Hydrogel tumor whole-cell vaccine as well as preparation method and application thereof

Abstract

The invention discloses a hydrogel tumor whole-cell vaccine as well as a preparation method and application thereof. The preparation method comprises the following steps: S1, collecting tumor cells, performing hypochlorous acid treatment and inactivation treatment on the tumor cells to obtain a tumor whole-cell antigen solution with immunogenicity death, and the tumor whole-cell antigen solution contains 1 * 10 < 7 >-5 * 10 < 7 > tumor cells; s2, dropwise adding a CaCl2 solution into the sodium alginate aqueous solution in a stirring state, and uniformly stirring to obtain calcium alginate hydrogel; s3, uniformly mixing the calcium alginate hydrogel, a tumor whole-cell antigen solution and IL2 to obtain the hydrogel tumor whole-cell vaccine, wherein the dosage ratio of the calcium alginate hydrogel to the tumor whole-cell antigen solution is 1g: 1mL, and the final concentration of IL2 in the hydrogel tumor whole-cell vaccine is 0.01-100 [mu] g / mL. The preparation process is simple, the consumed time is short, and the slowly released tumor whole-cell antigen and IL2 can play a synergistic immune activation effect, so that the effect of remarkably inhibiting tumor recurrence or metastasis is played.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a hydrogel tumor whole-cell vaccine, its preparation method, and its application. Background Technology

[0002] Malignant bone tumors, including primary bone tumors and other solid tumors that metastasize to the bone marrow, are malignant diseases that seriously threaten human life and health. These tumors not only destroy the normal structure of bones, causing severe pain, pathological fractures, spinal cord compression, and other complications that significantly reduce patients' quality of life, but they also have extremely strong invasiveness and metastasis, making recurrence after treatment very likely, posing a huge challenge to clinical treatment.

[0003] Currently, the main clinical treatments for malignant bone tumors include surgical resection, chemotherapy, and radiotherapy. Surgical treatment reduces the risk of local recurrence by removing tumor-invaded bone tissue, but it is difficult to completely eliminate micrometastases, resulting in a still relatively high probability of postoperative tumor recurrence and distant metastasis. While chemotherapy and radiotherapy can inhibit tumor progression, they suffer from poor targeting, significant systemic toxicity, and substantial damage to the patient's immune system and normal tissues. Therefore, developing novel treatments that effectively prevent postoperative recurrence and metastasis of bone tumors, while possessing both safety and specificity, has become an urgent need in the field of oncology treatment.

[0004] In recent years, immunotherapy has become a research hotspot in cancer treatment due to its advantages such as high specificity and few side effects. Among them, cancer vaccines, as an immunotherapy strategy that activates the body's own immune system to specifically recognize and eliminate tumor cells, have shown promising application prospects. Existing cancer vaccines are mainly classified into the following categories based on their components and preparation methods:

[0005] (1) Dendritic cell (DC) vaccines: These vaccines utilize the efficient antigen-presenting capacity of DC cells. Patient-derived DC cells are loaded with tumor antigens in vitro and then reinfused into the body to activate T-cell immune responses. For example, Sipuleucel-T has been approved for the treatment of prostate cancer, but the preparation process of this type of vaccine is complex, time-consuming, and costly, making it difficult to achieve large-scale application.

[0006] (2) Peptide vaccines: These vaccines use short peptide fragments of tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs) as the core, which activate specific T cell responses by binding to MHC molecules. However, due to the significant heterogeneity of tumors, the antigenic spectrum of a single or a few peptide fragments is narrow and it is difficult to cover all the antigenic characteristics of tumors; at the same time, their immunogenicity is weak, requiring combination with adjuvants, and they are limited by the patient's HLA genotype, thus limiting the applicable population.

[0007] (3) Nucleic acid vaccines: including DNA vaccines and mRNA vaccines, which introduce nucleic acid sequences encoding tumor antigens and use host cells to express the antigens to stimulate an immune response. The success of the COVID-19 mRNA vaccine has verified the technical feasibility of nucleic acid vaccines, but DNA vaccines have the potential risk of genome integration and their safety is a major concern; mRNA vaccines, on the other hand, have poor stability and require special delivery systems and strict storage and transportation conditions, which limits their clinical translation and popularization.

[0008] (4) Viral vector vaccines: These use adenoviruses, poxviruses, etc., as vectors to carry tumor antigen genes into the body and express antigens. However, viral vectors are prone to triggering antiviral immune responses in the body, which not only reduces the efficacy of the vaccine but may also lead to safety issues such as fever and inflammation. In addition, their preparation process is complex and the production cost is high.

[0009] (5) Tumor whole-cell vaccine: The whole tumor cells are processed and used as antigens, which can cover all the antigenic components of the tumor. However, in the existing technology, the preparation process is complicated. It is necessary to obtain tumor samples from patients and then separate, inactivate and extract antigens, which is time-consuming and difficult to standardize. At the same time, the immunogenicity of unoptimized whole tumor cells is weak, and it is necessary to combine gene modification or immune stimulation molecules to enhance the effect, which makes large-scale production difficult.

[0010] In summary, existing tumor vaccines have limitations in terms of ease of preparation, immunogenicity, safety, and large-scale production, making it difficult to meet the clinical treatment needs of malignant bone tumors (especially for the prevention of postoperative recurrence and metastasis). Therefore, developing a tumor vaccine with a simple preparation process, strong immunogenicity, high safety, and the ability to be mass-produced is of great significance for improving the treatment effect of malignant bone tumors and improving patient prognosis. Summary of the Invention

[0011] To address the aforementioned technical problems, this invention provides a hydrogel tumor whole-cell vaccine, its preparation method, and its application. This invention improves the preparation method of tumor whole-cell antigen, shortening the preparation cycle and enhancing its immunogenicity. Furthermore, the prepared tumor whole-cell antigen is co-loaded with IL2 (recombinant human interleukin-2 protein) into a calcium alginate hydrogel to obtain the hydrogel tumor whole-cell vaccine described in this invention. Experiments have demonstrated that the tumor whole-cell antigen and IL2 loaded in the hydrogel exhibit an unexpected synergistic immune activation effect after sustained drug release, thereby significantly inhibiting tumor recurrence or metastasis.

[0012] To achieve the above objectives, the present invention first provides a method for preparing a hydrogel tumor whole-cell vaccine, comprising the following steps:

[0013] S1, collect tumor cells, which are then treated with hypochlorous acid and inactivated to obtain an immunogenic dead tumor whole-cell antigen solution, wherein the tumor whole-cell antigen solution contains 1×10 7 -5×10 7 One tumor cell;

[0014] S2, CaCl2 solution is added dropwise to sodium alginate aqueous solution under stirring, and calcium alginate hydrogel is obtained after stirring evenly.

[0015] S3, mix the calcium alginate hydrogel, tumor whole cell antigen solution and IL2 evenly to obtain the hydrogel tumor whole cell vaccine;

[0016] The ratio of the calcium alginate hydrogel to the tumor whole-cell antigen solution is 1g:1mL, and the final concentration of IL2 in the hydrogel tumor whole-cell vaccine is 0.01-100μg / mL.

[0017] Preferably, the concentration of hypochlorous acid is 50-500 μM, more preferably 100-200 μM, and most preferably 140-160 μM;

[0018] Preferably, the hypochlorous acid treatment time is 0h to 2h, more preferably 0 to 1h, and most preferably 0.15 to 0.3h.

[0019] Preferably, the specific steps of the inactivation treatment are: repeated freeze-thaw cycles with liquid nitrogen twice.

[0020] Preferably, the volume ratio of the CaCl2 solution to the sodium alginate aqueous solution is 1:1.

[0021] Preferably, the sodium alginate in the aqueous solution has a molecular weight of 1 kDa to 1000 kDa and a concentration of 10 mg / mL to 30 mg / mL, more preferably 15 to 25 mg / mL, and most preferably 19 to 21 mg / mL.

[0022] Preferably, the concentration of the CaCl2 solution is 1 mg / mL to 3 mg / mL, more preferably 1.5 to 2.5 mg / mL, and most preferably 1.9 to 2.1 mg / mL.

[0023] Preferably, the tumor cells include at least one of breast cancer cells, colorectal cancer cells, melanoma cells, and bone cancer cells.

[0024] In another aspect, the present invention provides a hydrogel tumor whole-cell vaccine, which is prepared according to the aforementioned preparation method.

[0025] In another aspect, the present invention provides the application of the aforementioned hydrogel tumor whole-cell vaccine in the preparation of antitumor drugs, wherein the tumor includes primary bone tumors or bone metastases of tumors.

[0026] Preferably, the hydrogel tumor whole-cell vaccine is administered via intramedullary injection.

[0027] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0028] 1. The effective active ingredients of the hydrogel tumor whole-cell vaccine provided by the present invention include tumor whole-cell immunogen and IL2. After the vaccine is injected locally or intraosseously into the target site, it can release the effective active ingredients in a sustained manner, achieving localized, long-term, high-concentration drug delivery, fully leveraging the immune activation effect of the tumor microenvironment, and reducing the burden of multiple or systemic administration. More importantly, experiments have shown that the tumor whole-cell antigen and IL2 can also exert an unexpected synergistic immune activation effect, thereby significantly inhibiting tumor recurrence or metastasis.

[0029] 2. The preparation process of this invention is simple, and the tumor whole cell antigen solution can be prepared in only 0.5-2 hours. Then, it can be mixed evenly with IL2 solution and calcium alginate hydrogel (or hydrogel lyophilized powder). No other complex or special equipment is required for processing. Therefore, it is particularly suitable for rapid intraoperative sampling and immediate preparation of hydrogel tumor whole cell vaccines to meet the needs of personalized clinical treatment.

[0030] 3. The hydrogel tumor whole-cell vaccine of the present invention has a clearly defined and quantifiable composition and proportion, has excellent production and quality control standards, and has the potential to achieve large-scale preparation and application. Attached Figure Description

[0031] Figure 1 This represents the results of flow cytometry detection of tumor cell apoptosis under different hypochlorous acid treatment conditions.

[0032] Figure 2 Scanning electron microscopy images showing the surface morphology of tumor cells. The left side shows normal tumor cells, and the right side shows tumor cells that have been treated with hypochlorous acid and frozen with liquid nitrogen.

[0033] Figure 3 The image shows the morphology of the calcium alginate hydrogel lyophilized powder under a scanning electron microscope.

[0034] Figure 4 This indicates the rheological properties of calcium alginate hydrogel.

[0035] Figure 5 This indicates that the treatment process with hypochlorous acid and liquid nitrogen enhanced the immunogenicity of tumor cells.

[0036] Figure 6This represents the release curve of the protein-loaded drug in the calcium alginate hydrogel.

[0037] Figure 7 This indicates the retention status of the gel vaccine in the bone marrow cavity of mice.

[0038] Figure 8 This indicates the proportion of cDC1 in the culture medium of each group as detected by flow cytometry.

[0039] Figure 9 This indicates the detection of Th1 and CD8 in the culture medium of each group by flow cytometry. + and CD8 + IFN-γ + The proportion of T cells.

[0040] Figure 10 This indicates the therapeutic efficacy of the hydrogel tumor whole-cell vaccine of the present invention against the growth of melanoma tumors in B16F10 mice. In this context, PBS represents the intraosseous injection group of the same volume of PBS, IL2+Ag represents the intraosseous administration group of free IL2+Ag, Gel represents the intraosseous injection group of blank gel, Gel-IL2+Ag represents the intraosseous injection group of hydrogel tumor whole-cell vaccine, and Gel-IL2+Ag sc represents the subcutaneous injection group of hydrogel tumor whole-cell vaccine.

[0041] Figure 11 This invention demonstrates the effect of the hydrogel tumor whole-cell vaccine of the present invention on cDC1 in the bone marrow immune microenvironment of B16F10 mouse melanoma model mice.

[0042] Figure 12 This indicates that the hydrogel tumor whole-cell vaccine of the present invention has an inhibitory effect on the tail vein metastasis model of 4T1 mouse breast cancer cells. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0044] Terminology

[0045] In this invention, "intraosseous cavity injection" refers to the injection site of the hydrogel tumor whole-cell vaccine being the cavity where the bone marrow tissue is located.

[0046] In this invention, "cDC1 cells" refers to traditional type 1 dendritic cells, a highly specialized subset of dendritic cells that play a key role in immune responses, especially in anti-tumor immunity.

[0047] In this invention, "CD8" +T cells are a key type of cytotoxic lymphocyte in the adaptive immune system. They play a central role in anti-infection, anti-tumor immunity, and immune regulation by recognizing and eliminating abnormal cells such as cells infected by pathogens and mutated tumor cells.

[0048] In this invention, "room temperature" generally refers to 4 to 30°C, and more preferably 15 to 25°C.

[0049] As described in the background section, existing tumor vaccines have limitations in terms of ease of preparation, immunogenicity, safety, and large-scale production, making it difficult to meet the clinical treatment needs of malignant bone tumors (especially for the prevention of postoperative recurrence and metastasis). Developing a tumor vaccine with a simple preparation process, strong immunogenicity, high safety, and the ability to be mass-produced is a pressing technical challenge that needs to be addressed in this field.

[0050] To this end, the present invention has conducted extensive research and experiments. By improving the preparation method of tumor whole-cell antigen, the preparation cycle of tumor whole-cell antigen has been shortened and its immunogenicity has been improved. Furthermore, the prepared tumor whole-cell antigen and IL2 (recombinant human interleukin-2 protein) were co-loaded into calcium alginate hydrogel to prepare the hydrogel tumor whole-cell vaccine of the present invention. Among them, recombinant human interleukin-2 protein (IL2) is a clinically approved therapeutic cytokine with significant immunomodulatory effects. However, the currently marketed IL2 preparations are only available in the form of injection, and the administration methods are limited to intramuscular injection, intravenous injection and subcutaneous injection. These traditional administration methods have many shortcomings: (1) IL2 has a short half-life and requires frequent administration to maintain an effective blood drug concentration, which increases the treatment burden on patients; (2) Systemic administration may lead to serious side effects, such as capillary leakage syndrome, hypotension and immune-related toxicity, which limits its clinical application; (3) Traditional administration methods are difficult to achieve local high-concentration drug delivery and cannot fully exert the immune activation effect of IL2 in the tumor microenvironment.

[0051] This invention combines IL2 and tumor whole-cell antigen via a hydrogel. Experiments have shown that this hydrogel tumor whole-cell vaccine, when injected into the target site, not only sustains the release of tumor whole-cell antigen and IL2, achieving sustained high-concentration drug delivery locally, but more importantly, tumor whole-cell antigen and IL2 also exert an unexpected synergistic immune activation effect, including increasing the number and function of cDC1 and CD8. + The function and proliferation capacity of T cells ultimately play a significant role in inhibiting tumor recurrence or metastasis.

[0052] The following section will describe the research process and results of this invention in detail with reference to experimental data. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0053] Unless otherwise specified, the experimental methods used in the following examples were performed under conventional or manufacturer-recommended conditions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Sodium alginate (30164428) and anhydrous calcium chloride (10005817) used in the embodiments of this invention were purchased from Sinopharm Chemical Reagent Co., Ltd.; Hank's balanced salt solution (HBSS) (calcium, magnesium ions-free and phenol red-free, 1×) (60148ES76) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; sodium hypochlorite (S953735) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; PBS, 1× (Phosphate Buffered Saline) (G4202-500ML) was purchased from Wuhan Saiwei Biotechnology Co., Ltd.; Annexin V-FITC / PI apoptosis detection kit (MA0220) was purchased from Dalian Meilun Biotechnology Co., Ltd.; mouse IL2 recombinant protein (5μg, 212-12) was purchased from Thermo Fisher Scientific; Zombie Aqua™ Fixable Viability Kit, Alexa 700anti-mouse CD45 antibody, APC / Cyanine7 anti-mouse CD3 antibody, PE / Cyanine7 anti-mouse CD8a antibody, Brilliant Violet 605 TM anti-mouse IFN-γantibody and FITC anti-mouse Ki-67antibody, FITC anti-mouse CD3 antibody, FITCanti-mouse CD19 antibody, FITC anti-mouse CD49B antibody, FITC anti-mouse LY6Gantibody, FITC anti-mouse TER119 antibody, Brilliant Violet 605 TManti-mouseXCR1 antibody, PE / Cyanine7 anti-mouse CD172A antibody, PerCP / Cy5.5anti-mouseCD11C antibody, PE anti-mouse CD80 antibody, Brilliant Violet 785 TM anti-mouseCD86 antibody, Brilliant Violet 650 TM anti-mouse F480 antibody, Brilliant Violet421 TM The flow cytometry antibody for the anti-mouse IA / IE antibody was purchased from BioLegend, Inc., USA.

[0054] In this embodiment of the invention, a magnetic heating stirrer (LC-MSA-D, Shanghai Lichen) was used to dissolve sodium alginate solution and stir to prepare hydrogels; the morphology of the hydrogels was observed and photographed using a low-vacuum ultra-high resolution field emission scanning electron microscope (NOVA NanoSEM230, USA); the rheological properties of the hydrogels were measured using a rotational rheometer (DHR20, USA); and cDC1 and CD8 were examined using a flow cytometer (BD Fortessa, USA). + The proportion and function of T cells and the immune response in mice were examined; the distribution of hydrogels in the bone marrow cavity of mice was investigated using a small animal in vivo imaging system (Tanon ABL-X3, Shanghai Tianneng).

[0055] (I) Preparation and performance characterization of hydrogel tumor whole-cell vaccines

[0056] 1. Preparation method and optimization

[0057] The preparation method of the hydrogel tumor whole-cell vaccine in this embodiment is as follows:

[0058] (1) Collect tumor cells, wash with PBS solution, resuspend in hypochlorous acid solution, incubate at 37°C for a period of time, wash the cells by centrifugation with PBS solution, and then freeze and thaw twice with liquid nitrogen to obtain tumor whole cell antigen solution (containing 1×10⁻⁶ cells / mL). 7 -5×10 7 One tumor cell, in this embodiment 1×10 7 indivual).

[0059] (2) Prepare a sodium alginate solution with a concentration of 20 mg / mL, stir overnight to dissolve completely, then add the sodium alginate solution to a beaker, and add a CaCl2 (calcium chloride) solution with a concentration of 2 mg / mL dropwise with a syringe while stirring magnetically. After the addition is complete (the volume ratio of CaCl2 solution to sodium alginate solution is 1:1), continue stirring at room temperature for 2 to 5 minutes to obtain calcium alginate hydrogel.

[0060] It should be noted that in other embodiments, the calcium alginate hydrogel obtained above can also be processed into freeze-dried powder for later use by the following treatment: after stopping stirring, let it stand for 5 minutes, place the obtained calcium alginate hydrogel at -80℃ for full freezing, and then dehydrate and dry it under low temperature and low pressure conditions to obtain calcium alginate hydrogel freeze-dried powder; wherein, the low temperature and low pressure conditions are: pressure 5~150Pa, temperature -80℃~+40℃, preferably, pressure 10~50Pa, temperature -50℃~-30℃.

[0061] (3) Add whole-cell tumor antigen solution and IL2 solution (in this example, the purchased IL2 powder is dissolved in PBS solution) to the calcium alginate hydrogel obtained in step (2) and stir evenly. 1 mL of whole-cell tumor antigen solution is added to every 1 g of calcium alginate hydrogel. A hydrogel whole-cell tumor vaccine with a final IL2 concentration of 0.01–100 μg / mL is obtained (0.3 μg / mL in this example).

[0062] It should be noted that the hypochlorous acid in step (1) is used to induce immunogenic death of tumor cells. In this embodiment, the concentration and time of hypochlorous acid treatment have been optimized. The specific experimental process is as follows:

[0063] 4T1 cells (mouse breast cancer cells) were injected at a rate of 5 × 10⁻⁶. 5 Cells were seeded at a density of cells / well in six-well plates and cultured overnight. Then, different concentrations of hypochlorous acid solution (prepared by Hanks) (0, 75, 100, 150, 200, 250, 300 μM) were added and incubated at 37°C for 20 min. Cells were then collected. After washing with PBS and centrifuging, the cells were resuspended in 200 μL of staining buffer and Annexin V and PI staining solutions were added. After incubation at room temperature for 5-10 min, cell apoptosis was detected by flow cytometry. Annexin V+ / PI+ cells located in the Q2 quadrant were late apoptotic cells.

[0064] The results are as follows Figure 1 As shown, when the concentration of hypochlorous acid solution was 150 μM and the treatment time was 20 min, the proportion of late apoptotic cells reached more than 90%, indicating a better effect.

[0065] 2. Performance Characterization

[0066] Based on the above preparation method, the hydrogel tumor whole-cell vaccine of this embodiment was prepared using a hypochlorous acid solution concentration of 150 μM and a treatment time of 20 min, and then subjected to a series of characterizations, including:

[0067] (1) Morphological observation of immunogenic tumor cells

[0068] Tumor cells were treated with 150 μM hypochlorous acid solution for 20 min, followed by two freeze-thaw cycles in liquid nitrogen to obtain whole-cell tumor antigens. Untreated cells were included as a control. After collection, the cells were first fixed with 4% paraformaldehyde for 2 h, then treated sequentially with 30%, 50%, 70%, and 90% ethanol for 15 min each, followed by centrifugation to gradually dehydrate them. Finally, the tumor cells were dropped onto a single-crystal silicon wafer (5×5 mm) and freeze-dried. Their morphology was observed using a scanning electron microscope.

[0069] The results are as follows Figure 2 As shown, compared with normal tumor cells (left), tumor cells treated with hypochlorous acid and frozen with liquid nitrogen (right) are shrunken, have rough surfaces, and have lost their original smooth shape.

[0070] (2) Characterization of the porous structure of calcium alginate hydrogel freeze-dried powder

[0071] The morphology of the hydrogel lyophilized powder was observed and photographed using a low-vacuum ultra-high resolution field emission scanning electron microscope (NOVA NanoSEM 230, USA).

[0072] The results are as follows Figure 3 As shown, the calcium alginate hydrogel lyophilized powder exhibits a complete, regular, and uniform porous internal structure, which is beneficial for the slow release of the loaded drug components.

[0073] (3) Rheological detection of hydrogel tumor whole-cell vaccine

[0074] The rheological properties of the final hydrogel tumor whole-cell vaccine were determined using a rotational rheometer. The results are as follows: Figure 4 As shown, the relationship between elastic modulus G' and viscous modulus G” and frequency can reflect the strength of hydrogel. According to the rheometer test results, the hydrogel tumor whole cell vaccine obtained in this embodiment has G'>G” in the tested frequency range (0.1-100 rad / s), and the change of G' with frequency is not significant, which indicates that it has been fully gelled.

[0075] (4) Immunogenicity characterization of tumor cells

[0076] Mouse bone marrow-derived dendritic cells (BMDCs) were co-cultured with untreated tumor cells and tumor whole-cell antigens obtained in the above examples for 24 h. BMDCs were collected, and the expression levels of co-stimulatory molecules CD80 / CD86 on the surface of BMDCs were detected by flow cytometry after antibody staining.

[0077] The results are as follows Figure 5 As shown, compared with untreated tumor cells, tumor whole-cell antigens can significantly increase the expression of co-stimulatory molecules CD80 / CD86 on the surface of BMDCs, suggesting that tumor cell antigens express antigens that can be recognized by DCs or release danger signals, thereby activating BMDCs.

[0078] (5) Detection of sustained-release properties of loaded drugs

[0079] In this embodiment, BSA solution was added to the lyophilized calcium alginate hydrogel powder to prepare a calcium alginate hydrogel loaded with a protein drug, with a final protein drug concentration of 1 mg / mL. The prepared gel was added to a 100 kDa dialysis bag, placed at the bottom of a 15 mL centrifuge tube, and then 1.5 mL of PBS solution was added. The centrifuge tube was placed in a shaker at 37°C. Each group was repeated in triplicate. The supernatant was collected at preset time points (0, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120 h), and immediately replenished with fresh PBS solution at the same temperature. Finally, the concentration of the protein drug was measured, and a release curve was plotted.

[0080] The results are as follows Figure 6 As shown, the calcium alginate hydrogel has a significant sustained-release effect on the loaded protein drug, maintaining a high drug concentration for up to 5 days, which further confirms the structural stability of the hydrogel prepared in this embodiment.

[0081] (6) In vivo imaging to observe bone marrow retention of gel vaccine

[0082] In this embodiment, a Cy5-labeled tumor cell antigen solution and a DiR-labeled OVA protein solution (model protein drug) were added to the lyophilized calcium alginate hydrogel powder to prepare a fluorescently labeled gel vaccine. The gel vaccine was injected bilaterally into the bone marrow cavity of C57BL / 6 mice, and the distribution of the hydrogel within the mouse bone marrow cavity was examined using a small animal in vivo imaging system (Tanon ABL-X3, Shanghai Tianneng).

[0083] The results are as follows Figure 7As shown, after 21 days, Cy5-labeled tumor cell antigen (left) and DiR-labeled OVA protein (right) could still be observed in the bone marrow cavity of mice, indicating that the gel carrier has a good retention effect on the loaded drug.

[0084] (II) Evaluation of the immune activation effect of the hydrogel tumor whole-cell vaccine of the present invention

[0085] 1. The hydrogel tumor whole-cell vaccine of this invention can significantly increase the proportion of cDC1 cells.

[0086] A mouse melanoma cell B16F10 tumor whole-cell antigen solution (containing 1×10⁻⁶ cells) was prepared according to the method described in Experiment (I) above. 7 (One tumor cell), for later use.

[0087] Bone marrow was collected from the tibia and femur of male C57BL / 6 mice, and single-cell suspensions were prepared after lysing red blood cells. The prepared single-cell suspensions were added to 24-well cell culture plates and divided into three groups: PBS group, IL2 group (final concentration 300 ng / mL), Ag group (i.e., antigen, tumor whole-cell antigen group, used at a ratio of tumor cells to bone marrow cells = 1:1), and free IL2+Ag group. After incubation for 48 hours, cells were collected, centrifuged at 300g for 5 min, and dead cells were labeled using the ZombieAqua™ Fixable Viability Kit. Cells were then diluted with antibody dilution buffers (FITC anti-mouse CD3 antibody, FITC anti-mouse CD19 antibody, FITC anti-mouse CD49B antibody, FITC anti-mouse LY6G antibody, FITC anti-mouse TER119 antibody, and BrilliantViolet 605). TM anti-mouse XCR1 antibody, PE / Cyanine7 anti-mouse CD172A antibody, PerCP / Cy5.5 anti-mouse CD11C antibody, PE anti-mouse CD80 antibody, BrilliantViolet 785 TM anti-mouse CD86 antibody, Brilliant Violet650 TM anti-mouse F480antibody, Brilliant Violet 42 TMThe anti-mouse IA / IE antibody was incubated at 4°C for 30 min. After incubation, the proportion of cDC1 cells in each group of culture medium was measured by flow cytometry to evaluate the effect of the effective active ingredient of the hydrogel tumor whole cell vaccine of the present invention on the number of cDC1 cells.

[0088] The results are as follows Figure 8 As shown, compared to the PBS group, both the IL2 group and the Ag group effectively increased the proportion of cDC1 cells in bone marrow cells. More importantly, when IL2 and tumor whole-cell antigen are used in combination, they exert a synergistic effect, significantly increasing the proportion of cDC1 cells in bone marrow cells. These results indicate that the effective active ingredients of the hydrogel tumor whole-cell vaccine provided by this invention can enhance antigen presentation capabilities and activate the body's immune response.

[0089] 2. The hydrogel tumor whole-cell vaccine of this invention can significantly enhance CD8. + The function of T

[0090] After obtaining single-cell suspensions of spleen from C57BL / 6 mice, they were inoculated at 1×10⁻⁶ cells per cell. 6 Cells were seeded at a density of [number] cells / mL in 24-well plates, specifically divided into PBS, IL2, Ag, and IL2+Ag groups. After 48 hours of culture, cells were collected, centrifuged at 300g for 5 minutes, and dead cells were labeled using the Zombie Aqua™ Fixable Viability Kit. Cells were then diluted with antibody dilution buffer (Alexa). 700anti-mouse CD45antibody, APC / Cyanine7 anti-mouse CD3 antibody, Brilliant Violet 650 TM anti-mouse CD4, PE / Cyanine7 anti-mouse CD8a antibody, Brilliant Violet 605 TM Anti-mouse IFN-γ antibody was incubated at 4°C for 30 min; after incubation, the levels of Th1 and CD8 in each culture medium were measured by flow cytometry. + and CD8 + IFN-γ + The proportion of T cells was used to evaluate the effectiveness of the active ingredient in the hydrogel tumor whole-cell vaccine of this invention on Th1 and CD8 cells. + The impact on T cell function.

[0091] The results are as follows Figure 9 As shown, compared to the PBS group, both the IL2 group and the Ag group can effectively enhance Th1 and CD8 levels. +and CD8 + IFN-γ + The proportion of T cells; more importantly, the two can exert a synergistic effect, significantly increasing Th1 and CD8 levels. + and CD8 + IFN-γ + The proportion of T cells. These results indicate that the effective active ingredient of the hydrogel tumor whole-cell vaccine provided by this invention can enhance CD8. + T cells enhance the function of immune killing.

[0092] 3. Evaluation of therapeutic effect on tumor growth in mice and analysis of the immune microenvironment

[0093] Based on the aforementioned optimized method for preparing hydrogel tumor whole-cell vaccines, antigens were prepared using B16F10 mouse melanoma cells, and simultaneously loaded with the cytokine IL2 to obtain a hydrogel tumor whole-cell vaccine containing IL2 / antigen (denoted as Gel-IL2+Ag). B16F10 cells were subcutaneously injected into the right hind limb of C57BL / 6 mice at a concentration of 5 × 10⁶ cells. 6 A mouse melanoma model was established using 6 mice per group, with day 0. C57BL / 6 mice underwent bilateral intramedullary injection of Gel-IL2+Ag in the tibia on days 2, 7, and 12. A subcutaneous injection group of Gel-IL2+Ag (indicated in the figure) was also included. PBS, free IL2+Ag, and blank gel were used as controls. Each group was replicated (6 mice per group). Tumor growth and body weight were recorded, and survival was monitored. Simultaneously, a mouse melanoma model was established, and tibias from each group were collected, single-cell suspensions were prepared, and staining analysis was performed on cDC1 cells.

[0094] The results are as follows Figure 10 As shown, compared with the PBS group, free IL2+Ag group, blank gel group, and subcutaneous injection group, the intraosseous administration group of Gel-IL2+Ag showed the best inhibitory effect on mouse tumor growth, indicating that the hydrogel whole-cell tumor vaccine prepared in this invention can effectively inhibit tumor growth after intraosseous administration. Meanwhile, as... Figure 11 As shown, the proportion of cDC1 cells in the tumor tissue of tumor-bearing mice in the Gel-IL2+Ag group was higher than that in the subcutaneous injection group. Based on the clinical treatment of bone tumor patients, one method involves removing tumor-containing bone tissue, freezing it in liquid nitrogen, and then re-entering it into the patient. It is hypothesized that hydrogel whole-cell tumor vaccines could be used for postoperative intraosseous injection in bone tumor patients as an effective adjuvant therapy.

[0095] 4. Evaluation of the efficacy of hydrogel-based whole-cell tumor vaccine in preventing tumor metastasis in mice

[0096] Based on the aforementioned optimized preparation method for the hydrogel tumor whole-cell vaccine, antigens were prepared using 4T1 mouse breast cancer cells, and simultaneously loaded with the cytokine IL2 to obtain a hydrogel tumor whole-cell vaccine containing IL2 / antigen (denoted as Gel-IL2+Ag). Balb / c mice were injected bilaterally into the tibial bone marrow cavity with Gel-IL2+Ag (Gel-IL2+Ag is shown in the figure) on days 0, 5, and 10, with PBS and subcutaneous injection of Gel-IL2+Ag (Gel-IL2+Ag sc) groups serving as controls. Each group was replicated in groups of 6 mice. On day 15, 4T1 cells (3 × 10⁶ cells) were injected into the tail vein of Balb / c mice. 5 A mouse model of breast cancer metastasis was established using one mouse per mouse. The survival status of the mice was recorded.

[0097] The results are as follows Figure 12 As shown, compared with the PBS group and the Gel-IL2+Ag subcutaneous injection group, intraosseous injection of Gel-IL2+Ag significantly prolonged the survival of mice, indicating that the hydrogel tumor whole-cell vaccine prepared in this invention can effectively inhibit tumor metastasis after intraosseous administration.

[0098] In summary, this invention improves the preparation method of tumor whole-cell antigen, which not only shortens the preparation cycle but also enhances its immunogenicity. Furthermore, the prepared tumor whole-cell antigen and IL2 (recombinant human interleukin-2 protein) are co-loaded into calcium alginate hydrogel to prepare the hydrogel tumor whole-cell vaccine described in this invention. Experiments have shown that the tumor whole-cell antigen and IL2 loaded in the hydrogel can exert an unexpected synergistic immune activation effect, thereby significantly inhibiting tumor recurrence or metastasis.

[0099] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for preparing a hydrogel tumor whole-cell vaccine, characterized in that, Includes the following steps: S1, collect tumor cells, which are then treated with hypochlorous acid and inactivated to obtain an immunogenic dead tumor whole-cell antigen solution, wherein the tumor whole-cell antigen solution contains 1×10 7 -5×10 7 One tumor cell; S2, CaCl2 solution is added dropwise to sodium alginate aqueous solution under stirring, and calcium alginate hydrogel is obtained after stirring evenly. S3, mix the calcium alginate hydrogel, tumor whole cell antigen solution and IL2 evenly to obtain the hydrogel tumor whole cell vaccine; The ratio of the calcium alginate hydrogel to the tumor whole-cell antigen solution is 1g:1mL, and the final concentration of IL2 in the hydrogel tumor whole-cell vaccine is 0.01-100μg / mL.

2. The method for preparing the hydrogel tumor whole-cell vaccine as described in claim 1, characterized in that, The concentration of hypochlorous acid is 50–500 μM, and the treatment time is 0–2 h.

3. The method for preparing the hydrogel tumor whole-cell vaccine as described in claim 1, characterized in that, The specific steps of the inactivation treatment are as follows: repeated freezing and thawing twice with liquid nitrogen.

4. The method for preparing the hydrogel tumor whole-cell vaccine as described in claim 1, characterized in that, The volume ratio of the CaCl2 solution to the sodium alginate aqueous solution is 1:

1.

5. The method for preparing the hydrogel tumor whole-cell vaccine as described in claim 4, characterized in that, The sodium alginate in the aqueous solution has a molecular weight of 1kDa to 1000kDa and a concentration of 10mg / mL to 30mg / mL.

6. The method for preparing the hydrogel tumor whole-cell vaccine as described in claim 4, characterized in that, The concentration of the CaCl2 solution is 1 mg / mL to 3 mg / mL.

7. The method for preparing the hydrogel tumor whole-cell vaccine as described in claim 1, characterized in that, The tumor cells include at least one of breast cancer cells, colorectal cancer cells, melanoma cells, and bone cancer cells.

8. A hydrogel tumor whole-cell vaccine, characterized in that, The hydrogel tumor whole-cell vaccine is prepared by the method according to any one of claims 1-7.

9. The application of the hydrogel tumor whole-cell vaccine according to claim 8 in the preparation of antitumor drugs, characterized in that, The tumors include primary bone tumors or bone metastases.

10. The application as described in claim 9, characterized in that, The hydrogel tumor whole-cell vaccine can be administered via intramedullary injection.

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