A manganese dioxide nanovaccine for dendritic cell targeted delivery of tumor neoantigens and a preparation method and application thereof

Abstract

The application provides a manganese dioxide nanovaccine for dendritic cell targeted delivery of tumor neoantigens and a preparation method and application thereof, and belongs to the technical field of biological medicines. Hollow mesoporous MnO2 nanoparticles are prepared; the MnO2 nanoparticles and 10K-Adpgk aqueous solution are mixed, stirred, and centrifuged to obtain MA nanoparticles; DLin-MC3-DMA, DSPC, DSPE-PEG-Dcpep, cholesterol and chloroform are mixed, first ultrasonic water bath is carried out, rotary evaporation is carried out, MA sterile enzyme-free water is added, second ultrasonic water bath is carried out, and then extrusion and filtration are carried out to obtain MA@DC-NLP, namely the manganese dioxide nanovaccine for dendritic cell targeted delivery of tumor neoantigens. The obtained nanovaccine can accurately and efficiently co-deliver tumor antigens and immune adjuvants to the immune system, activate a strong and long-lasting anti-tumor immune response, and provide a controllable scheme for tumor immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a manganese dioxide nanovaccine that targets and delivers neoantigens to tumors via dendritic cells, its preparation method, and its application. Background Technology

[0002] For a long time, cancer has seriously threatened human health due to its high metastasis and recurrence rates. Despite the continuous development of traditional methods such as surgery, radiotherapy, and chemotherapy, the treatment effect for advanced and metastatic solid tumors remains unsatisfactory, and the five-year survival rate of patients urgently needs to be improved. With the rapid development of biotechnology and in-depth research on the mechanisms of tumor occurrence and progression, immunotherapy has become a new model for comprehensive cancer treatment. Among them, cancer vaccines, as a promising immunotherapy, aim to eradicate malignant tumors and induce tumor-specific immune responses to recognize and destroy tumor cells while preserving normal tissues. However, traditional cancer vaccines often suffer from key bottlenecks such as weak antigen immunogenicity, low delivery efficiency, and inability to effectively activate cellular immunity, resulting in limited clinical response rates. Therefore, effective vaccines must train the immune system to recognize and attack tumor cells by administering tumor antigens and adjuvants. To date, cancer vaccines have used a variety of antigens, including tumor-associated antigens (TAAs), whole-cell antigens, and the latest tumor neoantigens. Tumor neoantigens, due to their high tumor specificity and strong immunogenicity, are considered ideal targets for breaking immune tolerance and achieving precise killing.

[0003] Adjuvants can enhance vaccine efficacy, stimulate specific immune responses, and reduce antigen and vaccine dosages, playing an important role in cancer vaccines. 2+ It has been found that it can act on immune cells through the cGAS-STING pathway, enhancing anti-tumor immune responses and improving the efficacy of clinical immunotherapy. 2+ It can significantly promote the maturation of dendritic cells (DCs) and macrophages and the presentation of tumor-specific antigens, and improve CD8. + T cell differentiation and activation indicate Mn 2+ It is an effective adjuvant for enhancing immune responses. The rapid development of nanotechnology provides a potential strategy for improving the efficacy of cancer vaccines. Manganese can be designed into nanomaterials with different functionalities. Among these manganese-based nanomaterials, manganese dioxide nanoparticles (MnO2NPs) are among the most stable and functionally diverse, possessing important biological activities and immune response regulation activities. Under acidic conditions, MnO2NPs can release Mn through interaction with H2O2. 2+ This allows for the efficient catalysis of O2 generation. Furthermore, MnO2NPs can interact with intracellular glutathione (GSH) to release Mn... 2+These capabilities not only enable MnO2NPs to regulate cellular oxidative stress, but also endow MnO2NPs with the ability to interact with Mn through oxidation-related mechanisms. 2+ The potential of related mechanisms to regulate multiple immune responses.

[0004] Therefore, there is an urgent need in this field to develop a manganese dioxide nanovaccine that can simultaneously achieve precise delivery of tumor neoantigens and release of adjuvants. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a manganese dioxide nanovaccine for targeted delivery of neotumor antigens by dendritic cells, its preparation method and application, so as to solve the problems of low delivery efficiency and weak immunogenicity of traditional tumor vaccines, and to achieve precise and efficient co-delivery of tumor antigens and immune adjuvants to the immune system, thereby activating a strong and long-lasting antitumor immune response.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a manganese dioxide nanovaccine that targets and delivers neoantigens to dendritic cells, comprising the following steps: (1) Mix tetraethyl orthosilicate and ethanol to obtain a mixture. Mix ethanol, water and ammonia, stir for the first time, add the mixture dropwise, stir for the second time, centrifuge to collect the precipitate, and obtain the product. (2) After mixing the product with water, add potassium permanganate, sonicate, stir, centrifuge to collect the precipitate, then disperse the precipitate in Na2CO3 solution for etching, centrifuge again to obtain MnO2 nanoparticles. (3) Mix the MnO2 nanoparticle solution and the 10K-Adpgk aqueous solution, stir, and centrifuge to obtain MA nanoparticles; (4) Mix DLin-MC3-DMA, DSPC, DSPE-PEG-Dcpep, cholesterol and chloroform, sonicate in a water bath for the first time, then evaporate by rotary evaporation, add MA sterile enzyme-free water, sonicate in a water bath for the second time, and then extrude and filter to obtain MA@DC-NLP, which is a manganese dioxide nanovaccine that targets and delivers neoantigens to dendritic cells.

[0007] Preferably, in step (1), the volume ratio of tetraethyl orthosilicate to ethanol is 3~8:10~20, and the volume ratio of ethanol, water and ammonia is 60~80:5~9:2~4; the temperature of the first stirring is 40~60℃, and the time of the first stirring is 20~40min; the temperature of the second stirring is 40~60℃, and the time of the second stirring is 3~5h.

[0008] Preferably, in step (2), the mass-to-volume ratio of the product to water is 180~220mg:40~60mL, the mass ratio of the product to potassium permanganate is 1:1~3; the frequency of the ultrasound is 20~30kHz, the temperature of the ultrasound is 20~30℃, and the time of the ultrasound is 1~3h; the stirring speed is 200~400rpm, and the stirring time is 12~20h; the concentration of the Na2CO3 solution is 0.1~0.3M; the etching temperature is 50~70℃, and the etching time is 8~16h.

[0009] Preferably, in step (3), the concentration of the MnO2 nanoparticle solution is 2 mg / mL, the concentration of the 10K-Adpgk aqueous solution is 0.5~4 mg / mL, the volume ratio of the MnO2 nanoparticle solution to the 10K-Adpgk aqueous solution is 1:1, the stirring time is 18~30 h, the centrifugation speed is 10000~14000 rpm, and the centrifugation time is 10~20 min.

[0010] Preferably, in step (4), the molar ratio of DLin-MC3-DMA, DSPC, and DSPE-PEG-Dcpep is 50:10:1~2, the molar ratio of DLin-MC3-DMA to cholesterol is 3~5:1, and the volume of chloroform is 5~15mL.

[0011] Preferably, in step (4), the temperature of the first water bath ultrasound is 20~30℃, and the time of the first water bath ultrasound is 20~40min; the rotation speed of the rotary evaporation is 50~150rpm, the temperature of the rotary evaporation is 30~50℃, and the time of the rotary evaporation is 1~3h; the concentration of the MA sterile enzyme-free water is 1~3mg / mL, and the volume of the MA sterile enzyme-free water is 3~8mL; the temperature of the second water bath ultrasound is 40~60℃, and the time of the second water bath ultrasound is 20~40min.

[0012] The present invention also provides a manganese dioxide nanovaccine for targeted delivery of tumor neoantigens to dendritic cells prepared by the aforementioned preparation method.

[0013] The present invention also provides the application of the manganese dioxide nanovaccine that targets and delivers tumor neoantigens to dendritic cells in the preparation of antitumor formulations.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention synthesizes hollow manganese dioxide nanoparticles using silica as a hard template, then encapsulates the tumor neoantigen 10K-Adpgk within them, and finally coats the surface with dendritic cell-targeting cationic liposomes, thereby obtaining a manganese dioxide nanovaccine for dendritic cell-targeted delivery of tumor neoantigens. The nanovaccine obtained by this invention can precisely and efficiently co-deliver tumor antigens and immune adjuvants to the immune system, activating a strong and durable anti-tumor immune response, providing a controllable approach for tumor immunotherapy. Attached Figure Description

[0015] Figure 1 These are images of the morphology of SiO2 and SiO2@MnO2 nanoparticles captured by scanning electron microscopy (SEM). Figure 2 The images show the morphology of nanoparticles obtained by transmission electron microscopy (TEM) using MnO2, MA, and MA@DC-NLP synthesized in Example 1. Figure 3 This is a graph showing the results of energy-dispersive X-ray spectroscopy analysis; Figure 4 This is an X-ray photoelectron spectroscopy result diagram; Figure 5 This is a graph showing the results of dynamic light scattering (DLS) measurements; Figure 6 The graph shows the drug loading rate of 10K-Adpgk and the encapsulation efficiency of MnO2. Figure 7 This is a graph showing the results of an in vitro phagocytosis test; Figure 8 This is a graph showing the results of IFN-β cytokine detection; Figure 9 This is a graph showing the results of IL-6 cytokine detection; Figure 10 It is the cytokine IL-12 p40 Test result image; Figure 11 It's CD40 + Image showing cell proportion detection results; Figure 12 It's CD80 + Image showing cell proportion detection results; Figure 13 It's CD80 + / CD86 + Image showing cell proportion detection results; Figure 14 It is CD86 + Image showing cell proportion detection results; Figure 15 It is MHC II + Image showing the results of cell proportion detection. Detailed Implementation

[0016] This invention provides a method for preparing a manganese dioxide nanovaccine that targets and delivers neoantigens to dendritic cells, comprising the following steps: (1) Mix tetraethyl orthosilicate and ethanol to obtain a mixture. Mix ethanol, water and ammonia, stir for the first time, add the mixture dropwise, stir for the second time, centrifuge to collect the precipitate, and obtain the product. (2) After mixing the product with water, add potassium permanganate, sonicate, stir, centrifuge to collect the precipitate, then disperse the precipitate in Na2CO3 solution for etching, centrifuge again to obtain MnO2 nanoparticles. (3) Mix the MnO2 nanoparticle solution and the 10K-Adpgk aqueous solution, stir, and centrifuge to obtain MA nanoparticles; (4) Mix DLin-MC3-DMA, DSPC, DSPE-PEG-Dcpep, cholesterol and chloroform, sonicate in a water bath for the first time, then evaporate by rotary evaporation, add MA sterile enzyme-free water, sonicate in a water bath for the second time, and then extrude and filter to obtain MA@DC-NLP, which is a manganese dioxide nanovaccine that targets and delivers neoantigens to dendritic cells.

[0017] In this invention, tetraethyl orthosilicate and ethanol are mixed to obtain a mixture. Ethanol, water and ammonia are mixed and stirred for the first time. The mixture is then added dropwise to the mixture and stirred for the second time. The precipitate is collected by centrifugation to obtain the product. The volume ratio of tetraethyl orthosilicate to ethanol is preferably 3~8:10~20, more preferably 4~7:12~18, and even more preferably 5:15; the volume ratio of ethanol, water, and ammonia is preferably 60~80:5~9:2~4, more preferably 65~75:6~8:2.5~3.5, and even more preferably 70:7:3; the temperature of the first stirring is preferably 40~60℃, more preferably 45~55℃, and even more preferably 50℃; the time of the first stirring is preferably 20~40min, more preferably 25~35min, and even more preferably 30min; the temperature of the second stirring is preferably 40~60℃, more preferably 45~55℃, and even more preferably 50℃; the time of the second stirring is preferably 3~5h, more preferably 3.5~4.5h, and even more preferably 4h.

[0018] In this invention, the product and water are mixed, potassium permanganate is added, and the mixture is ultrasonically stirred overnight. The precipitate is then collected by centrifugation, dispersed in Na₂CO₃ solution for etching, and centrifuged again to obtain MnO₂ nanoparticles. The preferred mass-to-volume ratio of the product to water is 180-220 mg:40-60 mL, more preferably 190-210 mg:45-55 mL, and even more preferably 200 mg:50 mL; the preferred mass ratio of the product to potassium permanganate is 1:1-3, more preferably 1:1.5-2.5, and even more preferably 1:1; the preferred ultrasonic frequency is 20-30 kHz, more preferably 22-28 kHz, and even more preferably 25 kHz; the preferred ultrasonic temperature is 20-30 °C, more preferably 22-28 °C, and even more preferably 25 °C; the preferred ultrasonic time is 1-3 h, and even more preferably... The stirring time is preferably 2 hours; the stirring speed is preferably 200-400 rpm, more preferably 250-350 rpm, and even more preferably 300 rpm; the stirring time is preferably 12-20 hours, more preferably 14-18 hours, and even more preferably 16 hours; the concentration of the Na2CO3 solution is preferably 0.1-0.3 M, more preferably 0.15-0.25 M, and even more preferably 0.2 M; the etching temperature is preferably 50-70℃, more preferably 55-65℃, and even more preferably 60℃; the etching time is preferably 8-16 hours, more preferably 10-14 hours, and even more preferably 12 hours.

[0019] In this invention, a MnO2 nanoparticle solution and a 10K-Adpgk aqueous solution are mixed, stirred, and centrifuged to obtain MA nanoparticles. The concentration of the MnO2 nanoparticle solution is 2 mg / mL; the concentration of the 10K-Adpgk aqueous solution is preferably 0.5~4 mg / mL, more preferably 1~3 mg / mL, and even more preferably 2 mg / mL; the volume ratio of the MnO2 nanoparticle solution to the 10K-Adpgk aqueous solution is 1:1; the stirring time is preferably 18~30 h, more preferably 21~27 h, and even more preferably 24 h; the centrifugation speed is preferably 10000~14000 rpm, more preferably 11000~13000 rpm, and even more preferably 12000 rpm; the centrifugation time is preferably 10~20 min, more preferably 12~18 min, and even more preferably 15 min.

[0020] In this invention, DLin-MC3-DMA, DSPC, DSPE-PEG-Dcpep, cholesterol and chloroform are mixed, subjected to a first water bath sonication, followed by rotary evaporation, and then added with sterile, enzyme-free water containing MA. After a second water bath sonication, the mixture is extruded and filtered to obtain MA@DC-NLP, a manganese dioxide nanovaccine that targets and delivers neoantigens to dendritic cells. The molar ratio of DLin-MC3-DMA, DSPC, and DSPE-PEG-Dcpep is preferably 50:10:1~2, more preferably 50:10:1.5; the molar ratio of DLin-MC3-DMA to cholesterol is preferably 3~5:1, more preferably 4:1; the volume of chloroform is preferably 5~15mL, more preferably 8~12mL, and even more preferably 10mL; the temperature of the first water bath ultrasound is preferably 20~30℃, more preferably 22~28℃, and even more preferably 25℃; the time of the first water bath ultrasound is preferably 20~40min, more preferably 25~35min, and even more preferably 30min; the rotation speed of the rotary evaporation is preferably 50~150rpm, more preferably 80~120rpm, and even more preferably 100rpm; the temperature of the rotary evaporation is preferably 30~50℃, and even more preferably 35~45℃. The preferred temperature is 40℃; the preferred rotary evaporation time is 1-3 h, more preferably 1.5-2.5 h, and even more preferably 2 h; the preferred concentration of the MA sterile enzyme-free water is 1-3 mg / mL, more preferably 2 mg / mL; the preferred volume of the MA sterile enzyme-free water is 3-8 mL, more preferably 4-7 mL, and even more preferably 5 mL; the preferred temperature of the second water bath ultrasonication is 40-60℃, more preferably 45-55℃, and even more preferably 50℃; the preferred time of the second water bath ultrasonication is 20-40 min, more preferably 25-35 min, and even more preferably 30 min; the extrusion uses a liposome extruder, the preferred pore size of the liposome extruder is 0.2-0.6 μm, more preferably 0.4 μm, and the preferred number of extrusions is 4-6 times, more preferably 5 times; the preferred filtration uses a needle filter, the preferred pore size of the needle filter is 0.22 mm, and the preferred number of filtrations is 2-4 times, more preferably 3 times.

[0021] The present invention also provides a manganese dioxide nanovaccine for targeted delivery of tumor neoantigens to dendritic cells prepared by the aforementioned preparation method.

[0022] The present invention also provides the application of the manganese dioxide nanovaccine that targets and delivers tumor neoantigens to dendritic cells in the preparation of antitumor formulations.

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1

[0025] A method for preparing a manganese dioxide nanovaccine that targets and delivers tumor neoantigens via dendritic cells, comprising the following steps: (1) Measure 70 mL of ethanol, 7 mL of water, and 3 mL of ammonia and add them to a 100 mL round-bottom flask. Stir the mixture magnetically at 50°C for 30 min. Then, add a mixture of 5 mL of tetraethyl orthosilicate (TEOS) and 15 mL of ethanol dropwise and continue stirring magnetically at 50°C for 4 h. Centrifuge the resulting precipitate and wash it twice with ethanol and water to obtain the product SiO2. Disperse 200 mg of the product in 50 mL of water, add 400 mg of potassium permanganate (KMnO4), and sonicate (25 kHz, 25°C) for 2 h. Then, stir the mixture magnetically (300 rpm) at room temperature for 16 h. Centrifuge the product at 8000 × g and wash the precipitate twice with ethanol and water to obtain SiO2@MnO2. Then disperse SiO2@MnO2 in 0.2 M Na2CO3 solution, etch it at 60°C for 12 h, collect the product by centrifugation, and wash it twice with ethanol and water. Hollow mesoporous MnO2 nanoparticles with a diameter of approximately 150 nm were finally prepared.

[0026] (2) MnO2 nanoparticles were dissolved in water to obtain a MnO2 nanoparticle solution with a concentration of 2 mg / mL. Then, an equal volume of 10K-Adpgk aqueous solution was added dropwise while stirring. After 24 h, the nanoparticles were collected by centrifugation (12,000 rpm, 15 min) to obtain MA nanoparticles.

[0027] (3) DC-targeted liposomes DC-NLP and MA@DC-NLP were synthesized via a thin-film hydration method. The specific steps are as follows: ① Calculate the material usage based on the ratio of ionizable phospholipids (DLin-MC3-DMA) to cholesterol at a molar ratio of 4:1.

[0028] ②Weigh DLin-MC3-DMA, DSPC, DSPE-PEG-Dcpep and cholesterol precisely using an electronic balance according to the molar ratio of DLin-MC3-DMA:DSPC:DSPE-PEG-Dcpep=50:10:1.5, dissolve them in 10mL of chloroform, and sonicate in a water bath (25kHz, 25℃) for 30 min to completely dissolve the lipid material.

[0029] ③ Install the round-bottom flask on the rotary evaporator, ensuring that the liquid inside the flask is completely submerged below the liquid surface.

[0030] ④ Evaporate at 100 rpm and 40℃ for 2 hours. After evaporation, there should be no residual liquid or chloroform odor inside the flask.

[0031] ⑤ Remove the round-bottom flask and place it in a fume hood. Add 5 mL of sterile, enzyme-free water containing 2 mg / mL MA and shake to dissolve the lipid film inside the flask.

[0032] ⑥ Sonicate in a water bath at 50℃ for 30 min.

[0033] ⑦ The sonicated solution was passed through a liposome extruder with a pore size of 0.4 μm five times to obtain MA@DC-NLP.

[0034] ⑧ Pass the above-mentioned MA@DC-NLP through a 0.22 mm syringe filter three times in a biosafety cabinet to obtain sterile MA@DC-NLP.

[0035] Example 2

[0036] A method for preparing a manganese dioxide nanovaccine that targets and delivers tumor neoantigens via dendritic cells, comprising the following steps: (1) Measure 60 mL of ethanol, 5 mL of water and 2 mL of ammonia and add them to a 100 mL round-bottom flask. Stir thoroughly with a magnetic stirrer at 40°C for 20 min. Then, add dropwise a mixture of 3 mL of tetraethyl orthosilicate (TEOS) and 10 mL of ethanol and continue stirring with a magnetic stirrer at 40°C for 3 h. Centrifuge the resulting precipitate and wash it twice with ethanol and water to obtain the product SiO2. Disperse 180 mg of the product in 40 mL of water, add 180 mg of potassium permanganate (KMnO4), and sonicate (20 kHz, 20 °C) for 1 h. Then, stir the mixture with a magnetic stirrer at room temperature (200 rpm) for 20 h. Centrifuge the product at 7000 × g, wash the precipitate twice with ethanol and water to obtain SiO2@MnO2. Then disperse SiO2@MnO2 in 0.1 M Na2CO3 solution, etch at 50°C for 16 h, collect the product by centrifugation, and wash it twice with ethanol and water. Hollow mesoporous MnO2 nanoparticles were finally prepared.

[0037] (2) MnO2 nanoparticles were dissolved in water to obtain a MnO2 nanoparticle solution with a concentration of 2 mg / mL. Then, an equal volume of 10K-Adpgk aqueous solution with a concentration of 0.5 mg / mL was added dropwise while stirring. After 18 h, the nanoparticles were collected by centrifugation (10,000 rpm, 20 min) to obtain MA nanoparticles.

[0038] (3) DC-targeted liposomes DC-NLP and MA@DC-NLP were synthesized via a thin-film hydration method. The specific steps are as follows: ① Calculate the material usage based on the ratio of ionizable phospholipids (DLin-MC3-DMA) to cholesterol at a molar ratio of 4:1.

[0039] ②Weigh DLin-MC3-DMA, DSPC, DSPE-PEG-Dcpep and cholesterol precisely using an electronic balance in a molar ratio of DLin-MC3-DMA:DSPC:DSPE-PEG-Dcpep=50:10:1, dissolve in 5mL chloroform, and sonicate in a water bath (20kHz, 20℃) for 20 min to completely dissolve the lipid material.

[0040] ③ Install the round-bottom flask on the rotary evaporator, ensuring that the liquid inside the flask is completely submerged below the liquid surface.

[0041] ④ Evaporate at 50 rpm and 30℃ for 3 hours. After evaporation, there should be no residual liquid or chloroform odor inside the flask.

[0042] ⑤ Remove the round-bottom flask and place it in a fume hood. Add 3 mL of sterile, enzyme-free water containing 1 mg / mL MA and shake to dissolve the lipid film inside the flask.

[0043] ⑥ Sonicate in a water bath at 40℃ for 40 min.

[0044] ⑦ The sonicated solution was passed through a liposome extruder with a pore size of 0.2 μm four times to obtain MA@DC-NLP.

[0045] ⑧ Pass the above-mentioned MA@DC-NLP through a 0.22 mm syringe filter twice in a biosafety cabinet to obtain sterile MA@DC-NLP.

[0046] Example 3

[0047] A method for preparing a manganese dioxide nanovaccine that targets and delivers tumor neoantigens via dendritic cells, comprising the following steps: (1) Measure 80 mL of ethanol, 9 mL of water and 4 mL of ammonia and add them to a 100 mL round-bottom flask. Stir thoroughly with a magnetic stirrer at 60°C for 40 min. Then, add dropwise a mixture of 8 mL of tetraethyl orthosilicate (TEOS) and 20 mL of ethanol and continue stirring with a magnetic stirrer at 60°C for 5 h. Centrifuge the resulting precipitate and wash it twice with ethanol and water to obtain the product SiO2. Disperse 220 mg of the product in 60 mL of water, add 220 mg of potassium permanganate (KMnO4), and sonicate (30 kHz, 30 °C) for 3 h. Then, stir the mixture with a magnetic stirrer at room temperature (400 rpm) for 12 h. Centrifuge the product at 9000 × g, and wash the precipitate twice with ethanol and water to obtain SiO2@MnO2. Then disperse SiO2@MnO2 in 0.3 M Na2CO3 solution, etch at 70°C for 8 h, collect the product by centrifugation, and wash it twice with ethanol and water. Hollow mesoporous MnO2 nanoparticles were finally prepared.

[0048] (2) MnO2 nanoparticles were dissolved in water to obtain a 2 mg / mL MnO2 nanoparticle solution. Then, an equal volume of 10K-Adpgk aqueous solution with a concentration of 4 mg / mL was added dropwise while stirring. After 30 h, the nanoparticles were collected by centrifugation (14,000 rpm, 10 min) to obtain MA nanoparticles.

[0049] (3) DC-targeted liposomes DC-NLP and MA@DC-NLP were synthesized via a thin-film hydration method. The specific steps are as follows: ① Calculate the material usage based on the ratio of ionizable phospholipids (DLin-MC3-DMA) to cholesterol at a molar ratio of 4:1.

[0050] ②Weigh DLin-MC3-DMA, DSPC, DSPE-PEG-Dcpep and cholesterol precisely using an electronic balance in a molar ratio of DLin-MC3-DMA:DSPC:DSPE-PEG-Dcpep=50:10:2, dissolve in 15mL chloroform, and sonicate in a water bath (30kHz, 30℃) for 40 min to completely dissolve the lipid material.

[0051] ③ Install the round-bottom flask on the rotary evaporator, ensuring that the liquid inside the flask is completely submerged below the liquid surface.

[0052] ④ Evaporate at 150 rpm and 50℃ for 1 hour. After evaporation, there should be no residual liquid or chloroform odor inside the flask.

[0053] ⑤ Remove the round-bottom flask and place it in a fume hood. Add 8 mL of sterile, enzyme-free water containing 3 mg / mL MA and shake to dissolve the lipid film inside the flask.

[0054] ⑥ Sonicate in a water bath at 60℃ for 20 min.

[0055] ⑦ The sonicated solution was passed through a liposome extruder with a pore size of 0.6 μm six times to obtain MA@DC-NLP.

[0056] ⑧ Pass the above-mentioned MA@DC-NLP through a 0.22 mm syringe filter four times in a biosafety cabinet to obtain sterile MA@DC-NLP.

[0057] Comparative Example 1

[0058] The difference from Example 1 is that the 10K-Adpgk load and the DC-LNP wrapping are omitted; otherwise, it is the same as Example 1. MnO2 is obtained.

[0059] Comparative Example 2

[0060] The difference from Example 1 is that the DC-LNP encapsulation is omitted; otherwise, it is the same as Example 1. MnO2 particles loaded with 10K-Adpgk were obtained, denoted as MA.

[0061] Comparative Example 3

[0062] The difference from Example 1 is that DSPE-PEG-Dcpep was replaced with DSPE-PEG2000, otherwise it is the same as Example 1. Non-targeting liposome-encapsulated nanoparticles were obtained, denoted as MA@LNP.

[0063] Experimental Example 1

[0064] 1. Scanning electron microscopy (SEM) was used to image the morphology of nanoparticles.

[0065] ① The SiO2 and SiO2@MnO2 prepared in Example 1 were dropped onto the stage, air-dried for 24 hours, sputtered with gold 3 times, and observed by SEM.

[0066] Experimental results: such as Figure 1 As shown. By Figure 1 It can be seen that SiO2 is a smooth nanosphere, while the surface of SiO2@MnO2 becomes rough, indicating that a manganese dioxide shell was synthesized in situ on the surface of SiO2.

[0067] ② MnO2 from Comparative Example 1, MA from Comparative Example 2, and MA@DC-NLP synthesized in Example 1 were respectively added to the surface of a copper mesh carbon film, and observed by TEM after negative staining.

[0068] Experimental results: such as Figure 2As shown. Figure 2 The left image shows that after etching the internal SiO2 with sodium carbonate, the MnO2 becomes hollow, indicating the successful synthesis of hollow MnO2. Figure 2 The middle figure shows the successful encapsulation of the 10K-Adpgk peptide. Figure 2 The right image shows a thin film structure on the surface of the nanoparticles, indicating that the DC-targeted liposomes were successfully encapsulated.

[0069] 2. Energy-dispersive X-ray spectroscopy analysis

[0070] Energy-dispersive X-ray spectroscopy (EDS) analysis was performed on the Mn, O, C, N, P and S in the MA@DC-LPN nanoparticles synthesized in Example 1.

[0071] Experimental results: such as Figure 3 As shown. By Figure 3 It can be seen that in the EDS of MA@DC-LPN, Mn and O elements from MnO2, as well as C, O, N and S elements from 10K-Adpgk antigen peptide, were detected, and P element from cationic liposomes could also be clearly observed, indicating the successful loading of 10K-Adpgk antigen and the successful encapsulation by cationic liposomes.

[0072] 3. X-ray photoelectron spectroscopy

[0073] The MA@DC-LPN nanoparticles synthesized in Example 1 were uniformly sprinkled onto double-sided copper adhesive, pressed into tablets, and then scanned.

[0074] Experimental results: such as Figure 4 As shown. By Figure 4 It can be seen that the double peaks of Mn (Mn 2p3 / 2, 642.5 eV; Mn2p1 / 2, 654.2 eV) in the X-ray photoelectron spectrum confirm the presence of Mn element, and the Mn 2p3 / 2 peak is located at 642.5 eV, indicating that Mn element is tetravalent, that is, the valence state of manganese element in manganese dioxide.

[0075] 4. Dynamic Light Scattering (DLS) Measurement

[0076] DLS particle size and zeta potential analysis were performed. The SiO2 and SiO2@MnO2 of Example 1, the MnO2 of Comparative Example 1, the MA of Comparative Example 2 and the MA@DC-NLP synthesized in Example 1 were each diluted to 1 mL with ultrapure water, and the concentration was kept to 1 mg / mL. They were then added to a quartz glass dish and a zeta potential detection cell for DLS measurement.

[0077] Experimental results: such as Figure 5As shown in the figure, the hydration size and zeta potential of MA@DC-LPN were determined by dynamic light scattering (DLS). The hydration size results show that the synthesized nanoparticles have a uniform size and are consistent with the electron microscopy results. The change in zeta potential indicates successful loading of 10K-Adpgk and successful encapsulation of cationic liposomes.

[0078] 5. Drug loading rate of 10K-Adpgk and encapsulation efficiency of MnO2

[0079] Using a 3000 Da ultrafiltration tube, the synthesized product MA@DC-NLP with a 10K-Adpgk:MnO2 mass ratio of 0.25:1, 0.5:1, 0.75:1, 1:1, 1.5:1, and 2:1 (different concentrations of 10K-Adpgk were added dropwise to a 2 mg / mL MnO2 nanoparticle solution in equal volumes, with other conditions consistent with Example 1) was centrifuged at 4°C (5000 r / min) for 30 min. The peptide concentration of the filtered liquid was determined, and the free peptide content in the remaining liquid was detected. The encapsulation efficiency (%) was calculated according to "(total peptide amount (C0) - free peptide amount (C1)) / total peptide amount (C0) × 100%", and the drug loading rate was calculated according to "(total peptide amount (C0) - free peptide amount (C1)) / total feed weight (W0) × 100%".

[0080] Experimental results: such as Figure 6 As shown, when the mass ratio of 10K-Adpgk / MnO2 is 1:1, the encapsulation efficiency (EE%) reaches a near-peak of approximately 60%, while the drug loading (DE%) is approximately 38%. This phenomenon can be attributed to the hollow mesoporous structure of MnO2. With increasing 10K-Adpgk concentration, the drug loading shows a slight upward trend, while the encapsulation efficiency decreases significantly. Considering cost-effectiveness, a 1:1 mass ratio of 10K-Adpgk / MnO2 was selected for subsequent experiments.

[0081] 6. In vitro phagocytosis test

[0082] Mouse bone marrow-derived dendritic cells (BMDCs) and macrophage lines (BMDMs) were cultured in vitro (extracted and cultured from the tibia and femur of 6-8 week old female C57BL / 6 mice). After 12 h of culture, PBS, MnO2 (Comparative Example 1), Free 10K-Adpgk, MA (Comparative Example 2), MA@LNP (Comparative Example 3), and MA@DC-LNP synthesized in Example 1 were added respectively. After 24 h of further culture, flow cytometry analysis was performed.

[0083] Experimental results: such as Figure 7As shown, the fluorescence intensity of 10K-Adpgk in the MA@DC-LNP group increased significantly, indicating that the synthesized nanovaccine has good phagocytic uptake ability. Simultaneously, within the MA@DC-LNP group, compared to the macrophage cell line, the fluorescence intensity of 10K-Adpgk was significantly increased in bone marrow-derived dendritic cells, indicating that the synthesized nanovaccine has good dendritic cell targeting ability.

[0084] 7. Cytokine level detection

[0085] Mouse bone marrow-derived dendritic cells were seeded into 6-well plates and tested in 6 groups: PBS group, MnO2 group (comparative Example 1), Free 10K-Adpgk group, MA group (comparative Example 2), MA@LNP group (comparative Example 3), and MA@DC-LNP group synthesized in Example 1. After 24 hours of culture, the supernatant was collected, and ELISA was performed to detect INF-β, IL-6, and IL-12. p40 Cytokine levels.

[0086] Experimental results: such as Figures 8-10 As shown. By Figures 8-10 It can be seen that, compared with the control group, the MA@DC-LNP group had higher levels of IL-6 and IL-12. p40 The expression levels of MA@DC-LNP and IFN-β were significantly upregulated, indicating that MA@DC-LNP can effectively promote the increase of cytokine levels and induce a better immune response.

[0087] 8. In vitro dendritic cell activation experiment

[0088] Mouse bone marrow-derived dendritic cells were seeded into 24-well plates. The experiment was divided into six groups: PBS group, MnO2 group (Comparative Example 1), Free 10K-Adpgk group, MA group (Comparative Example 2), MA@LNP group (Comparative Example 3), and MA@DC-LNP group synthesized in Example 1. Staining was performed in 100 μL systems. The amounts of mouse flow cytometry antibodies used were: anti-CD16 / CD32 blocking antibody (0.25 μg), mouse FITC-anti-CD11c flow cytometry antibody (0.25 μg), mouse APC-anti-CD80 flow cytometry antibody (0.25 μg), mouse PerCP / Cy5.5-anti-CD86 flow cytometry antibody (0.25 μg), mouse PE-anti-IA / IE flow cytometry antibody (0.25 μg), and mouse APC-anti-CD40 flow cytometry antibody (0.25 μg). CD40 levels within the CD11c population were detected by flow cytometry. + CD80 + / CD86 + and MHC II + Cell ratio.

[0089] Experimental results: such as Figures 11-15 As shown. By Figures 11-15 It can be seen that, compared with the control group, the MA@DC-LNP group had a higher CD40 level. + CD80 + / CD86 + and MHC II + The significantly increased cell proportion indicates that MA@DC-LNP can effectively promote the maturation of BMDCs.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a manganese dioxide nanovaccine for targeted delivery of tumor neoantigens by dendritic cells, characterized in that, Includes the following steps: (1) Mix tetraethyl orthosilicate and ethanol to obtain a mixture. Mix ethanol, water and ammonia, stir for the first time, add the mixture dropwise, stir for the second time, centrifuge to collect the precipitate, and obtain the product. (2) After mixing the product with water, add potassium permanganate, sonicate, stir, centrifuge to collect the precipitate, then disperse the precipitate in Na2CO3 solution for etching, centrifuge again to obtain MnO2 nanoparticles. (3) Mix the MnO2 nanoparticle solution and the 10K-Adpgk aqueous solution, stir, and centrifuge to obtain MA nanoparticles; (4) Mix DLin-MC3-DMA, DSPC, DSPE-PEG-Dcpep, cholesterol and chloroform, sonicate in a water bath for the first time, then evaporate by rotary evaporation, add MA sterile enzyme-free water, sonicate in a water bath for the second time, and then extrude and filter to obtain MA@DC-NLP, which is a manganese dioxide nanovaccine that targets and delivers neoantigens to dendritic cells.

2. The manganese dioxide nanovaccine according to claim 1, characterized in that, In step (1), the volume ratio of tetraethyl orthosilicate to ethanol is 3~8:10~20, and the volume ratio of ethanol, water and ammonia is 60~80:5~9:2~4; the temperature of the first stirring is 40~60℃, and the time of the first stirring is 20~40min; the temperature of the second stirring is 40~60℃, and the time of the second stirring is 3~5h.

3. The manganese dioxide nano-vaccine according to claim 1, characterized in that, In step (2), the mass-to-volume ratio of the product to water is 180-220 mg: 40-60 mL, and the mass ratio of the product to potassium permanganate is 1:1-3; the frequency of the ultrasound is 20-30 kHz, the temperature of the ultrasound is 20-30 °C, and the duration of the ultrasound is 1-3 h; the stirring speed is 200-400 rpm, and the stirring time is 12-20 h; the concentration of the Na2CO3 solution is 0.1-0.3 M; the etching temperature is 50-70 °C, and the etching time is 8-16 h.

4. The manganese dioxide nano-vaccine according to claim 1, characterized in that, In step (3), the concentration of the MnO2 nanoparticle solution is 2 mg / mL, the concentration of the 10K-Adpgk aqueous solution is 0.5~4 mg / mL, the volume ratio of the MnO2 nanoparticle solution to the 10K-Adpgk aqueous solution is 1:1, the stirring time is 18~30 h, the centrifugation speed is 10000~14000 rpm, and the centrifugation time is 10~20 min.

5. The manganese dioxide nano-vaccine according to claim 1, characterized in that, In step (4), the molar ratio of DLin-MC3-DMA, DSPC, and DSPE-PEG-Dcpep is 50:10:1~2, the molar ratio of DLin-MC3-DMA to cholesterol is 3~5:1, and the volume of chloroform is 5~15mL.

6. The manganese dioxide nanovaccine according to claim 1, characterized in that, In step (4), the temperature of the first water bath ultrasound is 20~30℃, and the time of the first water bath ultrasound is 20~40min; the rotation speed of the rotary evaporator is 50~150rpm, the temperature of the rotary evaporator is 30~50℃, and the time of the rotary evaporator is 1~3h; the concentration of the MA sterile enzyme-free water is 1~3mg / mL, and the volume of the MA sterile enzyme-free water is 3~8mL; the temperature of the second water bath ultrasound is 40~60℃, and the time of the second water bath ultrasound is 20~40min.

7. The manganese dioxide nanovaccine for targeted delivery of tumor neoantigens to dendritic cells prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the manganese dioxide nanovaccine for targeted delivery of tumor neoantigens by dendritic cells as described in claim 7 in the preparation of antitumor formulations.

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