toxic eects of metal particles such as CuO, TiO2,
CuZn Fe2O4, Fe3O4, Fe2O3, were compared with those of SWCNTs and
MWCNTs using human lung epithelium cell lines. e highest toxic
eects were observed for copper nanoparticles and lowest for iron
particles (Fe3O
4, Fe2O3) [110]. CNTs were cytotoxic and also caused
DNA damage [135].
In order to identify the aected genes by SWCNTs in Human
Normal Lung Cells (HNBE), gene expression proles of untreated and
SWCNTs treated HNBE cells have been studied [45]. e microarray
analysis leads to the identication of marked changes in the gene
expression of 14,294 genes, with 7,029 genes being upregulated
and 7,265 being downregulated. SWCNTs treatment results in cell
apoptosis of HNBE cells through the activation of transcription factors
2, 3, 7 and 15. e gene responsible for cell survival, growth arrest, and
DNA damage, protein phosphatase 1 was overexpressed on SWCNTs
treatment in HNBE cells. Transcription factor 3 gene, responsible for
cell apoptosis and carcinogenesis was overexpressed in HNBE cells
treated with SWCNTs. e interleukin receptor 1 gene, responsible
for cell signaling of airway inammation through its activation by
interleukin 33, was overexpressed in HNBE cells treated with SWCNTs.
Downregulation of the hyaluronan-mediated motility receptor gene,
which is involved in cell motility and cell growth was observed in
HNBE cells with SWCNTs treatment. e identication and proling
of the gene expression in HNBE cells on SWCNTs treatment adds
to our understanding of the mechanism of the eect of SWCNTs on
human health. A preliminary protein prole of MWCNTs exposure
to Human Epidermal Keratinoctes (HEKs) shows that MWCNTs are
capable of altering the protein expression in a target epithelial cell
[76]. Proteins associated with metabolism, cell signaling, stress and
cytoskeletal elements and vesicular tracking are signicantly aected.
Another study aimed to assess the toxicity exhibited by MWCNTs and
to explore possible molecular mechanisms underlying the biological
eects of MWCNTs exposure in A549 cells was conducted [43]. Results
clearly indicated that 200 µg/ml concentration of MWCNTs decreased
cell viability considerably in A549 cells. MWCNTs stimulated the
generation of ROS which released the pro-inammatory cytokines and
caused the translocation of transcription factor NF-ƙB, to the nucleus,
which regulates pro-inammatory gene for IL-8. Proteomics study was
conducted to evaluate the eects of MWCNTs on protein expression
prole in human monoblastic leukemia cells (U937) [136]. Dierence in
protein expression pattern was observed on treatment with MWCNTs
with dierent impurities within the CNTs by thermally treating
highly contaminated As-grown MWCNTs at 1800ºC (HTT1800) and
2800ºC (HTT2800). Alteration of protein expression of 45 proteins,
that play key roles in metabolism, biosynthesis, stress related response
and cell dierentiation was observed aer treatment with HTT1800
and HTT2800 tubes. HTT2800 did not inhibit cell proliferation and
were found to be non-toxic to U937 cells. e biomarker protein of
Parkinson’s disease, DJ-1, also related to cancer, was identied aer
exposure to both MWCNTs. e results concluded that cytotoxicty of
MWCNTs depends on impurities such as iron, although MWCNTs
themselves cause cytotoxic responses in vitro [136].
Risks from accidental exposure, regulatory concerns and
commercial market potential of CNTs
e wide variety of applications of CNTs has revolutionized the
eld of electronics, energy storage, material science and engineering,
diagnostics and biomedicine. However, challenges for safe use of CNTs
because of their potential toxicity have slowed down the eorts for
further innovation in their applications, especially in the biomedical
eld. As the large scale production and utilization of nanomaterials
increase, so does the risk to human health and environment. e toxic
eects of CNTs and other nanomaterials are not fully characterized,
and as the eld grows, the need for standardization and categorization
will increase so that results can be compared and analyzed for potential
risks [137]. As per the US Environmental Protection Agency (EPA),
CNTs are regarded as a “chemical substance” under the toxic substances
control act (TSCA). EPA permits only limited manufacturing, use and
environmental release of CNTs as they could create a serious threat
to environmental safety [138]. According to recent (June, 2011) EPA
reports, MWCNTs have been designated as a chemical substance
hazardous to human health.
ere is a complex array of issues surrounding toxicity of CNTs
including the route of entry, chemical modication and others (Figure
5). e most common routes of entry of CNTs inside the human body
are inhalation, dermal exposure or ingestion. Dermal exposure could
lead to skin irritation, sensitization, skin cancer or produce systemic
eects aer absorption. Functionalized CNTs with hydrophilic
functionalities prevent their aggregation and allows their circulation
in the respiratory system for longer duration aer inhalation. Several
questions are relevant to the health risk assessment of CNTs such as
how quickly the inhaled nanotubes are cleared from the lungs, the
fate of the persisting nanotubes and how shape, size, surface charge,
metal impurities, surface functionalization and the degree to which
they agglomerate inuence toxicity of CNTs. CNTs functionalized
simultaneously with various molecules of particular interest display
unique properties that may impact human health and thus require
reliable characterization, standardization and evaluation of their in
vitro toxicity behavior in representative cell lines. e chronic exposure
to these engineered CNTs could make the situation more complicated
as their toxicity data base management and documentation is dicult.
e in vitro and in vivo impact of CNTs may vary depending on surface
functionalization. ey have been shown to cross the blood-brain-
Citation: Jain S, Singh SR, Pillai S (2012) Toxicity Issues Related to Biomedical Applications of Carbon Nanotubes. J Nanomed Nanotechol 3:140.
doi:10.4172/2157-7439.1000140
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ISSN:2157-7439 JNMNT an open access journal
barrier which could lead to adverse health and associated risks [139].
Much has been written speculatively on the potential health risks of
carbon nanotubes, mostly as an aerthought to variety of application
prospects. Recently, the dose-response and time course of pulmonary
toxicity of Baytubes®, a more exible MWCNTs type has been examined
in previous single and repeated exposure 13 week rat inhalation studies
[140]. Also, kinetic endpoints and potential to translocate to extra
pulmonary organs studies provide better understanding of dosimetric
response and etiopathology of pulmonary inammation. Baytubes®
show remarkable similarity in retention half-time (t1/2) with poorly
soluble and agglomerated carbon black (carbon black at 7 mg/m3
has t1/2=329 days; MWCNTs at 6 mg/m3 has t1/2=375days). e study
supports the conclusion that the dierence in the pulmonary toxic
potency is related to the low density (≈ 0.1-0.3 g/m3) of MWCNTs
assemblage [21]. Hence, the nanoparticles, attain the volumetric lung
overload-related inammatory response conditions earlier than the
conventional particles. e pulmonary overload triggers the cascade of
events to slow down the clearance and consequently increase MWCNTs
concentration that leads to sustained pulmonary inammation. Based
on NOAEL (No-Observed Adverse Eect Level) from the 13 week
inhalation study on rats, an occupational exposure limit (OEL) of 0.05
mg Baytubes/m3 (time weighed average) is considered reasonably safe
and does not cause lung injury on accidental inhalation. A promising
new method for detailed safety evaluation using proteomics-based
approach has also been used for assessment of biological responses
to nanomaterials [136]. If the protein database related to all diseases
is established, protein matching will be an ecient method for
evaluation of the possibility of whether MWCNTs cause disease. us,
proteomics based approach allows safety evaluation of CNTs and
other nanomaterials and also provides information about secondary
inuence, impurities and response mechanism.
ere are other issues that may come in the way of evaluation
of CNTs toxicity and the formulation of reasonable regulatory
guidelines, such as the correct determination of cytoxicity end-
points. Carbon based materials interact with the dye markers used in
cell-culture cytotoxicity assays such as Neutral Red, NR (3-amino-7-
dimethylamino-2 methylphenazine hydrochloride) and MTT (3-[4,
5-Dimethyl-2-thiazol]-2, 5-phenyl-2H-tetrazolium bromide) that are
widely utilized for cell viability assays. e adsorption of these dyes
from the cell culture media interferes with the absorption spectra
causing false readings and incorrect evaluation of the toxicity of CNTs
[105]. us accurate identication of CNTs toxicity requires a matrix
based on a set of representative cell lines and in vitro cytotoxicity assays
measuring dierent cytotoxicity endpoints.
Despite toxicity, safety and regulatory concerns, CNTs have
received considerable attention from scientists because of their
immense potential for commercialization in a variety of applications.
e visionaries and the futurists are thrilled by the properties of CNTs
as in theory they could be used to build incredibly strong structures
such as, ‘space elevators’ that would stretch thousands of miles from
earth. Nanocomp® technologies Inc. are trying to bring the future
closer by commercially producing yarns and sheets made of CNTs
[141]. Bayer Materials Science synthesized a multilament yarn coated
with CNTs to make new fabric heater with improved electrical and
thermal conductivity that shows long lasting bending resistance [142].
Bayer used epoxy gel coat modied with Baytubes®, the CNTs from
Bayer for reinforcing the outer surface of a kayak. e coating makes
the surface resistant to abrasion and easy to clean [143]. Nanoledge®
manufactures tennis rackets with the material reinforced with CNTs.
e racket bends less during ball impact and improves the performance
of the player [144]. Recently Ensysce Bioscience® developed SWCNTs
conjugated siRNA for treatment of cancer. ey received regulatory
approval to conduct clinical trials on siRNA delivery into tumor cells
[11,145,146]. Recently FDA has approved the use of CNTs to improve
colorectal cancer imaging. e research is focused on clinical trials
on ovarian and non-small cell lung cancer diagnostics using CNTs.
Even though CNTs play a lead role in the eld of nanomedicine to get
clinical approval as a drug delivery vehicle, more research is required to
guarantee safety. In a breakthrough discovery by a team of Swedish and
American scientists, CNTs have been shown to break down to carbon
dioxide and water by an enzyme-meyloperoxidase found in white
blood cells [147]. is contradicts the previous assumptions that CNTs
were non-biodegradable and cannot be broken down in the body or in
nature. is discovery will probably change the face of nanomedicine
with increased use of CNTs for diagnosis and treatment.
Conclusions
Although the potential of CNTs as a useful nanomaterial for
biomedical applications cannot be denied, the failure to reach a
consensus on their toxicity hampers the future prospects for clinical
trials as safe drug delivery vehicles. Functionalization of CNTs allows
for improved solubility and biocompatibility and opens new avenues
for their utilization in the eld of nanomedicine. Only through a relative
comparison can one understand the dangers of functionalized CNTs
administration against other treatment options available. e toxicity
of CNTs is likely to be inuenced by the method used to manipulate
their surfaces. Characterization, uptake by cells and elimination
mechanisms need to be understood properly and new test systems
are needed in order to systematically study the nanotoxic behavior
of CNTs and reveal important features necessary for risk assessment.
It is recommended that the issues regarding toxicity of CNTs be
ameliorated by functionalization before biomedical application. With
increasing production of CNTs, there is an urgent need to rene
strategies to assess their possible eects on employees, who represent
the main exposed population so that appropriate safety regulation
can be formulated. Standardized protocols to evaluate the toxicity of
CNTs and categorize their associated risks would help to accelerate
their routine application. Current toxicity reports on biological risk
may have to be evaluated in the context of exposure due to ambient
environment. Additionally, the hazards of treatment with CNTs need
to be compared with other treatments to arrive at safe doses and relative
benets of CNTs over current regimens. If the risk/benet aspects are
satisfactorily answered, then the use of CNTs in biological systems
might be feasible. Continued eorts of researchers and scientists, on
CNTs may result in safer products to enhance the quality of human
health and environment.
Acknowledgement
This work was supported by NSF-CREST (HRD-07342321). We would like
to thank Ms. Eva Dennis for creating some of the gures used in the manuscript.
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