Nitrogen-containing heterocyclic micromolecule modified polyethyleneimine polymer derivative as well as preparation method and application thereof

Abstract

The invention provides a preparation method and application of a nitrogen-containing heterocyclic micromolecule modified polyethyleneimine polymer derivative. Compared with a polyethyleneimine polymer, the nitrogen heterocyclic ring micromolecule modified polyethyleneimine polymer derivative provided by the invention has immunostimulatory activity. Besides, the polymer derivative material provided by the invention can be simply mixed with a protein antigen to form nanoparticles, so that efficient loading of the protein antigen is realized, the delivery efficiency of the protein antigen to lymph nodes is improved, and the polymer derivative material can be used for preparing a novel nano vaccine preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a polyethyleneimine polymer derivative modified by a nitrogen-containing heterocyclic small molecule and a preparation method and application thereof. BACKGROUND

[0002] Polyethyleneimine (PEI) is a cationic polymer. Due to its positive charge and multiple sites for modification, PEI has been developed for the delivery of negatively charged protein antigens and nucleic acid molecules to achieve anti-tumor immunotherapy. In addition to its delivery function as a carrier material, recent studies have found that PEI polymers have certain immunostimulatory activity. For example, PEI polymers are found to stimulate myeloid-derived suppressor cells (MDSCs) including tumor-associated macrophages (TAM) to repolarize from the immunosuppressive M2 phenotype to the M1 phenotype with tumor-killing activity. Due to its immunostimulatory activity, PEI polymers can be used as active carrier components to prepare nanoparticles with immunostimulatory effects for anti-tumor therapy.

[0003] However, PEI polymers still have the problems of insufficient immunostimulatory activity, low protein antigen loading efficiency, and low lymph node enrichment efficiency of the formed antigen-loaded nanoparticles, which greatly hinders the application of PEI as an anti-tumor immunonanocarrier. Therefore, it is a key technical problem to be solved to find a suitable structural modification strategy to improve the immunostimulatory activity of PEI polymers and the loading efficiency and lymph node delivery efficiency of PEI for antigens. SUMMARY

[0004] To solve the above key technical problems, the present application aims to provide a PEI polymer derivative modified by a nitrogen-containing heterocyclic small molecule and a preparation method and application thereof. The PEI polymer derivative modified by a nitrogen-containing heterocyclic small molecule has a multivalent effect due to the bonding of nitrogen-containing heterocyclic groups at multiple sites, so that the polymer derivative itself has immunostimulatory activity and can be used as a carrier and / or active component to prepare nanoparticles with immunostimulatory effects. In addition, the PEI polymer derivative modified by a nitrogen-containing heterocyclic small molecule can improve the loading efficiency of negatively charged protein antigens through multiple site interactions and electrostatic interactions, form nanoparticles with protein antigens through simple physical mixing to achieve efficient loading of protein antigens, and improve the delivery efficiency of protein antigens to lymph nodes, and can be used to prepare novel nanovaccines.

[0005] In one aspect, the present application provides a polyethyleneimine (PEI) polymer derivative modified by a nitrogen-containing heterocyclic small molecule.

[0006] In some embodiments, the polyethyleneimine is a hyperbranched polyethyleneimine or a linear polyethyleneimine, preferably a hyperbranched polyethyleneimine.

[0007] In some embodiments, the hyperbranched polyethyleneimine is CAS 9002-98-6.

[0008] In some embodiments, the polyethyleneimine has a molecular weight of 10,000 to 40,000 Dalton (Da), preferably 25,000 Da. Here, the molecular weight refers to the molecular weight measured by gel permeation chromatography.

[0009] In some embodiments, the nitrogen-containing heterocyclic small molecule is a nitrogen-containing heterocyclic molecule containing a benzimidazole, quinoline, pyrimidine or purine structure and containing a carboxyl group, preferably any one or more of 2-pyridin-4-yl-3H-benzimidazole-5-carboxylic acid, 2-phenyl-4-quinoline carboxylic acid, 2-(6-(((benzyloxy)carbonyl)amino)-9H-purin-9-yl)acetic acid and 2-(6-(((dibenzyl oxy)carbonyl)amino)-9H-purin-9-yl)acetic acid. More preferably, any one or more of 2-pyridin-4-yl-3H-benzimidazole-5-carboxylic acid and 2-(6-(((dibenzyl oxy)carbonyl)amino)-9H-purin-9-yl)acetic acid.

[0010] The modification rate of the nitrogen-containing heterocyclic small molecule in the nitrogen-containing heterocyclic small molecule modified polyethyleneimine polymer derivative is 5% to 65%, preferably 10 to 45%. Here, the modification rate refers to the ratio of the characteristic peak area of the nitrogen-containing heterocyclic small molecule to the characteristic peak area of the polyethyleneimine polymer after correction by the number of non-active hydrogens in the structure in the nuclear magnetic hydrogen spectrum of the nitrogen-containing heterocyclic small molecule modified polyethyleneimine polymer derivative when heavy water is used as the solvent.

[0011] In some embodiments, the nitrogen-containing heterocyclic small molecule modified polyethyleneimine polymer derivative prepared by the present application itself has immunostimulatory activity and can induce TAM to repolarize from the immunosuppressive M2 phenotype to the M1 phenotype with tumor killing activity.

[0012] In another aspect, the present application provides a method for preparing the nitrogen-containing heterocyclic small molecule modified polyethyleneimine polymer derivative.

[0013] The nitrogen-containing heterocyclic small molecule is bonded to the amino group of the polyethyleneimine through amide condensation reaction.

[0014] In another aspect, the present application provides a protein nanovaccine preparation comprising the above-mentioned nitrogen-containing heterocyclic small molecule modified PEI polymer derivative and a protein antigen. Preferably, the mass ratio of the PEI polymer derivative to the protein antigen is 1:1. Preferably, the initial concentration of the protein antigen is 1 mg / mL. The above-mentioned physical mixing process requires that the PEI polymer derivative solution is slowly added dropwise to the protein antigen solution under stirring, and the stirring is continued for 5 minutes.

[0015] In some embodiments, the prepared protein nanovaccine formulation has a particle size of 50-350 nm, preferably 100-250 nm.

[0016] Here, the particle size refers to the particle size measured by dynamic light scattering method. The above prepared protein nanovaccine formulation can be efficiently enriched in the lymph nodes of mice after subcutaneous injection.

[0017] In some embodiments, the above nitrogen-containing heterocyclic small molecule modified PEI polymer derivative and protein antigen are obtained by simple physical mixing.

[0018] On the other hand, the present application provides the use of the above nitrogen-containing heterocyclic small molecule modified PEI polymer derivative in the preparation of a drug or vaccine for treating and / or preventing breast cancer, especially triple-negative breast cancer.

[0019] Advantages The present application utilizes nitrogen-containing heterocyclic small molecule modified PEI polymer, and the obtained PEI polymer derivative has significant immune stimulating activity compared with PEI polymer, which can induce TAM to repolarize from immunosuppressive M2 phenotype to tumor-killing M1 phenotype, and can be used as a carrier component to prepare nanoparticles with immune stimulating effect for realizing antitumor immunotherapy. In addition, the nitrogen-containing heterocyclic small molecule modified PEI polymer derivative provided by the present application can be used in the field of vaccines. Compared with PEI, the nitrogen-containing heterocyclic small molecule modified PEI polymer derivative has both positively charged amine groups and nitrogen-containing heterocyclic groups in its structure, which can realize efficient loading of negatively charged protein antigens through electrostatic interaction and the multivalent effect of the bonded nitrogen-containing heterocyclic groups. In addition, the preparation method of the protein nanovaccine formulation based on the nitrogen-containing heterocyclic small molecule modified PEI polymer derivative provided by the present application can realize the preparation of the protein nanovaccine formulation through simple physical mixing of the PEI polymer derivative and the protein antigen, which is simple and mild in operation. In addition, the above protein nanovaccine formulation can be efficiently enriched in the lymph nodes after subcutaneous injection, which effectively improves the delivery efficiency of antigens to the lymph nodes. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Nuclear magnetic resonance hydrogen spectrum of the nitrogen-containing heterocyclic small molecule 2-pyridin-4-yl-3H-benzimidazole-5-carboxylic acid modified PEI polymer derivative P18 prepared in Example 1 of the present application.

[0021] Figure 2 Nuclear magnetic resonance hydrogen spectrum of the nitrogen-containing heterocyclic small molecule 2-phenyl-4-quinoline carboxylic acid modified PEI polymer derivative P27 prepared in Example 1 of the present application.

[0022] Figure 3NMR spectrum of the hydrogen of the PEI polymer derivative P80 modified by the nitrogen-containing heterocyclic small molecule 2-(6-(((benzyloxy)carbonyl)amino)-9H-purin-9-yl)acetic acid prepared in Example 1 of the present application.

[0023] Figure 4 NMR spectrum of the hydrogen of the PEI polymer derivative P81 modified by the nitrogen-containing heterocyclic small molecule 2-(6-(((dibenzyl oxy)carbonyl)amino)-9H-purin-9-yl)acetic acid prepared in Example 1 of the present application.

[0024] Figure 5 Flow cytometry characterization chart of the PEI polymer derivative modified by the nitrogen-containing heterocyclic small molecule in Example 2 of the present application in inducing M2 type macrophages to polarize to M1 type macrophages in vitro; single factor ANOVA (ANOVA) was used for comparison among multiple groups, *p<0.05, **p<0.01 and ***p<0.001.

[0025] Figure 6 Dynamic light scattering particle size distribution chart of the protein nano vaccine prepared by mixing the PEI polymer derivative modified by the nitrogen-containing heterocyclic small molecule in Example 3 of the present application and ovalbumin (OVA) antigen.

[0026] Figure 7 Characterization results of the enrichment of the protein vaccine nano preparation prepared in Example 4 of the present application in the mouse lymph nodes after subcutaneous injection (the left chart is the imaging results chart of the mouse lymph node ex vivo tissue, and the right chart is the quantitative results of the fluorescence intensity in the mouse lymph node ex vivo tissue); single factor ANOVA (ANOVA) was used for comparison among multiple groups, *p<0.05. DETAILED DESCRIPTION

[0027] The present application is further described below in conjunction with examples, but the present application is not limited to the following examples. The concentration, volume, etc. of the solution used can be adjusted as needed.

[0028] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0029] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0030] 2-pyridin-4-yl-3H-benzimidazole-5-carboxylic acid, 2-phenyl-4-quinolinecarboxylic acid, 2-(6-(((benzyloxy)carbonyl)amino)-9H-purin-9-yl)acetic acid, 2-(6-(((dibenzyl oxy)carbonyl)amino)-9H-purin-9-yl)acetic acid, hyperbranched polyethyleneimine (CAS No. 9002-98-6) and OVA were purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were purchased from Shanghai Shaoyuan Technology Co., Ltd. Anti-mouse APC-CD80 flow antibody was purchased from Dakewe Biotechnology Co., Ltd. RMPI 1640 and DMEM medium were purchased from Shanghai Weibi Biological Technology Co., Ltd. CY5-NHS was purchased from Dalian Milen Biotechnology Co., Ltd. Other reagents used in the experiment were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. if not specifically specified.

[0031] Eppendorf centrifuge (Eppendorf, Germany); 600 megahertz nuclear magnetic resonance spectrometer (AVANCE III 600, Swiss); laser particle size analyzer (ZEN3690, Malven, USA); electronic balance (BP190S, Sartorius); cell counter (Invitrogen, USA); biological safety cabinet (LABCONCO, USA); pure water instrument (Millipore, USA); carbon dioxide incubator (Forma Series II, Thermo-Fisher Scientific, USA); freeze dryer (12L, LABCONCO, USA); flow cytometer (Fortessa, BD Bioscience, USA); small animal live imaging instrument (IVIS Spectrum, USA).

[0032] Example 1 Preparation of polyethyleneimine polymer derivatives modified with nitrogen-containing heterocyclic small molecules The 30 times equivalent nitrogen-containing heterocyclic small molecules (as shown in Table 1) were dissolved in dimethyl sulfoxide (DMSO) and added to the reaction bottle, then a DMSO solution containing 45 times equivalent N-hydroxysuccinimide (NHS) and 45 times equivalent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) was added, and the reaction was stirred for half an hour to activate the carboxyl group. Then, 1 equivalent of hyperbranched PEI polymer with a molecular weight of 25000 Da was dissolved in DMSO, and the PEI polymer solution was added dropwise into the above reaction bottle, and the reaction was stirred overnight at room temperature, then the reaction solution was diluted with methanol, precipitated with ethyl ether to obtain the PEI polymer derivative product. The obtained PEI polymer derivative product was redissolved after precipitation, and was dialyzed in a cellulose dialysis bag with a molecular weight of 3500 Da in pure water medium for 48 hours, and then freeze-dried to obtain a solid product, which was characterized by nuclear magnetic resonance hydrogen spectrum using heavy water as a solvent. The modification rate of the nitrogen-containing heterocyclic small molecule modified PEI polymer derivative was calculated according to the characteristic peak area of the nitrogen-containing heterocyclic small molecule and the characteristic peak area of the PEI polymer in the nuclear magnetic resonance hydrogen spectrum, and then corrected by the number of non-active hydrogens in the structure.

[0033] The nuclear magnetic hydrogen spectrum of the 2-pyridin-4-yl-3H-benzimidazole-5-carboxylic acid (No. 18) modified PEI polymer derivative (denoted as P18) is shown in Figure 1 The peaks at 1.47-3.49 ppm are characteristic peaks of the PEI part, and the peaks at 6.96-8.40 ppm are characteristic peaks of the nitrogen-containing heterocyclic small molecule 18, indicating that the nitrogen-containing heterocyclic small molecule and PEI are successfully connected, and the modification rate of the product P18 is 41.14% according to the calculation of the characteristic peak area of the nuclear magnetic hydrogen spectrum.

[0034] The nuclear magnetic hydrogen spectrum of the 2-phenyl-4-quinoline carboxylic acid (No. 27) modified PEI polymer derivative (denoted as P27) is shown in Figure 2 The peaks at 1.45-3.53 ppm are characteristic peaks of the PEI part, and the peaks at 7.22-8.34 ppm are characteristic peaks of the nitrogen-containing heterocyclic small molecule 27, indicating that the nitrogen-containing heterocyclic small molecule and PEI are successfully connected, and the modification rate of the product P27 is 43.20% according to the calculation of the characteristic peak area of the nuclear magnetic hydrogen spectrum.

[0035] The nuclear magnetic hydrogen spectrum of the 2-(6-(((benzyloxy)carbonyl)amino)-9H-purin-9-yl)acetic acid (No. 80) modified PEI polymer derivative (denoted as P80) is shown in Figure 3 The peaks at 1.49-3.56 ppm are characteristic peaks of the PEI part, and the peaks at 6.25-8.40 ppm are characteristic peaks of the nitrogen-containing heterocyclic small molecule 80, indicating that the nitrogen-containing heterocyclic small molecule and PEI are successfully connected, and the modification rate of the product P80 is 30.54% according to the calculation of the characteristic peak area of the nuclear magnetic hydrogen spectrum.

[0036] 2-(6-(((dibenzyl oxy) carbonyl) amino)-9H-purin-9-yl) acetic acid (No. 81) modified PEI polymer derivative (denoted as P81) was prepared. The 1H NMR spectrum of the product is shown in Figure 4 Figure 2. The peaks at 1.42-3.63 ppm are characteristic peaks of the PEI moiety, and the peaks at 6.98-8.02 ppm are characteristic peaks of the nitrogen-containing heterocyclic small molecule 81, indicating that the nitrogen-containing heterocyclic small molecule and PEI are successfully connected. According to the characteristic peak area of the 1H NMR spectrum, the modification rate of the product P81 is calculated to be 12.80%.

[0037] Table 1. Number, Chinese name and structure of nitrogen-containing heterocyclic small molecules in Example 1 of the present application;

[0038] Example 2 Evaluation of the immune stimulating activity of nitrogen-containing heterocyclic small molecule modified polyethyleneimine polymer derivatives on macrophages in vitro The nitrogen-containing heterocyclic small molecule modified PEI polymer derivatives (P18, P27, P80 and P81) prepared in Example 1 were evaluated for their ability to induce M2 macrophages to polarize to M1 macrophages in vitro by flow cytometry to characterize the immune stimulating activity of the polymer derivatives themselves. RAW264.7 macrophages (from the Shanghai Cell Bank of the Chinese Academy of Sciences) were seeded at 200,000 cells per well in a 24-well plate and incubated overnight in DMEM medium containing 10% 4T1 cell culture supernatant to induce M2 macrophages. After overnight incubation, the old medium was discarded and DMEM medium containing 5 μg / mL PEI, P18, P27, P80 and P81 was added, respectively. After 24 hours of incubation, the cells were collected and stained with APC-CD80 flow cytometry antibody, and the proportion of M1 macrophages was analyzed by flow cytometry, wherein M1 macrophages were labeled as CD80 + macrophage population. The flow cytometry characterization results are shown in Figure 5 Figure 3. The results show that the nitrogen-containing heterocyclic small molecule modified PEI polymer derivatives P18, P80 and P81 can significantly induce M2 macrophages to polarize to M1 macrophages.

[0039] Example 3 Preparation of protein nanovaccine formulations based on nitrogen-containing heterocyclic small molecule modified polyethyleneimine polymer derivatives The PEI and the nitrogen-containing heterocyclic small molecule modified PEI polymer derivatives (P18, P27, P80 and P81) prepared in Example 1 were respectively mixed with the protein antigen OVA to prepare the protein nanovaccine formulations loaded with OVA. The OVA was dissolved in water to prepare a 1 mg / mL solution, and the PEI derivatives were dissolved in water to prepare a 10 mg / mL solution. Under stirring, 100 μL of the polymer solution was added dropwise to 1 mL of the OVA solution (OVA and PEI or PEI polymer derivative mass ratio was 1:1), and the stirring was continued for 5 minutes. Then the particle size and the polymer dispersibility index (PDI) were determined by dynamic light scattering (DLS) particle size instrument (ZEN3690, Malven, USA), and the results were shown in Table 2 and Table 3, respectively. Under the condition that the mass ratio of the added protein antigen OVA to the polymer was 1:1, compared with the protein nanovaccine formulation PEI-OVA prepared based on PEI, the protein nanovaccine formulations P18-OVA, P27-OVA, P80-OVA and P81-OVA prepared based on the nitrogen-containing heterocyclic small molecule modified PEI polymer derivatives had smaller particle size and smaller PDI value. Figure 6

[0040] Table 2 Particle size and PDI of the protein nanovaccine formulations prepared based on the nitrogen-containing heterocyclic small molecule modified PEI polymer derivatives in Example 3 of the present application

[0041] Example 4 Evaluation of the enrichment of the protein nanovaccine formulations based on the nitrogen-containing heterocyclic small molecule modified polyethyleneimine polymer derivatives in lymph nodes Preparation of Cy5 fluorescently labeled OVA (OVA-Cy5). A 10 mg / mL DMSO solution of Cy5-NHS was prepared. 300 mg of OVA was dissolved in 15 mL of pure water, and 200 μL of the Cy5-NHS solution and 45 μL of triethylamine (TEA) were added under stirring in the dark. After overnight reaction at room temperature in the dark, the product was freeze-dried after dialysis with pure water for one day to obtain a blue loose powder.

[0042] ​Cy5 fluorescently labeled protein nanovaccine formulations (PEI-OVA-Cy5, P18-OVA-Cy5, P27-OVA-Cy5, P80-OVA-Cy5, and P81-OVA-Cy5) were prepared by physical mixing with PEI and the nitrogen-containing heterocyclic small molecule modified PEI polymer derivatives (P18, P27, P80-OVA-Cy5, and P81-OVA-Cy5) prepared above. Specifically, OVA-Cy5 was dissolved in water to prepare a 1 mg / mL solution, and PEI or nitrogen-containing heterocyclic small molecule modified PEI polymer derivatives were dissolved in water to prepare a 10 mg / mL solution. Under stirring conditions, 100 μL of the above polymer solution was added dropwise to 1 mL of OVA-Cy5 solution (the mass ratio of added OVA-Cy5 to PEI or PEI polymer derivative was 1:1), and stirring was continued for 5 minutes to prepare Cy5 fluorescently labeled protein nanovaccine formulations.

[0043] Balb / c mice: 4-6 weeks old, 18-22 g, purchased from Beijing Huafukang Biotechnology Co., Ltd., and the breeding conditions and experimental procedures were in accordance with the relevant requirements and standards of the Ethics Committee for the Management and Use of Laboratory Animals of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0044] In healthy Balb / c mice, the prepared OVA-Cy5 and Cy5 fluorescently labeled protein nanovaccine formulations were administered subcutaneously at a dose of 36.8 μg Cy5 per mouse. Eight hours after administration, the mice were euthanized, and the inguinal lymph nodes were harvested and images were acquired using a live imaging system. The fluorescence distribution of the inguinal lymph nodes after subcutaneous injection of the OVA-Cy5 and Cy5 fluorescently labeled protein nanovaccine formulations is shown below. Figure 7 As shown, the results indicate that, compared with the protein nanovaccine formulation PEI-OVA-Cy5 prepared based on PEI, the protein nanovaccine formulations P18-OVA-Cy5, P27-OVA-Cy5, and P81-OVA-Cy5 prepared based on PEI polymer derivatives modified with nitrogen-containing heterocyclic small molecules can effectively accumulate in lymph nodes, thus effectively improving the delivery efficiency of antigen to lymph nodes.

Claims

1. A nitrogen-containing heterocyclic small molecule modified polyethyleneimine polymer derivative.

2. The polymer derivative according to claim 1, wherein, The polyethyleneimine is hyperbranched polyethyleneimine or linear polyethyleneimine, preferably hyperbranched polyethyleneimine.

3. The polymer derivative according to claim 1, wherein, The molecular weight of the polyethyleneimine can be 10000 to 40000 Dalton (Da), preferably 25000 Da.

4. The polymer derivative according to claim 1, wherein, The nitrogen-containing heterocyclic small molecule is a nitrogen-containing heterocyclic molecule containing a benzimidazole, quinoline, pyrimidine or purine structure and containing a carboxyl group, preferably any one or more of 2-pyridin-4-yl-3H-benzimidazole-5-carboxylic acid, 2-phenyl-4-quinoline carboxylic acid, 2-(6-(((benzyloxy)carbonyl)amino)-9H-purin-9-yl)acetic acid and 2-(6-(((dibenzyl oxy)carbonyl)amino)-9H-purin-9-yl)acetic acid, more preferably any one or more of 2-pyridin-4-yl-3H-benzimidazole-5-carboxylic acid and 2-(6-(((dibenzyl oxy)carbonyl)amino)-9H-purin-9-yl)acetic acid.

5. The polymer derivative according to claim 1, wherein, The modification rate of the nitrogen-containing heterocyclic small molecule in the nitrogen-containing heterocyclic small molecule modified polyethyleneimine polymer derivative is 5% to 65%, preferably 10 to 45%. 6.A method for preparing the polymer derivative according to any one of claims 1 to 5, wherein, The nitrogen-containing heterocyclic small molecule is bonded to the amino group of the polyethyleneimine through an amide condensation reaction. 7.A protein nanovaccine preparation comprising the polymer derivative according to any one of claims 1 to 5 and a protein antigen, preferably, the mass ratio of the polyethyleneimine polymer derivative to the protein antigen is 1:

1.

8. The protein nanovaccine formulation according to claim 7, wherein, The particle size of the protein nanovaccine preparation is 50 to 350 nm, preferably 100 to 250 nm. 9.Use of the polymer derivative according to any one of claims 1 to 5 in the preparation of a drug or vaccine for treating and / or preventing breast cancer, in particular triple-negative breast cancer.

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