Nano-drug of grapefruit extracellular vesicle loaded trifunctional tetravalent platinum compound as well as preparation method and application of nano-drug

Abstract

The invention provides a trifunctional tetravalent platinum compound of a captopril-aspirin ligand and a reference drug captopril difunctional tetravalent platinum compound of the trifunctional tetravalent platinum compound. The invention also develops a nano-drug Tf-GEVs (at) Pt (IV) of an aspirin-captopril three-function tetravalent platinum compound entrapped by grapefruit extracellular vesicles (GEVs). The Tf-GEVs (at) Pt (IV) is prepared from a trifunctional tetravalent platinum compound serving as an active component, grapefruit extracellular vesicles (GEVs) serving as a carrier, DSPE-PEG serving as a carrier, and a transferrin modified targeting carrier DSPE-PEG-Tf. The Tf-GEVs (at) Pt (IV) can release an active component, namely a trifunctional tetravalent platinum compound in vivo. The trifunctional tetravalent platinum compound as a nano-drug active component can cause DNA damage, and can also effectively regulate inflammatory, fibrosis and immunosuppressive TME. The nano-drug is remarkable in anti-tumor capacity, has an excellent treatment effect on metastatic malignant tumors, is expected to provide a new candidate drug for clinical treatment of tumors, and provides a new direction for research and development of novel platinum drugs and anti-metastatic malignant tumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a nanomedicine based on grapefruit extracellular vesicles (GEVs) loaded with a trifunctional tetravalent platinum conjugate, a preparation method thereof, and an application thereof in tumor treatment drugs. Background Art

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

[0003] Platinum drugs are the most representative metal 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 to overcome the above shortcomings by introducing a variety of functional ligands in the axial position to construct new platinum drugs. Aspirin (ASP) is a non-steroidal anti-inflammatory drug with great potential in inhibiting TAI. Captopril (CTP), as an effective angiotensin-converting enzyme (ACE) inhibitor, has been shown to effectively inhibit fibrosis by downregulating MMPs and TGF-β1. In view of this, the present invention uses CTP-ASP as a functional ligand to introduce a tetravalent platinum system and develops a series of novel trifunctional tetravalent platinum conjugates. These conjugates can inhibit fibrosis and inflammatory TME, damage DNA and further reverse immunosuppressive TME, thereby showing strong anti-tumor activity.

[0004] Tumor-targeted drugs are a hot topic in new drug development. The use of nanodrug 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 risks, a wide range of sources, and low costs. More importantly, they can easily cross the fibrotic barrier of tumors. Recently, some PDVs-based drugs have been developed and entered the clinical trial stage. However, the application of PDVs loaded with quadrivalent platinum drugs has not been studied and deserves further discussion.

[0005] Grapefruit-derived exosomes (GEVs), as a typical type of PDVs, show superiority in anti-tumor cells and have low toxicity. GEVs can also effectively inhibit inflammation by reducing the expression of pro-inflammatory factors. Therefore, the study 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 efficacy. Despite this, the poor targeting of GEVs nanomedicines is still a restrictive factor in its development. Modifying GEVs is an effective strategy to enhance tumor targeting properties. Tumor cells have a high demand for iron for unlimited proliferation and overexpress transferrin receptor (TfR) on their surface. Transferrin (Tf) has good tumor targeting ability and is often used for nano-drug modification. So far, the modification of PDVs by Tf has not been studied. Therefore, the present invention designs and develops a Tf-modified GEVs system for the first time, hoping to improve the tumor targeting properties of anti-tumor drugs. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention designs and develops a trifunctional tetravalent platinum compound with captopril-aspirin ligands and a bifunctional tetravalent platinum compound of captopril; develops a nanomedicine GEVs@Pt(IV) containing aspirin-captopril trifunctional tetravalent platinum compound encapsulated in grapefruit extracellular vesicles (GEVs) and a nanomedicine Tf-GEVs@Pt(IV) modified with transferrin, a tumor targeting group. As an active component of nanomedicine, the trifunctional tetravalent platinum compound can not only cause DNA damage, but also effectively regulate inflammatory, fibrotic and immunosuppressive TME; GEVs in nanomedicines promote the tumor tissue permeability of the drug; transferrin (Tf) enhances its tumor targeting properties and improves the anti-tumor activity of the active component. Through in vivo and in vitro test methods, the anti-tumor proliferation and anti-metastasis effects of the target compound were verified, and the anti-tumor mechanism of the drug was studied. The results confirmed that this type of drug has significant anti-tumor ability and excellent therapeutic effect on metastatic malignant tumors. It is expected to provide new candidate drugs for the clinical treatment of tumors and provide a new direction for the research and development of new platinum drugs and anti-metastatic malignant tumor drugs.

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

[0008]

[0009] in, Selected from cisplatin or oxaliplatin; L is hydroxyl or

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

[0011]

[0012] Furthermore, the trifunctional tetravalent platinum compound of the captopril-aspirin ligand of the present invention is selected from compounds 1 and 2:

[0013]

[0014] Another object of the present invention is to provide a method for preparing a trifunctional tetravalent platinum compound of a captopril-aspirin ligand as shown in general formula (I). The synthesis route of the compound is shown below.

[0015] The synthesis route of Ia and Ib is as follows:

[0016]

[0017] Compound II and compound 6 undergo coupling reaction to prepare trifunctional tetravalent platinum derivatives Ia or Ib. When the molar ratio of compound II to compound 6 is 1:1.0-1.5, an asymmetric monosubstituted trifunctional modified tetravalent platinum compound Ia is obtained; when the molar ratio of compound II to compound 6 is 1:2.0-5.0, a symmetric disubstituted trifunctional modified tetravalent platinum compound Ib is obtained.

[0018] Furthermore, in the synthetic route 1, the preparation steps of the trifunctional tetravalent platinum compound of captopril-aspirin ligand are:

[0019] Under an inert gas atmosphere, compound 6, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent for reaction, compound II is added, and after the reaction is protected from light, a trifunctional tetravalent platinum derivative I is separated through post-treatment;

[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] Furthermore, the inert gas is nitrogen, helium or argon; the condensing agent is TBTU, HATU or EDCI; the organic base is triethylamine, N,N-diisopropylethylamine or 4-dimethylaminopyridine; and the organic solvent is DMF or DMSO.

[0022] The preparation process can be specifically as follows: adding a condensing agent and compound 6 to a reaction vessel, replacing the air in the system with an inert gas, adding an anhydrous organic solvent, and then adding an anhydrous organic base to the reaction system, stirring the reaction for 5-60 minutes at room temperature, and then adding a tetravalent platinum compound II to the reaction system, replacing the air in the system with an inert gas again, and placing the reaction system at 25-120° C. in the dark for 24-72 hours. After the reaction is completed, the solvent is removed under reduced pressure, and column chromatography is performed to obtain an asymmetric monosubstituted trifunctional modified tetravalent platinum compound Ia, or a symmetric disubstituted trifunctional modified tetravalent platinum compound Ib.

[0023] Furthermore, in the synthetic route, the preparation steps of compound 6 are:

[0024]

[0025] Preparation method of compound 6: 1H-benzotriazole is dissolved in dichloromethane, thionyl chloride is added under ice bath, and stirring is continued at room temperature. Aspirin is added, and the mixture is stirred at room temperature. Then, the suspension is filtered, and the organic solution is washed with saturated Na2CO3 solution; then, hexane is added to precipitate white solid compound 5. Compound 5 is dissolved in acetonitrile, an aqueous solution containing CTP and triethylamine are added, and the mixture is stirred. Acetonitrile is removed under reduced pressure; the residue is extracted with ethyl acetate, and evaporated to obtain white solid compound 6.

[0026] Specific method for preparing compound 6: 1H-benzotriazole (H-Bt, 28g, 240mmol) was dissolved in 200mL of dichloromethane, thionyl chloride (4mL, 60mmol) was added under ice bath, and stirring was continued at room temperature for 15min. Aspirin (10.8g, 60mmol) was added, and the mixture was stirred at room temperature for 2.5h. Then, the suspension was filtered, and the organic solution was washed with saturated Na2CO3 solution; then, hexane (200mL) was added to precipitate white solid compound 5 (6g, 50%). Compound 5 (0.65g, 2.3mmol) was dissolved in acetonitrile (20mL), 10mL of aqueous solution containing CTP (0.5g, 2.3mmol) and triethylamine (0.23g, 2.3mmol) were added, and the mixture was stirred for 20h. Acetonitrile was removed under reduced pressure; the residue was extracted with ethyl acetate, and white solid compound 6 (0.31g, 35.6%) was obtained after evaporation.

[0027] Further, the compound II is composed of After being oxidized by hydrogen peroxide, the product is prepared. The specific preparation process is as follows:

[0028]

[0029] Divalent platinum compounds The dihydroxy tetravalent platinum compound II is prepared by oxidizing with hydrogen peroxide at 60-70°C for 1-8h.

[0030] In order to achieve another object of the present invention, we designed and prepared a captopril bifunctional tetravalent platinum compound III. Its structure is:

[0031]

[0032] in, Selected from cisplatin or oxaliplatin.

[0033] Preferably, the captopril bifunctional tetravalent platinum derivative III of the present invention is selected from compound 3:

[0034]

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

[0036]

[0037] Compound II undergoes a coupling reaction with acetyl captopril compound 7 to obtain a symmetrical bifunctional captopril tetravalent platinum compound III; wherein the molar ratio of compound II to compound 7 is 1:2.0-5.0.

[0038] Furthermore, in the synthetic route 3, the preparation steps of the bifunctional captopril tetravalent platinum compound III are:

[0039] Under an inert gas atmosphere, compound 7, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent for reaction, compound II is added, and after the reaction is protected from light, a bifunctional captopril tetravalent platinum compound III is separated through post-treatment;

[0040] Among them, the feeding molar ratio of compound II, compound 7, condensation agent and organic base is 1:2.0-5.0:2.0-5.0:2.0-5.0; the feeding relationship between compound II and organic solvent is that 10-100 ml of organic solvent is added for every 1 g of compound II.

[0041] Furthermore, the inert gas is nitrogen, helium or argon; the condensing agent is TBTU, HATU or EDCI; the organic base is triethylamine, N,N-diisopropylethylamine or 4-dimethylaminopyridine; and the organic solvent is DMF or DMSO.

[0042] The preparation process can be specifically as follows: first, a condensing agent and compound 7 are added to the reaction vessel, then the air in the system is replaced with an inert gas, then an anhydrous organic solvent is added, and then 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 to 120°C in the dark for 24 to 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 adjuvants thereof.

[0044] The pharmaceutically acceptable excipients of the present invention include one or more carriers, excipients, diluents, etc., such as various types of adhesives. The excipients can be aqueous or non-aqueous, and conventional ones include gelatin and other colloids, corn starch and other starches, lactose and other sugars, sodium carboxymethyl cellulose and other cellulose materials and mixtures thereof. It also includes tragacanth powder, etc., and oils, alcohols, esters, buffers, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, etc. can also be used as excipients. The trifunctional tetravalent platinum compound or pharmaceutical composition of the present invention has a variety of dosage forms such as tablets, and there are also sustained-release, controlled-release or nano-preparations prepared according to common knowledge of pharmacy.

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

[0046] The administration route of the trifunctional tetravalent platinum compound of the present invention can be intravenous administration, including intravenous injection, intramuscular injection, intratumor injection, subcutaneous injection and acupoint injection.

[0047] The method of preparing the active ingredient into a drug in the present invention can be prepared by methods known to ordinary technicians in the field, 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 the general formula (I) or the 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] Furthermore, the anti-tumor is anti-lung cancer, anti-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 invention also provides a combined preparation, comprising a compound or pharmaceutical composition as shown in general formula (I) or general formula (III), and an anti-tumor drug of the paclitaxel type, fluorouracil type, gemcitabine type, vinca alkaloid type, or antibody type.

[0052] The trifunctional derivative tetravalent platinum compound of the present invention can be used alone or in combination with a variety of marketed drugs to prepare anti-tumor combined preparations, including platinum, paclitaxel, etc. The combined preparations have various dosage forms such as tablets, capsules, injections, etc.

[0053] In order to achieve another object of the present invention, the technical scheme of the present invention also includes the preparation of a nanomedicine of a trifunctional tetravalent platinum compound loaded with grapefruit extracellular vesicles: the nanomedicine is prepared from a trifunctional tetravalent platinum derivative I as an active ingredient, a carrier DSPE-PEG, a carrier grapefruit extracellular vesicle GEVs, and a 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] Furthermore, the preparation method of the nano drug is to use an anti-solvent precipitation method combined with an ultrasonic method, which includes the following specific steps:

[0055] (1) dispersing a tetravalent platinum derivative (I) and DSPE-PEG in DMF at a ratio of 1:(0.1-100), and injecting into PBS at 1-20° C. and 100-1000W ultrasonic conditions; 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-encapsulated tetravalent platinum drug nanoparticles;

[0056] (2) The above-mentioned DSPE-PEG-encapsulated tetravalent platinum drug and carrier GEVs are incubated at a ratio of 1: (0.1-100) [I: GEVs, (w / w)] under ultrasonic conditions of 100-1000W at 0-40°C for 1-300 minutes to obtain a non-targeted grapefruit extracellular vesicle-loaded trifunctional tetravalent platinum compound nanodrug.

[0057] (3) The above-mentioned non-targeted grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound nanodrug and the targeting carrier DSPE-PEG-Tf are incubated in a 0-50°C water bath for 0.1-5 hours at a ratio of 1:(0.1-100) [I:DSPE-PEG-Tf, (w / w)] to obtain transferrin-modified targeted grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound nanodrug.

[0058] The specific preparation process is as follows: (1) the trifunctional tetravalent platinum derivative 1 (20 mg) and DSPE-PEG (20 mg) are dispersed in 0.4 mL DMF at a ratio of 1:1 (w / w), and injected into PBS under ultrasonic conditions of 200 W at 10°C; the resulting mixture is further stirred for 15 minutes; high pressure homogenization is performed for 30 minutes, and the solution is dialyzed twice with PBS to obtain DSPE-PEG-encapsulated tetravalent platinum drug nanoparticles DSPE-PEG@Pt(IV);

[0059] (2) The above-mentioned DSPE-PEG-encapsulated tetravalent platinum drug and carrier GEVs 20 mg were incubated at a ratio of 1:1 (compound 1:GEVs, w / w) under ultrasonic conditions of 300 W at 10°C for 3 minutes to obtain non-targeted grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound nanodrug GEVs@Pt(IV).

[0060] (3) The above-mentioned non-targeted grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound nanodrug GEVs@Pt(IV) and the targeted carrier DSPE-PEG-Tf 20 mg were incubated in a 30°C water bath at a ratio of 1:1 (compound 1:GEVs, w / w) for 1 hour to obtain transferrin-modified targeted grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound nanodrug Tf-GEVs@Pt(IV).

[0061] Furthermore, the use of grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound nanomedicine in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs.

[0062] Furthermore, the anti-tumor proliferation is anti-human lung adenocarcinoma, anti-cisplatin-resistant human lung adenocarcinoma, anti-human liver cancer or anti-mouse breast cancer.

[0063] Furthermore, the anti-tumor metastasis is anti-mouse breast cancer cells.

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

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

[0066] (2) The present invention has developed a nanomedicine containing a trifunctional tetravalent platinum compound encapsulated by grapefruit extracellular vesicles (GEVs), and introduced transferrin Tf as a targeting group to prepare a nanomedicine containing a trifunctional tetravalent platinum compound loaded with transferrin-modified targeted grapefruit extracellular vesicles. GEVs promote the penetration of Tf-GEVs@Pt(IV) into tumor tissues; transferrin (Tf) further enhances its tumor targeting properties; the trifunctional tetravalent platinum active component can reshape the tumor microenvironment and has significant anti-tumor activity.

[0067] (3) The structural innovation of the compounds of Formula I and Formula III described in the present invention is expected to obtain a variety of lead molecules that are effective against tumors, opening up new avenues for the development of platinum compounds. Such source-based innovative drug research will have important theoretical value and practical significance for national economic and social development and people's health.

[0068] (4) The grapefruit extracellular vesicles (GEVs)-encapsulated trifunctional tetravalent platinum compound nanomedicine described in the present invention is a new attempt in the field of pharmaceutical preparations, and is expected to obtain new, highly efficient, and tumor-targeted nanomedicines, providing new candidate drugs for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0070] Figure 2 Characteristics of DSPE-PEG@Pt(IV), GEVs, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) were 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 3The stability of nanoparticles DSPE-PEG@Pt(IV), GEVs, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) stored at 4°C for 7 days (n=3) was determined by DLS. (a) Particle size change. (b) PDI change.

[0072] Figure 4 .In vitro drug release curves of free compound 1, nanoparticle 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 μM) at 37°C for 24 h. ***P<0.001.

[0074] Figure 6 In vivo antitumor activity of candidate tetravalent platinum compound 1 and nanodrugs GEVs@Pt(IV) and Tf-GEVs@Pt(IV) against 4T1 tumors in female BALB / c mice, with GEVs, CDDP and the mixture CDDP+6 as reference drugs (n=5). (a) Schematic diagram of experimental design. (b) Relative body weight of mice during treatment. (c) Graph of tumor volume changes. (d) Tumor weight of mice in each group at the end of the experiment. The TGI of the tested drugs compared with blank is shown above [TGI=(1-tumor weight of drug-treated group / tumor weight of blank)×100%]. (e) Uptake of platinum in tumor tissue. (f) Tumor image 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, GEVs, CDDP, CDDP+6 mixture, nanoparticle GEVs@Pt(IV) and Tf-GEVs@Pt(IV) groups. Tissues were obtained from in vivo antitumor experiments.

[0076] Figure 8 In vitro Transwell assay was used to evaluate the migration inhibition properties of compound 1, GEVs, CDDP, CDDP+6, and nanoparticles GEVs@Pt(IV) and Tf-GEVs@Pt(IV) (5 μM) on 4T1 cells. Tumor cells were treated with drugs for 24 h. (a) Representative images. (b) Relative mobility analysis. ***P<0.001.

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

[0078] Fig.10 .Inhibitory properties of compound 1 and nanoparticles GEVs@Pt(IV) and Tf-GEVs@Pt(IV) on lung metastasis of 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 experimental design. (b) Representative photos of the front and back of the lungs of each group 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] Fig.11 RNA-seq analysis of 4T1 tumor tissues in the blank, GEVs, and Tf-GEVs@Pt(IV) treated groups in the in vivo antitumor experiment. (a) Gene heat map. (b) Venn diagram of differentially expressed genes in the three groups. (c) Volcano plot of DEGs in the blank vs Tf-GEVs@Pt(IV) groups. (d) KEGG enrichment in the blank vs Tf-GEVs@Pt(IV) groups. (e) GSEA plot of the blank vs Tf-GEVs@Pt(IV) groups.

[0080] Fig.12 .Ability to cause DNA damage. (ab)GO enrichment and GSEA analysis of DEGs related to DNA damage in blank vs Tf-GEVs@Pt(IV) cells. (cd)Western blot analysis of γ-H2AX and P53 in 4T1 cells treated with CDDP (5μM), CDDP+6 (5μM / 10μM), compound 1 (5μM), GEVs@Pt(IV) (5μM) and Tf-GEVs@Pt(IV) (5μM) at 37℃ for 24h, and cells treated with PBS and GEVs were used as controls. **P<0.01, ***P<0.001.

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

[0082] Fig.14 .Properties leading to mitochondrial-mediated apoptosis. (ab) GO enrichment and GSEA analysis of mitochondrial-mediated apoptosis-related DEGs in Blank vs Tf-GEVs@Pt(IV). (c) Quantification of apoptosis in 4T1 cells by flow cytometry using Annexin V-FITC / PI staining. (de) Western blot analysis of Bcl-2, Bax, caspase3, and c-caspase-3 in 4T1 cells. 4T1 tumor cells were incubated with 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) for 24 h, with PBS and GEVs as controls. **P<0.01, ***P<0.001.

[0083] Fig.15 JC-1 staining was used to quantify mitochondrial damage in 4T1 cells. 4T1 tumor cells were cultured with 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) for 24 h, with PBS and GEVs as controls.

[0084] Fig.16 .Properties of reducing tumor fibrosis. (ab)GO enrichment and GSEA analysis of DEGs related to tumor fibrosis in Blank vs Tf-GEVs@Pt(IV). (cd) Immunohistochemical staining of α-SMA and FSP1, Picrosirius red and Masson staining of collagen fibers in in vivo anti-tumor experimental tissues. **P<0.01, ***P<0.001.

[0085] Fig.17 .(ab) Western blot analysis of TGF-β1, MMP2 and MMP9 in 4T1 cells. 4T1 tumor cells were cultured with CDDP (5μM), CDDP+6 (5μM / 10μM), compound 1 (5μM), GEVs@Pt(IV) (5μM) and Tf-GEVs@Pt(IV) (5μM) for 24 h, with PBS and GEVs as controls. (cd) Immunohistochemical staining of TGF-β1 and MMP9 in tumor tissues in the in vivo antitumor experiment. ***P<0.001.

[0086] Fig.18.Reversal of the characteristics of inflammatory TME. (ab) GO enrichment and GSEA analysis of DEGs related to inflammation in Blank vs Tf-GEVs@Pt(IV). (cd) Western blot analysis of COX-2, TNF-α, and IL-6 in 4T1 cells. 4T1 tumor cells were cultured with CDDP (5μM), CDDP+6 (5μM / 10μM), compound 1 (5μM), GEVs@Pt(IV) (5μM), and Tf-GEVs@Pt(IV) (5μM) for 24 h, with PBS and GEVs as controls. (e) Elisa analysis of TNF-α and IL-6 in tumor tissues in in vivo antitumor experiments. (fg) Immunohistochemical staining of COX-2 in tumor tissues in in vivo antitumor experiments. **p<0.01, ***p<0.001.

[0087] Fig.19 .Reversing the immunosuppressive properties of the TME. (ab) GO enrichment and GSEA analysis of immune-related DEGs in Blank vs Tf-GEVs@Pt(IV). (cd) Western blot analysis of PD-L1, STING, p-STING, TBK1, p-TBK1, IFR3, and p-IFR3 in 4T1 cells. (ef) Immunohistochemical staining of PD-L1 expression, CD3+ and CD8+ T cells, CD206+M2 and CD86+M1 macrophages in tumor tissues in the in vivo anti-tumor experiment. ***P<0.001.

[0088] Fig. 20 . Schematic diagram of the mechanism of action of grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound nanodrug Tf-GEVs@Pt(IV). DETAILED DESCRIPTION

[0089] In order to make the objectives, technical solutions and advantages of the present invention more clear, representative embodiments of the present invention are described in detail below, but are not limited thereto.

[0090] Example 1.

[0091] 1. Preparation of tetravalent platinum shown in structural formula II

[0092] Synthesis of Dihydroxycis-tetravalent Platinum IIa

[0093]

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

[0095] 2. Preparation of aspirin-captopril tetravalent platinum shown in structural formula I

[0096] 1. Preparation of Symmetric Aspirin-Captopril Tetravalent Platinum Compound 1

[0097]

[0098] Compound 6 (341 mg, 0.90 mmol) and TBTU (289 mg, 0.90 mmol) were dissolved in 5 mL of 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, dihydroxycis-tetravalent platinum IIa (100 mg, 0.30 mmol) was added. The mixture was stirred at 50 ° C and protected from light for 48 h under nitrogen protection. After the reaction was completed, the solvent was removed in vacuo, and the crude product was purified by silica gel column chromatography to obtain a yellow solid compound 1 (101 mg, 32%). The purity was 98.9% as determined by HPLC (MeOH / H2O=70 / 30).

[0099] 1 H NMR(500MHz,DMSO-d6)δ7.95–7.60(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(126MHz,DMSO-d6)δ172.8,167.7,157.8,137.7,135.5,134.6,129.3,122 .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 of 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, dihydroxycis-tetravalent platinum IIa (100 mg, 0.3 mmol) was added. The mixture was stirred at 50 ° C and protected from light for 48 h under nitrogen protection. After the reaction was completed, the solvent was removed in vacuo and the crude product was purified by silica gel column chromatography to obtain yellow solid compound 2 (58.3 mg, 28%). The purity was 97.1% as determined by HPLC (MeOH / H2O=70 / 30).

[0103] 1 H NMR(500MHz,Methanol-d4)δ7.97–7.79(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(126MHz,Methanol-d4)δ174.8,174.1,170.1,166.3,150.8,133.5,131.5,12 5.7,123.7,123.5,58.7,42.2,30.8,28.9,26.8,24.3,19.7,15.7.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] 3. Preparation of Captopril Bifunctional Tetravalent Platinum Represented by Structural Formula III

[0105] Preparation of Captopril tetravalent platinum compound 3

[0106]

[0107] Compound 7 (233 mg, 0.90 mmol) and TBTU (289 mg, 0.90 mmol) were dissolved in 5 ml of 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 oxide IIa (100 mg, 0.30 mmol) was added. The mixture was stirred at 50 ° C and protected from light for 48 h under nitrogen protection. After the reaction was completed, the solvent was removed in vacuo and the crude product was purified by silica gel column chromatography to obtain yellow solid compound 3 (90 mg, 37%). The purity was 95.2% as determined by HPLC (MeOH / H2O=70 / 30).

[0108] 1 H NMR(500MHz,DMSO-d6)δ4.56–4.15(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(126MHz,DMSO)δ195.9,195.8,173.3,66.0,58.8,46.9,37.8,31.0,29.5,25.7,16.9.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] 4. Preparation of grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound nanodrugs

[0110] Preparation of grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compounds as nanopharmaceuticals Figure 1 shown.

[0111] 1. Preparation and Characterization of Grapefruit Extracellular Vesicles (GEVs)

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

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

[0114] The 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 at 10°C under ultrasonic conditions of 200 W; the resulting mixture was further stirred for 15 minutes; high pressure homogenization was performed for 30 minutes, and the solution was dialyzed twice with PBS to obtain DSPE-PEG-encapsulated tetravalent platinum drug nanoparticles DSPE-PEG@Pt(IV);

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

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

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

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

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

[0120] DiD (DSPE-PEG2000: compound 1 = 1%) and DSPE-PEG2000 were dissolved in DMF to prepare nanomedicines, and the subsequent steps were the same as above to prepare DiD-labeled nanomedicines DiD-GEVs@Pt(IV) and DiD-Tf-GEVs@Pt(IV).

[0121] 5. Characterization of Nanomedicine

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

[0123] Dynamic laser light scattering (DLS) and transmission electron microscopy (TEM, Figure 2bd) Characterization of nanoparticles: The particle size of DSPE-PEG@Pt(IV) is 68.1±0.4nm (PDI=0.16±0.01, ζ=-22.54±1.26). The particle size of GEVs is 223.8±5.2nm (PDI=0.33±0.03, ζ=-14.64±3.81), which is larger than DSPE-PEG@Pt(IV) and is easy to encapsulate DSPE-PEG@Pt(IV). The particle sizes of GEVs@Pt(IV) and Tf-GEVs@Pt(IV) are 146.0±1.9nm (PDI=0.27±0.04, ζ=-20.22±0.69) and 190.3±6.3nm (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 contained contents, which further confirmed that DSPE-PEG@Pt(IV) was embedded in GEVs.

[0124] Stability assay: The stability of nanodrugs (including DSPE-PEG@Pt(IV), GEVs, GEVs@Pt(IV), and Tf-GEVs@Pt(IV)) cultured at 4 °C for 7 days was monitored by measuring the changes in particle size using DLS. Figure 3 The results showed that DSPE-PEG@Pt(IV), GEVs, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) remained stable within 7 days. These facts indicate that the nanomedicine prepared by encapsulating trifunctional tetravalent platinum drugs with GEVs as carriers is stable in aqueous media.

[0125] Release behavior: The release behavior of GEVs@Pt(IV) and Tf-GEVs@Pt(IV) was examined in PBS medium. The nanoparticles (5 mL) were placed in a dialysis bag (8-14 kDa), immersed in 100 mL of PBS, and stirred at 37 °C (100 rpm). The release solution (3 mL) was withdrawn regularly and supplemented with fresh PBS (3 mL). The drug concentration was determined by atomic absorption spectroscopy (AAS) to measure the platinum content. 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 hours, which was significantly slower than the free drug (88% released within the first 6 hours). This indicates that GEVs@Pt(IV) and Tf-GEVs@Pt(IV) have a significant sustained release effect.

[0126] 6. Pharmacological Activity Experiment

[0127] 1. In vitro antiproliferative activity

[0128] The antiproliferative activities of tetravalent platinum compounds 1-4 and nanomedicines 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), mouse breast cancer (4T1), human liver cancer (HepG2)) and a human normal liver cell line (LO2) were determined by MTT assay, 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 (5×10 3 / well) and pre-cultured overnight. Then, 100 μL of culture medium containing different concentrations of drugs was added. Tumor cells were treated with drugs for 48 h. 20 μL MTT solution (5 mg / mL) was added and cultured for another 4 h. Then, the culture supernatant was removed and 150 μL dimethyl sulfoxide (DMSO) was added. The absorbance of each well was measured using a microwell spectrophotometer (490 nm). Antiproliferation data were expressed as IC 50 Value representation.

[0129] At the same time, we tested the aspirin tetravalent platinum compound 4 As a control.

[0130] Table 1. Antiproliferative activities of tetravalent platinum compounds 1-4 and 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: a mixture of CDDP and acid 6, with a molar ratio of 1:2.

[0134] The results showed that: first, the trifunctional tetravalent platinum compounds with CTP-ASP ligands were more active than bifunctional compounds. The activity of trifunctional compound 1 was significantly stronger than the corresponding bifunctional tetravalent platinum drugs 3 (CTP tetravalent platinum) and 4 (ASP tetravalent platinum). Secondly, the number of ligands in the tetravalent platinum had an effect on its activity, among which the activity of the dual-ligand compound 1 was significantly higher than that of the single-ligand compound 2. It is worth noting that compound 1 showed the strongest antitumor activity against all tumor cell lines tested, and its IC 50 The value was lower than 4.18 μM, showing a significantly stronger activity advantage compared to the reference drugs CDDP, ASP, CTP and the mixture CDDP + 6. Based on the above results, compound 1 has good application potential and was selected as a candidate drug for further preparation of nanomedicines.

[0135] Compared with free compound 1, nanoparticles GEVs@Pt(IV) and Tf-GEVs@Pt(IV) prepared with GEVs as carriers showed superior antitumor properties. In particular, Tf-GEVs@Pt(IV) with tumor targeting groups was more than 1.9 times more active against all tumor cell lines than free compound 1. The uptake of drugs in tumor cells is a key factor affecting the antitumor efficacy. We detected the drug uptake level in 4T1 cells ( Figure 5 The results showed that Tf-GEVs@Pt(IV) accumulated more in tumor cells compared with GEVs@Pt(IV) (P < 0.001) and free compound 1 (P < 0.001), which is also a key factor in its significant antitumor activity.

[0136] Drug resistance is one of the clinical challenges 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 showed that all prepared tetravalent platinum compounds 1-4 were able to effectively reduce drug resistance, and their RF = 1.05-3.78 was lower than that of the reference drug CDDP and the mixture CDDP+6 (RF = 6.08, 5.06). In addition, the nanodrugs GEVs@Pt(IV) and Tf-GEVs@Pt(IV) also had low RF values ​​(1.89 and 0.70), indicating that they also have great potential in overcoming the drug resistance of CDDP.

[0137] Serious toxicity to normal cells is another problem faced by platinum drugs. We also calculated the selectivity index (SI: LO2 IC 50 Values ​​and HepG2 IC 50The SI of compound 1 was 2.21, which was significantly higher than that of free compound 1 and GEVs@Pt(IV) (SI=2.21, 2.63), compared with CDDP (SI=1.68) and the mixture CDDP+6 (SI=1.77), which effectively reduced the toxicity. The SI of nanomedicine Tf-GEVs@Pt(IV) was 6.88, which was 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, trifunctional tetravalent platinum compounds with CTP-ASP functional groups have excellent anti-tumor activity; the grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compounds constructed with it as the active component have better activity; Tf-GEVs@Pt(IV) prepared by modifying it with the tumor targeting group Tf has the strongest activity and can effectively overcome drug resistance and reduce the toxicity of platinum drugs.

[0139] 2. In vivo antitumor activity

[0140] Female BALB / c mice (18-20 g) were used as the model. Tumor cells (1×10 6 ) tumor model was established. The mice were divided into 7 groups (Blank, CDDP, CDDP+6, 1, GEVs, GEVs@Pt(IV) and Tf-GEVs@Pt(IV), n=5) on day 4. Then, the drug was injected into the tail vein at a dose of 2 mg Pt / kg ( Figure 6 a). Tumor volume (V=W) was monitored daily. 2 ×L / 2, where W is the width of the tumor and L is the length), and the weight of the mice was recorded. Then, the mice were killed on day 12. 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)) × 100%]. The heart, liver, spleen, lung, kidney and tumor were collected and fixed with 4% paraformaldehyde for 48 hours. H&E staining and immunohistochemistry experiments were then performed.

[0141] Figure 6 The results showed that GEVs had a certain inhibitory effect on tumors, with a TGI of 51.4%, indicating that they were not only drug carriers, but also enhanced the anti-tumor activity of nanomedicines ( Figure 6 c, d). Compared with the reference drug CDDP and the mixture CDDP+6, candidate compound 1 can more effectively inhibit the growth of tumors in vivo, and its trend is similar to the in vitro MTT results. In addition, the efficacy of nanodrugs GEVs@Pt(IV) and Tf-GEVs@Pt(IV) was significantly higher than that of compound 1 and GEVs group (P < 0.01). Tf-modified nanodrug Tf-GEVs@Pt(IV) (85.5 mm 3, TGI = 83.7%) than GEVs@Pt(IV)(138.3mm 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 based on the results of hematoxylin and eosin (H&E) staining. Figure 7 The images in the figure show that significant apoptosis and necrosis areas were observed in the tumors of the aspirin-captopril trifunctional tetravalent platinum compound 1, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) groups, which were even stronger than those of the CDDP group and CDDP+6 group. This indicates that the use of GEVs to encapsulate the aspirin-captopril trifunctional tetravalent platinum compound 1 to prepare the corresponding nanomedicine can effectively improve the anti-tumor activity in vivo.

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

[0143] Therefore, the anti-tumor efficacy of aspirin-captopril trifunctional tetravalent platinum compound 1 was significantly improved and the in vivo toxicity was reduced by adding CTP-ASP ligand. Then, nanomedicine Tf-GEVs@Pt(IV) was prepared using GEVs as carriers and Tf as tumor targeting groups, which further improved the accumulation of aspirin-captopril trifunctional tetravalent platinum compound 1 in tumors and enhanced 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 , 0.2mL) was added to the upper chamber. 0.6mL 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 (5μM), CDDP+6 (5μM / 10μM), compound 1 (5μM), GEVs@Pt(IV) (5μM) and Tf-GEVs@Pt(IV) (5μM), and PBS and GEVs were set as control groups. After 24h of cell culture, the cells were fixed with 4% paraformaldehyde for 20min. Subsequently, the cells were stained with 0.1% crystal violet for 20min. Gently scrape away 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. After that, 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 h, and the scratches of the cells were photographed using an inverted microscope at 0, 12 and 24 h.

[0147] Female BALB / c mice (18-20 g) were used as subjects for the in vivo anti-metastasis experiment. 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). Then, the mice were 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 nanomedicine. On the 12th day, the mice were killed, the 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 nanomedicines 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 ( Fig. 9 ), compared with blank, CDDP, CDDP+6 and GEVs groups, compound 1, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) can more effectively inhibit the scratch healing of tumor cells. This proves that the trifunctional tetravalent platinum compound 1 and its nanomedicines GEVs@Pt(IV) and Tf-GEVs@Pt(IV) have the effect of inhibiting the metastasis of tumor cells in vitro.

[0149] In vivo tumor lung metastasis model experiments showed that ( Fig.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 performance. 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 the active ingredient 1, but also participate in the inhibition of tumor metastasis, which further explains the excellent anti-metastatic performance 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 of 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 performed by Qingke Biotechnology Co., Ltd. (Beijing, China). Total RNA was collected from fresh frozen tumor samples of GEVs, GEVs@Pt(IV), and Tf-GEVs@Pt(IV) treatment groups in the anti-tumor proliferation experiment. 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 cDNA libraries were evaluated using an Agilent 4200 bioanalyzer, and sequencing was performed using an Illumina novaseq6000.

[0152] Experimental results: RNA sequencing was performed on tumor tissues of blank, GEVs and Tf-GEVs@Pt(IV) groups to analyze the potential anti-tumor mechanism ( Fig.11 ). Tf-GEVs@Pt(IV) induced a large number of differentially expressed genes (DEGs) in tumor cells compared with blank (1952) and GEVs groups (1860). In addition, 1241 DEGs appeared simultaneously in 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 the 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

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

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

[0156] Experimental results: GO enrichment analysis of DEGs in tumor tissues showed that Tf-GEVs@Pt(IV) had significant effects on cell components (CC): cytoplasmic ribosomes; biological processes (BP): DNA replication, deamination base formation and DNA chain elongation. GSEA further confirmed that genes of DNA replication and base excision repair were significantly affected ( Fig.12 ab). These facts suggest that Tf-GEVs@Pt(IV) significantly regulated genes involved in DNA damage, which was mainly attributed to the active tetravalent Pt component 1.

[0157] The uptake of platinum drugs in the cell nucleus directly affects their ability to damage DNA. Therefore, the AAS method was used to determine the sub-distribution of platinum drugs in 4T1 cells ( Fig.13 ). The trifunctional tetravalent platinum compound 1 and its nanodrugs GEVs@Pt(IV) and Tf-GEVs@Pt(IV) showed excellent antitumor properties, 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 the free compound 1 and the nanodrug GEVs@Pt(IV), the nanoparticle Tf-GEVs@Pt(IV) with the tumor targeting group Tf had a higher accumulation in DNA, which may be related to its improved tumor targeting ability.

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

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

[0160] 6. Mitochondria-mediated apoptosis

[0161] The changes in mitochondrial membrane potential after drug treatment were measured by flow cytometry using a JC-1 staining kit (Solarbio). 54T1 cells were seeded into six-well plates and pre-cultured overnight. CDDP (5 μM), CDDP+6 (5 μM / 10 μM), 1 (5 μM), GEVs@Pt(IV) (5 μM) and Tf-GEVs@Pt(IV) (5 μM) were then added, with PBS and GEVs as controls, and the cells were cultured for another 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 (Biyuntian) was used to detect cell apoptosis after drug treatment by flow cytometry. 5 4T1 cells were seeded into six-well plates and pre-cultured overnight. CDDP (5 μM), CDDP+6 (5 μM / 10 μM), 1 (5 μM), GEVs@Pt(IV) (5 μM) and Tf-GEVs@Pt(IV) (5 μM) were then added, with PBS and GEVs as controls, and the cells were cultured for another 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 for platinum-based chemotherapy drugs to kill tumor cells, and it is closely related to mitochondrial damage. This paper studies the characteristics of the prepared agents in inducing mitochondrial-mediated apoptosis from different aspects. GO results show that compared with blank, genes related to apoptotic cell clearance (BP), mitochondria, mitochondrial inner membrane, and mitochondrial ribosome regulation (CC) in tumors treated with Tf-GEVs@Pt(IV) were significantly changed. GSEA also confirmed that pathways related to apoptosis and cell cycle were activated ( Fig.14 ab). Fig.14 The apoptosis induction results in c showed that the proportion of cell apoptosis caused by the 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 is basically consistent with the antitumor activity in vitro and in vivo. Then, the JC-1 assay further verified their effect of disrupting mitochondria ( Fig.15 ). A severe collapse of mitochondrial membrane potential (ΔΨm) was observed in tumor cells treated with Tf-GEVs@Pt(IV), with a greater proportion 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 induce mitochondria-mediated apoptosis, providing strong evidence for its anti-tumor effect.

[0164] The Bcl-2 pathway is a key pathway that regulates the process of cell apoptosis, so we measured the expression of Bcl-2 pathway proteins by Western blot. Fig.14 As shown in Figure 5, the anti-apoptotic protein Bcl-2 was significantly downregulated (P<0.001), while the pro-apoptotic protein Bax was upregulated (P<0.001). In addition, compared with the control group, the expression of apoptotic execution protein c-caspase-3 increased, and the ratio of c-caspase-3 / caspase-3 was significantly increased (P<0.001). Therefore, the nanodrug Tf-GEVs@Pt(IV) can induce tumor cell apoptosis through mitochondria-mediated Bcl-2 / Bax / caspase-3 signaling, thereby effectively inhibiting tumor proliferation.

[0165] 7. Reduce tumor fibrosis

[0166] Immunohistochemistry, picrosirius red and Masson staining assays: Tumor tissues of blank, CDDP, CDDP+6, compound 1, GEVs, GEVs@Pt(IV) and Tf-GEVs@Pt(IV) groups were obtained from the in vivo anti-tumor growth assay. These tumor tissues were embedded in paraffin 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 the secondary antibody at room temperature for 50 min. 3,3′-diaminobenzidine (DAB) was used for color development and co-stained with hematoxylin. Finally, the sections were photographed with an inverted microscope, and the protein expression was quantified using Image J based on five randomly selected fields of view.

[0167] Tumor fibrosis is closely related to the 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, immunohistochemistry, picrosirius red and Masson staining were used to evaluate the effects of trifunctional tetravalent platinum compound 1, nanomedicine GEVs@Pt(IV) and Tf-GEVs@Pt(IV) on tumor fibrosis.

[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 ( Fig.16 ab). Sirius red staining and Masson staining results ( Fig.16cd) showed that Tf-GEVs@Pt(IV), GEVs@Pt(IV) and compound 1 could significantly reduce the level of collagen fibrosis in tumor sections compared with the blank group (P<0.001), while the effect of divalent platinum drugs was minimal. In addition, the expression of proteins α-SMA and FSP1 related to collagen fibrosis was also inhibited (P<0.001), indicating that compound 1 and its nanodrugs Tf-GEVs@Pt(IV) and GEVs@Pt(IV) have significant efficacy in inhibiting tumor fibrosis.

[0169] The protein TGF-β is a key protein that induces ECM fibrosis. MMPs, as key enzymes regulating ECM, play a synergistic role with TGF-β in promoting tumor metastasis and fibrosis. CTP can effectively reduce ECM deposition and 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. Fig.17 ab showed that compared with blank, Tf-GEVs@Pt(IV) simultaneously inhibited the expression of TGF-β1, MMP2, and MMP9 in tumor cells (P<0.001), and was more effective than free compound 1 (P<0.001). These inhibitory effects were also observed in in vivo antitumor experiments ( Fig.17 cd).

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

[0171] 8. Reversing the inflammatory TME

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

[0173] GO and GSEA analysis based on RNA-seq data ( Fig.18 ab) also showed that Tf-GEVs@Pt(IV) had a significant effect on tumor inflammatory response and cytokine secretion compared with blank. Subsequent Western blot and immunohistochemical staining detection ( Fig.18cd, fg) showed that Tf-GEVs@Pt(IV) effectively inhibited the expression of COX-2 (P<0.001), a key enzyme in inflammation. Western blot and Elisa assay results further showed that ( Fig.18 e), the secretion of inflammatory cytokines TNF-α and IL-6 in tumor cells in vitro and tumor tissues in vivo was significantly inhibited (P<0.001). It is worth noting that compared with blank, carrier GEVs can also effectively reduce tumor inflammation. Therefore, the nanodrug Tf-GEVs@Pt(IV) effectively inhibits tumor inflammation by inhibiting COX-2, TNF-α and IL-6, which is mainly attributed to the synergistic effect of the functional base ASP and exosomal GEVs.

[0174] 9. Reversing the immunosuppressive TME

[0175] Immunosuppression is considered another characteristic of cancer. The presence of fibrosis in tumors constitutes an immunosuppressive barrier to T cell infiltration; and the inflammatory TME is also an important driver of immunosuppression. Given the role of the target drug in inhibiting tumor fibrosis and tumor inflammation, we examined its activation of immunity.

[0176] RNA-seq analysis results ( Fig.19 ab) showed that Tf-GEVs@Pt(IV) could effectively regulate genes related to immune response, T cell activation, and macrophage activation. In addition, the key checkpoints PD-L1 / PD-1 and cGAS / STING pathway genes closely related to immunity were 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 ( Fig.19 ef). Compared with blank, the Tf-GEVs@Pt(IV)-treated group significantly increased CD3 + and CD8 + The number of T cells increased to 22.7 and 20.8 times of the blank group (P<0.001). + M2 macrophages decreased (15%, P<0.001), and tumor-killing CD86 + The number of M1 macrophages increased (19.11%, P<0.001), indicating that Tf-GEVs@Pt(IV) treatment promoted the polarization of macrophages from M2 to M1 in tumors. We further studied the mechanism by western blot and immunohistochemical staining ( Fig.19cf). The immune checkpoint PD-L1 was significantly inhibited in both in vitro tumor cells (P<0.001) and in vivo tumor tissues (P<0.001). Subsequently, the dsDNA released after DNA damage by divalent platinum compounds activated the cGAS / STING pathway, and the expression of STING, p-STING, TBK1, p-TBK1, IFR3, and p-IFR3 in 4T1 cells was also increased (P<0.001), which proved that the cGAS / STING pathway was activated and promoted immunity during Tf-GEVs@Pt(IV) treatment. It is worth noting that free compound 1 also had a similar immune activation trend in vitro and in vivo. In summary, the nanodrug Tf-GEVs@Pt(IV) can inhibit the expression of PD-L1, activate T cell immunity, and induce macrophage polarization from M2 to M1 by activating the cGAS / STING pathway, thereby effectively activating anti-tumor immunity.

[0177] 10. Mechanism of Action

[0178] Based on the above RNA-seq and experimental results, the mechanism of action of Tf-GEVs@Pt(IV) was drawn Fig. 20 . Nanomedicines with GEVs as carriers can effectively penetrate the fibrotic barrier to reach tumor tissues; the targeting group Tf significantly improves its tumor targeting performance. Then, the active ingredient 1 is reduced in the reductive TME and further releases divalent platinum, CTP and ASP fragments. Divalent platinum compounds bind to DNA, causing severe DNA damage and further increasing the expression of γ-H2AX and p53. Subsequently, mitochondria-mediated apoptosis is activated by activating the Bcl-2 / Bax / caspase3 pathway. CTP inhibits extracellular matrix fibrosis in tumor tissues by inhibiting the TGF-β1 and MMPs (MMP2, MMP9) pathways, reducing the levels of α-SMA and FSP1. Subsequently, the fibrotic barrier around the tumor is broken, promoting the penetration of drugs and immune cells into the tumor. After Tf-GEVs@Pt(IV) treatment, the inflammatory TME was significantly reversed, which was mainly attributed to the synergistic effect of ASP and GEVs, which inhibited the secretion of the key enzyme COX-2 and the inflammatory cytokines TNF-α and IL-6. In addition, with the suppression of fibrosis and inflammation in the 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 polarized from M2 to M1. The development of the above-mentioned grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compounds nanomedicines with multiple anti-tumor mechanisms provides a new strategy for the future design of new and potent tetravalent platinum anti-tumor drugs.

[0179] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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 I containing a captopril-aspirin ligand according to claim 1, characterized in that: 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 reference drug of the trifunctional tetravalent platinum compound containing captopril-aspirin ligand according to claim 1, characterized in that: The reference drug is a bifunctional tetravalent platinum compound (III) containing a captopril ligand, and its structure is as follows: in, Selected from cisplatin or oxaliplatin.

4. A grapefruit extracellular vesicle-loaded nanomedicine of a trifunctional tetravalent platinum compound, characterized in that: The nano drug is prepared from a trifunctional tetravalent platinum compound I 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 tetravalent platinum compound (I), the carrier DSPE-PEG, the carrier GEVs, and the targeting carrier DSPE-PEG-Tf in the nano drug is 1:(0.1-100):(0.1-100):(0.1-100).

5. The method for preparing the nanomedicine of grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound as claimed in 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 tetravalent platinum compound (I) and DSPE-PEG in DMF at a mass ratio of 1:(0.1-100), and injecting into PBS under ultrasonic conditions; further stirring the resulting 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-encapsulated tetravalent platinum drug nanoparticles; (2) The above-mentioned DSPE-PEG-encapsulated tetravalent platinum drug and carrier GEVs are incubated at a ratio of 1: (0.1-100) [I: GEVs, (w / w)] under ultrasonic conditions of 100-1000W at 0-40°C for 1-300 minutes to obtain a non-targeted grapefruit extracellular vesicle-loaded trifunctional tetravalent platinum compound nanodrug. (3) The above-mentioned non-targeted grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound nanodrug and the targeting carrier DSPE-PEG-Tf are incubated in a 0-50°C water bath for 0.1-5 hours at a ratio of 1:(0.1-100) [I:DSPE-PEG-Tf, (w / w)] to obtain transferrin-modified targeted grapefruit extracellular vesicles loaded with trifunctional tetravalent platinum compound nanodrug.

6. Use of the trifunctional tetravalent platinum compound (I) containing captopril-aspirin ligand according to any one of claims 1 to 2 in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs.

7. Use of the bifunctional tetravalent platinum compound (III) of captopril ligand according to claim 3 in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs.

8. Use of the grapefruit extracellular vesicle-loaded nanomedicine of trifunctional tetravalent platinum compound as claimed in claim 4 in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs.

9. The use according to any one of claims 6 to 8, 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.

10. The use according to any one of claims 6 to 8, characterized in that: The anti-tumor metastasis is anti-mouse breast cancer metastasis.

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