A sonopiezoresponse COF-based smart nanoreactor orchestrating in situ bioorthogonal chemistry and cuproptosis for enhanced tumor piezocatalytic therapy

A sonopiezoresponse COF-based smart nanoreactor orchestrating in situ bioorthogonal chemistry and cuproptosis for enhanced tumor piezocatalytic therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A sonopiezoresponse COF-based smart nanoreactor orchestrating in situ bioorthogonal chemistry and cuproptosis for enhanced tumor piezocatalytic therapy Chunyuan Hou, Yu Zhang, Jun Gu, Jun Wan, Ziyao Zhao, Peicheng Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8806647/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Piezocatalytic therapy (PCT) has garnered increasing interest in the field of cancer treatment. However, its therapeutic efficacy is hampered by the limitations of current piezoelectric materials and the intrinsic therapeutic resistance of tumors. Here, leveraging elaborately designed copper-coordinated covalent organic frameworks (CuCOF-2N) as novel piezocatalysts, a smart ultrasound-controlled nanoreactor (Cu2N@D-FA) is constructed by co-encapsulating with doxorubicin prodrug into folic acid-modified liposomes, to enhance the antitumor efficacy of PCT through the combination of cuproptosis and bioorthogonal catalysis. The structure-property comparison with related COFs underscores the important role of highly polar triazine rings and symmetry-disrupting bidentate ligands in enhancing piezoelectric performance of CuCOF-2N. Upon ultrasound irradiation, CuCOF-2N not only produces hydroxyl radicals independently of oxygen but also enables a self-sustained oxygen supply for generating superoxide anions and singlet oxygen, which surmount constraints of traditional piezoelectric materials to trigger strong PCT. Moreover, the mediated-copper valence switching in CuCOF-2N permits spatiotemporally precise bioorthogonal catalysis and triggers cuproptosis, overcoming therapeutic limitations and amplifying the PCT effect. In vivo studies demonstrate that Cu2N@D-FA selectively accumulates in the tumor and effectively eliminates the tumor without side-effects under US stimulation. Therefore, this study highlights the great promise of COFs in piezocatalysis and offers novel insights for enhancing PCT. Materials Chemistry covalent organic framework bioorthogonal chemistry cuproptosis piezocatalytic therapy tumor treatment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Piezocatalytic therapy (PCT) has emerged as a promising ultrasound (US) mediated antitumor strategy, distinguished by its noninvasiveness, deep tissue penetration, and low oxygen dependence. 1 Unlike conventional sonodynamic therapy (SDT), which is limited by rapid charge recombination, 2 PCT utilizes the intrinsic piezoelectric field generated under ultrasonic mechanical stress to dramatically improve electron-hole (e⁻-h⁺) pair separation efficiency, thereby facilitating robust reactive oxygen species (ROS) generation, including hydroxyl radicals (·OH), superoxide anions (O 2 •− ), and singlet oxygen ( 1 O 2 ). 3 However, current research on piezoelectric antitumor materials remains largely confined to inorganic semiconductors (such as BaTiO 3 , 4 ZnO, 5 ZnIn 2 S 4 , 3 g-C 3 N 4 , 1 and 2D Bi-based materials oxygen-vacancy-rich BiO 2−x nanosheets 6 ), which still face critical limitations including suboptimal piezoelectric response, insufficient ROS production, and compromised biocompatibility. 7 Therefore, there is an urgent need to develop novel piezoelectric sonosensitizers with strong piezoelectric response and favorable biocompatibility, while simultaneously devising multimodal synergistic strategies to counteract the intrinsic defense mechanisms of cancer, enhancing the clinical translation potential of PCT. Covalent organic frameworks (COFs), an emerging class of porous crystalline materials, have recently been recognized as attractive candidates for piezoelectric materials owing to their precisely designable non-centrosymmetric architectures, programmable band gap structures, and superior charge transport properties. 8 COFs demonstrate unique advantages over conventional inorganic piezoelectric materials. For instance, their porous structures readily undergo deformation under ultrasonic mechanical stress, amplifying local polarization effects to enhance piezoelectric response. 9 In addition, the conjugated frameworks and nitrogen-rich moieties (such as triazine rings) may facilitate charge separation and transfer, thereby boosting ROS generation efficiency. 10 Particularly, their symmetry can be further broken through metal single-atom anchoring or donor-acceptor (D-A) structural design to optimize piezocatalytic activity. 11 – 13 Importantly, their inherent biocompatibility and biodegradability provide safety assurance for their in vivo application. 14 Although piezoelectric applications based on COFs remain in nascent stages, they have demonstrated considerable promise in US-controlled ROS production and the construction of multimodal therapeutic platforms, which opens avenues for creating news piezoelectric sonosensitizers with higher piezoelectric responsiveness and lower systemic toxicity to enhance piezocatalytic treatment efficacy. Metal homeostasis represents a fundamental biological regulatory network, with copper ions serving an essential role in critical physiological processes including enzymatic catalysis and intracellular signaling. 15 However, tumor cells often hijack metal metabolic pathways to meet their abnormal proliferation needs, and this “metal addiction” characteristic makes metal homeostasis a highly promising anti-cancer target. 16 In recent years, studies have found that cuproptosis exerts anti-tumor effects by disrupting the mitochondrial TCA cycle and triggering proteotoxic stress. 17 Noatbly, cuproptosis is fundamentally distinct from most other forms of programmed and non-programmed cell death, which demonstrates significant promise in overcoming resistance to conventional therapies. 18 In fact, multiple studies have indicated that cuproptosis can amplify the antitumor efficacy of ROS-dependent therapies, including SDT, photodynamic therapy (PDT), and chemodynamic therapy (CDT), through various molecular events such as ROS accumulation, proteotoxic stress, and metabolic regulation. 19 – 24 These mechanistic synergies suggest that cuproptosis can serve as an ideal adjunctive therapy to enhance PCT. To fully exploit the synergistic potential of combining PCT and cuproptosis while minimizing side effects, the precise regulation of copper ions in vivo is crucial. Bioorthogonal catalysis enables the execution of exogenous chemical reactions within complex biological systems without interfering with endogenous physiological processes, offering an unprecedented solution to this challenge. 25 Notably, recent studies have demonstrated that US can drive bioorthogonal catalysis to achieve valence transition of transition metal ions, which enables the remote and precise regulation of copper ions to induce cuproptosis. 26 – 28 Importantly, bioorthogonal catalysis can also achieve the in situ activation of biomolecules and the specific deprotection of prodrugs. 29 This capability holds promise for creating multi-dimensional synergy with PCT, further enhancing the antitumor efficacy of the combination. Therefore, integrating bioorthogonal catalysis, cuproptosis, and piezocatalytic therapy may offer a rational design strategy for enhancing therapeutic efficacy. Here, by incorporating nitrogen-rich monomers and anchored metal designs into COFs, we successfully constructed piezocatalytically active frameworks (CuCOF-2N) and combined it with a DOX prodrug (DOX-Pro) and folic acid (FA)-modified liposomes further developed an intelligent US-controlled nanoreactor (Cu2N@D-FA) for enhancing antitumor efficacy of PCT by combining cuproptosis with bioorthogonal catalysis. Notably, the comparative study of structurally related COFs underscores the crucial roles of highly polar triazine rings and symmetry-breaking bidentate ligands in enhancing the piezoelectric catalytic performance of CuCOF-2N. The predesigned CuCOF-2N, as a novel piezoelectric catalyst, could break through oxygen limitations to generate massive ·OH under ultrasonic excitation. Moreover, CuCOF-2N could autonomously regulate the tumor microenvironment to enhance generation of O 2 •− and 1 O 2 by catalyzing endogenous H 2 O 2 to produce oxygen. This allows Cu2N@D-FA to overcome the limitations of convertional piezoelectric materials and triggers strong PCT. Most importantly, CuCOF-2N enabled US-intelligent regulation of copper valence conversion, allowing for spatiotemporally controllable bioorthogonal catalysis and the induction cuproptosis, which synergistically enhances the antitumor activity of PCT. To our knowledge, this work represents the first successful application of COF materials in piezocatalytic therapy and pioneers the “one stone, two birds” utilization of copper ions simultaneously driving bioorthogonal catalysis and inducing cuproptosis, thereby establishing a novel paradigm for precision oncotherapy. 2. Results and Discussion 2.1. Synthesis and characterization of COFs To endow COF frameworks with intrinsic piezoelectric properties, we predesigned nitrogen-rich triazine ring TTA (4,4’,4’’-(1,3,5-triazine-2,4,6-triyl)trianiline) as the amino ligand, with TAPB (1,3,5-tris(4-aminophenyl)benzene) as the benzene-ring control. Besides, in order to increase metal active sites, we strategically selected the bidentate ligand DBPy (6,6’-diformyl-3,3’-bipyridine) as an aldehyde ligand for anchoring copper ions (Cu(OTf) 2 ), and BPA (2,2'-bipyridine-5,5’-dicarboxaldehyde) as a monodentate ligand. Through systematic combination of these ligands, we successfully constructed four piezoelectric-responsive Cu-coordinated COFs (Fig. 2 a) via Schiff-base condensation: CuCOF-1C(TAPB-BPA), CuCOF-1N(TTA-BPA), CuCOF-2C(TAPB-DBPy), and CuCOF-2N(TTA-DBPy). The specific reaction routes can be found in Scheme S1 and S2 . Notably, all four COFs can be synthesized in only ten minutes and produced on a gram scale. The chemical structures of the synthesized COFs were verified by Fourier transform infrared spectroscopy (FT-IR). As shown in Fig. 2 b and Figure S1 , the characteristic absorption peak corresponding to the stretching vibration of the imine bond (C = N) at approximately 1590 cm − 1 was observed, while the disappearance of the C = O stretching vibration peak at 1700 cm − 1 further confirmed the successful formation of imine linkage. 30 , 31 X-ray photoelectron spectroscopy (XPS) analysis revealed that these COFs were primarily composed of four elements: C, N, O, and Cu (Fig. 2 c). The high-resolution of Cu 2p confirmed the existence of Cu 2+ ( Figure S2 ). N 2 adsorption-desorption measurements at 77 K demonstrated the porous nature of the Cu-coordinated COFs, with Brunauer-Emmett-Teller (BET) specific surface areas calculated to be 407 m 2 ∙g − 1 (CuCOF-1C), 621 m 2 ∙g − 1 (CuCOF-1N), m 2 ∙g − 1 (CuCOF-2C), and m 2 ∙g − 1 (CuCOF-2N), respectively ( Figure S3 ). Powder X-ray diffraction (PXRD) analysis confirmed the high crystallinity of all four COFs (Fig. 2 d), with experimental diffraction patterns showing excellent agreement with the simulated data based on the A-A stacking model. 32 Optimized structures with the AA stacking mode were shown in Figure S4 . Reasonable unit cell parameters were obtained through Pawley refinement ( Table S3-6 ). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed these Cu-coordinated COFs can be converted into nanosheets from bulk COFs via simple ultrasonic exfoliation (Fig. 2 e,f). Energy-dispersive X-ray spectroscopy (EDX) elemental mapping confirmed the homogeneous distribution of C, N, O, and Cu throughout the CuCOF-2N (Fig. 2 g). Inductively coupled plasma mass spectrometry (ICP-MS) was used to detect the Cu content in these COFs, which was 5.9 wt.% for CuCOF-1C, 5.5 wt.% for CuCOF-1N, 8.3 wt.% for CuCOF-2C, and 8.1 wt.% for CuCOF-2N, respectively. 2.2. Piezoelectricity and band structure of COFs Next, we employed scanning probe piezoelectric force microscopy (PFM) to evaluate the piezoelectric properties of the Cu-coordinated COFs. The polarization behavior of piezoelectric domains was clearly identified through the amplitude and phase patterns of Cu-coordinated COFs ( Figure S5 ), confirming their intrinsic piezoelectric characteristics. Notably, upon the application of a ± 10 V bias voltage, these COFs displayed a characteristic butterfly-shaped amplitude loop and an approximately 180° phase-reversal hysteresis loop (Fig. 3 a), which are typical features of piezoelectric materials. 33 From the amplitude loop, CuCOF-2N had amplitude of 3.0 nm, while the amplitudes of CuCOF-1C, CuCOF-1N, and CuCOF-2C were 1.0 nm, 1.7 nm, and 2.0 nm, respectively, indicating that CuCOF-2N possessed a stronger piezoelectric response. 10 The enhanced piezoelectric property can be attributed to the strong polarity of the triazine rings, which promotes the formation of intrinsic dipole moments, while the increased copper coordination sites further disrupt the structural symmetry. To explore the influence of piezoelectric properties on the band structure, the band structures of Cu-coordinated COFs were analyzed using ultraviolet-visible diffuse reflectance spectroscopy (UV-vis DRS) and valence band X-ray photoelectron spectroscopy (VB-XPS). Figure S6 shows that all four COFs exhibited a broad light absorption range in the UV-vis region, while CuCOF-2C/2N further broadened this absorption. Tauc plots derived from (αhν)² versus photon energy (hν) reveal that the bandgap energy ( E g ) of CuCOF-1C, CuCOF-1N, CuCOF-2C, and CuCOF-2N were determined to be 2.12 eV, 2.3 eV, 1.99 eV, and 2.11 eV, respectively ( Figure S7 ). The VB-XPS results indicate that the valence band potential ( E VB, XPS ) of CuCOF-1C, CuCOF-1N, CuCOF-2C, and CuCOF-2N were 1.36 eV, 1.23 eV, 0.93 eV, and 1.10 eV, respectively ( Figure S8 ). Based on the E g and E VB,XPS data, the band position can be determined. Figure 3 b schematically illustrates the intrinsic and tilted band structures of the Cu-coordinated COFs under US irradiation. The intrinsic band diagram reveals that all four COFs can reduce O 2 to O 2 •− under ultrasonic irradiation, which subsequently may react with holes to generate 1 O 2 . Notably, the inherent band characteristics of these COFs intrinsically limit the production of ·OH. However, CuCOF-2C/2N, with exceptional piezoelectric properties, demonstrated obvious band tilting under US irradiation, which allowed CuCOF-2C/2N to oxidize water to produce ·OH. According to the band theory, piezoelectric materials can generate directional electric fields under mechanical excitation, which drives charge carriers toward the material surface. 34 To evaluate the charge separation abilities of Cu-coordinated COFs, electrochemical impedance spectroscopy (EIS) and time-resolved fluorescence (TRF) spectroscopy were employed. EIS measurements reveal that CuCOF-1C/1N exhibited a larger arc radius compared to CuCOF-2C/2N ( Figure S9 ), which could be attributed to their relatively wider bandgap. Meanwhile, TRF spectroscopy demonstrates that the average radiative lifetimes of the Cu-coordinated COFs were 1.76 ns, 2.72 ns, 2.72 ns, and 3.36 ns, respectively ( Figure S10 ). The prolonged fluorescence lifetime observed in CuCOF-2N suggested increased electron-hole pairs. These results demonstrate that the meticulously designed triazine ring and copper site effectively inhibit recombination of electron-hole pairs and enhance carrier transport, thereby improving piezocatalytic activity. 2.3. US-mediated ROS generation of COFs The tumor microenvironment (TME) is characterized by hypoxia, mild acidity, and elevated H 2 O 2 levels. 35 However, the generation of O 2 •− and 1 O 2 is oxygen-dependent. Considering the ability of Cu 2+ to decompose H 2 O 2 for O 2 generation, we evaluated the CAT-like activity of Cu-coordinated COFs by measuring dissolved oxygen generation during H 2 O 2 decomposition. As shown in FigureS11 , all Cu-coordinated COFs exhibited significant dissolved oxygen production, demonstrating their remarkable CAT-like activity. The introduction of US can improve the amount and rate of O 2 generation. This property can effectively overcome the oxygen-dependent limitation of conventional sonodynamic therapy, suggesting great potential for therapeutic applications in hypoxic TME. Subsequently, we investigated the ROS generation capability of Cu-coordinated COFs under US irradiation using 1,3-diphenylisobenzofuran (DPBF) fluorescence probe, which undergoes a characteristic color change from yellow to colorless upon reaction with ROS. 36 As expected, DPBF absorbance decreased significantly after 5 min of US irradiation (1 W·cm − 2 , 1 MHz, 50% duty cycle). Particularly, CuCOF-2C/2N exhibited more pronounced absorbance reduction (Fig. 3 c and Figure S12 ), which could be attributed to their narrower bandgap. Furthermore, we employed specific fluorescent probes, including singlet oxygen sensor green (SOSG), 37 dihydroethidium (DHE), 38 and 3,3’,5,5’-tetramethylbenzidine (TMB), 39 to detect the production of 1 O 2 , O 2 •− , and ·OH, respectively. As shown in FigureS13 , the fluorescence intensity of DHE and SOSG gradually increased with prolonged US irradiation time, indicating the generation of O 2 •− and 1 O 2 . Notably, CuCOF-2C/2N demonstrated stronger fluorescence signals than CuCOF-1C/1N, consistent with the DPBF results. In addition, the absorbance of TMB in the CuCOF-1C/1N group remained nearly unchanged under US irradiation, whereas that in CuCOF-2C/2N increased significantly (Fig. 3 d). This observation indicated that US-induced band structure modification in CuCOF-2C/2N enabled direct water oxidation to generate ·OH, with CuCOF-2N showing superior ·OH yield compared to CuCOF-2C. Notably, CuCOF-2N still generated a substantial amount of ·OH even under oxygen-deficient condition (Fig. 3 e), which indicated that the generation of ·OH is independent of oxygen participation. These results demonstrate that the energy band tilting caused by the excellent piezoelectric property of CuCOF-2N enabled the generation of an additional type of radical, which endowed CuCOF-2N with a unique advantage for its application in the antitumor therapy. Electron spin resonance (ESR) spectroscopy was employed to further characterize the ROS species generated under US irradiation. Using 2,2,6,6-tetramethylpiperidine (TEMP) as the spin trap, we detected the distinctive 1:1:1 triplet signal corresponding to 1 O 2 formation (Fig. 3 f). Furthermore, when using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trap, the six peaks shown in Fig. 3 g demonstrated the O 2 •− generation. Besides, we observed the characteristic signal with an intensity ratio of 1:2:2:1 (Fig. 3 h), which confirmed the ·OH production of CuCOF-2N under US irradiation. 2.4. ROS generation mechanism revealed by DFT calculation We performed density functional theory (DFT) calculations on the electron transfer in Cu-coordinated COFs and the possible mechanism of ROS generation under US irradiation (Fig. 4 a and b ). The generation mechanisms of O 2 •− and 1 O 2 were first investigated. The Gibbs free energy of the products is lower than that of the reactants, and the energy drop further confirmed that the reaction favors the generation of ROS. 40 Under US irradiation, COFs are excited to the singlet excited state (COFs*) and may generate electron-hole (e⁻-h⁺) pairs. The resulting e⁻ may be captured by O 2 to generate O 2 •− . Furthermore, O 2 •− is likely oxidized by holes to produce 1 O 2 . As shown in Fig. 4 d, compared to CuCOF-1C/1N, CuCOF-2C/2N is more favorable for the generation of O 2 •− and 1 O 2 . Next, we investigated the mechanism of ·OH generation by CuCOF-2C/2N. Firstly, CuCOF-2C/2N exhibited excellent H 2 O absorption capacity, which ensures efficient capture of H 2 O molecules at the active sites. Subsequently, H 2 O captures holes to generate ·OH. 41 The energy required for CuCOF-2N to generate ·OH is 1.93 eV, which is significantly lower than that of CuCOF-2C (2.3 eV) (Fig. 4 e). This indicates that CuCOF-2N is more favorable for the generation of ·OH under US irradiation. Based on these findings, it can be inferred that the mechanism of ROS generation involves the absorption of O 2 and H 2 O, the formation of O 2 •− via ultrasonically induced electrons, followed by the generation of 1 O 2 and ·OH through oxidation reactions (Fig. 4 c). 2.5. US-mediated copper valence transition for bioorthogonal catalysis To investigate whether US irradiation can induce the generation of Cu(I) active sites, Cu(I)-specific chelating agent neocuproine was used as an indicator (Fig. 4 f). As expected, yellow chromophore exhibiting a characteristic absorption maximum at 452 nm (Fig. 4 g) was detected, corresponding to the Cu(I)-neocuproine complex [Cu(neocuproine) 2 ] + , which provided strong evidence for the formation of Cu(I) active sites on the Cu-coordinated COFs after US irradiation. Notably, CuCOF-2N demonstrated a significantly higher Cu(I) content compared to other COFs. Furthermore, the characteristic absorption peak of chromophore showed a marked enhancement with increasing US irradiation time or power, indicating the generation of Cu(I) active sites is dependent on US power and time ( Figure S14 ). Shao et al. synthesized CuO-PMA nanosheets and utilized their electron delocalization property: US triggered the directional migration of electrons in CuO-PMA, facilitating the conversion of Cu(II) to Cu(I). 28 They attributed this to the enhanced orbital overlap exhibited by sub-nanoscale materials due to surface atomic rearrangement, which promotes electron delocalization between surface atoms, and the delocalization can be traced to photoinduced electron transfer. 42 , 43 Therefore, in situ XPS measurements were performed under xenon lamp irradiation. After 10 min of light irradiation, high-resolution Cu 2p spectra revealed a significant increase in Cu(I), which confirmed that photoexcited electrons induced charge accumulation at Cu sites and a subsequent valence transition, providing direct evidence for electron transfer in CuCOF-2N ( Figure S15 ). In addition, static XPS analyses were carried out on CuCOF-2N before and after US treatment, and the results were consistent with the in situ XPS results (Fig. 4 h). These findings not only illustrated the favorable responsiveness of Cu-coordinated COFs to external stimuli such as US and light, but also reaffirmed that upon ultrasonic stimulation-induced electron-hole separation, electrons migrate to Cu(II) sites, ultimately leading to a change in Cu valence, which provides possibility for bioorthogonal catalysis. Previous studies have reported that Cu(I) exhibits favorable bioorthogonal catalytic properties, enabling the creation of powerful multifunctional biocatalysts for biomedical applications. 44 Therefore, we subsequently evaluated the in vitro bioorthogonal catalytic effect of Cu-coordinated COFs using a fluorescence-quenched coumarin derivative (cd-Pro) as the reaction substrate. The synthetic route of cd-Pro was shown in Scheme S3 . After 5 min of US irradiation, the cleavage efficiency was evaluated by measuring the fluorescence recovery over a period of 24 h. The fluorescence signal gradually increased over time, with CuCOF-2N demonstrating the highest catalytic efficiency ( Figure S16 ). Doxorubicin (DOX) is a widely used drug for treating tumors, but its clinical utility is limited by non-specific targeting and considerable side effects. 45 Substantial research has been directed toward developing prodrug strategies to reduce the systemic toxicity of DOX. 46 Thus, we synthesized a DOX prodrug (DOX-Proc) by protecting its amino group ( Scheme S4 ). We further confirmed that US-triggered CuCOF-2N induced the conversion of DOX-Proc to DOX by monitoring high-performance liquid chromatography (HPLC). Figure 4 i and Figure S17 show that CuCOF-2N demonstrated the highest catalytic efficiency, with a DOX yield of 80% within 24 h. 2.6. Preparation and characterization of intelligent US-controlled nanoreactor Given the excellent US-driven piezoelectric property and bioorthogonal catalytic capability of CuCOF-2N, we further constructed an intelligently US-responsive nanoreactor, termed Cu2N@D-FA, consisting of CuCOF-2N, DOX-Proc, and FA-modified lipids, which was prepared via the thin-film hydration method. 47 As shown in Figure S18 , TEM revealed a spherical morphologies, while dynamic light scattering (DLS) measurements indicated an average size of 135.12 nm ( Figure S19 ) and a zeta potential of -16.28 mV ( Figure S20 ). Importantly, Cu2N@D-FA exhibited excellent colloidal stability over 48 hours in various media (deionized water, PBS, DMEM, 10% FBS-DMEM), with no significant changes in particle size, indicating biological applications potential ( Figure S21 ). Furthermore, HPLC measurements indicated a drug loading rate (LR) of 25.8% for DOX-Proc in Cu2N@D-FA ( Table S1 ), confirming the successful encapsulation of DOX-Proc within the Cu2N@D-FA. In addition, the control nanoreactors based on the CuCOF-1C, CuCOF-1N, and CuCOF-2C, namely Cu1C@D-FA, Cu1N@D-FA, and Cu2C@D-FA, as well as the control nanocomposites based on CuCOF-2N, including Cu2N@D-PEG (composed of CuCOF-2N, DOX-Proc, and PEG-modified lipids) and Cu2N-FA (composed of CuCOF-2N and FA-modified lipids), were prepared by the same method. The characterization data for them are detailed in Table S1 . 2.7. In vitro antitumor effect The mammary carcinoma cell line 4T1 was used to investigate the in vitro therapeutic efficacy of Cu2N@D-FA. The folate receptor (FR) is a membrane protein overexpressed in tumor cells. Folate (FA) is regarded as a promising and highly efficient tumor-targeting agent, primarily attributed to its strong interaction with FR. 48 Therefore, we first examined whether FA could enhance its cellular uptake in cancer cells, with Cu2N@D-PEG (PEG instead of FA) serving as the control. We fluorescently labeled Cu2N@D-PEG and Cu2N@D-FA using fluorescein isothiocyanate (FITC), followed by culturing with 4T1 cells and monitoring with fluorescence microscopy. As shown in Fig. 5 a and Figure S22 , the fluorescence signals of 4T1 cells treated with Cu2N@D-PEG and Cu2N@D-FA reached a plateau at 8 h, respectively. Importantly, the accumulated concentration of Cu2N@D-FA in 4T1 cells was 1.3-fold higher than that of Cu2N@D-PEG after 12h incubation, indicating that surface functionalization with FA promoted the cellular uptake of the nanoreactor in 4T1 cells. Notably, the superior lysosomal escape capability of nanoparticles is crucial for exerting antitumor effects. 49 Benefiting from the piezoelectric property of Cu-incorporated COFs, ROS generated by US irradiation in endolysosomes can enhance the permeability of the endolysosomal membrane and facilitate the escape of Cu2N@D-FA from these vesicles. Therefore, the US-driven endolysosomal escape performance of Cu2N@D-FA was evaluated. After 8 hours of incubation with FITC-labeled Cu2N@D-FA, 4T1 cells were treated with US irradiation for 3 min (1 W·cm − 2 , 1 MHz, 50% duty cycle). Subsequently, endolysosomes were stained with LysoTracker™ Red and visualized using confocal laser scanning microscopy (CLSM). Without US stimulation, the LysoTracker™ Red signal showed extensive overlap with FITC green fluorescence (Fig. 5 b), yielding a Pearson’s correlation coefficient (Rr) of 0.85 (Fig. 5 c), indicating that Cu2N@D-FA was largely trapped within endolysosomes. In contrast, the FITC signal diffused uniformly throughout the cytoplasm under US irradiation, and the degree of colocalization was significantly reduced (Rr = 0.22), demonstrating that US effectively promoted the endolysosomal escape of Cu2N@D-FA. We further assessed the antitumor effects of Cu2N@D-FA using the methyl thiazolyl tetrazolium (MTT) assay, with Cu1C@D-FA, Cu1N@D-FA, Cu2C@D-FA, and Cu2N-FA serving as controls. As illustrated in Fig. 5 d, the modified DOX prodrug (DOX-Proc) exhibited reduced cytotoxicity (half-maximal inhibitory concentration, IC 50 = 7.9 µM) compared to DOX (IC 50 = 0.15 µM). This dramatic reduction led to negligible cytotoxicity for all formulations in the absence of US irradiation (Fig. 5 e). In contrast, they demonstrated dose-dependent antitumor effects under US exposure (Fig. 5 f). The IC 50 of Cu2N@D-FA (56.2 µg · mL − 1 ) was significantly lower than those of Cu1C@D-FA (162.4 µg·mL⁻¹), Cu1N@D-FA (153.3 µg·mL⁻¹), and Cu2C@D-FA (94.8 µg·mL⁻¹), indicating its superior antitumor potency. Subsequently, we evaluated the intracellular bioorthogonal catalytic performance of Cu2N@D-FA. As shown in Figure S23 , US irradiation endows nanoreactors with bioorthogonal catalytic activity. Notably, a stronger DOX fluorescence intensity was detected in 4T1 cells treated with Cu2N@D-FA, indicating more efficient cleavage of DOX-Proc and superior bioorthogonal catalytic performance. Importantly, Cu2N@D-FA induced significantly higher cytotoxicity than Cu2N-FA, suggesting that bioorthogonal catalysis effectively enhances PCT (Fig. 5 g). Furthermore, live/dead staining was performed to evaluate the antitumor effects of Cu2N@D-FA. As shown in Fig. 5 h and Figure S24 , compared with other groups, Cu2N@D-FA showed the most remarkable antitumor efficacy, which was consistent with the MTT results, further indicating the favorable bioorthogonal catalytic performance of Cu2N@D-FA significantly enhances its piezocatalytic antitumor activity. To verify the anticancer role of cuproptosis, we validated Cu2N@D-FA-mediated cell death. The intracellular concentration of copper ions after different treatments was detected using Rhodamine B hydrazide (RBH), and the red fluorescence intensity was proportional to the copper ion concentration. 50 FigureS25 indicates that the intracellular copper ion level was significantly elevated after US irradiation. Cuproptosis is characterized by the aggregation of lipoylated proteins and the loss of Fe-S cluster proteins in the mitochondria. 22 Immunofluorescence results reveal that all drugs exhibited obvious aggregation of lipoylated protein Dihydrolipoamide dehydrogenase (DLAT), as observed via confocal microscopy ( Figure S26 ), confirming the contribution of cuproptosis on nanoreactor-induced cell death. Additionally, western blot analysis shows Cu2N@D-FA US+ effectively induced the loss of Fe-S cluster proteins in 4T1 cells, including ferredoxin 1 (FDX1) and lipoyl synthase (LIAS) (Fig. 5 i and Figure S27 ), indicating Cu2N@D-FA can enhance the combined antitumor efficacy of cuproptosis and piezocatalytic therapy. Hypoxia is one of the features of the tumor microenvironment, while the generation of ROS critically relies on the involvement of oxygen. Copper ion offers a promising solution to overcome this hypoxia barrier by catalyzing the conversion of endogenous hydrogen peroxide into oxygen. To monitor intracellular oxygen levels during treatment, we employed the oxygen-sensitive probe tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride ([Ru(dpp) 3 ]Cl 2 , RDPP), which exhibits increased fluorescence quenching in response to elevated oxygen concentrations. 51 The control group displayed the strongest red fluorescence, whereas the treatment group showed lower red fluorescence intensity, providing direct evidence of the self-supplied oxygen capability ( Figure S28 ). We also employed the fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) to investigate the generation of ROS in 4T1 cells treated with different formulations. As shown in Fig. 5 j, only minimal ROS was produced in 4T1 cells without US irradiation, which may be attributed to their modest chemodynamic effect. In contrast, Cu2N@D-FA US+ group exhibited the strongest green fluorescence signal, which was 1.71-fold, 1.97-fold, 1.33-fold and 1.07-fold higher than that of Cu1C@D-FA, Cu1N@D-FA, Cu2C@D-FA, and Cu2N-FA ( Figure S29 ), respectively, demonstrating its superior ROS-generating capability. To identify the specific ROS types, we used three fluorescent probes: Singlet Oxygen Sensor Green (SOSG) for 1 O 2 , dihydroethidium (DHE) for O 2 •− , and hydroxyphenyl fluorescein (HPF) for ·OH. As shown in Figure S30 and S31 , 1 O 2 and O 2 •− were observed, indicating their band structures endowed them intrinsic ROS generation capacity under US irradiation. In the absence of US irradiation, minimal ·OH was observed. However, Cu1C@D-FA and Cu1N@D-FA showed no significant change in ·OH generation with or without US irradiation. Remarkably, Cu2N@D-FA US+ group produced more ·OH than that of Cu2C@D-FA ( Figure S32 ). These results suggest that CuCOF-2N possesses excellent piezoelectric property, which can alter its band structure and facilitate water oxidation to generate ·OH, confirming its promising potential for antitumor application. The accumulation of intracellular ROS can trigger severe mitochondrial damage and a reduction in mitochondrial membrane potential (MMP). Therefore, we employed 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolocar bocyanine iodide (JC-1) assay to evaluate the ability of Cu2N@D-FA to induce MMP changes in 4T1 cells. As shown in Fig. 5 k, 4T1 cells exhibited strong red fluorescence and minimal green fluorescence in the absence of US treatment, indicating intact mitochondria with high MMP. Under US irradiation, 4T1 cells showed a significant increase in green fluorescence, suggesting MMP depolarization. Notably, Cu2N@D-FA induced the most pronounced decrease in MMP, with green fluorescence intensities approximately 2.1-fold, 1.8-fold, 1.2-fold, and 1.1-fold higher than that in Cu1C@D-FA, Cu1N@D-FA, Cu2C@D-FA, and Cu2N-FA groups (Fig. 5 m), respectively. These results demonstrated that Cu2N@D-FA efficiently promoted mitochondrial damage through the combination of PCT, cuproptosis, and bioorthogonal catalysis. 2.8. In vivo antitumor effect Given the excellent in vitro antitumor activity of Cu2N@D-FA, we further evaluated its efficacy in vivo . We first investigated its pharmacokinetic profile and tumor-targeting capability. Free DOX or Cu2N@D-FA was intravenously injected into rats, followed by monitoring drug concentrations in blood at different time points. As shown in Fig. 6 a, DOX was rapidly cleared from the bloodstream, whereas DOX delivered via Cu2N@D-FA exhibited a significantly prolonged circulation time. Detailed pharmacokinetic parameters were summarized in Table S2 . The AUC 0−inf and MRT 0−inf of DOX in Cu2N@D-FA were 174.8 µg/mL·h and 23.5 h (Fig. 6 b), respectively, which were 17-fold and 10.7-fold longer than that of free DOX, indicating that Cu2N@D-FA combines the advantages of both prodrug and liposomal formulations, substantially extending the systemic circulation duration of the drug. The tumor-targeting capacity of Cu2N@D-FA was evaluated in 4T1 tumor bearing mice. DiR-labeled Cu2N@D-FA or Cu2N@D-PEG was intravenously injected, and in vivo fluorescence was monitored for 24 hours using a Maestro EX imaging system. Both formulations exhibited time-dependent tumor accumulation, with fluorescence signals peaking at 12 hours post-injection (Fig. 6 c). Notably, Cu2N@D-FA demonstrated superior tumor accumulation, showing a 1.58-fold higher fluorescence intensity at 12h compared to Cu2N@D-PEG (Fig. 6 d), highlighting the critical role of FA-mediated active targeting in enhancing tumor specific accumulation. After 24 h of administration, the tumor and major organs were dissected to further characterize the biodistribution of Cu2N@D-FA. As shown in Fig. 6 e and Fig. 6 f, Cu2N@D-FA was rapidly cleared from major organs and selectively accumulated in tumor tissue, confirming its excellent tumor-targeting efficiency and favorable tissue selectivity. Next, to evaluate the in vivo antitumor efficacy of Cu2N@D-FA, the subcutaneous 4T1 bearing mice were randomly divided into four treatment groups: Saline US+, DOX, Cu2N-FA US+, and Cu2N@D-FA US+. As illustrated in Fig. 7 a, the subcutaneous 4T1 bearing mice were intravenously injected drugs every two days for 14 days. In the US treatment group, mice received 3 min of US irradiation (1 W·cm − 2 , 1 MHz, 50% duty cycle) at 12 h post-injection. Figure 7 b-d show that the Cu2N@D-FA US+ group exhibited the most potent antitumor effect, significantly outperforming the therapeutic effect of free DOX. The tumor size in the Cu2N@D-FA US+ treatment group was approximately 26.6-, 21-, and 7.4-fold smaller than that in Saline US+, DOX, and Cu2N-FA US+ groups, respectively, confirming US-driven cuproptosis and bioorthogonal catalysis can overcome intrinsic tumor resistance and substantially enhance the PCT efficacy. Notably, despite the low-dose regimen, a significant reduction in body weight was observed in the DOX-treated mice (Fig. 7 e). However, the body weight of mice treated with Cu2N-FA US + and Cu2N@D-FA US+ showed no significant difference compared to control mice. These results confirmed the prodrug was well tolerated and significantly reduced the systemic toxicity of DOX. Furthermore, Cu2N@D-FA did not cause pathological abnormalities in major organs (heart, liver, spleen, lungs, and kidneys), indicating its satisfactory biocompatibility ( Figure S33 ). Importantly, mice treated with Cu2N@D-FA US+ also demonstrated the longest survival time among all groups (Fig. 7 f). Finally, immunofluorescence and immunohistochemistry were conducted to evaluate the in vivo antitumor efficacy of Cu2N@D-FA (Fig. 7 g). HIF-1α protein expression level in tumor tissues indicated Cu2N@D-FA significant alleviated tumor hypoxia, as evidenced by a markedly lower green fluorescence intensity in the Cu2N@D-FA group compared to the control group. Besides, Cu2N@D-FA led to decreased expression of FDX1 and LIAS, along with aggregation of DLAT in tumor tissue, confirming the activation of cuproptosis in vivo . Importantly, Cu2N@D-FA generated the highest level of ROS among the groups, suggesting that US-mediated multidimensional combination therapy significantly increases oxidative stress in tumor tissues. Furthermore, hematoxylin and eosin (H&E) staining and TUNEL assays confirmed that Cu2N@D-FA significantly induced apoptosis in cancer cells. These results underscore the therapeutic advantage of combining US-induced piezoelectric effects with bioorthogonal catalysis and cuproptosis. Thus, Cu2N@D-FA represents a novel combinatorial antitumor platform capable of effectively inhibiting tumor proliferation. 3. Conclusion In summary, we successfully constructed a smart US-controlled nanoreactor (Cu2N@D-FA) comprising novel piezoresponsive COFs (CuCOF-2N), DOX prodrug (DOX-Proc) and folic acid (FA)-modified liposomes, which enhances PCT by synergizing cuproptosis with bioorthogonal catalysis. Notably, the comparative study of structurally related COFs highlighted the significance of strongly polar triazine rings and symmetry-disrupting bidentate ligands in enabling CuCOF-2N to overcome the limitations of conventional piezoelectric materials and enhance piezoelectric catalytic performance. Under US irradiation, Cu2N@D-FA could generate ·OH independent of oxygen, while also enabling self-supplied oxygen for the production of O 2 •− and 1 O 2 , triggering strong PCT. Moreover, US triggered directional electron migration in CuCOF-2N, facilitating the reduction of Cu(II) to Cu(I), and thereby in-situ activated the DOX prodrug to achieve bioorthogonal catalysis. Additionally, the accumulated copper ion in the mitochondrion could further induce aggregation of lipoylated proteins and the loss of Fe-S cluster proteins, thereby activating the cuproptosis pathway. In vivo studies confirmed that Cu2N@D-FA exhibited excellent biocompatibility and selectively accumulated in tumor tissue after intravenous administration, resulting in significant tumor suppression. Therefore, this work not only reveals the structure-property relationship of piezoelectric COFs in US-driven catalysis but also proposes a novel strategy that augments PCT efficacy by coupling bioorthogonal catalysis with metal-induced cell death to circumvent treatment barriers, providing new insights for the development of smart responsive nanomedicines. 4. Experimental Section/Methods All materials, methods, and additional data can be found in Supporting Information. All animal experiments were conducted in compliance with the National Institute of Health Guidelines under protocols approved by the Animal Ethical and Welfare Committee of Nanjing University of Science and Technology (Approval No: ACUC-NUST2023012). Declarations AUTHOR INFORMATION Corresponding Author Yu Zhang: School of Chemistry and Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China. Email: [email protected] Shujun Feng : State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, Nanjing, 210009, China E-mail: [email protected] Jun Luo: School of Chemistry and Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China. Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT C.H. and Y.Z. contributed equally to this work. The work was supported by the National Natural Science Foundation of China (22301136, 82502555, and 22075144), the Natural Science Foundation of Jiangsu Province (BK20230942 and BK20251560) and the Fundamental Research Funds for the Central Universities (No.30924010910). Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. References Yang Z et al (2025) Dual-Defect Regulated G-C 3 N 4 for Piezoelectric Catalytic Tumor Therapy with Enhanced Efficacy. Adv Mater 37:2412069. https://doi.org/10.1002/adma.202412069 Li G et al (2025) Alternating Interlayered Piezoelectric Self-Heterojunction Boosts Sono-Piezocatalytic Pyroptosis Oncotherapy. Adv Mater 37:2508941. https://doi.org/10.1002/adma.202508941 Yang M et al (2025) Multipath ROS Storm and Immune Activation via Sulfur Vacancy-Optimized ZnIn2S4 Nanosheets for Piezocatalytic Tumor Therapy. Angew Chem Int Ed 64:e202507502. https://doi.org/10.1002/anie.202507502 Zhang T, Zheng Y, Xiang H, Chen Y, Wu R (2024) Cascade piezocatalytic nanoprodrug for synergistic piezocatalytic therapy and sono-activated chemotherapy-augmented immunotherapy. Nano Today 58:102453. https://doi.org/https://doi.org/10.1016/j.nantod.2024.102453 Tian B et al (2023) Doping Engineering to Modulate Lattice and Electronic Structure for Enhanced Piezocatalytic Therapy and Ferroptosis. Adv Mater 35:2304262. https://doi.org/https://doi.org/10.1002/adma.202304262 Yang L et al (2023) Oxygen-Vacancy-Rich Piezoelectric BiO 2-x Nanosheets for Augmented Piezocatalytic, Sonothermal, and Enzymatic Therapies. Adv Mater 35:2300648. https://doi.org/10.1002/adma.202300648 Zheng H et al (2024) Steering Piezocatalytic Therapy for Optimized Tumoricidal Effect. Adv Funct Mater 34:2400174. https://doi.org/https:// doi.org/10.1002/adfm.202400174 Li Z et al (2024) Covalent Organic Frameworks for Boosting H 2 O 2 Photosynthesis via the Synergy of Multiple Charge Transfer Channels and Polarized Field. Angew Chem Int Ed 64:e202420218. https://doi.org/10.1002/anie.202420218 Guan L, Mehdi D, Li H, Chen F, Jin S (2025) Covalent organic frameworks: An emerging class of piezoelectric materials for mechanical energy transfer application. Chin Chem Lett 111389. https://doi.org/https://doi.org/10.1016/j.cclet.2025.111389 Guan L et al (2024) Bottom-up Synthesis of Piezoelectric Covalent Triazine-based Nanotube for Hydrogen Peroxide Production from Water and Air. Angew Chem Int Ed 64:e202419867. https://doi.org/10.1002/anie.202419867 Jiao Y et al (2025) P-P Hybrids Antimony Single-Atom Anchored Covalent Organic Framework for Efficient High-Selectivity H2O2 Piezosynthesis. Adv Funct Mater 35:2500501. https://doi.org/10.1002/adfm.202500501 Ghosh A et al (2025) Ferrielectric Dipolar Ordering in a Donor-Acceptor Based Covalent-Organic Framework for Piezocatalytic Water Splitting. Adv Funct Mater 35:2502787. https://doi.org/10.1002/adfm.202502787 Liang Z et al (2024) A Direct Z-Scheme Single-Atom MOC/COF Piezo-Photocatalytic System for Overall Water Splitting. ACS Catal 14:10447–10461. https://doi.org/10.1021/acscatal.4c02243 Shi C et al (2026) Covalent organic frameworks for ferroptosis cancer therapy. Coord Chem Rev 551:217457. https://doi.org/https://doi.org/ 10.1016/j.ccr.2025.217457 Chen X, Wang Q, Zhang H, Wang Y, Zhu H (2026) Metal homeostasis as a therapeutic lever: advancing metalloimmunology to remodel the tumor microenvironment and enhance cancer immunotherapy. Theranostics 16:1350–1373. https://doi.org/10.7150/thno.121988 Chen K et al (2024) Cellular Trojan Horse initiates bimetallic Fe-Cu MOF-mediated synergistic cuproptosis and ferroptosis against malignancies. Sci Adv 10:eadk3201. https://doi.org/doi:10.1126/sciadv.adk3201 Bing J et al (2025) Nanomedicine-enabled concurrent regulations of ROS generation and copper metabolism for sonodynamic-amplified tumor therapy. Biomaterials 318:123137. https://doi.org/10.1016/j.biomaterials.2025.123137 He G et al (2024) Microfluidic Synthesis of CuH Nanoparticles for Antitumor Therapy through Hydrogen-Enhanced Apoptosis and Cuproptosis. ACS Nano 18:9031–9042. https://doi.org/10.1021/acsnano.3c12796 He X et al (2024) Copper peroxide and cisplatin co-loaded silica nanoparticles-based trinity strategy for cooperative cuproptosis/chemo/chemodynamic cancer therapy. Chem Eng J 481:148522. https://doi.org/10.1016/j.cej.2024.148522 Lu S, Li Y, Yu Y (2024) Glutathione-Scavenging Celastrol-Cu Nanoparticles Induce Self-Amplified Cuproptosis for Augmented Cancer Immunotherapy. Adv Mater 36:2404971. https://doi.org/10.1002/adma.202404971 Wang Z et al (2026) Disrupting intracellular redox homeostasis through copper-driven dual cell death to induce anti-tumor immunotherapy. Biomaterials 324:123523. https://doi.org/10.1016/j.biomaterials.2025.123523 Yang L et al (2024) A singular plasmonic-thermoelectric hollow nanostructure inducing apoptosis and cuproptosis for catalytic cancer therapy. Nat Commun 15:7499. https://doi.org/10.1038/s41467-024-51772-1 Chu C et al (2025) Modifying metabolic and immune hallmarks of cancer by a copper complex. Sci China Chem 68:1051–1066. https://doi.org/10.1007/s11426-024-2316-4 Yan R et al (2025) Cuproptosis nanoprodrug-initiated self-promoted cascade reactions for postoperative tumor therapy. Biomaterials 318:123176. https://doi.org/10.1016/j.biomaterials.2025.123176 Liu Z, Sun M, Zhang W, Ren J, Qu X (2023) Target-Specific Bioorthogonal Reactions for Precise Biomedical Applications. Angew Chem Int Ed 62:e202308396. https://doi.org/10.1002/anie.202308396 Huang J et al (2025) Ultrasound-Triggered Nanoparticles Induce Cuproptosis for Enhancing Immunogenic Sonodynamic Therapy. Adv Mater 37:2504228. https://doi.org/10.1002/adma.202504228 Xia L et al (2022) Spatiotemporal Ultrasound-Driven Bioorthogonal Catalytic Therapy. Adv Mater 35:2209179. https://doi.org/10.1002/adma.202209179 Shao J et al (2025) Sub-1 nm CuO‐Phosphomolybdic Acid Nanosheets for Ultrasound-Controlled Pyroptosis Activation and Tumor Immunotherapy. Angew Chem Int Ed 64:e202508544. https://doi.org/10.1002/anie.202508544 Sun M et al (2023) Bioorthogonal-Activated In Situ Vaccine Mediated by a COF-Based Catalytic Platform for Potent Cancer Immunotherapy. J Am Chem Soc 145:5330–5341. https://doi.org/10.1021/jacs.2c13010 Feng T et al (2022) Ambient synthesis of metal-covalent organic frameworks with Fe-iminopyridine linkages. Chem Commun 58:8830–8833. https://doi.org/10.1039/d2cc03148e Zhang Y, Cao L, Bai G, Lan X (2023) Engineering Single Cu Sites into Covalent Organic Framework for Selective Photocatalytic CO2 Reduction. Small 19:2300035. https://doi.org/10.1002/smll.202300035 Feng T, Hao Q, Sun B, Wang D, Metal-Covalent (2023) Organic Frameworks Linked by Fe-Iminopyridine for Single-Atom Peroxidase-Mimetic Nanoenzymes. J Phys Chem C 127:3228–3234. https://doi.org/10.1021/acs.jpcc.2c07887 Gu Q et al (2024) High-Performance Piezoelectric Two-Dimensional Covalent Organic Frameworks. Angew Chem Int Ed 63:e202409708. https://doi.org/https://doi.org/10.1002/anie.202409708 Jiang T et al (2025) Piezocatalysis for water treatment: Mechanisms, recent advances, and future prospects. Environ Sci Ecotechnology 23:100495. https://doi.org/https://doi.org/ 10.1016/j.ese.2024.100495 Wu J et al (2024) Tensile Strain-Mediated Bimetallene Nanozyme for Enhanced Photothermal Tumor Catalytic Therapy. Angew Chem Int Ed 63:e202403203. https://doi.org/https://doi.org/10.1002/anie.202403203 Chen X et al (2025) Ultrafast energy-neutral molecular oxygen activation via atomically-adjacent bimetallic catalytic sites. Nat Commun. https://doi.org/10.1038/s41467-025-67706-4 Wang A et al (2025) Self-Generative Singlet Oxygen ( 1 O 2 )-Initiated Chemical Modification of Nuclear DNAs Combats Tumor Drug Resistance. J Am Chem Soc 147:20534–20547. https://doi.org/10.1021/jacs.5c02826 Jiang J et al (2025) Bismuth Sulfide Microneedle Patch for MRSA Biofilm Removal via Oxidative Stress Amplification. Adv Funct Mater 35:07540. https://doi.org/https://doi.org/10.1002/adfm.202507540 Zhu X et al (2024) Reactive Oxygen-Correlated Photothermal Imaging of Smart COF Nanoreactors for Monitoring Chemodynamic Sterilization and Promoting Wound Healing. Small 20:2310247. https://doi.org/10.1002/smll.202310247 Li X et al (2024) Nanoscale covalent organic framework-mediated pyroelectrocatalytic activation of immunogenic cell death for potent immunotherapy. Sci Adv 10:eadr5145. https://doi.org/doi:10.1126/sciadv.adr5145 Liu Y et al (2024) Single-Site Nanozymes with a Highly Conjugated Coordination Structure for Antitumor Immunotherapy via Cuproptosis and Cascade-Enhanced T Lymphocyte Activity. J Am Chem Soc 146:3675–3688. https://doi.org/10.1021/jacs.3c08622 Liu Q, Wang X (2022) Sub-nanometric materials: Electron transfer, delocalization, and beyond. Chem Catal 2:1257–1266. https://doi.org/https://doi.org/ 10.1016/j.checat.2022.03.008 Nie S, Wu L, Wang X (2023) Electron-Delocalization-Stabilized Photoelectrocatalytic Coupling of Methane by NiO-Polyoxometalate Sub-1 nm Heterostructures. J Am Chem Soc 145:23681–23690. https://doi.org/10.1021/jacs.3c07984 Wang X et al (2019) Copper-Triggered Bioorthogonal Cleavage Reactions for Reversible Protein and Cell Surface Modifications. J Am Chem Soc 141:17133–17141. https://doi.org/10.1021/jacs.9b05833 Yang Y et al (2020) Trisulfide bond–mediated doxorubicin dimeric prodrug nanoassemblies with high drug loading, high self-assembly stability, and high tumor selectivity. Sci Adv 6:eabc1725. https://doi.org/doi:10.1126/sciadv.abc1725 Yang Y et al (2021) Iron-doxorubicin prodrug loaded liposome nanogenerator programs multimodal ferroptosis for efficient cancer therapy. Asian J Pharm Sci 16:784–793. https://doi.org/https://doi. org/10.1016/j.ajps.2021.05.001 Wang G et al (2024) Transcytosable and Ultrasound-Activated Liposome Enables Deep Penetration of Biofilm for Surgical Site Infection Management. Adv Mater 37. https://doi.org/10.1002/adma.202411092 Li L et al (2024) Chemiluminescent Conjugated Polymer Nanoparticles for Deep-Tissue Inflammation Imaging and Photodynamic Therapy of Cancer. J Am Chem Soc 146:5927–5939. https://doi.org/10.1021/jacs.3c12132 Feng S et al (2023) A Gene-Editable Palladium-Based Bioorthogonal Nanoplatform Facilitates Macrophage Phagocytosis for Tumor Therapy. Angew Chem Int Ed 62:e202313968. https://doi.org/10.1002/anie.202313968 Xue P et al (2025) Biodegradable ionic nanoregulators for synchronous modulation of copper and iron ion homeostasis in breast cancer therapy. Chem Eng J 511:162041. https://doi.org/https://doi.org/10.1016/j.cej.2025.162041 Zhang Y et al (2021) An Adenovirus-Mimicking Photoactive Nanomachine Preferentially Invades and Destroys Cancer Cells through Hijacking Cellular Glucose Metabolism. Adv Funct Mater 32:2110092. https://doi.org/10.1002/adfm.202110092 Additional Declarations The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8806647","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":586956656,"identity":"068ae8eb-d96b-46b9-ac0f-c51f00216111","order_by":0,"name":"Chunyuan Hou","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Chunyuan","middleName":"","lastName":"Hou","suffix":""},{"id":586956657,"identity":"a14f504a-b2aa-4b76-ba55-2ac2b2a96e53","order_by":1,"name":"Yu Zhang","email":"","orcid":"https://orcid.org/0000-0003-3141-1243","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhang","suffix":""},{"id":586956658,"identity":"b1f24a26-2d97-4161-9a24-8a6123b1cff6","order_by":2,"name":"Jun Gu","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Gu","suffix":""},{"id":586956659,"identity":"ee22725b-7ed5-4c15-8768-e046373569cb","order_by":3,"name":"Jun Wan","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Wan","suffix":""},{"id":586956660,"identity":"26efc5bc-939d-40cd-9b42-a18094c5fb50","order_by":4,"name":"Ziyao Zhao","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ziyao","middleName":"","lastName":"Zhao","suffix":""},{"id":586956661,"identity":"c891db02-746c-407d-bcd6-fd69a1149bf5","order_by":5,"name":"Peicheng Wang","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Peicheng","middleName":"","lastName":"Wang","suffix":""},{"id":586956662,"identity":"afb1140e-c6d3-4e84-bd32-519061662ccd","order_by":6,"name":"Shujun Feng","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Shujun","middleName":"","lastName":"Feng","suffix":""},{"id":586956663,"identity":"abc87854-20f5-476f-81d4-17fa548c678d","order_by":7,"name":"Jun Luo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYBACPiA2YGCwYWBsAPOZCWthg2hJA2kB6SJSCxAcBhHEapFIPlDMu+N8HvOM9OcPGCqsExvYzx4goCUtwZj3zO1ixhk5hg0MZ9ITG3jyEghoyTEw5m27ndg4I4exgbHtcGKDBI8BMVrOAbWkP2xg/Ee8lgNALQmGDYwNxGjheZZgOLctObGx543hjIRj6cZtPDn4tfCzJx8zeNtml7ixPf3Bhw811rL97GfwawFZBFYBDC4GhgQGeEzhBcwPQKQ8ESpHwSgYBaNghAIArgBCNysNO1UAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-8093-5345","institution":"Nanjing University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Luo","suffix":""}],"badges":[],"createdAt":"2026-02-06 11:55:47","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-8806647/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8806647/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102168676,"identity":"2f404440-0209-4b34-9888-840dec618df8","added_by":"auto","created_at":"2026-02-09 03:31:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":361427,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram illustrating the synthesis steps, piezocatalytic mechanism and antitumor mechanism of Cu2N@D-FA. Cu2N@D-FA featured a piezocatalytic activity and US controlled effect. Under US irradiation, mechanical deformation of nanosheets generates piezoelectric fields, tilting band structures to enhance electron-hole (e\u003csup\u003e−\u003c/sup\u003e-h\u003csup\u003e+\u003c/sup\u003e) pair separation and drive ROS generation (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e•−\u003c/sup\u003e, \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, and ·OH), and controls the valence transition of Cu(II)/Cu(I), triggering biorthogonal catalysis. The copper ions accumulated in the mitochondria induce cuproptosis.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8806647/v1/692293c95defb9afa97b80df.png"},{"id":102168669,"identity":"73ef3305-c818-4302-8364-7041346672ff","added_by":"auto","created_at":"2026-02-09 03:31:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":748915,"visible":true,"origin":"","legend":"\u003cp\u003eStructure and characterization of Cu-coordinated COFs. (a) Structure; (b) FT-IR spectra; (c) XPS spectra; (d) XRD patterns; (e) SEM images; (f) TEM images of Cu-coordinated COFsnanosheets after ultrasonic exfoliation; (g) EDX elemental mapping of CuCOF-2N.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8806647/v1/a6e48c85c6530ef7438467ef.png"},{"id":102168675,"identity":"963c406a-8571-4faf-b733-fa18d21ae260","added_by":"auto","created_at":"2026-02-09 03:31:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":244556,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Amplitude-voltage curves and phase hysteresis loops of Cu-coordinated COFs; (b) Diagram of the intrinsic energy band of Cu-coordinated COF nanosheets and the tilted energy band of the piezoelectric field induced by US. (1C: CuCOF-1C, 1N: CuCOF-1N, 2C: CuCOF-2C, 2N: CuCOF-2N); (c) Changes of absorbance at 425 nm of DPBF under US irradiation for 5 min; (d-e) The absorbance changes of TMB after different treatments; (f) ESR spectra of ·OH for different groups by DMPO; (e) ESR spectra of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e•−\u003c/sup\u003e for different groups by DMPO; (g) ESR spectra of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e for different groups by TEMP; (h) ESR spectra of ·OH for different groups by DMPO.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8806647/v1/e76fe0323c8c999175199556.png"},{"id":102168671,"identity":"60a243a8-7949-453d-a2a7-2167b861f5a7","added_by":"auto","created_at":"2026-02-09 03:31:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":267212,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the formation process of (a) O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e•−\u003c/sup\u003e and \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, and (b) ·OH; (c) Scheme of ROS generation by Cu2N@D-FA under US irradiation; Reaction free energy plots of the (d) O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e•−\u003c/sup\u003e and \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e and (e) ·OH generation process; (f) The reaction mechanism of Cu(I) detection using neocuproine; (g) UV-Vis absorbance spectra of 1C: CuCOF-1C, 1N: CuCOF-1N, 2C: CuCOF-2C and 2N: CuCOF-2N incubated with neocuproine; (h) Cu 2p XPS pattern of CuCOF-2N before and after US irradiation; (i) Time-course study of the decaging efficiency of Dox-DMProc in the presence of CuCOF-2N as monitored by HPLC. The peaks of the reactant Dox-Proc (pink) and the product Dox (blue) are shown.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8806647/v1/24afd228f75750cc949ca508.png"},{"id":102168673,"identity":"f28f935f-9cd1-443d-9348-023a8660e3da","added_by":"auto","created_at":"2026-02-09 03:31:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1854987,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Cellular uptake of Cu2N@D-PEG and Cu2N@D-FA in 4T1 cells; (b) Endosomal escape analysis of Cu2N@D-FA in 4T1 cells; (c) Colocalization of FITC-labeled Sonozyme and LysoTracker Red-stained endosomes in 4T1 cells with or without US irradiation; (d) Cell viability of 4T1 cells after treatment with different concentrations of DOX and DOX-Proc for 24 h; Anticancer effects of CuX@D-FA and Cu2N-FAevaluated by the MTT assay (e) without US irradiation, (f) with US irradiation; (g) Cell viability of 4T1 cells after treatment with different concentrations of Cu2N-FA and Cu2N@D-FA for 24 h; (h) Live/Dead staining of 4T1 cells after different treatments; (i) Western blot analysis of the FDX1 and LIAS expression after different treatments; (j) Fluorescence images of ROS in 4T1 cells after different treatments; (k) Mitochondrial membrane potential of 4T1 cells measured by JC-1 assay; (m) Relative JC-1 monomer fluorescence intensity analysis.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8806647/v1/e142e48d5a3e98e91c3e25a5.png"},{"id":102296909,"identity":"e6d6c505-9446-49b8-84ab-8c6dc2a2e878","added_by":"auto","created_at":"2026-02-10 10:22:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":271697,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Plasma concentration-time curves of DOX and the released DOX from Cu2N@D-FA; (b) Pharmacokinetic parameters of DOX and the released DOX-Proc after intravenous injection of free DOX or Cu2N@D-FA for 48 h; (c) Time-dependent \u003cem\u003ein vivo\u003c/em\u003e fluorescent images of 4T1 tumor bearing mice after intravenous injection of DiR-labeled Cu2N@D-PEG or Cu2N@D-FA for 24 h; (d) Quantification of the fluorescence intensity in the tumor area with time after intravenous injection of DiR-labeled Cu2N@D-PEG or Cu2N@D-FA for 24 h; (e)Ex vivo fluorescence images of major organs and tumors after 24 h intravenous injection of DiR-labeled Cu2N@D-PEG or Cu2N@D-FA; (f) Quantification of the fluorescence intensity in major organs and tumors after 24 h intravenous injection intravenous injection of DiR-labeled Cu2N@D-PEG or Cu2N@D-FA.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8806647/v1/e37b48b74bbbfa05a6ab6e35.png"},{"id":102168672,"identity":"4a2aaa3b-cf43-46a4-a4cb-fed1ac961d1d","added_by":"auto","created_at":"2026-02-09 03:31:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2230135,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The treatment regimen for 4T1 tumor-bearing mice; (b) Tumor growth curves over the course of the 14-day treatment; (c) Average tumor weights after 14-day treatment; (d) Representative image of tumors after 14-day treatment; (e) Body weights of mice during 14-day treatment; (f) Survival curves of 4T1 bearing mice during 60-day treatment; (g) Histological analysis of tumor tissues after 14-day treatment, including HIF-1α, FDX1, LIAS, DLAT, ROS, H\u0026amp;E and TUNEL.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8806647/v1/2e050d38ebc4d627cef0f5db.png"},{"id":102299208,"identity":"6a8607c3-8028-4690-b43f-e33284f8a064","added_by":"auto","created_at":"2026-02-10 11:03:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6387394,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8806647/v1/78f54bf3-6fa2-4275-a764-3482f3bae1f0.pdf"},{"id":102168674,"identity":"b6faecdb-c860-495c-afd2-0dc3c7d7fa18","added_by":"auto","created_at":"2026-02-09 03:31:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16979312,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8806647/v1/3a523b154cb30afbd352d8f7.docx"},{"id":102296547,"identity":"aa8a6c64-efb1-48df-aa0b-2df0d4f629b6","added_by":"auto","created_at":"2026-02-10 10:20:03","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":406234,"visible":true,"origin":"","legend":"","description":"","filename":"SYNOPSIS.docx","url":"https://assets-eu.researchsquare.com/files/rs-8806647/v1/d611bbaf54a9dbf522d76942.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eA sonopiezoresponse COF-based smart nanoreactor orchestrating in situ bioorthogonal chemistry and cuproptosis for enhanced tumor piezocatalytic therapy\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePiezocatalytic therapy (PCT) has emerged as a promising ultrasound (US) mediated antitumor strategy, distinguished by its noninvasiveness, deep tissue penetration, and low oxygen dependence.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Unlike conventional sonodynamic therapy (SDT), which is limited by rapid charge recombination,\u003csup\u003e2\u003c/sup\u003e PCT utilizes the intrinsic piezoelectric field generated under ultrasonic mechanical stress to dramatically improve electron-hole (e⁻-h⁺) pair separation efficiency, thereby facilitating robust reactive oxygen species (ROS) generation, including hydroxyl radicals (\u0026middot;OH), superoxide anions (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e), and singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e).\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e However, current research on piezoelectric antitumor materials remains largely confined to inorganic semiconductors (such as BaTiO\u003csub\u003e3\u003c/sub\u003e,\u003csup\u003e4\u003c/sup\u003e ZnO,\u003csup\u003e5\u003c/sup\u003e ZnIn\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e4\u003c/sub\u003e,\u003csup\u003e3\u003c/sup\u003e g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e,\u003csup\u003e1\u003c/sup\u003e and 2D Bi-based materials oxygen-vacancy-rich BiO\u003csub\u003e2\u0026minus;x\u003c/sub\u003e nanosheets\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e), which still face critical limitations including suboptimal piezoelectric response, insufficient ROS production, and compromised biocompatibility.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Therefore, there is an urgent need to develop novel piezoelectric sonosensitizers with strong piezoelectric response and favorable biocompatibility, while simultaneously devising multimodal synergistic strategies to counteract the intrinsic defense mechanisms of cancer, enhancing the clinical translation potential of PCT.\u003c/p\u003e \u003cp\u003eCovalent organic frameworks (COFs), an emerging class of porous crystalline materials, have recently been recognized as attractive candidates for piezoelectric materials owing to their precisely designable non-centrosymmetric architectures, programmable band gap structures, and superior charge transport properties.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e COFs demonstrate unique advantages over conventional inorganic piezoelectric materials. For instance, their porous structures readily undergo deformation under ultrasonic mechanical stress, amplifying local polarization effects to enhance piezoelectric response.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e In addition, the conjugated frameworks and nitrogen-rich moieties (such as triazine rings) may facilitate charge separation and transfer, thereby boosting ROS generation efficiency.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Particularly, their symmetry can be further broken through metal single-atom anchoring or donor-acceptor (D-A) structural design to optimize piezocatalytic activity.\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Importantly, their inherent biocompatibility and biodegradability provide safety assurance for their \u003cem\u003ein vivo\u003c/em\u003e application.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Although piezoelectric applications based on COFs remain in nascent stages, they have demonstrated considerable promise in US-controlled ROS production and the construction of multimodal therapeutic platforms, which opens avenues for creating news piezoelectric sonosensitizers with higher piezoelectric responsiveness and lower systemic toxicity to enhance piezocatalytic treatment efficacy.\u003c/p\u003e \u003cp\u003eMetal homeostasis represents a fundamental biological regulatory network, with copper ions serving an essential role in critical physiological processes including enzymatic catalysis and intracellular signaling.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e However, tumor cells often hijack metal metabolic pathways to meet their abnormal proliferation needs, and this \u0026ldquo;metal addiction\u0026rdquo; characteristic makes metal homeostasis a highly promising anti-cancer target.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e In recent years, studies have found that cuproptosis exerts anti-tumor effects by disrupting the mitochondrial TCA cycle and triggering proteotoxic stress.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Noatbly, cuproptosis is fundamentally distinct from most other forms of programmed and non-programmed cell death, which demonstrates significant promise in overcoming resistance to conventional therapies.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e In fact, multiple studies have indicated that cuproptosis can amplify the antitumor efficacy of ROS-dependent therapies, including SDT, photodynamic therapy (PDT), and chemodynamic therapy (CDT), through various molecular events such as ROS accumulation, proteotoxic stress, and metabolic regulation.\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e These mechanistic synergies suggest that cuproptosis can serve as an ideal adjunctive therapy to enhance PCT.\u003c/p\u003e \u003cp\u003eTo fully exploit the synergistic potential of combining PCT and cuproptosis while minimizing side effects, the precise regulation of copper ions \u003cem\u003ein vivo\u003c/em\u003e is crucial. Bioorthogonal catalysis enables the execution of exogenous chemical reactions within complex biological systems without interfering with endogenous physiological processes, offering an unprecedented solution to this challenge.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Notably, recent studies have demonstrated that US can drive bioorthogonal catalysis to achieve valence transition of transition metal ions, which enables the remote and precise regulation of copper ions to induce cuproptosis.\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Importantly, bioorthogonal catalysis can also achieve the in situ activation of biomolecules and the specific deprotection of prodrugs.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e This capability holds promise for creating multi-dimensional synergy with PCT, further enhancing the antitumor efficacy of the combination. Therefore, integrating bioorthogonal catalysis, cuproptosis, and piezocatalytic therapy may offer a rational design strategy for enhancing therapeutic efficacy.\u003c/p\u003e \u003cp\u003eHere, by incorporating nitrogen-rich monomers and anchored metal designs into COFs, we successfully constructed piezocatalytically active frameworks (CuCOF-2N) and combined it with a DOX prodrug (DOX-Pro) and folic acid (FA)-modified liposomes further developed an intelligent US-controlled nanoreactor (Cu2N@D-FA) for enhancing antitumor efficacy of PCT by combining cuproptosis with bioorthogonal catalysis. Notably, the comparative study of structurally related COFs underscores the crucial roles of highly polar triazine rings and symmetry-breaking bidentate ligands in enhancing the piezoelectric catalytic performance of CuCOF-2N. The predesigned CuCOF-2N, as a novel piezoelectric catalyst, could break through oxygen limitations to generate massive \u0026middot;OH under ultrasonic excitation. Moreover, CuCOF-2N could autonomously regulate the tumor microenvironment to enhance generation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e by catalyzing endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to produce oxygen. This allows Cu2N@D-FA to overcome the limitations of convertional piezoelectric materials and triggers strong PCT. Most importantly, CuCOF-2N enabled US-intelligent regulation of copper valence conversion, allowing for spatiotemporally controllable bioorthogonal catalysis and the induction cuproptosis, which synergistically enhances the antitumor activity of PCT. To our knowledge, this work represents the first successful application of COF materials in piezocatalytic therapy and pioneers the \u0026ldquo;one stone, two birds\u0026rdquo; utilization of copper ions simultaneously driving bioorthogonal catalysis and inducing cuproptosis, thereby establishing a novel paradigm for precision oncotherapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Synthesis and characterization of COFs\u003c/h2\u003e \u003cp\u003eTo endow COF frameworks with intrinsic piezoelectric properties, we predesigned nitrogen-rich triazine ring TTA (4,4\u0026rsquo;,4\u0026rsquo;\u0026rsquo;-(1,3,5-triazine-2,4,6-triyl)trianiline) as the amino ligand, with TAPB (1,3,5-tris(4-aminophenyl)benzene) as the benzene-ring control. Besides, in order to increase metal active sites, we strategically selected the bidentate ligand DBPy (6,6\u0026rsquo;-diformyl-3,3\u0026rsquo;-bipyridine) as an aldehyde ligand for anchoring copper ions (Cu(OTf)\u003csub\u003e2\u003c/sub\u003e), and BPA (2,2'-bipyridine-5,5\u0026rsquo;-dicarboxaldehyde) as a monodentate ligand. Through systematic combination of these ligands, we successfully constructed four piezoelectric-responsive Cu-coordinated COFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) via Schiff-base condensation: CuCOF-1C(TAPB-BPA), CuCOF-1N(TTA-BPA), CuCOF-2C(TAPB-DBPy), and CuCOF-2N(TTA-DBPy). The specific reaction routes can be found in \u003cb\u003eScheme S1\u003c/b\u003e and \u003cb\u003eS2\u003c/b\u003e. Notably, all four COFs can be synthesized in only ten minutes and produced on a gram scale.\u003c/p\u003e \u003cp\u003eThe chemical structures of the synthesized COFs were verified by Fourier transform infrared spectroscopy (FT-IR). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cb\u003eFigure S1\u003c/b\u003e, the characteristic absorption peak corresponding to the stretching vibration of the imine bond (C\u0026thinsp;=\u0026thinsp;N) at approximately 1590 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was observed, while the disappearance of the C\u0026thinsp;=\u0026thinsp;O stretching vibration peak at 1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e further confirmed the successful formation of imine linkage.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e X-ray photoelectron spectroscopy (XPS) analysis revealed that these COFs were primarily composed of four elements: C, N, O, and Cu (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The high-resolution of Cu 2p confirmed the existence of Cu\u003csup\u003e2+\u003c/sup\u003e (\u003cb\u003eFigure S2\u003c/b\u003e). N\u003csub\u003e2\u003c/sub\u003e adsorption-desorption measurements at 77 K demonstrated the porous nature of the Cu-coordinated COFs, with Brunauer-Emmett-Teller (BET) specific surface areas calculated to be 407 m\u003csup\u003e2\u003c/sup\u003e∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (CuCOF-1C), 621 m\u003csup\u003e2\u003c/sup\u003e∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (CuCOF-1N), m\u003csup\u003e2\u003c/sup\u003e∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (CuCOF-2C), and m\u003csup\u003e2\u003c/sup\u003e∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (CuCOF-2N), respectively (\u003cb\u003eFigure S3\u003c/b\u003e). Powder X-ray diffraction (PXRD) analysis confirmed the high crystallinity of all four COFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), with experimental diffraction patterns showing excellent agreement with the simulated data based on the A-A stacking model.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Optimized structures with the AA stacking mode were shown in \u003cb\u003eFigure S4\u003c/b\u003e. Reasonable unit cell parameters were obtained through Pawley refinement (\u003cb\u003eTable S3-6\u003c/b\u003e). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed these Cu-coordinated COFs can be converted into nanosheets from bulk COFs via simple ultrasonic exfoliation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee,f). Energy-dispersive X-ray spectroscopy (EDX) elemental mapping confirmed the homogeneous distribution of C, N, O, and Cu throughout the CuCOF-2N (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Inductively coupled plasma mass spectrometry (ICP-MS) was used to detect the Cu content in these COFs, which was 5.9 wt.% for CuCOF-1C, 5.5 wt.% for CuCOF-1N, 8.3 wt.% for CuCOF-2C, and 8.1 wt.% for CuCOF-2N, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Piezoelectricity and band structure of COFs\u003c/h2\u003e \u003cp\u003eNext, we employed scanning probe piezoelectric force microscopy (PFM) to evaluate the piezoelectric properties of the Cu-coordinated COFs. The polarization behavior of piezoelectric domains was clearly identified through the amplitude and phase patterns of Cu-coordinated COFs (\u003cb\u003eFigure S5\u003c/b\u003e), confirming their intrinsic piezoelectric characteristics. Notably, upon the application of a\u0026thinsp;\u0026plusmn;\u0026thinsp;10 V bias voltage, these COFs displayed a characteristic butterfly-shaped amplitude loop and an approximately 180\u0026deg; phase-reversal hysteresis loop (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), which are typical features of piezoelectric materials.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e From the amplitude loop, CuCOF-2N had amplitude of 3.0 nm, while the amplitudes of CuCOF-1C, CuCOF-1N, and CuCOF-2C were 1.0 nm, 1.7 nm, and 2.0 nm, respectively, indicating that CuCOF-2N possessed a stronger piezoelectric response.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e The enhanced piezoelectric property can be attributed to the strong polarity of the triazine rings, which promotes the formation of intrinsic dipole moments, while the increased copper coordination sites further disrupt the structural symmetry.\u003c/p\u003e \u003cp\u003eTo explore the influence of piezoelectric properties on the band structure, the band structures of Cu-coordinated COFs were analyzed using ultraviolet-visible diffuse reflectance spectroscopy (UV-vis DRS) and valence band X-ray photoelectron spectroscopy (VB-XPS). \u003cb\u003eFigure S6\u003c/b\u003e shows that all four COFs exhibited a broad light absorption range in the UV-vis region, while CuCOF-2C/2N further broadened this absorption. Tauc plots derived from (αhν)\u0026sup2; versus photon energy (hν) reveal that the bandgap energy (\u003cem\u003eE\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e) of CuCOF-1C, CuCOF-1N, CuCOF-2C, and CuCOF-2N were determined to be 2.12 eV, 2.3 eV, 1.99 eV, and 2.11 eV, respectively (\u003cb\u003eFigure S7\u003c/b\u003e). The VB-XPS results indicate that the valence band potential (\u003cem\u003eE\u003c/em\u003e\u003csub\u003eVB, XPS\u003c/sub\u003e) of CuCOF-1C, CuCOF-1N, CuCOF-2C, and CuCOF-2N were 1.36 eV, 1.23 eV, 0.93 eV, and 1.10 eV, respectively (\u003cb\u003eFigure S8\u003c/b\u003e). Based on the \u003cem\u003eE\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e and \u003cem\u003eE\u003c/em\u003e\u003csub\u003eVB,XPS\u003c/sub\u003e data, the band position can be determined. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb schematically illustrates the intrinsic and tilted band structures of the Cu-coordinated COFs under US irradiation. The intrinsic band diagram reveals that all four COFs can reduce O\u003csub\u003e2\u003c/sub\u003e to O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e under ultrasonic irradiation, which subsequently may react with holes to generate \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e. Notably, the inherent band characteristics of these COFs intrinsically limit the production of \u0026middot;OH. However, CuCOF-2C/2N, with exceptional piezoelectric properties, demonstrated obvious band tilting under US irradiation, which allowed CuCOF-2C/2N to oxidize water to produce \u0026middot;OH.\u003c/p\u003e \u003cp\u003eAccording to the band theory, piezoelectric materials can generate directional electric fields under mechanical excitation, which drives charge carriers toward the material surface.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e To evaluate the charge separation abilities of Cu-coordinated COFs, electrochemical impedance spectroscopy (EIS) and time-resolved fluorescence (TRF) spectroscopy were employed. EIS measurements reveal that CuCOF-1C/1N exhibited a larger arc radius compared to CuCOF-2C/2N (\u003cb\u003eFigure S9\u003c/b\u003e), which could be attributed to their relatively wider bandgap. Meanwhile, TRF spectroscopy demonstrates that the average radiative lifetimes of the Cu-coordinated COFs were 1.76 ns, 2.72 ns, 2.72 ns, and 3.36 ns, respectively (\u003cb\u003eFigure S10\u003c/b\u003e). The prolonged fluorescence lifetime observed in CuCOF-2N suggested increased electron-hole pairs. These results demonstrate that the meticulously designed triazine ring and copper site effectively inhibit recombination of electron-hole pairs and enhance carrier transport, thereby improving piezocatalytic activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. US-mediated ROS generation of COFs\u003c/h2\u003e \u003cp\u003eThe tumor microenvironment (TME) is characterized by hypoxia, mild acidity, and elevated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e However, the generation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e is oxygen-dependent. Considering the ability of Cu\u003csup\u003e2+\u003c/sup\u003e to decompose H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for O\u003csub\u003e2\u003c/sub\u003e generation, we evaluated the CAT-like activity of Cu-coordinated COFs by measuring dissolved oxygen generation during H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition. As shown in \u003cb\u003eFigureS11\u003c/b\u003e, all Cu-coordinated COFs exhibited significant dissolved oxygen production, demonstrating their remarkable CAT-like activity. The introduction of US can improve the amount and rate of O\u003csub\u003e2\u003c/sub\u003e generation. This property can effectively overcome the oxygen-dependent limitation of conventional sonodynamic therapy, suggesting great potential for therapeutic applications in hypoxic TME.\u003c/p\u003e \u003cp\u003eSubsequently, we investigated the ROS generation capability of Cu-coordinated COFs under US irradiation using 1,3-diphenylisobenzofuran (DPBF) fluorescence probe, which undergoes a characteristic color change from yellow to colorless upon reaction with ROS.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e As expected, DPBF absorbance decreased significantly after 5 min of US irradiation (1 W\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 1 MHz, 50% duty cycle). Particularly, CuCOF-2C/2N exhibited more pronounced absorbance reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cb\u003eFigure S12\u003c/b\u003e), which could be attributed to their narrower bandgap. Furthermore, we employed specific fluorescent probes, including singlet oxygen sensor green (SOSG),\u003csup\u003e37\u003c/sup\u003e dihydroethidium (DHE),\u003csup\u003e38\u003c/sup\u003e and 3,3\u0026rsquo;,5,5\u0026rsquo;-tetramethylbenzidine (TMB),\u003csup\u003e39\u003c/sup\u003e to detect the production of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e, and \u0026middot;OH, respectively. As shown in \u003cb\u003eFigureS13\u003c/b\u003e, the fluorescence intensity of DHE and SOSG gradually increased with prolonged US irradiation time, indicating the generation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e. Notably, CuCOF-2C/2N demonstrated stronger fluorescence signals than CuCOF-1C/1N, consistent with the DPBF results. In addition, the absorbance of TMB in the CuCOF-1C/1N group remained nearly unchanged under US irradiation, whereas that in CuCOF-2C/2N increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). This observation indicated that US-induced band structure modification in CuCOF-2C/2N enabled direct water oxidation to generate \u0026middot;OH, with CuCOF-2N showing superior \u0026middot;OH yield compared to CuCOF-2C. Notably, CuCOF-2N still generated a substantial amount of \u0026middot;OH even under oxygen-deficient condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), which indicated that the generation of \u0026middot;OH is independent of oxygen participation. These results demonstrate that the energy band tilting caused by the excellent piezoelectric property of CuCOF-2N enabled the generation of an additional type of radical, which endowed CuCOF-2N with a unique advantage for its application in the antitumor therapy.\u003c/p\u003e \u003cp\u003eElectron spin resonance (ESR) spectroscopy was employed to further characterize the ROS species generated under US irradiation. Using 2,2,6,6-tetramethylpiperidine (TEMP) as the spin trap, we detected the distinctive 1:1:1 triplet signal corresponding to \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Furthermore, when using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trap, the six peaks shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg demonstrated the O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation. Besides, we observed the characteristic signal with an intensity ratio of 1:2:2:1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh), which confirmed the \u0026middot;OH production of CuCOF-2N under US irradiation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. ROS generation mechanism revealed by DFT calculation\u003c/h2\u003e \u003cp\u003eWe performed density functional theory (DFT) calculations on the electron transfer in Cu-coordinated COFs and the possible mechanism of ROS generation under US irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cb\u003eb\u003c/b\u003e). The generation mechanisms of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e were first investigated. The Gibbs free energy of the products is lower than that of the reactants, and the energy drop further confirmed that the reaction favors the generation of ROS.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e Under US irradiation, COFs are excited to the singlet excited state (COFs*) and may generate electron-hole (e⁻-h⁺) pairs. The resulting e⁻ may be captured by O\u003csub\u003e2\u003c/sub\u003e to generate O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e. Furthermore, O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e is likely oxidized by holes to produce \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, compared to CuCOF-1C/1N, CuCOF-2C/2N is more favorable for the generation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e. Next, we investigated the mechanism of \u0026middot;OH generation by CuCOF-2C/2N. Firstly, CuCOF-2C/2N exhibited excellent H\u003csub\u003e2\u003c/sub\u003eO absorption capacity, which ensures efficient capture of H\u003csub\u003e2\u003c/sub\u003eO molecules at the active sites. Subsequently, H\u003csub\u003e2\u003c/sub\u003eO captures holes to generate \u0026middot;OH.\u003csup\u003e41\u003c/sup\u003e The energy required for CuCOF-2N to generate \u0026middot;OH is 1.93 eV, which is significantly lower than that of CuCOF-2C (2.3 eV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). This indicates that CuCOF-2N is more favorable for the generation of \u0026middot;OH under US irradiation. Based on these findings, it can be inferred that the mechanism of ROS generation involves the absorption of O\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO, the formation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e via ultrasonically induced electrons, followed by the generation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e and \u0026middot;OH through oxidation reactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. US-mediated copper valence transition for bioorthogonal catalysis\u003c/h2\u003e \u003cp\u003eTo investigate whether US irradiation can induce the generation of Cu(I) active sites, Cu(I)-specific chelating agent neocuproine was used as an indicator (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). As expected, yellow chromophore exhibiting a characteristic absorption maximum at 452 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg) was detected, corresponding to the Cu(I)-neocuproine complex [Cu(neocuproine)\u003csub\u003e2\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e, which provided strong evidence for the formation of Cu(I) active sites on the Cu-coordinated COFs after US irradiation. Notably, CuCOF-2N demonstrated a significantly higher Cu(I) content compared to other COFs. Furthermore, the characteristic absorption peak of chromophore showed a marked enhancement with increasing US irradiation time or power, indicating the generation of Cu(I) active sites is dependent on US power and time (\u003cb\u003eFigure S14\u003c/b\u003e). Shao et al. synthesized CuO-PMA nanosheets and utilized their electron delocalization property: US triggered the directional migration of electrons in CuO-PMA, facilitating the conversion of Cu(II) to Cu(I).\u003csup\u003e28\u003c/sup\u003e They attributed this to the enhanced orbital overlap exhibited by sub-nanoscale materials due to surface atomic rearrangement, which promotes electron delocalization between surface atoms, and the delocalization can be traced to photoinduced electron transfer.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e Therefore, in situ XPS measurements were performed under xenon lamp irradiation. After 10 min of light irradiation, high-resolution Cu 2p spectra revealed a significant increase in Cu(I), which confirmed that photoexcited electrons induced charge accumulation at Cu sites and a subsequent valence transition, providing direct evidence for electron transfer in CuCOF-2N (\u003cb\u003eFigure S15\u003c/b\u003e). In addition, static XPS analyses were carried out on CuCOF-2N before and after US treatment, and the results were consistent with the in situ XPS results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). These findings not only illustrated the favorable responsiveness of Cu-coordinated COFs to external stimuli such as US and light, but also reaffirmed that upon ultrasonic stimulation-induced electron-hole separation, electrons migrate to Cu(II) sites, ultimately leading to a change in Cu valence, which provides possibility for bioorthogonal catalysis.\u003c/p\u003e \u003cp\u003ePrevious studies have reported that Cu(I) exhibits favorable bioorthogonal catalytic properties, enabling the creation of powerful multifunctional biocatalysts for biomedical applications.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Therefore, we subsequently evaluated the \u003cem\u003ein vitro\u003c/em\u003e bioorthogonal catalytic effect of Cu-coordinated COFs using a fluorescence-quenched coumarin derivative (cd-Pro) as the reaction substrate. The synthetic route of cd-Pro was shown in \u003cb\u003eScheme S3\u003c/b\u003e. After 5 min of US irradiation, the cleavage efficiency was evaluated by measuring the fluorescence recovery over a period of 24 h. The fluorescence signal gradually increased over time, with CuCOF-2N demonstrating the highest catalytic efficiency (\u003cb\u003eFigure S16\u003c/b\u003e). Doxorubicin (DOX) is a widely used drug for treating tumors, but its clinical utility is limited by non-specific targeting and considerable side effects.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e Substantial research has been directed toward developing prodrug strategies to reduce the systemic toxicity of DOX.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e Thus, we synthesized a DOX prodrug (DOX-Proc) by protecting its amino group (\u003cb\u003eScheme S4\u003c/b\u003e). We further confirmed that US-triggered CuCOF-2N induced the conversion of DOX-Proc to DOX by monitoring high-performance liquid chromatography (HPLC). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei and \u003cb\u003eFigure S17\u003c/b\u003e show that CuCOF-2N demonstrated the highest catalytic efficiency, with a DOX yield of 80% within 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Preparation and characterization of intelligent US-controlled nanoreactor\u003c/h2\u003e \u003cp\u003eGiven the excellent US-driven piezoelectric property and bioorthogonal catalytic capability of CuCOF-2N, we further constructed an intelligently US-responsive nanoreactor, termed Cu2N@D-FA, consisting of CuCOF-2N, DOX-Proc, and FA-modified lipids, which was prepared via the thin-film hydration method.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e As shown in \u003cb\u003eFigure S18\u003c/b\u003e, TEM revealed a spherical morphologies, while dynamic light scattering (DLS) measurements indicated an average size of 135.12 nm (\u003cb\u003eFigure S19\u003c/b\u003e) and a zeta potential of -16.28 mV (\u003cb\u003eFigure S20\u003c/b\u003e). Importantly, Cu2N@D-FA exhibited excellent colloidal stability over 48 hours in various media (deionized water, PBS, DMEM, 10% FBS-DMEM), with no significant changes in particle size, indicating biological applications potential (\u003cb\u003eFigure S21\u003c/b\u003e). Furthermore, HPLC measurements indicated a drug loading rate (LR) of 25.8% for DOX-Proc in Cu2N@D-FA (\u003cb\u003eTable S1\u003c/b\u003e), confirming the successful encapsulation of DOX-Proc within the Cu2N@D-FA. In addition, the control nanoreactors based on the CuCOF-1C, CuCOF-1N, and CuCOF-2C, namely Cu1C@D-FA, Cu1N@D-FA, and Cu2C@D-FA, as well as the control nanocomposites based on CuCOF-2N, including Cu2N@D-PEG (composed of CuCOF-2N, DOX-Proc, and PEG-modified lipids) and Cu2N-FA (composed of CuCOF-2N and FA-modified lipids), were prepared by the same method. The characterization data for them are detailed in \u003cb\u003eTable S1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. \u003cem\u003eIn vitro\u003c/em\u003e antitumor effect\u003c/h2\u003e \u003cp\u003eThe mammary carcinoma cell line 4T1 was used to investigate the \u003cem\u003ein vitro\u003c/em\u003e therapeutic efficacy of Cu2N@D-FA. The folate receptor (FR) is a membrane protein overexpressed in tumor cells. Folate (FA) is regarded as a promising and highly efficient tumor-targeting agent, primarily attributed to its strong interaction with FR.\u003csup\u003e48\u003c/sup\u003e Therefore, we first examined whether FA could enhance its cellular uptake in cancer cells, with Cu2N@D-PEG (PEG instead of FA) serving as the control. We fluorescently labeled Cu2N@D-PEG and Cu2N@D-FA using fluorescein isothiocyanate (FITC), followed by culturing with 4T1 cells and monitoring with fluorescence microscopy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cb\u003eFigure S22\u003c/b\u003e, the fluorescence signals of 4T1 cells treated with Cu2N@D-PEG and Cu2N@D-FA reached a plateau at 8 h, respectively. Importantly, the accumulated concentration of Cu2N@D-FA in 4T1 cells was 1.3-fold higher than that of Cu2N@D-PEG after 12h incubation, indicating that surface functionalization with FA promoted the cellular uptake of the nanoreactor in 4T1 cells.\u003c/p\u003e \u003cp\u003eNotably, the superior lysosomal escape capability of nanoparticles is crucial for exerting antitumor effects.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e Benefiting from the piezoelectric property of Cu-incorporated COFs, ROS generated by US irradiation in endolysosomes can enhance the permeability of the endolysosomal membrane and facilitate the escape of Cu2N@D-FA from these vesicles. Therefore, the US-driven endolysosomal escape performance of Cu2N@D-FA was evaluated. After 8 hours of incubation with FITC-labeled Cu2N@D-FA, 4T1 cells were treated with US irradiation for 3 min (1 W\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 1 MHz, 50% duty cycle). Subsequently, endolysosomes were stained with LysoTracker\u0026trade; Red and visualized using confocal laser scanning microscopy (CLSM). Without US stimulation, the LysoTracker\u0026trade; Red signal showed extensive overlap with FITC green fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), yielding a Pearson\u0026rsquo;s correlation coefficient (Rr) of 0.85 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), indicating that Cu2N@D-FA was largely trapped within endolysosomes. In contrast, the FITC signal diffused uniformly throughout the cytoplasm under US irradiation, and the degree of colocalization was significantly reduced (Rr\u0026thinsp;=\u0026thinsp;0.22), demonstrating that US effectively promoted the endolysosomal escape of Cu2N@D-FA.\u003c/p\u003e \u003cp\u003eWe further assessed the antitumor effects of Cu2N@D-FA using the methyl thiazolyl tetrazolium (MTT) assay, with Cu1C@D-FA, Cu1N@D-FA, Cu2C@D-FA, and Cu2N-FA serving as controls. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, the modified DOX prodrug (DOX-Proc) exhibited reduced cytotoxicity (half-maximal inhibitory concentration, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.9 \u0026micro;M) compared to DOX (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.15 \u0026micro;M). This dramatic reduction led to negligible cytotoxicity for all formulations in the absence of US irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). In contrast, they demonstrated dose-dependent antitumor effects under US exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). The IC\u003csub\u003e50\u003c/sub\u003e of Cu2N@D-FA (56.2 \u0026micro;g\u003cb\u003e\u0026middot;\u003c/b\u003emL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was significantly lower than those of Cu1C@D-FA (162.4 \u0026micro;g\u0026middot;mL⁻\u0026sup1;), Cu1N@D-FA (153.3 \u0026micro;g\u0026middot;mL⁻\u0026sup1;), and Cu2C@D-FA (94.8 \u0026micro;g\u0026middot;mL⁻\u0026sup1;), indicating its superior antitumor potency. Subsequently, we evaluated the intracellular bioorthogonal catalytic performance of Cu2N@D-FA. As shown in \u003cb\u003eFigure S23\u003c/b\u003e, US irradiation endows nanoreactors with bioorthogonal catalytic activity. Notably, a stronger DOX fluorescence intensity was detected in 4T1 cells treated with Cu2N@D-FA, indicating more efficient cleavage of DOX-Proc and superior bioorthogonal catalytic performance. Importantly, Cu2N@D-FA induced significantly higher cytotoxicity than Cu2N-FA, suggesting that bioorthogonal catalysis effectively enhances PCT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). Furthermore, live/dead staining was performed to evaluate the antitumor effects of Cu2N@D-FA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh and \u003cb\u003eFigure S24\u003c/b\u003e, compared with other groups, Cu2N@D-FA showed the most remarkable antitumor efficacy, which was consistent with the MTT results, further indicating the favorable bioorthogonal catalytic performance of Cu2N@D-FA significantly enhances its piezocatalytic antitumor activity.\u003c/p\u003e \u003cp\u003eTo verify the anticancer role of cuproptosis, we validated Cu2N@D-FA-mediated cell death. The intracellular concentration of copper ions after different treatments was detected using Rhodamine B hydrazide (RBH), and the red fluorescence intensity was proportional to the copper ion concentration.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e \u003cb\u003eFigureS25\u003c/b\u003e indicates that the intracellular copper ion level was significantly elevated after US irradiation. Cuproptosis is characterized by the aggregation of lipoylated proteins and the loss of Fe-S cluster proteins in the mitochondria.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Immunofluorescence results reveal that all drugs exhibited obvious aggregation of lipoylated protein Dihydrolipoamide dehydrogenase (DLAT), as observed via confocal microscopy (\u003cb\u003eFigure S26\u003c/b\u003e), confirming the contribution of cuproptosis on nanoreactor-induced cell death. Additionally, western blot analysis shows Cu2N@D-FA US+ effectively induced the loss of Fe-S cluster proteins in 4T1 cells, including ferredoxin 1 (FDX1) and lipoyl synthase (LIAS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei and \u003cb\u003eFigure S27\u003c/b\u003e), indicating Cu2N@D-FA can enhance the combined antitumor efficacy of cuproptosis and piezocatalytic therapy.\u003c/p\u003e \u003cp\u003eHypoxia is one of the features of the tumor microenvironment, while the generation of ROS critically relies on the involvement of oxygen. Copper ion offers a promising solution to overcome this hypoxia barrier by catalyzing the conversion of endogenous hydrogen peroxide into oxygen. To monitor intracellular oxygen levels during treatment, we employed the oxygen-sensitive probe tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride ([Ru(dpp)\u003csub\u003e3\u003c/sub\u003e]Cl\u003csub\u003e2\u003c/sub\u003e, RDPP), which exhibits increased fluorescence quenching in response to elevated oxygen concentrations.\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e The control group displayed the strongest red fluorescence, whereas the treatment group showed lower red fluorescence intensity, providing direct evidence of the self-supplied oxygen capability (\u003cb\u003eFigure S28\u003c/b\u003e). We also employed the fluorescent probe 2\u0026rsquo;,7\u0026rsquo;-dichlorodihydrofluorescein diacetate (DCFH-DA) to investigate the generation of ROS in 4T1 cells treated with different formulations. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej, only minimal ROS was produced in 4T1 cells without US irradiation, which may be attributed to their modest chemodynamic effect. In contrast, Cu2N@D-FA US+ group exhibited the strongest green fluorescence signal, which was 1.71-fold, 1.97-fold, 1.33-fold and 1.07-fold higher than that of Cu1C@D-FA, Cu1N@D-FA, Cu2C@D-FA, and Cu2N-FA (\u003cb\u003eFigure S29\u003c/b\u003e), respectively, demonstrating its superior ROS-generating capability. To identify the specific ROS types, we used three fluorescent probes: Singlet Oxygen Sensor Green (SOSG) for \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, dihydroethidium (DHE) for O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e, and hydroxyphenyl fluorescein (HPF) for \u0026middot;OH. As shown in \u003cb\u003eFigure S30\u003c/b\u003e and \u003cb\u003eS31\u003c/b\u003e, \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e were observed, indicating their band structures endowed them intrinsic ROS generation capacity under US irradiation. In the absence of US irradiation, minimal \u0026middot;OH was observed. However, Cu1C@D-FA and Cu1N@D-FA showed no significant change in \u0026middot;OH generation with or without US irradiation. Remarkably, Cu2N@D-FA US+ group produced more \u0026middot;OH than that of Cu2C@D-FA (\u003cb\u003eFigure S32\u003c/b\u003e). These results suggest that CuCOF-2N possesses excellent piezoelectric property, which can alter its band structure and facilitate water oxidation to generate \u0026middot;OH, confirming its promising potential for antitumor application.\u003c/p\u003e \u003cp\u003eThe accumulation of intracellular ROS can trigger severe mitochondrial damage and a reduction in mitochondrial membrane potential (MMP). Therefore, we employed 5,5\u0026rsquo;,6,6\u0026rsquo;-tetrachloro-1,1\u0026rsquo;,3,3\u0026rsquo;-tetraethylbenzimidazolocar bocyanine iodide (JC-1) assay to evaluate the ability of Cu2N@D-FA to induce MMP changes in 4T1 cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek, \u003cb\u003e4T1\u003c/b\u003e cells exhibited strong red fluorescence and minimal green fluorescence in the absence of US treatment, indicating intact mitochondria with high MMP. Under US irradiation, 4T1 cells showed a significant increase in green fluorescence, suggesting MMP depolarization. Notably, Cu2N@D-FA induced the most pronounced decrease in MMP, with green fluorescence intensities approximately 2.1-fold, 1.8-fold, 1.2-fold, and 1.1-fold higher than that in Cu1C@D-FA, Cu1N@D-FA, Cu2C@D-FA, and Cu2N-FA groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003em), respectively. These results demonstrated that Cu2N@D-FA efficiently promoted mitochondrial damage through the combination of PCT, cuproptosis, and bioorthogonal catalysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. \u003cem\u003eIn vivo\u003c/em\u003e antitumor effect\u003c/h2\u003e \u003cp\u003eGiven the excellent \u003cem\u003ein vitro\u003c/em\u003e antitumor activity of Cu2N@D-FA, we further evaluated its efficacy \u003cem\u003ein vivo\u003c/em\u003e. We first investigated its pharmacokinetic profile and tumor-targeting capability. Free DOX or Cu2N@D-FA was intravenously injected into rats, followed by monitoring drug concentrations in blood at different time points. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, DOX was rapidly cleared from the bloodstream, whereas DOX delivered via Cu2N@D-FA exhibited a significantly prolonged circulation time. Detailed pharmacokinetic parameters were summarized in \u003cb\u003eTable S2\u003c/b\u003e. The AUC\u003csub\u003e0\u0026minus;inf\u003c/sub\u003e and MRT\u003csub\u003e0\u0026minus;inf\u003c/sub\u003e of DOX in Cu2N@D-FA were 174.8 \u0026micro;g/mL\u0026middot;h and 23.5 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), respectively, which were 17-fold and 10.7-fold longer than that of free DOX, indicating that Cu2N@D-FA combines the advantages of both prodrug and liposomal formulations, substantially extending the systemic circulation duration of the drug. The tumor-targeting capacity of Cu2N@D-FA was evaluated in 4T1 tumor bearing mice. DiR-labeled Cu2N@D-FA or Cu2N@D-PEG was intravenously injected, and \u003cem\u003ein vivo\u003c/em\u003e fluorescence was monitored for 24 hours using a Maestro EX imaging system. Both formulations exhibited time-dependent tumor accumulation, with fluorescence signals peaking at 12 hours post-injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Notably, Cu2N@D-FA demonstrated superior tumor accumulation, showing a 1.58-fold higher fluorescence intensity at 12h compared to Cu2N@D-PEG (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), highlighting the critical role of FA-mediated active targeting in enhancing tumor specific accumulation. After 24 h of administration, the tumor and major organs were dissected to further characterize the biodistribution of Cu2N@D-FA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef, Cu2N@D-FA was rapidly cleared from major organs and selectively accumulated in tumor tissue, confirming its excellent tumor-targeting efficiency and favorable tissue selectivity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, to evaluate the \u003cem\u003ein vivo\u003c/em\u003e antitumor efficacy of Cu2N@D-FA, the subcutaneous 4T1 bearing mice were randomly divided into four treatment groups: Saline US+, DOX, Cu2N-FA US+, and Cu2N@D-FA US+. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, the subcutaneous 4T1 bearing mice were intravenously injected drugs every two days for 14 days. In the US treatment group, mice received 3 min of US irradiation (1 W\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 1 MHz, 50% duty cycle) at 12 h post-injection. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-d show that the Cu2N@D-FA US+ group exhibited the most potent antitumor effect, significantly outperforming the therapeutic effect of free DOX. The tumor size in the Cu2N@D-FA US+ treatment group was approximately 26.6-, 21-, and 7.4-fold smaller than that in Saline US+, DOX, and Cu2N-FA US+ groups, respectively, confirming US-driven cuproptosis and bioorthogonal catalysis can overcome intrinsic tumor resistance and substantially enhance the PCT efficacy. Notably, despite the low-dose regimen, a significant reduction in body weight was observed in the DOX-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). However, the body weight of mice treated with Cu2N-FA US\u0026thinsp;+\u0026thinsp;and Cu2N@D-FA US+ showed no significant difference compared to control mice. These results confirmed the prodrug was well tolerated and significantly reduced the systemic toxicity of DOX. Furthermore, Cu2N@D-FA did not cause pathological abnormalities in major organs (heart, liver, spleen, lungs, and kidneys), indicating its satisfactory biocompatibility (\u003cb\u003eFigure S33\u003c/b\u003e). Importantly, mice treated with Cu2N@D-FA US+ also demonstrated the longest survival time among all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eFinally, immunofluorescence and immunohistochemistry were conducted to evaluate the \u003cem\u003ein vivo\u003c/em\u003e antitumor efficacy of Cu2N@D-FA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). HIF-1α protein expression level in tumor tissues indicated Cu2N@D-FA significant alleviated tumor hypoxia, as evidenced by a markedly lower green fluorescence intensity in the Cu2N@D-FA group compared to the control group. Besides, Cu2N@D-FA led to decreased expression of FDX1 and LIAS, along with aggregation of DLAT in tumor tissue, confirming the activation of cuproptosis \u003cem\u003ein vivo\u003c/em\u003e. Importantly, Cu2N@D-FA generated the highest level of ROS among the groups, suggesting that US-mediated multidimensional combination therapy significantly increases oxidative stress in tumor tissues. Furthermore, hematoxylin and eosin (H\u0026amp;E) staining and TUNEL assays confirmed that Cu2N@D-FA significantly induced apoptosis in cancer cells. These results underscore the therapeutic advantage of combining US-induced piezoelectric effects with bioorthogonal catalysis and cuproptosis. Thus, Cu2N@D-FA represents a novel combinatorial antitumor platform capable of effectively inhibiting tumor proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eIn summary, we successfully constructed a smart US-controlled nanoreactor (Cu2N@D-FA) comprising novel piezoresponsive COFs (CuCOF-2N), DOX prodrug (DOX-Proc) and folic acid (FA)-modified liposomes, which enhances PCT by synergizing cuproptosis with bioorthogonal catalysis. Notably, the comparative study of structurally related COFs highlighted the significance of strongly polar triazine rings and symmetry-disrupting bidentate ligands in enabling CuCOF-2N to overcome the limitations of conventional piezoelectric materials and enhance piezoelectric catalytic performance. Under US irradiation, Cu2N@D-FA could generate \u0026middot;OH independent of oxygen, while also enabling self-supplied oxygen for the production of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, triggering strong PCT. Moreover, US triggered directional electron migration in CuCOF-2N, facilitating the reduction of Cu(II) to Cu(I), and thereby in-situ activated the DOX prodrug to achieve bioorthogonal catalysis. Additionally, the accumulated copper ion in the mitochondrion could further induce aggregation of lipoylated proteins and the loss of Fe-S cluster proteins, thereby activating the cuproptosis pathway. \u003cem\u003eIn vivo\u003c/em\u003e studies confirmed that Cu2N@D-FA exhibited excellent biocompatibility and selectively accumulated in tumor tissue after intravenous administration, resulting in significant tumor suppression. Therefore, this work not only reveals the structure-property relationship of piezoelectric COFs in US-driven catalysis but also proposes a novel strategy that augments PCT efficacy by coupling bioorthogonal catalysis with metal-induced cell death to circumvent treatment barriers, providing new insights for the development of smart responsive nanomedicines.\u003c/p\u003e"},{"header":"4. Experimental Section/Methods","content":"\u003cp\u003eAll materials, methods, and additional data can be found in Supporting Information. All animal experiments were conducted in compliance with the National Institute of Health Guidelines under protocols approved by the Animal Ethical and Welfare Committee of Nanjing University of Science and Technology (Approval No: ACUC-NUST2023012).\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorresponding Author\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYu Zhang:\u0026nbsp;\u003c/strong\u003eSchool of Chemistry and Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China.\u003c/p\u003e\n\u003cp\u003eEmail:\u0026nbsp;[email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShujun Feng\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eState Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, Nanjing, 210009, China\u003c/p\u003e\n\u003cp\u003eE-mail: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJun Luo:\u0026nbsp;\u003c/strong\u003eSchool of Chemistry and Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China.\u003c/p\u003e\n\u003cp\u003eEmail:\u0026nbsp;[email protected]\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eThe manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. \u0026Dagger;These authors contributed equally.\u003c/p\u003e\n\u003cp\u003eNotes\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.H. and Y.Z. contributed equally to this work. The work was supported by the National Natural Science Foundation of China (22301136, 82502555, and 22075144), the Natural Science Foundation of Jiangsu Province (BK20230942 and BK20251560) and the Fundamental Research Funds for the Central Universities (No.30924010910).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. \u0026Dagger;These authors contributed equally.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYang Z et al (2025) Dual-Defect Regulated G-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e for Piezoelectric Catalytic Tumor Therapy with Enhanced Efficacy. Adv Mater 37:2412069. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adma.202412069\u003c/span\u003e\u003cspan address=\"10.1002/adma.202412069\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi G et al (2025) Alternating Interlayered Piezoelectric Self-Heterojunction Boosts Sono-Piezocatalytic Pyroptosis Oncotherapy. Adv Mater 37:2508941. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adma.202508941\u003c/span\u003e\u003cspan address=\"10.1002/adma.202508941\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang M et al (2025) Multipath ROS Storm and Immune Activation via Sulfur Vacancy-Optimized ZnIn2S4 Nanosheets for Piezocatalytic Tumor Therapy. Angew Chem Int Ed 64:e202507502. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/anie.202507502\u003c/span\u003e\u003cspan address=\"10.1002/anie.202507502\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang T, Zheng Y, Xiang H, Chen Y, Wu R (2024) Cascade piezocatalytic nanoprodrug for synergistic piezocatalytic therapy and sono-activated chemotherapy-augmented immunotherapy. Nano Today 58:102453. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.nantod.2024.102453\u003c/span\u003e\u003cspan address=\"10.1016/j.nantod.2024.102453\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian B et al (2023) Doping Engineering to Modulate Lattice and Electronic Structure for Enhanced Piezocatalytic Therapy and Ferroptosis. Adv Mater 35:2304262. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1002/adma.202304262\u003c/span\u003e\u003cspan address=\"10.1002/adma.202304262\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L et al (2023) Oxygen-Vacancy-Rich Piezoelectric BiO\u003csub\u003e2-x\u003c/sub\u003e Nanosheets for Augmented Piezocatalytic, Sonothermal, and Enzymatic Therapies. Adv Mater 35:2300648. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adma.202300648\u003c/span\u003e\u003cspan address=\"10.1002/adma.202300648\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng H et al (2024) Steering Piezocatalytic Therapy for Optimized Tumoricidal Effect. Adv Funct Mater 34:2400174. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://\u003c/span\u003e\u003cspan address=\"https://doi.org/https://\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.1002/adfm.202400174\u003c/span\u003e\u003cspan address=\"10.1002/adfm.202400174\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z et al (2024) Covalent Organic Frameworks for Boosting H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Photosynthesis via the Synergy of Multiple Charge Transfer Channels and Polarized Field. Angew Chem Int Ed 64:e202420218. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/anie.202420218\u003c/span\u003e\u003cspan address=\"10.1002/anie.202420218\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuan L, Mehdi D, Li H, Chen F, Jin S (2025) Covalent organic frameworks: An emerging class of piezoelectric materials for mechanical energy transfer application. Chin Chem Lett 111389. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.cclet.2025.111389\u003c/span\u003e\u003cspan address=\"10.1016/j.cclet.2025.111389\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuan L et al (2024) Bottom-up Synthesis of Piezoelectric Covalent Triazine-based Nanotube for Hydrogen Peroxide Production from Water and Air. Angew Chem Int Ed 64:e202419867. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/anie.202419867\u003c/span\u003e\u003cspan address=\"10.1002/anie.202419867\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiao Y et al (2025) P-P Hybrids Antimony Single-Atom Anchored Covalent Organic Framework for Efficient High-Selectivity H2O2 Piezosynthesis. Adv Funct Mater 35:2500501. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adfm.202500501\u003c/span\u003e\u003cspan address=\"10.1002/adfm.202500501\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhosh A et al (2025) Ferrielectric Dipolar Ordering in a Donor-Acceptor Based Covalent-Organic Framework for Piezocatalytic Water Splitting. Adv Funct Mater 35:2502787. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adfm.202502787\u003c/span\u003e\u003cspan address=\"10.1002/adfm.202502787\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang Z et al (2024) A Direct Z-Scheme Single-Atom MOC/COF Piezo-Photocatalytic System for Overall Water Splitting. ACS Catal 14:10447\u0026ndash;10461. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acscatal.4c02243\u003c/span\u003e\u003cspan address=\"10.1021/acscatal.4c02243\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi C et al (2026) Covalent organic frameworks for ferroptosis cancer therapy. Coord Chem Rev 551:217457. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/\u003c/span\u003e\u003cspan address=\"https://doi.org/https://doi.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ccr.2025.217457\u003c/span\u003e\u003cspan address=\"10.1016/j.ccr.2025.217457\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Wang Q, Zhang H, Wang Y, Zhu H (2026) Metal homeostasis as a therapeutic lever: advancing metalloimmunology to remodel the tumor microenvironment and enhance cancer immunotherapy. Theranostics 16:1350\u0026ndash;1373. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7150/thno.121988\u003c/span\u003e\u003cspan address=\"10.7150/thno.121988\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen K et al (2024) Cellular Trojan Horse initiates bimetallic Fe-Cu MOF-mediated synergistic cuproptosis and ferroptosis against malignancies. Sci Adv 10:eadk3201. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/doi:10.1126/sciadv.adk3201\u003c/span\u003e\u003cspan address=\"doi:10.1126/sciadv.adk3201\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBing J et al (2025) Nanomedicine-enabled concurrent regulations of ROS generation and copper metabolism for sonodynamic-amplified tumor therapy. Biomaterials 318:123137. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biomaterials.2025.123137\u003c/span\u003e\u003cspan address=\"10.1016/j.biomaterials.2025.123137\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe G et al (2024) Microfluidic Synthesis of CuH Nanoparticles for Antitumor Therapy through Hydrogen-Enhanced Apoptosis and Cuproptosis. ACS Nano 18:9031\u0026ndash;9042. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acsnano.3c12796\u003c/span\u003e\u003cspan address=\"10.1021/acsnano.3c12796\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe X et al (2024) Copper peroxide and cisplatin co-loaded silica nanoparticles-based trinity strategy for cooperative cuproptosis/chemo/chemodynamic cancer therapy. Chem Eng J 481:148522. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cej.2024.148522\u003c/span\u003e\u003cspan address=\"10.1016/j.cej.2024.148522\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu S, Li Y, Yu Y (2024) Glutathione-Scavenging Celastrol-Cu Nanoparticles Induce Self-Amplified Cuproptosis for Augmented Cancer Immunotherapy. Adv Mater 36:2404971. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adma.202404971\u003c/span\u003e\u003cspan address=\"10.1002/adma.202404971\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z et al (2026) Disrupting intracellular redox homeostasis through copper-driven dual cell death to induce anti-tumor immunotherapy. Biomaterials 324:123523. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biomaterials.2025.123523\u003c/span\u003e\u003cspan address=\"10.1016/j.biomaterials.2025.123523\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L et al (2024) A singular plasmonic-thermoelectric hollow nanostructure inducing apoptosis and cuproptosis for catalytic cancer therapy. Nat Commun 15:7499. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-024-51772-1\u003c/span\u003e\u003cspan address=\"10.1038/s41467-024-51772-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChu C et al (2025) Modifying metabolic and immune hallmarks of cancer by a copper complex. Sci China Chem 68:1051\u0026ndash;1066. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11426-024-2316-4\u003c/span\u003e\u003cspan address=\"10.1007/s11426-024-2316-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan R et al (2025) Cuproptosis nanoprodrug-initiated self-promoted cascade reactions for postoperative tumor therapy. Biomaterials 318:123176. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biomaterials.2025.123176\u003c/span\u003e\u003cspan address=\"10.1016/j.biomaterials.2025.123176\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Z, Sun M, Zhang W, Ren J, Qu X (2023) Target-Specific Bioorthogonal Reactions for Precise Biomedical Applications. Angew Chem Int Ed 62:e202308396. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/anie.202308396\u003c/span\u003e\u003cspan address=\"10.1002/anie.202308396\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang J et al (2025) Ultrasound-Triggered Nanoparticles Induce Cuproptosis for Enhancing Immunogenic Sonodynamic Therapy. Adv Mater 37:2504228. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adma.202504228\u003c/span\u003e\u003cspan address=\"10.1002/adma.202504228\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia L et al (2022) Spatiotemporal Ultrasound-Driven Bioorthogonal Catalytic Therapy. Adv Mater 35:2209179. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adma.202209179\u003c/span\u003e\u003cspan address=\"10.1002/adma.202209179\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShao J et al (2025) Sub-1 nm CuO‐Phosphomolybdic Acid Nanosheets for Ultrasound-Controlled Pyroptosis Activation and Tumor Immunotherapy. Angew Chem Int Ed 64:e202508544. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/anie.202508544\u003c/span\u003e\u003cspan address=\"10.1002/anie.202508544\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun M et al (2023) Bioorthogonal-Activated In Situ Vaccine Mediated by a COF-Based Catalytic Platform for Potent Cancer Immunotherapy. J Am Chem Soc 145:5330\u0026ndash;5341. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jacs.2c13010\u003c/span\u003e\u003cspan address=\"10.1021/jacs.2c13010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng T et al (2022) Ambient synthesis of metal-covalent organic frameworks with Fe-iminopyridine linkages. Chem Commun 58:8830\u0026ndash;8833. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/d2cc03148e\u003c/span\u003e\u003cspan address=\"10.1039/d2cc03148e\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Cao L, Bai G, Lan X (2023) Engineering Single Cu Sites into Covalent Organic Framework for Selective Photocatalytic CO2 Reduction. Small 19:2300035. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/smll.202300035\u003c/span\u003e\u003cspan address=\"10.1002/smll.202300035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng T, Hao Q, Sun B, Wang D, Metal-Covalent (2023) Organic Frameworks Linked by Fe-Iminopyridine for Single-Atom Peroxidase-Mimetic Nanoenzymes. J Phys Chem C 127:3228\u0026ndash;3234. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jpcc.2c07887\u003c/span\u003e\u003cspan address=\"10.1021/acs.jpcc.2c07887\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGu Q et al (2024) High-Performance Piezoelectric Two-Dimensional Covalent Organic Frameworks. Angew Chem Int Ed 63:e202409708. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1002/anie.202409708\u003c/span\u003e\u003cspan address=\"10.1002/anie.202409708\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang T et al (2025) Piezocatalysis for water treatment: Mechanisms, recent advances, and future prospects. Environ Sci Ecotechnology 23:100495. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/\u003c/span\u003e\u003cspan address=\"https://doi.org/https://doi.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ese.2024.100495\u003c/span\u003e\u003cspan address=\"10.1016/j.ese.2024.100495\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu J et al (2024) Tensile Strain-Mediated Bimetallene Nanozyme for Enhanced Photothermal Tumor Catalytic Therapy. Angew Chem Int Ed 63:e202403203. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1002/anie.202403203\u003c/span\u003e\u003cspan address=\"10.1002/anie.202403203\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X et al (2025) Ultrafast energy-neutral molecular oxygen activation via atomically-adjacent bimetallic catalytic sites. Nat Commun. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-025-67706-4\u003c/span\u003e\u003cspan address=\"10.1038/s41467-025-67706-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang A et al (2025) Self-Generative Singlet Oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e)-Initiated Chemical Modification of Nuclear DNAs Combats Tumor Drug Resistance. J Am Chem Soc 147:20534\u0026ndash;20547. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jacs.5c02826\u003c/span\u003e\u003cspan address=\"10.1021/jacs.5c02826\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang J et al (2025) Bismuth Sulfide Microneedle Patch for MRSA Biofilm Removal via Oxidative Stress Amplification. Adv Funct Mater 35:07540. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1002/adfm.202507540\u003c/span\u003e\u003cspan address=\"10.1002/adfm.202507540\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu X et al (2024) Reactive Oxygen-Correlated Photothermal Imaging of Smart COF Nanoreactors for Monitoring Chemodynamic Sterilization and Promoting Wound Healing. Small 20:2310247. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/smll.202310247\u003c/span\u003e\u003cspan address=\"10.1002/smll.202310247\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X et al (2024) Nanoscale covalent organic framework-mediated pyroelectrocatalytic activation of immunogenic cell death for potent immunotherapy. Sci Adv 10:eadr5145. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/doi:10.1126/sciadv.adr5145\u003c/span\u003e\u003cspan address=\"doi:10.1126/sciadv.adr5145\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y et al (2024) Single-Site Nanozymes with a Highly Conjugated Coordination Structure for Antitumor Immunotherapy via Cuproptosis and Cascade-Enhanced T Lymphocyte Activity. J Am Chem Soc 146:3675\u0026ndash;3688. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jacs.3c08622\u003c/span\u003e\u003cspan address=\"10.1021/jacs.3c08622\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Q, Wang X (2022) Sub-nanometric materials: Electron transfer, delocalization, and beyond. Chem Catal 2:1257\u0026ndash;1266. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/\u003c/span\u003e\u003cspan address=\"https://doi.org/https://doi.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.checat.2022.03.008\u003c/span\u003e\u003cspan address=\"10.1016/j.checat.2022.03.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNie S, Wu L, Wang X (2023) Electron-Delocalization-Stabilized Photoelectrocatalytic Coupling of Methane by NiO-Polyoxometalate Sub-1 nm Heterostructures. J Am Chem Soc 145:23681\u0026ndash;23690. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jacs.3c07984\u003c/span\u003e\u003cspan address=\"10.1021/jacs.3c07984\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X et al (2019) Copper-Triggered Bioorthogonal Cleavage Reactions for Reversible Protein and Cell Surface Modifications. J Am Chem Soc 141:17133\u0026ndash;17141. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jacs.9b05833\u003c/span\u003e\u003cspan address=\"10.1021/jacs.9b05833\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y et al (2020) Trisulfide bond\u0026ndash;mediated doxorubicin dimeric prodrug nanoassemblies with high drug loading, high self-assembly stability, and high tumor selectivity. Sci Adv 6:eabc1725. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/doi:10.1126/sciadv.abc1725\u003c/span\u003e\u003cspan address=\"doi:10.1126/sciadv.abc1725\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y et al (2021) Iron-doxorubicin prodrug loaded liposome nanogenerator programs multimodal ferroptosis for efficient cancer therapy. Asian J Pharm Sci 16:784\u0026ndash;793. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.\u003c/span\u003e\u003cspan address=\"https://doi.org/https://doi.\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eorg/10.1016/j.ajps.2021.05.001\u003c/span\u003e\u003cspan address=\"10.1016/j.ajps.2021.05.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang G et al (2024) Transcytosable and Ultrasound-Activated Liposome Enables Deep Penetration of Biofilm for Surgical Site Infection Management. Adv Mater 37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adma.202411092\u003c/span\u003e\u003cspan address=\"10.1002/adma.202411092\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi L et al (2024) Chemiluminescent Conjugated Polymer Nanoparticles for Deep-Tissue Inflammation Imaging and Photodynamic Therapy of Cancer. J Am Chem Soc 146:5927\u0026ndash;5939. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jacs.3c12132\u003c/span\u003e\u003cspan address=\"10.1021/jacs.3c12132\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng S et al (2023) A Gene-Editable Palladium-Based Bioorthogonal Nanoplatform Facilitates Macrophage Phagocytosis for Tumor Therapy. Angew Chem Int Ed 62:e202313968. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/anie.202313968\u003c/span\u003e\u003cspan address=\"10.1002/anie.202313968\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXue P et al (2025) Biodegradable ionic nanoregulators for synchronous modulation of copper and iron ion homeostasis in breast cancer therapy. Chem Eng J 511:162041. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.cej.2025.162041\u003c/span\u003e\u003cspan address=\"10.1016/j.cej.2025.162041\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y et al (2021) An Adenovirus-Mimicking Photoactive Nanomachine Preferentially Invades and Destroys Cancer Cells through Hijacking Cellular Glucose Metabolism. Adv Funct Mater 32:2110092. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adfm.202110092\u003c/span\u003e\u003cspan address=\"10.1002/adfm.202110092\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"5fd8481d-0a57-4f77-8f11-9340080f9c26","identifier":"10.13039/501100001809","name":"National Natural Science Foundation of China","awardNumber":"22301136, 82502555, 22075144","order_by":0},{"identity":"a37790f1-a4a1-4831-b024-1de9bd0052d1","identifier":"10.13039/501100004608","name":"Natural Science Foundation of Jiangsu Province","awardNumber":"BK20230942, BK20251560","order_by":1},{"identity":"b3ebd7bf-ad00-4484-bb4a-4e2a1084869a","identifier":"10.13039/501100012226","name":"Fundamental Research Funds for the Central Universities","awardNumber":"30924010910","order_by":2}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Nanjing University of Science and Technology","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"covalent organic framework, bioorthogonal chemistry, cuproptosis, piezocatalytic therapy, tumor treatment","lastPublishedDoi":"10.21203/rs.3.rs-8806647/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8806647/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePiezocatalytic therapy (PCT) has garnered increasing interest in the field of cancer treatment. However, its therapeutic efficacy is hampered by the limitations of current piezoelectric materials and the intrinsic therapeutic resistance of tumors. Here, leveraging elaborately designed copper-coordinated covalent organic frameworks (CuCOF-2N) as novel piezocatalysts, a smart ultrasound-controlled nanoreactor (Cu2N@D-FA) is constructed by co-encapsulating with doxorubicin prodrug into folic acid-modified liposomes, to enhance the antitumor efficacy of PCT through the combination of cuproptosis and bioorthogonal catalysis. The structure-property comparison with related COFs underscores the important role of highly polar triazine rings and symmetry-disrupting bidentate ligands in enhancing piezoelectric performance of CuCOF-2N. Upon ultrasound irradiation, CuCOF-2N not only produces hydroxyl radicals independently of oxygen but also enables a self-sustained oxygen supply for generating superoxide anions and singlet oxygen, which surmount constraints of traditional piezoelectric materials to trigger strong PCT. Moreover, the mediated-copper valence switching in CuCOF-2N permits spatiotemporally precise bioorthogonal catalysis and triggers cuproptosis, overcoming therapeutic limitations and amplifying the PCT effect. In vivo studies demonstrate that Cu2N@D-FA selectively accumulates in the tumor and effectively eliminates the tumor without side-effects under US stimulation. Therefore, this study highlights the great promise of COFs in piezocatalysis and offers novel insights for enhancing PCT.\u003c/p\u003e","manuscriptTitle":"A sonopiezoresponse COF-based smart nanoreactor orchestrating in situ bioorthogonal chemistry and cuproptosis for enhanced tumor piezocatalytic therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-09 03:31:10","doi":"10.21203/rs.3.rs-8806647/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ae595588-151a-4e63-b28d-238322306026","owner":[],"postedDate":"February 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":62452682,"name":"Materials Chemistry"}],"tags":[],"updatedAt":"2026-02-09T03:31:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-09 03:31:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8806647","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8806647","identity":"rs-8806647","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}