An antigen delivery carrier targeting dendritic cells to promote antigen lysosomal escape and activate the immune system, and its preparation method and application

Abstract

The present invention discloses an antigen delivery carrier that targets dendritic cells, promotes antigen lysosomal escape, and activates the immune system, and its preparation method and application. The present invention provides a carrier for delivering antigens into the body, which is a metal skeleton complex obtained by functionally modifying a metal skeleton material with polyethyleneimine and mannose. The antigen delivery carrier provided by the present invention has uniform particle size, good dispersibility, high biosafety, and a large antigen loading capacity. It exhibits high targeting to dendritic cells, increases the uptake of antigens by dendritic cells, promotes the maturation of dendritic cells, and promotes the cross-presentation of antigens in dendritic cells, and can efficiently activate CD4 + and CD8 + T cells, causing a strong immune response and killing cancer cells. This carrier has a wide range of applications and can be used to encapsulate single or multiple antigens, and has the ability to kill common tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to an antigen delivery carrier that targets dendritic cells to promote antigen lysosome escape and activate the immune system, and a preparation method and application thereof. Background Art

[0002] Dendritic cells (DC) are the most powerful antigen-presenting cells known to date. They are at the center of initiating, regulating, and maintaining immune responses, and can effectively activate immune responses. DC-based immunotherapy is one of the most promising immunotherapy strategies. Currently, DC immunotherapy has made major breakthroughs in the treatment of major cancers. The ilixadencel DC vaccine announced by Aivita Biomedical has excellent performance in the treatment of malignant tumors such as metastatic renal cell carcinoma, glioblastoma, and melanoma, greatly improving patients' progression-free survival rate, overall survival rate, and complete remission rate, and is safer. DC-based immunotherapy has achieved exciting results in improving the treatment effect of tumor patients. Accelerating the research of efficient DC cell therapy will push cancer treatment into a new era.

[0003] However, DC therapy still has some limitations. First of all, from the perspective of the treatment process, DC therapy uses the patient's own monocytes to culture in vitro to induce the generation of DCs, and then loads the corresponding tumor antigens to make DCs loaded with tumor antigens. These DCs are then injected into the body to stimulate the proliferation of tumor-killing lymphocytes in the body to achieve the purpose of eliminating the tumor. The entire process is complex and time-consuming, and there is a risk of infection in in vitro culture. After being re-infused into the human body, it is easily rejected by the patient's own immune cells. Secondly, from the perspective of antigen loading capacity, since the antigen itself is not targeted, the DC's antigen loading capacity is not high. Thirdly, DC takes up exogenous antigens through endocytic vesicles, guides them to the MHC class II complex through the lysosomal pathway, and activates helper CD4 + T cells and cannot efficiently directly activate cytotoxic CD8 + The above three points limit the effect of DC immunotherapy. Developing methods to directly target DC in vivo, increase DC uptake of antigens, promote cross-presentation of antigens in DC, and enhance DC's ability to bind to CD8 + The T cell-activated antigen delivery system can comprehensively enhance the DC-induced immune response and become a key way to improve the efficacy of DC immunotherapy. Summary of the invention

[0004] The current problems of DC cell therapy are: 1. The treatment process is complicated and time-consuming; 2. DC has low efficiency in antigen uptake and presentation; 3. It cannot efficiently activate CD8 +The present invention provides a method for preparing a specific targeting DC with a metal skeleton material as the main body, improving DC antigen uptake, promoting antigen lysosome escape and activating CD8 + Preparation method and application of in vivo antigen delivery carrier (metal skeleton complex) for T cell function. The carrier directly encapsulates antigen (ovalbumin, OVA, including but not limited to) and immune adjuvant (CpG-ODN, including but not limited to) during the preparation process, and forms an in vivo antigen delivery system with the carrier to induce a strong immune response and kill cancer cells.

[0005] In a first aspect, the present invention claims a vector for delivering an antigen into the body.

[0006] The carrier for delivering antigens in vivo claimed by the present invention is a metal skeleton complex obtained by functionally modifying a metal skeleton material with polyethyleneimine and mannose. The metal skeleton material is well known in the art and is composed of zinc ions and 2-methylimidazole ligands.

[0007] The metal skeleton composite has a regular polygonal cube shape and a particle size of 100-120 nm. In a specific embodiment of the present invention, the particle size of the metal skeleton composite is 118 nm.

[0008] In some cases, the vector can be prepared according to a method comprising the following steps:

[0009] (A1) dissolving zinc ions (such as zinc nitrate hexahydrate Zn(NO3)2·6H2O) and polyethyleneimine (PEI MW 600) in water to obtain solution 1;

[0010] (A2) dissolving 2-methylimidazole (2MeIM) in water to obtain solution 2;

[0011] (A3) subjecting the solution 1 to magnetic stirring, pouring the solution 2 into the solution 1 under magnetic stirring, maintaining the magnetic stirring reaction for 10-15 min (e.g., 15 min), centrifuging the mixture at 10000-11000 rpm (e.g., 10000 rpm) for 15 min, aspirating the supernatant and retaining the precipitate, and then adding water to the precipitate to obtain a solution 3;

[0012] (A4) dissolving D-(+)-mannose in water to obtain solution 4;

[0013] (A5) subjecting the solution 3 to magnetic stirring, pouring the solution 4 into the solution 3 under magnetic stirring, maintaining the magnetic stirring reaction for 25-30 min (e.g., 30 min), centrifuging the mixture at 10000-11000 rpm (e.g., 10000 rpm) for 15 min, and vacuum drying the precipitate at 40-45° C. (e.g., 45° C.) to obtain the carrier;

[0014] The ratio of zinc nitrate hexahydrate and polyethyleneimine in the solution 1, 2-methylimidazole in the solution 2, and D-(+)-mannose in the solution 4 is 0.17 mmoL: 0.0833 mmoL: 11.8 mmoL: 2.77 mmoL.

[0015] In a second aspect, the present invention claims a method for preparing a carrier for delivering an antigen into the body.

[0016] The method for preparing a carrier for delivering an antigen into the body claimed in the present invention may include the above steps (A1) to (A5).

[0017] In a third aspect, the present invention claims an in vivo antigen delivery system loaded with antigen and / or immune adjuvant.

[0018] The in vivo antigen delivery system loaded with antigen and / or immune adjuvant claimed in the present invention can be prepared according to a method comprising the following steps:

[0019] (B1) dissolving zinc ions (such as zinc nitrate hexahydrate Zn(NO3)2·6H2O) and polyethyleneimine (PEI MW 600) in water to obtain solution 1;

[0020] (B2) dissolving the following 1) and 2) in water to obtain solution 2;

[0021] 1) 2-Methylimidazole;

[0022] 2) antigens and / or adjuvants;

[0023] (B3) subjecting the solution 1 to magnetic stirring, pouring the solution 2 into the solution 1 under magnetic stirring, maintaining the magnetic stirring reaction for 10-15 min (e.g., 15 min), centrifuging the mixture at 10000-11000 rpm (e.g., 10000 rpm) for 15 min, aspirating the supernatant and retaining the precipitate, and then adding water to the precipitate to obtain a solution 3;

[0024] (B4) dissolving D-(+)-mannose in water to obtain solution 4;

[0025] (B5) subjecting the solution 3 to magnetic stirring, pouring the solution 4 into the solution 3 under magnetic stirring, maintaining the magnetic stirring reaction for 25-30 min (e.g., 30 min), centrifuging the mixture at 10000-11000 rpm (e.g., 10000 rpm) for 15 min, aspirating the supernatant and retaining the precipitate, and vacuum drying the precipitate at 40-45° C. (e.g., 45° C.), thereby obtaining the in vivo delivery system loaded with the antigen and / or immune adjuvant;

[0026] Among them, the zinc ions in solution 1, polyethyleneimine, 2-methylimidazole, the antigen and / or adjuvant in solution 2, and D-(+)-mannose in solution 4 have a ratio of 0.17mmoL zinc ions: 0.0833mmoL polyethyleneimine: 11.8mmoL 2-methylimidazole: 0.0052mmoL antigen: 0.0000248mmoL adjuvant: 2.77mmoL D-(+)-mannose.

[0027] In some cases, the antigen delivery system is a vacuum dried powder.

[0028] In some cases, the antigen delivery system powder is dissolved in saline / cell culture medium.

[0029] In a fourth aspect, the present invention claims a method for preparing an in vivo antigen delivery system loaded with an antigen and / or an immune adjuvant.

[0030] The method for preparing the in vivo antigen delivery system loaded with antigen and / or immune adjuvant claimed in the present invention may include the above steps (B1) to (B5).

[0031] In a fifth aspect, the present invention claims protection for any of the following applications:

[0032] (C1) Use of the vector described in the first aspect or the system described in the third aspect for delivering antigens and / or immune adjuvants into the body;

[0033] (C2) Use of the carrier described in the first aspect above in preparing the delivery system described in the third aspect above;

[0034] (C3) Use of the vector described in the first aspect or the delivery system described in the third aspect in the preparation of a dendritic cell vaccine;

[0035] (C4) Use of the vector described in the first aspect or the delivery system described in the third aspect in the preparation of an immunostimulant;

[0036] (C5) Use of the vector described in the first aspect or the delivery system described in the third aspect in the preparation of a product for killing cancer cells.

[0037] In the above aspects, the antigen can be selected from any one or more of the following: OVA, tumor-specific antigen, tumor stem cell antigen, tumor cell lysate (tumor whole cell antigen), etc.

[0038] In the above aspects, the immune adjuvant can be selected from any one or more of the following: cell-macrophage colony stimulating factor, interleukin-1, interleukin-2, interferon-γ, Toll-like receptor agonists, bacteria or their products, inorganic adjuvants, etc.

[0039] Furthermore, the Toll-like receptor agonist may be peptidoglycan, CpG-ODN, imiquimod or resiquimod, etc.; the bacteria or their products may be mycobacteria (such as tuberculosis bacteria, BCG), Corynebacterium parvum, Bordetella pertussis or Gram-negative bacillus endotoxin, etc.; the inorganic adjuvant may be aluminum hydroxide, alum or aluminum phosphate, etc.;

[0040] In a sixth aspect, the present invention claims protection for any of the following products:

[0041] (D1) a dendritic cell vaccine, which is obtained by targeting the antigen delivery system described in the third aspect to dendritic cells;

[0042] (D2) an immunostimulant obtained by co-incubating the antigen delivery system described in the third aspect with dendritic cells;

[0043] (D3) A product for killing cancer cells, which is obtained by co-culturing the antigen delivery system described in the third aspect with dendritic cells, and then co-culturing the dendritic cells with T cells.

[0044] In the above aspects, the cancer cells can be cells of a wide range of cancer types, such as common cancer types such as lung cancer, breast cancer, gastric cancer, liver cancer, cervical cancer, esophageal cancer, colorectal cancer, lymphoma, etc.

[0045] In the above aspects, the antigen delivery system is used as an immunostimulant, wherein the immunostimulation includes cellular immune activation, humoral immune activation and / or tumor immune regulation. In particular, the antigen delivery system of the present invention can promote antigen lysosomal escape and activate CD8 + T cells.

[0046] The antigen delivery carrier provided by the present invention has uniform particle size, good dispersibility, high biosafety, and large antigen loading capacity, and exhibits high targeting to dendritic cells, increases the uptake of antigens by dendritic cells, promotes the maturation of dendritic cells, and promotes the cross-presentation of antigens in dendritic cells, and can efficiently activate CD4 + and CD8 +T cells, causing a strong immune response and killing cancer cells. The carrier is widely used and can achieve the coating of single or multiple antigens, and has the ability to kill a wide range of cancer cells. The present invention solves the problem that the dendritic cell vaccine antigen uptake and presentation efficiency is low and the CD8 + T cells and other difficult problems, providing a new nanoscale antigen delivery carrier for the preparation of safe and efficient anti-tumor dendritic vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the preparation process and function of the in vivo antigen delivery system.

[0048] Figure 2 The morphology, particle size and surface potential of the prepared metal skeleton material (named Zn-2MeIM) and antigen delivery carrier Zn-2MeIM@PEI@Man (abbreviated as ZPM) were characterized.

[0049] Figure 3 Characterization of the stability and dispersibility of Zn-2MeIM and ZPM. a: The vacuum-dried ZPM is in white powder form; b: The dispersion of Zn-2MeIM and ZPM in water; c: The UV absorption spectrum of Zn-2MeIM in water for 7 days; d: The UV absorption spectrum of ZPM in water for 7 days.

[0050] Figure 4 Characterization of surface groups and crystal forms of Zn-2MeIM and ZPM carriers. a: Infrared spectrum characterization of surface groups; b: X-ray diffraction spectrum characterization of crystal forms.

[0051] Figure 5 Characterization of the functionalized antigen delivery system (named OVA-CpG-ZPM) loaded with antigen and immune adjuvant. a: Planar morphology observed by transmission electron microscopy; b: Three-dimensional morphology observed by scanning electron microscopy; c: Particle size statistics; d: Hydration particle size and surface potential statistics.

[0052] Figure 6 The loading and acid response release efficiency of ZPM for antigen OVA. a: The highest encapsulation efficiency of ZPM for different concentrations of OVA; b: The antigen release efficiency of OVA-CpG-ZPM at different pH values ​​over time.

[0053] Figure 7 DCs were isolated from human peripheral blood mononuclear cells (PBMCs) and cultured in vitro.

[0054] Figure 8 To isolate T cells from PBMCs for in vitro culture.

[0055] Fig. 9To characterize the cytotoxicity of Zn-2MeIM, ZPM vector and delivery system OVA-CpG-ZPM.

[0056] Fig.10 The uptake of OVA and the delivery system OVA-CpG-ZPM by DCs, the intracellular localization of antigens, and their lysosomal escape. White arrows: DCs, purple arrows: antigen lysosomal escape.

[0057] Fig.11 This is a study on the release of cytokines by stimulating T cell activation with the antigen delivery system OVA-CpG-ZPM. The concentrations of related cytokines released in the supernatant were detected after each experimental group was co-cultured with DCs and T cells for 24 hours. a: INF-γ concentration; b: IL-4 concentration; c: TNF-α concentration. *P<0.05, **P<0.01, compared with the blank group; #P<0.05, ##P<0.01, compared with the OVA group.

[0058] Fig.12 LDH method was used to detect the killing ability of T cells activated by the antigen delivery system OVA-CpG-ZPM on cancer cells. Each experimental group was co-cultured with DC for 12 hours and then co-cultured with T cells for 24 hours, and then co-cultured with human lung adenocarcinoma cells A549 and H1975 for 24 hours. The apoptosis of cancer cells was detected using the LDH detection kit. a: A549; b: H1975. *P<0.05, **P<0.01, compared with the blank group; #P<0.05, ##P<0.01, compared with the OVA group.

[0059] Fig.13 The effects of the antigen delivery system OVA-CpG-ZPM on the physiological indicators of mice. ad: changes in blood biochemical indicators, where a is alanine aminotransferase, b is aspartate aminotransferase, c is urine creatinine, and d is lactate dehydrogenase; e: changes in body weight; f: changes in organ indexes. *P<0.05, compared with the blank group.

[0060] Fig.14 This is a study on the antigen delivery system stimulating antibody production in mice. The content of OVA-specific immunoglobulin IgG produced in the serum of C57BL / 6 mice after three subcutaneous injections of different doses of each component into the tail. a: injection dose 10μg; b: injection dose 25μg; c: injection dose 50μg. *P<0.05, **P<0.01, compared with the blank group; #P<0.05, compared with the OVA group. &P<0.05, &&P<0.01, compared with the OVA-CpG group.

[0061] Fig.15 For in vivo imaging and fluorescence imaging of isolated organs, the antigen delivery system can be used for in vivo imaging and fluorescence imaging of isolated organs. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0063] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0064] Example 1. Preparation process and action process of the in vivo antigen delivery vector / system of the present invention

[0065] like Figure 1 As shown, the preparation process and function diagram of the in vivo antigen delivery system are presented.

[0066] 1. Preparation process

[0067] 1. Preparation process of Zn-2MeIM metal skeleton material with uniform particle size, regular morphology, good dispersion and stability

[0068] 0.05 g Zn(NO3)2·6H2O (equivalent to 0.17 mmol zinc ion, Sigma-Aldrich 228737-100G) was dissolved in 3 mL deionized water to form solution 1, and 0.97 g 2-methylimidazole (Sigma-Aldrich M50850-100G) was dissolved in 2 mL deionized water to form solution 2. Solution 1 was transferred to a 25 mL round-bottom flask and poured into solution 2 under magnetic stirring at 500 rpm. After adding solution 2, the mixed solution immediately changed from clear to white turbid liquid. The magnetic stirring was maintained and the reaction was carried out for 15 min. After the reaction, the mixed solution was transferred to a 50 mL centrifuge tube and centrifuged at 10000 rpm (centrifugal radius 12 cm) for 15 min. After the reaction, the supernatant was aspirated and the bottom precipitate was retained. Subsequently, 5 mL deionized water was added to the centrifuge tube, and the precipitate was redispersed to a uniform liquid state under ultrasound. The centrifugation was repeated once (10000 rpm (centrifugal radius 12 cm) for 15 min), and then the supernatant was sucked out, and the white precipitate at the bottom was vacuum dried at 45°C until it turned into white powder for storage. Zn-2MeIM carriers with uniform particle size and regular morphology were obtained.

[0069] 2. Process of preparing antigen delivery carrier ZPM by functional protonation modification and targeting ligand modification of Zn-2MeIM carrier

[0070] 0.05 g Zn(NO3)2·6H2O (equivalent to 0.17 mmol zinc ion) and 0.05 g (equivalent to 0.0833 mmol) polyethyleneimine (PEI MW 600, aladding E107077-100 g) were mixed and dissolved in 3 mL deionized water to form solution 1, and 0.97 g (equivalent to 11.8 mmol) 2-methylimidazole was dissolved in 2 mL deionized water to form solution 2. Solution 1 was transferred to a 25 mL round-bottom flask, and solution 2 was poured into it under magnetic stirring at 500 rpm. The magnetic stirring was maintained and the reaction was carried out for 15 min. After the reaction was completed, the mixed solution was transferred to a 50 mL centrifuge tube and centrifuged at 10000 rpm (centrifugal radius 12 cm) for 15 min. After the reaction was completed, the supernatant was aspirated and the bottom precipitate was retained. Subsequently, 3 mL of Deionized water, the precipitate was redispersed to a uniform liquid state under ultrasound to form solution 3, 0.5g (equivalent to 2.77mmol) D-(+)-mannose (Sigma-Aldrich M2069-25G) was dissolved in 2mL deionized water to form solution 4, solution 3 was transferred to a 25mL round-bottom flask, and solution 4 was poured under 500rpm magnetic stirring, and the magnetic stirring reaction was maintained for 30min; after the reaction, the mixed solution was transferred to a 50mL centrifuge tube, centrifuged at 10000rpm (centrifugal radius 12cm) for 15min, and the supernatant was aspirated after the reaction, and the white precipitate at the bottom was vacuum dried at 45℃ to white powder for storage. The functionalized antigen delivery carrier modified with targeting ligand Man and protonated PEI was obtained and named ZPM.

[0071] 3. Preparation process of in vivo antigen delivery system coated with antigen and immune adjuvant (taking model antigen OVA and immune adjuvant CpG-ODN as examples)

[0072] 0.05 g Zn(NO3)2·6H2O (equivalent to 0.17 mmol zinc ion) and 0.05 g (equivalent to 0.0833 mmol) polyethyleneimine (PEI MW 600) were mixed and dissolved in 2.5 mL deionized water to form solution 1, 0.97 g (equivalent to 11.8 mmol) 2-methylimidazole, 0.005 g (equivalent to 0.0052 mmol) OVA (concentration equivalent to 2 mg / mL, Sigma-Aldrich A5503-5G), 150 μg (equivalent to 0.0000248 mmol) CpG-ODN (Guangzhou Aiji Biotechnology Co., Ltd.) were dissolved in 2.5 mL of deionized water to form solution 2, solution 1 was transferred to a 25 mL round-bottom flask, and solution 2 was poured in under magnetic stirring at 500 rpm, and magnetic stirring was maintained for 15 min; after the reaction, the mixed solution was transferred to a 50 mL centrifuge tube and centrifuged at 10000 rpm for 15 min. After the reaction, the supernatant was aspirated and the bottom precipitate was retained. Subsequently, 3 mL of deionized water was added to the centrifuge tube, and the precipitate was redispersed to a uniform liquid state under ultrasound to form solution 3, 0.5 g (equivalent to 2.77 mmol) D-(+)-mannose was dissolved in 2 mL of deionized water to form solution 4, solution 3 was transferred to a 25 mL round-bottom flask, and solution 4 was poured in under magnetic stirring at 500 rpm, and magnetic stirring was maintained for 30 min; after the reaction, the mixed solution was transferred to a 50 mL centrifuge tube and centrifuged at 10000 rpm for 15 min. rpm (centrifugal radius 12 cm) for 15 min, and then the supernatant was aspirated, and the white precipitate at the bottom was vacuum dried at 45°C until it turned into white powder for storage. The in vivo antigen delivery system coated with antigen and immune adjuvant was obtained, named OVA-CpG-ZPM.

[0073] 2. Action process

[0074] 1. The delivery system is precisely targeted to DC in vivo by utilizing the specific binding of the mannose (Man) receptors expressed in large quantities on the surface of dendritic cells (DC) and the Man modified on the surface of the delivery system. The delivery system enters the DC through receptor-mediated endocytosis, passes through endocytic vesicles, endosomes, and finally enters lysosomes.

[0075] 2. Utilize the acid-responsive cleavage properties of the metal skeleton carrier Zn-2MeIM to release the immune adjuvant of the internal antigen.

[0076] 3. Utilize the protonation effect of polyethyleneimine (PEI) to cause osmotic swelling of lysosomes, thereby promoting the lysosomal escape of antigens and adjuvants.

[0077] 4. Antigens are cross-presented within DCs and presented through MHC I complexes, activating CD8 + T cells; CD8 +T cells induce a strong immune response, killing cancer cells that express tumor antigens.

[0078] Example 2: Morphological observation, particle size and surface potential statistics of prepared Zn-2MeIM and ZPM

[0079] The morphology, particle size and surface potential of the prepared Zn-2MeIM and ZPM were observed and counted. Figure 2 As shown, the prepared Zn-2MeIM carrier was observed by transmission electron microscopy to have a regular polygonal cubic structure, with a relatively uniform size and good dispersibility, an average particle size of 105nm, and a positive surface potential of 24.55±1.31mV. After Zn-2MeIM was modified with PEI and Man, the morphology of the Zn-2MeIM metal skeleton material was not changed, and it still had a regular three-dimensional structure, but the hydrated particle size increased. After ZPM was modified with PEI and Man, the hydrated particle size increased to 118nm. The surface potential also changed accordingly, and the potential was reduced to 1.23±1.04mV. The changes in particle size and potential show that the present invention is effective for each step of modification of the Zn-2MeIM carrier.

[0080] Example 3: Characterization of Stability and Dispersibility of Zn-2MeIM and ZPM

[0081] The stability and dispersibility of Zn-2MeIM and ZPM were characterized as follows: 1 g of vacuum-dried powdered Zn-2MeIM and ZPM were weighed and dispersed in water. The changes in the UV absorption peaks of the two carriers were detected by UV spectrophotometer every day for 7 consecutive days. Figure 3 As shown in Figure a, the ZPM after vacuum drying is in the form of white powder, which is convenient for long-term storage. Zn-2MeIM and ZPM powder are dispersed in water, as shown in Figure a. Figure 3 As shown in b, the carrier can be evenly dispersed in water and has good dispersibility. To characterize its stability, the Zn-2MeIM and ZPM aqueous solutions were stored for 7 days, and their absorption peaks were characterized by UV spectrophotometer every day, as shown in FIG. Figure 3 As shown in c and d, the intensity of the absorption peak remained stable from day 2 to day 6, and the intensity of the absorption peak decreased slightly on the 7th day. This result shows that Zn-2MeIM and ZPM have high stability.

[0082] Example 4: Characterization of surface groups and crystal forms of Zn-2MeIM and ZPM carriers

[0083] Characterize the surface groups and crystal forms of Zn-2MeIM and ZPM carriers. Figure 4As shown in a, the surface groups of each carrier were characterized by infrared spectrometer. From the infrared spectrum, it can be seen that both carriers have obvious Zn-N bonds, CN bonds, C=C bonds and CN bonds, indicating the formation of self-assembled structures coordinated by Zn ions and 2-methylimidazole, while PEI and Man did not form covalent bonds with Zn-2MeIM. Figure 4 From the X-ray diffraction pattern of Figure b, it can be seen that the diffraction peaks of the two carriers are sharp and symmetrical, indicating that the carrier has a high degree of crystallinity and the internal atomic particles are arranged regularly. The above characterization data show that the modification of Zn-2MeIM will not affect its skeleton structure, but will change the hydrated particle size and surface potential.

[0084] Example 5 Characterization of the functionalized delivery system OVA-CpG-ZPM loaded with antigen and immune adjuvant

[0085] Characterization of the antigen-loaded functionalized delivery system OVA-CpG-ZPM. OVA-CpG-ZPM was observed using transmission electron microscopy and scanning electron microscopy, respectively. Figure 5 The transmission electron microscopy image in middle a shows that compared with the ZPM vector, the periphery of OVA-CpG-ZPM is rough, indicating the coating of the antigen OVA. Figure 5 The scanning electron microscope in middle b shows more intuitively and three-dimensionally that the interior and surface of OVA-CpG-ZPM are coated with OVA, but still maintain a relatively uniform size and shape. Figure 5 Image c in the figure shows that the particle size distribution of OVA-CpG-ZPM is relatively concentrated. Figure 5 Figure d shows that the hydrated particle size after coating with OVA increases to 143 nm. Since OVA exhibits a negative charge, the surface potential of OVA-CpG-ZPM becomes -22.09±1.35 mV. This figure shows the effective loading of ZPM for OVA and immune adjuvants.

[0086] Example 6: Study on the loading and acid-responsive release efficiency of the functional carrier ZPM for the antigen OVA

[0087] The loading and acid response release efficiency of the functionalized delivery carrier ZPM for the antigen OVA were studied. According to the preparation process in Example 1 (I. Preparation process-3), the OVA input concentration was changed to 0.3 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, and 2.5 mg / mL, respectively, and the other steps remained the same. After the preparation is completed, the supernatant is collected by centrifugation, and the uncoated OVA content in the supernatant is detected by BCA. The maximum loading capacity of ZPM for the antigen is calculated using the formula "coating rate % = (total amount - free amount in supernatant) / total amount × 100%". Figure 6As shown in a, when the OVA concentration is as low as 0.3 mg / mL, the loading efficiency of ZPM for OVA is very low, but with the increase of OVA input dose, the loading efficiency of ZPM for OVA gradually increases. When the OVA input concentration reaches 1 mg / mL, the loading efficiency tends to be stable, reaching a maximum coverage rate of 75% at 2 mg / mL, and then the coverage rate begins to decline. This result shows that when the OVA input dose reaches 2 mg / mL, ZPM has the highest loading efficiency for OVA.

[0088] Subsequently, the present invention explored the acid response antigen release efficiency of the functional delivery system OVA-CpG-ZPM. PBS solutions with pH values ​​of 7.4 (normal extracellular pH), 6.5 (endosome pH) and 5 (lysosome pH) were prepared using a pH meter. 6 mg of OVA-CpG-ZPM powder was weighed and added to 3 mL of PBS solutions with pH values ​​of 7.4, 6.5 and 5, respectively. After different time periods, the solution was centrifuged and the efficiency of antigen release in the solution was determined using the BCA method. Figure 6 As shown in b, the release efficiency of OVA from OVA-CpG-ZPM is low at pH 7.4, and the release rate of OVA increases with the increase of solution acidity, reaching a maximum of about 92% at pH 5. This result indicates that OVA-CpG-ZPM has acid-responsive properties and has acid-responsive release properties under acidic conditions of endosomes and lysosomes.

[0089] Example 7: Isolation of DCs from PBMCs for in vitro induction culture

[0090] DCs were isolated from PBMCs and cultured in vitro. The specific steps were as follows: 1) PBMCs were revived; 2) the cells were resuspended in AIM-V medium and the cell concentration was adjusted to 3-5×10 6 / well (24-well plate, culture medium volume 800μL / well); 3) After culturing in a 37℃ incubator for 2h, carefully shake the plate to make the bottom flocculent layer float up, aspirate the supernatant, leave the cells attached to the bottom, and add culture medium (800μL / well culture medium: AIM-V+200ng / mL GM-CSF+100ng / mL IL-4) to the 24-well plate again; 4) Change half the volume of the medium every 2-3 days and supplement cytokines; 5) On the 4th day of culture, add TNF-a to induce cell maturation (change culture medium: AIM-V+200ng / mlTNF-a); 6) On the 6th day of culture, harvest the cells. Figure 7As shown, the DCs on day 0 were round, and on day 3, as shown by the arrows, the DCs began to grow in clusters, and dendritic branches began to appear around the cells, which was consistent with the DC growth conditions reported in the literature. On day 6, the DCs grew in clusters more obviously, presenting a semi-suspended state, with dendritic branches clearly seen around the cells. Semi-suspended growth in clusters is also a sign of DC maturity, indicating that mature DCs were successfully induced and cultured in vitro.

[0091] Example 8: Isolation of T cells from PBMCs for in vitro culture

[0092] T cells were isolated from PBMC and cultured in vitro. The specific operation was as follows: 1. PBMC were resuscitated; 2. AIM-V medium was used to resuspend the cells and adjust the cell concentration to 3-5×10 6 / well (24-well plate, medium volume 800ul / well), 3. After culturing in a 37℃ incubator for 2h, carefully shake the plate to collect the upper flocculent layer as T cells, 4. T cells are cultured in a 24-well plate (800μL / well culture medium: X-VIVO medium containing 20ng / mL IL-2). Figure 8 As shown, the T cells on day 0 were in a round suspension state, and the T cells on day 7 were still in a round suspension state without much change, which was consistent with the growth characteristics of T cells. The above results show that T cells were successfully collected and cultured in vitro.

[0093] Example 9. Characterization of cytotoxicity of carriers and delivery systems

[0094] The cytotoxicity of the vector and delivery system was characterized using the CCK-8 kit (Beyotime Biotechnology C0037). The specific operation was as follows: 2×10 4 DCs were seeded in 96-well plates. After DCs were induced to mature, different concentrations of Zn-2MeIM, ZPM, and OVA-CpG-ZPM were added to DCs, and cultured overnight in a cell culture incubator at 37°C. Cell survival rate was detected after 24 hours. Fig. 9 As shown, after co-culture with DCs at a concentration of 150 mg / L (calculated as the mass concentration of Zn-2MeIM, ZPM, and OVA-CpG-ZPM) for 24 hours, the cell survival rate was still above 85%, indicating that the antigen delivery system has low cytotoxicity and provides a good basis for clinical translational applications.

[0095] Example 10. Study on the uptake of OVA and OVA-CPG-ZPM by DCs, intracellular localization of antigens and lysosomal escape

[0096] The uptake of OVA and OVA-ZPM by DC, the intracellular localization of antigens and their lysosomal escape were studied. The specific operation was as follows: mature DCs were induced according to the protocol in Example 7, and then DCs (1×10 5 100 mg / L FITC-OVA (product of Xi'an Haoran Biotechnology Co., Ltd.) and 100 mg / L FITC-OVA-CpG-ZPM (according to the preparation process in Example 1 (I. Preparation Process-3), OVA was replaced with FITC-OVA, and other steps remained unchanged) were added to the cells / well), and incubated with DC cells in a 37°C incubator for 12 hours, and then centrifuged (3000 rpm, centrifugal radius 10 cm, 5 min) to collect DC, remove the unabsorbed FITC-OVA or FITC-OVA-CpG-ZPM, and the collected DC was resuspended with 200 μL of AIM-V culture medium, and 2 μL of Lyso-Tracker Red dye (Beyotime Biotechnology C1046) was added to mark lysosomes, and then the cells were transferred to a confocal dish and observed using a laser confocal microscope. Fig.10 , the white arrows mark DCs. As can be seen from the figure, in the OVA group alone, only one of the three marked DCs took up OVA, and the amount taken was small. Some DC cells hardly took up OVA, indicating that OVA alone lacks targeting to DCs and DCs have low efficiency in taking up OVA. In the OVA-CpG-ZPM group, all four marked DCs took up OVA, and the amount of OVA taken up was significantly higher than that in the OVA group alone, indicating that OVA-CpG-ZPM has good targeting to DCs and significantly increases DCs' uptake of antigens. Lysosomes were labeled with Lyso-Tracker Red to observe the co-localization of lysosomes and OVA. The purple arrows in the last column of the figure indicate that the localization of OVA in DCs does not overlap with the red, indicating that OVA-CpG-ZPM can effectively promote the lysosomal escape of OVA.

[0097] Example 11: OVA-CpG-ZPM antigen delivery system stimulates T cell activation and releases cytokines

[0098] The antigen delivery system OVA-CpG-ZPM stimulated T cell activation and cytokine release to study the experimental groups were divided into a blank control group, an OVA-only incubation group, and an OVA-CpG-ZPM incubation group. Mature DCs were induced according to the protocol in Example 7, and then DCs (1×10 5cells / well), added 20, 50, and 100 mg / L of each experimental group, and co-incubated with DC cells in a 37°C incubator for 24 hours. Then, the DCs were collected by centrifugation (3000 rpm, centrifugal radius 10 cm, 5 min), and counted. The collected DCs were resuspended in 200 μL of AIM-V culture medium for later use. T cells were separated and cultured according to the scheme in Example 8. The above-mentioned DCs and T cells were mixed at a cell number ratio of 1:10, co-cultured in a 37°C incubator for 24 hours, and then the content of related cytokines released by T cell activation was detected using Elisa reagents (enzyme-linked biological: human tumor necrosis factor α (TNF-α), human gamma interferon (IFN-γ), human interleukin 4 (IL-4) Elisa kit). Fig.11 As shown, the OVA group can promote the secretion of IFN-γ and IL-4 cytokines, but has no effect on the secretion of TNF-α. The OVA-CpG-ZPM concentration groups can significantly increase the secretion of IFN-γ, IL-4 and TNF-α, and the OVA-CpG-ZPM group has significant differences compared with the OVA group. This result shows that OVA-CpG-ZPM has a more significant ability to activate T cells and promote T cells to secrete more immune cytokines.

[0099] Example 12: Detection of the ability of the antigen delivery system OVA-CpG-ZPM to kill cancer cells after activating T cells

[0100] The ability of the antigen delivery system OVA-CpG-ZPM to kill cancer cells after activating T cells was tested. The experimental groups were divided into a blank control group, an OVA-only incubation group, and an OVA-CpG-ZPM incubation group. Mature DCs were induced according to the protocol in Example 7, and then DCs (1×10 5 25, 50, and 75 mg / L of each experimental group were added to the cells (cells / well) and co-incubated with DC cells in a 37°C incubator for 12 h. DCs were then collected by centrifugation (3000 rpm, 5 min) and counted. The collected DCs were resuspended in 200 μL of AIM-V medium for later use. T cells were isolated and cultured according to the protocol in Example 8. The above DCs and T cells were mixed at a cell number ratio of 1:10 and co-cultured in a 37°C incubator for 24 h for later use. Human lung adenocarcinoma cells A549 (5×10 3 cells / well) and H1975 (5×10 3 cells / well) were cultured in a 96-well plate. 4 Cells / well) were added to A549 and H1975, respectively, and co-cultured in a 37°C incubator for 24 h. The apoptosis of cancer cells was detected using the LDH detection kit (Elabscience, E-BC-K046-M). Fig.12Compared with the OVA group alone, the LDH released in the solution of the OVA-CpG-ZPM group was significantly increased, indicating that the OVA-ZPM group had a significantly enhanced ability to kill cancer cells.

[0101] Example 13: Detection of the effects of the antigen delivery system OVA-CpG-ZPM on the physiological parameters of mice

[0102] Female 6-week-old C57BL / 6 mice (Zhuhai Baishitong Biotechnology Co., Ltd.) were selected as experimental mice. The experimental groups were divided into: blank group, Zn-2MeIM, ZPM, OVA, OVA-CpG (preparation method is as follows: 0.00485 g (equivalent to 0.005044 mmol) OVA, 150 μg (equivalent to 0.0000248 mmol) CpG-ODN were dissolved in 10 mL deionized water to prepare an injection with a concentration of 500 μg / mL), OVA-ZPM (according to the preparation process in Example 1 (I, Preparation Process-3), 150 μg (equivalent to 0.0000248 mmol) CpG-ODN was not added during the preparation process, and other steps remained unchanged) and OVA-CpG-ZPM group. Weigh 5 mg of each experimental group, dissolve in 10 mL of deionized water, prepare an injection solution with a concentration of 500 μg / mL, and inject 100 μL of the injection solution subcutaneously into the tail of each C57BL / 6 mouse, once every 7 days, for a total of 3 injections. On the 21st day, weigh the body weight of each group of mice, collect blood by removing the eyeballs, collect serum by centrifugation for later use, dissect and collect the organs of the mice, weigh and calculate the organ index. The collected serum was tested for changes in blood biochemical indicators such as alanine aminotransferase, aspartate aminotransferase, urine creatinine, and lactate dehydrogenase using a kit (Elabscience: E-BC-K046-M, E-BC-K236-M, E-BC-K235-M; ELISA kit: mouse urine creatinine Elisa kit). Fig.13 Only the OVA injection group resulted in an increase in aspartate aminotransferase in mice ( Fig.13 b), the physiological indicators of mice in the other injection groups (body weight changes are shown in Fig.13 Middle e; organ index see Fig.13 f; blood biochemistry Fig.13 There was no significant effect in a, c and d). The results showed that OVA-CpG-ZPM has good biosafety.

[0103] Example 14: Study on the Antigen Delivery System OVA-CpG-ZPM Stimulating Antibody Production in Mice

[0104] Female 6-week-old C57BL / 6 mice (Zhuhai Baishitong Biotechnology Co., Ltd.) were selected as experimental mice, and the experimental groups were divided into: blank group, Zn-2 MeIM, ZPM, OVA, OVA-CpG, OVA-ZPM and OVA-CpG-ZPM group. Weigh 1 mg, 2.5 mg, and 5 mg of each experimental group, respectively, dissolve in 10 mL of deionized water, and prepare injection solutions with concentrations of 100, 250, and 500 μg / mL. 100 μL (mass of 10, 25, and 50 μg) of each experimental group was subcutaneously injected into the tail of C57BL / 6 mice, once every 7 days, for a total of 3 injections. On the 21st day, blood was collected by removing the eyeballs, and the serum was collected by centrifugation. The content of OVA-specific antibody immunoglobulin IgG produced in the mouse body was detected using an Elisa kit (enzyme-linked biological, Mouse OVA-sIgG Elisa Kit). Fig.14 , 10μg and 25μg OVA groups failed to significantly stimulate the production of OVA-IgG, only the high-dose 50μg injection group promoted the production of OVA-IgG; while the low-concentration 10μg OVA-CpG-ZPM injection group could significantly stimulate the production of OVA-IgG antibodies in mice. This result shows that the antigen delivery system OVA-CpG-ZPM has a more effective immune activation effect.

[0105] Example 15: Aggregation of the antigen delivery system OVA-CpG-ZPM in mice

[0106] C57BL / 6 mice were subcutaneously injected with 100 μL of 50 μg FITC-OVA and FITC-OVA-CpG-ZPM, and the aggregation in vivo was observed using a small animal in vivo imaging device at different time points. Fig.15 , OVA and OVA-CpG-ZPM can be observed in mice 0.5h after injection. 6h after injection, the aggregation of OVA-CpG-ZPM in mice is significantly higher than that in the OVA group. 24h after injection, OVA is almost not observed in mice, but it can still be clearly observed in the OVA-CpG-ZPM group mice, and most of OVA-CpG-ZPM is accumulated in the inguinal lymph nodes of mice. This result shows that the antigen delivery system OVA-CpG-ZPM can effectively target the lymph nodes in mice and increase the retention time of the antigen in the body. No obvious fluorescence signal was observed in the in vivo imaging of the ex vivo organs, indicating that OVA / OVA-CpG-ZPM does not accumulate in the main organs.

[0107] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A carrier for delivering the antigen OVA into the body, characterized in that: The carrier is a metal skeleton complex obtained by functionalizing a metal skeleton material with polyethyleneimine and mannose; The metal skeleton material is Zn-2MeIM, which is composed of zinc ions and 2-methylimidazole ligands.

2. The carrier according to claim 1, characterized in that: The metal skeleton complex is in the shape of a regular polygonal cube, and has a particle size of 100-120 nm.

3. The carrier according to claim 1 or 2, characterized in that: The carrier is prepared according to a method comprising the following steps: (A1) dissolving zinc ions and polyethyleneimine in water to obtain solution 1; (A2) dissolving 2-methylimidazole in water to obtain solution 2; (A3) subjecting the solution 1 to magnetic stirring, pouring the solution 2 into the solution 1 under magnetic stirring, maintaining the magnetic stirring reaction for 10-15 minutes, centrifuging the mixture at 10000-11000 rpm for 15 minutes, aspirating the supernatant and retaining the precipitate, and then adding water to the precipitate to obtain a solution 3; (A4) dissolving D-(+)-mannose in water to obtain solution 4; (A5) subjecting the solution 3 to magnetic stirring, pouring the solution 4 into the solution 3 under magnetic stirring, maintaining the magnetic stirring reaction for 25-30 minutes, centrifuging the mixture at 10000-11000 rpm for 15 minutes, and vacuum drying the precipitate at 40-45° C. to obtain the carrier; The ratio of zinc ions in solution 1, polyethyleneimine, 2-methylimidazole in solution 2, and D-(+)-mannose in solution 4 is 0.17 mmoL zinc ions: 0.0833 mmoL polyethyleneimine: 11.8 mmoL 2-methylimidazole: 2.77 mmoL D-(+)-mannose.

4. A method for preparing a carrier for delivering the antigen OVA into the body, comprising steps (A1) to (A5) of claim 3.

5. An in vivo antigen delivery system loaded with an antigen and an immune adjuvant, prepared by a method comprising the following steps: (B1) dissolving zinc ions and polyethyleneimine in water to obtain solution 1; (B2) dissolving the following 1) and 2) in water to obtain solution 2; 1) 2-Methylimidazole; 2) Antigens and adjuvants; (B3) subjecting the solution 1 to magnetic stirring, pouring the solution 2 into the solution 1 under magnetic stirring, maintaining the magnetic stirring reaction for 10-15 minutes, centrifuging the mixture at 10000-11000 rpm for 15 minutes, aspirating the supernatant and retaining the precipitate, and then adding water to the precipitate to obtain solution 3; (B4) dissolving D-(+)-mannose in water to obtain solution 4; (B5) subjecting the solution 3 to magnetic stirring, pouring the solution 4 into the solution 3 under magnetic stirring, maintaining the magnetic stirring reaction for 25-30 minutes, centrifuging the mixture at 10000-11000 rpm for 15 minutes, aspirating the supernatant and retaining the precipitate, and vacuum drying the precipitate at 40-45° C. to obtain the in vivo antigen delivery system loaded with the antigen and / or immune adjuvant; in, The zinc ions in the solution 1, the polyethyleneimine, the 2-methylimidazole in the solution 2, the antigen and / or the adjuvant, and the D-(+)-mannose in the solution 4 are in a ratio of 0.17 mmoL zinc ions: 0.0833 mmoL polyethyleneimine: 11.8 mmoL 2-methylimidazole: 0.0052 mmoL antigen: 0.0000248 mmoL adjuvant: 2.77 mmoL D-(+)-mannose; The antigen is OVA; The immune adjuvant is: CpG-ODN.

6. A method for preparing an in vivo antigen delivery system loaded with an antigen and an immune adjuvant, comprising steps (B1) to (B5) of claim 5; The antigen is OVA; The immune adjuvant is: CpG-ODN.

7. Any of the following applications: (C1) Use of the carrier according to any one of claims 1 to 3 in preparing the delivery system according to claim 5; (C2) Use of the vector according to any one of claims 1 to 3 or the delivery system according to claim 5 in the preparation of a product for killing cancer cells; the cancer cells are human lung adenocarcinoma cells.

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