Nanopreparations, methods of making and uses thereof

Abstract

This application presents a nanoprodrug, its preparation method, and its uses. Based on the redox imbalance characteristics of the TNBC microenvironment and the overexpression of the TROP2 receptor in TNBC, this application constructs a redox-sensitive / TROP2-targeting nanoprodrug. This nanoprodrug has advantages such as specific targeting of tumor cells, achieving stable, spatiotemporally controlled, and long-term circulating drug release and activation at the target site. It overcomes microenvironment delivery barriers, exhibits redox-sensitive release characteristics, and enters TNBC cells through receptor-mediated endocytosis, which helps improve drug delivery efficiency and achieve rapid release at the tumor site. This suggests that the nanoprodrug has promising applications in the preparation of drugs for treating TNBC.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical formulation technology, specifically to a nanoprodrug, its preparation method, and its uses. Background Technology

[0002] Triple-negative breast cancer (TNBC) refers to a subtype of breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), accounting for approximately 15%-20% of all breast cancer types. TNBC is highly invasive, exhibits high histological grade, widespread heterogeneity, and ductal histological features; metastatic TNBC is associated with low overall survival. Treatment options for TNBC primarily include surgery, chemotherapy, radiotherapy, and immunotherapy, with chemotherapy being the most widely used. However, the poor water solubility and unsatisfactory biodistribution of chemotherapy drugs hinder their application in cancer treatment. There is an urgent need for new TNBC treatments to provide clinicians with more effective and safer treatment options.

[0003] Prodrugs often lack biological activity or have very low activity. In vivo, they can undergo fission to form the original drug, which then binds to the target protein to exert its biological activity. The ability of a drug to be transported to the target organ is fundamental to its efficacy. Targeted modification of the drug structure to achieve selective tissue delivery can enhance therapeutic activity and reduce side effects. The targeted design of prodrugs is often achieved by coupling structural units of the target tissue or cell with cytotoxic molecules. For example, peptide-drug conjugated prodrugs use different linkers to covalently link peptides to the drug, forming multifunctional PDCs that target tumor cells. These PDCs can selectively accumulate in tumor cells, extending their half-life and enhancing efficacy. Antibody-drug conjugated prodrugs utilize the high affinity of monoclonal antibodies for tumor cell surface antigens, targeting tumor cells and releasing the active drug through receptor-mediated endocytosis. Esterification is the most widespread application in prodrug carrier design, with ester drugs accounting for more than 50% of marketed prodrugs. Ester bonds can be hydrolyzed by esterases widely present in blood, liver, and other organs or tissues, releasing the drug from its prodrug molecule. Although simple esters are preferred as prodrug carriers, the biotransformation of some simple alkyl or aryl esters cannot be mediated by esterases or the esterase hydrolysis efficiency is low, resulting in insufficient system exposure and thus affecting drug delivery efficiency.

[0004] Therefore, the development of a prodrug with high drug delivery efficiency has become an urgent need. Summary of the Invention

[0005] The purpose of this application is to provide a nanoprodrug, its preparation method, and its use.

[0006] To achieve the above objectives, the embodiments of this application propose the following technical solutions:

[0007] In a first aspect, embodiments of this application provide a nanoprodrug having the structure shown in Formula I or a pharmaceutically acceptable salt thereof:

[0008]

[0009] The molecular weight of the nanoprodrug is 47820.28.

[0010] Secondly, embodiments of this application provide a method for preparing a nanoprodrug as described in the first aspect, the method comprising:

[0011] Under an inert gas atmosphere, 2-ethoxy-1,3,2-dioxaphosphane 2-oxide, dihydroxy disulfide and Sn(Oct)2 were added to anhydrous dichloromethane to react and obtain polymer I.

[0012] Baicalin was dissolved in an organic solvent, and EDC and a catalyst were added to react. The reactants were added to a dimethylformamide solution of polymer I, and the mixture was purified by dialysis after the reaction to obtain polymer II.

[0013] Bromoacetic acid was dispersed in PBS buffer solution, EDC and N-hydroxysuccinimide were added and stirred, and the mixture was centrifuged and the supernatant was discarded. The activated bromoacetic acid was stirred with Trop-2 monoclonal antibody, centrifuged and purified, dried and dialyzed to obtain polymer III.

[0014] Potassium carbonate, polymer II, and polymer III were reacted in a dimethylformamide solution, purified by centrifugation, and lyophilized to obtain nano-prodrugs.

[0015] The structural formulas of polymers I, II, and III are shown below:

[0016]

[0017]

[0018] In one embodiment, the organic solvent is dimethyl sulfoxide.

[0019] In one embodiment, the catalyst is 4-dimethylaminopyridine.

[0020] In one embodiment, the inert gas is nitrogen.

[0021] In one embodiment, the molar ratio of 2-ethoxy-1,3,2-dioxaphosphane 2-oxide to dihydroxy disulfide is 16 to 22:1.

[0022] In one embodiment, the molar ratio of baicalin to polymer I is 1:1 to 1.5.

[0023] Thirdly, embodiments of this application provide the use of the nanoprodrug described in the first aspect in the preparation of a medicament for the prevention, relief or treatment of triple-negative breast cancer.

[0024] In one implementation, the drug is a drug used to promote the active uptake ability of breast cancer cells.

[0025] The drug is an injectable solution or a drug for oral administration.

[0026] The embodiments of this application have at least the following beneficial effects:

[0027] This application utilizes nanotechnology to modify traditional drug molecules by screening biocompatible polymer materials. Introducing a nanoprodrug strategy with protecting groups can improve the physicochemical properties of baicalin, enhancing stability and water solubility, increasing bioavailability, and reducing adverse reactions. Polyphosphate (PPE) materials, due to their adjustable properties, biodegradability, and biocompatibility, have promising application prospects. In this application, the PPE polymer backbone contains many repeating phosphate ester bonds, allowing the introduction of various functional groups. This application modifies the water solubility and functionality of PPE by polymerizing monomers with various side groups. Furthermore, by conjugating TROP-2 antibodies to the surface of baicalin and enhancing the recognition between TROP-2 antibodies and receptors on the cell membrane, the active uptake capacity of TNBC cells is improved, resulting in a redox-sensitive / TROP-2-targeted nanoprodrug with potential applications. This provides a new strategy for developing efficient and low-toxicity nanodrug delivery systems.

[0028] Therefore, this application presents a redox-sensitive / TROP2-targeting nanoprodrug based on the redox imbalance characteristics of the TNBC microenvironment and the overexpression of the TROP2 receptor in TNBC. This nanoprodrug has the advantages of specifically targeting tumor cells, achieving stable, spatiotemporally controllable drug release and activation at the target site over a long period of in vivo circulation. It can overcome microenvironment delivery barriers, has redox-sensitive release characteristics, and enters TNBC cells through receptor-mediated endocytosis, which helps to improve drug delivery efficiency and achieve rapid release at the tumor site. This suggests that the nanoprodrug has good application prospects in the preparation of drugs for the treatment of TNBC.

[0029] Additional aspects and advantages of this application will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of this application. Attached Figure Description

[0030] Figure 1 The image shows the proton NMR spectrum of the compound PEP.

[0031] Figure 2 The 1H NMR spectrum of the copolymer OH-ss-PPEP is shown.

[0032] Figure 3 The 1H NMR spectrum of baicalin BA;

[0033] Figure 4 The 1H NMR spectrum of the polymer BA-ss-PPEP;

[0034] Figure 5 The 1H NMR spectrum of the polymer Trop2-BA-ss-PPEP;

[0035] Figure 6 The infrared spectrum obtained by infrared spectroscopy in Example 2;

[0036] Figure 7 This is a TG curve of the sample obtained from the thermogravimetric analysis test in Example 2;

[0037] Figure 8 This is a transmission electron microscope (TEM) morphology diagram of Trop2-BA-ss-PPEP in Example 2;

[0038] Figure 9 This is a schematic diagram of the particle size distribution of Trop2-BA-ss-PPEP in Example 2;

[0039] Figure 10 This is a schematic diagram of the potential distribution of Trop2-BA-ss-PPEP in Example 2;

[0040] Figure 11 This is a schematic diagram of the stability test results of Trop2-BA-ss-PPEP in Example 2;

[0041] Figure 12 This is a schematic diagram of the test results of the positive control group in the in vitro hemolysis test of Example 2;

[0042] Figure 13 This is a schematic diagram of the test results of the control group in the in vitro hemolysis test of Example 2.

[0043] Figure 14 This is a schematic diagram of the test results of Trop2-BA-ss-PPEP in the in vitro hemolysis test of Example 2;

[0044] Figure 15 This is a schematic diagram of the response release curve of TROP2-BA-SS-PPEP in Example 2;

[0045] Figure 16 This is a schematic diagram of the Boltzmann curve fitting for the detection of critical micelle concentration using the pyrene fluorescent probe method in Example 2;

[0046] Figure 17This is a schematic diagram illustrating the qualitative uptake of C-6, C-6-BSP, and C-6-AN-TBSP by MDA-MB-231 cells in the cell uptake experiment of Example 3.

[0047] Figure 18 This is a schematic diagram illustrating the quantitative uptake of C-6, C-6-BSP, and C-6-AN-TBSP by MDA-MB-231 cells in the cell uptake experiment of Example 3.

[0048] Figure 19 This is a schematic diagram of the average fluorescence intensity of MDA-MB-231 cells uptake of C-6, C-6-BSP, and C-6-AN-TBSP in the cell uptake experiment of Example 3.

[0049] Figure 20 This is a schematic diagram of the test results of the cell viability experiment in Example 3;

[0050] Figure 21 This is a schematic diagram of the test results of the cell scratch experiment in Example 3;

[0051] Figure 22 This is a schematic diagram of the test results of the cell invasion experiment in Example 3;

[0052] Figure 23 This is a schematic diagram of in vivo imaging of the in vivo distribution experiment in Example 3;

[0053] Figure 24 This is a schematic diagram of mouse metastasis imaging in the antitumor effect experiment of Example 3;

[0054] Figure 25 This is a schematic diagram of the body weight of the mice used in the anti-tumor effect experiment in Example 3. Detailed Implementation

[0055] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0056] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0057] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of this application. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0058] The following explanations of some of the terms and materials used in this embodiment will be provided to facilitate understanding by those skilled in the art.

[0059] PEP: 2-ethoxy-1,3,2-dioxaphosphane 2-oxide; the structural formula of PEP is... .

[0060] BA: Baicalin, the structural formula of BA is .

[0061] TROP2: also known as TROP-2, the structural formula of TROP2 is mAb-NH2.

[0062] Trop2-BA-ss-PPEP: This refers to nano-prodrugs.

[0063] EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide.

[0064] DMSO: Dimethyl sulfoxide.

[0065] DMAP: 4-Dimethylaminopyridine.

[0066] NHS: N-hydroxysuccinimide.

[0067] The nanoprodrugs of this application, their preparation methods, and their uses will be described in detail below.

[0068] First, let me explain the nanoprodrug of this embodiment.

[0069] Nanoprodrugs

[0070] This embodiment provides a nanoprodrug; specifically, the nanoprodrug has the structure shown below for compounds or pharmaceutically acceptable salts thereof:

[0071] .

[0072] The molecular weight of the nanoprodrug is 47820.28.

[0073] It is understood that the nanoprodrugs provided in this embodiment improve traditional drug molecules using nanotechnology by screening biocompatible polymer materials. Introducing a nanoprodrug strategy with protecting group modifications can improve the physicochemical properties of baicalin, enhancing stability and water solubility, increasing bioavailability, and reducing adverse reactions. Among these, polyphosphate (PPE) materials have promising applications due to their adjustable properties, biodegradability, and biocompatibility. In this embodiment, the PPE polymer backbone contains many repeating phosphate ester bonds, allowing the introduction of various functional groups. This embodiment modifies the water solubility and functionality of PPE by polymerizing monomers with various side groups, and enhances the active uptake capacity of TNBC cells by conjugating TROP-2 antibodies to the surface of baicalin and their recognition with receptors on the cell membrane. This yields a redox-sensitive / TROP-2-targeting nanoprodrug with potential applications, providing a new strategy for developing efficient and low-toxicity nanodrug delivery systems.

[0074] Therefore, this embodiment constructs a redox-sensitive / TROP2-targeting nanoprodrug based on the redox imbalance characteristics of the TNBC microenvironment and the overexpression of the TROP2 receptor in TNBC. This nanoprodrug has the advantages of specifically targeting tumor cells, achieving stable long-term circulation in vivo, and spatiotemporally controllable target site drug release and activation. It can overcome microenvironment delivery barriers, has redox-sensitive release characteristics, and enters TNBC cells through receptor-mediated endocytosis, which helps to improve drug delivery efficiency and achieve rapid release at the tumor site. This suggests that the nanoprodrug has good application prospects in the preparation of drugs for the treatment of TNBC.

[0075] It is understood that the pharmaceutically acceptable salts of the nanoprodrugs provided in this embodiment as described above can be: sodium salts, potassium salts, ammonium salts, amino acid salts, lactates, hydrochlorides, phosphates, acetates, malates, citrates, or aspartates, etc. This embodiment does not specifically limit the drug salts.

[0076] Next, the preparation method of the above-mentioned nanoprodrug will be described.

[0077] Preparation method

[0078] This embodiment also provides a method for preparing the above-mentioned nanoprodrug, the method comprising:

[0079] (1) Under an inert gas atmosphere, 2-ethoxy-1,3,2-dioxaphosphane 2-oxide, dihydroxy disulfide and Sn(Oct)2 were added to anhydrous dichloromethane and reacted to obtain polymer I;

[0080] (2) Dissolve baicalin in an organic solvent, add EDC and catalyst to react; add the reactants to a dimethylformamide solution of polymer I, and dialysis to purify the polymer II after the reaction;

[0081] (3) Disperse bromoacetic acid in PBS buffer solution, add EDC and N-hydroxysuccinimide and stir to react, centrifuge the mixture and discard the supernatant; stir activated bromoacetic acid with Trop-2 monoclonal antibody and centrifuge to purify, dry and dialyze to obtain polymer III;

[0082] (4) Potassium carbonate, polymer II and polymer III were reacted in dimethylformamide solution, purified by centrifugation, and lyophilized to obtain nano-prodrugs.

[0083] The structural formulas of polymers I, II, and III are shown below:

[0084]

[0085] The following will further explain each step of the above preparation method.

[0086] In step (1), the inert gas is nitrogen, preferably under a dry nitrogen atmosphere. The reaction process in step (1) can be found in the following synthetic route:

[0087] .

[0088] In this step, polymer I, namely polyphosphite-based random copolymer OH-ss-PPEP, is obtained by reacting 2-ethoxy-1,3,2-dioxaphosphatane 2-oxide and dihydroxy disulfide.

[0089] In step (1), the molar ratio of 2-ethoxy-1,3,2-dioxaphosphane 2-oxide to dihydroxy disulfide is 16 to 22:1, for example 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1.

[0090] Next, based on the polyphosphate-based random copolymer OH-ss-PPEP, it was modified onto BA to obtain the polymer BA-ss-PPEP.

[0091] The reaction process in step (2) can be found in the following synthetic route:

[0092] .

[0093] In step (2), the organic solvent is dimethyl sulfoxide and the catalyst is 4-dimethylaminopyridine.

[0094] The molar ratio of baicalin to polymer I is 1:1 to 1.5, for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, and 1:1.5.

[0095] Next, in step (3), polymer III is prepared, and in step (4), polymer III is conjugated to the surface of polymer BA-ss-PPEP.

[0096] The reaction process in step (3) can be found in the following synthetic route:

[0097] .

[0098] The reaction process in step (4) can be found in the following synthetic route:

[0099] .

[0100] Therefore, this embodiment modulates the water solubility and functionality of PPE by polymerizing monomers with various side groups, and enhances the active uptake capacity of TNBC cells by conjugating TROP-2 antibody on the surface of baicalin to recognize receptors on the cell membrane.

[0101] For example, after obtaining the nanoprodrug, the nanoprodrug can be dissolved in ethanol. Under stirring conditions, the ethanol solution of the prodrug is slowly added dropwise to water, and the prodrug spontaneously forms a solution containing uniform nanoparticles. The ethanol in the solution containing uniform nanoparticles is removed by vacuum rotary evaporation to obtain a nanomicelle solution without organic solvents.

[0102] The synthesis process of nanoprodrugs will be further clarified below, in conjunction with specific preparation methods.

[0103] Specifically, the preparation methods of nanoprodrugs include:

[0104] (1) Synthesis of polyphosphate-based random copolymer OH-ss-PPEP (or PPEP)

[0105] Under a dry nitrogen atmosphere, 2-ethoxy-1,3,2-dioxaphosphatane 2-oxide (PEP, 1080.84 mg, 6.00 mmol) and dihydroxy disulfide (46.27 mg, 0.30 mmol) and 10 mL of anhydrous dichloromethane were added to a 50 mL round-bottom flask equipped with a magnetic stirrer. Then, 0.2 mL of Sn(Oct)₂dichloromethane solution (0.486 mg, 0.0012 mmol) was added, and the mixture was reacted at 45 °C for 2 h. The crude product was precipitated twice in cold diethyl ether:methanol = 10:1 V / V. The precipitate was concentrated under reduced pressure at room temperature to give OH-ss-PPEP.

[0106] (2) Synthesis of polymer BA-ss-PPEP (BSP)

[0107] BA (446.36 mg, 1 mmol) was dissolved in 10 mL of DMSO solution and added to a 50 mL round-bottom flask. EDC (299.50 mg, 1.5 mmol) was slowly added under ice bath conditions. Then DMAP (24.43 mg, 0.2 mmol) was added, and the mixture was reacted at 0 °C for 30 min. The reactants were then added to 2 mL of DMF solution containing OH-ss-PPEP (380 mg, 1.2 mmol), and the mixture was reacted overnight at room temperature. The mixture was purified by dialyzing (MWCO 3500) for 48 h using a mixed solvent of ethanol-Milli-Q water (v / v ratio from 3 / 1 to 0 / 1) for lyophilization. The resulting pale yellow viscous product, BA-ss-PPEP, was obtained after lyophilization.

[0108] (3) Synthesis of antibody conjugates

[0109] Bromoacetic acid (11.116 mg, 0.08 mmol) was dispersed in 1 mL of PBS buffer (pH 5.0) and stirred with EDC (9.585 mg, 0.05 mmol) and NHS (5.755 mg, 0.05 mmol) at 25 °C for 30 min. The mixture was centrifuged at 8000 rpm for 15 min, and the supernatant was discarded. The activated bromoacetic acid was stirred with 1 mL of PBS (pH 7.0) and Trop-2 monoclonal antibody (12 μL, 0.5 mg / mL) at 4 °C for 12 h. The mixture was purified by ultrafiltration and centrifugation, and then lyophilized to obtain the antibody conjugate bromo-mAb.

[0110] (4) Synthesis of Trop2-BA-ss-PPEP (TBSP)

[0111] The reaction of brominated mAb with BA-ss-PPEP: Potassium carbonate (276.22 mg, 2.0 mmol), 1 mL of bromoacetyl mAb (45-65 g, 1.0 mmol), and BA-ss-PPEP (446.36 mg, 1.5 mmol) were reacted overnight at 35°C in 10 mL of DMF solution. The mixture was purified by ultrafiltration and centrifugation, and then lyophilized to obtain the pale yellow viscous product Trop2-BA-ss-PPEP, i.e., the nano-prodrug.

[0112] Secondly, the uses of the aforementioned nanoprodrugs will be explained.

[0113] Applications of nanoprodrugs

[0114] As mentioned above, this embodiment constructs a redox-sensitive / TROP2-targeting nanoprodrug based on the redox imbalance characteristics of the TNBC microenvironment and the overexpression of the TROP2 receptor in TNBC. This nanoprodrug has the advantages of specifically targeting tumor cells, achieving stable long-term circulation in vivo, and spatiotemporally controllable target site drug release and activation. It can overcome microenvironment delivery barriers, has redox-sensitive release characteristics, and enters TNBC cells through receptor-mediated endocytosis, which helps to improve drug delivery efficiency and achieve rapid release at the tumor site. This suggests that the nanoprodrug has good application prospects in the preparation of drugs for the treatment of TNBC.

[0115] Based on this, the nanoprodrug provided in this embodiment can be used to prepare drugs for the prevention, relief or treatment of triple-negative breast cancer.

[0116] Specifically, the aforementioned drugs are used to promote the active uptake ability of breast cancer cells.

[0117] For example, the above-mentioned drug is an injectable solution or a drug for oral administration.

[0118] Typically, when the aforementioned nanoprodrugs are formulated into corresponding drugs, the drugs also include pharmaceutically acceptable carriers and / or excipients.

[0119] The drug described in this embodiment uses nano-prodrugs as its active ingredient. However, variations in the formulation system and administration method, pharmaceutical salts derived from simple chemical modifications of the nano-prodrugs, and the combined use of multiple compounds are not excluded.

[0120] For example, in this embodiment, one or more compounds in the nano-prodrug of this embodiment can be formulated as active ingredients into a non-toxic, inert, and pharmaceutically acceptable carrier and / or excipient; the formulated drug can be administered via conventional routes, including but not limited to oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or local administration.

[0121] For example, when the dosage form of the drug in this embodiment is a drug for oral administration, it contains a safe and effective amount of nano-prodrug and a pharmaceutically acceptable carrier and / or excipient. The drug for oral administration can be made into commonly used dosage forms such as tablets, pills, powders, granules, capsules, emulsions, syrups, ointments, and suppositories. In this embodiment, no specific limitation is made on the carrier and / or excipient. The carrier and / or excipient can be adaptively adjusted according to the specific drug dosage form.

[0122] Typically, the “effective amount” of a compound (nanoprodrug) refers to the amount sufficient to elicit a response in the target organism. As those skilled in the art will understand, the effective amount of the compound in this embodiment can vary depending on factors such as the drug’s mediators and the subject’s age, health status, and symptoms of triple-negative breast cancer.

[0123] The effective dose includes the therapeutic effective dose and the preventive effective dose.

[0124] Unless otherwise stated, the “therapeutic effective amount” of the compounds used in this embodiment is an amount sufficient to provide benefit in the treatment of triple-negative breast cancer, or a minimum amount that improves or alleviates one or more symptoms (manifestations) associated with the triple-negative breast cancer state. The “preventive effective amount” of the compounds used in this embodiment is an amount sufficient to prevent the occurrence of triple-negative breast cancer, or an amount sufficient to prevent one or more symptoms associated with the occurrence of triple-negative breast cancer state.

[0125] It is understood that the drug in this embodiment can also be formulated into an injection. For example, the nano-prodrug can be combined with water for injection, physiological saline, and glucose solution to form a corresponding injection under aseptic conditions. The above-mentioned injection can be prepared by conventional methods.

[0126] The present application will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustration / explanation only and are not intended to limit the scope of the present application.

[0127] Unless otherwise specified, all materials, reagents and instruments used in the following embodiments are commercially available.

[0128] Example 1

[0129] The synthetic route for the nanoprodrug Trop2-BA-ss-PPEP is shown below:

[0130]

[0131] Specifically, it includes the following steps:

[0132] (1) Synthesis of polyphosphate-based random copolymer OH-ss-PPEP (or PPEP)

[0133] Under a dry nitrogen atmosphere, 2-ethoxy-1,3,2-dioxaphosphatane 2-oxide (PEP, 1080.84 mg, 6.00 mmol) and dihydroxy disulfide (46.27 mg, 0.30 mmol) and 10 mL of anhydrous dichloromethane were added to a 50 mL round-bottom flask equipped with a magnetic stirrer. Then, 0.2 mL of Sn(Oct)₂dichloromethane solution (0.486 mg, 0.0012 mmol) was added, and the mixture was reacted at 45 °C for 2 h. The crude product was precipitated twice in cold diethyl ether:methanol = 10:1 V / V. The precipitate was concentrated under reduced pressure at room temperature to give OH-ss-PPEP.

[0134] (2) Synthesis of polymer BA-ss-PPEP (BSP)

[0135] BA (446.36 mg, 1 mmol) was dissolved in 10 mL of DMSO solution and added to a 50 mL round-bottom flask. EDC (299.50 mg, 1.5 mmol) was slowly added under ice bath conditions. Then DMAP (24.43 mg, 0.2 mmol) was added, and the mixture was reacted at 0 °C for 30 min. The reactants were then added to 2 mL of DMF solution containing OH-ss-PPEP (380 mg, 1.2 mmol), and the mixture was reacted overnight at room temperature. The mixture was purified by dialyzing (MWCO 3500) for 48 h using a mixed solvent of ethanol-Milli-Q water (v / v ratio from 3 / 1 to 0 / 1) for lyophilization. The resulting pale yellow viscous product, BA-ss-PPEP, was obtained after lyophilization.

[0136] (3) Synthesis of antibody conjugates

[0137] Bromoacetic acid (11.116 mg, 0.08 mmol) was dispersed in 1 mL of PBS buffer (pH 5.0) and stirred with EDC (9.585 mg, 0.05 mmol) and NHS (5.755 mg, 0.05 mmol) at 25 °C for 30 min. The mixture was centrifuged at 8000 rpm for 15 min, and the supernatant was discarded. The activated bromoacetic acid was stirred with 1 mL of PBS (pH 7.0) and Trop-2 monoclonal antibody (12 μL, 0.5 mg / mL) at 4 °C for 12 h. The mixture was purified by ultrafiltration and centrifugation, and then lyophilized to obtain the antibody conjugate bromo-mAb.

[0138] (4) Synthesis of Trop2-BA-ss-PPEP (TBSP)

[0139] The reaction of brominated mAb with BA-ss-PPEP: Potassium carbonate (276.22 mg, 2.0 mmol), 1 mL of bromoacetyl mAb (45-65 g, 1.0 mmol), and BA-ss-PPEP (446.36 mg, 1.5 mmol) were reacted overnight at 35°C in 10 mL of LMF solution. The mixture was purified by ultrafiltration and centrifugation, and then lyophilized to obtain the pale yellow viscous product Trop2-BA-ss-PPEP.

[0140] (5) Antisolvent precipitation method

[0141] The nanoprodrug was dissolved in ethanol. Under stirring, the ethanol solution of the prodrug was slowly added dropwise to water, and the prodrug spontaneously formed a solution containing uniform nanoparticles. The ethanol in the solution containing uniform nanoparticles was removed by rotary evaporation under reduced pressure to obtain a nanomicelle solution without organic solvents (hereinafter referred to as TROP2-BA-SS-PPEP solution).

[0142] Example 2

[0143] The effectiveness of Trop2-BA-ss-PPEP prepared in Example 1 will be verified below.

[0144] Specifically, it includes:

[0145] 2.1. Proton nuclear magnetic resonance spectroscopy (¹H-NMR)

[0146] The Bruker AC-400 Fourier transform nuclear magnetic resonance spectrometer (FT-NMR) was used at 400 MHz at room temperature, with chloroform-d1 (CDCl3) as solvent and tetramethylsilane (TMS) as internal standard.

[0147] 2.2 Mass spectrometry detection of polymer molecular weight (SEC)

[0148] Polymer molecular weight (SEC) was determined using a three-in-one mass spectrometer, ranging from 350 to 15000 m / z. Data analysis was performed using Xcalibur software, and the Xtract function within Xcalibur was used to deconvolve the mass spectra.

[0149] 2.3. Fourier Transmission Infrared Spectroscopy (FTIR)

[0150] Weigh the sample and grind it with dry potassium bromide at a ratio of 1:100 until homogeneous. Press the mixture into a sample and scan it with an infrared spectrometer at 4000-500 cm⁻¹. -1 Infrared spectrum within the range.

[0151] 2.4 Thermogravimetric Analysis (TG)

[0152] The thermal properties of BA, PPEP, BA-SS-PPEP, and TROP2-BA-SS-PPEP were studied using a TG analyzer (PerkinElmer, USA). TG data were recorded under conditions of 30℃-600℃ and a nitrogen flow rate of 10℃ / min.

[0153] 2.5 Observation of the microscopic morphology of nanoparticles

[0154] Take TROP2-BA-SS-PPEP aqueous solution, drop it onto a coated copper mesh for adsorption and sample preparation, stain with 2% phosphotungstic acid, and air dry to prepare sample slides. Observe particle morphology with transmission electron microscopy.

[0155] 2.6 Determination of Zeta Potential and Particle Size of Nanoparticles

[0156] The TROP2-BA-SS-PPEP nanomicelle solution was taken, and the particle size and zeta potential were measured using a particle size analyzer to investigate the distribution of particle size and zeta potential.

[0157] 2.7 Measurement of Load Capacity (DLC) and Load Efficiency (DLE)

[0158] Take 200 μL of BA-SS-PPEP and TROP2-BA-SS-PPEP solutions into a 10 KD ultrafiltration tube, centrifuge at 12000 rpm / min for 5 min, dilute the supernatant with methanol and bring to volume with a 2 mL volumetric flask, filter through a 0.45 μm organic filter membrane, and determine the free BA content (Wt) by HPLC.

[0159] 2.8. In vitro stability study of nanoparticles

[0160] To evaluate the in vitro stability of the nanoparticles, TROP2-BA-SS-PPEP was redispersed in deionized water and PBS (pH 7.4) to simulate the nanoparticle storage environment. The stability of the nanoparticles was investigated over three months at 4℃ and 25℃, using particle size and potential as evaluation indicators.

[0161] 2.9 In vitro hemolysis test

[0162] Blood was collected from healthy mice and placed in centrifuge tubes containing an anticoagulant (whole blood: 2.5% sodium citrate (volume ratio) = 9:1). After washing, the resulting red blood cells were prepared into a 4% suspension using 0.9% sodium chloride solution. 900 μL of the red blood cell suspension was added to each centrifuge tube, followed by 100 μL of BA, PPEP, and BA-SS-PPEP (4.76 μg / mL based on BA), 0.9% sodium chloride solution (negative control), and different concentrations of Trop2-BA-SS-PPEP (9.52, 7.14, 4.76, 2.38, 1.19, and 0.60 μg / mL based on BA). The positive control (Triton-X-100) was set at 4%, 2%, 1%, 0.5%, 0.25%, and 0.125%. The tubes were incubated at 37℃ for 1 h, centrifuged at 1000 rpm for 15 min, and hemolysis was observed.

[0163] 2.10. Investigation on the in vitro redox-sensitive release of nanoparticles

[0164] BA and TROP2-BA-SS-PPEP were used to evaluate in vitro drug release in various PBS media: (1) pH 7.4, (2) pH 7.4, containing 2 mM GSH, (3) pH 5.5, (4) pH 5.5, containing 2 mM GSH, (1) pH 6.5, (2) pH 6.5, containing 2 mM GSH, (3) pH 6.5, containing 10 mM GSH. Release was achieved by constant-rate shaking in an air bath at 37°C. 0.5 mL of the external solution was taken at set times, and the same volume of PBS buffer was added to the dialysate. The in vitro drug release rate was calculated.

[0165] 2.11 Self-assembly of polymer prodrugs

[0166] A predetermined amount of pyrene was added to a series of ampoules, and the acetone was completely removed under vacuum. Then, 5 mL of polymer solutions of different concentrations were added to each ampoule, maintaining a pyrene concentration of 6 × 10⁻⁶ mol / L in each ampoule. The solutions were stirred at 25 °C for 24 h to reach equilibration. Fluorescence spectra were recorded using a microplate reader; excitation was performed at 335 nm, and emission spectra were recorded from 350–500 nm. The slit width for both excitation and emission was set to 1 nm.

[0167] The test results are as follows:

[0168] (1) Proton nuclear magnetic resonance spectroscopy (¹H-NMR)

[0169] Please see Figure 1 and Figure 2 The chemical structures of PPEP and PEP were characterized by 1H NMR analysis, revealing the corresponding chemical shifts of the protons in the initiator and copolymer. We observed that the original chemical shifts at δ4.70 ppm (peaks 3 and 4), δ4.17 ppm, and δ4.26 ppm in PEP disappeared after the reaction, replaced by new proton signals (peaks 5, 8, and 9) at δ3.45 ppm, δ3.69 ppm, and δ3.84 ppm. This indicates that the hydroxyl group at the C-1′ position of the bis(2-hydroxyethyl) disulfide successfully initiated the PEP monomer to yield PEEP.

[0170] Please refer to Figure 3 and Figure 4 BA-ss-PPEP is a product generated by the Steglich esterification reaction of BA and PPEP. By comparing the two spectra, the chemical shift of peak 7 changed from δ3.79ppm to δ4.20ppm due to the influence of the ester group, and the characteristic peak of the benzene ring of BA appeared in the range of 6.0-8.0ppm. New proton signals appeared at δ5.86ppm, δ6.57ppm, δ6.73ppm, δ7.95ppm, and δ8.12ppm. Figure 3The original chemical shifts (peak k) at δ8.69 ppm and δ12.59 ppm after the reaction were observed at... Figure 4 The disappearance of the group indicates that the carboxyl group at the C-6′ position of BA has been coupled with PPEP to obtain BA-ss-PPEP.

[0171] Please see Figure 4 The novel proton signals of TROP2-BA-ss-PPEP appeared at (peaks 10 and 11) δ4.67 ppm and δ6.94 ppm, respectively, representing the methylene hydrogen signal at the C-2′ position of bromoacetic acid and the amino hydrogen signal of the monoclonal antibody Trop-2. The original chemical shifts (peak f) at δ8.69 ppm and δ12.59 ppm disappeared after the reaction, indicating that the phenolic hydroxyl groups at the C-5 and C-6 positions of BA had been coupled with Trop-2 to obtain TROP2-BA-ss-PPEP. That is, the 5′-OH and C-6′-OH of BA have less steric hindrance and higher activity than 14′-OH, 15′-OH, and 16′-OH. TROP2-BA-ss-PPEP could find all the peaks from the PEEP block, BA, and Trop-2 moieties, proving the successful conjugation of BA and Trop-2 with PEEP.

[0172] (2) Polymer molecular weight (SEC)

[0173] Data calculated based on 1H NMR results. Please refer to Table 1 below. Based on the 1H NMR spectra, the calculated molecular weights of BSP and TBSP are 1124.84 g / mol and 47820.28 g / mol, respectively. Further analysis by biomass spectrometry revealed molecular weights of BSP and TBSP of 1349.14 g / mol and 49154.5 g / mol, respectively. Deconvolution analysis revealed a regular interval, consistent with the molecular weight (152.09) of a PEEP repeating unit. The apparent discrepancy between the molecular weights of polyphosphates determined by 1H NMR and biomass spectrometry may be attributed to the reduced sensitivity of 1H NMR detection for number-average molecular weights (Mn) above 3000, thus affecting the accuracy of molecular weight assessment.

[0174] Table 1: Molecular weights of BSP and TBSP

[0175]

[0176] (3) Fourier Transmission Infrared Spectroscopy (FTIR)

[0177] Please see Figure 6 The infrared spectrum of the BA molecule is shown at 3418.81 cm⁻¹. -1 The intermolecular hydrogen bond stretching vibration (OH) is observed, resulting in a broad absorption peak at 2968.81 cm⁻¹. -1It is a CH stretching vibration; 1600~1450cm -1 It is a C=C skeletal vibration of the benzene ring, with out-of-plane bending vibration absorption of CH in the 880–680 cm⁻¹ range; 1726.55 cm⁻¹ -1 Carboxylic acid C=O absorption at 920 cm⁻¹ -1 The out-of-plane bending vibration of the OH bonds leads to the formation of carboxylic acid dimers; 1660.08 cm -1 A strong peak appears at the C=O stretching vibration of the ketone group. Conjugation of the carbonyl group with an alkene bond or an aromatic ring will decrease the absorption frequency. The out-of-plane bending vibration of CH in olefins (1000–675 cm⁻¹) -1 ); 1150~1060cm -1 The stretching vibration exhibits a strong absorption peak (CO), 1050–1000 cm⁻¹. -1 It is absorbed by the two CO stretching vibrations of the aromatic ether.

[0178] The PPEP molecule is at 3401.66 cm⁻¹. -1 The broad absorption peak is due to the intermolecular hydrogen bond stretching vibration of OH: CH stretching vibration (2981.36 cm⁻¹). -1 ), 1453.79cm -1 The out-of-plane bending vibration is observed in CH; the stretching vibration of PC is absorbed at 1394.96 cm⁻¹. -1 The stretching vibration of P=O occurs at 1255.12 cm. -1 The antisymmetric stretching vibration of POC is at 1165.26 cm. -1 The stretching vibration of PO is at 962.54 cm. -1 CO stretching vibration absorption at 1013.68 cm⁻¹ -1 A strong absorption peak appears, with out-of-plane bending vibration of OH at 769-659 cm⁻¹; 544.74 cm⁻¹ -1 It has a characteristic absorption peak of disulfide bonds.

[0179] BSP is 3334.78cm -1 The broad absorption peak is due to the intermolecular hydrogen bond stretching vibration of OH: CH stretching vibration (2959.39 cm⁻¹). -1 ), 1440.18cm -1 It is an out-of-plane bending vibration of CH; 1562.52 cm -1 1440.18cm -1 It is a C=C skeletal vibration of the benzene ring, 801.02 cm⁻¹ -1 729.63cm -1 699.07cm -1 Absorption occurs due to the out-of-plane bending vibration of the benzene ring (CH); 1716.13 cm⁻¹ -1 The ester C=O absorption at 1655.13 cm⁻¹-1 The ketone C=O absorption and CO stretching vibration absorption occur at 1017.2 cm⁻¹. -1 A strong absorption peak appears; the stretching vibration absorption of PC is at 1392.18 cm⁻¹. -1 The stretching vibration of P=O occurs at 1258.23 cm. -1 The antisymmetric stretching vibration of POC is at 1097.08 cm⁻¹. -1 The stretching vibration of PO is at 978.61 cm. -1 510.09cm -1 It has a characteristic absorption peak of disulfide bonds.

[0180] Understandably, due to the large number of amide bonds introduced by the monoclonal antibody, TBSP exhibits a broad and large NH stretching vibration absorption peak at 3423.76 cm⁻¹ and 1085.39 cm⁻¹. -1 Absorbs CN stretching vibrations; 2964.54cm -1 This is a CH stretching vibration, 880–680 cm. -1 Out-of-plane bending vibration of the benzene ring CH; 1651.45cm -1 A strong peak appears at C=O stretching vibration, while CO stretching vibration absorption occurs at 1045.33 cm⁻¹. -1 PC absorbs stretching vibrations at 1400.97cm. -1 The stretching vibration of P=O occurs at 1216.47 cm. -1 The stretching vibration of PO is at 952.93 cm. -1 561.79cm -1 It has a characteristic absorption peak of disulfide bonds.

[0181] (4) Thermogravimetric analysis

[0182] Please see Figure 7 The TG curves of the samples showed no significant weight loss between 100 and 300 °C, but weight loss was observed in the 400-600 °C range, possibly related to dehydroxylation and decarbonization. At the end of the measurement at 600 °C, the residual masses of BA, PPEP, BSP, and TBSP were 40.5%, 24.0%, 21.7%, and 46.8%, respectively. Compared to BSP, the polyphosphate groups increased the thermal stability of BA. Because TBSP is conjugated with the antibody, and the antibody has low thermal stability, the thermal loss of TBSP increased.

[0183] (5) Micromorphology of nanoparticles

[0184] Please see Figure 8 Under a transmission electron microscope, TROP2-BA-SS-PPEP particles were found to be uniform in size and well-dispersed, with a particle size of approximately 100 nm.

[0185] (6) Particle size and potential

[0186] Please see Figure 9 The diagram shows the particle size distribution of Trop2-BA-SS-PPEP. The particle size of TROP2-BA-SS-PPEP is (127.9±8.52) nm, and the PDI is 0.145±0.17, indicating that the prepared nanoparticles have uniform particle size, good dispersibility, and good stability. Please refer to [link / reference]. Figure 10 The potential distribution diagram of Trop2-BA-SS-PPEP is shown. The Zate potential of the Trop2-BA-SS-PPEP aqueous solution is (-36.2±8.48) mV. The nanoparticles with negative potential tend to maintain a stable state of mutual repulsion.

[0187] (7) Measurement of load capacity (DLC) and load efficiency (DLE)

[0188] Three batches of TROP2-BA-SS-PPEP were prepared in parallel, and their loading capacity (DLC) and loading efficiency (DLE) were determined. Please refer to Table 2 below. The results show that the optimized nanoparticles have relatively stable DLC (22.78±0.006)% and DLE (71.09±0.002)%, indicating that the optimized preparation method has good reproducibility.

[0189] Table 2: DLC and DLE (n=3)

[0190]

[0191]

[0192] (8) In vitro stability study

[0193] Please see Figure 11 The diagram illustrates the stability test results, with the left side showing the particle size stability test results and the right side showing the potential stability test results. When TROP2-BA-SS-PPEP aqueous solution was stored at 4℃ and 25℃, the particle size change trend within 12 weeks was relatively gradual. The particle size increase trend of TROP2-BA-SS-PPEP after incubation with the same volume of PBS was greater than that of aqueous solution, and the increase was significant at room temperature. The absolute value of the potential of TROP2-BA-SS-PPEP gradually decreased with increasing storage time. The absolute value of the initial potential of TROP2-BA-SS-PPEP in PBS solution was lower than that in aqueous solution, possibly because the surface charge of the nanoparticles was destroyed by ions in the PBS solution, making them prone to aggregation and sedimentation, thus affecting the stability of the nanoparticles. Therefore, the TROP2-BA-SS-PPEP prepared in this embodiment showed good stability in aqueous solution at 4℃.

[0194] (9) In vitro hemolysis test

[0195] Please see Figures 12-14 , Figure 12 This is a schematic diagram of the test results for the positive control group (Triton-X-100); Figure 13 This is a schematic diagram showing the test results for the control group (from left to right: 0.9% sodium chloride solution, BA, PPEP, and BA-SS-PPEP). Figure 14 This is a schematic diagram of the test results for Trop2-BA-ss-PPEP; combined with... Figure 13 As shown, in the negative control group, red blood cells settled and the supernatant was colorless and transparent, indicating no hemolysis. Figure 12 As shown, the positive control group appeared clear and red, with only a small number of red blood cells remaining at the bottom of the tube, indicating significant hemolysis; combined with Figure 14 As shown, red blood cells sank in each tube of the sample group, and the supernatant was colorless and transparent, with no hemolysis or agglutination observed.

[0196] (10) Response release of nanoparticles

[0197] Please see Figure 15 The schematic diagram of the response-release curve is shown. To evaluate the response-release characteristics of TROP2-BA-SS-PPEP, its in vitro drug release under different pH and GSH conditions was studied, and the BA release rate was calculated by HPLC. In the absence of GSH, TROP2-BA-SS-PPEP slowly releases BA; compared with 2 mM GSH, 10 mM GSH significantly accelerated the release of TROP2-BA-SS-PPEP within 12 h, indicating that high concentrations of GSH induce the reduction-responsive drug release behavior of TROP2-BA-SS-PPEP. Low pH conditions can accelerate the release behavior of TROP2-BA-SS-PPEP. At pH 6.5 and 5.5, BA can be completely released after 36 h of incubation, while at pH 7.4, only about 72.7% of BA is released. The reason for these phenomena may be that the acidic environment leads to the breakage of disulfide bond joints, resulting in a loose nanoparticle structure and accelerating BA release.

[0198] Therefore, TROP2-BA-SS-PPEP can achieve responsive drug release in high-concentration GSH or acidic environments. The release trend of monomeric BA did not change significantly under different GSH concentrations; BA was relatively stable under acidic conditions, exhibiting a burst release at pH 7.4, while release was slower at pH 5.5 and 6.5, indicating that monomeric BA has low stability under physiological conditions and is prone to premature leakage.

[0199] (11) Detection of critical micelle concentration using pyrene fluorescent probe method

[0200] Please see Figure 16The fluorescence intensity of pyrene, I3 / I1, is plotted on the ordinate, and the logarithm of the nanosolution concentration, logC, is plotted on the abscissa. A1 and A2 are the maximum and minimum values ​​of I3 / I1, respectively, and x0 is the midpoint of the curve abrupt change, corresponding to the critical micelle concentration of the sample.

[0201] The variation of the I3 / I1 value with the surfactant solution concentration conforms to the Boltzmann curve: y=A2+(A1-A2) / 1+exp((x-x0) / dx)). Curve fitting of the results yielded the midpoint of the abrupt change x0, determining the critical micelle concentration to be 15.44μg / mL.

[0202] Example 3

[0203] The efficacy of the nanoprodrug prepared in Example 1 in inhibiting breast cancer metastasis was evaluated.

[0204] (1) Qualitative and quantitative cell uptake experiments

[0205] Qualitative analysis of cellular uptake was performed using coumarin-6 (C6)-loaded pH-responsive nanoparticles at a final C6 concentration of 10 μg / mL to investigate the uptake of TROP2-BA-SS-PPEP by MDA-MB-231 cells. C6, C6-AN-FA, and C6-AN-FA@mPEG were added, with a blank control group included. Cells were incubated at 37°C for 2 h in cell culture medium at pH 7.4 or 6.5. 500 μL of LDAPI was added for staining for 10 min. The uptake fluorescence intensity within MDA-MB-231 cells was observed using laser confocal microscopy. Cells were collected by centrifugation after trypsin digestion (without EDTA), and washed with PBS to remove fluorescent dye from the cell surface. After centrifugation, the cells were resuspended in flow cytometry tubes containing 500 μL of PBS, and the uptake fluorescence intensity was detected using flow cytometry (FACSCalibur, BD, USA).

[0206] (2) Cell viability experiment

[0207] MDA-MB-231 cell suspension was seeded at 4 × 10⁴ cells / well in 96-well plates and cultured for 24 h. Then, 100 μL / well of different concentrations of drug-containing culture medium were added: BA (18.0, 35.0, 72.0, 90.0, 108.0, 126.0, 144.0, 162.0 μg / mL), PPEP (1.8, 3.5, 7.2, 10.8, 14.4, 28.8, 57.6, 115.2 μg / mL), and BA-ss-PPE. P (based on BA, 1.8, 3.5, 5.4, 7.2, 9.0, 10.8, 12.6, 14.4 μg / mL), Trop2-BA-ss-PPEP (based on BA, 1.8, 3.5, 5.4, 7.2, 9.0, 10.8, 12.6, 14.4 μg / mL), positive control PTX (1.8, 3.5, 7.2, 10.8, 14.4, 28.8, 57.6, 115.2 μg / mL), and a blank control were also included. After incubation, 10 μL of MTT solution was added and the cells were cultured for 4 h. The MTT reaction solution and culture medium were then removed, and 150 μL of DMSO solution (cell grade) was added to dissolve formazan. The cells were incubated at 37°C with shaking for 20 min, and the OD value at 490 nm was measured using a microplate reader.

[0208] (3) Cell scratch test

[0209] MDA-MB-231 cell suspension was seeded at 1.2 × 10⁶ cells / well in 6-well plates and cultured for 24 h. Then, BA and BA-SS-PPEP (4.76 μg / mL based on BA), different concentrations of Trop2-BA-SS-PPEP (2.38, 4.76, and 9.52 μg / mL based on BA), and PTX were added as positive controls for 24 h. Using a pipette tip or other hard object, lines were drawn in the central region of the monolayer of cells, and the central portion of cells was removed. Cells were then cultured for the set experimental time to assess cell growth and migration ability.

[0210] (4) Cell invasion assay

[0211] Matrigel was melted at 4°C beforehand, and the pipette tip was pre-cooled. Matrigel was diluted 1:8 (8 parts Matrigel to 64 parts serum-free medium) on ice. 60 μL of Matrigel was vertically added to the upper surface of the membrane at the bottom of the Transwell chamber, and the mixture was incubated at 37°C for 3 hours to allow Matrigel to polymerize into a gel. The basement membrane was then hydrated at 37°C for 30 minutes. Cells were starved of serum for 24 hours. Cells were digested and added to the Transwell chamber, with 500 μL of complete medium added to the lower chamber. BA and BA-SS-PPEP (4.76 μg / mL based on BA), different concentrations of Trop2-BA-SS-PPEP (2.38, 4.76, and 9.52 μg / mL based on BA), and PTX were added as a positive control for 24 hours. The bottom of the chamber was stained with 0.1% crystal violet, and cells were immediately observed and counted under a microscope.

[0212] (5) Distribution in the body

[0213] Subcutaneous inoculation of 4T1 cells (2×10⁻⁶) 6 A mouse model of breast cancer was established using cells, and the tumor grew to 150 mm. 3 The experiment began around 10:00 AM. Twenty-one female nude mice (3-5 weeks old) were randomly divided into three groups: ICG saline, ICG-BA-SS-PPEP, and ICG-Trop2-BA-SS-PPEP, with three mice in each group, numbered 1-3. Administration was via tail vein injection, administered once. In vivo imaging was performed at 0h, 3h, 6h, 12h, 24h, and 48h post-administration; mice were sacrificed after 48h, and tumors and imaging of various organs (heart, liver, spleen, lung, and kidney) was performed.

[0214] (6) In vivo anti-tumor metastasis effect

[0215] 4T1-Luc cells (1.5 × 10⁻⁶) were injected via tail vein. 6 Mice were randomly divided into 7 groups of 12 mice each: blank control group, model group, PTX group, PPEP group, BSP group, and TBSP group (high, medium, and low). Mouse weight was measured and recorded every three days, and mouse activity was observed (n=5). Mouse transfer was observed using a small animal in vivo imaging system. D-fluorescein potassium salt (15 mg / mL, 200 mL) was injected intraperitoneally, and imaging was performed using the IVIS small animal in vivo imaging system.

[0216] The test results are as follows:

[0217] (1) Cell uptake experiment

[0218] Please see Figures 17-19 , Figure 17A schematic diagram illustrating the qualitative uptake of C-6, C-6-BSP, and C-6-AN-TBSP by MDA-MB-231 cells; Figure 18 A schematic diagram illustrating the quantitative uptake of C-6, C-6-BSP, and C-6-AN-TBSP by MDA-MB-231 cells; Figure 19 This is a schematic diagram showing the average fluorescence intensity of MDA-MB-231 cells uptake of C-6, C-6-BSP, and C-6-AN-TBSP.

[0219] Figure 18 The fluorescence intensity of breast cancer MDA-MB-231 cells in cell culture medium at pH 7.4 or 6.5 is shown in the figure. Group (A) shows the test results at pH 7.4. In Group (A), light green represents the control group, yellow represents C-6, blue represents C-6-BSP, and pink represents C-6-AN-TBSP. Group (B) shows the test results at pH 7.4. In Group (B), light green represents the control group, yellow represents C-6, blue represents C-6-BSP, and dark green represents C-6-AN-TBSP.

[0220] Figure 19 The figure shows the average fluorescence intensity of uptake of C-6, C-6-BSP, and C-6-AN-TBSP by breast cancer MDA-MB-231 cells in cell culture medium at pH 7.4 or 6.5, respectively; with the horizontal axis as the reference, the uptake fluorescence intensity in cell culture medium at pH 7.4 or 6.5 is shown from bottom to top.

[0221] Combination Figure 17-19 The test results showed that, compared with the control group, C-6-TROP2-BA-SS-PPEP (147313) exhibited stronger fluorescence signals in cells than C-6 (48414) and C-6-BA-SS-PPEP (81192), indicating that C-6-TROP2-BA-SS-PPEP is beneficial for actively targeting breast cancer MDA-MB-231 cells to promote drug uptake; and the fluorescence intensity was stronger in the pH 6.5 medium than in the pH 7.4 medium. These results indicate that pH 6.5 (a weakly acidic microenvironment) can promote the uptake of C-6-TROP2-BA-SS-PPEP by MDA-MB-231 cells.

[0222] (2) Cell viability experiment

[0223] Please see Figure 20The diagram illustrates the cell viability assay results. At a concentration of 5.4 μg / mL, TROP2-BA-SS-PPEP exhibited high cytotoxicity against MDA-MB-231 cells, resulting in a cell viability rate as low as 31.21%. At the same concentration, BA-SS-PPEP maintained a cell viability rate of 67.55% for MDA-MB-231 cells. Compared to monomeric BA, TROP2-BA-SS-PPEP significantly reduced cell viability within the concentration range of 1.8-14.4 μg / mL. Specifically, the IC50 for BA was 120.2 μg / mL, for BA-SS-PPEP it was 12.34 μg / mL, and for TROP2-BA-SS-PPEP it was 4.76 μg / mL. Polyphospholipid PPEP showed no significant toxicity within the concentration range of 1.8-115.2 μg / mL, and the viability of MDA-MB-231 cells was greater than 70%.

[0224] Compared with the blank control, the positive control drug PTX showed significant cytotoxicity with an IC50 of 198.5 ng / mL, and TROP2-BA-SS-PPEP showed similar effects in the control experiment.

[0225] Therefore, the TROP2-BA-SS-PPEP prepared in this embodiment can promote the active uptake of drugs by MDA-MB-231 cells. Based on the IC50 of TROP2-BA-SS-PPEP, 2.38, 4.76, and 9.52 μg / mL were selected as low, medium, and high doses for subsequent experimental administration. PTX was administered at a dose of 198.5 ng / mL based on the IC50 for subsequent experimental administration.

[0226] (3) Cell scratch test

[0227] Please see Figure 21 The schematic diagram shows the results of the cell scratch assay. Compared with the blank control, Trop2-BA-SS-PPEP significantly reduced the wound healing rate of MDA-MB-231 cells, and the inhibitory effect gradually increased with increasing concentration. Within 0-48h, Trop2-BA-SS-PPEP did not significantly inhibit the migration ability of MDA-MB-231 cells, and had an inhibitory effect similar to that of the positive control group PTX.

[0228] Among them, BA-SS-PPEP can also inhibit the migration of MDA-MB-231 cells, with a cell migration rate of only 17.81% within 48 hours. BA has the ability to inhibit the migration of MDA-MB-231 cells, but the inhibitory effect decreases with time, from 21.48% migration rate at 12 hours to 36.75% at 24 hours, and to 65.07% at 48 hours. This indicates that Trop2-BA-SS-PPEP can significantly increase the ability of BA to inhibit the migration of MDA-MB-231 cells.

[0229] (4) Cell invasion assay

[0230] Please see Figure 22 The diagram shows the results of the cell scratch assay. Compared with the blank control, Trop2-BA-SS-PPEP treatment significantly reduced the ability of MDA-MB-231 cells to invade Matrigel, with high, medium, and low concentrations reducing cell invasion by 55.0%, 39.73%, and 33.03%, respectively. BA inhibited the invasion rate of MDA-MB-231 cells by 71.64% after 24 hours, while the cell invasion rate of the positive control group PTX was 30%, and the cell invasion rate of BA-SS-PPEP was 47.67%. This indicates that the nano-prodrug formulation in this embodiment can effectively enhance the efficacy of baicalin in inhibiting the invasion of MDA-MB-231 cells.

[0231] (5) Distribution in the body

[0232] Please see Figure 23 The in vivo imaging diagram of the in vivo distribution experiment shows that, compared with free ICG, ICG-labeled Trop2-BA-SS-PPEP and BA-SS-PPEP nanoparticles rapidly accumulated in the tumor site after tail vein injection in mice, and their fluorescence signal slowly decreased over time, indicating that the nanoparticles not only have good targeting in tumor tissue, but can also remain stably in tumor tissue.

[0233] At the experimental endpoint, major organs (heart, liver, spleen, lung, and kidney) and tumor tissues of mice were collected. ICG-labeled Trop2-BA-SS-PPEP and BA-SS-PPEP mainly accumulated in tumor tissues, with the highest concentrations found in tumor tissues, consistent with in vivo monitoring results.

[0234] (6) Anti-tumor effect

[0235] Please see Figure 24 and Figure 25 ,in, Figure 24 This is a schematic diagram of mouse transfer imaging. Figure 25This is a schematic diagram of mouse body weight. The changes in fluorescence in mouse lung metastases indicate that tumors in the model group and the PPEP nanocarrier group grew rapidly, while tumors in the sample group grew more slowly. The fluorescence in the lungs of the nanoprodrug and PTX groups was significantly lower than that in the BA group, suggesting that the nanoprodrug can effectively improve the tumor-suppressive effect of BA and enhance its anti-breast cancer lung metastasis effect.

[0236] Specifically, Figure 25 The changes in mouse body weight showed that although the body weight of mice in the model group and the nanocarrier PPEP group fluctuated, it increased slightly overall; no significant changes in body weight were observed in the other sample groups, which preliminarily indicates that the nanoprodrug has good biosafety.

[0237] In summary, the nanoprodrug provided in this embodiment is based on the redox imbalance characteristics of the TNBC microenvironment and the overexpression of the TROP2 receptor in TNBC to construct a redox-sensitive / TROP2-targeting nanoprodrug. This nanoprodrug has the advantages of specifically targeting tumor cells, achieving stable, spatiotemporally controllable drug release and activation at the target site in vivo. It can overcome the delivery barriers of the microenvironment, has redox-sensitive release characteristics, and enters TNBC cells through receptor-mediated endocytosis, which helps to improve drug delivery efficiency and achieve rapid release at the tumor site. This suggests that the nanoprodrug has good application prospects in the preparation of drugs for the treatment of TNBC.

[0238] The technical solutions provided by the embodiments of this application have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of this application. The description of the above embodiments is only for helping to understand the principles of the embodiments of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A nanoprodrug, characterized in that, The nanoprodrug has a compound of Formula I or a pharmaceutically acceptable salt thereof: Formula I, Among them, mAb is a Trop-2 monoclonal antibody; The molecular weight of the nanoprodrug is 47820.

28.

2. A method for preparing a nanoprodrug as described in claim 1, characterized in that, The preparation method includes: Under an inert gas atmosphere, 2-ethoxy-1,3,2-dioxaphosphane 2-oxide, dihydroxy disulfide and Sn(Oct)2 were added to anhydrous dichloromethane to react and obtain polymer I. Baicalin was dissolved in an organic solvent, and EDC and a catalyst were added to react. The reactants were added to a dimethylformamide solution of polymer I, and the mixture was purified by dialysis after the reaction to obtain polymer II. Bromoacetic acid was dispersed in PBS buffer solution, EDC and N-hydroxysuccinimide were added and stirred, and the mixture was centrifuged and the supernatant was discarded. The activated bromoacetic acid was stirred with Trop-2 monoclonal antibody, centrifuged and purified, dried and dialyzed to obtain polymer III. Potassium carbonate, polymer II, and polymer III were reacted in a dimethylformamide solution, purified by centrifugation, and lyophilized to obtain nano-prodrugs. The structural formulas of polymers I, II, and III are shown below: Polymer I, Polymer II Polymer III.

3. The preparation method according to claim 2, characterized in that, The organic solvent is dimethyl sulfoxide.

4. The preparation method according to claim 2, characterized in that, The catalyst is 4-dimethylaminopyridine.

5. The preparation method according to claim 2, characterized in that, The inert gas is nitrogen.

6. The preparation method according to claim 2, characterized in that, The molar ratio of 2-ethoxy-1,3,2-dioxaphosphane 2-oxide to dihydroxy disulfide is 16~22:

1.

7. The preparation method according to claim 2, characterized in that, The molar ratio of baicalin to polymer I is 1:1 to 1.

5.

8. Use of the nanoprodrug according to claim 1 in the preparation of a medicament for the prevention or treatment of triple-negative breast cancer.

9. The use according to claim 8, characterized in that, The drug is used to promote the active uptake ability of breast cancer cells.

10. The use according to claim 8, characterized in that, The drug is an injectable solution or a drug intended for oral administration.

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