**2. Human exposure routes to nanomaterials**

Nanomaterials or nano-sized particles can enter the body through various routes such as skin, olfactory tracts, weak or damaged skin, intestinal tracts, respiratory tract, and intravenous or intramuscular routes. Their entry can have adverse biological effects such as tumors, cardiac diseases, skin problems, allergic reactions, and respiratory diseases. As a result of human industrial activities, dust storms or volcanic eruptions, humans get exposed to nanoparticles continuously and their toxicity depends on the chemical composition of the nanoparticles. Based on the composition, nanoparticles are divided into inorganic, metallic and metal oxide, where their biological effects and toxicity level are explained. The body parts easily exposed to nanoparticles are skin, gastrointestinal tract and lungs.

#### **2.1 Skin as the entry route**

Human skin which has an average surface area of approximately 2 m2 has a thick outer layer of keratinized dead cells and which does not absorb any essential elements other than solar radiation. Skin appendages and stratum corneum are the major routes of entry through the skin. The root of the hair or follicles gives space for nanoparticles to accumulate; during the follicle opening, they enter into deep layers of skin. Intercellular spaces along the lipid layers are the easiest route for the nanoparticles to penetrate the skin. The classical diffusion theory can understand the diffusion of aggregated particles through stratum corneum. Even though the stratum corneum is porous and allows facile entry of nanoparticles, the dose and exposure time affects the entry. Follicular penetration varies at different sites on the body. It is observed that the follicle morphology on the lateral forehead is the area of maximum penetration as it has maximum surface coverage. Follicle area is considered the best site for particle storage as it is not exposed to washing or fabric contact. This area is considered suitable for drug release and a shortcut to the systemic circulation [5, 6].

Polymeric nanoparticles or drug carriers cannot penetrate the skin without mechanical stress. Several studies have been conducted to study the drug release from polymeric particles through the skin. It has been found that the particles did not penetrate the skin without any mechanical stress. Studies on hairless animals showed that the drug could not penetrate and thus proved that hair follicles pave the way for entry. Most topical applicants or cosmetics include zinc oxide (ZnO) or titanium dioxide (TiO2) nanoparticles, which are mostly studied for skin penetration. It is shown that the size of the particles and exposure time effect the penetration. Titanium dioxide nanoparticles (TiO2 NP) of size 20 nm showed their presence in the first 3–5 layers of corneocytes. Very small sized TiO2 NP 4 nm size showed presence in the deep epidermis layer of pig's ear after 30 days of exposure. ZnO nanomaterials of size 30–40 nm could not penetrate deep into epidermis layer. Liquid type liposomes can penetrate epidermis layer, whereas gel type cannot. Metallic nanoparticles show

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

higher penetration rates. Another important aspect is the physical state of the skin. The damaged skin easily allows the nanoparticles whereas intact skin only paves the way for tiny nanoparticles below 10 nm to penetrate the epidermis layer [7, 8].

The shape of the nanoparticles also affects toxicity. Differently shaped silver nanoparticles penetrated differently into the various layers of skin. Tak et al. [9] showed that the intercellular penetration pathway plays a central role in the shapedependent penetration of AgNPs through the lipid matrix between corneocytes. The possible skin penetration pathways are shown in **Figure 1**.

The rate and depth of penetration of nanoparticles affect the toxicity level. Metallic nanoparticles can enter systemic circulation easily and thus can add to the toxicity depending on their dose. Thus, safety evaluation must be done before using nanoparticles in cosmetic formulations and drug delivery. Rapid penetrating nanoparticles pose more toxicity than slow penetrating ones as the latter can have efficient drug delivery and will be attacked by the immunological system of the body. In contrast, rapid penetrating NP can circumvent the macrophages and cause systemic toxicity. Argyria is a diseased condition due to the toxicity of silver nanoparticles leading to permanent pigmentation of eyes and skin [9].

#### **2.2 Inhalation as the entry route**

Airborne nanoparticles enter the lungs and cause respiratory disease by accumulating in the alveolar cells. They not only affect the lungs but also affect the

#### **Figure 1.**

*The schematic diagram of possible skin penetration pathways of three differently shaped AgNPs. (A) Two main possible skin penetration pathways are illustrated: (i) enters via hair follicles (the follicular penetration pathway); and (ii) diffuses through the gaps between corneocytes (the intercellular penetration pathway). Reproduced with Creative Commons Attribution 4.0 International License (CC BY. 4.0) from Ref. [9].*

extrapulmonary organs like the heart and liver. Metallic oxide and hydroxide NP cause pulmonary inflammation. The nanoparticles enter through olfactory tubes, pass the pharynx, and reach the alveoli. They have a good retention time, and the diameter of the particles affects the toxicity level. Some NP travel through the alveolar epithelium and capillary endothelial cells and reach the cardiovascular system and other internal organs [10]. Chronic obstructive pulmonary disease (COPD) is one of the major cause of death in the world as suggested by World Health Organization (WHO). Sarcoidosis and pulmonary fibrosis are other respiratory diseases caused by airborne pollutants. Wildfire, dust winds and volcanoes are sources of natural nanoparticles whereas engineered nanoparticles are found in gasoline exhausts, cosmetics and drug delivery carriers. These nanoparticles cause direct injury to lung tissues and cause inflammation. These nanoparticles then move to systemic circulation from the alveolar airspaces and thus cause toxicity to other internal organs. The high surface area of nanoparticles help them interact with enzymes or proteins of the immune system and thus causes inflammation and subsequent injury to the tissues, thus being detrimental to tissues [11].

The level of injury caused by nanoparticles is different in different regions of the lungs based on the clearance mechanism and cell types. In lung alveoli, there is only a single layer of cells; in bronchi and bronchioles, there is a layer of mucus for protection. Thus the nanoparticles which reach the alveoli can easily diffuse into blood capillaries and enter the systemic circulation, causing toxicity. Alveolar epithelial cells (AEC) are critical in protecting the respiratory tract and AEC in alveoli gets easily damaged by NP. AEC I gets damaged easily by NP and to replace it to maintain the alveolar structure, AEC II undergoes hyperplastic proliferation and repeated exposure to NP results in its aggregation in systemic circulation and thus leads to cardiovascular diseases. As cited above, the shape of nanoparticles determines the level of toxicity.

#### **Figure 2.**

*Systemic and pulmonary administrations of nanoparticles to target respiratory cell types. Reproduced with Creative Commons Attribution 4.0 International License from Ref. [13].*

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

Needle-like and rod-shaped NP cause more cellular destruction than spherical and flake-like structures. Other crucial factors to be considered are NP's degradation, solubility and chemical composition. The more they persist in the system, the more toxic they are to the cells. Metal NP like gold and silver persist in lungs for more than 7 days. This causes toxicity to all organs by creating oxidative stress, thus leading to cellular toxicity [12, 13] (**Figure 2**).

#### **2.3 Nanoparticle ingestion**

Metallic NP, carbon-based NP, ceramic NP, polymeric NP or dendrimers are the classes of nanoparticles that enter the gastrointestinal tract through ingestion. Implant materials have become very common, and orthopedic and dental implants use nanoparticle coatings to enhance their performance and bioactivity. However, intentionally or unintentionally, these implants release ions and NP over time, which cause adverse effects in the intestine and its related organs. The gastrointestinal tract (GIT) has a huge surface area of approximately 200 m2 , which is very much amenable to NP interaction. NP are absorbed by the GIT and thus enter the systemic circulation. Nanoparticles also damage the microbiome of the gut and thus affects digestion [14].

Targeted drug delivery through nanocarriers has been used in treating skeletal infections. But in many cases, these engineered nanomaterials create cytotoxicity. In order to achieve native bone tissue structure and more biocompatibility, nanomaterials have been used in orthopedic implants. Dental implants use a wide range of engineered materials, including nanomaterials. Titanium is used as dental implants, which can release titanium NP due to many activities like wear and tear caused by chewing or bacterial activity and chemical or physical deterioration. Titanium oxide nanoparticles from dental implants can dissolve in saliva reaching the intestine and other organs like the liver, spleen, kidney, and heart. Risk assessment of the engineered nanomaterials should be very focused on criteria like physicochemical characterization and thorough biological evaluation as per the regulatory agencies' guidelines [15]. Most implant failures occur due to wear and the release of particles which cause a chronic inflammatory response. A cobalt-chromium hip implant faced rejection due to the release of cobalt and chromium nanoparticles triggering an inflammatory response. A study conducted by Posada, 2015 showed the involvement of lymphocytes in such implant allergies, and within 48 hours of treatment, the metal nanoparticles caused apoptosis in the cells [16].

The most common nanoparticles that enter GIT are silver, iron oxide, titanium dioxide, zinc oxide etc. Organic NP, liposomes, engineered protein NP etc. pose little concern about their toxicity as many of them have been used by humans for centuries. Not many studies have been done in this area of nanotoxicology, and very little information is known about the toxicity of such nanoparticles. But the most important fact to be noted is that the properties of each type of nanoparticle differs on many factors and that these factors determine their toxicity. Dimension, morphology, composition, surface charge, aggregation state affects the fate of the nanoparticles in vivo. Most of the nanoparticles, after passing through various parts of the GIT change their properties due to interaction between the proteins and varying pH conditions. Thus, their fate is altered when compared with that in vitro. The properties of pristine nanoparticles will be very much different from the nanoparticles in vivo and thus, the studies related to their toxicity should be taken into consideration only for in vivo results. Most of the studies which show cytotoxicity in cell lines do not show any toxic effects when performed in animal models unless it is at a very high dose. Thus the in vivo studies should be concentrated more on the physiological aspects of the cells rather than on the organs as a whole [17, 18].
