European Observatory for Nanomaterials

Background 

Engineered nanomaterials (ENMs) have the ability to revolutionize many industries and are already used in e.g., cosmetics, electronics, batteries, fuels, fuel cells, solar cells, automobile components, etc. In medicine, they are used for the detection of biological molecules, imaging of diseased tissues and innovative therapeutics as well as drug delivery systems. Safety and targeting ability are important factors in developing sustainable nanomaterials. Designing safe and efficient ENMs for different applications requires an understanding of how ENMs behave in organisms’ bodies and humans and how they interact with the surrounding cells and tissues.

Analytical challenges 

Metal-bearing ENMs (M-ENMs) of different shapes and sizes are taken up by organisms through, e.g., gills/lungs and stomach, or via attachment to the outer membranes. When entering the biological medium, e.g., in the blood, M-ENM are prone to dissolution and agglomeration. It is also possible that the M-ENMs acquire an evolving layer of proteins and other biomolecules.

Tracking and analysing ENMs in cells and tissues is difficult. This difficulty arises because there is a limitation in analytics for characterization and quantification of ENMs in bio- and physiological matrices due to their presence at trace levels, and because of the presence of different background materials which interfere with the measurement of ENMs. Thus, researchers must apply an approach that is not only capable of measuring ENMs at trace levels but also of measuring the form of the particles at a specific moment in time. There are no standard and straightforward methods and analytical techniques available that are able to comprehensively characterize and quantify ENMs in situ in (micro)organisms. 

Developing a protocol 

We developed the first protocol, which combines different methods and fit-for-purpose techniques, for characterizing and quantifying metal-bearing ENMs (M-ENMs) in biological samples including cells, microorganisms, and higher organisms. The protocol is applicable to measurement of M-ENMs in tissues from both laboratory and field experiments. We have used this protocol previously to understand the bioaccumulation and biodistribution of M-ENMs in organisms (e.g., fish), which are important factors for understanding the toxicity of ENMs. The method was also used to quantify the cellular uptake of M-ENMs on a cell-by-cell basis in microorganisms (algae). The workflow is optimized to cover cells and microorganisms such as bacteria as well as tissues and organs of different organisms including plants.

Application of the protocol 

• > The protocol covers the detection, characterization, and quantification of M-ENMs in cells and (micro)organisms for use with in-vitro and in-vivo tests. The availability of this protocol will open new research horizons for further studies in e.g., the fields of environmental science, risk assessment, and nanomedicine. For example, the protocol can be applied for the following purposes:  
• > By toxicologists to understand the dose-response behavior following exposure to M-ENMs and to determine the mode of action in terms of particle effects or impacts related to release of ions following M-ENM dissolution;
• > By ecotoxicologists and environmental scientists to quantify the uptake, biodistribution, bioaccumulation, biotransformation and trophic transfer of M-ENMs;
• > By medical researchers to understand the ADME (absorption, distribution, metabolism, and excretion) and cellular uptake of M-ENMs and to perform bioimaging;
• > By the industry sector to adopt safe by design strategies for the production of M-ENM by considering ADME and safety;
• > By regulatory authorities to propose in their guidelines the use of the method as the way to detect, characterize, and quantify M-ENMs, and based on the potential risks identified develop restrictions and/or propose risk management measures.

Example of application 

For example, we have applied the protocol to understand whether silver (Ag) ENMs transform and transport into and through the human blood-brain barrier (BBB) cell layer. The optical density images show the shape of the cells. The results suggest the silver ENMs are transformed following uptake into the human BBB cells.

Recommendation and perspective 

Unlike previous methods, the protocol uses no fluorescent dyes or radiolabels to trace M-ENMs in biota and enables analysis of most M-ENMs at cellular, tissue and organism levels. Method development and validation should continue in order to optimize the workflows for different M-ENMs and also for carbon-based ENMs such as nanoplastics and carbon nanotubes and to facilitate direct analysis of these materials in organisms collected from the field. The protocols can be applied by a wide variety of users e.g., to correlate toxicity with a specific particle form, or to understand the ADME (Absorption, Distribution, Metabolism, and Excretion) of M-ENMs, which will open a new horizon for medical applications and risk assessment of ENMs as well as adaptation of safe by-design strategies for ENMs. In conclusion, this protocol can provide comprehensive information about the dynamic behavior and biological fate of M-ENMs in laboratory or field-based studies.

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