A manganese-doped protein immune agonist assembly, construction method and application thereof

Abstract

The present invention provides a manganese-doped protein immune agonist assembly BSA-Man@Mn 2+ -Ft@Lap, construction method and application thereof, the assembly is composed of BSA-Man@Mn 2+ Assembled with Ft@Lap, the BSA-Man@Mn 2+ It is a mannose-modified bovine serum albumin BSA-Man and manganese ion Mn 2+ The Ft@Lap is formed by the complexation of ferritin and labaquinone. The immune agonist assembly delivers Mn 2+ Activation of the cGAS-STING signaling axis promotes DC maturation, and tumor cell-induced immunogenic apoptosis synergistically triggers powerful CTL-mediated immunotherapy. Enhancing DC-mediated cross-priming of CTLs in the TME improves the anti-tumor immune response in tumor models with poor immunogenicity, providing a new way to improve the effectiveness of immunotherapy for solid tumor indications in the clinic.

Description

Technical Field

[0001] The present invention belongs to the interdisciplinary field of medicinal chemistry and immunology, and specifically relates to a manganese-doped protein immune agonist assembly BSA-Man@Mn2+-Ft@Lap, a construction method and an application thereof. Background Art

[0002] In recent years, the research of immunotherapy has developed rapidly and has become a new hot spot in tumor treatment. Immunotherapy will become a promising anti-tumor treatment. Immunotherapy refers to the activation of the body's immune system and the induction of anti-tumor effects by relying on its own immune function. From a mechanistic point of view, the therapeutic effect of immunotherapy mainly depends on the mobilization of tumor-specific effector T cells, during which T cells are activated by interacting with antigen-binding class I major histocompatibility complex (MHC-1) expressed on antigen-presenting cells (APCs). However, in actual situations, the tumor microenvironment is in a highly immunosuppressive state to escape the attack of T cells, which makes tumor immunotherapy challenging. Specifically, tumor cells can greatly interfere with the antigen presentation process by inhibiting the maturation and activation of dendritic cells (DCs). Dendritic cells are the main type of APCs in the human body. The resulting dendritic cell dysfunction will lead to the failure of T cell priming and ultimately immune tolerance. Therefore, stimulating the maturation and function of dendritic cells has become a promising strategy to reshape T cell-mediated anti-tumor immunity.

[0003] Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) signaling is an evolutionarily conserved in vivo DNA sensing mechanism that plays a key role in regulating the relationship between tumor cells and surrounding immune cells, and has become a promising target for enhancing tumor immunotherapy. Specifically, when dsDNA from tumor cells is captured by dendritic cells and binds to cGAS dimers in their cytoplasm, its enzymatic activity is activated, catalyzing the reaction between adenosine triphosphate (ATP) and guanosine triphosphate (GTP) to generate 2'3' cyclic GMP-AMP (cGAMP). cGAMP acts as a second messenger and binds to the STING protein anchored in the endoplasmic reticulum. The STING protein conforms and moves from the endoplasmic reticulum to the Golgi apparatus. In the process, TANK-binding kinase 1 (TBK1) is recruited. The activated TBK1 phosphorylates interferon regulatory factor 3 (IRF3), ultimately inducing the release of type I interferon. Currently, several STING agonists have been developed in clinical trials, but the effects are not good. The tumor microenvironment is a physiologically complex niche with different cell populations, which makes it extremely challenging to specifically deliver STING agonists to DCs. On the other hand, the minimum concentration threshold of tumor-derived dsDNA is required for effective dsDNA sensing and may kinetically hinder the activation of cGAS-STING in DCs. In addition, solid tumor tissues have dense extracellular matrix and high interstitial fluid pressure, which severely limits the diffusion rate of STING agonists in the tumor matrix and weakens their potential immunotherapy efficacy. In summary, it has become a very urgent scientific issue to construct a safe and effective STING agonist delivery system and implement anti-tumor immune efficacy evaluation.

[0004] Manganese is an essential trace element for the human body and has good toxicological properties. A variety of oral manganese supplements have been approved for the prevention or treatment of manganese deficiency-related diseases. 2+ Ions play a key role in cGAS-STING-mediated DNA sensing, demonstrating that Mn 2+ It is the second cGAS activator besides DNA. 2+ It can directly bind to cGAS, enhancing its sensitivity to dsDNA and its enzymatic activity to produce cGAMP. Therefore, it can stimulate downstream STING signaling even at low levels of tumor-derived dsDNA. On the other hand, Mn 2+-cGAMP complexation can greatly enhance the STING binding affinity of cGAMP and promote the activation of STING. Based on the unique advantages and broad application prospects of manganese ions as activating factors of the STING pathway, researchers have carried out some basic research and application exploration. Based on the previous results, the research team of Jiang Zhengfan from the School of Life Sciences of Peking University and the research team of Han Weidong from the First Medical Center of the General Hospital of the People's Liberation Army have developed a nanomanganese adjuvant that is both an immune activator and a delivery system. The results showed that the manganese adjuvant exhibited excellent immune effects both in vivo and in vitro. The team of Dai Yunlu from the University of Macau constructed a new type of metal-polyphenol network complex by using chemically synthesized polyphenol derivatives coordinated with a variety of metal ions to activate the STING pathway to assist in tumor radiotherapy.

[0005] How to build a Mn by delivering 2+ Immune agonists that activate the cGAS-STING signaling axis to promote DC maturation and induce immunogenic apoptosis in tumor cells synergistically trigger powerful CTL-mediated immunotherapy, becoming a new idea for immunotherapy. Summary of the invention

[0006] Based on this, in order to overcome the shortcomings of the prior art, the present invention provides a nanoassembly based on natural bovine serum albumin (BSA) and ferritin (Ft) activated in the tumor microenvironment, which delivers Mn 2+ Activation of the cGAS-STING signaling axis promoted DC maturation and induced immunogenic apoptosis of tumor cells, synergistically triggering powerful CTL-mediated immunotherapy.

[0007] The present invention provides a manganese-doped protein immune agonist assembly BSA-Man@Mn 2+ -Ft@Lap, the assembly is composed of BSA-Man@Mn 2+ Assembled with Ft@Lap, the BSA-Man@Mn 2+ It is a mannose-modified bovine serum albumin BSA-Man and manganese ion Mn 2+ The Ft@Lap is formed by the complexation of ferritin and labaquinone.

[0008] Furthermore, the ratio of bovine serum albumin to ferritin in the assembly is 1:1-9:1.

[0009] The present invention also provides the manganese-doped protein immune agonist assembly BSA-Man@Mn 2+ -Ft@Lap construction method, the method is to BSA-Man@Mn 2+After adding Ft@Lap dropwise, NaOH was added. Anhydrous ethanol was added dropwise to the mixture. When the mixture changed from a solution to a colloid, the addition of anhydrous ethanol was stopped. Then CHO-PEG2000-CHO was added for cross-linking. BSA-Man@Mn was obtained after reaction at room temperature. 2+ -Ft@Lap.

[0010] Furthermore, the room temperature reaction time is 5-7 hours.

[0011] Furthermore, the BSA-Man@Mn 2+ The method is prepared by dissolving BSA-Man and MnCl2 in deionized water and stirring them, removing excess manganese ions with a dialysis bag, and freeze-drying the mixture at low temperature.

[0012] Furthermore, the Ft@Lap is obtained by dissolving ferritin Ft and labaquinone Lap in deionized water and stirring.

[0013] Furthermore, the synthesis of the CHO-PEG2000-CHO is as follows: polyethylene glycol OH-PEG2000-OH with an average molecular weight of 2000, 4-carboxybenzaldehyde, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC·HCl, and p-dimethylaminopyridine DMAP are mixed, dichloromethane DCM is added to dissolve all the reagents, the DCM is removed by stirring and evaporating, and then the mixture is washed with a saturated sodium chloride solution and then with a 5% sodium chloride solution. The organic layer is collected, filtered with anhydrous magnesium sulfate, and then evaporated to recover the product, which is purified with excess ether, dialyzed with ultrapure water, and freeze-dried to obtain the obtained product.

[0014] Furthermore, the BSA-Man is prepared by dissolving mannose and sodium hydroxide in water and stirring, then adding chloroacetic acid dropwise, stirring in an oil bath, and then adding hydrochloric acid to adjust the pH value to 2-3 to obtain carboxymannan Man-COOH; then dissolving the Man-COOH, carbodiimide EDC, and N-hydroxysuccinimide NHS in water and stirring to obtain a carboxyl-activated solution, then dissolving bovine serum albumin BSA in water and adding it to the carboxyl-activated solution, continuing to stir, dialyzing the product, and then freeze-drying it.

[0015] The present invention also provides the manganese-doped protein immune agonist assembly BSA-Man@Mn 2+ - Application of Ft@Lap in activating anti-tumor immune effects.

[0016] Furthermore, the application includes intravenous injection into a tumor patient.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention utilizes multiple modification sites and metal binding sites in the protein structure, first functionalizes BSA with DC-targeted mannose (Man) groups, and then loads Mn by a simple ion diffusion method. 2+ Ion (BSA-Man@Mn 2+ ). At the same time, ferritin can not only efficiently coat Lap in the central cavity (Ft-Lap) through hydrophobic interaction, but also bind to transferrin receptor 1 (TfR1) overexpressed on the tumor cell membrane to achieve targeted delivery of Lap. BSA-Man@Mn 2+ and Ft-Lap through an acidic Schiff base reaction to provide a physiologically stable nanoassembly (BSA-Man@Mn 2+ These assemblies can be rapidly decomposed into single protein units with smaller nanoparticle size in the slightly acidic TME, thereby enhancing the penetration into solid tumor tissues.

[0019] 2. Specifically, Ft-Lap is selectively taken up by tumor cells through TFR1-mediated endocytosis, inducing immunogenic apoptosis, thereby releasing a large amount of tumor-derived dsDNA into the TME for cGAS-mediated dsDNA sensing in DCs. 2+ It is effectively internalized by DCs through Man-mediated ligand-receptor binding and releases Mn after enzymatic lysosomal degradation. 2+ Ions activate the cGAS-STING axis, ultimately triggering downstream immunostimulatory transcriptional activity. These functions can work synergistically to enhance DC-mediated cross-priming of CTLs in the TME, improve anti-tumor immune responses in tumor models with poor immunogenicity, and provide a new way to improve the effectiveness of immunotherapy for solid tumor indications in the clinic. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : H NMR spectra of Man-COOH and CHO-PEG2000-CHO; wherein a is the H NMR spectrum of Man-COOH, and b is the H NMR spectrum of CHO-PEG2000-CHO;

[0021] Figure 2 : Schematic diagram of the assembly of the immune agonist assembly protein of the present invention;

[0022] Figure 3 : Schematic diagram of the mechanism of action of the immune agonist assembly protein of the present invention;

[0023] Figure 4 : Morphological and physicochemical characterization of synthesized manganese-doped protein immunostimulatory assemblies, namely BSA-Man@Mn 2+ - Physicochemical properties of Ft@Lap nano-components;

[0024] Among them, a. TEM image of BSA-Man-Ft nanocarrier,

[0025] b / c.BSA-Man@Mn 2+ TEM images of Ft@Lap nanoassemblies at low (b) and high (c) magnifications to illustrate morphological details.

[0026] d. Scanning transmission electron microscopy (STEM) and BSA-Man@Mn 2+ - EDS image of Ft@Lap,

[0027] e. Ft@Lap, BSA-Man@Mn in solution 2+ 、BSA-Man@Mn 2+ - DLS analysis of particle size distribution of Ft@Lap,

[0028] f. Comparative analysis of UV-visible absorption of Lap, Ft, and Ft@Lap,

[0029] g.BSA-Man@Mn 2+ In different Mn 2+ Changes in fluorescence emission under feed ratio,

[0030] h.Ferritin@Lap、BSA-Man@Mn 2+ 、BSA-Man@Mn 2+ - UV-visible absorption comparative analysis of Ft@Lap,

[0031] i.Mn 2+ The release information comes from BSA-Man@Mn 2+ - Ft@Lap,

[0032] j. BSA-Man@Mn after incubation under different conditions 2+ - Time-dependent release of Ft@Lap Lap;

[0033] Figure 5 . Manganese-doped protein immune agonist assemblies induce effective immune responses in solid tumors to inhibit tumor growth;

[0034] a / d. The distribution of BSA-Man@Mn in the whole body of mice at different times of 6 / 12 / 18h after tail vein injection. 2+ -Ft@Lap@Cy5 (cross-linked with COOH-PEG2000-COOH); II: Tumor response BSA-Man@Mn 2+ -Ft@Lap@Cy5 (cross-linked with CHO-PEG2000-CHO);

[0035] b / e. Fluorescence distribution in major organs and tumors after euthanasia (heart, liver, spleen, lung, and kidney tumors); Figure b / e. The corresponding quantitative analysis results of each group, scale bar = 2 cm;

[0036] c. Undissociable BSA-Man@Mn 2+ - Fluorescence statistics of Ft@Lap@Cy5 nanoprotein assemblies in the main mouse tissues of heart, liver, spleen, lung and kidney tumors;

[0037] f. Fluorescence statistics of tumor microenvironment-responsive COOH-PEG2000-COOH cross-linked nanoprotein assemblies in the main mouse tissues of heart, liver, spleen, lung and kidney tumors;

[0038] g. Bioluminescence analysis of 4T1-Luc tumor-bearing mice after different treatments. (I) control, (II) BSA-Man-Ft, (III) BSA-Man-Ft@Lap, (IV) BSA-Man@Mn 2+ -Ft and (V)BSA-Man@Mn 2+ -Ft@Lap.

[0039] h. Changes in tumor size during the latent period after different treatments;

[0040] i. Mice were treated with (II) BSA-Man-Ft, (III) BSA-Man-Ft@Lap, (IV) BSA-Man@Mn 2 + -Ft and (V)BSA-Man@Mn 2+ - Survival curve analysis after Ft@Lap treatment;

[0041] j. Western blot analysis of the expression of damage-associated molecular patterns (HMGB1 and CRT), apoptosis markers (BAX, Bcl-2), and key cGAS / STING signaling pathway makers (STING, p-STING, IRF-3, p-IRF-3, TBK1, and p-TBK1) in tumors of 4T1 tumor-bearing mice after different sample treatments;

[0042] k. Results of H&E staining and TUNEL staining of tumor tissue specimens after different treatments (I) control, (II) BSA-Man-Ft, (III) BSA-Man-Ft@Lap, (IV) BSA-Man@Mn 2+ -Ft and (V)BSA-Man@Mn 2+ - Ft@Lap;

[0043] l. H&E apoptosis staining and TUNEL staining of tumor tissue sections. DETAILED DESCRIPTION

[0044] Example 1: Preparation of albumin-mannose (BSA-Man):

[0045] Mannose (100 mg) and sodium hydroxide (3M, 1 mL) were dissolved in a round-bottom flask and stirred for 20 min. 1.2 mL of chloroacetic acid (160 g / L-1) was gradually added to the above solution. After stirring in an oil bath at 55 °C for 7 h, 1 M hydrochloric acid was added to adjust the pH to 2-3 to obtain carboxymannan (Man-COOH); then Man-COOH (0.42 mmol, 0.1 g), EDC (0.84 mmol, 0.161 g) and NHS (0.84 mmol, 0.097 g) were dissolved in water. The solution was mixed and stirred for 4 hours to activate the carboxyl group, and bovine serum albumin (BSA, 500 mg) was dissolved in water and added to the above solution. After stirring for 24 hours, the product was dialyzed in a dialysis bag (MWCO 500 kDa) for 4 days. Finally, it was placed in a freeze dryer and lyophilized to obtain a white powder, which was BSA-Man.

[0046] The H NMR spectrum of Man-COOH is as follows Figure 1 As shown in a.

[0047] Example 2: Synthesis of tumor microenvironment responsive bond CHO-PEG2000-CHO

[0048] OH-PEG2000-OH (2.5 g), 4-carboxybenzaldehyde (1.125 g), EDC·HCl (4.793 g), and DMAP (0.122 g) were dissolved in a 250 mL round-bottom flask, and 150 mL of dichloromethane (DCM) was added to dissolve the above reagents. The mixture was stirred at 25°C for 48 h. DCM was removed by rotary evaporator, and the mixture was washed with saturated sodium chloride solution 5 times and 5% sodium chloride solution 3 times. The organic layer was collected and filtered with 50 mg of anhydrous magnesium sulfate to remove moisture. The product was recovered by rotary evaporator and purified twice with excess ether. Then, the product was dialyzed with ultrapure water (MWCO=1000 Da) for 2 days and freeze-dried to obtain the product OHC-PEG2000-CHO (yield 75.2%), which was characterized by hydrogen nuclear magnetic resonance (400 MHz, D2O, ppm).

[0049] The H NMR spectrum of CHO-PEG2000-CHO is as follows Figure 1 As shown in b.

[0050] Example 3: Manganese-doped protein immune agonist BSA-Man@Mn 2+ - Preparation of Ft@Lap assemblies

[0051] Dissolve BSA-Man (3 mg / mL, 10 mL) and MnCl2 (16 ug / mL, 3 mL) in a 25 mL round-bottom flask with deionized water (10 mL) and stir for 12 h. Use a dialysis bag (MWCO = 1000 Da) to remove excess manganese ions from the obtained product and dry it with a low-temperature freeze dryer to obtain BSA-Man@Mn 2+ Ft (5 mg / mL, 5 mL) and Lap (100 ug / mL, 3 mL) were dissolved in a 25 mL round-bottom flask with deionized water (10 ml) and stirred for 12 h to obtain the complex ferritin-labaquinone (Ft@Lap); 50 mg BSA-Man@Mn 2+ Mix with 10mL ultrapure water and dissolve by ultrasound. Then stir the solution at 300rpm, add Ft@Lap (5mg / mL, 2mL) dropwise, then add NaOH (1M, 20μL) and then add anhydrous ethanol dropwise. When the mixture changes from a solution to a colloid, stop adding anhydrous ethanol, then add CHO-PEG2000-CHO (100μL) for crosslinking, and react at room temperature for 6h to obtain BSA-Man@Mn. 2+ -Ft@Lap.

[0052] The present invention modifies albumin (BSA) with a group (Man) targeting DC cells and then loads Mn 2+ , and finally get BSA-Man@Mn 2+ Then, ferritin (Ft) was loaded with prodrug (Lap) to obtain Ft@Lap, and then under pH 7.4, BSA-Man@Mn was finally formed under tumor microenvironment responsive bond (aldehyde PEG cross-linking). 2+ -Ft@Lap. Taking advantage of multiple modification sites and metal binding sites in the protein structure, BSA was first functionalized with DC-targeted mannose (Man) groups, and then Mn was loaded by a simple ion diffusion method. 2+ Ion (BSA-Man@Mn 2+ ). At the same time, ferritin can not only efficiently coat Lap in the central cavity (Ft-Lap) through hydrophobic interaction, but also bind to transferrin receptor 1 (TfR1) overexpressed on the tumor cell membrane to achieve targeted delivery of Lap. BSA-Man@Mn 2+ and Ft-Lap through an acidic Schiff base reaction to provide a physiologically stable nanoassembly (BSA-Man@Mn 2+ -Ft-Lap). Figure 2 shown.

[0053] These assemblies can be rapidly decomposed into single protein units with smaller nanoparticle size in the slightly acidic TME, which enhances the penetration process into solid tumor tissues. Specifically, Ft-Lap is selectively absorbed by tumor cells through TFR1-mediated endocytosis, inducing immunogenic apoptosis, thereby releasing a large amount of tumor-derived dsDNA into the TME for cGAS-mediated dsDNA sensing in DCs. 2+ It is effectively internalized by DCs through Man-mediated ligand-receptor binding and releases Mn after enzymatic lysosomal degradation. 2+ Ions activate the cGAS-STING axis, ultimately triggering downstream immunostimulatory transcriptional activity. These functions can work synergistically to enhance DC-mediated cross-priming of CTLs in the TME and improve antitumor immune responses in tumor models with poor immunogenicity. Figure 3 shown.

[0054] The morphology and physicochemical characterization of the synthesized manganese-doped protein immune agonist assemblies were as follows Figure 4 As shown. That is, BSA-Man@Mn 2+ - Physical properties of Ft@Lap nanocomponents.

[0055] Example 4: BSA-Man@Mn 2+ - Assembly and optimization of Ft-Lap protein

[0056] The co-assembly process of bovine serum albumin and ferritin with different ratios (ratios of 5:5; 6:4; 8:2; 9:1) was investigated, and the morphology and uniformity of the assembled nanoparticles were analyzed. The successful loading of Mn ions and their distribution in the nanoparticles were confirmed by UV absorption, fluorescence spectroscopy, and EDS elemental analysis. The components of the assembly (Mn 2+ , and labaquinone) during the assembly process and the coordination-induced assembly mechanism. Atomic force scanning electron microscopy, field emission scanning electron microscopy, high-resolution transmission electron microscopy, etc. were used to observe the morphology and microstructure of the protein assembly; Zeta potential instrument was used to measure the surface charge characteristics and particle size distribution of the assembly.

[0057] Example 5: Study on the anti-tumor effect of manganese-doped protein immune agonist assembly

[0058] Sixty male Balb / c mice, with an average age and weight of 4-6 weeks and an average weight of 18 g, were injected with PBS solution containing 4T1-luc cells to establish a tumor animal model. When the tumor volume grew to 100 mm 3At about 1:10 pm, the nude mice were randomly divided into 5 groups (6 mice in each group), and the body weight and tumor volume of each group were kept at the same level. There were 5 experimental groups, namely (I) control, (II) BSA-Man-Ft, (III) BSA-Man-Ft@Lap, (IV) BSA-Man@Mn 2+ -Ft and (V)BSA-Man@Mn 2+ -Ft@Lap. The tumor size was measured every 3 days, and the calculation formula was Vtumor = L × W2 / 2 (L: longitudinal diameter of the tumor, W: transverse diameter of the tumor). After 21 days of treatment, the mice were euthanized, and the tumors and major tissues and organs were extracted and washed with PBS three times. The major organs and tumors were prepared into paraffin sections and stained with H&E and TUNEL.

[0059] In summary, we prepared and synthesized the manganese-doped protein immune agonist assembly BSA-Man@Mn2+-Ft@Lap. After the nanoparticles were targeted to the tumor microenvironment, they released albumin and ferritin with smaller particle sizes. BSA-Man@Mn2+ targeted DCs and further activated DCs that were immunosuppressive in the tumor microenvironment, while Ft@Lap achieved targeted delivery of Lap and induced immunogenic death of tumor cells by binding to transferrin receptor 1 (TfR1) overexpressed on the tumor cell membrane. The two can work synergistically to enhance the cross-priming of CTLs in the TME mediated by DCs, improve the anti-tumor immune response of tumor models with poor immunogenicity, and provide a new way to improve the effectiveness of immunotherapy for anti-solid tumor indications in the clinic.

[0060] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A manganese-doped protein immune agonist assembly BSA-Man@Mn 2+ -Ft@Lap, characterized by: The assembly consists of BSA-Man@Mn 2+ Assembled with Ft@Lap, the BSA-Man@Mn 2+ It is mannose-modified bovine serum albumin BSA-Man and manganese ion Mn 2+ The Ft@Lap is formed by the complexation of ferritin and labaquinone; The assembly method is to combine BSA-Man@Mn 2+ After adding Ft@Lap dropwise, NaOH was added. Anhydrous ethanol was added dropwise to the mixture. When the mixture changed from a solution to a colloid, the addition of anhydrous ethanol was stopped. Then CHO-PEG2000-CHO was added for cross-linking. BSA-Man@Mn was obtained after reaction at room temperature. 2+ -Ft@Lap; The BSA-Man@Mn 2+ The product is prepared by dissolving BSA-Man and MnCl2 in deionized water, stirring, removing excess manganese ions with a dialysis bag, and freeze-drying at low temperature. The Ft@Lap is obtained by dissolving ferritin Ft and labaquinone Lap in deionized water and stirring.

2. The manganese-doped protein immune agonist assembly BSA-Man@Mn according to claim 1 2+ -Ft@Lap, characterized by: The ratio of bovine serum albumin to ferritin in the assembly is 1:1-9:

1.

3. The manganese-doped protein immune agonist assembly BSA-Man@Mn according to claim 1 2+ -Ft@Lap, characterized by: The room temperature reaction time is 5-7 hours.

4. The manganese-doped protein immune agonist assembly BSA-Man@Mn according to claim 1 2+ -Ft@Lap, characterized by: The synthesis of the CHO-PEG2000-CHO is as follows: polyethylene glycol OH-PEG2000-OH with an average molecular weight of 2000, 4-carboxybenzaldehyde, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC·HCl, and p-dimethylaminopyridine DMAP are mixed, dichloromethane DCM is added to dissolve all the reagents, DCM is removed by stirring and evaporating, and then washed with a saturated sodium chloride solution, and then washed with a 5% sodium chloride solution, the organic layer is collected, filtered with anhydrous magnesium sulfate, and then evaporated to recover the product, purified with excess ether, dialyzed with ultrapure water, and freeze-dried to obtain the product.

5. The manganese-doped protein immune agonist assembly BSA-Man@Mn according to claim 1 2+ -Ft@Lap, characterized by: The BSA-Man is prepared by dissolving mannose and sodium hydroxide in water and stirring, then adding chloroacetic acid dropwise, stirring in an oil bath, and then adding hydrochloric acid to adjust the pH value to 2-3 to obtain carboxymannan Man-COOH; then dissolving the Man-COOH, carbodiimide EDC, and N-hydroxysuccinimide NHS in water and stirring to obtain a carboxyl-activated solution, then dissolving bovine serum albumin BSA in water and adding it to the carboxyl-activated solution, continuing to stir, dialyzing the product, and then freeze-drying it.

6. The manganese-doped protein immune agonist assembly BSA-Man@Mn according to claim 1 2+ -Application of Ft@Lap in the preparation of anti-breast cancer drugs.

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