**8. In vivo nano biomaterial cytotoxicity assessment**

### **8.1 Experimental models used**

#### *8.1.1 Animal models*

Animal models are particularly useful in studying those aspects of nanomaterials that cannot be mimicked by in vitro systems, such as particokinetics (absorption, distribution, metabolism, elimination: ADME), which are caused by cellular responses of the nanomaterials in vivo, and nanomaterial-caused intercellular communication, multicellular interactions, and immune regulation, all of which may be quite difficult to study in vitro [81]. Therefore animal experiments are indispensable in nanotoxicology.

The selection of the animal models is the first and critical step, as a predictive animal model is still underdetermined, although many models have been built. Mice, rats, zebrafish, rabbits, and *Caenorhabditis elegans* are used in nanotoxicology studies, while mice and rats are more appropriate models due to their explicit genome for toxicity tests of organs. Laboratory mice are the most used mammalian research model, popular because of their availability, size, relatively low cost, and ease of handling. Hamsters, as they have wide cheek pouches and mouths that can be widely opened, are used to study pulmonary clearance and toxicity of nanomaterials. Rabbits are also frequently used as they are mildly tempered, easy to confine, and breed. Their ease of handling benefits intravenous injections, blood sample collection, and dermal tests. Zebrafish (*Danio rerio*), a tropical freshwater fish, have a small size, short life cycle, high gene homology with superior animals (also human), and an availability of a wealth of genetic databases, which makes them effective in vivo models to study the toxicity of nanomaterials. Adult zebrafish may be exploited to study nanomaterial transport, biodistribution, bioaccumulation, and chronic toxicity [82].

For the selection of suitable animal models for the assessment of nanomaterial toxicity, Christop et al. [83], suggested the following: the animal model chosen for the toxicity assessment should be suitable in such a way that the pathways of NP penetration into the organism and excretion from the organism conform from the biochemical and anatomical perspective. For the assessment of nanosafety, using a healthy animal may not be suitable when the results are to be extrapolated to the general population, as interorgan compensatory mechanisms may lower the toxic effects of the nanoparticles. Therefore, they suggested that the suitable test animal model choice should be based on the intended application of engineered nanomaterial. Healthy animal models are suitable only for nanoparticle-based therapeutics, and special animal models with unique features should be used when nanoparticles for therapeutic approaches.

#### *8.1.2 Organs*

In vivo assessments include analysis of tissue structure changes, apoptosis, and inflammation infiltration in main organs (kidney, spleen, lung, brain, and heart). Target organ systems include those that may concentrate nanoparticles due to their structural specificities, such as hepatic sinusoid and Kupffer cells and the renal filtration membrane [84].

Hepatic assessment: Immunohistochemistry to detect liver fibrosis and inflammation, serum enzymology for hepatic function analysis, hematology and chemistry analyzer for comprehensive analysis.

Renal system: Histopathology and immunohistochemistry, kidney indices for assessment of renal function, and renal variables assessed from blood and urine.

Gastrointestinal system: Histological assay of the GIT, absorption function measured indirectly by the evaluation of metal content and electrolyte.

Pulmonary system: MRI to visualize the pulmonary accumulation of nanoparticles, intratracheal instillation for long-term and short-term toxicity assessments, LDH index, and assessment of oxidative stress.

Cardiovascular system: Assessment for signs and symptoms of phlebitis, hemolysis, thrombosis and signs of cardiac injury.

Nervous system: Assessment of drug delivery of solid lipid nanoparticles across the blood-brain barrier, by using various radiographic techniques such as PET and PET/CT.

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

Immune and reproductive system: Any modifications of the nanoparticles are assessed to understand their immunogenicity, the generation and release of inflammatory mediators are examined upon the use of nanoparticles, histological changes of major immune organs are assessed by H&E staining, long term studies on the reproductive organs, reproductive index and offspring survival, growth and development are assessed [84].

#### **8.2 In vivo toxicity evaluation methods**

In vivo methods of assessment of nanomaterial toxicity have certain advantages over in vitro testing methods. The advantages lie in the fact that in most of the in-vitro testing methods, the growth medium that is used for culturing the cells is supplemented with the lowered concentration of serum proteins as the source of nutrition, and usually, serum of bovine origin is used. The concentration of the serum proteins in the medium influences the nanoparticle-cell interaction, which may range from mild effects on the cells to cell death. A study to assess the role of serum concentration on nanoparticle-cell interactions was conducted by Kim et al. [85]. They suggested that while comparing the results of various in-vitro studies, the serum concentration in the medium used to culture the cell lines should be considered. Moreover, when serum of bovine origin is being used, it may not represent a condition relevant to real in vivo exposure situations.

In vivo toxicity assessment methods assess the toxicity and reactions of the nanomaterials in the organism from the point of administration through the subcutaneous vein, inhalation, skin, oral administration, and intraperitoneal routes; interaction with biological components (such as cells and proteins); spread to different organs, metabolism, penetration into the cells of organs and their excretion [86]. The nanomaterial is introduced into the body of the test animal, and the biodistribution, clearance, hematology, serum chemistry, and histopathology are monitored. This provides more practical data on the interaction of the nanoparticles with the immune system, proteins, and dynamic body fluids at the systemic level.

In order to evaluate the acute *in vivo* toxicity of nanomaterials, the Organization for Economic Cooperation and Development (OECD) guidelines recommend oral toxicity test, eye irritation, corrosion and dermal toxicity, and lethal Dose 50 (LD50).

OECD (Organization for Economic Cooperation and Development) guideline 423 provide data on the methods to be used to determine the LD50 (lethal dose 50%) of the nanoparticles being studied. The vehicle used to carry the nanoparticle dose must be non-reactive, and particles must disperse appropriately in it.

#### *8.2.1 Acute oral toxicity assessment*

Different concentrations of the nanoparticles may be administered to the test animals orally. Changes in body weight, behaviors, and other toxic signs and symptoms may be recorded regularly. The animals may be observed daily for 14 days for skin symptoms like edema, erythema, ulcers, body scabs, discoloration, and scars. Toxic signs like weight loss, water and food consumption, and the animals' behavior are also assessed. Skin biopsies and blood may be taken periodically for histopathological evaluation and biochemical and hematological investigations, respectively. After 14 days, the animal may be sacrificed, and all organs collected. The following formula may be used for the calculation of organosomatic index: [weight (g) of the organ/ total body weight (g)] × 100 [87, 88].

#### *8.2.2 Acute dermal toxicity assessment*

Tests may be performed on animals using the test guideline 404 published by the Organization for Economic Co-operation and Development [89, 91]. The substance to be tested may be applied to a small area of the skin of the experimental animal in a single dose for an exposure period of 4 h. The animals will to be examined for signs of erythema edema during the next 14 days. Skin biopsies are taken periodically, and after 14 days, the animal is sacrificed, and the skin is collected for histopathological examination.

### *8.2.3 Acute eye irritation and corrosion assessment*

The standard eye irritation test established by the Organization for Economic Co-operation and Development (OECD) for the testing of chemicals is used as the standard to measure nanomaterials eye toxicity. Five minutes prior to the application of the test substance, 2 drops of a topical ocular anesthetic is to be applied to minimize pain or distress and then the test. One eye may serve as a as a reference control. A small amount of the test substance (0.1 g or 0.1 ml of its colloidal suspension) may be applied to the conjunctival sac of the test eye. The animals are to be observed for toxic symptoms periodically at 1, 24, 48 and 72 h, and grading for ocular lesions of cornea, iris, conjunctivae, and chemosis may be done over 14 days according to OECD test guideline TG 405 [90, 91].

#### *8.2.4 Biodistribution studies*

Biodistribution of the nanoparticles may be detected in live or killed animals after conjugating with a radioactive label or organic dye and tracking in blood and tissue at different periods. Some metallic nanoparticles intrinsic properties may be probed by using specific instruments. These tests are especially important for nanomaterials used for drug delivery. Suspension of drug loaded nanoparticles may be radio-labeled and administered intra-venously into the test animal. Blood is collected at regular periods for 24 h, plasma is separated, and radioactivity levels of the residues may be measured. Main organs and tissues (lung, liver, kidney, heart, spleen, pancreas, brain, fat, and muscle) may also be collected, weighted and radioactivity may be measured [87, 92].

### *8.2.5 Changes in serum chemistry and cell type*

Blood from the test animals may be collected for biochemical (triglyceride, cholesterol, glucose, glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT)) and hematological investigations [88].

Chrishtop et al. [83], in their review on nanosafety versus nanotoxicology, in 2021, referred to studies that have demonstrated animal models with chronic diseases being sufficiently susceptible to nanomaterials even with low toxicity. Considering that chronic diseases, like bronchial asthma, are widely prevalent in various populations, they suggested that nanosafety should also be considered along with toxicity assessment of nanomaterials [83].
