Nano indium adjuvant as well as preparation method and application thereof

Abstract

The invention discloses a nano indium adjuvant as well as a preparation method and application thereof, and belongs to the technical field of nano medicines. The preparation method comprises the following steps: mixing indium trichloride with a PVP (Polyvinyl Pyrrolidone) solution, and carrying out metal ion chelation on the mixture and a gallic acid solution to obtain the uniform and stable nano indium adjuvant. Then, the antigen ovalbumin and the nano indium adjuvant form the nano indium adjuvant vaccine, humoral immunity and cellular immunity are activated at the same time, the presentation capacity and immunogenicity of the antigen are enhanced, and the requirements of clinical anti-tumor vaccines are met.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, and in particular relates to a nano indium adjuvant, its preparation method and application. Background Technology

[0002] In recent years, tumor immunotherapy has gradually attracted widespread attention as an emerging treatment approach. While traditional tumor treatments such as surgery, radiotherapy, and chemotherapy are effective, they often come with significant side effects and a risk of recurrence. Therefore, developing novel immunotherapy strategies has become a hot research topic.

[0003] Currently, various immune adjuvants are used in the development of tumor vaccines, among which aluminum adjuvants are widely used due to their good safety and immunomodulatory activity. However, traditional immune adjuvants often fail to effectively activate dendritic cells, resulting in insufficient antigen presentation capacity, which in turn affects the activation and proliferation of T cells. Furthermore, traditional aluminum adjuvants have limited effectiveness in enhancing humoral and cellular immunity, making it difficult to meet clinical needs. Existing methods for preparing nano-indium adjuvants are diverse, but they often suffer from complex preparation processes and poor stability, limiting their clinical application. In addition, most existing anti-tumor vaccines rely on a single immune mechanism, making it difficult to simultaneously activate humoral and cellular immunity. Therefore, developing a nano-indium adjuvant capable of simultaneously enhancing both immune responses and its application method is particularly important. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a nano-indium adjuvant, its preparation method and application, wherein the nano-indium adjuvant has good stability and uniformity, can effectively improve antigen presentation ability, activate dendritic cells, and promote T cell activation and proliferation.

[0005] Another objective of this invention is to provide a nano-indium adjuvant vaccine, its preparation method, and its application. The nano-indium adjuvant vaccine prepared using the nano-indium adjuvant can simultaneously activate humoral immunity and cellular immunity, significantly enhance immunogenicity, and meet the clinical needs for anti-tumor vaccines.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a nano-indium adjuvant, which is obtained by mixing a PVP solution with an indium trichloride solution and then reacting it with a gallic acid solution via a crosslinking reaction.

[0007] Preferably, the concentration ratio of the PVP solution, indium trichloride solution and gallic acid solution is (6.5-8.5):(90-110:(2-8), and the volume ratio is (6.8-10.8):(0.5-1.5):(1-3).

[0008] Preferably, the solvent for the PVP solution, indium trichloride solution, and gallic acid solution is water.

[0009] The present invention provides a method for preparing the nano-indium adjuvant, comprising the following steps: mixing a PVP solution with an indium trichloride solution, then stirring and reacting the mixture with a gallic acid solution, ultrafiltration and centrifugation, and collecting the precipitate.

[0010] Preferably, the mixing temperature is room temperature, the rotation speed is 500 rpm-1200 rpm, and the time is 1-4 h; the stirring reaction temperature is room temperature, the rotation speed is 500 rpm-1200 rpm, and the time is 20-28 h.

[0011] Preferably, the parameters for the ultrafiltration centrifugation include: a molecular weight cutoff of 8-12 kDa for the ultrafiltration tube, centrifugation at 7500-8500 rpm for 10-20 min at 2-6℃, and centrifugation 2-4 times.

[0012] This invention provides a method for preparing a nano-indium adjuvant vaccine, comprising the following steps: mixing an antigen solution with the nano-indium adjuvant or the nano-indium adjuvant obtained by the preparation method and incubating to obtain a nano-indium adjuvant vaccine.

[0013] Preferably, the mass ratio of the antigen solution to the nano-indium adjuvant is 1:0.5-4; the incubation temperature is room temperature, the rotation speed is 500-1200 rpm, and the time is 0.5-2.5 h.

[0014] The present invention provides a nano-indium adjuvant vaccine obtained by the preparation method described above.

[0015] The present invention provides the application of the nano-indium adjuvant, or the nano-indium adjuvant obtained by the preparation method, or the nano-indium adjuvant vaccine obtained by the preparation method, or the nano-indium adjuvant vaccine in the preparation of tumor immunotherapy products.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to mix a PVP solution with an indium trichloride solution, followed by a cross-linking reaction with a gallic acid solution to obtain indium nanoparticles as adjuvants. These indium nanoparticles are then mixed with an antigen to obtain an indium nanoparticle adjuvant vaccine. This invention employs a strategy combining metal ion chelation and a metallophenol network method, achieving efficient preparation and antigen loading of indium nanoparticles. It is the first time that indium nanoparticles have been applied to the field of vaccine adjuvants, opening up new applications for indium in immunotherapy.

[0017] The preparation process of this invention is simple and efficient: a one-step method is used to synthesize indium nanoparticles under mild reaction conditions, requiring no complex equipment and suitable for large-scale production. Experiments show that the indium nanoparticle adjuvant prepared by this invention can significantly promote DC cell maturation and antigen cross-presentation, resulting in significant immune enhancement. Simultaneously, the vaccine complex constructed via the metallophenol network method exhibits good stability, maintaining good dispersibility even after 30 days of storage at room temperature. PVP modification improves the biocompatibility of the nanoparticles, and cytotoxicity experiments show good safety. Experimental verification shows that the indium nanoparticle adjuvant can achieve an antigen loading rate of over 85%, demonstrating high antigen loading efficiency, significantly higher than traditional adjuvant systems. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the preparation process in Example 1.

[0019] Figure 2 The images show the comparison of transmission electron microscopy (TEM) images for each group of samples. In the images, A represents sample A-PVP / GA, B represents sample B-BSA / GA, C represents sample C-BSA / TA, and D represents sample D-PVP / TA. The scale bar is 100 nm.

[0020] Figure 3 The following are DLS analysis chromatograms for each group of samples. A represents sample A-PVP / GA, B represents sample B-BSA / GA, C represents sample C-BSA / TA, and D represents sample D-PVP / TA.

[0021] Figure 4 The graph shows the comparison results of the dispersion coefficients of each group of samples. In the graph, A represents sample A-PVP+GA, B represents sample B-BSA / GA, C represents sample C-BSA / TA, and D represents sample D-PVP / TA.

[0022] Figure 5 The results show the effect of different In NPs to OVA ratios on antigen loading rate.

[0023] Figure 6 These are transmission electron microscopy (TEM) images of OVA@In NPs. The scale bar is 100 nm (left image) and 50 nm (right image).

[0024] Figure 7 This is a comparison diagram of the activation of DC cells by different groups of samples.

[0025] Figure 8 This is a comparison chart of the neutralizing antibody titers induced in animals for each group of samples.

[0026] Figure 9 This is a comparison chart of CD8+ T cell proliferation in different groups of samples during the cell immunoassay.

[0027] Figure 10This is a comparison chart of the anti-tumor efficacy of different groups of samples in animals. Detailed Implementation

[0028] This invention provides a nano-indium adjuvant, obtained by mixing a polyvinylpyrrolidone (PVP) solution with an indium trichloride (InCl3) solution, followed by a crosslinking reaction with a gallic acid (GA) solution. This invention utilizes a metal ion chelation method to prepare the nano-indium adjuvant. Unless otherwise specified, the PVP solution, indium trichloride solution, and gallic acid solution described in this invention can be obtained through commercially available channels or preparation methods well-known in the art.

[0029] In this invention, the concentration ratios of the PVP solution, indium trichloride solution, and gallic acid solution are (6.5-8.5):(90-110):(2-8) and the volume ratios are (6.8-10.8):(0.5-1.5):(1-3), preferably (7-8):(95-105):(3-7) and (7.8-9.8):(0.8-1.2):(1.5-2.5), and even more preferably (7.5:100:5) and (8.8:1:2). Water is used as the solvent for the PVP solution, indium trichloride solution, and gallic acid solution. The preferred component concentrations are determined based on a combination of the nano-indium particle size and the amount of product synthesized.

[0030] This invention provides a method for preparing the aforementioned nano-indium adjuvant, comprising the following steps: mixing a PVP solution with an indium trichloride solution, then reacting the mixture with a gallic acid solution by stirring, ultrafiltration, centrifugation, and collecting the precipitate. This invention synthesizes the nano-indium adjuvant via metal ion chelation, utilizing PVP as a stabilizer to ensure the uniformity and stability of the nano-indium adjuvant; and utilizing gallic acid as a reducing agent to provide indium ion chelation sites.

[0031] In this invention, the mixing temperature is room temperature, the rotation speed is 500 rpm-1200 rpm, and the time is 1-4 h, preferably room temperature, the rotation speed is 800 rpm-1100 rpm, and the time is 1.5-3 h, more preferably room temperature, the rotation speed is 1000 rpm, and the time is 2 h; the stirring reaction temperature is room temperature, the rotation speed is 500 rpm-1200 rpm, and the time is 20-28 h, preferably room temperature, the rotation speed is 1000 rpm, and the time is 24 h.

[0032] In this invention, the ultrafiltration centrifugation parameters include: a molecular weight cutoff of 8-12 kDa for the ultrafiltration tube, centrifugation at 7500-8500 rpm for 10-20 min at 2-6℃, repeated 2-4 times; preferred parameters include: a molecular weight cutoff of 9-11 kDa for the ultrafiltration tube, centrifugation at 7700-8300 rpm for 12-18 min at 3-5℃, repeated 2-4 times; further preferred parameters include: a molecular weight cutoff of 10 kDa for the ultrafiltration tube, centrifugation at 8000 rpm for 15 min at 4℃, repeated 3 times. The ultrafiltration centrifugation of this invention removes unreacted small molecule impurities and free metal ions, yielding stable indium nanoparticles.

[0033] This invention provides a method for preparing an indium nanoparticle adjuvant vaccine (OVA@In NPs), comprising the following steps: mixing and incubating an antigen solution with the indium nanoparticle adjuvant or an indium nanoparticle adjuvant obtained by the preparation method to obtain the indium nanoparticle adjuvant vaccine. The antigen used in this invention is preferably ovalbumin (OVA). This invention further stabilizes the binding between the antigen and the nanoparticle adjuvant through a metallophenol network, ultimately obtaining a uniform OVA@In NPs nanoparticle vaccine formulation. The indium nanoparticle adjuvant vaccine prepared by this novel preparation method significantly enhances immunogenicity, providing a more effective solution for tumor immunotherapy and promoting the development of related research and clinical applications.

[0034] In this invention, the mass ratio of the antigen solution to the nano-indium adjuvant is 1:0.5-4, preferably 1:0.8-2, and more preferably 1:1; the incubation temperature is room temperature, the rotation speed is 500-1200 rpm, and the time is 0.5-2.5 h, preferably room temperature, the rotation speed is 800-1100 rpm, and the time is 0.8-2 h, and more preferably room temperature, the rotation speed is 1000 rpm, and the time is 1 h.

[0035] This invention provides a nano-indium adjuvant vaccine obtained by the aforementioned preparation method. The OVA@In NPs vaccine prepared by this invention can simultaneously activate humoral and cellular immunity, significantly enhance immunogenicity, and meet the clinical demand for anti-tumor vaccines.

[0036] This invention provides the application of the aforementioned indium nanoparticle adjuvant, or the indium nanoparticle adjuvant vaccine obtained by the aforementioned preparation method, or the indium nanoparticle adjuvant vaccine, in the preparation of tumor immunotherapy products. The tumors mentioned in this invention include, but are not limited to, solid tumors such as breast cancer (e.g., the 4T1 model) and melanoma (e.g., the B16 model).

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Unless otherwise specified, the following embodiments are all conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] Example 1 1. Preparation of Indium Nanoparticles (In NPs) (1) Preparation of PVP working solution: Weigh polyvinylpyrrolidone (PVP, CAS: 9003-39-8, molecular weight 10000) and dissolve it in ultrapure water to obtain 50 mL of PVP aqueous solution with a concentration of 30 mg / mL, and store at 4℃.

[0041] Preparation of indium trichloride working solution: Dissolve 5g of indium trichloride (CAS:10025-82-8) in 50mL of ultrapure water to obtain 50mL of indium trichloride working solution with a concentration of 100mg / mL, and store at 4℃.

[0042] Gallic acid working solution preparation: Dissolve 250 mg GA (CAS: 149-91-7) in 50 mL of ultrapure water to obtain 50 mL of gallic acid aqueous solution with a concentration of 5 mg / mL, and store at 4 °C.

[0043] (2) Take 8.8 mL of 7.5 mg / mL PVP aqueous solution and place it in a 25 mL round bottom flask. Add 1.0 mL of 100 mg / mL indium trichloride aqueous solution slowly at a stirring speed of 1000 rpm. Stir continuously at room temperature for 2 h to fully disperse the metal ions and PVP to obtain a mixed solution.

[0044] (3) Add 2.0 mL of 5 mg / mL gallic acid aqueous solution dropwise to the mixture using a 1 mL pipette. Stir continuously at 1000 rpm for 24 h at room temperature to ensure that the metal ions are fully chelated and cross-linked to form stable nanoparticles. After the reaction is complete, add 1.0 mL of deionized water to resuspend the mixture, centrifuge at 8000 rpm for 15 min at 4 °C, and then use a Millipore 10 kDa ultrafiltration tube to remove unreacted small molecule impurities and free metal ions. Take the bottom precipitate, repeat the above steps 3 times, and take the bottom precipitate to obtain a stable and highly uniform aqueous dispersion of indium nanoparticles. Figure 1 This is a schematic diagram illustrating the synthesis of a dispersion of nano-indium particles.

[0045] 2. OVA@In NPs vaccine synthesis (metallophenol network method) (1) Preparation of OVA solution: Weigh 10mg of OVA powder (manufacturer: InvivoGen; batch number: vac-stova; dosage form: 1.0g), dissolve and mix thoroughly with 1mL of PBS, filter through a 0.22μm sterile membrane on a clean bench, and store at 4℃ for later use.

[0046] (2) Mix the OVA solution (10 mg / mL, 1 mL) with the In NPs prepared above at a ratio of 1:1 (w / w) and incubate at room temperature at a fixed speed of 1000 rpm for 1 h to obtain OVA@In NPs nano-indium adjuvant vaccine.

[0047] Example 2 1. Preparation of Indium Nanoparticles (In NPs) (1) Preparation of PVP working solution, indium trichloride working solution, and gallic acid working solution: Same as in Example 1.

[0048] (2) Take 6.8 mL of 6.5 mg / mL PVP aqueous solution and place it in a 25 mL round bottom flask. Add 0.5 mL of 90 mg / mL indium trichloride aqueous solution slowly at a stirring speed of 500 rpm. Stir continuously at room temperature for 1 h to fully disperse the metal ions and PVP to obtain a mixed solution.

[0049] (3) Add 1.0 mL of 2 mg / mL gallic acid aqueous solution dropwise to the mixture using a 1 mL pipette. Stir continuously at 500 rpm for 22 h at room temperature. After the reaction is complete, add 1.0 mL of deionized water to resuspend the mixture, centrifuge at 7500 rpm for 10 min at 3 °C, and then use an 8 kDa ultrafiltration tube from Millipore to remove unreacted small molecule impurities and free metal ions. Take the bottom precipitate, repeat the above steps twice, and take the bottom precipitate to obtain a stable and highly uniform aqueous dispersion of indium nanoparticles.

[0050] 2. OVA@In NPs vaccine synthesis (metallophenol network method) (1) OVA solution preparation: Same as in Example 1.

[0051] (2) Mix the OVA solution (10 mg / mL, 1 mL) with the In NPs prepared above at a ratio of 1:0.5 (w / w), and incubate at room temperature at a fixed speed of 500 rpm for 0.5 h to ensure that the In NPs and OVA are fully mixed to obtain the OVA@In NPs nano-indium adjuvant vaccine.

[0052] Example 3 1. Preparation of Indium Nanoparticles (In NPs) (1) Preparation of PVP working solution, indium trichloride working solution, and gallic acid working solution: Same as in Example 1.

[0053] (2) Take 10.8 mL of 8.5 mg / mL PVP aqueous solution and place it in a 25 mL round bottom flask. Add 1.5 mL of 110 mg / mL indium trichloride aqueous solution slowly at a stirring speed of 1200 rpm. Stir continuously at room temperature for 4 h to fully disperse the metal ions and PVP to obtain a mixed solution.

[0054] (3) Add 3.0 mL of 8 mg / mL gallic acid aqueous solution dropwise to the mixture using a 1 mL pipette. Stir continuously at 1200 rpm for 26 h at room temperature. After the reaction is complete, add 1.0 mL of deionized water to resuspend the mixture, centrifuge at 8500 rpm for 20 min at 6 °C, and then use a Millipore 12 kd ultrafiltration tube for ultrafiltration to remove unreacted small molecule impurities and free metal ions. Take the bottom precipitate, repeat the above steps 4 times, and take the bottom precipitate to obtain a stable and highly uniform aqueous dispersion of indium nanoparticles.

[0055] 2. OVA@In NPs vaccine synthesis (metallophenol network method) (1) OVA solution preparation: Same as in Example 1.

[0056] (2) Mix the OVA solution (10 mg / mL, 1 mL) with the In NPs prepared above at a ratio of 1:4 (w / w) and incubate at room temperature at a fixed speed of 1200 rpm for 2.5 h to ensure that the In NPs and OVA are fully mixed to obtain the OVA@In NPs nano-indium adjuvant vaccine.

[0057] Example 1: Comparative Experiment on the Preparation of Nano-Adjuvants with Different Stabilizer / Reducing Agent Combinations 1. Grouping and corresponding samples Experimental Sample A (labeled as Sample A-PVP / GA): prepared by the method in Example 1.

[0058] Comparative Sample B (labeled as Sample B-BSA / GA): The preparation method is exactly the same as that of A-PVP / GA, except that the stabilizer PVP is replaced with bovine serum albumin (BSA) at an equal mass concentration (10 mg / mL).

[0059] Comparative sample C (labeled as sample C-PVP / TA): The preparation method is exactly the same as that of A-PVP / GA, except that the reducing agent GA aqueous solution is replaced with an aqueous solution of tannic acid (TA) of the same mass concentration (5 mg / mL).

[0060] Comparative sample D (labeled as sample D-BSA / TA): The preparation method is exactly the same as that of A-PVP / GA, except that the stabilizer PVP is replaced with BSA (10 mg / mL) and the reducing agent GA is replaced with tannic acid (TA, 5 mg / mL).

[0061] 2. Product characterization and effect comparison Morphology and Dispersion (TEM Analysis): The morphology, size, and stability of the four samples (samples A, B, C, and D) were characterized, and the results are as follows: Figure 2-4 The comparison is as follows: Sample A contains well-dispersed, uniformly sized, nearly spherical indium nanoparticles with an average particle size of approximately 15±3 nm. No obvious agglomeration is observed, and the polydispersity index (PDI) is 0.12, indicating excellent monodispersity. Sample B (BSA / GA) shows partial agglomeration, forming irregularly sized aggregates of particles, making it impossible to obtain single, stable dispersed nanoparticles. Sample C (PVP / TA) produces a small amount of nanoparticles, but their morphology is uneven, and vacuolar structures are present. Sample D (BSA / TA) produces a large amount of amorphous precipitate in the reaction system. After centrifugation, it is a two-phase system with separated supernatant and precipitate, showing almost no formation of a stable nanocolloid solution.

[0062] Experiment Example 2 Following the OVA@In NPs vaccine synthesis method in Example 1, the mass ratio of In NPs to OVA was 0.25:1, 0.5:1, 1:1, 2:1, and 4:1 (w / w). The antigen loading rate was determined as follows... Figure 5 As shown, although when In NPs are in excess (e.g., at a ratio of 2:1 or 4:1), more OVA may be adsorbed, and the loading rate of In NPs per unit mass may reach its maximum, a large amount of free, unloaded In NPs adjuvant will also be present. These free adjuvants may cause nonspecific inflammation, increasing the risk of systemic toxicity, and adjuvants that are not physically bound to the antigen are difficult to ensure "co-delivery," which will reduce vaccine efficacy. Therefore, based on the comprehensive evaluation of antigen loading in this experiment, the structural stability and dispersion uniformity of the experimental complex, and the expected subsequent synergistic immune efficacy, the optimal mass ratio of In NPs to OVA was determined to be 1:1 (w / w). Transmission electron microscopy (TEM) analysis was performed on the OVA@In NPs vaccine prepared with an In NPs to OVA mass ratio of 1:1 (w / w), and the results are as follows. Figure 5 As shown, in a 1:1 ratio, In NPs are tightly encapsulated by OVA protein, forming a structurally stable and homogeneous complex. This "protein crown" structure effectively prevents the aggregation of nanoparticles, ensuring their stable dispersion in the physiological environment, which is the basis for their function.

[0063] Experiment Example 3: OVA@In NPs Vaccine Efficacy Verification Experiment 1. DC Cell Activation Assay: The effects of PBS (blank control), OVA (antigen alone), OVA@Al-NPs (aluminum adjuvant nanovaccine, synthesis method as described in Example 1, OVA@In-NPs preparation method), and OVA@In-NPs (prepared in Example 1) on DC cell activation were evaluated. The samples from each group were co-cultured with mouse bone marrow-derived DC cells for 24 h, and the expression of maturation markers such as CD80, CD86, and MHC-II was detected by flow cytometry. The results showed that compared with the OVA-only group, the expression of maturation markers in DC cells was significantly upregulated in the OVA@In-NPs group (p<0.01). Figure 7 ).

[0064] 2. Humoral Immunity - IgG Antibody Level Detection: The experiment evaluated the kinetic effects of PBS (blank control), OVA (single antigen), OVA@Al-NPs (aluminum adjuvant nanovaccine, synthesis method referred to Example 1 OVA@In-NPs preparation method), and OVA@In-NPs (prepared in Example 1) on the time-dependent changes in ovalbumin-specific IgG antibody concentration in Balb / C mouse serum. Serum samples were collected at 0, 6, 24, 48, and 72 h after subcutaneous injection of the corresponding tumor vaccine into normal Balb / C mice, and antibody concentrations were detected using ELISA. The results showed that the OVA@In-NPs treatment group induced the highest level of specific IgG antibodies at all time points, especially at 24 h, where its concentration was significantly higher than all other groups. While the OVA@Al-NPs group also induced a strong humoral immune response, its peak antibody level and persistence were lower than those of the OVA@In-NPs group. This indicates that the antigen delivery system using indium nanoparticles as a carrier is superior to traditional nano-aluminum adjuvants in rapidly and efficiently stimulating humoral immune responses. Figure 8 ).

[0065] 3. Cellular Immunotherapy - T Cell Proliferation Assay: The experiment evaluated the effects of PBS (blank control), OVA (single antigen), OVA@Al-NPs (aluminum adjuvant nanovaccine, synthesis method as described in Example 1, OVA@In-NPs preparation method), and OVA@In-NPs (prepared in Example 1) on cellular immunotherapy - T cell proliferation. The samples from each group were co-cultured with mouse bone marrow-derived dendritic cells (BMDCs) for 24 h, and the supernatant was collected. Lymphocytes isolated from the spleen of normal mice were purified into CD8+ T cells using a CD8+ sorting kit. The obtained BMDCs supernatant was then co-incubated with CD8+ T cells for 24 h. Finally, the proliferation of CD8+ T cells was detected by flow cytometry. The results showed that compared with the OVA-only group and the OVA@Al-NPs group, the CD8+ T cell proliferation level in the OVA@In-NPs group was significantly increased (p<0.01). Figure 9 ).

[0066] 4. Animal Immunization Experiment: A 4T1-OVA subcutaneous xenograft tumor model was established in Balb / C mice. Mice were randomly divided into five groups, receiving local subcutaneous injections of PBS (blank control), OVA (antigen alone), OVA@Al-NPs (aluminum adjuvant nanovaccine, synthesized as described in Example 1), and OVA@In-NPs (prepared in Example 1), respectively. Tumor growth was observed after the treatment period to visually and qualitatively evaluate the antitumor effects of different formulations. The results showed that the OVA@In-NPs group had the smallest tumor volume, indicating that this nanovaccine produced the most significant tumor growth inhibition effect in this model. Figure 10 ).

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nano-indium adjuvant, characterized in that, The PVP solution was mixed with an indium trichloride solution and then crosslinked with a gallic acid solution to obtain the product.

2. The nano-indium adjuvant as described in claim 1, characterized in that, The concentration ratios of the PVP solution, indium trichloride solution, and gallic acid solution are (6.5-8.5): (90-110): (2-8), and the volume ratios are (6.8-10.8): (0.5-1.5): (1-3).

3. The nano-indium adjuvant as described in claim 1, characterized in that, The solvents for the PVP solution, indium trichloride solution, and gallic acid solution are water.

4. The method for preparing the nano-indium adjuvant according to any one of claims 1-3, characterized in that, The process includes the following steps: mixing PVP solution with indium trichloride solution, then stirring and reacting with gallic acid solution, ultrafiltration and centrifugation, and collecting the precipitate.

5. The preparation method according to claim 4, characterized in that, The mixing temperature is room temperature, the rotation speed is 500rpm-1200rpm, and the time is 1-4h; the stirring reaction temperature is room temperature, the rotation speed is 500rpm-1200rpm, and the time is 20-28h.

6. The preparation method according to claim 4, characterized in that, The parameters for ultrafiltration centrifugation include: a molecular weight cutoff of 8-12 kDa for the ultrafiltration tube, centrifugation at 7500-8500 rpm for 10-20 min at 2-6℃, and centrifugation 2-4 times.

7. A method for preparing a nano-indium adjuvant vaccine, characterized in that, The method includes the following steps: mixing and incubating an antigen solution with the nano-indium adjuvant as described in any one of claims 1-3 or the nano-indium adjuvant obtained by the preparation method described in any one of claims 4-6 to obtain a nano-indium adjuvant vaccine.

8. The preparation method according to claim 7, characterized in that, The mass ratio of the antigen solution to the nano-indium adjuvant is 1:0.5-4; the incubation temperature is room temperature, the rotation speed is 500-1200 rpm, and the time is 0.5-2.5 h.

9. The nano-indium adjuvant vaccine obtained by the preparation method according to claim 7 or 8.

10. The use of the nano-indium adjuvant as described in any one of claims 1-3, or the nano-indium adjuvant obtained by the preparation method as described in any one of claims 4-6, or the nano-indium adjuvant vaccine obtained by the preparation method as described in any one of claims 7-8, or the nano-indium adjuvant vaccine as described in claim 9, in the preparation of tumor immunotherapy products.

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