Skip to main content
NCBI home page
Wiley Open Access Collection logoLink to Wiley Open Access Collection
. 2026 Jul 9;65(37):e6633730. doi: 10.1002/anie.6633730

COF@DNAzyme Empowering Endogenous Copper for One‐Stitch Bioorthogonal Catalysis‐Based Anticancer Therapy

Minhao Jiang 1,2, Fang Pu 1,2,✉, Yinuo Shu 1,2, Guoshi Xu 1,2, Jiawei Zhu 1,2, Yinghua Peng 3, Yanjie Zhang 1,2, Anjun Song 1,2, Jinsong Ren 1,2,✉, Xiaogang Qu 1,2,✉
PMCID: PMC13549064  PMID: 42424162

ABSTRACT

Bioorthogonal chemistry, particularly the copper‐catalyzed azide‐alkyne cycloaddition (CuAAC) reaction, holds great promise for in situ prodrug activation to minimize systemic toxicity in disease treatments. However, existing approaches mainly rely on exogenous copper catalysts, which pose a risk of disrupting copper homeostasis and suffer from catalyst deactivation caused by cellular components. Moreover, the staggered administration of catalysts and prodrugs complicates clinical translation and leads to unpredictable reaction time windows. Herein, we construct a “one‐stitch” bioorthogonal catalytic therapy system that harnesses endogenous copper without the need for copper supplements or external reductants. By integrating the DNAzyme CLICK‐17 and the tumor‐targeting aptamer AS1411 onto covalent organic framework (COF) nanoparticles, co‐delivery of both the catalyst and the prodrugs in a single nanoplatform (COF‐P@C‐A) is achieved. Upon internalization by tumor cells, high intracellular glutathione (GSH) and an acidic environment triggered the degradation of COF nanoparticles to simultaneously release CLICK‐17 and the prodrugs while consuming excess GSH, thereby alleviating copper sequestration and enhancing the availability of endogenous Cu(I). CLICK‐17 then efficiently catalyzes the CuAAC reaction for localized drug synthesis, maximizing therapeutic efficacy and minimizing off‐target effects. This strategy offers a safe, synchronized, and clinically translatable approach to bioorthogonal prodrug activation.

Keywords: bioorthogonal catalysis, covalent organic framework, DNAzyme, endogenous copper, one‐stitch

By using COF and DNAzyme to empower endogenous copper, a one‐stitch bioorthogonal catalytic therapy platform is constructed. In response to elevated GSH and acidity in tumor cells, the system disassembles to simultaneously release DNAzyme and prodrugs, harnessing endogenous copper to catalyze bioorthogonal reactions for in situ anticancer drug synthesis. This work offers a safe and efficient bioorthogonal catalysis‐based therapeutic strategy.

graphic file with name ANIE-65-e6633730-g005.webp

1. Introduction

Bioorthogonal chemistry has emerged as a powerful tool for labeling intracellular targets and modulating biological processes without disturbing native biomolecules or functions [1, 2, 3, 4, 5, 6, 7, 8, 9]. Its potential is particularly evident in prodrug activation therapy, where it enables the in situ synthesis of drugs to reduce systemic side effects [9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19]. As one of the classic reactions in bioorthogonal chemistry, the copper‐catalyzed azide‐alkyne cycloaddition (CuAAC) reaction enables rapid and efficient coupling under mild conditions [20, 21, 22, 23]. Its high selectivity and efficiency facilitate chemical synthesis and prodrug activation, holding great potential to reduce off‐target toxicity and address multidrug resistance. Currently, a variety of copper‐containing complexes and nanomaterials have been directly delivered into living systems for catalyzing CuAAC reactions [24, 25, 26, 27, 28, 29, 30, 31]. However, the extensive introduction of exogenous copper‐containing catalysts can disrupt intracellular copper homeostasis, potentially inducing side effects on human health, such as allergies, acute or chronic toxicity, and neurological abnormalities [21, 32, 33]. In addition, some cellular components may deactivate these copper catalysts. For example, overexpressed glutathione (GSH) in tumor cells, which serves as a chelating agent for Cu(II) as well as a reducing agent to form stable GSSG‐Cu(I) complexes, often compromises catalytic efficiency [34, 35, 36]. Another key concern is that the staggered administration of prodrugs and catalysts is adopted in in vivo applications to avoid premature activation of the prodrugs, which causes significant operational difficulty in clinical translation and an unpredictable temporal window for reaction occurrence [37, 38]. These challenges motivate ongoing efforts to explore new bioorthogonal chemistry‐based prodrug activation strategies.

Recently, leveraging endogenous copper to catalyze the CuAAC reaction was explored to minimize side effects [39, 40, 41, 42, 43, 44]. Owing to the limited intracellular copper pool and the sequestration by endogenous ligands, the concentration of free Cu(I) ions in living cells is insufficient to efficiently catalyze the CuAAC reaction. As a result, endogenously catalyzed CuAAC reactions strongly depend on the additional administration of external chemical reductants, such as sodium ascorbate, which can boost the level of Cu(I) [40, 42, 45]. The asynchronous dynamics of prodrug delivery and the valence state transformation of intracellular copper significantly influence the efficiency of drug activation. Therefore, approaches that directly harness intracellular copper with cell selectivity would be highly desirable. DNAzymes, known for their sequence‐specific and cofactor‐dependent catalytic features and low cost, were widely used for biosensing and bioimaging, as well as potential therapeutic tools for gene silencing [46, 47, 48]. CLICK‐17, a DNAzyme, which can mediate CuAAC reaction utilizing ultra‐low concentrations of either Cu(I) or Cu(II) without reductants, has been used for Cu(II) detection [49, 50]. Motivated by the properties of CLICK‐17, we used COF@DNAzyme to empower endogenous copper, constructing a “one‐stitch” bioorthogonal catalytic therapy system.

As shown in Scheme 1, CLICK‐17 and tumor cell‐targeting aptamer AS1411 are adsorbed onto the surface of tumor‐microenvironment‐responsive COF nanoparticles, which serve as nanocarriers of nucleic acids and protect them from degradation by nucleases within the circulation system. Concurrently, prodrugs are loaded within the pores of the COF nanoparticles, where the adsorbed nucleic acids can act as gatekeepers to prevent premature leakage of the prodrugs. After being engulfed by tumor cells, the disulfide bond (S‐S) and imine bond (–C═N–) incorporated COF nanoparticles can be gradually degraded by high concentrations of glutathione (GSH) and acid within tumor cells, releasing DNAzyme CLICK‐17 and the prodrug molecules simultaneously. This release process instantly increases the local concentrations of both the prodrugs and CLICK‐17, thereby avoiding premature activation and metabolism of prodrugs. Furthermore, it consumes the overexpressed GSH in tumor cells, which in turn reduces its sequestration effect on free copper ions and significantly enhances the utilization of endogenous copper. CLICK‐17 directly utilizes intracellular copper to efficiently catalyze the CuAAC reaction, enabling in situ drug synthesis for elimination of cancer cells. Meanwhile, the poor internalization by normal cells, along with their relatively low levels of GSH and copper, enables the system to perform cell‐selective catalysis, thereby avoiding damage to normal cells. Consequently, it maximizes the chemotherapy efficacy mediated by the CuAAC reaction while reducing side effects in anticancer therapy. This study provides a new avenue for developing safe and efficient bioorthogonal catalytic technologies.

SCHEME 1.

SCHEME 1

Open in a new tab

Design and fabrication of a one‐stitch bioorthogonal catalysis system for cancer therapy. (a) Illustration of the construction of COF‐P@C‐A. (b) In response to GSH and acid, COF‐P@C‐A disassembles to release DNAzyme and prodrugs, harnessing endogenous copper for in situ bioorthogonal antitumor drug synthesis.

2. Results and Discussion

2.1. Synthesis and Characterization of COF‐P@C‐A

First, COF nanoparticles were synthesized at room temperature via an imine condensation reaction between 5,10,15,20‐tetrakis(4‐formylphenyl)porphyrin (TFPP) and 4‐(2‐(4‐aminophenyl)disulfanyl)benzenamine (APDSBA), catalyzed by a Lewis acidic organocatalyst tris(pentafluorophenyl)boron (TPFB) (Figure 1a). The resulting COF nanoparticles were loaded with two prodrugs, 5‐azidobenzene‐1,3‐diol (PN3) and 4‐ethynylphenol (PYnyl), to obtain COF‐P. Then, a DNAzyme CLICK‐17 and an aptamer AS1411 were sequentially coated onto COF‐P through π–π stacking interactions to form COF‐P@C‐A. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed that COF‐P@C‐A were uniform spherical nanoparticles with a diameter from 120 to 180 nm (Figure 1b,c and Figures S1 and S2). Next, Fourier transform infrared (FTIR) spectroscopy was employed to investigate the formation of COF nanoparticles. As shown in Figure 1d, COF exhibited a characteristic signal at 1622 cm–1, corresponding to a C═N bond, confirming the formation of an imine linkage and the successful synthesis of the imine‐linked disulfide‐based COF. Powder x‐ray diffraction (PXRD) was performed to investigate the phase purity of COF. As shown in Figure 1e, COF exhibited characteristic peaks at 4.4°, 6.2°, and 8.6°, consistent with theoretical values [51]. N2 adsorption/desorption measurement at 77 K confirmed the permanent porosity of COF (Figure 1f). Its sorption profile can be described as a type IV isotherm, which is characteristic of mesoporous materials. N2 adsorption revealed an absorption capacity of 435.58 cm3 g−1 for the COF, and the BET surface area of the COF was calculated to be 376.9 m2 g−1 (Figure S3a). The aperture distribution curves showed that the pore size of COF was 1.2 nm (Figure S3b). The regular microporous structure and large surface area of COF make it suitable for loading small molecular compounds and adsorbing nucleic acids. High‐performance liquid chromatography (HPLC) was used to measure the loading content values of prodrugs PN3 and PYnyl, which were 12.23% and 13.72%, respectively (Figure S4). After sequentially loading the prodrugs, CLICK‐17, and AS1411, the zeta potential of the COF changed from −9.63 to −30.42, −34.68, and −40.96 mV, respectively, demonstrating their efficient adsorption (Figure 1g). The decrease in UV‐Vis absorption at 260 nm also confirmed the coating of CLICK‐17 onto the COF (Figure S5), with a loading amount of 0.1027 mg per mg of COF‐P and a loading efficiency of 9.31%. And then, a carboxyfluorescein (FAM)‐labeled AS1411 aptamer was used as a fluorescence probe to verify the adsorption of the aptamer (Figure S6). The loading amount was 0.005 mg per mg of COF‐P@C, with a loading efficiency of 0.5%. The above results demonstrated the successful preparation of COF‐P@C‐A.

FIGURE 1.

FIGURE 1

Open in a new tab

(a) Scheme of the synthesis of COF. (b) SEM image of COF‐P@C‐A. Scale bar = 200 nm. (c) TEM image of COF‐P@C‐A. Scale bar = 100 nm. (d) FT‐IR of TFPP (blue), APDSBA (red), and COF (black). (e) XRD patterns of COF. (f) N2 absorption and desorption curves of COF. (g) Zeta potentials of COF, COF‐P, COF‐P@C, and COF‐P@C‐A, respectively. Data were expressed as mean ± standard deviation (SD) (n = 3).

Subsequently, we investigated the dissociation behavior of COF‐P@C‐A in the presence of GSH (10 mM) at pH 5.5. To mimic the reductive tumor microenvironment, the GSH concentration was set at 10 mM because the intracellular GSH level in tumor cells is significantly higher than that in normal cells, typically reaching 10 mM. As shown in the TEM images, treatment with GSH at different pH values for 0, 1, 2, 4, and 8 h resulted in partial disintegration of COF‐P@C‐A, and the degree of dissociation increased with incubation time (Figure 2a–d and Figure S7), confirming that COF‐P@C‐A collapsed due to the cleavage of disulfide bonds in response to GSH and the cleavage of imine bonds under acidic conditions. The GSH‐responsive and acid‐triggered disassembly of COF‐P@C‐A and the consequent release of the loaded cargos were assessed by encapsulating Rhodamine B (RhB) as a fluorescent reporter within COF (termed COF‐RhB@C‐A). COF‐RhB@C‐A was treated with different concentrations of GSH for 4 h, followed by fluorescence measurements. As shown in Figure 2e, the fluorescence intensity of RhB released from COF‐RhB@C‐A in the absence of GSH was low. With the increase of GSH concentration, the fluorescence intensity of RhB increased, suggesting efficient release of RhB from COF‐RhB@C‐A in response to GSH. We then evaluated the time‐dependent cargo release under various conditions. As showed in Figure 2f and Figure S8, COF‐RhB@C‐A without GSH treatment at pH 7.0 exhibited the lowest release rate of RhB. The release rate at pH 5.5 was higher than that at pH 7.0. This pH‐dependent release is attributed to the acid‐triggered hydrolysis of imine bonds within the COF framework. Upon treating COF‐RhB@C‐A with GSH at pH 7.0, the release rate of RhB also increased, resulting from GSH‐caused cleavage of disulfide bonds. Under simulated tumor microenvironment conditions (10 mM GSH, pH 5.5), approximately 74.55% of RhB was released from COF‐RhB@C‐A within 12 h. These results directly demonstrate the GSH‐ and acidity‐responsive drug release behavior of the nanocomposite. Furthermore, the time‐dependent release of RhB from COF‐RhB@C‐A and COF‐RhB in PBS (pH = 7.0) was tested (Figure 2g). The release profiles showed that COF‐RhB@C‐A loaded with CLICK‐17 and AS1411 exhibited a much slower RhB release rate compared to COF‐RhB, demonstrating that the adsorbed nucleic acids can act as gatekeepers to prevent premature leakage of the prodrugs. Furthermore, we evaluated the protective ability of COF for CLICK‐17 using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). As shown in Figure S9, the band in lane 2 disappeared after DNase I treatment, indicating complete degradation of CLICK‐17. In contrast, the band in lane 4 showed only a decrease in intensity compared to lane 3, confirming that COF acts as an effective carrier to protect CLICK‐17 from nuclease degradation. Collectively, these results demonstrate that COF functions as an effective nanocarrier capable of loading prodrugs and accurately controlling release in response to dual biological stimuli.

FIGURE 2.

FIGURE 2

Open in a new tab

(a) TEM image of COF‐P@C‐A treated with PBS (pH = 7.0) for 2 h. (b) TEM image of COF‐P@C‐A treated with PBS (pH = 5.5) for 2 h. (c) TEM image of COF‐P@C‐A treated with GSH (10 mM, pH = 7.4) for 2 h. (d) TEM image of COF‐P@C‐A treated with GSH (10 mM, pH = 5.5) for 2 h. (e) The release of RhB from COF‐RhB@C‐A under different concentrations of GSH. (f) Time‐dependent release of RhB from COF‐RhB@C‐A after treatment with PBS (pH = 7.0 and 5.5) and 10 mM GSH in PBS (pH = 7.0 and 5.5) (n = 3). (g) Time‐dependent release of RhB from COF‐RhB@C‐A and COF‐RhB treated with PBS (pH = 7.0) (n = 3) (h) GSH consumption treated with different concentration of COF‐P@C‐A (n = 3) (i) GSH consumption treated with COF‐P@C‐A at pH = 7.0 or 5.5 for 0, 1, and 2 h (n = 3). (j) Fluorescence spectra of the COF‐F@C‐A‐catalyzed click reaction with different GSH concentrations (0, 2.5, 5, 7.5, and 10 mM) in the absence of Cu(II) for 2 h. (k) Fluorescence spectra of the COF‐F@C‐A‐catalyzed click reaction with different Cu(II) concentrations (0, 2.5, 5, 10, and 20 µM) in the absence of GSH for 2 h. (l) Fluorescence spectra of the COF‐F@C‐A‐catalyzed click reaction with different Cu(II) concentrations (0, 2.5, 5, 10, and 20 µM) in the presence of GSH (10 mM) for 2 h. (m) Fluorescence spectra of the COF‐F@A‐catalyzed click reaction with different Cu(II) concentrations (0, 2.5, 5, 10, and 20 µM) in the presence of GSH (10 mM) for 2 h. (n) Fluorescence spectra of the COF‐F@C‐A‐catalyzed click reaction at different reaction times (0, 10, 30, 60, 90, 120, and 240 min) in the presence of Cu(II) (20 µM). (o) Fluorescence spectra of the COF‐F@C‐A‐catalyzed click reaction with different GSH concentrations (0, 2.5, 5, 7.5, and 10 mM) in the presence of Cu(II) (20 µM) for 2 h. (p) Fluorescence spectra of the CLICK‐17‐catalyzed click reaction at different conditions: (I) FYnyl + FN3 + Cu2+; (II) FYnyl + FN3 + CLICK‐17 + GSH + Cu2+; (III) FYnyl + FN3 + CLICK‐17 + GSH+Cu2+ + COF; (IV) FYnyl + FN3 + CLICK‐17 + Cu2+. All data were expressed as mean ± standard deviation (SD).

GSH can trigger the cleavage of disulfide bonds, and GSH itself is also consumed by generating glutathione disulfide (GSSG). Therefore, COF not only mediated GSH‐responsive drug release but also depleted the elevated GSH in tumor cells. The ability of COF to deplete GSH was quantitatively confirmed by Ellman's assay. As shown in Figure 2h and Figure S10, the consumption of GSH increased with the concentration of COF from 0 to 1 mg mL−1. The extent of GSH consumption exhibited a time‐dependent increase during incubation (Figure S11). Furthermore, the GSH consumption rate was markedly higher at pH 5.5 than that at pH 7.4, which can be attributed to the accelerated degradation of COF under acidic conditions (Figure 2i). This degradation exposed additional disulfide bonds, thereby enhancing their reaction with GSH. To the end, this depletion might reduce the sequestration of free copper ions by GSH, thereby significantly enhancing the bioavailability of endogenous copper.

2.2. Catalytic Performance of the COF‐P@C‐A in Vial

Next, the catalytic activity of the obtained nanoparticles in the absence and presence of Cu(II) in solution was investigated. To enable direct monitoring of the catalytic reaction, 3‐azido‐7‐hydroxycoumarin (FN3) and phenylacetylene (FYnyl), which can form the fluorescent triazole product (FTrz), were loaded into COF, followed by coating with CLICK‐17 and AS1411 (named as COF‐F@C‐A). COF loaded with precursors and coated with AS1411 (named COF‐F@A) was used as a control. As shown in Figure 2j, the supernatant of COF‐F@C‐A mixed with different concentrations of GSH did not exhibit fluorescence changes in the absence of Cu(II). It is reported that copper ions are highly accumulated in tumor cells as they exert a crucial role in cell proliferation, angiogenesis, and metastasis, with the concentrations in patients ranging from 0.3 to 20 µM. In the absence of GSH, COF‐F@C‐A was exposed to varying concentrations of Cu(II) (0, 2.5, 5, 10, and 20 µM). Little fluorescence was observed (Figure 2k), indicating that no fluorescent product was formed. This may be attributed to the fact that the precursors and CLICK‐17 loaded in the COF cannot be released in the absence of GSH. As a result, a bioorthogonal reaction could not occur. After co‐incubating COF‐F@C‐A with GSH (10 mM) for 120 min, different concentrations of Cu(II) were added. As shown in Figure 2l, the fluorescence intensity increased significantly with the increase of copper concentration. The results indicated that the precursors and CLICK‐17 could be released from COF‐F@C‐A in response to GSH and CLICK‐17 directly utilized Cu(II) to catalyze the formation of fluorescent product FTrz without the need for external reducing agents. As a comparison, COF‐F@A was treated with GSH (10 mM) and then incubated with different concentrations of Cu(II). As shown in Figure 2m, little fluorescence was observed. The result suggested that COF‐F@A without CLICK‐17 could not catalyze bioorthogonal reactions, even in the presence of Cu(II). CLICK‐17 is the key component that endows the system with bioorthogonal catalytic ability. Subsequently, we investigated the reaction kinetics and catalytic efficiency of COF‐F@C‐A. As shown in Figure 2n, the fluorescence intensity of product FTrz increased in a time‐dependent manner within 240 min. These indicated that the DNAzyme efficiently harnessed Cu(II) to catalyze CuAAC reactions and continued for a relatively long period of time. Furthermore, we investigated the influence of GSH levels on the bioorthogonal rate. The results indicated that the fluorescence intensity of product FTrz depended on the GSH concentration and dropped to a negligible level in the absence of GSH (Figure 2o). The catalytic differences indicated that GSH led to the dissociation of COF and subsequent release of CLICK‐17 and precursors.

We have demonstrated that COF not only served as a GSH‐responsive carrier but also consumed the elevated GSH in tumor cells. Furthermore, we evaluated the effect of GSH depletion on catalytic reactions. As shown in Figure S12, the catalytic activity increased with increasing concentrations of CLICK‐17 and Cu(II) and exhibited time‐dependent behavior. To demonstrate the inhibitory effect of GSH on the CuAAC reaction and the “detoxifying” role of COF, we evaluated the catalytic ability of CLICK‐17 in the presence of Cu(II) under GSH and GSH + COF conditions. As shown in Figure 2p, only weak product fluorescence was observed in the presence of GSH, whereas a significant fluorescence signal was restored upon addition of COF. These results indicated that GSH could sequester Cu(II), thereby limiting its availability for CLICK‐17‐mediated catalysis, while COF rescued catalytic activity by depleting GSH and releasing bioavailable copper. To investigate CLICK‐17‐induced valence conversion of copper, neocuproine was used as an indicator. As shown in Figures S13 and S14, neocuproine exhibited a characteristic UV‐vis absorption peak at 457 nm upon adding Cu(II) and did not respond to other metal ions (magnesium, manganese, calcium, etc.), suggesting that CLICK‐17 selectively reduces Cu(II) to Cu(I). Taken together, these results demonstrated the capability of COF‐F@C‐A to exploit intracellular copper for efficient bioorthogonal catalysis.

2.3. Endocytosis and Intracellular Catalytic Behavior of Nanoparticles

After demonstrating the in vitro catalytic activity of COF‐F@C‐A, its intracellular performance was explored. To monitor the internalization and distribution of COF‐F@C‐A in cancer cells, COF‐RhB@C‐A as a reporter was co‐incubated with 4T1 cells for fluorescence tracking. As shown in confocal laser scanning microscopy (CLSM) images, 4T1 cells treated with COF‐RhB@C‐A exhibited strong red fluorescence in the cytoplasm, reaching a maximum intensity at 4 h (Figure 3a and Figure S15). Meanwhile, strong red fluorescence in the cytoplasm of 4T1 cells treated with COF‐RhB@C was observed at 12 h, confirming the aptamer AS1411 enhances cellular uptake of COF‐RhB@C‐A by specifically targeting nucleolin of cancer cells (Figure S16). However, normal 3T3 cells (embryonic fibroblasts) treated with COF‐RhB@C‐A presented weak fluorescence at 4 h (Figure 3b and Figure S17). The result confirmed that COF‐RhB@C‐A could be efficiently internalized by cancer cells, resulting from the targeting ability of the aptamer AS1411. It is reported that the copper levels in tumor cells are higher than those in normal cells. To investigate the differential copper levels in tumor cells and normal cells, Rhodamine B hydrazide (RBH), a fluorescent probe of Cu(II), was added to the cells after different treatments. As shown in Figure 3c–f, 4T1 cells displayed strong red fluorescence, while 3T3 cells exhibited minimal fluorescence. The results indicated that tumor cells have a higher concentration of copper enrichment compared to normal cells. The result of inductively coupled plasma mass spectrometry (ICP−MS) further confirmed that intracellular copper levels in 4T1 tumor cells were significantly higher than those in 3T3 normal cells (Figure S18). The differential copper levels are a prerequisite for cell‐selective catalysis and biosafety.

FIGURE 3.

FIGURE 3

Open in a new tab

(a) CLSM images of COF‐RhB@C‐A internalized by 4T1 cells at different times (0‐24 h). Scale bar = 100 µm. (b) CLSM images of COF‐RhB@C‐A internalized by 3T3 cells at different times (0–24 h). Scale bar = 100 µm. (c) Schematic diagram of the intracellular CuAAC reaction. (d) Schematic diagram of RBH for detecting endogenous copper (II). (e) CLSM images of 4T1 cells after different treatments and corresponding statistical analysis of relative fluorescence intensity. Scale bar = 100 µm. (f) CLSM images of 3T3 cells after different treatments and corresponding statistical analysis of relative fluorescence intensity. Scale bar = 100 µm.

Then, the CuAAC reaction was determined after the addition of COF‐F@C‐A to 4T1 cells. As shown in Figure 3c–f, 4T1 cells treated with only precursors or COF‐F@A exhibited negligible blue fluorescence, implying that high levels of copper in tumor cells could not effectively catalyze bioorthogonal reactions in the absence of CLICK‐17. Cells treated with COF‐F@C‐A showed strong blue fluorescence, indicating that COF‐F@C‐A could significantly catalyze the intracellular CuAAC reaction. To further verify the adaptive ability of COF‐F@C‐A, a comparative experiment was conducted by co‐incubating COF‐F@C‐A with 3T3 cells. Compared with 4T1 cells, 3T3 cells treated with COF‐F@C‐A exhibited negligible emission (Figure 3e,f). The finding, on the one hand, suggested that COF was more easily degraded in 4T1 cells due to their higher endogenous GSH levels, leading to more release of precursors and DNAzyme. On the other hand, it indicated that COF‐F@C‐A could harness the high levels of endogenous copper within cancer cells to catalyze the CuAAC reaction. The poor internalization ability of normal cells, as well as their low levels of GSH and copper, make it difficult for bioorthogonal reactions to occur.

The differential degradation of the disulfide‐COF carrier, driven by the disparate endogenous GSH concentrations between tumor and normal cells, resulted in superior catalytic selectivity and enhanced biosafety of the COF nanoparticles for tumor treatment. In turn, the intracellular degradation of COF was accompanied by depletion of GSH. As shown in Figure 4a, compared with the PBS group, COF‐F@C‐A treatment led to a significant decrease in intracellular GSH levels and an increase in GSSG levels. Furthermore, intracellular Cu(I) levels were using the Cu(I)‐specific fluorescent probe CopperSensor‐1 (CS1). As shown in Figure S19, after treatment with COF‐P@C‐A, the intracellular Cu(I) level increased significantly. It results not only from the ability of CLICK‐17 to activate Cu(II) to Cu(I) but also from GSH depletion by COF, which enhances the bioavailability of Cu(I). This concurrently eliminated the inhibitory effect of GSH on the CuAAC reaction, thereby promoting the formation of the desired product.

FIGURE 4.

FIGURE 4

Open in a new tab

(a) Intracellular levels of GSH and GSSG following treatment under different conditions (n = 3). (b) Schematic diagram of in situ RESV analog synthesis by endogenous Cu. (c) Viability of 4T1 cells treated with PYnyl at different concentrations (from 0 to 200 µM) (n = 3). (d) Viability of 4T1 cells treated with PN3 at different concentrations (from 0 to 200 µM) (n = 3). (e) Viability of 4T1 cells treated with PYnyl+PN3 at different concentrations (from 0 to 200 µM) (n = 3). Statistical significance was calculated by one‐way ANOVA with Tukey's post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. (f) Viability of 4T1 cells treated with COF at different concentrations (from 0 to 250 mg mL−1) (n = 3). (g) Viability of 4T1 cells treated with COF‐P@A at different concentrations (from 0 to 250 mg mL−1) (n = 3). (h) Viability of 4T1 cells treated with COF‐P@C‐A at different concentrations (from 0 to 250 mg mL−1) (n = 3). (i) CLSM images of live/dead staining and corresponding statistical analysis of relative fluorescence intensity, scale bar = 100 µm. (j) Flow cytometry analysis of the apoptosis rate of 4T1 cells via Annexin V‐FITC and 7‐AAD. All data were expressed as mean ± SD.

After confirming the intracellular catalytic ability of COF‐F@C‐A, a resveratrol analog ([5‐(4‐(4‐hydroxyphenyl)‐1H‐1,2,3‐triazol‐1‐yl)benzene‐1,3‐diol], PRES), which has been proved to possess antitumor properties, was used as a model compound for investigating in situ drug synthesis catalyzed by COF‐P@C‐A (Figure 4b). We synthesized prodrugs PN3 (Figure S20) and PYnyl and then tested their cytotoxicity using the MTT assay. As shown in Figure 4c–e, the cytotoxicity of PYnyl, PN3, or their combination on 4T1 cells was negligible even at a concentration of 200 µM. COF and COF‐P@A also exhibited minimal toxicity at a concentration of 250 µg mL− 1, confirming the biocompatibility of the inactive components (Figure 4f, g). Notably, treatment with COF‐P@C‐A resulted in a significant decrease in the viability of 4T1 cells, indicating that PRES was successfully synthesized and exhibited anti‐tumor effects (Figure 4h). In contrast, 3T3 cells exposed to the same concentration of COF‐P@C‐A exhibited a high survival rate (Figure S21). This indicated that COF‐P@C‐A could selectively generate drugs in tumor cells through bioorthogonal reactions, achieving efficient tumor cell killing. This selectivity arises not only from the targeting function of the AS1411 aptamer but also from the differential levels of GSH and endogenous copper between tumor cells and normal cells. The effect of different formulations on cells was further determined by double staining experiments, in which live and dead cells were indicated by green and red fluorescence, respectively. As shown in Figure 4i and Figure S22, the COF and COF‐P@A groups presented green fluorescence, indicating minimal cell death, while the COF‐P@C‐A group exhibited red fluorescence, suggesting a significant cell‐killing effect. In addition, fluorescein annexin V and 7‐aminoactinomycin D (7‐AAD) staining assays were performed (Figure 4j and Figure S23). The result indicated that the apoptosis rate of cancer cells dramatically increased after incubation with COF‐P@C‐A compared to the cells in the other groups. Furthermore, the LC−MS result confirmed the generation of product PRES, demonstrating that the CuAAC reaction occurred in cells treated with COF‐P@C‐A (Figures S24–S26). Taken together, these results demonstrated that the co‐delivery of prodrugs and DNAzyme using COF could empower endogenous copper within tumor cells to facilitate an intracellular CuAAC reaction, thereby achieving the in situ synthesis of the active drug. Specifically, in normal cells with low endogenous GSH and copper levels, the bioorthogonal reaction was constrained even in the presence of DNAzyme. This dual requirement for elevated endogenous GSH and copper ensures precise activation and potent cytotoxic effects within cancer cells, while simultaneously minimizing off‐target toxicity in healthy tissues.

2.4. In Vivo Anticancer Effect

Encouraged by the excellent therapeutic effects at the cellular level, we further investigated the in vivo anticancer efficacy of COF‐P@C‐A. First, we established a mouse model to evaluate the toxicology of COF‐P@C‐A. As shown in Figure S27, the low hemolytic activity of COF‐P@C‐A demonstrates its high biocompatibility and suitability for intravenous administration. Hematological analyses and blood biochemical tests revealed no significant differences between the control group and the group treated with COF‐P@C‐A (Figures S28, S29). In addition, the injection of COF‐P@C‐A did not significantly affect the body weight of the mice throughout the entire treatment period (Figure S30). Hematoxylin and eosin (H&E) staining of major organs showed no evidence of organ damage or adverse reactions after the administration of COF‐P@C‐A (Figure S31). These results indicated favorable biocompatibility of COF‐P@C‐A, supporting its potential for further biological applications.

After intravenous injection of COF‐RhB@C‐A into 4T1 tumor‐bearing mice, the biodistribution was monitored at 0 and 24 h (Figure 5a,b). The result indicated a selective accumulation of COF‐RhB@C‐A in the tumor tissue. To evaluate the in vivo therapeutic performance of COF‐P@C‐A, we established a 4T1 tumor‐bearing mouse model and randomly assigned the mice to five groups: (1) PBS; (2) COF; (3) COF‐P@A; (4) PYnyl + PN3; and (5) COF‐P@C‐A. Different formulations were administered through intravenous injection. Since the prodrugs and catalyst were co‐delivered by a targeted carrier and there is no need for additional reducing agents, the administration is a “one‐stitch” injection. The single administration of the nanoformulation, which contains all the components required for the in situ bioorthogonal reaction, rather than separately injecting the catalyst and prodrugs as previously reported, greatly simplified the operation procedure and improved drug bioavailability. Tumor size of the mice was measured every 2 days for each group. As shown in Figure 5c–e and Figure S32, compared to the control groups, treatment with COF‐P@C‐A significantly suppressed tumor growth and demonstrated significant antitumor efficacy. There were no notable weight differences among the groups (Figure 5f). Furthermore, H&E staining of tumors from the different treatment groups confirmed the potent antitumor efficacy of COF‐P@C‐A (Figure 5g), while H&E images of major organs showed minimal side effects on mice after treatment (Figure S33). TUNEL staining revealed that apoptotic cells increased obviously after treatment with COF‐P@C‐A, suggesting that the cancer cells were seriously damaged by the in situ‐synthesized product PRES (Figure 5h and Figure S34). These results demonstrated potent antitumor efficacy of COF‐P@C‐A in vivo without inducing significant adverse effects.

FIGURE 5.

FIGURE 5

Open in a new tab

Distribution of RhB‐labeled COF‐P@C‐A in major organs and tumors at (a) 0 h and (b) 24 h, respectively. (c) Images of the dissected tumors after various treatments (n = 5). (d) Weight of the dissected tumors after various treatments (n = 5). (e) Volume of the dissected tumors after various treatments (n = 5). (f) Body weight of the mice after various treatments (n = 5). (g) H&E staining images of tumors from mice treated with different groups. Scale bar = 50 µm. (h) TUNEL staining images of primary tumor slices with different treatments. Scale bar = 50 µm. All data were expressed as mean ± SD.

3. Conclusion

In summary, we developed a one‐stitch bioorthogonal catalytic platform for tumor therapy, which selectively synthesized a highly effective anti‐cancer drug at the tumor site using endogenous copper, without disrupting copper homeostasis and requiring an external metal‐containing catalyst. The intracellular activation of COF‐P@C‐A was initiated with its response to highly expressed GSH, leading to GSH consumption and degradation of the carrier, which subsequently induced the simultaneous release of the prodrugs and DNAzyme. CLICK‐17 could empower endogenous copper in tumors for in situ bioorthogonal reactions. Collectively, the platform combines good biocompatibility, high selectivity for intracellular synthesis, potent antitumor activity, and negligible adverse effects in both in vitro and in vivo experiments. Our findings offer a novel strategy for developing safe and efficient bioorthogonal catalytic systems for therapeutic applications.

Author Contributions

Minhao Jiang: conceptualization, investigation, validation, writing – original draft. Fang Pu: conceptualization, funding acquisition, writing – review and editing, project administration, writing – original draft. Yinuo Shu: investigation. Guoshi Xu: investigation. Jiawei Zhu: investigation. Yinghua Peng: investigation, resources. Yanjie Zhang: investigation. Anjun Song: investigation. Jinsong Ren: conceptualization, funding acquisition, project administration, writing – review and editing. Xiaogang Qu: funding acquisition, writing – review and editing, supervision.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Additional supporting information can be found online in the Supporting Information section. Supplementary Information including Materials and Methods, Figures S1–S34, and supporting references. Supporting File 1: anie73537‐sup‐0001‐SuppMat.docx.

ANIE-65-e6633730-s001.docx (4.4MB, docx)

Acknowledgments

This work was supported by the National Natural Science Foundation of China (22477119, 22277117, 22237006, and T2495262), the National Key Research and Development Program of China (2022YFA1205804), the Natural Science Foundation of Jilin Province (20240101190JC), and the Changchun Science and Technology Development Plan Project (2024GZZ11).

Contributor Information

Fang Pu, Email: pufang@ciac.ac.cn.

Jinsong Ren, Email: jren@ciac.ac.cn.

Xiaogang Qu, Email: xqu@ciac.ac.cn.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  • 1. Prescher J. A. and Bertozzi C. R., “Chemistry in Living Systems,” Nature Chemical Biology 1 (2005): 13–21, 10.1038/nchembio0605-13. [DOI] [PubMed] [Google Scholar]
  • 2. Yusop R. M., Unciti‐Broceta A., Johansson E. M. V., Sánchez‐Martín R. M., and Bradley M., “Palladium‐Mediated Intracellular Chemistry,” Nature Chemistry 3 (2011): 239–243, 10.1038/nchem.981. [DOI] [PubMed] [Google Scholar]
  • 3. Tonga G. Y., Jeong Y., and Duncan B., “Supramolecular Regulation of Bioorthogonal Catalysis in Cells Using Nanoparticle‐Embedded Transition Metal Catalysts,” Nature Chemistry 7 (2015): 597–603, 10.1038/nchem.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Wang J., Cheng B., and Li J., “Chemical Remodeling of Cell‐Surface Sialic Acids Through a Palladium‐Triggered Bioorthogonal Elimination Reaction,” Angewandte Chemie International Edition 54 (2015): 5364–5368, 10.1002/anie.201409145. [DOI] [PubMed] [Google Scholar]
  • 5. Konč J., Sabatino V., and Jiménez‐Moreno E., et al., “Controlled In‐Cell Generation of Active Palladium(0) Species for Bioorthogonal Decaging,” Angewandte Chemie International Edition 61 (2022): e202113519. [DOI] [PubMed] [Google Scholar]
  • 6. Liu Z., Liu Z., Sun M., Zhang W., Ren J., and Qu X., “An Antibody Engineered Metalloenzyme for Mediating Cell–Cell Communication and Activation of Immuno‐ and Chemotherapy,” Nano Letters 23 (2023): 6424–6432, 10.1021/acs.nanolett.3c01186. [DOI] [PubMed] [Google Scholar]
  • 7. Garcia‐Peiro J. I., Ortega‐Liebana M. C., and Adam C., “Dendritic Platinum Nanoparticles Shielded by Pt‐S Pegylation as Intracellular Reactors for Bioorthogonal Uncaging Chemistry,” Angewandte Chemie International Edition 64 (2025): e202424037, 10.1002/anie.202424037. [DOI] [PubMed] [Google Scholar]
  • 8. Zhang Y., Lei F., and Qian W., “Designing Intelligent Bioorthogonal Nanozymes: Recent Advances of Stimuli‐Responsive Catalytic Systems for Biomedical Applications,” Controlled Release 373 (2024): 929–951, 10.1016/j.jconrel.2024.07.073. [DOI] [PubMed] [Google Scholar]
  • 9. Wu J., Cheng T., and Xie J., “Anti‐CD24 Antibody‐Nitric Oxide Donor Conjugates Bearing a Self‐Bioorthogonal Cleavable Linker,” Acta Pharmaceutica Sinica B 15 (2025): 5366–5386, 10.1016/j.apsb.2025.07.037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Wang X., Liu Y., and Fan X., “Copper‐Triggered Bioorthogonal Cleavage Reactions for Reversible Protein and Cell Surface Modifications,” Journal of the American Chemical Society 141 (2019): 17133–17141, 10.1021/jacs.9b05833. [DOI] [PubMed] [Google Scholar]
  • 11. Chen Z., Li H., and Bian Y., “Bioorthogonal Catalytic Patch,” Nature Nanotechnology 16 (2021): 933–941, 10.1038/s41565-021-00910-7. [DOI] [PubMed] [Google Scholar]
  • 12. Huang C., Zhao C., and Deng Q., “Hydrogen‐Bonded Organic Framework‐Based Bioorthogonal Catalysis Prevents Drug Metabolic Inactivation,” Nature Catalysis 6 (2023): 729–739, 10.1038/s41929-023-00999-0. [DOI] [Google Scholar]
  • 13. Zhang L., Sang Y., and Liu Z., “Liquid Metal as Bioinspired and Unusual Modulator in Bioorthogonal Catalysis for Tumor Inhibition Therapy,” Angewandte Chemie International Edition 62 (2023): e202218159, 10.1002/anie.202218159. [DOI] [PubMed] [Google Scholar]
  • 14. Feng S., Zhang Y., and Gao Y., “A Gene‐Editable Palladium‐Based Bioorthogonal Nanoplatform Facilitates Macrophage Phagocytosis for Tumor Therapy,” Angewandte Chemie International Edition 62 (2023): e202313968, 10.1002/anie.202313968. [DOI] [PubMed] [Google Scholar]
  • 15. Zhang Y., Zhang L., and Wang W., “A DNA‐Gated and Self‐Protected Bioorthogonal Catalyst for Nanozyme‐Assisted Safe Cancer Therapy,” Angewandte Chemie International Edition 62 (2023): e202306395, 10.1002/anie.202306395. [DOI] [PubMed] [Google Scholar]
  • 16. Liu Z., Zhang W., and Zhao H., “Light‐Controlled Bioorthogonal Chemistry Altered Natural Killer Cell Activity for Boosted Adoptive Immunotherapy,” Angewandte Chemie International Edition 63 (2024): e202411905, 10.1002/anie.202411905. [DOI] [PubMed] [Google Scholar]
  • 17. Yu S., Jang D., and Kang H., “Photoinduced Plasmon Electron Transfer‐Based Bioorthogonal Cleavage Reaction for Precision Tumor Therapy,” Advanced Materials 37 (2025): 2418134, 10.1002/adma.202418134. [DOI] [PubMed] [Google Scholar]
  • 18. Gao Z., Qin K., and He W., “Chemocatalytic Cell Tagging Platform for Recording Cell‐Cell Interactions via Engineered Palladium‐Based Artificial Metalloenzymes,” Angewandte Chemie International Edition 64 (2025): e202424738, 10.1002/anie.202424738. [DOI] [PubMed] [Google Scholar]
  • 19. Zhang Y., Song A., and Pu F., “A Logic‐Gated Nanogenerator as a Bioorthogonal Catalysis‐Mediated Multi‐Functional Immune Modulator for Antitumor Therapy,” Chemistry 12 (2026): 102792, 10.1016/j.chempr.2025.102792. [DOI] [Google Scholar]
  • 20. Agard N. J., Prescher J. A., and Bertozzi C. R., “A Strain‐Promoted [3 + 2] Azide−Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems,” Journal of the American Chemical Society 126 (2004): 15046–15047, 10.1021/ja044996f. [DOI] [PubMed] [Google Scholar]
  • 21. Clavadetscher J., Hoffmann S., and Lilienkampf A., “Copper Catalysis in Living Systems and In Situ Drug Synthesis,” Angewandte Chemie International Edition 55 (2016): 15662–15666, 10.1002/anie.201609837. [DOI] [PubMed] [Google Scholar]
  • 22. Xia L., Chen M., and Dong C., “Spatiotemporal Ultrasound‐Driven Bioorthogonal Catalytic Therapy,” Advanced Materials 35 (2022): 2209179, 10.1002/adma.202209179. [DOI] [PubMed] [Google Scholar]
  • 23. Prakasham A. P., Singh H., and Palmans A. R. A., “Enhancing the Cuaac Efficiency of a Cu(I)–Nhc Complex in Biological Media by Encapsulation,” Chemical Communications 61 (2025): 9697–9700, 10.1039/D5CC01891A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Wang F., Zhang Y., and Liu Z., “A Biocompatible Heterogeneous Mof–Cu Catalyst for In Vivo Drug Synthesis in Targeted Subcellular Organelles,” Angewandte Chemie International Edition 58 (2019): 6987–6992, 10.1002/anie.201901760. [DOI] [PubMed] [Google Scholar]
  • 25. Liu C., Niu J., Cui T., Ren J., and Qu X., “A Motor‐Based Carbonaceous Nanocalabash Catalyst for Deep‐Layered Bioorthogonal Chemistry,” Journal of the American Chemical Society 144 (2022): 19611–19618, 10.1021/jacs.2c09599. [DOI] [PubMed] [Google Scholar]
  • 26. You Y., Deng Q., and Wang Y., “DNA‐Based Platform for Efficient and Precisely Targeted Bioorthogonal Catalysis in Living Systems,” Nature Communications 13 (2022): 1459, 10.1038/s41467-022-29167-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Huang H., Xuan W., and Hai J., “Nir‐Ii Light‐Activated and Cu Nanocatalyst‐Enabled Bioorthogonal Reaction in Living Systems for Efficient Tumor Therapy,” Nano Today 59 (2024): 102483, 10.1016/j.nantod.2024.102483. [DOI] [Google Scholar]
  • 28. Man X., Li W., and Zhu M., “Anticancer Tetranuclear Cu(I) Complex Catalyzes a Click Reaction to Synthesize a Chemotherapeutic Agent In Situ to Achieve Targeted Dual‐Agent Combination Therapy for Cancer,” Angewandte Chemie International Edition 63 (2024): e202411846, 10.1002/anie.202411846. [DOI] [PubMed] [Google Scholar]
  • 29. Wu L., Lin H., and Cao X., “Bioorthogonal Cu Single‐Atom Nanozyme for Synergistic Nanocatalytic Therapy, Photothermal Therapy, Cuproptosis and Immunotherapy,” Angewandte Chemie International Edition 63 (2024): e202405937, 10.1002/anie.202405937. [DOI] [PubMed] [Google Scholar]
  • 30. Ma M., Yuan W., Zhong W., Cheng Y., Yao H., and Zhao Y., “In‐Situ Activation of Biomimetic Single‐Site Bioorthogonal Nanozyme for Tumor‐Specific Combination Therapy,” Biomaterials 312 (2025): 122755, 10.1016/j.biomaterials.2024.122755. [DOI] [PubMed] [Google Scholar]
  • 31. Ouyang D., Yang R., and Yao Y., “In Situ Protac Synthesis Enabled by Pathologically Activated Bioorthogonal Catalysis for Precision Cancer Therapy,” Journal of the American Chemical Society 147 (2025): 39987–39998, 10.1021/jacs.5c15945. [DOI] [PubMed] [Google Scholar]
  • 32. Taiariol L., Chaix C., Farre C., and Moreau E., “Click and Bioorthogonal Chemistry: The Future of Active Targeting of Nanoparticles for Nanomedicines?” Chemical Reviews 122 (2021): 340–384, 10.1021/acs.chemrev.1c00484. [DOI] [PubMed] [Google Scholar]
  • 33. Tsvetkov P., Coy S., and Petrova B., “Copper Induces Cell Death by Targeting Lipoylated TCA Cycle Proteins,” Science 375 (2022): 1254–1261, 10.1126/science.abf0529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Singleton W. C. J., McInnes K. T., and Cater M. A., “Role of Glutaredoxin1 and Glutathione in Regulating the Activity of the Copper‐Transporting P‐Type Atpases, Atp7a and Atp7b,” Journal of Biological Chemistry 285 (2010): 27111–27121, 10.1074/jbc.M110.154468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Bhattacharjee A., Chakraborty K., and Shukla A., “Cellular Copper Homeostasis: Current Concepts on Its Interplay With Glutathione Homeostasis and Its Implication in Physiology and Human Diseases,” Metallomics 9 (2017): 1376–1388, 10.1039/C7MT00066A. [DOI] [PubMed] [Google Scholar]
  • 36. Chung C. Y.‐S., Posimo J. M., and Lee S., “Activity‐Based Ratiometric Fret Probe Reveals Oncogene‐Driven Changes in Labile Copper Pools Induced by Altered Glutathione Metabolism,” Proceedings of the National Academy of Sciences 116 (2019): 18285–18294, 10.1073/pnas.1904610116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Wang L., Jing P., and Tan J., “'One‐Stitch' Bioorthogonal Prodrug Activation Based on Cross‐Linked Lipoic Acid Nanocapsules,” Biomaterials 273 (2021): 120823, 10.1016/j.biomaterials.2021.120823. [DOI] [PubMed] [Google Scholar]
  • 38. Wu S., Zhang L., Wei Y., Cui T., Ren J., and Qu X., “An All‐in‐One Bioorthogonal System for Precise Cancer Therapy,” Chemistry of Materials 34 (2022): 8544–8550, 10.1021/acs.chemmater.2c01130. [DOI] [Google Scholar]
  • 39. Xue X., Qian C., and Tao Q., “Using Bio‐Orthogonally Catalyzed Lethality Strategy to Generate Mitochondria‐Targeting Anti‐Tumor Metallodrugs In Vitro and In Vivo,” National Science Review 8 (2021): nwaa286, 10.1093/nsr/nwaa286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Zhu J., You Y., Zhang W., Pu F., Ren J., and Qu X., “Boosting Endogenous Copper(I) for Biologically Safe and Efficient Bioorthogonal Catalysis via Self‐Adaptive Metal–Organic Frameworks,” Journal of the American Chemical Society 145 (2023): 1955–1963, 10.1021/jacs.2c12374. [DOI] [PubMed] [Google Scholar]
  • 41. Zhu J., You Y., and Zhang W., “Sensitizing Cuproptosis by Endogenous Copper‐Triggered Bioorthogonal Nanoremodeler,” Nano Today 55 (2024): 102196, 10.1016/j.nantod.2024.102196. [DOI] [Google Scholar]
  • 42. Nian K., Liu Y., and Qiu Y., “Incu‐Click: DNA‐Enhanced Ligand Enables Live‐Cell, Intracellular Click Chemistry Reaction With Copper Catalyst,” Nature Communications 16 (2025): 4788, 10.1038/s41467-025-60143-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Zhu J., Zhang W., and You Y., “Disruption of Self‐Protection Pathways Enables Endogenous Copper‐Evoked Cuproptosis via Bioorthogonal Nanoblocker,” Nano Today 61 (2025): 102608, 10.1016/j.nantod.2024.102608. [DOI] [Google Scholar]
  • 44. Liu G., Ren H., and Li J., “Covalent Organic Framework Eliciting a Click‐Based Intracellular Copper Accumulation Cascade for Cuproptosis Cancer Therapy,” Cell Biomaterials 1 (2025): 100126, 10.1016/j.celbio.2025.100126. [DOI] [Google Scholar]
  • 45. Wei Y., Zhao H., Liu Z., Yang J., Ren J., and Qu X., “Mofs Modulate Copper Trafficking in Tumor Cells for Bioorthogonal Therapy,” Nano Letters 24 (2024): 1341–1350, 10.1021/acs.nanolett.3c04369. [DOI] [PubMed] [Google Scholar]
  • 46. Wang Z., Niu J., Zhao C., Wang X., Ren J., and Qu X., “A Bimetallic Metal–Organic Framework Encapsulated With Dnazyme for Intracellular Drug Synthesis and Self‐Sufficient Gene Therapy,” Angewandte Chemie International Edition 60 (2021): 12431–12437, 10.1002/anie.202016442. [DOI] [PubMed] [Google Scholar]
  • 47. You Y., Liu H., and Zhu J., “A Dnazyme‐Augmented Bioorthogonal Catalysis System for Synergistic Cancer Therapy,” Chemical Science 13 (2022): 7829–7836, 10.1039/D2SC02050E. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48. Shao X., Yang Z., and Xu Z., “Simultaneous Imaging of Cu+ and Cu2+ in Neural Cells Using Dnazyme Probes Reveals Mechanistic Link Between Copper Redox Imbalance and Amyloid Pathology,” Angewandte Chemie International Edition 65 (2026): e24237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Sen D., Yu H.‐Z., Lat P. K., and Liu K., “Click‐17, a DNA Enzyme That Harnesses Ultra‐Low Concentrations of Either Cu+ or Cu2+ to Catalyze the Azide‐Alkyne ‘Click’ Reaction in Water,” Nucleic Acids Research 48 (2020): 7356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Liu K., Chen K. S., Sen D., and Yu H.‐Z., “Ultrasensitive Detection of Total Copper With an Electrochemical Biosensor Built on the in Cis Coupling of Hexynyl Click‐17 Dnazyme With Azido Self‐Assembled Monolayers,” Electrochimica Acta 379 (2021): 138125, 10.1016/j.electacta.2021.138125. [DOI] [Google Scholar]
  • 51. Li W. Y., Wan J. J., and Kan J. L., “A Biodegradable Covalent Organic Framework for Synergistic Tumor Therapy,” Chemical Science 14 (2023): 1453–1460, 10.1039/D2SC05732H. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional supporting information can be found online in the Supporting Information section. Supplementary Information including Materials and Methods, Figures S1–S34, and supporting references. Supporting File 1: anie73537‐sup‐0001‐SuppMat.docx.

ANIE-65-e6633730-s001.docx (4.4MB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Angewandte Chemie (International Ed. in English) are provided here courtesy of Wiley

RESOURCES