**3. Pathways of cellular uptake**

In drug delivery applications, the nanocarriers should go inside the cells to efficiently deliver drugs to target organelles or cells. Thus, the uptake route or mechanism of NP into cell should be considered in designing a nanocarrier system. Cell membrane is the protective coating of a cell and is not permeable to all particles as it maintains homeostasis. The lipid molecules of the cell membrane have hydrophilic heads and hydrophobic tails. Lot of small molecules enter the cell by passive diffusion. The cell uptake of molecules with the help of energy from ATP (Adenosine triphosphate) is called active diffusion [19].

There are a lot of factors which influence the internalization of the NP and their interaction with intracellular components such as size, surface modifications, net charge, hydrodynamic volume and stiffness. The persistence of nanoparticles in the cell and their release of free radicals to induce oxidative stress bring toxicity and cell death. Endocytosis, an active transport, is the most important pathway by which molecules enter the cell and trigger responses. The main types of endocytosis are phagocytosis (cell eating) and pinocytosis. In the process of endocytosis, the particle

#### **Figure 3.**

*Schematic diagram showing the types of nanomaterials and their impact on various organ systems. Created with the help of Biorender.com [18].*

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

to be internalized with be covered by a part of cell membrane which the buds off inside the cell to form vesicle containing the particle [20].

Phagocytes are immune cells that protect from infections and pollution by engulfing foreign bodies. The 3 main phagocytes are macrophages, neutrophils and monocytes. In phagocytosis opsonins (group of proteins found in serum) play a major role by adsorbing onto NP surface and form protein corona. Phagocytes recognize these and a signaling cascade is triggered which internalizes the particles forming phagosome [18] (**Figures 3** and **4**).

Different cell types use different uptake mechanisms for the same NP. It can occur through phagocytosis, macropinocytosis, clathrin and caveolin mediated endocytosis, non-clathrin and non-caveolin mediated endocytosis are some among them. Nanoparticles less than 100 nm in size can enter the cells and size less than 40 nm can enter the nucleus. The surface charge of the NP are a crucial factor for cell internalization where the negatively charged cell membrane is attracted towards positively charged NP. Small NPs are engulfed by F-actin mechanism, dynamin and lipid rafts which are energy-dependent. NP of size more than 500 nm are engulfed by phagocytosis and macropinocytosis. Aggregated NPs, TiO2 or carbon black undergo opsonization in the biological fluid and undergo phagocytosis or macropinocytosis [19].

#### **3.1 Adverse effects of the cellular uptake**

The bio resistance of certain nanomaterials to degradation is the root cause of toxicity. These nanoparticles retain in the endosomal compartment of cell. Certain other nanoparticles like ZnO, when taken into the acidic part of lysosome will get dissolved into Zn2+ ions which in excess amounts will cause cytokine production leading to cytotoxicity. Non-soluble nanoparticles like TiO2 and carbon black NP were found to be free in cytoplasm and even in the D.N.A. The reasons for this toxicity could be that these NP diffuse through the cell membrane through transient holes or they may

#### **Figure 4.**

*Schematic representation showing the mechanisms of nanoparticle cellular internalization such as clathrinmediated; caveolin-mediated; clathrin- and caveolin-independent; phagocytosis and macropinocytosis pathways. Reproduced with creative commons attribution 4.0 (CC-BY-4.0) license from Augustine et al. [19].*

be accumulated in the lysosome where later on lead to membrane rupture and release in the cytoplasm. These cytoplasmic NP during mitosis can enter the nucleus through microtubules and are found in the DNA [21].

The engineered NP persisting in the cell result in oxidative stress leading to apoptosis and inflammation. Oxidative stress is a result of imbalance between reactive oxygen species [ROS] and antioxidant capacity of cell. NP trigger the ROS production and thus leads to the imbalance and oxidative stress. ROS has various cellular roles by acting as secondary messengers. If the stress caused by ROS is at a higher level, it can result in cell membrane and organelle injury leading to necrosis or apoptosis. The smaller the size of the nanoparticles, the higher the surface area and thus produce more ROS. Thus, the oxidative stress caused by the small NP affect lipid membrane and cause their disorganization in structure and function. Inactivation of certain sensitive proteins which have methionine or cysteine in their active site are also the toxic effects of the NP which entered the cells. Genotoxicity is also a serious effect of NP which damages DNA because of intrachain adducts and strand breakage [22].
