Polyester-docetaxel nano-coupling drug targeting PSMA as well as preparation method and application of polyester-docetaxel nano-coupling drug

Abstract

The invention relates to the technical field of drug synthesis, in particular to a PSMA-targeted polyester-docetaxel nano-coupling drug as well as a preparation method and application of the PSMA-targeted polyester-docetaxel nano-coupling drug. Comprising the following steps: mixing dimercapto polyethylene glycol, a diacrylate monomer, n-hexylamine and tetrahydrofuran to obtain PET-STrt; the preparation method comprises the following steps: mixing PET-STrt, trifluoroacetic acid, triethyl silane and dichloromethane to react, so as to obtain PET-SH; the preparation method comprises the following steps: mixing PET-SH, maleimide modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl) ureido) glutaric acid and N, N-dimethylformamide, and carrying out a reaction so as to obtain PET-DCL-SH; the preparation method comprises the following steps: mixing PET-DCL-SH, pyridyldisulfide modified docetaxel and N, N-dimethylformamide to react, so as to obtain the polyester-docetaxel nano conjugate, and the polyester-docetaxel nano conjugate has good water solubility and high drug loading capacity.

Description

Technical Field

[0001] The present invention relates to the technical field of drug synthesis, and in particular to a PSMA-targeted polyester-docetaxel nano-coupled drug and a preparation method and application thereof. Background Art

[0002] Prostate cancer (PCa) is the most common malignancy in the male urinary system. Advances in the molecular biology of prostate cancer have shown that prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer and is a specific biomarker for prostate cancer. PCa can be roughly divided into three subtypes: AR-dependent PCa (ADPC), cancer stem cells such as PCa, and neural PCa (NEPC). Generally speaking, ADPC accounts for 80-90% and is PSMA-positive, which helps to develop PMSA-based diagnostic or therapeutic tools to manage this type of disease. For example, indium 111 caprokusumab or Scan, a radiolabeled PMSA antibody (mAb7E11), has been approved as a diagnostic method for visualizing metastatic prostate cancer, which exceeds current computed tomography (CT) scans or magnetic resonance imaging (MRI) in terms of accuracy. In addition, the therapeutic value of PSMA has been observed in a clinical trial (NCT04227275) in which a cell-based therapy called CART-PSMA-TGFbRDN cells (CART) was tested in patients with advanced PCa. In addition, in recent years, several PSMA-based delivery systems have been reported for the treatment and diagnosis of prostate cancer.

[0003] It is well known that androgen receptor (AR) plays a key role in the occurrence and progression of prostate cancer, leading to the development of androgen deprivation therapy (ADT) as the golden treatment for prostate cancer. However, as PCa progresses to castration-resistant disease, the treatment time of ADT only lasts for 2 to 3 years. Chemotherapy is the universal treatment for castration-resistant prostate cancer (CRPC). Docetaxel (DTX), an anti-microtubule chemotherapy drug, has been approved by the FDA for the treatment of metastatic CRPC or the management of castration-sensitive prostate cancer combined with ADT. Clinical evidence shows that DTX can prolong the survival of patients by about 1 year. However, the severe side effects caused by DTX, including fatigue, diarrhea, and anemia, limit the clinical application of this chemotherapy drug, which may be related to the lack of tumor targeting of DTX and its unexpected distribution to other tissues in the body. In addition, DTX also has some problems, such as insolubility and poor pharmacokinetic properties.

[0004] Regarding these issues, the use of multifunctional carriers to deliver small molecules is a good strategy. These carefully designed drug delivery systems (DDSs) can self-assemble into various nanomorphologies in aqueous environments. Smart polymer-drug conjugates are a unique type of nano-DDS. They make drug molecules invisible in the circulatory system, but when targeted to lesions, release cargo to exert effects under internal or external stimuli. Biodegradability is a core safety indicator of polymers in DDS. In this regard, polyesters have become a good candidate because they can be catalyzed and hydrolyzed by carboxylesterases that are ubiquitous in the body. However, the inherent stubborn hydrophobicity of polyesters greatly limits their application.

[0005] Based on this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to develop a polyester-docetaxel nanoconjugate with good water solubility and high drug loading, and specifically relates to a PSMA-targeted polyester-docetaxel nanoconjugate drug and a preparation method and application thereof.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a PSMA-targeted polyester-docetaxel nanoconjugate, comprising the following steps:

[0009] (1) mixing bis-mercapto polyethylene glycol, diacrylate monomer, n-hexylamine and tetrahydrofuran, and reacting for 4 to 6 hours to obtain a polyester containing trityl mercapto groups;

[0010] (2) mixing a polyester containing trityl mercapto groups, trifluoroacetic acid, triethylsilane and dichloromethane, reacting at room temperature for 5 to 7 hours, removing the trityl group, and obtaining a polyester containing mercapto groups;

[0011] (3) mixing the thiol-containing polyester, maleimide-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid and N,N-dimethylformamide, and reacting for 5 to 7 hours to obtain a polyester containing a PSMA targeting group and a thiol group;

[0012] (4) A polyester containing a PSMA targeting group and a thiol group, pyridyldisulfide-modified docetaxel, and N,N-dimethylformamide are mixed, reacted for 11 to 13 hours, dissolved in deionized water, and dialyzed to obtain a polyester-docetaxel nanoconjugate.

[0013] Preferably, the mass ratio of the bis-mercapto polyethylene glycol to the diacrylate monomer is 580-620:155-160;

[0014] The mass ratio of the bis-mercapto polyethylene glycol to n-hexylamine is 580-620:5.8-6.2;

[0015] The mass volume ratio of the mixture of bis-mercapto polyethylene glycol and tetrahydrofuran is 580-620 mg: 1.5-2.5 mL;

[0016] The reaction temperature in step (1) is 23-27°C.

[0017] Preferably, the mass volume ratio of the polyester containing trityl mercapto group and dichloromethane is 520-540 mg: 37-39 mL;

[0018] The mass volume ratio of the trityl mercapto-containing polyester and trifluoroacetic acid is 520-540 mg: 1.5-2.5 mL;

[0019] The mass volume ratio of the trityl mercapto-containing polyester and triethylsilane is 520-540 mg: 1.1-1.3 mL.

[0020] Preferably, the mass volume ratio of the mercapto-containing polyester and N,N-dimethylformamide is 280-320 mg: 2.5-3.5 mL;

[0021] The mass ratio of the thiol-containing polyester to maleimide-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid (Mal-DCL) is 280-320:3;

[0022] The reaction temperature in step (3) is 23-27°C.

[0023] Preferably, the preparation method of the maleimide-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)pentanedioic acid is:

[0024] Mix di-tert-butyl (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid ester, 3-maleimidopropionic acid hydroxysuccinimide ester and N,N-diisopropylethylamine with dichloromethane, and react for 4.5 to 5.5 hours to obtain an intermediate product; mix the intermediate product, trifluoroacetic acid and dichloromethane, and react for 0.8 to 1.2 hours to obtain maleimide-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid;

[0025] The mass ratio of the (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid di-tert-butyl ester and 3-maleimidopropionic acid hydroxysuccinimide ester is 300:195-200;

[0026] The mass ratio of the mixture of (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid di-tert-butyl ester and N,N-diisopropylethylamine is 300:320-322;

[0027] The mass volume ratio of the mixture of (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid di-tert-butyl ester and dichloromethane is 300 mg:3-5 mL;

[0028] The mass volume ratio of the intermediate product and trifluoroacetic acid is 200-220 mg: 3-5 mL;

[0029] The mass volume ratio of the intermediate product and dichloromethane is 200-220 mg:2 mL.

[0030] Preferably, the mass volume ratio of the polyester containing PSMA targeting groups and thiol groups and N,N-dimethylformamide is 90-110 mg: 1.5-2.5 mL;

[0031] The mass ratio of the polyester containing PSMA targeting group and thiol group to the docetaxel modified with pyridyldisulfide group is 90-110:38-42;

[0032] The reaction temperature in step (4) is 23-27°C.

[0033] Preferably, the preparation method of the pyridyldisulfide-modified docetaxel is:

[0034] Docetaxel, 3-(2-pyridyldithio) propionic acid, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and dichloromethane are mixed and reacted for 7.8 to 8.2 hours to obtain pyridyldithio modified docetaxel;

[0035] The mass ratio of docetaxel and 3-(2-pyridyldithio)propionic acid is 1100:250-350;

[0036] The mass ratio of docetaxel and N,N'-dicyclohexylcarbodiimide is 1100:550-600;

[0037] The mass ratio of docetaxel and 4-dimethylaminopyridine is 1100:150-190;

[0038] The mass volume ratio of the mixture of docetaxel and dichloromethane is 1100 mg:18-22 mL.

[0039] The present invention also provides a polyester-docetaxel nanoconjugate prepared by the preparation method, wherein the molecular weight of the polyester-docetaxel nanoconjugate is 22.52 kDa, and the drug loading amount is ≥6.99%.

[0040] The present invention also provides the use of the polyester-docetaxel nanoconjugate in the preparation of a drug targeting prostate-specific membrane antigen-positive prostate cancer.

[0041] The present invention also provides the use of the polyester-docetaxel nanoconjugate prepared by the preparation method in the preparation of a drug targeting prostate-specific membrane antigen-positive prostate cancer.

[0042] The solution of the present invention has the following advantages:

[0043] The PSMA-targeted polyester-docetaxel nanoconjugate (PET-DCL-DTX) of the present invention is prepared by combining water-soluble polyester with an antitumor drug (DTX) and a prostate-specific membrane antigen (PSMA) ligand (DCL). The prepared PSMA-targeted polyester-docetaxel nanoconjugate is used to treat PSMA-positive prostate cancer. In the preparation process of polyester, short-chain polyethylene glycol is directly involved in polymerization as a monomer, which not only overcomes the problem of poor water solubility of polyester, but also does not affect the drug-carrying capacity. Benefiting from the ideal hydrophilic and hydrophobic balance, PET-DCL-DTX self-assembles into a nearly spherical 57-nanometer aqueous solution, and the external hydrophilic PEG and DCL disguised by hydrophobic DTX promote the target delivery of a large amount of DTX tumors and reduce the invasion of normal tissues. DCL enables the nanoconjugate to have a specific binding ability to PSMA-positive prostate tumor cells, thereby showing the selectivity of tumor subtypes. After entering the tumor cell, the reduction-sensitive cleavage of the disulfide bond causes DTX to detach from the polymer and revive. In addition, compared with DTX, the nanoconjugate has lower toxic side effects.

[0044] The present invention also prepares a polyester with good water solubility and high drug loading capacity, and uses it to construct a polymer-drug nanoconjugate for dual-targeted treatment of tumor tissue and subtypes, which is used to treat PSMA-positive prostate cancer. The nanoconjugate shows high efficiency and safety both in vivo and in vitro, and has potential clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of the synthesis of PET-DCL-DTX.

[0046] Figure 2The physicochemical properties of PET-DCL-DTX.

[0047] Figure 3 This is the effect of PET-DCL-DTX on the growth of PSMA-positive PCa cells.

[0048] Figure 4 The effect of PET-DCL-DTX on the proliferation and apoptosis of PSMA-positive PCa cells.

[0049] Figure 5 The effect of PET-DCL-DTX on the migration and invasion of PSMA-positive PCa cells.

[0050] Figure 6 This is the effect of PET-DCL-DTX on the ubiquitin-proteasome degradation pathway.

[0051] Figure 7 The effect of PET-DCL-DTX on PSMA-positive PCa tumor growth was examined in a mouse model. DETAILED DESCRIPTION

[0052] The preparation method of maleimide-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid (Mal-DCL) is as follows: (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid di-tert-butyl ester (DCL-O t Bu), 3-maleimidopropionic acid hydroxysuccinimide ester and N,N-diisopropylethylamine are mixed in dichloromethane and reacted for 5 hours to obtain an intermediate product; the intermediate product is mixed with trifluoroacetic acid in dichloromethane and reacted for 1 hour to obtain maleimide-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)pentanedioic acid;

[0053] The mass ratio of the (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid di-tert-butyl ester and 3-maleimidopropionic acid hydroxysuccinimide ester is preferably 300:197;

[0054] The mass ratio of the (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid di-tert-butyl ester and N,N-diisopropylethylamine is preferably 300:321;

[0055] The mass volume ratio of the mixture of (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid di-tert-butyl ester and dichloromethane is preferably 300 mg:4 mL;

[0056] The mass volume ratio of the intermediate product and trifluoroacetic acid is 200-220 mg:3-5 mL, preferably 210 mg:4 mL;

[0057] The mass volume ratio of the intermediate product and dichloromethane is 200-220 mg:2 mL, preferably 210 mg:2 mL.

[0058] In the present invention, the (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)pentanedioic acid di-tert-butyl ester reacts with 3-maleimidopropionic acid hydroxysuccinimide ester to introduce maleimide to obtain the intermediate product Mal-DCL-3O t Bu, the intermediate product is mixed with trifluoroacetic acid in dichloromethane (DCM), the tri-tert-butyl ester is removed, and the tricarboxylic acid structure of the PSMA targeting ligand DCL is restored, thereby obtaining maleimide-modified DCL (Mal-DCL).

[0059] Among the hydroxyl groups of docetaxel (DTX), the hydroxyl group on the straight chain has the highest esterification activity and has been successfully used to introduce DTX into various carriers through ester bonds. Specifically, the present invention mixes DTX with 3-(2-pyridyldithio) propionic acid (SPDP-COOH) for esterification to obtain pyridyldithio (SPDP)-modified DTX (SPDP-DTX).

[0060] During the preparation of the polyester support, the diacrylic acid ester monomer (diAC-STrt) was synthesized through a two-step reaction: first, N-boc-serine alcohol was diesterified with acryloyl chloride to obtain diAC-NHBoc, and then the Boc protecting group of diAC-NHBoc was removed with trifluoroacetic acid, followed by condensation with TrtS-COOH to finally obtain diAC-STrt.

[0061] In order to overcome the problem of poor water solubility of polyester as a carrier, the present invention adopts short-chain polyethylene glycol as a hydrophilic monomer to participate in polymerization. First, the selection principle of polyethylene glycol chain length should not only improve water solubility, but also ensure the ratio of polymerization degree and functional monomer. After preliminary screening and testing, polyethylene glycol with MW of 2kDa was finally determined. Dithiol polyethylene glycol (SH-PEG-SH) and diacrylate monomer (diAC-STrt) are catalyzed by n-hexylamine to carry out mercapto-ene click polymerization. Since both monomers are soluble in ether, the crude product can be washed with ether to obtain a pure target copolymer, a thiol-containing polyester (PET-STrt).

[0062] Using PET-SH as a carrier, DCL and DTX were introduced sequentially through covalent bonds. First, maleimide-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)pentanedioic acid (Mal-DCL) was combined with PET-SH through thiol-ene click chemistry to prepare PET-DCL-SH. Here, the content of Mal-DCL was controlled at 10% of the carrier, which not only met the targeting requirements but also retained more thiol groups to achieve subsequent high drug loading. After the introduction of DCL, DTX was coupled to the carrier through a thiol-disulfide exchange reaction between pyridine disulfide-modified docetaxel (SPDP-DTX) and PET-DCL-SH, and finally the target drug delivery system, polyester containing PSMA targeting groups and thiol groups (PET-DCL-DTX) was prepared.

[0063] The preparation method of pyridyldisulfide-modified docetaxel is as follows: docetaxel, 3-(2-pyridyldisulfide) propionic acid, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine are mixed in dichloromethane, reacted for 8 hours, and pyridyldisulfide-modified docetaxel (SPDP-DTX) is obtained. The mass ratio of docetaxel to 3-(2-pyridyldisulfide) propionic acid is preferably 1100:300; the mass ratio of docetaxel to N,N'-dicyclohexylcarbodiimide is preferably 1100:575; the mass ratio of docetaxel to 4-dimethylaminopyridine is preferably 1100:170; the mass volume ratio of docetaxel to dichloromethane is preferably 1100 mg:20 mL.

[0064] The solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0065] The bis-thiol polyethylene glycol described in the examples of the present invention was purchased from Beijing Baikai Biotechnology Co., Ltd. N-boc-serine alcohol and acryloyl chloride were purchased from Tokyo Chemical Industry Co., Ltd. TCI.

[0066] Example 1

[0067] Mix bisthiopolyethylene glycol (SH-PEG-SH) (600 mg, 0.30 mmol), diacrylate monomer (diAC-STrt) (158.9 mg, 0.30 mmol), and n-hexylamine (6.1 mg, 0.06 mmol) in tetrahydrofuran (2 mL), react at 25 ° C for 5 h to obtain a reaction solution, and add a trace amount of methyl acrylate to quench the polymerization. Then the reaction solution was added dropwise to ether (80 mL), and some solids appeared. After filtering, the solid was washed with ether (20 mL × 3) to obtain a PET-STrt copolymer as a white solid (630 mg, yield: 83.0%), MW is 18.85 kDa, PDI is 1.60, 1 HNMR confirmed its chemical structure.

[0068] In the mixture of 530 mg of PET-STrt copolymer, 2.0 mL of trifluoroacetic acid and triethylsilane (Et 3 1.2 mL of SiH) was mixed with 38 mL of dichloromethane, followed by stirring at room temperature for 6 h, and then washed with ether (20 mL×3) to obtain a thiol-containing polyester (PET-SH) as a white solid (464 mg) with a MW of 17.11 and a PDI of 1.62. 1 HNMR confirmed its chemical structure.

[0069] PET-SH 300 mg and 3 mg of maleimide-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)pentanedioic acid (Mal-DCL) were mixed in 3 mL of N,N-dimethylformamide and reacted at room temperature for 6 h. Under ice bath cooling, the reaction solution was slowly added to 300 mL of RO-water, dialyzed (MWCO=8 kDa), filtered and freeze-dried to obtain a white solid (270 mg) of polyester (PET-DCL-SH) containing PSMA targeting groups and thiol groups, with a MW of 19.20 and a PDI of 1.84. 1HNMR confirmed its chemical structure. The preparation method of maleimido-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)pentanedioic acid is as follows: di-tert-butyl (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)pentanedioate (DCL-OtBu) (300 mg, 0.62 mmol), 3-maleimidopropionic acid hydroxysuccinimide ester (197 mg, 0. 74mmol) and N,N-diisopropylethylamine (321mg, 2.48mmol) were mixed in 5mL of dichloromethane and reacted at room temperature for 5h to obtain an intermediate product; 210mg of the intermediate product was mixed with 4mL of trifluoroacetic acid in dichloromethane (2mL), and reacted at room temperature for 1h to obtain maleimide-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)pentanedioic acid.

[0070] PET-DCL-SH (100 mg) and pyridyldisulfide-modified docetaxel (SPDP-DTX) (40 mg) were mixed in N,N-dimethylformamide (2 mL), stirred at 25°C for 12 h, and methyl acrylate (20 mg) was added to quench the reaction. Under ice bath cooling, the reaction solution was slowly added to deionized water (20 mL), then dialyzed (MWCO = 8 kDa), filtered and freeze-dried to obtain PSMA-targeted polyester-docetaxel nanoconjugate (PET-DCL-DTX) as a white solid (78 mg), with a MW of 22.52 and a PDI of 1.67. 1 HNMR confirmed its chemical structure.

[0071] The preparation method of pyridinedisulfide-modified docetaxel is as follows: docetaxel (1.1 g, 1.39 mmol), 3-(2-pyridinedisulfide) propionic acid (300 mg, 1.39 mmol), N,N'-dicyclohexylcarbodiimide (575 mg, 2.78 mmol) and 4-dimethylaminopyridine (170 mg, 1.39 mmol) are mixed in dichloromethane (20 mL), and the mixture is reacted at room temperature for 8 h to obtain pyridinedisulfide-modified docetaxel.

[0072] The synthesis diagram of PET-DCL-DTX is as follows: Figure 1 shown.

[0073] 1 HNMR spectrum Figure 2 A. DLS was used to determine the molecular weight and polymer dispersion index of PET-DCL-DTX. The results are shown in Figure 2B; the content of docetaxel (DTX) in PET-DCL-DTX was determined by ultraviolet spectrophotometer (METASH, Shanghai) at an ultraviolet absorption wavelength of 230 nm. The results are shown in Figure 2 C; DLS method was used to detect the particle size of the conjugate, and the results were as follows Figure 2 D; TEM method was used to detect the morphology of the nanoconjugate, and the results were as follows Figure 2 E; DLS method was used to measure the surface potential of the nanoconjugate. The results are shown in Figure 2 As shown in F.

[0074] Figure 2 The molecular weight of PET-DCL-DTX is 22.52 kDa, the PDI is 1.67, the DTX content is as high as 6.99%, the water phase particle size is 57 nm, the spherical morphology is 40 nm in diameter, and the surface potential is 0 mV.

[0075] Example 2

[0076] In order to study the reduction-sensitive DTX release of PET-DCL-DTX, two different physiological reducing conditions were simulated in vitro, (PBS containing 10mM glutathione (GSH), pH 5.4, 37°C) or (PBS containing 10μM GSH, pH 7.4, 37°C), PET-DCL-DTX (4.30mg, 300μg of DTX) was dissolved in the above two DTT solutions and then incubated at 37°C for 48h. During this process, 200μL samples were taken at different time points (0h, 1h, 2h, 4h, 8h, 12h, 24h, 48h). The sample was then diluted with 400μL methanol and divided into three times. The DTX concentration was detected by HPLC to investigate the release of DTX in PET-DCL-DTX. The same method was used to study the effects of different acidic environments (PBS, pH 7.4, 37°C or PBS, pH 5.4, 37°C) on drug release. The results are shown in Figure 2 As shown in G.

[0077] Figure 2 G shows that the release behavior of DTX was obtained by HPLC detection of the change in DTX concentration during the 48h incubation. From the result graph, it can be seen that under high concentration of GSH, DTX can be rapidly released from the polymer, reaching 53% in the first 12h and 65% after 48h. In contrast, under low concentration of GSH, the release of DTX was significantly slow, 12% and 17% at 12h and 48h, respectively. The above results show that PET-DCL-DTX can maintain high stability under normal physiological reducing conditions, and can release drugs quickly and effectively under high reducing conditions.

[0078] PET-DCL-DTX is relatively stable under normal physiological pH conditions, with a total release of less than 3%. However, under weak acid conditions, due to the hydrolysis of ester bonds, the release efficiency is improved, with a total release of 9%. These results indicate that the hydrolysis of ester bonds caused by slightly acidic conditions can also promote drug release.

[0079] Example 3

[0080] PCa cell lines including C4-2, CWR22Rv1 and PC3 were purchased from Xiamen Immune Cell Co. PCa cells were cultured in 10% fetal bovine serum RPMI medium (VivaCell, Shanghai, China) supplemented with 2 mM-glutamine, 100 IU / mL penicillin and 100 μg / mL streptomycin.

[0081] CWR22Rv1 cells were treated with 10 nM PET-DCL-DTX for 4 h to construct PMSA-specific recognition of PET-DCL-DTX. Immunofluorescence was used to detect PMSA in the treated CWR22Rv1 cells. The results are shown in Figure 3 As shown in A (scale 5mm).

[0082] C4-2, CWR22Rv1 and PC3 cells were treated with different concentrations of PET-DCL-DTX, PET-DCL-SH and DTX for 4 h, and the growth of different cells was observed. Figure 3 As shown in B.

[0083] CWR22Rv1 cells and PC3 cells were treated with 10 nM PET-DCL-DTX and PET-DCL-SH for 24 h, and the colonies of cells were observed. Figure 3 C, the statistical results are as follows Figure 3 As shown in D.

[0084] F-actin staining was used to observe the cytoskeleton of CWR22Rv1 cells treated with different drugs. Figure 3 As shown in E (scale 5mm).

[0085] Immunofluorescence showed that PET-DCL-DTX specifically recognized PSMA. Compared with PET-DCL-SH, PET-DCL-DTX significantly inhibited the cell growth of CWR22Rv1 and C4-2 in a dose-dependent manner. In contrast, PET-DCL-DTX had little inhibitory activity against PSMA-negative PC3 cells. DTX significantly inhibited the viability of PCa cells without selecting PSMA ( Figure 3 B). PET-DCL-DTX significantly inhibited the colony formation ability of CWR22Rv1 cells, but had no significant difference in its effect on PC3 cells compared with PET-DCL-SH ( Figure 3 C and Figure 3 D) These data collectively indicate that PET-DCL-DTX is able to specifically recognize PSMA antigen, thereby exerting its anticancer activity.

[0086] Since DTX is a clinically well-known drug that disrupts the cytoskeletal structure by binding to b-tubulin, we investigated whether DTX-packaged PET-DCL-DTX still has this ability. As a result, compared with PET-DCL-SH, the nanoconjugate PET-DCL-DTX strongly disrupted the multi-branched microtubule network of CWR22Rv1 cells ( Figure 3 E), indicating that the destruction of the cytoskeleton network is one of the reasons for PET-DCL-DTX-induced cell death. In summary, the anticancer activity of PET-DCL-DTX on PCa cell growth depends on its recognition of PMSA.

[0087] Example 4

[0088] Apoptosis and proliferation are two key biological events related to cells. After treating CWR22Rv1 and PC3 cells with 10nM PET-DCL-DTX and PET-DCL-SH for 24h, Edu staining (DAPI was used to monitor the cell nucleus) was used to detect the proliferation of PCa cells treated with different drugs. Figure 4 A and Figure 4 B (scale 50 mm) shows the bar graph for statistical analysis. Figure 4 A shows the proliferation of CWR22Rv1 treated with different drugs. Figure 4 B shows the proliferation of PC3 cells treated with different drugs. PMSA-positive CWR22Rv1 cells and PMSA-negative PC3 cells were exposed to 10 nM PET-DCL-DTX and PET-DCL-SH for 48 h, respectively, and collected for annexin V. Flow cytometry was used to detect the apoptosis of CWR22Rv1 and PC3 cells treated with different drugs. Figure 4 C, Figure 4 The upper part of C shows the apoptosis of CWR22Rv1, and the lower part shows the apoptosis of PC3 cells. The bar graphs are their statistical analysis diagrams.

[0089] Western blot assay to detect cleaved PARP in CWR22Rv1 cells treated with different drugs Figure 4 D.

[0090] The results showed that the number of positive cells after PET-DCL-DTX treatment (39.6%) was significantly reduced compared with that after PET-DCL-SH treatment (61.6%). The percentage of Edu-positive cells was comparable in PSMA-deficient PC3 cells treated with or without PET-DCL-DTX, indicating that this PET-DCL-DTX specifically recognized the PSMA antigen and exerted its cell proliferation inhibitory effect.

[0091] The apoptosis effect of PET-DCL-DTX on CWR22Rv1 cells (9.2%) was stronger than that of PET-DCL-SH on CWR22Rv1 cells (2.3%), but there was no significant difference between the two components in the apoptosis effect on PC3 cells.

[0092] This was also confirmed by immunoblotting to detect the apoptosis marker PARP cleavage.

[0093] Taken together, these data suggest that PET-DCL-DTX, by targeting the DCL moiety, specifically inhibits cell viability of PSMA-positive PCa cells by triggering apoptosis, blocking cell cycle entry, and slowing down the cell proliferation rate.

[0094] Example 5

[0095] The wound healing assay was used to detect the migration of CWR22Rv1 cells and PC3 cells after treatment with 10 nM PET-DCL-DTX and PET-DCL-SH for 24 h. Figure 5 A) The left picture shows the migration of CWR22Rv1 cells, and the right picture shows the migration of PC3 cells. Transwell invasion assay detected the cell invasion inhibition of CWR22Rv1 cells and PC3 cells after 24h treatment with 10nM PET-DCL-DTX and PET-DCL-SH ( Figure 5 B), the bar graph is the statistical result of cell invasion inhibition.

[0096] Immunoblotting was used to detect the secretion of E-cadherin and N-cadherin in CWR22Rv1 cells treated with different concentrations of PET-DCL-DTX for 24 h. Figure 5 C). Epithelial-mesenchymal transition (EMT) is a key step in tumor migration and invasion. Fluorescence PCR was performed to detect the mRNA levels of EMT-related genes in CWR22Rv1 cells after treatment with different concentrations of PET-DCL-DTX for 24 hours ( Figure 5 D).

[0097] The results showed that PET-DCL-DTX could effectively inhibit the migration of CWR22Rv1 cells. This inhibition of cell migration was not observed in PSMA-deficient PC3 cells. Transwell invasion assays showed that PET-DCL-DTX treatment had a strong inhibitory effect on the invasion of CWR22Rv1 cells. PET-DCL-DTX lost its effect on the invasion of PC3 cells. The expression level of E-Cadherin (epithelial marker) increased, while the expression levels of PSMA (mesenchymal marker) and N-Cadherin (N-Cadherin) decreased after PET-DCL-DTX treatment. ET-DCL-DTX inhibited the EMT process of PSMAPCa cells. PET-DCL-DTX treatment led to EMT blockade, as shown by changes in the mRNA levels of EMT-related genes. In summary, these results indicate that the DCL portion of PET-DCL-DTX and packaged DTX have a strong inhibitory effect on the migration and invasion of PSMA-positive PCa cells.

[0098] Example 6

[0099] Given that AR signaling is a major driving force determining PCa development and previous reports have suggested that AR is also a target of DTX, the following studies were performed.

[0100] After CWR22Rv1 cells were treated with different concentrations of PET-DCL-DTX and PET-DCL-SH for 24 h, the AR protein levels in cells treated with different drugs at different concentrations were detected by stem cell blotting. Figure 6 A). GAPDH was used as a control. After CWR22Rv1 cells were treated with 10 nm PET-DCL-DTX and PET-DCL-SH for 24 h, qPCR was used to detect the expression levels of AR target genes in CWR22Rv1 cells treated with different drugs ( Figure 6 B). PET-DCL-DTX reduced AR in the proteasome because its inhibitor MG132, but not the lysosomal inhibitor chloroquine, prevented AR degradation ( Figure 6 C). After CWR22Rv1 cells were treated with 10 nm PET-DCL-DTX and PET-DCL-SH for different time periods, qPCR was used to detect the degradation rate of AR in CWR22Rv1 cells treated with different drugs, and GAPDH was used as a control ( Figure 6 D). After CWR22Rv1 cells were treated with 10 nm PET-DCL-DTX and PET-DCL-SH for 24 h, the ubiquitination level of AR in the cells was detected ( Figure 6 E) The interaction between DTX and AR in PET-DCL-DTX is shown in Figure 6 F.

[0101] The results showed that PET-DCL-DTX significantly led to a dose-dependent decrease in AR in CWR22Rv1 cells. However, PET-DCL-SH had no effect on AR protein levels. AR signaling pathways are presented through transcriptional regulation of its downstream genes. Consistent with AR reduction, PET-DCL-DTX also significantly suppressed the mRNA levels of AR downstream targets including PSA, TMPRSS2, and FKBP5, while PET-DCL-SH treatment did not. PET-DCL-DTX treatment significantly increased the degradation rate of AR. When CWR22Rv1 cells were exposed to PET-DCL-DTX treatment, more ubiquitin was coupled to AR, indicating that the reduction of AR by PET-DCL-DTX was mediated by the ubiquitin-proteasome degradation pathway. DTX released by PET-DCL-DTX nanomedicine may directly interact with the predicted Autodock of AR and change its conformation. In summary, PET-DCL-DTX inhibits AR signaling to inhibit the progression of PSMA PCa.

[0102] Example 7

[0103] Nude mice aged 6 to 8 weeks were purchased from Chongqing Tengxin Experimental Animal Co., Ltd. (Chongqing, China) and injected subcutaneously with 1×10 6 CWR22Rv1 cells (1:1 with growth factor-reduced matrix gel). After 4 weeks, the mice were divided into groups and given 1) PET-DCL-DTX, 2) PET-DCL-SH and 3) DTX. The effective concentration of DTX was 5 mg / kg. The administration method was intraperitoneal injection every other day. Tumor size and body weight were monitored weekly to check the efficacy of different drug treatments and potential side effects. Mice were killed at the end of treatment, and the tumors were isolated for image capture and weighting. The formula for calculating tumor volume is V=ab2 / 2 (cm 3 ). Tumor images at the end of treatment (7A, circles represent disappeared tumors, and error symbols represent prostate cancer tumors in dead mice). Growth curves of tumors treated with different drugs ( Figure 7 B). Tumor weight at treatment endpoint ( Figure 7 C). Body weight of mice at the end of treatment ( Figure 7 D). HE staining of mouse liver histology with different drug assistants, 200mm( Figure 7 E-7F). Immunohistochemical staining of AR and Ki67 in tumors of mice treated with different drugs ( Figure 7 G).

[0104] The results showed that PET-DCL-DTX and DTX treatment significantly limited the growth of CWR22Rv1 tumors compared to PET-DCL-SH. Notably, 1 mortality (false symbol) and 1 tumor-free mouse (circular symbol) were observed during PET-DCL-DTX treatment. In contrast, DTX treatment resulted in 3 deaths (false symbols), suggesting that DTX is more toxic than PET-DCL-DTX. Mice treated with PET-DCL-DTX showed improved health compared to mice treated with DTX. To further confirm the health benefits of PET-DCL-DTX treatment, liver damage was measured by histological and molecular analysis. ALT and AST, two key biomarkers of damaged liver, were significantly increased in the DTX-treated group compared to the PET-DCL-DTX-treated group ( Figure 7 E), Histological analysis of mouse livers also showed that there was less immune cell infiltration in the livers of PET-DCL-DTX mice compared with the DTX-treated control group (red arrows) ( Figure 7 F). Taken together, all these data indicate that PET-DCL-DTX can significantly inhibit the growth of PSMAPCa tumors in vivo but with reduced side effects.

[0105] The above data also suggest that PET-DCL-DTX inhibits the growth of PCa cells, at least in part, by downregulating AR signaling and cell proliferation. To confirm these findings in vivo, the expression levels of AR and proliferation marker Ki67 in xenograft mouse models were detected by IHC staining. As shown in the figure. Compared with PET-DCL-SH, PET-DCL-DTX and DTX significantly reduced the IHC scores of AR and Ki67, which is highly consistent with our in vitro data. Taken together, these findings enhance the anticancer activity of PET-DCL-DTX against PMSA-positive PCa in vivo, which is largely due to its DTX loading capacity and functional DCL group.

[0106] As can be seen from the above embodiments, the present invention provides a PSMA-targeted polyester-docetaxel nano-conjugate drug and its preparation method and application. The present invention is prepared by combining water-soluble polyester with anti-tumor drug (DTX) and prostate-specific membrane antigen (PSMA) ligand (DCL). The prepared polyester-docetaxel nano-conjugate is used to treat PSMA-positive prostate cancer. In the preparation process of polyester, short-chain polyethylene glycol directly participates in polymerization as a monomer, which not only overcomes the problem of poor water solubility of polyester, but also does not affect the drug loading capacity. Benefiting from the ideal hydrophilic and hydrophobic balance, PET-DCL-DTX self-assembles into a nearly spherical aqueous solution of about 57 nanometers, and the external hydrophilic PEG and DCL disguised by hydrophobic DTX promote the target delivery of a large amount of DTX tumors and reduce the invasion of normal tissues. DCL enables the nano-conjugate to have a specific binding ability to PSMA-positive prostate tumor cells, thereby showing the selectivity of tumor subtypes. After entering the tumor cell, the reduction-sensitive cleavage of the disulfide bond causes DTX to detach from the polymer and revive. In addition, compared with DTX, the toxic side effects of the nano-conjugate are lower.

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a PSMA-targeted polyester-docetaxel nanoconjugate, characterized in that: The steps include: (1) mixing bis-mercapto polyethylene glycol, diacrylate monomer, n-hexylamine and tetrahydrofuran, and reacting for 4 to 6 hours to obtain a polyester containing trityl mercapto groups; (2) mixing a polyester containing trityl mercapto groups, trifluoroacetic acid, triethylsilane and dichloromethane, reacting at room temperature for 5 to 7 hours, removing the trityl group, and obtaining a polyester containing mercapto groups; (3) mixing the thiol-containing polyester, maleimide-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid and N,N-dimethylformamide, and reacting for 5 to 7 hours to obtain a polyester containing a PSMA targeting group and a thiol group; (4) A polyester containing a PSMA targeting group and a thiol group, pyridyldisulfide-modified docetaxel, and N,N-dimethylformamide are mixed, reacted for 11 to 13 hours, dissolved in deionized water, and dialyzed to obtain a polyester-docetaxel nanoconjugate.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the bis-mercapto polyethylene glycol to the diacrylate monomer is 580-620:155-160; The mass ratio of the bis-mercapto polyethylene glycol to n-hexylamine is 580-620:5.8-6.2; The mass volume ratio of the mixture of bis-mercapto polyethylene glycol and tetrahydrofuran is 580-620 mg: 1.5-2.5 mL; The reaction temperature in step (1) is 23-27°C.

3. The preparation method according to claim 2, characterized in that: The mass volume ratio of the trityl mercapto-containing polyester and dichloromethane is 520-540 mg: 37-39 mL; The mass volume ratio of the trityl mercapto-containing polyester and trifluoroacetic acid is 520-540 mg: 1.5-2.5 mL; The mass volume ratio of the trityl mercapto-containing polyester and triethylsilane is 520-540 mg: 1.1-1.3 mL.

4. The preparation method according to claim 3, characterized in that: The mass volume ratio of the mercapto-containing polyester and N,N-dimethylformamide is 280-320 mg: 2.5-3.5 mL; The mass ratio of the thiol-containing polyester to maleimide-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid (Mal-DCL) is 280-320:3; The reaction temperature in step (3) is 23-27°C.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The preparation method of the maleimide-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)pentanedioic acid is as follows: Mix di-tert-butyl (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid ester, 3-maleimidopropionic acid hydroxysuccinimide ester and N,N-diisopropylethylamine with dichloromethane, and react for 4.5 to 5.5 hours to obtain an intermediate product; mix the intermediate product, trifluoroacetic acid and dichloromethane, and react for 0.8 to 1.2 hours to obtain maleimide-modified (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid; The mass ratio of the (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid di-tert-butyl ester and 3-maleimidopropionic acid hydroxysuccinimide ester is 300:195-200; The mass ratio of the mixture of (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid di-tert-butyl ester and N,N-diisopropylethylamine is 300:320-322; The mass volume ratio of the mixture of (S)-2-(3-((S)-amino-1-tert-butoxy-1-oxohexane-2-yl)ureido)glutaric acid di-tert-butyl ester and dichloromethane is 300 mg:3-5 mL; The mass volume ratio of the intermediate product and trifluoroacetic acid is 200-220 mg: 3-5 mL; The mass volume ratio of the intermediate product and dichloromethane is 200-220 mg:2 mL.

6. The preparation method according to claim 5, characterized in that: The mass volume ratio of the polyester containing PSMA targeting groups and thiol groups and N,N-dimethylformamide is 90-110 mg: 1.5-2.5 mL; The mass ratio of the polyester containing PSMA targeting group and thiol group to the docetaxel modified with pyridyldisulfide group is 90-110:38-42; The reaction temperature in step (4) is 23-27°C.

7. The preparation method according to claim 6, characterized in that: The preparation method of the pyridine disulfide-modified docetaxel is: Docetaxel, 3-(2-pyridyldithio) propionic acid, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and dichloromethane are mixed and reacted for 7.8 to 8.2 hours to obtain pyridyldithio modified docetaxel; The mass ratio of docetaxel and 3-(2-pyridyldithio)propionic acid is 1100:250-350; The mass ratio of docetaxel and N,N'-dicyclohexylcarbodiimide is 1100:550-600; The mass ratio of docetaxel and 4-dimethylaminopyridine is 1100:150-190; The mass volume ratio of the mixture of docetaxel and dichloromethane is 1100 mg:18-22 mL.

8. The polyester-docetaxel nanoconjugate prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The molecular weight of the PSMA-targeted polyester-docetaxel nanoconjugate is 22.52 kDa; the drug loading is ≥6.99%.

9. Use of the polyester-docetaxel nanoconjugate according to claim 8 in the preparation of a drug targeting prostate-specific membrane antigen-positive prostate cancer.

10. Use of the polyester-docetaxel nanoconjugate prepared by the preparation method according to any one of claims 1 to 7 in the preparation of a drug targeting prostate-specific membrane antigen-positive prostate cancer.

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