Commentary: Revisiting nanoparticle-assay interference: There's plenty of room at the bottom for misinterpretation

https://doi.org/10.1016/j.cbpb.2021.110601Get rights and content

Highlights

  • Nanotoxicology research has expanded with the proliferation of nanotechnology.

  • Nanomaterials can interfere with biochemical assays through a variety of mechanisms.

  • Assay interference can lead to erroneous conclusions regarding nanomaterial safety.

  • Recommendations for validating biochemical assays in nanotoxicology studies are presented.

Abstract

Engineered nanomaterials (ENMs) are a diverse class of materials whose distinct properties make them desirable in a multitude of applications. The proliferation of nanotoxicology research has improved our understanding of ENM toxicity, but an under appreciation for their potential to interfere with biochemical assays has hampered progress in the field. The physicochemical properties of ENMs can promote their interaction with membranes or biomacromolecules (e.g. proteins, genomic material). This can influence the activity of enzymes used as biomarkers or as reagents in biochemical assay protocols, bind indicator dyes in cytotoxicity tests, and/or interfere with the cellular mechanisms controlling the uptake of such dyes. The spectral characteristics of some ENMs can cause interference with common assay chromophores, fluorophores, and radioisotope scintillation cocktails. Finally, the inherent chemical reactivity of some ENMs can short circuit assay mechanisms by directly oxidizing or reducing indicator dyes. These processes affect data quality and may lead to significant misinterpretations regarding ENM safety. We provide an overview of some ENM properties that facilitate assay interference, examples of interference and the erroneous conclusions that may result from it, and a number of general and specific recommendations for validating cellular and biochemical assay protocols in nanotoxicology studies.

Introduction

Engineered nanomaterials (ENMs) are a diverse class of materials sharing at least one dimension between 1 and 100 nm (Stone et al., 2010). Their small size and large surface area confer physicochemical properties distinct from their bulk chemical counterparts, which are desirable in consumer, industrial, agricultural, and clinical products (Chan et al., 2016; Iqbal et al., 2016; Salama et al., 2021). The nanotechnology revolution, arguably inspired by Richard Feynman's seminal lecture “There's plenty of room at the bottom” (Feynman, 1960), prompted concerns about unforeseen health and environmental risks; consequently, dedicated toxicological research on ENMs, coined nanotoxicology (Donaldson, 2004), intensified exponentially over the past 15 years (Fig. 1). The proliferation of nanotoxicology research has alleviated some concern but has failed to keep pace with the growth of nanotechnology and its applications. The field has also suffered from growing pains, most related to the interdisciplinary nature of the topic. Due to their particulate nature, the adsorption, distribution, metabolism, and excretion (ADME) of ENMs is complex and difficult to predict. Exposure dosimetry is equally complicated; surface area to volume ratio increases with decreasing particle size and surface chemistry largely dictates ENM fate and bioactivity, meaning size, surface character, and reactivity are inextricably linked. Standard mass-based dose metrics alone thus lack sufficient detail to accurately quantify exposure without additional information on ENM characteristics (e.g. size, aspect ratio, etc.). ENMs can also form colloids in aqueous media (e.g. surface waters, biological fluids) and their colloidal stability is a critical determinant of bioavailability and fate (Handy et al., 2008; Klaine et al., 2008; Lead et al., 2018). Colloidal stability can be challenging to assess in an environmentally-meaningful way and is best quantified using multiple approaches (Domingos et al., 2009). Even the rate at which ENMs are introduced to a biological or environmental system can have profound effects on the fate and distribution of particles and their potential for bioactivity (Baker et al., 2016; Chen et al., 2020).

Pleas for the reporting of detailed materials characterization data in nanotoxicology studies have come repeatedly (Bouwmeester et al., 2011; Faria et al., 2018; Powers et al., 2006) as it is important for understanding and contextualizing toxicological endpoints. Unlike many contaminants, the physicochemical characterization of ENMs is complicated by its sensitivity to sample formulation (size, shape, and surface functionalization), preparation (purification, dissolution), and environment (colloidal stability, opsonization). An under appreciation for the importance of these parameters has likely impacted data quality in a large number of studies.

The diverse and sometimes unique physicochemical properties of ENMs can also influence data quality by different means. Early in the development of ‘nanotoxicology’, we recognized the potential for ENMs to interfere with biochemical assays used in toxicological assessments (MacCormack et al., 2012; Ong et al., 2014). Although interference has been noted many times (Breznan et al., 2015; Ciofani et al., 2010; Coccini et al., 2010; Ferraro et al., 2016; Han et al., 2011; Pichla et al., 2020; Semisch and Hartwig, 2014; Tournebize et al., 2013), the majority of new studies still fail to address it. It can occur through a variety of chemical and biochemical mechanisms (Fig. 2), some of which we cover below. The resulting data artefacts range from minor under or overestimates of toxicity, to entirely erroneous data and significant misinterpretations regarding ENM safety (Ong et al., 2014). Our intention herein is not to discredit studies lacking validation data, but to encourage the critical assessment of existing data and the incorporation of validation steps in new experimental designs.

Section snippets

Overview of relevant ENM properties

As particle size decreases, a greater proportion of molecules are on the surface and available to perform chemistry, so surface reactivity increases. Optical properties of ENMs are highly dependent upon size, shape, and environment, as discussed below. For many formulations, the particle core is ‘functionalized’ with a layer of ligands to add new chemical or biological functionalities or to confer colloidal stability by providing steric and electrostatic repulsion between approaching particles.

Direct physical interactions with biomacromolecules or superstructures

Weak electrostatic forces facilitate the adsorption of biomacromolecules to ENM surfaces, changing their structure in the process (Fei and Perrett, 2009; Satzer et al., 2016; Tsai et al., 2011; Xu et al., 2012). Colloidal ENMs and biomacromolecules often exhibit a surface (zeta) potential, which is highly influential in this process. The opsonization of ENMs leads to the development of a “biomolecular corona”, the properties of which have profound implications for ADME in environmental and

Multiparametric approaches to nanotoxicity assessment

Multiparametric assessment of cellular and tissue phenotypes following ENM exposure uses a collection of assays to generate a more comprehensive picture of ENM toxicity. For example, we have shown that ENMs presenting as non-toxic in the MTT viability assay demonstrate a > 50% reduction in capacity for clonogenic growth, 20-fold increase in intracellular reactive oxygen species, and altered cellular morphology (unpublished data). Conducting just the MTT assay in this case could lead to the

Regulatory testing considerations

Regulatory agencies generally apply existing risk assessment paradigms to evaluate the safety of ENM-enabled products. As the field of nanotoxicology has evolved to more effectively evaluate ENM hazards, regulators have begun incorporating specific testing considerations into risk assessment. They have recognized that unique considerations are required for assessing ENM hazards, including their potential to interfere with biochemical assays and toxicity tests (NIST, 2015; OECD, 2020). However,

Perspectives and recommendations

Nanotoxicology is a maturing field and many of the initial barriers to progress have been overcome, at least to some extent. Insufficient materials characterization remains a significant challenge, limiting the utility of many studies (Bouwmeester et al., 2011; Faria et al., 2018). At environmentally-relevant concentrations, the risk of ENM-related data artefacts is likely low, but many studies, including our own (Callaghan et al., 2018; Ong et al., 2014; Rundle et al., 2016; Schultz et al.,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

TJM, M-VM, and JLR are supported by Natural Sciences and Engineering Research Council of Canada Discovery Grants. CAD is supported by the National Science Foundation, Division of Chemical, Bioengineering, Transport, and Systems (CBET) programs. We thank Drs. Jonathan Veinot and Greg Goss for contributing unpublished data and for their support of earlier efforts on this topic. JDE and KJO thank Dr. Jo Anne Shatkin and Vireo Advisors, LLC for support of this work.

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