Nanosheet-based tumor antigen complex tumor vaccine and preparation method thereof

Abstract

The invention belongs to the technical field of nano materials, and particularly relates to a nanosheet-based tumor antigen complex tumor vaccine and a preparation method thereof, the preparation method comprises the following steps: synthesizing nanosheets and preparing the vaccine, after a part of tumor tissues are obtained, co-culturing the nanosheets and tumor cells, and enabling the tumor cells to fully take in the nanosheets. Near-infrared laser is irradiated, and through the photocatalytic effect of the nanosheets, tumor cells generate a strong immune effect, break and release a large number of tumor antigens. And due to the physical properties of multilayer stacking and roughness of the nanosheets, tumor antigens can be adsorbed on the surfaces of the nanosheets, so that the antigen uptake of immune cells is improved, and the immune escape effect is reduced. According to the method provided by the invention, the preparation difficulty of the tumor vaccine is greatly reduced, the uptake rate of the tumor antigen is improved, and a simple and effective preparation is provided for preparing the convenient and safe tumor vaccine inoculation.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials, and in particular relates to a nanosheet-based tumor antigen complex tumor vaccine and a preparation method thereof. Background Art

[0002] Malignant tumors are the main cause of death in patients. Efficient tumor treatment and delayed tumor recurrence are crucial to improving global life expectancy. Immunotherapy (including immune checkpoint inhibitors, adoptive cell therapy, cytokines and cancer vaccines) has become the trend and mainstream of tumor treatment. In particular, tumor vaccines have inherent advantages in preventing and treating tumor recurrence and tumor metastasis, and have the characteristics of preventing tumor recurrence and forming long-term immune memory. Slightly different from traditional vaccines, tumor vaccines stimulate specific cellular immune responses to prevent tumor growth and ultimately eradicate tumor cells. It is based on the principle that tumor cells themselves contain multiple tumor-associated antigens. Under normal circumstances, the human immune system may not be able to effectively recognize tumor cells. Tumor vaccines process tumor cells, such as inactivating tumor cells, extracting tumor cell lysates, or using genetic engineering technology to transform tumor cell-related components. These processed tumor-associated antigens can activate the body's immune system, including the activation of immune cells such as T cells and B cells, enabling the immune system to recognize and kill tumor cells, whether primary tumors or potential metastatic tumors, opening up a new direction for tumor treatment. At present, tumor vaccines have become a research hotspot in the field of tumor treatment.

[0003] However, existing tumor vaccines have the following difficulties: (1) Preparation difficulties: The preparation process of tumor vaccines is complicated, requiring the patient's tumor tissue to be obtained, tumor antigens to be extracted, and then processed and prepared into vaccines. This process is not only time-consuming and labor-intensive, but may also be limited by the quality and quantity of tumor tissue. The reason is that the acquisition of tumor tissue requires surgery or biopsy, which has certain risks and burdens for patients, and not all tumor tissues are suitable for vaccine preparation. (2) Low stability: Tumor antigens in tumor vaccines may be unstable and easily degraded or inactivated, thus affecting the effectiveness of the vaccine. The reason is that tumor antigens are usually proteins or peptides, which are easily affected by factors such as temperature and pH in the in vitro environment and undergo denaturation or degradation. (3) Insufficient immunogenicity: Many tumor-related antigens are self-antigens, and the human immune system is tolerant to them. This results in the immune response induced by tumor vaccines being insufficient and making it difficult to effectively eliminate tumor cells. Immune cells activated by vaccines alone may not be able to proliferate and play a killing role in large quantities and continuously, resulting in the difficulty of the immune response to fight tumors for a long time. Therefore, there is an urgent need to develop a tumor vaccine with a simple preparation method, good stability and improved immunogenicity. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a nanosheet-based tumor antigen complex tumor vaccine and a preparation method thereof.

[0005] The purpose of the present invention is achieved through the following technical solution: a method for preparing a tumor antigen complex tumor vaccine based on nanosheets, which comprises the following steps:

[0006] S1. Synthesis of nanosheets: dilute hydrochloric acid is added dropwise to a potassium hydroxide solution of meso-tetra(4-carboxyphenyl)porphine, the reaction vessel is sealed, and the reaction is heated and stirred for 70 to 74 hours. The reaction product is a nanosheet;

[0007] S2. Prepare the vaccine: add the nanosheets synthesized in step S1 to the single-cell suspension of tumor tissue, and incubate them in a 37°C incubator for 2.5 to 3.5 hours. After the incubation, irradiate the cells with a 660nm near-infrared laser for 15 to 25 minutes and then place them in a 37°C incubator for 1.5 to 2.5 hours. Filter to remove large flocs, and the resulting liquid is a nanosheet-based tumor antigen complex tumor vaccine.

[0008] As a preferred technical solution, the concentration of the potassium hydroxide solution in step S1 is 0.3-0.7 mol / L, the mass volume ratio of meso-tetrakis(4-carboxyphenyl)porphine to the potassium hydroxide solution in meso-tetrakis(4-carboxyphenyl)porphine is 0.04-0.06 g:1 ml; and the concentration of the dilute hydrochloric acid is 0.08-0.12 mol / L.

[0009] As a preferred technical solution, the volume ratio of the potassium hydroxide solution of meso-tetrakis(4-carboxyphenyl)porphine to dilute hydrochloric acid is 1:4-5.

[0010] As a preferred technical solution, the heating temperature in step S1 is 75-85° C., and the stirring rate is 1100-1300 rpm / min.

[0011] As a preferred technical solution, after the reactants in step S1 are heated and stirred for reaction, the liquid in the container is cooled to room temperature and centrifuged at a speed of 5500-6500 rpm / min for 13-18 minutes. The obtained precipitate is washed with double distilled water, the precipitate is resuspended, and then centrifuged at a speed of 280-330 rpm / min for 4-6 minutes to remove the precipitate. The liquid is placed in a rotary evaporator at a temperature of 55-65°C and rotary evaporated until completely dry, and the obtained powder is a nanosheet powder.

[0012] As a preferred technical solution, the concentration of the single cell suspension in step S2 is 1×10 6 ~1×10 8 After adding the nanosheets into the single cell suspension, the final concentration of the nanosheets is 90-110ug / ml.

[0013] As a preferred technical solution, the power of the near-infrared laser in step S2 is 0.4-0.6w / cm 2 .

[0014] As a preferred technical solution, the tumor cells are breast cancer cells.

[0015] The nanosheet-based tumor antigen complex tumor vaccine is prepared by the above method.

[0016] The present invention has the following advantages: the present invention designs a tumor antigen complex tumor vaccine which uses nanosheets as immune adjuvants and can be simply prepared, and applies the complex to the immunotherapy and prevention of tumors. After obtaining part of the tumor tissue, the nanosheets are co-cultured with tumor cells, so that the tumor cells fully absorb the nanosheets. Then, the near-infrared laser is irradiated, and the tumor cells produce a strong immune effect through the photocatalytic effect of the nanosheets, and break and release a large amount of tumor antigens. Because of the multi-layer stacking and rough physical properties of the nanosheets themselves, tumor antigens will be adsorbed on the surface of the nanosheets, which improves the uptake of antigens by immune cells and reduces the immune escape effect. The nanosheets are self-assembled and stacked by meso-tetra(4-carboxyphenyl)porphine monomers in an aqueous phase. In the preparation process of the vaccine, the nanosheets not only use their excellent photocatalytic effect to break tumor cells and release a large amount of antigens, but also do not need to be separated, and continue to exist as immune adjuvants in the tumor vaccine. The method disclosed by the present invention greatly reduces the difficulty of preparing tumor vaccines and improves the uptake rate of tumor antigens, providing a simple and effective preparation for convenient and safe tumor vaccination. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 3 is a characterization diagram of the structure of the nanosheet synthesized by the present invention; wherein, Figure a is a diagram of the nanosheet showing a stacked fan-shaped sheet structure under a scanning electron microscope, Figure b is the nanosheet morphology under a transmission electron microscope, Figure c is the lattice stripes of the nanosheet under a high-resolution transmission electron microscope, Figure d is a nanosheet particle size diagram, Figure e is a potential diagram of the nanosheet, Figure f is a nanosheet fluorescence spectrum, Figure g is a nanosheet ultraviolet absorption spectrum and a ultraviolet absorption spectrum of its synthesized monomer, Figure h is a powdered nanosheet after synthesis, and Figure i is a prepared nanosheet tumor antigen complex tumor vaccine.

[0018] Figure 2 This is the effect of the nanosheet-based tumor antigen complex tumor vaccine of the present invention on a mouse model; in the figure, a is the mouse vaccination and subsequent evaluation process, b is the size of tumor growth in different groups of mice after inoculation with 4T1 tumor cells, c is the weight of different groups of mice, and d is a photo of tumor growth in different groups of mice 20 days after inoculation with tumor cells. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with the accompanying drawings and examples. The protection scope of the present invention is not limited to the following: Example 1: A method for preparing a tumor antigen complex tumor vaccine based on nanosheets, which comprises the following steps:

[0020] S1. Synthesis of nanosheets: Dilute hydrochloric acid was added dropwise to a potassium hydroxide solution of meso-tetra(4-carboxyphenyl)porphine, the reaction vessel was sealed, heated to 75°C, stirred at 1100 rpm / min for 74 hours, the liquid in the container was cooled to room temperature, centrifuged at a speed of 5500 rpm / min for 13 minutes, the obtained precipitate was washed with double distilled water, the precipitate was resuspended, and then centrifuged at a speed of 280 rpm / min for 4 minutes, the precipitate was removed, and the liquid was placed in a rotary evaporator at a temperature of 55°C and rotary evaporated until completely dry, and the obtained powder was a nanosheet powder;

[0021] Wherein, the concentration of the potassium hydroxide solution is 0.3 mol / L, the mass volume ratio of meso-tetra(4-carboxyphenyl)porphine to the potassium hydroxide solution in meso-tetra(4-carboxyphenyl)porphine is 0.04 g:1 ml; the concentration of the dilute hydrochloric acid is 0.08 mol / L; the volume ratio of the meso-tetra(4-carboxyphenyl)porphine potassium hydroxide solution to the dilute hydrochloric acid is 1:4;

[0022] S2. Vaccine preparation: Breast cancer tissue was taken and prepared into a concentration of 1×10 6 The suspension was prepared into a single cell suspension of 100 μg / ml. The nanosheets synthesized in step S1 were added to the suspension to make the final concentration of the nanosheets 90 μg / ml. The suspension was incubated in a 37°C incubator for 3.5 h. After the incubation, the power was 0.4 W / cm 2 , 660nm near-infrared laser irradiated the cells for 25 minutes, then placed in an incubator at 37°C for 2.5 hours, filtered to remove large flocs, and the resulting liquid was a nanosheet-based tumor antigen complex tumor vaccine.

[0023] Example 2: A method for preparing a tumor antigen complex tumor vaccine based on nanosheets, comprising the following steps:

[0024] S1. Synthesis of nanosheets: Dilute hydrochloric acid was added dropwise to a potassium hydroxide solution of meso-tetra(4-carboxyphenyl)porphine, the reaction vessel was sealed, heated to 85°C, stirred at 1300 rpm / min for 70 hours, the liquid in the container was cooled to room temperature, centrifuged at a speed of 6500 rpm / min for 18 minutes, the obtained precipitate was washed with double distilled water, the precipitate was resuspended, and then centrifuged at a speed of 330 rpm / min for 6 minutes, the precipitate was removed, and the liquid was placed in a rotary evaporator at a temperature of 65°C and rotary evaporated until completely dry, and the obtained powder was a nanosheet powder;

[0025] Wherein, the concentration of the potassium hydroxide solution is 0.7 mol / L, the mass volume ratio of meso-tetra(4-carboxyphenyl)porphine to the potassium hydroxide solution in meso-tetra(4-carboxyphenyl)porphine is 0.06 g:1 ml; the concentration of the dilute hydrochloric acid is 0.12 mol / L; the volume ratio of the meso-tetra(4-carboxyphenyl)porphine potassium hydroxide solution to the dilute hydrochloric acid is 1:5;

[0026] S2. Vaccine preparation: Breast cancer tissue was taken and prepared into a concentration of 1×10 8 The suspension was prepared into a single cell suspension of 10 μg / ml. The nanosheets synthesized in step S1 were added to the suspension to make the final concentration of the nanosheets 110 μg / ml. The suspension was incubated in a 37°C incubator for 2.5 h. After the incubation, the power was 0.6 w / cm 2 , 660nm near-infrared laser irradiated the cells for 15 minutes, then placed in an incubator at 37°C for 1.5 hours, filtered to remove large flocs, and the resulting liquid was a nanosheet-based tumor antigen complex tumor vaccine.

[0027] Example 3: A method for preparing a tumor antigen complex tumor vaccine based on nanosheets, comprising the following steps:

[0028] S1. Synthesis of nanosheets: Dilute hydrochloric acid was added dropwise to a potassium hydroxide solution of meso-tetra(4-carboxyphenyl)porphine, the reaction vessel was sealed, heated to 80°C, stirred at 1200 rpm / min for 72 hours, the liquid in the container was cooled to room temperature, centrifuged at a speed of 6000 rpm / min for 15 minutes, the obtained precipitate was washed with double distilled water, the precipitate was resuspended, and then centrifuged at a speed of 300 rpm / min for 5 minutes, the precipitate was removed, and the liquid was placed in a rotary evaporator at a temperature of 60°C and rotary evaporated until completely dry, and the obtained powder was a nanosheet powder;

[0029] Wherein, the concentration of the potassium hydroxide solution is 0.5 mol / L, the mass volume ratio of meso-tetra(4-carboxyphenyl)porphine to the potassium hydroxide solution in meso-tetra(4-carboxyphenyl)porphine is 0.05 g:1 ml; the concentration of the dilute hydrochloric acid is 0.1 mol / L; the volume ratio of the meso-tetra(4-carboxyphenyl)porphine potassium hydroxide solution to the dilute hydrochloric acid is 1:4.5;

[0030] S2. Vaccine preparation: Breast cancer tissue was taken and prepared into a concentration of 1×10 7 The suspension was prepared into a single cell suspension of 100 μg / ml. The nanosheets synthesized in step S1 were added to the suspension to make the final concentration of the nanosheets 100 μg / ml. The suspension was incubated in a 37°C incubator for 3 h. After the incubation, the power was 0.5 w / cm 2, 660nm near-infrared laser irradiated the cells for 15 to 25 minutes, then placed in an incubator at 37°C for 1.5 to 2.5 hours, filtered to remove large flocs, and the resulting liquid was a nanosheet-based tumor antigen complex tumor vaccine.

[0031] The beneficial effects of the present invention are described below by experiments:

[0032] 1. Preparation of Nanosheet-based Tumor Antigen Complex Tumor Vaccine

[0033] 1.1 Nanosheet synthesis steps

[0034] 1) Add 10 ml of 0.5 M potassium hydroxide aqueous solution into a flask, heat it in an oil bath at 80°C for 20 min, then add 0.5 g of meso-tetrakis(4-carboxyphenyl)porphine (TCPP) into the flask, and add a rotor to start stirring at 1200 rpm / min;

[0035] 2) Stir until TCPP is completely dissolved, and use a constant pressure funnel to slowly drip 46.5 ml of 0.1 M dilute hydrochloric acid into the flask.

[0036] 3) Wait until the dilute hydrochloric acid is added, seal the flask, reduce the speed to 600 rpm / min, and heat at 80°C for 72 hours;

[0037] 4) After 72 hours, remove the liquid and cool it naturally to room temperature;

[0038] 5) Centrifuge the liquid at 6000 rpm / min for 15 min to obtain a precipitate, then rinse with ddH 2 O Wash the precipitate 5 times;

[0039] 6) resuspend the precipitate, centrifuge again at 300 rpm / min for 5 min, remove the precipitate to obtain a liquid;

[0040] 7) Place the liquid in a rotary evaporator and evaporate it at 60°C until it is completely dry. Then collect the powder and store it in a dry and dark environment.

[0041] 2.2 Preparation and storage conditions of antigen complex tumor vaccines

[0042] 1) Take 0.2 g of tumor tissue and prepare a single cell suspension;

[0043] 2) Add the nanosheets to the single cell suspension and mix well so that the final concentration of the nanosheets is 100 ug / ml;

[0044] 3) Place the single cell suspension mixed with the nanosheets in a 37°C incubator and incubate for 3 hours;

[0045] 4) After incubation, irradiate the cells with a 660nm near-infrared laser at a power of 0.5w / cm2 for 20 minutes;

[0046] 5) After irradiation, place in a 37°C incubator for 2 hours;

[0047] 6) Filter to remove large flocs, collect the liquid, and freeze it at -80°C overnight to obtain the tumor antigen complex tumor vaccine. The vaccine can be stored in liquid nitrogen or a -80°C refrigerator.

[0048] 2. Product Preparation and Characterization

[0049] like Figure 1 As shown, the nanosheets are in powder form after preparation ( Figure 1 h), and the nanosheets can be stored for a long time in a light-proof and dry environment. Scanning electron microscopy (SEM) showed that the nanosheets self-aggregated to present a stacked fan-shaped sheet structure ( Figure 1 a). Transmission electron microscopy (TEM) shows that it presents a uniformly sized flake structure ( Figure 1 b), High-resolution TEM (HRTEM) ( Figure 1 c) shows that the lattice distance of the nanosheets is 0.48nm and 0.56nm. The particle size of the synthesized nanosheets is 169.9nm ( Figure 1 d), its potential is -46.54mV( Figure 1 e), which is relatively stable. And the fluorescence spectrum of the nanosheets was detected ( Figure 1 f) and UV absorption spectra ( Figure 1 g), it can be seen that it has a fluorescent reaction and has a high absorption at 660nm. Then, we took out and weighed 200mg of tumor tissue, and prepared 0.5ml of tumor cell single cell suspension, then added 0.5ml of 200ug / ml nanosheet solution dissolved in PBS to the single cell suspension and mixed it thoroughly, and then placed it in a cell culture incubator at 37℃ for 2h to allow the cells to fully absorb the nanosheets, and then used 0.5W / cm 2 The 660nm laser was irradiated for 20 minutes, and then left to stand at 37 degrees Celsius for 2 hours to obtain the vaccine. This method makes the preparation of tumor vaccines easier. The rough multi-layer physical properties of the nanosheets can absorb a large amount of tumor cell antigens, enhancing their immunogenicity. This allows tumor antigens to be more efficiently taken up and presented, activating the memory effect of the body's immune system.

[0050] 3. The therapeutic effect of the product

[0051] In order to explore the preventive effect of nanosheet tumor antigen complex on breast cancer tumorigenesis in mice, mice were subcutaneously inoculated with tumor antigen complex tumor vaccine. The inoculation method was as follows:

[0052] 1) Take the vaccine out from -80℃ and slowly thaw it at 37℃; for mice weighing 18-22g, inoculate 90-120ul of the vaccine; inoculate again after 2 days, multiple stimulations, the purpose of re-inoculation is to supplement antigens and enhance immune effects. In this experiment, mice weighing 20g were inoculated with 100ul of the vaccine, and inoculated again after 2 days, multiple stimulations;

[0053] 2) Two days later, 1×10 5 4T1 cells were cultured and the growth of tumors in mice was observed.

[0054] Experimental groups: blank group, broken tumor cell group, nanosheet tumor antigen complex group. The product treatment process and results are as follows Figure 2 shown.

[0055] Figure 2 a is the process of vaccinating mice with tumor vaccines and verifying the experiment. After the three groups of experimental mice were inoculated with tumor cells, the tumors of the mice inoculated with the nanosheet tumor antigen complex tumor vaccine were significantly smaller than those of the other two control groups ( Figure 2 b). Figure 2 c shows the changes in body weight of the three groups of experimental mice after inoculation of tumor cells. It can be seen that the inoculation of the nanosheet tumor antigen complex tumor vaccine will not cause the mice to lose weight. Figure 2 d shows the size of the tumors of the three groups of experimental mice on the 20th day. It can be clearly seen that the tumors of the mice vaccinated with the nanosheet tumor antigen complex tumor vaccine did not grow. Therefore, the nanosheet tumor antigen complex tumor vaccine has a strong inhibitory effect on tumor growth. It is a tumor vaccine with a simple preparation method, good stability and improved immunogenicity.

[0056] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims

1. A method for preparing a tumor antigen complex tumor vaccine based on nanosheets, characterized in that: It includes the following steps: S1. Synthesis of nanosheets: dilute hydrochloric acid is added dropwise to a potassium hydroxide solution of meso-tetra(4-carboxyphenyl)porphine, the reaction vessel is sealed, and the reaction is heated and stirred for 70 to 74 hours. The reaction product is a nanosheet; S2. Prepare the vaccine: add the nanosheets synthesized in step S1 to the single-cell suspension of tumor tissue, and incubate them in a 37°C incubator for 2.5 to 3.5 hours. After the incubation, irradiate the cells with a 660nm near-infrared laser for 15 to 25 minutes and then place them in a 37°C incubator for 1.5 to 2.5 hours. Filter to remove large flocs, and the resulting liquid is a nanosheet-based tumor antigen complex tumor vaccine.

2. The method for preparing a nanosheet-based tumor antigen complex tumor vaccine according to claim 1, characterized in that: The concentration of the potassium hydroxide solution in step S1 is 0.3-0.7 mol / L, the mass volume ratio of meso-tetra(4-carboxyphenyl)porphine to the potassium hydroxide solution in meso-tetra(4-carboxyphenyl)porphine is 0.04-0.06 g:1 ml; the concentration of the dilute hydrochloric acid is 0.08-0.12 mol / L.

3. The method for preparing a nanosheet-based tumor antigen complex tumor vaccine according to claim 2, characterized in that: The volume ratio of the potassium hydroxide solution of meso-tetrakis(4-carboxyphenyl)porphine to dilute hydrochloric acid is 1:4-5.

4. The method for preparing a nanosheet-based tumor antigen complex tumor vaccine according to claim 1, characterized in that: The heating temperature in step S1 is 75-85° C., and the stirring rate is 1100-1300 rpm / min.

5. The method for preparing a nanosheet-based tumor antigen complex tumor vaccine according to claim 1, characterized in that: Step S1: After the reactants are heated and stirred for reaction, the liquid in the container is cooled to room temperature, centrifuged at a speed of 5500-6500 rpm / min for 13-18 minutes, the obtained precipitate is washed with double distilled water, the precipitate is resuspended, and then centrifuged at a speed of 280-330 rpm / min for 4-6 minutes to remove the precipitate, and the liquid is placed in a rotary evaporator at a temperature of 55-65° C. and rotary evaporated until completely dry, and the obtained powder is a nanosheet powder.

6. The method for preparing a nanosheet-based tumor antigen complex tumor vaccine according to claim 1, characterized in that: The concentration of the single cell suspension in step S2 is 1×10 6 ~1×10 8 After adding the nanosheets into the single cell suspension, the final concentration of the nanosheets is 90-110ug / ml.

7. The method for preparing a nanosheet-based tumor antigen complex tumor vaccine according to claim 1, characterized in that: The power of the near-infrared laser in step S2 is 0.4-0.6w / cm 2 .

8. The method for preparing a nanosheet-based tumor antigen complex tumor vaccine according to claim 1, characterized in that: The tumor cells are breast cancer cells.

9. A nanosheet-based tumor antigen complex tumor vaccine prepared by the method of any one of claims 1-8.

Images