Yavuz Oz

Alexandre Barras

Rana Sanyal

Rabah Boukherroub

Sabine Szunerits

Amitav Sanyal

Functionalization of Reduced Graphene Oxide via Thiol-Maleimide "Click" Chemistry: Facile Fabrication of Targeted Drug Delivery Vehicles

Keywords: ACS Appl. Mater. Interfaces -Post-print cellular targeting, click chemistry, DOX delivery, graphene oxide, photothermal effect, thiol-maleimide conjugation

Materials based on reduced graphene oxide (rGO) have shown to be amenable to noncovalent functionalization through hydrophobic interactions. The scaffold, however, does not provide sufficient covalent linkage given the low number of reactive carboxyl and alcohol groups typically available on the rGO. The integration of clickable groups, particularly the ones that can undergo efficient conjugation without any metal catalyst, would allow facile functionalization of these materials. This study reports on the noncovalent association of a maleimide-containing catechol (dopa-MAL) surface anchor onto the rGO. Thiol-maleimide chemistry allows thereafter the facile attachment of thiol-containing molecules under ambient metal-free conditions. Although the attachment of glutathione and 6-(ferrocenyl)hexanethiol Post-print

Introduction

Nanosystems that release cancer drugs and induce toxicity in response to an external stimulus offer unique advantages over the commonly employed conventional carriers that release their payload in a passive fashion. Reduced graphene oxide (rGO) nanosheets with restored sp 2 network have most lately shown to be ideally suited for drug-loading-and drug-release-based applications using electrical(1-3) as well as thermal induced triggers. [START_REF] Hu | Photoresponsive Protein-Graphene-Protein Hybrid Capsules with Dual Targeted Heat-Triggered Drug Delivery Approach for Enhanced Tumor Therapy[END_REF][START_REF] Wang | A Chitosan-Modified Graphene Nanogel for Noninvasive Controlled Drug Release[END_REF][START_REF] Teodorescu | Photothermally Triggered On-demand Insulin Release from Reduced Graphene Oxide Modified Hydrogels[END_REF][START_REF] Teodorescu | Transdermal Skin Patch Based on Reduced Graphene Oxide: A New Approach for Photothermal Triggered Permeation of Ondansetron Across Porcine Skin[END_REF][START_REF] Goenka | Graphene-based Nanomaterials for Drug Delivery and Tissue Engineering[END_REF][START_REF] Matteini | Graphene as a Photothermal Switch for Controlled Drug Release[END_REF][START_REF] Turcheniuk | Plasmonic Photothermal Cancer Therapy with Gold Nanorods/Reduced Graphene Oxide Core/Shell Nanocomposites RSC Adv[END_REF] Although the majority of the aromatic scaffold of rGO is devoid of any functional groups, a few carboxylic acid and alcohol groups are present at the edges and other defect points. This limits the effective functionalization of the rGO through covalent transformations. To circumvent this limitation, the noncovalent π-π stacking-based functionalization route of the rGO scaffold has been extensively exploited. [START_REF] Guo | RGD-Peptide Functionalized Graphene Biomimetic Live-Cell Sensor for Real-Time Detection of Nitric Oxide Molecules[END_REF][START_REF] Xu | Functionalization of Reduced Graphene Oxide Nanosheets via Stacking Interactions with the Fluorescent and Water-Soluble Perylene Bisimide-Containing[END_REF][START_REF] Kaminska | Preparation of a Responsive Carbohydrate-Coated Biointerface Based on Graphene/Azido-Terminated Tetrathiafulvalene Nanohybrid Material ACS[END_REF][START_REF] Kaminska | Reduction and Functionalization of Graphene Oxide Sheets Using Biomimetic Dopamine Derivatives in One Step ACS[END_REF][START_REF] Kaminska | Preparation of Graphene/Tetrathiafulvalene Nanocomposite Switchable Surfaces[END_REF][START_REF] Kaminska | Thiolyne Click Reaction on Alkynyl-Dopamine Modified Reduced Graphene Oxide[END_REF] This approach is effective for hydrophobic/aromatic molecules that are able to undergo efficient noncovalent interactions. Conjugation of molecules unable to undergo such an association with the rGO, however, becomes challenging. An approach that would allow facile and effective covalent attachment in a modular manner to the rGO would be rather useful.

Aromatic anchoring groups like pyrene, [START_REF] Guo | RGD-Peptide Functionalized Graphene Biomimetic Live-Cell Sensor for Real-Time Detection of Nitric Oxide Molecules[END_REF][START_REF] Qu | Noncovalent Functionalization of Graphene Attaching [6,6]-Phenyl-C61-butyric Acid Methyl Ester (PCBM) and Application as Electron Extraction Layer of Polymer Solar Cells[END_REF] tetrathiafulvalene [START_REF] Kaminska | Preparation of a Responsive Carbohydrate-Coated Biointerface Based on Graphene/Azido-Terminated Tetrathiafulvalene Nanohybrid Material ACS[END_REF][START_REF] Kaminska | Preparation of Graphene/Tetrathiafulvalene Nanocomposite Switchable Surfaces[END_REF] and dopamine derivatives [START_REF] Kaminska | Reduction and Functionalization of Graphene Oxide Sheets Using Biomimetic Dopamine Derivatives in One Step ACS[END_REF][START_REF] Kaminska | Thiolyne Click Reaction on Alkynyl-Dopamine Modified Reduced Graphene Oxide[END_REF]) have shown to be of high interest for rGO functionalization. Some of us have reported surface functionalization of the rGO with dopamine-containing clickable alkyne and azide groups. [START_REF] Kaminska | Reduction and Functionalization of Graphene Oxide Sheets Using Biomimetic Dopamine Derivatives in One Step ACS[END_REF][START_REF] Kaminska | Thiolyne Click Reaction on Alkynyl-Dopamine Modified Reduced Graphene Oxide[END_REF] The azide-containing rGO could be further modified with alkynylterminated molecules such as ferrocene via the Cu(I)-catalyzed azide-alkyne Huisgen-type click reaction. Although the azide-alkyne-based rGO functionalization was effective, the requirement to use copper as a catalyst can be a concern for the biomedical applications because any trace of copper adds to the overall cytotoxicity of the nanostructures. This necessitates the expansion of the tool box to include click reactions that do not need metal catalysts, like the strain-promoted azide-alkyne cycloaddition (SPAAC) [START_REF] Manova | Copper-Free Click Biofunctionalization of Silicon Nitride Surfaces via Strain-Promoted Alkyne-Azide Cycloaddition Reactions[END_REF] and the nucleophilic or radical thiol-ene reactions. [START_REF] Kaminska | Thiolyne Click Reaction on Alkynyl-Dopamine Modified Reduced Graphene Oxide[END_REF][START_REF] Zhang | Immobilization of Cysteine-Tagged Proteins on Electrode Surfaces by Thiol-Ene Click Chemistry[END_REF] Although approaches based on SPAAC have gained interest lately, [START_REF] Escorihuela | Metal-Free Click Chemistry Reactions on Surfaces[END_REF] the multistep synthesis or high cost of the cyclooctyne-based materials limits the widespread utilization of this approach. Nucleophilic thiol-ene-based reactions have been extensively employed in the bioconjugations using the thiol-maleimide functional group dyad and seem advantageous. [START_REF] Mazur | Iron Oxide Magnetic Nanoparticles with Versatile Surface Functions Based on Dopamine Anchors[END_REF][START_REF] Hoyle | Thiol-Click Chemistry: A Multifaceted Toolbox for Small Molecule and Polymer Synthesis[END_REF][START_REF] Oz | Modular Fabrication of Polymer Brush Coated Magnetic Nanoparticles: Engineering the Interface for Targeted Cellular Imaging ACS[END_REF][START_REF] Arslan | Bioinspired Anchorable Thiol-Reactive Polymers: Synthesis and Applications Toward Surface Functionalization of Magnetic Nanoparticles[END_REF] The maleimide-thiol addition reaction is a facile process that does not require any catalyst and can be carried out under mild conditions. Although thiol groups can be readily incorporated in various molecules, they are also present in the cysteine residue of various peptides and biomolecules, or can be installed using standard protocols during or after synthesis.

Herein, we report the synthesis of a maleimide functional group containing the rGO, which is amenable to facile functionalization by thiol group containing molecules (Scheme 1). The conjugation of thiol-bearing molecules proceeds through the thiol-ene click-type conjugation reaction, under metal-free conditions. Maleimide functionalized rGO was easily prepared through π-π interactions under ultrasonication of the rGO with a maleimide-containing dopamine (dopa-MAL) ligand. To demonstrate the utility of this clickable rGO, we took the advantage of the covalent conjugation of a thiol-bearing targeting peptide, c(RGDfC), and the noncovalent loading of the rGO-c(RGDfC) nanostructures with the anticancer drug doxorubicin (DOX). The two-dimensional structure of graphene sheets serves as an excellent drug carrier due to its high drug loading capacity arising from the π-π stacking and other hydrophobic interactions. Attachment of a targeting group on the rGO through covalent conjugation further expands its application to the targeted drug delivery. Selective delivery of anticancer agents to the tumor site has become important for efficient cancer therapy by minimizing the negative side effects. Synthetic cyclic peptides with arginine-glycine-aspartic acid sequences (RGDs) are known to possess high affinity to integrin α v β 3 , a cell adhesion domain highly expressed on the tumor cells. [START_REF] Waite | PAMAM-RGD Conjugates Enhance siRNA Delivery Through a Multicellular Spheroid Model of Malignant Glioma Bioconjugate Chem[END_REF][START_REF] Xiong | Enhanced Intracellular Delivery and Improved Antitumor Efficacy of Doxorubicin by Sterically Stabilized Liposomes Modified with a Synthetic RGD Mimetic[END_REF] Their integration into the nanostructures and polymers for targeted drug delivery has thus found growing interest. [START_REF] Zhu | RGD-Modified PEG-PAMAM-DOX Conjugate: In Vitro and In Vivo Targeting to Both Tumor Neovascular Endothelial Cells and Tumor Cells[END_REF][START_REF] Zhang | RGD-Modified PEG-PAMAM-DOX Conjugates: In Vitro and In Vivo Studies for Glioma Eur[END_REF] Scheme 1. Schematic Illustration of the Synthesis of Thiol-Ene "Clickable" Maleimide-rGO and its Subsequent Functionalization Finally, the excellent photothermal properties of DOX-loaded rGO/dopa-MAL-c(RGDfC) allow for chemophototherapy; the combination of photothermal therapy with chemotherapeutic release of DOX is often beneficial specifically for solid tumors. [START_REF] Luo | Chemophototherapy: An Emerging Treatment Option for Solid Tumors[END_REF] The cytotoxicity of targeted and nontargeted rGO under normal and photothermal conditions was assessed on two cancer cell lines, human breast MDA-MB-231 and human cervical cancer cells (HeLa), to highlight the advantages of the construct. Overall, facile fabrication and functionalization of a maleimide group containing rGO-based functional material with potential for applications in many areas is outlined.

Experimental Section

Materials

N-Hydroxysuccinimide

(NHS) was purchased from Alfa Aeser. N,N′-Dicyclohexylcarbodiimide (DCC), 4-(dimethylamino)pyridine (DMAP), dopamine hydrochloride, triethylamine (TEA), γ-aminobutyric acid, l-glutathione reduced, 6-(ferrocenyl)hexanethiol, graphite powder (<20 μm) and hydrazine monohydrate were purchased from Sigma-Aldrich. 4-(exo-3,5-Dioxo-10-oxa-4-aza-tricyclo[5.2.1.0]dec-8-en-4yl)-butyric acid (furan-protected maleimide containing acid) was synthesized according to a previous report. [START_REF] Maynard | Synthesis of Norbornenyl Polymers with Bioactive Oligopeptides by Ring-Opening Metathesis Polymerization[END_REF] Bags with regenerated cellulose dialysis membranes (Spectra/Por Biotech, molecular weight cut-offs 10 kDa) were obtained from Spectrum Laboratories. Purification by silica gel column was done using silica gel 60 (Merck). Purity of the compounds was determined using thin-layer chromatography on the silica-coated aluminum plates (Kieselgel 60 F254, Merck). Methanol, tetrahydrofuran, and dimethylformamide (DMF) were bought from Merck. Anhydrous toluene from SciMatCo system was used, and other solvents were stored on the molecular sieves when needed. Graphene oxide was bought from Graphenea, Spain.

Preparation of Reduced Graphene Oxide (rGO)

GO aqueous suspension (150 mg, 3 mg mL -1 ) was sonicated and hydrazine hydrate (50 μL, 1.03 mmol) added to this suspension. The mixture was heated for 24 h at 100 °C and rGO slowly precipitated out. The desired material was obtained by filtering with a poly(vinylidene difluoride) membrane (0.45 μm pore size), rinsed with water (20 mL × 5) and methanol (20 mL × 5), followed by drying in an oven.

Synthesis of Maleimide-Containing Dopamine (dopa-MAL)

Protected Maleimide-Containing Dopamine (dopa-pMAL)

Furan-protected maleimide-containing acid (2.45 g, 9.76 mmol), NHS (1.18 g, 10.25 mmol), and DMAP (0.24 g, 1.95 mmol) were dissolved in DMF (15 mL) in a round-bottom flask at 0 °C. DCC dissolved in 5 mL of DMF was subsequently added to this solution dropwise. After stirring at 0 °C for 15 min, the reaction was left to proceed at room temperature for 16 h. The solvent was removed in vacuo and the product (NHS ester of the acid) was extracted with NaHCO 3 . This activated ester was used without further purification. Dopamine, HCl (1.43 g, 7.54 mmol), and TEA (1.09 g, 10.8 mmol) were mixed in 10 mL of DMF with magnetic stirring for 15 min. The DMF solution of the activated ester (2.5 g, 7.18 mmol) was added to the mixture and stirred at room temperature for 24 h. The solvent was partially evaporated (≈1-2 mL) and 1 M HCl solution (50 mL) was added to the concentrated solution. It was extracted with EtOAc. The organic phase was collected and dried over Na 2 SO 4 . Column chromatography using CH 2 Cl 2 /methanol (10:1) was employed to obtain the pure product (1.28 g, yield: 82%).

Maleimide Group Activation via Retro Diels-Alder Reaction (dopa-MAL)

Furan-maleimide adduct-containing dopamine (1.0 g) in anhydrous toluene (150 mL) was heated under nitrogen protection at 110 °C for 10 h. The solution was then cooled down to ambient temperature and toluene was removed via rotary evaporation, yielding maleimideappended dopamine (dopa-MAL) (0.80 g, yield: 97%).

Preparation of rGO/dopa-MAL

To a solution of rGO in DMF (1 mg mL -1 ), dopa-MAL (6.3 mM) was added and the mixture ultrasonicated for 3 h at room temperature. Maleimide-functionalized rGO was collected by centrifugation at 13 000 rpm for 30 min and washed with DMF to remove all of the unbound dopa-MAL compounds. The functionalized rGO was then dried under vacuum.

Functionalization of rGO/dopa-MAL via Thiol-Ene Michael Addition Reactions

Glutathione and 6-(Ferrocenyl)hexanethiol Linking

Dispersion of rGO/dopa-MAL in DMF (1 mg mL -1 ) was mixed with glutathione (10 mM) and sonicated for 6 h at room temperature. The product rGO/dopa-Mal-glutathione was collected by centrifugation at 13 000 rpm for 30 min. Similarly, 6-(ferrocenyl)hexanethiol (10 mM) was attached to rGO/dopa-MAL (1 mg mL -1 DMF solution) under ultrasonication for 6 h at room temperature. Purified rGO/dopa-MAL-ferrocene was obtained by centrifugation at 13 000 rpm for 30 min.

c(RGDfC) Attachment to rGO/dopa-MAL

c(RGDfC) (4 mM) was added to 1 mg mL -1 DMF dispersion of rGO/dopa-MAL and the mixture sonicated for 12 h at room temperature. The nonconjugated peptide was removed by dialyzing (using MWCO 10 000 Da membrane) against water for 48 h to yield rGO/dopa-MAL-c(RGDfC).

Quantification of Peptide and DOX Loading

Peptide Quantification Using Bicinchoninic Acid (BCA) Assay

The amount of peptide c(RGDfC) in rGO/dopa-MAL-c(RGDfC) was measured using a BCA kit (ThermoScientific) using a protocol provided by the manufacturer.

Drug Loading

Peptide-modified rGO was dispersed in water at 0.5 mg mL -1 . Doxorubicin hydrochloride (4.2 mM) was added to the dispersion and left to stir for 12 h at ambient temperature. The solution was centrifuged to remove free DOX from the DOX-loaded rGO/dopa-MALc(RGDfC) nanoconjugate.

Characterization

Full details of the characterization techniques and instrumentation used in this study can be found in the Supporting Information.

Cell Viability and Internalization

The HeLa cell line derived from cervical carcinoma (ATCC CCL-2, ECACC, Sigma-Aldrich, Saint-Quentin Fallavier, France) and the MDA-MB-231 cell line derived from human breast adenocarcinoma (ATCC HTB-26, ECACC, Sigma-Aldrich, Saint-Quentin Fallavier, France) were used. Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) were used to culture and maintain the cells in a humidified incubator at 37 °C and 5% CO 2 .

Cell Viability upon Treatment with rGO/dopa-MAL-c(RGDfC)

A 96-well plate was used to seed cells at a density of 10 4 cells/well and grown for 24 h prior to assay. A fresh medium containing rGO/dopa-MAL-c(RGDfC) was used to replace the culture medium. After 48 h, the old medium was aspirated, washed with phosphate-buffered saline (PBS), and replaced with new medium. Cellular viability was measured using Uptiblue (Interchim) method. Briefly, 10 μL of the Uptiblue solution were added to the wells containing 100 μL of DMEM with 10% FBS and the plate was incubated for 4 h in the incubator. The fluorescence of each well (exc. 540 nm/em. 590 nm) was obtained with a plate reader. Experimental runs were repeated five times. The average absorbance of the untreated cells was considered as 100% viability.

Cell Viability of DOX-Loaded rGO/dopa-MAL-c(RGDfC) under Passive and Active Conditions

Cells were seeded in a 24-well plate (10 5 cells/well) and grown for 24 h prior to assay. For passive release of DOX, the cell culture medium was renewed with a fresh medium that contains DOX-loaded rGO/dopa-MAL-c(RGDfC) at pH 7.4. After 48 h, the old medium was removed, washed with PBS, and replaced with a fresh medium. The cell viability was measured using Uptiblue method as described above. For active release of DOX, the culture medium was replaced with the medium containing DOX-loaded rGO/dopa-MAL-c(RGDfC). After 6 h, each well was illuminated with a continuous laser at 980 nm at various power densities (1-4 W cm -2 ) for 10 min. During the same time, thermal images were taken with an IR camera. The cell viability was evaluated after additional 18 h using Uptiblue method as described above.

Results and Discussions

Synthesis of Dopamine-Maleimide-Modified reduced graphene oxide (rGO/dopa-MAL)

The aromatic structure of catechol units allows dopamine to develop strong π-stacking interactions with other aromatic bearing units such as rGO. Additionally, dopamine can be easily functionalized by exploiting its amine terminal functions as used here for the synthesis of dopa-MAL (Figure 1). showed the expected resonances, particularly those from the two distinct carbonyl groups, one from the amide and the other from the imide group (Figure S1C). Maleimide-functionalized rGO was obtained by mixing DMF dispersion of the rGO with dopa-MAL at room temperature for 3 h using a mass ratio of 2:1 (dopa-MAL/rGO). Fourier transform infrared (FT-IR) spectroscopy (Figure 2A) and X-ray photoelectron spectroscopy (XPS, Figure 2B) were performed for establishing the successful formation of rGO/dopa-MAL. The FT-IR spectrum of rGO/dopa-MAL shows a broad band at ∼3400 cm -1 due to the vibration of hydroxyl groups of the dopamine ligand and the remaining OH groups on the rGO. The characteristic vibration band of the maleimide carbonyl group at 1696 cm -1 is clearly observed. The band at ∼1570 cm -1 is due to the C═C stretching modes of the aromatic network, whereas the band at 1223 cm -1 is due to the C-O (alkoxy) stretching modes of the rGO and the ligand. The absorption bands at 2922 and 2859 cm -1 correspond to the CH and CH 2 stretching vibrations, respectively. X-ray photoelectron spectroscopy (XPS) analysis was performed in addition to gain additional information on the rGO/dopa-MAL chemical composition (Figure 2B). The XPS survey spectrum displays bands due to C 1s , O 1s , and N 1s (Table 1), in good agreement with the composition of the functionalized rGO. The C 1s high-resolution spectrum can be deconvoluted into bands at 284.

Thiol-Ene Michael Addition Reaction on rGO/dopa-MAL

The possibility to integrate thiol-containing ligands onto rGO/dopa-MAL was investigated by the conjugation of glutathione and 6-(ferrocenyl)hexanethiol via Michael addition reaction (Scheme 1). Both conjugations were carried out in DMF at room temperature for 6 h, and the attachment was confirmed by XPS and FT-IR spectroscopy. The presence of S 2p indicates the successful linking of glutathione or ferrocene onto the rGO/dopa-MAL nanocomposite (Table 1). The exact binding constant is not determined for the rGO/dopa-MAL system due to its heterogeneous nature. No significant decrease in nitrogen atom content was observed in the XPS analysis of the product obtained after conjugation with ferrocene thiol, thus indicating that the maleimide-containing ligands do not detach from the rGO surface during these steps (Table 1). Figure 3A depicts the high-resolution XPS spectrum of the S 2p core-level spectra of rGO/dopa-MAL-glutathione and rGO/dopa-MAL-ferrocene. Both spectra can be fitted with two peaks at 164.2 eV (S 2p3/2 ) and 165.3 eV (S 2p1/2 ) due to S-C bond formation with no oxidized sulfur being present, underlining the covalent integration of the thiol-bearing ligands.

As a control experiment, the rGO was reacted with glutathione under the same conditions and the product was analyzed by XPS. The absence of S 2p component in the XPS spectrum clearly indicates that glutathione physisorption does not take place (or very limited) under our experimental conditions. The same is true for 6-(ferrocenyl)hexanethiol-modified rGO/dopa-MAL. The presence of Fe 2p at ≈710 eV shows in addition the integration of ferrocene units onto the matrix (Figure 3B). The Fe 2p high-resolution XPS spectrum comprises two bands at 711. In addition, the ferrocene-modified rGO/dopa-MAL was probed by cyclic voltammetry (CV) in 0.1 M tetraethyl ammonium hexafluorophosphate (TEAPF 6 ) in DMF (Figure 3C). The presence of the redox peak with a peak separation of 164 mV and E 0 ≈ 0.18 V versus Ag/AgCl evidences the incorporation of ferrocene groups on the rGO/dopa-MAL. The peak current change recorded as a function of the scan rate suggests that the ferrocene units are immobilized on the surface rather than being adsorbed. Figure 3D shows that the anodic and cathodic peak currents scale linearly with the scan rate, indicating a surface redox process. The loading of attached ferrocene onto the rGO surface via thiol-ene reaction was calculated according to Γ = Q A /nFA, where the Faraday constant is represented by F, n is the number of exchanged electrons (n = 1), A is the area of the surface (A = 0.12 cm 2 ), and Q A is the transferred anodic charge. A surface coverage of Γ = 4.5 × 10 -10 mol cm -2 (2.71 × 10 14 molecules cm -2 ) is determined, being similar to that determined for the ferrocene "clicked" to diamond interfaces(31) and 6-(ferrocenyl)hexanethiol immobilized on rGO/alkynyldopamine through thiol-yne reaction.( 16)

Fabrication of Multifunctional Scaffold: Doxorubicin-Loaded rGO/dopa-MALc(RGDfC)

After the successful demonstration of facile covalent functionalization of the maleimidecontaining rGO, this scaffold was investigated for its potential to target cancer cells by covalently linking to the maleimide-functionalized rGO a cyclic peptide containing the sequence c(RGDfC), known to target the integrin receptors that are often overexpressed in various types of cancer (Figure 4A). Attachment of the thiol-containing peptide was undertaken in DMF under ambient conditions, followed by dialysis against water to remove any unbound peptide. The peptide attachment was confirmed by using the BCA protein assay kit. The amount of attached peptide on rGO/dopa-MAL was determined to be 263 μg mg -1 by using the calibration plot obtained from the UV absorbance data of the peptide solution standards at 562 nm (see SI, Figure S2). The cytotoxicity of rGO/dopa-MAL-c(RGDfC) was assessed using the Uptiblue assay on the human breast MDA-MB-231 and human cervical HeLa cancer cells (Figure 4B). The rGO/dopa-MAL-c(RGDfC) material does not show damage to the two cell lines at the concentration of 0-100 μg mL -1 after 48 h incubation, illustrating the suitability of the material as a drug delivery platform. The chemotherapy drug doxorubicin (DOX) was next loaded onto the rGO/dopa-MALc(RGDfC) nanosheets by exploiting the various interactions (π-stacking, H-bonding, hydrophobic) between DOX and rGO. The loading was carried out in water in the dark, and the unbound drug was removed through centrifugation of the dispersion at 13 000 rpm (Figure 4A). The successful loading of DOX on rGO/dopa-MAL-c(RGDfC) was evidenced by the reddish color of the solution attributed to DOX showing an absorption peak at 480 nm (Figure 4C). The amount of DOX loaded on the matrix was determined by monitoring the UV-vis absorbance of the supernatant (see SI, Figure S3) and determined to be as 170 μg DOX loaded onto 1 mg of rGO/dopa-MAL-c(RGDfC). We next evaluated the viability of HeLa and MDA-MB-231 cells upon the incubation with DOX-loaded rGO/dopa-MAL-c(RGDfC) nanostructure for 48 h. Figure 5B suggests that under physiological pH, the nanostructure does not show any cell-killing ability for the HeLa cells. However, 100 μg mL -1 of rGO/dopa-MAL-c(RGDfC) (corresponding to 17 μg DOX) induced cell killing with an estimated IC 50 = 58 μg mL -1 for DOX-loaded rGO/dopa-MALc(RGDfC). Considering that 17 μg DOX is integrated onto 100 μg of the rGO nanosheets, this corresponds to an IC 50 = 9.9 μg mL -1 for DOX in the matrix, comparable to the IC 50 = 10.5 μM of free DOX for the same cell line (Figure 5A). The targeting effect is even more clearly pronounced, as HeLa cells are more sensitive to DOX with free DOX showing a ≈4 times lower IC 50 value when compared with the MDA-MB-231 cells. The optical images of both cell lines after treatment with DOX-loaded rGO/dopa-MAL-c(RGDfC) (100 μg mL -1 ) (Figure 5C) demonstrate in addition the targeting effect of DOX-loaded rGO/dopa-MAL-c(RGDfC). The cell morphology of HeLa cells is preserved, whereas that of MDA-MB-231 cells is compromised. Interestingly, most DOX molecules remained attached to the rGO/dopa-MALc(RGDfC) nanostructure and only <1% of DOX was released from the structures after 24 h incubation in the physiological solution with the same ionic strength but different pH values. We were thus intrigued to see if the photothermal properties of the rGO/dopa-MALc(RGDfC) nanosheets can further improve the cell-killing ability without losing its targeting benefit.

Targeted Photothermal Cell-Killing Ability of DOX-Loaded rGO/dopa-MALc(RGDfC)

Graphene nanosheets, due to their good photothermal properties together with excellent loading capacity of water-insoluble cancer drugs(10) and drugs with aromatic structures(7) such as DOX, are considered as an ideal nanomaterial for the development of combined therapeutic approaches. [START_REF] Sneider | Remotely Triggered Nano-Theranostics For Cancer Applications Nanotheranostics[END_REF][START_REF] Chen | Functionalized Graphene Nanocomposites for Enhancing Photothermal Therapy in Tumor Treatment[END_REF] Particularly, the photothermal properties are appealing, as the generated heat will result in the photothermal ablation of cancer cells, which is known to increase the sensitivity of chemotherapy.(34) Figure 6A depicts the heat profiles of different concentrations of DOX-loaded rGO/dopa-MAL-c(RGDfC) solutions upon irradiation for 10 min with a continuous NIR laser at 980 nm at various laser power densities. The temperature increase is dependent on concentration and laser power, with a 100 μg mL -1 DOX-loaded rGO/dopa-MAL-c(RGDfC) heating from 22 to 45 °C in 10 min using a laser power of 4 W cm -2 , being sufficient to induce cancer cell death. In addition, the generated heat gradient changes the affinity of DOX to rGO/dopa-MAL-c(RGDfC). As seen from Figure 6B, a pHand laser-power-dependent DOX release takes place. The release is rather moderate from 0.17 μg mL -1 DOX (no light) to 2.1 μg mL -1 upon illumination at 4 W cm -2 . Looking at cell viability upon light irradiation (Figure 6C,D), it becomes additionally clear that the laser power densities of 4 W cm -2 override the targeting approach, resulting in nontargeted cell killing. With a laser power of 2 W cm -2 , the DOX-loaded rGO/dopa-MAL-c(RGDfC) shows a somewhat improved IC 50 = 48 μg mL -1 for MDA-MB-231 cells, corresponding to DOX IC 50 = 8.2 μg mL -1 and keeping a complete targeting capability.

Conclusions

In conclusion, we have demonstrated the interest of a maleimide-containing catechol (dopa-MAL) ligand as a versatile surface anchor onto the rGO nanosheets. The rGO/dopa-MALmodified nanosheets can be easily modified with the thiol-containing molecules under ambient metal-free conditions. We could show that this approach is not only effective for model molecules such as glutathione and ferrocene containing a thiol group, but it is also adaptable to the covalent linking of thiol-containing tripeptides, such as the cancer cell targeting peptide c(RGDfC). The specific cell targeting and ablation properties of DOXloaded rGO/dopa-MAL-c(RGDfC) nanosheets was confirmed through the cell viability of human breast MDA-MB-231 adenocarcinoma cells after 48 h incubation with the nanostructures and of the human cervical HeLa cancer cells as the negative control. The HeLa cells remained unaffected by even high concentrations of DOX-loaded rGO/dopa-MALc(RGDfC). The nanostructures were, however, effective on MDA-MB-231 cells with an IC 50 = 58 μg mL -1 for the DOX-loaded rGO/dopa-MAL-c(RGDfC), corresponding to an IC 50 = 9.9 μg mL -1 for DOX in the matrix. Slight improvements were achieved upon 980 nm irradiation of the DOX-loaded rGO/dopa-MAL-c(RGDfC) in the presence of MDA-MB-231 cells for 10 min at 2 W cm -2 , whereas high laser power densities proved detrimental for the targeting ability of the c(RGDfC) peptide-containing construct.

  4-(exo-3,5-Dioxo-10-oxa-4-aza-tricyclo[5.2.1.0]dec-8-en-4-yl)butyric acid (1) was obtained according to a previously reported literature procedure.(30) Dopamine group conjugation was achieved through the reaction between NHS-activated acid of maleimide derivative (2) and dopamine. The 1 H NMR spectroscopy of dopa-pMAL (3) is seen in FigureS1A. Furan deprotection was achieved by heating dopa-pMAL in toluene followed by NMR spectroscopy (FigureS1B). Loss of proton resonances at 2.84, 5.23, and 6.51 ppm belonging to the bicyclic core and emergence of a new proton resonance at 7.0 ppm belonging to the proton on maleimide ring were indicative of effective furan deprotection. The 13 C NMR spectrum for unmasked maleimide-containing dopamine (dopa-MAL, 4)

Figure 1 .

 1 Figure 1. Synthesis of dopa-MAL (4) and its conjugation onto rGO through π-π stacking interactions

Figure 2 .

 2 Figure 2. (A) FT-IR spectra of rGO/dopa-MAL (black) and dopa-MAL (gray). (B) C 1s widescan XPS spectrum of rGO/dopa-MAL. (C) N 1s wide-scan XPS spectrum of rGO/dopa-MAL. (D) UV-vis of rGO/dopa-MAL.

  5 eV (C-C sp 2 ), 285.0 eV (C-H/C-C), 286.1 eV (C-O), 288.2 eV (N-C═O, C═O), and 290.3 eV (COOH). The presence of N 1s is attributed to the incorporation of maleimide onto the rGO sheets. The band at 400.2 eV in the N 1s high resolution (Figure2C) corresponds to the N-C═O bonds in the rGO/dopa-MAL nanocomposite. The UV-vis spectrum of a water dispersion of rGO/dopa-MAL (Figure2D) exhibits an absorption maximum at 269 nm, characteristic of the sp 2 hybridized structure together with a prolonged absorption tail in the NIR.

Figure 3 .

 3 Figure 3. (A) S 2p high-resolution XPS spectra of rGO/dopa-MAL-glutathione (blue) and rGO/dopa-MAL-ferrocene (black). (B) Fe 2p high-resolution XPS spectrum of rGO/dopa-MAL-ferrocene. (C) Cyclic voltammogram of rGO/dopa-MAL-ferrocene in tetraethyl ammonium hexafluorophosphate (TEAPF 6 )/DMF (0.1 M), scan rate 100 mV s -1 . (D) Change in the current density as a function of scan rate.

Figure 4 .

 4 Figure 4. Formulation of doxorubicin-loaded rGO/dopa-MAL-c(RGDfC): (A) Schematic of the attachment of targeting peptide to rGO/dopa-MAL via Michael addition followed by the noncovalent integration of doxorubicin. (B) Cell viability of different concentrations (μg mL -1 ) of rGO/dopa-MAL-c(RGDfC) on HeLa and MDA-MB-231 cells after incubation for 48 h. (C) UV-vis spectrum of the DOX-loaded rGO/dopa-MAL-c(RGDfC).

3. 4 .

 4 Targeted Cell-Killing Ability of DOX-Loaded rGO/dopa-MAL-c(RGDfC)We next evaluated the cell-killing ability of DOX-loaded rGO/dopa-MAL-c(RGDfC) on the human cervical HeLa and the human breast MDA-MB-231 cancer cells at pH 7.4. The HeLa cells were used as a negative control, as the nanostructures are designed to target essentially the MDA-MB-231 cancer cell line. The dose-response curves of the free DOX for HeLa and MDA-MB-231 cancer cells (Figure5A) allow to determine the IC 50 values of 2.35 and 10.5 μM for HeLa and MDA-MB-231 lines, respectively. It is, thus, clear that DOX is much less effective on the MDA-MB-231 cell line than on the HeLa and will preferentially kill HeLa cells.

Figure 5 .

 5 Figure 5. Dose-response curves of HeLa (black) and MDA-MB-231 (blue) cancer cells to various concentrations of (A) free DOX for 24 h and (B) DOX-loaded rGO/dopa-MALc(RGDfC) nanostructures for 48 h. (C) Optical images showing the targeting effect of DOXloaded rGO/dopa-MAL-c(RGDfC) at two different concentrations (50 and 100 μg mL -1 ) after 6 h incubation before and after washing with PBS.

Figure 6 .

 6 Figure 6. (A) Photothermal heating profiles of DOX-loaded rGO/dopa-MAL-cRGDfC (100 μg mL -1 ) using a 980 nm laser at different laser power densities. (B) DOX amount released from the DOX-loaded rGO/dopa-MAL-cRGDfC (17 μg DOX for 100 μg rGO/dopa-MAL-cRGDfC) matrix. (C) Cell viability of MDA-MB-231 cells after irradiation for 10 min at 980 nm with different laser power densities after 6 h incubation and then additional 18 h incubation. (D) Cell viability of HeLa cells after irradiation at 980 nm for 10 min at different laser power densities after 6 h incubation and then additional 18 h incubation.

Table 1 . Determination of the Atomic Percentages of Elements by the XPS Analysis composite C 1s atom % O 1s atom % N 1s atom % S 2p atom % Fe 2p atom

 1

	%

Acknoledegment

The Centre National de la Recherche Scientifique (CNRS), the University Lille 1, the Hautsde-France region, the CPER "Photonics for Society," the Agence Nationale de la Recherche (ANR), and the EU union through FLAG-ERA JTC 2015-Graphtivity, and the Marie Sklodowska-Curie action (H2020-MSCA-RISE-2015, PANG-690836) are acknowledged for the financial support.


Rebecca Fribourg
email: rebecca.fribourg@inria.fr

Ferran Argelaguet
email: ferran.argelaguet@inria.fr

Ludovic Hoyet
email: ludovic.hoyet@inria.fr

Anatole L Écuyer
email: anatole.lecuyer@inria.fr

Studying the Sense of Embodiment in VR Shared Experiences

Keywords:

: Setup of the experiment: each user was able to interact in the virtual environment with his own avatar, while the physical setup provided both a reference frame and passive haptic feedback. From left to right: experimental conditions Alone, Mirror and Shared; Physical setup of the experiment.

INTRODUCTION

With the development of consumer-grade Virtual Reality (VR) equipment, more and more high-quality shared VR experiences are now being released by VR developers. Such progresses reinvigorate research interests in shared virtual environments (VE), e.g. [START_REF] Brown | Coordinating attention and cooperation in multi-user virtual reality narratives[END_REF][START_REF] Kuszter | Exploring stereoscopic multi-user interaction with individual views[END_REF][START_REF] Sharma | Multi-user vr classroom with 3d interaction and real-time motion detection[END_REF], and therefore in exploring the questions raised by such experiences. How do people interact with each other in VR? How do we represent users in VR? How do users perceive their virtual representation in VR? How does this representation influence their virtual experience? Among these topics, questions related to the avatar (i.e. the user's representation in the VE) are becoming more and more important because of their potential influence on shared experiences, especially as avatars have been demonstrated to improve perception in VR [START_REF] Mohler | The effect of viewing a self-avatar on distance judgments in an hmdbased virtual environment[END_REF], to influence behaviour [START_REF] Peck | Putting yourself in the skin of a black avatar reduces implicit racial bias[END_REF], and to provide a certain level of embodiment in the virtual world [START_REF] Kilteni | The sense of embodiment in virtual reality[END_REF]. In particular, the notion of Sense of Embodiment (SoE) is becoming more and more exploited to evaluate the extent to which users claim ownership of their avatar, e.g., to demonstrate that a virtual limb or a full virtual body can create illusions of body ownership [START_REF] Slater | Towards a digital body: the virtual arm illusion[END_REF][START_REF] Slater | First person experience of body transfer in virtual reality[END_REF], even to the extent of controlling virtual bodies differing from the participants' own body [START_REF] Hoyet | wow! i have six fingers!": Would you accept structural changes of your hand in vr[END_REF][START_REF] Won | Homuncular flexibility in virtual reality[END_REF]. However, such studies only focus on single-user experiences. It is therefore unclear how sharing virtual experiences with another user embodied in an avatar might influence one's own SoE, which is the focus of this paper.

At this point, it seems important to mention the concept of Presence in VR, which has been widely studied for shared experiences (e.g., [START_REF] Heeter | Being there: The subjective experience of presence[END_REF][START_REF] Schroeder | Social Interaction in Virtual Environments: Key Issues, Common Themes, and a Framework for Research[END_REF]). Particularly related to this paper, it was demonstrated that the sight of a human-like representation in the VE (apart from user's own avatar) had a positive effect on users' sense of presence [START_REF] Wilcox | Personal space in virtual reality[END_REF]. However, Presence focuses on "the feeling of being in the virtual world" [START_REF] Schuemie | Research on presence in virtual reality: A survey[END_REF], and not specifically on "the feeling of being embodied in one's avatar" [START_REF] Kilteni | The sense of embodiment in virtual reality[END_REF], and therefore differs from the SoE. This is why we focused in this paper on the SoE and decided to evaluate if the presence of other users embodied in the VE influences a user's own SoE.

To explore this question, ten pairs of male participants volunteered for a VR experiment where they sat in front of a table, with colocalized physical and virtual setups. They were embodied in a co-localized avatar (see Figure 1) and were asked to perform a gamified task. Each participant performed the experiment both alone and facing another embodied user. In order to assess users' SoE, we collected subjective questionnaires during and after the experiment, as well as physical reactions to the presence of a visual threat introduced in the form of sharp spikes at the edges of the table in half of the experimental conditions. Our results show that users experienced comparable levels of embodiment when immersed alone or with another user, but were more engaged when sharing the virtual experience, in particular during more competitive tasks. In this paper, the term "engagement" is related to the user implication and involvement in the VE, which was suggested to be strongly influenced by the notion of embodiment [START_REF] Przegalińska | Embodiment, engagement and the strength virtual communities: Avatars of second life in decay[END_REF].

The remainder of the paper is structured as follow. Section 2 provides an overview of related work regarding the SoE and shared experiences in VR. Section 3 presents the experiment conducted to evaluate the SoE in shared experiences along with the results, which are discussed in Section 4. Finally, Section 5 provides the concluding remarks.

RELATED WORK 2.1 Embodiment in VR

With the recent technological developments, avatars are now highly used in VR for research purposes, e.g., for 3D graphics and games research [START_REF] Trepte | Avatar creation and video game enjoyment[END_REF], behavioral research in psycholinguistic [START_REF] Heyselaar | In dialogue with an avatar, language behavior is identical to dialogue with a human partner[END_REF], or psychological research in general [START_REF] Zhang | Body ownership and response to threat[END_REF]. Indeed, avatars can be altered in numerous ways to assess changes in users' behaviors. It becomes possible to control the realism [START_REF] Petkova | If i were you: Perceptual illusion of body swapping[END_REF], the shape [START_REF] Piryankova | Owning an overweight or underweight body: Distinguishing the physical, experienced and virtual body[END_REF] or even the morphology of the avatar [START_REF] Hoyet | wow! i have six fingers!": Would you accept structural changes of your hand in vr[END_REF]. But how can the effects of these changes be measured? As mentioned by Jacquelyn Ford Morie, when a user enters a VE, he has the simultaneous perception of two distinct bodies "whether there is a virtual body image or whether there is direct or interpreted mappings of navigation movements" [START_REF] Morie | Performing in (virtual) spaces: Embodiment and being in virtual environments[END_REF]. However, we can wonder if it is possible to experience the same sensations across a virtual body in an immersive VE as we would experience them through the biological body. This can be measured by studying the SoE, a complex phenomenon which can be further subdivided in three dimensions according to Kilteni et al. [START_REF] Kilteni | The sense of embodiment in virtual reality[END_REF]: the sense of self-location, the sense of agency and the sense of body ownership. While De Vignemont [START_REF] Vignemont | Embodiment, ownership and disownership[END_REF] also provided a similar division by three main dimensions (Spatial, Motor and Affective), we decided to use the definition of Kilteni et al. in our study.

Self-location -"The sense of self-location refers to one's spatial experience of being inside a body and it does not refer to the spatial experience of being inside a world" [START_REF] Kilteni | The sense of embodiment in virtual reality[END_REF]. How do we perceive ourselves located in a virtual space? What can alter this sense of self-location? Among other studies, the famous rubber-hand illusion experiment [START_REF] Botvinick | Rubber hands 'feel' touch that eyes see[END_REF] revealed that the sense of self-location can be modified when synchronous visuo-tactile correlations are applied to a fake (rubber) and a real hand. This experiment has also been extended to virtual reality [START_REF] Slater | Towards a digital body: the virtual arm illusion[END_REF] increasing the possibilities to elicit a sense of embodiment by a more flexible approach, with for example the possibility to easily move the virtual arm according to the participant's movements.

Agency -The sense of Agency can be described as a motor activity control, and refers to the fact of experiencing an action, intention or selection toward a body. When proprioception is defined as the sense "that people know where the parts of their body are", the sense of agency could be defined as the sense that people have of knowing which action they can do, which control they have over this body and to which extent [START_REF] Blanke | Full-body illusions and minimal phenomenal selfhood[END_REF]. For example, it was questioned whether the virtual representation of a hand alters the sense of agency [START_REF] Argelaguet | The role of interaction in virtual embodiment: Effects of the virtual hand representation[END_REF], which was demonstrated to be more related to the virtual hand control and the task efficiency than to the virtual hand representation.

Ownership -The sense of ownership can be described as one's self-attribution of a body [START_REF]On agency and body-ownership: Phenomenological and neurocognitive reflections[END_REF] and a number of studies have explored how ownership can be elicited. For example, Botvinick et al. [START_REF] Botvinick | Rubber hands 'feel' touch that eyes see[END_REF] showed that synchronous visual and tactile stimuli can elicit the illusion of the ownership of a fake limb (rubber hand) into one's body representation. However, most studies only manipulate a limited number of factors. According to Slater et al. [START_REF] Slater | First person experience of body transfer in virtual reality[END_REF], the power of immersive virtual reality (IVR) had not been exploited yet to create various transformations in body ownership. Indeed, IVR gives the possibility to another kind of experimental design which allow to easily test different factors, such as first person point of view (1PPOV) to third person point of view (3PPOV) [START_REF] Petkova | When right feels left: Referral of touch and ownership between the hands[END_REF], changing the visual aspect of a part of the virtual body [START_REF] Won | Homuncular flexibility in virtual reality[END_REF] or altering the morphology of the virtual body [START_REF] Hoyet | wow! i have six fingers!": Would you accept structural changes of your hand in vr[END_REF][START_REF] Laha | Evaluating control schemes for the third arm of an avatar[END_REF].

Evaluating VR Shared Experiences

As people commonly interact and collaborate with each other in real life, the need to enable such collaborations to create more immersive VR is nowadays increasing. Historically, such questions paved the way to the development of Collaborative Virtual Environments (CVEs) [START_REF] Sharma | Multi-user vr classroom with 3d interaction and real-time motion detection[END_REF], telepresence platforms, and led to several types of experiments, e.g., regarding social interaction and group behavioral studies [START_REF] Slater | Meeting People Virtually: Experiments in Shared Virtual Environments[END_REF]. For instance, multi-user immersion was used to evaluate whether users in a small group would be more efficient in realizing a task in real world or in virtual reality [START_REF] Slater | Small-group behavior in a virtual and real environment: A comparative study[END_REF], with results suggesting that the immersed person tended to emerge as the leader in virtual groups, but not in real meetings.

To evaluate and enhance the quality of such shared experiences, the concept of Presence was originally explored. However, because of the complexity of such experiences, a user's sense of presence can be influenced by numerous factors [START_REF] Schuemie | Research on presence in virtual reality: A survey[END_REF]. In particular, it was demonstrated that seeing other users in the VE could be taken as an evidence of one's proper existence in the VE, and could increase the sense of presence [START_REF] Heeter | Being there: The subjective experience of presence[END_REF]. This supported the necessity to differentiate new notions, such as co-presence and social presence, from personal presence [START_REF] Slater | The influence of body movement on subjective presence in virtual environments[END_REF]. Those terms became highly employed when conducting studies on CVEs, for example in [START_REF] Poeschl | Measuring co-presence and social presence in virtual environments -psychometric construction of a german scale for a fear of public speaking scenario[END_REF]. It is indeed quite interesting to wonder how being with others in the same VE might influence the way we perceive it. It was for instance showed in [START_REF] Slater | Public speaking in virtual reality: facing an audience of avatars[END_REF], that co-presence in VR has for consequence to amplify users' reaction, making a "bad" situation worse and a "good" situation better.

Following these results, several studies naturally focused on the effects of the user representation on the sense of presence in shared VEs. In particular, they demonstrated that embodying users in anthropomorphic and realistic avatars also increase their own sense of presence [START_REF] Nowak | The effect of the agency and anthropomorphism on users' sense of telepresence, copresence, and social presence in virtual environments[END_REF], and more generally enhance their whole VR experience [START_REF] Bailenson | The effect of behavioral realism and form realism of real-time avatar faces on verbal disclosure, nonverbal disclosure, emotion recognition, and copresence in dyadic interaction[END_REF][START_REF] Roth | Socially immersive avatar-based communication[END_REF]. For instance, it was demonstrated that changing the avatar representation had a direct effect on the quality of social interaction in shared VEs, and more precisely that social interactions tend to be impeded with non-realistic avatars [START_REF] Roth | Avatar realism and social interaction quality in virtual reality[END_REF].

However, while measuring the quality of VR shared experiences with multi-user immersion has clearly required to explore new concepts related to the sense of presence, such as co-presence and social-presence, the sense of embodiment which is widely studied for single-user experiences is seldom explored in this context. For this reason, our contribution in this paper is to explore the influence of sharing a VR experience on the sense of embodiment.

EXPERIMENT

We hypothesized that being immersed in the same VE while sharing a common task together with another user will reinforce the SoE. In particular we made the assumption that seeing another user's avatar will reinforce the user experience, and in particular, that it will enable users to experience a higher sense of ownership and agency. In order to test this hypothesis, we designed an experiment in which users could perform a specific task, i.e. a whac-a-mole game (see Figure 1), alone or together with another user. To ensure that potential differences would not only be due to additional visual cues due to the presence of another body, we also introduced a condition where users were immersed alone in front of a mirror and therefore saw their own reflection. In order to assess users' SoE, we collected subjective questionnaires during and after the experiment. We also introduced a visual threat in half of the trials, in the form of sharp spikes at the edges of the table, and measured users' behavioral changes while performing the task. Steps are in order: 1) avatar initial resting pose, 2) rotation of the torso to align the avatar's head with the HMD, 3) elbow flexion to satisfy the distance between the shoulder joint and the target wrist (inferred from the controller transformation), and 4) final pose after rotating the shoulder to align the wrist with the target wrist.

Participants

Twenty male unpaid participants from the university campus took part in the experiment (age: min=21, max=33, and avg=26±2), recruited both among general students and staff. They were all naive with respect to the purpose of the experiment, had normal or corrected-to-normal vision, and gave written and informed consent. The study conformed to the declaration of Helsinki. Participants took part in the experiment in pairs. Among the participants, 9 subjects had none to very limited previous experience with VR, 6 had some previous experience, and 5 were familiar with VR. All participants were right-handed male Caucasians, to match the visual appearance of the virtual avatar as much as possible. In order to avoid any gender interaction bias, we always used same-gender avatars for each participant pair, with the assumption that mixing genders in pairs could have influenced interaction between users.

Technical Details

We developed a collaborative platform in Unity in which two users could share the same virtual and real environment, and interact in real time. Our setup was based on two HTC Vive Head-Mounted Displays (HMDs) with four HTC Vive controllers, to immerse participants in the VE. Users were embodied in anthropomorphic virtual avatars in 1PPOV (see Figure 4 left). In the center of the tracking zone, two chairs and a table were placed. A thin foam layer covered the table to avoid impacts of the HTC Vive controllers. The physical furniture had its virtual counterpart in the VE providing both a reference frame and passive haptic feedback (see Figure 1). Finally, the experiment took place inside a standard virtual office.

In order to elicit high levels of embodiment, we chose to use realistic human avatars in our experiment. Because sharing experiences with other embodied users means that people do not only observe their own virtual body, but also others', a lot of attention was given to the animation quality of the avatars, i.e. on the way avatars moved according to their user's movements. In particular, animation and control quality are strongly linked to the sense of agency, and are therefore extremely important to measure the SoE. We then detail two main aspects of the animation of the avatars: 1) the calibration of the avatar size to the user's and 2) the animation of the avatar according to the user controls (i.e. HTC Vive head and hands tracking).

Avatar Calibration

In order to provide the best experience, it is important to match the participants' height with their avatar's, in particular to ensure that the camera viewpoint is located near the head of the avatar, and at a correct height from the floor. Before starting the experiment, participants were therefore asked to sit upright on their chair while wearing the HMD and to place their hands on the table while holding the controllers. Then, the avatar's torso was automatically scaled to align the vertical position of the HMD with the avatar's eye height.

Avatar Animation

Avatars were controlled by user movements through the use of the HTC Vive HMD (head) and controllers (hands). However, as users were sitting in a chair, we only needed to animate the upper part of the avatar body, which was performed using a two-step process. First, we used the HMD position to drive the torso of the avatar by rotating the torso (from spine to head) around the pelvis (Figure 2.2), thus ensuring the alignment of the HMD position with the avatar's eyes (i.e. leaning based on the user's movements). Yet, during pilot studies we noticed that such alignment was not sufficient when users looked behind them, which often occurs when users want to explore a new virtual environment. As shoulders were not tracked, only the head of the avatar turned in such cases, which created visual skinning artifacts around the neck. As real life people would actually twist their spine to look behind them, we therefore included an additional linear combination of the head rotation along the spine which minimized skinning artifacts.

As a second step, the arms of the avatar were then driven with a standard analytical Inverse Kinematics method using the position and orientation of the Vive controllers. The rig of the character hands were modified ensuring that the character grasped the controller as naturally as possible. As the character rig and the current position and orientation of the controllers were known, the position and orientation of the characters' wrist could be inferred (hereafter referred as target wrist). At that stage we make two assumptions: first that the predefined relative transformation locating the Vive controller in the hand coordinate system is the same for all subjects and second, that it remains constant during the experiment, i.e. subjects do not modify their initial controller grasping posture. This approach provided satisfying results.

Moreover, characters were manually posed at rest (before animation) with the arms at a 10 • abduction angle from the vertical of the trunk (see Figure 2.1). During run-time, forearms were first flexed so that the distance between wrist and shoulder joints matched the distance between the shoulder joint and the target wrist (Figure 2.3). Then, we computed the normal vector to the plane defined by the shoulder, wrist and target wrist positions, and rotated the arm around this vector to align the wrist with the target wrist (Figure 2.4). This method allows us to avoid elbow singularities, while creating arm poses driven by the original abduction angle of the avatar at rest. While the elbow location might not match the users', this is a solution commonly used in interactive applications [START_REF] Kulpa | Morphology-independent representation of motions for interactive human-like animation[END_REF] [START_REF] Hecker | Real-time motion retargeting to highly varied user-created morphologies[END_REF]. It is also important to point out that the avatars' static hand postures matched a natural grasping of the virtual HTC controllers. We address the reader to the accompanying video to appreciate the avatar animation.

Experimental Protocol

Upon their arrival, participants read and signed the experiment consent form and filled in a demographic questionnaire. Then, they were briefed about the experiment and immersed into the VE (occupying one of the two chairs). As some experimental conditions (see Section 3.4) required one user and others two users, we scheduled the experiment so that 1) the first user performed all single-user conditions, then 2) the second user arrived and the two-users conditions were performed, and finally 3) the first user left and the second user performed the single-user conditions (see Figure 3).

Before each condition participants performed a short training session, in which they were asked to grasp virtual cubes and to place them at specified locations to become familiar with the system and the environment (see Figure 4 right). Using the original 3D model of the HTC Vive controller, we attached a 3D claw model on top, which was animated when pressing the trigger button of the controller. The virtual claw was used to pick up the cubes and to move them. When two users shared the same environment, they performed this task together by positioning successively one cube at a time.

After the training, participants performed the main task which consisted in a whac-a-mole game. A virtual foam hammer was attached to the virtual HTC Vive controller of the user's dominant hand, which participants used to hit the moles. Moles appeared at random time intervals and at random spots on the table (4x3 spots), and stayed visible from 0.8 to 2.6 seconds. They were also color-coded to indicate to participants which moles they had to hit (see Section 3.4). A score panel displayed the accumulated score for each round. Hitting the right mole increased the score by one, and hitting a wrong mole decreased the score by one. The task was moderately demanding in terms of attention and required fast reaction. Furthermore, while the non-dominant was not actively used in the task, users were still holding a controller tracking their non-dominant hand location. This information was used both for analysis of embodiment and animation purposes. Finally, participants filled in a subjective questionnaire at the end of each block of the experiment in order to gather subjective impressions on presence and embodiment.

Experimental Design

In our within-subject design, three independent variables were considered: Experience, Target and Danger. The main independent variable (Experience) considered whether there was a shared experience or not, and had three levels (Figure 1): 1) the user performed the task alone (Alone), 2) two users performed the task at the same time, sitting in front of each other (Shared), and 3) a control condition in which the user performed the task alone, but a mirror in front of him enabled to see his avatar (Mirror). The second independent variable (Target) was the difficulty of the whac-a-mole task, which had two levels: 1) users could hit all the moles (AllTargets) and 2) users could hit only half of the moles (OwnTargets). In OwnTargets, users were asked to hit only the moles corresponding to their color (matching the color of their shirt, green or purple), and hitting the wrong mole decreased their score. In AllTargets, all moles had the same color (white). This variable allowed to create two different situations. One more competitive, where users had to compete for the same moles, and another less competitive, where they only focused on their moles. Finally, the addition of potentially harmful elements in VEs is commonly used in embodiment studies to assess behavioral responses [START_REF] Hoyet | wow! i have six fingers!": Would you accept structural changes of your hand in vr[END_REF][START_REF] Zhang | Body ownership and response to threat[END_REF]. Thus, we considered the additional independent variable (Danger) whether there was a potential threat to the virtual avatar (Danger) or not (Safe). The potential threat were 25cm-height sharp spikes placed around the table (Figure 5).

The overall organization of the experiment is summarized in Figure 3, and further described below. The experiment was divided into 3 blocks, corresponding to the three Experience conditions. The Experience conditions were not fully-counterbalanced due to practical reasons, as single conditions were always done together.

Yet, half of the users did the shared condition first and half did it last. The Alone and Mirror conditions were counterbalanced for each pair of users. Each block included the training task and eight rounds of the whac-a-mole task (2 Target x 2 Danger x 2 repetitions). Each round had a duration of 1 minute and the threat always appeared 3 seconds after the beginning of the round and remained present until the end of the round. Target and Danger levels were fully counterbalanced. There were 32 moles for each round. At the end of each block, users removed the HMD and filled a subjective questionnaire to gather their subjective impressions. In total the experiment lasted approximately one hour.

The measured data (dependent variables) took into account performance and behavioral measurements which might show changes on the sense of embodiment (mainly ownership). Regarding performance, we only measured the mean selection time. It considered the time required to hit the mole after its appearance (in seconds). For each user, only trials in which they successfully hit a mole were considered. We did not consider the user score because performance was close to 100% in most conditions. Regarding behavioural measures, we mainly focused on the mean elevation of the dominant and non-dominant hands (in meters), which could be influenced by the virtual threat. Finally, there is also the subjective responses for the final questionnaire (see Table 1). The questionnaire was inspired from previous work [START_REF] Botvinick | Rubber hands 'feel' touch that eyes see[END_REF][START_REF] Kalckert | The spatial distance rule in the moving and classical rubber hand illusions[END_REF][START_REF] Longo | What is embodiment? a psychometric approach[END_REF] and divided in three groups: presence, ownership and agency. For each question, participants were asked to rate their answer on a 7-point Likert scale. Participants also reported general comments and feedback at the end of each questionnaire.

In summary, considering our experimental design, our main hypotheses were: H1 The more competitive the task is, the lower the mean selection time will be.

H2

The mean elevation of the dominant hand will be higher when the Danger is visible.

H3

The mean elevation of the non-dominant hand will be higher when the Danger is visible.

H4 Presence ratings will be higher when sharing the VE.

H5 Body ownership ratings will be higher when sharing the VE.

H6 Agency ratings will be higher when sharing the VE.

Results

Three-way Repeated Measures ANOVA analyses were performed to test the significance of the Experience, Danger and Target levels for each dependent variable. When main or interaction effects were found (p < 0.05), they were explored using pairwise Tukey post-hoc tests (α > 0.05). Only significant results are discussed. Anderson-Darling normality tests were performed to ensure a normal distribution of the data. Effect size was computed using partial eta squared (η 2 p ). Selection Time: The ANOVA analysis showed three main effects regarding Experience (F 2,32 = 47.31, p < 0.001, η 2 p = 0.75), Danger (F 1,16 = 22.08, p < 0.001, η 2 p = 0.58) and Target (F 1,16 = 232.46, p < 0.001, η 2 p = 0.94), Figure 6 (Left) shows the summary of the results. Posthoc tests showed that participants were significantly faster in the Shared condition compared to the Alone or the Mirror conditions. They were also significantly faster in AllTargets compared to Own-Targets, as well as in the Safe compared to Danger stages. Furthermore a two-way interaction was found between Experience and Target (F 2,32 = 35.75, p < 0.001, η 2 p = 0.69), where post-hoc tests showed that users were the fastest in the Shared × AllTargets combination. The interaction effect supports H1, as the most competitive condition Shared × AllTargets had the lowest selection time. H1 is further supported by the main effects of Experience and Target.

Dominant Hand Elevation:

The ANOVA analysis only showed a main effect of Danger (F 1,16 = 33.18, p < 0.001, η 2 p = 0.67). Post-hoc tests showed that users placed their dominant hand higher when the danger was visible than when it was not (M = 1.03m; SD = 0.08m vs M = 0.93m; SD = 0.04m; table height: 0.8m; see Figure 6, Right). This result supports H2 and showed an adaptation of users' behaviour due to the appearance of the virtual spikes.

Non-Dominant Hand Elevation: The ANOVA analysis showed a main effect of Experience (F 2,32 = 11.03, p < 0.05, η 2 p = 0.19) and a two-way interaction effect between Target and Danger (F 1,16 = 10.61, p < 0.01, η 2 p = 0.4). However, post-hoc tests did not show any significant effect, and mean differences were not higher than a few centimeters overall. The mean elevation was M = 0.81m; SD = 0.05m which shows that it remained very close to the height of the table (0.8m). In summary, this result does not support H3.

Questionnaires: Data from the questionnaires was structured into three groups (presence, ownership and agency). For each group and user, the scores were added (control questions were included by inverting their score), and normalized between 1 and 100 to improve readability (see Figure 7). In order to enable the analysis of the interaction effects (mixed ANOVA analysis) due to the non-continuous nature of the data, unaligned rank transform [START_REF] Wobbrock | The aligned rank transform for nonparametric factorial analyses using only anova procedures[END_REF] was applied. The ANOVA analysis considered the within-subjects variable Experience and the between-subjects variable Order (Shared first vs Shared last). Regarding presence, the ANOVA showed a main effect of Experience (F 2,32 = 8.56, p < 0.001). Post-hoc tests showed that the overall

Variable Question

Presence -Please rate your sense of being in the virtual office space, on the following scale from 1 to 7, where 7 represents your normal experience of being in a place. I had a sense of "being there" in the virtual office space -To what extent were there times during the experience when the virtual office space was the reality for you? There were times during the experience when the office space was the reality for me... -When you think back about your experience, do you think of the office space more as images that you saw, or more as somewhere that you visited? The office space seems to me to be more like ... -When you think back about your experience, do you think more as being elsewhere, or more as being in the office space? I thought more as...

-Consider your memory of being in the office space. How similar in terms of the structure of the memory is this to the structure of the memory of other places you have been today? By 'structure of the memory' consider things like the extent to which you have a visual memory of the office space, whether that memory is in colour, the extent to which the memory seems vivid or realistic, its size, location in your imagination, the extent to which it is panoramic in your imagination, and other such structural elements. I think of the office space as a place in a way similar to other places that I've been today... -During the time of the experience, did you often think to yourself that you were actually in the office space? During the experience I often thought that I was really seated in the office space... Ownership -I felt that the virtual body was my own body.

-I felt that the virtual arms were part of my body.

-I felt that the virtual could be harmed.

-I felt that my real arms could be harmed.

-I felt that virtual arms were not part of my body.

-I felt as if the virtual arms were from someone else's body.

Agency -I felt as if the virtual body moved just like I wanted it to, as if it was obeying my will.

-I expected the virtual body to react in the same way as my own body.

-I felt like I controlled the virtual body as if it was my own body.

sense of presence was higher for the Shared condition compared to the Mirror condition (p < 0.05) and also for the Alone condition (p < 0.05). This result supports H4. Regarding ownership, an interaction was found between Experience and Order (F 2,32 = 5.35, p < 0.01), which was not confirmed by the post-hoc analysis. Still, a deeper analysis seems to suggest that participants who started with the Shared condition gave a lower ownership score for the Alone and Mirror conditions compared to the users finishing with the Shared condition. Yet, the results are inconclusive and do not support H5.

Finally, for agency the ANOVA showed a significant main effect of Experience (F 2,32 = 3.63, p < 0.05). Post-hoc tests showed that agency ratings were lower for the Mirror condition compared to the Shared condition (p < 0.05). This result does not support H6.

DISCUSSION

The main objective of the experiment was to evaluate the influence of sharing a VE with another user also embodied in an avatar on each other's SoE. In this Section we discuss how the results can be interpreted in terms of body ownership and agency but also provide additional insights regarding user engagement and presence. We further illustrate those results with written user feedback, either supporting our results analysis or highlighting other aspects that did not arise from the variables observed during the experiment.

User Performance and Engagement

The results on selection time show that users were significantly more "efficient" in performing the task when sharing the VE and in particular in the competitive level (AllTargets). First, the main effect of Target shows that participants required less time to select the targets in the AllTargets level vs the OwnTargets level. This result can be explained by the increased cognitive load for the OwnTargets level as users had to determine whether the target had to be selected or not. Second, the main effect of Experience could be explained by an increased user engagement during the competitive (Shared) condition, leading to decreased selection times. In particular, this effect was stronger in the AllTargets level (significant interaction effect). This explanation is supported by Lalmas et al. [START_REF] Lalmas | Measuring user engagement[END_REF] who stated that user engagement depends on time, and that challenge is an element that influences engagement. Moreover, it is important to highlight that when users had to compete for the same moles, the evaluated selection time is actually the best out of two participants, rather than their individual performance. Despite the fact that selection time was significant lower in the Shared condition compared to the other conditions, it is still possible that this observation could have influenced this result. However, our result is also supported by the other subset of trials where users had to hit their own moles, in which a relevant change in the selection time was also observed depending on Experience, a result also supported by the increased presence ratings in the Shared condition.

User feedback was also in line with this interpretation. Users expressed a positive feeling towards the fact of sharing the VE with another user: "This is more enjoyable and realistic with a partner", "The feeling of incarnating the avatar is globally better with a second user in front", or "It is better with another person during the experiment".

Finally, users were also faster when the danger was not displayed. While it is difficult to separate selection time from the fact that their dominant hand was closer to the table in the Safe stage, or from the fact that they might have been more careful in the Danger stage, it is nonetheless important to take into consideration that users displayed different "motor strategies".

Body Ownership

First of all, subjective results on body ownership did not show any significant differences at the level of Experience. On average, participants reported a medium level of body ownership M = 52.0; SD = 15.7. Yet, participants starting with the Shared condition demonstrated a tendency to report lower ownership ratings for the Alone and Mirror conditions. This suggests that the Shared condition might have provided an upper bound sense of ownership depending on whether users started with the Shared condition or not. Nevertheless additional experiments would be required to validate this assumption.

Regarding the behavioural measurements, we found that participants placed their dominant hand higher in the presence of a virtual threat. Several hypotheses may explain this phenomenon: is this reaction due to the fact that they feared the threat? Or is it just because they avoided the collision? As it is established that a response to a threat testifies of a high sense of ownership, we make the assumption that participants were really punctually afraid for their virtual body to be harmed. On the contrary, it appeared that participants nearly did not raise their non-dominant hand when the threat was introduced, independently of the condition tested. It is however unclear why participants would react to a threat with their dominant-hand and not with their non-dominant hand. As participants did not need to interact using their non-dominant hand, it is therefore possible that this absence of interaction could be a reason why participants seemed to less appropriate their non-dominant virtual hand as their own. It is also possible that the non-dominant hand was less present in the field of view of participants, which could have influenced their reaction. In either way, participants were never asked to maintain their non-dominant hand on the table. This observation opens the question whether body ownership is uniform regarding the entire virtual body, or depends on whether a body part is active or not.

In addition, comments from users also testified of a reaction toward the virtual threat: "I felt strange when I moved my arm through the spikes" or "When the table was surrounded with spikes, it took me several seconds to be at ease with them and realize I could not be harmed". These remarks support the results of the dominant-hand height regarding the sense of ownership towards the virtual body. It has been considered the possibility that participants would actually move their hand thinking that touching the spikes would decrease their score, as the game was quite competitive, but the way most participants quickly reacted, surprised by the danger appearing, testifies of a basic instinct to a threat toward their body.

It is also interesting to acknowledge that we did not observe a significant increase in ownership in the mirror condition, which is contradictory to previous work where the presence of such a mirror was found to enhance the sense of ownership [START_REF] González-Franco | The contribution of real-time mirror reflections of motor actions on virtual body ownership in an immersive virtual environment[END_REF][START_REF] Spanlang | How to build an embodiment lab: Achieving body representation illusions in virtual reality[END_REF]. One possible explanation is that the mirror might have been distracting for participants, and have possibly highlighted small animation artifacts, which was however not reported by any participant. Furthermore, this result can also be explained due to the uncanny valley effect [START_REF] Mcdonnell | Render me real?: Investigating the effect of render style on the perception of animated virtual humans[END_REF]. The choice of a realistic anthropomorphic avatars might have influenced how participants accepted the avatar as their virtual representation, which could be further explored in future experiments.

Agency

Overall the agency score was high (M = 78.09; SD = 15.24), which shows that the avatar control was realistic and efficient. We took great care in providing a high quality to the visual rendering of the virtual scene, both in terms of appearance and avatar animation. Users were immersed in a realistic environment, similar to a real office, and embodied in realistic anthropomorphic avatars.

Interestingly, the analysis of Agency scores showed that the levels of agency were lower for the Mirror condition. Indeed, three participants communicated a negative feeling towards the presence of a mirror in the VE: "It is better without the mirror", "The mirror effect creates a loss of the sense of presence, I couldn't say why, but it installs a discomfort", and "I felt more immersed without the mirror". The possibility to look to one's own avatar motions in the mirror could have increased the chances to detect imperfections of the avatar control scheme. Also, the fact that we used inverse kinematics to animate the upper body of the avatar might have induced a lack of accuracy at the origin of those results.

Unlike the sense of ownership, to our knowledge the sense of agency had not yet been studied in relation to the presence or not of a mirror in virtual reality. For instance, while Slater et al. [START_REF] Slater | First person experience of body transfer in virtual reality[END_REF] explored the influence on agency of synchronous or asynchronous mirror reflections in IVR, they did not compare it to a control case without a mirror. Yet their results appeared to be in conflict with previous studies that suggested the importance of motor cues for the sense of self [START_REF] Jeannerod | The mechanism of self-recognition in humans[END_REF]. While our results suggest differences in the agency scores between the mirror and single conditions, such differences were small, showing the need to ensure accurate avatar control to maximize the sense of agency.

Limitations and Future Works

One of our verified hypotheses was that competition has an impact on user performance, showing an increase in user engagement. Indeed, the wack-a-mole task had a clear competitive dimension, which had for consequence that users were more attentive and efficient. However, the increase in engagement could have reduced the awareness of participants about their virtual body. Thus, it would be interesting to consider other tasks, reducing the ambiguity between engagement and embodiment. For instance, relevant tasks could involve higher awareness of one's virtual body and of others, such as users collaborating to achieve common goals while finely controlling their virtual body.

In addition, the interaction capabilities of the task were strongly constrained. For example, a participant reported that remaining seated, without being able to explore the room, reduced the ability of considering the virtual office as an actual real room. Further studies could explore increasing the interaction capabilities by providing the possibility to walk/navigate, or to interact with a wider range of virtual objects. Our results are also limited by the fact that we used only male participants, and further studies could be conducted using cross-gender or female participants. Finally, another aspect that requires additional research is the fact that we chose to have users sharing both the same virtual and physical environment. This implied that users eventually saw each other physically and could potentially talk and hear each other directly, which could have introduced additional implications in terms of social interactions. Our study could also be extended by involving more than two users.

CONCLUSION

In this paper, we explored how sharing a virtual environment with another user could generate changes in the behaviour and the perception of the virtual experience such as influencing the sense of embodiment. Our results show that shared experiences increased user engagement and the sense of presence, which is supported by performance and subjective measurements. In addition, all experimental conditions generated a strong sense of embodiment. Taken together, our results lead the way for VR applications designers to identify the important features to consider in order to develop multi-user VE. It can now be taken as an established fact that if users are immersed embodied in respective avatars, their SoE remains quite high, and so does the quality of their experience. It is also well-known that VR finds a large public in the entertainment area, and that multi-user games are quite popular in the gaming community. It is therefore relevant in this area to consider the influence of the competitive dimension existing in these applications on users' quality of experience.

Figure 2 :

 2 Figure 2: Main steps of the Inverse Kinematics based avatar animation. The physical position and orientation of the Vive HMD and controllers are displayed in red, and used as targets for our IK method.Steps are in order: 1) avatar initial resting pose, 2) rotation of the torso to align the avatar's head with the HMD, 3) elbow flexion to satisfy the distance between the shoulder joint and the target wrist (inferred from the controller transformation), and 4) final pose after rotating the shoulder to align the wrist with the target wrist.

Figure 3 :

 3 Figure 3: (Top) Experiment organization for each pair of users, according to the three conditions: Alone, Mirror, Shared. Alone or Mirror conditions were randomly presented first. (Bottom) Details of each condition organization: training followed by the main task, where Own-Targets or AllTargets were presented in random order. Danger and Safe stages were also presented in random order.

Figure 4 :

 4 Figure 4: 1PPOV when performing the whac-a-mole task (left) and 3PPOV when performing the Training (right).

Figure 5 :

 5 Figure5: Danger: virtual spikes appeared around the virtual table in order to threaten the virtual body.

Figure 6 :Figure 7 :

 67 Figure 6: Results summary. (Left) Boxplot of the selection time grouped by Target and Experience. (Right) Boxplot of the dominant hand elevation when hitting the mole grouped by Danger.

Table 1 :

 1 Questionnaire used in the experiment.

ACKNOWLEDGMENTS

We wish to thank all the reviewers for their constructive comments, as well as the participants who took part in our experiments. This work was sponsored by the Région Bretagne.