Calcium-doped manganese phosphate engineered red blood cells based on biomimetic mineralization technology and its application in immunotherapy

Abstract

The application belongs to the technical field of cellular immunity, and particularly relates to calcium-doped manganese phosphate engineered red blood cells based on biomimetic mineralization technology and application thereof in immunotherapy. Calcium ions, manganese phosphate, acidic polypeptides and carboxyl activators are deposited on the surface of red blood cells, the acidic polypeptides provide a negative electric environment, increase the concentration of calcium and manganese on the red blood cell membrane, and form a mineralization layer of calcium ions and manganese ions; the carboxyl activators can activate the carboxyl of amino acids on the acidic polypeptides and covalently combine with other molecules; calcium can improve the stability of manganese phosphate crystals, optimize the release of manganese ions, and activate the cGAS-STING pathway in DCs; the natural half-life of red blood cells is 120 days, which can avoid rapid clearance by the liver, and senescent red blood cells are phagocytosed by spleen DCs to promote antigen cross-presentation. The calcium-doped manganese phosphate engineered red blood cells provided by the application can promote the maturation, migration, homing and antigen presentation function of dendritic cells, and enhance the ability of dendritic cells to activate CD8 + T cells.

Description

Technical Field

[0001] This invention belongs to the field of cellular immunotherapy, specifically relating to calcium-doped manganese phosphate engineered erythrocytes based on biomimetic mineralization technology and their application in immunotherapy. Background Technology

[0002] Immunotherapy has become the fourth major cancer treatment modality after surgery, radiotherapy, and chemotherapy. Its core idea is to eliminate pathogens or diseased cells by regulating the body's immune system. The success of this strategy highly depends on the effective initiation and maintenance of antigen-specific T-cell immune responses, and a key step in this process is antigen presentation. In cellular immunotherapy, dendritic cells (DCs) are key antigen-presenting cells, and their functional status directly determines whether T cells can be effectively activated and an anti-tumor immune response can be triggered. However, the tumor microenvironment often leads to impaired dendritic cell function in patients, manifested as reduced expression of co-stimulatory molecules, abnormal cytokine secretion, and weakened ability to migrate to lymphoid tissues, thus severely limiting the effectiveness of immunotherapy. Restoring and reconstructing the immune function of damaged DCs in the host through intervention is crucial for the success of immunotherapy. However, existing vaccine adjuvants typically contain a combination of IL-1β, IL-6, TNF-α, and PGE2 (CC), which induces dendritic cell dysfunction, including low homing ability and lack of IL-12 secretion, thus limiting their effectiveness in clinical immunotherapy.

[0003] In recent years, the rise of metal immunotherapy, which utilizes metal ions to enhance immunotherapy, has provided entirely new avenues for adjuvant development. Among these, manganese ions (Mn)... 2+ Manganese ions can target and activate the unique cGAS-STING pathway, promoting the secretion of type I interferon, which is key to enhancing cytotoxic CD8+ T cell responses. Therefore, manganese ions are a highly promising candidate novel adjuvant. However, although manganese ions can address the insufficient ability of vaccines to initiate immune effector cell responses, their clinical application still faces many limitations, including a short in vivo half-life (<2h), rapid in vivo metabolism (low utilization), inefficient antigen loading methods, and low co-delivery efficiency. These issues urgently require efficient integration through vector engineering strategies. Summary of the Invention

[0004] The purpose of this invention is to develop calcium-doped manganese phosphate engineered erythrocytes based on biomimetic mineralization technology and their application in immunotherapy, thereby promoting the maturation, migration, and antigen presentation function of dendritic cells (DCs), and significantly enhancing the homing ability and CD8+ of DCs. + T-cell activation is used as an immune adjuvant in vaccine preparation for immunotherapy.

[0005] The present invention provides a calcium-doped manganese phosphate engineered red blood cell, wherein the calcium-doped manganese phosphate engineered red blood cell comprises red blood cells and calcium ions, manganese phosphate, acidic polypeptides and carboxyl activators deposited on the surface of the red blood cells.

[0006] Preferably, the amino acid sequence of the acidic polypeptide is shown in SEQ ID NO:1;

[0007] The carboxyl activator includes 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide.

[0008] This invention provides a method for preparing calcium-doped manganese phosphate engineered erythrocytes as described above, comprising the following steps:

[0009] Red blood cells, acidic polypeptides, carboxyl activator and first physiological saline were mixed and incubated at 20~25℃ for 0.5~1h to obtain the first incubation product;

[0010] The first incubation product was subjected to a first solid-liquid separation, and the precipitate was collected to obtain pretreated red blood cells.

[0011] The pretreated red blood cells, manganese chloride, calcium chloride, second physiological saline and phosphate buffer were mixed and incubated at 20-25°C for 15-30 min to obtain the second incubation product.

[0012] The second incubation product was subjected to a second solid-liquid separation, and the precipitate was collected to obtain the calcium-doped manganese phosphate engineered red blood cells.

[0013] Preferably, the concentration ratio of red blood cells to acidic peptides in the mixture of red blood cells, acidic peptides, carboxyl activator, and first physiological saline is 1×10⁻⁶. 7 ~2×10 8 Cells / mL: 50~100μg / mL; the molar ratio of acidic polypeptide to carboxyl activator in the mixture of red blood cells, acidic polypeptide, carboxyl activator and first physiological saline is 10~1:20~1;

[0014] The concentration ratio of pretreated red blood cells to manganese chloride in the mixture of pretreated red blood cells, manganese chloride, calcium chloride, second physiological saline, and phosphate buffer is 1×10⁻⁶. 7 ~2×10 8 Cells / mL: 50~100mM; the concentration ratio of pretreated red blood cells to calcium chloride in the mixture of pretreated red blood cells, manganese chloride, calcium chloride, second physiological saline and phosphate buffer is 1×10⁻⁶. 7 ~2×10 8 Cells / mL: 10~20μM;

[0015] The total volume ratio of the pretreated red blood cells, manganese chloride, calcium chloride, and the second physiological saline to the volume ratio of the phosphate buffer is 1:(1~2).

[0016] Preferably, the first solid-liquid separation and the second solid-liquid separation methods each include centrifugation; the centrifugation temperature is 2~6℃, the centrifugation force is 400~500×g, and the time is 5~10min.

[0017] This invention provides the application of calcium-doped manganese phosphate engineered red blood cells as described in the above technical solution, or calcium-doped manganese phosphate engineered red blood cells prepared by the preparation method described in the above technical solution, as an immune adjuvant in the preparation of vaccines.

[0018] This invention provides the application of the calcium-doped manganese phosphate engineered erythrocytes described in the above-described technical solution, or the calcium-doped manganese phosphate engineered erythrocytes prepared by the preparation method described in the above-described technical solution, in one or more of the following:

[0019] (1) Prepare products that promote the maturation of dendritic cells;

[0020] (2) Prepare products that promote dendritic cell migration and / or homing;

[0021] (3) Prepare products that promote the presentation of dendritic cell antigens;

[0022] (4) Preparation of enhanced dendritic cells to activate CD8 + Products that enhance T-cell capabilities;

[0023] (5) Prepare products that enhance one or more of the following abilities of dendritic cells: antitumor, anti-infection, anti-autoimmune disease and anti-graft-host disease.

[0024] Preferably, promoting dendritic cell maturation includes promoting the expression of dendritic cell co-stimulatory molecules; the co-stimulatory molecules include one or more of CD80, CD86, and CD40;

[0025] The promotion of dendritic cell migration and / or homing includes promoting the expression of dendritic cell chemokine receptors; the chemokine receptors include CCR7;

[0026] The function of promoting dendritic cell antigen presentation includes promoting the expression of the MHCⅠ-SIINFEKL complex on the surface of dendritic cells;

[0027] The enhanced dendritic cells activate CD8 + T cell capabilities include promoting CD8+ + The expression of one or more molecules of CD44, CD25, and CD69 in T cells.

[0028] This invention provides an immune enhancer comprising an antigen and calcium-doped manganese phosphate engineered red blood cells; wherein the calcium-doped manganese phosphate engineered red blood cells are the calcium-doped manganese phosphate engineered red blood cells described in the above technical solution or calcium-doped manganese phosphate engineered red blood cells prepared using the preparation method described in the above technical solution.

[0029] The present invention provides a method for preparing the immune enhancer described in the above technical solution, comprising the following steps: mixing the calcium-doped manganese phosphate engineered red blood cells and the antigen, incubating at 20~25℃ for 0.5~1h, obtaining the incubated product, performing solid-liquid separation, collecting the precipitate, and obtaining the dendritic cell immune enhancer.

[0030] Beneficial effects:

[0031] This invention deposits calcium ions, manganese phosphate, acidic polypeptides, and carboxyl activators on the surface of erythrocytes. The acidic polypeptides provide a negatively charged environment, increasing the local concentration of calcium and manganese ions on the erythrocyte membrane and simultaneously forming Ca2+ on the erythrocyte surface. 2+ and Mn 2+ The mineralized layer, carboxyl activators can activate the carboxyl groups of amino acids on acidic peptides, causing them to react with primary amines to form stable amide bonds, achieving covalent bonding with other molecules; calcium doping (Ca 2+ To improve the stability of manganese phosphate crystals and optimize Mn 2+ Release and activation of the cGAS-STING pathway within dendritic cells (DCs); the natural half-life of erythrocytes reaches 120 days, enabling them to evade rapid liver clearance; senescent erythrocytes are phagocytosed by splenic DCs, promoting antigen cross-presentation. The calcium-doped manganese phosphate engineered erythrocytes provided by this invention can act as an immune adjuvant for dendritic cells, promoting dendritic cell maturation, migration and / or homing, antigen presentation function, and enhancing CD8 activation by dendritic cells. + T cell capacity. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0033] Figure 1 The following are the characterization results of different erythrocyte-based immune enhancers in Test Example 1; where a is the scanning electron microscope (SEM) result; b is the SEM-EDS (energy-dispersive spectroscopy) result, and the table in the figure shows the relative elemental content results; c is the calcium content of different erythrocyte-based immune enhancers; d is the manganese content of different erythrocyte-based immune enhancers; *** is... p <0.001;

[0034] Figure 2To test the expression levels of co-stimulatory molecules CD80, CD86, and CD40 on the surface of dendritic cells in different treatment groups of Example 2; where * indicates p <0.05;

[0035] Figure 3 To test the expression levels of chemokine receptor CCR on the surface of dendritic cells in different treatment groups of Example 2; where * indicates p <0.05, ** is p <0.01;

[0036] Figure 4 To test the expression levels of the MHCⅠ-SIINFEKL complex on the surface of dendritic cells in different treatment groups of Example 3; where *** represents p <0.001;

[0037] Figure 5 For test example 4, different treatment groups CD8 + The positive rates of CD25, CD44, and CD69 in T cells; where * indicates p <0.05. Detailed Implementation

[0038] The present invention provides a calcium-doped manganese phosphate engineered red blood cell, wherein the calcium-doped manganese phosphate engineered red blood cell comprises red blood cells and calcium ions, manganese phosphate, acidic polypeptides and carboxyl activators deposited on the surface of the red blood cells.

[0039] As one embodiment, the amino acid sequence of the acidic polypeptide is shown in SEQ ID NO:1. The acidic polypeptide of this invention provides a negatively charged environment, thereby increasing the local concentration of calcium and manganese ions on the erythrocyte membrane and promoting the deposition of calcium ions and manganese phosphate in erythrocytes. This invention does not have strict requirements on the amino acid sequence of the acidic polypeptide; any polypeptide rich in acidic amino acid residues is acceptable. This invention uses a polypeptide with the amino acid sequence shown in SEQ ID NO:1 as an example in the embodiments, but this should not be construed as representing the entire scope of protection of this invention.

[0040] In one embodiment, the carboxyl activator comprises 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide. The carboxyl activator of this invention can activate the carboxyl group on acidic polypeptides, causing it to covalently bind to other molecules. For example, after activation, the carboxyl group on an acidic polypeptide can react with a primary amine to form a stable amide bond.

[0041] This invention provides a method for preparing calcium-doped manganese phosphate engineered erythrocytes as described above, comprising the following steps:

[0042] Red blood cells, acidic polypeptides, carboxyl activator and first physiological saline were mixed and incubated at 20~25℃ for 0.5~1h to obtain the first incubation product;

[0043] The first incubation product was subjected to a first solid-liquid separation, and the precipitate was collected to obtain pretreated red blood cells.

[0044] The pretreated red blood cells, manganese chloride, calcium chloride, second physiological saline and phosphate buffer were mixed and incubated at 20-25°C for 15-30 min to obtain the second incubation product.

[0045] The second incubation product was subjected to a second solid-liquid separation, and the precipitate was collected to obtain the calcium-doped manganese phosphate engineered red blood cells.

[0046] In this invention, red blood cells, acidic polypeptides, carboxyl activators and a first physiological saline are mixed and incubated at 20-25°C for 0.5-1 h to obtain a first incubation product.

[0047] In one embodiment, the concentration ratio of red blood cells to acidic peptides in the mixture of red blood cells, acidic peptides, carboxyl activator, and first physiological saline described in this invention is 1 × 10⁻⁶. 7 ~2×10 8 Cells / mL: 50~100 μg / mL; As another embodiment, the concentration ratio of erythrocytes to acidic peptides in the mixture of erythrocytes, acidic peptides, carboxyl activator, and first physiological saline described in this invention is 2×10⁻⁶. 8 Cells / mL: 100 μg / mL. This invention limits the concentration ratio of erythrocytes to acidic peptides, which has the advantages of product stability, easy quality control, and cost savings.

[0048] In one embodiment, the molar ratio of acidic peptides to carboxyl activator in the mixture of erythrocytes, acidic peptides, carboxyl activator, and first physiological saline is 10~1:20~1; in another embodiment, the molar ratio of acidic peptides to carboxyl activator in the mixture of erythrocytes, acidic peptides, carboxyl activator, and first physiological saline is 20:1. This invention limits the molar ratio of acidic peptides to carboxyl activator, which has the advantage of high coupling efficiency.

[0049] In one embodiment, the temperature of the first incubation in this invention is 25°C. In another embodiment, the time of the first incubation in this invention is 1 hour. This invention limits the temperature and time of the first incubation, offering advantages such as product stability and ease of quality control.

[0050] After obtaining the first incubation product, the present invention performs a first solid-liquid separation on the first incubation product, collects the precipitate, and obtains pretreated red blood cells.

[0051] In one embodiment, the first solid-liquid separation method of the present invention includes centrifugation. In one embodiment, the centrifugation temperature is 2-6°C; in another embodiment, the centrifugation temperature is 4°C. In one embodiment, the centrifugal force is 400-500 × g; in another embodiment, the centrifugal force is 500 × g. In one embodiment, the centrifugation time is 5-10 min; in another embodiment, the centrifugation time is 5 min.

[0052] After obtaining the pretreated red blood cells, the present invention mixes the pretreated red blood cells, manganese chloride, calcium chloride, second physiological saline and phosphate buffer, and incubates them at 20~25℃ for 0.5~1h to obtain the second incubation product.

[0053] In one embodiment, the concentration ratio of pretreated red blood cells to manganese chloride in the mixture of pretreated red blood cells, manganese chloride, calcium chloride, second physiological saline, and phosphate buffer is 1×10⁻⁶. 7 ~2×10 8 Cells / mL: 50~100mM; In another embodiment, the concentration ratio of pretreated red blood cells to manganese chloride in the mixture of pretreated red blood cells, manganese chloride, calcium chloride, second physiological saline, and phosphate buffer is 2×10⁻⁶ mM. 8 Cells / mL: 80mM. This invention limits the concentration ratio of pretreated red blood cells to manganese chloride, which has the advantages of rapid reaction and minimal hemolysis.

[0054] In one embodiment, the concentration ratio of pretreated red blood cells to calcium chloride in the mixture of pretreated red blood cells, manganese chloride, calcium chloride, second physiological saline, and phosphate buffer is 1×10⁻⁶. 7 ~2×10 8 Cells / mL: 10~20 μM; As another embodiment, the concentration ratio of pretreated red blood cells to calcium chloride in the mixture of pretreated red blood cells, manganese chloride, calcium chloride, second physiological saline and phosphate buffer is 2×10⁻⁶. 8 Cells / mL: 16mM. This invention limits the concentration ratio of pretreated red blood cells to calcium chloride, which has the advantages of rapid reaction and low hemolysis.

[0055] In one embodiment, the total volume ratio of the pretreated red blood cells, manganese chloride, calcium chloride, and second physiological saline to the phosphate buffer solution is 1:(1~2); in another embodiment, the total volume ratio of the pretreated red blood cells, manganese chloride, calcium chloride, and second physiological saline to the phosphate buffer solution is 1:1. This invention limits the total volume ratio of the pretreated red blood cells, manganese chloride, calcium chloride, and second physiological saline to the phosphate buffer solution, which has the advantages of rapid reaction and easy quality control.

[0056] In one embodiment, the second incubation temperature of the present invention is 25°C. In another embodiment, the second incubation time of the present invention is 30 minutes. The present invention limits the temperature and time of the second incubation, which has the advantages of being easy to control and requiring no additional cooling operation.

[0057] After obtaining the second incubation product, the present invention performs a second solid-liquid separation on the second incubation product, collects the precipitate portion, and obtains the calcium-doped manganese phosphate engineered red blood cells.

[0058] In one embodiment, the second solid-liquid separation method of the present invention includes centrifugation. In one embodiment, the centrifugation temperature is 2-6°C; in another embodiment, the centrifugation temperature is 4°C. In one embodiment, the centrifugal force is 400-500 × g; in another embodiment, the centrifugal force is 500 × g. In one embodiment, the centrifugation time is 5-10 min; in another embodiment, the centrifugation time is 5 min.

[0059] As one implementation method, the present invention uses physiological saline to resuspend the calcium-doped manganese phosphate engineered red blood cells to obtain a suspension of calcium-doped manganese phosphate engineered red blood cells.

[0060] The calcium-doped manganese phosphate engineered erythrocytes provided by this invention can serve as immune adjuvants, particularly as dendritic cell adjuvants, promoting dendritic cell maturation, migration and / or homing, antigen presentation, and CD8 activation. + T cell capacity.

[0061] Given the advantages of the calcium-doped manganese phosphate engineered erythrocytes of this invention, the application of the calcium-doped manganese phosphate engineered erythrocytes described in the above-described technical solution of this invention, or the calcium-doped manganese phosphate engineered erythrocytes prepared using the preparation method described in the above-described technical solution, as an immune adjuvant in the preparation of vaccines also falls within the scope of protection of this invention. As one embodiment, the vaccine of this invention includes a dendritic cell immunotherapy vaccine.

[0062] Given the advantages of the calcium-doped manganese phosphate engineered erythrocytes of the present invention, the application of the calcium-doped manganese phosphate engineered erythrocytes described in the above-described technical solutions of the present invention, or the calcium-doped manganese phosphate engineered erythrocytes prepared by the preparation method described in the above-described technical solutions, in one or more of the following applications also falls within the scope of protection of the present invention: (1) preparing products that promote dendritic cell maturation; (2) preparing products that promote dendritic cell migration and / or homing; (3) preparing products that promote the antigen presentation function of dendritic cells; (4) preparing products that enhance the activation of CD8 by dendritic cells. + (5) Products that enhance the T-cell capabilities; (6) Products that enhance one or more of the following capabilities of dendritic cells: antitumor, anti-infection, anti-autoimmune disease and anti-graft-host disease.

[0063] In one embodiment, the promotion of dendritic cell maturation according to the present invention includes promoting the expression of dendritic cell co-stimulatory molecules. In one embodiment, the co-stimulatory molecules include one or more of CD80, CD86, and CD40. In one embodiment, the promotion of dendritic cell migration and / or homing according to the present invention includes promoting the expression of dendritic cell chemokine receptors; the chemokine receptors include CCR7. In one embodiment, the promotion of dendritic cell antigen presentation function according to the present invention includes promoting the expression of the MHCⅠ-SIINFEKL complex on the surface of dendritic cells. In one embodiment, the enhancement of dendritic cell activation of CD8... + T cell capabilities include promoting CD8 + The expression of one or more molecules of CD44, CD25 and CD69 in T cells.

[0064] This invention provides an immune enhancer comprising an antigen and calcium-doped manganese phosphate engineered red blood cells; wherein the calcium-doped manganese phosphate engineered red blood cells are the calcium-doped manganese phosphate engineered red blood cells described in the above technical solution or calcium-doped manganese phosphate engineered red blood cells prepared using the preparation method described in the above technical solution.

[0065] In one embodiment, the antigen of this invention includes chicken ovalbumin. This invention uses chicken ovalbumin as an example for illustration, but it should not be construed as representing the entire scope of protection of this invention. In one embodiment, each 1×10 7 ~2×10 8 The calcium-doped manganese phosphate engineered red blood cells were incubated with an antigen at a concentration of 100 μg / mL, the number of which was measured in red blood cells.

[0066] The present invention provides a method for preparing the immune enhancer described in the above technical solution, comprising the following steps: mixing the calcium-doped manganese phosphate engineered red blood cells and the antigen, incubating at 20~25℃ for 0.5~1h, obtaining the incubated product, performing solid-liquid separation, collecting the precipitate, and obtaining the dendritic cell immune enhancer.

[0067] In one embodiment, the incubation temperature of the present invention is 25°C. In one embodiment, the incubation time of the present invention is 1 hour. In one embodiment, the oscillation frequency of the incubation of the present invention is 50~100 rpm; in another embodiment, the oscillation frequency of the incubation of the present invention is 80 rpm.

[0068] This invention utilizes the aforementioned dendritic cell immune enhancer to treat dendritic cells. Compared to a single antigen, it can upregulate the expression of the MHCⅠ-SIINFEKL complex on the surface of dendritic cells, thereby promoting the enhancement of the antigen presentation capacity of dendritic cells.

[0069] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes the calcium-doped manganese phosphate engineered red blood cells based on biomimetic mineralization technology and their application in immunotherapy, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0070] The materials and their sources used in the embodiments of this invention are as follows: Dabrafenib Mesylate (MCE); Dimethyl sulfoxide (DMSO, Sigma); RPMI 1640 medium (Gibco); Fetal bovine serum (Gibco); Penicillin-streptomycin (Gibco); Glutamine (Gibco); Granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech); IL-4 (Peprotech); Flow cytometry antibodies against CD11c, CD80, CD86, CCR7, MHC I-SIINFEKL, CD8, CD25, CD69, and CD44 were purchased from Biolegend; Naïve CD8 + T-cell sorting kit (Miltenyi Biotec).

[0071] The complete culture medium used in the embodiments of the present invention is RPMI 1640 medium containing GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin and glutamine, and the final concentrations of GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin and glutamine are 15ng / ml, 10ng / ml, 10%, 100U / ml, 100μg / ml and 2mM respectively.

[0072] The polypeptide OVA used in the embodiments of the present invention 257-264The sequence is SIINFEKL, purchased from InvivoGen.

[0073] The method for preparing bone marrow cells used in this embodiment of the invention is as follows: (1) Take the femur and tibia of a mouse, remove the muscle, disinfect and clean them, rinse the bone marrow cells into a 50ml centrifuge tube with PBS that has been sterilized by high pressure and pre-cooled at 4℃, centrifuge at 400g speed for 5 minutes, and remove the supernatant; (2) Resuspend the cells with PBS, filter the resuspended cells with a 40μm filter membrane to remove impurities such as bone fragments, centrifuge at 400g speed for 5 minutes, and remove the supernatant; (3) Resuspend the cells with complete culture medium and count them for later use.

[0074] Example 1

[0075] Preparation of calcium-doped manganese phosphate engineered red blood cells

[0076] 1. Place mouse whole blood anticoagulated with EDTA into a low-speed centrifuge, set the temperature to 22℃, the centrifugal force to 150×g, and centrifuge for 20 min to obtain the bottom layer of red blood cells.

[0077] 2. Aspirate the red blood cells, add 3 times the volume of physiological saline, mix well, and place in a low-speed centrifuge. Set the temperature to 4℃ and the centrifugal force to 500×g, and centrifuge for 5 minutes to obtain purified red blood cells.

[0078] 3. Adjust the red blood cell concentration to 2 × 10⁻⁶ using physiological saline. 8 Add 100 μg / mL of a polypeptide with 12 amino acid residues (SEQ ID NO:1: EENEDENNEDN), then add 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) to make the molar ratio of polypeptide to EDC 20:1. After mixing, incubate at 25°C for 1 h.

[0079] 4. Place in a low-speed centrifuge, set the temperature to 4℃, the centrifugal force to 500×g, centrifuge for 5 minutes, discard the supernatant and resuspend in physiological saline.

[0080] 5. Adjust the red blood cell concentration to 2 × 10⁻⁶ using physiological saline. 8 Add 80 mM manganese chloride and 16 mM calcium chloride per mL, mix well, then add an equal volume of phosphate-balanced saline (PBS), and incubate at 25°C for 30 min.

[0081] 6. Place in a low-speed centrifuge, set the temperature to 4℃, the centrifugal force to 500×g, centrifuge for 5 minutes, discard the supernatant, and obtain calcium-doped manganese phosphate engineered red blood cells, which can be used as vaccine adjuvants.

[0082] Example 2

[0083] Preparation of Dendritic Cell Immunostimulants Based on Calcium-Doped Manganese Phosphate Engineered Erythrocytes

[0084] 1. The concentration of the calcium-doped manganese phosphate engineered erythrocytes obtained in Example 1 was adjusted to 2 × 10⁻⁶ using physiological saline. 8 Add 100 μg / mL of ovalbumin (OVA), mix well, and incubate at 25°C with shaking at 80 rpm for 1 h.

[0085] 2. Place in a low-speed centrifuge, set the temperature to 4℃, the centrifugal force to 500×g, centrifuge for 5 minutes and discard the supernatant to obtain a dendritic cell immune enhancer based on calcium-doped manganese phosphate engineered erythrocytes.

[0086] Comparative Example 1

[0087] Preparation of erythrocyte-based immune enhancers

[0088] 1. Adjust the concentration of purified red blood cells obtained in step 2 of Example 1 to 2 × 10⁻⁶ using physiological saline. 8 Add 100 μg / mL of ovalbumin (OVA), mix well, and incubate at 25°C with shaking at 80 rpm for 1 h.

[0089] 2. Place in a low-speed centrifuge, set the temperature to 4℃, the centrifugal force to 500×g, centrifuge for 5 minutes and discard the supernatant to obtain the erythrocyte-based immune enhancer.

[0090] Test Example 1

[0091] Characterization of Dendritic Cell Immunostimulants Based on Calcium-Doped Manganese Phosphate Engineered Erythrocytes

[0092] The engineered erythrocyte-based immunostimulant (RBC@MnCaP) obtained in Example 2 and the erythrocyte-based immunostimulant (RBC) obtained in Comparative Example 1 were resuspended in 4% paraformaldehyde. After incubation at room temperature for 15 min, the cells were centrifuged at 4°C and 500×g for 5 min. The supernatant was discarded, and different concentration gradients (30%, 50%, 70%, 90%, 100%) of ethanol were added for gradient dehydration. Finally, the erythrocytes were resuspended in deionized water and detected by scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) and inductively coupled plasma mass spectrometry (ICP-MS). The detection results are as follows: Figure 1 As shown.

[0093] according to Figure 1 As can be seen, compared with the RBC obtained in Comparative Example 1, the surface of the RBC@MnCaP obtained in Example 2 becomes rougher and nanoscale mineral particles are visible deposited. Figure 1 (a) EDS and ICP-MS results also confirmed Ca2+ and Mn 2+ Deposited on red blood cells ( Figure 1 (bd). Example 2 successfully constructed an engineered erythrocyte-based immune enhancer.

[0094] Comparative Example 2

[0095] Preparation of calcium-engineered red blood cells

[0096] Same as Example 1, except that 80mM manganese chloride is not added in step 5.

[0097] Comparative Example 3

[0098] Preparation of manganese-engineered red blood cells

[0099] Same as Example 1, except that 16mM calcium chloride is not added in step 5.

[0100] Comparative Example 4

[0101] Preparation of calcium-engineered erythrocyte-based immune enhancers

[0102] Same as Example 2, except that the calcium-doped manganese phosphate engineered red blood cells were replaced with the calcium-engineered red blood cells obtained in Comparative Example 2.

[0103] Comparative Example 5

[0104] Preparation of manganese-engineered erythrocyte-based immune enhancers

[0105] Same as Example 2, except that the calcium-doped manganese phosphate engineered red blood cells were replaced with the manganese engineered red blood cells obtained in Comparative Example 3.

[0106] Test Example 2

[0107] Effects of engineered erythrocyte-based immune enhancers on the expression levels of dendritic cell-related factors

[0108] 1. Mouse bone marrow cells were cultured in complete culture medium at a concentration of 1.5 × 10⁻⁶. 6 The cells were resuspended at a concentration of 1 cell / ml and seeded at 2 ml / well in 6-well culture plates. The plates were then incubated at 37°C with 5% carbon dioxide for 7 days. The medium was changed on days 3 and 5. On day 7, immature dendritic cells with low immunogenicity were obtained.

[0109] 2. Resuspend the above immature dendritic cells in fresh complete culture medium at a concentration of 1.5 × 10⁻⁶. 6 The samples were collected at a concentration of 100 cells / ml and randomly divided into four treatment groups, labeled CON, CaCl2, MnCl2, and RBC@MnCaP, respectively. The following treatments were then performed:

[0110] Control group (CON): No treatment or intervention was given to dendritic cells;

[0111] CaCl2: Add 5×10 7 Comparative Example 4, which used a calcium-engineered erythrocyte-based immune enhancer, treated dendritic cells for 24 h.

[0112] MnCl2: Add 5×10 7 Comparative Example 5, which used manganese-engineered erythrocyte-based immune enhancers, was used to treat dendritic cells for 24 h.

[0113] RBC@MnCaP: Add 5×10 7 Dendritic cell immunostimulants based on calcium-doped manganese phosphate engineered erythrocytes obtained in Example 2 were used to treat dendritic cells for 24 hours.

[0114] 3. CD11c in living cells + The cells were dendritic cells. The proportions of CD80, CD86, and CD40-positive cells in dendritic cells reflected the expression levels of the co-stimulatory molecules CD80, CD86, and CD40, respectively. The proportion of CCR7-positive cells in dendritic cells reflected the expression level of the chemokine receptor CCR7. Results are as follows... Figure 2 and Figure 3 As shown.

[0115] according to Figure 2 It can be seen that the positive ratios of CD80, CD86, and CD40 on dendritic cells in the RBC@MnCaP treatment group were significantly higher than those in the control group. The dendritic cell immune enhancer based on calcium-doped manganese phosphate engineered erythrocytes obtained in Example 2 can upregulate the expression of co-stimulatory molecules CD80, CD86, and CD40 on the surface of dendritic cells, and promote dendritic cell maturation.

[0116] according to Figure 3 It can be seen that the positive ratio of CCR7 in dendritic cells of the RBC@MnCaP treatment group, CaCl2 treatment group, and MnCl2 treatment group was significantly higher than that in the control group, and the positive ratio of CCR7 in dendritic cells of the RBC@MnCaP treatment group was significantly higher than that in the MnCl2 treatment group. The dendritic cell immune enhancer based on calcium-doped manganese phosphate engineered erythrocytes obtained in Example 2 can upregulate the expression of chemokine receptor CCR on the surface of dendritic cells and promote the improvement of lymphocyte homing ability of dendritic cells.

[0117] Test Example 3

[0118] Effects of engineered erythrocyte-based immunostimulants on the expression level of the MHCⅠ-SIINFEKL complex on the surface of dendritic cells

[0119] 1. Mouse bone marrow cells were cultured in complete culture medium at a concentration of 1.5 × 10⁻⁶. 6 The cells were resuspended at a concentration of 1 cell / ml and seeded at 2 ml / well in 6-well culture plates. The plates were then incubated at 37°C with 5% carbon dioxide for 7 days. The medium was changed on days 3 and 5. On day 7, immature dendritic cells with low immunogenicity were obtained.

[0120] 2. Resuspend the above immature dendritic cells in fresh complete culture medium at a concentration of 1.5 × 10⁻⁶. 6 The samples were collected per ml and randomly divided into three treatment groups, labeled CON, OVA, and RBC@MnCaP, respectively. The following treatments were then performed:

[0121] Control group (CON): No treatment or intervention was given to dendritic cells;

[0122] OVA: Add OVA to a final concentration of 1 μg / ml. 257-26 Dendritic cells were treated with a peptide (sequence: SIINFEKL, SEQ ID NO:2) for 24 h.

[0123] RBC@MnCaP: Add 5×10 7 Dendritic cell immunostimulants based on calcium-doped manganese phosphate engineered erythrocytes obtained in Example 2 were used to treat dendritic cells for 24 hours.

[0124] 3. CD11c in living cells + The cells were dendritic cells, and the proportion of SIINFEKL-positive dendritic cells reflected the expression level of the MHCⅠ-SIINFEKL complex. Cells were harvested after treatment in each group, and the expression level of the MHCⅠ-SIINFEKL complex on the surface of dendritic cells was detected by flow cytometry staining. Results are as follows: Figure 4 As shown.

[0125] according to Figure 4 It can be seen that the positive ratio of CCR7 in dendritic cells of the RBC@MnCaP treatment group was significantly higher than that in dendritic cells of the OVA treatment group. The dendritic cell immune enhancer based on calcium-doped manganese phosphate engineered erythrocytes obtained in Example 2 can upregulate the expression of the MHCⅠ-SIINFEKL complex on the surface of dendritic cells and promote the enhancement of the antigen presentation capacity of dendritic cells.

[0126] Test Example 4

[0127] Engineered erythrocyte-based immune enhancers activate CD8 dendritic cells + The impact of T cell function

[0128] 1. Mouse bone marrow cells were cultured in complete culture medium at a concentration of 1.5 × 10⁻⁶. 6 The cells were resuspended at a concentration of 1 cell / ml and seeded at 2 ml / well in 6-well culture plates. The plates were then incubated at 37°C with 5% carbon dioxide for 7 days. The medium was changed on days 3 and 5. On day 7, immature dendritic cells with low immunogenicity were obtained.

[0129] 2. Resuspend the above immature dendritic cells in fresh complete culture medium at a concentration of 1.5 × 10⁻⁶. 6 The samples were collected per ml and randomly divided into two treatment groups, labeled CON and RBC@MnCaP respectively, and then processed as follows:

[0130] Control group (CON): No treatment or intervention was given to dendritic cells;

[0131] RBC@MnCaP: Add 5×10 7 Dendritic cell immunostimulants based on calcium-doped manganese phosphate engineered erythrocytes obtained in Example 2 were used to treat dendritic cells for 24 hours.

[0132] 3. Through the initial CD8 + T-cell sorting kit for purifying initial CD8+ cells from OT-I mice + T cells were resuspended in T cell culture medium and counted for later use.

[0133] 4. The dendritic cells obtained in different treatment groups from step 3 were then compared with naïve CD8+. + T cell co-culture, each co-culture system consisting of 500 μl and containing 5 × 10⁶ cells. 5 Dendritic cells, 2.5 × 10 6 An initial CD8 + T cells and 10 ng / ml OVA257-264 (amino acid sequence SIINFEKL, SEQ ID NO:2).

[0134] 5. After co-culturing for 24 hours, cells were harvested, and CD8+ staining was detected by flow cytometry. + Expression levels of T cell surface activation-related markers CD25, CD44, and CD69. CD8 expression levels in living cells. + The cells are CD8 + T cells, CD8 + The proportion of CD25, CD44, and CD69-positive cells in T cells reflects the expression levels of CD25, CD44, and CD69. The detection results are as follows: Figure 5 As shown.

[0135] according to Figure 5 It can be seen that the CD8 of the RBC@MnCaP treatment group +The positive rates of CD25, CD44, and CD69 in T cells were significantly higher than those in the control group. The dendritic cell immunostimulant based on calcium-doped manganese phosphate engineered erythrocytes obtained in Example 2 enhanced the activation of CD8 by dendritic cells. + The ability of T cells.

[0136] As can be seen from the above, the calcium-doped manganese phosphate engineered erythrocytes provided by this invention can act as an immune adjuvant for dendritic cells, promoting dendritic cell maturation, migration and / or homing, antigen presentation, and CD8 activation. + T cell capacity.

[0137] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing calcium-doped manganese phosphate engineered erythrocytes, characterized in that, Includes the following steps: Red blood cells, acidic polypeptides, carboxyl activator and first physiological saline were mixed and incubated at 25°C for 1 hour to obtain the first incubation product; The first incubation product was subjected to a first solid-liquid separation, and the precipitate was collected to obtain pretreated red blood cells. The pretreated red blood cells, manganese chloride, calcium chloride, second physiological saline and phosphate buffer were mixed and incubated at 25°C for 30 min to obtain the second incubation product; The second incubation product was subjected to a second solid-liquid separation, and the precipitate was collected to obtain the calcium-doped manganese phosphate engineered erythrocytes. The concentration ratio of red blood cells to acidic peptides in the mixture of red blood cells, acidic peptides, carboxyl activator, and first physiological saline is 2 × 10⁻⁶. 8 Cells / mL: 100 μg / mL; the molar ratio of acidic peptides to carboxyl activator in the mixture of red blood cells, acidic peptides, carboxyl activator and first physiological saline is 20:1; The concentration ratio of pretreated red blood cells to manganese chloride in the mixture of pretreated red blood cells, manganese chloride, calcium chloride, second physiological saline, and phosphate buffer is 2 × 10⁻⁶. 8 Cells / mL: 80mM; The concentration ratio of pretreated red blood cells to calcium chloride in the mixture of pretreated red blood cells, manganese chloride, calcium chloride, second physiological saline, and phosphate buffer is 2×10⁻⁶. 8 Cells / mL: 16μM; The total volume ratio of the pretreated red blood cells, manganese chloride, calcium chloride, and the second physiological saline to the volume ratio of the phosphate buffer is 1:

1. The calcium-doped manganese phosphate engineered red blood cells include red blood cells and calcium ions, manganese phosphate, acidic polypeptides, and carboxyl activators deposited on the surface of the red blood cells. The amino acid sequence of the acidic polypeptide is shown in SEQ ID NO:1; The carboxyl activator is 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide.

2. The preparation method according to claim 1, characterized in that, The first solid-liquid separation and the second solid-liquid separation methods each include centrifugation; the centrifugation temperature is 2~6℃, the centrifugation force is 400~500×g, and the time is 5~10min.

3. Calcium-doped manganese phosphate engineered red blood cells prepared by the preparation method according to claim 1 or 2.

4. The application of the calcium-doped manganese phosphate engineered erythrocytes as described in claim 3 as an immune adjuvant in the preparation of vaccines.

5. The use of the calcium-doped manganese phosphate engineered erythrocytes according to claim 3 in one or more of the following: (1) Prepare products that promote the maturation of dendritic cells; (2) Prepare products that promote dendritic cell migration and / or homing; (3) Prepare products that promote the presentation of dendritic cell antigens; (4) Preparation of enhanced dendritic cells to activate CD8 + Products that enhance T-cell capabilities.

6. The application according to claim 5, characterized in that, The promotion of dendritic cell maturation includes promoting the expression of dendritic cell co-stimulatory molecules; the co-stimulatory molecules include one or more of CD80, CD86, and CD40; The promotion of dendritic cell migration and / or homing includes promoting the expression of dendritic cell chemokine receptors; the chemokine receptors include CCR7; The function of promoting dendritic cell antigen presentation includes promoting the expression of the MHCⅠ-SIINFEKL complex on the surface of dendritic cells; The enhanced dendritic cell activation CD8 + T cell capabilities include promoting CD8 + The expression of one or more molecules of CD44, CD25 and CD69 in T cells.

7. An immune enhancer, characterized in that, It includes antigens and calcium-doped manganese phosphate engineered red blood cells; the calcium-doped manganese phosphate engineered red blood cells are those described in claim 3.

8. The method for preparing the immune enhancer according to claim 7, characterized in that, The process includes the following steps: mixing the calcium-doped manganese phosphate engineered red blood cells with the antigen, incubating at 20-25°C for 0.5-1 h, obtaining the incubated product, performing solid-liquid separation, collecting the precipitate, and obtaining the dendritic cell immune enhancer.

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