Biomedical Engineering Reference
In-Depth Information
method (Oostingh et al. 2011, Zhang et al. 2006). As damage to the DNA is highly correlated with
an increased risk of cancer, it is critical to assess such damages by any NP that is likely to come in
contact with humans. The comet assay (single-cell gel electrophoresis assay), which is utilized to
measure the number of single-strand breaks in DNA, is the most common method to assess DNA
damage. This assay has been used to assess DNA damage in cells exposed to cerium oxide (CeO
2
)
(Auffan et al. 2009), Ag (AshaRani et al. 2009), and SiO
2
NPs (AshaRani et al. 2009). Other meth-
ods to assess DNA damage include checking for the presence of micronuclei or other chromosomal
aberrations and measuring the expression of proteins implicated in DNA repair. An increase in
the expression and activation of DNA repair-related proteins was found upon cellular exposure to
MWCNTs (Zhu et al. 2007).
The general DNA microarray and more specific PCR analyses are being utilized to assess the
activity of functional genes involved in various cellular processes. They have been used to assess
changes in gene expression upon exposure to gold (Au) nanorods (Hauck et al. 2008), SWCNTs
(Nygaard et al. 2009), and SiO
2
-coated CdSe/ZnS QDs (Zhang et al. 2006). PCR was utilized to
study the effects of antimony trioxide (Sb
2
O
3
) NPs in erythroblasts (Bregoli et al. 2009) and the
impact of cesium dioxide (CeO
2
) NPs on the expression of genes related to oxidative stress and cell
structure (Park et al. 2008).
1.7.2.2 Immunogenicity
The ability of a given NP to evoke an immune response is a vital parameter in demonstrating its
toxicity on physiological systems, and it may not be explored by standard cellular toxicity studies.
ELISA can accurately detect cytokine levels at picograms levels. Many investigators have studied
the formation of proinflammatory cytokines (e.g., interleukin-6 and -8) following the exposure to
metal oxide NPs in various cell types by this technique (Veranth et al. 2007, Schanen et al. 2009).
1.7.2.3 Oxidative Stress
An elevated amount of ROS, either due to an innate immune response, to a NP or from the ability of
a specific NP (e.g., a fullerene or a metal oxide) to autocatalyze ROS formation in the cellular envi-
ronment, has the potential to damage or disrupt key cellular processes (Xia et al. 2006). Generally,
the presence of ROS can be directly assessed by quantifying the amount of ROS present in a given
cell population or indirectly assessed by monitoring the secondary effects of prolonged oxidative
stress. The spectrofluorimetry/FCM or spectrophotometry-based system can directly measure and
monitor the ROS-induced formation of the fluorescent product, fluorescein, from 2,7-dihydrodichlo-
rofluorescein diacetate (DCFDA), the superoxide-induced conversion of dihydroethidium (DHE)
from the blue fluorescent form to the red fluorescent form, or the superoxide-induced conversion of
nitroblue tetrazolium (NBT) to blue formazan. The DCFDA and DHE assays have experimentally
shown to change ROS levels in MPMCs (Marquis et al. 2011) or human fibroblasts (AshaRani et al.
2009), which were exposed to Au or Ag NPs with different surface functionalities. The effects of
ultra-small superparamagnetic iron oxide NPs (AshaRani et al. 2009) and cationic lipid-coated
Fe
3
O
4
NPs (Soenen et al. 2009) in human monocyte macrophages and 3T3 cells were assessed by
NBT assay. The determination of lipid peroxidation or antioxidant depletion is the measurement
of the secondary effects of increased cellular ROS levels. These can be done with the detection of
8-hydroxy deoxyguanosine (8-OHdG) and superoxide dismutase (SOD) activities. A green fluores-
cent dye, which turns red in the presence of oxidized lipids, was utilized to assess lipid peroxidation
in the presence of Cd/Te QDs (Choi et al. 2007).
1.7.2.4 Cell Proliferation
The rate of cell growth is an important indicator of overall cell integrity and of the potential for
NPs to interfere with proliferative processes. There are two quantitative assays commonly utilized
as the standard for assessing cell proliferation: (a) cell counting by FCM or high-content image ana-
lyzers, and (b) the colony-forming efficiency (CFE) assay. The effect of SWCNTs (Mu et al. 2009)
