A docetaxel nano-prodrug targeting tumor cells and tumor stem cells and a preparation method and application thereof

Abstract

The application discloses a docetaxel nano prodrug targeting tumor cells and tumor stem cells and a preparation method and application thereof. A docetaxel compound is synthesized through esterification and thiol-maleimide addition reaction, and a nano drug-loaded particle is prepared based on the docetaxel compound, so that the problems of low drug loading, poor targeting and tumor stem cell drug resistance in the prior art are solved, meanwhile, the nano drug-loaded particle can realize precise enrichment of the drug at a tumor site, improve the anti-tumor effect and reduce toxicity.

Description

Technical Field

[0001] This invention belongs to the technical field of pharmaceutical technology, specifically relating to a docetaxel nanoprodrug targeting tumor cells and tumor stem cells, its preparation method, and its application. Background Technology

[0002] Docetaxel reduces the number of free microtubules by promoting microtubule polymerization and inhibiting tubulin depolymerization within tumor cells, thereby suppressing tumor cell mitosis and leading to tumor cell death. Docetaxel's anti-microtubule depolymerization ability is twice that of paclitaxel, and it also exhibits some activity against paclitaxel-resistant cell lines. In vivo studies have demonstrated that it is more cytotoxic than paclitaxel against most transplanted tumors in humans.

[0003] After intravenous injection, docetaxel is difficult to specifically accumulate in tumor tissue. Its systemic distribution leads to common adverse reactions, including bone marrow suppression, gastrointestinal reactions, fluid retention, and allergic reactions. Severe adverse reactions often prevent patients from successfully completing first-line treatment, especially the severe granulocytopenia caused by docetaxel, which is often directly related to infection and even death.

[0004] Docetaxel itself exhibits high cytotoxic activity, is highly lipophilic (LogP value 4.3), and is virtually insoluble in water (solubility <20 ng / mL). Furthermore, it serves as a substrate for efflux transport proteins such as P-glycoprotein and metabolic enzymes such as CYP3A4, resulting in low oral bioavailability and significant individual variability. In clinical applications, docetaxel is administered intravenously. Commercially available docetaxel injections often use polysorbate 80 and dehydrated ethanol as the solvent system; however, polysorbate 80 may cause hypersensitivity reactions and reduce drug uptake by tumor tissue.

[0005] Currently available docetaxel injections are widely used to treat various types of cancer, such as breast cancer, gastric cancer, ovarian cancer, lung cancer, osteosarcoma, non-small cell lung cancer, and prostate cancer. Although docetaxel has contributed to improving the quality of life and prolonging survival for cancer patients, cellular resistance, toxicity, and adverse reactions severely limit its clinical application.

[0006] Due to its significant hydrophobic properties, docetaxel faces considerable challenges in efficient loading into PLGA nanoparticles, typically resulting in low drug loading levels (generally below 10%). While ligand modification with folic acid, antibodies, or other ligands can achieve tumor cell targeting, single-targeting methods struggle to simultaneously act on both tumor cells and cancer stem cells (CSCs), and CSCs are difficult to eliminate, leading to tumor recurrence and metastasis. Reduction-sensitive polymer prodrugs leverage the unique concentration differences of reducing agents and reactive oxygen species in tumors to achieve more precise drug release, increasing local drug concentration at the tumor site, but not ensuring specific recognition of CSCs. Therefore, developing docetaxel nanoparticle prodrugs that target both tumor cells and cancer stem cells can not only improve drug targeting and therapeutic efficacy but also significantly reduce drug toxicity, holding significant clinical value for improving patients' quality of life and prognosis. Summary of the Invention

[0007] In view of the aforementioned problems in the prior art, the primary objective of this invention is to provide a docetaxel compound.

[0008] The second objective of this invention is to provide a method for preparing docetaxel compounds.

[0009] A third objective of this invention is to provide a docetaxel nanoprodrug.

[0010] A fourth objective of this invention is to provide the use of a docetaxel compound or a docetaxel nanoprodrug in the preparation of a drug that targets and inhibits tumor cells and / or tumor stem cells.

[0011] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention claims protection for a docetaxel compound having the structural formula shown in formula (I):

[0012] (I) Among them, 15-30.

[0013] Preferably, n is 20-24; more preferably, n is 22-23.

[0014] Preferably, the docetaxel compound has the following structural formula: .

[0015] Furthermore, this invention claims protection for a method for preparing docetaxel compounds, comprising the following steps: esterification of docetaxel with polyethylene glycol containing a maleimide ring and a carboxyl group; subsequent addition reaction of neuropeptide Y1 analog PBL with the maleimide ring; and post-treatment to prepare the docetaxel compound shown in formula (I); the reaction formula is shown below:

[0016] Among them, 15-30.

[0017] Preferably, the esterification reaction is carried out in an EDCI / DMAP catalytic system.

[0018] Preferably, the post-processing includes at least one of a purification step and a dialysis step.

[0019] Preferably, the purification step involves column chromatography purification using an eluent system composed of dichloromethane and methanol.

[0020] Preferably, the molecular weight cutoff used in the dialysis step is 2000-3500.

[0021] Preferably, n is 20-24; more preferably, n is 22-23. The inventors discovered through research that when the molecular weight of polyethylene glycol is large, it hinders the grafting of neuropeptide Y1 analogs, resulting in a lower yield of the final product, docetaxel-like compounds; while when the molecular weight of polyethylene glycol is low, its water solubility is poor, which is not conducive to the subsequent preparation of prodrugs.

[0022] Furthermore, this invention claims protection for a docetaxel nanoprodrug comprising a docetaxel compound and a stabilizer; wherein the mass-to-volume ratio of the docetaxel compound and the stabilizer is 5-20:1-3 mg / mL.

[0023] Specifically, the mass-to-volume ratio of the docetaxel compound and the stabilizer is 10-20:2-3 mg / mL, 15-20:2.5-3 mg / mL, or any range formed by the above values, but the present invention is not limited thereto.

[0024] Preferably, the stabilizer is selected from at least one of poloxamer (such as poloxamer 188), povidone (such as PVPK30), Tween 80, sodium lauryl sulfate, glycyrrhizic acid, lecithin, hydroxypropyl methylcellulose, and vitamin E polyethylene glycol succinate.

[0025] Preferably, the stabilizer is selected from at least one of glycyrrhizic acid and vitamin E polyethylene glycol succinate.

[0026] Preferably, the average particle size of the docetaxel nanoprodrug is 90-105 nm; and / or the PDI of the docetaxel nanoprodrug is 0.15-0.25.

[0027] Furthermore, this invention claims protection for a method for preparing a docetaxel nanoprodrug, the method comprising the following steps: using a precipitation method, injecting a mixture of stabilizer and solvent into the docetaxel compound and mixing evenly to prepare the docetaxel nanoprodrug.

[0028] Furthermore, this invention claims protection for the use of the docetaxel compounds or the docetaxel nanoprodrugs in the preparation of drugs that target and inhibit tumor cells and / or tumor stem cells.

[0029] Preferably, the tumor is at least one of breast cancer, colon cancer, glioma, liver cancer, and lung cancer.

[0030] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a docetaxel-like compound, which uses a neuropeptide Y1 analog to modify PEGylated docetaxel. This allows for tumor tissue enrichment by targeting the neuropeptide Y1 receptor on cancer cells, while PEGylation enhances drug stability. The docetaxel nanoparticle prodrug prepared based on this invention addresses the problems of low drug loading, poor targeting, and drug resistance in tumor stem cells in existing technologies. It can simultaneously target both tumor cells and tumor stem cells, achieving precise drug enrichment at the tumor site, improving anti-tumor efficacy, and reducing systemic toxicity. Attached Figure Description Figure 1 MALDI-TOF-MS images of PBL-PEG-DTX and mPEG-DTX are shown. Figure 1 In the image, A represents the MALDI-TOF-MS image of PBL-PEG-DTX; Figure 1 B in the image is the MALDI-TOF-MS image of mPEG-DTX.

[0031] Figure 2 This is a schematic diagram showing the SEM images and particle size distribution of PBL-PEG-DTX@NPs and mPEG-DTX@NPs. Among them, Figure 2 In the image, A and B are SEM images of PBL-PEG-DTX@NPs and mPEG-DTX@NPs, respectively. Figure 2 C and D in the diagram represent the particle size distribution of PBL-PEG-DTX@NPs and mPEG-DTX@NPs, respectively.

[0032] Figure 3 Morphological diagram of enriched tumor stem cells. Among them, Figure 3 A in the image is an optical microscope photograph of 4T1 cells; Figure 3 B in the image is an optical microscope photograph of 4T1 CSCs; Figure 3 C and D in the equation are CD44. + / CD24 - Phenotypic proportions of breast cancer stem cells. C represents 4T1 cells; D represents 4T1-CSC cells. Figure 3 In the figure, E and F represent the proportion of breast cancer stem cells in the ALDH1+ phenotype. Specifically, E represents the 4T1 cell group; and F represents the 4T1-CSC cell group.

[0033] Figure 4 The results of intervention with DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs in 4T1 tumor cells and tumor stem cells. Figure 4 In this context, A represents the ability of DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs to inhibit the formation of spheroids in breast cancer stem cells; Figure 4 In this context, B represents the ability of DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs to inhibit the regeneration of breast cancer stem cells. Figure 4 C in the figure represents the reduction of ALDH1 in each group. + A schematic diagram of the proportion of breast cancer stem cells in the phenotype.

[0034] Figure 5 This study investigated the uptake of breast cancer cells and breast cancer stem cells. Among them, Figure 5 In the image, A represents the image of 4T1 cells taking up CU-6, CU6 / mPEG-DTX@NPs, and CU6 / PBL-PEG-DTX@NPs; Figure 5 In this context, B represents the result of flow cytometry quantitative analysis. Figure 5 C in the figure represents the breast cancer stem cell uptake experiment, showing up images of CU-6, CU6 / mPEG-DTX@NPs and CU6 / PBL-PEG-DTX@NPs taken up by 4T1 CSCs. Figure 5 In this context, D represents the result of flow cytometry quantitative analysis. Figure 5 E in the figure represents the experiment on the interference of neuropeptide Y1 on the uptake of breast cancer cells, in which images of 4T1 cells pretreated with neuropeptide Y1 uptake of CU-6, CU6 / mPEG-DTX@NPs and CU6 / PBL-PEG-DTX@NPs are shown. Figure 5 In this context, F represents the flow cytometry quantitative analysis results of the three treatment groups before and after treatment with neuropeptide Y1; Figure 5G in the figure represents the experiment on the interference of neuropeptide Y1 on the uptake of breast cancer stem cells, and G represents the uptake images of CU-6, CU6 / mPEG-DTX@NPs and CU6 / PBL-PEG-DTX@NPs by 4T1 CSCs pretreated with neuropeptide Y1. Figure 5 H in the figure represents the flow cytometry quantitative analysis results of the three treatment groups before and after treatment with neuropeptide Y1.

[0035] Figure 6 The results show the in vivo inhibition of breast cancer by DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs. Figure 6 In this context, A represents the tumor volume of each group; Figure 6 In this context, B represents the tumor weight of each group; Figure 6 C in the figure represents the distribution of nanoparticles in each group over time; Figure 6 D in the figure represents the enrichment of fluorescence in each organ by each group over 24 hours.

[0036] Figure 7 The inhibition of tumor cell proliferation and the half-maximal inhibitory rate (ICP-C) for each group are shown. Figure 7 In this context, A represents GL261 glioma cells; Figure 7 B in the text represents CT26 colon cancer cells; Figure 7 C in the text represents HepG2 liver cancer cells; Figure 7 D in the text represents A549 lung cancer cells; Figure 7 E in the text represents 4T1 breast cancer cells; Figure 7 F in the figure represents the measured IC50 value.

[0037] Figure 8 The inhibition of tumor stem cell proliferation and the half-maximal inhibitory rate (IC50) for each group. 50 ).in, Figure 8 In this context, A represents GL261 glioma tumor stem cells; Figure 8 B in the text represents CT26 colon cancer tumor stem cells; Figure 8 C in the text represents HepG2 liver cancer stem cells; Figure 8 D in the text represents A549 lung cancer tumor stem cells; Figure 8 E in the text represents 4T1 breast cancer tumor stem cells; Figure 8 F in the figure represents the measured IC. 50 value. Detailed Implementation

[0038] The present invention will be further described below with reference to the specification and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0039] Materials: Docetaxel (DTX, 99%) was purchased from Dalian Meilun Biotechnology Co., Ltd.; Mal-PEG-COOH (95%) and mPEG-COOH (95%) were purchased from Xi'an Kaixin Biotechnology Co., Ltd.; 4-Dimethylaminopyridine (DMAP, AR) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, AR) were purchased from Aladdin Reagent Co., Ltd.; Dichloromethane (DCM, SafeDry, ultra-dry) and N,N-dimethylformamide (DMF, SafeDry, ultra-dry) were purchased from Adamas Beta (Shanghai) Chemical Reagent Co., Ltd.; Diethyl ether (AR) was purchased from Chuandong Chemical Co., Ltd.; INBRN (PBL peptide, 96.86%) was purchased from Shanghai Qiangyao Biotechnology Co., Ltd.

[0040] Example 1 PBL-PEG 1000 Synthesis and Characterization of DTX 1. Synthesis of PBL-PEG-DTX conjugate (1) First, 5 mL of anhydrous DCM was placed in a three-necked flask, and an anhydrous and oxygen-free environment was created by evacuating and purging nitrogen three times. Then, Mal-PEG-COOH and DTX were dissolved in DCM and injected into the three-necked flask using a syringe. The flask was placed on an ice bath and stirred for 10 min. Then, DMAP was dissolved in DCM and injected into the three-necked flask using a syringe. Finally, EDCl was added, and the mixture was stirred on an ice bath for another 15 min. After that, the ice bath was removed, and the mixture was reacted at room temperature with stirring for 24 h. The product was separated by column chromatography using silica gel (after washing away DTX with dichloromethane:methanol = 22:1, the product was eluted with dichloromethane:methanol = 11:1), and the eluent was evaporated to dryness to obtain a pale yellow oily substance, Mal-PEG. 1000 -DTX.

[0041] The proton and carbon spectra of PBL-PEG-DTX are as follows: 1HNMR (600 MHz, DMSO) δ= 8.02 (t,1H), 7.98 (d, 1 H), 7.85 (d,1 H), 7.72 (d,1 H), 7.65 (t, 2 H), 7.41 (t, 1 H),7.35 (d, 2 H), 7.29 (t, 1 H), 7.17 (t, 1 H), 6.99 (s, 1 H), 5.85 (m, 1 H), 5.77 (t, 1 H), 5.39 (d, 1 H), 5.16 (d, 1 H), 5.06 (t, 2 H), 4.95 (s, 1 H), 4.99 (d, 1 H), 4.52 (s, 1 H), 4.35 (s, 1 H), 4.24 (dd, 3 H), 4.01 (m, 3 H), 3.86 (s, 1 H), 3.75 (m, 4 H), 3.50 (s, 80 H), 3.14 (t, 3 H), 2.61 (s, 1 H), 2.59 (m, 1 H), 2.32 (t, 1 H), 2.23 (s, 1H), 1.86 (m, 2 H), 1.81 (t,4 H), 1.70(s, 1 H), 1.62 (s, 1 H), 1.50 (s, 1 H), 1.34 (s, 1 H), 1.22 (s, 1 H), 0.97(s, 1H), 0.84 (t, 1H). 13 CNMR(600MHz, DMSO)δ= 13 CNMR(600MHz,DMSO)δ=171.22,165.77,130.04,129.16,128.67,127.65,84.22,80.73 ,78.67,77.36,71.27,70.44,69.95,58.50,57.46,43.35,28.62,26.89,22.87,21.22. (2) Under nitrogen protection, Mal-PEG was subjected to... 1000 -DTX and PBL (molar ratio 1:2) were dissolved in anhydrous DMF, and the resulting solution was stirred at room temperature for 24 h. The resulting product was dialyzed against ultrapure water for 12 h (molecular weight cutoff 2000) to remove unreacted PBL. Finally, a pale yellow viscous product, PBL-PEG, was obtained by freeze-drying. 1000 -DTX (hereinafter referred to as PBL-PEG-DTX), yield 73.8%. The reaction formula for preparing PBL-PEG-DTX is shown below.

[0042] .

[0043] Comparative Example 1mPEG 1000 -DTX synthesis First, place 5 mL of anhydrous DCM into a three-necked flask and create an anhydrous and oxygen-free environment by evacuating the flask and purging nitrogen three times. Then add mPEG. 1000 -COOH and DTX were dissolved in DCM and injected into a three-necked flask using a syringe. The flask was placed on an ice bath and stirred for 10 min. Then, DMAP was dissolved in DCM and injected into the three-necked flask using a syringe. Finally, EDCl was added, and the mixture was stirred on an ice bath for another 15 min. The ice bath was then removed, and the reaction was carried out at room temperature with stirring for 24 h. Separation was performed by column chromatography using silica gel (dixochloromethane:methanol = 22:1 to wash away DTX, then dichloromethane:methanol = 11:1 to elute the product). After evaporating the eluent, the product was lyophilized to obtain a white, slightly viscous product, i.e., mPEG. 1000 -DTX (hereinafter referred to as mPEG-DTX), yield 85.1%. The reaction formula is shown below: .

[0044] The proton and carbon spectra of mPEG1000-DTX are as follows: 1HNMR (600 MHz, DMSO) δ= 8.02 (t,1H), 7.98 (d, 1 H), 7.85 (d, 1 H), 7.72 (d,, 1 H), 7.65 (t, 2 H), 7.41 (t, 1H), 7.35 (d, 2 H), 7.29 (t,1 H), 7.17 (t, 1 H), 6.94(d, 1 H),6.06(t, 2 H),6.00(t, 2 H),5.85 (m, 1 H), 5.77 (t, 1 H), 5.39 (d,1 H), 5.24(s, 1 H),5.16(d, 1 H), 5.05 (td, 2 H), 4.95 (s, 1 H), 4.99 (d, 1 H), 4.52 (d,1 H), 4.35(s, 1 H), 4.24 (dd, 3 H), 4.01 (m, 3 H), 3.75 (m, 4 H), 3.50 (s, 64 H),3.23(s, 1 H),3.16 (s, 1 H), 3.01( (ddd, 3 H),2.73 (s, 1 H),2.61 (s, 1 H), 2.59(m, 1 H), 2.23 (s, 1 H), 1.86 (m, 2 H), 1.81 (t, 1 H), 1.70 (s, 1H), 1.62 (s,1 H), 1.50 (s, 1 H), 1.34 (s, 1H), 1.22 (s, 1 H), 0.97 (s,1H), 0.84 (t, 1 H). 13 CNMR(600 MHz, DMSO) δ= 13C NMR (600MHz, DMSO) δ=171.22, 165.77,130.04,129.16,128.67,127.65,84.22,80.73,78.67,77.36,71.27,70.44,69.95,58.50,57.46,43.35,28.62,26.89,22.87,21.22.。

[0045] Test Example 1 Structural Characterization of PBL-PEG-DTX and mPEG-DTX (1) Take 10 mg of each of the two products PBL-PEG-DTX and mPEG-DTX, dissolve them in a mixed solution of 20% acetonitrile and 80% pure water, and characterize their molecular weight using a matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer (MALDI-TOF-MS). The test matrix is ​​DHB.

[0046] (2) Take 10 mg of each of the two products and use deuterated DMSO with tetramethylsilane (TMS) as an internal standard as a solvent. Determine the structure by measuring the NMR spectrum using a nuclear magnetic resonance spectrometer.

[0047] Figure 1 MALDI-TOF-MS images of PBL-PEG-DTX and mPEG-DTX. Figure 1 It can be seen that the average molecular weight of PBL-PEG-DTX (3343.55) is basically consistent with the theoretical molecular weight (3338.06), proving that the target conjugate was successfully synthesized. The average molecular weight of mPEG-DTX (1581.99) is basically consistent with the theoretical molecular weight (1583.88), proving that the target conjugate was successfully synthesized.

[0048] Test Example 2: Preparation and Quality Evaluation of PBL-PEG-DTX Nanoparticles 1. Study on preparation and quality evaluation of nanoparticles (1) Investigation of nanoparticle preparation methods 1) Preparation of PBL-PEG-DTX@NPs by precipitation method Accurately weigh 15 mg of PBL-PEG-DTX, add it to 15 mL of anhydrous ethanol solution containing 3 mL of stabilizer (vitamin E polyethylene glycol succinate, D-α-Tocopherol polyethylene glycol succinate, TPGS) to dissolve it, add it to ultrapure water with constant stirring at room temperature, and dry the anhydrous ethanol with cold air to obtain PBL-PEG-DTX@NPs nanoparticle solution.

[0049] 2) Preparation of PBL-PEG-DTX@NPs by thin film dispersion method 15 mg of PBL-PEG-DTX was accurately weighed and dissolved in 15 mL of anhydrous ethanol. The solution was then dialyzed with 150 mL of ultrapure water for 4 h (molecular weight cutoff of 2000), vortexed for 5 min, and sonicated for 5 min to obtain the PBL-PEG-DTX@NPs solution. Particle size and polydispersity index were used as indicators for evaluation.

[0050] 15 mg of PBL-PEG-DTX was accurately weighed and dissolved in 15 mL of anhydrous ethanol. The anhydrous ethanol was removed by rotary evaporation at 40 °C to form a thin film. 15 mL of ultrapure water was added to dissolve the film, and the dissolved film was vortexed for 5 minutes, followed by sonication for 5 minutes to obtain the PBL-PEG-DTX@NPs solution. Particle size and polydispersity index were used as indicators for evaluation.

[0051] 3) Preparation of PBL-PEG-DTX@NPs by dialysis 15 mg of PBL-PEG-DTX was accurately weighed and dissolved in 15 mL of anhydrous ethanol. The solution was then dialyzed with 150 mL of ultrapure water for 4 h (molecular weight cutoff of 2000), vortexed for 5 min, and sonicated for 5 min to obtain the PBL-PEG-DTX@NPs solution. Particle size and polydispersity index were used as indicators for evaluation.

[0052] The PBL-PEG-DTX@NPs solution prepared by precipitation method did not precipitate after prolonged standing. The PBL-PEG-DTX@NPs solution prepared by thin-film dispersion method readily precipitated after standing for 2 hours. The PBL-PEG-DTX@NPs solution prepared by dialysis produced a small amount of precipitate after standing for approximately 4 hours. Table 1 shows the effect of different preparation methods on the results of PBL-PEG-DTX@NPs solutions (X±s, n=3).

[0053] Table 1

[0054] As shown in Table 1 above, the PBL-PEG-DTX@NPs prepared by the precipitation method of this invention have smaller particle size and polydispersity index than the other three methods. Therefore, the precipitation method was chosen as the method for preparing nanoparticle solutions.

[0055] (2) Investigation of stabilizer types The effects of stabilizers poloxamer 188, PVPK30, Tween 80, sodium dodecyl sulfate, glycyrrhizic acid, lecithin, hydroxypropyl methylcellulose, and vitamin E polyethylene glycol succinate (TPGS) on PBL-PEG-DTX@NPs solutions were investigated using particle size, polydispersity index, and stability index as indicators. The particle size of the stabilized nanoparticle solution was measured after centrifugation at 3000 r / min for 30 min, and the stability index was calculated. Stabilizers with a stability index close to 1 were selected. The stability index (SI) is calculated as follows: Stability index (SI) = Average particle size of the unprecipitated upper layer after centrifugation / Average particle size of the solution before centrifugation. Table 2 shows the effect of stabilizer type on the results of PBL-PEG-DTX@NPs solutions (X±s, n=3).

[0056] Table 2

[0057] As shown in Table 2, due to the high specific surface area and surface energy of nanoparticles, they tend to aggregate in the solution, affecting the properties and effectiveness of the nanoparticle solution. Adding a stabilizer can effectively reduce surface tension and prevent solute particle aggregation. When vitamin E polyethylene glycol succinate (TPGS) or glycyrrhizic acid is used as a stabilizer, the prepared PBL-PEG-DTX@NPs solution has a smaller particle size and polydispersity index, and the overall system stability is better than with other stabilizers. Therefore, one of these stabilizers is chosen.

[0058] (3) Nanoparticle encapsulation efficiency and drug loading Nanoparticle morphology observation: 10 μL of each of PBL-PEG-DTX@NPs solution and mPEG-DTX@NPs solution (prepared by precipitation method according to step (1) of Example 2) were accurately measured, dropped onto carbon film copper grid, dried at room temperature, and after negative staining, the sample was placed under HITACHI H-7650 transmission electron microscope to observe the morphology of nanoparticles.

[0059] Nanoparticle encapsulation efficiency and drug loading: Accurately measure 500 μL each of PBL-PEG-DTX@NPs solution and mPEG-DTX@NPs solution into two 5 mL EP tubes, add 1.5 mL of ultrapure water to each tube, mix well, and centrifuge at 10000 rpm for 10 min. Take an appropriate amount of the supernatant after centrifugation, filter it through a 0.45 μm microporous membrane, and inject it into a high-performance liquid chromatograph (HPLC). Record the peak area and calculate the free DTX content (w1) based on the peak area. Accurately measure 500 μL each of PBL-PEG-DTX@NPs solution and mPEG-DTX@NPs solution into two 5 mL EP tubes, add 1.5 mL of methanol to each tube, sonicate to break emulsion for 10 min, filter it through a 0.45 μm microporous membrane, and inject it into an HPLC. Record the peak area and calculate the total DTX content (w0) based on the peak area. The encapsulation efficiency (EE%) and drug loading (DL%) are calculated as follows: EE% = (w0 - w1) / w0 × 100%; DL% = (w0 - w1) / ws × 100%; ws is the total weight of nanoparticles.

[0060] Figure 2 This is a schematic diagram showing the SEM images and particle size distribution of PBL-PEG-DTX@NPs and mPEG-DTX@NPs. Both PBL-PEG-DTX@NPs and mPEG-DTX@NPs are spherical in shape, relatively uniform in size, and have a particle size of less than 200 nm.

[0061] After investigating the optimization factors of the nanoparticle preparation method and the type of stabilizer, PBL-PEG-DTX@NPs and mPEG-DTX@NPs were prepared using the optimized parameters. The particle size, PDI, encapsulation efficiency, and drug loading results are shown in Table 3. Table 3 shows the particle size, PDI, encapsulation efficiency, and drug loading of PBL-PEG-DTX@NPs and mPEG-DTX@NPs.

[0062] Table 3

[0063] Test Example 3: Culture and Characterization of Breast Cancer Stem Cells (1) Suspension culture and microscopic observation of tumor stem cells Prepare serum-free culture medium: Add 10 mL of B27, 0.4% BSA, 5 μg / mL insulin, and 20 ng / mL of basic fibroblast growth factor (b-FGF) and epidermal growth factor (EGF) supplements to 500 mL of DMEM / F12 culture medium, mix well, and dispense for use.

[0064] 4T1 breast cancer cells in good logarithmic growth phase were collected, digested, centrifuged at 1000 rpm for 5 min, counted, and resuspended in serum-free medium. 1 x 102 cells were then seeded per well in a 6-well ultra-low adhesion plate. 4 Four T1 breast cancer cells were transferred to a cell culture incubator (37℃, 5% CO2) and cultured for approximately 7 days. Cells and cell microspheres in good growth condition were selected and observed and photographed under an inverted microscope to obtain microscopic images of both.

[0065] Figure 3 Morphological diagram of enriched tumor stem cells. Figure 3 In the A group, the 4T1 cells exhibited an epithelial-like morphology and adhered to the wall. Figure 3 B in the figure represents the morphology of 4T1 tumor stem cells (CSCs) under serum-free suspension culture conditions, and the volume of 4T1-CSCs microspheres gradually increases with the extension of culture time.

[0066] (2) Breast cancer stem cell markers (CD44) + / CD24 - ) detection CD44 + / CD24 - Breast cancer cells with this phenotype are considered to be breast cancer stem cells, CD44 + / CD24 -It can be used as one of the biomarkers for breast cancer stem cells. Logarithmically growing 4T1 cells and third-generation cell microspheres were collected, digested with trypsin into single-cell suspensions, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were washed with PBS, resuspended, and counted. Approximately 1 × 10⁻⁶ cells from each of the 4T1 cells and third-generation suspension microspheres were collected. 6 Each sample was placed in a centrifuge tube. The experimental group received 5 μL each of CD44-FITC and CD24-PE, while the isotype control group received 5 μL each of PE-IgG2aκ and FITC-IgG2bκ. All samples were thoroughly mixed by pipetting. After incubation at 4°C in the dark for 30 min, the samples were centrifuged at 1000 rpm for 5 min, washed and resuspended with PBS, and transferred to flow cytometry tubes for analysis. CD44 was detected by flow cytometry. + / CD24 - Percentage of phenotypic breast cancer stem cells.

[0067] Figure 3 C in the text refers to CD44 in 4T1 cells. + / CD24 - The phenotypic proportion was 15.03%. Figure 3 In this context, D represents CD44 in suspended microsphere cells. + / CD24 - The proportion of 4T1-CSC cells in the breast cancer stem cell phenotype was 44.02%. The enrichment of breast cancer stem cell populations in suspension culture increased significantly. This indicates that the suspension microsphere cells cultured using this method can serve as a model for further investigation of breast cancer stem cells.

[0068] (3) Acetaldehyde dehydrogenase activity assay ALDH1 + It is also recognized as one of the biomarkers for breast cancer stem cells. Logarithmically growing 4T1 cells and third-generation cell microspheres were collected, digested with trypsin, and prepared into single-cell suspensions with PBS. Approximately 1 × 10⁻⁶ cells from each of the 4T1 cells and third-generation suspension microspheres were collected. 6 Two flow cytometry tubes were used to divide the cells into an experimental group and a control group. 5 μL of DEAB inhibitor was added to the control group. 5 μL of ALDEFLUOR™ activator was added to the experimental group, and the mixture was thoroughly mixed.

[0069] Add 500 μL of the experimental group mixture to the control group EP tube and mix thoroughly. Incubate at 37°C for 30 min, then centrifuge at 1000 rpm for 5 min. Collect the supernatant, add 200 μL of PBS, and analyze ALDH1 using flow cytometry. + Percentage of phenotypic breast cancer stem cells.

[0070] Figure 3 E in the text represents ALDH1 in the suspended microsphere cells. +The phenotype was present in 18.59% of 4T1 cells; Figure 3 F in the text represents ALDH1 in the suspended microsphere cells. + The proportion of 4T1-CSC cells with the ALDH1 phenotype was 57.74%. + The increased proportion of viable cells further indicates that the suspended microspheres cultured by this method can be used as a model to investigate breast cancer stem cells.

[0071] Test Example 4: In vitro anti-breast cancer stem cell activity study of PBL-PEG-DTX nanoparticles (1) Inhibits the ability of breast cancer stem cells to form spheroids The ability of tumor cells to form spheroids is an important indicator for evaluating tumor stem cells. Serum-free culture medium was used to set up a control group and three treatment groups. The control group used standard culture medium, while the three treatment groups contained 10 μg / mL DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs, respectively. Breast cancer cells seeded in ultra-low adhesion 96-well plates were cultured in suspension using the above culture media, and the diameter of cancer cell spheroids was observed in the control group, DTX treatment group, mPEG-DTX@NPs treatment group, and PBL-PEG-DTX@NPs treatment group.

[0072] Figure 4 In the figure, A represents the ability of DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs to inhibit the formation of spheroids in breast cancer stem cells. After 7 days of treatment with PBL-PEG-DTX@NPs, the diameter of the PBL-PEG-DTX@NPs-treated group was smaller than that of the control group, the DTX-treated group, and the mPEG-DTX@NPs-treated group. The diameter of the tumor spheroids in the control group was approximately twice that of the PBL-PEG-DTX@NPs-treated group, and the diameter of the tumor spheroids in the DTX-treated group was approximately 1.5 times that of the PBL-PEG-DTX@NPs-treated group.

[0073] (2) Inhibits the ability of breast cancer stem cells to regenerate into spheres. The ability of tumor cells to regenerate into spheroids is an important indicator for evaluating tumor stem cells. Serum-free culture medium was used, with a control group and three treatment groups. The control group used standard culture medium, while the three treatment groups contained 10 μg / mL DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs, respectively. Breast cancer cells seeded in ultra-low adhesion 24-well plates were cultured in suspension using the above culture media. The size and number of cancer cell spheroids were observed in the control group, DTX treatment group, mPEG-DTX@NPs treatment group, and PBL-PEG-DTX@NPs treatment group. The number of tumor spheroids larger than the diameter of cancer cell microspheres in the spheroidization ability group was counted.

[0074] Figure 4 In the figure, B represents the ability of DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs to inhibit the regeneration of breast cancer stem cells. Four days after treatment with PBL-PEG-DTX@NPs, the number of regenerated tumor spheroids in the PBL-PEG-DTX@NPs treatment group was significantly lower than that in the control group, DTX treatment group, and mPEG-DTX@NPs treatment group. The number of regenerated tumor spheroids in the control group was approximately 6 times that in the PBL-PEG-DTX@NPs treatment group, while the number of regenerated tumor spheroids in the DTX treatment group and mPEG-DTX@NPs treatment group was approximately 4 times that in the PBL-PEG-DTX@NPs treatment group.

[0075] (3) Inhibit ALDH + The ratio of phenotypic cells Third-generation cell microspheres treated with culture medium containing 20 μg / mL DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs were digested with trypsin, and single-cell suspensions were prepared with PBS. Approximately 1 × 10⁶ cells were collected. 6 Two EP tubes were used to divide the cells into an experimental group and a control group. 5 μL of DEAB inhibitor was added to the control group. Single-cell suspension and 5 μL of LALDEFLUOR™ activator were added to the experimental group and mixed thoroughly. 500 μL of the experimental group mixture was then added to the control group EP tube and mixed thoroughly. The cells were incubated in a 37°C water bath for 30 min, followed by centrifugation at 1000 rpm for 5 min. The supernatant was collected and 200 μL of flow cytometry staining solution was added for ALDH1 detection using a flow cytometer. + Percentage of phenotypic breast cancer stem cells.

[0076] Figure 4 C in the figure represents the reduction of ALDH in each group. + Phenotypic phenotype: Proportion of breast cancer stem cells. Compared with the blank control group, free DTX treatment group, and mPEG-DTX@NPs treatment group, the ALDH1 level in the PBL-PEG-DTX@NPs treatment group was significantly higher. + The number of breast cancer stem cells was significantly reduced to 12.2%. Therefore, PBL-PEG-DTX@NPs reduced the proportion of breast cancer stem cells with the ALDH1+ phenotype.

[0077] Test Example 5: In vitro targeted uptake of PBL-PEG-DTX nanoparticles by breast cancer cells and breast cancer stem cells. (1) Breast cancer cell uptake experiment Qualitative analysis: 4T1 cells in logarithmic growth phase were divided into groups of 10 cells per well. 5Cells were seeded at a density of 1000 g / mL in 12-well plates and incubated overnight until adherence. After removing the old culture medium, 2 mL of coumarin-6 (CU-6) medium, CU6 / mPEG-DTX@NPs, and CU6 / PBL-PEG-DTX@NPs were added at a drug concentration of 50 ng / mL. Three replicates were prepared, and the cells were incubated in a cell culture incubator (37°C, 5% CO2) for 2 h. The original culture medium was removed, and the cells were washed twice with PBS. After fixation with 1.0 mL of 4% paraformaldehyde for 10 min, and washing three times with PBS, the cell nuclei were stained with 10 μg / mL DAPI in the dark for 10 min. After washing three times with PBS, the cells were observed under a laser confocal microscope.

[0078] Quantitative analysis: 4T1 cells in logarithmic growth phase were divided into groups of 10 cells per well. 6 Cells were seeded at a density of [number] cells / well in 6-well plates and incubated for 24 h until adherence. After removing the old culture medium, 2 mL of coumarin-6 (CU-6) medium, CU6 / mPEG-DTX@NPs, and CU6 / PBL-PEG-DTX@NPs were added at a drug concentration of 50 ng / mL. Three replicates were prepared, and the cells were incubated in a cell culture incubator (37℃, 5% CO2) for 2 h. The original medium was then removed, and the cells were washed twice with PBS. The resulting cell suspension was collected, and the fluorescence intensity of the FITC channel of CU-6 in the cells was detected using flow cytometry.

[0079] like Figure 5 As shown in Figure A, after 1 h of cell incubation, the fluorescence intensity of the CU6 / PBL-PEG-DTX@NPs treatment group was the strongest, significantly greater than that of the CU6 / mPEG-DTX@NPs treatment group and the CU6 treatment group. This indicates that 4T1 cells take up CU6 / PBL-PEG-DTX@NPs much more readily than CU6 / mPEG-DTX@NPs and free CU6. Figure 5 The B-flow cytometry results were consistent with the uptake image results, and the 4T1 cells showed significant differences in uptake results among the three groups.

[0080] (2) Breast cancer stem cell uptake experiment Qualitative and quantitative analyses were performed using the same method as the breast cancer cell uptake assay, with the difference being that third-generation breast cancer stem cell microspheres were used in both qualitative and quantitative analyses.

[0081] like Figure 5As shown in Figure C, after 1 h of incubation with breast cancer stem cells, the fluorescence intensity of the CU6 / PBL-PEG-DTX@NPs treatment group was the strongest, significantly greater than that of the CU6 / mPEG-DTX@NPs treatment group and the CU6 treatment group. This indicates that the uptake of CU6 / PBL-PEG-DTX@NPs by 4T1CSCs is much greater than that by CU6 / mPEG-DTX@NPs and free CU6. Furthermore, the flow cytometry results are consistent with the uptake image results. Figure 5 The D, 4T1 CSCs showed significant differences in intake outcomes among the three groups.

[0082] (3) Experiment on interference of neuropeptide Y1 on breast cancer cell uptake Logarithmic growth phase 4T1 cells were cultured at 10 per well. 6 Cells were seeded at a density of 100 μg / mL in 6-well plates and incubated for 24 h until adherence. After removing the old medium, 2 mL of medium containing 200 μg / mL neuropeptide Y1 was added and incubated for 1 h. Then, 2 mL of coumarin-6 (CU-6) medium, CU6 / mPEG-DTX@NPs, and CU6 / PBL-PEG-DTX@NPs were added at a drug concentration of 50 ng / mL. Three replicates were set up and incubated in a cell culture incubator (37°C, 5% CO2) for 2 h.

[0083] Qualitative analysis: After the above steps, the original solution was aspirated and the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min, washed three times with PBS, and then the cell nuclei were stained with 10 ug / mL DAPI in the dark for 10 min. After washing three times with PBS, the cells were observed under a laser confocal microscope.

[0084] Quantitative analysis: After the above steps, the original solution was aspirated, the cells were washed twice with PBS, digested and collected, and the fluorescence intensity of the FITC channel of CU-6 in the cells was detected by flow cytometry.

[0085] like Figure 5 As shown in Figure E, after pre-binding neuropeptide Y1 to the neuropeptide Y1 receptor on 4T1 breast cancer cells, the fluorescence intensity change was greatest in the CU6 / PBL-PEG-DTX@NPs treatment group, while the fluorescence intensity was smaller in the CU6 / mPEG-DTX@NPs treatment group and the CU6 treatment group. Furthermore, the flow cytometry results... Figure 5 The F-resistance fluorescence image results indicate that PBL-PEG-DTX@NPs are highly selective for 4T1 cells expressing the neuropeptide Y1 receptor.

[0086] (4) Experiment on interference of neuropeptide Y1 on breast cancer stem cell uptake The procedure was similar to the experiment on the interference of neuropeptide Y1 on the uptake of breast cancer cells, except that the logarithmic growth phase 4T1 cells were replaced with third-generation breast cancer stem cell microspheres.

[0087] like Figure 5 As shown in G, after pre-binding neuropeptide Y1 to the neuropeptide Y1 receptor on 4T1 CSCs, the fluorescence intensity change was greatest in the CU6 / PBL-PEG-DTX@NPs treatment group, while the fluorescence intensity was smaller in the CU6 / mPEG-DTX@NPs treatment group and the CU6 treatment group. Furthermore, as shown in G... Figure 5 The H flow cytometry results showed that PBL-PEG-DTX@NPs had high selectivity for 4T1 CSCs expressing the neuropeptide Y1 receptor.

[0088] Test Example 6: In vivo targeted anti-breast cancer evaluation of PBL-PEG-DTX nanoparticles (1) Establishment of 4T1 subcutaneous xenograft and determination of tumor size Healthy BALB / c female mice aged 6–8 weeks were randomly divided into several groups, with the same number of mice in each group. Each mouse was subcutaneously inoculated with 4 × 10⁴ mg of the mammary fat pad or the right back. 6 For each of the 4T1 breast cancer cells, the length and width of the tumor were measured every three days using calipers, and the tumor volume was calculated using the formula: Tumor volume = 1 / 2 × length × width. 2 Tumor volume was calculated. The treatment groups received different treatments, including 5 mg / kg equivalent of DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs, while the control group received an equal volume of saline. At the end of the experimental period, mice were sacrificed, and the tumors were dissected and weighed. Tumor growth curves were plotted to compare the differences between the treatment and control groups, and the treatment efficacy was assessed using a tumor weight inhibition rate formula.

[0089] Figure 6 In the figure, A represents the tumor-inhibiting effects of DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs on 4T1 breast cancer-bearing mice. The images clearly show that the tumor volume in the PBL-PEG-DTX@NPs group was significantly reduced, and its inhibitory effect was superior to that in the DTX and mPEG-DTX@NPs groups.

[0090] Figure 6 The tumor inhibition effect of each group was further analyzed by weighing the isolated tumor tissue. The average tumor weight after administration in the PBL-PEG-DTX@NPs group was 0.59 g, which was more than 42% lower than that in the DTX group (1.15 g) and the mPEG-DTX@NPs group (1.03 g), and significantly lower than that in the saline control group (1.29 g).

[0091] (2) Targeting assay of 4T1 subcutaneous xenograft Healthy BALB / c female mice aged 6–8 weeks were randomly divided into several groups, with the same number of mice in each group. Each mouse was subcutaneously inoculated with 4 × 10⁴ mg of the mammary fat pad or the right back. 6 Each of the four T1 breast cancer cells was used until the tumor volume reached approximately 500 mm. 3 Subsequently, Dir, Dir / mPEG-DTX@NPs, and Dir / PBL-PEG-DTX@NPs were injected via tail vein at a concentration of 50 ng / mL. Three tumor-bearing mice were used in parallel, and Pekirn Element Living Image was used to dynamically photograph the enrichment of fluorescence in the tumor at 0h, 1h, 2h, 4h, 8h, and 24h. Twenty-four hours after drug administration, the heart, liver, spleen, lungs, kidneys, and tumors of the tumor-bearing mice were dissected, and fluorescence intensity was calculated.

[0092] Figure 6 In the figure, C represents the distribution of fluorescence in each group throughout the tumor-bearing mouse. In vivo fluorescence molecular imaging experiments were conducted to evaluate the targeting distribution of targeted nanoparticles Dir / PBL-PEG-DTX@NPs, non-targeted nanoparticles Dir / mPEG-DTX@NPs, and free fluorescein Dir in vivo. In the experiment, 4T1 tumor-bearing mice were intravenously injected with 0.1 mg of Dir-containing Dir / mPEG-DTX@NPs, Dir / PBL-PEG-DTX@NPs, and free Dir. The results showed that Dir / PBL-PEG-DTX@NPs accumulated significantly more at the tumor site than Dir / mPEG-DTX@NPs and free fluorescein Dir, and the accumulation of targeted nanoparticles in the tumor gradually increased over time. Furthermore, the detected strong fluorescence signal indicated that these nanoparticles were mainly metabolized by the liver, leading to a more pronounced liver retention in the isolated organ. These results further demonstrate the advantages of targeted nanoparticles in terms of tumor targeting and retention time.

[0093] Figure 6 In the figure, D represents the drug distribution in the heart, liver, spleen, lung, kidney, and tumor organs 24 hours after drug administration. Among them, Dir-loaded nanoparticles remained in the tumor for a longer time, and Dir / PBL-PEG-DTX@NPs accumulated significantly more in the tumor site than Dir / mPEG-DTX@NPs and free fluorescein Dir.

[0094] Test Example 7: Evaluation of the in vitro inhibitory effect of PBL-PEG-DTX nanoparticles on the proliferation of various tumor cells and tumor stem cells. (1) Cytotoxicity test of DTX, mPEG-DTX@NPs and PBL-PEG-DTX@NPs against various tumor cells 1) Cell seeding: 4T1, HepG2, CT26, GL261, and A549 cells in logarithmic growth phase were harvested, removed from their original culture medium, washed twice with PBS, digested with trypsin, centrifuged at 1000 rpm for 5 min, and the supernatant was aspirated with a Pasteur pipette. The cell pellet was resuspended in complete culture medium and seeded at 3 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates. The cells were cultured overnight at 37°C and 5% CO2 until adherence.

[0095] 2) Experimental grouping and drug preparation: Blank control group (no cells seeded in wells, blank culture medium added); negative control group (cells seeded in wells, blank culture medium added); experimental group (cells seeded in wells, different concentrations of drug-containing culture medium added): drug-containing culture medium with concentration gradients of 0.005-100 μM for DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs was prepared. Each group had 3 replicates.

[0096] 3) Drug administration and assay: After cell adhesion, remove the old culture medium and add the corresponding culture medium to each group. Incubate in an incubator (37℃, 5% CO2) for 48 h, then add 20 μL of 5 mg / mL MTT solution to each well and incubate in an incubator (37℃, 5% CO2) for 4 h. After removing the 96-well plate, gently shake it and measure the absorbance at 490 nm using a microplate reader, recording the OD value. Calculate the cell viability (%) using the following formula, and plot the cell viability against the drug concentration to calculate the IC50. 50 Cell viability (%) = (OD value of experimental group - OD value of negative control group) / (OD value of blank control group - OD value of negative control group) × 100%.

[0097] Figure 7 The inhibition of tumor cell proliferation and the half-maximal inhibitory rate (ICP-C) for each group are shown. Figure 7 As shown in Figures A to E, within the drug concentration range of 0.1–100 μg / mL, cell viability gradually decreased with increasing DTX concentration. Free DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs all exhibited concentration-dependent cytotoxicity against tumor cells, such as… Figure 7 The IC50 of PBL-PEG-DTX@NPs was calculated from F. 50 The levels were lower than those of free DTX and mPEG-DTX@NPs in different tumor cell lines.

[0098] (2) Cytotoxicity test of DTX, mPEG-DTX@NPs and PBL-PEG-DTX@NPs against tumor stem cells 1) Cell seeding: Enriched 4T1, HepG2, CT26, GL261, and A549 tumor stem cells were collected, the original culture medium was aspirated, the cells were washed twice with PBS, digested with trypsin, centrifuged at 1000 rpm for 5 min, the supernatant was aspirated using a Pasteur pipette, and the cell pellet was resuspended in complete culture medium. Cells were then seeded at 5 × 10⁶ cells per well. 3 Individual cells were inoculated at a density of 1,000 cells / well in 96-well ultra-low adhesion plates and incubated overnight at 37°C and 5% CO2.

[0099] 2) Experimental grouping and drug preparation: Blank control group (no cells seeded in wells, blank culture medium added); negative control group (cells seeded in wells, blank culture medium added); experimental group (cells seeded in wells, different concentrations of drug-containing culture medium added): drug-containing culture medium with concentration gradients of 0.005-100 μM for DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs was prepared. Each group had 3 replicates.

[0100] 3) Drug administration and assay: Add the corresponding culture medium to each group, incubate in an incubator (37℃, 5% CO2) for 48 h, then remove the old culture medium using a Pasteur pipette. Add 100 μL of the prepared CCK-8 dilution buffer (containing 10 μL CCK-8 and 90 μL culture medium) to each well and incubate in an incubator (37℃, 5% CO2) for 3 h. After removing the 96-well plate, gently shake it and measure the absorbance at 450 nm using a microplate reader, recording the OD value. Calculate the cell viability (%) using the following formula, and plot the breast cancer cell viability against the drug concentration to calculate the IC50. 50 value.

[0101] Figure 8 Results of DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs intervention in tumor stem cells. Figure 8 As shown in Figures A to E, within the drug concentration range, cell viability gradually decreases with increasing DTX concentration. Free DTX, mPEG-DTX@NPs, and PBL-PEG-DTX@NPs all exhibit concentration-dependent toxicity to tumor stem cells, such as... Figure 8 From F, we can see that the IC of PBL-PEG-DTX@NPs can be calculated. 50 The levels were lower than those of free DTX and mPEG-DTX@NPs in different tumor stem cell lines.

[0102] The foregoing examples are merely illustrative, used to explain some features of the method described in this invention. The appended claims are intended to claim the broadest possible scope, and the embodiments presented herein are demonstrated by the applicant's actual experimental results. Therefore, the applicant intends that the appended claims are not limited by the selection of examples illustrating the features of the invention. Some numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A docetaxel compound, characterized in that, The docetaxel compounds have the structural formula shown in formula (I): （I） Where n is between 15 and 30.

2. The docetaxel compound according to claim 1, characterized in that, n is 20-24.

3. The docetaxel compound according to claim 1, characterized in that, The docetaxel compounds have the following structural formula: 。 4. The method for preparing the docetaxel compound according to claim 1, characterized in that, The process includes the following steps: polyethylene glycol containing a maleimide ring and a carboxyl group undergoes an esterification reaction with docetaxel, followed by an addition reaction between the neuropeptide Y1 analog PBL and the maleimide ring. After post-processing, the docetaxel compound shown in formula (I) is prepared; its reaction formula is shown below: Where n is between 15 and 30.

5. A docetaxel nanoprodrug, characterized in that, It includes the docetaxel compound of claim 1 and a stabilizer; the mass-to-volume ratio of the docetaxel compound and the stabilizer is 5-20:1-3 mg / mL.

6. The docetaxel nanoprodrug according to claim 5, characterized in that, The stabilizer is selected from at least one of poloxamer, povidone, Tween 80, sodium lauryl sulfate, glycyrrhizic acid, lecithin, hydroxypropyl methylcellulose, and vitamin E polyethylene glycol succinate.

7. The docetaxel nanoprodrug according to claim 6, characterized in that, The stabilizer is selected from at least one of glycyrrhizic acid and vitamin E polyethylene glycol succinate.

8. The docetaxel nanoprodrug according to claim 5, characterized in that, The docetaxel nanoprodrug has an average particle size of 90-105 nm; and / or the PDI of the docetaxel nanoprodrug is 0.15-0.

25.

9. The use of the docetaxel compound of any one of claims 1-3 or the docetaxel nanoprodrug of any one of claims 5-8 in the preparation of a drug that targets and inhibits tumor cells and / or tumor stem cells.

10. The application according to claim 9, characterized in that, The tumor is at least one of the following: breast cancer, colon cancer, glioma, liver cancer, and lung cancer.

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