Hydrogel as well as preparation method and application thereof

Abstract

The invention discloses hydrogel as well as a preparation method and application thereof, and relates to the technical field of hydrogel. The gel forming material of the hydrogel, the ATP or the ATP salt and the anthracycline antibiotic derivative are innovatively combined, and the gel forming material of the hydrogel, the ATP or the ATP salt and the anthracycline antibiotic derivative can be self-assembled to form the hydrogel which is stable in property and obvious in curative effect; according to the scheme, the toxic and side effects of the anthracycline antibiotic derivative such as adriamycin can be reduced while the curative effect of the anthracycline antibiotic derivative is ensured or improved, and postoperative recurrence is inhibited.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogels, and in particular to a hydrogel and a preparation method and application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC), a specialized type of breast cancer lacking estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 (HER2), is refractory to targeted and endocrine therapy. Furthermore, it is highly aggressive and prone to early metastasis, leading many patients to miss the optimal opportunity for surgery. Chemotherapy remains a common treatment for TNBC, but it is prone to developing drug resistance, resulting in a poor prognosis and high recurrence rates. Exploring novel treatments for TNBC is of great clinical significance.

[0003] The anthracycline drug doxorubicin (DOX) is a broad-spectrum antitumor drug used to treat a variety of malignancies. Doxorubicin's antitumor mechanisms primarily involve: interfering with DNA replication and transcription by intercalating into DNA, thereby inhibiting cancer cell proliferation; inhibiting topoisomerase II to unwind the double-stranded DNA helical structure; and generating reactive oxygen species (ROS) during intracellular metabolism, further damaging tumor cell membranes and DNA. However, doxorubicin also has significant cytotoxic effects on normal cells. One of the most serious side effects is cardiotoxicity, including myocardial damage and heart failure, which increases the risk of sudden cardiac death in patients and significantly hinders its clinical application.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a hydrogel and a preparation method and application thereof.

[0006] The present invention is achieved in that: In a first aspect, an embodiment of the present invention provides a composition comprising: a hydrogel-forming material, ATP or an ATP salt, and an anthracycline antibiotic derivative.

[0007] In a second aspect, an embodiment of the present invention provides a hydrogel, the raw materials of which include the composition described in the above embodiment.

[0008] In a third aspect, embodiments of the present invention provide use of the composition described in the preceding embodiments or the hydrogel prepared by the preparation method described in the preceding embodiments in the preparation of a drug.

[0009] The present invention has the following beneficial effects: (1) This application innovatively combines hydrogel-forming materials, ATP or ATP salts, and anthracycline antibiotic derivatives. The hydrogel-forming materials, ATP or ATP salts, and anthracycline antibiotic derivatives can self-assemble to form a hydrogel with stable properties and significant therapeutic effects. The hydrogel can ensure or improve the efficacy of anthracycline antibiotic derivatives such as doxorubicin while reducing their toxic side effects and effectively inhibiting postoperative tumor recurrence.

[0010] (2) ATP or ATP salts have immunomodulatory effects and can work together with hydrogel-forming materials such as monoammonium glycyrrhizate to activate DCs and T cells, thereby enhancing anti-tumor immune responses. (3) Hydrogel-forming materials such as monoammonium glycyrrhizate can self-assemble to form a gel. ATP or ATP salt can be combined with it to improve its stability and form a stable hydrogel. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0012] Figure 1 The formation of gels with different formulation processes; among them, (A) glycyrrhizic acid monoammonium salt / ATP- / doxorubicin hydrochloride hydrogel formation; (B) Formation of glycyrrhizic acid monoammonium salt / puerarin / doxorubicin hydrochloride hydrogel at different preparation temperatures; Figure 2 is the injectability of AG-DOX hydrogel; where A is different ATP- Inverted images of GAA hydrogels with different contents (ATP- The contents are 0, 10, 20, 30, 40, and 50 mg / mL, respectively); B is the injectability and morphological image of AG-DOX at room temperature; Figure 3 This is an in vitro immune activation experiment of AG hydrogel (n=3); A is a flow cytometry analysis of the activation effect of different drugs on BMDCs; B is Histogram of cell proportions; *P <0.05 and **P <0.01; Figure 4 The results of 4T1 cell uptake of hydrogels are shown in Figure 1. A is a representative histogram of DOX uptake; B is a statistical graph of the MFI of DOX uptake. Figure 5 The MTT results of 4T1 cells treated with different concentrations of drugs (n=6); A is the cell viability after treatment in each group; B is the IC50 value obtained after treatment of 4T1 cells in the DOX group and AG-DOX group; *p <0.05 and **p <0.01; Figure 6 Figure 3 MTT results of HL-1 cells treated with different concentrations of drugs (n=6); A is the cell viability after treatment with DOX, DOX-GAA, DOX-ATP, and AG5-DOX; B is the cell viability after treatment with GAA, ATP, and AG; C is the IC50 value of HL-1 cells treated with DOX and AG5-DOX groups. *p <0.05 and **p <0.01; Figure 7 The apoptosis results of 4T1 cells after treatment with different concentrations of drugs; A is a representative scatter plot; B is a statistical chart of the percentage of apoptotic cells after treatment in each group; *p <0.05 and **p <0.01; Figure 8 Mitochondrial activity of 4T1 cells after treatment with different concentrations of drugs; A is a representative scatter plot; B is a statistical graph of the low potential percentage after treatment in each group; *p <0.05 and **p <0.01; Figure 9 Figure 3 shows the in vivo efficacy of AG-DOX hydrogel in treating triple-negative breast cancer model mice; A is the treatment timeline of triple-negative breast cancer model mice; B is the tumor image of triple-negative breast cancer model mice after AG-DOX hydrogel treatment; C is the change in tumor volume during AG-DOX hydrogel treatment of triple-negative breast cancer model mice; D is the tumor weight of triple-negative breast cancer model mice treated with AG-DOX hydrogel; *p <0.05 and **p <0.01; Figure 10 This is the in vivo safety evaluation of AG-DOX hydrogel in treating triple-negative breast cancer model mice; A represents the weight change of triple-negative breast cancer model mice during treatment; B represents the organ coefficient; *p <0.05 and **p <0.01; Figure 11Results of AG-DOX hydrogel in preventing postoperative recurrence of triple-negative breast cancer (n = 5); (A) Animal experimental treatment plan for AG-DOX hydrogel in preventing postoperative recurrence of triple-negative breast cancer; (B) Photos of 4T1 tumors in different dosing groups; red circles indicate mouse deaths, and green circles indicate mice without recurrent tumors; (C) Weight changes of mice during treatment; (D) T1 tumor volume; (E) 4T1 tumor weight; *P < 0.05 and **P < 0.01. DETAILED DESCRIPTION

[0013] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0014] Definition of noun "ATP" herein is the abbreviation for adenosine triphosphate.

[0015] The "doxorubicin" in this article is an anthracycline antibiotic chemotherapy drug, the chemical name of which is doxorubicin, which is a natural anti-tumor antibiotic (derived from Streptomyces Streptomyces peucetius Fermentation), molecular formula: .

[0016] The term "doxorubicin hydrochloride" herein refers to the hydrochloride salt form of doxorubicin, which is obtained by removing the amino group ( ) reacts with hydrochloric acid (HCl) to form a water-soluble salt, Doxorubicin Hydrochloride, with the molecular formula: .

[0017] As used herein, "treating" includes preventing or alleviating a condition, reducing the rate at which a condition develops or progresses, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination thereof.

[0018] For cancer, "treating" can mean inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treating" includes eliminating all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the progression of a tumor, or some combination thereof.

[0019] In one aspect, an embodiment of the present invention provides a composition comprising: a hydrogel-forming material, ATP or an ATP salt, and an anthracycline antibiotic derivative.

[0020] In some embodiments, the composition comprises, by weight: 10-50 parts of a hydrogel-forming material, 10-80 parts of ATP or ATP salt, and 0.5-5 parts of anthracycline antibiotic derivative.

[0021] In some embodiments, the weight percentage of the hydrogel-forming material can be in the range of any one or any two of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50 parts. The weight percentage of ATP or ATP salt can be in the range of any one or any two of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80 parts. The weight percentage of the anthracycline antibiotic derivative can be in the range of any one or any two of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 parts.

[0022] In some embodiments, the composition comprises, by weight: 10-50 parts of a hydrogel-forming material, 10-80 parts of ATP or ATP salt, and 0.5-5 parts of anthracycline antibiotic derivative.

[0023] In some embodiments, the gel-forming material of the hydrogel includes any one or more of glycyrrhizic acid or a glycyrrhizic acid derivative, glycyrrhizic acid, puerarin, rhein, and ursoleic acid.

[0024] In some embodiments, the glycyrrhizic acid derivative includes one of ammonium glycyrrhizate, ammonium glycyrrhizate salt, monopotassium glycyrrhizate, dipotassium glycyrrhizate, and trisodium glycyrrhizate.

[0025] In some embodiments, the ammonium glycyrrhizate salt includes glycyrrhizic acid ammonium salt (GAA). Glycyrrhizic acid ammonium salt is a type of glycyrrhizic acid ammonium salt, which is a mixture of glycyrrhizic acid and ammonia. ) or ammonium ions ( ) reacts to form a monoammonium salt. Chemical structure: A carboxyl group (-COOH) in the glycyrrhizic acid molecule reacts with an ammonium ion ( ) to form a salt, while the other carboxyl group remains free. Monoammonium glycyrrhizate can self-assemble into a colloid, but its properties are unstable. Adding disodium adenosine-5'-triphosphate allows GAA to form a stable hydrogel.

[0026] In some embodiments, the ATP salt includes any one or more of ATP sodium salt, ATP magnesium salt, and ATP potassium salt.

[0027] In some embodiments, the ATP sodium salt comprises adenosine-5'-triphosphate disodium salt. or ATP) is the sodium salt form of ATP, usually a stabilized preparation for laboratory or medical use. Structural features: The two free phosphate groups (α and β positions) in the ATP molecule are connected to the sodium ion ( ) to form salts, the chemical formula is often written as .

[0028] In some embodiments, the anthracycline antibiotic derivative comprises doxorubicin or a water-soluble salt of doxorubicin.

[0029] In some embodiments, the water-soluble salt of doxorubicin includes doxorubicin hydrochloride.

[0030] In some embodiments, the composition further comprises: any one or more of water and a buffer.

[0031] In some embodiments, the buffer comprises PBS or PB. PBS (Phosphate-Buffered Saline) is an isotonic buffer solution, and PB (Phosphate Buffer) consists only of phosphate ( or ) buffer, without NaCl or other salts.

[0032] On the other hand, an embodiment of the present invention provides a hydrogel, the raw materials of which include the composition described in any of the above embodiments.

[0033] In some embodiments, the preparation method comprises: mixing a hydrogel-forming material, ATP or ATP salt, and an anthracycline antibiotic derivative.

[0034] In some embodiments, the step of mixing comprises: dissolving the anthracycline antibiotic derivative in water to obtain a drug-containing solution; adding the hydrogel-forming material to the drug-containing solution to obtain a mixed solution; The ATP or ATP salt is added to the mixed solution to obtain a hydrogel.

[0035] In some embodiments, in the hydrogel, the effective concentration of the hydrogel-forming material is 10-50 mg / mL, the effective concentration of the ATP or ATP salt is 10-80 mg / mL, and the effective concentration of the anthracycline antibiotic derivative is 0.5-5 mg / mL.

[0036] In some embodiments, the effective concentration of the hydrogel-forming material can be in the range of any one or any two of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50 mg / mL. The effective concentration of ATP or ATP salt can be in the range of any one or any two of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80 mg / mL. The effective concentration of the anthracycline antibiotic derivative can be in the range of any one or any two of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 mg / mL.

[0037] In some embodiments, the mixing further comprises: first dissolving ATP or ATP salt in the buffer solution described in the above embodiments, and then adding the solution to the mixture.

[0038] In some embodiments, the mixing temperature includes 50-70°C, specifically any one of 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 and 70°C or a range between any two thereof.

[0039] In some embodiments, the preparation method further comprises: cooling the product after adding ATP or ATP salt to the mixed solution to obtain the hydrogel.

[0040] In addition, embodiments of the present invention further provide use of the composition described in any of the foregoing embodiments or the hydrogel prepared by the preparation method described in any of the foregoing embodiments in the preparation of a drug.

[0041] In some embodiments, the drug comprises an anti-tumor drug.

[0042] In some embodiments, the anti-tumor drug has the effect of treating tumors and / or inhibiting tumor recurrence.

[0043] In some embodiments, the tumor is an indication for doxorubicin.

[0044] In some embodiments, the tumor includes any one of hematological malignancies, solid tumors and other tumors; the hematological malignancies include leukemia, lymphoma and multiple myeloma, the solid tumors include breast cancer, soft tissue sarcoma, osteosarcoma, ovarian cancer, small cell lung cancer and gastric cancer; the other tumors include neuroblastoma, bladder cancer and liver cancer.

[0045] In some embodiments, the tumor comprises triple-negative breast cancer.

[0046] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0047] Example 1: Co-loading of doxorubicin hydrochloride and ATP- Preparation and characterization of monoammonium glycyrrhizinate hydrogel.

[0048] 1.1 Co-loading of doxorubicin hydrochloride and ATP Preparation of Glycyrrhizic Acid Monoammonium Salt Injectable Hydrogel (AG-DOX) Preheat a metal bath to 60°C. Place a 2 mL centrifuge tube containing 500 μL of pure water in the bath and heat for 10 minutes. Weigh 2 mg of doxorubicin hydrochloride into the tube and allow it to fully dissolve to obtain a drug-containing solution. 30 mg of glycyrrhizic acid monoammonium salt powder was added to the drug-containing solution, and the solution was heated in a 60°C metal bath, with multiple shakes to dissolve the solution, to obtain a mixture of glycyrrhizic acid monoammonium salt and doxorubicin hydrochloride. Prepare 500 μL of phosphate buffered saline (PBS, 2×, 4 mM , 274 mM NaCl, 20 mM , 5.4 mM KCl), weigh 50 mg of adenosine-5'-triphosphate disodium salt (ATP- ) powder is fully dissolved therein to obtain ATP- solution (abbreviated as ATP solution); The obtained ATP- The solution was added to a mixture of glycyrrhizic acid monoammonium salt and doxorubicin hydrochloride. After heating was stopped and the mixture was allowed to cool, the solution transformed into a gel, resulting in an injectable hydrogel containing doxorubicin hydrochloride and ATP (AG-DOX). In the above experimental steps, no ATP was added, representing the blank injectable hydrogel containing ATP (AG).

[0049] According to the preparation principle, verify different preparation parameters: (1) Different preparation temperatures, specifically as follows: add 30 mg of glycyrrhizic acid monoammonium salt and 2 mg of doxorubicin hydrochloride powder to 500 μL of pure water, place in a metal bath at 40, 50, and 60 °C and heat, shaken several times to fully dissolve them, and obtain a mixture of glycyrrhizic acid monoammonium salt and doxorubicin hydrochloride; add 500 μL of ATP- The solution (PBS, 2×; 100 mg / ml) was mixed well and then heating was stopped. After cooling, the EP tube was inverted and the image was acquired.

[0050] (2) Different gel materials, as follows: 30 mg of ammonium glycyrrhizinate and 2 mg of doxorubicin hydrochloride powder were added into 500 μL of pure water, and were placed in a 40, 50, 60 °C metal bath for heating, during which they were fully dissolved by multiple oscillations, to obtain a mixture of ammonium glycyrrhizinate and doxorubicin hydrochloride; 500 μL of puerarin solution (PBS, 2x; 30 mg / ml, 90 °C heating and dissolution) was added at 40, 50, 60 °C, respectively, and after uniform mixing, heating was stopped, and after cooling, the EP tube was inverted to obtain an image.

[0051] The results, as shown in Figure 1 , showed that the ammonium glycyrrhizinate / ATP- / doxorubicin hydrochloride hydrogel was uniformly formed into a gel at least at 60 °C, and no stable gel was formed at 40 and 50 °C. When ATP- was replaced by puerarin, the ammonium glycyrrhizinate / puerarin / doxorubicin hydrochloride hydrogel could also be prepared at 60 °C, indicating that the preparation method requires 60 °C to form a uniform gel, and other gel materials can be replaced, and the applicability is relatively wide.

[0052] 1.2 Injectable property of AG-DOX hydrogel To evaluate the injectability of the AG-DOX hydrogel, the prepared hydrogel sample was loaded into a 1 mL syringe (needle size 22 G), and the AG-DOX hydrogel was injected onto a whiteboard, and the shape of the AG-DOX hydrogel after injection was observed with the naked eye, and any shape was drawn to indicate the injectability of the AG-DOX hydrogel.

[0053] According to the method described in step 1.1, after ammonium glycyrrhizinate was dissolved in water at 60 °C, an equal volume of 2x PBS solution with different ATP- contents was added, and as the temperature decreased, a stable hydrogel was formed (A in Figure 2 ). The hydrogel was sucked into a 1 mL syringe, and the hydrogel could be smoothly pushed out, and any shape could be drawn at will, indicating that the hydrogel had injectability (B in Figure 2 ).

[0054] Example 2, in vitro pharmacodynamics of ammonium glycyrrhizinate injectable hydrogel co-loaded with doxorubicin hydrochloride and ATP- (AG-DOX).

[0055] 2.1 Culture of 4T1 cells Preparation of 4T1 cell culture medium: 10% fetal bovine serum and 1% penicillin-streptomycin solution were added to RPMI 1640 medium. All cells were cultured in a 37 °C, 5% incubator.

[0056] Preparation of HL-1 cell culture medium: Add 10% fetal bovine serum and 1% penicillin-streptomycin solution to DMEM / F-12 culture medium. All cells were cultured at 37°C, 5% Culture in an incubator.

[0057] 2.2 Hydrogel in vitro immune activation experiment Extraction and induction of primary dendritic cells from mice: Before the experiment, prepare 6-8 week-old Balb / c mice, ophthalmic forceps, ophthalmic scissors, a beaker, a 200-mesh nylon screen, a 1 mL syringe, RPMI 1640 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin), red blood cell lysis buffer, GM-CSF stimulator, 75% ethanol, sterile 1× PBS, and sterile water. Ensure the sterility of experimental equipment. Use the UV sterilizer in a biosafety cabinet for 30 minutes to ensure a sterile environment. Pre-cool the centrifuge at 4°C.

[0058] Balb / c mice were euthanized and sterilized by soaking in 75% ethanol for 10 minutes. The hind legs were cut open with sterile surgical scissors, and the tibia, fibula, and femur were separated. These bones were placed in a culture dish containing 75% ethanol (taking care not to cut the bones) and soaked for 2-3 minutes. The bones were then placed in a culture dish containing sterile 1× PBS. Excess tissue was carefully removed using scissors and forceps. The femur and tibia were removed and soaked in 75% ethanol for 1 minute. The bones were then placed in a dish containing 1× PBS. Subsequently, the femur or tibia was separated by grasping the middle of the bone with forceps and cutting the ends. Sterile 1× PBS was aspirated using a 1 mL syringe and vigorously flushed, starting at the thicker end, until the bone contents were completely flushed out and the bone appeared white. The cells were then gently pipetted into a single-cell suspension using a 1 mL pipette and filtered through a 200-mesh nylon mesh into a 15 mL centrifuge tube. The suspension was centrifuged at 500 g for 3 minutes at 4°C. The supernatant was discarded, and 5 mL of red blood cell lysis buffer (1×) was added to gently resuspend the cells. After standing for 5 minutes, 5 mL of sterile 1× PBS was added to terminate the lysis, and the cells were centrifuged at 4°C, 500 g for 3 minutes, and the supernatant was discarded. Finally, the cells were resuspended in 1640 medium (10% FBS + 1% P / S + GM-CSF with a final concentration of 20 ng / mL), transferred to a cell culture dish with a diameter of 10 cm, and placed at 37°C, 5% Culture in a cell culture incubator.

[0059] On the second and fourth days of culture, perform a half-medium change with 1640 medium (10% FBS + 1% P / S + GM-CSF with a final concentration of 20 ng / mL). Continue culture until the sixth day, at which time obvious cell colonies, namely bone marrow-derived dendritic cells (BMDCs), can be observed. Collect the cells and count them at 5× / well density 0.5 mL per well was inoculated into a 12-well plate, and GAA, ATP- and AG induction medium, in which GAA and ATP- The amounts of LPS were 60 μg / mL and 100 μg / mL respectively, and 1 μg / mL LPS was added to the positive group, 0.5 mL per well, and the cells were gently mixed and incubated in the incubator for 48 h. All cells were scraped off with a cell scraper, and the cells were collected by centrifugation (2000 rpm, 3 min), washed three times with 1× PBS, and CD86-PE, MHCⅡ-PerCP / Cy5.5, and CD11c-EV450 were prepared with antibody diluent, 100 μL was added to each tube, incubated at 4°C for 30 min, washed three times with 1× PBS, and detected by flow cytometry.

[0060] Bone marrow-derived dendritic cells (BMDCs) are important immune cells that can capture, process, and present antigens to T cells. Immature BMDCs are highly migratory, able to migrate from peripheral tissues to lymph nodes, transmit antigen information to T cells, and initiate immune responses against abnormal cells (such as cancer cells) in the body. Mature BMDCs are highly efficient in antigen presentation, activating and stimulating T cell differentiation, initiating and amplifying immune responses. Overexpression of CD86 co-stimulatory molecules and MHCII antigen-presenting molecules on the surface of mature BMDCs can be detected and analyzed by flow cytometry.

[0061] The experimental results are as follows Figure 3 As shown in A and B, compared with PBS, GAA, ATP- Both ATP and AG can stimulate the maturation of DC cells; compared with simple ATP- Compared with the GAA group, the AG group could significantly increase the proportion of CD86 and MHCII double-positive cells, indicating that GAA and ATP- The combination of the two can effectively promote the maturation of BMDCs, enhance the antigen presentation ability and immune activation ability of BMDCs. AG is a potential new immune adjuvant.

[0062] 2.3 Uptake of AG-DOX hydrogel by 4T1 cells AG-DOX hydrogel was prepared based on the method of 1.1 of Example 1. Two experimental groups were set up. Experimental group 1 was the same as Example 1, and ATP- The mass ratio of ATP-DOX to GAA was 5:3, and the prepared AG-DOX hydrogel was recorded as AG5-DOX. Experimental group 2 was similar to Example 1 except that The amount of ATP added is different. The mass ratio of AG to GAA was 3:3, and the prepared AG-DOX hydrogel was recorded as AG3-DOX.

[0063] Since doxorubicin hydrochloride has autofluorescence, it usually emits fluorescence at a wavelength of about 590 nm when the excitation wavelength is 480 nm. The uptake of doxorubicin hydrochloride by 4T1 cells can be sensitively detected using flow cytometry. 4T1 cells in the logarithmic growth phase were digested and collected, and 2× The cells were seeded at a density of 100 μg / well in a 6-well plate and cultured in a cell culture incubator overnight. The supernatant was discarded and DOX, AG3-DOX, and AG5-DOX were added to the cells in the 12-well plate at a concentration of 2 μg / mL. The cells were incubated for 6 h, and then trypsinized and centrifuged at 2000 rpm for 3 min at 4°C. The supernatant was discarded and the cells were washed three times with 1× PBS before being analyzed by flow cytometry.

[0064] See the results Figure 4 There was no significant difference in the uptake of AG3-DOX and AG5-DOX hydrogels by 4T1 cells compared with DOX. Loading DOX into AG gel failed to affect the ability of 4T1 cells to uptake DOX.

[0065] AG-DOX hydrogel inhibits 4T1 cell proliferation The toxicity of drugs to 4T1 cells was evaluated by MTT assay. 4T1 cells in the logarithmic growth phase were trypsinized for 3 min. The cell number was 5×10 4 / mL were seeded in 96-well plates, each well contained 100 μL of cell suspension, and cultured in a cell culture incubator. After overnight adherence, the cells were grouped as follows: DOX, GAA, ATP- (abbreviated as ATP), AG, AG3-DOX and AG5-DOX, the maximum administration concentrations were: DOX (4 μg / mL), GAA (60 μg / mL), ATP- (100 μg / mL), which was then diluted in half and administered to cells in a series of drug-containing media at different concentrations. After 48 hours of co-culture, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated for another 4 hours. After incubation, the medium was aspirated and 100 μL of DMSO was added to each well. The cells were shaken in the dark at room temperature for 15 minutes to completely dissolve the formazan precipitate in DMSO. The OD value was measured at 490 nm using a microplate reader. Cell viability was calculated as follows: ; in, is the absorbance value of the drug group, is the absorbance value of the normal group, is the absorbance of the solvent.

[0066] HL-1 cells are a cardiomyocyte cell line derived from mouse hearts and are used to evaluate the effects of drugs on cardiac function. This experiment used the MTT assay to evaluate the toxicity of HL-1 cells, which reflects the safety of AG-DOX hydrogel. HL-1 cells in the logarithmic growth phase were digested with trypsin for 1 min. The cell number was 2× / mL were seeded in 96-well plates, each well contained 100 μL of cell suspension, and cultured in a cell culture incubator. After overnight adherence, the cells were grouped as follows: DOX, GAA, ATP- (abbreviated as ATP), AG, AG3-DOX and AG5-DOX, the maximum administration concentrations were: DOX (2 μg / mL), GAA (30 μg / mL), ATP- The cells were then treated with a series of drug-containing media (50 μg / mL) and diluted in half. After 24 h of co-culture, the MTT assay was performed and cell viability was calculated according to the above steps.

[0067] The results are as follows Figure 5 As shown in A and B, the DOX group, AG3-DOX group and AG5-DOX group can effectively inhibit the rapid proliferation of 4T1 cells, and the AG5-DOX The value is lower, suggesting that GAA and ATP are co-loaded. It can significantly enhance the inhibitory effect of DOX on 4T1 cell activity.

[0068] The results are as follows Figure 6 As shown in A~C, GAA, ATP- The toxicity of AG5-DOX hydrogel to mouse myocardial HL-1 cells (normal cells) was significantly weaker than that to 4T1 cells (tumor cells), and the myocardial cytotoxicity of AG5-DOX was significantly lower than that of the DOX group, suggesting that the co-loading of GAA and ATP- It can significantly enhance the inhibitory effect of DOX on 4T1 cell activity while reducing the cardiotoxicity caused by DOX.

[0069] 2.5 AG-DOX hydrogel induces apoptosis of 4T1 cells.

[0070] Flow cytometry was used to detect the effect of drugs on cell apoptosis. Preparation of DAPI (apoptosis) stock solution: accurately weigh 5 mg / mL of DAPI powder, add 1 mL of PBS to dissolve, dispense 100 μL into each tube, and store in a -20°C refrigerator away from light for later use. Preparation of apoptosis detection reagent (flow cytometry): first dilute the 10× Annexin V binding solution to the working concentration with ultrapure water, dilute the Annexin V solution and DAPI (apoptosis) stock solution with Annexin V binding solution at a ratio of 1:1000, and mix equal volumes for later use. The above reagents should be prepared before use. Take 4T1 cells in the logarithmic growth phase, digest them with trypsin, and then incubate them at 3× The cells were seeded into 6-well plates at a density of 100 μg / well and placed in a cell culture incubator for overnight culture. (abbreviated as ATP), AG, AG-DOX (Example 1), the dosage concentrations were: DOX (4 μg / mL), GAA (60 μg / mL), ATP- (100 μg / mL), the drug content of AG and AG-DOX was consistent with that of free drug. After incubation for 48 h, the floating cells in the supernatant were collected by centrifugation, the cells in the wells were trypsinized, and the cells were centrifuged at 4°C and 2800 rpm for 3 min. The supernatant was discarded and washed three times with 1×PBS. The supernatant and cells in the well plate were combined, and 300 μL Annexin V-DAPI working solution (1:200 Annexin V, 1:1000 DAPI, 1× Binding buffer) was added to each tube for resuspending and incubated at 4°C for 30 min.

[0071] The results are as follows Figure 7 As shown in Figures A to B, early and late apoptosis occurred in the different drug treatment groups. Compared with the free DOX group, the proportions of early, late and total apoptotic cells in the AG-DOX hydrogel group were significantly increased.

[0072] AG-DOX hydrogel inhibits mitochondrial activity in 4T1 cells Changes in mitochondrial membrane potential are often related to the health status, apoptosis and pathological processes of cells. It is a sign of early apoptosis and can therefore reflect the health status of cells. In this experiment, mitochondrial membrane potential (MMP, ΔΨm) was used to evaluate mitochondrial function after AG-DOX hydrogel acted on cells. The JC-10 fluorescent probe is membrane potential-dependent. When the mitochondrial membrane potential is normal or high, JC-10 will accumulate in the inner membrane and form aggregates (called "J-aggregates"), showing strong red fluorescence (about 590 nm); conversely, when the mitochondrial membrane potential decreases, JC-10 will exist in a monomeric form and emit green fluorescence (about 525 nm), which is a manifestation of mitochondrial dysfunction in the cell. The fluorescence intensity was detected by flow cytometry. 4T1 cells in the logarithmic growth phase were taken, digested with trypsin, and then incubated at 3× The cells were seeded at a density of 100 μg / mL / well in a 6-well plate and placed in a cell culture incubator for overnight culture. Then, DOX, GAA, ATP, AG, and AG-DOX were added to the cells in the 6-well plate at the following concentrations: DOX (2 μg / mL), GAA (30 μg / mL), ATP- (50 μg / mL), the drug content of AG and AG-DOX (Example 1) was consistent with the free drug. After incubation for 48 h, the floating cells in the supernatant were collected by centrifugation, the cells in the wells were trypsinized, and the cells were centrifuged at 4°C and 2000 rpm for 3 min. The supernatant was discarded, and the cells were washed three times with 1×PBS. The supernatant and the cells in the well plate were combined, and 300 μL of JC-10 working solution (1:200 diluted with 1640 complete culture medium) was added to each tube. The cells were incubated at 37°C for 30 min, washed three times with 1×PBS, and then detected by flow cytometry.

[0073] The experimental results are as follows Figure 8 As shown in A~B, the transition from red fluorescence to green fluorescence was observed in all drug treatment groups, indicating that the drug can cause functional damage to mitochondria in 4T1 cells. Among them, the positive rate of cell MMP dissipation in the AG-DOX hydrogel group was significantly higher than that in other groups.

[0074] Example 3, Co-loading of doxorubicin hydrochloride and ATP- In vivo pharmacodynamics of glycyrrhizic acid monoammonium salt injectable hydrogel (AG-DOX).

[0075] 3.1 Experimental cells and animals Experimental cells: Mouse triple-negative breast cancer epithelial 4T1 cells were selected for animal tumor bearing experiments. 4T1 cells were purchased from Pronose and cultured in complete RPMI Medium 1640 basal medium (1×) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). The culture conditions were: normoxic environment (5% , 37℃).

[0076] Balb / c female mice weighing 18 ± 2 g were purchased from the Guangdong Provincial Laboratory Animal Center. They were housed in an environmental temperature of 22 ± 2°C, a relative humidity of 50 ± 5%, and a 12-h light / dark cycle. Sterile feed, water, and bedding were provided and replaced promptly. The animal experiment protocol was approved by the Laboratory Animal Ethics Committee of Guangzhou University of Chinese Medicine and was conducted in accordance with the guidelines for the care and use of laboratory animals of Guangzhou University of Chinese Medicine.

[0077] 3.2 Experimental methods 3.2.1 Preparation of Balb / c mouse transplant tumor model Take 4T1 cells in the logarithmic growth phase, digest the cells with trypsin, centrifuge at 4°C, 2800 rpm for 3 min, discard the supernatant, resuspend in PBS, centrifuge again, collect the cells, resuspend in PBS, and adjust the cell concentration to 1× Under sterile conditions, 0.1 mL of the cell suspension was inoculated subcutaneously in the first pair of mammary fat pads of the mouse abdomen. After tumor cell implantation, the long and short diameters of the transplanted tumors were measured daily using a vernier caliper. The volume of the transplanted tumors was calculated using the following formula: ; Among them, V ( ) is the tumor volume, where L (mm) represents the long diameter and W (mm) represents the short diameter. Pharmacological experiments can be carried out.

[0078] The experimental timeline is as follows Figure 9 As shown in A. Figure 9 As shown in Figures B and C, after the model group animals were transplanted with 4T1 cells, their tumor volume increased over time. DOX and AG-DOX (Example 1) were able to effectively inhibit the growth of solid tumors in vivo. The free DOX group was able to effectively reduce the volume of 4T1 tumors, while the AG-DOX hydrogel group showed statistically significant differences from the free DOX group. Furthermore, on the 14th day of treatment, the tumor weight was recorded, and it was found that the tumor weight of the AG-DOX group was significantly smaller than that of the other groups ( Figure 9 (D) shows that AG-DOX hydrogel can significantly inhibit the growth of primary triple-negative breast cancer.

[0079] 3.2.2 In vivo safety evaluation of AG-DOX hydrogel Tumor-bearing mice were randomly divided into the following 6 groups: DOX, GAA, ATP- (abbreviated as ATP), AG, AG-DOX (Example 1), Model. The dosage was DOX (80 μg / kg), GAA (1.2 mg / kg), ATP- (2 mg / kg), the drug content of AG and AG-DOX was consistent with that of free drug, and the drug was injected into the tumor once every two days for a total of 7 times. The tumor volume and mouse body weight were monitored and recorded at the same time. At the end of the experiment, the mice were euthanized and the tumors and major organs, including the heart, liver, spleen, lungs, and kidneys, were removed. The tumors of the mice were weighed, photographed, and the data recorded. The weights of the major organs of the mice were weighed and recorded, and the organ index was calculated. The organ index can be calculated according to the following formula: Organ index = (organ weight / body weight) × 100%.

[0080] The weight changes of mice in each group were recorded during the treatment period. Figure 10 As shown in Figure A, there was no significant difference in the body weight of the mice in each group. Changes in organ weight usually occur earlier than changes in organ morphology and are one of the commonly used indicators for measuring drug toxicity. Figure 10 As shown in Figure B, compared with the model group, there was no significant difference in the weight changes of the heart, lungs, and kidneys in each group after drug treatment; however, there were significant differences in the organ coefficients of the spleen in each group, especially the changes in the spleen, suggesting that there may also be significant changes in the immune level in the body. The spleen is an important immune organ. Figure 10 The results showed that AG-DOX hydrogel treatment successfully reduced the tumor burden in mice, possibly improved the systemic immune status, and led to a reduction in spleen volume; compared with the normal group, there was no significant difference in the coefficients of other organs in the AG-DOX hydrogel group, indicating that AG-DOX hydrogel has good biocompatibility.

[0081] 3.2.3 AG-DOX in the treatment of recurrent breast cancer The steps for tumor inoculation are the same as those in 3.2.1. After tumor cell implantation, the long and short diameters of the transplanted tumors were measured daily using a vernier caliper. The volume of the transplanted tumors was calculated using the following formula: V= ; Among them, V ( ) is the tumor volume, where L (mm) represents the long diameter and W (mm) represents the short diameter. The mice were anesthetized for about 24 hours, and the tumors were partially removed. The remaining tumors were sutured with sterile needles and thread to simulate clinical tumor resection surgery. Appropriate analgesia and warming treatment were given after surgery. The mice were randomly divided into three groups: PBS, DOX, and AG-DOX (Example 1). After surgery, PBS, DOX, and AG-DOX (Example 1) were injected into each mouse, respectively, with a 50 μL injection. The dosage of DOX (5 mg / kg), GAA (75 mg / kg), ATP- (125 mg / kg), administered every 7 days for a total of 3 times. During the experiment, mice may experience tumor growth, weight loss, loss of ability to drink or eat, or even death. Regularly monitor the weight of mice and tumor recurrence, measure the size of recurrent tumors, and when the volume of recurrent tumors exceeds 1000 The experiment was terminated at 4 hr and the mice were euthanized by cervical dislocation under deep anesthesia.

[0082] In order to further verify the effect of AG-DOX hydrogel on the postoperative recurrence of triple-negative breast cancer, this example established a mouse model of postoperative recurrence of triple-negative breast cancer. The experimental timeline is as follows: Figure 11 As shown in Figure A. At the end of the experiment, the recurrent tumors of mice in each treatment group were collected. It can be seen intuitively that compared with the PBS group, the recurrent tumor volumes of the free DOX group and the AG-DOX (Example 1) group were reduced, while the tumor volume of the AG-DOX group was significantly different from that of the free DOX group, and two mice did not develop recurrent tumors. In addition, mice died in the PBS group, while no mice died in the DOX group or the AG-DOX hydrogel group ( Figure 11 (B) shows that AG-DOX hydrogel can effectively prevent and inhibit the growth of postoperative recurrent tumors. During the treatment process, the weight changes of mice in each treatment group were regularly monitored, such as Figure 11 As shown in Figure C, the weight of mice in each group was at a normal level, indicating that the mice were in good growth condition. At the same time, the changes in tumor volume of mice in each drug-treated group during the treatment process were also recorded, as shown in Figure 4. Figure 11 As shown in Figure D, compared with the PBS group and the free DOX group, the AG-DOX hydrogel group did not develop recurrent tumors until 10 days after surgery, and the volume of recurrent tumors in the AG-DOX hydrogel group was significantly reduced, indicating that AG-DOX hydrogel can effectively inhibit the postoperative recurrence of triple-negative breast cancer. In addition, the collected postoperative recurrent tumors of each drug group were weighed, and the results are shown in Figure 3. Figure 11 Middle E shows that the weight of recurrent tumors in the AG-DOX hydrogel group was significantly lower than that in the PBS group and the free DOX group, indicating that AG-DOX hydrogel can inhibit the growth of recurrent tumors after surgery for triple-negative breast cancer.

[0083] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A composition, characterized in that It includes: Hydrogel-forming material, ATP or ATP salt and anthracycline antibiotic derivatives.

2. The composition according to claim 1, characterized in that The composition comprises, by weight, 10 to 50 parts of a hydrogel-forming material, 10 to 80 parts of ATP or ATP salt, and 0.5 to 5 parts of an anthracycline antibiotic derivative; Optionally, the composition comprises, by weight: 10 to 50 parts of a hydrogel-forming material, 10 to 80 parts of ATP or ATP salt, and 0.5 to 5 parts of anthracycline antibiotic derivatives.

3. The composition according to claim 2, characterized in that The gel-forming material of the hydrogel includes any one or more of glycyrrhizic acid or glycyrrhizic acid derivatives, glycyrrhizic acid, puerarin, rhein and ursoleic acid; Optionally, the glycyrrhizic acid derivative includes one of ammonium glycyrrhizate, ammonium glycyrrhizate salt, monopotassium glycyrrhizate, dipotassium glycyrrhizate and trisodium glycyrrhizate; Optionally, the ammonium glycyrrhizate salt includes monoammonium glycyrrhizate.

4. The composition according to claim 2, characterized in that The ATP salt includes any one or more of ATP sodium salt, ATP magnesium salt, and ATP potassium salt; Optionally, the ATP sodium salt comprises adenosine-5'-triphosphate disodium salt.

5. The composition according to claim 2, characterized in that The anthracycline antibiotic derivatives include doxorubicin or a water-soluble salt of doxorubicin; Optionally, the water-soluble salt of doxorubicin includes doxorubicin hydrochloride.

6. The composition according to any one of claims 1 to 5, characterized in that The composition further comprises: any one or more of water and buffer; Optionally, the buffer comprises PBS or PB.

7. A hydrogel, characterized in that The raw materials include the composition according to any one of claims 1 to 6.

8. The method for preparing the hydrogel according to claim 7, wherein: The preparation method comprises: mixing a hydrogel-forming material, ATP or ATP salt and an anthracycline antibiotic derivative; Optionally, the mixing step comprises: dissolving the anthracycline antibiotic derivative in water to obtain a drug-containing solution; adding the hydrogel-forming material to the drug-containing solution to obtain a mixed solution; adding the ATP or ATP salt to the mixed solution to obtain a hydrogel; Optionally, in the hydrogel, the effective concentration of the hydrogel-forming material is 10-50 mg / mL, the effective concentration of the ATP or ATP salt is 10-80 mg / mL, and the effective concentration of the anthracycline antibiotic derivative is 0.5-5 mg / mL; Optionally, the mixing further comprises: first dissolving ATP or ATP salt in the buffer solution according to claim 6, and then adding the ATP or ATP salt into the mixed solution.

9. The preparation method according to claim 7 or 8, characterized in that The mixing temperature includes 50~70℃; Optionally, the preparation method further comprises: cooling the product after adding ATP or ATP salt to the mixed solution to obtain the hydrogel.

10. Use of the composition according to any one of claims 1 to 6 or the hydrogel prepared by the preparation method according to any one of claims 7 to 9 in the preparation of a drug; Optionally, the drug includes an anti-tumor drug; Optionally, the anti-tumor drug has the effect of treating tumors and / or inhibiting tumor recurrence; Optionally, the tumor includes an indication for doxorubicin; Optionally, the tumor includes any one of hematological malignancies, solid tumors and other tumors; the hematological malignancies include leukemia, lymphoma and multiple myeloma; the solid tumors include breast cancer, soft tissue sarcoma, osteosarcoma, ovarian cancer, small cell lung cancer and gastric cancer; the other tumors include neuroblastoma, bladder cancer and liver cancer; Optionally, the tumor comprises triple-negative breast cancer.

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