Astragalus polysaccharide-loaded nano-drug as well as preparation method and application thereof
The nanomedicine loaded with astragalus polysaccharide prepared by PLGA nanocarrier has solved the problems of delivery and enrichment of astragalus polysaccharide in the treatment of colorectal cancer, realizing tumor-targeted delivery and immune activation, significantly inhibiting tumor growth and metastasis, and providing a new treatment option for colorectal cancer.
Patent Information
- Application Number
- CN202511745167.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-25
- Publication Date
- 2026-02-27
AI Technical Summary
In the existing technology, astragalus polysaccharide, as a macromolecular polysaccharide, has problems such as high water solubility, poor in vivo stability, low bioavailability, and difficulty in effectively accumulating in tumor sites, which limits its application in immunotherapy for colorectal cancer.
Nanomedicine loaded with astragalus polysaccharide (APS@PLGA) was prepared by using polylactic acid-glycolic acid copolymer (PLGA) nanocarriers via a complex emulsion solvent evaporation method. This method achieved efficient encapsulation and stable delivery of astragalus polysaccharide, which was then targeted and enriched in tumor tissues using the EPR effect. The astragalus polysaccharide was then released slowly in the tumor microenvironment, activating an adaptive immune response.
It significantly inhibits the growth and metastasis of colorectal cancer, promotes T cell activation and infiltration, and provides a green and controllable method for preparing nanomedicines, suitable for large-scale production, thus enhancing the therapeutic effect of colorectal cancer.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the fields of biomedicine and nanotechnology, and in particular to a astragalus polysaccharide (APS) loaded polylactic-co-glycolic acid (PLGA) nanomedicine APS@PLGA, a preparation method thereof and an application thereof in the treatment of colorectal cancer. BACKGROUND
[0002] Colorectal cancer (CRC) is one of the most common malignant tumors in the world, and its morbidity and mortality rank first among all types of cancers. Although certain progress has been made in traditional treatment methods such as surgery, chemotherapy, and radiotherapy, the prognosis of patients with advanced colorectal cancer is still not ideal, and traditional chemotherapeutic drugs have serious side effects and drug resistance problems. In recent years, tumor immunotherapy has attracted much attention because it can activate the body's own immune system and produce a lasting anti-tumor effect. However, immunotherapy still faces challenges such as insufficient immune cell infiltration and low immune response rate.
[0003] Astragalus polysaccharide (APS) is one of the main active components of traditional Chinese medicine Astragalus membranaceus, and has significant immunomodulatory, anti-inflammatory, and anti-tumor biological activities. Studies have shown that astragalus polysaccharide can activate T cells, NK cells, macrophages, and other immune cells to enhance the body's anti-tumor immune function. However, as a macromolecular polysaccharide, astragalus polysaccharide has high water solubility, poor in vivo stability, low bioavailability, and difficulty in effective enrichment in tumor sites, which severely limits its clinical application.
[0004] Nanomedicine delivery systems provide an effective strategy to solve the above problems. Polylactic-co-glycolic acid (PLGA) is a biodegradable polymer material approved by the US FDA, and has good biocompatibility, controllable degradation rate, and excellent drug-forming properties. Polylactic-co-glycolic acid nanocarriers can protect drugs from in vivo enzymatic degradation, prolong circulation time, and achieve tumor-targeted delivery through the enhanced permeability and retention (EPR) effect. Double emulsion solvent evaporation method is a classic method for loading water-soluble macromolecular drugs, and has the advantages of stable process and controllable drug loading.
[0005] Currently, there is no related report on using polylactic-co-glycolic acid nanosystems to load astragalus polysaccharide for colorectal cancer immunotherapy. Developing an APS@PLGA nanomedicine to achieve efficient delivery and tumor-targeted release of astragalus polysaccharide is of great significance for improving the anti-tumor efficacy of astragalus polysaccharide and promoting the modern application of active components of traditional Chinese medicine. SUMMARY
[0006] The present application aims to overcome the deficiencies of the prior art, and provides a polysaccharide of Astragalus membranaceus loaded poly (lactic-co-glycolic acid) nanomedicine and a preparation method thereof. The nanomedicine can realize efficient encapsulation and stable delivery of the polysaccharide of Astragalus membranaceus, target enrichment in tumor tissues through the EPR effect, slow release of the polysaccharide of Astragalus membranaceus in the tumor microenvironment, effectively activate adaptive immune response, and significantly inhibit the growth and metastasis of colorectal cancer.
[0007] Another object of the present application is to provide use of the nanomedicine in the preparation of a drug for immunotherapy of colorectal cancer.
[0008] The polysaccharide of Astragalus membranaceus loaded nanomedicine uses poly (lactic-co-glycolic acid) as a carrier and loads the polysaccharide of Astragalus membranaceus as a core immunologically active component.
[0009] Preferably, the nanomedicine has a molecular weight of 10,000-100,000 Da of the poly (lactic-co-glycolic acid), a molar ratio of lactic acid to glycolic acid of 50:50-75:25, and a molecular weight of 5-500 kDa of the polysaccharide of Astragalus membranaceus.
[0010] Preferably, the nanomedicine has a smooth near-spherical particle morphology observed under a transmission electron microscope.
[0011] A preparation method of the polysaccharide of Astragalus membranaceus loaded nanomedicine is as follows: poly (lactic-co-glycolic acid) is dissolved in an organic solvent to form an oil phase, the polysaccharide of Astragalus membranaceus is dissolved in an aqueous phase, then the aqueous phase is added to the oil phase to form an initial emulsion, the initial emulsion is added to an external aqueous phase to form a complex emulsion by secondary emulsification, finally the organic solvent is volatilized, the nanoparticles are collected by centrifugation and washed, and then freeze-dried to obtain the nanomedicine.
[0012] Preferably, in the preparation method, the organic solvent is dichloromethane or ethyl acetate, and the emulsification is performed by ultrasonic or high-pressure homogenization.
[0013] The polysaccharide of Astragalus membranaceus loaded nanomedicine is used in the preparation of a drug for treating colorectal cancer.
[0014] Preferably, the administration scheme of the nanomedicine is as follows: immunotherapy is started 7 days after tumor inoculation, subcutaneous injection is performed once every 7 days, a total of 3 times; each injection is 100 μL of the nanomedicine (wherein the polysaccharide of Astragalus membranaceus has a concentration of 5 μg / μL), and the administration dose is 0.1-10 mg / kg.
[0015] Preferably, the nanomedicine is in the form of a freeze-dried powder.
[0016] The disclosed astragalus polysaccharide-loaded nanomedicine and its preparation method can achieve the following beneficial effects: through the loading of polylactic acid-hydroxyacetic acid copolymer, the nanomedicine can realize the lymphatic system targeting delivery of astragalus polysaccharide. The nanomedicine exhibits the following significant advantages in the treatment of colorectal cancer: the nanomedicine shows significant inhibition of tumor growth and significant promotion of the activation and infiltration of T cells (especially cytotoxic CD8 + T cells) in in vitro and in vivo experiments. In addition, the nanomedicine preparation method is green, controllable, and suitable for large-scale production. In summary, as a new type of combined treatment strategy, the nanomedicine not only provides a new solution for the treatment of colorectal cancer, but also provides a new idea for the synergistic treatment of other types of tumors.
[0017] Other features and advantages of the present application will become apparent from the following detailed description of exemplary embodiments thereof, taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS
[0018] The accompanying drawings incorporated in and forming a part of the specification, illustrate embodiments of the application and, together with the description, serve to explain the principles of the application.
[0019] Figure 1 is a nanomedicine preparation process flowchart provided by the embodiments of the present application; Figure 2 is an electron microscope morphology of the nanomedicine provided by the embodiments of the present application; Figure 3 is the inhibition effect of the nanomedicine on the migration and invasion of CT-26 tumor cells provided by the embodiments of the present application; Figure 4 is the in vivo treatment effect of the nanomedicine provided by the embodiments of the present application; Figure 5 is a graph showing the enhancement of the number and function of tumor infiltrating CD8 + T cells by the nanomedicine provided by the embodiments of the present application; Figure 6 is a safety evaluation result graph of the nanomedicine provided by the embodiments of the present application. DETAILED DESCRIPTION
[0020] In order to make the purposes, technical solutions and beneficial technical effects of the present application clearer, the present application will be described in detail below in combination with specific embodiments. It should be understood that the embodiments described in the specification are only for the purpose of explaining the present application, and are not intended to limit the present application.
[0021] For simplicity, this paper only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can serve as its own lower or upper limit and be combined with any other point or individual value, or with other lower or upper limits, to form an unspecified range.
[0022] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.
[0023] The foregoing description of this invention is not intended to describe every disclosed embodiment or implementation. Instead, the following description provides more specific examples of exemplary embodiments. Throughout the application, guidance is provided through a series of embodiments that can be used in various combinations. The examples listed are merely representative and should not be construed as exhaustive.
[0024] Example 1: Preparation of Nanomedicines See Figure 1 Since the loaded Astragalus polysaccharide is water-soluble, the APS@PLGA nanomedicine was prepared by a water-in-oil-in-water emulsion solvent evaporation method. The specific preparation method is as follows: 1.1 Preparation of oil phase: Weigh 100 mg of polylactic acid-glycolic acid copolymer (molecular weight 50,000 Da, lactic acid:glycolic acid = 50:50) and dissolve it in 5 mL of dichloromethane as the oil phase.
[0025] 1.2 Preparation of aqueous phase: Dissolve 20 mg of Astragalus polysaccharide in 2 mL of ultrapure water to prepare the aqueous phase.
[0026] 1.3 Ultrasonic emulsification: The aqueous phase is slowly added to the oil phase and ultrasonically emulsified (200 W, 2 min) to form a primary emulsion.
[0027] 1.4 Preparation of double emulsion: Pour the primary emulsion into 20 mL of an aqueous solution containing 2% polyvinyl alcohol (PVA) and mechanically stir (1000 rpm) to form a double emulsion.
[0028] 1.5 Evaporation of organic solvents: Stir at room temperature for 4 h to evaporate organic solvents.
[0029] 1.6 Centrifugation and washing: Collect nanoparticles by centrifugation (12,000 rpm, 20 min) and wash three times with ultrapure water.
[0030] 1.7 Freeze-drying to obtain white powder-like nanomedicine APS@PLGA.
[0031] Example Two: Morphological characterization of nanomedicine 2.1 Experimental method Morphological characterization of the APS@PLGA nanomedicine prepared in Example One: 2 mg of nanomedicine freeze-dried powder was weighed into an EP tube, and 500 μL of deionized water was added to resuspend the sample to an appropriate concentration. The sample was dropped onto a copper mesh for electron microscopy, and after the sample was dried, 2% (W / V) phosphotungstic acid staining solution was added for negative staining for 15 minutes. Excess phosphotungstic acid solution was absorbed with filter paper at the edge of the copper mesh, and after drying, the sample was placed under a transmission electron microscope (TEM) for observation of its morphology.
[0032] 2.2 Experimental results See Figure 2 Under the observation of a transmission electron microscope (TEM), it was observed that the nanomedicine APS@PLGA was a smooth, nearly spherical particle, which was consistent with the ultrastructure characteristics of nanomedicine.
[0033] Example Three: In vitro anti-migration and anti-invasion efficacy of nanomedicine 3.1 Experimental method The effect of APS@PLGA nanomedicine on the migration and invasion ability of mouse colorectal cancer cells CT-26 was evaluated by a Transwell experiment, and the specific experimental steps were as follows: 3.1.1 Cell treatment: CT-26 cells were seeded in a 6-well plate (density 5 x 10 5 cells / well), and PBS (control) and APS@PLGA were added, respectively, and incubated for 24 hours.
[0034] 3.1.2 Migration experiment: The treated cells were resuspended with serum-free medium, and the density was adjusted to 2 x 10 5 / mL, 200 μL was added to the upper chamber of the Transwell, and 600 μL of medium containing 10% FBS was added to the lower chamber, and incubated for 24 hours.
[0035] 3.1.3 Invasion experiment: Matrigel matrix gel (50 μg / well) was pre-coated in the upper chamber, and the remaining steps were the same as the migration experiment.
[0036] 3.1.4 Staining and counting: The chamber was removed, fixed with 4% paraformaldehyde for 15 minutes, and stained with 0.1% crystal violet for 15 minutes. The cells on the upper surface were wiped off with a cotton swab, and 5 fields were randomly selected under a microscope for counting.
[0037] 3.2 Experimental results See Figure 3, the number of cell migration and invasion of the APS@PLGA nanodrug treatment group were significantly reduced, which proved that the nanodrug could effectively inhibit the metastatic potential of tumor cells.
[0038] Example Four: Evaluation of the in vivo tumor treatment effect of the nanodrug 4.1 Experimental animals Fifteen SPF 4-5-week-old male BALB / c mice were raised in the Experimental Animal Center of Ningxia Medical University. The experimental protocol was approved by the Animal Ethics Committee of the school (Approval No. IACUC-NYLAC-2023-161), and all operations were in accordance with the “Regulations on the Management of Experimental Animals”.
[0039] 4.2 Establishment of subcutaneous colon cancer xenograft model Each mouse was subcutaneously inoculated with 5×10 6 CT-26 cells on the right side of the back, and when the tumor volume reached 100mm 3 , about 7 days, they were randomly divided into 2 groups (n=5): ① PBS group (blank control); ② free APS group; ③ APS@PLGA group.
[0040] 4.3 Drug inoculation After the successful construction of the subcutaneous colon cancer model in mice, three treatment processes were started on the 7th day, see Figure 4 A timeline, subcutaneously injecting 100 μL PBS, 100 μL APS, or 100 μL APS@PLGA nanodrug every 7 days, for a total of 3 immunotherapies.
[0041] 4.4 Observation and measurement results From the 3rd day after inoculation of colorectal cancer cells, the mice's body weight, behavior, and injection site reactions were monitored every other day. After the tumor was palpable, the long diameter (a) and short diameter (b) were measured every 2 days with a digital vernier caliper, and the volume was calculated according to the formula V=a×b 2 / 2. On the 28th day after inoculation, euthanasia was performed by CO2 asphyxiation. The tumor was completely peeled off, the surface blood was absorbed with filter paper, and then photographed, weighed, and the maximum a, b values were measured to calculate the final volume; at the same time, the draining lymph nodes and heart, liver, spleen, lung, kidney were collected for subsequent immunological and safety analysis.
[0042] 4.5 Experimental results See Figure 4 , the subcutaneous tumor volume of the mouse treated with APS@PLGA nanodrug was reduced, and the tumor weight was significantly reduced.
[0043] Example Five: Study of the immune mechanism of the nanodrug 5.1 Tissue sampling and single nuclear cell separation The tumor-bearing mice were euthanized by CO2 asphyxiation and dissected to obtain the spleen, mesenteric lymph nodes and tumor tissues of the mice at day 28 after CT-26 cell injection.
[0044] Obtaining mononuclear cells: the spleen, mesenteric lymph nodes and tumor of the mouse were placed on a 200-mesh screen, ground with a syringe, and the ground suspension was filtered into a 15-mL centrifuge tube with a 200-mesh screen. The spleen and tumor cell suspension was added to the mouse spleen mononuclear cell separation solution (Solarbio) and mouse tumor infiltrating lymphocyte separation solution (Solarbio) at a ratio of 1:1, respectively. After centrifugation at 2000 rpm for 20 minutes, the white membrane layer was aspirated, and 5 times the volume of PBS was added. After mixing well, centrifugation was performed at 1500 r / min for 10 minutes, and the supernatant was discarded. The mononuclear cell suspension was resuspended with 1 mL of PBS, counted, and then divided into tubes. The suspension of the mesenteric lymph nodes was resuspended with PBS to 5 mL, centrifuged at 1500 rpm for 10 minutes, and resuspended with PBS to obtain the mononuclear cell suspension.
[0045] 5.2 T cell activation markers and detection Surface staining: 1x10 6 cells / tube were taken, surface antibody CD8-PE was added, 4 °C incubation for 30 minutes, PBS washing for 2 times, and immediately collected by CytoFLEX flow cytometer, and analyzed by FlowJo v10.
[0046] Intracellular factor staining: according to the eBioscience fixation / breaking membrane kit instructions, TNF-α-PE, IFN-γ-PerCP-Cy5.5 were added in sequence, 4 °C incubation for 35 min, and then resuspended in 300 μL PBS after washing. Immediately collected by CytoFLEX flow cytometer, and analyzed by FlowJo v10.
[0047] 5.3 Experimental results See Figure 5 , the flow cytometry detection results showed that in the mouse spleen, lymph node and tumor tissue, the proportion of CD8 + T cells, IFN-γ + and CD8 + TNF-α + T cells in the APS@PLGA drug treatment group was significantly increased, indicating that the APS@PLGA nanodrug successfully triggered an effective cellular immune response.
[0048] Example Six: Safety Evaluation of Nanodrug (H&E Staining) 6.1 Experimental method 6.1.1 After the mice are euthanized, the heart, liver, spleen, lung, kidney and other major organs are taken out by dissection and fixed with 4% paraformaldehyde, dehydrated, immersed in wax, embedded, sliced (4 μm).
[0049] 6.1.2 Hematoxylin staining solution is added dropwise on the slice, and dyed for 5 min, and then the staining solution is washed away with distilled water. After the slice is spun dry, differentiation solution is added dropwise, and then distilled water is used for washing after 30 s.
[0050] 6.1.3 Eosin staining solution is added dropwise on the slice tissue, and dyed for 2 min, and then washed with distilled water after the end. Finally, dehydration and mounting are performed. The steps are the same as those in Example Eight.
[0051] 6.1.4 Microtissue morphology is observed under a microscope.
[0052] 6.2 Experimental results See Figure 6 The results show that the main organ tissue structure of the APS@PLGA drug treatment group of mice is complete, the cell morphology is normal, the staining is uniform, and there is no obvious inflammation, necrosis and other pathological changes.
[0053] The above examples show that the APS@PLGA nanodrug provided by the present application amplifies its immune regulation function through a high-efficiency nanodelivery system, is simple to prepare, has remarkable effects, and has good clinical application prospects and combined drug potential as a basic immune regulator.
Claims
1. A nano-drug loaded with astragalus polysaccharide, characterized in that, The nano-drug takes polylactic acid-glycolic acid copolymer as a carrier and carries astragalus polysaccharide as a core immune active component.
2. The nanomedicine according to claim 1, characterized in that, The polylactic acid-glycolic acid copolymer has a molecular weight of 10,000-100,000 Da, and a molar ratio of lactic acid to glycolic acid of 50:50-75:25; the astragalus polysaccharide has a molecular weight of 5-500 kDa.
3. The nanomedicine according to claim 1, wherein, The morphology observed under a transmission electron microscope is smooth near-spherical particles.
4. The preparation method of the astragalus polysaccharide-loaded nanomedicine according to any one of claims 1-3, characterized in that, The polylactic acid-glycolic acid copolymer is dissolved in an organic solvent to form an oil phase, and the astragalus polysaccharide is dissolved in an aqueous phase, then the aqueous phase is added to the oil phase to emulsify and form a primary emulsion, the primary emulsion is added to an external aqueous phase to perform secondary emulsification and form a complex emulsion, finally the organic solvent is volatilized, the nanoparticles are collected by centrifugation and washed, and then freeze-dried to obtain the nano-drug.
5. The production method according to claim 4, characterized by, The organic solvent is dichloromethane or ethyl acetate, and the emulsification is performed by ultrasonic or high-pressure homogenization.
6. Use of the astragalus polysaccharide-loaded nano-drug of any one of claims 1-3 in the preparation of a drug for treating colorectal cancer.
7. The use according to claim 6, characterized in that, The immunotherapy is started 7 days after tumor inoculation, and is subcutaneously injected once every 7 days, with a total of 3 administrations; each injection is 100 μL of the nano-drug (with an astragalus polysaccharide concentration of 5 μg / μL), and the administration dose is 0.1-10 mg / kg.
8. The use according to claim 6, characterized in that, The drug is in the form of a freeze-dried powder.