A method for preparing a high molecular polymer nanozyme for regulating tumor immunosuppressive microenvironment

Abstract

A method for preparing a polymer nanoenzyme for regulating the tumor immunosuppressive microenvironment belongs to the field of nanomedicine preparation. The present invention designs a polymer nanocarrier that responds to the microacidic environment of the tumor and encapsulates lactate oxidase for selectively consuming lactic acid in tumor tissues to improve the tumor immunosuppressive microenvironment and enhance the tumor immunotherapy effect. The nanodrug delivery platform greatly improves the circulation time and stability of lactate oxidase by encapsulating lactate oxidase into a polymer nanocarrier. In addition, by specifically changing the properties such as permeability in tumor tissues, the nanocarrier can significantly reduce the toxic side effects of lactate oxidase. The drug lactate oxidase of the present invention catalyzes the oxidation of lactic acid into pyruvic acid and hydrogen peroxide, thereby improving the tumor immunosuppressive microenvironment, enhancing the immunotherapy effect, etc.

Description

Technical Field

[0001] The present invention belongs to the field of nano drug preparation, and in particular relates to a method for preparing a high molecular polymer nanozyme for regulating a tumor immunosuppressive microenvironment. Background Art

[0002] Tumor immunotherapy has been applied to the clinical treatment of a variety of malignant solid tumors. However, due to the existence of tumor immunosuppressive microenvironment, only less than 20% of patients can respond well to immunotherapy. The accumulation of a large amount of lactic acid in tumor tissues through the "Warburg effect" due to the vigorous metabolism of tumor cells is an important reason for the formation of tumor immunosuppressive microenvironment. Excessive lactic acid can cause the inactivation of effector T cells and provide energy for the activity of regulatory T cells, thereby significantly inhibiting the response of tumors to immunotherapy. Therefore, reducing the lactic acid level in the tumor microenvironment is of great value in alleviating or even reversing the tumor immunosuppressive microenvironment and thus improving the effect of immunotherapy.

[0003] Lactate oxidase is a flavoprotein that catalyzes the oxidation of lactate to produce pyruvate and hydrogen peroxide. The degradation of lactate in tumors by lactate oxidase can effectively alleviate the immunosuppressive function of the tumor microenvironment. However, due to the lack of circulatory stability of lactate oxidase, it is easily degraded and inactivated by proteases in the human body, so its clinical application in tumor treatment is greatly limited.

[0004] Safety and efficacy are the main indicators for judging drug efficacy, and the biosafety of lactate oxidase itself is another key factor limiting its application in tumor treatment. Since lactate is the carbon source for most normal tissues, the nonspecific lactate removal caused by lactate oxidase may cause serious side effects. Therefore, how to use lactate oxidase to selectively remove lactate in tumors without affecting the lactate content in other tissues and organs is the key to determining whether lactate oxidase can be used in clinical tumor treatment. Summary of the invention

[0005] The purpose of the present invention is to improve the circulatory stability of lactate oxidase and avoid the toxic side effects of lactate oxidase, solve the problem of low selectivity of lactate oxidase, and provide a method for preparing a high-molecular polymer nanozyme loaded with lactate oxidase for regulating the tumor immunosuppressive microenvironment.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing a high molecular polymer nanozyme for regulating a tumor immunosuppressive microenvironment, the method comprising:

[0008] Step 1: Preparation of 2-(azepan-1-yl)ethyl methacrylate monomer responsive to tumor microacid environment

[0009] N-(2-hydroxyethyl)hexamethylenediamine and triethylamine are dissolved in anhydrous tetrahydrofuran, and then methacryloyl chloride is added dropwise to the above solution. After heating and refluxing, the insoluble triethylamine hydrochloride is removed by filtration, and then purified to obtain 2-(azepan-1-yl)ethyl methacrylate (CA7) monomer that responds to the slightly acidic environment of tumors;

[0010] Step 2: Preparation of hydrogen peroxide-responsive NLG919 monomer

[0011] Dissolve oxalyl chloride in anhydrous dichloromethane, stir at -5~5℃ for 25~35 minutes, then add 2-hydroxyethyl methacrylate dropwise and continue stirring for 2~3h; remove excess oxalyl chloride and solvent by rotary evaporation, add the mixture to anhydrous dichloromethane solution of NLG919, add triethylamine to adjust the pH value of the solution to 7-8, stir at -5~5℃ overnight; then filter the solution and rotary evaporate to obtain hydrogen peroxide-responsive NLG919 monomer;

[0012] Step 3: Preparation of PEG-PCA7-PNLG919 amphiphilic polymer that responds to both tumor microacidic environment and hydrogen peroxide

[0013] 2-(Azepan-1-yl)ethyl methacrylate monomer, hydrogen peroxide-responsive NLG919 monomer, polyethylene glycol-4-cyano-4-(phenylcarbonylthiothiothio)pentanoate and AIBN were dissolved in N,N-dimethylformamide solution for reaction, and then dialyzed, precipitated with deionized water and freeze-dried to obtain PEG-PCA-PNLG919 amphiphilic polymer;

[0014] Step 4: Preparation of polymer nanozymes loaded with lactate oxidase

[0015] The PEG-PCA7-PNLG919 amphiphilic polymer was dissolved in tetrahydrofuran, and deionized water containing lactate oxidase was added dropwise during stirring, and stirring was continued at room temperature. Since THF is volatile, as THF evaporates, the amphiphilic polymer in the solution self-assembles and encapsulates the lactate oxidase into the hydrophilic core, thereby obtaining a spherical nanozyme encapsulating lactate oxidase.

[0016] Furthermore, in step 1, the mass ratio of N-(2-hydroxyethyl)hexamethylenediamine, triethylamine and methacryloyl chloride is 2~3:2~3:2~3.

[0017] Furthermore, in step 1, the purification is a combination of silica gel column chromatography (eluent methanol / ethyl acetate = 3 / 1 v / v) and suspension evaporation.

[0018] Furthermore, in step 2, the mass ratio of oxalyl chloride, 2-hydroxyethyl methacrylate, and NLG919 is 5~7:2~4:0.5~1.

[0019] Furthermore, in step three, the molar ratio of the 2-(azepan-1-yl)ethyl methacrylate monomer, the hydrogen peroxide-responsive NLG919 monomer, polyethylene glycol-4-cyano-4-(phenylcarbonylthiothiothio)pentanoate and AIBN is 25~35:25~35:0.8~1.2:0.1~0.5.

[0020] Furthermore, in step three, the reaction conditions are: nitrogen protection for 30 minutes and then reaction at 75-85° C. for 24 hours.

[0021] Furthermore, in step 4, the mass ratio of the PEG-PCA7-PNLG919 amphiphilic polymer to lactate oxidase is 0.1-10:0.06-6.

[0022] The beneficial effects of the present invention relative to the prior art are as follows: the present invention designs a high molecular polymer nanocarrier that responds to the microacid environment of the tumor and carries lactate oxidase for specific delivery of lactate oxidase to the tumor site to alleviate the inhibitory effect of the tumor microenvironment on the immune system. The nano drug delivery platform can protect the encapsulated lactate oxidase from contact with proteases during the in vivo circulation process, thereby improving the circulatory stability of lactate oxidase. In addition, the nano drug can change its own permeability by responding to the lower pH environment of the tumor tissue than that of normal tissue, selectively catalyzes and oxidizes lactic acid in the tumor microenvironment without affecting the lactic acid level in normal tissue, thereby reducing the potential toxic side effects of lactate oxidase. The drug lactate oxidase of the present invention can effectively improve the tumor immunosuppressive microenvironment, enhance the activity of effector T cells and promote the loss of regulatory T cell activity, thereby improving the immunotherapy effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of polymer nanozymes used to regulate tumor immunosuppressive microenvironment. DETAILED DESCRIPTION

[0024] The technical scheme in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work belong to the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0025] The present invention actively innovates the method and approach of using lactate oxidase to treat malignant tumors. It adopts a model of preparing high-molecular polymer nanozymes loaded with lactate oxidase, which improves the circulatory stability of lactate oxidase while allowing the nanozymes to specifically catalyze the oxidation of lactic acid in the tumor microenvironment, enhance the activity of effector T cells and inhibit the function of regulatory T cells, thereby improving the tumor immunosuppressive microenvironment and improving the immunotherapy effect.

[0026] Figure 1 The present invention is a schematic diagram of a polymer nanozyme loaded with lactate oxidase designed and prepared by the present invention for regulating the tumor immunosuppressive microenvironment. An intelligent polymer nanozyme with slightly acidic pH responsiveness can be obtained by assembling an amphiphilic block copolymer and encapsulating lactate oxidase. In the slightly acidic environment of the tumor, the polymer nanozyme is protonated, resulting in increased permeability. Subsequently, lactic acid enters the nanozyme and contacts with lactate oxidase and is catalytically oxidized into pyruvic acid and hydrogen peroxide, thereby selectively consuming lactic acid in the tumor microenvironment, improving the tumor immunosuppressive microenvironment, and ultimately achieving the purpose of improving the effect of tumor immunotherapy.

[0027] Embodiment 1:

[0028] A method for preparing a high molecular polymer nanozyme for regulating a tumor immunosuppressive microenvironment, the method comprising:

[0029] Step 1: Preparation of 2-(azepan-1-yl)ethyl methacrylate monomer responsive to tumor microacid environment

[0030] N-(2-hydroxyethyl)hexamethylenediamine (2.86 g, 20 mmol) and triethylamine (2.02 g, 20 mmol) were dissolved in anhydrous tetrahydrofuran (THF, 20 ml), and then methacryloyl chloride (2.08 g, 20 mmol) was added dropwise to the above solution. After heating under reflux for 2 hours, the insoluble triethylamine hydrochloride was removed by filtration, and then the 2-(azacycloheptan-1-yl)ethyl methacrylate (CA7) monomer that responds to the slightly acidic environment of the tumor was obtained by silica gel column chromatography (eluent methanol / ethyl acetate = 3 / 1 v / v) and suspension evaporation;

[0031] Step 2: Preparation of hydrogen peroxide-responsive NLG919 monomer

[0032] Oxalyl chloride (5.9 g, 46 mmol) was dissolved in anhydrous dichloromethane (CH2Cl2, 40 ml), and after stirring at 0 degrees Celsius for 30 minutes, 2-hydroxyethyl methacrylate (3 g, 23 mmol) was added dropwise and the stirring was continued for 2 hours; the excess oxalyl chloride and solvent were removed by rotary evaporation, and then the mixture was added into an anhydrous dichloromethane solution (30 ml) of NLG919 (0.82 g, 2.9 mmol), and triethylamine (1 g, 10 mmol) was added to adjust the pH value of the solution to 7-8, and the mixture was stirred at 0 degrees Celsius overnight; the solution was then filtered and rotary evaporated to obtain hydrogen peroxide-responsive NLG919 monomer;

[0033] Step 3: Preparation of PEG-PCA7-PNLG919 amphiphilic polymer that responds to both tumor microacidic environment and hydrogen peroxide

[0034] 2-(Azepan-1-yl)ethyl methacrylate monomer, hydrogen peroxide-responsive NLG919 monomer, polyethylene glycol-4-cyano-4-(phenylcarbonylthiothiothio) pentanoate and AIBN were dissolved in N, N-dimethylformamide (DMF, 3 ml) solution at a molar ratio of 30:30:1:0.2, and reacted at 75-85°C for 24 h after nitrogen protection for 30 minutes. After dialysis, the deionized water precipitate was freeze-dried to obtain the PEG-PCA-PNLG919 amphiphilic polymer;

[0035] Step 4: Preparation of polymer nanozymes loaded with lactate oxidase

[0036] Take 0.1-10 mg of PEG-PCA7-PNLG919 amphiphilic polymer and dissolve it in 900 μL of tetrahydrofuran (THF), and add 100-1000 μL of deionized water dissolved with lactate oxidase (LOX, 0.06-6 mg) dropwise while stirring with a magnetic stirrer, and keep stirring at room temperature. Since THF is volatile, as THF evaporates, the amphiphilic polymer in the solution self-assembles and encapsulates lactate oxidase into the hydrophilic core to obtain spherical nanozymes encapsulating lactate oxidase.

[0037] Lactate oxidase shows poor stability and low tumor specificity during the in vivo circulation process. In response to these problems, the present invention has developed a polymer nanozyme loaded with lactate oxidase that responds to the micro-acid environment of the tumor to enhance the effect of tumor immunotherapy. The mouse tumor model was established experimentally and the fluorescently labeled free drug (free lactate oxidase) and the polymer nanozyme prepared in Example 1 were injected into the mouse body by intravenous injection. There were five mice in both the experimental group and the control group. By detecting the fluorescent signal in the blood, it was found that the in vivo circulation half-life of the polymer nanozyme was significantly improved compared with the free drug. In addition, free lactate oxidase had no significant effect on the lactic acid level in the tumor site, while the polymer nanozyme could significantly reduce the lactic acid concentration in the tumor 24 hours after intravenous injection, indicating that the polymer nanozyme can selectively catalyze and degrade lactic acid in the tumor. The effects of different groups of drugs on tumor growth in tumor-bearing mice were evaluated by monitoring tumor volume and the proportion of related immune cells in the tumor. PBS, free lactate oxidase and polymer nanozymes were intravenously injected into tumor-bearing mice, and the tumor volume of the mice was recorded every two days. After 14 consecutive days of recording, the mice were killed and the tumor tissues of the mice were taken for immunofluorescence labeling. The proportion of different immune cells was determined by flow cytometry. The results showed that there was no significant difference in the tumor volume and the proportion of related immune cells in mice treated with PBS and free lactate oxidase, while the tumor growth of mice treated with polymer nanozymes was significantly inhibited, and the proportion of effector T cells in the tumor increased while the proportion of regulatory T cells decreased, indicating that polymer nanozymes can improve the immunosuppressive microenvironment by selectively consuming lactic acid in tumor tissues and improve the effect of tumor immunotherapy.

[0038] The high molecular polymer nanozyme loaded with lactate oxidase designed and prepared by the present invention can be used to improve the tumor immunosuppressive microenvironment and thus enhance the immunotherapy effect. It is recommended that tumor patients be intravenously injected with nanomedicines once a day, with each dose of 0.1 mg / kg-0.5 mg / kg. The time to stop administration is determined by observing the growth of the patient's tumor. The improvement of the toxic and side effects of the nanomedicine is determined by detecting the lactic acid content in the patient's blood. Mouse experiments have found that compared with free lactate oxidase drugs, high molecular polymer nanozymes can significantly improve the circulation stability of lactate oxidase and reduce its potential toxic and side effects. Therefore, compared with the control group mice treated with free lactate oxidase, the tumor-bearing model mice treated with high molecular polymer nanozymes showed obvious tumor inhibition and immune activation effects.

Claims

1. A method for preparing a polymer nanozyme for regulating tumor immunosuppressive microenvironment, characterized in that: The method is: Step 1: Preparation of 2-(azepan-1-yl)ethyl methacrylate monomer responsive to tumor microacid environment N-(2-hydroxyethyl)hexamethylenediamine and triethylamine are dissolved in anhydrous tetrahydrofuran, and then methacryloyl chloride is added dropwise to the solution, and after heating under reflux, insoluble triethylamine hydrochloride is removed by filtration, and then 2-(azepan-1-yl)ethyl methacrylate monomer responsive to the slightly acidic environment of tumors is obtained by purification; Step 2: Preparation of hydrogen peroxide-responsive NLG919 monomer Dissolve oxalyl chloride in anhydrous dichloromethane, stir at -5~5℃ for 25~35 minutes, then add 2-hydroxyethyl methacrylate dropwise and continue stirring for 2~3h; remove excess oxalyl chloride and solvent by rotary evaporation, add the mixture to anhydrous dichloromethane solution of NLG919, add triethylamine to adjust the pH value of the solution to 7-8, stir at -5~5℃ overnight; then filter the solution and rotary evaporate to obtain hydrogen peroxide-responsive NLG919 monomer; Step 3: Preparation of PEG-PCA7-PNLG919 amphiphilic polymer that responds to both tumor microacidic environment and hydrogen peroxide 2-(Azepan-1-yl)ethyl methacrylate monomer, hydrogen peroxide-responsive NLG919 monomer, polyethylene glycol-4-cyano-4-(phenylcarbonylthiothiothio)pentanoate and AIBN were dissolved in N,N-dimethylformamide solution for reaction, and then dialyzed, precipitated with deionized water and freeze-dried to obtain PEG-PCA7-PNLG919 amphiphilic polymer; Step 4: Preparation of polymer nanozymes loaded with lactate oxidase The PEG-PCA7-PNLG919 amphiphilic polymer was dissolved in tetrahydrofuran, and deionized water containing lactate oxidase was added dropwise during stirring, and stirring was continued at room temperature. The amphiphilic polymer in the solution self-assembled and encapsulated the lactate oxidase into the hydrophilic core, thereby obtaining a spherical nanozyme encapsulating lactate oxidase.

2. The method for preparing a polymer nanozyme for regulating tumor immunosuppressive microenvironment according to claim 1, characterized in that: In step 1, the mass ratio of N-(2-hydroxyethyl)hexamethylenediamine, triethylamine and methacryloyl chloride is 2-3:2-3:2-3.

3. The method for preparing a polymer nanozyme for regulating tumor immunosuppressive microenvironment according to claim 1, characterized in that: In step 1, the purification is a combination of silica gel column chromatography and rotary evaporation, and the eluent of the silica gel column chromatography is a mixed solution of methanol and ethyl acetate in a volume ratio of 3:

1.

4. The method for preparing a polymer nanozyme for regulating tumor immunosuppressive microenvironment according to claim 1, characterized in that: In step 2, the mass ratio of oxalyl chloride, 2-hydroxyethyl methacrylate, and NLG919 is 5-7:2-4:0.5-1.

5. The method for preparing a high molecular polymer nanozyme for regulating tumor immunosuppressive microenvironment according to claim 1, characterized in that: In step three, the molar ratio of the 2-(azepan-1-yl)ethyl methacrylate monomer, the hydrogen peroxide-responsive NLG919 monomer, polyethylene glycol-4-cyano-4-(phenylcarbonylthiothiothio)pentanoate and AIBN is 25-35:25-35:0.8-1.2:0.1-0.

5.

6. The method for preparing a high molecular polymer nanozyme for regulating tumor immunosuppressive microenvironment according to claim 1, characterized in that: In step 3, the reaction conditions are: nitrogen protection for 30 minutes and then reaction at 75-85° C. for 24 hours.

7. The method for preparing a high molecular polymer nanozyme for regulating tumor immunosuppressive microenvironment according to claim 1, characterized in that: In step 4, the mass ratio of the PEG-PCA7-PNLG919 amphiphilic polymer to lactate oxidase is 0.1-10:0.06-6.

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