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Graphene and other 2D materials, such as molybdenum disulfide, have been increasingly used in electronics, composites, and biomedicine. In particular, MoS2 and graphene hybrids have attracted a great interest for applications in the biomedical research, therefore stimulating a pertinent investigation on their safety in immune cells like macrophages, which commonly engulf these materials. In this study, M1 and M2 macrophage viability and activation are mainly found to be unaffected by few‐layer graphene (FLG) and MoS2 at doses up to 50 µg mL−1. The uptake of both materials is confirmed by transmission electron microscopy, inductively coupled plasma mass spectrometry, and inductively coupled plasma atomic emission spectroscopy. Notably, both 2D materials increase the secretion of inflammatory cytokines in M1 macrophages. At the highest dose, FLG decreases CD206 expression while MoS2 decreases CD80 expression. CathB and CathL gene expressions are dose‐dependently increased by both materials. Despite a minimal impact on the autophagic pathway, FLG is found to increase the expression of Atg5 and autophagic flux, as observed by Western blotting of LC3‐II, in M1 macrophages. Overall, FLG and MoS2 are of little toxicity in human macrophages even though they are found to trigger cell stress and inflammatory responses.

Carbon-based materials (CBMs), such as graphene, nanodiamonds, carbon fibers, and carbon dots, have attracted a great deal scientific attention due to their potential as biomedical tools. Following exposure, particularly intravenous injection, these nanomaterials can be recognized by immune cells. Such interactions could be modulated by the different physicochemical properties of the materials (e.g. structure, size, and chemical functions), by either stimulating or suppressing the immune response. However, a harmonized cutting-edge approach for the classification of these materials based not only on their physicochemical parameters but also their immune properties has been missing. The European Commission-funded G-IMMUNOMICS and CARBO-IMmap projects aimed to fill this gap, developing a functional pipeline for the qualitative and quantitative immune characterization of graphene, graphene-related materials (GRMs), and other CBMs. The goal was to open breakthrough perspectives for the definition of the immune profiles of these materials. Here, we summarize our methodological approach, key results, and the necessary multidisciplinary expertise ranging across various fields, from material chemistry to engineering, immunology, toxicology, and systems biology. G-IMMUNOMICS, as a partnering project of the Graphene Flagship, the largest scientific research initiative on graphene worldwide, also complemented the studies performed in the Flagship on health and environmental impact of GRMs. Finally, we present the nanoimmunity-by-design concept, developed within the projects, which can be readily applied to other 2D materials. Overall, the G-IMMUNOMICS and CARBO-IMmap projects have provided new insights on the immune impact of GRMs and CBMs, thus laying the foundation for their safe use and future translation in medicine.

In the last decade, graphene-based materials have received increasing attention for both academic research and industrial uses. However, products containing graphene must meet the same standards for quality, safety and efficacy as products not containing nanomaterials. Our aim is to shed light on the toxicological characterization of few-layer graphene (FLG) dispersions. In the present study, graphene was easily dispersed in water using biocompatible riboflavin-5′-phosphate sodium salt (Rib). A highly concentrated FLG dispersion (G-Rib) was stable for months. G-Rib suspension was characterized by HR-TEM, Raman spectroscopy and ζ-potential. Our results showed that even at high concentration of G-Rib, cell survival was always above 85%. Then, we investigated the tissue distribution and toxic effects of G-Rib in mice up to 30 days after intravenous injections. Histological analysis of the tissues revealed hepatic accumulation and excretion through the kidneys. The biochemical and hematological parameters remained within the reference range showing no hematotoxicity. Finally, no signs of inflammation were detected in the cells isolated from the lymph nodes and spleen. The results of the study show that highly water dispersed FLG is a material with a very low toxic profile, which is one of the first requirements for the future development of biomedical applications.

While the interaction between 2D materials and cells is of key importance to the development of nanomedicines and safe applications of nanotechnology, still little is known about the biological interactions of many emerging 2D materials. Here, an investigation of how hexagonal boron nitride (hBN) interacts with the cell membrane is carried out by combining molecular dynamics (MD), liquid‐phase exfoliation, and in vitro imaging methods. MD simulations reveal that a sharp hBN wedge can penetrate a lipid bilayer and form a cross‐membrane water channel along its exposed polar edges, while a round hBN sheet does not exhibit this behavior. It is hypothesized that such water channels can facilitate cross‐membrane transport, with important consequences including lysosomal membrane permeabilization, an emerging mechanism of cellular toxicity that involves the release of cathepsin B and generation of radical oxygen species leading to cell apoptosis. To test this hypothesis, two types of hBN nanosheets, one with a rhomboidal, cornered morphology and one with a round morphology, are prepared, and human lung epithelial cells are exposed to both materials. The cornered hBN with lateral polar edges results in a dose‐dependent cytotoxic effect, whereas round hBN does not cause significant toxicity, thus confirming our premise.

Graphene has been covalently functionalized throughaone-pot reductive pathway using graphite intercalation compounds (GICs),i np articularK C 8 ,w ith three different orthogonally protected derivatives of 4-aminobenzylamine. This novel multifunctional platform exhibits excellent bulk functionalization homogeneity (H bulk )a nd degree of addition whilepreserving the chemical functionalities of the organic addends throughd ifferent protecting groups, namely: tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz) and phthalimide (Pht). We have employed( temperature-dependent) statisticalR aman spectroscopy (SRS), X-ray photoelec-tron spectroscopy (XPS), magic angle spinning solid state 13 CNMR (MAS-NMR), and ac haracterizationt ool consisting of thermogravimetric analysisc oupledw ith gas chromatography and mass spectrometry (TG-GC-MS) to unambiguously demonstrate the covalentb inding and the chemical nature of the different molecular linkers. This work paves the way for the development of smart graphene-based materials of great interesti nb iomedicine or electronics, to name af ew, and will serve as ag uide in the designo fn ew 2D multifunctionalm aterials.

Graphene‐based materials (GBMs) have promising applications in various sectors, including pulmonary nanomedicine. Nevertheless, the influence of GBM physicochemical characteristics on their fate and impact in lung has not been thoroughly addressed. To fill this gap, the biological response, distribution, and bio‐persistence of four different GBMs in mouse lungs up to 28 days after single oropharyngeal aspiration are investigated. None of the GBMs, varying in size (large versus small) and carbon to oxygen ratio as well as thickness (few‐layers graphene (FLG) versus thin graphene oxide (GO)), induce a strong pulmonary immune response. However, recruited neutrophils internalize nanosheets better and degrade GBMs faster than macrophages, revealing their crucial role in the elimination of small GBMs. In contrast, large GO sheets induce more damages due to a hindered degradation and long‐term persistence in macrophages. Overall, small dimensions appear to be a leading feature in the design of safe GBM pulmonary nanovectors due to an enhanced degradation in phagocytes and a faster clearance from the lungs for small GBMs. Thickness also plays an important role, since decreased material loading in alveolar phagocytes and faster elimination are found for FLGs compared to thinner GOs. These results are important for designing safer‐by‐design GBMs for biomedical application.

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