Investigating the Toxicity of Iron(III) Oxide Nanoparticles, Zinc(II) Oxide
Nanorods and Multi-Walled Carbon Nanotubes on Red Blood Cells
Stephanie Katharine Loeb, AshaRani P.V. Nair, Suresh Valiyaveettil
Department of Chemistry, Faculty of Science, National University of Singapore
Abstract
In recent years there has been much research into the use of nanomaterials in
biological systems. Hemolysis of erythrocytes is a useful method to examine the effects of
particles on the cell membrane. This study investigated the toxic effects of multi walled
carbon nanotubes (MWCNT), zinc (II) oxide (ZnO) and iron(III) oxide (Fe2O3) nanomaterials
on human red blood cells (RBC). Cell morphology was studied before and after exposure to
nanomaterials, using optical microscope, and AFM, which revealed distinct morphological
aberrations. Ultra high resolution imaging systems were employed for studying the
interaction of nanoparticles and RBC that unveiled attachment of nanoparticles to RBC and
their cross linking effects. The hemolytic and hemagglutinating activities of these
nanomaterials were investigated in detail. Our results showed that ZnO nanorods were able to
induce hemolysis and hemagglutination in treated RBC whereas Fe2O3 displayed only
hemagglutination, and MWCNT showed only hemolysis. This study evidenced that
MWCNT, ZnO and Fe2O3 are toxic to human red blood cells irrespective of the blood group.
Introduction
Nanoparticles are a relatively new
class of biomedical products. These
materials offer the potential to result in
novel approaches for combating complex
disorders
including
cancers
and
neurodegenerative
disorders
(M.
Dobrovolskaia et al., 2008). Before this
can be done careful investigation is
required to study the effects that these
nano-sized materials will have on human
bodies.
Hemolysis is the destruction of red
blood cells resulting in the release of
hemoglobin into the surrounding fluid.
This release of hemoglobin can be
detected through spectroscopic methods. If
hemolysis occurs to a significant number
of red blood cells in the body it can lead to
dangerous
pathological
conditions.
Therefore, all biomedical products which
have
intent
to
be
administered
intravenously should be evaluated for
hemolytic properties (M. Dobrovolskaia et
al., 2008).
Titanium dioxide is a common
substance known for its wide range of
applications including paint and cosmetics.
TiO2 has generally been regarded as
harmless and inert, but in recent research,
it was discovered that TiO2 nanoparticles
induced human erythrocyte hemolysis. (Y.
Aisaka, et al, 2008).
The
series
of
experiments
described in this report were conducted
with the objective of obtaining information
on the toxic effects of zinc(II) oxide (ZnO)
and iron(III) oxide (Fe2O3) nanomaterials
on human RBCs. Fe2O3 nanoparticles,
ZnO nanorods as well as Multi-Walled
Carbon Nanotubes (MWCNT) are
examined for their hemolytic properties as
well as their effects on cell morphology.
Both the shape and composition of these
particles were studied as major factors
contributing to the resulting level of
damage to the cell.
Methods and Materials
Nanomaterial Synthesis: MWCNTs were
purchased from Shenzhen Nanotech and
functionalized as described in previous
reports (P.V. AshaRani et al. 2008). For
the synthesis of Fe2O3 nanoparticles and
ZnO nanorods, NaOH (2M) was added
dropwise to 50ml of 0.1M ZnCl2 and
FeCl3 stirring until a pH of 8 and 10 was
obtained respectively.
(i) ZnCl2 + 2NaOH  Zn(OH)2 + 2NaCl
(ii) FeCl3 + 3NaOH  Fe(OH)3 + 3NaCl
Zn(OH)2
and
Fe(OH)3
are
precipitates which were separated by
centrifugation and subsequently washed
with milli-Q water by centrifugation at
5000 rpm. Washing was repeated 3 times.
Varying amount of PVP were dissolved in
100 ml of milli-Q water. Each precipitate
was then added to a polymer solution and
mixed and refluxed under inert condition
for 3 hours. After the reactions were
completed, the mixtures were centrifuged
to obtain the nanomaterials, which were
dried in an oven at 100C to remove any
traces of water present.
Collection and Separation of RBC from
whole blood: Human blood was collected
fresh, from healthy volunteers (A,B and O
groups) in to lithium heparin vacutainers
(BD Biosciences, USA) . Blood (3 mL)
was diluted to 15 mL with Dulbecco's
phosphate buffered saline (DPBS, Mg++
and Ca++ free, Sigma-Aldrich, USA).
RBCs were separated by centrifugation
(Jouan centrifuge, model number BR4i).
Red blood cell counts were determined
with a hemocytometer. 70 million RBCs in
1 mL DBPS was added to each tube.
Nanoparticle preparation: Depending on
the solubility of the nanomaterials to be
tested, different carrier solvents were
employed.
Each
nanoparticle
was
dissolved in their respective solutions:
(i) ZnO: 2 parts DMSO to 3 parts DPBS
(ii) FeO: 4 parts DI water to 1 part DMSO
(iii)MWCNT: de-ionized water
Each mixture was sonicated until
the particles were completely dissolved in
the solution. Concentrations of 25, 50,
100, 200 and 400 mg/mL were inserted by
micropipette in each 1mL test tube. A
positive control was made by adding
0.01% of Triton X-100 (Sigma-Aldrich,
USA).
The test tubes were incubated in a CO2
incubator, (Sanyo, MCO-18AIC, UV), at
37°C for 3 hours. After 3 hours, the
samples were removed and observed for
hemagglutination. Those sedimented as
buttons were considered as negative and
those showing a mat-like appearance were
considered
as
positive
for
hemagglutination. The pellet was used for
imaging purposes including SEM, AFM
and Cytoviva.
The supernatant was centrifuged at
20,000 rpm for 45 min to remove the
nanoparticles from the solution. This is
essential as nanoparticles can interfere
with the absorbance based methods for
hemoglobin quantification.
Hemolysis Testing: Taking the final
product from the blood processing, 3 x 100
μL aliquots from each sample were added
to 96 well plates (Nunclon Delta,
Denmark). Drabkin’s reagent (100 μL)
was added to each sample in the absence
of direct light. The reagent and sample
were allowed to sit for 15 minutes, before
recording the absorbance at 540 nm using
aμQuant Bio-Tek spectrophotometer.
Values were compared to a known
standard curve which was constructed
from various concentrations of human
hemoglobin
samples
(Sigma-aldrich,
USA) as described by M. Dobrovolskaia et
al., 2008.
Results
Hemolysis
The concentration of hemoglobin
present in the samples was calculated from
the absorbance values.
Figure 1: Hemolysis values of Fe2O3, ZnO and
MWCNT in human RBC. As the concentration
increases in most cases so does the hemolytic
activity
As the graph in figure 1 displays,
overall the rods were showing far more
lysing than the nanoparticles. ZnO
exhibited the most hemolysis. Although
Fe2O3 did not show significant hemolysis,
it did prove to have morphological effects
on the red blood cells.
Hemagglutination
The effect of hemagglutination was seen
most strongly in the samples treated with
Fe2O3 nanoparticles beginning at 50
μmg/mL. It was also observable in the
ZnO treated samples but only at the
highest concentration of 400 μg/mL. The
samples treated with the multi-walled
carbon nanotubes did not exhibit
hemagglutination.
Cytoviva
Figure 2: Cytoviva images of nanomaterials
interacting with RBCs (a) control sample of
untreated cells (b) RBC treated with Fe2O3
nanoparticles at 400 μg/mL: a patch of
hemoglutinated damaged red blood cells is visible
(c) RBC treated with ZnO nanorods at 400 μg/mL:
the ZnO nanorods are clustered on an RBC (d)
RBC treated with ZnO nanorods at 400μg/mL: a
large area of damaged cells
The Cytoviva images show the
nature of the interactions between the
nanomaterials and the RBC. As in Figure
2B, the samples treated with Fe2O3 show
hemagglutinated effects, but the images do
not show evidence that the nanoparticles
have lysed the membrane or entered the
cell. The samples treated with ZnO shows
large areas of aggregated nanorods on the
RBC. As seen in figure 2D, agglomerated
clusters have caused heavy damage and
hemolysis to an area of RBCs.
AFM
Figure 3: AFM images of RBCs treated with
nanomaterials (a) control sample of untreated cells
(b) RBC treated with Fe2O3 nanoparticles at
50μg/mL: the nanoparticles can be seen as small
circle on the surface of the RBC (c) RBC treated
with ZnO nanorods at 400μg/mL: the large white
spots are agglomerated ZnO nanorods (d) RBC
treated with MWCNT at 400 μg/mL: the
biconcave structure is disrupted
The images captured by AFM
support those captured from Cytoviva. The
RBC shown in Figure 3B is treated with
Fe2O3 nanoparticles. The particles are on
the surface of the RBC and not inside the
cell. Figure 3C shows the agglomerated
ZnO clusters. Figure 3D depicts a sample
treated with MWCNTs. The MWCNTs
have not agglomerated in that way that the
ZnO nanorods have, but the RBC shows
considerable damage.
Summary of Results
Figure 4: Summary
Hemagglutination Results
Nanomaterial
Zinc(II)
Oxide
Nanorods
Iron(III)
Oxide
Nanoparticles
Multi-walled
Carbon
Nanotubes
of
Hemolysis
Yes, 0.37
mg/mL at
400 μg/mL
No, 0.04
mg/mL at
400 μg/mL
Yes, 0.32
mg/mL at
400 μg/mL
Hemolysis
and
Hemagglutination
Yes, but beginning
at 400 μg/mL
Yes, beginning at
concentrations of
50μg/mL
No appreciable
hemagglutination
observed
Discussion
Examining the Cytoviva images of the
Fe2O3 effected RBC’s, there appears to be
a strong affinity for the nanoparticles to
bind to the cell, and a weaker, but still
substantial, attraction of the nanoparticles
to aggregate and bind together. This
combination of attractions causes the
RBCs to clump together creating larger
amounts of cell aggregation, producing
heavily hemagglutinated samples. In the
samples treated with ZnO, the nanorods
show a strong attraction to bind with each
other and aggregate in much larger clumps
which begins to approach larger
micrometer sizes. These larger particles
have the ability to move about and cause
large amounts of damage to the RBC’s.
The
samples
treated
with
MWCNTs displayed hemolysis, but no
hemagglutination. The AFM images give
no indication that the MWCNTs were
agglomerating into larger micron sized
particles. Since elemental carbon is a
naturally non-evasive biological material
and the carbon nanotubes had so such
hemolytic activity, it can be concluded that
the main contributing factor to its toxicity
is the shape of the MWCNT and not its
composition.
Conclusions
The toxicity of nanomaterials on
RBCs is a result of a combination of shape
and composition
The effect of shape is based on the
degrees of free rotation. Damage caused to
the red blood cell is likely to increase
when introduced to nanomaterials which
have greater degrees of freedom like
nanorods, nanotubes or even asterisk and
star shaped nanoparticles. The effects of
composition are based on the affinity for
the nanomaterial to itself and to the red
blood cells. The greater the nanomaterial
affinity to agglomerate to itself without
precipitating out of the solution, the more
hemolytic activity will be detected.
Further Experimentation
Reversing the factors of composition and
shape in the metal oxide particles (i.e. ZnO
nanoparticles and Fe2O3nanorods) should,
by the hypothesis which has so far been
formulated, result in hemolysis by both the
zinc particles (due to their composition)
and the iron nanorods (due to their shape).
Comparing the levels of hemolysis in both
these cases could lead to an evaluation of
whether shape or composition is the
leading factor in determining toxicity.
The toxicity of the C60 buckyball
has to some extent been studied and very
little evidence has been gathered to
indicate that C60 is toxic. The issue with
current testing of toxicity is the lack of
standardized procedures for toxicological
studies on nanoparticles (M. Arruebo et al,
2008). Due to this lack of standardization,
a test on the hemolytic proprieties of
buckyballs conducted under the same
parameters as the testing on the nanotubes
performed in this experiment could result
in well documented and comparable
literature on the difference in the toxic
effects of buckyballs versus carbon
nanotubes.
Acknowledgements
Thank-you
to
Professor
Suresh
Valiyaveettil, Asharani P V Nair, Sajini
Vadukumpully and the rest of the research
group. Also to Shoaping Zong for helping
produce the AFM images
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