Construction of tumor targeted delivery system for in-situ synthesis of apoptosis inducer and application of tumor targeted delivery system in apoptosis-copper death synergistic treatment

Abstract

The invention belongs to the field of biological medicine, and particularly relates to construction of a tumor targeted delivery system for in-situ synthesis of an apoptosis inducer and application of the tumor targeted delivery system in apoptosis-copper death synergistic treatment. The synergistic mitochondrial injury is realized through combined treatment of apoptosis-copper death mediated by small molecule CTT for inducing tumor cell apoptosis. CTT is synthesized in situ in a tumor through a biological orthogonalization method. In order to improve the tumor selective synthesis efficiency, reduce the off-target effect and overcome the problems of rapid removal and metabolism of the drug, the corresponding precursor drug is delivered by adopting a ZIF-8-based nano material. Under the acidic condition of a tumor microenvironment, PVP (at) CuP (at) ZIF is decomposed and releases Cu (I) ions and a prodrug, and in-situ synthesis of CTT is realized by means of a Cu (I) catalytic azide-alkyne cycloaddition reaction. Based on in-situ synthesis with high tumor selectivity, the invention provides a novel method for overcoming tumor drug resistance through apoptosis-copper death combined pathway mediated synergistic mitochondrial damage.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the construction of a tumor-targeted delivery system for in situ synthesis of apoptosis inducers and its application in apoptosis-copper death synergistic therapy. Background Technology

[0002] Chemotherapy is one of the most traditional methods for treating cancer, and its mechanism of action often involves inducing tumor cell apoptosis through small molecule drugs, which can regulate the mitochondrial apoptosis pathway. However, developing novel anticancer drugs based on this pathway still faces challenges such as acquired mutations and persistent drug resistance. As a non-apoptotic cell death mechanism, copper death has recently been revealed to regulate cell death through mitochondrial metabolism-related mechanisms and is less sensitive to conventional drug resistance. When copper accumulates in mitochondria, it promotes the oligomerization of lipoylated proteins and disrupts the stability of iron-sulfur cluster proteins, ultimately leading to protein toxicity stress and cell death. Therefore, combining mitochondrial apoptosis with mitochondrial copper death holds promise for causing synergistic and irreversible damage to the mitochondrial region, potentially overcoming treatment resistance. However, this faces challenges in the delivery of small molecule compounds that induce tumor cell apoptosis and in the synergistic mechanism of apoptosis-copper death therapy. Furthermore, the efficacy of any treatment method is limited by the shortcomings of traditional drug delivery, including rapid clearance, poor tumor specificity, unsatisfactory pharmacokinetics, and off-target toxicity to normal tissues. Summary of the Invention

[0003] To address the problems existing in the prior art, the present invention first provides a small molecule CTT that induces tumor cell apoptosis.

[0004] The present invention also provides a tumor-targeted delivery system for in situ synthesis of apoptosis inducers, namely, a tumor-selective delivery nanomaterial PVP@CuP@ZIF.

[0005] This invention also provides a method for constructing the above-mentioned tumor-selective delivery nanomaterial PVP@CuP@ZIF.

[0006] Another objective of this invention is to provide the application of the aforementioned tumor-selective delivery nanomaterial PVP@CuP@ZIF.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides a small molecule CTT that induces tumor cell apoptosis, with the following structural formula: .

[0008] The present invention also provides a method for preparing the above-mentioned small molecule CTT that induces tumor cell apoptosis, comprising the following steps: stirring 3-azido-7-hydroxycoumarin and 2-ethynylthiophene in acetonitrile and refluxing the mixture; then cooling the solution to room temperature, concentrating under reduced pressure, purifying the crude product, and obtaining CTT.

[0009] Preferably, the molar ratio of 3-azido-7-hydroxycoumarin to 2-ethynylthiophene is 2.46:1.14; the reflux reaction is carried out by heating at 82°C for 24 hours; and the purification is carried out by column chromatography using petroleum ether / ethyl acetate as eluent.

[0010] This invention further provides a tumor-selective delivery nanomaterial PVP@CuP@ZIF for in situ synthesis of small molecule CTT that induces tumor cell apoptosis. The PVP@CuP@ZIF is formed by encapsulating Cu2O and 3-azido-7-hydroxycoumarin into ZIF-8 nanoparticles to form the intermediate Cu@PI@ZIF; subsequently, 2-ethynylthiophene is anchored on the surface of Cu@PI@ZIF to obtain the intermediate CuP@ZIF; finally, PVP is coated on the outermost layer of CuP@ZIF to obtain the delivery nanomaterial PVP@CuP@ZIF.

[0011] Another object of the present invention is to provide a method for preparing the above-mentioned tumor-selectively delivered bioorthogonalized nanomaterial PVP@CuP@ZIF within a tumor, comprising the following steps: (1) Dissolve CuI and NaAsc in water; then add sodium hydroxide to the mixture, stir for a period of time, wash the Cu2O nanoparticles, centrifuge and collect the precipitate to obtain Cu2O nanoparticles; (2) Synthesis of Cu@PI@ZIF nanoparticles: Cu2O nanoparticles were dispersed in methanol and sonicated; under argon protection, methanol solutions containing 3-azido-7-hydroxycoumarin and Zn(NO3)2·6H2O were added sequentially; after stirring evenly, methanol solutions containing 2-methylimidazole were slowly added; the mixture was stirred to carry out the reaction, and then the precipitate was collected by centrifugation, washed and dried to obtain Cu@PI@ZIF; (3) Synthesis of PVP@CuP@ZIF nanoparticles: Cu@PI@ZIF was ultrasonically dispersed in a mixed solution of methanol and water. Then, 2-ethynylthiophene dissolved in methanol was slowly added to the Cu@PI@ZIF solution. After stirring for a period of time, the solid was collected by centrifugation, washed and dried to obtain CuP@ZIF nanoparticles. Ethanol was added to the nanoparticles to obtain CuP@ZIF ethanol solution. CuP@ZIF ethanol solution was added to the PVP solution and stirred. After centrifugation and washing, PVP@CuP@ZIF nanoparticles were obtained.

[0012] Preferably, in step (1), the molar ratio of CuI to NaAsc is 1:5; the mass ratio of CuI to sodium hydroxide is 0.025:1; and the stirring is carried out at room temperature for 5-6 minutes until the solution turns orange. Preferably, in step (2), the mass ratio of Cu2O nanoparticles to 3-azido-7-hydroxycoumarin, calculated as CuI, is 1:2; the concentration of the methanol solution containing 3-azido-7-hydroxycoumarin is 2.5 mg / mL; the concentration of the methanol solution containing Zn(NO3)2·6H2O is 25 mg / mL; the concentration of the methanol solution containing 2-methylimidazole is 30 mg / mL; the molar ratio of 3-azido-7-hydroxycoumarin, Zn(NO3)2·6H2O, and 2-methylimidazole is 0.2-0.3:1.6-1.7:7.0-7.5; and the reaction is carried out under argon protection and stirred at room temperature for 0.5-2 h.

[0013] Preferably, in step (3), the concentration of Cu@PI@ZIF in the mixed solvent is 2.5 mg / mL; the mixed solvent is composed of methanol and water in a ratio of 8-9:1-2 (v / v); the mass ratio of Cu@PI@ZIF to 2-ethynylthiophene is 1:5-8; the concentration of 2-ethynylthiophene in methanol is 30 mg / mL; the mass ratio of CuP@ZIF nanoparticles to polyvinylpyrrolidone is 1:40; the concentration of CuP@ZIF ethanol solution is 50 mg / mL; after adding the 2-ethynylthiophene solution, the mixture is stirred at room temperature for 4-5 h; the CuP@ZIF nanoparticles are coated with the PVP solution by stirring at room temperature for 2-4 h.

[0014] This invention also provides the application of the above-mentioned PVP@CuP@ZIF in the preparation of drugs that achieve tumor-selective delivery to synthesize CTT in situ at the tumor site and for anti-tumor purposes.

[0015] Preferably, the PVP@CuP@ZIF is used as a delivery precursor to synthesize a small molecule CTT that induces tumor cell apoptosis at the tumor site via a CuAAC reaction.

[0016] The reaction formula for the small molecule CTT synthesized in this invention that induces tumor cell apoptosis is as follows: .

[0017] In the formula, R is .

[0018] This invention utilizes nanomaterials (PVP@CuP@ZIF) to deliver precursors, synthesizing small-molecule CTT that induces tumor cell apoptosis at the tumor site via a CuAAC reaction, achieving synergistic mitochondrial damage under apoptosis-copper death therapy. This invention synthesizes a small molecule, 1,2,3-triazole derivative (CTT), that induces tumor cell apoptosis. To achieve tumor-selective delivery for in-situ synthesis of CTT at the tumor site, nanomaterials PVP@CuP@ZIF based on the zeolite imidazolium ester backbone-8 (ZIF-8) were prepared. First, Cu@PI@ZIF was prepared to load Cu(I) and precursor I (3-azido-7-hydroxycoumarin), followed by surface loading of precursor II (2-ethynylthiophene) to obtain CuP@ZIF. Then, stable nanomaterials PVP@CuP@ZIF were obtained by coating with polyvinylpyrrolidone (PVP) for subsequent treatment. In the bloodstream, PVP@CuP@ZIF accumulates at the tumor site due to its enhanced permeability and retention (EPR) effect, and enters cancer cells for therapeutic application through a proton sponge effect. Subsequently, in the acidic tumor microenvironment, PVP@CuP@ZIF decomposes to release Cu(I) ions and prodrugs, and CTT is synthesized in situ via the CuAAC reaction catalyzed by Cu(I). The resulting small-molecule CTT, which induces tumor cell apoptosis, then enhances mitochondrial membrane permeability and releases apoptosis factors (such as cytochrome c) to activate the apoptosis pathway through apoptosis therapy. Simultaneously, the released Cu(I) accumulates in the mitochondria and induces copper death for non-apoptotic therapy. In the mitochondria, Cu(I) binds to lipoylated proteins in the tricarboxylic acid (TCA) cycle, inducing oligomerization of dihydrolipoamide S-acetyltransferase (DLAT) and loss of iron-sulfur (Fe-S) cluster proteins, ultimately leading to protein toxicity stress and cell death. This invention not only enhances tumor-specific therapy through in-situ synthesis but also opens up a new strategy for anti-drug resistance therapy through synergistic mitochondrial damage via the apoptosis-copper death pathway.

[0019] The beneficial effects of this invention are as follows: (1) The delivery nanomaterial PVP@CuP@ZIF constructed in this invention not only enhances the stability of the nanomaterial in blood circulation, but also promotes the selective enrichment of PVP@CuP@ZIF at the tumor site by means of the EPR effect.

[0020] (2) This invention achieves synergistic mitochondrial damage through apoptosis-copper death combined therapy mediated by the small molecule CTT that induces tumor cell apoptosis. CTT was synthesized in situ within the tumor using a bioorthogonalization method. To improve tumor-selective synthesis efficiency, reduce off-target effects, and overcome the problems of rapid drug clearance and metabolism, the corresponding prodrug was delivered using ZIF-8-based nanomaterials. Under the acidic conditions of the tumor microenvironment, PVP@CuP@ZIF decomposes and releases Cu(I) ions and the prodrug. The in situ synthesis of CTT is achieved by using Cu(I) to catalyze a copper-catalyzed azide-alkyne cycloaddition reaction. After CTT is localized in the mitochondria, it initiates the mitochondrial apoptosis pathway characterized by mitochondrial damage and the release of apoptosis factors. Simultaneously, the released Cu(I) accumulates in the mitochondria and induces copper death. Based on highly tumor-selective in situ synthesis, this invention proposes a new method to overcome tumor drug resistance by mediating synergistic mitochondrial damage through the apoptosis-copper death combined pathway. Attached Figure Description

[0021] Figure 1 The ¹H NMR spectrum of CTT (d-DMSO); Figure 2 The image shows the high-resolution mass spectra (HR-MS) spectra of CTT. Figure 3 The IC50 value of compound CTT in HeLa cells; Figure 4 Characterization and in-situ synthesis of PVP@CuP@ZIF; (a) Transmission electron microscopy (TEM) image of CuP@ZIF; (b) TEM image of PVP@CuP@ZIF; (c) Elemental distribution map of CuP@ZIF; (d) Fourier transform infrared (FTIR) spectra of ZIF-8, PVP@CuP@ZIF, CuP@ZIF, and PVP@CuP@ZIF NPs; (e) Fluorescence spectra of PVP@CuP@ZIF and its precursor in PBS at pH 6.5 at 37°C for 24 hours; (f) Fluorescence spectra of PVP@CuP@ZIF at different pH values; (g) Mass spectra of the in-situ synthesis system after 24 hours of reaction at pH 7.4 and 6.5, and mass spectra of the CTT standard product (m / z: 312.06). Figure 5For the in vitro evaluation of PVP@CuP@ZIF; (a) Schematic diagram of in situ synthesis of CTT in live cells; (b) Mass spectra of HeLa cells after treatment with CTT and PVP@CuP@ZIF; (c) Confocal laser scanning microscopy images of HeLa cells in different treatment groups (scale bar: 50 μm); (d) Confocal laser scanning microscopy images of Cu ions in HeLa cells after different treatments; (e) Cytotoxicity of different treatments on HeLa cells (data are expressed as mean ± standard deviation, n = 6); (f) Confocal laser scanning microscopy images of HeLa cells after different treatments: live cells stained with calcein AM (green), dead cells stained with propidium iodide (red); (g) Flow cytometry apoptosis analysis of HeLa cells after 24 hours of treatment in different groups (cells were double-stained with Annexin V-FITC and propidium iodide). Figure 6 Detection of apoptosis and copper death mitochondrial damage induced by PVP@CuP@ZIF; (a) Schematic diagram of synergistic mitochondrial damage; (b) Detection of mitochondrial membrane potential in HeLa cells treated with PBS control, CTT and PVP@CuP@ZIF using the JC-1 probe; (c) Transmission electron microscopy images of mitochondria in HeLa cells treated with PBS control, CTT and PVP@CuP@ZIF (red arrows indicate mitochondria); (d) Statistical analysis of the green / red signal intensity ratio in (b); (e) Statistical analysis of the aspect ratio of mitochondria in (c); (f) Detection of intracellular ATP levels in HeLa cells treated with PBS control, CTT and PVP@CuP@ZIF; (g) Western blot analysis and quantitative statistics of anti-apoptotic regulatory proteins MCL-1 and BCL-2 after different treatments; (h) Western blot analysis and quantitative statistics of expression levels of apoptosis effector protein Casp-3 and signaling protein cytochrome C after different treatments; (i) Western blot analysis and quantitative statistics of expression levels of copper death-related proteins DLAT and FDX1 after different treatments; p<0.05, p<0.01, p<0.001, p<0.0001); Figure 7 For the assessment of tumor suppression ability; (a) Schematic diagram of the establishment and treatment process of HeLa tumor xenograft model. (b) Curves of mouse body weight change after different treatments; (c) Changes in tumor volume within 14 days after treatment with PBS control group, CTT, Cu@PI@ZIF and PVP@CuP@ZIF; (d) Statistics of ex vivo tumor weight after 14 days of different treatments (data are expressed as mean ± standard deviation, n=3 for each group; ns indicates no significant difference). p<0.05, p<0.01, p<0.001, (p<0.0001); (e) Representative tumor photographs after different treatments; (f) Histochemical analysis of tumor tissue 14 days after treatment: H&E staining (I), TUNEL staining (II) and immunohistochemical analysis of DLAT expression (III); Fluorescence images of ex vivo serum at different time points. Detailed Implementation

[0022] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0023] The CCK-8 cell proliferation and toxicity assay kit, copper assay kit, Annexin V-FITC apoptosis assay kit, Calcein-AM / PI live / dead cell double staining kit, and JC-1 mitochondrial membrane potential assay kit used in this invention were purchased from Beijing Solarbio Science & Technology Co., Ltd. All other reagents were of analytical grade and used directly without further purification. Ultrapure water (Millipore, Milli-Q, 18.2 megohms) was used in all experiments. Female BALB / c nude mice (4-6 weeks old, 18-22 g) used in animal experiments were provided by Beijing Spefolk Biotechnology Co., Ltd. Female BALB / c nude mice (5 weeks old, 18-20 g) used in animal experiments were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0024] Example 1: Synthesis of compound CTT Under nitrogen protection, 3-azido-7-hydroxycoumarin (500 mg, 2.46 mmol), 2-ethynylthiophene (114 mg, 1.14 mmol), and cuprous iodide (100 mg) were added to 500 mL of acetonitrile, and the mixture was heated under reflux at 82 °C with stirring for 24 hours. After the reaction was complete, the mixture was cooled to room temperature and concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 50:1, isogradient elution) to give 281 mg of colorless oily product CTT, in 82% yield. like Figure 1 and Figure 2shown. ¹H NMR (600 MHz, DMSO-D6) δ 10.92 (s, 1H), 8.88 (d, J= 1.2 Hz, 1H), 8.62 (s, 1H), 7.74 (d, J = 8.6 Hz, 1H), 7.60 – 7.50 (m, 3H), 6.89 (dd, J = 8.8, 2.2 Hz, 1H), 6.84 (d, J = 2.1 Hz, 1H). HR-MS m / z 312.0527[M + H] + . Example 2: Preparation of PVP@CuP@ZIF (1) Synthesis of Cu2O nanoparticles: CuI (25 mg, 0.1 mmol) and NaAsc (88 mg, 0.5 mmol) were dissolved in 50 mL of water. NaOH (1 g) was then added to the mixture, and the solution immediately turned orange. The mixture was stirred at room temperature for 5 minutes. The resulting orange Cu2O nanoparticles were washed twice with water and then collected by centrifugation at 8000 rpm for 3 minutes. The precipitate was finally collected by centrifugation and washed with water at least three times. The Cu2O nanoparticles were vacuum sealed and stored at -20°C for later use.

[0025] (2) Synthesis of Cu@PI@ZIF nanoparticles: The prepared Cu2O nanoparticles were dispersed in methanol (20 mL) and sonicated for 5 minutes. Under argon protection, three 20 mL methanol solutions were added sequentially: one containing 3-azido-7-hydroxycoumarin (50 mg, 0.217 mmol), another containing Zn(NO3)2·6H2O (0.5 g, 1.68 mmol), and the third containing 2-methylimidazole (0.6 g, 7.3 mmol). The mixture was then stirred at room temperature for 30 minutes under argon protection, the precipitate was collected by centrifugation, washed with methanol at least three times, and finally dried in a vacuum oven (60 °C).

[0026] (3) Synthesis of PVP@CuP@ZIF nanoparticles: First, 50 mg of Cu@PI@ZIF was dispersed in a mixed solvent of 20 mL methanol and water. Then, an appropriate amount of sodium hydroxide was added to achieve uniform dispersion. Next, 300 mg of 2-ethynylthiophene dissolved in 10 mL methanol was added to the solution, and the mixture was stirred at room temperature for 4 hours to obtain a black solid. The solid was collected by centrifugation, washed thoroughly several times with methanol and water, and then vacuum dried overnight at 60°C to finally obtain the product CuP@ZIF. To obtain the final product PVP@CuP@ZIF, 0.2 g of PVP (polyvinylpyrrolidone) was dissolved in 1.5 mL of water. After the PVP was sonicated until completely dissolved, 100 µL of a 50 mg / mL CuP@ZIF ethanol solution was added to the PVP solution and stirred for 4 hours for coating. The final product was collected after centrifugation and washing with water three times to obtain PVP@CuP@ZIF nanoparticles.

[0027] Example 1 (I) Design and evaluation of small molecule CTTs that induce tumor cell apoptosis: To develop a small-molecule CTT that induces tumor cell apoptosis, a polycyclic skeleton for screening was constructed by integrating a key pharmacophore fragment (coumarin) with the major group of the CuAAC reaction (1,2,3-triazole moiety). Aromatic substituents were introduced at the R position to design coumarin-triazole hybrid compounds. To evaluate its therapeutic efficacy, its cytotoxicity against HeLa cells was assessed using the CCK-8 assay. The results showed that compound CTT exhibited the most significant antitumor activity against HeLa cells, while also achieving good yields under simulated tumor microenvironment conditions. Furthermore, purified CTT showed an IC50 concentration of 2.280 μg / mL in HeLa cells. 50 The value confirms its potent cytotoxicity, meeting the needs of cancer treatment. Figure 3 ).

[0028] Example 2: Characterization and in-situ synthesis of PVP@CuP@ZIF.

[0029] The transmission electron microscope image of the PVP@CuP@ZIF constructed in Example 2 shows that CuP@ZIF ( Figure 4 a) and PVP@CuP@ZIF ( Figure 4 b) Both exhibit a uniform, ZIF-8-like morphology, the latter being slightly larger in size due to PVP surface modification. Furthermore, energy-dispersive X-ray spectroscopy (EDS) Figure 4 (c) This demonstrates the uniform distribution of Cu, O, and S elements in the CuP@ZIF nanostructure, further confirming the successful integration of the precursor into the nanoplatform. Powder X-ray diffraction pattern ( Figure 5This confirms that the characteristic crystal framework of ZIF-8 is well preserved in all intermediates and the final nanomaterial, indicating that precursor loading does not compromise the intrinsic structural integrity of the zeolite imidazole ester framework. Furthermore, Fourier transform infrared spectroscopy (FTIR) confirms that... Figure 4 d) The analysis provides further evidence for the stepwise preparation of PVP@CuP@ZIF. Complementary zeta potential measurements further validated the sequential modification process: the zeta potential changed from +26.37 mV in Cu@PI@ZIF to -34.2 mV after 2-ethynylthiophene coating, and then slightly increased to -18.77 mV after PVP functionalization. These results are consistent with the size increase observed in dynamic light scattering measurements.

[0030] In vitro degradation behavior of PVP@CuP@ZIF: Different concentrations of PVP@CuP@ZIF nanoparticles were added to PBS buffer solutions with pH values ​​of 7.4, 6.5, and 5.8, respectively. The resulting test solutions were incubated with gentle magnetic stirring at 37°C. At specified time points, the degradation solutions were centrifuged to separate the precipitate and supernatant. Changes in the precipitate were observed by transmission electron microscopy, and changes in the supernatant were detected by fluorescence spectroscopy and electrospray ionization mass spectrometry.

[0031] ESI-MS analysis of intracellular drug CTT generation in live HeLa cells: HeLa cells were cultured at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and cultured for 24 hours. Subsequently, the cells were divided into two groups: one treated with PVP@CuP@ZIF, and the other with PBS, both for 24 hours. Cells were then collected using a cell scraper and resuspended in water. The cell suspension was lysed by sonication for 30 minutes. The lysate was centrifuged at 13,000 rpm for 10 minutes to obtain the supernatant. The supernatant was mixed with pre-chilled acetone and stored overnight at -20°C. The mixture was again centrifuged at 13,000 rpm for 30 minutes to obtain the solid residue. After vacuum drying, the residue was analyzed by ESI-MS and compared with a standard of the target product to determine whether the cell lysate contained the target substance.

[0032] PVP@CuP@ZIF-mediated intracellular CuAAC click reaction: To investigate the cellular uptake and CuAAC click reaction efficiency of PVP@CuP@ZIF, cells were seeded in 6-well plates (2 × 10⁶ cells per well). 5 HeLa cells (number of cells) were treated with PVP@CuP@ZIF, Cu@PI@ZIF, prodrug (I+II) (100 µg mL⁻¹), and PBS for 24 hours, respectively. The old culture medium was then discarded, and the cells were washed three times with PBS. Finally, fluorescence images were acquired using a confocal fluorescence microscope.

[0033] like Figure 4As shown in the figure, based on fluorescence analysis, the product CTT exhibited significant fluorescence emission at approximately 475 nm, while no significant emission was observed in other substrates or precursors. Therefore, the formation of CTT can be quantitatively monitored by the time-dependent enhancement of fluorescence intensity and the concentration-dependent fluorescence response of PVP@CuP@ZIF. Based on this, the pH-responsive biodegradation of the delivered nanomaterial PVP@CuP@ZIF was evaluated by fluorescence response. The results showed that after incubating PVP@CuP@ZIF NPs in PBS at different pH values ​​for 24 hours, significant enhancement of fluorescence emission was observed under weakly acidic conditions at pH 5.8 and 6.5. Mass spectrometry analysis ( Figure 4 g) This directly confirmed the formation of CTT, with the characteristic ion [CTT-H] recorded at m / z 312.06 at pH 6.5. - No significant product was observed under neutral pH 7.4 conditions. The results indicate that the encapsulated substrate and precursor can be released under acidic conditions, driving the in-situ CuAAC reaction to synthesize CTT, accompanied by blue fluorescence emission. Therefore, this pH-responsive nanoplatform holds great potential for site-specific synthesis of CTT in acidic tumor microenvironments, enabling highly selective tumor therapy.

[0034] Example 2: In vitro cell evaluation and cytotoxicity assessment of PVP@CuP@ZIF.

[0035] To evaluate the feasibility of in-situ synthesis of CTT for selective synergistic mitochondrial damage in cancer therapy ( Figure 5 a) Cell experiments were conducted using HeLa cells as a model.

[0036] Cell culture: HeLa cells were cultured in DMEM medium containing 10% (v / v) fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO2.

[0037] Intracellular Cu 2+ Level assessment: HeLa cells were prepared at 1.0 × 10⁶ cells per dish. 5 Cells were seeded at a density of [number] cells per confocal culture dish and cultured for 24 hours. Subsequently, cells were treated with PBS, CTT, Cu@PI@ZIF, and PVP@CuP@ZIF (100 μg / mL) for 12 hours each. Afterward, the treatment solutions were discarded, and the cells were incubated for another 12 hours with the RBH fluorescent probe, followed by observation using a confocal laser scanning microscope.

[0038] Flow cytometry apoptosis assay: HeLa cells were cultured at 5 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in 6-well plates and cultured for 24 hours. Subsequently, cells were treated for 24 hours with medium containing different samples (PVP@CuP@ZIF, Cu@PI@ZIF, CTT, PBS, prodrug I+II)). Cells were collected by trypsin digestion with EDTA-free solution and washed with PBS. Then, 2 μL of Annexin V-FITC solution was added to the cell suspension and incubated at 4°C in the dark for 5 minutes. Afterward, 2 μL of PI solution was added at 4°C. Finally, the cells were analyzed using flow cytometry.

[0039] Live / dead cell staining assay: HeLa cells were seeded in 35 mm glass-bottomed cell culture dishes and cultured at 37°C for 24 hours. Afterward, the old culture medium was discarded and replaced with medium containing PBS, prodrugs (I+II), Cu@PI@ZIF, and PVP@CuP@ZIF (100 μg / mL). After co-culturing for 6 hours, the culture medium was aspirated. Cells were then washed three times with cold PBS buffer and imaged using a confocal laser scanning microscope.

[0040] First, mass spectrometry analysis of HeLa cells treated with PVP@CuP@ZIF for 12 hours confirmed the in situ synthesis of CTT within HeLa cells. Figure 5 b). Subsequently, confocal laser scanning microscopy was used to evaluate the cellular uptake of PVP@CuP@ZIF for in situ synthesis. Figure 5 As shown in Figure c, compared with cells treated with the prodrug or Cu@PI@ZIF, the PVP@CuP@ZIF treatment group showed a significant intracellular blue fluorescence signal, indicating its effective intracellular memory function and subsequent in situ synthesis. Furthermore, using rhodamine B hydrazide as a probe, intracellular copper ions released from cancer cells were monitored by fluorescence imaging. The results showed ( Figure 5 d) Cells in both the CTT-treated and Cu@PI@ZIF-treated groups exhibited relatively weak red fluorescence signals. In contrast, the PVP@CuP@ZIF-treated group showed the most significant red fluorescence emission, confirming the efficient delivery and release of Cu(I) ions within the cells required to drive the CTT treatment process.

[0041] To directly assess the cytotoxicity of the current PVP@CuP@ZIF, CCK-8 assays were performed on cancer cells (HeLa cells) and normal cells (HEK-293T cells), and the results were compared with different prodrugs or nanomaterials.

[0042] Cell viability assay: The cytotoxicity of the test compounds and nanoparticles was assessed using the CCK-8 assay. Briefly, HeLa cells were seeded at a density of 10,000 cells per well in 96-well plates, with 100 µL of culture medium added to each well, and cultured for 24 hours. For cytotoxicity screening, cells were treated with the reaction product solution corresponding to the compound under investigation for 24 hours. The IC50 of compound CTT was determined. 50 Cells were treated with CTT medium containing concentration gradients for 24 hours. Furthermore, the cytotoxicity of the nanocomposites (ZIF-8, Cu@PI@ZIF, PVP@CuP@ZIF nanoparticles and prodrug (I+II)) was evaluated by treating cells with an equivalent concentration of each material at 100 µg / mL for 24 hours. After treatment, cells were incubated with CCK8 reagent (CCK8 reagent: medium = 1:10) for approximately 4 hours, followed by absorbance measurement using a microplate reader.

[0043] Live / dead cell staining assay: HeLa cells were seeded in 35 mm glass-bottomed cell culture dishes and cultured at 37°C for 24 hours. Afterward, the old culture medium was discarded and replaced with fresh medium containing PBS, prodrugs (I+II), Cu@PI@ZIF, and PVP@CuP@ZIF (100 μg / mL). After co-culturing for 24 hours, the cells were stained with calcein acetoxymethyl ester (1.5 μL) and propidium iodide (1.5 μL) for 20 minutes, followed by imaging using a confocal laser scanning microscope.

[0044] like Figure 5 As shown in Figure e, compared with the control group, prodrug substrate group, ZIF-8 group, and Cu@PI@ZIF group, HeLa cells treated with PVP@CuP@ZIF exhibited significantly higher cytotoxicity. However, no significant cytotoxicity was recorded in HEK-293T cells, which fully confirms the selective cytotoxic effect of PVP@CuP@ZIF on cancer cells. Complementary live / dead cell staining experiments were performed after treating HeLa cells with different nanomaterials. Figure 5 As shown in f, a significant green fluorescence signal (a characteristic of live cells) was detected in HeLa cells treated with the prodrug substrate, indicating minimal cytotoxicity to cancer cells in the absence of in situ CTT synthesis. In contrast, the PVP@CuP@ZIF treatment group showed the strongest red fluorescence signal (corresponding to dead cells), validating the potent cytotoxicity of PVP@CuP@ZIF to HeLa cells. This cytotoxicity originates from apoptosis, which was verified by flow cytometry apoptosis analysis, showing that the PVP@CuP@ZIF group had the highest apoptosis rate, reaching 95.4%, higher than the other groups ( Figure 5g). Therefore, by efficiently delivering Cu(I) and prodrug substrates to cancer cells via PVP@CuP@ZIF, in-situ synthesis of CTT was achieved, thereby selectively and efficiently killing cancer cells.

[0045] Example 3: Detection of mitochondrial damage induced by apoptosis and copper death.

[0046] The mechanism by which it efficiently kills cancer cells was further evaluated. For example... Figure 6 As shown in Figure a, in-situ synthesized CTT initiates apoptosis by inhibiting BCL-2 and MCL-1 in mitochondria. This induces mitochondrial damage, manifested as decreased mitochondrial membrane potential and reduced ATP expression. When mitochondria are damaged, the apoptosis factor cytochrome c is released to cleave Caspase-3, ultimately inducing mitochondrial apoptosis. Simultaneously, Cu(I) released from mitochondria induces DLAT oligomerization and loss of Fe-S cluster proteins, ultimately leading to copper death. A series of mechanistic experiments were conducted to verify the synergistic mitochondrial damage induced by the combined pathways of apoptosis and copper death.

[0047] Mitochondrial membrane potential measurement: The JC-1 assay kit is widely used as an ideal fluorescent probe for measuring mitochondrial membrane potential. First, tumor cells are prepared at 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells in confocal culture dishes and incubated at 37°C for 24 hours. Cells received four different treatments (PBS, CTT, PVP@CuP@ZIF), followed by staining with JC-1 staining working solution for 20 minutes. Finally, the cells were observed and photographed using a fluorescence confocal microscope.

[0048] To directly assess mitochondrial damage, mitochondrial membrane potential was detected using the JC-1 probe. The results are as follows: Figure 6 As shown in b, compared with HeLa cells directly incubated with CTT, cells treated with PVP@CuP@ZIF showed a significant decrease in mitochondrial membrane potential (increased green / red fluorescence ratio). Figure 6 (d) This demonstrates that PVP@CuP@ZIF can effectively cause mitochondrial damage. This is consistent with the significant morphological changes in HeLa cells observed during transmission electron microscopy, namely, the obvious swelling, vacuolation, and cristae breakage of cells after incubation with PVP@CuP@ZIF. Figure 6 (c and 6e). Furthermore, HeLa cells treated with PVP@CuP@ZIF also showed the most significant ATP depletion, providing further evidence for mitochondrial damage induced by PVP@CuP@ZIF.

[0049] To further confirm the mitochondrial damage caused by the copper death and apoptosis co-pathway, the expression of the corresponding proteins was detected by Western blot analysis.

[0050] Western blot analysis: HeLa cells were seeded in 6-well plates and cultured for 24 hours. Cells were then treated for an additional 24 hours with different formulations (PBS, CTT, and PVP@CuP@ZIF). Proteins were extracted using RIPA lysis buffer and quantified using a BCA protein assay kit. Protein concentrations were normalized, and proteins were separated by SDS-PAGE gradient gel electrophoresis and transferred to PVDF membranes. The membranes were blocked for 0.5 hours at room temperature with TBST buffer containing 5% skim milk. Then, the membranes were incubated overnight at 4°C with the corresponding primary antibody diluted in TBST buffer containing 5% skim milk. After washing three times with TBST (5 minutes each time), the membranes were incubated for 0.5 hours at room temperature with secondary antibody diluted in TBST buffer containing 5% skim milk. After washing three more times with TBST (5 minutes each time), protein expression was detected using a highly sensitive chemiluminescence system and imaged using an automated chemiluminescence imaging system.

[0051] First, the expression levels of BCL-2 and MCL-1 in cells were assessed. The results showed that the expression levels of both proteins decreased most significantly after treatment with PVP@CuP@ZIF. Figure 6 (g) This confirms that the in-situ synthesized small molecule CTT, which induces tumor cell apoptosis, effectively inhibits BCL-2 and MCL-1, initiating subsequent apoptosis therapy. The significant increase in cytochrome c levels in the cytoplasm further confirms the induction of apoptosis, suggesting its release from damaged mitochondria. Furthermore, the decrease in Caspase-3 expression is consistent with the Caspase-3 cleavage process accompanying cytochrome c release, ultimately inducing apoptosis. Simultaneously, copper death therapy was also confirmed by the significant downregulation of key regulatory proteins DLAT and ferroredoxin 1. In addition, cell scratch assays validated the highly efficient apoptosis-copper death combined therapy mediated by PVP@CuP@ZIF, showing that this material can significantly inhibit cell migration. Therefore, PVP@CuP@ZIF exhibits significant inhibition of BCL-2 and MCL-1 and accumulation of Cu ions in mitochondria, inducing synergistic mitochondrial damage during apoptosis and copper death.

[0052] Example 4: In vivo experimental evaluation of the effect Using HeLa tumor-bearing mice as a model, the therapeutic efficacy of current PVP@CuP@ZIF was evaluated in vivo.

[0053] Mouse xenograft tumor model: All animal experimental protocols were reviewed and approved by the Animal Protection and Use Committee of Beijing Normal University and complied with all relevant ethical regulations. Female BALB / c nude mice were purchased and housed in a specific pathogen-free laboratory. Approximately 5 × 10⁵ xenografts were subcutaneously injected into the right back of the BALB / c nude mice. 6 A tumor-bearing mouse model was established using HeLa cells. Mouse weight was measured every 3 days for 14 days post-injection.

[0054] Hemolysis assay of PVP@CuP@ZIF NPs: 1 mL of whole blood was collected from the orbital venous plexus of BALB / c mice using blood collection tubes pre-filled with lithium heparin anticoagulant. Subsequently, 300 μL of the collected blood was mixed with 1 mL of physiological saline, centrifuged at 2500 rpm for 5 minutes, and the supernatant was carefully discarded. This washing process was repeated 3–4 times until the supernatant was completely colorless and transparent, indicating the removal of plasma components. The precipitated red blood cells were then resuspended in physiological saline to prepare a red blood cell suspension. PVP@CuP@ZIF NPs were added to the red blood cell suspension to final concentrations of 1, 10, 50, and 100 μg / mL. For negative control samples, the red blood cell suspension was diluted with physiological saline; for positive control samples, sonication and gentle shaking were performed to ensure complete lysis of the red blood cells. The tubes were incubated at room temperature for 8 hours, during which time hemolysis was systematically monitored and recorded. Simultaneously, the specific spectrophotometric absorbance of hemoglobin at 540 nm was analyzed.

[0055] The formula for calculating hemolysis rate is as follows: Hemolysis rate = (A 样品 - A 阴性 ) / (A 阳性 - A 阴性 ) 100% Among them, A 样品 A 阳性 and A 阴性 These represent the absorbance values ​​of the sample, positive control, and negative control, respectively.

[0056] Tumor growth inhibition: For in vivo anti-tumor studies, mice were randomly divided into four groups until the tumor volume reached approximately 100-120 mm. 3 Treatment began at that time. Each nude mouse was injected every two days via tail vein with 200 μL of the excipient, therapeutic nanoformulations PVP@CuP@ZIF, Cu@PI@ZIF, and compound CTT for 14 days, while animal weight and size were monitored. Tumor size was measured using calipers, and tumor volume was calculated using the following formula: Tumor volume = Tumor long axis (Tumor short axis) 2 / 2.

[0057] After the 14th treatment, tumors and major organs were taken for hematoxylin-eosin staining analysis and TUNEL immunofluorescence assay.

[0058] Mice were randomly divided into four groups and administered 200 μL of PBS (control group), CTT, Cu@PI@ZIF, and PVP@CuP@ZIF (all at a dose of 10 mg / kg) intravenously, respectively. Figure 7 As shown in Figure a, different therapeutic drugs or nanomaterials were injected via the tail vein every 3 days, and tumor volume and body weight were monitored every 3 days during the 14-day treatment period. Results showed that no significant changes in mouse body weight were recorded during the 14-day treatment period in any of the different treatment groups. Figure 7 b). However, the PVP@CuP@ZIF treatment group showed a significant tumor suppression effect, with a tumor volume (b) significantly lower than other groups. Figure 7 c) and weight ( Figure 7 d) were all the lowest. This is consistent with the results of representative tumor photographs after different treatments, showing that the tumor size was smallest 14 days after PVP@CuP@ZIF treatment ( Figure 7 e). Therefore, the current PVP@CuP@ZIF can achieve selective tumor suppression.

[0059] Finally, the antitumor activity and biosafety of PVP@CuP@ZIF were evaluated by hematoxylin-eosin staining, terminal deoxynucleotidyl transferase (dUTP) nick-end labeling, and immunohistochemical staining. Figure 7 As shown in f: H&E staining results showed that the PVP@CuP@ZIF group caused the most significant damage to tumor tissue, with reduced tumor cell density and increased chromatin aggregation compared to other groups. Furthermore, no significant pathological abnormalities were observed after 14 days of treatment with the PVP@CuP@ZIF nanomaterials provided by this invention, indicating extremely low off-target toxicity. Consistently, TUNEL staining confirmed the apoptosis-inducing effect of PVP@CuP@ZIF through significantly enhanced green TUNEL signal, while DLAT staining demonstrated the PVP@CuP@ZIF-induced copper death process through downregulation of blue DLAT signal. Hemolysis experiments further supported its good biocompatibility, showing negligible hemolytic activity even at high concentrations of PVP@CuP@ZIF, confirming its excellent blood compatibility. Therefore, PVP@CuP@ZIF demonstrates highly selective, significantly effective, and low-side-effect cancer treatment performance by synergistically inducing mitochondrial damage to initiate the apoptosis-copper death process.

Claims

1. A small molecule CTT that induces tumor cell apoptosis, characterized in that, The structural formula of the small molecule CTT that induces tumor cell apoptosis is: 。 2. A method for preparing the small molecule CTT that induces tumor cell apoptosis as described in claim 1, characterized in that, Includes the following steps: 3-Azide-7-hydroxycoumarin and 2-ethynylthiophene were stirred in acetonitrile and refluxed. The solution was then cooled to room temperature, concentrated under reduced pressure, and the crude product was purified to obtain CTT.

3. The preparation method according to claim 2, characterized in that, The molar ratio of 3-azido-7-hydroxycoumarin to 2-ethynylthiophene is 2.46:1.14; the reflux reaction is carried out by heating at 82°C for 24 hours; the purification is carried out by column chromatography using petroleum ether / ethyl acetate as eluent.

4. A tumor-selective delivery nanomaterial PVP@CuP@ZIF for in situ synthesis of the small molecule CTT as described in claim 1, characterized in that, The PVP@CuP@ZIF is formed by encapsulating Cu2O and 3-azido-7-hydroxycoumarin into ZIF-8 nanoparticles to form the intermediate Cu@PI@ZIF; subsequently, 2-ethynylthiophene is anchored on the surface of Cu@PI@ZIF to obtain the intermediate CuP@ZIF; finally, PVP is coated on the outermost layer of CuP@ZIF to obtain the delivery nanomaterial PVP@CuP@ZIF.

5. A method for preparing the tumor-selective delivery nanomaterial PVP@CuP@ZIF as described in claim 4, characterized in that, Includes the following steps: (1) Dissolve CuI and NaAsc in water; then add sodium hydroxide to the mixture, stir for a period of time, wash the Cu2O nanoparticles, centrifuge and collect the precipitate to obtain Cu2O nanoparticles; (2) Synthesis of Cu@PI@ZIF nanoparticles: Cu2O nanoparticles were dispersed in methanol and sonicated; under argon protection, methanol solutions containing 3-azido-7-hydroxycoumarin and Zn(NO3)2·6H2O were added sequentially; after stirring evenly, methanol solutions containing 2-methylimidazole were slowly added; the mixture was stirred to carry out the reaction, and then the precipitate was collected by centrifugation, washed and dried to obtain Cu@PI@ZIF; (3) Synthesis of PVP@CuP@ZIF nanoparticles: Cu@PI@ZIF was ultrasonically dispersed in a mixed solution of methanol and water. Then, 2-ethynylthiophene dissolved in methanol was slowly added to the Cu@PI@ZIF solution. After stirring for a period of time, the solid was collected by centrifugation, washed and dried to obtain CuP@ZIF nanoparticles. Ethanol was added to the nanoparticles to obtain CuP@ZIF ethanol solution. CuP@ZIF ethanol solution was added to the PVP solution and stirred. After centrifugation and washing, PVP@CuP@ZIF nanoparticles were obtained.

6. The preparation method according to claim 5, characterized in that, In step (1), the molar ratio of CuI to NaAsc is 1:5; the mass ratio of CuI to sodium hydroxide is 0.025:1; and the stirring is carried out at room temperature until the solution turns orange.

7. The preparation method according to claim 5 or 6, characterized in that, In step (2), the mass ratio of Cu2O nanoparticles to 3-azido-7-hydroxycoumarin, calculated as CuI, is 1:2; the concentration of the methanol solution containing 3-azido-7-hydroxycoumarin is 2.5 mg / mL; the concentration of the methanol solution containing Zn(NO3)2·6H2O is 25 mg / mL; the concentration of the methanol solution containing 2-methylimidazole is 30 mg / mL; the molar ratio of 3-azido-7-hydroxycoumarin, Zn(NO3)2·6H2O, and 2-methylimidazole is 0.2-0.3:1.6-1.7:7.0-7.5; and the reaction is carried out under argon protection and stirred at room temperature for 0.5-2 h.

8. The preparation method according to claim 5, characterized in that, In step (3), the concentration of Cu@PI@ZIF in the mixed solvent is 2.5 mg / mL; the mixed solvent is composed of methanol and water in a ratio of 8-9:1-2 (v / v); the mass ratio of Cu@PI@ZIF to 2-ethynylthiophene is 1:5-8; the concentration of 2-ethynylthiophene in methanol is 30 mg / mL; the mass ratio of CuP@ZIF nanoparticles to polyvinylpyrrolidone is 1:40; the concentration of CuP@ZIF ethanol solution is 50 mg / mL; after adding the 2-ethynylthiophene solution, the mixture is stirred at room temperature for 4-5 h; the CuP@ZIF nanoparticles are coated with PVP solution by stirring at room temperature for 2-4 h.

9. The use of the PVP@CuP@ZIF as described in claim 1 in the preparation of a drug for selective tumor delivery to synthesize CTT in situ at the tumor site and for antitumor purposes.

10. The application according to claim 3, characterized in that, The PVP@CuP@ZIF is used as a delivery precursor to synthesize a small molecule CTT that induces tumor cell apoptosis at the tumor site via a CuAAC reaction.

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