Glucan-modified bionic nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a glucan modified bionic nanoparticle as well as a preparation method and application thereof. Preparing nanoparticles based on PLGA as an inner core; the tumor cell membrane is extracted from B16F10 melanoma cells and is used as an outer capsid; glucan is coupled with a hydrophobic long chain through reductive amination and is modified on the surface of a biomimetic nanoparticle cell membrane through a hydrophobic effect. The dextran nano delivery system is successfully constructed, cell membrane specific antigens and dextran are efficiently and jointly delivered to lymph nodes, tumor cell membranes provide epitopes for immune activation of nanoparticles, and beta-dextran provides molecular support for nanoparticle targeting immune cells. In-vivo and in-vitro activity experiments prove that the glycolipid modified bionic nanoparticles can effectively activate innate immunity through macrophages and NK cells, can promote dendritic cell antigen presentation, and initiate potent adaptive immune response. The bionic nanoparticles have huge development potential of novel tumor vaccines, and a new thought is provided for immunotherapy of cancers.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a dextran-modified biomimetic nanoparticle and a preparation method and application thereof. Background Art

[0002] Tumors seriously threaten human life and health. As the second leading cause of death in the world, the number of deaths and cases is increasing year by year. With the aging of the population, the incidence and mortality of cancer in the world will continue to rise. Therefore, finding a suitable and effective tumor treatment method has become an important issue that people need to solve urgently.

[0003] Immunotherapy is an emerging and effective tumor treatment method after surgery, radiotherapy and chemotherapy. In 2013, it was selected as the top ten scientific breakthroughs of the year by Science magazine, and in 2018, its research on immune checkpoints won the Nobel Prize in Physiology or Medicine. Tumor vaccines have become a very attractive type of tumor immunotherapy due to their high specificity and few side effects. Tumor vaccines are composed of tumor antigens and vaccine adjuvants. How to find suitable antigens and adjuvants is the key to tumor vaccine research and development.

[0004] Functional proteins such as specific tumor antigen epitopes expressed on tumor cell membranes can be used as key antigens in the development of tumor vaccines. At the same time, β-glucan, as a natural immunologically active carbohydrate substance, has been widely used in tumor adjuvant therapy, especially in terms of exerting anti-tumor activity by regulating innate immunity. If tumor cell membranes, β-glucan and nanoparticles are combined to construct a nano-delivery system, efficient co-delivery of antigens and adjuvants can be achieved, resulting in a good immune effect. Summary of the invention

[0005] The purpose of the present invention is to provide a dextran modified bionic nanoparticle and a preparation method and application thereof.

[0006] A dextran-modified biomimetic nanoparticle comprises a tumor cell membrane vesicle and dextran, wherein the dextran is anchored on the phospholipid bilayer of the tumor cell membrane vesicle based on hydrophobic interaction.

[0007] The preparation method of the dextran modified biomimetic nanoparticles is carried out according to the following steps: (1) Tumor cells are subjected to hypotonic treatment or freeze-thaw cycles to break them, and the cell membranes are separated by differential centrifugation. The cell membrane suspension is washed and redissolved with a buffer containing protease inhibitors to obtain cell membrane suspension, which is then homogenized by ultrasound or extruded through a porous polycarbonate membrane to obtain cell membrane vesicles; (2) PLGA nanoparticles are prepared by an emulsified solvent evaporation method: 40-80 mg of polylactic acid-glycolic acid copolymer is dissolved in 10-50 mL of dichloromethane; the mixture solution is dropped into 0.2-1.0 L of polyvinyl alcohol aqueous solution; the obtained solution is stirred for 12-36 hours to evaporate the dichloromethane solvent, and then dialyzed with double distilled water for 8-36 hours, with the water replaced every 3 hours, and the PLGA nanoparticles are obtained by centrifugation at a speed of 8000-16000 rpm / min for 20-60 minutes, which are then washed with double distilled water and freeze-dried to obtain PLGA nanoparticles; (3) Preparation of tumor cell membrane-coated bionic nanoparticles based on mechanical extrusion method: Cell membrane vesicles and PLGA nanoparticles were co-extruded through a porous polycarbonate membrane, and then passed through 400 nm, 200 nm, and 100 nm polycarbonate porous membranes in sequence. Through repeated extrusion, the cell membrane was wrapped onto the surface of the nanoparticles to obtain bionic nanoparticles; (4) Covalently bind dextran to phospholipids by reductive amination: dextran and phospholipids are placed in a reaction bottle, dissolved with a mixed solution of dimethyl sulfoxide and chloroform, and then sodium cyanoborohydride is added. A magnetic bar is added, and the reaction is heated in an oil bath at 50-80°C for 12-48 hours. The reaction bottle is taken out and cooled to room temperature. The chloroform in the system is removed by rotary evaporation. Anhydrous ethanol saturated with sodium chloride is added, and the mixture is centrifuged at a speed of 6000-12000 rpm / min for 10-30 minutes. The supernatant is discarded, and the resulting precipitate is the crude glycolipid. The mixture is dialyzed with double distilled water (molecular weight cutoff (MWCO) = 3500 Da) for 12-48 hours, and freeze-dried to obtain the glycolipid. The glycolipid is resuspended in PBS to obtain a glycolipid solution with a concentration of 5-20 mg / mL. The glycolipid solution is added to the biomimetic nanoparticles, stirred at 4-10°C for 2-12 hours, and centrifuged at 8000-16000 rpm / min. rpm / min, centrifuge for 15-30 min, discard the supernatant, and the precipitate is the dextran-modified biomimetic nanoparticles.

[0008] The pore sizes of the porous polycarbonate membrane in step (1) are 50 nm, 100 nm, 200 nm, and 400 nm.

[0009] Furthermore, the particle size of the dextran-modified biomimetic nanoparticles is 100-300 nm, and the β-glucan content is 5-15%.

[0010] The tumor cells in step (1) are B16-F10 mouse melanoma cells, the centrifugal forces of the differential centrifugation are 1000-2000 g, 6000-10000 g, 40000-50000 g, and the centrifugation time is 30-60 min.

[0011] The volume ratio of chloroform to dimethyl sulfoxide in the mixed solution of dimethyl sulfoxide and chloroform is 1:(2-4).

[0012] Preferably, the volume ratio of chloroform to dimethyl sulfoxide in the mixed solution of dimethyl sulfoxide and chloroform is 1:2.

[0013] The molecular weight of the glucan is 2-8 kDa; the monosaccharide composition is mainly glucose, and its content is above 95%, and the connection method is that the main chain is β-1,3 glucan with β-1,6 branches.

[0014] The molar ratio of the dextran to the phospholipid is 1:(2-20); the molar ratio of the dextran to sodium cyanoborohydride is 1:(2-20).

[0015] Preferably, the molar ratio of dextran to phospholipid is 1:10, and the molar ratio of dextran to sodium cyanoborohydride is 1:10.

[0016] The dextran modified bionic nanoparticles are used in the preparation of anti-tumor growth and anti-tumor metastasis drugs.

[0017] Furthermore, the dextran-modified biomimetic nanoparticles can significantly promote the secretion of IL-12, TNF-α, and IFN-γ, enhance the phagocytic ability of macrophages and the natural killing ability of NK cells, and effectively activate the innate immune response; increase the expression of co-stimulatory molecules on the surface of dendritic cells, enhance the antigen presentation of dendritic cells, promote T cell proliferation and differentiation, and induce a strong adaptive immune response; effectively enhance the infiltration of cytotoxic T cells in tumor tissues, reduce the tumor infiltration of MDSCs inhibitory immune cells, and play a role in inhibiting tumor growth and tumor metastasis.

[0018] Beneficial effects of the present invention: The present invention designs and successfully prepares dextran-modified biomimetic nanoparticles with immunotherapeutic effects, which can effectively activate macrophages and NK cells, induce innate immunity, enhance DC cell antigen presentation, activate T and B lymphocytes, promote adaptive immunity, and effectively exert immune anti-tumor activity through the dual pathways of innate immunity and adaptive immunity. Melanoma is a highly prevalent malignant tumor of the skin, mucous membranes and pigment membranes, and its growth, metastasis and recurrence have become the main causes of death in melanoma patients. Activating immunity to fight melanoma is one of the effective methods for treating and improving the quality of life of patients. In the present invention, an in situ mouse tumor model and a lung metastasis mouse tumor model based on B16-F10 cells were selected to evaluate the immuno-anti-tumor and anti-metastasis effects of dextran-modified biomimetic nanoparticles. The present invention is based on seaweed-derived β-glucan (DAG), and after lipid modification and tumor cell membrane anchoring, biomimetic nanoparticles are successfully constructed, which show excellent immunomodulatory anti-tumor activity at the molecular, cellular and animal levels, and have great potential for development into a new type of tumor vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Schematic diagram of the preparation and structure of dextran-modified bionic cell membrane nanoparticles (NPs@CL1-M). Figure 2 This is the H NMR spectrum of a series of β-glucan lipids (CL1). Figure 3 High-resolution mass spectra of a series of β-glucan lipids (CL1). Figure 4 This is the structural characterization diagram of dextran modified bionic cell membrane nanoparticles (NPs@CL1-M). Figure 5 This is a diagram showing the regulatory effect of dextran-modified bionic cell membrane nanoparticles (NPs@CL1-M) on innate immune cells. Figure 6 This diagram shows the regulatory effect of dextran-modified bionic cell membrane nanoparticles (NPs@CL1-M) on adaptive immune cells. Figure 7 This is a diagram showing the inhibitory effect of dextran-modified bionic cell membrane nanoparticles (NPs@CL1-M) on the B16F10 in situ tumor model. Figure 8 This is a graph showing the inhibitory effect of dextran-modified bionic cell membrane nanoparticles (NPs@CL1-M) on the B16F10 metastatic tumor model (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. Example 1

[0021] The glucan used in this Example 1 is seaweed-derived β-glucan (DAG), and the preparation and structural diagram of glucan-modified biomimetic cell membrane nanoparticles (NPs@CL1-M) (see FIG. Figure 1 The specific steps are as follows: (1) Dissolve β-glucan (DAG, 1 e.q.) and phospholipid DPPE (L1, 10 e.q.) in 1.5 mL of dimethyl sulfoxide / chloroform mixed reaction solvent (2:1, v / v) and place in a 10 mL reaction bottle. Add sodium cyanoborohydride (NaBH3CN, 10 e.q.) to the above reaction solution, place the reaction bottle in an oil bath, and react at 80°C for 24 h. Remove the reaction bottle, cool to room temperature, and remove chloroform by rotary evaporation. Transfer the reaction solution to a 15 mL centrifuge tube and add 4 mL of anhydrous ethanol for alcohol precipitation. Centrifuge at 10,000 rpm / min for 15 min to precipitate the crude glycolipid. Redissolve the alcohol precipitate, collect the precipitate by centrifugation, repeat twice, dialyze (molecular weight cutoff (MWCO) = 3500 Da) for 48 h, and freeze-dry to obtain β-glucan lipid (CL1).

[0022] (2) Culture B16-F10 melanoma cells in 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C 5% carbon dioxide incubator. When the cells grow to almost cover the bottom of the culture dish, digest and subculture. Take B16F10 melanoma cells in the logarithmic growth phase, remove the old culture medium, wash twice with 3 mL 1×PBS, add trypsin, and digest the adherent cells. Transfer the cell suspension to a 50 mL centrifuge tube, centrifuge at 1300 rpm for 3 min, remove the supernatant, and re-dissolve in PBS to a cell density of 1x10 6 / mL. Take 5 mL of cell suspension in a 10 mL centrifuge tube, wash the cells 3 times with 5 mL 1×PBS, suspend the cells in 5 mL of hypotonic lysis buffer containing a mixture of protease and phosphatase inhibitors, and break the cells with a homogenizer (15 times). Centrifuge the homogenized solution at 3000 rpm for 30 min at 4°C. Discard the precipitate, and centrifuge the supernatant again at 8000 rpm for 30 min at 4°C. Discard the precipitate, and centrifuge the supernatant again at 45000 g for 60 min at 4°C. After centrifugation, collect the precipitate, which is the cell membrane. The obtained cell membrane is re-dissolved and then physically extruded through 200nm and 100nm porous polycarbonate membranes to form tumor cell membrane vesicles.

[0023] (3) Dissolve 50 mg of poly(lactic acid-co-glycolic acid) copolymer in 20 mL of dichloromethane; drip the mixture solution into 0.6 L of poly(vinyl alcohol) aqueous solution; stir the obtained solution for 24 h to evaporate the dichloromethane solvent, then dialyze with deionized water for 24 h, replace the water every 3 h, centrifuge at 12,000 rpm / min for 40 min to obtain PLGA nanoparticles, wash with deionized water, and freeze-dry to obtain PLGA nanoparticles; based on a liposome extruder, pass the prepared tumor cell membrane vesicles and PLGA nanoparticles through 400 nm, 200 nm, and 100 nm polycarbonate porous membranes in sequence for 20 extrusion cycles, and the cell membrane is wrapped onto the surface of the nanoparticles to obtain biomimetic nanoparticles NPs@M. Wash the obtained NPs@M three times with PBS and resuspend it in PBS. Weigh 10 mg of β-glucan lipid CL1 and add 1 mL of PBS to dissolve it. Add the glycolipid solution to the biomimetic nanoparticle solution and stir overnight at 4°C. The mixture was centrifuged at 14,000 rpm / min for 30 min, and the supernatant was discarded. The precipitate was the dextran-modified biomimetic nanoparticles (NPs@CL1-M). Example 2

[0024] (1) Nuclear magnetic resonance hydrogen spectrum characterization: Take 5 mg of glycolipid (CL1), add 500 μL of heavy water, freeze-dry, repeat the heavy water exchange 3 times, add 500 μL of heavy water and transfer to a nuclear magnetic resonance tube, and perform one-dimensional 1H-NMR detection and analysis at 25°C using a Bruker BioSpin GmbH 400-MHz nuclear magnetic resonance spectrometer. After data acquisition, data processing was performed using MestReNova 9.0 software. The results are shown in Figure 2. Figure 2 As shown, the glycolipid NMR spectrum clearly has characteristic peaks of lipid substances. At 1.2 ppm, it is the continuous methylene of lipid substances, and at 0.9 ppm, it is the methyl at the end of lipid substances, indicating that the glycolipid is successfully constructed.

[0025] (2) High-resolution mass spectrometry characterization: The glycolipid (CL1) was dissolved in a 50% acetonitrile / water solution (containing 5 mM ammonium acetate) and directly entered the LTQ ORBITRAP XL mass spectrometer through the electrospray protonation (ESI) source in the negative ion mode of the FT-MS detector. After data acquisition, the data was processed using Xcalibur 2.2 (Thermo Scientific Foundation 2.0) software. The results are shown in Figure 2. Figure 3 As shown in the figure, the fragment ions 1579.23 and 1633.25 with z=3 are all ion fragments of covalent coupling of β-glucan with different polymerization degrees and lipid DPPE. The molecular weight of each glycolipid increases in order compared with β-glucan DAG, indicating that the glycolipid (CL1) is prepared by covalent coupling of glucan and lipid rather than a physical mixture.

[0026] (3) Transmission electron microscopy characterization: Take the prepared nano solution (1 mg / mL) and use a pipette to spot it on the sample plate (20 μL). Place the copper mesh on the droplet on the sample plate and soak for 3 min. Take out the copper mesh with tweezers and irradiate it with a fluorescent lamp for 3 min. Immerse the copper mesh in phosphotungstic acid again and soak for 3 min. Take out the copper mesh with tweezers, dry it with a fluorescent lamp, and examine it with a transmission electron microscope. Figure 4 a is a transmission electron microscopy image of the biomimetic nanoparticle preparation. It can be seen from the figure that the prepared biomimetic nanoparticles NPs@M have a typical core-shell structure, and the biomimetic nanoparticles are successfully constructed.

[0027] (4) Malvern particle size analyzer characterization: Accurately weigh a series of nano samples, prepare them to a concentration of 1 mg / mL, and mix them evenly. Take the sample detection pool, clean it, and rinse it with the nano sample three times. Use a pipette to draw 1 mL of the nano sample solution, select the particle size analysis program of the Malvern particle size analyzer, and perform the test. Figure 4b is the particle size detection diagram of Malvern particle size analyzer. It can be seen that the particle size of dextran-modified biomimetic nanoparticles is larger than that of unmodified biomimetic nanoparticles, indicating that dextran lipids are effectively modified on the outside of the nanoparticle cell membrane. Due to the good particle size and morphology, dextran-modified biomimetic nanoparticles containing tumor antigens are helpful to enrich to lymph nodes through lymphatic circulation.

[0028] (5) Characterization of protein content: Take the BCA kit, dissolve the protein standard (BSA) at room temperature, and dilute it with PBS to a final concentration of 0.5 mg / mL to obtain the standard solution. Take solution A and solution B in the kit and mix them at a ratio of 50:1 (v:v) to obtain the BCA working solution. Take the standard solution (0.5 mg / mL) and add it to the standard wells of a 96-well plate at a volume of 0, 1, 2, 4, 8, 12, 16, and 20 µL, respectively, and add PBS to 20 µL. Take 20 µL of the series of samples and add them to the sample wells of a 96-well plate. Add 200 µL of the working solution to each well, mix well, and incubate at 37°C for 30 min, or leave at room temperature for 2 h. Figure 4 c is the protein content detection diagram. The protein content of bionic nanoparticles NPs@M is significantly higher than that of nanoparticles NPs, indicating that the cell membrane is effectively wrapped on the outside of the nanoparticles (NPs).

[0029] (6) Characterization of sugar content: Accurately weigh dextran and dissolve it in double distilled water to obtain a 0.25 mg / mL standard solution. Pipette 0, 0.1, 0.2, 0.3, 0.5, 0.7 and 0.9 mL of the standard solution respectively, and add double distilled water to 1.0 mL to obtain a series of standard solutions with concentrations of 0, 0.025, 0.050, 0.075, 0.125, 0.175, and 0.225 mg / mL. Take numbered glass test tubes and add 200 µL of the series of standard solutions and sample solutions in sequence. Add 200 μL of 6% phenol solution to each glass tube, and then add 1 mL of concentrated sulfuric acid and mix well. Place the glass tube in a 100℃ water bath, heat in the water bath for 10 min, and cool in cold water. Pipette 200 µL and add it to the corresponding wells of a 96-well plate in sequence according to the number, and detect with an enzyme reader at a wavelength of 490 nm. Figure 4 d is the sugar content detection diagram. Compared with the unmodified (NPs@M), the sugar content of glycolipid-modified bionic nanoparticles (NPs@CL-M) is significantly increased, further indicating that the glycolipid is effectively coated on the surface of the bionic nanoparticles. Example 3

[0030] (1) Neutral red phagocytosis characterization: Prepare 50 mL of complete culture medium: 45 mL of basal culture medium + 5 mL of fetal bovine serum + 500 μL of penicillin-streptomycin. Take RAW264.7 cells in good condition and adjust the cell density to 3x10 with complete culture medium. 5 / mL, and obtain RAW264.7 cell suspension. Take the RAW264.7 cell suspension and add it to a 96-well plate at a volume of 100 μL per well. Place the 96-well plate in a 5% carbon dioxide incubator and incubate at 37°C for 24 h. Take out the 96-well plate, add the corresponding series of nanomaterials (NPs, NPs@M, NPs@CL1-M) to the corresponding wells, and set the final concentration of each material to (25, 50, 100 μg / mL), and continue to incubate in the incubator for 24 h. Centrifuge at 1300 rpm for 3 min, remove the supernatant, and then add neutral red solution (100 μL) to each well. Put the 96-well plate back into the incubator and continue incubating at 37°C for 30 min. Centrifuge at 1300 rpm for 3 min, remove the supernatant, and wash the cell pellet with PBS three times. Take acetic acid and ethanol and prepare cell lysis solution in a volume ratio of 1: 1. Add 200 μL of cell lysis solution to each well and incubate at 4°C overnight. Take a 96-well plate and measure the OD value with a microplate reader (570 nm). Figure 5 a is a macrophage neutral red phagocytosis detection diagram. It can be seen from the figure that dextran-modified biomimetic nanoparticles can enhance the phagocytic ability of macrophages, and the effect increases with the increase of drug concentration.

[0031] (2) Cytokine characterization: Take the cultured RAW264.7 cells, centrifuge at 1300 rpm for 3 min, remove the supernatant, and adjust the cell density to 5x10 per ml with complete culture medium. 5 To obtain RAW264.7 cell suspension. Take a 96-well plate, add RAW264.7 cell suspension at a volume of 100 μL per well, place it in a carbon dioxide (5%) incubator, and culture it overnight at 37°C. Add a series of nano-samples to be tested (NPs, NPs@ M, NPs@CL1-M) to the 96-well plate in a biosafety cabinet and pipette evenly. Incubate in the biosafety cabinet for 24 h, and obtain the cell supernatant by centrifugation.

[0032] According to the instructions of the NO kit, take the standard and dilute it with DMEM complete medium to 0, 1, 2, 5, 10, 20, 40, 60, 100 μM. Add different concentrations of standard and series of cell supernatants to a 96-well plate, 50 μL per well. Then add 50 μL of Griess Reagent Ⅰ and Griess Reagent Ⅱ that have been restored to room temperature to each well. Place the 96-well plate in an ELISA reader and measure the absorbance of each well at a wavelength of 540 nm.

[0033] According to the instructions of the Elisa kit, set up blank wells, standard wells and sample wells. Except for the blank wells, add 100 μL of enzyme-labeled reagent to each well, seal the plate with a sealing film and incubate at 37°C for 60 min. Remove the sealing film, spin dry, fill each well with washing solution, let it stand for 30 s and then discard, repeat 5 times. First add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake and mix, and color at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well, adjust to zero with the blank well, and measure the absorbance of each well in sequence at a wavelength of 450 nm.

[0034] Figure 5 b, 5c, and 5d are the detection diagrams of NO, IL-12, and TNF-α content after RAW264.7 cells were treated with nanomaterials. The increase in endogenous NO secretion leads to the disruption of the tricarboxylic acid cycle, which in turn triggers the M1 phenotype polarization, which is conducive to the occurrence of anti-tumor immunity. IL-12 is an important pro-inflammatory cytokine that can enhance the tumor killing of NK cells and T cells. TNF-α can act indirectly on T cells and also cause tumor cell necrosis through direct killing. Figure 5 b, 5c, and 5d show that dextran-modified biomimetic nanoparticles can effectively activate RAW264.7 cells, promote the secretion of NO, IL-12, and TNF-α, produce a strong immune response, and have good anti-tumor potential. Example 4

[0035] (1) Characterization of antigen presentation ability: C57BL / 6 mice were euthanized by carbon dioxide asphyxiation. The femur and tibia of the mice were dissected and disinfected by soaking in ethanol (70%). The two ends of the femur and tibia were cut off, and the bone marrow cells were flushed out of the medullary cavity with a sterile syringe. This was repeated twice to obtain a cell suspension. The cell suspension was centrifuged at 300 g for 5 min and the supernatant was discarded. 1 mL of red blood cell lysis buffer was added, lysed for 5 min, centrifuged at 300 g for 5 min, the precipitate was collected, and washed twice with PBS. The cells were resuspended in culture medium (containing 15 ng / ml GM-CSF and 10 ng / ml IL-4) and transferred to a cell culture incubator for culture. On the 7th day, the cells loosely adhered to the culture dish were bone marrow-derived dendritic cells. The isolated dendritic cells were counted on a counting plate, and RPMI 1640 complete culture medium (10% FBS) was taken to adjust the cell suspension density to 5×10 5 / mL, add to 6-well plate. Transfer the 6-well plate to a cell culture incubator and culture overnight at 37℃. Add a series of nano samples (NPs, NPs@M, NPs@CL1-M) to a 6-well plate, with a final sample concentration of 100 μg / mL, and record the sample number. Incubate in a cell culture incubator for 24 h. Discard the supernatant, resuspend the dendritic cells with 500 μL PBS to obtain a cell suspension, and block with blocking solution on ice for 20 min. Discard the supernatant and resuspend with 200 μL PBS. Add premixed antibodies (CD11c, CD80, CD86) and incubate in a 4℃ refrigerator away from light for 40 min. Centrifuge at 300 g for 5 min to collect the cell pellet. Take 0.5mL PBS and wash the cells twice. Resuspend the dendritic cells with PBS containing the live-dead dye 7-AAD to obtain the upper fluid. Pass the upper fluid through a 40 μm mesh, transfer to a flow tube, and detect by flow cytometry. Figure 6 a is a flow cytometry image of the maturation of dendritic cells induced by nanoparticles. CD80 and CD86 are co-stimulatory molecules for antigen presentation and are the second signal for T cell activation, which is crucial for antigen presentation. When dendritic cells recognize antigen molecules, they turn into a mature and activated state, and the expression of CD80 and CD86 co-stimulatory molecules increases. Figure 6 a It can be seen that after co-incubation of glycolipid-modified biomimetic nanoparticles and dendritic cells, the proportion of dendritic cells highly expressing co-stimulatory molecules increased significantly, significantly more than that of the normal control group, which indicates that glycolipid-modified biomimetic nanoparticles are effectively recognized by dendritic cells to achieve antigen presentation.

[0036] (2) Characterization of tumor killing ability: C57BL / 6 mice were euthanized and the spleen was removed by dissection. A sieve was placed on a plate and a small amount of PBS was added. The spleen was placed on the sieve and ground with a sterile syringe piston. After grinding, the sieve was rinsed with PBS and the cell suspension was collected. The sieve was filtered to obtain a single cell suspension of the spleen. A centrifuge tube was taken and a separation solution was added. The spleen single cell suspension was added to the surface of the separation solution. The centrifuge tube was centrifuged at 500 g for 30 min at room temperature. After centrifugation, the centrifuge tube was divided into four layers from top to bottom. The second layer was a ring-shaped milky white layer, which was the lymphocyte layer. The milky white lymphocyte layer was aspirated into another centrifuge tube and 10 mL of cell washing solution was added. The centrifuge tube was centrifuged at 250 g for 10 min and the supernatant was discarded. The PBS was washed twice and the obtained cell pellet was the mouse spleen lymphocyte. Dendritic cells stimulated by a series of nanomaterials were co-incubated with spleen lymphocytes to obtain dendritic cell-stimulated lymphocytes. B16 melanoma cells and 4T1 breast cancer cells in good growth state were taken and counted and plated respectively using a counting plate. According to a 10:1 effector-target ratio, the obtained dendritic cell-stimulated lymphocytes were co-incubated with B16 cells and 4T1 cells, respectively, to detect the killing of related tumor cells. Figure 6 b and Figure 6 c is a test chart of the killing of different tumor cells by spleen lymphocytes activated by nano-preparation. The tumor killing effect of the bionic nanoparticles modified with dextran is better than that of the group without dextran modification. It is speculated that the bionic nanoparticles modified with dextran can better activate dendritic cells and induce the best antigen presentation, so the T cells are activated effectively. In addition, spleen lymphocytes co-incubated with different tumor cells can better kill B16 tumor cells, but no obvious effect is seen on 4T1 cells, indicating that bionic nanoparticles based on specific cell membranes can induce specific immunity and kill tumors. Example 5

[0037] (1) Male C57BL / 6J mice aged 6-8 weeks were housed in an SPF-grade ventilated animal room for 2 weeks, with regular changes of food and drinking water to acclimate to the environment. The mice were administered subcutaneously at a concentration of 10 mg / mL and a dose of 100 μL / mouse, once every 7 days for 3 times. Seven days after the third administration, B16F10 mouse melanoma cells in good growth condition were obtained and resuspended in PBS to adjust the cell density to 2×10 5 / mL, and a tumor cell suspension was prepared. Take the tumor cell suspension and inoculate the tumor subcutaneously into the right armpit of C57BL / 6J mice at a dose of 100 μL per mouse. Observe the mice every day and record the changes in tumor volume. When the tumor grows to 1500 mm 3 The experiment was stopped when . The eyeballs were removed, blood was collected in a centrifuge tube, and allowed to stand at room temperature for 2 hours. The mice were centrifuged at 3000 rpm for 10 min, and the precipitate was discarded to obtain mouse serum. The secretion of factors such as IgG and IFN-γ in mouse serum was detected by enzyme-linked immunosorbent assay kit. The mice were killed by dislocating the cervical vertebrae, and the mouse tumor tissue was obtained by dissection. A small amount of mouse tumor tissue was taken, placed in a cryopreservation tube, and quickly frozen with liquid nitrogen. The samples were sent on dry ice for transcriptome detection. Figure 7 a is a picture of the tumors of mice in each group. From the picture, we can see that the tumor volume of the dextran-modified biomimetic nanoparticle group is the smallest and has the best immune anti-tumor effect. Figure 7 b and Figure 7 c is the secretion of cytokines and antibodies in the serum of mice in each group. The secretion levels of IFN-γ and IgG in the dextran-modified biomimetic nanoparticle group were significantly upregulated, which suggests that the T and B lymphocytes in the mice were activated. Figure 7 d, 7e, 7f, and 7g are mouse transcriptome data analysis diagrams. There are a large number of differentially expressed genes in the dextran-modified bionic nanoparticle group compared with the model group. Combined with STING pathway analysis, it was found that the expression of related signaling molecules STING-1, IFR-3, and TBK1 was significantly increased. The activation of the STING pathway may be an important reason for the immune anti-tumor effect of dextran-modified bionic nanoparticles.

[0038] (2) Male C57BL / 6J mice aged 6-8 weeks were kept in an SPF-grade ventilated animal room for 2 weeks to adapt to the environment. In accordance with the experimental ethics requirements, the mice were regularly fed with cage bedding, food and water. The drug was administered by subcutaneous injection once every 7 days for a total of 3 times (10 mg / mL, 100 μL / mouse). Seven days after the third administration, B16F10 mouse melanoma cells were obtained and resuspended in PBS to adjust the cell density to 2×10 5 / mL, and a tumor cell suspension was prepared. C57BL / 6J mice were fixed with a mouse fixator, and the tumor cell suspension was taken. The tumor was inoculated into the tail vein at a dose of 100 μL per mouse. The mice were killed by dislocating the cervical vertebrae, and the mouse organs were obtained by dissection, and the number of lung tumor nodules in the mice was measured. Part of the tumor tissue was taken, dewaxed and hydrated, and H&E staining and immunofluorescence staining were performed. Figure 8 a is the picture of lung tumor in each group of mice. Figure 8 b is a statistical chart of lung tumor nodule counts in each group of mice. From the picture, we can see that the dextran-modified biomimetic nanoparticles group has the least number of tumor nodules and has the best anti-tumor metastasis effect. Figure 8 c is the H&E staining image of mouse lung tumor. The dextran-modified biomimetic nanoparticle group not only had significantly fewer tumor nodules than the model group, but also had significantly reduced lung tissue congestion and edema, indicating that it can effectively protect mice. Figure 8 d is the result of immunofluorescence detection of T cells in tumor tissue. It can be seen from the figure that the proportion of T cells in tumor tissue in the dextran-modified bionic nanoparticle group is significantly higher than that in the model group, indicating that dextran-modified bionic nanoparticles can effectively activate the adaptive immune response and promote the tumor infiltration of T cells.

[0039] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A dextran-modified biomimetic nanoparticle, characterized in that: The invention comprises tumor cell membrane vesicles and dextran, wherein the dextran is anchored on the phospholipid bilayer of the tumor cell membrane vesicles based on hydrophobic interaction.

2. The method for preparing the dextran-modified biomimetic nanoparticles according to claim 1, characterized in that: Follow these steps: (1) Tumor cells are subjected to hypotonic treatment or freeze-thaw cycles to break them, and the cell membranes are separated by differential centrifugation. The cell membrane suspension is washed and redissolved with a buffer containing protease inhibitors to obtain cell membrane suspension, which is then homogenized by ultrasound or extruded through a porous polycarbonate membrane to obtain cell membrane vesicles; (2) PLGA nanoparticles are prepared by an emulsified solvent evaporation method: 40-80 mg of polylactic acid-glycolic acid copolymer is dissolved in 10-50 mL of dichloromethane; the mixture solution is dropped into 0.2-1.0 L of polyvinyl alcohol aqueous solution; the obtained solution is stirred for 12-36 hours to evaporate the dichloromethane solvent, and then dialyzed with double distilled water for 8-36 hours, with the water replaced every 3 hours, and the PLGA nanoparticles are obtained by centrifugation at a speed of 8000-16000 rpm / min for 20-60 minutes, which are then washed with double distilled water and freeze-dried to obtain PLGA nanoparticles; (3) Preparation of tumor cell membrane-coated bionic nanoparticles based on mechanical extrusion method: Cell membrane vesicles and PLGA nanoparticles were co-extruded through a porous polycarbonate membrane, and then passed through 400 nm, 200 nm, and 100 nm polycarbonate porous membranes in sequence. Through repeated extrusion, the cell membrane was wrapped onto the surface of the nanoparticles to obtain bionic nanoparticles; (4) Covalently bind dextran to phospholipids by reductive amination: put dextran and phospholipids into a reaction bottle, dissolve them with a mixed solution of dimethyl sulfoxide and chloroform, then add sodium cyanoborohydride, add a magnet, heat in an oil bath at 50-80°C for 12-48h, take out the reaction bottle, cool to room temperature, remove the chloroform in the system by rotary evaporation, add sodium chloride saturated anhydrous ethanol, centrifuge at 6000-12000 rpm / min for 10-30min, discard the supernatant, and the resulting precipitate is the crude glycolipid; dialyze with double distilled water for 12-48h, freeze-dry to obtain the glycolipid; resuspend the glycolipid in PBS to obtain a glycolipid solution with a concentration of 5-20mg / mL; take the glycolipid solution, add it to the biomimetic nanoparticles, stir at 4-10°C for 2-12h, and centrifuge at 8000-16000 rpm / min, centrifuge for 15-30 min, discard the supernatant, and the precipitate is the dextran-modified biomimetic nanoparticles.

3. The method for preparing the dextran-modified biomimetic nanoparticles according to claim 2, characterized in that: The pore sizes of the porous polycarbonate membrane in step (1) are 50 nm, 100 nm, 200 nm, and 400 nm.

4. The method for preparing the dextran-modified biomimetic nanoparticles according to claim 2, characterized in that: The particle size of the dextran-modified bionic nanoparticles is 100-300 nm, and the content of β-glucan is 5-15%.

5. The method for preparing the dextran-modified biomimetic nanoparticles according to claim 2, characterized in that: The tumor cells in step (1) are B16-F10 mouse melanoma cells, the centrifugal forces of the differential centrifugation are 1000-2000 g, 6000-10000 g, 40000-50000 g, and the centrifugation time is 30-60 min.

6. The method for preparing the dextran-modified biomimetic nanoparticles according to claim 2, characterized in that: The volume ratio of chloroform to dimethyl sulfoxide in the mixed solution of dimethyl sulfoxide and chloroform is 1:(2-4).

7. The method for preparing the dextran-modified biomimetic nanoparticles according to claim 2, characterized in that: The molecular weight of the glucan is 2-8 kDa; the monosaccharide composition is mainly glucose, and its content is above 95%, and the connection method is that the main chain is β-1,3 glucan with β-1,6 branches.

8. The method for preparing dextran-modified biomimetic nanoparticles according to claim 2, characterized in that: The molar ratio of the dextran to the phospholipid is 1:(2-20); the molar ratio of the dextran to sodium cyanoborohydride is 1:(2-20).

9. Use of the dextran modified biomimetic nanoparticles according to claim 1 in the preparation of anti-tumor growth and anti-tumor metastasis drugs.

10. The use according to claim 9, characterized in that: The dextran-modified biomimetic nanoparticles can significantly promote the secretion of IL-12, TNF-α, and IFN-γ, enhance the phagocytic ability of macrophages and the natural killing ability of NK cells, and effectively activate the innate immune response; It increases the expression of co-stimulatory molecules on the surface of dendritic cells, enhances antigen presentation by dendritic cells, promotes T cell proliferation and differentiation, and triggers a strong adaptive immune response; it effectively enhances the infiltration of cytotoxic T cells in tumor tissues, reduces the tumor infiltration of MDSCs inhibitory immune cells, and plays a role in inhibiting tumor growth and tumor metastasis.

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