Biomedical Engineering Reference
In-Depth Information
endpoints. There is not a single method that is satisfactory for obtaining all the information on the
toxicity. Since different NPs elicit different biological responses, to study mechanisms underlying
toxicity, a combination of assays is often required.
19.9.1.1 Detection of Mitochondrial Activity
The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is
a widely used cell viability assay based on the reduction of the yellow tetrazolium salt MTT to a
purple water-insoluble formazan by succinic dehydrogenase in cells bearing intact mitochondria
[108]. It has been applied in numerous cytotoxicity studies and employed to validate other meth-
ods and to determine NP toxicity [108]. The MTT assays technique has been utilized for assess-
ment of different NPs such as NPs composed of titanium dioxide, iron oxide, zinc oxide, chitosan,
silica, for fullerenes, and for naked or chitosan-coated QD [109]. The absorption spectrum of
reduced MTT is pH dependent, and metal ions such as Zn
2+
interfere with the MTT reduction
react ion [110].
19.9.1.1.1 Drawbacks of MTT Assay
1. The SWCNTs may interact with the substrate, thereby depleting free MTT and causing
false-negative results [111].
2. Owing to optical properties of NPs, and their presence in the reaction mixture, in or on cell
culture, cells may directly influence the readout by increasing the light absorption [112].
3. It has been found that the cytotoxic effects of carbonaceous nanomaterials on human epi-
dermal keratinocytes using classical dye-based assays such as the MTT assay produce
invalid results due to nanomaterial/dye interactions and/or nanomaterial adsorption of the
dye/dye products [113].
4. MTT assay failed to report toxicity of certain porous silica microparticles due to sponta-
neous redox reactions where the MTT is reduced and the particle surfaces are oxidized
simultaneously. However, for other completely oxidized particles, the assay yielded the
expected results [114].
19.9.1.2 Detection of LDH Release upon Necrosis
The lactate dehydrogenase (LDH) release from damaged cells was measured using a colorimetric
assay. LDH is a stable cytosolic enzyme that is released upon cell lysis. This method is based on
the oxidation of the yellow tetrazolium salt of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazo-
lium chloride (INT) to a red formazan to evaluate the amounts of LDH released from the cytosol
upon cellular necrosis [115]. The intensity of the color produced is proportional to the number of
lysed cells. The LDH leakage (%) relative to the control without NPs is calculated as [test] − [con-
trol] × 100, where [test] is the absorbance of the test sample and [control] is the absorbance of the
control sample [116].
LDH assays using INT as the substrate have been applied to assess the cytotoxic potential of
various NPs. LDH is significantly deactivated under low pH conditions, whereas a high basic pH
destabilizes the substrate [117]. Furthermore, metal ions (e.g., copper) have been shown to interfere
with the LDH assay [118]. SWCNTs do not interact with the substrate INT [111].
19.9.1.3 Fluorescein Diacetate Test
Cell viability is also determined by the fluorescein diacetate (FDA) test [119]. In this method, the
cells are treated with 1:1 solution of FDA and ethidium bromide and observed under fluorescence
microscope with an excitation filter of 488 nm (blue light). Living cells are stained in green while
dead cells exhibit their nucleus stained in orange. The survival percentage is obtained dividing the
number of living cells by the total number of cells.
