Preparation method of compound for treating malignant tumors

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of a compound for treating malignant tumors. According to the compound (EV-CuET), a copper ion solution and a dimethyl dithiocarbamate solution are sequentially added into an extracellular vesicle solution, and CuET is formed on the modified extracellular vesicle, so that an anti-tumor effective component CuET is wrapped in a lipid bilayer of the extracellular vesicle. Therefore, the compound (EV-CuET) disclosed by the invention can be used for effectively carrying an antitumor drug CuET, endowing the CuET with solubility and high drug loading capacity, and can stably exist in vivo.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a method for preparing a compound for treating malignant tumors. Background Art

[0002] Despite significant advances in cancer diagnosis and treatment, cancer remains a major threat to human health. Chemotherapy is one of the most commonly used clinical cancer treatments, but its application is limited by the emergence of multidrug resistance in tumor cells and the inevitable severe side effects. Repurposing existing drugs for their potential anticancer effects offers a cost-effective, rapid, and feasible alternative.

[0003] Disulfiram (DSF) is a dimer of dimethyldithiocarbamic acid (DCT). It is an alcohol addiction treatment that has been used clinically in many countries for sixty years. At the FDA's recommended dosage, it has clear pharmacokinetics, good safety, and good body tolerance. The anti-cancer effect of disulfiram was first reported in the 1970s. Subsequently, a large number of studies have confirmed that disulfiram has a significant inhibitory effect on tumor growth, invasion, and metastasis. The anti-tumor effect of disulfiram is copper-dependent. Some studies have suggested that the anti-tumor mechanism of disulfiram may lie in the interaction between its metabolic monomer DTC and copper ions (Cu 2+ ) can synthesize a metal chelate CuET, which can interfere with the proteasome system in tumor cells, causing tumor cells to accumulate a large amount of defective proteins and ultimately induce cell apoptosis (Nature, 2017, 552(7684):194-199). Experiments have also shown that the chelate of disulfiram and copper ions can inhibit tumor cell proliferation by increasing the level of intracellular reactive oxygen species and inhibiting the NF-κB signaling pathway (Mol. Oncol. 2015, 9, 1155–1168), inhibiting tumor angiogenesis (Cancer Lett. 2015, 369, 86–96), and antagonizing ALDH (British Journal of Cancer, 2011, 104(10):1564-1574). Although disulfiram combined with copper ions has shown good anti-tumor effects in experiments, due to some characteristics of disulfiram and copper ions, its application still faces great challenges.

[0004] Disulfiram is a hydrophobic molecule currently used clinically primarily through oral administration. However, oral disulfiram is rapidly reduced to its monomer, DCT, by glutathione in red blood cells. This monomer is then rapidly metabolized in the liver, making it unstable in the body. Studies have shown that even at oral doses of 500 mg / kg, blood concentrations remain below detection, making it difficult to accumulate sufficient drug concentrations at the tumor site. Copper ions are primarily administered orally in the form of copper gluconate in combination with disulfiram for cancer treatment. Copper gluconate has a short serum half-life, and while tumors accumulate more copper ions than other tissues, the concentration is still too low for the copper-disulfiram chelate (CuET) to achieve an effective inhibitory concentration at the tumor site. Furthermore, this systemic administration of copper ions can lead to excessive copper accumulation in patients without copper deficiency. Consequently, this oral route of administration performed poorly in early clinical trials.

[0005] To address these issues, patents have attempted to address disulfiram's water solubility and stability issues by using liposomes and other methods to create disulfiram nanoformulations (CN201680077511, CN201910170699, and CN201510422492). However, these disulfiram formulations fail to incorporate the crucial copper ion, and thus remain unable to achieve effective concentrations of both disulfiram and copper ions at the tumor site. Researchers have encapsulated disulfiram and copper ions in a silica nanoparticle-PEG complex, but this approach has a short serum half-life (only 1.8 hours), resulting in low drug accumulation at the tumor site (DOI: 10.1021 / jacs.9b03503). Some researchers (ACS Appl. Mater. Interfaces, 2018, 10, 48, 41118–41128, DOI: 10.1021 / acs.biomac.9b00367) have attempted to encapsulate disulfiram and copper ion chelate (CuET) in nanomicelles, demonstrating promising anti-tumor efficacy in vitro. However, no reports of in vivo application of CuET have been reported. Currently, there are few reports of successful in vivo anti-tumor efficacy using disulfiram and copper ion chelate in the literature. Therefore, the efficient co-delivery of disulfiram and copper ions to tumor sites remains a significant challenge. Summary of the Invention

[0006] The first aspect of the present invention aims to provide a method for preparing a composite for treating tumors.

[0007] The second aspect of the present invention aims to provide a method for preparing a complex for targeted tumor treatment.

[0008] The third aspect of the present invention aims to provide use of the complexes described in the first and second aspects of the present invention in the preparation of drugs for treating tumors.

[0009] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a method for preparing a tumor treating composite, comprising the following steps:

[0011] 1) Incubating carboxylate-modified liposomes with copper ions;

[0012] 2) Add disulfiram monomer and mix.

[0013] In some embodiments of the invention, the liposomes comprise extracellular vesicles (EVs).

[0014] In some embodiments of the present invention, the extracellular vesicles include animal-derived extracellular vesicles and / or plant-derived extracellular vesicles.

[0015] In some embodiments of the present invention, the animal-derived extracellular vesicles include but are not limited to humans, cows, pigs, horses, donkeys, mice, rabbits, sheep, etc.

[0016] In some embodiments of the present invention, the plant-derived extracellular vesicles include but are not limited to lemon extracellular vesicles, grapefruit extracellular vesicles, ginger extracellular vesicles, grape extracellular vesicles, kudzu root extracellular vesicles, tomato extracellular vesicles, ginseng extracellular vesicles, carrot extracellular vesicles, watermelon extracellular vesicles, olive extracellular vesicles, pineapple extracellular vesicles, corn extracellular vesicles, etc.

[0017] In some embodiments of the present invention, in addition to extracellular vesicles, the liposomes also include carboxylate-modified liposomes, including but not limited to DSPE-PEG-COOH.

[0018] In some embodiments of the present invention, the disulfiram monomer (DCT) is dimethyldithiocarbamate.

[0019] In some embodiments of the present invention, the disulfiram monomer (DCT) is specifically sodium dimethyldithiocarbamate trihydrate (C3H6NNaS2·3H2O).

[0020] In some embodiments of the present invention, the ratio of the copper ion to the carrier is 1 μg: (1-20) nM; preferably, the ratio of the copper ion to the liposome is 1 μg: (1-10) nM.

[0021] In some embodiments of the present invention, the molar ratio of the copper ion to the disulfiram monomer is 1:(1-5); preferably, the molar ratio of the copper ion to the disulfiram monomer is 1:(1-3).

[0022] The first aspect of the present invention is to solve the problem of the reaction between disulfiram monomer DTC and copper ion (Cu 2+ The technical problem of the insolubility of the metal chelate CuET synthesized by ) in water is solved by designing and applying the extracellular vesicles with carboxyl groups on their surface to adsorb CuET by taking advantage of the fact that metal ions can form ionic bonds with carboxylate ions and that the lipid bilayer on the surface of extracellular vesicles can be used to encapsulate hydrophobic drugs. 2+ EV-Cu is formed, and then water-soluble disulfiram monomer DCT is added to the Cu 2+ While CuET chelates with DCT to form hydrophobic water, the hydrophobic CuET is encapsulated in the lipid bilayer of the extracellular vesicle, thereby preparing a drug delivery system EV-CuEt that can carry both disulfiram monomer DCT and copper ion chelate CuET. This strategy can effectively solve the hydrophobicity and stability problems of disulfiram / copper ion chelate. The principle diagram is shown in the figure below. Figure 1 shown.

[0023] Those skilled in the art will understand that the specific plant species does not affect the realization of the technical effects of the present invention.

[0024] A second aspect of the present invention provides a method for preparing a complex for targeted tumor therapy. This method, based on the method of the first aspect of the present invention, further incorporates modifications to the tumor-targeting peptide and introduces more carboxyl groups onto the surface of the extracellular vesicles, thereby increasing the CuET loading capacity. The method comprises the following steps:

[0025] 1) mixing a carboxylate-modified tumor targeting peptide with liposomes, adding a catalyst for reaction, and obtaining liposomes modified with the tumor targeting peptide;

[0026] 2) incubating the tumor-targeting peptide-modified liposomes with copper ions;

[0027] 3) Add disulfiram monomer and mix.

[0028] In some embodiments of the invention, the liposomes comprise extracellular vesicles (EVs).

[0029] In some embodiments of the present invention, the extracellular vesicles include animal-derived extracellular vesicles and / or plant-derived extracellular vesicles.

[0030] In some embodiments of the present invention, the animal-derived extracellular vesicles include but are not limited to humans, cows, pigs, horses, donkeys, mice, rabbits, sheep, etc.

[0031] In some embodiments of the present invention, the plant-derived extracellular vesicles include but are not limited to lemon extracellular vesicles, grapefruit extracellular vesicles, ginger extracellular vesicles, grape extracellular vesicles, kudzu root extracellular vesicles, tomato extracellular vesicles, ginseng extracellular vesicles, carrot extracellular vesicles, watermelon extracellular vesicles, olive extracellular vesicles, pineapple extracellular vesicles, corn extracellular vesicles, etc.

[0032] In some embodiments of the present invention, in addition to extracellular vesicles, the liposomes also include carboxylate-modified liposomes, including but not limited to DSPE-PEG-COOH.

[0033] In some embodiments of the present invention, the disulfiram monomer (DCT) is dimethyldithiocarbamate.

[0034] In some embodiments of the present invention, the disulfiram monomer (DCT) is specifically sodium dimethyldithiocarbamate trihydrate (C3H6NNaS2·3H2O).

[0035] In some embodiments of the present invention, the ratio of copper ions to liposomes is 1 μg: (1-20) nM; preferably, the ratio of copper ions to liposomes is 1 μg: (1-10) nM.

[0036] In some embodiments of the present invention, the molar ratio of the copper ion to the disulfiram monomer is 1:(1-5); preferably, the molar ratio of the copper ion to the disulfiram monomer is 1:(1-3).

[0037] In some embodiments of the present invention, the tumor targeting peptide includes at least one of RGD peptide, NGR peptide, Angiopep-2, HK peptide, LyP-1 peptide, and YIGSR peptide.

[0038] In some embodiments of the present invention, the tumor targeting peptide comprises cRGD, CAS No.: 161552-03-0.

[0039] In some embodiments of the present invention, the cross-linking agent includes at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide.

[0040] In some embodiments of the present invention, the method for preparing the tumor targeting peptide modified with a carboxylate group comprises the following steps:

[0041] The carboxylated heparin, the tumor targeting peptide and the cross-linking agent are mixed and reacted.

[0042] In some embodiments of the present invention, the mass ratio of the carboxylated heparin to the tumor targeting peptide is (5-20):1.

[0043] In some embodiments of the present invention, the catalyst includes at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide.

[0044] In some embodiments of the invention, the complex is detectably labeled.

[0045] In some embodiments of the present invention, the detectable label comprises a radionuclide, a fluorescent protein, or a fluorescent group.

[0046] In some embodiments of the present invention, the fluorescent group includes Cy7.

[0047] The third aspect of the present invention provides use of the complexes described in the first and second aspects of the present invention in the preparation of drugs for treating tumors.

[0048] In some embodiments of the present invention, the medicament includes a pharmaceutically acceptable carrier.

[0049] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0050] The above-mentioned pharmaceutically acceptable excipients are generally recognized for this purpose and as inactive ingredients of pharmaceutical preparations. A compilation of pharmaceutically acceptable excipients can be found in reference books such as the Handbook of Pharmaceutical Excipients (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Gess, London, 1994); and the Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients.

[0051] The beneficial effects of the present invention are:

[0052] The EV-CuET complex described in this invention is formed by sequentially adding a copper ion solution and a dimethyldithiocarbamate solution to an extracellular vesicle solution. This forms CuET on the modified extracellular vesicles, encapsulating the anti-tumor active ingredient, CuET, within the lipid bilayer of the extracellular vesicles. Consequently, the EV-CuET complex effectively carries the anti-tumor drug CuET, making it soluble, highly loaded, and stable in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0054] Figure 1 It is a schematic diagram of the principle of the present invention.

[0055] Figure 2 The dissolution of CuET obtained by different preparation methods.

[0056] Figure 3 These are the characterization and identification results of the complex (EV-CuET) prepared in Example 1.

[0057] Figure 4 This is the protein distribution expression result of the complex (EV-CuET) prepared in Example 1.

[0058] Figure 5 These are the stability test results of the complex (EV-CuET) prepared in Example 1.

[0059] Figure 6 The figures show the survival rate of different compositions on ovarian cancer cell lines SKOV-3 and A2780.

[0060] Figure 7 Figure 3 shows the drug distribution of the EHR-CuET complex in a nude mouse orthotopic ovarian cancer model.

[0061] Figure 8 These are the experimental results of the tumor inhibition effect of the complex EHR-CuET in the nude mouse orthotopic ovarian cancer model. DETAILED DESCRIPTION

[0062] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0063] Example 1 Preparation of the complex (EV-CuET)

[0064] (1) Weigh 2.5 mg of copper sulfate pentahydrate (CuSO4·5H2O) powder and 1.97 mg of sodium dimethyldithiocarbamate trihydrate (C3H6NNaS2·3H2O) powder and dissolve them in 1 mL of deionized water to obtain 10 μM / mL copper ion solution and 10 μM / mL dimethyldithiocarbamate solution (DCT), respectively.

[0065] (2) Take 100 μg of extracellular vesicles (EV), add 55.5 μL of the copper ion solution obtained in (1), mix thoroughly, and then add 111 μL of the dimethyldithiocarbamate solution obtained in (1) at a molar ratio of copper ion: dimethyldithiocarbamate ion (DTC) = 1:2 based on the molar amount of the added copper ion, and mix thoroughly to obtain the complex (EV-CuET).

[0066] The extracellular vesicles in this example are lemon extracellular vesicles. Fresh lemons are peeled and juiced, and gradient centrifugation is performed. The precipitate is collected and resuspended in PBS buffer. The precipitate is then extracted using sucrose as an extractant by density gradient centrifugation. The extracellular vesicles are then frozen at -80°C for later use (the method for extracting lemon exosomes has been published in patent CN108186772A).

[0067] See also Figure 2 In the presence of extracellular vesicles (EVs) as drug carriers, Cu 2+ and DCT solution, the CuET produced by the chelation of the two obtained water solubility and presented a clear and transparent light brown solution.

[0068] Example 2 Preparation of the complex (EV-CuET)

[0069] (1) Weigh 2.5 mg of copper sulfate pentahydrate (CuSO4·5H2O) powder and 1.97 mg of sodium dimethyldithiocarbamate trihydrate (C3H6NNaS2·3H2O) powder and dissolve them in 1 mL of deionized water to obtain 10 μM / mL copper ion solution and 10 μM / mL dimethyldithiocarbamate solution (DCT), respectively.

[0070] (2) Take 100 μg of extracellular vesicles (EV), add 55.5 μL of the copper ion solution obtained in (1), mix thoroughly, and then add 111 μL of the dimethyldithiocarbamate solution obtained in (1) at a molar ratio of copper ion: dimethyldithiocarbamate ion (DTC) = 1:2 based on the molar amount of the added copper ion, and mix thoroughly to obtain the complex (EV-CuET).

[0071] The extracellular vesicles in this example are grapefruit extracellular vesicles. Fresh grapefruits are peeled and juiced, and gradient centrifugation is performed. The precipitate is collected and resuspended in PBS buffer. The precipitate is then extracted using sucrose as an extractant by density gradient centrifugation. The extracellular vesicles are then frozen at -80°C for later use (the method for extracting grapefruit exosomes has been published in patent CN 109731106 A).

[0072] See also Figure 2 In the presence of extracellular vesicles (EVs) as drug carriers, Cu 2+ and DCT solution, the CuET produced by the chelation of the two obtained water solubility and presented a clear and transparent light brown solution.

[0073] Exosomes from different plant sources can all be obtained as clear, transparent, light brown solutions (EV-CuET).

[0074] Example 3 Preparation of tumor-targeting complex (EHR-CuET)

[0075] 1. Preparation of a tumor tissue-targeted complex (cRGD-EV-CuET)

[0076] (1) Preparation of cRGD peptide-targeted extracellular vesicle complex EHR

[0077] Weigh 50 mg of carboxylated heparin (Heparin) and 4 mg of tumor-targeting cyclic peptide cRGD, dissolve them in 2 mL of dimethyl sulfoxide, add 7.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC·HCL) and 7.5 mg of N-hydroxysuccinimide (NHS), mix thoroughly at room temperature, and after reacting for at least 12 h, dialyze in deionized water for 48 h using a dialysis bag (MW=3500) to obtain Heparin-cRGD, seal it and set aside.

[0078] The extracellular vesicles (EV) obtained in Example 1 and Heparin-cRGD were synthesized at a mass ratio of 100 μg:1 mg. 50 μg of extracellular vesicles (EV) and a solution containing 0.5 mg of HR were taken, and the catalysts 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC·HCl) and N-hydroxysuccinimide (NHS) were added, respectively. The mixture was reacted overnight at room temperature in the dark. Ultracentrifuge at 100,000 g for 1 hour twice, the supernatant was discarded, and the mixture was thoroughly resuspended in PBS buffer to obtain the lemon extracellular vesicle complex EHR modified with Heparin-cRGD.

[0079] 2) Preparation of drug-loaded extracellular vesicle complex EHR-CuET

[0080] Weigh 2.5 mg of copper sulfate (CuSO4·5H2O) and 1.97 mg of sodium dimethyldithiocarbamate trihydrate (DCT·3H2O) and dissolve them in 1 mL of deionized water (10 μM / mL). Add 50 μL of CuSO4 solution and 100 μL of DCT to 100 μg of extracellular vesicles (EHRs) and mix thoroughly to obtain the EHR-CuET complex.

[0081] EHR was prepared by introducing Heparin and tumor targeting peptide cRGD containing a large number of carboxyl groups on the surface of EVs. Cu 2+ and DCT solution, and the two are chelated to produce EHR-CuET. This method can significantly increase the loading capacity of EV carrying CuET and has tumor targeting characteristics.

[0082] Example 4 Preparation of Fluorescently Labeled Cy7-EHR-CuET

[0083] (1) 250 μg / mL EHR (prepared in Example 3) and 100 μg of Cy7 were mixed in aqueous solution, and 10 μg each of EDC and NHS were added to react overnight. The mixture was centrifuged at 100,000 g, washed with PBS, and resuspended.

[0084] (2) The same as step (2) of Example 3, the extracellular vesicle HER was replaced with Cy7-HER to prepare Cy7-EHR-CuET.

[0085] Comparative Example 1 Preparation of DTC-Cu

[0086] (1) Weigh 2.5 mg of copper sulfate pentahydrate (CuSO4·5H2O) powder and 1.97 mg of sodium dimethyldithiocarbamate trihydrate (C3H6NNaS2·3H2O) powder and dissolve them in 1 mL of deionized water to obtain 10 μM / mL copper ion solution and 10 μM / mL dimethyldithiocarbamate solution (DCT), respectively.

[0087] (2) 55.5 μL of copper ion solution was mixed with 111 μL of DTC solution at a molar ratio of copper ion to dimethyldithiocarbamate ion (DTC) = 1:2.

[0088] See also Figure 2 When the monomer dimethyldithiocarbamate (DCT) solution of disulfiram is mixed with copper sulfate solution, a brown insoluble flocculent is immediately produced, which is the chelate of disulfiram and copper ions (CuET).

[0089] Experimental Example 1 Characterization and Identification of the Complex (EV-CuET)

[0090] Taking the complex (EV-CuET) prepared in Example 1 as an example, characterization and identification were performed.

[0091] like Figure 3 As shown in Figure 2, under transmission electron microscopy, the complex (EV-CuET) has a lipid bilayer structure, with different small ring-shaped protrusions on the surface of the lipid layer, slightly less smooth edges, and a slightly irregular spherical shape as a whole. Dynamic light scattering (DLS) detection showed that the average particle size of the complex (EV-CuET) was 226.3±1.64nm. Western blotting experiments were used to detect extracellular vesicles and complexes, and the results were as follows: Figure 4 As shown, Alix, CD9, and TSG101, three extracellular vesicle marker proteins, were expressed in both extracellular vesicles (EVs) and EV-CuETs, or in the extracellular vesicle complex. This indicates that the complex (EV-CuET) has the same protein expression as pure extracellular vesicles and that the complex synthesis process did not change the structural properties of the extracellular vesicles. Scanning spectra of EV-CuET and its components (DCT, CuET, EV) were measured using an ultraviolet spectrophotometer. The results showed that CuET has a characteristic absorption peak at a wavelength of 435 nm. The spectral curve of the complex (EV-CuET) also exhibited a similar absorption peak at 435 nm, indicating that CuET was successfully loaded onto the modified extracellular vesicle (EV) carrier.

[0092] Experimental Example 2 Stability test of the complex (EV-CuET)

[0093] Taking the complex (EV-CuET) prepared in Example 1 as an example, stability testing was performed.

[0094] In order to detect the stability of the extracellular vesicle complex, the polydispersity coefficient and particle size (such as 0h, 24h, 48h, 72h, and 96h) of the complex EV-CuET prepared in Example 1 were detected by dynamic light scattering (DLS). Figure 5 The results showed that the polydispersity coefficient of EV-CuET during synthesis was 0.198±0.0187, and it was still maintained near 0.2 at various time points within the following 96 hours; its particle size remained between 210-230 nm at various time points until 96 hours after synthesis, and remained basically unchanged, indicating that the extracellular vesicle complex EV-CuET constructed above maintains a stable structure in terms of water solubility, and the extracellular vesicles as drug carriers can effectively protect the in vivo delivery of the anti-tumor component CuET.

[0095] Experimental Example 3 Cytotoxicity of the EHR-CuET complex in ovarian cancer cell lines SKOV3 and A2780

[0096] Ovarian cancer cell lines SKOV-3 and A2780 in logarithmic growth phase were digested into cell suspension and counted. 3×10 3 The cells were seeded into 96-well plates and cultured overnight in a constant temperature incubator. After the cells adhered to the wall, 5 concentration gradients were set in advance based on the disulfiram concentration, and each group of drugs (EHR-CuET, DSF / Cu 2+ and EHR, using CuET concentrations of 0.02, 0.04, 0.06, 0.08, and 0.12 μg / mL as standards, and the same amount of HER for comparison). A blank control group was also set up, and each group was set up with 5 replicates, and incubated in a constant temperature incubator for another 24 hours. MTT was added and incubated in the dark for another 4 hours, after which the cell culture medium was aspirated, dimethyl sulfoxide was added, and the cells were incubated in the dark on a shaker for 10-15 minutes. The absorbance (OD) of each well at a wavelength of 490 nm was measured on a microplate reader, and the average OD value of each replicate was calculated. The relative survival rate of each group of cells at each drug concentration gradient was calculated according to the formula: relative cell survival rate (%) = (mean OD value of the drug group / mean OD value of the control group) × 100%.

[0097] The results are as follows Figure 6 As shown in Figure 2, the cytotoxicity of the EHR-CuET complex to ovarian cancer cell lines SKOV3 and A2780 was stronger than that of DSF / CuET. 2+ group, while the modified extracellular vesicles EHR alone had no significant inhibitory effect on the two ovarian cancer cell lines.

[0098] Experimental Example 4 In vivo biological function experiment of the EHR-CuET complex

[0099] 1. Drug distribution of the EHR-CuET complex in a nude mouse orthotopic ovarian cancer model

[0100] The firefly luciferase-labeled SKOV3-Luc cell line was selected and injected into the ovaries of nude mice to construct a nude mouse ovarian carcinoma in situ model, and tumor fluorescence was detected using a live imaging device. The ovarian cancer nude mouse model was intraperitoneally injected with the near-infrared fluorescent dye Cy7-labeled complex Cy7-EHR-CuET (quantified by Cy7, 0.1μg of Cy7 per mouse), and small animal live imaging was performed on the tumor-bearing mice at time points of 0.5h, 3h, 6h, 12h, 24h, 48h, and 72h after administration. The mice were killed under anesthesia 72h later, and the tumors and other major organs (heart, liver, spleen, lungs, and kidneys) of the tumor-bearing mice were removed. The fluorescence intensity of the Cy7-labeled complex and the tumor fluorescence were detected. The results are as follows. Figure 7As shown in the figure, the fluorescence value of the complex (EHR-CuET) specifically accumulated at the tumor site 0.5 hours after drug injection, and drug fluorescence could still be observed at the tumor site 72 hours later, indicating that the drug can accumulate at the tumor site after entering the body and stay there for a long time; fluorescence detection in isolated organs also confirmed that the complex (EHR-CuET) can accumulate well at the tumor site, indicating that the complex (EHR-CuET) can be specifically taken up by tumor cells. (The difference test level is set to * P < 0.05 (two-sided), ** P<0.01, *** P<0.001, P<0.05 were considered statistically significant.)

[0101] 2. Experimental study on the tumor inhibition effect of the EHR-CuET complex in a nude mouse orthotopic ovarian cancer model

[0102] The in vivo tumor inhibition effect of the complex was tested using a nude mouse orthotopic ovarian cancer metastasis model. About 72 hours after SKOV3 cell lines were implanted in the nude mouse ovaries, the cells were randomly divided into a control group (PBS group), a DSF / Cu group, and a control group (PBS group). 2+ There were 5 tumor-bearing mice in each group. The experimental group received disulfiram or disulfiram equivalent at a concentration of 5 μg / g and the control group received the same volume of PBS buffer via tail vein injection. The administration frequency was once every 3 days. The tumor proliferation in the tumor-bearing mice was dynamically monitored every 4 days using a live imaging device. The tumor-bearing mice were sacrificed under anesthesia on the 16th day after treatment, and the tumor tissues were taken to measure the proliferation. Figure 8 As shown in the figure, the fluorescence signal of the tumor in the control group began to increase exponentially on the 4th day of treatment. 2+ It can reduce the tumor growth rate, and the composite (EHR-CuET) is better than DSF / Cu 2+ It has a significantly better inhibitory effect on tumors. In summary, the composite EHR-CuET prepared by the present invention can effectively inhibit the proliferation of ovarian cancer. (The difference test level is set to * P < 0.05 (two-sided), ** P<0.01, *** P<0.001, P<0.05 were considered statistically significant.)

[0103] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A method for preparing a tumor treating compound, comprising the following steps: 1) Incubating carboxylate-modified liposomes with copper ions; 2) Add disulfiram monomer and mix.

2. A method for preparing a complex for targeted tumor therapy, comprising the following steps: 1) mixing a carboxylate-modified tumor targeting peptide with liposomes, adding a catalyst for reaction, and obtaining liposomes modified with the tumor targeting peptide; 2) incubating the tumor-targeting peptide-modified liposomes with copper ions; 3) Add disulfiram monomer and mix.

3. The preparation method according to claim 1 or 2, characterized in that: The liposomes include extracellular vesicles.

4. The preparation method according to claim 3, wherein: The extracellular vesicles include animal-derived extracellular vesicles and / or plant-derived extracellular vesicles.

5. The preparation method according to claim 1 or 2, characterized in that: The ratio of the copper ion to the liposome is 1 μg: (1-20) nM.

6. The preparation method according to claim 1 or 2, characterized in that: The molar ratio of the copper ion to the disulfiram monomer is 1:(1-5).

7. The preparation method according to claim 2, characterized in that: The tumor targeting peptide includes at least one of RGD peptide, NGR peptide, Angiopep-2, HK peptide, LyP-1 peptide, and YIGSR peptide.

8. The preparation method according to claim 2, wherein: The catalyst includes at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide.

9. Use of the complex obtained by the preparation method according to any one of claims 1 to 8 in the preparation of drugs for treating tumors.

10. The use according to claim 9, characterized in that: The drug includes a pharmaceutically acceptable carrier.

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