Keywords

7.1 Introduction

The field of nanotechnology and nanoscience gained much attention since the last decade. Significant advancements in such fields can be attributed to their utilization in biological sciences (Liu 2006). The idea of using nanoparticles in biomedical research has drawn much attention and interest due to the fact that nanoparticles (NPs) can be easily engineered to attain unparalleled functions and unique compositions, thus rendering novel tools and techniques useful for biological research (Wang and Wang 2014). The manipulation and subsequent engineering of matter at atomic, molecular, and supramolecular levels to produce different nanomaterials useful for the advancement of various fields are comprehensively termed as nanotechnology. The merging of biology and nanotechnology has conceived a new field, nanobiotechnology, which involves the utilization of different organisms including bacteria, fungi, yeast, and plants to produce NPs at an economical and environmentally friendly manner when compared with chemical processes (Shah et al. 2015). These nanoparticles play a significant role in diverse applications ranging from biological sciences to mechanics. A rise in the usage of nanoparticles has led to the evolution of novel fields such as nanobiotechnology and bionanotechnology apart from their usage in biomedicine and biosensors where NPs are engineered and manipulated to achieve desired results (Anirudh et al. 2018).

7.2 Types of Nanoparticles

On a broad scale, nanoparticles are classified into metallic, semiconductor, and polymeric nanoparticles. Based on their application on whether they are used for diagnosis or for therapeutic purposes or for their usage in imaging, nanoparticles are categorized into inorganic and organic nanoparticles. Organic nanoparticles include micelles, liposomes, dendrimers, and polymeric and albumin-bound nanoparticles. Inorganic nanoparticles include silver, gold, iron oxide, alloy, magnetic, and quantum dots.

Unilamellar liposomes are one of the first platforms used due to their small size ranging from 100 to 800 nm. Usage of liposomes as a model for cell membranes in biophysical research was first reported in the year 1965, after which they were used for gene delivery as platforms for nanoparticles. Liposomes have the unique ability of carrying and delivering many types of biomolecules through biological membranes into cells, thus establishing themselves as a widely used type of transfection agents in biological research. Liposomes also offer several advantages such as being leak-proof, economical, non-toxic, and non-immunogenic apart from being biocompatible and biodegradable (Orive et al. 2009). Micelles are composed of either polymers or lipids that work on the principle of hydrophilic-hydrophobic interactions. Biomolecules that are water-insoluble can be embedded into the hydrophobic part of micelles, whereas water-soluble molecules can be loaded into the hydrophilic part. When transferred into the bloodstream, they have a higher life due to the presence of a hydrophilic shell surrounding them. The biomolecules bound to albumin (albumin-bound nanoparticles) serve as a useful nanoparticle drug delivery vehicle (Hawkins et al. 2008). Non-covalent binding and endogenous albumin pathways are utilized for carrying the hydrophobic molecules such as Abraxane into the bloodstream, for treating metastatic breast cancer (Harries et al. 2005). Dendrimers are branched structures with an internal core surrounded by branched structures made up of amino acids, sugars, and nucleotides followed by an external surface (Medina and El-Sayed 2009). Based on the number of generations formed over these cores, the size, shape, and extent of branching can be described. These dendrimer cores can be embedded with small-sized biomolecules with the help of hydrogen bonding or hydrophobic interactions. The only disadvantage of these nanoparticles is their synthesis, which is time-consuming and the loading of biomolecules into the core (Adair et al. 2010). Polymeric nanoparticles are synthesized by utilizing the copolymers of different hydrophobic levels. A unique feature of these nanoparticles is that they are synthesized from biodegradable and biocompatible polymeric substances. These NPs can be loaded with small biomolecules, which may be either hydrophilic or hydrophobic in nature and has an added advantage of being more leak-proof and stable than liposomes. In general, metal nanoparticles can be made thermodynamically stable by adding capping agents such as oligosaccharides and polysaccharides, which helps to increase their solubility and also prevents aggregation (Sanjay et al. 2009).

Silver nanoparticles are categorized into two different types, uncapped and capped varieties. The size of uncapped NPs ranges from 20 to 50 nm, while capped NPs have a size of ~25 nm (Lima et al. 2012). These are the most widely used inorganic nanoparticles in textile industries and cosmetic applications due to their antimicrobial properties against microbes such as bacteria and viruses (Sharma et al. 2015). Gold nanoparticles are widely preferred as they are biocompatible and have a wide array of optical and chemical properties, thus offering themselves a part in bio-imaging, biochemical sensing and detection, diagnostics, and therapeutic applications. These nanoparticles are used as a tracer in the detection of DNA during DNA fingerprinting and also used for identifying protein interactions in immunological studies (Hasan 2015). Magnetic nanoparticles are produced from magnetized materials or from materials that can be attracted by magnets such as Fe, Co, Ni, etc. Some of the widely used magnetic nanoparticles are maghemite (Fe2O3) and magnetite (Fe3O4), which are biocompatible. Alloy nanoparticles are mainly structural and have a high electrical conductivity compared to other metals and thus are widely used. Some alloy nanoparticles are bimetallic in nature and are advantageous over other metallic nanoparticles such as silver, gold, etc. (Mohl et al. 2011).

When quantum effects take place in nanocrystals with extremely small diameters in addition to entrapment of electron carriers and holes at a size range lesser than Bohr’s radius, they are referred to as quantum dot nanoparticles. Their size ranges from 3 to 12 nm in diameter. These are neither solid structures nor individual molecules as the number of atoms in the quantum dot nanoparticle ranges from 1000 to 100,000 making it a unique nanoparticle. There are different types of quantum dots based on III–V semiconductors, II–VI semiconductors, and silicon atoms included at the core part (Ghaderi et al. 2011). The overall structure of a quantum dot nanoparticle can be described as having a secondary shell on the exterior followed by a primary shell with a core part at the centre. Most of the quantum dot nanoparticles are capped by ZnS, and their core is made up of CdSe. As the quantum dots are insoluble in water, they are not suitable for the transfer of biomolecules in an aqueous medium. Iron oxide nanoparticles belong to the paramagnetic type of nanoparticles. Due to the presence of paramagnetic nature, the iron oxide nanoparticles are widely used as a contrasting agent in magnetic resonance imaging due to increased magnetic susceptibility. The core of SPION is generally made up of iron oxide and is layered with a hydrophilic substance such as dextran so as to enhance its stability. Currently, there are two super paramagnetic iron oxide nanoparticles (SPION) with size 60–180 nm, namely, ferumoxides and ferucarbotran, respectively, that are used for MRI and molecular imaging (Weissleder 2006).

7.3 Structure of Nanoparticles

Nanoparticles generally exist in nature either in non-crystalline or in a low symmetry form. On the basis of nanoparticle size and composition of the aggregates, determination of the most stable form is important which in turn will determine the physical and chemical properties of the nanoparticles. Some of the cardinal factors that affect how the nanoparticles accumulate and interact with the body are size, shape, solubility, structural properties, and chemical composition (Vinita et al. 2010). Nanoparticles exhibit distinct and unique properties due to the presence of unusually high ratio of surface area to volume. Huge surface area of nanoparticles are attributed to a distinct surface chemistry in relation to their core properties. The surface properties that are highly specific and unique to each nanoparticle are generally lost in the process of aggregation and accumulation.

Structurally a nanoparticle can be divided into three components, namely, functionalized surface, shell, and core which is the cardinal part in determining the properties of the nanoparticle.

7.3.1 Surface

In order for nanoparticles to dissolve in an aqueous medium, their surface needs to have a charge. This charged surface can be obtained by covalently binding it with biomolecules having certain charged functional groups. This covalent binding of nanoparticle surface with biomolecules, metal ions and polymeric substances is referred to as the functionalization of nanoparticle surface (Christian et al. 2008). The three-dimensional structure of the nanoparticles can be measured by using the coherent X-ray diffraction technique (Moyu et al. 2011). A stable nanoparticle aggregate can be formed by using surfactants such as SDS, where the surfactant goes and binds to the nanoparticle formed in the micelle core.

7.3.2 Shell

The outer layer of an inorganic nanoparticle may have different chemical and surface properties when compared to the core part of a nanoparticle. This distinctive layer is considered to be the shell of a nanoparticle. Some nanoparticles may have either a single shell surrounding the core or two shells one layering over the other as seen in a core-shell quantum dot. The quantum dot nanoparticles having CdSe alloy in the core and ZnS in the shell are known to exhibit a quantum yield of greater than 50% (Ding et al. 2017). An event where the formation of a shell layer occurs indirectly without deliberately preparing it can be seen in the formation of an iron oxide layer during iron nanoparticle formation (Santos et al. 2017).

7.3.3 Core

All the properties exhibited by a nanoparticle can be attributed to the composition of the core. Some of the physicochemical and toxicological properties manifested by a nanoparticle are due to its core composition. During the preparation of a nanoparticle, a definite phase form may not sound plausible because an inorganic nanoparticle may have its existence in different phases and two distinct phases showing up may be common, which might reflect the physicochemical properties of a nanoparticle to deviate entirely from what is expected.

7.4 Properties of Nanoparticles

Fundamentally the amount of stability of nanoparticles during production and its efficiency in any operation can be attributed to its diverse properties, each having its own significance.

7.4.1 Physicochemical Properties

On a broader point of view, the physicochemical properties include optical, magnetic, mechanical and thermal properties.

7.4.1.1 Optical Properties

For measuring optical absorbance and other such properties, researchers have used spectrophotometers. Apart from such experimental methods, some theoretical methods such as Mie scattering method, effective medium theory (EMT) and Monte Carlo methods are also most widely used for the theoretical determination of the optical properties of a nanoparticle (Rastar et al. 2012). For the characterization of nanostructures and particles, several techniques such as Raman, differential and surface-enhanced Raman spectroscopy are used.

7.4.1.2 Magnetic Properties

The presence of a magnetic property in nanoparticles is due to the uneven electronic distribution. When the size of a nanoparticle is less than 10–20 nm, the magnetic property of the particle dominates. Thus, these nanoparticles are useful in catalysis, MRI applications, etc. Some of the properties which can be tuned and modified to suit different applications are coercivity, blocking temperature, saturation magnetization and Neel and Brownian relaxation time (Arati et al. 2013).

7.4.1.3 Mechanical Properties

There are many kinds of forces that play a crucial role in establishing the mechanical properties of NPs. They are van der Waals forces, capillary forces, electrostatic force and electrical double layer force, solvation and structural and hydration forces (Dan et al. 2014). In order to test the mechanical properties of nanoparticles such as hardness, elasticity, adhesion, friction, etc., various methods are used such as atomic force microscopy (AFM), transmission electron microscopy and particle tracking velocimetry (PTV).

7.4.1.4 Thermal Properties

The thermal conductivity of metal nanoparticles is much higher when compared to liquids. This advantage can be used by suspending the metal nanoparticles in liquids such as ethylene glycol, oils, water, etc. and preparing the nanofluids. These nanofluids exhibit higher conductivity due to the presence of high surface area to volume ratio as the amount of heat transfer is proportional to the surface area present. This also enhances the overall stability of the suspension (Khan et al. 2017).

7.4.1.5 Antibacterial Properties

The need for using nanoparticles in the treatment of bacterial infections arose because of the fact that, as resistance against a drug increases, the dosage of antibiotics also increases drastically enhancing the toxicity. Another advantage is the species sensitivity of a particular nanoparticle to a bacterial strain (Mohammad et al. 2012).

7.4.1.6 Thermodynamic Properties

Some of the cardinal properties which determine the thermodynamic status of a nanoparticle are melting temperature, elastic moduli, enthalpy and entropy of melting, specific heat capacity, etc. By obtaining the Gibbs free energy using surface free energy model, the melting and superheating behavior of nanoparticle structures can be extrapolated. Due to the suppression of the thermal vibration of atoms at the interface between the nanoparticle and Nano cavity, superheating is provoked (Xiong et al. 2011).

7.4.1.7 Immunological Properties

Once binding of nanoparticles to the proteins in the blood takes place, these proteins determine the quantity of uptake of nanoparticles into the cells, which either stimulates or suppress the immune response. One beneficial property of nanoparticles is its ability to reduce the toxicity of a drug by increasing their solubility. All immunogenic properties mainly depend on their thickness, zeta potential, size, etc.

7.4.1.8 Melting Properties

As melting is a surface property, it is dependent on the surface area to volume ratio. As the size of the particle decreases, the ratio increases dramatically; thus the surface energy also increases resulting in an increase in the melting point. X-ray diffraction or electron diffraction is widely used to determine the melting properties.

7.5 Size and Shape of Nanoparticles

Particle size and its distribution are mainly important characteristics of a nanoparticle system. These characteristics will determine the in vivo distribution, biological destiny, toxicity and the targeting capacity of a nanoparticle system. In addition, they can also control the drug loading, drug release and strength of nanoparticles. Dispersion and distribution of nanoparticles, biological effects and amount of immune response are dependent on the size of a nanoparticle. The capacity of intracellular uptake and in vivo mobility of nanoparticles are more compared to micro particles due to their reduced size. Another beneficial feature of the nanoparticles in drug targeting and delivery is their small size, with maximum surface area, providing a large space for carrying the drug on its surface. In the case of micro particles, due to the huge surface area, the drug might diffuse through the outer shell towards the core. Some of the shapes that nanoparticles possess are cubes, nanostars, triangles and prisms (Calum 2017).

7.5.1 Surface Chemistry

Some of the most important properties such as charge, reactivity and hydrophobicity hold a crucial role in the interaction between nanoparticles and the biological systems, which can be modified upon attachment of nanoparticles. The main reason why the surface chemistry of small nanoparticles is significantly different from the bulk nanomaterial or larger nanoparticles is because of the disparities in the number of binding sites and the effect of quantum confinement. Another important surface property is zeta potential, which allows for the aggregations of nanoparticles as they carry specific charge, specific to each medium (Wang et al. 2013).

7.5.2 Surface Tension

Earlier, Du-Noüy ring method using an automatic surface tensiometer was employed for testing the surface tension valve in nanofluids. Surface tension is a major phenomenon that plays a major role in determining the heat transfer of a thermal system (Bhuiyan et al. 2015). It is basically defined as the amount of free surface energy present per unit area of the liquid droplet. One aspect in which surface tension plays a negative role by causing core-shell interface defect is by building up of nanosized impurity particles (Shabarovaa et al. 2018). By utilizing a thermodynamic model that includes surface tension property dependent on the size of the nanoparticle, the behavior of the NPs can be elucidated. This surface tension is generally considered as the interface between the nanoparticle and the surface of the fluid, where it is associated, providing us with the required information regarding the stability of the biomolecule at that interface. It is generally reported that there are innumerable changes in the adsorption properties of Nano-sized materials because of surface tension that is resulting from applied surface strain. Another point to note is that the relative binding of adsorbents gets decreased when compared to their bonding on extended surfaces due to the effect of surface tension of a nanoparticle cluster and this is more evident on close-packed structures. As the size and concentration of nanoparticles in a nanofluid increases the overall surface tension increases (Lin et al. 2015). Surface tension plays a cardinal role in heat transfer applications where optimal utilization of nanofluids take place (Prasad et al. 2018).

7.6 Nanoparticle-Biomolecule Interactions

Application of nanoparticles can invariably improve the way in which many diseases caused by tumor cells are diagnosed and treated. They also show a promising result in the treatment of lung and heart diseases. It all depends on the interaction or interface of nanoparticle and biomolecule, where the nanoparticles come in contact with the biological components. Examples of some biological components are proteins, DNA, phospholipids, organelles and other biological membranes (Ghosh and Paria 2012). The nano-bio interface includes various physicochemical interactions, kinetics and also thermodynamic changes between the surface of biological components and the surface of nanoparticles. It makes highly essential to study these interactions that occur at the interface for attaining a keen insight regarding the utilization of nanoparticles for human health benefits. There are three main components that contribute to the interactions at the interface:

  1. (a)

    The surface of the nanoparticle.

  2. (b)

    The interaction of the solid particle with the surrounding liquid medium resulting in the formation of solid liquid interface.

  3. (c)

    The interaction of the solid liquid interface with the biological surface.

All these interactions are dictated by surface properties of the nanoparticles. A detail of the interaction of the nanoparticles was provided in Table 7.1. Surface properties of nanoparticles listed in the table help in the interaction of nanoparticle with the medium by adsorbing proteins and various ion molecules, by dissolution in the medium and finally by the formation of double layer (Gilbert et al. 2004). The medium in which the nanoparticles get suspended may be composed of acids and bases, water molecules, detergents, ions, salts and some other large molecules. Once the nanoparticle is placed in any medium, the properties of the material get modified to a great extent, and then they form an interface, which is in metastable state. This interface further will lead to the formation of a highly unstable nano-bio interface, which in turn undergoes many changes with certain changes in the composition of the surrounding medium. For example, with the release of any biological product into the medium, the nanoparticle interacts with the product and undergoes some changes that will influence the interface.

Table 7.1 Characterization of different surface properties of all the three interacting components
Full size table

The interaction at the nano-bio interface includes many types of forces. The forces like van der Waals forces, electrostatic or ionic forces occur mainly between the colloidal particles are also applicable here but with slight variation (Min et al. 2008). During the interaction between two individual nanoparticles such as silicon dioxide when placed in water, attractive van der Waals forces occur due to the mutual induction of dipole moment in both the particles. Because of the surface negative charge, electrostatic forces are formed, which are repulsive in nature. Solvation forces occur when the water molecules form layers on the surface of the particles which retards their adherence thus stabilizing the particle. In contrast, when the same nanoparticle begins to interact with a living cell, all fundamental forces will be redefined by minor differences like (a) there is a chance for the cell membrane to get deformed, as it is non-rigid, (b) the non-uniform charge on the surface may change the energy of the surface and (c) the non-passive nature of the cell membrane may hinder the movement of silicon dioxide particles (Dobrovolskaia and McNeil 2007). Due to the changes that occur in the biological systems, it is presumed that these interactions are complicated and also difficult to predict.

To clearly investigate and acquire knowledge on how nanomaterials interact with cells, various cell lines such as THP-1 have been used as models for conducting experiments. THP-1 tumor cells are collected from patients with acute monocytes leukemia are used to test polystyrene nanoparticles. These nanoparticles do not degrade easily and their size is nearly 110 nm. It was identified that these particles get internalized very well by the cell lines through pinocytosis, when compared to nanoparticles of size 50 nm which got phagocytosed by the phagocytic cells like macrophages and monocytes (Cornelia Loos et al. 2014). Just like polystyrene nanoparticles, gold nanoparticles are also used to study the interactions. Size and shape of gold nanoparticles can be easily altered according to our interest. Coupled with this, the inertness of gold made researchers use it for various works (Alaaldin et al. 2013). Many different cell lines such as HELA cells and mammalian cells are also used to understand interactions occurring at the nano-bio interface. These interactions will further affect the uptake, transportation and cytotoxicity of the nanoparticles by different kinds of cell lines.

7.7 Biological Effects of Nanoparticles

Nanoparticles and their interactions with the living cells and biomolecules serve as precursors for various biological effects. When a nanoparticle passes through the cell membrane overcoming all the barriers at the nano-bio interface and reaches the cytosol, it elicits numerous responses. These biological effects are both beneficial and detrimental to human health. Cancer is the leading life-threatening disease currently, and a large number of deaths have been caused by breast carcinoma. Biodegradable nanoparticles like PLGA-PEG loaded with chrysin are used for testing against breast cancer cells. PLGA is a copolymer of polyglycolic acid and polylactic acid. Chrysin is an anti-cancer drug that induces cell apoptosis. This drug is loaded on to PLGA-PEG nanoparticles and tested against the breast cell lines. This resulted in an enhanced inhibitory effect when compared to the effect caused by chrysin alone (Makadia and Siegel 2011). Radiation therapy is gaining importance day by day due to the incidence of cancer, and larger doses of radiation are needed to destroy tumor cells, but the healthy tissues cannot tolerate it. So an alternative was devised in which gold nanoparticles (AuNPs) are being used. When the level of dosage was relatively reduced, it was found out that a large number of cells got killed. Similarly, metallic nanoparticles made up of copper, gold and silver also showed some important biological responses within organisms. Copper nanoparticles are stable and can be mixed with polymers very easily. LCuNPs are one of the nanoparticles synthesized from plant latex that are efficient in apoptosis of lung cancer cells (Mayur et al. 2011). Human bones and teeth are made up of hydroxyapatite, which is used as a coat over the prostheses and also has many biomedical applications. Initially micro-sized hydroxyapatite particles (m-HAP) are prepared and tested with osteoblast cells like MG-63 cells. Later on nano-sized HAP was synthesized, and the same test was repeated. It was found that nano-HAP is many times more effective in inducing growth and inhibiting apoptosis in MG-63 cell lines (Shi et al. 2009). Further, due to the presence of glutamic acid in the surrounding medium, reactive oxygen species (ROS) are produced inside the nerve cells, and the presence of these ROS leads to cell death. To avoid this, antioxidants are used to reduce the free radicals. Nanoparticles made of cerium oxide and yttrium oxide were made to react with nerve cells like HT22 cell line to test their antioxidant nature. Nano sizes of yttrium oxide particles showed better results in reducing the oxidative stress when compared to the ones made with cerium oxide, thus depicting their neuroprotective nature (Schubert et al. 2006).

Silver nanoparticles (AgNPs) are well known for their antimicrobial activity against many viruses. In order to assess their anti-tumor activity, analyses were performed on Dalton’s lymphoma ascites (DLA) cell lines. AgNPs induced an enzyme called caspase3, which caused apoptosis of tumor cells, confirmed by the nuclear fragmentation. These nanoparticles also assisted in reducing the ascetic fluid in mice affected with cancer, which helped in getting back their body weight to normal (Muthu et al. 2010). Additionally silver nanoparticles were proved to be effective against colon cancer cells. They invigorated G0/G1 cell arrest causing DNA destruction. This leads to reduced viability due to apoptosis of colon cancer cells (Wan et al. 2009). Utilization of magnetic nanoparticles has been given much importance as they can be modified easily. Dendrimers are mixed with oligonucleotides and coated on the nanoparticles and incubated with breast cancer cells. This brought into light the ability of magnetic nanoparticles to deliver any gene into cells effectively, thus proving their potential to be used for cancer treatment (Pan et al. 2007). The biological function of nanoparticles on different cell lines has been listed in Table 7.2.

Table 7.2 Biological function of nanoparticles on different cell lines
Full size table

In spite of having numerous advantages, nanoparticles have equally considerable disadvantages. Understanding these disadvantages is very crucial as most of them are related to human health. Cobalt nanoparticles (CoNPs) are known to enter inside the cells very rapidly when compared to many other potential nanomaterials. Once they enter, they increase the reactive oxygen species instead of depleting them resulting in many undesirable reactions in the body (Elena et al. 2009). Titanium dioxide nanoparticles reduce cell activity and viability by depleting ATP when given in low doses, and there is an observed decrease in mitochondria when given in higher dosages (Roslyn et al. 2011). SiO2, TiO2 and ZnO nanoparticles are predominantly used in the food-processing industries. The major cause for gastrointestinal toxicity and immunological side effects caused by these food borne nanoparticles is due to a large production of reactive oxygen species inside the cells causing significant DNA damage and cell death by cell cycle arrest.

7.8 Toxic Effects of Nanoparticles

The size of nanoparticles is the main characteristic feature that allows them to easily pass through the cell membrane and perform important functions by eliciting biological responses. The integration of these biological effects may be both advantageous and deleterious to the human body. There are numerous mechanisms that cause nanoparticle toxicity. Usually very small quantity of reactive oxygen species (ROS) is produced in different organs, and their level is retrieved by the action of antioxidant enzymes and glutathione. But the production of these ROS is gradually enhanced in organs such as lungs, when nanoparticles are inhaled. During this process, glutathione gets oxidized and the ratio of glutathione to oxidized glutathione, a regulator of equilibrium in the cell decreases resulting in no effect on the depletion of ROS. When oxidative stress is in moderate levels, MAPK and NF-kB cascades get activated resulting in inflammatory responses. In case of high stress, electron transfer is disrupted, and apoptosis of cells takes place. This is the mechanism through which nanoparticles cause toxicity to the human body. This toxicity may be cytotoxicity, in which the cells are forced to undergo programmed cell death or genotoxicity where the genetic material like DNA and RNA is affected.

Even though this is the basic mechanism used by the nanoparticles, different types of nanomaterials modify it according to their ease. Carbon nanotubes are a type of nanomaterials known for many unique properties. These are of two types: single- and multiple-walled carbon nanotubes. Both of them follow different mechanisms of toxicity. Single-walled carbon nanotubes activate CN-KB in human keratinocytes to produce oxidative stress, whereas multiple-walled carbon nanotubes result in apoptosis of T-cells (Oberdorster 2004). Metal nanoparticle results in the intracellular toxicity, and the mechanism responsible for it is found in many experiments. The lysosomal compartment of the cell is acidic in nature due to which the particles get internalized, and release of ions takes place leading to the intracellular toxicity. This mechanism is commonly referred to as ‘lysosome-enhanced Trojan horse effect’ (Stefania et al. 2014). Endoplasmic reticulum releases calcium ions due to oxidative stress and results in the mitochondrial deformation and cell death. Upon exposure to ZnO, there was a significant increase in calcium ion release (Xia et al. 2008). The mechanism underlying silver nanoparticles involves the oxidation of the nanoparticle surface by oxygen molecules, releasing silver ions that are toxic to the cell (Mc Shan et al. 2014). When silica nanoparticles are exposed to lung epithelial cells, lipid peroxidation occurs due to production of ROS, leading to enhanced activation of transcription factor and regulation of interleukin 6, 8 and MMP-9, finally leading to PCD using caspase-3 (McCarthy et al. 2012).

7.8.1 Cytotoxicity of the Nanoparticles

The interactions between nanoparticles with cells and tissues can sometimes be detrimental, causing cell cycle arrest, DNA damage, production of ROS, etc. This phenomenon where the nanoparticles elicit an intense immune response by causing toxic effects on cells is called as cytotoxicity. Intracellular organelles may also get affected due to cytotoxicity when oxidative stress, mitochondrial dysfunction and lipid peroxidation take place (Schanen et al. 2009). In a study, positively charged nanoceria caused toxicity in the MCF-7 breast cell line and negatively charged nanoceria caused toxic effects in A549 lung carcinoma cell line. Based on light scattering experiments, it was found that the size of nanoceria particles ranged from 5 to 14 nm in diameter (Asati et al. 2010). In a similar work done by Connor et al. (2005) on the leukemia cell line, K562, by application of gold nanoparticles, it was found that, if the concentration of the nanoparticles was greater than 25 μM, it was toxic. A549 cell lines of human lung carcinoma had shown cytotoxicity upon inducing silver nanoparticles of concentration 20 μg/mL. It caused necrosis and apoptosis in the affected cells and inhibited the normal functioning of mitochondria (Rasmus et al. 2011). Gold nanoparticles also were found to induce cytotoxicity in HeLa cell lines when the size was 1–2 nm. Synthesis of spherical silver nanoparticles of 17.6–41 nm using Piper longum leaf extract and introduction into Hep-2 cell lines showed intense cytotoxic effects (Justin et al. 2012). Zinc oxide nanoparticles of 60 nm size were synthesized by subjecting to ultrasound irradiation and were tested on different human glioma cell lines U87, A172, U251, LNZ308, LN18 and LN229. It was reported that a concentration of 10 mmol/L of zinc oxide concentration was sufficeint enough to induce apoptosis and cell death. It was also found that the reactive oxygen species are responsible for decreasing the viability of these cells (Stella et al. 2009). Silica nanoparticles of different sizes such as 104 nm and 335 nm showed less cytotoxicity effects on the human endothelial cell line EAHY926 when compared to smaller nano-sized silica particles, thus establishing the fact that smaller particles showed higher cytotoxicity (Dorota et al. 2009).

In a study, Chitosan nanoparticles (size of 65 nm and surface charge of 52 mV) were used against human gastric carcinoma cell line MGC803, which led to DNA degradation and necrosis (Li-Feng et al. 2005). In another study, silica nanoparticles of sizes 20 and 50 nm have shown significant nuclear condensation, cell shrinkage and high production of ROS when employed on human embryonic kidney stem cells, HEK293 cells (Fen et al. 2009). Silver nanoparticles of 15 nm size was used and tested against human lung epithelial carcinoma cell line, namely, A549, where there was a significant decrease in mitochondrial transmembrane potential, increased ROS and leakage of LDH (Jae et al. 2014). DNA damage, arrest of cell cycle and apoptosis of Hep-G2 cells occurred when silica particles of size 498 nm were used (Yang et al. 2010). Human hepatocyte (L02) and human embryonic kidney (HEK293) cells were tested for cytotoxicity by using zinc oxide nanoparticles (particle size of 50 nm), and it was found that morphological modifications, mitochondrial dysfunction, reduction of super oxide dismutase and glutathione and DNA damage have occurred (Rongfa et al. 2012). A549 lung and MCF-7 breast cancer cell lines were tested by using silver nanoparticles of 27 nm, and severe cytotoxic effects were observed (Ivan et al. 2014). Cerium oxide nanoparticles were toxic towards prostate cancer PC-3 cell lines. Testing of 15.6–1000 μg/mL nickel-zinc (NiZn) ferrite nanoparticles against MCF7 breast cancer, human HT29colon cancer and HepG2 liver cancer cell lines resulted in induction of apoptosis and cell death (Renu et al. 2012).

7.8.2 Genotoxicity of the Nanoparticles

Genotoxicity of nanoparticles leads to chromosomal changes, breakage of DNA strands and mutations. Genotoxicity may be direct or indirect depending on the mechanism applied. In the direct genotoxicity, the nanoparticles directly enter into the cells by passing through the nuclear pores and interact with the DNA present in the chromosomes during interphase. This nanoparticle interrupts transcription process by binding to DNA (Jin et al. 2011). In the indirect method, NPs do not interact directly with the DNA, but they cause toxicity in the following ways: (a) interaction with mitotic spindle, (b) interaction with nuclear proteins, (c) disturbing cell cycle checkpoints, (d) ROS production and (e) inhibition of antioxidant activity (Magdolenova et al. 2014). When human epidermal cells were exposed to titanium dioxide nanoparticles, they were readily taken up by the cells, and after 6 h of exposure, there is a significant damage done to the DNA. There is also a direct relationship between the concentration of nanoparticles and damage of DNA (Ritesh et al. 2011). Similarly, the entry of ZnONPs into the human body causes DNA damage by inducing oxidative stress in the human liver cells HepG2 (Vyom et al. 2011). Hepatic cancer cells (HepG2) and peripheral blood mononuclear cells have rendered varying levels of DNA damage by gold nanoparticles. Cobalt nanoparticles produce cobalt ions which compete with magnesium ions and impair enzyme proteins in binding with DNA when they are incubated with human leukocyte cells (Colognato et al. 2008). Copper oxide nanoparticles have the potential to effect DNA damage in the A549 human lung cells, when exposed for a brief period of time (Maqusood et al. 2010). Genotoxicity of silver nanoparticles was studied using comet assay and cytokinesis-block micronucleus cytome assay. During mitosis, micronuclei found in chromosomes cannot combine with the mitotic spindle. In the comet assay, the extent of DNA damage was shown. In cancer cells, there was an increase in the DNA damage with an increase in nanoparticle concentration. But in fibroblast cells, the damage was not significant beyond certain concentration of nanoparticle (Asha Rani et al. 2009). Nickel oxide nanoparticles resulted in the nuclear translocation of proteins in the human epithelial lung cell lines (Laura et al. 2014). In a study, metal cobalt nanoparticles were readily taken up by the human peripheral blood cancer cell lines causing severe DNA fragmentation revealing its genotoxic nature.

7.9 Assays and Models for Testing the Nanoparticles

Nanotechnology is one of the rapidly developing fields, which has applications in numerous fields related to human health and welfare. Presently, nanoparticles are assayed for the cyto-, immuno- and genotoxicities. Determination of cell viability is done by using tetrazolium-based assays such as MTT, MTS and WST-1. Cell inflammation caused by the nanoparticles is analysed by testing for IL-8, IL-6 inflammatory biomarkers and tumor necrosis factor using ELISA. The solidarity of the cell membrane is obtained by using lactate dehydrogenase (LDH) assay. Nanoparticles are used in fields like electronics, biotechnology, medicine, etc. because of their tiny size. Nanoparticles can enter into the human body very easily and cause severe damage. Hence, various methods have been proposed for testing the safety of nanoparticles. US FDA has considered the issue of nanoparticle toxicity and issued that they are not completely harmless or safe for humans and each product must be subjected to regulation (Bahadar et al. 2016). Kathleen et al. (2011) presented different assays which can be used for studying the suitability of nanomaterials in cardiovascular applications, including in vitro blood compatibility studies. Nanoparticle-induced hemolysis can be evaluated by spectrophotometry, platelet activation and complement activation can be evaluated by thromboelastography (Kathleen et al. 2011). Toxicity assessment of aerosol nanoparticles in human lung cells (A549) was carried out by cell viability using MTT assay, extent of membrane damage by LDH assay and temporal dose response by measuring the size distribution of silicone nanoparticles using dynamic light scattering technique (Irfan et al. 2014).

Alzheimer’s disease (AD) is the most common type of dementia which results in progressive loss of memory and thinking skills. AD rats are treated by transferring the neural stem cells and observing their improved learning and memory function. Nerve growth factor, poly (ethylene glycol)-poly(lactic-co-glycolic acid) nanoparticles (NGF-PEG-PLGA-NPs), can be used for the differentiation of neural stem cells in vitro. A study was conducted on AD rat models to evaluate the efficacy of NGF-PEG-PLGA-NPs combined with transplantation. This type of transplantation can specifically improve learning and memory functions of AD rats by replenishing basal forebrain cholinergic neurons and can help in the formation of hippocampal synapses and AchE-positive fibers (Chen et al. 2015). Wistar rats are used as models to study the dose-dependent in vivo toxicity of silver nanoparticles and examined hematological parameters such as WBC count, platelet count and RBC count. These parameters were found to be changing with the amount of dosage (Dhermendra et al. 2011). Genotoxicity testing of Tio2 anatase nanoparticles was performed using comet assay and wing-spot test in Drosophila. Nanoparticles are reported to cause cytotoxicity on midgut and imaginal disc of larvae, but there is no genotoxicity reported in wing-spot test. But, amount of DNA damage in was identified by comet assay (Erico et al. 2015). Human embryonic stem cell derived three-dimensional in vitro model allowed us to test the neurotoxicity of nanoparticles. For testing the specific toxicity of nanoparticles, chemically inert polyethylene was chosen. They found to penetrate deep into the three-dimensional structures and impacted gene expression at non-cytotoxic concentrations (Hoelting et al. 2013). Blood-brain barrier model is useful to test the permeability of nanoparticles for easy and reproducible assay. Reconstruction of this model is done by culturing both primary rat brain endothelial cells and pericytes to support the tight junction of endothelial cells (Hanada et al. 2014).

7.10 Applications of Nanoparticles in Biological Systems

Nanobiotechnology emerged as a mature biomedical field which possesses several applications in molecular biology, genetic engineering, diagnosis (biomarkers) and cancer treatment (De Jong and Borm 2008; Rathi Sre et al. 2015). Nanomedicines built on nanocarriers are used to treat microbial infections and diseases like cancer (Wesselinova 2011). Due to their physicochemical properties, they are widely used in food industry, cosmetology, agriculture and medicine for protection of human health. Metal-based nanoparticles are used in electron microscopy, bio-imaging, magnetic resonance, computed tomography for visualization due to their optical density and paramagnetic properties (Rai et al. 2016; Veronesi et al. 2016; Liu et al. 2013).

7.10.1 Pharma and Healthcare

The use of nanoparticles in medicine and more specifically drug delivery is currently gaining momentum. In pharmaceutical sciences, the use of nanoparticles mainly focused on to minimize the side effects and toxicity associated with the drugs. The use of nanoparticles made of biopolymers and polymeric biomaterials has become popular to overcome the toxic effects during the drug delivery (Duncan 2003). Biodegradable polymeric nanoparticles reach the diseased site in the body more specifically and exert their action effectively compared to other drug delivery systems (Ferrari 2005). Since the nanoparticles possess several therapeutic properties, research is going on to find the novel biomaterial-based nanoparticles for targeted drug delivery.

7.10.2 Environment

Nanoparticles have potential applications in various areas with environmental inferences. Selective adsorption of certain compounds is needed for removing contaminants in landfills and industrial waste stream. We can use adsorbents, fabricated by nanoparticles. Nanoparticles, used as catalyst for effective oxidation, exhibit high specific surface areas, obtained by sol-gel method (Ward and Ko 1995). Photocatalysis is used for environmental remediation where there is total oxidation of compounds in gas and aqueous phase systems (indoor air problems, purifying air in industrial settings). Separations mediated by inorganic nanoparticulate filtration media, which overcome the limitations caused by ceramic membranes and polymeric membranes for activity of reverse osmosis. Another application of nanoparticles that benefits the environment is the usage of nanoparticle oxides for fabricating thin-film batteries, ultra capacitors and fuel cells, which are used as energy storage and power devices (Zeltner and Anderson 1996).

7.10.3 Industry

Mass production of nanoparticulate carbon improves strength. Abrasion resistance tyres are used as fillers and TiO2 aerosols as key ingredient in white paints. Pigments with high refractive index such as titania allow high color depth and strong optical. Inorganic pigments based on titania and zinc oxide absorb UV radiations and thus are used in cosmetics. Nanometer-thick active ingredients applied as a coating on window glass will have self-cleaning property (Manning et al. 2011). Catalysis by nanoparticles like Ni, Pd and Pt is with industrial relevance. Nanoparticulate forms are used in improving the healing, implant adhesions and tissue response, delivering materials to the site of application due to their rapid mobility (Stark et al. 2015).

7.11 Conclusions

Nanoparticles are being used in designing different types of products, and their unique properties will guide us to attain new technological breakthroughs. Products produced by using nanoparticles are utilized in electronics, healthcare, chemicals, cosmetics and energy. Besides its advantages, there are limitations due to its toxicity towards health, which means the capacity of a substance to cause illness, injury or death of any organism. If a product contains toxic nanoparticles, it may create health and safety risks through all its stages of life cycle. As nanotechnology is an emerging field now, all the producers are focusing on safer and effective production by integration of nanoparticles into products. The five principles of SAFER Guides are the following: (1) size, surface and structure, (2) alternative material, (3) functionalization, (4) encapsulation and (5) reduction of the quantity used as a framework for safe nanotechnology. To benefit society and acquire acceptance, there is a need of systemic investigations by considering environment and human health. To build the investigation, we need to understand the interactions with biological system. Our approach should start with the synthesis of nanoparticles by using nanoparticle libraries with combination of green chemistry. Evaluation is performed using in vivo models to assess the biological activity and toxic potentials of nanoparticles at various levels of organization of organisms. Nanomedicines are developed and evaluated by European Medicines Agency giving priority to patient safety. It is also needed to ensure next-generation nanomedicine to enter the market with time in a safe way. It is also important to assess the biological effects of manufactured nanoparticles by using a tool like Quantitative Nanostructure-Activity Relationship (QNAR).