Method for preparing hydrogel based on molecules containing single boric acid groups and application

Abstract

The invention discloses a method for preparing hydrogel based on molecules containing single boric acid groups and application, and belongs to the technical field of biological medicine. The preparation method comprises the following steps: (1) dissolving a compound containing a single boric acid group and an aromatic hydrophobic group in a solvent to prepare a solution A; (2) dissolving polyvinyl alcohol in the aqueous phase solution to prepare a solution B; and (3) mixing the solution A and the solution B, and reacting to form the hydrogel. According to the invention, a prodrug modified by a boric acid group structure or a drug carrying a boric acid group structure can be directly used as a cross-linking agent for preparing hydrogel to form a drug sustained-release agent; the hydrogel can also be used as a carrier to load a second drug, and the prodrug in the hydrogel needs to release active ingredients after response of active oxygen, so that differential slow release of the two drugs can be realized.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a method for preparing a hydrogel based on molecules containing a single boronic acid group and application thereof in preparing a drug sustained-release gel. Background Art

[0002] Most common drug preparations release drugs at a rate greater than the rate at which they are absorbed through biological membranes and enter the body fluid circulation, which has the advantage of rapid onset of action, but the duration of drug efficacy is short. In order to maintain long-term drug efficacy, high-frequency administration (3 to 4 times per day) is often required, which easily leads to "peaks and valleys" in blood drug concentrations and concentration fluctuations, that is, when the concentration is too low, the drug cannot exert its therapeutic effect; when the concentration is too high, adverse reactions are likely to occur.

[0003] In order to solve the above problems, drug sustained-release technology came into being. Its purpose is to release the drug for a longer period of time after the patient ingests it. This requires the use of materials that can be gradually decomposed in the human body, have high biosafety and will not cause rejection reactions as drug carriers. After the drug is loaded with a suitable carrier, it can not only achieve the purpose of sustained release, but also change the way the drug is administered, thereby reducing the number of administrations, reducing adverse drug reactions, and improving the bioavailability of the drug.

[0004] In recent years, drug sustained-release systems have made rapid progress in theory and technology. At present, the more common drug sustained-release systems mainly include nanoparticles, microspheres and hydrogels. Among them, hydrogels can be used as excellent long-acting sustained-release materials due to their unique three-dimensional network structure and the high cross-linking of hydrophilic groups and mesh chains on their skeletons. Hydrogels can regulate the release rate of drugs by controlling the network pore size and cross-linking density. They have the advantages of good biocompatibility and bioresponsive degradation. However, the reported hydrogel materials usually require complex chemical synthesis processes, a variety of non-functional additives, no therapeutic activity or function themselves, and cannot achieve differential release between different drugs carried, which limits their clinical transformation.

[0005] Polyvinyl alcohol (PVA) is a water-soluble polymer. The hydroxyl groups in the polymer material have strong water absorption, which causes water molecules to gather around it to form a gel. Previous reports have provided methods for preparing gels by covalently crosslinking small molecules or polymers containing diboric acid or polyboric acid groups with polyvinyl alcohol (ACS nano, 2018, 12 (3): 2466-2473; ACS Applied Materials & Interfaces, 2020, 12 (45): 50248-50259; CN114957714A). However, there are no reports of materials containing one boronic acid group in the crosslinker molecule and capable of forming a gel with polyvinyl alcohol. Summary of the invention

[0006] The purpose of the present invention is to provide a new method for preparing hydrogels, by modifying small molecule drugs with a single boronic acid group, forming a gel with polyvinyl alcohol to prepare a gel drug, and releasing the drug after responding to a high content of reactive oxygen species (ROS); on the other hand, the hydrogel can be used as a carrier to encapsulate small molecule or macromolecular drugs to achieve differential sustained release of the two drugs.

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

[0008] The present invention provides a method for preparing a hydrogel based on molecules containing a single boronic acid group, comprising the following steps:

[0009] (1) dissolving a compound containing a single boronic acid group and an aromatic hydrophobic group in a solvent to prepare a liquid A;

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

[0011] (3) Liquid A and Liquid B are mixed and reacted to form a hydrogel.

[0012] In the above-mentioned hydrogel formation process, a single boric acid group in the molecular structure of the compound works together with an aromatic hydrophobic group to realize the gelation of the polyvinyl alcohol aqueous solution. Specifically, the boric acid group bonds with the ortho-dihydroxyl group of polyvinyl alcohol, and the aromatic hydrophobic group aggregates through hydrophobic interaction and intermolecular π-π stacking and hydrogen bonding, resulting in cross-linking of the polymer, thereby 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 may be, but is not limited to, a benzene ring, an anthracene ring, a pyridine ring, a pyrimidine ring, a furan ring, a thiophene ring, a pyrrole ring, a benzopyridine ring, a benzopyrimidine ring, a benzopyrrole, a benzofuran, or a benzothiophene.

[0014] The boric acid group and polyvinyl alcohol are coordinated and cross-linked to form a reversible covalent boric acid ester bond, which is a reactive oxygen species (ROS) response bond. When the hydrogel is exposed to a high concentration of reactive oxygen species, the boric acid ester bond breaks to release the active compound. Therefore, the compound can be a prodrug with a modified boric acid group structure or a drug that carries a boric acid group structure. The prodrug or drug itself has no biological activity or has very low activity. After the reactive oxygen species response bond breaks, the active ingredient with biological activity will be released, which can be used to achieve the preparation of controlled-release drugs.

[0015] Preferably, the compound is a dipyridamole prodrug modified with a phenylboronic acid group. Specifically, the phenylboronic acid group structure of 4-(bromomethyl)phenylboronic acid is connected to the tertiary amine group of dipyridamole by an amination reaction of benzyl bromide and tertiary amine. The prodrug component plays a decisive role in the formation of the gel and the ROS response of the gel. Moreover, the activity itself is relatively low, and it can become an active original drug after being metabolized in the body, which can reduce the systemic toxic and side effects of the drug.

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

[0017]

[0018] The present invention's research shows that the above-mentioned small molecule prodrug will slowly activate and release dipyridamole under high concentrations of reactive oxygen media, thereby promoting the polarization of tumor-associated macrophages from M2 type to M1 type.

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

[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] Ixazomib 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] As a key component of the gel, polyvinyl alcohol has the advantage of high biological safety. The molecular formula of the polyvinyl alcohol is [-CH2CHOH-] n , the structural formula is shown in formula (IV):

[0025]

[0026] The present invention uses polyvinyl alcohol with a molecular weight of 10000-200000. Preferably, the molecular weight of the polyvinyl alcohol is 14600-18600. 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 phase solution to form a hydrogel by using a solution mixing method such as a two-liquid simultaneous injection method or a two-liquid sequential injection method.

[0028] Preferably, the mass ratio of the compound to polyvinyl alcohol in the mixed system is 1:1-5. The present invention can obtain gel materials with different hardnesses by a simple physical mixing method and adjusting the mass concentrations of liquid A and liquid B. The gelling efficiency and speed of the gel increase with the increase of the amount of polymer used, but excessive amount of polymer causes waste of polymer. Within this mass ratio range, faster gel formation and polymer utilization can be guaranteed. More preferably, the mass ratio of the compound to polyvinyl alcohol in the mixed system is 1:1-2.

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

[0030] The present invention also provides the use of the hydrogel in preparing a drug sustained-release agent, wherein the hydrogel is used as a drug carrier, and / or the compound used to prepare the hydrogel is a prodrug modified with a boronic acid group structure or a drug carrying a boronic acid group structure.

[0031] The hydrogel provided by the present invention can be used as a drug delivery carrier to further encapsulate small molecule or macromolecular drugs to achieve 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 macromolecular drug in a good solvent respectively, and then mixing them by physical mixing method to instantly form a hydrogel encapsulating the drug.

[0033] The present invention can directly use the prodrug with a boronic acid group structure modified or the drug with a boronic acid group structure as a cross-linking agent for the preparation of the hydrogel to form a drug sustained-release agent. The hydrogel can also be used as a carrier to load a second drug. Since the prodrug in the hydrogel needs to release the active ingredient after the active oxygen response, this solution can achieve differential sustained release of the two drugs.

[0034] The present invention also provides the use of the hydrogel in preparing tumor immunotherapy drugs, wherein the compound is a dipyridamole prodrug modified with a phenylboronic acid group, 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 uses or activates immunity to kill tumors. However, due to the existence of an immunosuppressive tumor microenvironment (TME), the response rate of tumor immunotherapy is still low. Tumor-associated macrophages (TAMs) are the most common immune cells in the tumor microenvironment. Usually, due to changes in tumor cell metabolism and their regulation of the tumor microenvironment, they are prone to differentiate into the immunosuppressive M2 phenotype and damage the proliferation and differentiation function of tumor-infiltrating lymphocytes by secreting a variety of active substances. Among the strategies for TAMs regulation, the macrophage polarization strategy can reprogram TAMs into a pro-inflammatory M1 phenotype, thereby effectively reversing the immunosuppressive TME and improving the efficacy of immunotherapy.

[0036] The present invention has found that dipyridamole has the activity of promoting macrophage polarization, and can polarize tumor-associated macrophages from M2 to M1. The dipyridamole prodrug modified with the phenylboronic acid group has low activity. Only under the action of highly expressed reactive oxygen mediators in tumor cells, the dipyridamole original drug is released and activated, promoting macrophage polarization, thereby reversing the tumor immunosuppressive microenvironment and exerting the anti-tumor immunotherapy effect.

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

[0038] The drug can be administered by injection near the tumor. After the gel drug is injected near the tumor, the high level of ROS in the tumor tissue microenvironment can trigger the macrophage polarization prodrug in the gel to slowly degrade into macrophage polarization original drug, and continuously deliver it to the tumor tissue, greatly improving the accumulation and retention time of the drug in the tumor, thereby achieving continuous macrophage polarization in the tumor tissue. In addition, local injection can reduce the chance of the drug entering normal tissue, significantly improve the tumor selectivity of the drug, and achieve precise treatment of tumors.

[0039] The present invention has the following beneficial effects:

[0040] (1) The present invention provides a crosslinking agent material for gelation of a polyvinyl alcohol aqueous solution using a compound containing a single boronic acid group and an aromatic hydrophobic group, which forms a gel instantly after physical mixing. The present invention can directly use a prodrug with a boronic acid group structure modified or a drug with a boronic acid group structure as a crosslinking agent for the preparation of a hydrogel to form a drug sustained-release agent; the hydrogel can also 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) The present invention uses the dipyridamole prodrug modified with the phenylboronic acid group structure as a crosslinking agent material for the gelation of polyvinyl alcohol aqueous solution to prepare gel drugs. The gel component gives the drug the ability to slowly and long-term deliver and accumulate in the tumor. When the drug reaches the tumor tissue, due to the high expression of reactive oxygen components in tumor cells, it can trigger the degradation of dipyridamole prodrug to release dipyridamole original drug, promote the polarization of tumor-associated macrophages in tumor tissue from M2 type to M1 type, and then reverse the tumor immunosuppressive microenvironment, exert anti-tumor immunotherapy effects, and achieve precise tumor treatment. Since the polarized drug only works in tumor tissue, the systemic toxicity and side effects of the gel drug are reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : is the hydrogen nuclear magnetic resonance spectrum of the dipyridamole prodrug DIPPGel in the example.

[0043] Figure 2 : is the mass spectrum of dipyridamole prodrug DIPPGel in the examples.

[0044] Figure 3 This is a picture of dipyridamole prodrug and polyvinyl alcohol mixed into a gel DIPPGel in the example.

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

[0046] Figure 5 This is the rheological properties diagram of the gel DIPPGel in the example.

[0047] Figure 6 This is a picture of bortezomib and polyvinyl alcohol mixed into a gel in the example.

[0048] Figure 7 This is a picture of ixazomib and polyvinyl alcohol mixed into a gel in the examples.

[0049] Figure 8 This is a picture of S-(2-boronoethyl)-L-cysteine ​​mixed with polyvinyl alcohol in the example.

[0050] Fig. 9 This is a picture of Talabostat mixed with polyvinyl alcohol in the example.

[0051] Fig.10 This is the drug release curve of the 3BP-encapsulated gel DIPPGel in the example.

[0052] Fig.11 The expression levels of CD80, iNOS, CD206 and Arg-1 in BMDM after treatment with different concentrations of gel DIPPGel for 24 hours were analyzed by western blot in the example.

[0053] Fig.12 This is the ELISA quantitative analysis of the release of IFN-γ, IL-6, TGF-β1 and IL-10 cytokines in the BMDM culture medium after stimulation with DIPPGel (containing 20 μM DIP) for 24 hours in the example.

[0054] Fig.13 This is a graph showing the tumor growth curve in the experiment in which the gel DIPPGel inhibits tumors in Balb / c mice bearing 4T1 breast cancer cells.

[0055] Fig.14 This is a curve chart of the weight changes of Balb / c mice during the experiment of the gel DIPPGel inhibiting tumors in Balb / c mice bearing 4T1 breast cancer cells in the example. DETAILED DESCRIPTION

[0056] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0057] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels 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 purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. 4T1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0059] The induced differentiated M2 BMDM was prepared by extracting bone marrow-derived macrophages from the peritoneal cavity of BALB / c mice and differentiating them. The preparation method was as follows: 6-8 week-old female BALB / c mice were used as the source of bone marrow mononuclear cells. Before the experiment, hand scissors, ophthalmic forceps, filter gauze, and beaker were soaked in alcohol and sterilized with other materials in a clean bench under ultraviolet irradiation for 30 minutes. After the mice were killed by dislocating the neck, they were soaked in sterile 75% ethanol for 10 minutes. In a sterile large dish filled with 75% ethanol, the tibia of the lower limbs, humerus and femur of the upper limbs of the mice were separated, and the muscles were carefully removed. The clean muscles were removed in two small dishes filled with 75% ethanol (the treatment time in 75% ethanol should not exceed 30 minutes to prevent the bones from being easily broken due to too long time, resulting in bone marrow exposure and infection), and then the residual muscles were removed in four small dishes filled with sterile PBS, and clean bones without muscles were obtained as much as possible. After cleaning, use sterile scissors to cut off both ends of the tibia, femur and humerus, use a 1mL syringe to absorb serum-free culture medium, flush out the bone marrow, blow it evenly, transfer it to a 15mL centrifuge tube, centrifuge it at 1500rpm for 5min, discard the supernatant, add 3-5mL sterile red blood cell lysis buffer (1×) to resuspend, let it stand at room temperature for 5min, add an equal amount of sterile culture medium to terminate red blood cell lysis, pass it through a sterile filter to a new sterile 15mL centrifuge tube, centrifuge it at 1500rpm for 5min, discard the supernatant, and the cell pellet is the bone marrow-derived cells. Use BMDM induction culture medium (DMEM culture medium with 20% fetal bovine serum, 1% penicillin-streptomycin and 20ng / mL M-CSF) to blow it evenly and plate it. After 3-4 days, a large number of cells adhere to the wall. 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 dimethoate prodrug gel DIPPGel

[0061] 1. Preparation of dipyridamole prodrug containing boronic acid bond: 4-(bromomethyl)phenylboronic acid (0.2g, 1mmol) and dipyridamole (DIP, 1g, 2mmol) were dissolved in 30mL of anhydrous DMF solution and ultrasonically mixed. The system was then stirred at 45°C for 6h. After the reaction was completed, 200mL of ether was added for filtration and washed three times with 200mL of ether. After vacuum drying overnight, a 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 dipyridamole prodrug was successfully prepared by nuclear magnetic resonance spectroscopy and mass spectrometry characterization.

[0064] 2. Prepare dipyridamole prodrug gel DIPPGel by physical mixing: Dissolve dipyridamole prodrug (50 mg) in 1 mL of pure water solution to prepare a dipyridamole prodrug solution with a mass concentration (w / w) of 5%, namely, solution A. Dissolve polyvinyl alcohol (50 mg) in 1 mL of pure water solution, and heat at 100°C for half an hour to prepare a polyvinyl alcohol solution with a mass concentration (w / w) of 5%, namely, solution B. Then, solution A and solution B are respectively loaded into a double-barreled syringe, and solution A and solution B can be injected simultaneously or separately to make the two contact and mix with each other, and DIPPGel gel can be formed instantly.

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

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

[0067] like Figure 5 As shown, from the rheological properties of DIPPGel during its formation, it can be observed that when the DIPP solution and the 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 is prepared by a physical mixing method: Bortezomib (50 mg) is dissolved in 1 mL of methanol to prepare a bortezomib solution with a mass concentration (w / w) of 5%, namely, solution A, and polyvinyl alcohol (50 mg) is dissolved in 1 mL of aqueous solution, and heated at 100° C. for half an hour to prepare a polyvinyl alcohol solution with a mass concentration (w / w) of 5%, namely, solution B. Subsequently, solution A and solution B are respectively loaded into a double-barreled syringe, and solution A and solution B are injected simultaneously or sequentially so that the two are in contact and mixed with each other, and bortezomib gel can be formed instantly.

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

[0071] Example 3: Preparation of Ixazomib Gel

[0072] The ixazomib gel is prepared by a physical mixing method: ixazomib (50 mg) is dissolved in 1 mL of methanol to prepare an ixazomib solution with a mass concentration (w / w) of 5%, namely, solution A, and polyvinyl alcohol (50 mg) is dissolved in 1 mL of aqueous solution, and heated at 100° C. for half an hour to prepare a polyvinyl alcohol solution with a mass concentration (w / w) of 5%, namely, solution B. Subsequently, solution A and solution B are respectively loaded into a double-barreled syringe, and solution A and solution B are injected simultaneously or sequentially so that the two are in contact and mixed with each other, and the ixazomib gel can be formed instantly.

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

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

[0075] BEC gel was prepared by physical mixing: BEC hydrochloride (50 mg) was dissolved in 1 mL of pure water to prepare a 5% BEC solution (w / w), i.e., solution A. At the same time, 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% polyvinyl alcohol solution (w / w), i.e., solution B. Then, solution A and solution B were respectively loaded into a double-barreled syringe, and solution A and solution B were injected simultaneously or sequentially so that the two were in contact with each other and mixed. No matter how long they were mixed, no gel could be formed. Figure 8 shown.

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

[0077] Talabostat gel was prepared by physical mixing: Talabostat mesylate (50 mg) was dissolved in 1 mL of pure water to prepare a 5% Talabostat solution (w / w), i.e., solution A. 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% polyvinyl alcohol solution (w / w), i.e., solution B. Solution A and solution B were then loaded into a double-barreled syringe, and solution A and solution B were injected simultaneously or sequentially so that the two were in contact with each other and mixed. No matter how long they were mixed, gel could not be formed. Fig. 9 Test Example 1: In vitro differential drug release of DIPPGel gel

[0078] The ability to respond to active oxygen species is a very important part of evaluating the in vivo application of DIPP gel. Good and slow response release ability is the guarantee for the full activation of macrophage polarization drugs and the subsequent promotion of macrophage polarization.

[0079] In order to explore the differentiated release behavior of the two drugs in the gel, 3-bromopyruvic acid (3BP) was used as the encapsulated drug.

[0080] First, dipyridamole prodrug (50 mg) was dissolved in 1 mL of pure water solution to form a 5% dipyridamole prodrug solution, i.e., liquid A. Polyvinyl alcohol (50 mg) and 3-bromopyruvic acid (1 mg) were dissolved in 1 mL of pure water solution at the same time, and polyvinyl alcohol solution (50 mg) and 3-bromopyruvic acid (1 mg) were heated at 100° C. for half an hour to form a 5% polyvinyl alcohol solution, i.e., liquid B. The above liquid A and liquid B were mixed to prepare gel. 100 μL of gel (dipyridamole content of 5 mg) 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 containing or not containing 1 mM hydrogen peroxide (H2O2). 500 μL of dialyzate outside the dialysis bag was collected at regular intervals, and the concentrations of dipyridamole and 3-bromopyruvic acid were then measured by HPLC.

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

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

[0083] 1. To explore the effect of DIP released from DIPPGel on macrophage polarization, the induced differentiated M2 BMDM were plated in 6-well plates with a cell density of 2×10 5 First, 1 mL of the mixed prepared gel DIPPGel (liquid A is DIPP: 1 mL, 5%, w / w; liquid B is PVA: 1 mL, 10%, w / w, mass ratio is 1:1) was taken and added to hydrogen peroxide (1 mM, 5 mL) for 7 days. The collected solution was lyophilized into powder to obtain the degraded dipyridamole original drug. Then different masses of powder were weighed and dissolved in DMSO to prepare dipyridamole solution (5 μM, 10 μM and 20 μM, and then the drug was added, with three replicates in each group. After incubation in a 37°C cell culture incubator for 24 hours, the expression of polarization-related proteins in BMDM was detected by western blot.

[0084] like Fig.11 As shown in the figure, using western blot to examine the ability of DIPPGel to polarize macrophages, 5μM and 10μM DIPPGel can significantly upregulate the expression of CD80 in BMDM cells. On the contrary, the protein expression of Arg-1 also showed a concentration-dependent decreasing trend. The two low concentrations of DIPPGel did not significantly change the expression of iNOS and CD206 proteins. For DIPPGel at a concentration of 20μM, the expression levels of M1 phenotype-related biomarker proteins CD80 and iNOS were greatly increased, and the M2 phenotype-related biomarker proteins CD206 and Arg-1 also showed a significant downward trend, indicating that the DIP released after DIPPGel was degraded by H2O2 showed a strong macrophage polarization effect, and showed a concentration-dependent trend.

[0085] 2. The supernatant of cells treated with DIPPGel was analyzed for pro-inflammatory cytokines using an ELISA kit.

[0086] like Fig.12 As shown in the figure, the expression of pro-inflammatory cytokines IFN-γ and IL-6 in the cell supernatant after DIPPGel treatment increased significantly. On the contrary, the expression of anti-inflammatory cytokines TGF-β and IL-10 decreased significantly compared with the control group, and the expression of TGF-β was almost completely inhibited. The results show that after DIPPGel polarizes M2 macrophages to M1, it can also secrete a large number of pro-inflammatory cytokines and inhibit the release of anti-inflammatory cytokines.

[0087] The above results prove that DIPPGel gel has the effect of promoting macrophage polarization, thereby playing a strong positive regulatory role in the complex tumor microenvironment.

[0088] Test Example 3: Tumor-suppressing effect of DIPPGel in the 4T1 tumor model of Balb / C mice

[0089] Balb / C mice were injected subcutaneously with 50×10 4 4T1 cells. Tumor volume reaches 100mm 3 At about 24 hours, mice were randomly assigned to 2 treatment groups (n=5): PBS and DIPPGel. The gel injection method was peritumoral injection, and the gel injection dose was DIPP: 200μL, 5%, w / w; PVA: 200μL, 10%, w / w; A liquid and B liquid were injected into the gel at the same time. The drug was administered once in total. The tumor volume and mouse body weight were measured every 4 days. The tumor volume (mm 3 ): tumor volume = (shortest diameter) 2 ×(longest diameter)×0.5.

[0090] The inhibition of tumor was evaluated in the 4T1 subcutaneous tumor model of Balb / C mice. Fig.13 As shown in the figure, compared with the PBS group, the DIPPGel group showed a significant tumor inhibition effect. At the same time, there was no significant change in the body weight of the mice in the DIPPGel group ( Fig.14 ).

Claims

1. A method for preparing a hydrogel based on molecules containing a monoboronic acid group, characterized in that: The following steps are involved: (1) dissolving a compound containing a single boronic acid group and an aromatic hydrophobic group in a solvent to prepare a liquid A; (2) dissolving polyvinyl alcohol in an aqueous solution to prepare liquid B; (3) Liquid A and Liquid B are mixed and reacted to form a hydrogel.

2. The method according to claim 1, characterized in that The compound is a dipyridamole prodrug modified with a phenylboronic acid group.

3. The method according to claim 2, characterized in that The dipyridamole prodrug structural formula is shown in formula (I), 4. The method according to claim 1, characterized in that The compound is bortezomib or ixazomib.

5. The method according to claim 1, characterized in that The molecular weight of the polyvinyl alcohol is 10,000-200,000.

6. The method according to claim 1, characterized in that The mass ratio of the compound to polyvinyl alcohol in the mixed system is 1:1-5.

7. A hydrogel prepared by the method according to any one of claims 1 to 6.

8. Use of the hydrogel according to claim 7 in preparing a drug sustained-release agent, characterized in that: The hydrogel is used as a drug carrier, and / or the compound used to prepare the hydrogel is a prodrug with a modified boronic acid group structure or a drug that carries a boronic acid group structure.

9. Use of the hydrogel according to claim 7 in preparing a tumor immunotherapy drug, characterized in that: The compound is a dipyridamole prodrug modified with a phenylboronic acid group, and the hydrogel promotes the polarization of tumor-associated macrophages from M2 type to M1 type.

10. The use according to claim 9, characterized in that The tumor is breast cancer.

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