**2.** *In vitro* **models**

#### **2.1 Proliferation assays**

Proliferation assays are assays used to check for cellular metabolism in active metabolic cells. They help to ascertain the viability of cells when treated with NPs to check for the toxic effects of the particles on the cells. One of the commonly used assays is the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It is a colorimetric assay where 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyltetrazolium bromide (MTT) is enzymatically reduced by mitochondrial succinate dehydrogenase to formazan crystals, an insoluble product, in the mitochondria of live cells. The breakdown is spectrophotometrically measured to estimate cell viability. In this assay, there is a linear relationship between the color formed when the breakdown to formazan occurs or the absorbance when measured and the viability of the cells. It is a very sensitive and quantitative assay [13, 14]. The MTT assay proves to be advantageous because it produces results quickly under a maximum of 3 to 4 hours. It is also reproducible and does not require manipulation of the target cell [15].

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

The Alamar Blue Assay is also a colorimetric indicator assay characterized by the reduction of a blue, non-fluorescent dye to a pink-colored substance known as Resazurin [16]. It is both a qualitative and quantitative assay where colorimetric measurement can be taken or physical observation of color change in assessing cell viability or treatment toxicity. It is taken at a wavelength of 570 and 600 nm or 540 and 630 nm using the spectrophotometer. It is excited and emitted at wavelengths of 530–560 and 590 nm respectively [17]. Alamar Blue assay is highly sensitive, requires low cost, it's easy and safe to use, non-radioactive, and can be used for a large number of sample. It can also be used for both quantitative and qualitative analysis. Another method used to assess proliferation is the incorporation of [3 H] thymidine into the DNA of proliferating cells during the S phase of the cell cycle, with the use of autoradiography [18]. As the cells proliferate, new DNA strands are formed. The tritiated thymidine, a radioactive nucleoside, then enters into the new chromosomal strands as the cells divide [19]. [3 H] thymidine incorporation method is also widely used in immunological studies because of its high throughput and direct measurement of cell proliferation. It is also sensitive [19]. Another commonly used assay, clonogenic assay is used to assess cell survival and reproductive ability after treatment. The ability of a single cell to reproduce a colony is checked [20].

As shown in **Figure 2**, MTT assay has its limitations that affect the interpretation of data. Due to the formation of formazan, an insoluble dye, in MTT assay, other assays like XTT (2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) are used. With XTT, in the presence of mitochondrial dehydrogenase enzymes, a soluble water product is produced. However, both assays also faced the limitation of reaction under other conditions like acidic media, additives found in culture media, polyphenols, the presence of NPs and O<sup>−</sup> 2. These gives a false positive result of cell viability [22–24]. In a study carried out on Chinese hamster ovary cells (CHO-K1) after exposure to nano-TiO2, which increases the formation of O<sup>−</sup> 2, MTT and XTT assay were found to produce an inaccurate analysis of cell viability, because of the ability of the superoxide produced by the NPs to reduce the two salts [25]. Other factors such as cell number, the concentration of the MTT reagent, the treatment applied to the cells, and the toxic effect of the MTT itself has to be critically considered when analyzing the results from the assay. When prostate cancer cell lines (PC-3) were treated with polyethylene glycol-coated gold NPs (Au-PEG-NPs) at a concentration of 5 nm, there was an observed significant optical interference with the MTT assay. However, this effect could be minimized by washing the cells and removing the supernatant before MTT incubation and using appropriate blanks [21].

A study on the *in vitro* assessment of the toxicity of carbon based NPs HiPco single-walled carbon nanotubes (SWCNT), arc discharge SWCNT and Printex 90 carbon black NPs was done to initiate a more reliable method with the use of clonogenic assay [26]. This study was performed to avoid the interaction of carbon based NPs with the indicator dyes used in colorimetric assays, leading to inaccurate interpretations on toxicity studies [27, 28]. Graphene oxide and TiO2 NPs have also been observed to interact with the dyes [29, 30]. This assay rules out the possibility of the nanoparticle reacting with the assay itself. It is recommended as a very useful tool for testing cytotoxicity since colony number and size are taken into account. This makes it an effective differentiating tool between cell viability and cell proliferation [26]. It is also a sensitive method since colony size depicts the division rate and proliferation after the cells are treated [31].

#### **Figure 2.**

*Factors affecting the final optical density (OD) measurements in the MTT assay. These include the concentration of MTT reagent and the proportion that actually enters the cell, cellular metabolic activity (which is highly dependent on a multitude of variables including treatments to the cells, biological effect of culture media, cell density, and impedance of cell metabolism due to toxic effects of MTT), cell number, timing of formazan crystals extrusion (which could impede further MTT uptake), chemical interference such as abiotic reduction of MTT by culture media, the tested treatment, or released cellular content, optical interference by all the background components, time of incubating cells with MTT reagent and/or tested treatment, and ultimately the optical measurement. Chemical structure of MTT and formazan are illustrated inside the cell: MTT consists of a tetrazole ring core containing four nitrogen atoms (1) surrounded by three aromatic rings including two phenyl moieties (2) and one thiazolyl ring (3). Reduction of MTT results in disruption of the core tetrazole ring and the formation of formazan. Red arrows and the "-" sign indicate disruption of MTT reduction on the normal metabolic activity of the cells and the impeding effect of the formazan crystals (when presenting on the cell surface) on further uptake of MTT reagent by cells [21].*

For Alamar Blue assay, because of its photosensitivity, it is affected by light exposure and has to be done in the dark. Cell density is also a factor that can affect the assay reading, hence cells must be cultured in high population to prevent slow growth and allow adequate dye reduction. The assay is also limited to a pH range of 7.0 and 7.4, and optimum temperature of 37°C. Longer incubation times could also lead to the reduced dye being bleached to a pink color. This quenches the fluorescence and could cause misinterpretation of the fluorescent signals. There is also the need for a positive and negative control to rule out non-specific interaction with the chemistry [17]. Specifically, with the use of nanomaterials, it is necessary to check toxicity using a combination of other assays because of the diversity and physiochemical properties of the nanomaterials, which could affect the cell and also interfere with the chemistry of the assay [32, 33].

The [3 H] thymidine proliferation method is toxic and also requires radioactive facilities, which can be very expensive. Also, there is the need for proper waste management of the radioactive materials used with this method [34, 35]. It has also been found to interfere with the target cells by inducing cell cycle arrest, apoptosis and fragmenting the DNA [34, 36, 37].

One of the major cons of Clonogenic assay is that it is time-consuming as it takes about 10–14 days to perform. This is quite long compared to the other assays described above which take only a few hours to a day [38].

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

#### **2.2 Apoptosis assays**

Apoptosis Assay is another assay carried out to evaluate the cytotoxicity of nanomaterials. It measures the extent of DNA damage and cell death when cells are treated. The release of free radicals due to oxidative stress is a pointer to DNA damage and nanomaterials have been found to elicit such reactions [39, 40]. One of the methods used to study apoptosis is the Annexin-V assay. This assay works on the principle of annexin-V binding to phosphatidylserine (PS) which is externalized on the plasma membrane due to activation of caspase-dependent pathway. In combination with Propidium Iodide (PI) which stains the nucleus indicating the last stage of cell death, both dead and apoptotic cells can be identified [41, 42]. Annexin-V and PI assays have been used to detect apoptosis in HeLa and human HepG2 hepatoma cells treated with gold and silica NPs respectively [43, 44]. Annexin-V does not penetrate the cell but only binds to phosphatidylserine on the extracellular membrane of the cell; hence it does not cause cell damage or affect the intracellular components of the cell. It is highly sensitive as it binds to only PS amidst other molecules on the cell surface. It produces bright fluorescent signals and it's easy to perform [41]. There are different options available for its labeling and also it can be performed both *in vitro* and *in vivo* [45].

Comet assay is used to determine DNA damage, single- and double-stranded DNA breaks and the mutagenicity of treatment both *in vivo* and *in vitro* [46–50]. It is one of the commonly used assay to assess the genotoxic effects of engineered nanomaterials like TiO2, SiO2, Zinc oxide NPs [51–55]. Damaged bases are detected when nucleoids are incubated with endonuclease III and formamidopyrimidine DNA glycosylase (FPG), specific to oxidized pyrimidine and purines, respectively [56]. Comet assay is a sensitive technique that is quick, cheap and easy to do [57]. It has been used both *in vitro* and *in vivo* and hence can be used to check the toxic effects of treatment in a particular organ or tissue and on any animal model [49, 58]. It can also be done on the first or specific site of contact.

TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) assay is a commonly, wisely used tool for the detection of DNA damage caused by both apoptosis, DNA fragmentation and necrosis, caused by exposure to toxic materials [59–61]. When DNA fragmentation occurs, the 3′ hydroxyl termina becomes free. Then the enzyme terminal deoxynucleotidyl transferase incorporates labeled dUTP into the free end, which causes the staining observed in the assay [62]. It can be detected by light microscopy, fluorescence microscopy or flow cytometry [63]. TUNEL assay is fast as it can be completed within 3 hours. It has high sensitivity and versatile with the use of various techniques, as single cells can be detected using fluorescence microscopy or few cells using flow cytometry [63, 64].

Flow Cytometry is a very common and versatile technique used in the toxicity analysis of nanomaterials when applied to cells. It works on the principle of detecting fluorescently labeled cells when light beams are passed through the cell suspension. The technique analyses both the size and granularity of the cell populations, which gives a characteristic of the type of cells being studied [65]. With the use of flow cytometry, large populations can be analyzed with valid statistical results and this is done within a short period of time. This method can be used to evaluate the uptake of NPs by the cells, cell death, and expression of certain proteins [66].

However, these assays have their own set of limitations. For example, Annexin-V assay cannot be effectively used to obtain high throughput due to strong background signals when there are unbound labeled annexin-V. It can be difficult to optically

image tissues when stained with annexin-v because of its short lifetime and slow diffusion in the body [67]. It can produce false positive results when it binds to negatively charged aldehyde adducts [68]. Also, Engineered NPs have been found to interfere and hinder the detection of DNA damaged from oxidation with the comet assay when ions are released when the particles are dissolved [69]. Cell toxicity, even in the absence of DNA damage, can lead to false-positive results in Comet Assay. There are also variability of protocols from the assay which can make it difficult to compare results with other laboratories, as there is no standardization [57]. For TUNEL assay, there is the limitation of non-specific staining because it labels all the free 3′ hydroxyl termini end irrespective of the cause of it. Hence, it can label nonapoptotic cells, which gives a false positive result. This means it can stain cells undergoing DNA repair, cells secretes factors causing proliferation in neighboring cells, or cells damaged through other means [70]. It is also costly. For flow cytometry, freshly prepared samples are required. If samples are kept for a long period of time before analysis, it could affect the cell properties and the analysis. Also, the use of the flow machine requires competent and skillful hands to accurately analyze and interpret the data [71]. NPs smaller than 100 nm experience low light scattering which could reduce the sensitivity of the detection [72].
