Tumor vaccine adjuvant and application thereof

Abstract

The invention belongs to the technical field of nano medicine, and particularly relates to a tumor vaccine adjuvant and application thereof. The tumor vaccine adjuvant is a ferrite particle adjuvant prepared by reacting ferrite nanoparticles with an organic polymer in a mass ratio of 1: (1-200) at 50-60 DEG C. The invention provides a novel tumor vaccine adjuvant, which can enhance the stimulation and activation of a tumor vaccine on dendritic cells, efficiently activate anti-tumor immune cells, and improve the tumor immunity of the tumor vaccine. The immune response of an organism to an antigen is improved, and the treatment effect of the vaccine is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomedicine, and in particular relates to a tumor vaccine adjuvant and application thereof. Background Art

[0002] Cancer is one of the leading causes of death. With the continuous advancement of medical technology, tumor immunotherapy, as a new generation of treatment after surgery, radiotherapy and chemotherapy, has shown broad prospects for clinical application. Among them, tumor vaccines, as an important immunotherapy strategy, activate the autoimmune system and induce specific immune responses by introducing inactivated tumor cells or fragments containing tumor-associated antigens or tumor-specific antigens into the body, thereby achieving effective clearance of tumor cells. However, the immunogenicity and specificity of current tumor vaccines are weak, and the stimulation of antigen-presenting cells (such as dendritic cells) is insufficient, which limits the cross-presentation of antigens and the activation of T cell immunity, resulting in a low response rate of patients in clinical trials. These bottlenecks have seriously hindered the clinical transformation and promotion of tumor vaccines.

[0003] Adjuvants, as key components of vaccines, are usually added to enhance the immunogenicity and immune response of vaccines. According to their mechanism of action, adjuvants can be roughly divided into two categories: ① immunopotentiators, including pathogen-associated molecular patterns, damage-associated molecular patterns, and chemically synthesized small molecule Toll-like receptor (TLR) agonists. They mainly promote the maturation and activation of antigen-presenting cells by targeting TLRs and other pattern recognition receptors, thereby enhancing the production of antigen presentation signals and co-stimulatory signals, and ultimately stimulating a stronger adaptive immune response; ② delivery vehicles, such as lipid nanoparticles and encapsulated protein nanoparticles, are mainly used to improve the stability and delivery efficiency of antigens. They can bind to antigens, prevent antigens from being degraded by enzymes, and achieve slow release of antigens, thereby prolonging the retention time of antigens in the body and improving their bioavailability. In addition, delivery vehicles can more efficiently deliver antigens to antigen-presenting cells by mimicking the size and spatial structure of pathogens, or directly targeting specific receptors on the surface of antigen-presenting cells, thereby promoting cross-presentation of antigens and activation of immune responses. By rationally designing and optimizing adjuvants, the immune efficacy of tumor vaccines can be significantly improved, providing a new solution to overcome the challenge of weak immunogenicity of current tumor vaccines.

[0004] At present, most commercial vaccines, such as the COVID-19 vaccine and HPV vaccine, are prevented by inducing B cell-mediated humoral immunity. The neutralizing antibodies produced in this immune process can bind to and eliminate invading exogenous pathogens and produce immune memory. Usually, these vaccines are used in combination with vaccine adjuvants to enhance the immune response and improve the overall efficacy of the vaccine. However, the formation and development of tumors mainly come from the mutation of proto-oncogenes and tumor suppressor genes in cells, which cannot be recognized and eliminated by humoral immune responses, but can be killed by T cell-mediated anti-tumor cell immunity. Aluminum adjuvants (such as aluminum hydroxide and aluminum phosphate) that are widely used in clinical practice mainly enhance humoral immunity by promoting the sustained release of antigens and stimulating inflammatory responses, but their ability to induce cellular immunity is weak and it is difficult to effectively activate T cell killing. Therefore, for tumor vaccines, there is an urgent need to develop adjuvants that can target antigen-presenting cells, enhance the immunogenicity of tumor vaccines, promote anti-tumor cell immunity, and have good safety, so as to provide a more effective strategy for tumor immunotherapy. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a tumor vaccine adjuvant and application thereof.

[0006] A tumor vaccine adjuvant is a ferrite particle adjuvant prepared by reacting ferrite nanoparticles and an organic polymer at a mass ratio of 1:1 to 200 at 50°C to 60°C for 2 h to 12 h; The ferrite nanoparticles are Fe 3 O 4 Or metal-doped Fe 3 O 4 ; The organic polymer is any one of polyethylene glycol, polyacrylic acid, polyvinyl pyrrolidone, citric acid, 3-(3,4-dihydroxyphenyl)propionic acid, 2,3-dimercaptosuccinic acid, mannose and 3,4-dihydroxyhydrocinnamic acid.

[0007] Preferably, the size of the ferrite nanoparticles is 2 nm to 500 nm.

[0008] Preferably, the metal-doped Fe 3 O 4 MnFe 2 O 4 、ZnFe 2 O 4 、CoFe 2 O 4 MgFe 2 O 4 .

[0009] Preferably, the ferrite particle adjuvant targeting dendritic cells is obtained by modifying the ferrite particle adjuvant with mannose.

[0010] The application of the tumor vaccine adjuvant in the preparation of tumor vaccines.

[0011] Preferably, the method for preparing the tumor vaccine is: the ferrite particle adjuvant and the tumor antigen are mixed in a volume ratio of 1:1-5 to obtain the tumor vaccine.

[0012] Preferably, the tumor is any one of liver cancer, melanoma, colorectal cancer, pancreatic cancer, gastric cancer and breast cancer.

[0013] Preferably, the tumor antigen is a tumor cell autoantigen or a tumor cell formed after tumor cell inactivation and its lysate.

[0014] Preferably, the tumor cell is any one of Hepa1-6 cells, 4T1 cells, Pan02 cells and LLC cells.

[0015] Preferably, the tumor vaccine is any one of whole tumor cell vaccine, dendritic cell vaccine, mRNA vaccine, and peptide vaccine.

[0016] Compared with the prior art, the present invention is beneficial in that: The present invention provides a new type of tumor vaccine particle adjuvant, which can efficiently stimulate and activate dendritic cells, thereby activating immune cells, enhancing the body's immune response to antigens, and improving vaccine efficacy. The ferrite nanoparticles used in the present invention have excellent biosafety and biodegradability, ensuring their good metabolic properties in the body. At the same time, these nanoparticles can be used as vaccine adjuvants to promote the processing and presentation of tumor antigens by dendritic cells, further enhancing the immune effect of tumor vaccines. Compared with traditional vaccine adjuvants, the ferrite nanoparticles provided by the present invention are expected to overcome clinical challenges such as low immunogenicity of tumor vaccine antigens and weak induced immune responses, and provide a more efficient, safe and more selective adjuvant system for the research and development of tumor vaccines, which will help the breakthrough development of tumor immunotherapy.

[0017] The present invention found that ferrite nanoparticles can directly activate dendritic cells, increase their activation level, and further promote maturation after binding to antigens. In vivo experiments have shown that the adjuvant can enhance the immune effect mediated by tumor antigens, significantly inhibit tumor growth, and improve the anti-tumor efficacy of vaccines. In addition, the synthesis strategy of the ferrite nanoparticles of the present invention is simple, providing a safe and efficient adjuvant solution for vaccine development, which is expected to break through the bottleneck of low immunogenicity and weak induced immune response of existing tumor vaccines.

[0018] The experiment further confirmed that the ferrite particle adjuvant can promote the maturation of dendritic cells alone and significantly enhance their immunogenicity after binding to tumor antigens, thereby effectively enhancing the anti-tumor effect of tumor vaccines and significantly inhibiting tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the transmission electron micrograph of the prepared ferrite particle adjuvant.

[0020] Figure 2 This is a graph showing the cytotoxicity results of ferrite particle adjuvants.

[0021] Figure 3 Figure 1 is a quantitative result of ferrite particle adjuvant stimulating BMDC activation, where A is the ferrite particle adjuvant Fe 3 O 4 -DHCA stimulation, B is ferrite particle adjuvant Fe 3 O 4 -Man stimulation, C is ferrite particle adjuvant MnFe 2 O 4 -Man stimulation, D is antigen and ferrite particle adjuvant Fe 3 O 4 -DHCA stimulation, E is antigen and ferrite particle adjuvant Fe 3 O 4 -Man is stimulating.

[0022] Figure 4 Schematic diagram of in vivo treatment with ferrite particle adjuvant combined with tumor antigen.

[0023] Figure 5 is the tumor growth curve of mice after in vivo treatment with ferrite particle adjuvant combined with tumor antigen, where A is the ferrite particle adjuvant Fe 3 O 4 -DHCA, B is ferrite particle adjuvant Fe 3 O 4 -Man. DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0025] Example 1. Preparation of ferrite nanoparticles 1. Fe 3 O 4 The preparation of ferrite nanoparticles comprises the following steps: (1) Take 0.8 mL of 0.50 M FeCl 3solution, 0.72 mL 0.02 M NH 4 H 2 PO 4 The solution and 40 mL of deionized water were placed in a reactor, stirred for 20 min, and then the reactor was sealed. The reactor was placed in an oven at 220 °C for 280 min. After the reaction, it was naturally cooled, the brick-red reaction liquid at the bottom was aspirated, and washed by centrifugation with anhydrous ethanol to obtain α-Fe 2 O 3 ; (2) Dry the α-Fe 2 O 3 After being ground into fine powder, it was placed in a tube furnace and H 2 / Ar (volume ratio of 8:92) mixed gas, heated to 450℃ for 30 min, reacted at 450℃ for 120 min, and obtained Fe 3 O 4 .

[0026] (3) Accurately weigh 30 mg Fe 3 O 4 Black powder, 0.4 mL oleic acid, and 10 g octadecene were placed in a centrifuge tube and mixed evenly by ultrasonication for 20 min. The mixed liquid was added to a three-necked flask and rapidly heated to 280 °C under argon protection for 50 min. After the reaction, it was cooled to room temperature and finally washed by centrifugation at 8000 rpm / min for 10 minutes using n-hexane as a dispersant and anhydrous ethanol as a precipitant to obtain Fe 3 O 4 Ferrite nanoparticles.

[0027] 2. MnFe 2 O 4 The preparation of ferrite nanoparticles comprises the following steps: (1) Preparation of ferric erucate: 2.7 g of ferric chloride and 10.2 g of erucic acid were dissolved in 50 mL of methanol and heated to 40°C under magnetic stirring to form solution A. 1.2 g of sodium hydroxide was dissolved in 100 mL of methanol to form solution B. Solution B was added dropwise to solution A to react slowly. After the reaction, an ferric erucate complex was obtained. The complex was then repeatedly washed with deionized water and methanol and finally dried under vacuum at 45°C for 12 h.

[0028] (2) Preparation of manganese oleate: 1.98 g of manganese chloride and 5.65 g of oleic acid were dissolved in 50 mL of methanol and heated to 40°C under magnetic stirring to form solution A. 0.8 g of sodium hydroxide was ultrasonically dissolved in 100 mL of methanol to form solution B. Solution B was added dropwise to solution A for slow reaction. After the reaction, manganese oleate complex was obtained. The complex was then repeatedly washed with deionized water and methanol and finally dried in vacuum at 45°C for 12 h.

[0029] (3) MnFe 2 O 4 Preparation: 1.07 g of erucate, 0.62 g of manganese oleate, 0.57 g of oleic acid, and 1.61 g of oleyl alcohol were placed in a three-necked flask, and 10 g of benzyl ether was added. A magnetic stirring device was set up, and the mixture was heated to 110 °C under argon protection and maintained for 30 min. The reaction temperature was then raised to 265 °C at a rate of 5 °C / min and maintained at 265 °C for 30 min, and then rapidly cooled to room temperature. Finally, n-hexane was used as a dispersant and anhydrous ethanol was used as a precipitant and the mixture was centrifuged at 10,000 rpm / min for 10 minutes for washing to obtain MnFe 2 O 4 Ferrite nanoparticles.

[0030] 3. ZnFe 2 O 4 The preparation of ferrite nanoparticles comprises the following steps: (1) Preparation of ferric erucate: 2.7 g of ferric chloride and 10.2 g of erucic acid were dissolved in 50 mL of methanol and heated to 40°C under magnetic stirring to form solution A. 1.2 g of sodium hydroxide was dissolved in 100 mL of methanol to form solution B. Solution B was added dropwise to solution A to react slowly. After the reaction, an ferric erucate complex was obtained. The complex was then repeatedly washed with deionized water and methanol and finally dried under vacuum at 45°C for 12 h.

[0031] (2) ZnFe 2 O 4 Preparation of ferrite: 1.07 g of erucate, 0.12 g of zinc carbonate, 0.57 g of oleic acid, and 1.61 g of oleyl alcohol were placed in a three-necked flask, 10 g of benzyl ether was added, a magnetic stirring device was set up, and the reaction was heated to 110 ° C under argon protection and maintained for 30 min. Then, the reaction temperature was raised to 265 ° C at 5 ° C / min and maintained at 265 ° C for 30 min, and then quickly cooled to room temperature. Finally, n-hexane was used as a dispersant and anhydrous ethanol was used as a precipitant. Centrifugation was performed at 10000 rpm / min for 10 minutes to wash and obtain ZnFe 2 O 4 Ferrite nanoparticles.

[0032] 4. CoFe 2O 4 The preparation of ferrite nanoparticles comprises the following steps: (1) Preparation of ferric erucate: 2.7 g of ferric chloride and 5.65 g of erucic acid were dissolved in 50 mL of methanol and heated to 40°C under magnetic stirring to form solution A. 0.8 g of sodium hydroxide was dissolved in 100 mL of methanol to form solution B. Solution B was added dropwise to solution A to react slowly. After the reaction, ferric erucate complex was obtained. The complex was then repeatedly washed with deionized water and methanol and finally dried under vacuum at 45°C for 12 h.

[0033] (2) Preparation of cobalt oleate: 2.38 g of cobalt chloride and 10.2 g of oleic acid were dissolved in 50 mL of methanol and heated to 40°C under magnetic stirring to form solution A. 0.8 g of sodium hydroxide was dissolved in 100 mL of methanol to form solution B. Solution B was added dropwise to solution A to react slowly. After the reaction, an oleic manganese complex was obtained. The complex was then repeatedly washed with deionized water and methanol and finally dried in a vacuum at 45°C for 12 h.

[0034] (3) CoFe 2 O 4 Preparation: 1.07 g of ferric erucate, 0.62 g of cobalt oleate, 0.57 g of oleic acid, and 1.61 g of oleyl alcohol were placed in a three-necked flask, 10 g of benzyl ether was added, and a magnetic stirring device was set up. The mixture was heated to 110 °C under argon protection and maintained for 30 min. The temperature was then raised to 265 °C at 5 °C / min and maintained at 265 °C for 30 min. The mixture was then quickly cooled to room temperature. Finally, n-hexane was used as a dispersant and anhydrous ethanol was used as a precipitant. The mixture was centrifuged at 10,000 rpm / min for 10 minutes to obtain CoFe 2 O 4 Ferrite nanoparticles.

[0035] 5. MgFe 2 O 4 The preparation of ferrite nanoparticles comprises the following steps: (1) Preparation of ferric erucate: 2.7 g of ferric chloride and 10.2 g of erucic acid were dissolved in 50 mL of methanol and heated to 40°C under magnetic stirring to form solution A. 1.2 g of sodium hydroxide was dissolved in 100 mL of methanol to form solution B. Solution B was added dropwise to solution A to react slowly. After the reaction, an ferric erucate complex was obtained. The complex was then repeatedly washed with deionized water and methanol and finally dried under vacuum at 45°C for 12 h.

[0036] (2) Preparation of magnesium oleate: 1.0 g of magnesium chloride and 2.04 g of oleic acid were dissolved in 50 mL of methanol and heated to 40°C under magnetic stirring to form solution A. 0.8 g of sodium hydroxide was dissolved in 100 mL of methanol to form solution B. Solution B was added dropwise to solution A to react slowly. After the reaction, manganese oleate complex was obtained. The complex was then repeatedly washed with deionized water and methanol and finally dried in vacuum at 45°C for 12 h.

[0037] (3) MgFe 2 O 4 Preparation: 1.07 g of ferric erucate, 0.62 g of cobalt oleate, 0.57 g of oleic acid, and 1.61 g of oleyl alcohol were placed in a three-necked flask, 10 g of benzyl ether was added, a magnetic stirring device was set up, and the mixture was heated to 110°C under argon protection and maintained for 30 min. Then, the reaction temperature was raised to 265°C at 5°C / min and maintained at 265°C for 30 min, and then the mixture was rapidly cooled to room temperature. Finally, n-hexane was used as a dispersant and anhydrous ethanol was used as a precipitant and the mixture was centrifuged at 10,000 rpm / min for 10 minutes for washing to obtain MgFe 2 O 4 Ferrite nanoparticles.

[0038] 2. Tumor Vaccines The tumor vaccine is prepared by ferrite particle adjuvant and tumor antigen.

[0039] 1. Preparation of ferrite particle adjuvant Take the Fe prepared above 3 O 4 Ferrite nanoparticles and DHCA powder were placed in a three-necked flask, 10 mL of tetrahydrofuran was added, a magnetic stirring device was set up, condensation reflux was introduced, and the temperature was raised to 55 ° C under argon protection for 5 h. After the reaction, n-hexane was used as a dispersant and anhydrous ethanol was used as a precipitant to wash by centrifugation at 8000 rpm / min for 10 minutes to obtain ferrite particle adjuvant Fe 3 O 4 -DHCA, of which Fe 3 O 4 The mass ratio of ferrite nanoparticles to DHCA was 1:10.

[0040] Take the MnFe prepared above 2 O 4 Ferrite nanoparticles and DHCA powder were placed in a three-necked flask, 10 mL of tetrahydrofuran was added, a magnetic stirring device was set up, condensation reflux was introduced, and the temperature was raised to 55 ° C under argon protection for 5 h. After the reaction, n-hexane was used as a dispersant and anhydrous ethanol was used as a precipitant to wash by centrifugation at 8000 rpm / min for 10 minutes to obtain ferrite particle adjuvant MnFe 2 O4 -DHCA, MnFe 2 O 4 The mass ratio of ferrite nanoparticles to DHCA was 1:10.

[0041] In the same way, ZnFe 2 O 4 Preparation of Ferrite Nanoparticles Ferrite Particle Adjuvant ZnFe 2 O 4 -DHCA.

[0042] In the same way, CoFe 2 O 4 Ferrite Nanoparticles Preparation Ferrite Particle Adjuvant CoFe 2 O 4 -DHCA.

[0043] In the same way, MgFe 2 O 4 Preparation of Ferrite Nanoparticles Ferrite Particle Adjuvant MgFe 2 O 4 -DHCA.

[0044] Here, DHCA is the abbreviation of 3,4-dihydroxyhydrocinnamic acid.

[0045] 2. Preparation of tumor vaccines Preparation of tumor antigens after magnetic thermal inactivation: Hepa1-6 cells were inoculated in a 35 mm cell culture dish. After reaching the logarithmic growth phase, DMEM complete medium containing 75 μg / mL magnetic hyperthermia agent was added in the form of cell replacement medium for 8 hours. The original medium was discarded, washed 3 times with PBS and replaced with fresh medium. The cell culture dish was placed in a 365 kHz and 350Oe magnetic field generating device, and the magnetic field was treated for 10 minutes. The cell suspension was centrifuged at 1000 rpm for 3 minutes in a biosafety cabinet after blowing a few times. The bottom precipitate was cells and their lysates, which were tumor antigens. Among them, the magnetic thermal inactivation technology can refer to patent PCT / CN2024 / 074225.

[0046] Fe 3 O 4 、MnFe 2 O 4 、ZnFe 2 O 4 、CoFe 2 O 4 MgFe 2 O 4 The ferrite particle adjuvant and the above-prepared tumor antigen were vortex-mixed at a ratio of 1:2 to obtain a liver cancer tumor vaccine.

[0047] 3. Mannose receptors exist on the surface of dendritic cells. The various types of ferrite particle adjuvants prepared above have carboxyl groups on their surfaces. Therefore, ferrite particle adjuvants that can target dendritic cells can be obtained by chemically reacting EDC with mannose with amino groups, which is expected to further improve the processing and presentation efficiency and activation level of dendritic cells for tumor antigens.

[0048] The ferrite particles adjuvant Fe 3 O 4 -DHCA and EDC were mixed in a mass ratio of 1:2 and added to MES buffer, and placed on a shaker at room temperature for 15 min to activate the carboxyl groups on the surface of the ferrite. Then mannose (mannose and ferrite particle adjuvant Fe 3 O 4 -DHCA mass ratio of 1:1) was shaken for 120 min, and after the reaction, PBS was used for washing by centrifugation at 10000 rpm / min for 10 min to obtain the ferrite particle adjuvant Fe that can target dendritic cells 3 O 4 -Man.

[0049] The same method was used to add ferrite particles adjuvant MnFe 2 O 4 -DHCA was used to prepare the ferrite particle adjuvant MnFe 2 O 4 -Man.

[0050] Here, EDC is the abbreviation of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0051] Effect verification 1. Characterization of ferrite particle adjuvant The ferrite particle adjuvant dispersed in deionized water was diluted to a suitable concentration, dropped onto the copper grid covered with the carbon support film, and allowed to stand until naturally dry before TEM characterization. Figure 1 It shows that the sizes of various ferrite particles prepared are uniform, among which Fe 3 O 4 The morphology is ring-shaped and the size is about 70 nm. The ultra-small MnFe 2 O 4 、ZnFe 2 O 4 、CoFe 2 O 4 and MgFe 2 O 4 The size is about 3 nm.

[0052] 2. Cytotoxicity Evaluation of Ferrite Particle Adjuvant Mouse hepatocellular carcinoma cells Hepa1-6 were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured at 37°C with 5% CO 2 The cells were cultured in a cell culture incubator with DMEM high-glucose medium containing 10% FBS and 1% penicillin-streptomycin, and passaged every two days. The cells in the logarithmic growth phase with good growth conditions were used for the experiment, and the ferrite particle adjuvant Fe 3 O 4 -DHCA and ferrite particle adjuvant Fe 3 O 4 -Man was used for subsequent experiments.

[0053] Hepa1-6 cells were inoculated into 96-well plates. After the cells adhered to the plate, the old culture medium was discarded and the adjuvant Fe containing different concentrations of ferrite particles was added. 3 O 4 -DHCA and ferrite particle adjuvant Fe 3 O 4 -Man complete medium was co-incubated with Hepa1-6 cells for 24 h, and the cell activity was detected by CCK-8 method. 3 O 4 The concentrations of the ferrite particle adjuvant were 0 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL.

[0054] Cell viability = (OD 实验 -OD 对照 ) / (OD 对照 -OD 空白 )× 100%.

[0055] The experimental results are as follows Figure 2 , two different concentrations of Fe 3 O 4 After the ferrite nanoadjuvant was co-incubated with Hepa1-6 cells, it was found that when the concentration of the nanoadjuvant was 100 μg / mL, the cell survival rate was greater than 80%, and there was no significant change in cell activity compared with the control group, indicating that the prepared ferrite nanoadjuvant has good biocompatibility.

[0056] 3. Experiment on the effect of ferrite particles adjuvant on promoting dendritic cell maturation in vitro The specific extraction operation of mouse bone marrow-derived dendritic cells (BMDC) is as follows: take the leg bones of mice, disinfect them with 70% ethanol, use a syringe to draw RPMI 1640 culture medium to flush out the bone marrow until the bones turn completely white, and blow the bone marrow fluid to separate the bone marrow clumps. The bone marrow fluid is centrifuged at 1500 rpm for 5 min, and red blood cell lysis solution is added to lyse the red blood cells, and then the cell pellet is cultured in RPMI 1640 complete culture medium containing induction factors for one week to obtain induced differentiated BMDC, where the induction factors are 20 ng / mL GM-CSF and 50 ng / mL IL-4.

[0057] Next, the induced differentiated BMDCs were co-incubated with ferrite particle adjuvant for 24 h, and the activation of BMDCs was detected by flow cytometry after staining with CD11c, CD80, and CD86 antibodies.

[0058] like Figure 3 As shown, three kinds of ferrite particle adjuvants Fe 3 O 4 -DHCA, Fe 3 O 4 -Man, MnFe 2 O 4 -Man can promote BMDC activation alone; at the same time, the ferrite particle adjuvant has a greatly enhanced BMDC activation ability after being mixed with magnetically inactivated tumor antigens (the ratio of adjuvant to antigen is 1:2), which indicates that ferrite nanoparticles can be used as vaccine particle adjuvants to enhance the immunogenicity of tumor antigens, improve tumor antigen-mediated immune stimulation, and promote the processing, presentation and maturation of tumor antigens by dendritic cells.

[0059] 4. Treatment of mouse liver cancer with ferrite particle adjuvant combined with tumor antigen C57BL / 6 (6-8 weeks, male) were purchased from Chengdu Jicui Pharmaceutical Co., Ltd. and maintained in a standard environmental control room (23°C, humidity 55±5%, light-dark cycle 12h-12h).

[0060] The specific treatment plan is as follows: First, 1×10 7 Hepa1-6 cells, and wait until the tumor grows to about 100 mm 3 The mice were randomly divided into groups and inoculated with tumor antigens subcutaneously on the left flank at 14:00 pm, which was recorded as day 0. They were then treated once on day 7 and day 14, for a total of 3 treatments. The changes in tumor size in the mice were observed every other day.

[0061] The results are as follows Figure 4 and Figure 5As shown in the figure, the ferrite particle adjuvant significantly enhanced the anti-tumor effect of the tumor vaccine. Using tumor antigen alone can inhibit tumor growth with an inhibition rate of 24.48%, and when the ferrite particle adjuvant is used in combination with tumor antigen, the tumor inhibition rate is further improved. 3 O 4 -DHCA granule adjuvant increased the tumor inhibition rate from 24.48% to 72.60%, Fe 3 O 4 -Man particle adjuvant increased the inhibition rate from 24.48% to 89.59%. These results show that ferrite particle adjuvant can not only enhance the immunogenicity of tumor antigens, but also promote the processing, presentation and maturation of antigens by dendritic cells, thereby enhancing the immune stimulation of vaccines and improving anti-tumor efficacy.

[0062] In summary, the novel tumor vaccine adjuvant provided by the present invention can effectively promote the stimulation and activation of dendritic cells by tumor vaccines by targeting antigen-presenting cells, thereby efficiently activating immune cells, enhancing the body's immune response to antigens, and significantly improving the therapeutic effect of the vaccine.

[0063] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0064] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0065] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A tumor vaccine adjuvant, characterized in that: It is a ferrite particle adjuvant prepared by reacting ferrite nanoparticles with organic polymers at a mass ratio of 1:1 to 200 at 50°C to 60°C for 2 h to 12 h; The ferrite nanoparticles are Fe3O4 or metal-doped Fe3O4; The organic polymer is any one of polyethylene glycol, polyacrylic acid, polyvinyl pyrrolidone, citric acid, 3-(3,4-dihydroxyphenyl)propionic acid, 2,3-dimercaptosuccinic acid, mannose and 3,4-dihydroxyhydrocinnamic acid.

2. The tumor vaccine adjuvant according to claim 1, characterized in that The size of the ferrite nanoparticles is 2 nm to 500 nm.

3. The tumor vaccine adjuvant according to claim 1, characterized in that The metal-doped Fe3O4 is MnFe2O4, ZnFe2O4, CoFe2O4 or MgFe2O4.

4. The tumor vaccine adjuvant according to claim 1, characterized in that The invention discloses a ferrite particle adjuvant targeting dendritic cells, which is obtained by modifying the ferrite particle adjuvant by using mannose.

5. Use of the tumor vaccine adjuvant according to claim 1 or claim 4 in the preparation of tumor vaccines.

6. The use according to claim 5, characterized in that: The preparation method of the tumor vaccine is as follows: the ferrite particle adjuvant and the tumor antigen are mixed in a volume ratio of 1:1-5 to obtain the tumor vaccine.

7. The use according to claim 5, characterized in that: The tumor is any one of liver cancer, melanoma, colorectal cancer, pancreatic cancer, gastric cancer and breast cancer.

8. The use according to claim 5, characterized in that: The tumor antigens are tumor cell autoantigens or tumor cells and their lysates formed after tumor cells are inactivated.

9. The use according to claim 5, characterized in that: The tumor cell is any one of Hepa1-6 cells, 4T1 cells, Pan02 cells and LLC cells.

10. The use according to claim 5, characterized in that: The tumor vaccine is any one of whole tumor cell vaccine, dendritic cell vaccine, mRNA vaccine and peptide vaccine.

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