# Cellular Impact and Biodegradability of S- and N-Doped Graphene Quantum Dots on Human Monocytes and Macrophages

Zheng Mei Song, Jun Gong, Rym Soltani, Jean-Daniel Fauny, Cécilia

Ménard-Moyon, Peng Chen, Alberto Bianco

► To cite this version:

Zheng Mei Song, Jun Gong, Rym Soltani, Jean-Daniel Fauny, Cécilia Ménard-Moyon, et al. Cellular Impact and Biodegradability of S- and N-Doped Graphene Quantum Dots on Human Monocytes and Macrophages. Advanced Functional Materials, 2024, pp.2405856. 10.1002/adfm.202405856. hal-04699539

HAL Id: hal-04699539

https://hal.science/hal-04699539v1

Submitted on 1 Sep 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Cellular impact and biodegradability of S- and N-doped graphene quantum dots on human monocytes and macrophages

Zhengmei Song,¹ Jun Gong,² Rym Soltani,¹ Jean-Daniel Fauny, Cécilia Ménard-Moyon,¹,* Peng Chen,²,* Alberto Bianco¹,*

¹CNRS, Immunology, Immunopathology and Therapeutic Chemistry, UPR 3572, University of Strasbourg, ISIS, 67000 Strasbourg, France

²School of Chemical & Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore

**Abstract**

Graphene quantum dots (GQDs), small graphene domains with lateral dimensions of less than 10 nm, have been increasingly used in electronics, composites and biomedicine. Chemical doping of GQDs allows tuning their optical properties. Immune cells are among the first cells exposed to nanomaterials entering a living body, rapidly triggering downstream immune responses. However, the assessment of the impact of chemically-doped GQDs on the immune system remains rather limited if not absent. In this context, we evaluated the effects and the biodegradability of sulfur-doped and nitrogen-doped GQDs (S-GQDs and N-GQDs) on human monocytes and macrophages. We studied the metabolic activity, membrane integrity, apoptosis and intracellular reactive oxygen species generation. In parallel, we investigated *in test tube* degradation of GQDs using human myeloperoxidase and a peroxynitrite-mediated system. We also followed their degradation in macrophages. High resolution transmission electron microscopy, fluorescence spectroscopy, Raman and flow cytometry were used to confirm the degradation. Overall, both GQDs exert little activation on both monocytes and macrophages although they decreased the metabolic viability in a dose-dependent manner. The loss of the native GQD structure and crystal lattice provides evidence of their biodegradability. Both the safety and biodegradability of S-GQDs and N-GQDs ensure their potential in biomedical applications.

**Keywords:** Carbon nanomaterials, immune system, toxicity, human peroxidases, peroxynitrite
# 1. Introduction

Graphene quantum dots (GQDs) are a class of graphene family materials made of small graphitic domains with lateral dimensions lower than 10 nm (e.g., average 5 nm).[1, 2] Owing to their diverse surface area, strong photoluminescence, excellent electrical properties, superior chemical inertness and biocompatibility, GQDs have potential applications in photovoltaics, bioimaging, biosensing, as antimicrobials and for drug delivery.[3, 4] Nowadays, the heteroatom-doping of GQDs has been developed as an appropriate approach for tailoring the properties of GQDs. For instance, doping with heteroatoms such as nitrogen or sulfur can modulate their physicochemical properties, such as chemical reactivity, optical activity and electronic structure.[5-10]

GQDs have been investigated in a wide range of safety studies,[11-13] with a multitude of assays conducted on different cell lines, whole animals and cultured tissues. Chemically-doped GQDs usually exhibit larger quantum yield than GQDs, which was exploited to visualize the presence of intracellular glutathione in HeLa cells.[14] N-GQDs were proved to be highly biocompatible on vascular smooth muscle cells, which can be considered beneficial for imaging experiments in living cells.[15] In another study, it was demonstrated that N-GQDs were nontoxic to HeLa cells and exhibited high photostability under repeated laser irradiation.[16]

While the safety of GQDs has been investigated, relatively limited effort has been made to assess their degradation that may be responsible for their systemic elimination and safety. Previous studies have reported the enzymatic biodegradation of carbon nanotubes (CNTs) or graphene oxide (GO) by recombinant myeloperoxidase (MPO), eosinophil peroxidase (EPO) or activated human neutrophils releasing MPO.[17-22] Researchers have also described peroxynitrite-dependent biodegradation of CNTs.[23, 24] Our group studied the enzymatic biodegradation of GQDs by MPO and EPO, showing the first biodegradability evidence of GQDs, thus emphasizing their real potential for biomedical applications and offering great opportunities in nanomedicine.[25] However, the biodegradation of doped GQDs has not been investigated yet and there is a dearth of information on their immune effect.

Monocytes are a type of leukocyte or white blood cell that can differentiate into macrophages and monocyte-derived dendritic cells, that are the most prominent phagocytes in the blood and are the body's first defense against foreign organisms or materials. Macrophages can be found in all
tissues and are fundamental in host defense. They phagocytose dead cells and debris, shape inflammatory response and modulate adaptive immunity.[26]

In this study, we systematically evaluated the biocompatibility and biodegradability of sulfur- and nitrogen-doped GQDs (S-GQDs and N-GQDs) on human THP-1 monocytes and THP-1-derived macrophages. We assessed cell viability by examining metabolic activity, membrane integrity, apoptosis and intracellular ROS generation. Spinning disc confocal microscopy and flow cytometry were employed to validate the cellular uptake by these immune cells. Additionally, cytokine secretions were measured to assess inflammatory responses. We also conducted investigations on the *in test tube* degradation of the doped GQDs using two systems: 1) an enzyme-mediated degradation system involving human MPO and 2) peroxynitrite-mediated degradation. Furthermore, we monitored their degradation in macrophages by high resolution transmission electron microscopy (HRTEM), flow cytometry and Raman spectroscopy.

## 2. Results and discussion

### 2.1. Preparation and characterization of S-GQDs and N-GQDs

S-GQDs and N-GQDs were prepared by hydrothermal treatment of 1,3,6-trinitropyrene in the presence of different doping precursors under alkaline condition. Na₂S was used as the doping source to synthesize S-GQDs and L-tryptophan was used for the N-doping of the GQDs. S-GQDs displayed a blue fluorescence under 365-nm UV light illumination and achieved the maximum emission wavelength at 480 nm, when excited at 390 nm (Figure 1A). In Figure 1B, the transmission electron microscopy (TEM) images showed that the lateral size of S-GQDs was around 5 nm. The HRTEM analysis revealed a lattice distance of 0.25 nm corresponding to the graphene (100) planes. The atomic force microscopy (AFM) analysis evidenced that the thickness of S-GQDs was around 1 nm, suggesting a single- or double-layered structure (Figure 1C). In contrast to S-GQDs, N-GQDs showed a yellow/green fluorescence under 365-nm light illumination and a red-shifted emission wavelength at 535 nm under 470 nm excitation (Figure 1D). Moreover, the average diameter of N-GQDs was around 10 nm (Figure 1E), indicating that the increased size may also contribute to the red-shifted fluorescence of N-GQDs owing to
quantum effects. The AFM images of N-GQDs confirmed the thickness of around 0.8 nm, indicating that most N-GQDs are single-layer (Figure 1F).

<figure><img src="image_2.png" /></figure>

**Figure 1.** Characterization of S-GQDs and N-GQDs. (A) Photoluminescence spectra of S-GQDs. Inset: photograph of an aqueous dispersion of S-GQDs under a 365-nm UV light illumination. (B) TEM and HRTEM (inset) images of S-GQDs. (C) AFM image of S-GQDs. Inset: height profile along the red line. (D) Photoluminescence spectra of N-GQDs. Inset: photograph of an aqueous dispersion of N-GQDs under a 365-nm UV light illumination. (E) TEM image of N-GQDs. (F) AFM image of N-GQDs. Inset: height profile along the red line.

## 2.2. Viability of immune cells treated with the doped GQDs

The immune system identifies and protects the body from infections and other exogenous molecules and materials. Therefore, understanding the interaction of the doped GQDs with the pivotal cells of the innate immune system is of particular importance.[12, 27] THP-1 is a human leukemia monocytic cell line. THP-1 cells in the monocyte state can be differentiated into a macrophage-like phenotype using phorbol-12-myristate-13-acetate (PMA) (Figure S1). THP-1 monocytes and derived macrophages have been used to study the activity of differential uptake of functionalized polystyrene nanoparticles by monocytes/macrophages,[28] and impact of
superparamagnetic iron oxide nanoparticles.[29] In this part, the effect of S-GQDs and N-GQDs was therefore assessed in human THP-1 monocytes and THP-1-derived macrophages to determine possible differences in the behavior of the tested materials towards the two types of human immune cells.

We evaluated the cell viability of THP-1-derived macrophages after incubation with S-GQDs or N-GQDs at concentrations up to 200 µg/mL for 8 h using the adenosine triphosphate (ATP) and lactate dehydrogenase (LDH) assays. As shown in **Figure S2**, the ATP viability was about 90% at the highest concentration indicating no membrane damages by S-GQDs and N-GQDs. Next, we studied the viability after 24-h incubation. We observed that the viability of the monocytes and macrophages assessed by the ATP assay decreased in a dose-dependent manner (**Figure 2A** and **2E**). At 50 and 100 µg/mL of S-GQDs, the viability of the THP-1 monocytes significantly diminished to about 80% and 40%, respectively (**Figure 2A**). In contrast, at 100 µg/mL of S-GQDs or N-GQDs, the viability of the macrophages remained over 80% (**Figure 2E**). Low doses (25 and 50 µg/mL) of S-GQDs or N-GQDs did not induce any change of the ATP concentration in the THP-1-derived macrophages. Indeed, the viability was over 90% compared with the untreated cells, even though there was a significant difference for the macrophages exposed to 50 µg/mL. Subsequently, the membrane integrity of the THP-1 monocytes and macrophages exposed to S-GQDs or N-GQDs for 24 h was measured using the LDH assay. No membrane damage in both cells was observed, except for the THP-1 monocytes incubated with S-GQDs at 100 µg/mL, which is consistent with the ATP assay in Figure 2A, indicating a cytotoxic effect of high dose S-GQDs on this type of immune cells (**Figure 2B** and **2F**).

To further understand the effects of the doped GQDs on the immune cell viability, we determined the percentage of apoptotic THP-1 monocytes and THP-1-derived macrophages after exposure to S-GQDs or N-GQDs for 24 h using Annexin V-APC staining and flow cytometry. As seen in **Figure 2C** and **2G**, only the dose of 100 µg/mL of S-GQDs significantly induced 17% apoptotic cells in the THP-1 monocytes. These results combined with the ATP and LDH assays evidenced that a high dose of S-GQDs is toxic for the THP-1 monocytes. S-GQDs and N-GQDs could instead slightly decrease the metabolic activity of the THP-1 macrophages without disrupting the cell membrane and inducing apoptosis. Our findings are in agreement with a previous study that reported the decrease of the viability evaluated by MTT assay of THP-1-derived macrophages incubated with GQDs without affecting the cell membrane integrity.[30]
We then evaluated the generation of intracellular reactive oxygen species (ROS) by the immune cells. For this purpose, ROS were measured by the CellROX deep red reagent using flow cytometry after 24 h of exposure to both doped GQDs. No significant changes were observed in both THP-1 monocytes and macrophages for S-GQDs and N-GQDs (Figure 2D and 2H). Our results are in agreement with our previous study in which GO induced a concentration-dependent decrease in cell viability but no significant difference in ROS generation on human monocyte derived macrophages.[31] Another study also reported similar results showing that multi-walled CNTs decreased the proliferation of SH-SY5Y cells after 2 weeks without inducing intracellular ROS generation.[32]

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>
**Figure 2.** Evaluation of the cytotoxicity of S-GQDs and N-GQDs on THP-1 monocytes (A, B, C, D) and THP-1-derived macrophages (E, F, G, H) after 24-h exposure: (A, E) cellular ATP assay, (B, F) cell membrane integrity assay, (C, G) apoptosis assay and (D, H) ROS generation. S-25, S-50 and S-100 in abscissa mean S-GQDs with a final concentration of 25, 50 and 100 µg/mL, respectively; N-25, N-50 and N-100 in abscissa mean N-GQDs with a final concentration of 25, 50 and 100 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.

### 2.3. Cytokine secretion by immune cells

Monocytes and macrophages are cells involved in the initiation, progression and resolution of inflammation.[33] Hence, to further evaluate the immunological impact of the doped GQDs in THP-1 monocytes and macrophages, various cytokines secreted by these cells were measured by ELISA after 24-h exposure to the doped GQDs at doses from 25 to 100 µg/mL. No secretion of the pro-inflammatory cytokine TNF-α was observed in the THP-1 monocytes with both doped GQDs (Figure 3A). However, the highest dose of N-GQDs significantly increased the secretion of IL-1β (Figure 3B). In the THP-1-derived macrophages, TNF-α and IL-1β were significantly upregulated following 24-h exposure to N-GQDs (Figure 3E and 3F), in a dose-dependent manner for IL-1β. The release of IL-6 in both THP-1 cells was instead not modified by both doped GQDs for all doses tested in comparison to the untreated control (Figure 3C and 3G).

The presence of foreign substances such as bidimensional materials can increase the production of IL-8, a chemoattractant cytokine that is produced by many cells in infection and trauma.[34] Indeed, both doped GQDs dose-dependently increased IL-8 generation in the case of the THP-1 monocytes (Figure 3D). In contrast, no significant changes of IL-8 level were observed in the THP-1-derived macrophages with both doped GQDs (Figure 3H). Our results are in agreement with previous studies showing that the IL-8 expression in THP-1 macrophages did not change after incubation with GO and reduced GO.[35]

Our data confirmed that N-GQDs were able to stimulate the production of pro-inflammatory cytokines TNF-α only in the THP-1 macrophages and IL-1β in both types of cells. In the THP-1 monocytes both doped GQDs modulated the secretion of IL-8. Similar trends were found in one of our previous studies showing that reduced GO triggered the production of TNF-α and IL-1β in THP-1-derived macrophages, suggesting a possible inflammasome activation.[36]
<figure><img src="image_7.png" /><figcaption>**Figure 3.** Cytokine generation by THP-1 monocytes (A, B, C and D) or THP-1-derived macrophages (E, F, G and H) after exposure to the doped GQDs for 24 h. S-25, S-50 and S-100 in abscissa mean S-GQDs with a final concentration of 25, 50 and 100 µg/mL; N-25, N-50 and N-100 in abscissa mean N-GQDs with a final concentration of 25, 50 and 100 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>

**2.4. Evaluation of the cellular uptake of the doped GQDs into macrophages**

Benefitting from their photoluminescence properties, the cellular internalization of S-GQDs and N-GQDs into THP-1-derived macrophages was evaluated at different time points (2, 4 and 8 h) and concentrations (25, 50 and 100 µg/mL) by flow cytometry (Figure 4) and confocal microscopy (Figure 5). The mean fluorescence intensity of the macrophages increased with increasing S-GQD and N-GQD concentrations and incubation times (Figure 4A and 4B). The mean fluorescence intensity of the THP-1 macrophages incubated with N-GQDs resulted much higher compared with S-GQD-incubated macrophages. As N-GQDs have similar absolute photoluminescence quantum yield to S-GQDs, corresponding to 24.8% and 20.1%, respectively,[37] N-GQDs likely have the capacity to penetrate the macrophages to a higher extent than S-GQDs.

To confirm the cellular uptake of the doped GQDs, the macrophages were observed under confocal microscopy after incubation with the non-toxic concentration of 25 µg/mL of N-GQDs for 30 min, 2, 4 and 8 h. CellMask™ was used to stain the cell membranes. Almost no fluorescence was
observed after 30 min of incubation with N-GQDs (Figure 5). After 2 h, a strong fluorescence was observed in the N-GQD-incubated macrophages, which increased up to 8 h (Figure 5 and Video S1-S4). The N-GQD fluorescence was observed throughout the entire cytoplasm, except the nucleus. These data indicated that a large amount of N-GQDs was internalized into the THP-1 macrophages after 2 h, and correlate with the flow cytometry results (Figure 4). Taken together, flow cytometry and confocal imaging showed that the cellular uptake was concentration- and time-dependent.

<figure><img src="image_8.png" /><figcaption>**Figure 4.** Mean fluorescence intensity of THP-1-derived macrophages after exposure to 25, 50 and 100 µg/mL of S-GQDs (A, C) or N-GQDs (B, D) for 2 h (black), 4 h (red) and 8 h (blue), respectively. The results obtained by flow cytometry are expressed as the means ± SD (n = 4). *p*<0.05, **p**<0.01, ***p**<0.001, ****p<0.0001 compared with the untreated control.</figcaption></figure>
was the visual decrease (naked eye) of the color intensity of the MPO-treated samples due to the material transformation after 25 h (Figure S4A and S4C). We analyzed the fluorescence of the samples at different time points. We observed a time-dependent decrease of the fluorescence intensity of the MPO-treated S-GQDs and N-GQDs compared to its respective control (Figure 6A, 6C, S4B and S4D), likely due to the decrease in the concentration of GQDs caused by the enzymatic degradation.

In order to provide additional evidence of the degradation of S-GQDs and N-GQDs, we utilized Raman spectroscopy and HRTEM analysis. Specifically, we tracked the spectral Raman changes in intensity from the D band at 1380 cm⁻¹ to the G band at 1601 cm⁻¹ of S-GQDs and from the D band at 1368 cm⁻¹ to the G band at 1580 cm⁻¹ of N-GQDs. These bands correspond to disordered carbon vibrations and the sp² tangential mode, respectively (Figure 6C and 6D).[³⁹] During the course of the treatment, the intensity and the form of the D and G bands of both S-GQDs (Figure 6C) and N-GQDs (Figure 6D) are noticeably affected, and the autofluorescence clearly increased, which is indicative of the loss of the doped GQD structures. No changes were instead observed for the two doped GQDs treated only with H₂O₂. Besides these qualitative observations, we also measured the ratio of the I<sub>D</sub>/I<sub>G</sub> intensities. The values of the I<sub>D</sub>/I<sub>G</sub> ratio increased from 0.76 to 0.83 and decreased from 0.94 to 0.89 for S-GQDs and N-GQDs, respectively. Although we expected more defect introduced to both doped dots leading to an overall increase of the relative intensity of the D band, the strong autofluorescence likely affects these data. However, the Raman band changes confirmed the gradual loss of the GQD structure due to degradation by the enzyme. To examine the alterations in the structure and morphology of S-GQDs and N-GQDs following their incubation with MPO, we also conducted HRTEM analyses. We observed that compared with the untreated doped GQDs, many fragments were present in both treated S-GQDs and N-GQDs, indicating that were not completely degraded by the enzyme. Interestingly, the size of S-GQDs and N-GQDs resulted smaller after being treated with MPO (Figure 6E and S5). Furthermore, we clearly noticed a partial loss of the crystal lattice structure of S-GQDs (Figure 6E and S5), certainly due to oxidation by MPO and consequent degradation of S-GQDs, leading to less graphitic structures. These results confirmed the degradability of the doped GQDs by MPO. The partial degradation of GQDs and GO was also observed in previous studies, mainly due to the rather short duration of the study, which is limited by the instability of MPO in test tube experiments.[²⁵, ³⁸, ⁴⁰]
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 6.** Enzyme-mediated degradation of the doped GQDs. (A, B) Fluorescence intensity of the MPO-treated (A) S-GQDs and (B) N-GQDs, and their respective control samples (H₂O₂-treated) at different incubation time points (the measurements were repeated three times). (C, D) Normalized Raman average spectra of MPO-treated (C) S-GQDs and (D) N-GQDs and their</figcaption></figure>
respective control samples (H₂O₂-treated) after 25 h of incubation. At least 30 different measurements were performed in several sample locations. (E) HRTEM images of the MPO-treated S-GQDs and N-GQDs after 25 h of incubation. Note: diminishing the amount of N-GQDs to half of S-GQDs (see Experimental Section) does not lead to their complete degradation.

*Peroxynitrite-mediated degradation.* The peroxynitrite-mediated degradation consists in the simultaneous production of superoxide radicals and NO by decomposition of 3-morpholinosydnonimine hydrochloride (SIN-1) or the combination of xanthine/xanthine oxidase (X/XO) as a superoxide-generating system with a NO donor, 1-(3-aminopropyl)-2-hydroxy-3-oxo-1-propyltriazane (PAPA NONOate). [23, 24] As common chemically-doped GQDs, S-GQDs were selected as a representative material to conduct this experiment and the degradation was monitored by fluorescence and Raman spectroscopy. As shown in **Figure 7A** and **7B**, the fluorescence of S-GQDs decreased of 20% and 50% compared to the respective controls (designated as 100%) after incubation for 2 and 5 days with PAPA NONOate, X and XO. The increase of the autofluorescence background on Raman spectra of the treated S-GQDs after 2 and 5 days further confirmed the transformation of the dots (**Figure 7C**). On the other hand, the fluorescence of the SIN-1-treated S-GQDs did not decrease even after incubation for 5 days (**Figure 7A** and **7B**). The changes in the Raman spectra were also negligible (**Figure 7D**). These results showed that the degradation of S-GQDs was more effective using PAPA NONOate/X/XO system than SIN-1. This is consistent with a previous study, where PAPA NONOate/X/XO system was found more effective to digest single-walled CNTs than SIN-1. [23]
<figure><img src="image_14.png" /><figcaption>**Figure 7.** Peroxynitrite-mediated degradation of S-GQDs. (A) Fluorescence intensity of S-GQDs after treatment with PAPA NONOate/X/XO or SIN-1 for 2 and 5 days. The results are expressed as the means ± SD (n=3). \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 compared with the PBS-treated control. (B) Fluorescence spectra of S-GQDs after treatment with PAPA NONOate/X/XO or SIN-1 for 5 days. (C, D) Normalized Raman average spectra of S-GQDs after incubation with PAPA NONOate/X/XO (C) and SIN-1 (D) for 2 and 5 days. At least 30 measurements were performed in different sample locations.</figcaption></figure>

## 2.6. N-GQD degradation in macrophages

It is well-known that some nanoparticles can remain into the organs after being taken up by macrophages, increasing the potential for unintended acute or chronic toxicity.[41, 42] We have seen that N-GQDs had the capacity to be internalized into human THP-1 macrophages within 4 h in a larger quantity compared to S-GQDs (Figure 5). Understanding the fate of N-GQDs after entering
macrophages is crucial in the study of their immunological effects. Therefore, we evaluated their
degradation by the human THP-1 macrophages. For this purpose, the THP-1 macrophages were
incubated with a non-cytotoxic concentration of N-GQDs (1, 5, and 10 µg/mL) for 24 h, then
washed 3 times to remove extracellular N-GQDs, replacing the solution with fresh medium to keep
the macrophages alive for another 3 or 5 days.

Since exocytosis is an important process for cells to excrete waste and foreign matter from the
cytoplasm to the outside of the cell, we collected the supernatants in the culture medium at day 0,
3 and 5, and measured fluorescence by a microplate reader to assess if exocytosis happened.
Compared to the untreated control group, the fluorescence of the supernatant did not change,
indicating that exocytosis did not occur, at least to an extent detectable by fluorescence (Figure
S6).

The biodegradation of N-GQDs within the macrophages was studied at day 0, 3 and 5 by flow
cytometry and Raman mapping analysis. As shown in **Figure 8A**, the fluorescence intensity
increased with increasing concentrations (1, 5 and 10 µg/mL), further proving that the cellular
uptake was dose-dependent (**Figure 4**). The mean fluorescence intensity of the THP-1
macrophages decreased after incubation for 3 and 5 days in culture medium in a time-dependent
manner, indicating a change of N-GQDs inside the macrophages. The Raman analysis showed a
decrease in the intensity, a modification of the form of the D and G bands at day 5, and an increase
of the autofluorescence background after incubation of the THP-1 macrophages (**Figure 8B** and
**Figure S7**). However, the value of I<sub>D</sub>/I<sub>G</sub> ratio only slightly changed from 0.99 to 0.97, likely due
again to the influence of the strong autofluorescence. We verified that the THP-1-derived
macrophages did not display any specific Raman band (**Figure S7**).

We next focused on the possible mechanism of N-GQD cellular degradation. As serum starvation
can inhibit the cell metabolic activity, we studied the capacity of the macrophages to degrade N-
GQDs in these conditions. In short, THP-1 macrophages were incubated with 10 µg/mL of N-
GQDs for 24 h (the macrophages at this step correspond to day 0). They were then washed 3 times
to remove extracellular N-GQDs, replacing the solution with fresh culture medium or fetal bovine
serum (FBS)-free medium for 3 days (the macrophages at this step correspond to day 3). After
these steps, the culture medium or FBS-free medium was replaced with fresh culture medium and
the macrophages incubated for another 2 days (the macrophages at this step correspond to day 5). We first observed the cell morphology under serum starvation. As shown in **Figure S8**, compared with cells cultured in serum medium, the cell morphology under starvation did not change, proving that three days of serum starvation did not cause cell death. Then, we compared the mean fluorescence intensity of the macrophages maintained in culture medium or FBS-free medium after the incubation with N-GQDs at day 0, 3 and 5 by flow cytometry (**Figure 8C**). The results showed that the degradation did not begin in the first 3 days of serum starvation, but after the two additional days of incubation in normal serum conditions, when the cell metabolic activity resumed and degradation started. Surprisingly, a significant increase was found in the fluorescence intensity of N-GQDs within THP-1 macrophages when incubated in FBS-free medium after three days (**Figure 8C**). We speculated that when degradation is inhibited, the intracellular environment (e.g., change of intracellular pH) might lead to this increased fluorescent signal. However, further future studies are needed to demonstrate this observation.

Few previous studies reported that THP-1 macrophages are involved in the “digestion” of CNTs through superoxide/NO peroxynitrite-driven oxidative pathway. [23, 24] The THP-1-derived macrophages are known to generate peroxynitrite [43] and NO is an important intermediate substance in the production of peroxynitrite generated by cells. To support the role of NO in the biodegradation process, we monitored the presence of NO in the THP-1 macrophages under normal medium or in FBS-free medium by using the fluorescent probe 4, 5-diamino-rhodamine B (DAR-1). We found that the NO fluorescence signal was very strong in normal conditions, while the NO generation was inhibited in FBS-free medium (**Figure 8D**). These results indicated that nitric oxide radicals might be one of the important pathways used by macrophages to degrade N-GQDs.
<figure><img src="image_15.png" /><figcaption>**Figure 8.** Degradation of N-GQDs in human macrophages. (A) Average mean fluorescence intensity of THP-1 macrophages, which were maintained in culture medium for 3 or 5 days after incubation with N-GQDs for 24 h. The results are expressed as the means ± SD (n=3). **p < 0.01, ****p < 0.0001 compared with 1 µg/mL treated macrophages at day 0; ###p < 0.0001 compared with 5 µg/mL treated macrophages at day 0; &&&p < 0.001, &&&&p < 0.0001 compared with 10 µg/mL treated macrophages at day 0. (B) Normalized Raman average spectra of N-GQDs (10 µg/mL) in the THP-1 macrophages, which were maintained in culture medium for 5 days after incubation with N-GQDs for 24 h. (C) Average mean fluorescence intensity of THP-1 macrophages, which were maintained in culture medium (wine red) or FBS-free medium (orange) for 3 days (day 3), then the solution was replaced with fresh culture medium and incubated for another 2 days (day 5) after initial incubation with 10 µg/mL N-GQDs for 24 h (day 0). The results are expressed as the means ± SD (n=3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with related sample. (D) NO generation level by the THP-1 macrophages in complete cell culture medium or FBS-free medium. The images were analyzed by the Image J software.</figcaption></figure>
### 3. Conclusions

Chemical doping of GQDs tunes their optical properties with applications in biosensing or bioimaging. We evaluated the effects of S-GQDs and N-GQDs on human monocytes and macrophages, and assessed their biodegradability. We measured the metabolic activity, membrane integrity, apoptosis, cellular uptake and cytokine secretions to assess the impact of these materials on human immune cells. We also investigated the biodegradation of the doped GQDs in test tubes and in macrophages. Overall, both S-GQDs and N-GQDs exerted little activation on both THP-1 monocytes and THP-1-derived macrophages, even though they decreased their metabolic viability in a dose-dependent manner. The loss of the GQD structure and crystal lattice with the decrease in fluorescence provided evidence of their biodegradability. Our study brings new information on both safety and biodegradability of S-GQDs and N-GQDs, confirming their potential in the biomedical domain.

### 4. Experimental Section

*Synthesis and characterization of S-GQDs and N-GQDs.* Based on the previous reports,[44, 45] a mixture of pyrene (2.5 g) and 200 mL concentrated nitric acid was treated under continuous stirring for 13 h at 80°C, and 1,3,6-trinitropyrene was collected after filtering and full drying at 60°C. Four hundred mg of 1,3,6-trinitropyrene and 2 g of Na₂S were dispersed in 200 mL NaOH (5 mM) and then sealed into a 500-mL Teflon autoclave for 6-h hydrothermal treatment at 200°C. To remove large particles and aggregates, the obtained S-GQD dispersion was dialyzed for 3 days (MWCO 500 Da) and filtered using a 0.1 mm syringe filter. The S-GQD powder was isolated by freeze-drying. The synthesis of N-GQDs followed the same procedure, except that L-tryptophan (3.06 g) was used as source of nitrogen to obtain N-GQDs.

The structure and morphology of the doped GQDs was revealed by AFM (Bruker ICON) and TEM (JEM 2100PLUS). The photoluminescence properties were characterized using a Fluorolog® PL spectrometer (Horiba Scientific). The photoluminescence quantum yield was measured using FL 3C-11 spectrofluorimeter (Hariba Scientific).

*Cell culture:* Human monocytic (THP-1) cells were cultured in RPMI 1640 supplemented with 50 µg/mL penicillin, 50 µg/mL streptomycin and 10% FBS at 37°C in a 5% CO₂ incubator. The cells were passaged at a cell density up to maximum 8.0×10⁵/mL every 3-4 days. For the THP-1 macrophage experiments, the THP-1 cells were seeded in a plate 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 the doped GQDs. The cells were only used up to 20 passages.

*Cell metabolic viability and cell membrane integrity assays.* The cell viability was evaluated by the ATP assay kit (CellTiter-Glo® 2.0 Cell Viability Assay, Promega, USA) and the cell membrane integrity was assessed by the LDH test kit (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega, USA). THP-1 monocytes (1.5×10⁴ cells/well) mixed with S-GQDs and N-GQDs at 0, 25, 50 and 100 µg/mL were plated in 96-well plates and incubated for 24 h. For the THP-1 macrophage experiments, PMA-derived macrophages were exposed to S-GQDs or N-GQDs with concentrations at 0, 25, 50 and 100 µg/mL for 24 h. Subsequently, the culture medium was collected for LDH measurement and the cells for the ATP assay following the manufacturer instruction. For the LDH assay, the positive control was prepared by adding 10 µL lysis solution to the control cells for 30 min. The luminescence of the ATP assay and optical density of LDH test of each well was recorded on a microplate reader (Varioskan Flash, Thermo, USA).

*Cell apoptosis assay.* APC Annexin-V apoptosis detection reagent (BD Biosciences, USA) was employed to detect the apoptotic cells. THP-1 monocytes (15×10⁴ cells/well) mixed with S-GQDs and N-GQDs at 0, 25, 50 and 100 µg/mL were plated in 24-well plates and incubated for 24 h. For the THP-1 macrophage experiments, PMA-derived THP-1 macrophages were exposed to S-GQDs or N-GQDs with concentrations at 0, 25, 50 and 100 µg/mL for 24 h. The untreated cells were incubated in a 56°C water bath for 10 min to induce cell apoptosis as the positive control. Finally, the cells were collected and stained with APC Annexin-V following strictly the manufacturer protocol. The stained cells diluted by the binding buffer were analyzed by the flow cytometry (Gallios, Beckman Coulter, USA).

*Intracellular ROS generation.* ROS were detected using CellROX deep red reagent (Invitrogen, USA). THP-1 monocytes (15×10⁴ cells/well) mixed with S-GQDs and N-GQDs at 0, 25, 50 and 100 µg/mL were plated in 24-well plates and incubated for 24 h. For the THP-1 macrophage experiment, the THP-1 monocytes were pretreated with PMA to induce differentiation and then exposed to S-GQDs or N-GQDs with concentrations at 0, 25, 50 and 100 µg/mL for 24 h. Then, the ROS levels of both THP-1 monocytes and macrophages were determined following the protocol. Briefly, cells were collected and washed with PBS and then 1 µL CellROX reagent was added. The positive controls were prepared by exposing untreated cells to 200 mM H₂O₂ for 30 min. Thirty minutes later, the cells were washed 3 times and soaked with PBS for flow cytometry
analysis (Ex: 644 nm; Em: 665 nm). The ROS level is presented as the ratio of $F_{test}/F_{control} \times 100\%$, where $F_{test}$ is the mean fluorescence intensity of the cells exposed to S-GQDs or N-GQDs or the positive control, and $F_{control}$ is the mean fluorescence intensity of the control.

**Cytokine assay.** THP-1 monocytes (15×10⁴ cells/well) mixed with S-GQDs and N-GQDs at 0, 25, 50 and 100 µg/mL were plated in 24-well plates and incubated for 24 h. For the THP-1 macrophage experiments, the derived THP-1 macrophages were exposed to S-GQDs or N-GQDs with concentrations at 0, 25, 50 and 100 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 lipopolysaccharide (LPS) plus 20 ng/mL IFN-γ for 24 h as positive control. The supernatants were collected, and the concentrations of TNF-α, IL-6, IL-1β and IL-8 were determined using ELISA kits (BD Biosciences, USA), following the manufacturer instructions.

**Cellular uptake assessed by flow cytometry and confocal microscopy.** THP-1-derived macrophages were incubated with S-GQDs or N-GQDs at 25, 50, and 100 µg/mL concentration for 2, 4, and 8 h. Then the cells were gently washed and collected for flow cytometry analysis. The mean fluorescence intensity of the untreated control was normalized to 1. The macrophages were detached and resuspended in PBS and analyzed by the flow cytometry (Gallios, Beckman Coulter, USA). The data shown represents the average fluorescence intensity from 4 independent experiments analyzing 10000 cells per experiment.

For confocal microscopy, THP-1-derived macrophages were incubated with 25 µg/mL N-GQDs for 30 min, 2, 4 and 8 h. Then, the membranes were stained with CellMask™ plasma membrane stains (Invitrogen, C10046, deep red). Untreated macrophages were used as control. Confocal images were obtained with a ZEISS Axio Observer Z1 spinning disk confocal microscope. The images were treated with ImageJ software. The fluorescence signal from CellMask was obtained using a 649-nm laser excitation, whereas N-GQDs were recorded using a 488-nm laser excitation. The fluorescence of doped GQDs was set to red and the membrane to blue.

**Enzyme-mediated degradation of S-GQDs and N-GQDs.** Our group previously studied the enzymatic degradation of graphene quantum dots by human peroxidases,[25] In that work 160 µg of dots were used to evaluate the MPO- and EPO-mediated degradation. In order to better compare the enzymatic degradation of GQDs and doped GQDs, we first selected the same amount of 160 µg for S-GQDs and we decided then to use the half dose of 80 µg/mL for N-GQDs to evaluate if a lower starting amount of dots could lead to a complete degradation. In details, for the degradation
of doped GQDs by human enzyme-mediated degradation, a mixture of 160 µg of S-GQDs or 80 µg of N-GQDs, and 80 µg of MPO was suspended in 1 mL of 50×10⁻³ M PBS containing 140×10⁻³ M NaCl and 100×10⁻⁶ M DTPA. H₂O₂ was added at a rate of 200×10⁻⁶ M/h. MPO was renewed every 5 h (5 additions). The reaction mixtures were maintained at 37°C for 25 h. The control was performed by incubating GQDs with only H₂O₂. Different aliquots (200 µL) were withdrawn from the samples at incubation time points of 0, 15 and 25 h for further analysis.

*Peroxynitrite-mediated degradation of S-GQDs and N-GQDs.* S-GQDs or N-GQDs (160 µg) suspended in PBS were incubated with peroxynitrite for 5 days. Peroxynitrite was generated by SIN-1 (Sigma), simultaneously producing nitric oxide and superoxide, or a NO donor (PAPA NONOate, Sigma), combined with superoxide-generating X/XO (Sigma). PAPA NONOate and X/XO alone were used as controls. SIN-1, PAPA NONOate and xanthine were added 5 times per day (every 1.5 h) and the final concentration was 300 µM. Additionally, 1.5 µL of solution containing 0.125 mU of xanthine oxidase were added in the morning and in the evening. After 5 days of treatment, the samples were stored at -20°C for further analysis.

*Fluorescence spectroscopy.* The fluorescence of the samples was analyzed by using a JASCO FP-8300 fluorescence spectrometer. Briefly, 5 µL of each fresh aliquot were diluted with Milli-Q water® to 1 mL. The 100% fluorescence intensity at the different incubation time points corresponds to the fluorescence of the control sample.

*Raman spectroscopy.* Ten µL of each initial aliquot were dropped on silicon substrates and dried for 24 h before the analysis. The Raman spectra were acquired using a Renishaw inVia microscope equipped with a 532-nm laser (exposure time: 1s; laser power: 1%; accumulations: 1). A minimum of 30 measurements per sample in different sample locations was recorded.

*HRTEM.* Six µL of each aliquot were dropped on a TEM grid made of a holey carbon membrane of 10 nm thickness on copper grids and dried for 30 min under a lamp. Milli-Q® water was dropped on the grids to wash and dried for 24 h before the analysis. The HRTEM analyses were performed on a JEOL 2100F TEM/STEM electron microscope operating at 200 kV.

*N-GQD degradation in macrophages.* N-GQDs at different concentrations (1, 5 or 10 µg/mL) were added to THP-1 macrophages and incubated for 24 h, then the macrophages were washed three times with pre-warmed PBS (at 37°C) to remove the extracellular N-GQDs, and incubated with fresh culture medium or FBS-free medium for another 0, 3 and 5 days (named day 0, day 3 or day 5). The macrophages at day 0, day 3 or day 5 were washed and collected to measure the mean
fluorescence intensity by flow cytometry. The untreated controls at day 0, 3 and 5 underwent the same washing and incubation steps as the treated macrophages at day 0, 3 and 5. For Raman analysis, the macrophages were washed 3 times, collected by centrifugation and then resuspended in 1 mL of PBS. The cells were disrupted by probe sonication (Sonics Vibra-Cell, 150 W) for 1 min to break the cells and obtain well-dispersed suspensions before Raman analysis.

*Measurement of NO production by THP-1 macrophages.* Intracellular NO was determined by live cell permeable sensitive fluorescent indicator DAR-1 (Sigma-Aldrich, USA, 09014) that reacts specifically with NO and generates intense red fluorescence that can be imaged using green filter.
[46-48] Briefly THP-1 macrophages were cultured in complete culture medium with 10% FBS or FBS-free medium for 5 days. The medium was refreshed at day 3. Then, the cells were labeled with DAR-1 at a final concentration of 15 µM. After 90 min, the cells were carefully washed with cold PBS two times and imaging was conducted using a green filter in a fluorescence microscope (ZEISS, Axiovert 200 M) at 10×/0.3 Plan Neofluar magnification and analyzed by Image J software.

*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 $p<0.05$.

**Declaration of competing interest**

The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

**Acknowledgements**

The authors gratefully acknowledge the financial support from the EU Graphene Flagship project (no. 881603), the Ministry of Europe and Foreign Affairs (MEAE) for the support through the PHC MERLION 2020 program (Project N°45242TL), the Interdisciplinary Thematic Institute SysChem via the IdEx Unistra (ANR-10-IDEX-0002) within the program Investissement d'Avenir. The Centre National de la Recherche Scientifique (CNRS), and Jean-Marie Lehn Foundation. The authors are indebted to Cathy Royer and Valérie Demais from the Plateforme Imagerie in Vitro of ITI Neurostra CNRS UAR 3156, University of Strasbourg (Strasbourg, France) for TEM analyses,
Dr. Iwona Janica for Raman analyses, Dr. Dris Ihiawakrim from the Transmission Electron Microscopy Platform at the IPCMS (Strasbourg, France) for HRTEM analyses, and Prof. Fengna Xi for kindly providing the value of photoluminescence quantum yield of doped GQDs.

## References

[1] M. J. Im, J. I. Kim, S.-K. Hyeong, B. J. Moon, S. Bae, *Small* **2023**, *19*, 2304497.

[2] V. Meunier, C. Ania, A. Bianco, Y. Chen, G. Choi, Y. A. Kim, N. Koratkar, C. L., J. M.D. Tascon, M. Terrones, *Carbon* **2022**, *195*, 272.

[3] D. Iannazzo, I. Ziccarelli, A. Pistone, *J. Mater. Chem. B* **2017**, *5*, 6471.

[4] M. C. Biswas, M. T. Islam, P. K. Nandy, M. M. Hossain, *ACS Materials Lett.* **2021**, *3*, 889.

[5] S. Yang, J. Sun, X. Li, W. Zhou, Z. Wang, P. He, G. Ding, X. Xie, Z. Kang, M. Jiang, *J. Mater. Chem. A* **2014**, *2*, 8660.

[6] R. Liu, D. Wu, X. Feng, K. Muellen, *J. Am. Chem. Soc.* **2011**, *133*, 15221.

[7] W.-W. Liu, Y.-Q. Feng, X.-B. Yan, J.-T. Chen, Q.-J. Xue, *Adv. Funct. Mater.* **2013**, *23*, 4111.

[8] Y. Li, Y. Zhao, H. Cheng, Y. Hu, G. Shi, L. Dai, L. Qu, *J. Am. Chem. Soc.* **2011**, *134*, 15.

[9] K. Li, J. Chen, Y. Yan, Y. Min, H. Li, F. Xi, J. Liu, P. Chen, *Carbon* **2018**, *136*, 224.

[10] C. Shen, S. Ge, Y. Pang, F. Xi, J. Liu, X. Dong, P. Chen, *J. Mater. Chem. B* **2017**, *5*, 6593.

[11] N. Sohal, B. Maity, S. Basu, *RSC Adv.* **2021**, *11*, 25586.

[12] H. Lin, Z. Song, A. Bianco, *J. Environ. Sci. Health-B* **2021**, *56*, 333.

[13] S. Wang, I. S. Cole, Q. Li, *RSC Adv.* **2016**, *6*, 89867.

[14] J. Ju, R. Zhang, S. He, W. Chen, *RSC Adv.* **2014**, *4*, 52583.

[15] I. J. Gómez, M. V. Sulleiro, A. Dolečková, N. Pizúrová, J. Medalová, R. Roy, D. Nečas, L. Zajíčková, *J. Phys. Chem. C* **2021**, *125*, 21044.

[16] Q. Liu, B. Guo, Z. Rao, B. Zhang, J. R. Gong, *Nano Lett.* **2013**, *13*, 2436.

[17] B. Ma, C. Martin, R. Kurapati, A. Bianco, *Chem. Soc. Rev.* **2020**, *49*, 6224.

[18] V. E. Kagan, N. V. Konduru, W. Feng, B. L. Allen, J. Conroy, Y. Volkov, I. I. Vlasova, N. A. Belikova, N. Yanamala, A. Kapralov, Y. Y. Tyurina, J. Shi, E. R. Kisin, A. R. Murray, J. Franks, D. Stolz, P. Gou, J. Klein-Seetharaman, B. Fadeel, A. Star, A. A. Shvedova, *Nat. Nanotechnol.* **2010**, *5*, 354.
[19] F. T. Andón, A. A. Kapralov, N. Yanamala, W. Feng, A. Baygan, B. J. Chambers, K. Hultenby, F. Ye, M. S. Toprak, B. D. Brandner, A. Fornara, J. Klein-Seetharaman, G. P. Kotchey, A. Star, A. A. Shvedova, B. Fadeel, V. E. Kagan, *Small* **2013**, *9*, 2721.

[20] K. Bhattacharya, R. El-Sayed, F. T. Andón, S. P. Mukherjee, J. Gregory, H. Li, Y. Zhao, W. Seo, A. Fornara, B. Brandner, M. S. Toprak, K. Leifer, A. Star, B. Fadeel, *Carbon* **2015**, *91*, 506.

[21] R. Kurapati, J. Russier, M. A. Squillaci, E. Treossi, C. Ménard-Moyon, A. E. Del Rio-Castillo, E. Vazquez, P. Samorì, V. Palermo, A. Bianco, *Small* **2015**, *11*, 3985.

[22] S. P. Mukherjee, A. R. Gliga, B. Lazzaretto, B. Brandner, M. Fielden, C. Vogt, L. Newman, A. F. Rodrigues, W. Shao, P. M. Fournier, M. S. Toprak, A. Star, K. Kostarelos, K. Bhattacharya, B. Fadeel, *Nanoscale* **2018**, *10*, 1180.

[23] V. E. Kagan, A. A. Kapralov, C. M. St. Croix, S. C. Watkins, E. R. Kisin, G. P. Kotchey, K. Balasubramanian, I. I. Vlasova, J. Yu, K. Kim, W. Seo, R. K. Mallampalli, A. Star, A. A. Shvedova, *ACS Nano* **2014**, *8*, 5610.

[24] G. Peng, M. F. Montenegro, C. N. M. Ntola, S. Vranic, K. Kostarelos, C. Vogt, M. S. Toprak, T. Duan, K. Leifer, L. Bräutigam, J. O. Lundberg, B. Fadeel, *Nanoscale* **2020**, *12*, 16730.

[25] C. Martín, G. Jun, R. Schurhammer, G. Reina, P. Chen, A. Bianco, C. Ménard-Moyon, *Small* **2019**, *15*, 1905405.

[26] R. Weissleder, M. Nahrendorf, M. J. Pittet, *Nat. Mater.* **2014**, *13*, 125.

[27] D. Boraschi, P. Italiani, R. Palomba, P. Decuzzi, A. Duschl, B. Fadeel, S. M. Moghimi, *Semin. Immun.* **2017**, *34*, 33.

[28] O. Lunov, T. Syrovets, C. Loos, J. Beil, M. Delacher, K. Tron, G. U. Nienhaus, A. Musyanovych, V. Mailander, K. Landfester, T. Simmet, *ACS Nano* **2011**, *5*, 1657.

[29] C. Polasky, T. Studt, A.-K. Steuer, K. Loyal, K. Ludtke-Buzug, *Front. Mol. Biosci.* **2022**, *9*, 811116.

[30] Y. Qin, Z.-W. Zhou, S.-T. Pan, Z.-X. He, X. Zhang, J.-X. Qiu, W. Duan, T. Yang, S.-F. Zhou, *Toxicology* **2015** 327, 62.

[31] J. Russier, E. Treossi, A. Scarsi, F. Perrozzi, H. Dumortier, L. Ottaviano, M. Meneghetti, V. Palermo, A. Bianco, *Nanoscale* **2013**, *5*, 11234.

[32] O. Vittorio, V. Raffa, A. Cuschieri, *Nanomedicine.* **2009**, *5*, 424.

[33] H. Zhou, K. Zhao, W. Li, N. Yang, Y. Liu, C. Chen, T. Wei, *Biomaterials* **2012**, *33*, 6933.

[34] M. Baggiolini, B. Dewald, B. Moser, *Adv. Immunol.* **1994**, *55*, 97.
[35] E. D. Ianni, P. Møller, U. B. Vogel, N. R. Jacobsen, *Mutat. Res.-Genet. Toxicol. Environ. Mutagen.* **2021**, *872*, 503405.

[36] S. Chortarea, O. C. Kuru, W. Netkueakul, M. Pelin, S. Keshavan, Z. Song, B. Ma, J. Gomes, E. V. Abalos, L. A. V. Luna, T. Loret, A. Fordham, M. Drummond, N. Kontis, G. Anagnostopoulos, G. Paterakis, P. Cataldi, A. Tubaro, C. Galiotis, I. Kinloch, B. Fadeel, C. Bussy, K. Kostarelos, T. Buerki-Thurnherr, M. Prato, A. Bianco, P. Wick, *J. Hazard. Mater.* **2022**, *435*, 129053.

[37] Y. Mao, C. Zhao, S. Ge, T. Luo, J. Chen, J. Liu, F. Xi, J. Liu, *RSC Adv.*, **2019**, *9*, 32977.

[38] R. Kurapati, J. Russier, M. A. Squillaci, E. Treossi, C. Ménard-Moyon, A. E. Del Rio-Castillo, E. Vazquez, P. Samorì, V. Palermo, A. Bianco, *Small* **2015**, *11*, 3985.

[39] A. C. Ferrari, *Solid State Commun.* **2007**, *143*, 47.

[40] R. Kurapati, S. P. Mukherjee, C. Martín, G. Bepete, E. Vázquez, A. Pénicaud, B. Fadeel, A. Bianco, *Angew. Chem.* **2018**, *57*, 11722.

[41] J. P. M. Almeida, A. L. Chen, A. Foster, R. Drezek, *Nanomedicine* **2011**, *6*, 5.

[42] Ngo, S. Ahmed, C. Blackadar, B. Bussin, Q. Ji, S.M. Mladjenovic, Z. Sepahi, W.C.W. Chan, *Adv. Drug Deliv. Rev.* **2022**, *185*, 114238.

[43] C. Prolo, M. N. Álvarez, R. Radi, *Biofactors* **2014**, *40*, 2.

[44] L. Wang, Y. Wang, T. Xu, H. Liao, C. Yao, Y. Liu, Z. Li, Z. Chen, D. Pan, L. Sun, M. Wu, *Nat. Commun.* **2014**, *5*, 1.

[45] J. Gong, Z. Zhang, Z. Zeng, W. Wang, L. Kong, J. Liu. P. Chen, *Carbon* **2021**, *184*, 554.

[46] H. Li, A. Wan, *Analyst* **2015**, *140*, 7129.

[47] O. B. Halbach, *Nitric Oxide Biol. Chem.* **2003**, *9*, 217.

[48] M. J. Akhtar, M. Ahamed, H. A. Alhadlaq, S. Kumar, S. A. Alrokayan, *BBA – Gen. Subj.* **2020**, *1864*, 129452.