Doxorubicin and shikonin co-delivery nanomicelles and preparation method and application thereof

Abstract

The invention discloses a doxorubicin and shikonin co-delivery nano-micelle, and its preparation method comprises the following steps: first, FA-PEG-COOH, doxorubicin, N,N'-carbonyldiimidazole, 4-lutidine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride are used as raw materials to synthesize PEG grafted with folic acid and doxorubicin, and then shikonin is loaded on the PEG grafted with folic acid and doxorubicin, that is, the doxorubicin and shikonin co-delivery nano-micelle of the present invention has pH sensitivity, can realize the localized release of drugs, has good tumor targeting specificity, and at the same time, it can significantly increase the uptake of DOX by tumor cells, maintain the toxic concentration of DOX in the tumor cytoplasm, thereby effectively improving the therapeutic efficiency of tumor drugs, and can be applied to the preparation of drugs for treating breast cancer, especially triple-negative breast cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a doxorubicin and shikonin co-delivery nano-micelle capable of simultaneously delivering doxorubicin and shikonin, and a preparation method and application thereof. Background Art

[0002] Currently, breast cancer is still the most common tumor in women. Among them, triple-negative breast cancer (TNBC) has limited efficacy in treatment with classical drugs (such as doxorubicin) and has a poor prognosis due to the lack of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER-2). At the same time, the treatment of TNBC is prone to drug resistance.

[0003] Doxorubicin (DOX) is a first-line small molecule drug for breast cancer, and its therapeutic effect is mainly exerted by upregulating the epithelial-mesenchymal transition (EMT) pathway. Clinical studies have shown that DOX has dose-related cardiotoxicity and is prone to drug resistance when used to treat breast cancer. The cardiotoxicity of DOX can be effectively reduced by encapsulating DOX in liposomes and administering it in the form of liposome nanomedicines. However, the efficacy of the liposomes in the treatment of breast cancer is only comparable to that of free doxorubicin, and the efficacy still needs to be improved.

[0004] Shikonin (SKN) is a natural bioactive substance present in the root of Lithosperraum erythrorhizon Sieb.et Zucc., a plant of the Boraginaceae family, and plants such as Arnebia euchroma (Royle) Johnst. It is mainly used clinically to treat acute and chronic hepatitis. Chinese patent CN113181117B discloses a shikonin and anthracycline chemotherapy drug co-loaded liposome, which has the effect of enhancing efficacy and reducing toxicity when the co-loaded liposome is used to treat melanoma by encapsulating shikonin and anthracycline drugs mitoxantrone or doxorubicin in liposomes at the same time. However, due to the lack of drug targeting, the dose required to reach the effective dose is still very high, and its effect for treating breast cancer is unknown. In addition, the effect of promoting tumor cell uptake when the co-loaded liposome is used to treat melanoma is not obvious, and its therapeutic effect for tumors still needs to be further improved. Summary of the invention

[0005] The first object of the present invention is to provide a doxorubicin and shikonin co-delivery nanomicelle to solve at least one of the above technical problems.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned doxorubicin and shikonin co-delivery nanomicelles to solve at least one of the above-mentioned technical problems.

[0007] The third object of the present invention is to provide the use of the above-mentioned doxorubicin and shikonin co-delivery nano-micelles in the preparation of drugs for treating breast cancer, so as to solve at least one of the above-mentioned technical problems.

[0008] According to a first aspect of the present invention, there is provided a doxorubicin and shikonin co-delivery nanomicelle, the preparation method of which comprises the following steps:

[0009] Dissolve doxorubicin and N,N'-carbonyldiimidazole (CDI) in DMSO, and stir at 35-45°C for 2-6 hours to obtain a mixed solution I;

[0010] Dissolve FA-PEG-COOH in DMSO containing 4-dimethylpyridine (DMAP) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), activate for 20-45 minutes, and obtain a mixed solution II;

[0011] Mixing the mixed solution I and the mixed solution II, reacting at a temperature of 35-45° C. for 20-30 h, dialyzing the reaction product in deionized water (3000 Da) for 4-8 h, and freeze-drying to obtain FA-PEG-DOX;

[0012] FA-PEG-DOX and shikonin were dissolved in DMSO and dialyzed in deionized water (7000Da) for 3-6 hours to obtain doxorubicin and shikonin co-delivery nanomicelles.

[0013] The results of drug release experiments show that the drug release rate of the doxorubicin and shikonin co-delivered nanomicelles provided by the present invention is low in PBS at pH = 7.4, but the drug release is significantly increased under acidic conditions, and it is pH sensitive, and can achieve localized release of the drug. The results of in vitro anti-tumor efficacy tests show that the therapeutic effect of the doxorubicin and shikonin co-delivered nanomicelles provided by the present invention on tumors is significantly higher than that of the free double drugs, and can effectively inhibit the vitality and metastasis of TNBC. In addition, the results of cell uptake experiments show that the doxorubicin and shikonin co-delivered nanomicelles provided by the present invention can significantly increase the cell uptake of DOX, and can maintain the toxic concentration of DOX in the tumor cytoplasm, achieving long-lasting cell uptake.

[0014] In some embodiments, the mass ratio of doxorubicin to shikonin may be 1:3 to 1:1.

[0015] In some embodiments, the mass ratio of doxorubicin to shikonin is 1: 1. Thus, the synergistic effect of the drugs is most significant and the anti-tumor effect is best.

[0016] In some embodiments, the mass ratio of doxorubicin to N,N'-carbonyldiimidazole may be 1:3 to 1:2.

[0017] In some embodiments, the mass ratio of FA-PEG-COOH to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride can be 2:1 to 5:1; the mass ratio of 4-lutidine to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride can be 1:2 to 1:1.

[0018] In some embodiments, the mass ratio of doxorubicin to FA-PEG-COOH can be 1:10 to 1:4. The particle size of the nanomicelles can be adjusted by adjusting the ratio of DOX to FA-PEG-COOH to prepare nanomicelles with a suitable particle size.

[0019] In some embodiments, the mass ratio of doxorubicin to FA-PEG-COOH can be 1: 10. Thus, the hydrodynamic size of the prepared nanomicelles is small, not greater than 200 nm, and the prepared nanomicelles have stronger tumor targeting.

[0020] In some embodiments, the molecular weight of FA-PEG-COOH may be 2000-3000 Da, preferably 2000 Da.

[0021] In some embodiments, the preparation method of FA-PEG-COOH may include the following steps:

[0022] Folic acid (FA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) were added to DMSO and activated at 35-45°C for 20-45 minutes; then NH 2 -PEG-COOH to obtain a mixed solution, dialyze the mixed solution in deionized water (3000 kDa) for 4-8 h, and freeze-dry to obtain FA-PEG-COOH.

[0023] The doxorubicin and shikonin co-delivery nanomicelles provided by the present invention have higher anti-TNBC activity than free drugs at the same drug concentration, can significantly increase the uptake of DOX by triple-negative breast cancer cells, maintain the toxic concentration of DOX in the tumor cytoplasm, and inhibit the activity and metastasis of triple-negative breast cancer cells, have significant TNBC targeted therapeutic effects, and can be used to prepare drugs for treating breast cancer, especially triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 FA, NH 2- Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR) of PEG-COOH, FA-PEG-COOH, and FA-PEG-DOX;

[0025] Figure 2-4 The toxicity of free DOX, free SKN, and free DOX+free SKN combined drugs to MDA-MB-231 cells at different concentrations is shown in order. * P<0.05, ** P<0.01, *** P < 0.001 and *** P<0.0001 indicated a significant difference;

[0026] Figure 5 The results of the combined synergy score analysis were calculated using SynergyFinder 3.0;

[0027] Figure 6 Characterization diagrams of FPD NMs and SKN@FPD NMs, wherein (A) and (B) are the particle size and zeta potential results of FPD NMs measured by DLS, respectively; (C) and (D) are the particle size and zeta potential results of SKN@FPD NMs measured by DLS, respectively; (E) and (F) are TEM images of FPD NMs and SKN@FPD NMs, respectively;

[0028] Figure 7 The release graphs of DOX and SKN under in vitro physiological conditions (PBS, pH=7.4) and simulated tumor endosomal microenvironment (PBS, pH=5.5);

[0029] Figure 8 The cytotoxicity of free DOX, FA-PEG-DOX, free DOX+free SKN dual drug and SKN@FPD NMs to MDA-MB-231 cells, among which, * P<0.05, ** P<0.01, *** P < 0.001 and *** P<0.0001 indicated a significant difference;

[0030] Figure 9-10 When the concentration of DOX is 3 μg / mL and the concentration of DAPI is 1 μg / mL, the uptake effect of cells on free DOX, free DOX+free SKN dual drugs and SKN@FPD NMs is shown in Figure 2. * P<0.05, ** P<0.01, *** P < 0.001 and ***P<0.0001 indicated a significant difference;

[0031] Figure 11-12 This is the result of the cell migration experiment, where * P<0.05, ** P<0.01, *** P < 0.001 and *** P<0.0001 indicates a significant difference. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below with reference to specific embodiments, but the embodiments of the present invention are not limited thereto.

[0033] The materials involved in the following examples, except for those specially noted, can be obtained from commercial channels. For process parameters not specially noted, conventional techniques can be used.

[0034] In the present invention, doxorubicin (DOX), folic acid (FA), dimethyl sulfoxide (DMSO), N,N'-carbonyldiimidazole (CDI) and 4-dimethylpyridine (DMAP) were purchased from Aladdin (Shanghai, China); NH 2 -PEG-COOH (2000Da) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; cell counting kit-8 (CCK-8) and 4',6-diamidino-2-phenylindole (DAPI) were purchased from Beyotime Biotechnology Co., Ltd.; DMEM-H and fetal bovine serum were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0035] Cell culture: Triple negative breast cancer cells (MDA-MB-231) were inoculated into 25T culture flasks and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The culture flasks were placed in a carbon dioxide incubator (37°C, 5% CO 2 ) were used for passaging using trypsin containing EDTA.

[0036] Example 1 Preparation of FA-PEG-COOH

[0037] The steps include:

[0038] 1 g FA and 1 g EDCI were added to 30 mL DMSO and activated at 40 °C for 30 min. Then 2 g NH 2 -PEG-COOH to obtain a mixed solution, which was dialyzed in deionized water (3000Da) for 6 hours and then freeze-dried to obtain FA-PEG-COOH.

[0039] Example 2 Preparation of FA-PEG-DOX Nanomicelles

[0040] The steps include:

[0041] (1) Dissolve 0.1 g DOX and 0.20 g CDI in 10 mL DMSO and stir at 40 °C for 4 h to obtain a mixed solution I;

[0042] (2) 1.0 g of FA-PEG-COOH prepared in Example 1 was dissolved in DMSO containing 0.1 g of DMAP and 0.2 g of EDCI, and activated for 30 minutes until the mixture was uniformly mixed to obtain a mixed solution II;

[0043] (3) Mixed solution I and mixed solution II were stirred and mixed, and reacted at 40° C. for 1 day; finally, the reaction product was dialyzed in deionized water (3000 Da) for 6 hours and freeze-dried to obtain FA-PEG-DOX;

[0044] (4) 10 mg of FA-PEG-DOX was dissolved in 5 mL of DMSO and dialyzed in deionized water (7000 Da) for 4 h to obtain FA-PEG-DOX nanomicelles (FPD NMs).

[0045] Example 3 Preparation of SKN@FA-PEG-DOX Nanomicelles

[0046] The steps include:

[0047] (1) Dissolve 0.1 g DOX and 0.20 g CDI in 10 mL DMSO and stir at 40 °C for 4 h to obtain a mixed solution I;

[0048] (2) 1.0 g of FA-PEG-COOH prepared in Example 1 was dissolved in DMSO containing 0.1 g of DMAP and 0.2 g of EDCI, and activated for 30 minutes until the mixture was uniformly mixed to obtain a mixed solution II;

[0049] (3) Mixed solution I and mixed solution II were stirred and mixed, and reacted at 40° C. for 1 day; finally, the reaction product was dialyzed in deionized water (3000 Da) for 6 hours and freeze-dried to obtain FA-PEG-DOX;

[0050] (4) FA-PEG-DOX and 0.1 g SKN were dissolved in 50 mL DMSO and dialyzed in deionized water (7000 Da) for 4 h to obtain SKN@FA-PEG-DOX nanomicelles (SKN@FPD NMs).

[0051] Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy ( 1H NMR) to detect the structure of nanomaterials. The sample was mixed with dry potassium bromide solid, ground and pressed into thin sheets for FTIR measurement; dissolved in deuterated DMSO for measurement 1 H-NMR, infrared spectrum scanning range is 400~4000cm -1 The results are as follows Figure 1 shown.

[0052] For FA-PEG-COOH, FITR spectroscopy confirmed its -1 There is an ether bond COC stretching vibration peak at 1685cm -1 and 1718cm -1 There are stretching vibration peaks of polyamide and free carbonyl from FA at 8.65ppm. Meanwhile, the signals of FA and FA-PEG-COOH at 8.65ppm come from heterocyclic rings, the signals at 7.65ppm and 6.64ppm come from hydrogen on the benzene ring, and the signal at 6.94ppm comes from secondary amine substituted at the ortho position on the benzene ring. These results prove the successful synthesis of FA-PEG-COOH. For FA-PEG-DOX, FITR spectrum proves that the peak at 1281cm -1 There is a vC-O peak of phenol at 1728 cm -1 The carbonyl stretching vibration peak of DOX at 5.00 ppm proved that DOX was successfully grafted. Meanwhile, the unreacted hydroxyl signals and OC signals of hydroxyl at 5.82 ppm, 5.34 ppm, 5.05 ppm and 4.12 ppm, as well as the characteristic methyl band from DOX at 4.00 ppm indicated that the synthesis of FA-PEG-DOX was successful.

[0053] Experimental Example 1 Cytotoxicity and synergistic effect of free doxorubicin (DOX) and shikonin (SKN)

[0054] MDA-MB-231 cells were seeded into 96-well plates (5000 cells per well), and then DOX and SKN were dispersed in complete medium, and the cells were treated with equal volumes of complete medium containing different concentrations of drugs for 2 days. Then, the above medium was replaced with fresh complete medium containing 10% CCK-8 reagent, and the absorbance (OD) value at 490nm was detected after treating the cells for 1 hour. In subsequent experiments, similar methods were used to evaluate the efficacy of free drugs and nanomicelles.

[0055] The cell activity was calculated as follows:

[0056]

[0057] Among them, a culture medium containing 10% CCK-8 reagent was added to the wells without cells as a blank group, and the same culture medium containing CCK-8 reagent was added to the wells with cells not treated with drugs as a control group.

[0058] In addition, to explore the synergistic efficacy of DOX and SKN in the treatment of TNBC, the synergistic effect of free DOX and SKN was evaluated by treating with different concentration combinations of the two drugs, detecting cell activity using CCK-8 reagent, and calculating the synergistic effect score using SynergyFinder 3.0.

[0059] The results are as follows Figure 2-5 shown.

[0060] The experimental results show that with the increase of drug concentration, the activity of MBA-MD-231 cells gradually decreases. 50 0.1356±0.0100μg / mL, less than the IC of SKN 50 (0.3434±0.0203μg / mL).

[0061] When MBA-MD-231 cells were treated with DOX and SKN together, the cell survival rate decreased to 0.4328±0.068 when the concentrations of DOX and SKN were 0.1μg / mL and 0.1μg / mL, respectively, while the cell survival rate was 0.2151±0.0267 under the treatment of 0.2μg / mL DOX and 0.3μg / mL SKN. Figure 5 As shown in the figure, DOX and SKN have a certain synergistic effect, with a synergistic effect score of 3.75. When the concentration ratio of DOX and SKN is 1:1, the synergistic effect of the drugs is more significant.

[0062] Experimental Example 2 Characterization of NMs

[0063] Dynamic light scattering (DLS) was used to measure the hydrodynamic size and zeta potential of the nanomicelles prepared in Example 2 and Example 3. The parameters during the measurement were set as follows:

[0064] Hydrodynamic dimensions: wavelength 658 nm, temperature 25 ± 0.1 °C, DLS angle 90°;

[0065] Zeta potential: 1.4 V / cm, 13.0 mA, 25 ± 0.1 °C.

[0066] For morphological measurements, NMs were dropped on a copper grid, dried, scanned by a transmission electron microscope (TEM), and photographed.

[0067] The results are as follows Figure 6 shown.

[0068] The hydrodynamic size of FPD NMs was 106.8±0.8nm, and the PDI was 0.226±0.009; the hydrodynamic size of SKN@FPD NMs was 121.8±1.1nm, and the PDI was 0.216±0.020. Compared with blank NMs, the size of drug-loaded NMs increased.

[0069] Transmission electron microscopy images show that the two NMs have regular shapes and uniform sizes. At the same time, the two NMs have similar zeta potentials, with the zeta potential of FPD NMs being -6.44±0.23mV and that of SKN@FPD NMs being -6.33±0.16mV.

[0070] Moreover, FPD NMs and SKN@FPD NMs exhibited high stability at low temperature for 3 days.

[0071] Experimental Example 3 Evaluation of drug loading and drug release of NMs

[0072] In order to detect the drug loading, the degree of substitution of DOX in FA-PEG-DOX was first detected. DOX hydrochloric acid solutions (pH = 5.5) with concentrations of 2, 4, 6, 8 and 10 μg / mL were prepared, and their absorbance at 480nm was detected on a UV-visible absorption instrument, and then a standard curve was drawn. The absorbance of 20 μg / mL FA-PEG-DOX solution (pH = 5.5) at 488nm was detected, and the substitution amount of DOX was calculated according to the following formula:

[0073]

[0074] The dual-wavelength spectrophotometry method was used to determine the concentration of the dual drugs in SKN@FPD NMs. By detecting the absorbance of the dual drugs at 480nm and 565nm, a standard curve was drawn and the drug loading was calculated according to the following formula:

[0075]

[0076] According to the above formula, it can be calculated that the content of DOX in FA-PEG-DOX prepared in Example 2 is 9.78±0.14%. At the same time, the drug loadings of DOX and SKN in SKN@FPD NMs prepared in Example 3 are 8.86±0.21% and 9.43±0.13%, respectively, and the total drug loading of SKN@FPD NMs is close to 20%.

[0077] In order to detect the drug release of SKN@FPD NMs, 5 mL of NMs were placed in a dialysis bag (7000 Da), dialyzed in 30 mL of PBS (pH = 7.4 or pH = 5.5), and shaken on a shaker (37 ° C, 75 rpm). At 0, 0.5, 2, 4, 8, 16, 24 and 48 h, 30 mL of fresh PBS was used to replace the old PBS, and the pH of the old PBS was adjusted to 5.5, and then the absorbance was detected at 480 nm and 565 nm. The release amount of dual-drug loaded drugs was calculated as follows:

[0078]

[0079] Among them, Q t is the drug release rate at t hours; V t and C t is the volume of PBS outside the dialysis bag and the drug concentration in PBS at t hours; V db is the volume of PBS in the dialysis bag; Cdb(Drug) is the initial concentration of the nanosample. t=0,0.5,1,……,n,……,48 hours; V 0 , C 0 Both are 0.

[0080] The results are as follows Figure 7 As shown in the figure, in the first 8 h, free DOX and SKN were rapidly released in PBS at pH = 7.4, with release rates of 67.57 ± 1.45% and 72.12 ± 2.61%, respectively; while the release rate of free DOX in PBS at pH = 5.5 increased significantly to 77.36 ± 1.85%, and the release rate of free SKN in PBS at pH = 5.5 was 65.32 ± 2.08%. For SKN@FPD NMs, in PBS at pH = 7.4 and pH = 5.5, the release rates of DOX were 38.56 ± 1.05% and 43.96 ± 1.42%, respectively; the release rates of SKN were 50.11 ± 1.33% and 71.72 ± 1.65%, respectively. The results showed that the acidic environment significantly increased the release rate of SKN. SKN@FPD NMs could effectively delay the release of drugs under physiological conditions, while significantly increasing the release of drugs in the tumor microenvironment or tumor endosomes.

[0081] Experimental Example 4 In vitro cytotoxicity of NMs

[0082] CCK-8 kit was used to detect the in vitro toxicity of NMs and free drugs on TNBC.

[0083] The results are as follows Figure 8As shown, at the maximum free DOX concentration (0.094 μg / mL), the cell survival rate of MBA-MD-231 was 0.5706±0.0283, while at the maximum FA-PEG-DOX concentration, the cell survival rate was 0.6394±0.0202. The PEG bond does not seem to have a significant effect on the toxicity of DOX. In addition, the survival rate of MBA-MD-231 cells at the maximum concentration of dual free drugs was 0.4252±0.0246; in comparison, the survival rate of cells at the corresponding drug concentration of SKN@FPD NMs was significantly reduced to 0.3730±0.0096. The results show that the SKN@FPD NMs provided by the present invention have higher anti-TNBC activity than free drugs at the same drug concentration.

[0084] Experimental Example 5 In vitro cellular uptake evaluation

[0085] MDA-MB-231 cells were seeded into 24-well plates (2 × 10 5 cells). When these cells grew to 50%, free DOX, free DOX+free SKN, and SKN@FPD NMs with a DOX concentration of 3 μg / mL were added for treatment. The treated cells were stained with DAPI (1 μg / mL) and photographed at 2 hours, 4 hours, and 6 hours, and the cell uptake was calculated using Image J software.

[0086] The results are as follows Figure 9-10 As shown. The cells in the free DOX group continuously took up DOX within 4 hours. The free dual-drug group had a similar DOX cellular uptake effect as the free DOX group, and the free DOX mainly entered the cell nucleus. Compared with the free drug, SKN@FPD NMs had a higher continuous cellular uptake effect within 4 hours. In addition, the DOX in SKN@FPD NMs was partially distributed in the cytoplasm near the cell nucleus, forming a weak red light around the cell nucleus. The above results indicate that SKN@FPD NMs can significantly increase the cellular uptake of DOX and maintain the toxic concentration of DOX in the tumor cytoplasm.

[0087] Test Example 6 Cell migration test

[0088] MDA-MB-231 cells were seeded into 24-well plates (5 × 10 5 When these cells grew to 80%, they were treated with free DOX, free DOX+free SKN, and SKN@FPD NMs at the corresponding drug concentrations, and cells without drug treatment were used as controls. The cells were photographed (100×) at 0, 6, and 12 hours, and their migration rates were calculated using Image J software.

[0089] The results are as follows Figure 11-12 As shown. The control group cells showed obvious metastasis at 6 and 12 hours. However, the cells treated with free DOX showed no obvious metastasis within 12 hours. Meanwhile, the dual free drug group and NMs group showed almost no metastasis compared with the DOX group or the control group within 12 hours. In addition, the cell morphology of MDA-MB-231 changed significantly after 6h or 12h of treatment with synergistic drugs and SKN@FPD NMs.

[0090] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing doxorubicin and shikonin co-delivery nanomicelles, characterized in that: The steps include: Step 1: dissolving doxorubicin and N,N'-carbonyldiimidazole in DMSO to obtain a mixed solution I; Step 2: dissolving FA-PEG-COOH in DMSO containing 4-lutidine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, activating for 20-45 minutes to obtain a mixed solution II; Step 3: Mix the mixed solution I and the mixed solution II, react at a temperature of 35-45° C. for 20-30 hours, dialyze the reaction product in deionized water for 4-8 hours, and freeze-dry to obtain FA-PEG-DOX; Step 4: Dissolve FA-PEG-DOX and shikonin in DMSO and dialyze in deionized water for 3-6 hours to obtain doxorubicin and shikonin co-delivery nanomicelles; The preparation method of FA-PEG-COOH comprises the following steps: Folic acid and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride are added to DMSO and activated at a temperature of 35-45°C for 20-45 minutes; then NH2-PEG-COOH is added to obtain a mixed solution, the mixed solution is dialyzed in deionized water for 4-8 hours, and freeze-dried to obtain FA-PEG-COOH.

2. The method for preparing doxorubicin and shikonin co-delivery nanomicelles according to claim 1, characterized in that: The mass ratio of doxorubicin to shikonin is 1:3 to 1:

1.

3. The method for preparing doxorubicin and shikonin co-delivery nanomicelles according to claim 2, characterized in that: The mass ratio of doxorubicin to shikonin is 1:

1.

4. The method for preparing doxorubicin and shikonin co-delivery nanomicelles according to any one of claims 1 to 3, characterized in that: The mass ratio of doxorubicin to N,N'-carbonyldiimidazole is 1:3 to 1:

2.

5. The method for preparing doxorubicin and shikonin co-delivery nanomicelles according to claim 4, characterized in that: The mass ratio of FA-PEG-COOH and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in step 2 is 2:1 to 5:1; the mass ratio of 4-lutidine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:2 to 1:

1.

6. The method for preparing doxorubicin and shikonin co-delivery nanomicelles according to claim 5, characterized in that: The mass ratio of doxorubicin to FA-PEG-COOH is 1:10 to 1:

4. 7 . The doxorubicin and shikonin co-delivery nanomicelles prepared according to the method for preparing doxorubicin and shikonin co-delivery nanomicelles according to any one of claims 1 to 6 .

8. Use of the doxorubicin and shikonin co-delivery nanomicelles according to claim 7 in the preparation of a drug for treating breast cancer.

9. The use according to claim 8, characterized in that: The breast cancer is triple-negative breast cancer.

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