# A comparative study on cytotoxicity of different types of commercial single- and few-layer graphenes on human monocytes and macrophages

Zhengmei Song, Luna Boulbet-Friedelmeyer, Marvin Motay, Antonio Esau del Rio Castillo, Housseinou Ba, Rym Soltani, Carlos Drummond, Yannick Lafue, Alain Penicaud, Francesco Bonaccorso, et al.

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Zhengmei Song, Luna Boulbet-Friedelmeyer, Marvin Motay, Antonio Esau del Rio Castillo, Housseinou Ba, et al. A comparative study on cytotoxicity of different types of commercial single- and few-layer graphenes on human monocytes and macrophages. Carbon, 2025, 244, pp.120685. 10.1016/j.carbon.2025.120685. hal-05337698

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# A comparative study on cytotoxicity of different types of commercial single- and few-layer graphenes on human monocytes and macrophages

Zhengmei Song<sup>a</sup>, Luna Boulbet-Friedelmeyer<sup>b,c</sup>, Marvin Motay<sup>d</sup>,

Antonio Esau del Rio Castillo<sup>e</sup>, Housseinou Ba<sup>d</sup>, Rym Soltani<sup>b</sup>, Carlos Drummond<sup>c, ID</sup>,

Yannick Lafue<sup>d</sup>, Alain Penicaud<sup>c</sup>, Francesco Bonaccorso<sup>e</sup>, Alberto Bianco<sup>a, * ID</sup>

## ARTICLE INFO

### Keywords:

- Carbon materials

- Human immune cells

- Immune activation

- Cell interaction

- Safety

## ABSTRACT

Few-layer graphene (FLG) were prepared via liquid phase exfoliation using surfactants like polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), or sodium deoxycholate (SDC). Alternatively, surfactant-free graphene water dispersions were obtained by oxidizing graphenide (i.e., KC8) in tetrahydrofuran, mixing with water, and evaporating the solvent to yield single-layer (SLG). Immune cells are the first responders to exogenous materials, rapidly initiate immune responses, but little is known about the immune effects of commercialized FLG and SLG. This study aims to compare the impact of one SLG and three FLG on THP-1 monocytes and derived macrophages in terms of viability, apoptosis, and reactive oxygen species (ROS) generation. While no significant effects were observed on monocytes, the three FLG dose-dependently reduced macrophage viability, while no effects were observed for SLG. To further investigate the cellular interactions, transmission electron microscopy and scanning electron microscopy confirmed the uptake of FLG/PVA, FLG/SDC, and SLG, and their membrane interactions with macrophages. All three types of FLG increased IL-8 secretion in monocytes and slightly increased IL-1β secretion in macrophages and CD14 expression in monocytes, at 50 µg/mL (the maximum dose tested). FLG/SDC was found to reduce CD163 and CD206 expression in macrophages. SLG exposure (up to 50 µg/mL) caused no significant immune activation. Overall, FLG and SLG exhibited low toxicity, although the three kinds of FLG triggered little immune activation and the production of IL-8 by THP-1 monocytes.

## 1. Introduction

Graphene-related materials have been increasingly used in electronics, composites, and biomedicine [1]. Few-layer graphene (FLG) refers to graphene-related materials with less than 4–10 layers of nanosheets [2], FLG can be prepared by liquid phase exfoliation (LPE) of graphite using different types of surfactants, including for example polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or sodium deoxycholate (SDC) [3]. Surfactants are widely used in industrial production of FLG, allowing to enhance the exfoliation efficiency and to modify their surface properties. Additionally, they can improve the dispersion and the colloidal stability in various solvents. An alternative method to prepare surfactant-free graphene water dispersions consists instead on

the oxidation of a graphenide (negatively charged graphene) solution in tetrahydrofuran [4]. After mixing this solution with water and evaporating the organic solvent, water-dispersed single-layer graphene (SLG) can be isolated [4,5]. For industrial applications, FLG can serve as a versatile platform for various applications across multiple sectors such as aerospace (for resistive heating coatings), energy conversion [6] and storage [7] (for electrification of catalytic processes [8] and to improve the performances of silicon-dominant anode for Li-ion batteries [9]), construction (as concrete additive), photovoltaics (to formulate highly conductive carbon-pastes rivalling the performances of metallic-based blends [10,11]), and defense (for electromagnetic absorption). Similarly, single-layer graphene enriched formulations are useful as additives in applications ranging from mechanical reinforcement or thermal

<footnote><sup>a</sup> CNRS, Immunology, Immunopathology and Therapeutic Chemistry, UPR 3572, University of Strasbourg, ISIS, 67000, Strasbourg, France</footnote>

<footnote><sup>b</sup> Carbon Waters, 33600, Pessac, France</footnote>

<footnote><sup>c</sup> University of Bordeaux, CNRS, CRPP, UMR 5031, 33600, Pessac, France</footnote>

<footnote><sup>d</sup> Blackleaf, 67400, Illkirch-Graffenstaden, France</footnote>

<footnote><sup>e</sup> BeDimensional, 16163, Genova, Italy</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: a.bianco@ibmc-cnrs.unistra.fr (A. Bianco).</footnote>
improvement of composites, to protective/smart coatings [12].

Numerous studies have demonstrated that FLG, within a certain concentration range, are relatively safe. Both FLG and SLG have been extensively investigated for safety and biodegradability through a variety of *in vitro* and *in vivo* tests [1,13–18]. While the impact of FLG and SLG has been investigated mainly using academic/laboratory samples, relatively partial efforts have been made to assess the toxicity profile of commercial graphene materials, especially considering the presence of surfactants during the production processes. A survey published in 2022 on 36 commercially available graphene materials (some of them containing surfactants) tried to correlate their physicochemical properties to cytotoxicity. This study reported that the cellular toxicity was independent of the defects found on the graphene layers of the tested products, but was essentially due to the presence of impurities. This finding highlights the importance in the quality of a determined graphene material when selected for a specific application [19]. Studies on safety of FLG in the presence of surfactants compared to surfactant-free graphene, especially on human immune responses, remain limited.

Monocytes are one of the most abundant phagocytic cells in the blood and are the body's first defense against foreign organisms or materials, while macrophages can be found in all tissues of living organisms and are fundamental in host defense [20]. THP-1 is a human monocytic cell line derived from a patient with an acute monocytic leukemia, and has been widely used in research.

In this study, we conducted a comparative safety analysis of four commercial graphene materials: FLG with 10 % PVA (FLG/PVA) and FLG with 10 % PVP (FLG/PVP) from BLACKLEAF (France), FLG with 10 % SDC (FLG/SDC) from BeDimensional (Italy), and surfactant-free SLG from Carbon Waters (France). We systematically evaluated the biocompatibility of these four materials on human THP-1 monocytes and THP-1-derived macrophages. We assessed the cell viability by examining metabolic activity, apoptosis, and intracellular ROS generation. The expression of specific M1 markers (i.e., CD80, CD86) and M2 markers (i.e., CD163, CD206) was measured on THP-1-derived macrophages to check their activation. Cytokine secretions were measured to assess the inflammatory responses in both THP-1 monocytes and THP-1-derived macrophages. Additionally, we also conducted the investigation on the cellular uptake by THP-1-derived macrophages using transmission electron microscopy (TEM) and the interaction of FLG and SLG with the macrophage membrane using scanning electron microscopy (SEM). Our results revealed a minimal impact on cytotoxicity and activation, although the three FLG induced some cell activation and a limited inflammatory response.

## 2. Experimental section

### 2.1. Materials and methods

**Synthesis of BLACKLEAF few-layer graphene.** FLG was synthesized through an advanced LPE technique [21] starting from high-purity graphite from Imerys (France). Polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP) were obtained from Across Chemicals. One gram of graphite was combined with 0.1 g of PVA or PVP in 1 L of distilled water. Each homogeneous suspension was then subjected to ultrasonication employing an 80 W probe sonicator, ensuring consistent mechanical agitation throughout 2 h, to achieve exfoliation. FLG/PVA consisted of flakes of 2–5 layers by TEM, with a lateral size of D90 ≈ 6 µm by SEM, and an $I_{2D}/I_G$ ratio = 0.40 by Raman. FLG/PVP consisted of flakes with 2–5 layers, as determined by TEM, with lateral size D90 ≈ 10 µm by SEM, and an $I_{2D}/I_G$ ratio of 0.40 measured by Raman. Post-exfoliation, the master solutions were diluted to obtain the appropriate concentrations for the cytotoxicity experiments. The stock dispersions of FLG/PVA and FLG/PVP for the biological experiments were prepared at a concentration of 1 mg/mL in water.

**Synthesis of BeDimensional few-layer graphene.** FLG stabilized by sodium deoxycholate (SDC, Merk) was obtained using a high-pressure

homogenizer, as previously reported [22,23]. Briefly, 10 g of graphite (from Graphit Kropfmühl GmbH, Germany) were dispersed in 1 L of Milli-Q® water, mixed with 1 g of SDC and 0.1 g of N-methyl-2-pyrrolidone (NMP) (the latter represents the amount of solvent mass residuals found in the commercial powder produced by BeDimensional). FLG/SDC consisted of flakes with a lognormal thickness distribution, consisting of a mode of 4.6 nm ($\mu$ = 2.25 and $\sigma$ = 0.81, in which $\mu$ represents the mean of the natural logarithm of the data, and $\sigma$ represents the standard deviation of the natural logarithm of the data) determined by a statistical analysis of AFM data. Similarly, the lateral size of FLG flakes show a mode peaking at 95 nm determined statistically measuring 112 individual flakes in TEM images. Finally, the obtained material shows an average $I_D/I_G$ ratio centered around 0.25 by Raman. The dispersion was processed in a high-pressure homogenizer at 200 MPa and stored at room temperature for the cytotoxicity tests. A stock dispersion of FLG/SDC was prepared at a concentration of 1 mg/mL in water.

**Synthesis of Carbon Waters single-layer graphene.** The aqueous graphene dispersion was prepared by LPE of graphite intercalated compound (GIC) $KC_8$ [24–26]. GIC was synthesized by mixing molten potassium and graphite flakes at C/K = 8 (with 3 % excess potassium) at 180 °C under an inert atmosphere for 5 h. GIC was then exfoliated in distilled anhydrous tetrahydrofuran (THF) at room temperature, under inert atmosphere and constant stirring for 5 days. Then, the large undissolved particles of $KC_8$ were separated from the graphenide solution by centrifugation. The graphenide solution was oxidized with dry air and transferred into water. THF was evaporated under ambient conditions. The final water formulation corresponded to a dispersion of neutral single layer graphene containing also a little amount (not quantified) of few-layer graphene. The mean graphene flake size, as determined by dynamic light scattering, was around 800 nm, while TEM allowed to measure a size distribution around 400 nm. In a detailed Raman study reported in the past [25], graphene produced by this method showed a basal plane defect concentration between 200 and 650 ppm and a major presence of single layer graphene, as deduced from the narrow ($28 \pm 2 \text{ cm}^{-1}$) and symmetric 2D band obtained in the Raman spectra. The proportion between single layer graphene and few-layer graphene was reduced in the industrial scale sample explored in this work, which present a larger ($42 \text{ cm}^{-1}$), albeit still symmetrical, 2D band. A stock dispersion of SLG for the biological experiments was prepared at a concentration of 0.8 mg/mL in water.

**Scanning electron microscopy.** BLACKLEAF samples were examined using a Zeiss 2600F scanning electron microscope with a resolution of 5 nm. To mitigate charging effects during the imaging process, specimens were carefully mounted onto a double-sided graphite tape.

**Transmission electron microscopy.** All samples were observed on a Hitachi 7500 transmission electron microscope (Hitachi High Technologies Corporation, Tokyo, Japan) equipped with an AMT Hamamatsu digital camera (Hamamatsu Photonics, Hamamatsu City, Japan). Additional microscopy data were provided separately by each company according to their internal criteria of characterization and applied techniques. TEM analyses of BLACKLEAF samples were also conducted using a JEOL 2100F transmission electron microscope, operating at an accelerated voltage of 200 kV, and equipped with a probe corrector designed to counteract spherical aberrations, facilitating a point-to-point resolution of 0.2 nm. To prepare the samples for analysis, a suspension of FLG was sonicated for 5 min to achieve a homogeneous, stable suspension that would not settle. This suspension was then evaporatively cast onto a copper grid coated with a holey carbon membrane for observation. TEM analyses of the BeDimensional FLG dispersions were also performed in a JEOL JEM-1011 transmission electron microscope, operated at an acceleration voltage of 100 kV. The FLG samples were diluted 1:50 and drop-cast onto ultrathin holey Formvar film 400 mesh Cu grids, from Ted Pella Inc. The grids were stored under vacuum at room temperature.

**Raman spectroscopy.** Raman spectra were recorded using an inVia,
Renishaw equipped with a 532 nm laser, using 100× objective lens, from 1000 to 3000 nm. The samples were prepared by dropping 10 µL of each suspension on a Si substrate (Tedpella, USA, Canada) and they were irradiated with the laser power of 0.1 %. At least 3 accumulations, each lasting 10 s, were recorded, and finally 2 to 4 points were averaged. Additional microscopy data were provided separately by each company according to their internal criteria of characterization and applied techniques. Raman spectral analyses of BLACKLEAF samples were performed using the LabRAM ARAMIS system from Horiba. The spectra were acquired between 500 and 4000 $cm^{-1}$ using a laser excitation wavelength of 532 nm. Before the spectroscopic assessments, the samples were uniformly deposited onto glass substrates via spin-coating and subsequently dried. The BeDimensional FLG dispersions were diluted 1:30 using the same solvent composition and drop-cast onto a Si wafer (LDB Technologies Ltd) with 300 nm thermally grown SiO$_2$. The Raman measurements were carried out on a Renishaw inVia spectrometer using a 50× objective with a numerical aperture of 0.7, a laser with a wavelength of 514.5 nm with an incident power of 5 mW on the sample. A total of ~300 points per sample were measured to perform the statistical analysis. OriginPro 2016 was used to perform deconvolution and statistics.

**Atomic force microscopy.** The BeDimensional FLG dispersions were diluted 1:30, and 100 µL of the dilution was drop-cast onto mica substrates and dried at 50 °C overnight. The atomic force microscopy (AFM) images were acquired with a Bruker® AFM instrument in tapping mode using silicon probes (frequency ¼ 300 kHz, spring constant ¼ 40 N m⁻¹). The thickness statistics are obtained by measuring >100 flakes from the AFM images. The statistical analyses were plotted following lognormal distributions. Statistical analyses were performed using the WSxM Beta 4.0 software.

## 2.2. THP-1 monocytes culture and differentiation

Human monocytic THP-1 cells were cultured in RPMI1640 supplemented with 50 µg/mL penicillin, 50 µg/mL streptomycin and 10% fetal bovine serum (FBS) at 37 °C in a 5 % CO$_2$ incubator. The cells were passaged at a cell density of up to a maximum 8.0 × 10⁵/mL every 3-4 days. For the THP-1 macrophage experiments, the THP-1 cells were seeded in 96-well plates (1.0 × 10⁴ cells/well) and pre-incubated for 72 h in the presence of 200 nM PMA. After 72 h, the differentiated THP-1 cells were washed with lukewarm PBS and exposed to FLG or SLG materials at different concentrations for further testing. All graphene samples displayed good colloidal stability in cell culture media. At the highest concentrations, we observed black clusters in the case of FLG, corresponding to materials in closed contact with the cellular membrane or inside the cells. The cells were only used up to 20 passages.

## 2.3. ATP assay

The cell metabolic viability assay was evaluated by ATP assay kit (CellTiter-Glo® 2.0 Cell Viability Assay, Promega, USA). The THP-1 monocytes (1.5 × 10⁴ cells/well) mixed with FLG materials (FLG/PVA, FLG/PVP and FLG/SDC) at 10, 25, 50 and 100 µg/mL or SLG at 10, 25, 50 and 80 µg/mL were plated in 96-well plates and incubated for 24 h. For the THP-1 macrophage experiment, the THP-1 derived-macrophages were exposed to FLG materials (FLG/PVA, FLG/PVP, and FLG/SDC) with concentrations at 10, 25, 50 and 100 µg/mL or SLG with concentrations at 10, 25, 50 and 80 µg/mL for 24 h. In this study, we tested SLG at a maximum concentration of 80 µg/mL instead of 100 µg/mL. This adjustment was made because our as-produced stock solution of SLG was 100 µg/mL, and dilution with 10 × culture medium resulted in a final concentration of 80 µg/mL. Before studying the viability of the FLG or SLG materials, we conducted an assessment of the viability of THP-1 monocytes or macrophages when exposed to the surfactants PVA (5, 10, 25, 50 and 100 µg/mL), PVP (5, 10, 25, 50 and 100 µg/mL), and the residual organic solvent NMP (0.001, 0.01, 0.1, 1, and 10 µg/mL)

and SDC (0.01, 0.1, 1, and 10 µg/mL) for 24 h. Subsequently, the cells were collected for the ATP assay following the manufacturer instructions. The luminescence of each well was recorded on a Microplate Reader (Varioskan Flash, Thermo, USA).

## 2.4. Cell apoptosis assay

APC Annexin V apoptosis detection reagent (BD Biosciences, USA) was used to detect the apoptotic cells. The THP-1 monocytes (50 × 10⁴ cells/well) mixed with FLG/PVA, FLG/PVP, FLG/SDC and SLG at 10 and 50 µg/mL were plated in 12-well plates and incubated for 24 h. For the THP-1 macrophage experiment, the cells were exposed to FLG/PVA, FLG/PVP, FLG/SDC and SLG with concentrations at 10 and 50 µg/mL for 24 h. Finally, the cells were collected and stained with APC Annexin V following the user manual protocol. The stained cells diluted by the binding buffer were analyzed by the flow cytometry (Gallios, Beckman Coulter, USA).

## 2.5. Intracellular ROS generation

ROS production was detected using CM-H$_2$DCFDA (Invitrogen, USA). The THP-1 monocytes (50 × 10⁴ cells/well) mixed with FLG/PVA, FLG/PVP, FLG/SDC and SLG at 10 and 50 µg/mL were plated in 12-well plates and incubated for 24 h. For the THP-1 macrophage experiment, the THP-1 monocytes were seeded in the 12-well plate (16 × 10⁴ cells/well) and pre-incubated for 72 h in the presence of 200 nM PMA. After 72 h, the differentiated THP-1 cells were washed with lukewarm PBS and incubated with FLG or SLG at concentrations of 10 and 50 µg/mL for 24 h. Then THP-1 cells were stained with 10 mM CM-H$_2$DCFDA working solution for 30 min at 37 °C. After staining, cells were washed twice with FACS buffer, resuspended in FACS buffer and analyzed on the flow cytometer.

## 2.6. Cell activation

The activation of monocytes or macrophages treated with FLG or SLG for 24 h was assessed using flow cytometry (Beckman Coulter Gallios). The THP-1 monocytes (50 × 10⁴ cells/well) mixed with FLG/PVA, FLG/PVP, FLG/SDC and SLG at 10 and 50 µg/mL were plated in 12-well plates and incubated for 24 h. For the THP-1 macrophages experiment, the THP-1 monocytes were seeded in the 12-well plate (16 × 10⁴ cells/well) and pre-incubated for 72 h in the presence of 200 nM PMA. After 72 h the differentiated THP-1 cells were washed with lukewarm PBS and exposed to FLG or SLG with concentrations of 10 and 50 µg/mL. After treating with FLG or SLG for 24 h, cells were collected and washed with 2 % FBS in PBS (FACS Buffer), then stained with the respective antibody mix at 4 °C for 30 min. The anti-human antibodies used were CD11b-PE (Biolegend, #301306), CD14-FITC (BD, #555397), CD80-FITC (Biolegend, #305205), CD86-APC (BD, #555660), CD163-APC (Biolegend, #326510), CD206-FITC (BD, #551135), to identify monocyte or macrophage populations.

## 2.7. Cytokine detection

The THP-1 monocytes (50 × 10⁴ cells/well) mixed with FLG and SLG at 10 and 50 µg/mL were plated in 12-well plates and incubated for 24 h. For the THP-1 macrophages experiment, the THP-1 monocytes were seeded in 12-well plates (16 × 10⁴ cells/well) and pre-incubated for 72 h in the presence of 200 nM PMA. After 72 h the differentiated THP-1 cells were washed with lukewarm PBS and exposed to FLG or SLG at 10 and 50 µg/mL for 24 h; the cells cultured in the medium without materials were taken as the control. The cells were treated with 1 µg/mL LPS (lipopolysaccharide) plus 20 ng/mL IFN-γ for 24 h as positive control. The supernatants were collected, and the concentrations of IL-6, IL-8, TNF-α, and IL-1β, were determined using ELISA kits (BD Biosciences, USA), respectively, following the manufacturer's instructions.
<figure><img src="image_3.png" /><figcaption>Fig. 1. TEM images of (a) FLG/PVA, (b) FLG/PVP, (c) FLG/SDC, and (d) SLG.</figcaption></figure>

## 2.8. Cellular uptake

The cellular uptake of FLG and SLG was studied by transmission electron microscopy (TEM) imaging. The THP-1 macrophages were exposed to FLG/PVA, FLG/SDC, and SLG at 25 µg/mL for 24 h along

with control untreated cells. After incubation, the cells were washed with cacodylate buffer twice and then fixed in 2.5 % glutaraldehyde in cacodylate buffer at 4 °C overnight. Following overnight fixation, the cells were rinsed thrice with cacodylate buffer. Later, the cells were post-fixed with 0.5 % osmium tetroxide for 30 min at room temperature and were washed thrice with MilliQ water. Cells were then dehydrated through a series of ethanol baths: 1 × 25 % ethanol for 10 min, 1 × 50 % ethanol for 15 min, 1 × 70 % ethanol for 15 min, 1 × 95 % ethanol for 10 min, and 3 × 100 % ethanol for 15 min. Following dehydration, the cells were soaked in a 1:1 ratio of 100 % ethanol and Epon overnight at 4 °C. The next day, the cells were rinsed (2 × 4 h) with Epon. After soaking, final inclusion of Epon into the cells was done by polymerizing Epon at 40 °C for 30 min then incubating at 60 °C for 48 h. Afterward, the polymerized blocks were removed and sliced into ultrathin sections using a diamond knife attached to an ultramicrotome cutter (Leica). The ultrathin sections were then collected on copper grids coated with Butvar B-98 and stained with 1 % uranyl acetate for 30 min followed by lead citrate staining for 2 min. Grids were then examined by TEM (Hitachi H600). Control cells without FLG or SLG underwent the same procedure.

## 2.9. Interaction of FLG and SLG with the macrophage membrane

The interaction of FLG and SLG with the human THP-1-derived macrophages was studied by scanning electron microscope (SEM) imaging. The THP-1 macrophages were exposed to FLG/PVA, FLG/SDC, and SLG at 25 µg/mL for 24 h along with control untreated cells. After incubation, the cells were washed with cacodylate buffer twice and then fixed in 2.5 % glutaraldehyde in cacodylate buffer at 4 °C overnight. Following overnight fixation, the cells were rinsed thrice with cacodylate buffer alone. Afterward, the cells were post-fixed with 0.5 % osmium tetroxide for 30 min at room temperature and were washed

<figure><img src="image_4.png" /><figcaption>Fig. 2. Raman spectra of (a) FLG/PVA, (b) FLG/PVP, (c) FLG/SDC, and (d) SLG.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** ATP assay (a, b), apoptosis assay (c, d), and ROS generation (e, f) of FLG and SLG on THP-1 monocytes (a, c, e) and THP-1-derived macrophages (b, d, f) after exposure 24 h. FLG/PVA-10, FLG/PVA-25, FLG/PVA-50, and FLG/PVA-100 in abscissa mean FLG/PVA at a final concentration of 10, 25, 50, and 100 µg/mL; FLG/PVP-10, FLG/PVP-25, FLG/PVP-50, and FLG/PVP-100 in abscissa mean FLG/PVP at a final concentration of 10, 25, 50, and 100 µg/mL; FLG/SDC-10, FLG/SDC-25, FLG/SDC-50, and FLG/SDC-100 in abscissa mean FLG/SDC at a final concentration of 10, 25, 50, and 100 µg/mL; SLG-10, SLG-25, SLG-50, and SLG-80 in abscissa mean SLG at a final concentration of 10, 25, 50, and 80 µg/mL respectively. The results are expressed as the means ± SD (n ≥ 3). \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001 compared with the untreated control.</figcaption></figure>

thrice with MilliQ water. Cells were then dehydrated through a series of ethanol baths: 1 × 25 % ethanol for 10 min, 1 × 50 % ethanol for 15 min, 1 × 70 % ethanol for 15 min, 1 × 95 % ethanol for 10 min, and 3 × 100 % ethanol for 15 min. Following dehydration, the samples were critical point-dried, metalized and analyzed in a JOEL 6400 JSM scanning electron microscope (JEOL Ltd., Tokyo, JP) operated at 35 kV.

## 2.10. Statistical analysis

All means were calculated from at least three independent experiments, and are expressed as means ± SD. The analysis of statistical significance was done using a one-way ANOVA followed by the Bonferroni's correction. The results were considered significant if p < 0.05.

# 3. Results and discussion

## 3.1. Preparation and characterization of the different FLG and SLG

In this work we have selected three different types of commercially available FLG manufactured in water by LPE in the presence of biocompatible surfactants, and one water-dispersible SLG prepared from a graphite intercalation compound.

### 3.1.1. BLACKLEAF material

BLACKLEAF applied its custom-made exfoliation protocol, to prepare FLG sheets, by treating a graphite suspension in the presence of PVA and PVP. These two FLG were characterized by TEM, SEM and high-resolution transmission electron microscopy (HRTEM) (Fig. 1a and b and Fig. S1), and Raman (Fig. 2a and b and Fig. S2). The TEM images revealed graphene flake structures with extended lateral dimensions, averaging around 5 µm, and composed of 4–5 distinctly stacked layers
(Fig. S1C and S1F).

To prove further the structural integrity and the overall quality of the FLG, Raman spectroscopy was used. The spectra of FLG/PVA and FLG/PVP featured the characteristic D and G bands, resonating at 1345 cm⁻¹ and 1590 cm⁻¹, respectively (Fig. 2a and b and Fig. S2) [27,28]. A low I<sub>D</sub>/I<sub>G</sub> intensity ratio was measured, corroborating the high level of graphitization of both exfoliated FLG. Additionally, the 2D Raman peak observed around 2690 cm⁻¹, indicative of the number of stacked graphene layers (Fig. S2B), confirmed the TEM analysis with the prevalence of graphene comprising less than 10 layers, in comparison to bulk graphite (Fig. S2A) [29].

### 3.1.2. BeDimensional material

BeDimensional has prepared the exfoliated FLG using their well-established LPE process [22,30]. Graphite was homogenized at high-pressure at the Laboratory scale in the presence of SDC in water with the addition of a low percentage of NMP to mimic the industrial product. TEM (Fig. 1c and Fig. S3c) and AFM (Fig. S3a) were used to assess the morphological characteristics of the FLG/SDC flakes. AFM images allowed to observe several flakes with the characteristic flat topographies of FLG and a thickness of 4.6 nm (with a lognormal standard deviation of 0.81 nm) (Fig. S3b). Similarly, TEM was used to examine the morphology of the exfoliated material, measuring a lateral size mode of 95 nm (with a lognormal standard deviation of 0.97) (Fig. S3d). The Raman spectra of FLG/SDC have shown the G peak at ~1585 cm⁻¹, a very small D peak at ~1350 cm⁻¹ and the 2D peak at ~2690 cm⁻¹, as superposition of multiple components, the main being the 2D<sub>1</sub> and 2D<sub>2</sub> components (Fig. 2c and Fig. S4) [29,31]. Considering the intensity ratios of the 2D<sub>1</sub> and 2D<sub>2</sub> bands, it was possible to estimate the thickness of the flakes and the corresponding number of layers (Fig. S4b-e). All spectra were normalized to their corresponding G peak intensities. The D band intensity variation (Fig. S4b) is associated with the presence of edge or in-plane defects [32-34]. Similarly, the increase in defects induces an increase in the FWHM(G) (Fig. S4c) [35]. In this manner, the combination of the I<sub>D</sub>/I<sub>G</sub> and FWHM(G) allows the discrimination between edge defects and structural defects produced in the exfoliation process. In the case of the exfoliated material, the creation of defects corresponds mainly to edge defects, and not to structural ones since no correlation (r² = 0.68) is found between the I<sub>D</sub>/I<sub>G</sub> and FWHM(G) (Fig. S4d). In general, for graphite, the intensity of the 2D<sub>2</sub> peak (I<sub>2D2</sub>) is roughly double compared to the intensity of the 2D<sub>1</sub> peak (I<sub>2D1</sub>). Furthermore, the intensity ratio (I<sub>2D2</sub>/I<sub>2D1</sub>) decreases as the flake thickness is reduced [35]. The normalized intensity ratios I<sub>2D1</sub>/I<sub>G</sub> vs I<sub>2D2</sub>/I<sub>G</sub> gives an insight into the thickness of the flake. The frontier between the few layers and graphitic or platelet-like flakes can be represented with the condition in which I<sub>2D1</sub>/I<sub>G</sub> = I<sub>2D2</sub>/I<sub>G</sub>. This frontier allows separating the Raman 2D data set into two groups: I<sub>2D1</sub>/I<sub>G</sub> < I<sub>2D2</sub>/I<sub>G</sub>, considered graphitic flakes, and I<sub>2D1</sub>/I<sub>G</sub> > I<sub>2D2</sub>/I<sub>G</sub> considered single- and few-layers [22]. The 2D band analysis of the processed sample indicated the presence of 65 % of FLG flakes in good agreement with the AFM analysis.

### 3.1.3. Carbon Waters material

Commercially available Carbon Waters SLG dispersion in pure water was prepared following the methodology originally reported by Bepete et al. [4] Shiny bronze-coloured flakes, characteristics of GIC KC<sub>8</sub>, were first obtained in molten potassium, (Fig. S5), and dissolved in dry THF. This graphenide solution, containing exfoliated and negatively charged graphene flakes, was then exposed to dry air. The oxidized graphenide became neutral graphene, known as organic graphene dispersion (OGD), once dispersed in THF. The OGD was then transferred into water and THF was evaporated in ambient conditions, affording a neutral dispersion of graphene at pH 7.8 in water (see Fig. S6 for the detailed entire process). The SLG flakes were observed by TEM and further analyzed by Raman. TEM micrographs allowed to observe the presence of layered graphene flakes similar to those of the three different FLG (Fig. 1d), with

an average size distribution around 400 nm (Fig. S7). The Raman spectrum of SLG is characterized by three main peaks at 1349, 1585 and 2698 cm⁻¹, corresponding to the D, G and 2D vibration modes (Fig. 2d). The calculation of the I<sub>2D</sub>/I<sub>G</sub> ratio, corresponding to 0.90, and the full-width at half maximum (FWHM) of 2D band of 42 cm⁻¹, are in agreement with the specifications of the commercial product provided by Carbon Waters [24].

## 3.2. Cell viability, apoptosis and ROS generation

To study the impact of the four materials, we initially measured the basal viability, apoptosis and cellular ROS generation on human THP-1 monocytes and THP-1-derived macrophages. Before performing the experiments on the three FLG and SLG samples, the ATP assay was used to test the effect of the surfactants PVA (5, 10, 25, 50, 100 µg/mL), PVP (5, 10, 25, 50, 100 µg/mL) and SDC (0.001, 0.01, 0.1, 1, 10 µg/mL), and the residual solvent NMP (0.01, 0.1, 1, 10 µg/mL) in these human immune cells after treatment for 24 h. The results have shown no significant changes in the viability of both immune cells compared with the untreated control cells (Fig. S8a and S8b). In the case of exfoliated FLG and SLG, the human THP-1 monocytes treated for 24 h with increasing doses of FLG/PVA (10, 25, 50, 100 µg/mL), FLG/PVP (10, 25, 50, 100 µg/mL), FLG/SDC (10, 25, 50, 100 µg/mL), and SLG (10, 25, 50, 80 µg/mL) remained unchanged in terms of viability compared to untreated control (Fig. 3a), while a dose-dependent increase of mortality was observed for FLG/PVA, FLG/PVP and FLG/SDC on human THP-1-derived macrophages (Fig. 3b). No change in viability was instead observed in THP-1 monocytes and macrophages for SLG, even at the highest dose used (80 µg/mL) (Fig. 3b).

To explore whether the decrease in ATP assay was due to a decreased cellular metabolism or apoptosis, cell apoptosis in THP-1 monocytes and macrophages treated for 24 h with FLG/PVA, FLG/PVP, FLG/SDC, and SLG was further investigated. This was the first experiment in this study. To gain a better understanding of the impact on the viability of the four materials, we tested concentrations up to 100 µg/mL. However, release or exposure to such high concentration is unlikely in real life or the environment. Therefore, for the subsequent experiments, we decided to select 50 µg/mL as the highest concentration. No cell apoptosis was observed for the four materials compared to untreated control (Fig. 3c and d) on both phagocytic cells, even at the highest dose tested, confirming that the decrease in viability on THP-1 macrophages measured by the ATP assay was due to cell metabolic perturbation. Previous studies have revealed that graphene oxide (GO) could reduce the viability of A549 cells and human neuroblastoma SH-SY5Y cells in a dose-dependent manner, but without inducing apoptosis [36,37]. Our recent work also proved that heteroatom-doped graphene quantum dots (GQDs) could slightly decrease the metabolic activity of the THP-1 macrophages again without inducing apoptosis [38]. The data in the current study agree with the previous reports; the three FLG decreased the cell viability of THP-1-derived macrophages in a dose-dependent manner without inducing any apoptosis.

We subsequently focused our attention on cellular ROS generation, measured by the commercial dye CM-H<sub>2</sub>DCFDA using flow cytometry. We first tested the cellular ROS in the THP-1 monocytes and macrophages treated with the surfactants. The results have shown no significant changes in the ROS generation of both immune cells compared with untreated control cells (Fig. S8c and S8d). Similarly, no significant changes on ROS generation were observed in both human THP-1 monocytes and macrophages, except measuring a significant increase in macrophages for FLG/PVP and FLG/SDC at 50 µg/mL (Fig. 3e and f). Previous studies have shown that multi-layer graphene slightly induced a ROS response [39,40]. The ROS generation has been regarded as an important response mechanism influencing cellular metabolism and viability [41,42]. In our study, FLG/PVP and FLG/SDC could induce a ROS-related stress in THP-1-derived macrophages at the highest concentration (50 µg/mL), but not in monocytes. These results were
<figure><img src="image_6.png" /><figcaption>Fig. 4. The activation. (a) CD11b expression, (b) CD14 expression, (c) CD80 expression, (d) CD86 expression, (e) CD163 expression, (f) CD206 expression with FLG, SLG or relevant surfactants PVA, PVP, SDC, NMP treated the THP-1 monocytes (a, b) or macrophages (c–f) for 24 h. FLG/PVA-10, FLG/PVA-50 in abscissa mean FLG/PVA at a final concentration of 10 and 50 µg/mL; FLG/PVP-10, FLG/PVP-50 in abscissa mean FLG/PVP at a final concentration of 10 and 50 µg/mL; FLG/SDC-10, FLG/SDC-50 in abscissa mean FLG/SDC at a final concentration of 10 and 50 µg/mL; SLG-10, SLG-50 in abscissa mean SLG at a final concentration of 10 and 50 µg/mL; PVA-5 in abscissa mean PVA at a final concentration of 5 µg/mL; PVP-5 in abscissa mean PVP at a final concentration of 5 µg/mL; SDC-0.05 in abscissa mean SDC with a final concentration of 0.05 µg/mL; NMP-0.005 in abscissa mean NMP at a final concentration of 0.005 µg/mL; SDC + NMP in abscissa mean SDC at a final concentration of 0.05 µg/mL plus NMP at a final concentration of 0.005 µg/mL respectively. The results are expressed as the means ± SD (n = 3). \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001 by one-way ANOVA with Bonferroni post-tests compared with the untreated control.</figcaption></figure>

expected and consistent with our ATP assay and apoptosis results. Previous studies have also demonstrated that, despite ROS production, no cell death or apoptosis damage were observed after treatment with single-walled carbon nanotubes and graphene [43,44] or titanium dioxide nanoparticles [42,45].

### 3.3. Cell activation

The major role of the immune cells is to respond to exogenous molecules or materials, being their activation a crucial endpoint. The activation of THP-1 monocytes was evaluated by the expression of the specific differentiation markers CD11b and CD14. The activation of the THP-1-derived macrophages was instead evaluated by the expression of the specific M1 (i.e., CD80, CD86) and M2 markers (i.e., CD163, CD206) measured after 24 h of treatment with FLG and SLG at doses of 10 and 50 µg/mL. The upregulation of CD11b and CD14 is crucial for THP-1 differentiation into macrophages. Compared with the untreated

control, the expression of CD11b on THP-1 monocytes was not influenced by FLG/PVA, FLG/SDC and SLG, even at the highest concentration, with only FLG/PVP significantly increasing CD11b at 50 µg/mL (Fig. 4a). The expression of CD14 was significantly increased by all three FLG materials at 50 µg/mL. FLG/PVP seems to have a more pronounced effect on CD14, significantly increasing this marker even at the lowest dose (10 µg/mL) (Fig. 4b).

The expression of the M1 macrophage markers CD80 and CD86 on THP-1-derived macrophages remained unchanged after 24 h at all concentrations for the four materials (Fig. 4c and d). The expression of the M2 markers CD163 and CD206 was downregulated in FLG/PVP and FLG/SDC, even at 10 µg/mL. In contrast, CD206 expression was upregulated in SLG, while CD163 expression remained unchanged at all concentrations in SLG (Fig. 4e and f). We found in a previous study a decrease in CD206 expression after 24 h incubation with FLG, obtained using riboflavin-5'-phosphate as exfoliating agent, at the highest dose (50 µg/mL) in human primary M2 macrophages [14]. Our results
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Cytokine secretion. Supernatants from 24 h FLG, SLG or the relevant surfactants PVA, PVP, SDC, NMP treated THP-1 monocytes (a, b, c, d) or THP-1-derived macrophages (e, f, g, h) were analyzed by ELISA to measure cytokine production. THP-1 monocytes cytokines (a) IL-6, (b) IL-1β, (c) TNFα, and chemokine (d) IL-8. THP-1-derived macrophages cytokines (e) IL-6, (f) IL-1β, (g) TNFα, and chemokine (h) IL-8. FLG/PVA-10, FLG/PVA-50 in abscissa mean FLG/PVA at a final concentration of 10 and 50 µg/mL; FLG/PVP-10, FLG/PVP-50 in abscissa mean FLG/PVP at a final concentration of 10 and 50 µg/mL; FLG/SDC-10, FLG/SDC-50 in abscissa mean FLG/SDC at a final concentration of 10 and 50 µg/mL; SLG-10, SLG-50 in abscissa mean SLG at a final concentration of 10 and 50 µg/mL; PVA-5 in abscissa mean PVA with a final concentration of 5 µg/mL; PVP-5 in abscissa mean PVP at a final concentration of 5 µg/mL; SDC-0.05 in abscissa mean SDC at a final concentration of 0.05 µg/mL; NMP-0.005 in abscissa mean NMP at a final concentration of 0.005 µg/mL; SDC + NMP in abscissa mean SDC at a final concentration of 0.05 µg/mL plus NMP with a final concentration of 0.005 µg/mL respectively. Cells exposed to 1 µg/mL LPS plus 20 ng/mL IFN-γ were used as positive control. The results are expressed as the means ± SD (n = 3). \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001 compared with the untreated control.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** THP-1-derived macrophage transmission electron microscopy analysis. THP-1-derived macrophages were treated with 25 µg/mL FLG/PVA, FLG/SDC, and SLG for 24 h. The arrows indicate the localization of the materials.</figcaption></figure>

evidenced that FLG/PVP increased CD14 expression in monocytes, while FLG/SDC decreased CD163 and CD206 expression in THP-1-derived macrophages.

In order to study the influence of the surfactants and residual organic solvent (PVA, PVP, SDC, NMP) present in the FLG and SLG materials in both human immune cells, the expression of surface markers were also measured after 24 h treatment with PVA (5 µg/mL, similar to the PVA concentration in 50 µg/mL FLG/PVA), PVP (5 µg/mL, of the order of the highest concentration in FLG/PVP), SDC (0.05 µg/mL, of the order of the highest concentration in FLG/SDC) and NMP (0.005 µg/mL, of the order of the highest concentration estimated in FLG/SDC). The results have shown no significant changes in both immune cells compared with untreated control cells (Fig. 4a-f), which is consistent with previous studies [46–48]. This allows to conclude that these components, necessary to achieve stable graphene dispersions, do not affect the cellular homeostasis.

### 3.4. Cytokine secretion

Following the assessment of the activation cellular markers, we measured the pro-inflammatory cytokines IL-6, IL-1β, TNF-α, and chemokine IL-8 by ELISA after 24 h of FLG or SLG exposure at concentrations of 10 and 50 µg/mL. IL-6 and TNF-α were non-significantly increased by both FLG and SLG in human THP-1 monocytes (Fig. 5a

and c) and macrophages (Fig. 5e and g). For IL-1β, only FLG/PVP at the highest dose (50 µg/mL) has shown a significant increase in THP-1 monocytes (Fig. 5b), whereas all FLG materials increased the secretion of IL-1β in macrophages (Fig. 5f). The chemokine IL-8 was dose-dependent and significantly increased by all FLG in monocytes (Fig. 5d) and increased in macrophages but non-significantly (Fig. 5h). SLG, however, has shown no effect on all tested cytokines IL-6, IL-1β, TNF-α or chemokine IL-8 either in human THP-1 monocytes or macrophages (Fig. 5a–h).

In order to study the influence of the surfactants and the residual NMP in FLG materials in both human immune cells, the cytokines were also tested after 24 h treatment with PVA, PVP, SDC and NMP at the same concentrations reported for the tests on cell marker activation. The results have shown no significant changes in both immune cells compared with untreated controls (Fig. 5a–h). The cells were also treated with 1 µg/mL LPS (lipopolysaccharide) plus 20 ng/mL IFN-γ for 24 h as positive control, and all cytokines and chemokines were significantly increased (Fig. 5a–h). Similar trends were found in our previous studies showing that GQDs triggered the production of IL-8 on THP-1 monocytes [38] and reduced graphene oxide and doped GQDs increased the generation of IL-1β on THP-1 macrophages [38,49], suggesting a possible inflammasome activation [50].
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** The interactions between THP-1 macrophages membrane and 25 µg/mL graphene. SEM images of THP-1-derived macrophages after 24 h's treatment: (a) Control, (b) FLG/PVA, (c) FLG/SDC, and (d) SLG. The arrows indicate the materials on the membrane surface.</figcaption></figure>

### 3.5. Cell uptake of FLG and SLG

Different interactions between materials and cells can have distinct effects on cell viability, oxidative stress generation, and cell activation. In order to possibly identify the mechanisms responsible for such effects, the uptake of FLG/PVA, FLG/SDC and SLG at 25 µg/mL in THP-1-derived macrophages were examined by TEM. The TEM micrographs revealed the presence of graphene inside the macrophages for all samples, an expected behavior as macrophages are phagocytic cells (Fig. 6). The materials were observed within phagocytic vacuoles or into cytoplasm, in different amounts and in varying degrees of aggregation. Compared to untreated control cells (Fig. 6), FLG/PVA was mainly present in phagosomes, with small flakes observable in the cytoplasm. On the contrary, FLG/SDC was found in large amounts in the cytoplasm of the macrophages, but rarely into phagosomes. In contrast, SLG was seldom found in other cellular compartments, with the most observable clusters present in the cytoplasm. Overall, the three materials seem not to accumulate in specific organelles, such as mitochondria, or the peri-nuclear region, nor did they cause damage to mitochondria.

The nanomaterial accumulation in the cytoplasm has been suggested to be caused by passive translocation through the cellular membrane, while those found in phagosomes could be taken up via phagocytosis or endocytosis [51]. The GO has been frequently investigated in other cell types using TEM. This type of graphenic material was visualized in both the phagosomes and the cytoplasm of RAW 264.7 macrophages [52] and in primary human and murine macrophages [53]. One of our previous studies demonstrated that GO mostly resided in phagosomes, and less often in the cytoplasm in human primary macrophages [14,44]. In the current study, FLG/PVA and FLG/SDC were found in both cytoplasm and phagosomes. Based on the above discussion, we can hypothesize that FLG/PVA and FLG/SDC likely translocate both passively and phagocyted by the THP-1 macrophages, while SLG most probably directly crosses the cell membrane.

Although we discovered a large amount of internalized FLG/PVA and FLG/SDC in THP-1-derived macrophages during TEM observations, they did not cause significant effects in cell viability and ROS levels. At 25 µg/ mL of FLG/PVA and FLG/SDC, the metabolic viability of THP-1 macrophages remained around 90 % compared to the untreated control. Even at 50 µg/mL, FLG/PVA and FLG/SDC did not induce apoptosis. This indicates that cellular endocytosis does not induce cell damage. This is consistent with our previous study showing that a large amount of graphenic materials (i.e., GQDs) were able to penetrate THP-1 macrophages without inducing the loss of cell viability or apoptosis [38].

### 3.6. Interaction of FLG and SLG with the macrophage membrane

The TEM imaging was useful to visualize graphene cellular internalization and localization, in order to further understand the entry mode. In addition to TEM, we carried out 24 h exposures of macrophages with 25 µg/mL of FLG or SLG to observe the interaction with the cellular membrane and to capture the uptake process, using SEM. Fig. 7 shows high-resolution images of cell surfaces untreated or exposed to graphene after 24 h. Untreated human THP-1-derived macrophages demonstrated typical macrophage morphology with developed projections and pseudopodia protruding from the cell surface (Fig. 7a). When treated with graphenes, the materials can be observed on the membrane surface of macrophages (Fig. 7b-d, arrows indicate the FLG or SLG). From Fig. 7b and c, it can be inferred that permeation through the membrane is occurring. It has been reported that this type of process starts from the roughness or the protrusions at the graphene edges, or from the corners of the graphene sheets [54]. The observation of this permeation mode allows us to further confirm the hypothesized passive translocation mechanism of FLG and SLG. Since a graphene sheet is planar, unlike the passive translocation mechanism of carbon nanotube nanoneedle cell penetration [55], its translocation mechanism might follow a direct cell membrane penetration from the edges or the corners as demonstrated using molecular dynamic simulations [56].

## 4. Conclusion

In this study, the effects of commercial FLG/PVA, FLG/PVP, FLG/SDC, and SLG on THP-1 monocyte viability, apoptosis, and ROS generation were compared. The different materials have shown no significant effects on THP-1 monocytes. However, when examining THP-1 macrophages, only FLG/PVA, FLG/PVP, and FLG/SDC were found to be dose-dependent on reducing macrophage viability. To further investigate the cellular interactions, transmission electron microscopy confirmed the uptake of FLG/PVA, FLG/SDC, and SLG by THP-1 macrophages. Additionally, the interactions of FLG/PVA, FLG/SDC, and SLG with the membrane of THP-1 macrophages was confirmed by scanning electron microscopy. Notably, FLG/PVA, FLG/PVP, and FLG/SDC exhibited dose-dependent increases in IL-8 secretion in THP-1 monocytes. At 50 µg/mL, FLG also slightly increased IL-1β secretion in macrophages, and increased CD14 expression in monocytes, while FLG/SDC decreased CD163 and CD206 expression in THP-1-derived macrophages. No significant changes were observed in activation or cytokine secretion of monocytes or macrophages after 24 h exposure to SLG, even at the highest concentration tested. Overall, FLG and SLG have shown very low toxicity in human monocytes and macrophages. Only the three FLG induced low activation and inflammatory responses. As commercially graphene materials might contain endotoxins, we cannot totally exclude that these impurities contribute to the low cellular activation found for FLG products. The uptake and elimination of the materials are important mechanisms determining their bioeffects. Material uptake can occur not only via internalization through passive diffusion but also through active absorption of substances by the living tissues [57]. In this context, studying cell exocytosis and biodegradation will be important challenges in future studies.
CRediT authorship contribution statement

**Zhengmei Song:** Writing – original draft, Investigation, Formal analysis, Data curation. **Luna Boulbet-Friedelmeyer:** Writing – review & editing, Investigation, Data curation. **Marvin Motay:** Writing – review & editing, Resources, Formal analysis. **Antonio Esau del Rio Castillo:** Writing – review & editing, Resources, Formal analysis. **Housseinou Ba:** Writing – review & editing, Resources, Formal analysis. **Rym Soltani:** Writing – review & editing, Resources, Methodology. **Carlos Drummond:** Writing – review & editing, Supervision. **Yannick Lafue:** Writing – review & editing, Supervision, Resources. **Alain Pénicaud:** Writing – review & editing, Supervision, Funding acquisition. **Francesco Bonaccorso:** Writing – review & editing, Supervision, Funding acquisition. **Alberto Bianco:** Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

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

This work was supported by the European Commission through the Graphene Flagship project (grant agreement no. 881603), and the Interdisciplinary Thematic Institute SysChem via IdEx Unistra (ANR-10-IDEX-0002) within the program Investissement d'Avenir. This project received funding from the European Union's GREENCAP Horizon Europe research and innovation program under Grant Agreement No. 101091572 and LAPERITIVO Horizon Europe research and innovation program under Grant Agreement No. 101147311. The authors also acknowledge the Centre National de la Recherche Scientifique (CNRS) and the International Center for Frontier Research in Chemistry (icFRC) for support. The authors wish to thank Valérie Demais and Cathy Royer from the "Plateforme Imagerie In Vitro de l'ITI Neurostra", CNRS UAR 3156, University of Strasbourg (Strasbourg, France), for TEM and SEM analyses, and Céline Corcelle for her help in recording Raman spectra. L. B.-F. wishes to thank l'Association Nationale de la recherche et de la Technologie (ANRT) and Carbon Waters for supporting her a PhD. C.D. and A.P. wish to thank l'Agence Nationale de la Recherche, (project 2D-MEMBA, ANR-21-CE09-0034-01).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.carbon.2025.120685.

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