TNBC-targeted multifunctional nano-micelle as well as preparation method and application thereof

Abstract

The invention provides a TNBC-targeted multifunctional nano-micelle. The TNBC-targeted multifunctional nano-micelle comprises a carrier skeleton and a loaded drug, the carrier skeleton is based on hyaluronic acid HA and is formed by connecting cystamine CYS and vitamin E succinate alpha-TOS through amido bonds; the loaded medicine is paclitaxel PTX and an IRE1alpha-XBP1 pathway inhibitor Kira6. The invention further discloses a preparation method of the paclitaxel-XBP1 pathway inhibitor. The invention also provides a preparation method of the TNBC-targeted multifunctional nano-micelle and application of the TNBC-targeted multifunctional nano-micelle in preparation of triple negative breast cancer treatment drugs. The preparation method has the advantages that the preparation can be used for treating triple negative breast cancer by targeting tumor stem cells, reversing an immunosuppression microenvironment and accurately delivering chemotherapeutic drugs; the compound has a strong killing effect on TNBC tumor cells, the in-vivo tumor inhibition rate reaches 78%, the system toxicity is remarkably reduced, and the compound has outstanding clinical transformation potential.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and nanotechnology, and in particular to a multifunctional nanomicelle targeting triple-negative breast cancer (TNBC) and a preparation method and application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) is a highly aggressive, metastatic, and drug-resistant subtype of breast cancer. Because it is negative for estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2, it lacks ideal targets for endocrine therapy and targeted therapy. More than 70% of women with metastatic TNBC will die within 5 years. Currently, clinical treatment mainly relies on surgery combined with chemotherapy. However, the presence of a large number of immunosuppressive cells (such as M2-tumor-associated macrophages) and tumor stem cells in the tumor microenvironment forms an inhibitory microenvironment, resulting in poor chemotherapy efficacy and prone to drug resistance and metastasis in patients. Existing targeted therapies are also difficult to improve prognosis, and there is an urgent need to explore new treatment strategies.

[0003] The concept of cancer stem cells (CSCs) is widely recognized in cancer biology. Although they constitute a small proportion of tumors, they possess robust self-renewal and differentiation capabilities, making them the root cause of tumor recurrence and metastasis. CD44 is a prototypical CSC surface marker. Its binding to hyaluronic acid (HA) or other extracellular matrix molecules in vivo has been shown to activate signaling pathways such as Nanog-Stat3, Oct4-Sox2-Nanog, and c-Src kinase, and upregulate miR-21 expression. This allows CSCs to sense microenvironmental changes and induces their stemness, promoting tumor progression and metastasis, and increasing their susceptibility to chemoradiation and acquired drug resistance. Therefore, blocking CD44 binding to HA can reverse CSC characteristics and inhibit tumor progression. For example, knocking out CD44 can induce CSC differentiation into non-CSCs, thereby increasing sensitivity to chemotherapy and radiotherapy. Using hyaluronic acid-modified nanocarriers to target CD44 and eliminate CSCs effectively inhibits the growth of breast tumor cell spheres in vitro.

[0004] CSCs can also evade immune surveillance through various pathways. They release inhibitory cytokines, promoting the accumulation of immunosuppressive M2 macrophages while simultaneously reducing the activity of dendritic cells (DCs) and T cells, creating an immunosuppressive environment and accelerating tumor growth. Therefore, while targeting CSCs, it is also necessary to reverse the severe immunosuppression seen in TNBC to more comprehensively dismantle the tumor-suppressive microenvironment and eradicate the tumor at its source.

[0005] Based on this, treatment for TNBC can improve the tumor immune microenvironment in two ways: first, effectively eliminating immunosuppressive cells; different immune cells rely on different metabolic patterns for energy, and tumor cells promote the enrichment of immunosuppressive cells by altering metabolism, inhibiting endoplasmic reticulum stress and oxidative stress, and targeted elimination of M2 macrophages, which can reverse immunosuppression. Second, enhancing the anti-tumor immune response; DCs are crucial for activating cytotoxic T cells (CTLs), but the tumor environment often weakens their function, necessitating enhanced DC activity to promote CTL activation.

[0006] Furthermore, traditional chemotherapy has significant side effects, difficulty in reaching tumor sites, and the tendency for tumors to develop resistance to chemotherapy drugs. Therefore, seeking novel nano-delivery strategies to improve the targeted delivery of chemotherapy drugs to tumor sites is crucial for effective cancer treatment.

[0007] Hyaluronic acid (HA) is a negatively charged glycosaminoglycan that is widely present in the body. It has unique properties such as good biodegradability, biocompatibility, and non-immunogenicity. It has a high affinity for the CD44 receptor. A large number of studies have shown that CD44 is also overexpressed on the cell membrane of most tumors (including TNBC), which provides a specific target for the delivery system to deliver active drugs to the tumor site. In addition, more and more studies have confirmed that receptor targeting can increase cellular uptake of drugs, thereby further improving the bioavailability of drugs while reducing toxic side effects on non-targeted cells. Therefore, by utilizing this highly sensitive cell surface receptor, the delivery system can be used to precisely target and deliver chemotherapy drugs to the lesion site, improve the efficacy of the drug, and reduce toxic side effects. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a multifunctional nanomicelle targeting triple-negative breast cancer (TNBC), as well as a preparation method and application thereof. The nanomicelle is based on hyaluronic acid (HA), connects cystamine (CYS) and vitamin E succinate (α-TOS) as a carrier skeleton, and simultaneously loads the chemotherapy drug paclitaxel (PTX) and IRE1α-XBP1 pathway inhibitor (Kira6); the nanomicelle is used to treat triple-negative breast cancer (TNBC) by targeting cancer stem cells (CSC), reversing the immunosuppressive microenvironment, and precisely delivering chemotherapy drugs.

[0009] The present invention adopts the following technical solutions to solve the above technical problems:

[0010] A multifunctional nanomicelle targeting TNBC comprises a carrier skeleton and a loaded drug; the carrier skeleton is based on hyaluronic acid (HA, molecular weight 5-20kDa) and is composed of cystamine (CYS) and vitamin E succinate (α-TOS) linked by amide bonds; the loaded drug is paclitaxel (PTX) and an IRE1α-XBP1 pathway inhibitor (Kira6).

[0011] As one of the preferred embodiments of the present invention, the hyaluronic acid is linked to CYS and α-TOS via an amide bond to form a redox-sensitive polymer HSST (disulfide bonds impart redox responsiveness) as the carrier skeleton.

[0012] As one of the preferred embodiments of the present invention, the particle size of the HSST is 100 to 150 nm, and the surface potential is -20 to -40 mV.

[0013] As one of the preferred embodiments of the present invention, in the loaded drug, PTX is used to directly kill tumor cells, and Kira6 is used to inhibit endoplasmic reticulum stress, and the mass ratio of the two is (1-3):1, more preferably 3:1.

[0014] As one of the preferred embodiments of the present invention, the nanomicelles loaded with drugs have a particle size of 150 to 200 nm and a surface potential of -20 to -40 mV.

[0015] A method for preparing the multifunctional nanomicelles targeting TNBC comprises the following steps:

[0016] (1) Carboxyl activation

[0017] HA, EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide), and NHS (N-hydroxysuccinimide) were dissolved in PBS and reacted at room temperature for 0.5-1.5 h;

[0018] (2) Cystamine coupling

[0019] Add CYS, react for 20-28 hours, dialyze and freeze-dry to obtain HA-CYS;

[0020] (3) α-TOS modification

[0021] α-TOS was activated by EDC / NHS, coupled with HA-CYS, and purified by ethanol precipitation to obtain HSST;

[0022] (4) Drug loading

[0023] PTX and Kira6 were dissolved in ethanol, mixed with an HSST aqueous solution, and freeze-dried to obtain the TNBC-targeting multifunctional nanomicelles (K-PTX-HSST).

[0024] As one of the preferred embodiments of the present invention, in step (1), HA, EDC and NHS are dissolved in PBS at pH 7.4 at a molar ratio of 1:5:5.

[0025] As one of the preferred embodiments of the present invention, in step (1), the reaction is carried out at room temperature for 1.0 h.

[0026] As one of the preferred embodiments of the present invention, in step (2), the molar ratio of CYS to HA is 10:1.

[0027] As one of the preferred embodiments of the present invention, in step (2), the reaction is carried out for 24 hours.

[0028] As one of the preferred embodiments of the present invention, in the step (3), in the EDC / NHS activation of α-TOS, the molar ratio of α-TOS, EDC and NHS is 1:5:5.

[0029] As one of the preferred embodiments of the present invention, in step (4), after PTX and Kira6 are dissolved in ethanol, the mass ratio of the mixed solution to the HSST aqueous solution is 1:5.

[0030] The multifunctional nanomicelles targeting TNBC are used in the preparation of drugs for treating triple-negative breast cancer, which inhibit tumor progression, metastasis and recurrence through multi-target synergistic effects.

[0031] The mechanism of action of the multifunctional nanomicelles of the present invention:

[0032] Precision chemotherapy: High concentrations of glutathione (GSH, 10 mM) in the tumor microenvironment trigger disulfide bond cleavage, with a PTX release rate ≥ 65% within 10 hours.

[0033] Targeting CSCs: HA targets CSCs via the CD44 receptor and inhibits the Nanog-Stat3 pathway;

[0034] Immune microenvironment remodeling: Kira6 inhibits the IRE1α-XBP1 pathway and combines with α-TOS to clear ROS and promote Polarize toward M1, activate DCs and enhance CTL function;

[0035] The advantages of the present invention over the prior art are:

[0036] The present invention uses hyaluronic acid (HA) as the basis, connects cystamine (CYS) and vitamin E succinate (α-TOS) as the carrier skeleton, and simultaneously loads the chemotherapy drug paclitaxel (PTX) and the IRE1α-XBP1 pathway inhibitor (Kira6) to prepare a multifunctional nanomicelle (K-PTX-HSST) targeting TNBC; the nanomicelle eliminates cancer stem cells (CSCs) by targeting the CD44 receptor and inhibits the Nanog-Stat3 pathway; by dually inhibiting endoplasmic reticulum stress and oxidative stress, it reverses M2 macrophage polarization and activates dendritic cells (DCs); at the same time, it releases PTX in response to the tumor microenvironment to accurately kill tumor cells.

[0037] In vitro experiments showed that the nanomicelles of the present invention have a strong killing effect on TNBC tumor cells, with an in vivo tumor inhibition rate of 78%, and significantly reduced systemic toxicity, showing outstanding clinical translation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The construction and characterization of hyaluronic acid polymer micelles HSST in Example 1 (Figure A shows the synthesis route of hyaluronic acid polymer micelles; Figure B shows the particle size results; Figure C shows the potential results; and Figure D shows a transmission electron microscope image);

[0039] Figure 2 This is a hydrogen NMR spectrum of the characteristic structure of the hyaluronic acid polymer micelle HSST in Example 1 (in the figure, from bottom to top are hyaluronic acid HA, redox-sensitive hyaluronic acid derivative HA-CYS, and redox-sensitive hyaluronic acid vitamin E succinate derivative HA-SS-TOS);

[0040] Figure 3 This is the verification result of the drug release characteristics of hyaluronic acid micelles in Experimental Example 1 (Figure A is the fluorescence image of NR@HSST drug-loaded micelles incubated in different reducing environments; Figure B is the corresponding quantitative image);

[0041] Figure 4 is the PTX drug release result of K-PTX@HSST drug-loaded micelles under different reducing environments (0 mM, 10 mM) in Experimental Example 2;

[0042] Figure 5 The comparison results of the toxicity of K-PTX-HSST on TNBC cells and normal cells in Experimental Example 3 (Figure A shows the toxicity results on normal cells LO2; Figure B shows the toxicity results on TNBC tumor cells 4T1);

[0043] Figure 6 This is the fluorescence imaging result of the targeted uptake of micelles by tumor cells in Experimental Example 4;

[0044] Figure 7This is the result of the in vivo tumor volume change in Experimental Example 5;

[0045] Figure 8 This is the result of the weight change of mice after treatment in Experimental Example 5. DETAILED DESCRIPTION

[0046] The following embodiments of the present invention are described in detail. These embodiments are implemented based on the technical solutions of the present invention, and detailed implementation methods and specific operating procedures are given. However, the scope of protection of the present invention is not limited to the following embodiments. At the same time, the reagents and experimental methods used in the following examples and experimental examples are conventional reagents or methods in the art unless otherwise specified, and will not be repeated here.

[0047] Example 1, Synthesis and Characterization of HSST:

[0048] (1) Accurately weigh 200.0 mg HA (10 kDa), 76.8 mg EDC, and 46.0 mg NHS into a 100 mL dry round-bottom flask (HA, EDC, and NHS molar ratio of 1:5:5), add 20 mL PBS solution, and stir until completely dissolved; react at room temperature for 1 h to activate the carboxyl groups on HA; add 760 mg CYS (HA to CYS molar ratio of 1:10) into the round-bottom flask and continue the reaction for 24 h; after the reaction, place the product in a dialysis bag (MWCO 3.5 kDa) and dialyze with deionized water for 48 h. Collect the liquid in the dialysis bag and pass it through a 0.45 μm water membrane to remove impurities; freeze-dry the sample to obtain the redox-sensitive hyaluronic acid derivative HA-CYS.

[0049] (2) Accurately weigh 106 mg of α-TOS, 115.2 mg of EDC and 69.0 mg of NHS and place them in a 100 mL dry round-bottom flask (the molar ratio of α-TOS, EDC and NHS is 1:5:5), add 20 mL of anhydrous ethanol, and stir until completely dissolved; react at room temperature for 1 hour to activate the carboxyl groups on HA; take another 200.0 mg of HA-CYS and place it in a 100 mL dry round-bottom flask, add 20 mL of pure water, and stir until completely dissolved. Slowly add the α-TOS activation reaction solution dropwise to the above solution and react at room temperature for 24 hours; pour the reaction solution into 500 mL of ice anhydrous ethanol, centrifuge at 4000 rpm, take the precipitate and re-dissolve it with pure water and pass it through a 0.45 μm water film to remove impurities, freeze-dry the sample to obtain hyaluronic acid polymer micelles HSST.

[0050] Dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements showed that the particle size of hyaluronic acid polymer micelles HSST was 114±11.6nm and the potential was -28.8±6.4mV( Figure 1 ).

[0051] At the same time, the structure of the obtained HSST was confirmed by nuclear magnetic resonance hydrogen spectrum (1H-NMR): HSST was dissolved in a mixed solvent of deuterated water (D2O) and deuterated methanol (CD3OD) (D2O:CD3OD=1:1, v / v) at a concentration of 10 mg / mL, and the nuclear magnetic resonance hydrogen spectrum was recorded under nuclear magnetic resonance detection (the characteristic peaks of HA, CYS, HMDA and α-TOS were used to confirm the structure of HSST.).

[0052] The results are as follows Figure 2 As shown in the figure, the proton peak with a chemical shift of 1.92 ppm is attributed to the methyl peak of the acetyl group of hyaluronic acid; the proton peak with a chemical shift of 2.85 ppm is attributed to the methylene group in the linear chain of cystamine (CYS), indicating that cystamine (CYS) has been successfully grafted onto the carboxyl group of hyaluronic acid; the proton peak with a chemical shift of 0.95 ppm is attributed to the multiple methyl groups on the long carbon chain of vitamin E succinate (α-TOS).

[0053] Example 2, Synthesis and Characterization of K-PTX-HSST:

[0054] 3.75 mg of PTX and 1.25 mg of Kira6 were dissolved in 1 ml of ethanol (PTX:Kira6 = 3:1) and slowly added dropwise to 25 mg of HSST (1 mL of deionized water) with stirring overnight. The mixture was centrifuged at 30% power for 6 minutes and then centrifuged at 4000 rpm for 10 minutes. The supernatant was filtered through a 0.45 μm filter to remove free purple PTX and Kira6. The supernatant was then lyophilized to obtain the TNBC-targeting multifunctional nanomicelles, K-PTX-HSST. The drug loading was 20% PTX and 15% Kira6.

[0055] Dynamic light scattering (DLS) and transmission electron microscopy (TEM) measured the particle size of the nanomicelle K-PTX-HSST of the present invention to be 168±14.6 nm and the potential to be -33.6±5.2 mV.

[0056] Experimental Example 1: Verification of drug release characteristics of hyaluronic acid polymer micelles:

[0057] Nile red (NR) was used as a model drug, and the drug loading method described in Example 2 was followed at a 5% (w / w) dosage to prepare a Nile red-loaded micelle solution (NR@HSST). Simultaneously, a series of reduced glutathione solutions (0 mM, 10 mM) in PBS were prepared using deoxygenated PBS buffer (pH = 7.4). Next, 90 μL of the NR@HSST solution was pipetted into a 96-well plate, followed by 10 μL of the reduced glutathione solution in PBS. The plates were incubated at 37°C for 10, 30, and 60 minutes, respectively. Fluorescence changes of the model drug, Nile red (Ex = 579 nm, Em = 610-640 nm), were observed using an in vivo imaging system to indirectly investigate drug release.

[0058] The results are as follows Figure 3 shown. Figure 3 The fluorescence intensity of NR@HSST incubated with PBS solutions containing different GSH concentrations (0 mM and 10 mM) showed a time- and concentration-dependent relationship: within the same incubation time, the fluorescence intensity in the wells increased with increasing GSH concentration; at the same incubation concentration, the fluorescence intensity in the wells increased with time. This suggests that the redox-sensitive hyaluronic acid derivative HSST is unstable in a reducing environment due to the reduction and cleavage of disulfide bonds, resulting in rapid drug release.

[0059] Experimental Example 2: In vitro release verification of drug-loaded micelles:

[0060] 1 M sodium salicylate PBS buffer solution (pH = 7.4) was used as the release medium to investigate the release behavior of paclitaxel from the drug-loaded micelles under normal physiological conditions and reducing conditions.

[0061] 1 mL of drug-loaded micelle (K-PTX@HSST) solution was transferred to a dialysis bag (MWCO 3.5 kDa) and placed in a centrifuge tube containing 15 mL of release medium containing reduced glutathione (0 mM and 10 mM). The mixture was shaken at 100 rpm at 37°C. Samples were taken at pre-determined time points (1, 3, 6, 10, 24, 36, and 48 h) and filtered through a 0.22 μm filter. After sampling, the entire release medium was replaced to ensure that paclitaxel sink conditions were achieved. High-performance liquid chromatography (HPLC) was used to determine the drug concentration in the samples, and the cumulative release amount and percentage of drug released were calculated. Chromatographic conditions were: C18 reversed-phase column; mobile phase: acetonitrile:water = 45:55 (v / v); flow rate: 1.0 mL / min; column temperature: 25°C; injection volume: 20 μL.

[0062] The results are as follows Figure 4 shown. Figure 4 The results showed that under the condition of 10 mM GSH, the PTX release rate reached 65% within 10 h.

[0063] Experimental Example 3: Verification of in vitro anti-tumor effect:

[0064] The CCK8 assay was used to detect the cytotoxicity of blank micelles and drug-loaded micelles to TNBC tumor cells (4T1) and normal cells (LO2).

[0065] Tumor cells 4T1 and normal cells LO2 in the logarithmic growth phase were digested with trypsin and the cells were divided into 5×10 3 Cells were seeded at a density of 100 μg / well in a 96-well cell culture plate and cultured overnight in a 37°C, 5% CO2 incubator. After the cells adhered and grew, 20 μL of HSST, PTX, Kira6, and K-PTX-HSST at different concentrations were added to the 96-well plate to achieve final PTX concentrations of 50, 25, 5, 2.5, 0.5, and 0.05 μg / mL, respectively. Five replicate wells were set up in each group and the cell viability was detected using the CCK8 kit.

[0066] The results are as follows Figure 5 As shown in the results, it can be seen that K-PTX-HSST has a strong killing effect on tumor cells and has a mild toxicity to normal cells.

[0067] Experimental Example 4: Validation of CD44 Active Targeting of Hyaluronic Acid Micelles in Tumor Mouse Models:

[0068] The CT26 tumor mouse model was used as the model animal to investigate the CD44 receptor-mediated active tumor targeting of hyaluronic acid micelles in the animal body.

[0069] (1) Accurately weigh 10 mg of ICG and dissolve it in 5 mL of DMSO to obtain a 2 mg / mL ICG / DMSO solution; aspirate 2.5 mL, add 8 mg of DCC and 5 mg of DMAP, and stir at room temperature for 1 hour to activate the sulfonic acid groups; prepare a 10 mg / mL hyaluronic acid micelle solution HSST, take 5 mL and slowly add it dropwise to the above reaction solution, stir magnetically for 24 hours, and dialyze (MWCO 3500) to remove unreacted ICG and impurities; after freeze-drying, ICG-HSST labeled hyaluronic acid micelles were obtained.

[0070] (2) Mice with tumors of appropriate size were divided into targeting and blocking groups.

[0071] Targeting group: mice were injected with 200 μL of 1 mg / mL ICG-HSST hyaluronic acid micelle solution via tail vein;

[0072] Blocking group: After the free hyaluronic acid solution (200 μL, 5 mg / mL) was injected 0.5 h in advance, 200 μL of 1 mg / mL ICG-HSST hyaluronic acid micelle solution was injected into the tail vein of the mice as in the targeting group.

[0073] (3) After injection of ICG-HSST hyaluronic acid micelle solution, the two groups were observed and photographed at the specified time (6, 24 and 48 hours) using a small animal in vivo imaging device (the filter was ICG, the excitation wavelength was 704nm, the emission wavelength was 740-950nm, and the intermittent wavelength was 10nm). Among them, the mice were killed after 6, 24 and 48 hours, and the organs (heart, liver, spleen, kidney, lung, brain, intestine, bone, tumor, blood) were photographed using an in vivo imaging device and weighed. The fluorescence distribution intensity of hyaluronic acid micelles in each tissue was calculated using a 5% injection dose as the control standard.

[0074] In this experiment, ICG-HSST was injected into the tail vein and the fluorescence distribution in the tumor mouse model was observed to evaluate the in vivo active targeting ability of HSST to the CD44 receptor.

[0075] The results are as follows Figure 6 As shown. Figure 6 As can be seen from the figure, as time goes on, the fluorescence in the tumor shows a trend of first increasing and then decreasing; and in the targeting group, the fluorescence intensity in the tumor is always stronger than that in the blocking group, indicating that the accumulation of hyaluronic acid micelles in the tumor is mediated by the CD44 receptor.

[0076] Experimental Example 5: Verification of anti-tumor effect in vivo:

[0077] A 4T1 orthotopic tumor mouse model was established. Treatment was initiated after tumor formation, and tumor growth was observed and recorded. After tumor metastasis, the mice were randomly divided into five groups and injected with saline, HSST, PTX, Kira6, and K-PTX-HSST, respectively. Tumor volume and body weight were recorded.

[0078] The results are as follows Figure 7 、 Figure 8 The results show that K-PTX-HSST has the strongest tumor inhibition effect (tumor inhibition rate in vivo reaches 78%), and the body weight of mice remains basically unchanged, indicating that K-PTX-HSST has good safety.

[0079] In summary, the multifunctional nanomicelles of this invention can be used to treat triple-negative breast cancer (TNBC) by targeting cancer stem cells (CSCs), reversing the immunosuppressive microenvironment, and precisely delivering chemotherapeutic drugs. They exhibit a strong killing effect on TNBC tumor cells, with an in vivo tumor inhibition rate of 78%, and significantly reduced systemic toxicity, demonstrating outstanding potential for clinical translation.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A multifunctional nanomicelle targeting TNBC, characterized in that: It includes a carrier skeleton and a loaded drug; the carrier skeleton is based on hyaluronic acid HA and is composed of cystamine CYS and vitamin E succinate α-TOS connected by amide bonds; the loaded drug is paclitaxel PTX and IRE1α-XBP1 pathway inhibitor Kira6.

2. The multifunctional nanomicelle targeting TNBC according to claim 1, characterized in that The hyaluronic acid is connected to CYS and α-TOS via an amide bond to form a redox-sensitive polymer HSST, which serves as the carrier skeleton.

3. The multifunctional nanomicelle targeting TNBC according to claim 2, characterized in that The particle size of the HSST is 100 to 150 nm, and the surface potential is -20 to -40 mV.

4. The multifunctional nanomicelle targeting TNBC according to claim 1, characterized in that In the loaded drug, the mass ratio of PTX to Kira6 is (1-3):

1.

5. The multifunctional nanomicelle targeting TNBC according to any one of claims 1 to 4, characterized in that The nano micelles loaded with drugs have a particle size of 150 to 200 nm and a surface potential of -20 to -40 mV.

6. A method for preparing a multifunctional nanomicelle targeting TNBC according to any one of claims 1 to 5, characterized in that: The steps include: (1) Carboxyl activation Dissolve HA, EDC, and NHS in PBS and react at room temperature for 0.5-1.5 h; (2) Cystamine coupling Add CYS, react for 20-28 hours, dialyze and freeze-dry to obtain HA-CYS; (3) α-TOS modification α-TOS was activated by EDC / NHS, coupled with HA-CYS, and purified by ethanol precipitation to obtain HSST; (4) Drug loading PTX and Kira6 are dissolved in ethanol, mixed with an HSST aqueous solution, and freeze-dried to obtain the TNBC-targeting multifunctional nanomicelles.

7. The method for preparing multifunctional nanomicelles targeting TNBC according to claim 6, characterized in that: In the step (1), HA, EDC and NHS are dissolved in PBS at pH 7.4 at a molar ratio of 1:5:

5.

8. The method for preparing multifunctional nanomicelles targeting TNBC according to claim 6, characterized in that: In the step (2), the molar ratio of CYS to HA is 10:

1.

9. The method for preparing multifunctional nanomicelles targeting TNBC according to claim 6, characterized in that: In the step (4), after PTX and Kira6 are dissolved in ethanol, the mass ratio of the mixed solution to the HSST aqueous solution is 1:

5.

10. Use of the TNBC-targeting multifunctional nanomicelle according to any one of claims 1 to 5 in preparing a drug for treating triple-negative breast cancer.

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