Dr. Eleonore Frohlich from LKH-Univ.Klinikum Graz looks at the latest issues surrounding nanomaterials and toxicology.
Naanotechnology is regarded as a key technology of the 21st century. The development of nanostructured materials enabled the most recent developments in technical, industrial, pharmaceutical and medical sectors. On the other hand, these materials may represent certain hazards for all living creatures including humans, as the ‘dark’ side of their useful characteristics. Carbon nanotubes are famous for their mechanical strength and stability but they are also almost not biodegradable and may persist and accumulate in cells, organs and the environment with yet unknown effects. The high reactive surface of nanoparticles provides higher catalytic reactivity but causes also a higher biological reactivity and cellular oxidative stress. The penetration of nanoparticles through barriers in the body improves drug delivery but the uncontrolled penetration of nanoparticles into the body may pose problems.
Researchers got aware of potential adverse effects of nanomaterials by studies on environmental particles. Upon exposure to high concentrations of ultrafine particles in the air, the incidence of inflammation and allergy in the lung and of cardiovascular problems was increased. These findings were the initiator of studies on nanotoxicity. After several years of intense research, much insight has been gained into the multifaceted action of nanomaterials but test systems and toxicological testing of nanomaterials is still not standardized. Scientists working in this field, therefore, avoid global statements on the toxicity of nanoparticles but many yellow press newspaper articles and advice booklets, in general ignoring the complex nature of nanotoxicity, discourage people from using nanoparticle-containing products and proclaim a general health risk by nanoparticle exposure. Such a categorical rejection of nanoparticle-containing products is understandable because guidelines for use and testing of nanomaterials are lacking and the declaration of nanomaterials in consumer articles and food is not regulated by law but appears not justified from a scientific point of view.
Biological definition of nanomaterials
Nanomaterials are defined by their size and comprise nanostructured materials and nanoobjects. Nanoscale is the 1-100 nm size range, in which unique phenomena enable novel applications. Nanostructured materials are materials containing nano-sized pores, nanocrystalline materials and complex fluids. Nanoobjects possess one (nanoplates), two (nanotubes) or three dimensions (nanoparticles) on the nanoscale. In biomedicine the term nanoparticle describes particles with a diameter of lesser than 1000 nm, instead of 100 nm, because nanoparticles in biological applications usually measure between 200-300 nm. Others refer to particles with a size between 100-1000 nm as mesoparticles or submicron particles. In the biological context nanoobjects range from smaller than antibodies to greater than virus (Fig. 1).
‘The nanoparticle’
The definition based on the size suggests that there is something like ‘the nanoparticle’, and that all particles of a certain size react similarly. This, however, is not the case and for this reason the existing definition of nanoparticles may not be the best one. Nanoparticles are a very heterogeneous group and consist of different materials; they have different sizes, surface properties and shapes, and all of these parameters influence their biological action. Common representatives of nanoobjects include non-biodegradable nanoparticles like carbon nanotubes, fullerenes, nanoparticles made from gold, silver, iron oxide, zinc oxide, titanium dioxide, polystyrene and biodegradable nanoparticles such as liposomes, dendrimers, polymeric nanoparticles and nanoparticles made from poly(lactic-co-glycolic acid) (Fig. 2). The predominant nanoparticles are often linked to the application. Cosmetic products mainly contain fullerenes, zinc oxide and titanium dioxide nanoparticles. In food, especially silicon dioxide, titanium dioxide and biodegradable nanoparticles can be found. Medical nanoparticles are mainly iron oxide, gold and biodegradable nanoparticles.
Fig. 2: Transmission and scanning electron microscopic images of nanoparticles differing in material and shape.
Risk: nanomaterial exposure
For risk assessment exposure and hazard of nanomaterials have to be known. Detection of nanoparticles in heterogeneous matrices is difficult. The typical dimensions of nanoparticles are below the diffraction limit of visible light and cannot be visualized by optical microscopy. In low concentration, however, single chromophore detection is possible and in Scanning Near Field Optical Microscopy sub-wavelength feature can be analysed. Chemical analysis of individual nanoparticles in a dilute environment was for a long time impossible due to their low mass, and only recently have methods become available for this purpose.
Exposure is further influenced by the penetration through the barriers of the human body. Out of these barriers (skin, lung, gastrointestinal tract, urogenital tract, eye), the skin is a very tight barrier but the lung a very leaky one. The passage is particle size-dependent and linked to a variety of surface properties. As a rule, small not charged hydrophobic nanoparticles in general have a higher chance to pass epithelia than large charged hydrophilic ones. The presence of mucus and other liquid layers covering the surface of most epithelial barriers, such as orogastrointestinal tract, urogenital tract etc., however, favors the penetration of not so small, charged particles. Aggregation of particles and adsorption of macromolecules and contaminants from the biological substrates additionally influence nanoparticle penetration. The absorption into the body is further influenced by the matrix in which the nanoparticles is applied. For nanoparticles in pharmaceutical applications such as creams interaction with macromolecules and aggregation is higher than for air-borne particles. Hence, nanomaterials in aerosols are more dangerous than those in other dermal applications or in food. The extent of penetration through the barriers of the human body is of eminent importance for the interpretation of cytotoxicity data and for the estimation of potential risks.
Risk: hazard of nanomaterials
Hazard is usually indicated as toxicity and the notion that “Dosis sola facit venenum” [The dose alone makes a poison] is known since Paracelsus (1493-1541). Although toxicological targets for nanoparticles, in essence, are similar to those of conventional chemicals and drugs, the assessment of nanoparticle toxicity is more complex. Toxicity of nanoparticles is influenced by size/ formation of aggregates, surface properties (charge, hydrophobicity, structure), shape, residues from the production (different content of metals and other contaminants).
Pronounced differences in the toxicity of identical particles between different cell types further complicate the assessment. Part of these problems is caused to the fact that single nanoparticles in suspension do not sediment but aggregates do. Cells cultured at the bottom of a culture well are therefore, exposed predominantly to aggregates not to single particles, whereas non-adherent cells are in contact mainly with single nanoparticles. All types of cells rapidly ingest particles smaller than 100 nm (Fig. 3A) but particle effects at the same mass per volume are markedly different. A difference in the size by a factor of two may cause an about 10-fold difference in cell viability (Fig. 3B), suggesting a much higher toxicity of smaller particles. There is an ongoing discussion if indication of mass/volume, like for conventional substances, or other measures such as mass/area of the exposed cells (e.g. µg/cm2), particle number (e.g. 1010 particles), or particle surface area (e.g. m2) is more appropriate to compare toxicity between different particles. For inhaled air-borne particles toxicity in the respiratory tract appears to be correlated to surface area. The situation is more complicated for nanoparticle in oral or parenteral applications because aggregation and coating with macromolecules render the calculation of surface area problematic. For these applications the toxicity of a nanoparticle cannot be deduced from the testing of a smaller or larger particle and every particle has to be evaluated separately. As agglomeration and binding of macromolecules to particles depends on both particle parameters and biological fluid, not only every particle has to be tested separately but also in different fluids to get a complete picture of the toxicity of the respective particle.
Fig. 3 A: Uptake of nanoparticles (green) into a cell, where mitochondria (red) and nucleus (blue) are stained with fluorescent dyes. B: Assessment of viability in cultured cells after exposure to different concentrations of 20 nm (blue diamonds) and 40 nm (pink squares) particles.
Standardization
As mentioned above the toxicity of nanoparticles is influenced by a variety of particle parameters, which in addition are not constant but influenced by the composition of the suspension fluids. Stability of nanoparticle suspensions and interference with the assay systems require the inclusion of additional controls. If these problems are not taken into account, misinterpretation of nanoparticle effects occurs leading to contradictory data for identical particles. For a meaningful interpretation of toxicity data and comparison between different particles standardization of the testing is required. Working groups of various organizations aim to define standards and develop Standard Operation Procedures for the testing of nanomaterials. Regulatory testing of nanomaterials will include a physicochemical characterization of the particles in the media used for the testing, the use of several assays and additional controls to identify potential interference with the detection system and realistic exposure systems. Relevant in-vitro systems are needed for an in-depth evaluation of particle effects to reduce the extent of animal exposures to a reasonable amount.
Interdisciplinary biological testing
Targets for nanotoxicity are all components of the cell including their genetic material, blood and immune system. In contrast to drugs and chemicals chronic effects of nanoparticle exposure have a higher importance because nanoparticles may accumulate in cells, organs and environment and, thereby, low doses of nanoparticles may persist and accumulate over months and years in the human body.
The study of nanotoxicity is highly interdisciplinary because the assessment of toxic effects and the development of nontoxic nanoparticle afford the interaction of chemists, physicists, biologists, pharmacists and medical doctors. To assess the biological actions of nanoparticles centers like for instance the Namur Nanosafety Centre in Belgium, the Nanotechnology Characterization Laboratory at the National Cancer Institute at Frederick in the United States of America and the Centre for Interdisciplinary Research in Great Britain were established. Another approach to improve testing of nanomaterials was taken in Austria. The BioNanoNet Forschungsgesellschaft mbH (www.bionanonet.at), as a network company, has initiated the build up of a national contact point for nanotoxicology, the European Centre for Nanotoxicology (“EURO-NanoTox”; www.euro-nanotox.eu ). This virtual center unites all Austrian experts in the field of nanotoxicology and helps to develop standardized methods. EURO-NanoTox initiates research projects together with its members and it links Austrian activities in the field of nanotoxicology to European activities.
In order to ensure the sustainable economic success of nanotechnology it is essential that the potential dangers of nanomaterials for human health can be assessed in a transparent manner. EURO-NanoTox provides a panel of standardized in vitro and in vivo toxicity tests that enables researchers in both academia and industry to gain an early insight into the potential toxicity of the materials being developed. Early warnings of potential toxicity will facilitate early product optimization (with respect to toxicity) and will thus help to avoid expensive late product developmental failures.
In addition to that a new journal in the biomedical field was founded that fills the gap between material science orientated and medical journals. The main aim of EURO-NanoTox-LETTERS (www.EURO-NanoTox-LETTERS.com) is to increase the knowledge in the field of nanotoxicology and to help to pave the way from the present case-to-case to a holistic approach. This journal should help to ensure a sustainable development of the entire field of nanotechnology. The journal will publish in vitro, ex vivo and in vivo studies elucidating the behavior of nanomaterials in physiological environment. It will describe absorption, distribution, metabolism and elimination of nanomaterials in order to find out to which extent toxicity testing guidelines for drug products can be used for the toxicological assessment of these materials.
Nanotechnology is not only one of the key technologies of the 21st century the biological evaluation of nanomaterials appears also to be one of the greatest challenges of this century.
