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A Tumor Microenvironment-Activated Metal-Organic Framework-Based Nanoplatform for Amplified Oxidative Stress-Induced Enhanced Chemotherapy

  • Author Footnotes
    † Bo Li, Xin Yao and Jiaqi Li contributed equally to this work
    Bo Li
    Footnotes
    † Bo Li, Xin Yao and Jiaqi Li contributed equally to this work
    Affiliations
    Institutes of Physics Science and Information Technology, Key Laboratory of Structure and Functional Regulation of Hybrid Materials, Ministry of Education, Anhui University, Hefei 230601, P. R. China
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  • Author Footnotes
    † Bo Li, Xin Yao and Jiaqi Li contributed equally to this work
    Xin Yao
    Footnotes
    † Bo Li, Xin Yao and Jiaqi Li contributed equally to this work
    Affiliations
    Institutes of Physics Science and Information Technology, Key Laboratory of Structure and Functional Regulation of Hybrid Materials, Ministry of Education, Anhui University, Hefei 230601, P. R. China

    Department of Chemistry, Key Laboratory of Functional Inorganic Material Chemistry of Anhui Province, Anhui University, Hefei 230601, P. R. China
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  • Author Footnotes
    † Bo Li, Xin Yao and Jiaqi Li contributed equally to this work
    Jiaqi Li
    Footnotes
    † Bo Li, Xin Yao and Jiaqi Li contributed equally to this work
    Affiliations
    Institutes of Physics Science and Information Technology, Key Laboratory of Structure and Functional Regulation of Hybrid Materials, Ministry of Education, Anhui University, Hefei 230601, P. R. China
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  • Xin Lu
    Affiliations
    Department of Chemistry, Key Laboratory of Functional Inorganic Material Chemistry of Anhui Province, Anhui University, Hefei 230601, P. R. China
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  • Wen Zhang
    Affiliations
    Department of Chemistry, Key Laboratory of Functional Inorganic Material Chemistry of Anhui Province, Anhui University, Hefei 230601, P. R. China
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  • Wenyao Duan
    Affiliations
    Institutes of Physics Science and Information Technology, Key Laboratory of Structure and Functional Regulation of Hybrid Materials, Ministry of Education, Anhui University, Hefei 230601, P. R. China
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  • Yupeng Tian
    Affiliations
    Department of Chemistry, Key Laboratory of Functional Inorganic Material Chemistry of Anhui Province, Anhui University, Hefei 230601, P. R. China
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  • Dandan Li
    Correspondence
    Corresponding author: D. Li
    Affiliations
    Institutes of Physics Science and Information Technology, Key Laboratory of Structure and Functional Regulation of Hybrid Materials, Ministry of Education, Anhui University, Hefei 230601, P. R. China

    Department of Chemistry, Key Laboratory of Functional Inorganic Material Chemistry of Anhui Province, Anhui University, Hefei 230601, P. R. China
    Search for articles by this author
  • Author Footnotes
    † Bo Li, Xin Yao and Jiaqi Li contributed equally to this work
Open AccessPublished:November 23, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102742

      Abstract

      Engineering a highly tumor microenvironment-responsive nanoplatform toward effective chemotherapy has always been a challenge in targeted cancer treatment. Metal-organic frameworks are a promising delivery system to reformulate previously approved drugs for enhanced chemotherapy, such as disulfiram (DSF). Herein, a tumor microenvironment-activated metal-organic framework-based nanoplatform [email protected]@FA has been fabricated to realize amplified oxidative stress-induced enhanced chemotherapy. Our results unveil that the copper ions and disulfiram released by [email protected]@FA in an acidic environment can be converted into toxic bis (N, N-diethyl dithiocarbamato) copper and then induce cell apoptosis. Simultaneously, we determined that the apoptosis outcome is further promoted by amplified oxidative stress through effective generation of reactive oxygen species and GSH elimination. In conclusion, this work provides a promising platform for effective anti-cancer treatment.

      Keywords

      Introduction

      Chemotherapy still leads to the most prevalent modalities among a variety of anticancer treatments in recent years although all kinds of treatment methods have developed [
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      ] and so on. In addition, as an old drug approved by the U. S. Food and Drug Administration (FDA), disulfiram (DSF) has been used for treating alcohol dependence for over six decades. Recent research illustrated that it can turn into toxic bis (N, N-diethyl dithiocarbamato) copper (Ⅱ) (CuET) after chelated with Cu(II) to induce heat shock response (HSR) and cancer-cell death [
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      ]. However, the serious side effect, high system toxicity and unsatisfactory therapeutic efficacy limited its application [
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      ]. Considering the high cost from the research of the pharmacokinetics and safety profiles of new drugs, searching for new anticancer drug formulations based on DSF is an attractive strategy to combat the above issue.
      Metal-organic frameworks (MOFs), consisting of metal nodes and organic ligand, have been widely used in various advanced fields, such as gas sorption and separation, catalysis, food safety, drug delivery and cancer therapy [
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      ]. In particular, MOFs recommended themselves as very promising hosts for old drugs loading to develop new anticancer drug formulations due to their high porosity [
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      ]. Of note, their metal nodes offer ample possibilities for fabricating tumor microenvironment (TME)-responsive platforms. For example, some Cu-based MOFs endow their particular merits toward TME-responsive therapy by releasing Cu(II) ions in acid TME. The resultant Cu(II) could trigger GSH depletion [
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      ] and Cu(I)-mediated ·OH generation via self-cyclic valence alternation [
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      ], giving rise to amplified oxidative stress and further improve the chemotherapeutic effect [
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      ]. In this sense, Cu(II)-based MOFs hold great advantages in promoting the CuET-mediated chemotherapeutic by the amplified intracellular oxidative stress.
      Bearing the above considerations in mind, we employed the acid-responsive MOF-199 as the main material to fabricate an intelligent delivery system ([email protected]@FA) to promote the chemotherapy effect. As illustrated in Scheme 1, the folic acid (FA) was wrapped on the surface of [email protected] to endow it ([email protected]@FA) with cancer cell-specific targeting ability [
      • Wang X.-S.
      • Zeng J.-Y.
      • Zhang M.-K.
      • Zeng X.
      • Zhang X.-Z.
      A Versatile Pt-Based Core–Shell Nanoplatform as a Nanofactory for Enhanced Tumor Therapy.
      ]. In addition, the TME-responsive system could release Cu(II) ions and DSF. Thereinto, DSF could convert into CuET (Fig. S1, S2) in situ to induce cell apoptosis. Besides, the released Cu(II) ions could be used to amplify intracellular oxidative stress by consuming intracellular GSH and generating ·OH (generated from the Fenton-like reaction) via self-cyclic valence alternation. Finally, this [email protected]@FA mediated amplified oxidative stress strategy provides a new paradigm to amplify the CuET chemotherapeutic effect.
      Figure thumbnail sc1
      Scheme 1Scheme illustration showing the preparation of [email protected]@FA, highlighting the amplified oxidative stress enhanced CuET-mediated chemotherapy.

      Results and Discussion

      [email protected]@FA was fabricated by loading DSF into the pores of defective MOF-199 through simply physical absorption and then wrapped with FA. As identified by Fig. 1a, the power X-ray diffraction (PXRD) pattern of MOF-199 showed the same characteristic peaks as the simulated one, illustrating the MOF-199 was successful synthesized. The crystal structure and crystallinity of [email protected], [email protected] and [email protected]@FA remained well compared with MOF-199. Compared with MOF-199, the obvious diffraction peaks at 2θ = 9.25°, 9.95° in [email protected] and [email protected]@FA. Besides, in terms of [email protected] and [email protected]@FA, the emerged small diffraction peaks (2θ = 9.25°, 9.95°) could be attributed to the DSF loading given the interaction between DSF molecules and Cu ions of MOFs, which was revealed by PXRD and XPS measurements (Fig. S3, S4). The scanning electron microscopy (SEM) image in Fig. 1b and transmission electron microscopy (TEM) image in Fig. S5 of [email protected]@FA displayed the unchanged octahedron morphology suggesting the absence of structure and morphology variation of MOF-199 after loading with DSF and coating with FA.
      Figure thumbnail gr1
      Fig. 1(a) PXRD patterns of simulated MOF-199, [email protected], and [email protected]@FA. (b) SEM image of [email protected]@FA. (c) Elemental mapping images of the ultra-thin slice of [email protected]@FA in TEM. (d) TGA curves of MOF-199 and [email protected] (e) UV-vis absorbance spectra of folic acid, DSF, MOF-199, [email protected]@FA. (f) Zeta potentials of MOF-199, [email protected], [email protected] and [email protected]@FA.
      Besides, the [email protected]@FA was sliced into ultra-thin slices and the elemental mapping images were collected, in which Cu was attributed to MOF-199, S was assigned to DSF, N contributed to DSF and FA (Fig. 1c), which demonstrated that the DSF was loaded into the pores of MOF-199. As shown in Fig. 1d, the weight loss between 200 °C and 300 °C was attributed to the DSF decompose [
      • Liu W.
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      • Jiang Q.
      • Yang L.
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      Nanomedicine Enables Drug-Potency Activation with Tumor Sensitivity and Hyperthermia Synergy in the Second Near-Infrared Biowindow.
      ], which further demonstrated that DSF was efficiently loaded into MOF-199. The loading efficiency of DSF was calculated to be 4.9% in the [email protected]@FA by ICP-AES measurement. The UV-vis absorption spectrum of [email protected]@FA displayed significant change after subsequent DSF loading and FA coating, a new absorption band appeared (230-350 nm) due to the DSF and FA absorption (Fig. 1e). Particularly, the absorption band around 260-320 nm showed a difference between [email protected] and [email protected]@FA which was attributed to the FA coating. Besides, the new peak around 1608 cm-1 in FTIR spectrum increased after modified by FA was contributed to the -NH stretching vibrations of -NH2 in FA (Fig. S6). In addition, as displayed in Fig. 1f, the zeta potential experiment indicated a change in the surface charge from a positive potential for MOF-199 (+8.04 mV) to a negative potential for [email protected] (-4.05 mV) and [email protected] (-4.92 mV) after FA modification and DSF loading, respectively. The potential was further reduced to -11.5 mV ([email protected]@FA) after loading with DSF and modifying with FA simultaneously. All the above results demonstrate the successful fabrication of [email protected]@FA.
      Motivated by the successful fabrication of [email protected]@FA, the ROS generation, GSH depleting and CuET formation performance were studied thoroughly by the in vitro experiments. Firstly, the acidity-responsive degradation performance of MOF-199 was evaluated by SEM observation. As shown in Fig. S7 and S8, the MOF-199 was physiologically stable after incubating with PBS in neutral condition (pH = 7.4) for 24 h, while degraded quickly in the acidic PBS solution (pH = 6.5), suggesting that the MOF-199 could degrade and release the copper ions and DSF for anti-cancer treatment in the acid environment. Furthermore, the polydispersity index (PDI) of [email protected]@FA maintained stable in serum over 7 days, which confirmed its good stability in the blood circulation (Fig. S9). Then, terephthalic acid (TA) which can react with ·OH radicals to form 2-hydroxyterephthalic acid (TAOH) was used to evaluate the ·OH (origin from the Fenton-like reaction) generation capability of [email protected]@FA. With the prolonged incubation time, a fluorescence enhancement around 450 ± 20 nm appeared, indicating that [email protected]@FA is capable of generating ·OH in the acidic TME efficiently (Fig. 2b and Fig. S10) while the [email protected]@FA or H2O2 alone do not generate any ·OH. The Fenton-like effect of [email protected]@FA was proved to be derived from free Cu2+ ions by the control experiments of CuET and Cu2+ (Fig. S11). Moreover, thiolite™ green was selected as the detection agent for GSH. As shown in Fig. S12, the solution showed the faint green fluorescence at 520 nm in the presence of H2O2 and [email protected]@FA, which proved that [email protected]@FA displayed excellent GSH consumption capacity (Fig. S12). Besides, the fluorescence was gradually faded (Fig. 2c) with the concentration of [email protected]@FA increased. In addition, compared with [email protected], the UV-vis spectrum of degradation product originating from [email protected]@FA presented a characteristic peak around 450 nm (the peak of CuET), suggesting that the [email protected]@FA could release copper ions and DSF in the acidic environment and then form CuET complex in suit for chemotherapy (Fig. 2d). With the extension of incubation time, the absorption at 450 ± 20 nm increased gradually with the extension of incubation time in the acidic environment, which proved the formation of CuET (Fig. S13, S14). Given [email protected]@FA could generate ·OH and consume GSH via self-cyclic valence alternation, it is implied that [email protected]@FA can promote the CuET-mediated chemotherapeutic effect by amplifying the intracellular oxidative stress.
      Figure thumbnail gr2
      Fig. 2(a) Illustration of ROS generation, GSH depleting and CuET formation process within [email protected]@FA. (b) Determination of the formation of ∙OH treated with [email protected]@FA by terephthalic acid as the fluorescent probe. Reaction conditions: [email protected]@FA (50 μg mL-1), TA (0.05 mM), pH = 6.5. (c) GSH depleting ability of [email protected]@FA at different concentration with thiolite™ green as the detection agent for GSH. (d) UV–vis spectrum of degradation product of [email protected] and [email protected]@FA.
      Encouraged by the above experiments, we decided to investigate the ·OH generation performance of [email protected]@FA in cancer cells via confocal laser scanning microscopy (CLSM) imaging, in which the 4T1 cells were stained with hydroxyphenyl fluorescein (HPF). As illustrated in Fig. 3a, compared with the blank group, the green fluorescence of HPF increased significantly for [email protected]@FA with/without H2O2 groups. Besides, the fluorescence almost disappeared upon ·OH scavenger (ascorbic acid, AA) added, indicating the ·OH generation ability of [email protected]@FA within 4T1 cells. Meanwhile, the enhanced fluorescence intensity of [email protected]@FA (Fig. 3b) with the addition of H2O2 demonstrating that the additional H2O2 is able to facilitate the Fenton-like reaction and further improve ·OH generation.
      Figure thumbnail gr3
      Fig. 3(a) Intracellular ·OH evaluation. CLSM fluorescence images of HPF-stained 4T1 cells treated with PBS, [email protected]@FA + AA, [email protected]@FA, [email protected]@FA + H2O2, reaction conditions: [email protected]@FA (50 μg mL-1), H2O2 (100 μM), AA (25 μg mL-1), scale bar = 20 μm. (b) HPF intensity in 4T1 cells after different treatments. (c) Intracellular GSH levels in 4T1 cells after different treatments. Data are presented as mean ± s.d; n.s.: not significant; **p<0.01, ***p<0.001
      Apart from ROS, GSH also plays an important role in amplifying oxidative stress. Therefore, thiolite™ green was utilized to assess the GSH level in the 4T1 cells after different treatments. Obviously, in contrast to the blank group, a decreased GSH concentration could be observed upon [email protected]@FA treated. As displayed in Fig. 3c, compared with the [email protected]@FA group, the concentration of GSH increased with AA addition which probably attributed to the inhibition of GSH consumption by AA. Moreover, GSH level could even be reduced further when H2O2 added. It demonstrated that [email protected]@FA can effectively consume the intracellular GSH due to the released Cu(II) was reduced to Cu(I) by GSH through redox reaction. The above results further confirmed that [email protected]@FA can be used to amplify oxidative stress by producing ·OH and consuming GSH for enhanced chemotherapy.
      To avoid side effects on normal cells, we modified [email protected] with FA to enhance its cancer cell-specific targeting capability. The cancer cell-specific targeting behavior of [email protected]@FA were evaluated by cell uptake experiments with FAR (folate receptor) abundant cells (HeLa: human cervical cancer cell) and FAR negative cells (HEK 293FT: human embryonic kidney cells). To characterize the process of the endocytosis of [email protected]@FA into cancer cells clearly, [email protected]@FA were labeled with FITC ([email protected]@FA-FITC) (Fig. S15). As illustrated in Fig. S16 and S17, [email protected]@FA-FITC preferentially accumulated in HeLa cells but not in HEK 293T cells, Hela cells which incubated with [email protected] and the HeLa cells which were incubated with free FA in advance. Then, cell uptake experiments with 4T1 cells (mouse breast cancer cells, FAR abundant cells) also demonstrated that FA-modified can effectively target FAR-overexpressing cancer cells (Fig. S18). Moreover, the cytotoxicity was evaluated via standard (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using HEK 293T cells (human embryonic kidney cell), SW480 cells (human colorectal carcinoma cell), Hela cells (human cervical cancer cell) and 4T1 cells (mouse breast cancer cell). As shown in Fig. S19, more than 90% of HEK 293T cells survived after being incubated with different concentration of [email protected]@FA (0-80 μg mL-1) for 12 h, suggesting their low cytotoxicity to healthy cells and excellent biocompatibility. In contrast, cell viability of 4T1 cells decreased along with an increase in the concentration of [email protected]@FA (Fig. 4a), the treated Hela cells and SW480 cells showed similar results (Fig. S20). The cell viability of 4T1 cells was further decreased after H2O2 addition because of the amplified oxidative stress trigged by H2O2. Notably, as shown in Fig. S21, H2O2 (100 μM) displayed ignorable cytotoxicity. These results reveal that [email protected]@FA can selectively induce cancer cells apoptosis and then avoid side effects effectively.
      Figure thumbnail gr4
      Fig. 4(a) Cell viability in 4T1 cells after 12 h of incubation with different treatments. (b) Cell viability after varied treatments, including blank, DSF only, [email protected], [email protected]@FA + AA, [email protected]@FA, and [email protected]@FA + H2O2 ([email protected]@FA (50 μg mL-1), [email protected] (47.5 μg mL-1), DSF (2.5 μg mL-1), H2O2 (100 μM), AA (25 μM)). (c) 3D fluorescence images of MCTs after different treatments. 4T1 MCTs incubated with AM (indicator of living cells)/PI (indicator of dead cells). Data are presented as mean ± s.d; n.s.: not significant; **p<0.01, ***p<0.001
      Strategically, we deployed a systematic protocol to evaluate toxicity of DSF after chelated with Cu(II) ions as well as amplified oxidative stress induced chemotherapy effect, including standard MTT and 3D multicellular tumor spheroids (3D MCTs) assays (Fig. 4). Evidently, in contrast to the ignorable cytotoxicity toward 4T1 cells and 3D MCTs treated with DSF or [email protected], [email protected]@FA (with equal concentration of DSF) displayed obviously cytotoxicity elaborating the in situ generated CuET could serve as a chemotherapeutic agent. [email protected]@FA with H2O2 treatment group displayed much lower cell viability due to the generated ·OH originating from the Fenton-like reaction amplified the intracellular oxidative stress. In addition, upon AA (reduced oxidative stress) added, the survival rates increased.
      Inspired by the amplified oxidative stress and CuET-mediated chemotherapy of [email protected]@FA, the systematic performance of therapeutic effect was further evaluated by CLSM observation (Fig. 5a). Red PI signal could be observed in the [email protected]@FA group demonstrating its chemotherapy outcome against tumor cells. Upon AA (reduce oxidative stress) or H2O2 (amplify oxidative stress) added, weakened or enhanced PI signal could be collected, respectively, corroborating the amplified oxidative stress induced enhanced chemotherapy performance. Moreover, the above results were further confirmed by flow cytometry (Fig. 5b). With the addition of H2O2, [email protected]@FA induced 99.43% apoptotic cells, which was obviously higher than that of with AA (82.18%) or without H2O2 (97.05%) treatment. All the above in vitro results showed that [email protected]@FA can be used to enhance CuET- mediated chemotherapy by the amplified oxidative stress.
      Figure thumbnail gr5
      Fig. 5(a) CLSM images of 4T1 cells stained with calcein AM/PI after different treatments (scale bar: 100 μm). (b) 4T1 cells treated with [email protected]@FA apoptosis analyzed by flow cytometry after different treatment using annexin V-FITC and PI as indicators of apoptosis.
      Based on the surprising in vitro therapeutic effect of amplified oxidative stress induced enhanced CuET-mediated chemotherapy, the in vivo biological behavior of [email protected]@FA, including circulation, biocompatibility and anti-cancer ability were investigated on the 4T1 breast tumors-bearing female BALB/C nude mice. Initially, the pharmacokinetic behavior of [email protected]@FA in blood circulation was studied by intravenous injection, and the half-life was calculated to be 2.47 h within the bloodstream (Fig. 6b). Afterward, the female BALB/C mice bearing 4T1 tumor were randomly divided into 5 groups (n = 5) and intravenous injected with PBS, DSF only, MOF-199 only, [email protected] only, and [email protected]@FA only, respectively. During the whole therapeutic period, the body-weight changes of the five groups mice showed an upward tendency and no damage was observed in the major organs (heart, liver, spleen, lung, and kidney) (Fig. 6c and Fig. S22). The high therapeutic biosafety of [email protected]@FA was further validated through blood routine examination (Fig. S23). Remarkably, compared with the other groups, [email protected]@FA group exhibited a significantly suppressed effect on 4T1 tumor growth (Fig. 6d, S24-25). Furthermore, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Ki-67 and H&E stained tumor pathological sections showed that the [email protected]@FA can induce cell necrosis in the tumor section, indicating the new anticancer drug formulations based on DSF has the better anti-cancer efficiency (Fig. 6e and Fig. S26).
      Figure thumbnail gr6
      Fig. 6(a) Schematic diagram of 4T1 subcutaneous tumor-bearing female BALB/C mice model development and treatment process. (b) Blood-circulation lifetime of [email protected]@FA after intravenous injection. (c) Curves of body weights of nude mice in various treatment groups (PBS, DSF, MOF-199, [email protected] and [email protected]@FA (n = 5)). (d) Curves of tumor volumes of the PBS, DSF, MOF-199, [email protected] and [email protected]@FA groups (n = 5). (e) H&E stained sections of tumors in different treatment groups. Data are presented as mean ± s.d; n.s.: not significant; **p<0.01, ***p<0.001

      Conclusion

      In summary, we have constructed a TME-active nanotheranostic platform, [email protected]@FA, for amplified oxidative stress induced enhanced CuET-mediated chemotherapy. The results show that the released DSF and Cu(Ⅱ) ions in acidic environment can form toxic CuET species in situ to induce chemotherapy outcome. Meanwhile, the therapeutic effect can be further enhanced by the copper ions mediated amplified oxidative stress through ·OH generating (origin from Fenton-like reaction) and GSH consuming. This work not only represents a distinctive paradigm of a TME-activated nanosystem for amplified oxidative stress induced enhanced CuET-mediated chemotherapy but also provides insight into repurposing FDA-approved drugs as versatile cancer therapeutics for effective cancer treatment.

      Experimental procedures

      Synthesis of defective MOF-199

      Cu(NO3)2 aqueous solution (0.9 mL, 0.1 M), CTAB aqueous solution (9.6 mL, 0.1 M), benzene-1,3,5-tricarboxylate (BTC) triethylammonium salt aqueous solution (0.6 mL, 0.1 M) were added in the mixture of ethanol (15 mL) and deionized water (15 mL). Next, the above mixture was stirred vigorously at the room temperature for 10 min, and then collected by centrifugation (5000 rpm, 1 min).

      Synthesis of [email protected]

      Disulfiram (DSF, 20 mg) was dispersed in the acetone (10 mL) first, and then MOF-199 (20 mg) was added under continuous sonication. The above mixture was stirred at room temperature for 4 hours. The light blue products were obtained by centrifugation, washing, and drying after the reaction finished.

      Synthesis of [email protected]/[email protected]@FA

      Folic acid (50 mg) was dispersed into DMF (50 mL) under sonication. After that, MOF-199/[email protected] (50 mg) was added into the above solutions and stirred at 30 °C overnight without light interference. Thereafter, the products were washed with DMF three times to remove the excess folic acid, and then washed with ethanol three times again, the light blue product was preserved in ethanol.

      Synthesis of [email protected]@FA-FITC

      FITC (2 mg) was dispersed into DMF (2 mL), and then [email protected]@FA (2 mg) was added into the above solutions and stirred overnight. Thereafter, the products were washed with DMF and ethanol three times respectively. The light blue product was preserved in ethanol.

      Synthesis of CuET

      87 mg DSF and 50 mg CuCl2 were added to 100 mL deionized water and stirred at room temperature for 24 h. The solution was extracted with chloroform and dried to form a black solid.

      Date availability

      All data generated or analyzed during this study are included in this published article and its additional files.
      MCTs: multicellular tumor spheroids; TA: Terephthalic acid.

      Supplementary Information

      This article contains supporting information

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgements

      We appreciate the support form National Natural Science Foundation of China (22171001) and Natural Science Foundation of Anhui Province of China (2108085MB49).

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