A multifunctional hydrogel and preparation method thereof

Abstract

The present invention belongs to the field of biomedical materials, and specifically relates to a multifunctional hydrogel and a preparation method and application thereof, and more specifically, to the preparation and application of an anti-tumor injectable hydrogel and a bone-supported hydrogel system. A multifunctional hydrogel of the present invention is mainly composed of PVA and rhein, according to the mass ratio of PVA: rhein = 0.2~2 g: 10~100 mg, preferably 0.48~1.5 g: 36~72 mg, and more preferably 0.64~1.2g: 36~72 mg. The anti-tumor hydrogel constructed by the present invention is injectable and can inhibit the growth of tumor cells through a single administration, and the constructed bone-supported hydrogel has a strong supporting strength.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and in particular relates to a multifunctional hydrogel and a preparation method thereof. Background Art

[0002] Osteosarcoma is the most common primary malignant bone tumor in adolescents. It is highly malignant, progresses rapidly, and is prone to distal metastasis. Its common feature is that tumor cells form immature bone or bone-like tissue, accounting for 20-40% of all bone tumors. The global incidence of osteosarcoma is about 3 / 1 million, with two peaks. The first peak occurs during adolescence when bones grow rapidly, with an incidence of about 4.4 / 1 million, and the second peak occurs in the elderly over 65 years old, with an incidence of about 4.2 / 1 million. Osteosarcoma most often occurs in the epiphysis of long bones near the growth plate. Two-thirds of tumors appear around the distal femoral knee joint, followed by the proximal tibia. The proximal humerus is the third most common tumor site, accounting for 10% of the total number of tumors. Osteosarcoma patients usually show local swelling or pain at the site of onset, which brings great pain and inconvenience to patients, especially tumors near the knee joint. Swelling and pain make patients unable to walk normally. The current dosing regimen for treating osteosarcoma will bring systemic toxicity due to the intravenous administration method, which is not conducive to the treatment of patients.

[0003] As a biomaterial that can be injected in situ, hydrogel has excellent biocompatibility. It can not only achieve continuous drug delivery at the tumor site, but also because its structure is similar to the extracellular matrix, it can provide a suitable growth environment for cell growth and meet the need to eliminate tumors during the treatment of osteosarcoma. Therefore, hydrogel systems have received increasing attention in the treatment of osteosarcoma. Considering biocompatibility, polyvinyl alcohol (PVA) is a suitable material for preparing hydrogels. The physical cross-linking method does not add chemical cross-linking agents and is safer, but its preparation process is time-consuming and needs to be improved. As a traditional Chinese medicine ingredient, rhein (RH) has good anti-inflammatory and anti-tumor functions. Its self-assembly can form hydrogels, but its stability is poor.

[0004] In order to cope with the current treatment bottleneck of osteosarcoma, it is necessary to design a shear-thinning multifunctional hydrogel system that can slowly release drugs, effectively kill tumor cells, is harmless to normal tissues, and is independent of gelation time. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the first purpose of the present invention is to provide a multifunctional hydrogel, which is formed by compounding PVA and rhein in a mass ratio to form a network system of PVA and rhein, thereby overcoming the shortcomings of existing preparations for treating osteosarcoma. The multifunctional hydrogel system has simple preparation process, injectability, single injection sustained release, and excellent drug loading properties. Before surgery, the drug-loaded hydrogel can be injected in situ with a single injection, so that the drug can be effectively accumulated near the tumor and slowly released over a period of up to two weeks, thereby effectively inhibiting tumor growth and reducing tumor volume.

[0006] The second object of the present invention is to provide a method for preparing a multifunctional hydrogel. The present invention improves the mechanical strength of the hydrogel and has a wide drug loading range. Different drugs loaded can have different therapeutic effects. Therefore, the multifunctional hydrogel system prepared by this method can effectively reduce the size of tumors and reduce the harm to patients, which has significant significance for the treatment of patients.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention protects a multifunctional hydrogel mainly composed of PVA and rhein (RH), with a mass ratio of PVA: rhein = 0.2-2g:10-100mg, preferably 0.48-1.5g:36-72mg, and more preferably 0.64-1.2g:36-72mg.

[0009] The PVA / RH hydrogel formed by cross-linking PVA and rhein in the present invention has a three-dimensional network structure, can efficiently load drug molecules and control the release rate of drug molecules, thereby achieving the effect of inhibiting tumor recurrence over a long period of time.

[0010] As a preferred technical solution of the present application, chemotherapy drugs and / or materials promoting bone repair are also added to the hydrogel;

[0011] When only chemotherapy drugs are added to the hydrogel, the mass ratio of PVA: rhein: chemotherapy drugs = 0.2-2 g: 10-100 mg: 5-35 mg, preferably 0.64-1.2 g: 36-72 mg: 10-25 mg;

[0012] When only the material promoting bone support is added to the hydrogel, the mass ratio of PVA: rhein: material promoting bone support = 0.2-2g: 10-100mg: 2-20mg; preferably 0.64-1.2g: 36-72mg: 5-15mg.

[0013] When chemotherapeutic drugs and materials promoting bone support are added to the hydrogel, the mass ratio of PVA: rhein: chemotherapeutic drugs: materials promoting bone support = 0.2-2g: 10-100mg: 5-35mg: 2-20mg; preferably 0.64-1.2g: 36-72mg: 10-25mg: 5-15mg.

[0014] The present invention protects an injectable hydrogel for treating osteosarcoma, comprising: PVA and rhein hydrogel and chemotherapy drugs; wherein the mass ratio of PVA: rhein: chemotherapy drugs = 0.2-2g: 10-100mg: 5-35mg, preferably 0.64-1.2g: 36-72mg: 10-25mg.

[0015] The present invention protects a bone-supporting hydrogel for treating osteosarcoma and promoting bone repair, comprising: PVA and rhein hydrogel, chemotherapy drugs, and materials that promote bone support; wherein, the mass ratio of PVA: rhein: chemotherapy drugs: materials that promote bone support = 0.2-2g: 10-100mg: 5-35mg: 2-20mg; preferably 0.64-1.2g: 36-72mg: 10-25mg: 5-15mg.

[0016] As a preferred technical solution of the present application, the chemotherapy drug is any one or more of doxorubicin hydrochloride, adriamycin, and albendazole, preferably doxorubicin hydrochloride.

[0017] As a preferred technical solution of the present application, the material promoting bone support is nano-hydroxyapatite.

[0018] As a preferred technical solution of the present application, the molecular weight of the PVA is: 89000-98000, such as 89000, 90000, 91000, 92000, 93000, 94000, 95000, 96000, 97000, 98000 and the like.

[0019] Optionally, the surface and / or interior of the hydrogel material is loaded with drugs for treating osteosarcoma.

[0020] In a second aspect, the present invention also protects a method for preparing the aforementioned multifunctional hydrogel, comprising the following steps:

[0021] Step 1, preparing a PVA solution: dispersing PVA in water, stirring under heating conditions to completely dissolve it and form a uniform solution; wherein the mass concentration of PVA in the PVA aqueous solution is 4-20%, preferably 8-15%, and can be 8%, 10%, 12%, 15%, etc.;

[0022] Step 2, preparing a rhein solution; dissolving rhein in an alkaline solution to make it completely dissolved, and then using hydrochloric acid to adjust the pH to 8.0-9.4, preferably 8.8-9.0; the concentration of the hydrochloric acid is: 1mmol / L-5mmol / L;

[0023] Step 3, adding the rhein solution of step (2) to the PVA solution of step (1), stirring evenly and then sonicating;

[0024] Step 4: After freezing at -80°C for 1 hour, take out the sample and thaw in a 37°C water bath for 30 minutes. This is one freeze-thaw cycle.

[0025] The present invention combines PVA with rhein, and the two substances interpenetrate each other when forming a gel network to form an interpenetrating network. A hydrogel can be formed by one freeze-thaw cycle, which not only shortens the preparation time but also enables the hydrogel to have good mechanical properties.

[0026] The present invention also protects a method for preparing the aforementioned injectable hydrogel for treating osteosarcoma, comprising the following steps:

[0027] Step 1, preparing a PVA solution: dispersing PVA in water, stirring under heating conditions to completely dissolve it and form a uniform solution; wherein the mass concentration of PVA in the PVA aqueous solution is 4-20%, preferably 8-15%, and can be 8%, 10%, 12%, 15%, etc.;

[0028] Step 2, preparing a rhein solution; dissolving rhein in an alkaline solution to make it completely dissolved, and then using hydrochloric acid to adjust the pH to 8.0-9.4, preferably 8.8-9.0; the concentration of the hydrochloric acid is: 1mmol / L-5mmol / L;

[0029] Step 3, preparing a doxorubicin hydrochloride solution: dispersing doxorubicin hydrochloride in the PVA aqueous solution described in step 1, and stirring or ultrasonicating to dissolve it; wherein the mass concentration of doxorubicin hydrochloride is 0.01-3 mg / mL;

[0030] Step 4, adding the rhein solution described in step 2 to the doxorubicin hydrochloride solution described in step 3, the process is accompanied by stirring, and after stirring evenly, ultrasonicating for 30 to 60 minutes;

[0031] Step 5, after freezing in a -20°C to -80°C refrigerator for 1 hour, take out and thaw in a 37°C water bath for 30 minutes, which is a freeze-thaw cycle; wherein, this freeze-thaw cycle ranges from 1 to 8 times, preferably 1 to 7 times, and more preferably 4 to 6 times.

[0032] The present invention also protects a method for preparing a bone-supporting hydrogel for treating osteosarcoma and promoting bone repair, comprising the following steps:

[0033] Step 1, preparing a PVA solution: dispersing PVA in water, stirring under heating conditions to completely dissolve it and form a uniform solution; wherein the mass concentration of PVA in the PVA aqueous solution is 4-20%, preferably 8-15%, and can be 8%, 10%, 12%, 15%, etc.;

[0034] Step 2, preparing a nano-hydroxyapatite solution: taking nano-hydroxyapatite, dissolving it with hydrochloric acid, the concentration of hydrochloric acid is: 1mmol / L-5mmol / L, and the mass concentration of nano-hydroxyapatite is: 0.1-167mg / mL;

[0035] Step 3, preparing a rhein solution; dissolving rhein in an alkaline solution to make it completely dissolved, and then using hydrochloric acid to adjust the pH to 8.0-9.4, preferably 8.8-9.0; wherein the hydrochloric acid is the hydrochloric acid solution obtained after dissolving the nano-hydroxyapatite in step 2;

[0036] Step 4, preparing a doxorubicin hydrochloride solution: dispersing doxorubicin hydrochloride in the PVA aqueous solution described in step 1, and stirring or ultrasonicating to dissolve it; wherein the mass concentration of doxorubicin hydrochloride is 0.01-3 mg / mL;

[0037] Step 5, preparing a synergistic osteosarcoma treatment and bone support hydrogel: adding the rhein solution to the doxorubicin hydrochloride solution, the process is accompanied by stirring, and after stirring evenly, ultrasonicating for 30 to 60 minutes;

[0038] Step 6, after freezing in a -20 to -80°C refrigerator for 1 hour, take out and thaw in a 37°C water bath for 30 minutes, which is a freeze-thaw cycle; wherein, the freeze-thaw cycle is from 1 to 8 times, preferably 1 to 7 times, and more preferably 4 to 6 times.

[0039] As a preferred technical solution of the present application, in step 1, the heating temperature is: 80-100°C, preferably 95-100°C, and more preferably 97°C; the heating and stirring time is: 20min-1h.

[0040] As a preferred technical solution of the present application, in the preparation of the rhein solution, the mass concentration of rhein is 4-25 mg / mL, preferably 4-10 mg / mL, and the alkali in the alkaline solution is any one of sodium hydroxide, sodium carbonate or sodium bicarbonate.

[0041] As a preferred technical solution of the present application, the number of freeze-thaw cycles mentioned above ranges from 1 to 8 times, preferably 1 to 6 times, and can be 1, 2, 3, 4, 5, or 6 times.

[0042] The present invention also protects the hydrogel prepared by the preparation method described above.

[0043] The present invention also protects the use of the hydrogel described above in preparing drugs for treating tumors.

[0044] The present invention also protects the use of the hydrogel described above in preparing drugs for treating tumors and / or promoting bone repair.

[0045] As a preferred technical solution of the present application, the tumor is osteosarcoma.

[0046] Beneficial Effects

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) PVA hydrogel needs to undergo multiple freeze-thaw cycles to form a hydrogel by strengthening hydrogen bonds. However, the present invention combines PVA with rhein, and the two substances interpenetrate when forming a gel network to form an interpenetrating network. At the same time, the salting-out effect of rhein sodium salt reduces the spacing between PVA molecular chains and increases the formation of hydrogen bonds, so that a hydrogel can be formed after one freeze-thaw cycle, which not only shortens the preparation time, but also makes the hydrogel have good mechanical properties;

[0049] (2) The PVA / RH hydrogel formed by cross-linking PVA and rhein in the present invention has a three-dimensional network structure, which can efficiently load drug molecules and control the release rate of drug molecules, thereby achieving the effect of inhibiting tumor recurrence over a long period of time;

[0050] (3) The PVA / RH hydrogel of the present invention can load drugs, such as the chemotherapy drug doxorubicin hydrochloride and the bone repair drug nanohydroxyapatite. Doxorubicin hydrochloride can effectively kill tumor cells and inhibit tumor growth. At the same time, rhein also has the effect of inhibiting tumor growth and reducing the multidrug resistance of doxorubicin hydrochloride. The combination of the two can be synergistically applied to the treatment of osteosarcoma. Nanohydroxyapatite can treat the bone defect process caused by osteosarcoma. By increasing the number of freeze-thaw cycles, the mechanical properties of the hydrogel network can be enhanced, which can support the defect site and effectively prevent the occurrence of more serious bone defects such as fractures.

[0051] (4) The PVA / RH hydrogel of the present invention can be used to prepare hydrogels with different mechanical strengths by changing the number of freeze-thaw cycles, so that it can be applied to different treatment requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The effect of PVA mass fraction on hydrogel formation was verified.

[0053] Figure 2 To verify whether the addition of RH has an effect on the formation of hydrogel.

[0054] Figure 3 The effect of PVA mass fraction and RH on the formation of hydrogel.

[0055] Figure 4 To screen the preparation conditions of PVA / RH / DOX (PRD) hydrogel through orthogonal experiments.

[0056] Figure 5 This is the orthogonal experimental results table of PRD hydrogel system.

[0057] Figure 6 This is a SEM experimental result diagram of the PVA / RH / DOX hydrogel of the present invention.

[0058] Figure 7 This is a SEM experimental result diagram of the PVA / RH / DOX / nHA hydrogel scaffold material of the present invention.

[0059] Figure 8 Schematic diagram and injectability of PVA / RH / DOX hydrogel of the present invention.

[0060] Fig. 9 This is to verify the supporting capacity of the PVA / RH / DOX / nHA hydrogel scaffold material of the present invention.

[0061] Fig.10 The tensile and compressive properties of PVA / RH / DOX / nHA hydrogel scaffold materials were tested.

[0062] Fig.11 These are the results of the cytotoxicity experiment of PVA / RH / DOX hydrogel on osteosarcoma cells K7M2, where Figures A, B, C, and D are the cytotoxicity of PVA, PVA / DOX, PVA / RH, and PVA / RH / DOX after co-incubation with K7M2 cells for 24 hours and 48 hours, respectively.

[0063] Fig.12 This is the flow cytometry apoptosis result of PVA / RH / DOX hydrogel on osteosarcoma K7M2 cells.

[0064] Fig.13 This is a graph showing the pharmacodynamic results of PVA / RH / DOX hydrogel in osteosarcoma model mice.

[0065] Fig.14 This is a graph showing the changes in tumor volume of PVA / RH / DOX hydrogel in osteosarcoma model mice. DETAILED DESCRIPTION

[0066] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0067] The invention firstly provides a multifunctional hydrogel. The hydrogel is prepared by compounding PVA and rhein in a mass ratio of 2-20 g:10-100 mg to form a network system cross-linking PVA and rhein.

[0068] Optionally, the hydrogel can carry different drugs alone, or different combinations of two drugs, three drugs, etc. Optionally, the hydrogel can be loaded with: doxorubicin hydrochloride, nano-hydroxyapatite, doxorubicin hydrochloride and nano-hydroxyapatite, etc.

[0069] Polyvinyl alcohol (PVA) has been used in drug delivery devices, artificial organs, wound dressings, contact lenses, antibacterial, skin care systems, protein adsorption, protein controlled release and other fields due to its non-toxicity, good biocompatibility, biodegradability, high mechanical strength, etc., and has been approved by the US Food and Drug Administration for clinical applications. The PRD hydrogel formed by self-assembly and cross-linking with rhein has a three-dimensional network structure. The surface and / or interior of the three-dimensional network structure can be loaded with anti-tumor drugs, and the loaded drugs can be released continuously, solving the problem of rapid diffusion of small molecule drugs and alleviating the strong side effects of chemotherapy drugs. In addition, compared with hard scaffolds that need to be preformed, soft hydrogels have good plasticity and can be filled in any irregularly shaped frame. They can be injected through the needle of a 1ml syringe to achieve injectable performance.

[0070] Rhein (4.5-dihydroxy anthraquinone, RH for short) is a monoanthracene 1,8-dihydroxyanthraquinone derivative, and is an effective component isolated and purified from a variety of traditional Chinese medicines such as rhubarb, Polygonum multiflorum, and Polygonum cuspidatum. The structural formula of RH, RH contains two hydroxyl groups and one carboxyl group, has a strong polarity, and has electrochemical redox properties. It has been reported in China that using the Chinese medicine monomer rhein as a gel factor, in the presence of NaHCO 3 In the solution, non-covalent bonds such as II-II bonds, hydrogen bonds, ionic bonds, and hydrophobic interactions are utilized to form a rhein supramolecular hydrogel with a three-dimensional network structure through self-assembly. The gel morphology of the rhein hydrogel described here is not stable enough and cannot achieve the performance of drug loading. The concentration and close stacking of polymers in the freezing process of PVA prepare polymer chains for the strong aggregation and crystallization induced by subsequent salting out, and the sodium salt of citric acid can be separated by salting out, strongly inducing the aggregation and crystallization of PVA to form nanofibrils. Rhein can also form sodium salt at a suitable pH, which promotes the aggregation of PVA molecular chains in the form of rhein sodium salt and helps PVA to form a hydrogel.

[0071] Doxorubicin hydrochloride belongs to the class of antibiotics, and its molecular formula is C 27 H 29 NO 11·HCI, it is an orange-red loose block or powder, easily soluble in water, DMSO, tetrahydrofuran, alcohol, insoluble in acetone, chloroform, benzene, ether. It is an anti-tumor antibiotic that inhibits the synthesis of nucleic acid, the genetic material of cancer cells, and has a wide anti-tumor spectrum, killing a variety of tumor cells.

[0072] Hydroxyapatite (HA) is the main inorganic mineral component of vertebrate bones and teeth. It is composed of hexagonal columnar single crystals with a molecular formula of Ca 10 (PO 4 ) 6 (OH) 2 , slightly soluble in pure water, weakly alkaline (pH 7-9), easily soluble in acid but insoluble in alkali. Hydroxyapatite is chemically similar to natural bone, has certain toughness and mechanical strength, uniformly distributed pore structure, large specific surface area, and good ability to load drugs and bioactive factors. It is an ideal bone tissue engineering scaffold material, but it is difficult to transplant into irregular bone defect areas and has poor degradation performance. In order to overcome the limitations of hydrogel and hydroxyapatite, hydrogel and hydroxyapatite are mixed to form a stable scaffold structure, which is expected to obtain a bone tissue engineering scaffold that meets the requirements.

[0073] The present invention composites PVA hydrogel with rhein hydrogel, and PVA / RH hydrogel can load drugs. By loading drugs for different therapeutic purposes and changing the number of freeze-thaw cycles, a synergistic osteosarcoma treatment and bone support hydrogel material (PVA / RH / DOX, PVA / RH / DOX / nHA scaffold material) can be prepared. The material has a porous structure of a three-dimensional network, can be coated with drugs, simulates the extracellular matrix, is conducive to cell proliferation, migration and differentiation, and can be used as a relatively ideal tissue engineering scaffold material. At the same time, the present invention further evaluates the tumor killing ability of the PVA / RH / DOX hydrogel material, and the results show that the material can be used as an ideal osteosarcoma treatment material.

[0074] The present invention is further explained below in conjunction with examples. It should be understood that the following examples are only used to explain the present invention, rather than to limit the scope of protection of the present invention.

[0075] Example 1 Preparation of hydrogel PVA / RH

[0076] (1) Preparation of PVA solution: 0.96 g of PVA was placed in a beaker, 8 mL of pure water was added, and the beaker was placed on a heating and stirring device. Under heating conditions of 97°C, stirring at 400 / min for 30 min allowed it to completely dissolve to form a uniform solution (mass concentrations were 4%, 6%, 8%, 10%, 12%, and 15%, respectively).

[0077] (2) Prepare rhein solution; take 36 mg of rhein, dissolve it in 0.5 mol / L NaOH solution, and use 2.5 mmol / L hydrochloric acid to adjust the pH to 8.8-9.0.

[0078] (3) Add the rhein solution of step (2) to the PVA solution of step (1), stir for 10 min, and ultrasonicate at 25° C. for 30 min.

[0079] (4) Freeze in a -80°C refrigerator for 1 hour, then take out and thaw in a 37°C water bath for 30 minutes. This is one freeze-thaw cycle.

[0080] Comparative Example 1

[0081] The other aspects are the same as Example 1, except that, in this comparative example, rhein was not added, and only PVA blank hydrogel was prepared, and the mass fractions of the PVA solutions were 4%, 6%, 8%, 10%, 12%, and 15%, respectively.

[0082] Experimental Example 2 Preparation of in situ injectable hydrogel PVA / RH / DOX for the treatment of osteosarcoma

[0083] (1) Preparation of PVA solution: 0.96 g of PVA was placed in a beaker, 8 mL of pure water was added, and the beaker was placed on a heating and stirring device. The PVA was completely dissolved at 97° C. and stirred at 400 / min for 30 min to form a uniform solution.

[0084] (2) Prepare rhein solution; take 36 mg of rhein, dissolve it in 0.5 mol / L NaOH solution, and use 2.5 mmol / L hydrochloric acid to adjust the pH to 8.8-9.0.

[0085] (3) Preparation of doxorubicin hydrochloride solution: Take 25 mg of doxorubicin hydrochloride and dissolve it in 8 mL of the PVA aqueous solution described in step (1), and stir for 15 minutes to dissolve it.

[0086] (4) Add the rhein solution described in step (2) to the doxorubicin hydrochloride solution described in step (3), and stir for 10 minutes to make the solution uniform, and then ultrasonicate at 25° C. for 30 minutes.

[0087] (5) After being frozen at -80°C for 1 hour, the sample was taken out and thawed in a 37°C water bath for 30 minutes. This is one freeze-thaw cycle. After one freeze-thaw cycle, an injectable gel PVA / RH / DOX scaffold with needle permeability can be obtained.

[0088] In other embodiments, the freeze-thaw cycle can be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, or 8 times.

[0089] Experimental Example 3 Preparation of in situ injectable hydrogel PVA / RH / DOX for treating osteosarcoma

[0090] (1) Preparation of PVA solution: 0.96 g of PVA was placed in a beaker, 8 mL of pure water was added, and the beaker was placed on a heating and stirring device. The PVA was completely dissolved at 97° C. and stirred at 400 / min for 30 min to form a uniform solution.

[0091] (2) Prepare rhein solution; take 72 mg of rhein, dissolve it in 0.5 mol / L NaOH solution, and use 2.5 mmol / L hydrochloric acid to adjust the pH to 8.8-9.0.

[0092] (3) Preparation of doxorubicin hydrochloride solution: Take 20 mg of doxorubicin hydrochloride and dissolve it in 8 mL of the PVA aqueous solution described in step (1), and stir for 15 minutes to dissolve it.

[0093] (4) Add the rhein solution described in step (1) to the doxorubicin hydrochloride solution, stirring for 10 minutes to make the solution uniform, and then ultrasonicate at 25° C. for 30 minutes.

[0094] (5) After being frozen at -80°C for 1 hour, the sample was taken out and thawed in a 37°C water bath for 30 minutes. This is one freeze-thaw cycle. After one freeze-thaw cycle, an injectable gel PVA / RH / DOX scaffold with needle permeability can be obtained.

[0095] In other embodiments, the freeze-thaw cycle can be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, or 8 times.

[0096] Experimental Example 4 Preparation of in situ injectable hydrogel PVA / RH / DOX for treating osteosarcoma

[0097] (1) Preparation of PVA solution: 0.96 g of PVA was placed in a beaker, 8 mL of pure water was added, and the beaker was placed on a heating and stirring device. The PVA was completely dissolved at 97° C. and stirred at 400 / min for 30 min to form a uniform solution.

[0098] (2) Prepare rhein solution; take 36 mg of rhein, dissolve it in 0.5 mol / L NaOH solution, and use 2.5 mmol / L hydrochloric acid to adjust the pH to 8.8-9.0.

[0099] (3) Preparation of doxorubicin hydrochloride solution: 15 mg of doxorubicin hydrochloride was dissolved in 8 mL of the PVA aqueous solution described in step (1), and stirred for 15 min to dissolve.

[0100] (4) Add the rhein solution described in step (1) to the doxorubicin hydrochloride solution, stirring for 10 minutes to make the solution uniform, and then ultrasonicate at 25° C. for 30 minutes.

[0101] (5) After being frozen at -80°C for 1 hour, the sample was taken out and thawed in a 37°C water bath for 30 minutes. This is one freeze-thaw cycle. After one freeze-thaw cycle, an injectable gel PVA / RH / DOX scaffold with needle permeability can be obtained.

[0102] In other embodiments, the freeze-thaw cycle can be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, or 8 times.

[0103] Experimental Example 5 Preparation of in situ injectable hydrogel PVA / RH / DOX for treating osteosarcoma

[0104] (1) Preparation of PVA solution: 0.96 g of PVA was placed in a beaker, 8 mL of pure water was added, and the beaker was placed on a heating and stirring device. The PVA was completely dissolved at 97° C. and stirred at 400 / min for 30 min to form a uniform solution.

[0105] (2) Prepare rhein solution; take 36 mg of rhein, dissolve it in 0.5 mol / L NaOH solution, and use 2.5 mmol / L hydrochloric acid to adjust the pH to 8.8-9.0.

[0106] (3) Preparation of doxorubicin hydrochloride solution: Dissolve 10 mg of doxorubicin hydrochloride in 8 mL of the PVA aqueous solution described in step (1) and stir for 15 min to dissolve it.

[0107] (4) Add the rhein solution described in step (1) to the doxorubicin hydrochloride solution, stirring for 10 minutes to make the solution uniform, and then ultrasonicate at 25° C. for 30 minutes.

[0108] (5) After being frozen at -80°C for 1 hour, the sample was taken out and thawed in a 37°C water bath for 30 minutes. This is one freeze-thaw cycle. After one freeze-thaw cycle, an injectable gel PVA / RH / DOX scaffold with needle permeability can be obtained.

[0109] In other embodiments, the freeze-thaw cycle can be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, or 8 times.

[0110] Experimental Example 6 Preparation of in situ injectable hydrogel PVA / RH / DOX for treating osteosarcoma

[0111] (1) Preparation of PVA solution: 0.8 g of PVA was placed in a beaker, 8 mL of pure water was added, and the beaker was placed on a heating and stirring device. The PVA was completely dissolved at 97° C. and stirred at 400 / min for 30 min to form a uniform solution.

[0112] (2) Prepare rhein solution; take 36 mg of rhein, dissolve it in 0.5 mol / L NaOH solution, and use 2.5 mmol / L hydrochloric acid to adjust the pH to 8.8-9.0.

[0113] (3) Preparation of doxorubicin hydrochloride solution: Take 25 mg of doxorubicin hydrochloride and dissolve it in 8 mL of the PVA aqueous solution described in step (1), and stir for 15 minutes to dissolve it.

[0114] (4) Add the rhein solution described in step (1) to the doxorubicin hydrochloride solution, stirring for 10 minutes to make the solution uniform, and then ultrasonicate at 25° C. for 30 minutes.

[0115] (5) After being frozen at -80°C for 1 hour, the sample was taken out and thawed in a 37°C water bath for 30 minutes. This is one freeze-thaw cycle. After one freeze-thaw cycle, an injectable gel PVA / RH / DOX scaffold with needle permeability can be obtained.

[0116] In other embodiments, the freeze-thaw cycle can be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, or 8 times.

[0117] Experimental Example 7 Preparation of in situ injectable hydrogel PVA / RH / DOX for treating osteosarcoma

[0118] (1) Preparation of PVA solution: 0.64 g of PVA was placed in a beaker, and 8 mL of pure water was added. The beaker was placed on a heating and stirring device, and heated at 97°C and stirred at 400 / min for 30 min to completely dissolve the PVA to form a uniform solution.

[0119] (2) Prepare rhein solution; take 36 mg of rhein, dissolve it in 0.5 mol / L NaOH solution, and use 2.5 mmol / L hydrochloric acid to adjust the pH to 8.8-9.0.

[0120] (3) Preparation of doxorubicin hydrochloride solution: Take 25 mg of doxorubicin hydrochloride and dissolve it in 8 mL of the PVA aqueous solution described in step (1), and stir for 15 minutes to dissolve it.

[0121] (4) Add the rhein solution described in step (1) to the doxorubicin hydrochloride solution, stirring for 10 minutes to make the solution uniform, and then ultrasonicate at 25° C. for 30 minutes.

[0122] (5) After being frozen at -80°C for 1 hour, the sample was taken out and thawed in a 37°C water bath for 30 minutes. This is one freeze-thaw cycle. After two freeze-thaw cycles, an injectable gel PVA / RH / DOX scaffold with needle permeability was obtained.

[0123] In other embodiments, the freeze-thaw cycle can also be 3 times, 4 times, 5 times, 6 times, 7 times, or 8 times.

[0124] Experimental Example 8 Preparation of in situ injectable hydrogel PVA / RH / DOX for treating osteosarcoma

[0125] (1) Preparation of PVA solution: 1.2 g of PVA was placed in a beaker, and 8 mL of pure water was added. The beaker was placed on a heating and stirring device. The PVA was completely dissolved at 97° C. and stirred at 400 / min for 30 min to form a uniform solution.

[0126] (2) Prepare rhein solution; take 36 mg of rhein, dissolve it in 0.5 mol / L NaOH solution, and use 2.5 mmol / L hydrochloric acid to adjust the pH to 8.8-9.0.

[0127] (3) Preparation of doxorubicin hydrochloride solution: Take 25 mg of doxorubicin hydrochloride and dissolve it in 8 mL of the PVA aqueous solution described in step (1), and stir for 15 minutes to dissolve it.

[0128] (4) Add the rhein solution described in step (1) to the doxorubicin hydrochloride solution, stirring for 10 minutes to make the solution uniform, and then ultrasonicate at 25° C. for 30 minutes.

[0129] (5) After being frozen in a -20°C refrigerator for 12 hours, the sample was taken out and thawed in a 37°C water bath for 30 minutes. This is a freeze-thaw cycle. After completing a freeze-thaw cycle, an injectable gel PVA / RH / DOX scaffold with needle permeability can be obtained.

[0130] In other embodiments, the freeze-thaw cycle can be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, or 8 times.

[0131] Experimental Example 9 Preparation of bone repair hydrogel PVA / RH / DOX / nHA for bone support

[0132] (1) Preparation of PVA solution: 0.96 g of PVA was placed in a beaker, 8 mL of pure water was added, and the beaker was placed on a heating and stirring device. The PVA was completely dissolved at 97° C. and stirred at 400 / min for 30 min to form a uniform solution.

[0133] (2) Preparation of nanohydroxyapatite solution: 15 mg of nanohydroxyapatite was taken and dissolved in 2.5 mmol / L hydrochloric acid.

[0134] (3) preparing a rhein solution: taking 36 mg of rhein, dissolving it in a 0.5 mol / L NaOH solution, and adjusting the pH to 8.8-9.0 using 2.5 mmol / L hydrochloric acid. The hydrochloric acid is the hydrochloric acid solution obtained by dissolving the nano-hydroxyapatite in the above step (2).

[0135] (4) Preparation of doxorubicin hydrochloride solution: Take 25 mg of doxorubicin hydrochloride and dissolve it in 8 mL of the PVA aqueous solution described in step (1), and stir for 15 minutes to dissolve it.

[0136] (5) Add the rhein solution described in step (1) to the doxorubicin hydrochloride solution, stirring for 10 minutes to make the solution uniform, and then ultrasonicate at 25° C. for 30 minutes.

[0137] (6) After being frozen at -80°C for 1 hour, the sample was taken out and thawed in a 37°C water bath for 30 minutes. This is one freeze-thaw cycle. After completing five freeze-thaw cycles, a hydrogel PVA / RH / DOX / nHA scaffold with bone-supporting strength was obtained.

[0138] In other embodiments, the freeze-thaw cycle can be 6 times, 7 times, or 8 times.

[0139] Experimental Example 10 Preparation of bone repair hydrogel PVA / RH / DOX / nHA for bone support

[0140] (1) Preparation of PVA solution: 0.8 g of PVA was placed in a beaker, 8 mL of pure water was added, and the beaker was placed on a heating and stirring device. The PVA was completely dissolved at 97° C. and stirred at 400 / min for 30 min to form a uniform solution.

[0141] (2) Preparation of nanohydroxyapatite solution: 10 mg of nanohydroxyapatite was taken and dissolved in 2.5 mmol / L hydrochloric acid.

[0142] (3) preparing a rhein solution: taking 36 mg of rhein, dissolving it in a 0.5 mol / L NaOH solution, and adjusting the pH to 8.8-9.0 using 2.5 mmol / L hydrochloric acid. The hydrochloric acid is the hydrochloric acid solution obtained by dissolving the nano-hydroxyapatite in the above step (2).

[0143] (4) Preparation of doxorubicin hydrochloride solution: Take 25 mg of doxorubicin hydrochloride and dissolve it in 8 mL of the PVA aqueous solution described in step (1), and stir for 15 minutes to dissolve it.

[0144] (5) Add the rhein solution described in step (1) to the doxorubicin hydrochloride solution, stirring for 10 minutes to make the solution uniform, and then ultrasonicate at 25° C. for 30 minutes.

[0145] (6) After being frozen at -80°C for 1 hour, the sample was taken out and thawed in a 37°C water bath for 30 minutes. This is one freeze-thaw cycle. After completing five freeze-thaw cycles, a hydrogel PVA / RH / DOX / nHA scaffold with bone-supporting strength was obtained.

[0146] In other embodiments, the freeze-thaw cycle can be 6 times, 7 times, or 8 times.

[0147] Experimental Example 11 Preparation of bone repair hydrogel PVA / RH / DOX / nHA for bone support

[0148] (1) Preparation of PVA solution: 0.96 g of PVA was placed in a beaker, 8 mL of pure water was added, and the beaker was placed on a heating and stirring device. The PVA was completely dissolved at 97° C. and stirred at 400 / min for 30 min to form a uniform solution.

[0149] (2) Preparation of nanohydroxyapatite solution: 5 mg of nanohydroxyapatite was taken and dissolved in 2.5 mmol / L hydrochloric acid.

[0150] (3) preparing a rhein solution: taking 36 mg of rhein, dissolving it in a 0.5 mol / L NaOH solution, and adjusting the pH to 8.8-9.0 using 2.5 mmol / L hydrochloric acid. The hydrochloric acid is the hydrochloric acid solution obtained by dissolving the nano-hydroxyapatite in the above step (2).

[0151] (4) Preparation of doxorubicin hydrochloride solution: 15 mg of doxorubicin hydrochloride was dissolved in 8 mL of the PVA aqueous solution described in step (1), and stirred for 15 min to dissolve.

[0152] (5) Add the rhein solution described in step (1) to the doxorubicin hydrochloride solution, stirring for 10 minutes to make the solution uniform, and then ultrasonicate at 25° C. for 30 minutes.

[0153] (6) After being frozen at -80°C for 1 hour, the scaffold was taken out and thawed in a 37°C water bath for 30 minutes. This is one freeze-thaw cycle. After completing 6 freeze-thaw cycles, a hydrogel PVA / RH / DOX / nHA scaffold with bone support strength can be obtained.

[0154] In other embodiments, the freeze-thaw cycle can be 7 or 8 times.

[0155] After the preparation of PVA / RH / DOX and PVA / RH / DOX / nHA scaffold materials, the present invention further performs characterization analysis, mechanical property analysis and other experiments on the prepared hydrogel system to verify that the material is an ideal osteosarcoma treatment system. According to the treatment plan for osteosarcoma, the selected preparation processes are: PVA / RH: Example 1; PVA / RH / DOX: Example 2; PVA / RH / DOX / nHA: Example 9. The specific methods and results are shown below:

[0156] 1. After preparing PVA blank hydrogel, the present invention first investigated the effect of PVA mass fraction (4-15%) on the formation of hydrogel. The results are as follows: Figure 1 As shown in the figure, 4% PVA cannot form hydrogel after multiple freeze-thaw cycles, and does not have the ability to obtain hydrogel through freeze-thaw cycles. When the mass fraction of PVA reaches 8% or above, the number of freeze-thaw cycles only needs to be 3.

[0157] 2. Effect of RH addition on hydrogel formation

[0158] The present invention further investigates the effect of adding RH on the formation of hydrogels, and the results are as follows Figure 2 As shown, after adding rhein to PVA within a PVA mass fraction of 10-15%, the number of times required for hydrogel formation is reduced from 3 times required for PVA alone to 1 time, and the hydrogel morphology can be obtained through one freeze-thaw cycle, and the number of freeze-thaw cycles required is reduced to one third of the original.

[0159] 3. Effect of PVA mass fraction and RH addition on hydrogel formation

[0160] like Figure 3 As shown, within the PVA mass fraction range of 6-8%, the number of freeze-thaw cycles required for the rhein-loaded group was reduced by 2 compared with the PVA group. Within the PVA mass fraction range of 10-15%, the blank preparation group required 3 or more freeze-thaw cycles to form a hydrogel, while the rhein-loaded group reduced the number of freeze-thaw cycles to 1 cycle to form a hydrogel. The two hydrogel systems cross-linked to form an interpenetrating network, effectively reducing the number of freeze-thaw cycles required to prepare injectable hydrogels, which is one-third of the number of freeze-thaw cycles required for PVA alone.

[0161] 4. Screening of preparation conditions of PVA / RH / DOX hydrogel by orthogonal experiment

[0162] like Figure 4 As shown, an orthogonal experiment was designed to verify several conditions such as PVA mass fraction, freeze-thaw times, DOX loading, and RH dosage range, proving the adjustability of the hydrogel system.

[0163] 5. Orthogonal experimental results of hydrogel system

[0164] like Figure 5 As shown in the figure, the 12 orthogonal experiments set up according to their respective conditions obtained the hydrogel system. By observing the results after each freeze-thaw cycle, it can be found that: (1) The RH drug loading concentration is greater than 6 mg / mL, which can participate in the cross-linking of the hydrogel and reduce the number of freeze-thaw cycles. In addition, as the mass fraction increases, the formed gel is more stable. (2) The DOX drug loading can reach 3 mg / mL, indicating that the hydrogel system has excellent drug loading performance and the drug loading can be adjusted according to actual needs. (3) When the mass fraction of PVA is greater than 10%, the number of freeze-thaw cycles required to form a gel is significantly reduced, and the larger the mass fraction, the fewer the number of freeze-thaw cycles required. (4) Multiple freeze-thaw cycles after gel formation will make the hydrogel harder and have better mechanical properties.

[0165] 6. SEM image of PVA / RH / DOX hydrogel

[0166] like Figure 6 As shown, SEM imaging of PVA / RH / DOX hydrogel shows that the injectable hydrogel will form an obvious cross-linked network structure, and the cross-linked network is uniform, indicating that the two gel networks are mutually transferred to form a uniform network.

[0167] 7. SEM image of PVA / RH / DOX / nHA hydrogel scaffold

[0168] like Figure 7 As shown, SEM imaging of the PVA / RH / DOX / nHA hydrogel scaffold shows that the hydrogel forms an obvious cross-linked network structure.

[0169] 8. Morphology and injectability analysis

[0170] like Figure 8 As shown, the PVA / RH / DOX of the present invention can form an injectable hydrogel by freeze-thawing once, and Figure AB verifies the successful formation of the hydrogel by the inversion method. Figure C is a hydrogel obtained by a 1mL syringe. The hydrogel is quickly formed by the syringe and can stand upright without slipping, showing the properties of the hydrogel. Figure D verifies the needle-passing property of the hydrogel.

[0171] 9. PVA / RH / DOX / nHA hydrogel scaffold support performance

[0172] like Fig. 9 As shown, the present invention places a 500 g weight on the PVA / RH / DOX / nHA hydrogel scaffold, and the hydrogel scaffold is able to support the weight with only slight deformation.

[0173] 10. Mechanical properties analysis

[0174] The present invention performs compression and tensile performance analysis on bone support PVA / RH / DOX / nHA with excellent support performance. Gel analysis is performed on a microcomputer-controlled electronic universal testing machine of model CMT4503.

[0175] like Fig.10 As shown, the tensile strength was tested at a speed of 50 mm / min, and the PVA / RH / DOX / nHA hydrogel scaffold broke after a deformation of 200%. The compressive strength was tested at a compression speed of 5 mm / min, and the PVA / RH / DOX / nHA hydrogel scaffold collapsed after a deformation of 80%. This mechanical strength performance has the ability to support bone damage structures.

[0176] 11. PVA / RH / DOX hydrogel can inhibit the growth of osteosarcoma cells (K7M2)

[0177] 1×10 4 Osteosarcoma cells (K7M2) were inoculated in a 96-well plate at a density of 100 cells / well, and a series of PVA, PVA / RH, PVA / DOX and PVA / DOX / RH solutions were prepared and co-cultured for 24h and 48h. The UV absorption of each well at 490nm was detected using a multifunctional microplate reader to calculate the survival rate of cells under different drug concentrations.

[0178] The calculation formula is as follows:

[0179] Cell survival rate,

[0180] like Fig.11 As shown, the PVA blank carrier has good biocompatibility, PVA / RH, PVA / DOX and PVA / RH / DOX all have obvious concentration-dependent tumor cell killing ability, and the final preparation group has better killing effect under the same conditions.

[0181] 12. PVA / RH / DOX hydrogel induces apoptosis of osteosarcoma cells (K7M2)

[0182] like Fig.12 As shown, the PVA / RH / DOX hydrogel had a better apoptosis-inducing effect on osteosarcoma cells (K7M2) under 24h conditions, as verified by Annexinv / PI double staining flow cytometry.

[0183] 13. PVA / RH / DOX hydrogel inhibits the development of osteosarcoma

[0184] like Fig.13As shown, an osteosarcoma model was constructed on mice, and PVA / RH / DOX hydrogel was used for single in situ injection. After 14 days of observation, the mice were killed and dissected to obtain tumor tissue. This experiment can verify that the PVA / RH / DOX hydrogel of the present invention can effectively inhibit the development of osteosarcoma.

[0185] 14. PVA / RH / DOX hydrogel inhibits the development of osteosarcoma volume

[0186] Based on the tumor tissue obtained, the volume of the tumor tissue was measured using a digital caliper to measure the apical (AP) and longitudinal (L) sides of the tumor. The AP measurement spanned the kneecap and the L measurement was located anterior to the tibia. The primary tumor volume (V) was measured according to the following formula (1).

[0187]

[0188] like Fig.14 As shown, the PVA / RH / DOX hydrogel group of the present invention has significant differences from other groups and has better osteosarcoma inhibition effect.

[0189] In summary, the present invention provides a scaffold material for osteosarcoma treatment (PVA / RH / DOX) and bone support hydrogel (PVA / RH / DOX / nHA) on the basis of providing PVA / RH hydrogel, combining PVA / RH hydrogel, chemotherapy drug DOX, and nHA, wherein PVA / RH hydrogel can load drugs, chemotherapy drug DOX has excellent tumor killing ability, and nHA can enhance the mechanical properties of hydrogel. PVA / RH hydrogel and chemotherapy drug DOX are combined to obtain PVA / RH / DOX hydrogel, which is simple to prepare, has excellent biocompatibility and needle permeability, and can obtain good therapeutic effects through a single injection. PVA / RH hydrogel, chemotherapy drug DOX, and nHA are combined to produce PVA / RH / DOX / nHA, which can be used as a bone support material, and has excellent support performance verified in vitro.

[0190] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A multifunctional hydrogel, characterized in that: It is mainly composed of PVA and rhein, with a mass ratio of PVA: rhein = 0.2~2 g: 10~100 mg; when preparing the multifunctional hydrogel, rhein is dissolved in an alkaline solution, wherein the alkali in the alkaline solution is any one of sodium hydroxide, sodium carbonate or sodium bicarbonate.

2. The multifunctional hydrogel according to claim 1, characterized in that: Mass ratio PVA: rhein = 0.48~1.5 g: 36~72 mg.

3. The multifunctional hydrogel according to claim 2, characterized in that: Mass ratio PVA: rhein = 0.64~1.2g: 36~72 mg.

4. The multifunctional hydrogel according to claim 1, characterized in that The hydrogel is also added with chemotherapeutic drugs and / or materials that promote bone support; When only chemotherapy drugs were added to the hydrogel, the mass ratio of PVA: rhein: chemotherapy drugs = 0.2~2 g: 10~100 mg: 5~35 mg; When only the material promoting bone support is added to the hydrogel, the mass ratio of PVA: rhein: material promoting bone support = 0.2~2 g: 10~100 mg: 2~20 mg; When chemotherapeutic drugs and materials promoting bone support are added to the hydrogel, the mass ratio of PVA: rhein: chemotherapeutic drugs: materials promoting bone support = 0.2~2 g: 10~100 mg: 5~35 mg: 2~20 mg.

5. The multifunctional hydrogel according to claim 4, characterized in that: The hydrogel is also added with chemotherapeutic drugs and / or materials that promote bone support; When only chemotherapy drugs were added to the hydrogel, the mass ratio of PVA: rhein: chemotherapy drugs = 0.64~1.2 g: 36~72 mg: 10~25 mg; When only the material promoting bone support is added to the hydrogel, the mass ratio of PVA: rhein: material promoting bone support = 0.64~1.2 g: 36~72 mg: 5~15 mg; When chemotherapeutic drugs and materials promoting bone support are added to the hydrogel, the mass ratio of PVA: rhein: chemotherapeutic drugs: materials promoting bone support = 0.64~1.2 g: 36~72 mg: 10~25 mg: 5~15 mg.

6. An injectable hydrogel, characterized in that: include: The multifunctional hydrogel and chemotherapeutic drug of claim 1; wherein the mass ratio of PVA: rhein: chemotherapeutic drug = 0.2~2 g: 10~100 mg: 5~35 mg.

7. An injectable hydrogel according to claim 6, characterized in that: Mass ratio PVA: rhein: chemotherapy drug = 0.64~1.2 g: 36~72 mg: 10~25 mg.

8. A bone-supporting hydrogel, characterized in that: include: The multifunctional hydrogel, chemotherapeutic drug, and material promoting bone support in claim 1; wherein the mass ratio of PVA: rhein: chemotherapeutic drug: material promoting bone support = 0.2~2g: 10~100 mg: 5~35 mg: 2~20 mg.

9. The bone-supporting hydrogel according to claim 8, characterized in that: Mass ratio PVA: rhein: chemotherapy drugs: materials promoting bone support = 0.64~1.2 g: 36~72 mg: 10~25 mg: 5~15 mg.

10. The hydrogel according to any one of claims 4 to 9, characterized in that: The chemotherapy drug is any one or more of doxorubicin hydrochloride, adriamycin, and albendazole.

11. The hydrogel according to claim 10, characterized in that The chemotherapy drug is doxorubicin hydrochloride.

12. The hydrogel according to any one of claims 4-5 or 8-9, characterized in that: The material promoting bone support is nano-hydroxyapatite.

13. The hydrogel according to claim 12, characterized in that The molecular weight of the PVA is 89,000-98,000.

14. The method for preparing an injectable hydrogel according to claim 6, characterized in that: The following steps are involved: Step 1, preparing a PVA solution: dispersing PVA in water, stirring under heating conditions to completely dissolve it and form a uniform solution; wherein the mass concentration of PVA in the PVA aqueous solution is 4-20%; Step 2, preparing a rhein solution; dissolving rhein in an alkaline solution to make it completely dissolved, and then adjusting the pH to 8.0-9.4 using hydrochloric acid; Step 3, preparing doxorubicin hydrochloride solution: dispersing doxorubicin hydrochloride in the PVA aqueous solution described in step 1, and stirring or ultrasonicating to dissolve it; Step 4, adding the rhein solution described in step 2 to the doxorubicin hydrochloride solution described in step 3, the process is accompanied by stirring, and then ultrasonicating after stirring evenly; Step 5, freeze in a -20℃~-80℃ refrigerator for 1 hour, take out, and thaw in a 37℃ water bath for 30 min. This is one freeze-thaw cycle; the freeze-thaw cycle ranges from 1 to 8 times.

15. The preparation method according to claim 14, characterized in that: In the step 1, the mass concentration of PVA is 8-15%.

16. The preparation method according to claim 14, characterized in that: In the step 2, the pH value is 8.8-9.

0.

17. The preparation method according to claim 14, characterized in that: In step 5, the freeze-thaw cycle is 1-7 times.

18. The preparation method according to claim 17, characterized in that: In step 5, the freeze-thaw cycle is 4 to 6 times.

19. The method for preparing a bone-supporting hydrogel according to claim 8, characterized in that: The following steps are involved: Step 1, preparing a PVA solution: dispersing PVA in water, stirring under heating conditions to completely dissolve it and form a uniform solution; wherein the mass concentration of PVA in the PVA aqueous solution is 4-20%; Step 2, preparing a nano-hydroxyapatite solution: taking nano-hydroxyapatite and dissolving it with hydrochloric acid; Step 3, preparing a rhein solution; dissolving rhein in an alkaline solution to make it completely dissolved, and then adjusting the pH to 8.0-9.4 using hydrochloric acid; wherein the hydrochloric acid is the hydrochloric acid solution obtained after dissolving the nano-hydroxyapatite in step 2; Step 4, preparing doxorubicin hydrochloride solution: dispersing doxorubicin hydrochloride in the PVA aqueous solution described in step 1, and stirring or ultrasonicating to dissolve it; Step 5, preparing a synergistic osteosarcoma treatment and bone support hydrogel: adding the rhein solution to the doxorubicin hydrochloride solution, the process is accompanied by stirring, stirring evenly and then ultrasonicating; Step 6, after freezing in a -20~-80℃ refrigerator for 1 hour, take out and thaw in a 37℃ water bath for 30 minutes, which is a freeze-thaw cycle; wherein, this freeze-thaw cycle ranges from 1 to 8 times.

20. The preparation method according to claim 19, characterized in that: In the step 1, the mass concentration of PVA is 8-15%.

21. The preparation method according to claim 19, characterized in that: In the step 3, the pH value is 8.8-9.

0.

22. The preparation method according to claim 19, characterized in that: In step 6, the freeze-thaw cycle is 1-7 times.

23. The preparation method according to claim 22, characterized in that: In step 6, the freeze-thaw cycle is 4 to 6 times.

24. The preparation method according to claim 14 or 19, characterized in that: In the step 1, the heating temperature is 80-100° C. and the heating stirring time is 20 min-1 h.

25. The preparation method according to claim 24, characterized in that: In the step 1, the heating temperature is 95-100°C.

26. The preparation method according to claim 25, characterized in that: In the step 1, the heating temperature is 97°C.

27. The preparation method according to claim 24, characterized in that In the preparation of rhein solution, the mass concentration of rhein is 4-25 mg / mL, and the concentration of hydrochloric acid is 1 mmol / L-5 mmol / L.

28. The preparation method according to claim 27, characterized in that: In the preparation of the rhein solution, the mass concentration of rhein is 4-10 mg / mL.

29. The preparation method according to claim 24, characterized in that When the doxorubicin hydrochloride solution is prepared, its mass concentration is 0.1-3 mg / mL.

30. Use of the hydrogel according to any one of claims 1-9 or 10 or 11 or 12 or 13 in the preparation of a drug for treating tumors and / or promoting bone support; the tumor is osteosarcoma.

Images