**4.1 Size**

The size of the nanoparticle is proportional to its surface area. The lesser the size, more is the surface area and more its reactivity. The size of the particle and its effect in vivo is difficult to observe as its structure changes in vivo. The ability of the nanoparticles to enter the cells and modify the macromolecules accounts for its level of toxicity. The nanomaterials below 100 nm can enter cells easily as their size is comparable with that of protein globules, DNA., and cell membrane thickness. Very small NP of size less than 20 nm which can enter the nucleus are more toxic than particles

#### **Figure 5.**

*Schematic diagram showing the various physicochemical properties of nanoparticles and their effect on cell [23].*

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

which cannot enter the nucleus. Studies have done on gold nanoparticles in relation to their size and toxicity in vivo. It was found that particles of size 1.4 nm whose size is comparable with that of a major groove of DNA will easily block transcription by interacting with the sugar-phosphate in DNA. It is shown by Zhang et al. that those particles of size less than 5 nm enter cells through translocation while those above 5 nm enter through specific pathways like macropinocytosis and phagocytosis [24]. Small sized particles circulate rapidly all around the body while particle above 50 nm were found in organs like liver and spleen. Star shaped nanoparticles have shown accumulation in lungs while larger nanoparticles are recognized by the immune system and eliminated from the body. Size and shape influence the kinetics of excretion and accumulation of the particles in vivo. The larger surface area of the particles help in adsorption onto the cell surface where particles above 600 nm destructed the cell membrane and caused haemolysis. In contrast, particles of size 100 nm did not cause membrane destruction [23–25].

Larger particles show negligible toxicity when compared with smaller particles. The shape of the particles also makes them behave differently. Spherical particles are easier to be engulfed by cells and they are the ones that cause less toxicity. Whereas rod shaped and fiber-shaped nanomaterials are difficult to eliminate and their toxicity is higher when compared to that of their spherical counterparts. Nanoparticles that enter the body can agglomerate or agglomerates can disperse into primary particles. When primary particles come in contact with lung fluid, they agglomerate. Agglomerates or particles of size >1 μm are up taken by phagocytosis and their clearance mechanism is easy. But the smaller the particles or the agglomerates, the more difficult it becomes to eliminate them and toxicity like genotoxicity happens. The more time they retain in the body organs like lungs, the chances of depositing in secondary organs is more and they damage the lungs. Clearance rate by translocation is less than 0.5% compared to their exposure and these can result in their persistence, causing toxicity and organ damage [26].

### **4.2 Morphology**

The shape of particles is very important in determining their fate inside the body. In nature we can see different shapes for microorganisms like spherical shaped HIV virus, star shaped bacteria and rod shaped TMV which confer different effects in vivo. Thus the function of a nanoparticle with a specific shape determines their effect in nature. The nanoparticle uptake, toxicity, biodistribution and inflammatory response is highly dependent on the structure of the nanoparticle. It is studied that rod shaped particles enter inside the cell easily even compared to spherical particles. But the shape alone does not add to this effect, we need to consider the aspect ratio where higher aspect ratio particles expose more to the cell surface thus easily engulfed into the cells. Nanowires and nano worms due to their length have higher aspect ratio and they enter cells with ease. Particles with highly curved and sharp edges are not very easily up taken by cells. High aspect ratio particles have shown proinflammatory effects leading to cytotoxicity when conducted studies in in vitro models. The persistence of nanoparticles in vivo depends on the geometry where it has been found that filament-shaped micelles prolong the persistent time compared to spherical-shaped micelles. In a study conducted on gold nanoparticles in ovarian cancer xenograft by Arnida et al., it was shown that rod-shaped gold particles were more in circulation and accumulated in tumor cells than sphericalshaped gold NP [27].

The behavior of nanoparticles in vivo cannot be related to just one physicochemical factor alone, whereas the toxicity or fate of a nanoparticle depends on many factors like size, shape, ligands, material, charge, route of exposure etc. Among these factors, the surface chemistry of a nanoparticle has more influence than the particle's geometry since receptor ligand binding strength is the limiting factor which determines the uptake and thus leads to toxicity of these NP. But when you consider the same surface chemistry of a rod shaped and sphere-shaped NP., the rod-shaped NP will be favored by the cells. Apart from this, the fluid motion of rod-shaped particles is favorable. Spheres and short rod NP accumulate in liver, higher aspect ratio NP are found in spleen and lungs. Nanofibers that enter through lungs have more cytotoxic effects where the asbestos particles inhalation is an example. They can disrupt the cell membrane's lipid bilayer and cause a pro-inflammatory response [28].

Nanoparticle toxicity strongly depends on their morphology. In studies conducted on gold nanostructures, it was found that gold nanostars bind to serum proteins better than gold rods. Spherical gold nanoparticles showed higher binding affinity when compared with branched particles. The radius of curvature of the nanoparticles is a factor for protein corona formation. A planar surface is provided by a large radius of curvature, thus resulting in more effective protein binding [29].

#### **4.3 Surface charge**

The cytotoxic effects of nanoparticles depend on their charge density and charge polarity. Positively charged nanoparticles of gold, zinc oxide and silica have more profound effects than their counter parts. For certain nanoparticles of polymers, the charge does not have much effect on their properties in vivo. Some nanoparticles which are porous does not depend on charge such as mesoporous silica nanoparticles. But phagocytic cells interact more with negatively charged particles. In case of nonphagocytic cells where they interact with cationic charges on NP surface, its cellular uptake is more and thus cause cytotoxicity by plasma membrane disruption. But the effect of serum reduces their uptake. Whereas phagocytic cells interact more with anionic NP and ingest them effectively and serum has a positive effect on their uptake [30].

Cell type is an important factor which determines the uptake of NP. The same NP behave differently in various cell types. Effect of surface charge on Mesoporous silica NP in human mesenchymal stem cells revealed that a strong positive charge on the NP showed a good uptake in mesenchymal stem cells whereas their uptake was inhibited in 3 T3 L1 cell lines. When the positive charge density is less, the uptake efficiency is low [31].

Ionic Interactions of NP and cell membrane is important in understanding the fate and effect of NP in the living system. Even though the cell membrane is negatively charged, it behaves differently to positively charged molecules depending on the charge density and other factors. Gold nanoparticles of different surface charges (positive, negative, neutral, zwitterionic) were applied to various cell types. It was shown that positively charged AuNPs depolarized the membrane more than their counterparts. The authors also suggested that changing or varying the surface charge density or applying both positive and negative charges on the nanoparticle may result in an organized cellular uptake and thus target various organelles [32].

The toxicity of silver nanoparticles in the environment and to biological systems is greatly influenced by the surface charge of silver nanoparticles. The electrostatic barrier between silver NP and cell membrane highly affects the fate of NP and influence of other factors such as shape and size become negligible. The authors suggest

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

that surface charge measurement of NP can be used as an analyzing tool to find the toxicity of the NP [33].

Curcumin which is considered as a boon for many diseases can become toxic to cells, especially alveolar macrophages when the surface charge is altered. Curcumin nanoparticles when given positive charge by coating with the polymer polyvinylpyrrolidone and was given negative charge by coating with polyvinyl alcohol and neutral with dextran. The surface charge varied from −20 mV to +5.5 mV. The positively charged curcumin NP resulted in lysosomal and mitochondrial destabilization leading to ROS generation and apoptosis. They entered the cells through clathrin-coated endocytosis and damaged the lysosomes. Thus the effect of surface charge on materials, especially nanomaterials are a very important aspect to be looked upon to determine cytotoxicity and during the designing of nanoparticles [34]. Apart from these facts, another important factor is the density of cationic charges. When the density of the cationic charge is less, the toxicity effect will also be less. The zeta potential of the Nanoparticles does not have any effect if the surface charge density is very high [35].

#### **4.4 Coating**

Nanoparticles are given coating with various materials depending on the result to be achieved. When they are used for cell targeting, the NP must be coated with protein ligands identified by the cell receptors or if targeted to cancer cells then attached with RGD sequence. These coatings can affect the properties of nanoparticles when used in pristine form. The coating materials determine the toxicity of the NP, where they can increase or decrease the toxicity levels. There are a plethora of coating mechanisms and some methods prefer coating the NP with more than one material. Silver nanoparticles tend to aggregate, thus, coating helps improve the NP's stability in suspension. Certain coatings make the silver NP more toxic such as citrate compared with PVP, or protein coated when compared to bare silver NP [36].

Type of coating material, whether they are hydrophilic or hydrophobic, influences the toxicity of metal-based nanoparticles. Hydrophobic coatings have proved to be safer than hydrophilic coatings and this also depends on the density of the coating. Zinc oxide used in various cosmetic formulations when coated with hydrophobic material showed very little toxicity and is recommended for commercial use. This can be explained by the reduced bioactivity of the hydrophobic particles. Theoretical models such as QASR quantitative structure-activity relationships should be applied to evaluate the effect of various coating on nanoparticles [37].
