Nano vaccine for promoting cross presentation of antigens as well as preparation method and application of nano vaccine

Abstract

The invention belongs to the field of biological medicine and materials, and particularly relates to a nano vaccine for promoting antigen cross presentation and a preparation method and application thereof, the method comprises the following steps: inducing tumor cell immunogenicity death to obtain a tumor cell immunogenicity death product; a polylactic acid-glycolic acid copolymer is mixed with alpha-galactosylceramide to serve as a carrier, a tumor cell immunogenicity death product, racimod and endosome-lysosome escape promoting peptide are wrapped, and the nano vaccine is prepared. The method for inducing immunogenic death of the tumor cells comprises the following steps: co-incubating the tumor cells and the iridium composite oligoarginine polypeptide and / or irradiating the tumor cells with the photosensitizer by laser. The nano vaccine has efficient antigen cross presentation capability, and can stimulate expression of dendritic cell co-stimulation signals and secretion of cell factors in multiple ways, so that the biological effect of synergistically activating cellular immunity is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and materials, and specifically relates to a nano vaccine for promoting antigen cross-presentation and a preparation method and application thereof. Background Art

[0002] Tumor vaccines use tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs) to activate the patient's immune system. In theory, vaccines can stimulate specific cellular and humoral immune responses, prevent tumor growth, and ultimately eradicate tumor cells. However, tumor antigens are endogenous and have low immunogenicity, and are often difficult to induce effective immune responses. In addition, traditional vaccines mainly induce humoral immunity, but CD8 + Cellular immunity mediated by cytotoxic T cells (CTLs) is essential for the elimination of tumor cells. Therefore, how to induce effective cellular immunity is a key issue in tumor vaccine therapy.

[0003] Antigen-presenting cells (APCs) play a crucial role in tumor antigen-induced immune activation. Dendritic cells (DCs) are the most important antigen-presenting cells as a key bridge connecting innate immunity and adaptive immunity. In the process of initiating antiviral and antitumor T cell-mediated immune responses, dendritic cells (DCs) need to cross-present exogenous antigens on major histocompatibility complex (MHC) class I molecules. Antigen cross-presentation depends on the abnormal "leakage" of endosomes in dendritic cells, that is, the internalized proteins escape into the cytoplasm for the proteasome-mediated generation of MHCⅠ-binding peptides. The peptides generated by the proteasome are then transported to the endoplasmic reticulum by antigen processing-related transporters TAP1 and TAP2, loaded on newly formed MHCⅠ class molecules, and then presented on the surface of DCs. The presented antigens activate T cells together with cytokines and co-stimulatory signals.

[0004] The efficiency of antigen cross-presentation depends on the type of DCs. DCs are present in very small amounts in the body and are mainly divided into plasmacytoid dendritic cells (pDCs), conventional dendritic cells cDC1s and cDC2s derived from rare bone marrow-resident progenitor cells, and monocyte-derived dendritic cells MoDCs. Due to the advantages of antigen cross-presentation, cDC1s usually lead to stronger CD8 +T cell immunity. Although cDC1s have good cross-presentation ability and higher secretion levels of immune activation-related cytokines, and are associated with good prognosis, the difficulty in obtaining them limits their research and application in anti-tumor treatment. The therapeutic effects of in vitro generated DCs vaccines currently undergoing clinical trials are not satisfactory. This is because naturally existing DCs subsets have stronger antigen presentation ability, higher MHC molecule expression and functional specialization than in vitro generated DCs. How to improve DCs' antigen cross-presentation, expression of co-stimulatory signals and secretion of corresponding cytokines, and break through the limitations of DCs types and numbers on immune effects, is a key issue in tumor vaccine therapy. Summary of the invention

[0005] The present invention aims to solve the problems of low maturation promotion and antigen cross-presentation rate of dendritic cells in tumor vaccines, resulting in low efficiency of cellular immune activation. The present invention provides a nano vaccine for promoting antigen cross-presentation, a preparation method and application thereof, promotes cross-presentation of antigen peptides in dendritic cells by promoting endosome-lysosome escape peptides, obtains antigen peptides and damage-associated molecular patterns (DAMPs) with good immune activation potential by inducing immunogenic death of tumor cells, promotes dendritic cell maturation through multiple pathways of a-galactosylceramide (a-Galcer) and immune adjuvant resiquimod, induces dendritic cells to cross-present tumor antigen peptides and highly express costimulatory signals, and increases the secretion of pro-inflammatory cytokines, thereby efficiently activating anti-tumor cellular immunity.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A method for preparing a nano vaccine for promoting antigen cross-presentation comprises the following steps: inducing immunogenic death of tumor cells to obtain immunogenic death products of tumor cells; using polylactic acid-glycolic acid copolymer mixed with α-galactosylceramide as a carrier to encapsulate the immunogenic death products of tumor cells, resiquimod and an endosomal-lysosomal escape-promoting peptide to prepare a nano vaccine; the method for inducing immunogenic death of tumor cells is co-incubating tumor cells with iridium-complexed oligoarginine polypeptides and / or laser irradiating tumor cells that have taken in a photosensitizer.

[0007] Furthermore, the peptide promoting endosomal-lysosomal escape is an iridium complex oligoarginine polypeptide or a GALA peptide.

[0008] Furthermore, the mass ratio of polylactic acid-glycolic acid copolymer, resiquimod, and α-galactosylceramide is 50~100:1~20:0.0004~0.05, and the working concentration of the endosomal-lysosomal escape peptide is the concentration at which 20% of cell growth is inhibited or below.

[0009] Furthermore, the method of using the polylactic acid-glycolic acid copolymer mixed with α-galactosylceramide as a carrier to encapsulate the immunogenic death products of tumor cells, resiquimod and endosomal-lysosomal escape peptides includes: dissolving the polylactic acid-glycolic acid copolymer, α-galactosylceramide and resiquimod in dichloromethane to obtain a mixed solution, adding an aqueous phase containing the immunogenic death products of tumor cells and the endosomal-lysosomal escape peptides to the mixed solution, performing a first ultrasonic treatment, adding a polyvinyl alcohol aqueous solution, performing a second ultrasonic treatment, and then dripping an isopropanol aqueous solution, stirring, washing and centrifuging to obtain a nano vaccine.

[0010] Furthermore, the first ultrasonic treatment and the second ultrasonic treatment are both intermittent ultrasonic treatments, and the intermittent ultrasonic treatment is ultrasonic treatment for 1 to 10 s and rest for 1 to 10 s.

[0011] Furthermore, the volume ratio of the polyvinyl alcohol aqueous solution to the mixed solution containing the tumor cell immunogenic death products and the endosomal-lysosomal escape-promoting peptide is 0.2-1:1.

[0012] Furthermore, the method includes: co-incubating tumor cells with a photosensitizer (Cypate) and then irradiating them with a 785 nm laser, using a photodynamic method to induce immunogenic death of tumor cells, or incubating tumor cells with an iridium-complexed oligoarginine polypeptide overnight to obtain tumor cell immunogenic death products including tumor antigen peptides, damage-associated molecular patterns, etc.; polylactic acid-glycolic acid copolymer (PLGA) mixed with α-galactosylceramide (α-Galcer) as a carrier to encapsulate the tumor cell immunogenic death products, the immune adjuvant resimod and the endosomal-lysosomal escape-promoting peptide to prepare a nanovaccine.

[0013] The present invention also provides a nano vaccine for promoting antigen cross-presentation, which is prepared by the above method.

[0014] The present invention also provides the use of the above-mentioned nano vaccine for promoting antigen cross-presentation in the preparation of a drug for increasing the expression of co-stimulatory signals on the surface of dendritic cells.

[0015] The present invention also provides the use of the above-mentioned nano vaccine for promoting antigen cross-presentation in the preparation of anti-tumor drugs and / or anti-tumor recurrence drugs.

[0016] The present invention also provides the use of the above-mentioned nano vaccine for promoting antigen cross-presentation in the preparation of drugs for improving the secretion of pro-inflammatory factors.

[0017] The present invention also provides the use of the above-mentioned nano vaccine for promoting antigen cross-presentation in the preparation of a drug for improving the cross-presentation of dendritic cell antigens on MHC class I molecules.

[0018] Beneficial Effects

[0019] Nano vaccines are internalized into dendritic cells. Tumor antigens escape into the cytoplasm through endosomal-lysosomal escape peptides. After being processed by cytoplasmic proteases, antigen peptides are cross-presented to major histocompatibility complex (MHC) class I molecules to activate cellular immunity. If they cannot escape, they can only be presented to MHC class II molecules and cannot activate effective cellular immunity. The α-galactosylceramide in the carrier is presented to the CD1d of dendritic cells (DC) to activate natural killer T cells (NKT cells). NKT cells secrete cytokines to promote DC maturation and CD8 + T cell activation. Resiquimod can promote the maturation of dendritic cells as an immune adjuvant. The nano vaccine prepared by the method of the present invention has efficient antigen cross-presentation ability, and multi-pathway stimulation of the expression of dendritic cell co-stimulatory signals and the secretion of cytokines, thereby achieving the biological effect of synergistically activating cellular immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a flow chart of the preparation of the nano vaccine for promoting antigen cross-presentation according to the present invention; Figure 2 This is a graph of the hydrated particle size of the nanovaccine prepared by method 1; Figure 3 is a transmission electron micrograph of the nanovaccine prepared by product 1 via method 1; Figure 4 This is the hydrated particle size diagram of the nano vaccine prepared by method 2; Figure 5 is a transmission electron microscopy image of the nanovaccine prepared by method 2; Figure 6 The toxicity of the nanovaccine prepared in Example 2 to mouse melanoma cells (B16F10) and mouse-derived dendritic cells (BMDC) is shown by the survival rate of mouse melanoma cells (B16F10) and mouse-derived dendritic cells (BMDC) under the action of the nanovaccine, reflecting the toxicity of the nanovaccine; Figure 7 The effect of the type and concentration of the polypeptide encapsulated in the nanovaccine prepared by the method of Example 5 on the presentation rate of the OVA257-264 antigen peptide on MHCⅠ, ****p<0.0001; Figure 8 The nanovaccine prepared by the method of Example 2 promoted the expression of CD80 and CD86 on BMDCs, ****p<0.0001; Fig. 9 The nano-vaccines (prepared by the method of Example 3) at different concentrations promote the secretion of interleukin-6 by BMDC (measured in terms of R848 concentration); Fig.10The nano-vaccines (prepared by the method of Example 3) at different concentrations promote the secretion of tumor necrosis factor-a by BMDC (measured in terms of R848 concentration); Fig.11 The nano-vaccines (prepared by the method of Example 3) at different concentrations promote the secretion of interleukin-6 by BMDC (measured in terms of R848 concentration); Fig.12 is the presentation rate of α-galactosylceramide (α-GC) on CD1d of BMDCs, ***p<0.001; Fig.13 is the weight change curve of mice; Fig.14 is the tumor growth curve of mice in each group; Fig.15 This is the tumor growth curve of mice cured by the nanovaccine (prepared by the method of Example 2) after being challenged with tumor again. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0022] Example 1

[0023] Preparation of immunogenic tumor cell death products: The structural formula of poly(lactic acid-co-glycolic acid) (PLGA, x=50, y=50, molecular weight 24000–38000 Da): ; The structural formula of α-galactosylceramide (a-Galcer): ; The structural formula of R848 (Resiquimod) is as follows: ; The structural formula of iridium complex oligo-arginine polypeptide is as follows: .

[0024] In order to obtain the immunogenic death product of tumor cells, 6 million 4T1 mouse breast cancer cells were incubated with iridium-complexed oligoarginine peptide (15 μM) overnight to induce cell apoptosis. The cells and supernatant products were collected the next day, and the cells were broken by ultrasonic disruptor. The supernatant was collected to obtain the immunogenic death product of tumor cells caused by apoptosis (referred to as product 1). The ultrasonic conditions were: under ice bath conditions, power 200~300 W, supersonication for 5s and rest for 5s, and treatment time 6~10 min.

[0025] Dissolve 12.5 mg of cyanine dye (Cypate) in 1 mL of dimethyl sulfoxide (DMSO), take 100 mL of 12.5 mg / mL clear DMSO stock solution and add it to 400 mL of PEG300, mix well; then add 50 mL of Tween-80 to the above solution and mix well; then add 450 mL of normal saline to obtain a clear solution with a cyanine dye concentration of 1.89 mM. In order to obtain the immunogenic death product of tumor cells caused by photodynamic therapy, 6 million 4T1 mouse breast cancer cells were taken, and the 1.89 mM cyanine dye solution was diluted to 20 μM with serum-free cell culture medium and added to the cells, incubated overnight, and the next day at 785 nm, 1.5 w / cm 2 The cells were irradiated with laser for 10 min to induce photodynamic cell death, and then placed back in the incubator overnight. The cells and supernatant products were collected the next day, and the cells were broken by ultrasonic disruptor. The cells were centrifuged at 14,000 g for 10 min, and the supernatant was collected to obtain the immunogenic death product of tumor cells caused by photodynamic cell death (referred to as product 2). Products 1 and 2 can be stored at -20°C for a long time after freeze-drying.

[0026] Example 2 Preparation of Nano Vaccines: Product 1 was quantified by BCA kit and diluted with ultrapure water to a cell product aqueous solution with a protein content of 6 mg / mL. PLGA, α-galactosylceramide and immune adjuvant resiquimod (R848) were taken and dissolved in dichloromethane to obtain a mixed solution. The prepared mixed solution contained 5 mg PLGA, 1 mg (0.1-2 mg) R848 and 50 μg (0.04-50 μg) α-galactosylceramide per milliliter. 100 μL of cell product aqueous solution and 6 μM iridium complex oligoarginine polypeptide (iPep) were added to 1 mL of the prepared solution, and the ultrasonic disruptor was ultrasonicated for 1 min 30 s (to obtain nanovaccines with a particle size of 150-200 nm) or 45 s (to obtain nanovaccines with a particle size of 100-150 nm) using method 1 (390 W, ultrasonication for 5 s, intermittent for 5 s). Then, polyvinyl alcohol PVA aqueous solution was added according to the volume ratio of polyvinyl alcohol PVA aqueous solution (50 mg / mL): mixed solution of dichloromethane = 1:1.1, and ultrasonication was performed for 1.5 min (390 W, ultrasonication for 3 s, intermittent for 3 s). The ultrasonicated sample was gradually dripped into a 2% isopropanol aqueous solution (volume / volume, v / v) under stirring and stirred overnight. After the dichloromethane was completely evaporated, the supernatant was discarded by centrifugation, and 10 mL of ultrapure water was added for washing 3 times, and the supernatant was discarded by centrifugation at 6000 g for 10 min to obtain a nanovaccine with a theoretical diameter of 100-200 nm. The nanovaccine was resuspended in 10 mL of pure water / 1xPBS / 4% trehalose aqueous solution / normal saline.

[0027] Example 3 Product 2 was prepared by the same method as above, except that the ultrasonic conditions were changed to method 2 (357.5W, ultrasonic for 3 s, intermittent for 3 s) for 30 s, iPep was changed to 7 μM GALA peptide (polypeptide sequence WEAALAEALAEALAEHLAEALAEALEALAA, SEQ ID NO. 1), and the ultrasonic conditions after adding polyvinyl alcohol aqueous solution were the same as method 2 to obtain a nanovaccine with a theoretical diameter of 200-300 nm. Some of the nanoparticles were taken and resuspended in ultrapure water, and the size, morphology and particle size dispersion index of the nanoparticles were detected by dynamic light scattering and transmission electron microscopy. Figures 2~5 As shown in the figure, the results of dynamic light scattering and transmission electron microscopy showed that the particle size of the nanovaccines prepared by the two methods was between 80-200 nm and 200-300 nm, and the morphology was a vesicle structure with an aqueous core and a lipid shell. The particle size dispersion index was <0.3, and the particle size distribution was relatively uniform.

[0028] Example 4 In order to confirm the exact content of residual iridium-complexed oligoarginine in product 1, 1x phosphate buffer solution (1xPBS) with concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 μM of iridium-complexed oligoarginine was first prepared, and the emission intensity at 520 nm was detected by a fluorescence spectrophotometer (excitation light 328 nm, excitation and emission slit width 10 nm, voltage 650 V), and a concentration-fluorescence emission intensity standard curve was established. Then 100 mL of product 1 was diluted to 1 mL, and its fluorescence intensity at 520 nm was detected under the same conditions. The concentration of residual iridium-complexed oligoarginine polypeptide was calculated according to the standard curve, which was calculated to be 0.97 μM. At this concentration, the iridium-complexed oligoarginine polypeptide did not produce obvious cytotoxicity.

[0029] Then, 2 mL of nanovaccine was taken, centrifuged at 10,000 g for 5 min, the supernatant was discarded, 2 mL of DMEM medium was added for resuspending, 100 μL of nanovaccine was added to each well of a 96-well plate with 8,000 4T1 cells / well, and the cells were incubated overnight at 37°C in 5% carbon dioxide. The solution was then discarded, CCK8 was added for incubation for 2 h, and the absorbance at 450 nm was detected by an enzyme reader. Figure 6 The results showed that the survival rates of mouse melanoma cells (B16F10) and mouse-derived dendritic cells (BMDC) were very high under the action of nanovaccines, indicating that the iridium-complexed oligoarginine polypeptide in the nanovaccine did not cause obvious cytotoxicity, and that the nanovaccine mainly exerted its anti-tumor effect by activating the immune function.

[0030] Example 5 Set the DMSO solution with resiquimod concentrations of 1, 2, 4, 8, 16, and 32 μg / mL, and establish a concentration-absorbance standard curve by liquid chromatography. Take 100 μL of the nanovaccine prepared in Example 2, add 900 μL of dimethyl sulfoxide, centrifuge at 1000 g for 10 min, take the supernatant, analyze the resiquimod content in the sample by high-performance liquid chromatography, and calculate the encapsulation efficiency and drug loading of resiquimod based on the standard curve. The chromatographic conditions are as follows: chromatographic column, InertSustain ®C18 (4.6 mm×150 mm, 5 μm); column temperature, 30℃; flow rate, 1.0 mL / min; injection volume, 10 μL; diode array detector detection wavelength, 254 nm; running time, 40 min; dilution solution, acetonitrile: water (V:V) = 60:40; needle washing solution, methanol; mobile phase A, 0.1% trifluoroacetic acid aqueous solution; mobile phase B, acetonitrile; elution gradient (%A, 0 min, 90%; 5 min, 90%; 25 min, 10%; 33 min, 10%; 35 min, 90%; 40 min, 90%. %B, 0 min, 10%; 5 min, 10%; 25 min, 90%; 33 min, 90%; 35 min, 10%; 40 min, 10%). The drug loading of resiquimod in the nanovaccine was 13.6% and the encapsulation efficiency was 82.7%.

[0031] Example 6 Nano-vaccines promote antigen presentation: Product 1 was replaced with ovalbumin (OVA) and the nanovaccine was prepared according to method 1. Then, the nanovaccine was co-incubated with 300,000 / well mouse bone marrow-induced dendritic cells for 24 h, the supernatant was discarded, the dendritic cells were collected, washed twice with PBS, and resuspended in cell staining buffer. Then, FITC-labeled anti-mouse CD11c, Percp / Cy5.5-labeled anti-mouse-H-2kd, and PE / Cy7-labeled anti-mouse H-2kb targeting OVA antigen peptide (SIINFEKL, SEQ ID NO.2) were added at a ratio of 1 μL dye per 1 million cells. The mixture was incubated on ice in the dark for 15 min, centrifuged at 1500 g for 5 min, washed three times with cell staining buffer, and resuspended in 500 μL of new buffer for flow cytometry detection. The results are shown in Figure 7 As shown, it shows that the polypeptide significantly affects the antigen cross-presentation effect of the nanovaccine. The cross-antigen presentation rate of OVA PLGA nanoparticles is only 1.13%, while with the increase of endosomal-lysosomal escape peptide (iPep), the cross-antigen presentation efficiency increases significantly. When the concentration of iridium-complexed oligoarginine polypeptide is 7 μM, the cross-antigen presentation rate is 48.73%, which increases by about 40 times. When the concentration of GALA peptide with lysosomal escape function is 7 μM, the cross-antigen presentation rate also increases to 27.86%, indicating that endosomal-lysosomal escape peptide significantly increases the presentation of antigen peptides on MHCⅠ, which is beneficial to the activation of tumor-specific cellular immunity.

[0032] Example 7 Nanovaccines promote the expression of co-stimulatory signals in bone marrow-derived mouse dendritic cells (BMDCs): The nanovaccine prepared in Example 2 was co-incubated with 300,000 / well mouse bone marrow-induced dendritic cells for 24 h. BMDCs incubated with lipopolysaccharide (LPS) were used as the positive control group. The supernatant was discarded, the dendritic cells were collected, washed twice with PBS, and resuspended in cell staining buffer. Then, FITC-labeled anti-mouse CD11c, APC-labeled anti-mouse CD86, and PE-labeled anti-mouse CD80 were added at a ratio of 1 μL dye per 1 million cells. The cells were incubated on ice in the dark for 15 min, centrifuged at 1500 g for 5 min, washed three times with cell staining buffer, and resuspended in 500 μL of new buffer for flow cytometry detection. The results are shown in Figure 2. Figure 8 As shown, the proportion of mature BMDC cells with CD80+CD86+ in the negative control group was only 10.35%, while that in the nanovaccine group reached 41.73%, an increase of about 4 times, indicating that the nanovaccine can significantly increase the expression of co-stimulatory signals on the surface of dendritic cells and promote the maturation of dendritic cells.

[0033] Example 8 Nanovaccines promote cytokine secretion of bone marrow-derived mouse dendritic cells (BMDCs): 100 μL of nanovaccine was added to 900 μL of culture medium and then incubated with 500,000 / well mouse bone marrow-induced dendritic cells for 24 h. The supernatant was collected and the secretion of interleukin-6, tumor necrosis factor α (TNF-α), and interleukin-12 (p40) in the cell supernatant was detected according to the steps of the enzyme-linked immunosorbent assay kit. Figures 9-11 As shown, the secretion of three proinflammatory cytokines by BMDCs is positively correlated with the dosage of nanovaccine. As the dosage of nanovaccine increases, the secretion of proinflammatory factors increases, which is beneficial to the activation of anti-tumor immunity when applied in vivo.

[0034] Example 9 The presentation effect of α-galactosylceramide of nanovaccine on CD1d of BMDCs: BMDCs were plated in 6-well plates, with 500,000 cells per well, and the nanovaccine was added for incubation for 24 h. The supernatant was then discarded, the cells were collected, and Fc was first blocked with anti-mouse CD16 / 32. After washing, anti-mouse CD11c-APC and anti-mouse α-GC:CD1d complex-PE were added at a ratio of 1 μL per 1 million cells, incubated at 4°C in the dark for 30 min, then washed 3 times, and the cells were resuspended in 500 μL cell staining buffer and detected by flow cytometry. The results are shown in Fig.12As shown, BMDCs that have taken up the nanovaccine effectively present α-galactosylceramide on CD1d on the cell surface. The presentation rate of the negative control group was only 1.17%, the presentation rate of the free α-galactosylceramide group was 18.35%, and the presentation rate of the nanovaccine group was 60.73%, indicating that loading α-galactosylceramide on nanovaccines with appropriate particle size is more conducive to the uptake and presentation of dendritic cells.

[0035] Example 10 Biosafety of Nanovaccines: C57 mice were subcutaneously injected with 100 μL of the nanovaccine of Example 2 for 5 consecutive days, and the control group was subcutaneously injected with the same volume of PBS, and the weight changes of the mice were detected. Fig.13 As shown, continuous injection of nanovaccine did not cause a significant decrease in mouse weight, and there was no significant difference compared with the PBS group, indicating that the nanovaccine has good biosafety.

[0036] Embodiment 11 Tumor therapeutic effect of nano vaccines: 6-8 week old C57BL6 / j mice were subcutaneously inoculated with 1 million B16F10 cells on the right back. 3 The time was set as day 1, and 100 μL of the nanovaccine of Example 2 was subcutaneously inoculated on days 1 / 4 / 7 / 12 / 17, respectively. The tumor size of the mice was measured with a vernier caliper according to the formula tumor volume = length * width 2 / 2 calculation. The result is as follows Fig.14 As shown, the nanovaccine has a good tumor treatment effect. On the 24th day, all tumors in the mice in the nanovaccine group were eliminated and no recurrence was observed during the experimental period.

[0037] Example 12 Nano vaccines provide long-term immune protection: The cured mice were selected and C57 mice of similar age and weight that had not received any treatment were used as controls. 3 million B16F10 cells were inoculated again on the left back of each mouse to observe the growth of the tumor. Fig.15 As shown, the mice cured by the nanovaccine developed long-lasting immune memory protection, which could resist the reinvasion of more homologous cells, and had good therapeutic and anti-recurrence potential in tumor treatment.

Claims

1. A method for preparing a nano vaccine that promotes antigen cross-presentation, characterized in that: The following steps are involved: Induce the immunogenic death of tumor cells and obtain the immunogenic death products of tumor cells; A polylactic acid-glycolic acid copolymer is mixed with α-galactosylceramide as a carrier to encapsulate the immunogenic death products of tumor cells, resiquimod and endosomal-lysosomal escape-promoting peptides to prepare a nano vaccine; the method for inducing immunogenic death of tumor cells is to co-incubate tumor cells with iridium complex oligoarginine polypeptides and / or laser irradiate tumor cells that have taken in photosensitizers.

2. The method for preparing a nanovaccine that promotes antigen cross-presentation according to claim 1, characterized in that: The endosomal-lysosomal escape-promoting peptide is an iridium complex oligoarginine polypeptide or a GALA peptide.

3. The method for preparing a nanovaccine that promotes antigen cross-presentation according to claim 1, characterized in that: The mass ratio of poly(lactic acid-co-glycolic acid) copolymer, resiquimod, and α-galactosylceramide is 50-100:1-20:0.0004-0.05, and the working concentration of the endosomal-lysosomal escape peptide is the concentration at which 20% of cell growth is inhibited or below.

4. The method for preparing a nanovaccine that promotes antigen cross-presentation according to claim 1, characterized in that: The method of using the polylactic acid-glycolic acid copolymer mixed with α-galactosylceramide as a carrier to encapsulate the immunogenic death products of tumor cells, resiquimod and endosomal-lysosomal escape peptide comprises: dissolving the polylactic acid-glycolic acid copolymer, α-galactosylceramide and resiquimod in dichloromethane to obtain a mixed solution, adding an aqueous phase containing the immunogenic death products of tumor cells and the endosomal-lysosomal escape peptide to the mixed solution, performing a first ultrasonic treatment, adding a polyvinyl alcohol aqueous solution, performing a second ultrasonic treatment, then dripping an isopropanol aqueous solution, stirring, washing and centrifuging to obtain a nano vaccine.

5. The method for preparing a nanovaccine that promotes antigen cross-presentation according to claim 4, characterized in that: The first ultrasonic treatment and the second ultrasonic treatment are both intermittent ultrasonic treatments, and the intermittent ultrasonic treatment is ultrasonic treatment for 1 to 10 s and rest for 1 to 10 s.

6. The method for preparing a nano vaccine that promotes antigen cross-presentation according to claim 4, characterized in that: The volume ratio of the polyvinyl alcohol aqueous solution to the mixed solution containing the immunogenic death products of tumor cells and the peptide promoting endosomal-lysosomal escape is 0.2~1:

1.

7. A nanovaccine that promotes antigen cross-presentation, characterized in that: Prepared by the method according to any one of claims 1 to 6.

8. Use of the nanovaccine that promotes antigen cross-presentation according to claim 7 in the preparation of anti-tumor drugs and / or anti-tumor recurrence drugs.

9. Use of the nanovaccine that promotes antigen cross-presentation according to claim 7 in the preparation of drugs for increasing the secretion of pro-inflammatory factors.

10. Use of the nano vaccine for promoting antigen cross-presentation according to claim 7 in the preparation of a drug for improving cross-presentation of dendritic cells antigens on MHC class I molecules.

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