Introduction

Nanotechnology holds great potential for creating new materials with enhanced properties. A number of nanotech based products are finding applications in industries like medical devices, imaging, sports, biosensing, electronics, drugs, environmental cleanup, cosmetics and sunscreens etc. [1, 2]. The global economy will be increasingly influenced by nanotechnology as more products containing nanomaterials move from research and development into production and commerce (Fig. 1).

Fig. 1
figure 1

Applications of engineered nanoparticles in the consumer products

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A nanometer (nm) is one billionth of a meter (10−9 m)—about half the size of the diameter of DNA. The prefix nano is derived from the Greek word for ‘dwarf’. The definition for the ‘Nanoparticle’ given in the new PAS71 document developed by the UK’s National Standards Body—British Standards Institution (BSI) is: “a particle having one or more dimensions of the order of 100 nm or less” [3]. Nanoparticles are also called as ultrafine particles by some toxicologists [2], aitken mode and nucleation mode particles by atmospheric scientists [4, 5], and engineered nanoparticles by the materials scientists [6].

Different Types of Nanoparticles

Nanoparticles can be classified into two main categories: natural and anthropogenic. Natural nanoparticles existed in the environment long before the nanotechnology era started. Examples of natural nanoparticles include soil colloids, airborne nano-crystals of sea salts, fullerenes, carbon nanotubes and biogenic magnetite etc. [7, 8]. Soil contains many kinds of inorganic and organic nanoparticles, including clay minerals, metal oxides and hydroxides, humic substances, allophane, and imogolite [9]. Natural organic nanoparticles can also be found in plants [10]. Anthropogenic nanoparticles can be divided into two categories: incidental, which are nanoparticles produced unintentionally in manmade processes (e.g. carbon black, carbon nanotubes and fullerenes, platinum- and rhodium-containing nanoparticles from combustion by-products [8], and engineered/manufactured, which are nanoparticles that are produced intentionally for their specific properties.

A variety of engineered nanomaterials are already known and more novel materials are being engineered. The ways to produce engineered nanoparticles can be categorised in two different classes: “top-down” or “bottom-up”. Top down techniques involve etching or milling down a block of material to desired shape whereas bottom up involves arranging smaller subunits into more complex assemblies [11].

Some of the most widely used nanomaterials are listed below.

Carbon-Based Nanomaterials

These nanomaterials are composed mostly of carbon, in the shape of hollow spheres or tubes. Spherical carbon nanomaterials are referred to as fullerenes, while cylindrical ones are called carbon nanotubes (CNT). There are two types of CNT-singlewalled (SWCNT) or multiwalled (MWCNT). Both of these are typically a few nanometer in diameter and several micrometers to centimeters long [11].

Metal/Metal Oxide Nanoparticles/Nanomaterials

Metal oxide nanoparticles have applications in cosmetics, textiles, fuels, drug delivery and paints. Examples include silver, gold, zinc oxide, aluminium oxide, cerium oxide and titanium dioxide nanoparticles.

Quantum Dots

Quantum dots (QD) are semiconductor nanocrystals used extensively in biomedical imaging. Fluorescent QDs can be conjugated with bioactive moieties (e.g. antibodies, receptor, ligands) to target specific biologic events and cellular structures [12].

Dendrimers

Dendrimers are spherical polymeric molecules, formed through a nanoscale hierarchical self-assembly process. Dendrimers can act as nanoscale carrier molecules and can be used in drug delivery [11].

Nanoparticle/Nanomaterials Toxicity

Nanomaterials have a high surface area to volume ratio compared to the same mass of material produced in larger form because the ratio of surface to total atoms or molecules increases exponentially with decreasing particle size [13]. This leads to high surface reactivity which affects their strength and physical properties.

The small size, and subsequent larger surface area of nanoparticles, endows them with some highly useful and specific properties but, it also renders them biologically more active leading to unexpected and unanticipated consequences on interaction with biological systems. Smaller size also imparts a different biokinetic behaviour and ability to reach more distal regions of the body [13]. The occupational exposure will also increase with the growing production and use of nanomaterials in society. Environmental contamination is yet another concern. These apprehensions have generated concerns about the potential adverse effects of engineered nanomaterials on human health and the environment.

Government/Regulatory Authorities and Environmental, Health and Safety (EHS) of Nanotechnology

Governments and scientific authorities all over the world are realizing the importance of nanomaterial risk assessment. The UK Government commissioned The Royal Society and The Royal Academy of Engineering in June 2003, to look into the ethical, health and safety issues related to nanotechnology. The Royal Society recommended in its report “Nanoscience and Nanotechnologies: Opportunities and Uncertainties” published in 2004, that “chemicals in the form of nanoparticles or nanotubes should be treated as new substances under the existing notification of new substances (NONS) regulations and in the registration, evaluation, authorisation and restriction of chemicals (REACH)” to trigger additional testing [11]. Committees on the Toxicity, Carcinogenicity and Mutagenicity of Chemicals in Food, Consumer Products and the Environment have also identified the risk assessment of nanomaterials as an area of interest in their ‘Joint Statement on Nanomaterials Toxicology’ [14]. United States Environmental Protection Agency (USEPA) while recognizing the potential benefits of nanotechnology has also stressed on the need for a responsible development of nanotechnology and a proactive approach. In its document—EPA 100/B-07/001 (Nanotechnology White Paper) published in 2007, it has stated “as the use of nanomaterials in society increases, it is reasonable to assume that their presence in environmental media will increase proportionately, with consequences for human and environmental exposure” [2]. The European Commission’s Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) has also reviewed the existing information/data and issues to be considered in conducting risk assessment on nanomaterials [15]. European Commission’s Scientific Committee on Consumer Products (SCCP) issued a document titled “Opinion On Safety of Nanomaterials in Cosmetic Products” and raised a concern about large data gaps, inappropriateness of existing methodologies for nanoparticle risk assessment and inadequate information regarding nanoparticles skin absorption in both normal and abnormal (diseased) skins [16]. Guidance documents on safe handling of nanomaterials are also being drafted by researchers [17]. Non-governmental organisations like the Friends of the Earth warned against nanotechnology in cosmetic and sunscreen products, since they may produce a possible uptake of particles by human skin: if nanoparticles penetrate the skin, they can join the bloodstream and circulate around the body with uptake by cells, tissues and organs [18].

Exposure to Nanoparticles

Exposure Sources

The exposure of general population to the nanoparticles can occur either directly or indirectly.

Occupational Exposure at Work Place

In the production processes there is a likelihood of exposure during synthesis and recovery phases. The nature and probability of the exposure would differ according to the specific stage of process. Nanoparticle synthesis and manufacturing can also contribute to environmental contamination from industrial effluents or spillage during shipping and handling.

Exposure Through Consumer Products

Nanoparticles are being used in personal care products such as cosmetics and sunscreens. This provides a direct source of exposure to humans and can also enter the environment from washing off of consumer products.

Exposure Through Unintentional Release

Nanoparticles being used in electronics, tyres, fuel additives and many other products may reach the environment either through accidental leakage or during their disposal [19].

Exposure Routes

Depending on the exposure source, nanoparticles can enter the organism through different routes and can have varied health effects. If nanoparticles contaminate the air, they will mainly enter the organism by inhalation and will interact with the respiratory system. In the case of dermal exposure, nanoparticles have to pass through the skin. When soils or waters are contaminated or nanoparticles are deliberately injected or swallowed, their absorption is mainly through the gastro-intestinal or circulatory route.

Inhalation Exposure

The most common route of exposure is through inhalation. This involves intake of airborne nanoparticles through the respiratory system. Inhalation exposure of airborne particles may occur while processing and/or packaging the dry powder during nanomaterials synthesis.

Studies on rats have shown that nanometer particles are more potent than the micrometer particles in inducing pulmonary toxicity [13]. The inhaled nanoparticles can target all three regions of the human respiratory tract—the nasopharyngeal, tracheobronchial, and alveolar regions. Any foreign body in the respiratory tract may face several clearance mechanisms. The mucociliary escalator dominates the clearance from upper airways (nasopharyngeal and tracheobronchial region); while clearance from the deep lung is predominantly by macrophage phagocytosis. The alveolar macrophage is the most important defense mechanism in the alveolar region for fine and coarse particles, yet inhaled singlet nanoparticles are not efficiently phagocytized by alveolar macrophages [13].

A study based on the inhalation exposure of rats to ultrafine (20 nm) and fine (200 nm) titanium dioxide particles, demonstrated that the ultrafine particles cleared significantly slower, showing more translocation to interstitial sites and to regional lymph nodes than the fine titanium dioxide particles [20]. The nanoparticles can be introduced into the blood circulation from the pulmonary interstitial sites. From the blood circulation they can be distributed to other target organs like liver and spleen.

Dermal Exposure

Dermal exposure to nanomaterials has received much attention, perhaps due to concerns with occupational exposure and the introduction of nanomaterials in cosmetics [21]. Dermal exposure may also occur during the equipment cleaning and maintenance.

Skin is the largest organ of the body and structured in three layers: the epidermis, the dermis, and the subcutaneous layer. It functions as a strict barrier to exogenous toxicants and micron sized particles. The efficiency of dermal barrier against nanoparticles however, is still to be investigated completely. The possible routes for nanoparticle entry through the skin are: inter-cellular, trans-cellular and trans-appendageal [22, 23]. In the intercellular route—the lipid-soluble particles may move through lipid medium passing between skin cells whereas in the trans-cellular route the substance enters the skin cells. The trans-appendageal route is through the sweat glands and hair follicles which are scattered all over the skin in different densities and may become portals for nanoparticles to get through deeper parts of the skin [24].

Gastrointestinal Exposure

Nanoparticles can be ingested directly when used in food, food packaging, drug delivery and cosmetics. Workers can be exposed by unintentional hand-to-mouth transfer of materials. Furthermore, because of their use in a wide array of consumer products, they may enter the environment by a variety of routes such as discharge of industrial waste water and disposal of nanoparticles containing products. From the environment they can enter the human body through food chain. In addition, some of the nanoparticles can be swallowed into the gastrointestinal tract when they are expelled from the mucociliary system of the lungs after inhalation exposure [13]. Nanoparticles could be translocated from the lumen of the intestinal tract into different organs. Jani et al. [25] found a particle size-dependent uptake (6.6 % of the administered 50 nm particles, 5.8 % of the 100 nm particles, 0.8 % of 1 μm particles, and 0 % for 3 μm particles) of polystyrene particles by the gastrointestinal mucosa. The particles translocated from the Pyer’s patches into the mesenteric lymph and then to systemic organs (i.e., liver, spleen, blood, bone marrow, and kidney). Bockmann et al. [26] also found the uptake of the TiO2 nanoparticles from the gastrointestinal tract into the blood.

Assessing the Biological Responses to Nanomaterial Exposure

The concerns and awareness related to health and safety aspects of nanoparticles have culminated in a number of scientific publications concerning toxicological evaluation of nanomaterials. Some of the major biological effects exhibited by different categories of nanomaterials and the methods undertaken to study them have been reviewed in this section.

Nanoparticle-Induced DNA Damage

Genotoxicity is a central element of risk assessment for any chemical compound to which humans may be routinely exposed to (e.g. in foods or personal care products). Information about genotoxicity is vital as DNA damage can not only initiate cancer development, but can also have an impact upon fertility and the health of subsequent generations if disturbances arise in reproductive cells [27]. Nanoparticles have been demonstrated to possess genotoxic potential [28] which may be attributed to following main reasons.

Direct Interactions with the DNA

Nanoparticles may gain direct access to DNA after transport into the nucleus [29]. The entry of nanoparticles into the nucleus has been demonstrated by several studies [30, 31]. The nanoparticles are unable to cross the nuclear membrane but accumulate in the cytoplasm, can gain access to the nucleus during mitosis when the nuclear membrane breaks down [27]. The direct interaction of nanoparticles with the DNA and DNA-related protein may lead to physical damage to the genetic material. Interference with the structure or function of DNA repair enzymes in the nucleus might be another reason for DNA damage.

Oxidative DNA Damage

The nanomaterials due to their high surface area to volume ratio are known to produce reactive oxidative species (ROS). A previous study showed that the perinuclear distribution of TiO2 nanoparticles in BEAS-2B cells correlated with the induction of ROS in the same region as visualized by a fluorescence dye. ROS can induce DNA damage in the form of single and double stranded DNA breaks, base modifications and DNA cross-links [32]. They may also produce ROS by interacting with the cellular organelles. It can be by particle-induced disturbance of the mitochondrial electron transport chain function [33]. The ROS can also be generated during particle elicited inflammation. Inflammation from activated phagocytes (macrophages, neutrophils) during the inflammation produce bursts of ROS in order to destroy invading pathogens [34]. These ROS can damage the DNA of invaded cells, and also nearby host cells that were not invaded.

The single cell gel electrophoresis, also known as Comet assay is a well established, simple, rapid, visual, sensitive procedure. It is an extensively used biomarker to assess DNA damage quantitatively as well as qualitatively in individual cell population [35]. It has gained wide acceptance as a valuable tool in fundamental DNA damage and repair studies [36], genotoxicity testing [37] and population biomonitoring [38].

The assay requires small number of cells per sample (~10,000). It can also be used to detect specific classes of DNA adducts (e.g. thymidine dimmers and oxidative damage) by using lesion specific antibodies or specific DNA repair enzymes. The collection of the data at the level of the individual cells allows robust types of statistical analysis. Over 60 studies in different cells have investigated the genotoxic effects of manufactured nanoparticles by the Comet assay and the majority of them found positive results in the form of DNA damaging potential [39–44]. Lin et al. [45] have shown the genotoxic potential of ZnO nanoparticles in the Comet assay in A549 cell line after 24 h exposure at concentrations which were significantly cytotoxic. However, the Comet assay guidelines by Tice et al. [46] suggests that in conducting in vitro Comet studies, care should be taken to avoid conditions that would lead to positive results that do not reflect genotoxicity but arise from DNA damage (i.e., double strand breaks) associated with cytotoxicity.

The micronucleus assay detects the chromosomal damage representing possible clastogenic or aneugenic activity of the test agent. When used with some actin inhibitor (e.g. cytochalasin B), it can differentiate between mononuclear and binucleated cells to give information about the background micronuclei levels. The use of some centromere specific probe in the micronucleus assay can further help in establishing the identity of micronucleus as aneugenic or clastogenic event.

The cytochalasin B which is an actin inhibitor may affect the process of endocytosis which is needed to for the cellular uptake of nanoparticles. Moreover, the potential interactions of nanoparticles with the cytochalasin B, therefore also need consideration while performing micronucleus assay with nanoparticles. When assessing the mutagenic effects of nanoparticles at high concentrations, the presence of nanoparticles on the prepared slides may hinder the visualization of micronucleus and thus disrupting the counting process [47].

The genotoxic effects of nanoparticles are not only confined to the human species but also extend to other species like bacteria. Therefore, the effects of nanomaterials on microbial systems also have been explored as they form an important component of the ecosystem and play a vital role in ecological balance. Ames test which is also known as salmonella reverse mutation assay is a well recognized genotoxicity test which has been applied to different nanoparticles [48–52]. Ames test detects mutation in single gene (point mutations) in prokaryotic cells with and without S9 fraction. The Ames test has been used for assessing the ecotoxicity of TiO2 and ZnO nanoparticles in the Salmonella typhimurium and Escherichia coli. These nanoparticles demonstrated a weak mutagenic potential in S. typhimurium strains TA98, TA1537 and E. coli [49, 50, 53]. Some other studies have reported negative results while assessing the mutagenic potential of nanoparticles by this test [51]. However, different sources of nanoparticles, their physico-chemical characteristics, methods of nanoparticle suspension preparation (concentration, sonication etc.) may give rise to these differences.

The testing of mutagenic potential of nanoparticles by Ames test has posed several questions. The structure of bacterial and mammalian cell membrane is different and the endocytosis is supposed to play an important role in the process of nanoparticles uptake which can subsequently cause mutations. The conventional Ames test strain were made for testing the chemical mutagenesis and hence do not account the physical interactions of nanoparticles with DNA.

Cytotoxicity of Nanoparticles

Different categories of nanoparticles have been reported to cause a decrease in the cell viability [54–59]. The in vitro test systems and assays being easy, time and cost effective are usually performed to check the effects on cell viability. Although different assays have been used to assess the cytotoxic potential of nanoparticles, however, the most commonly employed are mitochondrial dehydrogenase activity, neutral red uptake and lactate dehydrogenase (LDH) release assay.

The activity of mitochondrial dehydrogenase can be measured by a tetrazolium dye called 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide or simply MTT dye. The mitochondrial dehydrogenase activity of viable cells cleaves the tetrazolium ring, yielding purple colored crystals insoluble in aqueous solvents. After dissolving the crystals in acidified isopropanol or DMSO, the absorbance is measured by spectrophotometer [60]. The lysosomal membrane damage can be assessed by neutral red uptake assay in which the vital dye neutral red is actively transported into the lysosmes of viable cells. However in the non-viable cells the dye cannot be retained in the lysosomes and is therefore washed away [61]. The LDH release assay is based on the measurement of LDH activity in the extracellular medium. The loss of intracellular LDH and its release into the culture medium is an indicator of irreversible cell death due to cell membrane damage.

The nanoparticles display several unique physicochemical properties due to which they interfere with normal test systems and this has been well documented in the literature [62–65]. Examples of such properties include: high surface area, leading to increased adsorption capacity; different optical properties that interfere with fluorescence or visible light absorption detection systems; increased catalytic activity due to enhanced surface energy and magnetic properties that make them redox active and thus interfere with methods based on redox reactions [62]. A recent study by Zaquat et al. [66] have demonstrated that TiO2 nanoparticles bind to LDH, and consequently, TiO2 nanoparticle-induced toxicity could be underestimated by the LDH activity assay.

Nanoparticle Internalization in Biological Systems

It has been shown that size plays a critical role in cellular uptake of nano-structures [67]. A thorough understanding of the mechanisms of nanoparticles entering and leaving the cells could lead to a better understanding of NP toxicity as well as improvement in their bio-medical applications.

Earlier studies have demonstrated that when nanoparticles are exposed to cells at 4 °C or in ATP-depleted conditions, their entry into the cells is inhibited [67, 68]. Endocytosis, therefore has been proposed as the inferred pathway for cellular uptake of nanoparticles rather than the free diffusion. It can be clathrin mediated endocytosis [69], caveolae mediated endocytosis or clathrin and caveolae independent endocytosis [70].

Accumulation of nanoparticles in the perinuclear region or internalization via caveolae-mediated endocytosis to reach endoplasmic reticulum may facilitate physical contact of nanoparticles with nuclear pore complexes and subsequent transport into the nucleus [71]. The phagocytosis is unlikely to contribute to the nanoparticles uptake into the cells. This is evident from the fact that even non-phagocytic cells can efficiently internalize nanoparticles and the lower size cut-off described for phagocytosis is 500 nm [72].

Tracking nanoparticle internalization in cellular systems is of utmost importance for understanding and correlating the biological effects elicited by these nanoparticles.

Transmission electron microscopy provides a detailed view of the interaction of nanoparticles with cell structures. Due to its high resolution, transmission electron microscopy enables the imaging of membrane invaginations, vesicle formation, and organelles [73] which helps in understanding the mode of nanoparticle uptake. It also aids in understanding the ultrastructural changes that occur in cells subsequent to nanoparticle uptake [74, 75]. However, transmission electron microscopy is only a qualitative tool for assessing nanoparticle uptake, and is usually confined to imaging a few cells due to the complicated sample preparation and image analysis involved [76]. Further, the staining procedures generally used for electron microscopic preparations can introduce electron-dense artefacts that may be mistaken for nanoparticles.

Inductively coupled plasma mass spectroscopy (ICPMS) can be used as a sensitive and quantitative tool for the determination of trace amounts of nanoparticles. ICPMS becomes especially important in the context of an in vivo scenario, where it identifies the target organs for nanoparticles [77]. Using this technique, even trace amounts of nanoparticles that enter through a different route can be detected in various body organs.

Flow cytometry is a simple, easy and sensitive method used to study nanoparticle uptake in bacterial and mammalian cells [78–82]. It can be used for the detection of fluorescent as well as non-fluorescent nanoparticles inside the cells.

In Vivo Toxicity of Nanoparticles

The toxicity assessment of any novel entity in vivo is always desirable. This is due to the complex cell–cell, cell–matrix interactions, diversity of cell types, hormonal effects that are hard to mimic in the in vitro systems. In addition, studying long term chronic effects of the test compound is possible only by in vivo experiments. The importance of the in vivo studies in nanomaterial toxicology has been highlighted [83].

There are no existing guidelines and standard methodologies for risk assessment of nanomaterials. However, the “committees on toxicity (COT), mutagenicity (COM) and carcinogenicity (COC) of chemicals in food, consumer products and the environment, UK” have suggested extrapolating the in vitro nanotoxicity findings to in vivo experiments and confirm the results [14]. The same committees (COT, COM and COC) have also advised to consider appropriate route of exposure in the in vivo experiments.

Many investigations have studied the in vivo effects of nanoparticles by suitable systems and reported accumulation in organs like liver, adverse effects on DNA, immune system and neurobehavioral patterns [84–87].

In vivo studies with nanomaterials, unlike studies involving chemicals/compounds, are interlaced with many challenges. The in vivo dose used for experiments should be derived from the quantity of nanoparticles exposed to in the actual scenario. However, determining the quantity of nanoparticles in air, water, soil or any consumer product is a technical challenge due to their tiny size and the small quantity present. Even if the dose of nanoparticles is known, exceeding a certain dose in experiments is not advisable due to increased agglomeration of nanoparticles. The biodosimetry or biodistribution of nonfluorescent, nonradioactive, nonmagnetic nanoparticles is almost impossible to investigate with the presently available techniques.

Conclusion

The safety/toxicity aspects of nanomaterials have lagged far behind the rate at which they are being produced. A multidisciplinary team effort from material scientists, molecular biologists, toxicologists and physicists is necessary as it will facilitate the interlinking of different facets of nanotoxicology thus aiding in the understanding of cellular responses to nanomaterials exposure and mechanisms involved.

There is also a need for more comprehensive studies to fully understand and address the potential risks of engineered nanomaterials to human health and the environment. This will help in creating environment friendly and biologically safe nanoparticles.