European Observatory for Nanomaterials

The Graphene Flagship (GF), the largest ever research programme in the EU, has been running (evolving) for 10 years (2013-2023). Overall, more than 5000 scientific publications have been published. However, as the GF project as we know it comes to an end, the voyage continues in the form of several separately funded research projects under the umbrella of a so-called coordination and support action (CSA) (see: https://graphene-flagship.eu). One of the key goals of the GF has been to create a commercial ecosystem for graphene and other 2D materials in the EU, and we believe that safety assessment is integral to this effort. Here we present some of the lessons learned during the course of the past decade in the work package on Health and Environment (WP4). This is of course merely a snapshot of the results achieved.

 

 

 

Possible interactions of GBMs with human cells and organs and environmental species. From: Bianco A, Prato M. Safety concerns on graphene and 2D materials: a Flagship perspective. 2D Materials. 2015;2:030201.

 

 

There are a number of general lessons to be gleaned from this 10-year journey. The first lesson is that nanosafety is multidisciplinary. In other words, experts in chemistry and materials science, environmental science, toxicology, and occupational exposure assessment must work side by side. Indeed, safety assessment of any new material starts with a detailed understanding of the material properties [1]. This is why WP4 partners have been working hard to prepare graphene-based materials (GBMs) with well controlled properties (see, for example, reference [2]). The second lesson is that communication is key. Most projects do not run for more than a few years, barely allowing the project participants to get to know each other, but in the GF, members of WP4 could work side by side for several years, eventually learning to speak the same language. To foster communication, we have organized work package meetings every 6 months throughout the project (virtual meetings during the pandemic) to complement the annual general assembly meeting and the annual GF conference. The third lesson is that funding is fundamental because research takes time, not least if we are to develop and validate new test methods with which to assess (new) materials.

There are of course some more specific lessons in relation to the safety assessment of GBMs. WP4 addressed the hazard potential of GBMs and other 2D materials using a wide range of model systems and assays spanning both human health and the environment. As such, this represents the single largest effort to understand the health and environmental impact of GBMs. One take home message is that not all carbon-based nanomaterials are alike. Hence, studies in mice have shown that graphene oxide (GO) sheets with lateral dimensions similar to the length of multi-walled carbon nanotubes (MWCNTs) were less pathogenic than the MWCNTs [3]. Moreover, GO and other GBMs were found to be susceptible to degradation in vitro and in vivo (in mice) [4,5] and in the environment [6]. Thus, GBMs are not necessarily biopersistent like asbestos fibers. This has important implications for the risk assessment of GBMs.

Additionally, fundamental research on the biological interactions of 2D materials is needed if we are to understand the toxicological impact of the materials. One cannot a priori assume that all nanomaterials will interact in a similar manner with living systems, and it is important to disentangle the physicochemical properties that underlie the biological effects of 2D materials. Research in the GF has disclosed the importance of interactions between GBMs and proteins for the subsequent cellular impact of these materials [7]. Studies performed in collaboration with an associated project funded under the transnational FLAG-ERA call (see: https://www.flagera.eu) have shown that surface functionalization of GO governs the interactions with immune cells [8]. Furthermore, the lateral dimensions of GO seem to dictate the immunological impact in vivo as shown in a mouse model of intraperitoneal injection [3]. Moreover, recent studies have revealed that GBMs may affect the immune system indirectly via their effects on the microbiome, opening up a new frontier in the study of these materials [9].

However, while basic science is important, there is also an urgent need for harmonized and validated test protocols to support regulation. This work was initiated in WP4 and in the SafeGraph project (a so-called spearhead project within the GF which deals with regulatory issues of selected products), and these efforts should continue beyond the GF to enable the safe and sustainable implementation and commercialization of GBMs and other advanced materials.

Finally, it is important to consider the life cycle of 2D material-enabled products, as the risk of exposure to GBMs as well as other 2D materials may vary along the life cycle from production to use to disposal. Recent work in the GF has shown that reduced graphene oxide (rGO) displayed modest effects both in vitro and in vivo (in mice) whereas the respirable fraction of degraded composites reinforced with a few wt.% of rGO had a negligible impact [10]. WP4 partners have just completed a similar study on thermoplastic polyurethane reinforced with hexagonal boron nitride, another important 2D material, and the results will be published soon. 

 

References

1. 1. Wick P, et al. Classification framework for graphene-based materials. Angew Chem Int Ed Engl. 2014;53(30):7714-7718. 
2. 2. González-Domínguez JM, et al. Production of ready-to-use few-layer graphene in aqueous suspensions. Nat Protoc. 2018;13(3):495-506.
3. 3. Rodrigues AF, et al. Immunological impact of graphene oxide sheets in the abdominal cavity is governed by surface reactivity. Arch Toxicol. 2018;92(11):3359-3379. 
4. 4. Kurapati R, et al. Degradation of single-layer and few-layer graphene by neutrophil myeloperoxidase. Angew Chem Int Ed Engl. 2018;57(36):11722-11727. 
5. 5. Newman L, et al. Splenic capture and in vivo intracellular biodegradation of biological-grade graphene oxide sheets. ACS Nano. 2020;14(8):10168-10186.
6. 6. Candotto Carniel F, et al. Graphene environmental biodegradation: wood degrading and saprotrophic fungi oxidize few-layer graphene. J Hazard Mater. 2021;414:125553.
7. 7. Castagnola V, et al. Biological recognition of graphene nanoflakes. Nat Commun. 2018;9(1):1577.
8. 8. Orecchioni M, et al. Single-cell mass cytometry and transcriptome profiling reveal the impact of graphene on human immune cells. Nat Commun. 2017;8(1):1109. 
9. 9. Peng G, et al. Graphene oxide elicits microbiome-dependent type 2 immune responses via the aryl hydrocarbon receptor. Nat Nanotechnol. 2023;18(1):42-48. 
10. 10. Chortarea S, et al. Hazard assessment of abraded thermoplastic composites reinforced with reduced graphene oxide. J Hazard Mater. 2022;435:129053. 

 

Biographies of the authors

Bengt Fadeel, M.D., Ph.D., is a Professor of Medical Inflammation Research at the Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. He is a fellow of the Academy of Toxicological Sciences since 2012. He is chairman of the national nanosafety platform, SweNanoSafe, and he serves as an editor of several academic journals including Current Opinion in Toxicology, Frontiers in Toxicology, and Toxicological Sciences. He is a member of the Work Package on Health and Environment in the Graphene Flagship (2013-2023).

 

 

Alberto Bianco, Ph.D., is a Research Director at the CNRS in Strasbourg, France. He received his Ph.D. from the University of Padova (Italy). He has been a visiting scientist at the University of Lausanne (Switzerland), University of Tübingen (Germany), University of Padova, and Kyoto University (Japan). He is a fellow of the European Academy of Sciences and Academia Europaea. He was awarded the CNRS Silver Medal in 2019. He is an editor of Carbon. He is Deputy Leader of the Work Package on Health and Environment in the Graphene Flagship.

 

 

Maurizio Prato, Ph.D. (University of Padova), is a Professor of Chemistry at the University of Trieste, Italy, and Ikerbasque Professor at CIC biomaGUNE in San Sebastián, Spain. He is the recipient of two ERC Advanced Research Grants (in 2008 and 2020). He is a member of Accademia Nazionale dei Lincei since 2010. His research focuses on the synthesis of functional materials and their safety profile for applications in medicine and energy, in particular in spinal cord repair, splitting of water, and reduction of carbon dioxide into useful chemicals. He is Leader of the Work Package on Health and Environment in the Graphene Flagship.

 

 

 

 

 

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