Ultrasound‐Activated Piezoelectric MoS2 Enhances Sonodynamic for Bacterial Killing

Bacterial infections are a serious public health issue that threatens the lives of patients because of their ability to induce other lethal complications without prompt treatment. Conventional antibiotic therapy can cause bacterial resistance and other adverse effects. It is highly desirable to develop effective and antibiotic‐independent therapeutic strategies to treat various kinds of bacterial infections. Herein, sonodynamic‐enhanced piezoelectric materials MoS2 and Cu2Oheterostructure that responds to exogenous ultrasound (US) and generates reactive oxygen for Staphylococcus aureus elimination are developed. It is shown in the results that the polariton charge induced by piezoelectric MoS2 nanosheets under US irradiation can accelerate the transfer of electric in Cu2O. Furthermore, US irradiation induces valence conversion from Cu(I) to Cu(II), which can accelerate glutathione oxidation significantly and destroy the bacterial antioxidant defense system. Hence, the as‐prepared piezoelectric‐enhanced sonosensitizer possesses a highly effective antibacterial efficacy of 99.85% against S. aureus under US irradiation for 20 min, with good biocompatibility. Herein, effective ultrasonic piezocatalytic therapy is offered through constructing heterogeneous interfaces with ultrasonic piezoelectric response.

Bacterial infections are a serious public health issue that threatens the lives of patients because of their ability to induce other lethal complications without prompt treatment. Conventional antibiotic therapy can cause bacterial resistance and other adverse effects. It is highly desirable to develop effective and antibioticindependent therapeutic strategies to treat various kinds of bacterial infections. Herein, sonodynamic-enhanced piezoelectric materials MoS 2 and Cu 2 Oheterostructure that responds to exogenous ultrasound (US) and generates reactive oxygen for Staphylococcus aureus elimination are developed. It is shown in the results that the polariton charge induced by piezoelectric MoS 2 nanosheets under US irradiation can accelerate the transfer of electric in Cu 2 O. Furthermore, US irradiation induces valence conversion from Cu(I) to Cu(II), which can accelerate glutathione oxidation significantly and destroy the bacterial antioxidant defense system. Hence, the as-prepared piezoelectric-enhanced sonosensitizer possesses a highly effective antibacterial efficacy of 99.85% against S. aureus under US irradiation for 20 min, with good biocompatibility. Herein, effective ultrasonic piezocatalytic therapy is offered through constructing heterogeneous interfaces with ultrasonic piezoelectric response.
In particular, ultrasound (US)-responsive materials are currently mainly derived from the photosensitizer metalloporphyrin, noble-metal carbon materials, inorganic materials (TiO 2 ), curcumin with nanostructures, carbon materials, and piezoelectric nanoparticles. [14][15][16][17][18] Similar to photosensitizers, the catalytic performance of sonosensitizers depends on the electron-transfer efficiency of ultrasonic excitation. [19] Further modification of the sonosensitizer material is the most effective way to improve its catalytic performance. Currently, the main methods are the use of noble metal materials to modify the sonosensitizer; while improving the catalytic performance, its toxicity is also greatly increased. Therefore, it is important to develop a good biocompatible sonocatalytic material. With their unique piezoelectric effects, piezoelectric materials, such as barium titanate (BaTiO 3 ), [20] MoS 2 nanosheets, [21] and black phosphorus, can instantly create a built-in electric field when subjected to external mechanical stress. [22] Under ultrasonic excitation, a piezoelectric material is polarized, and electrons gather on one side but quickly neutralize the surrounding charge, which greatly limits their catalytic performance. [23] In particular, when piezoelectric materials and semiconductor materials are combined to form a heterojunction, the polarized electrons excited by ultrasound at the interface are efficiently migrated; thus, the yield of electrons can be increased to produce more ROS. [24] Cu(I), as a low-valent transition-metal cation, has the properties of a Fenton/Fenton-like agent that catalyzes the production of ROS from hydrogen peroxide. [25,26] Importantly, bacterial endogenous glutathione (GSH) can regulate the transition of the copper valence state and destroy the internal structure of bacteria. [27,28] However, these effects are far less than the requirements for the killing of bacteria. Therefore, the adoption of external energy, such as US excitation, the regulation of the reversible conversion of Cu(I) and Cu(II) valence states, and the depletion of GSH inside bacteria are potential treatment strategies to achieve the requirements of a rapid and precise treatment of bacterial infections. [29] Cuprous oxide (Cu 2 O), rich in large amounts of Cu(I), is a semiconductor with a narrow bandgap and is widely used in various catalytic reactions. [30][31][32] In addition, based on the US induced between separation electrons and holes, Cu 2 O are employed as sonosensitizers converting H 2 O and O 2 into ROS for SDT. [33][34][35] Herein, we created a piezoelectric-assisted, valence-adjustable sonosensitizer heterojunction structure with a strong USresponding ability to treat S. aureus infection through efficient SDT. Based on the previous analysis, the Cu 2 O nanocube was chosen as a sonosensitizer because it has not only good electrical conductivity but also a valence structure with ultrasonic excitation. MoS 2 and Cu 2 O heterojunctions were synthesized using a simple hydrothermal method. MoS 2 nanosheets with piezoelectric effects were modified on the surface of Cu 2 O, which could augment the antibacterial SDT ability of Cu 2 O through a sonopiezoelectric polarization effect and mechanical force. On the one hand, in the construction of heterojunctions, the electrons generated by piezoelectricity at the interface of heterojunctions are rapidly transferred so that their ultrasonic catalytic effect is enhanced. On the other hand, the US regulation of the conversion of Cu(I) and Cu(II) can rapidly oxidize GSH inside bacteria and further improve antibacterial efficiency. Consequently, the as-prepared MoS 2 /Cu 2 O (MC) exhibited highly effective bacterial killing efficacy due to the sonocatalytic, as well as piezocatalytic, properties, as schematically illustrated in Scheme 1.

Characterization of Morphology and Structure Materials
A schematic of the preparation process for MC is presented in Figure 1a. The morphologies of the materials (MoS 2 , Cu 2 O, MC) were observed by field-emission scanning electron microscopy (FE-SEM). The SEM image of MoS 2 is shown in Figure 1b. The images show that MoS 2 is composed of nanosheets with abundant active sites. The transmission electron microscopy (TEM) image of MoS 2 (Figure 1c) also confirmed the observed structure of MoS 2 is a lamellar. The lattice spacing of 0.62 nm observed by high-resolution TEM (HRTEM) (Figure 1d) is the (002) crystal plane belonging to MoS 2 . [5,11,36] Element mapping ( Figure S1a,b, Supporting Information) showed that Mo and S were uniformly distributed on the MoS 2 nanosheets. The prepared Cu 2 O exhibited a nanocube morphology (Figure 1e), which corresponded to the TEM image of Cu 2 O ( Figure 1f ). As shown in Figure 1g, the lattice spacing of 0.21 nm was consistent in the (200) crystal plane of Cu 2 O. [37,38] Element mapping ( Figure S2a, b, Supporting Information) showed that Cu and O were uniformly distributed on the Cu 2 O nanocube. The SEM and TEM images (Figure 1h,i) showed a similar core-shell structure of the synthesized MC (i.e., nanocubes were encapsulated by layer-like nanosheets). The presence of a heterojunction interface between Cu 2 O and MoS 2 was observed by HRTEM, and defects due to lattice distortion were also observed in molybdenum sulfide. Energy-dispersive spectroscopy (EDS) element mapping ( Figure 1k) showed that Cu and O were uniformly distributed on the Cu 2 O nanosheets, whereas the elements Mo and S Scheme 1. Sono-piezocatalysis mechanism of antibacterial.
www.advancedsciencenews.com www.small-science-journal.com were homogeneously distributed on the plane of MoS 2 . Figure S3 shows the content of each element of MC, indicating that Mo, S, Cu and O are 2.76%, 17.41%, 53.17%, and 26.11%, respectively. Inductively coupled plasma (ICP) detection corresponding to Table 1 (Supporting information) shows that the contents of Mo and Cu are 5.7884% and 59.7277%, respectively. Furthermore, as shown in Figure 2, the atomic force microscope (AFM) image and the corresponding height profiles agreed well with the TEM images of these different samples. Figure Figure 3a shows the X-ray diffraction (XRD) patterns of the asprepared of Cu 2 O, MoS 2 , and MC. The XRD pattern of MoS 2 exhibited diffraction peaks at 14.3°(002), 33.8°(100), and 58.78°(110), which agreed well with the diffraction patterns of the hexagonal 2H-phase of MoS 2 . [11,36] The diffraction peak of MoS 2 in MC was weaker than that of Cu 2 O, which may be caused by the relatively small amount of MoS 2 in MC. To further investigate the combinations on the valence state of elements in the as-prepared materials, we used the X-ray photoelectron spectroscopy (XPS) spectra. As shown in Figure 3b, the survey scan of the MC (red curve) indicated the existence of diffraction peaks of Mo, S, Cu, C, and O. Figure 3c shows that the highresolution spectrum of S 2p obtained from MoS 2 displayed two peaks at 163.18 (S 2p 1/2 ) and 161.70 eV (S 2p 3/2 ). [39] The S 2p was tested at lower binding energies in the MC than MoS 2 , which was the difference in electronegativity between S and Cu, the electron cloud density around S decreased. The Mo 3d XPS spectrum is shown in Figure 3d. The peaks of 228.9 and 232.4 eV belonged to Mo 2d 3/2 and Mo 2d 5/2 , respectively, indicating the presence of Mo (IV). [40] In contrast, the Mo 3d binding energies of MC were redshifted to lower binding energies compared to MoS 2 , indicating an interaction between MoS 2 and Cu 2 O in the MC hybrid. Bands at 932.46 and 952.36 eV were attributed to Cu(I) (Figure 3e). Moreover, the peaks at 933.93 and 953.96 eV were assigned to Cu 2þ 2p 1/2 and Cu 2þ 2p 3/2 , respectively. The O 1s spectrum in Figure 3f shows that the bands at 530.38 and 531.68 eV belonged to lattice oxygen and Cu 2 O, respectively. [36] The XPS spectrum results www.advancedsciencenews.com www.small-science-journal.com demonstrate that the MoS 2 and MC successfully composition, which is consistent with XRD and TEM results.

Investigation of Sonodynamic Effects
The XPS results indicated a strong electron interaction and charge transfer between Cu 2 O and MoS 2 . Therefore, the sonodynamic effects of MC were measured by the following experiments. As shown in Figure 4a, the UV-vis diffuse reflectance spectra of the different samples were obtained to measure changes in their optical properties. The MC showed large absorption compared with Cu 2 O, suggesting a higher tendency to be activated by exogenous stimuli with lower energy in MC. Figure 4b presents the bandgap values of the different materials   calculated by UV-vis spectra (Figure 4a). The bandgap value of Cu 2 O was calculated to be 2.31 eV, and the bandgap value of MoS 2 was calculated to be 1.42 eV. As shown in Figure S7, Supporting Information, the bandgap of MC is minimal compared to MoS 2 and Cu 2 O. Furthermore, the photoluminescence (PL) spectra ( Figure 4c) were used to measure the electron-hole combination efficiency of the various samples. Compared with MoS 2 , the MC showed lower intensity, indicating that the piezoelectric potential separating the electron-hole pair of the MC had the lowest recombination rate. [41] Figure 4d shows the electrochemical impedance spectroscopy (EIS) curves of the as-prepared samples.  Figure 4e. To further explore the catalytic mechanism, we performed piezoelectric force microscopy (PFM) to understand the piezoelectricity of MC. As shown in Figure 4f,g, a typical amplitude voltage butterfly loop and a phase change close to %180°were observed under the applied voltage of 8 eV, proving that MC possessed typical piezoelectric properties. Therefore, as a mechanical wave, US can stimulate MC to produce polarization (e.g., the piezoelectric effect). Furthermore, total ROS generation under US irradiation was measured using the dye 2 0 ,7 0 -dichlorofluorescein (DCFH) (Figure 4h). Under US (1.5 W cm À2 ) irradiation, the control group and MoS 2 showed very little ROS. The ROS yields of the Cu 2 O group were slightly www.advancedsciencenews.com www.small-science-journal.com Small Sci. 2023, 3, 2300022 higher than those of the control group. In contrast, the combination between MoS 2 and Cu 2 O induced the most yields of ROS from the synthesized MC under US treatment for 20 min, indicating the best US catalytic performance. In conclusion, the mechanism behind the sonocatalytic of MC is shown in Figure 4i. Given that the sheet of molybdenum sulfide had a good piezoelectric effect, the energy generated by US could excite molybdenum sulfide to produce a polarized charge. [42] After the formation of the heterojunction, the electrons and hole teams generated by ultrasonic excitation of cuprous oxide are separated, the electrons flow to the surface of the heterojunction, and the polarized charge is trapped. This causes more holes and polarized electrons to participate in the generation of ROS, thereby producing more ROS.

In Vitro Sonodynamic Antibacterial Performance of MoS 2 /Cu 2 O
Given the excellent ultrasonic response and piezoelectric catalytic ability of heterojunctions, we further explored the types of ROS under ultrasonic excitation. Electron-spin resonance www.advancedsciencenews.com www.small-science-journal.com spectroscopy (Figure 5a) was used to further verify the type of ROS produced by the as-prepared samples under 1.5 W cm À2 US excitation. By using 2,2,6,6-tetramethylpiperidine as a trapping agent, typical spectra of⋅ 1 O2 radical were detected in each group with US (1.5 W cm À2 ) treatment. Compared with group MC, group MC þ US showed the highest singlet oxygen signal. This indicates that MC can produce a large amount of ROS under US. As shown in Figure 5b, in the absence of ultrasonic excitation, there was no hydroxyl-free signal generation in the heterojunction, whereas after ultrasonic excitation, the hydroxyl radical signal was significantly enhanced. Singlet oxygen and hydroxyl radicals produced by US excitation could effectively remove bacterial infections. To demonstrate whether US can induce the transition between Cu(I) and Cu(II), we used neocuproine as the detection reagent. When Cu þ is present, Cu þ will combine with neocuproine to form [Cu(neocuproine) 2 ] þ to make the solution yellow, and when Cu þ decreases, the color of the solution decreases. Figure 5c shows that when the US is applied to MC, the color of the solution becomes lighter, and the absorption intensity at 450 nm decreases, indicating that Cu(I) has changed to Cu(II). Because of the piezoelectric action of MC, which causes an excess of ROS to be produced when US is excited, we employed the spread plate method to count the number of S. aureus colonies to assess the antibacterial performance of MC. Figure S9, Supporting Information, shows the antibacterial performance of MC at different concentrations, and after comparison, we determined that 500 ppm is the optimal antibacterial concentration for MC. As shown in Figure 5d, there was no significant reduction in colonies in all experimental groups without US excitation compared to the control group. On the contrary, under US excitation, the antibacterial efficiency of Cu 2 O group was 38.51% relative to the control group. This was due to the action of copper ions under US, which promotes the conversion of Cu(I) and Cu(II) in US. [20] Although MoS 2 exhibited piezoelectric performance under US excitation, due to the rapid recombination of polarized electrons and hole pairs, the ROS yield was low, so its antibacterial effect was not significant. In contrast, after US irradiation for 20 min, the antibacterial efficiency of MC was 99.85%, which was due to the synergistic effects of enhanced ultrasonic performance and Cu (II). Figure 5e shows a histogram of the antibacterial efficiency corresponding to Figure 5d. Figure 5f reveals the morphology of the bacteria, and the structure of the bacteria was relatively complete in each group without US irradiation. In contrast, the cell membranes of the bacteria were damaged to varying degrees after US irradiation for 20 min. Especially in the MC group, the cell membranes of the bacteria were severely folded, and the bacteria were deformed. The outcome corresponded well to the results shown in Figure 5d. To further investigate the antibacterial mechanism of MC under US irradiation, we conducted the following experiments.
A probe called 2 0 ,7 0 -dichlorofluorescein diacetate (DCFH-DA) can identify intracellular ROS, which can penetrate the bacteria's interior and hydrolyze into DCFH, which then emits  fluorescence. The more ROS within the bacteria, the brighter the fluorescence. Figure 5g shows the fluorescence intensity of intracellular ROS in each group under US at 525 nm. The results show that the MC group had the strongest intensity at 525 nm and produced the most intracellular ROS, which would cause severe oxidative stress in bacteria. The results of Figure 5h correspond well to those in Figure 5g. As shown in Figure 5i, compared to the control group, the protein leakage from S. aureus was negligent in the MoS 2 group, with or without US irradiation. In contrast, under US irradiation, the protein leakage of bacteria in the Cu 2 O group increased significantly. The protein leakage of bacteria with US (1.5 W cm À2 ) irradiation in the MC group was particularly obvious relative to that in the control group. As an endogenous antioxidant, GSH was used as an indicator of oxidative stress to assess the oxidative properties of all the groups. [43] As shown in Figure 5j, compared with the control group, GSH depletion in the MoS 2 group was negligible, with and without US excitation. In the absence of US, the depletion of Cu 2 O group compared with the control GSH was 8.81%, which was caused by the oxidation of Cu þ on the surface of Cu 2 O. The depletion of GSH in the Cu 2 O þ US group was 24.75%, indicating that US could convert Cu þ in Cu 2 O to Cu 2þ to participate in the depletion of GSH. In comparison, the MC þ US group had the largest depletion of 65.89%, which showed that the MC could effectively oxidize GSH under US irradiation. Figure 5k shows the antibacterial mechanism of MC.

In Vitro Cytocompatibility
NIH-3T3 is crucial for wound healing. It was used to assess the biocompatibility of different materials. Using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, we investigated the cytocompatibility of the materials. As shown in Figure 6a, after coculture of cells and materials for 1 day, the MoS 2 group showed good biocompatibility, which might be ascribed to the nanosheet structure of MoS 2 because this structure could provide sufficient active sites for the cell to proliferate and expand. The Cu 2 O group showed obvious toxic effects on the cells, which might be ascribed to excess Cu þ . [44] However, after 3 days of culture, the cell viability of MC increased up to over 80%, indicating that MC had good cytocompatibility. As shown in Figure 6b, the fluorescence staining images of the cells showed that the MoS 2 group exhibited more filopodia extensions around compared to the control group. This result indicates that the nanosheet structure of MoS 2 facilitated fibroblasts to migrate and proliferate. However, the large amount of Cu ions (Cu þ , Cu 2þ ) released induced the death of cells, which contributed to the shrinking in the shape of the cells in the Cu 2 O group. Furthermore, the cells showed filopodia extending around due to the good biocompatibility of MC. Figure 6b is in accordance with Figure 6a (MTT assay) and further demonstrates that MC had good cytocompatibility.

Discussion and Conclusion
In this work, we developed MC nanocomposites with piezoelectric and valence-adjustable heterostructures for sonodynamic antibacterial therapy. On the one hand, the establishment of the heterojunction interface prevented the recombination of the piezoelectrically polarized electrons and holes generated by the US excitation of MoS 2 , which improved the sonocatalytic ability of MC. On the other hand, the conversion of Cu(I) and Cu(II) in Cu 2 O due to US propelling increased the yield of electrons, and the generated Cu(II) ions could oxidize bacterial endo-GSH, thus reducing bacterial activity. Because of the high ROS production of MC, S. aureus could be effectively killed under ultrasonic excitation at 1.5 W cm À2 for 20 min. In vitro cytotoxicity tests revealed that MoS 2 @Cu 2 O had good cytocompatibility. The current study will provide new insights into developing not only piezoelectric US-excited bacteria-killing materials but also piezoelectric devices, such as piezoelectric pacemakers and piezoelectric skin.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.