# N -Acetylcysteine-Functionalized Gold Nanoparticles via Photoclick Reaction as Antioxidant Nanomedicine

Yang Zhang, Greta Malavasi, Philippe Nizard, Virginie Gueguen, Cédric Chauvierre, Fan Sun, Pauline Cornette, Miryana Hémadi, Aazdine Lamouri, Claire Mangeney, et al.

► To cite this version:

Yang Zhang, Greta Malavasi, Philippe Nizard, Virginie Gueguen, Cédric Chauvierre, et al. N -Acetylcysteine-Functionalized Gold Nanoparticles via Photoclick Reaction as Antioxidant Nanomedicine. ACS Applied Nano Materials, 2025, 10.1021/acsnanm.5c02940. [https://doi.org/10.1021/acsnanm.5c02940]

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
N-Acetylcysteine-Functionalized Gold Nanoparticle via Photoclick Reaction as
Antioxidant Nanomedicine

Yang Zhang ¹,², Greta Malavasi ¹,², Philippe Nizard ¹, Virginie Gueguen ²,
Cédric Chauvierre ², Fan Sun³, Pauline Cornette ¹, Miryana Hémadi ⁴, Aazdine Lamouri ⁴,
Claire Mangeney¹*, Graciela Pavon-Djavid²*, Yun Luo¹,*

¹ Université Paris Cité, CNRS, Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, F-75006 Paris, France.

² Université Sorbonne Paris Nord, Université Paris Cité, INSERM U1148, Laboratory for Vascular Translational Science, Nanotechnologies for Vascular Medicine and Imaging Team, F-75018 Paris, France

³ PSL Université, Chimie Paris Tech, IRCP, CNRS UMR 8247, 11, rue Pierre et Marie Curie, 75005 Paris, France.

⁴ Université Paris Cité, CNRS, ITODYS, F-75013, Paris, France.

*Corresponding authors: Graciela Pavon-Djavid: graciela.pavon@univ-paris13.fr; Claire Mangeney: claire.mangeney@u-paris.fr; Yun Luo: yun.luo@u-paris.fr

ABSTRACT

Reactive oxygen species (ROS) are highly reactive molecules regulated by a balance of enzymatic and non-enzymatic antioxidant systems. In the heart, ROS play crucial roles in maintaining cellular homeostasis, influencing processes such as cell proliferation, differentiation, and excitation-contraction coupling. However, when ROS levels surpass the antioxidant defense capacity, oxidative stress happens, leading to cellular and molecular damage that can ultimately result in cardiac dysfunction.

In this study, we developed an antioxidant nanomedicine based on gold nanoparticles (Au NPs) functionalized with N-acetylcysteine (NAC), designed to counteract oxidative stress in the heart. NAC is an essential endogenous antioxidant, which can effectively prevent local ROS formation and protects human endothelial and stem cells from oxidative stress.

Using a thiol-yne photo-click chemistry approach, NAC was efficiently grafted onto Au NPs pre-functionalized with 4-ethynylbenzene diazonium salts. The resulting Au NPs exhibit excellent biocompatibility, along with significant antioxidant activity, good compatibility with human endothelial cells and efficient cellular internalization.
Keywords: gold nanoparticles; surface functionalization; aryl diazonium salts; thiol-yne
photoclick chemistry; antioxidant nanoparticles; N-acetylcysteine.

# 1. INTRODUCTION

Energy production in eukaryotic cells is achieved through the respiratory chain within the mitochondria. In this process, various reactive oxygen species (ROS) are produced, including superoxide and peroxide. The concentration of ROS is typically kept at minimal levels in a healthy state to support crucial cellular functions. Nevertheless, when the level of ROS surpasses the endogenous regulatory capacity, these highly reactive compounds initiate oxidation of molecules like membrane lipids, proteins, and DNA, leading to a loss of their normal functions ¹. In situations of oxidative stress, characterized by elevated ROS levels, cellular and tissue malfunctions contribute to the development of pathologies such as cardiovascular diseases, diabetes, and neurodegenerative disorders ². In physiological conditions, the concentration of ROS is controlled through the regulation of various intracellular enzymes (such as superoxide dismutase, peroxidase, and catalase) and extracellular antioxidant compounds like vitamin E, vitamin C, and coenzyme Q. These enzymes and compounds function to eliminate oxygen radicals and reduce the harmful effects induced by ROS. However, their bioavailability is limited, and often insufficient in situations of oxidative stress due to inadequate production, chemical instability, low solubility, and inefficient cellular uptake.

N-acetylcysteine (NAC) is an antioxidant and a precursor of glutathione (GSH), one of the body's most crucial endogenous antioxidants. It is a FDA (U.S. Food and Drug Administration) approved compound widely used for the treatment of acetaminophen (paracetamol) overdose and more recently as a mucolytic agent in respiratory diseases ³. NAC's antioxidant action is twofold: it directly reacts with reactive oxygen and nitrogen species, and it indirectly boosts intracellular cysteine levels, promoting GSH synthesis. This dual action helps alleviate oxidative stress and compensates for GSH deficiencies, thereby enhancing the body's natural antioxidant defenses ³.

However, the administration efficacy of NAC and almost all exogenous antioxidants is limited due to their poor absorption, difficulty in crossing cell membranes and degradation during administration, all of which contribute to their limited bioavailability. According to the study of Olsson *et al*. ⁴, the bioavailability of free NAC is very low after oral
administration (< 10%), and only a very small quantity of the intact molecule reaches the plasma and tissues.

To solve these problems, one of the strategies developed so far is to covalently graft or to entrap antioxidants onto nanoparticles, in order to offer superior stability, gradual and prolonged release, and targeted delivery of antioxidants. This approach has garnered significant attention for its potential in treating oxidative damage-related pathologies due to their distinct advantages over conventional molecular antioxidants. Various nanoparticles loaded with NAC have been designed for diverse clinical uses.

Gold nanoparticles (Au NPs) are particularly promising candidates for grafting antioxidants due to their inert, nontoxic nature and excellent biocompatibility. As reported in literature, when NAC is bonded to substrates such as Au NPs, its antioxidant effect can be further enhanced through synergistic interactions, offering potential benefits beyond those of NAC or Au NPs alone⁵. Nishanthi Ezhumalai et al.⁶ reported the antibacterial properties of NAC-conjugated cholic acid (NAC-ACyCA)-stabilized spherical Au NPs in aqueous medium. NAC-ACyCA was first synthesized via thiol-yne click chemistry under UV light exposure and was then used as both a reducing and capping agent for the *in situ* preparation of Au NPs under sunlight. However, this *in situ* synthesis method resulted in poor control over NP size dispersion and promoted aggregation in physiological environments. Fajstavr et al.⁷ reported an alternative approach where the Au NPs were first synthetized by sputter deposition in polyethylene glycol and then modified using NAC, grafted via thiolate bonds. This strategy led to an increase in Au NP size during solution aging, and high NAC concentrations caused nanoparticle aggregation⁷. These insights from the literature highlight the necessity of developing a new strategy for grafting NAC onto Au NPs, aimed at strengthening the gold-NAC interface and, consequently, improving the nanohybrids' stability.

To address this issue, we propose herein an innovative strategy combining aryl diazonium salts and photo-click chemistry (see illustration in **Fig. 1**) to create a robustly anchored NAC coating on Au NPs and evaluate its potential as an antioxidant for cardiovascular treatments. It is worth noting that while NPs have been extensively investigated as potential antioxidants⁸⁻¹⁴, their combination with molecular antioxidants for cardiovascular therapy remains largely unexplored, likely due to challenges in achieving stable and robust covalent conjugation on NPs surface.
Aryldiazonium salts were selected as coupling agents for the attachment of NAC on AuNPs as they exhibit several advantages such as their ease of preparation, rapid grafting using environmentally-friendly conditions (in water, at room temperature, in the presence of air), and formation of strong covalent Au-carbon bonds, ensuring excellent stability of the molecular coating while preserving the colloidal properties of NPs ¹⁵⁻¹⁷. Moreover, these molecules offer a large choice of reactive functional groups, available for post-functionalization reactions. Recent studies have shown that the post-functionalization of gold gratings modified by 4-ethynylphenyl groups derived from diazonium salts can be achieved through thiol-yne reactions ¹⁸. However, it is noteworthy that aryl diazonium salts have never been used in conjunction with thiol-yne reactions for the surface grafting of NAC onto colloidal Au NPs. This proof-of-concept study thus offers new breakthroughs both for the surface engineering of colloidal Au NPs and for the design of antioxidant-loaded NPs.

The resulting NAC-loaded Au NPs were extensively characterized for size and shape using Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM), while their surface chemistry was analyzed using Surface-Enhanced Raman Spectroscopy (SERS) and X-ray Photoelectron Spectroscopy (XPS). Antioxidant activity was assessed through the Oxygen Radical Absorbance Capacity (ORAC) assay. Biocompatibility was evaluated *in vitro* using human endothelial cells by monitoring cell viability (MTT assay) and morphology (nucleus/cytoskeleton staining), and NPs uptake was assessed using fluorescence/SERS colocalization imaging.

## 2. EXPERIMENTAL SECTION

### 2.1 Materials

4-ethynylbenzenediazonium tetrafluoroborate (DCC) was synthesized as described previously ¹⁵. Trisodium citrate, Polyvinylpyrrolidone (PVP, 10 kDa), Potassium persulfate powder; 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), NaCl, (4,5-dimethylthiazol-2-yl)2,5diphenyltetrazolium bromide (MTT), N-acetylcysteine, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropriophenone (Irgacure 2959), and Fluorescein were purchased from Sigma-Aldrich (St. Louis, MO, USA). HUVECs (ATCC® CRL-1730, HUV-EC-C™)
were purchased from ATCC (Middlesex, TW11 0LY, UK). Dulbecco's modified Eagle's medium (DMEM) (1X) + GlutaMAXTM, Antibiotic (ATB), Fetal Bovine Serum (FBS), Trypsin, Phosphate-buffered saline (PBS) solution (1X), 4',6'-diamidino-2-phénylindole (DAPI), Alexa Fluor™ Plus 555 Phalloidin, and hydrogen tetrachloroaurate (III) trihydrate (HAuCl₄·3H₂O) were purchased from Thermo-Fisher Scientific SAS (Illkirch, France).

## 2.2 Synthesis and functionalization of gold nanoparticles with NAC

Spherical Au NPs (Cit-Au NPs) were prepared through the modified Turkevich method ¹⁹, by citrate reduction of HAuCl₄. In brief, 50 mL of HAuCl₄ (1 mmol.L⁻¹) was heated to boiling under continuous stirring, followed by the addition of 5 mL of trisodium citrate (39 mmol.L⁻¹). The mixture was maintained at boiling for ca. 10-15 min until the color changed to dark red. After cooling to room temperature, the colloidal Au NPs were stabilized with 0.001 wt% of PVP and stored at 4°C.

To prepare Au@DCC sample, Au NPs (300 µg.mL⁻¹) were incubated with 10 µmol.L⁻¹ of aqueous DCC solution (synthesis described in our previous publication ¹⁵) for 1h at 25°C. Then, 20 µmol.L⁻¹ of NAC and 20 µmol.L⁻¹ of the photoinitiator Irgacure 2959 were added to the Au@DCC aqueous dispersion, which was degassed with argon for at least 15 min. The thiol-yne photo-click chemistry was initiated under UV light (365 nm, power density = 20 mJ cm⁻²) for a duration ranging from 1 to 30 minutes. The resulting Au@DCC@NAC NPs were purified through three cycles of centrifugation and redispersion in deionized water. The Au NP reference samples consisting of bare citrate-capped NPs or NPs functionalized solely with either NAC or DCC, were prepared following the same protocol, as summarized in Table 1.

<table><caption>**Table 1:** Summary of preparation parameters for each sample. The concentration of Cit-Au NPs was fixed at 300 µg.mL⁻¹ for all samples.</caption><thead><tr><th>Sample</th><th>DCC<br>(µmol.L<sup>-1</sup>)</th><th>NAC<br>(µmol.L<sup>-1</sup>)</th><th>Photoinitiator<br>(µmol.L<sup>-1</sup>)</th><th>UV<br>exposure<br>(min)</th></tr></thead><tbody><tr><td>Cit-Au NPs</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Au@DCC</td><td>10</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
<table><tr><td>Au@DCC-UV</td><td>10</td><td>0</td><td>20</td><td>30</td></tr><tr><td>Au@NAC</td><td>0</td><td>20</td><td>0</td><td>0</td></tr><tr><td>Au@NAC-UV</td><td>0</td><td>20</td><td>20</td><td>30</td></tr><tr><td>Au@DCC@NAC</td><td>10</td><td>20</td><td>20</td><td>1-30</td></tr></table>

## 2.3 Physicochemical characterization methods

The extinction spectra of Au colloidal solutions were recorded by UV-vis spectroscopy (Shimadzu 2700 UV-vis spectrometer). The concentration of Au colloidal solutions was quantified according to Beer-Lambert law <sup>20</sup>. The hydrodynamic sizes were measured by DLS (CORDOUAN Visco Kin). The zeta potential values were obtained from the diluted Au NPs dispersions in distilled water (Cit-Au NPs, Au@DCC, Au@NAC and Au@DCC@NAC) using a Zeta potential analyzer (WALLIS) at room temperature. Each sample was measured in triplicate, and the experiment was repeated twice. The results were presented by mean ± SD.

Morphological investigations of both bare and functionalized Au NPs were performed with TEM using a Tecnai microscope operating at 120 kV equipped with a 4k × 4k Eagle camera (Thermofisher, USA). Prior to imaging, the Au colloidal solutions were deposited on carbon grids. The mean particle size for each sample was determined by analyzing at least 100 particles from various TEM images using ImageJ software. The results were presented by mean ± SD. The Raman spectra and images were obtained using a Horiba XploRA PLUS Raman microscope, utilizing a 638 nm laser. X-ray photoelectron spectra (XPS) of the dried samples were recorded using a Thermo VG Scientific ESCALAB 250 system, which was fitted with a micro-focused, monochromatic Al Kα X-ray source (1486.6 eV) and a magnetic lens. An X-ray beam of 650 µm was used (15 kV × 200 W). The pass energy was set at 150 and 40 eV for the survey and the narrow regions, respectively. An electron flood gun was used, under a 2.10<sup>-8</sup> mBar partial pressure of argon, for static charge compensation <sup>21</sup>. Data acquisition and processing were carried out using CasaXPS software.

## 2.4 Establishment of a calibration curve for the quantification of grafted NAC

The efficiency of the photo-click reaction was evaluated using SERS technology with a calibration curve constructed from Cit-Au NPs mixed with varying concentrations of NAC.
Briefly, 1 mL of Cit-Au NPs (300 µg.mL⁻¹) was combined with NAC at concentrations ranging from 0 to 30 µmol.L⁻¹, and then directly centrifuged at 13,000 rpm for 10 minutes. 1 µL of the resulting pellet was dried on a glass slide for Raman spectroscopy (Fig. S1a). As shown in Fig. S1b-c, in the spectral region of 200–500 cm⁻¹, the peak associated with Cit-Au NPs sample at ca. 240 cm⁻¹ (referring to Au-Cl⁻ vibration ²²) was replaced by a new peak at ca. 295 cm⁻¹, corresponding to the SERS signal of bonded NAC (likely due to Au-S vibration ²³). By determining the area ratio of these two peaks (I₂₉₅/I₂₄₀) after deconvolution, a calibration curve for NAC concentration was established (Fig. S1d). The raw data in the range of 200-400 cm⁻¹ for Cit-Au NPs mixed with various concentrations of NAC are provided in Fig. S1e.

## 2.5 Redox characterization - Oxygen Radical Absorbance Capacity test – ORAC

The antioxidant capacity of samples was assessed by the ORAC method as previously described ²⁴ with modifications ²⁵. Briefly, solutions of Fluorescein (1 µmol.L⁻¹), AAPH (20 mM), NAC (standard) (1-50 µmol.L⁻¹), Trolox (control, 0-40 µmol.L⁻¹), and Au NPs (Cit-Au NPs, Au@NAC and Au@DCC@NAC) were prepared in PBS. Fluorescein solution (150 µL) and either 25 µL of samples, controls, blank (PBS), or standard were added to each well of a 96-well microplate. Finally, pre-heated AAPH at 37°C (25 µL) was added to the wells to start the reaction. Fluorescence was monitored at 485/528 nm (Ex/Em) using a fluorescence plate reader (Varioskan), every minute for 60 min (3 × 8 measurements/well). The sample antioxidant activity was determined from their ability to keep the fluorescence signal of the indicator in the presence of peroxyl radicals. The AUC and AUCₙet were calculated using Eq. 1, Eq. 2., where f₁ is the initial fluorescence reading at 0 min and fₙ is the fluorescence measured at time n. Final ORAC values were calculated by using the regression equation between the Trolox concentration and the AUCₙet and were expressed as Trolox equivalents (TEAC) as millimolar per liter.

$$AUC = 0.5 + \left(\frac{f_2}{f_1}\right) + \left(\frac{f_3}{f_1}\right) + \cdots + 0.5 \left(\frac{f_n}{f_1}\right) \quad (1)$$

$$AUC_{net} = AUC_{sample} - AUC_{blank} \quad (2)$$

## 2.6 Cells viability
HUVEC were used for the biocompatibility experiments 26,27, where cells were grown in DMEM, with low glucose content, supplemented with 10% (v/v) FBS and 1% penicillin-streptomycin-amphotericin (PSA) (called complete media) at 37°C and 5% CO2. Cells were detached with trypsin after reaching 80% confluence and seeded at a density of 3×10⁴ cells/well (100 µL) in 96-well cell culture plates. After 24 hours culture, the medium was removed, and 100 µL of fresh complete medium containing the samples (Cit-Au NPs, Au@NAC and Au@DCC@NAC) at different concentrations (15-120 µg.mL⁻¹) were added. Positive (cells were treated with dimethyl sulfoxide; PC) and negative (cells without any treatment; NC) cytotoxicity controls were also included in each test. After another 24 h incubation, the medium was replaced with a PBS solution containing MTT reagent (0.5 mg.mL⁻¹). Cells were then cultured for 3 h away from light in a humidified incubator at 37°C to allow MTT to be metabolized by living cells. After discarding the supernatant, 2-propanol (100 µL/well) was added to dissolve the formazan crystals. The absorbance of the samples was recorded at a 570 nm wavelength with Varioskan. The percentage of proliferation was calculated with respect to control. The cytotoxicity of the agent was measured as follows:

$$ \text{Viability (\%)} = (\text{OD of treated cells} / \text{OD of NC}) \times 100 $$

## 2.7 Cells morphology

25×10³ cells/well (500 µL of cell suspension in complete DMEM) were seeded in 4-well glass slide chamber (Lab-Tek® II Chamber Slide™, Nunc™) and incubated for 24 h. The medium was then removed, and 500 µL of fresh complete medium containing the samples (Cit-Au NPs, Au@NAC and Au@DCC@NAC) at different concentrations (30-120 µg.mL⁻¹) were added and cells were cultured for 24 hours. After washing in PBS, cells were fixed using formaldehyde (4% in PBS), and permeabilized (triton 100X 0.1% in PBS). Cells were stained with Phalloidin solution (0.165 µmol.L⁻¹ in PBS) and DAPI (300 nmol.L⁻¹) and then mounted. Fluorescent signals were recorded using Zeiss fluorescence microscope.

## 2.8 Cellular uptake of nanoparticles assessed by colocalization of fluorescence imaging and Raman mapping
10x10⁴ cells/well (2 mL in complete DMEM) were seeded in a 6-well plate and incubated for 24 h. The medium was then removed and replaced by fresh complete medium containing the Cit-Au NPs or Au@DCC@NAC samples (50 µg.mL⁻¹). Cells were incubated with the NPs for 3 hours. After washing in PBS, cells were fixed using formaldehyde (4% in PBS) for 30 min, the slides were then stored in sodium azide (0.002%). Cell entrapment was observed and analyzed using an XploRA Plus Raman Microscope coupled to fluorescence imaging (stained with Hoechst and WGA) modality.

## 2.9 Statistical Analysis

All experiments were performed in at least three independent replicates. For ORAC assays, each condition was tested in triplicate wells per experiment. For cell viability assays, three independent experiments were conducted, each with six technical replicates. Data were first inspected for potential outliers and tested for normal distribution and homogeneity of variances. Results are expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey's post-hoc multiple comparison test to identify pairwise differences. Statistical significance was defined as p < 0.05. All analyses were conducted using JMP® 18.2.2 software.

# 3. RESULTS AND DISCUSSION

## 3.1 Preparation and chemical characterization of Au@DCC@NAC

The thiol-yne photo-click chemistry was performed on citrate-capped gold nanoparticles (Cit-Au NPs) using 4-ethynylbenzenediazonium salts (DCC) as a coupling agent ²⁸. Briefly, a colloidal solution of Cit-Au NPs (300 µg.mL⁻¹) was mixed with DCC (10 µmol.L⁻¹) at 25°C for 1 hour, resulting in surface-modified Au@DCC NPs (Fig. 1a). After removing dissolved oxygen by argon bubbling, the solution of Au@DCC NPs was then mixed with a photoinitiator (20 µmol.L⁻¹) and NAC (20 µmol.L⁻¹) and subjected to UV irradiation (365 nm) for 10 minutes. The final Au@DCC@NAC products were washed by 3 cycles of centrifugation and redispersion in water. TEM images (Fig. 1b-g) reveal minimal changes in the morphology and colloidal state of Au NPs throughout the chemical grafting and photo-click chemistry processes. The mean particle sizes, determined from distribution curves (Fig. S2a-c) and summarized in Table 2, are 12 ± 3 nm for Cit-Au NPs, 12 ± 3 nm for Au@DCC, and 12 ± 4 nm for Au@DCC@NAC.
DLS measurements (Table 2) showed a slight increase in the hydrodynamic size from 24 nm for Cit-Au NPs to 29 nm for Au@DCC. The zeta potential value also increased from -33 mV for Cit-Au NPs to -14 mV for Au@DCC, in line with the replacement of surface-adsorbed citrate (negatively charged) by grafted polyaryl layers (with neutral charges). Upon thiol-yne photo-click reaction with NAC, the hydrodynamic size increased to 42 nm, while the zeta potential slightly decreased (-15 mV), which can be explained by the surface immobilization of NAC, which presents carboxylate groups, around the AuNPs. The increase in hydrodynamic size of Au@DCC@NAC is likely due to the swelling of the surface layer caused by multiple functionalization of DCC and NAC. For comparison, the direct adsorption of NAC onto Au NPs, without addition of DCC, was also investigated (sample Au@NAC, reaction shown in Fig. S3a). Here, the mean particle size (12 nm, cf. Table 2 and Fig. S3b-d) and hydrodynamic diameter (30 nm) was close to that of Cit-Au NPs, while the zeta potential was comparable to that of Au@DCC@NAC, consistent with the direct replacement of citrate ligands by NAC through Au–S bonds.
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** (a) Schematic illustration of the thiol-yne photo-click chemistry process. Citrate-capped gold nanoparticles (Cit-Au NPs) were first functionalized with DCC to form Au@DCC, followed by UV irradiation in the presence of a photoinitiator and NAC, resulting in Au@DCC@NAC. TEM images of Cit-Au NPs (b, c), Au@DCC (d, e) and Au@DCC@NAC (f, g) samples, showing the absence of noticeable morphological alteration throughout the functionalization process.</figcaption></figure>

<table><caption>**Table 2.** Mean particle size (derived from TEM images), hydrodynamic size (measured by DLS) and Zeta potential of Cit-Au NPs, Au@DCC, Au@NAC and Au@DCC@NAC samples.</caption><thead><tr><th>Sample</th><th>Mean particle size<br>from TEM (nm)</th><th>Hydrodynamic size<br>from DLS (nm)</th><th>Zeta potential<br>(mV)</th></tr></thead><tbody><tr><td>Cit-Au NPs</td><td>12 ± 3</td><td>24 ± 1</td><td>-33 ± 4</td></tr><tr><td>Au@DCC</td><td>12 ± 3</td><td>29 ± 4</td><td>-14 ± 3</td></tr></tbody></table>
<table><tr><td>Au@NAC</td><td>12 ± 4</td><td>30 ± 3</td><td>-17 ± 2</td></tr><tr><td>Au@DCC@NAC</td><td>12 ± 4</td><td>42 ± 1</td><td>-15 ± 2</td></tr></table>

The surface composition of Cit-Au NPs, Au@DCC, Au@DCC@NAC was evaluated using XPS, and compared with Au@NAC sample (Fig. S4). The survey spectra of all samples (Fig. S4) display intense peaks related to Au4f (at ca. 84 eV), C1s (at ca. 284.8 eV) and O1s (at ca. 530 eV). The surface elemental compositions are summarized in Table 3. In Cit-Au NPs, the Au signal is strongly attenuated due to the presence of citrate ligands, which are rich in carbon and oxygen. Some contamination is also evident from the detection of sulfur and nitrogen signals unrelated to the citrate coating, which may originate from environmental exposure, or handling-related impurities. Upon functionalization with the diazonium salt DCC (Au@DCC sample), a marked increase in the nitrogen signal suggests that surface modification occurs, at least in part, via azophenyl radical pathways, as previously reported in other studies²⁹. Following the reaction with NAC, an increase in the sulfur signal was observed for both Au@NAC and Au@DCC@NAC samples, as expected from the immobilization of this sulfur-containing molecule. Notably, the S/N ratio is close to 1 for Au@NAC, consistent with the theoretical elemental ratio of NAC (C₅H₉NSO₃), whereas it decreases to approximately 0.6 for Au@DCC@NAC, likely due to the presence of azo bonds within the polyaryl coating.

<table><caption>**Table 3.** Surface chemical composition (at%) of Cit-Au NPs, Au@DCC, Au@DCC@NAC and Au@NAC samples, as determined by XPS analysis.</caption><thead><tr><td>Element</td><td>Cit-Au NPs</td><td>Au@DCC</td><td>Au@NAC</td><td>Au@DCC@NAC</td></tr></thead><tbody><tr><td>Au</td><td>1.9</td><td>35.8</td><td>38.7</td><td>37.4</td></tr><tr><td>S</td><td>0.3</td><td>0</td><td>2.6</td><td>2.8</td></tr><tr><td>C</td><td>62.6</td><td>48.5</td><td>42.2</td><td>43.4</td></tr><tr><td>O</td><td>35.2</td><td>9.9</td><td>14.0</td><td>12.0</td></tr><tr><td>N</td><td>0</td><td>5.8</td><td>2.5</td><td>4.4</td></tr></tbody></table>

Deconvolution of the C1s and S2p high-resolution spectra was performed to provide deeper insights into the surface chemistry of the AuNP-coated samples. The high-resolution C1s spectrum (Fig. 2) of the Cit-Au NPs revealed four components: C-C/C-H (284.8 eV), C-O (286.3 eV), O=C-O⁻ (288.1) and O=C-OH (289.6 eV) due to the citrate capping molecules and some surface contamination. The shape of the C1s signal for Au@DCC sample was notably different with four components assigned to C≡C/C=C/C-
C/C-H (284.8 eV), C-O (285.9 eV), C-N=N (carbon linked to azo bond, 287.9 eV) and O=C-OH (very weak peak at 289.7 eV due to surface contaminants or some remaining citrates). After thiol-yne photo-click chemistry, the C1s spectrum of Au@DCC@NAC further evolved, revealing five distinct peaks corresponding to C=C/C-C/C-H (284.8 eV), C-O/C-N/C-S (286.1 eV), C-N=N/N-C=O (287.9 eV), and O=C-OH (289.2 eV). The two latter components can be respectively attributed to the amide and carboxylic functional groups of NAC and are also observed in the C1s spectrum of Au@NAC.

Deconvolution of the S 2p core-level spectra (Fig. 2) for both Au@NAC and Au@DCC@NAC revealed distinct spectral profiles, indicating differences in sulfur speciation between the two samples. For Au@NAC, two peaks centered at 161.7 eV (S 2p<sub>3/2</sub>) and 162.9 eV (S 2p<sub>1/2</sub>), with a characteristic spin-orbit splitting of 1.2 eV, are observed, consistent with the formation of Au–S bonds, suggesting a predominantly thiolate-type attachment to the Au NPs, as typically reported for molecular thiols chemisorbed on gold surfaces<sup>30</sup>. An additional component appears at higher binding energy (around 169 eV), which can be attributed to trace oxidized sulfur species, likely contaminants, such as sulfonates or sulfates. These two components (Au–S and contaminants) can be also observed in Au@DCC@NAC. The detection of a thiolate–gold bond suggests that some NAC molecules manage to reach and bind directly to the gold surface, likely because the polyaryl DCC layer does not fully cover the entire AuNP surface, leaving accessible areas for direct attachment. In addition to these peaks, two additional components are observed at higher binding energies for Au@DCC@NAC - at 162.3/163.5 eV and 164.4/165.6 eV -which can be attributed to thioether groups formed via thiol-yne chemistry<sup>31</sup>, either coordinated to the gold surface or present as unbound species. These results suggest that NAC was grafted through both direct thiolate–gold bonds and post-functionalization of DCC via thiol–yne chemistry, leading to the formation of thioether (C–S–C) linkages. This dual grafting mechanism strengthens the surface chemistry of nanoparticles by combining thiolate-Au bonds with additional thioether linkages.
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Surface chemical composition of Au, S and C from survey analysis, and high-resolution C 1s and S 2p spectra for Cit-Au NPs, Au@DCC, Au@NAC and Au@DCC@NAC samples.</figcaption></figure>

The SERS spectra of pristine and functionalized Cit-Au NPs (Fig. 3a) confirmed the replacement of citrate ligands by a polyaryl coating, as evidenced by the emergence of characteristic vibrational features of phenyl derivatives originating from diazonium salts (highlighted in the yellow box), including peaks at 1593 cm⁻¹ (C=C stretching in the ortho-meta positions) and 1135 cm⁻¹ (in-plane CH bending), as well as a distinct –C≡CH stretching mode at 2006 cm⁻¹ (highlighted in the green box). Additional peak (highlighted in the blue box) around 1382 cm⁻¹ is attributed to the presence of diazenyl groups within the polyaryl coating. This observation supports the involvement of azo coupling reactions, where the electrophilic attack of the diazonium cation on pre-grafted aromatic rings facilitates the growth of the polyaryl layers on the surface of Au NPs, forming –Ph–N=N–Ph–R linkages. This mechanism is consistent with the XPS results and aligns with the findings of G. Deniau *et al.*³². Interestingly, the low wavenumber region of the spectra reveals new insights into the interfacial bonding with Au NPs. A strong peak at 240 cm⁻¹ is observed for Cit-Au NPs, corresponding to the Au-Cl stretching mode ²²,
originating from residual chloride ions present in the gold precursor salt. This peak significantly weakens after DCC grafting, clearly demonstrating the replacement of citrates by the polyaryl coating. Furthermore, a new low-intensity peak at 413 cm⁻¹ emerges in the SERS spectrum of Au@DCC, assigned to the Au–C stretch, which indicates the coordination of the aryl group to the gold surface. Following the photo-click reaction, the SERS spectra changed significantly, with the emergence of new peaks characteristic of NAC, located at 1558 cm⁻¹ (amide II) ³³, and at 1230-1300 cm⁻¹ (amide III), as confirmed by comparison with the Raman spectrum of Au@NAC (Fig. S5b). Simultaneously, a pronounced decrease in the intensity of the –C≡CH stretching peak was observed, consistent with the progress of thiol-yne hydrothiolation reactions. In addition, a new peak appeared at approximately 295 cm⁻¹, indicative of Au–S bond ²³ formation and supporting the presence of directly attached NAC molecules and thioether groups coordinated to the gold surface, an interpretation further validated by XPS analysis. Nevertheless, the Au–C stretching mode at 416 cm⁻¹ remained visible, confirming the persistence of the covalent aryl–gold bonding through the diazonium-derived polyaryl layer.

The kinetics of the photo-click reaction were evaluated by monitoring the reaction as a function of UV irradiation time. As shown in Fig. 3b, the intensity of the –C≡CH peak decreased with increasing irradiation time. Using the aryl peak at 1593 cm⁻¹ as a reference, the I<sub>C≡CH</sub>/I<sub>Aryl</sub> peak intensity ratio notably (Fig. 3c) decreased with irradiation time, reaching its minimum value after 30 minutes of UV exposure, reflecting the progressive transformation of alkynes into the thiol-yne adduct. It is worth noting that 30 min UV irradiation alone on the Au@DCC sample caused negligible changes to the –C≡CH peak, as shown in Fig. S5a, demonstrating the good stability of this group under the chosen experimental conditions, in the absence of NAC. However, the UV-vis spectra of Au@DCC@NAC (Fig. 3d) showed significant extinction loss and plasmon band broadening, indicating the agglomeration of Au NPs after 20 minutes of irradiation.

The evolution of the hydrodynamic size of Au@DCC@NAC under varying irradiation times was analyzed by DLS. As shown in Fig. S2d, the size was ca. 45 nm after 5-15 minutes of irradiation. However, for samples exposed to 20 and 30 minutes of UV, the size was increased to 77-84 nm, potentially caused by NP aggregation. The TEM image analysis (Fig. S2e) showed that extending the UV irradiation time did not affect the mean particle size. However, the histogram (Fig. S2f) displayed an increased counting frequency
of particles greater than 20 nm, which suggests aggregate formation with prolonged UV treatment. Therefore, the optimal UV irradiation time for the photo-click reaction was set at 10 minutes in this work.

The stability of Au@DCC@NAC NPs in physiological environment was evaluated in phosphate buffer and compared to NPs stored in H₂O. As depicted in Fig. S2g, the hydrodynamic size of the NPs remains stable in PBS for 2 days. However, their hydrodynamic size increased from 50 ± 3 nm on day 0 to 170 ± 20 nm on day 5, and further to 609 ± 47 nm on day 7. In contrast, in H₂O, the NPs maintained a constant hydrodynamic size at 42 ± 3 nm for 5 days, and slightly increases to 60 ± 9 nm on day 7.

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** (a) SERS spectra of Cit-Au NPs, Au@DCC and Au@DCC@NAC samples. The Au@DCC@NAC sample was obtained by adding the photoinitiator, followed by 30 min UV irradiation. (b) Evolution of the SERS spectra of Au@DCC@NAC, obtained after various times of irradiation, from 1 to 30 min during photo-click chemistry. All the spectrums were normalized to the 1593 cm⁻¹ peak, setting its intensity equal across all spectrums. (c) Peak intensity ratio of –C≡CH (2006 cm⁻¹) divided by that of aryl (1593 cm⁻¹) at</figcaption></figure>
different UV irradiation times. (d) UV-vis spectrum of Cit-Au NPs and Au@DCC@NAC (UV irradiation = 10, 20 and 30 min in presence of photoinitiator) samples.

As direct quantification of grafted NAC via SERS is hindered by the strong signal from the polyaryl groups, the grafted amount was indirectly determined by measuring the unreacted NAC remaining in the supernatant after centrifugation, using a calibration curve previously established from known NAC concentrations (see calibration curve in Fig. S1). The grafted amount was then estimated by subtracting the non-grafted molecules left in solution from the initial quantity, providing an estimate of the grafted species. The spectrum of the supernatant closely resembled that of Cit-Au NPs, indicating that no residual NAC was detectable and suggesting near-complete grafting onto the Au NP surface.

As depicted in **Fig. S6**, 1 mL of Au@DCC@NAC was centrifuged at 13000 rpm for 10 minutes. A 0.5 mL aliquot of the supernatant containing free NAC was then mixed with 1 mL of Cit-Au NPs, centrifuged at 13000 rpm for 10 minutes, and 1 µL of the resulting pellet was dried for Raman spectroscopy. **Fig. S6** compared the SERS spectra of the Au@DCC@NAC supernatant with those of Cit-Au NPs and the pellet of Au@DCC@NAC. The spectrum of the supernatant closely resembled that of Cit-Au NPs and was distinct from the pellet, indicating a high efficiency of the photo-click thiol-yne reaction. After deconvolution, no peak at ca. 295 cm⁻¹ was distinguished from the citrate reference peak at ca. 240 cm⁻¹. Since 20 µmol.L⁻¹ of NAC was initially used to prepare Au@DCC@NAC, it can be assumed that all the NAC was covalently attached to the Au@DCC NPs. Similarly, under the same conditions, full loading of NAC (20 µmol.L⁻¹) can be also considered for Au@NAC sample. It is important to note that the SERS spectrum of the Au@DCC@NAC sample (**Fig. 3b**), which was exposed to 10 minutes of UV irradiation, still showed signals corresponding to unreacted -C≡CH groups. Theoretically, 20 µmol.L⁻¹ of NAC should completely react with 10 µmol.L⁻¹ of -C≡CH to form thiol-yne. To explain this, we should consider the information from the XPS data in **Fig. 2**. This data reveals the presence of Au–S bonds in addition to thiol–yne bonds, suggesting that some NAC molecules are directly attached to the gold surface.

## 3.2 Redox characterizations

The ROS-scavenging capacity of Au@DCC@NAC was kinetically evaluated using the ORAC assay, a widely recognized method for assessing antioxidant activity³⁴. This assay
measures the ability to inhibit alkyl (R•), peroxyl (ROO•) and alkoxyl (RO•) radicals (where R = H₂N(HN)C) which are generated through AAPH decomposition. These radicals induce a decrease in fluorescence signal of fluorescein at 485/528 nm (Ex/Em) over time, enabling the quantification of antioxidant capacity <sup>35,36</sup>. A stronger antioxidant activity is reflected by a higher fluorescence signal, which can be quantified by measuring the area under the curve (AUC). Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) served as the reference antioxidant, while free NAC was used as the positive control to compare NAC's activity before and after grafting onto the nanoparticles.

The standard curves are presented in the supplementary data **Fig. S7**. The Trolox Equivalent Antioxidant Capacity (TEAC) of free NAC was 1.57, calculated by comparing the slopes of the standards curves (AUC<sub>net</sub> vs concentration) of NAC (**Fig. S7a and c**) and TROLOX (**Fig. S7b and d**). Free NAC has been shown to interact with various radicals produced by the thermal decomposition of AAPH used in ORAC assay, including R•, ROO•, and RO• radicals <sup>37</sup>.

The antioxidant activity of NAC-functionalized gold nanoparticles was evaluated for Au@NAC and Au@DCC@NAC, and compared to Cit-Au NPs, as shown in **Fig. 4**. Increasing the concentration of Au@NAC from 100 to 300 µg.mL⁻¹ led to an increase in antioxidant activity, with AUC<sub>net</sub> values ranging from 8.25 to 13.7. Au@DCC@NAC displayed even stronger antioxidant properties, with AUC<sub>net</sub> values increasing from 14.3 to 18.2 over the same concentration range. In comparison, Cit-Au NPs showed a low activity, which may be partially attributed to the intrinsic reducing properties of the citrate used during synthesis.

Although both NAC-modified nanoparticles contained the same NAC content (0.067 nmol of NAC per µg of Au NPs), Au@DCC@NAC showed superior performance. This suggests that in Au@DCC@NAC, NAC might be more exposed or accessible for ROS scavenging, thus improving its antioxidant efficacy. It is worth noting that, although NAC is at least partially grafted on the Au NPs via thioether linkages, its antioxidant activity remains preserved, in line with the well-established antioxidant properties of thioether-containing compounds reported in the literature <sup>38,39</sup>.
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Antioxidant activity (AUC<sub>net</sub> value) of Cit-Au NPs, Au@NAC and Au@DCC@NAC evaluated by Oxygen Radical Absorbance Capacity (ORAC) test, derived from the curves in Fig.S7e-g. Data are means ± SD of 3 experiments. Groups sharing the same letter are not significantly different (p > 0.05, Tukey's test). Comparisons were performed separately for each concentration.</figcaption></figure>

These results are in agreement with Razzaq *et al.* who showed a potentiation of activity when antioxidants were combined with Au NPs <sup>40</sup>. This improved activity could be due to the spatial arrangement of NAC on the nanoparticle surface, with molecular crowding potentially playing a role when NAC concentration is too high. In addition, a 10-fold increase in the half-life of NAC was measured when immobilized on Au NPs <sup>41</sup>.

This phenomenon was already observed with Au NPs functionalized with other
antioxidants, such as the tocopherol derivative, Trolox; a significantly enhanced
antioxidant activity was measured for Au@Trolox compared to the Trolox monomer in the
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test, with an increase of about
eightfold <sup>42</sup>. A similar trend was observed by Gubitosa *et al.* on Au NPs synthesized using
*Punica granatum* juice, which was known for its antioxidant activity. Their study
demonstrated a marked enhancement of the antioxidant activity of Au NPs, as the
concentration of Trolox required to achieve a comparable antioxidant activity was
2.5×10<sup>6</sup> times higher than the concentration of Au NPs <sup>43</sup>, highlighting the remarkable
efficiency of the nanoparticle-based system. The same properties were demonstrated in a
supramolecular system comprising Au NPs/chitosan/ellagic acid, where the amounts of Au
NPs and Trolox needed to inhibit 10% of DPPH were markedly different, with a much
higher concentration (5.0×10<sup>-5</sup> mol.L<sup>-1</sup>) required for Trolox compared to Au NPs
(2.0×10⁻¹² mol.L⁻¹)⁴⁴. This group also validated the ability of Au NPs to inhibit oxidative processes in the presence of H₂ O₂, used as a ROS model. These results suggest that the assembly of antioxidants on the surface of gold nanoparticles could enhance their chemical reactivity and antioxidant efficacy due to protection against degradation, improved bioavailability, synergistic effect, directed targeting, and surface and size effects ⁴⁵.

In addition, **Fig S7h** compares the ORAC results for Au@DCC@NAC samples, which were prepared with UV irradiation for 5, 10, and 30 minutes. A 10 min UV irradiation time yielded the highest AUCₙet value. Based on SERS spectra in **Fig. S3b-c**, a 5 min UV exposure resulted in few NAC molecules reacting and loading on Au NPs. Conversely, 30 min irradiation caused NPs aggregation (TEM in **Fig. S2e**). Therefore, a 10 min UV irradiation for NAC loading achieved optimal antioxidant activity. Moreover, Au@DCC sample exhibited an AUCₙet value greater than pristine Cit-Au, and even surpassed Au@NAC at 300 µg/mL. Report has indeed indicated that alkyne-based organic compounds possess radical scavenging activities⁴⁶. Our latest findings suggested Au@DCC@NAC bearing NAC through Au-S bonds and thioethers (XPS in **Fig. 2**) alongside unreacted C≡CH (SERS in **Fig. 3b**). Thioethers are known to primarily scavenge ROS by oxidizing to sulfoxides/sulfones⁴⁷. Furthermore, alkynes can undergo radical addition to their triple bond, where radical species attack the C≡C to generate a vinyl radical intermediate⁴⁸. This mechanism potentially consumes ROS, thus offering protection against tissue damage. This fact highlights a synergistic contribution of both DCC and NAC to the antioxidant activity observed in the Au@DCC@NAC sample.

### 3.3 Biological characterization

It is important to study the potential adverse effects of the modified Au NPs on the main cell types lining the inner wall of human blood vessels, *i.e.* endothelial cells. Human umbilical vein endothelial cells (HUVECs) are among the most widely used *in vitro* models to assess nanoparticle toxicity ²⁶. To this end, we studied the viability and the morphology of HUVECs in contact with different concentrations of Au NPs.

Cellular metabolic activity assessment was carried out by MTT test based on the reduction of a yellow tetrazolium salt to purple formazan crystals, by a mitochondrial enzyme NADPH (nicotinamide adenine dinucleotide phosphate hydrogen), in living cells. The
formazan crystals dissolve in isopropanol, producing a colored solution, the absorbance of which is directly proportional to the number of living cells. The results (Fig. 5a to d) demonstrated no observable toxicity for all tested samples at concentrations of 15 and 30 µg.mL⁻¹, as indicated by cell viability values exceeding 90%. According to the international organization for standardization ⁴⁹, samples are classified as toxic if cell viability is reduced to less than 70% compared to the control after 24 hours of incubation. The functionalized samples at concentrations of 15, 30 and 60 µg.mL⁻¹, exhibited cell viability comparable to the negative control (NC). At higher concentrations (120 µg.mL⁻¹), the samples showed significant deviations from the NC; however, the viability values remained above 70%, indicating no detectable cytotoxic effects by the MTT test at the studied concentrations.

The images in **Fig. 5e** show the morphology of HUVEC cells after 24 hours of incubation with pristine and functionalized Au NPs (30 µg.mL⁻¹) or the control medium (negative control). At this concentration, the cells maintained a normal morphology with well-defined actin filaments (orange) and clearly distinguishable nuclei (blue). Similar observations were recorded at concentrations of 60 µg.mL⁻¹. However, at 120 µg.mL⁻¹ notable morphological alterations were observed, including cellular rounding, deformation and the presence of extracellular material loss (Fig. S8). This apparent contradiction might be due by the fact that the MTT assay measures mitochondrial metabolic activity, which can remain high even in cells undergoing early apoptosis or showing compromised morphology. In such cases, metabolic activity may overestimate the number of functionally healthy cells. This test further confirmed that cell viability and morphology remained unaffected after 24 hours of incubation at concentrations below 60 µg.mL⁻¹. To ensure both robust viability and preserved morphology, we therefore selected an intermediate concentration of 50 µg.mL⁻¹ for subsequent internalization studies (experiments in section 3.4). At this dose, HUVECs exhibited normal morphology and high metabolic activity, confirming the overall good biocompatibility of the Au@DCC@NAC nanoparticles within the tested range.
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

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

<figure><img src="image_11.png" /><figcaption>**Fig. 5.** Endothelial cells in contact with the pristine and functionalized AuNPs. (a-d) Viability of HUVECs in contact with Cit-Au NPs, Au@NAC and Au@DCC@NAC (15, 30, 60 and 120 µg.mL⁻¹) samples. A, B and C indicated that the difference of the mean is significant at the 0.05 level. p < 0.05. (e) Morphology of HUVECs after 24h culture in contact with the pristine and functionalized Au NPs at 30 µg.mL⁻¹. The actin filaments are indicated by the yellow arrows and the nucleus by blue arrows.</figcaption></figure>
### 3.4 Nanoparticle cellular uptake studied by colocalized fluorescence imaging and Raman mapping

HUVECs were stained with Hoechst to visualize nuclei and WGA (wheat germ agglutinin) to label the cell membrane. This staining facilitated the localization of internalized NPs. After incubation with Au@DCC@NAC (50 µg.mL⁻¹) for 3 hours, the cells were rinsed and fixed and then analyzed via colocalization of fluorescence imaging and Raman mapping. As shown in **Fig. 6a**, the merged optical, fluorescence, and Raman images confirm the presence of NPs (red spots) within the cells, indicating efficient cellular uptake. Interestingly, the Raman spectra of the internalized NPs closely resembled those of the Au@DCC@NAC reference sample (before incubation with cells). However, the appearance of new peaks indicates slight spectral differences, potentially caused by interactions between the NPs and intracellular components.

The same investigation was conducted on HUVEC cells incubated with Cit-Au NPs. As shown in **Fig. 6b**, the merged optical, fluorescence, and Raman images revealed partial localization of Cit-Au NPs signals (green spots P1, P2, P3) within the cells, while other signals (P4, P5, P6) were detected outside the cells. This suggests less efficient cellular uptake compared to Au@DCC@NAC NPs. This observation underscores the importance of surface modification in altering nanoparticle interactions within biological environments.
<figure><img src="image_12.png" /><figcaption>**Fig. 6.** Optical, fluorescence and Raman imaging of HUVEC cells incubated with (a) Au@DCC@NAC NPs and (b) Cit-Au NPs for 3 hours ($C_{Au} = 50~\mu g.mL^{-1}$). The red and green dash cages in the merged (BF+RM), and merged (BF+Hoechst+WGA+RM) images indicate the boundary of the Raman mapping area. The SERS spectrum of Au@DCC@NAC prior to cell incubation was used as a reference for comparison with average spectra obtained in region P1-P6 from in Raman mapping in (a).</figcaption></figure>

Literature showed that the cellular mechanisms of Au NPs trapping are primarily mediated by endocytosis, and that it varies depending on the size, shape, and surface characteristics of the nanoparticles ⁵⁰ as well as incubation time. In HUVECs, it was shown that the uptake of Au NPs of size close to 120 nm occurred mainly by clathrin-mediated endocytosis and trafficking to membrane enclosures as single particles and agglomerations of 2-3 particles ⁵¹. The same authors showed that exposure to Au NPs did not result in cellular cytotoxicity, while the production of intracellular ROS slightly increased. As discussed at the beginning of this study, elevated levels of ROS can induce oxidative stress, potentially leading to cardiac dysfunction. Therefore, the development of
antioxidant Au NPs is of critical importance. Our results demonstrate that NAC-functionalized Au NPs can be efficiently internalized by HUVECs, highlighting their potential as a promising antioxidant nanomedicine for cardiac dysfunction.

4. CONCLUSION

Aryl diazonium salt chemistry was used to functionalize Au NPs by NAC for the design of new antioxidant nanoparticles. NAC was successfully conjugated onto Au NPs through a photo-click thiol-yne reaction following 4-ethynylbenzene diazonium salt pre-functionalization. The resulting Au@DCC@NAC NPs were fully characterized by complementary techniques, including TEM, DLS, XPS and SERS, which revealed that NAC was grafted via both direct thiolate–gold bonds and post-functionalization of DCC through thiol–yne chemistry. This dual grafting mechanism reinforces the nanoparticle surface chemistry by combining thiolate-Au bonds with additional thioether linkages and thus offers a promising alternative to the direct immobilization of NAC on Au NPs. Interestingly, ORAC measurements showed that the Au@DCC@NAC NPs preserve the antioxidant activity of NAC after conjugation, surpassing Cit-Au NPs and Au@NAC samples. The biological evaluations demonstrated the capacity of human endothelial cells to internalize Au@DCC@NAC after 3h, with no toxicity observed within the concentration range studied (up to 120 µg.mL⁻¹). This proof-of-concept study thus highlights the promising potential of Au@DCC@NAC as an antioxydant nanomedicine, which effectively preserves NAC's antioxidant activity while potentially improving its bioavailability and facilitating better cellular interaction. This strategy may allow for lower dosing while enhancing the therapeutic efficacy of NAC, though further studies are required to validate this hypothesis. In particular, the use of antioxidant nanomedicine for cardiovascular diseases may offer unique advantages, such as enabling targeted delivery agents (such as ICAM-1, VCAM-1, etc.) on Au@DCC@NAC via co-functionalization or/and adsorption, to inflamed vascular regions and thereby minimizing systemic exposure. This localized therapeutic action could enhance efficacy in mitigating oxidative stress related to cardiac dysfunction, strengthening its promise as a future cardiovascular therapy.

**Supporting Information**
Additional experimental details, supplementary SERS, TEM, DLS, XPS and ORAC data and analysis; cell morphology images.

## Acknowledgments

This research is funded by the French National Research Agency (Agence Nationale de la Recherche) under the project BIORESORB (ANR-21-CE18-0012). Yang Zhang is supported by a scholarship from the China Scholarship Council (CSC No. 202106340022). Greta Malavasi was supported by the European ERASMUS program between The University of Modena and Reggio Emilia (Italy) and Université Paris Cité (France). We extend our gratitude to Pascal Cheng, Zhendong Peng and Philippe Decorse from Le laboratoire Interfaces Traitements Organisation et DYnamique des Systèmes – ITODYS, UMR7086, Université Paris Cité for their valuable assistance with XPS analysis. We acknowledge the ImagoSeine core facility of the Institut Jacques Monod, member of the France BioImaging infrastructure (https://ror.org/01y7vt929) supported by the French National Research Agency (ANR-24-INBS-0005 FBI BIOGEN) and GIS-IBISA for TEM observation.

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