A tumor-targeting Bletilla striata polysaccharide polymer nanomicelle, its preparation method and application

Abstract

This invention provides a tumor-targeting Bletilla striata polysaccharide polymer nanomicelle, its preparation method, and its application. The invention involves the self-assembly of Bletilla striata polysaccharide and vitamin E succinate in water to form polymer nanomicelles—a tumor-targeting and biodegradable drug delivery system. Bletilla striata polysaccharide is the main active ingredient of the traditional Chinese medicine Bletilla striata, a natural biodegradable polymer material with antitumor activity, enabling a "drug-adjuvant" effect. Simultaneously, the biodegradability of the raw material addresses the safety issue of long-term retention of "ineffective" carriers in vivo. The vitamin E succinate is used as a hydrophobic modification of Bletilla striata polysaccharide, forming the polymer micelles, and also possesses antitumor activity, resulting in a synergistic effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a tumor-targeting Bletilla striata polysaccharide polymer nanomicelle, its preparation method, and its application. Background Technology

[0002] Cancer is one of the leading causes of death worldwide. In 2020, there were 19.29 million new cancer cases globally, and in 2021, this number exceeded 20 million, showing a year-on-year upward trend. With the increasing cancer incidence rate, people are no longer unfamiliar with various types of cancer. Cancer has become a major threat to human health and life, and cancer prevention and control has risen to the level of a national strategy in my country. Current cancer treatments mainly focus on symptom relief and disease mitigation through surgery and chemotherapy, making a complete cure very difficult. Moreover, many patients experience cancer metastasis or recurrence after surgery, leading to treatment failure. Furthermore, most chemotherapy drugs suffer from poor selectivity, significant toxic side effects, and low bioavailability, inevitably damaging normal cells while killing cancer cells. To address these issues and simultaneously improve the drug loading, stability, and bioavailability of anti-tumor drugs, achieving sustained-release and controlled-release effects, researchers have recently applied nanotechnology to tumor treatment, constructing various nano-drug delivery systems. These systems use passive or active targeting to allow drugs to accumulate at the tumor site, increasing drug concentration.

[0003] For example, in reference document 1: [Chinese Invention] CN202010694481.6, a tumor-targeting nanomicelle, its preparation method and its application as a drug carrier, is based on water-soluble chondroitin sulfate as the basic framework and uses rhein and lipoic acid as hydrophobic modifications. The resulting nanomicelles are small in size and have good drug loading and encapsulation efficiency. The disulfide bonds on the surface of the nanomicelles can be specifically broken after reaching the tumor site, thereby achieving targeted release. Under the stimulation of ultrasound, the nanomicelles can promote the ability of rhein to generate reactive oxygen species, effectively improving the utilization rate of anti-tumor drugs. However, in practice, the invention has the following defects: (1) Water-soluble chondroitin sulfate has side effects and can cause significant irritation to the upper gastrointestinal tract; (2) Rhein also has toxic side effects and requires external ultrasonic stimulation to affect the metabolic process of cells; (3) The drug loading, encapsulation efficiency, bioavailability and carrier biosafety are low.

[0004] Therefore, based on the original technology, the invention team further developed a drug delivery system with high tumor specificity to deliver anti-tumor drugs, which has important clinical significance for cancer treatment. Summary of the Invention

[0005] The purpose of this invention is to provide a tumor-targeting leukobacterium polysaccharide polymer nanomicelle.

[0006] Another object of the present invention is to provide a method for preparing tumor-targeting Bletilla striata polysaccharide polymer nanomicelles.

[0007] Another objective of this invention is to provide an application of tumor-targeting leucovorin polysaccharide polymer nanomicelles in tumor targeting.

[0008] This invention is achieved through the following technical solution:

[0009] The tumor-targeting Bletilla striata polysaccharide polymer nanomicelles of the present invention are composed of the following components: Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles, antitumor drugs, and deionized water; wherein the antitumor drugs are any one of andrographolide, coumarin-6, and cell membrane staining reagent Dir.

[0010] Preferably, the formulation of the components described in this invention comprises: 25-34 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles, 1-3 mg of andrographolide, and 8-12 mL of deionized water; or 25-34 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles, 180-220 μg of coumarin-6, 180-220 μg of cell membrane staining reagent Dir, and 8-12 mL of deionized water; or 25-34 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles, 180-220 μg of cell membrane staining reagent Dir, and 8-12 mL of deionized water.

[0011] Further preferably, the formulation of the components of the present invention comprises the following amounts: 29 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles, 2 mg of andrographolide, and 10 mL of deionized water; or 29 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles, 200 μg of coumarin-6, and 10 mL of deionized water; or 29 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles, 200 μg of cell membrane staining reagent Dir, and 10 mL of deionized water.

[0012] The Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles of this invention serve as a carrier, with hydrophobic andrographolide, coumarin-6, or cell membrane staining reagent Dir encapsulated in the hydrophobic core of the polymer micelles. The preparation method is as follows:

[0013] (1) Take vitamin E succinate, dissolve it evenly in dimethyl sulfoxide solution under ultrasound, then add 4-dimethylpyridine and 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and react under magnetic stirring to activate the carboxyl group on vitamin E succinate to obtain reaction solution A;

[0014] (2) Take Bletilla striata polysaccharide, dissolve it in dimethyl sulfoxide solution, add reaction solution A dropwise, continue to stir the reaction with magnetic force, then dialyze with deionized water, freeze dry, and obtain Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles.

[0015] Preferably, the preparation method of the Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles of the present invention is as follows:

[0016] (1) Weigh 100-600 mg of vitamin E succinate and 10-25 ml of dimethyl sulfoxide solution and dissolve them evenly under sonication. Then add 23-138 mg of 4-dimethylpyridine and 43-260 mg of 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride and react for 1.5 h under magnetic stirring at 38 °C to activate the carboxyl group on vitamin E succinate to obtain reaction solution A;

[0017] (2) Weigh 100-300 mg of Bletilla striata polysaccharide, dissolve it in 20-35 mL of dimethyl sulfoxide solution, add reaction solution A dropwise, continue to stir magnetically for 24 h, then dialyze with deionized water for 48 h, freeze dry at -80℃ to obtain 83-264 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles.

[0018] A further preferred embodiment of the preparation method of the tumor-targeting Bletilla striata polysaccharide polymer nanomicelles of the present invention is as follows: weigh Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles and dissolve them in deionized water. Add anhydrous ethanol solution containing andrographolide, coumarin-6 or cell membrane staining reagent Dir under stirring until turbidity is reached. Stir magnetically for 6-8 hours, then dialyze for 8 hours, and freeze-dry to obtain tumor-targeting Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles.

[0019] The preparation method of tumor-targeting Bletilla striata polysaccharide polymer nanomicelles of the present invention includes the following steps:

[0020] (1) Weigh out vitamin E succinate, dissolve it evenly in dimethyl sulfoxide solution under ultrasound, then add 4-dimethylpyridine and 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and react under magnetic stirring to activate the carboxyl group on vitamin E succinate to obtain reaction solution A;

[0021] (2) Weigh out Bletilla striata polysaccharide, dissolve it in dimethyl sulfoxide solution, add reaction solution A dropwise, continue to stir the reaction with magnetic force, then dialyze with deionized water, freeze dry, and obtain Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles.

[0022] (3) Weigh out Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles and dissolve them in deionized water. Add anhydrous ethanol solution containing antitumor drugs under stirring until turbidity is reached. Stir magnetically and then dialyze to obtain tumor-targeting Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles. Freeze-dry and store.

[0023] Preferably, the preparation method of the tumor-targeting Bletilla striata polysaccharide polymer nanomicelles of the present invention specifically includes the following steps:

[0024] (1) Weigh 100-600 mg of vitamin E succinate and 10-25 ml of dimethyl sulfoxide solution and dissolve them evenly under sonication. Then add 23-138 mg of 4-dimethylpyridine and 43-260 mg of 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride and react for 1.5 h under magnetic stirring at 38 °C to activate the carboxyl group on vitamin E succinate to obtain reaction solution A;

[0025] (2) Weigh 100-300 mg of Bletilla striata polysaccharide, dissolve it in 20-35 mL of dimethyl sulfoxide solution, add reaction solution A dropwise, continue to stir magnetically for 24 h, then dialyze with deionized water for 48 h, freeze dry at -80℃ to obtain 83-264 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles.

[0026] (3) Weigh 25-34 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles and dissolve them in deionized water. Add anhydrous ethanol solution containing antitumor drugs under stirring until turbidity is reached. Stir magnetically for 6-8 hours and then dialyze for 8 hours to obtain 8-10 mL of tumor-targeting Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles. Freeze-dry and store.

[0027] The molar ratio of vitamin E succinate, 4-dimethylpyridine and 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride in this invention is 1:1:1.2; the mass ratio of Bletilla striata polysaccharide to vitamin E succinate is 1:2.

[0028] The application of the Bletilla striata polysaccharide polymer nanomicelles described in this invention in the preparation of therapeutic or / preventive tumor-targeting drugs.

[0029] The beneficial effects of this invention are:

[0030] 1. This invention uses Bletilla striata polysaccharide, which has both pharmaceutical and excipient properties and is naturally biodegradable, as the basic framework, and performs hydrophobic modification. The results were obtained through investigation of the dosage and ratio of Bletilla striata polysaccharide, vitamin E succinate, catalyst, dialysis medium, and dialysis time.

[0031] (1) When other conditions are fixed, the ratio of Bletilla striata polysaccharide to vitamin E succinate is 1:2. The particle size of Bletilla striata polysaccharide vitamin E succinate polymer micelles is small, and the potential and PDI are the most stable. Therefore, the optimal ratio of Bletilla striata polysaccharide to vitamin E succinate is 1:2.

[0032] (2) When the catalyst ratio is 1:1:1.2, the particle size of the Bletilla striata polysaccharide vitamin E succinate polymer micelles is small and the PDI is the most stable. Therefore, the optimal ratio of catalyst is 1:1:1.2.

[0033] (3) When sodium carbonate and sodium bicarbonate buffer solution is used as dialysis medium, the particle size is large and the PDI value cannot be measured. On the contrary, the particle size of the sample prepared with deionized water as dialysis medium is uniform and stable. Therefore, deionized water is selected as the best dialysis medium. When the dialysis time is 24h, the dialysis is not complete, which may be due to incomplete synthesis of polymer micelles. The particle size, potential and distribution of micelles prepared by dialysis for 72h are not much different from those prepared by dialysis for 48h. Therefore, 48h is selected as the nearest dialysis time.

[0034] The optimal ratio of vitamin E succinate to Bletilla striata polysaccharide was 2:1, the catalyst ratio was 1:1:1.2, the dialysis medium was deionized water, and the dialysis time was 48 h. This method yielded the best Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelle carrier. This carrier was used to deliver andrographolide, improving the drug-likeness and bioavailability of the hydrophobic drug through micellar solubilization. The critical micelle concentration of the system was determined to be 48.3 μg / mL. In vivo targeted distribution experiments also showed that 48 hours after tail vein injection, a large amount of drug fluorescence was still observed at the tumor site, significantly prolonging the drug's half-life in vivo. This method effectively addresses the problems of poor water solubility, low bioavailability, and short in vivo half-life associated with the drug.

[0035] 2. Through experimental research on polymer nanomicelles, this invention has explored the self-assembly of Bletilla striata polysaccharide-vitamin E succinate to form polymer nanomicelles of about 50-100 nm, which can load poorly soluble drugs and have good affinity and tumor targeting, making it a new drug carrier with potential application value.

[0036] 3. This invention uses proton nuclear magnetic resonance spectroscopy to confirm the structure of the BSP-VES polymer. 1 1H NMR results confirmed that the hydrophobic chain of vitamin E succinate is linked to the backbone of Bletilla striata polysaccharide.

[0037] 4. The critical micelle concentration of the BSP-VES copolymer was determined by fluorescence spectroscopy in this invention. The results showed that the critical micelle concentration of the synthesized BSP-VES polymer was 48.3 μg / mL, indicating that the synthesized polymer has a good ability to self-assemble into micelles in water.

[0038] 5. In this invention, the drug loading and encapsulation efficiency of AG@BSP-VES polymer micelles were determined by high performance liquid chromatography (HPLC). The results showed that the drug loading of AG@BSP-VES was 6.6±0.43% and the encapsulation efficiency was 91.7±2.65%.

[0039] 6. The present invention uses HPLC to determine the concentration of the compound andrographolide and calculates the cumulative release of the drug. The results show that the synthesized polymer micelles have a certain sustained-release ability.

[0040] 7. The in vitro biocompatibility of BSP-VES polymer micelles was evaluated using the MTT assay. The results showed that the proliferation rate of BSP-VES polymer nanomicelles at a concentration of 1 mg / mL was about 80% after incubation in NCM460 and CT26 cells for 24 h or 48 h, indicating that the carrier has high biocompatibility.

[0041] 8. This invention uses a fluorescence inverted microscope to observe the uptake of C6@BSP-VES by gastric cancer cells (SGC7091) and normal cells (NCM460) for 2 hours. The experimental results clearly show that, compared with NCM460, SGC7091 cells uptake more C6@BSP-VES; and compared with the SGC7091 group blocked by Bletilla striata polysaccharide (BSP), it shows that BSP-VES polymer micelles have the ability to target gastric cancer cells.

[0042] 9. This invention conducted an in vivo distribution experiment in tumor-bearing mice. The results showed that a small amount of fluorescence signal was observed 1 hour after tail vein injection of ROI@BSP-VES nanoparticles. As time increased, the accumulation of fluorescence at the tumor site increased. By the 8th hour, a high level of fluorescence had accumulated in the tumor site. BSP-VES polymer nanomicelles were most abundant in the liver and less abundant in other organs. Obvious fluorescence was observed at the tumor site outside the body, indicating that BSP-VES polymer nanomicelles have a good ability to target CT26 tumors.

[0043] 10. This invention, through in vivo distribution experiments in tumor-bearing mice, showed that: 1 hour after tail vein injection of ROI@BSP-VES nanoparticles, a small amount of fluorescence signal was observed, and the accumulation of fluorescence at the tumor site increased with time; by the 8th hour, a high level of fluorescence had accumulated in the tumor site; BSP-VES polymer nanomicelles were most abundantly distributed in the liver, with less distribution in other organs, and showed significant fluorescence at the tumor site outside the body, indicating that BSP-VES polymer nanomicelles have a good ability to target CT26 and SGC7091 tumors.

[0044] 11. This invention uses the MTT assay to investigate the antitumor activity of andrographolide and drug-loaded micelles. The results show that in CT26 tumor cells, the inhibitory effect of AG@BSP-VES polymer nanomicelles is stronger than that of free andrographolide. This may be because the carrier BSP-VES has a better affinity for tumor cells, enabling better and faster delivery of the encapsulated, poorly soluble drug AG into CT26 cells.

[0045] 12. This invention uses the Live & Dead cell staining method to evaluate the in vitro antitumor activity of AG@BSP-VES polymer nanomicelles. The results showed that dead cells (red) appeared in both CT26 cells and NCM460 cells after administration of free andrographolide; nearly half of the dead cells (red) appeared in CT26 cells after administration of AG@BSP-VES polymer nanomicelles, while the number of dead cells in NCM460 cells was significantly less than that in CT26 cells. The experimental results indicate that free andrographolide has no selectivity for normal cells or cancer cells, while using the BSP-VES carrier to deliver the drug can increase the antitumor effect of the drug and may also improve its selectivity. Attached Figure Description

[0046] Figure 1 : H NMR spectrum of BSP-VES polymer nanomicelles

[0047] Figure 2 Size distribution of BSP-VES polymer nanomicelles

[0048] Figure 3 Critical micelle concentration of BSP-VES polymer nanomicelles

[0049] Figure 4 : Andrographolide standard song

[0050] Figure 5 In vitro release curve

[0051] Figure 6-A Biosafety analysis of BSP-VES polymer nanomicelles in NCM460 cells Figure 6-BBiosafety analysis of BSP-VES polymer nanomicelles in CT26 cells

[0052] Figure 7 Confocal microscopy images of C6@BSP-VES polymer nanomicelles in NCM460 and CT26 cells.

[0053] Figure 8 Confocal microscopy images of C6@BSP-VES polymer nanomicelles in NCM460 and SGC7091 cells.

[0054] Figure 9 Distribution of Dir@BSP-VES polymer nanomicelles in subcutaneous colon cancer

[0055] Figure 10 Distribution of Dir@BSP-VES polymer nanomicelles in subcutaneous gastric cancer

[0056] Figure 11 Cytotoxicity analysis of AG@BSP-VES polymer nanomicelles

[0057] Figure 12 Results of Live & Dead cell staining of CT26 and NCM460 cells Detailed Implementation

[0058] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0059] Example 1: Tumor-targeting Bletilla striata polysaccharide polymer nanomicelles

[0060] Ingredients: 29mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles, 2mg of andrographolide, and 10mL of deionized water.

[0061] 1) Weigh 600 mg of vitamin E succinate and dissolve it evenly in 25 ml of dimethyl sulfoxide solution under sonication. Then add 138 mg of 4-dimethylpyridine and 260 mg of 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride and react for 1.5 h under magnetic stirring at 38 °C to activate the carboxyl group on vitamin E succinate, to obtain reaction solution A;

[0062] 2) Weigh 300 mg of Bletilla striata polysaccharide, dissolve it in 35 mL of dimethyl sulfoxide solution, add reaction solution A dropwise, continue to stir magnetically for 24 h, then dialyze with deionized water for 48 h, and freeze dry at -80℃ to obtain Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles (264 mg).

[0063] 3) Weigh out the amount of the formula Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles and dissolve them in deionized water. Add anhydrous ethanol solution containing andrographolide under stirring until turbidity is reached. Stir magnetically for 8 hours and then dialyze for 8 hours to obtain 10 mL of tumor-targeting Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles. Freeze-dry and store.

[0064] Example 2: Tumor-targeting Bletilla striata polysaccharide polymer nanomicelles

[0065] Ingredients: Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles 25mg, Dir 200ug, deionized water 8mL.

[0066] 1) Weigh 200 mg of vitamin E succinate and dissolve it evenly in 15 ml of dimethyl sulfoxide solution under sonication. Then add 46 mg of 4-dimethylpyridine and 87 mg of 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride and react for 1.5 h under magnetic stirring at 38 °C to activate the carboxyl group on vitamin E succinate, to obtain reaction solution A;

[0067] 2) Weigh 100 mg of Bletilla striata polysaccharide, dissolve it in 20 mL of dimethyl sulfoxide solution, add reaction solution A dropwise, continue to stir magnetically for 24 h, then dialyze with deionized water for 48 h, and freeze dry at -80℃ to obtain Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles (83 mg).

[0068] 3) Weigh out the amount of the formula Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles and dissolve them in deionized water. Add anhydrous ethanol solution containing Dir while stirring until turbidity is reached. Stir magnetically for 8 hours and then dialyze for 8 hours to obtain 8 mL of tumor-targeting Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles. Store directly at -20℃.

[0069] Example 3: Tumor-targeting Bletilla striata polysaccharide polymer nanomicelles

[0070] Ingredients: 27 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles (obtained in Example 1), 200 ug of coumarin-6 (C6), and 8 mL of deionized water.

[0071] Weigh out Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles and dissolve them in deionized water. Add anhydrous ethanol solution containing coumarin-6 under stirring until turbidity is reached. Stir magnetically for 8 hours and then dialyze for 8 hours to obtain 9 mL of tumor-targeting Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles. Store directly at -20℃.

[0072] Example 4: Tumor-targeting Bletilla striata polysaccharide polymer nanomicelles

[0073] Ingredients: 27 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles (obtained in Example 2), 2 mg of Andrographolide, and 8 mL of deionized water.

[0074] Weigh out Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles and dissolve them in deionized water. Add anhydrous ethanol solution containing andrographolide under stirring until turbidity is reached. Stir magnetically for 8 hours and then dialyze for 8 hours to obtain 9 mL of tumor-targeting Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles. Store directly at -20℃.

[0075] Example 5: Tumor-targeting Bletilla striata polysaccharide polymer nanomicelles

[0076] Ingredients: 27 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles (obtained in Example 1), 200 ug of coumarin-6 (C6), and 8 mL of deionized water.

[0077] Weigh out Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles and dissolve them in deionized water. Add anhydrous ethanol solution containing coumarin-6 under stirring until turbidity is reached. Stir magnetically for 8 hours and then dialyze for 8 hours to obtain 9 mL of tumor-targeting Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles. Store directly at -20℃.

[0078] To further verify the feasibility of the present invention, the inventors conducted a series of experiments, as follows:

[0079] I. Screening Experiment for the Synthesis of Tumor-Targeting Bletilla striata Polysaccharide Nanomicelles

[0080] 1.1 Medicines and Reagents

[0081] Bletilla striata polysaccharide (BSP, Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., purity >95%, MV≈50000); D-α-tocopherol succinate (VES, purity 98%), andrographolide (AG, purity 98%), 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, purity 98%), 4-dimethylpyridine (DMAP, purity 99%), dimethyl sulfoxide (DMSO, purity 99%), and thiazolyl blue (MTT, purity 98%), Aladdin); fetal bovine serum from Sijiqing (FBS, Zhejiang Tianhang Biotechnology Co., Ltd.); RPMI 1640 culture medium, PBS (Zhejiang Senrui Biotechnology Co., Ltd.); 0.25% trypsin, penicillin-streptomycin solution (Gibco, USA); anhydrous ethanol, purity 99%, Chengdu Jinshan Chemical Reagent Co., Ltd.; methanol (chromatographic grade, Anhui Tiandi High Purity Solvent Co., Ltd.); water was ultrapure water; other reagents were analytical grade.

[0082] 1.2 Laboratory Animals

[0083] BALB / c mice, SPF grade, weighing 20 - 25 g, female, purchased from Beijing Huafukang Biotechnology Co., Ltd. (certificate number: SCXK(Beijing)2019 - 0008). Feeding conditions: temperature 20 - 26 °C, humidity 40% - 70%. During the experiment, the animals were allowed to eat and drink freely with a normal circadian rhythm. The animal experiment was approved by the Animal Experiment Ethics Committee of Guizhou University of Traditional Chinese Medicine (approval number: 20220061).

[0084] 1.3 Instruments

[0085] Agilent 1260 high performance liquid chromatography (HPLC) instrument (Hangzhou Ruixi Technology Co., Ltd.); fluorescence inverted microscope (Thermo Fisher, USA); DF - 101S type collecting heat - type constant temperature heating magnetic stirrer (Shanghai Qiuzuo Scientific Instruments Co., Ltd.); YXQ - S0SII type vertical pressure steam sterilizer (Shanghai Dongya Pressure Vessel Manufacturing Co., Ltd.); Beckman BECK MAN nano - particle size analyzer DelsaMax PRO (Xiamen Laichi Electronic Technology Co., Ltd.); IVIS Lumina II type in - vivo imaging system (Caliper LifeScience, Cold Spring Harbor, USA).

[0086] 1.4 Investigation on the synthesis method of BSP - VES

[0087] 1.4.1 Synthesis method one

[0088] (1) Weigh 600 mg of vitamin E succinate, dissolve it evenly in 25 ml of dimethyl sulfoxide solution under ultrasonic treatment, then add 138 mg of 4 - dimethylpyridine and 260 mg of 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide hydrochloride, and react for 1.5 h under magnetic stirring at 38 °C to activate the carboxyl group on vitamin E succinate, obtaining reaction solution A;

[0089] (2) Weigh 300 mg of Bletilla striata polysaccharide, dissolve it in 35 mL of dimethyl sulfoxide solution, drip reaction solution A, continue magnetic stirring for 24 h, then dialyze with deionized water for 48 h, and freeze - dry at - 80 °C to obtain 264 mg of Bletilla striata polysaccharide - vitamin E succinate polymer nanomicelles.

[0090] 1.4.2 Synthesis method two

[0091] 1) Weigh 200 mg of vitamin E succinate and dissolve it evenly in 15 ml of dimethyl sulfoxide solution under sonication. Then add 46 mg of 4-dimethylpyridine and 87 mg of 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride and react for 1.5 h under magnetic stirring at 38 °C to activate the carboxyl group on vitamin E succinate, to obtain reaction solution A;

[0092] 2) Weigh 100 mg of Bletilla striata polysaccharide, dissolve it in 20 mL of dimethyl sulfoxide solution, add reaction solution A dropwise, continue to stir magnetically for 24 h, then dialyze with deionized water for 48 h, freeze dry at -80℃ to obtain 83 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles.

[0093] To further optimize the preparation conditions of BSP-VES polymer micelles, the dosage of vitamin E succinate, catalyst, dialysis medium, and dialysis time were investigated. The effects of different reaction conditions on the particle size and distribution of the prepared micelles were examined. Specific dosages of each substance are shown in Tables 1-4 below.

[0094] Table 1. Formulations for the preparation of BSP-VES polymer micelles at different dosages of vitamin E succinate.

[0095]

[0096] Table 2. Formulations for the preparation of BSP-VES polymer micelles with different catalyst dosages.

[0097]

[0098] Table 3. Formulations for the preparation of BSP-VES polymer micelles under different dialysis media.

[0099]

[0100] Table 4. Formulations for the preparation of BSP-VES polymer micelles under different dialysis times.

[0101]

[0102] Table 5. Effects of the ratio of Bletilla striata polysaccharide to vitamin E succinate on the micelle size, zeta potential, and distribution of BSP-VES polymer (x±sn=10)

[0103]

[0104] The results (Table 5) show that when other conditions are fixed, the ratio of Bletilla striata polysaccharide to vitamin E succinate is 1:2, resulting in the smallest particle size, the most stable potential and PDI of the Bletilla striata polysaccharide-vitamin E succinate polymer micelles. Therefore, the initial screening of the ratio of Bletilla striata polysaccharide to vitamin E succinate is 1:2.

[0105] Table 6. Effects of catalyst dosage on micelle size, Zeta potential, and distribution (x±s, n=10)

[0106]

[0107] Experiments show (Table 6) that when the catalyst ratio is 1:1:1.2, the particle size of the Bletilla striata polysaccharide vitamin E succinate polymer micelles is small and the PDI is most stable. Therefore, the optimal catalyst ratio for the initial screening is 1:1:1.2.

[0108] Table 7. Effects of dialysis media on micelle size, Zeta potential, and distribution (x±s, n=10)

[0109]

[0110] Polymer micelles were prepared by dialysis. The preparation of BSP-VES polymer micelles using deionized water and sodium carbonate-sodium bicarbonate buffer solution as dialysis media were investigated. The results (Table 7) showed that the particles prepared using sodium carbonate-sodium bicarbonate buffer solution as dialysis media were larger and the PDI value could not be measured. In contrast, the particles prepared using deionized water as dialysis media were uniform and stable.

[0111] Table 8. Effects of dialysis time on micelle size, Zeta potential, and distribution (x±s, n=3)

[0112]

[0113] Table 8 shows that the optimal dialysis time is 48 hours. When the dialysis time is 24 hours, the dialysis is not complete, and the synthesis of polymer micelles may not be complete. The micelles prepared by dialysis for 72 hours are not significantly different in particle size, potential and distribution from those prepared by dialysis for 48 hours. Therefore, 48 hours is initially selected as the nearest dialysis time.

[0114] In summary, the optimal formulation was initially determined to be a 2:1 ratio of vitamin E succinate to Bletilla striata polysaccharide, a 1:1:1.2 ratio of catalyst, deionized water as the dialysis medium, and a dialysis time of 48 hours.

[0115] 1.5 Confirmation of the final method for synthesizing tumor-targeting Bletilla striata polysaccharide polymer nanomicelles

[0116] 1) Weigh 100-600 mg of vitamin E succinate and dissolve it evenly in 10-25 ml of dimethyl sulfoxide solution under sonication. Then add 28-168 mg of 4-dimethylpyridine and 53-324 mg of 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride. React at 38°C with magnetic stirring for 1.5 h to activate the carboxyl group on vitamin E succinate, to obtain reaction solution A. The molar ratio of vitamin E succinate: 4-dimethylpyridine: 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1:1.2.

[0117] 2) Weigh 100-300 mg of Bletilla striata polysaccharide, dissolve it in 20-35 mL of dimethyl sulfoxide solution, add reaction solution A dropwise, continue to stir magnetically for 24 h, then dialyze with deionized water for 48 h, and freeze dry at -80℃ to obtain Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles (83-264 mg); wherein, the ratio of Bletilla striata polysaccharide to vitamin E succinate is 1:2;

[0118] 3) Weigh out the amount of the formula Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles and dissolve them in deionized water. Add anhydrous ethanol solution containing antitumor drugs under stirring until turbidity is reached. Stir magnetically for 8 hours and then dialyze for 8 hours to obtain 10 mL of tumor-targeting Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles. Freeze-dry and store.

[0119] Experimental Example 1

[0120] 5 mg of BSP, VES, and BSP-VES were weighed into NMR tubes and dissolved using deuterated reagents D₂O or DMSO. The samples were characterized by 1H-NMR spectroscopy. The structure of the BSP-VES polymer was confirmed by 1H NMR spectroscopy. Figure 1 , 1 HNMR results confirmed that the hydrophobic chain of vitamin E succinate is linked to the backbone of Bletilla striata polysaccharide.

[0121] Experimental Example 2

[0122] BSP-VES micelles were diluted with distilled water to a concentration of 1 mg / ml. The average particle size, PDI, and Zata potential were measured using a DelsaMax PRO Beckman nanoparticle size analyzer. The results are shown in Table 9. Figure 2 .

[0123] Table 9. Size, potential, and PDI value of BSP-VES polymer nanomicelles

[0124]

[0125] Experimental Example 3

[0126] The critical micelle concentration of the BSP-VES copolymer was determined by fluorescence spectroscopy. A concentration of 1.2 x 10⁻⁶ was prepared using methanol. - 5 After placing a mmol / mL pyrene solution in a brown volumetric flask, nitrogen gas was purged to rapidly evaporate the methanol. Prepared BSP-VES polymer micelles (400, 200, 100, 25, 10, 2.5 μg / mL) were added to the aforementioned pyrene-containing brown volumetric flasks, and the solutions were sonicated in a water bath for 30 min under light-protected conditions, then equilibrated in a 37℃, 100 rpm water bath for 24 h. The fluorescence emission spectra of BSP-VES solutions with various concentrations of pyrene were scanned in the 300–500 nm range. The first peak (I = 374 nm) and the third peak (I = 385 nm) of the fluorescence emission spectra were recorded, and the ratio of the two peaks was calculated. The logarithm of the BSP-VES concentration (LogC) was plotted on the x-axis, and I... 374 / I 385 The values ​​are plotted on the ordinate, and the experimental data are analyzed and fitted. The BSP-VES concentration corresponding to the intersection of the fitted curves is the CMC value. Results are shown below. Figure 3 The graph is plotted with the logarithm of polymer micelle concentration on the x-axis and the I374 / I385 ratio on the y-axis. A clear inflection point, representing the critical micelle concentration (CMC), is observed. The calculated CMC of the synthesized BSP-VES polymer is 48.3 μg / mL, indicating that the synthesized polymer has good self-assembly ability to form micelles in water.

[0127] Test Example 4

[0128] The drug loading and encapsulation efficiency of AG@BSP-VES polymer micelles were determined by high performance liquid chromatography (HPLC). Chromatographic conditions: C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: methanol-water solution (60:40); flow rate: 0.8 mL / min; column temperature: 30 °C; detection wavelength: 226 nm; injection volume: 10 μL.

[0129] Establishment of the andrographolide standard curve: Accurately weigh 10.1 mg of andrographolide reference standard, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, and obtain a 0.202 mg / mL reference standard stock solution. Accurately take 0.1 mL, 0.5 mL, 1 mL, 2 mL, 4 mL, 6 mL, 8 mL, and 10 mL, and dilute to 10 mL with methanol, respectively, and determine by HPLC. Andrographolide was extracted by organic solvent extraction, and its content was determined by HPLC. The encapsulation efficiency (EE) and drug loading (DL) were calculated by formula.

[0130] The sample was injected and measured under the above chromatographic conditions. A linear regression was performed on the peak area (Y) against the concentration (X), and the linear regression equation was obtained as Y = 20.133X + 14.153(R²). 2 =0.9999), see Figure 4 The results showed that andrographolide exhibited good linearity in the concentration range of 2.02–202 μg / mL. Based on the linear regression equation, the drug loading of AG@BSP-VES was calculated to be 6.6 ± 0.43%, and the encapsulation efficiency was 91.7 ± 2.65%.

[0131] Experimental Example 5

[0132] Using phosphate-buffered saline (PBS) (pH 7.4) as the release medium, an appropriate amount of AG@BSP-VES was dissolved in PBS, and 1 mL was accurately measured and added to a dialysis bag (MWCO 3.5 kDa). This bag was then placed in a release tube containing PBS solution at pH 7.4. A solution of andrographolide at the same drug concentration was used as a control. Each sample was tested in triplicate. After sampling at pre-set time points, all old release media were discarded and replaced with fresh release media. The concentration of the compound andrographolide was determined by HPLC, and the cumulative drug release was calculated based on the standard curve. The results are as follows: Figure 5 This indicates that the synthesized polymer micelles have a certain sustained-release capacity.

[0133] Experimental Example 6

[0134] The in vitro biocompatibility of BSP-VES polymer micelles was evaluated using the MTT assay. NCM460 or CT26 cells were seeded in 96-well plates. After cell adhesion and growth, a series of different concentrations (0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.7 mg / mL, 0.8 mg / mL, and 1 mg / mL) of BSP-VES polymer micelle solutions were added, with untreated blank cells serving as a control. Four replicates were set per well. After culturing the treated cells for 24 or 48 hours, MTT solution was added, and the cells were incubated for another 4 hours. The supernatant was discarded, and 150 μL LDMSO was added to each well to dissolve the purple precipitate formazan. The cells were incubated at a constant temperature and shaken for 30 minutes to ensure complete dissolution. The absorbance at 490 nm was measured using a microplate reader.

[0135] The results are as follows Figure 6-A , Figure 6-B As shown, the proliferation rate of BSP-VES polymer nanomicelles at a concentration of 1 mg / mL was around 80% after incubation in NCM460 and CT26 cells for 24 h or 48 h, indicating that the vector has high biocompatibility.

[0136] Experimental Example 7

[0137] Cells in good condition were digested with trypsin and seeded into 24-well plates with coverslips at the bottom, then cultured for 24 hours. C6@BSP-VES cells were co-incubated for 2 hours, followed by staining with DAPI for 20 minutes, and then fixed with paraformaldehyde. After 20 minutes, the fixed cells were removed, placed on glycerol-soaked slides, sealed with nail polish, and observed under an inverted fluorescence microscope. Results are as follows: Figure 7 As shown, C6 exhibits green fluorescence, while nuclear staining (DAPI) shows blue fluorescence. The green fluorescence intensity of CT26 cells is significantly higher than that of NCM460 cells, indicating that CT26 cells take up more C6@BSP-VES.

[0138] Experimental Example 8

[0139] The uptake of C6@BSP-VES by gastric cancer cells (SGC7091) and normal cells (NCM460) for 2 hours was observed using a fluorescence inverted microscope. The experimental method was the same as above. Figure 8 As shown in the figure, the experimental results clearly indicate that SGC7091 cells take up more C6@BSP-VES compared to NCM460. Furthermore, compared to the SGC7091 group blocked by Bletilla striata polysaccharide (BSP), it is shown that the BSP-VES polymer micelles have the ability to target gastric cancer cells.

[0140] Experimental Example 9

[0141] To better evaluate the distribution of BSP-VES polymer nanomicelles in mice, BSP-VES nanoparticles labeled with near-infrared fluorescent dye (ROI) were injected into mice via the tail vein. In vivo imaging was performed at 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h. Mice were then sacrificed, and fluorescence imaging of various organs was conducted to analyze the time-dependent distribution of BSP-VES in tumor-bearing animals. Results are as follows: Figure 7 As shown, a small amount of fluorescence signal was observed 1 hour after tail vein injection of ROI@BSP-VES nanoparticles. With increasing time, the accumulation of fluorescence at the tumor site increased. By the 8th hour, a high level of fluorescence had accumulated within the tumor. Mice bearing CT26 tumors were sacrificed after 24 hours, and the fluorescence distribution in various tissues was as follows. Figure 9 The results showed that BSP-VES polymer nanomicelles were most abundant in the liver, with less distribution in other organs, and exhibited significant fluorescence at the tumor site in vitro. In vivo distribution experiments in tumor-bearing mice indicated that BSP-VES polymer nanomicelles had good targeting ability for CT26 tumors.

[0142] Experimental Example 10

[0143] To further evaluate the targeting ability of BSP-VES polymer nanomicelles at tumor sites, ROI-labeled BSP-VES nanoparticles were injected subcutaneously into mice with gastric cancer via the tail vein, and the distribution of systemic fluorescence signals was observed. The results are as follows: Figure 10 As shown, fluorescence accumulation at the tumor site increased over time. Mice bearing SGC7091 tumors were sacrificed after 24 hours, and the fluorescence distribution results of various tissues are as follows. Figure 7 The results showed that BSP-VES polymer nanomicelles were most abundant in the liver, with less distribution in other organs, and exhibited significant fluorescence at the tumor site in vitro. In vivo distribution experiments in tumor-bearing mice demonstrated that BSP-VES polymer nanomicelles have a good ability to target SGC7091 tumors.

[0144] Experimental Example 11

[0145] The antitumor activity of andrographolide and drug-loaded micelles was investigated using the MTT assay. CT26 cells were cultured at 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded at a density of [value missing] mL in 96-well plates. After cell attachment, AG and AG@BSP-VES polymer micelles were added at a specific concentration gradient, with untreated cells serving as a control. The absorbance of the cells at 490 nm was measured using a microplate reader.

[0146] The MTT assay was used to determine the proliferation inhibition rate and half-maximal inhibitory concentration (IC50) of AG@BSP-VES polymer nanomicelles against CT26 cells. 50 The inhibitory effects of AG and AG@BSP-VES on CT26 tumor cells are as follows: Figure 11 As shown, the inhibitory effect of free andrographolide and AG@BSP-VES on tumor cell proliferation is dose-dependent; that is, as the drug concentration increases, the inhibitory effect on cells strengthens and the cell proliferation rate decreases. The IC50 values ​​of free andrographolide and AG@BSP-VES polymer nanomicelles in CT26 cells were calculated. 50 The values ​​were 33.77 μg / mL and 19.49 μg / mL, respectively. MTT assay results showed that in CT26 tumor cells, the inhibitory effect of AG@BSP-VES polymer nanomicelles was stronger than that of free andrographolide. This may be because the carrier BSP-VES has a better affinity for tumor cells, enabling better and faster delivery of the encapsulated, poorly soluble drug AG into CT26 cells.

[0147] Experimental Example 12

[0148] The in vitro antitumor activity of AG@BSP-VES polymer nanomicelles was evaluated using Live & Dead cell staining. Logarithmically growing NCM460 or CT26 cells were seeded in 96-well cell culture plates. After cell adhesion, AG or AG@BSP-VES polymer nanomicelle solution at a concentration of 20 μg / mL was added, with untreated blank cells as a control. After culturing for 24 h, the cells were gently washed twice with 100 μL of PBS, and Live & Dead cell stain (EthD-1:Calcein AM = 20 μL * 0.5 mL : 0.5 μL * 0.5 mL PBS) was added. The cells were incubated for another 20 min, and images were taken under inverted fluorescence. The number of live and dead cells was statistically analyzed using Imaging J software.

[0149] The results of Live & Dead cell staining and statistical analysis of live and dead cells based on Imaging J software are shown in [reference needed]. Figure 12 As shown in the figure, both CT26 and NCM460 cells treated with free andrographolide exhibited dead cells (red). Nearly half of the CT26 cells treated with AG@BSP-VES polymer nanomicelles showed dead cells (red), while the number of dead cells in NCM460 cells was significantly lower than that in CT26 cells. The experimental results indicate that free andrographolide has no selectivity for either normal or cancer cells, while using the BSP-VES carrier to deliver the drug can increase its antitumor effect and may also improve its selectivity.

[0150] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, some modifications or improvements can be made to it based on the present invention, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A tumor-targeting Bletilla striata polysaccharide polymer nanomicelle, characterized in that: The tumor-targeting Bletilla striata polysaccharide polymer nanomicelles are composed of the following components: Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles, antitumor drugs, and deionized water; the antitumor drugs are either andrographolide or coumarin-6; the Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles serve as a carrier, encapsulating hydrophobic antitumor drugs in the hydrophobic core of the polymer micelles, and their preparation method is as follows: (1) Weigh out vitamin E succinate, dissolve it evenly in dimethyl sulfoxide solution under ultrasound, then add 4-dimethylpyridine and 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and react under magnetic stirring to activate the carboxyl group on vitamin E succinate to obtain reaction solution A; (2) Weigh out Bletilla striata polysaccharide, dissolve it in dimethyl sulfoxide solution, add reaction solution A dropwise, continue to stir the reaction with magnetic force, then dialyze with deionized water, freeze dry, and obtain Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles. (3) Weigh out Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles and dissolve them in deionized water. Add anhydrous ethanol solution containing antitumor drugs under stirring until turbidity is reached. Stir magnetically and then dialyze to obtain tumor-targeting Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles. Freeze-dry and store.

2. The tumor-targeting Bletilla striata polysaccharide polymer nanomicelles according to claim 1, characterized in that, The tumor-targeting Bletilla striata polysaccharide polymer nanomicelles are composed of the following components: 25-34 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles, 1-3 mg of andrographolide, and 8-12 mL of deionized water; or 25-34 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles, 180-220 μg of coumarin-6, and 8-12 mL of deionized water.

3. The tumor-targeting Bletilla striata polysaccharide polymer nanomicelles according to claim 2, characterized in that, The tumor-targeting Bletilla striata polysaccharide polymer nanomicelles are composed of the following components: 29 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles, 2 mg of andrographolide, and 10 mL of deionized water; or 29 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles, 200 μg of coumarin-6, and 10 mL of deionized water.

4. The tumor-targeting Bletilla striata polysaccharide polymer nanomicelles according to claim 1, characterized in that, The specific method for preparing the Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles is as follows: (1) Weigh 100-600 mg of vitamin E succinate and 10-25 ml of dimethyl sulfoxide solution and dissolve them evenly under sonication. Then add 23-138 mg of 4-dimethylpyridine and 43-260 mg of 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride and react for 1.5 h under magnetic stirring at 38 °C to activate the carboxyl group on vitamin E succinate to obtain reaction solution A; (2) Weigh 100-300 mg of Bletilla striata polysaccharide, dissolve it in 20-35 mL of dimethyl sulfoxide solution, add reaction solution A dropwise, continue to stir magnetically for 24 h, then dialyze with deionized water for 48 h, freeze dry at -80℃ to obtain 83-264 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles.

5. The tumor-targeting Bletilla striata polysaccharide polymer nanomicelles according to claim 1, characterized in that, The specific method for preparing the tumor-targeting Bletilla striata polysaccharide polymer nanomicelles is as follows: (1) Weigh 100-600 mg of vitamin E succinate and 10-25 ml of dimethyl sulfoxide solution and dissolve them evenly under sonication. Then add 23-138 mg of 4-dimethylpyridine and 43-260 mg of 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride and react for 1.5 h under magnetic stirring at 38 °C to activate the carboxyl group on vitamin E succinate to obtain reaction solution A; (2) Weigh 100-300 mg of Bletilla striata polysaccharide, dissolve it in 20-35 mL of dimethyl sulfoxide solution, add reaction solution A dropwise, continue to stir magnetically for 24 h, then dialyze with deionized water for 48 h, freeze dry at -80℃ to obtain 83-264 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles. (3) Weigh 25-34 mg of Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles and dissolve them in deionized water. Add anhydrous ethanol solution containing antitumor drugs under stirring until turbidity is reached. Stir magnetically for 6-8 hours and then dialyze for 8 hours to obtain 8-10 mL of tumor-targeting Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles. Freeze-dry and store.

6. The tumor-targeting Bletilla striata polysaccharide polymer nanomicelles according to any one of claims 4 or 5, characterized in that, In the preparation method of the Bletilla striata polysaccharide-vitamin E succinate polymer nanomicelles or the preparation method of the tumor-targeting Bletilla striata polysaccharide polymer nanomicelles: the molar ratio of vitamin E succinate, 4-dimethylpyridine and 1-yl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1:1.2; the mass ratio of Bletilla striata polysaccharide to vitamin E succinate is 1:

2.

7. The use of Bletilla striata polysaccharide polymer nanomicelles as described in any one of claims 1-6 in the preparation of targeted drugs for treating tumors.

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