**7. In vitro nano biomaterial cytotoxicity assessment**

The minimal ethical concerns associated with in vitro assays make them in demand for toxicity assays. It is much faster, and the cost is also reasonable. It is further subdivided into a few**.**

### **7.1 Experimental models used**

#### *7.1.1 Cell lines*

Various types of cell lines are used in the study of the cytotoxicity of nanoparticles. Using various cell lines for toxicity assay has added risks, which have been documented by Donaldson et al. [48]. This is because of the different toxic responses that the cells experience in vitro when compared to in vivo. Moreover, when carcinoma cell lines are used for in vitro toxicity testing of nanoparticles, the results may conflict with that of normal cells, as the carcinoma cell lines have different pathophysiology than normal cells [49]. The various types of cell lines that are used include murine cell lines (mouse fibroblast cells, mouse macrophage cells, rat mesenchymal cells, etc.), mammalian cells, human osteoblast cell lines, human alveolar and bronchial epithelial cells, human hepatocytes, human macrophages, cancerous cell lines (lung cancer cells, hepatocellular carcinoma cells, colon and cervix carcinoma cell lines, human epidermoid like carcinoma cells—HELA, etc.) and various hematic cells from murine, and mammalian species and humans [50].

#### **7.2 In vitro cytotoxicity assessment methods**

#### *7.2.1 Proliferation assay*

The cell viability assay is the most crucial investigation to understand a foreign agent's toxicity. Cell viability confirms the number of healthy cells in a sample indicated by their proliferation potential. It also gives information on cell death in the given sample and monitors cytotoxicity [51].

The most prevalent cell viability assays are based on the estimation of the metabolic activity of the cells. Examples are the MTT assay, protease activity assay, and the reduction of resazurin salts [52]. The mitochondrial and the cytoplasmic enzymes in the viable cells can react with these substrates to bring out colored products or fluorescence, which corresponds to the number of live cells.

The most commonly involved method is the MTT assay where a tetrazolium dye MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) penetrates the cells and due to the activity of mitochondrial enzymes get converted to formazan, a colored product. They are insoluble crystals and accumulate within the

#### *Toxicity Evaluation and Biocompatibility of Nanostructured Biomaterials DOI: http://dx.doi.org/10.5772/intechopen.109078*

cells, which are later solubilized using reagents like sodium dodecyl sulfate or isopropanol. The absorbance is then recorded, and maximum absorbance indicates higher survival [53, 54]. With an increasing number of viable cells, the intensity of color deepens. The advantages are quick results and the requirement of limited manipulations [55]. The positively charged MTT dye can readily penetrate the cell membrane. There are negatively charged tetrazolium dyes too, like the MTS (3-(4,5-dimethyl-2-thiazolyl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium), XTT, and WST-1(2-(4-iodophenyl)-3-(4-nitrophenyl)-5(2,4-disulfophenyl)-2Htetrazolium, monosodium salt) that can penetrate the cells only in the presence of an electron acceptor. Since the end products are soluble, they are also preferred.

In protease viability assay, a marker known as glycylphenylalanyl-aminofluorocoumarin (GF-AFC). The aminopeptidase enzyme in the cytoplasm of viable cells acts upon this substrate breaking down the compound into glycine, phenylalanine, and aminoflourocoumarin (AFC) that exhibits fluorescence corresponding to the number of viable cells [56].

Alamar blue/Resazurin dye (7-hydroxy-10-oxidophenoxazin-10-ium3-one) method is more sensitive, offers simpler sample preparation protocols and is inexpensive compared to MTT. The deep blue-colored resazurin gets converted to pinkcolored resorufin by the activity of inner mitochondrial enzymes. It is less successful due to difficulty in the biochemical assaying and unwanted reactions [53].

ATP cell assay is also a preferred method for assessing cell viability. The advantages include faster results, higher sensitivity, and lesser artifacts. The principle behind this is that cells with damaged membranes cannot synthesize ATP and cellular ATP concentration gets depleted faster by the endogenous ATPase.

The clonogenic cells assay is a qualitative assessment that investigates the proliferation capacity of cells. The cell sample is exposed to a known quantity of nanomaterials and allowed to proliferate. After 1–3 weeks, they are stained and quantified per growth in number or size. A survival curve is plotted based on percentage survival and increasing dose of nanomaterials. Increasing dosage gradually results in a lower number or size of colonies [57].

DNA synthesis cell proliferation assay is where a radioactive tracker like 3H-thymidine is incubated with a cell sample. This gets incorporated into the DNA of proliferating cells. The radioactivity of the DNA of the daughter cells is measured using a scintillation beta counter, thereby quantifying the number of viable cells. Another method is to use a non-radioactive compound which is 5 Bromo-2′ deoxyuridine (BrdU). BrdU-specific antibodies are then used to obtain a colorimetric estimation [58]. Developed thymidine analogues like 5-ethynyl-2′-deoxyuridine (EdU), 5-iodo-2′-deoxyuridine (IdU) and 5-chloro-2′deoxyuridine (CldU) are also sensitive methods that to have additional advantages over BrdU assay [59].

A precise cell viability assay available is flow cytometry. A cell suspension is introduced into the flow cytometer which allows particles of less than 150 μm to pass through. During the passage, a laser light beam of a known wavelength interrogates the solution, interacts with every single cell, and gets scattered. These scattered lights are converted to voltage signals, the intensity of which gives information on cell viability and cellular kinetics.

### *7.2.2 Apoptosis assay*

Another important parameter observed in nanoparticle toxicity assays is the occurrence of apoptosis. Studies have confirmed that exposure to silver nanoparticles has resulted in apoptosis and DNA damage with the release of apoptosis markers viz. caspase-3 and caspase-9 [60]. Various tests for assessing apoptosis in the cell culture systems are as follows.

Annexin-V and propidium iodide (PI.) are two cell death markers that give information on apoptosis. The activation of the caspase-dependent pathway during apoptosis causes externalization of the plasma membrane, which is indicated by increased fluorescence. This is because of the binding of Annexin-V to phosphatidylserine. PI is usually impermeable, but it can stain the nucleus when the integrity of the cell membrane is lost. This also relates to the later stages of apoptosis [61].

Comet assay, or the single cell gel electrophoresis assay (SCGE) is a sensitive test can be used both in vitro and in vivo. It detects the breaks in the DNA strands of individual cells [62]. The basic principle is that when an electric current is applied to the cell system, the damaged DNA fragments migrate out of the cell, whereas the undamaged DNA will remain within the nucleus. A comet shape can be seen with undamaged DNA forming the head and damaged DNA forming the tail. The size and shape of the comet give information about the extent of DNA damage. DNA-specific fluorescent dyes will stain the samples, and later the amount of fluorescence in the head and the tail portions, as well as the tail length, are analyzed [63, 64].

The morphological changes specific to apoptosis are another parameter of importance. Irregular reduction in the cell size and fragmentation of DNA confirms the presence of apoptosis. Typical ladder-like patterned DNA fragmentation and irregular cell sizes can be easily identified by Agarose gel electrophoresis [65].

TUNEL assay or the terminal deoxynucleotidyl transferase dUTP nick end labeling assay—it is a method of staining initially described for identifying cells that have undergone programmed cell death and DNA fragmentation [66]. Later it was confirmed that the test could also detect DNA damage due to non-apoptotic events like necrosis [67]. Hence it cannot distinguish between apoptosis and necrosis. It is based on the activity of enzyme terminal deoxynucleotidyl transferase on fluorescentlabeled dUTP. The cells with fragmented DNA will bind to the fluorescent labeled assay molecule and later be estimated by fluorescent microscopy or by immunohistochemical staining. This method gives a quantitative estimation of the viable cells [68].

#### *7.2.3 Necrosis assay*

Necrosis assay determines the viability of cells by identifying the loss of membrane integrity. The dye exclusion method is a preliminary method to identify dead or the dying cells. Dying cells exhibit a loss of membrane integrity, so there will be permeation of the dye within the cell. The most used dyes are trypan blue, eosin, or propidium iodide. The cell suspensions are added with the dyes and manual cell counting using a Neubauer hemocytometer conventionally to determine the number of live cells [69].

PI dye can stain the nucleus and binds to the DNA of nonviable cells forming a fluorescent complex. The amount of fluorescent light emitted is proportional to the voltage signal output. The higher the output, the higher the number of nonviable cells will be. Even though the method is faster and more convenient, it is time-consuming and requires expensive instrumentation. A recently designed microchip and microcell counter (Adam, Nanoentek, Seoul, Republic of Korea) is established on which PI stain can be used to distinguish viable and nonviable cells by direct cell counting technique [70].

Neutral Red is another dye that is weakly cationic and slightly acidic in nature. They can easily diffuse through the plasma membrane and get concentrated in the lysosomes binding to the anionic sites of the lysosomal matrix [71]. Exposure of cells to nanoparticles leads to cell surface alterations and increased lysosomal fragility that favors binding of neutral red thus helping in identifying viable and dead cells [72].

#### *7.2.4 Oxidative stress assay*

Exposure to nanoparticles leads to the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [73]. These compounds can be detected by X-band electron paramagnetic resonance (EPR) [74], fluorescent probes, and nonfluorescent probes. EPR use is limited due to its high cost. Fluorescent probes are costeffective but inefficient due to high reactivity, which gives misleading results [75].

Oxidative stress can also be assessed by measuring the lipid peroxidation BODIPY-C11 assay. ROS can induce membrane lipid peroxidation. BODIPY-C11 is a fluorescent dye that inserts into the lipid bilayers and helps in identifying the oxidized and unoxidized lipids by their respective green and red colors. Later these are quantified fluorimetrically [76].

ROS-induced membrane lipid peroxidation TBA assay for malondialdehyde [77] also gives information on oxidative stress. Various other assays like the measurement of lipid hydroperoxide using Amplex Red assay, antioxidant depletion by 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and superoxide dismutase activity by Nitro blue tetrazolium assay is also convenient procedures [78].

#### *7.2.5 Endotoxin assays*

Nanoparticles can absorb contaminants onto their surface, resulting in an exaggerated inflammatory response as seen in the case of endotoxins. This acute inflammatory response results in the activating the signaling cascades and releasing inflammatory mediators like cytokines.

The main assays in this regard are the gel clot assay, the coagulogen-based (turbidity) assay, and the chromogenic assay. In the gel clot method, the endotoxin sample solution is combined with LAL solution (Limulus amebocyte lysate) leading to cleavage of coagulogen, which is then checked for subsequent clotting. Chromogenic assay substitute chromogens for coagulogen and releases chromophores when cleaved. Commercial kits like Endosafe®-PTS (Portable Test System) based on chromogenic LAL assays are also available [79, 80].
