A phenylboronic acid copolymer for reshaping the tumor immune microenvironment and its application

Abstract

The present invention discloses a phenylboronic acid copolymer for reshaping the tumor immune microenvironment and its application. The phenylboronic acid copolymer is formed by copolymerizing phenylboronic acid (PBA), polymeric CXCR4 antagonist AMD3100, and cholesterol. First, the AMD3100 moiety is polymerized through a Michael addition reaction, and then PBA and cholesterol are further modified to obtain the copolymer. The phenylboronic acid copolymer of the present invention can self-assemble into ATP-hypersensitive nanogels, which can load chemotherapeutic drugs inside and synergistically deliver albumin nanoparticles through electrostatic adsorption outside, realizing ATP-sensitive hierarchical delivery. It can significantly inhibit the number and activity of TAMs in the tumor microenvironment, reduce the recruitment of Tregs, amplify the immune signals in the microenvironment, reshape the tumor immune microenvironment in multiple dimensions, and enhance the effect of anti-tumor immune response.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a phenylboronic acid copolymer for remodeling the tumor immune microenvironment and its application. Background Art

[0002] Malignant tumors are a major category of diseases that seriously endanger human life and health and have become one of the main causes of death worldwide. In recent years, the successful application of tumor immunotherapy represented by methods such as adoptive cell immunotherapy (CAR-T), immune checkpoint blockade (PD-L1), and tumor vaccines has brought new hope for humans to overcome cancer. To further improve the effect of immunotherapy, researchers have proposed a clinical development strategy from single-drug treatment to combination therapy, such as the combination of immunotherapy with chemotherapy, radiotherapy, etc. When chemotherapy drugs are combined with immunotherapy, a significant enhancement of tumor-specific immune responses is shown. Currently, the FDA has approved 11 chemotherapy-immunotherapy combination treatment regimens, showing remarkable prospects.

[0003] The immunosuppression of the tumor microenvironment often leads to significant differences in the efficacy of existing immunotherapy regimens among different patients. The tumor microenvironment is the "soil" on which tumor cells depend for survival and is conducive to the occurrence and development of tumors. The inhibitory microenvironment will hinder the aggregation of effector T cells towards tumors, promote tumor immune escape, and result in poor effects of T cell-mediated immunotherapy. The chemokine system plays a key role in all aspects of tumor growth and metastasis. Research shows that the expression of CXCR4 in tumor cells is significantly higher than that in normal tissues. By interacting with its ligand CXCL12, it can induce the migration of regulatory T cells (Tregs) into the tumor microenvironment, reduce the tumor infiltration of effector T cells, and promote tumor cell immune tolerance. Therefore, CXCR4 has become an ideal target for tumor immunotherapy. CXCR4 antagonists such as AMD3100 have been proven to effectively block the physiological function of the CXCR4 / CXCL12 axis, restore the tumor tropism and effector function of T cells, and inhibit the growth and metastasis of tumor cells.

[0004] Tumor-associated macrophages (TAMs) are an important component of the tumor microenvironment, participating in tumor immune escape and promoting tumor malignant progression. Inhibiting the recruitment and activity of TAMs by regulating colony-stimulating factor-1 (CSF-1) is a potential therapeutic approach to hinder tumor progression. BLZ-945 is an effective selective CSF-1R inhibitor, which can inhibit CSF-1R phosphorylation, significantly reduce the number of TAMs, activate the specific killing effect of cytotoxic T cells, and thus enhance the anti-tumor effect of chemotherapeutic drugs. Therefore, combining the immunotherapeutic drug BLZ-945 and AMD3100 with chemotherapeutic drugs for anti-tumor treatment can not only reshape the tumor immune microenvironment in multiple dimensions, enhance the anti-tumor immune response, but also make up for the disadvantages of large toxicity and easy drug resistance of chemotherapeutic drugs, playing a synergistic and enhancing therapeutic role.

[0005] Phenylboronic acid (PBA) is a class of excellent recognition agents for diol units, which can reversibly react with polyhydroxy compounds with adjacent diol or meta-diol structures in aqueous solution to form covalent complexes. Based on this property, PBA has a high affinity for the diol unit on the pentose ring of adenosine triphosphate (ATP), and realizes drug delivery by responding to the high concentration of ATP in the tumor microenvironment, expanding new ideas for the design of stimulus-responsive systems and tumor treatment. Summary of the Invention

[0006] The object of the present invention is to provide a phenylboronic acid copolymer for reshaping the tumor immune microenvironment. By Michael addition reaction, the CXCR4 antagonist AMD3100 moiety is polymerized, and further modified with phenylboronic acid (PBA) and cholesterol to obtain a phenylboronic acid-polymerized AMD3100-cholesterol cationic polymer. Among them, the polymerized AMD3100 can form a cationic "therapeutic carrier", and PBA and cholesterol are used for drug loading and ATP-sensitive drug release.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A phenylboronic acid copolymer for reshaping the tumor immune microenvironment, the structural formula of which is shown in formula (Ⅰ):

[0009]

[0010] Wherein: n is any natural number from 1 to 100; preferably, n is any natural number from 30 to 60.

[0011] In an embodiment of the present invention, the above phenylboronic acid copolymer is prepared by the following steps:

[0012] Step 1, 1,6 - hexanediamine and acryloyl chloride are subjected to a substitution reaction to obtain hexamethylenebisacrylamide (HMBA). The molar ratio of 1,6 - hexanediamine to acryloyl chloride is 1.0:(1.5 - 3.0). The substitution reaction temperature is - 5 - 10°C, and the reaction time is 2 - 6 h. Preferably, the molar ratio of 1,6 - hexanediamine to acryloyl chloride is 1.0:2.1, the substitution reaction temperature is 0 - 5°C, and the reaction time is 4 h.

[0013] Specifically, 1,6 - hexanediamine is dissolved in water, and then a dichloromethane solution of acryloyl chloride and a saturated sodium hydroxide solution are simultaneously added dropwise thereto. After the addition, stirring reaction is carried out.

[0014] Step 2, HMBA and AMD3100 are subjected to a Michael addition reaction to prepare polymerized AMD3100 (poly - AMD, hereinafter referred to as pAMD), and the polymer is capped. The molar ratio of HMBA to AMD3100 is 1.0:(0.5 - 2.0). The conditions for the Michael addition reaction are light avoidance and nitrogen protection. The reaction temperature is 20 - 50°C, the reaction time is 1 - 5 d, and the capping reaction time is 4 - 24 h. Preferably, the molar ratio of HMBA to AMD3100 is 1.0:1.0, the Michael addition reaction temperature is 37°C, the reaction time is 3 d, and the capping reaction time is 12 h.

[0015] Specifically, HMBA and AMD3100 are respectively weighed and dissolved in a mixed solvent of methanol and water, and reacted under nitrogen protection and light avoidance. Then AMD3100 is added, and the polymer is capped by reacting under nitrogen protection and light avoidance. The capped polymer is dialyzed to obtain pAMD.

[0016] Step 3, pAMD and cholesteryl chloroformate are subjected to an acylation reaction to prepare a pAMD - cholesterol (pAMD - Ch, hereinafter referred to as PC) cationic polymer. The molar ratio of pAMD to cholesteryl chloroformate is 1.0:(5.0 - 20.0). The acylation reaction includes two stages. The temperature of the first stage is - 5 - 10°C, the temperature of the second stage is 15 - 35°C, the time of the first stage is 0 - 4 h, and the time of the second stage is 12 - 48 h. Preferably, the molar ratio of pAMD to cholesteryl chloroformate is 1.0:11.1, the temperature of the first stage of the acylation reaction is 0 - 5°C, the temperature of the second stage is 25°C, the time of the first stage is 1 h, and the time of the second stage is 24 h.

[0017] Specifically, pAMD is weighed and dissolved in anhydrous dichloromethane, a solution of the acid - binding agent DIPEA is added, and then a solution of cholesteryl chloroformate in anhydrous dichloromethane is added dropwise. First, the reaction is carried out in an ice bath, and then the reaction is carried out at room temperature to obtain the product pAMD - Ch (PC).

[0018] Step 4: 4-Carboxyphenylboronic acid pinacol ester and N-boc-ethylenediamine (EDA-boc) are subjected to a dehydration condensation reaction, and then the boc protecting group is removed with a strong acid to prepare phenylboronic acid-ethylenediamine (PBA-EDA). The molar ratio of 4-carboxyphenylboronic acid pinacol ester to EDA-boc is 1.0:(1.0 - 2.0). The temperature of the dehydration condensation reaction is 15 - 35°C, and the reaction time is 12 - 48 h. Preferably, the molar ratio of 4-carboxyphenylboronic acid pinacol ester to EDA-boc is 1.0:1.2; the temperature of the dehydration condensation reaction is 25°C, and the reaction time is 24 h.

[0019] Specifically, 4-carboxyphenylboronic acid pinacol ester, EDC·HCl, HOBt, and DIEA are separately dissolved in anhydrous DMF, and the above solutions are mixed and reacted at room temperature to activate the carboxyl group of phenylboronic acid. Then N-boc-ethylenediamine is added for reaction. The reaction solution is transferred to a separatory funnel, ethyl acetate is added to dissolve the product, and then it is washed with HCl solution and saturated sodium bicarbonate solution respectively. The organic phase is taken and anhydrous sodium sulfate is added for water removal, and the PBA-EDA-boc product is obtained through silica gel column treatment. PBA-EDA-boc is taken and added to HCl-ethyl acetate solution to remove the boc protecting group, and the PBA-EDA product is obtained.

[0020] Step 5: PBA-EDA is grafted and modified onto pAMD-Ch to prepare the phenylboronic acid-pAMD-Ch (PBA-pAMD-Ch, hereinafter referred to as PPC) cationic polymer. The molar ratio of PBA-EDA to pAMD-Ch is (100 - 200):1. The reaction temperature is 15 - 35°C, and the reaction time is 1 - 6 d. Preferably, the molar ratio of PBA-EDA to pAMD-Ch is 155:1; the reaction temperature is 25°C, and the reaction time is 4 d.

[0021] Specifically, pAMD-Ch is dissolved in DMF, CDI is added for reaction, and then the DMF solution of PBA-EDA is added for reaction. The reaction solution is added to anhydrous ether, the precipitate is collected by centrifugation, dissolved in a mixed solvent of ethanol and water, and the solution is dialyzed and then freeze-dried to obtain the phenylboronic acid-pAMD-Ch (PPC) cationic polymer.

[0022] Use of the above phenylboronic acid copolymer for remodeling the tumor immune microenvironment in the preparation of tumor therapeutic drugs.

[0023] A tumor therapeutic drug, comprising a nanogel prepared from the above phenylboronic acid copolymer.

[0024] Furthermore, the nanogel encapsulates chemotherapeutic drugs. The chemotherapeutic drugs are one or more of cisplatin, oxaliplatin, paclitaxel, gemcitabine, doxorubicin, or other anti-tumor drugs.

[0025] In one embodiment of the present invention, the preparation method of the nanogel is as follows:

[0026] Step 1, dissolve the phenylboronic acid copolymer (PPC polymer) and the chemotherapeutic drug in an organic solvent, and form a film by rotary evaporation under reduced pressure;

[0027] Step 2, add deionized aqueous solution to disperse the film, and obtain drug-loaded nanogels with uniform particle size distribution by probe sonication.

[0028] Furthermore, the tumor therapeutic drug further includes albumin nanoparticles, and the albumin nanoparticles are adsorbed on the surface of the nanogel.

[0029] In one embodiment of the present invention, the albumin nanoparticles are albumin nanoparticles modified with the CSF-1R inhibitor BLZ-945. The composite nanogel formed by the nanogel made of the phenylboronic acid copolymer and the albumin nanoparticles can achieve the three-drug loading of the CXCR4 antagonist AMD3100, the CSF-1R inhibitor BLZ-945, and the chemotherapeutic drug paclitaxel and ATP-sensitive hierarchical delivery.

[0030] Specifically, the preparation of the composite nanogel includes the following steps:

[0031] Step 1, dissolve the phenylboronic acid copolymer (PPC polymer) and the chemotherapeutic drug in an organic solvent, and form a film by rotary evaporation under reduced pressure;

[0032] Step 2, add deionized aqueous solution to disperse the film, and obtain a drug-loaded nanogel core with uniform particle size distribution by probe sonication;

[0033] Step 3, mix the nanogel core in Step 2 and the albumin nanoparticle solution modified with the CSF-1R inhibitor BLZ-945 according to a certain volume ratio, vortex for a period of time and then let it stand to obtain the composite nanogel;

[0034] The organic solvent in Step 1 is one or more of dichloromethane, chloroform, ether, ethyl acetate, acetone, ethanol, or methanol; preferably, the organic solvent is methanol;

[0035] The albumin in Step 3 is one or more of human serum albumin (HSA), bovine serum albumin (BSA), ovalbumin (OVA), mouse serum albumin (RSA or MSA), or other albumins; preferably, the albumin is BSA.

[0036] The particle size of the composite nanogel described in Step 3 is 10 - 500 nm; preferably, the particle size of the composite nanogel is 100 - 200 nm.

[0037] Advantages of the present invention:

[0038] (1) The phenylboronic acid copolymer provided in the present invention for remodeling the tumor immune microenvironment can self-assemble to form an ATP-hypersensitive composite nanogel for realizing the three-drug loading and hierarchical delivery of the CXCR4 antagonist AMD3100, the CSF-1R inhibitor BLZ-945, and the chemotherapeutic drug paclitaxel, and can remodel the tumor immune microenvironment in multiple dimensions, amplify the immune signals in the microenvironment, and enhance the anti-tumor immune response, providing a novel and efficient chemotherapy-immunotherapy combined treatment plan for clinical anti-tumor treatment.

[0039] (2) The phenylboronic acid copolymer provided in the present invention for remodeling the tumor immune microenvironment is prepared by a mild and easy-to-perform polymer chemical reaction, is environmentally friendly, has simple raw materials, and a high yield. Then, the composite nanogel is prepared by the thin-film dispersion method and electrostatic adsorption, without complex equipment and operation methods, and has good industrial production value.

[0040] (3) The phenylboronic acid copolymer provided in the present invention for remodeling the tumor immune microenvironment shows good biocompatibility, can prepare a composite nanogel with low safety and toxicity, improves its deep penetration and cellular uptake in tumor tissues, and has potential clinical application prospects. Description of the drawings

[0041] Figure 1 1H NMR spectrum of HMBA 1 H NMR) spectrum.

[0042] Figure 2 1H NMR and mass spectrometry (MS) spectra of PBA-EDA-boc and PBA-EDA 1 H NMR) and mass spectrometry (MS) spectra.

[0043] Figure 3 1H NMR spectra of pAMD, pAMD-Ch(PC), and PBA-pAMD-Ch(PPC) 1 H NMR) spectra.

[0044] Figure 4 1H NMR and mass spectrometry (MS) spectra of carboxylated BLZ945 (BLZ-COOH)

[0045] Figure 5Transmission electron microscopy (TEM) planar images of albumin nanoparticles (BB) modified with CSF-1R inhibitor BLZ-945, PPC (PPC-PTX) nanogels loaded with paclitaxel, PPC (BB@PPC-PTX) composite nanogels loaded with both paclitaxel and electrostatically adsorbed albumin nanoparticles, and solutions of composite nanogels incubated with 0.1 mM ATP (BB@PPC-PTX + 0.1 mM ATP), and the diameters of the above-mentioned preparations shown in the TEM images. The scale bar is 100 nm.

[0046] Figure 6 In vitro release profiles of rhodamine B-labeled BSA (RhB-BSA) in each nanogel group treated with different concentrations of ATP.

[0047] Figure 7 Time-dependent release of paclitaxel (PTX) from PPC (PPC-PTX) nanogels loaded with paclitaxel in PBS at pH 5.0, 6.8, and 7.4.

[0048] Figure 8 To evaluate the apoptosis induced by PBS, PPC (20 μg / mL), albumin nanoparticles modified with CSF-1R inhibitor BLZ-945 (BB, 20 μg / mL), paclitaxel (PTX, 5 μg / mL), PPC (PPC-PTX) nanogels loaded with paclitaxel, and PPC (BB@PPC-PTX) composite nanogels loaded with both paclitaxel and electrostatically adsorbed albumin nanoparticles (PTX concentration equal to 5 μg / mL) using an Annexin V-FITC / PI apoptosis detection double staining kit.

[0049] Figure 9 CXCR4 antagonistic effects and quantitative analysis graphs of each preparation group. The scale bar is 20 μm.

[0050] Figure 10 To determine the effects of each preparation group on the expression of pCSF-1R protein by Western blot analysis (n = 3, ***P < 0.001, **P < 0.01). Detailed implementation manners

[0051] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps, or conditions of the present invention shall fall within the scope of the present invention. The experimental methods without specific conditions and the reagents without specific formulations described in the examples are all in accordance with the conventional conditions in the art.

[0052] The nuclear magnetic resonance hydrogen spectrum of the polymer obtained in the examples of the present invention 11H NMR) and mass spectrometry (MS) were detected by the Analysis and Testing Center of China Pharmaceutical University, and the solvent was D 2 O. The molecular weight of the obtained polymer was detected by the University of Nebraska Medical Center in the United States. Gel permeation chromatography (GPC) was used for detection. The sample was 5 mg / mL, the mobile phase was 0.3 M sodium acetate buffer (pH 5.0), the flow rate was 0.3 mL / min, and the GPC results were analyzed using Astra 6.1 software.

[0053] Example 1

[0054] 1. Preparation of hexamethylenebisacrylamide (HMBA)

[0055] Weighed 2.90 g of 1,6-hexanediamine and dissolved it in 20 mL of deionized water, and transferred the solution to a three-necked flask. Weighed 4.62 g of acryloyl chloride and dissolved it in 5 mL of dichloromethane, and transferred it to a dropping funnel. Another 10 mL of saturated sodium hydroxide (NaOH) solution was prepared and transferred to another dropping funnel. The solutions in the two funnels were simultaneously added dropwise to the three-necked flask, and the temperature of the reaction solution was maintained at 0 - 5 °C, and the addition was completed within 2 h. Stirring was continued at room temperature for 2 h, and the reactants were filtered by suction to obtain a white solid. The filter cake was washed 3 times with deionized water to obtain the product HMBA.

[0056] Figure 1 1H NMR spectrum of HMBA 1 1H NMR). As can be seen from the figure, double bond peaks appeared in HMBA at about δ = 5.6 and 6.1 ppm. The peaks at δ = 3.4 and 1.3 - 1.6 ppm were the H characteristic peaks on the methylene structure (-CH2-) of HMBA. The multiplet peak at δ = 6.3 ppm was the H characteristic peak on HMBA near the double bond, indicating that covalent bonding between hexanediamine and acryloyl chloride had occurred successfully.

[0057] 2. Preparation of polymerized AMD3100 (pAMD)

[0058] Weighed 112 mg of HMBA (0.5 mmol) and 250 mg of AMD3100 (0.5 mmol) respectively and dissolved them in 4 mL of methanol-water (7:3 v / v) mixed solvent. The reaction was carried out under nitrogen protection at 37 °C in the dark for 3 d. Then 25 mg of AMD3100 (0.05 mmol) was added, and the reaction was carried out under nitrogen protection at 37 °C in the dark for 12 h to cap the polymer. The capped polymer was directly dialyzed in methanol for 2 d, and then obtained pAMD after rotary evaporation under reduced pressure for the next reaction.

[0059] 3. Preparation of pAMD-Ch (PC) cationic polymer.

[0060] Weigh 200 mg of pAMD and dissolve it in 3 mL of anhydrous dichloromethane. Add 100 μL of DIPEA solution and stir for 5 min. Weigh 40 mg of cholesterol formyl chloride and dissolve it in 2 mL of anhydrous dichloromethane. Add it dropwise to the pAMD solution and react for 1 h in an ice bath, then react at room temperature for 24 h. The product pAMD-Ch(PC) is obtained by rotary evaporation under reduced pressure and washed three times with anhydrous ether to remove unreacted cholesterol formyl chloride.

[0061] 4. Preparation of PBA-EDA.

[0062] Weigh 1 g of 4-carboxyphenylboronic acid pinacol ester, 845 mg of EDC·HCl, 594 mg of HOBt, and 1.55 g of DIEA respectively, and dissolve each in 2 mL of anhydrous DMF. Mix the above solutions and react at room temperature for 1 h to activate the carboxyl group of the phenylboronic acid. Then add 768 mg of N-boc-ethylenediamine (EDA-boc) and continue to react for 24 h. Transfer the reaction solution to a separatory funnel, add 20 mL of ethyl acetate to dissolve the product, then wash it twice with 1 M HCl solution and twice with saturated sodium bicarbonate solution. Take the organic phase and add anhydrous sodium sulfate to remove water overnight. Concentrate the organic solvent by rotary evaporation to 1 - 2 mL, add it to a silica gel column, and develop it with a developing agent of dichloromethane:ethyl acetate = 1:2 to obtain the PBA-EDA-boc product. Weigh 500 mg of PBA-EDA-boc and add it to 10 mL of HCl-ethyl acetate solution, react for 12 h to remove the boc protecting group, and rotary evaporate to obtain the PBA-EDA product.

[0063] Figure 2 1H NMR and mass spectrometry (MS) spectra of PBA-EDA-boc and PBA-EDA. 1 As shown in Figure 2 A, the singlet peak at δ = 1.2 ppm (peak a) is the methyl peak on the phenylboronic acid pinacol ester of PBA-EDA-boc; the peak at δ = 1.4 ppm (peak b) is the characteristic methyl H peak on the single boc of PBA-EDA-boc; the multiplet at δ = 3.4 - 3.6 ppm (peak c) is the characteristic methylene peak on ethylenediamine; the multiplet at δ = 7.8 ppm (peak d) is the characteristic peak of the benzene ring on phenylboronic acid. This indicates that PBA and EDA-boc have successfully undergone covalent binding. As shown in Figure 2 C, after treatment with strong acid, the characteristic methyl H peak on the single boc at δ = 1.4 ppm disappears, proving the exposure of the active amino group and the successful preparation of PBA-EDA. As shown in Figure 2 B and 2D, 413.3 and 291.2 are the molecular weights of PBA-EDA-boc and PBA-EDA plus H respectively, which further proves the successful preparation of the two small molecule compounds.

[0064] 5. Preparation of PBA-pAMD-Ch(PPC) cationic polymer.

[0065] Weigh 90 mg of pAMD-Ch and dissolve it in 2 mL of DMF, then add 90 mg of CDI and react at room temperature for 4 h. Weigh 67.5 mg of PBA-EDA and dissolve it in 2 mL of DMF, then add it to the above reaction solution and react at room temperature for 4 d. Add the reaction solution to 10 times the amount of anhydrous ether and centrifuge at 5000 rpm for 10 min. Collect the white precipitate at the bottom of the centrifuge tube, dissolve it with an ethanol-water (1:1, v / v) mixed solvent, place the solution in a dialysis bag (MWCO 12 kDa), dialyze it in deionized water with pH 4.0 for 2 d, then change to pure water and dialyze 1 - 2 times. Finally, obtain the PPC cationic polymer by freeze-drying.

[0066] Figure 3 1H NMR spectra of pAMD, pAMD-Ch(PC), and PBA-pAMD-Ch(PPC). 1 In the figure, peak 1 (7.3 ppm) is the proton on the benzene ring of AMD3100, peak 2 (0.65 ppm) is the newly introduced cholesterol methyl peak, and peak 3 (7.7 ppm) is the characteristic peak of the benzene ring H on the newly introduced PBA. According to the NMR results, the cholesterol content in the polymer is 47.2%, and the PBA content is 51.6%. According to the analysis of the gel permeation chromatography experimental results, the average molecular weight (Mw) of the pAMD polymer is 25 kDa, the Mw of the PC polymer is 70 kDa, and the Mw of the PPC polymer is 95 kDa.

[0067] Example 2

[0068] Preparation of drug-loaded composite nanogel

[0069] 1. Preparation of albumin particles modified with CSF-1R inhibitor BLZ-945 (BLZ-BSA, BB)

[0070] Dissolve 50 mg of BLZ945, 50 mg of EDC·HCl, 30 mg of DMAP, and 60 mg of succinic anhydride (SA) in 5 mL of DMF and react at 37 °C for 24 h. Transfer the solution to a separatory funnel, add 20 mL of dichloromethane to dissolve the product, then wash it twice with 1 M HCl solution and twice with saturated sodium bicarbonate solution. Take the organic phase and dehydrate it with anhydrous sodium sulfate overnight. Rotate and evaporate to concentrate the organic solvent to 1 mL, add it to a silica gel column, and develop it with a developing agent of dichloromethane:ethyl acetate = 5:1 to obtain the carboxylated BLZ945 (BLZ-COOH) product. Dissolve 10 mg of BLZ-COOH in 200 μL of DMSO, add 15 mg of EDC·HCl and 9 mg of NHS to activate for 1 h. Dissolve 100 mg of BSA in 5 mL of PBS buffer at pH 7.4, and slowly add the activation solution to the BSA solution, keeping the reaction solution clear, and continue to react for 24 h. Place the solution in a dialysis bag with a molecular weight cut-off of 12 kDa and dialyze it in deionized water for 2 d, and finally obtain BLZ-BSA nanoparticles by freeze-drying. The grafting rate of BLZ is calculated from the elemental analysis results of C, N, and O.

[0071] Figure 4 Figures are the 1H NMR and MS spectra of BLZ-COOH. The results show that a new singlet appears at about δ = 2.7 ppm, which is the characteristic peak of the methylene (-CH2-) of succinic anhydride, indicating that BLZ945 and succinic anhydride have successfully undergone covalent binding. 499.1 in the MS spectrum is the molecular weight of BLZ-COOH plus H, which further proves the successful preparation of this small molecule compound. Immediately afterwards, BLZ-COOH was grafted onto BSA through the EDC / NHS reaction, and its grafting rate was calculated by elemental analysis of N. The N element content before grafting was 14.23%, and after grafting it was 15.05%, indicating that the grafting rate of BLZ was 9.8%.

[0072] 2. Preparation of drug-loaded composite nanogels

[0073] Weigh 5 mg of PPC polymer and 1 mg of paclitaxel (PTX) and dissolve them in 1 mL of methanol solution. Rotate-evaporate the solution for 45 min to remove the excess methanol and form a thin film. Add 5 mL of deionized water and sonicate for 15 min to disperse the thin film in water, and then probe sonicate for 20 min (sonicate for 1 s and stop for 2 s) to obtain PPC-PTX nanogels with a uniform particle size distribution. Place the nano-solution in an ultrafiltration tube (MWCO 3000) and centrifuge at 4000 rpm for 15 min to remove the free PTX. Take another 5 mL of deionized water to resuspend the supernatant of the ultrafiltration tube to obtain the final PPC-PTX nanogels. Weigh 5 mg of albumin nanoparticles modified with CSF-1R inhibitor BLZ-945 (BLZ-BSA, BB) and dissolve them in 5 mL of HEPES (10 mM) buffer at pH 7.4. Mix the PPC-PTX nanogels and the BB solution in a ratio of 1:1 (v:v), vortex for 30 s, and then let it stand for 30 min to prepare the final BB@PPC-PTX composite nanogels.

[0074] Figure 5 Transmission electron microscopy (TEM) images of BB, PPC-PTX, BB@PPC-PTX nanogels, and BB@PPC-PTX nanogel solution co-incubated with 0.1 mM ATP (BB@PPC-PTX + 0.1 mM ATP), and the diameters of the above-mentioned preparations shown in the TEM images determined by dynamic light scattering. The scale bar is 100 nm. The results show that the particle size of BB nanoparticles is 11.2 nm and they are small and round, while the PPC-PTX and BB@PPC-PTX nanogels are regular circles with a particle size of 98.9 nm. After adding 0.1 mM ATP, it was observed that the morphology of the nanoparticles was significantly disturbed and there were small free particles with a particle size of about 10 nm, presumably BB protein nanoparticles released due to charge reversal. The TEM results further demonstrated that ATP can effectively compete for BB protein nanoparticles and cause their release in the tumor microenvironment.

[0075] Example 3

[0076] Protein release of composite nanogels under different ATP conditions.

[0077] Prepare rhodamine B (RhB)-labeled BSA (RB) to replace BB for observing the in vitro release behavior. Weigh 10 mg of RhB and dissolve it in 200 μL of DMSO. Add 32 mg of EDC·HCl and 20 mg of NHS and activate for 1 h. Weigh 100 mg of BSA and dissolve it in 5 mL of PBS buffer at pH 7.4. Slowly add the activation solution to the BSA solution, keep the reaction solution clear, and continue the reaction for 24 h. Place the solution in a dialysis bag (MWCO 12 kDa) and dialyze it in deionized water for 2 d. Finally, obtain RhB-BSA nanoparticles by freeze-drying. Use RhB-BSA to prepare RB@PPC-PTX nanogels and measure the fluorescence intensity of RhB in them. Also measure the fluorescence intensity of an equal amount of RhB-BSA aqueous solution. Add different concentrations of ATP, incubate, and then measure the fluorescence intensity again after adding different concentrations of ATP. Calculate the release of RB protein particles according to formula (1-1):

[0078] I = I f + I n = I f0 × X / 100 + I n0 × (100 - X) / 100 (1-1)

[0079] Wherein, I f is the intensity of the free fluorescent dye, I n is the fluorescence intensity in the nanogel, and X is the percentage of the free fluorescence intensity.

[0080] Figure 6 The in vitro release diagrams of RhB-BSA in each nanogel group treated with different concentrations of ATP are shown. The results show that the RB@PC nanogel is almost insensitive to 0.1 - 0.4 mM of ATP, and the release rate of RB protein particles is only 6.5%. On the contrary, the release rates of RB@PPC and RB@PPC-PTX treated with a low concentration of 0.1 mM ATP are about 55% and 51%, respectively, while the release rates of RB@PPC and RB@PPC-PTX treated with 0.4 mM ATP can reach 91.1% and 93.4%. This shows the ultrasensitive property of the PBA-grafted polymeric cationic carrier to ATP.

[0081] Example 4

[0082] PTX release of the composite nanogel under different PBS conditions.

[0083] Take 1 mL of BB@PPC-PTX solution (PTX concentration is 500 μg / mL) and place it in a tightly tied dialysis bag (MWCO 8-12 kDa). Place the dialysis bag in 20 mL of PBS buffer with 0.1% Tween-80 at pH 7.4, pH 6.8, and pH 5.0. Stir mechanically at 100 rpm at 37°C. Take 1 mL of the solution at 0, 1, 2, 4, 8, 12, 24, 48, and 72 h, and then supplement 1 mL of the corresponding buffer and continue stirring. Determine the content of PTX in each solution by high performance liquid chromatography (HPLC), and calculate the cumulative release content of each preparation in different buffers accordingly.

[0084] Figure 7 Time-dependent release of paclitaxel (PTX) from PPC (PPC-PTX) nanogels loaded with paclitaxel in PBS at pH 5.0, 6.8, and 7.4. The results show that under physiological conditions of pH 7.4 and pH 6.8, the release amounts of PTX are generally the same, and only 13.9% and 17.1% are released at 72 h, indicating that the cationic nanogel core can maintain its stability in the blood and tumor microenvironment. The lower pH environment (pH 5.0) will interfere with the structure of cholesterol and the hydrophobic cavity of PTX, causing a large amount of PTX to be released from the nanogel (45.1% cumulative release at 72 h). It is proved that the nanogel can disintegrate under the acidic conditions of lysosomes and release PTX to act on the cell nucleus.

[0085] Example 5

[0086] Evaluate the apoptosis induced by nanogels by AnnexinV-FITC / PI double staining method.

[0087] Take 4T1 cells growing in the logarithmic growth phase, resuspend them after digestion with 0.25% trypsin, and seed the cells at 1×10 5 cells per well in a 6-well culture plate. After culturing in an incubator for 24 h, aspirate the culture medium, add 2 mL of the prepared solution of each preparation, incubate for 24 h, remove the medium and wash twice with ice-cold PBS, stain the cells of each group according to the operation method of the AnnexinV-FITC / PI staining kit, and analyze the samples on a flow cytometer.

[0088] Figure 8The Annexin V-FITC / PI apoptosis detection double staining kit was used to evaluate the apoptosis induced by PBS, PPC (20 μg / mL), albumin nanoparticles modified with CSF-1R inhibitor BLZ-945 (BB, 20 μg / mL), paclitaxel (PTX, 5 μg / mL), PPC nanogels loaded with paclitaxel (PPC-PTX) and PPC composite nanogels loaded with paclitaxel and electrostatically adsorbed albumin nanoparticles (BB@PPC-PTX) (PTX concentration equal to 5 μg / mL). The cells in the PBS and BB nanoparticle incubation groups were basically in the lower left quadrant, indicating that the cells were in good condition, proving that the protein carrier was safe. The PTX treatment group caused 3.76% early apoptosis and 22.4% late apoptosis after incubation with cells. The PPC carrier alone also showed certain cytotoxicity, with a total apoptosis of about 12.8%. After the preparation was formed, a large number of cells in the PPC-PTX nanogel group showed apoptosis, with a total amount of 48.03%. At the same time, the BB@PPC-PTX nanogel group also induced about 48.6% of total cell apoptosis, and the amount of apoptosis induced was greater than the total amount of pure PTX and PPC carriers, which further verified that the nanogel can effectively promote the hydrophobic drug PTX into cells and further kill tumor cells for anti-tumor treatment.

[0089] Example 6

[0090] Investigation of the CXCR4 antagonistic function of nanogel.

[0091] Human osteosarcoma cells U2OS expressing EGFP-CXCR4 coupled protein receptors were seeded in a 96-well black board (8000 cells / well), and then the cells were washed twice with 100 μL of detection solution (DMEM medium containing 2 mM L-glutamine, 1% FBS, 1% penicillin-streptomycin and 10 mM HEPES). pAMD, PC, PPC polymer carriers (1 μg / mL) were diluted with a detection solution containing 0.25% DMSO and incubated with cells at 37°C for 30 min, and free AMD3100 (300 nM) was used as a positive control. CXCL12 was then added to each well to a final concentration of 10 nM. U2OS cells were simply incubated with CXCL12 as a negative control. After incubation at 37°C for 1 h, U2OS cells were fixed with 4% paraformaldehyde for 20 min and washed 4 times with PBS. The fluorescence distribution of U2OS cells was photographed with an inverted fluorescence microscope at 20 times.

[0092] Figure 9CXCR4 antagonism and quantitative analysis graphs for each formulation group. Scale bar is 20 μm. The results showed that when U2OS cells were incubated with pAMD (1 μg / mL), PC (1 μg / mL), PPC (1 μg / mL), and PPC-PTX (1 μg / mL) nanogels, no obvious internalization of EGFP protein coupled to CXCR4 receptor (EGFP-CXCR4) was shown, and the results were the same as those of the positive control group with AMD3100 added. However, after U2OS cells were incubated only with the culture medium and then CXCL12 was added, obvious green fluorescence distribution was shown, indicating the internalization of EGFP-CXCR4 stimulated by CXCL12. The above results indicate that all AMD3100 series polymers can significantly antagonize the CXCR4 receptor and thus block the CXCR4 / CXCL12 biological axis.

[0093] Example 7

[0094] Evaluation of the inhibitory effect of BB protein particles on M2 macrophages

[0095] Take M2 macrophages at 5×10 5个 seeded in a 6-well plate and cultured in complete medium for 24 h. After the cell confluence reached about 80%, the culture medium was removed. Tumor cells were incubated with BLZ945 (0.25, 0.5 μM) and BB protein particles (BLZ945 concentration 0.25, 0.5 μM). After incubation for 12 h and 24 h, the protein levels of pCSF-1R and total protein in tumor cells were determined by Western Blot.

[0096] Figure 10 To determine the effect of each formulation group on the expression of pCSF-1R protein by Western blot analysis (n = 3, ***P < 0.001, **P < 0.01). The results showed that the PBS control group did not downregulate the pCSF-1R protein in TAMs. Incubation with 0.5 μM CSF-1R inhibitor BLZ-945 (positive control) for 24 h could produce a downregulation of about 75% in the pCSF-1R protein expression level. Incubation with albumin nanoparticles modified with BLZ-945 (BLZ-BSA, BB) containing the same concentration of BLZ-945 for 12 h or 24 h showed a downregulation of the pCSF-1R protein expression level to the same extent as the positive control. It indicates that the modification of BLZ-945 with BSA does not affect its high-efficiency and specific CSF-1R downregulation function. It is proved that BB protein particles can effectively kill TAMs cells in the tumor microenvironment and relieve the immunosuppressive effect of the microenvironment.

Claims

1. A phenylboronic acid copolymer for remodeling the tumor immune microenvironment, and its structural formula is shown as formula (Ⅰ): Wherein: n is any natural number from 30 to 60.

2. Use of the phenylboronic acid copolymer according to claim 1 as a drug delivery carrier in the preparation of a tumor therapeutic drug.

3. A tumor therapeutic drug, Characterized in that: It includes a nanogel made of the phenylboronic acid copolymer according to claim 1.

4. The tumor therapeutic drug according to claim 3, Characterized in that: The nanogel encapsulates a chemotherapeutic drug.

5. The tumor therapeutic drug according to claim 4, Characterized in that: The chemotherapeutic drug is selected from one or more of cisplatin, oxaliplatin, paclitaxel, gemcitabine or doxorubicin.

6. The tumor therapeutic drug according to claim 3, Characterized in that: The tumor therapeutic drug further includes albumin nanoparticles, and the albumin nanoparticles are adsorbed on the surface of the nanogel.

7. The tumor therapeutic drug according to claim 6, Characterized in that: The albumin nanoparticles are made of human serum albumin, bovine serum albumin, ovalbumin or mouse serum albumin.

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