Nanometer material for tumor treatment and recurrence prevention as well as preparation method and application of nanometer material

Abstract

The invention provides a nano material for tumor immunotherapy and a preparation method thereof, the nano material is prepared from a phenylalanine polymer and a stabilizer through a nano precipitation method, and the phenylalanine polymer is prepared from triethylamine, p-nitrophenol, L-phenylalanine and butanediol. The nano material provided by the invention can induce an organism to generate effective CD8 + T cell immune response, increase the proportion of CD8 + T cells and promote the immune function of the CD8 + T cells, so that the aim of preventing and treating tumors is finally achieved, and the nano material is a treatment scheme for effectively treating the tumors by influencing the functions of an immune system.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a nano material for tumor treatment and recurrence prevention, and a preparation method and application thereof. Background Art

[0002] Malignant tumors are one of the most serious threats to human life and health. How to effectively inhibit tumor progression, improve the treatment effect of tumor patients, and improve the prognosis of tumor patients is an important issue in the field of tumor research. The traditional tumor treatment is a combination of surgical resection and chemotherapy or radiotherapy, but this type of treatment often causes a huge physiological burden on tumor patients, and the prognosis is not satisfactory. In recent years, immunotherapy based on improving the function of immune cells and enhancing the body's anti-tumor immune ability has become an important part of the clinical treatment strategy of tumors. Compared with traditional treatment methods, immunotherapy can improve the treatment effect of tumor patients, reduce tumor recurrence and metastasis, and prolong the survival time of patients. It is a first-line treatment for many different types of tumors including melanoma, non-small cell lung cancer, and advanced esophageal squamous cell carcinoma. However, although immunotherapy represented by immune checkpoint inhibitors has achieved great success in the clinical treatment of malignant tumors, there are still defects such as low actual immune response rate, toxic side effects of drugs, and immune resistance of tumor tissues, which makes a considerable number of patients unable to benefit from existing immunotherapy. Therefore, how to overcome the above-mentioned defects and find new immunotherapy methods that can affect the function of immune cells and enhance the body's anti-tumor immunity has important clinical application value and significance.

[0003] Amino acid polymer materials are a type of biomacromolecule polymers that use natural amino acids as monomers and are synthesized by using amino acids as the main chain or by connecting amino acid residues to side chains. They can be divided into polyamides, polyesters, polyesteramides, polyurethanes, etc. The presence of amino acids often makes these polymers have good degradability and biocompatibility, and they can enter cells and be hydrolyzed by proteolytic enzymes such as α-chymotrypsin. These characteristics make amino acid polymers useful in tissue engineering, biosensors, biotherapy and other fields, and they are widely used and safe biomaterials. Under certain conditions, some amino acid polymer materials can also self-assemble to form nanoparticles and obtain additional immune functions, such as weakening the immunosuppressive function of myeloid suppressor cells. Therefore, whether amino acid polymer materials can be used for tumor immunotherapy, improve the immune microenvironment in tumors, and promote tumor regression is a scientific issue worthy of in-depth research and exploration.

[0004] In general, the existing tumor immunotherapy schemes are not perfect enough. As a type of biomaterial with potential immune function, whether amino acid polymer materials can be used for tumor immunotherapy requires more detailed research. Finding new and effective tumor immunotherapy methods is of great significance for the treatment of tumors and has broad application prospects. Summary of the invention

[0005] The purpose of the present invention is to provide a safe and effective nanomaterial that can be used for tumor immunotherapy or prevention of recurrence in response to the above technical problems.

[0006] Another object of the present invention is to provide a method for preparing the nanomaterial.

[0007] Another object of the present invention is to provide applications of the nanomaterial.

[0008] To this end, the present invention provides a nano material for tumor treatment and recurrence prevention. The nano material comprises a phenylalanine polymer and a stabilizer. The phenylalanine polymer is prepared from triethylamine, p-nitrophenol, L-phenylalanine and butanediol.

[0009] Preferably, the mass ratio of the phenylalanine polymer to the stabilizer is 5:1.

[0010] Preferably, the stabilizer includes but is not limited to distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000).

[0011] Preferably, the phenylalanine polymer is prepared by the following steps:

[0012] (1) Synthesis of Class I monomer: triethylamine and p-nitrophenol are mixed in a molar ratio of 32:31, acetone is added, ice bath is carried out, and then sebacyl chloride is added dropwise, the molar ratio of sebacyl chloride to p-nitrophenol is 2:1, the reaction temperature is 0°C, and stirring is carried out for 2 hours. After the reaction is completed, the generated p-nitrophenyl sebacate is left at room temperature overnight, then precipitated and washed with distilled water, and then dried under vacuum. The obtained p-nitrophenyl sebacate is then recrystallized three times in an ethyl acetate / dimethylformamide mixture at room temperature, wherein the volume ratio of ethyl acetate to dimethylformamide in the ethyl acetate / dimethylformamide mixture is 4:1, and finally a Class I monomer is obtained;

[0013] (2) Synthesis of Type II monomer: L-phenylalanine and butanediol were mixed in a molar ratio of 2:1, toluene and p-toluenesulfonic acid monohydrate were added, and the mixture was stirred and refluxed at 130° C. for 24 hours. The reaction mixture was cooled to room temperature, and after toluene was precipitated, the product was purified with isopropanol and finally vacuum dried to obtain Type II monomer;

[0014] (3) In dimethyl sulfoxide, a type I monomer and a type II monomer are added in a molar ratio of 1:1, and triethylamine is added after mixing until the monomers are dissolved. The mixture is reacted at 75° C. for 48 hours, and then the mixture is precipitated and purified in precooled ethyl acetate. After being dissolved in methanol, the mixture is repeatedly precipitated and purified, and finally dried under vacuum at 60° C. to obtain the phenylalanine polymer.

[0015] The present invention also provides a method for preparing the nano material, which comprises the following steps:

[0016] (1) dissolving the phenylalanine polymer in dimethyl sulfoxide at a concentration of 20 mg / ml, heating to 60°C until it is completely dissolved, and dissolving the stabilizer in dimethyl sulfoxide at a concentration of 10 mg / ml;

[0017] (2) adding phenylalanine polymer and stabilizer in sequence, vortex mixing, and obtaining a mixture;

[0018] (3) adding the mixture of step (2) dropwise into a corresponding volume of sterile water, with the volume ratio of the mixture to water being 1:10, and vortexing to mix after the addition is complete;

[0019] (4) standing for 10 minutes, and removing dimethyl sulfoxide by ultrafiltration and centrifugation. The mixture is washed with sterile water during ultrafiltration. The remaining liquid after ultrafiltration is the phenylalanine polymer nanomaterial.

[0020] Preferably, in step (4), ultrafiltration is performed using an ultrafiltration tube with a pore size of 100 kDa.

[0021] On the other hand, the present invention also provides the use of the nanomaterial in preparing a drug for preventing or treating tumors.

[0022] On the other hand, the present invention also provides a use of a phenylalanine polymer for tumor immunotherapy, wherein the phenylalanine polymer is prepared by reacting triethylamine, p-nitrophenol, L-phenylalanine and butanediol.

[0023] Preferably, the phenylalanine polymer is prepared by the following steps:

[0024] (1) Synthesis of Class I monomer: triethylamine and p-nitrophenol are mixed in a molar ratio of 32:31, acetone is added, ice bath is carried out, and then sebacyl chloride is added dropwise, the molar ratio of sebacyl chloride to p-nitrophenol is 2:1, the reaction temperature is 0°C, and stirring is carried out for 2 hours. After the reaction is completed, the generated p-nitrophenyl sebacate is left at room temperature overnight, then precipitated and washed with distilled water, and then dried under vacuum. The obtained p-nitrophenyl sebacate is then recrystallized three times in an ethyl acetate / dimethylformamide mixture at room temperature, wherein the volume ratio of ethyl acetate to dimethylformamide in the ethyl acetate / dimethylformamide mixture is 4:1, and finally a Class I monomer is obtained;

[0025] (2) Synthesis of Type II monomer: L-phenylalanine and butanediol were mixed in a molar ratio of 2:1, toluene and p-toluenesulfonic acid monohydrate were added, and the mixture was stirred and refluxed at 130° C. for 24 hours. The reaction mixture was cooled to room temperature, and after toluene was precipitated, the product was purified with isopropanol and finally vacuum dried to obtain Type II monomer;

[0026] (3) In dimethyl sulfoxide, a type I monomer and a type II monomer are added in a molar ratio of 1:1, and triethylamine is added after mixing until the monomers are dissolved. The mixture is reacted at 75° C. for 48 hours, and then the mixture is precipitated and purified in precooled ethyl acetate. After being dissolved in methanol, the mixture is repeatedly precipitated and purified, and finally dried under vacuum at 60° C. to obtain the phenylalanine polymer.

[0027] Preferably, the tumor includes but is not limited to melanoma or colon cancer.

[0028] In view of the problems that the existing tumor immunotherapy is not ideal and whether amino acid polymers are qualified as tumor immunotherapy drugs, the present invention provides a biomaterial prepared from phenylalanine polymer (codenamed 8p4), and combines the phenylalanine polymer 8p4 with a stabilizer through nanoprecipitation to form a nanomaterial (codenamed 8N). Experiments have shown that the nanomaterial 8N can significantly induce CD8 + T cell-related immune response affects the function of the immune system in the tumor, thereby preventing and treating the tumor. It is an effective tumor immunotherapy method. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the synthesis reaction principle of phenylalanine polymer 8p4.

[0030] Figure 2 This is the nuclear magnetic resonance hydrogen spectrum of the phenylalanine polymer 8p4 (A) and the transmission electron microscope scanning image of the nanomaterial 8N (B).

[0031] Figure 3 The nanomaterial 8N showed the effect of 8N on mouse CD8 + Effects of T cell response. A shows the experimental steps. B shows the CD45 + CD8 in immune cells + C shows the percentage of CD8 T cells in the two groups + Flow cytometry analysis of T cells. C shows CD8 + T cells and naive CD8 + Changes in the proportion of T cells.

[0032] Figure 4The preventive effect of nanomaterial 8N on different mouse tumors is shown. A shows the experimental steps of C57BL / 6 mice inoculated with B16-OVA cells. B shows the results of the B16-OVA tumor cell experiment. C shows the experimental steps of C57BL / 6 mice inoculated with MC38 cells. D shows the results of the MC38 tumor cell experiment. E shows the experimental steps of immunodeficient NSG mice inoculated with B16-OVA cells. F shows the results of the B16-OVA tumor cell experiment.

[0033] Figure 5 The therapeutic effects of nanomaterial 8N on different mouse tumors are shown. A shows the experimental steps of C57BL / 6 mice inoculated with CT26 cells. B shows the results of the CT26 tumor cell experiment. C shows the experimental steps of C57BL / 6 mice inoculated with B16 cells. D shows the results of the B16 tumor cell experiment. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following embodiments.

[0035] Unless otherwise specified, the reagents involved below can be purchased from commercial sources. For the sake of simplicity, some operations do not describe in detail the parameters, steps and instruments used, but it should be understood that these are well known and reproducible to those skilled in the art.

[0036] The phenylalanine polymer 8p4 used in the present invention is a neutral L-phenylalanine polymer with hydrophobic properties, and can self-assemble to form a nano material when mixed with a stabilizer for nanoprecipitation. The synthetic raw materials of the phenylalanine polymer 8p4 are triethylamine, p-nitrophenol, L-phenylalanine, and butanediol.

[0037] The preparation steps of phenylalanine polymer 8p4 are as follows:

[0038] (1) To synthesize the type I monomer, 0.32 mol of triethylamine and 0.31 mol of p-nitrophenol (molar ratio of 32:31) were mixed and added to a round-bottom flask containing 200 ml of acetone, placed in an ice bath, and then 0.62 mol of sebacyl chloride was added dropwise. The reaction temperature was 0°C and stirred for 2 hours. After the reaction was completed, the generated p-nitrophenyl sebacate was left at room temperature overnight, precipitated and washed with distilled water, and then dried under vacuum. At room temperature, the obtained p-nitrophenyl sebacate was recrystallized three times in a mixture of ethyl acetate / dimethylformamide (the volume ratio of ethyl acetate to dimethylformamide was 4:1) to finally obtain the type I monomer;

[0039] (2) To synthesize the type II monomer, 0.04 mol L-phenylalanine and 0.02 mol butanediol (molar ratio of 2:1) were mixed in a three-necked flask, 400 ml toluene and 0.082 mol p-toluenesulfonic acid monohydrate were added, and the mixture was stirred and refluxed at 130°C for 24 hours. The reaction mixture was cooled to room temperature and the precipitated toluene was poured out. 100 ml isopropanol was added, the product was dissolved at 75°C, and then precipitated at 4°C. The dissolution and precipitation steps were repeated three times for purification, and finally vacuum dried to obtain the type II monomer;

[0040] (3) Add 1.0 mmol of type I monomer and 1.0 mmol of type II monomer (molar ratio 1:1) to 1.5 ml of dimethyl sulfoxide (DMSO), stir and mix, heat to 75°C, then add triethylamine (2.2 mmol) dropwise, stir vigorously until the monomer is completely dissolved, react at 75°C for 48 hours, precipitate in 10 ml of pre-cooled ethyl acetate, decant, dry, dissolve in 10 ml of methanol, repeat the precipitation and purification steps. Finally, dry under vacuum at 60°C to obtain phenylalanine polymer 8p4 (light yellow powder).

[0041] The obtained phenylalanine polymer 8p4 was identified as a neutral hydrophobic L-phenylalanine amino acid polymer. Figure 1 The synthetic reaction principle of phenylalanine polymer 8p4 is shown. A is a type I monomer prepared by the reaction of triethylamine and p-nitrophenol, B is a type II monomer prepared by L-phenylalanine and butanediol, and C is a phenylalanine polymer obtained by the reaction of type I monomer and type II monomer.

[0042] The preparation process of the phenylalanine polymer nanomaterial of the present invention is as follows:

[0043] First, the amount of nanomaterials used was calculated according to the experiment; the phenylalanine polymer 8p4 (concentration of 20 mg / ml, solvent is dimethyl sulfoxide) was heated to 60°C before use to completely dissolve it; phenylalanine polymer 8p4 and stabilizer DSPE-PEG2000 (stabilizer DSPE-PEG2000 concentration of 10 mg / ml, solvent is dimethyl sulfoxide) were added in sequence according to the weight ratio (phenylalanine polymer 8p4: stabilizer DSPE-PEG2000 is 5:1); after the reagents were added, they were vortexed and mixed using an oscillator; after mixing, they were added dropwise to the corresponding volume of sterile water with water at a volume ratio of 1:10, and then vortexed and mixed again. After all the liquids were added and thoroughly mixed, they were allowed to stand for 10 minutes, and ultrafiltration was performed using an ultrafiltration tube with a pore size of 100 kDa to remove the reagents that did not form nanomaterials. During ultrafiltration, sterile water was used to wash the nano vaccine particles; the remaining liquid after ultrafiltration was the phenylalanine polymer nanomaterial (8N). When in use, phosphate buffered saline (PBS solution) can be added to resuspend the nanomaterial to 8N.

[0044] Figure 2 Figure A shows the H NMR spectrum of phenylalanine polymer 8p4, and Figure B shows the transmission electron microscope scanning image of nanomaterial 8N. The transmission electron microscope identified that the prepared nanomaterial 8N is a spherical nanomaterial with a particle size of 50-80 nm ( Figure 2 , B).

[0045] Figure 3 Phenylalanine polymer nanomaterial 8N shows that it affects CD8 in animals + Experimental results of T cell responses.

[0046] like Figure 3 In the step shown in A, the nanomaterial 8N was intravenously injected into the mouse tail vein for two consecutive weeks, with each mouse injected with 2 mg of nanomaterial each time. + T cell response. The experimental results showed that compared with the control group, after nanomaterial 8N treatment, CD8 + T cells will show a significant response, manifested by CD45 + CD8 in immune cells + T cells increased significantly ( Figure 3 , B). For the two groups of CD8 + T cells were further analyzed by flow cytometry ( Figure 3 , C), found that CD8 + T cells (CD44 + CD62L - ) increased significantly, while naive CD8 + T cells (CD44-CD62L + ) has significantly decreased ( Figure 3 , D). This result suggests that in vivo injection of nanomaterial 8N can significantly affect CD8 + T cells, promoting CD8 + Immune function of T cells.

[0047] In order to explore whether the nanomaterial 8N can play a role in preventing tumors, the immune-competent C57BL / 6 mice were divided into a phosphate buffered saline (PBS) injection group and a nanomaterial 8N group. The nanomaterial 8N was injected once every 7 days for a total of two times, with 1 mg of nanomaterial injected per mouse each time. Then, the mouse melanoma tumor cells B16-OVA were subcutaneously inoculated (B16-OVA cells were subcutaneously inoculated on the back, and 2×10 5 cells), the steps are as follows Figure 4 As shown in A. Figure 4The overall growth curve of B16-OVA tumors and the tumor growth curves of each group of mice shown in B show that 8N can significantly prevent tumor occurrence and inhibit tumor growth. In the animal model of mouse colon cancer MC38 tumors (MC38 cells were inoculated subcutaneously on the back, with 1×10 per mouse) 6 cells) Figure 4 , C), the overall growth curve of MC38 tumor and the tumor growth curve of each group of mice show that nanomaterial 8N can also effectively inhibit the growth of MC38 tumor and alleviate tumor progression ( Figure 4 , D). However, if immunodeficient NSG mice were used, the nanomaterial 8N could not effectively prevent B16-OVA tumors. This result indicates that the anti-tumor function of 8N depends on the participation of the immune system ( Figure 4 , E and F). Therefore, nanomaterial 8N inhibits tumor progression by affecting the immune system function. Figure 3 8N affects CD8 + Based on the results of T cells, it can be considered that the injection of nanomaterial 8N is an effective tumor immunotherapy method, which can inhibit tumor growth, curb tumor progression, and can be used to prevent tumors and their recurrence.

[0048] In order to explore whether the nanomaterial 8N can also play a role in treating tumors, C57BL / 6 mice were divided into PBS and 8N injection groups. Tumor cells were first inoculated on the back skin of mice, and then the nanomaterial 8N was injected on the 4th and 8th days after the tumor cell inoculation, with each mouse injected with 1 mg each time. By observing the overall tumor growth curve and the tumor growth curve in each group of mice, it can be found that the nanomaterial 8N can not only treat mouse colon cancer CT26 tumors (CT26 cells were inoculated subcutaneously on the back, and 1×10 6 cells) play an effective therapeutic role ( Figure 5 , A and B), and also effectively treat mouse melanoma B16 tumors ( Figure 5 , C and D). Therefore, nanomaterial 8N can also play a certain role in treating tumors and has good clinical application prospects.

Claims

1. A nanomaterial for tumor treatment and prevention of recurrence, characterized in that: The nano material comprises a phenylalanine polymer and a stabilizer, wherein the phenylalanine polymer is prepared from triethylamine, p-nitrophenol, L-phenylalanine and butanediol.

2. The nanomaterial according to claim 1, characterized in that The mass ratio of the phenylalanine polymer to the stabilizer is 5:

1.

3. The nanomaterial according to claim 1, characterized in that The stabilizer is distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

4. The nanomaterial according to claim 1, characterized in that The phenylalanine polymer is prepared by the following steps: (1) Synthesis of Class I monomer: triethylamine and p-nitrophenol are mixed in a molar ratio of 32:31, acetone is added, ice bath is carried out, and then sebacyl chloride is added dropwise, the molar ratio of sebacyl chloride to p-nitrophenol is 2:1, the reaction temperature is 0°C, and stirring is carried out for 2 hours. After the reaction is completed, the generated p-nitrophenyl sebacate is left at room temperature overnight, then precipitated and washed with distilled water, and then dried under vacuum. The obtained p-nitrophenyl sebacate is then recrystallized three times in an ethyl acetate / dimethylformamide mixture at room temperature, wherein the volume ratio of ethyl acetate to dimethylformamide in the ethyl acetate / dimethylformamide mixture is 4:1, and finally a Class I monomer is obtained; (2) Synthesis of Type II monomer: L-phenylalanine and butanediol were mixed in a molar ratio of 2:1, toluene and p-toluenesulfonic acid monohydrate were added, and the mixture was stirred and refluxed at 130° C. for 24 hours. The reaction mixture was cooled to room temperature, and after toluene was precipitated, the product was purified with isopropanol and finally vacuum dried to obtain Type II monomer; (3) In dimethyl sulfoxide, a type I monomer and a type II monomer are added in a molar ratio of 1:1, and triethylamine is added after mixing until the monomers are dissolved. The mixture is reacted at 75° C. for 48 hours, and then the mixture is precipitated and purified in precooled ethyl acetate. After being dissolved in methanol, the mixture is repeatedly precipitated and purified, and finally dried under vacuum at 60° C. to obtain the phenylalanine polymer.

5. The method for preparing the nanomaterial according to any one of claims 1 to 4, comprising the following steps: (1) dissolving the phenylalanine polymer in dimethyl sulfoxide at a concentration of 20 mg / ml, heating to 60°C until it is completely dissolved, and dissolving the stabilizer in dimethyl sulfoxide at a concentration of 10 mg / ml; (2) adding phenylalanine polymer and stabilizer in sequence, vortex mixing, and obtaining a mixture; (3) adding the mixture of step (2) dropwise into a corresponding volume of sterile water, with the volume ratio of the mixture to water being 1:10, and vortexing to mix after the addition is complete; (4) standing for 10 minutes, and removing dimethyl sulfoxide by ultrafiltration and centrifugation. The mixture is washed with sterile water during ultrafiltration. The remaining liquid after ultrafiltration is the nanomaterial.

6. The method according to claim 5, characterized in that In step (4), ultrafiltration is performed using an ultrafiltration tube with a pore size of 100 kDa.

7. Use of the nanomaterial according to claim 1 for preparing a drug for preventing or treating tumors.

8. Use of a phenylalanine polymer for preparing a drug for preventing or treating tumors, wherein the phenylalanine polymer is prepared by reacting triethylamine, p-nitrophenol, L-phenylalanine and butanediol.

9. The use according to claim 8, characterized in that The phenylalanine polymer is prepared by the following steps: (1) Synthesis of Class I monomer: triethylamine and p-nitrophenol are mixed in a molar ratio of 32:31, acetone is added, ice bath is carried out, and then sebacyl chloride is added dropwise, the molar ratio of sebacyl chloride to p-nitrophenol is 2:1, the reaction temperature is 0°C, and stirring is carried out for 2 hours. After the reaction is completed, the generated p-nitrophenyl sebacate is left at room temperature overnight, then precipitated and washed with distilled water, and then dried under vacuum. The obtained p-nitrophenyl sebacate is then recrystallized three times in an ethyl acetate / dimethylformamide mixture at room temperature, wherein the volume ratio of ethyl acetate to dimethylformamide in the ethyl acetate / dimethylformamide mixture is 4:1, and finally a Class I monomer is obtained; (2) Synthesis of Type II monomer: L-phenylalanine and butanediol were mixed in a molar ratio of 2:1, toluene and p-toluenesulfonic acid monohydrate were added, and the mixture was stirred and refluxed at 130° C. for 24 hours. The reaction mixture was cooled to room temperature, and after toluene was precipitated, the product was purified with isopropanol and finally vacuum dried to obtain Type II monomer; (3) In dimethyl sulfoxide, a type I monomer and a type II monomer are added in a molar ratio of 1:1, and triethylamine is added after mixing until the monomers are dissolved. The mixture is reacted at 75° C. for 48 hours, and then the mixture is precipitated and purified in precooled ethyl acetate. After being dissolved in methanol, the mixture is repeatedly precipitated and purified, and finally dried under vacuum at 60° C. to obtain the phenylalanine polymer.

10. The use according to claim 8, characterized in that The tumor is melanoma or colon cancer.

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