Multifunctional Hybrid MoS2-PEGylated/Au Nanostructures with Potential Theranostic Applications in Biomedicine

In this work, flower-like molybdenum disulfide (MoS2) microspheres were produced with polyethylene glycol (PEG) to form MoS2-PEG. Likewise, gold nanoparticles (AuNPs) were added to form MoS2-PEG/Au to investigate its potential application as a theranostic nanomaterial. These nanomaterials were fully characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), photoelectron X-ray spectroscopy (XPS), Fourier-transformed infrared spectroscopy (FTIR), cyclic voltammetry and impedance spectroscopy. The produced hierarchical MoS2-PEG/Au microstructures showed an average diameter of 400 nm containing distributed gold nanoparticles, with great cellular viability on tumoral and non-tumoral cells. This aspect makes them with multifunctional characteristics with potential application for cancer diagnosis and therapy. Through the complete morphological and physicochemical characterization, it was possible to observe that both MoS2-PEG and MoS2-PEG/Au showed good chemical stability and demonstrated noninterference in the pattern of the cell nucleus, as well. Thus, our results suggest the possible application of these hybrid nanomaterials can be immensely explored for theranostic proposals in biomedicine.


Introduction
Over the last decades the application of nanomaterials in biomedicine has been raised, especially for biosensing and drug delivery. However, the main feature that must be considered to a given material be used in biomedical proposals is its physicochemical stability and biological safety [1]. In this context, molybdenum disulfide (MoS 2 ) and its derivatives have attracted attention due to its excellent biocompatibility and simple synthesis [2]. The 2D nanomaterials have been used in the diagnosis and therapy of cancer [3] especially when they are combined with gold nanoparticles [4][5][6][7]. For instance, graphene The material was synthesized with hydrothermal method. In a typical experiment, ammonium molybdate (1.80 g) and PEG-2000 (1.0 g) were dissolved in 60 mL deionized water under stirring. Thioacetamide (1.80 g) was added into the solution and the mixture was stirred for 1 h. Then the mixture was transferred into a 100 mL autoclave and reacted for 3 h at 180 • C in an oven. After the autoclave cooling, the precipitate was collected by filtration and washed several times with Milli-Q water, ethanol and acetone. The material was dried in an oven at 100 • C for 4 h and MoS 2 microspheres were obtained.

Au Nanoparticles Production
The production of the gold nanoparticles (AuNPs) followed the method described by Meireles et al. [33]. The stock solution was prepared by mixing 5 mL of CTAB (0.2 M) and 5 mL of HAuCl 4 ·3H 2 O (0.0005 M). Then, 60 µL of ice-cold NaBH 4 (0.010 M) was added to the mixture, resulting in a brownish-yellow solution. Vigorous stirring was kept for 2 min at room temperature. The growing solution was prepared by adding CTAB (0.20 M), AgNO 3 (0.02 M) and HAuCl 4 ·3H 2 O (0.005 M). Then, 210 µL of L-ascorbic acid (C 6 H 8 O 6 ) (0.4 M) was added to the system as a moderate reducing agent changing the color of solution from brownish yellow to colorless. Finally, 300 µL of stock solution was added to the growing solution, kept stirring for 20 min, and gradually changing to a deep red color.

MoS 2 -PEG/Au Synthesis
The MoS 2 (500 mg) sample was dispersed in 70 mL of a mixture containing 350 mg of gold nanorods for 30 min in a bath ultrasound operating at power of 20 W. Then, the mixture was transferred for a polytetrafluoroethylene (PTFE) vessel. The reaction vessel was attached to an autoclave and the incorporation was carried out in an oven at a temperature of 180 • C for two hours.

Sample Preparation for Electrical Measurements
For sheet resistance measurements and contact angle measurements MoS 2 -PEG and MoS 2 -PEG/Au samples were prepared by mechanical compaction using a Shimadzu, SSP-10 A, P/N 200-64,175 hydraulic. The materials were pressured at 50 kN for 2 min, which resulted in disks of 13 mm in diameter and 2 mm in thickness, which can be seen in Figure S2 in the Supporting Information. For electrochemical measurements flexible graphite sheets, 1 mm thick, were cut into 2 × 4 cm 2 rectangles which served as conductive substrate for the electrodes. The electrodes were prepared for use through a procedure that included cleaning the graphite surface with the aid of a soft paper and washing with 92% ethanol solution (v/v), followed by drying with N 2 flow. Then, around 1 cm 2 of the graphite electrodes were modified by drop-casting 1 mL dispersions (1.0 mg mL −1 ) of MoS 2 -PEG and MoS 2 -PEG/Au in isopropyl alcohol. The samples were dried in an oven at 150 • C for 24 h.

Cellular Viability Based on Resazurin Reduction Assay
Cellular viability was assessed by performing viability method according to the manufacturer's instructions (Sigma, St. Louis, MO, USA). Briefly, the cells were seeded at 1 × 10 4 cells per well in 96-well flat-bottom plates (Kasvi, Curitiba, PR, Brazil) and incubated for 24 h at 37 • C with 5% CO 2 atmosphere. Cells were exposed to four different concentrations of MoS 2 -PEG and MoS 2 -PEG/Au, respectively, 100 µg/mL, 50 µg/mL, 10 µg/mL and 5 µg/mL. For death control Triton 100 × 10% was used. Each sample was dispersed in ultrasonic bath for 60 min and left exposed to each cell line for 24 h and 48 h. Subsequently, 10 µL per well of the reagent resazurin were added to the cells already cultured (1:10 final dilution). The cells were kept under biochemical reaction for 4 h at 37 • C in the dark and the absorbance at 580 nm was measured in a spectrophotometer (Multiskan GO, Thermo Fisher Scientific, Waltham, MA, USA). All tests were performed at least two times. The fraction of viable cells in treated groups was calculated as a percentage of the untreated control group that was considered 100% viable.

Hoechst Nuclear Staining Assay
To evaluate cell death patterns after nanomaterials treatment, a fresh staining solution containing Hoechst 33,342 (5 µg/mL) was used. Briefly, 1 × 10 4 FaDU cells were seeded in a 96 well plate and after monolayer cell formation, cells were treated with 100 µg/mL of MoS 2 -PEG/Au for 24 h and 48 h, respectively. Triton 100× was used as death control. Before staining, growth medium was removed and 50 µL of staining solution were added to each well. After 60 min at 37 • C, CO 2 incubator staining solution was removed and cells were fixed by formalin 10%. Cells were analyzed in an Evos FL cell image system microscope (Thermo Fischer, Waltham, MA, USA), (Belo Horizonte, Brazil). The stained cellular nuclei were counted by using ImageJ2 software (NIH, USA).

Characterization
FTIR measurements were made with Thermo Nicolet 6700 equipment (São Paulo, Brazil). The spectra were collected in the ATR mode from 64 accumulations in the transmission mode and were adjusted, taking baseline corrections into account. Micro-Raman measurements were carried out using a Horiba Jobin Yvon iHR 550 spectrometer (São Paulo, Brazil). The 532.0 nm line of an Ar-Kr laser was used as the excitation source. UV-visible spectroscopy measurement of gold nanoparticles was carried out in a UV-2550 spectrometer (Shimadzu, Japan) (São Paulo, Brazil). The spectrum was collected from 1000 to 200 nm in water suspension, where more details can be seen in Figure S1b in the Supporting Information. SEM characterization was made in a Carl Zeiss Field Emission Scanning Electron Microscope, model sigma VP equipped with an energy-dispersive X-ray (EDS) (São Paulo, Brazil). EDS spectra were obtained using a Bunker detector 410-M and software Quantax Espirit 1.9 (São Paulo, Brazil). The Peakfit v4 program was used to adjust the EDS spectra. The STEM images were carried out on a FEI TECNAI G2 (São Paulo, Brazil), microscope with a tungsten filament electron gun of 200 kV. XPS spectra were obtained using monochromatic Al Kα radiation (1486.6 eV). XPS spectra were acquired at room temperature on a VG Scientific Escalab-220-ixL (Belo Horizonte, Brazil). The detailed states of the different elements (C1s, O1s, Mo3p, Mo3d and S2p and Au4f) were obtained from fitting the XPS peaks by using Lorentzian and Gaussian functions. The sheet resistance and electrical conductivity of the nanostructures were measured in quintuplicate at room temperature by using an Ossila T2001A2 Four-Point Probe System (Sheffield, UK) (São Paulo, Brazil), with a probe space of 1.27 mm. Each material was evaluated by the smallest current signal that showed any response as voltage output. The applied current was 5 µA for both samples. The electrochemical measurements were carried out with a potentiostat Metrohm, model PGSTAT 128N (São Paulo, Brazil), controlled by software NOVA version 1.1.1, combined with a lab-made cell with three-electrode configuration, shown in Figures S2 and S3 in the Supporting Information section.

Characterization of Flower-Like MoS 2 -PEG/Au
The morphology and microstructure of the as-synthesized MoS 2 with polyethylene glycol (PEG) were characterized by SEM and STEM microscopy. SEM image (Figure 1a) shows that the structures are formed by uniform hierarchical microspheres of MoS 2 with average diameter of about 400 nm. According to the literature [34], PEG can provide template structure in the synthesis, obtaining flower-like MoS 2 microspheres. STEM images (Figure 2b,c) reveal that each microsphere is composed of several curled and ultra-thin nanosheets distributed on its surface, and the MoS 2 interlayer spacing has approximately 0.54 nm. glycol (PEG) were characterized by SEM and STEM microscopy. SEM image (Figure 1a) shows that the structures are formed by uniform hierarchical microspheres of MoS2 with average diameter of about 400 nm. According to the literature [34], PEG can provide template structure in the synthesis, obtaining flower-like MoS2 microspheres. STEM images (Figure 2b,c) reveal that each microsphere is composed of several curled and ultra-thin nanosheets distributed on its surface, and the MoS2 interlayer spacing has approximately 0.54 nm.  glycol (PEG) were characterized by SEM and STEM microscopy. SEM image (Figure 1a) shows that the structures are formed by uniform hierarchical microspheres of MoS2 with average diameter of about 400 nm. According to the literature [34], PEG can provide template structure in the synthesis, obtaining flower-like MoS2 microspheres. STEM images (Figure 2b,c) reveal that each microsphere is composed of several curled and ultra-thin nanosheets distributed on its surface, and the MoS2 interlayer spacing has approximately 0.54 nm.   (Figure 2b-d), it is noticeable that both structures appear as branched structure of aggregates with rough surface. However, when Au nanorods were added, microspheres exhibited less uniform shape showing a small difference in morphology in relation to the MoS 2 -PEG (see Figure 2d). Figure 3a-c shows EDS mapping were made to evaluate the element distribution of the MoS 2 -PEG/Au sample. It can be observed that the elements Mo, S and Au are uniformly distributed in the studied area. The EDS spectrum (Figure 3d,e) reveals the dominance of Mo and the primary component of MoS 2 , which is sulfur. The presence of C comes from the PEG used as a "covering agent" of the nanoflake during the synthesis [35]. The peaks of Si and Al, which can be considered a type of impurity, are probably associated to the elements present in the sample holder. After modification with AuNPs ( Figure 3e) a new peak referring to Au energy was identified [36]. Detailed discussion of the TEM and UV-vis of AuNPs can be seen in Figure S1 in Supporting Information.
MoS2PEG/Au by SEM (Figure 2b-d), it is noticeable that both structures appear as branched structure of aggregates with rough surface. However, when Au nanorods were added, microspheres exhibited less uniform shape showing a small difference in morphology in relation to the MoS2-PEG (see Figure 2d). Figure 3a-c show EDS mapping were made to evaluate the element distribution of the MoS2-PEG/Au sample. It can be observed that the elements Mo, S and Au are uniformly distributed in the studied area. The EDS spectrum (Figure 3d,e) reveals the dominance of Mo and the primary component of MoS2, which is sulfur. The presence of C comes from the PEG used as a "covering agent" of the nanoflake during the synthesis [35]. The peaks of Si and Al, which can be considered a type of impurity, are probably associated to the elements present in the sample holder. After modification with AuNPs ( Figure  3e) a new peak referring to Au energy was identified [36]. Detailed discussion of the TEM and UV-vis of AuNPs can be seen in Figure S1 in Supporting Information.  [16,37]. The band at 826.4 cm −1 represents the stretching mode band of the Mo=O bond. Previous studies reported that when in contact with air, the surface of MoS2 can be partially oxidized and contains adsorbed water, Mo=O groups, sulfate and thiosulfate anions, among others [16,[37][38][39]. At 931.5 cm −1 , the band originating from the vibrational mode is characteristic of S-S bond [35]. The contribution of PEG begins to appear at 1083.5 cm −1 , where the intense and sharp band points out to the C-O stretching mode, and at 1427.9 cm −1 , with less intense but even strong band from the bending mode vibration of C-H. At, 2880.8 cm −1 , a discrete band is showed from the weak stretching mode vibration of C-H. The results confirm that PEG acts as a wrapper on MoS2  [16,37]. The band at 826.4 cm −1 represents the stretching mode band of the Mo=O bond. Previous studies reported that when in contact with air, the surface of MoS 2 can be partially oxidized and contains adsorbed water, Mo=O groups, sulfate and thiosulfate anions, among others [16,[37][38][39]. At 931.5 cm −1 , the band originating from the vibrational mode is characteristic of S-S bond [35]. The contribution of PEG begins to appear at 1083.5 cm −1 , where the intense and sharp band points out to the C-O stretching mode, and at 1427.9 cm −1 , with less intense but even strong band from the bending mode vibration of C-H. At, 2880.8 cm −1 , a discrete band is showed from the weak stretching mode vibration of C-H. The results confirm that PEG acts as a wrapper on MoS 2 nanoparticles [16,[40][41][42]. As well the weak bands at 3021. 4 and 3179.1 cm −1 as the intense bands at 3616.5 and 3715 cm −1 correspond to the stretching mode of the inter and intramolecular hydrogen bonds and of the hydroxyl adsorbed from the atmosphere [40][41][42][43]. No significant changes were observed in the MoS 2 -PEG/Au spectrum related to MoS 2 -PEG, so it was suppressed in this work. For the MoS 2 -PEG nanomaterial, Raman spectroscopy ( Figure 4b) generated the characteristic bands of MoS 2 (at 371.5 and 462.8 cm −1 ) assigned to the E 2g and A 1g vibrational modes, respectively. Such modes, in turn, correspond to the E 2g (in-plane) vibrations of sulfur and molybdenum atoms, and to the A 1g (out-of-plane) vibrations of them [41]. In the regions of low frequency in Raman (from 329.4 cm −1 ), bands connected to vibrational modes of flexion between C-O and C-O-C were found, as well as low or medium PEG backbone vibrations. The band at 659.3 cm −1 is also derived from the C-C vibration of the PEG skeleton, as well as the intense band at 817.2 cm −1 [42,44,45]. The band at 992.7 cm −1 refers to the stretching and bending mode of the O-H bonds and the stretching of the C-C bonds, which is well defined and more intense for primary alcohols, as is the case with PEG [34,[46][47][48][49]. The MoS 2 -PEG/Au hybrid presented the same bands as the MoS 2 -PEG, but with the additional band at 1493.0 cm −1 ascribed to γ(C-C) from PEG which was intensified due the presence of gold nanostructures [50].
it was suppressed in this work. For the MoS2-PEG nanomaterial, Raman spectroscopy ( Figure 4b) generated the characteristic bands of MoS2 (at 371.5 and 462.8 cm −1 ) assigned to the E2g and A1g vibrational modes, respectively. Such modes, in turn, correspond to the in-plane vibrations of sulfur atoms in one direction and of molybdenum in the (E2g) direction and to the out-of-plane vibrations of sulfur atoms (A1g) [41]. In the regions of low frequency in Raman (from 329.4 cm −1 ), bands connected to vibrational modes of flexion between C-O and C-O-C are found, as well as low or medium PEG backbone vibrations. The band at 659.3 cm −1 is also derived from the C-C vibration of the PEG skeleton, as well as the intense band at 817.2 cm −1 [42,44,45]. The band at 992.7 cm −1 refers to the stretching and bending mode of the O-H bonds and the stretching of the C-C bonds, which is well defined and more intense for primary alcohols, as is the case with PEG [34,[46][47][48][49]. The MoS2-PEG/Au hybrid presented the same bands as the MoS2-PEG, but with the additional band at 1493.0 cm −1 ascribed to γ(C-C) from PEG which was intensified due the presence of gold nanostructures [50]. The surface chemical characterization of the produced nanomaterials was performed by XPS. As shown in the survey spectra, Figure 5a, both MoS2-PEG and MoS2-PEG/Au samples exhibit the elemental Mo, S components as well as carbon and oxygen peaks. The Au4f peak was under the detection limit for MoS2-PEG/Au, suggesting that Au has been covered by the other elements. Figure 5b shows the C1s peak, indicating that the amount of carbon has increased ca. five times after MoS2-PEG/Au synthesis, especially related to the C-O-C, C-OH and C-C species. The O1s peak for MoS2-PEG, see Figure 5c, exhibits a peak at 532.6 eV attributed to O-C, perfectly in line with the presence of PEG [51]. MoS2-PEG/Au shows, besides the O-C bond, a feature at 531 eV which can be attribute to a metal oxide (e.g., MoO3), for that sample. By examination of the Mo3d and S2p doublets of MoS2-PEG, the bottom spectra of Figure 5d,e, respectively, where it is possible to conclude that the MoS2 synthesis was successful. Peaks are very narrow as expected for a crystalline sample and the main peaks, Mo 4+ 3d5/2 at 229.5 eV and S 2-2p3/2 at 162.4 eV agree well with the binding energies found by other authors [52]. Besides that, only a very low contribution of Mo 6+ attributed to molybdenum oxide and a broad (and low) S2p feature at ~169 eV associated with sulfate (S 6+ ) species are observed. The synthesis with AuNPs, which involved bath ultrasound and thermal treatment in air, has importantly modified The surface chemical characterization of the produced nanomaterials was performed by XPS. As shown in the survey spectra, Figure 5a, both MoS 2 -PEG and MoS 2 -PEG/Au samples exhibit the elemental Mo, S components as well as carbon and oxygen peaks. The Au4f peak was under the detection limit for MoS 2 -PEG/Au, suggesting that Au has been covered by the other elements. Figure 5b shows the C1s peak, indicating that the amount of carbon has increased five times after MoS 2 -PEG/Au synthesis, especially related to the C-O-C, C-OH and C-C species. The O1s peak for MoS 2 -PEG, see Figure 5c, exhibits a peak at 532.6 eV attributed to O-C, perfectly in line with the presence of PEG [51]. MoS 2 -PEG/Au shows, besides the O-C bond, a feature at 531 eV which can be attribute to a metal oxide (e.g., MoO 3 ), for that sample. By examination of the Mo3d and S2p doublets of MoS 2 -PEG, the bottom spectra of Figure 5d,e, respectively, where it is possible to conclude that the MoS 2 synthesis was successful. Peaks are very narrow as expected for a crystalline sample and the main peaks, Mo 4+ 3d 5/2 at 229.5 eV and S 2− 2p 3/2 at 162.4 eV agree well with the binding energies found by other authors [52]. Besides that, only a very low contribution of Mo 6+ attributed to molybdenum oxide and a broad (and low) S2p feature at~169 eV associated with sulfate (S 6+ ) species were observed. The synthesis with AuNPs, which involved bath ultrasound and thermal treatment in air, has importantly modified the surface near region of the sample. Now the Mo 3d 3/2 , 3d 5/2 and S 2p 1/2 , 2p 3/2 doublets are highly attenuated at the MoS 2 binding energy positions, the Mo3d peaks were shifted to higher binding energies, where it can conclude that the surface has been oxidized. Two Mo3d 5/2 peaks with binding energies of 232.6 eV and 233.6 eV are assigned to molybdenum oxide (MoO 3 ) and molybdate species, respectively. The same occurs to S2p, it shrinks for MoS 2, and a sulfur doublet shows up at higher binding energy. The S2p 3/2 line appearing at 169.6 eV is attributed to S 6+ in sulfates (SO 4 ) 2 , which can be explained by either MoO 2 (SO 4 ) or Mo(SO 4 ) 3 formation at the surface of MoS 2 . Moreover, it was observed some sulfur loss at the surface near region, which may be due to the formation of volatile sulfur compounds during heat treatment.
Mo3d5/2 peaks with binding energies of 232.6 eV and 233.6 eV are assigned to molyb-denum oxide (MoO3) and molybdate species, respectively. The same occurs to S2p, it shrinks for MoS2, and a sulfur doublet shows up at higher binding energy. The S2p3/2 line appearing at 169.6 eV is attributed to S 6+ in sulfates (SO4) 2 , which can be explained by either MoO2(SO4) or Mo(SO4)3 formation at the surface of MoS2. Moreover, it was observed some sulfur loss at the surface near region, which may be due to the formation of volatile sulfur compounds during heat treatment. Before the capacitance measurements, the electrical characteristics for MoS2-PEG and MoS2-PEG/Au hybrid microspheres were evaluated by sheet resistance, resistivity and conductivity measurements using the four-point probe technique. As shown in Table 1, the two composites exhibit low electrical conductivity. However, as expected, the incorporation of AuNPs in MoS2-PEG significantly decreased the resistivity of the hybrid system. The best electrical conductivity of the MoS2-PEG/Au nanostructures can be associated with junctions between Au nanoparticles in which the electrons are transferred more easily [53]. Additionally, the sulfur atoms over the outer layer of the MoS2 structure are suitable for the formation of strong Au-S bonds, which improves the charge transfer between sulfur and AuNPs [54,55]. Before the capacitance measurements, the electrical characteristics for MoS 2 -PEG and MoS 2 -PEG/Au hybrid microspheres were evaluated by sheet resistance, resistivity and conductivity measurements using the four-point probe technique. As shown in Table 1, the two composites exhibit low electrical conductivity. However, as expected, the incorporation of AuNPs in MoS 2 -PEG significantly decreased the resistivity of the hybrid system. The best electrical conductivity of the MoS 2 -PEG/Au nanostructures can be associated with junctions between Au nanoparticles in which the electrons are transferred more easily [53]. Additionally, the sulfur atoms over the outer layer of the MoS 2 structure are suitable for the formation of strong Au-S bonds, which improves the charge transfer between sulfur and AuNPs [54,55]. Cyclic voltammetry (CV) measurements were performed to evaluate the charge storage capacity of both materials. electrolyte solution over a potential range of −0.2 to 0.4 V at different scan rates. The current signal at the CV curves measured with both electrode materials increases with the corresponding scan rate and the voltammograms show a nearly rectangular aspect, suggesting the charging current is the dominant signal due to reversible adsorption of electrolyte ions on the surface of electrodes. The pattern of specific capacitance decreases for both electrodes (Figure 6b-d) as the scanning rate increases, which can be attributed to lower interactions between electrolyte ions and active sites of the electrode at higher scan rates [56] and as expect the best capacitive results was achieved at 1 mV s −1 , being 181 F g −1 and 96 F g −1 for MoS 2 -PEG and MoS 2 -PEG/Au, respectively. Cyclic voltammetry (CV) measurements were performed to evaluate the charge storage capacity of both materials. Figure 6a-d shows the cyclic voltammograms obtained with (a) MoS2-PEG and (c) MoS2-PEG/Au electrodes (geometric area = 3.1 mm 2 ) in 1.0 M Na2SO4 electrolyte solution over a potential range of −0.2 to 0.4 V at different scan rates. The current signal at the CV curves measured with both electrode materials increases with the corresponding scan rate and the voltammograms show a nearly rectangular aspect, suggesting the charging current is the dominant signal due to reversible adsorption of electrolyte ions on the surface of electrodes. The pattern of specific capacitance decreases for both electrodes (Figure 6b-d) as the scanning rate increases, which can be attributed to lower interactions between electrolyte ions and active sites of the electrode at higher scan rates [56] and as expect the best capacitive results was achieved at 1 mV s −1 , being 181 F g −1 and 96 F g −1 for MoS2-PEG and MoS2-PEG/Au, respectively.   Table 2, it is verified that the coating MoS 2 -PEG on the graphite sheet surface electrode presents greater wettability than MoS 2 -PEG/Au electrode. This fact may be related to the low electroactive area of the MoS 2 -PEG/Au electrode due to its lower wettability, not allowing the effective access of the electrolyte in the specific electroactive area [57]. The electrochemical impedance spectroscopy (EIS) technique is an important tool for evaluating the behavior of electrode material at the interface electrolyte-electrode [58]. To examine the performance with both electrode materials, the impedance was measured at 1 MHz to 0.040 Hz at +0.  Figure 7 shows the Nyquist plots for both electrodes. At the highest frequency range, the value at the real axis corresponds to the equivalent series resistance (ESR) and represents the contribution of the electrolyte resistance and the intrinsic resistance of the electrode material. As both electrodes are in the same electrolyte, the ERS reflects the difference in the electrode material.
The low capacitance of MoS2-PEG/Au compared to MoS2-PEG was an unexpected fact, however when observing the values of the contact angle measurements of MoS2-PEG and MoS2-PEG/Au electrodes, Table 2, it is verified that the coating MoS2-PEG on the graphite sheet surface electrode presents greater wettability to MoS2-PEG/Au electrode. This fact may be related to the low electroactive area of the MoS2-PEG/Au electrode due to its lower wettability, not allowing the effective access of the electrolyte in the specific electroactive area [57]. The electrochemical impedance spectroscopy (EIS) technique is an important tool for evaluating the behavior of electrode material at the interface electrolyte-electrode [58]. To examine the performance with both electrode materials, the impedance was measured at 1 MHz to 0.040 Hz at +0.  Figure 7 shows the Nyquist plots for both electrodes. At the highest frequency range, the value at the real axis corresponds to the equivalent series resistance (ESR) and represents the contribution of the electrolyte resistance and the intrinsic resistance of the electrode material. As both electrodes are in the same electrolyte, the ERS reflects the difference in the electrode material. As a comparison, the MoS2-PEG electrode shows an ERS value of 256.4 Ω while MoS2-PEG/Au presents a higher value 339.2 Ω, which can justify the better capacitive performance of the MoS2-PEG electrode. The charge transfer resistance, which is measured from the semi-circle along the real axis from high to medium frequency is also lower in the MoS2-PEG electrode than in the MoS2-PEG/Au one. At low frequency, the Nyquist plots As a comparison, the MoS 2 -PEG electrode shows an ERS value of 256.4 Ω while MoS 2 -PEG/Au presents a higher value 339.2 Ω, which can justify the better capacitive performance of the MoS 2 -PEG electrode. The charge transfer resistance, which is measured from the semi-circle along the real axis from high to medium frequency is also lower in the MoS 2 -PEG electrode than in the MoS 2 -PEG/Au one. At low frequency, the Nyquist plots showed a linear range with a slope of near 45 • , known as Warburg resistance, where the frequency signal depends on the ion transportation in the electrolyte [57]. The examination of the curves at low frequency shows a smaller Warburg region for the MoS 2 -PEG electrode, suggesting a lower ion diffusion resistance in the structure of the electrode [59] while impedance performance of the MoS 2 -PEG/Au electrode shift along the x-axis toward more resistive values. Therefore, the better charge transmission and the ion diffusion through the electrode material structure help to explain the higher capacitance of the MoS 2 -PEG electrode.
The potential cytotoxicity of a given nanomaterial is a prerequisite for biomedical applications. In this study we assessed whether MoS 2 -PEG or MoS 2 -PEG/Au could be able to promote reduction of cellular viability in normal and tumoral cells under four different concentrations. The Green Monkey Kidney cell line VeroCCL-81 was used as non-tumoral model and the results applying MoS 2 -PEG showed discrete cellular viability reduction after 24 h when exposed to 10 µg/mL (2.1 ± 0.2)% and 5 µg/mL (5.2 ± 0.1)%, respectively, as shown in Figure 8a. On the other hand, cells exposed to MoS 2 PEG/Au demonstrated decreased cellular viability in all concentrations tested, such as (12.2 ± 0.3)% for 100 µg/mL, (19.9 ± 0.9)% for 50 µg/mL, (14.4 ± 0.4)% for 10 µg/mL and (2.5 ± 0.1)% for 5 µg/mL, respectively, compared to 65.5% ± 0.1% of the death control cells. After 48 h no cellular viability reduction was observed for cells exposed to MoS 2 -PEG and smaller decrease of cellular viability was observed in cells exposed to MoS 2 -PEG/Au at 100 µg/mL (3.2 ± 0.1)%, 50 µg/mL (5.5 ± 0.2)%, 10 µg/mL (5.0 ± 0.1)% and 5 µg/mL (6.9 ± 0.1)%, respectively, as shown in Figure 8c,d, suggesting a recovery of the cells in a time-dependent manner. Even though good dispersion in water small aggregations were observed over the cells as highlighted in Supporting information Figure S4.
show a linear range with a slope of near 45°, known as Warburg resistance, where the frequency signal depends on the ion transportation in the electrolyte [57]. The examination of the curves at low frequency shows a smaller Warburg region for the MoS2-PEG electrode, suggesting a lower ion diffusion resistance in the structure of the electrode [59] while impedance performance of the MoS2-PEG electrode shift along the x-axis toward more resistive values. Therefore, the better charge transmission and the ion diffusion through the electrode material structure help to explain the higher capacitance of the MoS2-PEG electrode.
The potential cytotoxicity of a given nanomaterial is a prerequisite for biomedical applications. In this study we assessed whether MoS2-PEG or MoS2-PEG/Au could be able to promote reduction of cellular viability in normal and tumoral cells under four different concentrations. The Green Monkey Kidney cell line VeroCCL-81 was used as non-tumoral model and the results applying MoS2-PEG showed discrete cellular viability reduction after 24 h when exposed to 10 μg/mL (2.1 ± 0.2)% and 5 μg/mL (5.2 ± 0.1)%, respectively, as shown in Figure 8a. On the other hand, cells exposed to MoS2PEG/Au demonstrated decreased cellular viability in all concentrations tested, such as (12.2 ± 0.3)% for 100 μg/mL, (19.9 ± 0.9)% for 50 μg/mL, (14.4 ± 0.4)% for 10 μg/mL and (2.5 ± 0.1)% for 5 μg/mL, respectively, compared to 65.5% ± 0.1% of the death control cells. After 48 h no cellular viability reduction was observed for cells exposed to MoS2-PEG and smaller decrease of cellular viability was observed in cells exposed to MoS2-PEG/Au at 100 μg/mL (3.2 ± 0.1)%, 50 μg/mL (5.5 ± 0.2)%, 10 μg/mL (5.0 ± 0.1)% and 5 μg/mL (6.9 ± 0.1)%, respectively, as shown in Figure 8c,d, suggesting a recovery of the cells in a time-dependent manner. Even though good dispersion in water small aggregations were observed over the cells as highlighted in supporting information Figure S4.   To study the effects of MoS2-PEG and MoS2-PEG/Au in the cellular viability of tumoral cells, the human squamous cell carcinoma A431 cell line and the human pharynx carcinoma FaDU cell line were used. For A431 cells no difference in the percentage of cellular viability was observed for MoS2-PEG in 24 h, as shown in Figure 9a. For 48 h there were a small decrease of the cellular viability with 100 μg/mL (5.2 ± 0.2)% and 50 μg/mL (5.8 ± 0.3)%, respectively, as observed in panel (b). A similar result was found when these cells were exposed to MoS2-PEG/Au at 48 h showing (3.2 ± 0.1)% decreasing for 100 μg/mL and (4.2 ± 0.2)% for 50 μg/mL, respectively, as shown in panels (c) and (d). No alterations in the cellular morphology were observed as shown in Supporting information Figure S5.  Another tumoral model was used and MoS 2 -PEG showed a decrease of 3.8 ± 0.3% for 100 µg/mL and (11.8 ± 0.2)% for 50 µg/mL at 24 h, as shown in Figure 10a. At 48 h there was only (2.4 ± 0.1)% for 50 µg/mL, suggesting a recovery of these cells in a time-dependent manner and a decrease of (7.3 ± 0.2)% was noticed for 5 µg/mL as shown in panel (b). For MoS 2 -PEG/Au the most pronounced cellular viability decreasing were observed for 100 µg/mL (6.5 ± 0.9)% and 50 µg/mL (11.61 ± 0.12)% at 48 h, as shown in panels (c) and (d). After contact with both nanomaterials FaDU cells demonstrated morphological changing when compared to control cells, as shown in Supporting information Figure S6. It is possible that these cells may present, somehow, plasma membrane reaction when in contact with charged materials, by promoting shape alterations. Then, due to the differences observed in their shapes and the decreasing of cellular viability the effect of 100 µg/mL of MoS 2 -PEG/Au in the number and shape of their nuclei were assessed by Hoechst 33,342 nuclear staining. Interestingly, no differences were observed neither in nuclei number nor in nuclei morphology even at the highest concentration applied, when compared to control cells as shown in Figure 11.
Another tumoral model was used and MoS2-PEG showed a decrease of 3.8 ± 0.3% for 100 μg/mL and (11.8 ± 0.2)% for 50 μg/mL at 24 h, as shown in Figure 10a. At 48 h there was only (2.4 ± 0.1)% for 50 μg/mL, suggesting a recovery of these cells in a time-dependent manner and a decrease of (7.3 ± 0.2)% was noticed for 5 μg/mL as shown in panel (b). For MoS2-PEG/Au the most pronounced cellular viability decreasing were observed for 100 μg/mL (6.5 ± 0.9)% and 50 μg/mL (11.61 ± 0.12)% at 48 h, as shown in panels (c) and (d). After contact with both nanomaterials FaDU cells demonstrated morphological changing when compared to control cells, as shown in Supporting information Figure S6. It is possible that these cells may present, somehow, plasma membrane reaction when in contact with charged materials, by promoting shape alterations. Then, due to the differences observed in their shapes and the decreasing of cellular viability the effect of 100 μg/mL of MoS2-PEG/Au in the number and shape of their nuclei were assessed by Hoechst 33,342 nuclear staining. Interestingly, no differences were observed neither in nuclei number nor in nuclei morphology even at the highest concentration applied, when compared to control cells as shown in Figure 11.   Our results demonstrated that both nanomaterials are biocompatible in normal cells, as well as in tumoral cells. Despite the high concentrations applied, for instance, 100 μg/mL and 50 μg/mL the cellular viability was kept above 80%. Moreover, at 48 h of contact with MoS2-PEG and MoS2-PEG/Au all cell lines presented very low damage what suggests the safety of these nanomaterials in vitro. Gold nanoparticles have been largely studied in biomedicine due to their well-known biocompatibility [60]. It seems that FaDU cell line changes its shape in contact with both MoS2-PEG and MoS2-PEG/Au. Nonetheless, this change in morphology did not alter cellular viability or nuclear number as demonstrated in Figure 11, corroborating the safety of MoS2-PEG/Au. Although both nanomaterials exhibited good dispersion in water they agglomerated in culture medium, maybe due to the presence of protein and salt. However, MoS2-PEG and MoS2-PEG/Au structures were able to adsorb in the cellular membranes and it is possible that a small amount of these nanomaterials carried into the cells which explains the alterations in cellular viability percentages. It was already shown that 2D MoS2 nanoparticles do not promote deleterious effects in cellular viability or induce genetic defects in HEK293f cells [20]. In this way, our data suggest the same behavior in other cellular models. Since neither MoS2-PEG and MoS2-PEG/Au themselves decrease cellular viability, they can be functionalized for theranostic purposes. We believe that MoS2-PEG/Au may possess potential application as a platform for theranostic, especially for cancer screening and photothermal therapy, because transition-metal dichalcogenides also enhance fluorescence through FRET and MoS2 nanosheets coated with folic acid had demonstrated efficient detection of Our results demonstrated that both nanomaterials are biocompatible in normal cells, as well as in tumoral cells. Despite the high concentrations applied, for instance, 100 µg/mL and 50 µg/mL the cellular viability was kept above 80%. Moreover, at 48 h of contact with MoS 2 -PEG and MoS 2 -PEG/Au all cell lines presented very low damage what suggests the safety of these nanomaterials in vitro. Gold nanoparticles have been largely studied in biomedicine due to their well-known biocompatibility [60]. It seems that FaDU cell line changes its shape in contact with both MoS 2 -PEG and MoS 2 -PEG/Au. Nonetheless, this change in morphology did not alter cellular viability or nuclear number as demonstrated in Figure 11, corroborating the safety of MoS 2 -PEG/Au. Although both nanomaterials exhibited good dispersion in water they agglomerated in culture medium, maybe due to the presence of protein and salt. However, MoS 2 -PEG and MoS 2 -PEG/Au structures were able to adsorb in the cellular membranes and it is possible that a small amount of these nanomaterials carried into the cells which explains the alterations in cellular viability percentages. It was already shown that 2D MoS 2 nanoparticles do not promote deleterious effects in cellular viability or induce genetic defects in HEK293f cells [20]. In this way, our data suggest the same behavior in other cellular models. Since neither MoS 2 -PEG and MoS 2 -PEG/Au themselves decrease cellular viability, they can be functionalized for theranostic purposes. We believe that MoS 2 -PEG/Au may possess potential application as a platform for theranostic, especially for cancer screening and photothermal therapy, because transition-metal dichalcogenides also enhance fluorescence through FRET and MoS 2 nanosheets coated with folic acid had demonstrated efficient detection of miRNA-21 expression in a unique MCF-7 and HeLa cancer cells lines, working as a fluorescent nanoprobe. Based on the lack of cellular injuries demonstrated by our data both nanocomposites could have their surfaces modified to improve their dispersibility in aqueous solution. As an example, MoS 2 -PEG could be investigated as a new contrast agent for radiological imaging, and MoS 2 -PEG/Au could be functionalized to promote drug-delivery. The carefully physicochemical and biological characterizations of nanomaterials for biomedical applications are the first steps for a such proposal [61], and our work provides data enough to indicate MoS 2 -PEG/Au for these purposes. Then, this initial study paves the way for other works in nanobiomedicine, for instance by incorporating dyes and/or biomolecules to MoS 2 -PEG/Au by taking advantages of the excellent optical properties of AuNPs and MoS 2 as well.

Conclusions
MoS 2 -PEG and MoS 2 -PEG/Au were successfully synthesized and widely characterized with different physicochemical techniques. By using TEM and SEM, it was possible to verify hierarchical flower-like structures, with a lateral size of up to 400 nm. Through the EDS and XPS studies, it was also observed that AuNPs were distributed throughout the material. MoS 2 -PEG/Au sample showed a lower capacitance value compared to MoS 2 -PEG; this fact may be associated with the low wettability of this electrode as observed in its contact angle. In this case, the presence of AuNPs could be related to increase the MoS 2 -PEG hydrophobicity, allowing only partial contact between the electrode and the electrolyte. However, in both cases the produced samples showed chemical stability and biocompatibility, due to the cell viability achieved above 80% for different times. Thus, its potential use as multifunctional nanomaterial for theranostic applications is an important subject that needs to be better explored.