A method for preparing hydrogels based on molecules containing monoboronic acid groups and its application

Abstract

This invention discloses a method for preparing hydrogels based on molecules containing monoboric acid groups and its application, belonging to the field of biomedical technology. The preparation method includes: (1) dissolving a compound containing a monoboric acid group and an aromatic hydrophobic group in a solvent to obtain solution A; (2) dissolving polyvinyl alcohol in an aqueous solution to obtain solution B; (3) mixing solution A and solution B and reacting to form a hydrogel. This invention can directly use prodrugs modified with boric acid group structures or drugs carrying boric acid group structures as crosslinking agents for the preparation of hydrogels to form drug sustained-release agents; it can also use this hydrogel as a carrier to load a second drug. Since the prodrug in the hydrogel needs to release the active ingredient after the reactive oxygen species response, differential sustained release of the two drugs can be achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing hydrogels based on molecules containing monoboronic acid groups and its application in the preparation of drug sustained-release gels. Background Technology

[0002] Most common drug formulations release the drug at a rate greater than the rate at which it is absorbed into the body fluids through biological membranes, giving them the advantage of rapid onset of action. However, their duration of effect is short. To maintain long-term efficacy, frequent dosing (3-4 times / day) is often required. This can easily lead to a "peak-trough" phenomenon in blood drug concentration, resulting in concentration fluctuations. That is, when the concentration is too low, it cannot exert its therapeutic effect; when the concentration is too high, it is prone to adverse reactions.

[0003] To address these issues, sustained-release drug technology has emerged. Its aim is to ensure a longer duration of drug release after ingestion, requiring the use of materials that are gradually biodegradable, biosafety-compliant, and do not trigger rejection reactions as drug carriers. By loading drugs onto suitable carriers, not only can sustained release be achieved, but the delivery method can also be altered, thereby reducing the frequency of administration, minimizing adverse drug reactions, and improving drug bioavailability.

[0004] In recent years, drug sustained-release systems have made rapid progress in both theory and technology. Currently, the most common drug sustained-release systems mainly include nanoparticles, microspheres, and hydrogels. Among them, hydrogels, due to their unique three-dimensional network structure and the highly cross-linked hydrophilic groups and network chains on their backbone, can serve as excellent long-acting sustained-release materials. The release rate of drugs can be controlled by adjusting the network pore size and cross-linking density, and hydrogels have advantages such as good biocompatibility and bioresponsive degradation. However, the reported hydrogel materials typically require complex chemical synthesis processes, multiple non-functional additives, lack therapeutic activity or function, and cannot achieve differentiated release between different loaded drugs, which limits their clinical translation.

[0005] Polyvinyl alcohol (PVA) is a water-soluble polymer. Due to the strong water absorption of the hydroxyl groups in the polymer, water molecules aggregate around it to form a gel. Previous reports have provided methods for preparing gels by covalently crosslinking small molecules or polymers containing diboronic acid or polyboronic acid groups with PVA (ACS nano, 2018, 12(3): 2466-2473; ACS Applied Materials & Interfaces, 2020, 12(45): 50248-50259; CN114957714A). However, materials containing one boric acid group in the crosslinking agent molecule that can form a gel with PVA have not been reported. Summary of the Invention

[0006] The purpose of this invention is to provide a novel method for preparing hydrogels, which involves modifying a small molecule drug with a single boric acid group and then forming a gel with polyvinyl alcohol to prepare a gel drug that releases the drug after responding to a high content of reactive oxygen species (ROS). On the other hand, the hydrogel can also serve as a carrier to encapsulate small or large molecule drugs, achieving differential sustained release of the two drugs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a method for preparing hydrogels based on molecules containing monoboronic acid groups, comprising the following steps:

[0009] (1) Dissolve a compound containing a single boric acid group and an aromatic hydrophobic group in a solvent to prepare solution A;

[0010] (2) Dissolve polyvinyl alcohol in an aqueous solution to prepare solution B;

[0011] (3) Mix liquid A and liquid B to form a hydrogel.

[0012] In the above-mentioned hydrogel formation process, the single boric acid group and the aromatic hydrophobic group on the molecular structure of the compound work together to achieve the gelation of polyvinyl alcohol aqueous solution. Specifically, the boric acid group coordinates and bonds with the ortho-dihydroxy group of polyvinyl alcohol, while the aromatic hydrophobic group aggregates through hydrophobic interaction, intermolecular π-π stacking and hydrogen bonding, resulting in cross-linking of the polymer and thus forming a gel.

[0013] The aromatic hydrophobic group is a type of hydrophobic group containing a benzene ring or a heterocyclic structure. The heterocyclic structure can be, but is not limited to, a benzene ring, anthracene ring, a pyridine ring, a pyrimidine ring, a furan ring, a thiophene ring, a pyrrole ring, benzopyridine, benzopyrimidine, benzopyrrole, benzofuran, and benzothiophene.

[0014] The boric acid group coordinates and crosslinks with polyvinyl alcohol to form a reversible covalent borate ester bond, which is a reactive oxygen species (ROS) responsive bond. When the hydrogel is exposed to high concentrations of ROS, the borate ester bond breaks, releasing the active compound. Therefore, the compound can be a prodrug modified with a boric acid group structure or a drug that inherently carries a boric acid group structure. The prodrug or drug itself has no biological activity or very low activity, but after the ROS responsive bond breaks, it releases a biologically active ingredient, which can be used to prepare controlled-release drugs.

[0015] Preferably, the compound is a dipyridamole prodrug modified with a phenylboronic acid group. Specifically, the phenylboronic acid group of 4-(bromomethyl)phenylboronic acid is linked to the tertiary amine group of dipyridamole via an amination reaction of benzyl bromide with a tertiary amine. This prodrug component plays a decisive role in gel formation and gel ROS response. Moreover, it has low intrinsic activity and can be metabolized in vivo to become the active drug, thus reducing systemic toxicity.

[0016] Furthermore, the structural formula of the dipyridamole prodrug is shown in formula (Ⅰ).

[0017]

[0018] The present invention demonstrates that the aforementioned small molecule prodrug can slowly activate and release dipyridamole in a high concentration of reactive oxygen species, thereby promoting the polarization of tumor-associated macrophages from M2 to M1.

[0019] Preferably, the compound is bortezomib or esazomib.

[0020] Specifically, the molecular formula of bortezomib is: C 19 H 25 BN4O4, molecular weight: 384.23, CAS number: 179324-69-7, structural formula as shown in formula (II):

[0021]

[0022] Isazomi molecular formula: C 14 H 19 BCl2N2O4, molecular weight: 361.03, CAS number: 1072833-77-2, structural formula as shown in formula (Ⅲ):

[0023]

[0024] Polyvinyl alcohol (PVA), as a key component of the gel, has the advantage of high biocompatibility. The molecular formula of PVA is [-CH₂CHOH-]. n The structural formula is shown in equation (IV):

[0025]

[0026] This invention uses polyvinyl alcohol with a molecular weight of 10,000-200,000. Preferably, the molecular weight of the polyvinyl alcohol is 14,600-18,600. Polyvinyl alcohol within this molecular weight range has suitable solubility and gelling properties.

[0027] In step (3), polyvinyl alcohol and the compound are self-assembled in a liquid solution to form a hydrogel using a solution mixing method such as simultaneous injection of two liquids or sequential injection of two liquids.

[0028] Preferably, the mass ratio of the compound to polyvinyl alcohol in the mixture is 1:1-5. This invention, through a simple physical mixing method and by adjusting the mass concentrations of solution A and solution B, can obtain gel materials with different hardnesses. The gelation efficiency and speed increase with increasing polymer dosage, but excessive polymer dosage leads to polymer waste. Within this mass ratio range, rapid gel formation and high polymer utilization are ensured. More preferably, the mass ratio of the compound to polyvinyl alcohol in the mixture is 1:1-2.

[0029] The present invention provides a hydrogel prepared by the above method.

[0030] The present invention also provides the application of the hydrogel in the preparation of drug sustained-release agents, wherein the hydrogel serves as a drug carrier, and / or the compound used to prepare the hydrogel is a prodrug modified with a boric acid group structure or a drug that carries a boric acid group structure itself.

[0031] The hydrogel provided by this invention can be used as a drug delivery carrier to further encapsulate small or large molecule drugs, thereby achieving local sustained release and delivery of drugs.

[0032] Specifically, the preparation method of the gel drug includes: dissolving the compound, polyvinyl alcohol and small molecule or large molecule drug in a good solvent, and then mixing them using a physical mixing method to instantly form a hydrogel encapsulating the drug.

[0033] This invention allows for the direct use of prodrugs modified with boric acid groups or drugs inherently carrying boric acid groups as crosslinking agents in the preparation of hydrogels to form sustained-release drug agents. Alternatively, this hydrogel can be used as a carrier to load a second drug. Since the prodrug in the hydrogel needs to release its active ingredient after a reactive oxygen species response, this approach can achieve differential sustained release of the two drugs.

[0034] The present invention also provides the application of the hydrogel in the preparation of tumor immunotherapy drugs, wherein the compound is a dipyridamole prodrug modified with phenylboronic acid groups, and the hydrogel promotes the polarization of tumor-associated macrophages from M2 type to M1 type.

[0035] Tumor immunotherapy is a treatment method in which the body utilizes or activates the immune system to kill tumors. However, due to the presence of the immunosuppressive tumor microenvironment (TME), the response rate of tumor immunotherapy remains low. Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment. Due to alterations in tumor cell metabolism and their regulation of the tumor microenvironment, they are prone to differentiating into the immunosuppressive M2 phenotype and impairing the proliferation and differentiation function of tumor-infiltrating lymphocytes by secreting various active substances. Among TAM regulation strategies, macrophage polarization strategies can reprogram TAMs into the pro-inflammatory M1 phenotype, thereby effectively reversing the immunosuppressive TME and improving the efficacy of immunotherapy.

[0036] This invention reveals that dipyridamole possesses macrophage polarization-promoting activity, capable of polarizing tumor-associated macrophages from M2 to M1 type. The dipyridamole prodrug modified with phenylboronic acid groups exhibits low activity; it is only under the influence of highly expressed reactive oxygen species (ROS) mediators in tumor cells that dipyridamole is released and activated, promoting macrophage polarization and thereby reversing the tumor's immunosuppressive microenvironment, thus exerting an anti-tumor immunotherapeutic effect.

[0037] The tumor is a solid tumor. Further, the tumor can be, but is not limited to, breast cancer. Compared to normal tissue, breast cancer contains a large number of M2 macrophages and reactive oxygen species, creating an immunosuppressive microenvironment in the tumor tissue, thereby promoting tumor growth and metastasis.

[0038] The drug can be administered via peritumoral injection. After the gel drug is injected near the tumor, the high level of ROS in the tumor microenvironment triggers the slow degradation of the macrophage polarization prodrug in the gel into the macrophage polarization agent, which is then continuously delivered to the tumor tissue. This significantly improves the drug's accumulation and retention time in the tumor, thereby achieving sustained macrophage polarization in the tumor tissue. Furthermore, local injection reduces the chance of the drug entering normal tissues, significantly improving the drug's tumor selectivity and enabling precise tumor treatment.

[0039] The beneficial effects of this invention are as follows:

[0040] (1) This invention provides a crosslinking agent material for the gelation of polyvinyl alcohol aqueous solution containing a single boric acid group and an aromatic hydrophobic group, which forms a gel instantaneously after physical mixing. This invention can directly use a prodrug modified with a boric acid group structure or a drug with a boric acid group structure as a crosslinking agent for the preparation of hydrogels to form a drug sustained-release agent; or the hydrogel can be used as a carrier to load a second drug. Since the prodrug in the hydrogel needs to release the active ingredient after the reactive oxygen species response, differential sustained release of the two drugs can be achieved.

[0041] (2) This invention utilizes dipyridamole prodrug modified with phenylboronic acid groups as a crosslinking agent for polyvinyl alcohol aqueous solution gelation to prepare a gel drug. The gel component endows the drug with the ability to be slowly and effectively delivered and accumulated in tumors. When the drug reaches the tumor tissue, due to the high expression of reactive oxygen species in tumor cells, it can promote the degradation of dipyridamole prodrug to release the original dipyridamole, promoting the polarization of tumor-associated macrophages in the tumor tissue from M2 type to M1 type, thereby reversing the tumor immunosuppressive microenvironment, exerting anti-tumor immunotherapy effects, and achieving precision tumor treatment. Since the polarizing drug only works in the tumor tissue, the systemic toxicity of the gel drug is reduced. Attached Figure Description

[0042] Figure 1 The image shows the 1H NMR spectrum of the dipyridamole prodrug DIPPGel in this example.

[0043] Figure 2 The mass spectrum of the dipyridamole prodrug DIPPGel in this example is shown.

[0044] Figure 3 The image shows a sample of dipyridamole prodrug mixed with polyvinyl alcohol to form a gel called DIPPGel.

[0045] Figure 4 The image shown is a field emission scanning electron microscope (FESEM) image of the gel DIPPGel in the example.

[0046] Figure 5 The rheological properties of the DIPPGel gel in this example are shown in the diagram.

[0047] Figure 6 This is an image of bortezomib mixed with polyvinyl alcohol to form a gel, as shown in the example.

[0048] Figure 7 This is an image of esazolidinone mixed with polyvinyl alcohol to form a gel, as shown in the example.

[0049] Figure 8 The image shows the mixture of S-(2-boronethyl)-L-cysteine ​​and polyvinyl alcohol in the example.

[0050] Figure 9 The image shown is of Talabostat mixed with polyvinyl alcohol in the example.

[0051] Figure 10 This is a drug release curve of the DIPPGel gel containing 3BP in the example.

[0052] Figure 11 In this example, Western blotting analysis was performed to determine the expression levels of CD80, iNOS, CD206, and Arg-1 in BMDM after treatment with different concentrations of DIPPGel gel for 24 hours.

[0053] Figure 12 In this example, ELISA was used to quantitatively analyze the release of IFN-γ, IL-6, TGF-β1 and IL-10 cytokines in BMDM culture medium 24 h after stimulation with DIPPGel (containing 20 μM DIP).

[0054] Figure 13 The image shows the tumor growth curve in the experiment of inhibiting 4T1 breast cancer cell tumors in Balb / c mice using gel DIPPGel, as shown in the example.

[0055] Figure 14 The graph shows the change in body weight of Balb / c mice during the experiment on the inhibition of 4T1 breast cancer cell-bearing Balb / c mice by gel DIPPGel in the example. Detailed Implementation

[0056] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0058] 4-(bromomethyl)phenylboronic acid (CAS No.: 68162-47-0), dipyridamole (CAS No.: 58-32-2), polyvinyl alcohol (CAS No.: 9002-89-5), and 3-bromopyruvic acid (CAS No.: 1113-59-3) were all purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. 4T1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0059] M2-type BMDM cells were induced to differentiate from bone marrow-derived macrophages extracted from the peritoneal cavity of BALB / c mice. The preparation method involved using 6-8 week old female BALB / c mice as the source of bone marrow mononuclear cells. Before the experiment, hand scissors, ophthalmic forceps, filter gauze, and beakers were soaked in alcohol and then sterilized together with other materials under ultraviolet light for 30 minutes in a laminar flow hood. Mice were euthanized by cervical dislocation and then immersed in sterile 75% ethanol for 10 minutes. In a sterile dish containing 75% ethanol, the tibia, humerus, and femur of the mice were separated, and the muscles were carefully removed. The remaining muscles were then removed in two small dishes containing 75% ethanol (the treatment time in 75% ethanol should not exceed 30 minutes to prevent bone fracture and subsequent exposure of the bone marrow for bacterial contamination). Finally, the remaining muscles were removed in four small dishes containing sterile PBS to obtain as much clean bone as possible without muscle. After cleaning, use sterile scissors to remove the ends of the tibia, femur, and humerus. Use a 1mL syringe to draw serum-free culture medium to flush out the bone marrow, mix it thoroughly, and transfer it to a 15mL centrifuge tube. Centrifuge at 1500rpm for 5min, discard the supernatant, add 3-5mL of sterile erythrocyte lysis buffer (1×) to resuspend the cells, let them stand at room temperature for 5min, then add an equal volume of sterile culture medium to stop erythrocyte lysis. Pass the cells through a sterile filter into a new sterile 15mL centrifuge tube, centrifuge at 1500rpm for 5min, discard the supernatant, and the cell pellet is the bone marrow-derived cells. Mix them thoroughly with BMDM induction medium (20% fetal bovine serum, 1% penicillin-streptomycin, and 20ng / mL M-CSF in DMEM medium) and plate them. After 3-4 days, a large number of cells will adhere to the plate. The adherent cells are M0 type BMDM cells. Add 40ng / mL IL-4 or 100ng / mL LPS and 20ng / mL IFN-γ respectively to obtain M2 or M1 type BMDM cells.

[0060] Example 1: Preparation of Dimethoprim Prodrug Gel (DIPPGel)

[0061] 1. Preparation of dipyridamole prodrug containing boric acid bonds: 4-(bromomethyl)phenylboronic acid (0.2 g, 1 mmol) and dipyridamole (DIP, 1 g, 2 mmol) were dissolved in 30 mL of anhydrous DMF solution and sonicated until homogeneous. The system was then stirred at 45 °C for 6 h. After the reaction was complete, 200 mL of diethyl ether was added, and the mixture was filtered and washed three times with 200 mL of diethyl ether. After vacuum drying overnight, the yellow solid DIP prodrug (DIPP) was obtained by silica gel column chromatography with a yield of 37%. The reaction process is as follows:

[0062]

[0063] like Figure 1 and 2 As shown, the successful preparation of dipyridamole prodrug was demonstrated by nuclear magnetic resonance spectroscopy and mass spectrometry characterization.

[0064] 2. Preparation of Dipyridamole Prodrug Gel (DIPPGel) via Physical Mixing: Dissolve 50 mg of dipyridamole prodrug in 1 mL of pure aqueous solution to prepare a 5% (w / w) dipyridamole prodrug solution, i.e., solution A. Dissolve 50 mg of polyvinyl alcohol in 1 mL of pure aqueous solution and heat at 100°C for half an hour to prepare a 5% (w / w) polyvinyl alcohol solution, i.e., solution B. Then, fill solutions A and B separately into a double-tube syringe and inject them simultaneously or sequentially to allow them to come into contact and mix, thus instantly forming the DIPPGel gel.

[0065] like Figure 3 As shown, the dimidamox prodrug gel prepared by the physical mixing method is yellow.

[0066] like Figure 4 As shown, field emission scanning electron microscopy (FESEM) revealed that the dipyridamole membrane prodrug gel is a gel with micron-sized pores.

[0067] like Figure 5 As shown, the rheological properties observed during the formation of DIPPGel reveal that when DIPP solution and polyvinyl alcohol solution are mixed, the elastic modulus (G′) increases rapidly and eventually exceeds the viscous modulus (G″), further confirming the formation of DIPPGel gel.

[0068] Example 2: Preparation of bortezomib gel

[0069] Bortezomib gel was prepared by physical mixing: 50 mg of bortezomib was dissolved in 1 mL of methanol to prepare a 5% (w / w) bortezomib solution, i.e., solution A. Simultaneously, 50 mg of polyvinyl alcohol was dissolved in 1 mL of aqueous solution and heated at 100°C for half an hour to prepare a 5% (w / w) polyvinyl alcohol solution, i.e., solution B. Then, solutions A and B were separately loaded into a double-tube syringe and injected simultaneously or sequentially to allow them to come into contact and mix, thus instantly forming the bortezomib gel.

[0070] like Figure 6 As shown, the bortezomib gel prepared by the physical mixing method is white.

[0071] Example 3: Preparation of Isazomib Gel

[0072] Ixazomib gel was prepared by physical mixing: Ixazomib (50 mg) was dissolved in 1 mL of methanol to prepare a 5% w / w ixazomib solution, i.e., solution A. Simultaneously, polyvinyl alcohol (50 mg) was dissolved in 1 mL of aqueous solution and heated at 100°C for half an hour to prepare a 5% w / w polyvinyl alcohol solution, i.e., solution B. Then, solutions A and B were separately loaded into a double-tube syringe and injected simultaneously or sequentially to allow them to come into contact and mix, thus instantly forming the ixazomib gel.

[0073] like Figure 7 As shown, the isazomi gel prepared by the physical mixing method is white.

[0074] Comparative Example 1: Preparation of S-(2-Boroethyl)-L-cysteine ​​(BEC) gel

[0075] BEC gel was prepared by physical mixing: 50 mg of BEC hydrochloride was dissolved in 1 mL of pure water to prepare a 5% (w / w) BEC solution, i.e., solution A. Simultaneously, 50 mg of polyvinyl alcohol was dissolved in 1 mL of aqueous solution and heated at 100°C for half an hour to prepare a 5% (w / w) polyvinyl alcohol solution, i.e., solution B. Then, solutions A and B were separately loaded into a double-tube syringe and injected simultaneously or sequentially to allow them to mix. Regardless of the mixing time, a gel could not form. Figure 8 As shown.

[0076] Comparative Example 2: Preparation of [(2R)-1-[(2S)-2-amino-3-methylbutyryl]pyrrolidine-2-yl]boronic acid (Talabostat) gel

[0077] Talabostat gel was prepared by physical mixing: Talabostat methanesulfonate (50 mg) was dissolved in 1 mL of pure water to prepare a 5% w / w Talabostat solution, solution A. Simultaneously, polyvinyl alcohol (50 mg) was dissolved in 1 mL of aqueous solution and heated at 100°C for half an hour to prepare a 5% w / w polyvinyl alcohol solution, solution B. Then, solutions A and B were separately loaded into a double-tube syringe and injected simultaneously or sequentially to allow them to mix. Regardless of the mixing time, a gel could not form. Figure 9 As shown. Test Example 1: In vitro differential drug release from DIPPGel gel.

[0078] The reactive oxygen species (ROS) response release capability is a very important part of evaluating the in vivo application of DIPP gel. A good and slow response release capability is a guarantee for the full activation of macrophage polarization drugs and subsequently promotes macrophage polarization.

[0079] To investigate the differential release behavior of the two drugs in the gel, 3-bromopyruvic acid (3BP) was used as the encapsulated drug.

[0080] First, a 5% (w / w) dipyridamole prodrug solution (solution A) was prepared by dissolving 50 mg of dipyridamole prodrug in 1 mL of pure aqueous solution. Simultaneously, 50 mg of polyvinyl alcohol and 1 mg of 3-bromopyruvic acid were dissolved in 1 mL of pure aqueous solution and heated at 100°C for half an hour to prepare a 5% (w / w) polyvinyl alcohol solution (solution B). Solutions A and B were then mixed to prepare a gel. 100 μL of the gel (containing 5 mg of dipyridamole) was sealed in a dialysis bag with a molecular weight cutoff of 3500 Da and incubated in 50 mL of PBS containing 1% Tween 80, with or without 1 mM hydrogen peroxide (H₂O₂). At regular intervals, 500 μL of dialysate was collected from outside the dialysis bag, and the concentrations of dipyridamole and 3-bromopyruvic acid were determined by HPLC.

[0081] like Figure 10 As shown, in 1 mM H2O2 (pH 7.4), after one day, approximately 82.5% of the 3BP encapsulated in the gel was released, while only 38.1% of the dipyridamole prodrug was reduced to the original dipyridamole and released. After 7 days, in the same 1 mM H2O2 (pH 7.4) environment, approximately 98.8% of the 3BP encapsulated in the gel was released, and approximately 82.5% of the dipyridamole prodrug was reduced to the original dipyridamole and released. This indicates that the prodrug gel can differentially release dipyridamole and the loaded 3BP.

[0082] Test Example 2: In vitro macrophage polarization promotion effect of DIPPGel gel

[0083] 1. To investigate the effect of DIP released from DIPPGel on macrophage polarization, M2-type BMDM cells that had been induced to differentiate were seeded in 6-well plates at a cell density of 2 × 10⁶ cells per well. 5 First, 1 mL of the prepared DIPP Gel mixture (Solution A: DIPP: 1 mL, 5%, w / w; Solution B: PVA: 1 mL, 10%, w / w, mass ratio 1:1) was added to hydrogen peroxide (1 mM, 5 mL) and treated for 7 days. The collected solution was freeze-dried into powder to obtain the degraded dipyridamole technical grade drug. Then, different masses of powder were weighed and dissolved in DMSO to prepare dipyridamole solutions (5 μM, 10 μM, and 20 μM before drug addition, with three replicates per group). After incubation in a 37°C cell culture incubator for 24 h, the expression of polarization-related proteins in BMDM was detected by Western blot.

[0084] like Figure 11 As shown, Western blot analysis was used to examine the ability of DIPPGel to polarize macrophages. 5 μM and 10 μM DIPPGel significantly upregulated CD80 expression in BMDM cells, while Arg-1 protein expression showed a concentration-dependent decreasing trend. The two low concentrations of DIPPGel did not significantly change the expression of iNOS and CD206 proteins. However, 20 μM DIPPGel significantly increased the expression levels of M1 phenotype-associated biomarkers CD80 and iNOS, while significantly downregulating M2 phenotype-associated biomarkers CD206 and Arg-1. This indicates that DIPPGel released after H2O2 degradation exhibits a strong macrophage polarizing effect in a concentration-dependent manner.

[0085] 2. The cell supernatant after DIPPGel treatment was analyzed for pro-inflammatory cytokines using an ELISA kit.

[0086] like Figure 12 As shown, the expression levels of pro-inflammatory cytokines IFN-γ and IL-6 in the cell supernatant after DIPPGel treatment were significantly increased. Conversely, the expression levels of anti-inflammatory cytokines TGF-β and IL-10 were significantly decreased compared to the control group, with TGF-β expression almost completely inhibited. These results indicate that DIPPGel polarizes M2 macrophages to M1 type, enabling them to secrete large amounts of pro-inflammatory cytokines and inhibit the release of anti-inflammatory cytokines.

[0087] The above results demonstrate that DIPPGel gel promotes macrophage polarization, thereby exerting a strong positive regulatory effect on the complex tumor microenvironment.

[0088] Test Example 3: The tumor-suppressing effect of DIPPGel in a Balb / C mouse 4T1 tumor model

[0089] Balb / C mice were subcutaneously injected with 50 × 10 4 4 T1 cells. Tumor volume reached 100 mm. 3 At approximately 4:00 PM, mice were randomly assigned to two treatment groups (n=5): PBS and DIPPGel. The gel was administered via peritumoral injection, with the following dosages: DIPP: 200 μL, 5%, w / w; PVA: 200 μL, 10%, w / w; solutions A and B were simultaneously injected to form a gel. The treatment was administered once. Tumor volume and mouse weight were measured every 4 days. Tumor volume (mm²) was calculated using the following formula. 3 Tumor volume = (shortest diameter) 2 ×(longest diameter)×0.5.

[0090] The tumor suppression was evaluated in a Balb / C mouse 4T1 subcutaneous tumor model. Figure 13 As shown, compared to the PBS group, the DIPPGel group exhibited a significant tumor-suppressive effect. Meanwhile, the body weight of mice in the DIPPGel group did not change significantly. Figure 14 ).

Claims

1. A method for preparing hydrogels based on molecules containing monoboronic acid groups, characterized in that, Includes the following steps: (1) Dissolve a compound containing a single boric acid group and an aromatic hydrophobic group in a solvent to prepare solution A; The compound is a dipyridamole prodrug modified with a phenylboronic acid group, and the structural formula of the dipyridamole prodrug is shown in Formula (I). (I)； Alternatively, the compound may be bortezomib; Alternatively, the compound may be esazolidinium; (2) Dissolve polyvinyl alcohol in an aqueous solution to prepare solution B; (3) Mix liquid A and liquid B to form a hydrogel. The mass ratio of the compound to polyvinyl alcohol in the mixed system is 1:1-5.

2. The method as described in claim 1, characterized in that, The molecular weight of the polyvinyl alcohol is 10,000-200,000.

3. The hydrogel prepared by the method according to claim 1 or 2.

4. The application of the hydrogel as described in claim 3 in the preparation of drug sustained-release agents, characterized in that, The hydrogel serves as a drug carrier.

5. The application of the hydrogel as described in claim 3 in the preparation of tumor immunotherapy drugs, characterized in that, The compound is a dipyridamole prodrug modified with phenylboronic acid groups, and the hydrogel promotes the polarization of tumor-associated macrophages from M2 type to M1 type.

6. The application as described in claim 5, characterized in that, The tumor is breast cancer.

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