Preparation method and application of targeting micromolecule (ACUPA) coupled nano drug delivery system (AMC) specifically binding to prostate specific membrane antigen (PSMA)

Abstract

The invention discloses a preparation method and application of a targeting small molecule (ACUPA) coupled nano drug delivery system (AMC) specifically combined with prostate specific membrane antigen (PSMA), and belongs to the technical field of biological medicine. The targeting micromolecule (ACUPA) coupled nano drug-loaded delivery system (AMC) is a targeting micromolecule coupled nano drug-loaded polymer micelle which can be specifically combined with prostate specific membrane antigen (PSMA). The micelle is formed by self-assembly through a film hydration method. The targeting micromolecule (ACUPA) specifically bound with the prostate specific membrane antigen (PSMA) is coupled with the polymer micelle through a chemical bond. The targeting micromolecule (ACUPA) can be specifically combined with prostate cancer cells expressing PSMA, a nano drug-loaded preparation is delivered to the prostate cancer cells, the active targeting property of combination with a specific antigen, the endothelial action of the antigen cells and the high-permeability long-retention effect of the nano preparation are fully exerted, multiple effects are synergistic, and the drug-loaded nano drug-loaded preparation is prepared. The toxicity of the medicine on prostate cancer cells is remarkably increased, and then the targeted therapy of prostate cancer is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of a targeted small molecule (ACUPA) conjugated nano-drug delivery system (AMC) that specifically binds to prostate specific membrane antigen (PSMA). Background Art

[0002] Prostatic carcinoma (PCa) is the most common malignant tumor in the male reproductive system. In recent years, the main clinical treatment methods for advanced PCa include radiotherapy, chemotherapy, endocrine therapy, etc. However, these treatment methods have problems such as poor drug compliance, poor tissue selectivity, large toxic reactions, and drug resistance. With the development of targeted drug delivery systems in recent years, targeted drug delivery systems have gradually become a research hotspot in the field of biotech drugs and are also considered the most promising research direction for cancer treatment.

[0003] The present invention is a targeted small molecule conjugated nano-drug delivery system developed for the treatment of prostate cancer. By conjugating a targeted small molecule, ACUPA (targeting prostate specific membrane antigen PSMA), on the surface of the nano-drug delivery system, a nano-drug delivery preparation that actively targets prostate cancer cells is created.

[0004] Prostate specific membrane antigen (PSMA) is an antigen specifically expressed on the surface of prostatic epithelial cells. Its expression is not affected by hormone secretion. Especially in advanced prostate cancer and metastatic prostate cancer, the elevation of PSMA is particularly obvious, which makes it an important target molecule for the diagnosis and treatment of prostate cancer. As a target molecule for therapeutic drugs, the most important feature of PSMA is that it can quickly enter cells through clathrin-coated pits through endocytosis, and then quickly return to the cell surface for the next cycle of endocytosis, which indicates that PSMA is a very ideal drug-directed target.

[0005] The ACUPA small molecule can specifically bind to prostate cancer cells expressing PSMA, deliver the nano-drug preparation to prostate cancer cells, and fully exert the active targeting of binding to specific antigens, the endocytosis of antigens by cells, and the high permeability and long retention effect of the nano-preparation. The multiple effects work together synergistically to significantly increase the toxicity of the drug to prostate cancer cells, thereby enhancing the targeted treatment of prostate cancer.

[0006] With the development of nanotechnology, targeted modification of nano-drug carriers to fully utilize the dual advantages of targeting and the nano-drugs themselves has become an important development direction in drug formulation research and development. Nano-drug carriers are drug delivery systems with sizes in the nanometer range, which can improve the bioavailability of drugs. For example, they can control the drug release process in the body, facilitate crossing biological barriers, have a longer half-life, and different biodistribution characteristics. At the same time, nano-carriers can accumulate in tumor tissues through the enhanced permeability and retention effect and the endocytosis of tumor cells, exerting a better anti-tumor effect while reducing the toxicity to normal cells.

[0007] The targeted small molecule (ACUPA) conjugated nano-drug delivery system can improve the accuracy of targeting tumor cells and the drug loading capacity, thereby enhancing the efficacy of tumor treatment, which is an innovation in drug delivery systems and disease treatment. However, up to now, no targeted small molecule (ACUPA) conjugated nano-drug delivery preparation has been marketed for the treatment of prostate cancer. Summary of the Invention

[0008] The object of the present invention is to provide a preparation method and application of a targeted small molecule (ACUPA) conjugated nano-drug delivery system that specifically binds to prostate-specific membrane antigen (PSMA). The targeted small molecule (ACUPA) conjugated nano-drug delivery system that specifically binds to prostate-specific membrane antigen (PSMA) can be used for the preparation and development of anti-tumor drugs, which is of great significance for the clinical treatment of tumors.

[0009] To achieve the above object, in the first aspect of the present invention, a targeted small molecule (ACUPA) conjugated nano-drug delivery system is provided. The targeted small molecule (ACUPA) conjugated nano-drug delivery system is assembled from poly(ethylene glycol)-poly(lactide) copolymer, a targeted small molecule that specifically binds to prostate-specific membrane antigen (PSMA), and a tumor treatment toxic drug; wherein: There are three types of poly(ethylene glycol)-poly(lactide) copolymers. One is formed by the poly(ethylene glycol) as the hydrophilic block binding to lactide as the hydrophobic block through a polycondensation reaction; one is formed by the poly(ethylene glycol) carrying the targeted small molecule (ACUPA) as the hydrophilic block binding to lactide as the hydrophobic block through a polycondensation reaction; and one is formed by the copolymer formed by the poly(ethylene glycol) as the hydrophilic block binding to lactide as the hydrophobic block through a polycondensation reaction and then connecting Fmoc-lysine through a reaction; and The targeted small molecule (ACUPA) can specifically bind to prostate-specific membrane antigen (PSMA) and target prostate tumor cells; Preferably, the targeted small molecule (ACUPA) conjugated nano-drug delivery system is in the form of a solution or a freeze-dried powder.

[0010] The targeted small molecule (ACUPA) conjugated nano-drug delivery system according to the first aspect of the present invention, wherein the anti-prostate cancer tumor toxic drugs are selected from one or more of the following: docetaxel, cabazitaxel, paclitaxel and other drugs.

[0011] Preferably, the anti-tumor toxic drugs are cabazitaxel and paclitaxel.

[0012] The targeted small molecule (ACUPA) conjugated nano-drug delivery system according to the first aspect of the present invention, wherein the nanoparticles are selected from one or two of the following: liposomes, polymeric micelles.

[0013] The targeted small molecule (ACUPA) conjugated nano-drug delivery system according to the first aspect of the present invention, wherein the targeted small molecule is: ACUPA modified with mercaptopropyl or amino group, and the chemical structural formula and molecular weight are as follows: Mercaptopropyl-modified ACUPA:

[0014] Molecular Weight 407.44 Amino-modified ACUPA:

[0015] Molecular Weight 319.31 Preferably, the targeted small molecule (ACUPA) conjugated nano-drug delivery system is a targeted small molecule (ACUPA) conjugated nano-drug delivery system self-assembled by poly(ethylene glycol)-poly(lactide) copolymer or poly(ethylene glycol)-poly(lactide) Fmoc-lysine copolymer, targeted small molecule (ACUPA) poly(ethylene glycol)-poly(lactide) copolymer and anti-tumor toxic drug by the thin film hydration method.

[0016] The targeted small molecule (ACUPA) conjugated nano-drug delivery system according to the first aspect of the present invention, wherein the molecular weight of the poly(ethylene glycol)-poly(lactide) copolymer is 1500 - 18000, preferably 1800 - 15000, more preferably 1800 - 13000; The particle size of the targeted small molecule (ACUPA) conjugated nano-drug delivery system is 10 - 100 nm, preferably 15 - 60 nm, more preferably 15 - 40 nm.

[0017] The molar ratio of the targeted small molecule (ACUPA), copolymer and anti-tumor toxic drug is 1 - 5:1 - 6:13 - 7:8 - 15.

[0018] The second aspect of the present invention provides a method for preparing the targeted small molecule (ACUPA)-conjugated nano-drug delivery system described in the first aspect, and the method includes the following steps: (1) Prepare poly(ethylene glycol)-poly(lactide) copolymer or poly(ethylene glycol)-poly(lactide)-Fmoc-lysine copolymer and targeted small molecule (ACUPA)-poly(ethylene glycol)-poly(lactide) copolymer respectively; (2) Self-assemble the above copolymers and tumor therapeutic toxic drugs into nano-polymer micelles by the thin film hydration method; (3) Preferably, dialyze and filter the targeted small molecule (ACUPA)-conjugated nano-drug-loaded polymer micelle solution obtained in step (2).

[0019] According to the preparation method of the second aspect of the present invention, wherein, in step (1): The catalyst used for preparing the copolymer is stannous octoate, and the catalyst ratio is 0.3‰ - 8‰ of the feeding amount. The solvents used are ethanol, ether, dichloromethane (ultra-dry), tetrahydrofuran, triethylamine, pivaloyl chloride, etc.; The molar ratio of polyethylene glycol to lactide used for preparing the copolymer is 1:8 - 30, preferably 1:8 - 25, and further preferably 1:10 - 20; The polymerization reaction temperature is 110 - 150 °C, preferably 110 - 140 °C, and further preferably 120 - 140 °C; The polymerization reaction time is 6 - 30 h, preferably 6 - 25 h, and further preferably 6 - 20 h; The refining temperature of the copolymer is -7 to -30 °C, preferably -7 to -25 °C, and further preferably -10 to -22 °C; The drying time of the copolymer is 15 - 70 h, preferably 20 - 70 h, and further preferably 35 - 70 h; The drying temperature of the copolymer is 20 - 50 °C, preferably 25 - 48 °C, and further preferably 25 - 45 °C; The molecular weight of the copolymer is 1500 - 18000, preferably 1800 - 15000, and further preferably 1800 - 13000.

[0020] According to the preparation method of the second aspect of the present invention, wherein, in step (2): The drug dissolution solvent is acetonitrile or ethanol, preferably acetonitrile; The hydration solvent is one or more of the following: ultrapure water, phosphate buffer solution, physiological saline, preferably phosphate buffer solution and ultrapure water, and most preferably phosphate buffer solution; The concentration of the tumor therapeutic toxic drug in the micelle solution is 1 - 25 mg / mL, preferably 1 - 20 mg / mL, and further preferably 3 - 15 mg / mL; The concentration of poly (ethylene glycol)-poly (lactide) copolymer or poly (ethylene glycol)-poly (lactide) Fmoc-lysine copolymer is 8 - 55 mg / mL, preferably 10 - 50 mg / mL, and more preferably 15 - 45 mg / mL; The concentration of the targeted small molecule (ACUPA)-poly (ethylene glycol)-poly (lactide) copolymer is 0.3 - 30 mg / mL, preferably 0.5 - 25 mg / mL, and more preferably 0.8 - 18 mg / mL; The temperature of the hydration water bath is 30 - 65 °C, preferably 35 - 60 °C, and more preferably 40 - 50 °C; The hydration time is 5 - 55 min, preferably 8 - 50 min, and more preferably 8 - 45 min.

[0021] According to the preparation method of the second aspect of the present invention, in step (3): the pore size of the dialysis membrane is 1000 - 12000 molecular weight, preferably 1000 - 8000 molecular weight, and more preferably 1500 - 6000 molecular weight; the pore size of the filtration membrane is 0.2 - 1.0 μm, preferably 0.2 - 0.5 μm, and more preferably 0.2 - 0.45 μm, and most preferably 0.22 μm.

[0022] The third aspect of the present invention provides the use of the targeted small molecule (ACUPA)-conjugated nano-drug delivery system described in the first aspect or the targeted small molecule (ACUPA)-conjugated nano-drug delivery system prepared according to the method described in the second aspect in the treatment of cancer drugs: Preferably, the cancer is prostate cancer; More preferably, the prostate cancer is characterized by prostate cancer cells or prostate cancer tissues with the expression or overexpression of prostate-specific membrane antigen (PSMA).

[0023] According to the application of the third aspect of the present invention, the types of the prostate cancer cells are selected from one or more of the following: DU145, LNCap, PC-3, 22Rv1, VCap, preferably LNCap or PC-3.

[0024] According to a particularly preferred embodiment of the present invention, a targeted small molecule (ACUPA)-conjugated nano-drug delivery system capable of targeting the PSMA target is provided. The targeted small molecule (ACUPA) brings the anti-tumor cytotoxic drug to the prostate cancer tissue by binding to PSMA, giving full play to the active targeting of binding to the specific antigen, the antigen intracellular uptake effect, and the high permeability and long retention effect of the nano-formulation. The multiple effects cooperate synergistically, significantly increasing the toxicity of the drug to the prostate cancer cells, thereby enhancing the targeted treatment of prostate cancer.

[0025] The targeted small molecule (ACUPA) conjugated nano-drug delivery system of the present invention may have, but is not limited to, the following beneficial effects: 1. The targeted small molecule (ACUPA) conjugated nano-drug delivery system improves the solubility and biological stability of anti-tumor toxic drugs, enhances the targeting and binding ability to prostate tumor tissues, thereby increasing the concentration of anti-tumor toxic drugs in tumor tissues and enhancing the inhibitory ability against the growth of prostate cancer cells.

[0026] 2. As shown by transmission electron microscopy, the targeted small molecule (ACUPA) conjugated nano-drug delivery system forms spherical polymer micelles with uniform particle sizes. The prepared micelles have a particle size distribution in the range of 10 - 35 nm, and an average particle size in the range of 15 - 30 nm.

[0027] 3. The targeted small molecule (ACUPA) conjugated nano-drug delivery system can inhibit the proliferation of prostate cancer cells. When the anti-tumor toxic drug is at a dose of 40 mg / kg, it can significantly inhibit the proliferation of prostate cancer cells (Figure - in vivo pharmacodynamic experiment). Description of the Drawings

[0028] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the following embodiments or the description of the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 : NMR spectrum of the targeted small molecule (ACUPA) poly(ethylene glycol)-poly(lactide) copolymer in Example 1; Figure 2 : NMR spectrum of the targeted small molecule (ACUPA) conjugated nano-drug polymer micelles in Example 1; Figure 3 : Electron micrograph of the targeted small molecule (ACUPA) conjugated nano-paclitaxel polymer micelles in Example 2; Figure 4 : Particle size spectrum of the targeted small molecule (ACUPA) conjugated nano-paclitaxel polymer micelles in Example 3; Figure 5 : HPLC spectrum for detecting the paclitaxel content of the targeted small molecule (ACUPA) conjugated nano-paclitaxel polymer micelles in Example 4; Figure 6 : Result graph of detecting the effect of the targeted small molecule (ACUPA) conjugated nano-paclitaxel polymer micelles on the proliferation of prostate cancer cells using the CCK8 cell proliferation assay provided in Example 5; Figure 7 : Results graph showing the effect of targeting small molecule (ACUPA)-conjugated nanopaclitaxel polymeric micelles on apoptosis of prostate cancer cells detected by AnnexinV staining and flow cytometry provided in Example 6; Figure 8 : Columnar schematic diagram of the results showing the effect of targeting small molecule (ACUPA)-conjugated nanopaclitaxel polymeric micelles on apoptosis of prostate cancer cells detected by AnnexinV staining and flow cytometry provided in Example 6; Figure 9 : Results graph showing the inhibitory effect of targeting small molecule (ACUPA)-conjugated nanopaclitaxel polymeric micelles on migration of prostate cancer cells detected by cell scratch assay provided in Example 7; Figure 10 : Columnar schematic diagram of the results showing the inhibitory effect of targeting small molecule (ACUPA)-conjugated nanopaclitaxel polymeric micelles on migration of prostate cancer cells detected by cell scratch assay provided in Example 7; Figure 11 : Results graph showing the inhibitory effect of targeting small molecule (ACUPA)-conjugated nanopaclitaxel polymeric micelles on invasion of prostate cancer cells detected by transwell invasion assay provided in Example 8; Figure 12 : Columnar schematic diagram of the results showing the inhibitory effect of targeting small molecule (ACUPA)-conjugated nanopaclitaxel polymeric micelles on invasion of prostate cancer cells detected by transwell invasion assay provided in Example 8; Figure 13 : Results graph showing the inhibitory effect of targeting small molecule (ACUPA)-conjugated nanopaclitaxel polymeric micelles on growth of prostate cancer cells detected by colony formation assay provided in Example 9; Figure 14 : Columnar schematic diagram of the results showing the inhibitory effect of targeting small molecule (ACUPA)-conjugated nanopaclitaxel polymeric micelles on growth of prostate cancer cells detected by colony formation assay provided in Example 9; Figure 15 : Results graph of the tumor volume curve of nude mice provided in Example 10; Figure 16 : Columnar schematic diagram of the tumor weight of nude mice provided in Example 10. Embodiment

[0030] The technical solution of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0031] This section gives a general description of the materials and test methods used in the experiments of the present invention. Although many materials and operation methods used to achieve the purpose of the present invention are well known in the art, the present invention still describes them in as much detail as possible herein. Those skilled in the art are aware that, in the context, unless otherwise specified, the materials and operation methods used in the present invention are well known in the art.

[0032] The reagents and instruments used in the following examples are as follows: Reagents: Mercaptopropyl- or amino-modified ACUPA was purchased from Xi'an Kaixin Biotech Co., Ltd., with a purity of over 95%. Polyethylene glycol was purchased from Beijing KeyGen Biotech Co., Ltd., with a purity of over 95%. Poly(lactic acid) was purchased from Sigma-Aldrich, with a purity of over 98%. Maleimide- or carboxyl-terminated polyethylene glycol was purchased from Beijing KeyGen Biotech Co., Ltd., with a purity of over 95%. Fmoc-Lysine was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with a purity of over 95%. Paclitaxel was purchased from Hainan Taxus Pharmaceutical Co., Ltd., with a purity of over 95%. The quality parameters of ultrapure water were resistivity 18.2 MΩ.cm @ 25°C and pH value 6.8 - 7.2. The pH value of PBS buffer was 7.0 - 7.5. Instruments: The reaction bath was purchased from Zhengzhou Great Wall Scientific Industry and Trade Co., Ltd., model HWCL-5. The refrigerator was purchased from Zhengzhou Great Wall Scientific Industry and Trade Co., Ltd., model DHJF-2005. The rotary evaporator was purchased from IKA GmbH, Germany, model RV10. The high performance liquid detector was purchased from Agilent Technologies, model Agilent Technologies 1260Ⅱ. The particle size detector was purchased from Hangzhou New Blue Technology Co., Ltd., model Zetasizer Nano ZS. Example 1

[0033] This example is used to illustrate the preparation method of the targeted small molecule (ACUPA) conjugated nano-drug-loaded polymer micelle preparation of the present invention.

[0034] (1) Preparation method of polyethylene glycol-poly(lactic acid) copolymer, maleimide- or carboxyl-terminated polyethylene glycol-poly(lactic acid) copolymer: A certain amount of polyethylene glycol or maleimide polyethylene glycol or carboxyl-terminated polyethylene glycol and a certain amount of lactide were polymerized under the action of the catalyst stannous octoate, and the obtained melt was refined and dried in ice ether or ethanol to obtain the product.

[0035] (2) Preparation method of targeted small molecule (ACUPA) polyethylene glycol-polylactide copolymer: A certain amount of maleimide or carboxyl polyethylene glycol-polylactide copolymer is dissolved in purified water solvent and reacted under the action of EDC.HCl and NHS. The obtained product is passed through a dialysis bag and freeze-dried to obtain. The nuclear magnetic detection results of the copolymer are as Figure 1 shown.

[0036] (3) Preparation method of polyethylene glycol-polylactide Fmoc-lysine copolymer: Fmoc-lysine is dissolved in tetrahydrofuran and reacted with a certain amount of triethylamine and pivaloyl chloride for 2-4 hours. After the solution is rotary evaporated, it is dissolved in dichloromethane solution; at the same time, a certain amount of polyethylene glycol-polylactide is dissolved in dichloromethane solution, then triethylamine and 4-pyrrolidinopyridine are added, and then the above-mentioned dichloromethane solution of Fmoc-lysine is added and reacted at room temperature for 15-36 hours. After the solution is rotary evaporated, it is refined 3 times with ice ethanol and dried to obtain.

[0037] (4) Preparation method of tumor toxic drug nanomicelles: A certain proportion of tumor toxic drug paclitaxel, polyethylene glycol-polylactide copolymer or polyethylene glycol-polylactide Fmoc-lysine copolymer, and targeted small molecule (ACUPA) polyethylene glycol-polylactide copolymer are dissolved in ethanol or acetonitrile. After complete dissolution, the solvent is removed by rotary evaporation in a rotary evaporator, and then an appropriate amount of PBS buffer is added and hydrated in a water bath at 42-55 °C for 10-40 min to obtain.

[0038] (5) The targeted small molecule-coupled drug-loaded nano-polymer micelle solution obtained in step (4) is filtered through a 0.22 μm filter membrane, and the filtrate is dispensed into 20 mL vials at 5 mL per bottle and freeze-dried. The nuclear magnetic spectrum of the copolymer is as Figure 2 shown. Example 2

[0039] Morphology analysis of targeted small molecule (ACUPA)-coupled nano-paclitaxel polymer micelles The morphology of the targeted small molecule (ACUPA)-coupled nano-paclitaxel polymer micelles was characterized by transmission electron microscopy. The micelle solution was diluted to 20 μM, 10 μL of the liquid was dropped on a copper grid, left standing overnight, 2 wt% uranyl acetate solution was dropped, after staining for 1 min, the liquid was sucked with filter paper, and after natural drying, it was observed and photographed under a transmission electron microscope. It can be seen from Figure 3 that the size of the targeted small molecule (ACUPA)-coupled nano-paclitaxel polymer micelles is about 20 nm. Example 3

[0040] Particle size analysis of targeted small molecule (ACUPA)-coupled nano-paclitaxel polymer micelles The particle size of the targeted small molecule (ACUPA)-conjugated paclitaxel polymer micelles was characterized using a particle size analyzer. Take 1 vial of the targeted small molecule (ACUPA)-conjugated paclitaxel polymer micelles, open the rubber stopper, and use a 10 mL pipette to transfer 5 mL of 0.9% sodium chloride injection along the bottle wall and add it to the container all at once to dissolve it, serving as the test sample. Use a 1 mL pipette to transfer 0.5 mL of the test sample into an EP tube, add 2 mL of 0.9% sodium chloride injection, invert and shake well 10 times, take 1 mL and place it in a clean and scratch-free particle size colorimetric cup, tighten the cup lid, serve as the test sample solution, and place it in the sample cell for measurement. Take another 1 vial and operate in the same way. Measure each vial three times, repeat twice, and take the average value. The measured particle size of the nano micelles was 18 - 22 nm, and PDI < 0.1. The particle size detection spectrum is as Figure 4 shown.

[0041] Table 1 Example 4

[0042] Content analysis of the targeted small molecule (ACUPA)-conjugated paclitaxel polymer micelles The content of the targeted small molecule (ACUPA)-conjugated paclitaxel polymer micelles was characterized using a high performance liquid detector. Weigh accurately about 0.1 g of the freeze-dried sample into a 25 mL volumetric flask, dissolve it with an appropriate amount of 0.9% sodium chloride solution, then make up the volume to the mark with acetonitrile and shake well. Centrifuge for 10 min (10000 rpm), take 1 mL of the supernatant into a 10 mL volumetric flask (prepare 2 portions), dilute to the mark with acetonitrile, shake well and filter to obtain the test sample solution. Take acetonitrile, paclitaxel system suitability solution, paclitaxel reference substance solution (200.4982 μg / mL), and the test sample solution for detection in turn, and record the spectrum data. It can be known that the content of paclitaxel in the targeted small molecule (ACUPA)-conjugated paclitaxel polymer micelles is about 27 mg / branch, which is 90% of the labeled amount. The high performance liquid spectrum of the content detection is shown in Figure 5. Example 5

[0043] Cell proliferation experiment The operation of the cell proliferation experiment in this example is as follows: Take PC-3 cells in the logarithmic growth phase, add serum-free medium to make a cell suspension (5000 / 100 μL), inoculate it on a 96-well plate, 5×10 3Tumor cells. The cells were treated in groups for 48 h (the drug solution was prepared with normal saline), and 180 μL of targeted small molecule (ACUPA)-coupled nanoscale paclitaxel polymer micelles (ACUPA-PM / PTX), paclitaxel polymer micelles, and docetaxel were added to the low-dose group (5 nM) and high-dose group (10 nM) respectively. After the drug action ended, CCK8 (prepared with fresh serum-free medium) was added; after incubation at 37 °C for 2 h, the OD value was measured at a wavelength of 490 nm, and the cell inhibition rate was calculated.

[0044] The results are as Figure 6 shown. As Figure 6 can be seen, compared with the solvent control group, after 48 h of drug treatment, the proliferation inhibition rates of PC-3 cells in the low-dose PTX micelle group, high-dose PTX micelle group, low-dose ACUPA-PM / PTX group, high-dose ACUPA-PM / PTX group, and positive drug (docetaxel) group were extremely significantly increased ( P <0.01). It can be seen from the figure that the cell proliferation inhibition rate of the ACUPA-PM / PTX group was significantly higher than that of other groups. The results indicate that the targeted small molecule (ACUPA)-coupled nanoscale paclitaxel polymer micelles can effectively inhibit the growth of prostate tumors. Example 6

[0045] Cell apoptosis experiment In this example, the cell apoptosis experiment was operated as follows: Cells in the logarithmic growth phase were seeded in a 6-well plate, and the cell seeding density was 1.2×10 6 cells per well. After culturing in the cell culture incubator for 24 h according to the grouping treatment in the above proliferation experiment, the cells were collected, washed twice with pre-cooled PBS, and the cell concentration was adjusted to 1×10 6 cells / mL. 500 μL of binding buffer was added, the supernatant was discarded by centrifugation, and then 100 μL of binding buffer was added and mixed evenly. Then, 5 μL of Annexin V-FITC and 10 μL of PI were added respectively and mixed well; the reaction was carried out at room temperature in the dark for 15 min. Finally, 400 μL of binding buffer was added, and the cell apoptosis rate was detected by flow cytometry within 1 h. The experiment was repeated 3 times.

[0046] The results are as Figure 7 and Figure 8 shown. As Figure 7 and Figure 8 can be seen, compared with the solvent control group, the low-dose PTX micelle group, high-dose PTX micelle group, low-dose ACUPA-PM / PTX group, and high-dose ACUPA-PM / PTX group were all extremely significantly increased ( P <0.01), and the apoptosis rate of PC-3 cells in the positive drug group was significantly increased ( P(<0.05). The apoptosis rate of the ACUPA-PM / PTX group in the figure was significantly higher than that of the control group and higher than that of the PTX micelle group, indicating that the targeted small molecule (ACUPA)-coupled nano-paclitaxel polymer micelle can effectively promote the apoptosis of prostate tumor cells. Example 7

[0047] Cell scratch assay In this example, the cell scratch healing experiment was operated as follows: Take a 6-well cell culture plate and evenly draw 5 horizontal lines on the back of the plate with a marker pen, with an interval of 0.5 cm. Inoculate each group of cells at a density of 5×10 5 cells per well. After culturing the cells until they covered the well holes, use a pipette tip to draw a straight line perpendicular to the bottom of the well hole along a ruler. Wash the cells 3 times with PBS buffer to wash away the cells under the scratch, add serum-free medium, continue to culture, observe the scratch healing situation and take pictures at 48 h, and use Image J software to analyze and calculate the scratch healing rate of each group of cells.

[0048] The results are as Figure 9 and Figure 10 shown. It can be seen from Figure 9 and Figure 10 that compared with the solvent control group, after 48 h of drug treatment, the cell migration rates of PC-3 in the low-dose PTX micelle group, high-dose PTX micelle group, low-dose ACUPA-PM / PTX group, high-dose ACUPA-PM / PTX group and positive drug group were all extremely significantly reduced ( P (<0.01), that is, the ability of cells to migrate horizontally was weakened. The cell migration rate of the ACUPA-PM / PTX group in the figure was lower than that of other groups, indicating that the targeted small molecule (ACUPA)-coupled nano-paclitaxel polymer micelle has an inhibitory effect on the migration of prostate tumor cells. Example 8

[0049] Cell invasion assay In this example, the cell invasion experiment was operated as follows: Dilute Matrigel with serum-free culture medium at a ratio of 3:1. Take 30 μl of the Matrigel dilution and evenly coat the Transwell chamber, and incubate overnight at 4 °C; place the Transwell chamber into a 24-well culture plate, add cells to the upper chamber, and culture in an incubator at 37 °C and 5% CO2 for 48 h. Wipe off the matrix gel and the cells on the bottom layer of the upper chamber with a cotton swab, fix with 4% paraformaldehyde for 10 min, wash 3 times with PBS, stain with 0.1% crystal violet staining solution for 30 min, and then take pictures and count the number of cells invading into the lower chamber under an inverted microscope; repeat the experiment 3 times.

[0050] The results are as Figure 11 and Figure 12 shown. It can be seen from Figure 11 and Figure 12It can be seen that, compared with the solvent control group, after 48 h of drug addition treatment, the number of invading cells of PC-3 in the low-dose PTX micelle group, high-dose PTX micelle group, low-dose ACUPA-PM / PTX group, high-dose ACUPA-PM / PTX group and positive drug group was extremely significantly reduced ( P <0.01), that is, the invasion ability was extremely significantly reduced. In the figure, the number of invading cells in the ACUPA-PM / PTX group was significantly lower than that in the control group and also lower than that in the PTX micelle group. It can be seen that the targeted small molecule (ACUPA) conjugated with nanoscale paclitaxel polymer micelles can effectively inhibit the invasion of tumor cells. Example 9

[0051] Cell colony formation assay The specific operation is as follows: (a) Trypsinize the cells of each experimental group in the logarithmic growth phase, resuspend them with complete medium to prepare a cell suspension, and count. (b) Inoculate the cells in a 24-well culture plate with a medium containing 30% FBS; after shaking the inoculated cells evenly, gently place them in an incubator for continued culture, and observe the clone size under a microscope. (c) Continue the culture until the number of cells in most single clones in the wells is greater than 50. Discard the supernatant and wash the cells with PBS. Add 1 mL of 4% paraformaldehyde to each well and fix the cells at 4°C for 60 min, then wash the cells with PBS. Add 1000 μL of clean and impurity-free crystal violet staining solution to each well and stain the cells for 2 min.

[0052] The results are shown in Figure 13 and Figure 14. It can be seen from Figure 11 and Figure 12 that, compared with the solvent control group, the cell colony formation ability of PC-3 in the low-dose PTX micelle group, high-dose PTX micelle group, low-dose ACUPA-PM / PTX group, high-dose ACUPA-PM / PTX group and positive drug group was extremely significantly reduced ( P <0.01). The inhibitory effect of the targeted small molecule (ACUPA) conjugated with nanoscale paclitaxel polymer micelles on the growth of tumor cells was detected by the colony formation assay. The results showed that the targeted small molecule (ACUPA) conjugated with nanoscale paclitaxel polymer micelles could significantly inhibit the ability of cancer cells to form colonies. Example 10

[0053] PC-3 prostate cancer nude mouse model The specific operations are as follows: Male Balb / C-nude mice were randomly divided into 6 groups under the armpits, including a solvent control group, a low-dose PXT micelle group (20 mg / kg / 3 days), a high-dose PXT micelle group (40 mg / kg / 3 days), a low-dose ACUPA-PM / PTX group (20 mg / kg / 3 days), a high-dose ACUPA-PM / PTX group (40 mg / kg / 3 days), and a positive control (docetaxel group), with 6 mice in each group. After adaptive feeding, PC-3 tumor tissues were taken, treated with PBS, and then inoculated subcutaneously into the armpits of mice with trocars. The volume of the tumor mass inoculated into each nude mouse was 10-15 mm 3 . The body weight and tumor data were measured every 3 days. After the tumor volume reached 100 mm³, the mice were given drugs via the tail vein in groups. After the drug administration was completed, the tumor weight was measured.

[0054] The results are as Figure 15 and Figure 16 shown. The results of the in-situ tumor volume measurement showed that the tumor volume of the mice in the ACUPA-PM / PTX group was significantly lower than that of the solvent control group and the positive control group, and was also lower than that of the PXT micelle group. The results of the tumor weight measurement showed that compared with the solvent control group and the positive control group, the tumor weight of the mice in each group was significantly reduced ( P <0.01). The decrease in tumor weight was more obvious in the test groups compared with the PXT micelle group. The experiment on the PC-3 prostate cancer nude mouse model showed that the targeted small molecule conjugate-loaded drug nanopolymer micelles also had good anti-tumor effects in animals.

[0055] Although the present invention has been described to a certain extent, obviously, various appropriate changes can be made without departing from the spirit and scope of the present invention. It can be understood that the present invention is not limited to the described embodiments, but belongs to the scope of the claims, which includes equivalent replacements of each factor described.

Claims

1. A targeted small molecule (ACUPA) conjugated nanomedicine delivery system (AMC) that specifically binds to prostate-specific membrane antigen (PSMA), characterized in that: The targeted small molecule (ACUPA)-conjugated nano-drug delivery system (AMC) is a nano-drug-loaded polymeric micelle conjugated with a targeted small molecule that can specifically bind to prostate-specific membrane antigen (PSMA). The micelle is self-assembled by the thin film hydration method; The targeted small molecule (ACUPA) that specifically binds to prostate-specific membrane antigen (PSMA) is conjugated to the polymeric micelle through a chemical bond. The targeted small molecule (ACUPA) can specifically bind to prostate cancer cells expressing PSMA, deliver the nano-drug preparation to prostate cancer cells, and fully exert the active targeting of binding to specific antigens, antigen endocytosis, and the enhanced permeability and retention effect of the nano-preparation. Multiple effects work together to significantly increase the toxicity of the drug to prostate cancer cells, thereby enhancing the targeted treatment of prostate cancer.

2. The nano-drug delivery system according to claim 1, characterized in that: The amphiphilic block copolymer contains the targeted small molecule (ACUPA), and the molecular weight of the polymer is 1800 - 13000.

3. The nano-drug delivery system according to claim 1, characterized in that: The amphiphilic block copolymer contains or does not contain Fmoc-lysine, and its molecular weight is 1000 - 9000.

4. The nano-drug delivery system according to any one of claims 1 to 3, characterized in that: The targeted small molecule (ACUPA) that can specifically bind to prostate-specific membrane antigen (PSMA) is ACUPA modified with mercaptopropyl or amino, and can react and link with maleimide or carboxyl poly(ethylene glycol)-poly(lactide) copolymer to modify the polymeric micelle.

5. The nano-drug delivery system according to claim 1, wherein: The nano-polymeric micelle encapsulates an anti-tumor drug.

6. The preparation method of the nano-drug delivery system according to claim 1, characterized in that: It includes the following steps: Step 1, prepare poly(ethylene glycol)-poly(lactide) copolymer or poly(ethylene glycol)-poly(lactide)-Fmoc-lysine copolymer and targeted small molecule (ACUPA)-poly(ethylene glycol)-poly(lactide) copolymer; Step 2, self-assemble the copolymer and the tumor treatment drug into a nano-drug-loaded polymeric micelle by the thin film hydration method.

7. Use of the nano-polymeric micelle prepared by the nano-drug delivery system described in claim 1 in the treatment of tumor diseases expressing prostate-specific membrane antigen (PSMA).

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