CN116803374A

CN116803374A - Carrier hydrogel for tumor treatment, preparation method and application thereof

Info

Publication number: CN116803374A
Application number: CN202310873042.5A
Authority: CN
Inventors: 甘璐; 李建业; 杨祥良; 雍土莹
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2023-07-14
Filing date: 2023-07-14
Publication date: 2023-09-26

Abstract

The invention belongs to the technical field of biomedical materials, and in particular relates to a liquid-carrying gel for postoperative tumor treatment, a preparation method and application thereof. The hydrogel scaffold material is prepared by grafting two reaction substrates dopamine and 3-aminophenylboronic acid onto a hyaluronic acid skeleton through an amide condensation reaction by a one-pot method. The flexible rheological properties and excellent bioadhesion ensure that they adhere stably to the resected wound site without displacement, the slow and pH-responsive release and the long-term retention of the drug in the body characteristics that enable the hydrogel to respond to the tumor microenvironment and maintain the tumor resected microenvironment at sufficient drug concentration for a long period of time. The hydrogel prepared by the invention can be loaded with anti-tumor chemotherapeutic drugs and drugs capable of improving tumor microenvironment simultaneously, so that the tumor microenvironment is obviously improved while the chemotherapeutic drugs inhibit tumors, and the anti-tumor chemotherapeutic drugs and the drugs are synergistic to promote the intervention inhibition of postoperative tumor recurrence and metastasis.

Description

Carrier hydrogel for tumor treatment, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and in particular relates to a liquid-carrying gel for tumor treatment, a preparation method and application thereof.

Background

Malignant tumors threaten human health, and traditional treatment means such as surgical excision, chemotherapy and radiotherapy are difficult to achieve complete cure of the malignant tumors, so that the treatment effect is poor. Surgical excision is the most important clinical treatment for malignant tumors, but often faces the problem of tumor recurrence and metastasis caused by residual tumor cells after surgery. Conventional clinical practice is to assist with systemic chemotherapy or radiotherapy after surgery, but with extreme toxicity and with a window of use due to patient weakness after surgery. In addition, tissue damage, changes in cells in the microenvironment, and inflammation from the post-operative procedure all contribute to the recurrence process, including activation of blood vessels and platelets recruited to the resection site by tissue damage, vascular neogenesis of damaged blood vessels, and stromal cells such as fat, all contribute to tumor recurrence and metastasis. Therefore, timely removal of residual tumor cells after surgery and combination with modification of the microenvironment after tumor surgery are important strategies for inhibiting recurrence and metastasis after tumor surgery and improving survival benefit of patients.

The in-situ administration system is a treatment means for in-situ administration by using materials which can be fixed on focus parts, and has remarkable advantages. In-situ drug delivery systems typically employ degradable and biocompatible materials as a matrix to carry one or more drugs, locally release the drug to the target site, increase the drug concentration at the target site, and simultaneously reduce the drug concentration in blood and systemic tissues, while ensuring therapeutic efficacy with low toxicity. The drug delivery system generally has the characteristic of slow release or controlled release, can carry out intelligent and accurate release of the drug according to the special microenvironment of the surgical excision part, plays the therapeutic effect to the maximum extent and reduces the side effect.

Hydrogels are a polymeric network system with a hydrophilic three-dimensional network of cross-linked structures, typically cross-linked by covalent or non-covalent bonds. The hydrogel may be made of polysaccharides (chitosan, hyaluronic acid, gelatin), polypeptides (poly (L-glutamic acid), silk fibroin), polymers (polyacrylamide and polyvinyl alcohol), etc. According to different materials and crosslinking methods, the hydrogel can have different physical characteristics such as elasticity, toughness, injectability, adhesiveness, anti-blocking property, conductivity, stretchability, plasticity, self-healing property and the like, so that various application requirements are met, and the application range of the hydrogel is wider from a drug-carrying system to tissue engineering to a wearable electronic element. However, the existing single hydrogel treatment means is difficult to obtain good curative effect, so that the more comprehensive treatment effect has better application prospect.

The largest proportion of adipocytes in all cell types constituting breast tissue is the key cell in the breast cancer tumor resection microenvironment. In the breast cancer tumor microenvironment, cancer-associated adipocytes are not only spatially adjacent to cancer cells, but also cross-talk with cancer cells, promoting tumor recurrence, progression and metastasis. Research shows that fat cells are not only used as energy storage cells, but also used as important endocrine cells, generate various bioactive molecules called as fat factors, such as leptin, adiponectin, IL-6, resistin and the like, participate in proliferation, diffusion, angiogenesis, invasion and metastasis of breast cancer through ways of fat factor regulation, metabolic reprogramming, microenvironment remodeling, immunoregulation and the like, but the research of preventing tumor recurrence and metastasis by inhibiting fat cells in the microenvironment after tumor operation is deficient at present. Furthermore, angiogenesis and remodeling are an important biological process in the tumor resection microenvironment that promotes tumor growth. The damaged blood vessels create new blood vessels to transport oxygen and nutrients, thereby promoting rapid recurrence and metastasis of the residual tumor. Therefore, it is expected to inhibit the function of adipocytes and angiogenesis while killing residual tumor cells, and inhibit tumor recurrence and metastasis after surgery.

Met is a clinically commonly used antidiabetic agent that regulates metabolism and inhibits angiogenesis by activating AMPK. Recent studies indicate that Met can delay tumor progression by affecting adipocytes, reducing adipokine secretion, to remodel fat-related tumor microenvironment. However, met has a short systemic half-life and low in vivo bioavailability, and it is difficult to achieve the desired effect.

The implantable drug-carrying platform can avoid the empty window period of the postoperative recovery of a patient by directly implanting the tumor excision part, increase the drug accumulation of the tumor excision part, prolong the drug release time, reduce the side effect of the whole body drug and provide a strategy with prospect for postoperative cancer treatment. Hydrogels are composed mainly of a three-dimensional macromolecular network and a large amount of water. The bioadhesive hydrogel has the characteristics of high porosity, strong plasticity and low toxicity, and simultaneously realizes controllable release of the drug and good tissue adhesion. Inspired by mussel adhesive proteins, adhesive hydrogel rich in catechol, dopamine (DA), gallic Acid (GA) and other polyphenols is applied to the fields of wound dressing, tissue engineering, flexible devices and the like. However, there is still a need for improvement in how to efficiently release drugs from hydrogels at tumor resection sites.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a carrier hydrogel for tumor treatment, a preparation method and application thereof, and the carrier hydrogel is used for in-situ administration of a chemotherapeutic drug and a tumor microenvironment modifying drug by constructing a hydrogel material with good biocompatibility so as to prevent postoperative recurrence and metastasis of tumors.

In order to achieve the above object, the present invention provides a method for preparing hydrogel for postoperative drug loading, comprising the steps of:

(1) Mixing and stirring 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and an aqueous solution of hyaluronic acid, keeping the solution to be slightly acidic, and activating carboxyl of the hyaluronic acid to obtain an activated precursor solution;

(2) Mixing the activated precursor solution obtained in the step (1) with 3-aminophenylboronic acid and dopamine hydrochloride, and reacting under the protection of inert gas and weak acidity, so that the 3-aminophenylboronic acid and the dopamine hydrochloride are grafted on the skeleton of the hyaluronic acid through an amide condensation reaction to obtain a crude product;

(3) Dialyzing the crude product obtained in the step (2) in acidic ultrapure water, regulating the pH of the liquid obtained by dialysis to be neutral, stirring, oxidatively polymerizing dopamine by using oxygen in air, and freeze-drying to obtain the hydrogel for carrying the medicine.

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

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

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

Preferably, step (3) transfers the crude product into a dialysis bag having a molecular weight cut-off of 10000-14000Da; the stirring time is 3-8h; and (3) freeze-drying the stirred product in vacuum for 24-72 hours to obtain the hydrogel for carrying the medicine.

According to another aspect of the present invention, there is provided a carrier hydrogel for inhibiting tumor postoperative recurrence and metastasis obtained by the preparation method.

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

According to another aspect of the invention there is provided the use of a loaded hydrogel as described in the manufacture of a medicament for post-operative treatment and/or inhibition of tumours.

According to another aspect of the invention, there is provided the use of the aqueous carrier gel in the manufacture of a medicament for inhibiting the regeneration of tumor adipocytes after surgery and/or inhibiting tumor angiogenesis after surgery.

According to another aspect of the invention there is provided the use of a loaded hydrogel as described in the manufacture of a medicament for inhibiting platelet activation after oncology.

In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art:

(1) According to the invention, two reaction substrates dopamine and 3-aminophenylboronic acid are grafted onto a hyaluronic acid skeleton through a one-pot amide condensation reaction, so that the hydrogel for carrying the medicine is prepared, and the preparation method is simple, convenient, safe and controllable.

(2) The hydrogel prepared by the invention adopts dopamine and 3-aminophenylboronic acid as reaction substrates, and has a double cross-linked network, so that the hydrogel has a tighter cross-linked network and better rheological property.

(3) The hydrogel has excellent bioadhesion performance due to the grafting of dopamine, and can be ensured to be adhered to biological tissues without displacement. The pH responsiveness of the phenylboronic acid ester bond formed between the dopamine and the 3-aminophenylboronic acid is achieved, and the prepared hydrogel can slowly release the pH responsiveness drug to the acidity of tissues. In addition, the invention controls the proper oxidation polymerization degree of the dopamine in the prepared gel material by using the oxygen in the air to carry out oxidation polymerization, thereby controlling the degradation speed of the gel material in vivo, namely, the corresponding proper drug release speed.

(4) The invention provides a medicine-carrying hydrogel for tumor postoperative treatment, which can wrap chemotherapeutic medicines or other intervention medicines. The hydrogel stent material provided by the invention has good biocompatibility, bioadhesion, swelling property and pH responsiveness. The flexible rheological properties and excellent bioadhesion ensure that they adhere stably to the resected wound site without displacement, the slow and pH-responsive release and the long-term retention of the drug in the body characteristics that enable the hydrogel to respond to the tumor microenvironment and maintain the tumor resected microenvironment at sufficient drug concentration for a long period of time.

(5) The hydrogel prepared by the invention can be loaded with anti-tumor chemotherapeutic drugs and drugs capable of improving tumor microenvironment simultaneously, so that the tumor microenvironment is improved while the chemotherapeutic drugs inhibit tumors, and the inhibition of postoperative tumor recurrence and metastasis is synergistically promoted. The carrier hydrogel which is prepared in the preferred embodiment and carries Doxorubicin (DOX) and metformin (Met) simultaneously is found through postoperative experiments, compared with a control group, the carrier hydrogel can remarkably inhibit postoperative recurrence and metastasis of tumors, inhibit the neogenesis of adipocytes and further inhibit the recurrence of tumors; but also can obviously inhibit the formation of blood vessels after tumor operation and reduce the oxygen and nutrition supply of residual tumor tissues. The invention is expected to provide theoretical basis and treatment strategy for postoperative treatment of tumors with more peripheral fat cells, such as breast cancer, pancreatic cancer, renal cell carcinoma and the like. In addition, the prepared DOX and aspirin (ASA) -loaded hydrogel can effectively inhibit platelet activation and effectively inhibit recurrence and metastasis after tumor operation when killing tumor cells.

Drawings

FIG. 1 is a flow chart of a preparation method of the hydrogel for carrying medicine for tumor treatment.

FIG. 2A shows the shear viscosity of HDP hydrogels with different dopamine/APBA ratios; content B is the change of G 'and G' of HDP hydrogel with different dopamine/APBA ratios along with the frequency; content C is the change of G 'and G' of HDP hydrogels with different dopamine/APBA ratios with the degree of strain.

FIG. 3A is a schematic diagram of a universal tester for measuring the adhesion ability of HDP hydrogels with different dopamine/APBA ratios; content B is a change curve of the adhesive strength of the HDP hydrogel with different dopamine/APBA ratios along with displacement; content C is the adhesion strength statistics of the HDP hydrogel adhesion strength with displacement for different dopamine/APBA ratios.

FIG. 4 content A is a photograph of a morphology of HDP hydrogel; content B is a schematic view of the HDP hydrogel self-healing principle; content C is a photograph of the self-healing process of the HDP hydrogel; content D is a photograph of HDP hydrogel adhered to a finger; content E is a photograph of HDP hydrogel malleable with finger bending; content F is a HDP hydrogel tissue adhesion photograph; content G is a photograph of HDP hydrogel tensile properties.

FIG. 5, content A, is an SEM observation of HDP (scale: 50 μm, right image is an enlarged image of a dashed box of left image); content B is the HDP statistical mean pore size by SEM image.

Fig. 6, panel a, shows the in vitro drug release behavior of DOX/met@hdp at ph=7.4 and 6.5; content B is Met in vitro drug release behavior at ph=7.4 and 6.5 for DOX/met@hdp.

FIG. 7, panel A, shows shear viscosity studies for HDP, DOX@HDP, met@HDP and DOX/Met@HDP; content B is the variation of G 'and G' of HDP, DOX@HDP, met@HDP and DOX/Met@HDP with frequency; content C is the change in G 'and G' of HDP, DOX@HDP, met@HDP and DOX/Met@HDP with the degree of strain.

FIG. 8 panel A is a fluorescence image of a representative in vivo Cy5.5 animal from mice treated with Cy5.5/ICG and Cy5.5/ICG@HDP after 4T1 in situ tumor resection; content B is in-vivo fluorescence quantification of Cy5.5 in mice treated by Cy5.5/ICG and Cy5.5/ICG@HDP after 4T1 in-situ tumor excision; in-vivo imaging fluorescence pictures of ICG representative small animals in mice treated by Cy5.5/ICG and Cy5.5/ICG@HDP after in-situ tumor excision of 4T 1; content D is in vivo ICG fluorescence quantification of mice treated with Cy5.5/ICG and Cy5.5/ICG@HDP after 4T1 in situ tumor resection.

FIG. 9 panel A is a representative fluorescence image of major organs and intratumoral Cy5.5 in mice at day 10 after in situ tumor resection of 4T1 after treatment with Cy5.5/ICG and Cy5.5/ICG@HDP; content B is fluorescence quantification of main organs of mice and Cy5.5 in tumors on day 10 after in-situ tumor excision of 4T1 and treatment of Cy5.5/ICG and Cy5.5/ICG@HDP; content C is representative fluorescence pictures of main organs of mice and ICG in tumors on day 10 after in-situ tumor excision of 4T1 and treatment of Cy5.5/ICG and Cy5.5/ICG@HDP; content D is the fluorescence quantification of major organs and intratumoral ICG in mice on day 10 after in situ tumor resection of 4T1 after treatment with Cy5.5/ICG and Cy5.5/ICG@HDP.

FIG. 10 panel A shows cell viability of 4T1 cells after 24h treatment with PBS, HDP releasing solution, DOX, met, DOX@HDP releasing solution, met@HDP releasing solution or DOX/Met@HDP releasing solution (wherein the free drug and releasing solution have different DOX and Met concentrations) as determined by CCK-8 method; content B is the apoptosis condition of an Annexin V apoptosis kit and 7-AAD detection 4T1 cells after treatment for 24 hours by PBS, HDP release liquid, DOX, met, DOX@HDP release liquid, met@HDP release liquid or DOX/Met@HDP release liquid; contents C are live dead cell staining to investigate the cytotoxicity of DOX/Met@HDP on 4T1 cells.

FIG. 11 Panel A shows cell viability of adipocytes treated with PBS, HDP, DOX, met, DOX@HDP, met@HDP or DOX/Met@HDP; content B is lipid content of adipocytes treated with PBS, HDP, DOX, met, dox@hdp, met@hdp or DOX/met@hdp; content C is the content of IL-6 in the supernatant of the fat cells after being treated by PBS, HDP releasing liquid, DOX, met, DOX@HDP releasing liquid, met@HDP releasing liquid or DOX/Met@HDP releasing liquid by ELISA method; content D is the expression level of the fat factor IL-6mRNA of the adipocytes after being treated by PBS, HDP releasing liquid, DOX, met, DOX@HDP releasing liquid, met@HDP releasing liquid or DOX/Met@HDP releasing liquid; content E is the expression level of adiponectin mRNA of fat factor of fat cells after treatment by PBS, HDP releasing liquid, DOX, met, DOX@HDP releasing liquid, met@HDP releasing liquid or DOX/Met@HDP releasing liquid; content F is the expression level of adipokine resistin mRNA of adipocytes treated with PBS, HDP releasing solution, DOX, met, DOX@HDP releasing solution, met@HDP releasing solution or DOX/Met@HDP releasing solution.

FIG. 12 panel A shows the effect of 4T1 cells on 4T1 cell proliferation after treatment with PBS, HDP releasing solution, DOX, met, DOX@HDP releasing solution, met@HDP releasing solution or DOX/Met@HDP releasing solution; content B is the quantification of scratch area in 4T1 cell scratch healing experiments 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 is a representative image of 4T1 cell scratch healing experiments (scale: 100 μm) 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 D is the number of crystal violet stained migrated cells 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; contents E are representative images of crystal violet staining of migrated 4T1 cells after co-incubation with adipocytes treated with PBS, HDP releasing solution, DOX, met, DOX@HDP releasing solution, met@HDP releasing solution or DOX/Met@HDP releasing solution (scale: 50 μm).

FIG. 13A is a flow chart of a 4T1 in situ tumor resection model and drug delivery therapy; content B is the average growth curve of tumors after in situ tumor resection and treatment by PBS, HDP, DOX, met, DOX/Met, DOX@HDP, met@HDP or DOX/Met@HDP; content C is the tumor curve of a single mouse treated by PBS after in situ tumor resection; content D is a tumor curve of a single mouse after in-situ tumor resection and HDP treatment; content E is a tumor curve of a single mouse after DOX treatment after in-situ tumor resection; content F is a tumor curve of a single mouse treated by Met after in-situ tumor resection; content G is the tumor curve of single mice treated by DOX/Met after in situ tumor resection; content H is the tumor curve of a single mouse after DOX@HDP treatment after in-situ tumor resection; content I is a tumor curve of a single mouse treated by Met@HDP after in-situ tumor resection; content J is the tumor curve of a single mouse after in situ tumor resection after DOX/Met@HDP treatment.

FIG. 14 panel A is a photograph of a tumor that was exfoliated 20 days after 4T1 in situ tumor resection, after PBS, HDP, DOX, met, DOX/Met, DOX@HDP, met@HDP or DOX/Met@HDP treatment; content B is the weight statistics of the tumor after in-situ tumor resection of 4T1 for 20 days after treatment with PBS, HDP, DOX, met, DOX/Met, DOX@HDP, met@HDP or DOX/Met@HDP; contents C are representative images of H & E stained sections of the tumor after 20 days post 4T1 in situ tumor resection with PBS, HDP, DOX, met, DOX/Met, DOX@HDP, met@HDP or DOX/Met@HDP.

FIG. 15 is the effect of treatment with PBS, HDP, DOX, met, DOX@HDP, met@HDP or DOX/Met@HDP on HUVECs cell tubule formation.

FIG. 16 is the effect of each sample group on IL-6 and CD31 expression in 4T1 in situ tumor post-surgery tumors.

FIG. 17 shows tumor volume detection after treatment of various sample groups (PBS control 1, HDP control 2, DOX control 3, ASA control 4, DOX@HDP control 5, ASA@HDP control 6, DOX/ASA@HDP experimental) after in situ tumor resection of 4T 1.

FIG. 18 is a comparison of lung nodule numbers after 4T1 in situ tumor resection in various sample groups (PBS control 1, HDP control 2, DOX control 3, ASA control 4, DOX@HDP control 5, ASA@HDP control 6, DOX/ASA@HDP experimental).

FIG. 19 shows the effect of treatment of various sample groups (PBS control 1, HDP control 2, DOX control 3, ASA control 4, DOX@HDP control 5, ASA@HDP control 6, DOX/ASA@HDP experimental) on the expression level of platelet-activating phenotype proteins in tumors after in situ tumor resection of 4T 1.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The preparation method of the drug-loaded hydrogel for inhibiting postoperative tumor recurrence and metastasis provided by the invention, as shown in figure 1, comprises the following steps:

(1) Mixing and stirring 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and an aqueous solution of hyaluronic acid, keeping the solution to be slightly acidic, and activating carboxyl of the hyaluronic acid to obtain an activated precursor solution;

(2) Mixing the activated precursor solution obtained in the step (1) with 3-aminophenylboronic acid (APBA) and dopamine hydrochloride, and reacting under the protection of inert gas and weak acidity, so that the 3-aminophenylboronic acid (APBA) and the dopamine hydrochloride are grafted onto the skeleton of the hyaluronic acid through an amide condensation reaction to obtain a crude product;

(3) Dialyzing the crude product obtained in the step (2) in acidic ultrapure water, regulating the pH of the liquid obtained by dialysis to be neutral, stirring in air, oxidatively polymerizing dopamine by using oxygen in the air, and freeze-drying to obtain the hydrogel for carrying the medicine.

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

In some embodiments, the weak acidity of step (1) and the weak acidity of step (2) have a ph=5-6.

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

In some embodiments, step (3) transfers the crude product to a dialysis bag having a molecular weight cut-off of 10000-14000Da, dialyses the product for 2-4 days with dilute hydrochloric acid added to ultrapure water to obtain acidic ultrapure water (ph=5), changes the acidic ultrapure water every 4-8 hours, then adjusts the pH of the liquid obtained by the dialysis to neutral by adding alkaline solution, then stirs the dialyzed solution on a stirrer, oxidizes the dialyzed solution for 3-8 hours by oxygen in air, freezes the hydrogel product to a solid, and then freeze-dries the solid in a vacuum freeze dryer for 24-72 hours to obtain the hydrogel for drug delivery.

The medicine-carrying hydrogel obtained by the preparation method can encapsulate fluorescent molecular model medicines such as ICG and Cy5.5 and simulate the retention condition of small molecular medicines in animals; the preparation method can also be used for encapsulating anti-tumor drugs and drugs capable of improving tumor microenvironment, wherein the anti-tumor drugs comprise one or more of doxorubicin, taxol, camptothecins and the like, and the drugs capable of improving tumor microenvironment comprise metformin, aspirin and the like. . The hydrogel and the antineoplastic and/or the medicine capable of improving the tumor microenvironment are mixed uniformly in PBS solution, and then the mixture is stood to obtain the water-carrying hydrogel. In some embodiments, the drug loading in the carrier hydrogel is less than or equal to 100 μg/mg.

Experiments prove that the water-carrying gel provided by the invention can be used for preparing a medicine for treating and/or inhibiting tumor after operation, can also be used for preparing a medicine for inhibiting tumor fat cell regeneration after operation and/or inhibiting tumor angiogenesis after operation, and can also be used for inhibiting platelet activation after tumor operation.

The invention provides a medicine-carrying hydrogel for tumor postoperative treatment and a preparation method thereof, which can wrap chemotherapeutic medicines or other intervention medicines. The hydrogel stent material provided by the invention has good biocompatibility, bioadhesion, swelling property and pH responsiveness. The flexible rheological properties and excellent bioadhesion ensure that they adhere stably to the resected wound site without displacement, the slow and pH-responsive release and the long-term retention of the drug in the body characteristics that enable the hydrogel to respond to the tumor microenvironment and maintain the tumor resected microenvironment at sufficient drug concentration for a long period of time.

In the preferred embodiment of the invention, dopamine and APBA are grafted on an HA skeleton by a one-pot method, and chemotherapeutic drugs DOX and Met are loaded at the same time, so that a liquid-carrying gel (DOX/Met@HDP) is constructed for postoperative recurrence and metastasis of breast cancer, the liquid-carrying gel performance, the effect of directly killing tumor cells at the cell level, inhibiting the growth and migration of tumor cells induced by fat cells and the influence on angiogenesis are studied, and the intervention effect of the liquid-carrying gel on recurrence and metastasis after tumor excision is evaluated at the animal level. Meanwhile, the chemotherapeutic drugs DOX and ASA are loaded, and the tumor postoperative recurrence and metastasis inhibition effect is also good.

The following are examples:

example 1

Hyaluronic acid hydrogel grafted with dopamine and 3-aminophenylboronic acid and having different dopamine/3-aminophenylboronic acid ratios and preparation method thereof

1. Experimental materials and reagents

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

2. Experimental procedure

1) 1g of HA powder was weighed and added to 100mL of ultrapure water, and the beaker was placed on a magnetic stirrer and stirred until the HA was completely dissolved, to obtain an HA solution with a mass fraction of 1%. The HA solution was placed in a round bottom flask, the flask was evacuated using a circulating water vacuum pump and argon purged to prevent oxidation.

2) 575mg of EDC and 345mg of NHS are weighed out and slowly added to the HA solution in sequence, the pH of the solution being maintained between 5 and 6 by the addition of hydrochloric acid and sodium hydroxide solution. After stirring and activating for 20min, weighing APBA and dopamine hydrochloride with the mass ratio of different substances, sequentially and slowly adding the APBA and the dopamine hydrochloride into a mixed system, and reacting the whole system for 24h under the protection of argon and the acidic pH of 5.5.

3) The resulting system was transferred to a dialysis bag (molecular weight cut-off 14000 Da) and dialyzed in acidic ultrapure water (ph=5) for 4 days, during which time the acidic ultrapure water was changed every 8 hours. After the pH of the dialyzed solution was adjusted to 7.4 by adding sodium hydroxide solution, the dialyzed solution was stirred on a stirrer, oxidized by oxygen in air for 4 hours, and the hydrogel product was frozen to a solid, which was then freeze-dried in a vacuum freeze-dryer for 72 hours to obtain a spongy product.

4) 10mg of the above product was weighed and 200. Mu.L of PBS was added to prepare 5% by mass of HDP hydrogel.

Other conditions are the same as those in the steps 1) to 4), except that in the synthesis process, only dopamine hydrochloride is added in the step 3), and APBA is not added, so that the HD hydrogel is prepared.

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

3. Experimental results

The shear viscosity of the HDP hydrogels of the five dopamine/APBA ratios is similar, and when the ratio of dopamine to APBA is 3:1, 2:1, 1:1, 1:2, 1:3, the shear viscosity is 5.42×10 respectively 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 (fig. 2 content a, content B, and content C). The HDP hydrogels with different dopamine/APBA ratios had G' consistently higher than G ", indicating stable viscoelastic states.

HDP hydrogels have stronger bioadhesive strength, with HDP hydrogels having the highest critical tensile strength (9.24 kPa) for dopamine to APBA ratios of 1:1, and lower for other ratios (critical tensile strengths for dopamine to APBA ratios of 3:1, 2:1, 1:2, 1:3 of 2.84kPa, 5.47kPa, 4.58kPa, 6.34kPa, respectively) (fig. 3, content a, content B, and content C).

Example 2

The hyaluronic acid hydrogel grafted with dopamine and 3-aminophenylboronic acid, prepared according to the method of the example, has a ratio of dopamine to 3-aminophenylboronic acid of 1:1.

(1) Hydrogel morphology

As can be seen from fig. 4, content a, the hydrogel was gelled at a concentration of 5%, and the hydrogel was able to maintain its original form in an inclined sample bottle, and had no fluidity, and had a basic form of hydrogel.

(2) Healable properties

As can be seen from fig. 4B and C, when the HDP hydrogel is cleaved, the reversible phenylboronic acid ester bond in the HDP is broken, and the cleavage surface is gradually reconnected after three minutes of contact, so that the HDP hydrogel can heal, and can accept a larger stretching force, thereby providing the HDP hydrogel with excellent healable performance. This ensures that the hydrogel is able to cope with possible shear stresses in the tumor resection cavity, maintaining the intact hydrogel morphology.

(3) Adhesion effect

As can be seen from fig. 4, panels D and F, the HDP hydrogel had better adhesion to both latex gloves and mouse liver, suggesting that the hydrogel had the potential to stably adhere to the tumor resection cavity without dislodging.

(4) Ductility

As can be seen from FIG. 4, the HDP hydrogel can adhere to the finger and deform accordingly according to the bending degree of the finger joint, and has better extensibility and adhesiveness. This feature ensures that the HDP hydrogel will conform to different resected surfaces and will stabilize against removal and rupture of the HDP hydrogel in a complex tumor resection cavity environment due to behavior of the implanted subject.

(5) Stretchability of

HDP can withstand large stretching forces, being stretched to 4.8 times the original length without breaking (fig. 4, content G). This better stretchability ensures that the HDP remains in a stable hydrogel state in the complex environment of the tumor resection cavity.

The result of the content A in fig. 5 shows that the HDP has a porous honeycomb network structure with uniform and compact holes, and the average pore diameter of the HDP is 39.7 μm (the content B in fig. 5) by counting the pore diameters in the electron microscope picture through Image J software, which creates a condition for the HDP to have better drug loading.

Comparative example 1

3) The resulting system was transferred to a dialysis bag (molecular weight cut-off 14000 Da) and dialyzed in acidic ultrapure water (ph=5) for 4 days, during which time the acidic ultrapure water was changed every 8 hours. After the pH of the liquid obtained by dialysis was adjusted to 7.4 by adding sodium hydroxide solution, the hydrogel product was frozen to a solid, and then freeze-dried in a vacuum freeze-dryer for 72 hours to obtain a spongy product.

4) Weighing 10mg of the above product, adding 170 μl of ultrapure water, mixing, and adding 10 μ L H ₂ O ₂ Solution (0.5 mol L) ^-1 ) And 20. Mu.L of HRP horseradish peroxidase solution (1 mg mL) ^-1 ) And stirring. The HDP-1 hydrogel with the mass fraction of 5% is prepared.

Comparative example 1 the other steps for preparing hydrogels were the same as in example 1 except that the HDP hydrogel was obtained by stirring and oxidizing in air in step (4) of example 1, whereas comparative example 1 prepared HDP-1 hydrogel using hydrogen peroxide and horseradish peroxidase. In experiments, the hydrogel obtained by adopting the oxidation mode of the comparative example 1 has very low degradation speed, and the drug release speed after the subsequent drug loading is affected, so that the drug release speed is too low. For this purpose, stirring is directly carried out in air for a plurality of hours, and the dopamine is oxidized by oxygen in the air, so that hydrogel HDP with moderate degradation speed is obtained.

The hydrogels obtained in example 1 and comparative example 1 were tested for hydrogel degradation rate in a shaking table at constant temperature of 100rpm at ph=7 and 37, respectively, and it was found that the time required for degradation of the HDP-1 hydrogel obtained in comparative example 1 was 95 days, whereas the time required for degradation of the HDP hydrogel obtained in example 1 was only 30 days. Therefore, the mode of the oxidative polymerization of the dopamine of the HDP hydrogel prepared by the one-pot method has a great influence on the degradation speed of the finally obtained gel. Air oxidation may make HDP have a low and controllable degree of oxidation of dopamine in it.

In addition, other experimental conditions were the same as in example 1, and the time of the oxidation treatment by oxygen in the air in step 3) was changed to 36 hours and 48 hours, respectively, and the degradation rate was found to be also slow by the test, and 56 days and 65 days were required, respectively.

Example 3

Drug loading capacity of hyaluronic acid Hydrogel (HDP) grafted with dopamine and 3-aminophenylboronic acid in a ratio of 1:1

1. Experimental materials and reagents

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).

2. Experimental procedure

1) Hydrogel preparation was the same as in example 1 (dopamine/3-aminophenylboronic acid ratio 1:1).

2) 10mg of HDP prepared in example 1 was weighed and 200. Mu.L of the mixture containing 60. Mu.g mL was added ^-1 DOX and 30mg mL ^-1 And (3) uniformly mixing the components in PBS of Met, and standing for 30min to obtain the double-carrier hydrogel DOX/Met@HDP.

3) A10 mL centrifuge tube was taken, 200. Mu.L DOX/Met@HDP was immersed in 5mL PBS solutions of different pH (pH=6.5, 7.4), and the release system was placed in a constant temperature shaker for drug release experiments, with a set temperature of 37℃and a shaker speed of 100rpm. Taking out 1mL of release liquid at different time points (1 h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, 72h and the like), supplementing 1mL of fresh PBS to a release system, measuring the concentration of DOX and Met of the release liquid by using a 1260 type high performance liquid chromatograph, and calculating the accumulated drug release amount.

3. Experimental results

The in vitro release properties of DOX/Met@HDP at different pH conditions were studied (FIG. 6, panel A and panel B). DOX/met@hdp at ph=7.4, the release of DOX was about 40.3% at 120h and 88.2% at ph=6.5 (fig. 6 content a); likewise, DOX/met@hdp released about 38.5% Met at 8h at ph=7.4 and 58.6% at ph=6.5 (fig. 6, panel B), indicating that DOX/met@hdp has pH-responsive drug release properties. The pH-responsive release of DOX/Met@HDP may be due to cleavage of phenylboronic acid ester bonds in the HDP hydrogel network at low pH, which facilitates 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, and pi-pi stacking or hydrogen bonding interactions between DOX and polydopamine.

The loading of DOX and Met had no significant effect on the shear viscosity of the HDP (FIG. 7, panel A) nor on the change in G 'and G' with frequency (FIG. 7, panel B) and strain level (FIG. 7, panel C), indicating that DOX@HDP, met@HDP and DOX/Met@HDP all had stable hydrogel properties. HDP is control group one, dox@hdp is control group two, met@hdp is control group three, DOX/met@hdp is experimental group.

Example 4: dopamine/3-aminophenylboronic acid ratio of 1:1, and drug loading capacity and in-vivo drug retention characteristics of hyaluronic acid hydrogel grafted with dopamine and 3-aminophenylboronic acid

1. Experimental materials and reagents

Hyaluronic Acid (HA), dopamine hydrochloride (DA), 3-aminophenylboronic acid (APBA), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), cyanine 5.5 carboxic acid (cy 5.5), indocyanine Green (ICG).

2. Experimental procedure

1) Hydrogel preparation was as in example 1.

2) 10mg of HDP was weighed and 200. Mu.L of 1. Mu.g mL was added ^-1 Cy5.5 and 1 μg mL ^-1 And (3) uniformly mixing the components in PBS of ICG to obtain the double-carrier hydrogel Cy5.5/ICG@HDP.

3) Selecting logarithmic phase 4T1 cells, performing cell count after digestion and resuspension, diluting with PBS to 1×10 density ⁷ mL ^-1 . Female BALB/c mice of 6 weeks old were anesthetized, the fourth pair of perimammary skin on the right side of the mice was cut off, 50. Mu.L of the homogenized cell suspension was injected into the fat pad and then sutured, followed by applying iodophor and taking thermal insulation measures to the mice until awakening. The surgical instruments are subjected to strict sterilization treatment. When the tumor volume reaches 300mm ³ Surgical excision is performed at that time. Firstly, performing skin preparation treatment on a mouse, pruning the hair near the tumor of the mouse, sterilizing by using iodophor, shearing the skin around the tumor after the mouse is anesthetized, removing about 90% of solid tumors, wiping blood by using cotton balls, placing a hydrogel material in a tumor excision cavity, suturing a wound, wiping with iodine, and keeping the body temperature until the mouse wakes up.

4) A4T 1 breast cancer surgical excision model is constructed and Cy5.5/ICG@HDP is implanted, and a control group of mice adopts a mode of administration that a mixed solution of free Cy5.5 and ICG, which is equal to that of an experimental group, is injected into an excision cavity. At 3h, 6h, 9h, 12h, 24h, 36h, 48h, 72h, etc., cy5.5 and ICG fluorescence in mice was imaged and photographed using an IVIS Lumina XR-type small animal imaging system, and the total amount of fluorescence was statistically analyzed. And 10 days after operation, dissecting the mice, taking out tumors and related organs (heart, liver, spleen, lung and kidney) for in vitro imaging, and carrying out statistical analysis on the total amount of Cy5.5 and ICG fluorescence in the tumors and different organs.

3. Experimental results

As can be seen from the fluorescence images of the In Vivo Imaging System (IVIS), the fluorescence signal decay rates of Cy5.5 (FIG. 8, panel A) and ICG (FIG. 8, panel C) of mice implanted with Cy5.5/ICG@HDP were significantly slower than those of mice injected with the free Cy5.5/ICG solution. The fluorescence of Cy5.5 in the free Cy5.5/ICG group was reduced to be invisible on day 10 after tumor resection, while the fluorescence of the Cy5.5/ICG@HDP group was clear and bright; at 48h, ICG fluorescence disappeared in the free Cy5.5/ICG group, while the fluorescence of the Cy5.5/ICG@HDP group was still more evident. From statistical data, the drug retention of cy5.5 (fig. 8, panel B) and ICG (fig. 8, panel D) was significantly better for the cy5.5/icg@hdp group than for the free cy5.5/ICG group. On day 10 post-implantation, the Cy5.5 fluorescence value of the Cy5.5/ICG@HDP group was 6.45X10 ⁸ Photons s ^-1 cm ^-2 sr ^-1 Whereas the free Cy5.5/ICG group was only 3.06X10 ⁸ Photons s ^-1 cm ^-2 sr ^-1 The method comprises the steps of carrying out a first treatment on the surface of the 48h post-implantation ICG fluorescence values for the Cy5.5/ICG@HDP group were 3.68X10 ⁹ Photons s ^-1 cm ^-2 sr ^-1 Whereas the free Cy5.5/ICG group was only 0.54×10 ⁹ Photons s ^-1 cm ^-2 sr ^-1 . From this, the 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 retain DOX and Met at the tumor site with high efficiency. The concentration of drug in tissues after administration was analyzed by euthanizing mice on day 10, taking out tumors and major organs and taking out photographs by fluorescence imaging. As can be seen from the fluorescence images of the ex vivo tissues (FIG. 9, panel A and panel C), the Cy5.5 and ICG fluorescence values of the tumor sites of the mice of the Cy5.5/ICG@HDP group were higher than those of the other organs (heart, liver, lung, spleen, kidney). And the fluorescence values of Cy5.5 and ICG of the tumor part of the Cy5.5/ICG@HDP group are obviously higher than those of free Cy5.5/ICG group (fig. 9 content B and content D). The data prove that the Cy5.5/ICG@HDP can improve the drug concentration of the tumor part and reduce the drug concentration in the main organ. The free Cy5.5/ICG group is the control group and the Cy5.5/ICG@HDP group is the experimental group.

Example 5: dopamine/3-aminophenylboronic acid ratio of 1:1 hyaluronic acid hydrogel grafted with dopamine and 3-aminophenylboronic acid loaded doxorubicin hydrochloride and metformin hydrochloride for killing tumor cells

1. Experimental materials and reagents

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), 4T1 breast cancer cells.

2. Experimental procedure

1) Hydrogel preparation was as in example 1.

2) 10mg of HDP was weighed and 200. Mu.L of 60. Mu.g mL was added ^-1 DOX and 30mg mL ^-1 And (3) uniformly mixing the components in PBS of Met, and standing for 30min to obtain the double-carrier hydrogel DOX/Met@HDP. In the same way, single drug-carrying DOX@HDP and Met@HDP are obtained.

3) The release solution of HDP, DOX@HDP, met@HDP, DOX/Met@HDP hydrogel was collected and released in PBS (shaking table at constant temperature of 37 ℃ C., 100 rpm) for 7 days. 4T1 cells in logarithmic growth phase were grown at 8X 10 ³ Density of/well inoculated in 96 well plates and cultured overnight, the original medium was discarded and 4T1 cells were treated with medium containing PBS, HDP releasing solution, DOX, met, DOX@HDP releasing solution, met@HDP releasing solution, DOX/Met@HDP releasing solution, respectively, wherein the drug concentrations of DOX and Met were DOX/Met=0.25 μg mL, respectively ^-1 /125μg mL ^-1 、0.5μg mL ^-1 /250μg mL ^-1 、1μg mL ^-1 /500μg mL ^-1 、2μg mL ^-1 /1000μg mL ^-1 . After 24h of treatment, the original culture medium is removed and replaced by a fresh culture medium containing a CCK-8 reagent for chromogenic reaction, after the chromogenic reaction is finished, the absorbance of the culture solution at the wavelength of 450nm is detected by using a full-wavelength enzyme-labeled instrument, and the survival rate of 4T1 cells after DOX/Met@HDP treatment is calculated.

(4) After 24h treatment of 4T1 cells in logarithmic growth phase (DOX and Met drug concentrations were 1. Mu.g mL, respectively) ^-1 And 500 μg mL ^-1 ) Collecting supernatant, collecting bottom adherent cells through pancreatin digestion, centrifuging for 5min at 300g, removing the supernatant, washing with PBS for three times, adding 100 mu L of staining working solution containing 5 mu L of Annexin V-FITC and 10 mu L of 7-AAD, re-suspending uniformly, incubating for 10-15 min in dark, adding 300 mu L of buffer solution after incubation is finished, placing on ice, detecting by a flow cytometer within 1h, and researching the apoptosis proportion condition of 4T1 cells induced by DOX/Met@HDP.

(5) After 24h of treatment of 4T1 cells in logarithmic growth phase, collecting supernatant, collecting adherent cells at the bottom layer through pancreatin digestion, centrifuging for 5min at 300g, removing the supernatant, washing with PBS three times, adding 300 mu L of staining working solution containing 2 mu M Calcein acetoxymethyl ester and 4.5 mu M propidium iodide, re-suspending uniformly, incubating for 15min at 37 ℃ in dark, centrifuging for 5min at 300g after incubation, removing the supernatant, washing with PBS twice, dripping the cell suspension re-suspended with PBS into a confocal dish, observing by a laser confocal microscope, marking living cells with Calcein-AM as green and dead cells with PI as red, and detecting 4T1 cell live-dead change induced by DOX/Met@HDP. PBS was control group 1, HDP was control group 2, DOX was control group 3, met was control group 4, DOX@HDP was control group 5, met@HDP was control group 6, and DOX/Met@HDP was experimental group.

3. Experimental results

As shown in fig. 10, the bar graph under each doxorubicin concentration condition corresponds to the experimental group of control group 1, control group 2, control group 3, control group 4, control group 5 and control group 6 from left to right, and each concentration of HDP does not affect the survival of 4T1 cells, further proving that HDP has good biosafety. Whereas for Met and met@hdp groups there was a slight effect on 4T1 cell survival at high concentrations of Met. The cell survival of DOX, DOX@HDP and DOX/Met@HDP groups under different DOX concentrations is lower, and the killing effect is more obvious along with the increase of the DOX concentration, so that the DOX-based composite material has strong concentration dependence. When DOX concentration is lower than 1. Mu.g mL ^-1 When DOX/Met@HDP released Met, the killing effect of DOX was slightly increased, resulting in the lowest cell viability. And when DOX concentration higher than 1. Mu.g mL ^-1 The effect is not obvious.

As shown in fig. 10, panel B (bar graph from left only corresponds to control group 1, control group 2, control group 3, control group 4, control group 5, control group 6 and experimental group), apoptosis was not apparent in PBS, HDP, met and met@hdp groups, DOX and dox@hdp caused apoptosis of 4T1 cells to the same extent, while DOX/met@hdp caused stronger apoptosis of 4T1 due to synergy of Met.

As shown in FIG. 10, content C, the 4T1 cells treated by PBS, HDP, met and Met@HDP are almost all green-labeled living cells, and only sporadic red-labeled dead cells prove to have almost no killing effect on the 4T1 cells. Whereas 4T1 cells treated with DOX, DOX@HDP and DOX/Met@HDP had more, denser red labeled dead cells, the above results indicate that DOX/Met@HDP has good killing properties for 4T1 cells.

Example 6

Dopamine/3-aminophenylboronic acid ratio of 1:1 hyaluronic acid hydrogel grafted with dopamine and 3-aminophenylboronic acid loaded doxorubicin hydrochloride and metformin hydrochloride for inhibiting adipocytes and related functions

1. Experimental materials and reagents

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), 3T3-L1 mouse embryo fibroblasts.

2. Experimental procedure

1) Hydrogel preparation was as in example 2.

2) 10mg of HDP was weighed and 200. Mu.L of 60. Mu.g mL was added ^-1 DOX and 30mg mL ^-1 And (3) uniformly mixing the components in PBS of Met, and standing for 30min to obtain the double-carrier hydrogel DOX/Met@HDP.

3) Adipocyte induction process: 3T3-L1 mouse embryo fibroblasts were cultured at 5X 10 ⁴ Inoculating the cells into 24-well plate, changing the culture medium into adipocyte differentiation culture medium for continuous culture after cell proliferation fusion degree reaches 100%, and performing fine culture every two daysCell replacement. After four days of differentiation, the medium was replaced with adipocyte maintenance medium, and cell exchange was performed every two days. The tenth day of differentiation, the medium was replaced with DMEM complete medium, and adipocytes were successfully induced.

4) The release solution of HDP, DOX@HDP, met@HDP, DOX/Met@HDP hydrogel was collected and released in PBS (shaking table at constant temperature of 37 ℃ C., 100 rpm) for 7 days. The original medium of the differentiated adipocytes in the 24-well plate was discarded, and a medium containing PBS, HDP releasing solution, DOX, met, DOX@HDP releasing solution, met@HDP releasing solution, DOX/Met@HDP releasing solution was added, respectively. After 48h of treatment, the supernatant was removed, the collected treated adipocytes were digested with pancreatin, then 200. Mu.L of PBS solution containing 2. Mu.M BODIPY 493/503 was added, incubated at 37℃for 15min in the absence of light, followed by centrifugation and washing with PBS three times, and the effect of DOX/Met@HDP on the lipid content of adipocytes was studied by detecting the BODIPY fluorescence intensity by flow cytometry. 5) The adipocytes were treated in the same manner as in 4) for 48 hours, collected into a 1.5mL coreless enzyme centrifuge tube, added with 1mL TRIzol, mixed well, then added with 200. Mu.L of chloroform and vigorously shaken for 15 seconds, left standing for 5min, centrifuged for 13800g for 15min (4 ℃) and mixed well with 500. Mu.L of isopropanol along the upper liquid surface, left standing for 15min and centrifuged for 13800g for 10min (4 ℃) and the supernatant was discarded, washed with 75% ethanol and centrifuged for 5400g for 5min (4 ℃) and the pellet was left standing until the pellet became transparent after repeating three times, and finally dissolved with 15 to 20. Mu.L of DEPC water to determine the RNA concentration. The RNA is preserved in a refrigerator at-80deg.C. 1. Mu.g of RNA was reverse transcribed into cDNA using HiScript O R R II Q RT SuperMix, and primers and cDNA were added as described in Taq Pro Universal SYBR qPCR Master Mix, and RT-qPCR was performed on a real-time quantitative PCR apparatus. The primers used are shown in the following table:

Primer sequences used in Table 1

5) Adipocytes were treated in the same manner for 48h, after discarding the medium, 300. Mu.L of serum-free medium was slowly added for further incubation for 12h, and after collecting the supernatant, the IL-6 content secreted by adipocytes was detected using the mouse IL-6 detection kit.

6) The adipocyte and 4T1 cell co-incubation system was performed using 24 well Transwell (0.4 μm) plates. The logarithmic phase of 4T1 cells was grown at 2X 10 ⁴ Density of wells/wells were seeded in 24-well plates and incubated overnight, after medium was discarded 600. Mu.L fresh serum-free medium was added. Adipocytes were treated in the same manner for 48 hours, collected by pancreatin digestion and resuspended in 200. Mu.L of serum-free medium, and after incubating the adipocytes with 4T1 cells cultured overnight in a Transwell upper chamber for 48 hours, the original medium was removed, and the medium was replaced with fresh medium containing CCK-8 reagent for chromogenic reaction, and after the end of the chromogenic reaction, the absorbance at a wavelength of 450nm was measured using a full wavelength microplate reader, and the effect of DOX/Met@HDP on proliferation of tumor cells was studied.

7) After establishing the 4T1 cell scratch model, adipocytes were treated for 48h in the same manner, pancreatin digested and the treated adipocytes were collected, resuspended in 200. Mu.L of serum-free medium, and the treated adipocytes were placed in a Transwell (0.4 μm) upper chamber and incubated with 4T1 cell scratches for 24h. The scratch healing condition of the 4T1 cells is observed and photographed by using an inverted microscope at 0h and 24h of the co-incubation culture, the scratch area is counted by Image J software, and the influence of DOX/Met@HDP on migration of the 4T1 cells after acting on the adipocytes is studied.

8) The adipocyte and 4T1 cell co-incubation system was performed using 24 well Transwell (8 μm) plates. Adipocytes were treated in the same manner for 48h and replaced with fresh serum-free medium after the original medium was discarded. Taking logarithmic phase 4T1 cells at 8×10 ⁴ Density of wells/wells were inoculated in a Transwell (8 μm) upper chamber and incubated with treated adipocytes for 10h. After the culture is finished, the upper chamber is taken out, cells on the upper surface are gently wiped and removed by a cotton swab, the upper chamber is washed by PBS, then 4T1 cells migrated at the bottom of the chamber are fixed by 4% paraformaldehyde fixing solution for 10min, after the upper chamber is washed by PBS, the upper chamber is dyed by 0.1% crystal violet, excess dye is removed by PBS, and the dyed 4T1 cells are observed and photographed by an inverted microscope.

The non-adipocyte co-incubation group is a control group, PBS is a control group 1, HDP is a control group 2, DOX is a control group 3, met is a control group 4, DOX@HDP is a control group 5, met@HDP is a control group 6 or DOX/Met@HDP is an experimental group. Fig. 11, content a to content F, each of which has a bar graph corresponding to control group 1, control group 2, control group 3, control group 4, control group 5, control group 6 and experimental group from left to right, respectively.

3. Experimental results

As can be seen from fig. 11, panel a, there was no significant difference in the viability of the treated adipocytes, indicating that DOX/met@hdp did not produce significant cytotoxicity to the induced adipocytes.

As shown in fig. 11, content B, in which BODIPY is a lipid dye, the fluorescence intensity can reflect the amount of lipid in adipocytes, DOX and dox@hdp have no significant effect on lipid in adipocytes compared to PBS group, whereas treatment with Met, met@hdp and DOX/met@hdp significantly reduced lipid content in adipocytes by 20.3%, 21.5% and 21.9%, respectively. DOX/Met@HDP therefore shows the potential to affect adipocyte function.

From FIG. 11, panel C, it can be seen that the HDP, DOX or DOX@HDP treatment had no significant effect on the secretion of IL-6 by adipocytes. IL-6 concentration in PBS group supernatant was 4.0ng mL ^-1 Whereas the IL-6 content in the supernatant after Met, met@HDP or DOX/Met@HDP treatment was only 1.86ng mL ^-1 、1.82ng mL ^-1 And 1.81ng mL ^-1 Compared with PBS group, the DOX/Met@HDP can obviously reduce, which indicates that DOX/Met@HDP can effectively inhibit the function of fat cells.

From fig. 11, panels D, E and F, it is evident that Met, met@hdp or DOX/met@hdp treatment significantly reduced the mRNA expression levels of the inflammation-associated factor IL-6 and resistin that promoted tumor cell proliferation and migration, with only 33.8%, 42.6%, 46.6% and 79.6%, 71.6%, 68.3% of the control PBS, respectively, with no significant trend between the other groups. The adiponectin can inhibit the development of tumors, and the expression of the adiponectin mRNA is obviously up-regulated after being treated by Met, met@HDP or DOX/Met@HDP, is about 1.7 times of the expression level of a PBS group, and has no obvious trend among other groups. However, the leptin expression levels were not significantly different after each treatment. DOX/Met@HDP can effectively regulate the expression level of adipocyte fat factor mRNA.

In fig. 12, contents a to E, 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. From fig. 12, panel a, it was shown that PBS group 4T1 cells incubated with adipocytes proliferated significantly more than the non-adipocyte group (1.9-fold), confirming that adipocytes promote 4T1 cell proliferation. The HDP, DOX or dox@hdp group treatments had no significant effect on 4T1 cell proliferation. The treatment of Met, met@HDP or DOX/Met@HDP group slows down the proliferation of 4T1 cells of adipocytes, which is only about 1.3 times that of the group without adipocytes, indicating that DOX/Met@HDP can effectively inhibit proliferation of tumor cells induced by adipocytes.

As can be seen from fig. 12B and C, the initial scratch distances of the groups at 0h are equivalent, the scratches of the non-adipocyte group remain larger gaps after incubation for 24h, the scratches of the PBS group are substantially healed, and the surface adipocytes can promote migration of 4T1 cells. HDP, DOX or dox@hdp treatment groups had no significant effect on adipocyte-promoted scratch healing. While the 4T1 cell scratches of the Met, met@HDP and DOX/Met@HDP treatment groups still remained large gaps. The results obtained after quantification of scratch area by Image J software remained consistent with the observations, indicating that DOX/met@hdp slowed down adipocyte-promoted 4T1 cell migration.

As can be seen from FIG. 12, panel D and panel E, the treatment of adipocytes with HDP, DOX or DOX@HDP had no significant effect on the number of migrating 4T1 cells, whereas the treatment group showed significantly less number of migrating 4T1 cells than the PBS group, and quantitative statistics of the number of migrating 4T1 cells gave consistent results.

Example 7

Anti-tumor effect study of dopamine/3-aminophenylboronic acid-grafted hyaluronic acid hydrogel loaded with doxorubicin hydrochloride and metformin hydrochloride with dopamine and 3-aminophenylboronic acid in a ratio of 1:1 after in-situ tumor resection of 4T1 breast cancer

1. Experimental materials and reagents

2. Experimental procedure

1) Hydrogel preparation was as in example 1.

3) Female BALB/c mice of 6 weeks of age were randomly divided into 8 groups of 6 animals each, and a 4T1 breast cancer surgical excision model was constructed. The HDP, DOX@HDP, met@HDP and DOX/Met@HDP were implanted separately, while the PBS, DOX, met, DOX/Met group was administered by injecting a drug solution into the tumor resection cavity (DOX dose was 0.6mg kg) ^-1 Met dosage is 300mg kg ^-1 ). Tumor volume was monitored continuously by vernier calipers (tumor volume = tumor long diameter x tumor short diameter x 0.5) while continuously monitoring mouse body weight as shown in fig. 13, panel B. PBS is control group 1, HDP is control group 2, DOX is control group 3, met is control group 4, DOX/Met is control group 5, DOX@HDP is control group 6, met@HDP is control group 7 or DOX/Met@HDP is experimental group.

3. Experimental results

As shown in fig. 13, content B, content C, content D, content E, content F, content G, content H, content I and content J, according to expectations, no significant tumor inhibition effect was observed for both HDP and Met treatments, whereas tumor recurrence was slightly slower in DOX and DOX/Met treated mice than in PBS groups, with tumor inhibition rates of 31.6% and 32.8%, respectively; the inhibition effect of DOX@HDP and Met@HDP is better than that of DOX, the inhibition rate is respectively improved to 61.2% and 48.9%, and compared with that of PBS (poly-styrene) group, the inhibition rate of DOX/Met@HDP group is as high as 89.8%, so that the DOX/Met@HDP group has the best tumor inhibition effect.

From the photographs of the tumors (content A in FIG. 14) and the weight statistics (content B in FIG. 14), it can be concluded that the tumor inhibition rates of DOX and DOX/Met are only 20.1% and 30.5%, the tumor inhibition rates of DOX@HDP and Met@HDP are 49.9% and 66%, respectively, and the tumor-bearing mice treated by DOX/Met@HDP have the slowest recurrence degree, have the best tumor inhibition effect compared with the PBS group, and the tumor inhibition rate is as high as 87.2%. It can also be seen from H & E stained sections of the tumor that the area of proliferating cells was minimal and the area of necrotic area was maximal after treatment with DOX/Met@HDP group (FIG. 14 content C).

Example 8

The hyaluronic acid hydrogel grafted with dopamine and 3-aminophenylboronic acid and having the ratio of dopamine to 3-aminophenylboronic acid of 1:1 is loaded with doxorubicin hydrochloride and metformin hydrochloride for inhibiting the related functions of HUVECs (human umbilical vein endothelial cells).

As shown in fig. 15, the effect of each group treatment on HUVECs cell tubule formation. The release solution of HDP, DOX@HDP, met@HDP, DOX/Met@HDP hydrogel was collected and released in PBS (shaking table at constant temperature of 37 ℃ C., 100 rpm) for 7 days. Through PBS (control group 1), HDP releasing liquid (control group 2), DOX (control group 3), met (control group 4), DOX@HDP releasing liquid (control group 5), met@HDP releasing liquid (control group 6) and DOX/Met@HDP releasing liquid (experimental group) (DOX concentration is 1 μg mL) ^-1 Met concentration of 500. Mu.g mL ^-1 ) The treated HUVECs cell tubules form experimental representative pictures (scale: 100 μm) (content a) and quantitative analysis of the number of grids (content B), the grid area (content C) and the total length of the tube (content D) (n=6). Effect of DOX/met@hdp treatment on scratch healing of HUVECs cells. Content E, content F was a solution released by PBS, HDP, DOX, met, DOX@HDP, met@HDP, or DOX/Met@HDP (DOX concentration 1. Mu.g mL) ^-1 Met concentration of 500. Mu.g mL ^-1 ) Representative images of scratch healing of treated HUVECs cells (scale: 100 μm) (content E) and scratch area statistics (F) (n=6).

In clinical tumor resection, a large number of blood vessels are destroyed and angiogenesis is a necessary condition for the reconstitution of nutrient supply and recurrence of tumors after resection. To investigate the effect of DOX/Met@HDP on angiogenesis, PBS, HDP releasing solution, DOX, met, DOX@HDP releasing solution, met@HDP releasing solution or DOX/Met@HDP releasing solution (containing 1. Mu.g mL) was used ^-1 DOX and 500. Mu.g Met) and 4h later observed for tubule formation. As shown in content A, PBS, HDP, DOX,The small pipe networks formed by HUVECs in DOX@HDP groups are many, dense, regular and highly intersected, while the small pipe networks formed by Met, met@HDP and DOX/Met@HDP groups are sparse and incomplete, and often cannot form a complete closed loop. Quantitative analysis (content B, content C and content D) of the formed grid number, grid coverage area and total pipe length is carried out by Image J software, and the statistical results of each index of Met, met@HDP or DOX/Met@HDP groups are obviously smaller than those of a control group, so that the DOX/Met@HDP has obvious inhibition effect on the formation of small pipes of HUVECs.

The migration ability of HUVECs was also an important indicator of angiogenesis by removing tubule formation, so that the migration ability of DOX/Met@HDP treated HUVECs was studied using a cell scratch healing assay. On the premise that the initial scratch areas of all groups are equal, the scratch healing speed of HUVECs cells in the PBS, HDP, DOX and DOX@HDP treatment groups is higher, and the HUVECs cells have smaller gaps (content E) at 24 hours; whereas the Met, met@HDP and DOX/Met@HDP treatment groups still had relatively large voids for 24h, the cell migration was slower. Quantitative analysis of the scratch areas of the cells of each group (content F) also led to a consistent conclusion, indicating that DOX/Met@HDP can significantly inhibit angiogenesis.

Example 9

The hyaluronic acid hydrogel grafted with dopamine and 3-aminophenylboronic acid and having the ratio of dopamine to 3-aminophenylboronic acid of 1:1 is loaded with doxorubicin hydrochloride and metformin hydrochloride to improve adipocytes and inhibit angiogenesis after in-situ tumor resection of 4T1 breast cancer.

1. Experimental materials and reagents

2. Experimental procedure

1) Hydrogel preparation was as in example 1.

3) Female BALB/c mice of 6 weeks of age were randomly divided into 8 groups of 6 animals each, and a 4T1 breast cancer surgical excision model was constructed. The HDP, DOX@HDP, met@HDP and DOX/Met@HDP were implanted separately, while the PBS, DOX, met, DOX/Met group was administered by injecting a drug solution into the tumor resection cavity (DOX dose was 0.6mg kg) ^-1 Met dosage is 300mg kg ^-1 ). The effect of DOX/Met@HDP on adipocytes and angiogenesis was investigated by immunofluorescent staining of treated mouse tumors, as shown in FIG. 16. PBS is control group 1, HDP is control group 2, DOX is control group 3, met is control group 4, DOX/Met is control group 5, DOX@HDP is control group 6, met@HDP is control group 7, DOX/Met@HDP is experimental group.

3. Experimental results

FIG. 16 is the effect of DOX/Met@HDP on IL-6 and CD31 expression in 4T1 in situ tumor post-surgery tumors. After 4T1 in situ tumor resection, the tumor was treated with PBS, HDP, DOX, met, DOX/Met, DOX@HDP, met@HDP or DOX/Met@HDP (DOX: 0.6mg kg) ^-1 ，Met：300mg kg ^-1 ) IL-6 and CD31 immunofluorescent staining representative images (Contents A and C) of tumor-bearing tumor cells after 20 days of treatment and IL-6 binding ⁺ 、CD31 ⁺ Statistical results (content B and content D) (scale: 50 μm) (n=6). It can be seen that the immunofluorescence positive areas of the fluorescent sections IL-6 and CD31 in the experimental group and the control group 7 are smaller than those of the PBS group, and the secretion and angiogenesis of the adipocyte IL-6 in tumor cells are obviously inhibited.

Example 10

The hyaluronic acid hydrogel grafted with dopamine and 3-aminophenylboronic acid and having the ratio of dopamine to 3-aminophenylboronic acid of 1:1 is loaded with doxorubicin hydrochloride and aspirin to inhibit tumor recurrence and platelet activation study after in-situ tumor resection of 4T1 breast cancer.

1. Experimental materials and reagents

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), 4T1 breast cancer cells.

2. Experimental procedure

1) Hydrogel preparation was as in example 1.

2) 10mg of HDP was weighed and 200. Mu.L of 60. Mu.g mL was added ^-1 DOX and 7.5mg mL ^-1 And uniformly mixing the components in PBS (phosphate buffer solution) of ASA, and standing for 30min to obtain the double-carrier hydrogel DOX/ASA@HDP.

3) Female BALB/c mice of 6 weeks of age were randomly divided into 8 groups of 6 animals each, and a 4T1 breast cancer surgical excision model was constructed. The HDP, DOX@HDP, ASA@HDP, DOX/ASA@HDP were implanted separately, while PBS, DOX, ASA groups were administered by injecting a drug solution into the tumor resection cavity (DOX dose was 0.6mg kg) ^-1 ASA dosage of 75mg kg ^-1 ). Tumor volume was monitored continuously by vernier calipers (tumor volume = tumor long diameter x tumor short diameter x 0.5) while continuously monitoring mouse body weight, CD41 and CD62P immunofluorescent staining was performed on the treated tumor tissue. Tumor tissues are sheared, digested and sieved to prepare single cell suspension, and platelet activation in tumors is detected after staining by CD41, CD62P and CD63 fluorescent antibodies. PBS control group 1, HDP control group 2, DOX control group 3, ASA control group 4, DOX@HDP control group 5, ASA@HDP control group 6, DOX/ASA@HDP control group.

3. Experimental results

As shown in FIG. 17, content A, content B, content C, content D, content E, content F, content G, content H, content I and content J, recurrence rates were faster in both PBS, HDP, DOX, ASA and ASA@HDP groups after tumor resection, and average tumor volumes were 1351mm after 25 days of treatment, respectively ³ 、1342mm ³ 、957mm ³ 、1306mm ³ And 1150mm ³ The method comprises the steps of carrying out a first treatment on the surface of the With no significant differences between the groups. The DOX@HDP group has a certain degree of tumor inhibition, and the tumor volume is smaller than that of the PBS group and is 372mm on average ³ And 380mm ³ . Whereas the DOX/ASA@HDP group had a minimum volume of tumor, an average of only 125mm3, and a gentle tumor growth rate. 25 days after the administration treatment, the DOX/ASA@HDP group had minimal tumor volume and weight as well as one mouse tumor elimination, as seen by the photograph and weight of the tumor dissected. The tumor growth curve trend of each group of single mice is consistent with the result, which indicates DOX/ASA@HDP can obviously inhibit tumor recurrence after operation of a 4T1 tumor-bearing mouse.

The lungs of the 4T1 tumor-bearing mice were dissected 25 days after treatment, stained fixedly in Bouin's fixative after washing, and counted for pulmonary nodules. As shown in fig. 18, content a, content B and content C, the mice treated with PBS, HDP, DOX and ASA had a higher number of pulmonary nodes, with an average of between 37 and 44; the number of lung nodules in the DOX@HDP group and the ASA@HDP group is small and equivalent, and the average number is between 17 and 22; while DOX/ASA@HDP had the least number of pulmonary metastasis nodules, with an average of about 7. Mouse lung H&E staining clearly observed pulmonary metastasis nodules, and the number and area were consistent with the above trend, indicating that DOX/ASA@HDP had good tumor metastasis inhibiting effect. To study the effect of DOX/ASA@HDP after tumor surgery on platelet inhibition in tumor microenvironment, mice tumors were stripped after 25 days of the above-described dosing treatment, and the tumor was ground and digested into single cell suspension, followed by analysis of intratumoral platelet activation using flow cytometry. From FIG. 19, panel A, panel B, panel C, panel D and panel E, it can be seen that the intratumoral platelet activation phenotype CD62P of the ASA@HDP and DOX/ASA@HDP groups is compared to the other control groups ⁺ And CD63 ⁺ Has the lowest expression and has the function of obviously inhibiting platelet activation. From tumor immunofluorescence sections, the same trend was seen for both CD41+ (representing platelets) and CD62P+ (representing platelet activation) cell fractions in the ASA@HDP and DOX/ASA@HDP groups of tumor sections compared to the other groups. In conclusion, DOX/ASA@HDP can effectively inhibit the quantity and activation state of platelets in the tumor microenvironment. It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. A method for preparing hydrogel for postoperative drug loading, which is characterized by comprising the following steps:

2. The method according to claim 1, wherein the mass ratio of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide to hyaluronic acid in step (1) is (0.4-0.8): (0.1-0.5): 1, the mass percentage of the hyaluronic acid in the aqueous solution of the hyaluronic acid is 0.5-1.5%; and (3) activating the catalyst in the step (1) for 10-40 minutes.

3. The preparation method according to claim 1, wherein the molar ratio of the 3-aminophenylboronic acid to the dopamine hydrochloride in the step (2) is 3:1-1:3, and the mass ratio of the dopamine hydrochloride to the hyaluronic acid is 0.28-1.1:1.

4. The method according to claim 1, wherein the inert gas in the step (2) is argon or nitrogen, and the reaction time is 12 to 36 hours.

5. The method of claim 1, wherein step (3) transfers the crude product to a dialysis bag having a molecular weight cut-off of 10000-14000Da; the stirring time is 3-8h; and (3) freeze-drying the stirred product in vacuum for 24-72 hours to obtain the hydrogel for carrying the medicine.

6. A hydrogel for postoperative drug delivery obtained by the production method according to any one of claims 1 to 5.

7. A postoperative loaded hydrogel according to claim 6, further comprising an anti-tumor drug and a drug capable of improving tumor microenvironment, wherein the anti-tumor drug comprises one or more of doxorubicin, paclitaxel and camptothecin, and the drug capable of improving tumor microenvironment comprises metformin and/or aspirin.

8. Use of the postoperative loaded hydrogel according to claim 7 for the preparation of a medicament for the postoperative treatment and/or inhibition of tumors.

9. Use of the postoperative loaded hydrogel according to claim 7 for the preparation of a medicament for inhibiting tumor adipocyte neogenesis and/or inhibiting tumor angiogenesis after surgery.

10. Use of the postoperative loaded hydrogel according to claim 7 for the preparation of a medicament for inhibiting platelet activation after tumor surgery.