Macrophage targeting type nano in-situ tumor vaccine and application thereof

Abstract

The invention relates to the technical field of tumor drugs, in particular to a macrophage targeting type nano in-situ tumor vaccine and application thereof. The nano tumor vaccine comprises nano particles; the nano-particles comprise astragalus polysaccharide, a PLGA (poly (lactic-co-glycolic acid)) complex and amino-polyethylene glycol mannose, amino-polyethylene glycol mannose is modified on the surface of the PLGA; the mass ratio of the amino-polyethylene glycol mannose to the astragalus polysaccharide to the PLGA is 1: (1-5): (10-20). The APS-coated PLGA is modified by amino-polyethylene glycol mannose, so that the anti-tumor effect of the nano-particles is remarkably improved, and the effect obviously exceeds the expectation. The APS-coated PLGA-man nano-particle provided by the invention can initiate PANoptosis to play a strong and lasting anti-cancer role, and can also have a synergistic effect with an immune checkpoint inhibitor to remarkably inhibit the growth of tumors, so that the APS-coated PLGA-man nano-particle has an important application value in the field of development of anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor drugs, and in particular to a macrophage-targeted nano in situ tumor vaccine and an application thereof. Background Art

[0002] As a malignant disease, tumors seriously endanger human health and shorten human life. Tumor clinical treatment is undergoing a revolution. The breakthrough results of immunotherapy have changed the pattern and future of cancer treatment. At present, tumor immunotherapy is mainly divided into two categories: active immunotherapy and passive immunotherapy. Compared with passive immunity represented by CAR-T, antibody and cytokine therapy, active immunity represented by therapeutic nano-tumor vaccines can not only kill primary tumors and metastases more effectively, but also more fully activate long-term anti-tumor immunity, and have a lower adverse reaction rate. Therapeutic nano-tumor vaccines are usually composed of nano-carriers, tumor antigens and immune agonists. Finding a suitable immune agonist is crucial for the efficacy of therapeutic nano-tumor vaccines. Astragalus polysaccharides (APS) are hydrophilic macromolecular mixtures composed of various acidic polysaccharides such as glucose, galactose, and arabinose. Because of its strong effects of improving immunity, anti-tumor, antioxidant, and antibacterial, it is widely used as an immunopotentiator in clinical anti-cancer treatment. At present, many researchers have combined the advantages of nanocarriers such as strong cell penetration and targeting ability, efficient pharmacokinetics, and good biocompatibility with the immune enhancement properties of APS to prepare a promising new anti-tumor immunotherapy drug, namely nano-astragalus polysaccharide. The commonly used methods for preparing nano-astragalus polysaccharide include: preparing astragalus polysaccharide nano-vaccines encapsulating OVA protein by microfluidics; using PLGA-PEG-COOH to encapsulate APS to form nanoparticles by double emulsification; using solvothermal method to prepare APS nanoparticles encapsulated by superparamagnetic iron oxide nanoparticles; preparing chitosan-encapsulated APS nanoparticles by ion crosslinking principle; preparing APS liposomes by thin film dispersion-microporous membrane extrusion method; or using the characteristics of APS self-assembly to form nano-aggregates with porous structure and large surface area. Through these methods, nano-APS with a particle size between 50-200nm can be prepared. Since the smaller the particle size, the easier it is to be taken up by cells through endocytosis, thereby enhancing the therapeutic effect of APS.

[0003] However, the nano-APS prepared at present lacks the targeting function to the target cells. After entering the body, the killing effect on tumor cells and the enhancement function on immune cells are weak, and large doses and long-term medication are often required in in vivo experiments. Due to the lack of cell targeting, high doses of drugs entering the body will be taken up by non-target normal cells in large quantities, causing serious adverse reactions to the body.

[0004] At present, in order to achieve good therapeutic effects, therapeutic nano-tumor vaccines require effective immune adjuvants and a large number of tumor-specific antigens of various types. There are two main ways to combine autologous nano-tumor vaccines with tumor antigens: one is to prepare antigens in vitro and then inject them into the patient's body. This method has low preparation efficiency, high price, limited tumor antigen loading, and is difficult to guarantee accuracy and popularity; the other is to combine local treatments such as tumor ablation to destroy tumors through local treatments, so that nano-tumor vaccines can obtain enough tumor antigens in the body and deliver them to immune cells. Due to the cumbersome technical means, expensive equipment, and high cost of treatment, and certain requirements for various body functions of patients, this combined treatment mode undoubtedly raises the threshold for future clinical transformation and reduces the accessibility and benefit of new medical technologies. Therefore, the design and preparation process of therapeutic nano-tumor vaccines still need to be improved. The current research and development focus is to design a multifunctional nano-tumor vaccine that can destroy tumor antigens, adsorb tumor antigens, target immune cells, and activate systemic specific anti-tumor immunity in the local tumor area in the body, and further improve the efficacy, preparation efficiency and popularity, which is the key to its entry into clinical application. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a macrophage-targeted nano in situ tumor vaccine and application thereof.

[0006] At present, autologous nano-tumor vaccines are mostly focused on enhancing the body's adaptive anti-tumor immunity while ignoring the huge tumor-suppressing effect of innate immunity. During the research process, the present invention found that the number of DCs and T cells in tumor tissues is small and mostly in a suppressed state, while tumor-associated macrophages (TAMs) are the innate immune cells with the highest proportion in the tumor microenvironment, accounting for up to 50% of the total volume of solid tumors. The innate innate immune response (various cells of the myeloid system, including monocytes, macrophages, polymorphonuclear cells, mast cells and natural killer cells, etc.) plays a significant role in the development, activation and functional maintenance of T cells. Innate immune cells represented by macrophages are the first line of defense of the body. By detecting the molecular changes of the tumor, they first initiate their own protective response and bridge the innate immunity and adaptive immunity through their own antigen presentation function, which is the key to triggering tumor-specific T cell function.

[0007] At present, the conventional technology for achieving active targeting of macrophages is to decorate the surface of nanoparticles with an agent (such as a ligand, an antibody, and a peptide) that can selectively interact and be recognized by specific cell types in certain tissues. However, the existing methods are cumbersome and expensive, which is not conducive to clinical popularization. Therefore, the present invention turns to studying the receptors contained on the surface of macrophage membranes. The surface of macrophage membranes contains many receptors, which determine the activity of macrophages, including growth, differentiation, activation, recognition, endocytosis, migration, and secretion. The present invention ultimately focuses on the mannose receptor, which is highly abundant in the tumor microenvironment and highly expressed on M2 macrophages. Mannose is its natural ligand. The present invention modifies amino-polyethylene glycol-mannose on the surface of poly(lactic-co-glycolic acid) (PLGA), effectively improving the targeting ability of PLGA nanoparticles to TAMs in tumor tissues. Specifically, the carboxyl group on the surface of PLGA can form a peptide bond with the amino group at the end of amino-polyethylene glycol-mannose (NH2-PEG-Man) through dehydration condensation, so that the surface of PLGA is modified with amino-polyethylene glycol-mannose to obtain a PLGA nano-astragalus polysaccharide in situ tumor vaccine (APS@PLGA-man, APM) with macrophage targeting ability. APM can promote the uptake of APS by macrophages and enhance the ability of macrophages to polarize to M1 and kill tumors.

[0008] After APM promotes the polarization of tumor-associated macrophages and kills tumors, the skeleton material PLGA of APM can also adsorb tumor protein antigens produced after tumor cell death through hydrophobic effects. APM can accurately deliver tumor antigens to macrophages, activate their antigen presentation properties, activate Th1 and CD8+T cells in the tumor area, activate anti-tumor adaptive immune responses, and produce strong and lasting anti-tumor immune responses.

[0009] In addition, the present invention also found in the study that the nanoparticles provided by the present invention also significantly promoted the occurrence of PANoptosis (there is no research report on activating M1 macrophage polarization while causing tumor PANoptosis), triggering multimodal tumor death. While releasing a large number of highly immunogenic tumor antigens, the tumor antigens are adsorbed, enriched, and delivered to macrophages in a targeted manner, and their antigen presentation function effectively activates specific anti-tumor immunity, thereby exerting a powerful and lasting anti-cancer effect. On this basis, it can be further combined with existing immunotherapy to reduce microenvironmental immunosuppression and achieve better therapeutic effects.

[0010] In a first aspect, the present invention provides a tumor vaccine, comprising: nanoparticles; The nanoparticles include: a complex of astragalus polysaccharide and PLGA, and amino-polyethylene glycol mannose; the amino-polyethylene glycol mannose is modified on the surface of the PLGA; and the mass ratio of the amino-polyethylene glycol mannose, astragalus polysaccharide and PLGA is 1:(1-5):(10-20).

[0011] Furthermore, the mass ratio of amino-polyethylene glycol mannose, astragalus polysaccharide and PLGA is 1:(1.5-3):(12-16).

[0012] Furthermore, the astragalus polysaccharide and PLGA complex is APS@PLGA nanoparticles, and the APS@PLGA nanoparticles are prepared by a double emulsification method.

[0013] Furthermore, the double emulsification method includes: using PVA solution as the external aqueous phase, using PLGA dissolved in an organic solvent as the organic phase, and using APS solution as the internal aqueous phase; The inner aqueous phase and the organic phase are mixed, and colostrum is obtained by ultrasonic treatment in an ice-water bath; the colostrum and the outer aqueous phase are mixed under stirring at room temperature, and then ultrasonically dispersed, stirred, filtered, and centrifuged in an ice-bath to obtain APS@PLGA nanoparticles.

[0014] Furthermore, the PVA concentration in the PVA solution is 0.1-10 w / v%, preferably 1 w / v%.

[0015] Furthermore, the organic solvent includes: one or more of dichloromethane, ethyl acetate or anhydrous ethanol, for example dichloromethane, or ethanol+dichloromethane (volume ratio is 1:2).

[0016] Furthermore, the ultrasonic time is 3 to 15 minutes, preferably 7 minutes.

[0017] Furthermore, the power is 75-120W, preferably 100W.

[0018] Furthermore, the volume ratio of the inner aqueous phase to the organic phase is 1:(2-9), preferably 1:5.

[0019] Furthermore, the volume ratio of the colostrum to the external aqueous phase is 1:(2-8), preferably 1:6.

[0020] Furthermore, the concentration of PLGA dissolved in the organic solvent is 5-15 mg / mL, preferably 14.6 mg / mL.

[0021] In a second aspect, the present invention provides a method for preparing the tumor vaccine, comprising: After APS@PLGA nanoparticles were activated by NHS and EDC, they were mixed with amino-polyethylene glycol mannose and stirred for more than 8 hours. The particles were filtered, centrifuged to obtain the precipitate, resuspended and freeze-dried.

[0022] Furthermore, the filtration is filtering through a 0.2-0.3 μm filter membrane; and / or the centrifugation is centrifuging at 18000-22000 g for more than 35 minutes in cold PBS.

[0023] In a third aspect, the present invention provides an anti-tumor drug comprising the aforementioned nanoparticles.

[0024] Furthermore, it also includes: immune checkpoint inhibitors, preferably PD-L1 inhibitors.

[0025] Immune checkpoint inhibitors can enhance the recognition and killing of tumors by CD8+T cells, and to a certain extent can also promote the polarization of TAMs to M1 type. The APM of the present invention can play a significant synergistic role with immune checkpoint inhibitors to further enhance the body's anti-tumor adaptive immunity and innate immunity. This combined treatment mode can significantly inhibit tumor growth.

[0026] In a fourth aspect, the present invention provides the aforementioned nanoparticles, or use of the anti-tumor drug in preventing or treating cancer.

[0027] Furthermore, the PD-L1 inhibitor is administered at a concentration of 0.5-2 mg / mL; preferably 1 mg / mL.

[0028] Furthermore, the administration concentration of the nanoparticles is 3 mg / mL.

[0029] Furthermore, the administration cycle is once every 3-5 days, and the total treatment course is 2 weeks.

[0030] Furthermore, the application includes: injecting the aforementioned nanoparticles into a tumor of a subject in need thereof, and simultaneously administering the PD-L1 inhibitor intravenously.

[0031] The present invention further provides use of the aforementioned nanoparticles in preparing a drug for preventing or treating cancer.

[0032] Furthermore, the cancer is lung cancer.

[0033] Furthermore, the prevention or treatment of cancer includes: Activate PANoptosis, enhance the body's anti-tumor immune response (increase the levels of TNF-α, INF-γ, and IL-6 inflammatory factors in the tumor), reduce the level of the intratumoral immunosuppressive factor IL-10, increase the proportion of M1 macrophages, reduce the volume and mass of the tumor, or increase the apoptosis rate of cancer cells.

[0034] The present invention has the following beneficial effects: 1. The present invention provides a nanoparticle APS@PLGA-man composited with astragalus polysaccharide, PLGA and amino-polyethylene glycol mannose. Amino-polyethylene glycol mannose modification improves the targeting ability of APS@PLGA to TAMs, promotes the uptake of APS by macrophages, and enhances the ability of macrophages to polarize to M1 and kill tumors.

[0035] 2. The nanoparticles APS@PLGA-man provided by the present invention can induce PANoptosis and effectively activate anti-tumor immunity. While releasing a large number of highly immunogenic tumor antigens, they adsorb and enrich tumor antigens and deliver them to macrophages in a targeted manner. Through their antigen presentation function, they effectively activate specific anti-tumor immunity, thereby exerting a powerful and lasting anti-cancer effect.

[0036] 3. The present invention further provides a treatment method of nanoparticles APS@PLGA-man combined with immune checkpoint inhibitors. The two synergistically further enhance the body's anti-tumor adaptive immunity and innate immunity, and have a significant inhibitory ability on tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 It is a schematic diagram of the characterization of the nanoparticles APS@PLGA and APS@PLGA-man provided in Example 2 of the present invention; wherein AC is the morphology of APS@PLGA-man at different sizes, D is the state of the APS@PLGA-man solution observed with the naked eye and the state of the solution showing the Tyndall effect under laser irradiation, EI is the particle size, Zeta potential and PDI comparison results of the nanoparticles APS@PLGA and APS@PLGA-man at different pH values ​​(5.6 and 7.4); J is the Fourier transform infrared spectrum image of APS@PLGA-man; in the figure, AP is APS@PLGA and APM is APS@PLGA-man.

[0039] Figure 2 This is a graph showing the release rates of the nanoparticles APS@PLGA and APS@PLGA-man provided in Example 4 of the present invention at different pH values; in the graph, APS is astragalus polysaccharide, and APM is APS@PLGA-man.

[0040] Figure 3 These are the results of the macrophage proliferation and toxicity experiment using cck8 as provided in Example 5 of the present invention; wherein A is the cell survival rate result of APS@PLGA-man and mouse macrophage RAW264.7 incubated at different concentrations; and B is the cell survival rate result of APS@PLGA-man and human monocyte THP-1 incubated at different concentrations.

[0041] Figure 4 It is the macrophage uptake experiment result provided in Example 6 of the present invention; wherein A is the fluorescence image of the control, APS, PLGA and APS@PLGA-man; B is the fluorescence level image corresponding to A.

[0042] Figure 5 These are the experimental results of tumor protein antigen adsorption of nanoparticles APS@PLGA and APS@PLGA-man provided in Example 7 of the present invention; wherein A is the potential change after APS@PLGA and APS@PLGA-man bind to the antigen, B is the particle size change after APS@PLGA and APS@PLGA-man bind to the antigen; C is the comparison of the adsorbed protein quality of nanoparticles PLGA-man, APS@PLGA and APS@PLGA-man.

[0043] Figure 6 It is the result of the macrophage polarization experiment provided in Example 8 of the present invention; wherein A is the change in the proportion of RAW264.7 M1 polarization marker CD86+ cells, a macrophage polarization marker of the control, APS, PLGA-man, APS@PLGA and APS@PLGA-man, B is the change in the proportion of CD86+ cells, a THP-1 M1 polarization marker, CD is the change in the cytokines TNF-α and INF-γ produced by M polarization, and E is the change in the level of IL-10 secreted by the M2 type.

[0044] Figure 7 It is the in vitro tumor cell inhibition ability detection result provided in Example 9 of the present invention; wherein A is a schematic diagram of the detection process; B is the change result of cell survival rate after co-incubation of different concentrations of nanoparticles APS@PLGA-man and LLC cells; CD is the effect of the conditioned medium after APS, AP and APM treatment of macrophages on the number of LLC cell colonies; EF is the scratch test result of the conditioned medium after APS, AP and APM treatment of macrophages; G is the result of combined treatment of conditioned medium after APS, AP and APM treatment of macrophages with different apoptosis inhibitors.

[0045] Figure 8It is the effect of the nanovaccine provided in Example 10 of the present invention on the in situ tumor of mice; wherein A is a schematic diagram of the detection process; BD is a diagram of the change of in situ tumor of mice after treatment with PLGA-man, APS, APS@PLGA-man, APS+αPD-L1 and APS@PLGA-man+αPD-L1, and E is the change of mouse weight; F is the growth curve of in situ tumor of mice after treatment with PLGA-man, APS, APS@PLGA-man, APS+αPD-L1 and APS@PLGA-man+αPD-L1; HG is the Tunel staining results of in situ tumors of mice in different treatment groups.

[0046] Fig. 9 It is the effect of the nanovaccine provided in Example 10 of the present invention on distal tumors in mice; wherein AC is a graph of changes in distal tumors in mice after treatment with PLGA-man, APS, APS@PLGA-man, APS+αPD-L1 and APS@PLGA-man+αPD-L1; D is a growth curve of distal tumors in mice after treatment with PLGA-man, APS, APS@PLGA-man, APS+αPD-L1 and APS@PLGA-man+αPD-L1.

[0047] Fig.10 It is the effect of APS@PLGA-man provided in Example 11 of the present invention on the in vivo tumor immune microenvironment; wherein A is the change in the proportion of M1 macrophages and M2 macrophages in mouse tumors after treatment with PLGA-man, APS, APM, APS+αPD-L1 and APM n+αPD-L1; B is the change in CD8+T cells in mouse tumors; C is the change in the proportion of Th1 cells in mouse spleen; D is the change in the proportion of CD8+ cells in mouse spleen.

[0048] Fig.11 It is the effect of the nanovaccine provided in Example 12 of the present invention on the level of inflammatory factors in mice; wherein A is the level of cytokine secretion in mice after treatment with PLGA-man, APS, APS@PLGA-man, APS+αPD-L1 and APS@PLGA-man+αPD-L1; wherein A, B, D are changes in TNF-α, INF-γ, and IL-6 concentrations in mouse tumors; C and F are changes in INF-γ and IL-6 concentrations in mouse serum; E is the change in the concentration of immunosuppressive factor IL-10 in mouse tumors.

[0049] Fig.12 This is a graph showing changes in PANoptosis-related gene expression in tumors of mice treated with the nanovaccine provided in Example 13 of the present invention.

[0050] Fig.13This is a graph showing changes in the expression of PANoptosis protein markers in tumors of mice treated with the nanovaccine provided in Example 13 of the present invention.

[0051] Fig.14 It is an in vivo biological targeting and metabolic evaluation diagram of the nanovaccine provided in Example 14 of the present invention; wherein A and C are time diagrams of APS, AP NPs and APM NPs staying in mouse tumors; and B is a distribution diagram of APS, AP NPs and APM NPs in different organs of mice.

[0052] Fig.15 These are the results of serum blood routine and organ safety tests after mice were treated with the nanoparticles APS@PLGA-man and APS@PLGA-man+αPD-L1 provided in Example 15 of the present invention.

[0053] Fig.16 These are the HE staining results of various organs of mice after being treated with the nanoparticles APS@PLGA-man and APS@PLGA-man+αPD-L1 provided in Example 15 of the present invention. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0056] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0057] Example 1

[0058] This embodiment provides a method for synthesizing a macrophage-targeted PLGA nano-astragalus polysaccharide in situ tumor vaccine, which specifically includes the following process: 1. Preparation of external aqueous phase: double emulsification method was used to prepare drug-loaded nanoparticles. 720 mg PVA was weighed and 72 ml ionized water was used as solvent to prepare 1% PVA solution. The two were then mixed and magnetically stirred at a speed of 600 rpm for 10 minutes. The temperature was then slowly raised to 85-99 degrees. After the PVA was completely dissolved, heating was stopped and the external aqueous phase (W2) was obtained after cooling. 2. Prepare the organic phase and the inner aqueous phase: Weigh 146 mg PLGA and dissolve it in 10 ml dichloromethane, and oscillate it in a warm water bath at 36°C with ultrasonic waves until it is completely dissolved to obtain the organic phase (O); dissolve 20 mg APS in 10 ml deionized water, and oscillate it in a warm water bath with ultrasonic waves until it is completely dissolved, and take 2 mL of APS solution to obtain the inner aqueous phase (W1); 3. Preparation of APS@PLGA nanoparticles: Add the inner aqueous phase to the organic phase and disperse it ultrasonically in an ice-water bath at 100W for 7 minutes to obtain colostrum (W1 / O); Under stirring at room temperature, inject the colostrum dropwise into the outer aqueous phase with a syringe, and then disperse it again at 100W ultrasound in an ice bath for 7 minutes. Subsequently, stir with a magnetic stirrer at room temperature for 4 hours to disperse the nanoparticles and evaporate the dichloromethane. Collect the solution, filter it through a syringe using a 0.22-micron filter membrane, and centrifuge it at 8000rpm for 20 minutes, discard the supernatant, wash the precipitate with deionized water 3 times, resuspend it in 10mL deionized water, and freeze-dry it for use; 4. Preparation of APS@PLGA-man nanoparticles: APS@PLGA nanoparticles (prepared in step 3) were dissolved in 10 mL MES buffer, pH = 5, and stirred for one hour; 10 mg 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (HCL·EDC) was added to the solution and stirred for one hour; 10 mg sulfo-N-hydroxysuccinimide (sulfo-NHS) was added to the NP solution at room temperature, and the suspension was stirred for 2 hours; 10 mg amino-polyethylene glycol mannose (NH2-PEG-Man) was added to the resulting solution and stirred overnight at room temperature. Filter again using a 0.22 μm filter membrane through a syringe, and then centrifuge the solution at 20,000 g for 40 minutes in cold PBS to remove excess reagents. The precipitate was resuspended in Milli-Q water, freeze-dried and stored at -20 °C until further use.

[0059] Similarly, PLGA-man and APS@PLGA nanoparticles were obtained by the above method, except that astragalus polysaccharide (APS) and amino-polyethylene glycol-mannose (NH2-PEG-Man) were not added.

[0060] Example 2

[0061] This example characterizes the nanoparticles prepared in Example 1, as follows: 1. The APS@PLGA-man solution showed a light blue opalescence with the Tyndall effect, indicating that a nanoparticle dispersion system was formed (e.g. Figure 1 as shown in D in the figure).

[0062] 2. The size and morphology of APS@PLGA-man at different pH values ​​were analyzed using transmission electron microscopy (TEM, HITACHI, HT7700) and scanning electron microscopy (SEM, Hitachi, S4800). The particle size, PDI and zeta potential of APS@PLGA-man at different pH values ​​were measured on a Malvern Zetasizer. A Fourier transform infrared spectrometer (BRUKER) was used to determine the chemical structure. After loading the drug, the PLGA carrier was modified with amino-polyethylene glycol-mannose. Electron microscopy images showed that under pH = 7.4, the nanoparticles were spherical, uniform in morphology, regular in surface, and had a particle size of approximately 150 nm. Under pH = 5.6, the particle size of the nanoparticles increased to approximately 200 nm, and their skeletons began to dissolve, forming cavities, indicating the release of the drug ( Figure 1 AC in Figure 1).

[0063] 3. The present invention observed the changes in particle size and zeta potential before and after amino-polyethylene glycol-mannose modification using Malvern Zetasizer.

[0064] (1) At pH = 7.4, the average particle size of APS@PLGA was 136.4±2nm, the average Zeta potential was -18.57±0.67mV, and the PDI was 0.086±0.02; the average particle size of APS-loaded PLGA-man (APS@PLGA-man) was 145±1.53nm, the average Zeta potential was -17.17±0.84mV, and the PDI was 0.132±0.01.

[0065] (2) At pH 5.6, the average particle size of APS@PLGA was 159.933±0.569nm, the average Zeta potential was -8.07±0.343mV, and the PDI was 0.253±0.05; the average particle size of APS@PLGA-man was 183.33±1.53nm, the average Zeta potential was -1.413±0.215mV, and the PDI was 0.262±0.01.

[0066] The above results show that the particle size of APS@PLGA-man increases after modification. Under acidic conditions of pH=5.6, the particle size of the nanoparticles increases significantly, and the absolute value of the potential decreases significantly, indicating that the nanoparticles may be structurally unstable under acidic conditions (simulating the intracellular environment) and are more likely to release drugs ( Figure 1 Chinese EI).

[0067] 4. The present invention further confirms the synthesis of nanoparticles using Fourier transform infrared spectroscopy (FTIR). The characteristic peak of APS is 1757 cm -1 ; PLGA characteristic peak is 1180-1150cm-1 ; The characteristic peaks of mannose are 843 and 1647 cm -1 ; Compared with the above components, APS@PLGA-man showed similar characteristic peaks, proving that each component was successfully loaded ( Figure 1 in J).

[0068] Example 3 Encapsulation efficiency determination

[0069] This example further measures the encapsulation efficiency of the nanoparticles obtained in Example 1, including the following process: In order to determine the amount of loaded drugs, the present invention uses the sulfuric acid-phenol method to determine the APS encapsulation efficiency of APS@PLGA and APS@PLGA-man, establishes an astragalus polysaccharide calibration curve using APS standards and sulfuric acid-phenol colorimetric method, and uses the sulfuric acid-phenol method to determine the absorbance of various indicators in the sample.

[0070] The nanoparticle precipitate after centrifugation was resuspended to 5 mL, and the sample absorbance was detected at a wavelength of 488 nm using an enzyme reader. The content of astragalus polysaccharide was obtained by substituting it into the regression equation: encapsulation efficiency EE (%) = (drug content encapsulated in nanoparticles / total amount of drug added) × 100%.

[0071] The results showed that the drug encapsulation efficiencies of APS@PLGA and APS@PLGA-man were approximately 77.81±0.01% and 91.02±0.921%, respectively.

[0072] Example 4 Drug Release Test

[0073] This example further conducts a drug release test on the nanoparticles obtained in Example 1, including the following process: In order to evaluate the drug release of APS, the present invention sealed free APS@PLGA and APS@PLGA-man in dialysis bags with a MWCO of 8000Da, and then placed the bags in different beakers. Appropriate amounts of PBS with pH 5.6 and pH 7.4 were added, and all dialysis bags were shaken at 300 rpm, and a portion of the supernatant buffer was collected at predetermined time intervals. Drug release was observed by measuring the APS content using the sulfuric acid phenol method and an ELISA reader.

[0074] The destruction of the PLGA skeleton and the release of APS in an acidic environment are key factors for the nanoparticles to function after cell uptake. The present invention tested the drug release of APS@PLGA-man in different environments and found that the nanoparticles slowed down the release of APS through PLGA encapsulation, which may prolong the stability and half-life of APS in the body. At the same time, when APS@PLGA-man enters macrophages and is destroyed in the intracellular acidic environment, targeted release can be achieved.

[0075] The present invention observed the release of APS at different pH values ​​and found that the release of APS in an acidic environment of pH 5.6 was significantly greater than that in a normal environment of pH 7.4. This is because the degradation of the polymer of nanoparticles with polyester materials such as PLGA as the skeleton is mainly the hydrolysis of ester bonds, and the acidic environment can accelerate this process. At the same time, acidic degradation products such as lactic acid and glycolic acid are generated during the hydrolysis process, and the generation rate of these acids is higher than the rate of diffusion and neutralization, so that a local acidic environment is generated inside the nanoparticles, which accelerates the degradation by self-catalysis (such as Figure 2 as shown).

[0076] Example 5 Macrophage proliferation and toxicity experiments

[0077] This example uses CCK8 to detect macrophage proliferation and toxicity experiments, which specifically include the following process: In order to evaluate the effect of APS@PLGA-man on macrophages in vitro, the present invention divided APS@PLGA-man into 6 concentration gradients of 0, 50, 100, 500, 750, and 1000 μg / mL, and incubated them with mouse macrophages RAW264.7 and human monocytes THP-1 for 24, 48, and 72 hours, respectively. Then, 10 μL of cck solution was added, and the absorbance at 450 nm was measured by an ELISA instrument for 4 hours, and the survival rate was calculated according to the formula: Cell viability (%) = [drug-blank] / [control-blank]*100.

[0078] The results showed that when the concentration of APS@PLGA-man was 50-1000μg / mL, it had a proliferative effect on macrophages within 3 days, and the optimal concentration was 500μg / mL. Compared with the control group (concentration of 0), the average survival rate of RAW264.7 cells treated with 500μg / mL APS@PLGA-man was 140.64-167.2% within 3 days ( Figure 3 The survival rate of THP-1 cells within 3 days was 138.86-165.85% ( Figure 3 This experiment shows that APS@PLGA-man significantly promoted the proliferation of macrophages.

[0079] Example 6 Macrophage uptake experiment

[0080] This example conducts a macrophage uptake experiment on the nanoparticles prepared in Example 1, which specifically includes the following process: RAW264.7 cells were cultured at 1.5 × 10 3The cells were seeded at a density of 10 cells / well in a confocal dish and incubated with APS@PLGA-man with fluorescent dye CY5 (calculated according to MOF, 50μg / mL) for 48 hours. The cell membrane was stained with PKH67, and then the culture medium was discarded, rinsed twice with PBS, and then fixed with 2% paraformaldehyde for 15 minutes. After fixation, DAPI was used to stain the cell nucleus, and the staining was protected from light for 15 minutes. The stain was removed by centrifugation, washed twice with PBS, and 500ul PBS was added, and observed under a confocal laser scanning microscope.

[0081] like Figure 4 A and B in the figure show that at 48h, the fluorescence of APS@PLGA-man nanoparticles entered the cells. Compared with the unmodified amino-polyethylene glycol-mannose nanoparticles and APS, the fluorescence ratio of APS@PLGA-man was higher, indicating that APS@PLGA-man can increase the uptake ratio of macrophages within 48 hours.

[0082] Example 7 Antigen Binding Ability Detection

[0083] This example is based on the antigen binding ability test of the nanoparticles prepared in Example 1, specifically including: The present invention took AP (APS@PLGA, the same below) and APM (APS@PLGA-man, the same below) prepared in Example 1, collected LLC cells in the logarithmic growth phase, and adjusted the cell concentration to 2×10 7 / ml, repeatedly frozen and thawed 3 times with liquid nitrogen, centrifuged at 100000r / min for 5min, filtered and sterilized to obtain the whole tumor antigen for use. APM was co-cultured with the whole tumor antigen at 1mg / mL for 8 hours, centrifuged at 3000r / min for 5min, filtered and sterilized, and the changes in the particle size, potential and adsorbed protein amount of the nano in situ tumor vaccine before and after adsorption were detected by ZETA potential analyzer and BCA kit respectively.

[0084] The results are shown in the figure. Figure 5 Figure A shows that the absolute value of the potential of AP and APM decreased after protein adsorption, from -22.97±1.66 to -17.1±1.15 and from -22.03±1.08 to -12.5±0.62, respectively. Figure 5 Figure B shows that the particle size increases from 146.37±1.96 to 191.23±7.67 and 156.33±1.95 to 217.45±17.17, respectively. This indicates that both nanoparticles can effectively adsorb tumor-derived protein antigens.

[0085] Subsequently, the present invention detected the amount of protein adsorbed by PLGA-man (PM), AP and APM by BCA protein quantitative analysis. The results are as follows Figure 5 As shown in C: PLGA-man (PM), AP and APM can effectively adsorb proteins through hydrophobic interaction, and the adsorption effect is not significantly weakened after surface modification with amino-polyethylene glycol-mannose. 1mgAPM can adsorb 664.29±1.51ug protein.

[0086] Example 8 Macrophage polarization experiment

[0087] The present invention conducted flow cytometry and ELISA experiments on the nanoparticles prepared in Example 1 to evaluate the effects of APS@PLGA-man on macrophage polarization and function, including the following process: The present invention seeded RAW264.7 in the logarithmic growth phase and THP-1 treated with 100 ng / mL PMA for 48 hours in a six-well plate, and treated the cells in each well with complete culture medium, 500 ug / mL concentration of PLGA-man, APS@PLGA-man, APS@PLGA, and APS for a total of 48 hours, then took the supernatant for ELISA experiment, and the cells were blown and centrifuged to prepare a single cell suspension for flow cytometry detection of macrophage polarization marker expression.

[0088] Flow cytometry results showed that compared with other groups, the proportion of CD86+ cells, a polarization marker of RAW264.7M1, treated with 500ug / mL APS@PLGA-man was significantly increased, with a statistically significant proportion of 19.2±2.29% ( Figure 6 The proportion of CD86+ cells, a marker of M1 polarization, in THP-1 treated with 500ug / mL APS@PLGA-man was also significantly increased ( Figure 6 The percentage of CD206+CD86- M2 cells in RAW264.7 and THP-1 was 21.03±0.737%, which was statistically significant.

[0089] ELISA results showed that in the supernatant of RAW264.7 cells treated with 500ug / mL APS@PLGA-man, the cytokines TNF-α and INF-γ produced by M1 polarization increased significantly, reaching 1598.19±47.254pg / mL and 72.706±5.939pg / mL, respectively, which was statistically significant compared with the control group ( Figure 6 C and D in the figure); in the supernatant of RAW264.7 cells treated with 500ug / mLAPS@PLGA-man, the level of IL-10, mainly secreted by M2 type, decreased by 4 times compared with the control group ( Figure 6 E in.

[0090] The above results collectively indicate that the nanoparticles APS@PLGA-man prepared in Example 1 can significantly increase the proportion of M1 macrophages and promote the secretion of anti-tumor cytokines.

[0091] Example 9 In vitro tumor cell inhibition ability detection

[0092] The nanoparticles prepared in Example 1 were tested for their ability to inhibit tumor cells in vitro, which specifically included the following process (eg Figure 7 (as shown in A): After incubation of RAW 264.7 cells with 0.5 mg / mL APM for 48 h to induce macrophage differentiation and maturation, conditioned medium (CM) was collected and filtered using a 0.22 μm filter. Logarithmically growing LLC cells were then cultured with conditioned medium containing different concentrations of APM in a humidified incubator at 37°C and 5% CO2 for several days and supplemented with fresh medium for further analysis.

[0093] Cell viability assay: After staining with CCK-8 kit, the relative viability of LLC at different APM NP concentrations was analyzed using a microplate reader. For colony formation assay, LLC cells in the logarithmic growth phase were collected and 1000 cells were seeded into each well of a 6-well plate. The cells were cultured for 10 days and then fixed with methanol for 20 minutes and stained with 0.1% crystal violet for 15 minutes. The number of colonies formed in each well was counted and photographed.

[0094] For the wound healing assay (scratch assay), LLC cells were cultured at 2 × 10 5 The cells were seeded at a density of 1.5 μg / mL in a 6-well plate. After 24 hours of incubation, a wound was made in the cell monolayer using a scratch tool, and the culture medium was replaced with serum-free culture medium. Images were taken at 0, 24, and 48 hours using a microscope. According to the CCK-8 results, the optimal concentration of CM for the scratch assay and the clone formation assay was set at 0.5 mg / mL. The clone formation assay showed that the supernatant after APM-induced polarization of RAW264.7 could significantly inhibit LLC proliferation and colony formation. Compared with the control group, CM containing APM could reduce the number of colony formation by about 10 times ( Figure 7 C and D in the figure). The results of the scratch experiment showed that the conditioned medium of RAW264.7 treated with APM could slow down the wound healing of LLC cells, indicating that the cytokines produced by RAW264.7 activated by APM significantly affected the migration of LLC ( Figure 7 E and F in the figure).

[0095] Example 10 Live / dead staining assay

[0096] The present invention further uses the conditioned medium in Example 9 to perform a live / dead staining test, including the following process: LLC cells were treated with (1) control (no additional treatment); (2) direct incubation with APM; (3) conditioned medium of APS-treated macrophages; (4) conditioned medium of AP nanoparticle-treated macrophages; (5) conditioned medium of APM nanoparticle-treated macrophages; (6) conditioned medium of APM nanoparticle-treated macrophages combined with apoptosis inhibitor ABT737 (5 μM); (7) conditioned medium of APM nanoparticle-treated macrophages combined with pyroptosis inhibitor VX765 (5 μM); (8) conditioned medium of APM nanoparticle-treated macrophages combined with necrosis inhibitor Nec-1 (10 μM). The above conditions were used for 48 h. Cell viability was assessed using a live / dead staining kit (Solarbio BioTech Co., Ltd., China) according to standard procedures.

[0097] The results are as follows Figure 7 As shown in G, by comparing the green fluorescence representing living cells and the red fluorescence representing dead cells, it was found that the red fluorescence of LLC cells treated with the conditioned medium group after APM treatment of macrophages far exceeded that of other groups, and it was proved that the nanoparticle APM has a strong killing ability on LLC lung cancer cells, and this mode of death involves multiple pathways, which is consistent with the characteristics of PANoptosis.

[0098] Example 11 In vivo tumor inhibition experiment

[0099] The present invention conducted an in vitro tumor inhibition experiment on the nanoparticles prepared in Example 1, which specifically includes the following process: All experimental procedures involving animals were approved by the Institute of Physics and Chemistry of the Chinese Academy of Sciences (IACUC-IPC-23080). The mice were divided into 6 groups: blank group, APS@PLGA-man group, APS@PLGA-man+αPD-L1 group, APS group, APS+αPD-L1 group, and PLGA-Man group, with 12 mice in each group. The mice were given corresponding treatments according to the group names (APS@PLGA-man group: 50ul of 3mg / ml APS@PLGA-man nanoparticles were injected orally into the tumor of mice; APS@PLGA-man+PD-L1 group: 50ul of 3mg / ml APS@PLGA-man nanoparticles were injected orally into the tumor of mice and 50ul of 1mg / ml PD-L1 was injected into the tumor of mice; APS+PD-L1 group: 50ul of 1mg / ml PD-L1 was injected into the tumor of mice and 100ul of 20mg / ml APS was injected into the tumor of mice; APS group: 100ul of 20mg / ml APS was injected into the tumor of mice; PLGA-Man group: 50ul of 3mg / ml APS@PLGA nanoparticles; blank group: mice were injected with 50ul normal saline intratumorally).

[0100] The specific scheme is as follows: In vivo experiments used a mouse LLC subcutaneous double tumor transplantation model. LLC cells in the logarithmic growth phase were collected and made into a single cell suspension using sterile saline. Female C57BL / 6J mice were injected with 1x10 6 LLC cells (orthotopic tumors), on day 3, mice were subcutaneously injected with 3x10 6 LLC cells (distal tumors) were used to establish a mouse double tumor model. The animals with a subcutaneous solid tumor of about 0.5 cm-0.7 cm in diameter were qualified animal models for future use. The specific intervention drugs were grouped. The tumor growth and mouse weight were continuously monitored until the 15th day (once every three days). After the monitoring was completed, all mice were killed and the corresponding organs of the mouse tissues were taken for testing ( Figure 8 A in the figure).

[0101] Compared with the control group, the size and weight of bilateral tumors in the APS group decreased to a certain extent, but the changes were small, indicating that the anti-tumor effect of APS alone may be weak. The PLGA-man group had no obvious tumor inhibition effect compared with the control group, indicating that the nanocarrier without APS loading had no anti-tumor function. Compared with the APS group, the tumor inhibition effect of APS+αPD-L1 was enhanced, which was because αPD-L1 further enhanced the activation function of APS for anti-tumor immunity. The tumor inhibition effect of APS@PLGA-man alone was slightly stronger than that of APS+αPD-L1, but there was no statistical difference, but it still showed that the astragalus polysaccharide nanotumor vaccine targeting macrophages can greatly enhance the original effect of APS. In addition, the reduction in the volume and mass of distal tumors shows that the anti-tumor specific immunity in vivo is fully activated by APS@PLGA-man and APS@PLGA-man+αPD-L1.

[0102] In summary, the APS@PLGA-man+αPD-L1 group showed the best anti-tumor and distal tumor effects compared with other groups. This demonstrates the superiority of the synergistic effect of the APS@PLGA-man+αPD-L1 treatment method in anti-tumor. The above findings can be seen in representative anatomical tumors ( Figure 8 B in Fig. 9 At the same time, compared with the drug-treated group, the weight of the control group decreased rapidly, indicating that the disease progressed faster, while there was no statistical difference in the weight of the other groups, indicating that the above-mentioned nanomaterials and drugs did not affect the weight of mice and had relatively balanced biosafety ( Figure 8 E in.

[0103] In order to study the anti-tumor mechanism of APS@PLGA-man and APS@PLGA-man+αPD-L1 group in mice, the present invention further performed Tunel staining on the above-obtained mouse tumor tissues ( Figure 8 H and G in Figure 3). The green fluorescence intensity of apoptosis in tumor tissue was significantly enhanced under this therapy, indicating that the tumor was inhibited under this therapy, and apoptosis was one of the main possible mechanisms.

[0104] Example 12 Evaluation of the ability to regulate the tumor immune microenvironment in vivo

[0105] The present invention conducted a flow cytometry experiment based on the experimental materials of Example 10 to evaluate the effect of APS@PLGA-man on the tumor immune microenvironment in vivo, which specifically includes the following process: The LLC subcutaneous transplanted tumor mice in each group treated for 14 days were killed, and their tumors were obtained. Subsequently, single cell suspensions were prepared for flow cytometry detection of the expression of related immune cell markers.

[0106] Flow cytometry results showed that compared with other groups, the proportion of M1 macrophages in the tumors of mice treated with APS@PLGA-man and APS@PLGA-man combined with αPD-L1 was significantly increased, with statistical significance, and the proportions were 25.33±0.808 and 44.03±13.86%, respectively. The proportion of M2 macrophages in the tumors of mice treated with APS@PLGA-man and APS@PLGA-man combined with αPD-L1 was significantly reduced ( Fig.10 A in the figure was statistically significant, with a ratio of 9.263±5.694% and 12.817±5.225%. At the same time, the proportion of CD8+T cells in the tumor and CD8+ cells in the spleen of mice treated with APS@PLGA-man and APS@PLGA-man combined with αPD-L1 increased significantly, with a ratio of CD8+T cells in the tumor of 2.503±0.169% and 2.887±0.211% ( Fig.10 B); the proportion of CD8+T cells in the spleen was 6.6±1.147% and 11.433±2.021% ( Fig.10 D); the proportion of Th1 cells in the spleen also increased significantly, reaching 3.701±0.09% and 4.983±0.967% ( Fig.10 C in.

[0107] These results suggest that APS@PLGA-man activated macrophage-mediated innate immunity and T lymphocyte-mediated adaptive immune responses in vivo to resist tumors, and its efficacy was further enhanced when combined with αPD-L1.

[0108] Example 13 Evaluation of Inflammatory Factor Levels in the Body

[0109] The present invention uses ELISA to detect the levels of inflammatory factors in mice based on the experimental materials of Example 10. The results show that the concentrations of TNF-α, INF-γ, and IL-6 in the tumors of mice treated with APS@PLGA-man and APS@PLGA-man combined with αPD-L1 increased ( Fig.11 A, B, and D in the figure), serum INF-γ and IL-6 concentrations also increased ( Fig.11 C, F in the figure) showed that the anti-tumor immune response in vivo was significantly enhanced, with statistical significance, while the concentration of the immunosuppressive factor IL-10 in the tumor decreased ( Fig.11 E in.

[0110] These results suggest that APS@PLGA-man can significantly increase the proportion of M1 macrophages and promote the secretion of anti-tumor cytokines.

[0111] Example 14 PANoptosis activation in vivo

[0112] Based on the experimental materials of Example 10, the present invention detected the expression of PANoptosis-related mRNA in tumor tissues by real-time fluorescence quantitative PCR to verify that APS@PLGA-man can promote PANoptosis in tumor cells through macrophage M1 polarization.

[0113] The results are as follows Fig.12 As shown in the figure, compared with the control group, the expression of PANoptosis-related genes in the tumor tissues of mice in the APS@PLGA-man and APS@PLGA-man+αPD-L1 groups was significantly increased. This proves that the synergistic therapy of APS@PLGA-man and APS@PLGA-man+αPD-L1 promoted PANoptosis of lung cancer cells in mice.

[0114] Subsequently, the present invention verified the protein expression of PANoptosis markers in tumor tissues through Western blotting experiments. The results showed that ( Fig.13 ), compared with the control group, APM and APM+αPD-L1 groups can promote the upregulation of protein expression of activation markers pRIPK1 and pMLKL representing necroptosis; upregulation of protein expression of apoptosis markers c-CASP3 and c-CASP8; upregulation of protein expression of N-terminal active fragment of GSDME and c-CASP1, pyroptosis markers. This further proves that APS@PLGA-man and APS@PLGA-man+αPD-L1 can promote PANoptosis in lung cancer.

[0115] Example 15 Biodistribution

[0116] The present invention further uses rhodamine B to label APS@PLGA-man and APS@PLGA to perform in vivo distribution detection and drug metabolism test. The specific process is as follows: Mature LLC tumor-bearing mice were randomly divided into three groups: APS group (1 mg / mL), APS@PLGA+Rhodamine B group (2 mg / mL), and APS@PLGA-man+Rhodamine B group (2 mg / mL). Then, 0.1 mL of different formulations (dissolved in saline) were injected into the tumor. After treatment (0.5, 3, 6, 9, 12, and 24 hours, respectively), the backs of the mice were imaged using an in vivo imaging system (IVISSPECTRUM, PerkinElmer). At the end of treatment (24 hours), the various organs of the mice were dissected and imaged. The results showed that the PLGA material itself has good sustained-release properties, and the benefit is that APS@PLGA-man can specifically bind to CD206 through mannose, so APS@PLGA-man can target tumor-associated macrophages, so APS@PLGA-man can stay in the tumor area of ​​mice for more than 24 hours. The APS@PLGA nanoparticles without amino-polyethylene glycol-mannose are metabolized faster. At 3 hours, the fluorescence intensity of APS@PLGA-man is significantly higher than that of APS@PLGA, and lasts for more than 24 hours ( Fig.14 A and C in the figure). Through in vivo organ imaging, the present invention found that APS@PLGA-man and APS@PLGA are mainly metabolized by the liver and kidney ( Fig.14 B in the figure).

[0117] Example 16 Biosafety Assessment

[0118] The present invention further conducts a biosafety assessment based on the experimental materials of Example 10, which specifically includes the following process: After killing the mice, serum was collected and related indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), creatine kinase (CK), blood glucose (GLU), red blood cells (RBC), white blood cells (WBC), and platelets (PLT) were detected using a small animal blood routine analyzer and an automatic biochemical analyzer to evaluate the health status of the mice.

[0119] The results are as follows Fig.15 As shown in the figure, the results showed that each treatment group would lead to an increase in WBC in mice, which was speculated to be a certain degree of inflammatory response after activating anti-tumor immunity, and had no effect on RBC and PLT; at the same time, it was proved that APS@PLGA-man and APS@PLGA-man combined with αPD-1 did not affect the blood sugar, heart, liver and kidney function of mice. Fig.16 As shown, HE staining showed that APS@PLGA-man and APS@PLGA-man combined with αPD-1 had no obvious organic damage to the main organs of liver, heart, lung, and kidney.

[0120] Overall, the results show that the therapy has good biosafety and compatibility.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tumor vaccine, characterized in that: include: Nanoparticles; The nanoparticles include: a complex of astragalus polysaccharide and PLGA, and amino-polyethylene glycol mannose; the amino-polyethylene glycol mannose is modified on the surface of the PLGA; and the mass ratio of the amino-polyethylene glycol mannose, astragalus polysaccharide and PLGA is 1:(1-5):(10-20).

2. The tumor vaccine according to claim 1, characterized in that The mass ratio of amino-polyethylene glycol mannose, astragalus polysaccharide and PLGA is 1:(1.5-3):(12-16).

3. The tumor vaccine according to claim 1 or 2, characterized in that The astragalus polysaccharide and PLGA complex is APS@PLGA nanoparticles, and the APS@PLGA nanoparticles are prepared by a double emulsification method.

4. The tumor vaccine according to claim 3, characterized in that The double emulsification method comprises: using PVA solution as the external aqueous phase, using PLGA dissolved in an organic solvent as the organic phase, and using APS solution as the internal aqueous phase; The inner aqueous phase and the organic phase are mixed, and colostrum is obtained by ultrasonic treatment in an ice-water bath; the colostrum and the outer aqueous phase are mixed under stirring at room temperature, and then ultrasonically dispersed, stirred, filtered, and centrifuged in an ice-bath to obtain APS@PLGA nanoparticles.

5. The method for preparing the tumor vaccine according to any one of claims 1 to 4, characterized in that: include: After APS@PLGA nanoparticles were activated by NHS and EDC, they were mixed with amino-polyethylene glycol mannose and stirred for more than 8 hours. The particles were filtered, centrifuged to obtain the precipitate, resuspended and freeze-dried.

6. The preparation method according to claim 5, characterized in that: The filtration is performed by filtering through a 0.2-0.3 μm filter membrane; and / or the centrifugation is performed in cold PBS at 18000-22000 g for more than 35 minutes.

7. An anti-tumor drug, characterized in that: The invention comprises the nanoparticles as claimed in any one of claims 1 to 4.

8. The anti-tumor drug according to claim 7, characterized in that: Also includes: Immune checkpoint inhibitors.

9. Use of the nanoparticles according to any one of claims 1 to 4, or the anti-tumor drug according to claim 7 or 8 in preventing or treating cancer.

10. Use of the nanoparticles according to any one of claims 1 to 4 in the preparation of a drug for preventing or treating cancer.

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