English

• The fabrication of nanomaterials enables the production of cytotoxic reactive oxygen species (ROS), including singlet oxygen (¹O₂), superoxide anion (^•O₂⁻), and hydroxyl radicals (^•OH), providing new methods to overcome the limitations of conventional tumor treatments [1–6]. One notable example is sonodynamic therapy (SDT), which utilizes sonosensitizers combined with ultrasound (US) stimulation to generate ROS, offering minimal invasiveness and high tumor specificity [7–11]. As a mechanical wave, US has advantages of precise orientation and long-distance propagation [12,13], thereby achieving deeper tissue penetration and higher spatial accuracy to reduce damage to normal tissues [14,15]. However, the development of SDT still encounter challenges related to low-efficiency ROS generation and the complicated tumor microenvironment (TME) [16,17]. Generally, overproduction of glutathione (GSH) in TME can consume the generated ROS, thereby discounting the efficacy of SDT [1,18,19]. Therefore, it is necessary to develop a more efficient sonosensitizers with TME responiveness to increase ROS generation and SDT therapeutic efficiency.

  Recently, piezoelectric semiconductor nanomaterials, such as titanium oxide (TiO₂) [20], zinc oxide (ZnO) [21], barium titanate (BaTiO₃) [22,23], bismuth tungstate (Bi₂WO₆) [24,25], and bismuth oxychloride (BiOCl) [26], have gained recognition as a novel class of sonosensitizers for enhancing sonodynamic therapy (SDT). These piezoelectric materials can generate an inherently dynamic electric field under periodic mechanical forces, such as ultrasonic vibrations [27–29], which facilitates the separation of electrons (e⁻) and holes (h⁺), promoting the catalysis of ROS production [30]. Compared to nonpiezoelectric sonosensitizers, this inherently dynamic electric field piezoelectric semiconductor nanomaterials can efficiently reduce the recombination of carriers. And the band bending under US irradiation also promote the carrier separation and ROS production. Zinc stannate (ZnSnO₃) has attracted much attention in various piezoelectric materials because of its piezoelectric and ferroelectric properties, with an electrical and structural ordering temperature up to 700 ℃ [31–33]. ZnSnO₃ boasts an impressive polarization of approximately 59 µC/cm² along the c-axis, surpassing other non-lead-based piezoelectric materials such as and barium titanate (BaTiO₃, 6 µC/cm²), zinc oxide (ZnO, 5 µC/cm²), potassium niobate (KNbO₃, 23 µC/cm²) [34,35]. Similar to other piezoelectric semiconductors, the challenge for ZnSnO₃ lies in the efficient separation of e⁻ and h⁺ as well as preventing their recombination [36]. Development of ZnSnO₃ based piezoelectric sonosensitizers with high efficiency of e⁻ and h⁺ seperation and ROS generation is highly desired.

  In this work, an urchin-like piezoelectric p-n heterojunction was designed and synthesized by integrating p-type ZnSnO₃ nanocubes (NCs) and n-type Cu₃P nanoneedles (NNs) to form p-n heterojunction for enhancing cancer SDT (Scheme 1). The fabricated ZnSnO₃/Cu₃P NCs effectively narrow the bandgap of ZnSnO₃ NCs, resulting in a higher redox activity to produce ROS. The urchin-like structure can further increase the US response to improve piezoelectric property. Additionally, Cu₃P NNs can respond to the TME, thereby consuming intracellular glutathione (GSH) and producing ROS through Fenton-like reaction to achieve CDT. Combining together, the piezoelectric p-n heterojunction realizes a 70% tumor growth inhibition rate on 4T1 tumor mice model by combining CDT and SDT. This piezoelectric p-n sonosensitizer opens the way for the construction and application of heterostructured nanomaterials in improving sonodynamic therapy. ZnSnO₃ NCs and Cu₃P NNs were prepared separately, and then ZnSnO₃/Cu₃P heterojunction was formed by hydrothermal method (Fig. 1A) [37]. Scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis revealed that ZnSnO₃ exhibited a uniform cubic shape, with average size of 71.9 ± 13.3 nm (Fig. 1B and Fig. S1 in Supporting information). Upon formation heterojunction with Cu₃P, the obtained ZnSnO₃/Cu₃P NCs displayed an urchin-like morphology, with numerous nanoneedles radiating outward from the center (Fig. 1C). High-resolution transmission electron microscopy (HRTEM) image displayed the lattice widths of 0.25 nm corresponding to (102) crystal plane of Cu₃P and 0.33 nm corresponding to (012) crystal plane of ZnSnO₃ (Figs. 1D and E). Elemental mapping analysis confirmed that Cu and P elements were uniformly distributed on the surface of ZnSnO₃ (Fig. 1F and Fig. S2 in Supporting information). The phase structures of ZnSnO₃ and ZnSnO₃/Cu₃P were characterized using X-ray diffraction (XRD), which revealed that only peaks corresponding to ZnSnO₃ (JCPDS No. 11-0274, Fig. S4 in Supporting information) was observed in the pattern of ZnSnO₃/Cu₃P, due to the low content of Cu₃P in the heterojunction (Fig. 1G). The chemical composition and valence state of the nanocubes were further analyzed by X-ray photoelectron spectroscopy (XPS). The XPS spectrum of ZnSnO₃/Cu₃P displayed characteristic peaks of Zn 2p, Sn 3d, Cu 2p and P 2p at 1021.6, 486.2, 933.8 and 129.6 eV, respectively (Fig. 1H and Table S1 in Supporting information). In the high-resolution Cu 2p spectrum (Fig. 1I), the peaks at 932.8 and 934.8 eV corresponded to Cu(I) and Cu(II), respectively. The calculation results of peak area showed that the ratio of Cu(I) to Cu(II) content was approximately 85:15 (Table S2 in Supporting information). The valence states of Zn 2p, Sn 3d and O 1s in ZnSnO₃/Cu₃P had no difference relative to ZnSnO₃ (Fig. 1J and Fig. S5 in Supporting information). Fourier transform infrared spectroscopy (FTIR) spectrum of ZnSnO₃/Cu₃P showed that the characteristic peak of P−O bond appeared at 1100 cm⁻¹. This result indicates that Cu₃P have been successfully synthesized on the surface of ZnSnO₃ (Fig. S6 in Supporting information).

  Scheme 1

  

  Scheme 1.  Schematic depiction of the synthesis process and mechanism in the antitumor process of ZnSnO₃/Cu₃P NCs.

  DownLoad: Full-Size Img PowerPoint

  Figure 1

  

  Figure 1.  Synthesis and characterizations of ZnSnO₃/Cu₃P. (A) Schematic diagram of the fabrication process of ZnSnO₃/Cu₃P. (B) SEM image of ZnSnO₃ NCs. (C) SEM image of ZnSnO₃/Cu₃P. (D) TEM image, (E) HRTEM image and (F) element mapping of ZnSnO₃/Cu₃P. (G) XRD, (H) XPS and high-resolution (I) Cu 2p and (J) O 1s XPS spectra of ZnSnO₃/Cu₃P. (K) hydrodynamic diameters and (L) zeta potential of ZnSnO₃ and ZnSnO₃/Cu₃P. The data represent means ± standard deviation (S.D.) (n = 5).

  DownLoad: Full-Size Img PowerPoint

  Dynamic light scattering (DLS) results illustrated that the mean hydrodynamic diameter of ZnSnO₃ was 215.7 nm, while that of ZnSnO₃/Cu₃P slightly increased to 327.1 nm (Fig. 1K). Zeta potential of ZnSnO₃ changed from positive to negative upon the formation of ZnSnO₃/Cu₃P heterojunction (Fig. 1L). Notably, the positive potential of ZnSnO₃ gradually decreased over time, while the zeta potential of ZnSnO₃/Cu₃P remained stable, indicating better aqueous stability of the heterojunction. After dispersion of ZnSnO₃/Cu₃P in phosphate buffer saline (PBS) solution at pH 6.5 for 24 h, a partial of nanoneedle on ZnSnO₃/Cu₃P were fallen off from the nanocubes (Fig. S3 in Supporting information).

  The ROS production of ZnSnO₃/Cu₃P through sonodynamic, chemodynamic, and their combination processes was investigated (Fig. 2A). To evaluate the sonodynamic performance of ZnSnO₃ and ZnSnO₃/Cu₃P, methylene blue (MB) was chosen as an indicator that can be degraded by ^•OH and superoxide anion (^•O₂⁻). After 10 min of US stimulation (1.0 MHz, 1.0 W/cm², 50% duty cycle), the presence of ZnSnO₃ or ZnSnO₃/Cu₃P in the MB solutions resulted in significant degradation of MB (Fig. 2B and Fig. S6 in Supporting information), indicating that both ZnSnO₃ and ZnSnO₃/Cu₃P NCs were capable of generating ROS under US stimulation. As shown in Fig. 2C, the first-order kinetic rate constant (k) of MB degradation by ZnSnO₃/Cu₃P + US was 0.036 (Table 1), higher than that by US (0.011) and ZnSnO₃ + US (0.031).

  Figure 2

  

  Figure 2.  Sonodynamic and chemodynamic performance of ZnSnO₃/Cu₃P. (A) Schematic representation illustrating the sonodynamic and chemodynamic processes of ZnSnO₃/Cu₃P. (B) Degradation curves of MB and (C) corresponding kinetic curves in SDT process. Degradation curves of NBT for ^•O₂⁻ detection in the SDT process. (E) Generation of ^•OH in the SDT process with TA as the indicator. (F) Production of ^•OH in the CDT process measured with TMB as the indicator. (G) Degradation curves of MB and (H) corresponding kinetic curves in the combination process of SDT and CDT. ESR spectra of (I) ^•OH and (J) ^•O₂⁻ trapped by DMPO under different conditions. (K) GSH oxidation by varying concentrations of ZnSnO₃/Cu₃P within 3 h. (L) Oxidation of GSH achieved by combining SDT and CDT.

  DownLoad: Full-Size Img PowerPoint

  Table 1

  

  Table 1.  First-order reaction kinetics of MB degradation under various treatments.

  DownLoad: CSV

  To identify the specific types of ROS produced during the SDT process, terephthalic acid (TA) and nitro tetrazolium blue chloride (NBT) were used as specific probes of ^•OH and ^•O₂⁻, respectively. After 10 min of US stimulation, the ^•OH generation by ZnSnO₃ and ZnSnO₃/Cu₃P NCs was approximately 1.82-fold and 2.34-fold higher than that in the mere US group (Fig. 2E and Fig. S8 in Supporting information), which correlated well with the results of MB degradation. Similarly, ZnSnO₃/Cu₃P NCs produced a substantial amount of ^•O₂⁻ under US stimulation (Fig. 2D and Fig. S7 in Supporting information). In contrast, the production of ^•O₂⁻ ZnSnO₃ was substantially lower, similar with that by mere US stimulation. These results demonstrated that ZnSnO₃/Cu₃P NCs can be excited by US irradiation to efficiently produce ROS, possessing superb SDT performance over ZnSnO₃. Also, the partial degradation of Cu₃P in a simulated tumor microenvironment (pH 6.5) did not affect the SDT performance of ZnSnO₃/Cu₃P (Fig. S10 in Supporting information). The specific reactions involved in the sonodynamic process are shown below. Firstly, upon US irradiation, ZnSnO₃/Cu₃P NCs, undergo polarization, leading to the segregation of electrons (e⁻) and holes (h⁺) (Eq. 1). Subsequently, nearby oxygen and water molecules react with these e⁻ and h⁺, resulting in the formation of ^•O₂⁻ and ^•OH (Eqs. 2 and 3).

  (1)

  (2)

  (3)

  As previous reported, Cu(I) can initiate Fenton-like reaction and efficiently catalyze H₂O₂ to produce ^•OH, enabling chemodynamic therapy [38–43]. Given the presence of Cu(I) in ZnSnO₃/Cu₃P NCs, we investigated the ROS generation in the presence of H₂O₂ under weakly acidic conditions. To assess this, 3, 3′, 5, 5′-tetramethylbenzidine (TMB) was selected as a probe, which can react with ^•OH to produce blue oxTMB. When mere H₂O₂ was added to the weakly acidic medium, the production of ^•OH was negligible. Upon the addition of ZnSnO₃/Cu₃P NCs, ^•OH generation significantly increased. Furthermore, as the pH values decreased towards a more acidic conditions, ^•OH production gradually increased (Fig. 2F). Hence, ZnSnO₃/Cu₃P can serve as a potent chemodynamic agent for generating ^•OH, as depicted in Eq. 4.

  (4)

  We further explored the synergistic effect of combining the sonodynamic and chemodynamic processes. We conducted a comparative analysis of MB degradation using ZnSnO₃ and ZnSnO₃/Cu₃P NCs with both H₂O₂ and US irradiation. In MB solution containing H₂O₂ (pH 6.5), ZnSnO₃/Cu₃P NCs induced simultaneous sonodynamic and chemodynamic processes under US irradiation. Remarkably, the integration of SDT and CDT exhibited a notable enhancement in ROS generation using ZnSnO₃/Cu₃P NCs compared to the single sonodynamic or chemodynamic process (Fig. 2G and Fig. S8 in Supporting information). Consequently, the degradation of MB exhibited a faster first-order kinetic rate constant of 0.098, which was 2.7 times higher than of single SDT (Fig. 2H and Table 2). These finding demonstrated the enhanced efficiency of ROS generation through the combination effect, surpassing either SDT or CDT alone.

  Table 2

  

  Table 2.  First-order reaction kinetics of MB degradation under various treatments.

  DownLoad: CSV

  Additionally, electron spin resonance (ESR) spectroscopy was utilized to confirm the specific oxygen radicals produced in the combination process. For this purpose, a spin-trapping agent, 5, 5-dimethyl-1-pyrroline N-oxide (DMPO), was utilized in either water or dimethyl sulfoxide to detect ^•OH and ^•O₂⁻, respectively. The DMPO/^•OH adduct exhibits a characteristic quadruple signal with a 1:2:2:1 amplitude ratio, while the DMPO/^•O₂⁻ adduct displays a characteristic multiple signals with a 1:1:1:1:1:1 amplitude ratio. In the presence of H₂O₂, ZnSnO₃/Cu₃P displayed the highest signal intensity for both ^•OH and ^•O₂⁻ under US irradiation (Figs. 2I and J), verifying its superb combination effect of SDT and CDT.

  In TME, GSH is overexpressed and acts as an antioxidant, which may hinder the therapeutic effect by ROS-induced oxidative damage [44]. Therefore, depletion of GSH in tumor tissue can enhance the oxidative stress and the efficacy of dynamic therapies. Considering the valence-variable nature of Cu, we assessed the capacity of ZnSnO₃/Cu₃P to consume GSH. Upon incubating the GSH solution with ZnSnO₃/Cu₃P NCs for 3 h, a significant decrease in GSH concentration was observed (Fig. 2K). Notably, GSH was depleted by 81% at a ZnSnO₃/Cu₃P concentration of 250 µg/mL, indicating oxidative activity of Cu(II) in ZnSnO₃/Cu₃P towards GSH. Furthermore, the addition of US stimulation further enhanced the degradation of GSH. When the concentration of ZnSnO₃/Cu₃P was 250 µg/mL, 10 min of US stimulation (1.0 MHz, 1.0 W/cm², 50% duty cycle) before 1 h of resting leading to a 1.82-fold improvement in GSH consumption (Fig. 2L). The SDT and CDT properties of ZnSnO₃/Cu₃P after 3 h of reaction with GSH were measured using MB and TMB as specific indicators. The reaction of ZnSnO₃/Cu₃P with GSH had negligible impact on the SDT performance of ZnSnO₃/Cu₃P (Fig. S12 in Supporting information), but improved its CDT performance (Fig. S13 in Supporting information). This may be due to the reduction of Cu(II) to Cu(I) in ZnSnO₃/Cu₃P NCs, which improved the Fenton-like activity. Therefore, the synergistic effect of SDT and CDT was increased after treated with GSH (Fig. S14 in Supporting information). Taking together, ZnSnO₃/Cu₃P NCs efficiently utilize the locally produced H₂O₂ in TME to catalyze the production of ^•OH through CDT. Second, the application of external US stimulation enhances the generation of both ^•OH and ^•O₂⁻ through SDT. Third, the GSH consumption capacity of ZnSnO₃/Cu₃P NCs can reduce the antioxidant activity of tumor cells. This combination holds great potential for augmenting cancer therapy.

  In order to understand the mechanism of the SDT performance of ZnSnO₃/Cu₃P NCs, the effect of piezotronic effect on carrier migration in ZnSnO₃/Cu₃P NCs is analyzed in detail (Fig. 3A). First, the band structures of ZnSnO₃ and ZnSnO₃/Cu₃P NCs were studied by ultraviolet−visible diffuse reflection spectra (UV−vis DRS). The bandgaps (E_g) of ZnSnO₃ and ZnSnO₃/Cu₃P NCs were determined by analyzing the intercepts of the tangent and the abscissa in the Kubelka-Munk conversion. The calculated bandgaps were found to be 3.7 eV for ZnSnO₃ and 3.5 eV for ZnSnO₃/Cu₃P (Figs. 3B and C). By examining the XPS valence band spectra, the valence band (VB) positions of ZnSnO₃ and ZnSnO₃/Cu₃P were estimated to be 3.0 eV and 2.2 eV, respectively (Figs. 3D and E). Consequently, the conduction band (CB) positions of ZnSnO₃ and ZnSnO₃/Cu₃P were determined as −0.7 eV and −1.3 eV, respectively.

  Figure 3

  

  Figure 3.  Mechanism underlying the improved sonodynamic performance of ZnSnO₃/Cu₃P NCs. (A) Schematic diagram illustrating the separation and migration of electron-hole pairs in both ZnSnO₃ NCs and ZnSnO₃/Cu₃P NCs. UV–vis diffuse reflection spectra and results of Kubelka–Munk conversion of (B) ZnSnO₃ NCs and (C) ZnSnO₃/Cu₃P NCs. XPS valence band spectra of (D) ZnSnO₃ NCs and (E) ZnSnO₃/Cu₃P NCs. PFM phase hysteresis loop, and amplitude butterfly loop of (F) ZnSnO₃ NCs and (G) ZnSnO₃/Cu₃P NCs. (H) COMSOL simulation of influence of surface nanoneedles (n = 0, 1, 3, and 5) on the electric potential under ultrasonic field and (I) statistics of the maximum surface potential.

  DownLoad: Full-Size Img PowerPoint

  The VB edge of pure ZnSnO₃ NCs (3.0 eV) was more positive than the H₂O/^•OH redox potential (2.01 eV vs. relative hydrogen electrode (RHE)), verifying that US-triggered h⁺ on VB have the ability to react with H₂O to produce ^•OH. Similarly, the CB level (−0.7 eV) of ZnSnO₃ NCs is more negative compared with the redox potential of O₂/^•O₂⁻ (−0.28 V vs. RHE), indicating that the e⁻ generated by US can react with O₂ to produce ^•O₂⁻. On the other hand, electron-hole pairs can rapidly recombine in pure ZnSnO₃ NCs, which leads to low efficiency of ROS production and SDT.

  For ZnSnO₃/Cu₃P, the creation of p-n heterojunction effectively inhibited the recombination of e⁻ and h⁺. More importantly, US can induce a built-in electric field that drives e⁻ and h⁺ to opposite surfaces, leading to a band bending, which further promotes ROS generation. The piezoelectric properties of the ZnSnO₃/Cu₃P heterojunction were assessed by piezoresponse force microscopy (PFM, Fig. S15 in Supporting information). The phase images acquired at an applied voltage of 20 V clearly demonstrate the presence of piezoelectricity of both ZnSnO₃ and ZnSnO₃/Cu₃P (Figs. 3F and G). When a DC voltage ranging from −20 V to +20 V is applied, the PFM phase hysteresis loop exhibits a 180° phase change, indicating a ferroelectric polarization switching process. To further quantify the piezoelectric behavior, the piezoelectric coefficients (d₃₃) of ZnSnO₃ and ZnSnO₃/Cu₃P NCs were measured using a typical amplitude butterfly loop method. The d₃₃ values of ZnSnO₃ and ZnSnO₃/Cu₃P NCs were about 200.0 and 219.5 pm/V, respectively. The results demonstrated that the piezoelectric response of ZnSnO₃/Cu₃P was slightly enhanced compared with that of ZnSnO₃.

  To investigate whether the needle-like structure on the surface of ZnSnO₃/Cu₃P NCs enhanced the piezoelectric potential under an ultrasonic field, a COMSOL simulation was carried out (Figs. 3H and I). The simulation results showed that the more needle-like structures on the surface of ZnSnO₃/Cu₃P, the higher the US-induced piezoelectric potential, which may be due to the enhancement of mechanical effect on ZnSnO₃ NCs surface caused by needle-like structures. Therefore, we can summarize that ZnSnO₃/Cu₃P p-n heterojunction can enhance the separation and migration of e⁻ and h⁺ under US irradiation, improving the piezoelectric properties and thus enhancing the sonodynamic properties.

  We validated the killing effect of ZnSnO₃/Cu₃P NCs on tumor cells through the combination of SDT, CDT, and GSH consumption (Fig. 4A). First, L929 mouse fibroblasts were utilized to assess the cytotoxicity of ZnSnO₃/Cu₃P NCs to normal cells. L929 cells remained an over 82% survival after 24 h incubation with ZnSnO₃/Cu₃P NCs when the concentration reached 200 µg/mL (Fig. 4B). The cell viability of 4T1 mouse-derived breast cancer cells decreased to 30% after 24 h incubation with 200 µg/mL ZnSnO₃/Cu₃P NC, which can be attributed to the CDT effect produced by higher H₂O₂ concentration in tumor cells. When US irradiation (1.0 MHz, 1.0 W/cm², 50% duty cycle) was imposed for 3 min, the cell killing rate remarkably increased to 80% (Fig. 4C). As shown in the biological TEM image (Fig. 4G), after endocytosis into the cells, ZnSnO₃/Cu₃P NCs nanoparticles located in both bilayer phospholipid membrane vesicles and cytoplasm of the cells.

  Figure 4

  

  Figure 4.  In vitro cell experiments demonstrating the combined SDT and CDT performance of ZnSnO₃/Cu₃P NCs for cancer cell killing. (A) Schematic representation depicting the mechanism of tumor cell killing by ZnSnO₃/Cu₃P NCs through the integration of SDT and CDT. (B) Viabilities of L929 cells after treatments with ZnSnO₃/Cu₃P NCs for 24 h. (C) Viability of 4T1 cells at 24 h post-treatment with different approaches. (D) Confocal images and green/red fluorescence (FL) ratios of Calcein AM/PI-stained 4T1 cells following various treatments. (E) Confocal images and corresponding quantitative analysis of intracellular ROS levels in 4T1 cells stained with DCFH-DA. (F) Confocal images and green/red FL ratios of JC-1-stained 4T1 cells. (G) Bio-TEM image of 4T1 cells after treatment with ZnSnO₃/Cu₃P NCs for 10 h. The data represent means ± S.D. (n = 5). ^***P < 0.001.

  DownLoad: Full-Size Img PowerPoint

  Calcein acetoxymethyl ester (calcein-AM) and propidium iodide (PI) were used for live/dead cell staining to further verify the cancer cell killing effect (Fig. 4D and Fig. S16 in Supporting information). The cell killing effect of mere US irradiation was negligible, while 31% of cells in the ZnSnO₃/Cu₃P group died due to the CDT effect. In ZnSnO₃/Cu₃P + US group, the percentage of dead cells was found to be 90%, which correlated with the results obtained from the MTT cell viability assay. 2′, 7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a fluorescence probe to evaluate the intracellular levels of reactive oxygen species (ROS) to gain further insights into the mechanism of cell killing. DCFH-DA can react with ROS to produce 2′, 7′-dichlorofluorescein (DCF) which has green fluorescence. Compared to the US group, the intracellular fluorescence intensity in the ZnSnO₃/Cu₃P and ZnSnO₃/Cu₃P + US groups increased by 2.22-fold and 3.25-fold, respectively, demonstrating the capacity of ZnSnO₃/Cu₃P NCs to generate abundant ROS via CDT and SDT especially under US activation (Fig. 4E).

  Furthermore, to assess mitochondrial damage, the fluorescent probe 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethyl-imidacarbocyanine iodide (JC-1) was employed to measure changes in mitochondrial membrane potential. Under normal conditions, JC-1 shows red fluorescence within intact mitochondrial membranes in the form of aggregates, while shows green fluorescence in damaged or depolarized mitochondria as monomers (Fig. S17 in Supporting information). As depicted in Fig. 4F, the ZnSnO₃/Cu₃P group exhibited both green and red fluorescence in the mitochondria of 4T1 cells, whereas the ZnSnO₃/Cu₃P + US group showed the strongest green fluorescence and red fluorescence, indicating significant mitochondrial oxidative damage.

  Prior to investigating the in vivo antitumor activity of ZnSnO₃/Cu₃P NCs, we evaluated their systemic toxicity. Acute toxicity experiments were performed by intravenously injecting female ICR mice with different doses of ZnSnO₃/Cu₃P NCs (50, 100, 200 mg/kg). Throughout the 14-day observation period, all mice displayed similar clinical manifestations. The trend of weight change in the treatment groups was similar to that of the control group (Fig. S18 in Supporting information).

  Blood biochemistry assay results showed that there were no abnormal changes in hepatic and renal function markers on the 14^th day post-injection, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine (CREA), and UREA, in mice injected with various doses of ZnSnO₃/Cu₃P NCs compared with those of mice in the control group. Blood routine test indicated normal levels of red blood cells (RBCs), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and platelets (PLT) in mice administered different doses of ZnSnO₃/Cu₃P NCs (Fig. S19 in Supporting information). Hematoxylin and eosin (H&E) staining revealed no histological abnormalities in major organs including heart, liver, spleen, lung and kidney in all mice, demonstrating the excellent biocompatibility of ZnSnO₃/Cu₃P NCs (Fig. S20 in Supporting information).

  The in vivo anticancer efficacy of ZnSnO₃/Cu₃P NCs under US stimulation was assessed using a subcutaneous 4T1 tumor-bearing BALB/c mice model. All the procedures during the experiment firmly stuck to the "Beijing Administration Rule of Laboratory Animals" and the national standards "Laboratory Animal Requirements of Environment and Housing Facilities (GB 14925-2001)". The animal experiments were approved by the Committee on Ethics of Beijing Institute of Nanoenergy and Nanosystems (Approval Number: 2022A044). Mice which had been inoculated with tumor were randomly divided into four groups (n = 5): (1) PBS group; (2) PBS + US group, which intertumoral (i.t.) injection of PBS was followed by exposure to US irradiation; (3) ZnSnO₃/Cu₃P group, which received i.t. injection of 10 mg/kg ZnSnO₃/Cu₃P NCs; (4) ZnSnO₃/Cu₃P + US group, which i.t. injection of 10 mg/kg ZnSnO₃/Cu₃P NCs was followed by exposure to US irradiation. During the therapeutic period of 16 days, ZnSnO₃/Cu₃P NCs were administered twice on day 0 and day 2. Additionally, US irradiation (1.0 MHz, 2.5 W/cm², 3 min, 50% duty cycle) was applied four times during the initial 4 days (Fig. 5A).

  Figure 5

  

  Figure 5.  In vivo cancer therapy on 4T1 tumor-bearing mice. (A) Schematic representation and timeline of the cancer treatment. (B–E) Individual tumor growth profiles and (F) average tumor growth profiles of mice following various treatments. (G) Average tumor weight and (H) representative tumor photographs from different groups on day 16. (I) H&E and Ki-67 staining of tumor sections in different groups. (J) Average body weight profiles. The data represent means ± S.D. (n = 5). ^***P < 0.001.

  DownLoad: Full-Size Img PowerPoint

  Based on the tumor volume growth curves recorded over the 16-day treatment period (Figs. 5B–F), these were no significant differences in tumor growth rate between the PBS + US group and the PBS group. By comparison, moderate inhibition of tumor growth was observed in the ZnSnO₃/Cu₃P group, mainly attributed to CDT effect from ZnSnO₃/Cu₃P NCs. In contrast, the ZnSnO₃/Cu₃P + US group exhibited the highest level of tumor suppression. At the end of the treatment, the ZnSnO₃/Cu₃P + US group exhibited a tumor growth inhibition rate of 70% based on tumor weights (Figs. 5G and H), which was the highest of all the groups.

  Histopathological H&E staining revealed extensive areas of tumor necrosis within the residual tissues of the tumors in the ZnSnO₃/Cu₃P + US group. Furthermore, Ki-67 immunohistochemical staining results showed a similar trend (Fig. 5I). Weight changes among all mice during the treatment exhibited no significant differences, indicating the compatibility and biosafety of the treatment modality (Fig. 5J). In addition, major organs such as the heart, liver, spleen, lung, and kidney exhibited no observable abnormalities or inflammation according to the results of H&E staining (Fig. S21 in Supporting information). These findings confirmed that ZnSnO₃/Cu₃P NCs, functioning as a sonosensitizer and chemodynamic agent, can achieve effective tumor therapy by combing SDT and CDT with a minimal systemic toxicity.

  In summary, an urchin-like piezoelectric ZnSnO₃/Cu₃P p-n heterojunction has been successfully synthesized as a sonosensitizer for enhancing cancer SDT. The p-n heterojunction optimizes the energy band structure of ZnSnO₃/Cu₃P NCs and promotes the US sensitization performance. Additionally, Cu₃P exhibits chemodynamic activity and ability of GSH depletion, enabling the combination treatment of SDT and CDT. Ultimately, the piezoelectric sonosensitizer effectively generates ROS, reduces GSH, disrupts the redox balance within tumor cells, and promotes cell death, achieving satisfactory antitumor properties on 4T1 tumor-bearing BALB/c mice model. This strategy paves the way for constructing heterogeneous nanomaterials for cancer SDT therapy.

  Declaration of competing interest

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

  Acknowledgments

  The work was supported by the National Nature Science Foundation (No. 82072065), the Fundamental Research Funds for the Central Universities (No. E2EG6802X2), and the National Youth Talent Support Program.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.109644.

  
  
• 1. [1]

     S. Bai, N.L. Yang, X.W. Wang, et al., ACS Nano 14 (2020) 15119–15130. doi: 10.1021/acsnano.0c05235

  2. [2]

     Y.M. Ding, Z. Wang, Z.Y. Zhang, et al., Nano Res. 15 (2022) 7304–7312. doi: 10.1007/s12274-022-4336-0

  3. [3]

     Y. Zhao, T. Huang, X. Zhang, et al., BMEMat 1 (2023) e12006. doi: 10.1002/bmm2.12006

  4. [4]

     D.D. Liu, X.L. Dai, W. Zhang, et al., Biomaterials 292 (2023) 121917. doi: 10.1016/j.biomaterials.2022.121917

  5. [5]

     Y. Zhu, Y.J. Wang, G.R. Williams, et al., Adv. Sci. 7 (2020) 2000272. doi: 10.1002/advs.202000272

  6. [6]

     X. Xue, H. Qu, Y. Li, Exploration 2 (2022) 20210134. doi: 10.1002/EXP.20210134

  7. [7]

     Y. Zhang, X.Q. Zhang, H.C. Yang, et al., Chem. Soc. Rev. 50 (2021) 11227–11248. doi: 10.1039/d1cs00403d

  8. [8]

     L.A. Sun, X.W. Wang, F. Gong, et al., Theranostics 11 (2021) 9234. doi: 10.7150/thno.58755

  9. [9]

     Y.Q. Yang, X.W. Wang, H.S. Qian, L. Cheng, Appl. Mater. Today 25 (2021) 101215. doi: 10.1016/j.apmt.2021.101215

  10. [10]

      M.F. Wang, P.A. Ma, J. Lin, Chin. Chem. Lett. 34 (2023) 108300. doi: 10.1016/j.cclet.2023.108300

  11. [11]

      T.T. Hu, W.C. Shen, F.Q. Meng, et al., Adv. Mater. 35 (2023) 2209692. doi: 10.1002/adma.202209692

  12. [12]

      A.B. Wu, L.D. Jiang, C. Xia, et al., Adv. Sci. 10 (2023) 2303016. doi: 10.1002/advs.202303016

  13. [13]

      A.Q. Ma, H.Q. Chen, Y.H. Cui, et al., Small 15 (2019) 1804028. doi: 10.1002/smll.201804028

  14. [14]

      S. Liang, X.R. Deng, P.A. Ma, Z.Y. Cheng, J. Lin, Adv. Mater. 32 (2020) 2003214. doi: 10.1002/adma.202003214

  15. [15]

      X.Q. Qian, Y.Y. Zheng, Y. Chen, Adv. Mater. 28 (2016) 8097–8129. doi: 10.1002/adma.201602012

  16. [16]

      Z.R. Gong, Z.F. Dai, Adv. Sci. 8 (2021) 2002178. doi: 10.1002/advs.202002178

  17. [17]

      X.H. Lin, J.B. Song, X.Y. Chen, H.H. Yang, Angew. Chem. Int. Ed. 59 (2020) 14212–14233. doi: 10.1002/anie.201906823

  18. [18]

      Y.F. Zhou, S.Y. Fan, L.L. Feng, X.L. Huang, X.Y. Chen, Adv. Mater. 33 (2021) 2104223. doi: 10.1002/adma.202104223

  19. [19]

      P.R. Zhao, H.Y. Li, W.B. Bu, Angew. Chem. Int. Ed. 62 (2023) e2022104.

  20. [20]

      S.P. Ning, X.L. Dai, W.W. Tang, et al., Acta Biomater. 152 (2022) 562–574. doi: 10.1016/j.actbio.2022.08.067

  21. [21]

      Q.T. Hoang, V. Ravichandran, T.G.N. Cao, et al., Chem. Eng. J. 435 (2022) 135039. doi: 10.1016/j.cej.2022.135039

  22. [22]

      P. Zhu, Y. Chen, J.L. Shi, Adv. Mater. 32 (2020) 2001976. doi: 10.1002/adma.202001976

  23. [23]

      Y.C. Zhao, S.B. Wang, Y.M. Ding, et al., ACS Nano 16 (2022) 9304–9316. doi: 10.1021/acsnano.2c01968

  24. [24]

      X.X. Deng, P. Chen, R.R. Cui, et al., Appl. Catal. B 339 (2023) 123148. doi: 10.1016/j.apcatb.2023.123148

  25. [25]

      H.Q. Ma, W.Y. Yang, S. Gao, et al., Chem. Eng. J. 455 (2023) 140471. doi: 10.1016/j.cej.2022.140471

  26. [26]

      J. Jang, K. Kim, J. Yoon, C.B. Park, Biomaterials 255 (2020) 120165. doi: 10.1016/j.biomaterials.2020.120165

  27. [27]

      Z.L. Wang, Nano Today 5 (2010) 540–552. doi: 10.1016/j.nantod.2010.10.008

  28. [28]

      Z.L. Wang, Adv. Mater. 24 (2012) 4632–4646. doi: 10.1002/adma.201104365

  29. [29]

      Z.L. Wang, W.Z. Wu, Natl. Sci. Rev. 1 (2014) 62–90. doi: 10.1093/nsr/nwt002

  30. [30]

      S. Chen, P. Zhu, L.J. Mao, et al., Adv. Mater. 35 (2023) 2208256. doi: 10.1002/adma.202208256

  31. [31]

      Z.J. Wang, J. Liu, F.J. Wang, et al., J. Phys. Chem. C 114 (2010) 13577–13582. doi: 10.1021/jp104733e

  32. [32]

      J. Zhang, K.L. Yao, Z.L. Liu, et al., Phys. Chem. Chem. Phys. 12 (2010) 9197–9204. doi: 10.1039/b920065g

  33. [33]

      Y. Inaguma, M. Yoshida, T. Katsumata, J. Am. Chem. Soc. 130 (2008) 6704. doi: 10.1021/ja801843v

  34. [34]

      K.Y. Lee, D. Kim, J.H. Lee, et al., Adv. Funct. Mater. 24 (2014) 37–43. doi: 10.1002/adfm.201301379

  35. [35]

      S. Shawon, Z.D. Carballo, V.S. Vega, et al., Nano Energy 92 (2022) 106653. doi: 10.1016/j.nanoen.2021.106653

  36. [36]

      X.S. Cao, M.X. Li, Q.Y. Liu, et al., Small 19 (2023) 2303195. doi: 10.1002/smll.202303195

  37. [37]

      G.R. Dillip, P.C. Nagajyothi, R. Ramaraghavulu, et al., Mater. Chem. Phys. 248 (2020) 122946. doi: 10.1016/j.matchemphys.2020.122946

  38. [38]

      B.J. Ma, S. Wang, F. Liu, et al., J. Am. Chem. Soc. 141 (2019) 849–857. doi: 10.1021/jacs.8b08714

  39. [39]

      S.J. Zhong, C. Xiong, Y.C. Zhao, et al., Adv. Funct. Mater. 33 (2023) 2305625. doi: 10.1002/adfm.202305625

  40. [40]

      Y.T. Zhao, L. Tao, Chin. Chem. Lett. 35 (2024) 108571. doi: 10.1016/j.cclet.2023.108571

  41. [41]

      S. Wang, S.Q. Yang, Z.L. Cui, et al., Chem. Eng. J. 457 (2023) 141186. doi: 10.1016/j.cej.2022.141186

  42. [42]

      H. Lei, Z. Pei, C. Jiang, L. Cheng, Exploration 3 (2023) 20220001. doi: 10.1002/EXP.20220001

  43. [43]

      D. Jana, Y. Zhao, Exploration 2 (2022) 20210238. doi: 10.1002/EXP.20210238

  44. [44]

      J.A. Malla, R.M. Umesh, S. Yousf, et al., Angew. Chem., Int. Ed. 59 (2020) 7944–7952. doi: 10.1002/anie.202000961

• 

  Scheme 1  Schematic depiction of the synthesis process and mechanism in the antitumor process of ZnSnO₃/Cu₃P NCs.

  下载: 全尺寸图片 幻灯片
  

  Figure 1  Synthesis and characterizations of ZnSnO₃/Cu₃P. (A) Schematic diagram of the fabrication process of ZnSnO₃/Cu₃P. (B) SEM image of ZnSnO₃ NCs. (C) SEM image of ZnSnO₃/Cu₃P. (D) TEM image, (E) HRTEM image and (F) element mapping of ZnSnO₃/Cu₃P. (G) XRD, (H) XPS and high-resolution (I) Cu 2p and (J) O 1s XPS spectra of ZnSnO₃/Cu₃P. (K) hydrodynamic diameters and (L) zeta potential of ZnSnO₃ and ZnSnO₃/Cu₃P. The data represent means ± standard deviation (S.D.) (n = 5).

  下载: 全尺寸图片 幻灯片
  

  Figure 2  Sonodynamic and chemodynamic performance of ZnSnO₃/Cu₃P. (A) Schematic representation illustrating the sonodynamic and chemodynamic processes of ZnSnO₃/Cu₃P. (B) Degradation curves of MB and (C) corresponding kinetic curves in SDT process. Degradation curves of NBT for ^•O₂⁻ detection in the SDT process. (E) Generation of ^•OH in the SDT process with TA as the indicator. (F) Production of ^•OH in the CDT process measured with TMB as the indicator. (G) Degradation curves of MB and (H) corresponding kinetic curves in the combination process of SDT and CDT. ESR spectra of (I) ^•OH and (J) ^•O₂⁻ trapped by DMPO under different conditions. (K) GSH oxidation by varying concentrations of ZnSnO₃/Cu₃P within 3 h. (L) Oxidation of GSH achieved by combining SDT and CDT.

  下载: 全尺寸图片 幻灯片
  

  Figure 3  Mechanism underlying the improved sonodynamic performance of ZnSnO₃/Cu₃P NCs. (A) Schematic diagram illustrating the separation and migration of electron-hole pairs in both ZnSnO₃ NCs and ZnSnO₃/Cu₃P NCs. UV–vis diffuse reflection spectra and results of Kubelka–Munk conversion of (B) ZnSnO₃ NCs and (C) ZnSnO₃/Cu₃P NCs. XPS valence band spectra of (D) ZnSnO₃ NCs and (E) ZnSnO₃/Cu₃P NCs. PFM phase hysteresis loop, and amplitude butterfly loop of (F) ZnSnO₃ NCs and (G) ZnSnO₃/Cu₃P NCs. (H) COMSOL simulation of influence of surface nanoneedles (n = 0, 1, 3, and 5) on the electric potential under ultrasonic field and (I) statistics of the maximum surface potential.

  下载: 全尺寸图片 幻灯片
  

  Figure 4  In vitro cell experiments demonstrating the combined SDT and CDT performance of ZnSnO₃/Cu₃P NCs for cancer cell killing. (A) Schematic representation depicting the mechanism of tumor cell killing by ZnSnO₃/Cu₃P NCs through the integration of SDT and CDT. (B) Viabilities of L929 cells after treatments with ZnSnO₃/Cu₃P NCs for 24 h. (C) Viability of 4T1 cells at 24 h post-treatment with different approaches. (D) Confocal images and green/red fluorescence (FL) ratios of Calcein AM/PI-stained 4T1 cells following various treatments. (E) Confocal images and corresponding quantitative analysis of intracellular ROS levels in 4T1 cells stained with DCFH-DA. (F) Confocal images and green/red FL ratios of JC-1-stained 4T1 cells. (G) Bio-TEM image of 4T1 cells after treatment with ZnSnO₃/Cu₃P NCs for 10 h. The data represent means ± S.D. (n = 5). ^***P < 0.001.

  下载: 全尺寸图片 幻灯片
  

  Figure 5  In vivo cancer therapy on 4T1 tumor-bearing mice. (A) Schematic representation and timeline of the cancer treatment. (B–E) Individual tumor growth profiles and (F) average tumor growth profiles of mice following various treatments. (G) Average tumor weight and (H) representative tumor photographs from different groups on day 16. (I) H&E and Ki-67 staining of tumor sections in different groups. (J) Average body weight profiles. The data represent means ± S.D. (n = 5). ^***P < 0.001.

  下载: 全尺寸图片 幻灯片

  Table 1.  First-order reaction kinetics of MB degradation under various treatments.

  下载: 导出CSV

  Table 2.  First-order reaction kinetics of MB degradation under various treatments.

  下载: 导出CSV
•