A nano-drug delivery system, a preparation method and application thereof

Abstract

This invention belongs to the field of tumor drug preparation technology, specifically disclosing a nanomedicine delivery system, its preparation method, and its application. The delivery system uses extracellular vesicles isolated from acid-treated tumor cells as a carrier. The surface of the carrier is coupled with a fungal polysaccharide active ingredient via a chemical coupling agent. This chemical coupling agent contains at least one carboxylic acid group and at least one boric acid group. The carboxylic acid group forms an amide bond with the amino group on the surface of the extracellular vesicle, while the boric acid group forms a borate ester bond with the hydroxyl group of the fungal polysaccharide. This invention addresses the problems of poor tumor targeting, low immunomodulatory efficiency, and unsatisfactory tumor treatment effects associated with fungal polysaccharides, providing a new approach for tumor immunotherapy through fungal polysaccharide targeting, and possesses significant clinical application potential.

Description

Technical Field

[0001] This invention belongs to the field of tumor drug preparation technology, and specifically discloses a nanomedicine delivery system, its preparation method and application. Background Technology

[0002] Macrophage polarization plays a crucial role in cancer treatment and is a hot topic in tumor immunology research. Tumor-associated macrophages (TAMs) are plastic and pluripotent immune cells, accounting for approximately 50% of the cells in malignant solid tumors. Numerous studies have shown that M1 and M2 macrophages play different roles in the tumor microenvironment. M1 macrophages have pro-inflammatory and tumor-killing effects, while M2 macrophages have anti-inflammatory and tumor-promoting effects. When the local microenvironment or mediators change, M1 and M2 macrophages can interconvert.

[0003] Fungal polysaccharides, as natural immunomodulators, can activate the immune system at multiple targets and have great potential in cancer treatment. However, their poor tumor targeting, low immunomodulatory efficiency, and unsatisfactory cancer treatment effects limit their practical application. For example, Ganoderma lucidum polysaccharide (GLP) is one of the most important active substances in Ganoderma lucidum. In recent years, researchers have found that GLP can not only significantly inhibit tumor angiogenesis and reverse tumor cell resistance, but also polarize M2 macrophages in the tumor microenvironment into M1, thereby achieving anti-tumor immunotherapy effects. GLP has a very broad application prospect in clinical treatment. However, GLP has a large molecular weight and lacks tumor targeting in vivo, and cannot autonomously target and accumulate at the tumor site. Therefore, it is necessary to develop a drug delivery system that can effectively deliver this class of fungal polysaccharides with therapeutic potential to the tumor site. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, this invention provides a nanomedicine delivery system, its preparation method, and its applications. This invention solves the problems of poor tumor targeting, low immunomodulatory efficiency, and unsatisfactory tumor treatment effects associated with fungal polysaccharides, providing a new approach for tumor immunotherapy through fungal polysaccharide targeting and possessing enormous clinical application potential.

[0005] In a first aspect, the present invention provides a nanomedicine delivery system, which uses extracellular vesicles isolated from acid-treated tumor cells as carriers, and the surface of the carriers is coupled with fungal polysaccharide active ingredients via a chemical coupling agent; the chemical coupling agent contains at least one carboxylic acid group and at least one boric acid group, wherein the carboxylic acid group of the chemical coupling agent is bonded to the amino group on the surface of the extracellular vesicles by forming an amide bond, and the boric acid group of the chemical coupling agent is bonded to the hydroxyl group of the fungal polysaccharide by forming a borate ester bond.

[0006] In some implementations, the particle size of the delivery system is 200–220 nm.

[0007] In some implementations, the potential of the delivery system is -17 to -19 mV.

[0008] In some implementations, the delivery system is shaped like a cup, matching the shape of the carrier.

[0009] In some embodiments, the mass ratio of plant polysaccharides to extracellular vesicles is 5 to 15:1, preferably 8 to 12:1.

[0010] In some embodiments, the mass ratio of the chemical coupling agent to the extracellular vesicle is 10 to 20:1, preferably 12 to 18:1.

[0011] In some embodiments, the acid-treated tumor cells are tumor cells incubated in an acidic complete culture medium, preferably tumor cells cultured in a complete culture medium with a pH of 3 to 5 for 10 to 20 minutes.

[0012] In some implementations, the tumor cells are colon cancer cells, preferably CT26 colon cancer cells.

[0013] In some embodiments, the chemical coupling agent is a carboxyl-containing phenylboronic acid, preferably 4-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 2-carboxyphenylboronic acid, or 3,5-dicarboxyphenylboronic acid.

[0014] In some implementations, fungal polysaccharides are a class of natural biological macromolecules extracted from fungi, and can be a variety of fungal polysaccharides with anti-tumor effects that have been extensively studied, including but not limited to Ganoderma lucidum polysaccharide, Lentinus edodes polysaccharide or Trametes versicolor polysaccharide, with Ganoderma lucidum polysaccharide being preferred.

[0015] Secondly, the present invention provides a method for preparing the aforementioned nanomedicine delivery system, comprising: incubating tumor cells in an acidic complete culture medium, centrifuging, discarding the supernatant, resuspending the cell pellet in a complete culture medium without extracellular vesicles, culturing, and separating extracellular vesicles from the culture medium; mixing a chemical coupling agent and a carboxyl activating agent in a solvent, reacting the reaction product with the extracellular vesicles first, then with fungal polysaccharides, purifying, and obtaining the nanomedicine delivery system.

[0016] In some implementations, the preparation of a complete culture medium without extracellular vesicles includes: ultracentrifuging fetal bovine serum, filtering the supernatant, and then adding the filtered supernatant to DMEM medium containing penicillin-streptomycin antibiotics.

[0017] In some embodiments, the carboxyl activating agent is used to activate the carboxyl group on the chemical coupling agent, selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and / or N -Hydroxysuccinimide.

[0018] In some implementations, the temperature of the three consecutive mixing reactions is room temperature, the reaction time is 2-4 hours, and the pH of the first mixing reaction is 6-7 and the pH of the third mixing reaction is 8-9.

[0019] In some embodiments, the mass ratio of chemical coupling agent, extracellular vesicles and plant polysaccharides is 0.1~0.2:0.005~0.015:0.05~0.15, preferably 0.15:0.01:0.1.

[0020] In some embodiments, the preparation method of the nanomedicine delivery system includes: incubating tumor cells in DMEM complete medium at pH 3-5 for 10-20 min, centrifuging, discarding the supernatant, resuspending the cell pellet in complete medium without extracellular vesicles, culturing at 37±0.5℃ in an incubator containing 5±0.5% CO2 for 20-30 hours and collecting the supernatant; centrifuging at 200-400 g for 10-20 minutes at 3-5℃ and collecting the supernatant; further centrifuging the obtained supernatant at 1,000-3000 g for 10-20 minutes at 3-5℃ and collecting the supernatant again; concentrating the obtained supernatant through a 40-60 kDa ultrafiltration tube, centrifuging at 80,000-120,000 g for 1-3 hours at 4℃, removing the supernatant, adding PBS to resuspend the pellet, and centrifuging at 80,000-120,000 g at 3-5℃. Centrifuge for 1-3 hours, remove the supernatant, resuspend the precipitate in PBS, and separate the extracellular vesicles. Dissolve the chemical coupling agent and carboxyl activating reagent in an organic solvent, adjust the pH to 6-7, and react at room temperature for 2-4 hours to obtain the coupling agent solution. Add the extracellular vesicles to the coupling agent solution and react at room temperature for 2-4 hours to obtain the intermediate coupling solution. Add fungal polysaccharide to the intermediate coupling solution, adjust the pH to 8-9, and react at room temperature for 2-4 hours. After ultrafiltration and washing, obtain the nanomedicine delivery system.

[0021] Thirdly, the present invention provides a pharmaceutical composition comprising the aforementioned nanomedicine delivery system, and may further comprise a pharmaceutically acceptable carrier.

[0022] Fourthly, the present invention provides the use of the aforementioned nanomedicine delivery system or pharmaceutical composition in the preparation of a medicament for treating tumors.

[0023] Compared to the traditional oral administration of Ganoderma lucidum polysaccharides (GLP) for tumor treatment, this invention utilizes a nanomedicine delivery system (GLP@LEV) formed by loading GLP onto the surface of a fungicide layer (LEV) for colorectal cancer treatment. Experimental results show that GLP@LEV, under the influence of LEV, can actively target and accumulate at homologous tumor tissues and tumor cells. GLP polarizes M2 macrophages to M1, improving the tumor microenvironment. This effectively solves the problems of poor tumor targeting and low therapeutic efficiency of GLP. As a regulator of immune cells, GLP can activate immune cells, while LEV can specifically deliver GLP to homologous tumor tissues, regulate the tumor microenvironment, further promote the reprogramming of M2 macrophages to M1, and enhance the therapeutic effect of colorectal cancer. The preparation method of this delivery system is simple and highly reproducible, suitable for loading and delivering other fungal polysaccharides. This research provides a new approach and method for GLP treatment of colorectal cancer, and is expected to solve the problem of poor efficacy of GLP in colorectal cancer treatment. It also provides technical reference and guidance for the treatment of colorectal cancer or other cancers with other polysaccharides, and can be applied to the development of various drugs for treating tumors.

[0024] The beneficial effects of this invention are as follows:

[0025] The delivery system provided by this invention, for the first time, uses extracellular vesicles isolated from acid-treated tumor cells as carriers and a bifunctional chemical coupling agent containing carboxylic acid and boric acid groups as a linker to load fungal polysaccharides onto the carrier surface. Extracellular vesicles are membrane-like vesicles with a phospholipid bilayer, possessing unique biological advantages compared to other nanoparticles, including high stability, non-toxicity, non-immunogenicity, and long in vivo circulation time. The vesicles derived from acid-treated tumor cells can be reprogrammed, exhibiting glycerol ester self-aggregation, significantly increasing targeting to homologous tumor tissue. After entering the body, they can spontaneously target and aggregate at homologous tumor sites without affecting the expression of CD9, CD63, and Alix characteristic proteins on the surface of the extracellular vesicles, demonstrating good biosafety and making them an excellent carrier for drug transfer and delivery. Loading fungal polysaccharides onto these vesicles enables the autonomous aggregation of fungal polysaccharides into tumor tissues, which previously had poor tumor targeting, polarizing M2 macrophages to M1, thereby achieving effective intervention against the tumor, successfully activating the anti-tumor immune response, and significantly inhibiting tumor growth. Attached Figure Description

[0026] Figure 1A is the Fourier transform infrared spectrum of the GLP-CPBA obtained in Example 1 of this invention;

[0027] Figure 1 B is the boron nuclear magnetic resonance spectrum of GLP-CPBA obtained in Example 1 of this invention;

[0028] Figure 2 Topographic images of LEV and GLP@LEV;

[0029] Figure 3 The particle size and potential of LEV and GLP@LEV nanoparticles;

[0030] Figure 4 The expression of CD63, Alix, and CD9 proteins on three types of nanoparticles: NEV, LEV, and GLP@LEV.

[0031] Figure 5 Flow cytometry analysis of the polarization effects of PBS, LEV, GLP and GLP@LEV nanomedicine delivery systems on M2 macrophages;

[0032] Figure 6 A represents mice carrying CT26 tumor cells that were intravenously injected with different concentrations of the GLP@LEV nanomedicine delivery system. Fluorescence imaging was performed using a small animal in vivo fluorescence imaging system 3, 6, 12, 24 and 48 h after injection.

[0033] Figure 6 B represents the fluorescence intensity at different time points in the tumor site;

[0034] Figure 7 A is a flowchart illustrating the treatment process in mice carrying CT26 tumor cells after intravenous injection of PBS, LEV, GLP, and GLP@LEV.

[0035] Figure 7 B represents the change in tumor volume in each group of tumor-bearing mice during the treatment period;

[0036] Figure 7 C represents the size of the tumors in each group of tumor-bearing mice at the end of treatment;

[0037] Figure 7 D represents the tumor inhibition rate of each group of tumor-bearing mice after the end of treatment. Detailed Implementation

[0038] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for more detailed and specific illustration and should not be construed as limiting the present invention in any way.

[0039] This section provides a general description of the materials and testing methods used in the experiments of this invention. While many of the materials and methods of operation used to achieve the objectives of this invention are well known in the art, the invention is still described in as much detail as possible herein. It will be apparent to those skilled in the art that, unless otherwise stated in the context, the materials and methods of operation used in this invention are well known in the art.

[0040] The reagents and instruments used in the following examples are as follows:

[0041] Reagents and materials:

[0042] CPBA and EDC·HCl were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0043] NHS was purchased from Beijing Solarbio Technology Co., Ltd.

[0044] DMSO was purchased from Sigma in the United States;

[0045] GLP was purchased from Shaanxi Ciyuan Biotechnology Co., Ltd.

[0046] Fetal bovine serum (FBS), Dulbecco's Modified Eagle's medium (DMEM) and penicillin-streptomycin bispecific antibody were purchased from Shanghai Xiaopeng Biotechnology Co., Ltd.

[0047] Cell lysis buffer was purchased from Beyotime Biotechnology Co., Ltd.

[0048] SDS-PAGE protein loading buffer was purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0049] High-efficiency Western blotting solution was purchased from Shanghai Jifu Life Science Co., Ltd.

[0050] The ECL chemiluminescence reagent kit was purchased from Lanjieke Technology Co., Ltd.

[0051] The BCA protein assay kit and protein marker were purchased from Novizan Biosciences Inc.

[0052] The PVDF membrane was purchased from Millipore, USA.

[0053] Alix mouse anti-mouse antibody, CD9 rabbit anti-mouse antibody and CD63 rabbit anti-mouse antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0054] PE-conjugated anti-mouse CD11b, FITC-conjugated anti-mouse CD86, and APC-conjugated anti-mouse CD206 antibodies were purchased from Thermo Fisher Scientific, Inc., USA.

[0055] The DiR cell membrane deep red fluorescent probe was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0056] CT26 cells were purchased from Peking Union Medical College.

[0057] instrument:

[0058] The flow cytometer was purchased from BD Systems, USA, model FACS Celesta.

[0059] pH meter, model PHS-25, purchased from Shanghai Leici Instruments Co., Ltd.

[0060] Transmission electron microscope, purchased from Hitachi, Japan, model HITACHI-HT7700;

[0061] The ultracentrifuge, purchased from Beckman Coulter, USA, model Optima XE-100;

[0062] Nuclear magnetic resonance spectrometer, purchased from Bruker Technology Co., Ltd., model AVANCE NEO 500M;

[0063] Fourier transform infrared spectrometer, purchased from Thermo Fisher Scientific, USA, model Nicolet 6700;

[0064] Chemiluminescence imager, purchased from Shanghai Tianneng Technology Co., Ltd., China, model Tanon-5200;

[0065] The Malvern laser particle size analyzer, model ZEN3690, was purchased from Malvern Instruments Ltd., UK.

[0066] The laser scanning confocal microscope, model STELLARIS 5, was purchased from Leica Instruments GmbH, Germany.

[0067] The microplate reader, model LabServ™ K3, was purchased from Thermo Fisher Scientific, USA.

[0068] The small animal fluorescence imaging system, model IVIS Spectrum, was purchased from PerkinElmer, USA.

[0069] Terminology definition:

[0070] The term "GLP" refers to Ganoderma lucidum polysaccharides.

[0071] The term "LEV" refers to extracellular vesicles extracted and purified from acid-treated CT26 cells.

[0072] The term "CPBA" refers to 4-carboxyphenylboronic acid.

[0073] The term "GLP@LEV" refers to the Ganoderma lucidum polysaccharide-extracellular vesicle nanomedicine delivery system.

[0074] Example 1: Preparation of GLP-CPBA and Detection of Boronate Bonds

[0075] 0.18 g GLP and 0.27 g CPBA were dissolved in 10 mL of anhydrous DMSO, and the pH was adjusted to 8.5. The reaction mixture was then incubated at 25 °C for 3 hours. The reaction solution was subsequently placed in a dialysis bag (MWCO 3.5 kDa) and dialyzed against PBS for 2 days to remove free CPBA, yielding the GLP-CPBA reaction solution. The solution was lyophilized for 12 hours to obtain GLP-CPBA powder (GLP and CPBA powders served as controls). The borate ester bonds between GLP-CPBA powders were analyzed using nuclear magnetic resonance spectroscopy (Bruker, Germany) and Fourier transform infrared spectroscopy (Thermo Fisher Scientific, USA).

[0076] like Figure 1 As shown in Figure A, GLP-CPBA was found to be at 1350 cm⁻¹ in the Fourier transform infrared spectrum. -1 The characteristic peak of the BO bond appears at 1080 cm⁻¹. -1 The characteristic peak of borate esters appears at 864 cm⁻¹. -1 Characteristic peaks of the benzene ring appear at the location; such as Figure 1 As shown in Figure B, the boron spectrum obtained from the nuclear magnetic resonance spectrum revealed that boron could still be detected in GLP-CPBA after dialysis to remove free CPBA, indicating that CPBA was successfully attached to GLP.

[0077] Example 2: Preparation method of GLP@LEV nanomedicine delivery system, including the following steps:

[0078] Step 1: Acid treatment of tumor cells and isolation of extracellular vesicles

[0079] (A) Culture medium for preparing extracellular vesicle-free cells

[0080] Fetal bovine serum (FBS) was balanced and centrifuged at 120,000 g for 4 hours. The supernatant was collected and filtered through a 0.22 μm filter to obtain FBS without extracellular vesicles. 10% FBS and 1% penicillin-streptomycin were added to DMEM medium to prepare a complete cell culture medium without vesicles, which was then stored at 4°C for later use.

[0081] (B) Acid treatment of CT26 cells and extraction and purification of extracellular vesicles (LEVs)

[0082] When CT26 cells reached a confluence of 90% in normal DMEM complete medium (DMEM medium containing 10% fetal bovine serum + 1% penicillin-dextrose antibody), discard the normal DMEM complete medium, wash once with PBS, add 1 mL of trypsin, incubate at 37°C for 3 minutes, discard the trypsin, add 3 mL of normal DMEM complete medium, pipette the CT26 cells into 15 mL centrifuge tubes, and centrifuge at 1000 rpm for 3 minutes. Discard the supernatant, resuspend the cells in DMEM complete medium (pH 4.0), incubate for 15 minutes, and centrifuge at 1000 rpm for 3 minutes. Discard the supernatant, resuspend the cells in DMEM complete medium (prepared in step A) with vesicles removed, aliquot into 10 cm culture dishes, and incubate at 37°C in a 5% CO2 incubator for 24 hours. Collect the supernatant, and then proceed with the following steps to isolate extracellular vesicles derived from CT26 cells:

[0083] a) Centrifuge the collected CT26 cell supernatant at 300 g for 15 minutes at 4°C and collect the supernatant. Centrifuge the resulting supernatant again at 2,000 g for 15 minutes at 4°C and collect the supernatant once more.

[0084] b) Concentrate the supernatant obtained in step a) through a 50 kDa ultrafiltration tube, aliquot it into 1 mL centrifuge tubes, ensuring no air bubbles are present, balance the tubes so that the weight difference between all centrifuge tubes is within 0.05 g, then place them in an ultracentrifuge (Beckman, USA), centrifuge at 100,000 g for 2 hours at 4°C, remove the supernatant, and retain the precipitate sample on the centrifuge tube wall;

[0085] c) Add PBS to each centrifuge tube to resuspend the precipitate in 1 mL of PBS, and centrifuge at 100,000 g for 2 hours at 4°C. After centrifugation, remove the supernatant, add 500 μL of PBS to the centrifugation tube to resuspend the precipitate, collect the extracellular vesicles on the centrifuge tube wall, aliquot and store them in a -80°C freezer.

[0086] Step 2: Preparation of Ganoderma lucidum polysaccharide-extracellular vesicle nanomedicine delivery system

[0087] 0.27 g CPBA (Shanghai Aladdin), 0.145 g EDC·HCl (Shanghai Aladdin), and 0.085 g NHS (Beijing Solarbio) were dissolved in 10 mL of anhydrous DMSO (Sigma), and the pH of the solution was adjusted to 6.5. The reaction was carried out at room temperature for 3 hours. 0.018 g LEV was added, and the amino group on LEV reacted with the carboxyl group on CPBA at 25 °C for 3 hours to obtain the intermediate conjugate LEV-CPBA. 0.18 g GLP (Shaanxi Ciyuan) was added to the above mixture, and the pH of the solution was adjusted to 8.5 with 0.05 M NaOH solution, and the borate group on CPBA reacted with the hydroxyl group on GLP at 25 °C for 3 hours. The reaction solution was loaded into a 100 kDa ultrafiltration tube, centrifuged at 5000 g for 10 minutes, and washed three times with PBS to obtain the GLP@LEV solution, which is the Ganoderma lucidum polysaccharide-extracellular vesicle nanomedicine delivery system.

[0088] Example 3: Physicochemical Detection of the GLP@LEV Nanomedicine Delivery System

[0089] 1. Morphology testing of LEV and GLP@LEV nanoparticles

[0090] First, the copper mesh was treated with glow discharge for 1 minute. Next, 10 μL of LEV and GLP@LEV (LEV concentration 1 μg / μL) were dropped onto the copper mesh, respectively, and allowed to stand for 1 minute. The solution was then gently blotted away with filter paper. 3 μL of acetic acid staining solution was added, and the mesh was allowed to stand for 1 minute. The negative staining solution was then gently blotted away with filter paper. Another 3 μL of acetic acid staining solution was added, and the staining was repeated once. The copper mesh was dried at room temperature, and the morphology of LEV and GLP@LEV was photographed using a transmission electron microscope (HITACHI, Japan).

[0091] like Figure 2 As shown, LEV appears cup-shaped under a transmission electron microscope. After loading GLP onto the surface, the morphology does not change and it remains cup-shaped, but the size increases significantly.

[0092] 2. Testing the particle size and potential of LEV and GLP@LEV nanoparticles.

[0093] Take 10 μg of LEV and GLP@LEV respectively, dilute with ultrapure water to 1 mL, and use Malvern laser particle size analyzer (Malvern, UK) to detect the particle size and potential of LEV and GLP@LEV.

[0094] like Figure 3 As shown, the LEV particle size is around 122 nm and the potential is around -20 mV. After surface loading with GLP, both the particle size and potential change to around 210 nm and -18 mV, respectively.

[0095] 3. Test the expression of CD9, Alix and CD63 proteins in two types of nanoparticles, LEV and GLP@LEV (NEV as a control).

[0096] First, take 100 μL (3 μg / μL) of NEV, LEV, and GLP@LEV suspension resuspended in PBS and add protein lysis buffer (Beyotime, P0013) containing 1 μL (v / v) protease inhibitor. After thorough vortexing, place the sample on ice for approximately 30 minutes for lysis. Then, place the sample in an ice-water bath and sonicate three times using an ultrasonic cleaner. Quantify the protein and calculate the protein concentration using a BCA protein assay kit (Vazyme, E112-01). Add 4× loading buffer (Proteintech, PR20003), mix thoroughly, heat at 100°C for 5 minutes, and store at -20°C until use.

[0097] Next, the expression of specific proteins on three nanoparticles, NEV, LEV, and GLP@LEV, was analyzed by Western blotting.

[0098] Lysed extracellular vesicles containing 45 μg of total protein were added to the wells of a 10% Tris-HCl gel plate. The total protein was separated by electrophoresis, and the proteins on the gel were transferred to a polyvinylidene fluoride (PVDF) membrane using a wet transfer method. After 1.5 hours, the PVDF membrane was blocked with high-performance Western spectroscopy blocking buffer (Genefist, GF1815) at room temperature for 20 minutes. The PVDF membrane was then transferred to 5 mL of primary antibody dilution buffer (the antibody dilution buffer was prepared with TBST solution and 5% skim milk powder) and incubated overnight on a shaker at 4°C. The membrane was then washed three times with TBST for 10 minutes each time. The PVDF membrane was then transferred to 5 mL of secondary antibody dilution buffer and incubated for 2 hours, followed by three washes with TBST for 10 minutes each time. Finally, developing solution (Biosharp, BL520A) was added to the PVDF membrane, and the membrane was exposed using a gel imaging system (Tanon, China).

[0099] like Figure 4 As shown, NEVs highly express CD9, CD63, and Alix characteristic proteins (β-actin as an internal control). The CD9, CD63, and Alix characteristic proteins on the surface of purified LEVs and GLP@LEV nanoparticles obtained after incubating CT26 cells in acidic medium did not change significantly after incubation. This indicates that incubation of CT26 cells with acidic medium for secreted LEVs and the loading of GLPs on LEVs does not affect the expression of these proteins.

[0100] 4. Test the polarization effects of PBS, LEV, GLP and GLP@LEV nanomedicine delivery systems on M2 macrophages.

[0101] M0 and M2 macrophages (5 × 10⁻⁶) 5 M2 cells (number of cells / well) were seeded in 24-well plates. After adhesion, the supernatant was discarded. PBS, GLP, LEV, or GLP@LEV were added to M2 cells, while LPS was added to M0 cells as a positive control. M2 cells served as a negative control. Cells were incubated at 37 °C for 24 hours. The supernatant was discarded, and complete culture medium was added. Cells were vigorously pipetted to detach completely. Cells were centrifuged at 1500 rpm for 3 minutes, and the supernatant was discarded. CD86-FITC, CD206-APC, and CD11b-PE antibodies were diluted 1:100 with PBS, and the cells were resuspended and incubated on ice in the dark for 30 minutes. Cells were centrifuged at 1500 rpm for 3 minutes, and the supernatant was discarded. Cells were washed twice with PBS. Finally, the cells were resuspended in PBS, and the expression of CD86 and CD206 in each group was detected by flow cytometry (BD FACS Celesta, USA). The percentage of positive cells in each group was recorded.

[0102] like Figure 5 As shown, LEV cannot polarize M2 macrophages, while GLP and GLP@LEV nanomedicine delivery systems can increase the expression of CD86 protein and decrease the expression of CD206 protein, thus having a polarizing effect on M2 macrophages.

[0103] Example 4: Experimental Study on the Application Effect of GLP@LEV Nanomedicine Delivery System

[0104] 1. Detection of the ability of the GLP@LEV nanomedicine delivery system to target colorectal cancer tumor tissue

[0105] The targeting ability of the GLP@LEV nanomedicine delivery system to mouse tumor tissue bearing CT26 tumor cells was tested. To further understand the blood circulation behavior of the nanomedicine delivery system prepared in Example 2 after intravenous injection in mice, different concentrations of GLP@LEV nanomedicine delivery system (25 mg / kg, 50 mg / kg and 100 mg / kg) were labeled with fluorescent DiR dye and injected intravenously into tumor-bearing mice. Fluorescence imaging was performed using a small animal in vivo fluorescence imaging system at 3, 6, 12, 24 and 48 h to screen for the optimal concentration of GLP@LEV for targeted enrichment of lesions in vivo.

[0106] like Figure 6As shown in Figure A, after injection of the GLP@LEV nanomedicine delivery system, the fluorescence signal value of the GLP@LEV nanomedicine delivery system at the tumor site gradually increased over time, reaching a maximum at 12 hours, and then slowly weakened. Fluorescence could still be detected at the tumor site after 48 hours. Figure 6 As shown in Figure B, the quantitative fluorescence results showed that the 100 mg / kg group had more GLP@LEV nanomedicine delivery system that was targeted and accumulated in tumor tissue than the 25 mg / kg and 50 mg / kg groups, indicating that the high concentration of GLP@LEV nanomedicine delivery system has a good tumor targeting effect after intravenous injection.

[0107] 2. Detection of the anti-colon cancer tumor efficacy of the GLP@LEV nanomedicine delivery system

[0108] To investigate the antitumor effect of the GLP@LEV nanomedicine delivery system, we first established a subcutaneous cell line xenograft model in Balb / c mice using CT26 cells and randomly divided them into 4 groups (n=7 per group). Each group of mice was intravenously injected with PBS (Control), LEV, GLP, and GLP@LEV (LEV dose: 10 mg / kg, GLP dose: 100 mg / kg; 100 μL per mouse). Injections were given every three days for a total of 5 times. During the injection process, mouse weight, body temperature, survival status, and tumor size were monitored. The tumor volume was calculated using the formula: Tumor volume = (length × width) / (length × width). 2 The tumor volume was calculated as (1 / 2). On day 20, the tumor-bearing mice were sacrificed, and the tumors and major organs (heart, liver, spleen, lungs, and kidneys) were dissected. The tumors were photographed and weighed, and the tumor inhibition rate was calculated.

[0109] like Figure 7 As shown in Figure A, tumors were treated with injections of PBS, LEV, GLP, and GLP@LEV on days 6, 9, 12, 15, and 18, as... Figure 7 As shown in B-7C, tumor suppression ended on day 20. Compared with other treatment groups, the GLP@LEV nanomedicine delivery system showed the most significant inhibitory effect on tumor size. Figure 7 As shown in Figure D, the tumor inhibition rate was optimal, demonstrating the therapeutic effect of the GLP@LEV nanomedicine delivery system on colon cancer.

[0110] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A nanomedicine delivery system, characterized in that, The delivery system uses extracellular vesicles isolated from acid-treated tumor cells as carriers, and the surface of the carriers is coupled with fungal polysaccharide active ingredients via a chemical coupling agent. The chemical coupling agent contains at least one carboxylic acid group and at least one boric acid group. The chemical coupling agent is bonded together with the amino group on the surface of the extracellular vesicle by an amide bond, and the boric acid group is bonded together with the hydroxyl group of the fungal polysaccharide by a borate ester bond. The tumor cells were colon cancer cells; The fungal polysaccharide is Ganoderma lucidum polysaccharide; The preparation method of the nanomedicine delivery system includes: Tumor cells were incubated in acidic complete medium, centrifuged, and the supernatant was discarded. The cell pellet was resuspended in complete medium without extracellular vesicles, cultured, and extracellular vesicles were isolated from the culture medium. The chemical coupling agent and carboxyl activating agent are mixed and reacted in a solvent. The reaction product is first mixed and reacted with extracellular vesicles, and then mixed and reacted with fungal polysaccharides. After purification, the nanomedicine delivery system is obtained. The temperature of the three mixing reactions was room temperature, the reaction time was 2-4 h, and the pH of the first mixing reaction was 6-7 and the pH of the third mixing reaction was 8-9. The mass ratio of the chemical coupling agent, extracellular vesicles, and fungal polysaccharide is 0.1~0.2:0.005~0.015:0.05~0.

15.

2. The nanomedicine delivery system according to claim 1, characterized in that, The particle size of the delivery system is 200~220 nm; And / or, the potential of the delivery system is -17 to -19 mV; And / or, the delivery system is cup-shaped.

3. The nanomedicine delivery system according to claim 1, characterized in that, The mass ratio of the fungal polysaccharide to the extracellular vesicles is 5-15:1; And / or, the mass ratio of the chemical coupling agent to the extracellular vesicles is 10~20:

1.

4. The nanomedicine delivery system according to claim 3, characterized in that, The mass ratio of the fungal polysaccharide to the extracellular vesicles is 8-12:1; And / or, the mass ratio of the chemical coupling agent to the extracellular vesicle is 12-18:

1.

5. The nanomedicine delivery system according to claim 1, characterized in that, The acid-treated tumor cells are tumor cells incubated in an acidic complete culture medium; And / or, the chemical coupling agent is phenylboronic acid containing a carboxyl group.

6. The nanomedicine delivery system according to claim 5, characterized in that, The acid-treated tumor cells are tumor cells incubated in a complete culture medium with a pH of 3-5 for 10-20 min. And / or, the tumor cells are CT26 colon cancer cells; And / or, the chemical coupling agent is 4-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 2-carboxyphenylboronic acid, or 3,5-dicarboxyphenylboronic acid.

7. A method for preparing a nanomedicine delivery system according to any one of claims 1 to 6, characterized in that, The preparation method includes: Tumor cells were incubated in acidic complete medium, centrifuged, and the supernatant was discarded. The cell pellet was resuspended in complete medium without extracellular vesicles, cultured, and extracellular vesicles were isolated from the culture medium. The chemical coupling agent and carboxyl activating agent are mixed and reacted in a solvent. The reaction product is first mixed and reacted with extracellular vesicles, and then mixed and reacted with fungal polysaccharides. After purification, the nanomedicine delivery system is obtained. The temperature of the three mixing reactions was room temperature, the reaction time was 2-4 h, and the pH of the first mixing reaction was 6-7 and the pH of the third mixing reaction was 8-9. The mass ratio of the chemical coupling agent, extracellular vesicles, and fungal polysaccharide is 0.1~0.2:0.005~0.015:0.05~0.

15.

8. The preparation method according to claim 7, characterized in that, The carboxyl activating agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and / or N -Hydroxysuccinimide.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the nanomedicine delivery system according to any one of claims 1 to 6.

10. The use of the nanomedicine delivery system according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating colon cancer.

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