Toward High‐Dimensional Single‐Cell Analysis of Graphene Oxide Biological Impact: Tracking on Immune Cells by Single‐Cell Mass Cytometry

Abstract

Graphene, a single layer of hexagonally arranged carbon atoms, is considered the most versatile material available to mankind: the thinnest, the strongest, the lightest, extremely flexible, highly electrically and thermally conductive material, [1,2] a versatile entity for multiple functionalization. Due to its unique combination of superior properties, graphene is the starting platform for new disruptive technologies across a wide range of fields [3,4] Various graphene-based technologies have now transformed into commercial products, in sports goods, automotive coatings, conductive inks, touch screens and several others. [5] The massive production and use of graphene materials will increase exponentially over the coming years. On the other hand, an ever-growing literature is related to the explorations of graphene materials for new diagnostic and therapeutic strategies. [6][7][8][9] The graphene material exposure, due to the different applications, might open possible threats Considering the potential exposure to graphene, the most investigated nanomaterial, the assessment of the impact on human health has become an urgent need. The deep understanding of nanomaterial safety is today possible by high-throughput single-cell technologies. Single-cell mass cytometry (cytometry by time-of flight, CyTOF) shows an unparalleled ability to phenotypically and functionally profile complex cellular systems, in particular related to the immune system, as recently also proved for graphene impact. The next challenge is to track the graphene distribution at the single-cell level. Therefore, graphene oxide (GO) is functionalized with AgInS 2 nanocrystals (GO-In), allowing to trace GO immune-cell interactions via the indium ( 115 In) channel. Indium is specifically chosen to avoid overlaps with the commercial panels (>30 immune markers). As a proof of concept, the GO-In CyTOF tracking is performed at the single-cell level on blood immune subpopulations, showing the GO interaction with monocytes and B cells, therefore guiding future immune studies. The proposed approach can be applied not only to the immune safety assessment of the multitude of graphene physical and chemical parameters, but also for graphene applications in neuroscience. Moreover, this approach can be translated to other 2D emerging materials and will likely advance the understanding of their toxicology.

Lateral dimension and amino-functionalization on the balance to assess the single-cell toxicity of graphene on fifteen immune cell types

“…Overall, CyTOF findings confirmed the data obtained by the classic flow cytometry method and allowed to evaluate the impact on the different immune subpopulations, revealing the differences among the four materials that were appreciable only by this high-dimensional single-cell technology. The specific effects of the materials on the distinct immune cell subpopulations could be related to the different uptake of the materials in the different typed of cells, as we previously reported for GO into human PBMC subpopulations, enabling the material detection at CyTOF through the functionalization with indium [38].…”

Graphene Oxide Nanoparticles in the Regulation of the Oxidative Activity of Human Monocytes

“…However, the data on the antioxidant activity of GO are contradictory. In 2019 studies, it was shown that GO exhibits low antioxidant activity against H 2 O 2 and OH*, while Qui et al considered about high antioxidant activity of GO (10 μg/ml) to OH*, as well as its ability to neutralize the superoxide anion (at a concentration of about 80 μg / treated / untreated ml) [4,8,13]. Chemical modification of GO (doping with nitrogen, the introduction of various functional groups) significantly increases its antioxidant activity, in this regard, it can be assumed that treatment with PEG affects the neutralization of ROS [6].…”

Graphene oxide activates B cells with upregulation of granzyme B expression: evidence at the single-cell level for its immune-modulatory properties and anticancer activity

“…20 We previously proposed the use of single-cell mass cytometry to dissect the immunological effects of GBMs on individual cells. 21,22 Recently, we characterized and described a new functionalization of GO with ZnS-doped AgInS2 quantum dots (GO-In) that enables the detection of the material in the indium channel ( 115 In), 23 allowing its high-throughput tracking by single-cell mass cytometry. 21 This new approach revealed the ability of GO-In to interact with human classical monocytes and, unexpectedly, B cells, showing high single-B-cell functional heterogeneity.…”

“…21,22 Recently, we characterized and described a new functionalization of GO with ZnS-doped AgInS2 quantum dots (GO-In) that enables the detection of the material in the indium channel ( 115 In), 23 allowing its high-throughput tracking by single-cell mass cytometry. 21 This new approach revealed the ability of GO-In to interact with human classical monocytes and, unexpectedly, B cells, showing high single-B-cell functional heterogeneity. 21 The interaction of GBMs with monocytes and monocyte-derived macrophages has been studied by us and others.…”