A method for preparing a polymeric prodrug nanoreactor for activating anti-tumor oxidative stress and long-term immune response

Abstract

A method for preparing a polymer prodrug nanoreactor for activating anti-tumor oxidative stress and long-term immune response belongs to the field of nano drug preparation. The present invention designs a polymer prodrug nanoreactor that responds to the tumor microenvironment for inducing tumor cell oxidative stress apoptosis and inhibiting the activity of indoleamine 2,3-dioxygenase in the tumor to improve the tumor immunosuppressive microenvironment and improve the tumor immunotherapy effect. In addition, the nanoreactor can effectively avoid the production of by-products such as phenol by introducing a linker containing oxalate, significantly reducing the toxic and side effects of oxalate derivatives, thereby achieving long-term inhibition of tumor recurrence and metastasis.

Description

Technical Field

[0001] The invention belongs to the field of nano drug preparation, and in particular relates to a method for preparing a polymer prodrug nanoreactor for activating anti-tumor oxidative stress and long-term immune response. Background Art

[0002] Tumor photodynamic therapy has gained widespread attention in tumor treatment due to its unique low invasiveness and spatiotemporal controllability. However, the insufficient tissue penetration of external light sources makes photodynamic therapy difficult to use in the treatment of deep tumors. In recent years, the treatment scheme that uses chemical energy to activate photosensitizers to produce tumor-site-specific oxidative stress to inhibit tumor growth is considered to be a new generation of tumor treatment strategy that can replace photodynamic therapy because it does not rely on external light sources. This scheme uses the large amount of chemical energy generated by the reaction of oxalate derivatives with hydrogen peroxide in the tumor to activate photosensitizers to produce reactive oxygen species, thereby inducing oxidative stress apoptosis of tumor cells in the absence of an external light source, successfully overcoming the challenge of insufficient tissue penetration of external light sources in traditional photodynamic therapy.

[0003] Safety and effectiveness are the main indicators for judging drug efficacy, and the biosafety issues of oxalate derivatives themselves are the key factors limiting their application in tumor treatment. Since the reaction of oxalate derivatives with hydrogen peroxide is often accompanied by the production of byproducts such as phenol, the use of oxalate derivatives for tumor treatment will inevitably cause serious side effects. Therefore, how to design a new carrier to activate photosensitizers without producing toxic byproducts is the key to whether chemical energy-based anti-tumor oxidative stress therapy can be applied clinically.

[0004] Although studies have shown that chemical energy-based anti-tumor oxidative stress therapy can effectively activate anti-tumor immune effects, the immune effects generated by tumor oxidative stress alone are not sufficient to suppress tumor recurrence and metastasis in the long term. This is because oxidative stress therapy can lead to the upregulation of indoleamine 2,3-dioxygenase in the tumor, which can catalyze the decomposition of tryptophan and the synthesis of kynurenine, thereby inhibiting the anti-tumor immune response. Therefore, combining chemical energy-based anti-tumor oxidative stress therapy with indoleamine 2,3-dioxygenase inhibitors can effectively activate long-term anti-tumor immune responses and achieve long-term suppression of tumor recurrence and metastasis. Summary of the invention

[0005] The purpose of the present invention is to improve the immune activation effect of chemical energy-based anti-tumor oxidative stress therapy and avoid the toxic side effects of oxalate derivatives, and to provide a method for preparing a polymeric prodrug nanoreactor for activating anti-tumor oxidative stress and long-term immune response.

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

[0007] A method for preparing a polymeric prodrug nanoreactor for activating anti-tumor oxidative stress and long-term immune response, the method comprising:

[0008] Step 1: Preparation of hydrogen peroxide-responsive NLG919 monomers

[0009] Dissolve oxalyl chloride in anhydrous dichloromethane, stir at -5 to 5°C for 25 to 35 minutes, then add 2-hydroxyethyl methacrylate dropwise and continue stirring for 2 to 3 hours; 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, and stir at -5 to 5°C overnight; then filter the solution and rotary evaporate to obtain hydrogen peroxide-responsive NLG919 monomer;

[0010] Step 2: Preparation of Ce6-modified PEG-PNLG919 amphiphilic polymer

[0011] The hydrogen peroxide-responsive NLG919 monomer, 2-aminoethyl methacrylate, polyethylene glycol-4-cyano-4-(phenylcarbonylthiothiothio) pentanoate and AIBN were dissolved in N,N-dimethylformamide solution for reaction, and the PEG-PNLG919 amphiphilic polymer was obtained by dialysis, deionized water precipitation and freeze-drying; then, the PEG-PNLG919 amphiphilic polymer was dissolved together with Ce6, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in anhydrous dimethyl sulfoxide, and the mixture was stirred at -5 to 5°C for 22 to 26 hours, filtered, dialyzed and freeze-dried to obtain the Ce6-modified PEG-PNLG919 amphiphilic polymer;

[0012] Step 3: Preparation of polymeric prodrug nanoreactors for activation of anti-tumor oxidative stress and long-term immune responses

[0013] 0.1-10 mg of Ce6-modified PEG-PNLG919 amphiphilic polymer was dissolved in 900 μL of tetrahydrofuran (THF), and 100-1000 μL of deionized water was added dropwise while stirring with a magnetic stirrer. The mixture was kept stirred at room temperature. Since THF is volatile, the amphiphilic polymer in the solution self-assembled as THF evaporated to obtain spherical nano-prodrug particles.

[0014] Furthermore, in step 1, the mass ratio of oxalyl chloride, 2-hydroxyethyl methacrylate, and NLG919 is 5-7:2-4:0.5-1.

[0015] Furthermore, in step 2, the molar ratio of the hydrogen peroxide-responsive NLG919 monomer, 2-aminoethyl methacrylate, polyethylene glycol-4-cyano-4-(phenylcarbonylthiothiothio) pentanoate and AIBN is 25-35:25-35:0.8-1.2:0.1-0.5.

[0016] Furthermore, in step 2, the reaction is specifically as follows: reacting at 75-85° C. for 24 hours after nitrogen protection for 30 minutes.

[0017] Furthermore, in step 2, the mass ratio of the PEG-PNLG919 amphiphilic polymer, Ce6, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 115-130:55-65:40-50:25-35.

[0018] The beneficial effects of the present invention relative to the prior art are as follows: the present invention designs a polymeric prodrug nanoreactor that responds to hydrogen peroxide in the tumor microenvironment to activate photosensitizers to produce oxidative stress, thereby inducing tumor cell apoptosis. The nanoreactor can release the indoleamine 2,3-dioxygenase inhibitor NLG919 while activating the photosensitizer, selectively improve the tumor immunosuppressive microenvironment, enhance the activity of effector T cells and promote the loss of regulatory T cell activity, thereby achieving long-term inhibition of tumor recurrence and metastasis. In addition, since the reaction of the oxalate linker with hydrogen peroxide does not produce byproducts such as phenol, the nanoreactor can effectively reduce the toxic side effects commonly seen in traditional oxalate derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the polymeric prodrug nanoreactor activating photosensitizers through chemical energy and regulating the tumor immunosuppressive microenvironment. DETAILED DESCRIPTION

[0020] 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.

[0021] The present invention actively innovates the anti-tumor oxidative stress therapy based on chemical energy. It adopts a model for preparing a polymer prodrug nanoreactor containing an oxalate linker, a photosensitizer Ce6 and NLG919. While activating oxidative stress to induce tumor cell apoptosis, it can prevent the production of reaction by-products such as phenol, etc., prompting the nanoreactor to selectively release NLG919 at the tumor site, inhibiting the activity of indoleamine 2,3-dioxygenase, enhancing the activity of effector T cells and inhibiting the function of regulatory T cells, thereby improving the tumor immunosuppressive microenvironment and achieving long-term immunotherapy effects.

[0022] Figure 1 The schematic diagram of the polymer prodrug nanoreactor designed and prepared by the present invention for activating anti-tumor oxidative stress and long-term immune response. A polymer prodrug nanoreactor with hydrogen peroxide responsiveness can be obtained by assembling amphiphilic block copolymer prodrugs. Under the condition of excessive hydrogen peroxide in the tumor microenvironment, the polymer prodrug nanoreactor reacts with hydrogen peroxide to release a large amount of chemical energy and NLG919. The former can activate Ce6 to produce reactive oxygen to induce tumor cell apoptosis, and the latter can effectively inhibit the activity of indoleamine 2,3-dioxygenase and improve the tumor immunosuppressive microenvironment. The two work together to successfully achieve the purpose of long-term activation of anti-tumor immune function and long-term inhibition of tumor recurrence and metastasis.

[0023] Embodiment 1:

[0024] A method for preparing a polymeric prodrug nanoreactor for activating anti-tumor oxidative stress and long-term immune response, the method comprising:

[0025] Step 1: Preparation of hydrogen peroxide-responsive NLG919 monomers

[0026] 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; after the excess oxalyl chloride and solvent were removed by rotary evaporation, 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;

[0027] Step 2: Preparation of Ce6-modified PEG-PNLG919 amphiphilic polymer

[0028] The hydrogen peroxide-responsive NLG919 monomer, 2-aminoethyl methacrylate, 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 hours after nitrogen protection for 30 minutes. After dialysis, the precipitate was freeze-dried with deionized water to obtain the PEG-PNLG919 amphiphilic polymer; then the PEG-PNLG919 amphiphilic polymer (123 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 46 mg, 0.24 mmol), and N-hydroxysuccinimide (NHS, 28 mg, 0.24 mmol) were dissolved in anhydrous dimethyl sulfoxide (DMSO, 5 ml), stirred at 0 degrees Celsius for 24 hours, filtered, dialyzed, and freeze-dried to obtain Ce6-modified PEG-PNLG919 amphiphilic polymer;

[0029] Step 3: Preparation of polymeric prodrug nanoreactors for activation of anti-tumor oxidative stress and long-term immune responses

[0030] 0.1-10 mg of Ce6-modified PEG-PNLG919 amphiphilic polymer was dissolved in 900 μL of tetrahydrofuran (THF), and 100-1000 μL of deionized water was added dropwise while stirring with a magnetic stirrer. The mixture was kept stirred at room temperature. Since THF is volatile, the amphiphilic polymer in the solution self-assembled as THF evaporated to obtain spherical nano-prodrug particles.

[0031] Traditional anti-tumor oxidative stress therapy based on chemical energy often has problems such as large side effects and insufficient immune activation ability. In view of these problems, the present invention has developed a polymeric prodrug nanoreactor for activating anti-tumor oxidative stress and long-term immune response to enhance the effect of tumor immunotherapy and reduce its toxic side effects. The mouse tumor model was established in the experiment and PBS and the polymeric prodrug nanoreactor 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. The mouse tumor volume was then recorded every two days. After 22 days of continuous recording, the mice were killed and the mouse tumor tissue was taken for immunofluorescence labeling, and the proportion of different immune cells was determined by flow analysis. The effect of different groups of drugs on tumor growth in tumor-bearing mice was evaluated by changes in tumor volume and the proportion of related immune cells in the tumor. The results showed that compared with mice treated with PBS, the tumor growth of mice treated with polymeric prodrug nanoreactors was significantly inhibited, and the proportion of effector T cells in the tumor increased while the proportion of regulatory T cells decreased, indicating that the polymeric prodrug nanoreactor can effectively improve the immunosuppressive microenvironment and improve the effect of tumor immunotherapy by inducing tumor cell oxidative stress apoptosis and inhibiting indoleamine 2,3-dioxygenase activity. In addition, the toxic and side effects of the nanoreactor on mice were evaluated by performing blood routine analysis and liver and kidney function tests on mice in the experimental and control groups. It was found that similar to the mice in the PBS group, the polymer prodrug nanoreactor had no obvious toxic and side effects on tumor-bearing mice.

[0032] The polymeric prodrug nanoreactor designed and prepared by the present invention can be used to activate anti-tumor oxidative stress and long-term immune response. It is recommended that tumor patients be intravenously injected with nanomedicines, once a day, with each dose of 0.1mg / kg-0.5mg / 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 phenol content in the patient's blood. Mouse experiments have found that the polymeric prodrug nanoreactor can induce anti-tumor immune response in tumor-bearing mice and reduce the potential toxic and side effects of oxalate derivatives. Therefore, compared with traditional tumor oxidative stress therapy, the polymeric prodrug nanoreactor can cause more significant tumor inhibition and immune activation effects.

Claims

1. A method for preparing a polymeric prodrug nanoreactor for activating anti-tumor oxidative stress and long-term immune response, characterized in that: The method is: Step 1: Preparation of hydrogen peroxide-responsive NLG919 monomers Dissolve oxalyl chloride in anhydrous dichloromethane, stir at -5 to 5°C for 25 to 35 minutes, then add 2-hydroxyethyl methacrylate dropwise and continue stirring for 2 to 3 hours; 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, and stir at -5 to 5°C overnight; then filter the solution and rotary evaporate to obtain hydrogen peroxide-responsive NLG919 monomer; Step 2: Preparation of Ce6-modified PEG-PNLG919 amphiphilic polymer The hydrogen peroxide-responsive NLG919 monomer, 2-aminoethyl methacrylate, polyethylene glycol-4-cyano-4-(phenylcarbonylthiothiothio) pentanoate and AIBN were dissolved in N,N-dimethylformamide solution for reaction, and the PEG-PNLG919 amphiphilic polymer was obtained by dialysis, deionized water precipitation and freeze-drying; then, the PEG-PNLG919 amphiphilic polymer was dissolved together with Ce6, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in anhydrous dimethyl sulfoxide, and the mixture was stirred at -5 to 5°C for 22 to 26 hours, filtered, dialyzed and freeze-dried to obtain the Ce6-modified PEG-PNLG919 amphiphilic polymer; Step 3: Preparation of polymeric prodrug nanoreactors for activation of anti-tumor oxidative stress and long-term immune responses 0.1-10 mg of Ce6-modified PEG-PNLG919 amphiphilic polymer was dissolved in 900 μL of tetrahydrofuran (THF), and 100-1000 μL of deionized water was added dropwise while stirring with a magnetic stirrer. The mixture was placed at room temperature and stirred continuously. The amphiphilic polymer in the solution self-assembled to obtain a spherical polymer prodrug nanoreactor.

2. The method for preparing a polymeric prodrug nanoreactor for activating anti-tumor oxidative stress and long-term immune response according to claim 1, characterized in that: In step 1, the mass ratio of oxalyl chloride, 2-hydroxyethyl methacrylate and NLG919 is 5-7:2-4:0.5-1.

3. The method for preparing a polymeric prodrug nanoreactor for activating anti-tumor oxidative stress and long-term immune response according to claim 1, characterized in that: In step 2, the molar ratio of the hydrogen peroxide-responsive NLG919 monomer, 2-aminoethyl methacrylate, polyethylene glycol-4-cyano-4-(phenylcarbonylthiothiothio) pentanoate and AIBN is 25-35:25-35:0.8-1.2:0.1-0.

5.

4. The method for preparing a polymeric prodrug nanoreactor for activating anti-tumor oxidative stress and long-term immune response according to claim 1, characterized in that: In step 2, the reaction is specifically as follows: nitrogen protection for 30 minutes and then reacting at 75-85° C. for 24 hours.

5. The method for preparing a polymeric prodrug nanoreactor for activating anti-tumor oxidative stress and long-term immune response according to claim 1, characterized in that: In step 2, the mass ratio of the PEG-PNLG919 amphiphilic polymer, Ce6, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 115-130:55-65:40-50:25-35.

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