An injectable hydrogel loaded with microcystis and its preparation method and application

Abstract

The present invention discloses an injectable hydrogel loaded with microcystis and a preparation method and application thereof, belonging to the technical field of medical materials. The preparation method comprises the following steps: S1, dispersing the microcystis in a dopamine solution, stirring in the dark at room temperature for 2 to 12 hours, centrifuging and washing the reaction solution for multiple times to obtain polydopamine-modified microcystis, which is retained for later use; S2, dispersing a thermosensitive material in sterile water to obtain a thermosensitive hydrogel solution with a mass fraction of 25% to 35%; S3, dispersing the polydopamine-modified microcystis in the thermosensitive hydrogel solution obtained in step S2, stirring the mixed solution overnight, and obtaining the injectable hydrogel loaded with microcystis. The hydrogel prepared by the method of the present invention has the ability to cause immunogenic death of tumor cells alone and capture antigens released by tumors. While resisting tumors, it adsorbs the generated antigens to form an "antigen library" to induce a long-term and sustained anti-tumor immune response.

Description

Technical Field

[0001] The invention belongs to the technical field of medical materials, and in particular relates to an injectable hydrogel loaded with microcystis, a preparation method and an application thereof. Background Art

[0002] Compared with traditional therapies, cancer immunotherapy is a promising treatment strategy that can activate the patient's own immune system to fight cancer or destroy cancer cells. As one of the main strategies for immunotherapy, tumor vaccines have great potential in inducing antigen-specific immune responses and long-term immune memory. However, low tumor vaccine immunogenicity and tumor heterogeneity are two major challenges, which may be due to insufficient antigens or different tumor characteristics in different patients or even at different development stages of the same tumor. Both lead to insufficient activation of antigen presenting cells (APCs) and low anti-tumor immune responses, resulting in preclinical tumor recurrence and metastasis.

[0003] In order to improve antigenic immunogenicity, many strategies have been developed. For example, immune adjuvants are often used to improve the maturity of APCs; various antigen delivery systems based on nanoparticles have been developed to improve the efficiency of antigen delivery; in addition, some specific ligands have been used to give antigen delivery vectors APC targeting capabilities, such as mannose ligands, major histocompatibility complex class II targeting peptides, and DEC205 antibodies. Although these strategies have improved antigen presentation and anti-tumor immune responses to a certain extent, the problem of poor immunogenicity has not been fundamentally solved, and the preparation process is relatively complicated.

[0004] Recent studies suggest that in situ exposure of large amounts of tumor antigens may be a promising approach. Certain chemotherapy, radiotherapy, photodynamic therapy, and photothermal therapy (PTT) can induce immunogenic death of tumor cells, release tumor-associated protein antigens, etc. These autologous tumor antigens can overcome the heterogeneity problem of tumor vaccines, but it is difficult to observe the body's anti-tumor immune response in clinical or research. This may be because these antigens produced by treatment are easily cleared by macrophages and the like as foreign bodies in the body, so they exist in the body for a short time and cannot induce a sustained or strong anti-tumor immune response. In order to prolong the retention time of antigens at the tumor site, researchers have developed some nanocarriers functionalized with protein adsorption groups, such as amino and maleimide, to capture antigens. However, due to their limited residence time, they still cannot achieve effective immune effects alone. Therefore, there is an urgent need in this field to develop a biomaterial with a simple preparation method and an efficient and lasting anti-tumor immune response effect.

[0005] Microcystis aeruginosa is an algae widely found in natural water bodies, and its sources are abundant. Some studies have reported that the extracellular acidic polysaccharides of Microcystis aeruginosa have a significant inhibitory effect on the growth of human cervical cancer cells and rat myeloma cells; some studies have also reported that microcystin can strongly inhibit the activity of protein phosphatases and is a strong liver tumor promoter. The effect of Microcystis aeruginosa on tumor immunotherapy is unknown. After reviewing relevant domestic and foreign literature, no research reports on the application of Microcystis in tumor immunotherapy have been found. Summary of the invention

[0006] The present invention aims to solve the problems of short antigen capture residence time, complex preparation method, poor immune response effect and the like in the prior art biomaterials used for immunotherapy, and provides an injectable hydrogel loaded with Microcystis and its preparation method and application. The injectable hydrogel loaded with Microcystis prepared by the method of the present invention has the ability to cause immunogenic death of tumor cells and capture antigens released by tumors alone. While resisting tumors, it adsorbs the generated antigens to form an "antigen library", which can induce long-term and sustained anti-tumor immune response. In addition, the preparation method of the present invention is simple and low-cost, which is conducive to clinical promotion and application.

[0007] The present invention first provides a method for preparing an injectable hydrogel loaded with Microcystis, comprising the following steps:

[0008] S1, preparing polydopamine-modified Microcystis: dispersing Microcystis in a dopamine solution, stirring for 2 to 12 hours at room temperature in the dark, and centrifuging and washing the reaction solution multiple times to obtain polydopamine-modified Microcystis, which is retained for later use;

[0009] S2, preparing a thermosensitive hydrogel solution: dispersing the thermosensitive material in sterile water to obtain a thermosensitive hydrogel solution with a mass fraction of 25% to 35%;

[0010] S3, dispersing the polydopamine-modified Microcystis obtained in step S1 in the thermosensitive hydrogel solution prepared in step S2, stirring the mixed solution overnight to obtain the injectable hydrogel loaded with Microcystis.

[0011] The microcystis includes Microcystis aeruginosa.

[0012] Preferably, in the mixed solution described in step S3, the concentration of Microcystis is 1×10 4 ~1×10 7 Pieces / mL.

[0013] Preferably, in step S1, the concentration of the dopamine solution is 7-10 mM.

[0014] Preferably, in step S1, after dispersing Microcystis in the dopamine solution, the pH of the solution is adjusted to 8 to 8.5.

[0015] Preferably, the temperature-sensitive hydrogel solution comprises any one or more combinations of Pluronic F-127 aqueous solution, chitosan-sodium glycerophosphate hydrogel solution, thiol chitosan hydrogel solution and chitosan-collagen hydrogel solution.

[0016] Another aspect of the present invention further provides an injectable hydrogel loaded with Microcystis prepared according to any of the preparation methods described above.

[0017] Another aspect of the present invention also provides the use of the aforementioned injectable hydrogel loaded with Microcystis in the preparation of tumor vaccine preparations and / or tumor immunotherapy drugs.

[0018] Preferably, the tumor comprises breast cancer.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] (1) The injectable hydrogel loaded with Microcystis described in the present invention can be used alone or in combination with other means such as chemotherapy, radiotherapy, photothermal therapy, etc. When used alone, the Microcystis in the hydrogel can cause the immunogenic death of tumor cells and release antigens. The catechol group contained in polydopamine has strong adhesion characteristics. Therefore, under the stimulation of Microcystis, tumor cells die and produce a large number of autologous tumor-derived protein antigens, which are then captured by polydopamine and hydrogel to form an "antigen library" that can induce long-term and sustained anti-tumor immune response. When used in combination with other means, more autologous tumor antigens will be produced, further improving the anti-tumor immune effect.

[0021] (2) The injectable hydrogel loaded with Microcystis described in the present invention captures autologous tumor antigens at the tumor site, thus overcoming the problem of tumor heterogeneity in tumor vaccines.

[0022] (3) The preparation method of the injectable hydrogel loaded with Microcystis provided by the present invention is simple, the raw materials are easily available and the cost is low, which is conducive to clinical promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The performance of the MA@PDA-F127 hydrogel prepared in Example 1 is characterized, wherein:

[0024] A represents the transmission electron microscopy (TEM) image of MA@PDA-F127 hydrogel;

[0025] B represents the photothermal effect diagram of MA@PDA-F127 hydrogel under near-infrared light;

[0026] C represents the temperature change of MA@PDA-F127 hydrogel under near-infrared light as a function of illumination time.

[0027] Figure 2 Flow cytometry was used to detect the double positive ratios of CD80 and CD86 and the positive ratio of MHC II in mouse dendritic cells in four groups: PBS control group (PBS), hydrogel control group (hydrogel), Microcystis hydrogel group (MA@PDA-F127), and Microcystis hydrogel illumination group (MA@PDA-F127+NIR), among which:

[0028] A is the flow cytometric graph of dendritic cells in each group;

[0029] B is the comparison of the double positive proportion of CD80 and CD86 in dendritic cells of each group;

[0030] C is the comparison of the MHC II positive proportion of dendritic cells in each group.

[0031] Figure 3 Flow cytometry was used to detect the CFSE positive ratio and mean fluorescence intensity (MFI) of mouse dendritic cells in four groups: PBS control group (PBS), hydrogel control group (hydrogel), microcystis hydrogel group (MA@PDA-F127), and microcystis hydrogel illumination group (MA@PDA-F127+NIR), where:

[0032] A is the flow cytometric graph of dendritic cells in each group;

[0033] B is the comparison of CFSE mean fluorescence intensity (MFI) of dendritic cells in each group;

[0034] C is the comparison of CFSE positive proportion of dendritic cells in each group.

[0035] Figure 4 PCR was used to detect the gene expression of IL-1β, IL-6, and TNF-α in mouse dendritic cells after co-culture with Microcystis hydrogel (hydrogel-MA) and hydrogel (hydrogel), where:

[0036] A is the expression results of il-1β mRNA in cells of each group;

[0037] B is the expression results of il-6β mRNA in cells of each group;

[0038] C shows the expression results of tnf-α mRNA in cells of each group.

[0039] Figure 5 The in vivo immunotherapy effect evaluation of breast cancer cell line subcutaneous tumor model mice treated with PBS control group (PBS), hydrogel control group (hydrogel), Microcystis hydrogel group (MA@PDA-F127), and Microcystis hydrogel illumination group (MA@PDA-F127+NIR), among which:

[0040] A is the comparison of the proportion of mature dendritic cells in the tumor-draining lymph nodes of mice in each group;

[0041] B is the gross view of the tumors of mice in each group;

[0042] C is the tumor weight curve of mice in each group;

[0043] D is the tumor growth curve of mice in each group.

[0044] Figure 6 These are the in vivo imaging and tissue (organ) fluorescence imaging images of breast cancer cell line subcutaneous tumor model mice after injection of MA@PDA-F127 hydrogel at different sites in the body, including:

[0045] A is a mouse in vivo imaging image;

[0046] B is the fluorescence imaging of tissue (organ). DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0048] Based on the fact that the existing biomaterials used for immunotherapy still have the problems of short antigen capture residence time, complex preparation method, poor immune response effect, etc., the present invention provides an injectable hydrogel loaded with Microcystis through extensive research, and also provides a preparation method of the hydrogel, comprising the following steps:

[0049] S1, preparing polydopamine-modified Microcystis: dispersing Microcystis in a dopamine solution, stirring for 2 to 12 hours at room temperature in the dark, and centrifuging and washing the reaction solution multiple times to obtain polydopamine-modified Microcystis, which is retained for later use;

[0050] S2, preparing a thermosensitive hydrogel solution: dispersing the thermosensitive material in sterile water to obtain a thermosensitive hydrogel solution with a mass fraction of 25% to 35%;

[0051] S3, dispersing the polydopamine-modified Microcystis obtained in step S1 in the thermosensitive hydrogel solution prepared in step S2, stirring the mixed solution overnight to obtain the injectable hydrogel loaded with Microcystis.

[0052] The microcystis includes Microcystis aeruginosa.

[0053] In some embodiments, in step S3, the concentration of Microcystis in the mixed solution is 1×10 4 ~1×10 7 Pieces / mL.

[0054] In some embodiments, in step S1, the concentration of the dopamine solution is 7-10 mM.

[0055] In some embodiments, in step S1, after dispersing Microcystis in the dopamine solution, the pH of the solution is adjusted to 8-8.5.

[0056] In some embodiments, the thermosensitive hydrogel solution includes any one of a Pluronic F-127 aqueous solution, a chitosan-sodium glycerophosphate hydrogel solution, a thiol chitosan hydrogel solution, and a chitosan-collagen hydrogel solution.

[0057] On the other hand, the invention also provides the use of the aforementioned injectable hydrogel loaded with Microcystis in the preparation of tumor vaccine preparations and / or tumor immunotherapy drugs.

[0058] The present invention evaluates the anti-tumor immunotherapy effect of the injectable hydrogel loaded with Microcystis through a series of in vitro and in vivo experiments. The experimental process and experimental results of the present invention are described in detail below.

[0059] The Microcystis aeruginosa used in the examples of the present invention is from Shanghai Guangyu Biotechnology Co., Ltd. (No. PCC7806); dopamine (PDA) is purchased from Sigma; Pluronic F-127 is a thermosensitive injectable hydrogel purchased from Biyuntian Biotechnology Co., Ltd. (No. ST501-100g). If the specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0060] Example 1 Preparation and characterization of an injectable hydrogel loaded with Microcystis

[0061] S1, 5 mL of Microcystis aeruginosa (concentration 1×10 5 -1×10 7 ) was dispersed in 10 mL of dopamine aqueous solution after centrifugation, the dopamine concentration was 9 mM, the solution pH was 8-8.5, and stirred for 2-12 hours in the dark at room temperature. The above solution was centrifuged and washed with water 3-5 times to fully wash away the unreacted dopamine aqueous solution to obtain polydopamine-modified Microcystis aeruginosa.

[0062] S2, Pluronic F-127 was dispersed in sterile water to prepare a 30% by mass aqueous solution of Pluronic F-127, and stirred at 4 degrees overnight in the dark.

[0063] S3, disperse the product after centrifugation in step S1 (i.e., polydopamine-modified copper-aluminum Microcystis) in 10 mL of the solution in step S2 (i.e., a 30% mass fraction Pluronic F-127 aqueous solution), and stir overnight to obtain a PDA-modified Microcystis hydrogel, named MA@PDA-F127.

[0064] like Figure 1 As shown in Figure A, the MA@PDA-F127 hydrogel prepared in this example was subjected to TEM transmission electron microscopy scanning. The results showed that the size of Microcystis aeruginosa in the hydrogel was about 5 μm. After PDA modification, the surface was smooth and had little effect on the dispersibility of the material.

[0065] like Figure 1 As shown in the BC diagram, the MA@PDA-F127 hydrogel prepared in this example was subjected to a photothermal response test. The PBS group was used as the control group, the near-infrared light irradiation wavelength was 808 nm, and the laser power was 1.5 W / cm 2 The irradiation time was 10 minutes. The results showed that short-term irradiation of hydrogel with near-infrared light could significantly increase the temperature of hydrogel, and the hydrogel produced a certain photothermal effect under near-infrared laser.

[0066] Example 2 The hydrogel of the present invention promotes the maturation and antigen uptake ability of mouse dendritic cells

[0067] The MA@PDA-F127 hydrogel prepared in Example 1 was used to demonstrate the immunotherapeutic effect of the hydrogel of the present invention through in vitro cell experiments.

[0068] (I) MA@PDA-F127 hydrogel promotes the maturation of dendritic cells

[0069] 1. Experimental methods

[0070] After obtaining the mouse breast cancer cell line 4T1, a 24-well plate was plated and treated after the density reached 80%. Grouping: blank control group (PBS), hydrogel control group (hydrogel), Microcystis hydrogel group (MA@PDA-F127) and Microcystis hydrogel illumination group (MA@PDA-F127+NIR). The corresponding materials of each group were added to the upper chamber of the transwell chamber with a volume of 600μL, and then the chamber was placed in the well plate; in the illumination group, the well plate was irradiated with infrared light after the hydrogel solidified (808nm, 1.0W / cm 2 , 5 min). The cells were placed in a 37°C incubator and cultured for 24 h.

[0071] After 24 hours of culture, mouse dendritic cells were added to the well plate, 200,000 per well, and co-culture was continued for 24 hours. After the culture was completed, the supernatant cells were collected and CD11C was detected by flow cytometry. + CD80 in cells (dendritic cells) + CD86 + and MHC II + The proportion of cells is the proportion of mature dendritic cells.

[0072] It should be noted that the hydrogel used in the hydrogel control group (hydrogel) described in the examples of the present invention is Pluronic F-127 hydrogel.

[0073] 2. Experimental results

[0074] The results are as follows Figure 2 As shown in the AC figure, compared with the PBS group and the hydrogel group, after treatment with the hydrogel loaded with Microcystis (MA@PDA-F127 group), the CD80 and CD86 double-positive ratios of mouse dendritic cells increased, and the MHC II-positive ratio increased, indicating that the MA@PDA-F127 hydrogel of the present invention can promote the maturation of dendritic cells; after combined PPT treatment (MA@PDA-F127+NIR), the mature ratio of mouse dendritic cells was further significantly improved.

[0075] (II) MA@PDA-F127 hydrogel promotes antigen uptake ability of dendritic cells

[0076] 1. Experimental methods

[0077] Obtain mouse breast cancer cell line 4T1 and stain it with CFSE dye (1μM, 37℃, 15min). 10mL PBS was added to terminate the staining. After centrifugation, the cell pellet was obtained. The cells were plated on a 24-well plate and treated after the density reached 80%. Grouping: blank control group (PBS), hydrogel control group (hydrogel), Microcystis hydrogel group (MA@PDA-F127) and Microcystis hydrogel illumination group (MA@PDA-F127+NIR). The corresponding materials of each group were added to the upper chamber of the transwell chamber with a volume of 600μL, and then the chamber was placed in the well plate; in the illumination group, the well plate was irradiated with infrared light (808nm, 1.0W / cm 2 , 5 min). The cells were placed in a 37°C incubator and cultured for 24 h.

[0078] After culturing for 24 h, mouse dendritic cells were added to the well plate, 2×10 5 After the culture, the supernatant cells were collected and CD11C + CFSE in cells (dendritic cells) + The proportion of cells is the proportion of dendritic cells that phagocytize tumor-associated antigens.

[0079] 2. Experimental results

[0080] The results are as follows Figure 3As shown in the AC figure, compared with the PBS group and the hydrogel group, after treatment with the hydrogel loaded with Microcystis (MA@PDA-F127 group), the CFSE-positive ratio of mouse dendritic cells increased and the mean fluorescence intensity (MFI) was enhanced, indicating that the MA@PDA-F127 hydrogel of the present invention can promote the delivery of tumor cell antigens to dendritic cells; after combined PTT treatment (MA@PDA-F127+NIR), the CFSE-positive ratio in mouse dendritic cells increased further significantly.

[0081] The above results indicate that the MA@PDA-F127 hydrogel of the present invention can promote the maturation of dendritic cells and the antigen uptake ability of tumor cells.

[0082] In addition, this example also uses PCR to detect the IL-1β, IL-6, and TNF-α gene expression levels of mouse dendritic cells after co-culture with Microcystis hydrogel (hydrogel-MA) and hydrogel (hydrogel) to evaluate the effect of Microcystis on the expression of inflammatory factors in dendritic cells. The results are as follows Figure 4 As shown in Figure AC: After mouse dendritic cells were co-cultured with Microcystis hydrogel, the mRNA expression levels of inflammatory factors il-1β, il-6, and tnf-α were significantly upregulated, indicating that Microcystis can stimulate the inflammatory polarization of dendritic cells and thus exert anti-tumor immune effects.

[0083] Example 3 In vivo therapeutic effect of the MA@PDA-F127 hydrogel of the present invention

[0084] The in vivo distribution and degradation of the MA@PDA-F127 hydrogel of the present invention, as well as the immunotherapy effect, were evaluated using a mouse breast cancer cell line 4T1 subcutaneous tumor model.

[0085] The mice in this example were obtained from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The research on experimental animals was approved by the Experimental Animal Ethics Committee of the Ninth People's Hospital Affiliated to Shanghai Jiaotong University School of Medicine. All animals were raised in the Animal Experiment Center of the Ninth People's Hospital Affiliated to Shanghai Jiaotong University School of Medicine, and all experimental procedures were carried out in accordance with the policies and ethics of animal research.

[0086] (A) MA@PDA-F127 hydrogel inhibits tumor growth in vivo

[0087] 1. Experimental methods

[0088] 10 6The breast cancer cell line 4T1 was injected subcutaneously into the right back of 6-week-old balb / c female mice to establish a subcutaneous tumor model of mouse breast cancer cell line 4T1. After 7 days, the mice were evenly divided into 4 groups: blank control group (PBS), hydrogel control group (hydrogel), Microcystis hydrogel group (MA@PDA-F127) and Microcystis hydrogel illumination group (MA@PDA-F127+NIR).

[0089] Treatment method: The blank control group was injected with 100 μL PBS, the hydrogel group was injected with 100 μL Pluronic F-127 hydrogel, and the Microcystis hydrogel group was injected with 100 μL MA@PDA-F127 sample. The illumination group was supplemented with tumor area illumination (808 nm, 1.5 W / cm 2 , 8min).

[0090] Tumor volume and weight were observed and measured every 2 days, and samples were collected after 1 week. Single cell suspension of mouse draining lymph nodes was prepared, and CD80 was detected by flow cytometry. + CD86 + CD11C + The proportion of cells, that is, the proportion of mature dendritic cells in the lymph nodes, is used to characterize the immune function of the tumor and lymph node areas of mice.

[0091] 2. Experimental results

[0092] like Figure 5 As shown in the figure, compared with the PBS group and the hydrogel group, the proportion of mature dendritic cells in the tumor-draining lymph nodes of mice was significantly increased after treatment with the hydrogel loaded with Microcystis (MA@PDA-F127 group) ( Figure 5 A), the volume and weight of mouse tumors were significantly reduced ( Figure 5 BD), and combined with PTT treatment showed stronger tumor inhibitory effect.

[0093] (II) Distribution and degradation of MA@PDA-F127 hydrogel in vivo

[0094] After the MA@PDA-F127 material was injected into the mouse tumor, in vivo imaging was performed to detect the distribution and degradation of the material of the present invention.

[0095] 1. Experimental methods

[0096] 10 6 The breast cancer cell line 4T1 was injected into the right back of 6-week-old female balb / c mice to establish a subcutaneous tumor model of mouse breast cancer cell line 4T1. After the tumor was formed in the mice, the tumor volume reached 100-150mm. 3The experiment was then conducted: After the mice were anesthetized, 20 μL of MA@PDA-F127 material was injected into the tumor. The fluorescence distribution was detected at 0, 10 min, 1 h, 24 h, and 48 h (excitation light: 555 nm, emission light: 600 nm). After the 48 h experiment, the mice were killed and the fluorescence images of the heart, liver, spleen, lung, kidney, and tumor were taken.

[0097] 2. Experimental results

[0098] like Figure 6 As shown, the MA@PDA-F127 material of the present invention is confined to the tumor and still maintains a certain content after 48 hours. The material maintenance time is ideal, providing the possibility for the "antigen library" to release antigens for a long time and continuously to induce anti-tumor immune response ( Figure 6 A); In addition, the MA@PDA-F127 material of the present invention is only distributed in the tumor site, and is not distributed in organs such as the heart and liver, which reflects the biosafety of the material of the present invention ( Figure 6 B).

[0099] In summary, the injectable hydrogel loaded with microcystis prepared by the method of the present invention has the ability to cause immunogenic death of tumor cells and capture antigens released by tumors alone. While resisting tumors, it adsorbs the generated antigens to form an "antigen library" and can induce a long-term and sustained anti-tumor immune response. In addition, the preparation method of the present invention is simple and low in cost, which is conducive to clinical promotion and application.

[0100] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A method for preparing an injectable hydrogel loaded with Microcystis, characterized in that: The following steps are involved: S1, preparing polydopamine-modified Microcystis: dispersing Microcystis in a dopamine solution, stirring for 2 to 12 hours at room temperature in the dark, and centrifuging and washing the reaction solution multiple times to obtain polydopamine-modified Microcystis, which is retained for later use; S2, preparing a thermosensitive hydrogel solution: dispersing the thermosensitive material in sterile water to obtain a thermosensitive hydrogel solution with a mass fraction of 25% to 35%; S3, dispersing the polydopamine-modified Microcystis obtained in step S1 in the thermosensitive hydrogel solution obtained in step S2, and stirring the mixed solution overnight to obtain the injectable hydrogel loaded with Microcystis; Wherein, the Microcystis includes Microcystis aeruginosa; The temperature-sensitive hydrogel solution includes any one or more combinations of Pluronic F-127 hydrogel solution, chitosan-sodium glycerophosphate hydrogel solution, thiol chitosan hydrogel solution and chitosan-collagen hydrogel solution.

2. The method for preparing the injectable hydrogel loaded with Microcystis according to claim 1, characterized in that: In the mixed solution described in step S3, the concentration of Microcystis is 1×10 4 ~1×10 7 Pieces / mL.

3. The method for preparing the injectable hydrogel loaded with Microcystis according to claim 1, characterized in that: In step S1, the concentration of the dopamine solution is 7-10 mM.

4. The method for preparing the injectable hydrogel loaded with Microcystis according to claim 1, characterized in that: In step S1, after dispersing Microcystis in the dopamine solution, the pH of the solution is adjusted to 8 to 8.

5.

5. An injectable hydrogel loaded with Microcystis prepared according to the preparation method of any one of claims 1 to 4.

6. Use of the injectable hydrogel loaded with Microcystis as claimed in claim 5 in the preparation of tumor vaccine preparations and / or tumor immunotherapy drugs, characterized in that: The tumor is breast cancer.

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