A microenvironment-responsive micelle, a preparation method thereof, and an application thereof in preparing a drug for inhibiting recurrence of ovarian cancer

Abstract

The present invention discloses a microenvironment-responsive micelle, a preparation method thereof, and an application thereof in the preparation of a drug for inhibiting ovarian cancer recurrence, relating to the technical field of pharmaceutical preparations. The preparation method comprises the following steps: dissolving polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, D-α-tocopheryl polyethylene glycol succinate, DSPE-PEG 2000 , DSPE-PEG 2000 -TK-PEG 5000 , paclitaxel, curcumol, and DSPE-PEG 2000 -FSHB in a solvent, transferring the solution to a rotary evaporation device, evaporating to remove the solvent to form a thin film; hydrating the thin film with a PBS solution, and then performing an extrusion treatment with a microporous membrane to obtain the microenvironment-responsive micelle. The microenvironment-responsive micelle can enhance the uptake of drugs by cancer stem cells, enabling the drugs to play a role in inhibiting tumor recurrence in vivo.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and particularly to a microenvironment-responsive micelle, a preparation method thereof, and an application thereof in the preparation of a drug for inhibiting ovarian cancer recurrence. Background Art

[0002] Ovarian cancer is a fatal gynecological malignancy. Since the ovaries are located deep in the pelvis, ovarian cancer usually has no typical clinical symptoms in the early stage, and most patients are diagnosed at an advanced stage. The standard treatment methods for ovarian cancer include first-line chemotherapy (carboplatin-paclitaxel regimen) and surgical debulking. Although this regimen is initially effective, its clinical treatment still poses great challenges due to intrinsic or acquired chemotherapy resistance and recurrence caused by local or distant lesions. Most ovarian cancer patients relapse within 12 to 18 months, and the five-year mortality rate of patients is as high as 70%. Therefore, there is an urgent need to solve the problem of recurrence in the treatment of ovarian cancer.

[0003] Curcumol is an anti-cancer active component of the traditional Chinese medicine Curcuma wenyujin. In recent years, it has shown significant potential in anti-tumor recurrence and reversal of drug resistance, such as showing good effects in inhibiting the recurrence of lung cancer, breast cancer, and nasopharyngeal carcinoma. Its anti-tumor mechanism is diverse, including inhibiting proto-oncogenes, activating tumor suppressor genes, inhibiting the nucleic acid metabolism of tumor cells, inhibiting the growth of tumor cells, enhancing the effect of chemotherapy drugs by regulating drug metabolism, changing the tumor microenvironment or affecting the signal pathway of tumor cells, and reversing the drug resistance of tumor cells. However, it still faces some challenges in clinical application, such as poor water solubility and low bioavailability.

[0004] Studies have shown that there is a certain relationship between tumor recurrence and cancer stem cells (CSCs). CSCs are the main culprit for the resistance of malignant tumors to chemotherapy, radiotherapy efficacy, and recurrence. As tumor-initiating cells, CSCs have high tumorigenicity and have self-renewal and differentiation abilities similar to normal stem cells. These malignant characteristics of CSCs are regulated by various factors, including stimuli from the internal tumor microenvironment and triggers from the external environment. By inhibiting the expression or activity of CSCs markers alone or in combination, the growth and dissemination characteristics of CSCs in vitro and in vivo can be effectively reduced, and the malignancy of CSCs can be weakened. Therefore, targeted therapy against CSCs is a feasible means for treating ovarian cancer recurrence.

[0005] Nanodrug delivery systems have broad prospects in biomedical applications due to their functions such as efficient drug loading, targeted drug delivery, and controlled release. Compared with traditional chemotherapeutic drugs, nanodrug delivery systems can utilize the enhanced permeability and retention (EPR) effect caused by the pathological leakage of tumor vasculature and poor lymphatic drainage to preferentially accumulate in tumor tissues, achieving passive targeting. With the continuous progress of nanomedicine, nanodrug delivery systems with active targeting, tumor microenvironment-sensitive drug release, and external energy-responsive drug release developed in recent years have further enhanced their accumulation effect at the site of malignant tumors. Their multi-target action mode has incomparable advantages in inhibiting tumor recurrence, enhancing the efficacy of anti-tumor drugs while reducing their side effects. Among them, polymeric micelles are a commonly used carrier in nanodrug delivery systems. The micelle core can provide a good encapsulation space for hydrophobic drugs, thus playing a solubilization role; the hydrophilic segments of the micelles extend outward to form a solvation shell, reducing the interaction force between micelles, thereby improving the stability of the entire micelle structure.

[0006] The tumor microenvironment (TME) is an important factor affecting tumor growth, invasion, and metastasis, in which reactive oxygen species (ROS) play an important role. ROS are oxygen-containing free radicals produced during cell metabolism, including superoxide anion, hydrogen peroxide, and hydroxyl radicals. In the tumor microenvironment, the level of ROS is usually higher than that in normal tissues, which provides potential targets for tumor treatment.

[0007] The present invention aims to develop a microenvironment-responsive nanomicelle drug delivery system to achieve targeted drug delivery and thus inhibit the recurrence of ovarian cancer. Summary of the Invention

[0008] The purpose of the present invention is to provide a microenvironment-responsive micelle, a preparation method thereof, and its application in the preparation of drugs for inhibiting the recurrence of ovarian cancer to solve the problems existing in the above-mentioned prior art. The microenvironment-responsive micelle can efficiently deliver the chemotherapeutic drug paclitaxel and the regulator curcumol, and by enhancing the uptake of drugs by cancer stem cells, enable the drugs to play roles of physiological stability, long circulation, toxicity reduction, efficacy enhancement, and tumor recurrence inhibition in vivo.

[0009] To achieve the above purpose, the present invention provides the following solutions:

[0010] The present invention provides a preparation method of a microenvironment-responsive micelle, comprising the following steps:

[0011] Dissolve poly(vinylcaprolactam)-poly(vinyl acetate)-poly(ethylene glycol) graft copolymer, D-α-tocopheryl polyethylene glycol succinate, DSPE-PEG 2000 、DSPE-PEG 2000 -TK-PEG 5000, paclitaxel, curcumol and DSPE-PEG 2000 After 2000 -FSHB is dissolved, it is transferred to a rotary evaporation device to evaporate and remove the solvent, forming a thin film;

[0012] The thin film is hydrated with PBS solution, and then extruded with a microporous filter membrane to obtain the microenvironment-responsive micelles;

[0013] The DSPE-PEG 2000 -FSHB is obtained by mixing and reacting DSPE-PEG 2000 -NHS, follicle-stimulating hormone polypeptide FSHB and triethylamine, and then dialysis;

[0014] The DSPE-PEG 2000 -TK-PEG 5000 is obtained by mixing and reacting DSPE-PEG 2000 -NHS, mPEG 5000 -TK-NH 2 and triethylamine.

[0015] Furthermore, the mass ratio of the polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, the D-α-tocopheryl polyethylene glycol succinate, the DSPE-PEG 2000 , the DSPE-PEG 2000 -TK-PEG 5000 , the paclitaxel, the curcumol and the DSPE-PEG 2000 -FSHB is 120:20:2:2:1:2.76:2.

[0016] Furthermore, the solvent is methanol.

[0017] Furthermore, the pore size of the microporous filter membrane is 0.22 μm.

[0018] Furthermore, the mixing reaction for preparing the DSPE-PEG 2000 -FSHB is carried out in DMF for 12 h.

[0019] Furthermore, the mixing reaction for preparing the DSPE-PEG 2000 -TK-PEG 5000 is carried out in chloroform for 0.5 h.

[0020] The present invention also provides a microenvironment-responsive micelle with the effect of inhibiting ovarian cancer recurrence prepared by the above preparation method.

[0021] The present invention also provides the application of the above microenvironment-responsive micelles in the preparation of drugs for inhibiting ovarian cancer recurrence.

[0022] The present invention also provides a drug for inhibiting recurrence of ovarian cancer, and the active ingredient comprises the above-mentioned microenvironment-responsive micelles.

[0023] Furthermore, the drug also comprises pharmaceutically acceptable excipients.

[0024] The present invention discloses the following technical effects:

[0025] The present invention designs a microenvironment-responsive micelle, which can deliver the encapsulated anti-cancer drugs paclitaxel and the traditional Chinese medicine monomer curcumol to the tumor site and achieve drug release, thereby inhibiting recurrence of ovarian cancer. The present invention uses a ketal (TK) that can be cleaved under ROS conditions as an intermediate chain to connect DSPE-PEG 2000 and PEG 5000 , to form a novel amphiphilic polymer (DSPE-PEG 2000 -TK-PEG 5000 ), which can self-assemble into nanoparticles with a diameter less than 100 nm, and can achieve long circulation in vivo and accumulate at the tumor site by the EPR effect. DSPE-PEG 2000 -TK-PEG 5000 can also achieve the stability transition of the nanocarrier, stably exist during the drug delivery process, and become unstable and rapidly release drugs after entering tumor cells. Follicle-stimulating hormone polypeptide FSHB is shielded by the dense PEG hydration layer during blood circulation. Once the TK bond is cleaved under the action of ROS highly expressed in tumor tissues, the long-chain PEG 5000 detaches, exposing the follicle-stimulating hormone polypeptide FSHB modified at the end of the short-chain PEG 2000 , realizing the active targeting of ovarian cancer stem cells (OCSCs) positive for follicle-stimulating hormone receptor FSHR, promoting the uptake of tumor cells, and enhancing the tumor-targeted delivery efficiency of drugs. The endocytosis mediated by FSHR enables the micelles to enter OCSCs and rapidly dissociate, releasing paclitaxel and curcumol to kill tumor cells. The oxidation-responsive "shell removal" strategy is proven to be an effective drug controlled-release method for realizing the transformation of nanodrugs from stealth to sticky. This tumor oxidation-specific biodegradable nanomicelle has potential application prospects for the delivery of chemotherapy drugs. By enhancing the uptake of drugs by tumor stem cells, it can more effectively inhibit recurrence of ovarian cancer. The present invention provides a new strategy for the treatment of tumors, which has important theoretical and clinical significance. Description of the Drawings

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only 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.

[0027] Figure 1 It is the 1H NMR spectrum of DSPE-PEG2000-FSHB;

[0028] Figure 2 It is the 1H NMR spectrum of DSPE-PEG2000-TK-PEG5000;

[0029] Figure 3 It is the transmission electron microscopy image of TK-NMs;

[0030] Figure 4 It is the particle size distribution diagram of TK-NMs;

[0031] Figure 5 It is the Zeta distribution diagram of TK-NMs;

[0032] Figure 6 It is the determination diagram of critical micelle concentration;

[0033] Figure 7 It is the change diagram of particle size and PDI value of TK-NMs acting at 4°C for 30 days;

[0034] Figure 8 It is for TK-NMs in 1 mM H 2 O 2 The particle size change diagram under the action;

[0035] Figure 9 It is for TK-NMs in 1 mM H 2 O 2 The PDI value change diagram under the action;

[0036] Figure 10 It is the particle size change diagram of TK-NMs under the action of three different media;

[0037] Figure 11 It is the PDI value change diagram of TK-NMs under the action of three different media;

[0038] Figure 12 It is the fluorescence microscope image of OCSCs identified by the mouse nestin antibody detection experiment; The scale bar is 25 μm;

[0039] Figure 13 It is the result of flow cytometry detecting the stem cell surface marker CD133;

[0040] Figure 14 Inverted fluorescence microscopy images showing the uptake of different micelles by OCSCs and OCSC spheres; scale bar: 100 μm;

[0041] Figure 15 Results of flow cytometry analysis of the uptake of different micelles by OCSCs and OCSC spheres;

[0042] Figure 16 Images showing the inhibitory effect of different micelles on the growth of OCSCs; scale bar: 100 μm;

[0043] Figure 17 Results of flow cytometry analysis of apoptosis induction of OCSCs by different micelles;

[0044] Figure 18 Microscopic images (A) and quantitative analysis graphs (B) showing the inhibitory effect of different micelles on the migration of OCSCs; scale bar in A: 50 μm;

[0045] Figure 19 Microscopic images (A) and quantitative analysis graphs (B) showing the inhibitory effect of different micelles on the invasion of OCSCs; scale bar in A: 50 μm;

[0046] Figure 20 Microscopic images (A) and quantitative analysis graphs (B) showing the inhibitory effect of different micelles on the mimicry angiogenesis of OCSCs; scale bar in A: 100 μm;

[0047] Figure 21 Microscopic images (A) and quantitative analysis graphs (B) showing the inhibitory effect of different micelles on the wound healing of OCSCs; scale bar in A: 100 μm;

[0048] Figure 22 Statistical graphs showing the P-gp levels in OCSCs after treatment with different micelles;

[0049] Figure 23 Statistical graphs showing the MRP1 levels in OCSCs after treatment with different micelles;

[0050] Figure 24 Statistical graphs showing the BCRP levels in OCSCs after treatment with different micelles;

[0051] Figure 25 Results of in vivo imaging of small animals showing the targeting of different formulations in tumor-bearing mice;

[0052] Figure 26 Representative images of the tumor-bearing mouse model;

[0053] Figure 27 Statistical analysis graphs of the tumor volumes of mice in different formulation groups;

[0054] Figure 28 HE staining images of tumors in relapsed mice after intervention with different preparations; the scale bar is 100 μm;

[0055] Figure 29 Immunofluorescence staining images of relapse-related proteins in tumor tissues; the scale bar is 50 μm. Detailed implementation manners

[0056] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0057] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0058] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0059] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are also obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0060] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0061] Example 1

[0062] I. Experimental instruments and materials

[0063] 1.1 Experimental instruments

[0064] XS105 type one in a hundred thousand balance (Mettler Toledo); DZKW-S-4 electrothermal constant temperature water bath (Medical Instrument Factory No. 5, Shanghai Medical Instrument Company); RE52CS type rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); Ti-S type fluorescence inverted microscope (Nikon Corporation, Japan); 1100 type liquid chromatograph (UV detector, Dalian Elite); C18 chromatographic column (250mm×4.6mm, 5μm, Shanghai Yuexu); Sephadex G-50 dextran gel column (Shanghai Hualan Chemical Technology Co., Ltd.); dialysis bag (Beijing Solarbio Science & Technology Co., Ltd., cut-off molecular weight 2000Da); polycarbonate membrane (Millipore Corporation, USA); 500 An type nano particle size and Zeta potential analyzer (Anton Paar GmbH, Austria); JEM-2000EX transmission electron microscope (JEOL Ltd., Japan); SW-CJ-1D single-person purification workbench (Shanghai Sujing Industrial Co., Ltd.); air-jacketed carbon dioxide cell incubator (Thermo Flesher Scientific (Asheville) LLC, USA); HBS-1096A type microplate reader (Nanjing Detie Experimental Equipment Co., Ltd.); FACSCalibur flow cytometer (BD Biosciences, USA); multispectral in vivo imaging system (Carestream Health, Inc., USA); RM2235 type paraffin slicer (Leica Microsystems GmbH, Germany); confocal microscope (Leica Microsystems GmbH, Germany).

[0065] 1.2 Experimental materials

[0066] Paclitaxel (PTX) (Dalian Meilun Biotechnology Co., Ltd., purity 99%); Curcumol (CC) (Chengdu Gelipu Biotechnology Co., Ltd., purity 98.65%); polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus) (BASF SE, Germany); P-glycoprotein inhibitor D-α-tocopheryl polyethylene glycol succinate (TPGS 1000 ), DSPE-PEG 2000 , DSPE-PEG 2000 -NHS, mPEG 5000 -TK-NH 2 (Xi'an Ruixi Biotechnology Co., Ltd.); 4',6-diamidino-2-phenylindole (DAPI) (Beijing Solarbio Science & Technology Co., Ltd.); EGF, FGF (Peprotech Inc., USA); B27 (Beijing Yiaobang Biotechnology Co., Ltd.); recombinant Alexa 594 Fluorescent Anti-CD133 antibody (Abcam, UK); Nestin antibody (Beijing Protein Innovation Co., Ltd., China); 1,1-Dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) (Nanjing KayGen Biotech Co., Ltd., China). Other reagents were of analytical grade, and water was purified water.

[0067] II. Experimental methods

[0068] 1 Preparation of micelles

[0069] 1.1 Preparation of DSPE-PEG 2000 -FSHB

[0070] Weigh 100 mg of DSPE-PEG 2000 -NHS and dissolve it in 3 mL of DMF. Add follicle-stimulating hormone polypeptide FSHB (110 mg) and triethylamine (300 μL) and dissolve completely. React at room temperature for 12 h. Transfer the reaction solution to a dialysis bag (cut-off molecular weight 2000 Da) and dialyze in pure water for 24 h. Collect the dialysis solution and freeze-dry to obtain the product DSPE-PEG 2000 -FSHB.

[0071] The amino acid sequence of follicle-stimulating hormone polypeptide FSHB is QCHCGKCDSDSTDCT.

[0072] 1.2 Preparation of DSPE-PEG 2000 -TK-PEG 5000

[0073] Weigh 100 mg of DSPE-PEG 2000 -NHS and dissolve it in 5 mL of chloroform. Add m PEG 5000 -TK-NH 2 (100 mg) and triethylamine (300 μL) and dissolve completely. After reacting at room temperature for 0.5 h, concentrate the reaction solution by rotary evaporation under reduced pressure. Precipitate with a large amount of ice ether, filter and collect the precipitate, and dry it under vacuum to obtain DSPE-PEG 2000 -TK-PEG 5000 .

[0074] 1.3 Preparation of microenvironment-responsive micelles

[0075] Prepare microenvironment-responsive micelles by the thin-film dispersion method. Weigh accurately 120 mg of Soluplus, 20 mg of TPGS, 2 mg of DSPE-PEG 2000 、2 mg of DSPE-PEG 2000 -TK-PEG 5000 、1 mg of PTX, 2.76 mg of CC and 2 mg of DSPE-PEG2000 - In a 15 mL centrifuge tube, dissolve the above materials with methanol as the solvent and transfer them to a 50 mL round-bottom flask. Under a water bath at 40 °C, perform rotary evaporation under reduced pressure to remove the solvent. After a thin film forms at the bottom of the round-bottom flask, add 5 mL of PBS solution for hydration, and extrude it twice through a 0.22 μm microporous filter membrane to obtain the microenvironment-responsive micelles (TK-NMs). During the preparation of the micelles, DSPE-PEG is not added. 2000 - TK-PEG 5000 、PTX, CC, and DSPE-PEG 2000 - FSHB to obtain blank micelles; without adding DSPE-PEG 2000 - TK-PEG 5000 and DSPE-PEG 2000 - FSHB to obtain ordinary micelles; without adding DSPE-PEG 2000 - TK-PEG 5000 to obtain FSHB-modified micelles (FSHB-NMs).

[0076] 1.4 Preparation of DiR micelles

[0077] DiR micelles were prepared using the same method as above, where PTX and CC were replaced with DiR.

[0078] 2 Characterization of micelles

[0079] 2.1 Detection of encapsulation efficiency and drug loading

[0080] Take 1 mL of TK-NMs, mix it evenly with methanol according to a volume ratio of 1:4, weigh it, ultrasonicate for 2 min, then let it stand for 30 min, reweigh it, and make up the weight difference before and after with methanol. Then centrifuge (rotation speed: 9000 r / min) for 2 min, take the supernatant, filter it twice through a 0.45 μm microporous filter membrane to remove impurities, and set it aside. The chromatographic system uses an Elite 1100 type high-performance liquid chromatography. The chromatographic column is C18 (4.6 mm × 250 mm, 5 μm). The mobile phase for paclitaxel: water, methanol, and acetonitrile, and the volume ratio of the three liquids is 30:28:42. The flow rate of the mobile phase is 1 mL / min; the detection wavelength is 227 nm, the injection volume each time is 20 μL, and the column temperature is 30 °C. The mobile phase for curcumol: acetonitrile and 0.1% phosphoric acid, and the volume ratio of the two liquids is 66:34. The flow rate of the mobile phase is 1 mL / min, the detection wavelength is 210 nm, the injection volume each time is 20 μL, and the column temperature is 30 °C. Calculate the encapsulation efficiency (EE%) and drug loading (DL%) of the drug in the micelles according to the following formula:

[0081] EE(%) = peak area of the drug after passing through the column / peak area of the drug before passing through the column × 100%;

[0082] DL(%) = (total drug mass - unencapsulated drug mass) / total micelle mass × 100%.

[0083] 2.2 Determination of particle size and zeta potential of microenvironment-responsive micelles

[0084] Take 1 mL of micelles and use a 500 An-type nano-particle size and zeta potential analyzer to measure the particle size, polydispersity coefficient, and zeta potential of different micelles by dynamic light scattering method. Each preparation is measured 3 times, and each time 6 measurements are set in a cycle.

[0085] 2.3 Observation of the morphology of microenvironment-responsive micelles by transmission electron microscopy

[0086] Observe the morphology of microenvironment-responsive micelles by transmission electron microscopy. Dilute an appropriate amount of micelles to a suitable concentration, drop one drop on a special copper grid for electron microscopy, stain with 2% phosphotungstic acid, and observe its morphology by transmission electron microscopy after air drying. The accelerating voltage of the transmission electron microscope is set to 120 kV.

[0087] 2.4 Determination of critical micelle concentration

[0088] Use pyrene as a fluorescence probe to determine the CMC of micelles (excitation wavelength 334 nm, emission wavelength 340 - 450 nm, excitation and emission slit widths are both 3.0 nm).

[0089] Precisely weigh 40.4 mg of pyrene and place it in a 10 mL volumetric flask, make up the volume with acetone, and then dilute it 100 times to obtain a stock solution of 2×10 - 4 mol / L. Prepare Blank-NMs into solutions with concentrations of 1×10 -6 , 2.5×10 -6 , 5×10 -6 , 1×10 -5 , 2.5×10 -5 , 5×10 -5 , 1×10 -4 , 2.5×10 -4 , 5×10 -4 , 1×10 -3 , 2.5×10 -3 , 5×10 -3 mg / mL. Add 80 μL of the pyrene stock solution to a test tube, and let it dry naturally overnight in the dark. Pipette 8 mL of each of the above-mentioned micelle solutions with different concentrations into the test tube, so that the final concentration of pyrene is 2×10 -6 mol / L. Seal the test tube and shake it gently to mix evenly. Let it stand overnight in the dark. Filter the liquid in the test tube through a 0.22 μm microporous filter membrane, add 200 μL to each well of a 96-well plate, and measure the fluorescence values at wavelengths of 372 and 383 nm with a fluorescence spectrometer. Take I 372 / I 384The critical aggregation concentration was calculated by cross - plotting the logarithm of the sample concentration C (mg / mL), logC.

[0090] 2.5 Evaluation of micelle stability

[0091] Three batches of microenvironment - responsive micelles were prepared in vials and sealed. The sealed micelles were stored in a 4 °C refrigerator. Samples were taken at 5, 10, 15, 20, 25, and 30 days to investigate the particle size and PDI value of the micelles.

[0092] 2.6 Evaluation of ROS responsiveness of microenvironment - responsive micelles

[0093] Hydrogen peroxide (H 2 O 2 ) was used to simulate the high - level ROS environment in tumor sites. Two portions of TK - NMs solution were prepared with PBS buffer. One portion of FSHB - NMs solution was prepared with PBS buffer. An appropriate amount of hydrogen peroxide (H 2 O 2 ) was added to one portion of the TK - NMs PBS solution to make the concentration of H 2 O 2 in the solution 1 mM; the other solution was diluted with an equal volume of pure water as a control group. At different time points after adding hydrogen peroxide, the particle size change of the micelles was monitored using a dynamic light scattering instrument, and 3 parallel experiments were set for each group.

[0094] 2.7 Evaluation of serum stability of microenvironment - responsive micelles

[0095] Take 3 mL of TK - NMs and add it to PBS (pH 7.4), PBS containing 10% FBS (pH 7.4), and DMEM culture medium containing 10% FBS at a volume ratio of 1:10. Incubate under constant temperature water bath conditions at 37 °C. Take 1 mL at 0, 2, 4, 6, 8, 12, 24, and 48 h, and analyze the changes in the particle size and PDI value of the micelles using a dynamic light scattering instrument to evaluate its serum stability.

[0096] 3 Evaluation of in vitro targeting of microenvironment - responsive micelles

[0097] 3.1 Culture of ID8 cells and stem cells

[0098] Mouse ovarian epithelial cancer cells (ID8) were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin), placed in a cell culture incubator at 37 °C, 5% CO 2 - 95% air, and passaged when the confluence reached 90%.

[0099] ID8 cells in the logarithmic growth phase were placed in DMEM-F12 medium containing 2% B27, 10% basic fibroblast growth factor, 20 ng / mL bFGF epidermal growth factor, 100 U / mL penicillin and 100 μg / mL streptomycin, and incubated in an incubator at 37°C and 5% CO 2 The cells were incubated in an incubator at 37°C and 5% CO₂. The medium was changed by half every 2 days, and mature stem cell spheres (OCSCs spheres) could be formed in 1-2 weeks. The mature stem cell spheres were transferred back to normal DMEM medium for culture to obtain OCSCs cells.

[0100] 3.2 Stem cell identification

[0101] The stem cell surface marker Nestin was observed by fluorescence inverted microscope. ID8 cells, OCSCs cells, and OCSCs spheres in the logarithmic growth phase were inoculated into 24-well plates and cultured in an incubator. After 12 h, the medium was discarded and the cells were fixed with 4% paraformaldehyde for 15 min. The cells were washed 3 times with PBS and incubated with 1% Triton X-100 at 37°C for 30 min. The Nestin antibody was diluted with 1% BSA and incubated overnight at 4°C. Fluorescein isothiocyanate (FITC)-labeled secondary antibody (dilution ratio 1:300) was added and incubated at 37°C in the dark for 1 h. After washing with PBS, images were taken with a fluorescence inverted microscope.

[0102] The stem cell surface marker CD133 was detected by flow cytometry. ID8 cells, OCSCs cells, and OCSCs spheres in the logarithmic growth phase were collected by centrifugation. The cells were fixed with 80% paraformaldehyde for 5 min, and the paraformaldehyde was removed by centrifugation. The cells were washed 3 times with PBS. The cells were permeabilized with 1% Triton X-100 at 37°C for 30 min and washed 3 times with PBS. The cells were blocked with 5% BSA for 1 h, and the fluorescent Anti-CD133 antibody was added and incubated overnight at 4°C. After washing with PBS, 300 μL PBS was added, and the cells were detected by flow cytometry.

[0103] 3.3 Ovarian cancer stem cell uptake experiment

[0104] The uptake of different micelles in OCSCs cells and OCSCs spheres was qualitatively analyzed by fluorescence inverted microscope. The cells in each group were inoculated into 24-well plates for culture, and 3 parallel replicates were set. After 24 h of culture, each group of cells was incubated with Cou-NMs, FSHB-Cou-NMs, and TK-Cou-NMs+H 2 O 2 (coumarin concentration 2 μM) for 2 h. After washing 3 times with PBS buffer, the cells were fixed with 4% paraformaldehyde for 10 min, and the cell nuclei were stained with DAPI staining solution in the dark for 6 min. The distribution of fluorescence in the cells was observed by fluorescence inverted microscope.

[0105] Flow cytometry was used to quantitatively analyze the uptake of micelles by cells in each group. Cells were treated in the same way. After washing the cells 3 times with PBS buffer, the cells were digested with trypsin and centrifuged, resuspended in 300 μL of PBS, and the average fluorescence intensity of coumarin taken up by the cells was quantitatively measured by FACScan flow cytometry.

[0106] 4 Ability of different micelles to induce apoptosis of ovarian cancer stem cells

[0107] OCSCs were passaged into 24-well plates and cultured overnight. After the cell state was stable, the medium was replaced by half and each group of micelles was added. After 4 days, the state of the stem cell spheres was observed under bright field of an inverted microscope and photographed.

[0108] The effects of each group of micelles on the apoptosis of OCSCs were detected by flow cytometry. OCSCs were seeded in 6-well plates at a density of 1×10 5 / well. When they grew to 70%, each group of micelles was added. After culturing for 48 h, the cells were digested with 0.25% trypsin, collected, washed with PBS, and the supernatant was discarded. According to the instructions, the cells were suspended in Binding Buffer, and double-stained with Annexin V-FITC and PI. The reaction was carried out at room temperature in the dark for 15 min and the apoptosis rate of the cells was detected by the machine within 1 h.

[0109] Calculation formula: Apoptosis rate = (number of early apoptotic cells + number of late apoptotic cells) / total number of cells × 100%.

[0110] 5 Ability of different micelles to inhibit the invasion and migration of OCSCs in vitro

[0111] 5.1 Stem cell migration experiment

[0112] Take a Transwell co-culture chamber. Add 600 μL of DMEM medium containing FBS to the lower chamber, and add 20 μL of drugs with different formulations and 180 μL of cell suspension resuspended in DMEM medium without FBS to the upper chamber. The cell density is 2×10 4 cells / well, and 3 replicate wells are set for each group. After continuing to culture for 24 h, the medium in the upper chamber was aspirated and the cells in the upper chamber were washed with PBS. The stem cells that did not migrate inside the chamber were gently wiped clean with a cotton swab. Take a new lower chamber and add 700 μL of 4% paraformaldehyde. The chamber was placed and fixed for 15 min, stained with 0.5% crystal violet solution at room temperature for 15 min, the unbound crystal violet was washed away with PBS, dried at room temperature, and observed and photographed under an inverted microscope. The cells in the photographed pictures were counted using Image J software. The calculation formula for cell migration rate is as follows:

[0113] Migration rate (%) = number of cells in the formulation group / number of cells in the blank control group × 100%.

[0114] 5.2 Stem cell invasion experiment

[0115] Under pre-cooling conditions, Matrigel was coated on Transwell chambers (Matrigel was diluted 1:4 with serum-free medium), and then 180 μL of the stem cell suspension was added to the chambers at a density of 3×10 4 cells / mL. Other steps were the same as those in the stem cell migration experiment.

[0116] Invasion rate (%) = number of cells in the formulation group / number of cells in the blank control group × 100%.

[0117] 5.3 Experiment on the inhibition of VM channel formation in OCSCs cells by different micelles

[0118] Add 50 μL of Matrigel to each well of a 96-well plate, shake well to ensure no bubbles, and immediately place the 96-well plate in a 37 °C cell incubator for 30 min to ensure the Matrigel solidifies. Inoculate ovarian cancer stem cells into the above 96-well plate at a concentration of 1.5×10 4 cells / well, and add micelles with different formulations to each well. Continue to incubate for 4 h, and observe the damage to mimic angiogenesis by different micelles under an inverted microscope. Use Image J software to count the nodes, junctions, branches, and segments of the mimic blood vessels in the captured pictures.

[0119] 5.4 Experiment on the inhibition of wound healing ability of OCSCs cells by different micelles

[0120] OCSCs cells were inoculated into a 6-well plate at a concentration of 1×10 5 / well. After incubation until the growth reached 80%, two parallel straight lines were gently scratched with the tip of a 20 μL sterile pipette in a laminar flow hood. Wash with PBS to remove cell debris, change to fresh medium, and add different micelles. Set 3 replicates for each group. Observe and photograph at 0 h, 12 h, and 24 h after drug administration using an inverted microscope. Use Image J software to statistically analyze the scratch area. The formula for calculating the wound healing rate: Wound healing rate (%) = (1 - A t / A 0 ) × 100%, where A t represents the scratch area measured at 12 h or 24 h, and A 0 represents the scratch area measured at 0 h.

[0121] 6 ELISA experiment to determine the expression of proteins related to tumor recurrence

[0122] Inoculate OCSCs into a 25 cm 2When the cells in the culture flask reached 80% confluence, different drug-loaded micelles were added respectively, and blank micelles were used as the blank control group. After 24 h, the cells were washed 3 times with cold PBS, and then 800 μL of cell lysate was added to each group to completely lyse the cells. The supernatant was collected by a refrigerated centrifuge at 4 °C. According to the ELISA kit instructions, the contents of P-gp, MRP-1, and BCRP in each sample were measured at 450 nm.

[0123] 7 Evaluation of in vivo targeting of different micelles

[0124] 7.1 Establishment of tumor-bearing mouse model

[0125] A tumor-bearing mouse model was established by injecting OCSCs subaxillary. 1.5×10 6 OCSCs were suspended in 200 μL of PBS, and the cell suspension was inoculated into the left anterior limb axilla of C57BL / 6 mice. The growth of the axillary tumors in the mice was observed daily, and the tumor size was measured every other day with a vernier caliper. When the tumor volume reached about 200 - 300 mm 3 it could be used for subsequent experiments.

[0126] 7.2 In vivo targeting study

[0127] In vivo targeting of different formulations was investigated by in vivo imaging experiments. The drug was replaced with the fluorescent reagent DiR. The tumor-bearing mice were divided into the following groups: (1) normal saline (Control); (2) free DiR (Free DiR); (3) DiR micelles (DiR-NMs); (4) follicle-stimulating hormone polypeptide-modified DiR micelles (FSHB-DiR-NMs); (5) microenvironment-responsive micelles (TK-DiR-NMs). The DiR dose in each administration group was the same, 2 μg / mouse. The mice were anesthetized with isoflurane, and the fluorescence signals in the mice were monitored using an in vivo imaging system. Fluorescence imaging was performed on the mice at different time points (3, 6, 9, 12, 24, 48, 72, 84 h) to observe the intensity of the fluorescence signals in vivo.

[0128] 8 Evaluation of the effect of different micelles on inhibiting ovarian cancer recurrence

[0129] 8.1 Grouping of tumor-bearing mice and intervention strategy

[0130] The method for establishing the tumor-bearing mouse model is described in detail in 7.1. The growth of the axillary tumors in the mice was observed daily, and the tumor size was measured every other day with a vernier caliper. When the tumor volume reached about 400 mm 3When performing tumor resection surgery on mice anesthetized with 7% chloral hydrate, 90% of the tumor tissue was resected and sutured layer by layer. The tumor-bearing mice were divided into: (1) normal saline (Blank Control); (2) free drug (Free PTX / CC); (3) ordinary micelles (PTX / CC-NMs); (4) follicle-stimulating hormone polypeptide-modified micelles (FSHB-NMs); (5) microenvironment-responsive micelles (TK-NMs). The above different preparations were respectively injected via the tail vein, once every 2 days for 8 consecutive times. The tumor size was measured at 4, 8, 12, and 16 days after surgery to evaluate the effect of different preparations on tumor recurrence in mice. The tumor volume calculation formula is: V(mm 3 ) = L × W 2 / 2 (where L and W are the length and width of the tumor, respectively).

[0131] On the 16th day after surgery, the mice were euthanized, and the tumor tissue was excised and embedded in paraffin. Each tissue was cut into 5-μm thick slices using a paraffin slicer. H&E staining of the tumor tissue was used to evaluate the destructive effect of different micelles on the axillary tumor tissue of tumor-bearing mice. Immunofluorescence was used to detect the expression levels of proteins Nestin, CD133, and ALDH1 related to tumor recurrence.

[0132] 9 Statistical analysis

[0133] Statistical analysis was performed using Graphpad Prism 9.0 software. The experimental data were all expressed as Mean ± SD. One-way ANOVA was used for comparison among multiple groups, and further pairwise comparison was performed using the LSD-t test. P < 0.05 indicated that the difference was statistically significant.

[0134] III. Results

[0135] 1. Characterization of micelles

[0136] The nuclear magnetic resonance hydrogen spectrum of the targeting molecule DSPE-PEG 2000 -FSHB is as Figure 1 shown. The characteristic peaks of the FSHB polypeptide are clearly visible in the spectrum, proving the successful synthesis of the targeting molecule DSPE-PEG 2000 -FSHB. Figure 2 This is the nuclear magnetic resonance hydrogen spectrum of DSPE-PEG 2000 -TK-PEG 5000 , proving the successful synthesis of DSPE-PEG 2000 -TK-PEG 5000 . The transmission electron microscope photograph of the microenvironment-responsive micelles ( Figure 3 ) shows that the morphology of TK-NMs is spherical, the surface is smooth, and the particle size is approximately 80 nm. The particle size and Zeta potential of the micelles were measured using a nanoparticle size and Zeta potential analyzer, and the results are shown in Figures 4 - 5。The particle size of TK-NMs was (78.04 ± 0.99) nm, and the polydispersity index was (PDI, 0.07 ± 0.01), indicating that the particle size was uniform, the dispersibility was good, there was no agglomeration phenomenon, and the particle size met the experimental expectations. The encapsulation efficiency and drug loading results of PTX and CC in TK-NMs are shown in Table 1. The encapsulation efficiency of PTX was (87.732 ± 1.617)%, and the encapsulation efficiency of CC was (79.914 ± 2.527)%; the drug loading of PTX was (0.586 ± 0.009)%, and the drug loading of CC was (1.455 ± 0.037)%. The micelles had a low critical micelle concentration. As Figure 6 shown, the critical micelle concentration was 12.078 mg / L, indicating that the micelles had strong anti-dilution ability and could avoid damaging their integrity due to body fluid dilution after entering the body, and had good dilution stability. Figure 7 Figure 4 shows the stability evaluation results of TK-NMs stored at 4 °C for 30 days. The particle size did not change significantly, and the PDI was less than 0.15, indicating that TK-NMs could be stably stored at 4 °C for at least 30 days.

[0137] Table 1 Encapsulation efficiency and drug loading of ROS-sensitive long-circulating targeted micelles

[0138]

[0139]

[0140] To evaluate the response ability of DSPE-PEG 2000 -TK-PEG 5000 to reactive oxygen species (ROS), the present invention detected the changes in the particle size and PDI values of TK-NMs in a 1 mM H 2 O 2 environment. The results are shown in Figures 8 - 9 Figure 5. As the incubation time extended, the particle size of TK-NMs in the H 2 O 2 group gradually decreased from the initial 79.45 ± 1.78 nm to 72.10 ± 1.59 nm after 4 h of incubation, approaching the particle size of the FSHB-NMs group, while the particle size of TK-NMs in the PBS group did not change significantly. It can be seen that the addition of hydrogen peroxide had a great impact on the particle size change of TK-NMs. This was because the TK bond had a strong ROS responsiveness and could be rapidly oxidized and broken in a ROS environment, causing the hydration film on the outer layer of the micelles to fall off, resulting in particle size shrinkage. This result indicated that TK-NMs had strong ROS response ability.

[0141] The present invention evaluated the stability of TK-NMs in three different media, as Figures 10 - 11As shown, the micelles were relatively stable in PBS solution within 48 h, with small changes in particle size and PDI value, meeting the requirements for intravenous injection. In the PBS + 10% serum group and the DMEM medium + 10% serum group, the particle size, PDI value, and zeta potential of the micelles increased over time. However, within 8 h, the particle size was still less than 100 nm and the PDI was less than 0.2, indicating that they still had a long-circulation effect within 8 h, which was beneficial for the passive targeting of micelles to the tumor site. The changes were more obvious after 24 h, which might be due to the aggregation of various proteins in the serum affecting the particle size and PDI of the micelles.

[0142] 2. Stem cell identification

[0143] Nestin is an intermediate cytoskeletal protein and mainly serves as a marker of pluripotent stem cells, participating in tissue development and regeneration. In many solid tumors, including ovarian cancer, Nestin is highly expressed, which is associated with tumor recurrence and poor prognosis. The expression of Nestin in OCSCs was identified by immunofluorescence staining with a mouse nestin antibody, and the results are as Figures 12 - 13 shown. Figure 12 The intensity of green fluorescence in the figure represents the fluorescence intensity of Nestin expression. The order of Nestin expression levels in cells was: OCSCs spheres > OCSCs > ID8 cells > blank control.

[0144] CD133 (Prominin-1) is a pentaspan transmembrane glycoprotein and is one of the recognized tumor stem cell markers. It is expressed on the membranes of various stem cells and tumor cells, participating in the regulation of cell polarity, migration, and cell-cell interaction, and is associated with tumor recurrence in many solid tumors. Detecting the distribution and abundance of CD133-positive cells in tumor stem cells or tumor sections can reflect the status of tumor stem cells. The order of the expression levels of the stem cell surface marker CD133 detected by flow cytometry was: OCSCs spheres > OCSCs > ID8 cells > blank control.

[0145] 3. Evaluation of in vitro targeting of micelles

[0146] Follicle-stimulating hormone receptor (FSHR) is present in the placenta, uterus, prostate, bone tissue, and ovarian tissue. FSHR has also been found to be selectively highly expressed on the surface of many tumor neovascular endothelial cells and is related to tumor recurrence. Some studies have shown that FSHR is highly expressed in ID8 stem cells. Figure 14Fluorescence images of the uptake of different micelles by OCSCs and OCSC spheres, showing the intracellular distribution of different coumarin micelles. The fluorescence signal of the micelles modified with FSHB (FSHB-Cou-NMs) was significantly stronger than that of the ordinary micelle group (Cou-NMs), indicating that FSHB-Cou-NMs can specifically target ovarian cancer stem cells highly expressing FSHR, and enhance the active uptake of micelles by cells through the endocytosis mediated by the specific binding of follicle-stimulating hormone polypeptide to follicle-stimulating hormone receptor. The measurement results of flow cytometry ( Figure 15 ) verified the above conclusion.

[0147] 4. Evaluation of the inhibitory effect of different micelles on the growth of OCSCs and their apoptotic induction

[0148] Cancer stem cells have become important drug treatment targets due to their significant self-renewal ability and characteristics of promoting tumor development, and are closely related to tumor drug resistance and recurrence. To evaluate the effect of TK-NMs nanomaterials on the stem cell characteristics of ovarian cancer stem cells, the ability and efficiency of single stem cells to form cell clusters were observed through sphere formation assays, so as to evaluate the in vitro regeneration potential of these cells. The experimental results ( Figure 16 ) showed that after 4 days of culture, compared with the control group, each micelle group had a certain inhibitory effect on the growth of OCSCs, and the number and volume of cell clusters formed by ovarian cancer stem cells treated with the TK-NMs and FSHB-NMs groups decreased most significantly.

[0149] The apoptotic results of OCSCs under the action of different micelles were detected by flow cytometry after double staining with AnnexinV-FITC. Normal living cells were lightly stained with both Annexin V and PI; apoptotic cells were highly stained with AnnexinV and lightly stained with PI; necrotic cells were highly stained with both Annexin V and PI. Figure 17 These are the results detected by flow cytometer. The proportions of total apoptotic cells in the CC-NMs group, PTX-NMs group, PTX / CC-NMs group, FSHB-NMs group, TK-NMs group and TK-NMs+H 2 O 2 group were 16.52%, 24.07%, 35.51%, 51.40%, 38.03% and 52.03% respectively, all significantly higher than 5.74% of the control group. This indicates that each administration group can induce apoptosis of OCSCs. The difference between the PTX-NMs group and the PTX / CC-NMs group confirmed that CC can synergistically increase the ability of paclitaxel to induce apoptosis. The difference between the PTX / CC-NMs group and the FSHB-NMs group was due to the endocytosis mediated by the specific binding of follicle-stimulating hormone polypeptide to follicle-stimulating hormone receptor, which resulted in more uptake of FSHB-NMs by OCSCs. TK-NMs+H 2 O 2The apoptosis ratio of the group was close to that of the FSHB-NMs group and significantly higher than that of the TK-NMs group. This was because under the action of ROS, the TK bond was broken, and the dense PEG hydration layer fell off, exposing the follicle-stimulating hormone polypeptide, achieving active targeting of follicle-stimulating hormone receptor-positive OCSCs and promoting the uptake of drugs by tumor stem cells.

[0150] 5. Ability of micelles to inhibit the invasion and migration of OCSCs in vitro

[0151] The Transwell experiment evaluated the inhibitory effect on the invasion and migration ability of OCSCs after treatment with different micelle groups. Figure 18 and Figure 19 are the images of ovarian cancer stem cells in each group after passing through the Transwell chamber after administration of different micelle groups, Figure 18 is the result of inhibiting stem cell migration, Figure 19 is the result of inhibiting stem cell invasion. As Figure 18 and Figure 19 shown, there were a large number of cells passing through the chamber in the BlankControl group, indicating that OCSCs have strong migration and invasion abilities. Compared with the BlankControl group, the number of cells passing through the Transwell chamber in each administration group was significantly reduced. Among them, the TK-NMs+H 2 O 2 group had the most significant effect on inhibiting the migration and invasion of OCSCs.

[0152] Figure 20 shows the destructive effect of different micelles on the mimic vascular (VM) channels. An obvious channel composed of cell elongation had been formed in the Blank Control group, and the grid was complete and the grid area was large. The VM channels formed by OCSCs treated with different micelle groups were damaged to varying degrees, mainly manifested as the formed VM channels being short and fragmented, the integrity of the grid being poor or no grid being formed, and the area of a single grid being small. The FSHB-NMs group and the TK-NMs+H 2 O 2 group could significantly inhibit the formation of VM channels.

[0153] The scratch images of each micelle administration group at different time points are as Figure 21 shown. After 24 h of administration, the scratch in the Blank Control group was nearly healed. The scratch width of each administration group was reduced compared with the scratch width at 0 h. The results showed that different micelles had a certain inhibitory effect on scratch healing. Among them, the FSHB-NMs group and the TK-NMs+H 2 O 2 group had the most obvious inhibitory degree.

[0154] 6. ELISA experiment to determine the expression of proteins related to tumor recurrence

[0155] The recurrence of tumors is closely related to the multidrug resistance of tumor cells. Multidrug resistance is a major obstacle in tumor treatment, which enables tumor cells to develop resistance to a variety of chemotherapeutic drugs with different structures and mechanisms of action, resulting in poor chemotherapy efficacy and promoting the recurrence and metastasis of tumors. Efflux transporters play an important role in the occurrence of multidrug resistance. P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP) are three efflux transporters, which belong to the members of the ATP-binding cassette (ABC) transporter superfamily. Their main function is to transport various endogenous and exogenous substances, including many anti-tumor drugs, from inside the cell to outside the cell, resulting in the inability of drugs to accumulate effectively to sufficient concentrations, thereby enabling tumor cells to develop resistance to chemotherapeutic drugs and further promoting the recurrence of tumors. Related studies have shown that P-gp and BCRP are two important mechanisms of ID8 cell resistance and play a key role in maintaining the stem cell characteristics and chemotherapy resistance of ID8 cells. The results of ELISA experiments are as Figures 22 - 24 shown. Due to the drug resistance of OCSCs, the expression levels of p-gp, MRP1, and BCRP in OCSCs are higher than those in ID8 cells. The FSHB-NMs and TK-NMs+H 2 O 2 groups can significantly down-regulate the expression levels of P-gp, MRP, and BCRP in OCSCs.

[0156] 7. Evaluation of in vivo targeting of micelles

[0157] An in vivo fluorescence imaging analyzer was used to observe the in vivo distribution patterns of different preparations at different time periods. The results are shown in Figure 25 . No fluorescence signal was detected in the blank group. The fluorescence signal of the Free DiR group was mainly concentrated in the liver region of mice. The fluorescence signal of the micelle group was distributed throughout the body and accumulated more in the tumor site. As time increased, the fluorescence signal intensity of each group decreased. In the Free DiR group of mice, a weak fluorescence signal could be observed in the tumor site during 3 - 9 h, and no fluorescence signal could be observed after 12 h. In different micelle groups of mice, obvious fluorescence signals could be observed in the tumor site between 3 - 84 h, and the TK-DiR-NMs group of mice had the strongest fluorescence signal at each time point in the tumor site. The order of DiR fluorescence intensity at different time points was as follows: TK-DiR-NMs > FSHB-DiR-NMs > DiR-NMs > Free DiR. The results of this experiment indicate that TK-DiR-NMs has better tumor targeting and long-circulation effects.

[0158] 8. Evaluation of the effect of different micelles on inhibiting the recurrence of ovarian cancer

[0159] According toFigure 26 It can be seen from the observation results that tumor recurrence occurred in the mice of the blank control group after the operation. The tumor recurrence rate in other treatment groups decreased significantly, and the growth rate of the recurrent tumors also slowed down significantly. The effects of inhibiting tumor recurrence in each micelle group were better than those in the free drug group, and the TK-NMs group showed the strongest inhibitory effect.

[0160] It can be seen from Figure 27 that after the intervention of different preparations, the recurrence of tumors can be inhibited. Among them, the inhibitory effect of each micelle group on tumor recurrence was significantly better than that of Free PTX / CC; and the inhibitory effect of TK-NMs was the best. The order of tumor size in tumor-bearing mice of different administration groups was: Blank Control>Free PTX / CC>PTX / CC-NMs>FSHB-NMs>TK-NMs. The H&E staining results of tumor tissues in different preparation groups are shown in Figure 28 . In the tumor tissues of the mice in the blank group, the cell arrangement was uniform and neat, and the cell and cell nucleus morphology were full; after treatment with other preparations, varying degrees of necrosis occurred in each group of cells, and the TK-NMs group caused the most serious damage to the tumor cells, with a large number of cell shrinkages and extensive necrosis and apoptosis.

[0161] The three protein markers Nestin, CD133, and ALDH1 are involved in the key characteristics of cancer stem cells and may play an important role in the recurrence of ovarian cancer tumors. Detecting their expression in tumor sections by immunofluorescence staining helps to evaluate the recurrence risk of tumors. As Figure 29 shown in the immunofluorescence staining for evaluating the expression of recurrence-related proteins in tumor tissues. The fluorescence intensity of the blank control group was the strongest, and the fluorescence intensity of each micelle group decreased, indicating a decrease in the expression of related protein markers, suggesting a reduction in the characteristics of cancer stem cells and the recurrence risk. Among them, the fluorescence intensity of the TK-NMs group was the weakest, indicating that the microenvironment-responsive micelles had the strongest regulatory effect on the characteristics of cancer stem cells.

[0162] In summary, it can be seen from the above implementation examples that the microenvironment-responsive micelle TK-NMs showed strong anti-cancer activity in 2D cancer stem cells and 3D cancer stem cell spheres in vitro, significantly inhibiting the invasion and migration of tumor cells; in the in vivo mouse tumor recurrence model, TK-NMs showed good anti-tumor recurrence effects. The step-by-step "passive targeting" and "active targeting" of microenvironment-responsive micelles can effectively achieve the drug-controlled release method of the transformation of nano-drugs from stealth to adhesion, significantly promoting the uptake of drugs by cancer stem cells, achieving physiological stability, long circulation, and attenuation of toxicity and enhancement of efficacy of drugs in vivo, and inhibiting tumor growth and recurrence.

[0163] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a microenvironment-responsive micelle having an effect of inhibiting the recurrence of ovarian cancer, characterized in that: The following steps are involved: Polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, D-α-vitamin E polyethylene glycol succinate, DSPE-PEG 2000 , DSPE-PEG 2000 -TK-PEG 5000 , paclitaxel, curcumol and DSPE-PEG 2000 -After FSHB is dissolved, it is transferred to a rotary evaporation device to evaporate the solvent to form a thin film; The film is hydrated with a PBS solution, and then extruded with a microporous filter membrane to obtain the microenvironment-responsive micelles; The DSPE-PEG 2000 -FSHB is DSPE-PEG 2000 -NHS, follicle stimulating hormone polypeptide FSHB and triethylamine are mixed and dialyzed to obtain; The DSPE-PEG 2000 -TK-PEG 5000 DSPE-PEG 2000 -NHS, mPEG 5000 -TK-NH2 and triethylamine are mixed to obtain; The polyethylene caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, the D-α-vitamin E polyethylene glycol succinate, the DSPE-PEG 2000 , the DSPE-PEG 2000 -TK-PEG 5000 , the paclitaxel, the curcumol and the DSPE-PEG 2000 -The mass ratio of FSHB is 120:20:2:2:1:2.76:2; The TK is thioketal.

2. The preparation method according to claim 1, characterized in that: The solvent is methanol.

3. The preparation method according to claim 1, characterized in that: The pore size of the microporous filter membrane is 0.22 μm.

4. The preparation method according to claim 1, characterized in that: Preparation of the DSPE-PEG 2000 The mixed reaction of -FSHB was carried out in DMF for 12 h.

5. The preparation method according to claim 1, characterized in that: Preparation of the DSPE-PEG 2000 -TK-PEG 5000 The mixed reaction was carried out in chloroform for 0.5 h.

6. A microenvironment-responsive micelle having an ovarian cancer recurrence-inhibiting effect prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the microenvironment-responsive micelle according to claim 6 in the preparation of a drug for inhibiting the recurrence of ovarian cancer.

8. A drug for inhibiting the recurrence of ovarian cancer, characterized in that: The active ingredient comprises the microenvironment-responsive micelle according to claim 6.

9. The drug according to claim 8, characterized in that The drug also includes pharmaceutically acceptable excipients.

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