**4. In vitro tests to evaluate the toxicity of nanoparticles**

The in vitro evaluation of the toxicity of NPs serves as a standard approach for identifying acute hazards associated with potentially harmful NPs during this screening process. However, these methods can only partially replace *in vivo* evaluations. At the same time, in vitro analyses report the immediate harmful effects of NPs in specific cellular settings, *in vivo* animal models monitor biodistribution and bioaccumulation pathways, which are not accessible through in vitro observations. A comprehensive understanding of NPs toxicity and potential risk requires using both methods [100]. In vitro, NPs toxicity assessment is a critical technique that offers benefits such as reduced cost, quicker results, and minimal ethical issues [101].

#### **4.1 Size and surface charge evaluation**

Various analytical approaches exist to examine the toxicological properties of NPs, with two key techniques frequently employed to provide crucial quantitative data: dynamic light scattering (DLS) and zeta potential (ZP) analysis. The ZP and, subsequently, the surface charge of a particle play an important role in inferring potential toxicity, promoting combined DLS-ZP systems as vital tools for preliminary biocompatibility assessments. Other in vitro tests utilised to determine the size and surface charge of NPs encompass scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force spectroscopy (AFM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), surface-enhanced Raman spectroscopy (SERS) and solid-state nuclear magnetic resonance spectroscopy (SSNMR) for compositional analysis, as well as fluorometry for photonic characteristics [102].

#### **4.2 Proliferation assay**

Proliferation analysis is conducted by evaluating cell metabolic function to determine cell metabolism using 3-(4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), the most commonly utilised tetrazolium bromide for evaluating NPs toxicity [103]. This test uses tetrazolium salt to gauge cellular metabolism. The intricate chemical preparation of the MTT assay has led to its substitution for the Alamar blue assay, which is simpler to prepare and measures the cellular redox potential [104]. However, its success has been hindered due to an unclear operating mechanism. Other evaluations include the [3H] thymidine incorporation method to assess cell proliferation, which is typically avoided due to its toxicity [105]. The cologenic assay is another method that counts proliferating cells by visually inspecting them after exposure to NPs [106].

#### **4.3 Apoptosis assay**

One primary indicator of nanoparticles (NPs)-induced cell toxicity is apoptosis. This process, triggered by NPs, can lead to cellular renewal and accelerated ageing

#### *Potential Toxicity of Nanoparticles for the Oral Delivery of Therapeutics DOI: http://dx.doi.org/10.5772/intechopen.111946*

in mammalian hosts [100]. Commonly used markers for apoptosis include phosphatidylserine (PS), which moves to the cell membrane's outer layer, and caspase activation. Research has shown that silver NP can induce apoptosis in cells such as mouse embryonic stem cells [107] and stimulate the activation of Caspase 3 and 9 when applied to Drosophila melanogaster larvae [108]. Once host signalling pathways are initiated, active caspases provide various assays, such as cleavable substrates with fluorogenic and chromogenic labels, immunoblotting, immunofluorescence, and affinity assays with connected reporter units [109].

Numerous in vitro tests evaluate NPs toxicity, such as the Annexin V assay, which binds to PS and is a barrier against coagulation cascades. There are also various other techniques for examining apoptosis in cells or tissues exposed to NPs, including the comet assay, the TUNEL (terminal deoxynucleotidyl transferase (TdT) Nick-End labelling) staining technique, and analysing morphological alterations [110–112]. A decrease in cell size and DNA fragmentation are signs of apoptosis in NPs-treated cells. DNA gel electrophoresis is the most straightforward test for identifying cellular abnormalities. DNA fragmentation with uneven DNA sizes in agarose gel signifies necrosis-mediated cell death, while ladder-like electrophoretic DNA patterns indicate apoptosis-induced cell death in NPs-treated cells [113, 114]. Such changes were observed in human HepG2 hepatoma cells exposed to silica NPs [115]. Furthermore, the single-cell gel electrophoresis assay (SCGE), also known as the comet assay, is used to detect the mutagenic potential of NPs-treated cells by identifying DNA breaks in *Drosophila tertiolecta* exposed to SiO and TiO NPs [70, 116].

#### **4.4 In vitro assessment of oxidative stress**

As a result of the high surface area-to-volume ratio imposed by NPs, enhanced reactivity promotes intracellular damage due to oxidative stress. Detection of oxidative events through ROS measurement, protein carbonyl content, genotoxicity, and inflammatory markers reveal the potential of NPs to produce harmful toxicants in the host [100]. Beyond the natural levels, ROS levels that are produced in the cytoplasm can be detected using ROS-sensitive dyes such as nonionic, non-polar, membrane permeable fluorophore 2′,7′-dichlorofluorescein diacetate (DCFH-DA) [117]. After cellular entry of DCFH-DA, the fluorophore is hydrolysed enzymatically by cytosolic esterase into non-fluorescent polar analogue dichlorofluorescein (DCFH). It is oxidised by cellular ROS into highly fluorescent dichlorofluorescein (DCF) that can be monitored [118]. The use of other agents, such as 2,2,6,6-tetramethylpiperidine (TEMP), detect ROS by reacting with the stable O2 radical, which can be detected using X-band electron paramagnetic resonance (EPR) [119]. However, the use of DCFH-DA is advantageous over that of TEMP because of its high cost. Other available assays to detect ROS induced due to NPs can be detected using lipid hydroperoxide, Amplex Red assay, measurement of antioxidant depletion by 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and superoxide dismutase (SOD) activity by Nitro blue tetrazolium assay [120].

#### **4.5 Necrosis assay**

Necrosis disrupts the integrity of the membrane and is commonly used to determine the viability of cells. Necrosis is measured using two dyes Neutral Red and trypan Blue. Neutral red (2-amino-3 methyl-7-dimethyl-amino-phenazoniumchloride) is a weakly cationic supravital dye that, at slightly acid pH, yields a deep red colour [121]. The mechanism of action of neutral red includes diffusion through the plasma membrane and concentrates on the binding of lysosomes through electrostatic, hydrophobic bonds [101]. Any disruption in the cell membrane brought about by NPs results in reduced uptake of neutral red, differentiating live and necrotic cells. Studies have used neutral red to detect necrotic cells induced when exposed to silver NPs [122]. The other dye used for detecting necrotic cells induced through NPs is trypan blue which enters dead cells while being excluded by live cells and thus is used to detect the stability of the cell membrane. Trypan blue has been used to detect the cytotoxicity of cells exerted by Zn NPs [123].

#### **4.6 Viability assays**

Lactate dehydrogenase (LDH) is an enzyme produced by living cells that regulates pyruvate and lactate levels through the oxidation of nicotinamide adenine dinucleotide (NAD). When cells are lysed due to the toxic effects of NPs and other agents, LDH is released into the surroundings and maintains the enzyme activity. The usual conversion of pyruvate to lactate in the presence of LDH follows the NADH + pyruvate NAD + + lactate. Spectroscopic methods can monitor the NADH complementary reaction with tetrazolium salts. This time-dependent change in spectroscopic absorbance through enzyme-linked immunosorbent assay (ELISA) tests infers the extent of cellular trauma [100]. Common tetrazolium salts include iodonitrotetrazolium (INT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)- 2-(4-sulfophenyl)-2H-tetrazolium (MTS) which rival the throughput of the 51Cr assay and exhibit a threefold increase in sensitivity over ultraviolet NADH assays [124]. In addition to these, luminogenic glycylphenylalanyl-aminofluorocoumarin (GF-AFC) and firefly luciferase ATP assays provide non-toxic and highly sensitive options for assessing cell viability [125].

### **5.** *In vivo* **tests to evaluate the toxicity of nanoparticles**

*In vivo* toxicity assessment methods provide more reliable data than in vitro models, and often yield conflicting results [126]. Some examples of *in vivo* toxicity assessment methods for NPs include biodistribution, clearance, haematology, serum chemistry, and histopathology [101]. For example, researchers have studied the toxicity of aluminium oxide NPs. These NPs have been analysed for their cell toxicity, immunotoxicity, and genotoxicity using *in vivo* models [126]. Tetrazolium-based assays such as MTT, MTS, and WST-1 have been used to evaluate the cytotoxicity of these NPs [126]. Manganese oxide nanomaterials have also been examined for their potentially harmful effects [127]. One study evaluated the toxicity of a newly synthesised nanomaterial called GNA35 and its precursor Mn3O4 using in vitro models representing the respiratory, GIT, and skin systems of the human body [127]. The study provided information on the potential health risks associated with these nanomaterials, highlighting the need for further evaluation using *in vivo* models [127].

*In vivo* toxicity assessment of various NPs is crucial for assessing their safety in biomedical and industrial applications. Here are some examples of *in vivo* toxicity assessment of various NPs:

**Polymer NPs**, often used for drug delivery systems, have been evaluated for toxicity using *in vivo* models. A study utilised zebrafish embryos as a model to investigate the toxicity of polystyrene NPs. The study found that NPs demonstrated

#### *Potential Toxicity of Nanoparticles for the Oral Delivery of Therapeutics DOI: http://dx.doi.org/10.5772/intechopen.111946*

dose-dependent toxicity, leading to developmental and behavioural abnormalities in zebrafish embryos [46].

**Lipid-based NPs** are commonly used in drug delivery systems because of their biocompatibility and low toxicity. In a study by Lama et al., researchers evaluated the *in vivo* toxicity of lipid NPs in rats by measuring their biodistribution, clearance, and histopathology [128]. They found lipid NPs were well tolerated, with no significant adverse effects observed [101].

**Protein NPs**, such as albumin-based NPs, have been developed for targeted drug delivery. *In vivo* studies in mice have shown that these NPs can accumulate in tumour tissues and effectively deliver drugs without causing significant systemic toxicity. However, further evaluation of protein NPs using *in vivo* models is necessary to ensure the safety of these drug delivery systems [129].

**Silica NPs** have many applications, from drug delivery to cosmetics. In a study by Yu et al., researchers assessed the *in vivo* toxicity of silica NPs in mice by assessing their biodistribution, clearance, and inflammatory response. They found that silica NPs were distributed primarily in the liver, spleen, and kidneys and induced a transient inflammatory response, suggesting potential risks associated with their use [130].

**AuNPs** have been studied for their potential applications in various fields, including drug delivery and diagnostics. *In vivo* toxicity evaluation of gold NPs has been carried out using different animal models. For example, a study investigated the biodistribution and toxicity of AuNPs in mice using various doses and sizes. The results showed that, depending on size and dose, AuNPs could accumulate in organs such as the liver, spleen, lungs, and kidneys, leading to potential toxicity problems [46, 129].

**AgNPs** have also been investigated for potential applications in medicine and industry. An example of *in vivo* toxicity assessment of AgNPs is a study that evaluated silver NPs stabilised with gum Arabic protein (AgNPs-GP) in Daphnia, a small freshwater crustacean model. The results indicated that AgNPs-GP exhibited dose-dependent toxicity, causing adverse effects on Daphnia's survival and reproduction [131].

**Iron oxide NPs (SPIONPs)** are widely used in biomedical applications, such as drug delivery and magnetic resonance imaging. In a study by Mahmoudi et al., the researchers evaluated the *in vivo* toxic effects of SPIONPs in mice by observing their biodistribution, retention, and clearance [132]. They found that SPIONPs did not exhibit significant toxicity, but NPs tended to accumulate in the liver and spleen, which can cause long-term adverse effects [57].
