Shikonin-targeted delivery micelles, and preparation method and use thereof

Abstract

The present application relates to a shikonin targeting micelle, which is coated by red blood cell membrane or modified red blood cell membrane containing loaded shikonin cationic nanoparticles, in which shikonin and block copolymer TPP-PEG n1 -PCL m1 , TPP-PEG n2 -Hyd-PCL m2 , mPEG n3 -PCL m3 form a hydrophobic inner core, and PEG forms a hydrophilic layer in the middle. The micelle of the present application can effectively enrich the active ingredient of shikonin at the treatment target, which is beneficial to improve the clinical treatment effect and compliance, and to increase efficacy and reduce toxicity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to a targeted delivery micelle of shikonin, its preparation method, and its uses. Background Technology

[0002] Triple-negative breast cancer (TNBC) is a highly aggressive, frequently recurrent, and poorly prognostic subtype of breast cancer. Currently available drugs lack effective targets, leading to TNBC being considered incurable. Therefore, new therapeutic strategies are urgently needed for TNBC treatment. Mitochondria, organelles, have been shown to influence almost all steps in tumorigenesis and are a promising target for developing anticancer drugs. In the case of TNBC, the most common metabolic phenotype in tumor cells is the mitochondrial type, followed by glutamine breakdown and glycolysis. Furthermore, the progression of TNBC is accompanied by enhanced mitochondrial biosynthesis, while inhibition of mitochondrial biosynthesis effectively reduces the invasive and metastatic capabilities of TNBC cells. Therefore, these clinical studies suggest that mitochondrial-targeted drug therapy may be a potential strategy to overcome non-targeted TNBC.

[0003] Triphenylphosphine (TPP), a cationic compound, is commonly used as a specific ligand to mediate mitochondrial targeting of nanoparticles. It has been reported to assist nanoparticles in passing through membrane pores under the guidance of a negative mitochondrial membrane potential, promoting nanoparticle accumulation within the mitochondria. However, a major obstacle to developing TPP-modified mitochondrial-targeting nanoparticles is overcoming elimination from circulation to maximize tumor lesion distribution. In the presence of biological fluids, cationic nanoparticles form a “protein crown” on their surface, typically leading to nanoparticle clearance from circulation via the reticuloendothelial system (RES) or the mononuclear phagocyte system (MPS). For decades, numerous strategies have been proposed to overcome these challenges, such as evading immune surveillance by incorporating polyethylene glycol (PEG) into the nanoparticle surface and combining tumor cell-targeting components to promote nanoparticle accumulation in tumors. However, mounting evidence shows limitations in PEGylation and ligand-mediated active targeting strategies, including the tendency of repeated injections of PEGylated nanoparticles to induce anti-PEG immune responses and low active targeting efficiency due to uncontrolled modification.

[0004] Shikonin (SK) is the main active ingredient extracted from the root of *Lithospermum erythrorhizon*. Due to its potent inhibitory activity against various cancers, it has become a promising candidate anticancer drug. SK interferes with mitochondrial energy production and ROS generation, leading to the induction of apoptosis. Studies have found that SK effectively inhibits the growth and metastasis of TNBC cells both in vitro and in vivo, indicating that SK is an attractive bioactive compound for the treatment of TNBC. However, SK has high lipophilicity and is easily oxidized and polymerized, resulting in poor stability and low oral bioavailability. Therefore, to achieve the best anticancer effect on TNBC, it is necessary to develop a delivery material that assists SK in overcoming in vivo barriers and achieving specific accumulation at tumor lesions, particularly in the mitochondrial region of cells, thereby more effectively exerting the anticancer effect of shikonin. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a shikonin-targeted micelle, wherein the micelle consists of erythrocyte membranes or modified erythrocyte membranes coating cationic nanoparticles loaded with shikonin (SK), wherein the shikonin-loaded cationic nanoparticles contain shikonin and the block copolymer TPP-PEG. n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 mPEG n3 -PCL m3 The PCL end forms a hydrophobic core, and the PEG forms a hydrophilic layer in the middle.

[0006] According to an embodiment of the present invention, the erythrocyte membrane or modified erythrocyte membrane forms a biomimetic shell on its outer side under the electrostatic adsorption force of TPP. TPP not bound to the erythrocyte membrane has a targeting function for mitochondria.

[0007] According to an embodiment of the present invention, TPP is an abbreviation for PPh3Br-(CH2)4-COOH. TPP is related to NH2-PEG via a carboxyl group. n1 -PCL m1 They are linked via a condensation reaction. The TPP-PEG... n1 -PCL m1 The structure is as follows:

[0008] The TPP-PEG n1 -PCL m1 In this context, n1 is selected from 1000-5000, preferably from 2000-3500, for example, 2000; m1 is selected from 5000-10000, preferably from 5000-8000, for example, 6600; According to an embodiment of the present invention, the TPP-PEG n2 -Hyd-PCL m2 The structure is as follows:

[0009] The TPP-PEG n2 -Hyd-PCL m2 In this context, n2 is selected from 1000-5000, preferably 2000-3500, for example 3400; m2 is selected from 5000-10000, preferably 5000-8000, for example 6600.

[0010] The mPEG n3 -PCL m3 The structure is as follows:

[0011] n3 is selected from 1000-5000, preferably 2000-3500, for example 2000; m3 is selected from 2000-10000, preferably 5000-8000, for example 6600.

[0012] According to an embodiment of the present invention, the modified erythrocyte membrane is produced by FA-PEG. n4 -FA-modified erythrocyte membrane; wherein FA represents folic acid, and the FA-PEG n4 The FA structure is shown below: ; Wherein, n4 is selected from 500-8000, preferably from 1000-5000, for example 1000, 2000, 3400.

[0013] According to an embodiment of the present invention, the method for preparing the modified erythrocyte membrane (abbreviated as RBCm) includes the following steps: Using FA-PEG with different PEG lengths n4 -FA and RBCm are incubated at a 1:1 mass ratio. Preferably, premixing at 37°C for 20-40 min allows FA-PEG to be formed. n4 -FA is inserted into the outer membrane of RBCm.

[0014] According to an embodiment of the present invention, the method for preparing the modified erythrocyte membrane specifically includes one or more of the following steps: (a1) Collect red blood cells and separate the red blood cell membranes, and resuspend them in PBS (1X); (b1) Before modification, RBCm was subjected to ultrasonic bath treatment (200 W) for 4-10 minutes; (c1) Using FA-PEG n4 - FA and RBCm are incubated at a 1:1 mass ratio and premixed at 37°C for 20-40 minutes to form FA-PEG. n4 -FA is inserted into the outer membrane of RBCm; (d1) Ultrafiltration removal of unbound FA-PEG n4 -FA.

[0015] According to an embodiment of the present invention, the protein concentration of RBCm is quantified using a BCA protein analysis kit prior to incubation; the protein concentration is 1.5 mg / mL. According to an embodiment of the present invention, the FA-PEG n4 -FA is dissolved in DMSO at a concentration of 50-100 mg / mL, for example, 80 mg / mL.

[0016] According to an embodiment of the present invention, the TPP-PEG n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 The preparation method includes the following steps: (a2) Dissolve TPP and EDCl in organic solvent A respectively; (b2) Dissolve HOBT in organic solvent B; (c2) Add the solutions from steps (a) and (b) to the reaction vessel and stir at room temperature for 20-60 minutes; (d2) NH2-PEG n1 -PCL m1 (0.86g, 0.1mmol) or NH2-PEG n2 -Hyd-PCL m2 Dissolve in organic solvent B and add dropwise to the solution obtained in step (c), and stir overnight at room temperature under nitrogen. (e2) Dialyze the reaction mixture obtained in step (d) with deionized water (MWCO, 3500 Da) for 12-36 hours and freeze-dry to obtain TPP-PEG. n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 .

[0017] According to an embodiment of the present invention, the organic solvent A is selected from one or two of dimethyl sulfoxide and acetone; the organic solvent B is selected from one or two of tetrahydrofuran and acetone.

[0018] According to an embodiment of the present invention, in the cationic nanoparticles loaded with shikonin, the TPP-PEG n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 mPEG n3 -PCL m3 The mass ratio is 1-10:1-10:1-10; preferably, it is 1:1:2.

[0019] According to an embodiment of the present invention, the cationic nanoparticles loaded with shikonin are prepared by the following method: (a3) The TPP-PEG n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 mPEG n3 -PCL m3 SK was dissolved in organic solvent C, and deionized water was added while stirring; (b3) Stir at room temperature for 0.2-1 hour, evaporate the solution to remove organic solvent, centrifuge and ultrafilter for 10-40 min, and separate and remove unused SK through a 0.45µm-0.8μm filter.

[0020] According to an embodiment of the present invention, the organic solvent C is selected from one or more of acetone, dimethyl sulfoxide, and tetrahydrofuran; the block polymer TPP-PEG n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 mPEG n3 -PCL m3 The total mass ratio of the solution to SK is 1:10-50, preferably 1:15-25, for example 1:20; the mass-to-volume ratio of SK to the organic solvent is 1:1-4 mg / ml, for example 1:2 mg / ml. The centrifugal ultrafiltration is preferably performed three times at 3500×g using an Amicon Ultra-15 tube (MWCO 100kDa).

[0021] According to an embodiment of the present invention, the red blood cell membrane or modified red blood cell membrane coating adopts the following scheme: (a4) Add the cationic nanoparticles loaded with shikonin to the erythrocyte membrane or the modified erythrocyte membrane solution in PBS solution, rotate for 20-40 seconds, and incubate at 37°C for 20-40 min. (b4) Sonicate the solution; (c4) Using an extruder to extrude through a 200 nm polycarbonate film to receive ThTM / SK@RBCm and ThTM / SK@FP-RBCm.

[0022] According to an embodiment of the present invention, the ultrasonic conditions are 200 W in a water bath for 4-6 minutes; the mass ratio of the cationic nanoparticles loaded with shikonin to the erythrocyte membrane or the modified erythrocyte membrane is 10-80:1, preferably 10:1.

[0023] In another aspect, the present invention provides a method for preparing the shikonin-targeted micelles, the method comprising the following steps: (1) Preparation of block copolymer TPP-PEG n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 Preferably, the method described above is used; (2) Block copolymer TPP-PEG is used n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 mPEG n3 -PCL m3 Cationic nanoparticles loaded with shikonin were prepared; preferably, the method described above was used. (3) The cationic nanoparticles loaded with shikonin are coated with erythrocyte membrane or modified erythrocyte membrane; preferably, the method described above is used.

[0024] In another aspect, the present invention provides the use of the shikonin-targeting micelles in the preparation of drugs for treating cancer.

[0025] In another aspect, the present invention provides the application of the shikonin-targeting micelles in the preparation of drugs that inhibit mitochondrial biosynthesis in cancer cells.

[0026] According to embodiments of the present invention, the cancer includes epithelial cell-derived cancers, such as breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, oral cancer, esophageal cancer, small bowel cancer, gastric cancer, colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, skin cancer, prostate cancer, and renal cell carcinoma; preferably, the breast cancer is triple-negative breast cancer.

[0027] Beneficial effects This invention utilizes an effective delivery carrier system to efficiently concentrate the active ingredient of shikonin at therapeutic targets, thereby improving clinical treatment efficacy and compliance, and enhancing efficacy while reducing toxicity. Attached Figure Description

[0028] Figure 1 (a) Kaplan-Meier analysis of the difference in distant metastasis-free survival (DMFS) among TNBC patients with different POLG expression levels (n=434); (b) Multiplex fluorescence staining analysis of the differences in Pan-CK, PGC-1α, and POLG expression in TNBC tumor tissue microarrays. Cell nuclei were localized using DAPI staining; (c) Levels of PGC-1α and POLG expression in TNBC tumor tissues (n=39); (d) A positive correlation between PGC-1α and POLG expression in TNBC tumor tissues (R=0.597, P<0.0001, n=39); (e) Molecular docking of shikonin and POLG; (f) MST assay to detect the affinity of POLG for binding to shikonin; (g) Effect of shikonin on the inhibition of mitochondrial, cytoplasmic, and total POLG expression in TNBC cells MDA-MB-231.

[0029] Figure 2 (a) Particle size distribution of RBCm-encapsulated micelles in phosphate buffer media at pH 7.4 and 5.5; (b) Zeta potential of RBCm-encapsulated micelles in phosphate buffer media at pH 7.4 and 5.5 (n=3); (c) Protein content in RBCm, erythrocyte lysates, ThTM / SK@RBCm, ThTM / SK@FP-RBCm and ThTM / SK detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis; (d) UV-Vis spectra of free SK, RBCm, free FA, ThTM@FP-RBCm and ThTM / SK@FP-RBCm: (e, f, g) diameter, PDI (e), zeta potential (f), and LR, LC (g) of NM / SK, TM / SK, ThTM / SK, ThTM / SK@RBCm and ThTM / SK@FP-RBCm (n=3); (h) Transmission electron microscopy imaging of ThTM / SK, Morphology and size of ThTM / SK@RBCm and ThTM / SK@FP-RBCm (scale bar = 100 nm, scale bar = 50 nm, data are mean ± standard deviation).

[0030] Figure 3(a, b) The co-localization of micelles containing c6 (green fluorescence, black line) with lysosomes (red fluorescence, gray line) after co-culturing with MDA-MB-231 cells for 2 hours (a) and 4 hours (b), as photographed using the Operetta CLS high-content imaging system (scale bar = 50µm); (c, d) The co-localization of micelles containing c6 (green fluorescence, black line) with mitochondria (red fluorescence, gray line) after co-culturing with MDA-MB-231 cells for 2 hours (a) and 4 hours (b) (overlapping black and gray lines indicate co-localization), as photographed using the Operetta CLS high-content imaging system (scale bar = 50µm).

[0031] Figure 4 (a) Effects of free shikonin and shikonin-encapsulated drug carriers on mitochondrial content in MDA-MB-231 cells; (b) Effects of free shikonin and shikonin-encapsulated drug carriers on mitochondrial DNA (mtDNA) content in MDA-MB-231 cells; (c) Effects of free shikonin and shikonin-encapsulated drug carriers on ATP content in MDA-MB-231 cells; (d) Effects of free shikonin and shikonin-encapsulated drug carriers on MDA-MB-231 cell viability; (e) Effects of free shikonin and shikonin-encapsulated drug carriers on POLG and PGC-1α expression in mitochondria and cytoplasm in MDA-MB-231 cells; (f) Effects of free shikonin and shikonin-encapsulated drug carriers on MDA-MB-231 cell migration and invasion.

[0032] Figure 5 (a) The presence of micelles encapsulated in erythrocyte membranes in vivo; (b) Western blot analysis of the content of the protein marker CD47 in erythrocyte membranes, ThTM / SK@RBCm, ThTM / SK@FP-RBCm, and ThTM / SK formulations, where membrane proteins were analyzed using Na+. + -K +- ATPase was used as an internal control; (c) Transmission electron microscopy images of ThTM / SK@RBCm and ThTM / SK@FP-RBCm (scale bar = 50 nm), labeled with immunogold-labeled CD47, indicated by black dots; (d) Plasma concentration-time curves of SK after intravenous injection of free SK, NM / SK, ThTM / SK, ThTM / SK@RBCm, and ThTM / SK@FP-RBCm in mice (n=5). Data are mean ± SD. (e) Changes in the diameter of NM / SK, ThTM / SK, ThTM / SK@RBCm, and ThTM / SK@FP-RBCm over 12 h in PBS containing 50% fetal bovine serum at pH 7.4 (n=3); (f) Binding rate of erythrocytes to NM / C6, ThTM / C6, ThTM / C6@RBCm, and ThTM / C6@FP-RBCm in PBS at pH 7.4 for 24 h (n=3); (g) Binding rate of leukocytes to NM / C6, ThTM / C6, ThTM / C6@RBCm, and ThTM / C6@FP-RBCm in PBS at pH 7.4 for 24 h (n=3); Figure 6 (a) The distribution of DiR in mice orally inoculated with TNBC cells was observed using an in vivo imaging system 2, 6, 12, and 24 h after tail vein injection of ThTM / DiR, ThTM / DiR@RBCm, and ThTM / DiR@FP-RBCm; (b) The distribution of ThTM / DiR, ThTM / DiR@RBCm, and ThTM / DiR@FP-RBCm in tumors, heart, liver, lungs, and kidneys 24 h after intravenous injection; (c, d) The intensity of ThTM / DiR, ThTM / DiR@RBCm, and ThTM / DiR@FP-RBCm in tumors and visceral organs was analyzed using an IVIS Lumina III in vivo imaging system. Data are presented as mean ± SD, *P < 0.05.

[0033] Figure 7(a) Treatment timeline of MDA-MB-231 cells injected into the mammary fat pads of NOD / SCID mice for tumor growth studies; (b) Analysis of the effect of free SK and drug carriers containing SK on tumor growth by plotting tumor volume growth curves (n=6); (c) Tumor weight measured 14 days after drug administration (n=6); (d) Tumor tissue photographed 14 days after drug administration; (e) Timeline of establishing a lung metastasis model in NOD / SCID mice by tail vein injection of MDA-MB-231 (Luc 1) cells; (f) In vivo imaging of the lung metastasis model 7, 14 and 21 days after drug treatment. (g) Representative images of lung tissue from different treatment groups; (h) Statistical results of the number of lung metastatic nodules in different treatment groups (n = 3); (i) Representative H&E staining results of metastatic lung tissue (100×; n = 6, scale bar = 100 μm); (j) Calculation of the average area of ​​lung metastases based on H&E stained section images (n = 6); (k) Immunohistochemical detection of POLG and PGC-1α expression in tumors and lung tissue (400×; n = 6, scale bar = 50 μm); (l) Quantitative analysis of immunohistochemical staining using ImageJ software, with results representing relative staining intensity (n = 5). Data are presented as mean ± SD, *P < 0.05, **P < 0.01.

[0034] Figure 8 The TPP-PEG-PCL synthesized in Example 2 is illustrated. 1 H-NMR and 31 P-NMR spectrum.

[0035] Figure 9 This illustrates TPP-PEG-Hyd-PCL. 1 H-NMR and 31 P-NMR spectrum.

[0036] Figure 10 This illustrates the optimization of the percentage of TPP-PEG-PCL in TM / SK micelles.

[0037] Figure 11 This illustrates the optimization of the polymer / RBCm ratio and PEG chain length.

[0038] Figure 12 This illustrates the behavior of ThTM / SK@FP-RBCm in releasing SK in PBS buffer (0.01M, containing 0.5% Tween 80) at pH 7.4, 6.8, and 5.5.

[0039] Figure 13 This illustrates the cytotoxicity of the blank vector on MDA-MB-231 cells.

[0040] Figure 14 This illustrates the characteristics of C6 micelles.

[0041] Figure 15 This illustrates the characteristics of DiR micelles.

[0042] Figure 16 This illustrates the effects of the SK formulation on body weight and major organ histopathology in female NOD / SCID mice. Detailed Implementation

[0043] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0044] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental materials, reagents, instruments, and related test descriptions involved in the examples are as follows: (a) Materials and reagents: NH2-PEG 2k -PCL 6.6k NH2-PEG 3.4k -Hyd-PCL 6.6k mPEG 2k -PCL 6.6k and FA-PEG-FA (PEG) 1k PEG 2k and PEG 3.4k The following were purchased from Xi'an Ruixi Biotechnology Co., Ltd. (Shaanxi, China). N-(carbonylmethoxy polyethylene glycol 2000)-1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE-PEG2000) and DOTAP chloride were obtained from NOF Corporation (Tokyo, Japan). SK (98.0% purity, lot number C12097235) and 1,1'-octadecyl-3,3,3',3'-tetramethylindole tricarbonine (DiR, ≥ 95.0% purity, lot number C11697305) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Coumarin 6 (C6, ≥ 98.0% purity, lot number J1916037) was obtained from Aladdin Reagent Co., Ltd. (Shanghai, China).

[0045] (II) Instruments and Testing Methods: Micelle particle size, polydispersity index (PDI), and zeta potential were measured using a NanoBrook 90PlusPALS (Brookhaven, New York, USA). All samples were diluted 10-fold with phosphate buffer (0.01 M) at pH 7.4 prior to assay. For pH sensitivity studies, ThTM / SK@RBCm and ThTM / SK@FP-RBCm were diluted 10-fold with phosphate buffer (0.01 M) at pH 5.5 or 7.4 and incubated for 3 hours prior to assay.

[0046] The morphology of ThTM / SK, ThTM / SK@RBCm, and ThTM / SK@FP- was observed by transmission electron microscopy (TEM) using negative staining. In short, micelles were adsorbed onto a copper grid and stained with 2% phosphotungstic acid solution (pH 7.0) for 30 seconds. The grid was observed by TEM (HT7800, Hitachi Ltd., Tokyo, Japan) at an accelerating voltage of 120 kV, and digital images were captured using a CCD camera.

[0047] (III) Detection of SK load rate, load capacity and release behavior The drug loading rate (LR) and drug loading amount (LC) of SK-loaded micelles were determined by HPLC (LC-16, Shimadzu, Suzhou, China) and UV detector (SPD-16, Shimadzu, Suzhou, China). Specifically, the micelle sample was dissolved in methanol at a volume ratio of 1:20 and sonicated for 20 minutes to break up the micelles and obtain free SK. A Hedera ODS-C18 column (250 × 4.6 mm, 5) was used. m. The sample was analyzed using Hanbon (Jiangsu, China) and detected at 516 nm using methanol:3% acetic acid (90:10, v / v) at a flow rate of 1 mL / min. The LC and LR of SK were calculated according to the following formulas.

[0048] (1) (2) The drug release behavior of SK in micelles was studied using membrane dialysis. In the preparation of the release media, physiological conditions, tumor microenvironments, and lysosomes were stimulated using 0.01 M phosphate buffers (pH 7.4, 6.8, and 5.5) containing 0.5% Tween 80. 300 μL of micelles were placed in a dialysis bag (MWCO, 12 kDa) and exposed to 20 mL of culture medium in the tube. The tubes were incubated in a 37°C water bath with shaking at 50 rpm / min. At appropriate time intervals, 200 μL of culture medium was removed and replenished with the same volume of fresh medium. Samples were analyzed using the same HPLC method.

[0049] (iv) In vitro intracellular transport of micelles Intracellular transport of micelles was captured using the Operetta CLS high-content imaging system (Perkinlemer, Berlin, Germany), and confocal information was quantified using ImageJ software (v.1.8.0). Specifically, MDA-MB-231 cells (6000 cells / well) were seeded in ViewPlate-96 microplates (Perkinlemer) for 24 hours, and then treated with NM / C6, TM / C6, ThTM / C6, ThTM / C6@RBCm, and ThTM / C6@FP-RBCm at equal concentrations of C6. After incubation for 2 or 4 hours, the cells were stained for 30 minutes with the mitochondrial tracer MitoTracker Deep Red FM (Thermo, 2179203600 nM) or the lysosomal tracer LysoDeep Red (Abcam, ab176829, 1:500), and finally stained with Hoechst 33342 (1 μg / mL) for 30 minutes.

[0050] Mitochondrial mass was labeled with MitoTracker Green FM dye. Cells were collected and incubated with 100 nM MitoTracker Green FM at 37°C for 35 min. Fluorescence intensity was detected at Ex / Em = 488 / 525 nM using a NovoCyte flow cytometer and analyzed using NovoExpress software (ACEA Biosciences, California, USA).

[0051] Cells were lysed using a lysis buffer containing a mixture of PMSF and protease inhibitors, and protein concentrations were measured using a BCA Protein Assay Kit (Thermo). ATP levels were assessed using a luminescent ATP assay kit (Invitrogen, Carlsbad, California, USA) and detected at 560 nm using a Varioskan multimode microplate reader (Thermo). ATP levels were normalized to protein concentrations.

[0052] Example 1: Shikonin inhibits mitochondrial biosynthesis in TNBC cells via targeted POLG. As a DNA polymerase in mitochondria, POLG is essential for mitochondrial DNA repair and replication, thus it is a key mediator regulating mitochondrial biosynthesis and function. This invention analyzed the correlation between POLG mRNA expression and prognosis in patients with TNBC (tumor nephrotic syndrome) breast cancer. Analysis using the Kaplan-Meier Plotter database showed that TNBC breast cancer patients with high POLG expression had shorter distant metastasis-free survival (DMFS) than those with low POLG expression. Figure 1a). High POLG expression in breast cancer patients is a predictor of poor prognosis. Furthermore, multiplex immunofluorescence staining using tumor tissue microarrays was used to determine whether POLG expression is associated with mitochondrial biosynthesis in TNBC tissues. Figure 1 b). Peroxisome proliferator-activated receptor gamma-coactivator (PGC-1α) expression is used to indicate mitochondrial biosynthesis, and pan-CK is used to distinguish tumor cells and stromal cells in TNBC tissue. The inventors discovered that PGC-1α protein expression in tumor cells (pan-CK) + The expression in CK cells is higher than in stromal cells (pan-CK). - This indicates that mitochondrial biosynthesis is more active in tumor cells than in stromal cells in TNBC tissue. Figure 1 c). Similarly, POLG expression was high in tumor cells but low in stromal cells. According to the nonparametric Spearman correlation test, we found a positive correlation between POLG and POLG expression in TNBC tumor cells (R=0.59, P<0.001). Figure 1 d).

[0053] The potential affinity of shikonin for POLG was analyzed using AutoDock Vina software. Among these residues in POLG, ASP-890 and ASP-1135 are catalytic residues involved in phosphate transfer; LYS-947 is a positively charged residue on the O-helical finger domain responsible for triphosphate binding; and TYR-951 is involved in ribose recognition. The results indicate that shikonin is predicted to form hydrogen bonds with the active residues of POLG, including ASP-890, ASP-1135, LYS-947, and TYR-951. Figure 1 e). Furthermore, the binding affinity between shikonin and POLG was further confirmed by MST analysis. Different concentrations of shikonin were incubated with cell lysates containing GFP-labeled POLG or free GFP. The results showed that shikonin could bind to GFP-POLG, forming a typical binding curve (Kd = 3.1 ± 0.4 × e). -5 M), which was not observed in GFP ( Figure 1 f). Furthermore, shikonin reduced mitochondrial POLG levels, as well as cytoplasmic and total POLG levels, in TNBC MDA-MB-231 cells (f). Figure 1 g). In summary, experiments show that POLG in tumor tissue predicts poor prognosis in breast cancer patients and is a promising target for reducing mitochondrial biosynthesis in TNBC cells. Shikonin targeting POLG demonstrates its inhibitory potential on TNBC cell mitochondria.

[0054] Example 2: Preparation and characterization of micelles containing FP-RBCm-coated shikonin 2.1 Shikonin Targeted Modification Strategy Folic acid (FA) is essential for the formation of heme from red blood cells, and heme is a component of hemoglobin. Folic acid receptors (FRs) are widely distributed on the red blood cell membrane (RBCm). Overexpression of FA receptor α (FRα) has also been detected in TNBC cells. Therefore, this invention employs an RBCm modification strategy to achieve targeted membrane encapsulation. FA-PEG-FA is synthesized by coupling polyethylene glycol (PEG) derivatives of different chain lengths to FA at both ends. Before membrane encapsulation, co-incubation of RBCm and FA-PEG-FA is expected to bind one end of the FA-PEG-FA to the FR of the RBCm through specific ligand-receptor interactions, thereby obtaining modified RBCm (FP-RBCm). The hydrated shell formed by the PEG chain on the RBCm prevents the attraction between the nanoparticle core and the RBC outer membrane, thus avoiding random adsorption and collapse of the cell membrane. FP-RBCm ensures the long-term circulation function of the RBCm, and the exposure of FA on the RBCm surface enables the nanoparticles to target TNBC cells by anchoring FRα.

[0055] 2.2 Micellar Preparation Method The preparation of micelles includes the preparation of ThTM / SK micelles and the extraction, modification, and coating of RBCm membranes. Details are as follows: 2.2.1 TPP-PEG 2k -PCL 6.6k and TPP-PEG 3.4k -hyd-PCL 6.6k Synthesis (4-Carboxybutyl)triphenylphosphine bromide (TPP, 0.09 g, 0.2 mmol) and (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.04 g, 0.2 mmol) were dissolved separately in 1 mL of dimethyl sulfoxide. 1-Hydroxybenzotriazole monohydrate (HOBT, 0.03 mg, 0.2 mmol) was dissolved in 1 mL of tetrahydrofuran. The solutions were added to a round-bottom flask and stirred at room temperature for 30 minutes. NH2-PEG 2k -PCL 6.6k (0.86g, 0.1mmol) or NH2-PEG 3.4k -Hyd-PCL 6.6k Dissolved in 2 mL of tetrahydrofuran (0.90 g, 0.1 mmol), the solution was added dropwise to activated TPP and stirred overnight at room temperature under nitrogen. The resulting reaction mixture was dialyzed against deionized water (MWCO, 3500 Da) for 24 hours and then lyophilized to obtain TPP-PEG as a white solid. 2k -PCL 6.6k and TPP-PEG 3.4k-hyd-PCL 6.6k The yields were 84.5% (0.765 g) and 82.3% (0.778 g), respectively. The obtained products were obtained through... 1 H NMR and 31 Characterized by P NMR (AVANCE NEO 600MHz, Bruker, Switzerland) (see [reference]). Figure 8 , Figure 9 ).

[0056] TPP-PEG 2k -PCL 6.6k : 1 H NMR (600 MHz, DMSO) δ 7.96 - 7.74 (m), 6.00 (m, 1H), 3.98 (t, J =6.3 Hz, 2H), 3.51 (s, 2H), 3.34 (s, 2H), 2.27 (t, J = 6.7 Hz, 2H), 1.52 (m),1.28 (m). 31 P-NMR (600 MHz, DMSO) δ 23.89 ppm (m). TPP-PEG 3.4k -hyd-PCL 6.6k : 1 H-NMR (600 MHz, DMSO): 9.01 - 8.96 (m, 1H), 8.70 (m,1H), 8.57 (s,1H), 7.49 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 6.98 - 6.94 (m),3.98 (t, J = 5.9 Hz, 2H), 3.51 (s, 4H), 3.35 (s, 1H), 2.27 (t, J = 6.3 Hz,2H), 1.53 (m2H), 1.28 (m, 2H). 31 P-NMR (600 MHz, DMSO) δ 23.90 ppm. 2.2.2 Preparation of ThTM / SK micelles Normal micelles (NM) loaded with SK were prepared by solvent diffusion, specifically including: adding 20 mg mPEG 2k -PCL 6.6k1 mg of SK was dissolved in 2 mL of acetone and added to 10 mL of deionized water with stirring. The mixture was then stirred at room temperature for 0.5 hours. The polymer solution was evaporated using a rotary evaporator (RE-52AA, Nanjing Beidi Experimental Instruments, China) to remove the organic solvent. The resulting solution was subjected to three cycles of ultrafiltration at 3500 × g for 20 min each using an Amicon Ultra-15 tube (MWCO 100 kDa) to replace the medium. Unused SK was separated and removed through a 0.45 µm filter.

[0057] Use TPP-PEG 2k -PCL 6.6k TM / SK micelles were prepared by partially replacing mPEG2k-PCL6.6k in different proportions. To obtain pH-sensitive micelles, TPP-PEG... 2k -PCL 6.6k and TPP-PEG 3.4k -hyd-PCL 6.6k Participated in the preparation of ThTM / SK micelles. The ratio of TPP-PEG-PCL to the total polymer weight was studied to obtain optimal particle size, polydispersity index (PDI), zeta potential, and drug loading capacity. Figure 10 When the mass ratio of TPP-PEG-PCL is 50%, the SK LR of TM / SK micelles is the highest.

[0058] 2.2.3 Extraction of RBCm The extracted and purified RBCm was quantified by BCA analysis to obtain the total protein concentration before micelle coating. When the RBCm was resuspended in an equal volume of whole blood in 1X PBS, the average protein concentration reached approximately 1.5 mg / mL.

[0059] 2.2.4 RBCm Modification FA-PEG-FA with different PEG lengths were used to modify RBCm to optimize the membrane camouflage process. RBCm was derived from whole blood collected from male ddY mice. Specifically, whole blood was received from the aorta of the mice and processed at 1,000 × 10⁻⁶ ppm. gCentrifuge for 15 minutes. Collect red blood cells after washing three times with PBS (1X) and hemolyze them with PBS (0.25X) by the hypotonic effect. Separate RBCm by centrifugation at 8000×g for 15 minutes and resuspend in PBS (1X). Sonicate the RBCm in a 200 W bath for 5 minutes before further application. For surface modification of RBCm, incubate with FA-PEG-FA of different PEG lengths at a 1:1 mass ratio to obtain FA-PEG-FA modified RBCm (FP-RBCm). Premixing at 37°C for 30 min allows FA-PEG-FA to insert into the outer membrane of the RBCm. Prior to incubation, the protein concentration of RBCm was quantified using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), with FA-PEG-FA dissolved in DMSO at a concentration of 80 mg / mL. Unbound FA-PEG-FA was removed by ultrafiltration at 10000×g for 15 minutes using Amicon Ultra-0.5 tubes (MWCO 10 kDa, Merck Millipore Ltd, Ireland).

[0060] 2.2.5 Preparation and Optimization of RBCm-Coated Micelles ThTM / SK micelles were prepared according to the method in 2.2.2 under a TPP-PEG-Hyd-PCL / TPP-PEG-PCL / mPEG-PCL weight ratio of 1 / 1 / 2. 20 mg of ThTM / SK micelles were added dropwise to PBS solutions of 2 mg RBCm and FP RBCm, respectively, followed by rotation for 30 s and incubation at 37 °C for 30 min. Subsequently, the solutions were sonicated in a water bath (200 W, 5 min) and extruded through a 200 nm polycarbonate membrane using an extruder (Avastin, Canada) to obtain ThTM / SK@RBCm and ThTM / SK@FP-RBCm.

[0061] Accordingly, C6 and DiR micelles were prepared using the same process described above, wherein SK was replaced by C6 and DiR, and the mass ratio of support to C6 was 20:0.1, and the mass ratio of support to DiR was 20:1. Regarding RBCm camouflage, the size of ThTM / SK@RBCm increased from 60 nm to 140 nm as the polymer / RBCm weight ratio decreased from 80 / 1 to 10 / 1. Figure 11Although cell membrane coating on cationic nanoparticles typically leads to cell membrane collapse and nanoparticle aggregation, no precipitation was observed until the polymer / RBCm weight ratio reached 5:1. It can be seen that ThTM / SK@FP-RBCm exhibits smaller size and more stable zeta potential at a polymer / RBCm weight ratio of 10:1.

[0062] 2.3 Characterization of micelles The pH sensitivity of micelles is determined by changes in their size. Figure 2 a) and zeta potential ( Figure 2 b) Evaluation was conducted. Charge reversal was observed in all samples at different pH values. ThTM / SK@RBCm showed a leftward shift in particle size distribution peaks between pH 7.4 and 5.5, while ThTM / SK@FP-RBCm showed the opposite size increase regardless of PEG chain length. Considering micelle size, stability, and pH sensitivity, experiments were conducted using ThTM / SK@RBCm and ThTM / SK@FP-RBCm (polymer / RBCm weight ratio of 10:1) prepared according to 2.2.5. SDS-PAGE and UV spectroscopy were performed to verify the successful coating of RBCm and encapsulation of SK. ThTM / SK@RBCm and ThTM / SK@FP-RBCm exhibited similar banding to RBCm in the polymer fraction of SDS-PAGE ( Figure 2 c). Simultaneously, a characteristic peak of RBCm was measured at 420 nm, which completely overlapped with the peaks on ThTM / SK@RBCm and ThTM@FP-RBCm. It can be reasonably concluded that RBCm was successfully and completely encapsulated on these micelles. Furthermore, the peak overlap between free SK and ThTM / SK@FP-RBCm in the 450–580 nm range indicates the effectiveness of SK encapsulation (…). Figure 2 d). The characteristic peak of free FA at 280 nm confirms that ThTM / SK@FP-RBCm and ThTM@FP-RBCm are modified by FP.

[0063] The particle size, PDI, zeta potential, and LR / LC of SK-supported micelles were determined. Except for ThTM / SK@RBCm, the diameter of all micelles was less than 80 nm. Figure 2 e). Charge reversal was observed after RBCm camouflage ( Figure 2 f). The LR of all SK groups was above 50%, and RBCm coating slightly reduced the LR of SK (f). Figure 2 g). It is speculated that RBCm coating may partially remove the SK adhering to the ThTM / SK surface. Consistent with the particle size analysis results, ThTM / SK at around 50 nm appears spherical under transmission electron microscopy (TEM). Figure 2 h). Using RBCm camouflage, irregularly shaped micelle aggregates up to 150 nm were observed in ThTM / SK@RBCm. The binding portion between micelle units was approximately 8 nm, similar in thickness to a cell membrane. After FA-PEG-FA modification, separated particles of approximately 60 nm with a clear core-shell structure were observed in ThTM@FP-RBCm. Given the opposite particle size trends of ThTM / SK@RBCm and ThTM@FP-RBCm between pH 7.4 and 5.5, it is speculated that the partial separation of micelle units from RBCm leads to a decrease in the particle size of ThTM / SK@RBCm. Since ThTM / SK@FP-RBCm exists as independent micelles rather than aggregates, it has a strong adsorption to RBCm and may require a certain amount of time to separate from RBCm, thus exhibiting as swollen nanoparticles. Accordingly, the pH-sensitive release behavior of SK was determined ( Figure 12 At pH 5.5, SK is released at a faster rate and with a higher cumulative release, which is closely related to the hydrazone bond breakage under acidic conditions.

[0064] Example 3 Intracellular transport of coumarin 6-encapsulated micelles To study micelle uptake and intracellular transport, coumarin 6 (C6) was encapsulated in micelles. Figure 14 The micelles were observed using confocal microscopy. Quantitative analysis showed colocalization of the micelles with fluorescently labeled organelles. The overlap of the green and red curves indicates the location of the micelles on the labeled organelles. Effective mitochondrial targeting of nanoparticles is typically limited by cellular uptake and subsequent endosomal pathways. We conducted time-dependent uptake studies using the lysosomal marker Lysotracker Red and the mitochondrial marker Mitotracker Red to investigate intracellular transport of micelles, including uptake, lysosomal fusion and escape, and mitochondrial accumulation. For micelles containing TPP-PEG-PCL, colocalization with lysosomes was observed at 2 hours (overlapping black and gray lines), and significantly decreased at 4 hours. Figure 3(a, b) This indicates that the efficient lysosomal escape of the micelles is attributed to the "proton sponge" effect of TPP, which prevents endosome / lysosomal acidification, leading to osmotic swelling and membrane rupture. Regardless of the group, the green fluorescence intensity did not change significantly over time, and ThTM / C6 adsorption on the membrane even decreased at 4 h. Assuming that most micelles are absorbed by the cells within 2 h, ThTM / C6 adsorbed on the membrane may be stripped away over time due to hydrazone bond breakage. RBCm-coated micelles showed little membrane adsorption and clear lysosomal escape, enabling subsequent mitochondrial accumulation. However, ThTM@FP-RBCs were observed to have no significant enhancement effect on cellular uptake compared to ThTM@RBCm. Mitochondrial targeting ability reflects the efficiency of micelle delivery and accumulation on mitochondria, which is key to SK's inhibition of mitochondrial biosynthesis. Colocalization of micelles and mitochondria was monitored at 2 h and 4 h. Figure 3 (c, d) All micelles showed similar cellular uptake at 2 and 4 hours. Mitochondrial aggregation of the tested substances is represented by overlapping red and green curves; significant red-green curve overlap was observed for ThTM@RBCm and ThTM@FP-RBCm. Consistent with lysosomal colocalization studies, TM / C6 and ThTM / C6 exhibited significant cell membrane uptake. Therefore, intracellular transport studies indicate that RBCm promotes cellular uptake of micelles by preventing TPP-induced membrane adsorption, thereby further promoting TPP-mediated lysosomal escape and subsequent mitochondrial translocation. The cellular uptake rate of ThTM@FP-RBCm was not higher than that of ThTM@RBCm, possibly due to the sufficiently small particle size of the micelles encapsulated by both RBCm types.

[0065] Example 4: Preparation of micelles to inhibit mitochondrial biosynthesis and their in vitro anti-TNBC proliferation and metastasis effects. The inhibitory effects of free SK and the SK micelles prepared in this invention on mitochondrial biosynthesis in MDA-MB-231 cells were evaluated, including their effects on mitochondrial DNA (mtDNA), mitochondrial content, and ATP production. Free SK reduced mitochondrial DNA, mitochondrial content, and ATP levels, while SK micelles containing the TPP moiety further enhanced the inhibitory effect on mitochondrial biosynthesis, especially ThTM / SK@FP-RBCm (…). Figure 4 a, b, c). To confirm that mitochondrial biosynthesis inhibition is mainly mediated by the regulation of POLG and PGC-1α, protein levels in MDA-MB-231 cells were measured after treatment with free SK and its micelles. Figure 4 e). Consistent with the inhibitory effect on mitochondrial biosynthesis, SK micelles downregulated the expression levels of mitochondrial or total POLG and PGC-1α proteins in a similar trend.

[0066] Furthermore, the antiproliferative activity of the SK micelles prepared in this invention was evaluated by cytotoxicity assays. Figure 4 d). ICs with RBCm-coated micelles 50 About 3 times lower than free SK ( Figure 4 d). Meanwhile, both ThTM@FP-RBCm and ThTM@RBCm blank vectors did not produce significant toxicity even at the highest concentrations. Figure 13 Although TM / SK and ThTM / SK also enhanced the cytotoxicity of MDA-MB-231 cells, the cytotoxicity of blank TM and ThTM was negligible. The anti-metastasis activity of SK micelles against TNBC cells was evaluated by cell invasion and migration assays; SK micelles showed a trend similar to cytotoxicity. Figure 4 f). It is worth noting that ThTM / SK@FP-RBCm and ThTM / SK@RBCm IC 50 The values ​​are similar, but ThTM / SK@FP-RBCm exhibits slightly higher anti-metastasis activity than ThTM / SK@RBCm. It is well known that the transport of nanoparticles through the inlet pores across the membrane and their distribution into the inner mitochondrial membrane are closely related to particle size and charge potential. It has been reported that the maximum absorption of nanoparticles is limited to 100 nm, while positive charge plays a decisive role in mitochondrial accumulation. Clearly, the smaller particle size of ThTM / SK@FP-RBCm (less than 70 nm) and the charge reversal under acidic conditions favor mitochondrial transport. Although the approximately 150 nm size of ThTM / SK@RBCm limits its transport within mitochondrial pores, the low pH conditions of lysosome-mediated micellar separation may promote mitochondrial accumulation. In summary, preferential transport to mitochondria can improve the inhibitory effect on mitochondrial biogeneration, thereby enhancing cytotoxicity and anti-metastasis activity.

[0067] Example 5: Pharmacokinetics and Biodistribution of Shikonin Micelles Micelles loaded with SK and DiR ( Figure 15 The pharmacokinetic characteristics and biodistribution of ThTM micellars were investigated. Theoretically, positively charged ThTM micellars are readily recognized and eliminated via phagocytosis, while the FP-RBCm camouflage on cationic micellars is expected to evade immune surveillance via the membrane protein CD47, leading to prolonged circulation. Figure 5 a). Western blot showed that CD47, the most important marker protein of RBCm, was completely transferred to ThTM / SK@RBCm and ThTM / SK@FP-RBCm, preserving the ability of erythrocytes to evade immune surveillance. Figure 5 b). Transmission electron microscopy results with immunogold as shown. Figure 5As shown in c, this further demonstrates that FA-PEG-FA achieves targeted encapsulation of RBCm. SK pharmacokinetic studies were conducted in healthy mice. After intravenous administration, serum samples were collected, and the plasma drug concentration of SK was measured. Figure 5 d). All samples were eliminated from circulation within 6 hours. Compared with free SK and ThTM / SK, NM / SK, ThTM / SK@RBCm, and ThTM / SK@FP-RBCm showed lower clearance and higher AUC, especially ThTM / SK@FP-RBCm. Blood retention of micelles is closely related to the interactions between nanoparticles, serum, and circulating cells. A high percentage of serum proteins in whole blood constitutes the first barrier to nanoparticle circulation. In up to 50% FBS (fetal bovine serum), changes in micelle diameter were detected to assess interference with the micelles. Figure 5 e). The diameter of ThTM micelles increased, while the diameters of NM / SK and ThTM / SK@FP-RBCm micelles did not change significantly, indicating higher stability. Furthermore, the binding of micelles to blood cells was detected to evaluate changes in the physiological distribution of micelles caused by blood cells. ThTM / C6@FP-RBCm showed approximately 30% erythrocyte binding, even lower than NM / C6 ( Figure 5 f). Compared to ThTM / C6 with a binding rate higher than 40%, ThTM / C6@FP-RBCm also had a leukocyte binding rate lower than 30% at 24 hours. Figure 5 g). It is reasonable to conclude that FP-RBCm camouflage prolongs micellar circulation through CD47-mediated immune surveillance escape, and that the PEG chain confers higher serum stability and lower cell binding rate.

[0068] TNBC cell in situ seeding model was established by inoculating MDA-MB-231 cells into the mammary fat pads of female NOD / SCID mice to evaluate the biodistribution of SK micelles. ThTM / DIR, ThTM / DiR@RBCm, and ThTM / DiR@FP-RBCm were intravenously injected, and in vivo images were acquired at 2, 6, 12, and 24 hours post-injection. Figure 6 a). In the ThTM / DiR@RBCm and ThTM / DiR@FP-RBCm groups, DiR aggregation was clearly visible at the tumor lesion, and the fluorescence intensity increased over time. Conversely, no fluorescence signal was detected in the ThTM / DiR group within 24 hours. Fluorescence intensity was quantified at the end of the experiment using the separator. Figure 6(b, c, d) Further confirmation showed that almost no signal was detected in the tumor lesions of the ThTM / DiR group. Compared with ThTM / DiR@RBCm, ThTM / DiR@FP-RBCm effectively accumulated at the tumor lesions and was accompanied by rapid clearance from the liver. This invention found that RBCm camouflage is conducive to tumor aggregation, and FA ligands further improve tumor targeting. In summary, RBCm prolongs micelle circulation, thereby improving accumulation at the tumor lesions, and FA-PEG-FA modified RBCm enhances this effect.

[0069] Example 6: Therapeutic effects of SK micelles on TNBC cell orthotopic seeding model and lung metastasis model. In vivo, mouse models of TNBC cell in situ and lung metastasis were constructed to explore the anti-proliferative and anti-metastatic effects of SK micelles. Figure 7 a, e). Compared with the control group, free SK and NM / SK moderately inhibited tumor growth, but ThTM / SK@FP-RBCm and ThTM / SK@RBCm showed more significant inhibitory effects on tumor growth compared with free SK or NM / SK. Figure 7 (b, c, d). The tumor weight inhibition rates of the free SK, NM / SK, ThTM / SK@RBCm, and ThTM / SK@FP-RBCm treatment groups were 22.73%, 32.15%, 43.01%, and 61.10%, respectively. Figure 7 c). Consistent with in vitro results, ThTM / SK@FP-RBCm exhibited the most effective inhibitory effect on tumor growth. Figure 7 b, c, d). Furthermore, free SK and NM / SK also moderately reduced the number of nodules formed by MDA-MB-231X cell metastasis in the lungs, and ThTM / SK@RBCm and ThTM / SK@FP-RBCm showed better inhibitory effects than free SK and NM / SK. Figure 7 e, f). The inhibitory effects of free SK, NM / SK, ThTM / SK@RBCm, and ThTM / SK@FP-RBCm on the number of lung metastatic nodules were 39.31%, 51.03%, 68.96%, and 88.97%, respectively. Figure 7 h). Histological analysis of lung sections showed that the metastatic lesion area in the PBS, blank vector, FreeSK, NM / SK, ThTM / SK@RBCm, and ThTM / SK@FP-RBCm treatment groups was 83.1±13.0 μm. 2 85.8±7.9 μm 2 33.5±7.3 μm 2 ,25.6±3.1 μm2,18.5±1.6 μm 2 8.3±1.7 μm 2( Figure 7 i, j). Similarly, ThTM / SK@FP-RBCm showed more pronounced anti-metastasis effects than other formulations (i, j). Figure 7 fj).

[0070] IHC staining of tumor tissue or lung tissue sections showed that, compared with the PBS treatment group, free SK reduced the expression of PGC-1α and POLG in tumor tissue or lung metastases. Furthermore, NM / SK, ThTM / SK@RBCm, and ThTM / SK@FP-RBCm showed superior inhibitory effects on PGC-1α and POLG expression compared to free SK, with ThTM / SK@FP-RBCm exhibiting the most effective effect. Figure 7 These results indicate that SK micelles inhibit the growth and metastasis of TNBC cells by interfering with POLG, reflecting the inhibition of mitochondrial biosynthesis.

[0071] SK micelles, including NM / SK, ThTM / SK@RBCm, and ThTM / SK@FP-RBCm, did not cause significant harm to body weight. Figure 16 a, b). HE staining of heart, lung, liver, spleen, and kidney sections showed that free SK and its formulations did not cause significant histopathological changes in the major organs. Figure 16 c, d).

[0072] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A shikonin-targeting micelle, characterized in that, The micelles consist of erythrocyte membranes or modified erythrocyte membranes coating cationic nanoparticles loaded with shikonin, wherein the shikonin-loaded cationic nanoparticles contain shikonin and the block copolymer TPP-PEG. n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 and mPEG n3 -PCL m3 The PCL end forms a hydrophobic core, and PEG forms a hydrophilic layer in the middle; the modified red blood cell membrane is produced by FA-PEG. n4 -FA-modified erythrocyte membrane, the FA-PEG n4 The FA structure is shown below: ； Among them, n4 is selected from 500-8000; The TPP-PEG n1 -PCL m1 The structure is as follows: The TPP-PEG n1 -PCL m1 In this context, n1 is selected from 1000-5000; m1 is selected from 5000-10000; The TPP-PEG n2 -Hyd-PCL m2 The structure is as follows: The TPP-PEG n2 -Hyd-PCL m2 In this context, n2 is selected from 1000-5000; m2 is selected from 5000-10000; The mPEG n3 -PCL m3 The structure is as follows: n3 is selected from 1000-5000; m3 is selected from 2000-10000.

2. The method for preparing shikonin-targeted micelles according to claim 1, characterized in that, Includes the following steps: (1) Preparation of block copolymer TPP-PEG n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 ; (2) Block copolymer TPP-PEG is used n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 and mPEG n3 -PCL m3 Preparation of cationic nanoparticles loaded with shikonin; (3) The cationic nanoparticles loaded with shikonin are coated with erythrocyte membrane or modified erythrocyte membrane; The preparation method of the cationic nanoparticles loaded with shikonin includes the following steps: (a1) The TPP-PEG n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 mPEG n3 -PCL m3 Shikonin is dissolved in organic solvent C, and deionized water is added while stirring; the organic solvent C is selected from one or more of acetone, dimethyl sulfoxide, and tetrahydrofuran; in the cationic nanoparticles loaded with shikonin, the TPP-PEG... n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 mPEG n3 -PCL m3 The mass ratio is 1:1:2; the block polymer TPP-PEG n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 mPEG n3 -PCL m3 The total mass ratio of the total mass to the mass of shikonin is 1:15-25; (b1) Stir at room temperature for 0.2-1 hour, evaporate the solution to remove organic solvent, centrifuge and ultrafilter for 10-40 min, and separate and remove unused shikonin through a 0.45µm-0.8μm filter; The red blood cell membrane or modified red blood cell membrane coating is performed using the following steps: (a2) Add the cationic nanoparticles loaded with shikonin to the erythrocyte membrane or the modified erythrocyte membrane solution in PBS solution, rotate for 20-40 seconds, and incubate at 37°C for 20-40 min; (b2) Sonicate the solution; (c2) Using an extruder to extrude through a 200 nm polycarbonate membrane to receive the erythrocyte membrane or modified erythrocyte membrane-coated cationic nanoparticles loaded with shikonin; the mass ratio of the cationic nanoparticles loaded with shikonin to the erythrocyte membrane or modified erythrocyte membrane is 10-80:

1.

3. The preparation method according to claim 2, characterized in that, The method for preparing the modified erythrocyte membrane includes the following steps: Using FA-PEG with different PEG lengths n4 -FA and RBCm were incubated at a mass ratio of 1:

1.

4. The preparation method according to claim 2, characterized in that, The block copolymer TPP-PEG n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 The preparation method includes the following steps: (a3) Dissolve TPP and EDCl in organic solvent A respectively; (b3) Dissolve HOBT in organic solvent B; (c3) Add the solutions from steps (a3) ​​and (b3) to the reaction vessel and stir at room temperature for 20-60 minutes; (d3) NH2-PEG n1 -PCL m1 or NH2-PEG n2 -Hyd-PCL m2 Dissolve in organic solvent B and add dropwise to the solution obtained in step (c3), and stir overnight at room temperature under nitrogen. (e3) Dialyze the reaction mixture obtained in step (d3) with deionized water for 12-36 hours and freeze-dry to obtain TPP-PEG. n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 ; The organic solvent A is selected from one or two of dimethyl sulfoxide and acetone; the organic solvent B is selected from one or two of tetrahydrofuran and acetone.

5. The preparation method according to claim 2, characterized in that, In the preparation step of the cationic nanoparticles loaded with shikonin, the block polymer TPP-PEG n1 -PCL m1 TPP-PEG n2 -Hyd-PCL m2 and mPEG n3 -PCL m3 The total mass ratio of the total mass to the mass of shikonin is 1:20; the mass-to-volume ratio of shikonin to organic solvent C is 1:1-4 mg / ml; the centrifugation and ultrafiltration are performed three times using Amicon Ultra-15 tubes, centrifuging and ultrafiltration at 3500×g.

6. The preparation method according to claim 2, characterized in that, In the red blood cell membrane or modified red blood cell membrane coating step, the ultrasonic conditions are 200 W in a water bath for 4-6 minutes; the mass ratio of the cationic nanoparticles loaded with shikonin to the red blood cell membrane or modified red blood cell membrane is 10:

1.

7. The application of the shikonin-targeting micelles according to claim 1 in the preparation of a drug for treating breast cancer.

8. The application according to claim 7, characterized in that, The breast cancer mentioned is triple-negative breast cancer.

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