Nano vaccine for in-situ antigen capture and lymph node targeted delivery as well as preparation method and application of nano vaccine

Abstract

The invention discloses a nano vaccine for in-situ antigen capture and lymph node targeted delivery as well as a preparation method and application of the nano vaccine. The nano vaccine comprises a polymer nano assembly and a drug, wherein the polymer nano assembly comprises an amphiphilic polymer and cationic liposome co-assembly; the amphiphilic polymer comprises a polyethylene glycol-polyphosphate segmented copolymer; the medicine comprises an immunologic adjuvant. The nano vaccine provided by the invention not only can be used for in-situ capture of tumor antigens, but also can carry antigens and immune agonists and deliver the antigens and immune agonists to tumor drainage lymph nodes in a targeted manner by utilizing the advantage of small size, so that the maturation of dendritic cells is promoted, the mature dendritic cells process the antigens and present the antigens to T cells, and CD8 + T cells are activated; and a strong anti-tumor immune response is induced.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a nano vaccine for in situ antigen capture and lymph node targeted delivery, and a preparation method and application thereof. Background Art

[0002] Therapeutic cancer vaccines that induce or enhance T cell-mediated anti-cancer immune responses are considered to be one of the most promising methods for destroying tumor cells and inhibiting tumor recurrence and metastasis. The purpose of therapeutic cancer vaccines is to stimulate the patient's adaptive immune system against specific tumor antigens to regain control of tumor growth, induce regression of established tumors, and eradicate minimal residual disease. The basic principles required for successful therapeutic cancer vaccination include delivering large amounts of high-quality personalized antigens to dendritic cells (DCs), optimizing DC activation, inducing strong and sustained CD4 + T helper and cytotoxic T lymphocyte (CTL) responses, infiltration of the tumor microenvironment (TME), and persistence and maintenance of responses. This can be achieved through several approaches, such as reversal of tumor-induced immune exhaustion by immune checkpoint inhibitors, activation of DCs and effector T cells by administering tumor-associated antigens with adjuvants, or vaccination with autologous DCs loaded with specific tumor antigens, which can also broadly activate the local immune environment of the tumor to induce tumor cell death.

[0003] Although a DC-focused cell vaccine, Sipuleucel-T, was clinically approved more than 10 years ago for the treatment of advanced prostate cancer, no other therapeutic cancer vaccines have been approved so far. And it was later found that the survival advantage of prostate cancer patients treated with Sipuleucel-T was small, which led to widespread disappointment with cancer vaccines. Most of the traditional cancer vaccines currently under study are related to some defined antigens. However, the efficacy of vaccines is largely limited by factors such as the difficulty in identifying tumor neoantigens, heterogeneity between and within different tumors, inaccurate identification of antigens, and antigen mutations. Therefore, in situ cancer vaccines that convert autologous whole tumor tissue into personalized antigens have become an attractive approach for cancer vaccine development in recent years. In addition to antigens, cancer vaccines also require adjuvants that can stimulate antigen-presenting cells (APCs) as active ingredients. In fact, the ideal cancer vaccine requires that tumor antigens and adjuvants be co-delivered to lymph nodes and internalized by antigen-presenting cells to initiate a series of systemic immune responses.

[0004] Therefore, multifunctional nanovaccines based on nanotechnology have attracted widespread attention. However, the current research on cancer vaccines still has certain limitations, and the therapeutic effects produced are far from the expected effects. Compared with traditional vaccine formulations, nanovaccine platforms based on nanotechnology have many advantages. First, nanovaccines can achieve the co-delivery of tumor antigens and adjuvants. In addition to solving the delivery and release problems of antigens and adjuvants, nanovaccines can also solve the problem of off-target side effects, thereby improving safety and enhancing immune regulation. Nanovaccines of appropriate size can enhance the accumulation of antigens and adjuvants in lymph nodes. It is worth noting that the tumor microenvironment is a key but complex target for cancer immunotherapy, which contributes to tumor cell invasion and inhibits T cell activation and proliferation. The use of personalized nanovaccines may effectively recruit immune cells such as T cells, which is particularly beneficial for solving insufficient T cell trafficking and overcoming the inhibitory tumor microenvironment. Summary of the invention

[0005] In view of this background, the present invention is committed to solving at least one of the above-mentioned technical problems of the prior art. To this end, the purpose of the present invention is to provide a nano vaccine and a preparation method and application thereof. The nano vaccine can efficiently capture the antigens produced in situ by the tumor. After capturing the antigens, the nano vaccine carries the antigens and the adjuvants carried by the nano vaccine for targeted delivery to the tumor-draining lymph nodes and is taken up by DC, producing a strong cancer vaccine effect. The mature DC cells process the antigens and present them to T cells, promoting antigen cross-presentation and causing a strong systemic anti-tumor immune response.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A nano vaccine comprises a polymer nano assembly and a drug, wherein the polymer nano assembly comprises a co-assembly of an amphiphilic polymer and a cationic liposome; the amphiphilic polymer comprises a polyethylene glycol-polyphosphate block copolymer; and the drug comprises an immune adjuvant.

[0008] In the present invention, after the nano-vaccine reaches the tumor lesion, the nano-vaccine captures a large amount of tumor antigens by virtue of the viscous flow state of the block copolymer and the positive charge of the cationic liposome. After capturing the antigen, the nano-vaccine carries the antigen and adjuvant to migrate to the lymph nodes by virtue of its small size advantage, stimulates DC maturation, promotes antigen cross-presentation to T cells, and activates CD8 + T cells, producing a powerful cancer nanovaccine effect.

[0009] In some embodiments of the present invention, the hydrophilic end polyethylene glycol constitutes the outer shell of the nanovaccine, and the hydrophobic end polyphosphate, cationic liposome and immune adjuvant constitute the inner core of the nanovaccine.

[0010] In some embodiments of the present invention, the nanovaccine is spherical or quasi-spherical, with an average diameter of 20 to 40 nm.

[0011] In some embodiments of the present invention, the immunoadjuvant encapsulation efficiency of the nanovaccine is 58.6%.

[0012] In some embodiments of the present invention, the mass ratio of the polyethylene glycol-polyphosphate block copolymer, the cationic liposome and the immune adjuvant is 10-15:1-2:1-2.

[0013] In some embodiments of the present invention, the cationic liposomes include at least one of (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP) and dioleoylpropyltrimethylammonium chloride (DOTMA).

[0014] In some embodiments of the present invention, the immune agonist (immune adjuvant) is one or more of a CpG oligonucleotide adjuvant, a co-stimulatory ligand adjuvant, a STING agonist, and a TLR agonist.

[0015] In some embodiments of the present invention, the TLR agonist is selected from TLR7 / 8 agonists, specifically R837 and / or R848.

[0016] In some embodiments of the present invention, when a tumor is stimulated exogenously to release antigens, the nanovaccine can capture a large amount of tumor antigens in situ in the tumor by relying on the viscous flow state of the block copolymer and the positive charge of the cationic liposome.

[0017] In some embodiments of the present invention, the nanovaccine captures a large amount of tumor antigens in situ in the tumor and then carries the antigens to the tumor-draining lymph nodes.

[0018] The second aspect of the present invention proposes a method for preparing the nano vaccine: a polymer nano assembly is reacted with a cationic liposome and an immune adjuvant to prepare the nano vaccine. Specifically, 10 mg of amphiphilic polymer mPEG-b-PHEP, 1 mg of cationic liposome DOTAP, 1 mg of R848 (purchased from MedChemexpress Biotechnology, CAS: 144875-48-9) are weighed, and 1 mL of chloroform is added to dissolve. The mixture is slowly added dropwise to a 50 mL centrifuge tube containing 10 mL of ultrapure water, and particles are prepared using an ultrasonic crusher. The power is set to 20%, the ultrasonic time is turned on for 3 s, turned off for 3 s, and the ultrasonic time is 10 min.

[0019] The third aspect of the present invention provides at least one of the following applications of the nanovaccine:

[0020] (1) Preparing products for capturing tumor antigens in situ in tumors;

[0021] (2) Preparation of nano-vaccine products with small size;

[0022] (3) preparing products that simultaneously deliver tumor antigens and immune adjuvants to lymph nodes;

[0023] (4) Improve the maturation ability of DC cells;

[0024] (5) preparing products that improve the maturation ability of DC cells;

[0025] (6) Improve antigen cross-presentation ability;

[0026] (7) Prepare products that improve antigen cross-presentation ability.

[0027] (8) Increase CD8 + T cell activation capacity;

[0028] (9) Preparation to increase CD8 + Products with the ability to activate T cells;

[0029] In some embodiments of the present invention, the product includes any one of a drug, a reagent or a kit.

[0030] In some embodiments of the invention, the T cells include CD4 + T and / or CD8 + T cells.

[0031] Compared with the prior art, the advantages of the present invention are as follows: the nano-vaccine provided by the present invention for in situ capture of antigens and targeted delivery to lymph nodes has an ultra-small particle size (~28nm) and can effectively deliver immune adjuvants to the antigen APCs in the lymph nodes, and because of its viscous flow properties, it can capture a large number of tumor-generated antigens. The in situ nano-vaccine with lymph node targeted delivery capability can actually stimulate DC maturation, promote DC to present antigens to T cells, trigger a strong anti-tumor immune response, completely inhibit or even eradicate some distal tumors, and also inhibit tumor recurrence and metastasis. A powerful therapeutic effect can be produced based on a simple and available production process. The nano-vaccine provided by the present invention solves the problems of antigen capture and difficult delivery of antigens and immune adjuvants, and provides an effective technical strategy for the development of personalized cancer nano-vaccines, with a high prospect for clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The invention discloses a synthetic route for the monomer HEP which constitutes the amphiphilic block copolymer of the nano vaccine of the invention.

[0033] Figure 2 This is the polymerization route of the amphiphilic block copolymer mPEG-b-PHEP of the nanovaccine of the present invention.

[0034] Figure 3 This is the GPC chart of the amphiphilic block copolymer mPEG-b-PHEP of the present invention.

[0035] Figure 4 The figure is the hydrogen nuclear magnetic spectrum of the amphiphilic block copolymer mPEG-b-PHEP of the present invention.

[0036] Figure 5 The nano vaccine VNP of the present invention R848 Particle size distribution diagram.

[0037] Figure 6 The nano vaccine VNP of the present invention R848 Transmission electron microscopy image.

[0038] Figure 7 The nano vaccine VNP of the present invention R848 Particle size change before and after antigen capture.

[0039] Figure 8 The nano vaccine VNP of the present invention R848 Graph showing the change in potential before and after antigen capture.

[0040] Fig. 9 The nano vaccine VNP of the present invention R848 Capture protein quantity graph.

[0041] Fig.10 The nano vaccine VNP of the present invention R848 Band graph of captured protein amount.

[0042] Fig.11 The relative abundance of tumor antigens captured by the nanovaccine was identified for the present invention.

[0043] Fig.12 It is the encapsulation rate of R848 before and after the nano vaccine of the present invention captures the antigen.

[0044] Fig.13 This is the distribution diagram of the nano vaccine of the present invention in small animals.

[0045] Fig.14 This is a diagram of lymph node enrichment in small animals of the nanovaccine of the present invention.

[0046] Fig.15 This is a flow cytometric graph showing the uptake of the nanovaccine of the present invention by DCs in lymph nodes.

[0047] Fig.16 This is a curve diagram showing that the nanovaccine of the present invention inhibits distal tumor progression in a distal animal model.

[0048] Fig.17 This is an in vitro diagram showing that the nanovaccine of the present invention inhibits distal tumors in a distal animal model.

[0049] Fig.18 This is a flow cytometric graph showing that the nanovaccine of the present invention stimulates DC maturation in lymph nodes in vivo.

[0050] Fig.19 The nano vaccine of the present invention stimulates CD8 in the tumor + Flow cytometry of T cell infiltration.

[0051] Fig. 20 Schematic diagram of the antigen capture and lymph node targeted delivery principle of the nanovaccine of the present invention. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0053] Example 1

[0054] This example prepares a nano vaccine, and the specific process is as follows:

[0055] mPEG-b-PHEP was synthesized by ring-opening polymerization as follows:

[0056] (1) Set up the apparatus according to the operating procedure of "vacuum distillation", connect phosphorus oxychloride (COP) together with the round-bottom flask directly to the distillation head, and evacuate for 3 to 5 minutes. When the bubbles in the round-bottom flask are significantly reduced, place the entire vacuum distillation apparatus in an oil bath; slowly increase the temperature of the oil bath to 120°C, collect the fraction when the steam temperature is stable (70°C), and the fractions at other temperatures are regarded as the front fractions, until there is no more fraction; after the distillation is completed, quickly seal it with a pre-dried rubber stopper, seal it again with a sealing film, and store it in a -20°C refrigerator for later use.

[0057] (2) Dehydration of n-hexanol: Add appropriate amount of calcium hydride to 60 ml of n-hexanol to dehydrate, stir overnight, and cover with a balloon. After dehydration, purify by vacuum distillation at 40°C, and collect the fraction at 30°C.

[0058] (3) Purification of triethylamine: Reflux phthalic anhydride (to remove water, primary and secondary amines) at 135°C for one day. Pour 500 ml into a reflux device and reflux for one day. Collect the solution when using.

[0059] (4) Synthesis of HEP monomers Figure 1): Take a dry and clean 500mL three-necked flask, slowly add n-hexanol (31.2g), triethylamine (31.3g) and THF (200mL) with a syringe. Take another dry and clean balanced pressure funnel, add COP (44.1g) and THF (50mL) with a syringe. Place the entire reaction system in a 0℃ constant temperature cold bath, slowly add the mixed solution in the balanced pressure funnel drop by drop into the three-necked flask under stirring, and after the addition is completed, continue to react in a 0℃ constant temperature cold bath overnight.

[0060] (5) After 24 hours, take a dry and clean sand core funnel, quickly pour the reaction solution into the sand core funnel, quickly filter under negative pressure to remove the by-product triethylamine salt, and concentrate THF using a vacuum oil pump. Purify the sample by vacuum distillation to obtain the polymer monomer HEP.

[0061] (6) Dehydration of mPEG: 10 g mPEG and 60 ml toluene were added to a normal pressure distillation apparatus, with the oil bath temperature at 135°C and the steam temperature at 108°C, until no more toluene can be evaporated (approximately 2 h), and then vacuumed for 8 h to obtain mPEG.

[0062] (7) mPEG-b-PHEP polymerization Figure 2 ): In the glove box, the azeotropic mPEG 5k (2.0g), HEP monomer (2.8g) after vacuum distillation was dissolved in anhydrous THE (15mL), and stirred at room temperature to dissolve evenly. Catalyst TBD (50mg) was added, and the reaction was carried out at 25°C for 10min, and then THF containing benzoic acid (0.5g) was added to terminate the polymerization reaction. After the reaction, the solvent was concentrated by rotary evaporator, and then precipitated three times with cold ether / methanol (volume ratio of 10:1), and the residual ether solvent was removed by negative pressure. The GPC and 1 H NMR characterization of the polymers confirmed that they were successfully synthesized ( Figure 3 )( Figure 4 ).

[0063] Example 2

[0064] This example prepares a nano vaccine, and the specific process is as follows:

[0065] Nanoparticles self-assembled with amphiphilic polyphosphates by single emulsification method have excellent viscosity and flowability. Take 10 mg of mPEG-b-PHEP, 1 mg of R848 (purchased from MedChemexpress Biotechnology, CAS: 144875-48-9), and 1 mg of DOTAP, dissolve these substances in 1 mL of chloroform, and inject the mixture dropwise into a 50 mL centrifuge tube containing 10 mL of ultrapure water. Under 0° environment, use the metal probe equipped with an ultrasonic cell disruptor for ultrasonic emulsification treatment for 10 minutes (power setting 20%, ultrasonic time on 3s, off 3s). After the ultrasonic treatment is completed, use a rotary evaporator to remove chloroform, and filter through a 220nm filter membrane to remove unencapsulated R848. The nanovaccine encapsulating R848 can be obtained. Because of its excellent viscosity and flowability, it is expressed as VNP. R848 .

[0066] Take the VNP prepared above R848 500 μL was freeze-dried using a freeze dryer, dissolved in 2 mL DMSO, and VNP was measured using a UV spectrophotometer. R848 and R848 standard curve (10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 μg mL -1 ) absorbance at 320 nm, calculate VNP R848 The encapsulation efficiency was 58.6% and the drug loading efficiency was 5%. The VNP prepared above was measured by a nanoparticle size analyzer. R848 The particle size is about 28nm±10nm( Figure 5 In addition, the transmission electron microscope (TEM) also showed that the particles had been successfully prepared ( Figure 6 ).

[0067] Example 3

[0068] This example is used to investigate the VNPs prepared in Example 2. R848 In terms of antigen capture capability, the specific process is as follows:

[0069] (1) LLC cells (2×10 5 The cells (cells / well) were treated with photothermal / photodynamic / magnetic thermal / chemotherapy methods to release tumor antigens and damage-associated molecular patterns (DAMPs).

[0070] (2) Then join VPN R848 (100 μg mL -1 ) and incubated with the treated cells for 24 h.

[0071] (3) The supernatant was collected and further purified three times using an ultrafiltration device (molecular weight cutoff 100K Da) at 1000 g, 3 min. In addition, the VNPs in the ultrafiltration device were measured by a nanoparticle size analyzer. R848 The particle size and zeta potential of VNPs R848 The particle size increased slightly from 28nm to 35nm ( Figure 7 ), its zeta potential decreased from +5.62mV to -1.15mV ( Figure 8 ). The total protein and VNP R848 The captured proteins were measured using the BCA protein quantification kit, and VNP R848 It can efficiently capture proteins released by tumor cells, with a capture amount of 83.2ug protein per mg nanoparticle ( Fig. 9 ).

[0072] (4) Further sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed, and protein bands were detected with Coomassie brilliant blue. The total protein produced by LLC cells and the captured protein bands were basically similar, proving that VNP R848 The antigen produced can be successfully captured ( Fig.10 ).

[0073] (5) To determine the number and type of proteins, the total protein and captured protein were broken down into peptide fragments by trypsin and further extracted for liquid chromatography / tandem mass spectrometry analysis. The mass spectrometry results showed that VNP R848 Effectively captured tumor-specific antigens including Eef1a1, Eef2, Gnas and damage-associated molecular patterns (DAMPs) Hsp90ab1, Hsp90aa1, Hspa8, Hmgb1, etc. ( Fig.11 ). And the nanoparticles can still effectively encapsulate R848 and VNP after capture R848 After capturing the antigen, the encapsulation rate was still 50.6%, which was not much different from that before capture (58.6%) ( Fig.12 ).

[0074] Example 4

[0075] This example is used to investigate the VNP prepared in Example 2. R848 The specific process is as follows:

[0076] (1) First, VNP nanoparticles loaded with fluorescent dye DID were prepared. DID, the method is similar to the preparation method of Example 2. Weigh 10 mg mPEG-b-PHEP, 1 mg DOTAP, 1 mg DID, dissolve in 1 mL chloroform, and then inject the mixture dropwise into a 50 mL centrifuge tube containing 10 mL ultrapure water. Under a 0° environment, use a metal probe equipped with an ultrasonic cell disruptor for ultrasonic emulsification treatment for 10 minutes (power setting 20%, ultrasonic time on 3s, off 3s). After the ultrasonic treatment is completed, use a rotary evaporator to remove chloroform, and filter through a 220 nm filter membrane to remove the unencapsulated DID.

[0077] (2) LLC cells (5×10 6 ) was injected into the back of female C57BL / 6 mice (6 weeks old) to establish a subcutaneous lung cancer model. When the tumor volume reached ∼100 mm 3 The mice were randomly divided into two groups (3 mice in each group). PBS and DiD-labeled VNPs were injected intratumorally. DID , DiD dose was 3.0 μg / mouse.

[0078] (3) 24 hours after administration, the tumor-draining lymph nodes were harvested and the DiD fluorescence signal was detected using a small animal in vivo imaging system. DID After 24 hours, it is mainly distributed in the tumor and lymph nodes ( Fig.13 ), VNP was shown by analyzing lymph nodes alone DID Can accumulate effectively in lymph nodes ( Fig.14 ).

[0079] (4) The collected lymph nodes were ground and dissociated into single cell suspensions, and then stained with BV510-anti-mouse CD45 and PE-anti-mouse CD11c for flow cytometric analysis. The analysis showed that DCs were more sensitive to VNPs than to PBS. DID Up to 26.8% ( Fig.15 ). This demonstrates that the nanovaccine has the ability to migrate to lymph nodes and be taken up by DCs, which is important for activating CD8 + A key prerequisite for T cell-mediated immune responses.

[0080] Example 5

[0081] This example is used to investigate the prepared VNP R848 The specific process is as follows:

[0082] (1) LLC cells (1×10 6 ) was injected subcutaneously on both sides of the back of female C57BL / 6 mice to establish a bilateral tumor model with a tumor volume of ∼100 mm 3The mice were randomly divided into 5 groups (5 mice in each group): PBS group, photothermal group, VNP group R848 Group, photothermal / VNP group, photothermal / VNP R848 The VNP group did not contain the immune adjuvant R848, and the left tumor was treated with photothermal therapy to produce tumor antigens. After the photothermal treatment, each mouse VNP R848 The injection volume is 30 mg kg -1 .

[0083] (2) Treatment was performed on the first and fourth days, and the right tumor volume was measured every two days to draw a tumor curve ( Fig.16 After the experiment, the right tumor tissue was collected and photographed ( Fig.17 ). PBS group, photothermal group, VNP R848 The photothermal / VNP group was unable to induce systemic anti-tumor immunity, so the tumor progressed rapidly. The photothermal / VNP group had a certain right-side or distal tumor inhibition effect because VNP could capture antigens and deliver them to lymph nodes, but lacked the ability of immune adjuvants to amplify the immune effect. R848 The distal tumor volume of the group was significantly reduced compared with the other groups, and was basically completely suppressed, indicating that a systemic immune response was induced. (3) In order to analyze the immune mechanism of the nanovaccine of the present invention, the tumor-draining lymph nodes were further separated, mechanically ground and passed through a 200-mesh nylon mesh to make a single cell suspension, which was incubated with BV510-anti-mouse CD45, PE-anti-mouse CD11c, FITC-anti-mouse CD80 and APC / Cy7-anti-mouse CD86 antibodies, and flow cytometry was performed to analyze the nanovaccine-stimulated DC maturation ( Fig.18 ). The analysis showed that in the PBS group, photothermal group, VNP R848 The photothermal / VNP group showed universal immunogenicity, stimulating DC maturation by approximately 21.1%, 20.8%, and 23.2%, respectively. It is worth noting that the photothermal / VNP group showed a higher DC maturation (28.5%), which may be because VNPs without immune adjuvants can capture antigens and deliver them to DCs, thereby stimulating DC maturation. Most importantly, the photothermal / VNP R848 The DC maturity of the group was the highest, at 36.1%, which indicates that the nanovaccine of the present invention can effectively stimulate and activate DC maturation.

[0084] (4) To further elucidate the potential immune mechanism of nanovaccines, distal tumor tissue was obtained from a distal lung cancer tumor model, minced, and treated with 10% fetal bovine serum and type IV collagenase (1 mg mL -1 ), hyaluronidase (100 μg mL -1 ) and DNase I (100 μg mL -1) in RPMI-1640 medium at 37°C, 150 rpm for 20 min, filter the cells with a nylon mesh (200 mesh), collect the cells by centrifugation (600×g, 5 min), and further purify them with 40% Percoll (GE) solution. Collect the obtained lymphocytes and incubate with BV510-anti-mouse CD45, FITC-anti-mouse CD3, APC / Cy7-anti-mouse CD4, PE-anti-mouse CD8a, and perform flow cytometric analysis ( Fig.19 ). Flow cytometry analysis showed similar results to the above results. The CD8 + T cell infiltration increased moderately, while photothermal / VNP R848 Tumor infiltrating CD8 + The proportion of T cells increased sharply to 73.0% compared with the PBS group (46.4%). This indicates that the nanovaccine of the present invention can activate CD8 + T cells, inducing a powerful anti-tumor immune response. Fig. 20 The VNP of the present invention is also shown R848 Schematic diagram of the capture antigen and lymph node-targeted delivery mechanism.

Claims

1. A nanovaccine for in situ antigen capture and lymph node targeted delivery, characterized in that: It comprises a polymer nanoassembly, a cationic liposome and a drug, wherein the polymer nanoassembly is an amphiphilic polymer assembly; the amphiphilic polymer assembly comprises a polyethylene glycol-polyphosphate block copolymer; the drug comprises an immune adjuvant; When the tumor is stimulated exogenously to release antigens, the nano-vaccine relies on the viscous flow state of the block copolymer and the positive charge of the cationic liposome to capture a large amount of tumor antigens in situ in the tumor; The nano vaccine captures a large amount of tumor antigens in situ in the tumor and then carries the antigens to the tumor-draining lymph nodes.

2. The nanovaccine for in situ antigen capture and lymph node targeted delivery according to claim 1, characterized in that: The hydrophilic end polyethylene glycol of the amphiphilic polymer assembly forms the outer shell of the nano vaccine, and the hydrophobic end polyphosphate of the amphiphilic polymer assembly, cationic liposome and immune adjuvant form the inner core of the nano vaccine.

3. The nanovaccine for in situ antigen capture and lymph node targeted delivery according to claim 1, characterized in that: The nano vaccine is in a spherical or quasi-spherical state, and the average diameter of the nano vaccine is 20 to 40 nm.

4. The nanovaccine for in situ antigen capture and lymph node targeted delivery according to claim 1, characterized in that: The cationic liposome comprises at least one of (2,3-dioleyloxypropyl)trimethylammonium chloride DOTAP and dioleoylpropyltrimethylammonium chloride DOTMA.

5. The nanovaccine for in situ antigen capture and lymph node targeted delivery according to claim 1, characterized in that: The immunoadjuvant encapsulation rate of the nano vaccine is 55-60%.

6. The nanovaccine for in situ antigen capture and lymph node targeted delivery according to claim 1, characterized in that: The mass ratio of the polyethylene glycol-polyphosphate block copolymer, the cationic liposome and the immune adjuvant is 10-15:1-2:1-2.

7. The nanovaccine for in situ antigen capture and lymph node targeted delivery according to claim 1, characterized in that: The immune adjuvant is one or more of a CpG oligonucleotide adjuvant, a co-stimulatory ligand adjuvant, a STING agonist, and a TLR agonist.

8. The method for preparing the nano vaccine for in situ antigen capture and lymph node targeted delivery according to any one of claims 1 to 7, characterized in that: The following steps are involved: The polymer nanoassembly, cationic liposome and drug are weighed, dissolved in chloroform, dropped into ultrapure water, and prepared by using an ultrasonic crusher.

9. The preparation method according to claim 8, characterized in that: The power of the ultrasonic disruptor was set to 20%, the ultrasonic time was on for 3 seconds, off for 3 seconds, and the ultrasonic time was 10 minutes.

10. At least one of the following uses of the in situ tumor vaccine according to any one of claims 1 to 9: (1) Preparing products for capturing tumor antigens at the tumor site; (2) Preparation of nano-vaccine products with small size; (3) Preparation of nano-vaccine products capable of loading immune adjuvants (4) preparing products that simultaneously deliver tumor antigens and immune adjuvants to lymph nodes; (5) preparing products that improve the maturation ability of DC cells; (6) Prepare products that improve antigen cross-presentation ability.

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