Tubulin disrupting agent dimer and copper death drug combined nano-particle as well as preparation method and application thereof

Abstract

The invention provides nano-particles combined with tubulin disrupting agent dimer and copper death medicine as well as a preparation method and application of the nano-particles, and belongs to the field of biological medicine. The invention provides a nanoparticle combining a tubulin disrupting agent dimer and a copper death drug. The microtubulin destroying agent can be used for enhancing the sensitivity of tumor cells to oxidative damage by destroying a cell microtubule structure and inducing oxidative stress and blood flow occlusion; the copper death drug promotes copper ion accumulation, causes mitochondrial protein aggregation and dysfunction, and finally induces cell death. The two medicines are combined for use, so that a remarkable synergistic anti-tumor effect can be generated, and the treatment efficiency is remarkably improved; meanwhile, after the two drugs are prepared into the nanoparticles, the selectivity index of the nanoparticles for resisting tumors is good, and the toxic and side effects on cells are small. The method has wide clinical transformation prospects and important social and economic values.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a nanoparticle comprising a microtubule-destroying agent dimer and a copper-death drug, as well as a preparation method and use thereof. Background Art

[0002] The occurrence and progression of tumors are closely related to abnormal cell proliferation. Microtubules, as an important component of the cytoskeleton system, play an indispensable role in mitosis. Therefore, microtubule-disrupting agents have been widely used in clinical practice as an important anti-tumor treatment strategy. Currently, a variety of anti-tumor drugs targeting microtubules, such as paclitaxel, vincristine, and irinotecan, have been approved for the treatment of various solid tumors. However, traditional microtubule-targeting drugs often develop increased drug resistance, severe side effects, and decreased therapeutic efficacy after long-term use. There is an urgent need to develop new, efficient, and low-toxic microtubule-regulating methods.

[0003] To overcome the limitations of traditional microtubule-disrupting drugs, tubulin-disrupting agent dimers (such as CA-4 dimers) have become a research hotspot in recent years. By covalently linking two microtubule-binding groups via a linker, dimeric drugs can achieve multivalent effects at the binding site, enhancing microtubule inhibitory activity and cytotoxicity, and helping to reduce drug tolerance. CA-4 (Combretastatin A-4), a natural product derivative, can specifically bind to the Vinca binding site of tubulin, inhibiting microtubule polymerization and causing cell cycle arrest at the G2 / M phase. Various dimeric structures developed based on CA-4 have demonstrated excellent antitumor activity in vitro and in vivo, and are also effective against multidrug-resistant (MDR) tumor cells.

[0004] In addition to CA-4 dimers, paclitaxel dimers have improved stability and drug resistance by optimizing their connection structures. Vinorelbine dimers exhibit excellent microtubule inhibitory activity, enhancing the killing effect on drug-resistant tumor cells. Epothilone B and its derivatives, as novel microtubule stabilizers, have overcome the problem of paclitaxel resistance. Eribulin, with its unique microtubule dynamics inhibition mechanism, has been successful in the treatment of advanced breast cancer. Furthermore, small molecules developed based on microtubule binding sites, such as MMAE (Monomethylauristatin E), have significantly improved the targeting and therapeutic window of microtubule drugs through the antibody-drug conjugate (ADC) platform.

[0005] On the other hand, copper ions (Cu) have important physiological functions within cells, but an imbalance in their homeostasis can lead to cell death. The recently proposed term "cuproptosis" is a newly discovered form of cell death directly linked to elevated copper ion density. Its mechanism involves the aggregation of mitochondrial fatty acylated proteins, the inactivation of iron-sulfur cluster proteins, and proteotoxic stress. Unlike traditional cell death mechanisms such as apoptosis, necrosis, and autophagy, cuproptosis represents a novel target for therapeutic intervention, particularly in tumor types resistant to traditional therapies.

[0006] Currently, a variety of small molecule drugs capable of inducing copper-induced cell death have been discovered. Elesclomol is a drug that delivers copper ions to mitochondria, inducing copper-related cell death by promoting oxidative stress. Disulfiram, originally used to treat alcoholism, binds to copper ions to form a CuET complex, significantly inducing tumor cell death. Ammonium tetrathiomolybdate (ATTM), a copper ion chelator, also achieves anti-tumor effects by regulating intracellular copper homeostasis. As the mechanisms of copper-induced cell death are gradually elucidated, an increasing number of small molecule copper-induced cell death agents or copper ion carriers have entered the research and development stage.

[0007] In addition to elesclomol, disulfiram, and ATTM, NSC319726 (APR-246) is a novel small molecule that can regulate copper ion metabolism while restoring the function of mutant p53 proteins. Cu-ATSM (Copper diacetyl-bis(N4-methylthiosemicarbazone)) is a copper complex that targets hypoxic tumor areas and is widely used in radiotherapy and copper apoptosis research. CuET (Bis(diethyldithiocarbamate)-copper complex), a metabolite of disulfiram, has been validated in multiple tumor models for its copper apoptosis-inducing effects. Tetrathiomolybdate (TM) inhibits tumor angiogenesis and growth by chelating excess copper ions.

[0008] Basic research has also revealed that D-penicillamine can, under specific conditions, induce copper homeostasis and promote cytotoxic effects. Furthermore, researchers have developed novel copper ion carriers based on a pyridimidazole backbone that effectively direct copper ion transport across membranes, thereby activating the copper death pathway. BCS (bathocuproinedisulfonic acid) and its derivatives have also been found to indirectly induce cell death by affecting intracellular copper ion levels.

[0009] Although the research on microtubule-destroying agent dimers and copper-death drugs has been continuously deepened, there has been no study on the combined use of microtubule-destroying agent dimers and copper-death drugs for anti-tumor purposes. The effect needs further study. Summary of the Invention

[0010] The purpose of the present invention is to provide a nanoparticle combining a tubulin disruptor dimer with a copper-depleting drug, a preparation method and use thereof, so as to overcome the problems of insufficient efficacy, strong drug resistance and large side effects of existing single treatment methods.

[0011] The present invention provides a nanoparticle comprising a tubulin-destroying agent dimer and a copper-death drug. The nanoparticle is prepared from the following raw materials in parts by weight and volume: 10 parts by weight of tubulin disrupting agent dimer, 4-10 parts by weight of copper-death drug, 5-10 parts by weight of surfactant, 0.5-1 part by weight of copper ion salt, 100-1000 parts by volume of organic solvent, and 2000-20000 parts by volume of water.

[0012] Here, the ratio of parts by weight to parts by volume is g / mL.

[0013] Furthermore, the nanoparticles are prepared from the following raw materials in parts by weight and volume: 10 parts by weight of tubulin disrupting agent dimer, 4.8 parts by weight of copper-deleting drug, 8 parts by weight of surfactant, 0.88 parts by weight of copper ion salt, 400 parts by volume of organic solvent, and 16,000 parts by volume of water.

[0014] Further, The tubulin disrupting agent dimer is selected from one or more of the following: CA-4 dimer, paclitaxel dimer, Vinorel dimer, Ibovan dimer, Eribulin dimer or salts or derivatives of the above dimers; The copper-killing drug is selected from one or more of the following: Elesclomol, Disulfiram, ammonium tetrathiomolybdate, NSC319726, Cu-ATSM, CuET, Tetrathiomolybdate, D-penicillamine or salts or derivatives of the above compounds.

[0015] Furthermore, the tubulin disrupting agent dimer is selected from the CA-4 dimer compound represented by Formula I, a salt thereof, or a stereoisomer thereof: Formula I Wherein, L is selected from or ; n and m are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; The copper-killing drug is selected from Elesclomol.

[0016] Furthermore, the tubulin disrupting agent dimer is selected from one of the following structural formulas: .

[0017] Further, The surfactant is selected from distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000; And / or, the copper ion salt is selected from CuCl2; And / or, the organic solvent is selected from tetrahydrofuran.

[0018] Furthermore, the method for preparing the nanoparticles comprises the following steps: (1) Preparing an organic phase: dissolving a tubulin-destroying agent dimer, a copper-death drug, a surfactant, and a copper ion salt in an organic solvent to obtain an organic phase; (2) Add the organic phase obtained in step (1) into water and stir to obtain the product.

[0019] Preferably, in step (2), the stirring temperature is 25-35° C., and the stirring time is 1-5 h.

[0020] Preferably, in step (2), the stirring is followed by dialysis.

[0021] The present invention also provides a method for preparing the aforementioned nanoparticles, which comprises the following steps: (1) Preparing an organic phase: dissolving a tubulin-destroying agent dimer, a copper-death drug, a surfactant, and a copper ion salt in an organic solvent to obtain an organic phase; (2) Add the organic phase obtained in step (1) into water and stir to obtain the product.

[0022] Preferably, in step (2), the stirring temperature is 25-35° C., and the stirring time is 1-5 h.

[0023] Preferably, in step (2), the stirring is followed by dialysis.

[0024] The present invention also provides use of the aforementioned nanoparticles in preparing drugs for preventing and / or treating cancer.

[0025] Furthermore, the cancer is selected from lung cancer, liver cancer, pancreatic cancer, breast cancer, melanoma, and glioblastoma.

[0026] Preferably, the cancer is a drug-resistant or highly aggressive cancer.

[0027] The anti-tumor nanoparticles of the present invention can exert their effects through the following mechanisms: 1. Tubulin-disrupting agent dimers can directly interfere with the assembly and stability of the microtubule skeleton in tumor cells, leading to abnormal mitosis, cell cycle arrest at the G2 / M phase, and inducing apoptosis or other forms of cell death; 2. Copper-killing drugs can increase the concentration of copper ions in cells, induce the aggregation of mitochondrial fatty acylated proteins, inactivate iron-sulfur cluster proteins, and activate specific copper-killing signaling pathways; After combined use, the intracellular stress environment induced by the tubulin-destroying agent can further amplify the toxic effects of copper ions. The two promote each other and synergistically induce rapid tumor cell death. 3. The combination strategy of the present invention can overcome the drug resistance of tumor cells to single drugs and reduce the risk of tumor metastasis and recurrence.

[0028] Compared with the prior art, the present invention has achieved the following beneficial effects: The present invention proposes a new tumor treatment strategy by combining a microtubule-destroying agent dimer with a copper-killing drug. Microtubule-destroying agents induce cell cycle arrest and endogenous stress responses by destroying the cytoskeleton and mitosis, while promoting the abnormal accumulation of copper ions in cells. Copper-killing drugs further amplify cellular stress, inducing fatty protein aggregation, mitochondrial dysfunction, and iron-sulfur cluster protein inactivation, ultimately leading to cell death. The synergistic effect of the two can achieve precise and efficient killing of tumor cells, especially showing excellent therapeutic effects on multidrug-resistant, hypoxic, or hypermetabolic tumors.

[0029] It is worth noting that the nanoparticles of the present invention achieve synergistic anti-tumor effects while reducing drug toxicity and side effects, significantly improving patients' tolerance to medication and their quality of life.

[0030] At the same time, based on the present invention, the types and proportions of tubulin disruptors and copper-destroying drugs can be flexibly adjusted according to different tumor types, gene mutation backgrounds and copper metabolism characteristics, reflecting the concept of individualized and precise treatment.

[0031] With the continuous deepening of research on the mechanism of copper death and the continuous emergence of new structures of microtubule-destroying agents, the strategy of combining microtubule-destroying agent dimers with copper death drugs proposed in this invention is expected to become an important direction in the future development and clinical application of anti-tumor drugs, with broad industrial prospects and social value.

[0032] The present invention also provides a treatment method using the above-mentioned anti-tumor nanoparticles, comprising the following steps: An effective dose of nanoparticles containing a microtubule-destroying dimer and a copper-death drug is administered to the subject, either once or multiple times, to inhibit tumor cell growth and promote tumor cell death, thereby achieving the purpose of treating the tumor.

[0033] Preferably, the administration route may be intravenous injection, intraperitoneal injection, oral administration, local injection or other pharmaceutically acceptable routes.

[0034] A large number of in vitro and in vivo experiments have verified that the combined use of the microtubule-destroying agent dimer of the present invention and the copper-death drug can exhibit a synergistic killing effect in a variety of tumor cell lines that is significantly higher than that of a single drug, and can effectively reduce the effective dose required for treatment and reduce the toxic effects on normal cells, and has significant clinical translation potential.

[0035] In summary, the present invention provides a novel and highly effective tumor treatment strategy, which fills the gap in the existing technology for the treatment of drug-resistant and highly invasive tumors, and has important scientific significance and broad application prospects.

[0036] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0037] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the H-NMR spectrum of the tubulin disrupting agent dimer CA-4S2 prepared in Example 1.

[0039] Figure 2 This is the C-NMR spectrum of the tubulin disrupting agent dimer CA-4S2 prepared in Example 1.

[0040] Figure 3 This is the H-NMR spectrum of the tubulin-disrupting agent dimer CA-4C6 prepared in Example 2.

[0041] Figure 4 This is the C-NMR spectrum of the tubulin-disrupting agent dimer CA-4C6 prepared in Example 2.

[0042] Figure 5UV-visible absorption spectra of the raw materials and products in the preparation of CA-4S2@ES-Cu and CA-4C6@ES-Cu: A is the UV-visible absorption spectra of ES, ES-Cu, CA-4S2, and CA-4S2@ES-Cu; B is the UV-visible absorption spectra of ES, ES-Cu, CA-4C6, and CA-4C6@ES-Cu.

[0043] Figure 6 Infrared spectra of ES, ES-Cu, CA-4S2, CA-4C6, CA-4S2@ES-Cu and CA-4C6@ES-Cu.

[0044] Figure 7 Figure 3 shows the particle size distribution results of CA-4S2@ES-Cu and CA-4C6@ES-Cu in ultrapure water and the zeta potential measurement results in ultrapure water (n=3): A is the particle size distribution result diagram; B is the zeta potential measurement result diagram.

[0045] Figure 8 TEM images of CA-4S2@ES-Cu and CA-4C6@ES-Cu: A is the TEM image of CA-4S2@ES-Cu; B is the TEM image of CA-4C6@ES-Cu.

[0046] Figure 9 Element distribution (Cu, O, N, and S) of CA-4S2@ES-Cu and CA-4C6@ES-Cu. The scale bar is 200 nm.

[0047] Figure 10 XPS results of CA-4S2@ES-Cu and CA-4C6@ES-Cu.

[0048] Figure 11 Figure 2 shows the comparison of selectivity index (SI) of different drug groups: A shows the selectivity index (SI) of different drugs by IC 50 (THLE-2) / IC 50 (Huh-7) SI results; B is the SI results of different drugs by IC 50 (AML12) / IC 50 (Hepa1-6) obtained SI results Figure.

[0049] Figure 12 The results of the observation of morphological changes of HCC cells (Huh-7 and Hepa1-6 cells) after treatment with different drugs (×10) are shown.

[0050] Figure 13 The figure shows the results of detecting the proliferation activity of HCC cells (Huh-7 and Hepa1-6 cells) after treatment with different drugs using EdU staining (×40).

[0051] Figure 14 The figure shows the effects of different drug treatments on the clone-forming ability of HCC cells (Huh-7 and Hepa1-6 cells).

[0052] Figure 15 The figures show the effects of different drug treatments on the cell cycle distribution of HCC cells (Huh-7 and Hepa1-6 cells) and the effects of different concentrations of CA-4 treatment on the cell cycle distribution of HCC cells: A shows the effects of different drug treatments on the cell cycle distribution of HCC cells; B shows the effects of different concentrations of CA-4 treatment on the cell cycle distribution of HCC cells.

[0053] Figure 16 Figure 2 shows the changes in microtubule morphology in HCC cells (Huh-7 and Hepa1-6 cells) after treatment with different drugs (×100). A is the result of Huh-7 cells; B is the result of Hepa 1-6 cells.

[0054] Figure 17 The figure shows the effects of different drug treatments on apoptosis of HCC cells (Huh-7 and Hepa1-6 cells).

[0055] Figure 18 The graph shows the copper ion content in HCC cells (Huh-7 and Hepa1-6 cells) after treatment with different drugs.

[0056] Figure 19 The graph shows the cuprous ion content in HCC cells (Huh-7 and Hepa1-6 cells) after treatment with different drugs.

[0057] Figure 20 Figure 2 shows the results of GSH levels in HCC cells (Huh-7 and Hepa1-6 cells) after treatment with different drugs.

[0058] Figure 21 Representative confocal microscopy images of DLAT accumulation in HCC cells (Huh-7 and Hepa1-6 cells) after treatment with different drugs.

[0059] Figure 22 The results of ROS levels in HCC cells (Huh-7 and Hepa1-6 cells) after treatment with different drugs.

[0060] Figure 23 Representative confocal microscopy images of mitochondrial membrane potential levels in HCC cells (Huh-7 and Hepa1-6 cells) after treatment with different drugs.

[0061] Figure 24Figure 2 shows the results of Western Blot analysis of the changes in FDX1 and DLAT proteins in HCC cells (Huh-7 and Hepa1-6 cells) after different treatments: A is the result of Huh-7 cells; B is the result of Hepa 1-6 cells. DETAILED DESCRIPTION

[0062] Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be purchased from chemical companies.

[0063] Abbreviations of the present invention include: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCI); 4-dimethylaminopyridine (DMAP); petroleum ether (PE); ethyl acetate (EA); dichloromethane (DCM); copper-killing drug Elesclomol (ES); distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE-PEG 2K ).

[0064] In the present invention, room temperature refers to 25-35°C.

[0065] Example 1. Preparation of tubulin disrupting agent dimer CA-4S2 Reaction route: The specific reaction steps are as follows: CA-4 (505.5 mg, 1.6 mmol), EDC·HCl (306.3 mg, 1.6 mmol), and DMAP (48.8 mg, 0.4 mmol) were weighed into a 50 mL round-bottom flask and dissolved in 25 mL of dichloromethane. The mixture was stirred for 15 min. 3,3'-dithiodipropionic acid (468.0 mg, 0.8 mmol) was added to the system and stirred at room temperature for 8 h. The reaction progress was monitored by TLC (PE:EA = 5:1, volume ratio). After the reaction was complete, 25 mL of saturated aqueous ammonium chloride was added to the system. The mixture was extracted three times with dichloromethane (25 mL). The organic layer was extracted twice with saturated brine (30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under pressure to obtain a white crude product. This product was separated and purified by silica gel column chromatography (PE:EA = 3:1, volume ratio) to obtain a white solid product, CA-4S2 (550.0 mg), with a yield of 85.3%. The H-NMR spectrum of CA-4S2 is shown below. Figure 1 As shown, the C-NMR spectrum is as Figure 2 shown.

[0066] Example 2: Preparation of tubulin disrupting agent dimer CA-4C6 Reaction route: At room temperature, suberic acid (137.7 mg, 0.8 mmol) was weighed into a 50 mL round-bottom flask and completely dissolved in 20 mL of dichloromethane. EDC·HCl (302.9 mg, 1.6 mmol) and DMAP (48.3 mg, 0.4 mmol) were then dissolved in the reaction solution and stirred for 10 minutes. After uniform mixing, CA-4 (500.0 mg, 1.6 mmol) was added to the reaction system. The reaction was allowed to proceed at room temperature for 12 hours, and the reaction progress was monitored by TLC (PE:EA = 1:1, volume ratio). After completion of the reaction, 30 mL of saturated aqueous ammonium chloride was added to the reaction solution, followed by extraction three times with dichloromethane (30 mL). The organic phase was collected and extracted three times with saturated brine (30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated at low temperature to remove dichloromethane to obtain the crude product. The crude product was separated and purified by column chromatography to obtain a white solid product, CA-4C6 (450.0 mg), with a yield of 73.8%. The H-NMR spectrum of CA-4C6 is shown in Figure 2. Figure 3 As shown, the C-NMR spectrum is as Figure 4 shown.

[0067] Example 3. Preparation of CA-4S2@ES-Cu and CA-4C6@ES-Cu Nanoparticles ES (6.0 mg), CA-4S2 (12.5 mg), and DSPE-PEG 2K A 10.0 mg portion of the solution was dissolved in tetrahydrofuran (THF, 500 μL). CuCl₂ (1.1 mg) was then added to the solution and stirred at room temperature for 2 hours. After the reaction, excess CuCl₂ was removed by filtration, and the resulting mixture was added dropwise to 20 mL of pure water with stirring. After stirring for another 2 hours, the mixture was placed in a dialysis bag (1000 Da MWCO) and dialyzed against deionized water, changing the water every 6 hours for a total of three times to remove THF and free small molecules. Finally, a yellow nanoparticle solution containing CA-4S₂@ES-Cu nanoparticles was obtained.

[0068] According to the above preparation method, only CA-4S2 (12.5 mg) was replaced by CA-4C6 (12.5 mg) to prepare a nanosolution containing CA-4C6@ES-Cu nanoparticles.

[0069] Comparative Example 1: Preparation of ES-Cu ES (6.0 mg) was dissolved in 1 mL of dichloromethane, and CuCl2 (1.1 mg) was added to the solution. The mixture was stirred at room temperature for 2 hours. After the reaction, the excess CuCl2 was removed by filtration, and the solvent was evaporated to obtain ES-Cu.

[0070] Comparative Example 2: Preparation of CA-4C6NPs CA-4C6 (12.5 mg) and DSPE-PEG 2K A 10.0 mg portion of CA-4C6 nanoparticles (10.0 mg) was dissolved in tetrahydrofuran (THF, 500 μL) and added dropwise to 20 mL of pure water with stirring. After stirring for 2 hours, the resulting mixture was placed in a dialysis bag (1000 Da MWCO) and dialyzed against deionized water, changing the water every 6 hours for a total of three times to remove THF and free small molecules. Finally, a white nanoparticle solution containing CA-4C6 nanoparticles was obtained. The solution was freeze-dried and stored at -20°C.

[0071] Comparative Example 3: Preparation of CA-4S2NPs CA-4S2 (12.5 mg) and DSPE-PEG 2K A 10.0 mg portion of CA-4S2 nanoparticles (10.0 mg) was dissolved in tetrahydrofuran (THF, 500 μL) and added dropwise to 20 mL of pure water with stirring. After stirring for 2 hours, the resulting mixture was placed in a dialysis bag (1000 Da MWCO) and dialyzed against deionized water, changing the water every 6 hours for a total of three times to remove THF and free small molecules. Finally, a white nanoparticle solution containing CA-4S2 nanoparticles was obtained. The solution was freeze-dried and stored at -20°C.

[0072] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0073] The CA-4S2, CA-4C6, CA-4S2@ES-Cu and CA-4C6@ES-Cu involved in the following experimental examples were prepared according to the methods described in Examples 1 to 3, and ES-Cu, CA-4C6 NPs and CA-4S2 NPs were prepared according to the methods described in Comparative Examples 1 to 3.

[0074] Test Example 1: Structural Characterization 1. Ultraviolet-visible absorption (UV-vis) spectroscopy UV-visible absorption spectroscopy was used to analyze the raw materials and products (ES, CA-4S2, CA-4C6, CA-4S2@ES-Cu, CA-4C6@ES-Cu) and ES-Cu during the preparation of CA-4S2@ES-Cu and CA-4C6@ES-Cu.

[0075] Ultraviolet-visible absorption spectroscopy (UV-Vis) analysis showed that ( Figure 5 ), CA-4S2@ES-Cu and CA-4C6@ES-Cu both exhibited significantly enhanced absorption peaks at 420 nm, consistent with the characteristic absorption wavelength of ES-Cu, indicating successful encapsulation of ES-Cu within the nanosystem. Furthermore, the absorption characteristics of CA-4S2@ES-Cu and CA-4C6@ES-Cu in the 300-500 nm range were similar to those of free ES-Cu, indicating that the encapsulation of ES-Cu by the nanocarrier did not significantly affect its optical properties.

[0076] 2. Infrared spectroscopy (FTIR) Fourier transform infrared spectroscopy was used to analyze the infrared spectra of ES, ES-Cu, CA-4S2, CA-4C6, CA-4S2@ES-Cu and CA-4C6@ES-Cu.

[0077] Fourier transform infrared spectroscopy further verified the successful assembly of ES-Cu, CA-4S2 and CA-4C6 components in the CA-4S2@ES-Cu and CA-4C6@ES-Cu systems ( Figure 6 Among them, the characteristic absorption peak of C=O stretching vibration was observed at 1600-1700 cm⁻¹, indicating that DSPE-PEG 2K The CA-4S2@ES-Cu and CA-4C6@ES-Cu were successfully incorporated and participated in the formation of the nanostructure. Characteristic peaks of CA-4S2 and CA-4C6 stretching vibrations were observed at 1000-1300 cm⁻¹, indicating the successful incorporation of the CA-4 dimer. The characteristic peak at 500-600 cm⁻¹ corresponds to that of ES-Cu, further demonstrating the successful encapsulation of ES-Cu. These results demonstrate that CA-4S2@ES-Cu and CA-4C6@ES-Cu possess good structural integrity and are successfully loaded with ES-Cu and the corresponding CA-4 dimer.

[0078] 3. Particle size, potential and transmission electron microscopy of CA-4S2@ES-Cu and CA-4C6@ES-Cu Dynamic light scattering (DLS) was used to measure the particle size and Zeta potential of CA-4S2@ES-Cu and CA-4C6@ES-Cu. Figure 7 The results show that the average particle sizes of CA-4S2@ES-Cu and CA-4C6@ES-Cu are 124.4 nm and 143.7 nm, respectively ( Figure 7 A), indicating that both nanoparticles are in the appropriate nanosize range, which is conducive to long-term circulation and accumulation in tumor sites. In addition, the zeta potentials of the two in ultrapure water are -13.6 mV and -12.8 mV, respectively ( Figure 7B), the negative charge property helps to improve the colloidal stability of nanoparticles and reduce nonspecific protein adsorption, thereby enhancing in vivo stability and biocompatibility. The morphology of the nanoparticles was further characterized using transmission electron microscopy (TEM), and the results showed that ( Figure 8 Both CA-4S2@ES-Cu and CA-4C6@ES-Cu exhibited uniformly distributed spherical nanostructures, demonstrating the structural integrity of the prepared nanoparticles. Furthermore, TEM images revealed clear nanoparticle boundaries with no apparent agglomeration. These results demonstrate that CA-4S2@ES-Cu and CA-4C6@ES-Cu were successfully prepared and exhibited excellent morphological uniformity.

[0079] 4. Elemental analysis of CA-4S2@ES-Cu and CA-4C6@ES-Cu Elemental spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) were performed on CA-4S2@ES-Cu and CA-4C6@ES-Cu.

[0080] The results of elemental spectrum analysis showed that ( Figure 9 ), CA-4S2@ES-Cu and CA-4C6@ES-Cu both contain Cu, O, N, and S elements, and the copper element is evenly distributed in the nanoparticles, which confirms the successful encapsulation of ES-Cu. At the same time, X-ray photoelectron spectroscopy analysis further verified the elemental composition of the two nanoparticles, clearly showing the presence of Cu, O, N, and S elements ( Figure 10 ), thus proving the successful construction of the nanoformulation.

[0081] Test Example 2: Cell Experiment 1. Cell safety evaluation The present invention used the MTT assay to detect the effects of each drug on tumor cell and normal cell activity, and further calculated the selectivity index (SI) of each drug to assess drug safety. The drugs included CA-4, CA-4C6 NPs, CA-4S2 NPs, ES-Cu, CA-4C6@ES-Cu, and CA-4S2@ES-Cu.

[0082] Take the cells of logarithmic growth phase (Huh-7 human liver cancer cells, Hepa1-6 mouse liver cancer cells, THLE-2 human liver immortalized cells and AML-12 mouse normal liver cells) and adjust the cell density to 1*10 5Cells were plated at 100 μL per well of the cell suspension in a 96-well cell culture plate. Three replicates were set up for each group, and the culture plate was placed in a cell culture incubator for 24 hours to allow the cells to adhere to the wall and grow. After the cells adhered to the wall, the culture medium was removed, and drug solutions of different concentration gradients were added to the 96-well plate. After 48 hours of culture, the cell activity was detected using the MTT method. The cell survival rate was calculated, and the relationship between cell survival rate and drug concentration was plotted to obtain the IC 50 Value, according to IC 50 The selectivity index (SI) of each drug was calculated using the IC 50 (Normal cells) / IC 50 (tumor cells).

[0083] The selectivity index (SI) results of each drug are shown in Table 1 and Figure 11 shown.

[0084] Table 1. Selectivity index (SI) results of each drug Table 1 and Figure 11 The results showed that the SI values ​​of CA-4, ES-Cu, CA-4C6 NPs, and CA-4S2 NPs ranged from 0.9 to 1.6, with no significant differences between the drug groups. This suggests that they lack specificity for tumor cells and, while inhibiting tumor cell activity, are likely to also inhibit normal cell activity, resulting in poor safety. In contrast, the SI values ​​of CA-4C6@ES-Cu and CA-4S2@ES-Cu were significantly improved, both exceeding 2.6. This demonstrates that the nanopharmaceuticals CA-4C6@ES-Cu and CA-4S2@ES-Cu prepared by the present invention are specifically selective for tumor cells and have good safety profiles.

[0085] 2. Cell morphology observation Take cells in the logarithmic growth phase (Huh-7 or Hepa 1-6 cells) and adjust the cell density to 2.5*10 5 Cells were seeded into 6-well cell culture plates at a concentration of 2 mL per well. The plates were incubated in a cell culture incubator for 24 hours to allow the cells to adhere and grow. After cell attachment, the medium was aspirated and the drugs were added to the 6-well plates. The cells were cultured for 24 hours and the cell morphology was observed microscopically after 24 hours of culture. Cell morphology at 0 hours served as a control. The drugs were ES-Cu, CA-4C6 NPs, CA-4S2 NPs, CA-4C6@ES-Cu, and CA-4S2@ES-Cu. The drug solutions were prepared using DMSO as the solvent. The ES-Cu concentration was 2 nM, and the other drugs were administered at a concentration of 2 nM based on the CA-4 active ingredient. Cells treated with solvent served as a control.

[0086] Microscopic observation showed that cells in different drug-treated groups showed corresponding morphological changes, including cell deformation, expansion and contraction ( Figure 12 ), indicating that drug treatment has a significant impact on cell structure. This indicates that the nanoparticles CA-4S2@ES-Cu and CA-4C6@ES-Cu of the present invention have a good ability to induce cell death.

[0087] 3. Cell proliferation assay (EdU detection method) Following the method described in step 2 of Experimental Example 2, Huh-7 and Hepa 1-6 cells were treated with ES-Cu, CA-4C6 NPs, CA-4S2 NPs, CA-4C6@ES-Cu, and CA-4S2@ES-Cu for 24 hours. Cell proliferation activity was then assessed using the EdU assay. Cells treated with solvent served as a control.

[0088] After 24 hours of drug treatment of HCC cells (Huh-7 and Hepa 1-6 cells), EdU assay results showed that ( Figure 13 ), CA-4C6@ES-Cu and CA-4S2@ES-Cu significantly reduced the proportion of EdU-positive cells, indicating that the cell DNA synthesis activity was inhibited. At the same time, the cell nuclear volume was significantly increased, indicating that CA-4C6@ES-Cu and CA-4S2@ES-Cu have a good inhibitory ability on tumor cell proliferation, and the effect is better than using ES-Cu, CA-4C6NPs and CA-4S2NPs alone, exerting a synergistic effect in inhibiting tumor proliferation.

[0089] 4. Clone formation experiment Logarithmically growing cells (Huh-7 or Hepa 1-6) were obtained, trypsinized, resuspended in the corresponding culture medium to form a single-cell suspension, and counted. The cell suspension was seeded at a density of 500 cells per well in a 6-well plate. ES-Cu, CA-4C6 NPs, CA-4S2 NPs, CA-4C6@ES-Cu, and CA-4S2@ES-Cu were then added to the cells. Each drug solution was prepared using DMSO as the solvent. The concentration of ES-Cu was 0.5 nM, and the concentration of the other drugs, calculated as the CA-4 active ingredient, was 0.5 nM. After drug addition, the 6-well plate was incubated at 37°C, 5% CO₂ for 12 days. The culture medium was changed every three days, and the cells were observed. After incubation, the cells were washed with PBS, fixed with 1 mL of 4% paraformaldehyde per well for 30 minutes, and then washed with PBS. Then, 1 mL of crystal violet stain was added to each well and stained for 10 minutes. The cells were washed several times with PBS, dried, and photographed. Cells treated with solvent served as controls.

[0090] Through the clone formation experiment, it was found that compared with ES-Cu, CA-4C6NPs and CA-4S2NPs, at the same dose, CA-4C6@ES-Cu and CA-4S2@ES-Cu nanoparticles were more effective in inhibiting cancer cell clones, indicating that CA-4C6@ES-Cu and CA-4S2@ES-Cu nanoparticles played a synergistic and synergistic role in inhibiting cancer cell clones. Among them, CA-4S2@ES-Cu showed the strongest ability to inhibit cell clones among multiple drug groups ( Figure 14 ), indicating that it has significant advantages in inhibiting long-term proliferation capacity.

[0091] 5. Cell Cycle Analysis Following the method described in step 2 of Experimental Example 2, Huh-7 and Hepa 1-6 cells were treated with ES-Cu, CA-4C6 NPs, CA-4S2 NPs, CA-4C6@ES-Cu, and CA-4S2@ES-Cu for 24 hours. After trypsinization, the cells were harvested, washed once with PBS, and slowly added to pre-chilled 70% ethanol. The cells were fixed overnight at 4°C. The next day, the supernatant was discarded by centrifugation and washed once with PBS. The cells were stained according to the instructions of a cell cycle detection kit (Beyotime), and the cell cycle was analyzed by flow cytometry. Cells treated with solvent served as a control. Control analyses were also performed using different concentrations of CA-4.

[0092] The results showed that all the above drugs containing CA-4 components could arrest the HCC cell cycle at the G2 / M phase, which was almost the same as the effect of CA-4 alone (2 nM). Figure 15 This result further confirms that CA-4 can maintain its original anti-tumor mechanism and biological function after structural dimerization and nano-delivery system encapsulation.

[0093] 6. Observation of microtubule morphology Microtubules disassemble and reassemble into the spindle during the prophase of cell division, participating in mitosis. Their unique dynamic instability makes them an important target for anti-tumor therapy. CA-4, a highly effective microtubule polymerization inhibitor, can inhibit mitosis by binding to the colchicine binding site on tubulin, blocking microtubule assembly.

[0094] To explore the effects of their prodrug forms, the present invention investigated the effects of CA-4C6NPs, CA-4S2NPs, and CA-4C6@ES-Cu and CA-4S2@ES-Cu co-assembled with ES-Cu on the microtubule structure of HCC cells (Huh-7 or Hepa 1-6 cells).

[0095] Following the method described in step 2 of Experimental Example 2, Huh-7 and Hepa 1-6 cells were treated with ES-Cu, CA-4C6 NPs, CA-4S2 NPs, CA-4C6@ES-Cu, and CA-4S2@ES-Cu for 24 hours. The supernatant was discarded, and the cells were washed once with PBS. Microtubules were stained according to the instructions of the Microtubule Staining Kit (Beyotime) and observed using a laser confocal microscope. Cells treated with solvent served as a control.

[0096] The results showed that all the above nanomedicines could significantly inhibit microtubule polymerization in HCC cells, showing the same effect trend as free CA-4 ( Figure 16 This result once again confirmed that CA-4 can still maintain its original microtubule polymerization inhibition ability after structural dimerization and nanodelivery system encapsulation.

[0097] 7. Cell apoptosis experiment Following the method described in step 2 of Experimental Example 2, Huh-7 and Hepa 1-6 cells were treated with ES-Cu, CA-4C6 NPs, CA-4S2 NPs, CA-4C6@ES-Cu, and CA-4S2@ES-Cu for 48 hours. The cells were then harvested and the ability of each drug to induce HCC cell apoptosis was assessed using an Annexin V-FITC / PI double staining kit (Beyotime). Cells treated with a solvent served as a control.

[0098] The results show that ( Figure 17 ), both CA-4S2NPs and CA-4C6NPs induced a certain degree of cell apoptosis, but their pro-apoptotic activity was significantly weaker than that of ES-Cu. Notably, when ES-Cu was co-loaded with CA-4S2 or CA-4C6, the resulting CA-4S2@ES-Cu or CA-4S2@ES-Cu induced a more pronounced apoptotic response, demonstrating a synergistic effect. Furthermore, CA-4S2@ES-Cu exhibited superior apoptosis-promoting activity compared to CA-4C6@ES-Cu.

[0099] 8. Intracellular copper ions (Cu 2+ ) content determination Following the method described in step 2 of Experimental Example 2, Huh-7 and Hepa 1-6 cells were treated with ES-Cu, CA-4C6 NPs, CA-4S2 NPs, CA-4C6@ES-Cu, and CA-4S2@ES-Cu for 24 hours. Intracellular copper ion concentrations were then quantified using a copper ion detection kit (Elabscience) to assess the copper ion delivery efficiency of the different treatment groups. Cells treated with solvent served as a control.

[0100] The results show that ( Figure 18 ), CA-4C6NPs and CA-4S2NPs themselves have weak copper ion transport capabilities. However, after treatment with ES-Cu, CA-4C6@ES-Cu, and CA-4S2@ES-Cu, copper ion levels in HCC cells increased significantly, approximately 1.5 to 2 times that of the control group. Since there was no significant difference in copper ion concentration among the three treatment groups, it shows that the encapsulation of CA-4 dimers did not affect the copper ion transport capacity of ES in the nanosystem.

[0101] 9. Intracellular cuprous ions (Cu + ) content detection FDX1 is a mitochondrial reductase that plays a key role in the formation of Fe-S clusters and can bind copper ions and reduce them to more toxic cuprous ions (Cu + ).

[0102] Following the method described in step 2 of Experimental Example 2, Huh-7 and Hepa 1-6 cells were treated with ES-Cu, CA-4C6 NPs, CA-4S2 NPs, CA-4C6@ES-Cu, and CA-4S2@ES-Cu for 24 hours. Intracellular cuprous ion concentrations were then quantified using a cuprous ion detection kit (Elabscience). Cells treated with solvent served as a control.

[0103] The results are as follows Figure 19 As shown, compared with copper ion levels, both CA-4C6@ES-Cu and CA-4S2@ES-Cu exhibited stronger cuprous ion generation capabilities, demonstrating a synergistic effect compared to ES-Cu, CA-4C6 NPs, and CA-4S2 NPs. In particular, the CA-4S2@ES-Cu treatment group further increased cuprous ion levels in HCC cells by approximately 1.5-fold compared to the ES-Cu group. This result suggests that CA-4S2@ES-Cu not only effectively delivers copper ions but also enhances their biotransformation efficiency within cells, potentially exerting a stronger effect in inducing copper-induced cell death.

[0104] 10. Glutathione (GSH) content detection Glutathione (GSH), as an important antioxidant in the body, plays a key role in maintaining cellular redox homeostasis. It can also slow down the occurrence of copper death by binding to cuprous ions and preventing them from interacting with lipoyl-dylated DLAT.

[0105] Following the method described in step 2 of Experimental Example 2, Huh-7 and Hepa 1-6 cells were treated with ES-Cu, CA-4C6 NPs, CA-4S2 NPs, CA-4C6@ES-Cu, and CA-4S2@ES-Cu for 24 hours. Intracellular glutathione levels were then analyzed using a Beyotime glutathione assay kit. Cells treated with a solvent served as a control.

[0106] The results are as follows Figure 20 As shown in the present invention, ES-Cu, CA-4C6 NPs, and CA-4S2 NPs all exhibited a certain degree of GSH depletion in HCC cells. CA-4S2 NPs exhibited a more pronounced GSH depletion effect, with a decrease in GSH levels of approximately 70%. CA-4C6@ES-Cu and CA-4S2@ES-Cu, however, caused even more significant GSH depletion, significantly outperforming ES-Cu, CA-4C6 NPs, and CA-4S2 NPs, demonstrating a synergistic effect.

[0107] 11. Cellular DLAT aggregation During the process of cuproptosis, FDX1 is not only responsible for reducing copper ions to cuprous ions (Cu⁺), but also promotes the lipoylation modification of various proteins, a process that relies on lipoic acid metabolism within mitochondria. Cuprous ions can induce oligomerization of lipoylated proteins of DLAT (dihydrolipoamide S-acetyltransferase), a key component of the pyruvate dehydrogenase complex. This interferes with the tricarboxylic acid (TCA) cycle, induces proteotoxic stress, and ultimately leads to cuproptosis. To verify this process, the degree of DLAT oligomerization was further examined by immunofluorescence imaging.

[0108] Following the method in step 2 of Experiment 2, Huh-7 and Hepa 1-6 cells attached to slides were treated with ES-Cu, CA-4C6 NPs, CA-4S2 NPs, CA-4C6@ES-Cu, and CA-4S2@ES-Cu for 24 hours. The cells were then washed once with PBS and fixed, then incubated with a DLAT primary antibody overnight at 4°C. The next day, the primary antibody was removed and the cells were incubated with a 640 goat anti-rabbit fluorescent secondary antibody. The slides were then mounted and the degree of DLAT oligomerization was examined using laser confocal microscopy. Cells treated with solvent served as a control.

[0109] The results show that ( Figure 21 The trend of DLAT oligomerization was highly consistent with the aforementioned cuprous ion generation results: CA-4C6 NPs and CA-4S2 NPs had essentially no DLAT oligomerization ability. Compared with the ES-Cu-treated group, both CA-4C6@ES-Cu and CA-4S2@ES-Cu exhibited stronger DLAT oligomerization abilities, with CA-4S2@ES-Cu in particular inducing the highest degree of DLAT oligomerization in HCC cells. This result further validates the enhancing effects of CA-4C6@ES-Cu and CA-4S2@ES-Cu in activating the copper death pathway.

[0110] 12. Reactive oxygen species (ROS) level detection Mitochondria are the primary site of copper-induced cell death. Copper death is triggered by the accumulation of mitochondrial fatty acylated proteins, which then leads to oxidative damage to the mitochondrial membrane and mitochondrial dysfunction. To investigate the effects of various drug treatments on cellular oxidative stress, this study used flow cytometry to measure intracellular reactive oxygen species (ROS) levels.

[0111] Following the method described in step 2 of Experimental Example 2, Huh-7 and Hepa 1-6 cells were treated with ES-Cu, CA-4C6 NPs, CA-4S2 NPs, CA-4C6@ES-Cu, and CA-4S2@ES-Cu for 24 hours. The cells were then stained using a Beyotime reactive oxygen species (ROS) detection kit, and intracellular ROS levels were measured by flow cytometry. Cells treated with a solvent served as a control.

[0112] The results show that ( Figure 22 ), all treatment groups induced ROS generation to varying degrees. The CA-4C6@ES-Cu and CA-4S2@ES-Cu groups elicited significantly higher ROS levels than the ES-Cu, CA-4C6NPs, and CA-4S2NPs groups, demonstrating a synergistic effect. The CA-4S2@ES-Cu group elicited the highest ROS levels, demonstrating the strongest oxidative stress activation capacity. Compared to the control group, CA-4S2@ES-Cu treatment increased ROS levels in HCC cells by approximately 3-4.5 times, further confirming the important role of the nanoparticles in activating the copper death mechanism by enhancing oxidative stress.

[0113] 13. Mitochondrial membrane potential (MMP) detection Mitochondria are core organs for cellular energy metabolism and redox regulation. Their dysfunction is often a key step in the cell death process. Especially during copper death, mitochondrial damage is considered a crucial process in triggering downstream death signals. Therefore, mitochondrial membrane potential was measured in HCC cells.

[0114] According to the method of "2" in Experimental Example 2, Huh-7 and Hepa 1-6 cells were treated with ES-Cu, CA-4C6NPs, CA-4S2NPs, CA-4C6@ES-Cu and CA-4S2@ES-Cu for 24 hours, and then the cells were stained with the mitochondrial membrane potential (MMP) detection kit (Beyotime), and the changes in cellular MMP were observed by laser confocal microscopy.

[0115] The results show that ( Figure 23 ): The downward trend of MMP (red fluorescence converted to green fluorescence) was consistent with the results of the effect of reactive oxygen species levels. CA-4S2@ES-Cu treatment significantly induced mitochondrial dysfunction, further confirming the deep stress response it triggered at the cellular level.

[0116] 14. Western blot protein expression detection To further verify the activation of copper-related death signaling pathways, Western blot was used to detect the protein expression levels of FDX1 and soluble DLAT.

[0117] According to the method of "2" in Experimental Example 2, Huh-7 and Hepa 1-6 cells were treated with ES-Cu, CA-4S2 NPs and different concentrations of CA-4S2@ES-Cu for 24 hours, 200 μL of RIPA lysis buffer was added to a 6-well plate, and the cells were completely lysed using a cell scraper, followed by denaturation treatment. The obtained protein samples were placed in a 4°C refrigerator for later use, and then the protein expression levels of FDX1 and soluble DLAT were detected by Western blot.

[0118] The results show that ( Figure 24 ), the expression of the above two key proteins in the CA-4S2@ES-Cu treatment group was significantly downregulated, suggesting that it can enhance intracellular proteotoxic stress by interfering with the mitochondrial reduction process of copper ions and promoting DLAT oligomerization (after oligomerization, the protein is insoluble in water and the soluble DLAT content is reduced), thereby more effectively inducing copper death.

[0119] In summary, the present invention provides a nanoparticle that combines a microtubule-destroying agent dimer with a copper-death drug. The microtubule-destroying agent destroys the cell microtubule structure, induces oxidative stress and blood flow blockage, and enhances the sensitivity of tumor cells to oxidative damage; the copper-death drug promotes the accumulation of copper ions, triggers mitochondrial protein aggregation and dysfunction, and ultimately induces cell death. The combination of the two types of drugs can produce a significant synergistic anti-tumor effect and significantly improve the treatment efficiency; at the same time, after the two types of drugs are prepared into nanoparticles, the selectivity index for anti-tumor use is good and the toxic side effects on cells are small. The present invention has broad prospects for clinical transformation and important social and economic value.

Claims

1. A nanoparticle comprising a tubulin-destroying dimer and a copper-death drug, characterized in that: The nanoparticles are prepared from the following raw materials in parts by weight and volume: 10 parts by weight of tubulin disrupting agent dimer, 4-10 parts by weight of copper-death drug, 5-10 parts by weight of surfactant, 0.5-1 part by weight of copper ion salt, 100-1000 parts by volume of organic solvent, and 2000-20000 parts by volume of water.

2. The nanoparticle according to claim 1, characterized in that: The nanoparticles are prepared from the following raw materials in parts by weight and volume: 10 parts by weight of tubulin disrupting agent dimer, 4.8 parts by weight of copper-deleting drug, 8 parts by weight of surfactant, 0.88 parts by weight of copper ion salt, 400 parts by volume of organic solvent, and 16,000 parts by volume of water.

3. The nanoparticle according to claim 2, characterized in that: The tubulin disrupting agent dimer is selected from one or more of the following: CA-4 dimer, paclitaxel dimer, Vinorel dimer, Ibovan dimer, Eribulin dimer or salts or derivatives of the above dimers; The copper-killing drug is selected from one or more of the following: Elesclomol, Disulfiram, ammonium tetrathiomolybdate, NSC319726, Cu-ATSM, CuET, Tetrathiomolybdate, D-penicillamine or salts or derivatives of the above compounds.

4. The nanoparticles according to claim 3, characterized in that: The tubulin disrupting agent dimer is selected from the CA-4 dimer compound shown in Formula I, its salt or stereoisomer thereof: Formula I Wherein, L is selected from or ; n and m are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; The copper-killing drug is selected from Elesclomol.

5. The nanoparticle according to claim 4, characterized in that: The tubulin disrupting agent dimer is selected from one of the following structural formulas: 。 6. The nanoparticle according to claim 2, characterized in that: The surfactant is selected from distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000; And / or, the copper ion salt is selected from CuCl2; And / or, the organic solvent is selected from tetrahydrofuran.

7. The nanoparticle according to any one of claims 1 to 6, characterized in that: The preparation method of the nanoparticles comprises the following steps: (1) Preparing an organic phase: dissolving a tubulin-destroying agent dimer, a copper-death drug, a surfactant, and a copper ion salt in an organic solvent to obtain an organic phase; (2) Add the organic phase obtained in step (1) into water and stir to obtain the product.

8. The method for preparing nanoparticles according to any one of claims 1 to 7, characterized in that: The preparation method comprises the following steps: (1) Preparing an organic phase: dissolving a tubulin-destroying agent dimer, a copper-death drug, a surfactant, and a copper ion salt in an organic solvent to obtain an organic phase; (2) Add the organic phase obtained in step (1) into water and stir to obtain the product.

9. Use of the nanoparticles according to any one of claims 1 to 7 in the preparation of a medicament for preventing and / or treating cancer.

10. The use according to claim 9, characterized in that: The cancer is selected from lung cancer, liver cancer, pancreatic cancer, breast cancer, melanoma, and glioblastoma.

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