A nano-drug of grapefruit extracellular vesicle loaded with trifunctional tetravalent platinum compound and preparation method and application thereof

Abstract

The application provides a trifunctional tetravalent platinum compound of captopril-aspirin ligand and a reference drug of a bifunctional tetravalent platinum compound of captopril; and develops a nanomedicine Tf-GEVs@Pt(IV) of the aspirin-captopril trifunctional tetravalent platinum compound loaded in grapefruit extracellular vesicles (GEVs). The Tf-GEVs@Pt(IV) is prepared from the trifunctional tetravalent platinum compound as an active ingredient, the carrier grapefruit extracellular vesicles (GEVs), the carrier DSPE-PEG and the transferrin modified targeting carrier DSPE-PEG-Tf. The Tf-GEVs@Pt(IV) can release the active component trifunctional tetravalent platinum compound in vivo. In addition to causing DNA damage, the trifunctional tetravalent platinum compound as the active component of the nanomedicine can effectively regulate inflammatory, fibrotic and immunosuppressive TME. The nanomedicine has a significant anti-tumor ability and has excellent therapeutic effect on metastatic malignant tumors, and is expected to provide a new candidate drug for clinical treatment of tumors and provide a new direction for research and development of new platinum drugs and anti-metastatic malignant tumor drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a nanomedicine based on grapefruit extracellular vesicles (GEVs) loaded with trifunctional tetravalent platinum conjugates, a preparation method thereof and application thereof in tumor treatment drugs. BACKGROUND

[0002] Cancer is a serious threat to human health. The complex tumor microenvironment (TME) is not only an important barrier to tumor, but also an important reason for the failure of chemotherapy. Remodeling TME is expected to improve the efficacy of anticancer drugs. Tumor fibrosis and tumor-associated inflammation (TAI) are important features of TME, which synergistically inhibit tumor immunity. Reversing fibrotic and inflammatory TME, and then changing the immune environment from "cold" to "hot", is expected to inhibit tumor growth and metastasis.

[0003] Platinum drugs are the most representative metal-based chemotherapy drugs. More and more studies have confirmed that inflammation, fibrosis and immunosuppressive TME are important reasons for the failure of platinum chemotherapy. As a prodrug of divalent platinum drugs, tetravalent platinum provides an important method for overcoming the above shortcomings by introducing various functional ligands into the axial position. Aspirin (ASP) is a non-steroidal anti-inflammatory drug, which has great potential in inhibiting TAI. Captopril (CTP) as an effective angiotensin-converting enzyme (ACE) inhibitor has been shown to effectively inhibit fibrosis by down-regulating MMPs and TGF-β1. In view of this, the present application uses CTP-ASP as a functional ligand to introduce into the tetravalent platinum system, and develops a series of new trifunctional tetravalent platinum conjugates. These conjugates can inhibit fibrotic and inflammatory TME, damage DNA and further reverse immunosuppressive TME, thereby showing strong anti-tumor activity.

[0004] Tumor-targeted drugs are a hot spot in new drug development. The use of nanomedicine delivery systems (NDDS) to deliver platinum compounds has important development prospects. Extracellular vesicles (EVs), especially plant-derived EVs (PDVs), have great development potential as NDDS. PDVs have low immunogenicity and toxicity risk, wide sources, low cost, and more importantly, they can easily cross the fibrotic barrier of tumors. Recently, some PDV-based drugs have been developed and are in the clinical trial stage. However, the application of PDVs loaded with tetravalent platinum drugs has not been studied and is worth further exploring.

[0005] Grapefruit-derived exosomes (GEVs) as a typical PDVs, show superiority in anti-tumor cells, and lower toxicity. GEVs can also effectively inhibit inflammation by reducing the expression of pro-inflammatory factors. Therefore, the research of GEVs as drug carriers has attracted more and more attention, and it shows great potential in reducing the side effects of encapsulated drugs and improving the therapeutic effect. However, the poor targeting of GEVs nanomedicine is still a constraint factor for its development. Modification of GEVs is an effective strategy to enhance the tumor targeting property. Tumor cells have high demand for iron due to their unlimited proliferation, and overexpress transferrin receptor (TfR) on the surface. Transferrin (Tf) has good tumor targeting ability and is often used for nanomedicine modification. So far, the modification of Tf on PDVs has not been studied. Therefore, the present application first designs and develops a Tf-modified GEVs system, which is expected to improve the tumor targeting property of anti-tumor drugs. SUMMARY

[0006] In view of the problems existing in the prior art, the present application designs and develops a trifunctional tetravalent platinum compound with captopril-aspirin ligand and a captopril bifunctional tetravalent platinum compound; develops a nanomedicine GEVs@Pt(IV) of grapefruit extracellular vesicles (GEVs) loaded with aspirin-captopril trifunctional tetravalent platinum compound and a nanomedicine Tf-GEVs@Pt(IV) of tumor targeting group transferrin modification. The trifunctional tetravalent platinum compound as the active component of the nanomedicine can not only cause DNA damage, but also effectively regulate inflammation, fibrosis and immunosuppressive TME; GEVs in the nanomedicine promote the tumor tissue permeability of the drug; transferrin (Tf) enhances the tumor targeting property and improves the anti-tumor activity of the active component. Through in vivo and in vitro experimental methods, the anti-tumor proliferation and anti-metastasis effects of the target compound are verified, and the anti-tumor mechanism of the drug is studied. The results confirm that the drug has a significant anti-tumor ability and excellent therapeutic effect on metastatic malignant tumors, which is expected to provide a new candidate drug for clinical treatment of tumors and a new direction for research and development of new platinum drugs and anti-metastatic malignant tumor drugs.

[0007] In order to achieve the purpose of the present application, the present application provides a trifunctional tetravalent platinum derivative, the structure general formula of which is shown as (I):

[0008]

[0009] Among them, selected from cisplatin or oxaliplatin; L is hydroxyl or

[0010] The trifunctional tetravalent platinum compound of the captopril-aspirin ligand is selected from: monosubstituted trifunctional tetravalent platinum derivative Ia, disubstituted trifunctional tetravalent platinum derivative Ib.

[0011]

[0012] Further, the tri-functional tetravalent platinum compound of captopril-aspirin ligand according to the present application is selected from the group consisting of compound 1, 2:

[0013]

[0014] Another object of the present application provides a preparation method of the tri-functional tetravalent platinum compound of captopril-aspirin ligand as shown in general formula (I), and the synthesis route of the compound is shown as follows.

[0015] The route one for synthesizing Ia and Ib is as follows:

[0016]

[0017] The coupling reaction of compound II and compound 6 produces the tri-functional tetravalent platinum derivative Ia or Ib. When the molar ratio of compound II and compound 6 is 1:1.0-1.5, the asymmetric monosubstituted tri-functional modified tetravalent platinum compound Ia is obtained; when the molar ratio of compound II and compound 6 is 1:2.0-5.0, the symmetric disubstituted tri-functional modified tetravalent platinum compound Ib is obtained.

[0018] Further, in the synthesis route one, the preparation steps of the tri-functional tetravalent platinum compound of captopril-aspirin ligand are as follows:

[0019] Compound 6, condensing agent, organic base are dissolved in anhydrous organic solvent under inert gas atmosphere, compound II is added, and the reaction is carried out in the dark, followed by post-treatment to obtain the tri-functional tetravalent platinum derivative I;

[0020] The molar ratio of compound II, compound 6, condensing agent, and organic base is 1:1.0-1.5:1.0-1.5:1.0-1.5; the feeding relationship of compound II and organic solvent is that 10-100 ml of organic solvent is added for every 1 g of compound II, and the obtained product is Ia. The molar ratio of compound II, compound 6, condensing agent, and organic base is 1:2.0-5.0:2.0-5.0:2.0-5.0; the feeding relationship of compound II and organic solvent is that 10-100 ml of organic solvent is added for every 1 g of compound II, and the obtained product is Ib.

[0021] Further, the inert gas is nitrogen, helium or argon; the condensing agent is TBTU, HATU or EDCI; the organic base is triethylamine, N,N-diisopropyl ethylamine or 4-dimethylamino pyridine; and the organic solvent is DMF or DMSO.

[0022] The preparation process can be specifically as follows: adding a condensing agent and compound 6 into a reaction container, replacing the air in the system with inert gas, adding anhydrous organic solvent, adding anhydrous organic base into the reaction system, stirring the reaction at room temperature for 5-60 min, adding tetravalent platinum compound II into the reaction system, replacing the air in the system with inert gas again, and placing the reaction system in the dark at 25-120°C for 24-72 h; after the reaction is completed, removing the solvent under reduced pressure, and column chromatography to obtain asymmetrically single-substituted trifunctionally modified tetravalent platinum compound Ia or symmetrically double-substituted trifunctionally modified tetravalent platinum compound Ib.

[0023] Further, in the synthesis route, the preparation step of compound 6 is as follows:

[0024]

[0025] The preparation method of compound 6 is as follows: dissolving 1H-benzotriazole in dichloromethane, adding sulfurous chloride under ice bath, and continuing to stir at room temperature; adding aspirin, and continuing to stir the mixture at room temperature; then filtering the suspension, washing the organic solution with saturated Na2CO3 solution; then adding hexane, and precipitating white solid compound 5; dissolving compound 5 in acetonitrile, adding an aqueous solution of CTP and triethylamine, and stirring the mixture; removing acetonitrile under reduced pressure; extracting the residue with ethyl acetate, and evaporating to obtain white solid compound 6.

[0026] The specific preparation method of compound 6 is as follows: dissolving 1H-benzotriazole (H-Bt, 28 g, 240 mmol) in 200 mL of dichloromethane, adding sulfurous chloride (4 mL, 60 mmol) under ice bath, and continuing to stir at room temperature for 15 min; adding aspirin (10.8 g, 60 mmol), and continuing to stir the mixture at room temperature for 2.5 h; then filtering the suspension, washing the organic solution with saturated Na2CO3 solution; then adding hexane (200 mL), and precipitating white solid compound 5 (6 g, 50%); dissolving compound 5 (0.65 g, 2.3 mmol) in acetonitrile (20 mL), adding an aqueous solution of CTP (0.5 g, 2.3 mmol) and triethylamine (0.23 g, 2.3 mmol), and stirring the mixture for 20 h; removing acetonitrile under reduced pressure; extracting the residue with ethyl acetate, and evaporating to obtain white solid compound 6 (0.31 g, 35.6%).

[0027] Further, the compound II is prepared from After being oxidized by hydrogen peroxide, the compound II is prepared, and the preparation process can be specifically as follows:

[0028]

[0029] The divalent platinum compound is prepared from the compound II. The bishydroxy tetravalent platinum compound II is prepared by oxidation with hydrogen peroxide at 60-70°C for 1-8h.

[0030] To achieve another object of the present application, we designed and prepared the Captopril bifunctional tetravalent platinum compound III. Its structure is as follows:

[0031]

[0032] wherein, selected from cisplatin or oxaliplatin.

[0033] Preferably, the Captopril bifunctional tetravalent platinum compound III according to the present application is selected from compound 3:

[0034]

[0035] The synthesis route III of the Captopril bifunctional tetravalent platinum III is as follows:

[0036]

[0037] The compound II is coupled with the acetyl Captopril compound 7 to obtain the symmetrical bifunctional Captopril tetravalent platinum compound III; wherein the material molar ratio of the compound II to the compound 7 is 1:2.0-5.0.

[0038] Further, in the synthesis route III, the preparation step of the bifunctional Captopril tetravalent platinum compound III is as follows:

[0039] The compound 7, the condensing agent, the organic base are dissolved in anhydrous organic solvent under inert gas atmosphere, the compound II is added, and after reaction in the dark, the bifunctional Captopril tetravalent platinum compound III is separated after post-treatment;

[0040] wherein the material molar ratio of the compound II, the compound 7, the condensing agent, the organic base is 1:2.0-5.0:2.0-5.0:2.0-5.0; and the material ratio of the compound II to the organic solvent is 1g of the compound II to 10-100ml of the organic solvent.

[0041] Further, the inert gas is nitrogen, helium or argon; the condensing agent is TBTU, HATU or EDCI; the organic base is triethylamine, N,N-diisopropyl ethylamine or 4-dimethylamino pyridine; and the organic solvent is DMF or DMSO.

[0042] The preparation process is as follows: first, a condensing agent and compound 7 are added to a reaction vessel, the air in the system is replaced with an inert gas, an anhydrous organic solvent is added, and an anhydrous organic base is added to the reaction system, and the reaction is stirred at room temperature for 5-60 minutes. Thereafter, a tetravalent platinum compound II is added to the reaction system, the air in the system is replaced with an inert gas again, and the reaction system is placed at 25-120°C in the dark for 24-72 hours. After the reaction is completed, the solvent is removed under reduced pressure, and finally the bifunctional captopril tetravalent platinum compound III is obtained by column chromatography.

[0043] The present invention provides a pharmaceutical composition comprising an effective therapeutic amount of a compound represented by general formula (I) or (III), and pharmaceutically acceptable excipients thereof.

[0044] Pharmaceutically acceptable excipients for the present invention include one or more carriers, excipients, and diluents, including various types of adhesives. Excipients can be aqueous or non-aqueous, with typical examples including gelatin and other colloids, starches such as corn starch, sugars such as lactose, cellulosic materials such as sodium carboxymethylcellulose, and mixtures thereof. Other excipients include tragacanth gum powder, as well as oils, alcohols, esters, buffers, alginic acid, pyrogen-free water, isotonic saline, and Ringer's solution. The trifunctional tetravalent platinum compounds or pharmaceutical compositions of the present invention can be formulated in a variety of dosage forms, including tablets, as well as sustained-release, controlled-release, or nanoformulations prepared according to conventional pharmaceutical practice.

[0045] The trifunctional tetravalent platinum compound of the present invention can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, etc.

[0046] The trifunctional tetravalent platinum compound of the present invention can be administered intravenously, including intravenous injection, intramuscular injection, intratumoral injection, subcutaneous injection, and acupuncture injection.

[0047] The method of preparing the active ingredient into a medicine in the present invention can be prepared by methods known to those skilled in the art. For example, the active ingredient can be diluted with a carrier or encapsulated in a carrier so that it can be quickly released, slowly released, or delayed released after administration to a subject.

[0048] Another object of the present invention is to provide the use of the compound or pharmaceutical composition represented by general formula (I) or general formula (III) in the preparation of anti-tumor drugs, specifically in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs.

[0049] The trifunctional tetravalent platinum derivatives described in the present invention can have a good therapeutic effect on metastatic malignant tumors, can effectively inhibit the tumor epithelial-mesenchymal transition process, and effectively activate the tumor immune response, and have a good therapeutic effect on metastatic malignant tumors.

[0050] Further, the anti-tumor is anti-lung cancer, anti-drug-resistant lung cancer, anti-liver cancer or anti-breast cancer, etc.; wherein the anti-tumor proliferation is specifically anti-human lung adenocarcinoma, anti-cisplatin-resistant human lung adenocarcinoma, anti-human liver cancer or anti-mouse breast cancer; the anti-tumor metastasis is anti-mouse breast cancer cells.

[0051] The application further provides a combined preparation comprising the compound or the pharmaceutical composition as shown in the general formula (I) and the general formula (III), and an anti-tumor drug of the combination of taxol, fluorouracil, gemcitabine, vinblastine and antibodies.

[0052] The trifunctional derivative tetravalent platinum compound of the application can be prepared into an anti-tumor combined preparation alone or in combination with various marketed drugs, including platinum, taxol, etc. The dosage forms of the combined preparation include tablets, capsules, injections and various forms.

[0053] In order to achieve another object of the application, the technical scheme of the application further comprises the preparation of the nanomedicine of the trifunctional tetravalent platinum compound loaded in grapefruit extracellular vesicles: the nanomedicine is prepared from the trifunctional tetravalent platinum derivative I as an active ingredient, the carrier DSPE-PEG, the carrier grapefruit extracellular vesicles GEVs and the targeting carrier DSPE-PEG-Tf; the mass ratio of the tetravalent platinum derivative (I), the carrier DSPE-PEG, the carrier GEVs and the targeting carrier DSPE-PEG-Tf in the nanomedicine is 1:(0.1-100):(0.1-100):(0.1-100).

[0054] Further, the preparation method of the nanomedicine is a combination of an anti-solvent precipitation method and an ultrasonic method, which comprises the following specific steps:

[0055] (1) dispersing the tetravalent platinum derivative (I) and DSPE-PEG in DMF at a ratio of 1:(0.1-100), and injecting into PBS under ultrasonic conditions of 1-20 DEG C and 100-1000 W; further stirring the obtained mixture for 5-60 minutes; high-pressure homogenization for 5-120 minutes, and dialyzing the solution with PBS for 1-5 times to obtain DSPE-PEG-loaded tetravalent platinum drug nanoparticles;

[0056] (2) mixing the DSPE-PEG-loaded tetravalent platinum drug and the carrier GEVs at a ratio of 1:(0.1-100)

I:GEVs, (w / w)

[0057] (3) The non-targeted grapefruit extracellular vesicle loaded with the trifunctional tetravalent platinum compound nanomedicine is incubated with the targeting carrier DSPE-PEG-Tf at a ratio of 1:(0.1-100)

I: DSPE-PEG-Tf, (w / w)

[0058] The preparation process can be specifically as follows: (1) dispersing the trifunctional tetravalent platinum derivative 1 (20 mg) and DSPE-PEG (20 mg) in 0.4 mL DMF at a ratio of 1:1 (w / w), and injecting into PBS under the condition of 200 W ultrasonic at 10℃; the obtained mixture is further stirred for 15 minutes; high-pressure homogenization for 30 minutes, and the solution is dialyzed twice with PBS, to obtain the DSPE-PEG loaded tetravalent platinum drug nanoparticles DSPE-PEG@Pt(IV);

[0059] (2) The above DSPE-PEG loaded tetravalent platinum drug is incubated with the carrier GEVs 20 mg at a ratio of 1:1 (compound 1:GEVs, w / w) under the condition of ultrasonic 300 W for 3 minutes at 10℃, to obtain the non-targeted grapefruit extracellular vesicle loaded with the trifunctional tetravalent platinum compound nanomedicine GEVs@Pt(IV).

[0060] (3) The non-targeted grapefruit extracellular vesicle loaded with the trifunctional tetravalent platinum compound nanomedicine GEVs@Pt(IV) is incubated with the targeting carrier DSPE-PEG-Tf 20 mg at a ratio of 1:1 (compound 1:GEVs, w / w) in a water bath at 30℃ for 1 hour, to obtain the transferrin-modified targeted grapefruit extracellular vesicle loaded with the trifunctional tetravalent platinum compound nanomedicine Tf-GEVs@Pt(IV).

[0061] Further, the grapefruit extracellular vesicle loaded with the trifunctional tetravalent platinum compound nanomedicine is used for preparing an antitumor proliferation and antitumor metastasis drug.

[0062] Further, the antitumor proliferation is against human lung adenocarcinoma, cisplatin-resistant human lung adenocarcinoma, human hepatocarcinoma or mouse breast cancer.

[0063] Further, the antitumor metastasis is against mouse breast cancer cells.

[0064] Compared with the prior art, the compound and nanomedicine with the trifunctional tetravalent platinum structure have the following advantages:

[0065] (1) Chemotherapy is weak to metastatic tumors, which is the main reason for the failure of chemotherapy. Tumor microenvironment (TME) plays a crucial role in promoting tumor proliferation and metastasis, and is a promising target for cancer treatment. The present application successfully synthesizes a series of novel trifunctional platinum (IV) compounds by combining aspirin-captopril with platinum (IV) system. In addition to causing DNA damage, the drug can effectively regulate inflammation, fibrosis and immunosuppressive TME. These compounds exhibit significant anti-tumor proliferation and anti-migration effects in anti-tumor activity experiments, and have great development prospects as anti-tumor metastasis drugs.

[0066] (2) The present application develops a nano drug loaded with trifunctional platinum (IV) compounds by grapefruit extracellular vesicles (GEVs), and introduces transferrin Tf as a targeting group to prepare a nano drug of transferrin modified targeted grapefruit extracellular vesicles loaded with trifunctional platinum (IV) compounds. GEVs promote the penetration of Tf-GEVs@Pt(IV) into tumor tissues; transferrin (Tf) further enhances its tumor targeting properties; the active components of trifunctional platinum (IV) can remodel the tumor microenvironment and have significant anti-tumor activity.

[0067] (3) The compound structures of general formula I and general formula III described in the present application are innovative, and are expected to obtain a variety of effective tumor lead molecules, opening up a new way for the development of platinum compounds. Such innovative drug research at the source will have important theoretical value and practical significance for national economic and social development and people's health.

[0068] (4) The nano drug of trifunctional platinum (IV) compounds loaded by grapefruit extracellular vesicles (GEVs) described in the present application is a new attempt in the field of pharmaceutical preparations, and is expected to obtain a new type of high-efficiency, tumor-targeting nano drug, providing a new candidate drug for cancer treatment. BRIEF DESCRIPTION OF DRAWINGS

[0069] Figure 1 . Structures of platinum (IV) compounds 1-4 and nanoparticles GEVs@Pt(IV) and Tf-GEVs@Pt(IV).

[0070] Figure 2 . The properties of DSPE-PEG@Pt(IV), GEVs, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) are detected by Western blot, DLS and TEM. (a) Western blot analysis of exosome marker proteins Alix, CD9 and TSG101. (b, c) Nanoparticle size and Zeta potential determined by DLS. (d) TEM image of nanoparticles.

[0071] Figure 3Stability of nanoparticles DSPE-PEG@Pt(IV), GEVs, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) was determined by DLS after 7 days of storage at 4°C (n=3). (a) Change in particle size. (b) Change in PDI.

[0072] Figure 4 In vitro drug release profile of free compound 1, nanoparticles GEVs@Pt(IV) and Tf-GEVs@Pt(IV) in PBS medium.

[0073] Figure 5 Uptake of platinum drugs in 4T1 cells in vitro. 4T1 cells were incubated with CDDP, CDDP+6, 1, 2, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) (5 mM) for 24 h at 37°C. ***P<0.001.

[0074] Figure 6 In vivo antitumor activity of candidate tetravalent platinum compound 1 and nanoparticles GEVs@Pt(IV) and Tf-GEVs@Pt(IV) against 4T1 tumors in female BALB / c mice, using GEVs, CDDP and mixture CDDP+6 as reference drugs (n=5). (a) Schematic representation of the experimental design. (b) Relative body weight of mice during treatment. (c) Tumor volume change graph. (d) Tumor weight of mice in each group at the end of the experiment. TGI of tested drugs is shown above [TGI = (1 - tumor weight of drug-treated group / tumor weight of blank) x 100%]. (e) Uptake of platinum in tumor tissue. (f) Tumor images at the end of the experiment. **P<0.01, ***P<0.001.

[0075] Figure 7 H&E staining of tumor, liver, spleen and kidney tissues of mice in blank, compound 1 group, GEVs group, CDDP group, CDDP+6 mixture group, nanoparticles GEVs@Pt(IV) group and Tf-GEVs@Pt(IV) group. Tissues were taken from in vivo antitumor experiment.

[0076] Figure 8 Inhibition of 4T1 cell migration by compound 1, GEVs, CDDP, CDDP+6 and nanoparticles GEVs@Pt(IV) and Tf-GEVs@Pt(IV) (5 mM) was evaluated using in vitro Transwell assay. Tumor cells were treated with drugs for 24 h. (a) Representative images. (b) Relative migration rate analysis. ***P<0.001.

[0077] Figure 9The migration inhibition properties of compound 1, GEVs, CDDP, CDDP+6, and nanoparticle GEVs@Pt(IV) and Tf-GEVs@Pt(IV) (5 mM) on 4T1 cells were evaluated using an in vitro scratch test. The tumor cells were treated with drugs for 24 h. (a) Representative images. (b) Relative migration rate analysis.

[0078] Figure 10 The lung metastasis inhibition properties of compound 1 and nanoparticle GEVs@Pt(IV) and Tf-GEVs@Pt(IV) on 4T1 tumors in female BALB / c mice, with GEVs, CDDP, and CDDP+6 as reference drugs (n = 5). ***P < 0.001. (a) Schematic diagram of the experimental design. (b) Representative photos of the front and back of the lungs at the end of the experiment. (c) Lung nodules in each group. The inhibition rate compared with blank is listed above. (d) H&E staining of lung metastatic nodules. Nodules are indicated by arrows.

[0079] Figure 11 RNA-seq analysis of 4T1 tumor tissues in blank, GEVs, and Tf-GEVs@Pt(IV) treatment groups in the in vivo anti-tumor experiment. (a) Gene heat map. (b) Wayne diagram of differentially expressed genes in the three groups. (c) Volcano plot of DEGs in blank vs Tf-GEVs@Pt(IV) groups. (d) KEGG enrichment of blank vs Tf-GEVs@Pt(IV) groups. (e) GSEA plot of blank vs Tf-GEVs@Pt(IV) groups.

[0080] Figure 12 The ability to cause DNA damage. (a-b) GO enrichment and GSEA analysis of DEGs related to DNA damage in blank vs Tf-GEVs@Pt(IV) cells. (c-d) Western blot analysis of g-H2AX and P53 in 4T1 cells treated with CDDP (5 mM), CDDP+6 mixture (5 mM / 10 mM), compound 1 (5 mM), GEVs@Pt(IV) (5 mM), and Tf-GEVs@Pt(IV) (5 mM) at 37°C for 24 h, with PBS and GEVs-treated cells as controls. **P < 0.01, ***P < 0.001.

[0081] Figure 13 Intracellular sub-distribution of platinum in tumor cells treated in vitro with CDDP (5 mM), CDDP+6 mixture (5 mM / 10 mM), tetravalent platinum compound 1 (5 mM), nanoparticle GEVs@Pt(IV) (5 mM), and Tf-GEVs@Pt(IV) (5 mM) for 24 h. **p < 0.01, ***p < 0.001.

[0082] Figure 14 Characteristics that cause mitochondrial-mediated apoptosis. (a-b) GO enrichment and GSEA analysis of DEGs related to mitochondrial-mediated apoptosis in Blank vs Tf-GEVs@Pt(IV). (c) Quantification of apoptosis in 4T1 cells by flow cytometry using Annexin V-FITC / PI staining. (d-e) Western blot analysis of Bcl-2, Bax, caspase3 and c-caspase-3 in 4T1 cells. 4T1 tumor cells were incubated with CDDP (5 mM), CDDP+6 (5 mM / 10 mM), Compound 1 (5 mM), GEVs@Pt(IV) (5 mM) and Tf-GEVs@Pt(IV) (5 mM) for 24 h, with PBS and GEVs as controls. **P < 0.01, ***P < 0.001.

[0083] Figure 15 Quantification of 4T1 cells mitochondrial damage using JC-1 staining. 4T1 tumor cells were incubated with CDDP (5 mM), CDDP+6 (5 mM / 10 mM), Compound 1 (5 mM), GEVs@Pt(IV) (5 mM) and Tf-GEVs@Pt(IV) (5 mM) for 24 h, with PBS and GEVs as controls.

[0084] Figure 16 Characteristics that reduce tumor fibrosis. (a-b) GO enrichment and GSEA analysis of DEGs related to tumor fibrosis in Blank vs Tf-GEVs@Pt(IV). (c-d) Immunohistochemical staining of a-SMA and FSP1, Picrosirius red and Masson’s staining of collagen fibers in tissues from in vivo anti-tumor experiments. **P < 0.01, ***P < 0.001.

[0085] Figure 17 (a-b) Western blot analysis of TGF-β1, MMP2 and MMP9 in 4T1 cells. 4T1 tumor cells were incubated with CDDP (5 mM), CDDP+6 (5 mM / 10 mM), Compound 1 (5 mM), GEVs@Pt(IV) (5 mM) and Tf-GEVs@Pt(IV) (5 mM) for 24 h, with PBS and GEVs as controls. (c-d) Immunohistochemical staining of TGF-β1 and MMP9 in tumor tissues from in vivo anti-tumor experiments. ***P < 0.001.

[0086] Figure 18Reversing the characteristics of inflammatory TME. (a-b) GO enrichment and GSEA analysis of DEGs related to inflammation in Blank vs Tf-GEVs@Pt(IV). (c-d) Western blot analysis of COX-2, TNF-a and IL-6 in 4T1 cells. 4T1 tumor cells were incubated with CDDP (5 mM), CDDP + 6 (5 mM / 10 mM), Compound 1 (5 mM), GEVs@Pt(IV) (5 mM) and Tf-GEVs@Pt(IV) (5 mM) for 24 h, with PBS and GEVs as controls. (e) Elisa analysis of TNF-a and IL-6 in tumor tissues in in vivo anti-tumor experiments. (f-g) Immunohistochemical staining of COX-2 in tumor tissues in in vivo anti-tumor experiments. **p<0.01, ***p<0.001.

[0087] Figure 19 Reversing the characteristics of immunosuppressive TME. (a-b) GO enrichment and GSEA analysis of DEGs related to immunity in Blank vs Tf-GEVs@Pt(IV). (c-d) Western blot analysis of PD-L1, STING, p-STING, TBK1, p-TBK1, IFR3 and p-IFR3 in 4T1 cells. (e-f) Immunohistochemical staining of PD-L1 expression, CD3+ and CD8+ T cells, CD206+ M2 and CD86+ M1 macrophages in tumor tissues in in vivo anti-tumor experiments. ***P<0.001.

[0088] Figure 20 Schematic diagram of the mechanism of action of the nanomedicine Tf-GEVs@Pt(IV) loaded with the trifunctional tetravalent platinum compound. DETAILED DESCRIPTION

[0089] In order to make the objects, technical solutions and advantages of the present application clearer, the representative embodiments of the present application are described in detail below, without being limited thereto.

[0090] Example 1.

[0091] I. Preparation of tetravalent platinum of structural formula II

[0092] Synthesis of dihydroxy cis tetravalent platinum IIa

[0093]

[0094] A suspension of CDDP (1.0 g, 3.3 mmol) in 30 mL distilled water was stirred at room temperature. Then 50 mL of 30% H2O2 was added dropwise and the suspension was stirred at 60 °C for 4 h. After filtration, the crude product was obtained as a yellow solid. Further recrystallization in water gave dihydroxy cis-platinum IVa as yellow crystals (0.78 g, 70%).

[0095] II. Preparation of Aspirin-Captopril Tetraplatin of Structural Formula I

[0096] 1. Preparation of Symmetrical Aspirin-Captopril Tetraplatin Compound 1

[0097]

[0098] Compound 6 (341 mg, 0.90 mmol) and TBTU (289 mg, 0.90 mmol) were dissolved in 5 mL dry N,N-dimethylformamide (DMF) and stirred at room temperature for 15 min. Then, triethanolamine (125 μL, 0.90 mmol) was added and the mixture was stirred for another 15 min. Then, dihydroxy cis-platinum IVa (100 mg, 0.30 mmol) was added. The mixture was stirred at 50 °C under nitrogen protection and in the dark for 48 h. After the reaction was completed, the solvent was removed under vacuum and the crude product was purified by silica gel column chromatography to give compound 1 as a yellow solid (101 mg, 32%). The purity was 98.9% as determined by HPLC (MeOH / H2O = 70 / 30).

[0099] 1 H NMR (500 MHz, DMSO-d6) δ 7.95 - 7.60 (m, 3H), 7.55 - 7.41 (m, 2H), 7.28 - 7.10 (m, 1H), 7.03 - 6.86 (m, 2H), 6.45 (br, 6H, NH3), 4.58 - 4.28 (m, 2H), 3.73 - 3.39 (m, 4H), 3.20 - 3.05 (m, 4H), 2.94 - 2.70 (m, 2H), 2.35 - 2.25 (m, 2H), 2.07 - 1.74 (m, 8H), 1.34 - 0.99 (m, 10H). 13 CNMR (126 MHz, DMSO-d6) δ 172.8, 167.7, 157.8, 137.7, 135.5, 134.6, 129.3, 122.7, 119.8, 118.1, 59.8, 46.0, 37.8, 32.1, 29.4, 24.4, 17.08, 9.1. MS-ESI: calcd for [M+H] + : 1058 (M = C 36 H 46Cl2N4O 12 PtS2), found: 1058. HRMS: calcd for [M+NH4] + : 1073.1917 (M = C 36 H 46 Cl2N4O 12 PtS2), found: 1073.1916.

[0100] 2. Preparation of asymmetric aspirin-captopril tetravalent platinum compound 2

[0101]

[0102] Compound 6 (114 mg, 0.3 mmol) and TBTU (96 mg, 0.3 mmol) were dissolved in 5 mL dry DMF and stirred at room temperature for 15 min. Then, triethanolamine (42 μL, 0.3 mmol) was added and the mixture was stirred for another 15 min. Then, dihydroxy cis-tetravalent platinum IIa (100 mg, 0.3 mmol) was added. The mixture was stirred at 50 °C under nitrogen protection and in the dark for 48 h. After the reaction was completed, the solvent was removed under vacuum and the crude product was purified by silica gel column chromatography to obtain compound 2 (58.3 mg, 28%) as a yellow solid. The purity was 97.1% as determined by HPLC (MeOH / H2O = 70 / 30).

[0103] 1 H NMR (500 MHz, Methanol-d4) δ 7.97 - 7.79 (m, 1H), 7.64 - 7.48 (m, 1H), 7.43 - 7.13 (m, 1H), 7.00 - 6.76 (m, 1H), 4.63 - 4.39 (m, 1H), 3.80 - 3.50 (m, 2H), 3.23 - 2.99 (m, 2H), 2.49 - 2.20 (m, 2H), 2.18 - 1.78 (m, 4H), 1.38 - 1.16 (m, 5H). 13 C NMR (126 MHz, Methanol-d4) δ 174.8, 174.1, 170.1, 166.3, 150.8, 133.5, 131.5, 125.7, 123.7, 123.5, 58.7, 42.2, 30.8, 28.9, 26.8, 24.3, 19.7, 15.7. MS-ESI: calcd for [M+H] + : 695 (M = C 18 H 27 Cl2N3O7PtS), found: 695. HRMS: calcd for [M+Na] +: 717.0487 (M = C 18 H 27 Cl2N3O7PtS), found: 717.0497.

[0104] III. Preparation of Captopril bifunctional tetraplatin of structural formula III

[0105] Preparation of Captopril tetraplatin compound 3

[0106]

[0107] Compound 7 (233 mg, 0.90 mmol) and TBTU (289 mg, 0.90 mmol) were dissolved in 5 mL dry DMF and stirred at room temperature for 15 min. Then, triethylamine (125 μL, 0.90 mmol) was added and the mixture was stirred for another 15 min. Then, platinum IIa oxide (100 mg, 0.30 mmol) was added. The mixture was stirred at 50 °C under nitrogen protection and in the dark for 48 h. After the reaction was completed, the solvent was removed under vacuum and the crude product was purified by silica gel column chromatography to obtain compound 3 (90 mg, 37%) as a yellow solid. The purity was 95.2% as determined by HPLC (MeOH / H2O = 70 / 30).

[0108] 1 H NMR (500 MHz, DMSO-d6) δ 4.56 - 4.15 (m, 2H), 3.66 - 3.40 (m, 4H), 3.04 - 2.71 (m, 6H), 2.59 (s, 3H), 2.43 - 2.30 (m, 3H), 2.23 - 2.07 (m, 2H), 1.96 - 1.83 (m, 4H), 1.28 - 1.20 (m, 2H), 1.14 - 0.98 (m, 6H). 13 CNMR (126 MHz, DMSO) δ 195.9, 195.8, 173.3, 66.0, 58.8, 46.9, 37.8, 31.0, 29.5, 25.7, 16.9. MS-ESI: calcd for [M] + : 816 (M = C 22 H 38 Cl2N4O8PtS2), found: 816. HRMS: calcd for [M + Na] + : 838.1054 (M = C 22 H 38 Cl2N4O8PtS2), found: 838.1052.

[0109] IV. Preparation of nanomedicine of grapefruit extracellular vesicles loaded with trifunctional tetraplatin compound

[0110] Preparation of grapefruit extracellular vesicles loaded with nano-drug of trifunctional tetravalent platinum compound as shown in Figure 1 .

[0111] 1. Preparation and characterization of grapefruit extracellular vesicles GEVs

[0112] Grapefruit was used as material, and GEVs were prepared by differential centrifugation using an ultracentrifuge (Beckman Optima XPN-100). Gradient centrifugation steps: remove the solid after 30 min centrifugation at 3000g; remove the solid after 1 h centrifugation at 15000g; filter through a 0.45 pm filter membrane; the solid precipitate after 2 h centrifugation at 100000g is GEVs. The concentration of GEVs was determined by BCA protein quantitative detection kit, and diluted with PBS to a concentration of 2 mg / mL. Western blot was used to detect marker proteins Alix, CD9 and TSG101. DLS and TEM were used to characterize the morphology.

[0113] 2. Preparation of DSPE-PEG loaded tetravalent platinum drug nanoparticles DSPE-PEG@Pt(IV)

[0114] Trifunctional tetravalent platinum derivative 1 (20 mg) and DSPE-PEG (20 mg) were dispersed in 0.4 mL DMF at a ratio of 1:1 (w / w), and injected into PBS under the condition of ultrasonic 200W at 10°C; the resulting mixture was further stirred for 15 min; high pressure homogenization for 30 min, and the solution was dialyzed twice with PBS to obtain DSPE-PEG loaded tetravalent platinum drug nanoparticles DSPE-PEG@Pt(IV).

[0115] 3. Preparation of non-targeted grapefruit extracellular vesicles loaded with nano-drug of trifunctional tetravalent platinum compound GEVs@Pt(IV)

[0116] The above DSPE-PEG loaded tetravalent platinum drug and carrier GEVs 20 mg were incubated at a ratio of 1:1 (compound 1:GEVs, w / w) under the condition of ultrasonic 300W at 10°C for 3 min to obtain non-targeted grapefruit extracellular vesicles loaded with nano-drug of trifunctional tetravalent platinum compound GEVs@Pt(IV).

[0117] 4. Preparation of targeted grapefruit extracellular vesicles loaded with nano-drug of trifunctional tetravalent platinum compound Tf-GEVs@Pt(IV)

[0118] The nanodrug GEVs@Pt(IV) of non-targeted grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compounds described above was incubated with the targeting carrier DSPE-PEG-Tf 20 mg at a ratio of 1:1 (compound 1:GEVs, w / w) in a 30°C water bath for 1 hour, i.e. to obtain the transferrin-modified targeted grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compounds nanodrug Tf-GEVs@Pt(IV).

[0119] 5. Preparation of DiD-labeled nanodrugs DiD-GEVs@Pt(IV) and DiD-Tf-GEVs@Pt(IV)

[0120] The nanodrug was prepared by dissolving DiD (DSPE-PEG2000: compound 1 = 1%) and DSPE-PEG2000 in DMF, and the subsequent steps were the same as above. DiD-labeled nanodrugs DiD-GEVs@Pt(IV) and DiD-Tf-GEVs@Pt(IV) were prepared.

[0121] V. Nanodrug characterization

[0122] The marker proteins of EVs were detected by Western blot Figure 2 a). The results showed that GEVs were rich in Alix, CD9 and TSG101, indicating that the vitality of the prepared exosomes was normal. These proteins were also highly expressed in nanodrugs GEVs@Pt(IV) and Tf-GEVs@Pt(IV), indicating that the addition of DSPE-PEG@Pt(IV) had no significant effect on the biological characteristics of GEVs.

[0123] Dynamic light scattering (DLS) and transmission electron microscopy (TEM, Figure 2b-d) Characterization of nanoparticles: The size of DSPE-PEG@Pt(IV) was 68.1 ± 0.4 nm (PDI = 0.16 ± 0.01, ζ = -22.54 ± 1.26). The size of GEVs was 223.8 ± 5.2 nm (PDI = 0.33 ± 0.03, ζ = -14.64 ± 3.81), which was larger than that of DSPE-PEG@Pt(IV) and was easy to encapsulate DSPE-PEG@Pt(IV). The sizes of GEVs@Pt(IV) and Tf-GEVs@Pt(IV) were 146.0 ± 1.9 nm (PDI = 0.27 ± 0.04, ζ = -20.22 ± 0.69) and 190.3 ± 6.3 nm (PDI = 0.39 ± 0.06, ζ = -15.64 ± 0.40), respectively. TEM images further showed that GEVs had the typical round vesicle structure reported in the literature, and the nanodrugs GEVs@Pt(IV) and Tf-GEVs@Pt(IV) were also approximately spherical and had contents, which further confirmed that DSPE-PEG@Pt(IV) was embedded in GEVs.

[0124] Stability detection: The stability of nanodrugs (including DSPE-PEG@Pt(IV), GEVs, GEVs@Pt(IV) and Tf-GEVs@Pt(IV)) was monitored by measuring the change in particle size using DLS at 4°C for 7 days. Figure 3 It was shown that DSPE-PEG@Pt(IV), GEVs, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) remained stable within 7 days. These facts indicated that the nanodrugs prepared by using GEVs as a carrier to encapsulate trifunctional platinum drugs were stable in aqueous media.

[0125] Release behavior: The release behavior of GEVs@Pt(IV) and Tf-GEVs@Pt(IV) was detected in PBS medium. The nanoparticles (5 mL) were loaded into a dialysis bag (8-14 kDa) and immersed in 100 mL of PBS, which was stirred (100 rpm) at 37°C. The release solution (3 mL) was drawn at a fixed time, and fresh PBS (3 mL) was supplemented. The drug concentration was determined by measuring the platinum content by atomic absorption (AAS). Figure 4 The results showed that both GEVs@Pt(IV) and Tf-GEVs@Pt(IV) could gradually release about 80% of compound 1 within 24 h, which was significantly slower than the free drug (88% was released within the first 6 h). This indicated that GEVs@Pt(IV) and Tf-GEVs@Pt(IV) had a significant sustained-release effect.

[0126] Six, pharmacological activity experiment

[0127] 1. In vitro anti-proliferative activity

[0128] The anti-proliferative activities of the Pt(IV) compounds 1-4 and the nanodrugs GEVs@Pt(IV) and Tf-GEVs@Pt(IV) against four tumor cell lines (i.e. human lung cancer (A549), CDDP-resistant human lung cancer (A549R), murine breast cancer (4T1), human liver cancer (HepG2)) and one human normal liver cell line (LO2) were determined by MTT method, and GEVs, CDDP, mixture CDDP+6 (1:2), acid 6, CTP and ASP were used as reference drugs. Briefly, tumor cells suspended in 100 μL of culture medium were added to 96-well plates (5 x 10 3 / well) and pre-cultured overnight. Then, 100 μL of culture medium containing different concentrations of drugs were added. Tumor cells were treated with drugs for 48 h. 20 μL of MTT solution (5 mg / mL) was added and incubated for another 4 h. Then, the culture supernatant was removed and 150 μL of dimethyl sulfoxide (DMSO) was added. The absorbance of each well was measured using a microplate spectrophotometer (490 nm). The anti-proliferative data were expressed as IC 50 values.

[0129] At the same time, we tested the anti-proliferative activities of the Pt(IV) compound 4 as a control.

[0130] Table 1. Anti-proliferative activities of the Pt(IV) compounds 1-4 and the nanodrugs GEVs@Pt(IV) and Tf-GEVs@Pt(IV).

[0131]

[0132]

[0133] a RF: resistance factor, RF = IC 50 (A549R) / IC 50 (A549); b SI: selectivity index, SI = IC 50 (LO2) / IC 50 (HepG2); c ND: not tested or not calculated; d CDDP+6: mixture of CDDP and acid 6 with a molar ratio of 1:2.

[0134] Results show that firstly, the activity of the trifunctional tetravalent platinum compound with CTP-ASP ligand is higher than that of the bifunctional compound. The activity of trifunctional compound 1 is significantly stronger than that of the corresponding bifunctional tetravalent platinum drugs 3 (CTP tetravalent platinum) and 4 (ASP tetravalent platinum). Secondly, the number of ligands in tetravalent platinum has an impact on its activity, in which the activity of the double-ligand compound 1 is significantly higher than that of the single-ligand compound 2. It is worth noting that compound 1 exhibits the strongest anti-tumor activity against all tumor cell lines involved in the test, with an IC 50 value lower than 4.18 μM, showing a significantly stronger activity advantage compared with the reference drugs CDDP, ASP, CTP and mixture CDDP+6. Based on the above results, compound 1 has good application potential and is therefore selected as a candidate drug to further carry out the preparation of nanodrugs.

[0135] Compared with free compound 1, the nanoparticles GEVs@Pt(IV) and Tf-GEVs@Pt(IV) prepared with GEVs as carriers show superior anti-tumor performance. In particular, Tf-GEVs@Pt(IV) with a tumor targeting group has an activity 1.9 times higher than that of free compound 1 against all tumor cell lines. The drug uptake in tumor cells is a key factor affecting the anti-tumor efficacy. We detected the drug uptake level of 4T1 cells Figure 5 ). The results show that compared with GEVs@Pt(IV) (P<0.001) and free compound 1 (P<0.001), Tf-GEVs@Pt(IV) has a higher accumulation in tumor cells, which is a key factor for its significant anti-tumor activity.

[0136] Drug resistance is one of the clinical problems faced by platinum drugs. In this paper, the resistance factor (RF) of A549R was calculated to study the effectiveness of these drugs in overcoming drug resistance. The results show that all the prepared tetravalent platinum compounds 1-4 can effectively reduce drug resistance, with RF = 1.05-3.78 lower than that of the reference drugs CDDP and mixture CDDP+6 (RF = 6.08, 5.06). In addition, nanodrugs GEVs@Pt(IV) and Tf-GEVs@Pt(IV) also have low RF values (1.89 and 0.70), indicating that they also have great potential in overcoming drug resistance of CDDP.

[0137] Severe toxicity to normal cells is another problem faced by platinum drugs. We also calculated the selectivity index (SI: LO2 IC 50 value and HepG2 IC 50The ratio of the values). Compared with CDDP (SI = 1.68) and mixture CDDP + 6 (SI = 1.77), compound 1 has a higher SI = 2.21, effectively reducing toxicity. The SI of nanomedicine Tf-GEVs@Pt(IV) = 6.88 is significantly higher than that of free compound 1 and GEVs@Pt(IV) (SI = 2.21, 2.63), which may be attributed to its excellent tumor targeting properties.

[0138] In summary, the trifunctional tetravalent platinum compound with CTP-ASP functional groups has excellent antitumor activity; the nanomedicine GEVs@Pt(IV) loaded with trifunctional tetravalent platinum compound constructed with it as an active component has better activity; the Tf-GEVs@Pt(IV) prepared by modifying it with the tumor targeting group Tf has the strongest activity, which can effectively overcome drug resistance and reduce the toxicity of platinum drugs.

[0139] 2. In vivo antitumor activity

[0140] Experiments were conducted using female BALB / c mice (18-20 g) as a model, and a tumor model was established by subcutaneously injecting tumor cells (1 x 10 6 ) into the left side of the mice. The mice were divided into 7 groups (Blank, CDDP, CDDP + 6, 1, GEVs, GEVs@Pt(IV) and Tf-GEVs@Pt(IV), n = 5) on the 4th day. Then, the drugs were injected through the tail vein on the 4th, 7th and 10th days, and the dose was 2 mg Pt / kg Figure 6 a). The tumor volume (V = W 2 x L / 2, where W is the width of the tumor and L is the length) was monitored every day, and the body weight of the mice was recorded. Then, the mice were sacrificed on the 12th day. The tumor weight was weighed, and the tumor growth inhibition rate TGI was calculated

TGI = (1-(tumor weight of drug treatment group / tumor weight of control group)) x 100%

[0141] Figure 6 The results show that GEVs have a certain inhibitory effect on tumors, with a TGI of 51.4%, which indicates that they are not only drug carriers but also enhance the antitumor activity of nanomedicines Figure 6 c, d). Compared with the reference drugs CDDP and mixture CDDP + 6, the candidate compound 1 can more effectively inhibit the growth of tumors in vivo, and the trend is similar to the in vitro MTT results. In addition, the efficacy of nanomedicines GEVs@Pt(IV) and Tf-GEVs@Pt(IV) is significantly higher than that of compound 1 and the GEVs group (P < 0.01). The nanomedicine Tf-GEVs@Pt(IV) modified with Tf (85.5 mm 3, TGI = 83.7%) than GEVs@Pt(IV) (138.3 mm 3 , TGI = 74.9%, P < 0.01) showed stronger anti-tumor ability. To further detect the intrinsic molecular mechanism of these drugs, we performed histological analysis according to hematoxylin and eosin (H&E) staining results. Figure 7 The images in Fig. 6 show that significant apoptosis and necrosis areas were observed in the tumors of the aspirin-captopril tri-functional quadruplex platinum compound 1, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) groups, even stronger than the CDDP group and CDDP+6 group. This indicates that the use of GEVs to encapsulate aspirin-captopril tri-functional quadruplex platinum compound 1 to prepare the corresponding nanodrugs can effectively improve the in vivo anti-tumor activity.

[0142] The uptake of chemotherapeutic drugs in tumors is a key factor affecting anti-tumor efficacy. In this paper, atomic absorption spectrometry (AAS) was used to measure the platinum drug uptake in tumor tissues. Figure 6 The drug uptake data in Fig. 7e are basically consistent with the anti-tumor ability of the drugs. The uptake levels of compound 1 and nanodrugs GEVs@Pt(IV) and Tf-GEVs@Pt(IV) in tumors were significantly higher than those of CDDP and CDDP+6 (P < 0.01 or P < 0.001). Notably, Tf-GEVs@Pt(IV) had a higher uptake in tumors than free compound 1 and GEVs@Pt(IV) (P < 0.001), which can be mainly attributed to its tumor targeting property. This indicates that the uptake of drugs in tumors has a great influence on the anti-tumor activity of the target nanodrugs in vivo. Body weight changes during the experiment Figure 6 b) and H&E staining Figure 7 are effective indicators for evaluating drug toxicity. The results show that compound 1 and its nanodrugs GEVs@Pt(IV) and Tf-GEVs@Pt(IV) have no significant toxicity in vivo. Compared with blank, these drugs do not cause significant weight loss in mice, and their toxicity is lower than that of CDDP (P < 0.001). No significant histological differences were observed in liver, spleen and kidney tissues.

[0143] Therefore, the construction of aspirin-captopril tri-functional quadruplex platinum compound 1 by adding CTP-ASP ligands significantly improves the anti-tumor efficacy and reduces the in vivo toxicity. Then, by using GEVs as the carrier and Tf as the tumor targeting group, the nanodrug Tf-GEVs@Pt(IV) is prepared, which further improves the accumulation of aspirin-captopril tri-functional quadruplex platinum compound 1 in tumors and enhances its anti-tumor efficacy.

[0144] 3. Anti-metastatic activity in vitro and in vivo

[0145] Transwell assay: The in vitro anti-metastatic properties of drugs were determined using 24-well Transwell plates (Costar 3422) containing chambers (8 μm pore size). Briefly, 4T1 cells were serum-deprived for 48 h and then suspended in RPMI1640 medium (5 × 10 4 , 0.2 mL) was added to the upper chamber. 0.6 mL of culture medium containing 10% FBS and different drugs was added to the lower chamber, and the concentrations of each drug were set to: CDDP (5 μM), CDDP+6 (5 μM / 10 μM), compound 1 (5 μM), GEVs@Pt(IV) (5 μM) and Tf-GEVs@Pt(IV) (5 μM), and PBS and GEVs were set as control groups. After 24 hours of cell culture, the cells were fixed with 4% paraformaldehyde for 20 minutes. Subsequently, the cells were stained with 0.1% crystal violet for 20 minutes. Gently scrape off the non-migrated cells in the upper chamber. Use an inverted microscope to photograph the migrating cells on the lower surface and calculate the migration rate (5 fields of view).

[0146] Scratch test: The anti-metastatic properties of the drug in vitro were further determined by the scratch test. 4T1 cells deprived of serum for 48 h were plated at 4×10 5 The cells were added to a six-well plate at a density of 100 μg / mL and incubated overnight in RPMI1640 medium containing 1% FBS. When the cells reached a density of 90%, scratches were made. Afterwards, the culture medium and dead cells were removed, and new culture medium containing CDDP (5 μM), CDDP+6 (5 μM / 10 μM), compound 1 (5 μM), GEVs@Pt(IV) (5 μM) and Tf-GEVs@Pt(IV) (5 μM) was added, and PBS and GEVs were used as controls. The cells were then cultured for another 24 hours, and the scratches of the cells were photographed using an inverted microscope at 0, 12, and 24 hours.

[0147] In vivo anti-metastasis experiments were conducted on female BALB / c mice (18-20 g). Tumor cells (1×10 5 , 0.2mL PBS) to establish a lung metastasis model. The mice were divided into seven groups (blank, CDDP, CDDP+6, 1, GEVs, GEVs@Pt(IV) and Tf-GEVs@Pt(IV), n=5). The mice were then intravenously injected with drugs through the tail vein on days 4, 7, and 10 at a dose of 2mg Pt / kg. The dosage of GEVs was the same as that of the nanodrug. On day 12, the mice were killed, lung tissues were collected, and the number of nodules in each group of mice was counted. The metastatic nodules inside the lung tissue were then observed using H&E staining.

[0148] Experimental results: Transwell assay showed that compound 1 could effectively inhibit the migration of tumor cells in vitro, and its effect was significantly better than that of CDDP and CDDP+6 (P<0.001) ( Figure 8 ). Then, the candidate compound was converted into nanomedicine GEVs@Pt(IV) and Tf-GEVs@Pt(IV), which further reduced the metastasis rate of tumor cells (P<0.001). Subsequently, the anti-metastatic effects of tetravalent platinum compound 1, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) were further verified in the scratch healing experiment ( Figure 9 ), compared with the blank, CDDP, CDDP+6, and GEVs groups, compound 1, GEVs@Pt(IV), and Tf-GEVs@Pt(IV) were more effective in inhibiting the scratch healing of tumor cells. This demonstrates that the trifunctional tetravalent platinum compound 1 and its nanoparticles GEVs@Pt(IV) and Tf-GEVs@Pt(IV) have the ability to inhibit tumor cell metastasis in vitro.

[0149] In vivo tumor lung metastasis model experiments showed that ( Figure 10 ), compound 1, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) showed significant inhibitory effects on 4T1 lung metastatic tumors in vivo. It is worth noting that the tetravalent platinum compound 1 has better activity than cisplatin and the mixture CDDP+6, which indicates that the introduction of the functional ligand CTP-ASP into the tetravalent platinum system to form an integral molecule is crucial for improving the anti-metastatic properties. In addition, GEVs also showed certain anti-metastatic activity in vivo (inhibition rate = 51.7%), which indicates that GEVs can not only serve as a carrier of active ingredient 1, but also participate in the inhibition of tumor metastasis, which further explains the excellent anti-metastatic properties of nanomedicines. Moreover, it can be found that the nanomedicines GEVs@Pt(IV) (inhibition rate = 85.6%) and Tf-GEVs@Pt(IV) (inhibition rate = 88.1%) showed more significant anti-metastatic activity than the free complex 1 (inhibition rate 72.6%, P < 0.001). Therefore, grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compounds are a promising class of anti-tumor metastasis drugs.

[0150] 4. RNA Sequencing

[0151] RNA sequencing (RNA-seq) was outsourced to GenScript Biotech Co., Ltd. (Beijing, China). Freshly frozen tumor samples from the GEVs, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) treatment groups in the anti-tumor proliferation experiment were selected to collect total RNA. Library preparation was performed according to the Illumina standard instructions (VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina). The concentration and distribution of the cDNA library were evaluated using an Agilent 4200 bioanalyzer, and sequencing was performed using an Illumina novaseq6000.

[0152] Experimental results: RNA sequence analysis was performed on the tumor tissues of the blank, GEVs and Tf-GEVs@Pt(IV) groups to analyze the potential anti-tumor mechanism. Figure 11 ) Compared with the blank (1952) and GEVs group (1860), Tf-GEVs@Pt(IV) induced a large number of differentially expressed genes (DEGs) in tumor cells. In addition, 1241 DEGs were present in both the Blank vs Tf-GEVs@Pt(IV) and GEVs vs Tf-GEVs@Pt(IV) groups. Subsequently, we performed KEGG, GO and GSEA analysis on Blank vs Tf-GEVs@Pt(IV) and found that DEGs were mainly involved in DNA damage and replication regulation, apoptosis, inflammation, tumor adhesion proteins and migration, immunology and tumor microenvironment pathways.

[0153] 5. DNA damage effect

[0154] Uptake experiment: The drug uptake of tumor cells or tissues was determined by the AAS method to detect the concentration of platinum: 4T1 cells (6 x 10 6 ) were placed in a 10 cm culture dish, then CDDP, CDDP+6, 1, 2, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) with a concentration of 5 μM were added, and incubated for 24 h. After that, the cells were collected and washed with PBS three times. Then the DNA, membrane and cytoplasm in the tumor cells were separated with a separation kit (Bi Yun Tian) according to the procedure. After digestion with 70% HNO3, the concentration of platinum was determined by AAS method.

[0155] DNA damage: The expression of DNA damage marker proteins γ-H2AX and P53 was detected by western blot method to detect DNA damage.

[0156] Experimental results: GO enrichment analysis of DEGs in tumor tissues showed that Tf-GEVs@Pt(IV) had a significant impact on cellular components (CC): cytoplasmic ribosomes; biological processes (BP): DNA replication, deaminated base formation, and DNA chain elongation processes. GSEA further confirmed that DNA replication and base excision repair genes were significantly affected Figure 12 a-b). These facts indicate that Tf-GEVs@Pt(IV) significantly regulates genes involved in DNA damage, which is mainly attributed to the active tetravalent platinum component 1.

[0157] The uptake of platinum drugs in the nucleus directly affects their ability to damage DNA, so the sub-distribution of platinum drugs in 4T1 cells was determined by AAS method Figure 13 ). The trifunctional tetravalent platinum compound 1 and its nanodrugs GEVs@Pt(IV) and Tf-GEVs@Pt(IV) showed excellent anti-tumor performance, and their accumulation levels in DNA were higher than those of the reference drugs CDDP and CDDP+6 (P<0.01). In addition, compared with free compound 1 and nanodrug GEVs@Pt(IV), the nanoparticles Tf-GEVs@Pt(IV) with tumor targeting groups Tf have higher accumulation in DNA, which may be related to the improvement of their tumor targeting ability.

[0158] The expression of DNA damage protein indicators γ-H2AX and P53 was also evaluated by Western blot Figure 12 c-d). Compared with blank, compound 1, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) can effectively increase the expression of γ-H2AX and P53 (P<0.001), and the trend is similar to that of the reference drugs CDDP and CDDP+6.

[0159] In summary, nanodrug Tf-GEVs@Pt(IV) can effectively induce DNA damage and up-regulate the expression of γ-H2AX and P53, which is mainly due to the active component 1 of trifunctional tetravalent platinum.

[0160] 6. Mitochondria-mediated apoptosis

[0161] The change of mitochondrial membrane potential after drug treatment was determined by flow cytometry using JC-1 staining kit (Solarbio). Briefly, 4×10 54T1 cells were seeded in six-well plates and pre-cultured overnight. Then CDDP (5 mM), CDDP + 6 (5 mM / 10 mM), 1 (5 mM), GEVs@Pt(IV) (5 mM) and Tf-GEVs@Pt(IV) (5 mM) were added, with PBS and GEVs as controls, and the cells were cultured for 24 h. Then, the cells were collected and stained with JC-1. Finally, flow cytometry was used for analysis.

[0162] Annexin V-FITC / PI staining kit (Bi Yun Tian) was used to detect apoptosis after drug treatment by flow cytometry. Briefly, 4 x 10 5 4T1 cells were seeded in six-well plates and pre-cultured overnight. Then CDDP (5 mM), CDDP + 6 (5 mM / 10 mM), 1 (5 mM), GEVs@Pt(IV) (5 mM) and Tf-GEVs@Pt(IV) (5 mM) were added, with PBS and GEVs as controls, and the cells were cultured for 24 h. Then, the cells were collected and stained with Annexin V-FITC / PI. Finally, flow cytometry was used for analysis.

[0163] Apoptosis is a major way of killing tumor cells by platinum-based chemotherapy drugs, which is closely related to mitochondrial damage. In this paper, the properties of the prepared agents in causing mitochondrial-mediated apoptosis were studied from different aspects. GO results showed that, compared with blank, the genes related to apoptosis cell clearance (BP), mitochondria, mitochondrial inner membrane, mitochondrial ribosome regulation (CC) were significantly changed in tumors treated with Tf-GEVs@Pt(IV). And GSEA also confirmed that the pathways related to cell apoptosis and cell cycle were activated Figure 14 a-b). Figure 14 The apoptosis induction results in c showed that the proportion of apoptosis caused by nanodrug Tf-GEVs@Pt(IV) (38.0%) was higher than that of free compound 1 (12.5%), GEVs@Pt(IV) without Tf ligand (22.7%), and reference drugs CDDP (10.1%), CDDP + 6 (10.2%) and GEVs (2.9%). This trend was basically consistent with the anti-tumor activity in vitro and in vivo. Then, JC-1 test further verified their role in damaging mitochondria Figure 15 ). Mitochondrial membrane potential collapse (ΔΨm) was observed in tumor cells treated with Tf-GEVs@Pt(IV), with a proportion greater than that of compound 1, GEVs@Pt(IV), CDDP, CDDP + 6 and GEVs. This indicates that the prepared nanodrug Tf-GEVs@Pt(IV) can effectively cause mitochondrial-mediated apoptosis, providing strong evidence for its anti-tumor effect.

[0164] The Bcl-2 pathway is a key pathway that regulates the process of apoptosis, so we determined the expression of Bcl-2 pathway proteins by Western blot. As shown in Fig. 9d-e, the anti-apoptotic protein Bcl-2 was significantly down-regulated (P < 0.001), while the pro-apoptotic protein Bax was up-regulated (P < 0.001). In addition, the expression of apoptosis execution protein c-caspase-3 was increased, and the ratio of c-caspase-3 / caspase-3 was significantly increased (P < 0.001) compared with the control group. Therefore, nanodrug Tf-GEVs@Pt(IV) can induce tumor cell apoptosis through mitochondrial-mediated Bcl-2 / Bax / caspase-3 signaling, thereby effectively inhibiting tumor proliferation. Figure 14 d-e, the anti-apoptotic protein Bcl-2 was significantly down-regulated (P < 0.001), while the pro-apoptotic protein Bax was up-regulated (P < 0.001). In addition, the expression of apoptosis execution protein c-caspase-3 was increased, and the ratio of c-caspase-3 / caspase-3 was significantly increased (P < 0.001) compared with the control group. Therefore, nanodrug Tf-GEVs@Pt(IV) can induce tumor cell apoptosis through mitochondrial-mediated Bcl-2 / Bax / caspase-3 signaling, thereby effectively inhibiting tumor proliferation.

[0165] 7. Reduce tumor fibrosis

[0166] Immunohistochemistry, picrosirius and Masson staining: Tumor tissues from blank, CDDP group, CDDP+6 group, compound 1 group, GEVs group, GEVs@Pt(IV) group and Tf-GEVs@Pt(IV) group were obtained from in vivo anti-tumor growth test. These tumor tissues were paraffin-embedded and cut into 5 μm sections. Then, the sections were deparaffinized, hydrated, and incubated with the primary antibody at 4°C overnight. The sections were treated with secondary antibody at room temperature for 50 min. Color development was performed with 3,3'-diaminobenzidine (DAB) and co-stained with hematoxylin. Finally, the sections were photographed with an inverted microscope, and the expression of proteins was quantified with Image J according to randomly selected five fields of view.

[0167] Tumor fibrosis is closely related to extracellular matrix ECM, which further affects the adhesion, invasion, migration and metastasis of tumor cells. It has been proven that CTP can effectively inhibit tumor fibrosis. In this paper, the effects of trifunctional tetravalent platinum compound 1, nanodrug GEVs@Pt(IV) and Tf-GEVs@Pt(IV) on tumor fibrosis were evaluated by immunohistochemistry, picrosirius and Masson staining.

[0168] GO enrichment results showed that Tf-GEVs@Pt(IV) caused significant changes in genes related to tumor cell fibrosis, adhesion and migration, while GSEA analysis showed that ECM-receptor interaction and cell adhesion molecule-related genes were also affected Figure 16 a-b). Picrosirius staining and Masson staining results Figure 16c-d) show that Tf-GEVs@Pt(IV), GEVs@Pt(IV) and Compound 1 can significantly reduce the level of collagen fibrosis in tumor sections compared with the blank group (P < 0.001), while the effect of the bivalent platinum drug is minimal. In addition, the expression of the proteins a-SMA and FSP1 related to collagen fibrosis is also inhibited (P < 0.001), which indicates that Compound 1 and its nanodrugs Tf-GEVs@Pt(IV) and GEVs@Pt(IV) have significant efficacy in inhibiting tumor fibrosis.

[0169] Protein TGF-β is a key protein that induces ECM fibrosis. As key enzymes that regulate ECM, MMPs play a synergistic role with TGF-β in promoting tumor metastasis and fibrosis. CTP can effectively reduce the deposition of ECM fibrosis by blocking the TGF-β1 and MMPs pathways. Therefore, we detected the expression of TGF-β1, MMP2 and MMP9 proteins by Western blot and immunohistochemical staining. Figure 17 a-b show that Tf-GEVs@Pt(IV) can inhibit the expression of TGF-β1, MMP2 and MMP9 in tumor cells compared with the blank (P < 0.001), which is more effective than free Compound 1 (P < 0.001). These inhibitory effects were also observed in the in vivo anti-tumor experiment Figure 17 c-d).

[0170] Accordingly, trifunctional quaternary platinum Compound 1 and its nanodrugs GEVs@Pt(IV) and Tf-GEVs@Pt(IV) can reduce tumor fibrosis by inhibiting key enzymes TGF-β1, MMP2 and MMP9, and further exert good anti-tumor metastasis activity in vitro and in vivo.

[0171] 8. Reversing inflammatory TME

[0172] Chronic inflammation is a hallmark of cancer and is a key factor in promoting the formation of TME. Tumor inflammation is closely related to the high expression of key enzyme COX-2 and the secretion of inflammatory cytokines such as TNF-α and IL-6. The introduction of ASP into trifunctional quaternary platinum compounds is expected to obtain drugs with anti-inflammatory properties. This paper studies the effect of the target drug on tumor inflammation.

[0173] GO and GSEA analysis based on RNA-seq data Figure 18 a-b) show that Tf-GEVs@Pt(IV) has a significant impact on tumor inflammatory response and cytokine secretion compared with the blank. Subsequent Western blot and immunohistochemical staining detection Figure 18c-d, f-g) show that Tf-GEVs@Pt(IV) effectively inhibited the expression of the key enzyme COX-2 (P < 0.001) of inflammation. Western blot and Elisa detection results further showed that Figure 18 e) the secretion of inflammatory cytokines TNF-a and IL-6 in tumor cells in vitro and tumor tissues in vivo was significantly inhibited (P < 0.001). Notably, the blank vector GEVs can also effectively alleviate tumor inflammation compared with the blank. Therefore, the nanodrug Tf-GEVs@Pt(IV) effectively inhibits tumor inflammation by inhibiting COX-2, TNF-a and IL-6, which is mainly attributed to the synergistic effect of functional group ASP and exosome GEVs.

[0174] 9. Reversing TME with immunosuppressive effect

[0175] Immune suppression is considered another feature of cancer. The presence of fibrosis in tumors constitutes an immunosuppressive barrier to T cell infiltration; while inflammatory TME is also an important driver of immune suppression. Given the role of the target drug in inhibiting tumor fibrosis and tumor inflammation, we investigated its activation effect on immunity.

[0176] RNA-seq analysis results Figure 19 a-b) show that Tf-GEVs@Pt(IV) can effectively regulate genes related to immune response, T cell activation and macrophage activation. In addition, the key checkpoint PD-L1 / PD-1 and cGAS / STING pathway genes closely related to immunity are also significantly affected. Subsequently, immunohistochemical staining of T cells and macrophages in tumor tissues confirmed the significant ability of Tf-GEVs@Pt(IV) in promoting anti-tumor immunity Figure 19 e-f). Compared with the blank, the Tf-GEVs@Pt(IV) treatment group significantly increased the number of CD3 + and CD8 + T cells by 22.7 times and 20.8 times (P < 0.001) compared with the blank group. At the same time, CD206 + M2 type macrophages that promote tumor growth decreased by 15% (P < 0.001), and CD86 + M1 type macrophages that kill tumors increased by 1911% (P < 0.001), indicating that Tf-GEVs@Pt(IV) treatment promoted the polarization of macrophages from M2 type to M1 type in tumors. We further carried out mechanism research by western blot and immunohistochemical staining Figure 19c-f). The immune checkpoint PD-L1 was significantly inhibited in both tumor cells in vitro (P < 0.001) and tumor tissues in vivo (P < 0.001). Subsequently, the dsDNA released after DNA damage of the bivalent platinum compound activated the cGAS / STING pathway, and the expressions of STING, p-STING, TBK1, p-TBK1, IFR3, p-IFR3 in 4T1 cells were also improved (P < 0.001), which proved that the cGAS / STING pathway was activated and promoted immunity during the treatment of Tf-GEVs@Pt(IV). Notably, the free compound 1 also had a similar trend of immune activation in vitro and in vivo. In summary, the nanodrug Tf-GEVs@Pt(IV) could effectively activate anti-tumor immunity by activating the cGAS / STING pathway, inhibiting the expression of PD-L1, and activating T cell immunity and inducing macrophages to polarize from M2 to M1.

[0177] 10. Mechanism of action

[0178] According to the above RNA-seq and experimental results, the mechanism of action of Tf-GEVs@Pt(IV) was drawn Figure 20 The nanodrug with GEVs as carriers can effectively penetrate the fibrotic barrier to reach the tumor tissue; the targeting group Tf significantly improves its tumor targeting performance. Then, the active ingredient 1 is reduced in the reducing TME and further releases bivalent platinum, CTP and ASP fragments. The bivalent platinum compound binds to DNA, causing severe DNA damage and further increasing the expression of γ-H2AX and p53. Subsequently, mitochondrial-mediated apoptosis is activated by initiating the Bcl-2 / Bax / caspase3 pathway. CTP inhibits the TGF-β1 and MMPs (MMP2, MMP9) pathways, reduces the levels of α-SMA and FSP1, and inhibits the fibrosis of the extracellular matrix in the tumor tissue. Subsequently, the fibrotic barrier around the tumor is broken, promoting the penetration of drugs and immune cells to the tumor. After Tf-GEVs@Pt(IV) treatment, the inflammatory TME is significantly reversed, which is mainly due to the synergistic effect of ASP and GEVs, which inhibits the secretion of key enzymes COX-2 and inflammatory cytokines TNF-α and IL-6. In addition, with the inhibition of fibrosis and inflammatory TME, tumor immunity is activated, the immune checkpoint PD-L1 is blocked, and the key pathway cGAS / STING is activated; then, CD3+ and CD8+ T cells are activated, and macrophages are also polarized from M2 to M1. The development of the nanodrug with multiple anti-tumor mechanisms of grapefruit extracellular vesicles loaded with a three-functional bivalent platinum compound provides a new strategy for designing new and powerful bivalent platinum anti-tumor drugs in the future.

[0179] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A trifunctional tetravalent platinum compound containing a captopril-aspirin ligand, the general structural formula of which is shown in (I): in, Selected from cisplatin or oxaliplatin; L is hydroxyl or 2. The trifunctional tetravalent platinum compound containing a captopril-aspirin ligand according to claim 1, wherein: The trifunctional tetravalent platinum compound is a monosubstituted trifunctional tetravalent platinum compound Ia or a disubstituted trifunctional tetravalent platinum compound Ib, and its structural formula is as follows:

3. A bifunctional tetravalent platinum compound containing a captopril ligand, wherein the compound has the general structural formula (III) shown below: in, Selected from cisplatin or oxaliplatin.

4. A grapefruit extracellular vesicle-loaded nanomedicine comprising a trifunctional tetravalent platinum compound, characterized in that: The nanomedicine is prepared from a trifunctional tetravalent platinum compound as an active ingredient, a carrier grapefruit extracellular vesicle GEVs, a carrier DSPE-PEG, and a targeting carrier DSPE-PEG-Tf; the mass ratio of the trifunctional tetravalent platinum compound, the carrier DSPE-PEG, the carrier GEVs, and the targeting carrier DSPE-PEG-Tf in the nanomedicine is 1:(0.1-100):(0.1-100):(0.1-100); wherein the trifunctional tetravalent platinum compound is the trifunctional tetravalent platinum compound according to claim 1 or 2.

5. The method for preparing the grapefruit extracellular vesicle-loaded nanomedicine containing trifunctional tetravalent platinum compounds according to claim 4, characterized in that: The anti-solvent precipitation method combined with the ultrasonic method is adopted, and the specific steps are as follows: (1) Dispersing a trifunctional tetravalent platinum compound and DSPE-PEG in DMF at a mass ratio of 1:(0.1-100) and injecting the mixture into PBS under ultrasonic conditions; further stirring the mixture for 5-60 minutes; homogenizing under high pressure for 5-120 minutes, and dialyzing the solution against PBS for 1-5 times to obtain DSPE-PEG-encapsulated tetravalent platinum drug nanoparticles; (2) The DSPE-PEG-encapsulated tetravalent platinum drug nanoparticles and carrier GEVs were mixed at a mass ratio of 1:(0.1-100) and incubated at 0-40°C for 1-300 minutes under ultrasonic conditions of 100-1000W to obtain non-targeted grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound nanoparticles; (3) The above-mentioned non-targeted grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound nanodrugs are mixed with the targeting carrier DSPE-PEG-Tf at a mass ratio of trifunctional tetravalent platinum compound to targeting carrier DSPE-PEG-Tf of 1:(0.1-100), and incubated in a 0-50°C water bath for 0.1-5 hours to obtain transferrin-modified tumor-targeted grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound nanodrugs.

6. Use of a trifunctional tetravalent platinum compound containing a captopril-aspirin ligand according to any one of claims 1 to 2 in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs, characterized in that: The anti-tumor proliferation is anti-human lung adenocarcinoma, anti-cisplatin-resistant human lung adenocarcinoma, anti-human liver cancer or anti-mouse breast cancer; the anti-tumor metastasis is anti-mouse breast cancer metastasis.

7. Use of the bifunctional tetravalent platinum compound of the captopril ligand according to claim 3 in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs, characterized in that: The anti-tumor proliferation is anti-human lung adenocarcinoma, anti-cisplatin-resistant human lung adenocarcinoma, anti-human liver cancer or anti-mouse breast cancer; the anti-tumor metastasis is anti-mouse breast cancer metastasis.

8. Use of the grapefruit extracellular vesicle-loaded trifunctional tetravalent platinum compound nanomedicine according to claim 4 in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs, characterized in that: The anti-tumor proliferation is anti-human lung adenocarcinoma, anti-cisplatin-resistant human lung adenocarcinoma, anti-human liver cancer or anti-mouse breast cancer; the anti-tumor metastasis is anti-mouse breast cancer metastasis.

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