Immune-activating nanosheets, methods of making and using the same

Abstract

The application belongs to the technical field of biological nanomaterials, and particularly relates to an immune activation nanosheet as well as a preparation method and application thereof. The immune activation nanosheet comprises a two-dimensional montmorillonite nanosheet with a sheet layer structure, and manganese ions and tumor antigens loaded in the interlayer of the two-dimensional montmorillonite nanosheet. The mass ratio of the manganese ions to the montmorillonite is 1:5-1:20, and the mass ratio of the tumor antigens to the montmorillonite is 1:1-1:10. The immune activation nanosheet has the characteristics of good oral stability, high mucosal targeting, strong immune efficacy, etc. The stability of the immune activation nanosheet in the gastrointestinal tract is good, the immune activation nanosheet can be effectively enriched in the small intestine, a vaccine inoculation pool is formed, the intestinal immune tolerance can be overcome, the immune effect is enhanced, and the immune activation nanosheet has a clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bionanomaterials technology, specifically relating to an immune-activating nanosheet, its preparation method, and its application. Background Technology

[0002] Mucosal cancer is a type of tumor with extremely high incidence and mortality rates. It occurs in mucosal tissue, which covers many organs in the body. Therefore, mucosal tumors involve multiple organs and sites, including lung cancer, head and neck cancer, digestive tract cancers (especially stomach cancer, colorectal cancer, etc.), and reproductive tract cancers, posing a major global disease challenge. Practice has shown that tumor vaccines can induce specific and durable anti-tumor immune responses in the body, demonstrating great potential in tumor prevention and treatment. The mucosal site has a rich mucosal immune system, and mucosal immune activation is key to the prevention and treatment of mucosal tumors. Clinically, extramucosal routes (subcutaneous, intramuscular, etc.) are commonly used for vaccination; however, this route has poor mucosal immune stimulation effects, mainly due to the significant compartmentalization and mucosal homing characteristics of the mucosal immune system. In contrast, oral mucosal vaccines are a promising alternative. The gut is the largest immune organ in the human body, with extensive mucosal distribution. Oral mucosal vaccines can induce strong mucosal and systemic immune responses. The resulting immune effector cells can reach the gut and other mucosal sites through mucosal homing, thereby inhibiting the growth and metastasis of mucosal tumors.

[0003] Although oral mucosal vaccines have many advantages for the treatment of mucosal tumors such as colorectal cancer, such as high safety, good patient compliance and low medical costs, there are still some serious problems in practical applications. For example, the complex gastrointestinal environment, including gastric acid and enzymes, affects vaccine stability; in addition, immune tolerance exists in the intestinal site, which limits the immune effect of the vaccine. Summary of the Invention

[0004] Based on the current state of the technology, this invention aims to provide a novel immune-activated nanosheet and its preparation method, along with its application in oral mucosal vaccines for the prevention and / or treatment of mucosal cancer. The immune-activated nanosheets of this invention possess characteristics such as good oral stability, high mucosal targeting, and strong immunogenicity. They exhibit good stability in the gastrointestinal environment, can effectively accumulate in the small intestine to form a vaccine library, and can overcome intestinal immune tolerance, enhancing the immune effect, thus showing promising clinical application prospects.

[0005] In a first aspect, the present invention provides an immune-activating nanosheet. The immune-activating nanosheet comprises a two-dimensional montmorillonite nanosheet with a layered structure, and manganese ions and tumor antigens loaded between the layers of the two-dimensional montmorillonite nanosheet; wherein the mass ratio of manganese ions to montmorillonite is 1:5 to 1:20, and the mass ratio of tumor antigen to montmorillonite is 1:1 to 1:10.

[0006] Preferably, the tumor antigen is one or more of the model antigen ovalbumin (OVA), ovalbumin polypeptide OVAAPeptide (257-264), and specific neotumor antigen Adpgk, with ovalbumin (OVA) being the most preferred.

[0007] Preferably, the two-dimensional montmorillonite nanosheets have a lateral dimension of 100–300 nm and a thickness of 1–10 nm.

[0008] Secondly, the present invention provides a method for preparing immune-activated nanosheets. The preparation method includes: Step (1) Purify the raw montmorillonite ore by differential centrifugation; Step (2) Add the purified montmorillonite to the manganese ion solution, adjust the pH to 4-5.5, stir the reaction, centrifuge to remove the supernatant, and obtain precipitate A; wash precipitate A with deionized water to obtain manganese-rich montmorillonite; Step (3) The manganese-rich montmorillonite is added to an aqueous solution of tumor antigen, and after ultrasonic oscillation and standing, the supernatant is removed by centrifugation to obtain precipitate B; Step (4) Disperse the precipitate B in deionized water, ultrasonically peel it off, centrifuge to remove the precipitate, and take the supernatant to obtain the aqueous dispersion of the immune-activating nanosheets.

[0009] Preferably, in step (2), the manganese ion solution is one or more of manganese sulfate aqueous solution, manganese chloride aqueous solution, and manganese nitrate aqueous solution; preferably, the concentration of the manganese ion solution is 0.2 to 0.6 mol / L.

[0010] Preferably, in step (2), the temperature of the stirring reaction is 50-100°C and the stirring reaction time is 4-6 hours.

[0011] Preferably, in step (3), the settling temperature is 0 to 10°C and the settling time is 12 to 24 hours.

[0012] Preferably, in step (4), the ultrasonic ablation power is 200-400W and the ultrasonic ablation time is 4-20min.

[0013] Thirdly, the present invention provides the application of the aforementioned immune-activating nanosheets in the preparation of an immunostimulant for oral delivery.

[0014] Fourthly, the present invention provides the application of the aforementioned immune-activated nanosheets in the preparation of tumor vaccines for the prevention and / or treatment of mucosal cancer.

[0015] The beneficial effects of this invention are:

[0016] This invention provides an immune-activating nanosheet and its preparation method, which features mild preparation conditions and simple operation. The immune-activating nanosheet provided by this invention exhibits good stability in the gastrointestinal environment, protecting tumor antigens from attack by gastric acid, enzymes, etc. The immune-activating nanosheet provided by this invention possesses good immunogenicity, effectively stimulating mucosal immunity and playing a role in the prevention and treatment of tumors. The immune-activating nanosheet provided by this invention repurposes montmorillonite, achieving tumor vaccine delivery via oral administration. It features good safety, high patient compliance, and low medical costs, and has excellent clinical application prospects. Attached Figure Description

[0017] Figure 1 A flowchart illustrating the preparation process of the immune-activating nanosheets according to the present invention is shown.

[0018] Figure 2 The particle size distribution of the immune-activating nanosheets prepared according to Example 1 of the present invention is shown.

[0019] Figure 3 The morphology of the immune-activated nanosheets prepared according to Example 1 of the present invention is shown under a transmission electron microscope.

[0020] Figure 4 XPS analysis of the immune-activating nanosheets according to the present invention is shown.

[0021] Figure 5 The manganese ion release curve of the immune-activating nanosheets according to the present invention is shown.

[0022] Figure 6 XRD analysis of the immune-activating nanosheets according to the present invention is shown.

[0023] Figure 7 The diagram shows the particle size variation of the immune-activated nanosheets in artificial gastric and intestinal fluids according to Embodiment 2 of the present invention.

[0024] Figure 8 The following are transmission electron micrographs of the immune-activated nanosheets according to Embodiment 2 of the present invention after being stored in artificial gastric fluid and artificial intestinal fluid for one week. Figures A and B are electron micrographs of the artificial gastric fluid and artificial intestinal fluid, respectively.

[0025] Figure 9 The following are the evaluation results of the effect of the immune-activated nanosheets in improving the function of intestinal dendritic cells according to Embodiment 3 of the present invention. Figure A is a flow cytometry analysis result of immune-activated nanosheets promoting dendritic cell antigen presentation; Figure B is a flow cytometry analysis result of immune-activated nanosheets promoting dendritic cell maturation.

[0026] Figure 10The diagram shows the flow cytometry results of the immune-activating nanosheets according to Example 4 of the present invention, which increased the level of specific T cells for OVA in the spleen. Detailed Implementation

[0027] The present invention is further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0028] The immune-activating nanosheets of this invention comprise two-dimensional montmorillonite nanosheets with a layered structure and manganese ions and tumor antigens loaded between the layers of the two-dimensional montmorillonite nanosheets. In other words, this invention uses uniform and stable two-dimensional nanosheets made primarily of montmorillonite as a carrier, and loads manganese ions and tumor antigens between the layers of these nanosheets to produce orally stable immune-activating nanosheets.

[0029] Existing technologies disclose the preparation of manganese dioxide-montmorillonite composite materials by co-precipitating manganese dioxide onto the surface of montmorillonite using montmorillonite as a growth matrix. These manganese dioxide nanozymes loaded onto the montmorillonite surface are primarily used for intestinal repair. However, loading manganese dioxide onto the montmorillonite surface affects its surface charge and other properties, thus impacting its adhesion to the intestinal mucosa. This invention introduces manganese ions into the interlayer of montmorillonite nanosheets, enabling slow release of manganese ions without affecting montmorillonite accumulation in the intestine. Furthermore, the immune-activating nanosheets of this invention can stimulate intestinal mucosal immunity, potentially for the treatment of mucosal-associated tumors. If montmorillonite nanosheets, tumor antigens, and manganese-containing substances are simply mixed, the tumor antigens are not located in the middle of the montmorillonite layers, leading to direct contact with the harsh gastrointestinal environment after oral administration, resulting in inactivation. Moreover, under simple mixing conditions, the slow release of manganese ions is not achieved, failing to effectively stimulate the immune response.

[0030] The two-dimensional nanosheets have a layered sheet structure. Preferably, the two-dimensional montmorillonite nanosheets have a lateral dimension of 100–300 nm and a thickness of 1–10 nm.

[0031] In the immune-activating nanosheets, the mass ratio of manganese ions to montmorillonite is 1:5 to 1:20. If the mass ratio of manganese ions to montmorillonite exceeds this range, the manganese ions may not be effectively loaded or the immune-enhancing effect may be poor.

[0032] Tumor antigens of proteins or peptides are applicable to this invention. For example, the tumor antigen is one or more of the model antigen ovalbumin (OVA), ovalbumin peptide (OVA Peptide 257-264), and specific neotumor antigen Adpgk, preferably ovalbumin (OVA).

[0033] In the immune-activating nanosheets, the mass ratio of tumor antigen to montmorillonite is 1:1 to 1:10. If the mass ratio of tumor antigen to montmorillonite exceeds this range, the tumor antigen may not be effectively loaded or the immune-enhancing effect may be poor.

[0034] Tests have shown that the immune-activating nanosheets have a sheet-like structure. In some embodiments, the average particle size (diagonal distance of the nanosheets) of the immune-activating nanosheets is 150–250 nm.

[0035] This invention also provides a method for preparing the aforementioned immune-activating nanosheets. The immune-activating nanosheets of this invention are prepared by purifying natural montmorillonite, then loading manganese ions into the interlayer of the purified montmorillonite using an ion exchange method to obtain manganese-rich montmorillonite. Subsequently, tumor antigens are efficiently loaded into the interlayer of the manganese-rich montmorillonite through intercalation, and the resulting nanosheets are obtained after ultrasonic exfoliation. The following exemplarily illustrates the method for preparing the immune-activating nanosheets of this invention.

[0036] The raw montmorillonite ore is purified. Differential centrifugation can be used to purify the raw montmorillonite ore. For example, the differential centrifugation method includes the following steps: Weigh the raw montmorillonite ore, mix it with deionized water, and stir thoroughly to achieve hydration and expansion. The mass-to-volume ratio of raw montmorillonite ore to deionized water can be 10–100 g / mL. The stirring speed can be 400–600 rpm, and the stirring time can be 4–8 h. Next, large impurities are removed by low-speed centrifugation. The low-speed centrifugation speed can be 800–1200 rpm, and the low-speed centrifugation time can be 1–3 min. After removing impurities, the suspension is centrifuged at high speed, and the precipitate is collected to obtain purified montmorillonite. The high-speed centrifugation speed can be 10000–15000 rpm, and the high-speed centrifugation time can be 5–10 min.

[0037] As an example, weigh out raw montmorillonite ore, mix it with deionized water, and stir evenly. Stir at 400–600 rpm for 4–8 hours to achieve hydration and expansion. Then, centrifuge at 800–1200 rpm for 1–3 minutes to remove large particles of impurities. Centrifuge the suspension after impurity removal at 10000–15000 rpm for 5–10 minutes, and collect the precipitate to obtain purified montmorillonite.

[0038] Purified montmorillonite was added to a manganese ion solution, and the pH was adjusted to 4–5.5. After stirring, the supernatant was removed by centrifugation to obtain precipitate A. Precipitate A was washed with deionized water to obtain manganese-rich montmorillonite. A pH adjuster commonly used in the art can be used. For example, sodium citrate can be used to adjust the pH. Existing technologies use alkaline conditions to prepare manganese dioxide nanozymes via in-situ growth, where montmorillonite provides nucleation sites. Manganese dioxide is generated through the hydrolysis and oxidation of manganese ions under alkaline conditions and then grown in situ on the montmorillonite surface. In contrast, this invention uses acidic conditions of pH 4–5.5, which allows manganese ions to be loaded between the montmorillonite layers via ion exchange reactions, rather than being easily deposited on the montmorillonite surface. Too low a pH may cause manganese ions to be further acidified or precipitated into other oxides, while too high a pH may reduce the exchange capacity between manganese ions and montmorillonite in the solution. The stirring reaction temperature can be 50–100°C, and the stirring reaction time can be 4–6 hours.

[0039] As an example, purified montmorillonite was added to a manganese ion solution, the pH was adjusted to 4–5.5, and the mixture was magnetically stirred at 80°C for 4–6 hours. After centrifugation at 10,000–15,000 rpm for 15–20 minutes, the supernatant was removed, and the precipitate was washed 3–4 times with deionized water to obtain manganese-rich montmorillonite.

[0040] The manganese ion solution may be one or more aqueous solutions of manganese sulfate, manganese chloride, and manganese nitrate, preferably an aqueous solution of manganese sulfate. The concentration of the manganese ion solution may be 0.2–0.6 mol / L. In some embodiments, the solid-liquid ratio (mass-volume ratio) of purified montmorillonite to the aqueous manganese sulfate solution is 1:5 to 1:15, preferably 1:10.

[0041] The manganese-rich montmorillonite is added to an aqueous solution of tumor antigen. After ultrasonic oscillation and settling, the supernatant is removed by centrifugation to obtain precipitate B. The ultrasonic oscillation frequency can be 400–600 W. The ultrasonic oscillation time can be 10–20 min. The settling temperature can be 0–10 °C, preferably 4 °C. The settling time can be 12–24 h. The concentration of the aqueous solution of tumor antigen is 0.5–1 g / mL. In some embodiments, the solid-liquid ratio (mass-volume ratio) of manganese-rich montmorillonite to the aqueous solution of tumor antigen is 1:1–1:20, preferably 1:5.

[0042] For example, the obtained manganese-rich montmorillonite product is added to an aqueous solution containing tumor antigen, ultrasonically vibrated for 10-20 min, allowed to stand for 12-24 h, centrifuged at 10000-15000 rpm for 15-20 min, and the supernatant is removed to obtain precipitate B.

[0043] The precipitate B is dispersed in deionized water, ultrasonically exfoliated, and centrifuged to remove the precipitate. The supernatant is then collected to obtain the aqueous dispersion of the immune-activating nanosheets. The ultrasonic exfoliation power can be 200–400 W, and the ultrasonic exfoliation time can be 4–20 min. For example, the obtained precipitate is dispersed in deionized water, ultrasonically exfoliated, centrifuged at 2000–8000 rpm for 3–10 min to remove the precipitate, and the supernatant is collected to obtain the aqueous dispersion of the immune-activating nanosheets. Since precipitate B has a relatively large particle size, manganese-rich montmorillonite loaded with tumor antigen can be obtained by centrifugation to remove the precipitate. However, after precipitate B is dispersed in deionized water and ultrasonically exfoliated, the particle size decreases. Therefore, after appropriate ultrasonic exfoliation and centrifugation, the supernatant form of the immune-activating nanosheets can be obtained.

[0044] The present invention also provides the application of the immune-activating nanosheets as an oral mucosal immune stimulant in the preparation of vaccines for the treatment and prevention of tumors such as colorectal cancer.

[0045] This invention provides the results of qualitative and quantitative expression of the system in vitro and in vivo, as well as the evaluation of its immune effects.

[0046] This invention demonstrates, through in vivo imaging technology, that the prepared immune-activated nanosheets possess gastrointestinal stability and can accumulate in the small intestine. Immunological efficacy evaluation verifies that the immune-activated nanosheets can alleviate intestinal immune tolerance and enhance immune efficacy. The immune-activated nanosheets constructed in this invention are administered orally, and in addition to their application in colorectal cancer as described in this invention, they are also suitable for the treatment and prevention of various mucosal tumors, such as head and neck cancer and reproductive tract cancer.

[0047] In summary, this invention provides orally stable, mucosal immune-specific activating nanosheets, their preparation method, and their application in the treatment and prevention of mucosal tumors such as colorectal cancer. Utilizing the properties of montmorillonite, the immune-activating nanosheets of this invention, after oral administration, can resist the harsh environment of the gastrointestinal tract, accumulating and releasing antigens in the intestine; simultaneously, through the immunostimulatory effect of manganese ions, they stimulate intestinal mucosal immunity, achieving a specific killing effect against mucosal tumors such as colorectal cancer.

[0048] This invention is illustrated by the following description and embodiments. It should also be understood that the following embodiments are only for further illustration of the invention and should not be construed as limiting the scope of protection of the invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the invention are within the scope of protection of the invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the range based on the description herein, and are not necessarily limited to the specific values ​​in the examples below.

[0049] Example 1: Preparation and characterization of immune-activated nanosheets

[0050] Weigh 50g of raw montmorillonite ore, mix it with 1L of deionized water, and stir evenly. Stir at 400rpm for 4 hours to achieve hydration and expansion. Then, centrifuge at 1000rpm for 1 minute to remove large particles of impurities. After removing impurities, centrifuge the suspension at 10000rpm for 5 minutes, and collect the precipitate to obtain purified montmorillonite.

[0051] The purified montmorillonite was then added to a manganese sulfate aqueous solution (0.4 mol / L, with a mass-to-volume ratio of purified montmorillonite to manganese sulfate aqueous solution of 1:10). The pH was adjusted to 5, and the mixture was magnetically stirred at 80°C for 4 hours. After centrifugation at 12,000 rpm for 15 minutes, the supernatant was removed, and the mixture was washed three times with distilled water to obtain manganese-rich montmorillonite (Mn-MMT).

[0052] Subsequently, manganese-rich montmorillonite was added to an aqueous solution of tumor antigen OVA (the concentration of the aqueous solution of tumor antigen was 1 g / mL, and the mass-to-volume ratio (g / mL) of manganese-rich montmorillonite to the aqueous solution of tumor antigen was 1:5). The solution was sonicated for 15 min at a frequency of 400 W, allowed to stand at 4°C for 12 h, and then centrifuged to separate the precipitate. The supernatant was collected (named Supernatant A). Supernatant A was used for subsequent determination of OVA protein content to calculate antigen loading.

[0053] The precipitate was dispersed in deionized water and sonicated at 300W for 4 minutes to remove montmorillonite. Then, it was centrifuged at 4000rpm for 3 minutes to remove unremoved montmorillonite, thus obtaining the final immune-activated nanosheets (Mn-NMMT@OVA).

[0054] The particle size of the nanovaccine was determined using a Malvern particle size analyzer. The results showed that the particle size of the immune-activating nanosheets was 182 nm, and the PDI was 0.144. Figure 2 ).

[0055] The OVA protein content in supernatant A was determined using a BCA protein concentration assay kit (Beyotime Biotechnology Co., Ltd., catalogue P0012), thereby calculating the OVA antigen protein loading of the immune-activated nanosheets. The calculation formula was (total OVA sample mass - OVA mass in supernatant A) / mass of Mn-NMMT@OVA × 100%. The results showed that the OVA antigen protein loading of the immune-activated nanosheets was 76.2%. The calculated mass ratio of manganese ions to montmorillonite in the product immune-activated nanosheets was 1:7.9.

[0056] Take 5g of Mn-NMMT@OVA, add 5mL of aqua regia and 5mL of deionized water, and place in a microwave digester for programmed temperature rise. After digestion, cool to room temperature, open the cap, and place on an acid-removing apparatus to near dryness. Then transfer to a 50mL plastic volumetric flask and dilute to the mark with high-purity water. After standing, take the supernatant and determine the manganese ion content using ICP-OES to calculate the manganese ion loading of the immune-activating nanosheets. The calculation formula is manganese ion mass / mass of Mn-NMMT@OVA × 100%. The results show that the manganese ion loading of the immune-activating nanosheets is 12.6%. After calculation, the mass ratio of tumor antigen to montmorillonite in the product immune-activating nanosheets is 1:1.3.

[0057] The nanovaccines were characterized using transmission electron microscopy (TEM). TEM results showed that the immune-activating nanosheets exhibited a sheet-like structure. Figure 3 ).

[0058] XPS analysis was performed on the immune-activated nanosheets Mn-NMMT@OVA. The results showed that ( Figure 4 In the nanosheets, manganese mainly exists in the form of divalent ions.

[0059] Immunoactivated nanosheets Mn-NMMT@OVA were dispersed in artificial gastric fluid (Shanghai Yuanye Biotechnology Co., Ltd., catalogue R41110) and artificial intestinal fluid buffer systems (Shanghai Yuanye Biotechnology Co., Ltd., catalogue R24021). Samples were collected at different time points (0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, and 48h), and the manganese ion content was determined by ICP-OES to calculate the cumulative release. The results showed ( Figure 5 Manganese ions exhibit a slow release characteristic in both simulated gastric and intestinal fluids.

[0060] like Figure 1 As shown, montmorillonite is a hydrous aluminosilicate two-dimensional layered mineral. Its structure consists of silicon-oxygen tetrahedra and aluminum-oxygen tetrahedra interspersed with aluminum-oxygen octahedra. Its crystals possess excess permanent negative charges, resulting in a large number of cations capable of ion exchange between its layers. Therefore, antigen protein molecules and manganese ions are loaded into the interlayer of montmorillonite sheets through interlayer ion exchange reactions. The sustained-release properties of manganese ions in the immune-activating nanosheets Mn-NMMT@OVA demonstrate that manganese ions are loaded into the interlayer of montmorillonite nanosheets. XRD analysis of the immune-activating nanosheets Mn-NMMT@OVA showed that ( Figure 6Compared to montmorillonite (NMMT), the d(001) peak of the immune-activating nanosheet Mn-NMMT@OVA shifted from 6.34° to a lower angle of 3.01°. Calculations based on the Bragg equation nλ=2dsinθ showed that the interlayer spacing of Mn-MMT@OVA expanded to 3.36 nm, indicating that the OVA protein was loaded into the montmorillonite sheets, thus increasing the interlayer spacing.

[0061] Example 2: Evaluation of the gastrointestinal stability of nano-mucosal tumors

[0062] Immunoactivated nanosheets Mn-NMMT@OVA were dispersed in simulated gastric fluid (Shanghai Yuanye Biotechnology Co., Ltd., catalogue R41110) and simulated intestinal fluid buffer systems (Shanghai Yuanye Biotechnology Co., Ltd., catalogue R24021) and incubated at 4°C for one week. The particle size changes of the immunoactivated nanosheets over one week were measured using a Malvern particle size analyzer, and the morphology of the immunoactivated nanosheets was observed under transmission electron microscopy on day eight to determine if it was affected.

[0063] The results showed that the particle size of the immune-activated nanosheets did not change significantly after being placed in simulated gastric and intestinal fluids for one week. Figure 7 Furthermore, its morphology remains intact, preserving its complete sheet-like structure. Figure 8 This indicates that the immune-activating nanosheets can remain stable in the gastrointestinal tract.

[0064] Example 3: Evaluation of the ability of immune-activating nanosheets to improve the intestinal immune microenvironment

[0065] C57BL / 6 mice were administered via gavage with immune-activating nanosheets (Mn-NMMT@OVA), conventional aluminum adjuvant vaccine (Alum@OVA), or saline (Mn 2+ : 1mg / kg, OVA: 2mg / kg, Al 3+(2.5 mg / kg). 24 hours later, the mice were dissected, the intestines were removed, the mesenteric tissue and fat were removed, the intestines were flushed, digested, and single-cell suspensions were prepared. According to the instructions of the fluorescently labeled antibody kit (Biolegend Biotechnology's FITC anti-mouse CD11c antibody: catalogue 117306, APC anti-mouse CD80 antibody: catalogue 104713, PE / Cy7 anti-mouse CD86 antibody: catalogue 105013, PE anti-mouse H-2Kb bound to SIINFEKL antibody: catalogue 141603), the single cells were stained with fluorescently labeled antibodies. The proportions of CD11c+ / CD80+ / CD86+ and CD11c+ / H-2Kb SIINFEKL+ cells were analyzed by flow cytometry to examine the maturation and antigen-presenting capacity of dendritic cells.

[0066] The results show that: Figure 9 As shown in Figure A, the cell proportions of CD11c+ / CD80+ / CD86+ in the saline group, aluminum adjuvant vaccine group, and immune-activated nanosheet group were 15.0%, 19.3%, and 33.6%, respectively. Figure 9 As shown in Figure B, the proportions of CD11c+ / H-2Kb SIINFEKL+ cells in the saline group, aluminum adjuvant vaccine group, and immune-activated nanosheet group were 4.22%, 7.04%, and 14.3%, respectively. Immune-activated nanosheets can more effectively increase the proportions of CD11c+ / CD80+ / CD86+ and CD11c+ / H-2Kb SIINFEKL+ cells, indicating that immune-activated nanosheets can more effectively promote dendritic cell maturation and antigen presentation, improve the intestinal immune microenvironment, and enhance vaccine efficacy.

[0067] Example 4: Evaluation of the effect of immune-activating nanosheets in inducing a systemic specific immune response

[0068] C57BL / 6 mice were administered via gavage on days 0, 3, and 8 with immune-activated nanosheets (Mn-NMMT@OVA), conventional aluminum adjuvant vaccine (Alum@OVA), or saline (Mn-NMMT@OVA). 2+ 1 mg / kg, OVA: 2 mg / kg, Al 3+:2.5 mg / kg). On day 12, mice were sacrificed, spleens were harvested, cut into small pieces, and immersed in a solution of collagenase IV (2 mg / mL) containing DNase I (0.2 mg / mL) for digestion at 37°C for 30 min. The tissue was then passed through a 70 μm filter and washed three times with PBS to obtain a single-cell suspension, which was stimulated with tumor antigen. According to the instructions for use of the fluorescently labeled antibody kits (Biolegend Biotechnology's APC anti-mouse CD3e antibody: catalogue 100235, FITC anti-mouse CD8a antibody: catalogue 100705; MBL Biotechnology's T-Select H-2Kb OVA tetramer-SIINFEKL-PE: catalogue TS-5001-1C), single cells were stained with fluorescently labeled antibodies, and the percentage of CD3+ / CD8+ / OVA tetramer+ was analyzed by flow cytometry to detect the level of specific T cells against OVA.

[0069] The results show that: Figure 10 As shown, the proportions of CD3+ / CD8+ / OVA tetramer+ cells in the saline group, aluminum adjuvant vaccine group, and immune-activated nanosheet group were 4.75%, 6.72%, and 13.0%, respectively. Immune-activated nanosheets can more effectively increase the proportion of CD3+ / CD8+ / OVA tetramer+ cells in the spleen, indicating that immune-activated nanosheets can more effectively increase the level of specific T cells against OVA and induce a systemic specific immune response.

Claims

1. An immune-activating nanosheet, characterized in that, The immune-activating nanosheets comprise two-dimensional montmorillonite nanosheets with a layered structure, and manganese ions and tumor antigens loaded between the layers of the two-dimensional montmorillonite nanosheets; wherein the mass ratio of manganese ions to montmorillonite is 1:5 to 1:20, and the mass ratio of tumor antigens to montmorillonite is 1:1 to 1:

10.

2. The immune-activating nanosheet according to claim 1, characterized in that, The tumor antigen is one or more of the following: the model antigen ovalbumin (OVA), ovalbumin peptide (OVA Peptide (257-264), and the specific neotumor antigen Adpgk, preferably ovalbumin (OVA).

3. The immune-activating nanosheet according to claim 1 or 2, characterized in that, The two-dimensional montmorillonite nanosheets have a lateral dimension of 100–300 nm and a thickness of 1–10 nm.

4. The method for preparing immune-activated nanosheets according to any one of claims 1 to 3, characterized in that, The preparation method includes: Step (1) Purify the raw montmorillonite ore by differential centrifugation; Step (2) Add the purified montmorillonite to the manganese ion solution, adjust the pH to 4-5.5, stir the reaction, centrifuge to remove the supernatant, and obtain precipitate A; wash precipitate A with deionized water to obtain manganese-rich montmorillonite; Step (3) The manganese-rich montmorillonite is added to an aqueous solution of tumor antigen, and after ultrasonic oscillation and standing, the supernatant is removed by centrifugation to obtain precipitate B; Step (4) Disperse the precipitate B in deionized water, ultrasonically peel it off, centrifuge to remove the precipitate, and take the supernatant to obtain the aqueous dispersion of the immune-activating nanosheets.

5. The preparation method according to claim 4, characterized in that, In step (2), the manganese ion solution is one or more of manganese sulfate aqueous solution, manganese chloride aqueous solution, and manganese nitrate aqueous solution; preferably, the concentration of the manganese ion solution is 0.2 to 0.6 mol / L.

6. The preparation method according to claim 4 or 5, characterized in that, In step (2), the temperature of the stirring reaction is 50-100℃, and the stirring reaction time is 4-6 hours.

7. The preparation method according to any one of claims 4 to 6, characterized in that, In step (3), the settling temperature is 0 to 10°C and the settling time is 12 to 24 hours.

8. The preparation method according to any one of claims 4 to 7, characterized in that, In step (4), the ultrasonic ablation power is 200-400W and the ultrasonic ablation time is 4-20min.

9. The use of the immune-activating nanosheets according to any one of claims 1 to 3 in the preparation of an immunostimulant for oral delivery.

10. The use of the immune-activated nanosheets according to any one of claims 1 to 3 in the preparation of tumor vaccines for the prevention and / or treatment of mucosal cancer.

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