Engineered nanomaterials and human health: Part 2. Applications and nanotoxicology (IUPAC Technical Report)

2.2.1 Limitations of small molecule drugs

Conventional small molecule drugs consist of single chemical entities with a dimension that usually ranges from about 0.6 to 2 nm [10]. Although the pharmacology of a small molecule drug can often be improved by designing analogues, some restrictions remain. For example, failure to solubilize when applied intravenously, unfavourable half-life, or failure to cross biological barriers will limit the delivery of a drug to the target organ or correct cellular location. Post-administration capture by the molecular and/or cellular elements of body fluids, lack of activation at the target site, and premature export from target cells via membrane carriers can further limit the therapeutic efficacy of the drug. It is a central goal of nanoparticle drug design to overcome such restrictions.

2.1.2 Types of nanomaterials in modern diagnostics and therapeutics

It is well-established that nano-sized substances have been used in pharmacology and diagnostics before the onset of nanoparticle engineering. Lipid emulsions were already infused as energy-rich nutrients in clinical settings [11] before the application of ENM and colloidal silver (e.g. Argyrol) has been applied to protect water and textiles against microorganisms for 100 years [12] and has been used to cure various diseases [13], [14], [15], [16]. Colloidal gold also has a history in disease treatment and polymeric gold compounds are used to treat otherwise therapy-resistant rheumatoid arthritis [17]. However, the engineering of more complex nanostructures with the aim to improve therapeutic and diagnostic possibilities started only some 20 years ago. In the search for useful and tolerable materials, a large number of different approaches are presently studied. The basic architecture used in many of these approaches consists of three components: scaffold, core, and surface. The ‘scaffold’ provides mechanical and physicochemical stability. It should have a defined size-range and a suitable surface that allows the addition of functional groups. The ‘core’ can be hollow, porous, or cavernous. It is used to accommodate therapeutic and diagnostic agents, alone or together with a solvent. Although the ‘core’ of the nanoparticles (a class of ENM, abbreviated as NP) may not interact directly with the biological environment, it is needed to stabilise the NP and protect and release the cargo. There are also requirements for it to be biocompatible. It will either have to degrade to smaller components, or else be small enough to be renally cleared. The particle ‘surface’ that interacts with cells, tissues, and fluids is usually modified to become biocompatible and immunoresistant. It can be conjugated with antibodies or other agents that direct the particle to a biological target.

Theranostic nanoparticles may have inorganic (e.g. metal), polymeric, lipid, carbohydrate, proteinaceous, or gaseous components [18], [19], [20]. Figure 3 in [9] depicts schematically some of the more common structures. They include nanospheres, nanocapsules, nanoshells, nanorods, liposomes, polymersomes, micelles, and virus-like-particles (the latter are not shown).

2.1.3 General features of theranostic nanomaterials

Theranostic nanoparticles are designed to exhibit special features, while being at the same time biocompatible. Many physicochemical features that are not directly related to the therapeutic or diagnostic aim must be controlled and possibly improved during development (see also [9]). These features include size distribution, shape, surface charge, surface functional groups, porosity, crystallinity, and solubility, as well as beneficial degradation or stability towards association in certain environments, as illustrated in Fig. 1.

Fig. 1:

Schematic illustration of the most important features of ENM for a rational design as theranostic material. VB: valence band; CB: conduction band; E_g: band gap energy (eV). Adapted from [21]. The particle size, shape, surface charge, and deformability/degradability of ENM and their effects on overcoming delivery barriers are reviewed in more details by Blanco et al. [22].

2.1.4 Scaffold influences

A variety of materials have been used as scaffolds, with polymers being the most prominent class. Commercially available polymers, such as poly(methyl 2-methylpropenoate), more commonly called poly(methyl methacrylate), have been used during the pioneering phase [23]. Currently, priority is given to degradable, and preferably bioresorbable, artificial polymers, such as lactic- and/or glycolic acid-based polymers and copolymers, etc. [24], [25]. Another class of scaffolds is made up of lipids and phospholipids that are able to form liposomes with different surfaces depending on the precursors used [26]. Such engineered particles can mimic the size and lipid composition of endogenous lipoproteins (low-density lipoprotein, high-density lipoprotein, for example ApoB-100 in LDL and Apo-AI and Apo-AII in HDL). They can accommodate lipophilic drugs in the lipid compartment and hydrophilic drugs in the aqueous core [27], [28]. Moreover, new techniques such as electrospinning are versatile methods to fabricate scaffolds. For example, nanofibers of materials such as polymers and ceramics with diameters from 3 nm to several micrometres are used as scaffolds for in vivo and in vitro tissue engineering [29]. Carbon fibres or carbon nanotubes and solid materials such as gold or mesoporous silica are also investigated for these purposes [30], [31]. The possibilities appear unlimited when one considers the number of parent materials and drugs that can be combined. However, in reality, the domain is very much limited, especially if the long list of prerequisites (e.g. reasonable chemistry; possibility of upscaling; easy formulation; sterilization, conditioning, and stability on storage; use-by date; cost; and approval by regulatory agencies) for effective application in humans is taken into account. This very much restricts the number of successful approaches.

2.1.5 Chemical surface modification and hydrophilization

From a physicochemical viewpoint, the particle surface must be compatible with the temperature, pH, ionic strength, and other parameters imposed by the surroundings and must help to prevent particle association. Conventional colloid techniques prevent the association of pristine NP by introducing capping agents (often charged) to the surface to induce steric or electrostatic repulsion between neighbouring NP [3], [32]. This is a popular means of controlling colloidal stability. Poly(oxyethylene) (the PEO abbreviation comes from the source-based name poly(ethylene oxide) [33]) can also be employed to create a steric barrier that reduces non-specific binding of the NP and has proven a popular surface coating in many drug delivery systems (Fig. 2). PEO is also referred to as poly(ethylene glycol) (PEG), which is a structural variation of PEO with two –OH groups, one at each terminus [34], [35]. The two terms are often used interchangeably, however the systematic term PEO is recommended by IUPAC to describe analogues with repeating fragments of –O–CH₂–CH₂–. We use the systematic name PEO in this document, but, for describing the process of attaching PEO to surfaces, we use the term ‘PEGylation’, which is widely used in the literature. PEO segments are available in a variety of forms (short or long chains or multiple chains in the same substituent). They are hydrophilic, neutral, and biocompatible, and can be conjugated to biomolecules [36]. Other macromolecules, such as dextran or dendrimers, have also been used to create sterically stabilised NP that are more biocompatible and which can be used for biomolecule attachment [37], [38], [39].

Fig. 2:

Surface coatings of engineered nanomaterials and their possible targets. Adapted from [45].

Surface PEO segments have been used in liposomal technologies because they prolong in vivo circulation lifetimes [40]. Improvements in the pharmacokinetics and bio-distribution of dye-doped silica NP upon PEGylation were shown by He et al. [41] in a study comparing 45 nm OH-, COOH- and PEO-coated NP in mice. Wiesner’s ultra-small dye-loaded silica NP, currently in clinical trials, also utilise PEO for enhancing NP circulation half-life [42], [43]. The effect of ‘PEGylation’ on these small NP (achieved using a PEO-siloxane) was recently elucidated using fluorescence correlation and cross-correlation spectroscopy [44].

2.1.6 Protein corona

Despite efforts to improve the colloidal stability of nanomaterials through grafting of sterically or electrostatically repelling coatings, additional surface modification often occurs inside the body by adsorption of substances present in body fluids [46], [47]. Such surface ‘fouling’ is primarily attributed to protein adsorption onto the NP surface to form a ‘protein corona’, also referred to as a ‘biomolecular corona’. Two types of protein corona, ‘hard’ and ‘soft’ (Fig. 2), have been shown to form on many types of NP, including gold, polystyrene, titanium, silica, and zinc oxide [48]. A hard corona is a near-monolayer of biomolecules tightly bound (not irreversibly but with possible denaturation effects) to the particle surface. Surrounding this layer is the ‘soft’ corona, which is composed of more loosely associated and rapidly exchanging biomolecules. According to the article ‘What the Cell “Sees” in Bionanoscience’, this corona provides the ‘extrinsic environmentally-derived identity’ that a cell would actually ‘see’ and interact with [49].

The emergence of the protein corona field highlights the importance of performing analytical characterization in complex biological media. For example, when developing assays using NP for biomarker detection, it is crucial to use whole blood, serum, urine, etc., which provide an environment comparable to real-life patient samples. Otherwise, the limits of detection, sensitivity, and signal-to-noise ratio reported may not reflect the true capability of the diagnostic test, as potential NP fouling would not have been considered. From a drug delivery viewpoint, Mahon et al. speculated that, if the nanoparticle corona could be precisely engineered, favourable cell targeting with more efficient and effective NP-based treatments would be possible [50]. A recent report has also shown that the ‘biomolecular’ corona composition may influence how NP are biologically processed and/or cleared. The epitopes of two major proteins in the serum corona, low-density lipoprotein and immunoglobulin G, were presented on the surface of silica NP and could be recognized by their respective receptors. The authors suggested that cells that exhibit such binding receptors may therefore be ‘mistakenly’ recognizing NP (based on their corona composition) as endogenous or exogenous objects, such as lipoproteins or viral infections, respectively [51], [52]. Coating surfaces with an albumin nanolayer was shown to be more efficient than coating them with PLA-PEO di-block copolymers for repelling major proteins under modelled physiological conditions [53]. The idea of corona engineering runs in parallel with the concept of ‘cell vision’ [54], where different cell types can elicit significantly different responses to nanomaterials based on their protein corona composition. Vast improvements in the understanding of nanoparticle-cell interactions are therefore needed for development of biological applications.

2.1.7 Surface modification with biomolecules

When an NP is engineered to target specific cell types, attachment of a targeting biomolecule is usually necessary (Fig. 2). The techniques for such functionalization are described in Part 1 [9]. Possible targeting moieties include ligands (usually peptides or proteins) for cell-surface receptors and membrane transporters, antibodies that are directed against specific tissues, antibacterial agents, surface components of microorganisms for vaccination, and others. Even after the successful synthesis of such functionalized NP, problems may become evident when they are introduced into physiological environments. Issues may include incorrect orientation of the ligand, unfavourable mobility of the biomolecule, non-specific binding en route to the target, instability towards degrading enzymes, or premature elimination. Therefore, a stepwise optimization of the surface functionalization method is usually necessary.

2.1.8 Drug release

Many new Nanosized Drug-Delivery Systems (NDDS) are currently focused on applications related to cancer therapy [22], [25], [27], [50], [55], [56], [57], [58], [59], [60], [61], [62]. NDDS were also reported to have the potential to significantly accelerate progress in the development of new therapies for other medical conditions, such as neurodegenerative disorders (Alzheimer’s [63] and Parkinson’s [64] disease), type 1 and 2 diabetes [65], and tuberculosis [66], [67], as well as cardiovascular [68] and infectious diseases [69]. In general, NDDS favor a slow release of a therapeutic agent that is either encapsulated in the core of the NP or attached on the surface of the nanocarrier. In some cases, for example in the new, hybrid, or multifunctional nanomaterials, the drug is incorporated inside a shell that surrounds a solid core. Very often, the core and the shell are made of combinations of organic and inorganic materials [70], [71], [72]. However, NDDS have little relevance for bioactive species that are very soluble in body fluids and diffuse out very rapidly. They are of more interest for hydrophobic species which usually have longer retention times [73], [74].

The aim of NDDS is to release their load progressively via diffusion or degradation phenomena (or both), and the speed of these processes must be adequate to achieve therapeutic drug concentrations that remain below toxic levels. The choice of whether the drug is encapsulated inside the core of the nanomaterial, inside the shell (usually up to a few tens of nm thick), or even attached on the surface, depends on the desired application, the nature of the drug, the composition of the ENM, whether the drug can diffuse through the shell or not, and whether the core/shell has a different function than transporting and protecting the drug (e.g. photothermal effects, imaging contrast, or magnetic properties). The size of the core and the thickness of the shell must be carefully considered. As a simple illustration, consider a 100 nm (diameter) core/shell nanoparticle with a core diameter of 50 nm and a shell thickness of 25 nm. The core volume is actually 7 times smaller than the volume of the 25 nm shell. Therefore, more drugs could be loaded in a suitable shell for such a nanoparticle, albeit at the expense of an enormous surface area that favors very fast release. On the other hand, although less drug can be loaded inside the core, the 25 nm shell may retard the release of the drug until it is needed. Suitable pharmacokinetic and pharmacodynamic characteristics for NDDS must be considered, as we will explain later.

2.1.8 Drug targeting

Current approaches for targeted drug delivery to diseased tissues rely on two targeting concepts: (i) active and (ii) passive. The active targeting route takes advantage of NDDS that are conjugated to a receptor-targeting ligand. Disease-specific receptors present on tumor cell membranes are reasonably well described in the literature [75], [76], [77], [78]. Targeting can result from specific transporter-mediated uptake by target cells or by toxic interactions with target-specific surface markers. There are two major concerns with this strategy: (1) the targeting receptors are not specific to diseased cells; although they may be overexpressed on the diseased cell surface, they are also typically available on healthy cells; and (2) the formulation of NP with ligands, such as antibodies, immobilized in their active form on the surface tends to be difficult due to antibody denaturation during long-term storage and challenges with large-scale production, corona formation and association, and/or nonspecific ionic interactions. In addition to these concerns, NP conjugated to targeting moieties are chemically complex species and a detailed characterization of their biological activity and their ability to specifically and quantitatively target the desired receptor is often lacking.

Passive targeting utilizes the enhanced permeability and retention (EPR) effect, a phenomenon observed in cancerous tissue [79], [80], including many solid tumors, which have defective blood vessels that exhibit increased vasculature permeability. Such conditions ensure that tumors are sufficiently supplied with nutrients and oxygen for rapid proliferation. The leaky blood vessels enable nano-sized material to diffuse into the tumor microenvironment. The EPR effect is believed to be responsible for the clinically successful nanoparticle-based therapeutic Doxil^® (doxorubicin encapsulated in a liposomal formulation) [81].

Despite the clinical success of Doxil^®, the EPR effect is complicated by many variables, such as interstitial fluid pressure, irregular blood vessel distribution, the type and location of the tumor, and limited blood flow in the tumor bed. As a result, many NP-based strategies designed to take advantage of the EPR effect were less successful than anticipated. In fact, a controversial 2016 review paper highlighted that only 0.7% (median) of an injected dose of NP arrive at the tumour site in mouse models and that no improvements on delivery efficiency have been reported for 10 years [82]. Several critical reviews highlight many differences between animal and human tumor models and summarize the principles, anomalies, and challenges of the EPR effect [83], [84], [85]. Therefore, the concept of Paul Ehrlich’s ‘Magic Bullet’, which surmises that the ultimate aim of targeted drug delivery is to develop a therapeutic that eliminates disease whilst avoiding interaction with healthy tissue, has started to raise many unanswered questions in the scientific community [86].

4.1.1 Molecular level

Different types of ENM have been shown to catalyze the formation of reactive oxygen species, a common first step of toxicity (Fig. 3, a). Metal-containing ENM may induce toxicity by releasing potentially toxic metal ions that bind to functional groups (e.g. thiol groups) on macromolecules (Fig. 3, b), disrupting their structure and function. Cationic ENM tend to be attracted by the negative surface charge of cells, thereby producing deleterious interactions (Fig. 3, c). Any proteins attached non-specifically to ENM (e.g. albumin, opsonin, fibrinogen etc.) will form a nanocoating or ‘corona’ that may disturb the functioning of the designed nanomaterial (Fig. 3, d).

Cell level

Large particles (about 500 nm) are taken up by phagocytosing cells, such as macrophages and hepatic Kupffer cells. Storage in these cells protects the organism from contact with ENM. However, if the phagocytosing cells are not able to degrade the ENM at a sufficient speed, then the cells will be overloaded (Fig. 3, e). There is also a risk that ENM-loaded phagocytosing cells will send out signalling molecules that cause local inflammation (Fig. 3, g). When ENM or their components are taken up by antigen presenting cells, a sensitizing immune reaction accompanied by intolerance may result (Fig. 3, f). Small ENM (1 to 10 nm) have been found to enter virtually all cell types, although usually at a low rate. There is some discussion on whether intracellular ENM may interact with the cell nucleus (Fig. 3, h).

4.1.3 Organ level

It is often believed that substances that do not enter cells will not exhibit toxic effects. This, however, is not correct. Extracellular ENM may affect blood flow in the capillaries, clog the renal filter apparatus (Fig. 3, i), disturb regular heartbeat (Fig. 3, j), or initiate chronic disease such as lung fibrosis (Fig. 3, k). There is a possibility that ENM can migrate from the nose to the brain via the olfactory tract (Fig. 3, l), which is otherwise protected by the blood-brain barrier; this route was recognized for environmental ultrafine particles [176], [177] before it was verified for ENM [178], [179]. Although not shown directly in Fig. 3, the liver is another important organ which must be considered for its uptake and toxicological response to ENM. The physical and chemical properties of ENM have a profound influence on their pharmacokinetic behaviour, which ultimately determines their ability to accumulate in the liver [180]. According to the review published by Zhang et al. 30 to 99% of administered NP will accumulate and sequester in the liver after administration into the body. The resulting effect is reduced delivery to the targeted diseased tissue and potentially increased toxicity at the hepatic cellular level. The authors review the inter- and intra-cellular interaction between nanoparticles and hepatic cells, the elimination mechanism of nanoparticles through the hepatobiliary system, and current strategies to manipulate liver sequestration [174], [175].

4.2.1 Transport-principle

The most important property for the transport of ENM in tissues and cells is their specific small size. Only small particles have access to the vesicular structures on the surface of cells, allowing use of the small vesicular transport pathways that are inaccessible to larger particles with sizes above 300 to 500 nm (Fig. 4). Excluding phagocytosis, which can be accomplished only by very specialized “phagocytes” in our body and is a mechanism for larger objects, only ENM can use the small vesicular transport pathways to enter the cell [181], [182].

Fig. 4:

Pathways for uptake of nanomaterials into cells. The importance of each pathway differs between cell types. For example, phagocytosis is characteristic mainly for macrophages and granulocytes. The specific route through which nanomaterials are internalized or transported inside the cell mostly depends on the material size and surface charge. Adapted from [45], [173].

As an example, in the human lung the alveolar epithelial cells have several thousands of so-called “caveolae” on their membrane surface at any given time, which means that there is a probability for inhaled ENM to enter epithelial cells via vesicular transport. Nevertheless, several studies have shown that the lung cleaning system (alveolar macrophages equals phagocytes) takes up most particles, largely independent of their size, and transports them out of the lung [183]. Other pathways of cellular uptake, such as receptor-coupled transport or passive diffusion, have been infrequently described and have not yet been confirmed by the community. The transport through small vesicles has been described as a “Trojan-horse-mechanism” (Fig. 5) [184], because this is an unintended uptake of foreign material which may have adverse effects in the interior of the cell.

Fig. 5:

Trojan-Horse-like transport of ENM into the interior of cells via vesicular pathways (caveolae). Small particles with a diameter of less than 100 nm can fit into vesicular structures, such as caveolae, and be transported into the cytoplasm. Within the cells, these vesicles may form a lysosome with an acidic interior, facilitating the dissolution of materials such as ZnO. The TEM image shows two nanoparticles (22 nm) located within caveolae of a lung epithelial cell (A549) in culture. Reproduced with permission from [173].

4.2.2 Surface-principle

The smaller the particle, the higher is its surface-to-volume ratio (Fig. 6). A one micrometre particle has less than 2% of its atoms located on the surface, whereas a five nanometer particle has more than 20% of its atoms in direct contact with the surrounding environment. As all safety rules at workplaces or in the environment are related to a certain mass per cubic meter of air or litre of water or kg of soil, the surface related dose has been often ignored so far. This question was raised in connection with animal studies on the pulmonary toxicity of ultrafine particles many years ago [185], [186] and it is currently more and more within the focus of researchers and regulators [187], [188]. However, it is still an open question if the mass per unit volume, the number, or the specific surface area is the best metric to describe a dose-response relationship for ENM [189].

Fig. 6:

An interesting analogy to illustrate the surface-to-volume ratio of engineered nanomaterials. If the particle (e.g. cube) in the figure has a size a=1 cm, the surface area of that particle is 6 cm². By decreasing its size to a=10 nm, while keeping the same overall volume and mass, the surface area now grows to 600 m². By reducing each particle further to a=1 nm, the surface area is proportional to the surface of four ice-hockey rinks. Source: Council of Canadian Academies.

Reducing the size of particles implies two important consequences for their interaction with the surrounding environment. First, the surface itself becomes larger and reactions (e.g. catalysis or electron transfer) can occur with a higher probability, making such particles more reactive compared to their larger counterparts. Second, the smaller the particles, the higher is the potential occurrence of surface defects within the crystalline structure or interatomic attractive forces facilitating direct interactions of neighbouring molecules with specific surface sites that are not present in the respective bulk material [153], [174]. These two surface properties enhance the possible interaction with biological molecules such as lipids, proteins, or nucleic acids and may induce adverse effects in cells and tissues [152], [190].

4.2.3 Material-principle

Variations in ENM composition (either bulk or surface) lead to differences in their reactivity and behaviour in biological systems [191]. Gold or silver metal particles; metal oxides, such as SiO₂, TiO₂, ZnO, Fe_xO_y, and CeO₂; and carbon-based materials, such as carbon black, nano-diamonds, carbon nanotubes, or graphene-based materials all have different binding capacities for biological molecules, different reactivity towards biological molecules such as proteins, lipids, or DNA, and different distribution patterns. Moreover, the formation of a molecular corona of biological substances, such as proteins, lipids, or other relevant molecules, is dependent on the material [8], [123] and the tendency of the material to associate strongly affects the behaviour and biological effects of ENM [29], [192], [193], [194], [195], [196], [197]. Thus, the material and its composition have a crucial role in determining the biological readout of exposure against ENM.

4.2.4 Fibre-principle

Besides the above described properties of ENM, the shape or aspect ratio is another important attribute with some predictive power for toxicity. There are several examples from the literature that describe the potential pathogenicity of “nanofibres” (HARN: high-aspect ratio nanomaterials [198], [199]) which might conform to the so-called ‘fibre pathogenicity paradigm’. This paradigm states that the pathogenicity of a fibre, especially after inhalation, can be predicted “on a continuum based on its length and biopersistence, as well as its aspect ratio” [200]. The fibre-paradigm applies only to fibres with a key length larger than 5 micrometres where the diameter does not determine the biological effect, as long as it is less than 1 to 3 micrometres and the aspect ratio is a minimum 3:1 (length over diameter) [201]. Carbon nanotubes with a length range that fulfils the nanofibre criteria and that belong to what are termed WHO-fibres, defined by the fibre-paradigm, have been shown to induce similar pathogenicity as asbestos fibres [198], [200], [201], [202]. This specific biological effect becomes more and more relevant as carbon nanotubes and other fibre-like nanomaterials are used in an increasing number of technical applications. Medical examples include carbon nanotubes as candidates for drug delivery systems, especially for neuro-regeneration [203], and the use of functionalised carbon nanotubes as tools for drug targeting, therapeutic applications, or imaging [204], [205].

The ‘fibre pathogenicity paradigm’ seems to be a broadly applicable rule, as it has been shown to apply not only to asbestos and carbon fibres (see above), but also to titanium dioxide nanobelts [206] and nickel nanowires [207]. As long as the aspect ratio is larger than 3:1 and the overall length is longer than 5 micrometres, either stiff or rigid nanofibres can induce the pathological effects described above and such nanomaterials should be handled with care during their whole life-cycle.

4.3.1 Biological barriers

Biological barriers are important for the protection of the organism from environmental influences and for its compartmentalization into organs. Barriers largely prevent the uptake of non-nutrient hydrophilic substances and particles, but allow the transport of nutrients (by membrane transporters). Major barriers that protect the organism from environmental influences are the skin, gastrointestinal tract, and the lung (Fig. 8). Some inner organs, such as the placenta and brain, are protected by additional barriers. Barriers commonly involve two structures: (1) a protecting layer of epithelial cells and (2) the adjacent cells that form the supplying blood capillaries. The barrier-function is achieved by dense tight junctions between neighbouring epithelial cells and/or between the capillary-forming endothelial cells preventing paracellular fluxes. In addition, the skin barrier has multilayered sheets of densely packed dead tissue on top of the living barrier [22]. Biological barriers are very important for three of the four toxicokinetics steps: absorption, distribution, and elimination.

Fig. 8:

Schematic drawing showing that biological barriers may be formed by either densely packed organ cells and/or by densely closed endothelial cells (forming capillaries). In the Intestinal barrier, the transcellular pathway is open for nutrients. The blood-brain barrier is impenetrable to large hydrophilic molecules and many ENM due to tight junctions between endothelial cells and a layer of surrounding pericytes. In the Placental barrier, the huge cell (Syncytiotrophoblast) leaves space for few paracellular pathways. The fetal endothelial cells are tightly connected, which means that particles smaller than 200 nm can cross the placenta, but particles above 300 nm remain in the mother’s body and do not cross the barrier.

4.3.2 Absorption

Applications of ENM in theranostics, food technology, cosmetics, or textiles all enable direct body contact. There are different pathways by which the uptake of ENM can occur (Fig. 7, absorption). A literature study evaluating publications between 2000 and May 2013 came to the conclusion that a substantial number of studies observed uptake via ingestion or inhalation, whereas dermal uptake is most unlikely for ENM [5] (Fig. 9).

Fig. 9:

Percentage of publications disclosing the uptake of NP into the mammalian body, classified either via oral (ingestion), pulmonary (inhalation) or transdermal (dermal) route. The results are from a literature survey for articles published between 2000 and 2013.

The small intestine, where the majority of orally taken drugs will be absorbed, constitutes a barrier between the intestinal lumen and the blood formed by densely connected epithelial cells covered by layers of mucus. Nanoparticles are, in general, too large to be absorbed by diffusion through the enterocyte membranes. Likewise, permeation through the intact paracellular pathway (between cells) is impossible for molecules or ions larger than approximately 1 nm. Additional possible uptake routes involve endocytosis through epithelial cells and passage via M-cells of the immune system, found in the gastrointestinal tract (transcellular pathway), or through a diseased and disrupted barrier [208].

For the skin, it has been shown in many different studies and systems that almost none of a given ENM-dose applied to normal or UV-stressed skin reaches living cells in the deeper layers [165], [209], [210], [211]. Thus, it is assumed that nanoobjects cannot reach the bloodstream via the skin barrier in any clinically relevant amounts unless they are specifically designed for this purpose [169].

Nanoparticles in the ambient air or in inhalation sprays can be so small that they behave like gas molecules in the respiratory system and can even localise in alveoli, the gas exchanging structures of the lungs. Some transition into the bloodstream may occur. However, the overall percentages of the initial dose delivered into the bloodstream are very low [212], [213], [214]. Most of the particles will be taken up by alveolar macrophages or reticulocytes. The macrophages migrate to the upper airways and transport the ENM out of the lung and the reticulocytes migrate to the adjacent lymph nodes.

4.3.3 Distribution

ENM used in nanomedicine (i.e. drug delivery systems and in vivo imaging probes) enter the body almost exclusively via the parenteral route. Recently, the use of the respiratory route for nanomaterial drug delivery has been explored because of the large, accessible surface area of the lungs and because the air-blood barrier is reasonably thin. Once an ENM crosses the air-blood barrier, the inhaled nanomaterial can enter the circulation and accumulate in secondary organs and tissues. Kreyling et al. studied the fate of nanoparticles that enter the lungs and performed a biokinetic analysis (dose-response study) as part of a comprehensive risk assessment (Fig. 10).

Fig. 10:

Biokinetics of nanoparticles after lung exposure. Scheme of translocation of nanoparticles from the lung epithelium to the regional lymph nodes and to the blood. Thereafter, they may be distributed to and accumulate in various secondary organs, or they may be excreted via the kidneys. AM, Alveolar Macrophage. Reprinted with permission from [213].

Once a small molecule drug or ENM has reached the blood, it will be distributed by the bloodstream to the tissues. However, it should not be assumed that all the ENM will be transported to the target tissue, since adhesion to the walls of the blood vessel may occur. ENM shape and size are major factors governing NP flow in blood and adhesion to blood vessel walls [22], [215]. A literature survey indicated that nearly 100% of all cell types tested in vitro (i.e. in a closed space) can take up ENM, with only minor exceptions (e.g. some graphene-based materials). Although ENM are taken up by all cell types, the uptake across internal tissue barriers, such as the placenta barrier and the blood-brain-barrier, is usually very restricted. A prominent example is the transport of drugs across the blood-brain barrier to cure brain diseases. Even after more than 30 years of research, it is still a problem to use nanocarriers to deliver a sufficient dose of a hydrophilic drug across this barrier [216], [217]. A somewhat different situation can be found at the placenta barrier. As this organ is specialized to transport various larger molecules, such as proteins in the nanometer size range, ENM can cross this organ after a relatively short time [218], [219], [220]. This transport is both size-dependent, as particles smaller than 240 nm can cross but larger particles cannot, and surface dependent, as negatively charged particles are transported much more slowly than neutral or positively charged ENM.

Other, usually less effective, uptake mechanisms may occur [45]. These involve vesicle involution, such as by pinocytosis or receptor mediated endocytosis (clathrin or caveolin-based). Engineered nanoparticle drugs equipped with adequate receptors can make use of endocytosis uptake routes. When particles are larger than 200 nm, they will preferentially be taken up by phagocytosing cells of the immune system, such as macrophages or the hepatic Kupffer cells. If ENM are found to be stored in the liver, they will often be confined to phagocytosing Kupffer cells. It has also been reported that airborne nanoparticles, inhaled via the nose, may travel from the nasal epithelium along the olfactory tract to the otherwise inaccessible brain [178].

4.3.4 Degradation

Except for permanent prostheses, all foreign materials, including ENM, that are introduced into the human body must be eliminated sooner or later via natural pathways to avoid undesired retention. Biochemical processes in phagocyting cells can be highly aggressive and degrade many types of organic compounds by the enzymatic formation of reactive oxygen and nitroxide species, thus releasing ions, molecules, or macromolecule debris. This is a major and unspecific biodegradation mechanism that often starts at the surface of particles. In parallel, degradation can be caused by abiotic chemical processes, especially hydrolysis and oxidation, in various tissues. The particular case of hydrolytically degradable polymers of the aliphatic polyester type, such as glycolic and lactic acid homopolymers and copolymers that are currently used as nanoobjects for drug delivery, has been extensively discussed, including changes in composition and morphologies [221], [222], [223]. An entrapped drug can play an important role in particle degradation, depending on its hydrophilicity or hydrophobicity. Targeting proteins on the surface will be degraded by proteases and released small molecules, such as drugs, may be subject to classic biotransformation reactions.

Intact ENM will rarely be subject to the classic biotransformation pathways that are known for small molecules, such as phase I (oxidation) and phase II (conjugation) reactions. This is because, first, ENM will not easily enter cells of the drug-metabolizing organs (predominantly liver cells), and second, their constituents are usually not typical substrates for the common biotransformation enzymes. Therefore, various other mechanisms must prevail.

Similarly, inorganic nanoparticles can also be degraded, as elegantly summarised in a review by Feliu et al. The authors reviewed in vivo degeneration and the fate of a variety of inorganic nanoparticles, and concluded that NP in vivo should no longer be considered as homogeneous entities, but rather as inorganic/organic/biological nano-hybrids with complex and intricately linked distribution and degradation pathways [224]. For insoluble inorganic particles (e.g. gold or magnetic particles) uptake and storage in phagocytosing cells may be followed by slow dissolution due to low pH in lysosomes or other solubility effects. Residue-free dissolution in the extracellular space is possible for soluble inorganic ENM (e.g. mesoporous silica, ZnO, CuO). Some carbohydrate and peptide polymers can be degraded in the extracellular space by amylases, peptidases, and hydrolases. Hydrolytically degradable polymers include poly(lactic acid)s as well as many other aliphatic polyesters, poly(anhydride)s, and some polyphosphazenes. Some products can enter the intermediary metabolism. ENM containing a mixture of constituents may be disassembled or biodegraded to smaller entities, which may be stored or further degraded by the above-mentioned mechanisms.

4.3.5 Elimination

If the degradation of ENM is not complete, then the remaining particles must be eliminated to avoid accumulation. The most common route of elimination/excretion is filtration in the kidneys. In the healthy kidney, filtration of nanoparticles can be nearly complete when the particle diameter is below 2 nm and is still efficient at a size of 5 nm, but tends to stop at particle sizes above 6 to 8 nm. Surface charge and interactions with biomolecules (corona formation and association) may attenuate renal filtration. Pulmonary particles can be excreted by ciliated epithelium of the airways [225]. Biliary excretion, a major route for small drug molecules, has been observed, but it is unclear if this can be a general route for particle elimination. Zhang et al. published the rather alarming finding that small (8 nm) TiO₂ nanoparticles were transferred from dams to pups through breastfeeding during lactation, likely through the disrupted blood-milk barrier [226]. However, this was an indirect conclusion and has yet to be unequivocally confirmed.