Biomedical Engineering Reference
In-Depth Information
TABLE 10.1
Summary of Selected Cytotoxicity Assays for Different Nanoparticles
Assay
Principle
Application
References
Tetrazolium salts
(MTT, MTS,
XT, WST)
Mitochondrial function
Ag-NP
TiO 2
CoCr
ZnO,Fe 2 O 3 , MCM-41
Ahamed et al. (2008)
Liu et al. (2010)
Papageorgiou et al. (2007)
Dua et al. (2011)
Lactate
dehydrogenase
Membrane integrity
CoCr
CI, CS, AS, a ZnO
Papageorgiou et al. (2007)
Sayes et al. (2007)
Trypan blue
Membrane integrity
Ag-NP
Hackenberg et al. (2011)
ATP assay
Actively growing cells
Ag-NP
AshaRani et al. (2008)
a CI, carbonyl iron; CS, crystalline silica; AS, amorphous silica.
Glutathione is a naturally occurring antioxidant in cells and biological fluids throughout the
body. GSH (reduced glutathione) can react directly with ROS to neutralize them, and then be rap-
idly converted back into GSH. The oxidative stress induced by Zn, Fe, and Si nanoparticles has
been assessed by measuring the glutathione production, demonstrating that the presence of these
nanoparticles barely increased the level of ROS (Cha and Myung, 2007). The concentration of GSH,
rather than the GSH: GSSG (oxidized) ratio, is selected because cells often actively export GSSG as
a protective mechanism, making GSSG concentrations very low and lowering the ability to measure
GSSG in cells.
Electron paramagnetic resonance (EPR) can quantify and specifically identify the free radical
species generated via the use of specific spin traps or probes in combination with specific reagents,
whereas DCFH assay does not possibly detect such levels of specificity. However, the measurement
may be confounded by chemical or physical interferences with spin-trapping agents.
The other methods to test the levels of ROS are through the measurements of lipid peroxidation,
the plasmid assay, the Trolox equivalent antioxidant capacity assay (TEAC), and the measurement
of mRNA expression changes in oxidative stress-dependent genes (Stone et al., 2009).
10.3.3 M IcroscopIc e valuatIoN of I Ntracellular l localIzatIoN
The microscopic evaluation techniques employed in nanotoxicology studies include scanning
electron microscope (SEM), transmission electron microscope (TEM), atomic force microscopy
(AFM), fluorescence spectroscopy, video-enhanced differential interference contrast micros-
copy (VEDICM), and so on. Figure 10.2 presents the images taken by TEM, showing that Ag-NP
agglomerates (100-300 nm in diameter) are within the cytoplasm (within compartments resembling
endosomes and lysosomes) and nuclei of HepG2 cells (Kim et al., 2009). Figure 10.3 is the SEM
of live, adherent fibroblasts, demonstrating that nanoparticles of cobalt-chromium (CoCr) are dis-
persed more randomly within the cytoplasmic space (a) as compared to the micron-sized particles
(b) (Papageorgiou et al., 2007). Figure 10.4 is the AFM images of the Ag NP-treated BHK21 and
HT29 cells showing higher degrees of surface roughness with pit-like structures spread over the
cell surface. These changes could be attributed to the aggregation of membrane proteins and the
accompanying randomization of membrane lipids, proving the toxic effects of Ag-NPs on the cell
membrane (Gopinath et al., 2010).
10.3.4 g eNe e xpressIoN a NalysIs
DNA microarrays can be used to measure changes in gene expression levels. Practically, the expres-
sion of ~40,000 gene spots and replicates can be simultaneously analyzed on a couple of glass arrays
 
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