A nano material for tumor treatment and prevention of recurrence, and a preparation method and application thereof

Abstract

This invention provides a nanomaterial for tumor immunotherapy and its preparation method. The nanomaterial is prepared by a nanoprecipitation method using a phenylalanine-based polymer and a stabilizer. The phenylalanine polymer is obtained from triethylamine, p-nitrophenol, L-phenylalanine, and butanediol. The nanomaterial provided by this invention can induce the body to produce effective CD8+. + T-cell immune response, increased CD8 + The proportion of T cells, promoting CD8 + T-cell immune function, ultimately aiming to prevent and treat tumors, is a treatment approach that effectively treats tumors by influencing the function of the immune system.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a nanomaterial for tumor treatment and recurrence prevention, its preparation method, and its application. Background Technology

[0002] Malignant tumors pose one of the most serious threats to human life and health. Effectively inhibiting tumor progression, improving treatment outcomes for cancer patients, and enhancing their prognosis have long been crucial issues in cancer research. Traditional cancer treatment combines surgical resection with chemotherapy or radiotherapy, but these approaches often impose a significant physiological burden on patients and have unsatisfactory prognoses. In recent years, immunotherapy, which improves immune cell function and enhances the body's anti-tumor immunity, has become an important part of clinical cancer treatment strategies. Compared to traditional treatments, immunotherapy can improve treatment outcomes, reduce tumor recurrence and metastasis, and prolong patient survival. It is a first-line treatment for various types of cancer, including melanoma, non-small cell lung cancer, and advanced esophageal squamous cell carcinoma. However, despite the significant success of immunotherapy, particularly immune checkpoint inhibitors, in the clinical treatment of malignant tumors, limitations remain, such as low actual immune response rates, drug toxicity, and the immune resistance of tumor tissues. These limitations prevent a considerable number of patients from benefiting from existing immunotherapy regimens. Therefore, overcoming the above-mentioned shortcomings and finding new immunotherapies that can affect the function of immune cells and enhance the body's anti-tumor immune capacity has important clinical application value and significance.

[0003] Amino acid polymer materials are a class of biopolymers synthesized using natural amino acids as monomers, either by using amino acids as the main chain or by linking amino acid residues to side chains. They can be classified into polyamides, polyesters, polyesteramides, polyurethanes, etc. The presence of amino acids often endows these polymers with good degradability and biocompatibility, allowing them to enter cells and be hydrolyzed by proteolytic enzymes such as α-chymotrypsin. These properties make amino acid polymers applicable in tissue engineering, biosensors, and biotherapy, making them versatile and safe biomaterials. Under certain conditions, some amino acid polymer materials can also self-assemble into nanoparticles to acquire additional immune functions, such as attenuating the immunosuppressive function of myeloid-derived suppressor cells. Therefore, whether amino acid polymer materials can be used for tumor immunotherapy, improving the tumor immune microenvironment, and promoting tumor regression are scientific questions worthy of in-depth research and exploration.

[0004] Overall, existing tumor immunotherapy regimens are not perfect. Whether amino acid polymer materials, as a class of biomaterials with potential immune functions, can be used for tumor immunotherapy requires further investigation. Finding novel and effective tumor immunotherapy methods is of great significance for cancer treatment and has broad application prospects. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned technical problems by providing a safe and effective nanomaterial that can be used for tumor immunotherapy or to prevent recurrence.

[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 nanomaterials.

[0008] Therefore, the present invention provides a nanomaterial for tumor treatment and recurrence prevention, the nanomaterial comprising a phenylalanine polymer and a stabilizer, wherein 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, distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000).

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

[0012] (1) Synthesis of Class I monomers: Triethylamine and p-nitrophenol were mixed in a molar ratio of 32:31, acetone was added, the mixture was placed in an ice bath, and then sebacate chloride was added dropwise. The molar ratio of sebacate chloride to p-nitrophenol was 2:1. The reaction temperature was 0℃, and the mixture was stirred for 2 hours. After the reaction was completed, the generated p-nitrophenyl sebacate was left at room temperature overnight, then precipitated and washed with distilled water, and then dried under vacuum. The obtained p-nitrophenyl sebacate was recrystallized three times at room temperature in an ethyl acetate / dimethylformamide mixture, wherein the volume ratio of ethyl acetate to dimethylformamide in the ethyl acetate / dimethylformamide mixture was 4:1. Finally, Class I monomers were obtained.

[0013] (2) Synthesis of Class II monomers: 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 then cooled to room temperature. After toluene precipitated, the product was purified with isopropanol and finally dried under vacuum to obtain Class II monomers.

[0014] (3) In dimethyl sulfoxide, type I monomer and type II monomer are added in a molar ratio of 1:1, mixed well, and then triethylamine is added until the monomer is dissolved. The reaction is carried out at 75°C for 48 hours, and then the product is purified by precipitation in pre-cooled ethyl acetate. After dissolving in methanol, the product is purified by precipitation again. Finally, the product is dried under vacuum at 60°C to obtain the phenylalanine polymer.

[0015] The present invention also provides a method for preparing the nanomaterial, the method comprising the following steps:

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

[0017] (2) Add phenylalanine polymer and stabilizer in sequence, vortex mix to obtain a mixture;

[0018] (3) Add the mixture from step (2) dropwise to the corresponding volume of sterile water. The volume ratio of the mixture to the water is 1:10. After the addition is complete, vortex to mix.

[0019] (4) Let stand for 10 minutes, then remove dimethyl sulfoxide by ultrafiltration and centrifugation. Wash with sterile water during ultrafiltration. The remaining liquid after ultrafiltration is the phenylalanine polymer nanomaterial.

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

[0021] On the other hand, the present invention also provides the use of the nanomaterials in the preparation of drugs for the prevention or treatment of tumors.

[0022] On the other hand, the present invention also provides the use of phenylalanine polymers for tumor immunotherapy, wherein the phenylalanine polymers are 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 monomers: Triethylamine and p-nitrophenol were mixed in a molar ratio of 32:31, acetone was added, the mixture was placed in an ice bath, and then sebacate chloride was added dropwise. The molar ratio of sebacate chloride to p-nitrophenol was 2:1. The reaction temperature was 0℃, and the mixture was stirred for 2 hours. After the reaction was completed, the generated p-nitrophenyl sebacate was left at room temperature overnight, then precipitated and washed with distilled water, and then dried under vacuum. The obtained p-nitrophenyl sebacate was recrystallized three times at room temperature in an ethyl acetate / dimethylformamide mixture, wherein the volume ratio of ethyl acetate to dimethylformamide in the ethyl acetate / dimethylformamide mixture was 4:1. Finally, Class I monomers were obtained.

[0025] (2) Synthesis of Class II monomers: 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 then cooled to room temperature. After toluene precipitated, the product was purified with isopropanol and finally dried under vacuum to obtain Class II monomers.

[0026] (3) In dimethyl sulfoxide, type I monomer and type II monomer are added in a molar ratio of 1:1, mixed well, and then triethylamine is added until the monomer is dissolved. The reaction is carried out at 75°C for 48 hours, and then the product is purified by precipitation in pre-cooled ethyl acetate. After dissolving in methanol, the product is purified by precipitation again. Finally, the product is 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] This invention addresses the shortcomings of existing tumor immunotherapy treatments, such as insufficient efficacy and uncertainty regarding the suitability of amino acid polymers for tumor immunotherapy. It provides a biomaterial prepared from a phenylalanine polymer (designated 8p4), and further develops a nanomaterial (designated 8N) by combining this 8p4 polymer with a stabilizer via nanoprecipitation. Experiments have demonstrated that this 8N nanomaterial significantly induces CD8+ in mice. + T-cell-related immune responses affect the function of the tumor's immune system, thereby preventing and treating tumors, and are an effective form of tumor immunotherapy. Attached Figure Description

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

[0030] Figure 2 The images show the 1H NMR spectrum of the phenylalanine polymer 8p4 (A) and the transmission electron microscopy (TEM) scan of the nanomaterial 8N (B).

[0031] Figure 3 The effects of the nanomaterial 8N on mouse CD8 were demonstrated. + The effect on T cell response. A shows the experimental procedure. B shows the effect of CD45 after experimental treatment. + CD8 in immune cells + The percentage of T cells is shown in C. This indicates the two groups of CD8... + Flow cytometry analysis results of T cells. D shows the effector CD8 cells. + T cells and immature CD8 + Changes in the proportion of T cells.

[0032] Figure 4The effects of the nanomaterial 8N on the prevention of different mouse tumors are shown. A shows the experimental procedure for inoculating C57BL / 6 mice with B16-OVA cells. B shows the results of the B16-OVA tumor cell experiment. C shows the experimental procedure for inoculating C57BL / 6 mice with MC38 cells. D shows the results of the MC38 tumor cell experiment. E shows the experimental procedure for inoculating immunodeficient NSG mice with B16-OVA cells. F shows the results of the B16-OVA tumor cell experiment.

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

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

[0035] Unless otherwise specified, all reagents mentioned below are commercially available. For simplicity, some procedures have not been detailed in terms of parameters, steps, and instruments used; it should be understood that these are well-known and reproducible to those skilled in the art.

[0036] The phenylalanine polymer 8p4 used in this invention is a neutral L-phenylalanine polymer with hydrophobic properties. When mixed with a stabilizer for nanoprecipitation, it can self-assemble to form nanomaterials. The raw materials for synthesizing 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 type I monomers, 0.32 mol of triethylamine and 0.31 mol of p-nitrophenol (molar ratio 32:31) were mixed and added to a round-bottom flask containing 200 ml of acetone. The mixture was placed in an ice bath, and then 0.62 mol of sebacyl chloride was added dropwise. The reaction temperature was 0℃, and the mixture was stirred for 2 hours. After the reaction was completed, the generated p-nitrophenyl sebacic acid was left at room temperature overnight, then precipitated with distilled water, washed, and dried under vacuum. At room temperature, the obtained p-nitrophenyl sebacic acid was recrystallized three times in a mixture of ethyl acetate / dimethylformamide (volume ratio of ethyl acetate to dimethylformamide was 4:1) to finally obtain type I monomers.

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

[0040] (3) In 1.5 ml of dimethyl sulfoxide (DMSO), 1.0 mmol of type I monomer and 1.0 mmol of type II monomer (molar ratio 1:1) were added, stirred and mixed, and heated to 75 °C. Triethylamine (2.2 mmol) was then added dropwise, and the mixture was stirred vigorously until the monomers were completely dissolved. The mixture was then reacted at 75 °C for 48 hours. The product was then precipitated in 10 ml of pre-cooled ethyl acetate, decanted, dried, and dissolved in 10 ml of methanol. The precipitation and purification steps were repeated. Finally, the product was dried under vacuum at 60 °C to obtain phenylalanine polymer 8p4 (pale yellow powder).

[0041] The obtained phenylalanine polymer 8p4 was identified as a neutral hydrophobic L-phenylalanine polymer. Figure 1 The synthesis reaction principle of phenylalanine polymer 8p4 is shown. In this diagram, A represents a type I monomer prepared by reacting triethylamine with p-nitrophenol; B represents a type II monomer prepared by reacting L-phenylalanine with butanediol; and C represents the phenylalanine polymer obtained by reacting type I and type II monomers.

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

[0043] First, calculate the amount of nanomaterials needed for the experiment. Phenylalanine polymer 8p4 (concentration 20 mg / ml, solvent: dimethyl sulfoxide) is heated to 60°C and completely dissolved before use. Phenylalanine polymer 8p4 and stabilizer DSPE-PEG2000 (concentration 10 mg / ml, solvent: dimethyl sulfoxide) are added sequentially according to a weight ratio (phenylalanine polymer 8p4: stabilizer DSPE-PEG2000 = 5:1). After the reagents are added, vortex the mixture. Then, add the mixture dropwise to the corresponding volume of sterile water at a volume ratio of 1:10, and vortex again. After all the liquid has been added and thoroughly mixed, let it stand for 10 minutes. Use a 100 kDa ultrafiltration tube to remove reagents that have not formed nanomaterials. During ultrafiltration, wash the nano-vaccine particles with sterile water. The remaining liquid after ultrafiltration is the phenylalanine polymer nanomaterial (8N). When using, the 8N nanomaterial can be resuspended in phosphate buffered saline (PBS solution).

[0044] Figure 2 Image A shows the 1H NMR spectrum of the phenylalanine polymer 8p4, and image B shows the transmission electron microscopy (TEM) image of the nanomaterial 8N. TEM analysis confirmed that the prepared nanomaterial 8N is a spherical nanomaterial with a particle size of 50-80 nm. Figure 2 B).

[0045] Figure 3 This study demonstrates the effect of the phenylalanine polymeric nanomaterial 8N on CD8 in animals. + Experimental results of T cell responses.

[0046] like Figure 3 As shown in step A, the nanomaterial 8N was injected intravenously into mice via the tail vein for two consecutive weeks, with each mouse receiving 2 mg of the nanomaterial per injection. The CD8+ levels in the mouse spleen were then analyzed. + T cell response. Experimental results showed that, compared with the control group, treatment with 8N nanomaterials increased CD8 levels in the spleen. + T cells will show a significant response, manifested as CD45 + CD8 in immune cells + T cells increased significantly ( Figure 3 B). For the two groups of CD8 + Further flow cytometry analysis of T cells ( Figure 3 (C) discovered that CD8 has an effective function. + T cells (CD44) + CD62L - The proportion of ) increased significantly, while the proportion of immature CD8 increased. + T cells (CD44) - CD62L+ The proportion of ) decreased significantly ( Figure 3 (D). This result suggests that in vivo injection of the nanomaterial 8N can significantly affect CD8. + T cells, promoting CD8 + T-cell immune function.

[0047] To investigate whether the nanomaterial 8N could play a role in preventing tumors, immunocompetent C57BL / 6 mice were randomly divided into a phosphate-buffered saline (PBS) injection group and a nanomaterial 8N group. The mice were injected with 8N nanomaterial twice, once every 7 days, with each injection containing 1 mg of nanomaterial. Following this, mouse melanoma tumor cells B16-OVA were subcutaneously inoculated (B16-OVA cells were subcutaneously injected into the back of the mouse, with 2 × 10⁶ cells per mouse). 5 (cells), steps as follows Figure 4 As shown in Figure A. From Figure 4 As shown in Figure B, the overall tumor growth curve of B16-OVA and the tumor growth curves in each group of mice demonstrate that 8N can significantly prevent tumor occurrence and inhibit tumor growth. In a mouse model of MC38 colon cancer (MC38 cells were subcutaneously injected into the back of each mouse, with 1×10⁶ cells injected per mouse),... 6 (cells) Figure 4 (C) As can be seen from the overall growth curve of MC38 tumors and the tumor growth curves in each group of mice, the nanomaterial 8N can also effectively inhibit the growth of MC38 tumors and alleviate tumor progression. Figure 4 (D). However, when using immunodeficient NSG mice, the nanomaterial 8N was ineffective in preventing B16-OVA tumors, indicating that the anti-tumor function of 8N depends on the participation of the immune system. Figure 4 (E and F). Therefore, the nanomaterial 8N inhibits tumor progression by affecting the function of the immune system, combined with Figure 3 The 8N effect shown in the diagram affects CD8 + The results of T-cell analysis suggest that injecting the nanomaterial 8N is an effective method of tumor immunotherapy, capable of inhibiting tumor growth, curbing tumor progression, and preventing tumors and their recurrence.

[0048] To investigate whether the nanomaterial 8N could also play a therapeutic role in tumors, C57BL / 6 mice were divided into PBS and 8N injection groups. Tumor cells were first inoculated into the skin on the back of the mice, and then 8N nanomaterial was injected on days 4 and 8, with each mouse receiving 1 mg of 8N each time. By observing the overall tumor growth curve and the tumor growth curves in each group of mice, it was found that the nanomaterial 8N could not only treat CT26 colon cancer tumors in mice (CT26 cells were subcutaneously inoculated on the back, with each mouse receiving 1 × 10⁶ cells), but also... 6 (each cell) plays an effective therapeutic role ( Figure 5 (A and B), which can also effectively treat mouse melanoma B16 tumors ( Figure 5 (C and D). Therefore, the nanomaterial 8N can also play a certain role in the treatment of tumors and has good prospects for clinical application.

Claims

1. The use of nanomaterials in the preparation of drugs for the prevention or treatment of tumors, characterized in that, The nanomaterial is composed of a phenylalanine polymer and a stabilizer. The phenylalanine polymer is prepared from triethylamine, p-nitrophenol, L-phenylalanine, and butanediol. The stabilizer is distearylphosphatidylethanolamine-polyethylene glycol 2000. The mass ratio of the phenylalanine polymer to the stabilizer is 5:

1. The phenylalanine polymer is prepared by the following steps: (1) Synthesis of Class I monomers: Triethylamine and p-nitrophenol were mixed in a molar ratio of 32:31, acetone was added, the mixture was placed in an ice bath, and then sebacate chloride was added dropwise. The molar ratio of sebacate chloride to p-nitrophenol was 2:

1. The reaction temperature was 0℃, and the mixture was stirred for 2 hours. After the reaction was completed, the generated p-nitrophenyl sebacate was left at room temperature overnight, then precipitated and washed with distilled water, and then dried under vacuum. The obtained p-nitrophenyl sebacate was recrystallized three times at room temperature in an ethyl acetate / dimethylformamide mixture, wherein the volume ratio of ethyl acetate to dimethylformamide in the ethyl acetate / dimethylformamide mixture was 4:

1. Finally, Class I monomers were obtained. (2) Synthesis of Class II monomers: 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 then cooled to room temperature. After toluene precipitated, the product was purified with isopropanol and finally dried under vacuum to obtain Class II monomers. (3) In dimethyl sulfoxide, type I monomers and type II monomers are added in a molar ratio of 1:1, mixed well, and then triethylamine is added until the monomers are dissolved. The reaction is carried out at 75°C for 48 hours. The mixture is then purified by precipitation in pre-cooled ethyl acetate, dissolved in methanol, and the precipitation and purification are repeated. Finally, the mixture is dried under vacuum at 60°C to obtain the phenylalanine polymer. The tumor is colon cancer; The preparation method of the nanomaterial includes the following steps: (1) Dissolve the phenylalanine polymer in dimethyl sulfoxide at a concentration of 20 mg / ml, heat to 60°C until it is completely dissolved, and dissolve the stabilizer in dimethyl sulfoxide at a concentration of 10 mg / ml; (2) Add phenylalanine polymer and stabilizer in sequence, vortex mix to obtain a mixture; (3) Add the mixture from step (2) dropwise to the corresponding volume of sterile water. The volume ratio of the mixture to the water is 1:

10. After the addition is complete, vortex to mix. (4) Let stand for 10 minutes, then remove dimethyl sulfoxide by ultrafiltration and centrifugation. Wash with sterile water during ultrafiltration. The remaining liquid after ultrafiltration is the nanomaterial. In step (4), an ultrafiltration tube with a pore size of 100 kDa is used for ultrafiltration.

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