Nano prodrug as well as preparation method and application thereof

Abstract

The embodiment of the invention provides a nano prodrug as well as a preparation method and application thereof. According to the embodiment of the invention, the redox-sensitive / TROP2 targeted nano prodrug is constructed based on the TNBC microenvironment redox imbalance characteristic and TNBC overexpression TROP2 receptor, and the nano prodrug has the advantages of specifically targeting tumor cells, realizing in-vivo long-circulation stability, time-space controllable target site drug release and activation and the like, can overcome microenvironment delivery obstacles, and can be used for preparing drugs for treating tumor tumors. The nano prodrug has the characteristics of redox sensitive release, enters TNBC cells through receptor-mediated endocytosis, is helpful for improving the drug delivery efficiency and realizing rapid release at tumor sites, and has a good application prospect in preparation of drugs for treating TNBC.

Description

Technical Field

[0001] The present application relates to the technical field of pharmaceutical preparations, and particularly relates to a nano-prodrug and its preparation method and use. Background Art

[0002] Triple-negative breast cancer (TNBC) refers to a subtype of breast cancer in which estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are negative, accounting for about 15%-20% of breast cancer types. TNBC is highly invasive, has a high histological grade, extensive heterogeneity, and ductal histological features. Metastatic TNBC is associated with a low overall survival rate. The main treatment methods for TNBC include surgery, chemotherapy, radiotherapy, and immunotherapy, among which chemotherapy is the most widely used. However, chemotherapy drugs have poor water solubility and unsatisfactory biodistribution, which hinder their application in tumor treatment. There is an urgent need for new TNBC treatment drugs to bring more effective and safe treatment options to the clinic.

[0003] Prodrugs do not have biological activity or have very low activity, and can be cleaved in vivo to form the original drug and bind to the target protein to exert biological activity. Among them, whether the drug can be transported to the target organ is the basis for the drug to exert its efficacy. Targeted modification of the drug structure to achieve selective tissue delivery can improve the therapeutic activity of the drug and reduce side effects. The targeted design of prodrugs often realizes by conjugating the structural unit targeting tissues or cells with cytotoxic molecules. For example, the carrier prodrugs of peptide-drug conjugates covalently link peptides to drugs through different linkers to form multifunctional PDCs targeting tumor cells, which can selectively accumulate in tumor cells, with an extended half-life and enhanced efficacy. The carrier prodrugs of antibody-drug conjugates utilize the high affinity of monoclonal antibodies for antigen proteins on the surface of tumor cells, and release active drugs targeting tumor cells through receptor-mediated endocytosis. Esterification is the most widely used type in the design of prodrug carriers, and ester drugs account for more than 50% of the marketed prodrugs. The ester bond 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 the first choice for prodrug carriers, the biotransformation of some simple alkyl esters or aryl esters cannot be mediated by esterases or the hydrolysis efficiency of esterases is low, resulting in insufficient systemic exposure and thus affecting the drug delivery efficiency.

[0004] In view of this, it has become an urgent need to develop a prodrug with high drug delivery efficiency. Summary of the Invention

[0005] The purpose of the present application is to provide a nano-prodrug and its preparation method and use.

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

[0007] In a first aspect, an embodiment of the present application provides a nano-prodrug, which is a compound having the structure shown in Formula I or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Among them, the molecular weight of the nano-prodrug is 47820.28.

[0010] In a second aspect, an embodiment of the present application proposes a preparation method of the nano-prodrug as described in the first aspect, and the preparation method includes:

[0011] Under an inert gas atmosphere, 2-ethoxy-1,3,2-dioxaphospholane 2-oxide, dihydroxydisulfide, and Sn(Oct)2 are added to anhydrous dichloromethane, and a reaction is carried out to obtain Polymer I;

[0012] Baicalin is dissolved in an organic solvent, EDC and a catalyst are added for reaction; the reactant is added to the dimethylformamide solution of Polymer I, and after reaction, dialysis purification is carried out to obtain Polymer II;

[0013] Bromoacetic acid is dispersed in PBS buffer solution, EDC and N-hydroxysuccinimide are added and stirred for reaction, and the mixture is centrifuged to discard the supernatant; the activated bromoacetic acid is stirred with Trop-2 monoclonal antibody and then centrifuged for purification, and dried and dialyzed to obtain Polymer III;

[0014] Potassium carbonate, Polymer II, and Polymer III are reacted in a dimethylformamide solution, centrifuged for purification, freeze-dried, and then a nano-prodrug is obtained;

[0015] Among them, the structural formulas of Polymer I, Polymer II, and Polymer III are shown as follows:

[0016]

[0017]

[0018] As an implementation manner, the organic solvent is dimethyl sulfoxide.

[0019] As an implementation manner, the catalyst is 4-dimethylaminopyridine.

[0020] As an implementation manner, the inert gas is nitrogen.

[0021] As an implementation manner, the molar ratio of 2-ethoxy-1,3,2-dioxaphospholane 2-oxide to dihydroxydisulfide is 16-22:1.

[0022] As an implementation manner, the molar ratio of baicalin to Polymer I is 1:1-1.5.

[0023] In a third aspect, an embodiment of the present application provides the use of the nano-prodrug described in the first aspect in the preparation of a drug for preventing, alleviating or treating triple-negative breast cancer.

[0024] As an implementation manner, the drug is a drug for promoting the active uptake ability of breast cancer cells.

[0025] Among them, the drug is an injectable solution or a drug for oral administration.

[0026] The embodiment of the present application has at least the following beneficial effects:

[0027] In the present application, by screening biocompatible polymer materials, traditional drug molecules are modified by nanotechnology. Introducing the nano-prodrug strategy after protecting group modification can improve the physicochemical properties of baicalin, enhance stability, water solubility, increase bioavailability and reduce adverse reactions, etc. Among them, polyphosphate (PPE) materials have good application prospects due to their adjustable properties, biodegradability and biocompatibility. In the present application, there are many repeating phosphate ester bonds in the PPE polymer backbone, allowing the introduction of various functional groups. The water solubility and functionality of PPE can be adjusted by polymerizing monomers with various side groups, and the active uptake ability of TNBC cells can be improved by conjugating TROP-2 antibody on the surface of baicalin and the recognition between receptors on the cell membrane, so as to obtain a redox-sensitive / TROP-2 targeted nano-prodrug with potential application prospects, providing a new strategy for the development of an efficient and low-toxic nano-drug delivery system.

[0028] Therefore, the embodiment of the present application constructs a redox-sensitive / TROP2-targeted nano-prodrug based on the characteristics of redox imbalance in the TNBC microenvironment and the overexpression of the TROP2 receptor in TNBC. This nano-prodrug has the advantages of specifically targeting tumor cells, achieving long-circulation stability in vivo, spatiotemporally controllable drug release and activation at the target site, etc. It can overcome the delivery obstacles in the microenvironment, has redox-sensitive release characteristics, enters TNBC cells through receptor-mediated endocytosis, helps to improve the drug delivery efficiency and achieve rapid release at the tumor site, indicating that this nano-prodrug has good application prospects in the preparation of drugs for treating TNBC.

[0029] Additional aspects and advantages of the present application will be given in part in the following description, and these will become apparent from the following description or be understood through the practice of the present application. Description of the Drawings

[0030] Figure 1 1H NMR spectrum of compound PEP;

[0031] Figure 2 1H NMR spectrum of copolymer OH-ss-PPEP;

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

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

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

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

[0036] Figure 7 TG curve of the sample obtained by thermogravimetric analysis in Example 2;

[0037] Figure 8 Schematic diagram of the morphology of Trop2-BA-ss-PPEP by transmission electron microscopy in Example 2;

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

[0039] Figure 10 Schematic diagram of the potential distribution of Trop2-BA-ss-PPEP in Example 2;

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

[0041] Figure 12 Schematic diagram of the test results of the positive control group in the in vitro hemolysis test of Example 2;

[0042] Figure 13 Schematic diagram of the test results of the control group in the in vitro hemolysis test of Example 2

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

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

[0045] Figure 16 Schematic diagram of the Boltzmann curve fitting for detecting the critical micelle concentration by pyrene fluorescence probe method in Example 2;

[0046] Figure 17Schematic diagram of 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 Schematic diagram of 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 Schematic diagram of the average fluorescence intensity of the 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;

[0049] Figure 20 Schematic diagram of the test results of the cell viability experiment in Example 3;

[0050] Figure 21 Schematic diagram of the test results of the cell scratch experiment in Example 3;

[0051] Figure 22 Schematic diagram of the test results of the cell invasion experiment in Example 3;

[0052] Figure 23 Schematic diagram of in vivo imaging of the in vivo distribution experiment in Example 3;

[0053] Figure 24 Schematic diagram of the metastatic imaging of mice in the anti-tumor effect experiment of Example 3;

[0054] Figure 25 Schematic diagram of the body weight of mice in the anti-tumor effect experiment of Example 3. Detailed implementation manners

[0055] Next, the technical solutions in the embodiments will be clearly and completely described in conjunction with the embodiments of the present application and the accompanying drawings. Obviously, the embodiments to be described below are only a part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present 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, wholes, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or their combinations.

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

[0058] First, some terms and materials involved in this embodiment will be explained below to facilitate the understanding of those skilled in the art.

[0059] PEP: 2-ethoxy-1,3,2-dioxaphospholane 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-NH 2 。

[0062] Trop2-BA-ss-PPEP: namely the nano-prodrug.

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

[0064] DMSO: dimethyl sulfoxide.

[0065] DMAP: 4-dimethylaminopyridine.

[0066] NHS: N-hydroxysuccinimide.

[0067] Next, the nano-prodrug of the embodiments of the present application, its preparation method and uses will be described in detail.

[0068] First, the nano-prodrug of this embodiment will be described.

[0069] Nanoprecursor drug

[0070] This embodiment provides a nano-prodrug; specifically, the nano-prodrug has a compound with the following structure or a pharmaceutically acceptable salt thereof:

[0071]

[0072] Among them, the molecular weight of the nano-prodrug is 47820.28.

[0073] It is understandable that the nano - prodrug provided in this embodiment modifies traditional drug molecules by screening biocompatible polymer materials and using nanotechnology. Introducing the nano - prodrug strategy after protecting - group modification can improve the physicochemical properties of baicalin, enhance stability, water - solubility, increase bioavailability, and reduce adverse reactions, etc. Among them, poly(phosphoester) (PPE) materials have good application prospects due to their adjustable properties, biodegradability, and biocompatibility. In this embodiment, there are many repeating phosphoester bonds in the PPE polymer backbone, allowing the introduction of various functional groups. In this embodiment, the water - solubility and functionality of PPE can be adjusted by polymerizing monomers with various side groups, and the active uptake ability of TNBC cells is improved by conjugating the TROP - 2 antibody on the surface of baicalin with the recognition between receptors on the cell membrane, so as to obtain a redox - sensitive / TROP - 2 - targeted nano - prodrug with potential application prospects, providing a new strategy for the development of highly efficient and low - toxic nano - drug delivery systems.

[0074] Therefore, based on the characteristics of redox imbalance in the TNBC microenvironment and the over - expression of the TROP2 receptor in TNBC, this embodiment constructs a redox - sensitive / TROP2 - targeted nano - prodrug. This nano - prodrug has the advantages of specifically targeting tumor cells, achieving long - circulating stability in vivo, spatiotemporally controllable drug release and activation at the target site, etc. It can overcome the delivery barriers in the microenvironment, has redox - sensitive release characteristics, enters TNBC cells through receptor - mediated endocytosis, helps to improve the drug delivery efficiency and achieve rapid release at the tumor site, indicating that this nano - prodrug has good application prospects in the preparation of drugs for treating TNBC.

[0075] It is understandable that the pharmaceutically acceptable salts of the above - mentioned nano - prodrug provided in this embodiment can be: sodium salt, potassium salt, ammonium salt, amino acid salt, lactate, hydrochloride, phosphate, acetate, malate, citrate or aspartate, etc. This embodiment does not make specific limitations on the drug salts.

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

[0077] Preparation method

[0078] This embodiment also provides a preparation method of the above - mentioned nano - prodrug, and this preparation method includes:

[0079] (1) Under an inert gas atmosphere, 2 - ethoxy - 1,3,2 - dioxaphospholane 2 - oxide, dihydroxy disulfide, and Sn(Oct)2 are added to anhydrous dichloromethane, and a reaction is carried out to obtain Polymer Ⅰ;

[0080] (2) Baicalin is dissolved in an organic solvent, EDC and a catalyst are added for reaction; the reactant is added to the dimethylformamide solution of Polymer Ⅰ, and after reaction, dialysis purification is carried out to obtain Polymer Ⅱ;

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

[0082] (4) React potassium carbonate, Polymer Ⅱ, and Polymer Ⅲ in dimethylformamide solution, centrifuge and purify, and obtain the nano-prodrug after freeze-drying.

[0083] Among them, the structural formulas of Polymer Ⅰ, Polymer Ⅱ, and Polymer Ⅲ are shown as follows:

[0084]

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

[0086] In step (1), the inert gas is nitrogen, preferably under a dry nitrogen atmosphere. Among them, the reaction process of step (1) can be referred to the following synthetic route:

[0087]

[0088] In this step, Polymer Ⅰ, namely polyphosphoester-based random copolymer OH-ss-PPEP, is obtained by the reaction of 2-ethoxy-1,3,2-dioxaphospholane 2-oxide and dihydroxy disulfide.

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

[0090] Then, based on the polyphosphoester-based random copolymer OH-ss-PPEP, it is modified on BA to obtain Polymer BA-ss-PPEP.

[0091] The reaction process of step (2) can be referred to the following synthetic route:

[0092]

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

[0094] Among them, the molar ratio of baicalin to Polymer Ⅰ is 1:1-1.5, such as 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 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] Among them, the reaction process of step (3) can be referred to the following synthetic route:

[0097]

[0098] The reaction process of step (4) can be referred to the following synthetic route:

[0099]

[0100] Thus, in this example, the water solubility and functionality of PPE can be adjusted by polymerizing monomers with various side groups, and the active uptake ability of TNBC cells can be improved by the recognition between the TROP-2 antibody conjugated on the surface of baicalin and the receptor on the cell membrane.

[0101] Exemplarily, after obtaining the nano-prodrug, the nano-prodrug 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 rotary evaporation under reduced pressure to obtain a nano-micelle solution without organic solvents.

[0102] Next, the synthesis process of the nano-prodrug will be further clarified in combination with specific preparation methods.

[0103] Specifically, the preparation method of the nano-prodrug includes:

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

[0105] Under a dry nitrogen atmosphere, 2-ethoxy-1,3,2-dioxaphospholane 2-oxide (PEP, 1080.84 mg, 6.00 mmol), dihydroxydisulfide (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 stir bar. 0.2 mL of Sn(Oct)2 dichloromethane 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 ether:methanol = 10:1 V / V), and the precipitate was concentrated under reduced pressure at room temperature to obtain OH-ss-PPEP.

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

[0107] Dissolve BA (446.36 mg, 1 mmol) in 10 mL of DMSO solution, add it to a 50 mL round-bottom flask, and slowly add EDC (299.50 mg, 1.5 mmol) under an ice bath; then add DMAP (24.43 mg, 0.2 mmol), and react at 0 °C for 30 min; add the reactant to 2 mL of a DMF solution of OH-ss-PPEP (380 mg, 1.2 mmol), and react overnight at room temperature; purify by dialysis (MWCO 3500) with a mixed solvent of ethanol-Milli-Q water (volume ratio v / v, from 3 / 1 to 0 / 1) for 48 h. After freeze-drying, a pale yellow viscous product BA-ss-PPEP is obtained.

[0108] (3) Synthesis of Antibody Conjugate

[0109] Disperse bromoacetic acid (11.116 mg, 0.08 mmol) in 1 mL of PBS buffer solution (pH 5.0), and stir it with EDC (9.585 mg, 0.05 mmol) and NHS (5.755 mg, 0.05 mmol) at 25 °C for 30 min; centrifuge the mixture at 8000 rpm for 15 min, and discard the supernatant; at 4 °C, stir the activated bromoacetic acid with 1 mL of PBS (pH 7.0) and Trop-2 monoclonal antibody (12 μL, 0.5 mg / mL) for 12 h, purify by ultrafiltration centrifugation, and obtain the antibody conjugate bromo-mAb after freeze-drying.

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

[0111] React bromo-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) react overnight at 35 °C in 10 mL of DMF solution. Purify by ultrafiltration centrifugation, and finally obtain a pale yellow viscous product Trop2-BA-ss-PPEP, namely the nano-prodrug, after freeze-drying.

[0112] Secondly, the use of the above nano-prodrug will be described.

[0113] Use of the nanoprecursor drug

[0114] As described above, based on the redox imbalance characteristics of the TNBC microenvironment and the overexpression of the TROP2 receptor in TNBC, a redox-sensitive / TROP2-targeted nano-prodrug was constructed in this example. This nano-prodrug has the advantages of specifically targeting tumor cells, achieving stable long-circulation in vivo, spatio-temporally controllable drug release and activation at the target site, etc. It can overcome the delivery barriers in the microenvironment, has redox-sensitive release characteristics, enters TNBC cells through receptor-mediated endocytosis, helps to improve the drug delivery efficiency and achieve rapid release at the tumor site, indicating that this nano-prodrug has good application prospects in the preparation of drugs for treating TNBC.

[0115] Based on this, the nano-prodrug provided in this example can be used to prepare drugs for preventing, alleviating or treating triple-negative breast cancer.

[0116] Specifically, the above-mentioned drug is a drug for promoting the active uptake ability of breast cancer cells.

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

[0118] Generally, when the above-mentioned nano-prodrug is made into the corresponding drug, the drug also includes a pharmaceutically acceptable carrier and / or adjuvant.

[0119] For the drug described in this example, the drug uses the nano-prodrug as the active ingredient, and does not exclude changes in the formulation system and administration method, pharmaceutical salts after simple chemical modification and adjustment of the above-mentioned nano-prodrug, and the combination of multiple compounds, etc.

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

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

[0122] Generally, the "effective amount" of a compound (nano-prodrug) refers to the amount sufficient to cause a target biological response. As understood by those of ordinary skill in the art, the effective amount of the compound in this example can be changed according to the following factors: for example, components such as the vehicle in the drug and the age, health condition of the subject and the symptoms of triple-negative breast cancer.

[0123] Among them, the effective amount includes a therapeutically effective amount and a prophylactically effective amount.

[0124] Unless otherwise stated, the "therapeutically effective amount" of the compound used in this example is an amount sufficient to provide a benefit during the treatment of triple-negative breast cancer, or the minimum amount that improves or alleviates one or more symptoms (manifestations) related to the triple-negative breast cancer state. The "prophylactically effective amount" of the compound used in this example is an amount sufficient to prevent the occurrence of triple-negative breast cancer, or an amount sufficient to prevent one or more symptoms related to the occurrence of the triple-negative breast cancer state.

[0125] It can be understood that the drug of this example can also be made into an injection. For example, the nano-prodrug can be made into the corresponding injection with water for injection, normal saline, and glucose solution under a sterile operating environment. The above-mentioned injection can be prepared by conventional methods.

[0126] The following will further elaborate on this application in combination with specific examples. It should be understood that these examples are only used to illustrate / explain this application and not to limit the scope of this application.

[0127] In the following examples, the materials, reagents, and instruments used can be obtained from commercial sources without special instructions.

[0128] Example 1

[0129] Synthesis of the nano-prodrug Trop2-BA-ss-PPEP, and its synthetic route is shown as follows:

[0130]

[0131] Specifically, it includes the following steps:

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

[0133] Under a dry nitrogen atmosphere, 2-ethoxy-1,3,2-dioxaphospholane 2-oxide (PEP, 1080.84 mg, 6.00 mmol), bis(hydroxyl) 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. 0.2 mL of a Sn(Oct)2 dichloromethane 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 ether:methanol = 10:1 V / V), and the precipitate was concentrated under reduced pressure at room temperature to obtain OH-ss-PPEP.

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

[0135] Dissolve BA (446.36 mg, 1 mmol) in 10 mL DMSO solution, add it to a 50 mL round-bottom flask, and slowly add EDC (299.50 mg, 1.5 mmol) under an ice bath; then add DMAP (24.43 mg, 0.2 mmol), and react at 0 °C for 30 min; add the reactant to 2 mL of a DMF solution of OH-ss-PPEP (380 mg, 1.2 mmol), and react overnight at room temperature; purify by dialysis (MWCO 3500) with a mixed solvent of ethanol-Milli-Q water (volume ratio v / v, from 3 / 1 to 0 / 1) for 48 h. After freeze-drying, a pale yellow viscous product BA-ss-PPEP is obtained.

[0136] (3) Synthesis of antibody conjugate

[0137] Disperse bromoacetic acid (11.116 mg, 0.08 mmol) in 1 mL of PBS buffer solution (pH 5.0), and stir it with EDC (9.585 mg, 0.05 mmol) and NHS (5.755 mg, 0.05 mmol) at 25 °C for 30 min; centrifuge the mixture at 8000 rpm for 15 min and discard the supernatant; at 4 °C, stir the activated bromoacetic acid with 1 mL of PBS (pH 7.0) and Trop-2 monoclonal antibody (12 μL, 0.5 mg / mL) for 12 h, purify by ultrafiltration centrifugation, and obtain the antibody conjugate bromo-mAb after freeze-drying.

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

[0139] React bromo-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) react overnight at 35 °C in 10 mL of DMF solution. Purify by ultrafiltration centrifugation, and finally obtain a pale yellow viscous product Trop2-BA-ss-PPEP after freeze-drying.

[0140] (5) Anti-solvent precipitation method

[0141] Dissolve the nano-prodrug in ethanol, and under stirring conditions, slowly drip the ethanol solution of the prodrug into water, and the prodrug spontaneously forms a solution containing uniform nanoparticles; use the reduced pressure rotary evaporation method to remove ethanol from the solution containing uniform nanoparticles to obtain a nano-micelle solution without organic solvents (hereinafter referred to as TROP2-BA-SS-PPEP solution).

[0142] Example 2

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

[0144] Specifically, it includes:

[0145] 2.1, Nuclear magnetic resonance hydrogen spectroscopy (1H-NMR)

[0146] A Bruker AC-400 Fourier transform nuclear magnetic resonance spectrometer (FT-NMR) was used to operate at 400 MHz at room temperature, and chloroform-d1 (CDCl 3 ) was used as the solvent, and tetramethylsilane (TMS) was used as the internal standard.

[0147] 2.2, Mass spectrometry for detecting polymer molecular weight (SEC)

[0148] A triple quadrupole mass spectrometer was used to detect the polymer molecular weight (SEC). The range was 350 - 15000 m / z. Data analysis was performed using Xcalibur software, and the mass spectrum was deconvoluted using the Xtract function in Xcalibur.

[0149] 2.3, Fourier transform infrared spectroscopy (FTIR)

[0150] The sample was weighed and ground and mixed evenly with dry potassium bromide at a ratio of 1:100, and then pressed into a sample. The infrared spectrum of the sample in the range of 4000 - 500 cm -1 was scanned using an infrared spectrometer.

[0151] 2.4, Thermogravimetric analysis (TG)

[0152] A TG analyzer (PerkinElmer, USA) was used to study the thermal properties of BA, PPEP, BA-SS-PPEP, and TROP2-BA-SS-PPEP, and TG data were recorded under the conditions of 30 °C - 600 °C and a nitrogen flow rate of 10 °C / min.

[0153] 2.5, Observation of the microscopic morphology of nanoparticles

[0154] An aqueous solution of TROP2-BA-SS-PPEP was taken, dropped on a copper mesh for coating and adsorption for sample preparation, negatively stained with 2% phosphotungstic acid, and then air-dried to prepare a sample slice. The particle morphology was observed using a transmission electron microscope.

[0155] 2.6, Determination of the Zeta potential and particle size of nanoparticles

[0156] An aqueous solution of TROP2-BA-SS-PPEP nanomicelles was taken, and the particle size and Zeta potential were measured using a particle size analyzer to investigate the particle size and Zeta potential distribution.

[0157] 2.7. Determination of drug loading content (DLC) and drug loading efficiency (DLE)

[0158] Take 200 μL of BA-SS-PPEP and TROP2-BA-SS-PPEP solutions and transfer them to a 10 KD ultrafiltration tube. Centrifuge at a high speed of 12,000 rpm for 5 min. Dilute the supernatant with methanol and make up the volume to 2 mL with a volumetric flask. Filter through a 0.45 μm organic filter membrane, and determine the content of free BA (Wt) by HPLC; Dilute with methanol and make up the volume to 2 mL with a volumetric flask. Centrifuge at a high speed of 12,000 rpm for 5 min. Filter the supernatant through a 0.45 μm aqueous filter membrane, and determine the content of BA by HPLC.

[0159] 2.8. Investigation of the in vitro stability of nanoparticles

[0160] To evaluate the in vitro stability of the nanoparticles, re-disperse TROP2-BA-SS-PPEP in deionized water and PBS (pH 7.4) solutions to simulate the storage environment of the nanoparticles. Take the particle size and zeta potential as evaluation indicators, and investigate the stability of the nanoparticles at 4 °C and 25 °C for three months.

[0161] 2.9. In vitro hemolysis test

[0162] Collect blood from healthy mice and put it into a centrifuge tube containing anticoagulant (whole blood: 2.5% sodium citrate (volume ratio) = 9:1). Wash the blood, and prepare a 4% suspension of the obtained red blood cells with 0.9% sodium chloride solution. Add 900 μL of the red blood cell suspension to each centrifuge tube, and respectively add 100 μL of BA, PPEP, and BA-SS-PPEP (4.76 μg / mL in terms of 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, 0.60 μg / mL in terms of BA); The content ratios of the positive control (Triton-X-100) are set to 4%, 2%, 1%, 0.5%, 0.25%, and 0.125%. Incubate in a 37 °C incubator for 1 h, centrifuge at 1000 rpm for 15 min, and observe the hemolysis phenomenon.

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

[0164] The in vitro drug release of BA and TROP2-BA-SS-PPEP was evaluated 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. The release was carried out with constant shaking in a 37 °C air bath. At set times, 0.5 mL of the external solution was taken out, and the same volume of PBS buffer was added to the dialysis solution. The in vitro release rate of the drug was calculated.

[0165] 2.11 Self-assembly of polymer prodrugs

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

[0167] The above test results are as follows:

[0168] (1) 1H-Nuclear Magnetic Resonance (1H-NMR)

[0169] Please refer to Figure 1 and Figure 2 , the chemical structures of PPEP and PEP were characterized by 1H NMR analysis, showing the chemical shifts corresponding to the protons of the initiator and the copolymer. We can find that the original chemical shift at δ 4.70 ppm (peaks 3, 4) in PEP shifted to δ 4.17 ppm and δ 4.26 ppm disappeared after the reaction, and new proton signals (peaks 5, 8, 9) at δ 3.45 ppm, δ 3.69 ppm, and δ 3.84 ppm appeared, indicating that the hydroxyl group at the C-1′ position of bis(2-hydroxyethyl) disulfide had successfully initiated the PEP monomer to obtain PEEP.

[0170] Among them, please refer to Figure 3 and Figure 4 , BA-ss-PPEP is the product formed by the Steglich esterification reaction of BA and PPEP. By comparing the two spectra, the chemical shift of peak 7 was affected by the ester group and changed from the original δ 3.79 ppm to δ 4.20 ppm, and characteristic benzene ring peaks of BA appeared at 6.0 - 8.0 ppm. New proton signals appeared at δ 5.86 ppm, δ 6.57 ppm, δ 6.73 ppm, δ 7.95 ppm, and δ 8.12 ppm. Figure 3The original chemical shifts at δ8.69 ppm and δ12.59 ppm (peak k) disappeared after the reaction in Figure 4 indicating that the carboxyl group at the C-6′ position on BA had been coupled with PPEP to obtain BA-ss-PPEP.

[0171] Please refer to Figure 4 , and new proton signals of TROP2-BA-ss-PPEP appeared at (peaks 10, 11) δ4.67 ppm and δ6.94 ppm, which were the methylene hydrogen signal at the C-2′ position on bromoacetic acid and the amino hydrogen signal of monoclonal antibody Trop-2, respectively. The original chemical shifts at δ8.69 ppm and δ12.59 ppm (peak f) disappeared after the reaction, indicating that the phenolic hydroxyl groups at the C-5 and C-6 positions on BA had been coupled with Trop-2 to obtain TROP2-BA-ss-PPEP, that is, the 5′-OH and C-6′-OH of BA had smaller steric hindrance and higher activity than the 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 the 1H NMR results. Please refer to Table 1 below. According to the 1H NMR spectra, the molecular weights of BSP and TBSP were calculated to be 1124.84 g / mol and 47820.28 g / mol, respectively. Further analysis by biopolymer mass spectrometry showed that the molecular weights of BSP and TBSP were 1349.14 g / mol and 49154.5 g / mol, respectively. Deconvolution analysis found that there was a regular interval, which was consistent with the molecular weight of one PEEP repeating unit (152.09). There seemed to be a difference in the molecular weights of the polyphosphates determined by 1H NMR and biopolymer mass spectrometry, which might be attributed to the reduced sensitivity of 1H NMR for detecting the number-average molecular weight (Mn) above 3000, thus affecting the accuracy of evaluating the molecular weight.

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

[0175]

[0176] (3) Fourier transform infrared spectroscopy (FTIR)

[0177] Please refer to Figure 6 the infrared spectrum of, in which, intermolecular hydrogen bond O-H stretching vibration appeared in BA molecules at 3418.81 cm -1 as a broad absorption peak; at 2968.81 cm -1is C-H stretching vibration; 1600 - 1450 cm -1 is the C=C skeletal vibration of the benzene ring, and there is an absorption of out-of-plane bending vibration of C-H at 880 - 680 cm-1; 1726.55 cm -1 is the absorption of carboxylic acid C=O at this position, 920 cm -1 is the out-of-plane bending vibration of the O-H bond in the formation of a bond, and a carboxylic acid dimer appears; 1660.08 cm -1 a strong peak appears at this position for the stretching vibration of the ketone C=O. Conjugation of the carbonyl group with an alkene bond or an aromatic ring will lower the absorption frequency. The out-of-plane bending vibration of alkene C-H (1000 - 675 cm -1 ); 1150 - 1060 cm -1 a strong absorption peak (C-O) appears for the stretching vibration, 1050 - 1000 cm -1 is the absorption of two C-O stretching vibrations of the aromatic ether.

[0178] A broad absorption peak appears at 3401.66 cm for the PPEP molecule, which is the stretching vibration of intermolecular hydrogen bond O-H: C-H stretching vibration (2981.36 cm -1 ); 1453.79 cm -1 is the out-of-plane bending vibration of C-H; The stretching vibration absorption of P-C is at 1394.96 cm -1 , the stretching vibration of P=O is at 1255.12 cm -1 , the antisymmetric stretching vibration of P-O-C is at 1165.26 cm -1 , the stretching vibration of P-O is at 962.54 cm -1 ; The stretching vibration absorption of C-O appears as a strong absorption peak at 1013.68 cm -1 ; The out-of-plane bending vibration of O-H at 769 - 659 cm-1; 544.74 cm -1 has a characteristic absorption peak of the disulfide bond. -1

[0179] A broad absorption peak appears at 3334.78 cm for BSP, which is the stretching vibration of intermolecular hydrogen bond O-H: C-H stretching vibration (2959.39 cm -1 ); 1440.18 cm -1 is the out-of-plane bending vibration of C-H; 1562.52 cm -1 、1440.18 cm -1 、1440.18 cm -1 are the C=C skeletal vibrations of the benzene ring, 801.02 cm -1 、729.63 cm -1 、699.07 cm -1 have the absorption of out-of-plane bending vibration of benzene ring C-H; 1716.13 cm -1 is the absorption of ester C=O at this position, 1655.13 cm-1 The ketone C=O absorption at -1 shows a strong absorption peak; the stretching vibration absorption of P-C is at 1392.18 cm -1 , the stretching vibration of P=O is at 1258.23 cm -1 , the antisymmetric stretching vibration of P-O-C is at 1097.08 cm -1 , the stretching vibration of P-O is at 978.61 cm -1 ; there is a characteristic absorption peak of disulfide bond at 510.09 cm -1 .

[0180] It can be understood that due to the introduction of a large number of amide bonds by monoclonal antibodies, TBSP shows a broad and large N-H stretching vibration absorption peak at 3423.76 cm-1, and the absorption at 1085.39 cm -1 is the C-N stretching vibration absorption; the absorption at 2964.54 cm -1 is the C-H stretching vibration, and the out-of-plane bending vibration of benzene ring C-H is in the range of 880 - 680 cm -1 ; a strong peak appears at 1651.45 cm -1 for the C=O stretching vibration, and the absorption of C-O stretching vibration is at 1045.33 cm -1 ; the stretching vibration absorption of P-C is at 1400.97 cm -1 , the stretching vibration of P=O is at 1216.47 cm -1 , the stretching vibration of P-O is at 952.93 cm -1 ; there is a characteristic absorption peak of disulfide bond at 561.79 cm -1 .

[0181] (4) Thermogravimetric analysis

[0182] Please refer to Figure 7 the TG curve of the sample. There is no obvious weight loss between 100 and 300 °C, but weight loss is shown in the range of 400 - 600 °C, which may be related to dehydroxylation and decarbonization. The residual masses of BA, PPEP, BSP, and TBSP at the end of the measurement at 600 °C are 40.5%, 24.0%, 21.7%, and 46.8% respectively. Compared with BSP, the polyphosphate group can increase the thermal stability of BA. Since TBSP is conjugated with the antibody and the antibody has low thermal stability, the thermal loss of TBSP increases.

[0183] (5) Microscopic morphology of nanoparticles

[0184] Please refer to Figure 8 , observed under a transmission electron microscope, the size of TROP2-BA-SS-PPEP is uniform, the dispersion is good, and the particle size is about 100 nm.

[0185] (6) Particle size and potential

[0186] Please refer to Figure 9 the schematic diagram of 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 Figure 10 the schematic diagram of the potential distribution of Trop2-BA-ss-PPEP. The Zate potential of the aqueous solution of TROP2-BA-SS-PPEP is (-36.2 ± 8.48) mV. The nanoparticles with negative potential tend to maintain the stability state of mutual repulsion.

[0187] (7) Determination of drug loading content (DLC) and drug loading efficiency (DLE)

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

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

[0190]

[0191]

[0192] (8) Investigation of in vitro stability

[0193] Please refer to Figure 11 the schematic diagram of the stability test results. Among them, the left side is the schematic diagram of the particle size stability test results, and the right side is the schematic diagram of the potential stability test results; when the aqueous solution of TROP2-BA-SS-PPEP is stored at 4 °C and 25 °C, the change trend of the particle size is relatively gentle within 12 weeks. The increasing trend of the particle size of TROP2-BA-SS-PPEP after incubation with the same volume of PBS is larger than that of the aqueous solution, and the increase amplitude of the particle size is obvious at room temperature; the absolute value of the potential of TROP2-BA-SS-PPEP gradually decreases with the increase of the storage time. The absolute value of the initial potential of the PBS solution of TROP2-BA-SS-PPEP is smaller than that of the aqueous solution, which may be because the surface charge of the nanoparticles is destroyed by the ions in the PBS solution, and agglomeration and sedimentation are likely to occur, affecting the stability of the nanoparticles. Therefore, the TROP2-BA-SS-PPEP prepared in this example has good stability in 4 °C aqueous solution.

[0194] (9) In vitro hemolysis test

[0195] Please refer to Figures 12 - 14 , Figure 12 which is a schematic diagram of the test results for the positive control group (Triton-X-100); Figure 13 which is a schematic diagram of the test results for the control groups (0.9% sodium chloride solution, BA, PPEP, and BA-SS-PPEP from left to right), Figure 14 which is a schematic diagram of the test results for Trop2-BA-ss-PPEP; Combining Figure 13 as shown, in the negative control group, red blood cells sank and the supernatant was colorless and transparent, and hemolysis did not occur; Combining Figure 12 as shown, the positive control group presented a red and clear state, with only a small amount of red blood cells remaining at the bottom of the tube, and obvious hemolysis occurred; Combining Figure 14 as shown, in each tube of the sample group, red blood cells sank, the supernatant was colorless and transparent, and no hemolysis or aggregation occurred.

[0196] (10) Responsive release of nanoparticles

[0197] Please refer to Figure 15 the schematic diagram of the responsive release curve. To evaluate the responsive 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 released BA; compared with 2 mM GSH, 10 mM GSH significantly accelerated the release of TROP2-BA-SS-PPEP within 12 h, and the results showed that high-concentration GSH induced the reduction-responsive drug release behavior of TROP2-BA-SS-PPEP. The release behavior of TROP2-BA-SS-PPEP could be accelerated under low pH conditions. At pH 6.5 and 5.5 conditions, BA could be completely released after 36 h of incubation, while only about 72.7% of BA was released at pH 7.4. The reason for these phenomena may be that the acidic environment can cause the disulfide bond linker to break, resulting in the loose structure of the nanoparticles and accelerating the release of BA.

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

[0199] (11) Detection of critical micelle concentration by pyrene fluorescence probe method

[0200] Please refer to Figure 16, with the fluorescence spectral intensity ratio I3 / I1 of pyrene as the ordinate and the logarithm of the nanosolution concentration logC as the abscissa, where A1 and A2 are the maximum and minimum values of I3 / I1 respectively, and x0 is the midpoint of the curve mutation, and the corresponding concentration is the critical micelle concentration of the sample.

[0201] Among them, the variation law 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 was performed on the results to obtain the midpoint of the mutation x0, and the critical micelle concentration of the micelle was determined to be 15.44 μg / mL.

[0202] Example 3

[0203] The anti-metastatic efficacy of the nano-prodrug prepared in Example 1 was evaluated.

[0204] (1) Cell qualitative and quantitative uptake experiments

[0205] For qualitative analysis of cell uptake, coumarin-6 (C6) was used to prepare coumarin-loaded pH-responsive nanoparticles with a final concentration of C-6 of 10 μg / mL, and the uptake of TROP2-BA-SS-PPEP by MDA-MB-231 cells was investigated. C-6, C-6-AN-FA, and C-6-AN-FA@mPEG were added respectively, and a blank control group was set. Incubate in cell culture medium at pH 7.4 or 6.5 at 37 °C for 2 h. Add 500 μL DAPI and stain for 10 min. Observe the fluorescence intensity of uptake in MDA-MB-231 cells with a laser confocal microscope; digest and centrifuge to collect cells with trypsin (without EDTA), and wash with PBS to remove the fluorescent dye on the cell surface. Centrifuge, resuspend in a flow tube containing 500 μL PBS, and detect the fluorescence intensity of cell uptake with a flow cytometer (FACSCalibur, BD, USA).

[0206] (2) Cell viability experiment

[0207] The MDA-MB-231 cell suspension was inoculated into 96-well plates at a density of 4×104 cells / well and cultured for 24 h. Different concentrations of drug-containing media (100 μL / well) were added, including 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), BA-ss-PPEP (calculated as BA, 1.8, 3.5, 5.4, 7.2, 9.0, 10.8, 12.6, 14.4 μg / mL), Trop2-BA-ss-PPEP (calculated as BA, 1.8, 3.5, 5.4, 7.2, 9.0, 10.8, 12.6, 14.4 μg / mL), the 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 was set. After incubation, 10 μL of MTT solution was added and cultured for 4 h. The MTT reaction solution and the medium were aspirated, 150 μL of DMSO solution (cell grade) was added to dissolve formazan, and it was shaken at a constant temperature of 37 °C for 20 min. The O.D. value at 490 nm was detected by an enzyme-linked immunosorbent assay (ELISA) reader.

[0208] (3) Cell scratch assay

[0209] The MDA-MB-231 cell suspension was inoculated into 6-well plates at a density of 1.2×106 cells / well and cultured for 24 h. Then, BA and BA-SS-PPEP (calculated as BA, 4.76 μg / mL), different concentrations of Trop2-BA-SS-PPEP (calculated as BA, 2.38, 4.76, 9.52 μg / mL), and PTX as the positive control were added and treated for 24 h. A pipette tip or other hard object was used to scratch the central area of the monolayer cells to remove the central cells, and then the cells were continuously cultured until the time set for the experiment to judge the cell growth and migration ability.

[0210] (4) Cell invasion assay

[0211] Matrigel was thawed in advance at 4°C, the pipette tips were pre-cooled, and Matrigel was diluted 1:8 (8 Matrigel: 64 serum-free medium) on ice. 60 μL of Matrigel was vertically added to the upper chamber surface of the bottom membrane of the Transwell chamber, and it was placed at 37°C for 3 h to polymerize Matrigel into a gel. Basement membrane hydration was carried out at 37°C for 30 min. The cells were serum-starved for 24 h. The cells were digested and added to the Transwell chamber, and 500 μL of complete medium was added to the lower chamber. BA and BA-SS-PPEP (4.76 μg / mL in terms of BA), different concentrations of Trop2-BA-SS-PPEP (2.38, 4.76, 9.52 μg / mL in terms of BA), and PTX were added as a positive control and treated for 24 h. The bottom of the chamber was immersed in 0.1% crystal violet for staining, and the cells were randomly observed under a microscope and counted.

[0212] (5) In vivo distribution

[0213] 4T1 cells (2×10 6 cells) were subcutaneously inoculated to establish a breast cancer-bearing mouse model, and the experiment was started when the tumor grew to about 150 mm 3 . Twenty-one female nude mice (3 - 5 weeks old) were randomly divided into 3 groups: ICG normal saline, ICG-BA-SS-PPEP, ICG-Trop2-BA-SS-PPEP, with 3 mice in each group, numbered 1 - 3 respectively. The administration method was intravenous injection through the tail vein, and the drug was administered once. In vivo imaging was performed on the mice at 0 h, 3 h, 6 h, 12 h, 24 h, and 48 h after drug administration; after 48 h, the mice were sacrificed, and the tumors and various organs (heart, liver, spleen, lung, kidney) were taken for imaging.

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

[0215] 4T1-Luc cells (1.5×10 6 cells / 0.15 mL / animal) were injected through the tail vein. The mice were randomly divided into 7 groups, with 12 mice in each group, namely the blank control group, the model group, the PTX group, the PPEP group, the BSP group, and the TBSP group (high, medium, low). The body weight was weighed and recorded every three days, and the activity status of the mice was observed (n = 5). The metastasis of the mice was observed using a small animal in vivo imager. D-luciferin potassium salt (15 mg / mL, 200 μL) 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 refer to Figures 17 - 19 , Figure 17Schematic diagram of qualitative uptake of C-6, C-6-BSP, and C-6-AN-TBSP by MDA-MB-231 cells; Figure 18 Schematic diagram of quantitative uptake of C-6, C-6-BSP, and C-6-AN-TBSP by MDA-MB-231 cells; Figure 19 Schematic diagram of the average fluorescence intensity of the uptake of C-6, C-6-BSP, and C-6-AN-TBSP by MDA-MB-231 cells.

[0219] Figure 18 In it, the fluorescence intensities of breast cancer MDA-MB-231 cells in cell culture medium with pH 7.4 or 6.5 are respectively shown. Among them, the test results of group (A) are for pH 7.4. In group (A), light green is the control group, yellow is C-6, blue is C-6-BSP, and pink is C-6-AN-TBSP; the test results of group (B) are for pH 7.4. In group (B), light green is the control group, yellow is C-6, blue is C-6-BSP, and dark green is C-6-AN-TBSP.

[0220] Figure 19 In it, the average fluorescence intensities of the uptake of C-6, C-6-BSP, and C-6-AN-TBSP by breast cancer MDA-MB-231 cells in cell culture medium with pH 7.4 or 6.5 are respectively shown; among them, based on the abscissa, from bottom to top are the uptake fluorescence intensities in cell culture medium with pH 7.4 or 6.5.

[0221] Combined with Figures 17 - 19 the test results, compared with the control group, C-6-TROP2-BA-SS-PPEP (147313) shows 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 to actively target breast cancer MDA-MB-231 cells to promote drug uptake; and the fluorescence intensity is stronger in the culture medium with pH 6.5 than that at pH 7.4. The results show that the uptake of C-6-TROP2-BA-SS-PPEP by MDA-MB-231 cells can be promoted at pH 6.5 (weak acidic microenvironment condition).

[0222] (2) Cell viability experiment

[0223] Please refer to Figure 20Schematic diagram of the test results of cell viability experiments; among them, when the concentration of TROP2-BA-SS-PPEP is 5.4 μg / mL, the cytotoxicity to MDA-MB-231 cells is relatively high, and only as low as 31.21% of the cell survival rate; at the same concentration, the cell survival rate of BA-SS-PPEP to MDA-MB-231 cells is 67.55%; compared with the monomer BA, TROP2-BA-SS-PPEP can significantly reduce cell viability in the concentration range of 1.8 - 14.4 μg / mL; among them, the IC50 of BA is 120.2 μg / mL, the IC50 of BA-SS-PPEP is 12.34 μg / M, and the IC50 of TROP2-BA-SS-PPEP is 4.76 μg / mL; polyphospholipid PPEP has no obvious toxicity in the concentration range of 1.8 - 115.2 μg / mL, and the activity of MDA-MB-231 cells is greater than 70%.

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

[0225] Therefore, the TROP2-BA-SS-PPEP prepared in this example can promote the active uptake of drugs by MDA-MB-231 cells. According to the IC50 of TROP2-BA-SS-PPEP, 2.38, 4.76, and 9.52 μg / mL were selected as the low, medium, and high doses for subsequent experimental drug administration. PTX used 198.5 ng / mL as the dose for subsequent experimental drug administration according to the IC50.

[0226] (3) Cell scratch assay

[0227] Please refer to Figure 21 Schematic diagram of the test 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 the increase in concentration; within 0 - 48 h, the change in the ability of Trop2-BA-SS-PPEP to inhibit the migration of MDA-MB-231 cells was not obvious, and it had a similar inhibitory effect to the positive control group PTX.

[0228] Among them, BA-SS-PPEP can also inhibit the migration of MDA-MB-231 cells, and the cell migration rate within 48 h is only 17.81%; BA has the ability to inhibit the migration of MDA-MB-231 cells, but the inhibitory effect decreases with time, increasing from the migration rate of 21.48% at 12 h to 36.75% at 24 h and to 65.07% at 48 h, indicating 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 refer to Figure 22 the schematic diagram of the test results of the cell scratch assay in. Compared with the blank control, Trop2-BA-SS-PPEP treatment significantly reduced the ability of MDA-MB-231 cells to invade Matrigel, and the cells at high, medium, and low concentrations were 55.0%, 39.73%, and 33.03% respectively; the inhibitory rate of BA on the invasion of MDA-MB-231 cells at 24 h was 71.64%, the cell invasion rate of the positive control group PTX was 30%, and the cell invasion rate of BA-SS-PPEP was 47.67%, indicating that the nano-prodrug preparation in this example can effectively improve the pharmacological effect of baicalin in inhibiting the invasion of MDA-MB-231 cells.

[0231] (5) In vivo distribution

[0232] Please refer to Figure 23 the in vivo imaging schematic diagram of the in vivo distribution experiment. Compared with free ICG, after intravenous injection of ICG-labeled Trop2-BA-SS-PPEP and BA-SS-PPEP nanoparticles into mice, they rapidly accumulated at the tumor site, and their fluorescence signals slowly decreased over time, indicating that the nanoparticles not only have good targeting in tumor tissues but also can stay stably in tumor tissues.

[0233] At the end of the experiment, the main organs (heart, liver, spleen, lung, kidney) and tumor tissues of the mice were collected. ICG-labeled Trop2-BA-SS-PPEP and BA-SS-PPEP mainly accumulated in the tumor tissues, and the concentration in the tumor tissues was the highest, which was consistent with the in vivo monitoring results.

[0234] (6) Antitumor effect

[0235] Please refer to Figure 24 and Figure 25 , among which, Figure 24 is the schematic diagram of mouse metastasis imaging, Figure 25It is a schematic diagram of the body weight of mice. From the fluorescence changes of lung metastases in mice, it can be seen that the tumors in the model group and the nano-carrier PPEP group grew rapidly, while the tumor growth rate of the mice in the sample group was slower. Among them, the fluorescence in the lungs of the nano-prodrug and PTX group was lower, significantly smaller than that in the BA group, indicating that the nano-prodrug can effectively improve the tumor inhibitory effect of BA and enhance the anti-breast cancer lung metastasis effect.

[0236] Specifically, Figure 25 The changes in the body weight of mice showed that although the body weights of the mice in the model group and the nano-carrier PPEP group fluctuated, they generally increased slightly. No obvious body weight changes were shown in the remaining sample groups, preliminarily indicating that the nano-prodrug has good biosafety.

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

[0238] The technical solutions provided in the embodiments of the present application have been introduced in detail above. Specific examples are used in this article to elaborate on the principles and implementation manners of the embodiments of the present application. The descriptions of the above embodiments are only applicable to help understand the principles of the embodiments of the present application. At the same time, for those of ordinary skill in the art, according to the embodiments of the present application, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present application.

Claims

1. A nano prodrug, characterized in that: The nanoprodrug has a compound having a structure shown in Formula I or a pharmaceutically acceptable salt thereof: Wherein, the molecular weight of the nano prodrug is 47820.

28.

2. A method for preparing a nano prodrug as claimed in claim 1, characterized in that: The preparation method comprises: 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; The baicalin is dissolved in an organic solvent, and EDC and a catalyst are added to react; the reactant is added to a dimethylformamide solution of polymer I, and after the reaction, the polymer II is purified by dialysis; Disperse bromoacetic acid in PBS buffer solution, add EDC and N-hydroxysuccinimide to stir and react, centrifuge the mixture and discard the supernatant; stir the activated bromoacetic acid with Trop-2 monoclonal antibody and centrifuge to purify, dry and dialyze to obtain polymer III; Potassium carbonate, polymer II and polymer III are reacted in a dimethylformamide solution, and the nano-prodrug is obtained after centrifugal purification and freeze-drying; The structural formulas of polymer I, polymer II and polymer III are as follows:

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 the 2-ethoxy-1,3,2-dioxaphosphane 2-oxide to the dihydroxy disulfide is 16 to 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 nano prodrug according to claim 1 in the preparation of a drug for preventing, alleviating or treating triple-negative breast cancer.

9. The use according to claim 8, characterized in that The drug is a drug 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 for oral administration.

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