Manganese boride whole-cell vaccine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of anti-tumor, in particular to a manganese boride whole-cell vaccine as well as a preparation method and application thereof. According to the preparation method, tumor cells are pretreated through photo-thermal nanoparticles MnB-coated beta-CD, near-infrared light irradiation is combined, and the pretreated tumor cells are loaded with injectable hydrogel, so that the broad-spectrum and high-efficiency tumor whole-cell vaccine is prepared on the basis, noninvasive and easy-to-operate near-infrared light irradiation is carried out at an inoculation part according to the tumor development condition, and the tumor whole-cell vaccine is prepared. And local inflammation can be caused by local warming under superficial irradiation, so that the recruitment of dendritic cells is promoted, and the cross excitation of anti-tumor effect T cells is enhanced. The tumor whole-cell vaccine has good biological safety and has relatively high protection efficiency on breast cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of anti-tumor, more particularly, it relates to a boronized manganese whole cell vaccine and a preparation method and application thereof. BACKGROUND

[0002] With the vigorous development of global tumor vaccine research and development, the existing vaccine types have their own characteristics but face significant limitations: dendritic cell vaccines have long preparation cycle, difficult to guarantee cell activity and low response rate (<15%); polypeptide vaccines are limited by single antigen epitope and high complexity of neoantigen screening; mRNA vaccines face challenges such as poor stability, delivery technology barriers and toxicity risks. Compared with the above, tumor whole cell vaccine has the innate advantages of overcoming tumor heterogeneity and immune escape because it carries the complete tumor-associated antigen spectrum and can stimulate multi-target immune response.

[0003] However, the preparation process of tumor whole cell vaccine often relies on repeated freeze-thawing, resulting in unstable vaccine potency and poor immune effect. In addition, how to enhance the immunogenicity of tumor cells is also an important technical problem. SUMMARY

[0004] In order to overcome the deficiencies of the prior art, the present application provides a boronized manganese whole cell vaccine and a preparation method and application thereof, which are used to solve the technical problem of low immunogenicity of tumor whole cell vaccine.

[0005] To achieve the above technical purpose, the present application adopts the following technical scheme: A preparation method of a boronized manganese whole cell vaccine, comprising the following steps: (1) incubating tumor cells with MnB@β-CD for 4 hours; (2) irradiating with near-infrared light at a power of 0.75 W / cm² for 20 minutes, and controlling the temperature to maintain 47-49°C; (3) continuing to incubate for 16 hours after stopping irradiation, and centrifuging to collect the pretreated tumor cells; (4) mixing the tumor cells obtained in step (3) with an injectable hydrogel.

[0006] Preferably, the injectable hydrogel is made by crosslinking 1% sodium alginate solution with 1.25% CaCl2 at a volume ratio of 100:4.5.

[0007] Preferably, the storage modulus of the injectable hydrogel is greater than the loss modulus, and it shows shear thinning behavior under shear strain.

[0008] Preferably, the tumor cells are breast cancer cells 4T1.

[0009] A boronized manganese whole cell vaccine prepared by the aforementioned preparation method.

[0010] Preferably, the tumor whole cell vaccine contains 5×10 5 MnB@β-CD and near-infrared light pretreated tumor cells per unit dose, and the tumor cells are suspended in 60 μL of injectable hydrogel.

[0011] A tumor immunotherapy kit preloaded with the tumor whole cell vaccine prepared by the aforementioned preparation method.

[0012] The tumor whole cell vaccine prepared by the aforementioned preparation method is used in tumor immunotherapy (specifically, in the prevention and treatment of breast cancer), which is administered by subcutaneous injection, and the injection site is irradiated with near-infrared light at a power of 0.75 W / cm² for 8 minutes.

[0013] The present application provides a preparation scheme for a photothermal tumor whole cell vaccine. Based on the concept of TCV, the tumor cells are co-incubated with photothermal nanoparticles MnB@β-CD and subjected to near-infrared light irradiation to induce the upregulation of heat shock proteins (HSPs) in the tumor cells. Subsequently, the prepared TCV is injected into the subcutis of mice through an injectable hydrogel, achieving in vivo immune prevention and enhancement of anti-tumor immune response.

[0014] The preparation method of the injectable hydrogel-encapsulated photothermal tumor whole cell vaccine system, as shown in Figure 1 , comprises the following steps: 1. Construction and characterization of photothermal nanoparticles MnB@β-CD 1) Preparation of MnB@β-CD An appropriate amount of β-cyclodextrin (β-CD) is mixed in ddH2O for standby; MnB is mixed in cyclohexane using ultrasonic mixing. Further, the MnB solution and the β-CD solution are stirred under the action of a motor stirrer for 20 h. After forming a uniform mixture, the cyclohexane is removed by rotary evaporation at 81 ℃, and the cooled aqueous solution is placed in a reaction kettle and reacted at 180 ℃ for 24 h. The precipitate collected by centrifugation is carbonized cyclodextrin-coated manganese boride (MnB@β-CD).

[0015] 2) Physical characterization External morphology: Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are used to observe the overall morphology and thickness of each sample.

[0016] Photothermal performance characterization: The photothermal performance of the material under different concentrations of MnB@β-CD, different near-infrared powers, and different irradiation times was characterized using an infrared thermal imager. The tumor cells were co-incubated with the photothermal nanoparticles, and the endocytosed photothermal nanoparticles were irradiated with near-infrared light to produce endogenous HSPs. The amount of HSP105 produced in the cells was determined using western-blot.

[0017] 2. Preparation and characterization of injectable hydrogel Dissolve 1% sodium alginate 10ml in physiological saline, stir with a glass rod and heat to 50℃ until completely dissolved. Add 1.25% CaCl2 in portions, 100ul each time, a total of 450ul. Stir while adding. Collect the hydrogel into a glass bottle and store in a refrigerator at 4℃. Test the material properties of the reaction hydrogel by rheology.

[0018] 3. Evaluation of biological safety In vitro biocompatibility: Take the peripheral blood of a rabbit, dilute it with physiological saline, and co-incubate it with different concentrations of MnB@β-CD for several hours. Observe the changes in red blood cell morphology and solution color. In vivo biological safety: Construct a tumor-bearing mouse model, inject MnB@β-CD whole cell vaccine subcutaneously, and monitor the mouse body weight for a long time. At the end of treatment, take important organ tissues from mice treated under different conditions for H&E staining to evaluate whether there are pathological changes.

[0019] 4. In vivo model validation of anti-tumor efficacy Preventive vaccine effect: Use the 4T1 cell line to construct a mouse subcutaneous tumor model. After random grouping, treat with MnB@β-CD hydrogel whole cell vaccine combined with NIR. Monitor the tumor size continuously, draw the tumor growth curve, and record the time of mouse death or ethical endpoint to draw the survival curve. Compare the effects of different doses of MnB@β-CD hydrogel whole cell vaccine and the irradiation frequency of NIR on the tumor growth curve to explore the best treatment mode. Inject the constructed whole cell vaccine into the subcutaneous part of the mouse and irradiate the injection site of the mouse. Monitor the tumor size continuously and draw the tumor growth curve. At the end of the experiment, take the mouse lymph nodes and spleen to evaluate the recruitment and activation of DCs at the injection site, the activation of T cells, and the infiltration of effector memory T cells using flow cytometry.

[0020] The present application has the following beneficial effects: the present application pretreats tumor cells by photothermal nanoparticles MnB@beta-CD, combines near-infrared light irradiation, and loads the pretreated tumor cells in injectable hydrogel, to prepare a broad-spectrum and efficient tumor whole cell vaccine, and according to the tumor progression, non-invasive and easy-to-operate near-infrared light irradiation is implemented at the inoculation site, so as to achieve the best pulse immune synergism, and local inflammation is caused by local heating under superficial irradiation, the recruitment of dendritic cells (DCs) is promoted, and the cross-priming of anti-tumor effector T cells is enhanced. The tumor whole cell vaccine has good biological safety, and has a high protection efficiency on breast cancer. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Schematic diagram of the construction scheme of the injectable hydrogel-encapsulated tumor whole cell vaccine system based on infrared light irradiation.

[0022] Figure 2 Transmission electron microscope and scanning electron microscope images of the photothermal nanoparticles in Example 1.

[0023] Figure 3 Temperature rising curve of the photothermal nanoparticles in Example 1 (showing concentration dependence and power dependence).

[0024] Figure 4 Thermal imaging diagram of the photothermal nanoparticles in Example 1.

[0025] Figure 5 Western-blot diagram of HSP105 in 4T1 cells in different treatment groups in Example 1.

[0026] Figure 6 Efficiency diagram of MnB@beta-CD in promoting T cell activation in vitro under NIR in Example 2.

[0027] Figure 7 Preparation characterization and subcutaneous absorption diagram of the injectable hydrogel in Example 3.

[0028] Figure 8 Tumor inhibition curve diagram of TCV in inhibiting 4T1 mouse breast cancer in Example 3.

[0029] Figure 9 Tumor diameter comparison diagram of TCV in inhibiting 4T1 mouse breast cancer in Example 3.

[0030] Figure 10 Mature situation of DCs in lymph nodes, activation situation of CD8+ memory T cells, and anti-tumor function situation of CD8+ memory T cells after TCV injection in Example 4.

[0031] Figure 11Figure for the maturation of DCs, the activation of CD8+ memory T cells, and the anti-tumor function of CD8+ memory T cells in the spleen after TCV injection in Example 4.

[0032] Figure 12 Figure for the infiltration of CD8+ T cells and Tregs in the tumor after TCV injection in Example 4.

[0033] Figure 13 Figure for the body weight change of mice during the treatment in Example 3.

[0034] Figure 14 Figure for the HE staining sections of heart, liver, spleen, lung, and kidney of mice at the endpoint in Example 3.

[0035] Figure 15 Figure for the in vitro hemolysis rate and safety verification of rabbit red blood cells in Example 5. DETAILED DESCRIPTION

[0036] In order to make the objects, features, and advantages of the present application more apparent, specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. Several embodiments of the present application are given in the accompanying drawings. However, the present application can be implemented in many different forms, and is not limited to the embodiments described herein.

[0037] In the following examples, the specific conditions not specified are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are conventional products that can be obtained by commercial purchase, unless the manufacturer is specified. Unless otherwise specified, the present application employs the existing technology in the field.

[0038] Example 1: Framework and characterization of photothermal nanoparticles MnB@β-CD (1) Preparation of photothermal nanoparticles 0.4 g of β-cyclodextrin was mixed with 60 ml of ddH2O to form a β-CD solution; 20 mg of MnB was mixed with 10 ml of cyclohexane, and ultrasonically mixed for 10 min to form a MnB solution; the β-CD solution and the MnB solution were mixed and stirred for 20 h using a dynamic stirrer; cyclohexane was removed by rotary evaporation at 81°C for 40 min, and the aqueous solution was cooled; the cooled aqueous solution was added to a reaction kettle, the reaction kettle was placed in an oven and reacted at 180°C for 24 h; after the reaction kettle was cooled, the aqueous solution was ultrasonically treated for 10 min, and then centrifuged at 12000 rpm for 5 min after aliquoting; the precipitate was collected to obtain carbonized cyclodextrin-coated manganese boride (MnB@β-CD).

[0039] (2) Transmission electron microscopy (TEM), scanning electron microscopy (SEM) were used to observe the overall morphology, thickness of MnB, β-CD, MnB@β-CD. β-CD was spherical, MnB was flaky, TEM of MnB@β-CD showed that the flaky MnB was wrapped by spherical β-CD, the material preparation was successful, and the results are shown in Figure 2 .

[0040] (3) The photothermal performance of the photothermal nanoparticles prepared in step (1) was characterized by using an infrared thermal imager. A series of different concentrations of photothermal nanoparticle aqueous solutions (0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL) were continuously irradiated for 600 s using an 808 nm laser (0.75 W / cm 2 ). During the near-infrared laser irradiation, the temperature was recorded every 30 s by the infrared thermal imager. As shown in Figure 3 , the results show that when the concentration of the nanoparticle aqueous solution is 50 μg / mL, the temperature will gradually rise under continuous light and stabilize at 47-49℃, which can make the tumor cells enter the heat shock state and produce a large amount of heat shock proteins.

[0041] The near-infrared thermal imager was used to evaluate the temperature change of the photothermal nanoparticles prepared in step (1) at a specific concentration and the corresponding relationship with the power of the 808 nm near-infrared laser. As shown in Figure 3 and Figure 4 , the results show that by adjusting the laser power (0 W / cm 2 , 0.32 W / cm 2 , 0.75 W / cm 2 , 1.0 W / cm 2 , 1.5 W / cm 2 ), the temperature of the irradiation area of the sample at this concentration can be effectively controlled.

[0042] Western-blot was used to characterize the expression of HSP105 in the cells after light treatment. The treated 4T1 cells (grouping: Ctrl group (regular 4T1 cell culture, no additional treatment), PTT group (simple infrared irradiation treatment: 0.75 W / cm 2 irradiate 4T1 cells for 20 min and continue to incubate in the cell incubator for 20 h), MnB@β-CD group (simple material treatment: 50 μg / mL of photothermal material is added to 4T1 cells, and incubated in the cell incubator for 20 h), MnB@β-CD+PTT group (infrared light irradiation and photothermal material co-treatment: 4T1 cells are pre-treated by MnB@β-CD for 4 h, and then 0.75 W / cm 2After the 4T1 cells were irradiated with infrared laser for 20 min, they were incubated in the cell incubator for another 16 h. Then, the cells were washed twice with ice-cold PBS, and RIPA lysis buffer was added for lysis on ice for 15 min. The lysis buffer was repeatedly blown and collected in a 1.5 mL centrifuge tube. Centrifugation was performed at 10,000 g for 10 min, and the supernatant was collected. The protein content was determined using a BCA detection kit. The supernatant of each group was diluted to the same protein concentration and mixed with SDS loading buffer, and then heated at 95°C for 5 min. The samples (20 μL per well) were added to LabPAGE 4-20% 15-well gels, electrophoretically separated, and transferred to a polyvinylidene fluoride transfer membrane. After the polyvinylidene fluoride transfer membrane was blocked in 5% skimmed dry milk for 2 hours, the primary antibody was incubated. The primary antibody was diluted as follows: GAPDH (1:5000), HSP105 (1:5000). Then, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:100,000), and imaging was performed using a multicolor fluorescence gel imaging system (DNRMFChemiBIS3.2). The results are shown in Figure 5 Fig. 6, which shows that the expression of HSP105 in 4T1 cells that endocytosed the photothermal nanoparticles was significantly up-regulated after irradiation for 20 min.

[0043] Example 2: Injectable hydrogel-encapsulated tumor whole-cell vaccine promotes T cell activation in vitro The lymph nodes and spleens of OT1 mice were taken, ground, and filtered through a 70-μm filter to obtain a single-cell suspension (the spleen was cracked red). The progenitor cells were resuspended with 100 IU / mL recombinant mouse IL-12 in complete 1640 medium, and the T cells were counted. The T cells were co-cultured with the treated B16F10-OVA at a ratio of 20:1 and 50:1 for 24 hours (B16-OVA cell groups: Ctrl group, vaccine group (incubated with the material for 4 h, NIR 0.75 W / cm 2 After 20 min, incubate for another 12 h). The T cells were stained with PerCP-Cy5.5-conjugated anti-mouse CD8 flow cytometry antibody, PE-conjugated anti-mouse CD44 flow cytometry antibody, APC-conjugated anti-mouse CD62L flow cytometry antibody, APC-conjugated anti-mouse IFN-γ flow cytometry antibody, BV510-conjugated anti-mouse TNFα flow cytometry antibody, and PE-conjugated anti-mouse granzyme B flow cytometry antibody to detect the function of the T cells and the activation of memory T cells. The results are shown in Figure 6 Fig. 5, which shows that the memory effect of CD8 + T cells was enhanced, and the anti-tumor effect of CD8 + T cells was enhanced.

[0044] Example 3: In vivo efficacy verification of injectable hydrogel-encapsulated tumor whole-cell vaccine Pre-preparation of the injectable hydrogel (INJ-Hydrogel): Dissolve 10 ml of 1% sodium alginate in physiological saline, stir with a glass rod, and heat to 50°C until completely dissolved. Add 1.25% CaCl2 dropwise, 100 μL each time, for a total of 450 μL, stirring continuously. After cooling, collect the hydrogel in a glass vial and store at 4°C. The hydrogel should have the following properties: Figure 7 As shown in (e), the material properties of the reactive hydrogel were tested by rheological analysis. Figure 7 (a) is a viscoelastic transition diagram of storage modulus G' and loss modulus G'' at different angular frequencies, reflecting the elastic response of the material at different angular frequencies. As the frequency increases, the elastic response of the material increases, the viscous behavior increases, and the fluidity decreases. G'>G'' indicates that this is an elastically dominated material with strong strain recovery and weak fluidity. Figure 7 (b) is a viscosity-shear strain diagram, which reflects the shear thinning phenomenon of the material under high shear strain, that is, the viscosity decreases as the shear strain increases, and the fluidity increases, which means that this is a liquid / soft material. Figure 7 (c) in the figure is the relationship between storage modulus and shear stress, which reflects the nonlinear viscoelastic behavior of the material. Under high shear stress, the elastic part of the hydrogel gradually disappears, exhibiting stronger fluidity. Figure 7 The results indicate that the hydrogel can remain under the skin and be gradually absorbed, continuously activating the immune system.

[0045] BALB / c mice (6 weeks old, female) were randomly divided into 4 groups (n=6): (1) NS group (subcutaneous injection of physiological saline), (2) ALG group (subcutaneous injection of injectable hydrogel INJ-Hydrogel), (3) MnB group (subcutaneous injection of injectable hydrogel-encapsulated pure material MnB@β-CD), (4) TCV vaccine group (subcutaneous injection of injectable hydrogel-encapsulated whole-cell vaccine; vaccine preparation: 4T1 cells were pretreated by co-incubation with MnB@β-CD for 4 h, then 0.75 W / cm 2 After irradiating 4T1 cells with infrared laser for 20 min, they were incubated in a cell incubator for 16 h, then centrifuged and mixed with injectable hydrogel to obtain the desired result.

[0046] The initial tumor seeding amount per mouse was 2.5 × 10⁻⁶. 5 Day 0 was designated as the first day of vaccination. Mice were given their first dose of vaccine on Day 5. In the NS group, each mouse received a subcutaneous injection of 60 μL of saline; in the ALG group, each mouse received a subcutaneous injection of 60 μL of injectable hydrogel; in the MnB group, each mouse received a subcutaneous injection of 60 μL of injectable hydrogel-encapsulated pure MnB@β-CD material; and in the TCV vaccine group, each mouse received a subcutaneous injection of 60 μL of the prepared hydrogel-encapsulated MnB@β-CD whole-cell vaccine (5 × 10⁶ cells per mouse). 5Day 11, the first near-infrared light irradiation (0.75 W / cm 2 of the infrared laser irradiated the whole cell vaccine inoculation site of the mice for 8 min). On Day 12, the second dose of cell vaccine was injected, and the grouping and operation were the same as the first dose of vaccine injection. On Day 18, the second near-infrared light irradiation (0.75 W / cm 2 of the infrared laser irradiated the whole cell vaccine inoculation site of the mice for 8 min). Day 22 was recorded as the end of the experiment. The changes in the tumor volume of the mice were measured with a vernier caliper throughout the process: volume (mm 3 ) = width 2 × length × 0.5, and the results are shown in Figure 8 to Figure 9 . The results show that the TCV vaccine group has the best tumor inhibition effect.

[0047] Example 4: Evaluation of the immune response of the whole cell vaccine in the lymph nodes and spleen BALB / c mice (6 weeks old, female) were randomly divided into 4 groups (n = 6): (1) NS group (subcutaneous injection of normal saline), (2) ALG group (subcutaneous injection of injectable hydrogel), (3) MnB group (subcutaneous injection of injectable hydrogel encapsulated simple material MnB@β-CD), (4) TCV group (subcutaneous injection of injectable hydrogel encapsulated whole cell vaccine, vaccine preparation: 4T1 cells were pretreated by co-incubation with MnB@β-CD for 4 h, and then 0.75 W / cm 2 of the infrared laser irradiated the 4T1 cells for 20 min, and then the cells were further incubated in the cell incubator for 16 h before centrifugation, mixed with the injectable hydrogel to obtain the vaccine). The corresponding treatment was given according to the method in Example 3. Then, on Day 24, the inguinal lymph nodes of each mouse were collected. After grinding, filtration, and washing, centrifugation at 400g for 5 min, a single cell suspension of the lymph nodes was obtained and divided into four equal parts; on Day 24, the spleen of each mouse was collected, ground, filtered, and washed, and then centrifuged at 400g for 5 min to obtain a single cell suspension of the spleen and divide it into three equal parts. On Day 24, the tumor of each mouse was collected, digested, ground, filtered, and washed, and then centrifuged at 400g for 5 min to obtain a single cell suspension of the tumor tissue.

[0048] The prepared single cell suspensions of the lymph nodes and spleen were each taken out and divided into an equal part, and PE / Cy7-conjugated anti-mouse CD11b flow cytometry antibody, PE-conjugated anti-mouse CD11c flow cytometry antibody, FITC-conjugated anti-mouse CD80 flow cytometry antibody, and PERCP-EF710-conjugated anti-mouse CD86 flow cytometry antibody were mixed and added to the suspension to stain the dendritic cells for 30 min to evaluate the degree of maturation of the dendritic cells.

[0049] Take out an equal part of the prepared lymph node and spleen single cell suspension, and mix FITC conjugated anti-mouse CD45 flow cytometry antibody, PE / Cy7 conjugated anti-mouse CD3 flow cytometry antibody, PerCP-Cy5.5 conjugated anti-mouse CD8 flow cytometry antibody, BV711 conjugated anti-mouse CD4 flow cytometry antibody, PE conjugated anti-mouse CD62L flow cytometry antibody, PC5.5 conjugated anti-mouse CD44 flow cytometry antibody into the suspension to stain T cells for 30 min, and detect the activation of memory T cells.

[0050] Take out an equal part of the prepared lymph node and spleen single cell suspension, and transfer it to a 48-well plate. Add 1640 complete culture medium and stimulation blocking agent, and culture in a cell incubator for 8 h. Then blow and digest to prepare a cell suspension for staining. Mix FITC conjugated anti-mouse CD45 flow cytometry antibody, PE / Cy7 conjugated anti-mouse CD3 flow cytometry antibody, and PerCP-Cy5.5 conjugated anti-mouse CD8 flow cytometry antibody into the prepared lymph node and spleen single cell suspension to stain T cells for 1 h. Then break the membrane with a membrane breaking reagent for 1 h and wash. Then mix APC conjugated anti-mouse IFN-γ flow cytometry antibody, BV510 conjugated anti-mouse TNFα flow cytometry antibody, and PE conjugated anti-mouse granzyme B flow cytometry antibody into the lymph node and spleen single cell suspension after membrane breaking to stain for 30 min, and detect the anti-tumor function of T cells.

[0051] Take the prepared tumor single cell suspension, mix FITC conjugated anti-mouse CD45 flow cytometry antibody, PE / Cy7 conjugated anti-mouse CD3 flow cytometry antibody, PerCP-Cy5.5 conjugated anti-mouse CD8 flow cytometry antibody, BV711 conjugated anti-mouse CD4 flow cytometry antibody, and APC conjugated anti-mouse CD25 flow cytometry antibody into the single cell suspension to stain T cells for 40 min. Then break the membrane with a membrane breaking reagent for 1 h and wash. Then mix PE conjugated anti-mouse Foxp3 flow cytometry antibody into the tumor single cell suspension after membrane breaking to stain for 30 min to detect the proportion of suppressor T cells. The results are shown in Figure 10-12 The results show that the maturation of DCs in the lymph nodes and spleen of the TCV vaccine group increases, the memory effect of CD8 + T cells in the lymph nodes and spleen is enhanced, the anti-tumor effect of CD8 + T cells in the lymph nodes and spleen is enhanced, the proportion of suppressor T cells Tregs in the tumor tissue of the TCV vaccine group decreases, and the infiltration of CD8 + T cells increases.

[0052] Example 5: Biological safety evaluation of tumor whole cell vaccine The body weight of the mice did not fluctuate significantly during the treatment in the above-mentioned Example 3, and the structure is shown in Figure 13 .

[0053] The end-point mice in Example 3 above were taken for inspection of heart, liver, spleen, lung and kidney, and as shown in the HE-stained sections, there was no significant difference between the vaccine group and normal tissue, and the mice had no significant weight fluctuation during treatment, indicating that the vaccine had good in vivo safety. Figure 14

[0054] Peripheral blood of rabbits was taken, diluted with physiological saline, and incubated with different concentrations of MnB@β-CD (12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) for 1 hour, and the red blood cell morphology and solution color change were observed, as shown in Figure 15 Compared with the negative control group, the 50 μg / mL material group had no obvious hemolysis.

[0055] In summary, the present application provides an injectable hydrogel-wrapped photothermal tumor whole-cell vaccine and its application, which addresses the low immunogenicity of existing whole-cell vaccines. The present application innovatively improves the immunogenicity of tumor cells (HSP105↑) through photothermal pretreatment (MnB@β-CD combined with 47℃ near-infrared light irradiation), and prolongs the subcutaneous retention time of the vaccine using shear-thinning hydrogel (sodium alginate / CaCl2 crosslinking), thereby achieving efficient recruitment of lymph node DCs, sustained activation of CD8⁺ T cells, and significant tumor inhibition. The present application provides a new vaccine strategy for solid tumor prevention and treatment that combines high efficiency and safety.

[0056] The above only describes the preferred embodiments of the present application, and the protection scope of the present application is not limited to the above examples. Any technical solutions falling within the scope of the present application should be considered within the protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, some improvements and refinements without departing from the principles of the present application should also be considered within the protection scope of the present application.​

Claims

1. A method for preparing a manganese boride whole-cell vaccine, characterized in that, Includes the following steps: (1) Tumor cells were co-incubated with MnB@β-CD for 4 hours; (2) Irradiate with near-infrared light at a power of 0.75 W / cm² for 20 minutes, and maintain the temperature at 47-49℃; (3) After stopping irradiation, continue incubation for 16 hours, and then collect the pretreated tumor cells by centrifugation; (4) Mix the tumor cells obtained in step (3) with the injectable hydrogel.

2. The preparation method according to claim 1, characterized in that, The injectable hydrogel is made by crosslinking 1% sodium alginate solution and 1.25% CaCl2 at a volume ratio of 100:4.

5.

3. The preparation method according to claim 2, characterized in that, The injectable hydrogel has a storage modulus greater than its loss modulus and exhibits shear thinning behavior under shear strain.

4. The preparation method according to claim 1, characterized in that, The tumor cells were breast cancer cells, specifically 4T1.

5. A manganese boride whole-cell vaccine, characterized in that, Prepared by the preparation method according to any one of claims 1-4.

6. The whole-cell vaccine according to claim 5, characterized in that, Each unit dose of the tumor whole-cell vaccine contains 5 × 10 5 Tumor cells pretreated with MnB@β-CD and near-infrared light were suspended in 60 μL of injectable hydrogel.

7. A tumor immunotherapy kit, characterized in that, The tumor whole-cell vaccine is pre-loaded with the preparation method described in any one of claims 1-4.

8. The application of a whole-cell tumor vaccine prepared by any one of claims 1-4 in tumor immunotherapy, characterized in that, The drug was administered via subcutaneous injection, followed by irradiation of the injection site with near-infrared light at a power of 0.75 W / cm² for 8 minutes.

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