A sugar-modified manganese-based nano-adjuvant, manganese-based nano-vaccine, and preparation method and application thereof

Abstract

The present application relates to a kind of sugar modified manganese-based nano adjuvant, manganese-based nano vaccine and its preparation method and application, the sugar modified manganese-based nano adjuvant includes manganese phosphate core and the phospholipid bilayer shell with positive charge with the surface modification of mannose.It can effectively adsorb immune antigen;It can effectively deliver immune antigen to antigen presenting cell, and greatly enhance antigen presenting cell uptake immune antigen, target improvement tumor microenvironment, so as to efficiently activate immune cell, obtain more excellent immunotherapy effect when using lower dosage adsorbing less antigen.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of vaccines, and relates to a sugar-modified manganese-based nano-adjuvant, a manganese-based nano-vaccine and a preparation method and application thereof, in particular to a sugar-modified manganese-based nano-adjuvant for double regulation of antigen-presenting cells, a manganese-based nano-vaccine and a preparation method and application thereof. BACKGROUND

[0002] In recent years, tumor immunotherapy has become a new treatment for tumors. Targeted regulation of antigen-presenting cells (APCs) is an important strategy. APCs mainly include dendritic cells and macrophages. Since the birth of tumor immunotherapy, the effect has not been satisfactory, mainly due to the weak immunogenicity of tumors, especially the low efficiency of antigen presentation and the existence of immunosuppressive microenvironments including M2-type TAM and MDSC. In order to solve the above two problems, multifunctional nanomaterials have entered the stage.

[0003] Nanomaterials can significantly enhance the antigen delivery efficiency of cancer vaccines, target immune cells and improve safety as delivery carriers; in addition, specific types of nanomaterials also play the function of immunomodulators, as adjuvant components other than antigens, further enhancing immune protection.

[0004] Nanovaccine is an organic, inorganic or composite nanocarrier that can adsorb antigens, with a particle size of 50-100 nm, which can more easily penetrate lymph nodes or tumors. Many metal ions, such as aluminum ions, calcium ions, bismuth ions, platinum ions, lipids or polymers, have been developed as nanoadjuvants to improve tumor immunogenicity and enhance immunotherapy effect.

[0005] Manganese adjuvant is a new type of adjuvant developed in recent years. At present, there have been some reports on the preparation of manganese adjuvant into nanovaccine, which has strong industrial production, transformation and application prospects. A team found that manganese elements can promote the generation of STING activator cGAMP molecules, can activate the interferon pathway in tumor-draining lymph node dendritic cells (tdLN DC), enhance the process of antigen recognition and presentation, and thus promote the proliferation of cytotoxic T lymphocytes and their migration to tumor tissues. In addition, manganese ions can also promote the reversal of tumor-associated macrophages (TAM) from M2 type to M1 type. M1-type TAM is a minority in malignant tumors, while M2-type TAM is a majority. M2-type TAM is involved in tumor invasion, metastasis and immunosuppression to promote tumor growth.

[0006] The functional modification of manganese-based nanoadjuvant can achieve efficient and safe delivery of the adjuvant. That is, compared with free manganese ions, manganese-based nanoadjuvant can achieve precise delivery of manganese elements and immune regulation, thereby reducing the influence of off-target toxic side effects. Therefore, it is very meaningful to develop a functional manganese-based nanoadjuvant and a manganese-based nanovaccine. SUMMARY

[0007] In view of the deficiencies of the prior art, the purpose of the present application is to provide a sugar-modified manganese-based nano-adjuvant, a manganese-based nano-vaccine, and a preparation method and application thereof, and in particular to provide a sugar-modified manganese-based nano-adjuvant for double-regulation of antigen-presenting cells, a manganese-based nano-vaccine, and a preparation method and application thereof.

[0008] To achieve the purpose of the present application, the following technical solutions are adopted in the present application:

[0009] In a first aspect, the present application provides a sugar-modified manganese-based nano-adjuvant, which comprises a manganese phosphate core and a phospholipid bilayer shell with mannose on the surface and with positive charges.

[0010] The manganese-based nano-adjuvant involved in the present application can effectively adsorb immune antigens; can effectively deliver immune antigens to antigen-presenting cells, and greatly enhance the uptake of immune antigens by antigen-presenting cells, target the improvement of tumor microenvironment, thereby efficiently activating immune cells, and obtain more excellent immunotherapy effect when using a lower dose to adsorb less antigens. The manganese-based nano-adjuvant can target APCs, positively enhance DC function and reversely regulate TAM phenotype, activate STING signal of DC, promote secretion of type I interferon, promote reprogramming of TAM from M2 type to M1 type, and enhance anti-tumor immunotherapy effect in an immune double-regulation manner when administered peritumorally in a tumor-bearing mouse model.

[0011] The mannose enhances nano-biological interaction (recognition and endocytosis) mediated by C-type lectin receptors (such as CD206), and can target antigen-presenting cells, including dendritic cells (DCs) and tumor-associated macrophages (TAMs), improve antigen presentation ability, trigger innate immune signals, including activation of DCs, remodeling of TAMs, and subsequent induction of strong T cell immune effect, thereby effectively inhibiting tumor growth.

[0012] The phospholipid bilayer shell with positive charges can effectively adsorb antigen molecules, and proton sponge effect occurs in the lysosomal acidic environment, which helps the release of manganese ions, thereby activating the immune signal STING pathway, promoting the generation of STING activator cGAMP molecules, activating the interferon pathway in tumor-draining lymph node dendritic cells (tdLN DCs), enhancing the antigen recognition and presentation process, and thereby promoting the proliferation and migration of cytotoxic T lymphocytes to tumor tissues. STING is a cytoplasmic protein, so the most important problem to be solved for the nano-material to activate the STING pathway is to achieve cytoplasmic release of the drug. For this purpose, we pay attention to a point in the material design. In addition, the released manganese ions can also promote the reversal of tumor-associated macrophages (TAMs) from M2 type to M1 type, and thereby have the potential to promote CD8 +T cell proliferation enhances the efficacy of anti-tumor immunotherapy through a dual immune regulation mechanism.

[0013] Preferably, the raw materials for preparing the phospholipid bilayer shell include phospholipid materials and cholesterol; the phospholipid materials include sodium dioleoylphosphatidyl salt, (2,3-dioleoyl-propyl)-trimethylamine, and mannose-modified phospholipid materials.

[0014] Preferably, the mannose-modified phospholipid material includes mannose-modified DSPE-PEG (abbreviated as Man-PEG-DSPE).

[0015] DSPE-PEG in phospholipid materials can enhance hydrophilicity, while DSPE-PEG modified with mannose molecules can improve the lymph node targeting of nano-vaccine particles. Compared with the traditional method of modifying mannose molecules separately after the synthesis of nanocarriers, mannose molecules are attached to the PEG chain in one step, which can achieve a simpler preparation.

[0016] Preferably, the molar ratio of sodium dioleoyl phospholipid, (2,3-dioleoyl-propyl)-trimethylamine, mannose-modified phospholipid material, and cholesterol is 1:(1-5):(0.1-1):(1-5).

[0017] Among them, the specific point values ​​in (1-5) can be 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, etc., and the specific point values ​​in (0.1-1) can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 0.8, 1, etc. All specific point values ​​within the above ranges can be selected, and will not be elaborated further here.

[0018] In this invention, drug in vivo escape, cytoplasmic release, antigen electrostatic adsorption, and targeted modification are achieved by selecting specific types and ratios of phospholipid materials. A phospholipid bilayer shell prepared from sodium dioleoylphosphatrate, (2,3-dioleoyl-propyl)-trimethylamine, mannose-modified phospholipid materials, and cholesterol in specific molar ratios exhibits good stability and biosafety. It can also more effectively adsorb antigen molecules and, in the acidic environment of lysosomes, undergo a proton sponge effect, facilitating the release of manganese ions. This, in turn, activates the STING immune signaling pathway and promotes the reversal of tumor-associated macrophages (TAMs) from M2 to M1, potentially promoting CD8+ activation. + T cell proliferation enhances the efficacy of anti-tumor immunotherapy through a dual immune regulation mechanism.

[0019] Preferably, the molar ratio of the manganese element to the phosphorus element is 5:(0.25-5), such as 5:0.25, 5:0.5, 5:0.75, 5:1, 5:1.5, 5:2, 5:2.5, 5:3, 5:3.5, 5:4, 5:4.5, 5:5, etc., and specific point values in the above numerical range can be selected, which will not be repeated here.

[0020] The molar ratio of the manganese element to the phosphorus element is specifically limited to 5:(0.25-5) because the nano-sized manganese-based adjuvant particles with a particle size in the range of 1-100 nm can be prepared to achieve lymph node penetration and targeted drug delivery.

[0021] In a second aspect, the present application provides a preparation method of the sugar-modified manganese-based nano-adjuvant according to the first aspect, which comprises:

[0022] (1) mixing an aqueous solution of a divalent manganese salt with an oil phase to form a first water-in-oil emulsion; mixing an aqueous solution of a phosphate or hydrogen phosphate with an oil phase, and then mixing with a dioleoylphosphatidic acid sodium salt solution to form a second water-in-oil emulsion;

[0023] (2) mixing the first water-in-oil emulsion with the second water-in-oil emulsion to form co-precipitated nanoparticles, then adding an organic solvent to destroy the oil phase, centrifuging to obtain a precipitate, and then dissolving the precipitate in an organic solvent again;

[0024] (3) mixing the product of step (2) with (2,3-dioleoyl-propyl)-trimethylamine, a mannose-modified phospholipid material, and cholesterol, and then performing a thin film hydration method to obtain the sugar-modified manganese-based nano-adjuvant.

[0025] The sugar-modified manganese-based nano-adjuvant according to the present application is formed by co-precipitation and gradual coordination self-assembly of Mn ions and phospholipids in a double oil phase microemulsion. Compared with the aqueous phase synthesis method of protein template mineralization, the reverse phase microemulsion method for preparing manganese-based nano-adjuvant in an oil phase can make the particles not only small in size, more uniform in morphology, and more controllable in particle size.

[0026] Preferably, the divalent manganese salt includes any one or a combination of at least two of manganese chloride, manganese nitrate, or manganese sulfate.

[0027] Preferably, the concentration of the aqueous solution of the divalent manganese salt is 5-1000 mmol / L, such as 5 mmol / L, 10 mmol / L, 30 mmol / L, 50 mmol / L, 100 mmol / L, 200 mmol / L, 500 mmol / L, 700 mmol / L, 1000 mmol / L, etc.

[0028] Preferably, the volume ratio of the aqueous solution of the divalent manganese salt to the oil phase is 1: (10-1000), such as 1:10, 1:30, 1:50, 1:100, 1:200, 1:400, 1:500, 1:700, 1:800, 1:1000, etc.

[0029] Preferably, the concentration of the aqueous solution of the phosphate or hydrogen phosphate is 1-200 mmol / L, such as 1 mmol / L, 10 mmol / L, 30 mmol / L, 50 mmol / L, 100 mmol / L, 120 mmol / L, 150 mmol / L, 180 mmol / L, 200 mmol / L, etc.

[0030] Preferably, the volume ratio of the aqueous solution of the phosphate or hydrogen phosphate to the oil phase is 1: (10-1000), such as 1:10, 1:30, 1:50, 1:100, 1:200, 1:400, 1:500, 1:700, 1:800, 1:1000, etc.

[0031] Other specific point values within the above-mentioned numerical ranges can also be selected, which will not be repeated here.

[0032] Preferably, the oil phase comprises any one or a combination of at least two of cyclohexane, cyclopentane, benzene or petroleum ether.

[0033] Preferably, a non-ionic surfactant is added to the oil phase. Adding a certain amount of non-ionic surfactant to the oil phase is conducive to the uniform dispersion of the aqueous phase in the oil phase and promotes emulsification.

[0034] Preferably, the non-ionic surfactant comprises any one or a combination of at least two of alkylphenol polyoxyethylene ether, octylphenol polyoxyethylene ether, polyoxyethylene nonyl phenyl ether.

[0035] Preferably, the volume ratio of the oil phase to the non-ionic surfactant is 1: (1-5), such as 1:1, 1:2, 1:3, 1:4, 1:5, etc.

[0036] Preferably, the mixing in step (2) is carried out under stirring, the stirring temperature is 37-60℃ (such as 37℃, 40℃, 42℃, 45℃, 50℃, 55℃, 60℃, etc.), the stirring time is 1-30 min (such as 1 min, 5 min, 10 min, 15 min, 20 min, 30 min, etc.), and the stirring speed is 200-500 r / min (such as 200 r / min, 250 r / min, 300 r / min, 400 r / min, 500 r / min, etc.).

[0037] Other specific point values within the above-mentioned numerical ranges can also be selected, which will not be repeated here.

[0038] Preferably, the organic solvent includes any one or a combination of at least two of methanol, ethanol, propanol, methyl ether, and ethyl ether.

[0039] Preferably, the thin film hydration method includes: evaporating the organic solvent of the mixed solution of step (3) to form a lipid film by a rotary evaporator, adding a buffer solution or deionized water, and ultrasonic treatment.

[0040] Preferably, the ultrasonic treatment is performed at 40-60℃ (for example, 40℃, 45℃, 50℃, 55℃, 60℃, etc.) for 5-10min (for example, 5min, 6min, 7min, 8min, 9min, 10min, etc.).

[0041] Other specific point values within the above-mentioned numerical ranges can also be selected, which will not be repeated here.

[0042] In a third aspect, the present application provides a sugar-modified manganese-based nanovaccine, which is formed by electrostatic adsorption and assembly of an antigen molecule and the sugar-modified manganese-based nanoadjuvant of the first aspect.

[0043] The antigen molecule can be selected according to actual needs, and thus the manganese-based nanoadjuvant and the manganese-based nanovaccine of the present application have wide clinical conversion and biomedical application prospects.

[0044] Preferably, the particle size of the sugar-modified manganese-based nanovaccine is 10-100nm, for example, 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, etc.

[0045] Preferably, the antigen molecule includes a tumor-associated antigen molecule.

[0046] Preferably, the mass ratio of manganese in the antigen molecule to the sugar-modified manganese-based nanoadjuvant of the first aspect is 1:(0.5-10), for example, 1:0.5, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc., preferably 1:(0.5-2). The particle size of the manganese-based nanovaccine prepared under this condition is about 50nm and is more uniform.

[0047] Preferably, the electrostatic adsorption assembly is incubated in a buffer at 25-37℃ (e.g., 25℃, 28℃, 30℃, 32℃, 35℃, 37℃, etc.) for 30-90 min (e.g., 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, etc.).

[0048] Other specific point values within the above-mentioned numerical ranges can also be selected, which will not be described here one by one.

[0049] In a fourth aspect, the application provides a use of the sugar-modified manganese-based nano-adjuvant according to the first aspect or the sugar-modified manganese-based nano-vaccine according to the third aspect in the preparation of a tumor immunotherapy drug.

[0050] Compared with the prior art, the application has the following beneficial effects:

[0051] The manganese-based nano-adjuvant involved in the application can effectively adsorb immune antigens; can effectively deliver the immune antigens to antigen-presenting cells, and greatly enhances the uptake of immune antigens by antigen-presenting cells, targets the improvement of the tumor microenvironment, thereby efficiently activates immune cells, and obtains more excellent immunotherapy effect when using a lower dose to adsorb less antigens.

[0052] Among them, the mannose enhances the nano-biological interaction (recognition and endocytosis) mediated by C-type lectin receptors (such as CD206), which can target antigen-presenting cells, including dendritic cells (DCs) and tumor-associated macrophages (TAMs), improve antigen presentation ability, trigger innate immune signals, including activating DCs, remodeling TAMs and subsequently inducing strong T cell immune effect, thereby effectively inhibiting tumor growth.

[0053] Among them, the positively charged phospholipid bilayer shell can effectively adsorb antigen molecules, and proton sponge effect occurs in the lysosomal acidic environment, which helps the release of manganese ions, thereby activating the immune signal STING pathway, promoting the generation of STING activator cGAMP molecules, activating the interferon pathway in tumor-draining lymph node dendritic cells (tdLN DCs), enhancing the antigen recognition and presentation process, thereby promoting the proliferation of cytotoxic T lymphocytes and their migration to tumor tissues. STING is a cytoplasmic protein, so the most important problem to be solved in the activation of the STING pathway by the nano material is to achieve cytoplasmic release of the drug. For this purpose, we pay attention to a point in the material design. In addition, the released manganese ions can also promote the reversal of tumor-associated macrophages (TAMs) from M2 type to M1 type, thereby having the potential to promote the proliferation of CD8 + T cells, and enhance the anti-tumor immunotherapy effect in an immune dual-regulation manner.

[0054] The sugar-modified manganese-based nanovaccine relates to the electrostatic adsorption and assembly of an antigen molecule and the above-mentioned sugar-modified manganese-based nanoadjuvant. The antigen molecule can be selected according to actual needs, and thus the manganese-based nanoadjuvant and the manganese-based nanovaccine both have wide clinical conversion and biomedical application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 is a TEM image of the manganese-based nanovaccine of the present application;

[0056] Figure 2 is an XPS image of the manganese-based nanovaccine of the present application;

[0057] Figure 3 is an element distribution image of the manganese-based nanovaccine of the present application;

[0058] Figure 4 is a biological toxicity test result and DC2.4 cell uptake result image of the manganese-based nanovaccine (oMan-LMP), wherein a is a biological toxicity test result image of the manganese-based nanovaccine (oMan-LMP); and b is a test result image of the manganese-based nanovaccine (oMan-LMP) being taken up and disassembled by DC2.4 cells;

[0059] Figure 5 is a result image of the DOTAP in the manganese-based nanovaccine promoting lysosome escape to enhance cytoplasmic release of drugs;

[0060] Figure 6 is an APC function modulation efficiency result image of the manganese-based nanovaccine (oMan-LMP), wherein a is a statistical chart of the expression amount analysis result of the interferon-related gene at the DC2.4 cell level; and b is a statistical chart of the Western Blot detection result of the expression level of the STING pathway-related protein;

[0061] Figure 7 is an accumulation situation result image of the manganese-based nanovaccine (oMan-LMP) in the lymph node, wherein a is an ex vivo imaging chart of the tumor-draining lymph node of the mouse after 12 hours of peritumoral administration in each group; and b is a statistical chart of the fluorescence signal intensity of the tumor-draining lymph node of the mouse after 12 hours of peritumoral administration in each group;

[0062] Figure 8 is a statistical result chart of the tumor volume in each group. DETAILED DESCRIPTION

[0063] The technical solutions of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application, and should not be regarded as specific limitations on the present application.

[0064] The antigen molecule model used in the following examples is OVA protein;

[0065] Example 1

[0066] This example provides a sugar-modified manganese-based nano-adjuvant, which is prepared as follows:

[0067] (1) 300 μL of 0.5M MnCl2·4H2O aqueous solution was added dropwise to 10 mL of oil phase containing cyclohexane and Igepal CO520 (volume ratio 7:3) under the action of water bath ultrasonic, to form a first water-in-oil emulsion;

[0068] 300 μL of 0.1M Na2HPO4 aqueous solution was added dropwise to 10 mL of oil phase containing cyclohexane and Igepal CO520 (volume ratio 7:3) under the action of water bath ultrasonic, and then 100 μL of 10 mM DOPA solution was added dropwise to the mixture under the action of water bath ultrasonic, to form a second water-in-oil emulsion;

[0069] (2) The first water-in-oil emulsion and the second water-in-oil emulsion were mixed and stirred at 25°C at 300 rpm for 10 min to form co-precipitated nanoparticles, 20 mL of ethanol was added under stirring to destroy the oil phase, and centrifugation was performed at 16,500 rpm for 30 min to obtain a precipitate, which was dissolved again in 5 mL of ethanol;

[0070] (3) 40 μL of 10 mM Man-PEG-DSPE, 690 μL of 10 mM DOTAP and 230 μL of 10 mM cholesterol were dissolved in 5 mL of CHCl3 and added to the product of step (2) above, rotary evaporation was performed at 37°C for 10 min, and then 5 min of 55°C hydration with normal saline was performed, to obtain the sugar-modified manganese-based nano-adjuvant (abbreviated as Man-LMP).

[0071] (4) According to the above method, the Man-LMP nano-adjuvant was synthesized, and a non-targeted manganese-based nano-adjuvant (abbreviated as LMP) was synthesized by replacing Man-PEG-DSPE with PEG-DSPE.

[0072] Example 2

[0073] This example provides a sugar-modified manganese-based nano-vaccine, which is prepared as follows:

[0074] The Man-LMP nano-adjuvant prepared in Example 1 was incubated with FITC-labeled OVA antigen in a buffer at 35°C for 60 min, the mass ratio of Mn element to OVA was 1:1, and finally a sugar-modified manganese-based nano-vaccine (abbreviated as oMan-LMP) was obtained; correspondingly, a non-targeted manganese-based nano-vaccine (abbreviated as o-LMP) was obtained.

[0075] The prepared oMan-LMP was subjected to basic performance characterization, and the results are shown in Figure 1 , Figure 2 , Figure 3 Among them, Figure 1 is a TEM image of the manganese-based nanovaccine of the application; Figure 2 is an XPS image of the manganese-based nanovaccine of the application; Figure 3 is an element distribution image of the manganese-based nanovaccine of the application.

[0076] As can be seen from Figure 1 , the average particle size of the constructed manganese-based nanovaccine is 24.1 nm.

[0077] As can be seen from Figure 2 , the constructed manganese-based nanovaccine contains elements such as C, N, O, Mn, and P, and the manganese element is divalent.

[0078] As can be seen from Figure 3 , the distribution of Mn, P, and O is consistent, indicating that the main structure of the particle is manganese phosphate structure.

[0079] Example 3

[0080] This example verifies that the manganese-based nanovaccine (oMan-LMP) has APC targeting due to mannose modification.

[0081] Cell viability detection: DC2.4 cells were seeded in a 96-well plate and treated with different concentrations (0-100 μg Mn / mL) of manganese-based nanovaccine (oMan-LMP) for 24 hours. CCK-8 kit was used to evaluate cell viability.

[0082] Confocal analysis experiment: DC2.4 cells were seeded in a six-well plate and cultured for 12 hours, then incubated with DiL-labeled oMan-LMP or DiL-labeled o-LMP for 1 hour and 4 hours, respectively. The cell nucleus was stained with Hoechst, and the OVA antigen was labeled with FITC.

[0083] The results are shown in Figure 4 . Figure 4 In the figure, a is the biological toxicity test result of the manganese-based nanovaccine (oMan-LMP); Figure 4 b is the test result of the manganese-based nanovaccine (oMan-LMP) being taken up and disassembled by DC2.4 cells.

[0084] As can be seen from Figure 4It can be seen that when the concentration of the nano-vaccine is below 10 μg / mL, the cell survival rate is above 80% after 24 hours of incubation, indicating that the preparation has good biological safety. After 1 hour of incubation with the nano-vaccine, stronger co-localization fluorescence signals of the antigen and the adjuvant were observed in DC2.4 compared with the o-LMP group without mannose modification, which indicates that the mannose modification can significantly promote the APC targeted delivery of the oMan-LMP nano-vaccine. After 4 hours of co-incubation, we found that the oMan-LMP nano-vaccine tends to disintegrate into free antigen OVA and nano-adjuvant in the cytoplasm, which is verified by the enhancement of the fluorescence signal, indicating that the components of the nano-vaccine have the possibility to function in the cytoplasm.

[0085] In order to verify the molecular mechanism of the APC targeted delivery of the vaccine, i.e. mediated by the surface overexpression of the C-type lectin family protein (mannose receptor CD206 molecule), we compared the mannose modified group with the o-LMP group without mannose modification and the free antigen group by labeling the antigen, and we used the competitive experiment method to pretreat the DC2.4 cells with 20 μM D-(+)-mannose for 30 minutes, and then incubate the oMan-LMP with the DC2.4.

[0086] The results are shown in Figure 5 . Figure 5 A result graph of DOTAP in the manganese-based nano-vaccine promoting lysosome escape to enhance cytoplasmic release of drugs.

[0087] From Figure 5 We can observe that after the pretreatment of the cells, the antigen uptake rate of the oMan-LMP nano-vaccine group is significantly reduced, indicating that the mannose receptor CD206 molecule on the surface of the DC has been competitively combined with D-(+)-mannose, so that the oMan-LMP cannot be quickly taken up. These experimental results further confirm that the modification of mannose promotes the DC targeted delivery of the nano-vaccine.

[0088] Example 4

[0089] This example verifies the cytoplasmic release and APC function regulation efficiency of the manganese-based nano-vaccine (oMan-LMP).

[0090] The expression amount of the interferon related genes of the DC2.4 cells after treatment with the nano-vaccine was standardized and analyzed by real-time quantitative PCR. The operation steps are as follows: 1) sample collection (on ice); 2) RNA extraction; 3) Nanodrop determination of RNA concentration; 4) RNA reverse transcription; 5) PCR reaction.

[0091] The expression levels of STING pathway-related proteins were detected by Western Blot: 1) extract sample protein; 2) prepare SDS-PAGE gel: according to the molecular weight of the target protein, prepare 12% separation gel and 5% concentrated gel; 3) SDS-PAGE gel electrophoresis: 4) detect target protein; 5) color reaction, film exposure; 6) result output: the band gray value is obtained by Quantity One v.4.6.2 software.

[0092] The results are shown in Figure 6 Figure 6 Figure a is a statistical chart of the expression level analysis of interferon-related genes of DC2.4 cells; Figure 6 Figure b is a statistical chart of the results of Western Blot detection of the expression levels of STING pathway-related proteins.

[0093] From the results Figure 6 It can be seen that oMan-LMP nano vaccine significantly promotes the up-regulation of the mRNA levels of interferon-related genes Tnf, Ifnb1, Iflt1, Ifltm10, Rsad2 and Irf7 by 2 times or even 3 times, which indicates that oMan-LMP can positively enhance and activate the immunological function of DC2.4 cells, is conducive to the release of interferon-related factors, and further activates innate immunity. Through Western blotting, it is further verified that oMan-LMP nano vaccine can significantly promote the expression of STING pathway-related proteins of DC2.4 cells, including STING and downstream phosphorylated TANK binding kinase 1 (p-TBK1) and interferon regulatory factor 3 (p-IRF3), which are increased by 2 times or even 3 times. This process is very important for anti-tumor immunity, because the activation of APC cell STING and its downstream signaling pathway can increase the production of type I interferon (I-IFN), such as β-IFN, and further stimulate the maturation and activation of APC, promote the activation of CD8 + T cells. In addition, type I IFN can directly act on CD8 + T cells to achieve proliferation and internal tumor migration. It has been reported that intratumoral injection of STING agonists can trigger the proliferation of tumor-specific CD8 + T cells. Traditional STING agonists adopt the mode of nano-carrier wrapping STING molecule agonist cGAMP, while here we adopt the mode of manganese ions directly coordinating with lipids to self-assemble into adjuvants, which simultaneously realizes the reasonable simplification of the dosage form and the improvement of the bioavailability of nano materials.

[0094] Example 5

[0095] This example verifies the accumulation of manganese-based nano vaccine (oMan-LMP) in lymph nodes. ​

[0096] OVA-B16F10 melanoma model mice were constructed: 3 x 10 5 OVA-B16F10 cells were subcutaneously inoculated into the right hind limbs of 6-week-old C57BL6N female mice. When the tumor volume of the mice reached 100 cubic millimeters, the drug was administered.

[0097] The manganese-based nanovaccine prepared in Example 2 was then administered to the constructed tumor model mice by peritumoral injection. The B16F10 melanoma model mice stably expressing OVA were divided into four groups: 1, blank group; 2, oMan-LMP group; 3, o-LMP group; 4, OVA group. When the average tumor volume reached 100 cubic millimeters, the peritumoral administration method was used, 1 group was injected with 100 μL of normal saline, 2 group was injected with 50 μg of Mn oMan-LMP nanovaccine, 3 group was injected with 50 μg of Mn o-LMP, and 4 group was injected with 100 μL of 50 μg of OVA.

[0098] The results are shown in Figure 7 Figure 7 wherein a is the ex vivo imaging graph of the tumor-draining lymph nodes of the mice in each group 12 hours after peritumoral administration, Figure 7 wherein b is the fluorescence signal intensity graph of the tumor-draining lymph nodes of the mice in each group 12 hours after peritumoral administration.

[0099] As can be seen from Figure 7 , the fluorescence signal of the oMan-LMP nanovaccine group is 2 times that of the non-targeted o-LMP group and 3 times that of the free OVA group, indicating that the mannose modification promotes the lymph node targeting of the nanovaccine, providing an important guarantee for the activation and regulation of tdLN DC.

[0100] Example 6

[0101] This example verifies the immunotherapy application prospect of the manganese-based nanovaccine (oMan-LMP).

[0102] The method for constructing OVA-B16F10 melanoma model mice is the same as that in Example 5.

[0103] ​The B16F10 melanoma model stably expressing OVA was divided into five groups, 1, blank group; 2, OVA group; 3, o-LMP group; 4, oMan-LMP group; 5, doxorubicin and oMan-LMP combined treatment group (DOX+oMan-LMP). When the average tumor volume grew to 100 cubic centimeters, three injections were performed on the first day (marked as -7d), the sixth day (marked as -2d), and the eighth day (marked as 0d), respectively, using peritumoral administration, 1 group was injected with 100 μL of normal saline; 2 group was injected with 100 μL of 50 μg of OVA; 3 group was injected with 50 μg of Mn o-LMP; 4 group was injected with 50 μg of Mn oMan-LMP nanovaccine; 5 group was injected with 50 μg of Mn oMan-LMP nanovaccine after DOX pretreatment. The growth of the tumor site and the change in tumor volume after three injections were recorded.

[0104] The results are shown in Figure 8 Figure 8 The tumor volume statistical results of each group are shown in the graph.

[0105] As can be seen from Figure 8 , compared with the tumor-bearing untreated group and the free OVA group, the growth of melanoma was significantly inhibited after three injections of oMan-LMP immunotherapy, and the survival of tumor-bearing mice was significantly prolonged. Notably, the o-LMP group of mice without mannose modification not only did not effectively inhibit the tumor, but also had a further tendency to grow worse, further emphasizing the significance of mannose modification and the importance of APC targeted regulation in inhibiting tumor growth. In order to take advantage of the endogenous neoantigens produced by tumor mutations and enhance tumor immunogenicity, we used a low-dose DOX injection to induce ICD effect, and then promote the release of neoantigens, better synergistically enhance the anti-tumor treatment effect of nanovaccine. Such immunotherapy and chemotherapy combined treatment strategy further improves the inhibition effect of melanoma, prolonging the survival of mice by about 10 days. In summary, the above results show that oMan-LMP can target and regulate APC in two mechanisms, enhance the STING pathway of DC cells, reverse M2 TAM to M1, significantly enhance tumor immunogenicity, and achieve high-efficiency tumor killing effect. At the same time, oMan-LMP nanovaccine shows the potential of a new type of cancer chemotherapy and immunotherapy strategy.

[0106] The applicant declares that the above-mentioned embodiments illustrate a sugar-modified manganese-based nano-adjuvant, manganese-based nano-vaccine, and its preparation method and application of the present application, but the present application is not limited to the above-mentioned embodiments, that is, it does not mean that the present application must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the scope of protection and disclosure of the present application.​

[0107] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details of the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0108] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not describe various possible combinations again.

Claims

1. A sugar-modified manganese-based nanoadjuvant, characterized in that, The sugar-modified manganese-based nanoadjuvant comprises a manganese phosphate core and a phospholipid bilayer shell with a surface modified with mannose and carrying a positive charge. The raw materials for preparing the phospholipid bilayer shell include phospholipid materials and cholesterol; the phospholipid materials include sodium dioleoyl phospholipidate, (2,3-dioleoyl-propyl)-trimethylamine, and mannose-modified phospholipid materials; the mannose-modified phospholipid materials include mannose-modified DSPE-PEG.

2. The sugar-modified manganese-based nanoadjuvant according to claim 1, characterized in that, The molar ratio of sodium dioleoyl phospholipid, (2,3-dioleoyl-propyl)-trimethylamine, mannose-modified phospholipid material to cholesterol is 1:(1-5):(0.1-1):(1-5).

3. The sugar-modified manganese-based nanoadjuvant according to claim 1, characterized in that, The molar ratio of manganese to phosphorus in the manganese-based nano-adjuvant is 5:(0.25-5).

4. The method for preparing the sugar-modified manganese-based nanoadjuvant according to any one of claims 1-3, characterized in that, The preparation method includes: (1) Mix the aqueous solution of divalent manganese salt with the oil phase to form a first water-in-oil emulsion; mix the aqueous solution of phosphate or hydrogen phosphate with the oil phase, and then mix with the sodium dioleoyl phosphatidyl salt solution to form a second water-in-oil emulsion; (2) The first water-in-oil emulsion and the second water-in-oil emulsion are mixed to form coprecipitated nanoparticles. Then, an organic solvent is added to destroy the oil phase, and the precipitate is obtained by centrifugation. The precipitate is then dissolved in the organic solvent again. (3) The product of step (2) is mixed with (2,3-dioleoyl-propyl)-trimethylamine, mannose-modified phospholipid material and cholesterol, and then subjected to thin film hydration to obtain the sugar-modified manganese-based nano-adjuvant.

5. The method for preparing the sugar-modified manganese-based nanoadjuvant according to claim 4, characterized in that, The divalent manganese salt includes any one or a combination of at least two of manganese chloride, manganese nitrate, or manganese sulfate.

6. The method for preparing the sugar-modified manganese-based nanoadjuvant according to claim 4, characterized in that, The concentration of the divalent manganese salt aqueous solution is 5-1000 mmol / L.

7. The method for preparing the sugar-modified manganese-based nanoadjuvant according to claim 4, characterized in that, The volume ratio of the divalent manganese salt aqueous solution to the oil phase is 1:(10-1000).

8. The method for preparing the sugar-modified manganese-based nanoadjuvant according to claim 4, characterized in that, The concentration of the aqueous solution of the phosphate or hydrogen phosphate is 1-200 mmol / L.

9. The method for preparing the sugar-modified manganese-based nanoadjuvant according to claim 4, characterized in that, The volume ratio of the aqueous solution of the phosphate or hydrogen phosphate to the oil phase is 1:(10-1000).

10. The method for preparing the sugar-modified manganese-based nanoadjuvant according to claim 4, characterized in that, The oil phase includes any one or a combination of at least two of cyclohexane, cyclopentane, benzene, or petroleum ether.

11. The method for preparing the sugar-modified manganese-based nanoadjuvant according to claim 4, characterized in that, The oil phase contains a nonionic surfactant.

12. The method for preparing the sugar-modified manganese-based nanoadjuvant according to claim 11, characterized in that, The nonionic surfactant includes any one or a combination of at least two of alkylphenol polyoxyethylene ether, octylphenol polyoxyethylene ether, and polyoxyethylene nonylphenyl ether.

13. The method for preparing the sugar-modified manganese-based nanoadjuvant according to claim 11, characterized in that, The volume ratio of the oil phase to the nonionic surfactant is 1:(1-5).

14. The method for preparing the sugar-modified manganese-based nanoadjuvant according to claim 4, characterized in that, The mixing in step (2) is carried out under stirring conditions, with a stirring time of 1-30 min and a stirring speed of 200-500 r / min.

15. The method for preparing the sugar-modified manganese-based nanoadjuvant according to claim 4, characterized in that, The organic solvent includes any one or a combination of at least two of methanol, ethanol, propanol, dimethyl ether, and diethyl ether.

16. The method for preparing the sugar-modified manganese-based nanoadjuvant according to claim 4, characterized in that, The thin film hydration method includes: evaporating the organic solvent of the mixed solution in step (3) using a rotary evaporator to form a lipid film, adding buffer solution or deionized water, and sonicating to obtain the final product.

17. The method for preparing the sugar-modified manganese-based nanoadjuvant according to claim 16, characterized in that, The ultrasound was performed at 40-60℃ for 5-10 minutes.

18. A sugar-modified manganese-based nanovaccine, characterized in that, The sugar-modified manganese-based nanovaccine is formed by electrostatic adsorption assembly of antigen molecules and sugar-modified manganese-based nanoadjuvants as described in any one of claims 1-3. The antigen molecule model is the OVA protein.

19. The manganese-based nanovaccine according to claim 18, characterized in that, The sugar-modified manganese-based nanovaccine has a particle size of 10-100 nm.

20. The manganese-based nanovaccine according to claim 18, characterized in that, The electrostatic adsorption assembly was incubated in a buffer solution at 25-37°C for 30-90 min.

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