**2.3 Necrosis assays**

Necrosis assays are generally used to check for membrane integrity of the cells after treatment. As shown in **Figure 3**, NPs can adhere to membranes causing changes in their structure and function, hence it's important to carry out these assays. Neutral Red (2-amino-3 methyl-7-dimethylaminophenazoniumchloride) uptake assay is a widely used assay used to measure cytotoxicity. The Neutral Red dye is a weak, cationic, cell permeable dye taken up by viable cells and localized in the lysosome. It permeates into the cell by nonionic passive dilution and binds to the phosphate groups found on the lysosome matrix. The dye is extracted from the viable cells and spectrophotometrically measured [74, 75]. It is a sensitive dye that measures the integrity

#### **Figure 3.**

*The toxicity mechanisms induced by nanoparticles [73].*

#### In Vitro*,* In Vivo *and* Ex Vivo *Models for Toxicity Evaluation of Nanoparticles: Advantages… DOI: http://dx.doi.org/10.5772/intechopen.111806*

of the cell membrane and measures the viability of the cells [76]. The Neutral Red uptake assay is very sensitive and requires less equipment. It does not face much interference and does not have unstable reagents like other assays like MTT, XTS, etc. [77]. It is simple and can be used to detect only viable cells. It can also be done together with estimation of total protein content [78]. However, the use of Neutral Red, a fluorescent-based assay, is prone to interference from the NPs, which could lead to quenching of the dye. In a study to check the toxicity effect of silver NPs, Neutral Red, amongst other dyes, were observed to face interference from the particles [79]. Also, when applied on certain compounds that are volatile and insoluble in water, it faces problems in analysis as it mainly works when soluble in water [76]. Some chemicals could also cause the transformation of the dye into insoluble crystals, which could increase the toxicity estimation of the assay, giving a false negative result. When total protein estimation is done after the neutral red uptake assay, it could lead to reduction of the amount of protein estimated [78].

Trypan Blue Exclusion Assay is used to measure cytotoxicity of nanoparticle treatment on cells. The Trypan Blue is a dye that is absorbed by dead cells, leaving out viable cells and giving the actual number of viable cells after treatment.

Trypan Blue Exclusion Assay is very simple and quick to do, and it does not require any technical know-how. However, Trypan Blue assay is less sensitive and less reliable compared to other assays. It can be tedious and time-consuming when done on large number of samples [80]. Because of the use of hemocytometer, the possibility of making counting errors is present. Also, if the cells are not properly diluted or poorly dispersed in the counting chamber, it can contribute to the error [81]. The assay is insensitive as it cannot distinguish between live cells and cells that are gradually losing cell function. Another disadvantage of this assay is it can cause toxicity to mammalian cells [82].

#### **2.4 Oxidative stress assays**

Due to the high surface area to volume ratio of NPs, they elicit the production of reactive oxidative species (ROS) from the cells as shown in **Figure 4**, which could lead to further cellular damage. The ROS produced is measured using some of the oxidative stress assays.

2′,7′-dichlorofluorescein diacetate (DCFH-DA) is a nonionic, nonpolar fluorophore that penetrates the membrane and is sensitive to ROS. When DCFH-DA is internalized by the cell, it is enzymatically hydrolyzed by cytosolic esterases into dichlorofluorescein (DCFH), which remains in the cytosol. The nonfluorescent DCFH is then oxidized by hydroxyl radicals and cellular ROS into highly fluorescent dichlorofluorescein (DCF), which is then analyzed by flow cytometry or fluorescence microscopy [84].

Tritiated borohydride and 2,4-dinitrophenylhydrazine (DNPH) are used to measure protein carbonyl levels, which are indicative of oxidative damage. Protein carbonyls are reduced to alcohols by borohydride. This reduction is measured spectrophotometrically at wavelength absorbance of 340 nm. Also, when protein carbonyls react with DNPH a stable 2,4-dinitrophenyl (DNP) hydrazone product is generated and its absorbance is read between 360 and 390 nm [85]. DNPH is highly sensitive and specific and result from the assay can further be improved with the use of highperformance liquid chromatography (HPLC) or Western blotting [86].

Another means of assessing the production of ROS is through analysis of lipid peroxidation products like malondialdehyde (MDA) and 4-hydroxyl-2-nonenal

**Figure 4.** *Induction of oxidative stress by nanomaterials [83].*

(4-HNE). MDA is formed when large polyunsaturated fatty acids (PUFAs) are broken down and during the metabolism of arachidonic acid (AA) [87]. The byproduct reacts with thiobarbituric acid (TBA) at pH 3.5 to produce MDA-TBA which is measured by a spectrophotometer at wavelengths of 532 nm and fluorescently at 553 nm [88]. 4-HNE is a by-product of AA and peroxidation of PUFAs. It reacts with primary amines to form Schiff 's bases and thiol. It also reacts with amino compounds to form Michael's adduct which is detected by HNE-protein adduct ELISA assays [89, 90].

DCFH-DA, However, is prone to inaccurate interpretation due to nonspecific enzymatic oxidation and photooxidation [84, 91]. With the use of DNPH, larger samples are required for the analysis [92]. The lipid peroxidation is nonspecific since TBA reacts with other molecules other than MDA, which could lead to false positive results.
