TOXICITY OF CARBON
NANOTUBES
Aziz Daabash
Brett Michalk
Amanda Mogollon
Derek Nelson
OUTLINE
 Introduction


Types
Properties
 Toxicity



Respiratory
Reactivity
Effect on humans
•Conclusions
•Recommendations
•Further Research
http://www.aip.org/png/images/ando.jpg
INTRODUCTION
A carbon nanotube (CNT)
is a tubular molecule
with axial simmetry and
diameter in the
nanometer range
(Muller).
It can be considered as a
rolled up graphene sheet.
However, it possesses
many properties that
leave no doubt this is not
just graphene.
http://www.nanotech-now.com/nanotube-buckyball-sites.htm
http://www.msm.cam.ac.uk/phasetrans/2005/paper/img19.png
TYPES
Single Walled CNT (SWCNT): one-atom-thick CNTs
 Multi Walled CNT (MWCNT): concentric layers of
CNTs

http://www-ibmc.u-strasbg.fr/ict/images/SWNT_MWNT.jpg
PROPERTIES
Among some of the properties
of the CNTs we can find:


Electrical: Both metallic and nonmetallic behaviors are observed,
while geometry plays a profound
part in determining the electronic
behavior. (Ebbesen)
http://www.studentsoftheworld.info/sites/family/img/27335_Electricity.jpg
Elastic: Tensile Young’s module
and torsion shear module
comparable to that of diamond
(Lu).
http://www.nanoshel.com/research-center/wp-content/uploads/2009/01/ballistic-impact.jpg
PROPERTIES
Mechanical: Carbon
nanotubes have high
strength plus
extraordinary flexibility
and resilience. (Salvetat)
 Thermal: Thermal
expansion of carbon
nanotubes will be
essentially isotropic that
is, uniform in all
directions (Ruoff).

http://brent.kearneys.ca/wp-content/uploads/2006/05/carbon_nanotube.jpg
TOXICITY
Due to the remarkable range of
properties that carbon
nanotubes posses, they have
numerous applications and
can be used in a variety of
industries that range from
electronics to food processing.
Since CNTs may be present in
our every day lives in a matter
of a few years, a concern on its
toxicity has been growing
recently.
http://www.artsandopinion.com/2007_v6_n6/volume_images/toxic-1.jpg
TOXICITY
http://www.lifemedicalsupplier.com/images/mask_1860.jpg
Why is it important to determine
CNTs toxicity?
 CNTs manufacturers and
suppliers, like Carbon
Nanotechnologies, Inc in
Houston, Texas, classified
CNTs as synthetic graphene
and using it as a reference for
the permissible inhalation and
exposure limit (PEL) (Lam).
TOXICITY

However, it is clear that CNTs do not have the
same properties as graphene, and thus this PEL
might not reflect the real toxicity and exposure
limits of CNTs.
http://cnx.org/content/m29187/latest/graphene.jpg
http://www.tcd.ie/Chemistry/staff/people/duesberg/ASIN%20
web%2027-10-09/images/CNT.jpg
RESPIRATORY TOXICITY OF CARBON
NANOTUBES: HOW WORRIED SHOULD
WE BE?
Julie Muller, François Huaux, Dominique Lison
*Note: All materials in the following section are obtained from the above research paper.
INTRODUCTION
Some physical
characteristics of
nanotubes, like their
length to diameter ratio,
low solubility in aqueous
media, propensity to
agglomerate, being light
and able to become
airborn, among others,
suggest that they may be
toxic.
http://romunov.blogsome.com/images/kozarec.jpg
INTRODUCTION
Residual catalytic
material like iron,
cobalt, and nickel, left
from different
manufacturing or
purification processes
may contribute to the
toxicity of CNTs.
http://www.mcgill.ca/files/_nea/106127_labSmall.jpg
INTRODUCTION
Inhalation exposures in
industrial settings
should be very low, but
since there is little
information on the
respiratory toxicity of
CNTs, it cannot be
concluded that the risk
is negligible.
http://images.lifescript.com/images/ebsco/images/inhaled_poison.jpg
ARE CNTS TOXIC?
In order to answer this question, the authors
gathered information from other experiments
and compared results
 Noticed a number of parameters that could have
affected the results of some experiments

Non-grinded CNTs tend to agglomerate
 Concentration of metal impurities

http://public.blu.livefilestore.com/y1pxaiFNCiK21KETuwhBtq0QlnoQQq9MkqWxmJhOOPK1PJ6ZMg7VstXtxs3Er1fERLLuQ
eBN4mCM5XiOx7XK15HHw/metales.jpg
EXPERIMENT COMPARISON
Muller et al.
RESULTS
Non-grounded MWCNTs agglomerate in large
airways, while ground ones disperse in the lungs.
 When MWCNTs reach the lungs, they are not
rapidly cleared. Intact MWCNTs are retained
longer than ground ones.

http://4.bp.blogspot.com/_vfImvyorvjQ/Sxr8EMfTESI/AAAAAAAAAbQ/gHWF8X3ToxI/s320/arkema+1.jpg
RESULTS
Sections of control
rat lung (A), and
immediately after
intratracheal
instillation of 2
mg/animal ground
MWCNTs (C,
arrows) which are
much
better dispersed in
the lung than
intact MWCNTs
which mainly
remained clumped
in large airways (B,
arrows).
Muller et al.
RESULTS
The lung toxicity of CNTs was of similar or
greater intensity than well-recognized lung
toxicants such as quartz or crocidolite fibers.
 Transition metals left as impurities might
contribute to the toxicity of CNTs.

Quartz
http://www.windows2universe.org/earth/geology/images/3quartz.JPG
Crocidolite
http://www.isitasbestos.com/images/crocidolite.jpg
CONCLUSIONS

If they reach the lungs, CNTs (SWCNTs and
MWCNTs) have potential to cause severe
inflammatory and fibrotic reactions.
FURTHER RESEARCH
CNTs should be tested in other animals to
determine the different toxic behaviors.
 Animals should be placed in an environment in
which CNTs are present, instead of placing the
CNTs intratracheally, to determine if they can
reach the lungs when inhaled.

http://www.dichotomistic.com/images/particles%20in%20box.jpg
NANOTUBES:
FREE RADICAL
GENERATION OR SCAVENGING
ACTIVITY?
Ivana Fenoglio a, Maura Tomatis a, Dominique Lison b, Julie Muller b,
Antonio Fonseca c, Janos B. Nagy c, Bice Fubini
Source:
http://nanotechnologytoday.blogspot.com/2009_11_01_archive.html
*Note: All materials in the following section are obtained from the above research paper.
INTRODUCTION
When passing from micro to nano scale,
most materials undergo remarkable
modifications in their chemical
properties, often associated with an
increase in their reactivity.
Because of their unique properties,
These properties make CNT suitable
for many industrial applications
including biomedical applications.
Dividing osteoblast on multi-walled CNTs
(Source: Dr. Laura Zanello, University of California-Riverside)
POTENTIAL TOXICITY
When inhaled, CNTs may constitute a
possible hazard to the health of
exposed workers or users.
The determinants of the toxicity of
inhaled particles involve three main
factors:
•form of the particle (e.g., fibrous vs.
isometric and smooth).
• surface reactivity, typically the
potential to generate free radicals.
• persistence in the respiratory tract
(low solubility and/or slow clearance).
Morphology changes of MSTO-211H cells after 3
days of exposure to 15 µg/ml of different fractions
of CNTs and asbestos. (a) Untreated MSTO-211H
cell culture and (b) cell culture exposed to asbestos.
Arrows indicate needles of asbestos. (c) Cells
treated with CNT agglomerates were roundshaped and lost their adherence on the cell culture
plate. Arrow point to CNT agglomeration. (d) Cells
exposed to CNT-bundles showed no visible
morphological changes compared to the control
cells. (e) Effect of CNT-pellet fraction. Non-tubes
material agglomerated during the incubation
period to micro-sized structures. (f) Cells incubated
with CNT raw material.
Source: www.nanowerk.com/spotlight
REACTIVITY
Recent researches reported that purified
MWCNT are active in inducing
inflammatory and fibrotic reactions in
the lung in a rat model.
Such reactions could contribute to
the generation of reactive oxygen
species (ROS).
ROS may react with extracellular
fluids and cellular products or
directly damage target cells.
Sources :Muller et al.
REACTIVITY (CONT.)
This report investigates whether the adverse
biological response to nanotubes reported in some
studies could be caused by surface reactivity.
How?
By examining the potential of these materials
to generate in aqueous suspension HOS or
carbon-centered radicals from hydrogen
peroxide and formate ions.
Such reactions did not take place, the
report subsequently investigated
whether nanotubes may act as
scavengers of oxygen-centered free
radicals.
INVESTIGATION
Unpurified SWCNT, containing a large
amount (30%) of iron, were able to
stimulate and induce the production of
ROS from human keratinocyte cells.
It has already been reported that
nanotubes exhibit antioxidant
properties in nanotube polymer
composites. Ex. Fullerenes (form of
crystalline carbon)
Fullerenes were recently reported to be
cytotoxic to human skin and liver
cells, the toxic effect being related to
the generation of ROS, which damage
cell membranes
Fullerenes
Source: http://www.surf.nuqe.nagoya-u.ac.jp/nanotubes/omake/fullerenes/fullerenese.html
GOAL OF INVESTIGATION
The scavenging activity of nanotubes was tested for
the hydroxyl radical and superoxide anion (O2*).
hydroxyl radical is the most reactive radical among ROS and the
major inducer of oxidative stress in pathological processes
What is oxidative stress ?
stress caused by an imbalance between the production of reactive oxygen and
a biological system's ability to readily detoxify the reactive intermediates or
easily repair the resulting damage
What is superoxide anion (O2 *)?
A relatively stable radical which is involved in both pathological and
physiological processes.
MATERIALS
Multiwall carbon nanotubes
•Multiwall carbon nanotubes (MCNT) were
synthesized by the decomposition of ethylene on an
alumina support doped with a cobalt–iron catalyst
mixture and purified by subsequent treatment with
NaOH.
•MCNT were grounded in an oscillatory agate ball
mill (Pulverisette 0, Fritsch) with a vertical vibration
of 1 mm applied for 6 hours.
Properties of purified MCNT:
•Small amounts of Co (0.29%), Fe (0.47%), and Al (0.05%).
•OD= 9.7 ± 2.1 nm mean length =5.9 ± 0.05 μm
•BET surface area = 378 m2/g , carbon content = 97.8%.
Multiwall carbon nanotubes
http://www.msm.cam.ac.uk/Department/DeptInfo/StaffProfiles/ResearchFigs/Windle.jpg
Along with the following materials:
DMPO (5,5-dimethyl-1-pyrroline-Noxide)
Pyrogenic amorphous silica Aerosil 300
Min-U-Sil 5 quartz
5,5-Dimethyl-1-pyrroline N-oxide
www.chemblink.com/structures/3317-61-1.gif
METHODS
1)- Free radical generation
(HO*) radicals:
Generated by suspending 5 mg of MWCNT in 50 μl of 5% sodium dodecyl sulfate (SDS)
diluted in 500 μl of 0.2 M potassium phosphate buffer, pH 7.4, containing 75 mM DMPO and
80 mM hydrogen peroxide.
(CO2*) radicals:
Generated by suspending of 5 mg of MWCNT in 50 μl of 5% SDS diluted in 625 μl of a
buffered 0.2 M potassium phosphate buffer, pH 7.4, containing 75 mM DMPO and 1 M
sodium formate.
2)- Scavenging activity toward hydroxyl radicals
Hydroxyl radicals (HO*) generated by adding 250 μl of a 0.2 M solution of hydrogen
peroxide in water to a solution containing 0.025 M DMPO and 1.7 mM FeSO4 in 0.17 M
potassium phosphate buffer, pH 7.4. The yield of HOS radicals was followed by measuring
the intensity of the spectrum of the DMPO/HOS adduct.
3)- Scavenging activity toward superoxide radicals
Superoxide radicals (O2*-) were generated in a buffered solution (1.2 mM phosphate
buffer, pH 7.8) containing 2.4 Μm EDTA, the enzyme xanthine oxidase (0.017 U/ml), and
at two different concentrations of xanthine. free radicals released from CNT in was
monitored by ESR spectroscopy with DMPO as trapping agent.
RESULTS
1)- Free radical generation by multiwall carbon
nanotubes
Under none of the conditions tested, however, did
nanotubes produce any detectable radicals.
Safe
Figure 1
RESULTS (CONT.)
2)- Scavenging activity of multiwall carbon
nanotubes
Hydroxyl radicals were generated by the Fenton reaction:
H2O2 þ Fe2þ → HOS þ OH þ Fe3þ
Tested reaction conditions for radicals generated by the Fenton
reaction :
Case (a): generation of HOS radicals by the Fenton
reaction only.
Case(c): presence of a suspension of 5mg of nanotubes in
50 μl of 5% SDS, signal was suppressed.
Case(b): presence of a high surface area amorphous silica,
signal wasn’t effected.
Indicates that the scavenging effect is not due to
the presence of small particles with a large surface
area, but to a more specific reaction ascribed to
MWCNT
RESULTS (CONT.)
To investigate if the observed effect was due to a
scavenging property toward radicals or to the
inactivation of iron ions
(HO*) radicals were also generated by photolysis of
hydrogen peroxide:
H2O2 → 2HOS
Figure.1
Tested reaction conditions for radicals generated by the
above photolysis reaction, note the linear increase of
radicals produced increased with the concentration of
(Figure1).
Figure.1
RESULTS (CONT.)
Tested reaction conditions for radicals
generated by the above photolysis reaction
(Figure2):
Case (a): generation of HOS radicals
by the photolysis of hydrogen peroxide.
Case(c): presence of a suspension of
nanotubes in 5% SDS, signal was
completely suppressed.
Case(b): presence of SDS alone, signal
was slightly effected.
Figure.2
Verifies that the surfactant SDS
did not substantially interfere
with the radical yield
RESULTS (CONT.)
Scavenging activity of multiwall carbon nanotubes on the
superoxide radical
Superoxide radicals (O2*) :
•Generated by the xanthine .
•Detected by spectrophotometry
(monitoring the reduction of cytochrome).
Effect of MWCNT on superoxide radicals at
two different concentrations of
xanthinein(Figure.1):
•Absence MWCNT (light gray bars)
•Presence of MWCNT (dark gray bars)
Verifies that MWCNT doesn’t
have
any
effect
on
the
concentration
of
superoxide
anions
Figure.1
RESULTS (CONT.)
Superoxide radicals (O2*) were also:
Generated by irradiating with a UV lamp a
solution of riboflavine and DMPO in phosphate
buffer (pH 7.4) located in the spectrometer
cavity.
Case (a): The generation of (O2*) by
the decomposition of its adduct.
Case(b): presence of MWCNT in 5%
SDS, signal almost completely
suppressed.
Case(c): the presence of surfactant
SDS only, presence of MWCNT in 5%
SDS, signal slightly effected.
Excludes the direct reaction of
MWCNT with cytochrome c
that may occur.
Figure.2
CONCLUSION
Present data indicate that purified
MWCNT are very effective
scavengers of ROS, both (HO*) and
(O2 *), no matter how such species
are generated
Inflammatory response elicited
in rats by purified MWCNT is
therefore ascribed to features
other than particle generated
radicals.
MWCNT represent the first case
of a potentially toxic particle
which exhibits scavenging
properties toward oxygen
radicals, instead of generating
them.
FURTHER RESEARCH
• The potential of purified single-wall or
multiwall nanotubes to generate per se free
radicals or ROS in acellular aqueous
suspension has not been tested so far, further
research should be implemented.
• Detailed investigation and description of
the precise mechanisms whereby MWNT can
quench ROS should be investigated to fully
understand the exact molecular mechanism
taking place.
Source:
http://internetservices.readingeagle.com/blog/gavel/archives/microscope.jpg
In vitro evaluation of cytotoxicity of
engineered carbon nanotubes in
selected human cell lines
Xiaoke Hu a, Sean Cook a, Peng Wang a, Huey-min Hwang a, Xi Liu
b, Quinton L. Williams b
*Note: All materials in the following section are obtained from the above research paper.
INTRODUCTION
Due to their
unique properties, carbon nanotubes
(CNTs) have been used in
various consumer, medical, and
industrial applications
Consequently, this expanding usage may
lead to widespread human
exposure via skin contact, ingestion,
intravenous injection (in medical
application) or by inhalation.
Divergent literature reports of CNTs'
toxicity make it difficult to conclude if
any health risks are associated
with CNTs exposure
http://sites.uclouvain.be/pcpm/themes/TRANSPORT_NT_files/image001.jpg
OBJECTIVE
Systematic approach to study and compare the in vitro cytotoxicity of
selected engineered carbon nanotubes (CNTs) to test cell lines
including human skin keratinocytes, lung cells and lymphocytes
http://www.babble.com/CS/blogs/strollerderby/2009/05/ivf.jpg
MATERIALS AND METHODS
A3 lymphocyte cell line
SWCNTs and MWCNTs
prepared without further purification in
distilled water to reach concentrations of 2, 5 and 10
ppm.
A3 lymphocyte cell line
MSTO-211H lung cell
HaCaT human keratinocyte cell line
Source: In vitro evaluation
MSTO-211H lung cell
Triad LT microplate reader
measure the fluorescence of the samples
in the FDA test
Source : in vitro cytotoxicity study with human cancer cells.
RESULTS
Single Wall Carbon Nanotube
(SWCNT)
Results of fluorescein diacetate (FDA)
uptake in T4-lymphocyte A3 cells
indicated cytotoxicity caused by (SWCNTs)
at 2, 5 and 10 ppm.
At 2 ppm, the SWCNT treatment group retained
71.3% viability compared to the PBS control
group. At 10 ppm, cellular viability further
decreased to 56.5% of the PBS control group.
In the skin keratinocyte HaCaT cells and
lung MSTO-211H cells, the SWCNT did not
demonstrate any cytotoxicity at
concentrations of 2 and 5 ppm but slightly
inhibited HaCaT cells and caused
significant toxicity to MSTO -211H cells at 10
ppm.
RESULTS (CONT.)
Multi-Wall Carbon Nanotube
(MWCNT)
Multi-walled carbon nanotube (MWCNT)
testing showed significant cytotoxicity to
A3 cells in a dose dependent manner. At
10 ppm the viability of the cells
decreased to 89.1% compared to the PBS
control.
In MSTO-211H cells, MWCNT caused
significant toxicity at concentrations of 2
ppm and higher
In HaCaT cells were inhibited by
MWCNT significantly only at 10 ppm
Figure 1 shows the normalized fluorescein
diacetate uptake by A3 cells following 60min treatment with 2, 5, and 10 ppm
MWCNT
Figure 1
CONCLUSIONS
Overall, the test CNTs inhibited cellular viabilities in a
concentration, cell type, and CNT type-dependent pattern. The
viabilities of the MWCNT-impacted cells are higher than the
corresponding SWCNT groups.
The greater availability of defects and contaminants for cellular
interaction may contribute to the higher cytotoxicity of SWCNT.
REFERENCES





http://www.nanotech-now.com/nanotube-buckyball-sites.htm
“In vitro evaluation of cytotoxicity of engineered carbon nanotubes in selected human
cell lines”. By Xiaoke Hu a, Sean Cook a, Peng Wang a, Huey-min Hwang a,⁎, Xi Liu b,
Quinton L. Williams .
“Potential in vitro effects of carbon nanotubes on human aortic endothelial cells” by
Valerie G. Walker a, Zheng Li a,1, Tracy Hulderman a, Diane Schwegler-Berry
b,Michael L. Kashon c, Petia P. Simeonova a. a Toxicology and Molecular Biology
Branch, Health Effects Laboratory Division.
“A Predictive Bayesian Dose-Response Assessment for Evaluating theToxicity of Carbon
Nanotubes Relative to Crocidolite Using a Proposed Emergent Model”. By Jeffrey J.
Iudicello a;James D. Englehardt a
J.-P. Salvetat, J.-M. Bonard, N.H. Thomson, A.J. Kulik, L. Forro, W. Benoit, L.
Zuppiroli. "Mechanical properties of carbon nanotubes." Appl. Phys. A (1999):
255–260.
REFERENCES





Lam, Chiu-Wing, et al. "Pulmonary toxicity of Single-Wall Carbon
Nanotubes in Mice 7 and 90 Days After Intratracheal Instillation."
Toxicological Sciences (2004): 126-134.
Lu, Jian Ping. "Elastic Properties of Carbon Nanotubes and Nanoropes."
Phys. Rev. Lett. (1997): 1297–1300.
Muller, J, F. Huaux and Lison D. "Respiratory toxicity of carbon
nanotubes: How worried should we be?" Science Direct (2006): 1048-1056.
Ruoff, R. S. and D. C. Lorents. "Mechanical and thermal properties of
carbon nanotubes." sciencedirect (1995): 925-930.
T. W. Ebbesen, H. J. Lezec, H. Hiura, J. W. Bennett, H. F. Ghaemi, T.
Thio. "Electrical conductivity of individual carbon nanotubes." Nature
(1996): 54-56.