A nano-vaccine for promoting antigen cross-presentation, its preparation method and application

Abstract

The present invention belongs to the fields of biomedicine and materials, and particularly relates to a nano-vaccine for promoting antigen cross-presentation, a preparation method thereof, and an application thereof. The method comprises the following steps: inducing immunogenic death of tumor cells to obtain tumor cell immunogenic death products; using poly(lactic-co-glycolic acid) copolymer mixed with α-galactosylceramide as a carrier to encapsulate the tumor cell immunogenic death products, resiquimod, and an endosome-lysosome escape peptide to prepare a nano-vaccine; the method for inducing immunogenic death of tumor cells is to co-incubate tumor cells with iridium complexed oligoarginine polypeptide and / or laser irradiate tumor cells that have taken up a photosensitizer. This nano-vaccine has high antigen cross-presentation ability, as well as multi-pathway stimulation of the expression of co-stimulatory signals and the secretion of cytokines by dendritic cells, so as to achieve the biological effect of synergistically activating cellular immunity.

Description

Technical Field

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

[0002] Tumor vaccines utilize tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs) to activate the immune system of patients. In theory, vaccines can stimulate specific cellular and humoral immune responses, prevent tumor growth, and ultimately eradicate tumor cells. However, tumor antigens are all endogenous and have low immunogenicity, often making it difficult to elicit an effective immune response. In addition, traditional vaccines mainly induce humoral immunity, but CD8 + Cytotoxic T lymphocytes (CTLs)-mediated cellular immunity is crucial for eliminating 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 the immune activation induced by tumor antigens. Dendritic cells (DCs), as the key bridge connecting innate immunity and adaptive immunity, are the most important antigen-presenting cells. During the initiation of antiviral and anti-tumor 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 generation of MHCⅠ-binding peptides mediated by the proteasome. The polypeptides generated by the proteasome are then transported to the endoplasmic reticulum by the transporter associated with antigen processing TAP1 and TAP2, loaded onto newly formed MHC class I molecules, and then presented on the surface of DCs. The presented antigen, together with cytokines and co-stimulatory signals, activates T cells.

[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 progenitors, and monocyte-derived dendritic cells MoDCs. Due to the advantages of antigen cross-presentation, cDC1s usually result in stronger CD8 +T cell immunity. Although cDC1s have good cross-presentation ability and higher levels of secretion of immune activation-related cytokines, and are associated with good prognosis, their difficult acquisition limits their research and application in anti-tumor therapy. Currently, the therapeutic effects of in vitro-generated DCs vaccines undergoing clinical trials are not satisfactory because naturally occurring DC subsets have stronger antigen presentation ability, higher MHC molecule expression, and functional specialization than in vitro-generated DCs. How to improve the antigen cross-presentation, co-stimulatory signal expression, and secretion of corresponding cytokines of DCs, and break through the limitations of DC type and quantity on immune effects, is a key issue in tumor vaccine therapy. Summary of the Invention

[0005] In view of the problem that existing tumor vaccines have low promotion of dendritic cell maturation and low antigen cross-presentation rate, resulting in low efficiency of cellular immune activation, the present invention provides a nano-vaccine for promoting antigen cross-presentation, its preparation method, and application. By promoting endosome-lysosome escape peptides, the cross-presentation of antigen peptides in dendritic cells is promoted. By inducing immunogenic death of tumor cells, antigen peptides with good immune activation potential and damage-associated molecular patterns (DAMPs) are obtained. Through α-galactosylceramide (α-Galcer) and the immune adjuvant resiquimod, dendritic cell maturation is promoted through multiple pathways, inducing dendritic cells to cross-present tumor antigen peptides, highly express co-stimulatory signals, and increase 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:

[0007] A preparation method of a nano-vaccine for promoting antigen cross-presentation, comprising the following steps: inducing immunogenic death of tumor cells to obtain tumor cell immunogenic death products; using poly(lactic-co-glycolic acid) copolymer mixed with α-galactosylceramide as a carrier to encapsulate tumor cell immunogenic death products, resiquimod, and endosome-lysosome escape peptides to prepare a nano-vaccine; the method for inducing immunogenic death of tumor cells is to co-incubate tumor cells with iridium-conjugated oligomeric arginine polypeptides and / or laser irradiate tumor cells that have taken up photosensitizers.

[0008] Further, the endosome-lysosome escape peptide is iridium-conjugated oligomeric arginine polypeptide or GALA peptide.

[0009] Further, the mass ratio of poly(lactic-co-glycolic acid) copolymer, resiquimod, and α-galactosylceramide is 50-100:1-20:0.0004-0.05, and the working concentration of the endosome-lysosome escape peptide is the concentration at which cell growth is inhibited by 20% or less.

[0010] Furthermore, the method for using poly(lactic-co-glycolic acid) copolymer mixed with α-galactosylceramide as a carrier to encapsulate tumor cell immunogenic death products, resiquimod, and endosome-lysosome escape peptides includes: dissolving poly(lactic-co-glycolic acid) copolymer, α-galactosylceramide, and resiquimod in dichloromethane to obtain a mixed solution, adding an aqueous phase containing tumor cell immunogenic death products and endosome-lysosome escape peptides to the mixed solution, performing the first ultrasonic treatment, adding an aqueous solution of polyvinyl alcohol, performing the second ultrasonic treatment, and then dropping it into an aqueous isopropanol solution, followed by stirring, washing, and centrifuging to obtain the nano-vaccine.

[0011] Furthermore, both the first ultrasonic treatment and the second ultrasonic treatment are intermittent ultrasonic treatments, and the intermittent ultrasonic treatment is ultrasonic treatment for 1 - 10 s with an interval of 1 - 10 s.

[0012] Furthermore, the volume ratio of the aqueous solution of polyvinyl alcohol to the mixed solution containing tumor cell immunogenic death products and endosome-lysosome escape peptides is 0.2 - 1:1.

[0013] Furthermore, the method includes: co-incubating tumor cells with a photosensitizer (Cypate) and then irradiating with 785 nm laser, using the method of photodynamic to cause immunogenic death of tumor cells, or incubating tumor cells with iridium-conjugated oligomeric arginine polypeptide overnight to obtain tumor cell immunogenic death products including tumor antigen peptides, damage-associated molecular patterns, etc.; using poly(lactic-co-glycolic acid) (PLGA) copolymer mixed with α-galactosylceramide (α-Galcer) as a carrier to encapsulate tumor cell immunogenic death products, the immune adjuvant resiquimod, and endosome-lysosome escape peptides to prepare a nano-vaccine.

[0014] The present invention also provides a nano-vaccine for promoting antigen cross-presentation, which is prepared by the above method.

[0015] The present invention also provides the application 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.

[0016] The present invention also provides the application of the above-mentioned nano-vaccine for promoting antigen cross-presentation in the preparation of an anti-tumor drug and / or an anti-tumor recurrence drug.

[0017] The present invention also provides the application of the above-mentioned nano-vaccine for promoting antigen cross-presentation in the preparation of a drug for increasing the secretion of pro-inflammatory factors.

[0018] The present invention also provides the application of the above-mentioned nano-vaccine for promoting antigen cross-presentation in the preparation of a drug for increasing the antigen cross-presentation of dendritic cells to MHC class I molecules.

[0019] Beneficial effects

[0020] The nano-vaccine internalized into dendritic cells, where tumor antigens escape into the cytoplasm through endosome-lysosome escape peptides. The antigen peptides processed by cytoplasmic proteases are cross-presented on major histocompatibility complex (MHC) class I molecules to activate cellular immunity. If they cannot escape, they can only be presented on MHC class II molecules and cannot activate effective cellular immunity. The α-galactosylceramide in the carrier is presented on CD1d of dendritic cells (DCs) to activate natural killer T cells (NKT cells). NKT cells secrete cytokines to promote DC maturation and CD8 + T cell activation. Resiquimod, as an immune adjuvant, can promote the maturation of dendritic cells. The nano-vaccine prepared by the method of the present invention has high antigen cross-presentation ability, and can stimulate the expression of co-stimulatory signals and the secretion of cytokines in dendritic cells through multiple pathways, so as to achieve the biological effect of synergistically activating cellular immunity. Brief Description of the Drawings

[0021] Figure 1 It is the preparation flow chart of the nano-vaccine for promoting antigen cross-presentation described in the present invention;

[0022] Figure 2 It is the hydrated particle size diagram of the nano-vaccine prepared by product 1 through method 1;

[0023] Figure 3 It is the transmission electron microscopy image of the nano-vaccine prepared by product 1 through method 1;

[0024] Figure 4 It is the hydrated particle size diagram of the nano-vaccine prepared by product 2 through method 2;

[0025] Figure 5 It is the transmission electron microscopy image of the nano-vaccine prepared by product 2 through method 2;

[0026] Figure 6 It is the toxicity of the nano-vaccine prepared in Example 2 to mouse melanoma cells (B16F10) and mouse-derived dendritic cells (BMDC). The toxicity of the nano-vaccine is reflected by the survival rate of mouse melanoma cells (B16F10) and mouse-derived dendritic cells (BMDC) under the action of the nano-vaccine;

[0027] Figure 7 It is the influence of the polypeptide types and concentrations encapsulated by the nano-vaccine prepared by the method of Example 5 on the presentation rate of OVA257-264 antigen peptide on MHC I, ****p < 0.0001;

[0028] Figure 8 It is the promotion of the expression of CD80 and CD86 on BMDCs by the nano-vaccine prepared by the method of Example 2, ****p < 0.0001;

[0029] Figure 9 The promotion of interleukin-6 secretion by BMDCs by nano-vaccines at different concentrations (prepared by the method of Example 3) (counted by the concentration of R848);

[0030] Figure 10 The promotion of tumor necrosis factor-α secretion by BMDCs by nano-vaccines at different concentrations (prepared by the method of Example 3) (counted by the concentration of R848);

[0031] Figure 11 The promotion of interleukin-6 secretion by BMDCs by nano-vaccines at different concentrations (prepared by the method of Example 3) (counted by the concentration of R848);

[0032] Figure 12 The presentation rate of α-galactosylceramide (α-GC) on CD1d of BMDCs, ***p < 0.001;

[0033] Figure 13 The body weight change curve of mice;

[0034] Figure 14 The tumor growth curve of mice in each group;

[0035] Figure 15 The tumor growth curve of mice cured by the nano-vaccine (prepared by the method of Example 2) after being challenged with tumors again. Detailed implementation mode

[0036] The present invention will be further described below with reference to the drawings and examples.

[0037] Example 1

[0038] Preparation of immunogenic cell death products of tumor cells:

[0039] The structural formula of poly(lactic-co-glycolic acid) (PLGA, x = 50, y = 50, molecular weight 24000–38000 Da):

[0040] ;

[0041] The structural formula of α-galactosylceramide (a-Galcer):

[0042] ;

[0043] The structural formula of R848 (resiquimod) is as follows:

[0044] ;

[0045] The structural formula of iridium complexed oligomeric arginine polypeptide is as follows:

[0046] 。

[0047] To obtain the immunogenic cell death products of tumor cells, 6 million 4T1 murine breast cancer cells were taken and incubated overnight with iridium complexed oligomeric arginine polypeptide (15 μM) to induce oncosis. The cells and supernatant products were collected the next day. The cells were disrupted by an ultrasonic cell disruptor, centrifuged at 14,000 g for 10 min, and the supernatant was collected to obtain the immunogenic cell death products of tumor cells caused by oncosis (abbreviated as Product 1). The ultrasonic conditions were as follows: under ice bath conditions, the power was 200 - 300 W, ultrasonic for 5 s and pause for 5 s, and the treatment time was 6 - 10 min.

[0048] 12.5 mg of cyanine dye (Cypate) was dissolved in 1 mL of dimethyl sulfoxide (DMSO). 100 mL of the clarified DMSO stock solution at 12.5 mg / mL was added to 400 mL of PEG300 and mixed evenly; then 50 mL of Tween - 80 was added to the above solution and mixed evenly; subsequently, 450 mL of normal saline was added to obtain a clarified solution with a cyanine dye concentration of 1.89 mM. To obtain the immunogenic cell death products of tumor cells caused by photodynamic therapy, 6 million 4T1 murine breast cancer cells were taken. The 1.89 mM cyanine dye solution was diluted to 20 μM with serum - free cell culture medium and then added to the cells, incubated overnight. The next day, the cells were irradiated with a laser at 785 nm and 1.5 w / cm 2 for 10 min to induce photodynamic death of the cells, and then returned to the incubator overnight. The cells and supernatant products were collected the next day. The cells were disrupted by an ultrasonic cell disruptor, centrifuged at 14,000 g for 10 min, and the supernatant was collected to obtain the immunogenic cell death products of tumor cells caused by photodynamic death (abbreviated as Product 2). Product 1 and Product 2 can be stored at - 20 °C for a long time after freeze - drying.

[0049] Example 2

[0050] Preparation of nano - vaccine:

[0051] The product 1 was quantified for protein using a BCA kit and diluted with ultrapure water to an aqueous cell product solution with a protein content of 6 mg / mL. Take PLGA, α-galactosylceramide, and the immune adjuvant resiquimod (R848), dissolve them in dichloromethane to obtain a mixed solution. The prepared mixed solution contains 5 mg of PLGA, 1 mg (0.1 - 2 mg) of R848, and 50 μg (0.04 - 50 μg) of α-galactosylceramide per milliliter. Add 100 μL of the aqueous cell product solution and 6 μM iridium-conjugated oligolysine polypeptide (iPep) to 1 mL of the prepared solution, and use an ultrasonic crusher to ultrasonically treat it for 1 min 30 s (to obtain a nano-vaccine with a particle size of 150 - 200 nm) or 45 s (to obtain a nano-vaccine with a particle size of 100 - 150 nm) according to Method 1 (390 W, ultrasonic for 5 s, intermittent for 5 s). Then, add an aqueous polyvinyl alcohol (PVA) solution according to a volume ratio of aqueous polyvinyl alcohol (PVA) solution (50 mg / mL): dichloromethane = 1:1.1, and ultrasonically treat it for 1.5 min (390 W, ultrasonic for 3 s, intermittent for 3 s). Gradually drop the ultrasonically treated sample into a 2% isopropyl alcohol aqueous solution (volume / volume, v / v) under stirring and stir overnight. After the dichloromethane has completely evaporated, centrifuge to discard the supernatant, add 10 mL of ultrapure water and wash 3 times, centrifuge at 6000 g for 10 min to discard the supernatant, and obtain a nano-vaccine with a theoretical diameter of 100 - 200 nm. Resuspend the nano-vaccine in 10 mL of pure water / 1xPBS / 4% trehalose aqueous solution / physiological saline.

[0052] Example 3

[0053] The product 2 was prepared using the same method above. The ultrasonic condition was changed to Method 2 (357.5 W, ultrasonic for 3 s, intermittent for 3 s) and ultrasonically treated for 30 s. The iPep was changed to 7 μM GALA peptide (polypeptide sequence WEAALAEALAEALAEHLAEALAEALEALAA, SEQ ID NO. 1). The ultrasonic condition after adding the aqueous polyvinyl alcohol solution was the same as Method 2, and a nano-vaccine with a theoretical diameter of 200 - 300 nm was obtained. Take a part of the nanoparticles, resuspend them in ultrapure water, and use a dynamic light scattering instrument and a transmission electron microscope to detect the size, morphology, and particle size dispersion index of the nanoparticles. As Figures 2 - 5 shown, the results of the dynamic light scattering instrument and the transmission electron microscope show that the particle sizes of the nano-vaccines prepared by the two methods are between 80 - 200 nm and 200 - 300 nm, the morphology is a vesicle structure with an aqueous core and a lipid shell, and the particle size dispersion index < 0.3, and the particle size distribution is relatively uniform.

[0054] Example 4

[0055] To confirm the exact content of the iridium complexed oligomeric arginine remaining in Product 1, 1x phosphate buffered saline (1xPBS) solutions with concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 μM of the iridium complexed oligomeric arginine were first prepared. The emission intensity at 520 nm was detected using a fluorescence spectrophotometer (excitation light 328 nm, excitation and emission slit widths 10 nm, voltage 650 V) to establish a standard curve of concentration - fluorescence emission intensity. 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 the remaining iridium complexed oligomeric arginine polypeptide was calculated according to the standard curve and was found to be 0.97 μM. At this concentration, the iridium complexed oligomeric arginine polypeptide did not cause obvious cytotoxicity.

[0056] Then, 2 mL of the nano - vaccine was taken, centrifuged at 10000 g for 5 min to discard the supernatant, resuspended in 2 mL of DMEM medium, and 100 μL of the nano - vaccine was added to each well of a 96 - well plate with 8000 4T1 cells / well. It was incubated overnight at 37°C in 5% carbon dioxide, then the solution was discarded, CCK8 was added, and it was incubated for 2 h. The absorbance at 450 nm was detected using an enzyme - linked immunosorbent assay (ELISA) reader. Figure 6 The results showed that the survival rates of mouse melanoma cells (B16F10) and mouse - derived dendritic cells (BMDC) were both very high under the action of the nano - vaccine, indicating that the iridium complexed oligomeric arginine polypeptide in the nano - vaccine did not cause obvious cytotoxicity, and the nano - vaccine mainly exerted its anti - tumor effect by activating the immune function.

[0057] Example 5

[0058] DMSO solutions with resiquimod concentrations of 1, 2, 4, 8, 16, and 32 μg / mL were set up, and a standard curve of concentration - absorbance was established by liquid chromatography. 100 μL of the nano - vaccine prepared in Example 2 was taken, 900 μL of dimethyl sulfoxide was added, centrifuged at 1000 g for 10 min, and the supernatant was taken. The content of resiquimod in the sample was analyzed by high - performance liquid chromatography, and the encapsulation efficiency and drug - loading capacity of resiquimod were calculated according to the standard curve. The chromatographic conditions were as follows: chromatographic column, InertSustain ®C18 (4.6 mm×150 mm, 5 μm); column temperature, 30 °C; flow rate, 1.0 mL / min; injection volume, 10 μL; detection wavelength of the diode array detector, 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%). After detection, the drug loading of resiquimod in the nano-vaccine was 13.6%, and the encapsulation rate was 82.7%.

[0059] Example 6

[0060] Promoting antigen presentation effect of the nano-vaccine:

[0061] Replace product 1 with ovalbumin (OVA) and prepare the nano-vaccine according to method 1. Then co-incubate it with dendritic cells induced by mouse bone marrow at 300,000 cells / well for 24 h. Discard the supernatant, collect the dendritic cells, wash them twice with PBS, resuspend them in cell staining buffer, and then add 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) according to the ratio of 1 μL dye per 1 million cells. Incubate in the dark on ice for 15 min, centrifuge at 1500 g for 5 min, wash three times with cell staining buffer and resuspend in 500 μL of a new buffer, and detect with a flow cytometer. The results are as Figure 7 shown, indicating that the polypeptide significantly affected the antigen cross-presentation effect of the nano-vaccine. The cross-antigen presentation rate of OVA PLGA nanoparticles was only 1.13%. With the increase of the endosome-lysosome escape peptide (iPep), the cross-antigen presentation efficiency increased significantly. When the concentration of iridium complex oligolysine polypeptide was 7 μM, the cross-antigen presentation rate was 48.73%, an increase of about 40 times. When the concentration of GALA peptide with the same lysosome escape function was 7 μM, the cross-antigen presentation rate also increased to 27.86%, indicating that the endosome-lysosome escape peptide significantly increased the presentation of antigen peptides on MHCⅠ, thus facilitating the activation of tumor-specific cellular immunity.

[0062] Example 7

[0063] Co - stimulation signal expression of nano - vaccine in mouse bone marrow - derived dendritic cells (BMDCs):

[0064] The nano - vaccine prepared in Example 2 was co - incubated with mouse bone marrow - induced dendritic cells at a density of 300,000 cells per well for 24 h. BMDCs incubated with lipopolysaccharide (LPS) were used as the positive control group. The supernatant was discarded, and the dendritic cells were collected, washed twice with PBS, resuspended in cell staining buffer, and 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 of dye per 1 million cells. The cells were incubated in the dark on ice for 15 min, centrifuged at 1500 g for 5 min, washed three times with cell staining buffer, and then resuspended in 500 μL of fresh buffer for flow cytometry detection. The results are as Figure 8 shown. It shows that the proportion of mature BMDC cells with CD80 + CD86 + in the negative control group is only 10.35%, while that in the nano - vaccine group reaches 41.73%, which is about 4 times higher. This indicates that the nano - vaccine can significantly increase the expression of co - stimulation signals on the surface of dendritic cells and promote the maturation of dendritic cells.

[0065] Example 8

[0066] Cytokine secretion of nano - vaccine in mouse bone marrow - derived dendritic cells (BMDCs):

[0067] 100 μL of nano - vaccine was added to 900 μL of culture medium, and then co - incubated with mouse bone marrow - induced dendritic cells at a density of 500,000 cells per well 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. The results are as Figures 9 - 11 shown. It shows that the secretion amounts of the three pro - inflammatory cytokines of BMDCs are positively correlated with the dosage of the nano - vaccine. As the dosage of the nano - vaccine increases, the secretion of pro - inflammatory factors increases, which is beneficial to the activation of anti - tumor immunity in in - vivo applications.

[0068] Example 9

[0069] Presentation effect of α - galactosylceramide of nano - vaccine on CD1d of BMDCs:

[0070] Seed BMDCs cells in a 6-well plate at a density of 500,000 cells per well, add the nano-vaccine and incubate for 24 h. Then discard the supernatant, collect the cells, first perform Fc blocking with anti-mouse CD16 / 32, and after washing, add anti-mouse CD11c-APC and anti-mouse α-GC:CD1d complex-PE at a ratio of 1 μL per 1,000,000 cells, incubate at 4 °C in the dark for 30 min, then wash 3 times, resuspend the cells in 500 μL of cell staining buffer, and detect by flow cytometry. The results are as Figure 12 shown, indicating that BMDCs that have taken up the nano-vaccine effectively present α-galactosylceramide on CD1d on the cell surface. The presentation rate of the negative control group is only 1.17%, the presentation rate of the free α-galactosylceramide group is 18.35%, and the presentation rate of the nano-vaccine group is 60.73%, indicating that loading α-galactosylceramide onto a nano-vaccine with a suitable particle size is more conducive to the uptake and presentation of dendritic cells.

[0071] Example 10

[0072] Bio-safety of the nano-vaccine:

[0073] Take C57 mice and subcutaneously inject 100 μL of the nano-vaccine of Example 2 for 5 consecutive days. The control group is subcutaneously injected with the same volume of PBS, and the body weight change of the mice is detected. The results are as Figure 13 shown, indicating that continuous injection of the nano-vaccine does not cause a significant decrease in the body weight of the mice, and there is no significant difference from the PBS group, indicating that the nano-vaccine has good bio-safety.

[0074] Example 11

[0075] Tumor treatment effect of the nano-vaccine:

[0076] Take 6-8-week-old C57BL6 / j mice and subcutaneously inoculate 1,000,000 B16F10 cells on the right dorsal side. When the tumor grows to about 30 mm 3 set it as day 1, and subcutaneously inoculate 100 μL of the nano-vaccine of Example 2 on days 1 / 4 / 7 / 12 / 17 respectively. Measure the tumor size of the mice with a vernier caliper, and calculate according to the tumor volume = length * width 2 / 2. The results are as Figure 14 shown, indicating that the nano-vaccine has good tumor treatment effect. On day 24, the tumors of the mice in the nano-vaccine group were all eliminated and no recurrence was observed during the experimental period.

[0077] Example 12

[0078] The nano-vaccine provides long-term immune protection:

[0079] Take the cured mice, and use C57 mice of similar age and weight that have not received any treatment as controls. Re-inject 3 million B16F10 cells into the left back of each mouse respectively, and observe the tumor growth. The results are as Figure 15 shown, indicating that the mice cured by the nano-vaccine have a long-lasting immune memory protection effect, can resist the re-invasion of more homologous cells, and have good potential for treatment and anti-relapse 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 nano vaccine is prepared by mixing polylactic acid-glycolic acid copolymer with α-galactosylceramide as a carrier to encapsulate the immunogenic death products of tumor cells, resiquimod and endosomal-lysosomal escape-promoting peptides; the method of inducing immunogenic death of tumor cells is to co-incubate tumor cells with iridium-complexed oligoarginine polypeptides and / or laser irradiate tumor cells that have taken up photosensitizers; The endosomal-lysosomal escape-promoting peptide is an iridium-complexed oligoarginine polypeptide; the structure of the iridium-complexed oligoarginine polypeptide is as follows: ； The mass ratio of poly(lactic-co-glycolic acid), 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; 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 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 peptides 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; 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 seconds with an interval of 1 to 10 seconds.

2. The method for preparing a nanovaccine that promotes antigen cross-presentation according to claim 1, 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.

3. The method for preparing a nanovaccine that promotes antigen cross-presentation according to claim 1, characterized in that: The laser irradiation conditions are 785 nm, 1.5 w / cm 2 .

4. The method for preparing a nanovaccine that promotes antigen cross-presentation according to claim 1, characterized in that: The laser irradiation time is 10 min.

5. The method for preparing a nano vaccine that promotes antigen cross-presentation according to claim 1, characterized in that: The power of the first ultrasonic treatment and the second ultrasonic treatment are both 390W.

6. The method for preparing a nanovaccine that promotes antigen cross-presentation according to claim 1, characterized in that: The concentration of the isopropanol aqueous solution is 2% v / v.

7. The method for preparing a nano vaccine that promotes antigen cross-presentation according to claim 1, characterized in that: The centrifugation time is 10 min.

8. The method for preparing a nano vaccine that promotes antigen cross-presentation according to claim 1, characterized in that: The washing is performed by washing with ultrapure water for 3 times.

9. A nanovaccine that promotes antigen cross-presentation, characterized in that: Prepared by the method according to any one of claims 1 to 8.

10. Use of the nano vaccine for promoting antigen cross-presentation according to claim 9 in the preparation of anti-tumor drugs and / or anti-tumor recurrence drugs.

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