Microenvironment response type micelle, preparation method and application of microenvironment response type micelle in preparation of medicine for inhibiting ovarian cancer recurrence

Abstract

The invention discloses a microenvironment response type micelle, a preparation method and application of the microenvironment response type micelle in preparation of a medicine for inhibiting ovarian cancer recurrence, and relates to the technical field of pharmaceutical preparations. The preparation method comprises the following steps: dissolving a polyethylene caprolactam-polyvinyl acetate-polyethylene glycol grafted copolymer, D-alpha-vitamin E polyethylene glycol succinate, DSPE-PEG2000, DSPE-PEG2000-TK-PEG5000, paclitaxel, curcumenol and DSPE-PEG2000-FSHB by using a solvent, transferring the solution into rotary evaporation equipment, and evaporating to remove the solvent so as to form a layer of film; carrying out hydration treatment on the thin film by adopting a PBS (Phosphate Buffer Solution), and then carrying out extrusion treatment by adopting a microfiltration membrane, so as to obtain the microenvironment response type micelle. According to the microenvironment response type micelle, the drug can play a role in inhibiting tumor recurrence in vivo by enhancing the drug uptake of tumor stem cells.

Description

Technical Field

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

[0002] Ovarian cancer is a fatal gynecological malignancy. Because the ovaries are located deep in the pelvic cavity, there are usually no typical clinical symptoms in the early stages of ovarian cancer, and most patients are already in the late stages when diagnosed. The standard treatment for ovarian cancer includes first-line chemotherapy (carboplatin-paclitaxel regimen) and surgical tumor reduction. Although this regimen was initially effective, its clinical treatment remains a great challenge 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 mortality rate of patients within five years is as high as 70%. Therefore, ovarian cancer treatment urgently needs to solve the problem of recurrence.

[0003] Curcumol is the anticancer active component of the traditional Chinese medicine Curcuma zedoariae. In recent years, it has shown significant potential in anti-tumor recurrence and reversal of drug resistance, such as good effects in inhibiting the recurrence of lung cancer, breast cancer, and nasopharyngeal carcinoma. Its anti-tumor mechanism of action is diverse, including inhibiting proto-oncogenes, activating tumor suppressor genes, inhibiting tumor cell nucleic acid metabolism, inhibiting tumor cell growth, enhancing the effect of chemotherapy drugs by regulating drug metabolism, changing the tumor microenvironment, or affecting the signaling pathways of tumor cells, and reversing tumor cell resistance. 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 culprit for malignant tumors to resist chemotherapy, radiotherapy and relapse. As tumor-initiating cells, CSCs are highly tumorigenic and have self-renewal and differentiation abilities similar to normal stem cells. These malignant characteristics of CSCs are regulated by multiple factors, including stimulation from the internal microenvironment of the tumor and triggering from the external environment. Inhibiting the expression or activity of CSCs markers alone or in combination can effectively reduce the growth and dissemination characteristics of CSCs in vitro and in vivo, and reduce the malignancy of CSCs. Therefore, targeted therapy for CSCs is a feasible means to treat ovarian cancer recurrence.

[0005] Nano drug delivery systems have broad prospects in biomedical applications due to their efficient drug loading, targeted drug delivery, and controlled release. Compared with traditional chemotherapy drugs, nano drug delivery systems can preferentially accumulate in tumor tissues by utilizing the enhanced permeability and retention (EPR) effect caused by the pathological leaky vasculature and poor lymphatic drainage of tumors, thereby achieving passive targeting. With the continuous progress of nanomedicine, the nano drug delivery systems developed in recent years with active targeting, tumor microenvironment-sensitive drug release, and external energy-responsive drug release have further enhanced their aggregation effect at the site of malignant tumors. Their multi-target action mode has incomparable advantages in inhibiting tumor recurrence, while enhancing the efficacy of anti-tumor drugs and reducing their side effects. Among them, polymer micelles are a commonly used carrier in nano drug delivery systems. The micelle core can provide a good encapsulation space for hydrophobic drugs, thereby playing a solubilizing role; the hydrophilic segments of the micelles extend outward to form a solvation shell, which reduces the interaction between micelles and 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, among which reactive oxygen species (ROS) play an important role. ROS are oxygen-containing free radicals produced during cell metabolism, including superoxide anions, hydrogen peroxide and hydroxyl radicals. In the tumor microenvironment, the level of ROS is usually higher than that in normal tissues, which provides a potential target for tumor treatment.

[0007] The present invention intends to develop a microenvironment-responsive nanomicelle drug delivery system to achieve targeted drug delivery, thereby inhibiting 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 and its application in the preparation of a drug for inhibiting the recurrence of ovarian cancer, so as to solve the problems existing in the above-mentioned prior art. The microenvironment-responsive micelle can efficiently deliver the chemotherapy drug paclitaxel and the regulator curcumol, and by enhancing the uptake of the drug by tumor stem cells, the drug can play the role of physiological stability, long circulation, attenuation, synergy and inhibition of tumor recurrence in the body.

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

[0010] The present invention provides a method for preparing microenvironment-responsive micelles, comprising the following steps:

[0011] 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;

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

[0013] The DSPE-PEG 2000 -FSHB is DSPE-PEG 2000 -NHS, follicle stimulating hormone polypeptide FSHB and triethylamine are mixed and dialyzed to obtain;

[0014] The DSPE-PEG 2000 -TK-PEG 5000 DSPE-PEG 2000 -NHS, mPEG 5000 -TK-NH2 and triethylamine are mixed to obtain the product.

[0015] Furthermore, 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.

[0016] Furthermore, the solvent is methanol.

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

[0018] Further, the DSPE-PEG is prepared 2000 The mixed reaction of -FSHB was carried out in DMF for 12 h.

[0019] Further, the DSPE-PEG is prepared 2000 -TK-PEG 5000 The mixed reaction was carried out in chloroform for 0.5 h.

[0020] The present invention also provides a microenvironment responsive micelle prepared according to the above preparation method and having the effect of inhibiting the recurrence of ovarian cancer.

[0021] The present invention also provides the use of the microenvironment responsive micelle in preparing a drug for inhibiting the recurrence of ovarian cancer.

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

[0023] Furthermore, the medicine also includes 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 anticancer drug paclitaxel and the Chinese medicine monomer curcumol to the tumor site and realize the release of the drugs, thereby inhibiting the recurrence of ovarian cancer. The present invention uses a thioketal (TK) that can be broken under ROS conditions as an intermediate chain to connect DSPE-PEG 2000 and PEG 5000 , forming a new type of amphiphilic polymer (DSPE-PEG 2000 -TK-PEG 5000 ), which can self-assemble into nanoparticles with a diameter of less than 100 nm, and can achieve long circulation in the body and be enriched in the tumor site using the EPR effect. 2000 -TK-PEG 5000 It can also achieve the stability transformation of nanocarriers, which are stable in the process of drug delivery, and become unstable and release drugs quickly after entering tumor cells. The follicle stimulating hormone peptide FSHB is shielded by a dense PEG hydration layer during blood circulation. Once the TK bond is broken under the action of ROS highly expressed in tumor tissue, the long-chain PEG 5000 Falling off, exposing the short chain PEG 2000 The terminally modified follicle stimulating hormone polypeptide FSHB achieves active targeting of ovarian cancer stem cells (OCSCs) that are positive for the follicle stimulating hormone receptor FSHR, promotes the uptake of tumor cells, and enhances the tumor-targeted delivery efficiency of drugs. FSHR-mediated endocytosis allows the micelles to enter OCSCs and quickly dissociate, releasing paclitaxel and curcumin to kill tumor cells. The oxidation-responsive "shell removal" strategy has been proven to be an effective drug controlled release method for achieving the transformation of nanomedicines from stealth to viscosity. This tumor oxidation-specific biodegradable nanomicelle has potential application prospects for the delivery of chemotherapeutic drugs. By enhancing the uptake of drugs by tumor stem cells, the recurrence of ovarian cancer can be more effectively inhibited. The present invention provides a new strategy for the treatment of tumors, which has important theoretical and clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

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

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

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

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

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

[0033] Figure 7 The graph showing the changes in particle size and PDI value of TK-NMs at 4°C for 30 days;

[0034] Figure 8 This is the particle size change diagram of TK-NMs under the action of 1mM H2O2;

[0035] Fig. 9 This is the PDI value change diagram of TK-NMs under the action of 1mM H2O2;

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

[0037] Fig.11 This is the PDI value variation diagram of TK-NMs under the action of three different media;

[0038] Fig.12 Fluorescence microscopy image of OCSCs identified by mouse nestin antibody detection experiment; scale bar is 25 μm;

[0039] Fig.13 The results are from flow cytometry detection of stem cell surface marker CD133;

[0040] Fig.14Inverted fluorescence microscopy measurement of the uptake of different micelles by OCSCs and OCSCs spheres; scale bar: 100 μm;

[0041] Fig.15 The results of flow cytometry determination of the uptake of different micelles by OCSCs and OCSCs spheres;

[0042] Fig.16 Images of the inhibitory effects of different micelles on the growth of OCSCs; scale bar is 100 μm;

[0043] Fig.17 The results of flow cytometry determination of OCSCs apoptosis induced by different micelles;

[0044] Fig.18 Microscopic detection images (A) and quantitative analysis images (B) of different micelles inhibiting OCSCs migration; the scale bar in A is 50 μm;

[0045] Fig.19 Microscopic detection images (A) and quantitative analysis images (B) of different micelles inhibiting OCSCs invasion; the scale bar in A is 50 μm;

[0046] Fig. 20 Microscopic detection images (A) and quantitative analysis images (B) of different micelles inhibiting OCSCs angiogenesis; the scale bar in A is 100 μm;

[0047] Fig.21 Microscopic detection (A) and quantitative analysis (B) of different micelles inhibiting OCSCs wound healing; the scale bar in A is 100 μm;

[0048] Fig. 22 Statistical graph of P-gp levels in OCSCs after treatment with different micelles;

[0049] Fig.23 Statistical graph of MRP1 levels in OCSCs after treatment with different micelles;

[0050] Fig.24 Statistical graph of BCRP levels in OCSCs after treatment with different micelles;

[0051] Fig.25 This is a result diagram of small animal in vivo imaging to observe the targeting effects of different preparations in recurrent mice;

[0052] Fig.26 Representative pictures of the relapse mouse model;

[0053] Fig. 27 This is a statistical analysis of the tumor volume of mice in different preparation groups;

[0054] Fig.28HE staining of recurrent mouse tumors after intervention with different preparations; scale bar is 100 μm;

[0055] Fig.29 Immunofluorescence staining images of recurrence-related proteins in tumor tissues; the scale bar is 50 μm. DETAILED DESCRIPTION

[0056] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0057] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0058] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0059] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0060] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0061] Example 1

[0062] 1. Experimental instruments and materials

[0063] 1.1 Experimental Instruments

[0064] XS105 one-hundred-thousandth balance (Mettler Toledo); DZKW-S-4 electric constant temperature water bath (Shanghai Medical Equipment Company Medical Equipment Factory No. 5); RE52CS rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); Ti-S inverted fluorescence microscope (Nikon Corporation, Japan); 1100 liquid chromatograph (UV detector, Dalian Yilite); C18 chromatographic column (250mm×4.6mm, 5μm, Shanghai Yuexu). Sephadex G-50 column (Shanghai Hualan Chemical Technology Co., Ltd.); dialysis bag (Beijing Solebold Technology Co., Ltd., molecular weight cutoff 2000Da); polycarbonate membrane (Milipore Co., Ltd., USA); 500A nanoparticle size and Zeta potential analyzer (Anton Paar, Austria); JEM-2000EX transmission electron microscope (Japan Electronics Co., Ltd.); SW-CJ-1D single-person clean bench (Shanghai Sujing Industrial Co., Ltd.); gas-jacketed carbon dioxide cell culture incubator (Thermo Flesher Scientific (Asheville) LLC, USA); HBS-1096A microplate reader (Nanjing Detie Experimental Equipment Co., Ltd.); FACSCalibur flow cytometer (BD Company, USA); multispectral in vivo imaging system (Carestream Health, Inc., USA); RM2235 paraffin sectioner (Leica, Germany); confocal microscope (Leica, 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%); polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus) (BASF, Germany); P-glycoprotein inhibitor D-α-tocopherol polyethylene glycol succinate (TPGS 1000 ), DSPE-PEG 2000 , DSPE-PEG 2000 -NHS, mPEG 5000 -TK-NH2 (Xi'an Ruixi Biotechnology Co., Ltd.); 4',6-diamidino-2-phenylindole (DAPI) (Beijing Solarbio Technology Co., Ltd.); EGF, FGF (Peprotech, USA); B27 (Beijing Yiaobang Biotechnology Co., Ltd.); recombinant Alexa 594 fluorescent Anti-CD133 antibody (Abcam, UK); Nestin antibody (Beijing Bioson Biotechnology Co., Ltd.); 1,1-dioctadecyl-3,3,3,3-tetramethylindole tricarbon cyanide iodide (DiR) (Nanjing Kaiji Biotechnology Development Co., Ltd.). The other reagents were of analytical grade, and water was purified water.

[0067] 2. Experimental Methods

[0068] 1 Preparation of micelles

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

[0070] Weigh DSPE-PEG 2000 -NHS 100 mg was dissolved in 3 mL DMF, follicle stimulating hormone polypeptide FSHB (110 mg) and triethylamine (300 μL) were added to dissolve completely, and the reaction was carried out at room temperature for 12 h; the reaction solution was transferred to a dialysis bag (molecular weight cutoff 2000 Da), dialyzed in pure water for 24 h, and the dialyzate was collected and freeze-dried to obtain the product DSPE-PEG 2000 -FSHB.

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

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

[0073] Weigh 100 mg DSPE-PEG 2000 -NHS was dissolved in 5 mL of chloroform and mPEG was added 5000 -TK-NH2 (100 mg) and triethylamine (300 μL) were completely dissolved, reacted at room temperature for 0.5 h, and the reaction solution was concentrated by rotary evaporation under reduced pressure, precipitated with a large amount of icy ether, filtered and collected, and vacuum dried to obtain DSPE-PEG 2000 -TK-PEG 5000 .

[0074] 1.3 Preparation of microenvironment-responsive micelles

[0075] The microenvironment-responsive micelles were prepared by thin film dispersion method. 120 mg Soluplus, 20 mg TPGS, and 2 mg DSPE-PEG were accurately weighed. 2000 、2mg DSPE-PEG 2000 -TK-PEG 5000 , 1 mg PTX, 2.76 mg CC and 2 mg DSPE-PEG 2000-FSHB was placed in a 15 mL centrifuge tube, and the above materials were dissolved in methanol as solvent and transferred to a 50 mL round-bottom flask. The solvent was removed by rotary evaporation under reduced pressure in a 40 °C water bath. After a thin film was formed on the bottom of the round-bottom flask, 5 mL of PBS solution was added to hydrate it, and it was extruded twice on a 0.22 μm microporous filter membrane to obtain microenvironment-responsive micelles (TK-NMs). In the process of preparing micelles, DSPE-PEG was not added. 2000 -TK-PEG 5000 , PTX, CC and DSPE-PEG 2000 -FSHB to obtain blank micelles; no DSPE-PEG was added 2000 -TK-PEG 5000 and DSPE-PEG 2000 -FSHB is used to obtain ordinary micelles; without adding DSPE-PEG 2000 -TK-PEG 5000 The FSHB modified micelles (FSHB-NMs) were obtained.

[0076] 1.4 Preparation of DiR micelles

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

[0078] 2 Characterization of micelles

[0079] 2.1 Encapsulation efficiency and drug loading test

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

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

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

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

[0084] Take 1 mL of micelles and use a 500A nanoparticle size and zeta potential analyzer to measure the particle size, polydispersity coefficient and zeta potential of different micelles by dynamic light scattering. Each preparation is measured 3 times, and each cycle is set to measure 6 times.

[0085] 2.3 Transmission electron microscopy observation of microenvironment-responsive micelle morphology

[0086] Observe the morphology of microenvironment-responsive micelles using a transmission electron microscope. Take an appropriate amount of micelles and dilute them to a suitable concentration. Drop a drop of the micelles on a copper mesh for electron microscopy, stain them with 2% phosphotungstic acid, and observe their morphology using a transmission electron microscope after drying. The acceleration voltage of the transmission electron microscope is set to 120 kV.

[0087] 2.4 Determination of critical micelle concentration

[0088] The CMC of micelles was determined using pyrene as a fluorescent probe (excitation wavelength 334 nm, emission wavelength 340-450 nm, excitation and emission slit widths were both 3.0 nm).

[0089] Accurately weigh 40.4 mg of pyrene and place it in a 10 mL volumetric flask, dilute to volume with acetone, and then dilute 100 times to obtain 2×10- 4 mol / L stock solution. Blank-NMs was prepared to 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 solution. Add 80 μL of pyrene stock solution to the test tube and dry it naturally overnight in the dark. Use a pipette to take 8 mL of the above micelle solution of different concentrations and add it to the test tube so that the final concentration of pyrene is 2×10 -6 mol / L. Seal the test tube and shake gently to mix evenly. Place in a dark place overnight. Filter the liquid in the test tube using a 0.22μm microporous filter membrane, add 200μL to each well of a 96-well plate, and measure the fluorescence value at 372 and 383nm using a fluorescence spectrometer. 372 / I 384The logarithmic value of the sample concentration C (mg / mL) was cross-plotted and the critical aggregation concentration was calculated.

[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 refrigerator at 4°C and taken out at 5, 10, 15, 20, 25 and 30 days to examine the micelle particle size and PDI value.

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

[0093] Hydrogen peroxide (H2O2) was used to simulate the high-level ROS environment in the tumor site. Two TK-NMs solutions were prepared using PBS buffer. One FSHB-NMs solution was prepared using PBS buffer. An appropriate amount of hydrogen peroxide (H2O2) was added to one of the TK-NMs PBS solutions to make the H2O2 concentration in the solution 1mM; the other solution was diluted with an equal amount of pure water as a control group. At different time points after the addition of hydrogen peroxide, a dynamic laser scattering instrument was used to monitor the change in the particle size of the micelles, and three parallel experiments were set up for each group.

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

[0095] 3 mL of TK-NMs were added into PBS (pH 7.4), PBS (pH 7.4) containing 10% FBS, and DMEM culture medium containing 10% FBS at a volume ratio of 1:10, and incubated at 37°C in a constant temperature water bath. 1 mL was taken out at 0, 2, 4, 6, 8, 12, 24, and 48 h, respectively, and the changes in micelle particle size and PDI value were analyzed by dynamic laser scattering to evaluate its serum stability.

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

[0097] 3.1 ID8 cell and stem cell culture

[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) in a 37°C, 5% CO2-95% air cell culture incubator 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 medium, 20ng / mL bFGF epidermal growth factor, 100U / mL penicillin and 100μg / mL streptomycin, and incubated in a 37°C, 5% CO2 incubator. The medium was changed by half every 2 days, and mature stem cell balls (OCSCs balls) were formed in 1 to 2 weeks. The mature stem cell balls were transferred back to ordinary DMEM medium for culture to obtain OCSCs cells.

[0100] 3.2 Stem cell identification

[0101] The stem cell surface marker Nestin was observed using a fluorescent 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 hours, the culture medium was discarded and fixed with 4% paraformaldehyde for 15 minutes. Washed with PBS three times and incubated with 1% Triton X-100 at 37°C for 30 minutes. Nestin antibody was diluted with 1% BSA and incubated at 4°C overnight. FITC-labeled fluorescent secondary antibody (1:300 dilution) was added and incubated at 37°C in the dark for 1 hour. Washed with PBS and imaged with a fluorescent inverted microscope.

[0102] Flow cytometry was used to detect stem cell surface marker CD133. ID8 cells, OCSCs cells and OCSCs spheres in the logarithmic growth phase were collected and centrifuged. 80% paraformaldehyde was used for fixation for 5 minutes, and the paraformaldehyde was removed by centrifugation. The cells were washed 3 times with PBS. 1% Triton X-100 was used for permeabilization at 37℃ for 30 minutes, and the cells were washed 3 times with PBS. 5% BSA was added for blocking for 1 hour, and fluorescent Anti-CD133 antibody was added, and the cells were incubated at 4℃ overnight. After washing with PBS, 300μL PBS was added and the cells were detected by flow cytometry.

[0103] 3.3 Ovarian cancer stem cell uptake experiment

[0104] Fluorescence inverted microscopy was used to qualitatively analyze the uptake of different micelles in OCSCs cells and OCSCs spheres. Each group of cells was inoculated in a 24-well plate and cultured, with 3 parallel wells. After 24 hours of culture, each cell was incubated with Cou-NMs, FSHB-Cou-NMs and TK-Cou-NMs+H2O2 (coumarin concentration was 2 μM) containing coumarin for 2 hours. After washing 3 times with PBS buffer, the cells were fixed with 4% paraformaldehyde for 10 minutes, and the nuclei were stained with DAPI staining solution in the dark for 6 minutes. The distribution of fluorescence in the cells was observed using a fluorescence inverted microscope.

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

[0106] 4. The ability of different micelles to induce apoptosis of ovarian cancer stem cells

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

[0108] Flow cytometry was used to detect the effect of each group of micelles on OCSCs apoptosis. 5 OCSCs at a density of 100 cells / well were seeded in a 6-well plate, and micelles of each group were added when the cells grew to 70%. After 48 hours of culture, the cells were digested and collected with 0.25% trypsin, washed with PBS, and the supernatant was discarded. Binding Buffer was added to suspend the cells according to the instructions, and Annexin V-FITC and PI were added for double staining. The reaction was carried out at room temperature in the dark for 15 minutes, and the apoptosis rate of the cells was detected within 1 hour.

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

[0110] 5. The ability of different micelles to inhibit OCSCs invasion and migration in vitro

[0111] 5.1 Stem cell migration assay

[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 of different formulas and 180 μL of DMEM medium without FBS to the upper chamber. The cell suspension was resuspended, and the cell density was 2×10 4 / well, with 3 replicate wells in each group. After continuing to culture for 24 hours, aspirate the culture medium in the upper chamber and wash the cells in the upper chamber with PBS, and use a cotton swab to gently wipe the unmigrated stem cells inside the chamber. Take a new lower chamber and add 700μL 4% paraformaldehyde. After the chamber is fixed for 15 minutes, it is stained with 0.5% crystal violet solution at room temperature for 15 minutes. The unbound crystal violet is washed away with PBS, dried at room temperature, and observed and photographed under an inverted microscope. Use Image J software to count the cells in the pictures taken. The formula for calculating cell migration rate is as follows:

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

[0114] 5.2 Stem cell invasion assay

[0115] In pre-cooled conditions, the Transwell chamber was coated with Matrigel (Matrigel was diluted 1:4 with serum-free medium), and then 3×10 4 180 μL of stem cell suspension was added into the chamber at a density of 10 cells / mL, and the other steps were consistent with the stem cell migration experiment.

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

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

[0118] Add 50 μL of matrix gel to each well of the 96-well plate, shake well to ensure that there are no bubbles, and immediately place the 96-well plate in a 37°C cell culture incubator for 30 min to ensure that the matrix gel solidifies. 4 Each well was inoculated with a concentration of 1000 cells / well in the above 96-well plate, and micelles of different formulations were added to each well. The incubation was continued for 4 hours, and the damage of different micelles to the formation of mimetic blood vessels was observed under an inverted microscope. The nodes, junctions, branches and segments of the mimetic blood vessels in the pictures were counted using Image J software.

[0119] 5.4 Inhibition of OCSCs wound healing ability by different micelles

[0120] OCSCs cells were counted at 1×10 5 / well concentration, each well was inoculated in a 6-well plate, incubated until the growth reached 80%, and then two parallel straight lines were gently drawn with a 20μL sterile pipette tip in a clean bench, washed with PBS to remove cell debris, replaced with fresh culture medium, and different micelles were added. Three replicate wells were set up in each group. An inverted microscope was used to observe and take pictures at 0h, 12h, and 24h of administration. Image J software was used to perform statistical analysis on the scratch area. Wound healing rate calculation formula: Wound healing rate % = (1-A t / A0)×100%, where A t A represents the scratch area measured at 12 h or 24 h, and A0 represents the scratch area measured at 0 h.

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

[0122] OCSCs were seeded in 25 cm 2In the culture flask, when the cells grew to 80%, different drug-loaded micelles were added, and blank micelles were used as blank control groups. After 24 hours, the cells were washed 3 times with cold PBS, and 800 μL of cell lysis buffer was added to each group to completely lyse the cells, and the supernatant was collected at 4°C in a refrigerated centrifuge. The P-gp, MRP-1 and BCRP contents in each sample were determined at 450 nm according to the instructions of the ELISA kit.

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

[0124] 7.1 Establishment of tumor-bearing mouse model

[0125] The tumor-bearing mouse model was established by injecting OCSCs into the armpit. 6 OCSCs were suspended in 200 μL PBS and inoculated into the left forelimb axilla of C57BL / 6 mice. The growth of the axilla tumor was observed daily and the size of the tumor was measured with a vernier caliper every other day. When the tumor volume reached about 200-300 mm 3 It can be used for subsequent experiments.

[0126] 7.2 In vivo targeting studies

[0127] In vivo imaging experiments were used to investigate the in vivo targeting of different preparations. The drug was replaced with the fluorescent reagent DiR. Tumor-bearing mice were divided into the following groups: (1) saline (Control); (2) free DiR (Free DiR); (3) DiR micelles (DiR-NMs); (4) follicle-stimulating hormone peptide-modified DiR micelles (FSHB-DiR-NMs); (5) microenvironment-responsive micelles (TK-DiR-NMs). The DiR dose in each dosing group was the same, 2 μg / mouse. Mice were anesthetized with isoflurane, and the fluorescence signals in each group of 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, and 84 h) to observe the intensity of the fluorescence signal in vivo.

[0128] Evaluation of the inhibitory effects of different micelles on 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 detailed in 7.1. The growth of the axillary tumor of the mice was observed daily, and the size of the tumor was measured with a vernier caliper every other day. 3At the time of the experiment, mice were anesthetized with 7% chloral hydrate and then tumor resection was performed. 90% of the volume of tumor tissue was removed and sutured layer by layer. The tumor-bearing mice were divided into: (1) normal saline (BlankControl); (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-mentioned different preparations were injected into 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 effects of different preparations on the recurrence of mice. The formula for calculating the tumor volume 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, the tumor tissues were removed and embedded in paraffin, and each tissue was cut into 5 μm thick slices using a paraffin slicer. H&E staining of tumor tissues was used to evaluate the destructive effects of different micelles on axillary tumor tissues of tumor-bearing mice. Immunofluorescence was used to detect the expression levels of proteins Nestin, CD133, and ALDH1 associated with tumor recurrence.

[0132] 9 Statistical analysis

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

[0134] 3. Results

[0135] 1. Characterization of Micelles

[0136] Targeting molecule DSPE-PEG 2000 -FSHB H NMR spectrum Figure 1 As shown in the figure, the characteristic peak of FSHB peptide is clearly visible in the spectrum, proving that the targeting molecule DSPE-PEG 2000 -FSHB synthesis was successful. Figure 2 DSPE-PEG 2000 -TK-PEG 5000 H NMR spectrum, proving DSPE-PEG 2000 -TK-PEG 5000 The synthesis was successful. Transmission electron microscopy photo of microenvironment-responsive micelles ( Figure 3 ) It can be seen that the TK-NMs are spherical in shape, with a smooth surface and a particle size of about 80nm. The particle size and Zeta potential of the micelles were measured using a nanoparticle size and Zeta potential analyzer. The results are shown in Figure 4-Figure 5. The particle size of TK-NMs is (78.04±0.99) nm, and the polydispersity index is (PDI, 0.07±0.01), indicating that the particle size is uniform and well dispersed, without agglomeration, and the particle size is in line with experimental expectations. The encapsulation efficiency and drug loading of PTX and CC in TK-NMs are shown in Table 1. The encapsulation efficiency of PTX is (87.732±1.617)%, and the encapsulation efficiency of CC is (79.914±2.527)%; the drug loading of PTX is (0.586±0.009)%, and the drug loading of CC is (1.455±0.037)%. The micelles have a low critical micelle concentration, such as Figure 6 As shown, the critical micelle concentration is 12.078 mg / L, which indicates that the micelle has a strong anti-dilution ability. After entering the body, it can avoid the destruction of its integrity due to dilution by body fluids and has good dilution stability. Figure 7 The stability evaluation results of TK-NMs at 4°C for 30 days show that the particle size has no significant change and the PDI is lower than 0.15, indicating that TK-NMs can 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 DSPE-PEG 2000 -TK-PEG 5000 The response ability to reactive oxygen species (ROS) was tested in the present invention for the changes in particle size and PDI value of TK-NMs in a 1 mM H2O2 environment. Figure 8-Figure 9 As shown in the figure, with the extension of incubation time, the particle size of TK-NMs in the H2O2 group gradually decreased from 79.45±1.78nm at the beginning to 72.10±1.59nm after 4h of incubation, which was close to 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 has a great influence on the particle size change of TK-NMs. This is because the TK bond has a strong ROS responsiveness, which can be quickly oxidized and broken in the ROS environment, causing the hydration film of the outer layer of the micelle to fall off, resulting in particle size shrinkage. This result shows that TK-NMs has a strong ROS responsiveness.

[0141] The present invention evaluated the stability of TK-NMs in three different media, such as Figure 10-11As shown in the figure, the micelles were relatively stable in PBS solution within 48 hours, and the particle size and PDI value changed little, which met the requirements for intravenous injection. The particle size, PDI value and zeta potential of the micelles in the PBS + 10% serum group and the DMEM medium + 10% serum group increased over time, but the particle size was still less than 100nm and the PDI was less than 0.2 within 8 hours, indicating that it still had a long circulation effect within 8 hours, which was conducive to the passive targeting of the micelles to the tumor site. The changes were more obvious after 24 hours, which may 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 that is mainly used as a marker of pluripotent stem cells and is involved in tissue development and regeneration. In many solid tumors, including ovarian cancer, Nestin expression is high and is associated with tumor recurrence and poor prognosis. The expression of Nestin in OCSCs was identified using mouse nestin antibody immunofluorescence staining. The results are shown in Figure 2. Figure 12-13 shown. Fig.12 The green fluorescence intensity indicates the fluorescence intensity of Nestin expression. The order of Nestin expression in cells is: OCSCs spheres>OCSCs>ID8 cells>blank control.

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

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

[0146] Follicle stimulating hormone receptor (FSHR) exists in the placenta, uterus, prostate, bone tissue and ovarian tissue. FSHR is also found to be selectively highly expressed on the surface of many tumor neovascular endothelial cells and is associated with tumor recurrence. Studies have shown that FSHR is highly expressed in ID8 stem cells. Fig.14The fluorescence images of OCSCs and OCSCs spheres taking different micelles show the distribution of different coumarin micelles in cells. The fluorescence signal of micelles modified with FSHB (FSHB-Cou-NMs) is significantly stronger than that of the ordinary micelle group (Cou-NMs), indicating that FSHB-Cou-NMs can specifically target ovarian cancer stem cells that highly express FSHR, and enhance the active uptake of micelles by cells through endocytosis mediated by the specific binding of follicle-stimulating hormone peptide to follicle-stimulating hormone receptor. The results of flow cytometry ( Fig.15 ) confirmed the above conclusion.

[0147] 4. Evaluation of the inhibitory effects of different micelles on OCSCs growth and apoptosis induction

[0148] Cancer stem cells have become an important target for drug therapy due to their remarkable self-renewal ability and the characteristics of promoting tumor development, and are closely related to tumor resistance and recurrence. In order to evaluate the effect of TK-NMs nanomaterials on the stemness characteristics of ovarian cancer stem cells, the ability and efficiency of single stem cells to form cell clusters were observed through spheroidization experiments, thereby evaluating the in vitro regenerative potential of these cells. Experimental results ( Fig.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, among which the number and volume of cell clusters formed by ovarian cancer stem cells treated with TK-NMs and FSHB-NMs groups were most significantly reduced.

[0149] Flow cytometry was used to detect the apoptosis of OCSCs under the action of different micelles after AnnexinV-FITC double staining. Normal living cells were lowly stained with Annexin V and PI; apoptotic cells were highly stained with Annexin V and lowly stained with PI; necrotic cells were highly stained with Annexin V and PI. Fig.17The results of flow cytometry showed that 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+H2O2 group were 16.52%, 24.07%, 35.51%, 51.40%, 38.03% and 52.03%, respectively, which were significantly higher than 5.74% in the control group. This showed that each drug-treated group could induce apoptosis of OCSCs. The difference between the PTX-NMs group and the PTX / CC-NMs group confirmed that CC could 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 OCSCs taking in FSHB-NMs. The cell apoptosis rate in the TK-NMs+H2O2 group was close to that in the FSHB-NMs group and significantly higher than that in the TK-NMs group. This was because the TK bond broke under the action of ROS, the dense PEG hydration layer fell off, and the follicle-stimulating hormone polypeptide was exposed, thus 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 OCSCs invasion and migration in vitro

[0151] Transwell assay was used to evaluate the inhibitory effect of different micelle groups on the invasion and migration ability of OCSCs. Fig.18 and Fig.19 These are the images of ovarian cancer stem cells in each group passing through the Transwell chamber after administration of different micelle groups. Fig.18 is the result of inhibiting stem cell migration. Fig.19 This is the result of inhibiting stem cell invasion. Fig.18 and Fig.19 As shown in the figure, a large number of cells in the BlankControl group passed through the chamber, 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 drug-treated group was significantly reduced, among which the TK-NMs+H2O2 group had the most significant effect in inhibiting the migration and invasion of OCSCs.

[0152] Fig. 20The destructive effects of different micelles on the mimetic vessel (VM) channels. The Blank Control group has formed obvious channels composed of elongated cells, and the grid is complete and the grid area is large. The VM channels formed by OCSCs treated with different micelle groups have been damaged to varying degrees, mainly manifested in the short and broken VM channels, poor grid integrity or no grid formation, and small single grid area. The FSHB-NMs group and the TK-NMs+H2O2 group can significantly inhibit the formation of VM channels.

[0153] The scratch images of each group of micelles at different time points are shown in Figure 2. Fig.21 As shown. 24 hours after drug administration, the scratches in the Blank Control group were almost healed, and the width of the scratches in each drug administration group was smaller than that at 0 hours. The results showed that different micelles had a certain inhibitory effect on scratch healing, among which the FSHB-NMs group and TK-NMs+H2O2 group had the most obvious inhibition.

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

[0155] Tumor recurrence is closely related to the multidrug resistance of tumor cells. Multidrug resistance is a major obstacle in tumor treatment. It makes tumor cells resistant to a variety of chemotherapy drugs with different structures and mechanisms of action, resulting in poor chemotherapy effects and promoting tumor recurrence and metastasis. 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. They are 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 effectively accumulate to sufficient concentrations, thereby making tumor cells resistant to chemotherapy drugs and promoting tumor recurrence. 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 the ELISA experiment are as follows Figure 22-Figure 24 As shown in the figure, due to the drug resistance of OCSCs, the expression levels of p-gp, MRP1, and BCRP in OCSCs were higher than those in ID8 cells. Both FSHB-NMs and TK-NMs+H2O2 groups could significantly downregulate the expression levels of P-gp, MRP, and BCRP in OCSCs.

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

[0157] The in vivo distribution patterns of different preparations and different time periods were observed using an in vivo fluorescence imaging analyzer. Fig.25. No fluorescence signal was detected in the blank group. The fluorescence signal of the Free DiR group was mainly concentrated in the liver area of ​​the mice. The fluorescence signal of the micelle group was distributed throughout the body and accumulated more in the tumor site. With the increase of time, the fluorescence signal intensity of each group decreased. A weak fluorescence signal could be observed in the tumor site of the Free DiR group mice during 3-9 hours, and no fluorescence signal was observed after 12 hours. Obvious fluorescence signals were observed in the tumor site of mice in different micelle groups between 3-84 hours, and the TK-DiR-NMs group mice had the strongest fluorescence signal in the tumor site at each time point. The order of DiR fluorescence intensity at different time points is as follows: TK-DiR-NMs>FSHB-DiR-NMs>DiR-NMs>Free DiR. The experimental results show that TK-DiR-NMs has better tumor targeting and long circulation effect.

[0158] 8. Evaluation of the inhibitory effects of different micelles on ovarian cancer recurrence

[0159] according to Fig.26 The observation results show that the blank control group mice had tumor recurrence after surgery. The tumor recurrence rate of other treatment groups decreased significantly, and the growth rate of recurrent tumors also slowed down significantly. The effect of each micelle group in inhibiting tumor recurrence was better than that of the free drug group, among which the TK-NMs group showed the strongest inhibitory effect.

[0160] Depend on Fig. 27 It can be seen that after intervention with different preparations, tumor recurrence can be inhibited. Among them, the inhibitory effect of each micelle group on tumor recurrence is significantly better than that of Free PTX / CC; and TK-NMs has the best inhibitory effect. The order of tumor size in tumor-bearing mice in different dosing groups is: Blank Control>Free PTX / CC>PTX / CC-NMs>FSHB-NMs>TK-NMs. The results of H&E staining of tumor tissues in different preparation groups are shown in Fig.28 The tumor tissue cells of the blank group mice were arranged evenly and neatly, and the cells and nuclei were plump in morphology; after treatment with other preparations, cells in each group showed varying degrees of necrosis, and the TK-NMs group had the most serious damage to tumor cells, with a large number of cells shrinking and large areas of necrosis and apoptosis.

[0161] Nestin, CD133 and ALDH1 are three protein markers that are involved in the key characteristics of tumor stem cells and may play an important role in the recurrence of ovarian cancer. Immunofluorescence staining to detect their expression in tumor sections is helpful in assessing the risk of tumor recurrence. Fig.29Immunofluorescence staining was used to evaluate 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 that the expression of related protein markers decreased, indicating that the characteristics of tumor stem cells and the risk of recurrence were reduced. 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 tumor stem cells.

[0162] In summary, through the above implementation examples, it can be seen that the microenvironment-responsive micelles TK-NMs exhibited strong anticancer activity in 2D tumor stem cells and 3D tumor stem cell spheroids in vitro, significantly inhibiting the invasion and migration of tumor cells; TK-NMs showed good anti-tumor recurrence effects in the in vivo mouse tumor recurrence model. The step-by-step "passive targeting" and "active targeting" of microenvironment-responsive micelles can effectively realize the drug controlled release method of nanomedicine from invisible to viscous transformation, significantly promote the uptake of drugs by tumor stem cells, achieve physiological stability, long circulation and toxicity reduction and efficacy enhancement of drugs in vivo, and inhibit tumor growth and recurrence.

[0163] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all 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 product.

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

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

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

5. 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.

6. 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.

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

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

9. A drug for inhibiting the recurrence of ovarian cancer, characterized in that: The active ingredient comprises the microenvironment-responsive micelle according to claim 7.

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

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