Toxicity studies of Zinc oxide Nanoparticles in Human cells
Chue, P.W.1 and S.Valiyaveettil.2
Department of Chemistry, Faculty of Science, National University of Singapore
3 Science Drive 3, Singapore 117543
ABSTRACT
Zinc oxide nanoparticles (ZnO-np) of different size and properties were synthesized using starch
and fluorescein isothiocyanate (FITC) as capping agents. Characterizations of the nanoparticles
were done using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy
(TEM) and Uv-Visble spectroscopy. Toxicity of the NPs was performed on Human cell lines (U251
and IMR-90). The cellular uptake was confirmed using confocal microscopy
The experiments demonstrated that the ZnO-np capped with FITC (ZnO-FITC, 50-100 nm)
exhibited more cytotoxic characteristic as compared to ZnO-np capped with starch (ZnO-starch,
100-200 nm). Furthermore, the effective internalization of Zno-FITC nanoparticles were detected
by confocal imaging. Our studies showed that the toxicity of Zno-np were size and dose dependant.
Also, capping agent employed played a crucial role by controlling the solubility.
INTRODUCTION
The ability to engineer materials at the nano or near-atomic scale has triggered rapid product
development due to their new interesting properties that were not previously seen at scales above
the micrometer. The vast industrial applications of nanoparticles (NPs) have resulted rapid growing
demand for nanosized materials and hence is the human exposure to NPs.
Unlike classical chemical compounds which are routinely subjected to toxicity studies prior to
release to the public, no such procedures currently exist for nanomaterials. In addition, little is
known about the toxicity of NPs except that they might not have the same chemical properties as
when in their bulk form. This is because the nanosized material has a much greater surface area
which can result in a higher reactivity.
ZnO-np possesses various functions for uses such as pigments, piezoelectric devices,
luminescent devices, gas sensors, catalyst, and cosmetic materials in our today society. (Fan and Lu,
2005) (Nohynek, et al., 2007). In particular, ZnO-np in the range of 100-200nm have been
commonly used in sunscreen products because of their ability to filter UVA as well as UVB light.
For this toxicity study, the two different types of ZnO-np, (ZnO-FITC and ZnO-starch), were
synthesized. The goal was to compare the activity of same metal oxide NPs with different sizes.
The bioactivity was investigated through in vitro studies on human glioblastoma cells (U251) and
human lung fibroblasts (IMR-90).
1
2
Student
Associate Professor
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RESULTS AND DISCUSSION
Synthesis and characterization of ZnO-np
The overview of the synthesis of ZnO-np can be summarized as below.
Zn(ClO4)2.6H2O(s)  Zn2+(aq) + 2(ClO4)-(aq)
Zn(ClO4)2(aq) + 2NaOH (aq)  Zn(OH)2(s) + 2Na(ClO4)(aq)
∆
Zn(OH)2(s)  ZnO(s) + H2O (aq)
B
Figure 1: TEM images of ZnO-FITC (A) and ZnO-Starch (B).
The ZnO-np synthesized was analysed using TEM and SEM. TEM analysis shows that the ZnOFITC nanoparticles showed a size distribution of 50-100 nm (Figure 1A) whereas the ZnO-starch
showed a size of 100-200 nm (Figure 2A).
Due to the high surface energy of the metal oxide NPs, the NPs will not be stable in aqueous
medium and agglomeration will occur. This will impose on the cellular uptake of NPs which is
carried out in aqueous medium. Therefore, the surface tailoring of the ZnO NPs is performed using
FITC and starch, to improve dispersion in aqueous medium and stability.
Both FITC and starch can cap around the ZnO, providing steric hindrance between ZnO NPs due
to their bulkiness. In addition, the free hydroxyl group present on starch and FITC can interact with
the aqueous medium and enhanced the solubility.
Starch was chosen because it is biocompatible with our study. The use of biocompatible capping
agent and hence water as solvent, offers numerous benefits such as safety and readiness for
integration to biologically relevant systems. On the other hand, FITC was chosen because the
fluorescence gives a clear indication of cellular uptake of NPs under the lens of confocal
microscope.
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Cytotoxicity studies
Figure 2: Confocal images of fibroblasts (A) and (B) are images of control cells which show no
fluorescence. Image of treated cells, showing fluorescence at different parts of the cells (C).
Figure 3: cytotoxicity of ZnO nanoparticles- ATP content: ZnO-FITC nanoparticles were more
toxic (A) compared to ZnO-starch (B). FITC controls showed less toxicity compared to the ZnOFITC nanoparticles (C). The statistical significance was calculated using students t test. P values
<0.05 were considered significant. * represents P <0.05, ** represents P<0.01.
When treated with ZnO-FITC, both cells showed a decrease in viability with increasing
concentration of the NPs. The data showed that only 3% IMR-90 cells were surviving as the
concentration of ZnO-FITC reaches 60 μg/mL. On the other hand, approximately half the number
of U251 cells still survived at this level of concentration (Figure 3A). Therefore, it can be
concluded that the ZnO-FITC toxicity is concentration dependent.
ZnO-starch does not seem to exhibit cytotoxic effect (Figure 3B). Both the cells’ viability was
not observed to change drastically as compared to that of ZnO-FITC.
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The size of ZnO-starch is bigger than ZnO-FITC as depicted from TEM images. Earlier reports
had established the dependence of zinc oxide nanoparticle toxicity on particle size. The ultrafine
particles are reported be more active than fine nanoparticles (Beckett et al., 2005).
Another interesting observation was the intercellular distribution of NPs. Microscopic
observation showed that the NPs can be found both on the surface of the cells and inside the cells
(Figure 2C). Therefore, it was assumed that the nanoparticle endocytosis by the cells was the main
event in toxicity. However the mechanism and kinetics of uptake and initiation of cytotoxicity
requires further study.
CONCLUSION
In summary, both nanosized ZnO capped with FITC and starch was successfully synthesized.
ZnO-FITC exhibited concentration dependant cytotoxicity on both U251 and IMR-90 cells.
Moreover, the toxicity was greatly dependant on particle size and surface functionalization
(Oberdorster et al., 2005), which were the major difference between the NPs. Whereas for ZnOStarch, no significant cytotoxicity was observed.
In addition, more investigation is called for further studies on (i) the intracellular distribution of
NPs that may contribute to the cytotoxic effect, (ii) organelles which are suspected to be involved
with the NPs. This could greatly facilitate the understanding of the mechanism by which ZnO
nanoparticle can induce cytotoxicity.
ACKNOWLEDGEMENT
I would like to thank my supervisor, Associate Professor Valiyaveettil Suresh and my mentor,
Asharani, for their patience guidance throughout my UROPS project.
REFERENCES
Beckett, W. S.; Chalupa, D. F.; Pauly-Brown, A.; Speers, D. M.; Stewart, J. C.; Frampton, M. W.;
Utell, M. J.; Huang, L. S.; Cox, C.; Zareba, W.; Oberdorster, G. Comparing inhaled
ultrafine versus fine zinc oxide particles in healthy adults: a human inhalation study.
Am. J. Respir. Crit Care Med. 2005, 171 (10), 1129-35.
Fan, Z.; Lu, J. G. Zinc oxide nanostructures: synthesis and properties. J. Nanosci.
Nanotechnol. 2005, 5 (10), 1561-73.
Nohynek, G. J.; Lademann, J.; Ribaud, C.; Roberts, M. S. Grey goo on the skin? Nanotechnology,
cosmetic and sunscreen safety. Crit Rev. Toxicol 2007, 37 (3), 251-77.
Oberdorster, G.; Maynard, A.; Donaldson, K.; Castranova, V.; Fitzpatrick, J.; Ausman, K.; Carter,
J.; Karn, B.; Kreyling, W.; Lai, D.; Olin, S.; Monteiro-Riviere, N.; Warheit, D.; Yang,
H. Principles for characterizing the potential human health effects from exposure to
nanomaterials: elements of a screening strategy. Part Fibre. Toxicol. 2005, 2, 8.
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