Improving nanotoxicological studies with focus on in vitro studies – Progress by the FNN project

Nanotechnology is one of the fastest growing scientific fields of both research and industry.^1,2 Its activity can be measured in terms of increase in research funding^2,3, expected economic impact^2,4 and emerging jobs⁴. The attractiveness of nanomaterials is mostly due to the enhancement of key properties of traditional materials by reducing their size to the nano-scale (about 1 – 100 nanometres) which results in new surface-related phenomena that can be exploited in many ways.  For example, nanomaterials have large surface area to volume ratios that introduces greater surface reactivity. In addition, the small size allows nanomaterials to be active at biological length scales. Along with the possibility of new products and better medicines, however, come concerns about potential hazards.  Understanding and reducing risks of nanoscale materials is of the upmost importance in order to ensuring their acceptance and use. The study of these risks is done through biological and environmental toxicology studies. It is important to understand that such testing is done to demonstrate that a material does not present a risk and can be safely used in commerce.

Defining the terms of nano-toxicological studies is not a trivial task. Nanomaterials have great variety in chemical composition, size, shape and form, surface conditions and coatings, and dispersion quality that makes a comprehensive study of any specific nanomaterial very challenging.  In generating a useful set of test results, one must have a clear definition of the study parameters and material quality as well as a systematic test approach.

One of the central goals of FutureNanoNeeds is to find and identify nanomaterial parameters that can potentially present health or environmental risks and  therefore require additional attention as part of a rigorous risk characterisation and assessment procedure.  To do this systematically, the FutureNanoNeeds project structure is made up of several work packages, divided into tasks and deliverables, that comprise a testing framework usable in testing and characterising future nanomaterials.  From the scientific perspective, this process pinpoints the specific criteria to be considered and reported in biological studies to enhance their usefulness for risk assessment and regulators.  As a result of this project, and its forerunner the QualityNano research infrastructure, several aspects of nanomaterials have been identified as having a profound effect on their interactions in biological systems. One of the most important variables assessing potential risks is how the nanomaterials are dispersed both in testing and in use. These materials are usually dispersed in a variety of fluids whose composition and concentrations vary widely. Below we summarise some of our finding with respect to the properties of nanomaterials in various dispersion conditions.

• Nanomaterial composition. Nanomaterials of different composition behave differently under similar dispersion conditions. For example, silver nanoparticles have been observed to dissolve in biological media⁵ while polystyrene nanoparticles do not⁶. Even the behaviour of the same nanomaterials can differ depending on surface coatings from the dispersion media, resulting in new properties compared to the bare nanoparticle. Changes of physico-chemical properties include mobility (as measured by the zeta potential), nanomaterial size and stability⁷, biological interactions,^8,9 and in some cases even interactions of bound entities on their surface¹⁰.
• Dispersion size and width. The effect of nanomaterial size on its processing in live systems (e.g. cells) is still under investigation; however it has been well documented that the size and size distribution (polydispersity) of the dispersion is a determining factor of the interaction.¹¹
• Stability of dispersion. As a consequence of the previous point, colloidal stability is also of great importance. By colloidal stability, we mean the original nanoparticle plus whatever coatings have been acquired through exposure to the dispersion media and its contents. If the nanomaterial is unstable in the selected media, the colloids presenting to a cell are of an unpredictable size. Additionally, instability could lead to agglomeration and settling of particles (often leading to suffocation and physical damage of cells) and thus an uncontrollable change in concentration, concentration gradients, etc.  Another form of instability is the dissolution of nanoparticles into ions or molecules that behave differently to the bulk material¹². Dissolution leads not only to confusing results but also to undesirable properties associated with the ions or molecules rather than the nanomaterial itself¹³.
• Nanomaterial shape. Shape has been observed to have an important role in the interactions with biological systems, though much study still needs to be done to determine the fundamental principles.¹⁴ Among the issues here are that spikes or needles in the shape can puncture cells, while edges and sharp angles can lead to defects in the structure, acting as sites for enhanced dissolution or binding of proteins or other biomolecules.
• Surface chemistry. Some materials, e.g. cationic polystyrene, are well known to reduce cell viability while others e.g. functionalized silica or carboxylic polystrene have more subtle effects on cell processes such as cell cycle arrest.^10,15 Thus, surface functionalization is one of the determining factors in nanomaterial – cell interactions, including being a key driver of the composition of the acquired protein and biomolecular corona.
• Sample contamination. Nanoparticle contamination, intentional or not, may have a large impact on product quality and toxicological properties. Intentional contaminants are molecules added either to stabilize the sample, control shape, or for other reasons. The most common example of unintentional contamination is from lipopolysaccharides that provide nanoparticles with (unwanted) immune activation properties. Both types of impurities have been shown to affect particle bioidentity¹⁶.
• Other concerns. In some cases, there are special considerations related to the specific system under study. For example, the protein to particle concentration, type of serum used, preincubation of particles to coat them with biomolecules, spin or sonication steps for isolation of the particles from the media, and other factors also need to be considered. It has been shown that there are a wide variety of experimental details that can have a large impact on the final results.  For example, the behaviour of a nanomaterials and its stability and protein corona are likely to be different in serum-free media than in the presence of 10% or higher concentration of serum¹⁷. As a result, the same (ostensibly) nanomaterial would have very different interactions and toxicities, that are not understandable in the absence of appropriate characterisation and sufficient description of the experimental set-up.

Summary: Addressing these points is critical to make test results more suitable for use in the broad context of risk assessment and especially for regulatory purposes.  Adopting a standardised system of parameters to be considered and addressed in each study would greatly enhance repeatability and data quality. Further it would help develop a framework allowing studies to be placed in the proper context.

References

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