Traditional Chinese medicine polysaccharide-mediated tumor treatment nano vaccine and preparation method thereof

Abstract

The invention relates to the technical field of medicines, in particular to a traditional Chinese medicine polysaccharide-mediated tumor treatment nano vaccine and a preparation method thereof, and the nano vaccine comprises the following components: chitosan oligosaccharide as a carrier; a whole cell tumor antigen; traditional Chinese medicine polysaccharide is used as an immunologic adjuvant; wherein the mass ratio of the chitosan oligosaccharide to the whole-cell tumor antigen to the traditional Chinese medicine polysaccharide is (0.5-2): (3-6): (3-6); the traditional Chinese medicine polysaccharide is selected from one or more of ginseng polysaccharide, ganoderma lucidum polysaccharide, astragalus polysaccharide, lycium barbarum polysaccharide, salvia miltiorrhiza polysaccharide and atractylodes macrocephala polysaccharide, and the content of mannose in the polysaccharide is not lower than 3 mu g / mg. The tumor growth can be obviously inhibited, the generation of far-end lung metastasis can be effectively inhibited, and the curative effect is obviously enhanced compared with the treatment by purely using the whole-cell tumor antigen.

Description

Technical Field

[0001] The invention relates to the field of medical technology, in particular to a traditional Chinese medicine polysaccharide-mediated tumor therapeutic nano vaccine and a preparation method thereof. Background Art

[0002] Tumor vaccines play an important role in the prevention and treatment of tumors. By activating the body's immune system, tumor vaccines can promote the formation of anti-tumor immune memory, thereby preventing the occurrence and recurrence of tumors, and are expected to induce tumor regression. At present, the tumor vaccines approved for marketing include two types: preventive and therapeutic. The therapeutic types are usually divided into RNA vaccines, antigen peptide vaccines, and whole tumor cell vaccines. Today, RNA vaccines and antigen peptide vaccines are most widely used in the research of personalized cancer vaccines and new immunotherapies, showing strong development potential. Whole tumor cell vaccines can provide a wide range of antigen coverage and stimulate a variety of immune responses, but they face challenges such as rapid antigen degradation, low delivery efficiency, and immunosuppression in the tumor microenvironment, making them difficult to develop.

[0003] Traditional immune adjuvants (such as aluminum salts and TLR agonists) are difficult to solve the above problems at the same time: aluminum adjuvants only enhance Th2 responses and are ineffective in activating cytotoxic T cells; TLR agonists (such as CpG) are prone to induce systemic inflammatory responses; and cationic liposome delivery systems are easily encapsulated and inactivated by serum proteins. Therefore, in view of the above status quo, it is urgent to develop a tumor treatment nanovaccine mediated by traditional Chinese medicine polysaccharides and its preparation method to overcome the shortcomings in current practical applications. Summary of the invention

[0004] The purpose of the present invention is to provide a traditional Chinese medicine polysaccharide-mediated tumor therapeutic nano vaccine and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions: A traditional Chinese medicine polysaccharide-mediated tumor treatment nano vaccine, comprising the following components: Chitosan oligosaccharide as a carrier; whole cell tumor antigen; Chinese herbal polysaccharides as immune adjuvants; Wherein, the mass ratio of the chitosan oligosaccharide, the whole cell tumor antigen and the traditional Chinese medicine polysaccharide is (0.5-2): (3-6): (3-6); The Chinese medicine polysaccharide is selected from one or more of ginseng polysaccharide, ganoderma lucidum polysaccharide, astragalus polysaccharide, wolfberry polysaccharide, salvia miltiorrhiza polysaccharide or atractylodes macrocephala polysaccharide, and the content of mannose in the polysaccharide is not less than 3 μg / mg.

[0006] As a further embodiment of the present invention: the molecular weight of the chitosan oligosaccharide is 1 kDa-10 kDa.

[0007] As a further aspect of the present invention: the whole cell tumor antigen is selected from one or more of the 4T1 breast cancer cell line, B16 melanoma cell line, LLC Lewis lung cancer cell line or CT26 colon cancer cell line.

[0008] A method for preparing a tumor treatment nano-vaccine mediated by traditional Chinese medicine polysaccharide according to the above, comprising the following steps: Step 1, provide a whole cell tumor antigen, which is prepared by photodynamic therapy, freeze-thaw lysis, irradiation inactivation, chemical inactivation or heat shock treatment; Step 2, mix the chitosan oligosaccharide solution with the whole cell tumor antigen solution, and magnetically stir at room temperature to self-assemble into a core-shell structure; Step 3, add the traditional Chinese medicine polysaccharide solution dropwise to the core-shell structure, and continue magnetic stirring to obtain a nano-vaccine suspension; Step 4, filter the nano-vaccine suspension through a 0.45 μm filter membrane to obtain the tumor treatment nano-vaccine.

[0009] As a further aspect of the present invention: in step 1, the steps for preparing the whole cell tumor antigen by photodynamic therapy include: Inoculate tumor cells in a culture plate and culture overnight; Treat the cells with an IR780 solution at a concentration of 100 μg / mL, after incubating for 2 hours, irradiate with an 808 nm laser for 5 minutes; Continue to culture for 24 hours, collect the cell lysate, and freeze-dry after centrifugation to obtain the tumor cell lysate.

[0010] As a further aspect of the present invention: in step 1, the steps for preparing the whole cell tumor antigen by freeze-thaw lysis include: Digest the tumor cells and adjust the density to 1×10 7 cells / mL, and aliquot into cryopreservation tubes; Place the cryopreservation tubes in liquid nitrogen for 5 minutes, then quickly transfer them to a 37°C water bath and heat for 5 minutes, and repeat the freeze-thaw cycle 5 times; Collect the supernatant after centrifugation, detect the protein content and store it.

[0011] As a further aspect of the present invention: in step 1, the steps for preparing the whole cell tumor antigen by irradiation inactivation include: Place the tumor cell suspension in an irradiation device and irradiate with a radiation dose of 50-200 Gy; After irradiation, adjust the cell concentration and directly use it as a vaccine or freeze and store it.

[0012] As a further solution of the present invention: In step 1, the steps of preparing whole-cell tumor antigen by chemical inactivation method include: Treat tumor cells with 10 μg / mL mitomycin C for 2 hours; After washing to remove residual chemicals, concentrate the cells and store them.

[0013] As a further solution of the present invention: In step 1, the steps of preparing whole-cell tumor antigen by heat shock treatment method include: Incubate the tumor cell suspension in a 37 °C water bath for 30 - 60 minutes, then quickly transfer it to an ice bath for cooling, and repeat this process 2 - 3 times; Directly use it as a vaccine or freeze it for storage.

[0014] An application of the tumor treatment nano-vaccine mediated by traditional Chinese medicine polysaccharide as described above in the preparation of anti-tumor drugs, wherein the drugs are used to treat in-situ tumors, recurrent tumors or metastatic tumors.

[0015] Compared with the prior art, the beneficial effects of the present invention are: The present invention provides a DCs-targeted whole-cell tumor nano-vaccine. By using effectively inactivated tumor cells as whole-cell tumor antigens, it self-assembles with chitosan oligosaccharide and immune adjuvant traditional Chinese medicine polysaccharide to form a nano-vaccine. This nano-vaccine is self-assembled by electrostatic adsorption, with a simple preparation process and low cost, having high operability and economy. Compared with traditional whole-cell tumor antigen vaccines, the nano-vaccine of the present invention can effectively target dendritic cells (DCs), promote the maturation of DCs and enhance their anti-tumor immune function. This mechanism not only overcomes the problem of weak immunogenicity of traditional whole-cell tumor antigens, but also significantly improves the intensity and persistence of immune responses. In addition, the experimental results of the whole-cell tumor nano-vaccine of the present invention in in-situ tumor, recurrent tumor and metastatic tumor mouse models show that it can significantly inhibit tumor growth and effectively inhibit the occurrence of distal lung metastasis. Compared with the treatment with only whole-cell tumor antigens, its curative effect has been significantly enhanced. Therefore, the present invention not only provides a new drug option for clinical tumor prevention and treatment, but also provides a new direction for the innovation of tumor treatment strategies, having strong clinical application prospects. Description of the Drawings

[0016] Figure 1 It is a schematic diagram of the transmission electron microscope (TEM) characterization of the tumor nano-vaccine in Example 1 of the present invention.

[0017] Figure 2 It is a growth curve graph of the tumor nano-vaccine for treating in-situ, recurrent and metastatic tumors in Test Example 1 of the present invention; Among them, A is the growth curve of in-situ tumors, B is the growth curve of recurrent tumors, and C is the growth curve of metastatic tumors.

[0018] Figure 3 This is a schematic diagram for the efficacy evaluation of the tumor nano-vaccine in treating in-situ, recurrent, and metastatic tumors in Test Example 1 of the present invention; Among them, A is 4T1 breast cancer, B is LLC lung cancer, and C is CT26 colon cancer.

[0019] Figure 4 This is a schematic diagram for the evaluation of the effect of the tumor nano-vaccine on the cytokine TNF-α in serum in Test Example 2 of the present invention; Among them, A is 4T1 breast cancer, B is LLC lung cancer, and C is CT26 colon cancer.

[0020] Figure 5 This is a schematic diagram for the evaluation of the effect of the tumor nano-vaccine on the cytokine IL-6 in serum in Test Example 2 of the present invention; Among them, A is 4T1 breast cancer, B is LLC lung cancer, and C is CT26 colon cancer. Detailed implementation manners

[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0022] The following describes the specific implementation of the present invention in detail in conjunction with specific embodiments.

[0023] Please refer to Figures 1 - 5 , to solve the technical bottleneck that the effect of whole-cell tumor vaccines is limited in tumor prevention and treatment, this study developed a whole-cell tumor nano-vaccine that can target dendritic cells (DCs). This vaccine significantly enhances the prevention and treatment effects of tumors by efficiently activating immune cells and inducing long-term immune memory, and has a significant inhibitory effect on the growth of in-situ tumors, recurrent tumors, and metastatic tumors.

[0024] Specifically, the present invention provides a DCs-targeted tumor nano-vaccine, which mainly comprises the following components: chitosan oligosaccharide as a carrier, whole-cell tumor antigen, and traditional Chinese medicine polysaccharide as an immune adjuvant, self-assembling to form a whole-cell tumor nano-vaccine. The selection of the whole-cell tumor antigen takes into account the adaptability of the experimental platform, the research needs of immune responses, as well as the convenience and cost of tumor sample sources. Specifically, in order to ensure the diversity and extensiveness of experimental research, the present invention selects multiple tumor cell lines as the whole-cell tumor antigen, including 4T1 breast cancer cell line, B16 melanoma cell line, LLC Lewis lung cancer cell line, and CT26 colon cancer cell line, ensuring that the experimental design can cover different types of tumors and has good applicability and operability.

[0025] Among them, the molecular weight of chitosan oligosaccharide is 1 kDa - 10 kDa; The preparation method of the whole-cell tumor antigen includes: 1. Preparation of antigen by photodynamic therapy: Inoculate tumor cells at a density of 1×10 5 cells per well in a 24-well plate and culture overnight. After cell adhesion, aspirate the culture medium, treat the cells with 1 mL of IR780 solution (dissolved in PBS) at a concentration of 100 μg / mL, incubate for 2 h, then irradiate with an 808 nm (2.0 W / cm 2 ) laser for 5 min, continue to culture for 24 h, transfer the cell lysate suspension to a 15 mL centrifuge tube, centrifuge for 15 min, collect the supernatant, and lyophilize the supernatant to obtain tumor cell lysate (TCL).

[0026] 2. Preparation of antigen by freeze-thaw lysis method: Culture tumor cells in a complete medium containing serum and double antibodies in a 37°C, 5% CO2 constant temperature incubator until the confluence is about 80%, digest with 0.25% trypsin for 1 - 2 minutes, and add medium containing serum to terminate digestion. Wash with PBS to remove residual medium, adjust the cell density to 1×10 7 cells / mL after cell counting, aliquot 1 mL into each cryopreservation tube; place the cryopreservation tube in liquid nitrogen for 5 min first, then quickly place it in a 37°C water bath for 5 min, and repeat freeze-thawing 5 times; transfer the liquid in the cryopreservation tube to a centrifuge tube, centrifuge at 4°C, 2000×g for 15 min; collect the supernatant, detect the protein content of the supernatant with a BCA kit, and store the supernatant at -20°C for short-term or -80°C for long-term.

[0027] 3. Preparation of antigen by irradiation inactivation method: Culture tumor cells until 80% - 90% confluence, wash with PBS, and then mix the tumor cells with 1×10 7 cells / mL, place the cell suspension in an irradiation device (such as 60In a Co source or X-ray device, irradiation is carried out using radiation with a dose of 50 - 200 Gy. After irradiation, the cells lose their ability to divide, but most of their membrane antigens and internal antigens are retained, which can effectively activate the immune system. Subsequently, the inactivated cells are adjusted to an appropriate concentration and directly prepared into a vaccine or cryopreserved for later use.

[0028] 4. Preparation of antigen by chemical inactivation method: After culturing tumor cells until 80% - 90% fusion, add 10 μg / mL of mitomycin C inactivation reagent to treat the cells for 2 hours. After treatment, wash the cells thoroughly with PBS to remove residual chemicals, and then concentrate the cells and directly use them as a vaccine or freeze them for later use.

[0029] 5. Preparation of antigen by heat shock treatment method: Adjust the concentration of the cultured tumor cells to 1×10 7 cells / mL, incubate them in a 37°C water bath for 30 - 60 minutes, then quickly transfer them to an ice bath for cooling, and repeat this process 2 - 3 times, and directly prepare them into a vaccine or cryopreserve them for later use.

[0030] Traditional Chinese medicine polysaccharides include traditional Chinese medicine polysaccharides such as ginseng polysaccharide, ganoderma lucidum polysaccharide, astragalus polysaccharide, wolfberry polysaccharide, salvia miltiorrhiza polysaccharide, and atractylodes macrocephala polysaccharide. The content of mannose in the polysaccharide should not be less than 3 μg / mg. Among them, the main components of the monosaccharide composition of traditional Chinese medicine include but are not limited to mannose, galactose, rhamnose, arabinose, fucose, and glucuronic acid, etc., which are immune adjuvants for whole-cell tumor nano-vaccines.

[0031] The mass ratio of chitosan oligosaccharide, whole-cell tumor antigen, and traditional Chinese medicine polysaccharide is (0.5 - 2):(3 - 6):(3 - 6).

[0032] In an embodiment of the present invention, when the traditional Chinese medicine polysaccharide is ganoderma lucidum polysaccharide, the preparation process of the DCs-targeted whole-cell tumor vaccine is self-assembly. Specifically: Add 4.0 mL of 1mg / mL COS solution to a vial, drop 1mL of 1mg / mL TCL solution into the COS solution, stir magnetically at room temperature for 60 min (600 rpm) to form a TCL-COS core-shell structure by self-assembly. After stopping stirring, add 4.0 mL of 1mg / mL GLP solution dropwise to the vial, stir magnetically at room temperature for 60 min (600 rpm), and size the nano-solution through a 0.45μm filter to prepare the GLP-COS-TCL (CGT) nano-vaccine. Prepare the GLP-COS-FOVA nano-vaccine in a similar method.

[0033] The application of the above DCs-targeted whole-cell tumor nano-vaccine in the preparation of tumor prevention and treatment drugs.

[0034] The present invention uses tumor cells treated by photothermal action as tumor antigens, and through the principle of electrostatic adsorption with chitosan oligosaccharide and traditional Chinese medicine polysaccharides, constructs a class of self-assembled DCs-targeted whole-cell tumor nano-vaccines. After subcutaneous injection, this nano-vaccine can be targeted and delivered to dendritic cells (DCs). In the lymph nodes (LNs), the nano-vaccine is internalized by DCs, promoting the maturation of DCs. The mature DCs present the tumor antigens on the cell surface for naive T cells to recognize, and further activate the differentiation of these T cells into cytotoxic T lymphocytes, ultimately triggering a potent anti-tumor immune response.

[0035] The whole-cell tumor nano-vaccine of the present invention can target DCs, promote the maturation of DCs and trigger a potent anti-tumor immune effect, and has significant therapeutic effects on in-situ, recurrent and metastatic tumors. Example

[0036] The preparation method of tumor cell lysate as an antigen is as described above. Add 4 mL of 1 mg / mL chitosan oligosaccharide solution into a vial, drop 1 mL of 1 mg / mL TCL solution into the chitosan oligosaccharide solution, stir magnetically at room temperature for 60 min (600 rpm) to form a core-shell structure by self-assembly. After stopping stirring, add 2 mL of 1 mg / mL Ganoderma lucidum polysaccharide solution dropwise into the vial, stir magnetically at room temperature for 60 min (600 rpm), and then size the nano-solution through a 0.45 μm filter to prepare the nano-vaccine. Example

[0037] The difference from Example 1 is that the immune adjuvant is ginseng polysaccharide. Example

[0038] The difference from Example 1 is that the immune adjuvant is astragalus polysaccharide. Example

[0039] The difference from Example 1 is that the immune adjuvant is wolfberry polysaccharide. Example

[0040] The difference from Example 1 is that the immune adjuvant is seaweed polysaccharide. Example

[0041] The difference from Example 1 is that the immune adjuvant is jujube polysaccharide. Example

[0042] The difference from Example 1 is that the immune adjuvant is lily polysaccharide. Example

[0043] The difference from Example 1 is that the immune adjuvant is chrysanthemum polysaccharide. Example

[0044] Different from Example 1, the immune adjuvant is Salvia miltiorrhiza polysaccharide. Example

[0045] Different from Example 1, the immune adjuvant is Atractylodes macrocephala polysaccharide.

[0046] Experimental example, single-factor experiment: This experiment investigated three factors: the dosage of antigen, the dosage of carrier, and the dosage of immune adjuvant. In order to observe the influence trend of a single factor on the particle size and considering that there is almost no interaction between factors, the method of single-factor analysis was finally selected for the experiment.

[0047] Experimental method: The preparation method of tumor cell lysate as antigen was as described above. Add a certain amount of 1 mg / mL chitosan oligosaccharide solution into a vial, drop a certain amount of 1 mg / mL TCL solution into the chitosan oligosaccharide solution, stir magnetically at room temperature for 60 min (600 rpm) to self-assemble into a core-shell structure. After stopping stirring, drop a certain amount of 1 mg / mL Ganoderma lucidum polysaccharide solution into the vial, stir magnetically at room temperature for 60 min (600 rpm), and then size the nano-solution through a 0.45 μm filter to obtain the nano-vaccine. The particle size of the above tumor nano-vaccine was characterized by a particle size analyzer, and the experimental results are shown in Table 1.

[0048] Table 1 Particle size results of different ratios Group Ratio (antigen ∶ carrier ∶ Chinese herbal polysaccharide) Particle size (nm) 1 0.5∶3∶5 431.8±2.7 2 0.5∶3∶6 397.2±6.5 3 0.5∶4∶3 381.5±7.4 4 0.5∶4∶5 182.0±5.6 5 0.5∶4∶6 173.9±4.9 6 0.5∶5∶3 176.9±3.2 7 0.5∶5∶4 213.8±4.3 8 0.5∶5∶6 206.8±5.1 9 0.5∶6∶3 235.2±6.5 10 0.5∶6∶4 410.0±5.6 11 0.5∶6∶5 414.3±6.1 12 0.5∶6∶6 417.9±7.1 13 1∶3∶5 439.5±4.9 14 1∶3∶6 416.4±2.3 15 1∶4∶3 277.3±8.0 16 1∶4∶5 199.6±5.3 17 1∶4∶6 195.2±4.6 18 1∶5∶3 202.7±6.3 19 1∶5∶4 282.6±5.9 20 1∶5∶6 418.4±3.9 21 1∶6∶3 313.7±7.3 22 1∶6∶4 323.6±2.0 23 1∶6∶5 334.8±6.0 24 1∶6∶6 340.1±8.2 25 2∶3∶5 435.4±1.8 26 2∶3∶6 469.5±5.2 27 2∶4∶3 299.3±2.7 28 2∶4∶5 212.7±8.3 29 2∶4∶6 245.2±3.7 30 2∶5∶3 233.6±6.9 31 2∶5∶4 308.4±2.8 32 2∶5∶6 364.5±5.1 33 2∶6∶3 351.1±4.1 34 2∶6∶4 364.7±7.5 35 2∶6∶5 381.2±4.5 36 2∶6∶6 388.8±4.7 The particle size results are shown in Table 1. The particle sizes of the obtained tumor nano-vaccines are all between 173.9 nm and 469.5 nm, and the morphological characterization results are as Figure 1 shown. The results show that the tumor nano-vaccines are spherical.

[0049] Test example 1: Pharmacodynamic evaluation of tumor nano-vaccine for preventing dryness-related drug-resistant tumors; 1. Antigen-site tumor pharmacodynamic experiment on 4T1 tumor-bearing mouse model; Digest the 4T1 cells in the logarithmic growth phase and prepare a cell suspension of 1×10 7 cells / mL. Inject 100 μL of the cell suspension into the right back of each mouse. Establish a 4T1 orthotopic tumor model. When the volume of the orthotopic tumor increases to 50 - 100 mm 3When the tumor-bearing mice were randomly divided into 3 groups (n = 5): (1) PBS group, (2) free TCL (200 μg / mL), (3) COS-GLP-TCL (TCL: 200 μg / mL, GLP: 400 μg / mL). Subcutaneous administration of 200 μL (right abdomen) every 2 days for a total of 3 times. On the 18th day after administration, blood was collected by eye socket puncture to obtain mouse blood samples. The mice were sacrificed, and the tumors, hearts, livers, spleens, lungs, kidneys, and lymph nodes of the mice were collected. The anti-tumor effect of the CGT nano-vaccine was evaluated by monitoring the growth of the primary tumor. The in-situ tumor growth curves of the mice in each group during the treatment period were as Figure 2 shown in A. The in-situ tumors in the PBS group and the free TCL group showed a rapid growth trend. It indicated that the free whole tumor cell antigen had no inhibitory effect on the in-situ tumors of tumor-bearing mice. While the growth of the in-situ tumors in the CGT group of tumor-bearing mice was significantly inhibited. After 18 days of treatment, the tumor inhibition rate of the in-situ tumors in the free TCL group was 19.09%, and the tumor inhibition rate of the in-situ tumors in the CGT group was 77.92%. CGT had a good inhibitory effect on the 4T1 in-situ tumors.

[0050] 2. Anti-relapse tumor pharmacodynamic experiment on the 4T1 tumor-bearing mouse model; SPF-grade 6-8-week-old female Balb / c mice were selected and randomly divided into 3 groups (n = 5): (1) PBS group, (2) free TCL (200 μg / mL), (3) COS-GLP-TCL (TCL: 200 μg / mL, GLP: 400 μg / mL). The drug was subcutaneously injected into the right abdomen of the mice, which was recorded as one immunization. Immunization was carried out once every 2 days for a total of 3 times. 7 days after the last immunization, 4T1 cells were subcutaneously inoculated into the mice to simulate tumor recurrence. When the tumor volume in the PBS group increased to 30 - 50 mm 3 it was counted as day 0, and the tumors of the mice were measured every 2 days. On the 18th day, blood was collected by eye socket puncture to obtain mouse blood samples. The mice were sacrificed, and the tumors, hearts, livers, spleens, lungs, kidneys, and lymph nodes of the mice were collected. The recurrence tumor growth curves of the mice in each group during the treatment period were as Figure 2 shown in B. The in-situ tumors in the PBS group and the free TCL group showed a rapid growth trend. It indicated that the free whole tumor cell antigen had no inhibitory effect on the in-situ tumors of tumor-bearing mice. While the growth of the recurrence tumors in the CGT group of tumor-bearing mice was significantly inhibited. After 18 days of treatment, the tumor inhibition rate of the in-situ tumors in the free TCL group was 18.79%, and the tumor inhibition rate of the recurrence tumors in the CGT group was 81.68%. CGT had a good inhibitory effect on the 4T1 recurrence tumors.

[0051] 3. Anti-metastatic tumor pharmacodynamic experiment on the 4T1 tumor-bearing mouse model; SPF-grade 8-week-old female Balb / c mice were randomly divided into 3 groups (n = 5): (1) PBS group, (2) free TCL (200 μg / mL), and (3) CGT (TCL: 200 μg / mL, GLP: 400 μg / mL). The drugs were subcutaneously injected into the right abdomen of the mice, which was recorded as one immunization. Immunization was performed once every 2 days for a total of 3 times. 7 days after the last immunization, 4T1 cells were subcutaneously inoculated into the mice to simulate tumor metastasis. When the tumor volume in the PBS group increased to 30 - 50 mm 3 it was counted as day 0, and the tumors of the mice were measured every 2 days. On the 18th day, blood was collected by eye enucleation to obtain mouse blood samples. The mice were sacrificed, and the tumors, heart, liver, spleen, lungs, kidneys, and lymph nodes of the mice were collected. The growth curves of metastatic tumors in each group of mice during the treatment period were as Figure 2 shown in Figure C. The in-situ tumors in the PBS group and free TCL both showed a rapid growth trend. It indicated that free whole tumor cell antigen had no inhibitory effect on metastatic tumors in tumor-bearing mice. While the growth of in-situ tumors in tumor-bearing mice in the CGT group was significantly inhibited. After 18 days of treatment, the tumor inhibition rate of metastatic tumors in the free TCL group was 19.45%, and that in the CGT group was 91.31%. CGT had a good inhibitory effect on 4T1 metastatic tumors.

[0052] 4. Anti-lung metastatic tumor pharmacodynamic experiment of 4T1 tumor-bearing mouse model; The excised lung tissues of each group of mice after treatment were as Figure 3 shown. In the in-situ, recurrence, and metastasis model mice, a large number of lung metastases were observed in the control group (PBS). The number of lung metastases in the TCL group decreased slightly, while almost no metastases were observed in the CGT group, indicating that the CGT nano-tumor vaccine strategy had a significant inhibitory effect on tumor cell metastasis and had a good effect on in-situ, recurrence, and metastasis tumor models.

[0053] Test Example 2: Cytokines in serum; ELISA kits were used to detect the levels of IL-6 and TNF-α in serum. TNF-α is a natural immune serum mediator that can cause tumor hemorrhagic necrosis, while IL-6 is a key regulatory factor for tumor accumulation and activation and is also a factor that promotes the survival, proliferation, metastasis, and invasion of tumor cells. As Figure 4 and Figure 5 shown, compared with the normal saline group and TCL, CGT promoted the secretion of TNF-α in the peripheral blood of mice and reduced the secretion of IL-6 in the peripheral blood of mice.

[0054] It should be noted that in the present invention, it should be understood that although this specification is described according to the implementation manners, not every implementation manner only contains an independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other implementation manners that can be understood by those skilled in the art.

Claims

1. A tumor treatment nano-vaccine mediated by Chinese herbal polysaccharides, characterized in that, It comprises the following components: Chitosan oligosaccharide as a carrier; Whole-cell tumor antigen; Traditional Chinese medicine polysaccharide as an immune adjuvant; Among them, the mass ratio of the chitosan oligosaccharide, the whole-cell tumor antigen, and the traditional Chinese medicine polysaccharide is (0.5 - 2):(3 - 6):(3 - 6); The traditional Chinese medicine polysaccharide is selected from one or more of ginseng polysaccharide, ganoderma polysaccharide, astragalus polysaccharide, wolfberry polysaccharide, salvia polysaccharide, or atractylodes polysaccharide, and the content of mannose in the polysaccharide is not less than 3 μg / mg.

2. The nano-vaccine for tumor treatment mediated by traditional Chinese medicine polysaccharide according to claim 1, wherein The molecular weight of the chitosan oligosaccharide is 1 kDa - 10 kDa.

3. The nano-vaccine for tumor treatment mediated by traditional Chinese medicine polysaccharide according to claim 1, characterized in that The whole-cell tumor antigen is selected from one or more of the 4T1 breast cancer cell line, B16 melanoma cell line, LLC Lewis lung cancer cell line, or CT26 colon cancer cell line.

4. A preparation method of a traditional Chinese medicine polysaccharide-mediated tumor treatment nano-vaccine according to any one of claims 1-3, characterized in that, It includes the following steps Step 1: Provide a whole-cell tumor antigen, which is prepared by photodynamic therapy, freeze-thaw lysis, irradiation inactivation, chemical inactivation, or heat shock treatment; Step 2: Mix the chitosan oligosaccharide solution with the whole-cell tumor antigen solution, and magnetically stir at room temperature to self-assemble into a core-shell structure; Step 3: Dropwise add the traditional Chinese medicine polysaccharide solution to the core-shell structure, and continue magnetic stirring to obtain a nano-vaccine suspension; Step 4: Filter the nano-vaccine suspension through a 0.45 μm filter membrane to size the particles and obtain the tumor treatment nano-vaccine.

5. The preparation method of the nano-vaccine for tumor treatment mediated by traditional Chinese medicine polysaccharide according to claim 4, wherein, In Step 1, the steps for preparing the whole-cell tumor antigen by photodynamic therapy include: Inoculate tumor cells in a culture plate and culture overnight; Treat the cells with an IR780 solution at a concentration of 100 μg / mL, after incubating for 2 hours, irradiate with an 808 nm laser for 5 minutes; Continue to culture for 24 hours, collect the cell lysate, and freeze-dry after centrifugation to obtain the tumor cell lysate.

6. The preparation method of the traditional Chinese medicine polysaccharide-mediated tumor treatment nano-vaccine according to claim 4, characterized in that, In Step 1, the steps for preparing the whole-cell tumor antigen by freeze-thaw lysis include: Digest the tumor cells and adjust the density to 1×10 7 cells / mL, and aliquot them into cryotubes; Place the cryotube in liquid nitrogen for 5 minutes, then quickly transfer it to a 37°C water bath and heat for 5 minutes, and repeat the freeze-thaw cycle 5 times; Collect the supernatant after centrifugation, detect the protein content and store it.

7. The preparation method of the nano-vaccine for tumor treatment mediated by traditional Chinese medicine polysaccharide according to claim 4, characterized in that, In Step 1, the steps for preparing the whole-cell tumor antigen by irradiation inactivation include: Place the tumor cell suspension in an irradiation device and irradiate with a radiation dose of 50 - 200 Gy; After irradiation, adjust the cell concentration and use it directly as a vaccine or freeze and store it.

8. The preparation method of the nano-vaccine for tumor treatment mediated by traditional Chinese medicine polysaccharide according to claim 4, characterized in that, In Step 1, the steps for preparing the whole-cell tumor antigen by chemical inactivation include: Treat tumor cells with 10 μg / mL of mitomycin C for 2 hours; Wash to remove residual chemicals, concentrate the cells and store them.

9. The preparation method of the traditional Chinese medicine polysaccharide-mediated tumor treatment nano-vaccine according to claim 4, wherein, In Step 1, the steps for preparing the whole-cell tumor antigen by heat shock treatment include: Place the tumor cell suspension in a 37°C water bath and incubate for 30 - 60 minutes, then quickly transfer it to an ice bath to cool, and repeat 2 - 3 times; Use it directly as a vaccine or freeze and store it.

10. Use of the traditional Chinese medicine polysaccharide-mediated tumor treatment nano-vaccine according to any one of claims 1-3 in the preparation of anti-tumor drugs, characterized in that, The drug is used to treat in-situ tumors, recurrent tumors, or metastatic tumors.

Images