A ginsenoside Rg3 drug-loading system, a preparation method and application thereof

Abstract

The present application relates to a ginsenoside Rg3 drug delivery system, and a preparation method and application thereof. White yam polysaccharides (BSPs) are extracted from white yam by water extraction and alcohol precipitation, and then subjected to defatting, decolorization, deproteinization and chromatographic purification. A small hydrophobic molecule SA is introduced to modify the structure of BSPs, and a dialysis method is used to prepare BSPs-SA polymer micelles, and an amphiphilic copolymer BSPs-SA is obtained as a carrier material. An emulsion solvent evaporation method is used to prepare G-Rg3@BSPs-SA polymer micelles, and the freeze-dried powder is loaded into a colon-soluble capsule shell to prepare a G-Rg3@BSPs-SA colon-soluble capsule with double targeting. The in vitro drug release behavior and preliminary stability of the capsule are evaluated. After oral administration of the delivery system, no drug is released in the upper gastrointestinal tract, and after entering the colon, the capsule shell is degraded in the specific pH environment of the colon to release G-Rg3@BSPs-SA polymer micelles. Through the dual targeting function on tumor cells and M2-TAMs, the delivery system can achieve multi-link regulation of "inhibiting tumor cell growth and regulating immune microenvironment" of colon cancer, and effectively inhibit the occurrence and development of colon cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of ginsenoside Rg3 drug preparation, and in particular relates to a ginsenoside Rg3 drug delivery system, a preparation method and an application thereof. Background Art

[0002] Colorectal cancer (CRC) is a common malignant tumor of the digestive tract. It is a malignant tumor that occurs in the colorectal mucosal epithelium and glandular parts under the combined effects of multiple pathogenic factors (such as genes, diet and environment). It has a high incidence of cancer worldwide and seriously endangers life and health. Its early diagnosis rate is low. Most patients are diagnosed in the middle and late stages, and some have already metastasized.

[0003] Ginsenoside Rg3 (G-Rg3) is a tetracyclic triterpenoid saponin extracted from ginseng. It is the main component of the new anticancer drug "Shenyi Capsule" and has good anticancer activity. G-Rg3 can not only "strengthen the body" by regulating TME and improving the body's immunity, but also directly act on tumor cells to inhibit proliferation and migration and promote apoptosis to "detoxify", thereby achieving multi-target and multi-link treatment of CRC. As a tetracyclic triterpenoid saponin component in ginseng, G-Rg3 has poor water solubility, weak fat solubility, and a short half-life. It is difficult to self-assemble in the shell-core structure. After oral administration, it enters the gastrointestinal tract and is metabolized by enzymes or intestinal bacteria therein. There is a first-pass effect and the blood drug concentration is low. The bioavailability is only 2.63%, which greatly limits its clinical application.

[0004] The oral colon-specific drug delivery system (OCDDS) can release drugs in the ileocecal region or colon instead of the upper gastrointestinal tract, exerting local or systemic therapeutic effects. It is often used to treat colon diseases such as ulcerative colitis and colon cancer. It is beneficial for drug accumulation in the diseased area, improving bioavailability and enhancing patient compliance.

[0005] Bletilla striata polysaccharides (BSPs) are water-soluble polysaccharides extracted from the dried tubers of the orchidaceae plant Bletilla striata (Thunb.) Reichb.f. BSPs are readily soluble in hot water, slightly soluble in cold water, and insoluble in ethanol. Their monosaccharide composition is relatively consistent, mostly consisting of mannose (Man) and glucose (Glc) linked by 1→4 glycosidic bonds, with a ratio of 4:1-2:1 and a molecular weight distribution of 1.0×10 4 ~1.0×10 5Da. BSPs themselves have multiple pharmacological activities such as hemostasis, wound healing, gastrointestinal mucosal protection, anti-inflammatory, antioxidant, anti-tumor and immune regulation. As a natural polymer material, BSPs are characterized by structural stability, biodegradability and high biosafety. They are often widely used in pharmaceutical raw materials and pharmaceutical excipients, playing the role of "drug-excipient integration". Because the main chain of BSPs contains a large number of active hydroxyl groups, it is easy to modify the structure. By using different design strategies to modify its structural matrix, carrier materials with different functions can be obtained.

[0006] BSPs are hydrophilic polymer materials. By introducing hydrophobic groups such as cholesteryl succinate, amphiphilic nanocarriers can be prepared. They can self-assemble in the aqueous phase to form nanomicelle systems with a core-shell structure. Their hydrophobic core is conducive to encapsulating poorly soluble anti-tumor drugs, improving drug bioavailability, and passively targeting tumor tissues through the enhanced permeability and retention effect (EPR). Summary of the Invention

[0007] The purpose of the present invention is to provide a G-Rg3@BSPs-SA colon-soluble capsule and a preparation method thereof. The G-Rg3@BSPs-SA colon-soluble capsule prepared by the present invention can be degraded in the specific pH environment of the colon to release G-Rg3@BSPs-SA polymer micelles. A large number of exposed Man molecules in BSPs bind to the highly expressed Man-R on colon cancer cells and M2-TAMs, respectively, promote cell uptake in an active targeted manner, and release drugs in the intracellular lysosomal environment (pH 5.0), respectively exerting their dual effects of "strengthening the body and detoxifying", improving the availability of drugs in the colon, and exerting anti-tumor effects, such as Figure 1 shown.

[0008] Ginsenoside Rg3 is a drug with the effect of "strengthening the body and detoxifying". Due to its weak lipid solubility, it is difficult to self-assemble in the shell-core structure. Therefore, the present invention adopts the emulsification solvent evaporation method to improve the preparation process. The present invention adopts the emulsification solvent evaporation method to prepare G-Rg3@BSPs-SA polymeric micelles and evaluates their in vitro drug release behavior. It is loaded into the colon-soluble capsule shell to prepare G-Rg3@BSPs-SA OCDDS, and its drug release behavior and preliminary stability are investigated. Through the dual targeting function of tumor cells and M2-TAMs, a multi-link regulatory effect of "inhibiting tumor cell growth and regulating the immune microenvironment" on colon cancer is achieved, thereby effectively inhibiting the occurrence and development of colon cancer.

[0009] In order to achieve the above tasks, the present invention is implemented through the following technical solutions:

[0010] A G-Rg3@BSPs-SA colon-soluble capsule, characterized in that it consists of the following components: ginsenoside Rg3 raw material, an amphiphilic Bletilla striata polysaccharide polymer carrier, and a colon-soluble capsule shell.

[0011] The amphiphilic Bletilla striata polysaccharide polymer carrier includes the following two compounds:

[0012] (a) Bletilla striata polysaccharides (BSPs), hydrophilic polymer materials.

[0013] (b), stearic acid (SA), hydrophobic group.

[0014] The G-Rg3@BSPs-SA colon-coated capsules are prepared by extracting BSPs from the traditional Chinese medicine Bletilla striata using a water-extraction and alcohol-precipitation method. The crude polysaccharide is purified by degreasing and color removal, followed by Savag deproteinization, and then purified using DEAE-52 and Sephadex G-200. The BSPs-SA polymer was synthesized using a ratio of n(SA):n(DMAP):n(EDC) of 1:1:1.2.

[0015] The above-mentioned G-Rg3@BSPs-SA polymer nanomicelles were prepared by dialysis method. BSPs-SA polymer micelles were self-assembled to obtain a clear and transparent polymer solution. TEM observation showed that the micelles were spherical and relatively evenly distributed. The particle size was determined by Malvern particle size analyzer to be 145.12±8.54nm, the PDI was 0.18±0.012 (PDI<0.3), and the ζ potential was -23.3±0.031mv. There was no significant difference in the particle size, dispersion index and ζ potential of the nanomicelles within 7 days (p>0.05). The BSPs-SA polymer nanomicelles have good stability and can be used for subsequent drug delivery.

[0016] The G-Rg3@BSPs-SA polymer nanomicelles, prepared by an emulsification and solvent evaporation method, exhibited spherical shape with a particle size of 185.43±1.34 nm, a PDI of 0.21±0.01, a zeta potential of -19.2±0.32 mV, a drug loading of 9.54±0.24%, and an encapsulation efficiency of 83.90±2.20%. The 48-hour cumulative release rates in three different media (pH 7.4, pH 6.5, and pH 5.0) were 65.32±3.22%, 79.56±4.28%, and 85.24±3.42%, respectively. The release rate was significantly greater than that of free G-Rg3. The drug was relatively stable in normal colonic fluid, with accelerated release in the acidic microenvironment of tumors. After intracellular entry, the drug was rapidly released in the acidic environment of lysosomes.

[0017] The above-mentioned G-Rg3@BSPs-SA colon-coated capsules do not release drugs in the stomach and small intestine. The cumulative drug release in simulated colonic fluid after 48 hours is 63.54±3.12%, which can increase the accumulation of drugs in the colon.

[0018] The above-mentioned G-Rg3@BSPs-SA polymer nanomicelles used Coumarin-6 as a fluorescent probe, and the cellular uptake was observed using a fluorescence microscope. Coumarin-6@BSPs-SA polymer nanomicelles were introduced into cells via the mannose receptor, and the uptake rates of CT-26 cells and M2-TAMs were 1.79 times and 1.46 times that of free Coumarin-6, respectively.

[0019] The BSPs-SA carrier in the G-Rg3@BSPs-SA polymer nanomicelles was tested for cytotoxicity using CCK-8. The results showed that the BSPs-SA carrier had no significant effect on the cell viability of CT-26 cells and M2-TAMs, indicating good biosafety.

[0020] The above-mentioned G-Rg3@BSPs-SA colon-coated capsule refers to a bidirectional colon-targeted drug for oral administration.

[0021] The preparation method of the above-mentioned G-Rg3@BSPs-SA colon-coated capsules is characterized by being carried out according to the following steps:

[0022] (a) Extraction of BSPs:

[0023] Place the Bletilla striata slices in a 60°C oven to dry thoroughly, then crush through an 80-mesh sieve. Take 100g of Bletilla striata coarse powder, add 20 times the volume of distilled water, and extract in a 90°C waterbath for 5 hours. Filter, collect the residue, and repeat the extraction three times. Filter through nylon gauze, collect the combined filtrate, and concentrate it to 1 / 3 of its original volume via rotary evaporation. Add 3 times the volume of anhydrous ethanol and let it stand overnight to allow for complete precipitation. Collect the precipitate and vacuum dry it to obtain the Bletilla striata polysaccharide extract.

[0024] Take an appropriate amount of Bletilla striata polysaccharide extract, add 10 times the volume of petroleum ether, reflux extract at 70℃ for 4 hours, filter and collect the residue. Add 10 times the volume of 80% ethanol, reflux extract for 4 hours, and filter to collect the residue.

[0025] Papain (0.5 mg / mL) was added to the defatted and decolorized polysaccharide aqueous solution. The pH was adjusted to 7.0 with NaOH solution (1 M). The solution was enzymatically hydrolyzed in a 50°C water bath for 2 hours, inactivated in a boiling water bath for 10 minutes, and cooled to room temperature. Protein removal was performed using the Sevag method. The polysaccharide solution was added to a separatory funnel and 20% Sevag reagent (chloroform: n-butanol, volume ratio 5:1) was added. The solution was shaken vigorously for 20 minutes and allowed to stand for 4 hours. The denatured protein at the interface of the layers was discarded. This process was repeated at least three times until no turbidity was observed at the interface. The upper layer was collected and concentrated by rotary evaporation to 1 / 3 of its original volume. Three volumes of anhydrous ethanol were added and the solution was allowed to stand overnight to fully decompose. The precipitate was collected and dried to obtain crude Bletilla striata polysaccharides (C-BSPs).

[0026] (b) Purification of BSPs:

[0027] Weigh 200 mg of C-BSPs and dissolve them in 20 mL of deionized water. Using a rubber-tipped pipette, slowly drip the Bletilla striata polysaccharide solution onto a DEAE-52 cellulose column (4 cm × 60 cm) for chromatography. Elute with a gradient of deionized water and 0.1, 0.2, and 0.5 mol / L NaCl solutions, sequentially, at a flow rate of 1 mL / min. Collect 5 mL of the solution in a test tube.

[0028] Weigh 100 mg of crude polysaccharide from Bletilla striata purified by DEAE-52 column chromatography, dissolve it in 10 mL of deionized water, and chromatograph it through a Sephadex G-200 column (2.3 cm × 90 cm). Elute with a 0.02 mol / L NaCl aqueous solution at a flow rate of 15 mL / min, collecting 5 mL of the solution in a test tube.

[0029] (c) Preparation of BSPs-SA:

[0030] 142.24 mg of SA, 61.08 mg of 4-dimethylaminopyridine (DMAP), and 93.14 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were dissolved in 3 mL of DMSO and activated with magnetic stirring at room temperature for 2 h to obtain an activation reaction solution. 400 mg of BSPs was dissolved in 4 mL of DMSO and added dropwise to the activation reaction solution. The reaction was allowed to proceed at 75°C for 4 h and then at room temperature for 24 h. After completion of the reaction, the reaction mixture was dialyzed in a 3500 Da dialysis bag for 24 h to remove DMSO. The mixture was then extracted three times with an equal volume of ethyl acetate to remove unreacted SA. The ethyl acetate was then removed by rotary evaporation at 40°C, and the product was freeze-dried to obtain BSPs-SA.

[0031] (d) Preparation of BSPs-SA polymer micelles:

[0032] BSPs-SA polymer micelles were prepared by dialysis. Accurately weigh 25.0 mg of BSPs-SA and dissolve it completely in 5 mL of DMSO. The solution was then transferred to an 8000 Da dialysis bag and dialyzed against 500 mL of pure water at room temperature, changing the water every 8 hours for a total of 48 hours. The solution was filtered through a 0.45 μm microporous membrane and transferred to a 25 mL volumetric flask to obtain a blank polymer micelle solution.

[0033] (e) Preparation of G-Rg3@BSPs-SA polymer nanomicelles:

[0034] Accurately weigh 25 mg of BSPs-SA and dissolve it completely in 4 mL of DMSO. Transfer the entire amount to a dialysis bag (8-14 kDa). Place the bag in 500 mL of pure water and dialyze at room temperature, changing the water every 8 hours for a total of 48 hours. Filter through a 0.45 μm microporous membrane and transfer to a 25 mL volumetric flask to obtain a 1 mg / mL blank micelle solution. Accurately weigh 5 mg of G-Rg3 and dissolve it in 2.5 mL of methanol to obtain a 2 mg / mL G-Rg3 solution. Add 1.6 mL of the G-Rg3 solution dropwise to the blank BSPs-SA micelles at 250 rpm. Stir at room temperature for 4 hours, evaporate the methanol, filter through a 0.45 μm microporous membrane, and freeze-dry to obtain the G-Rg3@BSPs-SA polymer micelle lyophilized powder.

[0035] (f) Preparation of G-Rg3@BSPs-SA colon-coated capsules:

[0036] Weigh an appropriate amount of G-Rg3@BSPs-SA polymer nanomicelle lyophilized powder and directly fill it into colon-soluble capsules for mice (provided by Huangshan Capsule Co., Ltd.). After filling, apply glue to the interface between the capsule cap and the capsule body and allow to dry. Each capsule contains 4 mg of polymer micelle powder and approximately 0.4 mg of G-Rg3.

[0037] The present invention is beneficial in that:

[0038] The present invention discloses a ginsenoside Rg3 colon-coated capsule and a preparation method thereof. The prepared G-Rg3@BSPs-SA colon-coated capsule is an oral bidirectional colon-targeted preparation. Ginsenoside Rg3@BSPs-SA OCDDS can, through the mediation of mannose receptors, target M2-TAMs to promote reprogramming to M1 type, reduce TGF-β, increase TNF-α, induce CD8+T cell infiltration, reverse the immunosuppressive environment, and reconstruct the tumor microenvironment to "restore the body's health"; it can target colon cancer cells to inhibit cell proliferation, migration and invasion, promote cell apoptosis, downregulate the expression of snail nuclear transcription factor to inhibit epithelial-mesenchymal transition, inhibit tumor growth and "detoxify", and effectively control the occurrence and development of colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the action of the ginsenoside Rg3 dual-targeted oral colon-localized drug delivery system.

[0040] Figure 2 A is the appearance of BSPs-SA polymer micelle solution, Figure 2 B is the transmission electron microscopy image of BSPs-SA polymer micelles.

[0041] Figure 3 A is the change of particle size and PDI of BSPs-SA polymer micelles within 7 days. Figure 3 B is the change of ζ potential of BSPs-SA polymer micelles within 7 days.

[0042] Figure 4 A is the particle size and dispersion index of G-Rg3@BSPs-SA polymer micelles prepared by different methods, Figure 4 B is the drug loading capacity and encapsulation efficiency of G-Rg3@BSPs-SA polymer micelles prepared by different methods.

[0043] Figure 5 A is the particle size and dispersion index of G-Rg3@BSPs-SA polymer micelles prepared at different G-Rg3 solution concentrations, Figure 5 B shows the drug loading and encapsulation efficiency of G-Rg3@BSPs-SA polymer micelles prepared with different G-Rg3 solution concentrations.

[0044] Figure 6A is the particle size and dispersion index of G-Rg3@BSPs-SA polymer micelles prepared with different G-Rg3 / BSPs-SA mass ratios, Figure 6 B shows the drug loading capacity and encapsulation efficiency of G-Rg3@BSPs-SA polymer micelles prepared with different G-Rg3 / BSPs-SA mass ratios.

[0045] Figure 7 A is the transmission electron microscopy image of G-Rg3@BSPs-SA micelles at pH = 7.4. Figure 7 B is the transmission electron microscopy image of G-Rg3@BSPs-SA micelles at pH = 5.0. Figure 7 C is a transmission electron microscopy image of G-Rg3@BSPs-SA micelles at a magnification of ×2 at pH = 5.0.

[0046] Figure 8 A is the particle size of G-Rg3@BSPs-SA polymer nanomicelles at pH = 7.4, Figure 8 B is the particle size of G-Rg3@BSPs-SA polymer nanomicelles at pH = 5.0, Figure 8 C is the zeta potential of G-Rg3@BSPs-SA polymer nanomicelles at pH = 7.4, Figure 8 D is the zeta potential of G-Rg3@BSPs-SA polymer nanomicelles at pH = 5.0.

[0047] Figure 9 In vitro release curves of G-Rg3 and G-Rg3@BSPs-SA polymer nanomicelles under different pH conditions.

[0048] Figure 10 In vitro release curves of G-Rg3 colon-coated capsules and G-Rg3@BSPs-SA colon-coated capsules.

[0049] Figure 11 Fluorescence microscopy was used to observe the effects of Coumarin-6 and

[0050] Uptake of Coumarin-6@BSPs-SA (Scale bar: 50 μm). Figure 11 A is a representative fluorescence image of CT-26 cells uptake of Coumarin-6 and Coumarin-6@BSPs-SA. Figure 11 B is the fluorescence intensity analysis of Coumarin-6 and Coumarin-6@BSPs-SA uptake by CT-26 cells. Figure 11 C is a representative fluorescence image of Coumarin-6 and Coumarin-6@BSPs-SA uptake by CT-26 cells after mannose treatment. Figure 11D is the expression of Coumarin-6 and

[0051] Fluorescence intensity analysis of Coumarin-6@BSPs-SA uptake (Scale bar: 50 μm).

[0052] Figure 12 A is a representative fluorescence image of Coumarin-6 and Coumarin-6@BSPs-SA uptake by M2-TAMs after pre-treatment with mannoside. Figure 12 B is the fluorescence intensity analysis of Coumarin-6 and Coumarin-6@BSPs-SA uptake by M2-TAMs after pre-treatment with mannoside (scale bar: 50 μm).

[0053] Figure 13 A shows the effects of G-Rg3 and G-Rg3@BSPs-SA polymer micelles on the survival rate of CT-26 cells. Figure 13 B shows the effects of G-Rg3 and G-Rg3@BSPs-SA polymer micelles on the survival rate of M2-TAMs cells.

[0054] Figure 14 Statistical analysis of early apoptosis, late apoptosis and total apoptosis rates of CT-26 cells in vitro (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

[0055] Figure 15 The scratch experiment was used to investigate the effects of each group of drugs on the wound healing ability of CT-26 cells.

[0056] Figure 16 The Transwell method was used to investigate the effects of drugs on the migration and invasion of CT-26 cells. Figure 16 A is a representative picture of CT-26 migrating cells; Figure 16 B is a representative picture of CT-26-invading cells; Figure 16 C is the migration rate of CT-26 cells; Figure 16 D is the invasion rate of CT-26 cells (scale bar: 50 μm, Mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

[0057] Figure 17 A is the cytotoxicity of G-Rg3 and G-Rg3@BSPs-SA polymer micelles at different concentrations to CT-26 cells in the Transwell co-culture model. Figure 17B shows the cytotoxicity results of each group of preparations (G-Rg3: 20 μM) on CT-26 cells in the Transwell co-culture model (Mean±SD, n=3, *p<0.05, ***p<0.0005, ****p<0.0001).

[0058] Figure 18 The figure shows the in vivo distribution of each group of drugs in CT-26 orthotopic colon cancer mice at different time points.

[0059] Figure 19 A is an in vitro fluorescence image of the intestine of a mouse with CT-26 orthotopic colon cancer. Figure 19 B is the in vitro fluorescence intensity analysis of the intestine of mice with CT-26 orthotopic colon cancer. Figure 19 C is the in vitro fluorescence image of the main organs of mice with CT-26 orthotopic colon cancer. Figure 19 D is the in vitro fluorescence intensity analysis of the main organs of mice bearing CT-26 orthotopic colon cancer.

[0060] Figure 20 A is a schematic diagram of CT-26 in situ colon cancer modeling and drug administration. Figure 20 B is the in vivo bioluminescent imaging of tumors in mice with CT-26 orthotopic colon cancer on days 0, 7, and 14 after administration. Figure 20 C is the curve of tumor bioluminescence intensity changes on days 0, 7, and 14 after administration of CT-26 orthotopic colon cancer mice. Figure 20 D is the tumor inhibition rate of each preparation in each group on the 14th day after administration to mice with CT-26 orthotopic colon cancer (mean±SD, n=15).

[0061] Figure 21 A is the survival time of CT-26 carcinoma in situ mice after drug treatment in each group. Figure 21 B is the weight change of CT-26 carcinoma in situ mice after drug treatment in each group. Figure 21 C shows the intestinal tumor morphology and number of CT-26 in situ carcinoma mice after drug treatment in each group. Figure 21 D is the weight of intestinal tumors in CT-26 carcinoma in situ mice after drug treatment in each group.

[0062] Figure 22 Pathological observation of tumor tissue and major organs (scale: 50 μm).

[0063] Figure 23 The TUNEL method was used to detect the apoptosis of tumor cells in CT-26 carcinoma in situ mice (scale bar: 50μm).

[0064] Figure 24 Immunofluorescence detection of Ki67 expression in tumor tissues of CT-26 carcinoma in situ mice (scale bar: 50 μm).

[0065] Figure 25 The fluorescence intensity of CD206 and CD86 in the tumor tissues of CT-26 orthotopic colon cancer mice in each group after drug administration (scale: 50 μm)

[0066] Figure 26 A is the relative fluorescence intensity of CD206 in the tumor tissues of CT-26 orthotopic colon cancer mice in each group after drug administration. Figure 26 B is the relative fluorescence intensity of CD86 in the tumor tissues of CT-26 orthotopic colon cancer mice in each group after drug administration.

[0067] Figure 27 A is a representative fluorescence image of CD8+ T cells in the tumor tissues of CT-26 orthotopic colon cancer mice in each group after drug administration. Figure 27 B is the relative fluorescence intensity analysis of CD8+ T cells in the tumor tissues of CT-26 orthotopic colon cancer mice in each group after drug administration. (Scale bar: 50μm)

[0068] Figure 28 A is the ELISA test of TNF-α levels in the supernatant of tumor tissues of CT-26 orthotopic colon cancer mice in each group after administration. Figure 28 B is the ELISA test of the TGF-β level in the tumor tissue supernatant of CT-26 orthotopic colon cancer mice in each group after administration.

[0069] Figure 29 A is the immunofluorescence assay of E-cadherin in the tumor tissues of CT-26 orthotopic colon cancer mice in each group after drug administration. Figure 29 B is the immunofluorescence assay of N-cadherin in the tumor tissues of CT-26 orthotopic colon cancer mice in each group after drug administration. Figure 29 C is the immunofluorescence assay of Vimentin in the tumor tissues of CT-26 orthotopic colon cancer mice in each group after drug administration. Figure 29 D is the immunofluorescence assay of Snail in the tumor tissues of CT-26 orthotopic colon cancer mice in each group after administration. (Scale bar: 50 μm) DETAILED DESCRIPTION

[0070] In the following examples, the ratios of SA, DMAP, and EDC when synthesizing BSPs-SA polymers, and the preparation process of BSPs-SA polymer nanomicelles are preferably:

[0071] BSPs-SA polymer was synthesized using a ratio of n(SA):n(DMAP):n(EDC) = 1:1:1.2. BSPs-SA polymer micelles were prepared by dialysis. The dialysis bag was placed in 500 mL of pure water and dialyzed at room temperature, with the water changed every 8 hours for a total of 48 hours.

[0072] In the following examples, the preparation process of G-Rg3@BSPs-SA polymer nanomicelles is preferably:

[0073] G-Rg3@BSPs-SA polymer micelles were prepared by an emulsification solvent evaporation method. The concentration of G-Rg3 was 2 mg / mL, the concentration of BSPs-SA polymer micelles was 1 mg / mL, the mass ratio of G-Rg3 to BSPs-SA was 1:8, and the stirring speed was 250 rpm at room temperature for 4 hours.

[0074] Example 1:

[0075] 1) Dissolve 142.24 mg of SA, 61.08 mg of 4-dimethylaminopyridine (DMAP), and 93.14 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in 3 mL of DMSO and activate with magnetic stirring at room temperature for 2 h to obtain an activation reaction solution.

[0076] 2) Weigh 400 mg of BSPs and dissolve them in 4 mL of DMSO. Add the solution dropwise to the activation reaction mixture and react at 75°C for 4 hours, then at room temperature for 24 hours. After the reaction, place the mixture in a dialysis bag (3500 Da) and dialyze for 24 hours to remove DMSO. Repeat extraction with an equal volume of ethyl acetate three times to remove unreacted SA. Rotary evaporate the ethyl acetate at 40°C and freeze-dry the product to obtain BSPs-SA.

[0077] 3) Accurately weigh 25.0 mg of BSPs-SA and dissolve it completely in 5 mL of DMSO. Transfer the entire amount to a dialysis bag (8000 Da). Place the dialysis bag in 500 mL of pure water and dialyze at room temperature, changing the water every 8 h for a total of 48 h. Pass the bag through a 0.45 μm microporous filter and transfer the diluted volume to a 25 mL volumetric flask to obtain blank BSPs-SA polymer micelles.

[0078] The BSPs-SA polymer micelles in Example 1 were observed by transmission electron microscopy, their particle size and zeta potential were measured, and their stability was investigated. The results showed that the prepared BSPs-SA polymer micelles were clear and transparent polymer solutions with regular spherical shapes, as shown in FIG. Figure 2The BSPs-SA polymer micelle particle size was 145.12 ± 8.54 nm, the PDI was 0.18 ± 0.012, the dispersion was good, and the zeta potential was -23.3 ± 0.031 mv. The particle size, dispersion index and zeta potential of the carrier micelles had no significant difference (p > 0.05) within 1 week. The amphiphilic polymer nanomicelle BSPs-SA prepared by dialysis had good stability, as shown in the figure. Figure 3

[0079] Example 2:

[0080] 1) The BSPs-SA polymer micelles of Example 1 were completely dissolved in DMSO and then transferred to a dialysis bag (8-14 kDa). The dialysis bag was placed in 500 mL of pure water at room temperature and dialyzed every 8 hours. The cumulative dialysis time was 48 hours. The solution was filtered through a 0.45 μm microporous filter and transferred to a 25 mL volumetric flask. The blank micelle solution was obtained.

[0081] 2) The prescribed amount of G-Rg3 was dissolved in methanol, and an appropriate amount was added dropwise to the blank BSPs-SA carrier micelles under stirring. The organic reagent methanol was removed by stirring at an appropriate temperature. After being filtered through a 0.45 μm microporous filter, G-Rg31@BSPs-SA was obtained by freeze-drying.

[0082] Example 3:

[0083] 1) The BSPs-SA polymer micelles of Example 1 were dispersed in DMSO, and an appropriate amount of G-Rg32 was dissolved in DMSO. The two were mixed overnight.

[0084] 2) The DMSO was removed by dialysis against deionized water for 32 hours, with fresh solution being replaced every 12 hours. The excess G-Rg3 was removed by freeze-drying to obtain G-Rg32@BSPs-SA.

[0085] Example 4:

[0086] 1) The BSPs-SA polymer micelles of Example 1 were dissolved in deionized water, and an appropriate amount of G-Rg33 was dissolved in DMSO.

[0087] 2) The G-Rg33 solution was added dropwise to the BSPs-SA aqueous solution using a syringe, and ultrasonicated for 30 min. The precipitate was collected by centrifugation after being filtered through a 0.45 um microporous filter to obtain G-Rg33@BSPs-SA.

[0088] ​The particle size, PDI, LC and EE of Example 2, Example 3 and Example 4 were measured to screen out the best preparation method. Flocculent precipitation occurred during the preparation of polymer nanomicelles in Example 4, while the polymer micelles prepared in Example 2 and Example 3 were clear and transparent. In contrast, the preparation prepared in Example 2 had a smaller particle size and PDI, a higher drug loading and encapsulation efficiency, as shown in Figure 2. Figure 4 shown.

[0089] Example 5:

[0090] 5 mg of G-Rg3 was dissolved in 2.5 mL of methanol, and 25 mg of BSPs-SA was dissolved in 4 mL of DMSO. After dialysis for 48 h, the solution was transferred to a 25 mL volumetric flask. Under magnetic stirring, other factors were kept constant, and the concentrations of G-Rg3 solution were selected to be 1.00 mg / mL, 2.00 mg / mL, and 3.00 mg / mL. The G-Rg3 methanol solution was slowly dripped into the BSPs-SA blank carrier micelles. The stirring time was 2 h, the temperature was 30°C, and the rotation speed was 100 rpm. G-Rg3@BSPs-SA polymer nanomicelles were prepared using the method described in Example 2.

[0091] Example 6:

[0092] 5 mg of G-Rg3 was dissolved in 2.5 mL of methanol, and 25 mg of BSPs-SA was dissolved in 4 mL of DMSO. After dialysis for 48 h, the solution was transferred to a 25 mL volumetric flask. Under magnetic stirring, while keeping other factors constant, different G-Rg3 / BSPs-SA mass ratios (1:3, 1:5, 1:8, 1:10, and 1:12) were selected. The G-Rg3 methanol solution was slowly added dropwise to the BSPs-SA blank carrier micelles. The stirring time was 2 h, the temperature was 30°C, and the rotation speed was 100 rpm. G-Rg3@BSPs-SA polymer nanomicelles were prepared using the method described in Example 2.

[0093] Example 7:

[0094] 5 mg of G-Rg3 was dissolved in 2.5 mL of methanol, and 25 mg of BSPs-SA was dissolved in 4 mL of DMSO. After dialysis for 48 h, the volume was transferred to a 25 mL volumetric flask. Under magnetic stirring, the G-Rg3 methanol solution was slowly added dropwise to the BSPs-SA blank carrier micelles. The stirring time was 2 h and the temperature was 30°C. Other factors were kept constant. Different rotation speeds (100 rpm, 250 rpm, 500 rpm) were selected to prepare G-Rg3@BSPs-SA polymer nanomicelles using the method described in Example 2.

[0095] Example 8:

[0096] 5 mg of G-Rg3 was dissolved in 2.5 mL of methanol, and 25 mg of BSPs-SA was dissolved in 4 mL of DMSO. After dialysis for 48 h, the volume was transferred to a 25 mL volumetric flask. Under magnetic stirring, the G-Rg3 methanol solution was slowly added dropwise to the BSPs-SA blank carrier micelles at a temperature of 30°C and a rotation speed of 100 rpm. While keeping other factors constant, different stirring times (2 h, 4 h, and 8 h) were selected to prepare G-Rg3@BSPs-SA polymer nanomicelles using the method described in Example 2.

[0097] Example 9:

[0098] 5 mg of G-Rg3 was dissolved in 2.5 mL of methanol, and 25 mg of BSPs-SA was dissolved in 4 mL of DMSO. After dialysis for 48 h, the volume was transferred to a 25 mL volumetric flask. Under magnetic stirring, the G-Rg3 methanol solution was slowly added dropwise to the BSPs-SA blank carrier micelles. The stirring time was 2 h and the rotation speed was 100 rpm. Other factors were kept constant. Different reaction temperatures (30°C, 50°C, and 80°C) were selected to prepare G-Rg3@BSPs-SA polymer nanomicelles using the method described in Example 2.

[0099] The changes in particle size, PDI, LC and EE were observed in Example 5, Example 6, Example 7, Example 8 and Example 9. When the concentration of G-Rg3 solution was 2.00 mg / mL, the particle size of polymer nano-micelles was smaller and the encapsulation efficiency was larger. Figure 5 As shown in the figure, the mass ratio of G-Rg3 / BSPs-SA is 1:8 as the prescription dosage, and the drug loading is the best. Figure 6 As shown in the figure, at a stirring speed of 250 rpm, the PDI value of G-Rg3@BSPs-SA polymer micelles was minimal, while drug loading and encapsulation efficiency were maximized. With increasing stirring time, particle size and PDI decreased. Compared with stirring for 8 hours, there was no difference in particle size after stirring for 4 hours. To save time, stirring for 4 hours was used to prepare drug-loaded polymer micelles. Stirring temperature had no significant effect on the results, so room temperature was selected as the stirring temperature for preparing polymer micelles.

[0100] According to the optimized prescription process, three G-Rg3@BSPs-SA polymer micelles were prepared in parallel, and their particle size, PDI, LC and EE were measured. The particle size, PDI, LC (%) and EE (%) of the G-Rg3@BSPs-SA polymer nanomicelles prepared by the optimal process and prescription were 183.68±4.82nm, 0.21±0.02, 9.54%±0.24 and 83.90%±2.20, respectively, indicating a stable process.

[0101] Example 10:

[0102] The G-Rg3@BSPs-SA polymer micelles in Example 2 were observed for their appearance, morphology, particle size and ζ potential, and in vitro release studies. The results showed that the prepared G-Rg3@BSPs-SA polymer micelles were spherical in shape with a regular particle size of about 180 nm at pH 7.4, which was about 35 nm larger than the blank micelle particle size and had a relatively uniform distribution. As the pH value decreased, at pH 5.0, the polymer micelle particle size increased slightly to about 205 nm. Figure 7 As shown. Under pH 7.4 conditions, the micelle size is 185.43±1.34nm, PDI is 0.21±0.01, and ζ potential is -19.2±0.32mV; under pH 5.0 conditions, the particle size is 205.65±2.03nm, and the distribution has a double peak, which may be due to the charge repulsion between particles causing the micelles to expand slightly and the ζ potential to increase slightly, as shown. Figure 8 As shown in Figure 2, the release rate of GRg3@BSPs-SA micelles was significantly greater than that of free G-Rg3 drugs, and the cumulative release rates were 65.32±3.22%, 79.56±4.28%, and 85.24±3.42%, respectively. Figure 9 shown.

[0103] Example 11:

[0104] An appropriate amount of the lyophilized polymer nanomicelle powder from Example 2 was weighed and directly filled into colon-coated capsules for mice (provided by Huangshan Capsule Co., Ltd.). After filling, the interface between the capsule cap and the capsule body was coated with glue and allowed to dry. This yielded G-Rg3@BSP-SA colon-coated capsules. Each capsule contained 4 mg of polymer micelle powder and approximately 0.4 mg of G-Rg3.

[0105] The G-Rg3@BSP-SA colon-coated capsules prepared in Example 11 were subjected to in vitro release studies and preliminary stability tests. Results: The prepared G-Rg3@BSP-SA colon-coated capsules began to release the drug when the release medium was set at pH 7.4. Within 48 hours, the cumulative drug release from G-Rg3 OCDDS was only 28.12±1.91%, while the cumulative drug release from G-Rg3@BSPs-SA OCDDS was 63.54±3.12%. Figure 10 After high temperature, low temperature, and strong light irradiation stability tests, the morphology of the G-Rg3@BSPs-SA micelle powder did not change significantly, but after 10 days of high humidity treatment, a small amount of powder was slightly sticky.

[0106] Example 12:

[0107] 1) Accurately weigh 25 mg of BSPs-SA and dissolve it completely in 4 mL of DMSO. Transfer the entire amount to a dialysis bag (8-14 kDa). Place the bag in 500 mL of pure water and dialyze at room temperature, changing the water every 8 h for a total of 48 h. Pass the bag through a 0.45 μm microporous filter membrane and transfer the solution to a 25 mL volumetric flask to obtain a 1 mg / mL blank micelle solution.

[0108] 2) Precisely weigh the prescribed amount of coumarin-6 solid powder in a dark place and dissolve it in an appropriate amount of methanol to prepare a stock solution with a final concentration of 2 mg / mL. Pour the solution dropwise into the blank BSPs-SA micelles at 250 rpm. Stir at room temperature for 4 h. Evaporate the methanol, filter through a 0.45 μm microporous membrane, and freeze-dry to obtain Coumarin-6@BSPs-SA.

[0109] The Coumarin-6@BSPs-SA of Example 12 was used to investigate the uptake of the polymer micelles by colon cancer CT-26 cells and M2-TAMs. The results showed that the Coumarin-6@BSPs-SA polymer micelles were mediated by the mannose receptors highly expressed on the surface of CT-26 cells, thereby improving the cellular uptake efficiency. The preparation has CT-26 cell targeting function, such as Figure 11 As shown. The mannose unit in the BSPs-SA carrier structure can bind to the mannose receptors highly expressed on the surface of M2-TAMs cells, targeting M2-TAMs without specifically targeting M1-TAMs in the tumor site. This lays the foundation for drug-loaded polymer micelles to regulate the depolarization of M2-TAMs toward M1-TAMs, relieve the immunosuppressive environment, and exert immunotherapy effects. Figure 12 shown.

[0110] Example 13:

[0111] Different concentrations of each drug or carrier were incubated with CT-26 cells and M2-TAMs for 24 hours, and the CCK-8 method was used to investigate the effects of free G-Rg3, G-Rg3@BSPs-SA polymer micelles and BSPs-SA on the viability of mouse colon cancer CT-26 cells and M2-TAMs cells. The results showed that both G-Rg3 and G-Rg3@BSPs-SA could effectively inhibit the proliferation of CT-26 cells, and G-Rg3@BSPs-SA polymer micelles showed stronger in vitro anti-tumor effect, as shown in Figure 3. Figure 13 shown.

[0112] Example 14:

[0113] Annexin V FITC and PI double staining was used to investigate the effects of G-Rg3, G-Rg3@BSPs-SA, BSPs-SA, and G-Rg3+BSPs-SA physical mixture on CT-26 cell apoptosis by flow cytometry. Figure 14 As shown in the figure, G-Rg3@BSPs-SA polymer micelles induced the most obvious apoptosis of CT-26 cells compared with other groups, and mainly induced early apoptosis.

[0114] Example 15:

[0115] The anti-metastatic ability of G-Rg3@BSPs-SA polymer micelles on CT-26 colon cancer cells was evaluated by scratch test. When the cells grew to a fused monolayer, a blank cell-free area was artificially created on the monolayer cells, which was recorded as the cell scratch width at 0h. The scratch healing was observed at 24h and 48h to determine the effect of the drug on cell migration ability. The results are shown in Figure 2. Figure 15 As shown, G-Rg3@BSPs-SA polymer micelles showed the strongest ability to inhibit the migration of CT-26 colon cancer cells compared with other groups.

[0116] Example 16:

[0117] Transwell assay was used to further verify the ability of G-Rg3@BSPs-SA to inhibit the migration and invasion of CT-26 cells. Based on the permeability of the cell membrane and the restrictive nature of the basement membrane, cells were seeded in the upper chamber of the Transwell, and culture medium containing chemoattractants or stimulatory factors was added to the lower chamber to promote cell migration to the target area. The migration and invasion abilities of cells were evaluated by observing and counting the number of CT-26 cells that passed through the micropores at the bottom of the upper chamber and reached the lower chamber and were stained with crystal violet. The results are shown in Figure 2. Figure 16 As shown in the results, G-Rg3@BSPs-SA polymer micelles significantly inhibited the migration and invasion abilities of CT-26 cells compared with other groups.

[0118] Example 17:

[0119] In order to simulate the tumor microenvironment in vitro, a non-contact co-culture model was established using a Transwell culture plate with a pore size of 0.4 μm. M2-TAMs were placed in the upper chamber of the chamber, and CT-26 cells were plated in the lower chamber of the chamber. The various cytokines, growth factors or chemokines in the upper and lower chambers of this model can smoothly pass through the micropores at the bottom of the chamber to exchange with each other, while the two cells remain in a non-contact independent state. The G-Rg3@BSPs-SA polymer micelles prepared in this study have better ability to induce tumor-associated macrophages to transform from M2 type to M1 type, upregulate the expression of pro-inflammatory cytokines, downregulate the expression of anti-inflammatory cytokines, relieve the immunosuppressive environment, reconstruct the tumor immune microenvironment, and thus enhance the anti-tumor effect.Figure 17 as shown.

[0120] Example 18:

[0121] 1) Precisely weigh 25 mg of BSPs-SA, add 4 mL of DMSO to completely dissolve, then transfer all into a dialysis bag (8-14 kDa), place the dialysis bag in 500 mL of pure water, dialyze at room temperature, change water every 8 h, accumulate dialysis for 48 h, pass through a 0.45 μm microporous filter, transfer to a 25 mL volumetric flask, and obtain a 1 mg / mL blank micelle solution.

[0122] 2) Precisely weigh an appropriate amount of DiR solid powder, dissolve with an appropriate amount of anhydrous ethanol to obtain a DiR ethanol solution with a final concentration of 2 mg / mL, store in the dark at 20°C. Drop by drop into the blank BSPs-SA micelles at a speed of 250 rpm, stir at room temperature for 4 h, evaporate the methanol, pass through a 0.45 um microporous filter, freeze-dry, and obtain DiR@BSPs-SA.

[0123] The DiR@BSPs-SA polymer micelle solution (containing an equal amount of DiR) in Example 18 was encapsulated into a mouse colon-soluble capsule shell, and each mouse was given 1 capsule for intragastrical administration as the test product group. Ten days after the in situ colon cancer modeling of Balb / c mice, the colon cancer in situ mice were randomly divided into 2 groups, 3 mice in each group. The control group was given 500 μL of free DiR ethanol solution (0.4 mg / mL) per mouse by intragastrical administration, and at 1, 2, 4, 8, 12 and 24 h after intragastrical administration, the mice were anesthetized with isoflurane and then placed on the IVIS Spectrum small animal live imaging system to observe the real-time distribution of fluorescence in the mice, as shown in Figure 18 As shown, the DiR@BSPs-SA colon-soluble capsules showed weak fluorescence within 4 h after intragastrical administration, and no drug was released in the stomach and small intestine. At the end of the experiment, the animals were euthanized, the intestines and main organs (heart, liver, spleen, lung, kidney) were dissected, and the tissue organs were imaged again using the small animal live imaging system to observe the 24 h ex vivo tissue distribution of the drug, as shown in Figure 19 As shown, the DiR@BSPs-SA colon-soluble capsule group showed the strongest fluorescence intensity at the tumor site compared with other groups, and fluorescence was only observed in the liver.

[0124] Example 19:

[0125] 1) Male Balb / c mice weighing 18-20 g were acclimated to room temperature for one week, fasted for 24 hours, weighed, and anesthetized intraperitoneally with 20% urethane at a rate of 0.006 mL / g. The abdomen was prepared and disinfected with iodine-containing cotton balls. The midline of the abdomen was incised using surgical scissors. The cecum was removed from the abdomen using alcohol-sterilized forceps. 50 μL of a CT-26Luc cell suspension (containing 10 μg / μL Matrigel) at a concentration of approximately 1 x 106 cells / mL was injected into the cecal wall using a 30G needle and a microinjector. The cecum was then returned to the peritoneal cavity, and the peritoneum and skin were closed with sutures to establish a CT-26 colon carcinoma in situ model.

[0126] 2) Ten days after colon carcinoma in situ modeling in Balb / c mice, the mice with colon carcinoma in situ were randomly divided into 6 groups, with 15 mice in each group, namely, PBS group, Shenyi capsule content group (referred to as Shenyi capsule group), G-Rg3 colon-soluble capsule group (referred to as free G-Rg3 group), BSPs-SA colon-soluble capsule group (referred to as BSPs-SA group), G-Rg3@BSPs-SA colon-soluble capsule group (referred to as G-Rg3@BSPs-SA group), and G-Rg3+BSPs-SA physical mixture colon-soluble capsule group (referred to as G-Rg3+BSPs-SA group). G-Rg3 was orally administered by gavage once a day, with a single dose of 20 mg / kg for 14 consecutive days.

[0127] 3) Pharmacodynamic studies were conducted using a Balb / c mouse CT-26 colon carcinoma in situ model. Dosing was initiated 10 days after model establishment, when the tumor bioluminescence intensity reached 109 p / sec / cm2 / sr. The intensity of mouse tumor bioluminescence is proportional to the number of tumor cells. Tumor growth can be monitored by changes in tumor bioluminescence intensity in each group. A small animal in vivo imaging system was used to observe changes in tumor fluorescence intensity in each group on days 0, 7, and 14. The results are shown in the table. Figure 20 The fluorescence intensity of the G-Rg3@BSPs-SA colon-dissolved capsule group increased at the slowest rate within 14 days. Compared with the positive control drug Shenyi capsule, it significantly improved the tumor inhibition rate of mice with orthotopic colon cancer.

[0128] 4) From day 0 of administration, the survival of Balb / c mice with colon carcinoma in situ was observed and recorded daily. The death date and survival time were recorded in a timely manner. The observation was continued for 60 days. The survival curve was analyzed and drawn using Graphpad Prism 10.0 software. The weight of the mice with colon carcinoma in situ was weighed every 3 days after administration, and the weight changes were recorded. The results are shown in Figure 21G-Rg3@BSPs-SA colon soluble capsule significantly prolonged the survival time of CT-26 orthotopic cancer mice, reduced the weight of intestinal tumors of CT-26 orthotopic cancer mice, and the size and number of intestinal tumors of orthotopic colon cancer mice in the G-Rg3@BSPs-SA colon soluble capsule group were the smallest;

[0129] 5) After the end of administration, the mice in each group were dissected, and the tumor tissues in the intestinal tract and the main organs (heart, liver, spleen, lung, kidney) were taken out and fixed with 4% paraformaldehyde for 24 h. Then the tested tissues were taken out and flattened with a scalpel, paraffin sections were prepared and HE stained, and placed under an inverted fluorescence microscope, observed and images were collected, and the results are shown in Figure 22 , A large number of tumor cell apoptosis occurred in the G-Rg3@BSPs-SA colon soluble capsule group, the cells were arranged loosely, and the other main organs were not significantly affected.

[0130] 6) The apoptosis of tumor cells after drug treatment in each group was detected by TUNEL method. When cell apoptosis occurs, the nuclear chromatin will be broken into fragments, and these fragments will be recognized and combined to DNA by its fluorescent enzyme-labeled DNA terminal transferase (Terminal Deoxynucleotidyl Transferase, TdT), forming terminal labeled DNA, and the apoptosis of cells was observed by fluorescence microscope, and the results are shown in Figure 23 , The BSPs-SA colon soluble capsule group had a weak pro-apoptotic effect, the number of cell apoptosis in the Shen Yi capsule and G-Rg3 colon soluble capsule group increased significantly, the fluorescence intensity of the G-Rg3@BSPs-SA colon soluble capsule group was significantly enhanced, and G-Rg3 and BSPs-SA showed superior synergistic effect.

[0131] 7) The cell proliferation in CT-26 colon cancer tissue after drug treatment in each group was detected by immunofluorescence, and the complex was formed by specific antibody and Ki67 protein, and then the complex was combined with fluorescent dye, and the influence on cell proliferation was investigated by observing the fluorescence intensity and distribution by fluorescence microscope, and the results are shown in Figure 24 Compared with the PBS control group, the red fluorescence intensity of the G-Rg3@BSPs-SA colon soluble capsule group was significantly reduced, and the preparation could effectively inhibit the proliferation of CT-26 tumor cells in vivo.

[0132] 8) In order to verify whether the preparation can effectively reverse M2-TAMs to M1-TAMs, the expression of CD206 (M2-TAMs marker protein) and CD86 (M1-TAMs marker protein) in the orthotopic colon tumor tissue of mice was detected by immunofluorescence, and the results are shown in Figure 25 、 Figure 26, BSPs-SA colon soluble capsule group induced M2-TAMs depolarization had no significant difference compared with the control group (p>0.05), but showed synergistic effect with G-Rg3 (p<0.001). Compared with the control group, the number of CD206+macrophages in G-Rg3@BSPs-SA colon soluble capsule tumor tissue decreased significantly, and the number of CD86+macrophages increased significantly, with significant difference (p<0.001).

[0133] 9) To further verify the regulation of M2-type tumor-associated macrophages depolarization on the immune suppression environment in vivo, the expression of CD8+T cells in tumor tissue was detected by immunofluorescence, and the results were as follows Figure 27 Compared with the PBS control group, the fluorescence intensity of the BSPs-SA colon soluble capsule group decreased, and the number of CD8+T cells in the other groups increased significantly (p<0.001). The fluorescence intensity of the G-Rg3@BSPs-SA colon soluble capsule group increased most significantly, about 9.29 times that of the PBS group (p<0.001).

[0134] 10) After 14 days of administration, the CT-26 orthotopic colon cancer mouse colon site tumor was quantitatively determined by ELISA kit for TNF-α and TGF-β levels in tumor tissue supernatant, and the results were as follows Figure 28 Compared with other groups, the G-Rg3@BSPs-SA colon soluble capsule group had the most significant effect, with TNF-α concentration increased by 134.83% and TGF-β concentration decreased by 63.61%.

[0135] 11) To further study the effect of the preparation on EMT process in vivo, the expression of E-cadherin, N-cadherin, Vimentin and snail protein in tumor tissue of each group after administration was determined by immunofluorescence, and the results were as follows Figure 29 Compared with the PBS control group, the fluorescence intensity of E-cadherin in each group was up-regulated to varying degrees, and the fluorescence intensity of N-cadherin, Vimentin and snail was down-regulated to varying degrees, indicating that each group could inhibit the occurrence of colon cancer EMT, and thus slow down the tumor metastasis. Among them, the G-Rg3@BSPs-SA colon soluble capsule group had the most significant inhibitory effect on EMT, and the carrier BSPs-SA showed synergistic effect with G-Rg3.

Claims

1. A ginsenoside Rg3 (G-Rg3) drug delivery system, characterized in that: The invention is composed of the following components: G-Rg3, an amphiphilic Bletilla striata polysaccharide polymer carrier; The amphiphilic Bletilla striata polysaccharide polymer carrier comprises Bletilla striata polysaccharides (BSPs) and stearic acid (SA) in a mass ratio of 1:0.1-2.5; The preparation method of the ginsenoside Rg3 drug delivery system comprises the following steps: S1: Stearic acid (SA) and Bletilla striata polysaccharides (BSPs) were dissolved in DMSO for reaction. After the reaction, DMSO and unreacted SA were removed and the product was freeze-dried to obtain BSPs-SA. S2: Take BSPs-SA and dissolve it in DMSO, transfer it to an 8000Da dialysis bag, place the dialysis bag in pure water and dialyze it at room temperature to obtain blank BSPs-SA polymer nanomicelles; S3: Take a blank BSPs-SA polymer nanomicelle solution, add DMSO to dissolve it, transfer it to an 8-14 kDa dialysis bag, place the dialysis bag in pure water and dialyze at room temperature, and adjust the volume to obtain blank BSPs-SA polymer nanomicelles; take G-Rg3 and dissolve it in methanol, take the G-Rg3 solution and add it dropwise to the blank BSPs-SA polymer micelles, remove the methanol, and freeze-dry to obtain G-Rg3@BSPs-SA polymer nanomicelle freeze-dried powder; The proportion of G-Rg3 in the drug delivery system is 8.5%-12.0%.

2. The ginsenoside Rg3 drug delivery system according to claim 1, characterized in that: The BSPs are composed of mannose and glucose in a molar ratio of 3.1-4.5:

1.

3. A method for preparing the ginsenoside Rg3 drug delivery system according to claim 1 or 2, characterized in that: The following steps are involved: S1: SA and BSPs are dissolved in DMSO for reaction. After the reaction, DMSO and unreacted SA are removed and the product is freeze-dried to obtain BSPs-SA. S2: Take BSPs-SA and dissolve it in DMSO, transfer it to an 8000Da dialysis bag, place the dialysis bag in pure water and dialyze it at room temperature to obtain blank BSPs-SA polymer nanomicelles; S3: Take the blank BSPs-SA polymer nanomicelle solution, add DMSO to dissolve it, transfer it to an 8-14kDa dialysis bag, place the dialysis bag in pure water for dialyzation at room temperature, and adjust the volume to obtain blank BSPs-SA polymer nanomicelles; take G-Rg3 and dissolve it in methanol, take the G-Rg3 solution and add it dropwise into the blank BSPs-SA polymer micelles, remove the methanol, and freeze-dry to obtain G-Rg3@BSPs-SA polymer nanomicelle freeze-dried powder.

4. The method for preparing the ginsenoside Rg3 drug delivery system according to claim 3, characterized in that: The reaction conditions in S1 are as follows: react at 75° C. for 4 h and then react at room temperature for 24 h.

5. The method for preparing the ginsenoside Rg3 drug delivery system according to claim 3, wherein: The mass ratio of G-Rg3 and BSPs-SA described in S3 is 1:7-11.

6. A pharmaceutical preparation, characterized in that Comprising the ginsenoside Rg3 drug delivery system according to claim 1 or 2.

7. Use of the drug delivery system according to claim 1 or 2 or the drug preparation according to claim 6 in the preparation of drugs for treating colon cancer.

8. The pharmaceutical preparation according to claim 6, wherein The pharmaceutical preparations are colon-soluble capsules, colon-soluble tablets and colon-soluble granules.

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