Drug-loaded hydrogel for tumor therapy, its preparation method and application

Abstract

The application belongs to the technical field of biomedical materials, and more particularly relates to a drug-loaded hydrogel for postoperative tumor treatment, a preparation method and application thereof. Two reaction substrates, dopamine and 3-aminobenzene boronic acid, are grafted onto a hyaluronic acid skeleton through an amide condensation reaction by a one-pot method to prepare a hydrogel scaffold material. The flexible rheological properties and excellent biological adhesion can ensure that the hydrogel is stably adhered to the resection wound without displacement, slow and pH-responsive release, and in-vivo drug retention for a long time, so that the hydrogel can respond to the tumor microenvironment and maintain the tumor resection microenvironment at a sufficient drug concentration for a long time. The hydrogel prepared by the application can simultaneously load an antitumor chemotherapeutic drug and a drug capable of improving the tumor microenvironment, so that the tumor microenvironment is significantly improved while the chemotherapeutic drug inhibits the tumor, and the two synergistically promote the intervention and inhibition of postoperative tumor recurrence and metastasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and more specifically, relates to a drug-loaded hydrogel for tumor treatment, its preparation method and application. Background Technology

[0002] Malignant tumors threaten human health. Traditional treatments such as surgical resection, chemotherapy, and radiotherapy are often insufficient to achieve a complete cure, resulting in unsatisfactory treatment outcomes. Surgical resection is the primary clinical treatment for malignant tumors, but it frequently faces the problem of residual tumor cells leading to tumor recurrence and metastasis. Conventional clinical measures involve adjuvant systemic chemotherapy or radiotherapy after surgery, but these are highly toxic and have a window of inactivity due to the patient's postoperative weakness. Furthermore, postoperative tissue damage, changes in the cellular microenvironment, and inflammation all contribute to the recurrence process. This includes the activation of platelets recruited to the resection site due to vascular and tissue damage, angiogenesis in damaged blood vessels, and the role of stromal cells such as adipose tissue in promoting tumor recurrence and metastasis. Therefore, timely removal of residual tumor cells after surgery, combined with modification of the postoperative tumor microenvironment, is a crucial strategy for inhibiting postoperative tumor recurrence and metastasis and improving patient survival.

[0003] In-situ drug delivery systems are a treatment method that utilizes materials that can be fixed to the lesion site for in-situ drug delivery, offering significant advantages. These systems typically employ biodegradable and biocompatible materials as a framework to load one or more drugs, releasing them locally to the target site. This increases drug concentration at the target site while reducing drug concentration in the blood and systemic tissues, ensuring therapeutic efficacy with low toxicity. Such delivery systems generally possess sustained-release or controlled-release characteristics, allowing for intelligent and precise drug release based on the unique microenvironment of the surgical site, maximizing therapeutic efficacy and minimizing side effects.

[0004] Hydrogels are hydrophilic, three-dimensional, cross-linked polymeric networks, typically cross-linked via covalent or non-covalent bonds. Materials composing hydrogels can include polysaccharides (chitosan, hyaluronic acid, gelatin), peptides (poly(L-glutamic acid), silk fibroin), and polymers (polyacrylamide and polyvinyl alcohol). Depending on the materials used and the cross-linking methods, hydrogels can possess various physical properties such as elasticity, toughness, injectability, adhesion, anti-adhesion, conductivity, stretchability, plasticity, and self-healing, meeting diverse application needs. From drug delivery systems to tissue engineering and wearable electronic components, the application range of hydrogels is expanding. However, single hydrogel treatments currently struggle to achieve satisfactory therapeutic effects; therefore, more comprehensive treatment approaches hold greater promise.

[0005] Adipocytes constitute the largest proportion of all cell types in breast tissue and are key cells in the tumor resection microenvironment of breast cancer. In the breast cancer tumor microenvironment, cancer-associated adipocytes not only spatially border cancer cells but also crosstalk with them, promoting tumor recurrence, progression, and metastasis. Studies have shown that adipocytes not only function as energy storage cells but also as important endocrine cells, producing various bioactive molecules called "adipokines," such as leptin, adiponectin, IL-6, and resistin. These adipocytes participate in the proliferation, spread, angiogenesis, invasion, and metastasis of breast cancer through adiponectin regulation, metabolic reprogramming, microenvironment remodeling, and immune regulation. However, research on preventing tumor recurrence and metastasis by inhibiting adipocytes in the postoperative tumor microenvironment is currently limited. Furthermore, angiogenesis and remodeling are important biological processes promoting tumor growth in the tumor resection microenvironment. Damaged blood vessels generate new blood vessels to transport oxygen and nutrients, thereby promoting rapid recurrence and metastasis of residual tumor cells. Therefore, inhibiting adipocyte function and angiogenesis while killing residual tumor cells holds promise for suppressing postoperative tumor recurrence and metastasis.

[0006] Metamide (Met) is a commonly used antidiabetic drug that regulates metabolism and inhibits angiogenesis by activating AMPK. Recent studies have shown that Met can delay tumor progression by influencing adipocytes and reducing adipokines secretion, thereby reshaping the adipose-associated tumor microenvironment. However, Met has a short half-life and low bioavailability when administered systemically, making it difficult to achieve the desired effects.

[0007] Implantable drug delivery platforms, by being directly implanted at the tumor resection site, can avoid the postoperative recovery window, increase drug accumulation at the tumor resection site, prolong drug release time, and reduce systemic drug side effects, providing a promising strategy for postoperative cancer treatment. Hydrogels are mainly composed of a three-dimensional macromolecular network and a large amount of water. Bioadhesive hydrogels are characterized by high porosity, strong plasticity, and low toxicity, while simultaneously achieving controlled drug release and good tissue adhesion. Inspired by mussel adhesion proteins, adhesive hydrogels rich in catechols and polyphenols such as dopamine (DA) and gallic acid (GA) have been applied in wound dressings, tissue engineering, and flexible devices. However, how to efficiently release drugs from hydrogels at the tumor resection site still needs improvement. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a drug-loaded hydrogel for tumor treatment, its preparation method, and its application. By constructing a biocompatible hydrogel material, it enables in-situ delivery of chemotherapeutic drugs and tumor microenvironment-modifying drugs to prevent postoperative tumor recurrence and metastasis.

[0009] To achieve the above objectives, the present invention provides a method for preparing a hydrogel for postoperative drug loading, comprising the following steps:

[0010] (1) Mix and stir an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and hyaluronic acid, and keep the solution weakly acidic to activate the carboxyl group of the hyaluronic acid to obtain an activated precursor solution.

[0011] (2) The activated precursor solution described in step (1) is mixed with 3-aminophenylboronic acid and dopamine hydrochloride and reacted under inert gas protection and weak acid conditions, so that the 3-aminophenylboronic acid and dopamine hydrochloride are grafted onto the backbone of the hyaluronic acid through an amide condensation reaction to obtain the crude product.

[0012] (3) The crude product described in step (2) is dialyzed in acidic ultrapure water. The pH of the liquid obtained by dialyzing is adjusted to neutral and then stirred. Dopamine is oxidized and polymerized using oxygen in the air, and then freeze-dried to obtain a hydrogel for drug loading.

[0013] Preferably, in step (1), the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and hyaluronic acid is (0.4-0.8):(0.1-0.5):1, and the mass percentage of hyaluronic acid in the aqueous solution is 0.5-1.5%; the activation time in step (1) is 10-40 minutes.

[0014] Preferably, the molar ratio of 3-aminophenylboronic acid and dopamine hydrochloride in step (2) is 3:1 to 1:3, and the mass ratio of dopamine hydrochloride to hyaluronic acid is 0.28 to 1.1:1.

[0015] Preferably, the inert gas in step (2) is argon or nitrogen, and the reaction time is 12-36 hours.

[0016] Preferably, in step (3), the crude product is transferred to a dialysis bag with a molecular weight cutoff of 10,000-14,000 Da; the stirring time is 3-8 hours; and the stirred product is freeze-dried under vacuum for 24-72 hours to obtain the hydrogel used for drug loading.

[0017] According to another aspect of the present invention, a drug-loaded hydrogel for inhibiting postoperative recurrence and metastasis of tumors is provided, prepared by the method described above.

[0018] According to another aspect of the invention, a postoperative drug-loaded hydrogel is provided, comprising the hydrogel, an antitumor drug, and a drug capable of improving the tumor microenvironment, wherein the antitumor drug comprises one or more of doxorubicin, paclitaxel, and camptothecin, and the drug capable of improving the tumor microenvironment comprises metformin and / or aspirin.

[0019] According to another aspect of the invention, the use of the drug-loaded hydrogel described herein in the preparation of medicaments for postoperative treatment and / or tumor suppression is provided.

[0020] According to another aspect of the invention, the use of the drug-loaded hydrogel described above in the preparation of a medicament for inhibiting postoperative tumor adipocyte regeneration and / or inhibiting postoperative tumor angiogenesis is provided.

[0021] According to another aspect of the invention, the use of the drug-loaded hydrogel described above in the preparation of a medicament for inhibiting platelet activation after tumor surgery is provided.

[0022] In summary, the technical solutions conceived by this invention have the following beneficial effects compared with the prior art:

[0023] (1) The present invention uses a one-pot amide condensation reaction to graft two reaction substrates, dopamine and 3-aminophenylboronic acid, onto a hyaluronic acid backbone to prepare a hydrogel for drug loading. The preparation method is simple, convenient, safe and controllable.

[0024] (2) The hydrogel prepared by the present invention uses dopamine and 3-aminophenylboronic acid as reaction substrates and has a double cross-linking network, which makes the hydrogel have a tighter cross-linking network and better rheological properties.

[0025] (3) The hydrogel of this invention exhibits excellent bioadhesion properties due to the grafting of dopamine, ensuring that the hydrogel adheres to biological tissues without displacement. Because the phenylboronic acid ester bond formed between dopamine and 3-aminophenylboronic acid is pH-responsive, the prepared hydrogel can slowly release drugs in response to the acidity of tissues. Furthermore, this invention utilizes oxygen from the air to oxidize and polymerize the dopamine in the prepared gel material, controlling the degree of oxidation and polymerization to achieve a suitable level, thereby controlling the degradation rate of the gel material in vivo, which corresponds to a suitable drug release rate.

[0026] (4) This invention provides a drug-loaded hydrogel for postoperative tumor treatment, which can encapsulate chemotherapy drugs or other interventional drugs. The hydrogel scaffold material provided by this invention has good biocompatibility, bioadhesion, swelling properties, and pH responsiveness. Its flexible rheological properties and excellent bioadhesion ensure stable adhesion to the surgical site without displacement. The slow and pH-responsive release and the ability to maintain drug retention in vivo for a long time enable the hydrogel to respond to the tumor microenvironment and maintain a sufficient drug concentration in the tumor surgical microenvironment for a long time.

[0027] (5) The hydrogel prepared by this invention can simultaneously load antitumor chemotherapeutic drugs and drugs that can improve the tumor microenvironment, so that while the chemotherapeutic drugs inhibit the tumor, the tumor microenvironment is also improved, synergistically promoting the inhibition of postoperative tumor recurrence and metastasis. In the preferred embodiment of this invention, the drug-loaded hydrogel simultaneously loaded with doxorubicin (DOX) and metformin (Met) was found through postoperative experiments to significantly inhibit postoperative tumor recurrence and metastasis compared with the control group, and to inhibit the new formation of adipocytes, thereby inhibiting tumor recurrence; moreover, it can significantly inhibit the formation of blood vessels after tumor surgery, reducing the oxygen and nutrient supply to residual tumor tissue. This invention is expected to provide a theoretical basis and treatment strategy for the postoperative treatment of tumors with a large number of surrounding adipocytes, such as breast cancer, pancreatic cancer, and renal cell carcinoma. In addition, the prepared drug-loaded hydrogel loaded with DOX and aspirin (ASA) can effectively inhibit platelet activation while killing tumor cells, effectively inhibiting postoperative tumor recurrence and metastasis. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating the preparation method of the drug-loaded hydrogel for tumor treatment according to the present invention.

[0029] Figure 2 Content A shows the shear viscosity of HDP hydrogels with different dopamine / APBA ratios; Content B shows the changes of G′ and G″ of HDP hydrogels with different dopamine / APBA ratios as a function of frequency; Content C shows the changes of G′ and G″ of HDP hydrogels with different dopamine / APBA ratios as a function of strain degree.

[0030] Figure 3 Content A is a schematic diagram of a universal testing machine for measuring the adhesion ability of HDP hydrogels with different dopamine / APBA ratios; Content B is a curve showing the change in adhesion strength of HDP hydrogels with different dopamine / APBA ratios as a function of displacement; Content C is a statistical analysis of the adhesion strength of HDP hydrogels with different dopamine / APBA ratios as a function of displacement.

[0031] Figure 4Content A shows a photograph of the morphology of HDP hydrogel; Content B shows a schematic diagram of the self-healing principle of HDP hydrogel; Content C shows a photograph of the self-healing process of HDP hydrogel; Content D shows a photograph of HDP hydrogel adhering to a finger; Content E shows a photograph of HDP hydrogel exhibiting extensibility as the finger bends; Content F shows a photograph of HDP hydrogel adhering to tissue; Content G shows a photograph of the tensile properties of HDP hydrogel.

[0032] Figure 5 Content A shows SEM observations of HDP (scale bar: 50 μm, right image is a magnified image of the dashed box in the left image); Content B shows the statistical average pore size of HDP through SEM images.

[0033] Figure 6 Content A describes the in vitro drug release behavior of DOX / Met@HDP at pH 7.4 and 6.5; Content B describes the in vitro drug release behavior of Met / Met@HDP at pH 7.4 and 6.5.

[0034] Figure 7 Content A presents the shear viscosity studies of HDP, DOX@HDP, Met@HDP, and DOX / Met@HDP; Content B presents the variations of G′ and G″ of HDP, DOX@HDP, Met@HDP, and DOX / Met@HDP with frequency; Content C presents the variations of G′ and G″ of HDP, DOX@HDP, Met@HDP, and DOX / Met@HDP with strain degree.

[0035] Figure 8 Content A shows in vivo fluorescence images of representative small animals of Cy5.5 in mice after Cy5.5 / ICG and Cy5.5 / ICG@HDP treatment following 4T1 in situ tumor resection; Content B shows the quantitative fluorescence of Cy5.5 in mice after Cy5.5 / ICG and Cy5.5 / ICG@HDP treatment following 4T1 in situ tumor resection; Content C shows in vivo fluorescence images of representative small animals of ICG in mice after Cy5.5 / ICG and Cy5.5 / ICG@HDP treatment following 4T1 in situ tumor resection; Content D shows the quantitative fluorescence of ICG in mice after Cy5.5 / ICG and Cy5.5 / ICG@HDP treatment following 4T1 in situ tumor resection.

[0036] Figure 9Content A shows representative Cy5.5 fluorescence images of major organs and tumors in mice on day 10 after Cy5.5 / ICG and Cy5.5 / ICG@HDP treatment following 4T1 in situ tumor resection; Content B shows quantitative fluorescence of Cy5.5 in major organs and tumors in mice on day 10 after Cy5.5 / ICG and Cy5.5 / ICG@HDP treatment following 4T1 in situ tumor resection; Content C shows representative ICG fluorescence images of major organs and tumors in mice on day 10 after Cy5.5 / ICG and Cy5.5 / ICG@HDP treatment following 4T1 in situ tumor resection; Content D shows quantitative fluorescence of ICG in major organs and tumors in mice on day 10 after Cy5.5 / ICG and Cy5.5 / ICG@HDP treatment following 4T1 in situ tumor resection.

[0037] Figure 10 Content A describes the CCK-8 assay for determining the cell viability of 4T1 cells after 24 hours of treatment with PBS, HDP release solution, DOX, Met, DOX@HDP release solution, Met@HDP release solution, or DOX / Met@HDP release solution (where the free drug and the release solution have different concentrations of DOX and Met); Content B describes the Annexin V apoptosis kit and 7-AAD assay for detecting apoptosis in 4T1 cells after 24 hours of treatment with PBS, HDP release solution, DOX, Met, DOX@HDP release solution, Met@HDP release solution, or DOX / Met@HDP release solution; Content C describes the staining of live and dead cells to study the cytotoxicity of DOX / Met@HDP on 4T1 cells.

[0038] Figure 11Content A shows the cell viability of adipocytes after treatment with PBS, HDP, DOX, Met, DOX@HDP, Met@HDP, or DOX / Met@HDP; Content B shows the lipid content of adipocytes after treatment with PBS, HDP, DOX, Met, DOX@HDP, Met@HDP, or DOX / Met@HDP; Content C shows the IL-6 content in the supernatant of adipocytes after treatment with PBS, HDP release solution, DOX, Met, DOX@HDP release solution, Met@HDP release solution, or DOX / Met@HDP release solution as detected by ELISA; Content D shows the IL-6 content in the supernatant of adipocytes after treatment with PBS, HDP release solution, DOX, Met ... t represents the expression level of adipokine IL-6 mRNA in adipocytes after treatment with DOX@HDP release solution, Met@HDP release solution, or DOX / Met@HDP release solution; E represents the expression level of adipokine adiponectin mRNA in adipocytes after treatment with PBS, HDP release solution, DOX, Met, DOX@HDP release solution, Met@HDP release solution, or DOX / Met@HDP release solution; F represents the expression level of adipokine resistin mRNA in adipocytes after treatment with PBS, HDP release solution, DOX, Met, DOX@HDP release solution, Met@HDP release solution, or DOX / Met@HDP release solution.

[0039] Figure 12 Content A shows the effect of 4T1 cells on 4T1 cell proliferation after co-incubation with adipocytes treated with PBS, HDP release solution, DOX, Met, DOX@HDP release solution, Met@HDP release solution, or DOX / Met@HDP release solution; Content B shows the quantification of scratch area in a scratch healing assay after co-incubation with adipocytes treated with PBS, HDP release solution, DOX, Met, DOX@HDP release solution, Met@HDP release solution, or DOX / Met@HDP release solution; Content C shows the effect of 4T1 cells on 4T1 cells treated with PBS, HDP release solution, DOX, Met, DOX@HDP release solution, Met@HDP release solution, or DOX / Met@HDP release solution. Representative images of 4T1 cells after co-incubation with adipocytes treated with release solution in a scratch healing assay (scale bar: 100 μm); Content D shows the quantitative data of 4T1 cells that migrated after co-incubation with adipocytes treated with PBS, HDP release solution, DOX, Met, DOX@HDP release solution, Met@HDP release solution, or DOX / Met@HDP release solution, stained with crystal violet; Content E shows representative images of 4T1 cells that migrated after co-incubation with adipocytes treated with PBS, HDP release solution, DOX, Met, DOX@HDP release solution, Met@HDP release solution, or DOX / Met@HDP release solution, stained with crystal violet (scale bar: 50 μm).

[0040] Figure 13 Content A is a flowchart of the 4T1 in situ tumor resection model and drug administration process; Content B is the average tumor growth curve after in situ tumor resection treated with PBS, HDP, DOX, Met, DOX / Met, DOX@HDP, Met@HDP, or DOX / Met@HDP; Content C is the tumor curve of a single mouse after in situ tumor resection treated with PBS; Content D is the tumor curve of a single mouse after in situ tumor resection treated with HDP; Content E is the tumor curve of a single mouse after in situ tumor resection treated with DOX; Content F is the tumor curve of a single mouse after in situ tumor resection treated with Met; Content G is the tumor curve of a single mouse after in situ tumor resection treated with DOX / Met; Content H is the tumor curve of a single mouse after in situ tumor resection treated with DOX@HDP; Content I is the tumor curve of a single mouse after in situ tumor resection treated with Met@HDP; Content J is the tumor curve of a single mouse after in situ tumor resection treated with DOX / Met@HDP.

[0041] Figure 14 Content A shows photographs of tumors removed 20 days after resection of 4T1 in situ tumors, treated with PBS, HDP, DOX, Met, DOX / Met, DOX@HDP, Met@HDP, or DOX / Met@HDP. Content B shows the weight statistics of tumors removed 20 days after resection of 4T1 in situ tumors, treated with PBS, HDP, DOX, Met, DOX / Met, DOX@HDP, Met@HDP, or DOX / Met@HDP. Content C shows representative H&E stained sections of tumors removed 20 days after resection of 4T1 in situ tumors, treated with PBS, HDP, DOX, Met, DOX / Met, DOX@HDP, Met@HDP, or DOX / Met@HDP.

[0042] Figure 15 The effects of treatment with PBS, HDP, DOX, Met, DOX@HDP, Met@HDP, or DOX / Met@HDP on tubule formation in HUVECs.

[0043] Figure 16 The effects of each sample group on the expression of IL-6 and CD31 in tumors after 4T1 in situ tumor surgery.

[0044] Figure 17 The tumor volume was measured after treatment of each sample group (PBS as control group 1, HDP as control group 2, DOX as control group 3, ASA as control group 4, DOX@HDP as control group 5, ASA@HDP as control group 6, and DOX / ASA@HDP as experimental group) following 4T1 in situ tumor resection.

[0045] Figure 18 The number of lung nodules was compared among the sample groups (PBS as control group 1, HDP as control group 2, DOX as control group 3, ASA as control group 4, DOX@HDP as control group 5, ASA@HDP as control group 6, and DOX / ASA@HDP as experimental group) after 4T1 in situ tumor resection.

[0046] Figure 19 The effects of different sample groups (PBS as control group 1, HDP as control group 2, DOX as control group 3, ASA as control group 4, DOX@HDP as control group 5, ASA@HDP as control group 6, and DOX / ASA@HDP as experimental group) on the expression of platelet activation phenotype proteins in tumors after 4T1 in situ tumor resection were investigated. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0048] This invention provides a method for preparing a drug-loaded hydrogel for inhibiting postoperative tumor recurrence and metastasis, such as... Figure 1 As shown, it includes the following steps:

[0049] (1) Mix and stir 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) with an aqueous solution of hyaluronic acid, and keep the solution weakly acidic to activate the carboxyl group of the hyaluronic acid to obtain an activated precursor solution.

[0050] (2) The activated precursor solution described in step (1) is mixed with 3-aminophenylboronic acid (APBA) and dopamine hydrochloride and reacted under inert gas protection and weak acid conditions, so that the 3-aminophenylboronic acid (APBA) and dopamine hydrochloride are grafted onto the backbone of the hyaluronic acid through an amide condensation reaction to obtain the crude product.

[0051] (3) The crude product described in step (2) is dialyzed in acidic ultrapure water. The pH of the liquid obtained by dialyz is adjusted to neutral and then stirred in air. The oxygen in the air is used to oxidize and polymerize dopamine. Then, the liquid is freeze-dried to obtain a hydrogel for drug loading.

[0052] In some embodiments, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) to hyaluronic acid in step (1) is (0.4-0.8):(0.1-0.5):1, and the mass percentage of hyaluronic acid in the aqueous solution of hyaluronic acid is 0.5-1.5%; the activation time in step (1) is 10-40 minutes.

[0053] In some embodiments, the weak acidity described in step (1) and the weak acidity described in step (2) have a pH of 5-6.

[0054] In some embodiments, the molar ratio of 3-aminophenylboronic acid (APBA) and dopamine hydrochloride in step (2) is 3:1 to 1:3, and the mass ratio of dopamine hydrochloride to hyaluronic acid is 0.28 to 1.1:1. The inert gas in step (2) is argon or nitrogen, and the reaction time is 12-36 hours.

[0055] In some embodiments, step (3) involves transferring the crude product into a dialysis bag with a molecular weight cutoff of 10,000-14,000 Da. The product is then dialyzed in acidic ultrapure water (pH=5) with dilute hydrochloric acid added to ultrapure water for 2-4 days, with the acidic ultrapure water being replaced every 4-8 hours. The pH of the dialysis solution is then adjusted to neutral by adding alkali. The dialyzed solution is then stirred on a stirrer and oxidized by oxygen in the air for 3-8 hours. The hydrogel product is then frozen into a solid and freeze-dried in a vacuum freeze dryer for 24-72 hours to obtain the hydrogel used for drug loading.

[0056] The drug-loaded hydrogel obtained by the above preparation method of the present invention can encapsulate fluorescent molecular model drugs such as ICG and Cy5.5 to simulate the retention of small molecule drugs in animals; it can also be used to encapsulate antitumor drugs and drugs that can improve the tumor microenvironment. The antitumor drugs include one or more of doxorubicin, paclitaxel, and camptothecin, and the drugs that can improve the tumor microenvironment include metformin and / or aspirin. The drug-loaded hydrogel is obtained by mixing the hydrogel with the antitumor drugs and / or drugs that can improve the tumor microenvironment in PBS solution and allowing it to stand. In some embodiments, the drug loading in the drug-loaded hydrogel is less than or equal to 100 μg / mg.

[0057] The drug-loaded hydrogel provided by this invention has been experimentally proven to be used to prepare drugs for postoperative treatment and / or tumor inhibition. It can also be used to prepare drugs that inhibit postoperative tumor adipocyte regeneration and / or tumor angiogenesis, and can also inhibit postoperative platelet activation.

[0058] This invention provides a drug-loaded hydrogel for postoperative tumor treatment and its preparation method, which can encapsulate chemotherapy drugs or other interventional drugs. The hydrogel scaffold material provided by this invention possesses good biocompatibility, bioadhesion, swelling properties, and pH responsiveness. Its flexible rheological properties and excellent bioadhesion ensure stable adhesion to the surgical site without displacement. The slow, pH-responsive release and long-term drug retention in vivo enable the hydrogel to respond to the tumor microenvironment and maintain a sufficient drug concentration in the tumor resection microenvironment over a long period.

[0059] In a preferred embodiment of this invention, dopamine and APBA are grafted onto the HA backbone using a one-pot method, while simultaneously loading the chemotherapeutic drugs DOX and Met to construct a drug-loaded hydrogel (DOX / Met@HDP) for postoperative recurrence and metastasis of breast cancer. The properties of this drug-loaded hydrogel, its direct killing of tumor cells at the cellular level, its inhibition of adipocyte-induced tumor cell growth and migration, and its effect on angiogenesis are investigated. Its intervention effect on postoperative tumor recurrence and metastasis is evaluated at the animal level. Simultaneous loading of the chemotherapeutic drugs DOX and ASA also exhibits the same good inhibitory effect on postoperative tumor recurrence and metastasis.

[0060] The following is an example:

[0061] Example 1

[0062] Hyaluronic acid hydrogels grafted with dopamine and 3-aminophenylboronic acid at different dopamine / 3-aminophenylboronic acid ratios and their preparation methods

[0063] 1. Experimental materials and reagents

[0064] Hyaluronic acid (HA), dopamine hydrochloride (DA), 3-aminophenylboronic acid (APBA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS).

[0065] 2. Experimental Procedure

[0066] 1) Weigh 1g of HA powder and add it to 100mL of ultrapure water. Place the beaker on a magnetic stirrer and stir until the HA is completely dissolved to obtain a 1% HA solution. Place the HA solution in a round-bottom flask, use a circulating water vacuum pump to evacuate the flask to a vacuum, and fill it with argon gas for protection to prevent oxidation.

[0067] 2) Weigh 575 mg EDC and 345 mg NHS, and slowly add them sequentially to the HA solution. Maintain the pH of the solution between 5 and 6 by adding hydrochloric acid and sodium hydroxide solution. After stirring and activating for 20 min, weigh APBA and dopamine hydrochloride in different molar ratios, and slowly add them sequentially to the mixed system. The entire system reacts for 24 h under argon protection and at an acidic pH of 5.5.

[0068] 3) The reaction mixture was transferred to a dialysis bag (molecular weight cutoff 14000 Da) and dialyzed in acidic ultrapure water (pH=5) for 4 days, with the acidic ultrapure water changed every 8 hours. The pH of the dialyzed liquid was adjusted to 7.4 by adding sodium hydroxide solution. The dialyzed solution was then stirred on a stirrer and oxidized with oxygen in the air for 4 hours. The hydrogel product was then frozen into a solid and freeze-dried in a vacuum freeze dryer for 72 hours to obtain a sponge-like product.

[0069] 4) Weigh 10 mg of the above product and add 200 μL of PBS to prepare a 5% HDP hydrogel.

[0070] Other conditions are the same as in steps 1) to 4), except that in the synthesis process, only dopamine hydrochloride is added in step 3), and APBA is not added, in order to prepare HD hydrogel.

[0071] Other conditions are the same as in steps 1) to 4), except that in the synthesis process, only APBA is added in step 3), and dopamine hydrochloride is not added to prepare HP hydrogel.

[0072] 3. Experimental Results

[0073] The shear viscosities of HDP hydrogels with five different dopamine / APBA ratios were similar. The shear viscosities were 5.42 × 10⁻⁶ when the dopamine / APBA ratios were 3:1, 2:1, 1:1, 1:2, and 1:3. 3 Pa·s, 5.59 × 10 3 Pa·s, 7.38 × 10 3 Pa·s, 7.56 × 10 3 Pa·s, 9.28 × 10 3 Pa·s( Figure 2 Contents A, B, and C). The G′ of HDP hydrogels with different dopamine / APBA ratios is always higher than G″, indicating that they all possess a stable viscoelastic state.

[0074] HDP hydrogels exhibit strong bioadhesion strength. The HDP hydrogel with a dopamine to APBA ratio of 1:1 has the highest critical tensile strength (9.24 kPa), while other ratios show lower values ​​(the critical tensile strengths for dopamine to APBA ratios of 3:1, 2:1, 1:2, and 1:3 are 2.84 kPa, 5.47 kPa, 4.58 kPa, and 6.34 kPa, respectively). Figure 3 Content A, Content B, and Content C).

[0075] Example 2

[0076] A hyaluronic acid hydrogel grafted with dopamine and 3-aminophenylboronic acid in a dopamine / 3-aminophenylboronic acid ratio of 1:1 was prepared according to the method in the examples.

[0077] (1) Hydrogel morphology

[0078] Depend on Figure 4 As can be seen from Content A, the hydrogel, after being gelled at a concentration of 5%, can maintain its original shape in a tilted sample vial, does not exhibit fluidity, and possesses the basic morphology of a hydrogel.

[0079] (2) Healing properties

[0080] Depend on Figure 4 As described in sections B and C, when HDP hydrogel is cut, the reversible phenylboronic acid ester bonds present in HDP break. After three minutes of continuous contact following cutting, the boronic acid ester bonds gradually reconnect, allowing for healing. This also enables the hydrogel to withstand significant tensile forces, giving HDP hydrogel excellent healing properties. This ensures that the hydrogel can cope with potential shear stress within the tumor resection cavity and maintain its intact morphology.

[0081] (3) Adhesion

[0082] Depend on Figure 4 As shown in contents D and F, HDP hydrogel has good adhesion to latex gloves and mouse livers, suggesting that the hydrogel has the potential to adhere stably within the tumor resection cavity without displacement.

[0083] (4) Extensibility

[0084] Depend on Figure 4 As described in section E, HDP hydrogel can adhere to the finger and deform accordingly based on the degree of bending of the finger joint, exhibiting good extensibility and adhesion. This property ensures that HDP hydrogel can stably adhere to different resection surfaces, preventing detachment and breakage of HDP hydrogel due to the behavior of the implantee in complex tumor resection cavities.

[0085] (5) Tensile properties

[0086] HDP can withstand large tensile forces and can be stretched to 4.8 times its original length without breaking. Figure 4 Content G). This good stretchability ensures that HDP maintains a stable hydrogel state in the complex environment of tumor resection cavities.

[0087] Figure 5 Results in section A show that HDP exhibits a porous honeycomb network structure with relatively uniform and dense pores. Statistical analysis of the pore size in the electron micrographs using ImageJ software revealed an average pore size of 39.7 μm. Figure 5 Content B) This creates conditions for HDP to have better drug loading capacity.

[0088] Comparative Example 1

[0089] 1) Weigh 1g of HA powder and add it to 100mL of ultrapure water. Place the beaker on a magnetic stirrer and stir until the HA is completely dissolved to obtain a 1% HA solution. Place the HA solution in a round-bottom flask, use a circulating water vacuum pump to evacuate the flask to a vacuum, and fill it with argon gas for protection to prevent oxidation.

[0090] 2) Weigh 575 mg EDC and 345 mg NHS, and slowly add them sequentially to the HA solution. Maintain the pH of the solution between 5 and 6 by adding hydrochloric acid and sodium hydroxide solution. After stirring and activating for 20 min, weigh APBA and dopamine hydrochloride in different molar ratios, and slowly add them sequentially to the mixed system. The entire system reacts for 24 h under argon protection and at an acidic pH of 5.5.

[0091] 3) The system obtained from the reaction was transferred to a dialysis bag (molecular weight cutoff 14000 Da) and dialyzed in acidic ultrapure water (pH=5) for 4 days, with the acidic ultrapure water changed every 8 hours. After adjusting the pH of the dialyzed liquid to 7.4 by adding sodium hydroxide solution, the hydrogel product was frozen into a solid and then freeze-dried in a vacuum freeze dryer for 72 hours to obtain a sponge-like product.

[0092] 4) Weigh 10 mg of the above product, add 170 μL of ultrapure water and mix well, then add 10 μL of H2O2 solution (0.5 mol / L). -1 ) and 20 μL of HRP horseradish peroxidase solution (1 mg / mL) -1 ), and stir well. A 5% (w / w) HDP-1 hydrogel was obtained.

[0093] The other steps in preparing the hydrogels in Comparative Example 1 and Example 1 were the same, except that in step (4) of Example 1, the HDP hydrogel was obtained by stirring and oxidizing in air, while in Comparative Example 1, the HDP-1 hydrogel was prepared using hydrogen peroxide and horseradish peroxidase. It was accidentally discovered during the experiment that the hydrogel obtained by the oxidation method in Comparative Example 1 degraded very slowly, which would inevitably affect the drug release rate after subsequent drug loading, resulting in a very slow drug release rate. Therefore, an attempt was made to directly stir the hydrogel in air for several hours to oxidize dopamine using oxygen in the air, resulting in a hydrogel HDP with a moderate degradation rate.

[0094] The hydrogels obtained in Example 1 and Comparative Example 1 were tested for degradation rate under constant temperature shaker conditions of pH=7, 37℃, and 100rpm. The experiment showed that the HDP-1 hydrogel obtained in Comparative Example 1 required 95 days to degrade, while the HDP hydrogel obtained in Example 1 required only 30 days. This indicates that the dopamine oxidative polymerization method of the HDP hydrogel prepared by the one-pot method in this invention has a significant impact on the degradation rate of the final gel. Air oxidation may result in a lower and more controllable degree of dopamine oxidation in HDP.

[0095] In addition, other experimental conditions were the same as in Example 1, except that step 3) was changed to oxidation treatment with oxygen in the air for 36 hours and 48 hours respectively. The tests showed that the degradation rate was also slow, requiring 56 days and 65 days respectively.

[0096] Example 3

[0097] Drug loading capacity and drug release characteristics of hyaluronic acid hydrogels (HDPs) grafted with dopamine and 3-aminophenylboronic acid at a dopamine / 3-aminophenylboronic acid ratio of 1:1.

[0098] 1. Experimental materials and reagents

[0099] Hyaluronic acid (HA), dopamine hydrochloride (DA), 3-aminophenylboronic acid (APBA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), doxorubicin hydrochloride (DOX), metformin hydrochloride (Met).

[0100] 2. Experimental Procedure

[0101] 1) The hydrogel was prepared in the same way as in Example 1 (the ratio of dopamine / 3-aminophenylboronic acid was 1:1).

[0102] 2) Weigh 10 mg of the HDP prepared in Example 1 and add it to 200 μL of a solution containing 60 μg / mL of HDP. -1 DOX and 30 mg mL -1After mixing Met in PBS and letting it stand for 30 minutes, the dual-drug-loaded hydrogel DOX / Met@HDP is obtained.

[0103] 3) Take a 10 mL centrifuge tube and immerse 200 μL of DOX / Met@HDP in 5 mL of PBS solution with different pH values ​​(pH = 6.5, 7.4). Place the release system in a constant temperature shaker for drug release experiments, setting the temperature to 37℃ and the shaker speed to 100 rpm. At different time points (1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, etc.), take 1 mL of the release solution and simultaneously add 1 mL of fresh PBS to the release system. Use a 1260 high performance liquid chromatograph to determine the concentrations of DOX and Met in the release solution and calculate the cumulative drug release amount.

[0104] 3. Experimental Results

[0105] The in vitro drug release performance of DOX / Met@HDP under different pH conditions was studied. Figure 6 Content A and Content B). At pH 7.4, the DOX / Met@HDP release rate over 120 hours was approximately 40.3%, while at pH 6.5 it was 88.2%. Figure 6 Content A); Similarly, with DOX / Met@HDP, the release of Met over 8 hours was approximately 38.5% at pH 7.4, compared to 58.6% at pH 6.5. Figure 6 Content B) indicates that DOX / Met@HDP exhibits pH-responsive drug release properties. This pH-responsive release of DOX / Met@HDP may be due to the cleavage of phenylboronic acid ester bonds in the HDP hydrogel network at low pH, facilitating the rapid release of DOX and Met in the acidic tumor microenvironment. The different release kinetics of DOX and Met in DOX / Met@HDP may be due to the different solubilities of DOX and Met in PBS, as well as the π-π stacking or hydrogen bonding interactions between DOX and polydopamine.

[0106] Loading DOX and Met affects the shear viscosity of HDP ( Figure 7 Content A) and G′ and G″ with frequency ( Figure 7 Content B) and degree of strain ( Figure 7 The changes in content C) had no significant impact, indicating that DOX@HDP, Met@HDP, and DOX / Met@HDP all possess stable hydrogel properties. HDP served as control group one, DOX@HDP as control group two, Met@HDP as control group three, and DOX / Met@HDP as the experimental group.

[0107] Example 4: Drug loading capacity and in vivo drug retention characteristics of hyaluronic acid hydrogels grafted with dopamine and 3-aminophenylboronic acid at a dopamine / 3-aminophenylboronic acid ratio of 1:1.

[0108] 1. Experimental materials and reagents

[0109] Hyaluronic acid (HA), dopamine hydrochloride (DA), 3-aminophenylboronic acid (APBA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Cyanine 5,5-carboxylic acid (Cy5.5), and Indocyanine Green (ICG).

[0110] 2. Experimental Procedure

[0111] 1) The hydrogel preparation is the same as in Example 1.

[0112] 2) Weigh 10 mg of HDP and add it to 200 μL of solution containing 1 μg / mL. -1 Cy5.5 and 1 μg mL -1 ICG was mixed in PBS to obtain the dual-drug-loaded hydrogel Cy5.5 / ICG@HDP.

[0113] 3) Select 4T1 cells in logarithmic growth phase, digest and resuspend them, and count the cells. Dilute the cells to a density of 1×10⁻⁶ with PBS. 7 mL -1 Six-week-old female BALB / c mice were anesthetized. The skin around the fourth pair of mammary glands on the right side of the mice was incised, and 50 μL of a well-mixed cell suspension was injected into the fat pad before suturing. Iodine solution was then applied, and the mice were kept warm until they regained consciousness. All surgical instruments used underwent strict sterilization. Tumors were treated until they reached a volume of 300 mm². 3 Surgical resection was performed. First, the mouse was prepared by trimming the hair around the tumor and disinfecting it with iodine. After anesthetizing the mouse, the skin around the tumor was cut open, and about 90% of the solid tumor was removed. Blood was wiped away with cotton balls, and hydrogel material was placed in the tumor resection cavity. The wound was sutured and wiped with iodine. The body temperature was maintained until the mouse woke up.

[0114] 4) A 4T1 breast cancer surgical resection model was established and Cy5.5 / ICG@HDP was implanted into the mice. Control group mice received the same amount of free Cy5.5 and ICG mixed solution injected into the resection cavity as the experimental group. At postoperative time points of 3h, 6h, 9h, 12h, 24h, 36h, 48h, and 72h, the fluorescence of Cy5.5 and ICG in the mice was imaged and photographed using the IVIS Lumina XR small animal imaging system, and the total fluorescence was statistically analyzed. Ten days postoperatively, the mice were dissected, and tumors and related organs (heart, liver, spleen, lung, and kidney) were removed for in vitro imaging. The total fluorescence of Cy5.5 and ICG in the tumors and different organs was statistically analyzed.

[0115] 3. Experimental Results

[0116] Fluorescence images obtained from an in vivo imaging system (IVIS) show that Cy5.5 implanted in mice with Cy5.5 / ICG@HDP... Figure 8 Content A) and ICG ( Figure 8 The fluorescence signal attenuation rate of the mice in the free Cy5.5 / ICG group (C) was significantly slower than that of mice injected with free Cy5.5 / ICG solution. Cy5.5 fluorescence in the free Cy5.5 / ICG group decreased to invisible by day 10 after tumor resection, while the fluorescence in the Cy5.5 / ICG@HDP group was clear and bright; at 48 hours, ICG fluorescence disappeared in the free Cy5.5 / ICG group, while fluorescence in the Cy5.5 / ICG@HDP group remained relatively obvious. Statistical data shows that the Cy5.5 (C) fluorescence in the Cy5.5 / ICG@HDP group... Figure 8 Content B) and ICG Figure 8 Content D) The drug retention rate was significantly better in the Cy5.5 / ICG group than in the free Cy5.5 / ICG group. On day 10 post-implantation, the Cy5.5 fluorescence value in the Cy5.5 / ICG@HDP group was 6.45 × 10⁻⁶. 8 Photons s -1 cm -2 sr -1 The free Cy5.5 / ICG group had only 3.06 × 10⁻⁶. 8 Photons s -1 cm -2 sr -1 At 48 hours post-implantation, the ICG fluorescence value in the Cy5.5 / ICG@HDP group was 3.68 × 10⁻⁶. 9 Photons s -1 cm -2 sr -1 The free Cy5.5 / ICG group had only 0.54 × 10⁻⁶. 9 Photons s -1 cm -2 sr -1Therefore, HDP hydrogel can significantly increase the retention of Cy5.5 and ICG at the tumor site, indicating that DOX / Met@HDP also has the ability to efficiently retain DOX and Met at the tumor site. Mice were euthanized on day 10, and tumors and major organs were removed for fluorescence imaging to analyze the drug concentration in the tissues after drug administration. The fluorescence images of the ex vivo tissues show that... Figure 9 (Contents A and C) In the Cy5.5 / ICG@HDP group, the fluorescence values ​​of Cy5.5 and ICG in the tumor sites of mice were higher than those in other organs (heart, liver, lung, spleen, and kidney). Furthermore, the fluorescence values ​​of Cy5.5 and ICG in the tumor sites of the Cy5.5 / ICG@HDP group were significantly higher than those in the free Cy5.5 / ICG group. Figure 9 (Contents B and D). The above data demonstrate that Cy5.5 / ICG@HDP can increase drug concentration at the tumor site and decrease drug concentration in major organs. The free Cy5.5 / ICG group served as the control group, and the Cy5.5 / ICG@HDP group served as the experimental group.

[0117] Example 5: A hyaluronic acid hydrogel grafted with dopamine and 3-aminophenylboronic acid at a dopamine / 3-aminophenylboronic acid ratio of 1:1, loaded with doxorubicin hydrochloride and metformin hydrochloride, was used to kill tumor cells.

[0118] 1. Experimental materials and reagents

[0119] Hyaluronic acid (HA), dopamine hydrochloride (DA), 3-aminophenylboronic acid (APBA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), doxorubicin hydrochloride (DOX), metformin hydrochloride (Met), and 4T1 breast cancer cells.

[0120] 2. Experimental Procedure

[0121] 1) The hydrogel preparation is the same as in Example 1.

[0122] 2) Weigh 10 mg of HDP and add it to 200 μL of solution containing 60 μg / mL of HDP. -1 DOX and 30 mg mL -1 After mixing Met in PBS and allowing it to stand for 30 minutes, the dual-drug-loaded hydrogel DOX / Met@HDP was obtained. The same method was used to obtain single-drug-loaded DOX@HDP and Met@HDP.

[0123] 3) Collect HDP, DOX@HDP, Met@HDP, and DOX / Met@HDP hydrogels in PBS (37℃ constant temperature shaker, 100 rpm) for 7 days as release medium. 4T1 cells in logarithmic growth phase were then injected with 8 × 10⁻⁶ cells / mL. 3The cells were seeded at a density of 1 / 2 well in 96-well plates and cultured overnight. The original culture medium was discarded, and the cells were treated with media containing PBS, HDP release medium, DOX, Met, DOX@HDP release medium, Met@HDP release medium, and DOX / Met@HDP release medium, respectively. The drug concentrations of DOX and Met were DOX / Met = 0.25 μg / mL. -1 / 125μg mL -1 0.5 μg mL -1 / 250μg mL -1 1 μg mL -1 / 500μg mL -1 2μg mL -1 / 1000μgmL -1 After 24 hours of cell treatment, the original culture medium was removed and replaced with fresh culture medium containing CCK-8 reagent for colorimetric reaction. After the colorimetric reaction, the absorbance of the culture medium at 450 nm was detected using a full-wavelength microplate reader, and the viability of 4T1 cells treated with DOX / Met@HDP was calculated.

[0124] (4) After treating 4T1 cells in the logarithmic growth phase for 24 h (the drug concentrations of DOX and Met were 1 μg / mL), -1 and 500 μg mL -1 The supernatant was collected, and the adherent cells at the bottom layer were collected by trypsin digestion. The cells were centrifuged at 300g for 5 min, the supernatant was removed, and the cells were washed three times with PBS. The cells were resuspended in 100 μL of staining working solution containing 5 μL Annexin V-FITC and 10 μL 7-AAD and mixed well. The cells were incubated in the dark for 10-15 min. After incubation, 300 μL of buffer was added and the cells were placed on ice. The cells were detected by flow cytometry within 1 h to study the proportion of 4T1 cells induced by DOX / Met@HDP.

[0125] (5) After treating 4T1 cells in logarithmic growth phase for 24 h, the supernatant was collected, and the adherent cells at the bottom layer were collected by trypsin digestion. The cells were centrifuged at 300 g for 5 min, the supernatant was removed, and the cells were washed three times with PBS. Then, 300 μL of staining working solution containing 2 μM calcein acetoxymethyl ester and 4.5 μM propidium iodide was added, and the cells were resuspended and mixed. The cells were incubated at 37 °C in the dark for 15 min. After incubation, the cells were centrifuged at 300 g for 5 min, the supernatant was removed, and the cells were washed twice with PBS. The cell suspension resuspended in PBS was added to a confocal dish and observed by laser confocal microscopy. Live cells were labeled green by Calcein-AM and dead cells were labeled red by PI. The changes in the liveness and death of 4T1 cells induced by DOX / Met@HDP were detected. PBS was the control group 1, HDP was the control group 2, DOX was the control group 3, Met was the control group 4, DOX@HDP was the control group 5, Met@HDP was the control group 6, and DOX / Met@HDP was the experimental group.

[0126] 3. Experimental Results

[0127] like Figure 10 As shown in Content A, the bar charts for each doxorubicin concentration condition, from left to right, correspond to control groups 1, 2, 3, 4, 5, and 6, respectively. All HDP concentrations did not affect the survival of 4T1 cells, further demonstrating the good biocompatibility of HDP. For the Met and Met@HDP groups, high Met concentrations had a slight impact on 4T1 cell survival. Cell survival was low in the DOX, DOX@HDP, and DOX / Met@HDP groups at different DOX concentrations, with the killing effect becoming more pronounced with increasing DOX concentration, exhibiting a strong concentration-dependent effect. When the DOX concentration was below 1 μg / mL... -1 At this time, the Met released by DOX / Met@HDP slightly increases the killing effect of DOX, resulting in the lowest cell survival rate. However, when the DOX concentration is higher than 1 μg / mL... -1 However, the effect is not obvious.

[0128] like Figure 10 As shown in Content B (the bars from left to right correspond to control group 1, control group 2, control group 3, control group 4, control group 5, control group 6 and experimental group respectively), apoptosis was not significant in the PBS, HDP, Met and Met@HDP groups. DOX and DOX@HDP induced apoptosis of 4T1 cells to the same degree, while DOX / Met@HDP, due to the synergistic effect of Met, led to stronger apoptosis in 4T1 cells.

[0129] like Figure 10As shown in section C, 4T1 cells treated with PBS, HDP, Met, and Met@HDP were almost entirely live cells marked in green, with only a few dead cells marked in red, demonstrating that these treatments had almost no killing effect on 4T1 cells. In contrast, 4T1 cells treated with DOX, DOX@HDP, and DOX / Met@HDP showed a larger and denser number of dead cells marked in red. These results indicate that DOX / Met@HDP has good killing activity against 4T1 cells.

[0130] Example 6

[0131] Hyaluronic acid hydrogels grafted with dopamine and 3-aminophenylboronic acid at a 1:1 ratio, loaded with doxorubicin hydrochloride and metformin hydrochloride, were used to inhibit adipocytes and their related functions.

[0132] 1. Experimental materials and reagents

[0133] Hyaluronic acid (HA), dopamine hydrochloride (DA), 3-aminophenylboronic acid (APBA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), doxorubicin hydrochloride (DOX), metformin hydrochloride (Met), and 3T3-L1 mouse embryonic fibroblasts.

[0134] 2. Experimental Procedure

[0135] 1) The hydrogel preparation is the same as in Example 2.

[0136] 2) Weigh 10 mg of HDP and add it to 200 μL of solution containing 60 μg / mL of HDP. -1 DOX and 30 mg mL -1 After mixing Met in PBS and letting it stand for 30 minutes, the dual-drug-loaded hydrogel DOX / Met@HDP is obtained.

[0137] 3) Adipocyte induction process: 3T3-L1 mouse embryonic fibroblasts were incubated at 5×10⁻⁶ cells / year. 4 The cells were seeded at a density of 1 / 2 well in 24-well plates. Once the cell proliferation and confluence reached 100%, the medium was replaced with adipocyte differentiation medium, and the cells were cultured for another two days, with the medium changed every two days. Four days after differentiation, the medium was replaced with adipocyte maintenance medium, and the cells were cultured for another two days. On the tenth day of differentiation, the medium was replaced with DMEM complete medium, and adipocytes were successfully induced.

[0138] 4) Release solutions were collected from HDP, DOX@HDP, Met@HDP, and DOX / Met@HDP hydrogels and released in PBS (37℃ constant temperature shaker, 100 rpm) for 7 days. The original culture medium of differentiated adipocytes in 24-well plates was discarded, and culture media containing PBS, HDP release solution, DOX, Met, DOX@HDP release solution, Met@HDP release solution, and DOX / Met@HDP release solution were added to each well. After 48 h of treatment, the supernatant was removed, and the treated adipocytes were collected by trypsin digestion. Then, 200 μL of PBS solution containing 2 μM BODIPY 493 / 503 was added, and the cells were incubated at 37℃ in the dark for 15 min. After centrifugation and washing three times with PBS, the BODIPY fluorescence intensity was detected by flow cytometry to investigate the effect of DOX / Met@HDP on the lipid content of adipocytes. 5) Treat adipocytes using the same method as in 4) for 48 h, collect adipocytes into 1.5 mL nuclease-free centrifuge tubes, add 1 mL TRIzol and mix well, then add 200 μL chloroform and shake vigorously for 15 s, let stand for 5 min, centrifuge at 13800 g for 15 min (4℃), aspirate 500 μL of supernatant along the liquid surface and mix with 500 μL isopropanol, let stand for another 15 min, centrifuge at 13800 g for 10 min (4℃), discard the supernatant, wash with 75% ethanol, centrifuge at 5400 g for 5 min (4℃), repeat three times, let the precipitate stand until it becomes clear, finally add 15–20 μL DEPC water to dissolve, and determine the RNA concentration. The RNA is stored at -80℃. Take 1 μg of RNA and reverse transcribe it into cDNA using HiScript® RII Q RT SuperMix. Add primers and cDNA according to the instructions for the Taq Pro Universal SYBR qPCR MasterMix, and perform RT-qPCR on a real-time quantitative PCR instrument. The primers used are shown in the table below:

[0139] Table 1 Primer sequences used

[0140]

[0141] 5) After treating adipocytes in the same way for 48 hours, the culture medium was discarded and 300 μL of serum-free culture medium was slowly added for 12 hours of incubation. The supernatant was collected and the IL-6 content secreted by adipocytes was detected using a mouse IL-6 detection kit.

[0142] 6) The co-incubation system of adipocytes and 4T1 cells was performed using 24-well Transwell (0.4 μm) culture plates. Logarithmic growth phase 4T1 cells were cultured at 2 × 10⁶ cells / well. 4Adipocytes were seeded at a density of / wells in 24-well plates and cultured overnight. After discarding the culture medium, 600 μL of fresh serum-free medium was added. Adipocytes were treated in the same manner for 48 h. The treated adipocytes were collected by trypsin digestion and resuspended in 200 μL of serum-free medium. The adipocytes were placed in the upper chamber of a Transwell plate and co-incubated with overnight 4T1 cells for 48 h. The original culture medium was then removed and replaced with fresh medium containing CCK-8 reagent for colorimetric reaction. After colorimetric reaction, the absorbance at 450 nm was measured using a full-wavelength microplate reader to investigate the effect of DOX / Met@HDP on tumor cell proliferation after treatment of adipocytes.

[0143] 7) After establishing the 4T1 cell scratch model, adipocytes were treated in the same manner for 48 h. The treated adipocytes were then digested with trypsin and collected, resuspended in 200 μL of serum-free medium, and placed in the upper chamber of a Transwell (0.4 μm) for co-incubation with the 4T1 cell scratch for 24 h. The healing of the 4T1 cell scratch was observed and photographed using an inverted microscope at 0 h and 24 h of co-incubation. The scratch area was statistically analyzed using ImageJ software to study the effect of DOX / Met@HDP on 4T1 cell migration.

[0144] 8) The co-incubation system of adipocytes and 4T1 cells was performed using 24-well Transwell (8μm) plates. Adipocytes were treated in the same manner for 48 hours, and the original culture medium was discarded and replaced with fresh serum-free culture medium. Logarithmic growth phase 4T1 cells were incubated at 8 × 10⁻⁶ cells / well. 4 The cells were seeded at a density of 8 μm in the upper chamber of a Transwell apparatus and co-incubated with treated adipocytes for 10 h. After culture, the upper chamber was removed, and the cells on the upper surface were gently wiped away with a cotton swab and washed with PBS. Subsequently, the 4T1 cells that had migrated to the bottom of the chamber were fixed with 4% paraformaldehyde fixative for 10 min. After washing with PBS, the cells were stained with 0.1% crystal violet, and excess dye was removed by washing with PBS. The stained 4T1 cells were observed and photographed using an inverted microscope.

[0145] The group without fat cells was used as the control group. PBS was used as the control group 1, HDP as the control group 2, DOX as the control group 3, Met as the control group 4, DOX@HDP as the control group 5, Met@HDP as the control group 6, or DOX / Met@HDP as the experimental group. Figure 11 Contents A through F, with each bar chart corresponding from left to right to control group 1, control group 2, control group 3, control group 4, control group 5, control group 6, and experimental group, respectively.

[0146] 3. Experimental Results

[0147] Depend on Figure 11 As shown in section A, there was no significant difference in the survival rate of adipocytes among the different groups after treatment, indicating that DOX / Met@HDP does not produce significant cytotoxicity to the induced adipocytes.

[0148] like Figure 11 In section B, BODIPY, a lipid dye, shows that its fluorescence intensity reflects the amount of lipids within adipocytes. Compared to the PBS group, DOX and DOX@HDP had no significant effect on adipocyte lipids. However, treatment with Met, Met@HDP, and DOX / Met@HDP significantly reduced the lipid content in adipocytes, with reduction rates of 20.3%, 21.5%, and 21.9%, respectively. Therefore, DOX / Met@HDP exhibits the potential to influence adipocyte function.

[0149] Depend on Figure 11 Content C indicates that HDP, DOX, or DOX@HDP treatment had no significant effect on IL-6 secretion from adipocytes. The concentration of IL-6 in the supernatant of the PBS group was 4.0 ng / mL. -1 The IL-6 content in the supernatant after treatment with Met, Met@HDP, or DOX / Met@HDP was only 1.86 ng / mL. -1 1.82 ng / mL -1 and 1.81 ng mL -1 The levels were significantly lower in the DOX / Met@HDP group compared to the PBS group, indicating that DOX / Met@HDP can effectively inhibit adipocyte function.

[0150] Depend on Figure 11 Contents D, E, and F show that treatment with Met, Met@HDP, or DOX / Met@HDP significantly reduced the mRNA expression levels of inflammatory factors IL-6 and resistin, which promote tumor cell proliferation and migration, to only 33.8%, 42.6%, 46.6%, and 79.6%, 71.6%, 68.3% of the control group PBS, respectively, while no significant trend was observed among the other groups. Adiponectin inhibited tumor development; the mRNA expression of adiponectin was significantly upregulated after treatment with Met, Met@HDP, or DOX / Met@HDP, approximately 1.7 times that of the PBS group, while no significant trend was observed among the other groups. However, the expression level of leptin did not show significant differences among the treatment groups. DOX / Met@HDP effectively regulated the expression level of adipocyte adipokine mRNA.

[0151] Figure 12In contents A to E, the samples from left to right correspond to the control group, adipocyte + control group 1, adipocyte + control group 2, adipocyte + control group 3, adipocyte + control group 4, adipocyte + control group 5, adipocyte + control group 6, and adipocyte + experimental group, respectively. Figure 12 Content A shows that the proliferation of 4T1 cells in the PBS group co-incubated with adipocytes was significantly higher than that in the group without adipocytes (1.9 times), confirming that adipocytes can promote the proliferation of 4T1 cells. HDP, DOX, or DOX@HDP treatments had no significant effect on 4T1 cell proliferation. However, treatment with Met, Met@HDP, or DOX / Met@HDP slowed down the adipocyte-induced proliferation of 4T1 cells, to approximately 1.3 times that of the group without adipocytes, indicating that DOX / Met@HDP can effectively inhibit adipocyte-induced tumor cell proliferation.

[0152] Depend on Figure 12 Contents B and C show that at 0h, the initial scratch distances in each group were similar. After 24h of co-incubation, the scratches in the adipocyte-free group still retained larger gaps, while the scratches in the PBS group were basically healed, indicating that surface adipocytes could promote the migration of 4T1 cells. HDP, DOX, or DOX@HDP treatments had no significant effect on adipocyte-promoted scratch healing. However, the scratches on 4T1 cells in the Met, Met@HDP, and DOX / Met@HDP treatment groups still retained larger gaps. The results obtained by quantifying the scratch area using ImageJ software were consistent with the observations, indicating that DOX / Met@HDP slowed down adipocyte-promoted 4T1 cell migration.

[0153] Depend on Figure 12 As shown in Contents D and E, HDP, DOX, or DOX@HDP treatment of adipocytes had no significant effect on the number of migrating 4T1 cells, while the number of migrating 4T1 cells in the Met, Met@HDP, or DOX / Met@HDP treatment groups was significantly reduced compared to the PBS group. Quantitative analysis of the number of migrating 4T1 cells also yielded consistent results.

[0154] Example 7

[0155] A study on the antitumor effects of dopamine / 3-aminophenylboronic acid grafted hyaluronic acid hydrogel with a dopamine / 3-aminophenylboronic acid ratio of 1:1, loaded with doxorubicin hydrochloride and metformin hydrochloride, after orthotopic resection of 4T1 breast cancer tumors.

[0156] 1. Experimental materials and reagents

[0157] Hyaluronic acid (HA), dopamine hydrochloride (DA), 3-aminophenylboronic acid (APBA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), doxorubicin hydrochloride (DOX), metformin hydrochloride (Met), and 4T1 breast cancer cells.

[0158] 2. Experimental Procedure

[0159] 1) The hydrogel preparation is the same as in Example 1.

[0160] 2) Weigh 10 mg of HDP and add it to 200 μL of solution containing 60 μg / mL of HDP. -1 DOX and 30 mg mL -1 After mixing Met in PBS and letting it stand for 30 minutes, the dual-drug-loaded hydrogel DOX / Met@HDP is obtained.

[0161] 3) Six-week-old female BALB / c mice were randomly divided into eight groups of six each to establish a 4T1 breast cancer surgical resection model. HDP, DOX@HDP, Met@HDP, and DOX / Met@HDP were implanted into the mice, respectively. Simultaneously, the PBS, DOX, Met, and DOX / Met groups were administered the drug solution via intratumoral injection (DOX dosage was 0.6 mg / kg). -1 The dosage of Met is 300 mg / kg. -1 The tumor volume was continuously monitored using calipers (tumor volume = tumor long diameter × tumor short diameter × tumor short diameter × 0.5), while the mouse's body weight was also continuously monitored. Figure 13 As shown in Content B. PBS is the control group 1, HDP is the control group 2, DOX is the control group 3, Met is the control group 4, DOX / Met is the control group 5, DOX@HDP is the control group 6, Met@HDP is the control group 7, or DOX / Met@HDP is the experimental group.

[0162] 3. Experimental Results

[0163] like Figure 13 As shown in contents B, C, D, E, F, G, H, I, and J, consistent with expectations, neither HDP nor Met treatments showed significant tumor-suppressing effects. However, the tumors in mice treated with DOX and DOX / Met recurred slightly slower than in the PBS group, with tumor inhibition rates of 31.6% and 32.8%, respectively. DOX@HDP and Met@HDP showed better inhibitory effects than DOX, with inhibition rates increasing to 61.2% and 48.9%, respectively. The DOX / Met@HDP group exhibited the best tumor-suppressing effect, with an inhibition rate as high as 89.8% compared to the PBS group.

[0164] Through tumor photos ( Figure 14 Content A) and weight statistics ( Figure 14 Content B) leads to conclusions consistent with the aforementioned tumor volume findings: DOX and DOX / Met showed tumor inhibition rates of only 20.1% and 30.5%, respectively, while DOX@HDP and Met@HDP showed inhibition rates of 49.9% and 66%, respectively. Furthermore, the tumor-bearing mice treated with DOX / Met@HDP exhibited the slowest recurrence rate, demonstrating the best tumor inhibition effect compared to the PBS group, with an inhibition rate as high as 87.2%. H&E staining of tumor sections also revealed that the DOX / Met@HDP group had the smallest area of ​​proliferating cells and the largest area of ​​necrotic cells. Figure 14 Content C).

[0165] Example 8

[0166] A hyaluronic acid hydrogel grafted with dopamine and 3-aminophenylboronic acid in a 1:1 ratio, loaded with doxorubicin hydrochloride and metformin hydrochloride, was used to inhibit the related functions of HUVECs (human umbilical vein endothelial cells).

[0167] like Figure 15 The effects of each treatment group on HUVEC cell tubule formation are shown in the figure. HDP, DOX@HDP, Met@HDP, and DOX / Met@HDP hydrogels were collected and released into PBS (37℃ constant temperature shaker, 100 rpm) for 7 days. The release solutions were obtained from PBS (control group 1), HDP release solution (control group 2), DOX (control group 3), Met (control group 4), DOX@HDP release solution (control group 5), Met@HDP release solution (control group 6), and DOX / Met@HDP release solution (experimental group) (DOX concentration 1 μg / mL). -1 The concentration of Met was 500 μg / mL. -1 Representative images of HUVECs cell tubule formation experiments treated with DOX (scale bar: 100 μm) (Content A) and quantitative analysis of grid number (Content B), grid area (Content C), and total tubule length (Content D) (n=6). Effect of DOX / Met@HDP treatment on scratch healing of HUVECs cells. Contents E and F show treatments with PBS, HDP release solution, DOX, Met, DOX@HDP release solution, Met@HDP release solution, or DOX / Met@HDP release solution (DOX concentration 1 μg / mL). -1 The concentration of Met was 500 μg / mL. -1 Representative images of scratch healing in HUVECs cells treated with 100 μm (scale bar: 100 μm) (content E) and scratch area statistics (F) (n=6).

[0168] During clinical tumor resection, a large number of blood vessels are destroyed. Angiogenesis is essential for the reconstruction of nutrient supply and prevention of recurrence after tumor resection. To investigate the effect of DOX / Met@HDP on angiogenesis, PBS, HDP release solution, DOX, Met, DOX@HDP release solution, Met@HDP release solution, or DOX / Met@HDP release solution (containing 1 μg / mL) were used. -1 HUVECs were treated with DOX and 500 μg Met, and tubule formation was observed after 4 hours. As shown in Content A, the tubule networks formed by HUVECs in the PBS, HDP, DOX, and DOX@HDP groups were more numerous, denser, more regular, and highly intersecting, while the tubule networks formed by the Met, Met@HDP, and DOX / Met@HDP groups were sparser and incomplete, and most could not form complete closed loops. Quantitative analysis of the number of grids, grid coverage area, and total tubule length using ImageJ software (Contents B, C, and D) revealed that the statistical results of the Met, Met@HDP, or DOX / Met@HDP groups were significantly lower than those of the control group in all indicators, indicating that DOX / Met@HDP has a significant inhibitory effect on tubule formation in HUVECs.

[0169] Besides tubule formation, the migration ability of HUVECs is also an important indicator of angiogenesis. Therefore, a scratch wound healing assay was used to study the migration ability of DOX / Met@HDP-treated HUVECs. Under the premise of equal initial scratch area in each group, the HUVECs in the PBS, HDP, DOX, and DOX@HDP treatment groups showed faster scratch wound healing speed and smaller gaps at 24 h (Content E); while the Met, Met@HDP, and DOX / Met@HDP treatment groups still had relatively larger gaps at 24 h, indicating slower cell migration. Quantitative analysis of the scratch area in each group (Content F) also yielded consistent results, indicating that DOX / Met@HDP can significantly inhibit angiogenesis.

[0170] Example 9

[0171] A study investigated the effects of dopamine and 3-aminophenylboronic acid grafted onto a hyaluronic acid hydrogel with a dopamine / 3-aminophenylboronic acid ratio of 1:1, loaded with doxorubicin hydrochloride and metformin hydrochloride, on adipocyte improvement and angiogenesis inhibition after orthotopic resection of 4T1 breast cancer.

[0172] 1. Experimental materials and reagents

[0173] Hyaluronic acid (HA), dopamine hydrochloride (DA), 3-aminophenylboronic acid (APBA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), doxorubicin hydrochloride (DOX), metformin hydrochloride (Met), and 4T1 breast cancer cells.

[0174] 2. Experimental Procedure

[0175] 1) The hydrogel preparation is the same as in Example 1.

[0176] 2) Weigh 10 mg of HDP and add it to 200 μL of solution containing 60 μg / mL of HDP. -1 DOX and 30 mg mL -1 After mixing Met in PBS and letting it stand for 30 minutes, the dual-drug-loaded hydrogel DOX / Met@HDP is obtained.

[0177] 3) Six-week-old female BALB / c mice were randomly divided into eight groups of six each to establish a 4T1 breast cancer surgical resection model. HDP, DOX@HDP, Met@HDP, and DOX / Met@HDP were implanted into the mice, respectively. Simultaneously, the PBS, DOX, Met, and DOX / Met groups were administered the drug solution via intratumoral injection (DOX dosage was 0.6 mg / kg). -1 The dosage of Met is 300 mg / kg. -1 The effects of DOX / Met@HDP on adipocytes and angiogenesis were investigated by immunofluorescence staining of tumors in treated mice. Figure 16 As shown in the figure. PBS is the control group 1, HDP is the control group 2, DOX is the control group 3, Met is the control group 4, DOX / Met is the control group 5, DOX@HDP is the control group 6, Met@HDP is the control group 7, and DOX / Met@HDP is the experimental group.

[0178] 3. Experimental Results

[0179] Figure 16 The effect of DOX / Met@HDP on IL-6 and CD31 expression in 4T1 orthotopic tumors after surgery. 4T1 orthotopic tumors were treated with PBS, HDP, DOX, Met, DOX / Met, DOX@HDP, Met@HDP, or DOX / Met@HDP (DOX: 0.6 mg / kg) after resection. -1 Met: 300mg / kg -1 Representative images of IL-6 and CD31 immunofluorescence staining of tumors excised 20 days after treatment (content A and content C), and the staining of IL-6... + CD31 + Statistical results (Contents B and D) (scale bar: 50 μm) (n = 6). It can be seen that the immunofluorescence positive area of ​​IL-6 and CD31 in the fluorescent sections of the experimental group and the control group (7 groups) was less than that in the PBS group, which significantly inhibited the secretion of IL-6 by adipocytes in tumor cells and angiogenesis.

[0180] Example 10

[0181] A study investigated the effects of dopamine / 3-aminophenylboronic acid grafted onto a hyaluronic acid hydrogel with a dopamine / 3-aminophenylboronic acid ratio of 1:1, loaded with doxorubicin hydrochloride and aspirin, on inhibiting tumor recurrence and platelet activation after orthotopic resection of 4T1 breast cancer.

[0182] 1. Experimental materials and reagents

[0183] Hyaluronic acid (HA), dopamine hydrochloride (DA), 3-aminophenylboronic acid (APBA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), doxorubicin hydrochloride (DOX), aspirin (ASA), and 4T1 breast cancer cells.

[0184] 2. Experimental Procedure

[0185] 1) The hydrogel preparation is the same as in Example 1.

[0186] 2) Weigh 10 mg of HDP and add it to 200 μL of solution containing 60 μg / mL of HDP. -1 DOX and 7.5 mg mL -1 After mixing ASA in PBS and letting it stand for 30 minutes, the dual-drug-loaded hydrogel DOX / ASA@HDP is obtained.

[0187] 3) Six-week-old female BALB / c mice were randomly divided into eight groups of six each to establish a 4T1 breast cancer surgical resection model. HDP, DOX@HDP, ASA@HDP, and DOX / ASA@HDP were implanted into the mice, respectively. Simultaneously, the PBS, DOX, and ASA groups were administered the drug solution via intratumoral injection (DOX dosage was 0.6 mg / kg). -1 The dosage of ASA is 75 mg / kg. -1 Tumor volume was continuously monitored using calipers (tumor volume = tumor long diameter × tumor short diameter × tumor short diameter × 0.5), and mouse body weight was also continuously monitored. The treated tumor tissue was subjected to CD41 and CD62P immunofluorescence staining. After being minced, digested, and sieved, the tumor tissue was used to prepare a single-cell suspension, which was then stained with CD41, CD62P, and CD63 fluorescent antibodies to detect platelet activation within the tumor. PBS was used as control group 1, HDP as control group 2, DOX as control group 3, ASA as control group 4, DOX@HDP as control group 5, ASA@HDP as control group 6, and DOX / ASA@HDP as the experimental group.

[0188] 3. Experimental Results

[0189] like Figure 17As shown in contents A, B, C, D, E, F, G, H, I, and J, the recurrence rates were faster in the PBS, HDP, DOX, ASA, and ASA@HDP groups after tumor resection, with an average tumor volume of 1351 mm² 25 days after treatment. 3 1342mm 3 957mm 3 1306mm 3 and 1150mm 3 Furthermore, there were no significant differences between the groups. The DOX@HDP group showed some degree of tumor inhibition, and the tumor volume was smaller than that in the PBS group, with an average of 372 mm. 3 and 380mm 3 The DOX / ASA@HDP group exhibited the smallest tumor volume, averaging only 125 mm³, and showed a slow tumor growth rate. Twenty-five days after treatment, photographs and weight analysis of the dissected tumors also confirmed that the DOX / ASA@HDP group had the smallest tumor volume and weight, with one mouse showing tumor elimination. The tumor growth curves of individual mice in each group were consistent with these results, indicating that DOX / ASA@HDP significantly inhibits postoperative tumor recurrence in 4T1 tumor-bearing mice.

[0190] Twenty-five days after treatment, the lungs of 4T1 tumor-bearing mice were dissected, washed, fixed and stained in Bouin's fixative, and the lung nodules were statistically analyzed. Figure 18 As shown in Contents A, B, and C, mice treated with PBS, HDP, DOX, and ASA had a higher number of lung nodules, averaging between 37 and 44. The DOX@HDP and ASA@HDP groups had fewer and similar numbers of lung nodules, averaging between 17 and 22. The DOX / ASA@HDP group had the fewest lung metastatic nodules, averaging only about 7. H&E staining of mouse lungs clearly revealed lung metastatic nodules, and their number and area were consistent with the above trends, indicating that DOX / ASA@HDP has a good inhibitory effect on tumor metastasis. To investigate the inhibitory effect of DOX / ASA@HDP on platelets in the tumor microenvironment after tumor surgery, 25 days after the above treatment, mouse tumors were dissected, ground and digested into a single-cell suspension, and intratumoral platelet activation was analyzed by flow cytometry. Figure 19 Contents A, B, C, D, and E show that, compared to other control groups, the ASA@HDP and DOX / ASA@HDP groups showed a higher intratumoral platelet activation phenotype, CD62P. + and CD63 +The expression level was lowest, indicating a significant inhibitory effect on platelet activation. Immunofluorescence analysis of tumor sections showed that, similarly, compared to other groups, the ASA@HDP and DOX / ASA@HDP groups had the lowest proportions of CD41+ (representing platelets) and CD62P+ (representing platelet activation) cells in tumor sections, and the statistical data also showed the same trend. In conclusion, DOX / ASA@HDP can effectively inhibit the number and activation state of platelets in the tumor microenvironment. It will be readily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A hydrogel for postoperative drug loading, characterized in that, Its preparation method includes the following steps: (1) Mix and stir an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and hyaluronic acid, and keep the solution weakly acidic to activate the carboxyl group of the hyaluronic acid to obtain an activated precursor solution; the mass percentage of hyaluronic acid in the aqueous solution of hyaluronic acid is 0.5-1.5%; (2) The activated precursor solution described in step (1) is mixed with 3-aminophenylboronic acid and dopamine hydrochloride and reacted under inert gas protection and weak acid conditions, so that the 3-aminophenylboronic acid and dopamine hydrochloride are grafted onto the backbone of the hyaluronic acid through an amide condensation reaction to obtain a crude product; the molar ratio of the 3-aminophenylboronic acid and dopamine hydrochloride is 3:1-1:3, and the mass ratio of the dopamine hydrochloride to the hyaluronic acid is 0.28-1.1:1; (3) Dialyze the crude product obtained in step (2) in acidic ultrapure water. Adjust the pH of the dialyzed liquid to neutral and stir it for 3-8 h. Use oxygen in the air to oxidize and polymerize dopamine, and then freeze-dry it to obtain a hydrogel for drug loading.

2. The hydrogel as described in claim 1, characterized in that, The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and hyaluronic acid in step (1) is (0.4-0.8):(0.1-0.5):1; the activation time in step (1) is 10-40 minutes.

3. The hydrogel as described in claim 1, characterized in that, The inert gas in step (2) is argon or nitrogen, and the reaction time is 12-36 hours.

4. The hydrogel as described in claim 1, characterized in that, Step (3) Transfer the crude product to a dialysis bag with a molecular weight cutoff of 10,000-14,000 Da; freeze-dry the stirred product under vacuum for 24-72 hours to obtain the hydrogel used for drug loading.

5. A postoperative drug-loaded hydrogel, characterized in that, It includes the hydrogel as described in any one of claims 1 to 4, and further includes a loaded antitumor drug and a drug capable of improving the tumor microenvironment, wherein the antitumor drug is doxorubicin and the drug capable of improving the tumor microenvironment is metformin.

6. The use of the postoperative drug-loaded hydrogel as described in claim 5 in the preparation of drugs for postoperative treatment and / or inhibition of breast cancer.

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