Ultrasound‐Activated Piezocatalyst Triggers Sulfate Radical Production for Augmented Oxidative Tumor Suppression

Sonopiezoelectric therapy (SPT) has gained ever‐increasing prominence in tumor therapy as a combination of ultrasound (US) and piezoelectric nanomaterials to generate cytotoxic reactive oxygen species (ROS) for tumor suppression. In this study, the facile hydrothermal method is used to construct the distinct 2D piezocatalyst Bi2Fe4O9 nanosheets (BFO NSs). The engineered BFO NSs can create a piezoelectric field when exposed to US irradiation, facilitating the separation of electron–hole (e−–h+) pairs to play a crucial role in Fe3+/Fe2+ circulation, which catalyzes the activation of peroxymonosulfate (PMS), resulting in an amplified production of cytotoxic sulfate radical (SO4•−). The produced SO4•− combines with ROS generated by SPT including hydroxyl radicals (•OH), and singlet oxygen (1O2), leading to the augmented oxidative tumor suppression. The BFO NSs and PMS are further integrated into a thermal hydrogel (BFO/PMS‐Gel) for in vitro and in vivo investigations on their catalytic antitumor effects. In both in vitro and in vivo studies, it is validated that BFO/PMS‐Gel, under US irradiation, significantly enhances radicals generation efficiency, which achieves superior antitumor performance while maintaining high therapeutic biocompatibility and biosafety, providing a representative paradigm of US‐activated multiple radicals production as enabled by piezoelectric nanomedicine for antitumor treatment.


Introduction
Tumors have emerged as a significant medical concern over recent decades, posing a substantial threat to human life and well-being. [1]Traditional antitumor modalities, such as surgery, radiotherapy, and chemotherapy, have made remarkable strides in clinical applications but remain burdened by their respective side effects. [2]Consequently, the quest for novel antitumor therapies persists.Among diverse emerging approaches, sonopiezoelectric therapy (SPT) has garnered considerable attention. [3]SPT harnesses ultrasound (US) to precisely deliver sonoenergy as a mechanical force to piezoelectric nanomaterials without causing harm and generates cytotoxic reactive oxygen species (ROS), imparting antitumor effects. [4]While both SPT and the widely studied sonodynamic effect involve US, they differ in their mechanisms.The US-triggered piezoelectric effect generates electric charges in response to mechanical stress, resulting in some optimized biological applications.In contrast, the sonodynamic effect involves the activation of sonosensitizers by ultrasonic cavitation effect or sonoluminescence to generate ROS for cancer cell killing.ROS encompass oxygen-derived free radicals like superoxide anion (O 2 À ), hydroxyl radicals (•OH), and singlet oxygen ( 1 O 2 ), known for their high chemical reactivity. [5]Under normal physiological conditions, an appropriate level of ROS is essential for cellular functions.However, a surge in ROS can lead to oxidative stress, a key mechanism by which SPT combats tumors. [6]iezoelectric nanomaterials, characterized by their noncentrosymmetric lattice structures, undergo deformation and polarization when subjected to mechanical forces, resulting in the generation of built-in piezoelectric fields and separation of electron-hole (e À -h þ ) pairs. [7]Piezocatalysts have received widespread attention due to their excellent catalytic activity.
Sonopiezoelectric therapy (SPT) has gained ever-increasing prominence in tumor therapy as a combination of ultrasound (US) and piezoelectric nanomaterials to generate cytotoxic reactive oxygen species (ROS) for tumor suppression.In this study, the facile hydrothermal method is used to construct the distinct 2D piezocatalyst Bi 2 Fe 4 O 9 nanosheets (BFO NSs).The engineered BFO NSs can create a piezoelectric field when exposed to US irradiation, facilitating the separation of electron-hole (e À -h þ ) pairs to play a crucial role in Fe 3þ /Fe 2þ circulation, which catalyzes the activation of peroxymonosulfate (PMS), resulting in an amplified production of cytotoxic sulfate radical (SO 4 •À ).The produced SO 4 •À combines with ROS generated by SPT including hydroxyl radicals (•OH), and singlet oxygen ( 1 O 2 ), leading to the augmented oxidative tumor suppression.
The BFO NSs and PMS are further integrated into a thermal hydrogel (BFO/PMS-Gel) for in vitro and in vivo investigations on their catalytic antitumor effects.
In both in vitro and in vivo studies, it is validated that BFO/PMS-Gel, under US irradiation, significantly enhances radicals generation efficiency, which achieves superior antitumor performance while maintaining high therapeutic biocompatibility and biosafety, providing a representative paradigm of US-activated multiple radicals production as enabled by piezoelectric nanomedicine for antitumor treatment.
When under mechanical forces such as electric current, US, lasers, etc., the separated e À /h þ catalyze the oxidation-reduction reaction of substrates and promote the production of active substances. [8]With the unique properties of nanoscale dimensions, piezocatalysts are highly suitable for biomedical applications and have made promising signs of progress in disease diagnosis and treatments. [9]eroxymonosulfate (PMS) exhibits the ability to selectively produce cytotoxic sulfate radical (SO 4

•À
), which is characterized by its prolonged half-life and higher-standard redox potential than other ROS. [10]Additionally, PMS-mediated radical generation remains independent of oxygen or H 2 O 2 levels and pH value, making it a desirable candidate for efficient oxidative tumor therapy, particularly well suited to the hypoxic and mildly acidic tumor microenvironment (TME). [11]The rational combination of SPT-triggered ROS production and PMS-involved sulfate radicals is expected to bring with a synergistic oxidative tumortherapeutic modality.
In this work, we rationally design and fabricate 2D Bi 2 Fe 4 O 9 nanosheets (BFO NSs) as an efficient piezocatalyst using a facile hydrothermal method. [12]The piezoelectric properties of these NSs were confirmed through the typical piezoresponse force microscopy (PFM). [13]With the SPT properties, BFO NSs can generate ROS including •OH and 1 O 2 under US irradiation.When combined with PMS, the Fe 2þ of BFO NSs acted as electron donators to activate the PMS for generation of cytotoxic SO 4 •À , •OH, and 1 O 2 .Delightfully, when exposed to US irradiation, the generated piezoelectric field facilitates the separation of e À -h þ pairs.The separated electrons reduce generated Fe 3þ back to Fe 2þ , thus continually activating PMS, leading to a substantial boost in radicals production for oxidative tumor suppression. [12,14]The integration of these NSs with a thermal hydrogel enabled the combination of the piezocatalyst and PMS, resulting in enhanced tumor eradication capabilities as demonstrated both in vitro and in vivo (Figure 1).This study combines the SPT effect and catalyzed generation of sulfate radicals by PMS activation for augmented radicals therapy.

Characterization of BFO NSs
The 2D BFO NSs were successfully synthesized using a facile hydrothermal method.Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images confirm the sheetlike morphology of BFO (Figure 2a,b).Energy-dispersive X-ray spectroscopy elemental mapping images reveals the uniform distribution of Bi, Fe, and O elements in the prepared NSs (Figure 2c).X-ray diffraction analysis confirms the successful synthesis, consistent with the standard Bi 2 Fe 4 O 9 phase (ICDD File Card No. 25-0090) (Figure 2d).High-resolution TEM image displays lattice spacings of 0.59 and 0.30 nm, corresponding to the (001) and (002) lattice orientations, respectively (Figure 2e).Furthermore, the selected-area electron diffraction pattern affirms the crystalline nature of the BFO NSs (Figure S1, Supporting Information).Atomic force microscopy image shows the thickness of BFO is 7 nm (Figure 2f,g).The dynamic light scattering test was carried out to get an accurate size of BFO NSs (Figure S2, Supporting Information), exhibiting the hydrodynamic diameter is 175.9 nm.Furthermore, the bandgap energy (E g ) is calculated to be 1.69 eV through the Tauc equation by ultraviolet-visible (UV-vis) diffuse reflectance spectroscopy measurement (Figure S3, Supporting Information).PFM serves as a vital method for assessing the piezoelectric properties of materials and detecting electroforming variables under external excitation voltage.PFM observations reveal a significant alteration in the characteristic butterfly loop in the amplitude, indicating the excellent piezoelectric properties of BFO NSs (Figure 2h).X-ray photoelectron spectroscopy (XPS) was employed to ascertain the chemical composition of BFO NSs, confirming the presence of Bi, O, and Fe elements (Figure 2i).Valence band (VB) XPS data provide the VB potential of 0.86 eV for BFO NSs (Figure S4, Supporting Information), with the conduction band potential calculated as À0.83 eV, indicating a more negative potential than the reduced potential of Fe 3þ /Fe 2þ (0.77 eV).This suggests that the redox between Fe 3þ /Fe 2þ is more favorable (Figure 2j).
To elucidate the role of sonopiezoelectric electrons for Fe 3þ /Fe 2þ circulation, we examined the Fe 2p spectra before and after US irradiation.The binding energy peaks at 709.7 and 710.8 eV represent Fe 2þ and Fe 3þ in BFO NSs, respectively (Figure 2k), with a calculated ratio of Fe 2þ :Fe 3þ as 26.3:73.7.
Significantly, after US irradiation, the ratio shifts to 63.7:36.3(Figure 2l), demonstrating the electrons separated by sonopiezoelectric effect boost the conversion from Fe 3þ to Fe 2þ .To rule out the possibility of Bi redox involvement, we also investigated the variation of Bi 4f spectra before and after treatment.The ratio of Bi 3þ to Bi 5þ remains relatively constant following activation (Figure S5, Supporting Information), indicating no significant redox between Bi 3þ /Bi 5þ during the catalytic process.

Catalytic Performance of BFO NSs for Radicals Generation
Based on the characterization results and analysis, we proceeded to investigate the synergistic enhancement of cytotoxic radicals through SPT combined with PMS activation catalyzed by BFO NSs.We established five experimental groups: 1) control, 2) BFO þ US, 3) PMS þ US, 4) BFO/PMS, and 5) BFO/ PMS þ US, and assessed ROS generation using a UV-vis spectrophotometer. [15]Initially, methylene blue (MB) was employed as a classical probe for •OH and SO 4 •À .When combines with •OH or SO 4 •À , the blue-colored MB turns colorless, resulting in a decrease in the UV-vis characteristic absorption peak at 663 nm. [16]Taking the BFO/PMS þ US group as an example, it is evident that under US irradiation, the characteristic peaks of MB consistently decrease, indicating the radicals generation (Figure 3a).Among all the groups, BFO NSs generate limited •OH under US irradiation.However, BFO NSs combined with PMS result in 16.0% MB degraded.This percentage increases to 39.5% in the BFO/PMS þ US group (Figure 3b), underscoring the superior catalytic performance of BFO NSs.Furthermore, the absorption peak of MB decreases rapidly with increased US intensity (Figure 3c), which is in a US powerdensity manner, indicating that piezoelectricity drives the catalytic performance.Subsequently, 1,3-diphenylisobenzofuran (DPBF) was further employed to detect the 1 O 2 generation.When 1 O 2 is captured, DPBF changes from yellow to colorless, and the UV-vis characteristic peak at 420 nm decreases. [17]With US irradiation, a significant decrease in the absorption peak is observed in the BFO/PMS þ US group, confirming the generation of 1 O 2 (Figure 3d).Across all the groups, these results align with the findings of the MB-probe experiments.BFO NSs or PMS alone under US irradiation have minimal impact compared to the control group.However, the employment of US effectively enhances the production efficiency of radicals generated by BFO/PMS (Figure 3e).Furthermore, the experiment of varying US intensities also demonstrates that higher US intensity boosts 1 O 2 generation, further confirming the sonopiezoelectric mechanism (Figure 3f ).Additionally, the overexpression of glutathione (GSH), a hallmark of the TME, plays a crucial role in counteracting oxidative damage.Ellman's Reagent (5,5 0 -dithiobis-[2-nitrobenzoic acid], DTNB) was used as the probe to detect GSH levels.Following co-incubation with BFO NSs, there was a notable decrease in DTNB absorption peaks, implying effective GSH consumption, and suggesting an enhancement in the oxidative antitumor effect (Figure S6, Supporting Information).
To identify the specific radicals generated during PMS activation and SPT, we conducted a quenching experiment.Ethanol (EtOH) is known as a quencher for SO 4 •À and •OH, as it can capture SO 4 •À and •OH more rapidly.Tert-butanol (TBA) serves as a •OH quencher because of the much faster binding rate with •OH than SO 4 •À .Upon the addition of these two quenchers, the characteristic absorbance peaks of MB only weakly diminish (Figure 3g,h).The degradation rate decreases from 39.5% to 11.4% when TBA is added to capture •OH, and drops to 7.4% when co-incubated with EtOH, which captures both •OH and SO 4 •À (Figure 3i).These results demonstrate the generation of these two radicals through BFO NSs-catalyzed PMS activation.Additionally, we utilized electron spin resonance analysis to quantify radicals generation using 2,2,6,6-tetramethyl-4-piperidino (TEMP) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as trapping agents for 1 O 2 and SO 4 •À /•OH, respectively. [18]The characteristic 1:1:1 absorbance peak of TEMP-1 O 2 confirms the generation of 1 O 2 , with the highest intensity observed in the BFO/PMS þ US group (Figure 3j).Furthermore, the characteristic absorbance peaks of DMPO-SO 4 •À and DMPO-•OH also substantiate the presence of SO 4 •À and •OH, with the assistance of US irradiation significantly enhancing the absorption peak intensity in the BFO/PMS þ US group compared to others (Figure 3k).These results elucidate the types of radicals generated during the PMS activation and SPT process and corroborate the potent catalytic effect of BFO NSs.
Based on the aforementioned results, the underlying mechanisms of SPT combined with PMS activation catalyzed by BFO NSs to enhance radicals generation can be outlined as follows: when the piezocatalyst BFO NSs are under US irradiation, the generated piezoelectric fields separate the e À -h þ pairs.The separated holes transfer H 2 O to generate •OH, while electrons react with O 2 for 1 O 2 generation (Figure 3l).Meanwhile, the Fe 2þ of BFO NSs provides electrons for PMS activation, resulting in the generation of SO 4 •À , •OH, and 1 O 2 (Equation ( 1)-( 4)).Subsequently, BFO NSs promote the separation of e À -h þ pairs under US irradiation (Equation ( 5)), with the separated electrons accelerating the Fe 3þ /Fe 2þ circulation (Equation ( 6)), thereby continually activating PMS, and resulting in an amplified production of cytotoxic radicals.

Augmented Antitumor Effect In Vitro
With the desirable catalytic performance of BFO NSs, we proceeded to investigate the in vitro antitumor effect.To optimize and enhance the catalytic performance both in vitro and in vivo, BFO NSs and PMS were integrated with a thermal hydrogel to form BFO/PMS-Gel.The corresponding in vitro experiments were conducted using transwells loaded with the hydrogel (Figure 4a).The thermal hydrogel underwent a transition from a solution state to a gel state upon exposure to a 37 °C water bath, resulting in an irregular porous structure, as demonstrated by SEM imaging (Figure S7 and S8, Supporting Information).Additionally, elemental mapping images confirm the uniform distribution of BFO NSs within the matrix of the thermal hydrogel (Figure S9, Supporting Information).
The experiments were categorized into six groups, including G1: control, G2: US, G3: blank hydrogel (Gel), G4: blank hydrogel along with US irradiation (Gel þ US), G5: hydrogel integrated with BFO NSs and PMS (BFO/PMS-Gel), and G6: hydrogel integrated with BFO NSs and PMS along with US irradiation (BFO/ PMS-Gel þ US).Initially, the biocompatibility of the antitumor platform was assessed after co-incubation with cells.We selected human umbilical vein endothelial cells (HUVECs) to represent normal cells and 4T1 murine breast tumor cells as representative tumor cells due to their similarity to human breast cancer in terms of growth and metastasis features.Cell viability was evaluated using the cell-counting kit 8 assay, a widely employed protocol for the sensitive detection of cell proliferation and cytotoxicity. [19]Following co-incubation with varying concentrations of BFO/PMS and BFO/PMS-Gel for 24 h, the cell viabilities of HUVECs and 4T1 cells were determined.Notably, HUVECs exhibit consistently high viability across all concentrations, underscoring the excellent biological safety of both pure and hydrogel-integrated BFO/PMS for normal cells.Furthermore, for 4T1 breast tumor cells, no significant decreases in viability are observed even at a high concentration of 200 μg mL À1 for both forms of BFO/PMS.When the concentration of the pure BFO/PMS platform was increased to 400 μg mL À1 , the cell viability starts to decrease to 77.1%, further affirming the high biocompatibility (Figure 4b,c).
Subsequently, we selected the concentration of 100 μg mL À1 , which exhibited low cytotoxicity, to assess the enhanced cytotoxicity based on SPT combined with PMS activation (Figure 4d).As observed, the viabilities in the control and blank gel groups experience negligible decreases.When US irradiation was applied, the viabilities in the US and Gel þ US groups decrease by approximately 10%.However, with the incorporation of BFO/PMS-Gel, cell viability decreases even further compared to the control and blank gel groups.Upon exposure to US irradiation, cell viability dramatically decreases to 42.3%, highlighting the excellent synergistic performance and the potential for antitumor applications.
Another characteristic indicator of cell death is the disruption of cell membrane integrity, leading to the release of intracellular enzymes into the culture medium.This includes the relatively stable enzyme lactate dehydrogenase (LDH), the detection of which can serve as an indicator of cytotoxicity.Different levels of LDH release were observed in all the groups subjected to US irradiation, likely due to the damages caused by the intensity of US.In the groups treated with BFO/PMS-Gel under US irradiation, there is a substantial increase in LDH release compared to the other groups (Figure 4e), providing further evidence of the cytotoxic effects induced by SPT combined with PMS activation.
Following this, a classic apoptosis assay was performed using flow cytometry with Annexin V-fluorescein isothiocyanate and propidium iodide (PI) to investigate the antitumor performance of BFO-NSs-mediated SPT and PMS activation (Figure 4f ).The upper right (Q2) quadrant represents nonviable apoptotic cells (Annexin Vþ/PIþ), the lower right (Q3) quadrant represents viable apoptotic cells (Annexin Vþ/PIÀ), and the sum of these quadrants represents the proportion of all apoptotic cells. [20]In the BFO/PMS-Gel þ US group, the proportion of apoptotic cells reaches up to 73.0%, significantly higher than those in other groups.
To directly visualize the cell death induced by the engineered antitumor platform, live/dead cell staining experiments were conducted using the calcein acetoxymethyl ester (calcein AM)/ PI cell viability assay kit.Calcein AM is a cell-staining reagent that can easily penetrate cell membranes and fluorescently stain living cells, and it is nonfluorescent by itself.Calcein AM is hydrolyzed by endogenous esterases in living cells to produce a polar molecule, calcein, which cannot permeate the cell membrane.Consequently, calcein becomes trapped within the cells and emits strong green fluorescence.In contrast, dead cells, due to the lack or very low activity of endogenous esterases, cannot efficiently stain with calcein AM or exhibit only weak staining.PI, a red fluorescent dye, is unable to penetrate the cell membrane of living cells and can only stain cells with compromised cell membrane integrity, such as dead cells.Therefore, the detection of intracellular esterase activity and cell membrane integrity using these two probes directly reflects cell activity and cytotoxicity induced by BFO-NSs-mediated SPT and PMS activation.Observations using a fluorescent inverted microscope reveal that the BFO/PMS-Gel þ US group exhibits the strongest red fluorescence compared to the other groups, indicating that the majority of cells in this group were dead (Figure 4g).
As previously established, the primary site for ROS production is intracellular mitochondria, which is also the main target of ROS attack.Mitochondrial damage can, in turn, lead to the production of more ROS, thereby creating a vicious cycle of oxidative stress.To assess mitochondrial damage, we utilized the commercial dye 5,5 0 ,6,6 0 -Tetrachloro-1,1 0 ,3,3 0 -tetraethyl-imidacarbocyanine iodide (JC-1) as a probe to measure changes in mitochondrial membrane potential (Figure 4h and S10, Supporting Information).The decline in mitochondrial membrane potential is a significant indicator of mitochondrial damage and signifies the early stages of apoptosis. [21]At a high mitochondrial membrane potential, JC-1 accumulates in the mitochondria's matrix to form polymer aggregates, which emit a red-colored fluorescence signal.Conversely, when the mitochondrial membrane potential is low, JC-1 cannot accumulate in the mitochondria's matrix and remains in a monomeric state, resulting in a green fluorescence signal.The strong red fluorescence signals in groups 1-4 indicate that US alone or in combination with blank gel caused minimal mitochondrial damages.In contrast, BFO/PMS-Gel induced some degree of damage, with the appearance of green fluorescence, resulting in an orange merged image.When US irradiation was added, strong green fluorescence signals were observed using a confocal laser scanning microscope (CLSM).This demonstrates that BFO NSs could cause severe mitochondrial damage and, consequently, augment oxidative tumor suppression.
Cytotoxic ROS have been demonstrated to be the primary causes of oxidative damages for tumors.We further investigated the in vitro ROS generation performance of BFO-NSs-mediated SPT combined with PMS activation.The 2,7-dichlorofluorescein diacetate (DCFH-DA) is a common ROS probe capable of freely crossing the cell membrane. [22]Once inside the cell, it is hydrolyzed to produce DCFH, which cannot permeate the cell membrane.Consequently, the probe is readily loaded into the cells.Intracellular ROS can oxidize nonfluorescent DCFH into fluorescent DCF, with the detection of this fluorescence revealing the level of intracellular ROS. [23]The green fluorescence signals observed by CLSM are significantly enhanced after US irradiation (Figure 4i), and a semiquantitative analysis reveals that the fluorescence signal intensity in the BFO/PMS-Gel þ US group is more than twice of that in the BFO/PMS-Gel group (Figure 4j).As expected, the same trend is observed in flow cytometry experiments (Figure S11, Supporting Information).Compared with the control group, the percentages of intracellular ROS increase only slightly in the US, Gel, and Gel þ US groups.However, when co-incubated with BFO/PMS-Gel, the percentage of generated ROS increases to 29.6%, and after US irradiation, this percentage more than doubles to 77.3%.This demonstrates that US-triggered piezocatalyst accelerates the generation of cytotoxic ROS, leading to the killing of tumor cells.All the aforementioned in vitro results strongly support the conclusion that the piezocatalyst BFO NSs can enhance PMS activation combined with SPT for the generation of cytotoxic ROS, resulting in highly effective antitumor performance in vitro.

Genetic Analysis of the Antitumor Effect
To further elucidate the detailed mechanism by which BFO NSs catalyzed PMS activation combined with SPT to achieve antitumor effects, we conducted RNA sequencing analysis on 4T1 tumor cells treated with BFO/PMS-Gel under US irradiation.Principal component analysis clearly shows differences in mRNA expression between the control and BFO/PMS-Gel þ US groups, a pattern supported by the heat map results (Figure 5a and S12, Supporting Information).The piezocatalytic process led to the differential expression of 326 genes, including 277 upregulated mRNAs and 49 downregulated mRNAs (Figure 5b).First, gene ontology analysis was performed to clarify the biological functions of the altered mRNAs and their corresponding action pathways across three categories: molecular function, biological process, and cellular components.The results are intuitively displayed in a circular diagram (Figure 5c), with the top 10 pathways in each category listed as well (Figure 5d).Subsequently, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted to identify the altered pathways after the piezocatalytic treatment (Figure 5e,f and S13, Supporting Information).Interestingly, in addition to classical pathways like necroptosis, we also found some inflammationrelated pathways among the top 20 enrichment pathways, such  as the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway and cytokine-cytokine receptor interaction.This suggests that mechanisms related to inflammation also contribute to the antitumor process.Traditional gene set enrichment analysis results indicate that BFO/PMS-Gel along with US irradiation significantly alters the expression of the NOD-like pathway (Figure 5g), as well as enrichment of cytokines such as caspase 4 and inflammation-related genes associated with pyroptosis.Although necroptosis and pyroptosis are common ways of cell death, there are still differences between them.Pyroptosis is a programmed cell death caused by inflammatory bodies, while necrosis is considered as an unregulated non-programmed one.Despite that pyroptosis and necrosis are different pathways of cell death, they constitute a coordinated cell death system.

Enhanced in Vivo Tumor Suppression
Motivated by the excellent performance of synergistic oxidative tumor suppression in vitro, we conducted further investigations into the therapeutic performance of BFO/PMS-Gel combined with US irradiation in vivo.All animal experimental protocols were performed following the relevant guidelines and approved by the ethics committee of Shanghai University.Specifically, subcutaneous xenografts of 4T1 breast tumors were established in female BALB/c mice.After 7 days, the tumor-bearing mice were randomly divided into four groups (n = 5 per group): G1: control, G2: US (1.0 W cm À2 , 50% duty cycle, 5 min), G3: BFO/PMS-Gel, and G4: BFO/PMS-Gel þ US (1.0 W cm À2 , 50% duty cycle, 5 min), and were sacrificed after 14 days following different treatments.To minimize systemic side effects and maximize antitumor efficacy, we injected the injectable BFO/PMS-Gel directly into the tumor at a dosage of 5 mg kg À1 on 0, 2, and 4 days.US irradiation was then applied 4 h after each injection (Figure 6a).
Prior to evaluating the augmented antitumor effect in vivo, it is ensured that the biosafety is essential for tumor therapeutic applications.All the mice in different groups were weighed every 2 days, and nearly no significant fluctuations were observed throughout the entire experimental process (Figure 6b).Furthermore, hematoxylin and eosin (H&E) staining of vital organs such as the heart, liver, spleen, lung, and kidney shows no significant damage caused by US, injectable BFO/PMS-Gel, or their combination, indicating the high biosafety of the engineered antitumor platform (Figure S14, Supporting Information).
Tumor volumes in different groups were measured every 2 days (Figure 6c).Compared with the control group, the growth of 4T1 tumors treated with US or BFO/PMS-Gel alone (G2 and G3) show some delay, likely due to the slight damages caused by US irradiation or hydrogel, respectively.Notably, the growth of 4T1 tumors treated with injectable BFO/PMS-Gel along with US irradiation (G4) are significantly inhibited (Figure 6d).Upon sacrificing the tumor-bearing mice, tumors were excised and weighed to support the conclusions drawn (Figure 6e).The tumor growth inhibition rate of the BFO/PMS-Gel þ US group reaches 86.1%, which is more than three times of the groups treated with US or BFO/PMS-Gel alone (Figure 6f ), demonstrating that the SPT combined with PMS activated by BFO NSs strongly enhanced the antitumor effect.Additionally, the calculated results of relative tumor volume provide additional evidence (Figure S15, Supporting Information).The ratio of tumor volumes after 14 days relative to the initial volume in the control group (G1) averaged to 22.8, which decreased to 13.1 and 14.3 in the US (G2) and BFO/PMS-Gel (G3) groups, respectively.In contrast, this ratio drops to 5.0 in the BFO/PMS-Gel along with US irradiation group (G4).
Digital images of the mice at different time points during the experiment and images of excised tumors at the end of the experiment were taken for a more intuitive demonstration of the superior antitumor effect (Figure 6g and S16, Supporting Information).Subsequently, H&E staining, terminal-deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining, and Ki67 immunofluorescence staining of tumor sections were conducted to confirm the catalyzed antitumor effect in vivo (Figure 6h,i, S17, and S18, Supporting Information).The results of H&E staining and TUNEL immunofluorescence staining show severe damages to tumor cells caused by BFO/ PMS-Gel under US irradiation compared with other groups.Furthermore, the Ki67 immunofluorescence staining results confirm maximal inhibition of tumor cell proliferation in the BFO/ PMS-Gel þ US group.Therefore, the fabricated piezocatalyst BFO NSs can catalyze the activation of PMS combined with SPT to enhance oxidative tumor suppression in vivo, with a high level of biosafety.

Conclusion
In conclusion, we have rationally engineered 2D piezocatalyst BFO NSs in conjunction with US irradiation to catalyze the activation of PMS, which combined with ROS generated through SPT for synergistic oxidative tumor suppression.Leveraging US irradiation as a mechanical force, the piezoelectric properties of BFO NSs were harnessed to generate the cytotoxic ROS including •OH and 1 O 2 .The piezoelectric-separated electrons played a crucial role in the circulation of Fe 3þ /Fe 2þ , thereby catalyzing the activation of PMS and accelerating the generation of SO 4 •À .The adopted RNA-seq analysis further shed light on the mechanisms underlying the enhanced antitumor performance achieved through the combination of BFO NSs, PMS, and US irradiation.This multifaceted approach led to tumor suppression through pathways involving necroptosis, pyroptosis, and various inflammation-related processes.Importantly, the comprehensive assessments of biosafety, conducted through both in vitro and in vivo experiments, affirm the favorable safety profile of the engineered antitumor platform.The results from these experiments collectively demonstrate the remarkable antitumor performance facilitated by BFO NSs in an augmented synergistic oxidative context.This research provides the paradigm for the broader application of piezocatalysts and offers the distinct avenues for combining SPT with other radicals to combat tumors with enhanced efficacy.

Figure 1 .
Figure 1.Schematic illustration of the synthesis of Bi 2 Fe 4 O 9 nanosheets (BFO NSs) and the catalyzed antitumor performance.The reactive oxygen species (ROS) generated by sonopiezoelectric therapy (SPT) combined with SO 4 •À generated by peroxymonosulfate (PMS) activation enhance the oxidative tumor suppression.

Figure 2 .
Figure 2. a) Scanning electron microscopy (SEM) image of BFO NSs.b) Transmission electron microscopy (TEM) image of BFO NSs.c) Scanning transmission electron microscopy and energy-dispersive X-ray spectroscopy mapping images of Bi, Fe, and O elements of BFO NSs.d) X-ray diffraction patterns of BFO NSs.e) High-resolution TEM image of BFO NS. f ) Atomic force microscopy image of BFO NS. g) The corresponding height profile of the white line marked in (f ).h) Amplitude butterfly loops and phase hysteresis loops of BFO NSs detected by piezoresponse force microscopy.i) Survey X-ray photoelectron spectroscopy (XPS) spectrum of BFO NSs.j) Diagram of the energy band of BFO NSs.k,l) XPS spectra of Fe 2p for BFO NSs (k) before and (l) after US irradiation.

Figure 3 .
Figure 3. a) UV-vis absorbance spectra of methylene blue (MB) oxidation by radicals generated from the piezocatalytic reaction of BFO NSs.b) MB degradation curves after different treatments (n = 3 independent experiments).c) US-intensity-dependent MB degradation curves in BFO/PMS þ US group.d) UV-vis absorption spectra of 1,3-diphenylisobenzofuran (DPBF) oxidation by 1 O 2 generated from the piezocatalytic reaction of BFO NSs.e) The DPBF degradation curves after different treatments (n = 3 independent experiments).f ) US-intensity-dependent DPBF degradation curves in BFO/ PMS þ US group.g,h) Quenching experiment using EtOH as a quencher for (g) •OH/SO 4 •À and (h) TBA for •OH.i) MB degradation curves after the addition of quenchers (n = 3 independent experiments).j,k) Electron spin resonance spectra of (j) •OH /SO 4 •À and (k) 1 O 2 .l) Schematic illustration of BFO NSs-catalyzed radicals generation.Data are presented as means AE SD.

Figure 4 .
Figure 4. a) Schematic illustration of the in vitro catalytic process of transwells-loaded BFO/PMS-Gel.b,c) Cell viabilities of normal cells (human umbilical vein endothelial cells) and tumor cells (4T1 breast tumor cells) after co-incubation with (b) BFO/PMS and (c) BFO/PMS-Gel (n = 6 biologically independent samples).d) Cell viabilities after different treatments (n = 6 biologically independent samples).e) Detection of lactate dehydrogenase release after different treatments (n = 3 biologically independent samples).f ) Representative flow cytometry analysis for cell apoptosis after different treatments.g) Representative calcein AM/propidium iodide staining of live/death cells after different treatments.h) Representative JC-1 staining for the changes of intracellular mitochondrial membrane potentials.i) Representative confocal images and j) semi-quantitative analysis of ROS generated in vitro stained by 2,7-dichlorofluorescein diacetate.Data are presented as means AE SD.P values were calculated with the one-way analysis of variance (ANOVA), ****p < 0.0001.

Figure 5 .
Figure 5. a) Principal component analysis and b) volcano plot of the differentially expressed genes in the control and BFO/PMS-Gel þ US groups.c) The circle diagram of gene ontology (GO) analyses.d) Top 30 differentially expressed genes by GO analysis between control and BFO/PMS-Gel þ US groups.e) Top 20 enrichment expressed genes by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis between control and BFO/PMS-Gel þ US groups.f ) KEGG chord diagram of the genes upregulated and downregulated in different signaling pathways.g) Traditional gene set enrichment analysis of the enrichment genes set of NOD-like receptor signaling pathway.

Figure 6 .
Figure 6.a) Schematic illustration of catalyzed antitumor effect in vivo.b) The body weights of mice at different time points after different treatments (n = 5 biologically independent samples).c) Individual tumor volume curves of mice in different groups.d) Growth curves of tumors after different treatments (n = 5 biologically independent samples).e) Weights of tumors excised after 14 days (n = 5 biologically independent samples).f ) Tumor growth inhibition of different groups (n = 5 biologically independent samples).g) Digital images of excised tumors grouped by different treatments.h) Hematoxylin and eosin staining of tumor slices in different groups.i) Transferase-mediated dUTP nick-end labeling and Ki67 immunofluorescence staining of tumor slices in different groups.Data are presented as means AE SD.P values were calculated with the one-way analysis of variance (ANOVA), **** p < 0.0001.